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© The Rockefeller University Press, 0022-1007/1997/9/955/ $5.00
The Journal of Experimental Medicine, Volume 186, Number 6, September 15, 1997 955-966

Articles

Rho is Required for the Initiation of Calcium Signaling and Phagocytosis by Fc{gamma} Receptors in Macrophages

David J. Hackam*,{ddagger}, Ori D. Rotstein{ddagger}, Alan Schreiber§, Wei-jian Zhang*,{ddagger}, and Sergio Grinstein*

From the * Division of Cell Biology, Hospital for Sick Children, Toronto, Ontario, M5G 1X8, Canada; {ddagger} Department of Surgery, The Toronto Hospital, Toronto, Ontario, M5G 2C4, Canada; and § Department of Medicine, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104-4283

Phagocytosis of bacteria by macrophages and neutrophils is an essential component of host defense against infection. The mechanism whereby the interaction of opsonized particles with Fc{gamma} receptors triggers the engulfment of opsonized particles remains incompletely understood, although activation of tyrosine kinases has been recognized as an early step. Recent studies in other systems have demonstrated that tyrosine kinases can in turn signal the activation of small GTPases of the ras superfamily. We therefore investigated the possible role of Rho in Fc receptor–mediated phagocytosis. To this end we microinjected J774 macrophages with C3 exotoxin from Clostridium botulinum, which ADP-ribosylates and inactivates Rho. C3 exotoxin induced the retraction of filopodia, the disappearance of focal complexes, and a global decrease in the F-actin content of J774 cells. In addition, these cells exhibited increased spreading and the formation of vacuolar structures. Importantly, inactivation of Rho resulted in the complete abrogation of phagocytosis. Inhibition of Fc{gamma} receptor–mediated phagocytosis by C3 exotoxin was confirmed in COS cells, which become phagocytic upon transfection of the Fc{gamma}RIIA receptor. Rho was found to be essential for the accumulation of phosphotyrosine and of F-actin around phagocytic cups and for Fc{gamma} receptor–mediated Ca2+ signaling. The clustering of receptors in response to opsonin, an essential step in Fc{gamma}-induced signaling, was the earliest event shown to be inhibited by C3 exotoxin. The effect of the toxin was specific, since clustering and internalization of transferrin receptors were unaffected by microinjection of C3. These data identify a role for small GTPases in Fc{gamma} receptor–mediated phagocytosis by leukocytes.

Address correspondence to Dr. Sergio Grinstein, Division of Cell Biology, Hospital for Sick Children, 555 University Avenue, Toronto, Ontario, Canada M5G 1X8. Phone: 416-813-5727: FAX: 416-813-5028; E-mail: sga{at}sickkids.on.ca

This research was funded by operating grants awarded to S. Grinstein and O.D. Rotstein by the Medical Research Council of Canada. D.J. Hackam is the recipient of a Medical Research Council of Canada Fellowship and an Ethicon-Society of University Surgeons Surgical Research Award. S. Grinstein is an International Scholar of the Howard Hughes Medical Institute.

1 Abbreviations used in this paper: [Ca2+]i, free cytosolic calcium; PAF, platelet-activating factor.


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