U.S. patent application number 12/929701 was filed with the patent office on 2011-06-09 for use of l-alpha-lysophosphatidylcholine to obtain the differentiation of monocytes in mature dendritic cells in vitro.
This patent application is currently assigned to BIOMERIEUX. Invention is credited to Patrice Andre, Vincent Lotteau.
Application Number | 20110135684 12/929701 |
Document ID | / |
Family ID | 27767084 |
Filed Date | 2011-06-09 |
United States Patent
Application |
20110135684 |
Kind Code |
A1 |
Lotteau; Vincent ; et
al. |
June 9, 2011 |
Use of L-alpha-lysophosphatidylcholine to obtain the
differentiation of monocytes in mature dendritic cells in vitro
Abstract
A method for treating and/or preventing certain ailments by
administering to a person in need thereof
L-.alpha.-lysophosphatidylcholine as an agent for activating the
immune system of the person. Also, vaccine compositions that
include L-.alpha.-lysophosphatidylcholine.
Inventors: |
Lotteau; Vincent; (Vourles,
FR) ; Andre; Patrice; (Lyon, FR) |
Assignee: |
BIOMERIEUX
Marcy L'Etoile
FR
INSTITUT NATIONAL DE LA SANTE ET DE LA RECHERCHE
MEDICALE
Paris
FR
|
Family ID: |
27767084 |
Appl. No.: |
12/929701 |
Filed: |
February 9, 2011 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12076591 |
Mar 20, 2008 |
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12929701 |
|
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10503402 |
Nov 1, 2004 |
7368287 |
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PCT/FR03/00741 |
Mar 7, 2003 |
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12076591 |
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Current U.S.
Class: |
424/204.1 ;
424/184.1; 424/234.1; 424/265.1; 424/274.1; 424/277.1; 514/44R;
514/76 |
Current CPC
Class: |
A61P 33/06 20180101;
A61P 31/20 20180101; A61P 35/00 20180101; A61P 43/00 20180101; A61P
37/00 20180101; A61P 31/14 20180101; A61K 9/0019 20130101; A61P
31/18 20180101; A61P 37/04 20180101; A61P 31/04 20180101; A61K
31/4439 20130101; A61P 35/02 20180101; A61K 47/10 20130101; A61P
31/00 20180101; A61P 31/10 20180101; A61P 29/00 20180101; A61P
33/00 20180101; A61P 31/12 20180101; A61K 47/24 20130101 |
Class at
Publication: |
424/204.1 ;
514/76; 424/184.1; 514/44.R; 424/234.1; 424/265.1; 424/277.1;
424/274.1 |
International
Class: |
A61K 39/12 20060101
A61K039/12; A61K 31/685 20060101 A61K031/685; A61K 39/00 20060101
A61K039/00; A61K 31/7088 20060101 A61K031/7088; A61K 39/02 20060101
A61K039/02; A61P 37/04 20060101 A61P037/04 |
Foreign Application Data
Date |
Code |
Application Number |
Mar 11, 2002 |
FR |
02/03499 |
Jan 30, 2003 |
FR |
03/01063 |
Claims
1. A method of treating and/or preventing, in a person, an
infection of bacterial, viral, fungal or parasitic origin or an
infection caused by a yeast, said method comprising administering
L-.alpha.-lysophosphatidylcholine as an agent for activating the
immune system of the person.
2. A method of treating or preventing cancer in a person, said
method comprising administering L-.alpha.-lysophosphatidylcholine
as an agent for activating the immune system of the person.
3. A method for preventing an inflammation and/or for combating an
inflammatory disease and/or an autoimmune disease in a person, said
method comprising: administering to the person an inhibitor of
L-.alpha.-lysophosphatidylcholine, in which the inhibitor is a
lipid emulsion comprising triglycerides, phospholipids and
glycerol.
4. The method of claim 3, wherein the lipid emulsion comprises:
from 15 to 25% of triglycerides, from 0.5 to 1.5% of phospholipids,
and from 1.8 to 2.6% of glycerol.
5. A method for preventing an inflammation and/or for combating an
inflammatory disease and/or an autoimmune disease in a person, said
method comprising administering to the person an inhibitor of
L-.alpha.-lysophosphatidylcholine, wherein the inhibitor is a
PPARgamma agonist.
6. A vaccine composition, comprising:
L-.alpha.-lysophosphatidylcholine as an agent for activating the
immune system, and a biological agent selected from the group
consisting of an antigen, a nucleic acid that encodes an antigen,
and a lysate of autologous or heterologous tumor cells.
7. The vaccine composition of claim 6, wherein said biological
agent is an antigen selected from the group consisting of a
bacterial antigen, a viral antigen, a yeast antigen, a parasite
antigen, a fungal antigen, and a tumor antigen.
8. The vaccine composition of claim 6, wherein said biological
agent is a nucleic acid that encodes an antigen selected from the
group consisting of a bacterial antigen, a viral antigen, a yeast
antigen, a parasite antigen, a fungal antigen, and a tumor
antigen.
9. The vaccine composition of claim 6, wherein said biological
agent is a lysate of autologous or of heterologous tumor cells.
10. The vaccine composition of claim 6, wherein said vaccine
composition is formulated for parenteral, subcutaneous,
intramuscular, intravenous, intradermal, topical, intratracheal,
rectal, or transdermal administration.
11. The vaccine composition of claim 10, wherein said biological
agent is an antigen selected from the group consisting of a
bacterial antigen, a viral antigen, a yeast antigen, a parasite
antigen, a fungal antigen, and a tumor antigen.
12. The vaccine composition of claim 10, wherein said biological
agent is a nucleic acid that encodes an antigen selected from the
group consisting of a bacterial antigen, a viral antigen, a yeast
antigen, a parasite antigen, a fungal antigen, and a tumor
antigen.
13. The vaccine composition of claim 10, wherein said biological
agent is a lysate of autologous or of heterologous tumor cells.
14. The vaccine composition of claim 10, wherein said vaccine
composition is formulated for parenteral administration.
Description
[0001] This is a Continuation of application Ser. No. 12/076,591
filed Mar. 20, 2008, which is a Division of application Ser. No.
10/503,402 filed Nov. 1, 2004, which in turn is a National Stage
Application of PCT/FR03/00741, filed Mar. 7, 2003, which in turn
claims the benefit of French Patent Application No. 02/03499, filed
Mar. 11, 2002 and French Patent Application No. 03/01063, filed
Jan. 30, 2003. The disclosure of the prior applications is hereby
incorporated by reference herein in its entirety.
[0002] The present invention relates to a method for
differentiating monocytes into mature dendritic cells, according to
which monocytes are provided in a suitable medium for their
differentiation, and L-.alpha.-lysophosphatidylcholine is added to
said medium. The present invention also relates to the use of at
least one inhibitor of L-.alpha.-lysophosphatidylcholine, for
producing a medicinal product for preventing an inflammation and/or
for combating an inflammatory disease and/or an autoimmune
disease.
[0003] Dendritic cells are involved in the development of an immune
response and in the initiation of a specific T-lymphocyte response
by recognition, uptake and presentation of antigens, in particular
of infectious agents (Steinman et al. 1997, Immuno. Rev., 156:
25-37; Cella et al. 1997, Curr. Opin. Immunol., 9: 10-16). While
immature dendritic cells take up and digest the antigens of
infectious agents very effectively, certain signals, such as
bacterial agents or inflammatory cytokines, can activate them by
inducing a process of maturation, which is the initial step in
triggering the adaptive immune response. In fact, during this
activation, dendritic cells acquire the ability to migrate to
lymphoid organs where T lymphocytes are found and the ability to
transmit costimulation signals that are essential to the activation
of naive T lymphocytes. During this maturation, dendritic cells
undergo functional and phenotypic modifications such as: [0004] an
increase in surface molecules involved in the activation of T
lymphocytes (such as CD40, CD80, CD83, CD86 and the molecules of
the major histocompatibility complex (MHC) class I and class II),
[0005] the production of proinflammatory cytokines (such as the
interleukins IL-12, IL-1.beta., TNF.alpha. and IL-6), [0006] a
decrease in their ability to take up and process the antigen.
[0007] By virtue of their abilities to develop an immune response
and to initiate a specific T lymphocyte response, mature dendritic
cells are of particular therapeutic interest, in particular in the
fields of anti-infectious and antitumor immunization and of
immunotherapy (Austin. 1998, Curr. Opin. Hematol. 5: 3-15; Reise
Sousa et al. 1999, Curr. Opin. Immunol. 11: 392-399). These
dendritic cells, by virtue of their ability to induce
immuniotolerance, are also an advantageous target when it is
desired to inhibit the immune response of a patient, in particular
in order to combat autoimmune or inflammatory diseases.
[0008] In vitro, mature dendritic cells can be obtained from
monocytes in culture. These monocytes are, in vivo, circulating
cells which, when crossing in particular the vascular endothelial
wall, come into contact with surrounding factors which influence
their outcome in a manner which is still poorly understood.
Schematically, three possibilities are then envisioned for these
monocytes: [0009] exiting the tissues and returning to the lymph
nodes, [0010] differentiating into macrophages [0011]
differentiating into immature dendritic cells.
[0012] The first step for obtaining mature dendritic cells from
monocytes in culture then consists in inducing the differentiation
of the monocytes into immature dendritic cells with, in particular,
the interleukin IL-4 and the factor GM-CSF (granulocyte
macrophage-stimulating factor). After 6 days, 95% of the cells in
culture are immature dendritic cells.
[0013] The second step then consists of induction of the maturation
of the immature dendritic cells into mature dendritic cells using
exogenous agents such as bacterial or viral agents. Thus, the
KpOmpA protein from Klebsiella pneumoniae is capable of inducing
the maturation of these immature dendritic cells into mature
dendritic cells (P. Jeannin et al., Nature Immunology, 2000, 1:
502-509). Mention may also be made of the maturation of immature
dendritic cells into mature dendritic cells by means of other
exogenous molecules, such as bacterial membrane lipopolysaccharides
(Dichman et al, Journal of Cellular Physiology, 2000, 185:
394-400). However, the use of exogenous molecules derived from
infectious agents induces problems of safety and of cost (direct or
indirect side effects in vivo; very important need for purification
according to very strict legal or regulatory requirements, etc.),
making the use of this type of molecule difficult in the context of
vaccinology and of immunotherapy.
[0014] More recently, it has been described, by Perrin-Cocon et
al., in 2001 (The Journal of Immunology, 167: 3785-3791), that the
production of mature dendritic cells from monocytes undergoing
differentiation can be induced with endogenous molecules, such as
certain oxidized plasma lipoproteins, and more particularly
oxidized low density lipoproteins (LDLs). However, oxidized LDLs
are complex particles made up of proteins, triacyl glycerols,
phospholipids, and free and esterified cholesterol, and are as a
result difficult to synthesize artificially.
[0015] It is also important to note that, while stimulation of the
differentiation of monocytes into dendritic cells is essential in
certain therapeutic applications (during immunization, during
stimulation of the immune response), it may also be essential to
inhibit such a differentiation of monocytes into dendritic cells,
or more generally to inhibit the maturation of immature dendritic
cells, in other therapeutic applications, such as in an autoimmune
or inflammatory disease. Treatments for combating inflammation,
such as the taking of corticoids or the taking of aspirin,
currently exist. The problem is that, when the inflammation is
chronic, such treatments have side effects that are very harmful
for the patient. The prolonged taking of corticoids can in
particular engender a Cushing's syndrome during which
demineralization, spontaneous fractures and diabetes are observed.
The prolonged taking of aspirin can, for its part, engender stomach
ulcers. The present invention proposes to solve the disadvantages
of the state of the art by also proposing an anti-inflammatory
molecule which is easy to synthesize and relatively inexpensive,
and which inhibits the differentiation of monocytes into mature
dendritic cells.
[0016] Thus, the present invention proposes to solve the
disadvantages of the state of the art by providing a
proinflammatory molecule, an endogenous molecule, which is easy to
synthesize and relatively inexpensive and which stimulates the
differentiation of monocytes into mature dendritic cells. The
present invention also relates to the use of a lipid emulsion
comprising triglycerides, phospholipids and glycerol, such as in
particular INTRALIPID.RTM., for inhibiting the differentiation of
monocytes to mature dendritic cells, by virtue of a direct action,
or an indirect action via inhibition of the action of
L-.alpha.-lysophosphatidylcholine.
[0017] Surprisingly, the present invention relates to the use of
L-.alpha.-lysophosphatidylcholine for differentiating monocytes
into mature dendritic cells in vitro.
[0018] The term "L-.alpha.-lysophosphatidylcholine" is intended to
mean a molecule for which the formula is as follows:
##STR00001##
in which represents a long chain of saturated fatty acids
containing from 12 to 20 carbon atoms, preferably from 16 to
18.
[0019] In fact, one of the active molecules generated during LDL
oxidation is L-.alpha.-lysophosphatidylcholine, hereinafter
referred to as LPC. However, it has been shown that the binding of
LPC to the G2A receptor, which is the high affinity receptor for
LPC, inhibits in particular the proliferation and the activation of
T lymphocytes, rather suggesting a role for LPC in inhibiting the
triggering of an immune reaction (Carson & Lo, 2001, Science,
293: 618-619). In addition, it has also been shown that a high
concentration of LPC (50 .mu.M) inhibits the activity of the
transcription factor NF-.kappa.B (nuclear factor .kappa.B), which
is however known to be activated during the maturation of dendritic
cells. In addition, LPC is present at high concentration in the
plasma, suggesting that it is not active in the plasma, which would
not predispose it to be chosen by those skilled in the art for
therapeutic use.
[0020] The invention relates to a method for the differentiation in
vitro of monocytes into mature dendritic cells, according to which:
[0021] A. monocytes are provided in a suitable culture medium,
[0022] B. the differentiation of the monocytes into dendritic cells
is induced in the presence of a differentiation factor, [0023] C.
L-.alpha.-lysophosphatidylcholine is added to said medium and
mature dendritic cells are obtained.
[0024] The expression "suitable culture medium" is intended to mean
a medium comprising all the elements required for cell viability.
By way of example, mention may be made of RPMI 1640 medium and its
derivatives, and any culture medium well known to those skilled in
the art. This medium comprises in particular at least one factor
for differentiating monocytes into dendritic cells. The factors for
differentiating monocytes into dendritic cells are well known to
those skilled in the art, and mention may in particular be made of
cytokines such as, without any implied limitation, the interleukin
IL-4, the factor GM-CFS (granulocyte macrophage-stimulating
factor), IL-13 or TNF (tumor necrosis factor).
[0025] In step C), the L-.alpha.-lysophosphatidylcholine is added
in particular to said medium at a final concentration in the medium
of between 10 and 80 .mu.M, preferably between approximately 20 and
60 .mu.M, and advantageously between 30 and 50 .mu.M. Still in step
C), the L-.alpha.-lysophosphatidylcholine is added to said medium
between the 3rd and 6th day of monocyte differentiation, preferably
between the 4th and 5th day of monocyte differentiation.
[0026] According to a particular embodiment of the invention, at
least one biological agent is also added to the culture medium, in
step C). The term "biological agent" is intended to mean a molecule
(or a set of molecules) which is the target of an immune response
or which allows the synthesis of this target. This biological agent
can thus be chosen from bacterial, viral, yeast, parasite or fungal
antigens, tumor antigens, and lysates of autologous and/or
heterologous tumor cells. The term "autologous tumor cells" is
intended to mean tumor cells belonging to the individual who
receives a given medicinal product. The tumor cells can be obtained
by taking a sample of cancerous tissues, in particular a biopsy or
a surgical resection. The term "heterologous tumor cells" is
intended to mean cells derived from tumors originating from an
individual who is different from the one receiving a given
medicinal product. The use of heterologous cells makes it possible
in particular to obtain a medicinal product for treating patients
suffering from cancer from whom it is not possible to obtain a
tumor cell sample. This also makes it possible to use a standard
source of tumor antigens. The term "cell lysate" is intended to
mean a mixture of intracellular and/or membrane antigens, obtained
by lysis of cells according to a protocol known to those skilled in
the art, such as mechanical, chemical or enzymatic lysis. This
biological agent may also be a nucleic acid which encodes at least
one antigen chosen from bacterial, viral, yeast, parasite or fungal
antigens, and tumor antigens. The term "tumor antigen" is intended
to mean an antigen derived from tumor cells, such as a
tumor-related peptide, in particular a peptide which interacts with
class 1 molecules and which is presented to CD8 T lymphocytes.
Mention may be made, in a known nonlimiting manner, of the
following tumor antigens: MAGE-2, MAGE-3, MART, MUC-1, MUC-2,
HER-2, GD2, carcinoembryonic antigen (CEA), TAG-72,
ovarian-associated antigens OV-TL3 and MOV18, TUAN,
alpha-fetoprotein (AFP), OFP, CA-125, CA-50, CA-19-9, renal
tumor-associated antigen G250, EGP-40 (or EpCAM), S100 (malignant
melanoma-associated antigen), p53, prostate tumor-associated
antigens (e.g. PSA and PSMA) and p21ras.
[0027] Thus, the addition of this biological agent to the culture
medium makes it possible to obtain mature dendritic cells which
then allow the activation of T lymphocytes which are directed
against a given antigen. These mature dendritic cells, obtained in
vitro, can then be reinjected in vivo.
[0028] In another embodiment of the invention, it is also possible
to use, in step C, a compound equivalent to
L-.alpha.-lysophosphatidylcholine, which is a molecule that acts
according to the same cellular mechanisms of action as
L-.alpha.-lysophosphatidylcholine, i.e. by the same membrane
receptors, in particular G protein-coupled membrane receptors, such
as the receptors G2A, GPR4 or the PAF (platelet activating factor)
receptor, and/or via the same nuclear receptors, such as the PPAR
receptors (peroxisome proliferator-activated receptor). This
equivalent compound can thus in particular be an agonist for the
above-mentioned receptors (such as in particular O-methyl-PAF,
carbamyl-PAF or 2-O-methyl-PAF), but also the PAF itself. This
compound may also be a PPAR.delta. agonist, such as in particular
L165041 (Merck Research), GW-5015616 (Glaxo) or carboprostacylin
(Cayman), or a PPAR.gamma. antagonist, such as in particular
bisphenol A diglycidyl ethyl (Sigma) or DICLOFENAC.RTM.
(2-[(2,6-dichlorophenyl)amino]benzeneacetic acid). This compound
equivalent to L-.alpha.-lysophosphatidylcholine can be used alone
or in synergy with L-.alpha.-lysophosphatidylcholine. According to
a preferred embodiment of the invention, the
L-.alpha.-lysophosphatidylcholine is used in synergy with platelet
activating factor (PAF).
[0029] The invention also relates to a method for activating T
lymphocytes in vitro, according to which: [0030] A. monocytes are
provided in a suitable culture medium, [0031] B. the
differentiation of the monocytes into dendritic cells is induced in
the presence of a differentiation factor, [0032] C.
L-.alpha.-lysophosphatidylcholine is added to said medium and
mature dendritic cells are obtained, [0033] D. a biological agent,
as defined above, is added to said medium and the mature dendritic
cells obtained in step B are directed against said biological
agent, [0034] E. the mature dendritic cells directed against the
biological agent according to step C are brought into contact with
T lymphocytes, and T lymphocytes directed against the biological
agent are obtained.
[0035] Step C) can be carried out as described above. Such a method
thus makes it possible to obtain, in vitro, T lymphocytes directed
against a given biological agent, which can then be reinjected in
vivo into a patient, in particular an immunodepressed patient.
[0036] The invention also relates to a method for maturing
dendritic cells in vitro, according to which: [0037] A. immature
dendritic cells are provided in a suitable culture medium, [0038]
B. L-.alpha.-lysophosphatidylcholine is added to said medium and
mature dendritic cells are obtained.
[0039] Those skilled in the art are well aware of dendritic cells
and their various stages of maturation.
[0040] In step B), the L-.alpha.-lysophosphatidylcholine is added
in particular to said medium at a final concentration in the medium
of between approximately 10 and 80 .mu.M, preferably between
approximately 20 and 60 .mu.M, and advantageously between 30 and 50
.mu.M. According to a particular embodiment of the invention, at
least one biological agent as defined above is also added to the
culture medium, in step B).
[0041] The invention also relates to a culture medium,
characterized in that it comprises
L-.alpha.-lysophosphatidylcholine and at least one differentiation
factor as defined above.
[0042] In a preferred embodiment of the invention,
L-.alpha.-lysophosphatidylcholine is at a final concentration in
the medium of between approximately 10 and 80 .mu.M, preferably
between approximately 20 and 60 .mu.M, and advantageously between
30 and 50 .mu.M. According to a particular embodiment of the
invention, the culture medium also comprises a biological agent as
defined above, which is in particular a bacterial, viral, yeast,
parasite or fungal antigen, an autologous and/or heterologous tumor
cell lysate antigen, a tumor antigen, a nucleic acid which encodes
a bacterial, viral, yeast, parasite or fungal antigen, or a nucleic
acid which encodes a tumor antigen.
[0043] The invention also relates to the use of
Ira-lysophosphatidylcholine as an agent for activating the immune
system. The term "agent for activating" is intended to mean a
molecule which, in a pharmaceutical composition, induces the
effects of a medication or reinforces or completes the effects of
the main medication. In the case of a vaccine composition, the
L-.alpha.-lysophosphatidylcholine then plays the role of adjuvant
which stimulates the host organism's immune response against a
given antigen. Thus, the invention relates to a vaccine
composition, characterized in that it comprises
L-.alpha.-lysophosphatidylcholine as an agent for activating the
immune system, which then plays the role of adjuvant, and a
biological agent as defined above, against which it is desired to
stimulate the patient's immune response.
[0044] According to a preferred embodiment of the invention,
L-.alpha.-lysophosphatidylcholine is used as an agent for
activating the immune system, for producing a medicinal product for
the treatment and/or the prevention of an infection of bacterial,
viral, fungal or parasitic origin or an infection caused by a
yeast, and/or for the production of a medicinal product for the
treatment and/or the prevention of cancers.
[0045] As bacterial infection, mention may in particular be made of
infections induced by staphylococci, mycobacteria, bacteria of the
Nisseria genus, legionellae, salmonellae, etc.
[0046] As viral infection, mention may in particular be made of
infections induced by HIV (human immunodeficiency virus), hepatitis
viruses, the measles virus, the rubella virus, polio viruses,
flavin viruses, etc.
[0047] As fungal infection, mention may in particular be made of
aspergillosis, candidosis, etc.
[0048] As parasitic infection, mention may in particular be made of
malaria, leishmaniasis, etc.
[0049] The term "cancer" is intended to mean all diseases due to an
abnormal multiplication of cells, and in particular, in a
nonlimiting manner, melanomas, lymphomas, leukemias, kidney, brain,
colon, prostate, rectal, pancreatic, ovarian, lung, liver and
breast carcinomas, skin cancers chosen from keratinomas and
carcinomas, and melanomas.
[0050] The medicinal product according to the invention may be
provided in the form of a pharmaceutical composition in combination
with at least one pharmaceutically acceptable excipient well known
to those skilled in the art. In the pharmaceutical compositions
according to the invention, for oral, sublingual, subcutaneous,
intramuscular, intravenous, topical, intratracheal, rectal or
transdermal administration, the L-.alpha.-lysophosphatidylcholine
can be administered in unit administration form or as a mixture
with conventional pharmaceutical supports, and when intended for
oral administration, for example in the form of a tablet, a gel
capsule, an oral solution, etc., or for rectal administration, in
the form of a suppository, for parenteral administration, in
particular in the form of an injectable solution, especially by
intravenous, intradermal or subcutaneous injection, etc., according
to conventional protocols well known to those skilled in the art.
For topical application, the L-.alpha.-lysophosphatidylcholine can
be used in creams, ointments, lotions or eye lotions.
[0051] When a solid composition is prepared in the form of tablets,
the L-.alpha.-lysophosphatidylcholine is mixed with a
pharmaceutically acceptable excipient, also called pharmaceutical
vehicle, such as gelatin, starch, lactose, magnesium stearate,
talc, gum arabic or the like. The tablets may be coated with
sucrose, with a cellulose derivative or with other suitable
materials. They can also be treated such that they have prolonged
or delayed activity and such that they continuously release a
predetermined amount of L-.alpha.-lysophosphatidylcholine. It is
also possible to obtain a preparation of gel capsules by mixing the
L-.alpha.-lysophosphatidylcholine with a diluent and pouring the
mixture into soft or hard gel capsules. It is also possible to
obtain a preparation in the form of syrup or for administration in
the form of drops, in which the L-.alpha.-lysophosphatidylcholine
is present together with a sweetener, an antiseptic, such as in
particular methylparaben and propylparaben, and also a suitable
flavor enhancer or dye. Water-dispersible powders or granules can
contain the Ira-lysophosphatidylcholine as a mixture with
dispersing agents or wetting agents, or suspending agents, well
known to those skilled in the art. For parenteral administration,
use is made of aqueous suspensions, isotonic saline solutions or
sterile and injectable solutions which contain dispersing agents or
wetting agents which are pharmacologically compatible, such as in
particular propylene glycol or butylene glycol.
[0052] The invention also relates to the use of at least one
inhibitor of L-.alpha.-lysophosphatidylcholine, for producing a
medicinal product for preventing an inflammation and/or for
combating an inflammatory disease and/or an autoimmune disease. The
term "inhibitor of L-.alpha.-lysophosphatidylcholine" is intended
to mean a molecule (or a set of molecules) which blocks the
inflammatory and/or immunostimulant activity of the
L-.alpha.-lysophosphatidylcholine, in particular by blocking the
differentiation of mature dendritic cells by
L-.alpha.-lysophosphatidylcholine. It is also intended to mean a
module having effects opposite to those of
L-.alpha.-lysophosphatidylcholine, by modulating in particular the
PPAR.delta./PPAR.gamma. ratio. By way of indication, mention may be
made of a lipid emulsion comprising triglycerides, phospholipids
and glycerol, such as in particular lNTRALIPID.RTM., LIPO D
E-80.RTM., a PPARgamma agonist. PPARgamma agonists are well known
to those skilled in the art, and mention may be made, in a
nonlimiting manner, of molecules of the thiazolidinedione class,
such as in particular ciglitazone, troglitazone, pioglitazone,
rosiglitazone, etc. Such inhibitors could thus be used in vivo, for
producing a medicinal product for decreasing an inflammatory
response, in particular an inflammatory response of the joints, or
especially during a transplant. According to a preferred embodiment
of the invention, the inhibitor of Ira-lysophosphatidylcholine is a
lipid emulsion comprising triglycerides, phospholipids and
glycerol. Preferably, the triglycerides are extracted from plant
oil such as in particular soybean oil. Even more preferably, the
lipid emulsion comprises from 15 to 25% of soybean oil, preferably
20% of soybean oil, from 0.5 to 1.5% of egg phospholipids,
preferably 1.2%, and from 1.8 to 2.6% of glycerol, preferably 2.2%
of glycerol. According to another preferred embodiment of the
invention, the inhibitor is a PPARgamma agonist as defined
above.
[0053] As inflammatory disease, mention may be in particular be
made of sarcomatosis, lupus, rheumatoid arthritis,
spondylarthritis, uveitis, etc.
[0054] As autoimmune disease, mention may in particular be made of
type 1 diabetes, multiple sclerosis, psoriasis, contact
hypersensitivities, spondylarthritis, rheumatoid arthritis,
etc.
[0055] FIG. 1 shows the titration curves giving the absorbance
(Abs) as a function of the logarithm of the dilution of the serum
of a batch of mice which are given LPC mixed with hen egg lysozyme
(HEL, 1 mg/ml), or HEL alone.
[0056] The following examples are given by way of explanation and
are in no way limiting in nature. They will make it possible to
understand the invention more thoroughly.
EXAMPLE 1
Differentiation of Monocytes in Culture into Dendritic Cells in the
Presence or Absence of L-.alpha.-Lysophosphatidylcholine (LPC)
[0057] Isolation, placing in culture and initiation of the
differentiation of monocytes--The monocytes are isolated from human
peripheral blood by means of first density gradient centrifugation
(620 g; 20 minutes) in Ficoll-Hypaque, followed by second
centrifugation (770 g; 20 minutes) in a 50% Percoll solution. The
monocytes are then purified by immunomagnetic depletion (Dynal,
Oslo, Norway), using a cocktail of anti-CD 19 (hydridoma 4G7)
(Becton Dickinson, Francklin Lakes, N.J., USA), anti-CD3 (OKT3,
American Type Culture Collection, Rockeville, Md.) and anti-CD56
(NKH1, Beckman Coulter, Fullerton, Calif., USA) monoclonal
antibodies. The monocytes thus obtained are then purified to at
least 90%, as shown by the absence of CD1a markers and the presence
of the CD14 marker. The differentiation of the monocytes into
dendritic cells is initiated with 40 ng/ml of recombined human
GM-CSF (Granulocyte-Macrophage Colony Stimulating Factor) and 250
U/ml of recombined human interleukin IL-4.
[0058] The monocytes are placed in culture in RPMI 1640 medium
(Life Technologies, Rockeville, Md., USA) enriched with 2 mM of
glutamine (Life Technologies), 10 mM of Hepes (Life Technologies),
40 ng/ml of gentamycin (Life Technologies) and 10% of
lipoprotein-depleted fetal calf serum (LPDS, Sigma, St
Quentin-Fallavier, France).
[0059] It should be noted that the culture medium presented in this
example is an LPDS culture medium. Comparable results can be
obtained using other culture media, such as an FCS medium, i.e. a
medium containing 10% of non-lipoprotein-depleted fetal calf serum,
or could be obtained using any synthetic culture medium known to
those skilled in the art.
[0060] Treatment of the monocytes with LPC--5 days after the
beginning of the differentiation of the monocytes, 40 .mu.M of LPC
(L-.alpha.-lysophosphatidylcholine; Sigma, St Quentin Fallavier,
France) are added to the culture medium for 24 hours. Control cells
are also obtained in the absence of LPC in the culture medium.
[0061] "Control" cells and "LPC" cells are then obtained.
EXAMPLE 2
Phenotype of Cells Obtained According to Example 1
[0062] The cells used in this analysis are those obtained on the
6th day of differentiation according to the protocol described in
example 1.
[0063] The phenotype of the "control" and "LPC" cells is analyzed
by flow cytometry on a FACSCalibur (Becton Dickinson, Francklin
Lakes, N.J., USA) using FITC (fluorosceine
isothiocyanate)-conjugated anti-CD14, anti-HLA-DR and anti-CD80 and
PE (phycoerythrin)-conjugated anti-CD1a, anti-CD83, anti-CD86 and
anti-CD40 (Beckman Coulter). According to the state of the art,
monocytes preferentially have a CD14+CD1a-phenotype, immature
dendritic cells preferentially have a CD14-CD1a+CD86-phenotype, and
mature dendritic cells preferentially have a CD14-CD1a
intermediate-CD86+phenotype.
[0064] The results obtained are given in table 1.
TABLE-US-00001 TABLE 1 Expression of the CD83, HLA-DR and CD86
markers in the absence (control) or in the presence of LPC CD83
HLA-DR CD86 Control 5.79 78.76 117.77 LPC 11.74 146.31 759.69
[0065] The "LPC" cells obtained after initiation of the
differentiation of the monocytes in the presence of LPC exhibit a
phenotype comparable to that of mature dendritic cells, as
demonstrated in particular by the induction of CD86 markers and the
increase in HLA-DR compared with the phenotype of the "control"
cells.
EXAMPLE 3
Internalization Capacity of the Cells Obtained According to Example
1
[0066] The "control" and "LPC" cells used in this analysis are
those obtained after 6 days of differentiation according to the
protocol described in example 1. These cells are incubated at
37.degree. C.: [0067] for 30 minutes with 1 mg/ml of
FITC-T70-Dextran (Sigma) in order to estimate the capacity of these
cells for internalization by endocytosis, [0068] for 30 minutes
with 1 mg/ml of Lucifer Yellow (ref. L0259, Sigma, St
Quentin-Fallavier, France) in order to estimate the capacity of
these cells for internalization by pinocytosis, [0069] for 3 hours
with carboxylate-modified yellow-green FluoSpheres (trade name,
0.45 .mu.m, Molecular Probes, Leiden, The Netherlands) in order to
estimate the capacity of these cells for internalization by
macropinocytosis.
[0070] The internalization is stopped on ice with a cold PBS buffer
containing 0.1% of BSA (Bovine Serum Albumin) and 0.05% of
NaN.sub.3. The "control" and "LPC" cells are washed 3 times at
4.degree. C. in this same buffer and the fluorescence is quantified
by FACScalibur (trade name, Becton Dickinson).
[0071] As shown in table 2, the capacities for internalization by
endocytosis, pinocytosis and macropinocytosis are greatly decreased
by the addition of LPC to the culture medium on the 5th day of
differentiation of the monocytes, compared to the control cells
differentiated in the absence of LPC.
TABLE-US-00002 TABLE 2 Capacity for internalization by endocytosis,
pinocytosis and macropinocytosis of the monocytes differentiated in
the absence (control) or in the presence of LPC Endocytosis
Pinocytosis Macropinocytosis Control 100% 100% 100% LPC 53% 49%
66%
[0072] The decrease in internalization capacities is one of the
characteristics of mature dendritic cells. These results show that,
in the presence of LPC, the monocytes have a strong tendency to
differentiate into mature dendritic cells.
EXAMPLE 4
Ability of the Cells Obtained According to Example 1 to Stimulate T
Lymphocytes
[0073] The "control" and "LPC" cells used in this analysis are
those obtained after 6 days of differentiation according to the
protocol described in example 1.
[0074] Naive allogenic T lymphocytes are isolated from human
peripheral blood. Peripheral blood mononuclear cells are isolated
by density gradient centrifugation (600 g, 20 minutes) in the
presence of Ficoll-Hypaque. After elimination of the monocytes on a
Percoll gradient, the peripheral blood lymphocytes are found in the
dense fraction. The T lymphocytes are purified by immunomagnetic
depletion using a cocktail of anti-CD19 (antibody 4G7) (Becton
Dickinson, Francklin Lakes, N.J., USA), anti-CD16 (antibody 3G8),
anti-CD56 (antibody NKH1), anti-glycophorin A (antibody 11E4B7.6)
and anti-CD14 (antibody RMPO.sub.52), monoclonal antibodies sold by
Beckman Coulter.
[0075] The purified T lymphocytes are cultured in flat-bottomed
96-well culture plates with the "control" or "LPC" cells.
[0076] 2.times.10.sup.5 T cells are cultured in 200 .mu.l of
culture medium according to a monocytes differentiated in the
presence or absence of LPC/T cells ratio (DC/LT ratio) of 1:5, 1:10
or 1:20. After 4 days, 50 .mu.l of the culture supernatant are used
to determine the secretion of IL-2 (interleukin 2) and of
.gamma.IFN (gamma interferon) using an ELISA kit (Endogen, Woburn,
Mass., USA).
[0077] As shown in table 3, the secretion of IL2 and of .gamma.IFN
by the T lymphocytes, conventionally expressed according to the
dendritic cells/T lymphocyte ratio (DC/LT ratio) is greatly
stimulated by the cells originating from the differentiation of
monocytes in the presence of LPC added 5 days after the initiation
of monocyte differentiation ("LPC" cells), compared to the control
results ("control" cells).
TABLE-US-00003 TABLE 3 Stimulation of the secretion of IL2 and of
.gamma.IFN by T lymphocytes, induced by the monocytes
differentiated in the absence (control) or in the presence of LPC
DC/LT ratio 0 0.05 0.1 0.2 Control IL-2 0 6 .+-. 8 15 .+-. 6 35
.+-. 11 .gamma.IFN 0 41 .+-. 14 51 .+-. 4 119 .+-. 9 LPC IL-2 0 23
.+-. 1 47 .+-. 26 173 .+-. 31 .gamma.IFN 0 73 .+-. 47 439 .+-. 108
795 .+-. 19
[0078] These results show an increase in the abilities of the
monocytes differentiated in the presence of LPC to stimulate
allogenic T lymphocytes, which is a characteristic of mature
dendritic cells. By way of indication, comparable results are
obtained whether the LPC is dissolved in ethanol or is in the form
of a lipid emulsion.
EXAMPLE 5
Properties of the Cells Obtained According to Example 1: Comparison
with Mature Dendritic Cells Obtained by Differentiation of
Monocytes in the Presence of Oxidized LDLs
[0079] The aim of this example is to demonstrate that the
mechanisms of action involved in the differentiation of monocytes
into mature dendritic cells in the presence of LPC could be
different from those involved in the differentiation of monocytes
into mature dendritic cells in the presence of oxidized LDLs
(Perrin-Cocon et al. The Journal of Immunology, 167: 3785-3891,
2001).
[0080] In this example, the cells used are "control" and "LPC"
cells as obtained in example 1, and also "LDLox" cells obtained as
described in the scientific publication (Perrin-Cocon et al. The
Journal of Immunology, 167: 3785-3891, 2001), i.e. after
differentiation of the monocytes in culture in the presence of
oxidized LDLs added to the medium 5 days after the initiation of
differentiation.
[0081] The inventors investigated whether the inhibitors of the
action of oxidized LDLs on the differentiation of monocytes into
mature dendritic cells also inhibited the action of LPC on the
differentiation of monocytes into mature dendritic cells.
[0082] Thus, a solution of INTRALIPID.RTM. (50 .mu.g/ml of
phospholipids, Fresenius Kabi, Sevres, France), or a solution of
synthetic molecules that are lipid in nature (LIPOID E-80.RTM.,
Lipoid Ag, Ludwigshafen, Germany, 50 .mu.g/ml of phospholipids) was
added to the culture medium on the 5th day of differentiation. The
expression of CD86 by the "LPC" and "LDLox" cells in the presence
of these inhibitors is analyzed according to the protocol described
in example 2, and the results are given in table 4.
TABLE-US-00004 TABLE 4 Inhibition (in %) of the expression of CD86
by the LCP and LDLox cells, by INTRALIPID .RTM. and lipoid E-80
INTRALIPID .RTM. LIPO D E-80 .RTM. LDLox 88% .+-. 10 81% .+-. 14
LPC 86% .+-. 12 24% .+-. 19
[0083] The addition of INTRALIPID.RTM. inhibits the differentiation
of monocytes into mature dendritic cells by means of oxidized LDLs
and by means of LPC, in a comparable manner (inhibition of the
order of 80%).
[0084] On the other hand, surprisingly, the addition of LIPOID
E-80.RTM. induces an 81% inhibition of the expression of CD86 when
the monocytes are cultured in the presence of oxidized LDLs,
although this inhibition is only 24% when the monocytes are
cultured in the presence of LPC.
[0085] These results suggest that the mechanisms of action of LPC
for the differentiation of monocytes into mature dendritic cells is
different from the mechanisms of action of oxidized LDLs.
EXAMPLE 6
Cellular Mechanisms Involved in the Differentiation of Monocytes
into Mature Dendritic Cells in the Presence of LPC
[0086] In order to advance further in the search for the cellular
mechanisms of the action of LPC in the differentiation of monocytes
into mature dendritic cells, the inventors investigated which
receptors could be involved.
[0087] Initially, in order to determine whether the receptors
involved in the action of LPC in the differentiation of monocytes
into mature dendritic cells belonged to the family of G
protein-coupled receptors, PTX (pertussis toxin; 100 ng/ml), known
to block G1 proteins, was added to the culture medium for 3 hours.
The culture medium was then changed and the LPC was added as
described in example 1.
[0088] The results given in table 5 show that preincubation of the
monocytes with PTX blocks the increase in CD86 induced by LPC,
suggesting that the action of LPC involves G1 protein-coupled
receptors.
TABLE-US-00005 TABLE 5 Inhibition of the expression of CD86 by the
"LPC" cells, with PTX Induction of CD86 LPC 100% LPC + PTX 10%
[0089] These receptors could in particular be the following
receptors: [0090] G2A receptor (G2A-R): it has recently been
demonstrated that LPC is a high-affinity ligand for the G2A
protein, a G protein coupled to a receptor expressed in
lymphocytes. In addition, the stimulation of G2A by LPC induces
phosphorylation of an extracellular kinase (ERK1/2: extracellular
signal-related kinases). This phosphorylation can, moreover, be
observed when LPC is added to the culture medium, suggesting the
involvement of the G2A receptor in the differentiation of monocytes
into mature dendritic cells by means of LPC, [0091] PAF receptor
(PAF-R) as described above; certain effects of LPC could involve
the PAF receptor in various types of cells, [0092] GPR4 receptor:
LPC is also a ligand for the GPR4 protein, another G protein
coupled to a receptor having a high affinity for
sphingosylphosphorylcholine.
[0093] Next, the inventors investigated the transcription factors
involved in the maturation process induced by LPC. In order to
determine whether PPARs are involved in the LPC-induced maturation,
the ability of the two transcription factors PPAR.gamma. and
PPAR.delta. to bind was analyzed using the gel shift technique. 3
isotypes of PPAR nuclear receptors (peroxisome
proliferator-activated receptor) exist: .alpha., .gamma. and
.delta.. PPAR.gamma.s are the targets for molecules of the
thiazolidinedione class, such as ciglitizone, used in the treatment
of type II diabetes. PPARs have a broader expression and can be
repressors of PPAR.gamma.s. The monocytes undergoing
differentiation were incubated for two hours with a solution of LPC
(40 .mu.M) ("LPC" cells). "Control" cells are also obtained in the
absence of LPC in the culture medium. After having harvested the
cells, the nuclear proteins were extracted with the Nuclear extract
Kit (Sigma). The nuclear proteins were then brought into contact
with a ratioactively labeled probe containing a response element
recognized by PPARs. After migration on a nondenaturing gel, a band
containing PPAR.gamma. and a doublet containing PPAR were
identified using antibodies, by the Supershift technique. The PPAR
binding activity was determined by measuring the intensity of these
bands. These activities are expressed as a percentage with respect
to the "control" cells.
TABLE-US-00006 TABLE 6 Modulation of the activity of PPAR.gamma.
and PPAR.delta. by LPC PPAR.gamma. PPAR.delta. Control 100% 100%
LPC 0% 263%
[0094] The treatment with LPC induces a large decrease in the
binding activity of PPAR.gamma., which can result in complete
disappearance, and greatly stimulates the activity of
PPAR.delta..
[0095] The inventors then used a PPAR.gamma. agonist, ciglitizone.
A solution of ciglitizone (Sigma, 50 .mu.M) was added to the
culture medium on the fifth day of monocyte differentiation, as
described in example 4, minutes before the addition of a solution
of LPC (40 .mu.M, for 2 hours). The cells obtained are
"LPC+ciglitizone" cells. "Ciglitizone" cells were obtained
according to the same protocol, but in the absence of LPC, and
"LPC" cells were obtained as described in example 1, in the absence
of ciglitizone.
[0096] The activity of the PPAR.gamma. and PPAR.delta.
transcription factors was measured in these cells by the gel shift
technique, as described above.
TABLE-US-00007 TABLE 7 Ciglitizone blocks the inactivation of
PPAR.gamma. and reduces the LPC-induced increase in PPAR.delta.
PPAR.gamma. PPAR.delta. Control 100% 100% Ciglitizone 237% 106% LPC
+ ciglitizone 115% 147%
[0097] These results indicate that ciglitizone, which activates
PPAR.gamma., greatly inhibits the effect of LPC on PPAR.gamma. and
PPAR.delta., and as a result blocks the LPC-induced maturation of
dendritic cells.
[0098] The functional ability of these cells ("control",
"ciglitazone" and "LPC+ciglitazone" cells) to stimulate T
lymphocytes (LT) were then analyzed. These cells were cultured as
described in example 4, in the presence of purified allogenic T
lymphocytes. The secretion of .gamma.IFN was measured as in example
4.
TABLE-US-00008 TABLE 8 Mixed leukocyte reaction: measurement of the
dendritic cell-induced secretion of .gamma.IFN by T lymphocytes
DC/LT ratio 0 0.05 0.1 0.2 Control 0 54 .+-. 82 69 .+-. 4 364 .+-.
27 LPC 0 750 .+-. 184 899 .+-. 124 1326 .+-. 248 LPC + ciglitizone
0 93 .+-. 7 169 .+-. 105 484 .+-. 229
[0099] All these results emphasize an important role for PPARs in
the LPC-induced maturation of dendritic cells and also the
importance of the PPAR.gamma./PPAR.delta. ratio in the production
of functionally mature dendritic cells. This ratio is modulated by
LPC and ciglitizone.
EXAMPLE 7
Differentiation of Monocytes into Mature Dendritic Cells in the
Presence of Compounds Equivalent to LPC
[0100] The inventors determined whether compounds equivalent to
LPC, i.e. involving the same cellular mechanisms of action via the
same membrane or intracellular receptors, could also induce the
differentiation of monocytes into mature dendritic cells.
[0101] For this, the inventors investigated whether the addition of
PAF to the culture medium could increase the action of LPC on the
differentiation of monocytes into mature dendritic cells.
[0102] Thus, a solution of PAF (Sigma, 5 .mu.M) was added to the
culture medium on the 5th day of monocyte differentiation. The
expression of CD86 by the "control" cells and by the "LPC" cells,
obtained in the presence or in the absence of PAF, was analyzed
according to the protocol described in example 2, and the results
are given in table 9.
TABLE-US-00009 TABLE 9 Expression of the CD86 marker (mean
fluorescence intensity) by the "control" and "LPC" cells in the
presence or in the absence of PAF CD86 Control 49.02 PAF 102.88 LPC
287.11 LPC + PAF 676.88
[0103] These results suggest that PAF alone induces a slight
overexpression of CD86, unlike LPC, which induces a 470% increase
in the expression of CD86. On the other hand, it is important to
note that PAF acts in synergy with LPC, since the action of the two
compounds induces a 1200% increase in the expression of CD86.
[0104] In order to reinforce the idea that the PAF receptor is
involved, the inventors studied the action of an antagonist for
this receptor on the differentiation of monocytes into mature
dendritic cells by means of LPC.
[0105] Thus, a solution of the PAF antagonist BN52021 (Biomol,
Plymouth Meeting, USA, 100 .mu.M) was added to the culture medium
on the 5th day of monocyte differentiation. The expression of CD86
by the "control" and "LPC" cells, obtained in the presence or in
the absence of BN52021, was analyzed according to the protocol
described in example 2, and the results are given in table 10.
TABLE-US-00010 TABLE 10 Expression of CD86 by the "LPC" cells in
the presence of the antagonist BN52021 CD86 LPC 100% LPC + BN52021
54%
[0106] These results suggest that the action of LPC clearly
involves the PAF receptor, but also suggest that the action of LPC
could involve other receptors.
[0107] By way of indication, the inventors also demonstrated that
the antagonist BN52021, added to the culture medium on the 5th day
of differentiation of the monocytes into mature dendritic cells
induced by oxidized LDLs, induced a 100% inhibition of the action
of the oxidized LDLs, suggesting here again that the mechanisms of
action of LPC and of oxidized LDLs are different.
EXAMPLE 8
In Vivo Stimulation of the Immune Response Against an Antigen, by
LPC
[0108] The aim of this example is to show that LPC is a molecule
which is an adjuvant of the immune system and which can be used in
the context of immunization in order to increase the specific T
response against an antigen.
[0109] LPC is an inflammatory product--In this example, a solution
of 100 to 500 nmol of LPC dissolved in 50 .mu.l of PBS is injected
into the plantar foot pad of BALB/c mice (Charles River
Laboratories) on day 0. The plantar foot pads are measured using a
micrometer before and after injection, up to the 10th day, and
compared to the plantar foot pads of "control" mice, obtained by
injection of PBS (50 .mu.l). The intensity of the inflammation is
reflected by the thickening of the foot. The maximum thickening is
observed on the 1st day, 24 h after the injection.
TABLE-US-00011 TABLE 11 Inflammation of the plantar foot pad
induced by LPC-thickening of the foot after 24 h (in mm) Thickening
of the foot (mm) PBS 0.025 LPC 100 nmol 0.35 LPC 200 nmol 0.55 LPC
300 nmol 0.725 LPC 400 nmol 1.125 LPC 500 nmol 1.075
[0110] These results show that LPC induces an inflammation
dependent on the dose injected.
[0111] In order to show that LPC induces the maturation and
therefore the migration of dendritic cells to the draining lymph
nodes, in vivo, a solution of LPC (0.1 M in dibutyl phthalate) was
applied to the skin of the ears of BALB/c mice (Charles River
Laboratories), 10 minutes before application of a solution of FITC
(Sigma) at 1.5% in 1:1 dibutyl phthalate/acetone. This fluorescent
label is taken up by the dendritic cells of the skin. After 24 h,
the auricular and maxillary lymph nodes are removed and the cells
are placed in suspension. The cell suspension is enriched in
dendritic cells by metrizamide (Sigma) gradient centrifugation. The
cells are labeled with an antibody (Pharmingen) which recognizes
Major Histocompatibility Complex (MHC) class I molecules and
analysis by flow cytometry makes it possible to quantify the
percentage of large cells (dendritic cells) expressing the MHC
class II molecules and containing FITC. These cells are presenting
cells which have taken up the FITC in the periphery and have
migrated to the lymph nodes.
TABLE-US-00012 TABLE 12 Stimulation of dendritic cell migration by
LPC FITC.sup.+ dendritic cells FITC 7.1 .+-. 1.7% LPC + FITC 19.3
.+-. 3.3%
[0112] These results show that the application of LPC stimulates
the migration of dendritic cells of the skin to the draining lymph
nodes.
[0113] LPC stimulates the T response specific for a co-injected
soluble antigen. LPC dissolved in PBS (250 or 500 nmol in 50 .mu.l)
is mixed with hen egg lysozyme (HEL, 50 .mu.g) and this mixture is
injected into the plantar foot pad of BALB/c mice. After 7 days,
the popliteal lymph nodes are removed and their cells are
restimulated in triplicate in vitro, in a culture medium containing
30 .mu.M of HEL antigen, or in the absence of HEL. After 3 days,
the T lymphocyte proliferation is measured by incorporation of
triturated thymidine for 16 h.
TABLE-US-00013 TABLE 13 Proliferation of HEL-specific T cells
(incorporation of triturated thymidine in CPM; mean of triplicates)
Immunization Proliferation in the Restimulation with conditions
absence of HEL HEL (30 .mu.M) HEL 1315 .+-. 275 5370 .+-. 1983 HEL
+ LPC 250 nmol 1608 .+-. 7 10885 .+-. 2861 HEL + LPC 500 nmol 2101
.+-. 87 23739 .+-. 4265
[0114] These results show that LPC co-injected with the antigen
promotes the activation in vivo of T lymphocytes specific for this
antigen.
[0115] LPC stimulates the production of specific antibodies against
a co-injected soluble antigen. LPC dissolved in PBS (350 nmol in 50
.mu.l) is mixed with hen egg lysozyme (HEL, 1 mg/ml) and this
mixture is injected into the plantar foot pad of BALB/c mice. The
"HEL+LPC" batch of mice receives 50 .mu.l of this mixture in the
foot pads of the two hind feet. The "HEL" batch of mice receives 50
.mu.l of HEL solution (1 mg/ml in PBS) in the two feet. After 15
days, a booster injection is given subcutaneously on the 2 flanks.
The "HEL+LPC" batch receives, on each flank, 100 .mu.l of a
solution of LPC (5 mM) and of HEL (1 mg/ml) in PBS. The "HEL" batch
receives, on each flank, 100 .mu.l of a solution of HEL (1 mg/ml)
in PBS. Two weeks later, blood is taken from the mice and the IgGs
specific for the HEL antigen are assayed by ELISA with respect to a
standard IgG solution range. The titration curves giving the
absorbance (Abs) as a function of the logarithm of the serum
dilution are represented in FIG. 1.
[0116] These results suggest that LPC co-injected with the antigen
promotes activation of the immune system and induces the synthesis
of antibodies specific for the antigen, whereas injection of the
antigen alone does not induce a response. LPC induces a humoral
response against the antigen, demonstrating its adjuvant
capacity.
[0117] LPC can induce a CD8+T lymphocyte response in vivo. In this
example, a test for delayed contact hypersensitivity to haptens was
used. In this model, BALB/c mice are sensitized by application of a
hapten, DNFB (1-fluoro-2,4-dinitrobenzene, 0.5% solution in 1:1
olive oil/acetone), to the back. Five days later, a non-irritant
dose of DNFB (0.2% solution in 1:1 olive oil/acetone) is applied to
the left ear, whereas the right ear receives the solvent. In this
elicitation phase, CD8+T lymphocytes specific for the haptenized
proteins are recruited and infiltrate the ear, secreting .gamma.IFN
and producing an edema.
[0118] A "control" batch of mice was sensitized with DNFB alone and
another batch ("LPC" mice) received 500 nmol of LPC by application
to the skin of the back of 20 .mu.l of an ethanolic solution of
LPC, 10 minutes before the application of DNFB. Five days later,
the two batches were treated in the same way for the elicitation
phase and the thickening of the ears was measured using a
micrometer 48 h after this application.
TABLE-US-00014 TABLE 14 Measurement of the ear edema Thickening of
the Thickening of the right ear (.mu.m) left ear (.mu.m) Control 0
.+-. 4 67 .+-. 32 LPC 0 .+-. 3 149 .+-. 31
[0119] These results show that the application of LPC 10 minutes
before the hapten during the sensitization phase increases the
edema induced during the 2nd application of the hapten
(elicitation). This means that LPC stimulates the response relayed
by CD8+T lymphocytes.
EXAMPLE 9
Action of a Lipid Emulsion Such as INTRALIPID.RTM. on the
Differentiation of Monocytes into Mature Dendritic Cells
[0120] Since it was shown in example 5 that a lipid emulsion such
as INTRALIPID.RTM. blocks the LPC-induced production of mature
dendritic cells, the inventors also investigated whether this
blocking came from an indirect action of this lipid emulsion via
inhibition of the action of LPC and/or from a direct action of this
lipid emulsion.
[0121] INTRALIPID.RTM. regulates PPARs in vitro. Initially, a
solution of INTRALIPID.RTM. (50 .mu.g/ml of phospholipids) was
added to the culture medium on the 5th day of monocyte
differentiation, according to a protocol comparable to that
described in example 6. The cells were harvested after 1 h, 2 h or
8 h of incubation with INTRALIPID.RTM., the nuclear proteins were
extracted and the activity of PPAR.gamma. and PPAR.delta. was
determined as described in example 6.
TABLE-US-00015 TABLE 15 Modulation of the activity of PPAR.gamma.
and PPAR.delta. of monocytes undergoing differentiation, by
INTRALIPID .RTM. Incubation time with Intralipid (h) PPAR.gamma.
PPAR.delta. 0 100% 100% 1 130% 94% 2 160% 94% 8 170% 77%
[0122] These results show that INTRALIPID.RTM. alone activates
PPAR.gamma. and inhibits the activity of PPAR.delta. This action of
INTRALIPID.RTM. is opposite to that of LPC.
[0123] Secondly, a solution of INTRALIPID.RTM. (50 .mu.g/ml of
phospholipids) was added to the culture medium on the 5th day of
monocyte differentiation, 15 minutes before the addition of a
solution of LPC (40 .mu.M) ("LPC+Intralipid" cells). "Intralipid"
cells were also obtained in the absence of LPC. "LPC" cells were
obtained in the absence of INTRALIPID.RTM.. The cells were
harvested after 2 h of incubation, the nuclear proteins were
extracted and the activity of PPAR.gamma. and PPAR.delta. was
determined as described in example 6.
TABLE-US-00016 TABLE 16 INTRALIPID .RTM. blocks the LPC-induced
generation of mature dendritic cells by modulating the activity of
PPAR.gamma. and PPAR.delta. PPAR.gamma. PPAR.delta. Control 100%
100% Intralipid 156% 99% LPC 1% 160% LPC + Intralipid 40% 80%
[0124] INTRALIPID.RTM. also blocks the action of LPC by modulating
the activity of PPAR.gamma. and PPAR.delta..
[0125] INTRALIPID.RTM. blocks the inflammation and the immune
response induced by LPC, in vivo. In this example, 3 batches of
BALB/c mice are immunized against the hen egg lysozyme (HEL)
antigen by injection of 50 .mu.l of solution into the plantar foot
pad. The "HEL" control batch receives 50 .mu.g of HEL, the
"HEL+Intralipid" batch receives a mixture of INTRALIPID.RTM. (45
.mu.l) and of 50 .mu.g of HEL (5 .mu.l of a solution at 10 mg/ml).
The "BEL+LPC" batch receives 50 .mu.g of HEL with 350 nmol of LPC
dissolved in PBS. The "HEL+LPC+Intralipid" batch receives 50 .mu.g
of HEL mixed with 350 nmol of LPC dissolved in 45 .mu.l of
INTRALIPID.RTM.. After 24 hours, the thickness of the foot pads is
measured using a micrometer, and compared with the measurement
taken before injection. The difference in thickness is proportional
to the intensity of the inflammation.
TABLE-US-00017 TABLE 17 Intralipid reduces the inflammation induced
in vivo by LPC Average thickness of the foot at 24 h (mm) HEL +
Intralipid 0.19 .+-. 0.04 HEL + LPC 1.07 .+-. 0.19 HEL + LPC +
Intralipid 0.45 .+-. 0.04
[0126] These results show that injecting INTRALIPID.RTM. at the
same time as LPC reduces the LPC-induced inflammation of the
plantar foot pad by 70%.
[0127] Seven days after the injection, the popliteal lymph nodes
are removed and their cells are restimulated in vitro in
triplicate, in a culture medium containing 10 .mu.M of HEL or in
the absence of HEL. After 3 days, the proliferation of HEL-specific
T lymphocytes is measured by incorporation of triturated thymidine
for 16 h.
TABLE-US-00018 TABLE 18 Proliferation of HEL-specific T cells (mean
of the mice) Proliferation in the Restimulation with Immunization
conditions absence of HEL (cpm) HEL (10 .mu.M) (cpm) HEL 370 .+-.
212 738 .+-. 408 HEL + LPC 2568 .+-. 1570 11340 .+-. 4426 HEL + LPC
+ Intralipid 1124 .+-. 541 3959 .+-. 2103
[0128] These results show that INTRALIPID.RTM. injected at the same
time as LPC blocks the LPC-induced stimulation of the T
response.
[0129] All these results show that INTRALIPID.RTM. blocks the
LPC-induced generation of mature dendritic cells by means of an
indirect action of INTRALIPID.RTM. via inhibition of the action of
LPC, but also by means of a direct action of INTRALIPID.RTM. on
PPARs, antagonist for LPC. These results show that INTRALIPID.RTM.
alone has an anti-inflammatory role.
[0130] All these results show the importance of the
PPAR.gamma./PPAR.delta. ratio in the generation of functionally
mature dendritic cells. This ratio is modulated by LPC, but also,
inversely, by a lipid emulsion such as INTRALIPID.RTM. or
ciglitizone.
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