The JI
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     
 

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Pricop, L.
Right arrow Articles by Salmon, J. E.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Pricop, L.
Right arrow Articles by Salmon, J. E.
The Journal of Immunology, 1999, 162: 7041-7048.
Copyright © 1999 by The American Association of Immunologists

Reactive Oxygen Intermediates Enhance Fc{gamma} Receptor Signaling and Amplify Phagocytic Capacity1

Luminita Pricop, Jayashree Gokhale, Patricia Redecha, Sonia C. Ng and Jane E. Salmon2

Department of Medicine, Hospital for Special Surgery and New York Presbyterian Hospital, Graduate Program in Immunology, Weill Medical College of Cornell University, New York, NY 10021


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Receptors for the Fc region of IgG (Fc{gamma}R) mediate internalization of opsonized particles by human neutrophils (PMN) and mononuclear phagocytes. Cross-linking of Fc{gamma}R leads to activation of protein tyrosine kinases and phosphorylation of immunoreceptor tyrosine-based activation motifs (ITAMs) within Fc{gamma}R subunits, both obligatory early signals for phagocytosis. Human PMN constitutively express two structurally distinct Fc{gamma}R, Fc{gamma}RIIa and Fc{gamma}RIIIb, and can be induced to express Fc{gamma}RI by IFN-{gamma}. We have previously shown that stimulation of PMN through Fc{gamma}RIIIb results in enhanced Fc{gamma}RIIa-mediated phagocytic activity that is inhibited by catalase. In the present study, we have tested the hypothesis that reactive oxygen intermediates (ROI) have the capacity to regulate Fc{gamma}R responses and defined a mechanism for this effect. We show that H2O2 augmented phagocytosis mediated by Fc{gamma}RIIa and Fc{gamma}RI in PMN and amplified receptor-triggered tyrosine phosphorylation of Fc{gamma}R-associated ITAMs and signaling elements. Generation of endogenous oxidants in PMN by cross-linking Fc{gamma}RIIIb similarly enhanced phosphorylation of Fc{gamma}RIIa and Syk, a tyrosine kinase required for phagocytic function, in a catalase-sensitive manner. Our results provide a mechanism for priming phagocytes for enhanced responses to receptor-driven effects. ROI generated in an inflammatory milieu may stimulate quiescent cells to rapidly increase the magnitude of their effector function. Indeed, human monocytes incubated in the presence of stimulated PMN showed oxidant-induced increases in Fc{gamma}RIIa-mediated phagocytosis. Definition of the role of oxidants as amplifiers of Fc{gamma}R signaling identifies a target for therapeutic intervention in immune complex-mediated tissue injury.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Receptors for the Fc portion for IgG (Fc{gamma}R)3 provide a crucial link between cellular and humoral components of the immune cascade. Characterizing their function is critical to understanding IgG-triggered effector events. For phagocytes, engagement of Fc{gamma}R by opsonized microbes or immune complexes stimulates phagocytosis, the release of inflammatory mediators, and generation of reactive oxygen intermediates (ROI). To initiate these responses, cross-linking of Fc{gamma}R activates intracellular signaling events analogous to those induced by engagement of T and B cell Ag receptors. Fc{gamma}R aggregation leads to phosphorylation and subsequent activation of tyrosine kinases of the Src and Syk families, which become associated with the immunoreceptor tyrosine-based activation motifs (ITAMs) within Fc{gamma}R subunits (1, 2). For Fc{gamma}RIIa, a single chain receptor, the ITAM is contained within cytoplasmic domain (3, 4). For other Fc{gamma}Rs, such as Fc{gamma}RI, the ITAM is present in associated FcR{gamma} subunits (2, 3). Activated tyrosine kinases phosphorylate the ITAM, as well as downstream substrates that lead to intracellular Ca2+ concentration ([Ca2+]i) transients, cytoskeletal changes, and ultimately transcriptional activation (5, 6).

The importance of phosphotyrosine accumulation to Fc{gamma}R function is underscored by observations that mutations of the tyrosines within the ITAM domains alter phagocytic capacity and inhibitors of protein tyrosine kinases block Fc{gamma}R-stimulated responses (7, 8, 9, 10). Further evidence of the critical role of tyrosine phosphorylation is derived from studies of macrophages deficient in Syk, which have defective Fc{gamma}R-mediated phagocytosis and signal transduction, and of macrophages deficient in Src family kinases, which show poor Syk activation and delayed Fc{gamma}R-mediated phagocytosis (11).

Tyrosine phosphorylation is regulated in part by the competing activities of protein tyrosine kinases and phosphatases. Indeed, the magnitude of Fc{gamma}R signaling and effector function is determined by the interplay between these kinases and phosphatases (2, 12). Increased kinase activity induced by overexpression of Syk or by inhibition of tyrosine phosphatases by vanadate has been shown to potentiate Fc{gamma}R signal transduction and phagocyte responses (13, 14). Conversely, coligation of Fc{gamma}R with the protein tyrosine phosphatase CD45 is associated with diminished Fc{gamma}R-triggered calcium mobilization, respiratory burst, and degranulation (12, 15, 16, 17). Therefore, one would predict that mediators that alter this balance to favor kinase activity would amplify Fc{gamma}R-triggered functions.

Recent observations suggest that ROI serve as intracellular signaling molecules and promote increased tyrosine phosphorylation in a wide range of cells, including PMN, T lymphocytes, platelets, vascular smooth muscle cells, and cardiac myocytes (18, 19, 20, 21, 22, 23, 24). Although ROI were once regarded simply as toxic agents implicated in antimicrobial defense, there is now considerable evidence that they act as intra- and extracellular messengers. ROI have been shown to inhibit the activity of certain tyrosine phosphatases, including CD45, and to induce the phosphorylation and activation of Src and Syk family kinases (19, 23, 25, 26). In contrast, the enzymatic activity of SHP-1, another phosphatase essential to monocytes and PMN, is not sensitive to modulation by oxidants (27). In as much as tyrosine phosphorylation is essential to Fc{gamma}R-triggered responses, and since phagocytes are prodigious producers of ROI, we hypothesized that ROI could play an important role in the regulation of Fc{gamma}R responses at sites of inflammation through autocrine and paracrine effects.

Human PMN constitutively express two types of Fc{gamma}R: Fc{gamma}RIIa, the predominant phagocytic receptor, and Fc{gamma}RIIIb, a glycosyl phosphatidylinositol-linked protein. Fc{gamma}RI is expressed on mononuclear phagocytes and its expression can be induced in PMN by IFN-{gamma} (2, 3, 4). We have previously shown that stimulation of PMN through Fc{gamma}RIIIb or PMA leads to enhanced Fc{gamma}RIIa-mediated phagocytic activity, and that this enhancement is inhibited by catalase (28). Based on these data, we proposed a model of oxidant-dependent activation of Fc{gamma}RIIa and Fc{gamma}RI. In the present study, we examine the direct effects of oxidants on Fc{gamma}R function and signaling capacity in human phagocytes. We show that H2O2 augments internalization mediated by Fc{gamma}R in PMN and monocytes, and that endogenously and exogenously generated ROI amplify receptor-triggered tyrosine phosphorylation of Fc{gamma}R-associated ITAMs and signaling elements.


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

Anti-Fc{gamma}RII mAb IV.3 Fab, anti-Fc{gamma}RIIIb mAb 3G8 F(ab')2 and anti-Fc{gamma}RI mAb 22.2 F(ab')2 fragments were obtained from Medarex (Annandale, NJ). Purified human IgG2{kappa} myeloma protein was obtained from The Binding Site (Birmingham, U.K.). Affinity pure F(ab')2 fragments of goat anti-mouse IgG (GAM) were obtained from Jackson ImmunoResearch (Westgrove, PA). Anti-Fc{gamma}RII receptor blotting mAb (II1A.5) was a generous gift from Dr. Jurgen Frey (Universitat Bielefeld, Bielefeld, Germany) (29). Rabbit polyclonal Abs to Syk were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Polyclonal Abs to the FcR{gamma} subunit were a gift from Dr. Jean-Pierre Kinet (Harvard University, Boston, MA). Antiphosphotyrosine mAb 4G10 and PY-20 were purchased from Upstate Biotechnology (Lake Placid, NY) and Santa Cruz Biotechnology, respectively. HRP-linked sheep anti-mouse and donkey anti-rabbit Abs as well as the ECL Western blotting detection reagents were obtained from Amersham (Arlington Heights, IL). Protein G-Sepharose beads were obtained from Pharmacia Biotech (Uppsala, Sweden). Aminotriazole (AT), hydrogen peroxide, catalase, and PMA were purchased from Sigma (St. Louis, MO).

Cells

Leukocytes were isolated from the venous blood of healthy volunteers by centrifugation on a discontinuous two-step Ficoll-Hypaque gradient with specific gravities of 1.078 and 1.119 g/ml (30). PMN were isolated from the lower interface and washed in HBSS. Contaminating erythrocytes were lysed. Cells were resuspended to 5 x 106/ml in RPMI 1640 + 10% FCS. Mixed mononuclear cells were isolated from the upper interface, washed, and resuspended as above. Adherent monocyte monolayers were prepared from mixed mononuclear cells as described previously (31). Human monocytic cell lines U937 and THP-1 were obtained from the American Type Culture Collection (Manassas, VA) and cultured in RPMI 1640 medium supplemented with 10% FCS.

For induction of Fc{gamma}RI, PMN were cultured overnight in medium (RPMI 1640 with 5% FCS and 0.7 mM 2-ME) supplemented with 400 U/ml of IFN-{gamma} (Genzyme, Cambridge, MA). For induction of Fc{gamma}RI and FcR{gamma} subunits, U937 cells were cultured for 4 days in medium supplemented with 400 U/ml of IFN-{gamma} (32). For activation by oxidants, cells were pretreated with AT (20 mM) for 30 min at room temperature followed by incubation with H2O2 (500 µM) for 10 min at room temperature.

Phagocytosis assay

Fc{gamma}R-specific probes were prepared as previously described (33). Briefly, bovine erythrocytes (E), human IgG2 (hIgG2) and anti-Fc{gamma}RI mAb 22.2 F(ab')2 were biotinylated. Erythrocytes were saturated with streptavidin, washed, and coated with biotinylated Abs. E-hIgG2 and E-22.2 were labeled with PKH26 lipophilic dye as described (34), and washed and resuspended in RPMI 1640 + 10% FCS to a final concentration of 1 x 108 erythrocytes/ml.

The capacity of PMN to internalize the target particles was measured using a flow cytometric assay (34). PMN (100 µl at 5 x 106 cell/ml) were mixed with E-hIgG2 or E-22.2 (125 µl at 1 x 108 E/ml). The cell mixture was centrifuged at 44 x g for 3 min at room temperature, incubated for 10 min at 37°C in RPMI 1640 + 20% FCS, followed by hypotonic lysis to remove noninternalized erythrocytes. Cells were washed three times in 1% BSA-PBS to remove lysed erythrocyte fragments. Quantification of phagocytosis of PKH26-labeled E-hIgG2 or E-22.2 by flow cytometry was performed using a FACScan (Becton Dickinson Immunocytometry Systems, San Jose, CA) equipped with a standard optical filter set as described (34). PKH26 fluorescence was detected in the FL2 channel and displayed on a logarithmic scale. The mean fluorescence intensity (MFI) for PKH26-labeled erythrocytes was standardized in each experiment. Increasing multiples of this MFI correspond to phagocytes with increasing numbers of internalized erythrocytes, as described (34). The phagocytic index (PI) was calculated by multiplying the percentage of cells that internalized PKH26-labeled erythrocytes (%P) and the MFI of phagocytes with internalized erythrocytes (PI = %P x MFI/100).

To study the capacity of stimulated PMN to alter monocyte phagocytic function a 12-well format cell culture insert with 0.4 µM pore size (Becton Dickinson, Franklin Lakes, NJ) was used. E-hIgG2 (125 µl at 1 x 108 E/ml) were added to wells with adherent monocytes (~1 x 105 cells/well), allowed to settle for 2 min, followed by the addition of medium to fill the well. PMN (1.5/ml at 5 x 106/ml) treated with or without PMA (20 ng/ml) were added to the upper chamber. After incubation for 15 min at 37°C, phagocytosis by adherent monocytes was measured by light microscopy as previously described (31). In preliminary experiments, it was determined that PMA alone had no effect on the internalization of E-hIgG2 by adherent monocytes.

Fc{gamma}RIIA genotyping

Genomic DNA was isolated using the Puregene DNA isolation kit (Gentra Systems, Minneapolis MN), and assays to determine the genotype of Fc{gamma}RIIA were performed as previously described (35).

Immunoprecipitations

Cells were suspended in buffer containing 125 mM NaCl, 5 mM KCl, 8 mM Na2HPO4, 2 mM NaH2PO4, 5 mM glucose (pH 7.35) and opsonized with the appropriate anti-Fc{gamma}R mAb fragments (5 µg/ml) for 10 min at room temperature, then washed and resuspended in the same buffer supplemented with 1.09 mM CaCl2 and 1.62 mM MgCl2. Fc{gamma}R aggregation was initiated by cross-linking the surface-bound mAb with goat anti-mouse F(ab')2 (30 µg/ml) at 30°C for varying times. Cells (1–2 x 107 cells/lane) were then washed in ice cold buffer, pelleted, and solubilized in lysis buffer (1% Nonidet P-40, 1% Triton X-100, 10% glycerol, 70 mM NaCl, 50 mM NaF, 16 mM Na2HPO4, 4 mM NaH2PO4, 5 mM EDTA, 0.4 mM Na3VO4, 10 µg/ml each aprotinin, leupeptin, soybean trypsin inhibitor, and pepstatin A, and 500 µg/ml pefabloc (pH 7.4)) for 1 h at 4°C. The lysates were centrifuged at 16,000 x g for 10 min at 4°C and the supernatants were immunoprecipitated using specific Abs (0.5–1 µg purified Ab) and protein G-Sepharose beads (15 µl) for 16 h at 4°C. The immune complexes were washed four times in PBS plus 1% Nonidet P-40, mixed with 2x Laemmli buffer plus 2-ME, heated for 5 min at 95°C, and separated on 10% SDS-PAGE. The samples were electrophoretically transferred to nitrocellulose membranes, blocked in PBS supplemented with 5% BSA, and analyzed for phosphotyrosine content with 4G10 mAb (0.5 µg/ml) and PY-20 (1 µg/ml) and for protein content with 1 µg/ml of the appropriate Ab followed by peroxidase-conjugated second Ab. Proteins were visualized using the enhanced chemiluminiscent detection (Amersham) and Kodak X-Omat radiographic film (Eastman Kodak, Rochester, NY).

Quantitation of radiograms was performed by densitometry (Molecular Dynamics, Sunnyvale, CA). All measurements were normalized by subtracting the background value for each blot. Readings for individual phosphorylated bands (detected by antiphosphotyrosine mAbs) and nonphosphorylated protein bands (detected by the Abs for the specific proteins) were obtained. For comparison of extent of tyrosine phosphorylation under different experimental conditions, the ratios of values for phosphorylated to nonphosphorylated bands were calculated and indicated on the figures.

Statistical analysis

Experiments were performed in a matched-triplet design for assessment of the effects of oxidants on the phagocytic capacity of individuals with each of the three different Fc{gamma}RIIA genotypes. Accordingly, in each experiment PMN from donors of different genotypes were studied simultaneously. The data are displayed as mean ± SEM. The effects of oxidant treatment were compared with control conditions using a paired t test (two-tailed). A probability of 0.05 was used to reject the null hypothesis that there is no difference between the conditions.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
ROI augment Fc{gamma}RIIa-mediated phagocytosis in PMN and monocytes

Activation of PMN by cross-linking of Fc{gamma}RIIIb or with PMA leads to a 2- to 4-fold increase in Fc{gamma}RIIa-mediated phagocytic activity (28). We have shown that priming of Fc{gamma}RIIa is transferable to resting PMN by supernatants from stimulated cells and that both the direct and supernatant-mediated effects are inhibited by catalase (28). To provide independent proof that it is the generation of oxidants that amplify Fc{gamma}RIIa function, we treated PMN with H2O2, a membrane-permeable ROI released at inflammatory sites. Phagocytosis mediated by Fc{gamma}RIIa was probed with an Fc{gamma}RIIa-specific native ligand, hIgG2 coupled to erythrocytes (36). Treatment of PMN with H2O2 (500 µM) in the presence of AT (20 mM), an inhibitor of catalase that can be released from endogenous PMN stores and which metabolizes H2O2, resulted in a 221 ± 22% increase in E-hIgG2 internalization (p < 0.0003, n = 35). The enhancing effect of H2O2 was maximal at 500 µM (Fig. 1Go, inset). Treatment of E-hIgG2 with H2O2 did not alter their internalization, indicating that the oxidants are acting on the phagocytes rather than on the target particles. The kinetics of phagocytosis of E-hIgG2 by PMN in the presence H2O2 and AT were similar to those in untreated cells (Fig. 1Go). Phagocytosis was initiated within 1 min of adding E-hIgG2 and was completed by 10 min. At 1 min, the oxidant-treated PMN had a nearly 2-fold increase in internalization as compared with control PMN (PMNH2O2 vs PMNcontrol: 43 ± 3 vs 26 ± 1 E-hIgG2 internalized/100 PMN, p = 0.02). This relative difference was maintained at the peak phagocytic index, which was reached at 10 min under both conditions (PMNH2O2 vs PMNcontrol: 91 ± 11 vs 45 ± 5, p = 0.015, n = 10). Thus, oxidant treatment increases Fc{gamma}RIIa phagocytic capacity (number of E-hIgG2 internalized/PMN) without altering kinetics.



View larger version (17K):
[in this window]
[in a new window]
  		FIGURE 1. Kinetics of Fc{gamma}RIIa phagocytic function in PMN treated with and without oxidants. Freshly isolated PMN were treated with AT (20 mM) followed by H2O2 (500 µM) (•) or medium ({circ}) for 10 min at room temperature and then combined with E-hIgG2. The phagocytic index was measured at the indicated times by flow cytometry as described in Materials and Methods (n = 4–10 experiments for each time point). Inset, PMN treated with AT and H2O2 at concentrations ranging from 10 µM to 2 mM for 10 min were incubated with E-hIgG2 for 10 min. Phagocytic index was determined by flow cytometry. Data are expressed as percent control PI (PIH2O2 - PIcontrol/PIcontrol). Values represent mean ± SEM of five experiments.

 
Fc{gamma}RIIa has two codominantly expressed alleles, R131 and H131, which differ structurally at amino acid position 131 in the second extracellular domain and differ in their ability to bind human IgG2 (H131>>R131) (36, 37). If ROI act distal to receptor binding and alter intracellular signaling, then H2O2 should affect the function of both allelic variants of Fc{gamma}RIIa. To test this possibility, we compared the effect of H2O2 on PMN from healthy donors homozygous for R131 or H131 genes and heterozygotes. Treatment of PMN with H2O2 (500 µM) in the presence of AT resulted in an increase in phagocytosis for R131 homozygotes (227 ± 39%; p < 0.04, n = 12), R131/H131 heterozygotes (263 ± 53%; p < 0.02, n = 11), and H131/H131 homozygotes (176 ± 17%; p < 0.02, n = 12) (Fig. 2Go). Oxidant-induced enhancement of phagocytosis was evident for PMN expressing all three genotypes, but the relative differences in E-hIgG2 internalization among the alleles persisted in oxidant-activated PMN. These data are consistent with oxidant-driven changes in signaling capacity, but they do not exclude alterations in binding.



View larger version (13K):
[in this window]
[in a new window]
  		FIGURE 2. Oxidant-induced enhancement of Fc{gamma}RIIa-mediated phagocytosis. PMN were isolated from disease-free individuals homozygous for Fc{gamma}RIIa-R131 or H131 or heterozygotes (R131/H131). Cells were treated with medium ({square}) or AT (20 mM) and H2O2 (500 µM) ({blacksquare}) and incubated with E-hIgG2 for 10 min. The phagocytic index was determined by flow cytometric analysis. Data represent mean ± SEM of 12 experiments. Control vs H2O2: *, p = 0.04; **, p = 0.02.

 
That PMN-derived ROI can similarly modulate Fc{gamma}RIIa function in monocytes was demonstrated using a two-chamber apparatus. Adherent monocytes were incubated in the presence of PMA-activated PMN or resting PMN, and Fc{gamma}RIIa-mediated phagocytosis was probed with E-hIgG2. In monocytes incubated with activated PMN, phagocytosis was increased by 153 ± 26% compared with monocytes cultured with resting PMN (p < 0.03, n = 4). The effect of exposure to activated PMN was inhibited by 85% in the presence of catalase. Thus, oxidant-induced activation of Fc{gamma}RIIa can occur in resting "bystander" mononuclear phagocytes.

Exogenous and endogenously generated oxidants induce hyperphosphorylation of the Fc{gamma}RIIa ITAM in PMN

It has been previously shown that Fc{gamma}RIIa ligation leads to receptor tyrosine phosphorylation that provides an obligatory early signal for phagocytosis (6, 38). We sought to determine whether endogenous or exogenously generated oxidants affect this process. In PMN, enhanced phosphorylation of Fc{gamma}RIIa was observed following receptor triggering in the presence of H2O2 (500 µM) and AT, the same conditions used for the functional studies (Fig. 3GoA, lane 2). Cross-linking Fc{gamma}RIIIb on PMN, a known trigger of endogenous ROI generation (39), resulted in an even greater increase in receptor-initiated tyrosine phosphorylation of Fc{gamma}RIIa (Fig. 3GoB, lane 4). In contrast, 3G8 F(ab')2 alone had no effect on Fc{gamma}RIIa (lane 3). The addition of oxidants in the absence of Fc{gamma}RIIa cross-linking similarly did not result in receptor tyrosine phosphorylation (data not shown).



View larger version (17K):
[in this window]
[in a new window]
  		FIGURE 3. Exogenous and endogenously generated oxidants enhance phosphorylation of Fc{gamma}RIIa in PMN. A, Freshly isolated PMN (2 x 107/lane) were pretreated with medium (lane 1) or AT and H2O2 (500 µM) (lane 2). Subsequently, cells were opsonized with IV.3 Fab (5 µg/ml) and stimulated with goat anti-mouse F(ab')2 (30 µg/ml) for 15 s. B, PMN (2 x 107/lane) were pretreated with medium (lanes 1 and 2), 3G8 F(ab')2 (5 µg/ml) (lanes 3 and 4), or 3G8 F(ab')2 and catalase (10,000 U/ml) (lane 5), followed by IV.3 Fab and goat anti-mouse F(ab')2 treatment for 1 min (lanes 2, 4, and 5). Cells were lysed, and proteins were immunoprecipitated with anti-Fc{gamma}RII mAb IV.3 and immunoblotted with anti-PY mAb (top panels). The immunoblot was stripped and reprobed with anti-Fc{gamma}RII mAb II1A.5 (bottom panels). Phosphoprotein to protein ratios were determined by densitometric measurements as described in Materials and Methods.

 
To confirm that endogenously generated oxidants contributed to the Fc{gamma}RIIIb-mediated amplification of Fc{gamma}RIIa phosphorylation, we repeated the Fc{gamma}RIIIb cross-linking experiments in the presence of catalase (10,000 U/ml), which rapidly degrades H2O2. Under these conditions, hyperphosphorylation of Fc{gamma}RIIa in PMN stimulated with 3G8 F(ab')2 and goat anti-mouse F(ab')2 was clearly reduced (Fig. 3GoB, lane 5). Thus, both enhanced Fc{gamma}RIIa phosphorylation and the associated augmented phagocytic capacity are inhibited by catalase (28), indicating a key role for endogenously generated oxidants.

Oxidants enhance tyrosine phosphorylation of Syk following Fc{gamma}RIIa stimulation in PMN

The protein tyrosine kinase Syk is essential for Fc{gamma}R-mediated phagocytosis (11). Upon cross-linking Fc{gamma}RIIa, Syk associates with the phosphorylated ITAM and is itself phosphorylated on tyrosine, resulting in its activation (6, 15). Hyperexpression of Syk enhances phagocytosis by Fc{gamma}RIIa in a transfection system, suggesting that increased activity of Syk can amplify the magnitude of Fc{gamma}RIIa-mediated effector function (8). We hypothesized that enhancement of phagocytosis by ROI was associated with increased Fc{gamma}RIIa-triggered phosphorylation of Syk. To determine whether endogenously generated oxidants increase the tyrosine phosphorylation of Syk, PMN were stimulated by cross-linking Fc{gamma}RIIIb. Fc{gamma}RIIa-initiated phosphotyrosine accumulation on Syk was markedly increased in the cells pretreated with 3G8 F(ab')2 and goat anti-mouse F(ab')2 (Fig. 4GoA, lane 3). This enhancement was blocked by catalase, demonstrating its oxidant dependence (lane 4).



View larger version (17K):
[in this window]
[in a new window]
  		FIGURE 4. Exogenous and endogenously generated oxidants accelerate and amplify Fc{gamma}RIIa-stimulated phosphorylation of Syk in PMN. A, Freshly isolated PMN (2 x 107/lane) were pretreated with medium (lanes 1 and 2), 3G8 F(ab')2 (5 µg/ml) (lane 3), or 3G8 F(ab')2 and catalase (10,000 U/ml) (lane 4). Subsequently, cells were opsonized with IV.3 Fab and stimulated with goat anti-mouse F(ab')2 (30 µg/ml) for 15 s (lanes 2–4). Cells were lysed and proteins were immunoprecipitated with anti-Syk Ab and immunoblotted with anti-PY mAb (top panels). The immunoblot was stripped and reprobed with anti-Syk Ab (bottom panels). Phosphoprotein to protein ratios were determined by densitometric measurements. B, PMN were incubated with medium (left panel) or AT and H2O2 (500 µM) for 10 min (right panel) at room temperature. Cells were stimulated with IV.3 Fab and goat anti-mouse F(ab')2 for the indicated times. Cell lysates were immunoprecipitated with anti-Syk Ab and analyzed with anti-PY mAb (top panel). Immunoblots were stripped and reprobed with anti-Syk Ab (bottom panel).

 
To obtain direct and independent evidence that oxidants enhance Fc{gamma}RIIa-mediated phosphorylation of Syk, PMN were incubated under the same conditions as those used in the studies of phagocytosis. In the presence of 500 µM H2O2, Fc{gamma}RIIa clustering resulted in amplified and accelerated Syk phosphotyrosine accumulation. As shown in Fig. 4GoB, Syk was more rapidly and more intensely phosphorylated, followed by earlier dephosphorylation in oxidant-treated PMN. In the presence of AT and H2O2 without receptor cross-linking, there was no evidence of tyrosine phosphorylation Syk (data not shown). In experiments with human mononuclear phagocyte derived THP-1 cells, we obtained similar evidence for H2O2-induced increases in phosphorylation of Fc{gamma}RIIa ITAM and Syk (Fig. 5Go). These results demonstrate that oxidant-induced activation of Fc{gamma}RIIa is not cell-type specific, and occurs in both mononuclear phagocytes and PMN.



View larger version (19K):
[in this window]
[in a new window]
  		FIGURE 5. Exogenous oxidants enhance phosphorylation of Fc{gamma}RIIa and Syk in THP-1 monocytic cells. THP-1 cells (2 x 107/lane) were pretreated with medium (lane 1), stimulated with IV.3 Fab (5 µg/ml) and goat anti-mouse F(ab')2 (30 µg/ml) for 2 min (lane 2), or treated with AT and H2O2 (500 µM) followed by stimulation with IV.3 Fab and goat anti-mouse F(ab')2 (lane 3). A, Cells lysates were immunoprecipitated with anti-Fc{gamma}RII mAb IV.3 and analyzed by immunoblotting with anti-PY mAb (top panel). Blots were stripped and reprobed with anti-Fc{gamma}RII mAb II1A.5 (bottom panel). B, Cell lysates were immunoprecipitated with anti-Syk Ab and analyzed with anti-PY mAb (top panel). Immunoblots were stripped and reprobed with anti-Syk Ab (bottom panel). Phosphoprotein to protein ratios were determined by densitometric measurements.

 
Oxidants enhance tyrosine phosphorylation of FcR{gamma} and Syk following Fc{gamma}RI stimulation and augment Fc{gamma}RI-mediated phagocytosis

To see whether oxidants modify signal transduction by other Fc{gamma}R family members, we studied Fc{gamma}RI, the high affinity receptor for IgG expressed constitutively on mononuclear phagocytes (2, 3, 4). Phagocytosis by Fc{gamma}RI requires the presence of the ITAM-bearing FcR{gamma} subunit, which is tyrosine phosphorylated upon receptor cross-linking. Like Fc{gamma}RIIa, stimulation through Fc{gamma}RI leads to tyrosine phosphorylation and activation of Syk (15, 32, 40, 41). To determine whether oxidants amplify this tyrosine phosphorylation triggered by Fc{gamma}RI, we studied U937 monocytic cells cultured with IFN-{gamma} to increase Fc{gamma}RI expression. Consistent with the findings of Durden and coworkers (40, 42), we observed that nonphosphorylated Syk is constitutively associated with Fc{gamma}RI{gamma} in resting IFN-{gamma}-treated U937 cells (Fig. 6Go, lane 1). After cross-linking Fc{gamma}RI with anti-Fc{gamma}RI mAb 22.2 F(ab')2 and goat anti-mouse F(ab')2, FcR{gamma} and FcR{gamma}-associated Syk undergo tyrosine phosphorylation (lane 2). In U937 cells pretreated with AT followed by H2O2 (500 µM) this process was markedly amplified (lane 3). A similar oxidant-induced increase in Syk phosphorylation was evident in lysates from U937 cells immunoprecipated with anti-Syk Ab and immunoblotted with anti-PY Abs.



View larger version (44K):
[in this window]
[in a new window]
  		FIGURE 6. Fc{gamma}RI-stimulated phosphorylation of Syk and FcR{gamma} in mononuclear phagocytes is amplified in the presence of oxidants. U937 cells (2 x 107/lane) cultured with IFN-{gamma} (400 U/ml) for 4 days were treated with medium (lane 1), stimulated with anti-Fc{gamma}RI mAb 22.2 F(ab')2 (5 µg/ml) and goat anti-mouse F(ab')2 (30 µg/ml) for 1 min 37°C (lane 2), or treated with AT and H2O2 (500 µM) followed by stimulation with 22.2 F(ab')2 and goat anti-mouse F(ab')2 (lane 3). Cell lysates were immunoprecipitated with rabbit anti-FcR{gamma} Abs and analyzed by immunoblotting with anti-PY mAb (A and C). Blots were stripped and reanalyzed with anti-Syk Abs (B) or anti-FcR{gamma} Abs (D).

 
To determine whether oxidants amplify Fc{gamma}RI signaling in PMN, cells were cultured with IFN-{gamma} (400 U/ml) overnight. Under these conditions there was a consistent and marked increase in Fc{gamma}RI expression documented by staining with mAb 22.2 (43). In contrast, Fc{gamma}RIIIb expression decreased, preventing experiments to examine the effects of Fc{gamma}RIIIb-initiated ROI generation on Fc{gamma}RI function. We, therefore, studied the effects of exogenous oxidants on Fc{gamma}RI signaling in PMN. In the presence of H2O2 (500 µM), Fc{gamma}RI cross-linking resulted in accelerated and markedly increased tyrosine phosphorylation of FcR{gamma} (Fig. 7GoA). Under these conditions, FcR{gamma} phosphorylation was augmented at 15 s, peaked at 30 s, and was almost completely reversed by 1 min, whereas in PMN triggered by Fc{gamma}RI without exogenous oxidants there was a much smaller and slower increase in tyrosine phosphorylation.



View larger version (21K):
[in this window]
[in a new window]
  		FIGURE 7. Oxidants increase Fc{gamma}RI-mediated phosphorylation of FcR{gamma} and Fc{gamma}RI-mediated phagocytosis in PMN. PMN were cultured overnight with IFN-{gamma} (400 U/ml) to induce Fc{gamma}RI expression. A, Cells were treated with control medium (lanes 1–3) or AT (20 mM) and H2O2 (500 µM) (lanes 4–6) followed by stimulation with 22.2 F(ab')2 and goat anti-mouse F(ab')2 at 37°C for the indicated times. Cell lysates were immunoprecipitated with rabbit anti-FcR{gamma} Abs and analyzed by immunoblotting with anti-PY mAb. Blots were stripped and reanalyzed with anti-FcR{gamma} Abs. B, Erythrocytes coupled to anti-Fc{gamma}RI F(ab')2 (E-22.2) and labeled with PKH26 were used to probe Fc{gamma}RI phagocytic function. PMN treated with AT (20 mM) and H2O2 (500 µM) or medium were combined with E-22.2 for 10 min at 37°C, followed by hypotonic lysis of noninternalized E. The mean fluorescence intensities for phagocytic PMN treated with AT + H2O2 was 1184 arbitrary units compared with 873 for control PMN. PI, determined by flow cytometry as described in Materials and Methods, was 273 for PMN treated with AT + H2O2 and 100 for control PMN. These data are representative of seven experiments.

 
Given the data that oxidants can alter Fc{gamma}RI triggered signaling, we examined the possibility that phagocytosis mediated by Fc{gamma}RI might also be modulated by ROI. We selected PMN for these experiments because of their robust phagocytic response compared with U937 cells. PMN cultured with IFN-{gamma} were treated with AT and H2O2 (500 µM) or control medium and Fc{gamma}RI-mediated phagocytic function was probed with erythrocytes coupled to anti-Fc{gamma}RI 22.2 F(ab')2. Treatment with oxidants increased phagocytic capacity by 157 ± 17% (n = 7, p = 0.006) (Fig. 7GoB). These results indicate that ROI provide a common mechanism to enhance both Fc{gamma}RI and Fc{gamma}RIIa signaling and amplify effector function.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
ROI, such as superoxide anion and H2O2, are highly reactive, diffusible molecules generated by all eukaryotic cells, with PMN and mononuclear phagocytes the most robust sources. While at high concentrations ROI are toxic to cells, at lower concentrations ROI can serve as intra- or extracellular second messengers. In this report, we present evidence for oxidant-dependent amplification of function and signaling through specific receptors in human phagocytes. We demonstrate that H2O2 augments phagocytosis mediated by Fc{gamma}RIIa and Fc{gamma}RI, and that exogenously or endogenously generated oxidants amplify tyrosine phosphorylation of Fc{gamma}R-associated ITAMs and signaling elements, defining a mechanism for such enhanced function. ROI secreted by PMN within an inflammatory milieu may thus stimulate quiescent cells to rapidly increase the magnitude of their effector functions. Our experiments showing that oxidants derived from activated PMN augment "bystander" monocyte Fc{gamma}RIIa function underscore this point.

Several other lines of evidence support a role for oxidants as modulators of Fc{gamma}R function. We have previously reported that in PMN Fc{gamma}RIIa-mediated phagocytic function is augmented following cross-linking of Fc{gamma}RIIIb, a receptor capable of production of ROI, and that this enhancement is blocked in the presence of catalase (28). ROI have similarly been implicated in the amplification of PMN Fc{gamma}R phagocytic function by PMA (44). Patients with chronic granulomatous disease (CGD), characterized by genetic defects in the NADPH oxidase system that result in markedly diminished generation of ROI, have served as clinical paradigms that establish the importance of oxidants in phagocyte function (45). PMN from such patients show impaired PMA and cytokine-dependent amplification of Fc{gamma}R-mediated internalization (44), emphasizing the possibility that increased Fc{gamma}R responsiveness is mediated by products of the respiratory burst. However, the mechanism for these effects is not understood and the relative sensitivity of the Fc{gamma}R isoforms to oxidant-induced modulation is unknown.

The importance of tyrosine phosphorylation in Fc{gamma}R-mediated internalization, taken together with observations that oxidants influence phosphotyrosine accumulation, led us to systematically examine the influence of endogenously generated and exogenously added oxidants on the proximal events of Fc{gamma}RIIa signaling and on the essential catalytic molecule that is recruited to its cytoplasmic domain. Our results show that in the presence of H2O2 there is increased phosphorylation of the ITAM of Fc{gamma}RIIa following receptor triggering in both PMN and mononuclear phagocytes. This phosphorylated ITAM functions as a scaffold to recruit and organize effector molecules. Endogenous generation of ROI initiated by Fc{gamma}RIIIb triggering induced an even greater enhancement of Fc{gamma}RIIa phosphorylation in PMN, which may be related to higher and more sustained intracellular oxidant levels. Indeed, catalase inhibition of such hyperphosphorylation confirms its oxidant-dependence. Our results are at variance with those recently reported by Green et al. (46), showing that tyrosine phosphorylation of cross-linked Fc{gamma}RIIa was diminished when co-cross-linked with Fc{gamma}RIIIb in Jurkat T cells transfected with human receptors. While their findings are not directly comparable with ours, obtained in fresh human PMN which are efficient sources of ROI, Green et al. argue for multiple mechanisms of cooperation between Fc{gamma}RIIa and Fc{gamma}RIIIb. Although the ITAM motifs in Fc{gamma}RIIa and Fc{gamma}RI-associated FcR{gamma} subunit differ, phosphorylation of the FcR{gamma} ITAM is similarly amplified following receptor cross-linking in the presence of H2O2, consistent with our observations of rapidly increased Fc{gamma}RI-mediated phagocytic capacity under identical conditions. These results suggest that ROI provide a common mechanism to enhance Fc{gamma}R signaling and thereby amplify effector functions.

The initial step, ITAM phosphorylation, leads to the recruitment, phosphorylation and activation of Syk, which then phosphorylates downstream signaling targets. We focused on Syk because it has been found to be a required element in Fc{gamma}R-mediated phagocytosis in studies of macrophages from Syk-deficient mice (11). Transfected cells expressing an Fc{gamma}RIII-Syk chimera can internalize particles that cross-link Fc{gamma}RIII, indicating that Syk kinase is sufficient for initiating cytoskeletal coupling and phagocytosis (47). Moreover, alterations in Syk expression modify efficiency of phagocytosis (8, 38). Our data show that ROI increase the rate and magnitude of Fc{gamma}RIIa-triggered phosphorylation of this critical kinase in PMN. Inhibition of Fc{gamma}RIIIb-stimulated Syk hyperphosphorylation in PMN in the presence of catalase emphasizes the importance of oxidant generation as a means for synergism of Fc{gamma}RIIa and Fc{gamma}RIIIb. That H2O2 enhanced Syk phosphorylation triggered by Fc{gamma}RIIa was confirmed in mononuclear phagocytes. Thus, we propose increased phosphorylation of this critical signaling element to be a common mechanism to increase phagocytic efficiency. Previous workers, using different conditions, have shown that ROI activate Syk, but they did not consider the effects of oxidants on receptor-initiated signaling. Brumell et al. (19) demonstrated phosphorylation and Syk kinase activation in PMN which were electropermeabilized, vanadate treated, and stimulated with GTP-{gamma}S to activate NADPH oxidase. Shieven et al. (23) showed Syk activation in lymphocytes exposed to 10 mM H2O2, a 20-fold higher concentration than used in our experiments. Exposure of phagocytes to 500 µM H2O2, in the absence of Fc{gamma}R cross-linking, did not induce tyrosine phosphorylation of Syk. Our studies establish the precedent that ROI, at nontoxic concentrations, modify receptor-triggered signaling pathways in viable human phagocytes and alter receptor-mediated function.

The means by which ROI increase tyrosine phosphorylation is not clear. It has been suggested that endogenous or exogenous oxidants can promote tyrosine phosphorylation by combined activation of kinases and inhibition of phosphatases (18, 19, 26, 48). Tyrosine phosphatases may be inactivated by oxidants that target critical cysteine residues in their catalytic domains (49, 50, 51). As a consequence, constitutive autophosphorylation and stimulation of kinases, which is no longer offset by phosphatase activity, results in accumulation of phosphotyrosine. Indeed, CD45, a known inhibitor of Fc{gamma}RIIa signaling in PMN, is susceptible to inactivation by oxidants (26, 52). That CGD PMN have diminished inhibition of CD45 tyrosine phosphatase activity in response to activation of NADPH oxidase and show impaired PMA-induced amplification of Fc{gamma}R function provides indirect support for this mechanism of oxidant-induced modulation of Fc{gamma}R signaling (26, 44). The accelerated dephosphorylation of Syk in PMN exposed to H2O2 indicates that not all protein tyrosine phosphatase activity is blocked by oxidants.

Our observations provide the basis for a better understanding of the regulation of Fc{gamma}R function at sites of inflammation. Perhaps more importantly, our data indicate a mechanism for priming phagocytes for enhanced responses to receptor-driven effects. ROI generated in an inflammatory milieu act in an autocrine and paracrine manner to rapidly amplify the effector potential of Fc{gamma}R on phagocytes by altering the signal transduction. Indeed, for Fc{gamma}RIIa, exposure to oxidants enables uptake of an IgG2-opsonized particle by Fc{gamma}RIIa-R131 homozygotes, albeit to a lesser extent than that of other Fc{gamma}RIIa genotypes, and thus allows removal of IgG2-opsonized microbes and immune complexes despite relatively low binding capacity. Hence for antimicrobial defense, ROI-initiated increases in phagocytosis are protective. The importance of the interplay between ROI and Fc{gamma}R in host defense is underscored by the recent report that the risk for immune-mediated complications of CGD is associated with Fc{gamma}R allelic polymorphisms (53).

In contrast, at sites of immune complex deposition, such as the kidney in systemic lupus erythematosus, amplification of Fc{gamma}RI- or Fc{gamma}RIIa-triggered release of inflammatory mediators may directly promote tissue injury. Indeed, in the absence of PMN influx, renal injury is attenuated in murine models of autoimmune glomerulonephritis (54). Alternatively, Fc{gamma}R-driven phagocyte-derived ROI may act as second messengers to increase platelet aggregation, vascular smooth muscle cell proliferation, and mesangial cell proliferation (20, 21, 55, 56), all characteristic findings in diffuse proliferative glomerulonephritis. Our data showing that oxidants from activated PMN amplify monocyte Fc{gamma}R function support this paracrine mechanism. These findings, along with recent evidence that Fc{gamma}R-deficient mice are protected from autoimmune glomerulonephritis (57, 58), underscore the importance of identifying the factors which modulate the efficiency of Fc{gamma}R function. Definition of the role of oxidants as amplifiers of Fc{gamma}R signaling provides a novel target for therapeutic intervention in immune complex-mediated tissue injury.


   Acknowledgments
 
We thank Drs. Robert Kimberly and Jeffrey Edberg for helpful suggestions and Drs. Jurgen Frey and Jean-Pierre Kinet for providing Abs.


   Footnotes
 
1 This work was supported in part by Grant RO1-AR38889 (to J.E.S.) awarded by the National Institutes of Health. L.P. is supported by a Career Development Award from the SLE Foundation, Inc. J.G. is supported by National Institutes of Health Training Grant T32-AR07281. The Flow Cytometry Core Facility at the Hospital for Special Surgery is supported in part by the Cornell Multipurpose Arthritis and Musculoskeletal Diseases Center (P60-AR38520). Back

2 Address correspondence and reprint requests to Dr. Jane E. Salmon, Hospital for Special Surgery, 535 East 70th Street, New York, NY 10021; E-mail address: Back

3 Abbreviations used in this paper: Fc{gamma}R, receptors for Fc portion of IgG in human cells; ROI, reactive oxygen intermediates; ITAM, immunoreceptor tyrosine-based activation motif; PMN, neutrophils; AT, aminotriazole; CGD, chronic granulomatous disease; E, bovine erythrocytes; E-hIgG2, E coated with human IgG2; Fc{gamma}RI, 72-kDa high affinity receptor for Fc portion of IgG; Fc{gamma}RIIa, 40-kDa receptor for Fc portion of IgG; H131, allelic variant of Fc{gamma}RIIa that binds human IgG2 (histidine at amino acid 131); FcR{gamma}, {gamma} subunit of FcR; PI, phagocytic index; PY, phosphotyrosine; R131, allelic variant of Fc{gamma}RIIa that does not bind human IgG2 (arginine at amino acid 131). Back

Received for publication November 10, 1998. Accepted for publication March 29, 1999.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Cambier, J. C.. 1995. Antigen and Fc receptor signaling: the awesome power of the immunoreceptor tyrosine-based activation motif (ITAM). J. Immunol. 155:3281.[Medline]
  2. Daeron, M.. 1997. Fc receptor biology. Annu. Rev. Immunol. 15:203.[Medline]
  3. Hulett, M. D., P. M. Hogarth. 1994. Molecular basis of Fc receptor function. Adv. Immunol. 57:1.[Medline]
  4. Kimberly, R. P., J. E. Salmon, J. C. Edberg. 1995. Receptors for immunoglobulin G: molecular diversity and implications for disease. Arthritis Rheum. 38:306.[Medline]
  5. Huang, M. M., Z. Indik, L. F. Brass, J. A. Hoxie, A. D. Schreiber, J. S. Brugge. 1992. Activation of Fc{gamma}RII induces tyrosine phosphorylation of multiple proteins including Fc{gamma}RII. J. Biol. Chem. 267:5467.[Abstract/Free Full Text]
  6. Shen, Z., C. T. Lin, J. C. Unkeless. 1994. Correlations among tyrosine phosphorylation of Shc, p72syk, PLC-{gamma}1, and [Ca2+]i flux in Fc{gamma}RIIA signaling. J. Immunol. 152:3017.[Abstract]
  7. Van den Herik-Oudijk, I. E., P. J. Capel, T. van der Bruggen, J. G. Van de Winkel. 1995. Identification of signaling motifs within human Fc{gamma}RIIa and Fc{gamma}RIIb isoforms. Blood 85:2202.[Abstract/Free Full Text]
  8. Indik, Z. K., J. G. Park, S. Hunter, A. D. Schreiber. 1995. The molecular dissection of Fc{gamma}receptor mediated phagocytosis. Blood 86:4389.[Abstract/Free Full Text]
  9. Mitchell, M. A., M. M. Huang, P. Chien, Z. K. Indik, X. Q. Pan, A. D. Schreiber. 1994. Substitutions and deletions in the cytoplasmic domain of the phagocytic receptor Fc{gamma}RIIA: effect on receptor tyrosine phosphorylation and phagocytosis (published erratum appears in 1994, Blood 84:3252). Blood 84:1753.[Abstract/Free Full Text]
  10. Odin, J. A., J. C. Edberg, C. J. Painter, R. P. Kimberly, J. C. Unkeless. 1991. Regulation of phagocytosis and [Ca2+]i flux by distinct regions of an Fc receptor. Science 254:1785.[Abstract/Free Full Text]
  11. Crowley, M. T., P. S. Costello, C. J. Fitzer-Attas, M. Turner, F. Meng, C. Lowell, V. L. Tybulewicz, A. L. DeFranco. 1997. A critical role for Syk in signal transduction and phagocytosis mediated by Fc{gamma} receptors on macrophages. J. Exp. Med. 186:1027.[Abstract/Free Full Text]
  12. Hoffmeyer, F., K. Witte, U. Gebhardt, R. E. Schmidt. 1995. The low affinity Fc{gamma}RIIa and Fc{gamma}RIIIb on polymorphonuclear neutrophils are differentially regulated by CD45 phosphatase. J. Immunol. 155:4016.[Abstract]
  13. Harvath, L., J. A. Balke, N. P. Christiansen, A. A. Russell, K. M. Skubitz. 1991. Selected antibodies to leukocyte common antigen (CD45) inhibit human neutrophil chemotaxis. J. Immunol. 146:949.[Abstract]
  14. Indik, Z. K., J. G. Park, X. Q. Pan, A. D. Schreiber. 1995. Induction of phagocytosis by a protein tyrosine kinase. Blood 85:1175.[Abstract/Free Full Text]
  15. Kiener, P. A., B. M. Rankin, A. L. Burkhardt, G. L. Schieven, L. K. Gilliland, R. B. Rowley, J. B. Bolen, J. A. Ledbetter. 1993. Cross-linking of Fc{gamma} receptor I (Fc{gamma}RI) and receptor II (Fc{gamma}RII) on monocytic cells activates a signal transduction pathway common to both Fc receptors that involves the stimulation of p72syk protein tyrosine kinase. J. Biol. Chem. 268:24442.[Abstract/Free Full Text]
  16. Rankin, B. M., S. A. Yocum, R. S. Mittler, P. A. Kiener. 1993. Stimulation of tyrosine phosphorylation and calcium mobilization by Fc{gamma} receptor cross-linking: regulation by the phosphotyrosine phosphatase CD45. J. Immunol. 150:605.[Abstract]
  17. Vossebeld, P. J., J. Kessler, A. E. von dem Borne, D. Roos, A. J. Verhoeven. 1995. Heterotypic Fc{gamma}R clusters evoke a synergistic Ca2+ response in human neutrophils. J. Biol. Chem. 270:10671.[Abstract/Free Full Text]
  18. Fialkow, L., C. K. Chan, S. Grinstein, G. P. Downey. 1993. Regulation of tyrosine phosphorylation in neutrophils by the NADPH oxidase: role of reactive oxygen intermediates. J. Biol. Chem. 268:17131.[Abstract/Free Full Text]
  19. Brumell, J. H., A. L. Burkhardt, J. B. Bolen, S. Grinstein. 1996. Endogenous reactive oxygen intermediates activate tyrosine kinases in human neutrophils. J. Biol. Chem. 271:1455.[Abstract/Free Full Text]
  20. Sundaresan, M., Z. X. Yu, V. J. Ferrans, K. Irani, T. Finkel. 1995. Requirement for generation of H2O2 for platelet-derived growth factor signal transduction. Science 270:296.[Abstract/Free Full Text]
  21. Pignatelli, P., F. M. Pulcinelli, L. Lenti, P. P. Gazzaniga, F. Violi. 1998. Hydrogen peroxide is involved in collagen-induced platelet activation. Blood 91:484.[Abstract/Free Full Text]
  22. Aikawa, R., I. Komuro, T. Yamazaki, Y. Zou, S. Kudoh, M. Tanaka, I. Shiojima, Y. Hiroi, Y. Yazaki. 1997. Oxidative stress activates extracellular signal-regulated kinases through Src and Ras in cultured cardiac myocytes of neonatal rats. J. Clin. Invest. 100:1813.[Medline]
  23. Schieven, G. L., J. M. Kirihara, D. L. Burg, R. L. Geahlen, J. A. Ledbetter. 1993. p72syk tyrosine kinase is activated by oxidizing conditions that induce lymphocyte tyrosine phosphorylation and Ca2+ signals. J. Biol. Chem. 268:16688.[Abstract/Free Full Text]
  24. Schieven, G. L., R. S. Mittler, S. G. Nadler, J. M. Kirihara, J. B. Bolen, S. B. Kanner, J. A. Ledbetter. 1994. ZAP-70 tyrosine kinase, CD45, and T cell receptor involvement in UV- and H2O2-induced T cell signal transduction. J. Biol. Chem. 269:20718.[Abstract/Free Full Text]
  25. Monteiro, H. P., A. Stern. 1996. Redox modulation of tyrosine phosphorylation-dependent signal transduction pathways. Free Radical Biol. Med. 21:323.[Medline]
  26. Fialkow, L., C. K. Chan, G. P. Downey. 1997. Inhibition of CD45 during neutrophil activation. J. Immunol. 158:5409.[Abstract]
  27. Brumell, J. H., C. K. Chan, J. Butler, N. Borregaard, K. A. Siminovitch, S. Grinstein, G. P. Downey. 1997. Regulation of Src homology 2-containing tyrosine phosphatase 1 during activation of human neutrophils: role of protein kinase C. J. Biol. Chem. 272:875.[Abstract/Free Full Text]
  28. Salmon, J. E., S. S. Millard, N. L. Brogle, R. P. Kimberly. 1995. Fc{gamma} receptor IIIb enhances Fc{gamma} receptor IIa function in an oxidant-dependent and allele-sensitive manner. J. Clin. Invest. 95:2877.
  29. Weinrich, V., P. Sondermann, N. Bewarder, K. Wissel, J. Frey. 1996. Epitope mapping of new monoclonal antibodies recognizing distinct human FcRII (CD32) isoforms. Hybridoma 15:109.[Medline]
  30. Salmon, J. E., J. C. Edberg, R. P. Kimberly. 1990. Fc{gamma} receptor III on human neutrophils: allelic variants have functionally distinct capacities. J. Clin. Invest. 85:1287.
  31. Salmon, J. E., R. P. Kimberly. 1986. Phagocytosis of concanavalin A-treated erythrocytes is mediated by the Fc{gamma} receptor. J. Immunol. 137:456.[Abstract]
  32. Duchemin, A. M., L. K. Ernst, C. L. Anderson. 1994. Clustering of the high affinity Fc receptor for immunoglobulin G (Fc{gamma}RI) results in phosphorylation of its associated {gamma}-chain. J. Biol. Chem. 269:12111.[Abstract/Free Full Text]
  33. Edberg, J. C., R. P. Kimberly. 1992. Receptor specific probes for the study of Fc{gamma} receptor specific function. J. Immunol. Methods 148:179.[Medline]
  34. Pricop, L., J. E. Salmon, J. C. Edberg, A. J. Beavis. 1997. Flow cytometric quantitation of attachment and phagocytosis in phenotypically-defined subpopulations of cells using PKH26-labeled Fc{gamma}R-specific probes. J. Immunol. Methods 205:55.[Medline]
  35. Salmon, J. E., S. Millard, L. A. Schachter, F. C. Arnett, E. M. Ginzler, M. F. Gourley, R. Ramsey-Goldman, M. G. Peterson, R. P. Kimberly. 1996. Fc{gamma}RIIA alleles are heritable risk factors for lupus nephritis in African Americans. J. Clin. Invest. 97:1348.[Medline]
  36. Salmon, J. E., J. C. Edberg, N. L. Brogle, R. P. Kimberly. 1992. Allelic polymorphisms of human Fc{gamma} receptor IIA and Fc{gamma} receptor IIIB: independent mechanisms for differences in human phagocyte function. J. Clin. Invest. 89:1274.
  37. Parren, P. W., P. A. Warmerdam, L. C. Boeije, J. Arts, N. A. Westerdaal, A. Vlug, P. J. Capel, L. A. Aarden, J. G. van de Winkel. 1992. On the interaction of IgG subclasses with the low affinity Fc{gamma}RIIa (CD32) on human monocytes, neutrophils, and platelets: analysis of a functional polymorphism to human IgG2. J. Clin. Invest. 90:1537.
  38. Matsuda, M., J. G. Park, D. C. Wang, S. Hunter, P. Chien, A. D. Schreiber. 1996. Abrogation of the Fc{gamma} receptor IIA-mediated phagocytic signal by stem-loop Syk antisense oligonucleotides. Mol. Biol. Cell 7:1095.[Abstract]
  39. Crockett-Torabi, E., J. C. Fantone. 1990. Soluble and insoluble immune complexes activate human neutrophil NADPH oxidase by distinct Fc{gamma} receptor-specific mechanisms. J. Immunol. 145:3026.[Abstract]
  40. Durden, D. L., Y. B. Liu. 1994. Protein-tyrosine kinase p72syk in Fc{gamma}RI receptor signaling. Blood 84:2102.[Abstract/Free Full Text]
  41. Park, J. G., A. D. Schreiber. 1995. Determinants of the phagocytic signal mediated by the type IIIA Fc{gamma} receptor, Fc{gamma}RIIIA: sequence requirements and interaction with protein-tyrosine kinases. Proc. Natl. Acad. Sci. USA 92:7381.[Abstract/Free Full Text]
  42. Durden, D. L., H. M. Kim, B. Calore, Y. Liu. 1995. The Fc{gamma}RI receptor signals through the activation of hck and MAP kinase. J. Immunol. 154:4039.[Abstract]
  43. Pan, L. Y., D. B. Mendel, J. Zurlo, P. M. Guyre. 1990. Regulation of the steady state level of Fc{gamma}RI mRNA by IFN-{gamma} and dexamethasone in human monocytes, neutrophils, and U-937 cells. J. Immunol. 145:267.[Abstract]
  44. Gresham, H. D., J. A. McGarr, P. G. Shackelford, E. J. Brown. 1988. Studies on the molecular mechanisms of human Fc receptor-mediated phagocytosis: amplification of ingestion is dependent on the generation of reactive oxygen metabolites and is deficient in polymorphonuclear leukocytes from patients with chronic granulomatous disease. J. Clin. Invest. 82:1192.
  45. Smith, R. M., J. T. Curnutte. 1991. Molecular basis of chronic granulomatous disease. Blood 77:673.[Free Full Text]
  46. Green, J. M., A. D. Schreiber, E. J. Brown. 1997. Role for a glycan phosphoinositol anchor in Fc{gamma} receptor synergy. J. Cell Biol. 139:1209.[Abstract/Free Full Text]
  47. Greenberg, S., P. Chang, D. C. Wang, R. Xavier, B. Seed. 1996. Clustered syk tyrosine kinase domains trigger phagocytosis. Proc. Natl. Acad. Sci. USA 93:1103.[Abstract/Free Full Text]
  48. Zor, U., E. Ferber, P. Gergely, K. Szucs, V. Dombradi, R. Goldman. 1993. Reactive oxygen species mediate phorbol ester-regulated tyrosine phosphorylation and phospholipase A2 activation: potentiation by vanadate. Biochem. J. 295:879.
  49. Streuli, M., N. X. Krueger, A. Y. Tsai, H. Saito. 1989. A family of receptor-linked protein tyrosine phosphatases in humans and Drosophila. Proc. Natl. Acad. Sci. USA 86:8698.[Abstract/Free Full Text]
  50. Fischer, E. H., H. Charbonneau, N. K. Tonks. 1991. Protein tyrosine phosphatases: a diverse family of intracellular and transmembrane enzymes. Science 253:401.[Abstract/Free Full Text]
  51. Tonks, N. K., C. D. Diltz, E. H. Fischer. 1988. Characterization of the major protein-tyrosine-phosphatases of human placenta. J. Biol. Chem. 263:6731.[Abstract/Free Full Text]
  52. Fialkow, L., C. K. Chan, D. Rotin, S. Grinstein, G. P. Downey. 1994. Activation of the mitogen-activated protein kinase signaling pathway in neutrophils: role of oxidants. J. Biol. Chem. 269:31234.[Abstract/Free Full Text]
  53. Foster, C. B., T. Lehrnbecher, F. Mol, S. M. Steinberg, D. J. Venzon, T. J. Walsh, D. Noack, J. Rae, J. A. Winkelstein, J. T. Curnutte, S. J. Chanock. 1998. Host defense molecule polymorphisms influence the risk for immune-mediated complications in chronic granulomatous disease. J. Clin. Invest. 102:2146.[Medline]
  54. Suzuki, Y., I. Shirato, K. Okumura, J. V. Ravetch, T. Takai, Y. Tomino, C. Ra. 1998. Distinct contribution of Fc receptors and angiotensin II-dependent pathways in anti-GBM glomerulonephritis. Kidney Int. 54:1166.[Medline]
  55. Kuroki, M., E. E. Voest, S. Amano, L. V. Beerepoot, S. Takashima, M. Tolentino, R. Y. Kim, R. M. Rohan, K. A. Colby, K. T. Yeo, A. P. Adamis. 1996. Reactive oxygen intermediates increase vascular endothelial growth factor expression in vitro and in vivo. J. Clin. Invest. 98:1667.[Medline]
  56. Wilmer, W. A., L. C. Tan, J. A. Dickerson, M. Danne, B. H. Rovin. 1997. Interleukin-1ß induction of mitogen-activated protein kinases in human mesangial cells: role of oxidation. J. Biol. Chem. 272:10877.[Abstract/Free Full Text]
  57. Clynes, R., C. Dumitru, J. V. Ravetch. 1998. Uncoupling of immune complex formation and kidney damage in autoimmune glomerulonephritis. Science 279:1052.[Abstract/Free Full Text]
  58. Park, S. Y., S. Ueda, H. Ohno, Y. Hamano, M. Tanaka, T. Shiratori, T. Yamazaki, H. Arase, N. Arase, A. Karasawa, et al 1998. Resistance of Fc receptor-deficient mice to fatal glomerulonephritis. J. Clin. Invest. 102:1229.[Medline]



This article has been cited by other articles:


Home page
 BloodHome page
R. Shashidharamurthy, R. A. Hennigar, S. Fuchs, P. Palaniswami, M. Sherman, and P. Selvaraj
Extravasations and emigration of neutrophils to the inflammatory site depend on the interaction of immune-complex with Fc{gamma} receptors and can be effectively blocked by decoy Fc{gamma} receptors
Blood, January 15, 2008; 111(2): 894 - 904.
[Abstract] [Full Text] [PDF]