Isolation of Receptor-Bearing Plasma Membrane Vesicles from Guinea Pig Macrophages

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Abstract
The plasma membrane serves as an important site for the interaction of macrophages and lymphocytes in antigen recognition and of macrophages and foreign agents in inflammatory responses. Despite the critical role of the macrophage membrane in these reactions, studies on the biochemical and functional characteristics of macrophage membranes which need to be performed have been limited by the lack of suitable methods for the isolation of macrophage plasma membranes that maintain functional and antigenic membrane markers. Recently a new method for membrane isolation has been reported which is based on the observation that a variety of sulfhydryl blocking agents can induce the shedding of plasma membrane vesicles of monolayer cell lines. In this paper we demonstrate that guinea pig peritoneal exudate macrophages can also be induced to shed significant quantities of plasma membrane vesicles by such reagents, in particular with a low m.w. aldehyde, and that these vesicles can be isolated by centrifugation. Vesiculation in macrophages is potentiated by disulfide reducing agents, such as dithiothreitol, and by calcium ions. Maximum vesiculation is observed when well spread macrophage monolayers are incubated in a slightly hypertonic vesiculant at pH 7.0 for 2 hr at 37°C. Brief exposure of macrophages to a vesiculant for intervals as short as 30 min is also effective. Macrophage vesicles shed from the cell surface by this procedure have been found to contain receptors for concanavalin A, wheat germ agglutinin, phytohemagglutinin, and lentil lectin. Vesicles also contain Fc and complement (C3) receptors and form rosettes with thymic lymphocytes. It is suggested that macrophage plasma membrane vesicles isolated by this technique should serve as a valuable new system to study the biochemical characteristics of macrophage plasma membranes and their role in immunologic reactions.

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