CR3 (Mac-l, CgM 2, CDllb/CD18) and Fc /RIII Cooperate in Generation of a Neutrophil Respiratory Burst: Requirement for FcTRII and Tyrosine Phosphorylation

Cooperation among plasma membrane receptors in activating signal transduction cascades is not well understood. For almost 20 years, it has been clear that when a particulate foreign body is opsonized with complement as well as IgG, the efficiency of IgG effector functions is markedly enhanced. However, the molecular mechanisms involved in cooperation between IgG Fc receptors and complement receptors have not been elucidated. In this work, we show that when human neutrophils (PMN) are plated on a surface coated with both anti-CR3 and anti-Fc3~RIII antibodies, the respiratory burst which occurs is equivalent to that stimulated by anti-Fc3,RII. The CR3 ligand iC3b is as effective as anti-CR3 for cooperating with anti-Fc~RJII in generation of a respiratory burst. The synergy between CR3 and Fc'yRIII for activating the NADPH oxidase is abolished by Fab of anti-Fc-tRII. Nonetheless, the observed synergy is not an artifact of unintended Fc,yRII ligation, since (a) only this combination of antibodies works to generate H202; (b) coating plates with either of the antibodies alone cannot activate the respiratory burst at any dose; (c) LAD (CR3 deficient) cells, which are perfectly competent to mount a respiratory burst when Fc~RII is engaged, are incapable of activating the respiratory burst when adherent to wells coated with anti-Fc.yRIII and antiCR3; (d) direct engagement of Fc3,RII activates the respiratory burst by a pathway pharmacologically distinguishable from the synergistic respiratory burst. FcTRIII/CR3 synergy is abolished by cytochalasin B and herbimicin, suggesting that both the actin cytoskeleton and tyrosine phosphorylation are necessary for activation of the synergistic respiratory burst. Further analysis shows that CR3 and Fc3,RIII have distinct roles in activation of this Fc.gRII-dependent assembly of the NADPH oxidase. Ligation of CR3 is sufficient to lead to FeyRII association with the actin cytoskeleton on the adherent PMN surface. Coligation of Fc3,RIH is required for tyrosine phosphorylation of Fc3,RII. These data are consistent with a model in which phosphorylation of Fc-yRlI or a closely associated substrate initiates activation of a signal transduction pathway leading to oxidase assembly. These are the first data to demonstrate a molecular mechanism for synergy between IgG Fc and complement receptors in activation of phagocyte effector functions. INCE the work of Ehlenberger and Nussenzweig almost 20 years ago (17), it has been clear that when a particulate foreign body is opsonized with complement as well as IgG, the efficiency of IgG effector functions is markedly enhanced. The Ehlenberger-Nussenzweig hypothesis was that the role for complement interaction with complement receptors was to increase the efficiency of presentation of IgG to phagocyte Fc receptors. The implication of this hypothesis was that complement receptors did not contribute to the signal transduction which led to phagocytosis, degranulation, respiratory burst, and leukotriene proAddress all correspondence to Dr. Brown, Division of Infectious Diseases, Campus Box 8051, Washington University School of Medicine, 660 S. Euclid Avenue, St. Louis, MO 63110. duction which occurred upon Fc receptor ligation. The lack of a role for complement receptor signaling in these functions has been supported by several studies (1, 42). However, recent studies have suggested that complement receptors may have a direct role in signal transduction in some phagocyte Fc receptor-mediated functions (10, 22, 25, 38). Biophysical studies from Petty's laboratory have shown that complement receptor 3 (CR3), 1 a member of the/32 integrin family, cocaps with Fc'yRIII, the phosphoinositol glycan1. Abbreviations used in this paper: CR3, complement receptor 3; DFP, diisopropyfluorophosphate; DOC, deoxycholic acid; LAP, integrin-associated protein; KRPG, Krebs buffer with glucose; PIG, phosphoinositoi glycan; PBS Tween, phosphate buffered saline with 0.5% Tween 20; PMN, polymorphonuclear leukocytes; RM, reaction mixture. © The Rockefeller University Press, 0021-9525/94/06/1407/10 $2.00 The Journal of Cell Biology, Volume 125, Number 6, June 1994 1407-1416 1407 on July 9, 2017 jcb.rress.org D ow nladed fom linked Fc receptor on polymorphonuclear leukocytes (PMN), and that this cocapping can be inhibited by N-acetyld-glucosamine (43). From these data, Petty has hypothesized a direct physical interaction between Fc~,R/II and CR3 (38). Together, these experiments suggest that when CR3 is ligated alone, signal transduction pathways may not be activated, but it has an important and necessary, but unexplained, role in signal transduction resulting from Fc receptor ligation. Despite these advances, direct tests of complement receptor-dependent signal transduction have been lacking. PMN express several complement receptors which recognize similar or identical ligands and also express at least two Fc receptors with similar ligand specificity. The resultant complexity of ligand-receptor interactions has made dissection of collaboration in signal transduction extremely difficult. Recently, an assay system using PMN adhesion to specific monoclonal antibodies has been developed which allows dissection of signal transduction phenomena (4, 44). In this work, we have used this assay system to understand Fc receptor-complement receptor collaboration in generation of a respiratory burst. We have found that the combination of anti-Fc,yR/II and anti-CR3 (or the CR3 ligand iC3b) leads to activation of a PMN respiratory burst, while neither antibody alone, nor iC3b alone, can do so. Anti-Fc3,RII, on the other hand, is competent to stimulate a respiratory burst on its own. Surprisingly, the respiratory burst stimulated by synergy between Fc~,RIII and CR3 is completely inhibited by Fab of anti-Fc~,RII. The synergistic respiratory burst is abrogated by cytochalasin B and by herbimicin, an inhibitor of tyrosine phosphorylation. Further analysis reveals that ligation of CR3 is sufficient to cause Fc3,RII association with the actin cytoskeleton, but that Fc-yRII is phosphorylated on tyrosine only when both CR3 and Fc3,RIII are ligated. These data are the first to provide a mechanism for cooperation between Fc receptors and complement receptors in phagocyte effector functions. We hypothesize that FcTRIII and CR3 have distinct roles in this collaboration, that ligation of CR3 signals an association of Fc-vRII with the actin cytoskeleton, and ligation of Fc~,RIII on the same adherent surface leads to tyrosine kinase activity in proximity with Fc3,RII. We suggest that this two-receptordependent coordination off enzyme and substrate initiates a pathway for assembly of the NADPH oxidase. Materials and Methods Monoclonal Antibodies The following mAbs were used in these studies: IB4 (anti-CD18) (4D; 3D9 (anti-CD 35) (33); W6/32 (anti-HLA) (2); B6H12 and 2D3 (anti-lAP, CD47) (11, 24); 3G8 (anti-CD16) (20), KuFc79 (40), and IV.3 (anti-CD32) (31). IB4, 3D9, 3G8, Ku79Fc, and IV.3 IgG were purified from ascites using octanoic acid as described (22). W6/32, B6H12, and 2D3 IgG were prepared using an Amicon Bioreactor (Amicon Inc., Danvers, MA) according to manufacturer's instructions. SDS-PAGE of all purified IgG preparations showed them to be >90% IgG. 4G10 (anti-phosphotyrosine, IgG2b) was from UBI (Lake Placid, NY). Fab fragments of W6/32, IV.3 and OKM1, F(ab92 fragments of 3G8 and 2D3 were prepared as described (34). FITCconjugated antibody fragments were prepared as described (43). Immunoprecipitation studies have shown that there is no cross-reactivity between anti-FcTRII mAb and Fc~,RIII, or between anti-Fc,yR/II mAb and FcvRII (19, 31, 40). Buffers and Other Reagents Phosphate buffered saline was from Biowhittaker, Walkersville, MD. KrebsRinger buffer (KRPG) was 145 mM NaCI, 4.86 mM KCI, 1.22 mM MgSO4, 5.7 mM Na2HPO4, 0.54 mM CaCI2, and 5.5 mM glucose, pH 7.4. Reaction mixture (RM) consisted of 37.5/~M scopoletin, 1.25 mM NAN3, 1.25 U/ml HPO in KRPG. Other reagents were obtained from standard sources as previously described (44).