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The Journal of Immunology, 2004, 173: 1313-1320.
Copyright © 2004 by The American Association of Immunologists

TNF-Induced {beta}2 Integrin Activation Involves Src Kinases and a Redox-Regulated Activation of p38 MAPK1

Mohamed Bouaouina, Eric Blouin, Lise Halbwachs-Mecarelli, Philippe Lesavre and Philippe Rieu2

Institut National de la Santé et de la Recherche Médicale U507, Necker Hospital, Paris, France


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
We previously demonstrated that the TNF-{alpha}-induced inside-out signaling leading to {beta}2 integrin activation is redox regulated. To identify kinases involved in this pathway, the effects of kinase inhibitors on the expression of {beta}2 integrin activation neoepitope (clone 24) were investigated. We show that both p38 MAPK (inhibited by SB203580) and Src kinases (inhibited by PP2) are involved in {beta}2 integrin activation by TNF and oxidants in human neutrophils. Src kinases appeared constitutively active in resting neutrophils and not further activated by TNF or oxidants in nonadherent conditions. However, PP2 blocked both TNF-induced expression of the 24 epitope and cell adhesion promoted by the integrin activating anti-CD18 KIM185 mAb, showing that both the inside-out and the outside-in signaling involve Src kinases. p38 MAPK was activated by TNF and oxidants in nonadherent conditions i.e., with 10 mM EDTA. This activation in EDTA resulted in CD11b, CD35 and CD66 up-regulation and in an oxidative response, all blocked by SB203580 and PP2. p38 MAPK was not activated upon direct integrin activation by KIM185 mAb. Thus, p38 activation allows the study to distinguish the initial transduction pathway leading to {beta}2 integrin activation from the signaling resulting from integrin engagement. Finally, p38 MAPK activation by TNF was blocked by diphenylene iodonium, an inhibitor of flavoprotein oxidoreductase, and by the free radical scavenger N-acetylcystein. Taken together, these results demonstrate, for the first time, that constitutively activated Src tyrosine kinases and a redox-regulated activation of p38 MAPK are involved in TNF inside-out signaling leading to {beta}2 integrin activation.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Polymorphonuclear leukocytes play a central role in immunity against pathogens as well as being the major cellular effectors of inflammation and tissue injury. Over the last decade, it has been appreciated that {beta}2 integrin-mediated adhesive interactions are essential in regulating the functions of these inflammatory cells (1, 2).

The {beta}2 integrin CD11/CD18 subclass is composed of four heterodimeric leukocyte surface molecules (CD11a, CD11b, CD11c, CD11d/CD18) that contain a common {beta}-chain (CD18) noncovalently associated with a unique {alpha}-chain (CD11a, CD11b, CD11c, CD11d). CD11a/CD18 is expressed on all leukocytes. CD11b and CD11c expression is restricted to myelomonocytic cells and NK cells. {beta}2 integrins mediate cell contact to immobilized ligands but also "outside-in" signaling and integrin-cytoskeleton interactions required for stable cell adhesion and adhesion-dependent functions such as migration, oxidative burst, and phagocytosis.

{beta}2 integrins on the cell surface need to be activated to acquire their ligand-binding capacity. Integrin activation involves both a conformational change in the {alpha}{beta} heterodimer and the clustering in the plasma membrane of laterally diffusing integrins. The latter effect, which increases the valency (avidity) of integrins vs their substrate, has sometimes been proposed as the essential event regulating integrin activation (3, 4). In the last few years, however, the importance of conformational changes, increasing the affinity of integrins for their ligand, has been re-emphasized (reviewed in Refs. 5 , 6). Recent observations of integrin {alpha}V{beta}3 x-ray crystal structure and its appearance by electron microscopy (7, 8) have allowed us to draw a precise model for these conformational changes; in the inactive conformation, integrins show a very peculiar V-shape, with both {alpha}- and {beta}-chains appearing as two bent legs with intracellular tails very close together and head pieces facing down toward the membrane. Integrin activation results in the separation of {alpha} and {beta} intracellular tails, whereas the closed conformation of {alpha} and {beta} head pieces opens, with a motion like a "switchblade," to reach an extended conformation (9, 10). Site-directed mutagenesis experiments have shown that the switchblade model applies to {beta}2 integrins as well, and underscored the contribution of {alpha} and {beta} I and I-like domains (or {alpha}A and {beta}A domains) in the affinity regulation (11). Conformational changes upon {beta}2 integrin activation are evidenced by the expression of activation epitopes such as the 24Ag (12), recently mapped in the in {beta}2 I-like domain (13, 14). The intracellular pathway leading to {beta}2 integrin activation is referred to as integrin "inside-out" signaling (15).

Stimulation of neutrophils by TNF induces {beta}2 integrin activation and adhesion-dependent functions such as spreading, degranulation, and oxidative burst. Adhesion lead to outside-in signaling that may facilitate signals generated by TNF, transmit independent signals that act in concert with TNF, or transmit signals that act independently of TNF. These three possibilities are not mutually exclusive (2). Many intracellular kinases are known to be activated by TNF in neutrophils (2, 16, 17, 18, 19). It is not always clear, however, in the literature whether these kinases are activated by a signaling pathway generated by TNF itself or by an outside-in integrin signaling, with the role of TNF being simply to activate adhesion. In addition, many adhesion-dependent functions induced by TNF are blocked by specific kinase inhibitors (2, 16, 17, 18). Because of the bidirectional integrin signaling, it is not clear, however, whether these inhibitors block the signaling pathway that lead to {beta}2 integrin activation or the signaling pathway that result from {beta}2 integrin engagement. Therefore, to determine the kinases involved in TNF inside-out signaling leading to {beta}2 integrin activation, we used two complementary approaches. First, we tested specific kinase inhibitors on the TNF-induced appearance of 24Ag activation epitope. Second, we measured kinase activation stimulated by TNF in the absence of adhesion, i.e., in the presence of EDTA. In addition, we studied the kinases that are redox-regulated because we had shown that reactive oxygen intermediates (ROI)3 are potential signaling molecules that control tyrosine phosphorylations during TNF-{alpha}-induced {beta}2 integrin activation (20). Our results show that constitutively activated Src tyrosine kinases and a redox-regulated activation of p38 MAPK are involved in TNF inside-out signaling leading to {beta}2 integrin activation in human neutrophils.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Reagents

Diamide, gelatin, BSA, EDTA, diphenyleneiodonium chloride (DPI), 2'-7'-dichlorofluorescin hydrodiacetate (DCFHDA), PMA, sodium orthovanadate, aprotinin, leupeptin, PMSF, phenylarsine oxide (PAO), and N-acetylcysteine (NAc) were from Sigma-Aldrich (St. Louis, MO). TNF-{alpha} was from PeproTech (Rocky Hill, NJ). SB203580, SB202474, 4-amino-5-(-4-chlorophenyl)-7-(-t-butyl)pyrazolo[3,4-d]pyramidine (PP2), and PP3 (4-amino-7-phenylpyrazol[3,4-d]pyrimidine) were from Calbiochem (Darmstadt, Germany), Triton X-100 was from Pierce (Rockford, IL), and hydrogen peroxide was from Merck (Darmstadt, Germany). The 96-well plates were obtained from ATGC Biotechnologies (Noisy le grand, France). FITC-labeled anti-CD11b, anti-CD35, anti-CD66b, control FITC-IgG1, FITC-F(ab')2 of goat anti-mouse IgG, and HRP-labeled secondary Ab were from Immunotech (Beckman Coulter, Roissy, France). Monoclonal Ab 24 for the active conformation of CD11/CD18 and the integrin-activating anti-CD18 mAb KIM185 were gifts from N. Hogg (Imperial Cancer Research Fund, London, U.K.) and M. Robinson (CellTech, Cambridge, U.K.), respectively. Rabbit anti-p55/56lyn was from Chemicon (Temecula, CA); rabbit anti-p59hck and rabbit anti-p55fgr were from Santa Cruz Biotechnologies (Santa Cruz, CA); rabbit anti-p38 MAPK and anti-phosphorylated threonine and tyrosine of p38 MAPK, and rabbit anti-MAPK-activated protein kinase 2 (MK-2) and anti-phosphorylated MK-2 were from Cell Signaling, (Beverly, MA), whereas the polyclonal phospho-specific anti-Src (pY418) Ab was from BioSource International (Camarillo, CA).

Neutrophil isolation

Human neutrophils were prepared, as described (21) by sequential platelet depletion, centrifugation on Polymorphprep (Nycomed, Oslo, Sweden), and hypotonic lysis of residual erythrocytes. They were resuspended in PBS, as previously mentioned.

Adhesion assays

Adhesion assays were performed as previously described (20). A total of 2 x 105 neutrophils in TBS containing Mg2+/Ca2+ 1 mM were allowed to adhere in 96-well plates coated with 1% gelatin. Cells were first preincubated with 1 µM SB203580, 10 µM PP2, or buffer alone during 30 min at room temperature. TNF-{alpha} (20 ng/ml), diamide (1 mM), or KIM185 mAb (8 µg/ml) was then added and after 30 min at 37°C, nonadherent cells were removed and wells were washed with binding buffer. After examination by light microscopy, adhesion was quantified by titration of adherent cells with a Micro BCA Protein Assay (Pierce). Adhesion of diamide-treated cells was normalized for the adhesion obtained with TNF-{alpha}-treated (20 ng/ml) cells in the same microtiter plate, as previously described (20).

Flow cytometry

Expression of the 24 active conformation epitope. For efficient binding, the anti-24 mAb has to be preincubated with the cells at 37°C in Mg2+ buffer and to be present during the whole activation step with TNF. FcR{gamma} were not blocked in this assay because aggregated Igs during this incubation at 37°C would result in FcR{gamma} triggering and neutrophils activation. The anti-24 mAb was previously centrifuged for 15 min at 12,000 x g to remove aggregates and an irrelevant mouse IgG1 control was included as a negative control. A total of 2.5 x 105 neutrophils in 200 µl TBS 0.1% BSA, Mg2+ 1 mM were preincubated with 1 µM SB203580, 10 µM PP2, or buffer alone for 30 min at room temperature. Cells were then incubated with mAb 24 or IgG1 for 10 min at 37°C and finally treated with TNF-{alpha} (20 ng/ml), diamide (1 mM), or buffer for 10 min at 37°C. Cells were washed in ice-cold PBS, 1% BSA, 0.1% sodium azide, incubated with FITC-F(ab')2 of goat anti-mouse IgG at 4°C for 30 min, washed again, fixed with 1% formaldehyde and then analyzed for fluorescence on a FACScan flow cytometer (BD Immunocytometry Systems, Mountain View, CA). As for adhesion assays (20), the results of 24Ag expression, given as mean fluorescence intensity (MFI), were normalized for the expression obtained with TNF (without inhibitors) in the same experiment; 24Ag expression as a percentage of TNF control = (24Ag MFI obtained with TNF and inhibitors) x 100/(24Ag MFI obtained with TNF alone).

Analysis of CD11b, CD35, and CD66 up-regulation. A total of 5 x 105 neutrophils in 500 µl of TBS with 10 mM EDTA, preincubated or not with SB203590 or PP2 as previously described, were treated with 20 ng/ml TNF for 15 min at 37°C. Cells were washed with ice-cold PBS, 1% BSA, 0.1% sodium azide and incubated 20 min with heat-aggregated goat IgG (1 mg/ml) to block FcR{gamma}, at 4°C and in the presence of sodium azide to prevent FcR{gamma} redistribution and activation. Cells were then treated at 4°C with FITC-labeled anti-CD11b, anti-CD35, or anti-CD66 mAbs or a control FITC-labeled mouse IgG1 and analyzed by flow cytometry, as previously described.

Western blot analysis of active Src family kinase tyrosine phosphorylation in whole cell lysates

A total of 2 x 106/ml neutrophils were treated with TNF-{alpha} at 37°C and the reaction was stopped by transferring cells to precooled (–20°C) tubes as described (22). Cells were rapidly centrifuged, resuspended in boiling reduced sample buffer (0.1 M Tris pH 6.8, 4% SDS, 1.8 mM glycerol, 1.5 mM 2-ME, and bromophenol blue) and immediately boiled for 10 min. Proteins were then separated by SDS-PAGE electrophoresis (10% acrylamide) and transferred onto a nitrocellulose membrane. Nonspecific sites were blocked with a 5% (w/v) BSA solution. The active state of Src kinases was analyzed using a phospho-specific polyclonal anti-Src (pY418) Ab (23), directed against the positive autophosphorylation site common to all Src family kinases (24). An HRP-labeled anti-rabbit secondary Ab was then used and the bands were detected with the ECL system from Amersham Biosciences (Buckinghamshire, U.K.). To further characterize the Src-pY418 bands, blots were stripped for 2 h at 55°C in Tris pH 6.7, 2% SDS, 100 mM 2-ME, washed, and reprobed with rabbit anti-Src kinase (Fgr, Lyn, and Hck) Abs followed by an HRP-labeled anti-rabbit secondary Ab.

Analysis of threonine and tyrosine phosphorylation of p38 MAPK

Neutrophils (106) were either directly treated with TNF-{alpha}, diamide, or H2O2 for the indicated period of time, or preincubated with various inhibitors, then treated with TNF-{alpha} for 15 min. Cells were centrifuged and immediately boiled for 10 min in reduced sample buffer. Western blot analysis was performed as previously described using a rabbit Ab to phosphorylated threonine and tyrosine of p38 MAPK, followed by an HRP-labeled anti-rabbit secondary Ab. To quantify the total p38 MAPK, blots were stripped as described and reprobed with a phosphorylation-state-independent anti-p38 MAPK Ab.

To study p38 activation during adhesion and spreading triggered by KIM185 anti-CD18 activating Ab, 6 x 106 cells were allowed to adhere to gelatin-coated 12-well plates for 30 min at 37°C. Cell adherence and spreading were controlled by light microscopy. Nonadherent cells were collected, centrifuged, resuspended in boiling reduced sample buffer, then added again to adherent cells, and the total cell lysate was boiled for 10 min before analysis of p38 activation state by Western blot, as described earlier.

Measure of p38 kinase activity on MK-2

The assay was based on the specific phosphorylation of MK-2 by p38 (25). A total of 2 x 106/ml neutrophils in 10 mM EDTA containing TBS were preincubated for 30 min at room temperature without or with SB203580 at various concentrations. TNF (20 ng/ml) was then added for 20 min at 37°C. The reaction was stopped by transferring cells to precooled (–20°C) tubes, rapidly centrifuged, resuspended in boiling reduced sample buffer and boiled for 10 min. After PAGE electrophoresis on 10% acrylamide gel and transfer, blots were revealed with an anti-phosphorylated MK-2 followed by an HRP-labeled anti-rabbit secondary Ab. To quantify MK2 total amounts, blots were stripped as previously described and reprobed with a phosphorylation-state-independent anti-MK2 Ab. Results are expressed as phospho-MK-2 to total MK-2 ratios.

Kinase assay for Src kinases

Cell pellets from 107 neutrophils, activated with TNF-{alpha} or diamide in nonadherent conditions as earlier described, were resuspended in lysis buffer (25 mM Tris pH 7.5, 0.15 M NaCl, 1% Triton, 5 µg/ml leupeptin, 5 µg/ml aprotinin, 1 mM PMSF, 100 µM sodium orthovanadate, 0.25% deoxycholate, and 0.1% SDS), incubated for 30 min at 4°C and then centrifuged at 13,000 x g for 20 min. Supernatants were immunoprecipitated with the appropriate Ab and protein A-Sepharose (10 µg Ab per 10 µl beads; Amersham, Little Chalfont, U.K.) for 3 h at 4°C. Sepharose beads were washed three times in lysis buffer and immunoprecipitated Lyn, Fgr, and Hck were incubated in 50 µl of kinase buffer assay (25 mM HEPES, 10 mM MnCl2, and 10 µCi [{gamma}-32P]ATP (3000 mCi/mmol); NEN Life Sciences, Paris, France) for 10 min at 30°C. Reduced sample buffer was then added in each samples and immediately boiled for 10 min. The 32P-labeled proteins were separated on a 7.5% SDS-polyacrylamide gel, transferred onto a nitrocellulose membrane and visualized by autoradiography.

Assessment of the cell oxidative response

A total of 0.5 x 106 neutrophils in 500 µl of TBS with 10 mM EDTA were preincubated with SB203580, PP2, or buffer for 30 min at room temperature, then with DCFHDA (5 µM) for 10 min at 37°C and finally with TNF (20 ng/ml) or PMA (10 ng/ml). After a further 30 min incubation at 37°C, DCFHDA intracellular oxidation was assessed by flow cytometry, as previously described (20).

Statistical analysis

The amount of cell adhesion and 24Ag expression, the intensity of p38, PY, or Src kinase bands of Western blots in samples incubated with or without inhibitors, were compared using a paired t test. Statistical significance was defined as *, p < 0.05, **, p < 0.01, and ***, p < 0.001 as indicated.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
p38 MAPK and Src kinases are involved in {beta}2 integrin inside-out signaling induced by TNF and oxidants

Stimulation of human neutrophils with TNF-{alpha} (20 ng/ml) or diamide induced (1 mM) {beta}2 integrin activation, as shown both by CD11/CD18-dependent adhesion to immobilized gelatin (Fig. 1, A and B) and mAb 24 epitope expression (Fig. 1, C and D). Neutrophil adhesion to immobilized gelatin was blocked by clone 44 anti-CD11b Ab (Ref. 20 and data not shown). Monoclonal Ab 24 binds only to activated {beta}2 integrin (12, 14).



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  		FIGURE 1. {beta}2 integrin-dependent neutrophil adhesion and {beta}2 integrin activation by TNF-{alpha} and oxidant involve Src kinases and p38 MAPK. Neutrophils were preincubated (30 min at room temperature) with Src kinase inhibitor PP2 (10 µM or the indicated concentration) or p38 MAPK inhibitor SB203580 (1 µM or the indicated concentration) then activated with TNF-{alpha} (20 ng/ml) (A and C) or diamide (1 mM) (B and D). Cell adhesion to gelatin-coated plates was measured (A and B) and the expression of 24 Ag activation epitope was analyzed by flow cytometry (C and D). The dose response of SB203580 inhibition of p38 kinase activity was measured by Western blot analysis of phosphorylated MK-2 vs total MK-2 as described in Materials and Methods (Ea and Eb). The inhibition by SB203580 and PP2 of TNF-induced adhesion (Ec and Ed, {blacksquare}) and of TNF-induced {beta}2 integrin activation (Ec and Ed, {square}) was dose-related. Neutrophil adhesion and 24 Ag expression were expressed as a percentage of adhesion or 24 Ag expression induced by TNF-{alpha} in the absence of inhibitors and results are expressed as mean ± SD of three experiments. Each mAb24 flow cytometry histogram shows a representative experiment of three similar experiments.

 
Because we have previously observed that TNF- and oxidant-triggered {beta}2 integrin activation in neutrophils involves protein tyrosine phosphorylation (20), we tested the effects of p38 MAPK inhibitor (SB203580), MAPK/ERK-activating enzyme inhibitor (PD098059), and Src family tyrosine kinase inhibitor (PP2). No effect was observed with PD098059 up to 40 µM (data not shown). By contrast, nanomolar concentrations of SB203580 inhibited neutrophil TNF-{alpha}-induced adhesion to gelatin (Fig. 1, A and Ec), confirming previous data (16) and expression of mAb 24 epitope (Fig. 1, C and Ec). SB203580 at high concentration may inhibit other kinases than p38 (unlike PP2, considered as a specific Src kinase inhibitor). To attribute the inhibition of {beta}2 integrin activation to p38 blocking, we therefore compared the dose-related inhibition of TNF-induced adhesion and mAb 24 epitope expression (Fig. 1Ec) with a dose-response curve of SB203580 inhibition of the phosphorylation of MK-2, a p38-specific substrate (Fig. 1, Ea and Eb). The IC50 of SB203580 was similar (40–150 nm) for the inhibition of adhesion, 24Ag expression, and p38 kinase activity. As a negative control, up to 10 µM of the inactive analogue SB202474 was without effect on TNF-induced adhesion or 24Ag expression (data not shown). Similarly, PP2 (but not its inactive analogue PP3; data not shown) inhibited neutrophil adhesion to gelatin (Fig. 1A) and expression of mAb 24 epitope (Fig. 1C) induced by TNF-{alpha} in a dose-dependent manner (Fig. 1Ed). Furthermore, neutrophil adhesion to gelatin and expression of mAb 24 epitope induced by diamide (1 mM) (Fig. 1, B and D) and H2O2 (10 mM) (data not shown) were also inhibited by SB203580 (1 µM) and PP2 (10 µM). These results show that {beta}2 integrin activation by TNF and directly by oxidants involves p38 MAPK and Src kinases in human neutrophils.

p38 MAPK is activated by TNF and oxidants in nonadherent conditions

Because p38 MAPK is involved in {beta}2 integrin activation induced by TNF and oxidants, we analyzed the phosphorylation of p38 in response to these agonists. Neutrophils in suspension were activated in gelatin-coated tubes and in the absence of Mg2+ to prevent adhesion. Following stimulation, the activation of cellular MAPK was evaluated in cell lysates by Western blot using rabbit Abs to phosphorylated p38 MAPK. Cellular p38 MAPK became phosphorylated in nonadherent neutrophils in response to stimulation by TNF-{alpha} (20 ng/ml), diamide (1 mM), and H2O2 (10 mM) (Fig. 2). Phosphorylation of p38 was detected after 10 mn for TNF, 5 mn for diamide, and 1 mn for H2O2. The peak of p38 phosphorylation declined after 60 mn for TNF, 15 min for diamide, and 5 min for H2O2.



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  		FIGURE 2. Activation of p38 MAPK by TNF and oxidants. Neutrophils were stimulated with TNF-{alpha} (20 ng/ml), diamide (1 mM), or H2O2 (10 mM) for the indicated period of time. Proteins were separated on a 10% polyacrylamide SDS gel and analyzed by Western blot using a specific Ab to phosphorylated threonine and tyrosine of p38 MAPK (upper panel of each stimulus). Blots were then stripped and reprobed with a phosphorylation-state-independent anti-p38 MAPK Ab to show the amounts of p38 MAPK (lower panel of each stimulus). Each blot shows a representative experiment from at least three similar experiments.

 
Spontaneous activation of Src family tyrosine kinases and lack of activation by TNF and oxidants in nonadherent conditions

Because Src family tyrosine kinases were also involved in {beta}2 integrin activation, we tested the autophosphorylation activity of the three major Src family tyrosine kinases present in neutrophils, after immunoprecipitation of each kinase; Fgr (Fig. 3A), Hck (Fig. 3B), and Lyn (Fig. 3C) in response to TNF and oxidants in nonadherent conditions, i.e., in the absence of Mg2+ as described in Materials and Methods. All three Src kinases were constitutively active in resting neutrophils. Statistical analysis of the scanned bands from 10, five, and six experiments for Fgr (Fig. 3A), Hck (Fig. 3B), or Lyn (Fig. 3C) blots, respectively, resulted in no significant increase of the phosphorylated forms by TNF or diamide.



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  		FIGURE 3. Src family tyrosine kinases are constitutively active in neutrophils and do not appear to be further activated by TNF or oxidants in nonadherent conditions. Neutrophils were stimulated with TNF-{alpha} (20 ng/ml) for 15 min or diamide (1 mM) for 5 min. Cells were then solubilized and Lyn, Hck, and Fgr were immunoprecipitated and submitted to in vitro protein tyrosine kinase activity assay as described in Materials and Methods. Autoradiograms show the autophosphorylation of Lyn (A), Hck (B), or Fgr (C) immunoprecipitates (107 cell equivalents/lane) of one representative experiment, whereas the lower panels show the statistical comparison of the scanned bands (expressed as a percentage of the unstimulated control) from 10, 5, and 6 similar experiments for Fgr, Hck, and Lyn analysis respectively (NS = p > 0.05). D, Neutrophils, pretreated or not for 30 min with 10 µM PP2, were incubated for 15 min in 10 mM EDTA at 37°C, without (control) or with TNF-{alpha} (20 ng/ml). Cells were immediately boiled in sample buffer and cell lysates analyzed by Western blot, as described in Materials and Methods. Blots were revealed with a phospho-specific anti-Src (pY418) Ab (right, phospho-Src (pY418)), stripped and analyzed with anti-Lyn, anti-Frg, or anti-Hck Abs (left, total Src) shows the control lanes without PP2 or TNF. The lower part shows a statistical analysis of scanned bands, expressed as phospho-Src to total Src ratios, for each Src family kinase and calculated from seven experiments (mean ± SEM).

 
Naccache and group (22) recently emphasized the inadequacy of using standard cell solubilization methods to analyze protein tyrosine phosphorylation in neutrophils. This finding is particularly true for Src kinases, which redistribute in detergent-resistant membrane structures or lipid rafts upon neutrophil activation (26) and are poorly solubilized by common lysing buffers. To avoid these difficulties, we analyzed by Western blot analysis the tyrosine phosphorylation of Src family kinases in neutrophils, treated or not with TNF (20 ng/ml for 15 min) in nonadherent conditions (EDTA 10 mM), and immediately boiled in sample buffer containing protease and phosphatase inhibitors, to obtain a whole cell lysate (Fig. 3D). However, tyrosine phosphorylation in Src family kinases may represent the "activating" autophosphorylation of the active site tyrosine (Tyr418 for c-Src) but also the "inhibitory" phosphorylation of another tyrosine, located in the carboxyl-terminal tail that binds to the Src homology 2 domain of the molecule in its inactive form (23, 24). We thus used a phospho-specific polyclonal Ab (anti-Src (pY418)) that allows us to specifically detect the phosphorylated tyrosine located in the activation loop and required for Src family kinase activity. As shown in Fig. 3D, neutrophils lysates from control neutrophils, incubated for 15 min at 37°C in EDTA as previously described, contained several bands recognized by this Ab (phospho-Src (pY418)). They were identified as Fgr, Lyn, and Hck bands, after blots stripping and labeling with specific anti-Src kinase Ab (total Src, Fig. 3D, left). All these bands were significantly decreased if the Src inhibitor PP2 was present during neutrophil incubation. This shows that Src kinases are constitutively able to autophosphorylate their active site tyrosine. TNF had no effect whatsoever on the active Src kinase band intensities, as shown by the statistical analysis of the scanned bands from seven experiments (Fig. 3D, bottom panel).

p38 MAPK is not involved in the {beta}2 integrin outside-in signaling

To determine the role of p38 MAPK in the post binding events induced by {beta}2 integrin outside-in signaling that lead to strong neutrophil adhesion, we tested the effect of SB203580 on neutrophil adhesion directly promoted by anti-CD18-activating Abs (KIM185 mAb), without requiring an inside-out signaling. PP2 was used as a control because the central role of Src family tyrosine kinases in {beta}2 integrin outside-in signaling is well established (17, 27, 28). As shown in Fig. 4A, PP2 partially inhibits neutrophil adhesion induced by KIM185. By contrast, up to 10 µM SB203580 had no significant effect on the adhesion induced by KIM185, suggesting that p38 MAPK is not involved in the post binding events necessary for strong adhesion. Furthermore, TNF-induced p38 MAPK activation still takes place in the presence of EDTA (Fig. 4B and Fig. 1Ea) and we did not observe any activation of p38 MAPK when the inside-out signaling pathway was bypassed by KIM185 anti-CD18 activating Abs (added for 5, 15, or 30 min, data not shown) in suspension (Fig. 4B) or in adherence and spreading conditions (Fig. 4C). These results clearly show that p38 MAPK is not involved in the {beta}2 integrin outside-in signaling in human neutrophils.



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  		FIGURE 4. Srk kinases but not p38 MAPK are involved in the outside-in signaling promoted by integrin-engagement. A, Adhesion of neutrophils, preincubated or not with 10 µM SB203580 or PP2, then stimulated with KIM185 (8 µg/ml), was assessed after 30 min at 37°C, as described in Materials and Methods. B, p38 MAPK activation in suspension. Neutrophils were activated for 30 min at 37°C in BSA-coated tubes, either with TNF in 10 mM EDTA (TBSEDTA) or with TNF or KIM185, in Ca2+/Mg2+ containing buffer (TBSCa2+Mg2+). C, p38 MAPK activation in adherent neutrophils on gelatin-coated plates after stimulation by either TNF or KIM185 in Ca2+/Mg2+ containing TBS.

 
p38 MAPK and Src kinases are involved in adhesion-independent functions induced by TNF

To study putative adhesion-independent neutrophil functions induced by TNF, we analyzed the free radical production and degranulation in the presence of 10 mM EDTA. TNF adhesion-independent signaling, in the presence of 10 mM EDTA, resulted in oxidation of intracellular DCFHDA (Fig. 5A). This intracellular production of H2O2 was inhibited by SB203580 (1 µM) and PP2 (10 µM) and by the flavine oxidase inhibitor DPI (data not shown). TNF also triggered, in similar nonadherent conditions, the up-regulation of CD11b (Fig. 5B). This reaction was inhibited by SB203580 and PP2 and resulted from secretory vesicle and possibly secondary granule exocytosis, as shown by the up-regulation of complement receptor type 1 (CR1; CD35) and of the carcinoembryonic, Ag-related cell adhesion molecule 8 (CD66b), respectively (Fig. 5B). By contrast, the lysosomal tetraspanin CD63, azurophilic granule marker, was not mobilized to the plasma membrane (data not shown). These results demonstrate that p38 MAPK and Src family tyrosine kinases mediate adhesion-independent functions triggered by TNF.



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  		FIGURE 5. Adhesion-independent functions triggered by TNF. Neutrophils in TBS containing 10 mM EDTA, were preincubated for 30 min with 10 µM PP2 or 1 µM SB203580 or medium, then activated 15 min with TNF-{alpha}. A, The oxidative response was assessed by flow cytometry using DCFHDA, as described in Materials and Methods. It was compared with that obtained with 50 ng/ml PMA. Results are expressed as oxidative response index: (x – Ctr)/Ctr x 100. B, Up-regulation of neutrophil membrane markers CD11b, CD35, and CD66b was measured by flow cytometry with FITC-labeled anti-CD mAbs.

 
p38 MAPK activation by TNF is redox-regulated

We have previously shown that the signaling pathway leading to {beta}2 integrin activation after TNF-{alpha} stimulation is redox-regulated. To determine whether free radicals play a role in p38 MAPK activation during neutrophil stimulation by TNF-{alpha}, we either blocked their production with the flavoproteine oxidase inhibitor DPI (29) or we neutralized them with the free radical scavenger, NAc, or we blocked the oxidative S-thiolation with PAO. Activated p38 was assessed by Western blot using an anti-phosphorylated p38 Ab, as described, and the total amount of p38 was measured on the same blots, after stripping, with an anti-whole p38 Ab. p38 bands were scanned and phospho-p38 to total p38 ratios were statistically analyzed. As shown in Fig. 6, DPI (18 experiments), NAc (11 experiments), and PAO (15 experiments) significantly inhibited TNF-induced p38 MAPK activation. These results are consistent with the activation of p38 MAPK observed with oxidants (Fig. 2).



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  		FIGURE 6. Redox regulation of TNF-induced p38 MAPK activation. Neutrophils were preincubated with either DPI (10 µM) (A and D), NAc (25 mM) (B and D), or PAO (1 µM) (C and D), then activated with TNF-{alpha} for 15 min. Cells were lysed with boiling sample buffer and analyzed by Western blot for p38 phosphorylation, as described in Fig. 2, A–C. Statistical analysis of scanned bands, expressed as phospho-p38 to total p38 ratios, were calculated from 18 (A), 11 (B), and 15 (C) experiments. D, Two representative experiments to illustrate statistical data.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The present work shows that 1) Src kinases, which are known to be involved in TNF-activated {beta}2 integrin outside-in signaling and adhesion-dependent functions, also mediate the inside-out signaling leading to {beta}2 integrin activation; 2) p38 MAPK is involved in the {beta}2 integrin inside-out signaling triggered by TNF, but not in the outside-in signaling resulting from integrin engagement; and 3) p38 MAPK activation, in response to TNF, involves an oxidative step.

Role of Src family tyrosine kinases in {beta}2 integrin activation triggered by TNF

Evidence has been accumulated that Src family tyrosine kinases play an essential role in the outside-in signaling resulting from {beta}2 integrin engagement (2, 17, 28, 30, 31). Src family kinases are involved in adhesion-dependent degranulation (17), ROI production, and neutrophil spreading (30). The present study provides the first evidence that Src family kinases are also involved in the inside-out signaling leading to {beta}2 integrin activation. Indeed, we added to usual adhesion assays, which measure a multicomponent process involving cell spreading (a post binding event), the analysis of the expression of the 24 epitope, specific for {beta}2 integrin active conformation (12). This combination allows us to clearly distinguish the role of kinases in the integrin activation process from their involvement in the pathways triggered by {beta}2 integrins engagement. In this study we show that the Src inhibitor PP2 inhibits both the adhesion and the 24 epitope expression, in a dose-related manner. This new finding suggests a model in which Src family kinases are involved in a first signaling wave promoted by TNFR engagement and then in a second wave of outside-in signaling by {beta}2 integrin engagement. The known participation of Src kinases to this second wave is illustrated by our results obtained with the KIM185 anti-CD18 mAb. The epitope recognized by the activating anti-{beta}2 KIM185 Ab was recently mapped to the C-terminal "stalk" region of {beta}2 extracellular domain, and KIM185 is thought to activate integrins by artificially keeping {alpha} and {beta} subunit stalk-regions apart (13). One may reasonably postulate that this mechanical activation does not require intracellular signaling and KIM185 promotes some Src-independent adhesion in the presence of PP2 (Fig. 4A). PP2 partial inhibition of KIM185-induced adhesion thus probably relates to the participation of Src kinases to {beta}2 integrin outside-in signaling, leading to cytoskeleton reorganization, cell spreading, and thus stronger adhesion.

We were not able to characterize the Src kinases involved in the inside-out signaling and previous data showed no activation of Fgr or Lyn by TNF in the absence of Mg2+ or in the presence of anti-CD11b/CD18 blocking Abs (28). The analysis of autophosphorylation activities of immunoprecipitated Src kinases showed a constitutive activation of the three Src kinases, Fgr, Lyn, and Hck, which was not modified by TNF. This finding was confirmed by the specific measurement of Src kinase active site tyrosine phosphorylation in total cell lysate, obtained in conditions precluding inadequate Src kinase solubilization. This result may be due to difficulties in detecting a discrete activation by TNF in nonadherent conditions. Alternatively, TNF would not activate Src kinases but allow the access of constitutively activated kinases to a substrate involved in the inside-out signaling.

Role of p38 MAPKs in TNF-triggered signaling leading to {beta}2 integrin activation

The activation of p38 MAPK plays a central role in neutrophil activation. Indeed, p38 MAPK inhibitors have been shown to inhibit cell adhesion and chemotaxis in vitro (16, 18, 32) and to reduce in vivo neutrophil infiltration in numerous inflammatory processes (33, 34, 35, 36, 37). All agonists that promote {beta}2 integrin activation, such as fMetLeuPhe, TNF-{alpha}, LPS, or L-selectin cross-linking, have been shown to activate p38 in neutrophils, IL-8 being an interesting exception (38, 39). p38 MAPK was previously shown to be required for {beta}2 integrin-dependent neutrophil adhesion triggered by TNF, by using the MAPK inhibitor SB203580 (16, 18). We confirm through this study that SB203580 inhibits the adhesion triggered by TNF-{alpha}. More importantly, we show that, as with PP2, SB203580 inhibits the expression of the 24 epitope specific for {beta}2 integrin active conformation (Fig. 1). In addition, we show that p38 MAPK is still activated by TNF when neutrophil adhesion is prevented by EDTA 10 mM (Fig. 4B), unlike what had been previously described as an "adhesion-dependent" activation of p38 by TNF-{alpha} (16). These two results demonstrate that p38 is directly involved in TNF-triggered inside-out signaling leading to {beta}2 integrin activation.

By contrast, p38 is not activated when TNF triggering is bypassed by artificial activation of the integrin by the anti-CD18 KIM185 Ab. We show that the {beta}2 integrin-dependent adhesion process promoted by KIM185 is not affected by the MAPK inhibitor SB203580 (Fig. 4A).

We can conclude from these data that, although Src family kinases participate both to the inside-out signaling leading to {beta}2 integrin activation and to the secondary outside-in signaling wave resulting from integrin engagement and cell adhesion, p38 MAPK is specifically involved in the first wave of inside-out signaling and not in the outside-in pathway.

Adhesion-independent functions triggered by TNF

Several neutrophil functions promoted by TNF, such as cell spreading, oxidative burst, and degranulation have been shown to be adhesion-dependent; they require the outside-in signaling resulting from {beta}2 integrin ligation (40, 41). Because we found that TNF may induce activation of intracellular pathways in absence of adhesion, we addressed the question of possible adhesion-independent functions triggered by TNF. We thus looked for functions that would occur in EDTA 10 mM, preventing any integrin engagement, and that would be modulated both by p38 and Src inhibitors. We show that neutrophils stimulated by TNF-{alpha} in EDTA-containing buffer raise an oxidative response, as assessed by DCFHDA intracellular oxidation. This response is inhibited by PP2, by SB203580 (Fig. 5A), and by DPI (data not shown). These results are at odds with previous data showing an absence of oxidative response to TNF in nonadherent conditions (40, 42). This may be due to the extracellular assays used by these authors, which underestimate the cell production of ROI, as compared with the intracellular measure of DCFHDA peroxidation used in this study (43).

Similarly, the plasma membrane expression of intracellular granules membrane markers, such as the integrin CD11b/CD18 itself but also CD35 (CR1) and CD66b, markers of secretory vesicles and possibly tertiary granules (44), was enhanced after TNF stimulation in EDTA and this up-regulation was inhibited by SB203580 and PP2 (Fig. 5B). By contrast, CD63 expression was not modified, showing an absence of azurophilic granule mobilization, known to require a high degree of neutrophil activation (44). CD11b and/or CD35 up-regulation in the absence of extracellular Ca2+ (45) or in 5 mM EDTA (46) had previously been described when neutrophils are stimulated by fMLP, calcium ionophore A3187, or PMA. Again, one can speculate that it may be important for the cell to increase its functional potential before any firm adhesion. One should remind that this up-regulation does not reflect a specific granule exocytosis. Both processes result from different signaling pathways and differ by their Ca2+ requirements (45). Secondary granule exocytosis appears to be strictly adhesion-dependent and Syk–/– neutrophils, which are deficient in integrin signaling and adhesion-dependent functions, were recently shown to be defective in lactoferrin release but able to up-regulate CD11b expression in response to TNF (19).

Adhesion-independent functions and {beta}2 integrin activation triggered by TNF in neutrophils required activated Src kinases and activation of p38 MAPKs. Activation of other kinases such as Syk (19) and further activation of Src kinases generated by {beta}2 integrin engagement (27, 42) amplify the effect of TNF.

Redox regulation of p38 activation

We had previously shown that CD11b/CD18 activation by TNF-{alpha} is redox-regulated (20) and our study shows that the oxidative step is upstream of p38 MAPK activation. Indeed, p38 activation in response to TNF is inhibited by a free radical scavenger and by DPI (Fig. 6). Furthermore, we show that p38 MAPK is activated by oxidants in nonadherent neutrophils (Fig. 2) and is involved in oxidant-induced {beta}2 integrin activation (Fig. 1, C and F). p38 MAPK activation by TNF is regulated by redox state in endothelial cells (47). A very recent paper, describes the activation of p38 by H2O2 in murine bone-derived neutrophils (48). These observations suggest the occurrence of an oxidative step between TNF receptors and p38MAPKs.

The mechanism of oxidant-triggered activation of tyrosine or serine/threonine kinases may be the inhibition of protein phosphatases (49), and inactivation of phosphatases during the early events of TNF signal transduction have been reported (50). Redox sensitivity of the conserved cysteine in the common active site of tyrosine phosphatases provides a means by which these enzymes become inactivated by thiol-oxidizing agents (51). As far as serine/threonine phosphatases are concerned, their activity depends upon the oxidation state of an active site metal ion that is sensitive to redox regulation (52). Inactivation of serine/threonine phosphatases by oxidant may also involve the oxidation of conserved vicinal thiol groups (53).

In summary, we have demonstrated that Src family kinases and p38 MAPK participate to the {beta}2 integrin inside-out signaling, triggered by TNF, which precedes the adhesion process. Src and MAPK activities, in nonadherent conditions, also result in an oxidative response and an increased membrane expression of CD11b, CD35, and CD66b. p38 MAPK is specifically involved in the inside-out signaling leading to {beta}2 integrin activation and not in the outside-in signaling resulting from the adhesion process. P38 activation by TNF is redox regulated and this redox regulation may be important for the reversible switching of integrin adhesiveness, allowing the adhesion/de-adhesion cycles necessary for migration. Further investigations are required to determine electron transport systems involved in this pathway.

The mechanisms by which intracellular transduction pathways promote the integrin conformational changes are not known. As mentioned in the introduction, the separation of {alpha} and {beta} intracellular tails are a critical trigger for extracellular domains conformational changes (10, 11). Recent data reveal that integrin connections with the cytoskeleton, and in particular the interaction of the {beta} subunit cytoplasmic tail with talin, may directly participate to the switch to the active conformation (10, 54). Neutrophil stimulation by TNF-{alpha} triggers oxidation/reduction and phosphorylation/dephosphorylation events that presumably modulate the activity and/or subcellular localization of key enzymes and substrates involved in integrin activation. One could speculate that, among these events, the exposure of specific substrates to constitutively active Src kinases or oxidase(s) would result in cytoskeleton reorganization and modified interactions with integrins cytoplasmic tails, signals then transduced to the extracellular domains for a transition to the high affinity conformation.


   Acknowledgments
 
We are grateful to Dr. Nancy Hogg, Imperial Cancer Research Fund, London, U.K., and M. Robinson, CellTech, Cambridge, U.K., for the invaluable gifts of mAb 24 and mAb KIM185, respectively.


   Footnotes
 
1 This work was supported by a grant from the Association pour la Recherche sur la Polyarthrite Rhumatoïde, and by doctoral fellowships from the Ministère de la Recherche et de la Technologie (to E.B. and M.B.) and from the Association pour la Recherche contre le Cancer (to E.B.). Back

2 Address correspondence and reprint requests to Dr. Philippe Rieu, Department of Nephrology and Centre National de la Recherche Scientifique Unité Mixte de Recherche 6198, Hôpital Maison Blanche, 45 rue Cognacq-Jay, 51092 Reims, France. E-mail address: prieu{at}chu-reims.fr Back

3 Abbreviations used in this paper: ROI, radical oxygen intermediate; DPI, diphenyleneiodonium chloride; DCFHDA, 2',7'-dichlorofluorescin hydrodiacetate; NAc, N-acetylcysteine; PP2, 4-amino-5-(-4-chlorophenyl)-7-(-t-butyl)pyrazolo[3,4-d]pyramidine; PAO, phenylarsine oxide; MK-2, MAPK-activated protein kinase-2. Back

Received for publication July 22, 2003. Accepted for publication May 11, 2004.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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