Synergistic effect of lymphokines and lipopolysaccharides on macrophage chemiluminescence. Appearance of spontaneous chemiluminescence and its correlation with cytotoxicity.

A new type of chemiluminescence (CL) was observed in thioglycolate-elicited ICR mouse peritoneal macrophages which had been incubated for 24 h in medium containing lymphokines (LK) and lipopolysaccharides (LPS), and then exposed to a CL reagent solution containing luminol and phorbol myristate acetate (PMA). The new CL, which was clearly distinguishable from PMA-induced CL, appeared immediately after the addition of the CL reagent solution, reaching a maximal level at about 30 s, and disappeared rapidly. We have called the new CL 'spontaneous CL', since it appeared spontaneously without PMA triggering. When the macrophages were incubated with LK in the presence of 10 ng/ml of LPS, the spontaneous CL began to appear after about 4 h incubation, the maximal level being reached at about 12 h, after which it decreased gradually on further incubation. After 48 h, it could not be observed at all. The spontaneous CL could be observed only in macrophages simultaneously treated with LK and LPS, as in the case of cytotoxicity. The correlation between spontaneous CL and cytotoxicity was suggested by the following; they showed a similar dose dependency to LK, and neither of them could be induced in aged macrophages which had been incubated in vitro for 1 day before exposure to LK and LPS. These results suggest that spontaneous CL measurement could possibly replace the cytotoxicity test as a simple method for the determination of macrophage activating factor.