Monocyte Responses to Sulfatide from Mycobacterium tuberculosis ...

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Feb 13, 1991 - In monocytes, sulfatide, a lipid from Mycobacterium tuberculosis, blocked priming for enhanced release of superoxide (02-) by the macrophage ...
Vol. 59, No. 8

INFECTION AND IMMUNITY, Aug. 1991, p. 2542-2548 0019-9567/91/082542-07$02.00/0

Monocyte Responses to Sulfatide from Mycobacterium tuberculosis: Inhibition of Priming for Enhanced Release of Superoxide, Associated with Increased Secretion of Interleukin-1 and Tumor Necrosis Factor Alpha, and Altered Protein Phosphorylation JOHN P.

BROZNA,l* MARILEE HORAN,1t JONELLA M. RADEMACHER,2

KAREN M. PABST,2

AND MICHAEL J. PABST2

Department of Pathology and Microbiology, University of Nebraska Medical Center, Omaha, Nebraska 68198, and Veterans Administration Medical Center, Omaha, Nebraska 68105,1 and Dental Research Center and Department of Biochemistry, University of Tennessee, Memphis, Tennessee 381632 Received 13 February 1991/Accepted 1 May 1991

In monocytes, sulfatide, a lipid from Mycobacterium tuberculosis, blocked priming for enhanced release of superoxide (02-) by the macrophage activating factors lipopolysaccharide, gamma interferon, interleukin-l1 (IL-1,), tumor necrosis factor alpha (TNF-a), and muramyl dipeptide. Sulfatide, in the presence of lipopolysaccharide, also caused increased secretion of IL-1I and TNF-oa into monocyte culture medium. Sulfatide altered the pattern of phosphorylation of monocyte proteins. Cell lysates prepared from monocytes treated with sulfatide showed decreased activity of protein kinase C, but sulfatide did not directly inhibit protein kinase C activity when added to lysates. A known inhibitor of protein kinase C, staurosporine, also inhibited 02 release and caused increased secretion of IL-1,B. Thus, sulfatide appeared to indirectly affect protein kinase C, implicating protein kinase C as part of the mechanism of priming. Because sulfatide blocked priming for enhanced release of 02- which could interfere with monocyte bactericidal activity, while causing enhanced secretion of IL-1, and TNF-a, which could promote formation of granulomata, sulfatide might be an important factor in the pathogenesis of M. tuberculosis. whereas sulfatide blocks priming by LPS and IFN--y at all concentrations. Hence, in this article, we examine sulfatide as a probe for the mechanism of macrophage priming. Our experiments suggest that inhibition of LPS priming by sulfatide involves both inhibition and enhancement of phosphorylation of various monocyte proteins, associated with decreased activity of protein kinase C in monocyte extracts. We also observed that inhibition of priming by sulfatide was accompanied by increased secretion of interleukin-13 (IL1) and tumor necrosis factor alpha (TNF-ox).

The production of microbicidal oxygen radicals by phagocytic leukocytes is regulated by the process of activation in vivo or by what is known as priming in vitro. Activated or primed monocytes produce large amounts of oxygen radicals when they phagocytize microbes or are triggered by chemical stimuli like phorbol myristate acetate (PMA) or formylmethionyl-leucyl-phenylalanine (16). In contrast, unprimed or resident monocytes produce small amounts of oxygen radicals when stimulated. We reported previously that a sulfatide isolated from Mycobacterium tuberculosis blocks priming of monocytes by lipopolysaccharide (LPS) or gamma interferon (IFN--y) (15). Sulfatide is a major component of the bacterial outer lipid layer. It is composed of the disaccharide trehalose esterified with one sulfate group and four fatty acids, three of which are long-chain methyl-branched fatty acids (5). Others have reported that sulfatide does not block priming of neutrophils by LPS (22), an observation with which we concur. The difference in the effect of sulfatide on LPS priming of monocytes and its effect on priming of neutrophils

MATERIALS AND METHODS Reagents. PMA (LC Services, Woburn, Mass.) was prepared as a concentrated stock solution in dimethyl sulfoxide and stored at -20°C. In all experiments, dimethyl sulfoxide was always 95%), with minor amounts of sulfatide SL-II and sulfatide SL-III, which are closely related chemically (5). In a typical experiment, the suspension was diluted 1:100 into culture medium to a final concentration of 20 pug/ml. All materials cultured with cells were free from endotoxin as determined by the Limulus assay (Sigma Chemical Co., St. Louis, Mo.). Cytochrome c (type III) was from Sigma. Cell isolation and culture. Blood was obtained from healthy donors by venipuncture by using 0.38% citrate as the anticoagulant. Mononuclear cells were separated by a plasma-Percoll procedure by using a two-step discontinuous gradient (8). The mononuclear cell fraction contained approximately 68% lymphocytes, 29% monocytes, and 3% neutrophils by hematoxylin and eosin and esterase stains. Monocytes were cultured either in Teflon bags at 3 x 106 mononuclear cells per ml (18) or in 16-mm multiwell tissue culture plates (Costar, Cambridge, Mass.) at approximately 1 x 106 monocytes per well. Cells cultured on plates were washed after 20 min to remove nonadherent cells, resulting in 86% monocytes, 10% lymphocytes, and 4% neutrophils. Mononuclear cells or monocytes purified by adherence were cultured in endotoxin-free modified Earle balanced salt solution in 5% CO2 at pH 7.3 (18). Monocytes remained viable and functional for at least 4 days in this simple salt solution. Priming with LPS, IFN--y, MDP, IL-1l, or TNF-a. LPS, MDP, rIFN--y, rIL-1p, or rTNF-ot were added to cultures in volumes of 1 to 10 IlI usually at the start of culture or after 24 h. Assay of stock solutions of recombinant cytokines by the Limulus assay showed that they contained