Immunosuppressive factors from mastocytoma cells cultured in serum-free medium

Abstract

Immunosuppressive factors were isolated from culture fluids of DBA/2 mouse mastocytoma cells grown in serum-free RPMI-1640 medium by measurement of inhibitory activity on tritiated thymidine uptake of DBA/2 spleen cells responding to Escherichia coli lipopolisaccharide (LPS) and concanavalin A (ConA) and by comparison of the number of hemolytic antibody-forming cells in vitro after simultaneous addition of the factors with sheep red blood cells (SRBC). The culture fluids were separated into four fractions with immunosuppressive activities: F-I (mol wt, > 30,000), and F-II (mol. wt, 10,000-30,000), F-III (mol wt, 2,000-10,000), and F-IV (molwt, 700-2,000), F-IV specifically suppressed lymphocyte responses to E. coli LPS at concentrations of 50-200 microgram/ml. The other three fractions also showed immunosuppressive activities in both Con-A-induced and E. coli LPS-induced lymphocyte responses. The four fractions as well as crude culture fluids of mastocytoma cells expressed as immunosuppressive effect when injected into the peritoneal cavities of DBA/2 mice at doses of 24-180 microgram/day for 5 consecutive days. The fractions with a molecular weight equivalent to F-IV of mastocytoma culture supernatant were detected little, if any, in the supernatant from a nonmalignant cell culture. No suppressive effects of the fraction with a molecular weight greater than 2,000 (equivalent to F-I + F-II + F-III) from nonmalignant cell culture were found on lymphocyte responses to mitogens or SRBC at the concentrations used (100-200 microgram/ml).