Interferon-gamma stimulates lipid metabolism in human monocytes.

In nonactivated human monocytes, radiolabeled oleic, arachidonic, and palmitic acids are primarily incorporated into neutral lipids and phosphatidylcholine. Each of these fatty acids is also incorporated into phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, and sphingomyelin in characteristic proportions which do not differ between donors. The phospholipid head group precursors, choline and serine, are incorporated into phosphatidylcholine, and phosphatidylserine and serine is incorporated into phosphatidylethanolamine and sphingomyelin. The incorporation of these lipid precursors and the total lipid content of monocytes activated with interferon-gamma were compared to those of nonactivated monocytes. Fatty acid incorporation into interferon-gamma-activated monocytes was dramatically increased, particularly for palmitic acid. Palmitic acid incorporation into phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, and sphingomyelin was increased in activated cells by 167-387% at 2 hr and 215-274% at 4 hr compared to that of controls. The greatest increase in incorporation was for palmitic acid into sphingomyelin. Incorporation of arachidonic acid into phosphatidylinositol and serine into phosphatidylethanolamine was also increased in the interferon-gamma-activated monocytes. The total lipid content of activated and nonactivated monocytes did not differ. These results suggest that IFN-gamma activation induces a short-term stimulation of phospholipid metabolism which does not alter the gross lipid composition. Such modifications of phospholipid metabolism may be important in signal transduction as well as an indication of functional changes in the membranes of activated macrophages.

Human monocyte-derived macrophages are lysed by schistosomula of Schistosoma mansoni and fail to kill the parasite after activation with interferon gamma.

In this study was examined the interaction between schistosomula of Schistosoma mansoni and human monocyte-derived macrophages activated with interferon gamma (IFN-gamma). Peripheral blood monocytes were matured for 6 days and activated by further culture with IFN-gamma (600 U/ml). These IFN-gamma-treated monocyte-derived macrophages are cytotoxic for the tumor cell line K562, which is not killed by nonactivated monocyte-derived macrophages. Activated monocyte-derived macrophages were incubated with schistosomula at ratios of 10(3):1 and 10(4):1 in the presence of serum pooled from patients with schistosomiasis. This antiserum promoted an increased adherence of cells to the parasite. However, the activated monocyte-derived macrophages failed to kill the schistosomula under all conditions tested. On the contrary, the monocyte-derived macrophages were killed by schistosomula in a time-dependent and antibody-dependent manner, which was most evident at a lower effector/target ratio, 200:1. Electron microscopy showed that monocyte-derived macrophages were lysed on the surface of schistosomula. Further, both monocyte-derived macrophages and contaminating blood platelets fused with the parasite surface membrane, so that the cell plasma membrane and the outer tegumental membrane formed a hybrid membrane. The results indicate that matured human monocyte-derived macrophages activated by IFN-gamma are unable to kill schistosomula. Instead, the effector cells fuse with the parasites and are lysed by them.

T cell-derived migration-inhibitory factor and colony-stimulating factor share common structural elements.

Migration-inhibitory factor (MIF) is a lymphokine that acts to localize mononuclear phagocytes (monocytes and macrophages) and perhaps to activate them. Mo cells are a human T cell leukemia virus II-infected T cell line previously shown to secrete large quantities of MIF upon stimulation with phytohemagglutinin and phorbol myristate acetate. MIF was purified from Mo cell-conditioned medium by gel filtration, phenyl-Sepharose affinity chromatography, isoelectrofocusing, and reverse-phase high-performance liquid chromatography (RP-HPLC). Overall purification was 6,000-fold. The purified MIF fraction was found to display potent colony-stimulating factor (CSF) activity when assayed on human bone marrow cells. The double peak of MIF activity as shown by C 18-RP-HPLC coincided with the double peak of CSF activity. A monoclonal antibody selected for its anti-MIF activity absorbed both the CSF and the MIF activity. These findings indicate that MIF and CSF are either identical molecules or closely related molecules with common structural elements.

A monoclonal antibody against migration inhibitory factor (MIF) obtained by immunization with MIF from the human lymphoblast cell line Mo.

A monoclonal antibody-secreting hybrid cell line, E7, was constructed from myeloma cells and spleen cells from BALB/c mice immunized with partially purified human MIF from culture fluid of the human T-lymphoblast cell line Mo. The hybrid cell line E7 was selected by screening hybridoma cultures for their capacity to adsorb added MIF activity when assayed together with rabbit anti-mouse IgG-coupled to protein A-Sepharose. The monoclonal antibody produced by the cloned hybridoma E7 also directly neutralized MIF activity from Mo cells and two species of MIF from the culture fluid of human peripheral blood lymphocytes, but did not neutralize IFN-gamma from Mo cells and from human peripheral blood lymphocytes. This antibody reacted also with a component in phytohemagglutinin preparations with an apparent molecular weight of 60,000; however, it did not react with the active tetramer of phytohemagglutinin.

Activation of human eosinophils by monokines and lymphokines: source and biochemical characteristics of the eosinophil cytotoxicity-enhancing activity produced by blood mononuclear cells.

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.

Production of migration-inhibitory factor by a human T-lymphoblast cell line.

Migration-inhibitory-factor (MIF) activity was detected in culture supernatants of the human T-lymphoblast cell line Mo after stimulation with phytohemagglutinin and phorbol myristate acetate. MIF activity was not detected in unstimulated cultures reconstituted with phytohemagglutinin and phorbol myristate acetate. Conditioned medium from the cell line Mo was fractionated by Sephadex G-100 gel filtration. MIF-containing Sephadex fractions corresponding to a Mr of 60,000 to 70,000 were further fractionated by isoelectrofocusing, resulting in a sharp peak of activity with a pI of 4.6 to 5.2. This MIF species constitutes a major form secreted by Mo cells; it adheres to Con A-Sepharose, is trypsin-resistant, and is denser than pure protein as determined by CsCl density gradient centrifugation. These are the same physicochemical characteristics previously established for second-day pH5-MIF from peripheral blood mononuclear cells (W.Y. Weiser et al., J. Immunol. 126, 1958, 1981). In contrast, Sephadex fractions corresponding to larger molecules (Mr 70,000-90,000) contain at least two additional MIF species. These larger MIF forms have a pI of 3.0 to 3.5 and of 4.6 to 5.2 and lack affinity to Con A Sepharose. Thus, the Mo T-cell line produces large quantities of at least three different species of human MIF.

Purification of guinea pig pH 3 migration inhibitory factor.

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.

Two migration inhibitory factors with different chromatographic behavior and isoelectric points.

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).

Decrease of three lysosomal enzymes in guinea pig macrophages activated by lymphocyte mediators.

Guinea pig peritoneal macrophages incubated for 72 h with MIF-rich medium which results in an activation of certain physiological functions have decreased specific activity of the lysosomal enzymes, acid phosphatase,Β-glucuronidase, and cathepsin D, and decreased phagocytosis of denatured particulate hemoglobin in comparison to control cells. Enhanced release of enzymes does not account for the decrease in lysosomal enzyme activity because these enzymes cannot be detected in the supernatant of the cells. Furthermore, when macrophages are activated by whole sensitized and stimulated lymphocytes, the specific activities of the lysosomal enzymes are also decreased or unchanged. It is therefore unlikely that the defense mechanisms of macrophages which are enhanced by lymphocyte mediators include an increase in lysosomal enzyme activity.

Studies with hydroxyurea. VII. Hydroxyurea and the synthesis of functional proteins.

Hydroxyurea affected neither the synthesis nor the degradation of bacterial messenger-ribonucleic acid. The proteins made by hydroxyurea-treated cells were structurally intact and fully functional. Since the expression of the lethal action of hydroxyurea requires active protein production, the data indicate that treated cells do not die as the result of the synthesis of abnormal proteins.