Combined effects of ATP on the therapeutic efficacy of antimicrobial drug regimens against Mycobacterium avium complex infection in mice and roles of cytosolic phospholipase A2-dependent mechanisms in the ATP-mediated potentiation of antimycobacterial host resistance.

ATP, which serves as a mediator of intramacrophage signaling pathways through purinoceptors, is known to potentiate macrophage antimycobacterial activity. In this study we examined the effects of ATP in potentiating host resistance to Mycobacterium avium complex (MAC) infection in mice undergoing treatment with a drug regimen using clarithromycin and rifamycin and obtained the following findings. First, the administration of ATP in combination with the clarithromycin and rifamycin regimen accelerated bacterial elimination in MAC-infected mice without causing changes in the histopathological features or the mRNA expression of pro- or anti-inflammatory cytokines from those in the mice not given ATP. Second, ATP potentiated the anti-MAC bactericidal activity of macrophages cultivated in the presence of clarithromycin and rifamycin. This effect of ATP was closely related to intracellular Ca2+ mobilization and was specifically blocked by a cytosolic phospholipase A2 (cPLA2) inhibitor, arachidonyl trifluoromethylketone. Third, intramacrophage translocation of membranous arachidonic acid molecules to MAC-containing phagosomes was also specifically blocked by arachidonyl trifluoromethylketone. In the confocal microscopic observation of MAC-infected macrophages, ATP enhanced the intracellular translocation of cPLA2 into MAC-containing phagosomes. These findings suggest that ATP increases the host anti-MAC resistance by potentiating the antimycobacterial activity of host macrophages and that the cPLA2-dependent generation of arachidonic acid from the phagosomal membrane is essential for such a phenomenon.

Mucosal immunity of nasopharynx: an experimental study in TCR-transgenic (OVA23-3) mice.

The ideal vaccine therapy has been warranted for activation of the mucosal immune response in the upper respiratory tract against various types of microbial infection. However, the precise study in regard to the mucosal route of vaccine administration and its mechanism of action remains to be further investigated. Therefore, to better understand the exact mechanism of nasopharyngeal mucosal immunology, from T-cell aspects, the antigen-specific antibody response was investigated in T cell receptor transgenic (OVA23-3) mice (Tg-mice) and wild type BALB/c mice, in comparison, which were stimulated with repeated nasal antigen challenges of ovalbumin (OVA) together with cholera toxin (CT) or OVA alone. OVA-specific IgA and IgG antibodies were not detected in nasal washings of BALB/c mice when these mice were intranasally stimulated with OVA alone. But they were detected in those of BALB/c mice stimulated with OVA and CT, as we have already reported. Interestingly, OVA-specific IgA and IgG antibodies were significantly higher in nasal washings of Tg-mice stimulated with OVA and CT or OVA alone rather than those of BALB/c mice stimulated with OVA and CT. In line with data of the antibody response, OVA-specific IgA and IgG antibody-producing cells significantly increased in number in nasal passage (NP), nasopharyngeal-associated lymphoreticular tissue (NALT), cervical lymph node (CLN), and spleen (SP) of these mice. In nasal washings of Tg-mice, interferon (IFN)-gamma and interleukin (IL)-4 was detected even with a small amount of antigen. To see the cytokine profile of NALT, NP, CLN, and SP of these mice, various cytokine concentrations were measured in supernatants of these cells cultured in vitro with OVA. As a result, IFN-gamma was detected at significantly higher levels in culture supernatants of lymphocytes sampled from NP, CLN, SP as well as NALT of mice having increased antibody titers in nasal washings. On the other hand, Th2 type cytokines such as IL-4, IL-6 and IL-13 were efficiently detected in culture supernatants of NP, CLN, and SP cells from Tg-mice mice, but not in those from NALT cells of those mice. All these data taken together indicate that helper T cells recruited into nasal mucosa and locally activated in an antigen-specific fashion, as well as NALT T cells, are essential for mounting local antigen-specific antibody responses.