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Separation of Lymphocyte Mitogen from Lymphotoxin and Experiments on the Production of Lymphotoxin by Lymphoid Cells Stimulated with the Partially Purified Mitogen: A Possible Amplification Mechanism of Cellular Immunity and Allergy¹

From the Department of Microbiology, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205

Abstract

A mitogenic factor (MF) from guinea pig lymph node cells (LNC), which was produced by stimulating immune LNC with the specific antigen, ovalbumin, was partially purified by the sequential use of Sephadex G-200 gel filtration, CM-cellulose ion exchange chromatography, DEAE-cellulose ion exchange chromatography, and polyacrylamide disc gel electrophoresis. The product was purified at least 100-fold with regard to protein content. In addition, the purified MF was functionally pure with respect to the absence of lymphotoxin (LT), another guinea pig lymphokine.

The possibility that MF, or a substance closely associated with this factor, might serve to amplify cellular immune and allergic reactions by nonspecifically inducing lymphoid cells to produce other lymphokines was then tested by stimulating cells from various lymphoid tissues with the purified MF and assaying the culture fluids for LT activity as a measure of lymphokine production. All the cell types studied incorporated ³H-thymidine in response to purified MF, and some of them produced LT. Thus, spleen cells from normal guinea pigs produced some LT when stimulated with a large dose of MF, but mesenteric LNC produced none. However, mesenteric LNC from guinea pigs that had been immunized by footpad injection of complete Freund's adjuvant (CFA) 10 or 16 days previously produced LT in amounts that were dependent on the stimulating dose of MF. Substantial quantities of LT were produced by cells from peripheral lymph nodes draining the sites of immunization, as well as by spleen cells, the degree of response being dependent on the stimulating dose of MF and the time span after injection of CFA. These results indicate that purified MF, or a substance closely associated with this factor, is capable of inducing LT production by a subset of lymphoid cells that appears to be expanded after immunization. Electrophoretic observations indicated that the induction of LT production may be due to a factor which is distinct from MF although closely similar in molecular size and charge.

Footnotes

¹ This work was supported in part by a Contract with the Office of Naval Research N00014-75-C-0142, American Cancer Society Grant IM-64 and Grant 1 RO1 AI 12472-01 from the National Institutes of Health.