A comparative study on the activation of J-774 macrophage-like cells by gamma-interferon, 1,25-dihydroxyvitamin D3 and lipopeptide RP-56142: ability to kill intracellularly multiplying Mycobacterium tuberculosis and Mycobacterium avium.

The J-774 macrophage-like cell line has been established as a model for intracellular multiplication of pathogenic mycobacteria, permitting assessment of the intracellular bactericidal action of the macrophages after addition of both the drugs and immunomodulators. In this study, the action of immunomodulators was investigated. Significant morphological changes were demonstrated under the optical and scanning electron microscope (SEM), and the degree of macrophage activation was also measured by acid phosphatase (AcPase) cytochemistry, release of free oxygen radicals and by their ability to hinder the intracellular multiplication of virulent strains of Mycobacterium tuberculosis (M.tb) and Mycobacterium avium (M.av). For This purpose, the macrophages were left to multiply during 3 days in the presence of 50 U/ml of recombinant murine gamma-interferon (INF), 4 micrograms/ml of 1,25 dihydroxyvitamin D3 (D3) and 50 micrograms/ml of lipopeptide RP-56142 (RP) added separately or in various possible combinations, and these "activated" cells were then challenged with viable bacteria. Parallel controls included bacterial multiplication in nonactivated macrophages and also extracellularly but under the same experimental conditions as in the macrophage experiments. Transmission electron microscopy (TEM) using the AcPase marker to localize phagosome-lysosome fusion (PLF) in infected cells was also performed. Although all the immunomodulators used significantly changed the morphology of treated cells and increased the % of AcPase-positive cells, none had any effect on the release of oxygen radicals. On the other hand, guinea-pig alveolar macrophages which served as a parallel positive control, were activated by INF and D3 (but not RP) to release superoxide anions. Our data suggest that differential killing mechanisms for intracellular M.tb and M.av may exist. The results obtained also showed that established mycobactericidal mechanisms of the host could not solely account for the antimycobacterial effects observed. Consequently, mechanisms not yet revealed may account for some of the antimycobacterial effects observed.

Characterization of Mycobacterium paratuberculosis and "wood-pigeon" mycobacteria by isoenzyme profile and selective staining of immunoprecipitates.

Cell-free extracts of various strains belonging to Mycobacterium paratuberculosis (Ptb) and "wood-pigeon" (WP) mycobacteria were compared by polyacrylamide gel electrophoresis and the various protein bands obtained were tested for peroxidase enzyme activity. One strain of Mycobacterium avium served as a control. Bacterial extracts were also analysed by crossed immunoelectrophoresis (CRIEP) and fused rocket immunoelectrophoresis (FRIEP) using antisera raised in rabbit against M. paratuberculosis and WP mycobacteria. The immunoprecipitates obtained both in CRIEP and FRIEP plates were subsequently stained for selective peroxidase enzyme staining. Our results showed that, although Ptb and WP mycobacteria shared common peroxidase isoenzymes and antigens, they also had specific immunoprecipitates showing the differences between the two groups of bacteria.

Intracellular bactericidal activity of ciprofloxacin and ofloxacin against Mycobacterium tuberculosis H37Rv multiplying in the J-774 macrophage cell line.

Among new fluoroquinolone derivatives, the N-cyclopropylanalogs Ciprofloxacin (CIPRO) and ofloxacin (OFLO) have recently been found to have a high in vitro activity against a variety of mycobacteria (2). In this study, we have investigated their action against intracellularly growing Mycobacterium tuberculosis. For this purpose, the J-774 macrophage cell line was infected with the H37Rv type strain of M. tuberculosis, and drugs (at their serum level concentrations obtainable in healthy individuals (= 5 micrograms/ml; 8) were added after 2 days of intracellular growth of the bacteria. The bacterial growth during the experimentation was followed both electron microscopically and also by lysing the macrophages at various time intervals, and enumerating the bacterial viable counts after plating the lysate on appropriate media. Both drugs at the concentrations used did not affect macrophage viability (as assessed by trypan blue staining), and were highly bactericidal against the virulent tubercle bacilli multiplying in our J-774 macrophage model.

Ability of smooth and rough variants of Mycobacterium avium and M. intracellulare to multiply and survive intracellularly: role of C-mycosides.

A spontaneous rough (Rg) mutant of M. avium ATCC 15769 was mutagenized using N-methyl-N-nitro-nitrosoguanidine (MNNG). Out of 54 clones initially chosen on the basis of their morphological appearance on the 7H11 agar, seven Rg clones were selected on the basis of their response to current biochemical tests, and were later characterized for their cell wall amphiphilic contents (analysis of loosely-bound surface lipids for mycosides, peptidolipids, phospholipids, and mycolic acids by thin layer chromatography, and fatty acids by gas chromatography), and ability to grow intracellularly inside J-774 macrophages. A parallel study was also performed on a previously reported Rg mutant of M. intracellulare serovar 20 (W.W. Barrow and P.J. Brennan, J. Bact. 150 (1982) 381-384) which lacked the ability to synthesize mycosides C (MYC-). The results obtained were compared to parental smooth (Sm) colony-types of the respective M. avium-intracellulare strains. Our results showed that neither the ninhydrin-reacting lipids (probably peptidolipids) nor the glycopeptidolipids (mycosides C) were primary factors responsible for the intracellular survival and multiplication of these bacteria. Ultrastructural studies showed that although the polysaccharide-rich outer wall layer (POL) in case of MYC- Rg strain was not uniformly distributed and contained blebs, these bacteria formed the characteristic electron-transparent zone (ETZ) upon phagocytes by macrophages. Furthermore, the multiplication of MYC- Rg strain inside macrophages did not result in intracellular selection of MYC+ bacteria, nor in Rg to Sm transition.

Comparative intracellular growth of difficult-to-grow and other mycobacteria in a macrophage cell line.

We have recently developed a murine macrophage cell line (J-774) model which permits the growth of various mycobacteria (8). The purpose of the present investigation was to compare the intracellular growth of various difficult-to-grow mycobacteria (Mycobacterium paratuberculosis, M. ulcerans), and other pathogenic (M. tuberculosis H37Rv, M. kansasii, M. bovis) and nonpathogenic or avirulent (M. tuberculosis H37Ra, M. bovis BCG, M. gastri) mycobacteria. Electron microscopic studies were also performed to elucidate whether the formation of an electron-transparent zone (ETZ) around phagocytized bacilli was linked to their intramacrophagic survival. Furthermore, the comparison of intracellular growth of a pathogenic (M. kansasii) and nonpathogenic (M. gastri) mycobacteria sharing the same phenolic glycolipid antigen at their surface (Mycoside-A, 5), suggested that these antigens did not play a primary role in intracellular survival and multiplication of these bacteria. Also, we were unable to propagate M. ulcerans inside J-774 macrophages, which were massively lyzed after infection (due to a characteristic toxin secreted inside the macrophages?). These results are discussed in terms of the validity of the J-774 model for studying intracellular growth of mycobacteria.

Isoenzymes as tools to discriminate various subdivisions in the Mycobacterium fortuitum complex.

Methods for the characterization of catalase, peroxidase, beta-glucosidase, esterase, and beta-lactamase mycobacterial isoenzymes were described. These methods were applied to examine strains of the M. fortuitum complex. M. fortuitum, M. peregrinum, M. chelonae- M. abscessus and an unnamed species had distinct isoenzyme profiles. M. chelonae and M. abscessus could not be satisfactorily differentiated using the described methods.

Isoenzymes profiles in slowly-growing and difficult-to-grow mycobacteria.

The cell-free extracts of 13 slowly-growing mycobacteria were run on polyacrilamide gels (Page) and the various protein bands obtained were tested for peroxidase and catalase enzyme activities. The results obtained were compared to those obtained with M. fortuitum and M. chelonae. Based only on a limited numbers of strains employed, it is suggested that these isoenzyme patterns may permit a better separation of "Wood-pigeon" mycobacteria from both M. avium and M. paratuberculosis and also give distinct profiles for other species used. These results further suggest the potential of isoenzymes as taxonomic markers.