Isolation of Substances Responsible for Lymphokine Activity from Sensitized Mouse Spleen Cells

Abstract

Cultures of brucella-sensitized mouse spleen cells exposed to Brucella abortus antigens in vitro release macrophage migration inhibition factor (MIF) and macrophage spreading factor. Subjecting the supernatants from such cultures to preparative scale electrophoresis in acrylamide gel yields several fractions, one of which contains both MIF and macrophage spreading factor. This material has properties attributable to guinea pig MIF: it is nondialyzable, heat stable, nontoxic to macrophages from heterologous murine donors, and has a greater anodal electrophoretic mobility than guinea pig serum albumin. Another fraction from the gel column inhibits macrophage spreading; its electrophoretic mobility is similar to that of guinea pig gamma globulin. Neither brucella antigen nor skin reactive substances were detectable in any acrylamide gel column fraction when tested by the mouse footpad induration assay technique.

Murine macrophage-lymphocyte interactions: scanning electron microscopic study

“…The increase in macrophage spreading due to the MIF-containing fractions is similar to previously reported results (42,57,58). In addition, it appears as though this more rapid spreading occurs in the presence of spleen, thymic, or peritoneal-cavity lymphocytes and leads to an earlier attachment of the lymphocytes to the macrophages.…”

Alterations in mouse macrophage migration: a function of assay systems, lymphocyte activation product preparation, and fractionation

“…Consequently, a number of systems of assay and separation have evolved by which LAPs are detected and characterized in various species. Nevertheless, there are few papers dealing with the concurrent fractionation and/or assay of more than one mouse LAP (7,15,21). In this report we present evidence that several LAPs may be detected in the fractionated supernatants of antigen-stimulated cells from Brucella abortus-infected mice.…”

“…LAP production: (i) Spleen cell LAPs. Mouse spleen cells were obtained from Brucella-infected and uninfected mice as described previously (21). Erythrocytes were hypotonically disrupted, and the remaining leukocytes were washed two times in Hanks balanced salts solution (Grand Island Biological Co., Grand Island, N.Y.) prior to cultivation.…”

“…(iii) Cell supernatant preparation. After incubation, the supernatant fluids were collected, appropriately pooled, centrifuged, and filtered as described previously (21). Complement was destroyed by heat at 56 C for 40 min.…”

“…Complement was destroyed by heat at 56 C for 40 min. Some fluids were concentrated fivefold by ultrafiltration as described elsewhere (21). Otherwise, the filtrates were dialyzed in washed (glycerol-free) dialysis tubes for 48 h at 4 C against 300 volumes of double-distilled water (DDW), changed twice.…”