Apoptosis genes in human alveolar macrophages infected with virulent or attenuated Mycobacterium tuberculosis: a pivotal role for tumor necrosis factor

Tumor necrosis factor (TNF)-alpha-dependent apoptosis of alveolar macrophages (AM) after infection with avirulent Mycobacterium tuberculosis (Mtb) results in bacillary death and the destruction of a growth niche for the pathogen. This response is minimized after infection with virulent strains of Mtb. To study the genetic control of Mtb-induced apoptosis, we used microarrays to interrogate the expression profile of infected human AM. Although we found variation in gene expression between different donors of AM, a set of genes were constant for each condition. A group of proapoptotic genes were downregulated after infection by virulent Mtb strain H37Rv, whereas infection with avirulent Mtb H37Ra led to a gene expression profile that would favor macrophage apoptosis. Neutralizing TNF in macrophage cultures infected with H37Ra changed the gene expression profile to one that resembled the profile of macrophages infected with H37Rv. These data reveal that apoptosis-related genes are regulated differently by virulent or attenuated Mtb strains, and are consistent with the hypothesis that virulent Mtb interfere with TNF death signaling. Given the importance of TNF in host defense against tuberculosis, the ability to repress the expression of genes activated by TNF may constitute a bacillary virulence mechanism.     

Effect of mycobacteria on sensitivity to the cytotoxic effects of tumor necrosis factor

Unlike Mycobacterium leprae, Mycobacterium tuberculosis is not found inside cells other than macrophages and polymorphonuclear cells in vivo, yet previous work has revealed that in vitro it readily enters all cell lines tested. Moreover, these cells are not killed by the intracellular mycobacteria. We report here that when fibroblasts take up live (but not killed) M. tuberculosis H37Rv, they develop greatly increased sensitivity to the toxic effects of tumor necrosis factor (TNF) whether the cell line is inherently sensitive to TNF or not. Ultrasonically disrupted M. tuberculosis also has this property. The increased sensitivity is seen in the absence of metabolic inhibitors, although addition of emetine, an inhibitor of protein synthesis, causes the effect to manifest itself earlier and at a lower concentration of TNF. In contrast, infection with Mycobacterium bovis bacillus Calmette-Guérin induces little or no increased sensitivity to TNF, whereas Mycobacterium avium and M. tuberculosis H37Ra have intermediate sensitivities. We discuss the possibility that virulent tuberculosis strains produce a factor which distorts the normal protective function of TNF, rendering it toxic to host tissues and leading to the classical immunopathology of tuberculous lesions.     

Expression of Virulence of Mycobacterium tuberculosis within Human Monocytes: Virulence Correlates with Intracellular Growth and Induction of Tumor Necrosis Factor Alpha but Not with Evasion of Lymphocyte-Dependent Monocyte Effector Functions

We assessed the applicability of an in vitro model of low-level infection of human monocytes to the characterization of the virulence of strains of the Mycobacterium tuberculosis family. Peripheral blood monocytes were infected at a 1:1 ratio with the virulent M. tuberculosis strain H37Rv, the avirulent M. tuberculosis strain H37Ra, and the attenuated M. bovis strain BCG. Both the percentages of cells infected by the three strains and the initial numbers of intracellular organisms were equivalent, as were levels of monocyte viability up to 7 days following infection. Intracellular growth reflected virulence, as H37Rv replicated in logarithmic fashion throughout the assay, BCG growth reached a plateau at 4 days, and H37Ra did not grow at all. The same patterns of growth were observed following infection of human alveolar macrophages with H37Rv and H37Ra. Monocyte production of tumor necrosis factor alpha was significantly higher following infection with virulent H37Rv than with either BCG or H37Ra. In contrast, there was no clear correlation of interleukin 10 production with virulence. Nonadherent cells of purified-protein-derivative-positive donors mediated equivalent degrees of reduction of the intracellular growth of H37Rv, BCG, and H37Ra. Low-level infection of human monocytes with H37Rv, BCG, and H37Ra thus provides an in vitro model for assessment of the virulence of these M. tuberculosis family strains. Furthermore, it is suggested that the virulence of these strains is expressed primarily by their differing abilities to adapt to the intracellular environment of the mononuclear phagocyte.     

Mycobacterium tuberculosis H37Ra and H37Rv differential growth and cytokine/chemokine induction in murine macrophages in vitro.

The role of tumor necrosis factor-alpha (TNF-alpha) in controlling growth of Mycobacterium tuberculosis in murine peritoneal macrophages infected in vitro was studied. TNF-alpha was shown to be required but not sufficient, and the amount of TNF-alpha produced by the infected cells did not correlate with the extent of growth control. In this system, TNF-alpha-dependent control of growth of the avirulent strain H37Ra was independent of inducible nitric oxide synthase (iNOS) and interferon-gamma (IFN-gamma), as shown by the infection of macrophages from selected gene-disrupted mice. TNF-alpha-mediated bacteriostasis of H37Ra in the infected macrophages was associated with increased expression of selected Th1-type cytokines and chemokines. In contrast, growth of the virulent strain H37Rv in macrophages involved upregulation by infected cells of Th2-type cytokines, including interleukin-5 (IL-5), IL-10, and IL-13. Taken together, these results suggest that the particular nature of macrophage activation and the cytokine and chemokine response to infection with different M. tuberculosis strains determine the ability of the cells to control the growth of the intracellular bacilli.     

Macrophage apoptosis in mycobacterial infections

Mycobacterial diseases are a major public health concern. In the case of tuberculosis, the problem has been acerbated due to the emergence of drug-resistant strains of Mycobacterium tuberculosis, and Mycobacterium avium is the major opportunistic pathogen in HIV-1 infection in the United States. M. tuberculosis and M. avium replicate in human macrophages and induce apoptosis. Incubation of freshly added uninfected autologous macrophages with apoptotic M. avium-infected macrophages results in 90% inhibition of bacterial growth. Apoptosis also prevents the release of intracellular components and the spread of mycobacterial infection by sequestering the pathogens within apoptotic bodies. Consistent with the model that host cell apoptosis is a defense mechanism against mycobacteria is the finding that the virulent M. tuberculosis strain H37Rv induces substantially less macrophage apoptosis than the attenuated strain H37Ra. Evasion of apoptosis by this pathogen is achieved by enhanced release of sTNFR2 by H37Rv-infected macrophages and subsequent formation of inactive TNF-alpha-TNFR2 complexes. These observations contribute to the hypothesis that apoptosis of the host macrophage is an important defense mechanism in mycobacterial infections, which prevents the spread of the infection.     

Effect of lipoarabinomannan and mycobacteria on tumour necrosis factor production by different populations of murine macrophages.

Tumour necrosis factor (TNF) production is an important pathological mediator in mycobacterial infections, and yet little is known of the factors which influence its production. We have studied the influence of murine macrophage heterogeneity and activation state on TNF production following mycobacterial stimulation in vitro. Lipoarabinomannan (LAM) from strains of Mycobacterium tuberculosis and Myco. avium differentially stimulated TNF production in thioglycollate-elicited macrophages in a dose-dependent manner. In comparison, resident peritoneal macrophages produced much less TNF when stimulated with LAM, dead mycobacteria or lipopolysaccharide (LPS). In contrast, zymosan stimulated resident macrophages to a higher degree than thioglycollate-elicited cells. Another comparison between bone marrow and thioglycollate-elicited macrophages showed that both responded to LPS, but only the latter was stimulated significantly by H37Rv LAM. This may indicate that LAM stimulation of macrophages takes place through a different pathway than both zymosan- and LPS-stimulated TNF production. Also, in vitro activation of peritoneal macrophages with interferon-gamma (IFN-gamma), increased TNF response to several stimuli. Our studies indicate that the pathology of mycobacterial infections through TNF production may be influenced by the type and activation state of the macrophage which responds to that infection.     

Tumor necrosis factor and granulocyte macrophage-colony stimulating factor stimulate human macrophages to restrict growth of virulent Mycobacterium avium and to kill avirulent M. avium: killing effector mechanism depends on the generation of reactive nitrogen intermediates

An avirulent and a virulent strain of Mycobacterium avium were selected on the basis of their growth patterns in human monocyte-derived macrophages. The virulent 7497 M. avium grew progressively in untreated macrophages, whereas the avirulent LR/149 M. avium was killed to a moderate extent by untreated human macrophages (50% of the original infectious inoculum killed 7 days after infection). We set out to investigate the possibility of modulating these growth patterns by cytokine treatment. Application of tumor necrosis factor (TNF) (100 U/ml) led to macrophages restricting significantly the growth of virulent M. avium 7497 (tenfold decrease at 7 days). TNF was also effective at modulating positively the interaction between avirulent LR/149 M. avium and macrophages inasmuch as TNF-treated cells killed 99% of infecting mycobacteria at 7 days. Granulocyte macrophage-colony stimulating factor (GM-CSF) (100-10,000 U/ml) treatment led to macrophages being as mycobacteriostatic for virulent 7497 M. avium as TNF-alpha-treated cells (i.e., tenfold reduction in growth). Treatment of macrophages with both GM-CSF and TNF-alpha was shown to have additive effects on bacteriostatic activity on M. avium. The mechanism of killing of avirulent M. avium by TNF-alpha was shown to be dependent on the generation of reactive nitrogen intermediates, as seen by inhibition of effector mechanisms by NG-monomethyl-arginine and arginase. Moreover, there was a correlation between NO2- generation and mycobactericidal activity of macrophages. Addition of superoxide dismutase reversed the killing of avirulent M. avium by untreated or TNF-treated macrophages. This abrogation was also apparent in chronic granulomatous disease (CGD) macrophages, which were inefficient at generating reactive oxygen intermediates. Moreover, macrophages from CGD patients killed avirulent M. avium as efficiently as cells from normal individuals. We conclude from these results that 1) GM-CSF and TNF-alpha, alone or in combination, increase effector functions of macrophages against virulent and avirulent strains of M. avium; 2) reactive nitrogen intermediates seem to be involved in this effector mechanism; and 3) superoxide dismutase protected M. avium against macrophage effector function, seemingly by protecting the bacteria against endogenous superoxide anion. The implications of these findings for host resistance to atypical mycobacteria are discussed.     

Effect of Mycobacterium bovis BCG vaccination on interleukin-1 beta and RANTES mRNA expression in guinea pig cells exposed to attenuated and virulent mycobacteria

The effect of Mycobacterium bovis BCG vaccination on interleukin-1 beta (IL-1 beta) or regulated-upon-activation, normally T-cell-expressed and -secreted chemokine (RANTES) mRNA expression in guinea pig spleen cells stimulated with concanavalin A, lipopolysaccharide (LPS), phorbol myristate acetate (PMA) plus ionomycin, or purified protein derivative (PPD) was studied in vitro. Similarly, peritoneal exudate cell-derived macrophages from na?ve and BCG-vaccinated guinea pigs were infected with M. bovis BCG, Mycobacterium avium, the attenuated Mycobacterium tuberculosis H37Ra strain, or virulent strains H37Rv and Erdman of M. tuberculosis. Total RNA was subjected to Northern blot analysis using probes generated from guinea pig IL-1 beta or RANTES cDNA. Although IL-1 beta and RANTES mRNA could be detected in the spleen cells from na?ve animals stimulated with LPS or PMA plus ionomycin, the levels were significantly enhanced after BCG vaccination. mRNA expression was also elevated in macrophages infected with live mycobacteria after BCG vaccination. However, macrophages infected with the virulent H37Rv strain of M. tuberculosis showed 75 to 90% reductions in IL-1 beta expression and 25 to 60% reductions in RANTES mRNA expression compared with macrophages infected with the attenuated H37Ra strain. The IL-1 beta mRNA levels peaked as soon as 1 h after PPD stimulation and 4 h after M. tuberculosis H37Rv infection of macrophages. In contrast, RANTES mRNA expression was delayed until 48 h after infection. These results indicate that molecular mediators produced in response to various stimuli associated with protective immunity against mycobacteria are upregulated after BCG vaccination; however, a significantly weaker response was observed with virulent M. tuberculosis. These initial studies indicate that BCG vaccination has a positive effect on IL-1 beta and RANTES mRNA expression by host cells in a highly relevant animal tuberculosis model.     

Pathogenesis of tuberculosis: interaction of Mycobacterium tuberculosis with macrophages.

Central to understanding the pathogenesis of tuberculosis is the interaction between the pathogen and mononuclear phagocytes. A key question about that interaction is whether Mycobacterium tuberculosis exerts an effect on phagolysosome fusion. We have reexamined the dynamics of phagolysosome fusion and its effect on intracellular bacterial replication in M. tuberculosis-infected macrophages by performing an extensive study at the electron microscopic level. Thoria-labelled murine and human macrophages were infected with a virulent (H37Rv) or avirulent (H37Ra) strain of M. tuberculosis or with Mycobacterium bovis BCG vaccine for times ranging from 2 h to 7 days. In all cases, by 2 h postinfection, approximately 85% of the bacteria clearly resided in fused vacuoles. However, at 4 days postinfection, fusion levels for viable H37Rv and H37Ra were reduced by half, whereas the fusion profiles of BCG and of heat-killed H37Rv and H37Ra were unchanged. A comparison of the numbers of bacteria per fused and nonfused vacuoles suggests both a net transfer of bacteria out of fused vacuoles and preferential bacterial multiplication in nonfused vacuoles. H37Rv and H37Ra appeared to bud from the phagolysosomes into tightly apposed membrane vesicles that did not fuse with secondary lysosomes. In some cases, no such membrane was seen and the bacteria appeared to be free in the cytoplasm. Only viable H37Rv showed a significant increase in bacterial counts during the course of infection. Thus, both of the attenuated strains we examined differed from the virulent strain H37Rv in their abilities to replicate successfully within macrophages, but each diverged from H37Rv at a different point in the process. Viable tubercle bacilli H37Rv and H37Ra had the capacity to escape from fused vesicles as the infection progressed; BCG did not. After extrusion from the phagolysosome, H37Rv, but not H37Ra, was able to multiply. These results suggest a novel mechanism by which virulent M. tuberculosis eludes the microbicidal mechanisms of macrophages by escaping from fused phagolysosomes into nonfused vesicles or the cytoplasm.     

Mannosylated lipoarabinomannan balances apoptosis and inflammatory state in mycobacteria-infected and uninfected bystander macrophages

To assess the role of mannosylated lipoarabinomannan (ManLAM) in the inflammatory and apoptotic response of mycobacteria-infected and uninfected, bystander cells we applied a mouse macrophage model of infection with avirulent strains--Mycobacterium bovis BCG, Mycobacterium tuberculosis (MTB) H37Ra and compared with a virulent MTB H37Rv strain infection. ManLAM contributed to the infection of macrophages by protection from apoptosis with stabilized Bcl-2 expression and down-regulated Bax expression for infected cells (BCG) or with stabilized Bcl-2 expression for uninfected bystander target cells (H37Ra). Additionally, ManLAM up-regulated FasL expression on the infected cells. Active extracellular signal-regulated kinase (ERK1/2) in BCG and H37Rv infection provided an anti-apoptotic effect by stabilization of anti-apoptotic Bcl-2 expression in the infected cells. Inhibitors specific for c-Jun-NH2-terminal kinase or stress-activated kinase (JNK) and p38 kinase decreased apoptosis of infected cells (BCG, H37Ra) and of uninfected bystanders (H37Ra) by down-regulating Bax. ManLAM significantly down-regulated production of pro-inflammatory IL-12 and TNF-alpha and activation of JNK by both avirulent strains. We conclude that by stabilization of Bcl-2 expression, down-regulation of JNK activity and down-regulation of pro-inflammatory cytokines production ManLAM can contribute to suppression of apoptosis and inflammatory reaction of uninfected, bystander cells.     

Role of gamma interferon and tumor necrosis factor alpha during T-cell-independent and -dependent phases of Mycobacterium avium infection.

To design an effective immunotherapy for Mycobacterium avium infections, the protective host response to the infection must be known. Here we analyzed the role of gamma interferon (IFN-gamma) and tumor necrosis factor alpha (TNF-alpha) in the innate and acquired responses to M. avium infections in mice. T-cell depletion studies showed that CD4+ T cells were required for control of the infection. CD(4+)-depleted mice showed enhanced bacterial proliferation and at the same time showed a reduction in the level of expression of both IFN-gamma and TNF-alpha mRNAs in spleen cells. In contrast, M. bovis BCG immunization restricted M. avium proliferation and at the same time promoted expression of the mRNAs for the two cytokines. In vivo depletion studies using specific monoclonal antibodies showed that both IFN-gamma and TNF-alpha are involved in an early protection possibly involving NK cells, and furthermore, IFN-gamma is involved in the later T-cell-protective response to infection. In vivo neutralization of IFN-gamma during M. avium infection also blocked the priming for enhanced TNF-alpha secretion triggered by endotoxin. Both cytokines were found to be involved in the resistance expressed in BCG-immunized animals and exhibited additive bacteriostatic effects in vitro on bone marrow-derived macrophages infected with different strains of M. avium. These data suggest that both cytokines act in an additive or synergistic fashion in the induction of bacteriostasis and that IFN-gamma is also involved in priming TNF-alpha secretion.     

Use of in vivo complementation in Mycobacterium tuberculosis to identify a genomic fragment associated with virulence.

Novel molecular tools and genetic methods were developed to isolate genomic fragments of Mycobacterium tuberculosis that may be associated with virulence. We sought to restore virulence, a characteristic of M. tuberculosis that is correlated with growth rate in mouse spleen and lung tissue, to the avirulent strain H37Ra by complementation. A representative library of the virulent M. tuberculosis strain H37Rv was constructed and transformed into H37Ra. Enrichment for individual faster-growing recombinants was achieved by passage of pools of H37Ra transformants harboring the H37Rv library through mice. A molecular strategy was devised to isolate and clone the H37Rv genomic DNA fragment ivg, which conferred a more rapid in vivo growth rate to H37Ra.     

Strain Virulence and the Lysosomal Response in Macrophages Infected with Mycobacterium tuberculosis

Strains H37Ra and H37Rv, attenuated and virulent variants, respectively, of the original human strain H37 of Mycobacterium tuberculosis, were used to infect cultures of mouse peritoneal macrophages. Bacterial viability of each strain was assessed over a 2-week period, and the cellular response to H37Ra during the first week was observed using electron microscopy. Prelabeling of secondary lysosomes with ferritin was used to facilitate the estimation of fusion of the lysosomes with phagosomes containing the bacteria. Streptomycin was excluded from the medium of cell cultures infected with H37Ra. The intracellular viability of strain H37Rv (in the presence of streptomycin) showed a lag during the first week after infection and then rose progressively to a mean figure seven times the starting level. In contrast, the viability of strain H37Ra declined, on the average, to one-fifth of the starting level during the first week; moreover, this decline occurred in the absence of antibiotics. In the second week a variable rise in the viable count took place, usually regaining the starting level. Electron microscopy of macrophages infected with H37Ra revealed a higher proportion of "damaged" bacteria 5 days after infection than at 1 day, in keeping with the decline in viability. Phagosomes containing these "damaged" (and presumed dead) organisms showed virtually universal fusion with prelabeled lysosomes. Phagosomes containing "intact" bacteria of this strain showed a prevalence of fusion varying from 38 to 56%, somewhat higher than the level previously reported for "intact" organisms of H37Rv. Nevertheless, the lysosome-phagosome fusion response to "intact" H37Ra was still far less extensive than that observed previously towards "intact" M. lepraemurium (around 90%). In conclusion, a difference between the macrophage lysosome-phagosome fusion response towards viable organisms of strain H37Ra and to the virulent strain H37Rv was observed, but was not pronounced, and the present findings are in keeping with the increasingly held view that H37Ra should be regarded as a low-virulence or attenuated strain rather than truly avirulent.     

Virulent Mycobacterium fortuitum restricts NO production by a gamma interferon-activated J774 cell line and phagosome-lysosome fusion

The virulence of different isolates of Mycobacterium has been associated with two morphologically distinguishable colonial variants: opaque (SmOp) and transparent (SmTr). In this report we used an in vitro assay to compare macrophage (Mphi) responses to SmOp and SmTr Mycobacterium fortuitum variants, taking advantage of the fact that these variants were derived from the same isolate. Cells preactivated or not with gamma interferon (IFN-gamma) were infected with SmOp or SmTr M. fortuitum. We showed that SmOp and SmTr induced different levels of nitric oxide (NO) production by IFN-gamma-stimulated Mphi. Indeed, the amount of IFN-gamma-induced NO production by J774 cells was 4.8 to 9.0 times higher by SmOp (23.1 to 37.7 micro M) compared to SmTr infection (3.9 to 4.8 micro M) (P = 0.0332), indicating that virulent SmTr bacilli restricted NO production. In addition, IFN-gamma-induced NO production by Mphi was higher when correlated with reduction of only avirulent SmOp bacillus viability. SNAP (S-nitroso-N-acetyl-DL-penicillamine)-induced NO production did not modify SmTr viability, indicating its resistance to nitrogen radicals. Electron microscopy studies were performed to evaluate the capacity of phagosomes to fuse with lysosomes labeled with bovine serum albumin-colloidal gold particles. By 24 h postinfection, 69% more phagosome-containing SmOp variant had fused with lysosomes compared to the SmTr-induced phagosomes. In conclusion, these data indicate that virulent SmTr bacilli may escape host defense by restricting IFN-gamma-induced NO production, resisting nitrogen toxic radicals, and limiting phagosome fusion with lysosomes.     

THP-1 cell apoptosis in response to Mycobacterial infection

We previously reported that Mycobacterium tuberculosis infection primes human alveolar macrophages (HAM) for tumor necrosis factor alpha (TNF-alpha)-mediated apoptosis and that macrophage apoptosis is associated with killing internalized bacilli. Virulent mycobacterial strains elicit much less apoptosis than attenuated strains, implying that apoptosis is a defense against intracellular infection. The present study evaluated the potential for phorbol myristate acetate-differentiated THP-1 cells to mimic this response of primary macrophages. Consistent with the behavior of alveolar macrophages, attenuated M. tuberculosis H37Ra and Mycobacterium bovis BCG strongly induce THP-1 apoptosis, which requires endogenous TNF. THP-1 apoptosis is associated with reduced viability of infecting BCG. In contrast, virulent wild-type M. tuberculosis H37Rv and M. bovis do not increase THP-1 apoptosis over baseline. BCG induced early activation of caspase 10 and 9, followed by caspase 3. In contrast, wild-type M. bovis infection failed to activate any caspases in THP-1 cells. BCG-induced THP-1 apoptosis is blocked by retroviral transduction with vectors expressing crmA but not bcl-2. We conclude that differentiated THP-1 cells faithfully model the apoptosis response of HAM. Analysis of the THP-1 cell response to infection with virulent mycobacteria suggests that TNF death signals are blocked proximal to initiator caspase activation, at the level of TNF receptor 1 or its associated intracytoplasmic adaptor complex. Interference with TNF death signaling may be a virulence mechanism that allows M. tuberculosis to circumvent innate defenses leading to apoptosis of infected host cells.     

Relationship between virulence of Mycobacterium avium strains and induction of tumor necrosis factor alpha production in infected mice and in in vitro-cultured mouse macrophages.

We studied the ability of two Mycobacterium avium strains with different virulences to induce tumor necrosis factor alpha (TNF) synthesis by mouse resident peritoneal macrophages (RPM phi) in vitro in an experiment to look for a possible correlation between virulence and this TNF-inducing capacity. The low-virulence strain, 1983, induced significantly higher production of TNF by RPM phi than did the high-virulence strain, ATCC 25291. TNF neutralization during culture of infected RPM phi resulted in enhancement of growth of strain 1983 and had no effect on growth of strain ATCC 25291; TNF treatment of strain ATCC 25291-infected macrophages had no effect on mycobacterial growth. The extent of M. avium growth and the amount of TNF synthesis were independent of the presence of contaminating T cells or NK cells in the macrophage monolayers. Intraperitoneal administration of anti-TNF monoclonal antibodies to BALB/c mice infected intravenously with M. avium 1983 abrogated the elimination of the bacteria in the liver and caused a slight increase in bacterial growth in the spleen. Neutralization of TNF led to a minor increase in the proliferation of M. avium ATCC 25291 in the liver and spleen of BALB/c mice late in infection. Anti-TNF treatment did not affect the growth of the two M. avium strains in BALB/c.Bcgr (C.D2) mice, suggesting that restriction of M. avium strains to induce TNF production by macrophages may limit their ability to proliferate both in vitro and in vivo.     

Structural basis of capacity of lipoarabinomannan to induce secretion of tumor necrosis factor.

The results of this study show that lipoarabinomannans (LAM) isolated from a virulent strain and from an avirulent strain of Mycobacterium tuberculosis, which have recently been shown to differ markedly in terms of the structures of their nonreducing termini, also differ markedly in the capacity to induce the secretion of tumor necrosis factor from murine macrophages. It was found that LAM from the avirulent H37Ra strain was 100-fold more potent at inducing tumor necrosis factor secretion than LAM from the virulent Erdman strain, thus leading us to hypothesize that the structure of LAM from a given mycobacterial isolate may directly influence its ability to elicit, or avoid, cytokine-mediated mechanisms of host resistance.     

Differential Growth Characteristics and Streptomycin Susceptibility of Virulent and Avirulent Mycobacterium tuberculosis Strains in a Novel Fibroblast-Mycobacterium Microcolony Assay

The ability to spread from cell to cell may be an important virulence determinant of Mycobacterium tuberculosis. An in vitro assay was developed to characterize this ability among four strains of M. tuberculosis: the attenuated strain H37Ra, the virulent strains H37Rv and Erdman, and a virulent clinical isolate (Stew). Confluent monolayers of human skin fibroblasts were infected with these strains and overlaid with agar-medium. M. tuberculosis infection developed over 21 days as microcolonies originating within the plane of the fibroblasts. Microcolonies of the virulent strains had an elongated appearance and exhibited extensive cording. The cords appeared to invade adjacent cells within the plane of the monolayer. Microcolony diameter of the Erdman strain was significantly larger than that of the other virulent strains, indicating that virulent strains can have distinguishing phenotypes in this assay. In contrast, avirulent H37Ra microcolonies were rounded and noncorded. H37Ra microcolonies were significantly smaller than those of the virulent strains. Microcolony diameter of the virulent strains was not reduced by the extracellularly acting antibiotic streptomycin at concentrations of up to 5.0 μg/ml. In contrast, H37Ra microcolony size was reduced at concentrations as low as 0.5 μg/ml. Growth of all strains was similarly inhibited by 1.0 μg of streptomycin per ml in fibroblast-conditioned tissue culture medium alone. When fibroblasts were infected with the M. tuberculosis strains without an agar overlay, with and without streptomycin, numbers of CFU mirrored the changes observed in the microcolony assay. There was a statistically significant decrease in H37Ra CFU compared to virulent strains after treatment with streptomycin. These differences between H37Ra and virulent strains in human fibroblasts suggest that H37Ra may be lacking a virulence determinant involved in cell-to-cell spread of M. tuberculosis.     

Interleukin-1 or tumor necrosis factor-alpha augmented the cytotoxic effect of mycobacteria on human fibroblasts: application to evaluation of pathogenesis of clinical isolates of Mycobacterium tuberculosis and M. avium complex.

Mycobacteria-induced in vitro events reflecting human tuberculosis can contribute to the evaluation of the pathogenesis of Mycobacterium tuberculosis (MTB). In this study, we propose such an in vitro method based on live mycobacteria-induced cytotoxicity to human cell lines. When human lung-derived normal fibroblast cell line MRC-5 was infected with various strains of mycobacteria (M. tuberculosis H(37)Rv and H(37) Ra, Mycobacterium avium 427S and 2151SmO, and Mycobacterium bovis BCG Pasteur and Tokyo), the fibroblasts were killed by mycobacteria according to the degree of virulence. Other human originated macrophage (U-937, THP-1), myeloid (HL-60), and epithelial carcinoma (A549) cell lines exhibited a similar cytotoxic response to virulent mycobacteria. MRC-5 was most susceptible to virulent mycobacteria among various human cell lines examined. The cytotoxicity was enhanced by the proinflammatory cytokines, interleukin-1 (IL-1) and tumor necrosis factor-a (TNF-alpha), which in the absence of mycobacteria stimulate the growth of normal human fibroblasts. This in vitro evaluation system was applied to clinical isolates of drug-sensitive MTB (DS-MTB), drug-resistant MTB (DR-MTB) including multidrug-resistant (MDR-MTB), and M. avium complex (MAC). MTB strains (n = 24) exhibited strong cytotoxic activity, but MAC strains (n = 5) had only weak activity. Furthermore, there was no significant difference in cytotoxicity between DS-MTB (n = 11) and DR-MTB (n = 13). Collectively, these results suggest that this new in vitro system is useful for evaluating the pathogenesis of mycobacteria and that there was no difference in the pathogenesis between drug-susceptible and drug-resistant clinical isolates.     

Temporal effect of tumor necrosis factor alpha on murine macrophages infected with Mycobacterium avium.

Members of the Mycobacterium avium complex are a family of bacteria that persist within macrophages in the face of an immune response. Elimination of these organisms is likely due to cytokine-induced macrophage activation. Because macrophage activation by tumor necrosis factor alpha (TNF-alpha) appears critical for killing of intracellular M. avium, early downregulation of TNF-alpha levels in infected macrophages has been suggested as a survival mechanism for virulent strains of M. avium. We examined the relationship between TNF-alpha and growth of M. avium strains of differing virulence, as measured by their ability to grow in murine bone marrow-derived macrophages. When exogenous TNF-alpha was added immediately following macrophage infection, significant growth inhibition of virulent M. avium strains was observed. If TNF-alpha addition was delayed by 24 h or more, growth inhibition was abrogated. To determine if early downregulation of TNF-alpha levels could explain the differential growth of virulent and avirulent strains, levels of TNF-alpha and prostaglandin E2 (PGE2), which has been shown to suppress TNF-alpha production in uninfected macrophages, were quantified over time. Upregulation of both TNF-alpha and PGE2, as measured by enzyme-linked immunosorbent assay, was evident by 6 h postinfection, indicating that the ability of M. avium to replicate in macrophages was not directly correlated with early downregulation of TNF-alpha production. However, TNF-alpha bioactivity, as measured by cytotoxicity, was significantly decreased in virulent M. avium strains at all time periods examined. Treatment of infected macrophages with gamma interferon immediately after infection resulted in significantly increased levels of nitric oxide but did not affect the growth of virulent M. avium strains. These results suggest that while significant levels of TNF-alpha are present in supernatants from all M. avium strains, levels of biologically active TNF-alpha are significantly reduced in supernatants from virulent M. avium strains. Preliminary results suggest that upregulation of the soluble p75 TNF receptor may be one mechanism by which TNF-alpha bioactivity reduction occurs.