Mechanisms of allergic bronchopulmonary hyperreactivity (B. Boris Vargaftig)
The prevalence of pulmonary allergic diseases has grown dramatically. These diseasesP results from the interaction between endogenous (exposure to allergens, infections, pollutants) and exogenous factors (for instance, the intensity and nature of lymphocytic responses). We are involved with research concerning the immunological and pharmacological modulation of lung and airways inflammation after allergenic, environmental and infectious provocations. In case of allergy, we study the interactions between antigen presenting cells, T lymphocytes and eosinophils, which are the potential vector of the epithelium lesions. These cell interactions lead to and are induced by the production of pro-allergic cytokines, IL-4, IL-5, IL-9, IL-13 and a large number of chemokines. Three distinct subtypes of CD4+ lymphocytes can presently be distinguised : the Th1, which produce IL-2 and IFN-g, the Th2 which synthesie IL-4, IL-5, IL-9, IL-10 and IL-13, and Th0, which are less elective. Allergy involves Th2, the Th12 being rather protective. IL-4 plays an essential role in lymphocyte engagement into the Th2 and the production of IgE, whereas IL-5 is essential for the initation and perpetuation of eosinophils. We correlate the immunological lung lesions avec bronchopulmonary hyperreactivity (BHR) and vascular and airways permeability, two important component of the late asthmatic reactions. BHR is the increased airways capacity to respond to nons-epcific bronchoconstrictor agents, which characterizes asthma and correlates with airways eosinophilia and mucosal metaplasia. The increased permeability correspond to lung edema. Since the essential cytokine for the pro-allergic evolution of Th2 lymphocytes is IL-4, one would expect its absence of neutralisation to prevent allergy. Surprisingly, -/- mice for the IL4-Ra, (receptor chain a, common with IL-13), which were not educated to produce Th2 lymphocytes, still express BHR, produce abundant IL-5, express increased permeability and recruit eosinophils to the lungs, in absence of IL-4, of IgE and eosinophil presence ion the bronchoalveolar lavage fluid. It appears that despite the absence of IL-4, Th2 cells capable of producing IL-5 are available.
We had demonstrated that mucusal metaplasia and eosinophilic inflammation after allergenic provocation are distinct events. We have now shown that the oral administration of ovalbumin before or immediately after immunisation suppresses the expression of BHR, airways eosinophilia, the increased production of IL-4, IL-5 and of IgE and mucosal metaplasia, with no effect over production of IFNg or of IL-12. It is thus possible to tolerise for all the landmlarks of experimezntal airways allergy.
Mechanisms of non-allergic bronchopulmonary hyperreactivity (B. Boris Vargaftig)
Three major events follow the administration of LPS to mice airways: the in situ activation of alveolar macrophages, neutrophil recruitment and activation and a vascular leakage syndrome, similar to the Acute Respiratory Distress Syndrome (ARDS). The i.v./i.p. administration of LPS to C57Bl/6 mice is followed by an intense adhesion of neutrophils to vascular endothelium, in absence of migration to the alveolar compartment. Since there is no diapedesis, strictly speaking there is no "inflammation", but rather a state of "pre-inflammation". LPS did reach the lungs when delivered systemically, since TNF-a RNA was found in lungs. By contrast, TNF-a was not found in the BALF. Despite the paucity of respiratory mechanical changes following systemic LPS, a marked BHR was unmasked when the bronchoconstrictor agent methacholine was aerosolized. This was not due to TNF-a formation nor to the endo-vascular neutrophil sequestration, since BHR persisted in mice immunized to TNF-a or when neutrophils were depleted (vinblastine, anti-granulocyte serum).This allowed to hypothesize that LPS generates a local (pulmonary ?) and/or systemic signal for partial neutrophil recruitment, which is TNF-a independent. Furthermore, BHR after LPS is also not mediated by TNF-a. The effects of the intra-airways delivery of LPS differ substantially from those which follow its systemic administration. A very intense " direct ", bronchoconstriction was noted 70-90 minutes after LPS instillation/aerosolisation, with a massive migration of neutrophils into the airways and later to the BALF, and the production of TNF-a possibly by the alveolar macrophage. This recruitment was followed by the BALF enrichment in proteins and by the augmentation of the lung weight, two markers of alveolo-capillary disfunction. In separate studies with the Unité d'Immunogénétique cellulaire, we demonstrated that the vascular leakage syndrome which hampers the clinical use of IL-2 can be duplicated in mice, provided it is administered systemically for 4 days. This occurs in absence of neutrophil recruitment to lungs and is not accompanied by BHR.
Anti-inflammatory glucocorticosteroids, which suppress the production of TNF-a and prevent BHR due to i.p. LPS, failed to reduce it when induced by aerosolized LPS. Paradoxically, large doses of dexamethasone or budesonide augmented BHR. Similarly, a small protective effect of the glucocorticosteroids on protein exudation was replaced by its aggravation when larger doses were injected. LPS-induced "direct" bronchoconstriction and lung sequestration of neutrophils were also practically unaffected by the steroids, even though their BALF recruitment (and TNF-a production) were suppressed. By contrast, vinblastine suppressed lung recruitment of neutrophils and bronchoconstriction and BHR by LPS. Our results indicate that the mechanisms of vascular adhesion and further progression of neutrophils on one side, and of their subsequent passage to the alveolar compartment, on the other side, are glucocorticosteroid-independent and dependent, respectively. Most interestingly, the recognized difficulties to reach significant protection with glucocorticosteroids in ARDS is mimiked in our model.
Cellular interactions in pulmonary airspaces (Michel Chignard, Dominique Pidard and Mustapha Si-Tahar)
Macrophages and polymorphonuclear neutrophils participate to inflammatory and infectious pulmonary diseases. These pathologies include emphysema, acute respiratory distress syndrome, chronic obstructive pulmonary disease, cystic fibrosis, or invasive aspergillosis.
Concerning neutrophils, three 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 inflammation system. Thus, we have shown that elastase and cathepsin G inhibit specifically endothelial cell activation induced by thrombin through a proteolysis of its receptor, namely the "Proteinase-activated receptor-1" (PAR-1). Ongoing experiments reveal that these proteinases also cleave PAR-2, a receptor expressed at the surface of pulmonary epithelial cells. Such a receptor, for which the physiological agonist is unknown, is most probably involved in the mechanisms of innate defense and inflammation. Furthermore, we have just shown that elastase and cathepsin G prevent monocyte activation induced by LPS, a wall component of Gram negative bacteria, through the proteolysis of CD14, a specific membrane receptor for LPS. The latter being devoid of a transmembrane domain able to transduce intracellular signaling, acts in concert with newly identified receptors called "Toll-like receptors" (TLR). These receptors allow cells to sense pathogens and to trigger mechanisms of innate defense. We currently evaluate the expression and the role of these receptors at the level of pulmonary cells and more specifically epithelial cells. Studies also presently performed show an effect of elastase and cathepsin G on neutrophils per se. Thus, we observed that they lower the membrane expression of CD87/uPAR (urokinase-plasminogen activator receptor), a glycoprotein also expressed by monocytes and epithelial cells, and involved in mechanisms of adhesion and tissue repair. Using a murine experimental model, we showed that administration of LPS to 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 synthesized IL-10, an anti-inflammatory cytokine. A surfactant protein, 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 airspaces. Such a production may play a role in animal mortality. It is of note that in this animal model of invasive aspergillosis, neutrophils but not alveolar macrophages play a major defensive role.
Role of phospholipase A2 in lung inflammation (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 cloned and classified in several families including intracellular and secretory PLA2s. The type-II secretory PLA2 (sPLA2-II) is suggested to play a significant role in different human inflammatory diseases, such as allergic rhinitis, rhumatoïd arthritis, septic shock or acute respiratory distress syndrome (ARDS). We developed an animal model, which reproduces the anatomopathological criteria of human ARDS. This model is based on the intra-tracheal administration of LPS to guinea pigs. We showed that LPS causes an acute pulmonary inflammation accompanied by an increased expression of sPLA2-II and its release in the alveolar space. Alveolar macrophages (AM) 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 transcriptional level via a process involving NF-kB activation. Recent results demonstrated that AA down-regulates the expression of sPLA2-II in AM 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 pulmonary surfactant. This deterioration is a typical characteristic of ARDS, leading to the permanent affixing of the alveolar walls (alveolar collapse), thus blocking the diffusion of oxygen. We also showed that hydrolysis of surfactant phospholipids is controlled by a surfactant protein, i.e., surfactant protein A (SP-A) which inhibits the catalytic activity of sPLA2-II, via a specific and calcium-dependent interaction. This process may represent a mechanism by which the organism protects surfactant against the deleterious effect of sPLA2-IIA.
Signalling Pathways in Blood Platelets and Human Pulmonary Endothelial Cells (Mohamed Hatmi)
Endothelial cells which cover the vessel wall play a key role in the adhesion and activation of circulating blood cells, particularly platelets. The latter have various functions including hemostasis, thrombosis and inflammation. Signalling pathways leading to their recruitment and activation are complex and involve numerous enzymes including, cyclooxygenases, protein kinases and metalloproteinases.
Study of platelet metalloproteinases. Matrix metalloproteinases (MMPs) are a family of proteinases whose principal function appears to be the breakdown of extracellular matrix proteins during tissue remodeling processes associated with growth, development and repair. We have shown the existence of membrane type-1 matrix metalloproteinase (MT1-MMP) in both plasma and serum of healthy human volunteers and we have investigated human platelets as a possible source of MT1-MMP. On platelet membranes, MT1-MMP was found in an apparent 89 kDa form and the 45 kDa furin processed form. After platelet degranulation only the 45 kDa form was detected. RT-PCR experiments showed the expression of mRNA encoding for MMP2, tissue inhibitor of matrix metalloproteinase type 2 (TIMP2) and MT1-MMP in blood platelets. Flow cytometric analysis revealed that MT1-MMP and TIMP2 expressions were enhanced at the surface of activated platelets. Recombinant TIMP2 and a synthetic MMP inhibitor BB94, inhibited aggregation in a concentration-dependent manner, suggesting the modulatory role of MMPs in platelet function.
Modulation by cAMP and by proinflammatory cytokines of cyclooxygenase-2 expression. We studied the expression of the inducible isoform of cyclooxygenase (Cox-2) in human pulmonary microvascular endothelial cells (HPMEC) and particularly its modulation by extracellular cAMP. Exposure of HPMEC to phorbol myristate acetate (PMA) led to the increase of Cox-2 protein level which was significantly attenuated by extracellular cAMP. In addition, Cox-2 activity evaluated through 6 keto-PGF1alpha generation was also inhibited by extracellular cAMP. PKI, a specific PKA inhibitor, blocked this extracellular cAMP effect, suggesting the involvement of PKA signalling at the outer surface of HPMEC. Accordingly, we established the presence of an endothelial ecto-protein A kinase activity. RT-PCR analysis showed that PMA-induced Cox-2 mRNA was markedly reduced by extracellular cAMP. On the other hand, we sudied the endothelial Cox-2 gene regulation by TNF-alpha. We found that interleukin-1beta (IL-1beta) increases Cox-2 expression in HPMEC, whereas tumor necrosis factor-alpha (TNF-alpha) is inactive but, surprisingly, greatly potentiates the response to IL-1beta. Then, we examined the role of transcription nuclear factor-kappaB (NF-kappaB), an up-regulator element of various inflammatory genes. Although unable to induce Cox-2 expression by itself, TNF-alpha promoted a marked NF-kappaB activation. MG-132, a proteasome inhibitor, prevented NF-kappaB activation by both cytokines as well as more distal IL-1beta responses and their potentiation by TNF-alpha. SB 203580, a p38 mitogen-activated protein (MAP) kinase inhibitor, markedly suppressed Cox-2 expression in response to IL-1beta alone or combined with TNF-alpha. In addition, TNF-alpha, unlike IL-1beta, was unable by itself to promote the phosphorylation of p38 MAP kinase, indicating that the failure of TNF-alpha to induce Cox-2 expression is linked to its unability to activate p38 MAP kinase pathway. Furthermore, the potentiating effect of TNF-alpha on IL-1beta-induced Cox-2 expression in HPMEC is tightly depende nt from NF-kappaB activation.