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.

In monocytes, sulfatide, a lipid from Mycobacterium tuberculosis, blocked priming for enhanced release of superoxide (O2-) by the macrophage activating factors lipopolysaccharide, gamma interferon, interleukin-1 beta (IL-1 beta), tumor necrosis factor alpha (TNF-alpha), and muramyl dipeptide. Sulfatide, in the presence of lipopolysaccharide, also caused increased secretion of IL-1 beta and TNF-alpha 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 O2- release and caused increased secretion of IL-1 beta. 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 O2-, which could interfere with monocyte bactericidal activity, while causing enhanced secretion of IL-1 beta and TNF-alpha, which could promote formation of granulomata, sulfatide might be an important factor in the pathogenesis of M. tuberculosis.

Effects of polymyxin B on superoxide anion release and priming in human polymorphonuclear leukocytes.

We studied the effect of a potent inhibitor of protein kinase C, polymyxin B (PMXB), on superoxide anion (O2-) release by human polymorphonuclear leukocytes (PMNL). PMXB was compared with another inhibitor of protein kinase C, 1-(5-isoquinoline-sulfonyl)-2-methyl piperazine (H-7). Both PMXB and H-7 inhibited phorbol myristate acetate (PMA)-stimulated O2- release. Formyl-methionyl-leucyl-phenylalanine (FMLP)-stimulated O2- release by cytochalasin B-treated PMNL was not inhibited significantly by either PMXB or H-7. 1-Oleoyl-2-acetyl-glycerol (OAG,25-100 microM) stimulated PMNL to release O2- with a long lag-time (8-10 min). Although H-7 inhibited OAG-stimulated O2- release, PMXB augmented the OAG-stimulated response by increasing rate and reducing lag time. The augmenting effect of PMXB was evident only when added after stimulation by OAG, with maximum effect observed at 3 min after addition of OAG. The augmenting effect was also seen with PMXB immobilized on agarose beads. PMXB did not affect the respiratory burst response to 1,2-dioctanoylglycerol. PMXB-augmented, OAG-stimulated O2- release was inhibited by the addition of H-7 before OAG. In contrast to the effect on O2- release, OAG-stimulated protein phosphorylation was inhibited similarly by either PMXB or H-7, when these agents were added 3 min after stimulation by OAG. These results suggested that initial activation of protein kinase C by OAG is essential for O2- release, but that PMXB acts in a manner independent from protein kinase C to augment OAG-stimulated O2- release. When priming by OAG for enhanced O2- release (as opposed to direct stimulation of O2- release) in FMLP-stimulated PMNL was examined, PMXB inhibited O2- release in OAG-primed PMNL, suggesting that protein kinase C is involved in priming of PMNL by OAG.

Inhibition of thermolysin by bifunctional N-carboxyalkyl dipeptides.

A series of N-carboxyalkyl derivatives of L-leucyl-L-alanine was synthesized and tested as inhibitors of the zinc endoproteinase thermolysin. The purpose of the study was to determine whether bifunctional N-carboxyalkyl compounds with secondary metal coordinating groups are more potent inhibitors than analogs lacking such an additional binding function. Reductive condensation of L-leucyl-L-alanine (LA) with pyruvic, oxalacetic, alpha-ketoglutaric, 2-oxopentanoic, 4-ethyloxalacetic, or imidazoylpyruvic acids gave N-[1(R, S)-carboxyethyl]-LA (I), N-[1(R, S)-carboxy-2-carboxyethyl]-LA (II), N-[1(R, S)-carboxy-3-carboxypropyl]-LA (III), N-[1(R, S)-carboxy-n-butyl]-LA (IV), N-[1(R, S)-2-ethylcarboxyethyl]-LA (V), and N-[1(R, S)-carboxy-2-(4-imidazoyl-ethyl]-LA (VI), respectively. Values of KI determined with furylacryloyl-Gly-Leu-NH2 as substrate were 116 +/- 21, 7.4 +/- 1.8, 6.3 +/- 0.5, 19.7 +/- 1.5, 17.0 +/- 1.0, and 3.3 +/- 0.1 microM for compounds I-VI, respectively. Although bifunctional inhibitors II, III, and VI were indeed more potent than I, they were not much more effective than analogs IV and V that contained noncoordinating functionalities of comparable size. The results do not provide strong evidence for chelation of the active site zinc ion as proposed, although such interactions do not appear to be ruled out altogether.