Philippe Lesavre

TNF-Induced β2 Integrin Activation Involves Src Kinases and a Redox-Regulated Activation of p38 MAPK

We previously demonstrated that the TNF-α-induced inside-out signaling leading to β2 integrin activation is redox regulated. To identify kinases involved in this pathway, the effects of kinase inhibitors on the expression of β2 integrin activation neoepitope (clone 24) were investigated. We show that both p38 MAPK (inhibited by SB203580) and Src kinases (inhibited by PP2) are involved in β2 integrin activation by TNF and oxidants in human neutrophils. Src kinases appeared constitutively active in resting neutrophils and not further activated by TNF or oxidants in nonadherent conditions. However, PP2 blocked both TNF-induced expression of the 24 epitope and cell adhesion promoted by the integrin activating anti-CD18 KIM185 mAb, showing that both the inside-out and the outside-in signaling involve Src kinases. p38 MAPK was activated by TNF and oxidants in nonadherent conditions i.e., with 10 mM EDTA. This activation in EDTA resulted in CD11b, CD35 and CD66 up-regulation and in an oxidative response, all blocked by SB203580 and PP2. p38 MAPK was not activated upon direct integrin activation by KIM185 mAb. Thus, p38 activation allows the study to distinguish the initial transduction pathway leading to β2 integrin activation from the signaling resulting from integrin engagement. Finally, p38 MAPK activation by TNF was blocked by diphenylene iodonium, an inhibitor of flavoprotein oxidoreductase, and by the free radical scavenger N-acetylcystein. Taken together, these results demonstrate, for the first time, that constitutively activated Src tyrosine kinases and a redox-regulated activation of p38 MAPK are involved in TNF inside-out signaling leading to β2 integrin activation.

Apoptosis‐induced proteinase 3 membrane expression is independent from degranulation

Proteinase 3 (PR3) and human neutrophil elastase (HNE) are serine proteinases stored in the azurophilic granules of neutrophils. In contrast to HNE, PR3 is the target of antineutrophil cytoplasm antibodies (ANCA) in Wegeners granulomatosis. The mechanisms leading to the membrane expression of PR3 and HNE are still unclear and appear to be critical to understand the pathophysiological role of ANCA. Stably transfected rat basophilic cell lines (RBL) with PR3 or HNE were used to analyze the PR3 and HNE secretion mechanisms and differentiate between them. RBL cells were lacking endogenous PR3 and HNE. They were stably transfected with HNE or PR3 or an inactive mutant of PR3 (PR3S203A). Using the calcium ionophore A23187 as a secretagogue, higher serine proteinase activity was secreted in the supernatant of RBL/HNE than in RBL/PR3. It is interesting that PR3 and PR3/S203A were also expressed at the plasma membrane, thus demonstrating that serine protease activity was not required for plasma membrane expression. In contrast, no expression of plasma membrane HNE could be detected in RBL/HNE. Apoptosis induced by etoposide was evaluated by DNA fragmentation, the presence of cytoplasmic histone‐associated DNA fragments, and annexin V labeling. No membrane HNE was detected in RBL/HNE. In contrast, in RBL/PR3 and in RBL/PR3S203A, the membrane expression of PR3 and PR3S203A increased with etoposide concentrations and appeared closely related to annexin V labeling. Our data suggest that membrane PR3 originates from two distinct pools, the granular pool mobilized following degranulation or a plasma membrane pool mobilized upon apoptosis.

Distribution of Integrin Subunits in Normal Human Kidney

We evaluated on serial sections the distribution of a large number of integrin alpha and beta chains in normal adult human kidney: 1) the beta 1 chain and its corresponding alpha subunits (alpha 1, alpha 2, alpha 3, alpha 4, alpha 5, alpha 6), 2) alpha v and beta 3 chains, 3) the beta 2 chain and its corresponding alpha chains (alpha X, alpha M, alpha L), and 4) the beta 4 chain. We also evaluated ICAM-1, VCAM and ELAM and the major extracellular matrix components (ECM). A three step immunoperoxidase technique was used on frozen sections. Each cell of the kidney shows a specific distribution of these molecules. The relation with ECM and some of their ligands was evaluated. This immunohistochemical study shows that there is no strict colocalisation of a given ECM component with its specific receptor.

Human neutrophil integrin α9β1: up-regulation by cell activation and synergy with β2 integrins during adhesion to endothelium under flow

Neutrophil β1 integrin expression and contribution to cell adhesion were revisited in this study. α9β1 and α5β1 appeared here as the main β1 integrins expressed on the membrane of resting platelet‐depleted neutrophils—α6β1 representing <15% and α2β1 undetectable. Neutrophil activation slightly enhanced α5 expression, did not change α6, but resulted in a two‐ to threefold increase of α9β1, which then became the major β1 integrin of the neutrophil membrane. α9β1 was the only β1 integrin to be up‐regulated after transendothelial migration across TNF‐α‐activated HUVECs. As α9β1 binds VCAM‐1, we analyzed its participation to neutrophil adhesion to TNF‐α‐activated endothelial cells. Blocking anti‐α9 mAb had little effect on neutrophil static adhesion, contrasting with the strong inhibition by anti‐β2 mAb. Under flow conditions, the anti‐α9 mAb had no effect by itself on neutrophil adhesion to activated HUVECs but enhanced the blocking effect of anti‐β2 antibodies significantly and further enhanced the velocity of β2–blocked rolling neutrophils. In conclusion, we describe here for the first time a nearly exclusive up‐regulation of α9β1 expression among all β1 integrins during neutrophil activation and transendothelial migration and a possibly important synergy between α9β1 and β2 integrins in stabilizing neutrophil adhesion to endothelium under flow conditions.

Circulating microparticles in renal diseases

well-regulated process generating MP that present specific features, with significant implications on their functional activity. Indeed, MP behave as vectors of bioactive molecules able to disseminate biological information in the vascular compartment. Due to the expression of both membrane PhtdSer and functional tissue factor (TF), MP are catalytic procoagulant surfaces involved in thrombogenesis. They also harbour inflammatory components (LpA, IL-1 and chemokine receptor CCR5) [4,5], growth factors (TGF-beta and VEGF) and proteases (uPA and MMP) that are related to their involvement in inflammation, immune response, angiogenesis and cancer [6–8].

M‐ficolin and leukosialin (CD43): new partners in neutrophil adhesion

M‐ficolin specificity for sialylated ligands prompted us to investigate its interactions with the main membrane sialoprotein of human neutrophils, CD43. rM‐ficolin bound CD43 and prevented the access of anti‐CD43 mAb. Moreover, rM‐ficolin reacted exclusively with CD43 on Western blots of neutrophil lysate. We confirmed that M‐ficolin is secreted by fMLP‐activated neutrophils, and this endogenous M‐ficolin also binds to CD43 and competes with anti‐CD43 mAb. Anti‐CD43 antibody cross‐linking or fMLP resulted in M‐ficolin and CD43 colocalization on polarized neutrophils. The binding of rM‐ficolin to resting neutrophils induced cell polarization, adhesion, and homotypic aggregation as anti‐CD43 mAb. The M‐ficolin Y271F mutant, unable to bind sialic acid, neither reacted with neutrophils nor modulated their functions. Finally, rM‐ficolin activated the lectin complement pathway on neutrophils. These results emphasize a new function of M‐ficolin, different from ficolin pathogen recognition, i.e., a participation to neutrophil adhesion potentially important in early inflammation, as nanomolar agonist concentrations are sufficient to mobilize M‐ficolin to the neutrophil surface. This multivalent lectin could then endow the antiadhesive CD43, essentially designed to prevent leukocyte aggregation in the blood flow, with new adhesive properties and explain, at least in part, dual‐adhesive/antiadhesive roles of CD43 in neutrophil recruitment.

Distribution of alphavbeta3, alphavbeta5 integrins and the integrin associated protein--IAP (CD47) in human glomerular diseases.

The av integrins present on the membrane of numerous cells, mediate attachment to matrix proteins, cell proliferation, migration and survival. We studied the expression of av integrins and CD47 (a 03 chain integrin associated protein) in various forms of glomerulonephritis (GN) characterized by mesangial proliferation and/or increased mesangial matrix. In normal glomeruli, epithelial cells expressed αvβT3, αvβT5 and CD47; endothelial cells expressed α5βT1 and CD47; mesangial cells expressed αvβT5, CD47, and to a less extent αvβT3. In acute post infectious GN (APIGN), membranoproliferative GN (MPGN) and diabetic nephropathy (DN), we observed that the βT3 chain, normally expressed by mesangial cells, was not detectable in the mesangium while its expression by epithelial cells was not modified. Parallel to the disappearance of αvβT3, the CD47 expression was decreased on the mesangial cells in MPGN, APIGN and DN. The expression of αvβT5 was clearly increased on podocytes and on proliferating mesangial cells in APIGN. By contrast, the mesangial expression of αvβT5 normal or decreased in DN. The α5 chain of integrin, absent on normal mesangial cell, was expressed on proliferating mesangial cells in MPGN and APIGN. Thus, we observed modifications of avp3 and avp5 expression during human GN. The modulations of αvβT3 and αvβT5 expression differed according to the different glomerular cell types and were not parallel in glomerular cells: avp3 was decreased (and αvβT5 unchanged) on proliferating mesangial cells and αvβT5 was increased (and αvβT3 unchanged) in podocytes. This may reflect the existence of two distinct regulatory pathways.

The macrophage-derived neutrophil chemotactic factor, MNCF: A lectin with TNF-α-like activities on neutrophils

The macrophage-derived neutrophil chemotactic factor (MNCF) is an alpha-galactoside-binding lectin, known to induce dexamethasone-insensitive neutrophil recruitment. We further characterized MNCF effects on neutrophils and showed that it shares with TNF-alpha the ability to delay apoptosis and to trigger degranulation. MNCF and TNF-alpha effects show similar kinetics and involve Src kinases and MAPKinases dependent pathways. They were, however, clearly distinguished, since the soluble TNF-receptor etanercept prevented TNF but not MNCF effects, while melibiose disaccharide inhibited MNCF but not TNF effects. Absorption of MNCF on detoxi-gel did not alter its properties, precluding an LPS contamination effect. By contrast, galectin-3 required LPS to activate neutrophils. Specific antibodies allowed to further demonstrate that MNCF and galectin-3 are two distinct molecules. Finally, MNCF- and IL-8-induced neutrophil activation differed by their kinetic and sensitivity to pertussis toxin. In conclusion, MNCF is a distinct neutrophil agonist, with pro-inflammatory activities involving its carbohydrate recognition domain.

How Frequent Are Hepatitis B Virus Markers in Adult Patients with Glomerular Diseases in a Low Endemic Country

In order to appreciate the frequency of hepatitis B virus (HBV) infection in patients with glomerular diseases in France, a low endemic country, we reviewed the series of patients biopsied in the years 1983-1989 in 2 departments of nephrology differing by the characteristics of the population. In Saint-Brieuc, where the population is almost exclusively Caucasian, with nearly no immigrant, HBsAg was not detected in any of the 86 patients. In Paris, a large number of patients come from highly or intermediately endemic regions. HBsAg was detected in 3 of 209 patients, 2 of the 75 patients with membranous nephropathy and 1 of the 32 patients with minimal-change nephrotic syndrome. These patients came from Africa and Asia. Therefore, in low endemic countries, the role of HBV infection in the etiology of glomerulonephritis is minimal. But, because of the late severity of the disease, screening remains essential in patients belonging to the high-risk groups.