Adhesion-Dependent Degranulation of Neutrophils Requires the Src Family Kinases Fgr and Hck This information is current as Attila Mócsai, Erzsébet Ligeti, Clifford A. Lowell and of September 25, 2021. Giorgio Berton J Immunol 1999; 162:1120-1126; ; http://www.jimmunol.org/content/162/2/1120 Downloaded from References This article cites 36 articles, 23 of which you can access for free at: http://www.jimmunol.org/content/162/2/1120.full#ref-list-1 Why The JI? Submit online. http://www.jimmunol.org/ • Rapid Reviews! 30 days* from submission to initial decision • No Triage! Every submission reviewed by practicing scientists • Fast Publication! 4 weeks from acceptance to publication *average by guest on September 25, 2021 Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 1999 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Adhesion-Dependent Degranulation of Neutrophils Requires the Src Family Kinases Fgr and Hck1 Attila Mo´csai,2*† Erzse´bet Ligeti,† Clifford A. Lowell,‡ and Giorgio Berton3* Polymorphonuclear neutrophils (PMN) adherent to integrin ligands respond to inflammatory mediators by reorganizing their cytoskeleton and releasing reactive oxygen intermediates. As Src family tyrosine kinases are implicated in these responses, we investigated their possible role in regulating degranulation. Human PMN incubated on fibrinogen released lactoferrin in response to TNF-a and this response was inhibited by PP1, a Src family tyrosine kinase inhibitor. This drug had no effect on lactoferrin secretion induced by PMA, an adhesion-independent agonist of PMN degranulation. However, PP1 blocked secretion in PMN plated on plain tissue culture plastic, a surface inducing PMN spreading in the absence of any stimulus. Double knockout hck2/2fgr2/2 PMN adherent to collagen or fibrinogen failed to release lactoferrin in response to TNF-a but responded to PMA 2/2 2/2 as wild-type PMN. Degranulation induced by spreading over tissue culture plastic was also defective in hck fgr PMN. Downloaded from Defective adhesion-dependent degranulation required the absence of both kinases, because single knockout fgr2/2 or hck2/2 PMN responded as wild-type cells. Analysis of lactoferrin secretion in hck2/2fgr2/2 or PP1-treated, suspended PMN showed that Src kinases are not implicated in degranulation dependent on activation of protein kinase C or increase in intracellular free Ca21 but may play a role in the response to FMLP of cytochalasin B-treated PMN. These findings identify a role for Src family kinases in a signaling pathway leading to granule-plasma membrane fusion and suggest that Fgr and Hck would be targets for pharmaco- logical control of adhesion-dependent degranulation in the inflammatory site. The Journal of Immunology, 1999, 162: 1120–1126. http://www.jimmunol.org/ olymorphonuclear neutrophils (PMN)4 adherent to extra- hesion and spreading of PMN are tightly coupled to an increase in cellular matrix proteins or endothelial counterreceptors re- tyrosine phosphorylation of several proteins, and tyrosine kinase P spond to inflammatory mediators, such as cytokines and inhibitors block adhesion-dependent generation of ROI by PMN chemoattractants, releasing reactive oxygen intermediates (ROI) (7, 8). The Src family tyrosine kinases Fgr, Hck, and Lyn, as well and granule constituents (1–5). These secretory responses are as p72syk, have been recently implicated in signaling from adhe- tightly coupled with a rearrangement of the cytoskeleton that dic- sion receptors (16–19). tates spreading of the cell over the adhesive surface (5–8). Dif- Signals involved in triggering degranulation by adherent PMN ferent adhesion receptors have been implicated in induction of are poorly understood. The evidence that protein tyrosine phos- by guest on September 25, 2021 b b PMN responses. For example, both 2 and 3 integrins have been phorylation (7, 16) and the Src family kinases Fgr and Hck (18) demonstrated to regulate PMN responses (2, 3, 6, 9–11). The re- play a critical role in ROI generation prompted us to investigate lease of ROI and degranulation in the context of PMN-endothelial whether adhesion-dependent degranulation is also regulated by the cell interactions plays a central role in the development of inflam- same signaling pathways. In this report, we show that adhesion- matory reactions and certainly contributes to tissue damage asso- dependent release of lactoferrin is blocked by a newly described ciated with inflammation (12, 13). tyrosine kinase inhibitor the effect of which has been reported to be In the last few years, a great progress has been made in the selective for Src family tyrosine kinases (20). Investigations with understanding of signaling from adhesion receptors (14, 15). Ad- PMN isolated from mice with the double deficiency of Fgr and Hck allowed us to demonstrate that these kinases play an essential role in signaling for adhesion-dependent degranulation. These re- † *Institute of General Pathology, University of Verona, Verona, Italy; Department of sults provide the first direct evidence that Src family kinases func- Physiology and Laboratory of Cellular and Molecular Physiology, Semmelweis Uni- versity of Medicine, Budapest, Hungary; and ‡Department of Laboratory Medicine, tion in a signaling pathway leading to granule-plasma membrane University of California, San Francisco, CA 94143 fusion. Received for publication March 26, 1998. Accepted for publication October 6, 1998. The costs of publication of this article were defrayed in part by the payment of page Materials and Methods charges. This article must therefore be hereby marked advertisement in accordance PMN isolation with 18 U.S.C. Section 1734 solely to indicate this fact. 1 This work was supported by grants from the Italian Association for Cancer Research Human PMN were isolated from buffy coats of healthy volunteers as pre- and Minister of University and Scientific Research of Italy/University of Verona viously described (9, 16). Wild-type (C57BL/6), p59/61hck,orp58c-fgr- (Research Program: Inflammation: Biology and Clinic) to G.B.; National Institutes of deficient single knockout mice and double knockout mice deficient in both Health Grants DK 50267 and HL54476 to C.A.L.; and grants from the Hungarian p59/61hck and p58c-fgr were described previously (21). Bone marrow of National Research Fund (OTKA) and the Swiss National Science Foundation 6–12-wk-old mice of both sexes were used in this study. Murine PMN (7UNPJ048717) to E.L. were isolated essentially as described (18) except that a simplified three- 2 Recipient of a short term fellowship from the European Molecular Biology layer gradient was utilized to separate neutrophils from other bone marrow Organization. cells (i.e., bone marrow cells suspended in Ca21-/Mg21-free HBSS sup- 3 Address correspondence and reprint requests to Dr. Giorgio Berton, Istituto di Pa- plemented with 0.1% BSA were layered on the top of a 62/81% two-layer tologia Generale, Universita`di Verona, Strada Le Grazie, 37134 Verona, Italy. E-mail Percoll (Pharmacia, Uppsala, Sweden) gradient, and after centrifugation, address: [email protected] PMN were harvested from the 62/81% interface). At the end of the prep- 4 Abbreviations used in this paper: PMN, polymorphonuclear neutrophils; ROI, aration, PMN were suspended in ice-cold HBSS containing 0.5 mM CaCl2, reactive oxygen intermediates; TNF, TNF-a; PKC, protein kinase C; CB, 1 mM MgCl2,and5mMD-glucose (HBSS) and kept in ice until use. For 1 cytochalasin B. experiments in which the Mg2 dependence of the PMN response was Copyright © 1999 by The American Association of Immunologists 0022-1767/99/$02.00 The Journal of Immunology 1121 investigated, cells were isolated as above described but resuspended in 21 21 Results Ca /Mg -free HBSS supplemented with 5 mM EDTA and, after 10 min TNF-stimulated lactoferrin release by adherent human and of incubation, washed and suspended in HBSS either with or without 1 mM Mg21 (17). murine PMN is blocked by the tyrosine kinase inhibitor PP1 Human adherent PMN can be stimulated to mobilize lactoferrin- Coating of tissue culture plastic plates with proteins containing specific granules in response to granulocyte-macro- Flat bottom polystyrene tissue culture plates with 96 wells (Greiner, Frick- phage-CSF, FMLP, or the Ca21 ionophore A23187 (5). As shown enhausen, Germany) were left untreated or were covered with rat collagen, in Fig. 1A, TNF is also an effective agonist of lactoferrin secretion human fibrinogen, or FCS as described (18). After coating, the plates were washed once with PBS and once with HBSS. in adherent human PMN. As previously reported with other stimuli (5), TNF-induced degranulation by adherent PMN is delayed in its Cell stimulation onset and prolonged up to 60 min of incubation (Fig. 1A). In ac- Human and murine PMN were washed at 4°C and resuspended in ice-cold cord with previous studies (4, 23–25), we could not detect release HBSS at 1 3 106 or 2 3 106 cells/ml, respectively. For adhesion assays, of the primary granule marker b-glucuronidase in response to TNF cell suspensions (100 ml/well), added or not with 10 mM PP1 (Calbiochem- (data not shown). Additionally, we also found that the response to Novabiochem Int., La Jolla, CA) were dispensed in protein-coated plates TNF depended on adhesion (25, 26), since suspended PMN failed and prewarmed for 10 min at 37°C before addition of 20 ng/ml human or murine TNF-a (TNF, Peprotech, London, U.K.), 100 ng/ml PMA (Sigma, to release lactoferrin following TNF stimulation while such cells St.