Activation of Human Mononuclear Cells for the Killing of Mycobacterium tuberculosis by Pro-inflammatory Cytokines through a Nitric-oxide-department Mechanism

P. Farina Ph.D, F. Mohammadi MD, S.J. Tabatabai MD, M.R. Masjedi MD, A.A. Velayati MD

National Research Institute of Tuberculosis and Lung Disease (NRITLD), Shaheed Beheshti University of Medical Sciences and Health Services, Tehran, Iran

  • Abstract

    Objectives-Our aim was to find out if the peripheral blood monocytes and cultured macrophages from patients with pulmonary tuberculosis treatment failure could respond to pro-inflammatory cytokines to produce detectable nitric-oxide (NO) levels and whether the antimycobacterial activity of these NO-producing monocytes/macrophages could be correlated with the amount of nitric-oxide generated in vitro.
    Methods-We examined the peripheral blood monocytes of 20 healthy controls and 20 treatment-failure tuberculosis patients immediately or after culture for 7 days.
    Results-In vitro activation of mononuclear cells by cytokines induced the release of nitric-oxide in an amount sufficient to lead to mycobactericidal effects. Moreover, as mononuclear cells mature in culture medium, NO production also increases. This property has been demonstrated in cells removed from healthy subjects as well as from cases. Induced nitric-oxide release and mycobactericidal efficacy were found to be closely correlated in enhancement by addition of L-arginine in the culture medium or abrogation by N-monomethyl-L-arginine (NGMM arginine). The interesting and unexpected observation was that monocytes/macrophages from patients responded more efficiently as compared to healthy controls.
    Conclusion-Our observation suggests that human mononuclear cells from treatment failure patients are not functionally impaired, and it may be possible to suitably modulate these cells to become mycobactericidal.

    Keywords · Nitric-oxide · mononuclear phagocytes · cytokines · L-arginine

    • Introduction

    Although Mycobacterium tuberculosis was discovered by Koch1 over a century ago, the mechanism of killing and resistance to this facultative intracellular pathogen, that replicates and persists inside mononuclear phagocytes, remains unclear. Since the initial report of the killing of Mycobacterium microti by immunologically activated macrophages2, research efforts to understand antimycobacterial mechan-isms have focused on the toxic effects of reactive oxygen intermediates (ROI) generated by phagocytes during the respiratory burst.3,4,5

    Recent findings indicate that biosynthesis of nitrate occurs in mammalian cells5 and that macrophages are a major source of mammalian nitrate synthesis.6,7 This has spurred intensive research efforts leading to the discovery of the L-arginine-dependent metabolic pathway used to generate reactive nitrogen intermediates (RNI).8 In murine system it has been suggested that mycobacteriostatic activity involves generation of nitric-oxide and its stable derivatives (NO2, NO3). 9 This reaction is carried out by the inducible form of NO synthase (iNOS) using L-arginine as the substrate.10,11,12 The aim of this study was to find out if peripheral blood monocytes and cultured macrophages from treatment failure pulmonary tuberculosis patients (PTB) could respond to the pro-inflammatory cytokines and produce detectable amounts of nitric-oxide and whether the antimycobacterial activity of these NO-producing monocytes/macrophages could be correlated with the amount of nitrogen-oxide generated in vitro.

    Materials and Methods

    Patients and controls: Twenty healthy volunteers, from laboratory and nursing staff, 20 to 50 years of age, were taken as controls. Twenty patients with pulmonary tuberculosis aged between 22 to 56 years (16 males and 4 females) were included in this study. All were being treated for bacteriologically and radiologically documented pulmonary tuberculosis by durgs from the first-line regimen, namely isoniazid, rifampin, ethambutol, pyrazinamide and streptomycin in various combinations and were still sputum positive for A F B (acid fast bacilli) beyond 6 months (ranging between 6 to 18 months).

    Isolation and preparation of peripheral blood mononuclear cells: The peripheral blood (25ml) was collected from all subjects. Mononuclear cells were isolated from the peripheral blood by density gradient centrifugation on ficoll-hypaque. Interphase cells (mononuclear cells) were recovered, washed and then resuspended in recommended primary medium for cell culture (RPMI-1640) with 10% fetal calf serum (FCS). Cells were in 24 well culture plates at 3x106 cells/well containing approximately 0.15x105 monocytes and were allowed to adhere at 37° C in 5% CO2 atmosphere for 2 hours. The supernatant medium and non-adherent cells were washed off and the wells were recharged with medium. The freshly harvested cells were consistently >95% viable as assessed by their ability to exclude trypan blue. A set of plates was incubated for 7 days to allow maturation of monocytes to macrophages and the other set of culture plates was used immediately for experiments with monocytes.

    Monocyte cell culture and experiments: The monocytes were allowed to settle for 4 hours before beginning the experiments. After 4 hours, wells containing 0.15x105 monocytes were incubated with three different cytokines at 37° C in 5% CO2 atmosphere in supplemented RPMI-1640 with 2% FCS. The cytokines, namely IFN-g, TNF-a, and IL-1 were added to the previously marked wells at a concentration of 100U, 1ngm, and 50U per well, respectively. Lipopolysaccharide phenol-extracted E.coli, serotype 012 B: B12 (LPS) at the concentration of 1ng/ml was added to the cytokine conditioned cells after 48 hours of incubation.

    At 100 hours, NGM M arginine (a specific inhibitor of L-arginine) was added. The incubation period continued and an assay for nitrite was undertaken after another 24 hours (124hours).

    Macrophage cell culture and experiments: Two culture plates were seeded with monocytes (0.15x105 cells/well) and incubated for 7 days maturation by replenishing the medium. Stimulation and activation as described for monocyte culture were also repeated with the monocytes-derived macrophages. The time points described started from the 8th day until the end of the experiments (0-124 hours) and were compared with the time points used for experiments with fresh monocytes.

    Nitrite assay: Concentration of NO2 produced by the monocytes and the macrophages as a measure of the production of nitric-oxide (NO) were determined spectrophotometrically at 540 nm by the following method described by Dring et al.14 An amount of supernatant was removed from the culture well at the time intervals of 0, 4, 24, 48, 52, 76, 100, and 124 hours and was centrifuged at 400 gx 10 min to make it cell-free. Then it was incubated with an equal volume (100 µl) of Giess reagent (1% sulfanilamide, 0.1% napthylethylene diamin dihydrochloride, 2.5% H3PO4) at room temperature for 10 min and absorbance read at 540 nm, using sodium nitrite as standard. NO2 release is reported as nanomoles/105 cell/well.

    Cell-free medium alone contained 0.1 to 0.2 nanomoles of NO2/well. This value was deducted from all test results before calculation.

    Bactericidal assay: The monocytes and cultured macrophages at 0 hours and after 48, 76, 100, and 124 hours of stimulation and activation were infected with M. tuberculosis at the ratio of 10 bacteria per cell. After the infection period (of 48 hours), the extra-cellular bacteria from each well were removed by three washes.11 This procedure has been shown to be effective in removing at least 99.9% of the extracellular bacteria. The total bacterial suspension from each well was inoculated onto Lowenstein-Jensen medium, using one plate for each well. The plates were allowed to dry at room temperature for 15 min and were then incubated at 37° C under 5% CO2 and moist air for 2 weeks. The results are reported as mean Colony Forming Unit (CFU)/ml of suspension, obtained after 28 days of incubation.

    Statistical analysis: results are expressed as means with standard errors; student’s t-test and pair t-test were used to assess significance.

    Results

    Effect of cytokines on fresh peripheral blood monocytes: Freshly isolated monocytes from patients and controls released negligible amount of NO2 without the help of any stimulatory agents at this point and the value was 1.25±1.05 nmoles in controls and 1.82±2.3 nmoles in cases. These were used as baseline values for all subsequent calculations. Fresh monocytes from cases as well as controls, when stimulated by IFN-g, TNF-a or IL-1, showed insignificant (p>0.05) nitrite production after 4 hours of incubation. However, from 24 to 48 hours, the production of NO2 rose significantly (Fig. 1a). The mean value of monocyte nitrite production from cases registered 17.0±3.2 nmoles at the same time-point of experiments. Kinetics of the stimulatory effect of cytokines IFN-g, TNF-a and IL-1 were different; IFN-g proved to be the most efficient cytokine we tested (Fig. 2a). Furthermore, addition of LPS in cytokine pulsed monocytes culture medium after 48 hours and NO2 assay after another 28 hours of incubation (76 hours) demonstrated enhanced nitrite production, 13.2±3.1 nmole in control subjects, 21.72±2.5 nmoles in cases. After adding L-arginine to the culture medium, the mean NO2 production by the monocytes showed a highly significant rise. In control wells the level of NO2 production rose from 13.2±3.1 nmoles at 76 hours to 22.5±3.3 nmoles (p<0.05) after 100 hours, whereas in case of PTB patients the mean production of NO2 in response to L-arginine almost doubled within 24 hours. From 21.7±2.5 nmoles in 76 hours the production stood at 31.63±2.4 nmoles after 100 hours. Enhanced production of NO2 in response to L-arginine could be abrogated by addition of NGMM arginine (350µm) in the culture medium (Fig. 2b).

    Effect of cytokines on cultured macrophages: Unstimulated macrophages from patients produced 2.6±1.85 nmoles of nitrite. After 4 hours of incubation with cytokines, macrophages were capable of releasing significant (p<0.05) amounts of NO2 into the culture medium (Fig. 1b). It was interesting to note that the mean value of NO2 produced after 48 hours of incubation, irrespective of cytokines used, was higher in the macrophages from cases (24.7±3.36 nmoles) than from healthy subjects (15.1±3.9 nmoles) (p<0.05). The production of nitrite by macrophages in both healthy and diseased subjects both maintained an upward tendency in respect to the incubation time till the end of experiment (124 hours). Comparison of NO2 production of monocytes with that of macrophages in controls as well as in patients clearly showed that the macrophages release higher amount of NO2 in culture medium than monocytes (p<0.05) than when stimulated with cytokines alone or with LPS. After 48 hours, when LPS was added to the cell-culture medium, the spectro-photometric reading resigned a temporatory dip at 52 hours. The dip was not only restored, but there was a further rise in production of NO2. It was observed that after adding L-arginine at 76 hours, the mean value of NO2 production by macrophages rose from 30.7±3.6 to 42.4±4.1 nmoles at 100 hours.

    Bactericidal activity by activated monocytes/ macrophages: In order to relate the nitrite- mediated effector activity of monocytes/ macrophages, we determined the percentage of live Mycobacterium tuberculosis in the culture supernate of activated monocytes/ macrophages. In the culture supernate of IFN-g-stimulated monocytes/ macrophages multiplication of M.tuberculosis was less (p<0.05) than that in unstimulated cells. Subsequent addition of LPS at 76 hours further lowered the colony counts (Table). Since the experiments were run in parallel, this lowered mycobacterial colony count (bactericidal effects) correlated with the lag phase of NO2 release at 48 hours and 76 hours. Moreover, the in vitro cultured macrophages demonstrated more efficient mycobactericidal activity than fresh monocytes (p<0.05) (Table).

    It is well known that mycobacteria grow faster in fetal calf serum containing medium due to its optimum iron availability and that the normal replication time of the Mycobacterium tuberculosis is around 22±2 hours. Taking these into account, in the present experiment, the growth of Mycobacterium tuberculosis was checked in the supernate after 48 hours of incubation, because the aim was to look for the bactericidal efficacy of the release nitric-oxide in the extracellular compartment. Recently it has been demonstrated that it is probable that nitric-oxide is directly toxic to mycobacteria.15 Interestingly, our results also indicate that L-arginine and its analogue NGMM arginine directly influence the extracellular myco-bactericidal activity of the monocytes/macrophages that may in turn greatly depend on the release of nitric-oxide in the cellular environment (Fig. 2b).

    Discussion

    In order to assess the bactericidal capacities in treatment failure TB patients, we examined the level of nitric-oxide (NO) in the induced monocytes/macrophages in these patients. For this purpose, we primed and activated the mononuclear cells isolated from the peripheral blood of these patients by three different pro-inflammatory cytokines; IFN-g, TNF-a, and IL-1.These potent pro-inflammatory cytokines were selected because their production by human mononuclear phagocytic cells have been reported in various clinicopathological conditions, e.g. tuberculosis 16, AIDS 17, cerebral malaria 18 and asthma 19. It has been demonstrated that the raised level of these cytokines could be correlated with enhanced cytotoxicity and bactericidal efficacy of the immune effector cells. 8,10 In murine models it has been established that the mononuclear-phagocytes mediate their cytotoxic and bacteriocidal activity by producing immune-effector molecules such as reactive nitrogen intermediates (including nitric-oxide, nitrite and nitrate).6,7,9 The mycobacteri-ostatic efficacy of nitric-oxide released by the cytokine activated macrophages (in murine models) have been demonstrated and accepted as a fact.6,7,13 Also, several workers have shown that nitric-oxide produced in an all-free system exerts bacteriostatic effect on Mycobacterium tuberculosis.However, extrapolation of these observations in the human situation has evoked conflicting opinions. In our previous report, we have demonstrated that the human monocytes/ macrophages can be primed and activated to use the inductive nitric-oxide synthase (iNOS) pathway and release detectable amounts of nitric-oxide.20,21 The results of our present experiments demonstrate that mononuclear phagocytes from treatment failure cases of pulmonary tuberculosis, when appropriately activated in vitro, produce RNI in an amount sufficient to lead to mycobactericidal effect.

    Interestingly, the mycobactericidal efficacy was greater in the patients as compared to the healthy controls. This was surprising because although these patients did not improve bacteriologically or clinically, the monocytes removed from them could effectively demonstrate bactericidal activity in-vitro, being marginally better than that of normal healthy individuals. Thereby, we presume that since monocytes from these patients were already in primed stage at the time of isolation, they could respond better to the cytokine activation in-vitro.

    As it has been shown by other researchers, the activation of monocytes/ macrophages is involved two sequential steps, priming and triggering.23 IFN-g produced by T cells is usually considered as the priming signal, whereas the other signal has been shown to be either the bacterial endotoxin (LPS) or TNE-a. This is supported by the fact that recombinant TNE-a, a poor activator by itself, potentiates the mycobactericidal-activity and the NO synthesis of murine macrophages induced by IFN-g.24 In our previous experiments system19,20, we observed that the production of superoxide (O2) by mononuclear cells in cases of the patients was higher than healthy controls. It means that the mononuclear cells of patients were in primed stage due to infection. However, it required the additional signal to become fully activated. That is the reason we could detect more NO production in them than mononuclear cells of healthy controls. Similar results were obtained with recombinant cytokines in the activation of leishmanicidal activity of macrophages.25 In the experiments for production of RNI in response to cytokines, macrophages demonstrated significantly higher values (p<0.01) as compared to monocytes in patients as well as in controls. Thereby, our results confirm that production of nitric-oxide increase as the mononuclear cells mature in culture.

    In our experiments we observed that nitric-oxide production by fresh monocytes/and monocytes-derived macrophages can be significantly induced by cytokines with or without LPS. Of the three pro-inflammatory cytokines, tested by us, IFN-g was the most efficient inducer of nitric-oxide production. Significantly, the multiplication of Mycobacterium tuberculosis IFN-g pulsed monocytes/macrophages had inverse relation with nitric-oxide production (Table ). Thereby, our data suggests a critical role of IFN-g as a macrophage activating factor in mycobactericidal activity through a nitric-oxide dependet mechanism. We also demonstrated that the production of NO2 could be further enhanced after addition of L-arginine in culture wells. At this point there was an inverse relation between the amount of NO2 produced and the viable mycobacteria in cell-culture medium. This indicates that NO is one of the effector molecules of mononuclear cells capable of producing mycobactericidal effect. The possibility that the nitric-oxide released by Mycobacterium tuberculosis infected macrophages may be due to the presence of nitrate reductase in the bacteria, was eliminated by the experiment where the NO2 production could be abrogated after addition of NGMM arginine to the culture medium (Fig. 1,2b).

    Taken together, our observation suggests that human mononuclear cells from treatment failure patients are not functionally impaired, and it may be possible to suitably modulate these cells to become mycobactericidal. Though this molecule (NO) has other cellular effects at different sites and organs of the body, when produced by mononuclear phagocytic cells, it may be possible that nitric-oxide could be employed by cytokine induction pathway in the control of mycobacterial growth. However, this needs further investigation. At present, studies are underway in our laboratory to elucidate the detailed features of nitric-oxide production and its relation with mRNA expression of cytokines in different stages of tuberculosis infection.

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