Enhancement of Mycobacterium tuberculosis-induced tumor necrosis factor alpha production from primary human monocytes by an activated T-cell membrane-mediated mechanism.

Mycobacterium tuberculosis alone induces small, donor-variable amounts of tumor necrosis factor alpha (TNF-alpha) from primary human monocytes in vitro. However, TNF-alpha release is increased 5- to 500-fold when fixed activated T cells (FAT) or their isolated, unfixed membranes are added to this system. This FAT-induced synergy was at least as potent as that induced by gamma interferon (IFN-gamma) at 100 U/ml. FAT-enhanced TNF-alpha production is at least in part transcriptionally mediated, as reflected by quantitative changes in TNF-alpha mRNA between 2 and 6 h poststimulation. Unlike IFN-gamma-cocultured cells, FAT-treated monocytes appeared not to have enhanced TNF-alpha message stability, suggesting that de novo transcription may be involved in this effect. Furthermore, M. tuberculosis alone induced only minimal DNA binding of monocyte NF-kappaB, but cells treated with M. tuberculosis and FAT potentiated NF-kappaB activity more effectively. It is therefore possible that one mechanism by which FAT synergize with M. tuberculosis to stimulate TNF-alpha production is via NF-kappaB-enhanced transcription. These data strongly suggest that in the interaction of cells involved in the immune response to M. tuberculosis, T-cell stimulation of monocyte TNF-alpha production involves a surface membrane interaction(s) as well as soluble mediators.

T cell-monocyte contact enhances tumor necrosis factor-alpha production in response to Mycobacterium leprae.

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.

Growth hormone activation of human monocytes for superoxide production but not tumor necrosis factor production, cell adherence, or action against Mycobacterium tuberculosis.

We have previously demonstrated that growth hormone (GH) is a human macrophage-activating factor which primes monocytes for enhanced production of H2O2 in vitro. This report extends our observations to other monocyte functions relevant to infection. We find that GH also primes monocytes for O2- 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.

Selective deactivation of human monocyte functions by TGF-beta.

TGF-beta at 1 and 10 ng/ml inhibited H2O2 production and fibronectin adherence by human monocytes. Coculture with anti-TGF-beta Abs or with IFN-gamma, but not with growth hormone, abrogated these effects. Neither viability nor superoxide production were decreased by TGF-beta treatment. TGF-beta appeared to be inhibiting H2O2 production rather than inducing catalase as preincubation in azide was without effect. Also, TGF-beta did not inhibit activity against virulent Mycobacterium tuberculosis. Coculture of monocytes with IFN-gamma + TGF-beta in vitro moderately inhibited the growth of M. tuberculosis when compared with untreated cells. Phagocytosis was not inhibited. Treatment of monocytes with another combination of cytokines, IFN-gamma+ TNF-alpha + vitamin D3, markedly reduced bacterial viability, although this appeared to be due to decreased phagocytosis leading to extracellular death of the bacteria. We conclude that despite suppressing some monocyte functions such as H2O2 production and adherence, TGF-beta, in combination with other cytokines, leaves other antimicrobial functions of the monocyte unaffected or even enhanced.

Interleukin 1-induced phosphorylation of MAD3, the major inhibitor of nuclear factor kappa B of HeLa cells. Interference in signalling by the proteinase inhibitors 3,4-dichloroisocoumarin and tosylphenylalanyl chloromethylketone.

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.

Growth hormone is a human macrophage activating factor. Priming of human monocytes for enhanced release of H2O2.

Although many effects of growth hormone (GH) and related factors upon the immune system have been demonstrated, few studies have examined the capacity of these factors to modulate human monocyte function in vitro. Assaying a range of mediators, only GH and prolactin (PRL), at 0.3 to 1.0 micrograms/ml, and growth hormone-releasing hormone (GRH) at very high doses, primed monocytes for enhanced hydrogen-peroxide production (H2O2) in response to PMA. GH-induced priming was not caused by endotoxin, nor by production of lymphokines such as IFN-gamma. Exogenous insulin-like growth factor-I (IGF-I), alone or in combination with GH, was without effect, making it unlikely that GH mediates its effects on monocytes via an autocrine/paracrine action of IGF-I. Monocytes specifically bound radiolabeled GH and contained mRNA for the GH receptor and, in some donors, the PRL receptor. Therefore, GH probably exerts its effects as a human macrophage-activating factor through either GH or PRL receptors, without requiring production of IGF-I.

Apparent killing of Mycobacterium tuberculosis by cytokine-activated human monocytes can be an artefact of a cytotoxic effect on the monocytes.

A protocol using combined exposure to interferon-gamma (IFN-gamma), calcitriol and tumour necrosis factor-alpha (TNF-alpha) 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.