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(Journal of Leukocyte Biology. 2000;68:897-902.)
© 2000 by Society for Leukocyte Biology

Role of IL-12 in macrophage activation during intracellular infection: IL-12 and mycobacteria synergistically release TNF-{alpha} and nitric oxide from macrophages via IFN-{gamma} induction

Zhou Xing, Anna Zganiacz and Micheal Santosuosso

Department of Pathology and Molecular Medicine, and Division of Infectious Diseases, Centre for Gene Therapeutics, McMaster University, Hamilton, Ontario, Canada

Correspondence: Dr. Zhou Xing, Rm. 4H19, Health Science Centre, Department of Pathology and Molecular Medicine, McMaster University, 1200 Main St. West, Hamilton, Ontario L8N 3Z5, Canada. E-mail: xingz{at}fhs.mcmaster.ca


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 REFERENCES
 
IL-12 is believed to play an important role in cell-mediated immunity against intracellular infection primarily by acting on T and NK cells. Recent evidence has suggested, however, that IL-12 has broader cellular targets than previously thought. In this study, we examined the role of IL-12 in macrophage TNF-{alpha} and nitric oxide (NO) release by using an in vitro model of intracellular infection. IL-12 alone released relatively little TNF-{alpha} and NO, whereas live mycobacteria alone released TNF-{alpha} markedly but little NO from murine alveolar macrophages. However, IL-12 and mycobacteria together enhanced TNF-{alpha} and NO release synergistically. Because IL-12 and mycobacteria also released IFN-{gamma} from macrophages synergistically, and exogenous IFN-{gamma} with mycobacteria enhanced TNF-{alpha} and NO release synergistically, we examined the role of endogenous IFN-{gamma} in IL-12/mycobacteria-stimulated macrophage activation. Using macrophages from mice deficient in IFN-{gamma}, we found that IL-12/mycobacteria-enhanced macrophage TNF-{alpha} and NO release was mediated through endogenous IFN-{gamma}. We further demonstrated that IFN-{gamma} and mycobacteria together had a selective effect on macrophage cytokine release because they released TNF-{alpha} synergistically but not macrophage chemotactic protein-1 (MCP-1). These findings reveal that IL-12 can activate macrophages potently during intracellular infection, and this activating effect is mediated primarily through its effect on macrophage IFN-{gamma} release.

Key Words: nitric oxide • MCP-1 • cytokine • tuberculosis


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 REFERENCES
 
Experimental and human studies have demonstrated that type 1 cytokines, including interleukin (IL)-12, interferon-{gamma} (IFN-{gamma}), and tumor necrosis factor {alpha} (TNF-{alpha}), play an important role in the development of cell-mediated immune responses to intracellular infections, particularly those caused by intracellular bacteria [1 2 3 4 5 ]. Among these, IL-12 is released primarily by antigen-presenting cells and acts as a linker between the innate and acquired immunities by inducing the differentiation of antigen-specific T cells of Th1 phenotype and the release of IFN-{gamma} from activated T cells as well as natural killer (NK) cells [6 7 8 9 ]. T cell-derived IFN-{gamma} is considered crucial to the adequate activation of macrophages, the ultimate effector cells in host defense against intracellular infection [2 , 5 ]. IFN-{gamma}-activated macrophages demonstrate increased release of nitric oxide (NO), which accounts for subsequently enhanced mycobactericidal activities in macrophages [4 , 10 ]. Recent evidence from us and others has, however, revealed that IL-12 is capable of a broader spectrum of biologic activities in the cell-mediated immunity by acting on multiple immune cell types including macrophages [11 12 13 ].

TNF-{alpha} is primarily a macrophage-derived cytokine [3 ]. TNF-{alpha}, together with IL-12 and IFN-{gamma}, is markedly induced during mycobacterial infection [9 , 11 , 14 15 16 17 ]. Studies using anti-TNF-{alpha} antibodies or gene knock-out mice have demonstrated a critical role of TNF-{alpha} in the optimal expression of type 1 cell-mediated immune responses to intracellular infections [14 , 15 , 18 , 19 ]. In contrast to the tight control of macrophage IFN{gamma} response, macrophages appear to release TNF-{alpha} under a variety of in vitro stimulatory conditions [3 , 11 , 20 ]. However, the mechanisms by which macrophage TNF-{alpha} release is regulated during intracellular infection remain to be completely understood, and, in particular, the role of IL-12 in macrophage TNF-{alpha} and NO responses is still unclear. In our current study, we used a model of intracellular infection to study TNF-{alpha} and NO responses in macrophages upon the interaction with type 1 cytokines and intracellular pathogens. We have examined the effect of IL-12, IFN-{gamma}, and live mycobacteria, alone or in combination, on macrophage TNF-{alpha} and NO release and dissected the mechanisms by which IL-12 modulates macrophage TNF-{alpha} and NO release.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS AND DISCUSSION
 REFERENCES
 
Mice
Mice were 8–14 weeks old. C57BL/6 mice were purchased from Harlan (Indianapolis, IN). Male and female IL-12-/- mice with C57BL/6 background (the breeding pair was kindly provided by Dr. J. Magram, Hoffmann-La Roche, Inc., Basel, Switzerland) were bred and maintained in the Level B pathogen-free facility at McMaster University Animal Quarter (Hamilton, Ontario, Canada). The generation and characterization of IL-12-/- mice have been described elsewhere [21 ]. IFN-{gamma}-/- mice were purchased from the Jackson Laboratory (West Grove, PA; C57BL/6-ifngtmlTs-stock 002287).

Reagents
An attenuated live strain of Mycobacterium bovis (BCG) was obtained from Connaught Laboratories (North York, Ontario, Canada). RPMI 1640 medium was supplemented with 10% fetal calf serum (FCS), 100 U/ml penicillin, and 100 µg/ml streptomycin. Murine IL-12 or IFN-{gamma} was used as cytokine-containing supernatants generated by transducing an A549 epithelial cell line with a gene-transfer vector expressing murine IL-12 or IFN-{gamma} [22 , 23 ]. The high concentration of murine IL-12 or IFN-{gamma} in these supernatants was determined by enzyme-linked immunosorbent assay (ELISA). Approximately 60% of IL-12 protein was found to be in the bioactive form of IL-12 p70. A control empty vector was used to generate control supernatants, which contained no IL-12 or IFN-{gamma} and were shown to have no effect on macrophage activation at all. In some experiments, recombinant murine IL-12 and IFN-{gamma} proteins (R&D Systems, Minneapolis, MN) were also used. An antimurine macrophage chemotactic protein-1 (MCP-1) antibody [purified immunoglobulin G (IgG)] was purchased from R&D Systems.

Isolation of lung macrophages and culture conditions
A bronchoalveolar lavage procedure was carried out to isolate lung macrophages from naive C57BL/6, IL-12-/-, or IFN-{gamma}-/- mice, as previously described [11 ]. Briefly, the mouse lung was lavaged with a total of 2.6 ml phosphate-buffered saline (PBS) in six aliquots (0.3 mlx2 and 0.5 mlx4) through a polyethylene tube (Becton Dickinson, Sparks, MD) cannulated into the mouse trachea. Collected lavage fluids were centrifuged to pellet cells. Approximately 0.6 to 1 x 106 cells per mouse were obtained, and >98% of these cells were alveolar macrophages identified by differential staining. For each experiment, cells isolated from three to four mice of each strain were pooled. These cells were resuspended in RPMI culture media and cultured in 96-well plates at a density of 0.1 x 106 cells/well without or with various stimuli, including 50 cfu/cell of live M. bovis BCG [11 ], various concentrations of IL-12 or IFN-{gamma}, or a combination of BCG and IL-12 or IFN-{gamma} in a total volume of 0.25 ml for 72 h at 37°C. The supernatants were collected and stored at -20°C until cytokine measurement.

Cytokine measurements
The level of cytokines in culture supernatants was determined by using ELISA kits for murine TNF-{alpha}, IFN-{gamma}, MCP-1, or IL-12p70, purchased from R&D Systems. The sensitivity of detection for all of these ELISA kits was <5 pg/ml. Total IL-12 protein (IL-12p40 and p70) was measured by using an ELISA kit purchased from Biosource International (Montreal, Canada).

Measurement of NO production
The release of NO by macrophages was determined by measuring the concentration in culture supernatants of the end product of NO, nitrite [24 ]. Briefly, diluted supernatants were mixed at a 1:1 ratio with Griess reagent buffer (Sigma Chemical Co., St. Louis, MO). The absorbance was determined at 540 nm by using a spectrophotometer. The final concentrations of nitrite were calculated from a standard curve derived from prepared solutions of NaNO2 of known concentrations.

Data analysis
Whenever applicable, the difference comparison was made by using a Student’s t-test. The difference was considered statistically significant when p <= 0.05.


   RESULTS AND DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
REFERENCES
 
Release of IL-12 by macrophages
We measured the release of total IL-12 (IL-12p40 and IL-12p70) by macrophages. We found that although mycobacteria or IFN-{gamma} increased total IL-12 release, the combined stimulation by mycobacteria and IFN-{gamma} had a synergistic effect on total IL-12 release (Table 1 ). Because only IL-12p70 is the bioactive form of IL-12 [1 ], we also measured the content of IL-12p70 under these conditions. Of interest, minimal amounts of IL-12p70 were detected under all of these conditions studied (Table 1) . This finding is in agreement with recent studies that demonstrate macrophage release of IL-12p70 was under a much tighter control than IL-12p40, and intracellular infection of murine macrophages by B. abortus released only IL-12p40 but not IL-12p70 [25 , 26 ].


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  		Table 1. Release of IL-12 by Macrophages (pg/ml)

 
Effect of IL-12 and mycobacteria on TNF-{alpha} release by macrophages
To study the effect of IL-12 on macrophage TNF-{alpha} release, we used bioactive IL-12 to stimulate macrophages because macrophages themselves released little IL-12p70 (Table 1) . To this end, alveolar macrophages from naive C57BL/6 mice were isolated and cultured without or with IL-12 or live mycobacterial BCG. Freshly isolated alveolar macrophages released a small amount of TNF-{alpha} spontaneously during culture (120 pg/ml; Fig. 1 ). (No effect on TNF-{alpha} release was observed when macrophages were cultured with the control supernatant generated with the empty vector.) However, IL-12, when used at different concentrations, enhanced only marginally TNF-{alpha} release (200 pg/ml). This finding reveals that IL-12 by itself is not a strong inducer of macrophage TNF-{alpha} release. In contrast, upon stimulation with mycobacteria alone, macrophages released significant amounts of TNF-{alpha} (Fig. 1) , supporting the finding that macrophages release this cytokine in response to stimulation by a variety of infectious agents [3 ]. Because macrophages are exposed not only to mycobacteria but also to IL-12 during pulmonary mycobacterial infection [16 ], we examined the effect of combined stimulation by mycobacterial infection and IL-12. There was a remarkable synergistic effect on TNF-{alpha} release by mycobacteria and IL-12 (Fig. 1) . The level of induced TNF-{alpha} was 100–400% higher than by mycobacteria alone, depending on the dose of IL-12. These data provide strong evidence that IL-12 plays an active role in modulation of macrophage TNF-{alpha} responses during mycobacterial infection.



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  		Figure 1. Synergistic effect on macrophage TNF-{alpha} release by IL-12 and mycobacteria. Alveolar macrophages were isolated from the lung of wild-type C57BL/6 mice and cultured in the absence or presence of IL-12 (800 and 2000 pg/ml) alone, live mycobacteria BCG alone, or BCG with IL-12-containing supernatant for three days. The resultant supernatants were measured for TNF-{alpha} by ELISA. Cells cultured with BCG and control supernatant (C) released similar amounts of TNF-{alpha} as those cultured with BCG. Results for most conditions are expressed as mean ± SE from triplicate wells. The difference between no treat vs. BCG and BCG vs. BCG + IL-12 (2 ng/ml) is statistically significant (p<0.05). The results are representative of at least two to three independent experiments.

 
To verify the effect of IL-12-containing culture supernatants on macrophage TNF-{alpha} release, we used the same doses of recombinant murine IL-12 (rIL-12) protein also to stimulate macrophages. Similar to our findings obtained by using IL-12-containing supernatants, although rIL-12 released little TNF-{alpha}, rIL-12 and mycobacteria had a synergistic effect on TNF-{alpha} release (Table 2 ).


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  		Table 2. TNF-{alpha} Release by Macrophages (pg/ml)

 
Effect of IFN-{gamma} and mycobacteria on TNF-{alpha} release by macrophages
Because we and others have shown that macrophages can release IFN-{gamma} under appropriate stimulatory conditions [11 12 13 ], the synergistic effect on TNF-{alpha} release by IL-12 and mycobacteria may be mediated through endogenous IFN-{gamma}. To address this issue, we examined first the content of IFN-{gamma} in the supernatants from IL-12- or IL-12/mycobacteria-stimulated macrophages of C57BL/6 mice. Indeed, although mycobacteria and IL-12 alone released little or small amounts of IFN-{gamma}, respectively, IL-12 and mycobacteria together synergistically released significant amounts of IFN-{gamma} (Fig. 2A ). We then examined the effect of exogenously added IFN-{gamma} in the presence or absence of mycobacteria on TNF-{alpha} release from alveolar macrophages isolated from naive C57BL/6 mice. IFN-{gamma} alone, as compared with IL-12 alone, appeared to be more potent in releasing TNF-{alpha} (300–450 pg/ml), although this difference was not statistically significant (p=0.09; Figs. 1 and 2B ). Furthermore, we examined whether there was a synergistic effect on TNF-{alpha} release when macrophages were stimulated with IFN-{gamma} and mycobacteria. Indeed, IFN-{gamma} and mycobacteria together enhanced synergistically TNF-{alpha} release by 200–500% compared with that by mycobacteria alone (Fig. 2B) .



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  		Figure 2. (A) Synergistic effect on macrophage IFN-{gamma} release by IL-12 and mycobacteria. Alveolar macrophages were obtained from the lung of wild-type C57BL/6 mice and cultured in the absence or presence of IL-12 alone, BCG alone, BCG with control supernatant (C), or BCG with IL-12-containing supernatant for three days. The supernatants were measured for IFN-{gamma} by ELISA. (B) Synergistic effect on macrophage TNF-{alpha} release by IFN-{gamma} and mycobacteria. Alveolar macrophages were obtained from the lung of wild-type C57BL/6 mice and cultured in the absence or presence of IFN-{gamma} alone (800 and 2000 pg/ml), BCG alone, BCG with control supernatant (C), or BCG with IFN-{gamma}-containing supernatant. The supernatants were measured for TNF-{alpha} by ELISA. Results for most conditions are expressed mean ± SE from triplicate wells. The difference between no treat vs. BCG and BCG vs. BCG + IFN-{gamma} (2 ng/ml) is statistically significant (p<0.05). The results are representative of at least two to three independent experiments.

 
Again, to verify the effect of IFN-{gamma}-containing supernatant on TNF-{alpha} release by macrophages, we tested the effect of rIFN-{gamma}. Very similar to our findings presented in Fig. 2B , rIFN-{gamma} by itself enhanced macrophage TNF-{alpha} release only moderately, and rIFN-{gamma} + mycobacteria enhanced TNF-{alpha} release markedly in a synergistic manner (Table 2) .

Synergistic effect on TNF-{alpha} release by IL-12/mycobacteria is dependent on endogenous IFN-{gamma}
Having established that IL-12 and mycobacteria release synergistically not only TNF-{alpha} but also IFN-{gamma} and that exogenous IFN-{gamma} together with mycobacteria stimulate synergistically TNF-{alpha} release in macrophages, we set out to examine whether the potentiated release of TNF-{alpha} by IL-12/mycobacteria required endogenous IFN-{gamma}. To this end, we isolated alveolar macrophages from the lung of naive mice deficient in the gene coding for IFN-{gamma} and cultured them under different conditions. IL-12 alone stimulated little TNF-{alpha} release, whereas IFN-{gamma} alone stimulated a higher level of TNF-{alpha} release by IFN-{gamma}-/- macrophages (Fig. 3A ). Mycobacteria alone released significant amounts of TNF-{alpha} from these cells, comparable with those by cells from wild-type C57BL/6 mice (Fig. 1) . However, stimulation by IL-12 and mycobacteria failed to enhance TNF-{alpha} release further in the absence of endogenous IFN-{gamma} and as a positive control, exogenously added IFN-{gamma} with mycobacteria released synergistically TNF-{alpha} from these cells (Fig. 3A) . These findings reveal that IFN-{gamma} is required for IL-12/mycobacteria-enhanced TNF-{alpha} responses in macrophages.



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  		Figure 3. (A) Requirement of endogenous IFN-{gamma} for IL-12/mycobacteria-enhanced TNF-{alpha} response in macrophages. Alveolar macrophages were isolated from the lung of IFN-{gamma} gene knock-out mice and cultured in the absence or presence of IL-12, IFN-{gamma}, or BCG alone, or BCG with IL-12 or IFN-{gamma}. In the absence of IFN-{gamma}, IL-12/mycobacteria failed to enhance TNF-{alpha} release synergistically from macrophages. The difference between IL-12 + BCG and IFN-{gamma} + BCG is statistically significant (p<0.01). (B) Independence of endogenous IL-12 for IFN-{gamma}/mycobacteria-enhanced TNF-{alpha} response in macrophages. Alveolar macrophages were isolated from the lung of IL-12p40 gene knock-out mice and cultured in the absence or presence of IFN-{gamma} or BCG alone or BCG with IFN-{gamma}. The supernatants were measured for TNF-{alpha} by ELISA. The difference between BCG and BCG + IFN-{gamma} (800 pg or 2 ng/ml) is statistically significant (p<0.01). Results (A and B) are expressed as mean ± SE from triplicate wells/condition.

 
Synergistic effect of IFN-{gamma} and mycobacteria on macrophage TNF-{alpha} release is IL-12-independent
We have, thus far, demonstrated the requirement of endogenous IFN-{gamma} for IL-12/mycobacteria-enhanced TNF-{alpha} release. Although we have shown that macrophages under the current stimulatory conditions released little bioactive IL-12p70 (Table 1) , it cannot be ruled out that endogenous IL-12p70, which may present in a membrane-bound form on macrophages [27 ], may potentiate the effect by IFN-{gamma} and mycobacteria. To this end, we examined the response in alveolar macrophages isolated from mice deficient in IL-12. We found that mycobacteria alone released increased amounts of TNF-{alpha} in the absence of endogenous IL-12, similar to those by macrophages from wild-type C57BL/6 (Fig. 1 ; Fig. 3B ), and the lack of IL-12 did not compromise at all the synergistic effect of IFN-{gamma} and mycobacteria on TNF-{alpha} release (Fig. 3B) . These findings have thus established a cascade of macrophage type 1 cytokine responses during intracellular mycobacterial infection: 1) Infection of macrophages by mycobacteria triggers basal levels of TNF-{alpha} release, which is IL-12- or IFN-{gamma}-independent. 2) IL-12 and mycobacteria are required for macrophage IFN-{gamma} release. 3) It is IFN-{gamma} that, together with intracellular mycobacteria, enhances the level of TNF-{alpha} release synergistically; importantly, this enhanced TNF-{alpha} release results from direct stimulation by IFN-{gamma}/mycobacteria, which is independent of IL-12.

IFN-{gamma} and mycobacteria do not have a synergistic effect on MCP-1 release from macrophages
It was important to examine also whether IFN-{gamma} and mycobacteria have a synergistic effect on the release of cytokines other than TNF-{alpha} in macrophages. To this end, we have chosen to examine the release of MCP-1, a C-C mononuclear cell chemokine shown to play a role during mycobacterial infection [28 ]. IFN-{gamma} or mycobacteria alone only moderately enhanced MCP-1 release (Fig. 4 ). However, IFN-{gamma} and mycobacteria together did not impose a synergistic effect on MCP-1 release in macrophages obtained from naive C57BL/6 mice. These results suggest a restricted synergistic effect on macrophage cytokine responses by IFN-{gamma} and intracellular pathogens. We also cultured macrophages in the presence of IFN-{gamma}, mycobacteria, and an antimurine MCP-1 antibody, and measured TNF-{alpha} response. We found that MCP-1 antibody only minimally reduced TNF-{alpha} release, suggesting that MCP-1 is unlikely involved in IFN-{gamma} and mycobacteria-stimulated TNF-{alpha} release (unpublished results).



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  		Figure 4. IFN-{gamma} and mycobacteria cannot enhance MCP-1 response in macrophages synergistically. Alveolar macrophages were obtained from the lung of wild-type C57BL/6 mice and cultured in the absence or presence of IFN-{gamma} or BCG alone or BCG with IFN-{gamma} for three days. The supernatants were measured for MCP-1 by ELISA. Results are expressed as mean ± SE from triplicate wells. The difference among no treat vs. BCG, no treat vs. BCG + IFN-{gamma}, and BCG vs. BCG + IFN-{gamma} is all statistically significant (p<0.05, p<0.01, and p<0.05, respectively).

 
Synergistic effect of IFN-{gamma} and mycobacteria on macrophage NO release
Activated macrophages produce NO, which is required for effective killing of intracellular mycobacteria [29 , 30 ]. We examined the regulation of NO release by macrophages under various conditions. The amount of NO was evaluated by measuring the concentration of the end product of NO nitrite in supernatants of alveolar macrophages obtained from naive C57BL/6 mice. Unstimulated macrophages released minimal amounts of NO. Of interest, mycobacteria or IL-12 alone released little NO, and IFN-{gamma} alone enhanced NO release moderately (Fig. 5 ). The effect of IL-12 on macrophage NO release had not been investigated previously. It has been found that only IFN-{gamma} but not any other macrophage-activating cytokines including IL-1, TNF-{alpha}, and granulocyte-macrophage colony-stimulating factor (GM-CSF) can release NO from naive macrophages in vitro [31 ]. Our finding that IL-12 alone is unable to release NO from macrophages supports such highly selected activity of proimmune cytokines in macrophage NO release. In contrast, we observed a synergistic effect on NO release by combined stimulation with mycobacteria and IL-12 or IFN-{gamma}. However, in the absence of intrinsic IFN-{gamma}, mycobacteria and IL-12 failed to induce NO production, in sharp contrast to NO induction by mycobacteria and exogenously added IFN-{gamma} (Fig. 5) . These findings indicate that IL-12 and mycobacteria synergized to induce NO via the effect of endogenously released IFN-{gamma} and extend the previous observation that a maximum of NO release from macrophages requires IFN-{gamma} and lipopolysaccharide (LPS) [32 ]. Our data suggest also that intracellular pathogens and IFN-{gamma} together form a unique combination leading to the full activation of macrophages, which is characterized by markedly enhanced release of TNF-{alpha} and NO.



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  		Figure 5. Synergistic effect of mycobacteria and IL-12 or IFN-{gamma} on NO release by macrophages. Alveolar macrophages were obtained from the lung of C57BL/6 or IFN-{gamma}-deficient (IFN-{gamma}-/-) mice and cultured in the absence or presence of BCG, IL-12 (800 pg/ml), or IFN-{gamma} (800 pg/ml), or a combination of BCG and IL-12 or IFN-{gamma}. The concentration of NO was determined by measuring nitrite in culture supernatants. Results are expressed as average value from two to three wells/condition.

 
The above findings have enhanced our understanding of the role of IL-12 in macrophage activation during intracellular infection. They suggest that during intracellular bacterial infection, IL-12 and TNF-{alpha} are released from antigen-presenting cells. The initial macrophage TNF-{alpha} release is IFN-{gamma}-independent. IL-12, together with intracellular bacteria, is an ignitor of macrophage activation. IL-12-stimulated IFN-{gamma} release from macrophages and lymphocytes is an ultimate macrophage activator, which, together with intracellular pathogens, is capable of markedly potentiating macrophage release of TNF-{alpha} and NO, molecules crucial to mycobactericidal activities (Fig. 6 ).



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  		Figure 6. Schematic presentation of cytokine cascade during pulmonary mycobacterial infection. Intracellular pathogens release IL-12 and TNF-{alpha} from antigen-presenting alveolar macrophages (AM) and dendritic cells (DC). TNF-{alpha} release by these cells is IFN-{gamma}-independent. IL-12 acts on antigen-specific T cells and together with pathogens, acts on alveolar macrophages to release IFN-{gamma}. Macrophage IFN-{gamma} release requires intracellular pathogens and IL-12. IFN-{gamma} and mycobacteria synergistically activate macrophages to release antimicrobial molecules including TNF-{alpha} and NO.

 


   ACKNOWLEDGEMENTS
 
This study was supported by the Ontario Thoracic Society, McMaster University, Hamilton Health Sciences Corporation, and St. Joseph’s Hospital. Z. X. is a scholar of the Medical Research Council, Canada, and a recipient of Ontario Premier’s Research Excellence Award. The authors are grateful to Dr. Robin Harkness for providing M. bovis BCG, Dr. J. Magram for providing IL-12-/- mouse-breeding pairs, and Dr. Jonathan Bramson for providing recombinant cytokines. An adenoviral gene-transfer vector for murine IFN-{gamma} was kindly provided by Dr. Jay Kolls. An adenoviral gene-transfer vector for murine IL-12 was constructed by Drs. Jonathan Bramson and Mary Hitt at McMaster University.

Received April 6, 2000; revised June 29, 2000; accepted June 30, 2000.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 REFERENCES
 

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