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Immunology. 1999 July; 97(3): 414–419. doi: 10.1046/j.1365-2567.1999.00777.x. | PMCID: PMC2326867 |
Syk and paxillin are differentially phosphorylated following adhesion to the plastic substrate in rat alveolar macrophages S Hirano and S Kanno Regional Environment Division, National Institute for Environmental Studies, Tsukuba, Ibaraki, Japan Received October 20, 1998; Revised January 26, 1999; Accepted January 26, 1999. Adhesion is associated with tyrosine phosphorylation in many types of cells. Although macrophages are known to adhere and phagocytose foreign particles, the signal transduction pathway of macrophages in response to adhesion to the foreign substrate has not been fully investigated. In the present study we investigated tyrosine-phosphorylated proteins and phosphorylation of paxillin in alveolar macrophages (AMs) following adhesion to a plastic substrate. Adhesion to a plastic dish resulted in tyrosine phosphorylation of a 68 000 MW protein, which was shown, by immunoprecipitation and immunoblotting in the present study, to be a rat Syk kinase. Treatment with erbstatin reduced both tyrosine phosphorylation of Syk and adherence of AMs, while treatment with cytochalasin B inhibited spreading of AMs but did not inhibit tyrosine phosphorylation of Syk. These results suggest that tyrosine phosphorylation of Syk plays an important role in adhesion of AMs to the plastic substrate, but not in AM spreading. Paxillin is known to be tyrosine phosphorylated following adhesion to the extracellular matrix in many types of cells. However, paxillin appeared to be serine/threonine phosphorylated rather than tyrosine phosphorylated following adhesion of AMs to the plastic substrate. Treatment with A23187 (a calcium ionophore), but not phorbol 12-myristate 13-acetate (PMA; a protein kinase C stimulator), induced tyrosine phosphorylation of Syk in non-adherent AMs. Treatment with either A23187 or PMA caused electromobility changes of paxillin that were mainly a result of serine/threonine phosphorylation. These results suggest that adhesion to the plastic substrate leads to two differently regulated events in AMs: tyrosine phosphorylation of Syk and serine/threonine phosphorylation of paxillin, both of which are probably mediated by an increase in intracellular calcium. Alveolar macrophages (AMs) play a pivotal role in the host defence in the lung. Fine particles (less than 2·5 μm) penetrate below terminal bronchioles and deposit in the respiratory region of the lung. 1 AMs phagocytose those particulate substances and reduce the alveolar burden of foreign substances. At the start of phagocytosis, AMs adhere to the surface of the foreign particulate substance. Thus, investigating the mechanism of non-specific adhesion and the subsequent signal transduction pathway may provide a clue to the mechanism for attachment of particles to AMs, an initial step of phagocytosis. It is well known that macrophages adhere to opsonized and immunogloblin-coated particles through C3b and Fc receptors. 2 On the other hand, the mechanisms responsible for non-specific adhesion (to plastics or glass) of macrophages have not been fully investigated. To our knowledge three membrane-associated molecules are known to mediate non-specific adhesion in macrophages. They are: scavenger 3 and C3bi receptors 4 and ‘attachmin’. 5 Recently one of the present authors reported that an anti-β 2 integrin antibody inhibited adhesion of AMs, not only to alveolar epithelial cells but also to plastic dishes. 6 It has been reported that adhesion to the plastic substrate stimulates arachidonic acid metabolism in rat AMs. 7 Thus, it is natural to suppose that outside-in non-specific adhesion signals may be followed by a signal transduction cascade and activate the metabolic system in AMs. In the last decade, tyrosine phosphorylation of focal adhesion kinase (FAK) and its substrate, paxillin, has been shown to play an important role in the adhesion of fibroblastic cells to an extracelluar matrix. It is well known that vinculin and talin are also recruited to the focal adhesion complex following autophosphorylation of FAK. 10 However, mechanisms of integrin (α vβ 5)-dependent macrophage adhesion to vitronectin are probably different from those of fibroblastic cells because it has been shown that FAK is not expressed in macrophages and 99% of paxillin phosphorylation occurs on serine residues in response to adhesion to vitronectin. 11 It has also been reported that human monocytes express no FAK, and adhesion to plastic dishes stimulated tyrosine phosphorylation of a calcium-dependent tyrosine kinase. 12 In the present study we describe that Syk kinase was predominantly tyrosine phosphorylated and paxillin was serine/threonine phosphorylated following adhesion of rat AMs to plastic dishes. Chemicals The following chemicals and antibodies were used in the present study. Monoclonal anti-phosphotyrosine (25.2G4; Wako Chem., Osaka, Japan), anti-paxillin (Transduction Lab., Lexington, KY), polyclonal anti-Syk kinase (N-19), horseradish peroxidase (HRP)-conjugated goat anti-rabbit and antimouse IgG antibodies (Santa Cruz Biotech., Santa Cruz, CA), agarose-conjugated protein A/G and a bicinchoninic acid (BCA) protein assay kit (Pierce, Rockford, IL), ABC staining and 3,3′-diaminobenzicline (DAB) substrate kits (Vector Lab., Burlingame, CA), genistein (Wako Chem.), erbstatin analogue (RBI, Natick, MA), pansorbin, cytochalasin B and A23187 (Calbiochem, La Jolla, CA), protein phosphatase type-1 (PP-1) (UBI, Lake Placid, NY), poly (2-hydroxyethyl methacrylate) (poly HEMA) and phorbol 12-myrystate 13-acetate (PMA) (Sigma, St. Louis, MO), enhanced chemiluminescence (ECL) staining kit (Amersham, Little Chalfont, Bucks, UK), bovine plasma fibronectin (Wako Chem.), Block Ace® (Dainippon Pharm., Osaka, Japan) and Diff-Quik® staining kit (Kokusai Shiyaku Co., Kobe, Japan). Other chemicals of analytical grade were obtained from Sigma or Wako. Cytochalasin B, tyrosine kinase inhibitors, A23187 and PMA were dissolved in dimethyl sulphoxide (DMSO), and the stock solution was used at a final concentration of 0·2% DMSO. Collection of AMs Specific pathogen-free, male Sprague–Dawley rats, 7–9 weeks old (Clea Japan, Tokyo), were used. The rats were anaesthetized with an intraperitoneal (i.p.) injection of sodium pentobarbital (50 mg/kg body weight) and killed by exsanguination from abdominal aorta. The lungs were lavaged eight times with saline and the lavaged fluid was centrifuged at 400 g for 5 min at 4°. The pellet was washed in RPMI-1640 minus fetal bovine serum and resuspended in fresh RPMI-1640 at 1 × 106 viable cells/ml. The viability of the lavaged cells was greater than 95% as determined by the exclusion ability of Trypan Blue. More than 97% of the lavaged cells were AMs as determined by differential cell counting using Diff-Quik® staining. Cell culture To prevent adhesion of AMs to the plastic dish, culture dishes (60-mm diameter; Costar, Cambridge, MA) were treated with poly HEMA solution. Briefly, poly HEMA dissolved in 95% ethanol (10 mg/ml) was aliquoted into culture dishes and the solvent was allowed to evaporate by incubation overnight on a clean bench. The AM suspension in serum-free RPMI-1640 was aliquoted into untreated or poly HEMA-treated culture dishes, and cultured at 37° in an atmosphere of 5% CO2. After 0·5, 1, and 6 hr of culture, non-adherent cells were centrifuged at 400 g for 5 min at 4°. Adherent cells were washed with Ca2+ and Mg2+-free phosphate-buffered saline (PBS). In a reference experiment, tyrosine phosphorylation of paxillin in fibroblastic L-M cells (CCL-1.2; ATCC, Rockville, MO) was investigated. Briefly, plastic culture dishes were treated overnight with fibronectin (20 μg/ml) and washed with PBS and serum-free RPMI-1640. L-M cells were cultured in fibronectin- or poly HEMA-treated plastic dishes for 1·5 hr. Adhesion assay One-hundred microlitre volumes of AM suspension (1 × 106 viable cells/ml in serum-free RPMI-1640) were aliquoted into a 96-well culture dish (Costar) and incubated on ice for 20 min in the presence or absence of tyrosine kinase inhibitors and cytochalasin B. The cells were further incubated at 37° for 30 min. After washing the wells five times with PBS, cells remaining in the wells were stained with a × 10 dilution of Giemsa solution for 15 min. The wells were washed five times and the adherent cells were lysed with 100 μl of 50% ethanol. The adherence of AMs was evaluated by measuring optical density (OD) at 630 nm using a microtitre plate reader (CS 9300 PC; Shimadzu, Kyoto, Japan). Electrophoresis and Western blotting Both adherent and non-adherent cells in one well were lysed together with lysate buffer on ice. The lysate buffer contained 150 mm sodium chloride, 1% Nonidet P-40 (NP-40), 0·5% sodium deoxycholate, 50 mm N-[2-hydroxyethyl]piperazine-N′-[4-butanesulphonic acid] (HEPES), 1 mm phenylmethylsulfonyl fluoride (PMSF), 1 mm sodium orthovanadate, 50 mm sodium fluoride, 1 mm p-nitrophenyl phosphate, 10 μg/ml aprotinin, 5 mm benzamidine and 20 nM calyculin A. The lysate was centrifuged at 1000 g, for 10 min at 4°, and the protein concentration of the supernatant was adjusted to 1 mg/ml. Proteins in the supernatant were subjected to sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE; 5–20%) under reducing conditions and electroblotted onto a polyvinylidene difluoride (PVDF) membrane. The blots were preincubated in Block Ace®, probed with anti-phosphotyrosine or anti-paxillin antibody, and visualized using mouse ABC and DAB substrate kits. Immunoprecipitation and immunobotting For immunoprecipitation experiments, 50 μl of lysed samples (2·5 mg protein/ml) were cleared with pansorbin at 4° for 30 min. After centrifugation, 2 μl of anti-Syk or anti-paxillin antibodies was added to the supernatant. The immunocomplex was allowed to bind to protein A/G-agarose overnight at 4°. The immunoprecipitate was washed four times in the lysis buffer, and boiled for 5 min with an equal volume of 2 × SDS sample buffer. Proteins in the supernatant of the sample were subjected to SDS–PAGE (5–20%) under reducing conditions and electroblotted onto a PVDF membrane. After blocking with Block Ace®, the blot was sequentially probed with anti-Syk and HRP-conjugated anti-rabbit IgG antibodies and visualized using ECL. The membrane was stripped and the blot was reprobed with anti-phosphotyrosine followed by incubation with HRP-conjugated anti-mouse IgG antibodies. The immunoblot for paxillin was probed with anti-phosphotyrosine and visualized with mouse ABC and DAB substrate kits. The blots were alternatively probed with anti-paxillin to confirm that the same amount of paxillin was immunoprecipitated. Phosphatase treatment This experiment was performed, using a serine/threonine-specific phosphatase inhibitor, to investigate if the adhesion-dependent electromobility change of paxillin in the gel was caused by serine/threonine phosphorylation. AMs were suspended in RPMI-1640 at 1 × 106 viable cells/ml. The cells were cultured in both untreated and poly HEMA-treated culture dishes for 30 min. The cells were lysed for Western blotting as described above except that the protein phosphatase inhibitors (sodium orthovanadate, sodium fluoride, p-nitrophenyl phosphate, benzamidine and calyculin A) were omitted. Aliquots of the cell lysate supernatant obtained from adherent AMs were treated with PP-1, with or without 100 nm calyculin A, at 37° for 1 hr. The samples were electrophoresed by SDS–PAGE (5–20%) and electrotransferred onto a PVDF membrane. The blot was probed with anti-paxillin, and visualized with mouse ABC and DAB kits. The mobility of paxillin from the PP-1-treated lysate was compared with that from the untreated lysate. Statistical analysis In the adhesion assay, data represent mean±SEM of five experiments performed with duplicate replicates. Statistical analyses were carried out using one-way analysis of variance followed by Bonferroni’s post hoc comparison. Probability values less than 5% were accepted as indicative of statistical significance. shows that a 68 000 MW protein was strongly tyrosine phosphorylated and 38 000 and 42 000 MW proteins were weakly tyrosine phosphorylated following adhesion of rat AMs to the plastic culture dish. Tyrosine phosphorylation of the 68 000 MW protein was evident at 0·5 hr of culture and had decreased by 6 hr of culture. The 68 000 MW protein was not tyrosine phosphorylated when AMs were cultured in poly HEMA-treated culture dishes. Tyrosine phosphorylation of the 68 000 MW protein was caused by adhesion and not by spreading of AMs because treatment of AMs with cytochalasin B did not reduce the tyrosine phosphorylation (). Treatment of AMs with cytochalasin B (both 0·5 and 5 μg/ml) inhibited AM spreading, as determined by inverted microscopy (data not shown), but did not significantly reduce the adherence of AMs to the plastic dish (). also shows that the treatment of AMs with erbstatin, a tyrosine-kinase inhibitor, decreased the adherence, while another tyrosine-kinase inhibitor, genistein, did not change the adherence of AMs to the plastic dish, suggesting that erbstatin-inhibitable tyrosine kinases play an important role in non-specific adhesion of AMs to the plastic dish. Essentially no AMs adhered to the poly HEMA-treated dish. shows that erbstatin reduced the tyrosine phosphorylation of the 68 000 MW protein dose dependently, while genistein did not change the tyrosine-phosphorylation level of this protein. The reduction of tyrosine phosphorylation of the 68 000 MW protein (), and changes in adherence of AMs caused by those tyrosine kinase inhibitors (), are correlated, suggesting that tyrosine phosphorylation of the 68 000 MW protein may be requisite for a maximal non-specific adhesion of AMs. According to the molecular weight (68 000) and adhesion-dependent tyrosine phosphorylation, we first thought that the 68 000 MW protein might be paxillin. As described below, paxillin was not tyrosine phosphorylated, even in the adherent AMs. We investigated, using immunoprecipitation and immunoblotting, whether Syk was tyrosine phosphorylated because several lines of evidence indicate that Syk is tyrosine phosphorylated following adhesion to fibronectin and vitronectin. shows that although almost the same amount of Syk was immunoprecipitated by anti-Syk antibody from adherent and non-adherent AM lysates, only Syk obtained from the adherent AMs lysate was tyrosine phosphorylated. We also investigated whether paxillin was tyrosine phosphorylated following adhesion. As shown in , paxillin was not tyrosine phosphorylated, and yet the electromobility of paxillin in the SDS–PAGE showed a shift to that of the higher molecular weight following adhesion. On the other hand, paxillin was tyrosine phosphorylated following adhesion to fibronectin in L-M cells, a fibroblastic cell line, and there was no observable electromobility change of paxillin between adherent and non-adherent cells. These results suggest that serine or threonine residues of paxillin may be phosphorylated following non-specific adhesion of AMs. To examine the presence of adhesion-dependent serine/threonine phosphorylation, adherent AMs were lysed without phosphatase inhibitors, and the lysate was treated in vitro with PP-1 (a protein phosphatase specific to serine/threonine) in the presence or absence of calyculin A, a protein phosphatase inhibitor. clearly shows that the electromobility change of paxillin in adherent AMs was caused by serine/threonine phosphorylation. We further investigated tyrosine phosphorylation of Syk and the mobility change of paxillin caused by other stimulation ( A23187 and PMA) in non-adherent AMs to study what downstream signals may be involved in those cells following adhesion. shows that Syk was tyrosine phosphorylated by A23187, but not by PMA, whilst the paxillin mobility change was caused by both A23187 and PMA in non-adherent AMs. These results suggest that both tyrosine phosphorylation of Syk and the electromobility change of paxillin can be caused by an increase in intracellular calcium and the latter event may be caused by activation of protein kinase C (PKC). AMs play an important role in removing foreign particulate materials from the alveolar surface by phagocytosis. The first step in phagocytosis should be adhesion of AMs to the surface of particles. The outside-in adhesion signal may reorganize the cytoskelton of AMs, leading to the engulfment of particles. Poly HEMA treatment completely inhibited adhesion of AMs to the plastic (), which enabled us to investigate signal transduction pathways of those cells in response to adhesion. The most prominent tyrosine phosphorylation following adhesion to the plastic dishes occurred in a 68 000 MW protein (), which was demonstrated, by immunoprecipitation and immunoblotting, to be Syk kinase (). Syk kinase was first found in the porcine spleen and reported to be a 72 000 MW protein. 13 However, an apparent molecular weight of murine Syk seems to be 69 000. 14 Besides playing an important role in signal transduction from B-cell antigen receptors 15 and cytotoxicity of natural killer cells, 16 Syk kinase has been reported to be required for immunoreceptor tyrosine activation motif (ITAM)-dependent actin assembly, 17 spreading and H 2O 2 release in adherent neutrophils. 18 On the other hand it has been shown that cross-linking of Fc receptors and integrins activates Syk kinase in macrophages, 19 and monocytic (THP-1) 20 and myelomonocytic cell lines (HL60). 21 In the present study, erbstatin significantly reduced both tyrosine phosphorylation of Syk () and adherence of AMs (), suggesting that adhesion to the plastic substrate activates Syk and tyrosine phosphorylation of Syk may in turn strengthen adherence of AMs to the plastic substrate. Lin et al. reported that although cytochalasin D inhibited both cell spreading over fibronectin and tyrosine phosphorylation of FAK and paxillin in THP-1, the cytochalasin treatment did not block tyrosine phosphorylation of Syk. 20 Yan & Berton also reported that β 2 integrin engagement induced redistribution of tyrosine kinases, such as p58 c–fgr, p53/56 lyn and p72 syk, independently of de novo actin polymerization in human neutropihls. 22 The present study also shows that adhesion, but not spreading, induced tyrosine phosphorylation in rat AMs, because cytochalasin B treatment did not inhibit tyrosine phosphorylation (). It is well established that paxillin is tyrosine phosphorylated following adhesion to an extracellular matrix. As shown in , paxillin appeared not to be tyrosine phosphorylated in AMs, although the electromobility of paxillin obtained from adherent AMs was markedly reduced. On the other hand, paxillin was tyrosine phosphorylated in L-M cells following adhesion to fibronectin and no obvious change in the electromobility of paxillin was found between adherent and non-adherent L-M cells. We postulated that serine or threonine residues of paxillin in adherent AMs were phosphorylated and the serine/threonine phosphorylation may have reduced the electrophoretic mobility of paxillin. To examine this hypothesis, we first lysed the adherent alveolar macrophages with lysis buffer minus phosphatase inhibitors and the lysate was treated in vitro with PP-1 in the presence or absence of calyculin A, a PP-1 inhibitor. clearly demonstrated that the electromobility change of paxillin in adherent AMs was caused by serine/threonine phosphorylation. De Nichilo & Yamada reported that macrophage colony-stimulating factor (M-CSF)-treated macrophages were adherent to vitronectin, and paxillin was predominantly phosphorylated on serine residues in a PKC-dependent manner. 11 Their findings are consistent with ours, even though AMs were allowed to adhere to the different substrates (vitronectin versus plastic). We next examined whether other stimulators, such as A23187 (a calcium ionophore) and PMA (a PKC activator), cause tyrosine phosphorylation of Syk and the electromobility changes of paxillin in non-adherent AMs (). A23187 or PMA was added to AMs cultured in poly HEMA-treated culture dishes. Syk was tyrosine phosphorylated dose dependently in A23187-treated AMs, while PMA did not cause tyrosine phosphorylation of Syk. On the other hand, both A23187 and PMA caused an electromobility change of paxillin. These results indicate that tyrosine phosphorylation can be caused by intracellular calcium, and the electromobility change of paxillin can be mediated by PKC. It has been shown that adhesion of AMs to the plastic dish was mediated, at least in part, by β 2 integrin, 6 and calcium cation was released from an intracellular store following cross-linking of CD11b or CD18 in neutrophils. 23 Thus, it is reasonable to suppose that adhesion to the plastic substrate may cause a transient increase in intracellular calcium, which leads to tyrosine phosphorylation of Syk kinase and activation of PKC. The latter event further leads to slower electrophoretic mobility or serine/threonine phosphorylation of paxillin. In summary, adhesion to the plastic substrates caused tyrosine phosphorylation of Syk, and treatment with erbstatin, which inhibited Syk tyrosine phosphorylation, reduced the adherence of AMs to the plastic dish. 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