Culture at high density improves the ability of human macrophages to control mycobacterial growth

The mechanisms through which granuloma formation helps control mycobacterial infection are poorly understood, but it is possible that the accumulation of macrophages at high density at sites of infection promotes the differentiation of macrophages into cells with improved mycobactericidal activity. To test this possibility, varying numbers of monocytes were cultured in 96-well plates for 3 days, infected with Mycobacterium bovis bacillus Calmette-Guérin, and mycobacterial number was assessed 7 days after infection based on the measurement of luciferase activity expressed by a mycobacterial reporter strain or by counting CFU. Mycobacterial growth was optimal in cultures containing 5 x 10(4) cells/well, but increasing the number of cells to 2 x 10(5) cells/well resulted in complete inhibition of mycobacterial growth. This effect could not be explained by differences in mycobacterial uptake, multiplicity of infection, acidification of the extracellular medium in high density cultures, enhanced NO production, or paracrine stimulation resulting from secretion of cytokines or other proteins. The morphology of cells cultured at high density was strikingly different from that of monocytes cultured at 5 x 10(4) cells/well, including the appearance of numerous giant cells. The bacteriostatic activity of monocyte-derived macrophages was also dependent on cell number, but fewer of these more mature cells were required to control mycobacterial growth. Thus, the ability of human macrophages to control mycobacterial infection in vitro is influenced by the density of cells present, findings that may help explain why the formation of granulomas in vivo appears to be a key event in the control of mycobacterial infections.     

An in vitro dual model of mycobacterial granulomas to investigate the molecular interactions between mycobacteria and human host cells

In the majority of individuals infected with Mycobacterium tuberculosis, the bacilli cause a long-term asymptomatic infection called latent tuberculosis, a state during which the bacilli reside within granulomas. Latently infected individuals have around 10% risk of progression to clinical disease at a later stage. Determining the state of the mycobacteria and the host cells during this latent phase, i.e. within the granulomas, would greatly improve our understanding of the physiopathology of tuberculosis, and thus enable the development of new therapeutic means to treat the one-third of the world's population who are latently infected. We have developed an in vitro model of human mycobacterial granulomas, enabling the cellular and molecular analysis of the very first steps in the host granulomatous response to either mycobacterial compounds or live mycobacterial species. In vitro mycobacterial granulomas mimic natural granulomas very well, with the progressive recruitment of macrophages around live bacilli or mycobacterial antigen-coated beads, their differentiation into multinucleated giant cells and epithelioid cells, and the final recruitment of a ring of activated lymphocytes. Besides morphological similarities, in vitro granulomas also functionally resemble natural ones, with the development of intense cellular co-operation and intracellular mycobactericidal activities.     

Enhanced protection against fatal mycobacterial infection in SCID beige mice by reshaping innate immunity with IFN-gamma transgene.

Humans with immune-compromised conditions such as SCID are unable to control infection caused by normally nonpathogenic intracellular pathogens such as Mycobacterium bovis bacillus Calmette-Guérin. We found that SCID beige mice lacking both lymphocytes and NK cells had functionally normal lung macrophages and yet a selectively impaired response of type 1 cytokines IFN-gamma and IL-12, but not TNF-alpha, during M. bovis bacillus Calmette-Guérin infection. These mice succumbed to such infection. A repeated lung gene transfer strategy was designed to reconstitute IFN-gamma in the lung, which allowed investigation of whether adequate activation of innate macrophages could enhance host defense in the complete absence of lymphocytes. IFN-gamma transgene-based treatment was initiated 10 days after the establishment of mycobacterial infection and led to increased levels of both IFN-gamma and IL-12, but not TNF-alpha, in the lung. Lung macrophages were activated to express increased MHC molecules, type 1 cytokines and NO, and increased phagocytic and mycobactericidal activities. Activation of innate immunity markedly inhibited otherwise uncontrollable growth of mycobacteria and prolonged the survival of infected SCID hosts. Thus, our study proposes a cytokine transgene-based therapeutic modality to enhance host defense in immune-compromised hosts against intracellular bacterial infection, and suggests a central effector activity played by IFN-gamma-activated macrophages in antimycobacterial cell-mediated immunity.     

Effect of proton pump inhibitor alone or in combination with clarithromycin on mycobacterial growth in human alveolar macrophages

The effect of omeprazole, a clinically used proton pump inhibitor, alone or in combination with clarithromycin was evaluated against Mycobacterium avium, Mycobacterium intracellulare and Mycobacterium tuberculosis, using a human alveolar macrophage model of infection. Omeprazole exhibited no significant effect on the growth of the two M. avium complex strains or on the mycobactericidal activity of clarithromycin against them. In contrast, omeprazole significantly promoted the growth of Mycobacterium tuberculosis and the anti-mycobacterial activity of clarithromycin against it in human alveolar macrophages. It was speculated that intracellular acidic milieu around M. tuberculosis might be one reason for the lower activity of clarithromycin in the treatment of human tuberculosis.     

Tumor necrosis factor, alone or in combination with IL-2, but not IFN-gamma, is associated with macrophage killing of Mycobacterium avium complex

Organisms belonging to the Mycobacterium avium complex (MAC) are the most common bacterial pathogens in patients with AIDS but factors associated with the activation of cellular defense mechanisms against this atypical mycobacterium have not been defined. Peritoneal macrophages harvested from a chronic MAC infection in C57 black mice are able to kill approximately 86% of intracellular MAC in contrast to 0 to 20% killing by unstimulated human and mouse macrophages in vitro. The availability of human rTNF-alpha, rIFN-gamma, and rIL-2 permitted evaluation of the role of each of these lymphokines/monokines, alone or in combination, in activating macrophages in vitro to kill MAC. Human monocyte-derived macrophages were cultured in vitro, stimulated with rIL-2, rIFN-gamma, or rTNF, and then infected with MAC (serovars 1 and 8). Mouse peritoneal macrophages were harvested, cultured in vitro, and stimulated with rIFN-gamma. rTNF (10(4) U/ml) was associated with a modest increase of intracellular killing of MAC (58 +/- 5%) even when utilized 24 or 48 h after macrophage infection or when administered for 5 consecutive days after infection (78.1 +/- 4%). Both human and murine IFN-gamma were associated with increased intracellular growth of MAC (32 +/- 4% for murine and 38 +/- 3% for human macrophages). However, intracellular killing (53 +/- 6% compared with control) was observed after 6 days of treatment with IFN-gamma. This latter effect was fully blocked by anti-TNF antibody, whereas rIL-2 alone did not augment the intracellular killing of MAC by human macrophages. rTNF plus either rIFN-gamma or rIL-2 triggered significant increases in superoxide anion production, but subsequent MAC killing was no greater than with rTNF alone. Treatment of macrophages with 10 U/ml of rTNF followed by rIL-2 (200 U/ml) was associated with 68% of intracellular killing. TNF seems to be an important monokine, promoting activation of mycobactericidal mechanisms in human macrophages.     

Suppressor macrophages in lymphocyte proliferation of cynomolgus monkeys

The present study demonstrated the presence of cells belonging to monocyte/macrophage lineage which suppressed mitogen-induced blastogenesis of peripheral blood lymphocytes in cynomolgus monkeys. Depletion of adherent or phagocytic cells from peripheral mononuclear cells caused a substantial increase in the blastogenic response of cynomolgus monkey lymphocytes whereas the same treatment led to marked reduction rather than enhancement in human lymphocyte blastogenesis. Addition of thioglycollate-elicited peritoneal exudate adherent cells as macrophages suppressed the blastogenic response of nonadherent lymphocytes in a dose-dependent manner. The suppressive effect was observed not only in autologous but also in allogeneic macrophages to the responder lymphocytes. Treatment of macrophages with silica, carrageenan or freezing-thawing reduced their suppressive effect but there was no reduction with mitomycin C or indomethacin. No suppressive activity was detected in the cell-free supernatant of macrophages cultured in the presence or absence of mitogens for up to 4 days. From these findings, it appeared that monocyte/macrophage lineage might be responsible for the observed suppressive effect on mitogen-induced blastogenesis of cynomolgus monkey lymphocytes.     

Human monocyte activation for cytotoxicity against intracellular Leishmania donovani amastigotes: induction of microbicidal activity by interferon-gamma

Macrophages are pivotal cells in interactions of man and leishmania. Leishmanial disease results from intracellular infection of macrophages: parasitized cells are seen in smears or biopsy specimens of lesions; macrophages cultured in vitro support replication of parasites. Paradoxically, parasite destruction is also mediated by macrophages, which become highly cytotoxic after exposure to immune lymphocytes or their lymphokine (LK) products. The precise molecular mechanisms by which lymphocytes or LK induce macrophage activation for leishmanicidal activity, however, are not yet known. We analyzed interactions of leishmania amastigotes with human monocytes cultured in vitro as a nonadherent cell pellet. Leishmania donovani and L. major replicated in freshly isolated monocytes. Monocytes treated with greater than 200 IU/ml of the LK, human Interferon-gamma (IFN-gamma), destroyed tumor cells and L. donovani, but not L. major. Phorbol myristate acetate, endotoxic bacterial lipopolysaccharide, and recombinant human IFN-alpha and IFN-beta did not induce cytotoxicity. The time course for induction of cytotoxicity contrasted sharply with that of previously described monocyte antileishmanial activity: IFN-gamma induced cytotoxicity even when added after infection with L. donovani; induction of cytotoxicity did not require that IFN-gamma be present throughout the period of culture after infection: a 30-min preinfection pulse of IFN-gamma was sufficient to induce 70% of maximal activity; and freshly isolated monocytes and cells cultured for up to 4 days in vitro prior to infection and IFN-gamma treatment were equally responsive to IFN-gamma. These studies provide convincing evidence for intracellular cytotoxicity for L. donovani by freshly isolated human monocytes. This system provides an important base for further analysis of induction and expression of cytotoxic mechanisms against leishmania and other intracellular organisms that cause human disease.     

Suppression of anti-Candida activity of macrophages by a quorum-sensing molecule, farnesol, through induction of oxidative stress

Farnesol is well known as a quorum-sensing molecule of Candida albicans . To assess the pathological function of farnesol, its effects on macrophage viability and functions including growth inhibitory activities against C. albicans were examined in vitro . Murine macrophages, when cultured in the presence of 56–112 μM of farnesol for 1–2 hr, decreased their activity inhibiting the mycelial growth of C. albicans and lost their viability. This suppression of macrophage function by farnesol was neutralized by the coexistence of the anti-oxidants probucol and trolox. Macrophages cultured in the presence of farnesol for 2 hr displayed morphological change of nuclei and DNA fragmentation, which suggested apoptosis of the cells. Intracellular production of ROS in the farnesol-treated macrophages was shown by fluorescence of DCFH-DA and increase of peroxidized materials. These effects of farnesol were blocked by probucol or trolox. These results indicate that farnesol lowered viability of the murine macrophages and suppressed their anti- Candida activity, perhaps through induction of ROS.     

Plasmin-mediated macrophage reversal of low density lipoprotein aggregation

Evidence suggests that aggregated low density lipoprotein (AgLDL) accumulates in atherosclerotic lesions. Previously, we showed that AgLDL induces and enters surface-connected compartments (SCC) in human monocyte-derived macrophages by a process we have named patocytosis. Most AgLDL taken up by these macrophages in the absence of serum is stored in SCC and remains undegraded. We now show that macrophages released AgLDL (prepared by vortexing or treatment with phospholipase C or sphingomyelinase) from their SCC when exposed to 10% human lipoprotein-deficient serum (LPDS). Macrophages also took up AgLDL in the presence of LPDS, but subsequently released it. In both cases, the released AgLDL was disaggregated. Although the AgLDL that macrophages took up could not pass through a 0.45-micrometer filter, >60% of AgLDL could pass this filter after release from the macrophages. Disaggregation of AgLDL was verified by gel-filtration chromatography and electron microscopy that also showed particles larger than LDL, reflecting fusion of LDL that aggregates. The factor in serum that mediated AgLDL release and disaggregation was plasmin generated from plasminogen by macrophage urokinase plasminogen activator. AgLDL release was decreased >90% by inhibitors of plasmin (epsilon-amino caproic acid and anti-plasminogen mAb), and also by inhibitors of urokinase plasminogen activator (plasminogen activator inhibitor-1 and anti-urokinase plasminogen activator mAb). Moreover, plasminogen could substitute for LPDS and produce similar macrophage release and disaggregation of AgLDL. Because only plasmin bound to the macrophage surface is protected from serum plasmin inhibitors, interaction of AgLDL with macrophages was necessary for reversal of its aggregation by LPDS. The released disaggregated LDL particles were competent to stimulate LDL receptor-mediated endocytosis in cultured fibroblasts. Macrophage-mediated disaggregation of aggregated and fused LDL is a mechanism for transforming LDL into lipoprotein structures size-consistent with lipid particles found in atherosclerotic lesions.     

Role of macrophage phospholipase D in natural and CpG-induced antimycobacterial activity

The present study addresses the differential ability of macrophages to control intracellular growth of non-pathogenic Mycobacterium smegmatis (Msm) and pathogenic M. tuberculosis (MTB). Results reported herein show that 3 h post infection, intracellular Msm, but not MTB, was significantly killed by macrophages. As the role of human macrophage phospholipase D (PLD) in the activation of antimicrobial mechanisms has been documented, we hypothesised the role of such enzyme in antimycobacterial mechanisms. To this aim, macrophage PLD activity was analysed at different times after exposure with either pathogenic MTB or non-pathogenic Msm. Results showed that, starting from 15 min after mycobacterial exposure, MTB did not induce macrophage PLD activity, whereas the environmental non-pathogenic Msm stably increased it. The direct contribution of PLD in intracellular mycobacterial killing was also analysed by inhibiting enzymatic activity with ethanol or calphostin C. Results show that PLD inhibition significantly increases intracellular Msm replication. In order to see whether the innate PLD-mediated antimicrobial mechanisms against MTB are also induced after CpG ODN stimulation, the role of PLD has been analysed in the course of CpG-mediated intracellular MTB killing. CpG DNA increased PLD activity in both uninfected and MTB-infected macrophages, and the inhibition of PLD activity resulted in a significant reduction of CpG-induced MTB killing. Taken together, our data suggest a relationship between host PLD activation and the macrophage ability to control intracellular mycobacterial growth.     

CD40 ligation activates murine macrophages via an IFN-gamma-dependent mechanism resulting in tumor cell destruction in vitro

We have shown previously that agonistic anti-CD40 mAb induced T cell-independent antitumor effects in vivo. In this study, we investigated mechanisms of macrophage activation with anti-CD40 mAb treatment, assessed by the antitumor action of macrophages in vitro. Intraperitoneal injection of anti-CD40 mAb into C57BL/6 mice resulted in activation of peritoneal macrophages capable of suppressing B16 melanoma cell proliferation in vitro, an effect that was greatly enhanced by LPS and observed against several murine and human tumor cell lines. Anti-CD40 mAb also primed macrophages in vitro to mediate cytostatic effects in the presence of LPS. The tumoristatic effect of CD40 ligation-activated macrophages was associated with apoptosis and killing of tumor cells. Activation of macrophages by anti-CD40 mAb required endogenous IFN-gamma because priming of macrophages by anti-CD40 mAb was abrogated in the presence of anti-IFN-gamma mAb, as well as in IFN-gamma-knockout mice. Macrophages obtained either from C57BL/6 mice depleted of T and NK cells by Ab treatment, or from scid/beige mice, were still activated by anti-CD40 mAb to mediate cytostatic activity. These results argued against the role of NK and T cells as the sole source of exogenous IFN-gamma for macrophage activation and suggested that anti-CD40 mAb-activated macrophages could produce IFN-gamma. We confirmed this hypothesis by detecting intracytoplasmic IFN-gamma in macrophages activated with anti-CD40 mAb in vivo or in vitro. IFN-gamma production by macrophages was dependent on IL-12. Taken together, the results show that murine macrophages are activated directly by anti-CD40 mAb to secrete IFN-gamma and mediate tumor cell destruction.     

Stimulation of macrophage H2O2 release by resident thymocytes: effect of a soluble factor distinct from interferon-gamma

Activated T cells are known to stimulate macrophage oxidative metabolism and antimicrobial activity through release of interferon-gamma (IFN-gamma). In contrast, the role of nonactivated T cells in regulating macrophage effector functions is less well defined. We have previously reported that a low molecular weight soluble factor derived from resident (nonactivated) thymocytes enhances macrophage receptor-mediated phagocytosis. In the present study, we examined the capacity of resident murine thymocytes to stimulate the respiratory burst and microbicidal activity of peritoneal macrophages. Macrophages cultured for 1-2 days with cell-free thymocyte supernatant (TS) released two to three times more H2O2 in response to PMA or opsonized zymosan than did control macrophages. The H2O2-stimulating factor in TS was distinguished from IFN-gamma by its heat stability (100 degrees C, 20 min), approximate MW of 2400 Da (gel filtration high-pressure liquid chromatography), and absence of interferon activity in both antiviral and enzyme-linked immunosorbent assays. TS-treated macrophages, however, did not exhibit a greater capacity to kill or inhibit the intracellular growth of Toxoplasma gondii, indicating that the thymocyte factor did not fully activate macrophage microbicidal mechanisms. These data suggest that thymocytes can increase the respiratory burst capacity of macrophages in the absence of antigen-specific immune responses.     

Chronic infection due to Mycobacterium intracellulare in mice: association with macrophage release of prostaglandin E2 and reversal by injection of indomethacin, muramyl dipeptide, or interferon-gamma

As a model for the study of human atypical mycobacterial disease, we explored the basis for the prolonged mycobacteriosis in mice infected with Mycobacterium intracellulare. Two weeks after i.v. injection of mycobacteria, peritoneal macrophages were found to be activated, as indicated by their capacity to produce large amounts of superoxide anion (O2-) in response to phorbol myristate acetate (PMA) or viable M. intracellulare. However, 4 wk after infection, despite the continued presence of large numbers of mycobacteria in the spleen, macrophages from infected animals produced low amounts of O2-. Unfractionated spleen cells from mice infected 4 wk earlier produced increased amounts of interleukin 2 and interferon (IFN) when stimulated with the mitogen concanavalin A, but less of these lymphokines than unstimulated cells when exposed to antigens derived from M. intracellulare, suggesting production of an inhibitory factor. Spleen cells from infected mice were not stimulated to incorporate [3H]thymidine by exposure to mycobacterial antigens; but this unresponsiveness could be reversed by addition of indomethacin to the cultures. Additional investigation showed that macrophages from infected animals produced large amounts of prostaglandin E2 (PGE2) when stimulated by mycobacterial antigens. In vitro, such concentrations of PGE2 inhibited uptake of [3H]thymidine by stimulated spleen lymphocytes from infected animals. Thus, it seemed likely that in infected animals, macrophage-derived PG suppressed production of IFN-gamma by lymphocytes, which in turn prevented activation of the macrophages to full microbicidal activity. To test this hypothesis, we administered either indomethacin, IFN-gamma, or muramyl dipeptide to infected mice. Mice treated with each of these agents showed decreased spleen and lung weights, and decreased numbers of viable mycobacteria in these organs. These results support the concept that interaction between the host and M. intracellulare is modulated profoundly by PG and suggest that administration of agents that directly promote macrophage activation can enhance resistance to infection by this organism.     

Macrophage fibrinolytic activity: identification of two pathways of plasmin formation by intact cells and of a plasminogen activator inhibitor

Endotoxin-stimulated macrophages hydrolyze fibrin by a plasmin-mediated process in the absence of detectable soluble plasminogen activator (PAs). The data show that macrophages also activate plasmin by a membrane-associated plasminogen activator (PAm). In the presence of endotoxin, PAm activity increases, and plasmin is formed only by PAm. In addition, endotoxin stimulates macrophages to secrete a proteinase inhibitor that blocks PAs activity but not PAm or plasmin activity. The increased PAm activity and the PA inhibitor secretion in response to endotoxin explains the ability of intact macrophages to hydrolyze fibrin in the absence of detectable PAs. Endotoxin, 100 ng/ml, induced an intracellular PA inhibitor in cultured macrophages, and this correlated with accumulation of inhibitor in medium over the cells. The intracellular PA inhibitor was found to be 50--60 kilodaltons by gel chromatography, to be of anionic charge at pH 7.4 and to inhibit urokinase esterolytic and proteolytic activity but not preformed plasmin. These results define two pathways of plasmin formation by intact macrophages and identify the macrophage cell surface as a site of PA activity relatively protected from soluble proteinase inhibitors.     

ATP-induced killing of virulent Mycobacterium tuberculosis within human macrophages requires phospholipase D

The global dissemination of antibiotic-resistant Mycobacterium tuberculosis has underscored the urgent need to understand the molecular mechanisms of immunity to this pathogen. Use of biological immunomodulatory compounds to enhance antituberculous therapy has been hampered by the limited efficacy of these agents toward infected human macrophages and lack of information regarding their mechanisms of activity. We tested the hypotheses that extracellular ATP (ATPe) promotes killing of virulent M. tuberculosis within human macrophages, and that activation of a specific macrophage enzyme, phospholipase D (PLD), functions in this response. ATPe treatment of infected monocyte-derived macrophages resulted in 3.5-log reduction in the viability of three different virulent strains of M. tuberculosis. Stimulation of macrophage P2X7 purinergic receptors was necessary, but not sufficient, for maximal killing by primary macrophages or human THP-1 promonocytes differentiated to a macrophage phenotype. Induction of tuberculocidal activity by ATPe was accompanied by marked stimulation of PLD activity, and two mechanistically distinct inhibitors of PLD produced dose-dependent reductions in ATPe-induced killing of intracellular bacilli. Purified PLD restored control levels of mycobacterial killing to inhibitor-treated cells, and potentiated ATPe-dependent tuberculocidal activity in control macrophages. These results demonstrate that ATPe promotes killing of virulent M. tuberculosis within infected human macrophages and strongly suggest that activation of PLD plays a key role in this process.     

Xenogenic macrophage immunization reduces atherosclerosis in apolipoprotein E knockout mice

Atherosclerosis is a complex chronic inflammatory disease in which macrophages play a critical role, and the intervention of the inflammatory process in atherogenesis could be a therapeutic strategy. In this study, we investigated the efficacy of xenogenic macrophage immunization on the atherosclerotic lesion formation in a model of murine atherosclerosis. Apolipoprotein E knockout (apoE-KO) mice were repeatedly immunized with formaldehyde-fixed cultured human macrophages (phorbol ester-stimulated THP-1 cells), using human serum albumin as a control protein or HepG2 cells as human control cells, once a week for four consecutive weeks. The vehicle phosphate-buffered saline was injected in the nonimmunized controls. THP-1 immunization induced antibodies that are immunoreactive with mouse macrophages. Although the plasma lipid levels were unchanged by the immunization, the atherosclerotic lesion area in the aortic root was significantly reduced by >50% in 16-wk-old THP-1-immunized apoE-KO mice compared with that in control mice. THP-1 immunization reduced in vivo macrophage infiltration, reduced in vitro macrophage adhesion, and changed cytokine production by macrophages to the antiatherogenic phenotype. Xenogenic macrophage immunization protects against the development of atherosclerosis in apoE-KO mice by modulating macrophage function in which antibodies induced by the immunization are likely to be involved. This method is a novel and potentially useful cell-mediated immune therapeutic technique against atherosclerosis. antibody; THP-1 cells     

Activated lymphocytes depress phagocytosis of latex particles by human monocyte-macrophages

In this study, monocyte-macrophages of normal human donors were cultured with and without lymphocytes and antigen in order to define the effect of antigen-stimulated lymphocytes on phagocytosis by macrophages. Phagocytosis was assessed by uptake of radio-labeled latex particles by macrophage monolayers. Although no effect was noted when purified macrophage monolayers were cultured in the presence of antigen, marked inhibition of phagocytosis was observed when macrophages were cultured in the presence of autochthonous antigen-stimulated lymphocytes. The degree of phagocytic depression correlated with the delayed cutaneous hypersensitivity response of the donor and with the concentration of antigen present in the system.     

Inhibition of production of macrophage-derived angiogenic activity by the anti-rheumatic agents gold sodium thiomalate and auranofin

We have investigated the effect of gold sodium thiomalate and auranofin, gold compounds employed in the treatment of rheumatoid arthritis, on production of macrophage-derived angiogenic activity. Elicited mouse peritoneal macrophages were cultured in the presence or absence of gold compounds or thiomalic acid, and the macrophages or their conditioned media were then assayed for their angiogenic activity in rat corneas. Control macrophage conditioned medium was potently angiogenic. In contrast, conditioned medium from gold or thiomalic acid treated macrophages was not. Addition of gold compounds or thiomalic acid to control macrophage conditioned medium did not inhibit its angiogenic activity. Drug treatments did not significantly affect macrophage lactate dehydrogenase release, lysozyme release, or protein synthesis. We conclude that gold sodium thiomalate and auranofin potently reduce the detectable angiogenic activity produced by macrophages.     

Mycobacterial growth inhibition by interferon-gamma-activated bone marrow macrophages and differential susceptibility among strains of Mycobacterium tuberculosis

Growth inhibition of the intracellular bacterial pathogens Mycobacterium bovis and M. tuberculosis by lymphokine-activated murine bone marrow macrophages was studied. Mycobacterial growth was assessed by the uptake of 3H-uracil or by determination of colony-forming units. Stimulation of macrophages with recombinant interferon-gamma (r-IFN-gamma) or with IFN-gamma-containing supernatants from antigen- or mitogen-stimulated T cells markedly reduced growth of M. bovis strain BCG Phipps or M. tuberculosis strain H37Rv. In contrast, M. tuberculosis strain Middelburg proved resistant to lymphokine-stimulated macrophages, suggesting heterogeneous susceptibility toward lymphokine-activated macrophages among different M. tuberculosis strains. Stimulation could be blocked by anti-IFN-gamma antiserum, indicating that IFN-gamma was capable of activating antimycobacterial macrophage functions. Stimulation with r-IFN-gamma and subsequent phagocytosis of M. bovis did not lead to increased chemiluminescence responses by bone marrow macrophages, suggesting that mycobacterial growth inhibition was not paralleled by the release of reactive oxygen metabolites. We conclude that IFN-gamma-mediated macrophage activation represents a major step in acquired resistance against tuberculosis and that evasion from this mechanism contributes to mycobacterial virulence.