Assuntos
Fatores Inibidores da Migração de Leucócitos/isolamento & purificação , Linfocinas/isolamento & purificação , Fatores Inibidores da Migração de Macrófagos/isolamento & purificação , Animais , Células Cultivadas , Centrifugação com Gradiente de Concentração/métodos , Cromatografia em Gel/métodos , Meios de Cultura , Cobaias , Humanos , Focalização Isoelétrica/métodos , Fatores Inibidores da Migração de Leucócitos/biossíntese , Linfócitos/citologia , Linfócitos/imunologia , Fatores Inibidores da Migração de Macrófagos/biossíntese , Macrófagos/imunologia , Masculino , Monócitos/citologia , Monócitos/fisiologia , Especificidade da EspécieAssuntos
Esterases/metabolismo , Fatores Inibidores da Migração de Macrófagos/farmacologia , Macrófagos/enzimologia , Animais , Centrifugação com Gradiente de Concentração , Fracionamento Químico , Cromatografia em Gel , Concanavalina A/farmacologia , Esterases/farmacologia , Cobaias , Ponto Isoelétrico , Linfonodos/imunologiaRESUMO
Migration inhibitory factor (MIF), produced by stimulation of guinea pig lymph node cells with concanavalin A, was fractionated by Sephadex G-100 gel filtration, sucrose density gradient electrophoresis, and isoelectrofocussing. Two distinct species were identified and separated. One, pH 3-MIF, has an isoelectric point of 3.0 to 4.5 and elutes from Sephadex G-75 columns with molecules having an apparent m.w. of 65,000 (Kd of 0.05 to 0.12). The other, pH 5-MIF, has an isoelectric point of 5.0 to 5.5 and elutes with molecules having an apparent m.w. between 25,000 and 43,000 (Kd of 0.15 to 0.23).
Assuntos
Fatores Inibidores da Migração de Macrófagos/isolamento & purificação , Animais , Centrifugação com Gradiente de Concentração , Cromatografia em Gel , Eletroforese Descontínua , Cobaias , Focalização IsoelétricaRESUMO
Macrophage migration inhibitory factor (MIF) from the guinea pig was recently shown to reside in two discrete and separable proteins referred to as pH 3 MIF and pH 5 MIF. One subfraction of pH 3 MIF has now been purified to apparent homogeneity from supernatants of stimulated lymph node cells. To monitor purification, biosynthetically radiolabeled MIF was prepared. Sensitized lymphocytes were stimulated in the presence of [3H]leucine by concanavalin A to produce radiolabeled mediators. MIF was purified approximately 30,000-fold from the culture fluid by using gel filtration, sucrose density gradient electrophoresis, isoelectric focusing, and hydrophobic affinity chromatography. This procedure yielded a single 3H-labeled polypeptide with an apparent Mr of 35,000 that coincides with MIF activity.
Assuntos
Linfonodos/imunologia , Fatores Inibidores da Migração de Macrófagos/isolamento & purificação , Animais , Cromatografia de Afinidade , Cromatografia em Gel , Cobaias , Ponto Isoelétrico , Linfonodos/análise , MasculinoRESUMO
We and others have previously reported that human blood mononuclear cells release in culture certain substances that enhance the capacity of purified human blood eosinophils to kill the antibody-coated larvae of Schistosoma mansoni. The present study shows that this eosinophil cytotoxicity-enhancing activity (ECEA) is released by monocytes and T lymphocytes. Monocytes produce ECEA in resting and in LPS-stimulated cultures; T lymphocytes release such activity when stimulated by mitogens such as concanavalin A. Furthermore, the human monocytic line U-937 also releases ECEA-like activity when stimulated by LPS. The enhancing activity produced by monocytes has been partially characterized: it is sensitive to proteolysis by trypsin, relatively heat stable, and associated with molecules that have an apparent molecular weight of 14,000 to 65,000 daltons and isoelectric points of 3.8-3.9, 4.2, 4.5, 4.8-4.9. This shows that while ECEA produced by monocytes is heterogeneous in size and charge, it is probably different from interleukin 1.