PubMed1977
A quantitative rosette assay was employed in order to determine if through pharmacologic probes we could gain an insight into the nature of the interaction between C3b-coated particles and the macrophage C3b receptor. Rabbit alveolar macrophage monolayers were challenged with chromium-labeled, complement-coated (via cold agglutinin) human erythrocytes (HEC3b) and the per cent of bound counts determined in the distilled water lysate. With this assay system in which ingestion is negligible, the cytochalasins (A greater than E greater than D greater than B) produced the most marked inhibition of rosette formation compared to control treated monolayers. No agent examined produced consistent augmentation. Cytochalasin A at 10(-5), 10(-6), and 10(-7) M inhibited rosette formation by 77+/- 2, 44 +/- 4 and 15 +/- 7 (S.E.), per cent, respectively. Cytochalasin E was also markedly inhibitory, Cytochalasins B and D produced approximately 30% inhibition at 10(-5) M. The cytochalasin effect was not secondary to an interaction between these agents and complement-sensitized erythrocytes, although cytochalasin E was also able to reduce erythrocyte-bouund C3b reactivity. Cytochalasin A and E modulation of the macrophage C3b reactivity occurred within a few minutes and was only slightly reversible. Cytochalasins A and E could also disrupt performed rosettes but the effect was not as pronounced as when these agents were present before and/or during the actual adherence phenomenon. Vinblastine and colchicine (10(-5) and 10(-6) M) also produced significant inhibition of rosette formation, although the magnitude of the effect was less than that for cytochalasins A and E. Further characterization of the vinblastine and colchicine effect demonstrated that the inhibition was rapid, irreversible over a 60-min incubation, and not explained by an alteration in macrophage attachment or in HEC3b reactivity. Agents producing insignificant inhibition of rosette formation included the following: dibutyryl cAMP and cAMP agonists (PGE1, theophylline), 8-bromo cGMP and cGMP agonists (carbachol, asorbic acid), dimethylsulfoxide, heparin, ethanol, dextran sulfate, DEAE-dextran, and poly-L-lysine. The data suggest that cytochalasin, vinblastine and colchicine sensitive membrane structures, most likely microfilaments and microtubules, are important in the interaction of C3b-coated particles with the macrophage C3b receptor.