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Journal of Experimental Medicine, Vol 152, 1236-1247, Copyright © 1980 by Rockefeller University Press
ARTICLES |
CA Rouzer, WA Scott, AL Hamill and ZA Cohn
A method for the radiochemical assay of LTC production by mouse peritoneal macrophages in vitro is presented. The method involves labeling macrophages in culture with [5,6,8,9,11,12,14,15-3H]20:4 followed by stimulation of arachidonic acid (20:4) release under the experimental conditions desired. Radiolabeled leukotriene C (LTC) is recovered from the culture medium by extraction and silicic acid chromatography in 40% yield with full retention of biological activity. Because this LTC is radiochemically pure, the quantity of LTC release may be estimated from the amount of radioactivity in the sample. Use of the radioassay to study parameters affecting LTC synthesis by macrophages indicated that the time course of LTC synthesis and its relationship to the dose of a phagocytic stimulus (zymosan) were very similar to those of prostaglandin (PG) release. LTC release was also similar to that of PG in that lower levels of both metabolites were produced by Corynebacterium parvum-elicited macrophages than by resident cells. Finally, LTC release was stimulated in response to a challenge with antigen-antibody complexes, but lower maximal levels were attained than those with zymosan. The data presented here are consistent with the hypothesis that challenge of macrophages with a phagocytic stimulus leads to the release of 20:4 by an inducible phospholipase. Cyclooxygenase and lipoxygenase then compete for the released 20:4, leading to the production of PG, hydroxyeicosatetraenoic acids, and LTC.
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