FcγRs mediate immune complex-induced tissue injury. The hypothesis that FcγRIIa and FcγRIIIb control neutrophil responses by activating mitogen-activated protein kinases was examined. Homotypic and heterotypic cross-linking of FcγRIIa and/or FcγRIIIb resulted in a rapid, transient increase in ERK and p38 activity, with maximal stimulation between 1 and 3 min. FcγRIIa and FcγRIIIb stimulated distinct patterns of ERK and p38 activity, and heterotypic cross-linking failed to stimulate synergistic activation of either ERK or p38 activity. Both FcγRIIa and FcγRIIIb required activation of a nonreceptor tyrosine kinase and phosphatidylinositol 3-kinase for stimulation of ERK and p38. Inhibition of ERK activation with PD98059 enhanced H2O2 production stimulated by homotypic and heterotypic FcγR cross-linking. Inhibition of p38 with SB203580 attenuated H2O2 production stimulated by FcγRIIIb or heterotypic cross-linking, but had no effect on FcγRIIa-stimulated H2O2 production. On the other hand, PD98059 inhibited actin polymerization stimulated by FcγR cross-linking, while SB203580 had no effect. Inhibition of actin polymerization with cytochalasin D enhanced p38 activity stimulated by either FcγRIIa or FcγRIIIb, but cytochalasin D only enhanced H2O2 production stimulated by FcγRIIIb. Our data indicate that FcγRIIa and FcγRIIIb independently activate ERK and p38. The two receptors demonstrate different efficacies for ERK and p38 activation, and they do not act cooperatively. ERK and p38 provide stimulatory and inhibitory signals for neutrophil responses to immune complexes. In addition, these data indicate that actin reorganization may play a role in mediating p38-dependent activation of respiratory burst upon stimulation of FcγRIIIb in neutrophils.

Immune complex deposition produces diseases such as glomerulonephritis, rheumatoid arthritis, systemic lupus erythematosus, and vasculitis. Immune complexes are potent stimuli for neutrophil activation, leading to tissue damage through the release of reactive oxygen species generated by the respiratory burst and secretion of granular enzymes. Recent reports indicate that immune complex binding to receptors for the Fc region of IgG (FcγRs), is necessary for initiation of tissue injury. Mice in which the FcγR γ-chain gene was deleted (FcγRI and FcγRIIIa deficient) failed to develop immune complex-mediated vasculitis or glomerulonephritis (1, 2, 3, 4). Others showed that complement-deficient mice demonstrated normal inflammatory responses (5).

Human neutrophils express two structurally distinct FcγRs, FcγRIIa and FcγRIIIb (6, 7). FcγRIIa is a transmembrane receptor expressing an intracellular tyrosine activation motif (ITAM)3 in the cytoplasmic domain. This region is required for activation of intracellular signals (8, 9). ITAMs are recognized and phosphorylated by Src family of tyrosine kinases following cross-linking of FcγRs by immune complexes (10, 11). Phosphorylation of ITAMs results in activation of Syk family nonreceptor tyrosine kinases, which, in turn, activate several downstream effectors, including phosphatidylinositol 3-kinase (PI-3K) and phospholipase Cγ (12, 13). FcγRIIIb is GPI-linked to the plasma membrane and is expressed at a 10-fold higher density than FcγRIIa (14). Because of the absence of transmembrane and cytoplasmic domains (15), it has been proposed that FcγRIIIb acts cooperatively with complement receptor 3 (CR3) in the activation of respiratory burst activity (16) or with FcγRIIa in mediating phagocytosis or respiratory burst activity (17, 18, 19). However, cross-linking of FcγRIIIb alone stimulates intracellular calcium redistribution (20) and activates actin polymerization (17), respiratory burst activity (21), degranulation (22), and phagocytosis of Con A-coated particles (23). These results suggest that FcγRIIIb initiates stimulatory intracellular signals independent of FcγRIIa or CR3. However, the nature of these signals is unknown.

Mitogen-activated protein kinases (MAPKs) are a superfamily of proline-directed serine/threonine kinases. Three major families of MAPKs have been identified, c-Jun N-terminal kinases (JNKs), extracellular signal-regulated kinases (ERKs), and p38. ERK1/2 and p38 are activated in human neutrophils by cytokines (24), chemoattractants (25, 26, 27), and bacterial LPS (28). Although JNK is present in human neutrophils, proinflammatory stimuli do not increase JNK activity (24, 26). Pharmacologic inhibition of ERK and/or p38 MAPKs impairs neutrophil respiratory burst activity, adherence, and chemotaxis (24, 25, 26, 27, 29, 30, 31). Cross-linking of FcγRIIa activates ERK1/2 in neutrophils (32), and cross-linking of FcγRs activates all three MAPK families in murine macrophages (33). Our laboratory recently reported that phagocytosis of IgG-opsonized Staphylococcus aureus is associated with increased p38 and ERK activity in human neutrophils (31). Based on these observations, the present study examined the hypothesis that ERK and p38 play a central role in FcγR-dependent activation of neutrophils. Our results show that FcγRIIa and FcγRIIIb stimulate ERK and p38 with different efficacies. ERK activation is necessary for actin polymerization, but acts as a negative regulator of respiratory burst activity. Activation of p38 is necessary for respiratory burst activity stimulated by FcγRIIIb, but not by FcγRIIa.

PD98059 and SB203580 were obtained from Calbiochem (La Jolla, CA). PD98059 was used at a final concentration of 50 μM, and SB203580 was used at a final concentration of 10 μM. Cytochalasin D, wortmannin, and genistein were obtained from Sigma (St. Louis, MO) and were used at final concentrations of 3 μM, 100 nM, and 30 μM, respectively.

Neutrophils were isolated from healthy donors using plasma-Percoll gradients (34). After isolation, neutrophils were washed and resuspended with LPS-free Krebs-Ringer phosphate buffer (pH 7.2) containing 0.2% dextrose (Krebs). Microscopic evaluation of isolated cells by trypan blue exclusion indicated that >95% of cells were neutrophils, and they were 95% viable.

Cross-linking of FcγRs was performed as described previously (35, 36, 37). Cells were incubated for 5 min at 37°C with 5 μg/ml anti-FcγRIIa Fab mAb (IV.3), 5 μg/ml anti-FcγRIIIb F(ab′)2 mAb (3G8), or both in the case of heterotypic cross-linking. IV.3 Fab and 3G8 F(ab′)2 were obtained from Medarex (Annandale, NJ). Excess mAb was removed by washing cells twice in Krebs with calcium (Krebs+). Then 35 μg/ml of a goat anti-mouse IgG (GAM), which is F(ab′)2 specific (Jackson ImmunoResearch Laboratories, West Grove, PA), was added as the cross-linking agent for the indicated times. Heterotypic cross-linking by this method was shown in past studies to lead to colocalization FcγRIIa and FcγRIIIb in the same cluster on the cell surface (36, 37).

ERK activity was detected by assaying the ability of immunoprecipitated enzyme to phosphorylate a substrate, myelin basic protein (MBP) (32). Following stimulation of cells, reactions were terminated by the addition of RIPA lysis buffer (4 mM PMSF, 1 mM EDTA, 1 mM EGTA, 50 mM Tris (pH 8.0), 150 mM NaCl, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS, 10 mM NaF, and 10 mM sodium pyrophosphate). Samples were centrifuged for 20 min at 14,000 × g at 4°C. Supernatants were incubated with ERK1 and ERK2 Abs (Santa Cruz Biotechnology, Santa Cruz, CA) for 1 h and with Sepharose A beads for an additional 1 h. Beads were washed once each with cold lysis buffer and cold kinase buffer (20 mM HEPES (pH 7.4), 10 mM MgCl2, and 1 mM DTT) and were resuspended in 40 μl of reaction mixture (250 μg/ml MBP, 20 μM ATP, and 250 μCi [γ-32P]ATP) for 30 min at room temperature. Reactions were terminated by the addition Laemmli buffer, and samples were boiled before separation by 15% SDS-PAGE. Products were visualized by autoradiography and quantified using ImageQuant (Becton Dickinson, Mountain View, CA).

ERK activity was also detected by measuring tyrosine phosphorylation of the MAPKs with specific antisera (25). Following stimulation, cells were lysed with RIPA and centrifuged. Laemmli buffer was added to supernatants, and samples were boiled. Proteins were separated by 10% SDS-PAGE, transferred onto nitrocellulose membrane, and blocked with 5% milk overnight. Blots were probed with specific phospho-ERK antisera (New England Biolabs, Boston, MA) followed by a peroxidase-conjugated, secondary Ab. Products were visualized by chemiluminescence and quantified by densitometry. To ensure equal loading of proteins in each lane, the blots were stripped and reprobed for total ERK with anti-ERK2 Ab (Santa Cruz Biotechnology).

p38 MAPK activity was measured by assaying the ability of immunoprecipitated enzyme to phosphorylate its substrate, ATF2 (25). Briefly, 1 × 107 neutrophils was preincubated for 5 min at 37°C before activation of FcγRIIa, FcγRIIIb, or both by cross-linking. The reaction was terminated by centrifugation at 2,500 × g. Cells were then lysed with RIPA lysis buffer and centrifuged at 15,000 × g for 15 min at 4°C. Cleared lysates were incubated with 4 μl of anti-p38 antisera, produced as previously reported (25), for 1 h at 4°C. Lysates were incubated with protein-Sepharose beads for an additional hour. Beads were washed once in RIPA lysis buffer and kinase buffer (1 M HEPES, 1 M DTT, 1 M MgCl2, 0.5 M β-glycerol phosphate, and 0.2 M Na2VO4) and incubated in reaction mixture (5 μCi of [γ-32P]ATP and 3 μg of recombinant ATF2). Reactions were incubated at 30°C for 30 min and terminated by the addition of Laemmli buffer. The samples were boiled, and products were resolved by 10% SDS-PAGE. Products were visualized by autoradiography and quantified using ImageQuant.

p38 activity was also measured by detection of tyrosine phosphorylation of p38 with specific antisera (New England Biolabs, Boston, MA) (25). Following stimulation, cells were lysed and centrifuged. Laemmli buffer was added to supernatants, and samples were boiled. Proteins were separated with 10% SDS-PAGE, transferred onto nitrocellulose membrane, and blocked with 5% milk overnight. Blots were probed with specific phospho-p38 and peroxidase-conjugated, secondary Ab (Vector, Burlingame, CA). Detected products were visualized by chemiluminescence and quantified by densitometry. To ensure equal loading of proteins in each lane, the blots were stripped and reprobed for total p38 with anti-rabbit p38 Ab (Santa Cruz Biotechnology).

Respiratory burst activity was measured by the ability of hydrogen peroxide to hydrolyze dichlorofluorescein (DCF) to its fluorescent analogue. Isolated neutrophils were resuspended in Krebs+ to a final volume of 4.5 × 106 cells/ml. Then, 900 μl of cell suspension was removed, placed in microcentrifuge tubes, and prewarmed in a 37°C water bath for 5 min. Cells were incubated with 5 μg/ml mAb to FcγRIIa alone, FcγRIIIb alone, or mAbs to both receptors for 5 min at 37°C as described above. After washing off excess mAb and resuspending cells in Krebs+, 50 μM of DCF was added to the cells for 10 min at 37°C. Following stimulation of cells, reactions were terminated by centrifuging samples at 2000 × g for 2 min. Samples were washed twice in PBS containing 0.1% gelatin and 0.1% glucose (buffer A), and 0.1% azide followed by resuspension in 1% paraformaldehyde in buffer A. Samples were analyzed on an EPICS Profile flow cytometer (Coulter, Hialeah, FL) that was calibrated before analysis with Standard-Brite beads. Negative controls included cells preincubated with mAb(s) to FcγR(s) but not stimulated with GAM and cells stimulated with GAM alone.

Following cross-linking of FcγRs, reactions were terminated by centrifuging samples for 20 s at 2500 × g, and cells were fixed by adding 200 μl of 1% paraformaldehyde to samples for 30 min at room temperature. Cells were then washed twice in Krebs+. Cells were permeabilized with 2% saponin for 30 min at 4°C, washed twice with Krebs+, incubated with 0.8 U of fluorescein-labeled phalloidin (Molecular Probes, Eugene, OR) at 4°C for 1 h, washed twice in Krebs+, and placed in chambered, cover-glass wells (Nunc, Naperville, IL). Samples were examined using a confocal microscope (IMT-2, Olympus, New Hyde Park, NY) and Genomic Solutions software.

Statistical analysis by one- or two-way ANOVA was performed using GraphPad Instat (GraphPad Software, San Diego, CA.). Differences between groups were determined using Bonferroni’s post-test, and significance was defined as p < 0.05.

To determine the ability of FcγRs to activate MAPKs in human neutrophils, ERK and p38 activities were measured at various times following homotypic and heterotypic cross-linking. Homotypic cross-linking was accomplished by incubating cells with anti-FcγRIIa Fab mAb or anti-FcγRIIIb F(ab′)2 mAb and cross-linking agent, F(ab′)2 GAM. Heterotypic cross-linking was accomplished by incubating cells with both FcγR mAbs followed by the addition of F(ab′)2 GAM. Heterotypic cross-linking by this method leads to colocalization of FcγRIIa and FcγRIIIb on the cell surface (21, 36). The time course of ERK activation was determined by an in vitro kinase assay, which is shown in Fig. 1. Maximal ERK activation occurred at 3 min following homotypic and heterotypic cross-linking (Fig. 1, A and B). Homotypic cross-linking of FcγRIIa stimulated significantly greater ERK activity compared with cross-linking of FcγRIIIb (p < 0.05). Heterotypic cross-linking of both receptors did not induce a significant synergistic activation of ERK at any of the time points. Cells exposed to either FcγR mAb or GAM alone did not stimulate ERK activity above basal levels (data not shown). These results were confirmed by immunoblotting for phosphorylated ERK activity (Fig. 1 C). This blot were stripped and reprobed for total ERK, which confirmed equal protein loading (data not shown).

FIGURE 1.

Activation of ERK activity by homotypic and heterotypic cross-linking of FcγRs in neutrophils. A, Time course of ERK activity measured by an in vitro kinase assay that detects the ability of ERK to phosphorylate substrate, MBP. Results are expressed as the mean ± SEM of the fold increase in ERK activity, quantitated with ImageQuant. These data are an average of three separate experiments. B, Representative autoradiogram of cell lysates from which the results in A were calculated. C, Immunoblot of cell lysates stained with phospho-ERK antisera following FcγR cross-linking.

FIGURE 1.

Activation of ERK activity by homotypic and heterotypic cross-linking of FcγRs in neutrophils. A, Time course of ERK activity measured by an in vitro kinase assay that detects the ability of ERK to phosphorylate substrate, MBP. Results are expressed as the mean ± SEM of the fold increase in ERK activity, quantitated with ImageQuant. These data are an average of three separate experiments. B, Representative autoradiogram of cell lysates from which the results in A were calculated. C, Immunoblot of cell lysates stained with phospho-ERK antisera following FcγR cross-linking.

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The time course of p38 activation was determined by an in vitro kinase assay. Maximal p38 activation occurred between 1 and 2 min (Fig. 2, A and B). Homotypic cross-linking of FcγRIIIb stimulated significantly greater p38 activity than cross-linking of FcγRIIa (p < 0.05). Cells treated with either FcγR mAb or GAM alone did not stimulate p38 activity above basal levels (data not shown). Heterotypic cross-linking of both receptors did not lead to synergistic activation of p38 MAPK activity. These observations were confirmed by immunoblotting for phosphorylated p38 (Fig. 2 C). This blot were stripped and reprobed for total p38, which confirmed equal protein loading (data not shown).

FIGURE 2.

Activation of p38 activity by homotypic and heterotypic cross-linking of FcγRs in neutrophils. A, Time course of p38 activity measured by an in vitro kinase assay that detects the ability of p38 to phosphorylate substrate, ATF2. Results are expressed as the mean ± SEM of the fold increase in p38 activity, quantitated with ImageQuant. These data are an average of three separate experiments. B, Representative autoradiogram of cell lysates, from which results in A were calculated. C, Immunoblot of cells lysates stained with phospho-p38 antisera following 1 min of FcγR cross-linking.

FIGURE 2.

Activation of p38 activity by homotypic and heterotypic cross-linking of FcγRs in neutrophils. A, Time course of p38 activity measured by an in vitro kinase assay that detects the ability of p38 to phosphorylate substrate, ATF2. Results are expressed as the mean ± SEM of the fold increase in p38 activity, quantitated with ImageQuant. These data are an average of three separate experiments. B, Representative autoradiogram of cell lysates, from which results in A were calculated. C, Immunoblot of cells lysates stained with phospho-p38 antisera following 1 min of FcγR cross-linking.

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FcγRIIa activates signal transduction pathways that contain nonreceptor tyrosine kinases and PI-3K as components (12, 13). The signal transduction pathways activated by FcγRIIIb are poorly understood, but Zhou et al. (19) showed that FcγRIIIb activates the nonreceptor tyrosine kinase Hck. Chemoattractant receptors have been shown to activate ERK and p38 through signal transduction pathways using tyrosine kinases and PI-3K (25). Therefore, we examined the effects of tyrosine kinase and PI-3K inhibition on FcγR-mediated MAPK activation by pretreatment of cells with 30 μM genistein or 100 nM wortmannin before cross-linking. Fig. 3 shows that both pharmacologic inhibitors significantly attenuate activation of ERK and p38 by either FcγRIIa or FcγRIIIb cross-linking.

FIGURE 3.

Role of tyrosine kinases and PI-3K in MAPK activation by FcγRs. Neutrophils were pretreated with 100 nM wortmannin or 30 μM genistein before cross-linking FcγRIIa and FcγRIIIb. A and B, Autoradiograms representing ERK in vitro kinase assays. C and D, Autoradiograms representing p38 in vitro kinase assays. A and C, Cells stimulated by FcγRIIa cross-linking. B and D, Cells stimulated by FcγRIIIb cross-linking. E, Results are expressed as the mean ± SEM fold increase in ERK or p38 activity. The results shown are representative of three separate experiments and were quantitated with ImageQuant. ∗, p < 0.05; ∗∗, p < 0.01; ∗∗∗, p < 0.001.

FIGURE 3.

Role of tyrosine kinases and PI-3K in MAPK activation by FcγRs. Neutrophils were pretreated with 100 nM wortmannin or 30 μM genistein before cross-linking FcγRIIa and FcγRIIIb. A and B, Autoradiograms representing ERK in vitro kinase assays. C and D, Autoradiograms representing p38 in vitro kinase assays. A and C, Cells stimulated by FcγRIIa cross-linking. B and D, Cells stimulated by FcγRIIIb cross-linking. E, Results are expressed as the mean ± SEM fold increase in ERK or p38 activity. The results shown are representative of three separate experiments and were quantitated with ImageQuant. ∗, p < 0.05; ∗∗, p < 0.01; ∗∗∗, p < 0.001.

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Previous studies showed that ERK and p38 play a role in the generation of neutrophil functions, including respiratory burst activity (24, 25, 26, 27). The role of ERK and p38 MAPKs in the regulation of FcγR-dependent respiratory burst activity was determined using a flow cytometric assay that measures intracellular hydrogen peroxide (H2O2). When cells were incubated with mAb to FcγRIIa or FcγRIIIb alone, H2O2 production did not differ from basal levels. Following cross-linking of FcγRIIa, H2O2 production was increased 3-fold (Fig. 4,A), while cross-linking of FcγRIIIb stimulated a 5-fold increase in H2O2 production (Fig. 4,B). Heterotypic cross-linking did not lead to synergistic increase in H2O2 production (Fig. 4 C).

FIGURE 4.

Activation of respiratory burst activity by homotypic and heterotypic cross-linking of FcγRs in neutrophils. Respiratory burst activity was measured by the ability of hydrogen peroxide to hydrolyze DCF to its fluorescent analogue, dichlorofluorescein. Cells were pretreated with 10 μM SB203580 or 50 μM PD98059 for 1 h before receptor cross-linking. A, Cells that were FcγRIIa cross-linked. B, Cells that were FcγRIIIb cross-linked. C, Cells that were FcγRIIa and FcγRIIIb cross-linked. Results are expressed as the mean channel fluorescence of DCF ± SEM. These data are an average of nine separate experiments. ∗, p < 0.05; ∗∗, p < 0.01; ∗∗∗, p < 0.001; ∗∗∗∗, p < 0.0001.

FIGURE 4.

Activation of respiratory burst activity by homotypic and heterotypic cross-linking of FcγRs in neutrophils. Respiratory burst activity was measured by the ability of hydrogen peroxide to hydrolyze DCF to its fluorescent analogue, dichlorofluorescein. Cells were pretreated with 10 μM SB203580 or 50 μM PD98059 for 1 h before receptor cross-linking. A, Cells that were FcγRIIa cross-linked. B, Cells that were FcγRIIIb cross-linked. C, Cells that were FcγRIIa and FcγRIIIb cross-linked. Results are expressed as the mean channel fluorescence of DCF ± SEM. These data are an average of nine separate experiments. ∗, p < 0.05; ∗∗, p < 0.01; ∗∗∗, p < 0.001; ∗∗∗∗, p < 0.0001.

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To determine the role of ERK and p38 in FcγR activation of respiratory burst activity, cells were pretreated with specific inhibitors for each MAPK (25). Inhibition of p38 activity by 10 μM SB203580 did not alter basal H2O2 production, but inhibition of ERK activity by 50 μM PD98059 significantly enhanced basal H2O2 production (Fig. 4). SB203580 significantly reduced H2O2 production stimulated by homotypic cross-linking of FcγRIIIb or heterotypic cross-linking (Fig. 4, B and C). The small reduction in H2O2 production following pretreatment with SB203580 before FcγRIIa cross-linking was not statistically significant (Fig. 4 A). Cells pretreated with PD98059 exhibited a significant increase in H2O2 production following homotypic and heterotypic cross-linking of FcγRs.

Actin polymerization plays a role in the regulation of many cell activities, including cell motility, phagocytosis, and respiratory burst activity (18, 21, 37, 38). In addition, other studies have indicated that MAPKs play a role in actin polymerization induced by bacterial phagocytosis and FMLP (30, 31). Therefore, the role of ERK and p38 in the regulation of FcγR-dependent actin polymerization was determined by staining F-actin with FITC-labeled phalloidin and observing cells by confocal microscopy. Untreated cells exhibited diffuse staining of F-actin throughout the cytoplasm (Fig. 5,A). This characteristic was also observed in cells treated with mAb alone (Fig. 5,B) and in cells treated with GAM alone (data not shown). Following FcγRIIIb cross-linking, F-actin relocalized to the cell periphery, and fluorescent intensity was enhanced (Fig. 5,C). Pretreatment of cells with 10 μM SB203580 did not alter the actin polymerization and relocalization observed upon FcγRIIIb cross-linking (Fig. 5,D). On the other hand, pretreatment with 50 μM PD98059 prevented actin polymerization and relocalization following FcγRIIIb cross-linking (Fig. 6,E). Pretreatment of neutrophils with cytochalasin D inhibited F-actin polymerization and relocalization, as expected (Fig. 5 F). Similar observations were made with homotypic cross-linking of FcγRIIa and heterotypic cross-linking (data not shown).

FIGURE 5.

Role of MAPKs in FcγR activation of actin polymerization. Actin polymerization was determined by staining cells with FITC-labeled phalloidin following FcγR cross-linking of neutrophils. A, Cells alone. B, Cells incubated with anti-FcγRIIIb F(ab′)2 mAb. C, Cells cross-linked with anti-FcγRIIIb F(ab′)2 mAb and GAM F(ab′)2 for 15 min. D, Cells pretreated with 10 μM SB203580 before FcγRIIIb cross-linking. E, Cells pretreated with 50 μM PD98059 before FcγRIIIb cross-linking. F, Cells pretreated with of 3 μM cytochalasin D before FcγRIIIb cross-linking.

FIGURE 5.

Role of MAPKs in FcγR activation of actin polymerization. Actin polymerization was determined by staining cells with FITC-labeled phalloidin following FcγR cross-linking of neutrophils. A, Cells alone. B, Cells incubated with anti-FcγRIIIb F(ab′)2 mAb. C, Cells cross-linked with anti-FcγRIIIb F(ab′)2 mAb and GAM F(ab′)2 for 15 min. D, Cells pretreated with 10 μM SB203580 before FcγRIIIb cross-linking. E, Cells pretreated with 50 μM PD98059 before FcγRIIIb cross-linking. F, Cells pretreated with of 3 μM cytochalasin D before FcγRIIIb cross-linking.

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FIGURE 6.

Role of actin assembly in mediating FcγR-dependent activation of respiratory burst. Cells were pretreated with 3 μM cytochalasin D for 1 h before FcγR cross-linking. Then, H2O2 production was detected by measuring the ability of H2O2 to hydrolyze DCF. Results are expressed as the mean ± SEM fold increase in mean channel fluorescence of DCF. These data are an average of three separate experiments and were quantitated with ImageQuant. ∗∗, p <0.01; ∗∗∗, p < 0.001.

FIGURE 6.

Role of actin assembly in mediating FcγR-dependent activation of respiratory burst. Cells were pretreated with 3 μM cytochalasin D for 1 h before FcγR cross-linking. Then, H2O2 production was detected by measuring the ability of H2O2 to hydrolyze DCF. Results are expressed as the mean ± SEM fold increase in mean channel fluorescence of DCF. These data are an average of three separate experiments and were quantitated with ImageQuant. ∗∗, p <0.01; ∗∗∗, p < 0.001.

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Past studies have shown that inhibition of actin polymerization enhances FMLP stimulation of respiratory burst activity (39, 40), suggesting that actin reorganization can regulate this response. The present study shows that inhibition of ERK activity blocks FcγR stimulation of actin polymerization, while enhancing FcγR stimulation of respiratory burst activity (see Figs. 4 and 5). To determine whether ERK regulation of FcγR-mediated respiratory burst activity is due to its ability to stimulate actin assembly, the effect of cytochalasin D on FcγR-stimulated H2O2 production was examined. Pretreatment with 3 μM cytochalasin D significantly enhanced H2O2 production stimulated by homotypic FcγRIIIb cross-linking or heterotypic cross-linking, while pretreatment with cytochalasin D failed to alter FcγRIIa-stimulated H2O2 production (Fig. 6). Thus, inhibition of actin polymerization affects FcγRIIa and FcγRIIIb-stimulated H2O2 production differently than inhibition of ERK activation. FcγRIIIb stimulation of ERK-dependent actin assembly regulates the respiratory burst response. However, ERK regulation of the FcγRIIa-stimulated respiratory burst is not dependent on actin assembly.

It is possible that actin assembly regulates H2O2 production indirectly by regulating p38 activity, a site upstream of FcγRIIIb-stimulated respiratory burst response. Therefore, we determined whether actin polymerization could regulate FcγR stimulation of MAPK activity. Fig. 7 shows that pretreatment of cells with 3 μM cytochalasin D significantly enhanced p38 activity stimulated by both FcγRIIa and FcγRIIIb cross-linking. On the other hand, cytochalasin D had no effect on FcγRIIa or FcγRIIIb activation of ERK. These observations suggest that one mechanism by which actin rearrangement regulates respiratory burst activity is by modulating a upstream site in the pathway, p38 MAPK activation.

FIGURE 7.

Role of actin assembly in mediating FcγR-dependent activation of MAPKs. Cells were pretreated with 3 μM cytochalasin D for 1 h before FcγR cross-linking. Then, ERK and p38 in vitro kinase assays were performed at 3 and 1 min, respectively. Results are expressed as the mean ± SEM fold increase in ERK and p38 activity. The results shown are representative of three separate experiments and were quantitated with ImageQuant. ∗, p < 0.05.

FIGURE 7.

Role of actin assembly in mediating FcγR-dependent activation of MAPKs. Cells were pretreated with 3 μM cytochalasin D for 1 h before FcγR cross-linking. Then, ERK and p38 in vitro kinase assays were performed at 3 and 1 min, respectively. Results are expressed as the mean ± SEM fold increase in ERK and p38 activity. The results shown are representative of three separate experiments and were quantitated with ImageQuant. ∗, p < 0.05.

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The results of the present study indicate that cross-linking FcγRIIa and FcγRIIIb rapidly and transiently activate two MAPK pathways in human neutrophils, ERK1/2 and p38. FcγRIIa and FcγRIIIb stimulate distinct patterns of ERK and p38 activities. FcγRIIa cross-linking stimulates greater ERK activity than FcγRIIIb, while FcγRIIIb stimulates greater p38 activity than FcγRIIa. Heterotypic cross-linking of both receptors did not lead to synergistic activation of MAPKs or functional responses, indicating that FcγRIIIb does not act cooperatively with FcγRIIa. The possibility that FcγRIIIb interacts with CR3 to activate intracellular signals was not addressed in the present study. Despite the evidence for independent signaling, both FcγRs use signal transduction pathways containing nonreceptor tyrosine kinases and PI-3Ks to stimulate ERK and p38 activity. Zhou et al. (19) reported that FcγRIIa activates Src tyrosine kinase Fgr, but not Hck, while FcγRIIIb activates Hck, but not Fgr. In other studies we have reported that tyrosine kinase activity participates in chemoattractant stimulation of ERK, but not p38, activity (25). Previous reports indicate that FcγRs activate PI-3K in a tyrosine kinase-dependent manner, and PI-3K activation is necessary for FcγR-mediated phagocytosis (41, 42). Our data indicate that PI-3K is required for ERK and p38 activation by both FcγRs. Past reports suggest that chemoattractants and GM-CSF stimulate ERK and p38 in a PI-3K-dependent manner in neutrophils (24, 25, 43, 44), while stimulation by TNF-α is PI-3K independent (24). Thus, there are multiple proximal signal transduction pathways in neutrophils that lead to MAPK activation.

Both FcγRIIa and FcγRIIIb stimulated respiratory burst response in neutrophils, but FcγRIIIb stimulated greater H2O2 production than cross-linking of FcγRIIa. The differences in strength of the respiratory burst response induced by FcγRIIa vs FcγRIIIb may be due to the following: 1) a 10-fold greater expression of FcγRIIIb (14), 2) the ability of FcγRIIIb to stimulate greater p38 activity than FcγRIIa, or 3) the ability of FcγRIIa to activate a greater counter-regulatory ERK response than FcγRIIIb. Heterotypic cross-linking of both receptors failed to stimulate a synergistic increase in respiratory burst activity.

The present study shows that both p38 and ERK regulated FcγR-mediated respiratory burst activity. Inhibition of p38 activity attenuated FcγRIIIb, but not FcγRIIa, stimulation of respiratory burst activity. This observation is consistent with the ability of FcγRIIIb to stimulate greater p38 activity than FcγRIIa (see Fig. 1). The inability of SB203580 to completely block FcγRIIIb-stimulated H2O2 production or to affect FcγRIIa-stimulated H2O2 production indicates that a p38-independent pathway exists for FcγR activation of the respiratory burst. A role for p38 in chemoattractant and phagocytic stimulation of respiratory burst activity has been documented in several previous reports (24, 25, 26, 31), and p38 has been shown to phosphorylate a component of the NADPH oxidase, p47phox (45). Pretreatment of cells with PD98059 led to an enhanced respiratory burst response following FcγR cross-linking, suggesting that ERK plays a counter-regulatory role in FcγR stimulation of respiratory burst activity. These results contradict the finding of previous studies that ERK inhibition reduces the respiratory burst response (24, 25, 29, 30). However, these previous studies used chemoattractants as the agonist, not immune complexes, and measured respiratory burst as superoxide release. In a previous study we reported that PD98059 enhanced intracellular H2O2 production stimulated by bacterial phagocytosis (31). This finding is consistent with the results of the present study.

Inhibition of ERK activation by PD98059 was found to inhibit actin polymerization stimulated by homotypic or heterotypic cross-linking of FcγRIIa and FcγRIIIb. Thus, cytoskeletal rearrangement stimulated by FcγRs is dependent on ERK activation. This finding contrasts with the report of Downey et al. (30), in which the FMLP-stimulated increase in F-actin was not inhibited by PD98059. ERK inhibition does not attenuate phagocytosis of bacteria or zymosan (30, 31). Thus, the role of ERK in FcγR-stimulated actin polymerization may be unique, and the consequences of this finding remain to be determined.

Inhibition of ERK enhanced FcγR-stimulated H2O2 production while inhibiting actin polymerization. Others have observed that FMLP stimulation of respiratory burst response is up-regulated when cells are pretreated with an inhibitor of actin assembly, cytochalasin D (39, 40). These observations suggest that ERK-mediated actin assembly regulates respiratory burst activity. This hypothesis was tested by examining the effects of cytochalasin D on FcγR-mediated H2O2 production. Cytochalasin D enhanced H2O2 production stimulated by FcγRIIIb and heterotypic cross-linking, but failed to increase FcγRIIa-stimulated respiratory burst activity. Cytochalasin D also led to enhanced FcγRIIa and FcγRIIIb stimulation of p38, but not ERK. These data indicate that ERK and actin assembly function independently to regulate FcγR-stimulated respiratory burst activity. In addition, these data indicate that actin assembly regulates FcγRIIIb-stimulated respiratory burst response by controlling p38 activation. However, actin assembly does not regulate the FcγRIIa-stimulated respiratory burst response, probably because FcγRIIa stimulation of respiratory burst is not p38 dependent. Alternative mechanisms may exist for regulation of the FcγRIIa- and FcγRIIIb-mediated respiratory burst response. Previous reports indicate that actin associates with components of the NADPH-oxidase complex such as p47phox, p76phox, and cytochrome b558 (38, 39). Thus, another possible mechanism by which actin polymerization regulates respiratory burst activity may be through inhibition of assembly of the NADPH oxidase enzyme complex. The finding that actin polymerization was required for activation of NADPH oxidase in a cell-free model, however, argues against this proposed mechanism (46).

Neutrophils mediate tissue injury in many immune complex diseases. The ability of FcγRIIa and FcγRIIIb to independently activate neutrophils provides a mechanism by which immune complexes initiate this injury without activation of complement. FcγRIIa and FcγRIIIb stimulate different patterns of ERK and p38 activation, and these MAPKs regulate neutrophil responses to FcγR cross-linking. Our study suggests that the state of actin assembly and the level of ERK activity provide independent, inhibitory signals regulating H2O2 production during immune complex interaction with neutrophils. Strategies that enhance these counter-regulatory signals may limit neutrophil-mediated tissue injury in immune complex diseases.

We thank Suzanne Eades for her technical assistance.

1

This work was supported by grants from the Department of Veterans Affairs (to K.R.M. and J.B.K.) and the American Heart Association, Kentucky Affiliate (to K.R.M., M.J.R.), and by a postdoctoral fellowship from the American Heart Association (to P.Y.C.).

3

Abbreviations used in this paper: ITAM, intracellular tyrosine activation motif; CR3, complement receptor 3; MAPK, mitogen-activated protein kinase; ERK, extracellular signal-regulated kinase; JNK, c-Jun N-terminal kinase; Krebs+, Krebs with calcium; GAM, goat anti-mouse F(ab′)2-specific IgG; MBP, myelin basic protein; ATF2, activation transcription factor-2; DCF, dichlorofluorescein; PI-3K, phosphatidylinositol 3-kinase; F-actin, filamentous actin.

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