Purification of macrophage deactivating factor.

Abstract

Macrophage deactivation factor (MDF) in P815 tumor cell-conditioned medium was assayed by its suppression of the ability of activated mouse peritoneal macrophages to release hydrogen peroxide. MDF displayed properties of a soluble protein(s) associated with both low (8-25,000) and high (greater than 450,000) Mr fractions. MDF was purified 6,140-fold by a seven-step procedure: extraction with acid-ethanol; precipitation with ether; and fractionation on gel filtration, anion-exchange, diphenyl reversed-phase and C4 reversed-phase HPLC columns, the last column twice. The final preparation contained two species: (a) a approximately 13,000 Mr band on reducing or nonreducing SDS-PAGE and on autoradiograms after radioiodination with chloramine T, and (b) a 66,000 Mr species ranging from approximately 5% to approximately 50% of the protein detectable by silver strain. The 66,000 Mr species was identified as albumin from its NH2-terminal amino acid sequence. However, no amino acid sequence could be obtained for the approximately 13,000 Mr species, either in fluid phase or after electroelution of the corresponding SDS-PAGE band. Thus, approximately 13,000 Mr MDF associates tightly with albumin through a variety of separation techniques, and may have a blocked NH2 terminus. Purified MDF afforded 50% inhibition of activated macrophage H2O2 releasing capacity at a concentration of 1-10 nM. Separation of MDF from most higher Mr moieties was associated with disproportionately small increases in specific activity, suggesting MDF might be partially inactivated by purification. As purified, MDF was approximately 1,000-fold less potent at deactivating macrophages than TGF-beta. Antibodies that neutralized the macrophage-deactivating effect of TGF-beta did not inhibit deactivation by MDF.

Macrophage deactivation by interleukin 10.

“…Supernatants from COS7 cells transfected with mlL-10 eDNA (1,000 U/ml, where 1 U caused half-maximal response of the MC/9 mast cell line as described [20]; LPS content at 100 U/ml < 10 pg/ml) and control supernatants from mock transfected cells (LPS content at a 1:10 dilution < 10 pg/ml) were kindly provided by Dr. K. Moore (DNAX, Palo Alto, CA; 1 MC/9 U/ml is equal to 1 CSIF U/ml, personal communication). MDF was purified from the culture supernatants of P815 mouse mastocytoma cells as described (10) or directly extracted from these cells following a similar procedure (Y. Vodovotz, C. Bogdan, and C. Nathan, unpublished results). A unit of MDF is defined as that amount of MDF in a final culture volume of 0.125 ml that causes 50% suppression of PMA-triggered mO HzO2-releasing capacity after a 48-h incubation (10).…”

Tumour cell inhibition of macrophage cytokine activity

“…Preliminary experiments using the protease inhibitor aprotinin (up to 5.0 units per ml) have revealed that inhibitor instability (37°C, > 1.5 h) is probably a result of proteolytic cleavage although the inhibitor itself, while probably a protein, is unlikely to be a protease (data not shown). Transforming growth factor /3 (TGF/3) and macrophage deactivating factor (MDF) are molecules with anti-cytokine activities, however, neither can influence PKC translocation [18,19]. One possible candidate molecule is IL-10, related to TGF/3 and MDF and recently shown to downregulate TNF, IL-1 and IL-6 protein and mRNA production in murine macrophages [20].…”

Tumour cell inhibition of macrophage cytokine activity

“…3), inhibition appears to be exerted on cytokine activity following its release from macrophages although data showing inhibition of PKC translocation (Fig. Transforming growth factor /3 (TGF/3) and macrophage deactivating factor (MDF) are molecules with anti-cytokine activities, however, neither can influence PKC translocation [18,19]. This would affect signalling to release cytokines and, possibly, their subsequent effect on target cells.…”