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Abstract
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.
Highlights
The inflammation resolution involves, for example, the shedding of cytokine receptors and adhesion molecules, which down-regulates leukocyte adhesion to endothelium
We demonstrate that CD43 proteolysis includes three simultaneous or sequential proteolytic steps, schematized in Figs. 1C and 3D: clips A and C, distant from the membrane and a juxtamembranous clip B
This cleavage was observed upon polymorphonuclear neutrophils (PMN) incubation without stimuli, in buffers devoid of BSA, and it increased with the cell concentration. This suggests that it is mediated by proteases either constitutively present on neutrophils, but normally blocked by plasma inhibitors or albumin, or induced on PMN membrane by the isolation procedure. This cleavage was enhanced by phorbol myristate acetate (PMA) or by TNF/fMLP and we confirm that it results in CD43 fragments similar to those observed with exogenous elastase [27, 29]
Summary
The inflammation resolution involves, for example, the shedding of cytokine receptors and adhesion molecules, which down-regulates leukocyte adhesion to endothelium. Analysis of PMA-activated PMN (Fig. 1B) confirmed previous data, because it revealed cell-associated fragments complementary in size to the reported soluble CD43 fragments observed by immunoprecipitation of 125I-labeled neutrophils [15, 27]: (i) a cell-bound 90-kDa doublet, resulting from clip A (Fig. 1C) and
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