Cultured human monocytes require exposure to bacterial products to maintain an optimal oxygen radical response

Abstract

Freshly isolated human blood monocytes displayed a vigorous oxygen radical response, measured as release of superoxide anion (O2-), after stimulation with phorbol myristate acetate (PMA) or opsonized zymosan. High O2- release was observed with cells isolated by using a variety of procedures. Monocytes cultured in endotoxin-free medium M199 with or without 5% heat-inactivated autologous serum gradually lost this ability to produce O2- in response to PMA over the course of 4 days. The decreased responsiveness to PMA was accompanied by decreased adherence and viability. The loss of function, adherence, and viability was prevented by supplementing the culture medium with either bacterial lipopolysaccharide (LPS) or muramyl dipeptide (MDP). The O2- response of monocytes cultured for several days without bacterial products could be partially restored by the addition of LPS on day 2 or 3 of culture. Partial restoration could be detected in monocytes after only 1 hr of exposure to LPS, although a maximal response required a 2-day exposure. The minimum effective concentration of MDP was 1 ng/ml; stereoisomers of MDP, which are inactive as adjuvants, had no effect at 1 micrograms/ml. The minimum effective concentration of LPS was 1 pg/ml, corresponding to fewer than 10 molecules of LPS per monocyte. These results suggest that exposure to LPS or other bacterial products, represented here by MDP, may be required to preserve the microbicidal potential of human monocyte-macrophages in vivo.