NF-κB1 inhibits TLR-induced IFN-ß production in macrophages through TPL-2-dependent ERK activation.

Although NF-κB1 p50/p105 has critical roles in immunity, the mechanism by which NF-κB1 regulates inflammatory responses is unclear. In this study, we analyzed the gene expression profile of LPS-stimulated Nfkb1(-/-) macrophages that lack both p50 and p105. Deficiency of p50/p105 selectively increased the expression of IFN-responsive genes, which correlated with increased IFN-ß expression and STAT1 phosphorylation. IFN Ab-blocking experiments indicated that increased STAT1 phosphorylation and expression of IFN-responsive genes observed in the absence of p50/p105 depended upon autocrine IFN-ß production. Markedly higher serum levels of IFN-ß were observed in Nfkb1(-/-) mice than in wild-type mice following LPS injection, demonstrating that Nfkb1 inhibits IFN-ß production under physiological conditions. TPL-2, a mitogen-activated protein kinase kinase kinase stabilized by association with the C-terminal ankyrin repeat domain of p105, negatively regulates LPS-induced IFN-ß production by macrophages via activation of ERK MAPK. Retroviral expression of TPL-2 in Nfkb1(-/-) macrophages, which are deficient in endogenous TPL-2, reduced LPS-induced IFN-ß secretion. Expression of the C-terminal ankyrin repeat domain of p105 in Nfkb1(-/-) macrophages, which rescued LPS activation of ERK, also inhibited IFN-ß expression. These data indicate that p50/p105 negatively regulates LPS-induced IFN signaling in macrophages by stabilizing TPL-2, thereby facilitating activation of ERK.

Human neutrophil integrin alpha9beta1: up-regulation by cell activation and synergy with beta2 integrins during adhesion to endothelium under flow.

Neutrophil beta1 integrin expression and contribution to cell adhesion were revisited in this study. alpha9beta1 and alpha5beta1 appeared here as the main beta1 integrins expressed on the membrane of resting platelet-depleted neutrophils-alpha6beta1 representing <15% and alpha2beta1 undetectable. Neutrophil activation slightly enhanced alpha5 expression, did not change alpha6, but resulted in a two- to threefold increase of alpha9beta1, which then became the major beta1 integrin of the neutrophil membrane. alpha9beta1 was the only beta1 integrin to be up-regulated after transendothelial migration across TNF-alpha-activated HUVECs. As alpha9beta1 binds VCAM-1, we analyzed its participation to neutrophil adhesion to TNF-alpha-activated endothelial cells. Blocking anti-alpha9 mAb had little effect on neutrophil static adhesion, contrasting with the strong inhibition by anti-beta2 mAb. Under flow conditions, the anti-alpha9 mAb had no effect by itself on neutrophil adhesion to activated HUVECs but enhanced the blocking effect of anti-beta2 antibodies significantly and further enhanced the velocity of beta2-blocked rolling neutrophils. In conclusion, we describe here for the first time a nearly exclusive up-regulation of alpha9beta1 expression among all beta1 integrins during neutrophil activation and transendothelial migration and a possibly important synergy between alpha9beta1 and beta2 integrins in stabilizing neutrophil adhesion to endothelium under flow conditions.

The macrophage-derived neutrophil chemotactic factor, MNCF: a lectin with TNF-alpha-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.

The cleavage of neutrophil leukosialin (CD43) by cathepsin G releases its extracellular domain and triggers its intramembrane proteolysis by presenilin/gamma-secretase.

The highly negatively charged membrane sialoglycoprotein leukosialin, CD43, is shed during neutrophil activation. This is generally thought to enhance cell adhesion. We here describe two novel consequences of this shedding, during neutrophil activation by phorbol esters or by chemoattractants after TNF-alpha priming. CD43 proteolysis was investigated by Western blotting, using a polyclonal antibody to CD43 intracellular domain. Our data emphasize the importance of a juxtamembranous cleavage of about 50% of membrane CD43 molecules by cathepsin G. Indeed, it is inhibited by alpha1-antichymotrypsin and cathepsin G inhibitor I and is reproduced by exogenous purified cathepsin G. The resulting membrane-anchored C-terminal fragment, CD43-CTF, becomes susceptible to presenilin/gamma-secretase, which releases CD43 intracytoplasmic domain: preincubation with three different gamma-secretase inhibitors, before PMN treatment by agonists or by purified cathepsin G, results in the accumulation of CD43-CTF. Because CD43 binds E-selectin, we also investigated the effect of the soluble extracellular domain CD43s, released by cathepsin G juxtamembranous cleavage, on neutrophil adhesion to endothelial cells. A recombinant CD43s-Fc fusion protein inhibited neutrophil E selectindependent adhesion to endothelial cells under flow conditions, while it had no effect on neutrophil static adhesion. We thus propose that, in addition to its potential pro-adhesive role, CD43 proteolysis results in: (i) the release, by cathepsin G, of CD43 extracellular domain, able to inhibit the adhesion of flowing neutrophils on endothelial cells and thus to participate to the natural control of inflammation; (ii) the release and/or the clearance, by presenilin/gamma-secretase, of CD43 intracellular domain, thereby regulating CD43-mediated signaling.

Increased membrane expression of proteinase 3 during neutrophil adhesion in the presence of anti proteinase 3 antibodies.

We investigated membrane proteinase 3 (mPR3) expression during TNF-alpha-induced adhesion of neutrophils in the presence of anti-PR3 antibodies, a situation occurring during anti-neutrophil cytoplasmic autoantibodies (ANCA)-associated vasculitis. Three increasing levels of mPR3 expression were observed on the mPR3(+) neutrophil subset after stepwise cell activation. TNF-alpha activation without adhesion, TNF-alpha-induced adhesion, and adhesion in the presence of anti-PR3 mAb or human anti-PR3 ANCA resulted, respectively, in a two-, seven-, and 24-fold increase of mPR3 levels. In plasma, anti-PR3 antibodies poorly recognized suspended neutrophils, whereas they bound to mPR3 on adherent cells. mPR3 upregulation was also triggered by IL-8, formyl-methionyl-leucyl-phenylalanine (fMLP), and neutrophil adhesion to activated human umbilical vein endothelial cells. It involved beta2 integrins and Fcgamma receptor, because it was prevented by anti-CD18 antibodies and was not observed with anti-PR3 F(ab')(2). Furthermore, it was specific to anti-PR3 mAb, and no mPR3 upregulation was observed with anti-myeloperoxidase or anti-HLA-ABC mAb. Newly expressed mPR3 molecules, after TNF-induced adhesion, were mobilized from secretory vesicles (CD35(+)) and secondary granules (CD11b(+)). The adhesion- and antibody-dependent upregulations of mPR3 expression occurred with little azurophilic granule degranulation, no sign of apoptosis, and no further CD177 upregulation. In conclusion, this study describes an amplifying loop in polymorphonuclear neutrophil activation process, whereby ANCA are involved in the membrane expression of their own antigen during cell adhesion. This could explain the restriction of ANCA-associated vasculitis to small vessels, the main site of neutrophil adhesion.