Regulation of P-selectin binding to the neutrophil P-selectin counter-receptor P-selectin glycoprotein ligand-1 by neutrophil elastase and cathepsin G

Abstract

In the inflammatory response, leukocyte rolling before adhesion and transmigration through the blood vessel wall is mediated by specific cell surface adhesion receptors. Neutrophil rolling involves the interaction of P-selectin expressed on activated endothelium and its counter-receptor on neutrophils, P-selectin glycoprotein ligand-1 (PSGL-1). Here, it is reported that P-selectin binding to neutrophils is lost under conditions that cause the release of proteinases from neutrophil primary granules. Treatment of neutrophils with the purified neutrophil granule proteinases, cathepsin G and elastase, rapidly abolished their capacity to bind P-selectin. This inactivation corresponded to loss of the N-terminal domain of PSGL-1, as assessed by Western blot analysis. A loss of intact PSGL-1 protein from the surfaces of neutrophils after the induction of degranulation was also detected by Western blot analysis. Cathepsin G initially cleaved near the PSGL-1 N-terminus, whereas neutrophil elastase predominantly cleaved at a more C-terminal site within the protein mucin core. Consistent with this, cathepsin G cleaved a synthetic peptide based on the PSGL-1 N-terminus between Tyr-7/Leu-8. Under conditions producing neutrophil degranulation in incubations containing mixtures of platelets and neutrophils, the loss of PSGL-1, but not P-selectin, from plateletneutrophil lysates was detected. Cathepsin G-or neutrophil elastase-mediated PSGL-1 proteolysis may constitute a potential autocrine mechanism for downregulation of neutrophil adhesion to P-selectin. IntroductionRecruitment of neutrophils to inflammatory sites is initiated by rolling of circulating cells on activated endothelium before firmer adhesion and subsequent transmigration. [1][2][3][4] Neutrophil rolling is mediated predominantly by expression of adhesive receptors of the selectin family on activated endothelial cells. 5,6 P-selectin is stored on Weibel-Palade body membranes and rapidly expressed on the surface of activated endothelial cells; E-selectin expression is induced in response to inflammatory stimuli. The counter-receptor for P-selectin on neutrophils is P-selectin glycoprotein ligand-1 (PSGL-1), an approximately 220-kd glycoprotein homodimer. The mature protein consists of an extracellular N-terminal anionic sequence containing 1 to 3 sulfated tyrosine residues (Gln-1-Glu-20), a sialomucin domain, a transmembrane domain, and a cytoplasmic tail. 7,8 Several lines of evidence have shown that the sulfated N-terminal sequence of PSGL-1 is critical for P-selectin binding. We found that a cobra venom metalloproteinase, mocarhagin, abolished P-selectin binding to neutrophils by selectively cleaving P-selectin between Tyr-10 and Asp-11. 9 Further, P-selectin binding to recombinant PSGL-1 was dependent on coexpression with tyrosylsulfate sulfotransferase, whereas blocking sulfation of the recombinant receptor inhibited ligand binding. [10][11][12] After initial rolling, tight adhesion of arrested neutrophils is regulated by other adhesion receptors, in particul...

Regulation of platelet membrane levels of glycoprotein VI by a platelet-derived metalloproteinase

“…15 The loss of GPVI in this study 15 was induced by treatment of mouse platelets with the anti-GPVI rat monoclonal antibody JAQ1 16 and was proposed to be mediated primarily through receptor internalization mechanisms, although proteolytic shedding of GPVI was not excluded. Regulation of activated adhesion receptors through proteolysis is well recognized as a mechanism for modulation of cell surface proteins 17,18 and has been identified in a number of systems including proteolytic cleavage of Notch, 19 and adhesion proteins, [20][21][22] as well as numerous cytokines, growth factors, and their receptors. 17,[23][24][25] In the majority of cases, one or more membrane-bound metalloproteinases appear to be responsible for the extracellular processing observed.…”

“…48 In the case of CD40L, shedding was dependent on ␣ IIb ␤ 3 integrin and inhibitable by RGD mimetics. In contrast, GPVI shedding was not affected by RGD-dependent blockade of ␣ IIb ␤ 3 integrin.The proteolytic shedding of adhesion receptors is a welldocumented autocrine mechanism to control receptor activity 21,22,27,50 and, for platelets, functional autocrine loops mediated by secreted factors such as adenosine diphosphate (ADP) and CD40L are well established. 47,[51][52][53] Recent work by Bergmeier and coworkers examining a platelet model of mitochondrial injury has linked the profound proteolytic shedding of GPIb␣ observed in this system to a platelet-derived metalloproteinase.…”

Neutrophils phagocytose activated platelets in vivo: a phosphatidylserine, P-selectin, and β2 integrin–dependent cell clearance program

“…Moreover, it is necessary for integrin transactivation, assembly of the phagosome, and neutrophil degranulation. 5,10,21,22,51,52 Therefore, platelet adhesion and neutrophil degranulation are simultaneously blocked in our system by the antibodies for P-selectin and PSGL-1.All together, we propose that tethering is achieved via P-selectin recognition, leading to MPO release, and activating ␤2 integrins for internalization; if the tethered substrate was a phosphatidylserineexpressing platelet, phagocytosis swiftly removes the activated platelets, which represent a template for thrombin formation, from the circulating blood. 31,46,47 …”

“…Moreover, it is necessary for integrin transactivation, assembly of the phagosome, and neutrophil degranulation. 5,10,21,22,51,52 Therefore, platelet adhesion and neutrophil degranulation are simultaneously blocked in our system by the antibodies for P-selectin and PSGL-1.…”

Identification of Neutrophil Granule Protein Cathepsin G as a Novel Chemotactic Agonist for the G Protein-Coupled Formyl Peptide Receptor

“…It is referred to as a chymotrypsin-like enzyme because it hydrolyzes peptide bonds after leucine, methionine, and phenylalanine residues. In addition to its capacity to proteolytically degrade engulfed cell debris and its microbicidal activity (1-3), CaG displays a variety of pathophysiological effects, such as degradation of extracellular matrix and regulating bioactivity of cytokines and cytokine receptors (2,3), induction of proinflammatory cytokines in macrophages, enhancing hemopoietic progenitor cell mobilization by cleaving CD106 (4), disrupting interaction between CXCR4 and stromal cell-derived factor-1␣ (5), and regulation of neutrophil migration by modifying the P-selectin receptor P-selectin glycoprotein ligand-1 (6). Human CaG has been shown to promote specific Ab responses when injected in mice (7).…”