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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.

There has not yet been systematic studies which attempt to elucidate detailed profiles of the interaction between antimicrobial drugs and macrophage microbicidal mechanisms. We examined the effects of antisense oligo DNAs (AsDNAs) against oxyR and ahpC on the susceptibility of Mycobacterium avium complex (MAC) to the H2O2-halogenation system and combined antimycobacterial drugs [clarithromycin (CAM) + rifampicin (RFP)], both separately and in combination. It was found that AsDNA treatment of MAC did not affect the susceptibility of the organisms to any of the antimicrobial systems tested. Since the present AsDNAs did not efficiently reduce the expression of AhpC mRNA, attempts to increase bacterial uptake of AsDNAs are necessary to achieve significant increase in the drug susceptibility of MAC organisms due to AsDNA treatment.

The virulence of 5 strains of M. intracellulare and 6 strains of M. avium to mice were examined. The former bacteria were obtained from the patients with mycobacterial pulmonary disease and the latter were from AIDS patients respectively. C57BL/6 (NRAMP-1 susceptible) and its NRAMP-1 congenic mice (resistant) were used to evaluate the virulence of these bacteria. Three of the 5 strains of M. intracelllulare showed a relatively high virulence. They grew in the liver, spleen and lungs of susceptible mice and even in the lungs of resistant mice. On the other hand, none of 6 strains of M. avium could grow in either susceptible or resistant mice. In conclusion, our mouse model may be a useful tool for evaluating the virulence of bacteria isolated from mycobacteriosis patients.

Mycobacterium avium complex (MAC) is a typical intracellular parasite similar to M. tuberculosis and is one of the most important pathogens that coinfects AIDS patients. Attention has been focused on M. avium infection causing immunosuppression of hosts. Specific serotype-subspecies such as 1, -4 or -8 serotypes can be isolated frequently in humans infected with HIV. Furthermore, the prognosis after infection differs depending on the serotype. Serotype-4 in general shows unfavourable prognosis, while serotype-16 yields rapid recovery. Therefore, we have been interested in the immunomodifying activity of the surface glycopeptidolipid (GPL) antigen. However, no information has been available to date dealing on the virulent factor of MAC that is directly related with intracellular bactericidal activity. Recently, we have tried to test the effect of various GPLs purified from MAC on phagocytic processes of human peripheral blood monocytes (PBMC). We have used GPL-coated heat-killed staphylococcal cells to be phagocytosed by PBMC, and phagosome-lysosome fusion (P-L fusion) was estimated by the acridine orange staining of fused vesicles and bacteria. Results showed strong promotion of phagocytosis and marked inhibition of P-L fusion by serotype-4 GPL, while neither promotion of phagocytosis nor inhibition of P-L fusion in phagocytic cells were shown by serotype-16 GPL. Serotype-8 GPL showed concomitant stimulation of both phagocytosis and P-L fusion. These effects may be due to some unknown interaction between specific carbohydrate chain and organella membranes and serotype-4 GPL may be one of the possible virulent factors in MAC. Comparison with known possible virulent factors such as trehalose 6,6'-dimycolate (TDM), trehalose 6-monomycolate (TMM), glucose 6-monomycolate (GM) or sulfatide was also reported.

Mycobacterium avium complex (MAC) is one of the most important opportunistic pathogens co-infected with HIV (AIDS) and a typical intracellular parasitic bacteria similar to M. tuberculosis. It is also noticed that M. avium infection causes immunosuppression especially in the cellular immunity of host animals, and specific serotype-subspecies such as sero-2, -4 or -8 can be isolated frequently in human infection. Furthermore, the prognosis after infection differs by the serotypes and serotype-4 shows heavy infection in general, while serotype-16 shows rapid improvement. Therefore, we have been interested in the immunomodifying activity of surface glycopeptidolipid (GPL) antigen. However, to date, no information has been available on the virulence factor of MAC related directly with intracellular bactericidal activity. Recently, we have tested the effect of various GPLs purified form MAC complex on phagocytic processes of human peripheral blood monocytes (PBMC). We have used GPL-coated heat-killed staphylococcal cells to be phagocytosed by PBMC, and phagosome-lysosome (P-L) fusion was estimated by the acridine orange staining of fused vesicles including bacteria. It was revealed that the serotype-4, -12 and -17 GPLs showed strong phagocytosis promotion and marked inhibition of P-L fusion, while serotype-9, -13, -16 and -19 GPLs showed neither promotion of phagocytosis, nor inhibition of P-L fusion in phagocytic cells. Serotype-5, -7, -8 and -10 GPLs showed stimulation of both phagocytosis and P-L fusion, concomitantly. These effects may be due to unknown interaction between specific carbohydrate chain of MAC and phagocytic cell membranes, and serotype-4, -12 and -17 GPLs may be one of the possible virulence factors in MAC.

This paper reviews some recent studies which have been performed by us and other investigators, in order to clarify the reason why most mycobacterial infections such as due to Mycobacterium tuberculosis and M. avium complex infections are intractable, that is, why these organisms can escape from attack by microbicidal mechanisms of host macrophages and consequently persist for long time at sites of infection. This paper mainly dealt with the two major subjects, which were studied by using an experimental model for murine M. avium infection. The first subject is on the modes and mechanisms of mycobacterial killing in host macrophages and the mechanisms of bacterial escape from an onslaught by macrophages. The second is on the characteristics of immunosuppressive macrophages induced in M. avium complex infection and the role of the suppressor macrophages in the establishment of immune unresponsiveness of host mice in the progressed stage of infection.

The susceptibility of antituberculous agents against colonized M. intracellulare which were isolated from patients with pulmonary disease and infectious M. intracellulare (pathogens) isolated from atypical mycobacterial culture was investigated. Aminoglycosides were more potent against colonized organisms than against pathogens. Isoniazid, ethambutol and rifampicin showed less potent antimicrobial activity against both colonized organism and pathogens as compared to aminoglycosides. On the contrary susceptibility of cycloserine against colonized organisms was as potent as against pathogens.

The two types of colonial variants of Mycobacterium avium complex, SmT (smooth, transparent, irregular) and SmD (smooth, opaque, dome-shaped) variants, were examined for their triggering activity for macrophage (M phi) respiratory burst, based on chemiluminescence (CL). SmD variants elicited an intense CL from zymosan A-induced M phi s in a dose-dependent manner, although SmT variants induced much lower M phi CL. The M phi s could steadily phagocytose SmT variants, although the phagocytizing rate was considerably lower compared to the case of SmD variants. Treatments of SmD variants with Tween 80, pronase and some endoglycosidases such as alpha-amylase, cellulase, dextranase and pectinase, heating (100 degrees C, 15 min), and delipidation by CHCl3-methanol extraction resulted in a marked reduction in the M phi CL-triggering activity of the SmD variants. Thus, the M phi CL-triggering ligand(s) seems to possess glycolipoprotein-like moieties. Tween 80-treatment of SmT variants, which is known to deprive the polysaccharide outer layer specific to the colonial variants, failed to recover the M phi CL-triggering activity of SmT variants. Therefore, the remarkably reduced M phi CL-triggering ability of the SmT variants may be caused by the extremely lowered expression of the M phi CL-triggering ligands rather than by masking of the CL-triggering ligands by SmT-specific outer layer.