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  Director : Salah Mécheri (smecheri@pasteur.fr)


  abstract

 

Our main activity is focused on the study of the mechanisms of mast cell (MC) activation and the immunoregulatory properties of MC. We have demonstrated and characterized the participation of SNARE proteins in MC exocytosis. Furthermore, MC have been shown to activate B and T lymphocytes through a novel mechanism involving exosomes released by MC. These observations have been extended to the investigation of the relationships between MC and dendritic cells.



  report

cale

Role of mouse mast cells in the initiation and the amplification of immune response in vivo: S. Mécheri

Mouse, human, and rat mast cells have been shown to express MHC II molecules and present antigens to specific T cell hybridomas in vitro. The purpose of our investigation was to determine whether mouse mast cells are able to initiate specific immune responses in vivo. Induction of anti-DNP IgG1 and IgG2a antibodies was performed by transfering OVA-DNP-pulsed bone marrow-derived mast cells (BMMC), B cells or macrophages into naive mice which were boosted later with soluble antigen. Cultured spleen cells from immunized mice were tested for their cytokine content. Our data show that mast cells were by far better inducers of anti-DNP IgG1 antibodies than B cells and macrophages. In contrast, anti-DNP IgG2a response induced by macrophages was much stronger than that obtained with mast cells whereas B cells were completely unable to elicit this response. In addition to a high index of cell proliferation, spleen cells from mast cell-injected mice produced more IFN-g than those mice who received macrophages or B cells by two- to five-fold, and almost ten-fold, respectively. Our data suggest that mast cells have the potential to upregulate both humoral and cellular immune responses in vivo.


Biochemical and functional analysis of mouse mast cell exosomes: potential application to the modulation of specific immune responses: S. Mécheri

We have demonstrated that, in addition to their role in allergic reactions, mast cells can contribute to the regulation of specific immune responses. Furthermore, we provided evidence that mast cells can activate B and T lymphocytes in an antigen-independent manner resulting in the formation of large clusters of T cell and B cell blasts as well as IL-2, IL-12, and IFN-g production. This mast cell-mediated heterotypic aggregation as well as cytokine production were completely inhibited by anti-LFA-1 and anti-ICAM-1 mAbs. Using biochemistry and immunoelectron microscopy, we found that the mast cell immunostimulatory activity was constitutively secreted and consisted of membrane vesicles termed exosomes, originally stored in the mast cell cytoplasmic granules. Preliminary data show that several proteins were found to be associated with exosomes including MHC II, LFA-1, ICAM-1, actin, hsc 70, and hsp 60. Given the immunostimulatory potential of these molecules, exosomes loaded with bovine serum albumin (BSA) were injected into mice and anti-BSA antibody response was measured. Exosomes were highly efficient in inducing primary and secondary anti-BSA IgG1 and IgG2a antibody responses. This subcellular compartment is proven to be useful as a substitute for whole cells in stimulating the immune system in vivo and therefore can be used as cell free vaccine for various infectious diseases and cancer.


Interactions between mast cells (DC) and dendritic cells (DC): Salah Mécheri and Christian Demeure (in collaboration with Pr Guy Delespesse, Montreal)


After capturing Ag in peripheral tissues, immature dendritic cells (iDCs) undergo major functional and morphological changes (maturation) and migrate to the T cell zones of secondary lymphoid organs where they prime naive T cells (nT). The fate of the latter is decided by the signals received from Ag-loaded mature DCs (mDCs). The rate of DCs migration, their survival and the spectrum of co-signals that they deliver to nT is in turn dependent upon the nature of the inflammatory reaction at the site of Ag entry and capture by iDCs. Since MCs co-localize with iDCs and may be rapidly and directly activated by pathogens or Ags, we postulate that the MCs-dependent inflammatory reaction will shape both the function of DCs and the nature of the subsequent T cell response. Immature DCs are obtained exactly as in our previous reports by culturing blood monocytes for 5-7 days with GM-CSF and either IL-4 or IFN-a. Although human mast cells are difficult to obtain, we recently found the optimal conditions to derive mature mast cells from human cord blood progenitors. All the human MCs employed throughout this project are generated in vitro by culturing CD34+ c-kit+ (CD117) precursors in the presence of SCF and IL-6. Mast cells generated after 8 weeks of culture express the high affinity receptor for IgE (FceRI), CD117, and contain tryptase, a specific marker for mast cells.


Analysis of the molecular mechanisms of the secretory function in FceRI-stimulated mast cells: Ulrich Blank

Upon stimulation through their high affinity IgE receptors (FceRI), mast cells (MC) release their preformed granular content of allergic mediators by degranulation and also secrete numerous cytokines. In this group we study the molecules as well as the signaling events involved in the secretory function of MC. Using a rat mast cell line (RBL-2H3), we developed a novel assay that allows us to detect MC degranulation at the single cell level using cytofluorometry. The assay is based on the measurement of externalized phosphatidylserine that is closely correlated to exocytosis and occurs in the complete absence of apoptosis. We also characterized the implication of the t-SNARE protein syntaxin 4 in MC exocytosis providing evidence that MC, like neuronal cells, utilize the structural framework provided by SNARE proteins to overcome the fusion barrier between opposing granular and plasma membrane during exocytosis. We further studied how environmental factors such as hydrogen peroxide produced at the inflammatory site could affect FceRI-induced degranulation and cytokine production. In a collaborative work with Dr. J. Rivera (NIH) we have also investigated the function and the recruitment into lipid rafts of the signaling adaptor protein vav known to be a crucial regulator of JUN Kinase (JNK) activity and consequently cytokine production.



  publications

puce Publications of the unit on Pasteur's references database


  personnel

  Office staff Researchers Scientific trainees Other personnel
   

Dr. Salah Mécheri, Chef de laboratoire (smecheri@pasteur.fr)

Dr. Ulrich Blank CR1 (CNRS ) (ublank@pasteur.fr)

Dr. Claudine Guérin-Marchand CR1 ( CNRS )(clobert@pasteur.fr)

Dr M. Roa CR (mroa@pasteur.fr)

Dr C. Demeure CR (cdemeure@pasteur.fr)

Isabel Pombo: 4e année de thèse

Sophie Martin: 3e année de thèse

Irène Villa: 3e année de thèse

Dimitris Skokos: 2e année de thèse

Ludovic Deriano: DEA

Hany Goubran-Botros: ingénieur

Bruno Iannascoli: technicien

Françoise Marchand: technicienne

Roger Peronet: technicien animalier


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