Kinase Pathways in Chemoattractant-Induced Degranulation of Neutrophils: The Role of p38 Mitogen-Activated Protein Kinase Activated by Src Family Kinases1 Attila Mo´csai,2* Zolta´n Jakus,* Tibor Va´ntus,† Giorgio Berton,‡ Clifford A. Lowell,§ and Erzse´bet Ligeti* The aim of the present study was to investigate the role of tyrosine phosphorylation pathways in fMLP-induced exocytosis of the different secretory compartments (primary and secondary granules, as well as secretory vesicles) of neutrophils. Genistein, a broad specificity tyrosine kinase inhibitor, blocked the exocytosis of primary and secondary granules, but had only a marginal effect on the release of secretory vesicles. Genistein also inhibited the phosphorylation of extracellular signal-regulated kinase (ERK) and p38 mitogen-activated protein kinases (MAPK), raising the possibility that inhibition of ERK and/or p38 MAPK might be re- sponsible for the effect of the drug on the degranulation response. Indeed, SB203580, an inhibitor of p38 MAPK, decreased the release of primary and secondary granules, but not that of secretory vesicles. However, blocking the ERK pathway with PD98059 had no effect on any of the exocytic responses tested. PP1, an inhibitor of Src family kinases, also attenuated the release of primary and secondary granules, and neutrophils from mice deficient in the Src family kinases Hck, Fgr, and Lyn were also defective in .secondary granule release. Furthermore, activation of p38 MAPK was blocked by both PP1 and the hck؊/؊fgr؊/؊lyn؊/؊ mutation Taken together, our data indicate that fMLP-induced degranulation of primary and secondary granules of neutrophils is mediated by p38 MAPK activated via Src family tyrosine kinases. Although piceatannol, a reportedly selective inhibitor of Syk, also prevented degranulation and activation of p38 MAPK, no fMLP-induced phosphorylation of Syk could be observed, raising doubts about the specificity of the inhibitor. The Journal of Immunology, 2000, 164: 4321–4331. timulation of the chemoattractant receptors of neutrophils (4–6), adaptor proteins (3), and components of the small G protein evokes multiple effector responses, including chemotaxis, su- network (7). Despite clear evidence of phosphorylation (and acti- S peroxide production, and degranulation. The latter response vation) of these proteins, little is known about their role in trig- consists of the release of the different intracellular compartments (sev- gering the fMLP-induced effector responses. eral types of granules as well as the secretory vesicle compartment) Using broad specificity tyrosine kinase inhibitors, we and others (1) to the extracellular space or into the phagosome. suggested that tyrosine kinases might play a role in the fMLP- The receptor for the bacterial tripeptide fMLP, the most widely induced release of primary and secondary granules (Ref. 8 and studied chemoattractant of neutrophils, activates a pertussis toxin- references therein). In the last few years great progress has been sensitive, heterotrimeric G protein belonging to the Gi/o family. made in understanding the roles of the different tyrosine kinases in One of the most characteristic features of fMLP signal transduc- the regulation of neutrophil functions (9). Activation of the Src- tion is the rapid induction of tyrosine phosphorylation of several related kinase Lyn in response to fMLP stimulation has been re- intracellular proteins, including tyrosine kinases (2, 3), different ported by several authors (2, 3, 9, 10). Fgr, another member of the members of the mitogen-activated protein kinase (MAPK)3 family Src kinase family, has been shown to be associated with the sec- ondary granules and to translocate to the plasma membrane upon *Department of Physiology, Semmelweis University of Medicine, Budapest, Hun- fMLP stimulation of the cells (11). Similarly, Hck, the third Src gary; †Joint Research Organization of the Hungarian Academy of Sciences and De- partment of Medical Chemistry, Molecular Biology, and Pathobiochemistry, Sem- family member present in neutrophils, is associated with primary melweis University of Medicine, Budapest, Hungary; ‡Department of Pathology, granules and translocates to the phagosome during phagocytosis of § Section of General Pathology, University of Verona, Verona, Italy; and Department serum-opsonized zymosan (12). Our pharmacological and gene of Laboratory Medicine, University of California, San Francisco, CA 94143 knockout experiments performed on murine neutrophils showed Received for publication August 11, 1999. Accepted for publication February 11, 2000. that the fMLP-induced exocytosis of secondary granules is medi- The costs of publication of this article were defrayed in part by the payment of page ated by Src-related tyrosine kinases (13). However, these reports charges. This article must therefore be hereby marked advertisement in accordance did not provide any clue to identification of possible downstream with 18 U.S.C. Section 1734 solely to indicate this fact. targets of tyrosine kinases. In addition, they did not address in 1 This work was supported by the Hungarian National Scientific Research Fund (OTKA T25078) and the Hungarian Ministry of Education (FKFP 0041/1997; to detail the distinctive role of tyrosine phosphorylation in regulating E.L.), by National Institutes of Health Grants DK50267 and HL54476 (to C.A.L.), exocytosis of the different granule populations and of secretory and the Fondazione Cariverona (Progetto Sanita`; to G.B.). vesicles. 2 Address correspondence and reprint requests to Dr. Attila Mo´csai, Department of In the present report we analyzed the fMLP-induced exocytosis Physiology, Semmelweis University of Medicine, P.O. Box 259, 1444 Budapest, Hungary. E-mail address: [email protected] of primary and secondary granules as well as secretory vesicles in 3 Abbreviations used in this paper: MAPK or MAP kinase, mitogen-activated protein response to fMLP using inhibitors with reported selectivity for kinase; CB, cytochalasin B; DFP, di-isopropyl fluorophosphate; ERK, extracellular sig- different tyrosine kinases or tyrosine-phosphorylated proteins nal-regulated kinase; Fak, focal adhesion kinase; ␤-GU, ␤-glucuronidase; HSA, human serum albumin; Hsp27, heat shock protein 27; Lfr, lactoferrin; MAPKAPK2, MAPK- and studying mice deficient in Src family kinases expressed in activated protein kinase 2; MBP, myelin basic protein; MSA, mouse serum albumin. neutrophils. Copyright © 2000 by The American Association of Immunologists 0022-1767/00/$02.00 4322 p38 MAPK AND Src FAMILY KINASES IN NEUTROPHIL DEGRANULATION This study followed two major directions of investigation. First, ylenediamine. The OD of the samples was read using a Labsystems (Hel- to obtain information on the molecular identity of the kinases par- sinki, Finland) iEMS microplate reader. For determination of adhesion-dependence of the exocytic response, the ticipating in the fMLP-induced degranulation response, we fo- ϩ effect of withdrawal of Mg2 on the release of secondary granules was cused on 1) the ERK and p38 MAPK proteins, members of the tested (13, 25). Cells were resuspended in HBSS-HB without CaCl2, MAP kinase family shown to become rapidly phosphorylated on MgCl2, and BSA, supplemented with 5 mM Na-EDTA. After incubation adjacent Tyr and Thr residues and thereby activated upon che- for 10 min at room temperature, cells were pelleted and resuspended in moattractant stimulation (4–6, 14, 15); 2) the Src family of ty- ice-cold HBSS-HB with or without 1 mM MgCl2. The fMLP-stimulated release of Lfr was then determined as described above. Simultaneously, rosine kinases, the members of which have been implicated in cells were plated on tissue culture plates precoated with human fibrinogen several aspects of the degranulation process (see above); and 3) the (13) and stimulated with 20 ng/ml human TNF-␣ (PeproTech, Rocky Hill, Syk tyrosine kinase, the function of which is closely related to that NJ) for 30 min at 37°C. Spreading of the cells was assessed visually under of Src family kinases in several cellular systems (16–19). Our a phase contrast microscope. second objective was to compare the involvement of the above Immunoblotting of human cell lysates and kinase assays pathways in the fMLP-induced release of three exocytic compart- ϫ 7 ments of human neutrophils: primary granules, secondary gran- Cells at 2.5 10 /ml were pretreated exactly as described for degranula- tion experiments, followed by a 2-min stimulation with 100 nM fMLP at ules, and secretory vesicles. Previous reports indicated differences 37°C. Samples were then immersed in liquid N2 to stop the reaction and in calcium sensitivity (20) and inhibition by wortmannin (21) of were lysed by adding a Triton-based lysis buffer with protease and phos- fMLP-induced exocytosis among these three granule populations. phatase inhibitors (8) from a 5ϫ concentrated stock solution followed by All these differences warranted a comparative study on the role of a 10-min incubation on ice and spinning down at 16,000 ϫ g for 1 min at tyrosine kinases in the release of the three exocytic compartments. 4°C. The Triton-soluble supernatant was used in further determinations. For immunoblot experiments, the Triton-soluble lysate was mixed with In the present paper we provide evidence that p38 MAPK, ac- sample buffer, boiled for 10 min, run on 12% SDS-PAGE, and blotted onto tivated by Src family kinases has a central role in eliciting the nitrocellulose sheets. Blots were processed using phospho-specific rabbit