Phagosomal Maturation, Acidification, and Inhibition of Bacterial Growth in Nonphagocytic Cells Transfected with FcγRIIA Receptors

doi:10.1074/jbc.274.40.28436

Phagosomal Maturation, Acidification, and Inhibition of Bacterial Growth in Nonphagocytic Cells Transfected with FcγRIIA Receptors*

From the ^‡Division of Respirology, Department of Medicine and the ^‖Department of Biochemistry, University of Toronto M5S 1A8, ^§Toronto Hospital, Toronto M5G 2C4, the ^¶Programme in Cell Biology, Hospital for Sick Children, Toronto M5G 1X8, Ontario, Canada, and the ^‡Department of Medicine, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104-4283

Abstract

Phagocytosis and killing of microbial pathogens by professional phagocytes is an essential component of the innate immune response. Recently, heterologous transfection of individual receptors into nonmyeloid cells has been used successfully to elucidate the early steps that signal phagosome formation. It is unclear, however, whether the vacuoles formed by such transfected cells are bona fide phagosomes, capable of fusion with endomembranes, of luminal acidification, and of controlling the growth of microorganisms. The aim of the current study was to determine whether COS-1 and Chinese hamster ovary cells, rendered phagocytic by expression of human FcγRIIA receptors, express the cellular machinery required to support phagosomal maturation. Immunolocalization studies demonstrated that early endosomes, as well as late endosomes and/or lysosomes, fuse sequentially with phagosomes in the transfectants. Microfluorescence ratio imaging of particles labeled with pH-sensitive dyes revealed that maturation of the phagosome was accompanied by luminal acidification. The drop in pH, which attained levels comparable to those reported in professional phagocytes, was prevented by inhibitors of vacuolar-type H⁺-ATPases. Optimal phagosomal acidification required elevation of cytosolic [Ca²⁺], suggesting that it results from fusion of endomembranes bearing proton pumps. Moreover, the transfected cells effectively internalized live bacteria. Opsonization was essential for bacterial internalization, implying that it occurred by FcγRIIA-mediated phagocytosis, as opposed to invasion. Uptake into phagolysosomes was associated with inhibition of bacterial growth, due at least in part to the low intraphagosomal pH. These studies indicate that the biochemical events that follow receptor-mediated particle internalization in cells transfected with FcγRIIA receptors closely resemble the process of phagosomal maturation in neutrophils and macrophages. FcγRIIA-transfected cells can, therefore, be used as a model for the study of additional aspects of phagocyte biology.

Footnotes

↵* This work was supported by the Medical Research Council of Canada and by National Institutes of Health Grant AI-22193.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵** Recipient of a Studentship from the Natural Science and Engineering Research Council of Canada.
↵§§ International Scholar of the Howard Hughes Medical Institute and the current holder of the Pitblado Chair in Cell Biology. To whom correspondence should be addressed: Division of Cell Biology, Hospital for Sick Children, 555 University Ave., Toronto M5G 1X8, Ontario, Canada. Tel.: 416-813-5727; Fax: 416-813-5028; E-mail: sga@sickkids.on.ca.
Abbreviations:

CHO

Chinese hamster ovary

BAPTA

1,2-bis(aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid

BCECF

2′,7′-bis(2-carboxyethyl)-5(6)-carboxyfluorescein

FITC

fluorescein isothiocyanate

LAMP

lysosome-associated membrane protein

PBS

phosphate-buffered saline

pH_c

cytosolic pH

pH_p

phagosomal pH

RBC

red blood cells

Tfn

transferrin

V-ATPase

vacuolar-type H⁺-ATPase

ITAM

immunoreceptor tyrosine-based activation motif
- Received March 18, 1999.
- Revision received July 22, 1999.
The American Society for Biochemistry and Molecular Biology, Inc.