Acquisition of β-glucuronidase activity by deficient fibroblasts during direct contact with lymphoid cells

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Abstract
Fibroblasts deficient in β-glucuronidase acquired high levels of this enzyme when they were co-cultured with concanavalin A-stimulated lymphocytes. Acquired enzyme activity, determined using a single-cell cytochemical assay, was directly proportional to the number of lymphocytes added and persisted for several days in fibroblasts maintained at high density. Lymphocytes did not secrete significant levels of β-glucuronidase into their culture medium, and did not release other substances able to induce synthesis of the enzyme by the deficient fibroblasts. Nor did β-glucuronidase acquisition result from concanavalin A-mediated uptake of enzyme, since a-methylmannoside did not reduce acquired activity. Moreover, lymphocytes from various sources, whether unstimulated or activated by a different mitogen, bacterial lipopolysaccharide, were equally effective in promoting the appearance of β-glucuronidase. Deficient fibroblasts did not acquire β-glucuronidase by active endocytosis when co-cultured with lymphocytes, since enzyme extracted from lymphocytes was not itself effective in this respect. Furthermore, mannose 6-phosphate, which did inhibit endocytosis by deficient fibroblasts of exogenous β-glucuronidase prepared from 3T3 cells, had no effect on enzyme acquisition by fibroblasts during their co-culture with lymphocytes. Conversely, inhibitors of protein synthesis and energy metabolism, which did not interfere with endocytosis of exogenous enzyme, abolished the acquisition of βglucuronidase during co-culture. Deficient fibroblasts did not acquire β-glucuronidase when they were cultured together with lymphocytes but separated from them by Millipore membranes permeable to exogenous enzyme. Thus, although the mechanism of acquisition is still unclear, the present results suggest that β-glucuronidase is transferred from lymphocytes to deficient fibroblasts by a process in which direct cell-to-cell contact is obligatory.

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