WO2001005423A1 - Use of vasoactive intestinal peptide (vip) for modulating the development of a specific immune response - Google Patents
Use of vasoactive intestinal peptide (vip) for modulating the development of a specific immune response Download PDFInfo
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- WO2001005423A1 WO2001005423A1 PCT/FR2000/002037 FR0002037W WO0105423A1 WO 2001005423 A1 WO2001005423 A1 WO 2001005423A1 FR 0002037 W FR0002037 W FR 0002037W WO 0105423 A1 WO0105423 A1 WO 0105423A1
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- vip
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- use according
- medicament
- cells
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Classifications
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- C—CHEMISTRY; METALLURGY
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- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0634—Cells from the blood or the immune system
- C12N5/0639—Dendritic cells, e.g. Langherhans cells in the epidermis
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- A61K38/22—Hormones
- A61K38/2278—Vasoactive intestinal peptide [VIP]; Related peptides (e.g. Exendin)
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P37/00—Drugs for immunological or allergic disorders
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
- A61K2039/515—Animal cells
- A61K2039/5154—Antigen presenting cells [APCs], e.g. dendritic cells or macrophages
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/20—Cytokines; Chemokines
- C12N2501/25—Tumour necrosing factors [TNF]
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/30—Hormones
- C12N2501/35—Vasoactive intestinal peptide [VIP]; Pituitary adenylate cyclase activating polypeptide [PACAP]
Definitions
- the present invention relates to the use of a peptide, the peptide
- VIP intestinal vasoactive peptide
- VIP intestinal vasoactive peptide
- VIP is a peptide of 28 amino acids widely distributed in the peripheral and central nervous system (Goetzl EJ et al, Ann NY Acad Sci (1998) 840, 540 & Calvo JR et al, Adv Neurosci (1996) 6, 39) Nerve fibers containing VIP have been identified in the primary and secondary lymphoid organs, vessels, respiratory tracts
- VIP 15 and intestinal as well as in the skin VIP is released at functional concentrations in many immune and inflammatory responses VIP concentrations are increased in the sera of patients with septic shock, and in the nasal secretions and bronchoalveolar lavage of allergic subject after contact with a
- VIP acts by using two types of receptors coupled to G proteins (VIP-RI and VIP-RII) expressed on the cells of the system
- VIP is a powerful vasodilator which increases the venule-capillary permeability and therefore participates in the inflammatory process VIP also modulates the immune response and in particular recruitment and activation leukocytes.
- VIP modulates the production of lymphokines by T lymphocytes (Ganea D et al, J Neurosci (1993) 48, 59), inhibits the production of IL-6, IL-12 and TNF ⁇ by monocytes and macrophages induced by lipopolysaccharide (LPS) (Delgado M et al, J leukocyte Biol (1998) 63, 591; Xin Z et al J Neuroimmunol (1998) 89, 206; Dewit D et al Immunol Lett (1998) 60, 57) and protects LPS-induced shock mice by inhibiting the production of TNF ⁇ and IL-6 (Delgado M et al J Immunol (1999) 162, 1200).
- LPS lipopolysaccharide
- VIP also modulates the production of immunoglobulins by B lymphocytes (Kimata H et al J Immunol (1993) 150, 4630), induces degranulation of mast cells and is mitogenic for connective tissue, epitheal cells and keratinocytes (Pincelli C et al, J Invest Dermatol (1997) 98, 421).
- VIP and its receptor agonists are proposed as vasodilators but also in allergy and bronchospasm as bronchodilators (WO 9735561 & JP 56128721).
- Dendic cells play a role in the development of an immune response and in the initiation of a specific T lymphocyte response (Steinman RM et al Immuno Rev (1997) 156, 25 & Sella M et al Curr Opin Immunol (1997 ) 9, 10).
- Immature dendritic cells are located in non-lymphoid tissue. At the level of the skin, in all the epidermis except the intestine, they are then called Langerhans cells; dendritic cells, in smaller quantities, are also localized in organs such as the lung, liver, intestine. These immature dendritic cells capture and digest antigens very efficiently.
- the dendritic cells After antigenic stimulation in vivo or stimulation by proinflammatory molecules, the dendritic cells which have captured the antigens migrate into the secondary lymphoid organs. During this migration, the dendritic cells undergo functional and phenotypic modifications which are grouped under the term of maturation. This maturation is characterized by an increase on the surface of dendritic cells of molecules involved in the activation of T lymphocytes (such as CD40, CD54, CD58 and MHC class l / ll), the production of cytokines (such as TNF ⁇ and IL-12) and the induction of surface expression of molecules such as CD83 (for human cells).
- T lymphocytes such as CD40, CD54, CD58 and MHC class l / ll
- cytokines such as TNF ⁇ and IL-12
- CD83 for human cells.
- dendritic cells present the antigens to the T lymphocytes and initiate the specific T responses very efficiently, the costimulation molecules being expressed in large quantities on these cells. As cells mature, they lose their ability to capture and process the antigen. In the thymo-dependent areas of the lymphoid organs, the migrated dendritic cells have acquired powerful immunostimulatory properties and will therefore very effectively activate the circulating naive T lymphocytes.
- VIP induces the maturation of human dendritic cells, thus allowing the development of a specific T lymphocyte response.
- the present invention relates to the use of at least one compound chosen from the group formed by VIP, VIP receptor agonists, VIP receptor antagonists for the manufacture of a medicament intended to modulate the maturation of dendritic cells.
- This drug can be applied to the skin, in particular by the cutaneous, subcutaneous, transdermal, intra-epidermal route or to the mucous membranes, it then acts systemically by the maturation of the dendritic cells and locally by the maturation of the cells of Langerhans.
- This drug can be used to promote the generation and maturation of dendritic cells in vitro after contact with a biological agent, the mature cells then being reinjected in vivo.
- the dendritic cells are modified so that they express tumor antigens.
- Said medicament may be used in vitro, in order to promote the maturation of the dendritic cells before reinjecting them, or this medicament may be injected at the same time as the dendritic cells.
- This biological agent is then chosen from the group formed by nucleic acid, proteins, lipids, lipopeptides, polysaccharides.
- This medication is the treatment or prevention of psoriasis.
- the maturation of dendritic cells has the effect of amplifying the development of a specific immune response. It has been demonstrated that VIP and VIP receptor agonists can be used to enhance the development of a specific immune response while the use of VIP receptor antagonists can be used to prevent initiation or slow down the development of an unwanted specific immune response. Different uses of VIP and VIP receptor agonists have been considered, these uses are presented below.
- VIP and VIP receptor agonists as an adjuvant or immunostimulant in vaccinology in anti-HIV vaccines infectious and anti-cancer and this in combination with vaccinating antigens and / or other compounds which can be a bacterium, a virus, a yeast, a fungus in order to produce therapeutic vaccines.
- Administering VIP along with the antigen will increase the presentation of the antigen by dendritic cells and thereby the effectiveness of the vaccine.
- VIP and VIP receptor agonists as an immunostimulant in the treatment of acquired congenital immunodeficiencies or of pathological origin. It may especially be the treatment of congenital or acquired immune deficiencies, but also treatments in patients with an absent or deficient immune response, for example to a tumor or a pathogen.
- VIP and VIP receptor agonists may be used in particular for the treatment of: - microbial infections, in particular chronic infections associated with the development of an ineffective specific immune response (virus: for example the immunodeficiency virus human (HIV), hepatic viruses, in particular hepatic viruses A, B, C and D and the parainfluenza virus, bacteria, parasites and yeasts), - cancers, in particular in subjects carrying HIV and / or suffering from myelomas, lymphomas, leukemias, melanomas, carcinomas, kidney, brain, prostate, rectum, pancreas, ovaries, lung, for example.
- virus for example the immunodeficiency virus human (HIV), hepatic viruses, in particular hepatic viruses A, B, C and D and the parainfluenza virus, bacteria, parasites and yeasts
- - cancers in particular in subjects carrying HIV and / or suffering from myelomas, lymphomas, leukemias, melanomas, carcinomas, kidney
- the present invention particularly relates to the use of at least one compound chosen from the group formed by VIP and VIP receptor agonists with at least one biological agent for the manufacture of a medicament for increasing the immunological response vis with respect to this biological agent.
- This medication can be a vaccine for the treatment or prevention of infectious diseases of viral origin - such as in particular HIV, hepatic viruses and paraifluenza virus -, bacterial, fungal, or caused by is a yeast or a parasite.
- the present invention is also the use of at least one compound chosen from the group formed by VIP and VIP receptor agonists with at least one biological agent for the manufacture of a medicament for the treatment or prevention of cancers and particularly cancers among myelomas, lymphomas, leukemias, carcinomas of the kidney, brain, prostate, rectum, pancreas, ovaries, lung, and for the manufacture of a medicament for the treatment or prevention of skin cancers chosen from keratinomas and carcinomas.
- the cancer cells are in direct contact with the Langerhans cells, located in the epidermis, the use according to the present invention by skin application makes it possible to act directly, locally on Langerhans cells.
- an advantage of the present invention is that the association VIP (or VIP receptor agonist or VIP receptor antagonist) - biological agent can be in an easily administered form such as an ointment, a lotion, a solution or else in the form of an adhesive composition: plaster, "patch".
- the treatment of these cancers can also be envisaged by injecting autologous dendritic cells, and in particular cells
- the use of at least one compound chosen from the group formed by VIP and VIP receptor agonists with at least one biological agent associated with TNF ⁇ can be used for the manufacture of a medicament intended to increase the proliferation of T cells. Consequently, the present invention also covers the use of at least one compound chosen from the group formed by VIP and VIP receptor agonists with at least one biological agent which is a tumor biological agent for the manufacture of a drug to promote a specific anti-tumor immune response.
- VIP receptor antagonists can be used to prevent the development of an undesirable specific immune response to an endogenous or exogenous antigen.
- VIP receptor antagonists may in particular be used in autoimmune diseases (rheumatoid arthritis and type I diabetes) and pathologies of allergic origin (allergic asthma and atopic dermatitis).
- the VIP receptor antagonist molecules can be used by different routes of introduction depending on the route of entry of the antigen (exogenous) or its production site (endogenous) and this in order to act locally. by preventing the development of an immune response (in cosmetics in the case of atopic dermatitis and skin allergies).
- the present invention also relates to the use of at least one antagonist of the VIP receptors for the manufacture of a medicament intended to decrease the intensity of a specific immune response, in particular to decrease the intensity of an autoimmune disease, for example of the rheumatoid arthritis type or type I diabetes or for the manufacture of a medicament intended to treat pathologies of allergic origin such as allergic asthma and atopic dermatitis.
- VIP induces the maturation of immature human dendritic cells and gives them powerful stimulatory and antigen presenting properties.
- This example demonstrates the action of VIP as a modulator of the expression of molecules expressed on the surface of immature human dendritic cells by:
- CMN Mononuclear cells
- the monocytes are purified by positive selection using a magnetic cell separator (MACS TM; Miltenyi Biotex, Bergisch Gladbach, Germany) in accordance with the manufacturer's instructions. Briefly, the CMNs are incubated for 20 minutes at 4 ° C. with magnetic beads onto which a monoclonal anti-human CD14 antibody is fixed. After washing, the cell suspension plus beads is placed on a column and subjected to a magnetic field. After three washes, the column is no longer subjected to the magnetic field and the monocytes are collected by gravitation.
- MCS TM magnetic cell separator
- the purity of the monocytes is evaluated by cytofluorometry (FACScan cytofluorometer; Becton Dickinson, Erembodegem, Belgium) on the basis of the size-granulosity parameters of the cells. The purity is greater than 98%.
- the monocytes are then cultured at a concentration of 10 6 cells / ml in the following medium (hereinafter called complete culture medium): RPMI 1640 medium supplemented with 10% fetal calf serum (heating at 56 ° C for 30 minutes), 2 mM L-glutamine, 50 U / ml of penicillin and 50 ug / ml of streptomycin (Life technologies) in 6-well culture plates ( Nunc, Roskilde, Denmark) at a rate of 5 ml of medium per well.
- the cells are activated with 20 ng / ml of recombinant human IL-4 and 20 ng / ml of recombinant human GM-CSF (R&D Systems, Abingdon, United Kingdom).
- the phenotype of the cells is defined by cytofluorometry. Briefly, an aliquot of the cell suspension is removed. The cells are washed in FACS buffer (10 mM phosphate buffer pH 7.4 containing 1% bovine serum albumin and 0.01% sodium azide) and then distributed in wells of a 96-well culture plate at the bottom conical (Nunc) at the rate of 2 ⁇ 10 5 cells in a volume of 50 ⁇ l of FACS buffer.
- FACS buffer (10 mM phosphate buffer pH 7.4 containing 1% bovine serum albumin and 0.01% sodium azide
- FIG. 1 represents the percentage of cells expressing the CD83 molecule (whose expression is restricted to mature dendritic cells) as a function of the VIP concentration in the culture.
- the curve in Figure 1 shows that
- the CD83 molecule is not expressed by immature human dendritic cells, - VIP induces the expression of the CD83 molecule in a concentration-dependent manner (significant at 10 "8 M and maximum at 10 " 6 M) on a fraction of immature dendritic cells (46% after 4 days of stimulation).
- the six-day immature dendritic cells presented above were collected, washed and then re-cultured in complete medium at the concentration of 10 5 cells in a volume of 200 ⁇ l in 96-well flat-bottom culture plates (Costar, Cambridge , USA).
- the cells are activated with VIP (Sigma, Saint Louis, USA).
- the expression of the molecules CD40, CD54, CD80, CD83, CD86 and HLA-Dr is evaluated by cytofluorometry using the following monoclonal antibodies: anti-CD40, anti-CD54, anti-CD80 antibody, anti-CD86 and anti-HLA-Dr labeled with fluorescein (Becton Dickinson) and anti-CD83 antibody revealed by an anti-mouse immunoglobulin antibody labeled with fluorescein (Silenus, Hauworth, Australia).
- the control isotypic antibodies used come from Becton Dickinson.
- the cells are washed in FACS buffer and then distributed in wells of a 96-well culture plate with a conical bottom at the rate of 2 ⁇ 10 5 cells in a volume of 50 ⁇ l of FACS buffer. An antibody is added to each well. After 20 minutes of incubation at 4 ° C, the cells are washed three times with 200 ⁇ l of FACS buffer and then are resuspended in 200 ⁇ l of this same buffer. The analysis of the expression of the surface markers is evaluated by FACS. The results show that VIP increases the expression of CD40, CD54, CD80 and HLA-DR on immature dendritic cells and induces the expression of CD83 on a fraction of dendritic cells (Table 1).
- Example 2 TNF ⁇ potentiates the effects of VIP on human dendritic cells.
- TNF ⁇ a proinflammatory mediator inducing the differentiation of dendritic cells, potentiates the effects of VIP on the activation and differentiation of human dendritic cells.
- Immature human dendritic cells were generated as described in Example 1. Immature dendritic cells were collected, washed and then re-cultured in complete medium at the concentration of 10 5 cells in a volume of 200 ⁇ l in culture plates 96 flat-bottomed wells (Costar, Cabridge, USA). The cells are activated with VIP (Sigma, Saint Louis, USA) in the absence or in the presence of 0.2 ng / ml of recombinant human TNF ⁇ (R&D Systems, Abingdon,
- LPS lipopolysaccharide
- HLA-Dr is evaluated by cytofluorometry using the following monoclonal antibodies: anti-CD40, anti-CD54, anti-CD80, anti-CD86 and anti-HLA- antibodies
- FIG. 2 shows the percentage of cells expressing the CD83 molecule as a function of the VIP concentration with an addition of 0.2 ng / ml (curve) of TNF ⁇ to the culture medium and without this addition (curve 2). The results on these cells after 4 days of culture are recorded in Table 1.
- the measurements marked “a” are expressed in mean intensity of MFI fluorescence after subtracting the value obtained with the control antibody.
- the measurements marked “b” are expressed as a percentage and value of the MFI of the positive cells.
- CD83 - TNF ⁇ at a concentration of 0.2 ng / ml potentiates the induction of the CD83 molecule induced by VIP on dendritic cells: the expression of CD83 is significantly induced at 10 "10 M (16% of cells express the CD83 molecule) and maximum at 10 "6 M (95% of cells express the CD83 molecule) (FIG. 2).
- Example 3 VIP induces the production of interleukin 12 by human dendritic cells. Potential effect of TNF ⁇ .
- VIP induces the production of IL-12 by dendritic cells.
- TNF ⁇ gamma interferon
- Immature human dendritic cells were generated as described in Example 1. After 6 days of culture, the cells are returned to culture in complete medium at the concentration of 10 5 cells in a volume of 200 ⁇ l in culture plates 96 flat bottom well. The cells are activated with VIP (10 "10 -10 " 6 M) in the absence or in the presence of 0.2 ng / ml of recombinant human TNF ⁇ . After 48 hours of culture, the culture supernatants are centrifuged at 10,000 rpm for 15 minutes at 4 ° C. Supernatants are collected. Bioactive IL-12 (also called heterodimer p40 / p75) is quantified using a commercial assay kit type ELISA (R&D Systems) according to the manufacturer's instructions.
- VIP 10 "10 -10 " 6 M
- the culture supernatants are centrifuged at 10,000 rpm for 15 minutes at 4 ° C. Supernatants are collected.
- Bioactive IL-12 also called heterodimer p40 / p75
- FIG. 3 shows the production of IL-12 by human dendritic cells as a function of the VIP concentration with an addition of 0.2 ng / ml of TNF ⁇ to the culture medium (curve 1) and without this addition (curve 2).
- IL-12 induced by VIP potentiates the production of IL-12 induced by VIP: the production of IL-12 is significant at 10 "8 M (8 ⁇ 3 pg / ml) and maximum at 10 "6 M VIP (22 ⁇ 2.5 pg / ml).
- Example 4 VIP and TNF ⁇ cooperate to confer on human dendritic cells costimulation properties
- Immature human dendritic cells are generated as described in Example 1. After 6 days of culture, the cells are washed in RPMI 1640 medium and then re-cultured in complete medium at a concentration of 2.5 ⁇ 10 5 cells / well in a 6-well culture plate (5 ml / well). The cells are not stimulated or they are stimulated in the presence of 0.2 ng / ml TNF ⁇ ; 10 "6 M VIP; 0.2 ng / ml TNF ⁇ and 10 " 6 M VIP or 20 ng / ml TNF ⁇ . After three days of culture, the cells are collected and washed three times in RPMI 1640 medium. The cells are then irradiated at 3000 rad.
- Human T cells freshly isolated from peripheral blood were prepared by the rosette technique with sheep erythrocytes. Briefly, the mononuclear cells are isolated on a Ficoll-Hypaque gradient, as described in example 1. The cells mononuclear cells are resuspended in complete medium at a concentration of 200 ⁇ 10 6 cells / ml and mixed with 1 ml of a 50% suspension of sheep erythrocytes (BioMérieux, Marcy l'Etoile, France). The cell suspension is incubated at 4 ° C overnight. After gentle resuspension, the T cells are isolated by centrifugation on a Ficoll-Hypaque gradient (1500 rpm for 30 minutes at room temperature).
- the T cell / erythrocyte complexes are collected at the bottom of the tube.
- the red blood cells are lysed by two successive hypotonic shocks.
- the purity of the cells thus isolated is evaluated by cytofluorometry using a human anti-CD3 antibody labeled with fluorescein (Becton Dickinson). Purity is greater than 95%.
- the cells are resuspended in the complete culture medium at a concentration of 2.5 ⁇ 10 5 cells / ml.
- the activated and irradiated dendritic cells are cultured at the concentration of 10 4 cells / 200 ⁇ l in a 96-well culture plate in the presence or not of 5 ⁇ 10 4 allogenic lymphocytes. After 5 days of culture, the proliferation of T cells is evaluated by measuring the incorporation of tritiated thymidine ( 3 H-Thy) (Amersham, Amersham, United Kingdom). Briefly, 0.25 ⁇ Ci of 3 H-Thy is added to each culture well. The incorporation of 3 H-Thy is measured by a liquid scintillation counter (Packard Instruments, Australia). The results are presented in counts per minute (cpm). Mixed lymphocyte reactions are performed with T lymphocytes from two different healthy donors.
- 3 H-Thy tritiated thymidine
- Figure 4 shows the cell proliferation rate in cpm of these two donors in the presence of the following stimuli: (1) no stimulus; (2) VIP (10 "6 M); (3) TNF ⁇ (0.2 ng / ml); (4) VIP (10 '6 M) + TNF ⁇ (0.2 ng / ml); (5) TNF ⁇ 20ng / ml.
- the results presented in Figure 4 show that:
- Example 5 VIP receptor antagonists prevent the effect of VIP on CD maturation
- Immature human dendritic cells are generated as described in Example 1. After 6 days of culture, the cells are washed in RPMI 1640 medium and then re-cultured in complete medium at a concentration of 2.5 ⁇ 10 5 cells / well in a 6-well culture plate (5 ml / well).
- the cells are stimulated with 0.2 ng / ml TNF ⁇ , 10 "6 M VIP, 0.2 ng / ml TNF ⁇ and 10 " 6 M VIP or 100 pg / ml of LPS in the absence (clear column, located at left) or in the presence (gray column, located in the center) of 5 ⁇ g / ml polymixin B or 10 "6 M of a receptor VIP antagonist (VIP 6-28, reference V4508) (Sigma) (Fishbein et al, Peptide (1994) 15, 95) (blackened column, right) After 4 days, the cells are labeled with an anti-CD83 antibody revealed by a fluorescein labeled anti-mouse immunoglobulin antibody (Silenus, Hauworth, Australia The labeling of the cells as well as the analysis by cytofluorometry were carried out as described in Example 1.
- Figure 5 shows the percentage of cells expressing the CD83 molecule in the presence of the following stimuli: no stimulus; VIP (10 "6 M); TNF ⁇ (0.2 ng / ml); VIP (10 " 6 M) + TNF ⁇ (0.2 ng / ml); LPS (100 pg / ml) in the absence and presence of polymixin B (5 g / ml); receptor VIP antagonist (10 "6 M).
- VIP receptor antagonists the following are inhibited (i) the induction of CD83 expression, (ii) the increase in the expression of molecules involved in the activation of the T lymphocyte such as: CD40, CD54, CD80 and HLA-Dr, (iii) the induction of the production of IL-12 and (iiii) the increased capacity of dendritic cells to induce a mixed lymphocyte reaction (compared to these immature dendritic cells).
Abstract
The invention concerns a novel effect of the vasoactive intestinal response (VIP) on its receptors showing that VIP and its antagonists exhibit novel therapeutic uses. It is shown that VIP induces maturation of dendritic cells and thereby promotes the development of a specific immune response.
Description
UTILISATON DU PEPTIDE INTESTINAL VASOACTIF (VIP) POUR MODULER LE DEVELOPPEMENT D'UNE REPONSE IIW'JNE SPECIFIQUEUSE OF VASOACTIVE INTESTINAL PEPTIDE (VIP) TO MODULATE THE DEVELOPMENT OF A SPECIFIC IIW'JNE RESPONSE
La présente invention concerne l'utilisation d'un peptide, le peptideThe present invention relates to the use of a peptide, the peptide
5 intestinal vasoactif, généralement nommé VIP (vasoactive intestinal peptide) pour induire la maturation des cellules dendntiques et ainsi favoriser le développement d'une réponse immune spécifique5 intestinal vasoactive, generally called VIP (intestinal vasoactive peptide) to induce the maturation of dendntic cells and thus promote the development of a specific immune response
Dans la suite de la présente description, l'expression « VIP » sera utilisée pour nommer le peptide intestinal vasoactifIn the remainder of this description, the expression "VIP" will be used to name the intestinal vasoactive peptide.
10 Le VIP est un peptide de 28 acides aminés largement distribué dans le système nerveux périphérique et central (Goetzl EJ et al, Ann N Y Acad Sci (1998) 840, 540 & Calvo JR et al, Adv Neurosci (1996) 6, 39) Des fibres nerveuses contenant du VIP ont été mises en évidence dans les organes lymphoides primaires et secondaires, les vaisseaux, les tractus respiratoires10 VIP is a peptide of 28 amino acids widely distributed in the peripheral and central nervous system (Goetzl EJ et al, Ann NY Acad Sci (1998) 840, 540 & Calvo JR et al, Adv Neurosci (1996) 6, 39) Nerve fibers containing VIP have been identified in the primary and secondary lymphoid organs, vessels, respiratory tracts
15 et intestinaux ainsi qu'au niveau de la peau Le VIP est libéré à des concentrations fonctionnelles dans de nombreuses réponses immune et inflammatoire Les concentrations de VIP sont augmentées dans les serums de patients atteints de choc septique, et dans les sécrétions nasales et dans le lavage bronchoalvéolaire de sujet allergique après contact avec un15 and intestinal as well as in the skin VIP is released at functional concentrations in many immune and inflammatory responses VIP concentrations are increased in the sera of patients with septic shock, and in the nasal secretions and bronchoalveolar lavage of allergic subject after contact with a
20 allergene Des taux élevés de VIP sont également trouvés dans la peau de patients atteints de dermatite atopique ou de psoriasis comparativement a des sujets sains20 allergen Elevated VIP levels are also found in the skin of patients with atopic dermatitis or psoriasis compared to healthy subjects
Le VIP agit en utilisant deux types de récepteurs couplés à des protéines G (VIP-RI et VIP-RII) exprimées sur les cellules du systèmeVIP acts by using two types of receptors coupled to G proteins (VIP-RI and VIP-RII) expressed on the cells of the system
25 immunitaire (Harmar AJ et al, Pharmacological Reviews (1998) 50, 265) Le VIP est un puissant vasodilatateur qui augmente la perméabilité venule- capillaire et donc participe au processus inflammatoire Le VIP module aussi la réponse immune et en particulier le recrutement et l'activation des
leucocytes. Le VIP module la production des lymphokines par les lymphocytes T (Ganea D et al, J Neurosci (1993) 48, 59), inhibe la production d'IL-6, d'IL-12 et de TNFα par les monocytes et macrophages induite par le lipopolysaccharide (LPS) (Delgado M et al, J leukocyte Biol (1998) 63, 591 ; Xin Z et al J Neuroimmunol (1998) 89, 206 ; Dewit D et al Immunol Lett (1998) 60, 57) et protège des souris d'un choc induit par le LPS en inhibant la production de TNFα et d'IL-6 (Delgado M et al J Immunol (1999) 162, 1200). Le VIP module également la production d'immunoglobulines par les lymphocytes B (Kimata H et al J Immunol (1993) 150, 4630), induit la dégranulation des mastocytes et est mitogénique pour le tissu conjonctif, les cellules épithéiiales et les kératinocytes (Pincelli C et al, J Invest Dermatol (1997) 98, 421 ).25 immune system (Harmar AJ et al, Pharmacological Reviews (1998) 50, 265) VIP is a powerful vasodilator which increases the venule-capillary permeability and therefore participates in the inflammatory process VIP also modulates the immune response and in particular recruitment and activation leukocytes. VIP modulates the production of lymphokines by T lymphocytes (Ganea D et al, J Neurosci (1993) 48, 59), inhibits the production of IL-6, IL-12 and TNFα by monocytes and macrophages induced by lipopolysaccharide (LPS) (Delgado M et al, J leukocyte Biol (1998) 63, 591; Xin Z et al J Neuroimmunol (1998) 89, 206; Dewit D et al Immunol Lett (1998) 60, 57) and protects LPS-induced shock mice by inhibiting the production of TNFα and IL-6 (Delgado M et al J Immunol (1999) 162, 1200). VIP also modulates the production of immunoglobulins by B lymphocytes (Kimata H et al J Immunol (1993) 150, 4630), induces degranulation of mast cells and is mitogenic for connective tissue, epitheal cells and keratinocytes (Pincelli C et al, J Invest Dermatol (1997) 98, 421).
Dès 1996, Bellinger et al. dans « The significance of VIP in immunomodulation » (Advances In Neuroimmunology (Adv. Neuroimmunol), 1996, vol : 6(1 ), p. 5-27) note qu'il découle de plusieurs études que le VIP a une influence sur les fonctions immunitaires mais que son moyen d'action n'est pas expliqué.As early as 1996, Bellinger et al. in "The significance of VIP in immunomodulation" (Advances In Neuroimmunology (Adv. Neuroimmunol), 1996, vol: 6 (1), p. 5-27) notes that it follows from several studies that VIP has an influence on immune functions but that its means of action is not explained.
De par ces propriétés, le VIP et les agonistes de ses récepteurs sont proposés en tant que vasodilatateurs mais également dans l'allergie et bronchospasme en tant que bronchodilatateurs (WO 9735561 & JP 56128721 ).Due to these properties, VIP and its receptor agonists are proposed as vasodilators but also in allergy and bronchospasm as bronchodilators (WO 9735561 & JP 56128721).
Les cellules dend tiques jouent un rôle dans le développement d'une réponse immune et dans l'initiation d'une réponse lymphocytaire T spécifique (Steinman RM et al Immuno Rev (1997) 156, 25 & Sella M et al Curr Opin Immunol (1997) 9, 10). Les cellules dendritiques immatures sont localisées dans les tissus non lymphoïdes. Au niveau de la peau, dans tous les épidermes hormis l'intestin, elles sont alors appelées cellules de Langerhans ; des cellules dendritiques, en plus faible quantité, sont aussi
localisées dans les organes tels que le poumon, le foie, l'intestin. Ces cellules dendritiques immatures captent et digèrent les antigènes de façon très efficace. Après une stimulation antigénique in vivo ou stimulation par des molécules proinflammatoires, les cellules dendritiques qui ont capté les antigènes migrent dans les organes lymphoïdes secondaires. Durant cette migration, les cellules dendritiques subissent des modifications fonctionnelles et phénotypiques qui sont regroupées sous le terme de maturation. Cette maturation se caractérise par une augmentation à la surface des cellules dendritiques de molécules impliquées dans l'activation des lymphocytes T (telles que CD40, CD54, CD58, CD86 et MHC classe l/ll), la production de cytokines (telles que TNFα et IL-12) et l'induction de l'expression en surface de molécule telle que CD83 (pour les cellules humaines). Ces cellules dendritiques matures présentent les antigènes aux lymphocytes T et initient les réponses T spécifiques de façon très efficace, les molécules de costimulation étant exprimées en quantité importante sur ces cellules. Les cellules en devenant matures perdent leur capacité à capter et processer l'antigène. Dans les zones thymo-dépendantes des organes lymphoïdes, les cellules dendritiques qui ont migré ont acquis de puissantes propriétés immunostimulatrices et vont donc activer de façon très efficace les lymphocytes T naïfs circulants.Dendic cells play a role in the development of an immune response and in the initiation of a specific T lymphocyte response (Steinman RM et al Immuno Rev (1997) 156, 25 & Sella M et al Curr Opin Immunol (1997 ) 9, 10). Immature dendritic cells are located in non-lymphoid tissue. At the level of the skin, in all the epidermis except the intestine, they are then called Langerhans cells; dendritic cells, in smaller quantities, are also localized in organs such as the lung, liver, intestine. These immature dendritic cells capture and digest antigens very efficiently. After antigenic stimulation in vivo or stimulation by proinflammatory molecules, the dendritic cells which have captured the antigens migrate into the secondary lymphoid organs. During this migration, the dendritic cells undergo functional and phenotypic modifications which are grouped under the term of maturation. This maturation is characterized by an increase on the surface of dendritic cells of molecules involved in the activation of T lymphocytes (such as CD40, CD54, CD58 and MHC class l / ll), the production of cytokines (such as TNFα and IL-12) and the induction of surface expression of molecules such as CD83 (for human cells). These mature dendritic cells present the antigens to the T lymphocytes and initiate the specific T responses very efficiently, the costimulation molecules being expressed in large quantities on these cells. As cells mature, they lose their ability to capture and process the antigen. In the thymo-dependent areas of the lymphoid organs, the migrated dendritic cells have acquired powerful immunostimulatory properties and will therefore very effectively activate the circulating naive T lymphocytes.
Les auteurs de la présente invention ont mis en évidence que le VIP induit la maturation des cellules dendritiques humaines permettant ainsi le développement d'une réponse lymphocytaire T spécifique.The authors of the present invention have demonstrated that VIP induces the maturation of human dendritic cells, thus allowing the development of a specific T lymphocyte response.
La présente invention concerne l'utilisation d'au moins un composé choisi dans le groupe formé par le VIP, les agonistes des récepteurs du VIP, les antagonistes des récepteurs du VIP pour la fabrication d'un médicament destiné à moduler la maturation des cellules dendritiques.
Ce médicament peut être appliqué au niveau de la peau notamment par voie cutanée, sous-cutanée, transdermique, intra-épidermique ou au niveau des muqueuses, il agit alors de façon systémique par la maturation des cellules dendritiques et localement par la maturation des cellules de Langerhans.The present invention relates to the use of at least one compound chosen from the group formed by VIP, VIP receptor agonists, VIP receptor antagonists for the manufacture of a medicament intended to modulate the maturation of dendritic cells. . This drug can be applied to the skin, in particular by the cutaneous, subcutaneous, transdermal, intra-epidermal route or to the mucous membranes, it then acts systemically by the maturation of the dendritic cells and locally by the maturation of the cells of Langerhans.
Ce médicament peut servir à favoriser la génération et la maturation des cellules dendritiques in vitro après mise en contact avec un agent biologique, les cellules matures étant ensuite réinjectées in vivo. Les cellules dendritiques sont modifiées de sorte qu'elles expriment des antigènes tumoraux. Ledit médicament pourra être mis en œuvre in vitro, afin de favoriser la maturation des cellules dendritiques avant de les réinjecter, ou ce médicament pourra être injecté en même temps que les cellules dendritiques.This drug can be used to promote the generation and maturation of dendritic cells in vitro after contact with a biological agent, the mature cells then being reinjected in vivo. The dendritic cells are modified so that they express tumor antigens. Said medicament may be used in vitro, in order to promote the maturation of the dendritic cells before reinjecting them, or this medicament may be injected at the same time as the dendritic cells.
Cet agent biologique est alors choisi dans le groupe formé par l'acide nucléique, les protéines, les lipides, les lipopeptides, les polysaccharides.This biological agent is then chosen from the group formed by nucleic acid, proteins, lipids, lipopeptides, polysaccharides.
Une utilisation de ce médicament est le traitement ou la prévention du psoriasis.One use of this medication is the treatment or prevention of psoriasis.
La maturation des cellules dendritiques a pour conséquence d'amplifier le développement d'une réponse immune spécifique. Il a été mis en évidence que le VIP et les agonistes des récepteurs du VIP peuvent être utilisés pour amplifier le développement d'une réponse immune spécifique alors que l'utilisation des antagonistes des récepteurs du VIP peuvent être utilisés pour empêcher l'initiation ou ralentir le développement d'une réponse immune spécifique indésirable. Différentes utilisations du VIP et des agonistes des récepteurs du VIP ont été envisagées, ces utilisations sont présentées ci-dessous.The maturation of dendritic cells has the effect of amplifying the development of a specific immune response. It has been demonstrated that VIP and VIP receptor agonists can be used to enhance the development of a specific immune response while the use of VIP receptor antagonists can be used to prevent initiation or slow down the development of an unwanted specific immune response. Different uses of VIP and VIP receptor agonists have been considered, these uses are presented below.
L'utilisation de VIP et des agonistes des récepteurs du VIP en tant qu'adjuvant ou immunostimulant en vaccinologie dans les vaccins anti-
infectieux et anti-cancereux et ce en combinaison avec des antigènes vaccinants et/ou d'autres composés qui peuvent être une bactérie, un virus, une levure, un champignon afin de produire des vaccins thérapeutiques.The use of VIP and VIP receptor agonists as an adjuvant or immunostimulant in vaccinology in anti-HIV vaccines infectious and anti-cancer and this in combination with vaccinating antigens and / or other compounds which can be a bacterium, a virus, a yeast, a fungus in order to produce therapeutic vaccines.
L'administration de VIP en même temps que l'antigène permettra d'augmenter la présentation de l'antigène par les cellules dendritiques et par là même l'efficacité du vaccin.Administering VIP along with the antigen will increase the presentation of the antigen by dendritic cells and thereby the effectiveness of the vaccine.
L'utilisation de VIP et des agonistes des récepteurs du VIP en tant qu'immuπostimulant dans le traitement des immunodéficiences congénitales acquises ou d'origine pathologique. Il peut s'agir notamment du traitement des déficits immunitaires congénitaux ou acquis, mais également des traitements chez des patients ayant une réponse immune absente ou déficiente vis-à-vis par exemple d'une tumeur ou d'un pathogène.The use of VIP and VIP receptor agonists as an immunostimulant in the treatment of acquired congenital immunodeficiencies or of pathological origin. It may especially be the treatment of congenital or acquired immune deficiencies, but also treatments in patients with an absent or deficient immune response, for example to a tumor or a pathogen.
Le VIP et les agonistes des récepteurs du VIP pourront être utilisés notamment pour le traitement : - des infections microbiennes, en particulier les infections de type chronique associées au développement d'une réponse immune spécifique inefficace (virus : par exemple le virus de l'immunodéficience humaine (VIH), les virus hépatiques, en particulier les virus hépatiques A, B, C et D et le parainfluenza virus, bactéries, parasites et levures), - des cancers, en particulier chez les sujets porteurs du VIH et/ou atteints de myélomes, lymphomes, leucémies, mélanomes, carcinomes, du rein, du cerveau, de la prostate, du rectum, du pancréas, des ovaires, du poumon, par exemple.VIP and VIP receptor agonists may be used in particular for the treatment of: - microbial infections, in particular chronic infections associated with the development of an ineffective specific immune response (virus: for example the immunodeficiency virus human (HIV), hepatic viruses, in particular hepatic viruses A, B, C and D and the parainfluenza virus, bacteria, parasites and yeasts), - cancers, in particular in subjects carrying HIV and / or suffering from myelomas, lymphomas, leukemias, melanomas, carcinomas, kidney, brain, prostate, rectum, pancreas, ovaries, lung, for example.
Dans les vaccins anti-cancéreux et anti-infectieux en particulier, il est maintenant proposé en immunothérapie cellulaire de générer in vitro des cellules dendritiques autologues, d'y introduire un antigène tumoral et de les réinjecter. L'exposition des cellules dendritiques in vitro à la molécule VIP ou
à des agonistes des récepteurs à VIP permettra d'augmenter la maturation des cellules dendritiques et donc d'augmenter l'efficacité du vaccin.In anti-cancer and anti-infectious vaccines in particular, it is now proposed in cellular immunotherapy to generate in vitro autologous dendritic cells, to introduce a tumor antigen there and to reinject them. Exposure of dendritic cells in vitro to the VIP molecule or VIP receptor agonists will increase the maturation of dendritic cells and therefore increase the effectiveness of the vaccine.
Aussi, la présente invention concerne particulièrement l'utilisation d'au moins un composé choisi dans le groupe formé par le VIP et les agonistes des récepteurs du VIP avec au moins un agent biologique pour la fabrication d'un médicament pour augmenter la réponse immunologique vis-à-vis de cet agent biologique. Ce médicament peut être un vaccin pour le traitement ou la prévention des maladies infectieuses d'origine virale -comme notamment le VIH, les virus hépatiques et le paraifluenza virus-, bactérienne, fongique, ou provoquées par est une levure ou un parasite.Also, the present invention particularly relates to the use of at least one compound chosen from the group formed by VIP and VIP receptor agonists with at least one biological agent for the manufacture of a medicament for increasing the immunological response vis with respect to this biological agent. This medication can be a vaccine for the treatment or prevention of infectious diseases of viral origin - such as in particular HIV, hepatic viruses and paraifluenza virus -, bacterial, fungal, or caused by is a yeast or a parasite.
La présente invention est encore l'utilisation d'au moins un composé choisi dans le groupe formé par le VIP et les agonistes des récepteurs du VIP avec au moins un agent biologique pour la fabrication d'un médicament pour le traitement ou la prévention des cancers et particulièrement des cancers parmi les myélomes, lymphomes, leucémies, carcinomes du rein, du cerveau, de la prostate, du rectum, du pancréas, des ovaires, du poumon, et pour la fabrication d'un médicament pour le traitement ou la prévention des cancers cutanés choisi parmi les kératinomes et les carcinomes.The present invention is also the use of at least one compound chosen from the group formed by VIP and VIP receptor agonists with at least one biological agent for the manufacture of a medicament for the treatment or prevention of cancers and particularly cancers among myelomas, lymphomas, leukemias, carcinomas of the kidney, brain, prostate, rectum, pancreas, ovaries, lung, and for the manufacture of a medicament for the treatment or prevention of skin cancers chosen from keratinomas and carcinomas.
En effet, dans les cancers de la peau tels que les mélanomes et les kératinomes, les cellules cancéreuses sont en contact direct avec les cellules de Langerhans, situées dans l'épiderme, l'utilisation selon la présente invention par application cutanée permet d'agir directement, localement sur les cellules de Langerhans.Indeed, in skin cancers such as melanomas and keratinomas, the cancer cells are in direct contact with the Langerhans cells, located in the epidermis, the use according to the present invention by skin application makes it possible to act directly, locally on Langerhans cells.
Un avantage de la présente invention est que l'association VIP (ou agoniste des récepteurs du VIP ou antagoniste des récepteurs du VIP) - agent biologique peut se présenter sous une forme facilement administrable telle qu'une pommade, une lotion, une solution ou encore sous forme d'une composition adhésive : emplâtre, «patch ».
Cependant, le traitement de ces cancers, peut aussi être envisagé en injectant des cellules dendritiques autologues, et notamment des cellules deAn advantage of the present invention is that the association VIP (or VIP receptor agonist or VIP receptor antagonist) - biological agent can be in an easily administered form such as an ointment, a lotion, a solution or else in the form of an adhesive composition: plaster, "patch". However, the treatment of these cancers can also be envisaged by injecting autologous dendritic cells, and in particular cells
Langerhans autologues qui ont été modifiées afin d'exprimer des antigènes tumoraux. La maturation des cellules dendritiques sera assurée par le VIP et ses analogues.Autologous Langerhans that have been engineered to express tumor antigens. The maturation of the dendritic cells will be ensured by the VIP and its analogues.
L'utilisation d'au moins un composé choisi dans le groupe formé par le VIP et les agonistes des récepteurs du VIP avec au moins un agent biologique associé au TNF α peut être utilisé pour la fabrication d'un médicament destiné à augmenter la prolifération des lymphocytes T. Par conséquent, la présente invention couvre aussi l'utilisation d'au moins un composé choisi dans le groupe formé par le VIP et les agonistes des récepteurs du VIP avec au moins un agent biologique qui est un agent biologique tumoral pour la fabrication d'un médicament pour favoriser une réponse immune spécifique anti-tumorale. Les antagonistes des récepteurs du VIP seront utilisables pour prévenir le développement d'une réponse immune spécifique indésirable vis- à-vis d'un antigène endogène ou exogène. Les antagonistes des récepteurs du VIP pourront notamment être utilisés par exemple dans les maladies autoimmunes (type arthrite rhumatoide et diabète de type I) et les pathologies d'origine allergique (type asthme allergique et dermatite atopique).The use of at least one compound chosen from the group formed by VIP and VIP receptor agonists with at least one biological agent associated with TNF α can be used for the manufacture of a medicament intended to increase the proliferation of T cells. Consequently, the present invention also covers the use of at least one compound chosen from the group formed by VIP and VIP receptor agonists with at least one biological agent which is a tumor biological agent for the manufacture of a drug to promote a specific anti-tumor immune response. VIP receptor antagonists can be used to prevent the development of an undesirable specific immune response to an endogenous or exogenous antigen. VIP receptor antagonists may in particular be used in autoimmune diseases (rheumatoid arthritis and type I diabetes) and pathologies of allergic origin (allergic asthma and atopic dermatitis).
L'utilisation des molécules antagonistes des récepteurs du VIP pourra se faire par différentes voies d'introduction en fonction de la voie d'entrée de l'antigène (exogène) ou de son site de production (endogène) et ceci afin d'agir localement en prévenant le développement d'une réponse immune (en cosmétique dans le cas des dermatites atopiques et allergies cutanées).The VIP receptor antagonist molecules can be used by different routes of introduction depending on the route of entry of the antigen (exogenous) or its production site (endogenous) and this in order to act locally. by preventing the development of an immune response (in cosmetics in the case of atopic dermatitis and skin allergies).
Par conséquent, la présente invention vise encore l'utilisation d'au moins un antagoniste des récepteurs du VIP pour la fabrication d'un
médicament destiné à diminuer l'intensité d'une réponse immune spécifique, notamment pour diminuer l'intensité d'une maladie autoimmune, par exemple de type arthrite rhumatoide ou diabète de type I ou pour la fabrication d'un médicament destiné à traiter les pathologies d'origine allergique telles que l'asthme allergique et les dermatites atopiques.Consequently, the present invention also relates to the use of at least one antagonist of the VIP receptors for the manufacture of a medicament intended to decrease the intensity of a specific immune response, in particular to decrease the intensity of an autoimmune disease, for example of the rheumatoid arthritis type or type I diabetes or for the manufacture of a medicament intended to treat pathologies of allergic origin such as allergic asthma and atopic dermatitis.
Les exemples qui suivent démontrent l'action du VIP sur les cellules dendritiques : le VIP induit la maturation des cellules dendritiques humaines immatures et leur confère de puissantes propriétés stimulatrices et présentatrices d'antigène.The following examples demonstrate the action of VIP on dendritic cells: VIP induces the maturation of immature human dendritic cells and gives them powerful stimulatory and antigen presenting properties.
Exemple 1Example 1
Cet exemple démontre l'action du VIP en tant que modulateur de l'expression des molécules exprimées à la surface des cellules dendritiques humaines immatures par :This example demonstrates the action of VIP as a modulator of the expression of molecules expressed on the surface of immature human dendritic cells by:
- induction de l'expression de la molécule CD83 qui est un marqueur d'activation pour les cellules dendritiques humaines (figure 1 ),- induction of the expression of the CD83 molecule which is an activation marker for human dendritic cells (FIG. 1),
- induction de l'expression de la molécule CD86 qui est une puissante molécule de costimulation (tableau 1 ),- induction of the expression of the CD86 molecule which is a powerful costimulation molecule (Table 1),
- augmentation de l'expression de molécules impliquées dans l'activation du lymphocyte T telles que : CD40, CD54, CD80 et HLA-Dr (tableau 1 ).- increase in the expression of molecules involved in the activation of the T lymphocyte such as: CD40, CD54, CD80 and HLA-Dr (Table 1).
L'ensemble de ces modifications montrent que les cellules dendritiques traitées par le VIP acquièrent un phénotype de cellules dendritiques matures.All of these modifications show that the VIP-treated dendritic cells acquire a phenotype of mature dendritic cells.
Cet exemple montre que le VIP induit l'expression de la molécule CD83 dont l'expression est restreinte aux cellules dendritiques matures.
MéthodologieThis example shows that VIP induces the expression of the CD83 molecule whose expression is restricted to mature dendritic cells. Methodology
- Génération in vitro de cellules dendritiques humaines immatures.- In vitro generation of immature human dendritic cells.
- Les cellules dendritiques humaines sont générées à partir de monocytes isolés du sang périphérique. Le sang est prélevé par leucophérèse en présence d'anticoagulant comme par exemple l'héparinate de lithium. Les cellules mononucléées (CMN) sont isolées de sujets sains par centrifugation sur un gradient de Ficoll-Hypaque (densité=1 ,077) (Amersham Pharmacia Biotech, Uppsala, Suède). Brièvement, les cellules du sang sont centrifugées à 1500 rpm pendant 30 minutes à température ambiante. Les CMN, localisées à l'interface Ficoll-plasma, sont récupérées et lavées deux fois en présence de milieu RPMI 1640 (Life technologies, Cergy Pontoise, France). Les monocytes sont purifiés par sélection positive en utilisant un séparateur magnétique de cellules (MACS™; Miltenyi Biotex, Bergisch Gladbach, Allemagne) en accord avec les instructions du fabricant. Brièvement, les CMN sont incubées pendant 20 minutes à 4°C avec des billes magnétiques sur lesquelles sont fixées un anticorps monoclonal anti- CD14 humain. Après lavage, la suspension cellulaire plus billes est déposée sur une colonne et soumise à un champ magnétique. Après trois lavages, la colonne n'est plus soumise au champ magnétique et les monocytes sont collectés par gravitation. La pureté des monocytes est évaluée par cytofluorométrie (cytofluoromètre FACScan ; Becton Dickinson, Erembodegem, Belgique) sur la base des paramètres taille-granulosité des cellules. La pureté est supérieure à 98%. Les monocytes sont ensuite mis en culture à la concentration de 106 cellules/ml dans le milieu suivant (dénommé par la suite milieu de culture complet) : milieu RPMI 1640 supplémenté de
10% de sérum de veau foetal (chauffage à 56°C pendant 30 minutes), 2 mM de L-glutamine, 50 U/ml de pénicilline et 50 ug/ml de streptomycine (Life technologies) dans des plaques de culture 6 puits (Nunc, Roskilde, Danemark) à raison de 5 ml de milieu par puits. Les cellules sont activées avec 20 ng/ml d'IL-4 humaine recombinante et 20 ng/ml de GM-CSF humain recombinant (R&D Systems, Abingdon, Royaume Uni). Après 6 jours de culture (37°C, 5% CO2 en atmosphère humide), le phénotype des cellules est défini par cytofluorométrie. Brièvement, un aliquot de la suspension cellulaire est prélevé. Les cellules sont lavées dans du tampon FACS (tampon phosphate 10 mM pH 7,4 contenant 1 % de sérum albumine bovine et 0,01% d'azide de sodium) puis réparties dans des puits d'une plaque de culture 96 puits à fond conique (Nunc) à raison de 2x105 cellules dans un volume de 50 μl de tampon FACS. Dans chaque puits est ajouté soit un anticorps anti- CD1a humain marqué à la fluorescéine (Becton Dickinson) soit un anticorps anti-CD83 humain non marqué (Becton Dickinson) révélé par un anticorps anti-souris marqué à la fluorescéine (Silenus, Hauworth, Australie). Après 20 minutes d'incubation à 4°C, les cellules sont lavées trois fois avec 200 μl de tampon FACS puis sont remises en suspension dans 200 μl de ce même tampon. L'analyse de l'expression de CD1a versus CD83 est évaluée par FACS. Seules les cellules dendritiques immatures caractérisées par une expression de la molécules CD1a (intensité moyenne de fluorescence (IMF>100) et l'absence d'expression de la molécule CD83 ont été utilisées.- Human dendritic cells are generated from monocytes isolated from peripheral blood. The blood is taken by leukopheresis in the presence of an anticoagulant such as for example lithium heparinate. Mononuclear cells (CMN) are isolated from healthy subjects by centrifugation on a Ficoll-Hypaque gradient (density = 1.077) (Amersham Pharmacia Biotech, Uppsala, Sweden). Briefly, the blood cells are centrifuged at 1500 rpm for 30 minutes at room temperature. The CMNs, located at the Ficoll-plasma interface, are recovered and washed twice in the presence of RPMI 1640 medium (Life technologies, Cergy Pontoise, France). The monocytes are purified by positive selection using a magnetic cell separator (MACS ™; Miltenyi Biotex, Bergisch Gladbach, Germany) in accordance with the manufacturer's instructions. Briefly, the CMNs are incubated for 20 minutes at 4 ° C. with magnetic beads onto which a monoclonal anti-human CD14 antibody is fixed. After washing, the cell suspension plus beads is placed on a column and subjected to a magnetic field. After three washes, the column is no longer subjected to the magnetic field and the monocytes are collected by gravitation. The purity of the monocytes is evaluated by cytofluorometry (FACScan cytofluorometer; Becton Dickinson, Erembodegem, Belgium) on the basis of the size-granulosity parameters of the cells. The purity is greater than 98%. The monocytes are then cultured at a concentration of 10 6 cells / ml in the following medium (hereinafter called complete culture medium): RPMI 1640 medium supplemented with 10% fetal calf serum (heating at 56 ° C for 30 minutes), 2 mM L-glutamine, 50 U / ml of penicillin and 50 ug / ml of streptomycin (Life technologies) in 6-well culture plates ( Nunc, Roskilde, Denmark) at a rate of 5 ml of medium per well. The cells are activated with 20 ng / ml of recombinant human IL-4 and 20 ng / ml of recombinant human GM-CSF (R&D Systems, Abingdon, United Kingdom). After 6 days of culture (37 ° C, 5% CO 2 in a humid atmosphere), the phenotype of the cells is defined by cytofluorometry. Briefly, an aliquot of the cell suspension is removed. The cells are washed in FACS buffer (10 mM phosphate buffer pH 7.4 containing 1% bovine serum albumin and 0.01% sodium azide) and then distributed in wells of a 96-well culture plate at the bottom conical (Nunc) at the rate of 2 × 10 5 cells in a volume of 50 μl of FACS buffer. To each well is added either an anti-human CD1a antibody labeled with fluorescein (Becton Dickinson) or an anti-human anti-CD83 antibody (Becton Dickinson) revealed by an anti-mouse antibody marked with fluorescein (Silenus, Hauworth, Australia ). After 20 minutes of incubation at 4 ° C, the cells are washed three times with 200 μl of FACS buffer and then are resuspended in 200 μl of this same buffer. The analysis of the expression of CD1a versus CD83 is evaluated by FACS. Only immature dendritic cells characterized by expression of the CD1a molecules (average fluorescence intensity (IMF> 100) and the absence of expression of the CD83 molecule were used.
Les résultats de cette culture sont consignés à la figure 1 qui représente le pourcentage de cellules exprimant la molécule CD83 (dont l'expression est restreinte aux cellules dendritiques matures) en fonction de la concentration en VIP dans la culture. La courbe de la figure 1 montre queThe results of this culture are recorded in FIG. 1 which represents the percentage of cells expressing the CD83 molecule (whose expression is restricted to mature dendritic cells) as a function of the VIP concentration in the culture. The curve in Figure 1 shows that
- la molécule CD83 n'est pas exprimée par les cellules dendritiques humaines immatures,
- le VIP induit l'expression de la molécule CD83 de manière dépendante de la concentration (significative à 10"8 M et maximale à 10"6 M) sur une fraction des cellules dendritiques immatures (46% après 4 jours de stimulation).- the CD83 molecule is not expressed by immature human dendritic cells, - VIP induces the expression of the CD83 molecule in a concentration-dependent manner (significant at 10 "8 M and maximum at 10 " 6 M) on a fraction of immature dendritic cells (46% after 4 days of stimulation).
- Analyse par cytofluorometrie de l'expression des marqueurs de différenciation induite par le VIP sur des cellules dendritiques humaines.- Analysis by cytofluorometry of the expression of the markers of differentiation induced by VIP on human dendritic cells.
Les cellules dendritiques immatures de six jours présentées ci-dessus ont été collectées, lavées puis remises en culture en milieu complet à la concentration de 105 cellules dans un volume de 200 μl dans des plaques de culture 96 puits à fond plat (Costar, Cambridge, USA). Les cellules sont activées avec le VIP (Sigma, Saint Louis, USA). Après quatre jours de culture, l'expression des molécules CD40, CD54, CD80, CD83, CD86 et HLA-Dr est évaluée par cytofluorometrie à l'aide des anticorps monoclonaux suivants : anticorps anti-CD40, anti-CD54, anti-CD80, anti-CD86 et anti-HLA- Dr marqués à la fluorescéine (Becton Dickinson) et anticorps anti-CD83 révélé par un anticorps anti-immunoglobuline de souris marqué à la fluorescéine (Silenus, Hauworth, Australie). Les anticorps isotypiques contrôles utilisés proviennent de Becton Dickinson. Les cellules sont lavées dans du tampon FACS puis réparties dans des puits d'une plaque de culture 96 puits à fond conique à raison de 2x105 cellules dans un volume de 50 μl de tampon FACS. Dans chaque puits est ajouté un anticorps. Après 20 minutes d'incubation à 4°C, les cellules sont lavées trois fois avec 200 μl de tampon FACS puis sont remises en suspension dans 200 μl de ce même tampon. L'analyse de l'expression des marqueurs de surface est évaluée par FACS.
Les résultats montrent que le VIP augmente l'expression des CD40, CD54, CD80 et HLA-DR sur les cellules dendritiques immatures et induit l'expression de CD83 sur une fraction des cellules dendritiques (tableau 1 ).The six-day immature dendritic cells presented above were collected, washed and then re-cultured in complete medium at the concentration of 10 5 cells in a volume of 200 μl in 96-well flat-bottom culture plates (Costar, Cambridge , USA). The cells are activated with VIP (Sigma, Saint Louis, USA). After four days of culture, the expression of the molecules CD40, CD54, CD80, CD83, CD86 and HLA-Dr is evaluated by cytofluorometry using the following monoclonal antibodies: anti-CD40, anti-CD54, anti-CD80 antibody, anti-CD86 and anti-HLA-Dr labeled with fluorescein (Becton Dickinson) and anti-CD83 antibody revealed by an anti-mouse immunoglobulin antibody labeled with fluorescein (Silenus, Hauworth, Australia). The control isotypic antibodies used come from Becton Dickinson. The cells are washed in FACS buffer and then distributed in wells of a 96-well culture plate with a conical bottom at the rate of 2 × 10 5 cells in a volume of 50 μl of FACS buffer. An antibody is added to each well. After 20 minutes of incubation at 4 ° C, the cells are washed three times with 200 μl of FACS buffer and then are resuspended in 200 μl of this same buffer. The analysis of the expression of the surface markers is evaluated by FACS. The results show that VIP increases the expression of CD40, CD54, CD80 and HLA-DR on immature dendritic cells and induces the expression of CD83 on a fraction of dendritic cells (Table 1).
Exemple 2 : Le TNFα potentialise les effets du VIP sur les cellules dendritiques humaines.Example 2: TNFα potentiates the effects of VIP on human dendritic cells.
Cet exemple montre que le TNFα, un médiateur proinflammatoire induisant la différentiation des cellules dendritiques, potentialise les effets du VIP sur l'activation et la différentiation des cellules dendritiques humaines.This example shows that TNFα, a proinflammatory mediator inducing the differentiation of dendritic cells, potentiates the effects of VIP on the activation and differentiation of human dendritic cells.
Méthodologie :Methodology :
Des cellules dendritiques humaines immatures ont été générées comme décrit dans l'exemple 1. Les cellules dendritiques immatures ont été collectées, lavées puis remises en culture en milieu complet à la concentration de 105 cellules dans un volume de 200 μl dans des plaques de culture 96 puits à fond plat (Costar, Cabridge, USA). Les cellules sont activées avec le VIP (Sigma, Saint Louis, USA) en l'absence ou en présence de 0,2 ng/ml de TNFα humain recombinant (R&D Systems, Abingdon,Immature human dendritic cells were generated as described in Example 1. Immature dendritic cells were collected, washed and then re-cultured in complete medium at the concentration of 10 5 cells in a volume of 200 μl in culture plates 96 flat-bottomed wells (Costar, Cabridge, USA). The cells are activated with VIP (Sigma, Saint Louis, USA) in the absence or in the presence of 0.2 ng / ml of recombinant human TNFα (R&D Systems, Abingdon,
Royaume Uni) ou de 10 ng/ml de lipopolysaccharide (LPS) (puhfé à partir de la souche Escherichia coli isotype 0111 :B4; Sigma). Après quatre jours de culture, l'expression des molécules CD40, CD54, CD80, CD83, CD86 etUnited Kingdom) or 10 ng / ml of lipopolysaccharide (LPS) (puhfe from the Escherichia coli strain isotype 0111: B4; Sigma). After four days of culture, the expression of the molecules CD40, CD54, CD80, CD83, CD86 and
HLA-Dr est évaluée par cytofluorometrie à l'aide des anticorps monoclonaux suivants : anticorps anti-CD40, anti-CD54, anti-CD80, anti-CD86 et anti-HLA-HLA-Dr is evaluated by cytofluorometry using the following monoclonal antibodies: anti-CD40, anti-CD54, anti-CD80, anti-CD86 and anti-HLA- antibodies
Dr marqués à la fluorescéine (Becton Dickinson) et anticorps anti-CD83 révélé par un anticorps anti-immunoglobuline de souris marqué à la
fluorescéine (Silenus, Hauworth, Australie). Le marquage des cellules ainsi que l'analyse par cytofluorometrie ont été réalisés comme décrit dans l'exemple 1.Dr labeled with fluorescein (Becton Dickinson) and anti-CD83 antibody revealed by an anti-mouse immunoglobulin antibody labeled with fluorescein (Silenus, Hauworth, Australia). The labeling of the cells as well as the analysis by cytofluorometry were carried out as described in Example 1.
La figure 2 présente le pourcentage de cellules exprimant la molécule CD83 en fonction de la concentration en VIP avec un ajout de 0,2 ng/ml (courbel ) de TNFα dans le milieu de culture et sans cet ajout (courbe 2). Les résultats sur ces cellules après 4 jours de culture sont consignés dans le tableau 1.FIG. 2 shows the percentage of cells expressing the CD83 molecule as a function of the VIP concentration with an addition of 0.2 ng / ml (curve) of TNFα to the culture medium and without this addition (curve 2). The results on these cells after 4 days of culture are recorded in Table 1.
TABLEAU ITABLE I
Stimulus CD40 a CD54 a CD80 a CD83fa CD86b HLA-DR a Stimulus CD40 a CD54 a CD80 a CD83 fa CD86 b HLA-DR a
Aucun 55 50 14 <3%- 26 <10% -39 178None 55 50 14 <3% - 26 <10% -39 178
TNFα (0,2 ng/ml) 54 49 15 <3%- 27 <10% -42 170TNFα (0.2 ng / ml) 54 49 15 <3% - 27 <10% -42 170
VIP (10"6 ) 90 82 20 46 % - 114 52 % - 154 210VIP (10 "6 ) 90 82 20 46% - 114 52% - 154 210
VIP (10-6 M) +VIP (10- 6 M) +
210 819 44 95% - 110 100% - 179 460 TNFα (0,2 ng/ml)210,819 44 95% - 110 100% - 179,460 TNFα (0.2 ng / ml)
TNFα (20 ng/ml) 214 825 43 98% - 102 100% - 175 473TNFα (20 ng / ml) 214,825 43 98% - 102 100% - 175,473
Les mesures portant la mention « a » sont exprimées en intensité moyenne de fluorescence IMF après soustraction de la valeur obtenue avec l'anticorps témoin.The measurements marked “a” are expressed in mean intensity of MFI fluorescence after subtracting the value obtained with the control antibody.
Les mesures portant la mention « b » sont exprimées en pourcentage et valeur de l'IMF des cellules positives.The measurements marked “b” are expressed as a percentage and value of the MFI of the positive cells.
Les résultats présentés dans la figure 2 et le tableau 1 montrent que : - le TNFα à la concentration de 20 ng/ml induit la maturation des cellules dendritiques (induction de l'expression de la molécule CD83 sur toutes les cellules dendritiques, IMF=110) ainsi que l'activation des cellules
dendritiques objectivée par l'augmentation de l'expression des molécules CD40 (IMF=214), CD54 (IMF=825), CD80 (IMF=43) et HLA-Dr (IMF=473) ainsi que l'induction de l'expression de la molécule CD86 (IMF=175) (tableauThe results presented in Figure 2 and Table 1 show that: - TNFα at a concentration of 20 ng / ml induces the maturation of dendritic cells (induction of expression of the CD83 molecule on all dendritic cells, IMF = 110 ) as well as cell activation dendritics objectified by the increase in the expression of the molecules CD40 (IMF = 214), CD54 (IMF = 825), CD80 (IMF = 43) and HLA-Dr (IMF = 473) as well as the induction of expression of the CD86 molecule (IMF = 175) (table
1). - le TNFα à la concentration de 0,2 ng/ml ne module pas l'expression des molécules analysées (tableau 1 ).1). - TNFα at a concentration of 0.2 ng / ml does not modulate the expression of the molecules analyzed (Table 1).
- le TNFα à la concentration de 0,2 ng/ml potentialise l'induction de la molécule CD83 induite par le VIP sur les cellules dendritiques : l'expression de CD83 est significativement induite à 10"10 M (16% de cellules expriment la molécule CD83) et maximale à 10"6 M (95% de cellules expriment la molécule CD83) (figure 2).- TNFα at a concentration of 0.2 ng / ml potentiates the induction of the CD83 molecule induced by VIP on dendritic cells: the expression of CD83 is significantly induced at 10 "10 M (16% of cells express the CD83 molecule) and maximum at 10 "6 M (95% of cells express the CD83 molecule) (FIG. 2).
- le TNFα à la concentration de 0,2 ng/ml potentialise l'expression des molécules CD40 (IMF=210), CD54 (IMF=819), CD80 (IMF=44) et HLA-Dr (IMF=460) en présence de 10"6 M VIP. (tableau 1 ).- TNFα at a concentration of 0.2 ng / ml potentiates the expression of the molecules CD40 (IMF = 210), CD54 (IMF = 819), CD80 (IMF = 44) and HLA-Dr (IMF = 460) in the presence 10 "6 M VIP. (table 1).
Exemple 3 : Le VIP induit la production d'interleukine 12 par les cellules dendritiques humaines. Effet potentiateur du TNFα.Example 3: VIP induces the production of interleukin 12 by human dendritic cells. Potential effect of TNFα.
Cet exemple montre que le VIP induit la production d'interleukine 12 (IL 12) par les cellules dendritiques humaines et que cet effet est potentialise par le TNFα.This example shows that VIP induces the production of interleukin 12 (IL 12) by human dendritic cells and that this effect is potentiated by TNFα.
Le VIP induit la production d'IL-12 par les cellules dendritiques. L'IL-VIP induces the production of IL-12 by dendritic cells. The IL-
12 est une cytokine qui joue un rôle crucial dans le développement d'une réponse lymphocytaire T puisqu'elle va amplifier la production d'interféron gamma (IFNγ) par les lymphocytes T. Nous montrons que le VIP induit une production dépendante de la dose d'IFNγ par les cellules dendritiques
immatures. Cette production est potentialisée en présence d'une dose suboptimale de TNFα.12 is a cytokine which plays a crucial role in the development of a T lymphocyte response since it will amplify the production of gamma interferon (IFNγ) by T lymphocytes. We show that VIP induces a dose-dependent production d 'IFNγ by dendritic cells immature. This production is potentiated in the presence of a suboptimal dose of TNFα.
La production d'IL-12 par les cellules dendritiques étant associée au phénomène de maturation, ces observations confirment que le VIP favorise la maturation de cellules dendritiques humaines.Since the production of IL-12 by dendritic cells is associated with the maturation phenomenon, these observations confirm that VIP promotes the maturation of human dendritic cells.
Méthodologie :Methodology :
Des cellules dendritiques humaines immatures ont été générées comme décrit dans l'exemple 1. Après 6 jours de culture, les cellules sont remises en culture en milieu complet à la concentration de 105 cellules dans un volume de 200 μl dans des plaques de culture 96 puits à fond plat. Les cellules sont activées avec le VIP (10"10-10"6 M) en l'absence ou en présence de 0,2 ng/ml de TNFα humain recombinant. Après 48 heures de culture, les surnageants de culture sont centrifugés à 10000 rpm pendant 15 minutes à 4°C. Les surnageants sont collectés. L'IL-12 bioactive (également nommée hétérodimère p40/p75) est quantifiée à l'aide d'un kit de dosage commercial type ELISA (R&D Systems) selon les instructions du fabricant. Les résultats sont exprimés en pg/ml. Les dosages sont réalisés en duplicat. La figure 3 présente la production d'IL-12 par les cellules dendritiques humaines en fonction de la concentration en VIP avec un ajout de 0,2 ng/ml de TNFα dans le milieu de culture (courbe 1 ) et sans cet ajout (courbe 2).Immature human dendritic cells were generated as described in Example 1. After 6 days of culture, the cells are returned to culture in complete medium at the concentration of 10 5 cells in a volume of 200 μl in culture plates 96 flat bottom well. The cells are activated with VIP (10 "10 -10 " 6 M) in the absence or in the presence of 0.2 ng / ml of recombinant human TNFα. After 48 hours of culture, the culture supernatants are centrifuged at 10,000 rpm for 15 minutes at 4 ° C. Supernatants are collected. Bioactive IL-12 (also called heterodimer p40 / p75) is quantified using a commercial assay kit type ELISA (R&D Systems) according to the manufacturer's instructions. The results are expressed in pg / ml. The assays are carried out in duplicate. FIG. 3 shows the production of IL-12 by human dendritic cells as a function of the VIP concentration with an addition of 0.2 ng / ml of TNFα to the culture medium (curve 1) and without this addition (curve 2).
Les résultats présentés dans la figure 3 montrent que : - le VIP induit une production modérée d'IL-12 par les cellules dendritiques humaines (3 ± 1 et 9 ± 2 pg/ml en présence de VIP à la concentration de 10"7 et 10"6 M, respectivement)
- le TNFα à la concentration de 0,2 ng/ml n'induit pas la production d'IL-12 par les cellules dendritiquesThe results presented in FIG. 3 show that: - VIP induces a moderate production of IL-12 by human dendritic cells (3 ± 1 and 9 ± 2 pg / ml in the presence of VIP at the concentration of 10 "7 and 10 "6 M, respectively) - TNFα at a concentration of 0.2 ng / ml does not induce the production of IL-12 by dendritic cells
- le TNFα à la concentration de 0,2 ng/ml potentialise la production d'IL-12 induite par le VIP : la production d'IL-12 est significative à 10"8 M (8 ± 3 pg/ml) et maximale à 10"6 M de VIP (22 ± 2,5 pg/ml).- TNFα at a concentration of 0.2 ng / ml potentiates the production of IL-12 induced by VIP: the production of IL-12 is significant at 10 "8 M (8 ± 3 pg / ml) and maximum at 10 "6 M VIP (22 ± 2.5 pg / ml).
Exemple 4 : VIP et TNFα coopèrent pour conférer aux cellules dendritiques humaines des propriétés de costimulationExample 4 VIP and TNFα cooperate to confer on human dendritic cells costimulation properties
Cet exemple montre que la maturation des cellules dendritiques humaines induites par le VIP en présence d'une concentration suboptimale de TNFα leur confère des propriétés de costimulation.This example shows that the maturation of human dendritic cells induced by VIP in the presence of a suboptimal concentration of TNFα gives them costimulation properties.
Méthodologie : Réaction lymphocytaire mixte.Methodology: Mixed lymphocyte reaction.
Les cellules dendritiques humaines immatures sont générées comme décrit dans l'exemple 1. Après 6 jours de culture, les cellules sont lavées en milieu RPMI 1640 puis remises en culture dans le milieu complet à la concentration de 2,5x105 cellules/puits dans une plaque de culture 6 puits (5 ml/puits). Les cellules ne sont pas stimulées ou elles sont stimulées en présence de 0,2 ng/ml TNFα ;10"6 M VIP ; 0,2 ng/ml TNFα et 10"6 M VIP ou 20 ng/ml TNFα. Après trois jours de culture, les cellules sont collectées et lavées trois fois en milieu RPMI 1640. Les cellules sont ensuite irradiées à 3000 rad. Les lymphocytes T humains fraîchement isolés du sang périphérique ont été préparés par la technique des rosettes avec des érythrocytes de mouton. Brièvement, les cellules mononucléées sont isolées sur gradient de Ficoll-Hypaque, comme décrit dans l'exemple 1. Les cellules
mononucléées sont remises en suspension dans du milieu complet à la concentration de 200x106 cellules/ml et mélangées avec 1 ml d'une suspension à 50% d'érythrocytes de mouton (BioMérieux, Marcy l'Etoile, France). La suspension cellulaire est incubée à 4°C pendant une nuit. Après remise en suspension douce, les cellules T sont isolées par centrifugation sur un gradient de Ficoll-Hypaque (1500 rpm pendant 30 minutes à température ambiante). Les complexes cellules T/érythrocytes sont collectés au fond du tube. Les globules rouges sont lysés par deux chocs hypotoniques successifs. La pureté des cellules ainsi isolées est évaluée par cytofluorometrie à l'aide d'un anticorps anti-CD3 humain marqué à la fluorescéine (Becton Dickinson). La pureté est supérieure à 95%. Après lavage, les cellules sont remises en suspension dans le milieu de culture complet à la concentration de 2,5x105 cellules/ml.Immature human dendritic cells are generated as described in Example 1. After 6 days of culture, the cells are washed in RPMI 1640 medium and then re-cultured in complete medium at a concentration of 2.5 × 10 5 cells / well in a 6-well culture plate (5 ml / well). The cells are not stimulated or they are stimulated in the presence of 0.2 ng / ml TNFα; 10 "6 M VIP; 0.2 ng / ml TNFα and 10 " 6 M VIP or 20 ng / ml TNFα. After three days of culture, the cells are collected and washed three times in RPMI 1640 medium. The cells are then irradiated at 3000 rad. Human T cells freshly isolated from peripheral blood were prepared by the rosette technique with sheep erythrocytes. Briefly, the mononuclear cells are isolated on a Ficoll-Hypaque gradient, as described in example 1. The cells mononuclear cells are resuspended in complete medium at a concentration of 200 × 10 6 cells / ml and mixed with 1 ml of a 50% suspension of sheep erythrocytes (BioMérieux, Marcy l'Etoile, France). The cell suspension is incubated at 4 ° C overnight. After gentle resuspension, the T cells are isolated by centrifugation on a Ficoll-Hypaque gradient (1500 rpm for 30 minutes at room temperature). The T cell / erythrocyte complexes are collected at the bottom of the tube. The red blood cells are lysed by two successive hypotonic shocks. The purity of the cells thus isolated is evaluated by cytofluorometry using a human anti-CD3 antibody labeled with fluorescein (Becton Dickinson). Purity is greater than 95%. After washing, the cells are resuspended in the complete culture medium at a concentration of 2.5 × 10 5 cells / ml.
Les cellules dendritiques activées et irradiées sont mises en culture à la concentration de 104 cellules/200 μl dans une plaque de culture 96 puits en présence ou non de 5x104 lymphocytes allogéniques. Après 5 jours de culture, la prolifération des cellules T est évaluée par mesure de l'incorporation de thymidine tritiée (3H-Thy) (Amersham, Amersham, Royaume Uni). Brièvement, 0,25 μCi de 3H-Thy sont ajoutés dans chaque puits de culture. L'incorporation de 3H-Thy est mesurée par un compteur à scintillation liquide (Packard Instruments, Australie). Les résultats sont présentés en coups par minute (cpm). Les réactions lymphocytaires mixtes sont réalisées avec les lymphocytes T provenant de deux donneurs sains différents. La figure 4 présente le taux de prolifération des cellules en cpm de ces deux donneurs en présence des stimuli suivants : (1 ) aucun stimulus ; (2) VIP (10"6 M) ; (3) TNFα (0,2 ng/ml) ; (4) VIP (10'6 M) + TNFα (0,2 ng/ml) ;(5) TNFα 20ng/ml.
Les résultats présentés dans la figure 4 montrent que :The activated and irradiated dendritic cells are cultured at the concentration of 10 4 cells / 200 μl in a 96-well culture plate in the presence or not of 5 × 10 4 allogenic lymphocytes. After 5 days of culture, the proliferation of T cells is evaluated by measuring the incorporation of tritiated thymidine ( 3 H-Thy) (Amersham, Amersham, United Kingdom). Briefly, 0.25 μCi of 3 H-Thy is added to each culture well. The incorporation of 3 H-Thy is measured by a liquid scintillation counter (Packard Instruments, Australia). The results are presented in counts per minute (cpm). Mixed lymphocyte reactions are performed with T lymphocytes from two different healthy donors. Figure 4 shows the cell proliferation rate in cpm of these two donors in the presence of the following stimuli: (1) no stimulus; (2) VIP (10 "6 M); (3) TNFα (0.2 ng / ml); (4) VIP (10 '6 M) + TNFα (0.2 ng / ml); (5) TNFα 20ng / ml. The results presented in Figure 4 show that:
- les cellules dendritiques stimulées par le VIP seul induisent une faible prolifération des cellules T- dendritic cells stimulated by VIP alone induce a low proliferation of T cells
- les cellules dendritiques immatures stimulées avec le TNFα à la concentration de 0,2 ng/ml n'induisent pas de prolifération cellulaire T- immature dendritic cells stimulated with TNFα at a concentration of 0.2 ng / ml do not induce T cell proliferation
- les cellules dendritiques stimulées par le TNFα (0,2 ng/ml) + VIP (10"6 M) induisent une forte prolifération des cellules T provenant des deux donneurs (28x103 et 22x103 cpm)- the dendritic cells stimulated by TNFα (0.2 ng / ml) + VIP (10 "6 M) induce a strong proliferation of T cells from the two donors (28x10 3 and 22x10 3 cpm)
- la prolifération induite par les cellules dendritiques stimulées par 0,2 ng/ml TNFα plus 10"6 M VIP est équivalente à celle induite par des cellules dendritiques stimulées avec une dose optimale de TNFα de 20 ng/ml- the proliferation induced by dendritic cells stimulated by 0.2 ng / ml TNFα plus 10 "6 M VIP is equivalent to that induced by dendritic cells stimulated with an optimal dose of TNFα of 20 ng / ml
Exemple 5 : Des antagonistes des récepteurs de VIP préviennent l'effet du VIP sur la maturation des CDExample 5: VIP receptor antagonists prevent the effect of VIP on CD maturation
Cet exemple montre que la maturation des cellules dendritiques humaines induites par le VIP est inhibée par des antagonistes du VIPThis example shows that the maturation of human dendritic cells induced by VIP is inhibited by VIP antagonists
Méthodologie :Methodology :
Les cellules dendritiques humaines immatures sont générées comme décrit dans l'exemple 1. Après 6 jours de culture, les cellules sont lavées en milieu RPMI 1640 puis remises en culture dans le milieu complet à la concentration de 2,5x105 cellules/puits dans une plaque de culture 6 puits (5 ml/puits). Les cellules sont stimulées par 0,2 ng/ml TNFα, 10"6 M VIP, 0,2 ng/ml TNFα et 10"6 M VIP ou 100 pg/ml de LPS en absence (colonne claire, située à
gauche) ou en présence (colonne grisée, située au centre) de 5 μg/ml polymixine B ou de 10"6 M d'un antagoniste du VIP récepteur (VIP 6-28, référence V4508) (Sigma) (Fishbein et al, Peptide (1994) 15, 95) (colonne noircie, située à droite). Après 4 jours, les cellules sont marquées avec un anticorps anti-CD83 révélé par un anticorps anti-immunoglobuline de souris marqué à la fluorescéine (Silenus, Hauworth, Australie). Le marquage- des cellules ainsi que l'analyse par cytofluorometrie ont été réalisés comme décrit dans l'exemple 1.Immature human dendritic cells are generated as described in Example 1. After 6 days of culture, the cells are washed in RPMI 1640 medium and then re-cultured in complete medium at a concentration of 2.5 × 10 5 cells / well in a 6-well culture plate (5 ml / well). The cells are stimulated with 0.2 ng / ml TNFα, 10 "6 M VIP, 0.2 ng / ml TNFα and 10 " 6 M VIP or 100 pg / ml of LPS in the absence (clear column, located at left) or in the presence (gray column, located in the center) of 5 μg / ml polymixin B or 10 "6 M of a receptor VIP antagonist (VIP 6-28, reference V4508) (Sigma) (Fishbein et al, Peptide (1994) 15, 95) (blackened column, right) After 4 days, the cells are labeled with an anti-CD83 antibody revealed by a fluorescein labeled anti-mouse immunoglobulin antibody (Silenus, Hauworth, Australia The labeling of the cells as well as the analysis by cytofluorometry were carried out as described in Example 1.
La figure 5 présente le pourcentage de cellules exprimant la molécule CD83 en présence des stimuli suivants : aucun stimulus ; VIP (10"6 M) ; TNFα (0,2 ng/ml) ; VIP (10"6 M) + TNFα (0,2 ng/ml) ; LPS (100 pg/ml) en l'absence et en présence de polymixine B (5 g/ml) ; antagoniste du VIP récepteur (10"6 M).Figure 5 shows the percentage of cells expressing the CD83 molecule in the presence of the following stimuli: no stimulus; VIP (10 "6 M); TNFα (0.2 ng / ml); VIP (10 " 6 M) + TNFα (0.2 ng / ml); LPS (100 pg / ml) in the absence and presence of polymixin B (5 g / ml); receptor VIP antagonist (10 "6 M).
Ces résultats montrent que : - l'effet du VIP sur l'expression de la molécule CD83 n'est pas inhibé par la polymixine B ce qui montre que l'effet du VIP n'est pas dû à la présence de trace d'endotoxine contaminante présente dans la préparationThese results show that: - the effect of VIP on the expression of the CD83 molecule is not inhibited by polymixin B which shows that the effect of VIP is not due to the presence of trace of endotoxin contaminant present in the preparation
- l'effet du VIP est inhibé par un antagoniste du VIP récepteur ce qui montre que VIP agit en se fixant sur ses récepteurs.- the effect of VIP is inhibited by a receptor VIP antagonist which shows that VIP acts by binding to its receptors.
Nous avons observé que des cellules dendritiques humaines immatures expriment les récepteurs à VIP de type I et II. De plus, nous décrivons également que l'effet du VIP sur la maturation des cellules dendritiques, est inhibé par des antagonistes des récepteurs à VIP : sont inhibées (i) l'induction de l'expression CD83, (ii) l'augmentation de l'expression des molécules impliquées dans l'activation du lymphocyte T telles que : CD40, CD54, CD80 et HLA-Dr, (iii) l'induction de la production d'IL-12 et (iiii) la capacité accrue des cellules dendritiques à induire une
réaction lymphocytaires mixte (comparativement à ces cellules dendritiques immatures).
We have observed that immature human dendritic cells express type I and II VIP receptors. In addition, we also describe that the effect of VIP on the maturation of dendritic cells, is inhibited by VIP receptor antagonists: the following are inhibited (i) the induction of CD83 expression, (ii) the increase in the expression of molecules involved in the activation of the T lymphocyte such as: CD40, CD54, CD80 and HLA-Dr, (iii) the induction of the production of IL-12 and (iiii) the increased capacity of dendritic cells to induce a mixed lymphocyte reaction (compared to these immature dendritic cells).
Claims
1. Utilisation d'au moins un composé choisi dans le groupe formé par le VIP, les agonistes des récepteurs du VIP, les antagonistes des récepteurs du VIP pour la fabrication d'un médicament destiné à moduler la maturation des cellules dendritiques.1. Use of at least one compound chosen from the group formed by VIP, VIP receptor agonists, VIP receptor antagonists for the manufacture of a medicament intended to modulate the maturation of dendritic cells.
2. Utilisation selon la revendication 1 pour la fabrication d'un médicament destiné à moduler la maturation des cellules dendritiques par application cutanée, sous-cutanée, transdermique, intra-épidermique ou par application au niveau des muqueuses.2. Use according to claim 1 for the manufacture of a medicament intended to modulate the maturation of dendritic cells by cutaneous, subcutaneous, transdermal, intra-epidermal application or by application to the mucous membranes.
3. Utilisation selon la revendication 1 caractérisé en ce que la génération et la maturation des cellules dendritiques est effectuée in vitro après mise en contact avec un agent biologique, les cellules matures étant ensuite réinjectées in vivo.3. Use according to claim 1 characterized in that the generation and maturation of dendritic cells is carried out in vitro after contact with a biological agent, the mature cells then being reinjected in vivo.
4. Utilisation selon l'une des revendication précédentes d'au moins un composé choisi dans le groupe formé par le VIP et les agonistes des récepteurs du VIP et d'au moins un agent biologique pour la fabrication d'un médicament pour augmenter la réponse immunologique vis-à-vis de cet agent biologique.4. Use according to one of the preceding claims of at least one compound chosen from the group formed by VIP and VIP receptor agonists and at least one biological agent for the manufacture of a medicament for increasing the response immunological with respect to this biological agent.
5. Utilisation selon la revendication 4 pour la fabrication d'un médicament qui est un vaccin pour le traitement ou la prévention des maladies infectieuses d'origine virale, bactérienne, fongique ou provoquée par une levure, ou un parasite. 5. Use according to claim 4 for the manufacture of a medicament which is a vaccine for the treatment or prevention of infectious diseases of viral, bacterial, fungal origin or caused by a yeast, or a parasite.
6. Utilisation selon la revendication 5 pour la fabrication d'un vaccin pour lutter contre un virus choisi parmi le VIH, les virus hépatiques et le parainfluenza virus.6. Use according to claim 5 for the manufacture of a vaccine to fight against a virus chosen from HIV, hepatic viruses and parainfluenza virus.
7. Utilisation selon la revendication 4 pour la fabrication d'un médicament pour le traitement ou la prévention des cancers.7. Use according to claim 4 for the manufacture of a medicament for the treatment or prevention of cancers.
8. Utilisation selon la revendication 7 caractérisé en ce que l'agent biologique est un agent biologique tumoral pour la fabrication d'un médicament pour favoriser une réponse immune spécifique anti-tumorale.8. Use according to claim 7 characterized in that the biological agent is a tumor biological agent for the manufacture of a medicament for promoting a specific anti-tumor immune response.
9. Utilisation selon l'une des revendications 7 ou 8 pour la fabrication d'un médicament pour le traitement ou la prévention d'un cancer parmi les myélomes, lymphomes, leucémies, carcinomes du rein, du cerveau, de la prostate, du rectum, du pancréas, des ovaires, du poumon.9. Use according to one of claims 7 or 8 for the manufacture of a medicament for the treatment or prevention of cancer among myelomas, lymphomas, leukemias, carcinomas of the kidney, brain, prostate, rectum , pancreas, ovaries, lung.
10. Utilisation selon la revendication 7 ou 8 pour la fabrication d'un médicament pour le traitement ou la prévention d'un cancer cutané choisi parmi les kératinomes et les carcinomes.10. Use according to claim 7 or 8 for the manufacture of a medicament for the treatment or prevention of skin cancer chosen from keratinomas and carcinomas.
11. Utilisation selon l'une des revendications précédentes d'au moins un composé choisi dans le groupe formé par le VIP et les agonistes des récepteurs du VIP, associé au TNF α pour la fabrication d'un médicament destiné à augmenter la prolifération des lymphocytes T.11. Use according to one of the preceding claims of at least one compound chosen from the group formed by VIP and VIP receptor agonists, associated with TNF α for the manufacture of a medicament intended to increase the proliferation of lymphocytes T.
12. Utilisation selon l'une revendications 1 ou 2 d'au moins un antagoniste des récepteurs du VIP pour la fabrication d'un médicament destiné à diminuer l'intensité d'une réponse immune spécifique. 12. Use according to claim 1 or 2 of at least one VIP receptor antagonist for the manufacture of a medicament intended to reduce the intensity of a specific immune response.
13. Utilisation selon la revendication 12 pour la fabrication d'un médicament destiné à diminuer l'intensité d'une maladie autoimmune.13. Use according to claim 12 for the manufacture of a medicament intended to reduce the intensity of an autoimmune disease.
14. Utilisation selon la revendication 12 ou 13 pour la fabrication d'un médicament destiné à diminuer l'intensité d'une maladie autoimmune de type arthrite rhumatoide ou diabète de type I.14. Use according to claim 12 or 13 for the manufacture of a medicament intended to reduce the intensity of an autoimmune disease of the rheumatoid arthritis type or type I diabetes.
15. Utilisation selon la revendication 12 d'un antagoniste des récepteurs de la VIP pour la fabrication d'un médicament destiné à traiter les pathologies d'origine allergique.15. Use according to claim 12 of a VIP receptor antagonist for the manufacture of a medicament intended to treat pathologies of allergic origin.
16. Utilisation selon la revendication 15 d'un antagoniste des récepteurs du VIP pour la fabrication d'un médicament destiné à traiter les pathologies d'origine allergique telle que l'asthme allergique et les dermatites atopiques.16. Use according to claim 15 of a VIP receptor antagonist for the manufacture of a medicament intended to treat pathologies of allergic origin such as allergic asthma and atopic dermatitis.
17. Utilisation selon la revendication 1 ou 2 pour la fabrication d'un médicament pour le traitement ou la prévention du psoriasis. 17. Use according to claim 1 or 2 for the manufacture of a medicament for the treatment or prevention of psoriasis.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| AU65757/00A AU6575700A (en) | 1999-07-15 | 2000-07-13 | Use of vasoactive intestinal peptide (vip) for modulating the development of a specific immune response |
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| FR9909174A FR2796280B1 (en) | 1999-07-15 | 1999-07-15 | NEW USE OF THE VASOACTIVE INTESTINAL PEPTIDE (VIP), ITS AGONISTS AND ANTAGONISTS TO MODULATE THE DEVELOPMENT / INITIATION OF A SPECIFIC IMMUNE RESPONSE |
| FR99/09174 | 1999-07-15 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| WO2001005423A1 true WO2001005423A1 (en) | 2001-01-25 |
Family
ID=9548127
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| PCT/FR2000/002037 WO2001005423A1 (en) | 1999-07-15 | 2000-07-13 | Use of vasoactive intestinal peptide (vip) for modulating the development of a specific immune response |
Country Status (3)
| Country | Link |
|---|---|
| AU (1) | AU6575700A (en) |
| FR (1) | FR2796280B1 (en) |
| WO (1) | WO2001005423A1 (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2006064378A2 (en) * | 2004-12-13 | 2006-06-22 | Maria Teresa De Magistris | Adjuvant activity of gastrointestinal peptides |
| WO2007104493A1 (en) * | 2006-03-15 | 2007-09-20 | Grup D'afectats D'esclerosi Multiple (Gaem) | Use for vasoactive intestinal peptide (vip) and the isolated cells stimulated by said peptide |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5376637A (en) * | 1991-04-22 | 1994-12-27 | Sanwa Kagaku Kenkyusho Co., Ltd. | Pharmaceutical preparation containing vasoactive intestinal polypeptide or its analogue |
-
1999
- 1999-07-15 FR FR9909174A patent/FR2796280B1/en not_active Expired - Fee Related
-
2000
- 2000-07-13 AU AU65757/00A patent/AU6575700A/en not_active Abandoned
- 2000-07-13 WO PCT/FR2000/002037 patent/WO2001005423A1/en active Application Filing
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5376637A (en) * | 1991-04-22 | 1994-12-27 | Sanwa Kagaku Kenkyusho Co., Ltd. | Pharmaceutical preparation containing vasoactive intestinal polypeptide or its analogue |
Non-Patent Citations (5)
| Title |
|---|
| BANCHEREAU J & STEINMAN R M: "Dendritic cells and the control of immunity", NATURE, vol. 392, no. 6673, 19 March 1998 (1998-03-19), pages 245 - 252, XP002134557 * |
| DELNESTE Y ET AL: "Vasoactive intestinal peptide synergizes with TNF-alpha in inducing human dendritic cell maturation.", JOURNAL OF IMMUNOLOGY, vol. 163, no. 6, September 1999 (1999-09-01), pages 3071 - 3075, XP002134556 * |
| JABRANE-FERRAT N ET AL.: "Enhancement by vasoactive intestinal peptide of gamma-interferon production by antigen-stimulated type 1 helper T cells", FASEB JOURNAL, vol. 13, no. 2, February 1999 (1999-02-01), pages 347 - 353, XP002134558 * |
| LUGER T A & LOTTI T: "Neuropeptides: role in inflammatory skin diseases", JOURNAL OF THE EUROPEAN ACADEMY OF DERMATOLOGY AND VENEREOLOGY, vol. 10, no. 3, May 1998 (1998-05-01), pages 207 - 211, XP000891693 * |
| MISERY L: "Skin, immunity and the nervous system", BRITISH JOURNAL OF DERMATOLOGY, vol. 137, no. 6, December 1997 (1997-12-01), pages 843 - 850, XP000891708 * |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2006064378A2 (en) * | 2004-12-13 | 2006-06-22 | Maria Teresa De Magistris | Adjuvant activity of gastrointestinal peptides |
| WO2006064378A3 (en) * | 2004-12-13 | 2006-08-10 | Magistris Maria Teresa De | Adjuvant activity of gastrointestinal peptides |
| WO2007104493A1 (en) * | 2006-03-15 | 2007-09-20 | Grup D'afectats D'esclerosi Multiple (Gaem) | Use for vasoactive intestinal peptide (vip) and the isolated cells stimulated by said peptide |
| ES2315081A1 (en) * | 2006-03-15 | 2009-03-16 | Grup D'afectats D'esclerosi Multiple (Gaem) | Use for vasoactive intestinal peptide (vip) and the isolated cells stimulated by said peptide |
| ES2315081B1 (en) * | 2006-03-15 | 2009-12-30 | Grup D'afectats D'esclerosi Multiple (Gaem) | NEW USE OF THE VASOACTIVE INTESTINAL PEPTIDE (VIP) AND THE ISOLATED CELLS STIMULATED WITH SUCH PEPTIDE. |
Also Published As
| Publication number | Publication date |
|---|---|
| FR2796280B1 (en) | 2001-10-26 |
| FR2796280A1 (en) | 2001-01-19 |
| AU6575700A (en) | 2001-02-05 |
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