Note: Descriptions are shown in the official language in which they were submitted.
WO9~/0l465 PCT/U~9~/0527~
~ia~797~
--1--
INTERLEUKIN 8 INHIBITION OF C~KINE-INDUCED HISTAMINE
RELEASE F~OM ~OSOPHILS OR MAST OE LLS
FUNDING: Development of the present invention was aided in
part by finding from NIH grant no. AI27864. Accordingly,
the F~deral Government may own certain rights.
Histamine releasing factors ~HRF) represent a group of
cytokines that release mediators from basophils and mast
cells. Our prior related applications, Serial No., 143,094
and its continuation, International Application No. WO
89/06545 (both of which are expressly incorporated herein
by reference), describe cytokines that inhi~it histamine
release induced with mononuclear cell-derived HRF. We ha~e
now discovered that recombinant interleu~in 8 (IL-8) is a
potent inhibitor o~ HRF-induced histamine release. ~ethods
for inhibi~ing HRF-induced release of allergic mediators by
exposing proallergic cells, such as mast cells and
basophils, to an effective concentration of IL-8 are
; claim~d.
30 . Basophils and mast cells have been the subjects of
scientific investiqation since they were described by Paul
Ehrlich in the 1870s. Mast cells are generally found in
connective tissue; ba~ophils, in the blood. The two cell
types have ~any similarities. Both contain numerous
; 35 metachromatically staining granules, ~oth have cell sur~ace
receptors that bind IgE with high affinity, and both
contain a myriad of diverse allergic mPdiators that can
~ause symptoms ranging from itching of the skin to the most
life-threatening clinical sit~ation, anaphylaxis.
Basophils and mast cells collectively account for virtually
all of the total body histamine and are collectively
referred to as histamine-containing proallergic cells.
,;~
- .
: : . : . .
~: ' , ~ .
.
W~92/01465 P~T/US91/05~7~
~ 7 ~ 2-
There is convincing evidence that mast cells and
basophils are essential for induction of allergic
hypersensitivity reactions. For example, substantial
evidence suggests that mast cells are th~ primary effector
~, ~ 5 cell of such allergic diseases as asthma, allergic
f rhinitis, conjunctivitis, urticaria and anaphylaxis.
; Increased numbers of mast cells have been found in
bronchoalveolar lavage fluid, respiratory mucosa, nasal
mucosa, and biopsy specimens of urticarial lesions in these
disorders. In addition, mast cell mediators, such as
histamine and prostaglandin (PG) D2, have been recovered
from serum, bronchoalvPolar lavage fluid, nasal washings,
and skin blister fluid during natural and prov~ked allergic
reactions. ~ore recently, the mas~ cell granule-specific
enzyme tryptase (not found in basophils~ has ~een detected
in serum from patients with allergic and anaphylactoid
reactions. Finally, most mast cell-derived mediators,
alone or in combination, can evoke such typical allergic
symptoms as bronchospasm, angioedema, cough, mucu~
secretion, rhinorrhea, sneezing, and wheal-and-flare skin
reactions and mast cell-specifio degranulating agents can
induce allergic reactions in vivo.
In addition, histologic evidence suggests that mast
cells are involved in the pathogenesis of a number of
chronic inflammatory diseases. Increased numbers of mast
cells have been detectsd in biopsy specimens ~rom patients
with rheumatoid arthritis, ulcerative colitis, Crohn's
disease, sarcoidosis, hypersensitivity pneumonitis,
pulmonary fibrosis, nasal polyps, atopic dermatitis,
allergic contact dermatitis, bullous pemphigoid, keloid
formation, scleroderma and progressive systemic sclerosis,
acute and chronic graft vs. host disease, and parasitic
` infestation. Mast cells also have been implicated in
regulation of nerve growth, and regulation of mast cell
degranulation has proved useful in neuxofibromatosis.
Furthermore, increased levels of histamine were detected in
; :, ~ . .
wos2/ol46s PCT/US91/05274
_3~ 7~
the bronchoalveolar lavage fluid from patients with
sarcoidosis, hypersensitivity pneumonitis, and idiopathic
pulmonary fibrosis. Cromolyn sodium, a putative mast cell
sta~ilizer, has been found beneficlal to a subgroup of
patients with ulcerative colitis, particularly those with
proctitis.
Basophils appear to be particularly involved in some
orms of allergic contact dermatitis, especially to poison
ivy and, experimentally, to dinitrochlorobenzene. These
cells represent 5% t~ 15~ of the total inf iltrating cells
and are present within eight hours after application of the
allergen to the skin. Given the number of inf lammatory
mediators released by mast cells and basophils, it is
conceivable that sustained piecemeal degranulation of these
cells contributes to the chronic inflammatory nature of the
aforementioned disorders.
Un~ortunately, despite the convincing evidence lin~ing
mast c~lls and basophils to these and other disease states,
the precise pathogenesis of mast cell and basophil
dependent disorders, . including allergic disease, is
incompletely understood. For example, it is known that
mast cells and basophils are stimulated to release
histamine, leukotrienes, and other inflammatory mediators
by the bridging of cell surface-bound IgE antibodi~s by
appropriate allergens (or anti-IgE antibodies), but ~he
severity o~ a number of allergic diseases, for example
bronchial asthma, rhinitis, and conjunctivitis, does not
correlate with a patient's IgE l~vel. Moreover, although
mast cells are believed to play a role in various other
diseases such as infla~matory bowel disease, xheumatoid
arthritis, pulmonary fibrosis, and sarcoidosis, in the
majority of these diseases, IgE antibody cannot be found.
:~ 35
Therefore, it appears that other mechanisms of
allergic mediator release play a critical role in
'
WO92/01~65 ~CT/US91/0S27~
~797~ -4- `
:
- pathoqenesis of allergic diseases and o~her disorders
mentioned above in which basophils and mast cells have been
, implicated. Elucidation of such mechanisms has been and
remains the goal of many skilled medical scientists.
One of the most exciting developments in this area was
the discov~ry of histamine releasing activity (HRA),
cytokines designated herein as histamine releasing
factor(s) (HRF), by investigators working in the
laboratories of the present inventors. Thueson, et al.,
(J. Immunol., 123:626 (1979); J. Immunol., 123:633 (1979))
and Lett-Brown, et al., ~Cell Immunol., 87:434 (1~84~; Cell
Immunol., 87:445 (1984)) first reported that antigen or
mitogen stimulated human mononuclear cells secre~e a
proteinaceous factor that induces release of histamine from
basophils and mast cells. Other laboratories then
confirmed the synthesis of HRF by mononuclear cells. It
has now been shown that HRF is also synthesized by B-
lymphocytes and T-lymphocytes, alveolar macrophages,
platelets, neutrophils, and blood monocytes cultured n
vitro. The wide variety of cell types report~d to secrete
~RF suggests that it has considerable ~iologic importance.
In addition to mediating histamine release, HRF has been
shown to induae secretion of leukotrienes and to be
chemotactic for basophils and monocytes. (For a review,
see Grant, et al., Fed. Proc., 45:2653 ~1986), J. Allerqx
Clin. Immunol , 77:407 (1986), and Alam, Insights in
Allergy, Vol. 2, no. 6 (1987), CV Mosby, St. Louis, all
incorporated herein by reference.)
Numerous studies provide data directly supporting the
importance of HRF as a mediator of human allergic disease.
For example, an HRF-like material has been obtained from
skin blister fluid obtained during the late aliergic
reaction, now considered an important factor in the
pathogene~is of chronic asthma and other allergic
conditions. HRF has also been recovered in nasal washings.
:, . - . . . .. . -
WO92J01465 2 ~ ~ 7 9 7 ~ PCT~US91/05274
-5-
In addition, HRF induces bronchoconstriction on inhalation
by asthmatic subjects and a wheal and-flare reaction in
humans and non-human primates. ~ononuclear cells from
asthmatic patients have been shown to spontanPously produce
relatively large amounts of HRF, and HRF production is
enhanced on in vitro incubation with specific allergen.
Moreover, the magnitude of spontaneous HRF production
correlates with the severity o~ bronchial hyperreactivity
in asthmatic patients [Alam, et al., J. Allerqy Clin.
Immunol., 79:103 (1987)]. These findings suggest that in
asthmatic patient6, increased spontaneous HRF production
may cause a sustained release of all~rgic mediators from
mast cells or basophils, resulting in chronic inflammation
an~ ultimately leading to the d~velopment of br~nchial
hyperreaotivity.
In addition, although immunotherapy is a well accepted
modality for treating allergic diseases, the mechanism of
its action is still obscure. Changes in serum IgE antibody
do not correlate well with the efficacy of immunotherapy,
Serum IgG blocking antibody usually rises after prolonged
treatment. Although some investigators have shown a
correlation between the efficacy of immunotherapy and the
level of IgG antibody, the correlation is often too tenuous
25 . to imply a casual relationship. Recently, however, one of
the present inventors has discovered that immunotherapy
abrogates the ~ea~onal rise in HRF production and
diminishes spontaneous HRF production in patients with
clinical improvements. Thus, there is a high correlation
between symptom-medication sc~re and spontaneous HRF
production (r=0.92, p=0.0002~.
:
Given the important role played by HRF in the
pathogenesis of allergic diseases and other disorders
including the release of mediators from basophils and mast
cells, it is likely that an agent capable of inhibiting
HRF-induced mediator release could provide a valuable tool
.,
WO92/01~65 PCT/US91/~5274
2~7~7~ 6
in treating mast cell/basophil dependent disorders, which
include the allergic diseases. Our prior patent
applications described a human histamine release inhibitory
factor having a molecular weight of about 8, 000 - 10, 000
daltons.-We have now determined that recombinant human
In~erleukin 8 (IL-8) (m.w. about 8,000 daltons) is an
extremely potent inhibitor of HRF~ duced histamine
release. HRIF and I~-8 specifically antagonize cytokine
mediated histamine release~
IL-8 is secreted as a 79 amino acid peptide which then
undergoes proteolytic cleavages yielding either a 77 or a
72 amino acid peptide both being active as neutrophil
activator/attractant. (T. Yoshimura et al.~ Proc. Natl.
Acad. Sci. USA 84:9233 (1987~; Leonard, E.J., and T.
Yoshimura, Am. J. Respir. Cell Mo. Biol. 2:479 (1990); M.
Baggiolini et al., J. Clin. Invest. 84:1045 (1989)~.
Leonard and Yoshimura have proposed the terms IL-8/N~P1
(neutrophil activatin~ peptide) alpha, beta and gamma for
the ~9, 77 and 72 amino acid peptides respectively (T.
Yoshimura et al., Proc. Natl. Acad. Sci. USA 84:9233
(1987)), and the generic term IL-8 is adopted herein as
covering the each of those species. I~-8 is produced by
many cells including monocytes, macrophages, lymphocytes,
endothelial ~ells, fibroblasts and keratinocytes.
Recombinan~ human I~-8 beta, a 77 ami~o acid peptide with
alanine in its N-terminus, was used in the studies
described below.
According to a general embodiment of the present
invention, there is provided a method for inhibiting HRF-
induced release of an allergic mediator from pro~allergic
cells comprising exposing the pro-allergic cells to
interleukin 8. In specific embodiments, the pro-allergic
cells comprise mast cells, or basophils, and the allergic
mediator is histamine. It is contemplated that the method
may be performed either in vitro or in vivo, preferably
:~ , i,
~ .- ; -
~WO92/0~65 2 ~ ~ 7 ~ ~ ~ PCT/US91/05274
!, 7
.,
;using human interleukin 8 and human pro-allergic cells. In
a preferred embodiment the interleuXin 8 is pre~ent at ~
concentration of at least about 10-~. Although either thè
79, 77 or 72 amino acid species of IL-8 ~also Xnown as
neutrophil activating p~ptide or leukocyte ~ adhesion
inhibitor) may be employed in accordance with the
invention, use of the 77 amino acid species is preferred,
as is use of a recombinantly produced IL-8. The invention
also includes a method for inhibiting release of allergic
mediators from pro-allergic cells comprising exposing the
pro-allergic cells to an effective concentration of
interleukin 8 to inhibit histamine release. In preferred
embodiments, histamine release may be i~hibited by at least
about 10 to at least about 60 percent, at least about 15 to
at least about 60 percent, and more preferably, at least
about 30 to at least about 60 percent, when measured
according to the protocol set forth here. These a~d other
aspects of the invention will become more apparent from a
description of particular embodiments when read in
~onjunction with the drawin~s.
FIGURE 1. Inhibition of HRF-induced histamine release
from basophils by IL-8. Leukocytes from 20 donors (ten
allergic and ten normal subjects) were preincubated with
various concentrations of IL-8 for 5 min and then
challenged with mononuclear cell-derived HRF. The percent
inhibition of histamine release from cells incubated with
IL-8 as compared to buffer is shown. Mean histamine
release by HRF was 50+g% for cells fro~ allergic
individuals and 38~7~ ~or cells from non-allergic
indi~iduals. The difference in the inhibition of histamine
release between the two groups was s~atistically
significant (*) at p~0.04.
FIGURE 2. Effect of IL-8 on histamine release from
basophils by MNC-HRF, anti-IgE, FMLP and C5a. Leukocytes
were preincubated with IL-8 (lO~M) for 5 min and then
.
.
, . . .
'' ~` ' '' " ' ' , '
:, :
WO 92J0146~ PCr~US91~05274
~ J7 ~ 8-
challenged with a predetermined dose of various
secretagogues. ~he number of experiments (N) is 20 for
MNC-HRF, 10 for anti-IgE, and 3 for FMLP and C5a. As~erisk
indicates statistical difference compared to control
release at p<0.04.
FIGURE 3. The requirement for preincubation of
leukocytes with IL-8 was investigated. Cells were
separately preincubated with buffer or with IL-8 ~or 5
minutes and then challenged with HRF. In another set of
experiments, IL-8 and HRF were added simultaneously to the
cells or HRF was preincubated with cells f irst and IL-8 was
added 5 min later. Results of one of three experiments are
shown.
~5
FIGURE 4. The effect of removal of IL-8 after
preincubation on the inhibition of histamine release.
A~ter the preincubation with IL-8, leukocytes were washed
3x with 30 volumes of ~uffer. The cells were then
challenged with HRF. In control experiments cells were
preincubated with buffer and then treated as above.
Results of one of three experiments are shown.
FIGURE S. IL-8-induced histamine release from
hasophils. Leukocytes from 20 donors were incubated with
various concentrations of IL-8 for 45 min and the released
histamine was measured. Spontaneous histamine release in
the presence of buffer was su~tracted. Basophils from 6 of
tventy donors (two normals and four allergic subjects)
responded to IL-8 beta. Results shown are mean+SEM of
histamine release by basophils from the six responder
donors.
FIGURE 6. The effect of neutrophil depletion on IL-8-
induced histamine release from basophils. Neutrophil-rich
and neutrophil-depleted leukocytes from three donors were
separated using sedimentation with hydroxyethyl starch and
. . ~ ,
.
:.. , ~ . , .: .
W092/01465 ~ ~ 8 ~ PCT/US91J0~274
, .
~. .
Ficoll-Hypaque gradient (sp. gr. 1. 077) centrifugation
respectively. The cells wPre then incubated with IL-8 and
the released his~amine was assayed.
FIGURE 7. The effect of preincubation with IL-8 on
Aubsequent I~-s-induced histamine release from leukocytes.
Leukocytes from four preselected allergic donors were
preincubated with different concentrations of IL-8 for 5
min and then challenged with IL-8 (1O~M). Error bars were
omitted for clarity (SD<1%).
FIGURE 8. The combined action of IL-3, IL-~ and GM-
CSF on basophils and comparison with MNC-HRF. Leukocytes
from 7 allergic donors were incubated with IL-3 (1 ug/ml
final concentration), IL 8 (~`~M) and GM-CSF (1 ug/ml) added
simultaneously to the cells. The histamine release by
these three cytokines was compared with that by NNC-HRF.
The histamine release by IL-3, IL-8 and GM-CSF were 30%, 7~
and 7% in SC, 15%, 6% and 12% in TW and 12%, 6%, and o% in
BW respectively. The release by the cytokines in other
donors was negligible.
EXAMPLE
Io M~te~iai~ and ~etbod
: 25 A. Reagent~.
RPMI 1640 was obtained ~rom GIBCO Laboratories, Grand
Island, NY; human serum albumin, glutamine, Ficoll,
Hypaque, Concanavalin A (Con A), penicillin, streptomycin,
FNLP and recombinant C5a from Sigma Chemicals Co., St.
Louis, MO; Hepes from Research organics, Inc., Cleveland,
OH; hydroxyethyl starch (HetaStarch) from American Mc~aw,
Ir~ine, C~; human recom~inant IL-3 and human recom~inant
endothelial IL-8 (77 amino acid peptide with alanine in its
N-terminus) was obtained from Pepro Tech. Inc., Rocky
Hills, NJ. A sample of IL3 (5xlO~U/mg) and GM-CSF
(1.7xlO7U/mg) were obtained from the Genetics Institute,
Boston, MA, courtesy of Dr. Steven Clark; rabbit and-human
W~92/0146~ P~T/U~9t/05274
~87~7~ -lo-
IgE serum (460,000 IU/ml) was from Behring Diagnostics,
Somerville, NY.
B. Generation of ~RF-containing supernatant.
Leukocytes were isolated frGm buffy coats obtained
from normal blood bank d~nors. ~NC were isolated by
Ficoll-Hypaque gradient centrifugation as previously
described (R. Alam et al., J. Clin. Invest. 82:2056 (1988))
and pulsed with Con A (25ug/ml in RPMI 1640 medium) ~or 4
hr, washed twice with Hanks' balanced salt solution,
resuspended in RPMI 1640 medium, and then cultured for 72
hr. Supernatants were harvested and concentrated 50x using
an Amicon ultrafiltration chamber with YM-5 filters ~MW
cut-off 5,000) and ultracentrifuged. Recovery of the
activity was approximately ~0-60~. The ultracentrifuged
supernatant was applied to a TSK 2000 gel filtration
column. ~he fractions co~taining HRF activity (15-40 KD)
were pooled, and aliquoted. The aliquots were frozen at -
70'C and used as a source of HRF. The concentration of
: 20 protein of this preparation was 8 ug/ml.
C. Isolation of peripheral blood leukocytes.
Blood donors were selected from a large group of
aller~ic and non-aller~ic subjects that were screened in
our laboratory for histamine release to HRF, anti-I~E . and
~Sa. Allergic status was defined by the presence of
clinical s~mptoms, past allPrgic history and positive
reaction to prick skin testing to a panel of local
aeroallergens (32 allergens).
Venous blood from donors was anticoagulated with 10 mM
EDTA and sedimented with 1.5% hydroxyethyl starch for 30
min at room temperature (R. Alam et al., J. Ciin. Invest.
~ 82:2056 tl988)). The leukocyte-rich buffy coat was
`: 35 collected and washed three times in HA buffer (Hepes
~uffered-saline, pH 7.4 and 0.03~ human serum albumin) in
: a refrigerated centrifuge (4 C) at 300 x g. The washed
. ~ :
: . .
. . ~ . ~ , . .
.. ~ . .~ - . .. . .. . . . .
~ ... . . . . .
Wvs2/0~46s P~T/US91~05274
2~797~
leukocytes were suspended in HACM buffer ~Hepes buffered-
saline, pH 7.4 0.03% human serum albumin, 2 mM CaCl2 and 1
mM MgC12)-
D. ~istamine release assay.
Aliquots of 50 ul of HRF, anti-IgE ~1:3,000 of the
stock solution, 460,000 IU/ml) or recombinant IL-~ (final
concentrations: 10-ll to lO~M) were incubated with 50 ul of
leukocyte suspension for 45 min in a shaking water bath at
lo 37 C. Each experiment was done in duplicate. Four hundred
microliters of HA buffer was added to each tube at the end
of the incubation. A~ter incubation the supernatants were
separated from the cells by centrifugation at 600 x g for
5 min at 4'C. The histamine content of the supernatants
was measured using an automated fluormetric analyzer (R.
Alam et al., ~. Clin. Invest. 82:2056 (1988)). Spontaneous
histamine release was assessed by incubating the cells in
HACN buf f er alone . The total histamine content of the
cells was measured by lysing the cells with 3% perchloric
~ 20 acid. The percentage of histamine release was calculated
: according to the formula: [(histamine in the
supernatant)xlOO]/(total histamine in ~he cells).
~,
Spon~aneous histamine release from the cells was
usually less than 5~. The values o~ spontaneous histamine
release were subtracted from the calculated histamine
release.
E. Histamine release inhibition assay.
For the inhibition assay, 50 ul aliquots of cells were
first incubated with various dilutions of IL-8 t10-l~ to la-
K) ~or 5 min at room temperature and then challenged with
50 ul of HRF, anti-IgE (1:3000 dilution), IL3 (1 ug/ml),
C5a (1 ug/ml) or FM1P (1:3000 dilution) separately (all
concentrations shown are final). The cells were then
further incubated for 45 min in a water bath at 37 C and
- the supernatants were separated by centrifugation.
. . .
.... .- . '
.
WO 92/0146~; 2 ~ ~ 7 9 1~ PCI`/US91/0~;274
--12--
Histamine.content of the supernatant and to~al cellular
content were determined as described above.
A typical experimental protocol includes:
Preincubation Challenge
a. leukocytes ~ buffer t buffer
b. leukocytes + buffer + HRF*
c. leukocytes + IL-8 ~ buffer
d. leukocytes + IL-8 t HRF*
~ or other secretagogues
The percentage of inhibltion of histamine release was
calculated according to the formula (R. Alam et al., J.
-:: Clin. Invest. 82:2056 (1988)):
[tb-a)](d-c)]xloo/(b-a)
Results are expressed as mean+SEM. Statistical
analyses were done with Wilcoxon's rank sum ~est.
II. De~on~tr~tion o~ hi~tamine raleas~ inhibit~nG activi~y
of I~-8
In order to assess the inhibitory actiYity of IL-8,
the effect of preincubation of leukocytes with the cytokine
on histamine release by MNC-HRF was studied on cell~ from
10 allergic and 10 non-allergic donors. IL-8 inhibited
HRF-induced histamine release from ~asophils obtained from
17 o~ 20 subjects (Fig. 1). Two of the non-inhibited
leukocyte samples were obtained from allergic donors; the
other was obtained from a non-allergic donor~ As shown in
Section III below, however, leukocytes from the two
allergic donors released low levels of hi~tamine upon
exposure to IL-8 alone, but cells from the normal donor did
:: not. A significant inhibitory activity (~15%) was apparen~
at 10-~M, although in some donors this was apparent at 10-1lM.
The inhibition of histamine release was significantly
higher in normal subjects than in allergic patients ! '~9+9%
vs 31+7%, p,0.04, Fig. 1) at lO~M. IL-8 does not affect
histamine release induced with anti-IgE, FMLP and C5a (Fig.
2).
,
.
. - ~. . . .
- . : -
. . . . .
W092/0~465 2 ~ 3 7 9 7 ~ PCT/USgl/05274
-13-
IL-3 also causes histamine release from a subgroup of
allergic patients. We have identified three allergic
patients that respond to IL-3 among 30 routine blood donor~
in our laboratory. The following studies were performed to
investigate whether IL-8 inhibited IL-3-induced histamine
from basophils. Basophils from all three donors were
studied for the inhibition of IL-3-induced histamine
release by I~-8. IL-8 inhibited IL-3-induced histamine
release from two donors. The release by IL-3 (1 ug/ml)
from leukocytes preincubated with buffer were 40~1% and
15+0.5% from donor 1 and donor 2 respectively. When
leukocytes were preincubated with IL-8 (10-~lO~M), the
release of by IL-3 were 31+1% and 7~0.3% respectively at
the highest concentration of IL-8. The third donor did no~
show any inhibition. This particular ~onor is a possible
non-responder to IL-8 since there was no inhibition of HRF-
induced histamine release by IL-8 from this donor, either.
The requirem~nt of preincubation of basophils with
IL-8 for optimal inhibition of histamine release was also
investigated. In our in vitro system, IL-8 did not show
any inhibitory activity when added to the cells
simultaneously with MNC-HRF or added five min after MNC-HRF
(Fig. 3). In order to determine whether continued presence
of IL-8 is necessary after preincubation with the cells,
leukocytes were preincubated with IL-8 for five minutes,
washed 3 times with 30 volumes of buffer, and then
challenged with MNC-HRF. As shown in Figure 4, that
procedure did not abrogate the inhibitory effect of IL-8.
Thus, these results have established that IL-8 acts as
a strong inhibitor of cytokine-induced histamine release at
concentrations as low as 10-9-lO~M.
III. 8tu~ Qn-hi~tamine relea~in~ ~ctivity of IL-~
The results reported above are surprising in view of
reports from other investigators ~M.V. White et al.,
W092/01465 2 ~ ~ 7 ~ 7 ~ PC~IUS9~/05274
-14-
I~munol. Lett. 22:151 (1989); C.A. Dahinden et al., J. Exp.
Med. 170:1787 (lsæ9)) who have ~oncluded that IL-8 is
c~pable of inducing or potentiating histamine release from
pro-allergic cells. Therefore, we investigated the
S histamine releasing activity of IL-8 using basophils from
the same 20 donors described above. Using a concentration
range of 10l1 to lo~, we found that IL-8 released histamine
from basophils obtained from two of ten non-alleryic donors
and f~ur of ten allergic donors only at the highest
concentration tested (lO~M), which was 100 to lO00 ~old
higher than the concentrations at which histamine release
inhibition was observedO The histamine release ranged ~rom
6-16% and the mean was 8.7+0.8~ (Fig. 5). The experiment
was repeated on three responder donors on two separate
occa~ions, and the results were reproduced. By incrPasing
the concentration of IL-8 up to 3 x lO~M, were able to
observe a small increase in histamine relaase in the range
of 15-20%. By comparison, anti Ig~ and ~NC-HRF released
34+7% and 51+7% of histamine respectively from the same
donors.
Neutrophils have high affinity receptors for IL-8
(also described as neutrophil activating peptide 1) (T.
Yoshimura et al., Proc. Natl~ Acad. Sci. USA 84:9233
: 25 (1987)). Since our leukocyte preparation contained
neutrophils, we postulated that the low histamine release
~rom basophils by IL-8 might be due to the avid binding of
the cytokine to neutrophils. Therefore we have compared
IL-8 activity using neutrophil-depleted mononuclear cell
preparations that contained 2~3% of basophils with
leukocyte preparations containing approxim~tely 60%
neutrophils. Neutrophil-depleted mononuclear cells (less
~han 3% neutrophils) were purified by Ficoll-Hypaque
gradient (sp. gr. 1.077). As shown in Figure 6,
simultaneous experiments done with neutrophils-depleted and
neutrophil-rich preparations did not show any difference in
histamine release.
, . ~ ~, .
,
,
' ~ . ~ ' : '
: , :
WO92/01465 2 a 3 7 9 7 9 PCT~IJS91/05Z74
-15-
Since IL-8 did release a low amount of histamine from
cells of selected donors, we performed the following
experiment in order to determine whether inhibition of HRF-
induced histamine release by IL-8 could be due to specific
desensitization (down regulation of receptors). For that
experiment, leukocytes obtained from selected IL-8
responder donors were preincubated with various
concentrations of I~-8 and then challenged with lO~M of
IL-8. Results of four experiments are shown in Fig. 7.
Although a moderate desensitization was observed in two
donors at lower concentrations of IL-8, results were
equivocal in the other two subjects. Even among the
responders, however, very low histamine release was
obtained upon ~xposure of the cells to IL-8, thus,
complicating the interpretation of the result.
In other experiments, we studied the priming effect of
IL-3 on IL-8-induced histamine release described by
Dahinden et al (J. Exp. Med. 170:1787 (1989)). Leukocytes
from 7 donors, six who did not respond to IL-8 and one who
did, were preincubated with IL-3 (25 ng/~l) for 5 min, and
then challenged with IL-8 at concentrations 109 to lO~M. No
histamine release was observed with cells from the six non-
respdnder donors. Cells Prom the one responder donor
demonstrated a synergistic effect of IL-3 and IL-8. The
release by IL-8 was 5+0.1% and 8~0.1% at 10'7 and lO~M
concentrations respectively. IL-3 alone (25 ng/ml)
released 8+0.2% of histamine. Preincubation with IL 3
followed by incubation with IL-8 cause 20+0.5% and 32~1% of
~` 30 histamine release respectively. This particular allergic
donor is highest "releaser" of histamine among our donors,
and he is also one of few donors who responds to both IL-3
and IL-8.
We have demonstrated that IL-3 and GM~CSF release
histamine from basophils of selected donors at high
concentrations (l ug/ml) (R. Alam et al., J. Immunol.
, ~
.,
'.` - , . . '
WO 92~0146~ 2 ~ ~ 7 ~J 7 ~ P~/IJ!~;9l/0~274
--16--
142:3431 t1989)). We, therefore, asked whether the
combined action of IL-3, IL-8 and GM-CSF would mimic the
activity of mononuclear cell-derived HRF in cells from
those donors. Seven patients were studied using relatively
high concPntrations of IL-3 (final concentration of
ug/ml), IL-8(10~M) and GM-CSF (1 ug/ml). Three alleryic
patients relea~ed significant amounts of histamine,
although less than MNC-derived HRF. Four other allergic
subjects did not respond to IL-3, IL-8 and GM-CSF, but did
release histamine in response t~ MNC-derived ~RF (Fig. 8).
we did not perform any experiments with basophils from
healthy controls since their leukocytes, in general, do not
respond to IL-3, GM-CSF ( 2 ) and IL-8 as shown above . In
contr~st, MNC-derived HRF released histamine from basophils
obtained from most normal donors.
CLINICAL APPLICATIONS
Due to precautions necessarily attendant to
development of every new pharmaceutical, the present
invention has not yet been tested in a clinical setting in
human subjects. Therefore, the in vitro activity of
Interleukin 8 in inhibiting histamine release has been used
to demonstrate the utility of the present invention as a
pharmacologic agent since the histamine release assay is
accepted by those of skill in the art as a reliable
correlate ~f n vivo histamine release. The following
prophatic embodiments represent the best mode contemplated
by the present inventors for carrying out the practice of
the invention in various clinical settings.
First, it is believed that the Interleukin 8 will
prove to be useful in treating numerous diseases in which
mast cells and basophils are involved, especially the
allergic disorders. In particular, these in~lude, but are
not limited to, bronchial asthma, allergic rhinitis,
conjunctivitis, and urticaria. Although the best mode of
administering the factor will depend on the particular
: .,
. - .: ,
. -. ... . .
W092/02465 2 ~ ~ ~ 9 7 9 PCT/US91/0~274
-~7-
clinical situation, it is believed that the factor may bP
most easily administPred by formulating it together with a
suitable pharmaceutical excipient and administering the
formulation topically. For example, the factor could be
formulated as a component of an aerosol for intranasal or
intrabronchial administration. These delivery devices
might be modified to be powered by freon. This mode of
administration may be particularly useful in treating
certain allergic diseases, for example, allergic rhinitis
and bronchial asthma. The factor could also be
administered topically to the skin or eye; this foxmulation
might prove effective for allergic disorders at these
sites. Alternatively, the factor could be formulated for
intravenous, intramuscular, subcutaneous, intradermal, or
intraarticular injection; such injections might be used to
treat inflammatory reactions at these sites in which the
triggering of mediator release from basophils and mast
cells is involved in the pathogenesis of the illness. In
all these formulations, suitable excipients, for example,
saline or physiologic buffers, are known to those of skill
in the art and may be used. Of course, in some cases, it
may be desirable to incorporate a preservative into this
excipient. Methods for incorporating therapeutic agents
into pharmaceutical vehicles are believed to be well within
; 25 the skill of the art.
As stated above, the invention has not yet been used
in clinical settings. The inventors have relied upon the
published results with other cytokines in predicting the
acceptable pharmaceutical dosage for Interleukin 8. The
most relevant studies of an aerosol were the use o~
intranasal alpha 2-interferon to prevent viral upper
respiratory infections. Douglas, et al., (New Enal. J.
~ed., 314:65 (1986)) and Hayden, et al., (New Enal. J.
Med., 314:71 (1986)) administered 5 X 106 international
units ~IU) per day for an effective response. This
cytokine is currently licensed for treatment of hairy cell
:
' `
W~92/01465 PCT/US91/~5274
2a~7979-l8
leukemia at a dose of 3 X 106 IU per day by intramuscular or
subcutaneous administration. Nathan, et al., (New Engl. J.
~, 315:6 (1986)) administered 20,000 to 200,000 U of
interferon-gamma intradermally to persons with lepromatous
leprosy ~or a ther~peutic response. The development and
clinical use o~ interferons has recently been reviewed by
Baron, et al., ~he Interferon System: A Current Review to
1987, University of Texas Press, Austin. As reported in
New Enal. J. Med., 31~:1485 (1985), interleukin-2 has been
administered intravPnously to patients with cancer at doses
: o~ 104 to 105 units per kg over eight hours and maximal dose
of up to 3.3 X 106 per kg. Finally, Vakhan-Raj, et al.,
(New Enal. J. Med., 317:1545 (1987)) recently injected G/M
CSF by continuous infusion at doses of 1.5 to 25 X 106
1~ units/M2 of body surface for an effective response in
; :~ patients with myelodysplasia. Therefore, the inventors
would propose that the effective dose of Interleukin 8 will
be from about 104 to about 107 units. Furthermore, the
: inventors would define a unit in the traditional manner:
20 1 unit causes 50% inhibition of near maximal histamine
release from HRF-stimulated basophils. The exact doses of
r ~ " Interleukin 8 to be used in a particular clinical
application must be determined by accepted pharmaceutical
. ~ethods ~nown to those ~killed in the pharmaceutical arts.
,` 25
* * * * * * * * ~
The ~oragoing description of the invention has been
directed to particular preferred embodiments in accordance
with the requirements of the patent statutes and for
purposes of explanation and illustration. It will be
apparent, however, to those skilled in the art that many
modifications and changes may be made without departing
from the scope and the spirit of the invention.
REFERENCES
This application contains a number of references which
may ~acilitate understanding or practice of certain aspects
~: :
WO 92/01465 2 0 ~ 7 9 7 3 PCr/US91/05274
--19--
of the present invention. I~clusis~ o$ a re~r~nce in thi~
applic~tioal i~ not inten~l3d to ~nd doe~ not con~titut0 ~
admi3sion that such reS~re~ace rep~e~t~ prior art with
r~sp~-t to th~ pr~s~nt i~v~ntion.
- 5
.
"
..
:
~ . .
,:
; ~ . ..
