|Director : Vargaftig B. Boris (email@example.com)|
We studied the mechanisms of alllergen-induced mucosal metaplasia in the murine model of asthma, particularly the differences between allergen and IL-13 and we showed a role for leukotrienes. Studies concerning the mechanisms of the corticosteroid-resistant bronchoconstriction induced in certain murine strains by LPS showed a large dependence upon CD14 and TLR receptors. Additionally, we identified phospholipid scramblase as a molecular effector of mast cell activation, a triggering step in allergic asthma. Furthermore, we studied the Cox-2 expression, which is enhanced in inflammatory settings. We established that Cox-2 induction in human pulmonary endothelial cells require both NF-kappaB and p38 MAP kinase activation. In addition, we showed that EPA, a polyunsaturated fatty acid, was able to inhibit this enzyme induction through lipoxygenase pathways.
Mechanisms of allergic and non-allergic bronchopulmonary hyperreactivity (B. Boris Vargaftig)
We study the immunological and pharmacological control of pulmonary inflammation, due to allergic, environmental and toxic origins. Th2 lymphocytes play an essential role in triggering and perpetuating allergy, whereas the augmentation of the number or of the activity of Th1 lymphocytes, particularly the augmented production of Il-12 and of d'IFN-g, opposes to this effect has thus an anti-allergic potencial. We demonstrated that the intra-pulmonary administration of B.C.G., a recognized Th1 stimulator, inhibits eosinophilic inflammation of the airways, which characterizes allergy, as well as the accompanying bronchopulmonary hyperreactivit (BHR). The unraveling of the role of the Th1 cytokines and of alveolar macrophages and dendritic cells is under investigation. An ex vivo procedure to study the production of cytokines by lung explants was developed, which allows to show the simultaneous production of cytokines and other relevant mediators generated ex vivo or in vitro following different provocations in vivo or in vitro, whether allergic or not. This procedure led to the study of IL-4R a -/- mice, which, as shown earlier, produce IL-5, undergo BHR and an augmented vascular permeability and recruit eosinophils to the lungs in the absence of IL-4 or of IgE specific for ovalbumin to which they are initially immunized. In preliminary experiences, -/- mice for IL-4 displayed a comparable pattern.
We had shown that mucipare metaplasia and the eosinophilic inflammation which follow the allergenic provocation can be dissociated. We have now demonstrated that this metaplasia, strongly stimulated by the intra-pulmonary administration of IL-13 and of ovalbumin to immunized mice, is accompanied by the production of leucotrienes. A inhibitor of lipoxygenase, the enzyme which accounts for the production of leukotrienes, and leukotriene antagonists, reduced strongly the production of mucus as induced by IL-13, but less so when due to ovalbumin, suggesting an important dissociation and the potential participation of IL-13 in non-allergic lung diseases.
Another aspect of our investigations relate to the bronchopulmonary effects of bacterial endotoxins (LPS), particularly the relationships between macrophage activation and neutrophil recruitment, accompanied by the vascular leakage syndrome similar to the one which characterizes the Acute Respiratory Distress Syndrome (ARDS). The use of different murine strains has shown that those effects are strictly dependent upon CD14 and TLR, and relatively dependent on the " Lipopolyssacharide-binding protein " (LBP).
Signal transduction in mast cells (M. Benhamou)
Mast cells play an important role in the onset of allergic asthma through their activation by IgE and allergen. To identify new molecular effectors in this activation, we generated monoclonal antibodies against phosphoproteins that were purified from stimulated mast cells. Two monoclonal antibodies bound to a protein that was identified as phospholipid scramblase, a protein involved in the distribution of phospholipids across the plasma membrane. This protein is dramatically phosphorylated on tyrosine after IgE receptor aggregation on the mast cell. Our data together with data from the litterature suggest that this protein may be involved in membrane fusion, a late step leading to mast cell degranulation. This hypothesis is currently tested with mutated forms of phospholipid scramblase.
Signalling Pathways in Blood Platelets and Human Pulmonary Endothelial Cells (Mohamed Hatmi)
Endothelial cells and platelets play a key role in thrombosis and participate largely in the inflammatory process. Signalling pathways leading to their activation and interaction involve several enzymes including cyclooxygenases, protein kinases and metalloproteinases.
In earlier previous studoes,, we demonstrated that the expression of the inducible isoform of prostaglandin H synthase (PGHS-2) in human pulmonary microvascular endothelial cells (HPMEC) is enhanced by phorbol myristate acetate and down-regulated by extracellular cAMP.
Then, we sudied the endothelial PGHS-2 gene regulation by inflammatory cytokines, tumor necrosis factor-alpha (TNF-alpha) and interleukin-1beta (IL-1beta). We established here that IL-1beta but not TNF-alpha increased PGHS-2 expression in human pulmonary microvascular endothelial cells (HPMEC). However, associated to IL-1beta, TNF-alpha greatly potentiated this enzyme induction. Although unable to induce PGHS-2 expression by itself, TNF-alpha promoted a similar transcription nuclear factor-kB (NF-kB) activation than IL-1beta. This effect was more pronounced when cells were simultaneously exposed to both cytokines. As expected, interleukin-8 induction, used as a positive control of NF-kB activation, was significantly enhanced by TNF-alpha alone. HPMEC pretreatment with MG-132, a proteasome inhibitor, prevented NF-kappaB activation as well as more distal signalling response, indicating that NF-kappaB activation is required but not sufficient for PGHS-2 expression. Both IL-1beta and TNF-alpha failed to activate c-Jun NH2-terminal kinase (JNK). In addition, PD98059, an inhibitor of p42/44 mitogen-activated protein kinase (MAPK) phosphorylation did not suppress the expression of the PGHS-2. However, SB 203580, a p38 MAPK inhibitor, markedly suppressed PGHS-2 induction by IL-1beta alone or combined with TNF-alpha, indicating that p38 MAPK but not p42/44 MAPK or JNK cascades were required for PGHS-2 up-regulation. Finally, TNF-alpha, unlike IL-1beta, was unable to promote p38 MAPK phosphorylation, indicating that the failure of TNF-alpha to induce PGHS-2 expression is linked, at least in part, to its inability to activate p38 MAPK signalling pathway.
Together, these data enhanced our understanding on PGHS-2 regulation in HPMEC and outline the heterogeneity of cellular responses to pro-inflammatory cytokines.
Furthermore, we examined the effect of eicosapentaenoic acid (EPA), a polyunsaturated fatty acid widespreat in fish oil and known for its antithrombotic activity. We particularly established that EPA down-regulates IL-1beta-induced PGHS-2 gene induction. This inhibitory effect is dependent from lipoxygenase pathways and involved, at least in part, the p38 MAPK cascade. In contrast, neither NF-kappaB nor peroxisome proliferator-activated receptors (PPARs) are implicated in the EPA effect.
Finally, we studied the matrix metalloproteinases (MMPs), a family of proteinases involved in various physiopathologic process including angiogenesis and inflammation. We established that MMP2 is constitutively expressed in HPMEC. In contrast, MMP9 which is not expressed at the basal level, is induced by both IL-1beta and TNFalpha cytokines.
In summary, our activity is focused on the study of signalling pathways involved in platelet and endothelial cell activation, particularly those linked to proinflammatory inducible enzymes PGHS-2 and MMP9.
|Publications of the unit on Pasteur's references database|
|Office staff||Researchers||Scientific trainees||Other personnel|
Thépaut Sylvana, firstname.lastname@example.org
Villeneuve Josiane, email@example.com
Vargaftig B. Boris, I.P., (firstname.lastname@example.org)
Hatmi Mohamed, I.P., C.R. (email@example.com)
Benhamou Marc, Inserm, C.R. 1, (firstname.lastname@example.org)
Assier Eric, contractual-researcher, (email@example.com)
Hubeau Cédric, contractual-researcher, (firstname.lastname@example.org)
Elalamy Ismail, teacher-cum-researcher, (email@example.com)
Ait Said Fatima, PhD Student
Charles, Nicolas, PhD Student
El Mehdi, Delphine, DEA
Proust, Barbara, PhD Student
Joseph, Danielle, assistant-engineer, CNRS, (firstname.lastname@example.org)
Lefort, Jean, engineer position 2, (jvlefort@pasteur)
Mamas, Suzannne, engineer position 3, (email@example.com)
Nahori, Marie-Anne, engineer position 1, (firstname.lastname@example.org)
Singer, Monique, engineer IE2 INSERM, (email@example.com)
Gomard Marie-Thérèse, sup. technician. 1D, (firstname.lastname@example.org)
Motreff Laurence,. sup. technician. 1D, (email@example.com)
Riveron, Stéphanie, sup. technician.labo. 1D
Berman, Bruce, lab. agent
Membrillera, Antonio, animal technician
Sahli, Fatima, lab. agent