Abstract. The shape changes and membrane ruffling that accompany neutrophil activation are dependent on the assembly and reorganization of the actin cytoskeleton, the molecular basis of which remains to be clarified . A role of protein kinase C (PKC) has been postulated because neutrophil activation, with the attendant shape and membrane ruffling changes, can be initiated by phorbol esters, known activators of PKC . It has become apparent, however, that multiple isoforms of PKC with differing substrate specificities exist. To reassess the role of PKC in cytoskeletal reorganization, we compared the effects of diacylglycerol analogs and of PKC antagonists on kinase activity and on actin assembly in human neutrophils . Ruffling of the plasma membrane was assessed by scanning EM, and spatial redistribution of filamentous (F)-actin was assessed by scanning confocal microscopy. Staining with NBDphallacidin and incorporation of actin into the Triton X-100-insoluble ("cytoskeletal") fraction were used to quantify the formation of (F)-actin . [31P]ATP was used to detect protein phosphorylation in electroporated cells . Exposure of neutrophils to 4/3-PMA (an activator of PKC) induced protein phosphorylation, membrane ruffling, and assembly and reorganization of the xPOSURE of neutrophils to a variety of agents including chemotactic factors and phorbol esters initiates a coordinated series ofbiological responses . Several of these responses, including shape change, projection of membrane ruffles and pseudopodia, cell motility, and phagocytosis, are dependent on the mechanical displacement of part or all of the cell. This in turn requires the assembly and reorganization ofcytoskeletal microfilaments, which are composed primarily of actin (reviewed by Stossel, 1989) . Actin also plays a major structural role in resting and activated neutrophils (Worthen et al., 1989). Despite the importance of actin-containing microfilaments, the molecular basis of the processes that signal their assembly and reorganization

Abstract. Assembly of microfilaments involves the conversion of actin from the monomeric (G) to the filamentous (F) form. The exact sequence of events responsible for this conversion is yet to be defined and, in particular, the role of calcium remains unclear. Intact and electropermeabilized human neutrophils were used to assess more directly the role of cytosolic calcium ([Ca2+]3 in actin assembly. Staining with 7-nitrobenz-2-oxa-l,3-diazole-phallacidin and right angle light scattering were used to monitor the formation of F-actin. Though addition of Ca 2÷ ionophores can be shown to induce actin assembly, the following observations suggest that an increased [Ca2+]~ is not directly responsible for receptor-induced actin polymerization: (a) intact cells in Ca2+-free medium, depleted of internal Ca 2+ by addition of ionophore, responded to the formyl peptide fMLP with actin assembly despite the absence of changes in [Ca2÷]~, assessed with Indo-1; (b) fMLP induced a significant increase in F-actin content in permeabilized cells equilibrated with medium containing 0.1 /zM free Ca 2÷, buffered with up to 10 mM EGTA; (c) increasing [Ca2+]~ beyond the resting level by direct addition of CaCI2 to permeabilized cells resulted in actin disassembly. Conversely, lowering [Ca2+]~ resulted in spontaneous actin assembly. To reconcile these findings with the actinpolymerizing effects of Ca 2+ ionophores, we investigated whether A23187 and ionomycin induced actin assembly by a mechanism independent of, or secondary to the increase in [Ca2+]~. We found that the ionophore-induced actin assembly was completely inhibited by the leukotriene B4 (LTB4) antagonist LY-223982, implying that the ionophore effect was secondary to LTB4 formation, possibly by stimulation of phospholipase A2. We conclude that actin assembly is not mediated by an increase in [Ca2+]~, but rather that elevated [Ca2+]~ facilitates actin disassembly, an effect possibly mediated by Ca2÷-sensitive actin filamentsevering proteins such as gelsolin. Sequential actin assembly and disassembly may be necessary for functions such as chemotaxis.

L-selectin is a leukocyte cell surface glycoprotein involved in carbohydrate-specific ligand binding which mediates rolling of leukocytes along endothelial surfaces. In addition to its role in adhesion, an intracellular signaling role for L-selectin has recently been recognized. In particular, cross-linking L-selectin leads to increased cytosolic Ca2+ levels and potentiation of the oxidative burst. As several cell surface glycoproteins have been shown to be linked to tyrosine kinases, we examined the hypothesis that L-selectin may be linked to pathways involving tyrosine phosphorylation in human neutrophils. Ligation of L-selectin by three different antibodies recognizing separate epitopes led to increased tyrosine phosphorylation of several cellular proteins as judged by anti-phosphotyrosine immunoblots of whole cell lysates with prominent bands at 40-42, 55-60, 70-72, and 105-120 kDa. The 42-kDa band comigrated with mitogen-activated protein (MAP) kinase as determined by immunoblotting with anti-MAP kinase antibody. This effect was specific for L-selectin, because antibodies against CD18, CD45, and CD10 did not increase tyrosine phosphorylation. Phosphorylation was not due to Fc binding, since F(ab')2 fragments of the anti-L-selectin antibodies were similarly effective, and the response was unaffected by Fc receptor blockade. Cross-linking of L-selectin was not required for enhanced tyrosine phosphorylation, because monovalent Fab fragments also increased tyrosine phosphorylation. The response to L-selectin antibodies was not inhibited by cytochalasin, suggesting that reorganization of the actin cytoskeleton was not required for this response. Sulfatides, sulfated glycolipids which may be natural ligands for L-selectin, also induced a rapid, dose-dependent increase in tyrosine phosphorylation. In addition, sulfatides, but not control glycolipids, resulted in enhanced tyrosine phosphorylation of MAP kinase. Both sulfatides and anti-L-selectin antibodies increased kinase activity of MAP kinase as determined by gel renaturation assay. The tyrosine kinase inhibitor, genistein, blocked the transient increase in intracellular Ca2+ and the oxidative burst induced by sulfatides, suggesting that this tyrosine phosphorylation is functionally important. We conclude that L-selectin is able to transmit intracellular signals, including increased tyrosine phosphorylation and activation of MAP kinase in neutrophils. We speculate that these events may contribute to the activation of neutrophils during adhesion.

The tyrosine phosphorylation of several proteins induced in neutrophils by soluble and particulate stimuli is thought to be crucial for initiating antimicrobial responses. Although activation of tyrosine kinases is thought to mediate this event, the role of tyrosine phosphatases in the initiation and modulation of neutrophil responses remains largely undefined. We investigated the role of Src homology 2-containing tyrosine phosphatase 1 (SHP-1; also known as protein tyrosine phosphatase 1C (PTP1C), hematopoetic cell phosphatase, PTP-N6, and SHPTP-1), a phosphatase expressed primarily in hemopoietic cells, in the activation of human neutrophils. SHP-1 mRNA and protein were detected in these cells, and the enzyme was found to be predominantly localized to the cytosol in unstimulated cells. Following stimulation with neutrophil agonists such as phorbol ester, chemotactic peptide, or opsonized zymosan, a fraction of the phosphatase redistributed to the cytoskeleton. Agonist treatment also induced significant decreases (30 -60%) in SHP-1 activity, which correlated temporally with increases in the cellular phosphotyrosine content. Phosphorylation of SHP-1 on serine residues was associated with the inhibition of its enzymatic activity, suggesting a causal relationship. Accordingly, both the agonist-evoked phosphorylation of SHP-1 and the inhibition of its catalytic activity were blocked by treatment with bisindolylmaleimide I, a potent and specific inhibitor of protein kinase C (PKC) activity. Immunoprecipitated SHP-1 was found to be phosphorylated efficiently by purified PKC in vitro. Such phosphorylation also caused a decrease in the phosphatase activity of SHP-1. Together, these data suggest that inhibition of SHP-1 by PKC-mediated serine phosphorylation plays a role in facilitating the accumulation of tyrosine-phosphorylated proteins following neutrophil stimulation.These findings provide a new link between the PKC and tyrosine phosphorylation branches of the signaling cascade that triggers antimicrobial responses in human neutrophils.