Eosinophil cytotoxicity enhancing factor: purification, characterization and immunocytochemical localization on the...

The monokine eosinophil cytotoxicity enhancing factor (ECEF) increases antibody-dependent cytotoxicity of eosinophils towards helminth larvae. A monokine biochemically indistinguishable from ECEF increases the release of leukotriene C4 and other arachidonic acid metabolites by eosinophils. We have developed monoclonal antibodies (mAb) to these monokines by immunizing mice with ECEF made by the U-937 histiocytic lymphoma cell line. mAb 81.10.C9 (IgG2b) and 9A6G (IgG1) inhibit the effect of the monokine on release of AA products. Both mAb bind ECEF, which appears after affinity chromatography purification as a major 13-14-kDa and a minor 62-kDa component (13-14 kDa and 52 kDa after reduction) in silver-stained gels. An additional component of 30 kDa is detectable after radioiodination of the immunopurified material. The specificity of both mAb was studied in several ways. In immunoprecipitation, both recognize the 13-14-kDa and the 30-kDa components, while the 62-(52)-kDa protein is not significantly precipitated. Both mAb react in enzyme-linked immunosorbent assay with products secreted by peripheral blood mononuclear cells and monocytes, as well as with those secreted by phorbol 12-myristate 13-acetate and lipopolysaccharide-stimulated U-937 cells and with the immunopurified proteins. These were separated in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, electroeluted and assayed for ECEF activity. Activity was associated with the 13-14-kDa and the 30-kDa fractions, as seen by increased eosinophil antibody-dependent adherence to schistosomula and cytotoxicity. Granulocyte-monocyte-colony-stimulating factor and interleukin 1, but not tumor necrosis factor, could be detected in crude U-937 supernatants. However, active immunopurified ECEF has no activity in assays for granulocyte-monocyte-colony-stimulating factor, interleukin 1 or tumor necrosis factor. Immunocytochemical localization of ECEF employing the mAb shows strong surface staining of viable monocytes and U-937 cells, suggesting that ECEF is associated to the cell surface. These properties distinguish ECEF from other monokines previously reported to activate eosinophils.