The regulation of the inhibitor of nuclear factor kappa B (I kappa B) by interleukin 1 (IL1) was investigated in HeLa cells. Two forms of I kappa B were resolved by ion-exchange chromatography. The major form (75%) was identified as MAD3 by specific antisera. IL1 generated rapidly (6 min) an electrophoretically retarded form of MAD3 that was stable in acid and was converted into the unmodified form by phosphatase 2A. It thus corresponded to a phosphorylation of the protein on serine or threonine. IL1 also caused the disappearance of MAD3 from the cells, which was complete 15 min after stimulation and coincided with a 46% reduction of cellular I kappa B activity. Newly-synthesized MAD3 accumulated to pre-stimulation levels between 60 and 90 min after stimulation and this coincided with the down-regulation of the phosphorylating activity. The serine proteinase inhibitors 3,4-dichloroisocoumarin (DCI) and tosylphenylalanyl chloromethylketone (TPCK) prevented phosphorylation and disappearance of MAD3. At the same concentrations (10-100 microM), they also increased basal phosphorylation of the small heat shock protein (hsp27) and prevented the IL1- and phorbol 12-myristate 13-acetate-induced increases of its phosphorylation. The inhibitors were thus interfering with protein kinases when blocking degradation of MAD3. Recombinant MAD3 phosphorylated in vitro by protein kinase C was not electrophoretically retarded, suggesting that MAD3 was phosphorylated by another kinase in IL1-stimulated cells. Our results suggest that the IL1-induced phosphorylation of MAD3 on serine or threonine leads to its degradation. DCI and TPCK blocked phosphorylation mechanisms and it could not be concluded that serine proteinases were involved in the breakdown of MAD3.

Tumor necrosis factor (TNF)-alpha has been implicated as a key factor in inflammatory processes occurring in erythema nodosum leprosum (ENL). In the present study, the roles of soluble factors and contact-mediated interaction in the induction of enhanced TNF-alpha secretion in leprosy have been investigated. In vitro studies have demonstrated that Mycobacterium leprae per se is a poor stimulus for TNF-alpha production by purified monocytes obtained from normal subjects, although this could be enhanced by either exogenous interferon-gamma or cell contact with fixed activated T lymphocytes. Further investigations demonstrated that monocyte-T cell contact enhanced M. leprae-induced TNF-alpha production by peripheral blood mononuclear cells of ENL patients and was modulated by blocking antibodies to CD40L, CD69, and CD18. These results suggest that physical contact with T cells isolated from patients in a particular disease state (ENL) modulates monocyte function and may contribute to the secretion of proinflammatory cytokines described in ENL.

We have previously demonstrated that growth hormone (GH) is a human macrophage-activating factor which primes monocytes for enhanced production of H 2 O 2 in vitro. This report extends our observations to other monocyte functions relevant to infection. We find that GH also primes monocytes for O 2 ؊ production, to a degree similar to the effect of gamma interferon. Neither macrophage-activating factor alone stimulates monocytes to release bioactive tumor necrosis factor. However, GH, unlike gamma interferon, does not synergize with endotoxin for enhanced tumor necrosis factor production. In further contrast, GH does not alter monocyte adherence or morphology, while phagocytosis and killing of Mycobacterium tuberculosis by GH-treated monocytes are also unaffected. Therefore, despite the multiplicity of the effects of GH on the immune system in vivo, its effects on human monocytes in vitro appear to be limited to priming for the release of reactive oxygen intermediates.

SUMMARY A protocol using combined exposure to interferon‐gamma (IFN‐γ), calcitriol and tumour necrosis factor‐alpha (TNF‐α) has been reported to activate human monocytes in vitro to kill Mycobacterium tuberculosis. We have attempted to repeat the findings in two laboratories, with negative results: treated cells were no different from untreated cells in this respect. However, the treated cells were more sensitive to a toxic effect of the bacteria. We suggest that the reported dramatic mycobacterial killing may have been an illusory consequence of the toxicity leading to cell lysis and loss of the liberated bacteria from the assay.

No abstract