Activation of murine polymorphonuclear neutrophils for fungicidal activity with supernatants from antigen-stimulated immune spleen cell cultures

Abstract

An in vitro model of in vivo immunological activation of murine polymorphonuclear neutrophils (PMN) was developed. Culture supernatants of spleen cells from Blastomyces dermatitidis-immunized mice stimulated with B. dermatitidis antigens in vitro were studied. Incubation of the supernatants with thioglycolate-elicited PMN enabled the cells to significantly reduce (31 +/- 6%) B. dermatitidis inoculum CFU. Optimum production of active supernatants occurred after 4 to 6 days of stimulation in vitro and required 200 micrograms of nonviable B. dermatitidis cells per ml. Generation of activity by immune spleen cells was shown to be antigen specific in that stimulation with a heterologous antigen or stimulation of nonimmune spleen cells with B. dermatitidis antigen did not produce active supernatants. The activity in supernatants was dose dependent, nondialyzable (molecular weight greater than or equal to 14,000), and relatively heat labile (80 degrees C, 30 min). Activation of PMN by supernatants for fungicidal activity against B. dermatitidis required only a short incubation period (1 h) followed by a 2-h coculture (challenge) period. Stimulation of normal spleen cells with concanavalin A also resulted in the production of supernatants capable of activating PMN for significant fungicidal activity (31.1 +/- 8.5%). These findings demonstrate for the first time a link between soluble factors produced by antigen stimulation of sensitized lymphoid cells and activation of PMN for enhanced microbicidal activity. Such a process defines an additional immune defense mechanism whereby the immune host may clear specific microorganisms.