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METHODS AND PRODUCTS ~'CiR MODULATION OF REPRODUCTIVE PROCESSES
AND FOR DIAGNOSIS, PROGNOSTICATION AND TREATMENT OF RELATED
CONDITIONS
The invention .relates mE:thods and products whir_h
may be used to modu:Lat a .reproductive gr,~c.:esses, such as
fertility. The i.nventic>n f_u.rther relates to the diagnosis,
prognostication and tr.~atmernt ofv assoc gated conditions, such
as endometriosis and infertility.
In <~n aspect, the invention describes that the
levels of cer.i~ain markers c:orrel.ate wi.tO. conditions such as
endometriosis. In an ~mxnodiment, the marker. is interleukin-1
receptor type II ( IL-~~ R:i I ) . In .-r fu:rthez: embodiment, the
marker is morzocyte che:rnotactic protein-i (MCP-1) . In a
1_'~ further embodiment, the marker i_s rnac:rophage migratory
inhibitory factor (MIFF . l.zr emb:~dimen~:~, tree invention
provides diagnostic and prognostic methods involving
determining a concentr_zt~:ion or level. of c;ne or more of: these
markers in a sample. I~ an embodiment, the sample is a
tissue or body fluid c:~>i.~a:irced from a suk>ject. In an
embodiment, t:he subject is an animal; in a further
embodiment, a mammal, ~.rn a furtrrcer embodiment, a human. In
embodiments, t:he tissue or body fluid includes but is not
limited to blood, serum,, plasma, per:itor~eal f luid, monoc:ytes,
endometrial t__ssue, an~i endometrial ce=L~..s .
In an embodiment, an irncre<~se i.n t.hc=_ level of MCP-1
is used to diagnose endometriosis.
In an embadiment, a decrease -ire the level of IL-
1RII is used t:o diagnc.~e er.domet::rios:i;s .
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In embc>dimerut;, the l~-vEls of the above noted
markers may be dE=term:.r~c d by dete,vmining protein level or by
determining the level of= n~cl~~i~ acid (e. g. mRNA) encoding
the protein markers.
In another ~:s~.ect, the =invention provides methods
of treating c:ondwtion:; such as endometriosis, the method
comprising the modulai i;_:n, in af~ embcdi_ment, the inhibition,
of IL-1 acti~,Tity. In an embodiment, the method involves
administering to a sukvjf:ct an IL,-_. antagonist:.
In anot:her .:aspect, the invention provides methods
of treating c:onds_tion: such as enc~ometricosis, the method
involving admini:~ terir:~:a t.o a suK:>j cec t IL-:1_RI ~ .
The det:ermir~at:;~on of thE:~ markers, particularly IL-
1RII, as noted above, may also be used for predicting for
1!~ example the window of im.plantat i.or~. of t::le endometrium, and
the receptiv_i_ty <:f eml:r-yon.ic .implantation.
In another ;:3s~>ect, the =,_nvention x>rovides methods
and material> for cont:racepti.on, t:cr example based on the
prevention of: em~:;.ryon c implant~.~ti_on.
The inventic.n further relates to compositions,
comprising for ex;amplc an I:L~-1 antagonist or IL-1RII in
admixture with a pharruac;eu~ic:all.y acceptable carrier.
The inventic:~n further re_L<~tes tc»~ses and
commercial packages rt l Bating to true above-noted methods and
2'.~ products .
SUMMARY OF THE INVENTION
Acc:ordi.ng t~.~ ari aspect: cJf the present invention,
there is pro~.Tided a method of assessing a reproduction-
associated d_i_sease in a sub-ject, :;aid method comprising
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(a) determining a test: level of a parameter selected from the
group cc~nsisti.ng u1-
( i. ) MI F prote :i..o ; ~:nci,
(i.i) MIF encc:~linc~ RNA
p in a ti r; sue or b:_sdy f=luid frorr~ said subject; and
(b) comparing said te.t level t:o a stacodard selected from
the groLZp consi::: i_ag of a corresponding level of said
parameter i_n a t:essue or body fl.uia of a control.
subj ect; anc~ a c;~ rn:~ce:~pondi rng ~.evel of said parameter in
a tissue or body l:Luid obtained ~=rom sa~~d subject at an
earlier time; wherein an increase in said test 1_evel is
indicative of rei:~:~-oduction--assocJ_~ated d:LSease.
According to another as~:~ect of the present
invetion, there is prc~vided a metr~od of assessing endometrial
receptivity i.:z a subjE~;:;t:, said method c:~mpr:ising:
(a) determining, in s~.ic: subject, a test level of a parameter
selected from th~~~ group cc>nsist=i_nca of:
(i) MIF protEiri;
(ii) MIF' encc.::~:irig RNA;
(iiil IL.-1RI~~ ;~~r~~tein;
(iv) IL-1RII erj~~~:~dir~g RNA; an~:~
(v) LL-1RT=L ~c;ti.vi_t.y;
(b) comparing sa~_d te::t level. wi.ti'u a standa:rr: selected from
the group consist:i og o_E a co.rre:pc~ruding level of raid
2.~ parameter from a :.::~nt:rol_ subject=; and a corresponding
level of said parameter obtained 1 rom said subject. at an
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earlier time, wh<~rein a decrease cf said test level is
indicative of en.:iometxi.al re:;eptivit~~.
According tc: st.ill another aspect of the prensent
invention, there i_s pz:::~~~ idc~d a metrlc>c~ :~?. assessing a
~~ reproduction-~rsso.~i ate; disease ~,_n a suk» ect., said method
comprising .
(a)determining a test ~..evel. of a pa:ramet::er selected from the
group consisting of .
( i ) I L-1F~II. p:c~c:~teirl;
(ii) IL-iRII encoding R~dA: and
( iii ) IL-1RII activity;
in serum from sa :i c, ubj ect~; :mc~
(b)comparing said test Level to a standard selected from the
group consisting c>i:_ a c:orresporzdirig .level of said
1'.i parameter ir1 ser~.nn of- a control subject; and a
corresponding le~yel of said parameter in serum obtained
from said subject, -its an earl:i_er° time; wherein a decrease
in said test l.ev:~:L is indi~~a~.ive of reproduction-
associated disea.:-;e.
Ac<:ording tc~ yet another aspect of the present
invetion, there i s prcwi_ded use of IL-1RII for the prevention
or treatment c~f a repzwoiuction-a.~s,!ociat~~d di.s~ease in a
subject.
According tca ~~ fu.rt:her_- aspect of i~he present
2.'p invention, there is pzvevided a commercial package comprising
means for assessing tx~c~ level. of a pararneter selected from
the group corv,sist:ing c;:f
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(i) MIF ~.~rotein; and
( ii ) MI F encoc l.ing RNA
in a tissue or body fl,ai::l c;f a subject=; together with
instructions for diagn:~s:._s, prognostic~ition, or both, of
5 reproduction-~.ssoci_ate:::1 alisease :un said sLibje;~t.
Acc~~rding tc::~ ;Tf~t. a further a:>pect o= the present
invention, there is pr_~:.;~.~-._t~ed a cc~m:nerc~~_a1 pac)cage comprising
means for assessing tr~~== t_eve l_ of a pax:anieter ;elected .from
the group of:
( i ) _CL-1RII pvotein;
( ii ) IL-1RI I f=>ncoding RNA,: and
(iii; IL--1RII ac:~tivrity;
in serum of a subject, t:.ogether with instructions for the
diagnosis, or procanost_ic: ~tion, oz bot:h, of reproduction-
associated di~~ease in .aid subject.
Acc~~rdir~g tc;t::i.ll a further aspect of the present
invention, there is pr::;T,>:idec~ a comrnex:vial package comprising
means for assessirng trr~_i.eve~_ ef a p~iz:ameter :-Selected from
the group cont,isting c::
(i) MIF ~::rote.r:;
(ii) IL-_LRII )protein;
(iii; MIF~~ enc~:~ding RNA;
( iv) IL-1RII font-oding RNA,; ar_c~
(v) =CL-1RII a;vt:i_vi_ty;
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in an endometrial tis~.ue. of a subject, t=ogether with
instructions 1 or the c:ievermiriata~~r. of ~,ne er~dometrial
receptivity i_n said sL.bject.
According tc ~an-other as~:~c-~ct oa the present
invetion, there i s prc: vi.ded a comrner~;ia.~_ package compri sing
IL-1RII and instructic_;ns for the ~>reven~~icn or treatment of a
reproduction-associat~:d disease in a s abject .
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1. Dis1=ributior3 ~~'}f MCP-1 ~ooncentr~ations in peritoneal
fluid of normal c:ontrc:~:l_s (n = 4~~) and p~:~t:ieruts with
endometriosis (n = 57 ) ~ccordinc~ t:.o st=age of disease.
Horizontal bars, Medi~~.ns of MCP-l conca_nt.rati.ons; horizontal
dashed line, shutoff v~~.:Lue ( 100 pg%m-~ ) .
Figure 2. Chemotact:ic response of U93~7::ells to plasma from
normal controls and p~:.tients with endometriosis. 0937 cells
were stimulated with c:l:ibutryl. cyclic adenos:i_ne monophosphate
to induce ditferentiat:ion and used at 6.70 x 103 cells per
well. Results were exf7:ressed as mE:ean number of cells that
migrate to lower side of membrane per well ~ SEM. Biologic
activity of MCP-1.. was e~aaluatE~d by pre.i:ncubat:ing plasma
samples with polyclom:~:l rabbit anti-MrP-7. antibody (I:500
dilution) forty 30 minut:.e=s. at 3'7°cv 1>efore add:it:ion of
differentiated U937 cel;.s r_o top wells. Preim.mune rabbit
serum was ass~~yed in :canoe E-ashion w_itizoa.zt~ regression of
2'~ activity. N-formyl-met:~.:i.ony:1-leucyl-pher~yl.a:lanine (FMLP) . 10-'
mol/L and phc>sphate-buf'.ferec~ saline sol;ztion were used <~s
positive and negative ~.~c>nt.rol.s, respectively. N, Normal; E,
endometriosia. Asterisk, p : 0.i5, versus control; two
asterisks, p < 0.01, ~~e.rsus contrc;:l.
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Figure 3. Re~~:resentat:iv~:~ i;i...l.ust:ratio:n ~t:v MCi:'-1 immunostaining
in the endomei_rium of ,nrm~il controls (A, proliferative day
9, immunostaining scow: f~ 1 ; B, sE:,c::ret:ory day 19,
immunostaining score i' anci pat.:ier:ts ~r-'~~.::lv~ endometriosis (C,
_'~ proliferative day S, :i.rrrrrrmunostaining score 2; D, secretory day
24, immunostaining scc:~r_~c:~ 2 i . Not:.e t:hf~ i~iown positive
immunostaininc~ in endc::rnc~t.ria.l glands of women with
endometriosis,. particr.::L.~rly in ttlE: secrfr.tory phase, compared
with that of normal sl;.kyj ecr;t::s . Magni f :icaiv: ion, ;333 .
Figure 4. Detection of C~CP-1 mRNA :in the endometrium by in
situ hybridization. Se:aians were hybricii_zed with biotin-
labeled cDNA probes. E;'v_r::t:irn detection mars performed using a
rabbit polycl.onal anti.--r:ic~tin antibody, a bio~tinylated goat
anti-rabbit polyclonal_ antibody, and fluorescein
l~ isothiocyanate-conjugated ,treptavid:i.r~, r~espe~:tively. Slides
were treated with propidium iodine, which makes the nucleus
visible in ye7_low-orange upon UV excitation, and mounted in
the presence of an ant..:i.--nad~ng agent (p-pheny:lenediamine) .
Appearance of endometr i.a 1 glands ( d ) an<i strorna ( s ) at X167
(Al) and X666 (A2) magru::_:f::Lc.:,ation fo:liowi.ng hybridization, and
staining with propidil.z.m iodine. t~dote tl:e green-yellow
hybridization signal (m:row) that could only be observed at
X1665 magnification (Bi 1:>.redorninant:l~r :_.n endornetrial glands.
Figure 5. Representati.~uE. illust.rati_orl of MCP-v' mRNA
expression in the endo-netrium of normal controls (A,
proliferative day 13, :~.c::-~.i:~E: i; B, sec:rE,tcry day 22, score 1)
and patients with endoi:net::ric>sis (C, proliferat;ive day 12,
score 2; D, secretory c~a~n 2 ~~, score 2) . Note the positive
green-yellow :pots (ar:,ows) in endom.etrial glands of women
with endometriosis, par~t:ic.ularly in the secretory phase,
compared with that cf normal sub_ect:~. Magnification, X1665.
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Figure 6. Rep:resentat~ ve illustration ot= IL--1RII
immunostainir:~g in tile nu.nan endometriu~m. Sections of
endometrial tissue we:ae in,.ubatf~d with mouse monoclonal anti-
IL-lRiI antix:,ody (A, l:,r-:~l.iterat_ivEe day 1.::~; B, secretory day
'_~ 24; original magnific2~tz.c>n, _x_6g;~ or with an equivalent
concentration of normal mon.zse Ic~G~; (C _gild D, respectively;
original magr~:i_ficat=iorr, X63) . Sect. ions were then incubat=ed
successively with. biot.i:nylated goat anti-mouse polyclon<~l
antibody and <~vidin-b_i of inyl,3t:ec~ r:orser_ac~.isri peroxidase
complex. The immunore~:<:tiorl was re~veal~=c_~ witch
diaminobenzidine (brown stai_ruinc~ j and _mrrlatoxyl.in was used
for counterstaining (l:.L~.ae w7t:ainv,ng) . N~~t:e the brown fine
positive staining in v::.romal anc~ epit'ne~~_al c~ell.s (cellular
staining) (E-H; c.rigirva.l m<~gniflcati~:~n, X26F~) , and the brown
1_'> deposit (arrow) that ~ s pr:imari 1y :Located at the apical side
of glandular (E, secrE_t_c~~ry phase day %4 anti surface (F,
secretory pha:>e day 1~ rp:i_t:lzeli.:an,, or r.~ore spread within the
glands lumen (G, secrFt:c>ry phase d~~y i6. Positive
immunostaininc~ is also, c=i;wtec~ed :ir, i_;5o1<:rt:ed stromal cel7_s (c)
(G, secretory phase day .16;~ and microve=~sel=~ (v) (H,
secretory pha:~e day 24 j foi_mc~ in the s.~~oma in the secretory
phase of the r~ens~ruai c°~cl:Le. s -- st:roma, g = gland.
Figure 7. Dua=_ .immurnofluo:resc~ent~ staining of IL-1RII (A) and
IL-1(3 (B) i.n t=he enc~orreei:::ri<~1. t:issuc~ of= ricrmal worsen. Ti:~sue
2~~ sections were succ~essi~,rE_:Ly incubated w:i_t:r~ mouse monoclonal
anti-IL-1RII antibody, r_abb_ir poly<~I_ona~' anti-IL-1 antibody,
and biotinylat:ed goat _:rr:t~ i-r<~bbi t arWihc,dy ~;efore being
incubated sirnultaneou.l_~y-r with rhodam:ine--c:onj ugated goat anti-
mouse antibody and fla:~:resc:e:in i~~ot:.h:ioc~~~~anat:e-conjugated
streptavidin. Seri-al sec=t.ions incubated with normal mouse and
normal_ rabbit IgGs intE,~c~ «:E t.rie hr:imary ant ibodies were
included as negative c;~ntrol:~ (C anti D> (ori.ginal
magnification, X160) . ~Z«tf~ the co-exp:ression of IL-1RII (red
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color; and II_~~-1(3 (q:rec~~n--yellow c:olor_ ) within the lumina=L
deposit in er~dometria.: glands. Data from a normal woman in
the secretory phase of true menstrua'~ cy,le (day 23).
Figure 8. Re~~:resentattv~~~ ialustration of IL-1RII
_'> immunostainirrg in the t~::~dornetr_ium of women with and without
endometriosis. A: Norrr.al sf::cretory day f_4, iuminal staining
score 3, cel7.ular slvainirv~ score 2 in 5-_:rcma:l and epithelial
cells . B: Endomet riosi > st<_rge secret:ory day 26, luminal
staining score 0, cellca.:Car staining score 2 in stromal and
1C1 epithelial cea.ls. Nota the lack of en~:l,~rnetr~.a1 glands in B.
Original magnificatior x:160.
Figure 9. Wesi~ern blot: analysis :~f I::L-1RII expression in the
endometrial tissue. A: f~lo.rma.l women: d<~~~ 10 (lanes l and 3)
and day 24 (lanes 2 or d 4 j . B: Women wii~h endometriosis:
1 ~~ stage II, day I3 ( l.anE > 1 and 3 ) and s rage I:I, day 25 ( 7_anes
2 and 4 ) . Equal amount ~> of endometr i.a_L L~rote=ins (100 ugi lane)
were subjected to sods :.z:n dodecyl sul. fa ~e-pol.yacrylamide gel
electrophore~~=_s and tratos.ferred to n:it:rc>cell_ulose membranes.
IL-1RII was detected us:i~:g a mouse rrronoclonal antibody (lanes
20 1 and 2) and the immur .o:omplex was revearl_ed by
chemiluminescence. No irn:rmznoreac.!=ion was observed in negative
controls where the ant.i--IL-1RI:I anti.bod~,~~ was replaced by an
equal concent.ratic_>n of: rncmzse i.mm;znog:Lobolint> ~f the same
isotype (lanes 3 and 9.
2 C. Figure 10 . Immunohistc ~hern~i cal. detec:t:ior: of I L-1RI I in t:he
endometrium. A) Positi sTe brown immozno5taining in the glands
and the strorna (Day 24). B) Negative control: serial secaion
from the same endometr~_c:~::L tissue ir~cubat_ed with normal mouse
immunoglobuli.ns instead of the primary antibody. C-F)
3C Representative illu:~tr~i:~_Lons of ~I~--1.R:I:I intensity of staining
in the endomet:rium thr~~aaghout the menstrual cycle: early
proliferative phase, I:,~~e 6 (C) ; latee proliferative phase, Day
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13 (D) ; midsecretory )::r.use, Day 1((E) ; ~:~nd late secretary
phase, Day 2:3 (F) . Nct~e the markeet immunostainir~g in early
proliferativfe (C) and. l;=ate sEecr.etc:>z:y (F) endometrial tissues
and the reduc.~°d intensity of that staining :in the glands and
'.~ surface epith~~liuTr~ of ni:~dsecretorse phase endcmet.rial tissue
(E) . Magnificvation X2'~~~.
Figure 11. Graphical illustration of .im-nunostaining scores
and their di~,tribut.ior~ according t;o the clay of the mens trual
cycle . A) Luminal. sta ~ n~ ng in g l andular and surface
10 epithelia. B) Cel_lulat staining in glandular and surface
epithelia. C) Cellu.laa :~t.ai_ning in the st:rorna. Stromal
extracellular staininc; not represented in this figure) could
not be evaluaved anc~ u,:~~ detectable onl,~ in Late secretary
endometrial tissues. ~'ertical hatched lines represent
threshold day; se~parat :irig t_he cy~cl.e into different expression
periods according to the intensity of I~~-1RII immunostaining.
Figure 12. A) Western blot analysis of iL-1RII protein
expression ire the endc:m~~t.rium throughou°_: the menstrual cycle:
Day 6 ( lanes .L and 5 ) , Clay 13 ( lar__es 2 and 6 ) , Day 19 ( lanes
3 and 7 ) , and Day 2 6 ( l~~ne:> 9 arid 8 ) . The ant ibody
specifically recognized di.ff~?rent bands, the mol_ecular_
weights of which range from 68 t:o 31 kO.~. The immunoreactive
bands (lanes :L-4) werc_ absent when t;he ~~rimary mouse
monoclonal ant=i-IL-1R~1 antibody was replaced by an equal
2_'> concentration of norma:a. mouse Irx~:~s (.lanes 5--8 ) . Although
barely detectable in tim= early ~>roliferative phase (lane 1) ,
IL-1RII bands were marked.l~~ .intense at tree approach of
ovulation (lane 2), bLt t:~eix intensity clearly decreased in
the midsecret.ory phasF ( lane 3 ) arch .inc ceased again
thereafter in late secr_c~tory endon.et.r.ia=~ tissue (lane 4) . B)
Nitrocellulose membrar_e--uansferred proteins were incubated
with the antibody irn t: h:cE. ,absence ( lanes 7. and 2 ) or the
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presence (lan=.~s 3 and 4; of an excess of nI.:L-1RII (20 mg/ml).
Minor bands recognized by the ar:t_~body ~isap~~eared following
competitive _i_nhiiitio:i 1:,y :recomi;oinant I__~-IR:11:, whereas the
intensity of major hi~:tnE.,r rnolec::ular weight bands was
.'~ considerably reduced. '-ndornetrial tissues were at Day 1~4
(lanes 1 and 3) and Day 28 (1_anc-s 2 and 4) of the menstrual
cycle.
Figure 13. Localizatic,n of Ih-ll~.I '. mRNA _in the endometr:ium by
in situ hybridization. ~eclvic>ns wE>re hyxori_d_ized with biotin-
labeled cDNA :robes. ~;etecr_ion c.~f biotin was performed using
a rabbit polyclonal ant=ibic~t::n antibody, a 1~i_otinylated goat
anti-rabbit ~_»~lyc:lo:na_ <~nt ibody, ~~nd fl.zoresc:ein
isothiocyanat.e-conjugacted stz:epta~ridin, respectively. S:Lides
were treated with proL;iciurn iodi.nee, whi~:h m<~kes the nuc:Leus
visible in yellow-orarcq~= oru u_Ltraviolet excitation, and
mounted in th~~ pzvesencve of an ar:tztadin-g agent (p-
phenylenediarnine) . Ap~~E=~arance o.f endometri.a_L glands (A) ,,
surface epithelium (B) , anti strc:>ma (C) ire shown at X:16'7 (1)
and X666 (2) magnif:ic<-ctic>n follows.ng hybridization and
2() staining with propidi~_nn iodine. Note th~~ green-yellow
hybridization signal that could bE~ observed only at X1655
magnifi-catio:z (3), p,ec:aom:inantLy in the endometrial gl<~nds
(A) and surface epithcylium (B) as compared to the strom<~ (C) .
Figure 14. Aru~lysis of ~L-:LRII rnRT~A expression by RT-PCI~. A)
Graphical il1_ustratior~ c~f IL-:LRII mRNA relative levels (° of
control ~ SEMI) in the er~dornetri.um throughout the menstrual
cycle. B) Rep resentat.:.ve Southern blots of II_-iRII and GAPDH
(internal c,ontro7_) tr<~;~c.ript::s ~n the whole endcmetrial
tissue after RT-PCR. , Positive control (;cDNA preparation
from human eru~ometria_ tissue expz:essing I:L-1.RII) ; 2,
negative control (PCR ir, the absence of cDNA). Tissues were
at Days (d) 1_0, 1_3, 1 ', 21, and 24 of tine menstrual cycle.
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Note the elevated 7_evc=ls of 1L-:LRII mRNR in endometr.ial
tissues at Day 1 . ( lat: a proliferat. ive phase) and Day 24 ( late
secretory pha:~e) of. tl;e menstru~=~1 _:vcle and the decreased
levels at Day 21 (midt:ecretory phase}. C) Representative
'_i Southern blots of IL-i IZI T ,end G~~!?L>H fr_orv separated strornal
(S) and glandular epithelial (E} cells at different days (10,
14, 17, and 2.'7) of t;he rnenst~rua~. cyc::L~~.
Figure 15. Represent:at .~~ve Gdestern blot: analysis of MIF
expression in endometz ~~7tic tissuE . 'fo':al ~>roteins were
subjected to ;>DS-PACE a::a.:Ly~.is arnd GJesvern blotting using an
affinity purii=led ~:~ol~c,:l~~na1 goat anti--P~1TF antibody (lanes 1-
3) or an equivalent concentration of normal goat Ig instead
of the primary (lanes ~l--of . Lanes 1 and 4, 10 ~g total
proteins; lanes 2 and '>,, 2C; ~g total. p~~oteins; lanes 3 and 6,
40 ~g total proteins. Tile detected band has an estimated
apparent molec:ula.r wei~Jt~t= c>f app>rcximafi.e:ly 1.2.5 kDa.
Figure 16. Representative after RT-PCR and Southern blot
analysis of M7:F t:ransc:r_i_pts. Total RNA c>btai_ned from
endometriotic tissue wa~~ reverse trap:>~~nibed, amplified with
2C MIF (upper lanes) or° G;~fI:~H (lower lanes) primers, and
hybridized with 32P-labeled corresponding probes. Lane 1,
positive control (cL>NA x>reparat:i.cm from t: he Lw.zman hystiocytic
cell line U93~, known t.c:~ secrete MIF'; lane 2, negative
control ( PCR i.n the ab;sc~nc:e of c.DNA) ; ;!.apes 3 -5, linearity
test with different RT volumes.
Figure 17. Immunohistochemical analysis of MI:_, expression in
endometriotic tissue (biopsy of :3 red papular endometriotic
lesion from a 37-yr-old vaoman with st:.age T endometriosis~) .
Note the intense brawn.i:~ln immunc>staininc~ in the glands and
cell aggregates througlrc:».zt lJhe st::rorna __r~ the E:~resence of a
goat polyclonal anti-MIF antibody (A} and the absence of such
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staining in t: he presence of Goat :~-gGs used at concentration
equivalent to that c~f t;oe ~~rima.r.~y antib:.~dy (B) (negative
control) . Scale bar, _'!~~ Vim.
Figure 18. Dl:~al-immun;fiuorescent stair~.ing of MIF (A-C) and
CD3 (D) , CD68 (E) or s-tNF (F) =Ln andometriot:ic: tissue. Tissue
sections were inc;ubatEed with goat po:Lyclona_L anti-MIF
antibody and ~aitr: moue monoclonal anti-CD3, mouse monoclonal
anti-C:D68, or rabbit ~~olyc.Lor:al ~~nti-vi~~, ani~i.body. Seci~ions
were then incubated simultaneously with rhodamine-conjugated
sheep antimou;~e ant:ibc:~dy and fluoresceiro isothiocyanate--
conjugated donkey ant~goat antibody to detect coexpression of
MIF with CD3 or CI768 c:~,- with rhoda.mine-conjugated mouse
antirabbit ar~~~ibody arid fluorescein iso:.hiocyanate-conjugated
donkey antigoat ant:ibc:~:~y tc> detect coex~r:~ess.:ion of MIF with
1_'i vGVF. Note the=_ expres~:i~.on of MIFF (green:: in CD3-, CD68-,, and
vWF-positive T lympho<:~~tes, rnacrophagesi and endothelial
cells, respect=ively (red) . .~uper~~osition of fluoresce in
(green) and i:hodamine (.red) signals clearly shows
coexpression (yel low :_ i_;:~n,.~l ) of MI F with CD~3 (G = A + D) ,
CD68 (H = B + E) , and vWF (I = C: + F) . kale bars, 20 Vim.
Figure 19. Gxvaphical :i..Lustrat:ion o:f: MT:~~, corrcentrations
(ng/mg of total protein;:) ~zs measured by; ELISA in
endometriotic; tissue. i~, Endometriot.~~ biopsies were
classified according to heir appearance at laparoscopy (red,
n = 11; typic<~l, n =- E: ; ;ahitf~, n -- 71 (A) or' to endometz:iosis
stage (stage I, n = la:; stage II, n ~ 9; stage III-IV, n = 3)
(B) . The box,--and--whihe:r. p:Lot was use~~ t=o illustrate the
distribution of MIF ccnL..entrations. 'The box delimits values
falling between the 2'~:lw~ arnd t:he 7~t:h percentiles and the
horizontal line wittnir: 1~.~re box rr~fer;~ ~c:, the :median scores.
*, Significant. difference between endometriosis stages I and
CA 02377786 2003-06-20
14 E>5409-5
II using the unpaired t test (P < 0.05), **, Significant
difference wish the rc~;:~ l.es:ions (P <. 0. ):l.) .
Figure 20. SemiqLiantit:<~ti.ve RT-I?~~R anal~Y~sis crf MIF mRNA in
endometriotic: tis,suc~. The quantity of ~:rze F:'e~R products was
'_i determined by der~.sitonuetri~::v ana:l.ysis of the intensity of the
hybridization signa_i. 'I'rle r_eiat~ive lev<,i oi~ MIF mRNA
normalized to GAPDH mF:NA was ca_zculated, and the results were
expressed as ~~ercent cvt control (~~ositi~fe control) . A, Types
of endometriot=ic lesions (red, ru = 9; t~,rpical, n = 6; white,
n = 6) ; B, stage of erc~ometriosi.s (c>tage I, n =- 10; stage II,
n = 7; stage III-IV, r -- 3) . The box-,~rrd-whisker plot was
used to illustrate t,he: c:~.is~r:ibut:icn o.f IvlF mRNA levels. The
box delimits values f~::'i:Lirrc~ Letween the 25th and the 75th
percentiles and the hc:ri.zontal lane w.irhs_n the box refers to
1~~ the median scores. *, ~>:igni_ficant= di_:faeence with the red
lesions (P < 0.05).
Figure 21. I17_ustraticn of I:~-1R:II mRNA expression in situ in
the endometrium of nor::n;~l women (A) anc:~ women with
endometriosis (B) . Sect::i_ons were hybrid= zed with biotin-
labeled cDNA ~>robes . Bioi~:i n detect ion was performed using a
rabbit polycl.onal anti~.~:i_o~i.n antibody, a biotinylated goat
anti-rabbit polyclonal antibody, and fluorescein
isothiocyanate-conjugate-.d strept.<:rvid:i.n, respe~~tively.
Propidium iodine was usec:i to make them nucleus visible in
2~ yellow-orange on ultrav:i.o:LE~t Excitation. Note the appearance
of endometrial glands (g;~ and stroma (s) at X666
magnification (Al and 31 i . The Plybr:i.cl:i.yat~ion signals (c~reen-
yellow; arrow) were vi.:~:Lble at 1h6~ m<~gnifi.cation (A2 and
B2) . Note th.e greater ~~izmber of hybr_i.d~ zati.o.~ signals i.n a
part of an endometria:l c~:Lar,~d from a nor_ma.l woman (Day 13; A2)
compared to that from <~ woman with stage I endometriosis (Day
14 , B2 ) .
CA 02377786 2003-06-20
15 Ei5409-5
Figure 22. G.rapriical vllustration of I~-1RII mRNA
hybridization scores in the endometrium of normal controls (n
- 26) and of women wi.t z endometric>sis of different stages (n
- 53) . A) Hyb:ridizati.c,n scores (mean ~ ,:~EM) in the glands. B)
Hybridization scc:~res ~rn~,an ~_ SEM> in th.: sti°omal
compartment.
* P < 0.05, ** P < 0.::1.
Figure 23. R'7:'--PCR folly o~aed by Southern blot analysis of IL-
1RII transcriF~ts in tr c~ endometrial tissue. Total RNA
samples were extracteca r.rom endometriai biopsy samples of
women with (n = 10) ar c1 o:f women w:it:hovt (n =- 8)
endometriosis,, then reT.rerse transcribed, amplified with IL-
1RII or GAPDI-I primers, and h:ybri.c~i zed wuth 3'P-labeled
corresponding probes . .A; Rc_>I>.rese.zt <~tive :;out:hern blot
analysis. Lanes 1 and ~.': women with stage I endometriosis,
1_'> Days 13 and 1f3 of the ;nc~:ns~:rual ~yc:l_e, ~~espectively; lanes 3
and 4: normal. women, Gays 7.2 anc~ 16 of ':he menstrual cycle,
respectively. The GAP I: Ii was used as a control. B) The ~~box
and whisker" plot was used to illustra=a IL-1RII mRNA levels
using semiquantitative I<.'r-PCR. The box delimits values
falling between the 2~'r- and the 75t'' pezcenti=es, and the
horizontal line witrnin 1__he~ box rwfers rc; the median scores.
**Significant difference between tree endometriosis and
control groups ( p <: 0Ø1;.
Figure 24. So7_uble IL-~.RII concentrations in the serum of
25~ control subjects (C) aru::i we>men w:~th encic:~metri~:~sis stage's I-II
and III-IV (E7:II-IV) . 'fhF~ "box and wh:i.s~~er" plot was used to
illustrate the distr.il"..~l:.iorn of ::IL-l.R:I:I values. 'The box
delimits valuE:s falling between the I?5''' and the 75th
percentiles and the hct~:i_zontal. lane w:i_i~hi.n t:h~~ box refers to
3C the median. * P <0.U1 as r:ompared t:o control. subjects using
the unpaired t: test.
CA 02377786 2003-06-20
16 X5409-5
Figure 25 . Ih-la (A) ~:n: Il:~-1 (3 (B) conc~.~nt r<~t ions measured in
the serum of ~~ontrols (~~:;) -end women w:i_t~u different
endometriosis (EI-II <:~n~:~ EIII-IV) stages. Comparison of
control and E:~IIdOITletric:~s:is gr;~up:~ was performed with the
'_i unpaired t te:~t .
Figure 26. Se,~um-induce~::~ MCP-1 ;:~ec:reti.oru by L937 cells. :L06
cells/well in 24-well ~~ultur~~ plates weave exposed for 24
hours at 37°C serum from normal. c:ontrol~; and women with
endometriosis (5 o vi v ~:IU l ut: i.on ::.n E,I3S-f Y°ee F<PMI:
medium) ,. MCP-
1C) 1 secretion (pg/ml) was measured ivy ELISA ire the culture
supernatant . A) contro:~_:_~ (~;:) versus end~.ometriosis (E)
patients; B) clontrols ;c~') ~:re:rsu.> endomet:rios:is stages I--II
(EI-II) and III-IV (E1I:I-I~,') . Data werE=_ presented as mean ~
SEM and analy.~ed with t:.h~s unpaired t. test, which comparE:s the
1'_. control group to each ~:ndome~riosis group. ~ P <0.05; ** P
<0.01.
Figure 27 . Efj:ect of r 1L--1RI:I ('f ug%m1 ) on s erum-induced MCP-
1 secretion. Data were presented as mean ~ SEM and analyzed
with the paired t test , ~ahi ch compares ;;era treated with
2f human rIL-1RII to untr~:<~ted sera ir: E,actn group. C - controls;
EI-II = endometriosis stages I and II; E;III-I'~J =
endometriosis. stage;> I:G::f:-:I G'. *' P <().(.).'~; ** P ~. 0.01.
Figure 28. Effect of h.nn<~n r_IL-l.r_a ( 1t)0 ng/ml) on serum-
induced MCP-1 secxvet:.io,n., Dat:~ were presented as mean ~ SEM
25 and analyzed with the paired t test, which compares IL-lra-
treated to untreated sf-m~.:~ wit=hiri each croup. ~; = controls;
EI-II = endometriosi.s ..:f::~ges I and I1; E:,I iI-I'~J =
endometriosis stages I1:~-_IV.
Figure 29. Effect of r:I:T.~--1R.II on serum-induced MCP-1
30 secretion. C =- controls; E = endometr_iosis; EI-II = stages I
and II; EIII-I:V = stagE:v CI:C--IV.
CA 02377786 2003-06-20
'L7 85409-5
DETAILED DESCb:IPTION O:f THE INVENTION
Study Design: H~ift~v-set4ren yat.ients with
endometriosis at ~apar;~scopy done for ~r:ferti:Lity and pelvic
pain were compared wit.Li 44 W :mile womE.r: with no evidence of
endometriosis at tubal :..:Ltigatior~ key =l.aparoscopy. Monocyte
chemotactic protein-1 concentration in the plasma was
determined by enzyme-l:i.:rlkec~,v immunoscrbent assay and its
biologic activity was ,w3luated by measuring monocyte
chemotaxis with u:~e of ~:~ human h;~st:ioc::yt is c:eLl line (J937) .
1C Results: Mon~=>cyte chemotact:i.c protein-1
concentratior.:~ (median ,:end .range of ~~~ai.t:es) found in the
plasma were r:igher in :patients wltt~ endc~metri~~sis ( 163, 0 to
788 pg/ml) thin in norona:L r_ont:rol_s (0, (7 to 355 pg/ml) . This
elevation was significant only in the minimal stage of
l ~~ endometriosis ( revisecA.rne= icon Fert:i L:it. y Sc>c: iety stage I ) .
However, incrE:ased chemotactic a~ctivi-y mean number of
migrating cel.__s/mm' ~ ~~E:;M) was fc:~und in the stages I ( 1240 ~
141) , II (519 ~ 30) , ~n~;~ III-IV (5'.%3 .-_ a?3j c;f the disea=~e
compared with normal controls (205 ~ 20. A total of 35o to
2O 44 0 of this act:iv:ity wa:~, inh.ibited in ttue presence of an
antibody specific to monocyte chemota~~t:ic protein-1.
Conclusion: Endometriesi.s .is associated with
increased level and acti.vit:y of mono~~ytE~ chemotactic protein-
1 in the peripheral. blood. The elevation and activation of
2_'~ this cytokine could play a relevar:t role in the
immunoinflamn«~tory prc::,:~sss associated with t:he disease.
Key words: Endometrio:= is, chemotac:ti c f<.-ctors, cytokine s
Endomet.riosis is a gynecologic disorder
characterized by the a:~ct:opic grc~~wt:h of '~is.sue similar to that
30 of the endomevrium, pr i.;t~,ar:i.ly on t:he peritoneum and the
CA 02377786 2003-06-20
18 85409-5
organs of the pelvic c:..avvty. A Growing body of evidence
indicates that: the i.mm. r~r~ system is a* ~eoted Ln ~.aomen with
endometriosis (Dmowski. 'v~J.l~. et a~_, l~~i~~l,' . Locally, an
immunoinflammatory pro~:ess involving leukocyte recruitment
and activation is taki.:-,::~ p7.ace (~laney E~. f. eat al, 1991.;
Taketani Y. et: al, 199:'.; Leiva M.C. et al, 1.991) .
Endometriotic lesions ,~rc~ Likely to c.;ontr.ibutF~ to the
modulation of these in~..m.:mol.ogic react~:ior~s ( I saacson K. B. et
al, 1989; Akoum A. et .:~1, 1995a) . :y<el_icv reflax of menstrual
debris in the peritone._-3:L cavity may a.l_sc~ occur, thereby
exacerbating t;he l_oc:a.l. ~rz:flammatory respc>nse (Halme J. et a.1,
1984a).
HowE:ver, t;hc:, al~e:ratiorzs i.n :immune functions
observed in patients wii~h endometriosis are not restricted to
1'~ the peritonea__ cavity. ~:>ystemic .-~ltera~:_v.ons ar_ both humoral
and cellular =_evels ha~.Te been reported, including elevated
levels of ant_-_bodies :.pe~l:if:.i.c to endometrial. antigens (Badawy
S.Z. et al, 1.~~90) and i.oc~reased acti.va~lonal. status of_
peripheral blood monocytes ('teller ,J.M. et al, 1987) .
Monocytes play a c:entr.:~:1.. role in the ma ~..nter~ance of humoral
and cell-mediated imm~rii ty, and thrc».~gh a panoply of
secretory products they :pan play a s:ign__ficant role in the
pathogenesis of endomet~:riosis. A~::cordinc:) to recent reports,
peripheral blood monoc.~~~;_,.~;~ of women wv ~lv endo:metriosis
2_'> secrete elevat=ed level s ::f p:rainflamm.~tc~~ry mediators such as
interleukin-7. ( IL-1. ) ~rnc~ show thf~ ab.i ~ it y to stimulate
endometrial cE~ll growt=h in vitro, whereas monocytes of normal
fertile women suppress.- th~~ p.rol~.ferat:z_~:~tu of these cells
(teller J.M. et a1, 1':~r3-;%; E~raun D.P. E_et a.1, 1.994) .
A new class of structurally r<,lated small molecular
weight cytokines wi_t:h <i:i f fe.rent target: :elect ivity has been
characterizes in the Blast f_ew yearn (S~lzall T.J., 1991).
CA 02377786 2003-06-20
:i9 85409-5
Monocyte chemotactic p:rcvi-e.r7--1 (MC:F~-:l. ~ has been shown tc>
exert a potent: ef fect can monocyt a c:hemoattract ion and
activation (L~E:onar_c~ E. ~:~" et: al, .°~.~0; . ~~c~cord:ing to our
recent data, L~1CP-1 is present in the peri tor:eal fluid of
women and its level is t:i~~r.er in womc.=.n with endometriosi.s
than i.n normal. contro:l.~~ (Ak:ourn R. et a:L, 199Fpa) . The
objective of t:he current-- study was to examine the presence of
MCP-1 in peripheral b:1 ~.~~:~d and to irnvesi=igate S.ahet:her any
difference in. its level anti activity could be found between
1C women with and without endometri,:asis.
Material and Methods
Sub-iects. WC;nlE'u were rr~ca:uitect into the study after
they provided informed consent for a p-otocol approved by
Saint-Fran~oi:> d'Assise hospital. ethi.<is committee. Subjects
1~~ with endometri_osis (n -- '.~~7j were i;ientiii.ed after they
underwent laparoscopy f=or infertility and for pelvic pain.
These women of:herwise tua~:i r~o c>thPr ~>elvvc di.s~:~rders and were
not taking any antiinf i<~rnmatory ::or hormonal medications at
least 3 month:> before I_aparoscopy. ThP :stage of endometriosis
2C~ was determined accordirn<~ true revised c:lassif:i.:ation of t:he
American Fertility Society. Control subjects (n = 44) were
fertile women requesti.ri;~ tubal. litigation or reanastomo:~is
and with no visible evi:~ene;e of <~ndc>me ~x i_osi.s at laparo:~copy.
Menstrual cyc__e datinc was determined ac_-.cording to the
25 regularity of the cyci y, tt-~e dat:r~ of: th<-: previous mense:~, and
the levels of progesterone in the p.Lasma. The primary
clinical pararleters lisved in Table 1 iruclude age,
infertility, cycle pha>e, and st..aae of- endometriosis.
Coi_Lection ana pr_oces:~ ir~g of aW ooc~ samples. B7_ood
3C~ samples were drawn a f ew days before i.a~oaro::copy. For MC:P-1
assays blood was coll~:cted in sterile tubes containing
ethylenediami.netetracE:t: i c ac:.id anti immec:~iately centrifuged at
CA 02377786 2003-06-20
;?0 85409-5
Table 1. Plasma 1_E:~vE:l~ ~:_~:f_ MCP-1 ~;io p=ic-~ograms pe:r mill_il.iter)
and subject characteri;=~t i c~v> at laparosc--opy
A9CT'-I (
riml)
lVo. ofpatient,s._~_-9ge (yJy A7ediun and Signifecance*
(mean tSD) range
Controls X44 33.75.6 0 (0-355)
EndometriosisS7 31.2+7.:? 16, (0-7831 p=0.01
subjects
Stage I 27 31.9~t7.1 180 ((7-788;1p=0.04
Stage II a?0 30.1+8.3 158 (0-5850 p=0.601
Stage III-1V10 31.35.5 195 (0-413 p=0.31
o
Endometriosis
subjects
Fertile 31 2.07.1 163 (0-788)
Infertile :'6 30.27.4 170(0-640) p=0.79
*Versus control:; witlu Wilc~~x, n t~::~t and F,onterr<>ni correcaion
2000g for 10 minutes a~-: ~_1"C', and the plasma aliquoted and
stored at -8Q" C ~,~ntii ,:zssayed. f,or_ h<-~r rnonal assays blood was
collected i.n red-toppe~~:l tubes arid sent: to the biochemistry
laboratory for steroid clete~rrni.nat=ion.
MCF--1 e.nzym~: --_I.inkec~ inununosa.r~oent assay.
MCP-1_
concentration:> were me .-~s~.~rEed, as previously reported
(A)<:oum
1C A. et al, 199E>a) , ,_~~z e:n:~yme-linked immun~:~sorbent
wl_tio assay
procedure developed iri 1=he laboratory. "his assay uses a
mouse monoclonal antih ~_irnan MCP-1 antihoc~y (F;&D Systems,
Minneapolis) and a ra>;. !o:i. t ~~o_Lycl.onal. <~ntz.human MCP-1
antibody
previously used in our ;:~t~.zd.ie~> (Ak;>ram u. et al, 1995a;
Akoum
l~~ A. et al, 1995b; Hac:hi.,ha PM. et al, 1'a9 ~) . This latter
antibody does not cro::; ::~ rf~a<_t wi,=r: se,~rfy~-al cytokines
that are
closely related to MCF -:1, vi_ncluding MCP-2, MCP-3 interleukin-
8 (IL-8), regulated or. activation of normal T expressed and
secreted (RAN'~ES) and :m:r~:rophage irlf:lamrnatoz:y protein-1
a and
20 ~i (MIP-la and MIF-1~3)l-iachicha M. et a_., 1993) . The
sensitivity limit of t: tnfJ assay was 50 pexirnl with intraassay
and interassay coeffic:: i_c->.nts of variation < 6~.
Mor~oc.yte cht-rn~:>t.3~>>is assay. M~~mocyte chemotaxi s was
assayed with use of a Bc~ydc:n chamber (Niacleopore,
CA 02377786 2003-06-20
21 85409-5
Pleasant own, C:alif. ) ar~ci a human histioc:.ytic c:el:L line (U937)
as reported previously Akoum A. et al., '~99Eai. Briefly, four
separate pool; of p1 asrn~-~ <:c;x~respondinc~ t o th.e four grc>u~>s of
the study (contro:i and endometriosis stages T, II, and III-
IV) were prepared. An f_~cy.za~ vol.urne oj- plasma was taken from
all the patients incluc:iec:i in eachr group. Trip:Licate samples
of each pool Hlere plac:~~:c:i in the bot:torrl wells c:~f she Boyden
chamber (200 pl/well_) . folyr_arbonate membranes were then
fixed in placE: to separate bottom from t:op wells and 600 x 103
U937 cells in 200 pL. c:~I:' p:~hosphatr~-bufaE.rec~ saline solution
containing 1% bovine s~erurn albumin were added to the upper
well. After 9C) minutes ut :incubat:ie:~n at: ~.'7°f,, nonmigrati.ng
cells were removed by several washed i_n phosphate-buffered
saline solution, and t:n<~ membrane was nixed in absolute
methanol for 7.0 minute;: at room temperature and stained with
Wright-Giemsa (Fisher ..::~c::ierut.ific, Moni:::r_eai) . 'rhe number of
cells migrating through each membrane was determined with a
computerized image anal.ysi4; system (B:i_o~_)uant. TVTM, Meg X,, R &
M Biometrics, Nashville, Tenn.). Cells were counted three
2C times in three dif~fere::-ri:: areas r<~ndorn:l_y selected at the
membrane surface, and the mean number ofi migrating cells per
square centimeter ~ SEMI was detEerrr7ined ~vor t.wo independent
experiments. N-formyl-methionyl-:Leucyl-1>henylalanine (Si.gma,
St. Lc>uis) , a known chnruo~acti.c pept:i.de, was used as a
2'~ positive control at 1C ' moL/L, whereas phosphate-buffered
saline solution containing 1'< bovine serum albumin served as
negative control.
To appreciate MCP-:1 activ.i.ty :.n the plasma, samples
were incubated with d~ f:Le:rent diluti.ons c;f polyclonal rabbit
30 anti-MCP-1 ant:ibody or_ w:ith equal dilu~.ons of normal. rabbit
immunoglobulin for 30 m:i:nutes at 37"C before incubation with
U937 cells, and the cterriotacti.c act: ivi ty was measured as
described above.
CA 02377786 2003-06-20
?2 85409-5
Estradiol anw~ progest:E~rcr~e assays. 'The
concentrations of e:>tr <:~ao:'~ and i:orc.,ge;>t:e tune in the pl_a~sma
were measured by a c:ory::»:-'t.~t. ive irYun-~anoa;~say based on anti.body-
coated tubes ycomrnerc:i~.r', k~~ts, C.'c~at-1~--e:ountT"', Diagnostic
Products, Los AngelE:s ) . 'i'he intr<:ras:~ay coefic Tents of
variation mea=cured at ~~~>ca, rnediearr~, arw:d high levE=ls of the
standard curves were b~~r.ween 1 . 8 ~~ and 8 . s o foa all the
immunoassays. The inten<:~r~say coefic:lent=:~ of variation were
8 . 1 o for estr~idio:l. and 10 ~ for ~>roc~estez-one .
1C Stat:istical a,oalyses. CvCP-.L concentrations found in
the plasma dc. not fol.l~~>w a r:orma'~ dist_r_~bution; therefore
analysis was c:onduct:ed x:>~;~ nonparametric, metho<ls. Analysi.s of
intergroup differences ~~eas performed cc>nserva-tivfaly with
Kruskal-Walli_~ one-way ~~rzalysis ~::pf va~-:;.ance by ranks.
1~ Individual groups were i-ompared with thc: Wil.cc~xon rank-~>um
test (Mann-Writney-Wil~::~~a.~on test; , and twhe Bo:~ferroni
procedure (al:~o call.ec~ L?~rnrz's mu'itip_Le comparison procedure)
was applied when more t man two groups were compared.
Receiver-oper~rtor chaa:,_ic-teristic: c~urvE_5 analysis was performed
20 to examine the tradeof is betweeru s~,ns:it~vity ,:end specificity
under different cutoff- ~~a:Lues. Sensi.i~.i~~_~.ty was defined as the
proportion of positive ta:~st .:result: in patients who had the
disease. Spec=_ficity r~x~_esented the p>:,~c>~>ortion of negative
test results __n patient=:, who did not. lua~Te the disease. A
2C~ cutoff value refers tc i_he point that :~e~%arates negativE: and
positive test. results. ~~, c:utoff value fc~r MCP-1
concentrations giving :.y:~t.irna:l lensit:ivi_t.y arid specificity was
then selected, and the. ri,~mbe:~ otwomen with and without
endometriosis with MC)=--:' clon~~ent~rat:i_ons f>elc:w or above t:he
30 cutoff value was detez:rn:ined. Comparison of patient age was
performed with Student: t test betweE>n two gz:oups and by
analysis of variance ~tnf~n ~ever_~r:1 groups wez:e compared.
Statistical analysis c>t morrowyte chemo-tactic activity in the
CA 02377786 2003-06-20
<_'3 85409-5
different pools o:;- per:it::;>nea7_ fl~.~i:_l wa=> performed with.
analysis of v<~riaruce, c;'a_)_owec~ by t: he i'ukey's honestly
significant difference t::f~st for. rnu:ltiple comparisons. For all
analyses the aifference:=; were corasi.dered as statistically
significant f~:r p valu::~:-~ ~: 0.05.
Results
MCF-2 concenl.'_rations in the plasma. MCP-1
concentrations in the ra1.~sma varie:~l among pat-~ents, and their
distribution .in ncarmai <:c:u~i endometric;t:i.c womeru according to
the stage of she ~_~isea::~e i.s illu.~tzat:ed in Fi~:~. I. Because
MCP-1 concent:cations w ~r~.~ root: normail.y cistrihuted, we
determined and compare :1 i:rieir mecti,~~n:;, as shown ~n Table I.
Receiver-operator char~c;~t:eristic curve ana:lys=_s was
performed, and an opti ;a:n:l. cut.of.f v,:~luE: of :100 pg/ml was
selected. This cutoff era l..ue yields a sE~r.sitiv.-~ty of 65 ~ and a
specificity o.f 61=~. In c>ther words, 37 of the 57 patients
with endometr.iosis ( 65 .; had MCP-1 conc:entrat ~.ons >100 pg/ml,
whereas 27 of the 44 c:mtrc~l. subnects 1610 herd MCP-1
concentrations <100 pg!rnl.. Static tir_.al ana:lysv~s of the
results with the Wilco~<:>ri t.est indicates that MCP-1
concentrations were si.-~nificantly~ tuigher in women with
endometriosis compared wi..t.h normal women with no laparoscopic
evidence of the diseas:,~ ~:ont:rol(p <: C.05) . A significant
difference among the c:>r~t:rol and endometriosi~ stages I, II,
and III-IV gr~:ups was -~J.>o (sound ai t:er analys~_s of inter-
group differences by tirE:~ Kxut>kal.-W~l.li_t> te;~t !;p < 0.05) . Post
hoc comparisons of: ind:iv~i.dual_ gre>ups ~>y the W_~_1_coxon test and
the procedure of Bonferrc>ni. reveal a s~.gnific<~nt elevation o.f
MCP-1 concentrations crnl.,,~ i.n stage I d~~:ease compared with
the control group (p < 0.05). Also, no statistically
significant ccrrea_atio.n l:~etween the plasma levels of MCP-1
CA 02377786 2003-06-20
.?4 85408-5
and infertl.lit:y withi:~ t:he g=coup with endometriosis was
observed.
Because endc:nE--li.riotic Les:i.ons are influenced by
cyclic change:> in ovar i_:~rz .-;ex st:ero:i.c~, i.t was of interest to
determine whether the i.a=vets of MCP-1 fc>und in the plasma
varied accord__ng t:o trre phases of t: he meristrual ;:ycle. As
shown in Table II, the ;a:if:ference between the levels of MCP-1
in patients w__th endorrrt r.iosis arid con -rot srzbjects was
significant c>nly :in t~ L~~t:eal. phase o:E t:he menstrual cycle
(p < 0. 01) , whereas ir; I::.ie fol.7_~ c:u La:r phase no significant
difference wa:~ nosed. '>).:~f~rw:ise, there was r~o difference
between the follicu~_ar and tut:eat plnasE~ s within each control
or endometriosis grout:, rlor was there aruy correlation between
MCP-1 concentrations ~;ncJ the levels of F-strad.iol (R2 - 0. 007 )
or progesterone ( R'' - ~..~ . i)10 ) found ~.n t:r~e plasma of patients .
Table 2 . Leve_Ls of est r<:~dio:l , progesterone, and MCP-1 in
plasma of patients accc:>rding to pl<~;>e o~ m.enstrual cycle
Estradiol ( nrollL) .~_ Pro esteronc: ( ~mollL) MCP-7 ( lml)
No. of Menn ~-SF'!'-9 No. of Mcan -!-.SEM No. o/
patient nnc:l puticnt and I utient Median, range, and
.s sienificancc * s , ~ .si~nilicunco* ~ s si,~Jni/icanco*
Follicular
phase
Controls 23 239 32 23 2.4l .l 24 I S (0-355)
29 365 51 30 1.50.3 31 165 (0-788) (p=0.11)
Endometrios (p==0.04)y=0.42)
is
Luteal phase
Controls I 36'7 18 19.73.2 18 0 (0-220)
8 55
38:3 ~ 20 25.84.6 21 16:3 (0-450) (p=0.006)
45
Endometrios (p-0.83) (p--0.?9)
is
*Versus controls of same c:~,~c.: a yhase
20 Morv~ocyt.e c:hem~~>t.actic activity of MCP-1 in plasma.
The biologic activity :~fi Mi~P-1 way; eval..zated by measuring its
ability to inc~ucE= monoc~yte chemc~taxis b~.~ use of the human
histiocytic c~f~ll line IJ~37. Plasma from each of the four
groups (contrc:>1 and rc ~~:i.sed .Americ:an Fe~t.i.lity Society stages
CA 02377786 2003-06-20
25 85409-5
I, II, and III:-IV) we.ra pooled arid the monoc:yte chemotac:tie
activity in samples f:r~~> rn each pool w<xs assessed either i.n
the
presence or absence o.f -:i rabbit po~ycl c>r~a 1 anti-MCP-1
antibody. Thin antibod-~~ specifically recognizes MCP-1, as we
have previousi_y rf-~port~ec:~ (~koum aj. e~l~ a_ , 199;~a; Akoum
A. et
al, 1995b) . St:atisti.ca:i.;~nal~~si.s of t=:die resul_ts by analysis
of variance shows a si.~ n~ficant c:~iyfexverrce am~~nq the four
groups of the study (x, ~. 0 . 01 ) . ~?cs t noc~ mutt iple
pairwise
comparisons b~~ the Tukf, n"; r~onest:ly s:igr.ific:ant differences
1C test reveal a higYier rr;~cyte cr,emc;tact=ic activity in
ma
endometriosis stage; Z 01240 1 41 ce=L_L.s/mm') , II. (519
30
cells/mm2) , and I:II-IV ( ~~23 23 ca--Jlls/mm') compared with
the
control group (205 2~ i cel ls/mm' ) (~> <- 0 . 01 ) ( Fi_g.
2 ) .
Preincubation of ~~he ~~~i.a~:~rna witYu anti-1'9C:P-1 antibody
(1:500
dilution) resulted in >ic~nifi.c<~nt: :i.ruhibition (percent
<~
inhibition, mE:an - SEM; of monocyte chemotaxis found in the
stages I ( 41 0 8 0 ) , ( 35'~ 5 ~ ) and I I:L-IV ( 44 0 3
I I 0 ) of
the disease (.p < O.C)1), whereas no significant inhibition in
the control (--1 0 _t 9'~ was observed. Flabb.it p:reimmune serum
)
2C was assayed in the sane='La shion ~,aithout~ any detectable
repression of monocyte ~nercrotact.ic actin%ity (dat<~ not
shown) .
Comment
In t:he current study we have .v.own that women with
endometriosis had hi..ghc-:r circulating _leveis of MCP-1 compared
with normal women with a normal clyneco=Logic status at
laparoscopy. ~3y use of c~onservat_ve non--parametric
statistical analyse:, ~~ s:icxro:~fic:ant E:1evation of MCP-1
concentration was observed only '~n st<~gEe I of the disea~>e,
although there also was a trend for an z_ncre~ased level of
3C MCP-1 in the more adva:.uc-e:~ stages ( I:L and II I -IV) . The
failure to det=ect a szcatiificant elevat:Lan in Lhese latter
stages could k:e due tc: ,_~ Li_rn:ited st:at:i:~ti.cal. ?ower in these
CA 02377786 2003-06-20
?6 85409-5
series of patients. We :gave also shown t:rzat patients with
endometriosis had a significant increase in c_zemotactic
activity for monocyt:es :i_rr a.:l:L st:_~ges, tm.lt particularly i.n the
stage I of tre disease. Furthermore, 35'; to 44~ of the
monocyte chemotaxis oL.:v>erved was irlhuh_it:ed i.rn the presence of
anti-MCP-1 ar_t:ibody. TEnese results inc~:ic:ate treat MCP-1 is
biologically ~rctive bEc:,~use it:s pr:ima:ry biol.o~:~ic properties
known to date are the ctnemoattraction arnd the activation of
monocytes (Leonard E.~~. ~~~ a:L, 1990) . ~I'IiE:y also suggest that
endometriosis is more active in the ear~.y stages. In this
regard, however, avail .:~t.,:L<= data are sT l .J. contradictory. Some
studies have ctocumerrte;:~ thmt ~>erit.oneaL fluid inflammation is
inversely related to the extent of visible endometriosis
(Haney A. F. et: al, 19~i. ) and triat lE:ss extensive disease may
1_'~ be more biochemically active than older implants (Vernon M.W.
et al, 1986) , whereas o~r_her :findings incaicate that peritoneal
fluid concentrations of c=~remokines sash as RANTES and IL-8
correlate with the seu-e:rity of the disease (Khorram 0. et al,
1993; Ryan I.I?. et al, 1995).
Several recent studies have focused on the role of
peripheral blood monoc:ytes in the pathophysiologic mechanisms
of endometriosis. The~~F~ :~:e'yls seem to bce more activated in
women with endometrio~:is and secrete elevated levels of IL-1
(Dmowski W. P. et al, 7 9'=~4; Haney F,. E~,. e~_ al, 1991; Taket=ani
2.'i Y. et al, 199?; Leiva M.C. et al, 2991; Isaacson K.B. et al,
1989; Akoum A. et a.1, 1995a; Halme ~T. et al, 1984a; Badawy
S.Z, et al, 1990; Zelier J.M. et al, 198'J). 'they also show an
altered expre:;~sion of ir,tegrin mol.ecule.:~, which play an
active role i.n monocyt:e traf_f:icXing, adhesion, signal.
transduction, and act~.vation (Gebel H.M, et al, 1995).
Furthermore, monocyte:~ ~ rorn patients with endometriosi.s have
been shown to sti.mulat.e endometxial cell proliferation in
vitro, whereas those c:.f norma:L f:er:tile women suppress the
CA 02377786 2003-06-20
2i 85409-5
proliferation of endomFWrial cells (Brau;~ D.P. et al, 1994) .
On one hand, c>vr f:indirng of an increased concentration and
activity of MC:P-1 in tlm periphe:ra:J. blooe~ of patients with
endometriosis :may corrc.:~:,c~ra ~e these: observat ions because MCP-
1 is known to exert a l:~ot~ent action on monoc:yte activation
and only monoc:ytes have i~>een shown to express a significant
number of rect~ptor_~s fo ~: t.hi s chemol<ine (Yo shimura T. et al,
1990 ) . On the other hard, however , t he f indinc~ of a higher
chemotactic activity ire ': he peripheral blood of endometriosis
women is difficult to f_,x~>lain because, according to previous
studies, the percentage:: c~f per_ipr~eral :nonocytes seems to be
unchanged in these wom:~~r;. (Gl.eichE=r rd. eat al, 7.984 ) .
Numf=rou s facr:ors may account. for the increased
circulating level=> of ;~JC'l:'-1 i.n pat l ent::; wi th emdometriosis .
This cytokine is secrei:ecl by several t:y~;es of cells and its
secretion is induced b~~e runny inf:~a~TUriatory cytokines (Leonard
E.J. et al, 1990) . Accc::~rcting to our prE:vious data,
endometriotic cells se~:.~~-e::te MCP-.i. i.n culture and such a
secretion is t~timulate;=1 x:>y TL-1 anc_l t:umc;r necrosis factor-a
(Akoum A. et al, :1995a ) . These ~.,:r~o.inf lan~mator=~ cytokine~, have
been found in elevated .'bevels in the peritoneal >rluid of
patients with endometr.iosis (Taketani 'f, et al, 1992). NICP-1
is also produced by eutop=is endometria~ cells and,
interestingly, its secnc~~t:i.c>n wa~~ shown t_o be' ap-.regulate>d in
women with endometrio::is (Akoum A. et a:, 1995b) . Activated
monocytes have been sl::own t:o produc:e MCT-'-1 (Leonard E. J. et
al, 1990) and may also account for its release in the
peripheral blood of pG t. l ent s .
Our. results also indicate a significant elevation
of MCP-1 in the lutE=a:;_t~hase of tfue ~nen;~t,rual cycle in
patients wit;!, endometr:i:_,:;i:; compaz°ed wit-h control subjects. A
trend for an elevation in the follicular phase of patients
CA 02377786 2003-06-20
a?8 85409-5
with endometri.osis ver~s°.~s contro:':.s cou:..c~ also be observed,
albeit statist:ica.l.ly u:u.~ignificant. This would suggest a
continuous activity of tln-' disease :regardless of the cycle
phase. Beside~~, no stat::Lsi=zcally si.gnij.=icant difference in
MCP-1 levels between tl~e I=o:lliculaA anc~ luteal phases was
detected. Also, no c:or r-r~:l<~t:ior~ ~:~etween t.l:e le~~els of MCP-1
and those of estradi.oi c.~:r progest:erode hound in she pla~;ma of
patients was noted. Th~=~7E~ result; seem t.o ru.le out any
hormonal modulation of I~~9iP-1 levels =;_n t he peripheral blood
because it would have been expecied on the basis of previous
studies reporting higlu __~-~vels of h;~rnan serum proinflammatory
cytokines such as Ih-:i ~_n the sec:retory phase of the
menstrual cycle (Cannon ,:T.C~. et al, 1~~f3'~) and a reduced
intraperitoneal ir~flammai:=ory reaction of ter hormonal
treatment of patients ia..t:h endometri<?s=..~~ (Lei.~,ra M.C. et al,
1991; Haney A. F, et a1, '988) . It: is st=ill, luowe~;rer, to be
determined whE~ther_ cir~:vi_z:~at i_ng MC:P-? .Levels varied on
hormonal therapy of pa':. i.c~nt::s witt-z er,c~or-retrics:is with
gonadotropin-releasing h<:>rmone ac~on:ist:s or danazol because
gonadotropin-releasing lw::>rmone agonise effect: seem to be due
to ovarian suF>pre:>si.on ; l~errvay A. , 199?; , whereas danazol
seems to also exert a c_i:i_ rec.t inh i_bitor_v action on the immune
system (Dmowsk:i W.P. e~ a1, i994j.
In t:he currew;: study we found increased MCP-1
levels and activit=y iru t:.he plasma of worriers wi.:~h endometriosis
compared with normal. fE:r_t:il.e women wit=hout laparoscopi.c
evidence of endometrio:_:~_s. Similar results were obtained in
the peritoneal. fluid c%loaf=Lents with endomet:_riosis. Taken
together, these f indiny7 rna ke plaus.ibl a Mc:P-1 as a key
effector cell mediator ::..evolved i_n t:lee l>athogenesis of the
disease.
CA 02377786 2003-06-20
29 E35409-5
The patrx~~genes:is ~:»- endometriosis, a disease widely
believed to arise f.ronn an ~L>errant: growth o~ endometria:L
tissue out=side tree utr~:r~.s, is st:i~.~ unc.~ear. We have
previously ob:~erved tt .-~t ::vtokirle-st=imulated endometria:L
_'p cells of women with er.c omet~riosis secrete irn vitro increased
amounts of mc~nocyte ct~r~raotac:tic protein-1 (MCP-1) . Th:Ls
factor may be imp>ortar~t in the recruitment and activation of
peritoneal ma~lro~>hage~: ::;:served iru endornetriosis patieni=s.
The present s,~udy repc_ :r1_ '~ t:uat=, in t=he pr_ esence of the
disease, such an up-rc~galation of MCP-1 expression arises in
vivo and can be encour~>>e>red in sit a in ~:.he vntrauterine
endometrium. In women ~~ai t h endomet rips ~:_~, MC:i~-1. expression
was elevated :Ln endomf~t:ria'L. g'~arndt., both at the level of the
protein (immunohistocr:emist:ry) and t=he mRNA (in situ
1'.~ hybridization i . This s~a~ observed throw<~heut= the menstrual
cycle and var:Led according to thc~ stage of the disease. These
findings strc:~ngly arga:.e in Lavor c:~:f t.h~~ presence of
pathophysiolcc~ical ch~nyes i:n the eutop:i.c: endometrium of
patients with endometrri~:osis and make pLausil>:Le MCP-1 as a key
effector cell mediator involved iro t:he pathogenesis of t:he
disease.
Endometrioss~ is a gynecolc~gi~al disorder
characterized by the ~:uesence of endome?~rial-like tissue
outside the ui=eras, m~:irly in true peritc:>nea7_ .cavity. It
2'i affects women in their r~~~roduct:ivw age, causing abdominal
pain, dysmenc,~rhea, d~; :>~=>areunia, abnormal_ uterine bleed~_ng,
and infertilii=y bat ca.~n al s« be asymptomatic and found __'_n
women undergoing laparo;-c~~py for tubal_ ~_itic~ation. Its
prevalence arr~.ong the c:c~rue:ra_i.. population is cx:ifficult to
ascertain, bui= estirnate;~; suggest that tine reproductive health
of as many a:~ 10% of 1 tm~ female pc~pulat ion is affected in
this disorder ( Strath)a : ~ . H . ~~t a 1, 7_ 98.? ~ .
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30 85409-5
According t~<; i:vne most predom:iruant hypothesis,
endometriosi~, would an i_:,a ~ rom t;~ne i.mplantat:ion .and
proliferation of andorr.;i-ria:1 tissue t=lnat ~~an reach the
peritoneal ca~lity by t:~..zl~:a:1 ref:Lux (ret:_vc:~aracle menstruation)
': (Sampson J. , 7.927 ) . Trv:_~; pt-,enomenor~ _~~, however, common to
all menstruating women, and it. i:~ st i:1 ! uncl.e~:~r =yet how
endometrial cells coul_~_i implant ~rnci p-ro~~ iferate ectopi_cal_ly
only in certain p<~ti..er:t.~>. Women may have: a genetic
predisposition to develop the disease (Frey C.H., 1957;
Malinak L. R. et al., 19~~1~:); . hormonal fa<aors might be involved
in the maintenance and c::ievelopment of er~dometriotic lesions,
as both eutopi.c and er_:ta.~;~ is endometria_~ tissues depend on
ovarian steroids (DizeY~f,>ga G.S. et a_~, i980; Rock J.A. et al,
1992) . In recent years, tmst imrrmno.iocx.:,_c~al dysfunction has
been invoked as a causa__ factor i_n trre cAevelopment of
endometriosis, and it cnay be a cause of pain and reduced
fertility in ~;ome patif_>rats (Drnow >k:i GJ.I?. et a:! , 1994) . C~ne of
the most consistently rel:~orted irnmuno~ogic:al abnormalities is
that of monocyte activ:t-.i_ora and rvec:ru~t=ment into the
peritoneal cavity of pa.tient:> (Gel~_er ~.T.M. et al, 1987; Braun
D. P. et al, 1994; Hanevy ~~. F, et a1, 11381; Halme J. et al,
1983). Activated monocy-tes / macrophages are known to secrete
many angiogeni.c and ot.lr:e,~ growtri :fact:c~~-s (McLaren J. et al,
1996; Halme J. et al, 1988; Olive C).L. et ,al, 1991), which
may promote the growth c:~t: endcmei~ri.a1 expl_ants and numerous
proinflammatory mr.~lecu lE:e~ (Braun D. P. et al, -1996; Rana N. et
al, 1996) that may exa:_c>rbate the i_nf=~~_arr nator~,~ reaction
observed in tr,e peritor~~:~au_ cavity c>f endometr:'~osis patients.
We also believe that is E:mdometrio.~:is armses from the
endometrium tads tissu:= would be the ::it.e of l:~iological
changes that rrcay t:aci:l:i.t:<=~t;e its own development in ectop~ic
locations. In our prev:ico.is st:udic~s we :>r:owed t=hat ectopic
endometrial cells (isolated from endometriotic implants)
CA 02377786 2003-06-20
31 85409-5
secrete monoc~~te c;hemot:<:~,;t.:i:~ prot,e:~r.--:l MCP-1) in vitro in
response to interleukin---1~3 and turm.~rwec:rosin factor-a (Akoum
A. et al, 1995a) , cyt.c>k:::_ne~ whose .level:, are F~levated in the
peritoneal fluid (Pf) a~:fv women w~_tzn E~ndc~metriosia (Fakih H.
et al, 1987; Eisermann J. et al, 1x88;.. MCP-1 is a chemokine
of which the :major bicic,:~ic'a-~ property l~:nown T_o date i.s that
of monocyte ac:tiv<~tion crud recruitment s nto the site of
inflammation (Schall I'.,1.,, 1991; Leonard E.J. et al, 1990).
Subsequently, we found e.l_evated worrcentratior:s and biological
activity of MC:P-l, bot',o _n the PF''' a:nd ~.r~e "erum (Akoum A. et
al, 1996a) of pat_i_ents w:i_t~h endomet.rio:~.is. We also observed
that followinc stimulat: i-t>n with r>roi_nflamsnatory c:ytokines in
vitro, eutopi:~~ en;lomet~o_i-~-~:l_ epithelia7_ cells secreted MCF-l,
and such a secretion w:~:~ greater in c:e~-is from women with
endometriosis than in :~~E_.._1_s from women raving a normal
gynecological status a!:: i..aparoscopy yAl<:cum A. et al, 1995b) .
These results make play-:s i..ble MCP-1 a=~ ar_ impo.ntant cell
mediator involved in t:!re acti.vatiori of: ~;eripheral. blood
monocytes and peri_tcne _a='a ma<_ rophag~~~s observed in
endometriosis patients .. ~'riey also ~_~ive rise tr.~ the key
question of whether, ira r_rie presc_::n~:::e of disea:>e, such an up-
regulation of MCP-1 ex:>rc:essic>n may occur in w:avo and could be
encountered i.~ situ in t:~ie in~ral..t~:-Sri-nE~ endometrium.
Therefore, t vE_~ objective of the pre:>ent= study was
to examine thc~ in si to e~r;~~ressiorG ~f MCP-i in the endometrium
of women with and with ?i:~t. enclometriosi=~ and tc> investigate
whether that f~xpressioa ~::ould vary with the stage of the
disease and the phases c:~tthe mer:strual. cycle.
Materials and methods
Subjects
CA 02377786 2003-06-20
32 85409-5
Women were r~~,c:ru:i.ted into the study between
February 1999 and Junc= 199E~ after they providad informed
consent for a prot:oc:o7. ~~pproved by t.lue ,'>aint.-Eran~ois
d' Assise Hospital Ethi :..~ ~ Ccnruni.ttee on Ht.iman Research. Women
included in the study .nac~ ruo endometria~i hyperplasia or
neoplasia, anal they ha~::l not:. received t~y° ant.i-in:flammatc>ry or
hormonal medication du.r:i_ng a perLod o- at least 3 months
before laparo~>copy. Erzc~ometriosis was diagnosed at
laparoscopy fc>r infE:.rt.:i..:~vt:y and%or pelv~ c pain or at t:ubal
litigation. The stage e~f endometriosis was determined
according to the revi.sfecz c: s.ass.ifi.caton of the American
Fertility Society (The Ameeric:an Fertv~l~_t.y Society, 1985) .
Patients with endometr:icsis (n = 4'7) had no other pelvic
pathology. Contro:L sub;i cu=t.s n = 22 ) weir a fert=ile women
requesting tug>al litigaa=ion anal ha~~inc~ no visible evidence of
endometriosis at lapar::>:~copy. The a:ycle phase (proliferative
or secretory) was dete:rmi_nE:d acct>rcl:irog tc the cycle history,
progesterone levels in t:he serum, and histologic crlterla.
Table 3 summarizes the main clinical c'hara~~te.r~istics of the
subjects incl;::ded in tf~c: ~~tudy: ;=cge, infertil.:~ty, cycle
phase, and stage of endometriosis.
Table 3. Clinical Char~:~cteristics of Fatients at Time of
Laparoscopy
Number of subjects by cycle
_ phase
.~lge (Mean
Number of E SD) _ Proliferative Secretory
subjec.:_ts
~
Controls 22 32.4+6.3 9 13
Endometriosis47 31, 8-+ 1 30
i.5
(total)
Stage I 18 31.3+5.1 7 11
Stage II 17 31.6+5.0 ~ 11
Stage III-IV12 32.8 ~ 4 8
6.9
Fertile 26 31.8-+6.6 12 14
Infertile 21 31.8-+3.8 5 16
CA 02377786 2003-06-20
33 X35409-5
Collection o.t~ Enc~'omet.:~:-i.~-i1 B.iops:ies
Enca~metrial k:>> opsies were obt~.inec:~ during
laparoscopy i.isinc7 a sterile Novak's cam.z.la. Specimens were
placed at 4°C: in ster:.le Hanks' balanced salt sclution
containing 1Cn U~'mi pE:~rv~.ci:ili.n, 100 ~rg/mL streptomycin, and
0.25 Irg/mL ar<rphotericp n, immediately transported to the
laboratory, :.>nap-frozc::~r~ in liquid n:Ltrogen with 'tissue-Tek
OCTTM compoun;~ (M:i.les, E:i .hart, IN) , ar.d snored at -70°C
until
analyzed.
Immunohistoch~=mi stry
Serial 4- tc:: 5-arm ~cryosections were first fixed in
4% formaldehyde sol.ut.i or:~ ( hi skier .;c.ientu.f ic, Montreal,
Canada) for 20 minutes at room temperatm°e, then
permeabilized with Tr:i I:c::~n ?~;-100~'f 1's in phos~:~hat.e-buffered
saline (PBS) ~=or 20 mirw,_es at: room 'temperature), and treated
with 0. 3 o H20~~ in abso:~ute methanol for 20 minutes at room
temperature to elimin~~~:.a endogeneous pe:oxidase.
Immunostaining was performed using a mouse monoclonal anti-
MCP-1 antibody ( 10 pg/.rL: .i.n 'aBS clonta.ini.ng 1. ~ bovine serum
albumin) (R & D Systerrvs,, Minneapolis, MI~~) arid a Vectastain
Elite ABC kit.z~~ (Vector: Laboratories, Burlingame, CA) and
diaminobenzid~_ne (Sign':.=~,, :3t:. Louis, Mcj as c:hromogen and
hematoxylin fcr counterstaining. The specificity of the
immunoreactivity showrn key the anti-MCP'--.: antibody (primary
2 ~ antibody) was examined k;~,~ preabsorpt.:ion with an excess c~f
MCP-1 (50 ug/mL) prior t:o incubation with endometrial tissue
sections . Sect:ions i.nca. x;<~t:ed wi.t ruooat t hew pri.m;~ry antibody or
with mouse immunoglobu l.in of the same :~nununoglobulin class
and concentration as flue primary antibody were included as
negative controls ir: a l ~. experiments . ~~s far as possible,
each experiment includE~c:~ tissues from :~c>rmal ;subjects and
patients with different ~~ndometries:i;~ :>t:ages.. Slides were
CA 02377786 2003-06-20
34 85409-5
viewed using a Leir_a ruit:~z~oscr>pe (I.~e:ica mikr~:~skopie and
systeme GmbH, Model DL~~:PI=~) , and photomicrographs were made
with Kodak 1(10 ASA fi-r. MCP-1 imnuunostaining was evaluated
in a blinded fashion a;y two independent observers without
knowledge of laparoscc~p:i c f indir~g~~ . The :i_ntensity of staining
was evaluate<:l 3 tames ire 3 dif.ferent~ areas randomly selected
in the section and scc, r~-~d using aru arbi ~rary scale { 0 =
absent, 1 = Eight, 2 =- »moderate, and 3 -- intense). High
concordance between true t;ao observers was found as determined
by the kappa ( K) measl.:r~~~ of agreement ( ~'leiss ~I . L. , 1981 ) for
MCP-1 express:i_on scorE-~ i n t:he st:rc~ma ( K =- 0 . '70 ) and
epithelial glands ( K -- i:~ . 8' ) .
DNA Probe Labf~ling anc? .i~~ Situ Hybridization
1~~ cDNA for hurr._~n MCP-1, a °i38-base pair fragment
subcloned into the plasrn.id vector_ {pUC:lB), and the pUClB
plasmid were provided ~>~~r the American '.type Culture Collection
(Rockville, MD) . ~3ioti~~--Labeled %: DNA p_~obes were prepared by
nick translation from t:k~e entire pla:~rn:icvect:~:or with the MCP-
2C 1 cDNA (Lawrence ~.I.B. ~-:r: <~1., 1.985) us.inc~ a Bi~:oNickTM Labeling
System (Life Technoloc;~.e~:s, Burlington, Canada) . Serial.
cryosections were prepaxed and (fixed i.r: formaldehyde as
described earlier. Aft.ex:~ digestion with prote:inase K (2 ug/mL
Tris/ethylenediamine ta.~~;:racetic acid; during L5 minutes at
25 37°C, sectiona were post:.--fixed in 4 o formaldehyde, acetylated
by immersion i n 0 . 2_5% :~c.-.et=ic anhydride i n O . J. mo:1/L
triethanolamine, pH 8, f~:~r 10 minute:, and rinsed in PB~.
before progre~;sive dehydration i~r alc:ohc:l baths (50 to 100%) .
Sections were preclybri,,iiaec~ for' 0 minutes at 37°C with the
30 hybridization buffer c7c~voi_d of pro~~e containing 50 0 (v/v)
formamide, 10~ (v/v) dr~x~:ran sulphate, G.1~ sodium dodecyl
sulphate, 2X ~.SC, 1X Den'nardt:'s sol_utior. (C.Oi?o Ficoll
CA 02377786 2003-06-20
35 X35409-5
( Pharmacia, Q xebec, Ca:mada ) , 0 . Ci2aa human t~erum albumin (HSA) ,
0. 02 o polyvinylpyroli~_.ione (Sigma) , and 40 mmol/L monosodium
phosphate, pH 7) . They were then riybridized with 5 ng/u:~
biotinylated aerobe ancf dissolved in the hybridization buffer
for 18 hours at 37°C .in a ;mmidif~.ed chamber. Thereafterr,
slides were first: immc::rv;ed in 5ii'o formami.de; 2X SSC solui~ion
(2 baths for ;? minut=e:. :each at :~r7°C) , tiler. _i_n 2X SSC, and
finally in PBS cO.Cltai:a.ir~g (a . 25 ~ ~--ISA (2 baths for 5 minui_es
each at room temperat;., _~c ) . E3i of in was dt->.tect=ed by a series of
45-minute incubations ..~t 3 7"~~ witru a rabbit pol.yclonal anti-
biotin antibc:~dy (lo d1 Lut.ion in PBS/0.2v'-t: H:>A) (Fnzo
Diagnostics, 7~ong Tsl~~ncl, New York) , a bxotinylated goat.
anti-rabbit polyclona:! ,antibody (1'=o dilution in PBS/0.250
HSA) (Vector) ,, and fll_,cor~~sc:ein ~_soth.ioc.yanate-conjugated
1'i streptavidin (0.5~ in f?~S/0.2_'a~ HA) (Life Technologies),
respectively. Slides were then treat=ed ~1~~ th propidium iodine
(1.5 ug/mL distilled c~~<~ter) (:>igma), which makes the nucleus
visible in ye_Llow-orar;c~e upon LJV exc:itarion, and mounted with
Mowiol to whi_<,h p-phen:yle:ne-diamine (~J:ic)ma), an anti-fading
agent, was added at a final concentration of 1 mg/mL.
Sections were finally observed under t=he Leica microscope
equipped for f=luorescence with a 100-watt. UV lamp, and
photomicrographs were oc:~de with Kcdak 4()0 ASA film. As
negative controls, :>ec t;:io:m> from each ::~; ssue were incubated
without specij=is ._~.DNA probes or wish nonspecific DNA probes
prepared by n__ck tram Laticjn from t_he p_~asmi_d vector alone,
ie, without the MCP-1 c;l~l~A. The specificity of MCP-1 c: DNA
probes was al:~o examir,e;~ by northern blc>t using total RNA
extracted from endomet :r°:i.a L c;el7_.. 1i c>:r ~t~ese experiments 3'
P-
labeled DNA probes were prepared by ni~:k translation from the
entire plasmicis (with end without MC:~~-L cDNA insert) and by
nick translation r~r "c:=L:igo.l_abels_ng" (T7~uickPrimeTM Kit,
Pharmacia) of the isolated MCP-1 cDNA fzagment. As far as
CA 02377786 2003-06-20
36 85409-5
possible, each experiment included tiss,.zes from normal
subjects and ~~ati.ents ~a::th differcmt= encLc:~mel~:riosis stages.
The expressic:n of MCP--L mRIaA wa:~ c:~valuat:ed 1_r~ a blinded
fashion by two indeper~d~-nt observer:> as descvribed ear:tier
.'> using a similar arbit, ~r y scale. f-!i_gh int~erobserver
concordance r_f~garding 1'~1~'P-_expression gating in the stroma
and the gland: wa.s foi:~rn:~ ac~c:~~rd~.ng t:o ttm. K measures of
agreement (0.'78 and 0. 7 %, respectively) .
Statistical Analyses
MCF--1 score:. :follow an o.rdina:l scale. Therefore,
statistical analyses ~,~ere perfoz:me~d conserva tive:Ly using non-
parametric mel~hods. Ar:alysis of ir~tergroup differences was
performed using t:~e Kr ~.z;~ka1-Wa 1 '~is one-way analysis of
variance by r<~nks . Cnc.:~ ~ id~_za l groups were compared using the
Wilcoxon rank--sure test (Mann-Whi~r~.ey-Wilooxon test), and the
presence of tied scorE:~ was taken into ~~ccount in the
determination of the um and the var.i._rnce of ranks (Siegel S.
et al, 1988 ) . The l3onferrorn:i. proc~edurc~ (also called Dunn' s
multiple comparison poc~edi_zre) was appL:i_ed when more than two
groups were compared. '~'lr; a correlat ion between
immunohistochemistry ancy iru .situ rvybridization scores in
individual tissue spec~niens was evaluated using the Spearman
correlation coeff.icier t. Comparison of patient's age was
performed using Stuc~er; ~~ ' s t-test. h~et:w~~~sr~. two groups and one-
2_'> way analysis of varian,~:~e when several gm~oups were compared.
All analyses were perto:rmed using the statistical analysis
system (SAS Institute, ~ar,~, rdC) . Diffe~~ences were considered
as statistica:l.ly si.gnifi:;ant. f:or P va~~y~.ze: < 0.05.
Results
Positive imrr~ur~:~:rl.is tochernic:al st=aining of MCP-._ was
observed both in the :-.t~:roma and epi.t-heli_al g:Lands of the
CA 02377786 2003-06-20
8.7 85409-5
endometrium, and the i.~rtf:,rsity of staining widely varied
among patients. St:atis:i~::al. analysis of the results with the
Wilcoxon test did not r~~::~w a sigruificar_t difference in the
intensity of MCP-1 immr.«5taining found in thE: stroma between
women with anti wit-bout E~r.~::lometriosis, albeit i.n the presence
of the disease, triere ,~~~..> a markE d tendency fc:>r an increased
expression (P = 0.0518', . Iru add.it.i:>ri, r:c~ stati.stically
significant difference ~.w MCP-1 irru~rmnostainin<:~ between stroma
and endometrial glands i.ra t: rue ccratrol group was observed (P
0.0927), whereas ,~n th~~ E:ndometrio=~is group, MCP-1
immunostaining was sigr~i..fvicantly greater at the level of
endometrial glands (P -- 0.0001) (Table 4) .
CA 02377786 2003-06-20
38 85409-5
Table 4. Number of Norrru ::1. and Endomet:rwc;~sis Subjects
According to the ~nten:=.~_ty cf MCi' arot=.e~-n Staining in the
Endometrium
Stroma c~l ands
Intensity ~~:~t ~ intensity of
staining _ _sta:ining
0 i . ~ - F va.Lue ) 1 ,_. 3 P ~ralue
Controls 19 8 - - ' i.9 1, -
Endometriosis 18 ?~ 0 'J.C75:i8y ' v'3 1.6 6 0.0001*
(total)
Stage I 8 lCi _. ~) . % 107 ' 1 =3 ? 1 0 . 0048
Stage II 3 14 C~ 1. 01 ~~ 4 " - ~s ~a 5 0 . 0006*
Stage III- 7 ~ - - (J.1 zO'a" 1 F: ' - 0.0321*
Iv
Fertile 1Ci 16 -- - - 1.2 7.1 3
Infertile 8 13 -- - 0.9899' :-' 1.. 'i 3 0.2886'
Controls
~ - - 3 6 - -
Proliferative
phase
Secretory 9 4 -- - 054c:y 4 F.~ _ - 0..515$
phase
Endometriosis
7 1 C) - - 1 11 '' 1
Proliferative
phase
Secretory 11 i9 -- - 0.'7i:?4' 1 L~ l2 5 0.0760;
p h a s a _ _ _ _ ___ _ ___ _
*Comparison with control<>,rv~P :.°alues coi:~rected by the Boriferroni
_'> procedure; r, cony>ari.son with :I=ert-le t.~omeo wir..~ en domet.riosis;
',comparison of the F:rolif<:r~~:.av~~ phase with rne secrer.ory phase.
Based on thi s finding, we furt:.her i nvestigated
whether there was any association between MCP-1 expression by
endometrial c=Lands any; ~=~ndometriosis. ST~atistical analysis of
the results with the 4~i-'~~c:oxon test indi,:ate:> that the level
of MCP-1 immunostaining was signif:icant.iy higher in the
endometriosis group tt-.arl in the ~~c>ntrol group ( P = 0. 0001 ) .
Furthermore, when endc~rnetr.iosis patients were stratified by
severity of disease (,stages I, II, and rTI-I'J), a significant
difference among the four groups was observed following
analysis of i.:~tergroux~ r~ifferenc:es by the Kruskal-Wail.is test
(P = 0.0013). Post ho~_~ comparisons of individual groups using
the Wilcoxon test and the procedure of Bonferroni show that
CA 02377786 2003-06-20
39 85409-5
the intensit~~' of stairiirig was higher in eaclu endometrios.is
stage compared with ccantro:Ls, but the most significant
elevation of hICP-l imrro.anostaini.ng was f~m.md in the milder
stages ( I anc~ II ) ( P =-- C. 0048 ar,.d 0 . 000:x, respectively)
(Table 4).
Star~ist.ical analysis of the data regarding the
influence of ~~he rnenst real cycle ~~:~ase ,m the level of MCP-1
immunostainirv.g shows t h ~t within the control group there was
no significanl~ differEncve in MCP-1 expression between the
proliferative and the .secretcry phases tI? = 0.7515), whereas
in the endomei~riosis c:a r~:up, there was a marked trend for an
increased exp_~ession ~n the secretory phase (P = 0.0760)
(Table 4) . Ors the oth~-r hared, MCP-1 exvression was
significantly higher iri endometr:iosis p<it:ients than in
1_'> control subjects both i_::r; the proiifE:rat_i.ve (P <: 0.05) and the
secretory ( P ~, O . 00 i ) puases of tl-~.e ~~y~~.l_e~ .
The 47 patiE_wts with enc,ometriosis were also
stratified for infert ~::1_ity and MCP-l_ imrnunostaining scores
were compared.. Using thc~ '~lil~~oxon test, both fertile and
infertile patients with endometriosa_s nad elevated level of
immunostaininc~ compare~::~ w it:h con ~rol women ( P < 0 . 01 ) .
However, no st:atistic~:..Ly significant di_fvference between
fertile and infertile s~.~bjects having ervdomE:t~riosis was
observed (P =- 0.2886) ('rab:Le 4) .
2_'> Representats~,rf~ examplE~s of MCf-1 immunostaining in
the endometri.um of worr~eri with and without: endometriosis,
according to t:he phase of the menstrual cycle, are shown in
Figure 3: A, normal proaiferat:ive, day ~; B, normal
secretory, day 19; C:, endometriosis pro:'__iferative, day 8; and
D, endomet:rio:>is sec:ret.c~ry, day 24. No~E~ the brown positive
immunostaining in women ',~it:h endom_et:rio>is, which is
particularly rlarked or i he lcaminal s:i_de of endometrial elands
CA 02377786 2003-06-20
4 () f35409-5
in the secret=ory phaste c:~f the c~ycLe (i.mmunostaining score =
2) compared with that o~, normal subjects (irnrnunostaining
score = 1) . No ircununo::e~ZCtioru was observed iru negative
controls in which the anti-MC:P-1 t~ntibody w,_~~: replaced by an
~ equal content=ration o: nucuse imrnunoglobuiins of the same
isotype or preabsorbe;:l with an excess of MCP-1 prior to
incubation with endomc:~tr-i.al. tissue sections (data not shown) .
Expression o.f MCF~-1 m~~;~i'~, in t_he E'ndometrium
The ex~7ress ~.c~,rn of MCP-1 in the enc~ometrium was also
studied by i.1 situ hyx:~rldization in order to examine this site
of MCP-1 synthesis an~:; t o corrlpare the 1_evels of MCP-1 mRNA in
patients with ancz wittn~u.zt: endomet~_ iosis . Figure 4A show's the
appearance o:fr endometo:ial. stroma and glands at X167 and X666
magnifications follow .~a hybr-:idizat:ion ,end :; gained with
1.'~ propidium iodine. The '.r~ybridi.zatic..~n signal ;green-yellow)
could only bFe viaua.li:red at higher magnification (X1665) and
appeared to be mainly located .into the cel_1 cytcplasm, as
illustrated :i_n Figure 4fi, showi;ag a part of an endometr.ial
gland of a women with er,dometrios;_s with a hybridization
score equal t:~ 2.
As ~~escribecl earlier, an arbitrary score was used
in order to c~uant...ify t:hc=v hybridiza:~tion :~:i.gnal, and the
results were .~na7yzed ccanservatively ;sing ncnparametric
analysis of v,~riante Table 5) . High .levcal_s of mRNA werf=
detected in t.:ze c~pithc~lial glands of women with endomet:riosis
compared witO women w _.tr.out ev:iderrce of the disease ( P =-
0. 0001) , whereas no s~ gnif icant: d~~_fference _in mRNA expression
in the stroma:~ between women with and wit~hout~ endometrio;sis
was noted (P -- 0.2453';. Furthermore, a significant elevation
of MCP-1 expression ire e~ndometr~a~_ glands w<~:~ observed :in
endometriosis stages ;f - 0.0054j, II (P = 0.0261), and
I II-IV ( P = Ci . 0001 ) compared with the control group.
CA 02377786 2003-06-20
41 E35409-5
ThEe effect ;:f the rrlenstrual c~acle on the levels of.
MCP-1 mRNA found in tree endometriurrc was also evaluated.
Patients with endometuiosit road a higher MCP-1 mRNA
expression than contr~::.l subjects in both proliferative and
secretory phases of true :menstrual cycle ~; P ~~ 0 . 0001 ) . Within
the endometriosis group, a trend t=oward a higher expression
was observed in t:he sta:,zretory pha::e (P - 0. ()804) , whereas in
the control group, no significant difference between the
proliferative~ and the s~~cretory phases aa~; noted (P =
0.7262).
Stavistical analysis of MCP-1 mRNA expression in
fertile and ~.nfertile patients having endometriosis did not
show any significant cl:ifferer~ce between the two groups as
assessed by the Wilcot:o:n Mann--Whitney test ( P = 0 . 2904 ) .
CA 02377786 2003-06-20
42 85409-5
Table 5. Number of Normal and Endometriosis :=subjects
According to the Intelv:~:i.t:y c>f M~:~:P--1 mRNA Staining in the
Endometrium
Stroma __ __ _Clands ___
Intc~ns.ity o' Int ensity of
sta_nin<t _ _ st~<ai_rv_nc; _
0 - 7 . G' value 1 ~ . 3 P value
Controls 11 11 -- - LO 4 -
-
Endometriosis 20 - 0.2453* 1 11 17 0 0.0001*
1G
(total)
Stage I 6 11 - 0.70!:>6* 1 4 11 2 0.0054*
Stage II 13 __ 1 0.6327* - ~ 1 0.0261*
Stage III- 1 6 -- O.OO;sh* - -- 7 5 0.0001*
IV
Fertile 9 12 -- - ~ 16 _i
Infertile 11 7 -- U.2701~' 7. 6 1'L3 0.29041
Controls
4 ~ _. _ _
_
Proliferative
phase
Secretory '7 5 - 0 . 700 ~' 11 ,._- 0.7267'
- -
phase
Endometriosis
7 - __ 4 1 3
O
Proli.ferative
phase
Secretory 15 12 - 0.08E34' 1 , L'?5 0.0804$
_
phase _____
~
*Comparison with _ ;, theBonferro ni-
co ntro-L:P t,~alues wo~-rect:ed
by
5 procedure; t,cotry~ari-aon r ferile worker.f~rdometii~,>sis;t,comparison
wi mth
of the proliferativephasevri th l r,e phase.
sE wr~:~torw
Rep:resentat~v. examples of MCP-1 mRNA express=ion in
the endometrium c:~f wonders w:itr~ and withov.~t endometriosis
according to -she menst =ual <:yclE: phase are :shown in Figure 5:
A, normal pro.Liferati~:~r~, day 13; ~>, normal secretory, day 22;
C, endometrio;~is prol:if~::rative, da,y 12; and D, endometriosis
secretory, day 25. Note the green-yell-o'a spots (arrows) in
the endometri<~1 gland:, ~:>f women with endomet:riosis (scor_e =
1_'i 2) , particularly in t'tvE~ .sec~ret=ory phase, compared with that
of normal suk::=j ect s ( sc or a =- l ; . A very l..ow level of
hybridization was obsf~rvJed i_n nega.tive c:cantrols including the
omission of k;:iotinylat ec DNA probes prepared from the pLJCl8
CA 02377786 2003-06-20
43 (35409-5
plasmid containing MCi-w-:i c.DNA ir~_sert or the use of
biotinylated DNA probE.~~. c.;,btainec~ prom the p=Lasm.id alone. The
absence of nonspecifi_.nteraction between plasrnid DNA and
RNA was also con(: irmec; i;v Northerru blot using total RNA
:~ extracted fro:rt endomet:rial cells and 'rF-labe red DNA probes
prepared from the pUC ': plasm:i.d, t:he i.,U'.:18 piasmid containing
MCP-1 cDNA, or the is<;.lated cDNA insert. Finally, MCP-1 cDNA
probes were t:~asted on cL.r.omosomc~ preparat-i.on, and the
hybridization spots wF~re localized at band 1?q11.2-q2l.l as
expected (Mehrabian M. R.. et a_1_, 1991) (~:~at.a not. shown) .
Discussion
In vhe presf nt study, we have sr~own that women with
endometriosis had a h_~.gher level of MCP-1 expression in the
eutopic endometrium a:=ompared with nor_ma.l women having a
1_'i normal gynecc:_Logi.cal ~ tutus at lapar~os~o~~py. The highest level
of MCP-1 expz:ession w~ s observed in en~~c:~metr_ial. glands,
although a low level c:( expression could also be observed in
the stroma. In a prfaVS~ms >tudy, we found trv~at, following
stimulation with proir:f.lammatory cytokines it vitro,
epithelial ce=Lls isol~:t.f:~d from t:lne c-:ndometrium of women
suffering from endometr:ios:is secrete hicxher levels of MCP-1
than those of normal wc:>men (Akourn A et: al., 7.995b) . The
results of the present :=tudy clearly .incaicat:e that such an
up-regulation of MCP-~ :synthesis and secretion arises in vivo
2~~ and can be enc:ounterec; i.n s:i ~u i:i the uterine endometrium of
endometriosis patierot:. Furthermore, ~lney suggest that a
process of ce~_1 acti.vat:::ion would t:~ke i~_~ace at this level.
What: are the i..mpl..icati.on:; of <5ur findings with
respect to tr:e pathoprly~~iology of endometriosis?
3C Fir:~t, they ,_~ra consistent. w:i.t=h the basic and most
accepted hypothesis advanced by :;ampson in 1927 (Sampson
CA 02377786 2003-06-20
44 85409-5
J.A. , 1927) , who defi~vec~l endornet r_ iosi..s as an ectopic growth
of tissue that takes ~:m: i.c~in from, the uterine endometrium and
reaches the peritonea l ~.:avi.ty by '::ubal reflui: during
menstruation. Retrogr,.~de seeding i.s a common phenomenon in
most menstruating wom~vr~ (Blumenkr<~tz I~'.,~T. e~ al, 1981; Halme
J. et al, 1984b; Liu ::i.T.Y. et al, 1986>, and the presence of
viable endometri.al se l1_~ in the pE,ritcneal cav.it:y per se is
unlikely to be a causative factor. Genetic predisposition has
been invoked to e:xpla ~r: why endomc_~trial ;;ell:; would implant
1U ectopically into some particular patients and not into others
(Frey C.H., :L957; Mal:.na.k L.R. eat al, 198U). Hormonal factors
(Dizerega G.S. et. al, 1980), and immur:ological dysfunctions
observed bot~~n locally ilo. the pex:i_~.::o:neal cavity and
systematically in the peripheral blood of patients may also
have an impo.rtani~ rolf:~ .n the patloophysiology of the disease
(Dmowski W.P. et al, _994; Gieicher N. et. al, 1987; Halme J.
et al, 1987; Vigano P.. <~t: al, 1991_; Gcster_lynck D. J. et al,
1991; Gebel H.M. et a:!., 1995). However, to develop
endometriosi,s foci, tide,, "migrat:inc~" endometrial cells must
have the intrinsic ab:ili.ty to implant outside: the uterus and
to promote their own c:~rc:~wth. Int euestingly, recent data
suggest that uterine E.~ndcmetr_ia<_-.ells have an enhanced
ability to p:roliferat~~ (Wingfield M. et al, 1995) and to
escape immunosurveillanc~e (SOmigliana E. et al, 1996). 'They
2~ also abnorma~~ly expre::,s aromatase,. which is involved in
estrogen synthesis (Nc>>bl.e L.S, et al, 1996). The
overexpression of MCP-- 7. by endomet~rial tissue together 'with
these new observation:: make plausible that endometriosis
could be associated wi.tln spec:if:ic; alteration, at. the level of
eutopic endometrium.
Se<:ond, our findings provide an interesting
contribution in the uruderstanding of numerous previously
reported observat:ions oo. monocyte and macro.E~raage activation
CA 02377786 2003-06-20
45 85409-5
in endometricsis. Per.pLAeral. blood monocytes from women with
endometriosis are morE activated and secrete elevated levels
of proinflarrunatcry cy':o~::ines (Dmowski. W. P. :~t a1, 1994;
teller J.M. et al, 19f37'?.
They ~rlso :_;t_i.rnul.ate enc ometrial ::ell proliferation
in vitro, whf=~rea:> tho:_e from normal fertile women suppress
the proliferation of c~rdometrial_ cells (Braun D.P. et al,
1994). An increased ni:mber of a~t~.vated peritoneal
macrophages i_s a~..so oi~;-c-~rvec:l in ttue disease (Haney A. F. et
al, 1981; Ha:l_:me ~i . et ,:~ ~ , 1.983) . J:'hese cells secrete numerous
growth factox-s and cyt..~kinr~s that may contribute to ectopic
growth of endomet rial cc:==lls (Mc:l::aren vT. et <~i, 1996; Ha.lme J.
et al, 1988; dive D. ~!, et: a~., 1 9~~1 ) and perpet:uate the
pelvic inflarrvnatory rc:~ction observed in endcmetriosis
l.'s patients (Rang N. et ail., 199F~; FaL>ih H. et: al, 1987;
Eisermann J. et al, 1v38~j ) . MCP-1 ~_s a patent mediator o:f
monocyte inf=i_ltration into tumors and tissues (Schall T.J.,
1991; Leonard E.Ct. et a_, 1990), and only monocytes express a
significant wamber of :receptors fcr MCP-1. ('foshimura T. et
al, 1990) . Therefore, MCP-1 repre;>ents ~ plausible candidate
as an importa:~t factor involved in macrophage and monocyte
activation ire endometr .ic:sis. In si:~pport t:o this role, we
recently reported the presence of elevated c:oncentrat.ions and
biological ac~tivi.ties o~ MCP-1 in the PF and the peripheral
blood of patients w:i_tLa endometr:ic;>is (Akoum A. et al, 1'~96a;
Akoum A. et a:cl, 1996bj. MCP-1 could be secreted by
endometriotic: implant:. (Akoum A. et a.L, 199~ia) , by activated
monocytes and mac:roph~:ges, or by c;ther types of cells such as
endothelial <:~.r mesothel:i al c~e_L:Ls ~;Schal i_ T.,J. , 1991; Leonard
E. J. et al, 1 990; Aric:v i A. et a:1, 1997 ) . However, i_t could
also be postulated that uterine endomet r~i_al c:el 1s, by having
the intrinsic pot:entia,l t:o expres:~ high 1. evel.s of MCP-1,,
might be invc~Lved in ro~c:;r~ocyte ac.~tivation, arnd when reaching
CA 02377786 2003-06-20
46 85409-5
the peritoneal cavity by tubal refiux these cells may help to
initiate mono~syte recr uit:ment and activ.~tiorl. Interestingly,
according to .recent d«ta (Ota H, et al, 1.996), women with
endometriosi::~ present ,gin increased infi Lt:ration of monocytes
even in the eutopic erdometrium. ''his i.s in keeping with our
observations <~nd suggest that Mc::P-1 ~sou'_ca be involved in
enhanced monoc~yte rec_rw itment.
At the prote.:in level, MCP-1 expression was elevated
in the initial stages of tue disease, ~ar~ticularly in the
stage II, and decreased in more advanced stages (III-IV). The
expression of MCP-1. mF:.N;~ was also si_gni*::i.cantly elevated in
the initial st=ages ( I arcd .I I ) bu ~ rema iris high in more severe
disease compared wii~h ~:a:~ntrol. '.auch a ~_:.screpancy in MCF?-1
protein and rnRNA express ion is difficult to explain with
certainty, bui~ it migr t_ be due t.o a recinced translation of
MCP-1 and/or t=o a probable degra:~ati.on of the protein in the
endometrium i.n the more ,~dvanc:ec~ stages of the disease. On
the other hand, our results would suggest that endometri_osis
is more active in the ea:r.ly stages. Some-: studies have
documented that PF inf:l.ammation is i.nversel.y related to the
extent of vis__ble endc~Tretrlosis (Haney E~.F, et al, 1991) and
that less ext.msi~ae disease may be more bioc:hemically active
than older implants (Ve_-:lOr1 M.W. et a:1, 1986) . According to
Lessey et al ;Lessey E . ~~.. e3t al, 1994 ) , the d~afect of
2~~ integrin exprE:ssion in eutopic endometr~um is inversely
related to the stage c t: c~ndornetr ios.i.;~ . Lc>wever, it has also
been reported that the concentrations oi. chemokines, such as
interleukin-8 and RAN'I.E;;~~ ( x egulat,e~ or: activa ion normal- T
expressed and secreted), correlate with the severity of the
3C disease (Ryan I. P. et ~-~:L, '1995; ihorrarn 0. et al, 1993) .
MCP--1 expres.=_,:i_on was ruigher iri endometriosis
patients than in contra.:L subjects both _n the proliferative
CA 02377786 2003-06-20
47 85409-5
and the secret=ory pha~~~:~ of the menst:rualcycle phase. Within
the endometri.asis grovC>,, however:, there was a high trend for
an increased expressic::r1 :in the secrE:to:r~,~ phase, either_ at the
level of the protein c:u the mR)'dA. 'I'hf~se results reveal a
process of ce_~l activ~:t: ien that: ~ccJu:r:t-hrougnout the
menstrual cyc: _e in t:he andornetriarr. of pa:~t:ients but is
amplified in t:he sec~rr:~:.c:.r;y phase. Moreo~.Ter, an intense MCP-1
immunostainin<~ was frc_queni::ly lo~:~at~ed in the lumen of
endometrial guands in r:.!.e ~~ecrel:=~ry phase, indicating an
1C) increased release of N:::=-1 <~v thus period of the cycle. It
remains uncle<~r how MC: f---1 F:xpre:>sion ,~s regulated in the
endometrium and what: <:rf~ the me c.harui_sms that: gavern the
increased synthesis ar c~ secret:ion af: MCi?--1 in the eutopic
endometrium a._- women v, L vh endometz:iosis. Some proinflammatory
1_'~ cytokines can up-regu~ t:~ ~ = MCP-1 expres:~=~.an by endometrial
cells (Akoum E~. et al, 1 99:~a) , but it r<.mains to be
determined whether such-i an express i.an ~~could be modulated by
ovarian sterc;ids .
In summary, w~~~ found inc.reaseci expression of MCP-1
2C) in the eutopi-c endornetrium of women witLn endametriosis
compared with normal ierti7_e women wit.~~out laparoscopic
evidence of endom.etric::is.. Such ari increased expression was
dependent on i=he stage: c>f endometriosis and occurred
throughout the menstrL G:~:l cycle. 'these f_~.r~dings strongly
2_'~ suggest that endometr~c:oJi.s .is not only a local disease
restricted tr.: the peri !~~:~nea1 cavity but c.culd also be
associated wil~h patho~;tmysiological c:harle:~es at the level of
eutopic endorr~:etrium. >~ mc~ametria:L cells of women who can
develop endometri.asis might: be functionally different from
30 those of norm<~l women. ~_?nce preservt ect~:~pically, these cells
would have tl'-.e intrin~: i_:_~ ability t.o implant, proliferate, and
display a different rc=spanse to st imu_Li ~oresent: in their new
environment. Our data make also plausib:Le MCP-1 as a key
CA 02377786 2003-06-20
48 85409-5
effector cell. med:i_ateot in~~olved '~r. the. ~>athc;genesis of t:he
disease.
CA 02377786 2003-06-20
49 85409-5
Many of the :::,.i.o_ogical change. occurring in the
endometrium during the n:u~rmtr_u.ai c;yc~.~e ~:~ear a striking
resemblance to those a:;_;ociated with inflamrr~atory and
reparative pre>cesses. (e~nce, it wol.zld neat be surprising to
find that cytc>kinE:as krv::~~fJrn ior_ t.racir pr_o--inflarrunatory
properties, such as inlverleukin-1 ( II_~-=_ ) , could play a k:ey
role in the physi;~logy cW= thv~s t:i_ssue ar!d than, their action
would be tightly car~tr::~1_ Lecl by local mechanisms. In the
present study, immur~ohi;1=ochemical and GJestern b:Lot analyses
show that in normal worrlen n = 39) , the endomf?trial ti.s~~ue
expresses, in a cycle-c:lc~x:~eradent manner, the I:'~-1 receptor
type II (IL-1F;II), a mc:~:_c=rule of which tr:e only biological
property known to date . s t hat of ..:apt=ur ing I I~-l, inhi.bi.ting
thereby its binding tc_~ t_Ine func:t-.:.oval t::ype I IL-1 receptor.
1~ IL-RII immuno:~taining ~~a.~~s partic:ular_Ly ~i-ntense within the
lumen of the elands arw:I <~t °'he apical side of surface
epithelium. Interestingly, the intensity of staining ways
markedly less pronounc:~>.::~ iru the endomei=rium o women with
endometriosis (n = '_i4), ~ disease be=L:ieved t.o arise from the
abnormal deve~7_opment. of endc~metria:L i.~:i_ss,ue outside the
uterus, especiall=y in .ue early stage:_> of- the disease (outages
I and II) . Tr:is study ,..., the f:irst to show th~~ local
expression in endometr i_::~:L t:.istsuE: cf :I~I~--_'1RII, a potent and
specific down--regulate: r- ::~L I~-1 action and i.ts decreased
2~~ expression in women suf:~eri_ng from endometriosis.
Clterine endc;mer_rium, one of she most dynamic:
tissues of the human x::.>dxye, is are aclt:i_ve s ite of cytokine
production anc~ action. D;~:ring each menai..rua1_ cycle and
throughout the reprodl.:c:?v:LVe phase of wc~rnen' s life, the
endometrial t_:ssue i.znce:rgoes a caries ot= dynamic
physiological. processes ~f regenerat:ion, remodeling, and
differentiation, follc~wf_~d by necrosis and menstrual shedding
at the end of: the cycle should _..n~~Lant~~t::l.on not occur. It is
CA 02377786 2003-06-20
50 85409-5
well establis)ved t=hat t:.~aese ;:omp:Le~ everit:s are orchestrated
by the coincz_c~ent vari~~tions of e~trogeu and progesterone
levels in thE: periphe:c~~:'. circulation. H~:owever, many of t=he
biological changes occ ~_rr: ring in the human endometrium during
_'> the menstruaL_ cycle bc=ar a str_ikir,g resemblance to those
associated wii~h inflan~rn,~tor_y and repar~~ ive processes. Hence,
it is not sur~~rising t~> find that pro-irnf.lammatory cyto)cines
can be involved at aul o~,~rirle, para.crine, anti endocrine levels
in the modulation of a, ~~turzety of endomfet:rial functions
(Tabibzadeh :: . , 1991; ~inu~n ~~. et al, 1998b) .
Interleukin---1 ( II_~-1 ) :is one o;'~ the major pro-
inflammatory clytokine.found t=o act on <:~r~d to be produced by
endometrial t::i_ssue (S:imo::n ~::. et_ al-, 199~~>>; Simon C. et <~l,
1993; Simon C:. et a:L, L998c; Tabi~~zadeh :.'. et al, 1990) .
1'_> Circulating levels of CC;-l were st.own tw be variable during
the menstrual cycle ar,~.~ to reach rr~<~xima=_ levels during t=he
secretory pha:~e (afteY ~~>vulation) (Cawnon J.G. et al, 1985) .
The cytokine :is ~-r_oduc:.c~ci by tr_ophoblast'~c: cells, and is
believed to ~~c~t as an wmbr~ronic signal arid t:.o play an
important ro7_e during t.:ne implantation process (Simon. C. et
al, 1998b; Psychoyos Vie., 1993; Sheth K.'°l. et al, 1991) . IL-1
is produced 1 ocally ir, .::ndomet~rial. tissue as well, mainly in
the late secretory phc~s~~ (Simon C. et .~i, 1993; Kauma S.. et
al, 1990) , suggesting tf-cat beside it:s ~otent:ial role in
2_'> implantation <~nd embr~,-o:ni.c develop>ment, t: hi s cytokine may be
involved in t:he inf:Lanunatory-hike process that takes place in
the endometrium at th~> ~~nd of each mensrrual cycle.
Based on thc= above evidence, ~t: is reasonable to
believe that endometr:i<~l t~-ssue pc>ssessa:~ the appropriate
regulatory me~~hanisms that :pan operate l_ocal_l.y and mains=ain
tight contr_ol~ on the ioc:al level c>f pro-inflammatory
cytokines. Trv:i_s is crit_i.ca1 for maintaining the inflammatory-
CA 02377786 2003-06-20
51 85409-5
like process within sG physioi.oaic::~l~i.mit:s. Any defect
f~ ' in
such mechanis ms rr~ay t~:o endc~metrisldysfunr_tion and
lc-a_a
consequently 1:o endomet:.ri.um--relatc~a >rder_s affecting the
d-is~_
reproductive :=unctic:>ne, i.nfertilit~,~,endc>met.riosis,
( T
_'i dysfunctional_bleedinc, ~nc:t neoplasia)
.
Lit:i:le is kr-a:~wn about th.e mechanisms that modulate
the expression and thf acti.ou of pro-i~~lammatory cytok -nes
such as IL-1, in the f rm:x:~metriur,~. Cell ,~rtiVati.on by IL--1
results from its binding to ~~eli surfa~~e IL--1 receptor t:ype-1
1Cl (IL-1RI) that: in concc. rt w_t~~ Ih-7 receptor accessory protein
(IL-lRacP) is capable ;.~ transducing thf, activation signal
(Dinarello C.A., 1996; i:3oraschi C~. et a._, 1996) . Type I=~_ IL-1
receptor (IL-:LRII; ha::, in contrast to t:he type I recept:or,
no signaling properties, W.zt has rec:entl..y been described as a
1'> ~~decoy receptor". The eutracellula.r domain of the recept:or
can be shed f:=om the cell surface a:~ a soluble molecule that
is capable of captur_ir:g IL--l, thus. preventing i.ts interaction
with the fund=Tonal rFC~eptor. Thev.e stvc~i.es suggest that: IL-
1RII play an importani ~ary:~iological ro.e in the regulation
20 of IL-1 action in the i_mf lammatior. sites (Colotta, F. et: al,
1993; Colotta F. et al, 1994; Boss.u P. fit al, 1995; Orlando
S. et al, 199'7; Coultc_r ~.R. et al., 199'x) .
In the presernt study, we invest:.igated the
expression of IL-1 RI:I in t:he endc~metria of healthy women,
25 and women wit:h endometr:iosis, a very frequent endometrium-
dependent gyr:ecologic~,:1_ disorder. The disease i.s
characterized by an alr;.rm~rmal development of endometrial
tissue outside the utE-=ru..~s, mainly in tht_~ peritoneal cav_Lty,
and associated with ar, nLmuno-ir~fl_ammat,~ry process that has
30 been describE:~d in the b~~:trl ectopic and eutopic endometrial
sites (Witz C.A. et a.; , 199 ;'; McMaster 1'~I. 'I'. et al, 1998; Oral
CA 02377786 2003-06-20
'~2 85409-5
E. et al, 199E>; Ota H. <..'_ al., 19'6; Tsf~rlg J. F. et al, 1996;
Jolicoeur C. et al., l9Cas3i .
Our study resrea:Led that IL-1RII is indeed expressed
in endometria~_ tissue t~r.d .i.n a evade-dependent manner. The
expression wa:> omnipre::~en~ in both epitrmlial and stromal
compartments, and was more conspicuous ._n the secretory phase
of the menstrual c:yc:lE-, 'f'lhe most. irnt.erl:;Ee imrnunostaining was,
however, locat=ed in the: luminal side of endometrial glands
and surface epithelium., Int.erestingl.y, we fc>und out that. such
expression wa~~ strikirvg:Ly defic_ent. in women with
endometriosis, parti.cL7..ar.ly :in t:rne sec:re;~t.ory phase of the
menstrual cyc=_e .
The study provides f:or the f first tame evidence for
the local expression in human endometrial tissue of the IL-1
1~~ decoy receptor, one of ~ha most specif ic::: down-regulator: of
IL-1 action. Furthermore, it reveals a defect in that
expression in the intrauterine end.omet.r_z.um of women suffering
from endometriosis, tr:.at is, in the tis:;ue where the disease
is believed t:o take or_.:igin.
Materials and Methods
Study Participants
Women were Y e~~:ruited into the study after they
provided informed con:.-:erut. +'or a prot=oca i approved by the
Saint-Fran~ois d'Assi~:e Hospital Ethics Committee on Human
2_'~ Research. Women i.nc:Luc,led in the st.uc~y ('I able 6) had no signs
of endometrial hyperpiasia or neoplasia arid were not
receiving any anti-:inflammatory or hormonal medication <~t
least 3 months before laparoscopy. Endometriosis was
diagnosed during inve;t::i.gative l.a~>arosc~~~:~y for infertility
and/or pelvic pain, oi. at tubal litigation. The stage of
CA 02377786 2003-06-20
.'~3 85409-5
endometriosis was dete _rnined accc>:rr,:)ing t c the revised
classification of the %~rr:erican Fertility Society (American
Fertility Society, 1.98!:-; . Pat:ient.s wit-r1 endometriosis (ri =
54) otherwise had no ov:=ner pelvic pathology. Normal women (n
- 39) were fertile, rec,p.zest.ing tczbal 1_~t igati<m, and having
no visible evi.denc.;e of t:~ridornE~trie~s:is at= laparoscopy.
Menstrual cycle dating ~~~as determined by menstrual history
and confirmed by histr.:a.~c>gic~al exam:i_r~atic:n using t;he criteria
of Noyes and c:olleague,s (idoyes R.W. eat al, 1995) .
1C Table 6. Clinical Criar,~z::t~<~r_istic~ of Pat ient.s at Time of
Laparoscopy
I~lumbe.- vbjec~ts
of. ~~ by
~.:~~ic:le
phasr~
_
F.~~E
umber ~:vt roli'er:ati
"Mean Secretor
y
subj ec:t:- ve
'
:
. 1
',)
C on t r o .__ _ 1 --
l s - ----.. _
~
'~
.
~
3~a ~ 1. t:
Endometriosis :17.
,
C~
54 ?.' ~2
(total)
Stage I , _?c7.1
,
, l.() 7.3
~
E..B
Stage II -~~~'I
S
1y, ~ 10
4
.
'l
Stage III-IV ~....7i
7.'.~ Fi 9
'
_
.
(1
Fertile -::i.6
2 y H 16
6
.
~~
Infertile ='1.9+
3~-~ ~.,~
i
. 6
Collection of Endometx.ial Ricpsies
Endometrial biopsies were obtained during
1'> laparoscopy with the ~.:se o.f a Pipe~lle . ~Inimar Inc. , Prodimed,
Neuilly-En-Tc:helle, Fxazr~:,~e) . ;>pecimens were placed at 4"C in
sterile Hanks'' balancE~~~ salt. solution containing 100 U/ml
penicillin, 1()0 ~:g/ml ~~t.reptomycir,, and (a.25 ~,ag/ml
amphotericin, immedi.at e1 y transported to the laboratory,
20 snap-frozen in liquid nitrogen or embedded in Tissue-Tek OCT
CA 02377786 2003-06-20
59 X5409-5
compound (Miles Inc., ~,lkha:rt, ~1~1), and stored at -70°C until
analyzed.
Immunohisto~~:'n~amist.ry
Ser:Lal 4-~.zm c..ryosec:tions, were placed on poly-h-
lysine-coated glass m:icrosc~o~e slides and fixed for 20
minutes in fc::=maldernycie [4'v in phc~sphatE~-buffered saline
(PBS) ] (Fisher Scient i fic, M..;ntr~a.l, Quabec, Canada) . All
incubations were perf< rn~ed at room t:emperature in a
humidified cramber. Sf_ at ions were r~wn:_~e~:I in PBS, immersed in
PBS-1o Triton X-100~'~M !:ox 2.0 minutes at room temperature,
rinsed again :Ln PBS, ~.vc:i treated for_ ~0 minutes with hydrogen
peroxide (H20~) (0.3° n absolute methanol) tc~ eliminate
endogenous pe~:roxidase . After a PB~! r_ rose, immunostaining was
performed using a moue monoclonal anti-human IL-1RII
antibody (R & D Systenus, Minneapolis, MN) (primary antibody),
a Vectastain Mite AB(: i;it'r' (Vec:tc>r haboratozies, Burlingame,
CA) and diaminobenz:idan~_= (Sigma Chemic:al_ Co., St. Louis, MO)
as chromogen. Brief:Ly, after inc::ubat=ion with blocking serum
for 30 minute;, tectic~r~~. wc~r_e rinsed _n PBS, incubated for 90
minutes with an appr_oi:~~i.ate and prPdeterrnined dilution of
primary antibody (15 i.~;~/ml of PBS containing 1~ bovine serum
albumin) , rin;~ed in PFsS, and incubat=ed for 60 minutes with
the secondary antibody? cvonsisting of bi~ot:i.nylat;ed goat <~nti-
mouse polyclc>nal antil:~ody. Sections were then rinsed in PBS
and the avid_i.z-bi.otin~,,~lated horseradish peroxidase comp:Lex
was applied for 4 5 Tnirnut_es . After a PBS rinse followed by a
10-minute inc:ubat.ion v:~.ith diaminobenzidine: H~02 (0.5 mg/0.030
H20~ in PBS) sect.i_ons :were: washed '~n taP water , counterstained
with hematoxylin, and mounted witlu Mowi:~:1 (cJalbiochem-
Novabiochem (~~rp. , La Je:.I_la, CAj . Sections :incubated wi shout
the primary a:c:ztibody c:r_ with nonirunune mouse serum were
included as negative .~or~tro:ls ire al_L experiments. Slide, were
CA 02377786 2003-06-20
55 85409-5
viewed using a Leica rr~i~~roscope (I~ei~~a rnikroskopie and
systeme GmbH, Model DM;Z13; Postfa~~r~, W~=~t:~l.ar, Germany) and
photomicrographs were t-~~k~en with Kodak 1. C;0 ASA film (Kodak,
Toronto, Ontario, Cana~c~a ) . I 1-1 R I I immunostaining was
_'~ evaluated in a bl.indec.i m:~nner by two independent observers
having no knowledge of l_ap~zroscor~ic findings. The inten city
of staining w<~s evalu~ t~~~W t h.ree irnes ir: three different.
areas randoml_~~ selecte~c~l in the section <ind a :mean score was
given using an arbitra~~y scale (0, abserut; I_, light; 2,
moderate; and 3, inter-:~~~) . High c;oncorda:mce between the two
observers was found a~ determined 'try t:rlc~ K measure of
agreement ( rc =- 0 . 8 9 ) .
Dual Immunoflnorescen! ~>ta ~~.ning
Tissue se<:t_ioris were treat=ed and incubated at room
1_'i temperature with the rc.ro.~se monoc:lc:~nal ar~t:i-IL-1.RII antibody
as described earlier. ?after a PBS ri:~se, the sections were
incubated for: 60 minut: e,s wit":~ a rabbit= polyclonal anti-IL-1(3
antibody dilul=ed 8: 1, t:~()~~i in PB S-1° bow-le serum albumin (R &
D Systems) , washe~:l in i'BS, incubated fo60 minutes with a
biotinylated coat anti-rabbit antibody ;V'ect=.or Laboratories)
diluted 1: 100 in PBS-1 '; bovine ser~~rm albumin, washed again in
PBS, and fina_Lly incul;~ated simu=its.neously for 60 minus=es in
the dark witr~ fluores<:ein ~scthiocyanatE,-conjugated
streptavidin and a rhe~e~amine-conjugated goat: anti-mouse
2.'~ antibody (Sigma), which were used at a =final dilution of
l : 100 and 1: o ) in PBS- :1°; bovine serum a=ibumin, respect=ively.
Slides were then mounre~1 with Mows.o ~ t~ which p-
phenylenediarl:ine (S=igma, an anti i-fad:ing agent, was added at
a final concentration lit: 1 mq/ml, then observed under. the
Leica microsc~~pe equi~~ped for fluoresce;nc_:e with a 100 w<3tt UV
lamp and phot~~mic:rogr~,phs were rnac:le with Kodak 400 ASA .film.
In every expe::riment, ~~er_~tions from each endometrial tissue
CA 02377786 2003-06-20
56 85409-5
incubated with normal mouse and normal rabbit IaGs (used at
concentrations ecluiVa.Crut t~:> thc:se~ of the primary antibodies)
were included as negal:i~~F ~~ontrcl.s.
Western Blot Analysis
Frozen endor~uet.rial tissues were directly
homogenized with a mi.::r,:.;scal.e tissue grinder (Kontes,
Vineland, NJ> in a bu~rfer containing 0.5s Triton X-100, 10
mmol/L HEPES (pH 7.4), '~50 mmol/h NaCl, '._% mmol/L
ethyleneglycol.tet:raacc:-t.:a c <~ci_d, 2rnmollL
ethylenediaminetetraac.eti.c acid, 0. 02 o NaN3 and a mix of. anti-
proteases composed by 5 Lrmol%L aprotinin, 6.3 ~amol/L
leupeptin, and 3 mmol;':L, pheny:lmc:tr.y:lsulfony.l fluoride. Tissue
homogenate was then iro~_v~ba~.ec3 at ~l °C for 45 minutes und~sr
gentle shaking, and calirifuger~ at. 11, 000 X ~~ for 30 minutes
1.'~ to recover tree soluble: <~xt:ract, wL,~ose total protein
concentration wa:~ det<:rroined using the Bio-Rad DC Protein
Assay (Bio-Rad Laborat:~~r i.es Ltd. , M_ississauga, Ontario>,
Canada) . Pro~:ein:> (10i) Eg) from each extract were then heat-
denatured in a bc.~ilin<~ bath fo.r 3 minutes in 5X sodium
dodecyl sulfate :>ample: buf:Eer (1.25 mol/:L T:ris-HCI, pH 6.8,
50o glycerol, 25~ ~3-m~=r~:aptoethanol, 10° sodium dodecyl
sulfate, and 0. 0~. o brc:rro.~pheno:l blue ) , separated by sodium
dodecyl sulfate-polya~: r ylam:ide ge=i.. electrophoresis in 10 0
acrylamide li_neaz: gra;i~rvt gEVl slabs, a2d transferred onto
2.'~ 0 . 45-}zm nitrocel~.ulosF: rr~embrane~~ using a l ectrophoretic
transfer cell. (trans-~1<.~t, Bi.oRad. Nitr~~c:eilulose membranes
were then immersed in f:F3S containing 5o skirmued milk and O.lo
Tween 20 (blacking so_'.ur ~.on) for ~. hour at: 3 r°C., cut into
strips, and incubated c~~Terruight at. 4°~ -,aith a monoclonal
mouse anti-htz:man IL-1E<.I I antibody ( 2 }.zg/ml of blocking
solution) (R and D Systems) or with normal mouse
immunoglobul:ins (lgGs: ;f t~hE=' same immunoglo~:ulin class and
CA 02377786 2003-06-20
57 85409-5
concentration as the pr:irnary ant:i.body ;F: & D ;systems) . The
specificity of: the imm~znc.~rea<aion way: al so verified by pre-
absorpti.on of the antilooc:~y with <,rn excess of IL-1RII (20
ug/ml) . Thereafter, tr~f~ ::~trl~~s were ir~c:l~bated for 1 hour at
37°C with Fc-~>pec.ific ~~eroxidase-labeler goat anti-mouse
antibody (1:3000 c:lilut.i.on in the biockiria solution) (~,Tac:kson
ImmunoResearch Laborat~~ar.ie~ :Lnc. , West C-rove, PA) , washed
three times in PBS/0.1~ Tween 20, incuhat.ed with
chemiluminescE:nce reag~erz~t (Amersham, Oa~:zrill.e, Ontario,
1C Canada) for 1 minute, -z:i:_r-dried, wr_appecz in a plastic bag,
and exposed t.o a Kodak X-GMAT AR film (Eastman Kodak,
Rochester, NY) for 1 rr ir~ute.
Statistical Analysis
IL-1RII staining scores follow an ordinal scale.
I'_~ Statistical analysis ~~rs per:formed usinct Fi;~her' s exact test
(Siegel S. et al, 198;, and the Bon:fe.rroni procedure was
applied when more than two groups were compared. Comparison
of patient' s <~ge was L:erformed using o_ze-way analysis of
variance. All. ana.lyse:=; were performed .n._~zng the statistical
20 analyses system (SAS nstitute Inc., ~a~~y, North Carolina).
Differences were cons:ic~~erecf as statisti<.:a.lly significant for
P values < 0.05.
Results
Po" itive imnourrohistoclierrzica.L :~taini.ng of IL-1RII
25 was observed in many compartments of en~.~ometrial tissue. In
the stroma, i.mmunosta:~_n~ng was in general weak in the
proliferativE: phase arid more pronc:~unced :in the secretory
phase of the menstrual. cycle, mainly in isolated aggregates
and microvessels (Figure 6). However, the most marked
30 staining was prirzvaril~~,, located i.n epithelial cells.
Immunostaining was ob:..erved all around cells (cellular
CA 02377786 2003-06-20
58 85409-5
staining), but: also hacl the appearance of an intense brown
extracellular deposit ;.ru~~t= was predc>rn:i.nantly ! ocated within
the lumen of t: he gland=~ and t:he apic~al. :>ide of surface
epithelium (luminal stainir;g) (Figure 6).
According tc; o°c~cer~t findiric~;~, I:L-LRII is a decoy
receptor that can be .r~.eleased by proteo~.ysis from the
membrane-bound receptcz: extraceilular domain. The resulting
soluble receptor seem; to retain the same affinity for its
natural ligand IL-1~3 ,~':olotta F. et al, 1993; Colotta F. et
al, 1994; Bossu F. E:et :L, 1995; ~Iymon~ ~:n.A. et al, 1995) .
Dual immunofluorescenc;=. analysis using antibodies specific to
IL-1RII and I:1~-l~ clear°:l.y :~noweca thaboth antigens were co-
expressed within the Luminal deposit that makes plausible the
formation of :CL-1RII- _ I~~-:13 ~~omplex ( F~.gure ? ) .
1_'> Within the :ame endometr:ial ;~er_-tion, the intensity
of staining varied withc:~ut any discernible or apparent order
from one gland to anoi ht~~:r. Furthermore, great variations
between biops:ies taker. from different women at different
periods of the menstrn:a..l. cycle were notfed. The intensity of
IL-1RII cellu:Lar and _i,,.zminal stair~c.ing was scored in a blinded
manner by two indepenc~et observers using an arbitrary scale.
Statistical analysis c,f the data regarding the influence of
the menstruaa cycle wing the Fisher's exact test showed that
cellular staining was c~ffecti.vely more Lr~tense in the
secretory than ir; the px-oliferative phase of the cycle both
in stromal ( ~' _ ~X 1!::~ ~~' ~ and epi toel i.al ( P = 0 . 0310 ) cells
(Table 7 ) . However, ora:L yu a weak tendency fo~.r an increased
luminal staining in gar~.dular and surface epithelium was
noted in the secretor~,~ phase of tree cycle as compared to the
proliferative phase (.' -- 0. 13'70 > (Table 8 ) . Scattered brown
CA 02377786 2003-06-20
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Table 7. Number of No r:na_L and E:ndometr~osis Patients
According to the I:nt;er~.~i.~~y o~ II_-1R.I:L C:ellul.ar Immunostaining
in the Stroma, and in t_.Ze Crlands and :~unface epithelium
c_1 and
~nc~s
Stroma su, face
Intensit~~<~ ~-pvdueliun
~
sta._ni.ng Inr ensivy
of
s'~ ~i q
_ -__ .._____-_ nin
_. - J .. 3 P
0 1 3 C 1 '=
<
value* alue*
Controls 3 19 > 4 a: 13 1~ 7
Endometriosis 5 _ J 0.1120 4 ~ a:6 1 0.0660
25
(total)
Stage I 1 9 3 0 0.7_'7':0_ ? 13 0 0.1030
Stage II . 10 0 0.3240 C 10 i 1 0.2580
Stage III- 1 6 ~ 0 ().71%0 7 ~1 8 0 0.3680
~
IV
Fertile 1 1.0 3 0 C.1910 1 9 14 0 0.1280
Infertile 1 15 ? 0 Ci. 3~~0 ~ 'i212 1 0.3010
1
Controls
G Za ~ 1 ~: 9 E Z
!
Proliferative
phase
Secretory 1 4 :_'3 2:10--'r C: 9 71 6 0.0310
phase
Endometriosis
4 14 a :) _ 14 4 0
Proliferative
phase
Secretory 1 11. 9 0 0.0011' _ '% ~:2 1 0.0004
1
phase
F?roliferative
phase
Control 2 15 1 ~ 9 (. 1
4 14 0 0.2430 14 ~~ 0 0. E>060
Endometriosis
Secretory
phase
Control 1 4 ? 3 C~ 4 1 6
1 11 , 0 C' . 0910i , < 1 0 .
2 C)5:30
Endometriosis
*Comparison with controls, P valme~~ corrected b;~ the Bonferroni procedure.
'Comparison of the p-~oliferat.ive phase with the ;euretory phase.
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Table 8. Num;'»°r of Noznnal and Endometri~~sis Patients
According to the Inter.s:ity of I~T~-1.RII Lumi.nal Immunosta:i.ning
in the Glanda and surface Epithelium
Gland::;
~.~~i
~u
Mace
epithe
L~
~,am
intr:nsity
of -~_i-:wing
st J
0 3 P value
Controls ? ~~ ' !:i :!! 6 6
Endometri.os:is 37 ~ 6 10-~*
(total)
Stage I 17 _. ~ ~_' 0.0006*
Stage II 11. (? ~ 0.0015*
Stage III-IV 9 . ~ _ Ci.0360*
Fertile 16 . (? 4 0.0010*
Infertile ~ . _ ' 4 ?~ 10
1 s*
Contro~s
Proliferative 5 ... 9
phase
Secretory phase :' 8 ' 9 0,_~370t
Endometriosi.s
Proliferative 13 ~ _.
phase
Secretory phase 24 E: O '- 0.05901
Proliferative
phase
Control 5 .. 9 ?
Endometr.iosis I3 _ 9 0.0760
Secretory phase
Control 2 8 % 4
Endometriosis i_'9 6 0 ? 8 X 10-~*
Comparison with c.>ntroa ~; only si_gr:i.ficantvalues werecorrect=ed
P by
the Bonferre.~.i r.:>cedur;:~.
p
f Comparison. of prolifer~_'_i_:~:= phase ory phase.
the wit? the .secrat
deposits, wh_i_ch were emcounterec~ ~_n stron~:a, beit :less
the al
markedly than in lumi :aa:i an:d glandu_Larepithelium,were also
more frequent the ~~e<: retort' phasean in the
in th
proliferative ph ase, k;at: the differenr~edid riot
reach the
level of statist ical :,ic~nifican:_e 0.093Ci)
(,F' _ .
Ba>ed c>n these findings and the recently reported
down-regulatory propeotses of I~-1RII toward IL-1-mediated
l.'s inflammation and cell ,:~c~tivatiou, we further investigated
whether there way any ,~lte.ration ~_.n IL-1RII expression in the
endometrium of women crith endometi°iosis, a frequent
endometrium-related patrlology associated wilt-, an aberrant
inflammatory process ::~b;erved not only in ectopi.c sites where
CA 02377786 2003-06-20
61 85409-5
endometrial tissue abn~.>rmal.ly implantsr bat even in the
eutopic intrauterine endornetriunu. E~ndornetriai biopsies were
collected from 54 women presenting l.a~~aJ~oscopical and
histological evid~~nc:e .>:_ erudomet.r_io~;:i_:~>. As i.n normal women,
cellular staining in w~::>rnen with endometriosi.s was
significantly higher ~.ri thE: secretory pr~.ase than in the
proliferative phase of 1=.he menstrual. cycle, b~~th in stromal
and epithelial_ cells iP -- 0.0011. anct 0.0003, respectively).
However, when compares; to t: hat o ~ nc>rma_! women, this staining
1C~ showed a marked trend fc:~r a decreased :iratentsity in glandular
and luminal epitheliurc (P =- 0.0660), wlereat> in the strc>ma
the difference between women with and without was less
evident ( P = 0 . 11;> 0 ) . F a rthermore, t:he clecrea sed
immunostaininc~ observers in endorr~et.r.i_a:l. epithelial cells of
15 women with enc~ometrio:i..:~ compared to normal c:~ntrols appeared
to occur in the secret o:ry ( P = C? . 0 '~30 ) rather than in the
proliferative (P == 0. F(O=~J) phase of the menstrual cycle
(Table 7).
Scat=tered bxoorn deposits we.rf: also observed in the
20 stroma of women with endomet:riosis. a:~ ~.r~ normal women, they
were more obv=~~ous in t nE_ sec.retory phasF= of the menstrual
cycle and showed a corr~~~arable le~,rel of .>taining. The most
striking difference between women wi_t~~ endometriosis and
normal women was howe~;er detected at the _Level of IL-1RII
2,i staining in the lumen ~of endomet:rial_ gl<~nds and the apical
side of surface epit~heli.um. In fact, statistical analysis of
the data (Tab:Le 8 ) showed a cons.ic~erablc~ lack of staining in
women with endometrio~.i;: compared to :normal controls (P = 10-
Endometriosis pati:~r~t~:> were then stratified by severity of
30 disease (stage I, II, a::ud III--IV). Comparison of individual
groups using i~he Fisher' s exact test= an<:~ the procedure of
Bonferroni showed that the intensity of staining was
significantly lower ir~~~ach endometr_iosis stage compared to
CA 02377786 2003-06-20
62 85409-5
controls, but the most: ~.ignificant dec:re~ase in IL-1RII
immunostaininc~ was toy nc_.i irl the m1 l.der :st=ages ( I. and I I ', ( P =
0.0006 and 0.0015, re.pe~tively) . ~urt:~rez~more, statistical
analysis of t:he data t <~kin~~ into accountv the phase of the
_'~ menstrual cyc:_Le revea_ r,~ttn at the most marked drop in Ih-1RII
luminal staining .in er~,Icrn~~r::r.i.osis occ...zr~ed in the secret:ory
phase (P = 8 X 10-~) . "r1s watt ai.sc> observed in all stages of
endometriosis,. but wat mere pronounced :i.n the early ( I and
I I ) ( P = 0 . 00a? 0 ) than i.r: trie 7_at a st::~ges of the disease ( III-
IV) (P = O.OC)60) . In cc,u:t.rast, during the proliferative phase
of the menstrual cycla , th~~ di_ffert~nce ~ri IL-1RII luminal
immunostainin<I betweer women with and wr.thout endometriosis
was perceptible, but: ci..c~ not reach the ievel_ of statistical
significance (P = 0.0%~~0) . ThE: 54 pa ti.ent:s with endometx:iosis
l~~ were also stratified f;~:r :infertilit.y anc.l IL--1RII lumina7_
immunostainin<I scores Uaere compared. Usvng t:he Fisher' s exact
test, both fertile ancinfertz.le pat::it=nts wi_t:h endometriosis
had decreased levels c:t imrnunost:aining c_-c;mpared with control
women ( P = 0. 0010 and 4 ~ 10 ~~', respecti~~ely) , but more
significant d=_fference::~ in :inferti 1e wc~rnen with. endometriosis
was noted.
Repoesentati ~Te~ examples of Ia,-LRII= immunostaining
in the endomet:rium of Va:~,mer~ with and without endometrio~~is
are shown in Figure 8 (~, normal. secr_etcry, day 24; B,
2~~ endometriosis secretory%,, clay 26) . Node the fine brown
immunostaining around ::;el.Ls both in the stroma and glandular
epithelium, and the brosam depc>sit~ in tlnf-~ lumen of gland; in
normal women. No i_mmun:a:_eaction was observed in negative
controls in which the in 1. i-I:..-1RI I ant:ibc;dy was replaced by
an equal concentration ~.f mouse immun«c~-~cbulins of the ~~ame
isotype or pre-absorbe:I w:it: h an excess c>f IL-1RII before
incubation with endomet:~ la: tissue sec-_-ons (data not shown) .
CA 02377786 2003-06-20
E>3 85409-5
To ~ontirm ::.mm.unohs.st~w.chemical data regarding the
expression of IL-1RII i.n normal and endometriosis women and
to determine whether t:l;~~ mobi.lit y of the endometrial re~septor
corresponds t:c the knc:car~ mo_Lecular weight of this protein,
'~ equivalent arnsunt:.s of erndomet::rial proteins were analyzed by
Western blot . Enclomet~ :i~;l bic>ps Les were selec:ted from the
proliferative anc3 the sec:reto:ry phases.
Figure 9 sh<w:-~ that monoclonal anti-IL-1RII
antibody reacted prima r:i 1.y wz th a 68-kd banca and a doublet of
1c) 45- and 48-kd mol.eculacr weight bands. Sixty--eight and 45 kd
correspond to the rep::rted mo:lecu~_a_r weights of the membrane-
bound and the so)_uble forms o:>= the :IL-1.RLI receptor,
respectively (BoraseJh_ L~. et <~l, ;_996; ~c;lott:a F'. et al,
1993; Colotta F, et a-., 1994). Mir:or bands of lower molt=cular
15 weights recog~izEed spFecific:ally by the antibody have not been
reported previous>ly an:~ may presunuably correspond to
degradation products. Hawever, foi:- the same amount of total
endometrial ~~roteins, the intensity of If~-1RLL bands waa
clearly lower_ in biop.ies from women with endometriosis
20 included within the surne experiments.
Discussion
In the pres~:o:t study, wE: have shown t:hat IL-11~II
expression wa,s ubiquitous throughc.~ut the endometrial tissue,
and was in gE_>:-rera:l cye:le phase-dependent . The most marked
25 immunostainin~~, which ;~L:~peared rr;ieroscop:ic:ally as an
extracellular brown deposit, was observr~d in the gland',
lumen and the apical .aide of lumina:L epithe:Lium. The :luminal
secretion most likely corresponds to the soluble form o:f IL-
1RII. Western blot an<:,lysi:~ of IL--1RII in endometrial
30 biopsies have shown, n fact, the presence ofbands whoae
molecular wei.~~hts are c~quival_en?~ to those reported fo.r the
membrane-bouru:~ (68 kd; and the so~_uble 1,45 kd) forms of the
CA 02377786 2003-06-20
64 85409-5
IL-1RII receptor. NumErc:,us recent studies have reported that
IL-1RII is a decoy recveptor t:hat could be released in a
soluble form after en:~.yrr~atic clr~a~.Jage of the ext.racellular
domain of the membrane-found receptor. The soluble receptor
~ possesses the ability tc-; bind Ii..~-~i.)3, the circulating and most
active form c>f Ih-1, _ x:r-~ibiting t.lue.reby the interaction of
the latter w:i_th t:he fi.nc~t:.ional 7L--1RI anc:~, ~;cnsequentl.y, IL-
1-mediated cell activ.3t:ion (C;o:Lc:tt:a F. ;~t: al, 1.993; Colotta
F. et al, 19':4; E3ossu >'. et a.1, 1~:~95; Symon~ J.P_. et al,
1995). Dual i:mmunofluc:rescenc:e analysis showed that IL-1RII
and IL-1(3 wez~e both e. fc,ct:ive.ly co-expressed within the
luminal deposit, whicto makes plausible the formation of IL-
1RII-IL-1(3 co:nplex. These finding:> might be cf interesting
physiological. signi.fic:ar,ce. In fact IL-:i i_s cne of the major
1'.~ cytokines that are in~,rc~ved in the different cyclic events
occurring in human enclometrium (Tabibza:~eh S., 1991; Simon C.
et al, 1998b). The ~cyt:t~Jkine has been demonstrated as a key
mediator in t: he attacume:~nt of the embryo c>nt~; the endomfstrium
and the impla:ntat.ion ~.rc~cess (Simon C. rat a:1, 1998b;
Psychoyos A. , 1993; St~et:h K. V, et a_L, 1991. ) . In the
peripheral c:i.rcul_atior~, IL-1 leve.s increase after ovul<~tion
(Cannon J.G. et al, 1~?85), and, locally within endometr:ial
tissue, IL-1 :~roducticn ha:~ been >hown o considerably
increase in t:~e secret:ory phase, x:eaching its maximal levels
at the end of the menstrual cycle (Kauma :~. et al, 1990).
Hence the considerabli~ mpor_tanc:~e of the local availability
in the endomE~trium of a rec~uiatory mech::3ni.sm such as that of
IL-1RII that ~;an count e:rba:lance on- buffer the local action of
IL-1 and maintain its leve:Ls wi~t.hi_n the physiological limits
during the crucial per:.ic~d of_ implantation and during the
inflammatory-like pro::ess that tal<>es place .in the endametrium
at the end o~- eac:h me~::t rual cycle.
CA 02377786 2003-06-20
65 85409-5
To investig~:te the ro:Le of IL-1RII in endometrium-
related disorc~er~;, we .:assessed .Lta exprc~~ssion in the
endometrium of women ::.offering from endumetriosis. The
disease is as:~oci.ated w:i.t.h an immuno-_i_nflammatory proce ss
observed cons :LStently ~L:c~ t'ne per.it.oneal c:avv_ty where
endometrial t=issue abnormally develops (Kauma S. et al, 1990;
Witz C.A. et al, 1997; 'Jinatier D. et a.i., 1996) , and recently
noticed in the eutopic intrauterine endometriurn of patients
as well (Tseng J.F. et a.1, 1996; Isaat.~:s«r. K.B. et al, 1990;
Vigano P. et girl, 1998,' . Acc:ordirug to ~:~ur dat:a, the eutopic
endometrium of women ~,i~h endometriosis expresses in situ
increased levels of MC ~?~-:1 !Jolic:oeur C. et al, 1998) , a
chemokine endowed w~_tr ~..he potent: ar~:iiity of inducing
monocyte / macrophage cl~emoattract_ion and activation (Leonard
1~~ E.J. et al, 1.~~90) . Furt:herrno.re, cultu:rE~rl endometrial
epithelial cells from women with endometriosis displayed an
increased responsivene~s:~ to IL-~. in vi tx c~~ by secreting higher
amounts of MCF~-1 than :::c~:Lls from normal women after exposure
to the same concentrations of the proinflammatory cytokine
(Akoum A. et ail, 1995;;: ) .. I-ios~revex , t=he caause ( s ) of such an
exaggerated inflammatory reaction remain unknown. The present
study shows a dramatic :Lack in I:h-1RI:I expression in
endometrial tissue of women with endometriosi.:~. This ways
particularly obvious at the level of _Lh-1RII luminal
secretion, but: was alS~:> IlOtiCeab.:Le at the ievc~l of the
cellular expression bot:i'1 ir: epithelial and stromal cells.
Western blot analysis ~of .iL~-1RII expr_es:~ion i.n endometri.al
tissue confirmed :immurucoh.istochemical data as .:it showed that
the intensity of the 6ri--kd and lower rnoi.ecular weight bands
recognized specificall vy~ i:~y the anti-=CL--1 P.II antibody, was
markedly lower in women witi: endometr_Losis as compared to
normal controls.
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66 85409-5
The most stt .i~;.ing lack ~.n LL-1.RII l.uminal
expression wa:> observF-d i.n the earl=~est and initial stages of
the disease ( stages I ~~nd :~ I ) . (:)n one hand, t h.i.s in keeping
with numerous studies i=~dicating that aadometriosis is more
_'i active in the initial stages (Haney ,?~. E. et al, 1991; Vernon
M.W. et al, 1986; Les~ey B. et al, 1994) and is consistent
with the pattern o.f MO:'~'-1 expr.es.~iori t:~at we observed in the
endometrium oi= endometr:iosis patients that increased in
initial and decreased :.u-~ .l~~te endc;me~tri~osis stages (Jolicoeur
C. et al, 1998). On tre otrier hand, these results suggest
that abnormal_ IL-1RII Pxp.ressi_on rr~.ay 1:>e involved in the
initiation of the inflammatory process in the intrauterine
endometrial t~_ssue when-a tree disease .is believed to take
origin. Interestingly, our study also sruowed that defective
1~~ IL-1RII expre:~sion was rru~re si.gnific:ant i.n i.nfertile thin in
fertile women having endometriosis, which suggests an
involvement in endometr,:i~.~si.s--associated infertility.
The mechanisms underly~na the decreased expression
of IL-1RII in women wit:_~ endometr~i~:~s_Ls remain to be further
2C elucidated. The most si.c~nificant deficiency occurred at the
level of IL-1F;II l.umim:a:L aecreti.~on in epithelial cells, in
particular in the secrr~t=orl: phase cf the menstrual cycle.
This would suclgest an i_ruhibit=ed sheddi.nc~ of the receptor in
endometriosis occurring throughout the cycle, but to a
25 greater extent: in the :~ec~ond phase. At. the present time, it
is still unclE:ar what molecular and biochemical pathways
could be involved in glue <~eneraticr: of :,oluble IL-RII.
According to recent da~.a, matrix metal~o-proteases rather
than differential split_ i_nc~, play a key role in the production
30 of soluble decoy RII bay enzymatic cleavage from the cell
surface receptor (Orlaricio ~. Et al . , 1997 ) . The observation
of higher levels of ce_L l:zlar staining vri epithelial cells of
women with enclomet:ri.os.i..~; irv th.e secret:ory phase of the
CA 02377786 2003-06-20
67 8.'409-5
menstrual cycle as com?~:a:r-ed to the proliferative phase (P =
0.00037, Tabl a 7) , makc::~; ylau.sible a potentia, inhibition of
IL-1RII release from t:ne cell surface ir: t:he secretory phase.
However, our resu.l.ts a L.;:r~> show trra,: either cellular staining
in epithelial cells or tr~:e intenwit.y c>f the 6~ -kd band
corresponding to t:.he reported membrane-bound form of IL-1RII
receptor was reduced i a ~aomen w.it h endc>rnetriosi.s. This
suggests that beyond a p~t:ential aberrar:t release of sTL-1RII
from endometrial ~,:ell~; c:~_ women wii_h enGOmetr~osis, a
deficiency in IL-1RII ~:~rotein synthesis and/or a reduced IL-
1RII mRNA levels or gecoEe t:.ranscr:iptic:n rriight be involved. In
fact, our prelimiruary analyses ef IL-lRlI mRNA levels in the
endometria of women wi~:.rn and without endometr_osis tend to
support such ~. hypothe::~:is (data root: sr~own) .
In c:onc lut;i.or:, t~hi:~ i s the i ~z st study to show the
expression in endometr:i.al tissue of t:rie decoy IL-1 receptor
type II, a specific nav:,:~.aral inhi.t~it:on of IL-1. that plays an
important role in the c°egulat:ion of_ z1~--j ~ act ivity in the
uterine environment. Fn.~ra_hermore, our study revealed a
2C striking lack in f:L-lE~',l:f: exp~~ess.on i.re women :suffering from
endometriosis. This ma;y represent a plausible mechanism
underlying immuno-inf:l.,:-,zrrunat.ory changes c:~bserved in the
eutopic endometrium of women with endometriosis as well as in
endometrial t-_ssue abru:o_ma:i.ly implanted i_n ectopic sites.
CA 02377786 2003-06-20
68 85409-5
Abstract
Cytokines su;Li as inte~~leukiri-1 (IL--1) play a major
role in the reparative and inflarrunatorv-like i?rocesses that
occur in human endometri_~am during every menstrua:L cycle, but
they also seem to be iwl~'~~icat:ed i n c:Vrit~~ cal z:e~producti.ve
events such a~~ ov~~lati.c.->n avid impl.aritat~,yc>n. Interleuki.n-1 is
tightly regulated in t ire body by a Complex network of cc>ntrol
systems . In the ~>reseri;:: study, ~,ae ex~~rn~_ned the expression of
IL-1RII, a nat:ura.1 speavi.f_ic inhibitor of IL-l, in the human
IC endometrium arid found ~~-rro interesting distribution and
temporal pattern of ex~:~Lc:~ssion throughout the menstrual
cycle. Immur_oreacti.ve C:i~-1RII w<~s found in stromal as well
as epithelial cells, W.z1= it was predominant w:Lthin the 1_umen
of the glands and the v~;:ica.1 ss_de of snrfaCE' ~aplthelium. In
I~~ situ hybridiz~ition anc=everse tran~;cri_ption-,~olymerase chain
reaction (RT-F?CR) analyses showed hi.ghc=! levels of mRNA in
epithelial than in str.rrr~a:1 cells. Thf~ ='~L-1RII cellular and
luminal secretion foll~w~~d a regulated cycle ,phase-dependent
pattern of expression. ,although eleva_c~d in the late
20 proliferative/early sec_retory phase of the menstrual cycle,
IL-1RII luminal secret i_c~-n :>:ignit-icant.iy decreased in the
midsecretory phase, reaclZing it:> lowesr_ levels at Day 27_,
before augmenting markedly again during the late secretary
phase. This pattern c~f expressi.on was less obvious at t:he
2_'> level of cellular st~a:aruing, as examined by
immunohistocriemistry, but it was corr~:~borated by Western blot
analysis of 7:7~-lP:iI protein and se~mi~:~uarnt.itative RT-PCR of
IL-1RII mRNA in the wL~c:>l.a endometrial t:i_;~sue and separated
glandular epii~helial cells. The reduced ex~>ression of IL-
30 1RII within t=he implantation window suggest: the existence of
accurate regu_Latory mes:~rianisrns ':hat, by down-regulating IL-
1RII expression, al.LeT, i_~te IL~-1 iruhibit u_~n during this
crucial period and :Eac: L l.itate il,-I pro imp l.ant.ation actions .
CA 02377786 2003-06-20
69 85409-5
The elevated cxpressi<;~r «f I:h-1R'~.L observed da.zring the late
secretory phase suggest; an s_nve,~v~rnent: ef II~--1.RII in ccntrol
of the proinflammatory t;tat:e that ta~:es place in the
endometrium d~:.ring the p.nennernstrua'~ anc~ menstrual. periods.
Introduction
Interleukin-.. ;; IL-7_ ) i= the germ used 1~o describe
two polypepti~les (IL-l.ra. sand IL-1~3) that: play ~~ key role in
immune and inflammator~~- react:ions (Di.r~.arello ~:.A., 1996) .
Three receptors for IL--J., type I (:I:L---wRT ) , type II ( IL-1RII ) ,
and type III ( IL-J_ 1RI II ) , have l:>een ~..c~enti.fie~d i.n different
cell types ( Di.narello .:'1. , x_996; Arend W . P. , 1991 ) . Cell
activation in response to IL-1 appears t c be mediated
exclusively vi.a the IL-~.'tW (Suns J.F. et al, X988; Sims J.E.
et al, 1993) , with coe:~=~;ression of a rec:eptcr accessory
protein (IL-1F;-Ac I? or i.L,--1RIII) being crucial to IL-1-
mediated signaling evera:"; (Greenf:ec~er :>.R. et. al, 1995;
Wesche H. et al, '_997; Iv:m°herr C. et a-.~, 1997 i . In itself,
IL-1RII is nct: a signa.l.i.ng molecule but, in fact, is reported
to be a decoy target: ef IL-1 (Mcl~qahar: C:. ~ . et: al, 1991.;
Colotta F. et al, 1994) . ~a.c~dition_vl-'~y, IL-R.II could be shed
from the cell surface a~a sclub:Le mol.ec:ule that would then
capture IL-1 ~~nd :inhibi.t: its binoling t:c> IL-1RI (Girl J.G. et
al, 1990; Symons J.A. ~_ec: al, 199'p; Bossiz P. et a1, 1995) ,
thus suggesting an i.mp~ol:vt~<~r~:t rolE: t=c~r_ CI,-RII in regulating
the biological_ activiti.~~s r_~f IL-~.
The avail~ibl.~:~ ;data ind.cate t~ruat I:L-1 is involved
in the regulat:ion of F,°u:i~::mnetrial. flm:ct::ic>ns ('L'abibzadeh
:~. ,
1991 ) . It has been sru.>~wo that I.L-1 :i;.., :;ecret;ed by human
blastocysts, and it i>thought: t: o acts a; an embryonic si-gnal
3C~ (Baranao R. I. et al, 1'x'3?; . The cyt.okine wa s also detected
locally in the endometri.;~:1 tissue dm:r.i-nc~ the late secret:ory
CA 02377786 2003-06-20
f' 0 85409-5
phase of the menstrual c:vycle (KaiimaEt al, 1990) . This
~.
suggests a role in the t=:i_ssue ne~~rosisand disintegration
occurring in the endom~:= t: ri_unn at d of th<- menstrual
t he en
cycle in the ,~bserice o: .mpi_anta-tic~n,which is not surprising
considering tr,e simila~Pi_t:y betwef:~nse processes and those
trie
occurring during inflar mat: ion. lv~xE:~res~>i.on of the functional
receptor of I:L~-1 ( i . _=h-1 RI ) bias>er: detected in the
a . ,. be
human endometrium as w~_= _i_:. (Simon al, 19~~3; Bigonnesse
C. et=
F. et al, 2001a) , where:=:'t. ~~ppears play a key role in the
tc,
implantation ~~rocess .imon C. et 1994 ) .
( ' al,
Due to its p:LE::iotropic acti.v~_t y and potent
proinflammatoryeffects,, f:L-J_ is t:ight:~y regulated in the body
by complex control sys.t:ems. In r>articular, two :Lnhibitors
participate in these r pu.i-'~atory mechan~_s;ms : true =eceptor
antagonist (IL~-lra) , wf-~.i,h binds avi.d:~y to IL--1RI and
prevents IL-1 binding .:mc:~ signal trarrsc~uction; and IL-1R.II,
which is considered to r>e:e a natur~a.l_ scw-enger for IL-1. The
IL-1RII can vE~ry eff:ic:iently bind IL-1~3, whereas its affinity
for IL-la and IL-Ira i~ 10- too 1.00--fold lower_ (Boraschi D. et
al, 1996) .
In view of tine major r~>le of 1I~-1 in the regulation
of various endometrial aad repro~~uc:t-~ve funct:'~ons, knowledge
regarding the local ava~.'~abi~it.y arid rxcc:urate production of
specific inhibitors fc>.r- Il~-1 in t:he en<~ometri~zm Throughout
the menstrual cycle be~,:ornes. esserlt:ial_. The expression of IL-
lra in the human endom~.>t: r_ium has been previously reported
(Simon C. et ~:1, 1995x) .; IL;-lra immunoreactivity was elevated
during the proliferative pha:~e oi= the menstru<~l cycle,
whereas endometri_a.l ce~l._:1:~ <xppeared to express intracellular
3C IL-lra (icIL-1_ra) . Th~=~ objecti.ve of the pres~snt study was to
assess the local avai.l;~x:,i_Lity and expre.,sior~ of IL-1RII
CA 02377786 2003-06-20
71 85409-5
throughout the normal rnerlst_rual cy~:le t:c~ further elucidate
the mechanism; controli=i.ng LL-1 act.ivz.ty in the endometrium.
Materials and Methods
SurJ~ect.~,. Women who pa .rtic_~1-<rt ed in the study
provided infcrmed consE_~rit ~-or_ a protocol approved by they
Saint-Fran~oi~> d'l~ssisE.~ flosp;~tal Et:hic-r Comrrci.ttee on Human
Research. They>e women ;n -= 42) were a<~ec between 23 and 47 yr
(mean ~ SD, 3~:. 6 1: 5. G ;~:~_ ) . The~y~ wE r_e i ertile, requested
tubal ligation, and hac:~ a r.or_mai and regular menstrual cycle.
1C None had visible endonv:t=vial r~.yperpla:_~ia or nf.=oplasia,
inflarrrmatory disease, ~.m:~ endornet:ric>s=i:~ at the time of
clinical examination or laparoscopy. Women were not
receiving any anti-ina:i.a:~rnmGtory or hormonal medication at
least 3 mo bef_ore laparoscopy. ~1'he cyc~~e day was determined
l~~ according to t;he cycle tn istory and h_lstologi.cal writeria
(Noyes R.W. et~ al, 19t~'~;~ . Eighteen women wer~s in the
proliferative phase ar7~-i ?4 i.n t:he secret:c>ry phase.
Col._Lection c.~fi ~ndometria l Biopsy Specimens.
Endometrial b__opsy spe.~:imens were obtained using sterile
20 pipelle (Unimar, Inc . , I~c~u~_ 1:1y-en-The i i_w, France ) . Samples
were placed ate 4°;: in sl:~eril.e Hank balanced salt solut=ion
(HBSS; Gibco BRL, Bur~:W gton, ON, c'anada) cc;ntaining 100 U/ml
of penicillin,. 100 E.tg~'rra of streptomycin, and 0.25 ~g/ml_ of
amphotericin. Samples ;~r~rc: then imme~,ti~~~tel~r transported to
2-'i the laborator~~, washes:. Twice in HBSS at 4°C, then snap-f=rozen
on dry ice anti kept at --80''C in Ep~pendo-rf tubes for Western
blot and reve:=se tran.cription-polymera~e chain reaction (RT-
PCR) analyses or in Tissue-Tek OCT compound (Mi_les, Ins..,
Elkhart, IN) :Eor imrnur.orist:.o~hen;ica7_ st~adie:>.
30 Immunohistocmemi.stry. Serial ~="_~-~m cryosections were
placed on pole-L-lysine-coated glass mir:roscope slides and
CA 02377786 2003-06-20
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fixed for 20 min in fc~r_maldel-~yde (;4° [v/v] in PBS; Fisher
Scientific, Montreal, ~~, '.anada) . A.11 Lncubations were
performed at room tempt~rature irz a humiatified chamber.
Sections werE~ rinsed i n PB:~, imrner~sed in PBS/la> (v/v) T_riton
X-100 for 2 0 min at r~:~cw~-. temperature, rLnsec~ again in PBS,
and then treaved for ~'() n~in with r~ydroga~~rl peroxide (H~>O2, 0.30
[v/v] in absc>:LutEa metl_ar~ol; to eliminate endogenous
peroxidase. defter a r-'i3:; r_~nse, irrununos~:ainirng was performed
using a mouse mor~oc:lor;:~i ants.-human IL-1RII antibody (primary
antibody; R&I:~ Systems, l~linneapolis, MN) at 1~~ ~g,/ml in I?BS
containing 1« (w/v) B;A with a Vectastain Elite ABC kit
(Vector Laboratories, Burlingame, CA) arid diaminobenzidine
(Sigma Chemical fo., .'::t. Louis, r~IO) as ~:hromogen.
Sec:i~ions =ineul:~ated without= t.ne primary antibody or
1'_> with nonimmune mouse :serum were ir:.cludec~ as negative controls
in all experiments. ;=Lides were viewed using a Leica
microscope (rr~c~del DMRF!; Leica Mikroskopi_e and Systeme GmbH,
Postfach, Wetz lar, c;e:rm~:~ny) , and photomicrographs were taken
with Kodak 100 ASA film (Kodak, 'T c.ronto, GN, Granada) . The
IL-1RII immunostaininc ~,aas evaluated in a blinded fashion by
two independent observers slaving n.o knovaiedge of laparoscopic
findings. The intensi tvy~ of_ =,tai.ning way eva:Luated three
times in three dif_fererlt: ~~:eas that. werf= randomly selected in
the section, and a mean score was give's using an arbitrary
2_'> scale (0 = absent, 1 -- Light; 2 - moderate, and 3 = intense).
High concordance betw~eo, the two ebser_v~~rs was found as
determined by the kap~~a (K) measure o.f agreement (K = 0.89)
(Armitage P. et al, 1~~'la4 ) .
GJes~_ern B_Lot .~lr_~a'ysis. Frozen endomet.rial tissues
were directly homogen-i zE_ed with t.tle use c~f a microscale tissue
grinder (Kontes, Vine-~am:~, TJ~J) i..n a buffer containing 0..50
(v/v) Triton X-100, I(: mM Hepes (~~H '7 ~ 4' , 1'~0 mM NaCl, 2 mM
CA 02377786 2003-06-20
73 85409-5
EGTA, 2 mM EDTA, 0.02'=. (w/v) NaN3 (Shet~n KV,. et al, 1991) , and
a mixture of antiprotc_°a: es composed of 5 ~M aprotinin, 63 ~M
leupeptin, and 3 mM PI~I'I-". Tissue homogenate was then
incubated at 48°c~ for 4'_-; mi_ri undeo gentle shaking and
centrifuged art 11 000 k: <, for .3(~ ruin to rec~:wer the soluble
extract . Tot=al prote:i. r~ co~ncentrai_.i.on was determined using
the Bio-Rad DC Pr_oteir t~:ssay (B:io--Rad Laboratories Ltd. ,
Mississauga, ON, Canac:~a~ . One-r:urndred micro«rams of protein
from each extract wer_f:~ toeatec~ in a bciling bath for 5 min in
5X SDS samplf> bui=fer .;1 . f 5 M Tris--HCl [pH 6. 8 ] , 50 0 [v/v]
glycerol, 25'<> (3-mercax:ta>ethanol, x.0'0 [w/v] SDS, and 0.0:1
[w/v] bromophenoui. blur-) , separated by SDS-PACE in 10'~ (w/v)
acrylamide l ineai:-grac:::i.ent si.ab gels, and transferred onto
0.45-um nitrc>cel~..uloser~oeml~ranev ;Sci~.leic:her & Schuell,
l.~ Keene, NH) u.,~ing an a : et: t=rophoret__c: transfer cell (Bio-:Rad) .
Equal loading in each lane was confirmed by staining the
blots with Ponceau S ( 2=. [w/v] ) . N.itroce)_lulose membra:rLes
were then immersed in PBS contain_.,ng 50 (w/v) skimmed milk
and O.lo (v/v) Tween aC (blocking solution) for 1 h at 37°C,
cut into strips, and ncvubated c;vernignt at 48°f with a
monoclonal mouse antil:urr~an Ih-1RI:'= antibody (2 ~g/ml of
blocking solution; R&ie .systems) or with normal mouse
immunoglobul:i_ns (Ig) of the sam~~~ ammunogiobulin class a:rld
concentration as the I:~~ ima ry ant ik>ody ( R& D Systems ) . T:ize
2.~ specificity r:~f the .imt.mnoreactic;n was al:~o verified by
preabsorption of the <3nt i.body witri an excess of IL-1RII (20
~g/ml). Thez~eafter, ;:,:e s rips were in:ubated for 1 h at
37°C with Fc--specific oeroxidase-~'.abele~ goat anti-mous~s
antibody (1:3000 dilut:ic,n in the blocking solution; Jackson
ImmunoResearc:h Laborai:ories, In~;s:. , West Grove, PA) , washed
three times .i_n PBS/0.:': ~ (v/v) Tween 20, incui:,ated for 1 min
with an enhanced chem:..lumines~cence .system (BN(
chemiluminesc:ence blon:tng substrate [POD]; R.oche
CA 02377786 2003-06-20
74 85409-5
Diagnostics, Laval, P~:a>, c~anada) , <~nd exposed to BioMax film
(Eastman Kodak, Roche:~te:r, NY) fo3_ 5-30 v;ec for an optimal
detection (a11_ bawds v:i. sibl a but not overexpo'~ed) .
In Siti.i Hyb.~:.ic_~ization. In situ hybridization was
~ performed as describe,:L :in .~;~r pre~,rious studies (Jolicoeur C.
et al, 1998) . Bl:iefl~-, cDNA for luuman II:.-1RII, a 1.3-
kilobase .frac~:ment:, wa::; ~ub~Ylonec::~nto the plasmid vector
pcDNA3 (Bossla P. et a., 1995) . B:..otin-labelE:~d cDNA probes
were prepared by nick--t~ anslati:~~n .f:rom ~Ze er:tire plasmid
vector with *:he IL,-1R:!:I cDNA (L.aw7:ence B. et. al, 1985) or
from the pla~~mid vect~:~r alone (negative control) using a
BioNick Labe.Ling SystE::~m (Gibco FsRI:,) . Serial cryosectio:ns
were prepared and fixcci in formaldehyde as described earlier,
then progres:~ive~.y delvyc~rated in al~zohol batr:.s (50 0-100 0
l ~ [v/v] ) . Sec~:ions werE:~ ~rehybricized with the hybridization
buffer (50 0 [v/v] forrr~amide, 10« !~v/v] ;~ext.ran sulfate, 0. 1 0
SDS [w/v] , 2X SSC [single strength: 0. 15 M sodium chloride
and 0 . 015 M :>odium cit: rate ] , and !..X Denhardt solution [ 0 . 02 0
(v/v) Ficoll (Amersharn F~harmaci F3.iotech, Inc. , Baie d' Jrfe,
QC, Canada) , 0. 02 0 (w,'v) human serum altoamim (HSA) , 0. 02 0
(w/v) polyvinylpyroli:;lor~e (Sigma) , and 40 mM monosodium
phosphate (pH 7 ) ] ) , tlierv hybridi. zed with _'i ne~/m1 of
biotinylated probe in tie ~ybridi<.>.ation buffer. Biotin was
detected by ~~ series a~f 45-min incubations at 3%°C with a
2~ rabbit polyc~'_ona=L ant:~,.b:ictin antik:>ody ( 1[v; v] dilution in
PBS/ 0. 25 0 [w/v] HSA; E,n.zo l~iagr~o7t ics, Inc. , Farmingdale,
NY) , a biotinylat:ed goat antz_-rabkvit polyc:lor~~al antibody (1 0
[v/v] dilution in PBS,'C~.25~ [w/v] HSP_; Vector Laboratories),
and fluorescein isoth~_oc°yanat~e-conjvagated strept.avidin (0.50
[v/v] in PBS~'0.250 [w;'v) HSA; Ruche Diagnostics, Montreal,
PQ, Canada), respecti~.;e:l.y. Slider were then treated with
propidium iodine (1.5 ~~~~/ml of -~i:~tilled water; Sigma) which
makes the nu<:leus vis:..ble in yellow-orange oru ultraviolet
CA 02377786 2003-06-20
~75 X35409-5
excitation, ,_~nd mounto:;~l with Mowi.ol (Calbiocr~.em, San Diego,
CA), to which p-~>heny!.a-~rnediarnirre~ (Sigma), an antifading
agent, was ac:~ded at a f=i.na 1 c:on~.verut.ratic~=i of 1 rtlg/ml .
Sections were finally cbse:rved v.:ncle-r tile Leica microscope
'.~ equipped for fluc;resc~~r~eve and connected to an image analysis
system (ISIS; Metasystems, Altlusshe.im, Germany). As
negative controls, sec--clans from ear_h tissue were incubated
without specific cDNA r~rc>bes or with nonspecific DNA probes
prepared by nick-tran~::Lati;:n from the plasmid vector alone
(i.e., withou-t the IL--1RII cDNA).
ReL~~°rse Trar~.sa~ription--Pc;lymerase Chain Reaction.
Total RNA was. ext.ractec:~ from endomet:rial- homogenized tissue
with TrizolTM reagent <:~ca~crding to the manufacturer' s
instructions (Gibco BFv~). The cDNA was synthesized using 500
1.'> ng of total cellular IIJ,'~ and 2. 5 N,M ra~ldom hexamers in 2 0 ~l
of a solution containi?o~ 50 mM KC1, 10 mM Tris-HCl, 5 mM
MgCl2, 1 mM of each de:w:yribonucl.eotide triphc:~sphate (dNTP) ,
U of RNase inh.ibitc:r,, and 50 iJ of. reverse transcriptase
using Gene Amp PCR Core K.itTr' (Perkin-Elmer, foster City, CA).
20 The reaction was incux:nted a~ 25"C for 5 min, 42°C for 30
min, and 99°C for 5 mirl.
For PCR analy:.;:is, 2-~1 aliquot.s of ~sach cDNA were
amplified in ~~ final t%~,>:1..:.zme of '~0 ~1 c~orlt.ains.ng PCR buffer
(10 mmol/L of Tris, 5U rnrnol./ L ot. KC:L, alnd 1.5 mmol/L of
2~ MgCl) , 0.2 mrnol/L of_ dT~l':Chs, 2. 5 i:7 of Tack DNA Polymerase (New
England Biola.bs, Beverl.,~, MA, and 10c_> ~:>mol of each IL-1.RII
primer (forward primer, 'p' -TC;C A'fG T('JC AAA TC~~ TCT CTT-~' (SEQ
ID N0. : 1) ; reverse pr:imc:~~, 5' -TCC TGC c:C-~T TCA TCT CAT ACC-
3' (SEQ ID N0. :2) ; ampl amer_ a=~ze, 5?6 ba:e pairs [bp] ) . To
quantify the PCR produt.,t:.s r_omparativel~! and to confirm the
integrity of the RNA, we rc>amplified a hcusekc~eping gene,
glyceraldehyde-phospha'e dehydrogenase a:GAPDH), in a
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76 85409-5
companion tube (forwan~~ primer, 5' -TGA 'I'GA CAT CAA GAA GGT
GGT GAA G-3' (SEQ TD NC_~. : ~) ; rever~~e prinuer, 5' -TCC TTG GAG
GCC ATG TGG GCC AT-3' . ~E~'Q ~:D N0. : 4 ) ; am~:>=(.imE~r~ size, 240 bp) .
Amplification. of IL-1~~::I7: was achieved with 30 cycles of 1 min
.'l of denaturat~.c~n at 95'~, I min of ~:~nneal.ing at 60°C, and 1
min of primer ext.ensi~_~~~ at 72°C. Amplirication of GAPDH was
achieved witr:~ 30 cycl~aa of 30 sec of der°~aturation at 95"C, 30
sec of anneai.ing at 6:~:~°~:, and 1 min of primer extension at
72°C. These optimal c:ondii:ions were de:.ermined following
linearity tesvs using 1, 2, 5, anrl 10 ~.L of the RT reaction
volume and 2w, 3f, anc! 35 amplification cycles.
Amplification of genon7ic DNA with these primers did not
produce a signal, sugcxesting that the amplification sites
crossed at le:~~st one rlt:ron/exor: xooundar-~,~.
Twe~.zty perce-nt: of the PC;R volume was then ana:Lyzed
on a to (w/v) agarose gel in the presence of ethidium bromide
and transferred t:.o Qiab:rane Nylon Plusrr' membz.:apes (Qiagen,
Santa Clarita, CA). Membranes were dehydrated at 37°C for 30
min, prehybri.~~ized with a hybridization buffer comprised of
5X SSC, 5X De:~hardt sc:lution, 50 rlM NaH_>PO.~, 0 . 5 o SDS, 200
~g/ml of salm~~n :>perm DNA, and 'v0','s (v!v) form amide;
hybridized in the same: buffer, but without Denhardt solution
and with B2P-.Labeled I1_:-~1.RII or ~A~'D~i c:DNA; and washed with 1X
SSC, 0.2X SSC, and 0._.a (w/v) S%S, respectively, before being
2'.~ exposed to x-ray film (Eastman Kodak) for approximately 1 h.
Specificity of the amplification pzocess was
verified by ~~outhern l~l.c::t hyb.ridi.zation. A negative control
(PCR in the absence ov esD'NA) as well as a positive control
(cDNA prepar~~tion from rnuman endometria:l tissue expressing
IL-1RII) were included i.n each series of IL-1RII or GAPDH
amplification.
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77 85409-5
For eac~n enc~ometri_al biopsy, ?CR. was performed
three times. ~Che quant: ity ~oi thE: F-~CR pra>duct:s was determined
by densitomet.lic analog s i s~ or t=he i :nt:ens .ty of the
hybridization signal. The relat:_ive ~evc~1 of IL-1RII mRPJA
_'> normalized tcGAF'DH mi-t~~ was calcu.l_ated, and the results were
expressed as a percenta~ae ~of the c.c~n-troi value (positive
control).
Ce1_1 Separat.i'~rz. Endometrial l,issue was minced into
small pieces and dissc:;~.~:fated with ~~:oliagenase (Sigma) to
separate epithelial g_<~:suds i:rom fibroblast-7_ike cells as
previously reported (F~.k~.:~um A. et: a.1, 19'3.'~b) . These two cell
populations were further purified using PercollTM density
gradients (Arrcersham Ph~armac;ia Bi_otec:h, =ric. ) . The purity of
epithelial or fibrobl~~st-like strcrmal calls was verified
1_'> morphological_Ly; immur~o~;~ytochemica.lly on coversli.p cultures
using antibocz:i_es spec: f:~ c: t:o cytok:erat:in~ (epithelial cell
marker) , vimentin (str o»~.al cell mG.rker) , smooth muscle cx-
actin, and factor VIII i enc~othel iG.1 ~el:'~ marker) ; and by flow
cytometry for the preerme of leukocytes using anti-CD45
monoclonal arvtibodies :3s previous.l_y des~,ribed (Akoum A. et
al, 1995b) . C'c~lls were kept at -80"c: unt:il use.
Statistical Ar:alyses. 'rhe Ij~-iRII i.mrnunostain=Lng
scores followE=d an orct:final scale. 'Iheret-ore, statistica=L
analyses were performE~d using non~;aramevric methods. The
association b~E~tween in~munostain:inc:~ scorns and the periods of
IL-1RII expre;ssic>n .:Ln '.he menstrual cycle as well as
intergroup compar:ison:> c~f irnmunUst:.ain:ing scores were analyzed
using the Fisher exact test, and t:he Bonferroni procedure was
applied when more than two groups were :compared. The
correlation between t:~e day of menstrual cycle and the
immunohistocYu~mistry .:~cc.~res was e~~~aluatad using the Spearman
correlation c~~eff icietat . ':The threshold <bays between the
CA 02377786 2003-06-20
78 k35409-5
different lea~°ls of staining (0, ~., 2, prod 3) were determined
using the best combination of sensitivity and specificity
values for a series of: cut-off days wit:~in the menstrual
cycle. These thr-esho:ld days al:Lowed us to define or to
delimit different expression periods. analysis of IL-11ZII
mRNA levels a.s determ.ined by serniquantitative RT-PCR was
performed using one-wny ANOVA arid the '.Cakey test for post-hoc
multiple com~:~arisons. wll analyses were performed using
Statistical Analysis ._'ystem (SA:: Lnst:i.t ate, Inc. , Ca.ry, NC) .
Differences were considered to k>e statistically signifir_ant
at P < 0.05.
Results
Immunohistocr~f=mistry
A rr~onoclonal antibody was asec~ to detect IL-1RII
1_'~ protein in endometria~A -issue sect:i.ons. Different
concentrations of the antibody ~;5, 10, L5, and 20 ~g/ml) were
tested to determine tr_e optimal :concentration to use. This
experiment wa:~ perforn er; on Y'hree di. eY~ent series of biopsy
specimens frorn diffE~rcril~ phases of them menstrual. cycle. A
concentration of 15 kZglrr.:l was seLec;t.ed, because it allowed an
optimal ratio of speci.f:icit.y (minimal background) and
sensitivity ldetectable po~~itive signal;. Examples of
positive immunostainiri~.~ ~uait:ki ant: i-IL,--LIZ=~I ant:.ibody are ~~hown
in Figure 10A. Immuncc~:1_a:~bl.a.:Lins of t.hf~ :;ame isotype and
species, when used at an equivalent concentration as that of
the antibody ;15 ~g/ml;, dil not display any detectable
immunoreactivity (Fig. :I.~:)I3) . Trrununc~rf:.~a<:tive IL-1RII wa~>
detectable throughout cen:lornei=ri_a i t:i s:~ue, bot:;n in the st:roma
and the glands. Brown :Lmmunostaining could be seen around
3C cells (cellular stainl.nc~; and along 1=he apical border of
luminal and gI_and~.~lar ~~y~ ~t:kneliunu (:i.urn:i.nal staining) . This
CA 02377786 2003-06-20
79 85409-5
was also observed, alt.h~ough less markedi.~;, in microvessels
and isolated aggregate :; t.hroughc:~~~t. t=he st_rorna in sections
from late sec:=-etory er;dc>metrial tissues ;Fig. 10A) . It is
noteworthy that lumin~. L. sec:ret:irm was :~~c~t_ uniform for all
_'~ endometrial SE'Ctl.onS examined.
The IL-1RII nununo,staining way assessed
semiquantitat=~vely by t~.ro independent observers in a double-
blind manner, taking :~ ru~.,~ account the :int:en:>ity as well as
the distribut.__on of tee immunost:.aining as described above.
Cellular and extracell~r:la:r :~tain:ing wf=:r a. scored independently
in the stroma and in the glands and swr~ace epithelium.
Score distributions ac::;~.rding to the ~~a~~~ of the menstrual
cycle are shown graphi:._~11y :in t'igurEs :L=_ .
To better un.~e.rstand I:.~-1R:L:L o:ycli.c va.riation:~,
expression wa~~ defined throughout t=he menstrual cycle using
the best combination cf;7ens=iti.v~_ty <~nc~ ~pec:i.fic.ity values
for different cut-off days within the cycle. Our analysis
revealed that after Dace 8, both ce11u:1_ai se~n;~it:ivity =
100. 0%, specificity = ~3~~ .'~a:, P < 0. COL; and luminal
(sensitivity =- 55.6°-a, ;~p~:~cif_~city --- :L.Ot).0'>, P < 0.001)
immunostaininc~ became ,~~gnl.ficant.ly detectablE~ in epithelial
cells. A further increase in the intYen~ity of staining
occurred after Days 21 ~~nd 22 , but teals increase was more
obvious at the cellular (Day 21, sensitivity =- 88.60,
specificity = 71. 4 0, F t.. 0.0~~) than at: the lurninal (Day 22,
sensitivity = 86.1p, sE:~ecificity = 5().()<-, P = 0.07) level.
In stromal cells, cell u:l..a:~r. stain:i.ng remained. weak to absent
throughout the whole p:rc:>:lit:eratiVe t>hase of t.tne menstrual
cycle, but it increase:: sigroificantly after Day 15 at th.e
beginning of the secret<:a:ry phase (.P <~ 0.001) . Extracellular
staining was ~~irtually s.rnc:~etectable in the st.~roma, except
weakly after Cray f.1 ire t: i:7sues f z om mica to late secretory
CA 02377786 2003-06-20
60 85409-5
endometria (sf=nsiti.vit y _ ~~ ! .2'0, ~s~>ec:if icity = 85. 7 0, P <
0.001).
Sta~.~istical a:ialysis c:f the d<~ta, using the
Spearman correlation woh.f.f i_r_.lerlt:., rewea._ed a significant,
positive correlat ion k::<.:t ween cel..lu:l_ar s~ aini.ng scores and day
of the menstrual cycle, botri in epit:-relal (R = 0.59, P <
0 . 001 ) and stroma.l. (R - 0 . 9 6, P 5~ 0 . O1 ) cells . However, no
positive correlation k.~e,Vween epithelial lumin.al staining and
day of the menstrual c:yc=le was found (.R =- 0.17, P = 0.29),
most probably because :a:f: a more 2:luctuati_ng E.x~~ression
pattern. To better delineate these rf~:lationships, we
determined the mean va.l_oaf~s of i.mrnunosta_~.ninq scores for each
cycle day, and we found that they fo_Llow a third-order
polynomial curve ( .Y = <3 -t- F3X + C'.~= + DXB? 1 F.ir~ , 11 ) . This
curve shows that aft:e.r <~ rnax=imal. ir~c:_rf_~~~.e at: approximately
Day 12 in the proliferative phase of the menstrual cycle,
luminal staining of IL.~-:LI~II ciecl..ined gradually in the
secretory pha:~e, reacr:i.n:~ its minimal level at approximately
Day 22 before increasim~ ac;ain progress~.vely until the end of
2C the cycle. The midsecri:tory drag ir~ the intensity of IL-1RII
luminal immunostaining ( (Jays 1.9-~'_2 ) was statistically
significant a;> compare~a i~o lat.e ~>rc;liferative/early secretory
(Days 9-18; P < 0.05) anc~ late secretor<< (Days 23-28; P <
0 . O1 ) immunost:aini.ng 1e ~ik.=.,ls .
Representative examples of IL-1RII immunostaining
in the endomet:rium thrc.w.agticut= thc:~ menst:x ual cycle are shown
in Figure 10 f:or Days ~~; (Fig. 10c~) , 1:.3 ~ Fig. 10D) , 18 (Fig.
10E) , and 23 I Fig. LOf, ivot~e t.l~e reduction ~.~f IL-1RI:I
luminal secretion in tf-.e glands and surface epithelium of
specimens at L>ay 18 as c:c>mpared to Days 13 anc:~ 23.
Western Blot ~~nalysis
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To :furthe:r f::~x~~mine =II:~-1F;IT pr~:~t:ein expression
throughout truf~ menstr~:,al cycle, total e.nc:~ometrial proteins
were extracted and eq~_,iv.alent amounts wtrre subjected to
Western blot analysis . ~:W r resi.rlt s stuowed that the antibody
'.~ specifically =~ecognizt=d several major and minor bands (Fig.
12 ) . From the:>e, the c=a3 -~ arid 4 5-- kLn,~ barld~; are consistent with
the commonly ,-eported rtiolecular weights of t:he membrane--
bound and the soluble receptors, respectively. The
immunoreactive bands were absent when t rue primary mouse
1C1 monoclonal anti-IL-1RI- antibody was re~:o.aced by an equal
concentration of norma'~ mouse IgCs (Fig. 12A). Low molecular
weight bands ;<45 kDa) :aisappeaz:~ed when t:he antibody wa;>
preabsorbed w=_th an excess of recombinant IL-1RII (20 ~g/ml)
before being incubatecr with nitrrocel.:Lu:Lcase membrane-
1~~ transferred proteins, whereas the int=en si.ty of major higher-
molecular-weight bands was considerably reduced (Fig. 12B),
suggesting specific int:r~ract:ion with r_hEe anti-IL-1RII
antibody. As shown in figure 12A, al:L IL-1RII bands revealed
by the antibody were rr,-:~o kE~dl y intense at the a3pp:roach of
2C ovulation. Tree intensity of these band:; clearly decrea~~ed
during the midsecretory phase but increased again thereafter
in tissues from late sec~ret.ory endometr_:ia.
In Situ Hybridization
Expression c~f:- LL-1RII rnRIVP., in the endometrium was
25 first studied by in situ hybridization to localize the site
of synthesis. Figure L:~ shows the appearance of endomet.rial
glands and stroma at 1 ~:~7X (A1, B:L, and C".l ) and 666X (A2, B2,
and C2) magnification" 1=ollowing hybridization and staining
with propidium iodine i i.at:e proliferat=v~ve endometrial tissue,
30 Day 13). The hybridize;:ion signal (green-yel:Lowj could only
be visualized at .highe:r m<~gn=_fic.-~ti.on ;1665X) , as illustrated
in the same figure, an::I wa:> more pronc~~anced i.n glandular (A3)
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82 85409-5
and surface (33) epitluelia:i than ~.n stromal (C3) cells. No
hybridizatioru was observed in negative ,-~ont_rols including the
omission of ~~iotiny:La! e::I DNA pre~bes prepared from the p:Lasmid
containing Ih-1RI:I cDl'ia insert= or the use of biotinylated DNA
probes obtained from tire plasmic.~ alone.
Reverse Transcription -F'clyrnerase C'.hain :~Zeact-i.on
Expression e:~f IL-1R.I:I mF;NA throughout the menstrual
cycle was studied by ~:e~niquantitative R'L-PCR. This was
achieved by r::ormal.izir g t.hc~ Ih- LRI I mRNr'~ to the mRNA of the
coamplified rousekeep:i n~:a gene GAPI>H and x:~y including an equal
amount of thE:~ sane pre:~p.~~ration of posit'_ve control (RT
preparation of cDNA f.:~rrc human e.~clometrLal RNA) in every
series of amplifi.catir~n=;. The c-orutro:L, which was subjected
to the same e:~periment:;~_c~ondit:LOns from the amplificat:ion
reaction until Sc>uthe c n blot ana Lytsis and autoradiography,
allowed for rru~nit:orina ~_>f i:he ir:terassay vari.at.ion. Results
were expressed as a pE=~~~enfiage of the cont.roL. value (:i.e.,
the amount of- IL-~1RII rn~~l.~1 rela t ive to ,ruat cf the
corresponding GAPDH dl video :by the amount: of IL-1RII mRNA
relative to that of Gr~~'L~H ~.n 1_he control ~: :L00) . Result=s
from 20 endorru~trial bio->sies across the menstrual cycle (Fig.
14A) show thaw: IL-IRII rruRNA level; are i_ow in early
proliferative endomet.~ :i_.~~1 t:issues and foll.oca a kinetics of
expression comparable t _~ that f_ovarud for the protein by
immunohistochf~mi~stry ~:Luminal secretion) and Western blot
analysis. Ir:c fact, ml.l~.~~ levels were elevated in late
proliferative~, early ~~:ret-:ory, and lat~.a secretory
endometria, x:~ut they i_.~nifiicantl~~ decreased in tissues from
the midsecret.ory phases. Representative examples of RTPCR and
Southern blot: analyse: ~f IL-lRI:I mRNA in tissues from
different cyc~:Le phase:- are shown i.n F:igure :L4B. The RT--PCR
analysis of I:L-1RII mF:N,%~ irn separated ~.~ells .=_showing higher
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83 X35409-5
levels of expression n epithe.l.i.a1 than in :>trornal cells
confirmed the in situ h,,~~bridizatiorl dat_~ (F:ig. 14C) .
Discussion
Hurm~n endomt,_t'ium i s an act:iv~~ site of cytokine
production and action. Complex interac'i.ons of epithelial,
stromal, endc:vhel.ial, ::end lymphoid ce.Ll:~ occurring in the
human endomet:rium as ~~e:ll_ ~~s dy:~amic change:> orchestrated in
cyclic events of ce:l1 l:~:roliferatic>n, cii~i-erentiation, and
shedding requ.:ire a wel:l_-elaborated netws:~rk of intercellular
communicatiorv~ signa:Ls :~uc;h as cytokines. Many of these
events are reminiscent .~f t=hose a~:sociated with the
inflammatory and the reyarative processes, wrich make
plausible the involvement of proir~flarrL-natory cytokines.
Furthermore, cvytekine:_ ~-such as IL- l, wh.~ch have been
1_'~ considered ar: im~~ort~an~t immune rnecliator_s, ai.so seem to be
implicated in critica:. zepr_oductive ev:~nt:s such as ovulation
and implantation, and E~~.~i.denc:e inclicat~=> that they act as
endocrine and local rEc_~~lat=ors <f many cendornetrial functions
(Tabibzadeh . 1991; ~imon C. et. al, 19':)fb) .
The control ~:_~r cytokine action in the endometrium
may require the local av~ailabi:lity of specific regulatory
mechanisms. l,or Ih-l, this i s illu s-tra.~.ed k>y local
expression of IL-lra, ~ specific inhibitor t=hat binds to
functional IL,--1 recept or type I ( i . a. , ~L,-1RI ) and that
2_'> blocks IL-1 binding ar.d cell sic~nal_i:ng ~;Boraschi D. et al,
1996) . Our present st iz;~y revea=:.ec3 t:n~s wxpression in the
endometrium c:_ another irat~.zrai _~nr.ibit=o:_r for_ IL- I, the decoy
IL-1RII, which acts di i: ferently, ~~y se~~uestrating active as
well as inactive IL-I~~ a;zd, thereby, restricting the
availability of the ligand for_ the fun~~t~ional receptor and
inhibiting even its m~:t~~ar.ation (~ymons :.A. et al, 1995;.
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Immunoreact:, ~;~~ IL-1.RII ~,ras localized throughout
endometrial ':issue bot:r, in epit=~e=:_ial and stromal
compartments, buts it ~.,,~;~ more oi;>v~..ous in endometrial glands
and surface epitheliuru. This w~~s cionfirmed >';y ~.n situ
') hybridization showing ~G_.gh :Levels c:f IL-iRII mRhA in
endometrial elands anc.; :,urface epithelium. The RT-PCR of IL-
1RII mRNA expression a s eparatt. c;landu:iar e~~ithelial a::~d
fibroblast-l:i_ke wells i. solated from endomet:rial tissue
corroborated these fit~d~r:gs. Inrmunolocalizat:ion. further
revealed two levels or staining. The first, which was
located all a:rrour,d thc: cells, may correspond to the cellular
membranebound IL-1RII receptor. ",he second, which was more
intense and located par..~dom:inantly within the lumen of
endometrial cilands anci at T_he~ apic:a_L side o:f surface
epithelial cE:lls, is r r~.t probable% a lumina:i secretion
corresponding to the :~c,:ub:le and .~ecretec~ form of the
receptor (sIh-1RII) . Ir: fact:, rile:>tern ~.lc>t analysis of IL-
1RII protein in endomc:tria:l tissue shows the presence o:f a
68-kDa band, which ~~oTvr_a>sponds ~.o the membrane-bound IL-1RII
(Colotta F. et a~., 190.4; Bora.schi D. et a1_, 1996), and <~
number of lower molece~~l,_~r weic~h~ >;>ands, which may correspond
to different soluble i-orms. It is well known that sIL-:1RII
is released from the ; ernbrane-bc=>und receptor following
proteolysis, and that the released solub:l.e mc;lecules keep
their ability to bind anc~ neutralize IL-l, particularly the
circulating form (IL- ~3i (~::«loti_a F.. et al., 1.994; Symons J.A.
et al, 1995; Bos:~u P . et a:1, 1995; Orla:zdo ,~. et al, 19'97) .
To our knowledge, a f:..i1_~ charactez:i:zation of such molecules
has not been performed, but it c,:ould be proposed that other
3() forms of soluble IL-li' I: ~ could ex_l st, p.:~ssibl_y due to the
presence of diffE:rent, uniderati:fied cleavage sites. Neumann
et al (Neumann D. et ~cl., 2000) re~.>crted a 3~4-kDa soluble form
of IL-1RII in the cult!are medium of IL-1RII-t:ransfected
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85 85409-5
keratinocytes. ~~ccoroli~og to Orlando et .7_ (Orlando S. et al,
1997), lipopolysaccha-i.cze-treated monecyt=es release a 60-kDa
IL-1-binding molecule; wrni~:h was u.dentified as the IL-1 decoy
receptor.
Both cellul<~r and extracellular/luminal IL-1RII
immunoreactivity vari:~~ within the menst:rua.1 cycle and
followed a rE:gulated ,c.yc:l.e phase-dependent pattern. In
stromal cells>, the int-:=nsity of. staining remained weak to
absent durincr the who:.e proli.ferat:ive phase but increased
moderately after ovula:~tion. In epithet ial. cells, the
regulation ap,~eared tc:: be diffe:eer~t, as immunoreactive
cellular IL-1.:RII firs,- increased mode.ratel.y after Day 8 in
the midproliw4rat:ive K 'ease, then nuore markedly after Day 21
in the secret.ory phasre. The IL-1RIT luminal secretion
followed a mc:>re c:ompl~:x ~:inetics. Being undetectable until
Day 8 of the ~~ycle, l:aminal secret=ion reached a maximum at
the end of true pr olife:.rative phase, there dec~l_ined
progressively until Day 22 before undertaking a final,
gradual augme~ztation ~_lu:ring the late secretary phase.
The pat:te.rn of IL-1_RI:I i_mmunost:aining in
endometrial epithelia cells was quite unusual, and the
temporal exprn.=_ssi.on f~~:r th:is natural inizibitor of IL-1 :is
rather intere~;sting. 'That the receptor expression is down-
regulated in 'the rnidsEe~retory pha:~e, especially during l~he
implantation window, rind up-regulated a'~ the end of the
menstrual cycle sugge~;~s that T1.-n.RTI may have multiple
functions in human endo:metrium. Tr~estern blot analysis
appears to cc:~nfizm them results of immunohi.stc>chemical
staining showing that er~dornetrial epithelial cells possess
low amounts o.f IL-1RI~ in the midluteal phase. Epithelial
IL-1RII expre.;ssion and distr_i.butic;n in endornetrial tissue
across the menstrual <:~yc:le is remarkable for several reasons.
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First, IL-lFzl~ appeared to be expressed and
released precl~~minantly l~~y g~_andul~~r and .lum:inal epithelial
cells rather than by : :i oma'~ cel 1.;. Thi> is of additional
interest, becv~use fir.~.Y inter act:icrzs between the embryo and
the endometr_i.vm occur at the levee of the luminal epithelial
cells during the adhe:> ion process .
Second, the significant decli.ze in IL-1RII lurninal
staining, which s ta:rte~,~ .at the bectinni_n~ of the secreto:ry
phase and rea~~hed its rn.:inimr.rrri at approximately Day 22, .in the
midsecretory ~~hase, i:- ~ ather ir~tE~resting arud suggests 1=hat
IL-1RII secretion is u;jected to subtle chronological
regulation ir. endometv~ i~l epit~helia~ ce Ll.s. This is
remarkable, x~c~cause ti;e ;phase of madur_.ac:~ sec.ret:ion correlates
with a putative "impl<~ntat~_on window" t~nought to exist within
l.'> the midsecretc~ry phase 1_~etween Lays 18 <,rnd 22 (Tabibzadeh S. ,
1998) . It is importa:~': to point: out= t~,=rt: the physiologic
basis for thi:~ window has not yet been a.:learly established.
Also, no gene ral agrea::rn~~nt exists s regarc:~ing the dates o1= the
endometrial receptivit ~~ pe:ricd c>r the implantation window
during normal met=stru~.1_ cycJles (Ta.b.ibzadeh S., 1998) .
According to :~imon et al (;~~imon ~. et a.~_, 1998a) , the
implantation process t:~.rts at Day LH i ':, which is
consistent wii=h IL-1R_I::C decreased expression. According to
Bergh and Navot (Bergs f'..?~. et: al, 1992 ~ , first embryonic
2'i signal detect: ion (pre~:urned window of _mplant:anon) extends
between Days a?0 and 2~I. However, ot:ners have suggested that
the implantation windc.~w .is confined to postovulatory Days 5-7
(Psychoyos A. ,, 1993) . l~onetheless, tYie decz:eased IL-1RI=I
expression at: that spEC:;i:fic time cE the cycle, at which
embryonic attachment Grn~ imp.lantatiom rnay occur, is
suggestive of: a pos sil.~ _e role for I:L-FtI I in the initial
interactions between rnw~erria.l. and embryonic cells and the
establishment: of an erdc:>>metrial period of receptivity.
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In :fact, num-:r .pus studies: indicate that IL-1 acts
as an embryonic signal and is sec:ret:ed early by embryonic
cells (Barano R.I. et ::~:~, 1.992; :;teeth K.~J. et al, 1991) .
Interleukin 1 appears tc: be crucial fc;x successful
implantation, because t-.ht, blackacie of i_ts functional receptor
(i.e., IL-1RI) in viva L::~r~events i.mp:lantation x.oy interfering
with embryoni~~ attachm~;::nt: ~;.vimon f.. Eat al, 1994; Simon f, et
al, 1998c) . InterestirAc_~ iy, t: he IL-1 ~-y:,tem appears to
mediate the regulation o~= int.egr:Ln expressicn in human
endometrium. Simon et ~~-~ have reported that binding of
embryonic IL-l.a and IL-1(3 tc endometrial epithelial IL-1RI
up-regulates endometrial epithel.:ial ~3~ ::ubunit, which is
considered to be a marker of endomet.r_ial receptivity (Lessey
B.A. et al, 1992), anu :_acilitat:es adOesion of human
lf~ blastoeyst (Si_mon C. cat: a:1, 199~'i . Serl.zm IL-L levels
increase at o~rulation ~;c::anruon C1.C. eW a_., 1~>8S) ; the down-
regulation of IL-1RTI 7:~c:~:lease in endc~mf,t.rial_ glands and
surface epithelium suggests, therefore, the existence of
accurate regu_~ata.r_y mec:hani.sms ttzat ai.Lf~viate IL-1 inhibition
during this crucial per:i~d, thereby fa~;_'~litating IL-1
proimplantati.on actiors. Interestingly, our previous studies
have shown that the ex:p:ression of :IL-li~I, the functional and
signaling receptor .for I:h-~w, is modE:ratc:~ly up-regulated
during the same perioc~~. ;Bic~onnesse H. at al, 2001a) . This a
remarkable example of fine-tuned and weC_1_-orcteestrated
endometrial events, true object of which is to preserve the
reproductive function of this tissue.
The: dec:.rease~c:i expression of IL-1R=LI: protein, which
was more perc:~~pti_ble <at the level of luminal secretion by
immunohistoch~amistry, was detected by Western blot analysis
and semiquantitative F<.'T-PCR. This makes an inhibition at the
level of IL-1RII mRNA more likely than a trar:slational or
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posttranslational prot~~c::'_ ysis-depe:udent mechanism. Whether
this is due to inl.ibit i.<:~:of TL-:~.R~I gene transcription or to
decreased IL-:1RII mRNA st abil ity r~,rnai.r,s to be further
elucidated. However, ~::r~is require,=. the identification of the
regulatory mechanisms .rncaerly.ing Ih-LR;.I downregulation.
Third, IL-1R f :I expressi.ori l n endomet~rial tissue
markedly increased in t:.Poe late scc:~et:c;ry phase of the
menstrual cycle, loth ,:~t: t: toe lev~;~1 of= t:he protein and of the
mRNA. This wa.s quite ::~i::~~T~.ous ire tl-re g7..ands and l.uminal
epithelium, b;:a it was <=i i_so v.isibla i.r the st:.~-oma of some
late secretory-phase bi.~,.>l.~sy speci.menso This may have a
considerable ~cignificanue, because in t:he absence of
implantation, the endontetria~i tissue unc~ergoe:~ a process of
cell necrosis and disiut.~gration at the end of the menstrual
cycle (Tabibzadeh S. , 1':39:1; . The a ~ evated exp:ressi.on of IL-
1RII observed in the 1.:-rte secretory phase may, therefore,
play a key ro~.e in the control c>F sur_rL an inflammatory-like
process durir_g the premenstrual and men:; t.rua?. periods .
The potential involvement of IL-1RII in
implantation and regulation of local in~~_amrnatory-like
processes is supporter ky our recent data showing a marked
deficiency in the expression of this specific inhibitor of
IL-1 in the endometrium :~f women wit: endometriosis,
especially those suffEr.ing from ir.fertii.ity (Akoum A. et. al,
2'_i 2001b).
Anoi_her natural inhibitor for IL-1, IL-lra, has
been found ir_ the endc~mctrium, but more in the proliferative
than in the secretory phase of the menstrual cycle.
Furthermore, accordincf t:o Simon et al (~imon C. et al,
1995aj , endorcretri.al cells rather express the icIL-lra. In
view of the l.~~ca7.izat_.:~r and thr, t:ernpor.:~:L pattern of
expression o: IL-~1RII revealed i.n our sturdy, this is quite
CA 02377786 2003-06-20
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interesting, and It sl:~c~c~ests that. IL~- :LF:IT anrl IL-lra have
complementary roles ire t: ne control c:~f _;:L;--1 a.ci"ion in
endometrial tissues, aon:7 1~l:at: t=r.cey exert their inhibitory
effects at different a:;ud complementary levels. This is all
the more pLausib:le bec:<:;~vse, accor_di.ng to Symo:ns et al (Symons
J.A. et al, 2~a95) , IL-.l.l<:CI does uct: interfere with IL-lra
mediated inhibition of CL-1, and the Two mol.ecules have an
additive effect in inr .i..l.-~i.ting the bi.nd:ing of IL-1(3 to cell-
surface IL-1 ~°eceptors .
1C) The process wlr.ereby IT.~-1R7::I expression is regulated
in the endomei~riu.m is ynt estab..isue~:i, c:~nd it remains unknown
at present whf~ther estr<l:~iol, progesterone, or other hormones
of the reproductive c,yo,l a c:-an di rectly or indirectly affect
IL-1RII expression in e:ndometrial tissue. On the other hand,
it is quite possible that proinflammatory cytokines such as
IL-1 and tumor necrosis factor a cTNFa), which have been
found to be p.redominar~t in the endometr-ium during the late
secretory phase (Kauma ... et al, 1990; Iabibzadeh S. et al,
1995 ) , can be: involveel n the u~.>-regulation of IL-1RII
2() expression. Tn fact, bath IL-1 and TNFcx have been shown to
up-regulate I:L-1RII e:;pression or release in other types of
cells (Shlopo v B.V. e!: a1, 2000; Yu P.W. et al, 1997; Plata-
Salaman C.R. et al, 1;9'7) .
In summary, IL-1RII expression in the endometrium
is an interesting and dynamic process. It is tempting to
hypothesize that aber~:ar~.t expression cf such a natural
inhibitor of IL-:C may >;;c.~ related or assoc:iatE:d with
pathologic states or infertility. Additionally, IL-1RII
appears to be predict,.3b1 e, based on tre time in the menstrual
cycle, thereby a:l.lowi__ng diagnosti~.~ use of IL-~1RII as a stage-
specific marker .in this. tissue. Finally, an understanding of
the function of IL-1RII throughout the menstrual cycle and
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the means to regulate it.s expression ma~.~ prove to be of
future therapE>utic usF .
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The establis.ument c:f a new vascular supply is
essential for the surv:.~~a1 of endomet:rial. tissue and it~>
development in ectopi~: :Locat_LOns. We have previously shown
that ectopic endometr~.-r:L ~~~a.l.:Ls r-elea~e an irtrportant mitogenic
'. activity for human enci>thelial cel:Ls and identified
macrophage mic)ration :.nh Lb:i.tory ract:oc MIF) ~s one of_ t:he
principal bioactive mcle~~ule:~ involved .n endothelial cell
proliferation., Irl thcE,>ret;ent: study, immunohistochemical and
dual immunofluorescencc: analyses showed that MIF is
effectively e;~pressed b:~ endomet.ri.ot:i~~ tissue, particularly
in the glands,. and idErntified endothelial cells, macrophages,
and T lymphoc~,rtes as ce.:L.ls markedly exp:r.~essing MIF in the
stroma. Western blot analysis showed a single 12.5-kDa band
corresponding to the ~:rn.7wn m~J,'~ wt of_ the molecule. The
highest concentration: ~~~f MIf protein in endometriotic
tissue, as measured b~. ELI~A, werEf=oun~:~ in flame-like red
endometriotic lesions, ~vompared with typical black-bluish (P
< 0.01) or_ wivh white :Lesions (~ <:: 0.01, . interestingly, MIF
displayed a r:~arked ex~~ress:~on in 1_esions from the initial
stage of endometriosi-; !;stage I;~ . Semic~uanti.tative R'r-)?CR
analysis of MIF mRNA ~.e~rels in the same E:ndometriotic tissues
showed a patt.~=rn of el::p.ression c.onnparable with that of the
protein. In view of '~s potent proinflammatory and
angiogenic prv~pez:~ties, .ocal prc:~duction of MI:F within
endometrial implants, particularly in those that are highly
vascularized and repr~:sent:ing tile earliest: and most active
forms of the disease, make plausible the involvement of this
factor in the local ir.~mt:mo:inflarnmatory process observed in
endometriosis and the irvitial steps of endomGtriotic tissue
growth and development:.
Enc~omet.rios i.s, one of the commonest gynecological
conditions, is definec:l as the pre:>ence of ti~,sue
histologically si.mila:v t:c endometri.um. at sites outside the
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uterine cavit.v. This ::~:isease is intrigizi.ng and unique in
that it is them on:l_y kr,::~w:a beni_gr3 d i.sea5c= :in which autologous
cells, accord= ng t:o tre : widely a<;cepted transplantation
theory (Sampson J. , 1~?'i ) , can im~:vl.ant <~nd develop in ec:topic
_'> locations. The etiolc,gy of endometr:iosa_s is still not
clearly defined, c:;ernet;in~ or edispos:it:io::z, environmental
toxins, hormonal factors, and immune deficiency may
contribute tcthe susc~ei~~t ibi.lity o.f a woman to develop t=his
disease (McLa-=en J. , L.U~J~..~ ) . HowEwer, a key condition for
endometrial tissue 1..o svnrv_ve and grow f-yc:topically following
successful adhesion arid implantation is the establishment of
an effective new b1_ooci :~upz~ay, a ~>rocess involving the
generation of new blo<.~d vessels or angiogenesis (Healy D.L.
et al, 1998).
Ear:Ly and mc,st: act ive endomet ri.oti_c: 7_esions are
markedly vascularized; increased vascularization is seen at
the implant surface arud also in the surrounding peritoneal
tissue (Wiegeerinck M.~~,. et al., 19931 su~ygesti.ng that
endometriotic implant is capable c;f induci.ng its own
neovasculariz~~tion by c~triving _ioc:.a_L mi-.:rovasculature. It is
therefore of ~~ crucia::. interest tc> elucidate the mechanisms
underlying en~~omE~trio:-s.i~~-associated angiogenesis and to
identify the factors vevolved in t:.hat process. We have
recently found that iru:nuc_>rtal.i.zed a:~s wel.l as L:rimary e~ztopic
endometrial epithelia::. cells release an .important mitogenic
activity for human en~;.c>t.helia.l ~-e:'~._ls and identified
macrophage migration _inhibitory factor (MIF) as an important
mediator of endot.heli~~.l cell prolu.ferat.ion (Yang Y. et ,al,
2000). Originally de:,cribed as a product of activated T
3~~ lymphocytes that inhit,a.i.t:ed the random migration of cultured
macrophages, MIF is nc>w known as ~~n important. modulator for a
variety of cell funct::.or:.s, includ~i_ng inflammatory and immune
responses (Metz C..N. at al, 199?; Nishihira ..1., 2000) . The
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identification of MI:F ~as a mitogenic facvtor for endothelial
cells released by ectoy=i_ c endometr ~a1 cells i.s consist:ent
with recent d~~ta show:i:;o.;~r_ _impc>rtar,i~ :~c~l.e for MIF
in tumor
growth-associated ang.i canes is 1::z vi.v<_~ and i n vi tro
~ >r~ in
autocrine regulation cI: c?ndothelvai ce:L:.' prc>l iferation
(Chesney J. et al, 199r~;, (~gawa H. et al, 2000; Shimizu T.
et
al, 1999) . The presen-t: st~t~dy was undertaken to assess t:he
expression of MIF in e:ur~ ornetriot:~c les:ic>ns, identify the
sites) of expression, r. nd :investigate whether such an
1C~ expression varies acct; r~:l ing tc> t. he stage and the activity of
the disease.
Materials and Methods
Source and t~r:c~.l_i!~g of tissue. Endornetriotic t:issue
specimens used in this :study were cabt<~ined f:rom women who
l~~ provided informed cons~..nt~ f:or a resea:rct~ protocol approved by
Saint-Fran~:oi:> d'Assis:-: ~-tosp:itai Eth:ic;s Committee on Human
Research. These patient.:: were found to have endometriosis
during laparoscopy or :i.:~paYvotomy, had nc~ endo.netrial
hyperplasi.a c>r neopl_as i.~, and had no- rf=ceived arty
20 antiinflammat:ory or hcrrr,onal medic<~t:ic>n duri_ny a period of at
least 3 months before smrgery. Enc~ome~rloSl.s was staged
according to the reviseI Ame:ri_c:an Fert:.i:'.ity Society
classification system ;i'~nneri_can Fert_ii:ity Society, 1985) .
The cycle phase (prol~.I_c_rati~aEe or secrf=t.ory) was determined
2~~ according to l..he patie,nr ~' cycle h isto:ry and to the serum
progesterone. The mearu age was 34.6 pl~~:; or :minus 6.0 yr.
Endometriotic: biopsies= were immediately placed at 4°C in
sterile HBSS (Life Tec:tur~ologies, Inc., Burlington, Ontario,
Canada) containing IOC IU/m:1 penici_I_lin, 100 ~g/ml
30 streptomycin, and 0.2')..a~/ml. am~7hotericin tzansported to the
laboratory at: whi~~h tr c~.;, were= immediate:':_y washed in HBS;3 at
4°C, snap frozen on dr~~r icc:, or fixed in 10, formalin.
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For immunoh~.~rochemica~l analy:>is, 25 endometriotic
biopsies were includec:. F.i E peen wc~x~e fa czer; at -80°C in
Tissue-Tek OCR, ccmpour:rl (M:i.le:; Irlc . , ~,.lr;:hart:, IN) , and 7_0
were embedded in par_af r:in. '>?hese h:iop:~:.es were from women
~' with endometr__osis stac:~E~ L ( two typical black-blue, two red
flame-like) , :stage II ;~::wo tvY~pic<~1 bl~r~.r:-blue, three white,
and one from the inner w,al:L of o~Tari<~n endometrioma) , st:ages
III-IV (three typical 1:>:L~~Jk-blue, three white, and nine from
the inner wal.=_ of ovax::i..~;~ endometrioma) .
For Western b_1.~:~tt i.ng, EI I~>~, and RT-PCR analy~;es,
24 biopsies were taker:. These bs.opsi_es were from women with
endometriosis stage I vw~~ typic:a.l b:Lac;k-blue, seven red
flame-like, and three wl.i-te) , st:age II ;f: our typical black-
blue, two red flame-1 ~ .kE=, and three whiff=e) , and stages III-IV
(two red flame-like arch :one whit:e) . BioL:as ie;; were all snap
frozen and kept at; -8C"~:' in microcentrituge tubes (Eppendorf,
Gordon Technologies Irvc., M:isss.ssauga, c)ntario, Canada) until
used.
Immunohistoci'zra!nistry. Five-micrometer cryosect:ions
of Optimal Cut=ting Terr~.~erat ure-frc.zcen ~~ridometri.otic lesions
were mounted on poly-_i--lysin'=-coated mic,rosc:ope glass s7_ides,
fixed during <?0 min ir.,~ 10~ buffered forma7_in phosphate
solution ( Fisher Scier t:: i. f ia:, Mon'~~rea l, Quebec, Canada ) , and
washed in PBS . Five-rr~.i_'v.rometer se'~t:i cn:.> of paraffin-embedded
2_'i tissues were mounted on poly-1-~yss_ne-crated microscope glass
slides, depar<~ffinizec:l ir. t:oluene, rehy<~rated through graded
solutions of ethanol Gn~:~ water_, anal wasrned in PBS. The
subsequent steps were ~._:v,e same f-or- c:ryosections and paraffin-
embedded tissue secti<:au=:. Briefly, aftfer permeabilization
with Triton-X--100~rM (1''; in P~.S) anc:1 elimination of endogenous
peroxidase wii~h H,OZ (n. ~'o in absolute methancl) , tissue
sections were success:) ~aJl.y ~_ncuLvated at morn temperature for
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90 min with a goat pol .~~:::1 oval am t~ human--MIF antibody (RtxD
Systems, Minneapolis, T~~l'-J) [0.6'6 ~c~/n~1 in PB:>/0.2% BSA/O.Olo
Tween 20TM (PF3S/BSA/Twe~e:r~.) ] , 90 min with a biotin-conjugated
rabbit antigoat antibc:dy (Jackson ImmunoResearch
Laboratories, Inc. , WF~st ~.~rove, PA) ( 1: '.~00 irn PBS/ BSA/Tween,
45 min with peroxidasE~-r~onjugate~_1 strep~~avidin (Jackson
ImmunoResearclu Labor_ato:ries, Inc. ) ( 1: St)0 dilution in
PBS/BSA/Tween) and 20 min diami.nobenzi d~~ne used as chrornogen
( 3 mg diamincbenzidinc--! n . 0;3 °o Hy:>~ in PBS ) . Sections were
counterstaine<:~ with hE_rn<rto~;ylin arid moarit:ed in Mowiol~~M
(Calbiochem-Novab;~ocharn :~orp., L,a Joila, CA). Sections
incubated with goat Ic..t:~;~. ,at the same ~~o.~cent:r_ation as the
primary antibody were u,-;ed as negative c:ontro.ls in all
experiments. Slides wc::re ~~j:ieweci using <., microscope
1_'i (mikroskopie and systc me ~SmbH, ro,:~de.l_ DI~IRB, Leica Corp. ,
Postfach, Wet zlar, Germany) and phot~~~grrzphed with 100 A:>A
film (Eastman Kodak Cc~. , Rochester, N~) .
Dua_1 immunoi .ir~orescent ~ taini.og. Cryostat and
paraffin-embedded tis:_uE_- sf=ctiorns were tr-eat:ed and incubated
at room temperature for 120 min with a Boat polyclonal
antihuman-MIF antibod~~ tR&D Syst.ertcs, Mimr':eapolis, MN) at. 0.66
ug/m1 in PBS/f3SA/Tweer <~s ciescrv~ bed ~~ar.~_ier ( see the
Immunohistochemistry m.ethodo.logy> . Att~~r a PBS/0.05% Tween
20 rinse, seci~ions were incubated at. r_oc:~m temperature for 90
2_'i min with one of the fol_.l,~wing antibodies: mouse monoclonal
antihuman-CD6f3 (DAK(:> r:.'orp. Diagrnos tics (:anada Inc. ,
Mississauga, Ontario, ~:,inac~a) (d:iLuted .:50 in PBS/BSA/Tween)
to detect mar.rophages; ur-.~use monoc.:Lon.a1 antihuman-CD3
(diluted 1:100 in PBS/~3~A,/Tween;to deaf-:ct T lymphocytes; and
rabbit polyclonal antitnuman von Will_ebrand factor (vWF)
(Sigma-Aldrich Cor_p. C:,a-~a~:~a :LTD, C.~akvi 1 .''Le, C)ntario, Canada)
(dilut:ed 1:200 in. PBS/I3SA/'i'ween) to det~-?c;t endothelial cells.
After a subsequent wa~.:Cz in PBS/0. CS <'s 'Iwc-yen 20, tissue
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sections werF incubated simultaneous.Ly for 60 min at room
temperature in the daz K with flt~oresceir~ isothiocyanate--
conjugated donkey ant: gc at 3ni~ik:ody (,1a ;kson ImmunoResearch
Laboratories, Inc. ) (cliZuted L: 50 in PBS!BSA/Tween) and
rhodarnine-coryjugated :,t~c=ep antirnot~se an-~i.body (Roche
Diagnostics, lava:L, Ql!.c~=t:;ec, ~'anada) (di Luted 1: 10 in
PBS/BSA/TweerA) for ti,sues marked for MLf arnd CD68 or CD3 or
rhodamine-conjugated rnoc.zse antiralr:bit anl~.ibody (Jackson
ImmunoResearc.:h Laborat cries, :Cnc. ) (dilated L: 50 in
PBS/BSA/Tweer:~) for ti~;:~c.ies marked for ~ICF arud vWF. After a
final wash irv. PB~:/O.Oc;o 'Tween 2C, slide; were mounted in
Mowiol containing loo para-phenylenediamine (Sigma-Aldr_ich
Corp. Canada ::.~td. ) , ar antlfading agent, anc~ observed under
the microsco~.~E~ (heica i;~rp. ) equiF~ped f:or fluorescence with a
1'.> 100-W UV lam~:~. Photom~ crogr_aphs were taken with 400 ASA film
( Koda k ) .
We~~tern blot !..:.ng. Protei.n extraction from
endometriotic tit:sue u;~s perforrrceci according to our
previously de:~cri.bed ~~r~~cedure (Bzgonne>se F. et al, 2001a),
and total pro~~ein cone:ervtration was deternr~ined using the DC
protein assay° (Bi.o-Rac I~abor_at=or.ies htd., Mississauga,
Ontario, Canada) . Fow '~lest~ern blc>t ana L~,~sis, denatured
proteins were separated by SDS-PACE in L'>o acrylamide slab
gels and tran:~ferred c,nt.o 0.95-iam nitro':-.ellulose membranes.
Equal protein loading ~,a.~s ;.onfirmed by :~tair:ing the membrane
with Ponceau ;~ (20). Nitrocellulose membranes were then cut
into strips and i.ncub~~t,~: 'd overnight at . °C with a polyc:Lonal
goat antihuman MIF ant :it:odv (R&I>> :~ystern~ate 2 ug/ml of
blocking soltavion (t).7 ~a 'Tris buffer, 0, ~)° NaCl_/0.05~ Tween
20 containinG 5o nonf<:~t= dr°~ mild: jwt~/vol] ) or with normal
goat :Cg (R&D Systems) at the same concentration. Strips were
then washed _n TBS-0.1'0 'fween 'Z0, incubated for 1 h at room
temperature with a pez oxidase-conj ugateal rabbit; antigoai=
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97 85409-5
antibody (Jackson Immurn:~E~e.~earch Laboratories, Inc. ) , diluted
1: 10, 000 i.n the b:Loc:ka_rlc:1 sc::lut:ion, washed again in TBS-0. 1%
Tween 20, incubated fc:r 1 min with an enl-~anced
chemiluminescence syste~rn upping BM chern:i.~ uminescence blotsting
substrate (Roche Diagn~:>st~_ic:s), a:ld exposed tc> BioMax film
(Kodak) for several tuna intervals a:L:l.owing f~~r_ an optimal
detection (all bands wi:ibue but. not o~rerexposed) .
MIF ELISA. N F concent:rati.on i.n endometriotic
tissue protein extract wa:~ measured by H~L,ISA according t:o a
previously reported p.roc.edure ICalandra T, et al, 1995).
Briefly, this techni_qu~e ~~ses a c<:rpture mouse monoclonal
antihuman-MIF antibodS-~ ( R YD :3yst:ems ) , a rabbit polycional
antihuman-MIF anti_body- =or detect=ic>n, a~.kali.ne phosphatase-
conjugated go~rt anti.ra:~~a~it IgGs (Cherni_con International Inc.,
l~~ Temecula, CA) and para;i:ltrophenyi phosphate as substrate
(Sigma) . The optical c~~nsii::y wa.> me~asa~:ed ate 405 nm and MIF
concentrations were exta.~;~po1ated from <~ standard curve using
recombinant human MI: F.
RT--~?CR. Tota'_ 1~NA was ext~rac.ted from endometriotic
2C~ tissue with TRIzo.L~lM rt:aqeni~ (L.ife Technologies, Inc. )
according to t:he manuf ~r;:t~.zrer' s v_nst.rucai ons . The GeneAmpTM
PCR core kit ;Perkin-F l_rrier C'orp. , Fosteir City, CA) was used
to synthesize cDNA wit 1u X00 ng total. c:e~~ l.ular RNA and 2. 5
}rmol/liter random hexarncers in 2 0 ~i~ of a RT-P~~R solution ( 50
2~~ mmol/liter KC'__, 10 mmc:_i_!liter Tr-is-Hci.l_, 5 mrnol/liter MgC:l2, 1
mmol/ liter each of dM':~:,1~;;, >e~ ILi RNase :~nhi~:itor, and 50 IU
reverse transc:riptase) . The reaction was inc.:ubated at 25°C
for 15 min, 4a?°C for ~0 m:in, and 99°c:: for 5 m.in. For PC:R
analysis, we used 10~ ,>:C t=he reverse Transcription (RT)
30 reaction volume as temla_Late in a fina.L ~~olume of 50 1zL with
50 pmol of each MIF pr i_rner (:Forward p:rirner, 5' -
CTCTCCGAGCTCAC;CCAGCAG- 3' (:~EQ ID I~cJ. : ~ ) ; reverse primer, 5' -
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CGCGTTCATGTCG'rAATAGTT- 3' (SEQ ID NC~. : 61 ; amplimer size, 255
bp) , 0.2 mmoi/lit.er dP'C(', ~~ru~°; 2. ~ iU T.ac~- DNA polymerase
(QIAGEN, Santa Cl.ar:ita:,, CA. Ampl i.fic~t,ion was performed for
30 cycles composed of L min denaturation (at 94°C) , 1 min
.'> annealing (at 60°C:) , < ic~i i min L:~ri-rner e~t:ens:ion (at
72°Cl .
These optimal conditicn~~ were determined by performing
linearity tesi~s with ~~=.'.,, 10~, arid 20s ot~ the RT reaction
volume and 2'; and 30 ~:rnplifi~~ati.or. c:.y;~le.. Amplification of
genomic DNA w=ith thesF~ primers dig not ~or.cduce a signal,
suggesting th<~t the arrp:l..i f icat:ion 5i te.s crossed at least= one
intron/exon boundary. ~):Y the PCR volume, 20~ was fractionated
by electrophoresis in :~ 1 . ~'a: agarc.se ge:l in the presence of
ethidium browide and t :::<~nsfe:rred to a s~:i abrane~r'~ Nylon P7_us
membrane (QIAGEN) . Tren the membrane was dehydrated at 37°C
1_'~ for 30 min, prehybrida.fed with a r:ybridlzation buffer
composed of 5X SSC (0.~ mol/lit~er sod.irrm chloride and 0.015
mol/liter sodium cit=r~ ,:c-~ ) , 5X DE:.rlrardt:.' .:: solution, 50
mmol/liter NaH~POy, 0. ':« SDS, 20i) pg/ml salmon sperm DNA, and
50% formamide; hybridized wish ~''P-labeled MIf cDNA in the
same buffer except Der:'ruardt' s sa:Luti.on; washed with SSC
solutions cont=aining C . :L'~ SDS, 11X SSC, 0 . 2X SSC and 0 . 1X
SSC, respectively, anc; E=:xposed t:o x-:ray t ilm (Eastman Kodak
Co. ) for different t~irr=~ int:ervals al_:l_ow:i.ng fo.r an optimal
detection (signals visil:~le but not ove.re?xposed) . As control,
2~~ glyceraldehyde phosph~~t:s~ c:~ehydrogena:~f= ;GAPL>H) amplification
was used. For PCR ana:L~Y~s_i~s, we used :~'.5'~~ of the RT reaction
volume as template i_n r f.:ir;a:L vc>:Lume o:E _'~0 p1 with 25 pmol of
each primer ( j-orward ~ r:imer, 5' -':CGATGACATCAAGAAGGTGGTGAAG-
3'(SEQ ID N0.::3); reverse primer, ~'-'TCCTTGGA~~GCCATGTGGGCCAT-
3C~ 3' (SEQ iD N0. : 4) ; amplirner size, 290 bpj , 0.2 mmol/liter
dNTPs, and 1 ~=U Vent L: VA polymerise. Amplification was
performed f.or 30 c:yc:lc~~ c~f 30 sec: denarurati.o:n (at 95°C), 30
sec annealing (at 60°c :,. crud 1. m:i_n L>:r.i_rnez e~:t~ension (at
CA 02377786 2003-06-20
~') X35409-5
72°C) . ThesE_~ opt:.imal c°orrd:ition; were dc~t.ermined
following
linearity te:;ts using 1(W>>, 25° , 5C?° , and 75a of the RT
reaction volume. Spe~.vit~.city of t:he ampli.f:i_cation process
was verified icy Sowr_ht~r_ra blot: hybr,idizat:i.on. A negative
control (PCR .in the aL~sence of cD~.IA) as wel_:L as a positive
control (cDNA preparat wc:.n prom t:hE~ huma;l hyst.iocytic ce:l1
line U937, krv«wn to st~~::nete MI)='i u,rere included in each series
of MIF or GAPDH amp:lif ication. 'Tree intensity of the
hybridization signals was a:~eterrr~ined by computer-assi sted
densitometry, using QL,a.r~tit:y One Quanti~atic>r~ software (Bio-
Rad Laboratories, Inc.t. a'he quantity «f the PCR products
was determined by dens_i_tometric ar:,aly;~is of the intensity of
the hybridizat=ion s:igr;al. The relat=ive ieve:l of MIF mRNA
normalized to GAfDH mF:IV~~ was ca a.cu.lated, and the results were
1-'i expressed as percent c: control (positi~.re~ control) .
Stav:istical ~-~r;alys is. Mult:.iplc-~ comparisons of MIF
protein concentration:-, as measured by ELISA, and mRNA
levels, as det~e.rrr.ined l.~y sem:iquant:itatiTre RT-PCR, in
endometriotic lesions are-;:cording t:c t:h~J ~:.ype of the lesion or
to endometric>:~is stagy were perf~.~rrned using one-way ANOVA and
the Tukey's test. Corr:~~arison of twc> groups was performed
using the unpaired t tc>;~t . ~?:11 an<al.ysc~;-> were performed using
statistical analysis sy;~tern (SAS Institute, Inc., Cary, NC).
Differences were c:ons~ac~red as stati.st.:ic;ally significant: for
2~, a P value less> than 0. (i5.
Results
The first ox; jc~c~.i.ve of ttli.s study was to asses>s the
presence of MI=F i_rl end~::>n~et=r:iotic les~:i.on.:. Western blot
analysis of prote.i.ns eL:i=racted f~~orn endometriotic tissue
3C using a goat polyclona.L anti.hurnan-MIh antibody showed a
single specific l.'?.5-kL)a band corresponding t=o the known
molecular weight of MIEN' (Fig. 1.'>; . RT-t'CR anc~ Southern blot
CA 02377786 2003-06-20
100 85409-5
analysis showed specie i_;:~ MIF t: r.anscr:ipy::, thereby confirming
MIF expression in endcrn:~triotic tissue at the level of mRNA
(Fig. 16).
Immunohistoc:uern:ica:1 ar~alys:is of MI:F expression
showed a specific brow:o:Lsh :irnmuruosta_Lnirag localized to
specific compartments ,r i= endometricat=ic: t:issue. MIF was found
to be strongl~~ expres~;~:e~ in glanrtula.r e~.>ithel.i.al cells and in
cells scattered througluc.ut the st:roma (F'i.g. 1.~A) . Incubation
of tissue sections witl-i normal goat -lgG: used at
concentration equ~valew': t:o that of t:.he primary goat
polyclonal anti-MiF ant: i_l~:~ody (negative control) ciid not
result in any nonspecif:i_c: .immunostaini.nct (Fig. 17B) .
To identify ce:l_ls expressing MIF in the stroma,
dual immunofluorescenc_~~:= arnalysis was performed using
antibodies specific tc P~1.' F and to CD~i, CD68, and vWF.
Representative photomi~-roc~raphs exhik>it=ed in Fig. 18 shcw a
marked expression of MIF in CD3-positive T lymphocytes, CD68-
positive macrophages, zn;a vWF-po~;itlVE' endothE-elial cells .
To ~~:uantify '!II r' expres lion in endometriotic tissue
and examine w:nether MI F' ~,~xpress.ic:>n c:crrelates with the type
of endometriotic =t_esic,n and endometri.osis stacte, we measured
MIF concentrations> in c~t:a:~l protf~i:u E~~:t..racts key F~LISA and
determined the levels ~t mRNA in t':~e Name tis~~ues using a
semiquantitat.ive RT-PC':~; ~.:;nal_ysis. A:> shown in Fig. 19A, the
highest concentrations of MIF protai.n were fo~_rnd in flamelike
red endometric~tic lesi ras, ~:~:ompare ~ wi.th typi<:al black bluish
(P < 0.01) or with whir:e lesions (P <: Ci.01). Otherwise, no
significant d:iffe:r-ence f~c:t:ween typical. and white
endometriotic lesions ~.~~~as: found. Furthermore, MIF
concentrations appeared -c::o be significantly :higher in lesions
from endometriosis sta~:;e I, compared with those from
endometriosis stage II: (~> < C.05;i, whereas no significant
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101 85409-5
difference berween st~.gE7s I and I~:I-IV ,~~ II and III-IV was
noted. However, only t.r~ree biopsies fr~:~m endometri.osis
stages III-It%' were :inc _Lude-:i in t:.h:i s a;>s~~y, which may have
limited the power of ~:i~,atistical anal~~ses including these
.'> stages (Fig. :L98) . ATra:Lys:LS of MLF ~ro'.c=:in expression
according to i~he phase a;f the men~truaL cycle showed no
statistically significant c~ifferen.ce between lesions from the
proliferative and. the sf:~csretory pl~.asc~s.
Sem=_quantit~t:.:ive R'f-PC:".R ana.ly:ais of MIF mRNA 7_evels
in the same endometric~::ic T: issues _;1-a~we<~ a pattern of
expression corlparable with that of the protein, but the
difference in MIF' mRNF L~svels was tc_>unc~ to be significant
only between red and white endometriotic lesions (Fig. 20A).
On the other hand, ruo s:i.~~ni.f:icant~ c:~i.ffei:~ence in MIF mRNA
levels according t:o eru:viornet riosi.~ stage ( Fig. 20B) or to the
menstrual cyc~_e phase ~:aa5 mated.
Discussion
Endometriosi => rn:ight be a mul.ti fact.o:rial disea~ce,
and its etiology :rema;_,°~s hypothet;ical. The presence of
tissue structurally anal t=o some E-:xt:ent functionally
comparable with the er:~iornetrium :f_oan<i ot,tside the uterine
cavity suggests that ttue condition, at least peritoneal
endometriosis, results. ::::nom r_mplant:at=.i.on of e:~fo:Liated
endometrium following :retrograde menstruation (Sampson J.,
1927 ) . Accordingly, t iut~ ect.opi.c grcwt:h and dc:~velopment of
endometrial tissue Thai: i.s endowed w~~th the capability of
adhering to and implan=.ing into the pei:itoneal tissue
requires the :_tenesis c::f- rnew miczc>ves:~e7_~., a ~~-rocess called
angiogenesis (McLaren ,.?-. , 2000; 1-fealy D. I . et al, 1998;
Folkman J., 1~~95) 1 . ::~;=,~;rEeral growttl f_ac:tors, :including acidic
and basic fibroblast. g_:owth factors, platelet--derived
endothelial cell growtkfactor, and vascular endothelial
CA 02377786 2003-06-20
102 85409-5
growth facto:~~ (Vf~GF) , r~~me the ,~~b i1 ity to sts.mul.ate vascular_
endothelial cell growil_ in vitro and in vivo (Gordon J.D. et
a1, 1995) anct were fo~:r~,~ t<~ be rxL~resse~ by endometriotic
lesions (McLaren J. e! ~:~J_, 1996; honnez ~:T. et al, 199;
Ferriani R.A. et al, 993; Fuj.imoto J. et al, 1999). Oar
previous studies shove=d that endometriotic: :Lesicns express
IL-8 (Akoum A. et al, %ii0lc), a chemokine having endothelial
cell growth promoting a~:tivity .1.n v.ivo (Iloch A.E. et al,
1992). In an effort t::~ understand what enables endometrial
cells to grow ect:opic~=ally in some women, we have previously
assessed the ~~apabi:Lip y of enciornet:rioti.~ c:ell.s to release
mitogenic activity foi endothelial. cells and identified MIF
as one of the major f~,.a::t:or s re:Leatsed by t:.hese cells in
culture and having them ability to stimulate endothelial cell
proliferation in vitr:i,fang Y. et al, 'UC)0) . In the present
study, we found that ~~1IF' was effectively expressed by ectopic
endometrial t::issue, b<~t:h at: the mF;~IA and protein levels, as
assessed by R'I-PC:R anc:'Nestern blotting. Furthermore, we
showed that NI:IF was lcv~:;:~te~~ in thEe glan«s as well as in cell
aggregates sc:atte~red c ~.~~.>.r t~hc st:roma. Dual--
immunofluorescence an~:lysis identified er:dothelial cells,
macrophages, <~nd T :Lyrr,p'.nocytes as cells markedly expressing
MIF in the st=soma. Fl:rthermore, MIF was found to be highly
produced in the endomE~t:r.iot:.i~~ 7_eszons that were presenting
2'i noticeable vasculariz~tio:n and ,_eukocytce infiltration.
These findir:~:~s clearly c.emons~rate that MIF can be
produced locally in erdt~metrioti~tissue, and that by
different cel_:I_ types. Tn ~.Tif~w of it:s capability to stimulate
endothelial cell growth shown in our and other previous
3C) studies (Yang Y. et a:~ , 200; C;hesney .:T. et al, 1999; Shimizu
T. et al, 1999) and its faculty tc; actizurate and to inhibit
macrophage migration (B.Loom 3. R, et a l., 1966; David J. R. ,
1966) , it cou:Ld be su:.c~sstt~rl that MI:F lc_scal7.y produced within
CA 02377786 2003-06-20
~~03 85409-5
endometriotic: implant 1. ~raay contn:i~~ut:e to their growth and
development either by stimazlating endt~tlielial cell
proliferation or by ret:,.:.fining mamop:nages that have been
ShOWn t0 SeCrE?te ~JECiF, ci pi:)tnrlt ~~r',C~1~:7~:~~?I?1C f:~3C'.tOr
(MCLaI_"eW T.
_'> et al, 1996) , and numE=rt~~us growt:n :La~lt~~r-s for endometrial
cells (Olive D.L. et: z~l, 1'91; ~talme 'T. et al, 1988) .
Moreover, the marked E-~l~~rE~s sign of MIF l:ay endothelial cells
makes plausible than tPl:is i_a~~tor can s-ti.mulate endothelial
cell proliferaticn viG autoc.rine and pa=~acrine mechanisms,
amplifying thereby the- ang.i_ogeni.~,~~ process's .
It _~~s well Documented that: MIF is a major
multifunctional proinf:-~amrnatory :~y~tokine. The molecule has
been shown to be exprE-s;:~ed by inflanunatc>ry c:e.lls such as
macrophages and lymphoc::yte~ and to stsimulate cytokine
1_'> production by these ce=~ls (Me~tz ~._.N. eve al, 1997; Nishihira
J., 2000; Calandra T. <e:: al, 199<~; Bachc~r M. et al, 1996) .
Interestingly, it has l::o~en reporr_ec~ that MIFF overrides
glucocorticoi.c~ inhibit::.~:;n c)f monocyte .secret:ion of TNF-a, IL-
1f3, IL-6, and IL-8, wr i.c~h are i_mpcrtan~ inducers of
immunological. and infW:~mmar:o.ry re:spc_mse:~ (Cal andra T. et: al,
1995) . MIF-st:imulat=ecz m:~c:rophages a:re :.r~ turn shown to
secrete bioact:ive TNF-c:x and IL-iii (Caiad~wa T. at al, 199.'i) .
These data strongly sr.:gg~est. that f:IF ~:~~~uld funr_tion locally
to amplify the in:f:lamma-tory :reaction mhserved in and around
2_'> endometriotic lesions, mairWy in those considered to
represent the most actwae foams of t:he c~i_sease (Wiegerinck
M.A. et al, 1993; Koko~~:ine i. et, a1_, 19~>'7; :>haw R.W., 1993;
blitz C.A. et al, 1997;,
Endometriot i c; le:~i.ons can be c1_as~:ified according
to their appearance and to their activit~~%. Tn fact, le:~ions
of the peritoneal lin~:rng of= the pelvis leave various
macroscopic appearancE=s, ~wl:ich rc~f:Le~:.~t their- .age and/or
CA 02377786 2003-06-20
104 85409-5
activity. The red su~t_:l.e lesions are more vascularized and
have a higher epir_hel-i-i_1 rn~totic inctE~x t han the typical,
puckered black or bluish per_iton~_~a1 :1_es:i_ons, whereas the
vascularization and to_> m:Ltc>ti.c index <~re lower in the white
~~ lesions. Thu:~, red les:~:iont; are thoughi~ to c:o;rrespond to the
first, active stage of _~~ray i.mplar~t.a~_LCn of: endometrial_
glands and str_oma and ~a~::~.~_Lc~i evol vre t.c~wa~-d the typical black
or bluish les~_on after c~nc..l_osure beneath the peritoneal
lining. The white ie: i_:.rn s, whic:n <~re be-~l.ieve~ to corre;~pond
1C~ to fibroti.c quiescent lc~.sic>ns, snow :Le~~ vas cularization
and/or mitotic: activity.% anc:l reprc-.seni~ Less active forms of
the disease (Wiegerinc>~: M.l~. et al, L99:3; Kokori:ne I. et: al,
1997; Nisolle M. et al, L993; Nisoll.e M. et al, 1997).
Int.erestingL,~,, cm.i.~ present s~~udy revealed that. MIF
1~~ expression is sig:nific~z~t:Ly higher i.n :reJd subtle than in
typical blackblue or u~fu:i.tt:~ endometriot:LC~ lesions. The
protein expression, a~ measured by E~ISA, according to
endometriotic lesion t yl,:e was in keeping wit:h that of mRNA as
assessed by semiquant_i.t:at.iwe RT-PCR, <~li_houdh in the lat:ter
20 case statistical sic~nit=:i~ance was c>bser~.~ed c.~nly between red
and white endomet.rs_oti c les ions . a'h_is _~ndic:ates that reduced
production of: MIf i.n rrcor~s advanced endometriotic lesion~r
occurs at the mRNA le~~~1 arid is more likely owing to a
reduced mRNA ;~ynthesi: <.nd;'or to rr,RNA instability than t:o
2_'~ translational and/or_ ~~c~~,ttransl_ational event:s. Uur findings
are consistent: with tt c>:~~e reported. by :Oonnez. et al (Donnez J.
et al, 1998), who deteot.ed a difference in the expression of
VEGF between different types of Lesion,>, with the early,
highly vascu7_arized reel les:i.~~ns havi~mx a greater expression
30 of VEGF than i~he latex rrr:~rc.: inactive black. ~>owder-burn
lesions. Thus, the h_ic~ller expression of MIF in the red
lesions might reflect its role _Ln promor,~ng~'maintaining a
higher degree of vascL:, L,~r development a:ud gwT~e support t:o our
CA 02377786 2003-06-20
105 85409-5
hypothesis that MIF p l_z~r:a an irn~~ortan~: r ole in endometriosis-
associated active angi:_;genesis ana i.n:~.Lan~rnatory processes and
as a marker c>j= active ~:isf:ase.
Although anG i _agenesis appears as cr.itic:al for
~~ endometriosis and that Fact::o:rs t:rrat .s t imulate angiogene:~is,
such as VEGF ;Donnez :i . et a.L, 1.998 ) , <arid MIF in the present
study were found ~o be: ;verexpressed :~_ri i_nit:ial endometriotic
implants, the mechanis:~n:~~nderl~%irg the up-regulated
expression of these ar:,c~i~~gen.ic factors _~n endometriotic cells
remain to be rlore t.horc»;gh:ly invest~i_gawe=d. It is well known
that endometr=_osis is t-z hormone-dependent. disease, frequently
associated wit:h an imr~unoinf:lammatory pi:ocess both in eutopic
and ectopic locations (D:izerega c~.>. ~_~: al, 1980; Noble L.S.
et al, 1996; Oral E. E-t. ~.1, 1.996; Ot:a H. et al, 1996;
1_'> Jolicoeur C. et al, 1~'),.; 'I'seng ~J.r'. et al, 1996) . Recent
interesting ~>t:udies cia<::rly demo>nstrat:ec:~ that E2 up-regulates
VEGF gene transcription in endometr:i_ai cells (Taylor R.N. et
al, 2001; Lebovic D.I. ct al, 2000b) .~ncl that E2-induced gene
transcription is ER dep~:.;ldent and i_s ac?: ivated through a
variant estrc;gen respc:;cu~vive ~~lerner,.t:~ ioc,al:ized within this
upstream region of i~hc 'vEGF gene ~:~romoter (Mueller M. D. et
al, 2000) . 7.1. also a~:~:>~~ared that IL-1., a major
proinflammatc;_ry cytokine whose peritonea- levels increase in
endometriosis (Fa.kih I-L. et al., 198?; i'aKetani Y. et al,
1992), induce: an angsa~~en~~c phenc~type iri endometriotic cells
(Lebovic D.I. et al, f000a;. Hypoxia, which up-regulates
VEGF expression in enc:W rnetrial cell:>, is the>ught to be
involved in endornetri~~ L angiogene~;is anti to assist
revascularizavion o:E e:lesquamated endomet_rial explants when
they attach av ec:topic~ >i_tcJs (Sharkey A.M. et. al, 2000) .
These factors may activate VEGF er>press~on in endometriotic
implants. However, trnear role in the uv-regulation of MIF
expression irv: endometi ic~tir_ as well as ~~ndornetrial cells is
CA 02377786 2003-06-20
106 85409-5
unknown and currently ,n~aE~stigated iru of°,r labc:~ratory.
Furthermore, z.t woul.d loe :i_nterest:ir~g t:.o determine whether
common transcript iorz :f ,~::~T,or i s ) c-,3n be involved in MIF/VEGF
production ar_d activat i_;~n of angiogenesl.s.
It is notewc;rthy that MIF was more markedly
expressed in 1_esions from the initial stage of endometriosis
(stage I ) , compared wi the th:e more advanced stages, which
makes plausib_~e the it °,r~,:Lvemc~nt: of t.hi:~ factor in the initial
steps of endornetriotic ~:::i~s:_>ue growth and development.
Recent studio:-. identit:ied MIF in normal endometrial
tissue, predominant 1y i,r, tree glandular epithelium, and showed
no significant differfv:n<-es i.n MIF level: across the menstrual
cycle (Arcuri F. et a , 2001). cur current study with
ectopic endomE~trial tissue showed no ~~i:~ni.f_Lcant difference
1.'~ in MIF protein and mRh.l~-a express_i.or~ according to the menstrual
cycle phase. However, intense immunostaining for MIF was
detected both in the :~tromal and the epithelial compartments,
suggesting different expression of MIF in eutopic and
ectopic endometri.um. ~tud:ies eval.uatin~~ MIF expression in
the eutopic endometria:31 t.issi;re ;:~f women with endometrio;sis in
comparison with endomE;triot:ic: tissue from the same women or
from women having a n<:~rr,;al gynec~o-~ogical status are, however,
in progress in our lak:>oz at~~~ry, wh:i ch may shed more light on
the functional rc;le o:~ fll:f in endometrir~sis-associated
angiogenesis.
In conclusi.~~>n, t;ne present study snowed MIF
expression in various c~:e.l.l types Throughout endometriotic
tissue and ins marked expression in lesions representing the
earliest and the most ~~:tive stages c>f the d-~sease. This
suggests tha~~ MIE may ~wpresent a key e~ffector cell mediator
involved in the patho~:..~1~~Y~~;ic>l.ogy of Emdc:~metri.osis through its
proir.f lammat~~~ry and a ~:vc~ ic>genic act ivi t.i es .
CA 02377786 2003-06-20
i07 85909-5
Abstract
Interleukin ~;LL-1j is ,_~ naajc~r prc:inflammatory
cytokine that is belie;ne~~u1 tc> play a central :r<ale in the
pathophysiology of end:~rnc~triosis. ThE: II~-I receptor type II
(IL-1RII) is ~nowru to i:~,:i.mc~t to IL-1 and to inhabit its
biological effects. I:n :cur prev.i.ous st.udi.es, we showed that
human endometrium exprc-:::;ses TL-1RII, anc we cbserved reduced
expression of the prot~-.~.n i..n women with endometri.osis. The
aim of this study was tc:> ~_nvestic~ate IL-7.RII rnRNA in the
endometrial tissue of r;or_mal womc:m (r~ ~- ~6) and of patients
with various degrees ca endometriosis ;r: = 5 j;; . In situ
hybridization showed thar_ IL-1RI:1 mRNA expres:~ion was
significantly decreased :in endometrios::,v, parvicularly during
the early stages of th~, iisease stages I and II ) . This was
quite obvious in both calanduiar and strc:mal calls, and it was
corroborated by reverse ~ranscriptlon-pc~lymerase chain
reaction analysis of IL-1RI:I mRNA iri the endornetrial tissue
of women witr~ (n = 10; <~nd without (n =- 8) endometriosis.
The reduced levels of Il~-1RII mRNA i.n the endometrium of:
women suffering from Er:~cicm~:t.riosis reveals a profound dE:fect
in IL-1RII gene expr.e~sion anl, :~cnse~:~uE-:ntly, a reduced
capability of: endometn';.a~1 t.i.ssue tc~ down--regulate IL-1
activity. De=_'ect.ive !_T~-lR:I:I gene exprf~~;.;:ion during the early
stages of endometr_i.osis (st_ages I and I ) may contributE: to
2_'~ the etiology of the d; cease.
Introduction
Recf=nt evidEnce has demo>nstrated a direct
relationship between ,.he immune and the reproductive systems
(Adashi E.Y., 1990) . Cytok_ines produced by .Leukocytes <~nd
various other cell tyke's ha.~ve a wde range of biological
activities, i_:zcluding the abi..lity to rey .~1_ate immunolog.ical,
neuroendocrine, and reor.od;.~ct.iva tuncti.ons (e-oetzl E.J. et
CA 02377786 2003-06-20
108 85409-5
al, 1992. ) . Among these ~~~,rtokine;~, the nterLeukin 1 ( IL-1 )
system appears to be relevant to various r_epr,.~ductive
processes and to a brca:~ s~~ectrurn of pathopruysiological
responses associated w i_l::i:~ roost: defense ~~nd i.nflammation
(Dinarello C.A. , 1989; 1:~:~.rsen C. et ai_, 7.990) .
A c~~tok.ine r aiv:inc~ potent anti nflarnmatory
properties, I7~-1 was shcwe to be involved in numerous
immunological and repr:oc~u~:~tive activ.iti<~s occurring normally
in the human endometr.:i urr, during a normal. rn.enstrual cycle or
during embryonic impla;:ntat:i_on crud devel<y>ment (Simon C. et
al, 1995b) . ~~ growinc; bod~.a of evidence ind,~cates that IL-1
may play an important r~~~le in the pattnophyszology of
endometriosi=, a gynec:~~iog:ic:a1 c~i;ease :-;teat i.s believed to
arise from ectopi c gre:~wr.h of enc~ometria_~.. t.i ssue and .i.s
associated with a chrt.:~nic immunoinf_lamTnatory process (Halme
J. et al, 19f.~4a; Gros.~>xinsky C.I~z. -st al, 1993; Sahakian V. et
al, 1993) . I:z women ~,r.it h endometriosis, peripheral blood
monocytes (teller J.M, Ft ~~1, 1'a8'') as well as peritoneal
macrophages (l~Iori H. t:t al, 1992) were found to be more
activated than irn norr~~ai women and to sc~c::rer.Fe elevated .levels
of IL-1. Increased cc..rrcen ;ratir>n,~ of IL--7_ were detected in
the peritoneal fluid :,f women suffering .f_rom endometriosis
(Mori H. et al, 1992; fakih H. et al, 1987). According to
our previous dat<~, IL--1 enharuces the production of monocyte
chemotactic protein-1 (MCP-1.) b~r. r:um.an endome~tri.otic cells
(Akoum A, et al, 1995<y ) and by r:utois endometri.al cells of
women with endometrio;>i~. (:clicoe;~r C. et al, 1998) .
Moreover, these cells ai:peared t.o be rr:ore sensitive to the
action of IL--1 i.r,, wom~::r~ with than i.n women w:i thout
endometriosis (Akoum .~~. et al, 995b) .
The effects of IL-1 l_Lke.ly are str.i.ngently
controlled l ~ vivo. ,lumber of IL-1 inhibitors that can
CA 02377786 2003-06-20
109 85409-S
block the activity of 1I.--~ c:n targE>t celis ha;Te >"een
identified anc~ pair°t:.ia7.l;; charactE~r~zed (Larric:k ~T.W., 1989) .
Thrf~e recept:::~rv; f or IL-1., now designated as TL-1RI,
IL-1RII, and IL-1P,III (moz:~e commonly called IL-1R AcP
[accessory protein] ) , IvarrE> been c:iesc:ri_'oed. The relative
importance of these re:ept.ors in IL-1 signaling has been
recently clarified. F. c_:l:~itical role for tine iL-_LRI and IL-1R
AcP in IL-1-i::~.duced cle:l:~- ~ cta_vati.oru has been demonstrated by
several group's (Colott:~ r'. et: al., -99; Sims ~7.E. et al,
1993; Greenfeder S.A. =~t: al, 199':>) . In contrast, IL-1RII
appears to be dispensah:l_~-~ for II,-1 signaling and may act as a
decoy receptor (Colott:~~ h. et: al, 199::; Sims ~.T.E. et al,
1993) . Interleuk:i_n--7. r-.~,eytor arutagori:~~>t (IL-lr<~) is another
natural inhibitor of 7:C,--I, which competes with IL-la and IL-
l~ 113 for IL-1. RI (G-ranowit:~ E.V. et al, ira91 ) . Results of
several studies indicate tt-~at the IL-1 .:system is available
locally in the human e:cndomet.ri-al. tissue and may be an
important mediator. in :1 ~:~:a:i. cell.~_zlaxv :i_nt:eract=ions (Tabibzadeh
S. et al, 1990; Simon ,.. et al, 1993; B_i_gonnesse F. et al,
2001a; Kauma S. et al, 1 990; Bictonnesse F,. et al, 2001b) .
However, few :atudies f:a~Je i ocuswd on t:lze possible role of
this system i.n the pa t:tuophys.iology of ~=er~dometriosis.
According to Sahakian et ~.~_! (Sahakian 'J, et al, 1993) ,
ectopic endometrial tissue does not: express IL-lra.
2.'~ According to our previo-~m studies, the erndometrial tissue of
women with er:,dometr=io::::L~; ex~rE~sse~> row l.PVels of IL-1R.II
protein compared with t:fu.at of healthy women (Akoum A. e1~ al,
2001a) . The aim of tu:.is study wa:> to investigate the
expression of IL-1RII at the level of mi~NA in the endometrium
of women wit;u and of ~r,romen without endornetr:i_osis. Alteration
cf the IL-1RI_i gene a>:;~:~~es.siorr ma~% prov.ide mc;lecular evidence
for a deficient contrc;l of IL-1 in the eutopic endometr.ial
tissue of women with cndometriosis.
CA 02377786 2003-06-20
17.0 8.5409-5
Materials and Methods
Subjects and .T~~:;sue Ccllect_c;r:. Endometrial tissues
were obtained from 79 ~,ao:rnen aged betweE:r. 30 arid :36 yr who
were undergoing l,aparo,:c:«pic surgery f: or infertility, pelvic
pain, or tubal ligatic°i and who quad note receimed any anti-
inflammatory '~~r horrnon~_l. meclicat ion cu.zring a ~:~eriod of at
least 3 mo before ~_apa:~oscopy. nifty-t=r:ree women had
endometriosis of trario:_a:== stages (I, II, III, and IV)
according to the reviw,::~c~ hrnerican ~,er_t:,wiity Society
classificatioc~ (Rock ~.~., 1995) . Twenty-six women were
fertile and had no vi.,v.l:~:Le erndome~trioa_~a at laparoscopy
(Table 9) . The cycle I~>:riase was c~etermi.ned according t.o the
cycle history, progesterone levees in t~r~e serum, and
histologic criteria. ~='girl ~raformec ~~onserit was obtained from
each patient, and the st.;.zdy was approved by the ethical
committee of t:he "Cent r c~ H~~spitd lien Urm~versitaire de Quebec"
(CHUQ).
Table 9. Clinical char:<~~r_.erist:ics of: pat~.ent:s at laparo;>copy.
Subje<:ts cycle
by
~>ha:>e
(n)
Sub;~'~~t~: Proli~erati
(n) Age (yr)'~ ae Secretory
Controls 26 ?
3 :!-6 '~ 1
5 .
. ~;
~
Endomet.riosis '_.3 3-1 r 5. 9 29
, f.;
~
(total;
Stage I ~.'2 3~~, ; 6,;311 11
5
Stage II 7.9 30. .'5. ~ 1-~
5 t)
Stages ITI-Iz7 7.2 ~ ;-q,'7': 7
i,
5
Fertile 2~3 32.~ E.~ 'i 13
InfE:ertile a'a 30. -~4.'7-=3 16
~3
2C) ° Mean ~ SD.
Endometr_ial :~arnple~, wr~rE~ r_oll>>ct.ed with a curette
before laparoscopy. '7'~ne tissue was plagued in cold, sterile
Hanks buffer~~~~ saline :~ontairlin~~ ainl~il:~i~Y~:i.cs, then
CA 02377786 2003-06-20
111 85909-5
immediately t-ansportec:i t:.c thir laboratc.~ry and snap-frozen in
liquid nitrogen before x~F ~~ng stored at. -80"C.
Fluc:res~rence :boa ~~it:u Hyb.ricl.i.~;ation. The present
experiments were ~>erfcrrr«:eci as prevvou~;ly desct-ibed (Jolicoeur
C. et al, 1998). Brietl', bi.otiri-labeled IL-1RII cDNA probe
was prepared lay ni.-ck t. c~~n-m:lat:ion (:f_awrenc_e J. r3, et al, 1985)
from the entire p~-asmi::r ~:~ecto.r pc~DivA~.
Cryosections it::hickness, 5 arm) from i9 endometrial
tissues (Table 9)~were :i.xed witr: formaldehyde and dehydrated
with alcohol ~~efor_e be:i-net ruybrid.i.zed with 5 ng/ml of
biotinylated IL-1RII c(nNA probe. Bioti.r was then detected
using a rabbit antibior__i.ou antibody, ~~ ~;ictiny-'~ated goat
antirabbit antibody, ar:ca i-luorese:ei-n iscthiocyanate-
conjugated streptavi.din, respect-.vely. ~,'ections were finally
treated with ~>rop:~di.urn a..c.~clir~e, which ma~:es the nucleus
visible in yel-low-orange fel-towing u_t:.z~aviole~ excitation,
and were observed under a fluorescence microscope (Leica
mikroskopie and syst:erm~ ~'.~rnbH, Model DMRB, Postfach, Wet z;l.ar,
Germany). Serial secti-;>ns from each tissue inr_ubated without
2C IL-1RII cDNA probe or ~a-i.~~h norcspec~~fuc: L:~NA pr~:~bes prepared
from the plasmid vector '~:Lor~e were used as negative controls.
Eva_tuation c~ .Staining. Stain ~ng was evaluated
using an arbit:rary scale as previous_Ly reported (Jolicoeur C.
et al, 1998) . In br.iE:~:,, c_=ac:h er~dorne~:r:ial section was gz-aded
according to t_he inters:ity of staining ~.hree times in three
different, randomly sE, _f:;zted area. arid :cored from 0 to 3 (0
- absent, 7. _- light, ~. -- moderate, and - intense) . Grading
of each specimen was ~:e:rformed by two different observers who
had no knowledge of th;e clirlica:~ star_us of t=he patients
including laparos;_opic. ':liagnosis.
CA 02377786 2003-06-20
11~ 85409-5
Reverse Transcription-Palymeyase Chain Reactian.
Total RNA from the end'ametrial tissue of 8 normal women (3 in
the proliferative pr:as fe ,rnc~~ .'~ in t:oc~ ;ec:retor,>> phase) and of
women with stage I- l encxomet.r-ic>s.i: ~ 4 ir_ -i_he
5 proliferative phase anc::i ~ ~n the secvretc>ry ~>hase) was
extracted using a Triz,~:::L ~M rea.gent accor~.:iing to the
manufacturer'; instrucldions (Gibc:o f3RI,, Burlington, ON,
Canada) . Total RTJA ('::~;)',.i ng;~ was rever:~E-~ transcribed into
cDNA using 50 U of rev=:~::-;:~e trans~.:riptasE.~ in the presence of
1C~ random hexamer pr:imer~(:?.5 mM) , ciN'fPs !1 mM each) , 1 Uiml of
RNase inhibitor, 10 m1~ ':rr.is-HCl, 5C~ ml~~ I;Cl, and 5 mM MgCl
(Gene AmpTM PC;R Core K::.t; Perkin-Elmer, Foster City, CA) . The
reaction was -_ncubatec.: <~t ~'S°C for 1.5 m~,.n, 42°C for 30
min,
and 99°C for 5 min. 'C'wc~ microli.ters of the rF=verse
transcription (RT) r_ea,<~-; iorl were use~~ fcor po:iymerase chain
reaction (PCR;~ in a final volume of 50 ;ai with 200 pmol of
each IL-1RII primer ('~': TCC ATG TGC AAA TCC TCTCTT (SEQ ID
NO . : 1 ) ; 5 ' : TCC TGC CC'C T~: A 'rCT CA'T Ai:'~ ( SEQ I D NO . : 2 ) ;
expected ampl:imer lenc::at~~-~, 576 bp) , 0.'~' rnM dN'fPs, 2 mM MgCl2,
and 2.5 U of Taq po:Lyrruerase (GrovF~s R.W. et al, 1994) .
Amplificatiorv. was pert:ormec:i fa.r 3C) cycles cons.i.sting of 1 min
of denaturation (94°C), 30 sec of annea.l.ing (60°C), and 1 min
of primer extension (';<''"C). Glyceraldenyde phosphate
dehydrogenase (GAPDH) was used as a control. Four
mi_croliters of tr:e RT :react=ion were used for PCR in a final
volume of 50 p1 with ;:::!o pmol of each pr :i_mer ( 5' : TGA 'r GA CAT
CAA GAA GGT GGT GAA G ( ~EQ I D Nc:) . : 3 ) ; 5' : TCC TTG GAG GCC ATG
TGG GCC AT (SEQ ID N0,.:4); ampli.mer size 240 bp), 0.2 mM
dNTPs, and 1 J of= Vent DNA Polymerase (New England Biolabs,
Beverly, MA) . Amplif_.c:ation wa;~ performed fc;r 30 cycles
consisting of 30 sec c:~.f denaturat=:on (95°C:) , 30 sec of
annealing (6~:)°C) , and 7 min of primer ext=ension (72°C) .
These optimal conditic>n:= were determined following linearity
CA 02377786 2003-06-20
1i3 85409-5
tests using l, 2, 4, aue~ 8 ~.al of tree F<'1' react ion volume and
25, 30, and 35 amplifi-<:~t_:_cr~ cyc~.es. tlnvplificat_ion of
genomic DNA with these I:~-~_m,ers d:id not.. produce a signal,
suggesting that the amE:~.l...ifi~:.ation site:: crossed at least one
intron/exon boundary. ~~ total o1v ~'0'~, of the 'CR volume was
then analyzed on a 1.° (w!jr) agarosc: c~e_I_ i.n true presence of
ethidium bromide and t r-ansfer_red to Qi.abraneTh' Nylon Plus
membranes (Qi~~gen, S ar:r.f~ ~::~ ar_i.ta, C:P,;~ . Membr<~nea were
dehydrated at 37°C for 3t:) rrai.n, p:~:ehybrucFized with a
1C hybridization buffer, y.Wridized in the same buffer (without
Denhardt solution) with ''-P-iabeieci :CL-1.Z:II or GAPDH cDNA, and
washed in 1X 0.15 M scdrum chloride and 0.015 M sodium
citrate (SSC) , 0. 2X St' .',, arid 0. -~ a SD:~, respec'~ively, before
being exposed to x-i.~a~~ :~.ilm (E~asa:.mar~ Kodak, R~:achester, NY) .
Specificity >:f° the amp_Lificav ion process was
verified by Southern ~..lcu:t r-iybridizat:iona A negative control
(PCR in the absence of c~ DNA) as we:ll_ a~;~ a positive control
(cDNA preparation from human endomet:ria.L tissue expressing
IL-1RII) were inc.ludec:: i.n c:e.~ch :yF~r.ies ~~tr IL-1RII or GAPI)H
amplification . The qa.ar~tit;y of the PCR produ.~ts was
determined by densit=orr.et: r i<: arla~.: ysi:> of the .intensity of the
hybridization signal. 'T'he relative levee of IL-1RII mRNA
normalized to GAPDH mFvtdA was calculated, and the results were
expressed as a o of tt:e control t~~,:.lue (ioosit:ive control) .
Statistical A.rualysF~s. The intensity of IL-1RI_L mRNA
hybridizatior_ signals was expressed as arbitrary units.
Statistical analysis urt:~:~ performed by t:ne Fisher exact
probability t.,sst (Guz. ck D. S . , ~! 9~~6 j , and Bonferroni
correction was applie<a when more than twco groups were
compared. An,~lysis of '~L-3~II mRNA _Levels as determined by
semiquantitative RT-PC'R. was performed using one-way ANOVA and
the Tukey tent fc:r pc_: t-race rnult_ ipl a compar:i.sons . All.
CA 02377786 2003-06-20
114 85409-5
analyses were carried :-~ut: us.ing t.h~: ;>tat. istict~l Analysis
System; (SAS Institm_rte, Lrn c. , ~.~ary, I~dC. D:iffe:rem:es were
considered to be stat:i.c=,-:i<,.ally si_grrif=ic~ar~ at. P ~~ 0.05.
Results
G Analysis of Ih-IR.II GE~.~~:~ E.~Y:p_ress.:icr~ _i.n r-he F;ndometrium by In
Situ Hybridi~~ition
The expressi~.>o of :IL-1RII rnR:'JA in the endometrium
was studied by in sitr.; Iuv~b.ridi.zat.ion to examine the site of
IL-1RII synthesis and t;-. c~c>mpare the :Le~,~els of IL-1RII mRNA
in patients w=_th and without endomet:riosi_s. Figure 21 shows
the appearance of endc::metria:l. st:rorna anci glands at 666X
magnification (A1 arud 1311 fo:l.low:ing hyb~:~idization and
staining with prep.idiu.rn iodine. The t:ybridization signal
(green-yellow) could r..n.ly be visc.zalized at rrigher
1_'> magnification (1665X) and appearea to be located mainly in
the endometrial glared::, (A'2 and E32) .
As described ear-~ier, an arbitrary score was used
to quantify the IL-lRy :I rnRi~IA hyb:ri.dizat..:.on signal.
Statistical analysis of hybridizat ion sa~c:>res using the hisher
exact test showed a s.7.gnificant de~cr_easc:~ i.n women with
endometriosis. compareca =:~ normal. women, both in endometrial
glands (P < 0.0001) arid st_roma (P < 0.006) ('fable 10) .
Furthermore, when pat::_ents with endometri_osis were grouped
according to the stage: of disease, a significant decreaae in
IL-1RII mRNA expressic:n in the glandular (.P < 0.0003) as well
as the stromal (P < 0.0:.2) compartment was observed in stage
I. In stage II, a sic:3:nifi:.ant decrease in IL-1R.II mRNA
levels was a1_so obsersrec~, t;ut o~l.v in the glands (P < 0.042) ,
whereas in more advanc~:eci stages (-=I:I and IV) , no
statistically signific::arit difference was found. A graphical
illustration of IL-lRll mRNA scores in normal controls and in
CA 02377786 2003-06-20
115 85409-5
women at different:. sta:,~c~~ cf: endc>metriosis is shown in figure
22.
The effect. or t:he menst_ rua). cycle oo levels of IL-
1RII mRNA in the endorra:=tr~urr~ was a'~so ewal_uated. Statistical
analysis of the hybrids ~:a~t:ior~ scores slic:wed no s:ignifi.cant
difference between the prol.iferat.=.ive ancthe secretory phases
within the centro~. or -:.he endomet.ricsis group;~. However, the
decreased expression of ~L-1RLI TnRNA ob_>erVed in women with
endometriosis was more not~ceabl.r: ciur_im~ the secretory phase
1C of the menstrual cycle, either l:~u t.w.e c~l.ands (F' < 0.003) or
in the stroma, in wrii~.:r a:~ vi,~ati~~tica_~:1.~,~ significant
difference between worrev with anc.~ women without endometriosis
was seen only during t.tie secretory pruase ( P ~: 0 . 018 ) ( Table
10) .
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11.6 85409-5
Table 10 . Number ~:~f suL: _j ect y acc~rcii.ng t o .intensity of the
IL-1RII mRNA ::~ybr.i.dizat::i<~n si.gna~ i..n the endornetrium.
Stroman; Glands (n)
Inter:::~it In tersty
,~ of ~. of
stair:::z_:: st ain...r:g
Mum- -._0 ..__ j ~. , _ 1 2 3 P
G (1
l.f~r _
Controls :-r~ 8 ~I ~ 0 ~ I 6 10 9
Endometriosis '_~ 31 ., 1 ,, 0. (i06Q ~:5 :12 2 0. 0001b
3
(total)
Stage I~ ~:2 16 c; ~ 0 0.012--4 l~: 3 0 0.0003b
Stage IIb 79 8 .0 C (;.422 0 12 6 I 0.042b
Stagas III- 1.2 7 '. C (:~.4:~(.)8 3 1 0.186
IV
Fertile ~'.3 14 '~ a 0 ~~ , 2 13 7 1 0. 030b
CalB'-
Infertile i~ 17 ~. 0 0.76 ~_ ~._ 5 1 0.0004b
Proliferative
phase
Control j 2 _ _ () C 3 2 4
Endometriosis '4 13 1C n ~C~,2172 75 7 0 0.009b
Serretory
Phase
Control ._ 6 ~I C 1 3 8 5
%
Endomet.riosis <'9 18 ... ) 0 ('.018'.. 20 5 2 0.003b
''Fisher exact test.
Comparison with controls; i va!11E?a c: _rc..~c;ted > y the I?onferroni
c procedure.
Stat:isti.cal _~r~a_Lys:is c>f t: he hybridization scores
according to t:he fertil:Lty status of suh7ects showed that,
compared to normal ferr:.:i Lf~ women, fern :i~_ee women with
1G endometriosis had decreased expression of IL-1RII mRNA, both
in the glandu__ar ( P < (:) . J 3C) ) and iru -~hf~ ~~trornal ( P < 0 . 018 )
compartments of endom~t~rial_ tissue. However, in infertile
women with endometrio.ia, a significant decrease in Ih-1_RII
mRNA expression was o):~~~e rvF~d only i.n them glands ( P < 0 . 0004 )
15 (Table 10) .
RT-PCR Analysis of IL-l.RII mRNA E~press.ior. in the Endometrium
Exp:-ession c~_ IL-1RII mF;NA in t:.h.e endometrial
tissue was fu:rther ev4cluated by semiquantitative RT-PCR in 8
normal contro:Ls and 1f, women with erldo~nei~riosis (stages I and
20 II). A representative RT-FCR and Southern blot analysis of
IL-1RII mRNA .in the er:~dometrial tissue ~f women with
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1.17 85409-5
endometriosis and of rv~t-mai c:antrals is shown in Figure 23A.
Levels of mRN.F~ we:r_e s_i:rnific:antly :i_ower in the endomet.riosis
group than in the cons: rv::l_ clroup ( F <: 0. C'062) ( F lg. 23B) , which
corroborates t:he in si.~-.~ hybridization data.
Discussion
Our results :>r~awed a significant decrease in the
levels of IL-LRII mRNh _~m the endametr=ium of patients with
endometriosis. This w,u._ o)vw=ious in lulaE~ strama but was mare
significant i.n the g.la ud:~ as examined by in situ
hybridization. Semiquant~Lt.ative RT-1?c:R analysis of IL-1RII
mRNA levels in the end~:nr,f~trial. tis.,uc-~ a~ so showed a
significant decrease in wc:~men with endometri.osis compared to
normal women, which cor~obarates the ire situ hybridization
data and prov__des evic_-.r~ce for a pro.faund defact in IL-1RII
gene expression in the ::.utopic endomet:r:.um of women with
endometriosis,. The IT -:l.:f~:.II is syr.thes:i~.ed as a membrane
bound receptor that lads t:he signal--trarasducing cytoplasmic
domain found in the W.rmti~~ria7_ receptor: type I (Suns J.F. et
al, 1993) . The receptor cyan be cleaved and shed from the
cell surface by the proteolytic action of matrix
metalloproteases (O.r_l~nc:xo ~. et al., 19a'') . Both membrane-
bound and soluble forms ,~f Ih-1RII c,~r1 hind to IL-1 and
prevent its interactic:n with the signal~~ransducing IL-1RI
(Colotta F. ei~ al., 19~~:3) . Our ray: silts, demonstrating a
2.'> significant decrease in IL-7.RII m)~:NA expression in the
endometrial tissue of ea~omen with endome~;riosis, reveal a de-
ficiency in the ability of eutapic: endometrial cells of these
women to down-regulatf their response t~7 IL-1. In fact,
eutopic endoraetri.al cFVl1 s ;f women with endorrietriosis
appeared to be mare sensitive tc I:L-1 compared to those of
normal women, and they secreted higher zmaunt:s of MCP-1
CA 02377786 2003-06-20
J18 85409-5
following stimulation with IL--1 in vitr« (Akoum A, et a-,
1995B).
In this wori< , we observed t~e~at: defective IL-1RII
gene expression in the end<.~met:rial tisa~ae was significant at
_'~ stages I and .CI, but: r~.>-:_ in more advan~~ed stages (III and
IV), of endometriosis. These results point toward a process
of cell act:ivaticn t:ha:t~ -~a~:es place 'L~oc~a,~ly in the
intrauterine endomet:r~ u:nr~. a ~: the ear l_ iest:: stages of the
disease, and l:hey sugrev;t that; end.ometr.-iosis is more act=ive
during its first stages. Available data regarding the
correlation between tr:c: .°xt_ent of endometriosis and that of
the chronic z_nfla~nmatcr~y~ process cbservec< in the peritoneal
fluid of patients and ::_r: ~~c:v~r_~pic.- as w~~l_:_as eatopic
endometrial_ tissue r_erT~a ~~n c:ont:rovers ial . It: has been
reported that the conce~;~ratiorus of inflammatory cytokines,
such as interleukin 8 arid RANTES ( regulated upon activat=ion,
normal T cell expressed :~nc:l secreted) , ~.-orrelate with the
severity of disease (Fy~~.n I.P. et al, 1t)95; Khorram 0. et al,
1993) . However, other :studies have srlown that less extensive
endometriosis may be r~o:r a i>ioc:hemiaa~~l~ active than older
implants (Vernon M.W. et al, 1980 , and that: peritoneal
inflammation is more a<~t.ive dur3_na t:he Lnitial than the
advanced stages of the ~~isease (Verno-n M.W. et al, 1986;
Haney A. F. et. al, 1991 . accorc~:ir~g to =~essey et al (Le:>sey
B.A. et al, J_~~94) , t=hc: prefect: of int:egr.irv expression in the
eutopic endometrial ta:>;~ue i.s iro-,rerse~~~ related to the :stage
of endometricsis. Our ~~revious studies :;bowed that in :>itu
expression of MCF-1., f" ~;.;ctemt chemotact:i_c and activating
factor for mc;~nocyt:es ~n~:~ macroph<~yes, in the endometrial
tissue was ma__kedly elevated during the initial. stages (I and
II) of endomei~riosis ~;uei decreased during more advanced
stages (III and IV) . L:r,terestingly, we also showed that.
defective IL-:LRII pros:cin e_xpre~~si.on in t:he c~.ndometrial
CA 02377786 2003-06-20
119 85409-5
tissue was m ore marked ea.~ 1_y stages of the disease
:l~..lri
ng tr~e~
(Akourn A. et ~~1, ~_OOla;~wivir_h iri keeping with the
,. i5
findings of the p.reser~t,:~~t=udy, we also found a
:rid
significant negative r_el.atior~ith MCP-1 expression i.n
c~>r w the
same tissues (Kha:rfi E:~t: al, ~-.
A. ~:~.)0
The data of t.xle present study suggest that the
reduced expre~~sion of T:L-1F<.II in the uterine endometri.um of
women with enclometri.os :.,:~ is ~~elat:ed, at. least. in part, to a
defect at the mRNA I_evf=~~. , although tr_ans lational or
proteolysis dependent ErlecharW sms cannc>t be ex~~luded.
However, whether such :..; c:~efec:t i~~ due tc:~ decreased mRNA
synthesis or reduced rr~l?Nl-~ stabili_ty ._s Linclear. It is also
noteworthy to add that t_t1e decre,-use yn rrIRNA lt~ve_l_s in th.e
endometrial tissue of women with endometriosi_s was more
significant during the p~~ol._i_1=era~:i.~~~e t:l~an the ser_retory phase
of the menstrual cycle, w>"ui.ch ag~,ii_rn ~._s ccnsistent with the
cycle-dependent pattern. ~af defic _ent IL--1RII protein
expression (Ak:oum A. en <~ i_, 2001x) . The mechanisms
underlying th.t cycle-:Iependent, a!_>ei:rar:t IL-:LRI=L expression
remain unknown. Howevr,. t: his rnay have an interesting
significance, because it: suggest:; ~:h~~t= endomet=rial tissue
debris refluxed into tE.e peritonceal_ c:a~rity at: the end of the
menstrual cycle may corv~t:~~~_n low _Level:; c f IL-:LRI=L, which may
make the tissue less capable of down-rec;ulati.ng =LL-1-mediated
cell activati:~n a:nd le:~ca t:o an exaggerated pea~itoneal
inflammatory response.
The data of tare present: study also suggest that
defective IL-lRII expr_~:=>~:v>~~c;n dun ing t:he early stages of
endometriosis may play a:~ rc;ie in t:he initiation of the
immunoinflamm;:tory pro~:c:e:>:~ eutopically, in the uterine
endometrium w-.ere the ~::..i~ease is belz_eved to originate, and
ectopically, in ttze pe:~ i.;::oneal cavity wr.ere endometrial
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120 85409-5
tissue is r_ef::Luxed ane: r~eveloped. mhe lack cf statistically
significant c:~:iffe=rence: ~.n TL-1R_L I expre,~sion between women
with and women without ~ndomet=r_os;_is during the late stages
of the disease may, o:~ the other hand, be part o.f the
counterregulatory o_r rf~>parative mechanisnus t:.hat stabilize
endometriosis by 1i_mit L~;g c:~:Curonia inflarzunation and profound
tissue damage by restr ictin~ IL-1-rnedi~,_ed proinflammatory
actions (Ding=ello C.t= . 1939; Lars.en c.. et a.1, 1990) . It is
interesting to note trat hlo:ri_ et: a1 (i~Iorv H. et al, 1992)
demonstrated i=hat the ~~:~prE-e:~siorl c:,f IL-'~~~ by :peritoneal
macrophages was elevated ~:~uring th.e irlit:i_al stages of
endometriosi~~ and that: i L-i:ra, amo~.hr~r natural specific
inhibitor for IL-~, w:~~~ more elevated d?.zrincx the late stages
of the disease.
1_'~In conclusicry,
our results
demonstrate
that the
expression of: IL-1RII rnl~.:'~1;~ was dec_:reasf~ca in the
endometrium
of women with endometrie: s is, pa~r~=i cJuiar:~y during
the initial
stages of the disease.:'ue~h a defect:.iv~~ IL-IRII gene
expression by endometn:la I c_:e:Lls pc: rots _oward a profound
defects in the capabil~~_~;- of endometr:iaL cells to down-
regulate IL-1. actions,tan:i_c_~:~ may ~vlay a relevant role
in the
initiation of the immr:rn_> inf lammatcry~ p:rc~cess associated
with
endometriosis, both tie intrauterine endometrium and in
ir:
the peritonea=_cavity i=;.,:1.1c_>wing 1=uba:L rEeflux. It
remains,
2_'~however, to determi n<:,a~ whether su~~h a def:e~~t is
be due t:o
transcriptiona l and/orf;ostt.rans;~riptiorual events.
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121 85409-5
Capsule: Serum .from wco~nc~~rz with endomet~~osi~: induced MCP-1
secretion by L)937 monc_:~-tic cells and sknowed a significant
decrease i.n sI=L~-1RII i~:fr~~LL .
Abstract: The ability ~r:t 1~E~~r_Lphera_ k~>:Loc,d serum from women
with endometriosis t.o i.~lduce rnonocyte. c,hemotactic protein-1
(MCP-1) secretion by r«~orloca,~tes was assessed, as well as the
effects of recombinant ini.=E:rleukin-1.13 (rlh-113;; , IL-1 receptor
antagonist (rI:L-lira) , .-~rLd solublf~ I I_,-=1. receptor type II (rIL-
1RI I ) on MCP-I secret:i oru .
Design: Cultures of U9_>-% mc:~nocyti.c cell.:; exposed to serum
from normal or enciomet:c~_c:~si s worr~t-yn.
Setting: Gynecology ci in:Lc and human r_~eproducUion research
laboratory.
Patient (s) :79 women ha~,r:irlg endornetrios~s and. 38 normal. women
having no evidence of E.~r~~:Iomet=ric.~.~is at I aparo~copy.
Intervention (_.) : Peri.pha_c:a:rl Wood obt,~ainE-d a few days before
laparoscopy.
Main Outcome !Measure (s ~ ; MCP-1 secret=ion in t:lne culture
medium and serum c:,oncer~t~rations ~of sIL,-1R.II, IL-1f3 and IL-la
by enzyme-linked immun::~.~or_bent assay (EhISA) or by enzyme
immuncmetric .ssay (D1.%1j ..
Result (s) :Serum coracenr...~°ations of: sIL-IRII were
significantly
lower in women with en:~i~rnetriosi,~ staae~ I-II than in normal
women (P = 0.;X02), whereas those of I=L-1~ and IL-Ia were
comparable in women w:irh <:~rnd witluo;_~t: enciometr_ osis. Serum of
women with en~lometri.os i:_; v~nduced higher secretion of MCF~-1 by
U937 cells th~.n that o_L normal women ;P = 0.018),
particularly in the ira~.'t:i_al stagr~s oi= er~domet:~~iosis (stages
CA 02377786 2003-06-20
102 85409-5
I-II) (P = 0.002) , anc: -IL--11~IT sign:ifir_antly blocked that
secretion ( P =- 0 . .)008 ) .
Conclusions: 7.'hese f_-in~aings point t=c-~wa.-cd a deficiency in the
mechanisms involved ire the :~clwn-regu=iat~.c>n of iL-1 actions at
systemic leve7_ and rev.-'al :,:IL-1R~I as a key factor involved
in that process.
Key words: IL.--1, IL-1 rf,»::;<~~:,tor, ~:mdc:~rnet.z~iosi.s, MCP-l,
peripheral blood.
Introduction
Endomet:riosi;-~ is an irnrnune-related chronic
inflammatory disease, ~~:naracteri~ ed by the presence of
endometrial-like tissuf_: in ectopic locations, mainly i.n the
peritoneal cavity, and r,:~;~oc.~iatE~c~ with increased secretion of
proinflammatcry cytokiwes including IL-~, IL-6, IL-8, tumor
necrosis factor-a'.~pha 1'hl~dl~'-cx) an::l MCP-1 in the peritoneal
fluid (Senturk: L.M. e1. :I_, 1~~99; Mt.ala.y~_rn. N. e'_ al, 1999) .
These factors have bee~i postulated as being implicated in the
development and pr_ogre::~,:>:i_oru of thle d-._:>e~~se. Iznmuno-
inflammatory changes ok.:~~>~-~r_ved ir. patYent:s with endometriosis
are not restricted onl ~y to the per i.toneal cavity where
endometriotic lesions :~:f>_quently de~veio~~ (Senturk L.M. e:t al,
1999; Mulayim. N. et a:1.,, :1_999; Harada ~'. et al., 2001), bu.t
were also detected i.n i_he eutopic endometrium (Braun D.F. et
al, 1998; Sharpe-~himms H;.. L~. , 2001 ) ) , and the foer:ipheral blood
(Mathur S. P. , 2000; Drrn~_~c~~~~:i. W. P. , et a~_, 1.994 . . In
endometriosis, peritont~al macrophages are mare activates. and
secrete elevated conce:r~tuations of proinflammatory cytok:ines
(Mori H. et al, 1991; f;ana N. et al, 19~~6j . However, other
reports indicate that oervpheral bl.aod monocytes from women
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123 85409-5
with endometriosis ar~als~.a act i.v~,ted ('<eller ~T.M. et al,
1987; Braun D. P. et a:i , 1996) an:~ _,how '.he ability to
stimulate endometrial ~:c:ll growth ~r1 ~;~i~.:°-~~~, whereas monocytes
of normal feni_.ile women suppres:~ the pra~~ iferation of these
'.> cells (Braun I:~. P. et a::1., 1 994 ) , lfowever, the mechanisms
involved in t:he active l~ i on of t~hfJ~ a ~~e ins remain unknown.
IL--1, a maj< r pr<,i:~~flamrnat~:~ry cytokine, exerts its
biological ef~=ects viG the receptor t~lpe I ( IL-1RI ) , t=he
signaling functional receptor, which r~:~ expressed in
different cel_L types. ;~}.~e~~ific inhibition of= IL-I is ensured
by IL-lra which compete:. with IL-1 f~:~r >pecific binding to
IL-1RI, without triggei~:~.ng s.igna.L transducti_oi~ and cell
activation (Braun D. P. E-'~ al, 19'41 . I:G--I_RII, having no
signaling properties, has been reported as another natural
inhibitor of -_L-1 (Bor~~s~~hiD. et= al., 1~a96; Colotta F. et al,
1993) . It has been surgc:sted thar_ this ~ ecept~r, with a short
29-amino acid cytop-ias,~rui_:~ doznair:, tLmcW .ons as a non signal
transducting cell surf a:rce or soluh:lce '''c~eac:oy" target for IL-1
(Boraschi D. et al, IU~:~v; C:"olotta I~'. e~ al, 1993) . We have
previously der:ronstratei that I:L-RI:I presents defective
expression in the endc:-nf-:mum ofwomen with endometriosis
(Akoum A. et al, 2001~'~. Moreover, c»~:r recent findings :showed
a significant inverse c.,_,rrelat:ion between the decreased
expression of IL-:LRII -zu.:~ t_he irir.rc:a:~ec~ expression of MC:P-1
2~~ in the endomet:rium of wornert with er:domet:r iosis (Kharfi A. , et
al, 2001) . Alt:bough tme rnec:hanisrns i.mpa.~cated in the
regulation of IL-1. anc MCP-1. are cornple~., such an abnormal
expression of IL-1RII p~.at into promi.nenr:e a major defect: in
the mechanism: involve. in the c:ontro:l c~f lc>cal IL-I actions.
3f In ~Tiew of_ t h:~ numerous systemic i.mznuno-
inflammatory changes ck>:~e.rved i n endornetmviosis, the aim of
the present work was t:~ assess c-i_rc.u_L<~t~_ng levels of IL--1RII
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124 85409-5
and its liganc~ IL-1, Urn::A tc> ir;vestigate t:he putative r_o7_e of
this system in endomet.~~:io.sis-ass.~ci.a~ec~ peripheral blood
monocyte activation.
Material and Method:
Pat gents. Women were xecru:i.r:ec~ into the study after
they provided informed ~::onsezzt for a prc;tocol_ approved by
Saint-Fran~oi:~ d'Assis~.: hospital ethics committee on human
research. Endometriosi ~ ~,aa:=. ideritifieci during laparoscopy for
infertility and/o.r pelv~ icl pain c>r fo.r t~ubal ligation only.
1C The stage of endomet:r:i~::>~ ~s was de~t~~rrn:ined according to t:he
revised classificatior: a:~:l- i'he Amer;~can I'erti.l ity Society
(American fertility Sc~~:_~.ety, 1985) . Sub-~ects with
endometriosis (n -- ?9j ~:o~herwise had nc> ot:her_ pelvic
pathology and were not a,~k~nc~ any ar:t~:i-~ nfla.mmatory or
15 hormonal medication at aas t ~ rricmths be-fore iaparoscopy.
Control subjects (n -_ v,~,) wer_e fr.rt:ile women requesting tubal
ligation and having nr~ ;ri sirs=~e e~~~idence of endomE=triosi~; at
laparoscopy (Table 1.1). 'Che cycle phase (prol.iferative or
secretory) wa~~ determ:irud accord.i_ng t=<:> t:he patients' cycle
20 history and to the seri_an~ progesterone. '~'hirty three of
endometriosis women an,-:I i~? of contr-o=1. .<~ubject;~ were in the
proliferative phase of t:Ine menstrual cycle, whereas 46 of
endometriosis women an:~ ? 1 oj= cont.rul sub~ect.5 were in the
secretory pha~>e,
25 Table 11. Serum source arid clinica'1_ characteristics of
subjects at laparoscopy
Serum Source N~ambar of pati2nt:s A_ge (mean ~ SD)
Controls _ ~ 3c ~
Endometriosis ~i'? 3y ~_ =,
Stage I 34e 2 ~_
Stage II 2- 31
Stages I and II 6: jy ~- r-,
Stages III and IV 1r 3q ~ ._
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Co1_!ection arl~ .P3-oeessznc~ o.f .talooca Samples. B1_ood
samples were cYrawn a f~:~a da.ys befior_e :Laparoscopy in sterile
tubes containing etY~y.l~ur~c~diaminetetracetic acid and
immediately cE:ntrifuge~~~ are ~'OOOcr for 1.0 minutas at 4°C. The
serum was then rec.over~=d, :>eparated =into small aliquot:s and
stored at -8C°C urail ,a.:,~ay.
MCF--1 and Ii--_IR.II emyrne--1_inxec~ immanosorbent assay
(EhISA) . MCP-7. concentr.~zt:ions were measured using a
previously de:>cribed F;:fs_;:>A (Akoum A. et: al, 1996a) . This:
assay uses a mouse nuono:~orzal an'~i-hurn~zra MCf-i antibody (R &
D systems) anct a rabbi' Y:~oiyclon~rl ant:=~-human MCP-1 antibody.
This latter antibody dc:~c~:; not cross-react with several
cytokines that are clo;;eiy related to I~~IC:P--l, inc_Luding NICP-2,
MCP-3, IL-8, the prote:~.ru regulated on acts.vat_ion normal T
expressed and secreted RANTES) :end t:tze macrophage
inflammatory protein-1 cx and ~ (MIP-:'_cx and MIP-1~3) (Haehicha
M. et al, 199~~) .
sIL-1RII c:onc:ernt=rat:ior~:~ i_n serum were measured
using an ELIS.F, devel.opcJc~ irv t:he Laborat:cry. This assay uses
respectively ~. mouse m::~rioclonal anti--Y~urr~an IL--1RII antibody
(R & D system's) for cal:~t:~zre, a ac>at: polyclona_'~ anti-human IL-
1RII antibody (R & D svyst:erra) for det:ect.ion, peroxidase-
conjugated rah>bit ants.-clc><~t. immunoglok>ul.ins (2ymed
Laboratories, Inc. San Francisco, t:A) and 'TMB (3,3', 5,5',-
tetramethylbenzid:ine) 't;i.o-Rad L<~bor-at~e;ries Lt:d, Mississauga,
Ontario, Canada) as suk~~>I~r_ate fcr:v f:~er.oxidase. The optical
density (OD) r~~as deter!rined at 450 nm, and sIL-1RII
concentration's were ca.lcu~ated b°,~ int=er~~clation from the
standard curve.
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IL---ll~ a.nd I~~--la enzym<_ .immunornetr_ic: assay (EIA) .
The measurements were ,r.»~rformed ~.~:~ ing E iA cr~mmercial kit=s,
accordin to l~he manu~~~~tu~er :~ ir~_~ =ru~~-ion.;
g ~ r ' ~t <~ (Cayman Chemical
MI, USA).
Monc~cyte c,u~ .r.ure ar!d bic>lo~ic,3l assn-y. For these
studies, we h<~ve ,zsed ~.~ hyst.iocyr.ic: ~.~e 1i line (U937) . Cells
were culture <~t 37°C, '_~-" ''.G? in RPNCI medium supplement with
10o heat-inaci:ivated .fE~:,:~_L bovine :~e.rum fFB:.~) and 1%
antibiotics, and inc:ux~~~:_~d with L mM ::yv.ic adenosine
monophosphate (CAMP) (~~~..c~rna, St. l~cuis) 1-or 48 hours to induce
cell differentiation. ';ells were harv<.st-~ed by centrifugation,
then distribut=ed in 2~l-sa~~l1 culture plat-.es at 10~'
cells/ml/well- in fBS-tr:~E_c~ FtfMI medium :supplemented with
different serum dilut_ions ;5, 1Ci and '>0'~) , and incubated .in
duplicate for 24 hours at _,7''C. ~:u1tm:~e supernatants were then
collected and frozen ~~t -80"C: until. assay. Thc.: b_ological
assay was perf=ormed or! ~>>oo i ed sera f::rom norma 1 controls or
from women wi.t=h endomet::-iosi.s ac,.:e.rd:ing to endometriosi:~
stage (I, II, and III-_~'J) . An equal vo.Lume c;f serum was taken
from all the patients Inc_.luded iv each croup. The effect: of
serum-induced MCP-1 se : _ a ~i.on was also :studied in the
presence of human rIL-l-E~:L:I ('~ ug/ml) anc~ human rIL-lra 1;100
r~g/ml) (R & D systems, ~~!inneapo? is, MNi .
Statistical ~!:al,ys.is. Data are press=nted as mean ~
2~~ SEM. The unpa,'~red t test:: was used t=o compare the leve7_s of
MCP-1 secretion or cir.:a.o:lat._i.ng c:ytok:ine:in women with
endometriosis and conta-c1 :;object=s, ari;~ Bonf:err_oni correction
was applying ~=or multi>lE~ c~ornpaz::ison:~. ~-'o compare the effect
of one treatment on MCi-'--1 secretion, we used the paired t
3C~ test. Differences werF= :~on:~.idered a.~s statistically
significant for P valu:~;~ 0.05.
CA 02377786 2003-06-20
127 85409-5
Results
We first measured sIL-1RII ~~oncentrations in the
serum of nornr.al contr~~:ls and wor<~en with endorreetriosis stages
I-II and III-IV. Stat::st_ical analv~sis of the results with the
unpaired t test showed a significant de~.rease in the levels
of sIL-1RII in the enclometriosis c:~roup as compared to the
control grouch (P = 0. ~~~J ,~'E) . Furthermore, this decrease eaas
found to be :>:ignificar_t in the initial stages of
endometriosi:: (stages L-~II(P -- Ci.00~',' ;Figure 24) .
Statistical analysis c ~ our_ rata a.ccord:.~ng too the menstrual
cycle phase showed no si.gnific:ant difference in sIL-1.RI=
levels between they prc L i fer~ative phase and the secretory
phase, neithew in normal c<.>ntrol s, n~:~r_ .:.n endometriosis
stages I-II o~~ III-IV. 1-3owever., .AIL--RII 1_evels were
1_', significantly higher a.n endometriosi_~~ st:.ages I-II as compared
to normal controls boor in the pro-ii.ferat=ive phase (P =
0. 0184 ) and the secret:avy phase (P = 0 . ()261 ) .
We then measuoed the circu~_Lating :Levels of IL--1 in
its tow forms IL-la an~,t .I=~-1(3. Figure 25 shows the
2C distribution of IL-7.a ~=m<~ .CL--1.~'> ~:onc:enl=r:ations found in
normal and en:lometrios i.~~ women a~.:ccrdin.c) to t:he stage of the
disease. Statz.stical aiaa i.f~sis of data with the unpaired t.
test showed no signifi~:~ant~ difference r_n IL-l.cx or. IL-l.~i
concentration's between normal and endometrics:is women,
25 whether these latter were grouped t=oget:her (P = 0.955 and
0.667, respectively) o:c~ separated ~_nt~o ~ groups of
endometriosis stages I-7::C i,P - 0.7.x_0 and 0.706, respectively)
and III-IV ( P = 0 . 657 anc:~ 0 . 103, respect ivelyj .
Considering t:hE:e above results showing comparable
30 levels of cir~_ulating CI,-7. in normal and endometriosis women,
but reduced levels of ~I:~~-iRII i;v women having the disease,
we further assessed thc~ effect of- ~eri~>heral blood serum from
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128 85409-5
women with and w-,.thoui c=:ndometrio;>is on monocyte activation,
by measuring ~ICF-1 se~.: raetion by U~:~::~7 monocytic cells in
response to t:hese sera. As shown in Figure '?E, serum from
women with er~dometrio:iinduced a h.ighar secretion of MCP-1
by U937 cell: than thG:~~~ from nor.nal women (P = 0.018) .
Furthermore, this effc_.ct appeared to be significant in the
initial ( P = 0 . 002 ) ray t tner than in t he mc:~re advanced ( P =
0.238) stage: of the c:iaease.
Hurrc<~n rIL-1F:.I T (_':i ug/rnl ) was then added to each
pool of serum: prior tc:: incubatic~~n with 11937 cells. As shown
in Figure 2.7, rIL-1.RI:I ,~.igni ficant 1y intnibit:ed MCP-1
secretion by 1J937 cells in .responsE~ to c::ontrol and to
endometriosi~> worr~en-de r:i. ;reci sera . Howe~J<::r, t:he most
significant i.nh.ib:itior: ::.;f MCP-1 induced secretion was
observed in endom.etric: s i s stage I anc:~ I . ( P -- 0 . 0008 ) .
U93f~ cells weoe f~.rrthcer irucubat:ed for 24 hours at
37°C with each pool of ~:,~rum to which recombiruant human IL-lra
(100 ng/ml) w~~s added. ::-sir data ~~e~>icteci in Figure 28 show a
slight inhibit: ion of M~'P-- 1 secret ion induced by normal- as
2C well as by endometri.o~~..t> women-der:~ved ~aera. It is noteworthy
that IL-lra inhibitory effect was, to some extent, more
noticeable in serum fr:vm women with endometriosis stages I-
II, but no statistical.l.y significant difference between IL-
lra-treated and untreat~ee:~ sera was fount; (P = 0.101) .
Discussion
Although ret;:c:>cJrade menstr.uat=ion is the most
accepted theory for en :iornet.ri.osi, the pathogenesis of this
disease is po~~~rly undee~~toc>d. Growth factors ~rnd cytokines
that are secreted by a::t.a_~Tated immune cells have been
implicated in the control. of the implantation and growth of
endometrial cells outs:_c:le the uterine cavity, and in
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12,9 85409-5
endometriosis-associat:eci c:Linical symptoms (Senturk L.M. et
al, 199; Braun D. P. et. al, 1 994 ; ~lalme ~~ . et al, 1988 ) .
Cumulative evidences p:o;ant towards a relevant role
for IL-1. The concent~aat.ions of ~ruis c~yLokine were found to
.'> be increased :in the pf:. r:i t orue,~l ~: lu~.id ;>f women with
endometriosis,, and ap~::ea.red to affect women's fertility
(Fakih H. et al, 1987; Hill :~,~.A. e~ al, 1980; Taketani Y. et
al, 1992) , According t:o oux, previcr:~s wtrzdie:;, both ectopic
and eutopic endometria.'_ o~slls of women with endometrios~_s
show an increased sent=itivity to IL-1 i~: vitro and secrete
increased amounts of N~~:1~--1 in response to this cytokine
(Akoum A. et ail, 1.995x; Akc>urn A. e2_ a:1,, 1995b) . Eutopic
endometrial cells of wcarnen with endornel~riosis were further
shown to express low lc~~.%~J1;_, of IL~-:l.F;L_L, a receptor that acts
lc as a decoy fox' IL-1 an~:a L:irn:its I:h-:~-med~.ated ~~e11 activation,
which suggested a defica.enr:y in their ab:.~ lity 1=a down-regulate
IL-1 action (F~koum A. ~4t al, 200:Laa) . A soluble version for
IL-1RII (sIL-1.RIIha~~ k:SF~en ident.if:ied in the supernatants
from a number of diffe:reni~ cell types ;.'ymons J.A. et al,
1990; Giri J.~. et al, ~a90), ir: inflammatory synovial fluid
(Arend W. P, et al, 199~~ ) , and i.n hurr.an sverurn (Eastgate J.A.
et al, 1990).
Based on the:-a evidences, we rrueasured circulating
sIL-1RII in t~.e peripht~x~a~. blood of normal anc~ endometriosis
women, and fo~.nd a sigr~a_iicant decx°ease in its levels in
women having 'the ~;iisea:~~>. Furtherm_ore., IL-1RI:I levels were
significantly reduced :i.rr endometriosis stages I-II, and that
in both the p.roliferat:i.ve and the secretory phases of the
menstrual cyc.Le, which ~>r:ovi.de e~~~ic:~er~.ce fo:r a deficiency in
the regulation of IL-1 a~:tions at systemic lejrel in initial
endometriosis stages.
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130 85409-5
IL--1 exists ~.r°: 2 fc>rms (IL-la and IL-l.~i) . Both
forms of IL-_ trigger cell acti~~Taticn through the same
functional IL-1RI:, anti ir~tera~ct wits the decc:y IL-1RII
although with dii feretvt aff:initie;~ (Borasc:h_i D. et al, 1996) .
'~ We therefore assessed the levels c.>f I:L-icx anc. IL-l~ in the
peripheral blood and mound no sigr~ifican~ difference between
women with arid without endometriosis, wizether these were
grouped toget::zer or s~::(~az:ared :i.rotc> two ~r-oups according to
endometriosi~ stages I-II and III:-:IV) . ~~'hest=~ findings point
towards an unbalance in IL-1/Ii~-1RII c~ircula~ing levels in
endometriosis, which rnay result in increased cell reactivity,
and may account for tt;e reported act=ivaTvi.on of peripheral
blood monocyt:cps in e~nc.ometr.~.osis women iaelu.er J.M. et al,
1987; Braun I:'. P. et a:i , 1996) .
1_'i To ~rerify tt:i_. hypotheses, we first exposed U937
monocytic cells to sera from normal an:~ endometriosis women,
and estimated cell activation by measu.r~ng MCP-1 secretion by
these cells. Our :resu~t;~ ~~7,.ear.ly siuowed that: the U93 7
monocytic cel.=.s produced larger amounts of MCP-1 in response
to peripheral blood ser.;~ from women wish endometriosis. They
further revea~_ed that such a serum-i.nduc:ed h9CP-1 secretion
occurred in endometric;,,:Ls stages I-T::I, and that sera from
women with more advanco~ endomet:rios:i_s :>tages (III-IV) had no
significant ef=fect. These findings are ~_n keeping with our
2~ previous data showing ~ signifi.cant increase in MCP-1 levels
in the periphera:I blood ;~f endometriosis patients, which,
interestingly, was obs~.~.n~,Teci in init:i<~1 e-~r.dorne~Lriosis stages
(Akoum A. et al, 1996b)" T)'3e~~ also indicate the presence in
the peripheralblood of women wi;,h endometriosis of soluble
mediators that. are capaal:ula of ac t:ivating monoc:ytes and
stimulating MC:P-1 secrf~t~ion., and s~.aggest that such an
activation prc>cess is ;;iependent. on endometriosis stage.
However, while activat=:~c~ mcnocytes are known to increase MCP-
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131 85409-5
1 production (Leonard F; . ;1. et: a i , 1990 ) , it i s ~~till to be
determined whether pe:iphe.ra~_ blood monocytes secrete
elevated levels c~f MCI~-~ in endometriosi.s.
In orda;r to iY,vest=igate whether the reduced levels
of sIL-1RII c:~:~served '.i~: the perprlerai blood of women with
endometriosis may accc:unt for the increased reactivity of
U937 cells t<:~~aarr_is encl.::n,etr_ic>si:~-women .:.lc>rived sera, we added
human rIL-1RII to the different pools of serum prior to
incubation wivh U937 cells. Interestingly, we observed that
rIL-1RII sign::ificanr_1~ reduced L~CP-i ~~r~~duct=i.on by U937 cells
in response to serum 'rc:m normal and endometriosis women.
However, the most s igr it i cant decrease was observed in women
with endometz:iosis st~,de=s 1: and I1, whic:r~is in keeping with
our findings showing reduced levels of sIL-1RII in the
peripheral blood of trf-~:~e patients. 3ased on 'these findings
and the fact t=hat circ:c.z.lati.ng levels of IL-l.a and IL-1(3 were
comparable in women with,. and withcut endometriosis, it is
therefore tempting too Ey~pothesiae that t:he enhancement of
MCP-1 product~_on by mc:nc:cytes in response to endometriosis
2C~ women-derived sera i_s rn~;:re l:ikel.v duf-~ tca a dewrease in ~~IL-
1RII levels than to an increase i_n t.hc_~:~e of I~-1(3 or IL-la.
However, the mechanisrr,s underlying s-IL-:~RII decreased levels
in the peripheral bloo~:~ ~f women with endometriosis remain to
be further clearly elu~_;:ic~ated. Gn the other hand, although
IL-Ira appeared less eai~cient than rIL-1RII in inhibiting
serum-induced MCP-1 sea.:2:w~t.:ion in our -in vitro assay, there
was a noticeak>le tendency of inhibiticr~ in endometriosis
stages I-II, which rrcakE-~> plausib 1_e a def icienc:y :in the down-
regulati.on of IL-1 act:i_c~n in initial endcmetr:iosis stages of
the systemic level.
In ~;ummary, ,:w_zr f findings ir~dir.ate that serum of
endometriosis patient's w,:-zs able t_o i.rmluc:e MCP--1 secretion by
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132 .35409-5
monocytes and re~Teal v:to.r- IL~-lRTi as a keys fac:-tor involved in
that process. Monocyt~::- activation may play a significant role
in endometriosis pathaip-tnysievlog~,~ as these cells were shown to
stimulate endomet r:ial c:; E- 1.1 growt h and t ~:~ secr. ete numerous
proinflammatory cytok.in~-: s that ma~.~ nave a deleterious effect
on women's fertility. F,~rt~ermore, the reversal effect of
recombinant :AIL-1RII ~r: MCP-i production by monocytes may be
of a potential truerapE: u1 lc irntere:,>t .
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