Adhesion-dependent protein tyrosine phosphorylation in neutrophils treated with tumor necrosis factor

Human neutrophils (PMN) respond to tumor necrosis factor (TNF) by releasing their granules, reorganizing their cytoskeleton, and massively secreting hydrogen peroxide. This response is dependent on adhesion to extracellular matrix proteins and expression of CD11b/CD18 integrins (Nathan, C., S. Srimal, C. Farber, E. Sanchez, L. Kabbash, A. Asch, J. Gailit, and S. D. Wright. 1989. J. Cell Biol. 109:1341-1349). We investigated the role of tyrosine phosphorylation in the response of PMN to TNF. PMN adherent to protein-coated surfaces but not suspended PMN showed tyrosine phosphorylation of several proteins (approximately 150, approximately 115, approximately 75, and approximately 65 kD) in response to TNF. Tyrosine phosphorylation was evident 5 min after addition of TNF and lasted at least 2 h. The tyrosine kinase inhibitors K252a, genistein and ST638 suppressed tyrosine phosphorylation and blocked hydrogen peroxide production in a reversible manner at low concentrations. Tyrosine kinase inhibitors also blocked the spreading of PMN in response to TNF. Dihydrocytochalasin B did not inhibit tyrosine phosphorylation, but in its presence phosphorylation was rapidly reversed. By immunocytochemistry, the majority of tyrosine phosphoproteins were localized to focal adhesions. Thus TNF-induced tyrosine phosphorylation depends on adhesion of PMN to extracellular matrix proteins, and participates in the transduction of the signals that direct the cells to spread on a biological surface and undergo a respiratory burst.     

Complement receptor 3 (CR3, Mac-1, integrin alpha M beta 2, CD11b/CD18) is required for tyrosine phosphorylation of paxillin in adherent and nonadherent neutrophils

Expression of the leukocyte (beta 2) integrins is required for many functions of activated neutrophils (PMN), even when there is no recognized ligand for any beta 2 integrin. To investigate the hypothesis that beta 2 integrins may be involved in a signal transduction pathway related to cytoskeletal reorganization, we examined whether beta 2 integrins have a role in tyrosine phosphorylation of the cytoskeletal protein paxillin. Treatment of PMN in suspension with phorbol esters, f-Met-Leu-Phe, and TNF-alpha resulted in paxillin tyrosine phosphorylation. However, treatment of beta 2-deficient (LAD) PMN failed to induce paxillin tyrosine phosphorylation. Normal PMN phosphorylated paxillin in response to adhesion to immune complexes, while the LAD PMN did not. Adhesion of phorbol ester activated-LAD PMN to the extracellular matrix proteins fibronectin, laminin, and vitronectin failed to induce paxillin tyrosine phosphorylation. Treatment of activated normal PMN with mAb directed against the beta 2 integrin alpha chains demonstrated that CR3 (alpha M beta 2) was required for paxillin phosphorylation. Transfection of the cell line K562 with CR3 confirmed that CR3 ligation resulted in paxillin tyrosine phosphorylation. As a control, K562 transfected with CR2 (CD21) which bound equally avidly to the same complement C3-derived ligand (C3bi) as the CR3 transfectants, showed no enhanced tyrosine phosphorylation of paxillin upon receptor ligation. While both CR2 and CR3 transfectants showed efficient adhesion to a C3bi-coated surface, only the CR3 transfectants spread during adhesion and phosphorylated paxillin. Together these data demonstrate that CR3 is required for paxillin phosphorylation during activation of both adherent and nonadherent PMN. Even PMN activated in suspension or by adhesion to immune complexes, when no CR3 ligand is apparent, still require CR3 for a signal transduction pathway leading to paxillin tyrosine phosphorylation. This pathway is likely to be important for PMN function in inflammation and host defense.     

Deficiency of Src family kinases p59/61hck and p58c-fgr results in defective adhesion-dependent neutrophil functions

Cross-linking of the neutrophil-beta 2- or beta 3-related leukocyte response integrins by extracellular matrix (ECM) proteins or monoclonal antibodies (mAb) stimulates cytoskeletal rearrangement leading to cell spreading and respiratory burst. Tyrosin phosphorylation of a variety of proteins and activation of the Src family kinases within polymorphonuclear leukocytes (PMN) have recently been implicated in the intracellular signaling pathways generated by leukocyte integrins (Yan, S.R., L. Fumagalli, and G Berton. 1995. J. Inflammation. 45:217-311.) To directly test whether these functional responses are dependent on the Src family kinases p59/61hck and p58c-fgr, we examined adhesion- dependent respiratory burst in PMNs isolated from hck -/-, fgr -/-, and hck -/- fgr -/- knockout mice. Purified bone marrow PMNS from wild-type mice released significant amounts of O2- when adherent to fibrinogen-, fibronectin-, or collagen-coated surfaces, in the presence of activating agents such as tumor necrosis factor (TNF) or formyl- methionyl-leucyl-phenylalanine, as described for human PMNs. PMNs from hck-/-fgr-/- double-mutant mic, however, failed to respond. This defect was specific for integrin signaling, since respiratory burst was normal in hck-/-fgr-/-PMNs stimulated by immune complexes or PMA. Stimulation of respiratory burst was observed in TNF-primed wild-type PMN plated on surfaces coated with murine intracellular adhesion molecule-1 (ICAM-1), while hck-/-fgr-/- PMNs, failed to respond. Direct cross-linking of the subunits of beta 2 and beta 2 integrins by surface-bound mAbs was elicited O2- production by wild-type PMNs, while the double-mutant hck- /-fgr-/- cells failed to respond. Photomicroscopy and cell adhesion assays revealed that the impaired functional responses of hck-/-fgr-/- PMNs were caused by defective spreading and tight adhesion on either ECM protein- or mAb-coated surfaces. In contrast, hck-/-or fgr-/-single mutant cells produced O2- at levels equivalent to wild-type cells on ECM protein, murine ICAM-1, and antiintegrin mAb-coated surfaces. Hence, either p59/61 hck and p 58c-fgr is required for signaling through leukocyte beta 2 and beta 3 integrins leading to PMN spreading and respiratory burst. This is the first direct genetic evidence of the importance of Src family kinases in integrin signaling within leukocytes, and it is also the best example of overlapping function between members of this gene family within a defined signal transduction pathway.     

Beta 2 integrin-dependent tyrosine phosphorylation of paxillin in human neutrophils treated with tumor necrosis factor

The focal adhesion protein paxillin undergoes tyrosine phosphorylation in response to signals mediated by integrins, neuropeptides and oncogene products, possibly via activation of the focal adhesion- associated kinase, p125FAK. In the present work, tumor necrosis factor- alpha (TNF) stimulated tyrosine phosphorylation of paxillin in human neutrophils. Cell adhesion and participation of the beta 2 integrin CD18 were necessary, but not sufficient, for the response. Adherent neutrophils also tyrosine phosphorylated paxillin in response to phorbol ester, formylmethionyl-leucyl-phenylalanine and opsonized bacteria. In contrast, p125FAK was constitutively tyrosine phosphorylated in a manner unaffected by adherence and/or TNF. Thus, cytokines and microbial products are among the stimuli that can induce the tyrosine phosphorylation of paxillin, and kinases other than p125FAK may be responsible. This is the first identification of paxillin and p125FAK in human cells and neutrophils, and one of the few identifications of a specific protein that undergoes tyrosine phosphorylation in response to any agonist in neutrophils or in response to TNF in any cell.     

Regulation of the pp72syk protein tyrosine kinase by platelet integrin alpha IIb beta 3.

pp72syk is essential for development and function of several hematopoietic cells, and it becomes activated through tandem SH2 interaction with ITAM motifs in immune response receptors. Since Syk is also activated through integrins, which do not contain ITAMs, a CHO cell model system was used to study Syk activation by the platelet integrin, alpha IIb beta 3. As in platelets, Syk underwent tyrosine phosphorylation and activation during CHO cell adhesion to alpha IIb beta 3 ligands, including fibrinogen. This involved Syk autophosphorylation and the tyrosine kinase activity of Src, and it exhibited two novel features. Firstly, unlike alpha IIb beta 3-mediated activation of pp125FAK, Syk activation could be triggered by the binding of soluble fibrinogen and abolished by truncation of the alpha IIb or beta 3 cytoplasmic tail, and it was resistant to inhibition by cytochalasin D. Secondly, it did not require phosphorylated ITAMs since it was unaffected by disruption of an ITAM-interaction motif in the SH2(C) domain of Syk or by simultaneous overexpression of the tandem SH2 domains. These studies demonstrate that Syk is a proximal component in alpha IIb beta 3 signaling and is regulated as a consequence of intimate functional relationships with the alpha IIb beta 3 cytoplasmic tails and with Src or a closely related kinase. Furthermore, there are fundamental differences in the activation of Syk by alpha IIb beta 3 and immune response receptors, suggesting a unique role for integrins in Syk function.     

Diverse effects of neutrophil integrin occupation on respiratory burst activation.

Integrin occupation can alter the function of neutrophils (PMN), but the mechanism(s) involved is still unclear. This study demonstrated that the occupation of PMN integrins (especially those of the beta(3) subfamily) strongly enhances TNF stimulation of the respiratory burst but down-regulates that induced by PMA, fMLP, Con A, and serum treated zymosan. Treatment of PMN with genistein, staurosporine, and wortmannin, inhibitors of tyrosine kinases, protein kinase C, and phosphotidylinostol 3-kinase (PI 3-kinase) respectively, completely blocked the TNF-stimulated respiratory burst in PMN. Genistein and wortmannin enhanced the PMA-stimulated respiratory burst but only in cells adherent to RGD peptide. These findings suggest that PMN integrins (beta(3) subfamily) can generate signals that regulate the PMN agonist responses, probably through the activities of tyrosine kinases and PI 3-kinase.     

The role of protein tyrosine phosphorylation in integrin-mediated gene induction in monocytes

Integrin-mediated cell adhesion, or cross-linking of integrins using antibodies, often results in the enhanced tyrosine phosphorylation of certain intracellular proteins, suggesting that integrins may play a role in signal transduction processes. In fibroblasts, platelets, and carcinoma cells, a novel tyrosine kinase termed pp125FAK has been implicated in integrin-mediated tyrosine phosphorylation. In some cell types, integrin ligation or cell adhesion has also been shown to result in the increased expression of certain genes. Although it seems reasonable to hypothesize that integrin-mediated tyrosine phosphorylation and integrin-mediated gene induction are related, until now, there has been no direct evidence supporting this hypothesis. In the current report, we explore the relationship between integrin- mediated tyrosine phosphorylation and gene induction in human monocytes. We demonstrate that monocyte adherence to tissue culture dishes or to extracellular matrix proteins is followed by a rapid and profound increase in tyrosine phosphorylation, with the predominant phosphorylated component being a protein of 76 kD (pp76). Tyrosine phosphorylation of pp76 and other monocyte proteins can also be triggered by incubation of monocytes with antibodies to the integrin beta 1 subunit, or by F(ab')2 fragments of such antibodies, but not by F(ab) fragments. The ligation of beta 1 integrins with antibodies or F(ab')2 fragments also induces the expression of immediate-early (IE) genes such as IL-1 beta. When adhering monocytes are treated with the tyrosine kinase inhibitors genistein or herbimycin, both phosphorylation of pp76 and induction of IL-1 beta message are blocked in a dose-dependent fashion. Similarly, treatment with genistein or herbimycin can block tyrosine phosphorylation of pp76 and IL-1 beta message induction mediated by ligation of beta 1 integrin with antibodies. These observations suggest that protein tyrosine phosphorylation is an important aspect of integrin-mediated IE gene induction in monocytes. The cytoplasmic tyrosine kinase pp125FAK, although important in integrin signaling in other cell types, seems not to play a role in monocytes because this protein could not be detected in these cells.     

A role for beta(2) integrins (CD11/CD18) in the regulation of cytokine gene expression of polymorphonuclear neutrophils during the inflammatory response.

Growing evidence supports the idea that adhesion via beta(2) integrins not only allows cellular targeting, but also induces intracellular signaling, which in turn activates functional responses of adherent cells. This study investigates whether beta(2) integrin-mediated adhesion of human polymorphonuclear neutrophils (PMN) has a functional impact on cytokine production. Aggregation of the beta(2) integrin Mac-1 (CD11b/CD18) by antibody cross-linking was found to induce substantial de novo synthesis of IL-8 mRNA as measured by semiquantitative RT-PCR and Northern blotting technique, respectively. Induction of IL-8 mRNA was also observed upon adhesion of PMN to immobilized fibrinogen, a functional equivalent of its clotting product fibrin that serves as a native ligand of Mac-1. Results were confirmed using PMN derived from CD18-deficient mice, which were unable to produce MIP-2 mRNA, a homologue of human IL-8, in the presence of immobilized fibrinogen. In contrast, a substantial increase of MIP-2 mRNA was observed when wild-type PMN were incubated on immobilized fibrinogen. In human PMN, ELISA technique showed that the gene activation that required tyrosine kinase activity resulted in a substantial production and secretion of biologically active IL-8 and IL-1beta. In contrast, no TNF-alpha or IL-6 production was found, revealing that beta(2) integrins mediate differential expression of proinflammatory cytokines. The biological relevance of the present findings was confirmed in an in vivo model of acute inflammation. Altogether, the present findings provide evidence for a functional link between clotting and inflammatory responses that may contribute to the recruitment and/or activation of PMN and other cells at sites of lesion.     

Tumor necrosis factor and CD11/CD18 (beta 2) integrins act synergistically to lower cAMP in human neutrophils

The ability of neutrophils (PMN) to undergo a prolonged respiratory burst in response to cytokines such as tumor necrosis factor-alpha (TNF) depends on expression of CD11/CD18 (beta 2) integrins and interaction with matrix protein-coated surfaces (Nathan, C., S. Srimal, C. Farber, E. Sanchez, L. Kabbash, A. Asch, J. Gailit, and S. D. Wright. 1989. J. Cell Biol. 109:1341-1349). We tested the hypothesis that changes in cAMP mediate the joint action of cytokines and integrins. When plated on FBS- or fibrinogen-coated surfaces, PMN responded to TNF with a sustained fall in intracellular cAMP. This did not occur without TNF; in suspended PMN; in PMN treated with anti-CD18 mAb; or in PMN genetically deficient in beta 2 integrins. A preceding fall in cAMP appeared essential for TNF to induce a respiratory burst, because drugs that elevate cAMP blocked the burst if added any time before, but not after, its onset. Adenosine analogues and cytochalasins also block the TNF-induced respiratory burst if added before, but not after, its onset. Both also blocked the TNF-induced fall in cAMP. The effect of cytochalasins led us to examine the relationship between cAMP and actin reorganization. The same conditions that led to a sustained fall in cAMP led at the same time to cell spreading and the assembly of actin filaments. As with the respiratory burst, cAMP-elevating agents inhibited TNF-induced cell spreading and actin filament assembly if added before, but not after, spreading began. Thus, occupation of TNF receptors and engagement of CD18 integrins interact synergistically in PMN to promote a fall in cAMP. The fall in cAMP is closely related to cell spreading and actin reorganization. These changes are necessary for TNF to induce a prolonged respiratory burst. We conclude that integrins can act jointly with cytokines to affect cell shape and function through alterations in the level of a second messenger, cAMP.     

Adhesive ligand binding to integrin alpha IIb beta 3 stimulates tyrosine phosphorylation of novel protein substrates before phosphorylation of pp125FAK

Tyrosine phosphorylation of multiple platelet proteins is stimulated by thrombin and other agonists that cause platelet aggregation and secretion. The phosphorylation of a subset of these proteins, including a protein tyrosine kinase, pp125FAK, is dependent on the platelet aggregation that follows fibrinogen binding to integrin alpha IIb beta 3. In this report, we examined whether fibrinogen binding, per se, triggers a process of tyrosine phosphorylation in the absence of exogenous agonists. Binding of soluble fibrinogen was induced with Fab fragments of an anti-beta 3 antibody (anti-LIBS6) that directly exposes the fibrinogen binding site in alpha IIb beta3. Proteins of 50-68 KD and 140 kD became phosphorylated on tyrosine residues in a fibrinogen- dependent manner. This response did not require prostaglandin synthesis, an increase in cytosolic free calcium, platelet aggregation or granule secretion, nor was it associated with tyrosine phosphorylation of pp125FAK. Tyrosine phosphorylation of the 50-68-kD and 140-kD proteins was also observed when (a) fibrinogen binding was stimulated by agonists such as epinephrine, ADP, or thrombin instead of by anti-LIBS6; (b) fragment X, a dimeric plasmin-derived fragment of fibrinogen was used instead of fibrinogen; or (c) alpha IIb beta 3 complexes were cross-linked by antibodies, even in the absence of fibrinogen. In contrast, no tyrosine phosphorylation was observed when the ligand consisted of monomeric cell recognition peptides derived from fibrinogen (RGDS or gamma 400-411). Fibrinogen-dependent tyrosine phosphorylation was inhibited by cytochalasin D. These studies demonstrate that fibrinogen binding to alpha IIb beta 3 initiates a process of tyrosine phosphorylation that precedes platelet aggregation and the phosphorylation of pp125FAK. This reaction may depend on the oligomerization of integrin receptors and on the state of actin polymerization, organizational processes that may juxtapose tyrosine kinases with their substrates.     

Immune complex-stimulated neutrophil LTB4 production is dependent on beta 2 integrins

The beta 2 integrins (LFA-1, Mac-1, and p150,95) are critical for many adhesive functions of leukocytes. Although the binding of the IgG- opsonized particles occurs normally in the absence of beta 2 integrins, phagocytosis of IgG-opsonized particles by activated neutrophils (PMN) requires these integrins. This observation suggests a role for beta 2 integrins in phagocytosis subsequent to particle binding. To investigate the mechanism of involvement of beta 2 integrins in IgG- mediated functions, we examined the role of beta 2 integrins in adhesion to immune complex (IC)-coated surfaces. Initial adhesion and spreading on IC-coated surfaces were equivalent in control and beta 2- deficient phagocytes. However, both genetically beta 2-deficient PMN and PMN treated with the anti-beta 2 mAb IB4 subsequently detached from the IC-coated surfaces. To determine whether biochemical consequences of IgG activation were also affected by beta 2 deficiency, LTB4 production in response to Fc receptor ligation was assessed. LTB4 production by beta 2-deficient PMN adherent to IC-coated surfaces was markedly decreased in comparison with control PMN. Importantly, LTB4 production by PMN stimulated with fluid phase heat-aggregated IgG also required the beta 2 integrins, showing that the defect was not a simple consequence of abnormal adhesion. In contrast, superoxide production by IC-adherent PMN was equivalent in control and beta 2-deficient PMN. The initial rises in intracytoplasmic [Ca2+]i in response to aggregated IgG also were unaffected by inhibition of beta 2 integrins. These data show that lack of beta 2 integrins does not inhibit all FcR-dependent signal transduction. Finally, LTB4 production by normal PMN adherent to ICs was inhibited by antibodies to FcRII, but not FcRIII, showing that FcRII ligation was required for this effect. Together these data identify a role for the beta 2 integrins in a signal transduction pathway leading to sustained adhesion and LTB4 production in response to IC. Since both beta 2 integrins and FcRII are required for these effects, the data further suggest cooperation between these receptors in generating PMN activation in response to IC stimulation.     

The CD11/CD18 (beta2) integrins modulate neutrophil caspase activation and survival following TNF-alpha or endotoxin induced transendothelial migration

Neutrophils (PMN) are short-lived cells but their survival is often prolonged in inflammation. The beta2 (CD11/CD18) integrins are involved in PMN migration into inflammation but their role in PMN survival is not well understood. We investigated the role of beta2 integrins in PMN caspase activation, a key enzyme cascade in apoptosis. After 20 h, caspase activation (Western blotting) was markedly decreased in PMN cultured on fibrinogen, a ligand for Mac-1 (CD11b/CD18), but not on fibronectin or albumin. In the presence of TNF-alpha or endotoxin (LPS), blockade of CD18 (beta2 chain) with mAb markedly increased caspase activation in PMN on fibrinogen. PMN which migrated through endothelium in vitro in response to TNF-alpha, LPS, IL-1alpha, IL-8 or C5a contained 58% fewer active caspase positive PMN after 20 h than non-migrated PMN remaining on the endothelium. When beta2 (CD18) integrin or lymphocyte function antigen (LFA)-1 (CD11a) plus Mac1 (CD11b) were blocked by mAb (intact or Fab'), the proportion of migrated PMN (but not of non-migrated PMN) with active caspases was significantly increased (2-4-fold) and this was associated with accelerated PMN apoptosis and death. Thus, engagement of ligands on extracellular matrix and endothelium by the beta2 integrins Mac-1 and LFA-1 plays a role in delaying apoptosis in PMN recruited in response to LPS and TNF-alpha. Inhibition of beta2 integrin function may not only inhibit PMN infiltration, but also accelerate PMN clearance from inflamed tissue.     

Tumor necrosis factor-alpha activation of the c-Jun N-terminal kinase pathway in human neutrophils. Integrin involvement in a pathway leading from cytoplasmic tyrosine kinases apoptosis

The intensity and duration of an inflammatory response depends on the balance of factors that favor perpetuation versus resolution. At sites of inflammation, neutrophils adherent to other cells or matrix components are exposed to tumor necrosis factor-alpha (TNFalpha). Although TNFalpha has been implicated in induction of pro-inflammatory responses, it may also inhibit the intensity of neutrophilic inflammation by promoting apoptosis. Since TNFalpha is not only an important activator of the stress-induced pathways leading to p38 MAPk and c-Jun N-terminal kinase (JNK) but also a potent effector of apoptosis, we investigated the effects of TNFalpha on the JNK pathway in adherent human neutrophils and the potential involvement of this pathway in neutrophil apoptosis. Stimulation with TNFalpha was found to result in beta2 integrin-mediated activation of the cytoplasmic tyrosine kinases Pyk2 and Syk, and activation of a three-part MAPk module composed of MEKK1, MKK7, and/or MKK4 and JNK1. JNK activation was attenuated by blocking antibodies to beta2 integrins, the tyrosine kinase inhibitors, genistein, and tyrphostin A9, a Pyk2-specific inhibitor, and piceatannol, a Syk-specific inhibitor. Exposure of adherent neutrophils to TNFalpha led to the rapid onset of apoptosis that was demonstrated by augmented annexin V binding and caspase-3 cleavage. TNFalpha-induced increases in annexin V binding to neutrophils were attenuated by blocking antibodies to beta2 integrins, and the caspase-3 cleavage was attenuated by tyrphostin A9. Hence, exposure of adherent neutrophils to TNFalpha leads to utilization of the JNK-signaling pathways that may contribute to diverse functional responses including induction of apoptosis and subsequent resolution of the inflammatory response.     

H(2)O(2)-induced tyrosine phosphorylation of protein kinase cdelta by a mechanism independent of inhibition of protein-tyrosine phosphatase in CHO and COS-7 cells

It has been proposed that H(2)O(2) increases tyrosine phosphorylation of cellular proteins by inhibiting protein-tyrosine phosphatase through oxidation of the cysteine residue of the enzyme essential for its catalytic activity. Tyrosine phosphorylation of the delta isoform of protein kinase C (PKC) was induced by H(2)O(2) in CHO and COS-7 cells. H(2)O(2) also induced activation of mitogen-activated protein kinase. Vanadate and molybdate, which inhibit protein-tyrosine phosphatase by binding to its active site, did not induce tyrosine phosphorylation of PKCdelta, but enhanced H(2)O(2)-induced tyrosine phosphorylation of PKCdelta in the cell. The oxoanions, however, generated the active form of mitogen-activated protein kinase. Another protein-tyrosine phosphatase inhibitor, phenylarsine oxide, which bridges the thiol residues of the enzyme, induced tyrosine phosphorylation of PKCdelta, and the reaction was enhanced by vanadate. These results suggest that inhibition of protein-tyrosine phosphatase is insufficient for induction of tyrosine phosphorylation of PKCdelta in the cells, and that presumably activation of protein-tyrosine kinase may be essential for tyrosine phosphorylation of the PKC isoform.     

Cross-linking of beta2 integrins caused diminished responses of neutrophils to priming agents like lipopolysaccharide or tumor necrosis factor-alpha: possible involvement of tyrosine kinase Syk

Neutrophils up-regulate beta2 integrins like CD11b/CD18 in response to lipopolysaccharide (LPS). Up-regulation of beta2 integrins causes neutrophils to adhere to surfaces, and to release superoxide anion (O2-). When neutrophils are exposed to LPS plus plasma under conditions not favorable for adherence (absence of Mg2+), the cells do not spontaneously release O2-, but instead they are primed for enhanced release of O2- after subsequent triggering by fMLP. In the presence of Mg2+, neutrophils adhere in response to LPS but fMLP-triggered O2- release by LPS-primed neutrophils is diminished. To understand why adherence interferes with the response of neutrophils to N-formyl-methionyl-leucyl-phenylalanine (fMLP), beta2 integrins were cross-linked by mouse monoclonal antibodies that had been immobilized by surface-bound anti-mouse antibody. When unprimed neutrophils were trapped on the surface by these cross-linked monoclonal antibodies, O2- release was triggered, and priming by LPS for fMLP-triggered O2- release was diminished, indicating that this cross-linking of beta2 integrins mimicked adherence. Alkaline phosphatase is up-regulated by LPS or tumor necrosis factor-alpha, and this response was also diminished by the cross-linking antibodies. The diminished alkaline phosphatase up-regulation was reversed by genistein, a general inhibitor of tyrosine kinases, and by piceatannol, an inhibitor for Syk kinase. Piceatannol also inhibited the phosphorylation of Syk caused by cross-linking of beta2 integrins. These results suggested that adherence-induced triggering and Syk kinase activation might be responsible for the diminished response of LPS-primed neutrophils to fMLP when neutrophils were adherent.     

Characterization of the protein apparently responsible for the elevated tyrosine protein kinase activity in LSTRA cells.

The LSTRA murine thymoma cell line contains an elevated level of tyrosine protein kinase activity. When a microsomal preparation from these cells is incubated in vitro with ATP, the principal tyrosine protein kinase substrate is a 56,000-dalton protein, p56. We have found that an activity phosphorylating p56 on tyrosine can also be detected at low levels in microsomes from most, but not all, T lymphoma cell lines and from normal thymic tissue. Only 1 of 30 other lymphoma cell lines was found to contain an elevated level of such a tyrosine protein kinase. An activity that phosphorylated p56 in vitro was not detectable in the cells of other hematopoietic lineages. Anti-peptide antibodies reactive with the site of in vitro tyrosine phosphorylation of p56 allowed us to determine that the apparent abundance of the p56 polypeptide parallels closely the level of the tyrosine protein kinase activity in the cell lines examined. This suggests that p56 is the protein kinase responsible for the elevated tyrosine protein kinase activity in LSTRA cells and that the phosphorylation of p56 observed in vitro results from autophosphorylation. Two-dimensional tryptic peptide mapping revealed that p56 is distinct from the proteins encoded by the cellular genes which are the progenitors of retroviral tyrosine protein kinases, src, yes, fgr, abl, fes, and ros. Additionally, none of these proto-oncogenes was found to be transcribed at elevated levels in LSTRA or Thy19 cells. Like the catalytic subunit of the cyclic AMP-dependent protein kinase, the cellular and viral forms of p60src, and the protein phosphatase calcineurin B, p56 contains covalently bound fatty acid.     

The Raman detection of peptide tyrosine phosphorylation

Drop-coating-deposition-Raman (DCDR) is used to detect spectral changes induced by phosphorylation of tyrosine amino acid residues in peptides. Four peptides are investigated, with sequences derived from the human protein-tyrosine kinase, p60c-src, with Y-216, Y-419, and Y-530 phosphorylation sites. Although the spectra of the four peptides are quite different, tyrosine phosphorylation is found to invariably induce the collapse of a doublet at 820-850cm(-1) and the attenuation of a peak around 1205cm(-1). Moreover, amide III band shifts suggest that tyrosine phosphorylation may promote beta sheet formation, particularly in peptides that lack phenylalanine residues. The degree of tyrosine phosphorylation in peptide mixtures is determined using DCDR combined with partial least squares multivariate calibration with a 2% root mean standard error of prediction.     

A role for beta2 integrin (CD11/CD18)-mediated tyrosine signaling in extravasation of human polymorphonuclear neutrophils

During the recruitment of human polymorphonuclear neutrophils (PMN) to sites of inflammation, leukocyte adhesion molecules of the beta2 integrin (CD11/CD18) family mediate firm adhesion of these cells to the endothelial cell monolayer lining the vessel wall. This process is a prerequisite for shape change and spreading of PMN on the endothelium which eventually allows PMN emigration into the extravascular space. In order to elucidate the molecular mechanisms which mediate this sequence of events, intracellular protein tyrosine signaling was studied subsequent to beta2 integrin-mediated ligand binding. Using western blotting technique, beta2 integrin-mediated adhesion was found to induce tyrosine phosphorylation of different proteins. The effect was absent in PMN derived from CD18-deficient mice which lack any beta2 integrin expression on the cell surface demonstrating the specificity of the observed response. Inhibition of beta2 integrin-mediated tyrosine signaling by herbimycin A almost completely inhibited adhesion, shape change, and subsequent spreading of PMN. Herbimycin A also diminished chemotactic migration of these cells in response to the soluble mediator N-formyl-Met-Leu-Phe (fMLP). In contrast, treatment of PMN with cytochalasin D had no substantial effect on beta2 integrin-mediated signaling or adhesion but inhibited shape change, spreading, and chemotactic migration of PMN. This suggests that the signaling capacity exerted by beta2 integrins upon ligand binding was independent of an intact cytoskeleton. Moreover, the beta2 integrin-mediated activation of intracellular signal transduction pathways was critical for firm adhesion of PMN, the prerequisite subsequent shape change and spreading, which allows emigration of PMN into the extravascular space.     

Responses of neutrophils to anti-integrin antibodies depends on costimulation through low affinity Fc gamma Rs: full activation requires both integrin and nonintegrin signals

The relative contribution of integrin and nonintegrin signals to neutrophil activation is incompletely understood. Immobilized anti-integrin Abs were previously shown to induce robust activation of neutrophils without any additional stimulus, suggesting that cross-linking of integrins is sufficient for full activation of the cells. However, the possible contribution from other receptors has not been tested in this system. In this study, we show that neutrophil responses to anti-integrin Abs requires costimulation through low-affinity Fc gamma Rs. Murine neutrophils lacking the FcR gamma-chain or Fc gamma RIII failed to respond to immobilized Abs against beta(1), beta(2), or beta(3) integrins and the activation of wild-type cells could be prevented by blocking Abs against Fc gamma RII/III. Plate-bound anti-CD18 Abs initiated a respiratory burst from human neutrophils, but this response was abrogated when the F(ab')(2) of the same Abs were used or the cells were preincubated with Fc gamma RIIA-blocking Abs. Lack of Fc gamma RIII or administration of Fc gamma R-blocking Abs had no effect on responses of TNF-stimulated cells plated on fibrinogen or rICAM-1. TNF restored the respiratory burst of Fc gamma RIII-deficient neutrophils plated on anti-CD18 mAbs. The p38 MAPK inhibitor SB203580 attenuated the responses of neutrophils to anti-CD18 mAbs or TNF stimulation on a fibrinogen surface. Taken together, these results indicate that activation of low-affinity Fc gamma Rs is required for neutrophil responses induced by anti-integrin Abs and suggest that a second coactivation signal (e.g., through TNF or FcR ligation) is indispensable for full integrin-mediated activation of neutrophils. These second signals are interchangeable and they may converge on the p38 MAPK.     

Evidence for GTP-binding protein involvement in the tyrosine phosphorylation of the T cell receptor zeta chain.

The zeta subunit of the T cell receptor (TCR) is a prominent substrate for a TCR-activated tyrosine kinase. Tyrosine phosphorylation of the zeta subunit in response to antibody-mediated receptor cross-linking was synergized in permeabilized T cells by either of two non-hydrolyzable GTP analogues, guanosine 5'-[gamma-thio]triphosphate (GTP gamma S) or guanosine 5'-[beta, gamma-imido]triphosphate Gpp(NH)p. ATP analogues did not significantly affect antibody-induced tyrosine phosphorylation. Unlike the GTP analogues, the GDP analogue guanosine 5'-[beta-thio]diphosphate (GDP beta S) did not enhance phosphorylation of zeta. The effect induced by the GTP analogues required TCR occupancy and was independent of protein kinase C. Taken together these observations implicate a GTP-binding protein in the modulation of TCR-induced tyrosine phosphorylation.