Macrophage-Melanoma Cell Heterokaryons

Abstract

The plasma membrane of the mouse peritoneal macrophage has specific receptors which enable the cell to ingest antibody-coated sheep red .cells and is also rich in a divalent cation-dependent adenosine triphosphatase (ATPase) activity (1, 2). By fusing macrophages with a strain of mouse melanoma cells which lacks these receptors, one could not only trace the fate of the macrophage membrane in the heterokaryons, but also hope to learn about the expression of cell-specific traits in animal cells (3).Macrophage homokaryons obtained by Sendai virus-induced cell fusion continued to exhibit active phagocytosis of sensitized erythrocytes. The phagocytic receptor could also be detected in heterokaryons shortly after fusion, but was progressively lost over the next 12-24 hr. The ATPase activity disappeared from heterokaryons as well, and in addition, seemed to spread out over the cell surface.Several possible mechanisms could account for these observations. These include mixing of membrane components resulting in dilution and loss of function; endo-or exocytosis without further synthesis; and masking of the macrophage membrane markers, either by serum factors or by the heterokaryon itself. We report experiments to distinguish among these hypotheses. Materials and MethodsCell Culture and Cdl Fusion Technlque.--Carried out as described (3). The macrophage phagocytic receptor was assayed with minor modifications of our previous procedure. Cover slip preparations were exposed at room temperature for 15 rain to a 0.1% suspension of sheep red blood cells (SRBC) 1 which had been coated with a 1/~o00 dilution of a rabbit anti-sheep * This work was partially supported by Grant AI 07012 from the National Institutes of Health.1 Abbreviations used in this paper: AbSRBC, antibody-coated sheep red blood cells; BGG, bovine gamma globulin; BSA, bovine serum albumin; EAT, Ehrlich ascites tumor cells; IgG, immunoglobulin G; IPT-FCS, immunoprecipitin-tested fetal calf serum; NBCS, newborn calf serum; 199 M, medium 199 + 10% NBCS; SRBC, sheep red blood cells; STI, soybean trypsin inhibitor.

Dynamics of the Macrophage Plasma Membrance

“…Modulation of Fe receptor activity has been observed with MPM hetero karyons (89,91,93) and in the 1774.2 macrophage cell line (169,170). Gordon et al (89,91,93) demonstrated the loss of Fe receptor expression in heterokaryons derived fr om fu sion ofMPM with L cells, melanoma cells, and Ehrlich ascites tumor cells, but not with chicken erythrocytes.…”

“…Gordon et al (89,91,93) demonstrated the loss of Fe receptor expression in heterokaryons derived fr om fu sion ofMPM with L cells, melanoma cells, and Ehrlich ascites tumor cells, but not with chicken erythrocytes. Receptor activity was unmasked by trypsin, but this effect was reversible with further culture.…”

“…The fluidity of the macrophage membrane has been demonstrated by cell fusion (89)(90)(91)(92)(93), electron spin resonance (229), and ligand-induced redistribution of surface components (227).…”

Zanvil Alexander Cohn 1926-1993.

“…directly observing phagocyte chemotaxis and motility. Gordon and Cohn were utilizing several phagocyte differentiation markers in their macrophage-melanocyte fusion studies (63)(64)(65)(66). When Steinman and Cohn evaluated this repertoire of known macrophage traits in dendritic cells, not a single resemblance to macrophages was apparent (91,92).…”

“…Elliot Osserman had found a huge output of lysozyme in the urine of patients with monocytic leukemia. Gordon and Cohn, searching for differentiation markers for macrophages to be used in their somatic cell genetic studies (63)(64)(65)(66), noted that many different populations of macrophages released lysozyme continously for days and without a phagocytic challenge. The secretion rate amounted to 2.5% of total cell protein per day.…”