Lymphocyte binding of fluid phase mouse C3b.

Isolated fluid phase mouse C3b adhered to human and to mouse lymphocytes. On human, but not on mouse cells it could be stained by fluoresceinated F(ab')2 anti-mouse C3. Binding to mouse cells was, however, shown by inhibition of C3-dependent rosette formation and by direct staining with fluorescein-conjugated C3b (FITC-mouse C3b). Optimal staining of lymphocytes by FITC-mouse C3b depended on a sufficient intensity of fluorochrome conjugation and on a degree of aggregation of the C3b. FITC-mouse C3b preparations which initially stained weakly, stained strongly after being aggregated with glutaraldehyde. The failure of immunofluorescent techniques to demonstrate binding by mouse lymphocytes of mouse C3b which had not been so aggregated was apparently due to inaccessibility to anti-C3 of the receptor-bound C3b. Aggregated mouse C3b-FITC induced sequential patching and capping of lymphocyte complement receptors followed by endocytosis, all of which was inhibited completely by cold (4 degrees), sodium azide (2 x 10(-3) m), cytochalasin B (28 microgram/ml) and chlorpromazine (10(-4) m), and partially by lignocaine (2 x 10(-3) m), and colchicine (10(-4) m). That mouse C3 which has interacted with mouse complement receptors may not be demonstrable by anti-C3 antibody may have important implications for immunohistochemical localization of C3 bound in vivo to lymphoid cells.