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  Director : Chignard Michel (chignard@pasteur.fr)


  abstract

 

Our studies deal with innate defense and inflammation in the lung and are performed with different in vitro approaches as well as animal models. We investigate in such a pathophysiological context the role of epithelial cells, alveolar macrophages, neutrophils and their receptors (Toll-like receptors, proteinase-activated receptors and CD87). Another aspect of our research concerns the effect of surfactant on alveolar macrophages (synthesis of phospholipase A2, IL-10 and TNF-a) as well as the interaction of phospholipase A2 with the surfactant-protein A (see figure).



  report

cale

The lung is the site of various diseases for which the mechanisms of innate defense and lung inflammation play a major role. Acute respiratory distress syndrome (ARDS), chronic obstructive pulmonary disease (COPD), cystic fibrosis or invasive pulmonary aspergillosis are typical lung inflammatory diseases. The induction of innate defense is a beneficial process but its exacerbation may lead to a pathologic inflammatory status. Therefore, the major aim of our research is to contribute to the qualitative and quantitative understanding of the mechanisms involved in these diseases, which would allow to target the events enhancing innate immunity without exacerbating the inflammatory process.

Cellular interactions in pulmonary airspaces (Michel Chignard, Dominique Pidard and Mustapha Si-Tahar).

Macrophages and polymorphonuclear neutrophils participate actively to inflammatory and infectious pulmonary diseases. Concerning neutrophils, three secretable serine proteinases particularly account for their effects, namely elastase, cathepsin G and proteinase 3. We have focused our research on the analysis of the effects of these proteinases on different membrane receptors involved in the activation or the adhesion of cells of the innate defense and inflammatory process. Current studies show an effect of elastase and cathepsin G on neutrophils per se. Thus, we observed that these enzymes lower the membrane expression of CD87/uPAR (urokinase-type plasminogen activator receptor), a glycoprotein also expressed by monocytes and epithelial cells, and involved in mechanisms of cell adhesion and tissue repair. Ongoing experiments reveal that these proteinases also cleave the "Proteinase-activated receptor-2" (PAR-2), a receptor expressed at the surface of pulmonary epithelial cells. Such a receptor, for which the physiological agonist(s) is not characterized, is most likely involved in the mechanisms of innate defense and inflammation. Furthermore, we have recently established that elastase and cathepsin G prevent monocyte activation induced by lipopolysaccharides (LPS), a wall component of Gram negative bacteria, through the proteolysis of CD14, a specific membrane receptor for LPS. Being devoid of a transmembrane domain able to transduce intracellular signaling, CD14 acts in concert with newly identified receptors called "Toll-like receptors" (TLR). These receptors allow cells to sense pathogens and trigger mechanisms of innate defense. We are currently evaluating the expression and the role of these receptors within pulmonary tissues and more specifically in epithelial cells.

Using a murine experimental model, we showed that administration of LPS in the airways triggers neutrophil migration and elastase release in the airspaces. Paradoxically, the influx of neutrophils is correlated with an increase of an anti-elastase activity related to DNA released by locally injured cells. The neutrophil influx is due in part to TNF-a produced by LPS-activated alveolar macrophages. Unexpectedly, these activated macrophages are unable to synthesize IL-10, an anti-inflammatory cytokine. The surfactant protein A (SP-A), may explain this particular phenotype as we showed that SP-A is able to down-regulate IL-10 production by cells from the monocytic lineage. Interestingly, during an experimental lethal pulmonary infection with Aspergillus fumigatus, IL-10 is detected in the airspaces and such a production may play a role in animal mortality. It is of note that in this animal model of invasive pulmonary aspergillosis, neutrophils and to a lesser extent alveolar macrophages play a major defensive role.

Mechanisms of regulation and roles of phospholipase A2 in lung inflammatory diseases (Lhousseine Touqui)

Phospholipase A2 (PLA2) catalyses the hydrolysis of phospholipids at the sn-2 position, leading to the generation of cytotoxic lysophospholipids and free fatty acids such as arachidonic acid (AA). The latter is the precursor of leukotrienes and prostaglandins endowed with various biological activities and involved in a number of inflammatory diseases. Recent studies showed the existence of several types of PLA2 whose genes were characterized and classified in several families including intracellular and secretory PLA2. The type IIA secretory PLA2 (sPLA2-IIA) is suggested to play a significant role in different human inflammatory diseases, such as allergic rhinitis, rhumatoïd arthritis, septic shock or ARDS. We developed an animal model which reproduces the anatomopathological criteria of human ARDS. This model is based on the intratracheal administration of LPS to guinea pigs. We showed that LPS causes an acute pulmonary inflammation accompanied by an increased expression of sPLA2-IIA and its release in the alveolar space. Alveolar macrophages are the main source of this enzyme whose expression is induced by an autocrine/paracrine process mediated by TNF-a and inhibited by cAMP. Stimulation of sPLA2-IIA synthesis by LPS is due to induction of this enzyme at the transcriptional level via a process involving the activation of the transcriptional factor NF-kB. Recent results demonstrated that AA down-regulates the expression of sPLA2-IIA mainly via its cyclooxygenase-dependent metabolites and through the inhibition of NF-kB activation. Finally, we showed that sPLA2-IIA is involved in the hydrolysis of surfactant phospholipids and suggested that this process may play a role in the deterioration of the pulmonary surfactant. This deterioration is a typical characteristic of ARDS, leading to the permanent affixing of the alveolar walls, thus blocking the diffusion of oxygen. We also showed that hydrolysis of surfactant phospholipids is controlled by SP-A, which inhibits the catalytic activity of sPLA2-IIA, via a specific and calcium-dependent interaction. This process may represent a mechanism by which surfactant is protected against the deleterious effect of sPLA2-IIA.



  publications

puce Publications of the unit on Pasteur's references database


  personnel

  Office staff Researchers Scientific trainees Other personnel
 

Villeneuve Josiane (jvillene@pasteur.fr)

Chignard Michel Inserm DR1 (chignard@pasteur.fr)

Pidard Dominique CNRS CR1 (dpidard@pasteur.fr)

Si-Tahar Mustapha Inserm CR2 (sitahar@pasteur.fr)

Touqui Lhousseine IP CR (touqui@pasteur.fr)

Beaufort Nathalie DEA (started on 10/10/01)

Chabot Sophie PhD student (schabot@pasteur.fr)

Dulon Sophie PhD student (sdulon@pasteur.fr)

Guillot Loïc PhD student (lguillot@pasteur.fr)

Medjane Samir PhD student (smedjanet@pasteur.fr)

Salez Laurent PhD student (left on 31/10/01)

Wu Yongzheng Postdoc (wuyzh@pasteur.fr)

Balloy Viviane Technician Inserm (vballoy@pasteur.fr)

Leduc Dominique Technician IP (dleduc@pasteur.fr)

Thouron Françoise Technician IP (fthouron@pasteur.fr)


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