PARK2 mediates interleukin 6 and monocyte chemoattractant protein 1 production by human macrophages.

Leprosy is a persistent infectious disease caused by Mycobacterium leprae that still affects over 200,000 new patients annually. The host genetic background is an important risk factor for leprosy susceptibility and the PARK2 gene is a replicated leprosy susceptibility candidate gene. The protein product of PARK2, Parkin, is an E3 ubiquitin ligase that is involved in the development of various forms of Parkinsonism. The human macrophage is both a natural host cell of M. leprae as well as a primary mediator of natural immune defenses, in part by secreting important pro-inflammatory cytokines and chemokines. Here, we report that down-regulation of Parkin in THP-1 macrophages, human monocyte-derived macrophages and human Schwann cells resulted in a consistent and specific decrease in interleukin-6 (IL-6) and monocyte chemoattractant protein 1 (MCP-1/CCL2) production in response to mycobacteria or LPS. Interestingly, production of IL-6 at 6 hours by THP-1 cells stimulated with live M. leprae and M. bovis BCG was dependent on pretreatment with 1,25-dihydroxyvitamin D(3) (VD). Parkin knockdown in VD-treated cells blocked IL-6 induction by mycobacteria. However, IκB-α phosphorylation and levels of IκB-ξ, a nuclear protein required for IL-6 expression, were not affected by Parkin silencing. Phosphorylation of MAPK ERK1/2 and p38 was unaffected by Parkin silencing while JNK activation was promoted but did not explain the altered cytokine production. In a final set of experiments we found that genetic risk factors of leprosy located in the PARK2 promoter region were significantly correlated with M. leprae sonicate triggered CCL2 and IL6 transcript levels in whole blood assays. These results associated genetically controlled changes in the production of MCP-1/CCL2 and IL-6 with known leprosy susceptibility factors.

Thalidomide inhibited the synthesis of IgM and IgG whereas Thalidomide+Dexamethasone and Dexamethasone alone acted as co-stimulants with pokeweed and enhanced their synthesis.

Thalidomide (Thal) provides effective treatment for erythema nodosum leprosum (ENL). In combination with Dexamethasome (Dex) it is an effective treatment for multiple myeloma (MM) and Waldenström's macroglobulinemia (WM). Thal's mechanism(s) of action in the treatment of these diverse medical conditions is not known, but it could be suppression of immunoglobulin (Ig) synthesis. Mononuclear cells were stimulated with pokeweed (PWM), and treated with Thal, Thal+Dex or Dex. The cultures were assayed for IgM and IgG. The maximum synthesis was expected to occur in cultures stimulated with PWM at 0.5, 5.0 or 10 microg/ml. The test agents at 15 microM each were expected to alter the response. Compared to cultures stimulated with PWM alone, there was significantly less Ig in the cultures containing Thal+PWM, and significantly more Ig in the cultures containing Thal+Dex+PWM or Dex+PWM (Wilcoxon). The median % of maximum was 57 for cultures treated with Thal+PWM; 184 for cultures treated with Thal+Dex+PWM, and 139 for cultures treated with Dex+PWM. Thal also acted as a co-stimulant with PWM and enhanced the synthesis of IL-2, IL-6 and DNA; whereas, Thal+Dex or Dex enhanced Ig synthesis, but suppressed IL-2, IL-6 and cell proliferation. Thal's ability to suppress Ig may explain its activity in ENL, MM and WM. The enhancement of Ig by Dex does not help to explain a role for Dex alone or in combination with Thal for the treatment of MM and WM.

The flagellar fraction of Trypanosoma cruzi depleted of an immunosuppressive antigen enhances protection to infection and elicits spontaneous T cell responses.

The flagellar fraction (FF) of Trypanosoma cruzi can be separated by immunoaffinity chromatography in two fractions with balanced but opposite immunological effects. The immunoaffinity purified fraction has immunosuppressive activity mediated at least partially by TGF-beta (Hansen et al., submitted). Here we report that the fraction depleted of immunosuppresive antigens (FT) administered with iscom-matrix as adjuvant provides enhanced protection to an infection challenge in immunized mice. In vitro, the FT but not the FF stimulated resident peritoneal cells to produce IL-1 and IL-6. In immunized mice, the FT elicited higher levels of antigen-specific IgG2a than the FF as well as broader recognition of T. cruzi antigens. Splenocytes from mice immunized with FT proliferated spontaneously in vitro and secreted TH1 and TH2 cytokines. The protection provided by FT correlates with its capacity to enhance the secretion of IFN-gamma. We postulate that immunosuppressive antigens present in the FF prevent the development of memory cells secreting IFN-gamma through a TGF-beta dependent mechanism.

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.

IL-6 production in response to purified mycobacterial heat-shock proteins and to antigen 85 in leprosy.

IL-6 production was examined in PBMC cultures from healthy leprosy contacts and from leprosy patients stimulated with the purified mycobacterial 18-, 65-, and 70-kDa heat-shock proteins (hsp) and the secreted fibronectin-binding antigen 85 (Ag85). In lepromin-negative contacts, the 70-kDa hsp was the only antigen capable of eliciting significant IL-6 production. In lepromin-positive contacts, Ag85, the 65- and the 70-kDa hsp induced substantial IL-6 titers. IL-6 levels induced with the 70-kDa antigen were about fourfold higher than with the 65-kDa hsp or with Ag85. The 18-kDa antigen did not induce any IL-6 in these healthy contacts. PBMC from tuberculoid leprosy patients produced even more elevated levels of IL-6, and PBMC from lepromatous leprosy patients produced extremely high levels of IL-6. All antigens were capable of inducing IL-6 in leprosy patients. Highest levels were found in cultures stimulated with the 65-kDa hsp, and lowest levels were in cultures stimulated with the 18-kDa hsp.