Alma D. Mednis

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.

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.

A sensitive fluorometric assay for the determination of cathepsin D

Abstract A fluorometric assay is described for cathepsin D—determining the tyrosine released by measuring the fluorescence of its nitrosonaphthol coupling product. The advantage of this method is its high sensitivity.

Interferon-γ stimulates lipid metabolism in human monocytes

Abstract 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-γ were compared to those of nonactivated monocytes. Fatty acid incorporation into interferon-γ-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-γ-activated monocytes. The total lipid content of activated and nonactivated monocytes did not differ. These results suggest that IFN-γ 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.

[28] Migration inhibitory factor

Publisher Summary This chapter presents the overview of migration inhibitory factor (MIF). MIF production is considered to be an in vitro correlate for delayed-type hypersensitivity reactions because lymphocytes from patients lacking delayed hypersensitivity reactions fail to produce MIF in vitro. Purified MIF shows a distinct pattern of heterogeneity and seems to be biochemically related to other macrophage directed lymphokines, such as the macrophage activating factor which induces tumoricidal activity in macrophages. The chapter further explains production of human MIF; it is derived from Con A-stimulated peripheral blood lymphocytes contains 3 distinct molecular species. Supernatants from cells cultured for one day contain a single species with an isoelectric point of 4.3-5.2 which is trypsin sensitive and neuraminidase resistant. Supernatants from the second day of incubation (24-48 hr) contain two distinct MIF-species, both distinct from each other and from first day-MIF with a pI of 2.4-3.3 and 4.3-5.6 which are both trypsin-resistant and neuraminidase-sensitive. The production of the first day and second day-MIF species is further highlighted in the chapter.

The effect of tunicamycin on the molecular heterogeneity of guinea pig migration inhibitory factor

Abstract Migration inhibitory factor (MIF) was produced by guinea pig lymph node cells stimulated with concanavalin A in the absence and presence of the glycosylation inhibitor tunicamycin. The active supernatants were purified on Sephadex G-100 and fractionated into pH 3-MIF and pH 5-MIF using isoelectrofocusing. When produced in the absence of tunicamycin pH 3-MIF shows extensive charge heterogeneity with activity focusing from pH 3.0 to 4.5; it elutes from Sephadex G-75 with molecules of an apparent MW of 70,000. In contrast, pH 3-MIF produced in the presence of tunicamycin (TM-pH 3-MIF) focuses as a sharp homogeneous peak with a p I of 3.6 to 4.0 and elutes from Sephadex G-75 with molecules of an apparent MW of 25,000–35,000. TM-pH 3-MIF is trypsin sensitive and displays a buoyant density similar to that of proteins which contain little or no carbohydrate ( ϱ 25 1.26−1.34). Tunicamycin caused no detectable change in the characteristics of pH 5-MIF. This study indicates that lymphocytes stimulated in the presence of tunicamycin produce a novel species of pH 3-MIF with characteristics distinct from classical pH 3-MIF.

Studies on MIF and the Role of Macrophage Associated Esterases in the Response of the Macrophage to MIF

It has been known for some time that “activated” macrophages obtained from immune animals have altered morphology and metabolism, and that they show enhanced capacities to deal with a number of microorganisms (1,2). A number of these studies have shown that the lymphocyte is involved in these activation processes (3,4). Their action can be partially explained by the production of soluble mediators such as macrophage activating factor (MAF) which increases the capacity of macrophages to kill bacteria and tumor cells (5,6). This factor cannot be distinguished from migration inhibitory factor (MIF) (7,8,9).