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Phagocytosis in Macrophages Lacking Cbl Reveals an Unsuspected Role for Fc Receptor Signaling and Actin Assembly in Target Binding¹

Benjamin M. Dale^*, Daniel Traum, Hediye Erdjument-Bromage, Paul Tempst and Steven Greenberg^2,3,*,

^* Department of Pharmacology and Division of Pulmonary, Allergy and Critical Care, Department of Medicine, Columbia University College of Physicians & Surgeons, New York, NY, 10032; and Molecular Biology Program, Memorial Sloan-Kettering Cancer Center, and Weill Graduate School of Medical Sciences, Cornell University, New York, NY 10021

Fc receptor (FcR)-mediated phagocytosis is known to require tyrosine kinases (TKs). We identified c-Cbl and Cbl-b as proteins that undergo tyrosine phosphorylation during phagocytosis. Cbl-deficient macrophages displayed enhanced FcR-mediated signaling and phagocytosis. Surprisingly, binding of IgG-coated targets (EIgG) was also enhanced. c-Cbl-deficient macrophages expressed less FcRIIb, the inhibitory Fc receptor; however, this did not account for enhanced target binding. We isolated the function of one Fc receptor isoform, FcRI, using IgG2a-coated targets (EIgG2a). Cbl-deficient macrophages demonstrated a disproportionate increase in binding EIgG2a, suggesting that signal strength regulates binding efficiency toward opsonized targets. In resting cells, FcRI colocalized with the Src family TK Hck in F-actin-rich structures, which was enhanced in Cbl-deficient macrophages. Target binding was sensitive to TK inhibitors, profoundly inhibited following depletion of cholesterol, and ablated at 4°C or in the presence of inhibitors of actin polymerization. Sensitivity of EIgG binding to cytoskeletal disruption was inversely proportional to opsonin density. These findings challenge the view that FcR-mediated binding is a passive event. They suggest that dynamic engagement of TKs and the cytoskeleton enables macrophages to serve as cellular "Venus fly traps", with the capacity to capture phagocytic targets under conditions of limiting opsonin density.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ This work was supported by National Institutes of Health Grants HL054164 and AI067502 to S.G.

² Current address: Merck Research Laboratories, 126 East Lincoln Avenue, RY34B-348, Rahway, NJ 07065.

³ Address correspondence and reprint requests to Dr. Steven Greenberg, Merck Research Laboratories, 126 East Lincoln Avenue, RY34B-348, Rahway, NJ 07065. E-mail address: smg8{at}columbia.edu

⁴ Abbreviations used in this paper: TK, tyrosine kinase; BMDM, bone marrow-derived macrophages; EIgG, IgG-coated erythrocytes; WT, wild type.

⁵ The online version of the article contains Extended Methods.

⁶ The online version of this article contains supplemental material.