Note: Descriptions are shown in the official language in which they were submitted.
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USE OF AN OLIGOSACCHAR~DE AS AN ~MMUNOMODULAl~R ~N A DERMATO-COS~T[C
COMPOSmON
The present invention relates to compounds useful in the
field of ~ lnology and in particular for the treatment of
hypersensitivity reactions responsible for allergies and
various intolerance phenomena.
Immunological defence against infectious agents, toxins or
neoplasms operates via the recognition of the foreign nature of
an antigen (or allergen) and the activation of cellular or
humoral mediators aiming to eliminate the foreign antigen.
However, this mechani6m can sometimes be undesirable, when it
is too intense, or takes place on encountering environmental
antigens which are not intrinsically noxious; this is likewise
the case during organ transplantation or tissue grafts.
Immunological reactions at the root of allergy have been
classified into 4 types:
- Type I:The mast cells are bound to IgE antibodies by their Fc
receptor; the fixing of the antigen triggers the degranulation
of the mast cells and the liberation of mediators (histamine,
SRS-A, ECF-A).
- Type II:The specific antibodies (IgG or IgM) react with the
antigen on the surface of target cells; this causes cytolysis,
either by direct action of the K cells, or by activation of the
complement.
- Type III:The antibodies (IgG or Ig~) form, with the antigen
and the complement, immune complexes which are deposited in the
tissues and cause the production of chemotactic factor6 for
polynuclear neutrophils; local inflammation results.
_ Type IV:The T lymphocytes sensitized to the antigen react
with this and liberate lymphokines. The lymphokines induce an
inflammatory reaction and cause the influx of macrophage6.
The lymphocytes are thus key cells in ;mmllnlty with
cellular or humoral mediation, especially with the secretion of
antibodies.
Allergic ailments can be the expression of one or more
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types of immunological reaction~.
These reactions can likewise be at the root of intolerance
reactions to hygiene and care products, observed in skins said
to be "hyperreactive".
These phenomena are due to intrinsic genetic factors and
to acquired hyper~ensitivity caused by the environment.
Unexpectedly, the applicant has now found that these
hypersensitivity phenomena could be controlled by
oligosaccharides comprising from 2 to 6 oside residues, and
having a galactose residue in the non-reducing terminal
position, or derivatives of such oligosaccharides substituted
by a hydrophobic residue.
The present invention relates to the use of
oligosaccharides comprising from 2 to 6 oside residues and
having a galactose residue in the non-reducing te ;nal
po~ition, or of their derivatives, for the preparation of
immunomodulator compo6itions, and in particular of compositions
intended for the treatment or prevention of hypersensitivity
reactions.
Particularly appropriate oligosaccharides could be chosen
from the group formed by melibiose, lactoRe and their
derivatives capable of being obtained by addition of a
hydrophobic residue.
Hydrophobic substituents are especially understood as
meaning linear or branched C1-C1g alkyls, C1-C1g alkylamines,
linear or branched, optionally subQtituted Cl-C1g carboxylic
acids, linear or branched, primary, secondary or tertiary C1-
C1g amide~, and C1-C1g arylalkyls.
The oligosaccharide derivatives suited for carrying out
the invention can e~pecially belong to one of the categories
mentioned below, in which the oligosaccharide corresponds to
the following general formula:
galactose-n (a or ~) - (Hex)p
in which
n represents the position 1, 2, 3, 4 or 6,
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Hex represents an a- or ~-linked hexose or pentose,
p is a number between l and 5;
a) - glycosides corresponding to the formulae:
(I) oligosaccharide l-O-R, in which R i8 a linear or
branched alkyl residue of l to 18 carbon atoms,
~ (II) oligosaccharide l-O-R-O-l-oligosaccharide in which
R = (CH2)m, m being between 2 and lO,
b) - an osylamine acylated according to one of the following
formulae, in which the oligosaccharide is preferably lacto~e,
melibiose or stachiose:
- osylamines acylated corresponding to one of the following
formulae:
~ (III) oligosaccharide l-NH-CO-R, in which
R is an alkyl residue of 2 to 18 carbon atoms, containing
0, l or 2 double bonds,
~ (IV) oligofiaccharide l-NH-CO-R-CO-NH-l-oligosaccharide,
in which
R = (CH2)m, m being between 2 and 8,
c) - an alkylamine acylated by an aldonic acid obtained by
oxidation of an oligosaccharide
~ (V) oligosaccharide -CO-NH-R, in which
R has the same meaning as in formula (III),
~ (VI) oligosaccharide CO-NH-R-NH-CO-oligosaccharide,
where R has the same e~ning as in formula (III),
2~ d) - or a reduction product of Schiff bases formed by
oligo6accharides with aliphatic mono- or diamines, and
corresponding to one of the following formulae:
~ (VII) Gal-(Hex)n-X-HN-R,
~ (VIII) Gal-(Hex)n-X-HN-R-NH-X-(Hex)n-Gal,
in which:
Hex is a hexose or a pentose,
n = 0, l or 2,
X = l-NH2-hexitol, and
R has the same meaning as in (III).
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According to a particularly advantageous method, the
oligosaccharide or its derivative such as defined above will be
used to prepare a composition containing, in addition,
pharmaceutically acceptable excipients suited to administration
by the external topical route.
In fact, the applicant has found that the reactions
involved in the disorder6 associated with hyper6ensitivity can
be correlated with the fixation, on a 6pecific lymphocyte
receptor, of peptides re6ulting from the degradation of
elastin, especially kappa-elastin.
The activation of these receptors triggers the liberation
of lytic enzymes, ~-glucuronidase, elastases and free radicals
such a6 the 6uperoxide ion, or hydrogen peroxide, which
generates hydroxyl radical6. These products can involve the
degradation of macromolecules of the extracellular matrix,
fibronectin, collagen and hyaluronan. The fixing of the elastin
peptides likewise stimulates the proliferation of lymphocytes.
These phenomena play an important part in the
immunoallergic reactions of atopic origin, especially of atopic
dermatitides or anaphylactic reactions, urticaria and allergic
contact dermatitides.
The subjects having skins said to be "sensitive" or
hyperreactive are more and more numerous in the population.
Such a skin reddens or smarts easily, and its reactivity
threshold is lowered with respect to other skins.
The subject experiences cutaneous discomfort, associated
with erythema, and a fine de6quamation can take place.
All these symptoms will be ameliorated or even suppressed
by the use of compositions cont~;n;ng oligosaccharides
according to the invention; melibiose, lactose, or their
derivatives, partially or totally suppress cellular
proliferation, in particular of the previously stimulated
lymphocytes; they can li~ewise inhibit the potentialization and
liberation of lytic enzymes.
The compositions will preferably be formulated in vehicle6
not comprising any perfume or allergenic agents. The
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compositions could be in the form of solutions, gels, lotions,
creams, W/0 or 0/W emulsions, or multiple emulsions or in
liposomal form. They will be adapted by the person skilled in
the art, preferably using emollients or mild surfactants.
The compositions are particularly 6uited to the treatment
or to the prevention of intolerance and/or allergic reactions
of the 6kin and/or of the mucous membranes.
In particular, the compositions according to the invention are
intended to prevent or to decrease the formation of free
radicals.
The oligosaccharides or their derivative6 could be a6sociated
with other agents enabling the skin to be protected and/or
hydrated, such as hyaluronic acid, vitamin E, glycol extract of
G. biloba, 60rbitol, glucosaminoglycans, alginates, etc.; they
can likewise be a6sociated with vegetable oils to nourish the
skin and/or with active emollient6 and salves 6uch as, for
example, extracts of cat grape, althaea, oats, linden,
camomile, sweet clover, Ruscus, procyanidols, a-bi6abolol,
coconut oil, 18-~-glycyrrhetinic acid.
According to another aspect of the invention, the
oligo6accharides and their derivatives such as defined above
are used for the preparation of a compo6ition which
additionally contains pharm~ceutically acceptable excipients,
suited for administration by the parenteral or enteral route.
The efficacy of the oligosaccharides according to the
invention is based on their surprising activity on cell-
mediated ;mm--ne reactions.
The compositions prepared according to the invention are
particularly useful for the treatment or prevention of
hypersensitivity reactions mediated by lymphocytes.
They are particularly intended for the treatment or for
the prevention of symptoms of an ailment chosen from amongst
atopy, polymorphous erythemae, xerodermititides, lupus
erythematosus, pemphigus, dermatitides, psoriasis and eczema6.
The oligosaccharides having a galactose residue in the
non-reducing tPrm;n~l position, and their derivatives
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substituted by a hydrophobic residue, can be utilized as
adjuvants limiting the hypersensitivity reactions capable of
being provoked by another active principle.
The invention likewise relates to a method of co6metic
S treatment of hyperreactive skins, characterized in that a
composition containing at least one oligosaccharide comprising
from 2 to 6 oside re6idues, and having a galactose residue in
the non-reducing terminal position, or a derivative of such an
oligosaccharide substituted by a hydrophobic residue, is
applied by the topical route in a cosmetically acceptable
vehicle.
Particularly preferred compounds for putting this method
into practice are melibiose, lactose and their derivatives
capable of being obtained by addition of a hydrophobic residue.
The examples which follow are intended to illu~trate the
invention.
In these examples, reference will be made to the following
figures:
Fig. l:Inhibition by lactose and melibiose of the activity of
stimulation of lymphocyte proliferation by ~-elastin. The
concentrations of lactose and of melibiose are l ~g/ml, lO
~g/ml, lO0 ~g/ml, l mg/ml and 2 mg/ml.
Fig. 2:Inhibition by lactose and melibio~e of the expre~ion of
elastic activity by lymphocyte~, stimulated with 2 ~g/ml of ~-
elastin. Activity in the culture medium.
Fig. 3:Inhibition by lactose and melibiose of the expression of
cathepsin G activity of lymphocytes stimulated with 2 ~g/ml of
K-elastin. Activity in the culture medium.
Example l
l - Methods
SeParation of lymPhocYtes
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The lymphocytes used for these experiments were
obtained starting from circulating human blood and
likewise from human tonsil after tonsillectomy. The
isolation of the peripheral lymphocytes is carried out as
follows: 5 ml of blood were deposited (in 15 ml
centrifugation tubes) on Ficoll-paque plus (Pharmacia)
- with care before separating the two phases, then centri-
fuged at 3000 rpm (say 600 g) for 40 min at ambient
temperature (20~C). The layer containing the lymphocytes
and the monocytes is recovered and mixed with 10 ml of
RPMI medium and centrifuged at 2200 rpm (say 400 g) for
10 min at 20~C. The sediment is resuspended in 0.5 ml of
RPMI, then an additional 4.5 ml of RPMI are added and a
new centrifugation is carried out at 1500 rpm (say 400 g)
for 10 min at 20~C. The final se~im~nt is taken up in 10
ml of RPMI and the cells are counted. The monocytes and
macrophages are eliminated by adhesion on a plastic
surface (incubation for 2 hours in a CO2/O2 incubator).
- SeParation of the PolYnuclear cells (PMN)
After the first centrifugation at 3000 rpm (say
600 g), the residue cont~in;ng the red corpuscles and the
PMNs are resuspended in 1% polyvinyl alcohol (PVA) in
DPBS (Dulbecco's modified phosphate-buffered saline),
(two volumes per volume of sediment), then allowed to
sediment for 20 min at ambient temperature. The
supernatant is then centrifuged for 5 min at 400 g at
4~C, the sediment i~ washed with DPBS in order to
eliminate the PVA by gentle agitation, without activating
the PMNs, followed by centrifugation at 400 g at 4~C. The
supernatant is eliminated. The red corpuscles are lysed
by osmotic ~hock then an excess of DPBS is added in order
to reestablish osmotic equilibrium. After centrifugation
for 5 min at 400 g at 4~C, the sediment, containing the
PMNs, is taken up in a small volume of RPMI. The cells
are counted and can be lysed to liberate the lytic
enzymes (elastase, cathepsin) by addition of 0.1% Triton
X-100 in 1 M NaCl at 0~C for 20 min, followed by
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centrifugation for 20 min at 0~C. The supernatant con-
tains the enzymes.
Preparation of lYmPhocytes startinq from human tonsil~
Freshly remo~ed tonsils are cut into small pieces
under sterile conditionR, in 10 ml of ~PMI. The tissue
suspension is filtered on a coarse filter in order to
remove the largest tissue debris. The suspension i8
placed in a test tube and allowed to s~im~nt for 15 min
at A~hient temperature. The supernatant is placed on
Ficoll-paque plus and centrifuged as described for
lymphocytes derived from blood. The monocytes are elimin-
ated as described for the lymphocytes derived from blood.
The sediment containing the lymphocytes is re~uspended
and the cells are counted after two centrifugations as
described. The lymphocytes obtained starting from tonsils
are approximately 50% of the type T and 50% of the type
B. The lymphocytes of blood origin are approximately 80~o
of type T and 20% of type B.
Culture of the lYmphocytes
The lymphocytes isolated as described a~ove are
cultured in 24-well Costar plates, 500 ~1 of cell
suspension/well (5 x 105 cells/ml or 2.5 x 105 cells) in
RPMI 1640 medium (Eurobio) made up with 10% foetal calf
serum (ATGC), 2 mM glutamine (Gibco), penicillin and
streptomycin (500 ~/ml, 0.25 mg/ml), and phyto-
haemagglutinin (SIGMA) 5 ~g/ml. After incubation for 4
days under cell culture conditions (37~C, CO2/O2
incubator) the cell suspension is recovered with a
pipette, centrifuged at 400 g for 10 min at 20~C and the
cell sediment is taken up in 0.5 ml of RPMI (without
FCS), dispersed by agitation and then after addition of
10 ml of RPMI without FCS, centrifuged again at 1500 rpm
(say 400 g) for 10 min at 20~C. After two more washings
as described above, the sediment is taken up in 10 ml of
RPMI without FCS and the cells are again cultured in a
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g
24-well Costar plate.
Action of the elastin peptides
A sterile solution of K-elastin (75 kD, Solabia)
is added at the concentration indicated (for example
2 ~g/ml) to cells put back into culture in the medium
without FCS for 2.5 hours at 37~C. After incubation, the
plates are gently agitated, and the cells are recovered
~y pipetting and counted. The suspension is centrifuyed
for 10 min at 4~C at 1500 rpm (say 400 g), and the
supernatant is distributed in 1 ml Eppendorf tubes at a
rate of 500 ~1 per tube and kept at -40~C if it is not
u6ed immediately for enzymatic determinations. The cell
sediment is resuspended in extraction buffer (0.1% Triton
X-100, 1 M NaCl, 0.02% NaN3 0.01% 8rij 35, pH 8). 1 ml
per 106 cells, agitated for 15 min at 1600 g at 0~C and
the supernatant obtained by centrifugation as described
above is redistributed in Eppendorf tubes and kept at -
40~C for the enzymatic det~r~;n~tions.
Determination of the enzymatic activity of leucocyte
elastase-type
.
The synthetic substrate used for determining the
enzymatic activity of elastase-type i~ Me-O-Suc-Ala-
Ala-Pro-Val-pNA. 50 ~1 of culture medium or 20 ~1 of cell
lysate are mixed with the buffer (100 mM tris-HCl, 0.05%
CaCl2, 0.02% NaN3, 0.01% Brij 35, pH 8) up to 190 ~1, then
with 10 ~1 of 85 mM substrate solution (in N-methylpyr-
rolidone) for a total volume of 200 ~1. The optical
density is read immediately at 410 nm and then after 4,
24, 48 and 72 hours' incubation at 37~C. The elastase
activity i8 expre6sed in nM of substrate hydrolysed per
106 cells and per hour.
Determination of the enzymatic activitY of cathepsin G
25 mg of the su~strate, Me O-Suc-Ala-Ala-Pro-Met-
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pNA in 1 ml of N-methylpyrrolidone (40 nM) are used under
the same conditions as above.
2 - Re~ults
A) Effect of elastin peptides on the proliferation
of lymphocytes
The passages of calcium produced by the addition
of elastin peptides to the white cells of the blood can
be considered as an indication of the intracellular
signal triggering a set of cellular functions. One of
these i8 the entry of cells into proliferation. It ha6
been shown that thi~ was the case for human skin
fibroblasts in the presence of elastin peptides (Ghuysen-
Itard et al, C.R. Acad. Sci., 1992, 315 : 473-478). The
result~ reported in Table 1 show that a stimulation
analogous to the proliferation of lymphocytes was
observed.
Table 1
Concentration Number of experiments Percentage ~timulation,
of K-ela~tin mean + SD
2 ~g/ml 6 62.67 + 37.90
10 ~g/ml 13 35.76 + 29.50
A m~i mllm Rtimulation is observed at 2 ~g/ml of
elastin peptides and a little weaker ~timulation with
10 ~g/ml of elastin peptides.
B) Liberation of proteolytic activity by the elastin
peptides
When PMNs or lymphocytes are incubated under
culture condition~ in the presence of 2 ~g/ml of
K-elastin, a gradual increase in the elastase and
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cathepsin activity can be determined in the cell extract6
and the cell lysates. Thi6 increa6e i6 much more marked
in the cell6 derived from elderly athero6clerotic
6ubject6. Such an increaBe iB not observed in the absence
of K-elastin. Addition of elastin peptides increa6e6 the
proteolytic activity 6imultaneously in the culture medium
~salted-out enzyme) and in the cell extract (enzyme bound
to the cell) for elastase as well as for cathepsin G,
although this effect may be much more pronounced for the
liberation of enzymatic activity in the culture
supernatant.
C) ~ffect of elastin peptides on cell survival
Thi6 experiment is carried out under the culture
condition6 such a6 described above, in the pre6ence of
phytohaemaglutinin (PHA).
S ml of aliquots of lymphocyte suspension (2.5 x
106 cell6 in 5 ml) obtained starting from human tonsil6
- are di6tributed in te~t tubes and increa6ing concentra-
tion6 of ~-elastin (BPM, PM c 10 kDa) are added:
0.1 ~g/ml, 2 ~g/ml, 10 ~g/ml, 500 ~g/ml, 1 and 2 mg/ml.
After 4 day6' incubation under the culture conditions the
cell6 are recovered and counted. Table 2 gives the cell
1O6s a6 a function of the concentration of elastin
peptide6 added.
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Table 2
Concentration of elastin peptide~ Percentage of cell 1O8~
~g/ml det~rmined by Trypan Blue
exclusion
0 5.3
1 5.0
2 2.5
17.4
lO0 l9.6
500 28.9
0 lO00 39.3
2000 39.1
It appears that elastin peptides exert a
cytotoxic activity at high concentrations (a thousand
times higher than the concentration stimulating cell
proliferation and the salting-out of the lytic enzymes).
Example 2: Effect of lactose and of melibiose on
reactions ~ ted by the elastin
receptor of l~ ,hocytes
1) Inhibition of cell proliferation
As shown in Figure l, growing concentrations of
lactose and of melibiose progressively suppress the
growth stimulation effect of elastin peptides, followed
by a net inhibition of proliferation at the strongest
concentrations tested (l and 2 mg/ml), equivalent to
5.84 x 10-3 mM). The strongest inhibition of proliferation
is of the order of 16 to 30% for lactose and 26 to 58%
for melibiose.
2) Effect of lactose and of melibiose on the
salting-out of proteolytic enzyme triggered by the
elastin receptor
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As shown above, an increase of 40 to 120% in the
activity of proteolytic enzymes is observed in the
culture medium as well as in the cell lysate of the
cultivated human lymphocytes in the presence of 2 ~g/ml
of K-elastin. The stimulation of the liberation of a
lytic enzymatic activity i~ inhibited effecti~ely by
melibiose starting from 1 ~g/ml. This inhibition can be
demonstrated as well for the elastase activities as for
cathepsin G. Lactose likewise has an efficacy but it is
less strong than that of melibiose (Figures 2 and 3).
Example 3: Quantification of the elastin receptor in
subpopulations of ~ n ly, hocytes
Methods
Ant; hoAi es used:
- 1st antibody: 67 kD elastin/l~m;n;n anti-receptor
antibody (bovine) (type: IgM) produced in mice (Elastin
Products Company).
- 2nd antibody: mouse anti-IgG + IgM (H + L) antibody
produced in goats, labelled by rhodamine (TRITC) (Jackson
Tmmllnoresearch Laboratories) (for fluorescence
microscopy).
- 2nd antibody: anti-IgM antibody (F(ab' )2 Of mice
produced in goats, labelled by r-phycoerythrin
(Chemicon)) (for flow cytometry).
PROTOCOL:
a) Fluorescence microscopy
Human lymphocytes are isolated from tonsils (as
described in Example 1) and cultured in the presence of
5 ~g/ml of phytohaemagglutinin (to acti~ate them) and for
certain cultures with 2 ~g/ml of kappa-elastin of high
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molecular weight (75 kD~ in an RPMI 1640 medium with 10%
foetal calf serum (FCS). After 48 to 120 hours' incuba-
tion at 37~C, the lymphocytes are washed three times with
~PMI medium contA;ning 2% FCS and then centrifuged
(400 g, 10 minutes at 4~C). Next, the cells are counted
and the cell density is adjusted to 10' cells/ml with
RPMI medium cont~;n;ng 2% FCS.
Then a volume (100 ~1) of cell suspension of
human lymphocytes (approximately 106 cells) is incubated
with 10 ~1 of solution of the first antibody (diluted to
1:100) for 30 minutes in an ice bath. 1.5 ml of cold ~PMI
are added to 2% FCS and then the cells are washed in a
refrigerated centrifuge at 4~C (at 400 g for 10 minutes).
The solution of the second antibody (conjugated
to TRITC) (diluted in cold RPMI to 1:200) is added to the
plug. After gentle agitation of the su~pension, it is
incubated at 4~C for 30 minutes. Next, 1.5 ml of cold
RPMI are added to 2% FCS and the suspension is centri-
fuged at 400 g at 4~C for 10 minutes.
At the end of this last washing, the lymphocytes
are resuspended in the residual medium after having
removed the supernatant by decantation. Smears are then
made on slides and dried in the air. After fixing with
absolute ethanol for S minutes, the 61ides are rehydrated
by immersion in several baths of PBS and mounted in
buffered glycerol (9 volumes of glycerol and 1 volume of
PBS). This post-fixation procedure increases the
brightnes6 of the ;mml~nofluorescence.
To identify the labelled cells, microscopic
fields are ~mined alternatively under ultraviolet
illumination and in visible light.
b) Fluorimetry
At the end of the last washing, the lymphocytes
are suspended in 1 ml of PBS and the cells are analysed
by cytofluorimetry.
The cell populations characterized are the
following:
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- total T lymphocytes (CD3+)
- "helper" T lymphocytes (CD4+)
- suppressor T lymphocytes (CD8+)
- B lymphocytes (CD20+)
5 - T activated lymphocytes (CD25+)
- memory T lymphocytes (CDA+/CD45RO+)
- granulocytes (CD15+)
Description of the labels used
CD 3
The CD 3 complex is expressed by all mature hl7m~n
T cells. It has molecular weights of between 16 and 28 ~D
composed of 5 chains (~ ) a6sociated with the
T-cell receptor in a non-co~alent manner. It i~ involved
in the transmission of activation signals.
CD 4
CD 4 (T4) recognizes the class II MHC molecules
- during the interaction of CD4+ cells with cells present-
ing the antigen or target cells. It is a glycoprotein of
59 kD belonging to the ;~llnoglobulin superfamily. It is
found in the "helper/inducer~ subpopulation of T
lymphocytes (45% of the lymphocytes of peripheral blood).
CD 8
The CD 8 molecule (T8, 30/32 kD) is a
glycoprotein formed of two peptide ch~in~ It iB found in
the cytotoxic/suppressor subpopulation of the T
lymphocytes (20-35% of the lymphocytes of the peripheral
blood). It is also present on NK cells and on 30% of the
"null" cells of the peripheral blood.
CD 15
The CD 15 antigen (3FAL, X-haptene, SSEA) is a
lacto-N-fucopentose III (200-185 kD). At least 5 major
antigens of CD 15 are present on the surface of poly-
nuclear cells (approximately 90% of the circulating human
granulocytes) and on part of the circulating monocytes
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t30-60%). This antigen is absent from the 6urface of
normal lymphocytes.
CD 20
The CD20 antigen is a phosphoprotein of 35/37kD.
It is present on all normal B cells of the peripheral
blood, of the tonsil and of the bone marrow.
CD25
The CD25 molecule corresponds to the low-affinity
receptor of interleukin-2. It is a glycoprotein of 55 kD
expressed by activated lymphocytes (T and B) but also by
activated macrophages.
CD4SR0
This is a tr~n~ ~ hrane glycoprotein of 180 kD
(the isoform of low molecular weight of the leucocyte
common antigen) (LCA). It is present on the surface of T
lymphocytes, thymocytes, granulocytes, monocytes (but is
absent on the surface of macrophages) and on a small
population of B lymphocytes. The T lymphocytes expressing
CD45R0 are memory T lymphocytes (or primed T cells) (45%
of the T lymphocytes of the peripheral blood).
The CD4+/CD45R0+ cells produce "helper" signals.
These are early producers of IL-2 and IFN-r.
For labelling with anti-CDx antibodies (Sigma
products, excluding anti-CD25 antibody, from Serotec),
100 ~l of the lymphocyte su~pension (already labelled or
not) are taken with the 67 kD anti-receptor antibody of
elastin at a cell density of 107 cells/ml.
10 ml of the anti-CDx antibody are added and the
suspension is incubated for 30 minutes at 20~C (except
with anti-CD25 antibody: incubation takes place at 0~C).
Next, 2 ml of PBS are added. After two washings (two
centriguations at 400 g for 10 minutes at 20~C), the cell
plug i~ taken, 0.5 ml of PBS is added to it and the
suspension is analysed by cytofluorimetry.
The labellings of the cells are likewise carried
out by isotype-similar mouse ;mm--noglobulins (non-speci-
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fic myeloma proteins) (for control).
R~S~LTS
a) Fluoresce~ce microscopy
Demonstration of the elastin reCePtor on activated
lymphocytes
Part of the analysed lymphocytes - those which
express the 67 kD elastin/l~;n;n receptor - show a
specific ;~mllnofluorescence under the experimental
conditions described above (positive cells).
The Percentaqe of positive cells on the 4th day of
culture is evaluated bY fluorescence microscoPY:
- cells cultured in the presence of 2 ~g/ml of kappa-
elastin:
28.52 + 12.60%
- cells cultured without kappa-elastin:
28.09 _ 10.91%
There is no significant difference between the
two series, activation by PXA is a sufficient stimulus to
make the elastin receptor express.
The percentaqe of Positive cells on the 5th daY of
culture:
- cells cultured in the presence of 2 ~g/ml of kappa-
elastin: 77.2% + 6.7%.
Effect of washinq cells with lactose or melibiose:
- cells cultured in the presence of 2 ~g/ml of kappa-
elastin + washed at the end of cell culture with a
solution of 1 mg/ml of lactose: 26.6% + 6.7% (p<0.001).
Before washing, 77.2% of cells are positive
- cells cultured 5 days in the pre~ence of 2 ~g~ml of
kappa-elastin + washed at the end of cell culture with a
solution of 1 mg/ml of melibiose: lB.3% + 9.7% (p~0.001).
Fluorescent cells incubated only with the second
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antibody (negative controls) and not with the first
antibody do not show any fluorescence. It thus appears
that melibiose is more effective than lactose for
deRorbing the 67 kD subunit of the lymphocyte elastin
receptor: lactose desorbs 66% of the receptor and
melibiose 76% under the experimental conditions used.
b) Cytofluorimetry
Table 3
Percentage of 1 lymphocytes expressing the elastin
receptor on different culture days
D0: on the day of separation of the lymphocytes;
D2, D3, D5: on the second; third; fifth day after separa-
tion of the lymphocytes.
% of positive cells
Day without number of with 2 ~g/ml of P
kappa-elastin experiments kappa-elastin
D01.12 + 0.2% 6 1.15+ 0.1%
D222.33 + 5.2% 5 23.02 + 3.2%
D329.70 + 0.8% 7 32.40 + 3.5% 0.178
D559.91 + 4.9% 10 66.42 + 1.9% 0.006
It is evident from this experiment that the
incubation of cells with PHA as stimulant progressively
induces the expression of the elastin receptor. This
induction is again stimulated in the presence of elastin
peptides. However, this stimulation does not become
significant until the start of the 5th day of culture.
Under these conditions approximately two thirds of the
lymphocytes (266%) express the elastin receptor on their
surface.
- Results of double lab~ll;n~8
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Table 4
Double lab~ll i n~ iment of 1~ ~hocytes to
detr ;~ the nature of the subpopulations
expressing the elastin receptor
5% of cells expressing the elastin receptor
(67 kD) + the label CDx
without kappa elastin with kappa elastin
R67kD+ 59.9 66.4
CD4+R67kD+ 26.5 45.7
CD8+R67kD+ 11.9 16.1
CD15+R67kD+ 0.0 0.0
CD20+R67kD+ 36.7 36.3
CD25+R67kD+ 39.6 46.9
- CD45RO+R67kD+ 26.6 41.1
CDx+R67kD = cells labelled simultaneously with the
anti-CDx antibody and with anti-subunit antibody of the
67 kD elastin receptor.
Note: The sum of the percentages does not give 100%
since a cell can simultaneously expre6s several
receptors.
In this experiment, the nature of the lymphocyte
subclass expressing the elastin receptor in the presence
and in the absence of elastin peptides was determined by
double labelling.
It appears that the majority of the lymphocyte
subclasses e~m;ned express the elastin receptor, with
the exception of CD15+ cells which remain negative.
However, this CD15 label corresponds to the PMNs and to
a part of monocytes.
This expression is only stimulated in an import-
ant fashion by the presence of elastin peptides on the
CD4+ and CD45RO+ lymphocytes which in the presence of
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elastin peptides 6trongly increased the expression of the
elastin receptor. It is a matter in that case of a
subpopulation of cells considered as helper and memory
cell~. The coupling of the elastin receptor by positive
retroaction to its own synthesis after its activation
would thus be specific to these cells.
A le 4: Protection against the cytotoxic effect of
~appa-elastin of low ~ lecular weight on
ly, hr~Cyte8
1) Isolation of the ly ~hs~ytes
Isolation of the lymphocytes according to the
method described in Example 1.
The cell suspension is diluted with RPMI contain-
ing 5 mM glutamine and 10% FCS such that the concentra-
tion is 106 cells/2 ml.
2) Cell culture
(D0)
The lymphocytes are put into culture in 24-well
Costar plates (500 ~1 of cell suspension/well at a
concentration of 106 cells/2 ml) in complemented RPMI
1640 medium. The distribution of the cells is as follows:
- 10 ml of cell suspension without K-elastin;
- 10 ml of cell suspension with 2 ~g/ml K-elastin,
and 10 ml of cell suspension with 2 mg/ml of K-elastin
(controls with kappa-elastin, without lactose and
melibiose);
- 10 ml of cell suspension with 2 mg/ml K-elastin +
1 mg/ml of melibiose;
- 10 ml of cell suspension with 2 mg/ml K-elastin +
1 mg/ml of lactose
- 10 ml of cell suspension with 1 mg/ml melibiose
- 10 ml of cell su~pension with 1 mg/ml lactose
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3) Counting of the cell~ in the pre~ence of Trypan Blue
to determine the percentage of dead cell~
On the fifth day of culture (D4), the cell
~u~pension i~ reco~ered, the cells are counted in a
Mala~sez cell in the pre~ence of 0.1% Trypan Blue (Sigma)
(only the dead cells are ~tained ~lue with Trypan Blue).
Counting i~ carried out immediately after having added
the ~tain on account of the toxicity of Trypan Blue.
.... .
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R~S~LTS
T~R~ - number of % of dead cell6
cells/ml (Trypan Blue)
without ~rr~-elastin 5.8 _ 0.6 9.2 + 0.4
with 2 ~g/ml ~appa- lO.23 _ 3.5 6.6 ~ l.3
5 elastin (control)
with 2 mg/ml ~appa- 3.6 + l.0 41.3 + 3.l
elastin
with l mg/ml lactose 7.7 + l.5 14.5 _ l.9
with l mg/ml -lihiose 7.26 _ 1.3 15.4 _ 1.6
10 with 2 mg/ml ~appa-
elastin + 1 mg/ml 8.8 + O.7 lO.6 + 2.3
lactose
with 2 mg/ml ~appa-
elastin ~ 1 mg/ml 8.8 + 1.6 10.5 + 0.9
l5 meli~iose
The table Rhows the percentage of dead cells in
the presence of elastin peptides and the protection
against cell death by lactose and melibiose. This protec-
tion is close to 100%: the overmortality (compared with
the control without K-elastin) is 32.1% with 2 mg/ml of
elastin peptides. In the presence of lactose or of
melibiose this overmortality is l.3% (96% protection).
