Enhanced chemiluminescence production by purified macrophage monolayers from C. parvum-treated mice

Chemiluminescence (CL) of both Corynebacterium parvum (C. parvum) and saline elicited murine peritoneal macrophage monolayers was studied employing a luminol-dependent assay. Glycogen-induced macrophages were obtained from animals that had been injected 7-10 days previously with either C. parvum or phosphate buffered saline (PBS). Peritoneal exudate cells (3 x 10(6)) were allowed to attach to an 18 mm diameter glass cover slip, and then were cultured an additional 20-24 hr. The cover slips were removed from the culture medium, and were washed thoroughly with PBS to remove contaminating nonadherent lymphocytes and nonviable neutrophils. The macrophage monolayers were then placed in plastic scintillation vials with a suspension of luminol (5 x 10(-6 M in PBS) and opsonized zymosan particles. CL was measured at intervals over a 1-2 hr period with a scintillation counter in an out-of-coincidence mode. C. parvum-elicited macrophages demonstrated a 3- to 4-fold increase in CL when compared to control macrophages. Attachment of cells to cover slips did not in itself significantly elevate macrophage CL. Chemiluminescence production by purified macrophage monolayers correlated directly with glucosamine incorporation and inversely with phagocytic uptake. This sensitive, quantitative assay appears to be an excellent indicator of macrophage-specific metabolic activation.