[Phagocytosis of Mycobacterium leprae down-regulates anti-microbial activity of murine macrophages against Mycobacterium intracellulare].

Patients with highly bacillated lepromatous leprosy (LL) essentially lack T cell-mediated immune responses specific to Mycobacterium leprae (ML) antigens, resulting in severely impaired host resistance to leprosy bacilli. Such type of immune unresponsiveness characteristic of LL patients is mainly attributable to markedly depressed T cell ability to activate/expand in response to ML antigens. In this study, we examined profiles of antimycobacterial activity of macrophages, which phagocytized leprosy bacilli, because there is another possibility that, in LL patients, host macrophages in the leprosy lesions are impaired in their antimicrobial activity due to their interaction with infected leprosy bacilli, particularly cellular events through binding with and/or internalization of the pathogens, thereby causing the reduction in host resistance to ML pathogens. The present study indicated the following. First, the anti-M. avium complex activity of murine peritoneal macrophages was significantly reduced when they had phagocytosed heat-killed leprosy bacilli. Second, infection of macrophages with leprosy bacilli did not affect macrophage-mediated suppressor activity against T cell proliferative response to Concanavalin A. These findings indicate that macrophage's intracellular signaling pathways that are up-regulated in response to phagocytosis of leprosy bacilli are linked to the signaling cascades participating in macrophage antimicrobial functions, but not cross-talk with those allowing the expression of macrophage's suppressor activity against T cell functions.

[Novel type of antimicrobial mechanism in host macrophages against mycobacterial infections].

Macrophages (M(phi)s) play a central role as anti-microbial effector cells in the expression of host resistance to mycobacterial infections. With respect to antimicrobial effector molecules of host M(phi) against mycobacterial pathogens, recent studies suggest the possibility that the reactive nitrogen intermediates (RNI)--and reactive oxygen intermediates-independent antimycobacterial mechanism(s) may be crucial for the antimycobacterial function of host M(phi). In this context, we previously found that free fatty acids (FFAs) such as arachidonic acid (AA) and linolenic acid exhibited potent antimicrobial activity against mycobacterial organisms, including Mycobacterium tuberculosis (MTB) and Mycobacterium avium complex (MAC). In addition, FFAs in combination with RNI played critical roles in manifestation of the activity of M(phi) against mycobacterial organisms. Moreover, our recent studies have shown the following findings. First, anti-MTB activity of IFN-gamma-activated M(phi)s was specifically blocked by arachidonyl trifluoromethylketone (aTFMK), an inhibitor of cytosolic phospholipase A2 (cPLA2). Second, ATP potentiated the anti-MAC bactericidal activity of M(phi)s cultivated in the presence of clarithromycin and rifamycin. This effect of ATP was closely related to intracellular Ca2+ mobilization and was specifically blocked by aTFMK. Third, intramacrophage translocation of membranous AA molecules to MAC-containing phagosomes was also specifically blocked by aTFMK. In the confocal microscopic observation of MAC-infected M(phi)s, ATP enhanced the intracellular translocation of cPLA2 into MAC-containing phagosomes. These findings suggest that FFAs (especially AA) produced by the enzymatic action of cPLA2 play important roles as antimycobacterial effectors in the expression of M(phi) antimicrobial activity against mycobacterial pathogens.

Profiles of the mRNA expression by macrophages infected with mycobacterium leprae and mycobacterium avium complex

In the present study, we examined profiles of the interaction of Mycobacterium leprae and Mycobacterium avium complex (MAC) with murine peritoneal macrophages (M phi s) in terms of up-regulation of M phi expression of proinflammatory and immunosuppressing cytolines (CKs) after infection. First, the reverse transcription polymerase chain reaction (RT-PCR) assay revealed that both MAC and M. leprae infections up-regulated M phi mRNA expression IL-12, TNF-alpha, IL-10, and transformating growth factor-beta (TGF-beta), except that M. leprae-infected M phi s showed no increase in the IL-12 mRNA expression. Second, the RT-PCR assay also showed some differences between M. leprae- and MAC-infected M phi s with respect to the modes of IL-10 and inducible nitric oxide synthase (iNOS) mRNA expression. That is MAC, but not M. leprae, infection caused a prolonged increase in the expression of IL-10 and iNOS mRNAs. Third, a ribonuclease protection assay revealed that M phi s co-infected with MAC and M. leprae showed the Il-12, TNF-alpha and IL-10 mRNA expression in an intermediate mode of those of M phi s infected with either M. leprae or MAC alone. This implies that the CK expression of M. leprae-infected M phi s may be modified by co-infection with MAC. These findings may suggest differential interactions of M. leprae and MAC organisms with murine peritoneal M phi s in terms of the activation of signal transduction pathways for expression of some kinds of immunoregulatory cytokines and immunoprotective enzymes.

Profiles of the mRNA expression by macrophages infected with Mycobacterium leprae and Mycobacterium avium complex.

In the present study, we examined profiles of the interaction of Mycobacterium leprae and Mycobacterium avium complex (MAC) with murine peritoneal macrophages (M phi s) in terms of up-regulation of M phi expression of proinflammatory and immunosuppressing cytolines (CKs) after infection. First, the reverse transcription polymerase chain reaction (RT-PCR) assay revealed that both MAC and M. leprae infections up-regulated M phi mRNA expression IL-12, TNF-alpha, IL-10, and transformating growth factor-beta (TGF-beta), except that M. leprae-infected M phi s showed no increase in the IL-12 mRNA expression. Second, the RT-PCR assay also showed some differences between M. leprae- and MAC-infected M phi s with respect to the modes of IL-10 and inducible nitric oxide synthase (iNOS) mRNA expression. That is MAC, but not M. leprae, infection caused a prolonged increase in the expression of IL-10 and iNOS mRNAs. Third, a ribonuclease protection assay revealed that M phi s co-infected with MAC and M. leprae showed the Il-12, TNF-alpha and IL-10 mRNA expression in an intermediate mode of those of M phi s infected with either M. leprae or MAC alone. This implies that the CK expression of M. leprae-infected M phi s may be modified by co-infection with MAC. These findings may suggest differential interactions of M. leprae and MAC organisms with murine peritoneal M phi s in terms of the activation of signal transduction pathways for expression of some kinds of immunoregulatory cytokines and immunoprotective enzymes.

Roles of tumor necrosis factor-alpha and transforming growth factor-beta in regulating intercellular adhesion molecule-1 expression on murine peritoneal macrophages infected with M. Leprae

Profiles of intercellular adhesion molecule-1 (ICAM-1) expression on murine peritoneal macrophages (M phi s) infected with Mycobacterium leprae during cultivation were examined with special reference to the regulatory effects of tumor necrosis factor-alpha (TNF-alpha) and transforming growth factor-beta (TGF-beta). When M phi s were infected with M. leprae or stimulated with heat-killed M. leprae at day 0, their ICAM-1 expression, measured in terms of the ratio of M phi s positively stained with anti-ICAM-1 antibody (Ab), rapidly increased, peaking during days 1 to 3 and thereafter fell, returning to the normal level by day 7. The addition of TNF-alpha or anti-TGF-beta Ab inhibited the middle phase (day 7) downregulation of M phi ICAM-1 expression, although the late-phase (day 14) downregulation of ICAM-1 was not prevented by them. M. leprae-infected M phi s released small amounts of TNF-alpha and significant amounts of TGF-beta into the culture medium. This may indicate that M. leprae-infected M phi s produced the majority of TNF-alpha in a membrane-bound form. Alternatively, endogenous TNF-alpha might upregulate M phi ICAM-1 expression even at very low concentrations. In any case, these findings indicate the central roles of TNF-alpha and TGF-beta in the early phase upregulation and the middle-to-late phase downregulation, respectively, of ICAM-1 expression by M. leprae-infected M phi s.