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  Director : Mécheri Salah (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

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Role of mast cells and CD117 receptor (c-kit) in the regulation of specific antibody responses: S. Mécheri

C-kit (CD117) has been described as the receptor for Stem Cell Factor (SCF) in mice, human and rat as a transmembrane tyrosine kinase receptor. In adult mice, c-kit is expressed on hematopoietic progenitors in the bone marrow and on mast cells (MCs) in peripheral tissues. We have been interested in the investigation of the role of c-kit+ cells, particularly MCs in the modulation of specific immune responses. C-kit+ cells in mice were targeted in vivo by using a rat monoclonal antibody (Ab) against c-kit (ACK2 clone). Intraperitoneal injection of FITC-ACK2 results in the labelling of a small proportion of c-kit+ cells in the bone marrow and in the peritoneal cavity. Analysis of the specific immune response against rat Ab epitopes demonstrated a dose dependent Ab response in mice injected with ACK2, in contrast to control rat IgG-injected mice. This c-kit mediated Ab response is independent of the Fc portion of ACK2 Ab. To elucidate which c-kit+ cells targeted by ACK2 Ab in vivo were involved in this enhanced immune response, we compared Ab responses in wild type mice (C57BL/6 +/+) and C57BL/6-W/Wv which are well characterised for their c-kit mutation. This point mutation abolishes c-kit signal transduction pathway, which leads to mast cell deficiency. Enhanced specific Ab response is observed in ACK2 injected mice, in contrast to IgG-injected mice and no significant difference could be observed between W/Wv and normal mice. To investigate whether c-kit mutation in W/Wv mice would prevent c-kit mediated endocytosis, FACS analysis of surface expression of c-kit following the addition of ACK2 Ab was performed. Bone marrow derived MC from both wild type and W/Wv showed identical pattern of c-kit endocytosis, indicating that c-kit mutation did not interfere with c-kit-mediated endocytosis. Our data demonstrate that protein antigens targeted to c-kit elicit adjuvant-independent Ab responses and offers a new approach to establish protective immunity against weak immunogens. This mechanism seems to be more dependent on c-kit-mediated endocytosis than on intrinsic signalling potential and is more likely to occur at the level of c-kit positive cells in the bone marrow.

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

The development of an immune adaptative response adequate to eliminate a given pathogen is a cascade of cell signals started in infected tissues by cells of the innate immunity and resulting in Ag-specific effector T cell recruitment. Immature dendritic cells (iDCs) are the key cells of this process: they capture Ag in tissues, 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). Therefore the fate of the T cell response is critically influenced by the signals received from Ag-loaded DCs, including the nature of the inflammatory reaction at the site of Ag entry. 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 adaptative response. This hypothesis is being tested in vitro in the human using monocyte-derived iDCs and stem cell-derived mast cells. Although human mast cells are difficult to obtain, we recently found conditions to derive mature mast cells from human cord blood progenitors. In parallel we also developed a model using Wv/Wv mast cell-deficient mice to assess mast cell role in vivo .

Immunoregulatory functions of mast cell-derived exosomes : S. Mécheri.

Supernatants from bone marrow-derived mouse mast cells (BMMC) and mast cell lines, P815 and MC9, have recently been shown to non-specifically induce B and T lymphocyte activation and cytokine release (IFN-g and IL-2). Using biochemistry and immunoelectron microscopy procedures, 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, hsc 70, and hsp 60. Our studies aimed at to demonstrate whether antigens are associated with exosomes and whether they are present under their native conformation or as processed molecules and to test their potential to prime the immune system in vivo. Indeed, exosomes are located in the endocytic pathway since exogenous antigens accumulate into exosomes within 10 to 15 min after internalization. When subjected to SDS-PAGE analysis followed by immunobloting, exosome-associated antigens appeared as bands with lower molecular weights as compared to their original size, suggesting that they are present in exosomes in a processed form. Given the immunostimulatory potential of several molecules associated with exosomes, exosomes isolated from BMMC loaded with bovine serum albumin (BSA), transferrin, and filamentous hemagglutinin antigen (FHA) a protein derived from Bordetella pertussis, were injected into mice in the absence of adjuvants and specific antibody responses were measured. Exosomes were highly efficient in inducing primary and secondary IgG1 and IgG2a antibody responses. Our data suggest that secretion of exosomes by mast cells may account for a potential role of these cells in the development of specific immunity. Furthermore, 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.

Analysis of the secretory machinery in mast cells and its regulation by stimulation though the high affinity receptor for IgE (FceRI): Ulrich Blank

1- Study of the role of SNAREs and associated proteins in degranulation.

We study the molecular machinery as well as the late signaling events via FceRI involved in the secretory function of mast cells (MC). In particular, we examine the involvement of SNARE proteins during degranulation as well as regulation of this process by proteins such as the small GTPase Rab3D and Munc18 isoforms. In looking for effectors of Rab3D that respond to calcium elevations we detected an associated Ser/Thr kinase activity called Rak3D (= Rab3D-associated Kinase), capable to specifically phosphorylate the t-SNARE syntaxin 4 localized at the plasma membrane. Such phosphorylation affects considerably the ability of syntaxin 4 to interact with its partner SNAP-23 to form a functional SNARE complex and thus could explain how Rab3D can block membrane fusion in resting cells.

2- Effect of oxydative stress on mast cell exocytosis.

We have examined the effect of exposing RBL-2H3 cells to oxydative stress on mediator secretion. We found that incubation in the presence of H202 strongly inhibits FceRI-stimulated degranulation and production of TNF-a without affecting earlier signaling events. Inhibition is increased in the presence of pharmacological agents that increase the ratio between oxidized and reduced glutathion and diminished by agents that decrease this ratio. Thus, our results are in favor of a negative regulatory role of oxidative stress in MC secretory responses, an effect that seemed to depend on the redox buffering capacity of the cell. This type of regulation could be particularly important in an inflammatory environment where the presence of oxidative species is highly elevated.



  publications

puce Publications of the unit on Pasteur's references database


  personnel

  Office staff Researchers Scientific trainees Other personnel
   

Head: Dr. Salah Mécheri (smecheri@pasteur.fr)

Dr. Ulrich Blank DR2 (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: 4th year PhD student

Sophie Martin: 3d year PhD student

Irène Villa: 3d year PhD student

Dimitris Skokos: 2d year PhD student

Bettyna Cyprien DEA

Hany Goubran-Botros: engineer

Bruno Iannascoli: technician

Françoise Marchand: technician

Roger Peronet: technician


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