Role of the tyrosine kinase pyk2 in the integrin-dependent activation of human neutrophils by TNF (original) (raw)

Evidence that TNF-induced respiratory burst of adherent PMN is mediated by integrin αLβ2

Journal of Leukocyte Biology, 2002

Polymorphonuclear leukocytes (PMN) respond to tumor necrosis factor (TNF) with a respiratory burst (RB) only after adherence to surfaces coated with extracellular matrix proteins such as fibronectin and fibrinogen (permissive substrates) but not with others such as laminin or collagen (nonpermissive substrates). As PMN adherence to both types of surfaces is dependent on ␤ 2 integrins, we investigated the molecular basis of the different metabolic response to TNF. In particular, we evaluated the relative role of each ␤ 2 integrin (␣ L ␤ 2 , ␣ M ␤ 2 , and ␣ X ␤ 2) in adherence and O 2 ؊ production of PMN residing on fibronectinand laminin-coated surfaces, which were considered as models of permissive and nonpermissive surfaces, respectively. By using ␣ chain-specific monoclonal antibodies (mAb), we show that ␣ M ␤ 2 and ␣ X ␤ 2 mediate adherence to fibronectin and laminin; ␣ L ␤ 2 is not involved in adherence to laminin and has only a minimal contribution in adherence to fibronectin. Furthermore, production of O 2 ؊ in response to TNF was induced by immobilized anti-␣ L ␤ 2 but not anti-␣ M ␤ 2 or anti-␣ X ␤ 2 mAb. A strong correlation was also found between expression of ␣ L ␤ 2 and TNF-induced RB on fibronectin. Lastly, PMN responded to TNF on laminin with a RB after the inclusion of ␣ L-specific mAb in the laminin coat. Thus, we conclude that TNFinduced RB by PMN residing on fibronectin is mediated by ␣ L ␤ 2 and that ␣ M ␤ 2 and ␣ X ␤ 2 are likely to play an ancillary role to the signaling activity of ␣ L ␤ 2 by facilitating its recruitment to sites of adherence. The nonpermissiveness of laminin appears to be a consequence of its inability to act as a ligand for ␣ L ␤ 2 .

Evidence that TNF-induced respiratory burst of adherent PMN is mediated by integrin L2

Polymorphonuclear leukocytes (PMN) respond to tumor necrosis factor (TNF) with a respiratory burst (RB) only after adherence to surfaces coated with extracellular matrix proteins such as fibronectin and fibrinogen (permissive substrates) but not with others such as laminin or collagen (nonpermissive substrates). As PMN adherence to both types of surfaces is dependent on ␤ 2 integrins, we investigated the molecular basis of the different metabolic response to TNF. In particular, we evaluated the relative role of each ␤ 2 integrin (␣ L ␤ 2 , ␣ M ␤ 2 , and ␣ X ␤ 2 ) in adherence and O 2 ؊ production of PMN residing on fibronectinand laminin-coated surfaces, which were considered as models of permissive and nonpermissive surfaces, respectively. By using ␣ chain-specific monoclonal antibodies (mAb), we show that ␣ M ␤ 2 and ␣ X ␤ 2 mediate adherence to fibronectin and laminin; ␣ L ␤ 2 is not involved in adherence to laminin and has only a minimal contribution in adherence to fibronectin. Furthermore, production of O 2 ؊ in response to TNF was induced by immobilized anti-␣ L ␤ 2 but not anti-␣ M ␤ 2 or anti-␣ X ␤ 2 mAb. A strong correlation was also found between expression of ␣ L ␤ 2 and TNF-induced RB on fibronectin. Lastly, PMN responded to TNF on laminin with a RB after the inclusion of ␣ L -specific mAb in the laminin coat. Thus, we conclude that TNFinduced RB by PMN residing on fibronectin is mediated by ␣ L ␤ 2 and that ␣ M ␤ 2 and ␣ X ␤ 2 are likely to play an ancillary role to the signaling activity of ␣ L ␤ 2 by facilitating its recruitment to sites of adherence. The nonpermissiveness of laminin appears to be a consequence of its inability to act as a ligand for ␣ L ␤ 2 . J. Leukoc. Biol. 72: 718 -726; 2002.

Evidence that TNF-induced respiratory burst of adherent PMN is mediated by integrin alpha(L)beta(2)

Journal of leukocyte biology, 2002

Mechanisms of stimulation of the respiratory burst by TNF in nonadherent neutrophils: its independence of lipidic transmembrane signaling and dependence on protein tyrosine phosphorylation and cytoskeleton

The Journal of Immunology

This study concerns the controversial problem of whether the TNF-alpha (TNF) induces a respiratory burst in human neutrophils in suspension. The results have shown that in these cells TNF induces a classical respiratory burst. In fact, the production of oxygen free radicals 1) is linked to the translocation of NADPH oxidase components from cytosol to the plasma membrane, 2) does not take place in neutrophils from a patient lacking the cytochrome b558, and 3) does not involve other sources such as mitochondrial respiratory chain or xanthine oxidase. Signal transduction studies have demonstrated that this respiratory burst 1) is not accompanied by calcium transients, stimulation of phosphoinositide turnover, and phospholipase D activity (moreover, this burst is associated with the stimulation of the activity of phospholipase A2, but not of sphingomyelinase); 2) is strictly dependent on activation of tyrosine kinases, which is functional to the translocation to the plasma membrane of t...

Evidence that tumor necrosis factor alpha (TNF)-induced activation of neutrophil respiratory burst on biologic surfaces is mediated by the p55 TNF receptor

Blood, 1994

Polymorphonuclear leukocytes (PMN) residing on biologic surfaces respond with a vigorous respiratory burst when exposed to tumor necrosis factor alpha (TNF). PMN possess both the p55 and the p75 TNF receptors, but their role in the elicitation of the respiratory burst is not known. We addressed this problem by studying the effect of monoclonal antibodies (MoAbs) directed against the p55 TNF receptor (MoAb H398 and MoAb htr-9) and the p75 TNF receptor (MoAb utr-1) on TNF-induced production of O2- by PMN residing on fibronectin-coated surfaces. Neither the anti-p55 nor the anti-p75 MoAbs affected TNF-induced O2- production despite their known ability to competitively inhibit TNF binding to the corresponding receptor. Experiments with the antibodies alone showed that the anti-p55 MoAbs directly triggered PMN O2- production, whereas no response was elicited by the anti-p75 MoAb. PMN unresponsiveness to the anti-p75 MoAb could not be ascribed to low expression of p75 receptor, because bi...

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 TO APOPTOSIS

Journal of Biological Chemistry, 2001

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-␣ (TNF␣). Although TNF␣ has been implicated in induction of pro-inflammatory responses, it may also inhibit the intensity of neutrophilic inflammation by promoting apoptosis. Since TNF␣ 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 TNF␣ on the JNK pathway in adherent human neutrophils and the potential involvement of this pathway in neutrophil apoptosis. Stimulation with TNF␣ was found to result in ␤ 2 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 ␤ 2 integrins, the tyrosine kinase inhibitors, genistein, and tyrphostin A9, a Pyk2-specific inhibitor, and piceatannol, a Syk-specific inhibitor. Exposure of adherent neutrophils to TNF␣ led to the rapid onset of apoptosis that was demonstrated by augmented annexin V binding and caspase-3 cleavage. TNF␣؊induced increases in annexin V binding to neutrophils were attenuated by blocking antibodies to ␤ 2 integrins, and the caspase-3 cleavage was attenuated by tyrphostin A9. Hence, exposure of adherent neutrophils to TNF␣ 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.

Syk Activation Is Required for Spreading and H2O2 Release in Adherent Human Neutrophils

The Journal of Immunology

Chemoattractant-stimulated polymorphonuclear leukocytes (PMNs) that are adherent to extracellular matrix proteins exhibit a massive, sustained respiratory burst that requires cell spreading. However, the signaling pathways culminating in PMN spreading are not well characterized. Studies showing that protein tyrosine phosphorylation increases with PMN spreading suggest that phosphorylation is critical for this process. In the present study, we observed increased tyrosine phosphorylation of both focal adhesion kinase and Syk in FMLP-activated PMNs that had been plated onto fibrinogen; an increase in Syk activity, but not focal adhesion kinase activity, was apparent. The time course of Syk phosphorylation correlated with the initiation of cell spreading and H2O2 release. Pretreatment of PMNs with piceatannol, a Syk-selective inhibitor, blocked Syk activity, cell spreading, and H2O2 release, indicating that Syk activity was required for the activation of adherent PMNs. Paxillin is a cyt...

Critical Role of the Carboxyl Terminus of Proline-rich Tyrosine Kinase (Pyk2) in the Activation of Human Neutrophils by Tumor Necrosis Factor: Separation of Signals for the Respiratory Burst and Degranulation

Journal of Experimental Medicine, 2002

Transduction of Tat-tagged fusion proteins confirmed a hypothesis based on pharmacologic inhibitors (Fuortes, M., M. Melchior, H. Han, G.J. Lyon, and C. Nathan. 1999. J. Clin. Invest. 104:327–335) that proline-rich tyrosine kinase (Pyk2) plays a critical role in the activation of adherent human neutrophils, and allowed an analysis of individual Pyk2 domains not possible with chemical inhibitors. Acting as a dominant negative, the COOH terminus of Pyk2 fused to a Tat peptide (Tat-CT), but not other regions of Pyk2, specifically inhibited the respiratory burst of cells responding to tumor necrosis factor (TNF), Salmonella, or Listeria, while sparing responses induced by phorbol ester. Tat-CT suppressed TNF-triggered cell spreading and the phosphorylation of endogenous Pyk2 and the associated tyrosine kinase Syk without blocking the ability of neutrophils to degranulate and kill bacteria. Thus, separate signals control the respiratory burst and degranulation, and a normal rate of killi...

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

Journal of Cell Biology, 1994

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 adhesionassociated kinase, p125 FAz. In the present work, tumor necrosis factor-or (TNF) stimulated tyrosine phosphorylation of paxillin in human neutrophils. Cell adhesion and participation of the/~2 integrin CD18 were necessary, but not sufficient, for the response. Adherent neutrophils also tyrosine phosphorylated paxillin in response to phorbol ester, formylmethionylleucyl-phenylalanine and opsonized bacteria. In

Role of the tyrosine kinase pyk2 in the integrin-dependent activation of human neutrophils by TNF (original) (raw)

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