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• W2000536824 abstract "The adhesion molecules known as selectins mediate the capture of neutrophils from the bloodstream. We have previously reported that ligation and cross-linking of L-selectin on the neutrophil surface enhances the adhesive function of β2-integrins in a synergistic manner with chemotactic agonists. In this work, we examined degranulation and adhesion of neutrophils in response to cross-linking of L-selectin and addition of interleukin-8. Cross-linking of L-selectin induced priming of degranulation that was similar to that observed with the alkaloid cytochalasin B. Activation mediated by L-selectin of neutrophil shape change and adhesion through CD11b/CD18 were strongly blocked by Merck C, an imidazole-based inhibitor of p38 mitogen-activated protein kinase (MAPK), but not by a structurally similar non-binding regioisomer. Priming by L-selectin of the release of secondary, tertiary, and secretory, but not primary, granules was blocked by inhibition of p38 MAPK. Peak phosphorylation of p38 MAPK was observed within 1 min of cross-linking L-selectin, whereas phosphorylation of ERK1/2 was highest at 10 min. Phosphorylation of p38 MAPK, but not ERK1/2, was inhibited by Merck C. These data suggest that signal transduction as a result of clustering L-selectin utilizes p38 MAPK to effect neutrophil shape change, integrin activation, and the release of secondary, tertiary, and secretory granules. The adhesion molecules known as selectins mediate the capture of neutrophils from the bloodstream. We have previously reported that ligation and cross-linking of L-selectin on the neutrophil surface enhances the adhesive function of β2-integrins in a synergistic manner with chemotactic agonists. In this work, we examined degranulation and adhesion of neutrophils in response to cross-linking of L-selectin and addition of interleukin-8. Cross-linking of L-selectin induced priming of degranulation that was similar to that observed with the alkaloid cytochalasin B. Activation mediated by L-selectin of neutrophil shape change and adhesion through CD11b/CD18 were strongly blocked by Merck C, an imidazole-based inhibitor of p38 mitogen-activated protein kinase (MAPK), but not by a structurally similar non-binding regioisomer. Priming by L-selectin of the release of secondary, tertiary, and secretory, but not primary, granules was blocked by inhibition of p38 MAPK. Peak phosphorylation of p38 MAPK was observed within 1 min of cross-linking L-selectin, whereas phosphorylation of ERK1/2 was highest at 10 min. Phosphorylation of p38 MAPK, but not ERK1/2, was inhibited by Merck C. These data suggest that signal transduction as a result of clustering L-selectin utilizes p38 MAPK to effect neutrophil shape change, integrin activation, and the release of secondary, tertiary, and secretory granules. intracellular adhesion molecule 1 interleukin mitogen-activated protein kinase human serum albumin polyacrylamide gel electrophoresis myeloperoxidase extracellular signal-regulated kinase Neutrophils circulate in the vasculature in a passive state and become more adhesive upon stimulation at sites of inflammation. Margination to the vessel wall and subsequent transmigration and phagocytosis (1.Henricks P.A.J. Van der Tol M.E. Verhoef J. Immunology. 1984; 52: 671-678PubMed Google Scholar) requires a number of surface proteins, including the β2-integrins and the selectins, as mediators of adherence to the endothelium (2.Smith C.W. Kishimoto T.K. Abbass O. Hughes B. Rothlein R. McIntire L.V. Butcher E. Anderson D.C. J. Clin. Invest. 1991; 87: 609-618Crossref PubMed Scopus (348) Google Scholar, 3.Lawrence M.B. Springer T.A. Cell. 1991; 65: 859-873Abstract Full Text PDF PubMed Scopus (1872) Google Scholar, 4.Smith C.W. Marlin S.D. Rothlein R. Toman C. Anderson D.C. J. Clin. Invest. 1989; 83: 2008-2017Crossref PubMed Scopus (945) Google Scholar, 5.Kishimoto T.K. Jutila M.A. Berg E.L. Butcher E.C. Science. 1989; 245: 1238-1241Crossref PubMed Scopus (905) Google Scholar). A sequence of molecular and biophysical events has been identified that facilitates neutrophil activation and increased adherence during the acute inflammatory response in vivo. Neutrophils entering post-capillary venules adjacent to inflammatory foci develop transient rolling adhesive interactions with endothelium via selectins (6.Zimmerman G.A. Prescott S.M. McIntyre T.M. Immunol. Today. 1992; 13: 93-100Abstract Full Text PDF PubMed Scopus (706) Google Scholar). Following exposure to inflammatory cytokines such as tumor necrosis factor and interleukin-1, endothelial cells are induced to express E-selectin and P-selectin (6.Zimmerman G.A. Prescott S.M. McIntyre T.M. Immunol. Today. 1992; 13: 93-100Abstract Full Text PDF PubMed Scopus (706) Google Scholar). Several surface glycoproteins on neutrophils, including L-selectin and P-selectin glycoprotein ligand 1, present oligosaccharide moieties that serve as counter receptors for E-selectin and P-selectin. In conjunction with neutrophil membrane L-selectin, which recognizes oligosaccharides on endothelial cells, they promote tethering and rolling of neutrophils on endothelium under flow conditions (3.Lawrence M.B. Springer T.A. Cell. 1991; 65: 859-873Abstract Full Text PDF PubMed Scopus (1872) Google Scholar, 6.Zimmerman G.A. Prescott S.M. McIntyre T.M. Immunol. Today. 1992; 13: 93-100Abstract Full Text PDF PubMed Scopus (706) Google Scholar, 7.Hammer D.A. Apte S.M. Biophys. J. 1992; 63: 35-57Abstract Full Text PDF PubMed Scopus (465) Google Scholar). Following ligation, all three selectins have demonstrated the ability to signal into the cell (8.Crockett-Torabi E. J. Leukocyte Biol. 1998; 63: 1-14Crossref PubMed Scopus (123) Google Scholar).Neutrophil rolling is a prerequisite for the transition to a shear-resistant firm adhesion on the endothelium (2.Smith C.W. Kishimoto T.K. Abbass O. Hughes B. Rothlein R. McIntire L.V. Butcher E. Anderson D.C. J. Clin. Invest. 1991; 87: 609-618Crossref PubMed Scopus (348) Google Scholar, 3.Lawrence M.B. Springer T.A. Cell. 1991; 65: 859-873Abstract Full Text PDF PubMed Scopus (1872) Google Scholar, 4.Smith C.W. Marlin S.D. Rothlein R. Toman C. Anderson D.C. J. Clin. Invest. 1989; 83: 2008-2017Crossref PubMed Scopus (945) Google Scholar, 7.Hammer D.A. Apte S.M. Biophys. J. 1992; 63: 35-57Abstract Full Text PDF PubMed Scopus (465) Google Scholar). Neutrophil arrest is mediated by the β2-integrins whose expression level and avidity for ligands are increased by the binding of chemotactic receptors early in the process of emigration (3.Lawrence M.B. Springer T.A. Cell. 1991; 65: 859-873Abstract Full Text PDF PubMed Scopus (1872) Google Scholar). For example, the adhesivity through binding of Mac-1 (CD11b/CD18) and LFA-1 (CD11a/CD18) to ICAM-11(CD54) is increased by exposure of neutrophils to numerous chemotactic stimuli, including IL-8, which is synthesized and presented on the surface of inflamed endothelium (4.Smith C.W. Marlin S.D. Rothlein R. Toman C. Anderson D.C. J. Clin. Invest. 1989; 83: 2008-2017Crossref PubMed Scopus (945) Google Scholar, 6.Zimmerman G.A. Prescott S.M. McIntyre T.M. Immunol. Today. 1992; 13: 93-100Abstract Full Text PDF PubMed Scopus (706) Google Scholar). During neutrophil activation, L-selectin, which initially has a high basal expression, is shed while Mac-1 is increased 10–20-fold on the surface following mobilization of granule stores (5.Kishimoto T.K. Jutila M.A. Berg E.L. Butcher E.C. Science. 1989; 245: 1238-1241Crossref PubMed Scopus (905) Google Scholar). These changes in surface expression and affinity occur over seconds to minutes following stimulation (9.Simon S.I. Chambers J.D. Butcher E. Sklar L.A. J. Immunol. 1992; 149: 2765-2771PubMed Google Scholar).Once migrated to the site of tissue injury, the neutrophil's chief function becomes that of a secretory cell. In response to the ligation of L-selectin or chemotactic receptors, CD11b/CD18 is up-regulated from rapidly mobilized secretory granules (10.Todd III, R.F. Arnaout M.A. Rosin R.E. Crowley C.A. Peters W.A. Babior B.M. J. Clin. Invest. 1984; 74: 1280-1290Crossref PubMed Scopus (169) Google Scholar, 11.Petrequin P.R. Todd III, R.F. Devall L.J. Boxer L.A. Curnutte III, J.T. Blood. 1987; 69: 605-610PubMed Google Scholar, 12.Stevenson K.B. Nauseef W.M. Clark R.A. J. Immunol. 1987; 139: 3759-3763PubMed Google Scholar, 13.Sengelov H. Kjeldsen L. Diamond M.S. Springer T.A. Borregaard N. J. Clin. Invest. 1993; 92: 1467-1476Crossref PubMed Scopus (235) Google Scholar). Increasing intensity of stimulation results in the release of secondary (specific) and then primary (azurophil) granules, a process known as sequential degranulation (14.Bainton D.F. J. Cell Biol. 1973; 58: 249-264Crossref PubMed Scopus (158) Google Scholar, 15.Bentwood B.J. Henson P.M. J. Immunol. 1980; 124: 855-862PubMed Google Scholar). To a large extent, the extracellular release of specific and azurophil granules remains under separate control (14.Bainton D.F. J. Cell Biol. 1973; 58: 249-264Crossref PubMed Scopus (158) Google Scholar, 15.Bentwood B.J. Henson P.M. J. Immunol. 1980; 124: 855-862PubMed Google Scholar, 16.Niessen H.W.M. Verhoeven A.J. Cell. Signal. 1992; 4: 501-509Crossref PubMed Scopus (50) Google Scholar, 17.Zaman W. Mitsuyama T. Hatakenaka M. Kang D. Minakami S. Takeshige K. J. Biochem. (Tokyo). 1994; 115: 238-244Crossref PubMed Scopus (8) Google Scholar). Chemoattractants and other substances selectively elicit the release of specific granules under conditions wherein azurophilic granule enzymes are not discharged (16.Niessen H.W.M. Verhoeven A.J. Cell. Signal. 1992; 4: 501-509Crossref PubMed Scopus (50) Google Scholar, 18.Smolen J.E. Weissmann G. Biochim. Biophys. Acta. 1981; 672: 197-206Crossref PubMed Scopus (44) Google Scholar, 19.Hanlon W.A. Stolk J. Davies P. Humes J.L. Mumford R. Bonney R.J. J. Leukocyte Biol. 1991; 50: 43-48Crossref PubMed Scopus (40) Google Scholar). On the other hand, stimuli for azurophilic granule release also stimulate concomitant exocytosis of specific granules (20.Estensen R.D. White J.G. Holmes B. Nature. 1974; 248: 347-348Crossref PubMed Scopus (105) Google Scholar), with rare exceptions (17.Zaman W. Mitsuyama T. Hatakenaka M. Kang D. Minakami S. Takeshige K. J. Biochem. (Tokyo). 1994; 115: 238-244Crossref PubMed Scopus (8) Google Scholar). The resistance of azurophil granules to secretion may be due to a requirement for a biochemical signal in addition to Ca2+(21.Niessen H.W. Kuijpers T.W. Roos D. Verhoeven A.J. Cell. Signal. 1991; 3: 625-633Crossref PubMed Scopus (21) Google Scholar).There is evidence that adhesion supported by β2-integrins or selectins can sensitize neutrophils for superoxide generation (22.Walzog B. Seifert R. Zakrzewicz A. Gaehtgens P. Ley K. J. Leukocyte Biol. 1994; 56: 625-635Crossref PubMed Scopus (88) Google Scholar, 23.Dapino P. Dallegri F. Ottonello L. Sacchetti C. Clin. Exp. Immunol. 1993; 94: 533-538Crossref PubMed Scopus (39) Google Scholar, 24.Waddell T.K. Fialkow L. Chan C.K. Kishimoto T.K. Downey G.P. J. Biol. Chem. 1994; 269: 18485-18491Abstract Full Text PDF PubMed Google Scholar). In this regard, cross-linking of L-selectin or Mac-1 with monoclonal antibody results in secretion of tertiary, but not primary, granules (25.Wize J. Sopata I. Smerdel A. Maslinski S. Inflamm. Res. 1998; 47: 325-327Crossref PubMed Scopus (39) Google Scholar). Clustering of integrins and selectins triggers intracellular signals, including intracellular Ca2+ release and phosphorylation of several cytoplasmic tyrosine kinases (26.Crockett-Torabi E. Fantone J.C. Am. J. Physiol. 1997; 272: H1302-H1308PubMed Google Scholar, 27.Crockett-Torabi E. Sulenbarger B. Smith C.W. Fantone J.C. J. Immunol. 1995; 154: 2291-2302PubMed Google Scholar, 28.Waddell T.K. Fialkow L. Chan C.K. Kishimoto T.K. Downey G.P. J. Biol. Chem. 1995; 270: 15403-15411Abstract Full Text Full Text PDF PubMed Scopus (186) Google Scholar). Published data indicate that some elements of a signaling pathway involving the mitogen-activated protein kinases (MAPK) are involved in signaling through both L-selectin and Mac-1 (28.Waddell T.K. Fialkow L. Chan C.K. Kishimoto T.K. Downey G.P. J. Biol. Chem. 1995; 270: 15403-15411Abstract Full Text Full Text PDF PubMed Scopus (186) Google Scholar, 29.Hidari K.I.-P.J. Weyrich A.S. Zimmerman G.A. McEver R.P. J. Biol. Chem. 1997; 272: 28750-28756Abstract Full Text Full Text PDF PubMed Scopus (198) Google Scholar, 30.Steeber D.A. Engel P. Miller A.S. Sheetz M.P. Tedder T.F. J. Immunol. 1997; 159: 952-963PubMed Google Scholar). However, to date there are no data linking the processes of adhesion and degranulation to signaling events involving p38 MAPK in neutrophils.In previous studies, we examined how concurrent signaling through chemotactic factors and L-selectin affected adhesive function (31.Tsang Y.T.M. Neelamegham S. Burns A.R. Berg E.L. Smith C.W. Simon S.I. J. Immunol. 1997; 159: 4566-4577PubMed Google Scholar, 32.Simon S.I. Burns A.R. Taylor A.D. Gopalan P.K. Lynam E.B. Sklar L.A. Smith C.W. J. Immunol. 1995; 155: 1502-1514PubMed Google Scholar, 33.Gopalan P.K. Smith C.W. Lu H.F. Berg E.L. McIntire L.V. Simon S.I. J. Immunol. 1997; 158: 367-375PubMed Google Scholar). We showed that cross-linking of L-selectin, in the presence of IL-8, potentiated adhesion of Mac-1 and LFA-1 to ICAM-1 under physiologic conditions of shear flow (33.Gopalan P.K. Smith C.W. Lu H.F. Berg E.L. McIntire L.V. Simon S.I. J. Immunol. 1997; 158: 367-375PubMed Google Scholar). Subsequent transmigration on IL-1 stimulated human umbilical vein endothelial cells was also potentiated by L-selectin cross-linking (31.Tsang Y.T.M. Neelamegham S. Burns A.R. Berg E.L. Smith C.W. Simon S.I. J. Immunol. 1997; 159: 4566-4577PubMed Google Scholar, 32.Simon S.I. Burns A.R. Taylor A.D. Gopalan P.K. Lynam E.B. Sklar L.A. Smith C.W. J. Immunol. 1995; 155: 1502-1514PubMed Google Scholar). In the current study, our objective was to determine whether L-selectin could influence the secretory functions of neutrophils. We examined whether antibody-induced clustering of L-selectin could potentiate the extent of degranulation in response to chemotactic stimulation. We show that cross-linking of L-selectin leads directly to phosphorylation of p38 MAPK. This response preceded activation of adhesion and priming of degranulation. Like shape change and activation of adhesion, L-selectin-mediated priming for secretion of secondary and tertiary granules was blocked by an inhibitor of p38 MAPK.RESULTSIn previous studies, we examined the process of chemotactic signaling on β2-integrin adhesive function (31.Tsang Y.T.M. Neelamegham S. Burns A.R. Berg E.L. Smith C.W. Simon S.I. J. Immunol. 1997; 159: 4566-4577PubMed Google Scholar, 32.Simon S.I. Burns A.R. Taylor A.D. Gopalan P.K. Lynam E.B. Sklar L.A. Smith C.W. J. Immunol. 1995; 155: 1502-1514PubMed Google Scholar, 33.Gopalan P.K. Smith C.W. Lu H.F. Berg E.L. McIntire L.V. Simon S.I. J. Immunol. 1997; 158: 367-375PubMed Google Scholar). We reported that cross-linking of L-selectin and addition of PAF or IL-8 were synergistic in activation of resting neutrophils. Here we examined the release of primary, secondary, and tertiary granules from neutrophils in response to cross-linking of L-selectin and addition of IL-8.We first wished to determine if ligation of L-selectin could serve as a sensitizing agent for degranulation. Since CD11b/CD18 is contained in secondary, tertiary, and secretory granules in neutrophils (10.Todd III, R.F. Arnaout M.A. Rosin R.E. Crowley C.A. Peters W.A. Babior B.M. J. Clin. Invest. 1984; 74: 1280-1290Crossref PubMed Scopus (169) Google Scholar, 11.Petrequin P.R. Todd III, R.F. Devall L.J. Boxer L.A. Curnutte III, J.T. Blood. 1987; 69: 605-610PubMed Google Scholar, 12.Stevenson K.B. Nauseef W.M. Clark R.A. J. Immunol. 1987; 139: 3759-3763PubMed Google Scholar, 13.Sengelov H. Kjeldsen L. Diamond M.S. Springer T.A. Borregaard N. J. Clin. Invest. 1993; 92: 1467-1476Crossref PubMed Scopus (235) Google Scholar), the surface expression of the integrin should be enhanced if these labile pools are mobilized. In experiments not shown, we first determined that cross-linking of L-selectin enhanced degranulation induced by IL-8. We performed extensive dose-response studies with IL-8 and selected a concentration of 1 nm as optimal for further studies.As shown in Fig. 1 A, surface expression of Mac-1 was statistically greater than unstimulated control at 1 nm IL-8. Of greatest interest here is the observation that cross-linking of L-selectin, by treatment with two humanized antibodies that bind to distinct epitopes on the lectin domain (HuDREG200 and HuDREG55), also enhanced the display of Mac-1 (Fig.1 A). That is, background levels in the absence of IL-8 were significantly greater with cross-linking of L-selectin alone. Also, with cross-linking of L-selectin, the responses elicited by IL-8 were significantly greater than unstimulated levels.As Mac-1 is found in secondary, tertiary, and secretory granules, its expression at these low concentrations of agonist should reflect only the elaboration of the tertiary and secretory granules, the more labile of the pools (10.Todd III, R.F. Arnaout M.A. Rosin R.E. Crowley C.A. Peters W.A. Babior B.M. J. Clin. Invest. 1984; 74: 1280-1290Crossref PubMed Scopus (169) Google Scholar, 11.Petrequin P.R. Todd III, R.F. Devall L.J. Boxer L.A. Curnutte III, J.T. Blood. 1987; 69: 605-610PubMed Google Scholar, 12.Stevenson K.B. Nauseef W.M. Clark R.A. J. Immunol. 1987; 139: 3759-3763PubMed Google Scholar, 13.Sengelov H. Kjeldsen L. Diamond M.S. Springer T.A. Borregaard N. J. Clin. Invest. 1993; 92: 1467-1476Crossref PubMed Scopus (235) Google Scholar). To examine secondary granules in this process, we measured lactoferrin in the cell supernatants (Fig. 1 B). In the absence of any other condition, only a small amount of lactoferrin was released in response to IL-8. In contrast, cross-linking of L-selectin alone and in combination with 1 nm IL-8 produced significant release of lactoferrin.The granule type most resistant to release is the azurophil, from which we measured both myeloperoxidase (MPO) and β-glucuronidase. As can be seen in Fig. 1 C, release of MPO was not statistically significant except in the strongest stimulatory conditions of IL-8 plus cross-linking. Similar results were found with β-glucuronidase release (Fig. 1 D), with the exception of a statistically significant enhancement of degranulation induced by 1 nmIL-8 alone. Taken together, the data show that IL-8 is a weak inducer of degranulation (at these relatively low concentrations), but its effect is significantly enhanced by co-stimulation with L-selectin.We confirmed that cross-linking of L-selectin enhanced adhesive function. This was measured as an increase in the binding of albumin-coated latex beads to neutrophils, a process that is dependent on the activation of Mac-1 (Fig. 2). Unstimulated neutrophils sheared in suspension with beads did not increase their adhesiveness over time. In response to stimulation with IL-8 at a relatively low concentration of 0.1 nm, a significant increase in bead binding was detected over the time course of stimulation (Fig. 2). A much stronger response was elicited through cross-linking of L-selectin with a secondary antibody. The combination of co-stimulation through L-selectin and IL-8 tended to increase the extent of bead binding over activation through L-selectin alone.Figure 2Neutrophil adhesion to albumin-coated latex beads in response to stimulation through cross-linking of L-selectin and IL-8. Neutrophil adhesion was stimulated by addition of 0.1 nm IL-8 and/or L-selectin cross-linking (20 μg/ml HuDREG200 cross-linked with 10 μg/ml goat anti-human IgG). Data represent the number of beads bound to cells at each time point before and after stimulation relative to the number bound to untreated (typically 0.5–1 bead/PMN). Plotted are the kinetics of adhesion from one experiment representative of five separate donors.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Previous work had implicated MAP kinases in signaling through adhesion molecules and in chemotactic stimulation of superoxide production (45.Detmers P.A. Zhou D.H. Polizzi E. Thieringer R. Hanlon W.A. Vaidya S. Bansal V. J. Immunol. 1998; 161: 1921-1929PubMed Google Scholar,46.Ng-Sikorski J. Lindén L. Eierman D. Franzen L. Molony L. Andersson T. J. Cell Sci. 1996; 109: 2361-2369Crossref PubMed Google Scholar). In particular, p38 MAPK has been recently shown to be involved in signaling through β2-integrins (45.Detmers P.A. Zhou D.H. Polizzi E. Thieringer R. Hanlon W.A. Vaidya S. Bansal V. J. Immunol. 1998; 161: 1921-1929PubMed Google Scholar). We therefore examined whether the sensitization induced by cross-linking L-selectin by addition of both HuDREG200 and HuDREG55 involved activation of p38 MAPK in the signaling pathway. We tested this using a high affinity inhibitor of p38 MAPK synthesized by Merck Research Laboratories, designated Merck C (47.Liverton N.J. Butcher J.W. Claiborne C.F. Claremon D.A. Libby B.E. Nguyen K.T. Pitzenberger S.M. Selnick H.G. Smith G.R. Tebben R. Vacca J.P. Varga S.L. Agarwal L. Dancheck K. Forsyth A.J. Fletcher D.S. Frantz B. Hanlon W.A. Harper C.P. Hofsess S.J. Kotsura M. Lin J. Luell S. O'Neill E.A. Orevillo C.J. Pang M. Parsons J. Rolando A. Sahly Y. Visco D.M. O'Keefe S.J. J. Med. Chem. 1999; 42: 2180-2190Crossref PubMed Scopus (205) Google Scholar). Merck C is a potent and specific blocker of p38 MAPK, with an IC50 of 0.24 nm for isolated enzyme and an IC50 of ∼2.2 nm for inhibition of p38-induced cellular function. Merck A is a non-functional isomer that has a similar chemical composition (47.Liverton N.J. Butcher J.W. Claiborne C.F. Claremon D.A. Libby B.E. Nguyen K.T. Pitzenberger S.M. Selnick H.G. Smith G.R. Tebben R. Vacca J.P. Varga S.L. Agarwal L. Dancheck K. Forsyth A.J. Fletcher D.S. Frantz B. Hanlon W.A. Harper C.P. Hofsess S.J. Kotsura M. Lin J. Luell S. O'Neill E.A. Orevillo C.J. Pang M. Parsons J. Rolando A. Sahly Y. Visco D.M. O'Keefe S.J. J. Med. Chem. 1999; 42: 2180-2190Crossref PubMed Scopus (205) Google Scholar). To determine the effective concentration for inhibition of neutrophil activation, we analyzed cell shape change stimulated by cross-linking of L-selectin. As shown in Fig. 3, Merck C was a potent inhibitor of shape change at all concentrations tested. In contrast, Merck A elicited modest inhibition of shape change only at a concentration of 100 nm. Based upon these results, Merck C was used at concentrations of 3 nm or greater for inhibition of L-selectin induced activation. Merck A, which exhibited no inhibitory activity in this dose range, served as a nonspecific control for Merck C. Incubation of neutrophils with the Merck compounds at concentrations up to 50 nm for 1 h at 37 °C did not result in any increase in cell lysis or death (not shown; see “Experimental Procedures”).Figure 3Neutrophil shape change in response to cross-linking of L-selectin and inhibition of p38 MAPK. Neutrophils (106/ml) were preincubated with Merck compounds at the indicated concentrations for 45 min at 37 °C. Cell samples were stimulated by cross-linking L-selectin as described under “Experimental Procedures” and then fixed and the morphology was assessed using a phase-contrast microscope. Cells were scored based on their shape change response to cross-linking L-selectin. Maximum response was defined as the fraction of cells with extensive ruffling of the plasma membrane and pseudopod formation. Data shown are the fraction of cells responding to L-selectin stimulation in the presence of Merck compounds. The data were compiled from two experiments for Merck A and from four experiments for Merck C.View Large Image Figure ViewerDownload Hi-res image Download (PPT)We next assessed whether cross-linking L-selectin by the binding of the two humanized DREG mAbs would elicit phosphorylation of p38 MAPK that could be inhibited by Merck C. Using commercial antibodies specific for the dually phosphorylated state of p38 MAPK, we found that cross-linking of L-selectin enhanced phosphorylation within 1 min. Compared with the untreated control, an increase in the phosphorylation of p38 MAPK of as much as 7-fold was detected (Fig.4 a). The pattern of p38 MAPK phosphorylation showed a decrease at 2 min and a second peak at 3–5 min. These kinetics varied only slightly between experiments, as shown in the densitometry results from four separate experiments (Fig.4 b). As can be seen, the peak of phosphorylation at 1 min and the succeeding drop at 2 min were statistically significant. It should be noted that a minor proteolytic product of p38 was occasionally evident (see 1 min point in Fig. 4 a).Figure 4a, ligation of L-selectin and the kinetics of phosphorylation of the MAP kinases. Neutrophils (5 × 106/ml) were preincubated with Merck A or Merck C (10 nm) for 45 min at 37 °C and stimulated in the presence of HuDreg200 and HuDREG55 added together at 10 μg/ml for between 1 and 10 min. Reactions were stopped by centrifugation and cell pellets were rapidly frozen in liquid nitrogen and suspended in ice-cold lysis buffer. The supernatants were resolved using 12% discontinuous SDS-polyacrylamide gels, proteins were transferred to nitrocellulose membranes, and the dually phosphorylated forms of: A, p38 MAPK on Thr180/Tyr182, andB, p44/42 MAP kinase (ERK1/2) on Thr202/Tyr204 were detected by Western blot analysis. Shown are the kinetics from unstimulated cells and those incubated for the indicated time with anti-L-selectins. Merck A- and C-treated samples correspond to 5-min incubations with anti-L-selectins. b, densitometric analysis of the kinetics of phosphorylation of p38 MAPK. Four experiments were conducted in which the dually phosphorylated form of p38 MAPK was detected as ina. Equal loading of the lanes was verified using Western blots to p38 MAPK protein or ERK1/2 protein. The intensities of the bands for doubly phosphorylated p38 MAPK were measured with Adobe Photoshop and corrected for background intensities. The intensities in each experiment were normalized using the maximum point in each experiment (generally the 1 min point). The data are expressed as the mean (± S.E., n = 4) of these relative intensities. The p values were determined by one-way ANOVA with significance between indicated groups analyzed by a Newman-Keuls post-test. c, densitometric analysis of the kinetics of phosphorylation of ERK1/2. Two experiments were conducted in which double phosphorylation of ERK1/2 was detected as described ina. The intensities were measured and analyzed as detailed in the legend to a. The data are expressed as the mean (± S.E., n = 2) of the relative intensities. d,MAPK activity and Merck dependent inhibition. Activation of MAPK kinase in response to cross-linking of L-selectin was assayed as described under “Experimental Procedures” by measuring the incorporation of radiolabeled phosphate [γ-33P]ATP into heat shock protein-27 (hsp-27), the downstream substrate of p38 MAPK. Neutrophils were preincubated for 45 min at 37 °C in the absence or presence of Merck A or Merck C (5 nm). Phosphorylation of hsp-27 was resolved by SDS-PAGE and the gel was subjected to PhosphorImager quantitation. The intensities were normalized as fractions of unstimulated control samples. Data are shown as kinase activity corresponding to cross-linked L-selectin (X-Link) in the presence of Merck compounds relative to controls from five separate experiments (mean ± S.E., * represents p< 0.05 compared with X-Link alone).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Phosphorylation of ERK1/2 (p44/42 MAP kinase) also increased during this time period, but was delayed in onset (Fig. 4 a). The lower of the bands, ERK2, was the most prominent following ligation with L-selectin. Fig. 4 c shows the combined densitometry results from two experiments, indicating that peak phosphorylation of ERK1/2 occurred at 5–10 min. Merck C almost completely inhibited the phosphorylation of p38 MAPK, but not ERK1/2. We confirmed that Merck C, at concentrations up to 30 nm, had no effect upon the phosphorylation of ERK1/2, using a potent stimulus for these phosphoproteins (5 ng/ml granulocyte-macrophage colony stimulating factor; data not shown). The control compound, Merck A had no effect on either phosphoprotein.As shown in Fig. 4 d, we also confirmed that the tyrosine-phosphorylated p38 MAPK that was induced by cross-linking L-selectin was effective at phosphorylating its downstream substrate MAPK-activated protein (heat shock protein hsp27). Preincubation with Merck A did not alter the kinase activity, whereas Merck C blocked p38 MAPK activity to baseline values.We next determined whether stimulation of degranulation was dependent upon p38 MAPK and inhibitable by Merck C. As shown in Fig.5, Merck C did not significantly inhibit the release of azurophil granules (MPO and β-glucuronidase). However, up-regulation of Mac-1, the tertiary and secretory granule marker, showed statistically significant inhibition with Merck C. This inhibition occurred with stimulation through L-selectin alone, and thus paralleled the observed activation of Mac-1 adhesive function. Inhibition of the release of lactoferrin, the secondary granule marker, was significant for both cross-linking alone and co-stimulation with IL" @default.
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• W2000536824 creator A5012294917 @default.
• W2000536824 creator A5019384731 @default.
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• W2000536824 date "2000-05-01" @default.
• W2000536824 modified "2023-10-12" @default.
• W2000536824 title "L-Selectin Signaling of Neutrophil Adhesion and Degranulation Involves p38 Mitogen-activated Protein Kinase" @default.
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• W2000536824 doi "https://doi.org/10.1074/jbc.m906232199" @default.
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