Lise Halbwachs-Mecarelli

Redox regulation of β2-integrin CD11b / CD18 activation

Although the central role of β2‐integrin CD11bu2009/u2009CD18 in neutrophil functions is well recognized, signaling pathway that regulate integrin activation remain to be elucidated. We analyzed the contribution of oxido‐reduction mechanisms in this signaling. Exogenously added H2O2 induced CD11bu2009/u2009CD18‐dependent neutrophil adhesion and expression of an integrin activation neoepitope recognized by monoclonal antibody (mAb) clone 24. H2O2‐triggered β2‐integrin activation was inhibited by tyrosine kinase inhibitors and by complexing sulfhydryl groups with phenylarsine oxide (PAO). CD11bu2009/u2009CD18‐dependent adhesion and mAb 24 antigen expression triggered by physiological agonists such as TNF‐α were inhibited by diphenylene iodonium (DPI, an inhibitor of flavoprotein oxidoreductase), by free radical scavengers, by tyrosine kinase inhibitors and by PAO. No inhibition was observed when adhesion was induced by the integrin‐activating KIM 185 mAb. Taken together, these results emphasize the importance of an oxidative S‐thiolation step(s) in the tyrosine kinase‐dependent signaling pathway leading to β2‐integrin activation. H2O2 would directly mediate this oxidative reaction and bypass the initial agonistu2009/u2009receptor pathway to promote integrin‐dependent adhesion. The putative oxidase(s) involved in this process is not NADPH oxidase, since adhesion of neutrophils from patients with chronic granulomatous disease was normal and inhibited by scavengers and DPI. These data shed a new light on the regulation of integrin activation required for cell migration into inflamed organs.

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.

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.

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.

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.