Digital integration of granulocyte aggregation responses - A simple and reproducible method for the quantitation of granulocyte adhesiveness

The pulmonary leukostasis and lung dysfunction associated with intravascular complement activation results from C5a-mediated granulocyte (GR) aggregation, a phenomenon which can be reproduced in vitro using standard nephelometric techniques. To produce a more subjective measure of the extent and rate of GR aggregation responses we added a digital integrator to the system. The validity of this approach was substantiated by the close correlation between the aggregating and chemotactic activities of C5a and N-formyl-metionine-leucine-phenylalanine. Use of this technique enabled us to define the dose-response relationship of the aggregation produced by the cationophore A23187 and the inhibitory effect of tetracaine on divalent cationdependent aggregation responses. The aggregation produced by these three stimuli does not result primarily from simple cross-linking of surface charges because, unlike the passive cell-cell association produced by the cation poly-l-lysine, it is not inhibited by anionic poly-l-glutamic acid. The importance of microtubules as regulators of GR adhesiveness was substantiated by the inhibitory effects of colchicine (but not lumicolchicine) on aggregation in this system. These data suggest that this integration of light transmission increments is a useful adjunct to this basic technique, whether used as a bioassay for chemotactic stimuli or as a model to study the many factors which regulate GR adhesiveness.