Lipopolysaccharide Induces Actin Reorganization and of Pyk2 and Paxillin in Monocytes and Macrophages This information is current as of September 26, 2021. Lynn M. Williams and Anne J. Ridley J Immunol 2000; 164:2028-2036; ; doi: 10.4049/jimmunol.164.4.2028 http://www.jimmunol.org/content/164/4/2028 Downloaded from

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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 © 2000 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Lipopolysaccharide Induces Actin Reorganization and Tyrosine Phosphorylation of Pyk2 and Paxillin in Monocytes and Macrophages1

Lynn M. Williams* and Anne J. Ridley2*†

The bacterial endotoxin LPS is a potent stimulator of monocyte and macrophage activation and induces adhesion of monocytes. Morphological changes in response to LPS have not been characterized in detail, however, nor have the signaling pathways mediating LPS-induced adhesion been elucidated. We have found that LPS rapidly induced adhesion and spreading of peripheral blood monocytes, and that this was inhibited by the Src family kinase inhibitor PP1 and the phosphatidylinositide 3-kinase inhibitor LY294002. LPS also stimulated actin reorganization, leading to the formation of filopodia, lamellipodia, and membrane ruffles in Bac1 mouse macrophages. -rich 2 (Pyk2), a tyrosine kinase related to kinase, and Downloaded from paxillin, a cytoskeletal that interacts with Pyk2, were both tyrosine phosphorylated in response to LPS in monocytes and macrophages. Both tyrosine phosphorylation events were inhibited by PP1 and LY294002. Adhesion also stimulated tyrosine phosphorylation of Pyk2 and paxillin in monocytes, and this was further enhanced by LPS. Finally, Pyk2 and paxillin colocalized within membrane ruffles in LPS-stimulated cells. These results indicate that LPS stimulation of monocytes and macrophages results in rapid morphological changes and suggest that Pyk2 and/or paxillin play a role in this response. The Journal of

Immunology, 2000, 164: 2028–2036. http://www.jimmunol.org/

onocyte adhesion to endothelial cells is an essential the transmembrane protein Toll-like receptor 2 has also been im- prerequisite for transmigration leading to their recruit- plicated in LPS signaling in macrophages (10). A number of sig- M ment into tissue and subsequent differentiation into naling pathways can be activated by LPS, including phospho- macrophages (1, 2). This adhesion process can be induced by the lipases A (11) and C (12), protein kinase C isoforms (13, 14), and endotoxin LPS (3). LPS is an outer membrane component of the Src family tyrosine kinases Hck, Lyn, and Fgr (15, 16). In Gram-negative bacteria, and is a potent activator of monocytes and monocytes/macrophages, LPS induces the tyrosine phosphoryla- macrophages (4). The subsequent production and release of proin- tion of many , including Vav (17) and the mitogen-acti- 3 flammatory mediators including IL-1␣, TNF, IL-6, and arachi- vated protein kinase (MAPK) family members p42/p44 MAPK by guest on September 26, 2021 donic acid metabolites recruit and activate other leukocytes to help (18), stress-activated protein kinase 1/c-Jun N-terminal kinase fight infection (5). High levels of LPS are a major cause of Gram- (19), and p38/stress-activated protein kinase 2 (20). LPS also stim- negative septic shock, in which LPS induces numerous changes ulates phosphatidylinositide 3-kinase (PI 3-K) activity in mono- including endothelial injury and activation, leukocyte adhesion to cytes, resulting in a transient increase in the level of phosphati- endothelial cells, and generation of free radicals. Lower levels of dylinositol 3,4,5-trisphosphate (21). This process was reported to LPS produced during chronic infections may contribute to the de- involve the association of PI 3-K with the Src kinase Lyn and to velopment of other diseases: for example, the pathological changes act upstream of protein kinase C-␨ activation (22). involved in the initiation and development of atherosclerosis re- Of the signaling pathways activated by LPS, the p42/p44 MAPK semble those induced by endotoxin (6), and accumulating data and the p38 pathways have both been shown to be directly in- suggest that persistent Gram-negative periodontal disease can lead volved in the production of the cytokines TNF-␣ and IL-1 (20, 23). to the development of cardiovascular disease (7). The functions of other signaling pathways induced by LPS have The major cell surface receptor for LPS on macrophages is not been established, and in particular the signaling pathways in- CD14, a 55-kDa glycosylphosphatidylinositol-linked membrane volved in LPS-induced adhesion have yet to be characterized. In protein (8), although at higher concentrations LPS can bind to other cell types, focal adhesion kinase (FAK), initially identified as ␤ other receptors such as the 2 CD11b/CD18 (9). Recently, a unique cytoplasmic tyrosine kinase involved in focal adhesions, is thought to play a key role in integrin-mediated cell adhesion (24). Proline-rich tyrosine kinase 2 (Pyk2), also known as related *Ludwig Institute for Cancer Research, Royal Free and University College Medical adhesion focal tyrosine kinase or cell adhesion kinase ␤ (25–27), School, London, United Kingdom; and †Department of Biochemistry and Molecular is a cytoplasmic tyrosine kinase related to FAK. A Pyk2 splice Biology, University College London, London, United Kingdom variant has been identified, termed Pyk2-H or monocyte calcium- Received for publication September 15, 1999. Accepted for publication December dependent tyrosine kinase (28, 29). Pyk2 shows considerable se- 8, 1999. quence homology and structural similarity to FAK, including con- The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance sensus motifs in the catalytic domain. Pyk2 expression is more with 18 U.S.C. Section 1734 solely to indicate this fact. 1 This work was supported by a Human Frontiers Scientific Program Grant RG304/96. 3 Abbreviations used in this paper: MAPK, mitogen-activated protein kinase; FAK, 2 Address correspondence and reprint requests to Dr. Anne J. Ridley, Ludwig Institute focal adhesion kinase; HIFCS, heat-inactivated FCS; PI 3-K, phosphatidylinositide for Cancer Research, 91 Riding House Street, London W1P 8BT, U.K. E-mail ad- 3-kinase; PP1, 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine; dress: [email protected] Pyk2, proline-rich tyrosine kinase 2; TRITC, tetramethylrhodamine isothiocyanate.

Copyright © 2000 by The American Association of Immunologists 0022-1767/00/$02.00 The Journal of Immunology 2029 limited than FAK, and has been detected in epithelial cells, neu- Immunofluorescence and confocal microscopy ronal cells, T and B cells, megakaryocytes, platelets, mast cells, For immunofluorescence studies, cells were fixed with 4% formaldehyde in and monocytes and macrophages (26, 30, 31), whereas Pyk2-H is PBS for 20 min at room temperature. Cells were permeabilized with 0.2% only expressed in hemopoietic cells (28, 29). The stimulation of Triton X-100 in PBS for 5 min. For localization of F-actin filaments, cells many types of cell surface receptors results in the tyrosine phos- were incubated with 0.1 ␮g/ml TRITC-labeled phalloidin (Sigma) for 45 phorylation of Pyk2, including , cytokines, and immune min. For localization of paxillin and Pyk2, cells were blocked with 0.2% heat-inactivated goat serum (Sigma) for 30 min, washed, and then incu- receptors (30, 32–34); and diverse stimuli such as membrane de- bated with a 1/12.5 dilution of mouse anti-paxillin Abs and/or a 1/50 di- polarization, stress stimuli, angiotensin, and PMA have all been lution of rabbit anti-Pyk2 (600) Abs, washed three times with PBS, and shown to induce Pyk2 tyrosine phosphorylation, resulting in the then incubated with a 1/100 dilution of TRITC-conjugated goat anti-mouse activation of its kinase activity (26, 31). IgG and/or FITC-conjugated goat anti-rabbit IgG. Images of cells were obtained using a Zeiss LSM 510 confocal laser-scanning microscope (Wel- Many proteins localize to focal adhesion sites, in which trans- wyn, Garden City, U.K.), using the accompanying LSM 510 software, and membrane integrins are clustered and link the were processed in Adobe photoshop 4.0. with the cytoskeleton (35). Paxillin, a -binding protein, colocalizes with FAK and integrins in fibroblast focal adhesions Determination of cell spreading (36). Paxillin also binds to Pyk2 (31, 37), and PMA-dependent Monocytes (5 ϫ 105/ml) were allowed to adhere to glass coverslips for 30 tyrosine phosphorylation of paxillin in megakaryocytes has been min before stimulation with LPS (100 ng/ml). Upon termination of the shown to be inhibited using a kinase-dead mutant form of Pyk2, experiment, cells were fixed and stained with TRITC-phalloidin and im- suggesting a functional link between Pyk2 and paxillin (38). As aged using confocal microscopy, as described above. Cells with a clearly visible cortical F-actin ring and a diameter equal or less than 10 ␮m were yet, however, there has been no evidence that Pyk2 colocalizes Downloaded from defined as unspread, whereas cells without a clear cortical ring and a di- with paxillin. ameter greater than 10 ␮m were defined as spread. LPS is known to induce monocyte adhesion (3), but its effects on cytoskeletal organization have not been previously examined in Western blotting and immunoprecipitations detail. In this study, we have analyzed changes in the actin cy- Following stimulation, cells were lysed for 15 min in lysis buffer (0.5% toskeleton induced by LPS in monocytes and macrophages, and Nonidet P-40, 30 mM sodium pyrophosphate, 50 mM Tris-HCl (pH 7.6), correlated them with increased tyrosine phosphorylation of Pyk2 150 mM NaCl, 0.1 mM EDTA, 50 mM NaF, 1 mM Na3VO4, 1 mM PMSF, http://www.jimmunol.org/ and paxillin. 10 ␮g/ml leupeptin, 1 ␮g/ml pepstatin, and 1 ␮g/ml aprotinin). Lysates were clarified by centrifugation at 14,000 ϫ g for 10 min. The protein Materials and Methods concentration of the cell extracts was determined using the Bio-Rad protein assay kit (Bio-Rad, Hercules, CA). The clarified lysates were precleared Reagents with either anti-mouse IgG (for subsequent paxillin immunoprecipitations) LPS (Salmonella typhi and Escherichia coli serotype 026:B6) was obtained or rabbit serum (for Pyk2 immunoprecipitations) with protein A-Sepharose from Sigma-Aldrich (Poole, U.K.). It was reconstituted in pyrogen-free (Pharmacia, Uppsala, Sweden). Immunoprecipitations were performed us- PBS (sonicated for 5 min) and stored at Ϫ20°C in glass vials. Immediately ing either anti-paxillin or anti-Pyk2 Abs and protein A-Sepharose. The before adding to cells, LPS suspensions were sonicated for an additional 2 immunoprecipitates were then washed in lysis buffer before addition of 2ϫ ϫ gel sample buffer, heated to 95°C for 3 min, and subsequently analyzed by

min before diluting to 10 final concentration in RPMI (Life Technolo- by guest on September 26, 2021 gies, Paisley, U.K.) containing 1% heat-inactivated FCS (HIFCS) (Sigma, SDS-PAGE, followed by electrophoretic transfer to polyvinylidene diflu- Dorset, U.K.) (monocytes and Bac1 cells) or 0.5% HIFCS/RPMI (J774 oride membranes (Immobilon-P; Millipore, Bedford, MA). cells). LPS was protein free, as determined by Bio-Rad protein assay (Bio- Membranes were blocked in 5% BSA and probed with a 1/4000 dilution Rad, Hercules, CA). PP1 and LY294002 were obtained from Calbiochem of the anti-phosphotyrosine Ab PY99. Blots were visualized with HRP- (Nottingham, U.K.). The mouse mAb to paxillin was purchased from conjugated donkey anti-rabbit Ab (1/8000) (Amersham, Little Chalfont, Transduction Laboratories (Lexington, KY); rabbit polyclonal Pyk2 Abs U.K.) and developed using enhanced chemiluminescence (ECL; Amer- (600 and 623) were a gift from Dr. I. Dikic (Ludwig Institute for Cancer sham). Membranes were either stripped in 0.1 mM glycine, pH 2, and Research, Uppsala, Sweden); monoclonal phosphotyrosine Py99 was from reprobed with anti-paxillin Abs (Transduction Laboratories), or alterna- Santa Cruz Biotechnology (Santa Cruz, CA); and FITC-labeled anti-mouse tively stripped with 2% SDS, 25 mM Tris, pH 6.8, and 100 mM 2-ME, and IgG and tetramethylrhodamine isothiocyanate (TRITC)-labeled anti-rabbit subsequently reprobed with anti-Pyk2 (600) Abs. IgG were from Jackson ImmunoResearch (West Grove, PA). Isolation of peripheral blood monocytes and cell culture Results LPS induces morphological changes and actin reorganization in Single donor platelet phoresis residues were purchased from the North London Blood Transfusion Service (Collindale, U.K.). Mononuclear cells human monocytes and murine Bac1 macrophages were isolated by Ficoll-Hypaque centrifugation (specific density, 1.077 LPS induced distinct changes in cell morphology and actin orga- g/ml) preceding monocyte separation in a Beckman JE6 elutriator. Mono- nization in monocytes (Fig. 1, a–f) and Bac1 mouse macrophage cyte purity was assessed by flow cytometry using directly conjugated anti- CD45 and anti-CD14 Abs (Leucogate, Becton Dickinson, San Jose, CA) cells (Fig. 1, g–j). When unstimulated monocytes were allowed to and was routinely greater than 85%. Monocytes were purified in and main- adhere to glass coverslips for 90 min, the majority did not spread tained in RPMI containing 1% HIFCS. All media and sera were routinely significantly (Table I) and maintained a cortical ring of actin fil- tested for endotoxin using the Limulus amoebocyte lysate test (BioWhit- aments (Fig. 1, a). This adhesion was transient, and more than 90% taker, Walkersville, MD) and rejected if the endotoxin concentration ex- ceeded 0.1 U/ml. of cells detached again by 24 h. In the presence of LPS, however, J774 cells were cultured in RPMI containing 10% HIFCS and supple- monocytes displayed a considerably greater degree of spreading mented with 25 U/ml of streptomycin and penicillin. They were cultured in (Fig. 1, b–f; Table I), and this adhesion was stable and maintained RPMI containing 0.5% HIFCS for 48 h before stimulation with LPS. for over 24 h. LPS-induced spreading was morphologically indis- Bac1.2F5 cells were kindly provided by Richard Stanley (Albert Einstein tinguishable whether it was added to monocytes before or after College of Medicine, New York, NY). They were cultured in DMEM supplemented with 10% HIFCS and 20% L cell-conditioned media as a they were allowed to adhere to coverslips (data not shown). In source of CSF-1 (39), and were starved in 1% HIFCS/RPMI (no CSF-1) for addition to this increase in cell spreading, LPS induced cell polar- 24 h before LPS stimulation. ization indicative of cell migration (40), as defined by the asym- In some experiments, monocytes or macrophages were pretreated with metric morphology of LPS-treated cells. Typically, one region of 10 ␮M PP1 or 25 ␮M LY294002 for 15 min before stimulation with LPS. All cultures treated with these inhibitors were assessed for cell viability, as the cell periphery displayed membrane ruffles (indicated by the determined by trypan blue (0.04%) (Sigma) exclusion. In all cases, more arrowheads in Fig. 1, d and f) and lamellipodia, characteristic of than 99% of cells were viable. the leading edge of a motile cell, whereas the opposite edge was 2030 LPS-INDUCED ACTIN REORGANIZATION AND PROTEIN PHOSPHORYLATION

Table I. LPS enhances adhesion and spreading of monocytesa

Unstimulated LPS

Cells per field 61.66 Ϯ 7.2 110 Ϯ 3.5** % Spread cells 18 Ϯ 053Ϯ 10*

a Peripheral blood monocytes (5 ϫ 105) suspended in 1% HIFCS RPMI were allowed to adhere to glass coverslips for 30 min prior to stimulation with LPS (100 ng/ml, 60 min). Cells were then stained with TRITC-phalloidin, and the number of cells per field or percentage of spread cells determined as described in Materials and Methods. Figures shown are the mean of three independent experiments (using three separate donors), where in each individual experiment the mean Ϯ SD of four ran- p ϭ 0.01; determined using ,ء ;p ϭ 0.0001 ,ءء .domly chosen frames was calculated Students t test.

To control for the presence of contaminating bacterial toxins in the LPS, a sample was heat inactivated for 60 min. There were no detectable differences in the morphological responses observed be- tween non-heat-inactivated (Fig. 1, a–d) and heat-inactivated (Fig. 1, e and f) LPS, indicating that the responses observed were due to

LPS itself rather than to any heat-sensitive contaminant. Downloaded from When Bac1 macrophages are deprived of CSF-1, they take on a rounded morphology (Fig. 1g) (41). Upon LPS stimulation, the cells responded rapidly by extending filopodia (e.g., arrowhead in Fig. 1h) and lamellipodia (e.g., arrowhead in Fig. 1i). Membrane ruffles were also observed on the dorsal surface of cells (Fig. 7, i

and j). By 60 min (Fig. 1j), the cells were more polarized and some http://www.jimmunol.org/ cells displayed punctate F-actin staining (arrowhead) similar to that observed in monocytes (e.g., Fig. 1e), localized toward the presumptive leading edge of the cells. Bac1 cells were less sensi- tive to LPS than monocytes, and morphological changes were most clearly observed at 1 ␮g/ml LPS, whereas human monocytes re- sponded morphologically to LPS at both 100 ng/ml (Fig. 1) and 10 ng/ml, but showed little response at 1 ng/ml (data not shown). This difference between Bac1 cells and monocytes could reflect recep-

tor levels and/or different types of receptors. by guest on September 26, 2021

LPS induces tyrosine phosphorylation of Pyk2 and paxillin in human monocytes and the murine macrophage cell line J774 To investigate the signaling processes involved in LPS-induced adhesion and spreading, we initially examined the ability of LPS to activate Pyk2. Pyk2 was chosen as although it was previously re- ported that FAK is expressed in human peripheral monocytes (42), in agreement with other workers (28), we were unable to detect any FAK expression in either human monocytes or murine mac- FIGURE 1. LPS-induced morphological changes and actin reorganiza- rophage cell lines. We therefore concentrated our studies to Pyk2, tion in human monocytes and Bac1 macrophages. Actin cytoskeletal or- which is abundantly expressed in cells of the monocyte/macro- ganization is shown in human monocytes (a–f) and Bac1 macrophages (g–j). Human monocytes (5 ϫ 105/ml) were seeded onto glass coverslips phage lineage. and allowed to adhere for 30 min. Cells were then left unstimulated (a), or Freshly isolated human peripheral blood monocytes were stim- stimulated with either 100 ng/ml LPS (b–d) or 100 ng/ml heat-inactivated ulated in suspension with 100 ng/ml LPS for different lengths of LPS (e and f) for 60 min. The images in c and d are sections of the same time, and then analyzed for Pyk2 tyrosine phosphorylation (Fig. 2). cell, showing a basal (c) and more apical (d) section. Similarly, e and f The Ab used to immunoprecipitate Pyk2 (600) detects both of the show basal and apical sections of the same cell. Bac1 macrophages (5 ϫ Pyk2 isoforms expressed in monocytes (29). An increase in Pyk2 104/ml) were allowed to adhere to coverslips overnight before the removal tyrosine phosphorylation was consistently induced by LPS in of CSF-1. Twenty-four hours after CSF-1 removal, the cells were left un- monocytes, and maximum Pyk2 phosphorylation was observed be- ␮ stimulated (g) or stimulated with LPS (1 g/ml) for 20 min (h and i)or60 tween 15–30 min after LPS addition. The experiments were per- min (j). Cells were fixed and stained with TRITC-labeled phalloidin to formed in the presence of 1% HIFCS, to facilitate LPS binding to reveal actin filaments. Arrowheads indicate membrane ruffles (d, f); retrac- tion fibers (e); a filopodium (h); lamellipodia (i and j). Bar in a represents CD14 via LPS-binding protein present in serum (8). In the pres- 10 ␮m and refers to a–f; bar in g represents 10 ␮m and refers to g–j. ence of 10% HIFCS, Pyk2 tyrosine phosphorylation was induced in the absence of LPS (data not shown). This could be a conse- quence of lysophosphatidic acid present in serum, since lysophos- phatidic acid has been shown to stimulate Pyk2 phosphorylation in rounded or displayed retraction fibers (arrowhead in Fig. 1e), typ- PC12 cells (43). ical of the trailing edge of a motile cell. Time-lapse videomicros- As Pyk2 has been shown to bind to paxillin both in vitro and in copy confirmed that actively migrating cells were present in LPS- vivo, we investigated whether LPS also altered paxillin phosphor- stimulated monocyte cultures (data not shown). ylation. LPS stimulated tyrosine phosphorylation of paxillin with The Journal of Immunology 2031

FIGURE 2. Kinetics of LPS-induced tyrosine phosphorylation of Pyk2 and paxillin in human monocytes. Human monocytes were kept in suspen- FIGURE 3. Dose response for LPS-induced tyrosine phosphorylation of sion and were stimulated with 100 ng/ml of LPS for the times indicated.

Pyk2 and paxillin in human monocytes. Human monocytes were kept in Downloaded from Cell lysates were immunoprecipitated with either anti-Pyk2 (600) or anti- suspension or allowed to adhere to tissue culture plastic (for 30 min) before paxillin Abs and analyzed by immunoblotting with the anti-phosphotyrosine stimulation with LPS (0–100 ng/ml for 30 min). Cell lysates were immu- Ab PY99. Membranes were stripped and reprobed with anti-Pyk2 (623) or noprecipitated with either anti-Pyk2 (600) or anti-paxillin Abs and ana- anti-paxillin Abs. The data are representative of three experiments performed lyzed via immunoblotting with the anti-phosphotyrosine Ab PY99. Mem- using different donors. branes were stripped and reprobed with anti-Pyk2 (623) or anti-paxillin Abs. The data are representative of three experiments performed using

different donors. http://www.jimmunol.org/ similar kinetics to those observed with Pyk2 phosphorylation (Fig. 2). Reprobing with anti-paxillin Abs demonstrated that there was an increase in lower mobility forms of paxillin, suggesting that paxillin is also phosphorylated at multiple sites in response illin tyrosine phosphorylation was significantly decreased (Fig. to LPS stimulation, as has been observed following adhesion of 4b). LPS was incapable of stimulating increased phosphorylation macrophages to vitronectin (44). of either Pyk2 or paxillin in these suspended cells. This suggests Although CD14 is believed to be the principal receptor for LPS that Bac1 cells differ from monocytes and J774 cells in that Pyk2 on macrophages, at higher concentrations LPS has been shown to tyrosine phosphorylation is already high as a consequence of other, ␤ by guest on September 26, 2021 bind to other cell surface proteins, including the 2 integrin com- unknown, signals, and cannot be further enhanced by either LPS or plement receptor type 3 (9). As engagement of integrins has been adhesion. In contrast, paxillin phosphorylation is regulated by shown to result in both Pyk2 and paxillin tyrosine phosphorylation LPS, but only in adherent Bac1 cells. (30, 44), it was important to determine the sensitivity of this re- sponse to LPS concentrations. LPS was capable of stimulating the tyrosine phosphorylation of both Pyk2 and paxillin at concentra- The Src kinase inhibitor PP1 and the PI 3-K inhibitor tions below 100 ng/ml (Fig. 3), indicating that this response is via LY294002 inhibit LPS-induced Pyk2 and paxillin tyrosine binding to the high affinity receptor CD14. phosphorylation and LPS-induced cell spreading in human Pyk2 has been reported to become tyrosine phosphorylated upon monocytes monocyte adhesion to tissue culture plastic (28). We similarly ob- To investigate the signaling pathways leading to Pyk2 and paxillin served tyrosine phosphorylation of Pyk2 and also paxillin in re- phosphorylation, we have focused on two potential upstream sig- sponse to adhesion (Fig. 3). However, once the monocytes had nal transducers. LPS has been shown to activate PI 3-K (21) and been allowed to adhere to tissue culture plastic (in 1% HIFCS), the Src family members Hck, Lyn, and Fgr (15, 16) within minutes LPS stimulated a further increase in the level of tyrosine phos- of stimulation. In addition, PI 3-K physically associates with Lyn, phorylation of Pyk2 and paxillin (Fig. 3). suggesting that they may act coordinately to regulate downstream To determine whether LPS could also induce paxillin and Pyk2 signaling (21). Using the Src family kinase inhibitor PP1 (45) or phosphorylation in macrophages, we investigated LPS responses the PI 3-K inhibitor LY294002 (46), we determined the roles of the in two adherent murine macrophage cell lines. In J774 cells, some targets of these compounds in LPS responses. Preincubation of constitutive Pyk2 tyrosine phosphorylation was observed, even at monocytes with either PP1 or LY294002 strongly inhibited the low serum concentrations (0.5%) (Fig. 4a). However, as in adher- LPS-induced phosphorylation of paxillin and Pyk2 (Fig. 5). PP1 ent monocytes, LPS enhanced the levels of tyrosine phosphoryla- reduced both paxillin and Pyk2 tyrosine phosphorylation below tion of both Pyk2 and paxillin: increased tyrosine phosphorylation levels detected in unstimulated cells, suggesting that basal as well was detected within 5 min and remained elevated for up to 2 h. In as stimulated levels of tyrosine phosphorylation are regulated by Bac1 cells, both Pyk2 and paxillin were constitutively tyrosine Src family kinases. LY294002 reduced the level of phosphoryla- phosphorylated, and the level of Pyk2 tyrosine phosphorylation tion to that observed in unstimulated cells. Consistent with previ- was not significantly increased when stimulated with LPS, even at ous reports (31, 47, 48), we observed coimmunoprecipitation of high concentrations of 1 ␮g/ml (Fig. 4b, left panels). In contrast, Pyk2 with paxillin (Fig. 5a, left panel). Interestingly, LPS stimu- LPS stimulated an increase in paxillin phosphorylation (Fig. 4b, lation did not increase the amount of Pyk2 coimmunoprecipitating right panels). When Bac1 cells were gently detached from the with paxillin, despite inducing tyrosine phosphorylation of Pyk2. tissue culture plates, and kept in suspension for 3 h before stimu- In addition, PP1 and LY294002 did not alter the association of lation, the level of Pyk2 phosphorylation was unaltered, but pax- Pyk2 with paxillin, indicating that this interaction is not regulated 2032 LPS-INDUCED ACTIN REORGANIZATION AND PROTEIN PHOSPHORYLATION Downloaded from http://www.jimmunol.org/

FIGURE 4. Analysis of Pyk2 and paxillin tyrosine phosphorylation in response to LPS in murine macrophages. Adherent cultures of J774 cells FIGURE 6. PP1 and LY294002 inhibit LPS-induced cell spreading. (a) were cultured in 0.5% HIFCS for 48 h before stimulation with LPS (1 Human monocytes (5 ϫ 105/ml) were seeded in 1% HIFCS onto glass ␮ g/ml) for the times indicated. Bac1 cells (b) were incubated in medium coverslips. Cells were allowed to adhere to coverslips for 30 min before by guest on September 26, 2021 containing 1% HIFCS (no CSF-1) for 24 h, and then were either left at- incubation with either DMSO control (0.1%) (a and b), LY294002 (25 tached or detached and kept in suspension for 3 h before stimulation with ␮M) (c), or PP1 (5 ␮M) (d) for 15 min before stimulation with LPS (100 ␮ LPS (1 g/ml for 30 min). Cell lysates were immunoprecipitated with ng/ml) (b, c and d) for 60 min (unstimulated in a). Cells were fixed and either anti-Pyk2 (600) or anti-paxillin Abs and analyzed by immunoblot- stained with TRITC-labeled phalloidin to reveal actin filaments. Bar: 10 ting with the anti-phosphotyrosine Ab PY99. Membranes were stripped ␮m(a–d are at the same magnification). A quantitative analysis of cell and reprobed with anti-Pyk2 (623) or anti-paxillin Abs. adhesion and spreading efficiency was obtained by calculating the percent- age of spread cells (e and f). Data represent the mean of three independent experiments (using three separate donors), in which each individual exper- by tyrosine phosphorylation of Pyk2 or paxillin, in agreement with iment represents the mean of four randomly chosen frames. p values were p Ͻϭ ,ءءء ;p Ͻ 0.01 ,ءء ;p Ͻ 0.05 ,ء :other workers (31, 47, 48). determined using Student’s t test To determine whether Src family kinases and PI 3-K could be 0.005. Both inhibitors significantly decreased spreading (p values of PP1 involved in LPS-induced morphological changes, PP1 and (p ϭ 0.006) and LY294002 (p ϭ 0.002) compared with LPS alone) and the number of adherent cells (p values of PP1 (p ϭ 0.017) and LY294002 (p ϭ 0.012) compared with LPS alone).

LY294002 were incubated with monocytes before LPS addition. Both PP1 and LY294002 prevented LPS-induced monocyte spreading (Fig. 6, a–d and f). Even cells that did spread (diameter Ͼ10 ␮m) to a limited degree remained unpolarized. In addition, by 60 min after LPS stimulation, PP1 and LY294002 induced a sig- FIGURE 5. The Src family kinase inhibitor PP1 and the PI 3-K inhibitor nificant reduction in the number of adherent monocytes (Fig. 6, LY294002 inhibit LPS-induced Pyk2 and paxillin tyrosine phosphorylation a–d and e). Both inhibitors also prevented the transient adhesion of in human monocytes. Human monocytes were kept in suspension, and monocytes to glass coverslips in the absence of LPS (data not ␮ ␮ preincubated with either PP1 (10 M), LY294002 (25 M), or DMSO shown), consistent with a role for Src kinases and PI 3-K in ad- control (0.1%) for 15 min before stimulation with 100 ng/ml of LPS for 30 hesion-induced signaling. The effects of PP1 and LY294002 on min. Cell were lysed and immunoprecipitated with either a, anti-paxillin or b, anti-Pyk2 (600), and immunoprecipitates analyzed by immunoblotting monocyte morphology were reversible, as if the inhibitors were with the anti-phosphotyrosine Ab PY99. Membranes were stripped and removed from the cultures by washing; the cells were able to adhere reprobed with anti-Pyk2 (623) or anti-paxillin Abs. The data are represen- and respond normally to LPS, indicating that the inhibitors were not tative of three experiments performed using different donors. exerting some nonspecific toxic effect upon the monocytes. The Journal of Immunology 2033 Downloaded from

FIGURE 8. Localization of paxillin and Pyk2 in Bac1 macrophages. Growing Bac1 cells were fixed and dual stained to show paxillin (a and c) and Pyk2 (b and d). A basal section shows that paxillin, but not Pyk2, localizes to focal complexes (a and b), whereas both paxillin and Pyk2

colocalize in membrane ruffles in a medial section (c and d) through the http://www.jimmunol.org/ same cell. Bar: 10 ␮m.

not shown). In contrast, vinculin localized to , as pre- viously described (50), as did paxillin (data not shown). In medial sections of cells, Pyk2 localized within F-actin-rich membrane ruffles (Fig. 7, e and f; arrowhead indicates a membrane FIGURE 7. Localization of paxillin and Pyk2 in monocytes. Human ruffle). Paxillin (Fig. 7g) and Pyk2 (Fig. 7h) colocalized in the by guest on September 26, 2021 ϫ 5 monocytes (a–h) were seeded at 5 10 /ml in 1% HIFCS onto glass perinuclear area of cells and at the plasma membrane (e.g., arrows coverslips. Monocytes were allowed to adhere to coverslips for 30 min, and in Fig. 7, g and h), consistent with their coimmunoprecipitation then were left unstimulated (a and b) or stimulated with LPS (100 ng/ml) for 60 min (c–h). Cells were fixed and dual stained to show paxillin (a and from cell lysates (Fig. 5a). Paxillin also localized to small focal g) and Pyk2 (b and h) localization, or dual stained to show actin filaments complexes observed in some areas at the periphery of LPS-stim- (c and e) and Pyk2 localization (d and f). The images in c and d show a ulated monocytes (Fig. 7g; basal section, arrowheads indicate ex- basal section, and in e and f a more apical section of the same cells. Arrows amples of focal complexes). Pyk2 did not detectably colocalize in c and d indicate colocalization of Pyk2 and actin filaments; arrowheads with paxillin in these focal complexes (Fig. 7h), indicating that in e and f indicate an example of colocalization of actin filaments and Pyk2 Pyk2 and paxillin are not always associated. Similarly, paxillin and in membrane ruffles; arrows in g and h show colocalization of paxillin and Pyk2 colocalized in protrusions of Bac1 cells (Fig. 8, a and b), but Pyk2 at the plasma membrane; while arrowheads in g indicate paxillin- Pyk2 clearly did not localize to paxillin-containing focal com- containing focal complexes, in which Pyk2 does not colocalize. Bar: 10 plexes. As in monocytes, paxillin and Pyk2 colocalized in mem- ␮m(a and b, c–f, g and h are at the same magnification). brane ruffles of Bac1 cells (Fig. 8, c and d).

Localization of paxillin and Pyk2 in monocytes and Bac1 Discussion macrophages The effects of LPS on cytokine production in monocytes and mac- Our observation that PP1 and LY294002 inhibited both LPS-in- rophages have been extensively studied, whereas its effects on cell duced morphological changes and phosphorylation of Pyk2 and morphology have not been characterized in detail. We have shown paxillin suggested that Pyk2 and/or paxillin could be involved in that LPS rapidly stimulates actin reorganization in monocytes and the morphological responses to LPS. We therefore investigated the macrophages, and promotes monocyte adhesion and spreading. localization of Pyk2 and paxillin with respect to the actin cytoskel- These responses to LPS are likely to be important in stimulating eton. In unstimulated monocytes, both paxillin and Pyk2 were dis- monocyte adhesion to and transmigration across endothelial cells, tributed diffusely throughout the cytoplasm (Fig. 7, a and b). Upon as well as recruitment of macrophages to sites of infection. Iden- LPS stimulation and subsequent cell spreading, Pyk2 was found to tifying signaling pathways mediating LPS-induced morphological concentrate, with F-actin, at the leading edge of polarized cells (as responses is therefore central to our understanding of how LPS shown in the basal plane, Fig. 7, c and d). Pyk2 did not localize to acts in vivo. We have observed that two proteins extensively im- podosomes, identified as punctate foci of F-actin (Fig. 7c; podo- plicated in adhesion-induced signaling, Pyk2 and paxillin, are ty- somes indicated by arrows), which are adhesion sites found in cells rosine phosphorylated in response to LPS and are found within of monocytic origin (49, 50). Pyk2 also did not localize to podo- membrane ruffles and lamellipodia in LPS-stimulated monocytes. somes in human macrophages differentiated from monocytes (data The Src kinase family inhibitor PP1 and the PI 3-K inhibitor 2034 LPS-INDUCED ACTIN REORGANIZATION AND PROTEIN PHOSPHORYLATION

LY294002 prevent LPS-induced monocyte spreading and concom- limiting step involving auto/transphosphorylation of Pyk2, before Src itantly inhibit tyrosine phosphorylation of Pyk2 and paxillin, sug- kinases can bind and further stimulate tyrosine phosphorylation. gesting that Pyk2 and/or paxillin play a role in LPS-induced mor- We have found that the PI 3-K inhibitor LY294002 prevents phological changes. LPS-induced tyrosine phosphorylation of Pyk2 and paxillin. PI In monocytes, Pyk2 has been shown to be tyrosine phosphory- 3-K itself is activated by both receptor and nonreceptor tyrosine lated upon adhesion (28), and we have similarly observed that kinases (59). LPS-induced PI 3-K activation in monocytes is in- adhesion stimulates Pyk2 and paxillin tyrosine phosphorylation. hibited by the tyrosine kinase inhibitor herbimycin A, and Lyn When monocytes are kept in suspension, Pyk2 is not responsive to associates with PI 3-K (21), suggesting that it or a related kinase stimuli such as PMA, RANTES, and thapsigargin, although these mediates PI 3-K activation by LPS. The PP1 inhibitor may there- stimuli enhance tyrosine phosphorylation of Pyk2 in adherent fore target both a Src kinase involved in LPS-induced activation of monocytes (28). In contrast, we have found that LPS is able to PI 3-K and also a Src kinase(s) involved in phosphorylating Pyk2 induce Pyk2 and paxillin tyrosine phosphorylation in suspension and paxillin. Precisely how PI 3-K contributes to Pyk2 or paxillin as well as in adherent monocytes. This indicates that neither Pyk2 tyrosine phosphorylation is unclear, but it is possible that it in- nor paxillin requires signals provided by adhesion to become ty- volves a member of the Tec family of Src-related tyrosine kinases, rosine phosphorylated, but that adhesion enhances the response to which bind to and are activated by the PI 3-K lipid product phos- LPS. Interestingly, LPS induces morphological changes in adher- phatidylinositol 3,4,5-trisphosphate via their pleckstrin homology ent Bac1 macrophages without detectably altering Pyk2 tyrosine domains (59, 60). Pyk2 can interact with PI 3-K (61), and this phosphorylation. In contrast, LPS does increase paxillin phosphor- could facilitate Pyk2 and paxillin localization to sites of PI 3-K ylation, and therefore it is plausible that in these cells paxillin but activity, where a Tec family kinase could then tyrosine phosphor- Downloaded from not Pyk2 contributes to LPS-induced morphological changes. It is ylate them. PI 3-K may also contribute to cell spreading by acting also possible that Pyk2 is involved, but only in concert with pax- upstream of the GTPase Rac (62), which regulates the formation of illin and other proteins. Indeed, our observation that Pyk2 localizes lamellipodia and membrane ruffles in many cell types, including to membrane ruffles and lamellipodia suggests that its site of action Bac1 macrophages (63, 64). Interestingly, paxillin has recently is in these structures, where new adhesions are formed during cell been shown to interact with two components of the Rac signaling spreading and migration (40). As Pyk2 tyrosine phosphorylation pathway, PAK (p21-activated kinase) and PIX (PAK-interacting http://www.jimmunol.org/ remains elevated in Bac1 cells even in suspension, it is possible guanine nucleotide exchange factor) (65), and thus it is possible that these cells constitutively secrete one or more chemokine that that Pyk2 and paxillin are part of a large complex of proteins acts in an autocrine fashion to activate Pyk2, as several chemo- involved in regulating lamellipodium extension and the formation kines have been shown to activate Pyk2 (28, 29). of new adhesion sites. We have shown that the Src family-specific inhibitor PP1 pre- The subcellular localization of Pyk2 has not previously been vented LPS-induced tyrosine phosphorylation of Pyk2 and paxil- described in monocytes and macrophages. Studies in smooth mus- lin. Potential Src family members involved in this response are Ϫ Ϫ cle cells (66), fibroblasts (67), and FAK / fibroblasts (68) have Hck, Lyn, and Fgr, which are all activated within minutes of LPS

revealed a predominantly perinuclear localization of Pyk2. Simi- by guest on September 26, 2021 stimulation (15, 16). Pyk2 has been shown to associate directly larly, we have observed that Pyk2 and paxillin have a perinuclear with the Src kinases Fyn, Lck, and Src, and Fyn can phosphorylate distribution in monocytes and macrophages. In addition, in LPS- Pyk2 in vitro (33, 43, 51, 52). In T cells, Pyk2 tyrosine phosphor- stimulated cells, Pyk2 was observed at the plasma membrane, co- ylation is regulated by Lck or Fyn, depending on the stimulus (51, 52). Activated Pyk2 in turn may phosphorylate paxillin, as it in- localizing with F-actin and paxillin in lamellipodia and membrane teracts directly via its C terminus with paxillin and can phosphor- ruffles. Ectopic expression of hemagglutinin-tagged Pyk2 in J774 ylate it in vitro (37, 53). However, although a proportion of Pyk2 cells confirmed that Pyk2 was present in membrane ruffles (our is constitutively associated and colocalizes with paxillin in cells, unpublished data). As lamellipodia extend over the substratum, their tyrosine phosphorylation does not always correlate. For ex- new integrin-mediated adhesions known as focal complexes form ample, ligation of CD28 in Jurkat T cells stimulates Pyk2, but not (40), and many cytoplasmic proteins including FAK, vinculin, and paxillin tyrosine phosphorylation (54). In addition, CD3- and paxillin localize to these complexes (63, 64). Dual staining for CD45-induced phosphorylation of paxillin, but not Pyk2, is de- Pyk2 and paxillin demonstrated that although Pyk2 and paxillin pendent on the Src family kinase Lck in T cells (47). It is therefore colocalized in membrane ruffles, Pyk2 was not found within pax- likely that Pyk2 and paxillin tyrosine phosphorylation can be reg- illin-containing focal complexes. Pyk2 also did not localize to po- ulated independently of each other. Indeed, it appears that Src dosomes, which are punctate sites of adhesion commonly observed kinases rather than FAK are responsible for paxillin tyrosine phos- in cells of monocytic origin (49, 50). This suggests that Pyk2- phorylation during spreading of chicken embryo cells (55). Pyk2 paxillin interaction occurs in membrane ruffles and/or in the pe- may therefore act as an adaptor to bring paxillin and Src kinases rinuclear region rather than in focal complexes or podosomes. In together, as has been proposed for FAK (55). In the context of LPS fibroblasts, a small proportion of Pyk2 was shown to localize in the signaling, it would therefore be of interest to determine whether vicinity of focal adhesions (67, 68), although it had a patchy dis- Pyk2 or paxillin are direct substrates of Hck, Lyn, or Fgr. tribution unlike that of vinculin or paxillin. In contrast, Pyk2 has Pyk2 can be activated by a diverse spectrum of stimuli, but there been reported to colocalize with vinculin in focal adhesions in is substantial variation in the kinetics of its tyrosine phosphoryla- neutrophils (69). It will therefore be important to determine the tion. Chemokine and integrin receptor engagement leads to the precise role that paxillin, and other paxillin family members such phosphorylation of Pyk2 within minutes (29, 48, 56), whereas as Hic-5 and leupaxin (67, 70), plays in the localization and func- other stimuli such as IL-2 and fluoroaluminate require a longer tion of Pyk2 and associated proteins in different cell types. time period (greater than 10 min) to induce the maximum level of In conclusion, our observations that LPS-induced monocyte ad- Pyk2 phosphorylation (57, 58). The kinetics of LPS-induced Pyk2 hesion and spreading are accompanied by Pyk2 and paxillin ty- and paxillin tyrosine phosphorylation in monocytes fall into the rosine phosphorylation, and that PP1 and LY294002 inhibit both latter category, as maximum phosphorylation was not observed of these responses, suggest that Pyk2 and/or paxillin play an im- until after 15 min. This delay could be a consequence of a rate- portant role in LPS-mediated morphological changes. In addition, The Journal of Immunology 2035 the localization of Pyk2 and paxillin in membrane ruffles and la- protein kinase involved in the regulation of inflammatory cytokine biosynthesis. mellipodia is consistent with a role for these proteins in cell mo- Nature 372:739. 21. Herrera-Velit, P., and N. E. Reiner. 1996. Bacterial lipopolysaccharide induces tility. Paxillin in particular has been implicated as playing an im- the association and coordinate activation of p53/56lyn and phosphatidylinositol portant role in cell spreading (55). Future studies with mutants of 3-kinase in human monocytes. J. Immunol. 156:1157. Pyk2 and paxillin should delineate more precisely how each pro- 22. Herrera, V. P., K. L. Knutson, and N. E. Reiner. 1997. Phosphatidylinositol 3-kinase-dependent activation of protein kinase C-␨ in bacterial lipopolysaccha- tein contributes to LPS-induced monocyte and macrophage motil- ride-treated human monocytes. J. Biol. Chem. 272:16445. ity responses. As a variety of stimuli can induce Pyk2 and paxillin 23. Foey, A., S. L. Parry, L. M. Williams, F. M. Brennan, and B. M. J. Foxwell. 1998. tyrosine phosphorylation in monocytes, it is likely that in vivo Regulation of monocyte IL-10 synthesis by endogenous IL-1 and TNF␣: role of p38 and p42/p44 MAP kinases. J. Immunol. 160:926. different signals, including LPS, cytokines, and extracellular ma- 24. Zachary, I., and E. Rozengurt. 1992. Focal adhesion kinase (p125FAK): a point trix proteins, act coordinately to stimulate signaling pathways pro- of convergence in the action of neuropeptides, integrins, and oncogenes. Cell moting monocyte motility. 71:891. 25. Avraham, S., R. London, Y. Fu, S. Ota, D. Hiregowdara, J. Li, S. Jiang, L. M. Pasztor, R. A. White, J. E. Groopman, and H. Avraham. 1995. Identifica- Acknowledgments tion and characterization of a novel related adhesion focal tyrosine kinase (RAFTK) from megakaryocytes and brain. J. Biol. Chem. 270:27742. We are grateful to the staff at the Kennedy Institute of Rheumatology 26. Lev, S., H. Moreno, R. Martinez, P. Canoll, E. Peles, J. M. Musacchio, (London, U.K.), in particular Brien Foxwell and T. Green, for the use of G. D. Plowman, B. Rudy, and J. Schlessinger. 1995. 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