Characterization of four lymphocyte activation products of guinea pig and their association with macrophage Migration Inhibitory Activity (MIF)

Abstract

Guinea pig lymph node cells were stimulated with Concanavalin A and simultaneously labelled with radioactive leucine. The 24 hr culture supernatants were fractionated using repeated Sephadex chromatography and various electrophoretic techniques. Using an anti-iymphokine antibody raised against purified migration inhibitory factor (MIF)* containing fractions which had been characterized extensively before, three lymphocyte activation products were identified initially with mol. wt 60,000 ( a), 45,000 ( b) and 30,000 ( c), which all had similar isoelectric points of around 5.2. With these materials the following characterizations have been performed: a, b and c were inhibitory in the macrophage migration assay. The buoyant density in a CsCl gradient was ϱ 1.278 for all three molecules. Treatment with neuraminidase did not cause a shift to pI greater than 5.2. Reduction and alkylation or treatment with EDTA did not change the mol. wt of a, b or c. No proteolytic activity could be detected in any of the preparations. Because of the similarities in their chemical and biological properties, it was assumed that the three molecules are oligomers of a common subunit of a mol. wt of 15,000 and a pI of 5.2. A molecule of this size was detected in low amounts in stimulated culture supernatants which also exhibited marked migration inhibitory activity. It is concluded that MIF activity is not associated with a single molecule but rather with a group of structurally related molecules.