Neutrophil rolling altered by inhibition of L-selectin shedding in vitro

Abstract

The L-selectin adhesion molecule is involved in guiding leukocytes to sites of inflammation. L-selectin is cleaved by an unusual proteolytic activity at a membrane-proximal site resulting in rapid shedding from the cell surface. Although it has been demonstrated that L-selectin mediates, in part, the early event of leukocyte rolling under hydrodynamic flow, the contribution of shedding to L-selectin function has remained unknown. Here we show that hydroxamic acid-based metalloprotease inhibitors block L-selectin downregulation from the cell surface of stimulated neutrophils, without affecting Mac-1 mobilization or general neutrophil activation, and inhibit cleavage of L-selectin in a cell-free system. Unexpectedly, the hydroxamic acid-based inhibitors reduced neutrophil rolling velocity under hydrodynamic flow, resulting in increased neutrophil accumulation. These results suggest that L-selectin is cleaved in seconds--much faster than previously suspected--during the process of rolling under hydrodynamic flow, and that shedding of L-selectin may contribute significantly to the velocity of leukocyte rolling. L-selectin shedding during rolling interactions may be physiologically important for limiting leukocyte aggregation and accumulation at sites of inflammation.

Effects of selective protein kinase C inhibitors on the proteolytic down-regulation of L-selectin from chemoattractant-activated neutrophils

“…Hydroxamic acid-based metalloprotease inhibitors have been shown by us and others to inhibit activation-induced L-selectin proteolysis [13,[18][19][20]. We observed that the hydroxamic acid-based metalloprotease inhibitor KD-IX-73-4 also completely blocked rottlerin and calphostin C-induced L-selectin shedding ( Fig.…”

“…Staurosporine, wortmannin, H89, KT5823, genistein, and K252b were purchased from Alexia (San Diego, CA); Gö-7874, Ro-31-8220, bisindolylmaleimide 1, rottlerin, and calphostin C were purchased from Calbiochem (San Diego, CA). KD-IX-73-4, a hydroxamic acid-based metalloprotease inhibitor, has previously been shown to block activation-induced L-selectin shedding [13,18]. All kinase inhibitors and KD-IX-73-4 were dissolved in dimethyl sulfoxide (DMSO) and diluted at least 1000-fold for all whole-cell assays.…”

“…We and others have shown that blocking L-selectin shedding dramatically reduces leukocyte rolling velocities and enhances leukocyte accumulation on endothelial ligands of L-selectin in vitro [13] and in vivo [14], which indicates that L-selectin shedding contributes in regulating L-selectin adhesion. Kahn et al [15] have shown that L-selectin shedding occurs just extracellular to its transmembrane region, resulting in the release of the extracellular portion of L-selectin from the cell surface and retention of a 6-kDa fragment in the cell membrane.…”

Further evidence for a common mechanism for shedding of cell surface proteins

“…E-mail: mullbergj@immunex.com Abbreviations: TNFR-Fc, soluble form of the p80 TNFR linked to the constant region of the human Ig heavy chain cells [12] and shedding of the p60 TNFR and IL-6R from transfected cells and human primary monocytes [13]. A recent study demonstrated that TAPI blocks the release of L-selectin and TGFcc from transfected cells [14] and hydroxamates have also been shown to inhibit L-selectin shedding from primary cells [15][16][17]. Other proteins whose release is blocked by TAPI or related compounds include the thyrotropin receptor [18], Fas ligand [19], heparin-binding EGF-like growth factor [20] and angiotensin-converting enzyme [21].…”

The Monoclonal Antibody CHO-131 Identifies a Subset of Cutaneous Lymphocyte-Associated Antigen T Cells Enriched in P-Selectin-Binding Cells

“…1A) was initially examined using an in vitro shear flow assay that allows for cellular analyses under hydrodynamic conditions simulating the microvasculature (29,30,32). CHO-131 ϩ and CHO-131 Ϫ CLA ϩ CD3 ϩ T cells were first isolated from the peripheral blood of normal individuals by sorting, and then the shear resistance of each subset was assessed over Nonspecific Ab labeling was determined using the appropriate isotype negative control Abs, as indicated.…”