In vitro stimulation of antibody formation by peritoneal cells. I. Plaque technique of high sensitivity enabling access to the cells.

Abstract

An improved method for the short-term culture of mouse peritoneal cells in a medium containing carboxymethylcellulose (CMC), sheep erythrocytes (SRBC), and guinea pig complement is described. It involves preparation of microcultures, of thickness 12-15 micro and volume 3.6 microl, under paraffin oil. With such cultures, peritoneal cells from normal, unimmunized young male CBA mice give about 3000 hemolytic plaques per million cells cultured, this figure being attained within 24 hr. The plaque detection method is about four times as sensitive as the Jerne technique. A method is described whereby such plaque-forming cells (PFC) can be transferred, by micromanipulation, to fresh monolayer cultures containing SRBC, CMC, and complement. In this fashion, the secretory capacity and susceptibility to inhibitors of peritoneal PFC can be tested in detail. Using this technique, evidence is presented that the hemolytic substance responsible for plaque formation is actually secreted by the cell at the center of the plaque, and is not a complement component but probably an antibody. Studies on the time of plaque appearance after cell transfer, and the subsequent growth rate of the zone of hemolysis, have been performed. They speak against the idea that the PFC is either a reservoir of cytophilic antibody or a "background" PFC. Rather they suggest that active antibody secretion is induced in the cell at some defined time point in culture. Detailed kinetics of the rate of appearance of plaques in peritoneal cell cultures revealed an exponential phase lasting from about 3 to about 13 hr with a doubling time of 2 hr. The reasons for this are not known. A greatly heightened reactivity was shown in peritoneal cells of mice that had been pregnant several times. Cultures of such cells showed more rapid plaque appearance and a peak activity about 20 times higher than with cells from young male mice. Cultures in which 1 cell in 10 formed a plaque were not infrequent. A series of experiments on germ-free mice showed reactivity similar to that of conventional mice from the same strain and source. The significance of the findings for cellular immunology are discussed.