Chiaki Sano

Type II Alveolar Cells Play Roles in Macrophage-Mediated Host Innate Resistance to Pulmonary Mycobacterial Infections by Producing Proinflammatory Cytokines

Roles of type II pneumocytes in macrophage (Mphi)-mediated host resistance to pulmonary Mycobacterium tuberculosis (MTB) and M. avium complex (MAC) infections were studied. Electron microscopy of the lung sections of mice given intratracheal infection indicated that the organisms invaded both Mphis and type II pneumocytes. When Mono-Mac-6 Mphis(MM6-Mphis) and A-549 type II pneumocytes (A-549 cells) were cocultivated, bacterial growth in MM6-Mphis was reduced by A-549 cell-derived soluble factors, indicating the roles of type II pneumocytes in Mphi-mediated host resistance to mycobacteria. MTB- or MAC-infected A-549 cells showed increased mitochondrial RNA expression of cytokines and surfactant proteins (SPs), in the order tumor necrosis factor-alpha (TNF-alpha) > or = granulocyte-Mphi colony-stimulating factor (GM-CSF) > Mphi chemoattractant protein > or = interleukin-8 > SP-D. Anti-TNF-alpha and anti-GM-CSF antibodies attenuated A-549 cell-dependent inhibition of intramacrophage mycobacteria, indicating their crucial roles in A-549 cell-mediated potentiation of Mphi antimycobacterial activity.

Effector molecules in expression of the antimicrobial activity of macrophages against Mycobacterium avium complex: roles of reactive nitrogen intermediates, reactive oxygen intermediates, and free fatty acids.

We studied microbicidal activities of reactive nitrogen intermediates (RNI), free fatty acids (FFA), and reactive oxygen intermediates (ROI) against Mycobacterium aviumcomplex (MAC) and the mode of macrophage (mΦ) production of these effectors. (1) Intracellular growth of MAC in murine peritoneal mΦs was accelerated by scavengers for ROI or RNI and inhibitors of nitric oxide synthase or phospholipase A2, indicating roles of ROI, RNI, and FFA in mΦ anti‐MAC functions. (2) Acidified NaNO2‐derived RNI, FFA (linolenic and arachidonic acids), and the H2O2‐mediated halogenation system exhibited a significant anti‐MAC bactericidal activity. The combination of RNI with FFA showed a synergistic effect However, the H2O2‐halogenation system in combination with either RNI or FFA showed an antagonism. When Listeria monocytogenes(Lm) was used as a target organism, the combinations of RNI + FFA and RNI + H2O2‐halogenation gave a synergistic effect, whereas FFA + H2O2‐halogenation showed an antagonism in exerting bactericidal activity. In addition, when ROI generated by the xanthine oxidase‐acetaldehyde system was combined with RNI, anti‐Lm but not anti‐MAC activity was potentiated. (3) ROI production by murine peritoneal mΦs was observed immediately after contact with MAC organisms (MAC stimulation) and ceased within 2 h. FFA release was seen 1‐24 h after MAC stimulation. RNI production was initiated from 3 h and increased during the first 36 h and continued at least for 4 days. These findings suggest that RNI and FFA rather than ROI are important effectors of anti‐MAC functions of mΦs, and the collaborating action of RNI with FFA temporarily participates in mΦ‐mediated killing of MAC in the relatively early phase after MAC stimulation. J. Leukoc. Biol. 62: 795–804; 1997.

Effects of secretory leucocyte protease inhibitor on the production of the anti-inflammatory cytokines, IL-10 and transforming growth factor-beta (TGF-β), by lipopolysaccharide-stimulated macrophages

We studied the effects of secretory leucocyte protease inhibitor (SLPI) on the production of the anti‐inflammatory cytokines, IL‐10 and TGF‐β, by lipopolysaccharide (LPS)‐stimulated macrophages, using half‐sized SLPI (1/2 SLPI) containing the C‐terminal domain (Arg58‐Ala107). ELISA testing of macrophage culture fluids showed a temporary production of IL‐10 by the macrophages in the early phase (24 h) after LPS stimulation at low (1 ng/ml) or high (10 μg/ml) concentrations. On the other hand, TGF‐β production was initiated after day 3 and progressively increased. 1/2 SLPI significantly increased IL‐10 and TGF‐β production by macrophages in response to a low dose as well as a high dose of LPS. Reverse transcription‐polymerase chain reaction analysis showed that 1/2 SLPI caused a significant increase in the expression of both IL‐10 and TGF‐β mRNAs by LPS‐stimulated macrophages. Thus, although the profile of macrophage TGF‐β production by LPS‐stimulated macrophages is markedly different from that of their IL‐10 production, SLPI causes an up‐regulation of the production of these anti‐inflammatory cytokines by LPS‐stimulated macrophages.

Characteristics of Suppressor Macrophages Induced by Mycobacterial and Protozoal Infections in relation to Alternatively Activated M2 Macrophages

In the advanced stages of mycobacterial infections, host immune systems tend to change from a Th1-type to Th2-type immune response, resulting in the abrogation of Th1 cell- and macrophage-mediated antimicrobial host protective immunity. Notably, this type of immune conversion is occasionally associated with the generation of certain types of suppressor macrophage populations. During the course of Mycobacterium tuberculosis (MTB) and Mycobacterium avium-intracellulare complex (MAC) infections, the generation of macrophages which possess strong suppressor activity against host T- and B-cell functions is frequently encountered. This paper describes the immunological properties of M1- and M2-type macrophages generated in tumor-bearing animals and those generated in hosts with certain microbial infections. In addition, this paper highlights the immunological and molecular biological characteristics of suppressor macrophages generated in hosts with mycobacterial infections, especially MAC infection.

The modulating effects of proinflammatory cytokines interferon‐gamma (IFN‐γ) and tumour necrosis factor‐alpha (TNF‐α), and immunoregulating cytokines IL‐10 and transforming growth factor‐beta (TGF‐β), on anti‐microbial activity of murine peritoneal macrophages against Mycobacterium avium‐intracellulare complex

We assessed the roles of proinflammatory cytokines IFN‐γ and TNF‐α, and immunoregulatory cytokines IL‐10 and TGF‐β in the modulation of the anti‐microbial activity of murine peritoneal macrophages against Mycobacterium avium‐intracellulare complex (MAIC). First, both IFN‐γ and TNF‐α significantly reduced the bacterial growth in macrophages, indicating that these cytokines participate in up‐regulation of macrophage anti‐MAIC function. Second, although MAIC‐infected macrophages produced substantial amounts of IL‐10 and TGF‐β, neutralization of endogenous IL‐10 and TGF‐β with anti‐IL‐10 and anti‐TGF‐β antibodies, respectively, did not affect the intracellular growth of MAIC in macrophages from mice with BcgS (MAIC‐susceptible) or Bcgr (MAIC‐resistant) genotype, regardless of the virulence of test MAIC strains. The same result was also obtained for macrophages stimulated with IFN‐γ or TNF‐α. Third, in MAIC‐infected mice, the growth of organisms at the sites of infection (lungs and spleens) was not affected by administration of anti‐IL‐10 or anti‐TGF‐β antibodies. These findings indicate that, in the case of mice, endogenous IL‐10 and TGF‐β are essentially ineffective in down‐regulating macrophage anti‐MAIC functions not only in vitro but also in vivo.

Antimicrobial activities of clarithromycin, gatifloxacin and sitafloxacin, in combination with various antimycobacterial drugs against extracellular and intramacrophage Mycobacterium avium complex

We studied the activities of clarithromycin and fluoroquinolones (gatifloxacin, sitafloxacin, levofloxacin) in combination with other antimycobacterial drugs against extracellular and intramacrophage Mycobacterium avium complex (MAC). Clarithromycin potentiated the activities of rifampicin and rifalazil against both extracellular and intramacrophage MAC. In contrast, all the test quinolones exhibited antagonistic effects against extracellular MAC when combined with either clarithromycin or rifamycins. Such an antagonism was not observed for the activity of these combinations against intramacrophage MAC. Combined effects were observed with combinations of these fluoroquinolones with either ethambutol or streptomycin. Similar profiles were seen for the activities of two-drug combinations of clarithromycin or fluoroquinolones with other drugs against intramacrophage MAC isolated from pulmonary and disseminated MAC infections.

Development of new antituberculous drugs based on bacterial virulence factors interfering with host cytokine networks.

The worldwide increase in the prevalence of tuberculosis (TB), especially multidrug-resistant TB and extensively drug-resistant TB, is an important global health concern, and new effective drugs are urgently needed. Information on the genome of Mycobacterium tuberculosis (MTB) and various mycobacterial virulence genes is leading to the identification of genes that code for new drug targets. Mycobacterium tuberculosis (MTB) is resistant to the antimicrobial mechanisms of host macrophages and can survive and replicate in macrophages for long periods, resulting in a persistent infection. Mycobacterial virulence factors suppress macrophage bactericidal functions partly via their downregulatory effects on the host antimicrobial cytokine networks, consisting of proinflammatory, immunopotentiating, and Th1-inducing cytokines. Thus, for the development of unique drugs that exhibit antimycobacterial action through novel mechanisms, it is reasonable to search for targets among bacterial genes encoding virulence factors which interfere with the host cytokine responses protective to mycobacterial pathogens. In this review, we discuss the profiles of cytokine networks related to host resistance to mycobacteria, including the mechanisms of downregulation of host antimycobacterial immunity due to immunosuppressive cytokines, which are occasionally induced in the advanced stages of TB. We also highlight the development of antituberculous drugs based on bacterial virulence factors interfering with the host antimycobacterial cytokine network.

The role of B7 molecules in the cell contact‐mediated suppression of T cell mitogenesis by immunosuppressive macrophages induced with mycobacterial infection

We found previously that immunosuppressive macrophages (Mφs) induced by Mycobacterium intracellulare infection (MI‐Mφs) transmitted their suppressor signals to target T cells through cell contact with target T cells. In this study, we examined what kinds of Mφ surface molecules are required for such cell–to–cell interaction. First, it was found that a B7‐1‐like molecule (B7–1LM) recognizable with one of three test clones of anti‐B7‐1 monoclonal antibodies (mAbs) was required for expression of the Mφ suppressor activity. Neither anti‐B7‐2, anti‐ICAM‐1, nor anti‐VCAM‐1 mAb blocked the Mφ suppressor activity. Second, MI‐Mφs increased the expression of B7–1LM in parallel with the acquisition of the suppressor activity. Moreover, MI‐Mφs bound with target T cells in a B7–1LM‐dependent fashion. Third, mAb blocking of CTLA‐4 on target T cells did not reduce the suppressor activity of MI‐Mφs, suggesting the role of a putative molecule on target T cells other than CTLA‐4 as the receptor for B7–1LM of MI‐Mφs. Fourth, concanavalin A (Con A) stimulation of MI‐Mφs was needed for effective cell contact with target T cells and subsequent expression of the suppressor activity of MI‐Mφs. Fifth, the Con A‐induced increase in the suppressor activity of MI‐Mφs was inhibited by KN‐62 but not by herbimycin A, H‐7, nor H‐88, indicating that Con A‐induced up‐regulation of MI‐Mφ function is mediated by calmodulin‐dependent protein kinase II or ATP/P2Z receptors, but independent of protein tyrosine kinase, protein kinase C, and protein kinase A. These findings indicate that a B7/CTLA‐4‐independent mechanism is needed for the transmission of the suppressor signals from MI‐Mφs to target T cells.

Effector molecules of the host defence mechanism against Mycobacterium avium complex: the evidence showing that reactive oxygen intermediates, reactive nitrogen intermediates, and free fatty acids each alone are not decisive in expression of macrophage antimicrobial activity against the parasites

In this study, we evaluated the roles of reactive oxygen intermediates (ROI), reactive nitrogen intermediates (RNI), and free fatty acids (FFA) as effectors of the macrophage‐mediated host defence mechanism against Mycobacterium avium complex (MAC). First, M. avium (three strains) and M. intracellulare (two strains) were treated with the H2O2‐Fe2+‐mediated halogenation system, acidified NaNO2‐derived RNI, or FFA (linolenic acid) in sodium acetate buffer pH 5.5, and then counted for the number of residual colony‐forming units (CFU) of organisms. Although these effectors exerted strong bactericidal activity against the MAC, the susceptibility of test organisms markedly varied from strain to strain. There was no significant relationship between the degree of resistance of a given MAC strain to these effectors and its virulence in mice, indicating that ROI, RNI, and FFA each alone are not decisive as the effector components of the host defence mechanism against the MAC. Second, the increase in ROI‐producing ability in murine peritoneal macrophages due to tumour necrosis factor‐alpha (TNF‐α) treatment was not accompanied by parallel potentiation of anti‐MAC activity of the same macrophage population. This excludes the possibility that ROI play a central role in macrophage‐mediated killing and inhibition of MAC organisms. Third, anti‐MAC activity of BAM3 macrophage cell line was not significantly attenuated by NG‐monomethyl‐l‐arginine (NO synthase‐inhibitor causing reduction of RNI production) or by quinacrine (phospholipase A2‐inhibitor causing reduction of FFA release), indicating that RNI and FFA each alone do not play crucial roles in the expression of macrophage antimicrobial activity against the MAC. The present findings suggest important roles of collaborating actions of various antimicrobial effectors and/or the participation of other kinds of effectors in macrophage‐mediated killing and inhibition of MAC organisms.

Comparative in vitro and in vivo antimicrobial activities of sitafloxacin, gatifloxacin and moxifloxacin against Mycobacterium avium

Moxifloxacin exhibits therapeutic activity against Mycobacterium avium infection in mice. Since not only moxifloxacin but also another 8-methoxy quinolone, gatifloxacin, and a C-8-chloro quinolone, sitafloxacin, show favourable antimycobacterial activity in vitro, their anti-M. avium activities were compared in vivo. Minimum inhibitory concentrations (MICs), minimum bactericidal concentrations (MBCs) and mutant prevention concentrations (MPCs) of the test quinolones for M. avium were determined by microdilution in 7HSF broth. Antimicrobial activity against intracellular bacteria was measured using Mono Mac 6 human macrophages. Therapeutic efficacy of the quinolones when administered subcutaneously with or without clarithromycin plus ethambutol was assessed using mice intravenously infected with M. avium in terms of changes in bacterial loads in the lungs and spleen following infection. Based on the MICs, MBCs and MPCs, the in vitro activities of sitafloxacin and moxifloxacin were greater than that of gatifloxacin. Moxifloxacin exhibited the strongest activity against intramacrophage M. avium. When each test quinolone was administered alone to infected mice, sitafloxacin and gatifloxacin exhibited greater therapeutic efficacy than moxifloxacin based on intrapulmonary bacterial elimination. However, moxifloxacin exerted greater activity in killing bacteria in the spleen. Moxifloxacin and sitafloxacin exhibited combined effects on intrapulmonary bacterial elimination when administered to mice in combination with clarithromycin plus ethambutol. Sitafloxacin exerted the most marked combined effects in bacterial killing in the spleen. Levofloxacin displayed the lowest in vitro and in vivo activities amongst the tested quinolones. Taken together, these findings indicate that sitafloxacin and moxifloxacin exhibit favourable activities against M. avium in vitro and in vivo.