A deactivating factor (MDF) is released from granuloma-like lesions of mice (giant and epithelioid macrophages) to the surrounding medium. Test cells incubated in the presence of MDF display dramatic inhibition of superoxide anion ((I2-) release when stimulated. This failure to manifest 0, release is observed whether PMA, all-transretinal, or fMet-Leu-Phe is the stimulating agent. MDF acts on different cell types from different species; mouse macrophages as well as guinea pig, human, and mouse neutrophils. Such results suggest that it is a universal regulatory cytokine with high affinity for phagocytic lineages. The factor was subjected to various purification methods: ultrafiltration, gel chromatography, and reversed phase HPLC. A crude preparation that resulted from conditioning of medium by old macrophages (MCM) shows two peaks of activity when subjected to gel filtration. These correspond to molecular weights for the active principle of 3 and 1 1 kD. When the factor was obtained by extraction of the same cells after washing and sonication, only the former peak was seen. Fractions corresponding to a MW of 3 kD from several preparations were combined and subjected to HPLC. MDF activity then appeared in a single fraction. MDF is thus putatively a modulator of the cidal activity of phagocytic cclls that utilize release of reactive oxygen species for cytocidal activity. (c) 111w WiIcy-Liss, Inc.
Recently we have shown that giant and epithelioid macrophages secrete a novel cytokine to the surrounding medium . This has been called "macrophage deactivating factor" (MDF) because it is obtained from those cells. It inhibits the ability of activated macrophages and other phagocytic cells to release O2 . Several other factors produced by some tumor cell lines, microbes, lung macrophages, and fibroblasts have been reported to inhibit the respiratory burst of macrophages . Of those factors, none has been shown to inhibit 0,production by neutrophils, as does MDF.
In this article, we present some biochemical characteristics of MDF as well as observations of the activities of this factor with respect to neutrophils triggered by a variety of different stimuli. This was done to eliminate the possibility that the factor operates a t the level of the stimulating agent per se. Moreover, MDF was purified by various procedures and its activity was compared with that of some other cytokines, such as p-endorphin and calcitonin gene-related peptide (CGRP).
MATERIALS AND METHODS Animals
CD-1 strain female mice, 5-7 weeks old, were obtained from the Charles River Breeding Laboratories 0 1993 WILEY-LISS. INC (Wilmington, MA). Female guinea pigs, weighing 500-600g, were obtained from Elm Hill Farm (Chelmsford, MA). The animals were kept on nightiday cycles and fed with appropriate chow pellets and water ad libitum.
Chemicals and apparatus
Horse heart ferricytochrome c type VI, bovine blood superoxide dismutase type I, phorbol-12-myristate-13acetate, albumin, flavin mononucleotide, vitamin BIZ, D-glucose, all-transretinal, and met-Leu-Phe were purchased from the Sigma Co. (St. Louis, MO). Protein molecular weight standards (Insulin chain B, 3.4 kD; aprotinin, 6.5 kD; cytochrome c, 12.5 kD, and trypsin inhibitor from soybean, 21.5 kD) were obtained from Boehringer-Mannheim (Indianapolis, IN). Calcitoningene-related-peptide (CGRP, human) and p-endorphin (human) were purchased from Peninsula Laboratories