FR2879747A1 - In vitro evaluation of e.g. photoimmunosuppression/ photosensitization of a product, comprises applying the product on keratinocytes and Langerhan cells, evaluating the modulating capacity, analyzing and measuring gene expression rate - Google Patents

Abstract

In vitro evaluation of the capacity of at least one product to effect change to the cutaneous immune defences in a photoinduced condition, comprises applying the product before, during and/or after UV irradiation, and comparing the number, size and dendricity of the cells that are exposed and are not exposed, is new.

Description

In particular, the invention relates to a method of in vitro evaluation of the

  ability of at least one product to modulate skin immune defenses in photoinduced condition, characterized in that it is evaluated, on an epidermis equivalent comprising

  at least keratinocytes and Langerhans cells exposed to UV irradiation, the ability of said product to modulate (a) the number and / or morphology of Langerhans cells and / or (b) the variation of the level of gene expression specifically regulated in Langerhans cells during a contact hypersensitivity reaction (HSC).

  The activity of a combination of products or even the overall activity of a composition can also be evaluated according to the invention.

  This method makes it possible in particular to screen and / or evaluate either the protective and / or repairing activity of products vis-à-vis photonmunosuppression (eg UV filters) or the photosensitizing character (photoallergising or phototoxic) of products intended for compositions cosmetic or dermatological.

  The invention also relates to the use of an epidermis equivalent comprising at least keratinocytes and Langerhans cells subjected to UV irradiation, as an in vitro model for evaluating the protective and / or repairing potential of visceral products. photoimmunosuppression or the photosensitizing nature of products.

  The skin is recognized as one of the main active elements of the immune defense system. Three types of epidermal cells participate in this system: keratinocytes, melanocytes and Langerhans cells, which play a key role in the cutaneous immune response to external aggressions (chemicals, allergens, micro-organisms, UV radiation).

  Langerhans cells are bone marrow-derived dendritic cells that can be characterized by the presence of Birbeck granules and the expression of the CD1α antigenic marker (CD1α positive cells). They play a decisive role in the initiation of immune responses directed against the antigens introduced into the skin or newly generated by it. In contact with an allergen, Langerhans cells migrate to ganglia where they trigger specific T-cell responses; as such, they are considered antigen presenting cells, essential for the proper functioning of T lymphocytes. However, Langerhans cells are also the preferred target of UV radiation, which can alter their structure and / or function and thereby - even affect the immune system dermal (photoimmunosuppresssion), leading to a decrease in resistance to aggression and including an increase in the incidence of certain cancers.

  UV radiation is also responsible for photosensitization, which is a chemical process associated with a molecule or product, called a photosensitizer, whose presence in a system makes it sensitive to light.

  These photosensitized skin reactions generally correspond to undesirable side effects of the product and are manifested: in the short term, by phototoxicity or photoallergy reactions; - in the long term, by mutagenic or carcinogenic responses.

  These photosensitized skin reactions can induce the formation of photoadducts within the epidermal cells, the generation of reactive oxygen species, a phototoxicity in terms of apoptosis and necrosis and consequences on the phenotype and the functionality of the cells whose Langerhans cells.

  A phototoxicity reaction is a photochemical irritation resulting from the combined action of a product and light, which occurs during a first exposure, provided that the phototoxic agent is in sufficient quantity and the skin is exposed to appropriate wavelengths. Clinical lesions are limited to surfaces exposed to light.

  A photoallergic reaction also requires the combined action of a product and light: it is an immunological phenomenon that includes an induction phase and a revelation phase, that is to say that the reaction is triggered usually during subsequent exposure to UV and the photoallergising agent.

  These photosensitizing molecules can be contained in different products (drugs, plant essences, dyes, household products or cosmetics) and the reaction can occur following ingestion or contact.

  The importance of Langerhans cells in the cutaneous immune system is therefore understood from the foregoing and the need for an in vitro model for predicting in vivo UV effects, which can be used as a model for evaluating products capable of modulating the structure and / or the function of Langerhans cells under photoinduced conditions, in other words to modulate the cutaneous immune defenses under photoinduced conditions.

  Now the Applicant has just shown that the in vitro model of epidermis equivalent comprising at least keratinocytes and Langerhans cells described in EP 0 789 074 responded to UV (UVA and UVB or UVA alone) of the same in vivo model of human skin. Indeed, it was able to show that conditions of exposure to UV radiation altered similarly in both models the number and / or morphology of Langerhans cells, which are one of the manifestations of a photoimmunosuppression reaction (at level of Langerhans cell structure).

  By altering the number of Langerhans' cells is meant a change in the number of Langerhans cells, in particular a decrease in their number.

  By alteration of the Langerhans cell morphology is meant a change in the size of the Langerhans cells and / or their dendricity, i.e. the number and length of the dendrites; in particular means a decrease in the size of said cells and / or their dendricity.

  Moreover, she was able to show that this same model, put in contact with an allergen and subjected to a UV radiation, made it possible to highlight an alteration of the response to an allergen-induced contact hypersensitivity reaction, another manifestation. of the photoimmunosuppression reaction (at this time of the function of the Langerhans cells). This alteration of the contact hypersensitivity reaction (HSC) was visualized by a decrease in the variation in the level of expression of the IL-113 gene known to be normally specifically overexpressed in Langerhans cells during a hypersensitivity reaction. HSC contact.

  Alteration of the contact hypersensitivity reaction (HSC) is understood to mean a decrease in the response to a contact hypersensitivity reaction, which is manifested by a decrease in the level of expression of specifically regulated genes in Langerhans cells during an HSC reaction. This decrease in the variation of the level of expression may correspond to a decrease or, more rarely, an increase in the level of expression of the genes, depending respectively on whether the genes are normally overexpressed or conversely under-expressed during a reaction. HSC. Indeed, it is known that the HLA-DR, CD80, CD86, CD83, CD54 (ICAM-1), CD40, CD44, CD45, CD58 (LFA-3), CD32 / 16 (FcIIIIII), CD23 (FcERII) genes, CD24, CD11c, CCR7, CXCR4, IL-1 (3, IL-1 [3 Converting Enzyme (ICE), Matrix Metalloproteinase-9 (MMP-9), 5- Lipoxygenase (5-LO), F4 / 80 are normally susceptible to be overexpressed specifically at the level of Langerhans cells during a contact hypersensitivity reaction, whereas the CD1a and CCR6 genes are on the contrary likely to be specifically under-expressed at the level of Langerhans cells during of a contact hypersensitivity reaction.

  The use of this equivalent of epidermis comprising at least keratinocytes and Langerhans cells thus appears to be advantageous as an in vitro model for evaluating protective and / or repairing products with regard to photoimmunosuppression or photosensitization ( photoallergising or phototoxic) of products.

  The invention therefore relates to a process for the in vitro evaluation of the ability of at least one product to modulate the cutaneous immune defenses under photoinduced conditions, characterized in that it is evaluated, on an epidermis equivalent comprising at least keratinocytes and Langerhans cells exposed to UV irradiation, the ability of said product to modulate (a) the number and / or morphology of Langerhans cells and / or (b) the variation in the level of expression of specifically regulated genes in Langerhans cells during a HSC contact hypersensitivity reaction.

  The person skilled in the art will easily determine, by habit, the nature of the control necessary for carrying out the process.

  In particular, the control may be an epidermis equivalent comprising keratinocytes and Langerhans cells placed under the same conditions as the test condition but in the absence of the test product.

  Appropriate controls are also used to ensure the relevance of the results observed on our model. It will be equivalents of epidermis comprising keratinocytes and Langerhans cells placed under the following conditions: - non-irradiated; - non-irradiated + product to be tested; - non-irradiated + allergenic; - irradiated.

  According to a first embodiment, the method according to the invention is a method for evaluating the protective and / or repairing activity of at least one product with respect to photoimmunosuppression, comprising the following steps: contacting an epidermis equivalent comprising at least keratinocytes and Langerhans cells with a product to be tested, said product being applied before and / or during and / or after UV irradiation; the number and / or the size and / or the dendricity of the Langerhans cells are analyzed; this result is compared with a control that has not been brought into contact with said product; the products for which the number and / or the size and / or the dendricity of the Langerhans cells are selectively or slightly decreased compared with the control are selected.

  To evaluate the ability of a product to maintain (protective potential) skin immune defenses under photoinduced conditions, the product must be applied before UV irradiation.

  To evaluate the ability of a product to restore (skin repair potential) skin immune defenses under photoinduced conditions, the product must be applied during and / or after UV irradiation.

  The analysis of the number and / or the size of the Langerhans cells may be carried out according to the standard techniques of immunostaining and image analysis. The number of Langerhans cells expressed in cells / cm 2 of epidermis or in cells / mm of epidermis length may, for example, correspond to the count of the number of HLA-DR, Langerine or CD1 + cells after the implementation of conventional techniques. immunostaining.

  The general principle of the immunolabeling technique consists, for example, in incubating the epidermal layers, previously separated from the dermis, with a monoclonal rat antibody directed against the antigens of the major histocompatibility complex of class II: human HLA-DR (clone: YE2 / 36HL Harlan Seralab) present at the cell membranes of Langerhans cells, or with an antibody against Langerin (Immunotech). The presence of these antibodies is then revealed by means of a second biotinylated antibody directed against rat immunoglobulins and then fixation of a peroxidase-coupled streptavidin complex and revelation with AEC (3-aminoethyl carbazole) or a second antibody. directed against fluorescein-coupled rat immunoglobulin (FITC).

  The analysis of the dendricity of Langerhans cells can be carried out according to conventional techniques of image analysis and measurement of the number and length of dendrites.

  According to an alternative, this process may incorporate the presence of an allergen and comprise the following steps: an epidermal equivalent comprising at least keratinocytes and Langerhans cells is brought into contact with a product to be tested, said product being applied before and / or during and / or after UV irradiation; an allergen is applied; the variation in the level of expression of specifically regulated genes in the Langerhans cells is measured in response to said allergen; this measurement is compared with a witness that has not been put in contact with said product; the products for which the variation in the level of expression of specifically regulated genes in the Langerhans cells in response to said allergen is selectively or slightly decreased with respect to the control.

  The measurement of the variation of the level of expression of specifically regulated genes in Langerhans cells in response to an allergen can be carried out by measuring the amount of mRNA of said gene (eg IL-1 (3) according to the techniques conventional transcriptomic analysis, such as RNA-arrays or RT-PCR, preferably the RT-PCR technique will be used.

  For example, the RT-PCR technique may include the steps of: isolating total RNAs from epidermal equivalents; synthesizing the corresponding cDNAs by reverse transcription; amplifying, by PCR polymerization, the fragments corresponding to IL-113, using primers specific for IL-1 R and for HPRT (control); - Analyze PCR products by agarose gel electrophoresis and quantification by fluorometric detection, compared to the untreated control.

  It will thus be advantageous to test the protective and / or repairing activity of UV filters with respect to photoimmunosuppression. Indeed, it is known that the effectiveness of UV filters with respect to the erythema defined by the SPF (Sun Protection Factor), does not predict their effectiveness vis-à-vis the photoimmunosuppression (Kelly DA et al. , 2000, J. Exp Med., Vol 191, num 3, pp.561-566).

  The protective and / or restorative activity of compounds vis-à-vis photoimmunosuppression, known to prevent photoinduced alteration of the number and / or morphology of Langerhans (eg polyphenols extracted from green tea) and / or the photo-induced decrease in HSC reactions (eg plant isoflavonoids, vitamins).

  According to another embodiment, the method according to the invention is a method for evaluating the phototoxic nature of at least one product, comprising the following steps: an epidermal equivalent comprising at least keratinocytes and Langerhans cells with a product to be tested, said product being applied before and / or during a UV irradiation; the number and / or the size and / or the dendricity of the Langerhans cells are analyzed; this result is compared with a control that has not been brought into contact with said product; phototoxic character is attributed to the products for which the number and / or the size and / or the dendricity of the Langerhans cells is decreased compared with the control.

  According to an alternative, said method can integrate the presence of an allergen and combine the following steps: an epidermal equivalent comprising at least keratinocytes and Langerhans cells is brought into contact with a product to be tested, said product being applied before and / or during UV irradiation; an allergen is applied; the variation in the level of expression of specifically regulated genes in the Langerhans cells is measured in response to said allergen; this measurement is compared with a witness that has not been put in contact with said product; the products of which the variation of the level of expression of genes specifically regulated in Langerhans cells in response to an allergen are attributed to a 'phototoxic' character are diminished with respect to the control.

  According to another embodiment, the method according to the invention is a method for evaluating the photoallergising character of at least one product, comprising the following steps: contacting an epidermis equivalent comprising at least keratinocytes and Langerhans cells with a product to be tested, said product being applied prior to UV irradiation; the number and / or the size and / or the dendricity of the Langerhans cells are analyzed, as well as (ii) the variation of the level of expression of genes specifically regulated in the Langerhans cells during a HSC reaction. ; these results are compared with a control that has not been put in contact with said product; - a photoallergenic character is attributed to products for which (i) the number and / or size and / or dendricity of Langerhans cells is decreased relative to the control and / or (ii) the variation in the expression level of Specifically regulated genes in Langerhans celllules are increased relative to the control.

  According to an alternative, the method may incorporate the presence of an allergen and comprise the following steps: an epidermal equivalent comprising at least keratinocytes and Langerhans cells is brought into contact with a product to be tested, said product 5 being applied before UV irradiation; an allergen is applied; the variation in the level of expression of specifically regulated genes in Langerhans cells is measured during a HSC reaction; this measurement is compared with a control which has not been brought into contact with the said product and the allergen; an allergen-amplifiable photoallergic character is attributed to products for which the variation in the level of expression of genes specifically regulated in Langerhans cells during a HSC reaction is amplified relative to the control.

  By 'photoallergic amplifiable by an allergen' is meant a compound whose photoallergising character is amplified in the presence of an allergen.

  This alternative may be of interest in particular for visualizing additive or synergistic effects of compounds with (allergenic) character, said compounds possibly being present in the same composition or in different compositions.

  In the methods described above comprising a step of measuring the variation of the level of expression of a gene specifically regulated in Langerhans cells in response to an allergen, said gene can be chosen from the HLA-DR genes, CD80 , CD86, CD83, CD54 (ICAM-1), CD40, CD44, CD45, CD58 (LFA-3), CD32 / 16 (FcγR11 / III), CD23 (FcεRII), CD24, CD11c, CCR7, CXCR4, IL-1. 1 R, IL-1p Converting Enzyme (ICE), Matrix Metalloproteinase-9 (MMP-9), 5-Lipoxygenase (5-LO), F4 / 80, CD1a and CCR6.

  The measurement of the variation of the level of expression of specifically regulated genes in Langerhans cells in response to an allergen can be carried out by measuring the amount of mRNA of said gene (eg IL-1p) according to the conventional techniques of transcriptomic analysis, such as RNA-arrays or RT-PCR.

  Preferably, the RT-PCR technique will be used, which may comprise, for example, the following steps: isolating the total RNAs from the epidermis equivalents; synthesizing the corresponding cDNAs by reverse transcription; amplify, by PCR polymerization, the fragments corresponding to IL-113, using primers specific for IL-1 [3 and HPRT (control); - Analyze PCR products by agarose gel electrophoresis and quantification by fluorometric detection, compared to the untreated control.

  The allergen used in the process according to the invention is an agent capable of inducing on the epidermis equivalent a contact hypersensitivity reaction (HSC), characterized in particular by a variation in the level of expression of certain genes. , specifically at the level of Langerhans cells.

  The allergen may, for example, be chosen from oxazolone, 2,4-dinitrofluorobenzene, para-phenylenediamine, eugenol, benzocaine and nickel sulphate.

  Advantageously, the oxazolone will be used.

  The allergen is generally applied at a sub-toxic concentration less than or equal to 1/3 of its cytotoxic concentration. This cytotoxic concentration is variable according to the allergens and is determined, for example, by the observation under the microscope of cellular alterations at the keratinocyte level (ex: modification of the shape, loss of adhesion).

  In particular the allergen will be applied at a dose and for a time sufficient to trigger a contact hypersensitivity reaction, measurable by the variation of the expression level of certain genes known to be specifically regulated in Langerhans cells in the presence of an allergen. For IL-1 [3 for example, the allergen, especially oxazolone, can be applied for 24 hours.

  The epidermis equivalent used in the methods of the invention described above, is described in application EP 0 789 074. It comprises at least keratinocytes and at least Langerhans cells precursors induced or not. It may also include other cell types of the skin, particularly melanocytes.

  This equivalent of epidermis is usually prepared on a support; by way of example, mention may be made, as a support, of collagen / fibroblast fusion slates, dermis previously de-epidermalized, artificial membranes such as, for example, Millipores brand filters, subcutaneous substitutes based on collagen, in particular the EPISKIN biomaterial. , plastic or any other medium compatible with cell viability.

  Preferentially, the support consists of a dermis previously derepidermized and the protocol described in Prunieras et al., Ann. Chir. Plast., 1979, 24, No. 4, 357-362.

  The keratinocytes used in the epidermis equivalent may be prepared according to any known method of the prior art. By way of example, mention may be made of the culture from dissociated epidermis originating from normal or pathological skin sampling or the culture of keratinocytes derived from the sheath of normal or pathological hair follicles.

  Preferably, according to the invention, the keratinocytes used are prepared from dissociated epidermis originating from normal or pathological human skin samples, in particular according to the method described in Régnier et al., Frontier of Matrix Biology, Vol.9, 4-35 (Karger, Basel 1981) or from the sheath of normal or pathological human hair follicles.

  The precursors of Langerhans cells may be any stem cell capable of differentiating under the effect of induction into Langerhans cells, that is to say able to differentiate into CD1a positive cells.

  Advantageously, these precursors may be CD34 + hematopoietic cells (Caux et al., Nature, Vol 360, Nov. 1992, 258).

  Langerhans cell precursors can be purified from naturally occurring tissues, including bone marrow, peripheral blood, umbilical cord blood. Preferentially, the CD34 + cells obtained from umbilical cord blood marketed by Praxcell will be used.

  Other sources of Langerhans cell precursors can also be used in the epidermis equivalent used in the method according to the invention: CD14 + monocytes derived from blood marketed by Poïetics / Cambrex; human myeloid lines, such as the HL60 KG1, U937 and THP1 lines marketed by ATCC or the MUTZ-3 line; dendritic cells derived from CD34 + cord blood (CD34-D) marketed by MatTek.

  In particular, Langerhans cells originating from precursors of Langerhans cells selected from cells derived from CD34 + hematopoietic cells of human origin, CD34-D cells or cells of myeloid lines HL60 KG1 or MUTZ-3 will be used.

  The induction of the differentiation of Langerhans cell precursors can be carried out before or after they are co-cultured.

  This differentiation can be induced by cytokines such as colony stimulating factor (Granulocyte / Macrophage -Colony Stimulating Factor or GMCSF), tumor necrosis factor (TNF-α), stem cell factor (Stem Cell Factor or SCF), interleukin 3 or interleukin 4 or a mixture thereof.

  But it can also be spontaneously induced by the presence of keratinocytes or by a culture of these precursors in a medium in which keratinocytes have previously been cultured.

  The precursors of the Langerhans cells induced or not are co-cultured at any stage of development of the epidermis equivalent.

  The ratio between the number of keratinocytes and the precursors of Langerhans cells induced or not, co-cultured on the support, is generally between 95/5 and 25/75 as a percentage of the total number of cells in the co-culture. and preferably between 75/25 and 35/65 and even more preferentially this ratio is 50/50.

  The nutrient medium used may be any nutrient medium known for its ability to allow proliferation and differentiation of keratinocytes. An example of this is Eagle-modified Dulbecco's medium, or defined medium with varying calcium content, such as the medium described by Boyce ST and Ham RG (J. Tissue Cult, Meth., 1985, 9, 83- 93). Advantageously, it is possible to use a mixture of several nutrient media such as, for example, the Dulbecco medium modified Eagle medium / HAM medium F12 or Rheinwald medium and Green, (Cell, 1975, 6, 331334).

  According to one particular embodiment of the invention, the epidermis equivalent used in one of the processes described above is prepared as follows: a mixture of normal human keratinocytes, previously isolated, is prepared according to the method described in Régnier et al. (Front Matrix Biol, Vol.9, 4-35, 1981) with or without human melanocytes previously isolated according to the method described in Olsson et al., (Acta Derm, Venereol., 1994, 74, 226-268), in a proportion of 10 to 1.

  These cells are deposited on a derepidermized dermis (Regnier et al, Front Matrix Biol, Vol.9, 4-35, 1981, Regnier et al., Exp Cell Res, 165: 63-72, 1986) or on a typical collagen carrier Episkin (1.3 cm). . On the derepidermized dermis, 5 × 10 5 cells per cm 2 are deposited according to the method described in Prunieras et al., (Ann Chir Plast., 1979, 24, No. 4, 357-362) and the culture is carried out in a medium consisting of of a mixture of Eagle-modified Dulbecco's medium and HAM F12 medium at a ratio of 3 to 1, containing 10% fetal calf serum, 10 ng / ml epidermal growth factor (EGF), 400 ng / ml of hydrocortisone, 10-6 M Isoproterenol, 5 11g / ml transferrin, 2 x 10-9 M detriiodothyronine, 1.8 x 10-4 M adenine and 5 g / ml insulin (Rheinwald and Green, Cell, 1975 , 6, 331-334).

  The culture is thus kept submerged for 6 days. The culture is then placed at the air / liquid interface, said liquid then being composed of the same medium as previously of which isoproterenol, transferrin, triiodothyronine and adenine have been removed.

  In parallel, CD34 + cells are isolated from umbilical cord blood and cultured according to the method described in Caux et al., (Nature, Vol 360, 19 November 1992, pp. 258-260), for 6 days in the presence of colony stimulating factor (Granulocyte / Macrophage Colony Stimulating Factor or GM-CSF) at a concentration of 200 ng / ml and Tumor Necrosis Factor (TNF-α) at a concentration of 2.5 ng / ml .

  2 days after passage of the keratinocyte / melanocyte mixture at the air / liquid interface, 5 × 10 5 CD34 + cells, as previously prepared, are deposited on the epidermis in reconstruction.

  The culture is then maintained until a histologically satisfactory epidermis equivalent is obtained, that is to say an epidermis equivalent having the conventional cell layers, namely the basal, suprabasal, granular and horny layers.

  On an episkin-type collagenous medium (1.3 cm), 4x105 keratinocytes are inoculated simultaneously with dendritic cells derived from CD34 + (5 × 10 4 CDl +).

  The CD34 + cells were previously cultured and differentiated into a CD1a + dendritic subpopulation for 7 days in StemBio A medium (C.N.R.S.

  Villejuif, France) in the presence of 1000 U / ml GM-CSF, 50 U / ml TNF-α, 20 ng / ml of Stem Oeil Factor (SCF) replaced on the 3rd or 4th day with 0.5 ng / ml of Tumor Growth Factor ( TGF (3).

  The cell mixture is kept immersed for 3 days in Eagle-modified Dulbecco's medium and HAM F12 medium in a ratio of 3 to 1, containing 10% fetal calf serum, 10 ng / ml epidermal growth factor (EGF), 400 ng of hydrocortisone, 10-6 M Isoproterenol, 5 g / ml transferrin, 2 x 10-9 M triiodothyronine, 1.8 x 10-4 M adenine and 5 g / ml insulin (Rheinwald and Green, Cell, 1975, 6, 331-334) .The cells are then placed for 10 days at the air / liquid interface in the same medium as above in which isoproterenol, transferrin, triiodothyronine and adenine have been removed.

  The UV irradiation used in the method of the invention is an irradiation comprising UVA and UVB (RSS or simulated solar radiation) or UVA alone.

  The RSS irradiation condition can be reproduced with an Oriel Xenon lamp (1000W) equipped with at least one dichroic mirror and a Schott WG-320 (RSS) filter. The irradiation condition with UVA alone can be reproduced using an Oriel Xenon lamp equipped with at least one dichroic mirror and a Schott WG-335 filter.

  The UV irradiation is applied to an effective biological dose (DBE or BED) and for a time sufficient to induce the formation of sunburn cells in the epidermis equivalent, characterized in particular by a suprabasal position, a picnotic nucleus (condensed chromatin) and an eosinophilic cytoplasm (colored orange).

  For example, the BED dose used in the method of the invention is a dose of between 8.4J / cm 2 and 9.6 J / cm 2; and the UV irradiation may for example be applied for a period of 10-15 min. The invention further relates to the use of IL-1 expression rate variation (3 as a marker of Langerhans cell activity under photoinduced conditions in an in vitro potency evaluation method. protecting and / or repairing at least one product vis-à-vis the photoimmunosuppression or the photosensitizing potential of at least one product, said method being carried out on an epidermis equivalent comprising at least keratinocytes and Langerhans cells subjected to UV irradiation (UVA and UVB or UVA only) and to an allergen.

  The invention further relates to the use of an epidermis equivalent comprising at least keratinocytes and Langerhans cells subjected to UV irradiation (UVA and UVB or UVA alone), as an in vitro model for evaluating the protective potential. and / or repairing products vis-à-vis photoimmunosuppression or photosensitizing potential of products.

  The invention will now be illustrated by the following nonlimiting examples.

EXAMPLES

  Example 1 Comparison of in vivo skin model and in vitro model of reconstructed epidermis containing Langerhans cells against UV effects (SSR or UVA alone) In vivo model Spindle-shaped biopsies 1 cm long were taken on subjects with fair skin (phototype II and III) under local anesthesia (xylocaine 2% adrenalysed) 24 hours (UVA study) or 72 hours (SSR study == UVA / UVB) after the exposure .

  The UVA exposures were performed using a 10 cm x 10 cm Oriel xenon lamp equipped with a dichroic mirror and a Schott WG-335/3 mm filter.

  The RSS-2MED exposures were realized using an Oriel xenon lamp equipped with a dichroic mirror and a Schott WG-320 / 1.5 mm filter.

  The analysis of Langerhans cells is made by an immunolabeling technique according to the following protocol: the epidermal layers, previously separated from the dermis, were incubated for 40 hours at 37 ° C. with a monoclonal rat antibody directed against the antigens of the complex major class II histocompatibility: human HLA-DR (clone: YE2 / 36HL - Harlan Seralab) present at the cell membranes of Langerhans cells either at room temperature overnight with an antibody against Langerin diluted to 1/200 (Immunotech). The presence of these antibodies is then revealed by means of a second biotinylated antibody directed against rat immunoglobulins and then fixation of a peroxidase-coupled streptavidin complex and revelation with AEC (3-aminoethyl carbazole) or a second antibody. directed against fluorescein-coupled rat immunoglobulin (FITC).

  In vitro model 000 CD1a + dendritic cells, derived from CD34 + umbilical cord blood cells, and 400,000 normal human keratinocytes are inoculated on Episkin support (1.3 cm2). Cells are cultured for 3 days in DMEM / Ham F12 medium (3: 1) containing 10% fetal calf serum, 10 ng / ml EGF, 400 ng / ml hydrocortisone, 10-6 M isoproterenol, 5 μg / ml transferrin, 2x10-9 M triiodothyronine, 1.8x10-4 M adenine and 5 μg / ml insulin. The cells are then placed at the air-liquid interface for 10 days to induce epidermal differentiation in a culture medium without isoproterenol, transferrin, triiodothyronine and adenine.

  The samples were exposed under a 15 cm x 15 cm Oriel Xenon lamp equipped with a dichroic mirror and a Schott WG-335/3 mm (UVA) filter or a Schott WG-320 / 1.5 filter. mm (SSR). Twenty-four hours after irradiation, the reconstructed epidermis was detached with 2M sodium bromide and fixed with acetone.

  The immunolabelings of the epidermal layers were carried out with the LSAB 2 System HRP Kit (DAKO, Glostrup, Denmark). The anti-Langerin monoclonal antibody (DCGM4 clone, Beckmann Coulter / Immunotech, Marseille, France) was used at 1 / 300th and the anti-HLA-DR (/ DP / DQ) antibody (clone CR3 / 43, M0775, DAKO) at 1 / 50th - Density and morphology analyzes The density (number) of positive cells is evaluated by image analyzer and the results are expressed in number of cells per mm 2 of epidermis.

  The morphological alterations of Langerhans cells, in terms of their size and / or their dendricity (number and length of dendrites) are evaluated by image analysis.

  UVA results The analysis of the density of HLA-DR positive and Langerin positive cells, identified as Langerhans cells, revealed a significant linear global dose effect (p <0.01) for the 2 modes of detection implemented (anti-HLA-DR or anti-Langerine antibodies) and for the 2 models tested (in vitro / vivo): the number of Langerhans cells decreases linearly as a function of the dose of UVA delivered.

  Morphological alterations of Langerhans cells (HLA-DR positive) are also observed after UVA exposure: in the in vivo model, a decrease in the size of Langerhans cells after UVA exposure and the disappearance of dendrites (decrease in their number and their length); this effect is linear as a function of the dose of UVA. An identical trend is observed in vitro after exposure to 30 J.cm 2 of UVA.

  Results In vivo, there is a decrease in the number of Langerhans cells (density) after SSR exposure. The threshold dose to observe this effect is greater than 1 DEM. An identical trend is observed in vitro after exposure to 8.4 J-cm-2 SSR.

  In the 2 models evaluated, we observed a variability of the results concerning changes in the morphology of Langerhans cells after SSR exposure, but a tendency towards a decrease in size and / or dendricity of Langerhans cells.

  All these results show that the effect of UV (UVA and SSR) on the number and morphology of Langerhans cells is globally similar in the two models evaluated: in vitro (reconstructed epidermis containing Langerhans cells) and in vitro. vivo (human skin).

  This makes our in vitro model a good model for evaluating the effect of UVA and UVB or UVA alone on the cutaneous immune system involving Langerhans cells, and hence a good model for evaluating protective and protective active ingredients. / or repairers vis-à-vis the photoimmunosuppression or photosensitizing active (phototoxic or photoallergic).

  Such a method for evaluating the protective and / or repairing activity with respect to the photoimmunosuppression or the photosensitizing activity of products may thus be based on an analysis of the number of Langerhans cells and / or their dendricity, compared to a witness.

  EXAMPLE 2 Evaluation of the Protective Potential of Mexoryle SX with respect to the Photoinduced Inhibition of Overexpression of IL-15 Caused by Oxazolone (photoimm unosuppression) Preparation of the epidermis equivalent The equivalent of epidermis comprising at least keratinocytes and Langerhans cells is prepared according to the method described in application EP 0 789 074 (L'OREAL) or in Regnier et al (1997, the journal of investigative dermatology, 109, No. 4 , pp.510-512) or a derived method, as described below.

  A mixture of normal human keratinocytes previously isolated is prepared according to the method described in Régnier et al., (Front Matrix Biol, Vol.9, 4-35, 1981) with or without human melanocytes previously isolated according to the method described in Olsson et al. (Acta Derm, Venereol., 1994, 74, 226-268) in a proportion of 10 to 1.

  4x105 keratinocytes are inoculated simultaneously with dendritic cells derived from CD34 + (5x104 CD1 a +) on a collagenous support type Episkin (1.3 cm2).

  The CD34 + cells were previously cultured and differentiated into a CD1a + dendritic subpopulation for 7 days in StemBio A medium (CNRS Villejuif, France) in the presence of 1000 U / ml GM-CSF, 50 U / ml TNF-α, 20 ng / mL of Stem Cell Factor (SCF) replaced on the 3rd or 4th day with 0.5 ng / ml of Tumor Growth Factor (TGF- (3).

  The cell mixture is kept immersed for 3 days in Eagle-modified Dulbecco's medium and HAM F12 medium in a ratio of 3 to 1, containing 10% fetal calf serum, 10 ng / ml epidermal growth factor (EGF), 400 ng / ml hydrocortisone, 10-6 M Isoproterenol, 5 μg / ml transferrin, 2 x 10-9 M triiodothyronine, 1.8 x 10-4 M adenine and 5 gg / ml insulin (Rheinwald and Green, Cell, 1975, 6, 331-334). The cells are then placed for 10 days at the air / liquid interface in the same medium as above in which isoproterenol, transferrin, triiodothyronine and adenine have been removed.

  Treatment of epidermal equivalents Epidermis equivalents thus prepared will be treated separately according to the following conditions: control 1: non-irradiated; control 2: application of oxazolone 1% (allergen) for 24 hours; - control 3: RSS irradiation; - control: irradiation RSS (Simulated Solar Radiation) then application, for example 1 hour later, oxazolone 1% for 24 hours; test: application of the product to be tested, namely Mexoryl SX 4% (UV filter) 15 minutes before irradiation RSS then application, for example 1 hour later, of oxazolone 1% for 24 hours.

  The RSS irradiation condition or Simulated Solar Radiation is reproduced with an Oriel Xenon lamp (1000W, 15cm x 15cm) equipped with a dichroic mirror and a Schott WG-320 / 1.5mm filter (RSS 9.6J / cm2).

  Alternatively, the RSS irradiation condition can be replaced by an UVA irradiation condition alone, using a 15 cm x 15 cm Oriel Xenon lamp equipped with a dichroic mirror and a filter. Schott WG-335/3 mm.

  Measurement of the variation of the expression level of the IL-113 mRNAs by RT-PCR.

  The epidermal layers reconstructed as described above are detached from the support and the total RNAs are extracted with Trizol (Life Technologies / GibcoBRL) according to the protocol recommended by the supplier, and then reverse-transcribed into cDNA. A first strand of cDNA is synthesized from 1 μg of RNA using (a primer) an oligo (dT) 18 primer (First-strand synthesis kit, Amersham Pharmacia Biotech). The cDNA is then amplified in a total volume of 100 μl containing 50 μM KCl, 10 μM Tris-HCl (pH 8.3), 1.5 μM MgCl 2, 200 μM dNTPs, specific PCR primers (200 μMol IL-1 (3 or 5 μMol of hypoxanthine- guanine phosphoribosyltransferase HPRT), and 2.5 units of AmpliTaq DNA polymerase (Perkin Elmer).

  PCR polymerase chain reaction cycles (Gene Amp PCR system 2400, Perkin Elmer) are carried out under the following conditions: denaturation at 94 ° C. for 45 seconds, binding of the primers at 60 ° C. (IL1 (3 / HPRT) for 1 minute and polymerization at 72 C for 2 minutes.

  The specific IL-1 (3 and HPRT (control) primers used are as follows: IL-1-p (250 bp PCR product, Reutter et al., Toxicol in vitro (1997) 11, 619- 626) 5 '- (6-Fam) GAC ACA TGG GAT AAC GAG GC 5'-ACG CAG GAC AGG TAC AGA TT HPRT (housekeeping gene, PCR product 470 bp): 5' - (6-Fam) GGC GTC GTG ATT AGT GAT GAT GAAT GAAT CC 5'-CTT GCG ACC TTG ACC ATC TT GGA PCR products are analyzed by 2% agarose gel electrophoresis in Trisacetate EDTA buffer containing ethidium bromide and quantified by fluorometric detection. after separation by capillary electrophoresis, using an ABI PRISM 310 Genetic Analyzer Analyzer (Applied Biosystems).

  The IL-113 / HPRT fluorometric intensities in the treated samples are compared to the untreated control.

  The following results are obtained: Experimental conditions Ratio mRNA IL-1 (3 / non-irradiated HPRT (control) 1.0 Oxazolone 1% (control 2) 1.7 RSS + Oxazolone 1% (control) 0.8 Mexoryl SX 4% + RSS + Oxazolone 1% 1.8 RSS (control 3) 1.1 These results show that the epidermal equivalent model comprising Langerhans cells subjected to an allergen (control 2) responds by a variation (increase) in the expression of IL-1 ( 3, corresponding to the activity of Langerhans cells during the contact hypersensitivity reaction in the presence of an allergen.

  The decrease in the variation of the level of expression of IL-1 (3 in the RSS + oxazolone condition (control), corresponds to a photoinduced inhibition of the over-expression of IL-1 (3 normally induced by allergen, this inhibition corresponds to a photoimmunosuppression reaction affecting the functionality of Langerhans cells (ability to produce IL-1R).

  In the presence of the Mexoryl SX 4% (UV filter test), applied before the RSS radiation and the application of the allergen, a level of IL-1 expression level is observed (3 equivalents to the allergen condition alone). , which shows a protective effect of the UV filter vis-à-vis the photoimmunosuppression reaction observed in the absence of UV filter.The UV filter is thus able to protect the immune function of Langerhans cells against the effects of UV.

  The irradiation condition RSS (control 3) alone shows that there is no influence on the constitutive expression level of IL-113.

  Thus the equivalent of reconstructed epidermis containing at least keratinocytes and Langerhans cells is a good model to evaluate the protective potential of products (eg UV filters) vis-à-vis the photoimmunosuppression.

  Example 3 Evaluation of the Restorative Potential of a Product with respect to the Photoinduced Inhibition of Overexpression of IL-1f Caused by Oxazolone Evaluation of the Repair Potential of a Product or of a Drug a composition containing at least this product may be carried out according to a protocol similar to that described in Example 2 but with the application of the product after and / or during the UV irradiation.

  The test may consist of: a preparation of an epidermis equivalent comprising at least keratinocytes and Langerhans cells, as described in Example 2; an RSS irradiation of said epidermis equivalent; the application, for example 1h after (or at the same time as the irradiation), on this equivalent of epidermis, the product or the composition to be tested for 24 to 72 hours - the application of the allergen for 24 hours; the measurement of the expression level of the IL-113 mRNAs by RT-PCR, according to the protocol described in Example 2.

  If the product is photoreparator, an increase in the level of expression of the IL-1 mRNAs (3 induced by the allergen compared to the control is measured, whereas a non-photoreparative product does not prevent the UV inhibition of allergen-induced IL-113 overexpression.

  EXAMPLE 4 Evaluation of the photosensitizing (phototoxic or photoallergic) potential of a product The evaluation of the photosensitizing potential of a product or of a composition containing at least this product can be carried out according to a protocol similar to that described in the example 2.

  The test may consist of: a preparation of an epidermis equivalent comprising at least keratinocytes and Langerhans cells, as described in Example 2; UV irradiation of said epidermal equivalent in the presence of the product or of the composition to be tested, said product being applied before the UV irradiation; the application, for example 1 hour after irradiation, of the allergen for 24 h, the measurement of the expression level of the IL-1 mRNAs (3 by RT-PCR, according to the protocol described in FIG. Example 2

  For a photoxic product, a greater inhibition of the over-expression of IL-1 (3 than with the UV alone will be obtained: the phototoxic product enhances the UV effect If the difference in inhibition between "UV" and "UV + phototoxic" is weak, we can show that we have the same inhibition but at a lower UV dose with the photoxic product (from a range of doses RSS).

  For a photoallergenic product, which behaves like a photoinducible allergen (need to put it before irradiation), one will obtain an increase of the expression IL-1 [3 compared to a lack of treatment or an allergen alone. If the difference between over-expression with "1 allergen" and "allergen + photoallergic" is low, we can show that we have the same over-expression but at a concentration of the lower allergen with the photoallergic product (from a range of concentrations for the allergen).

Claims (16)

  1. Method for in vitro evaluation of the ability of at least one product to modulate the cutaneous immune defenses under photoinduced conditions, characterized in that it is evaluated, on an epidermis equivalent comprising at least keratinocytes and cells of Langerhans exposed to irradiationUV, the ability of said product to modulate (a) the number and / or morphology of Langerhans cells and / or (b) the variation of the level of expression of specifically regulated genes in Langerhans cells during of a hypersensitivity reaction of HSC contact.   2. In vitro evaluation method according to claim 1, characterized in that it is a method of evaluating the protective and / or repairing activity of at least one product with respect to the photoimmuriosuppression, comprising the following steps: an epidermal equivalent comprising at least keratinocytes and Langerhans cells is brought into contact with a product to be tested, said product being applied before and / or during and / or after a UV irradiation ; the number and / or the size and / or the dendricity of the Langerhans cells are analyzed; this result is compared with a control that has not been brought into contact with said product; - Products are selected for which the number and / or size and / or dendricity of Langerhans cells is not decreased compared to the control. 25 3. Method according to claim 2, characterized in that it further comprises the application of an allergen on said equivalent after the step of contacting the product to be tested, and wherein: - the variation is measured the level of expression of specifically regulated genes in Langerhans cells in response to said allergen; this measurement is compared with a witness that has not been put in contact with said product; the products for which the variation of the level of expression of specifically regulated genes in the Langerhans cells in response to said allergen is selected is not decreased with respect to the control.   4. In vitro evaluation method according to claim 1, characterized in that it is a method for evaluating the phototoxic nature of at least one product, comprising the following steps: - contacting a epidermis equivalent comprising at least keratinocytes and Langerhans cells with a product to be tested, said product being applied before and / or during UV irradiation; the number and / or the size and / or the dendricity of the Langerhans cells are analyzed; this result is compared with a control that has not been brought into contact with said product; - the 'phototoxic' character is attributed to products for which the number and / or size and / or dendricity of Langerhans cells is decreased compared with the control.   5. In vitro evaluation method according to claim 4, characterized in that it further comprises the application of an allergen to said equivalent after the step of placing in contact with the product to be tested, and wherein: the variation in the level of expression of specifically regulated genes in the Langerhans cells is measured in response to said allergen; this measurement is compared with a witness that has not been put in contact with said product; phototoxic character is attributed to products for which the variation in the level of expression of specifically regulated genes in Langerhans cells in response to an allergen is decreased compared with the control.   6. In vitro evaluation method according to claim 4, characterized in that it is a method for evaluating the photoallergising nature of at least one product, wherein said product is applied before a UV irradiation, and which further comprises the steps of: - measuring the rate of expression of specifically regulated genes in Langerhans cells during a HSC reaction; this measurement is compared with a witness that has not been put in contact with said product; a photoallergising character is attributed to products for which the variation in the level of expression of genes specifically regulated in Langerhans cells is increased relative to the control.   7. In vitro evaluation method according to claim 6, characterized in that it further comprises the application of an allergen after contacting said equivalent with the product to be tested, and wherein: variation in the level of expression of specifically regulated genes in Langerhans cells during a HSC reaction; this measure is compared with a control that has not been brought into contact with the said product and the allergen; an allergen-amplifiable photoallergic character is attributed to products for which the variation in the level of expression of genes specifically regulated in Langerhans cells during a HSC reaction is amplified relative to the control.   8. Method according to any one of claims 1, 3, 5, 6 and 7, characterized in that the genes whose variation of the expression level is measured are chosen from HLA-DR, CD80, CD86, CD83, CC ) 54 (ICAM-1), CD40, CD44, CD45, CD58 (LFA-3), CD32 / 16 (FcγR11 / III), CD23 (FcεR11), CD24, CD11c, CCR7, CXCR4, IL-1 (3, IL -1 (3 Converting Enzyme (ICE), Matrix Metalloproteinase-9 (MMP-9), 5-Lipoxygenase (5-LO), F4 / 80, CD1a and CCR6.   9. Method according to any one of claims 1, 3, 5, 6 and 7, characterized in that the expression rate of specifically regulated genes in the Langerhans cells of the HSC reaction is evaluated by the amount of MRNA of said gene measured by an RT-PCR technique.   10. Process according to any one of claims 3, 5, 6 and 7, characterized in that the allergen is selected from oxazolone, 2,4 dinitrofluorobenzene, para-phenylenediamine (pPD), eugenol, benzocaine and nickel sulphate.   11. The method of claim 10, characterized in that the allergen is applied to said reconstructed epidermis in an amount less than or equal to 1/3 of the cytotoxic concentration of said allergen.   12. Method according to any one of claims 1 to 11, characterized in that the equivalent of epidermis comprises keratinocytes obtained from dissociated epidermis from normal or pathological human skin sampling or from the sheath of the skin. normal or pathological human hair follicles.   13. Method according to any one of claims 1 to 12, characterized in that the Langerhans cells are derived from precursors of Langerhans cells selected from cells derived from hematopoietic cells CD34 + of human origin, CD34-D cells, or cells of myeloid lines HL60 KG1 or MUTZ-3.   14. Method according to any one of claims 1 to 13, characterized in that the UV irradiation comprises UVA and UVB or UVA alone.   15. The method of claim 14, characterized in that the UV irradiation is applied to an effective biological dose of between 8.4J / cm 2 and 9.6J / cm 2.   16. Use of an epidermis equivalent comprising at least keratinocytes and Langerhans cells subjected to UV irradiation, as an in vitro model for evaluating the protective and / or repairing potential of products with respect to the skin. photoimmunosuppression or the photosensitizing nature of products.