Polymorphism of the 5' flanking region of the IL-12 receptor beta2 gene partially determines the clinical types of leprosy through impaired transcriptional activity.

BACKGROUND: Individual differences in T cell responsiveness to interleukin 12 (IL-12), resulting from inherited factors, may be responsible for differences in the intensity of cell mediated immune (CMI) responses in patients with leprosy, a disease with a wide clinical spectrum. AIM: Polymorphisms in the 5' flanking region of the IL12RB2 gene were analysed to determine potential immunogenetic factors affecting CMI responses, using leprosy as a model. METHODS: Polymorphisms in the 5' flanking region of IL12RB2 were examined using direct sequencing techniques, and allele frequencies between patients with lepromatous leprosy and patients with tuberculoid leprosy were compared. The effect of these single nucleotide polymorphisms (SNPs) on IL12RB2 expression was estimated using the dual luciferase reporter gene assay in Jurkat T cells. RESULTS: Several SNPs, including -1035A>G, -1023A>G, -650delG, and -465A>G, were detected within the 5' flanking region of IL12RB2. The frequency of haplotype 1 (-1035A, -1023A, -650G, -464A) was high in the general Japanese population, but was significantly lower in lepromatous patients compared with tuberculoid patients and healthy controls. Reporter gene assays using Jurkat T cells revealed that all haplotypes carrying one or more SNP exhibited a lower transcriptional activity compared with haplotype 1. CONCLUSION: SNPs within the 5' flanking region of IL12RB2 affect the degree of expression of this gene and may be implicated in individual differences in CMI responsiveness to mycobacterial antigens, leading to lepromatous or tuberculoid leprosy.

Dharmendra antigen but not integral M. leprae is an efficient inducer of immunostimulant cytokine production by human monocytes, and M. leprae lipids inhibit the cytokine production.

Killed integral Mycobacterium leprae, Mitsuda antigen, and chloroform-treated M. leprae, Dharmendra antigen (Dh-Ag), have been used for the classification of leprosy patients based on cell-mediated immunity. Heat-killed M. leprae also were used as a component of the Convit vaccine. Human blood monocytes were stimulated with M. leprae or Dh-Ag and their cytokine-inducing ability was compared. Monocytes were cultured in the presence of fresh human serum because of the efficiency of cytokine induction and the phagocytosis of M. leprae have been shown to be optimal in the presence of fresh serum. M. leprae and Dh-Ag were equally phagocytosed by monocytes. Dh-Ag was more potent than M. leprae in the induction of immunostimulatory/proinflammatory cytokines, interleukin-1 (IL-1), IL-6 and tumor necrosis factor (TNF). In contrast, a comparable level of IL-1ra, an immunosuppressive cytokine, was induced by M. leprae and Dh-Ag. The lipids extracted from M. leprae induced none of these cytokines by monocytes. Nevertheless, when monocytes were pretreated with the lipids followed by stimulation with Dh-Ag, productions of IL-1, IL-6 and TNF were all inhibited in a dose-dependent manner. However, the lipids did not inhibit the cytokine production induced by other stimuli including BCG and lipopolysaccharide. Moreover the lipids did not affect the production of IL-1ra. These results suggest that the lipids from M. leprae are responsible for the poor cytokine-inducing ability of M. leprae, thus favoring their infection. These results also suggest that Dh-Ag rather than integral M. leprae may be useful as a vaccine candidate because Dh-Ag is able to induce a large amount of cytokines from monocytes.

Down regulation of Ia expression in macrophages following incubation with mycobacteria.

Macrophages are known to release cytokines in response to various kinds of stimulators. In the present study, peritoneal macrophages from C3H/He or C3H/HeJ mice were incubated in vitro with heat-killed M. lepraemurium, M. intracellulare or M. gordonare for 3 days followed by harvest culture supernatant to analyze cytokine activities. It, therefore, seems that macrophages phagocytizing these mycobacteria, released interleukin-1 (IL-1) and tumor necrosis factor (TNF) in culture media. The amount of release was dose dependent on mycobacteria employed. In addition, macrophages, as already have reported elsewhere, treated with IFN for 2 to 3 days showed enhanced expression of surface Ia; although the expression was inhibited if the cells phagocytized mycobacteria. Similarly, the reduced expression of Ia was observed in peritoneal macrophages from MRL/lpr mice after 3 day-culture with mycobacteria in vitro. More importantly, in the presence of the supernatant obtained from macrophages incubated with mycobacteria, IFN gamma-treated normal macrophages exhibited suppressed expression of Ia. These results demonstrate that cytokine release and reduced expression of surface Ia in macrophages are simultaneous phenomena after phagocytosis of mycobacteria. Suppression of Ia may be in part induced by Ia suppressive factor(s) released from mycobacterium-phagocytized macrophages.

Clofazimine-mediated augmentation of LPS-induced tumor necrosis factor production in macrophages.

Tumor necrosis factor (TNF) exerts multiple biological activities including immune response. It is also believed to play an important role in anti-bacterial response. In this study in vitro, we observed augmentation of LPS-induced TNF production from mouse macrophages by clofazimine treatment. Rifampicin, however, did not indicate such an activity. Clofazimine itself, on the other hand, did not have any TNF-inducing activity. Clofazimine is a well known anti-leprosy drug; in addition, from the results obtained here, this drug could induce anti-M. leprae response of host by way of the augmented immune response by enhanced cytokine production from macrophages.

Differential production of interleukin 1 (IL-1), IL-6, tumor necrosis factor, and IL-1 receptor antagonist by human monocytes stimulated with Mycobacterium leprae and M. bovis BCG.

Human blood monocytes cultured in various serum conditions were stimulated with Mycobacterium leprae or M. bovis BCG and their cytokine-inducing abilities were compared. BCG, either live or killed, induced production of interleukin 1 (IL-1), IL-6, tumor necrosis factor (TNF), and IL-1 receptor antagonist (IL-1ra). Live BCG at a lower bacterial number was more potent than killed BCG in the induction of IL-6 and TNF. In contrast to BCG, killed M. leprae induced few cytokines except for IL-1ra. Similar results were obtained when monocytes were cultured in the presence of untreated or heat-inactivated fetal bovine serum (FBS). When FBS and human serum (HS) were compared and the effect of heat inactivation was investigated, monocytes in HS produced the most cytokines, then those in FBS, irrespective of heat inactivation, and those in heat-inactivated HS produced the least cytokines. There were no differences between live and killed M. leprae, and BCG were far more potent than M. leprae in all of our experimental conditions, indicating that the poor cytokine (IL-1, IL-6 and TNF)-inducing ability of M. leprae was not due to their viability. Cytokine production was partially in parallel with the phagocytosis of the mycobacteria. These results suggest that M. leprae favor their infection by evoking little host reaction through the induction of only low levels of immunostimulatory or proinflammatory cytokines but a substantial amount of immunosuppressive cytokine.

Regulation of murine macrophage effector functions by lipoarabinomannan from mycobacterial strains with different degrees of virulence.

Lipoarabinomannan (LAM) is the major arabinose- and mannose-containing phosphorylated lipopolysaccharide (LPS) in mycobacterial cell walls. LAM preparations from a virulent strain (Erdman) (LAM(Erdman)) and an attenuated strain (H37Ra) (LAMH37Ra) of Mycobacterium tuberculosis, as well as from M. leprae (a virulent mycobacterium), were analyzed for their effects on various macrophage (M phi) effector functions. LAMH37Ra, like gram-negative LPS, exhibited a dose-dependent ability to induce tumor necrosis factor alpha (TNF-alpha) production in normal M phi, and gamma interferon (IFN-gamma) priming of the M phi greatly augmented the levels of TNF-alpha. However, the effects of LAMH37Ra were unaffected by polymyxin B, which totally abrogated the effects of LPS. LAM(Erdman) and LAM from M. leprae, on the other hand, induced virtually no TNF-alpha production. Analysis of M phi mRNA by reverse transcription-polymerase chain reaction revealed that the levels of production. Analysis of M phi mRNA by reverse transcription-polymerase chain reaction revealed that the levels of TNF-alpha mRNA induced by the various preparations correlated with the levels of TNF-alpha protein detected. Interestingly, both LAMH37Ra and LAM(Erdman) could block subsequent IFN-gamma- and LPS-induced M phi activation, a previously reported measure of the potent ability of LAM to down-regulate M phi effector functions. Two lines of evidence suggested, however, that M phi cyclooxygenase products did not play a role in this down-regulation. LAMH37Ra and LPS could induce the production of NO2- in both normal and IFN-gamma-primed M phi, whereas LAM(Erdman) could stimulate NO2- production only in primed M phi. Both LAMH37Ra and LAM(Erdman) could substitute for LPS as a triggering signal for IFN-gamma-primed M phi in a toxoplasma killing assay. The triggering ability of LAM(Erdman), however, was abrogated by an anti-TNF-alpha antibody, suggesting that sufficient TNF-alpha production was stimulated by LAM(Erdman) to drive a M phi function relevant in host resistance. Thus, mycobacterial LAM is a potent regulator of M phi functions, a fact that may have important consequences in mycobacterial disease.

[Monokine production by mouse peritoneal macrophages after phagocytosis of mycobacteria].

It is well known that monokines, IL-1 (Interleukin-1) and TNF (Tumor Necrosis Factor), are produced by macrophages after stimulated with various agents. These cytokines are involved in various aspects of the inflammatory process and immunological response in addition to their original activities to proliferate T lymphocytes and causing tumor necrosis, respectively. Recently, there have been reported that IL-1 and TNF also play an important role in mycobacterial infections such as granuloma formation. In the present study, IL-1 and TNF productions were observed by mouse peritoneal exudate and resident macrophages after incubation with heat-killed M. lepraemurium and M. avium in vitro. The production was enhanced by phagocytosis of these mycobacteria in a dose dependent manner, and the time course of the production was maximum within 24 hr after phagocytosis of these mycobacteria. It was also shown of morphological changes and enhanced glucose consumption in media by these macrophages. Above results suggest that phagocytosis of mycobacteria by macrophages leads to monokine production, which would not only causes well known immunological reactions but also makes characteristic phenomena to be observed in mycobacterial infections.