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American Journal of Respiratory and Critical Care Medicine Vol 166. pp. 178-186, (2002)
© 2002 American Thoracic Society

Original Article

Analysis of Nitric Oxide Synthase and Nitrotyrosine Expression in Human Pulmonary Tuberculosis

Hyung-Seok Choi, Pradeep R. Rai, Hong Wei Chu, Carlyne Cool and Edward D. Chan

Department of Medicine, Program in Cell Biology, National Jewish Medical and Research Center, Denver Veterans Administration Medical Center, Division of Pulmonary Sciences and Critical Care Medicine, and Department of Pathology, University of Colorado Health Sciences Center, Denver, Colorado

Correspondence and requests for reprints should be addressed to Edward D. Chan, M.D., K613e, Goodman Building, National Jewish Medical and Research Center, 1400 Jackson Street, Denver, CO 80206. E-mail: chane{at}njc.org


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The role of nitric oxide (NO) in the host-defense against human tuberculosis (TB) is controversial. Although experimental evidence indicates that NO may play an important role in controlling TB, its expression in human tuberculous lungs has not been systematically characterized. We therefore investigated the expression of NO synthases (NOS) and of nitrotyrosine, the latter a marker of NO expression, in surgically resected lungs of eight patients with TB. Immunohistochemical and morphometric analyses revealed that, compared with control subjects, inducible NOS, endothelial NOS, and nitrotyrosine, but not neuronal NOS, were significantly elevated in the inflammatory zone of the tuberculous granulomas, and in the nongranulomatous pneumonitis zone. Tumor necrosis factor-{alpha} (TNF-{alpha}) was also significantly increased in tuberculous lungs and was principally localized to the necrotic, and to a lesser extent, the inflammatory and fibrotic areas of the granulomas. The NOS isoforms, nitrotyrosine, and TNF-{alpha} were expressed by the epithelioid macrophages and giant cells within the granulomas and in alveolar macrophages and epithelial cells in pneumonitis areas. This descriptive study provides evidence that in human TB, NOS isoenzymes and NO are present in specialized areas of the tuberculous granulomas; their precise role in human TB remains to be determined.

Key Words: Mycobacterium tuberculosis • nitric oxide • reactive nitrogen intermediates • immunohistochemistry • morphometry


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Although a third of the world's population is infected with Mycobacterium tuberculosis, the vast majority of individuals infected hold the mycobacteria in check. This observation would support the notion that the innate and specific immune system, characterized predominantly by macrophage and T cell responses against the tubercle bacilli, is generally successful in containing, although not necessarily in eliminating, the pathogen. M. tuberculosis is known to persist in human macrophages within granulomas, distinct lesions represented by a central necrotic area surrounded by inflammatory cells consisting of epithelioid macrophages, multinucleated giant cells, T cells, B cells, and scattered foci of fibroblasts. Granuloma formation is important in host-defense against intracellular bacteria and other types of foreign antigens. Survival of the host against M. tuberculosis may depend on the ability to form effective granulomas that limit microbial proliferation (1). Formation of tuberculous granulomas is complex, dependent on the migration and activation of an array of immune cells, initiated by cytokine and chemokine expression in response to mycobacterial antigens. Among the cytokines, tumor necrosis factor-{alpha} (TNF-{alpha}) is known to be critical in the formation of tuberculous granulomas (2, 3).

Nitric oxide (NO) is produced by the catalytic action of NO synthases (NOS), of which there are three principal members in mammalian cells: inducible NOS (iNOS), endothelial NOS (eNOS), and neuronal NOS (nNOS). Mice with a genetic disruption of iNOS (iNOS-/-) have a higher risk of dissemination and mortality from intravenously infected tuberculosis (TB) (4). The relative importance of NO in mice challenged by aerosolized M. tuberculosis is controversial, and the debate is evolving (5, 6). In vitro, NO plays an essential role in killing, or at least in limiting the growth of M. tuberculosis by murine mononuclear phagocytes. In contrast, the role of NO and other reactive nitrogen intermediates in human defense against TB is even more contentious, a debate fueled by initial reports that human macrophages do not produce NO. However, there are now at least one hundred citations in the literature that human macrophages are capable of producing NO (7). Moreover, human macrophage cell lines, monocyte-derived macrophages, and human alveolar macrophages have all been shown to produce NO upon infection with M. tuberculosis (8). Nicholson and coworkers (9) demonstrated the presence of catalytically active iNOS in alveolar macrophages of TB patients. Alveolar macrophages isolated from TB patients also release increase amounts of spontaneous NO compared with normal control subjects (10). These studies, together with in vitro investigations showing that human macrophages can kill M. tuberculosis via NO (8), suggest that NO may, directly or indirectly, serve an antimycobacterial role in human TB. Although the precise role of NO in TB has not been clearly defined, NO has been found to be essential in granuloma formation of infectious (11–16) and noninfectious (17) animal models. However, morphologic characterization of NO and NOS isoforms in granulomatous lesions of human pulmonary TB has not been previously reported. Thus, we quantitated the expression of NOS isoenzymes and nitrotyrosine in the pneumonitis and granulomatous regions of human pulmonary TB. In addition, we quantitated the expression of TNF-{alpha} and of macrophages, T cells, and B cells, known constituents of tuberculous granulomas, to compare their relative expressions with NOS and nitrotyrosine. Because iNOS is able to catalyze the production of relatively large quantities of NO, we hypothesized that iNOS and nitrotyrosine expression would be increased in the inflammatory regions of the granulomas and the pneumonitis areas.


   METHODS
TOP
 ABSTRACT
 INTRODUCTION
 METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Materials
Lung tissues were obtained after Institutional Review Board approval from eight TB patients who underwent pulmonary resection at the University of Colorado Health Sciences Center, performed as adjunctive treatment for either multidrug-resistant M. tuberculosis (seven patients) or recalcitrant drug-susceptible TB (one patient). All the patients with TB were HIV-negative and were on various antimycobacterial regimens at the time of their surgeries. For controls, lung tissues were obtained from four randomly chosen individuals who had undergone resectional surgery for diseases other than TB: two had carcinoid tumors, one had adenocarcinoma of the lung, and one had a metastatic synovial tumor. The control lung tissues used for immunostaining were histologically normal. Polyclonal antibodies specific against human iNOS and nNOS, and mouse monoclonal anti-human eNOS antibody were purchased from Transduction Laboratories (San Diego, CA). Polyclonal anti-nitrotyrosine antibody was obtained from Upstate Biotechnology (Lake Placid, NY). Anti-human TNF-{alpha} antibody was purchased from R&D Systems (Minneapolis, MN). Vectastain Elite ABC immunoperoxidase system was purchased from Vector Laboratories (Burlingame, CA). Anti-CD3, anti-CD20, and anti-CD68 antibodies were purchased from DAKO Corporation (Carpinteria, CA). Mayer's hematoxylin, 3-amino-9-ethyl-carbazole, 3,3'-diaminobenzidine, and N,N'-dimethylforamide were purchased from Sigma Chemicals (St. Louis, MO).

Immunohistochemical Staining
Immunohistochemical staining was performed as previously described (18) for iNOS, eNOS, nNOS, nitrotyrosine, and TNF-{alpha}, as well as cellular markers for macrophages (CD68), T cells (CD3), and B cells (CD20). For each antigen, the controls and the tuberculous lungs were immunostained simultaneously. In brief, after formalin-fixed, paraffinized lung tissues were cut in 5-µm sections, the slides were deparaffinized and rehydrated. High-temperature epitope retrieval was performed as previously described. The Vectastain Elite ABC Kit was used according to manufacturer's instructions. Primary antibody in the following dilutions (1:250 for anti-iNOS and anti-nitrotyrosine, 1:125 for anti-nNOS, 1:400 for anti-eNOS, and 1:500 for anti-TNF-{alpha}) was then incubated with the lung tissues at 4°C overnight. For the negative control, normal blocking serum without primary antibody was used. The nuclei were counterstained with Mayer's hematoxylin.

To identify T cells, B cells, and macrophages, the slides were incubated with anti-CD3/HRP, anti-CD20/HRP, and anti-CD68/HRP antibody solution, respectively, as described by the manufacturer.

Morphometric Analysis
Two-dimensional morphometric analyses were performed for the eight antigens (iNOS, eNOS, nNOS, nitrotyrosine, TNF-{alpha}, CD68, CD3, and CD20) in each of the three granuloma zones (a central necrotic zone, an inflammatory zone, and an outer fibrous zone) and the surrounding nongranulomatous pneumonitis zone for the tuberculous lungs. For the control lungs, the analysis following the same method was performed on the morphologically normal lung tissues; the alveolar spaces free of any cells were subtracted from the total area. For each slide, 10 representative but random microscopic fields (magnification: x200) in each zone were analyzed with a Nikon Diaphot microscope. The ratio of positive staining area to the total area for each of the four zones was calculated using IP-Lab 3.1a software for the Macintosh (Scanlytics, Fairfax, VA) as described previously (19) and per manufacturer's instructions. In brief, positive staining areas in each zone were identified by microscopy and captured as digital pictures. For each antigen, the ratio of positive area to total microscopic area in the digital picture was calculated by the software. For each antigen, the mean percent of the positive staining area to the total microscopic area was calculated for 10 separate microscopic fields.

Statistical Analysis
Values for all measurements were expressed as mean ± SEM. The mean values were compared in each area between the patients' group and the normal control group. Group means were compared by Wilcoxon rank-sum test. Differences were considered significant when p values were less than 0.05.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Validation of the Specificity of the NOS Isoform Antibodies
To validate the specificity of the three NOS antibodies, immunoblotting was performed on whole cell lysates prepared from human endothelial cells, interferon-{gamma} (IFN{gamma}) + lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophages, and rat pituitary tumor, known to express eNOS, iNOS, and nNOS, respectively. As shown in Figure 1A, each of the antibodies was relatively specific for eNOS (140 kD), iNOS (130 kD), or nNOS (155 kD). To confirm the specificities of the antibodies in intact cells or tissues, we performed immunohistochemistry on an autopsy lung tissue and immunocytochemistry on unstimulated and IFN{gamma} + LPS–stimulated RAW 264.7 cells. As shown in Figures 1B–1D, endothelial cells on a blood vessel (bv) immunostained for eNOS (Figure 1B, arrowheads) but not for nNOS (Figure 1C) or iNOS (Figure 1D). Conversely, a nerve fiber immunostained for nNOS (Figure 1C, arrowheads) but not for eNOS (Figure 1B) or iNOS (Figure 1D). In contrast, there was no significant iNOS expression in either the blood vessel or nerve fiber (Figure 1D), but iNOS was strongly present in alveolar macrophages (Figure 1D, arrowheads). In unstimulated RAW 264.7 macrophages, there was no eNOS expression, whereas in IFN{gamma} + LPS–stimulated cells, there was a very slight increase in punctate eNOS staining (Figures 1E and 1F). In both unstimulated and stimulated macrophages, there was a modest amount of basal nNOS detected (Figures 1G and 1H), although in the absence of stimulation, there is no detectable NO production (data not shown). However, compared with unstimulated cells (Figure 1I), catalytically active iNOS showed the greatest induction with IFN{gamma} + LPS stimulation (Figure 1J). Hence, the differential expression of each of the NOS isoforms in these experiments illustrates that the NOS isoform antibodies are relatively specific for their respective NOS.



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  		Figure 1. Specificity of the NOS antibodies. The specificity of the three NOS antibodies, namely anti-eNOS, anti-iNOS, and anti-nNOS, (denoted {alpha}-eNOS, {alpha}-iNOS, and {alpha}-nNOS), was tested by two methods. In (A), Western blot was performed with each of the three antibodies against lysates prepared from human endothelial cells (ec, source of eNOS), IFN{gamma} + LPS–stimulated RAW 264.7 macrophages (MØ, source of iNOS), and rat pituitary tumor (pit, source of nNOS). As shown, each of the three NOS antibodies was relatively specific for eNOS (140 kD), iNOS (130 kD), or nNOS (155 kD). In (B–D), immunohistochemistry was performed with each of the three antibodies on an autopsy lung tissue. As can be seen in (B), {alpha}-eNOS immunostained for endothelial cells on a blood vessel (bv, arrowheads) but not a nerve fiber (n); in (C), the reverse pattern was seen in that the nerve fiber (n, arrowheads) but not the blood vessel immunostained with {alpha}-nNOS. In (D), there was little or no iNOS expression in either the blood vessel (bv) or nerve fiber (n), but iNOS was abundantly present in alveolar macrophages (arrowheads). In (E–J), unstimulated and IFN{gamma} + LPS–stimulated RAW 264.7 macrophages were immunostained with the NOS antibodies. There was no eNOS expression in unstimulated cells (E) and minimal increase in stimulated macrophages as shown by punctate eNOS immunostaining (F). There was modest basal nNOS staining in both unstimulated and stimulated cells (G–H). As shown in (I and J), iNOS showed the greatest induction with IFN{gamma} + LPS stimulation.

 
Total Expression of NOS Isoforms, Nitrotyrosine, TNF-{alpha}, and Cellular Markers
To determine whether expression of NOS isoforms was elevated in pulmonary TB, the lungs of eight TB patients and four control subjects were immunostained and quantitated for iNOS, eNOS, and nNOS. As shown in Figure 2, expressions of iNOS and eNOS were significantly elevated in the TB patients. In contrast, nNOS expression was not significantly different between TB patients and control subjects. Nitrotyrosine abundance, an indirect marker of NO expression, was also elevated in the tuberculous lungs. In addition, there was an overall increase in TNF-{alpha} expression as well as an increase in the number of macrophages (CD68), T cells (CD3), and B cells (CD20) in the tuberculous lungs (Figure 2). Immunohistochemistry with a nonimmune isotype antibody of each of the lungs showed no immunostaining (data not shown).



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  		Figure 2. Morphometric quantitation of NOS isoforms, nitrotyrosine, TNF-{alpha}, and cellular markers (CD68, CD3, CD20) in eight tuberculous lungs versus four control lungs. As described in the METHODS section, morphometric analysis was performed on 10 representative but random microscopic fields for each antigen in each TB patient. Similar calculations were performed for the four control lungs. The mean percent of the positive staining area ([ratio of positive staining area divided by the total microscopic area measured] x 100) was determined for each antigen and reported as percent expression. iNOS = inducible nitric oxide synthase; eNOS = endothelial NOS; nNOS = neuronal NOS; NT = nitrotyrosine; TNF-{alpha} = tumor necrosis factor-{alpha}. *p < 0.05, **p < 0.01, ns = not significant, compared with their respective control lungs.

 
Localization of NOS Isoforms and Nitrotyrosine
To further elucidate whether each of the NOS isoforms was preferentially localized to specific pathologic sites, their expressions were quantitated in each of the three granulomatous zones and the pneumonitis area. Well-defined granulomas such as that shown in Figure 3 were chosen for morphometric analysis. Necrotic zones (n) were readily identified as central amorphous areas. Inflammatory zones (i) were noted as regions containing a profusion of mononuclear cells surrounding the necrotic area. The fibrotic zone (f) was not as easily identified but was characterized by increased collagen staining in areas surrounding the inflammatory zone (Figure 3). Interestingly, as shown in Figures 3 and 4B, increased collagen was also occasionally found in the inflammatory zone that was immediately adjacent to the necrotic zone. Pneumonitis areas (p) were defined as abnormal lung characterized by chronic inflammation but without granulomas. As shown in Figure 4A, there was minimal staining for iNOS in the normal control lungs. In contrast, there was a significant increase in the amount of iNOS staining in the inflammatory zone of granulomas (Figure 4B) and in pneumonitis areas (Figure 4C). The principal cell types expressing iNOS were macrophages (inset of Figure 4B and arrowheads in Figure 4C) and multinucleated giant cells. As expected, lymphocytes did not express iNOS (Figures 4C, arrows). Similarly, compared with lung tissues of normal control subjects (Figure 5A), those of patients with TB showed increased expression of eNOS in the granulomatous (Figure 5B) and pneumonitis (Figure 5C) areas. As shown in Figures 4D and 5D, increases in iNOS and eNOS expression in both the inflammatory regions of the granulomatous and pneumonitis areas of the TB patients were significant. Although nNOS expression was also increased in the inflammatory region of the granuloma and the pneumonitis areas compared with normal controls (Figure 6A versus Figures 6B and 6C), this was not statistically significant (Figure 6D, Bars 3 and 5 versus Bar 1).



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  		Figure 3. Identification of the granuloma zones in tuberculous lungs stained with sirius red for collagen (original magnification: x400). The necrotic zone (n) is well-delineated as a central acellular region. The inflammatory zone (i) is characterized by a profusion of mononuclear inflammatory cells, consisting primarily of epithelioid macrophages, lymphocytes, and multinucleated giant cells. Occasionally, the margins between the inflammatory and fibrotic zone ( f ) are less distinct, but the fibrotic zone is defined as areas of granulomas with increased collagen deposition and decreased cellular infiltration. The pneumonitis area (p) is defined as an area of chronic inflammation in the lung parenchyma distinct from granulomas.

 


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  		Figure 4. iNOS expression. In (A), a representative control lung stained for iNOS (original magnification: x400). Shown in (B) is a granuloma with the necrotic, inflammatory, and fibrotic zones identified as n, i, and f, respectively (original magnification: x100). The inset (original magnification: x400), shows epithelioid macrophages in the inflammatory region with iNOS expression. Shown in (C) is a pneumonitis region revealing macrophages that are positive for iNOS (arrowheads) and lymphocytes with absent iNOS staining (arrows) (original magnification: x400). Figure (D) shows the percentage of iNOS expression for the four controls and the three granulomatous zones, and pneumonitis areas for the eight tuberculous lungs. c = control lungs, n = necrotic zone of granuloma, i = inflammatory zone of granuloma, f = fibrotic zone of granuloma, p = pneumonitis area. *p < 0.05 compared with control lungs.

 


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  		Figure 5. eNOS expression. In (A), a representative control lung stained for eNOS (original magnification: x400). Note that in this particular section, intra-alveolar macrophages constitutively express eNOS (arrows). Shown in (B) is a granuloma with necrotic (n), inflammatory (i), and fibrotic (f ) zones identified (original magnification: x100). The inset (x400) demonstrates a multinucleated giant cell with eNOS expression. Note also the intense staining of eNOS in the bronchial epithelium (arrowheads) in addition to the inflammatory (i) region. Shown in (C) is a pneumonitis area with increased eNOS expression in alveolar macrophages (original magnification: x400). In (D), the percentages of eNOS expression in the different regions for the four control lungs and eight tuberculous lungs. (See legend to Figure 4 for abbreviations). **p < 0.01 compared with control lungs.

 


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  		Figure 6. nNOS expression. In (A), a representative control lung stained for nNOS with intra-alveolar macrophages indicated by arrows (original magnification: x400). Shown in (B) is a granuloma with necrotic (n) and inflammatory (i) zones identified (original magnification: x400). The inset (x400) shows a multinucleated giant cell with positive staining for nNOS. Shown in (C) is a cluster of intra-alveolar macrophages (arrow) with positive nNOS staining in a pneumonitis area (original magnification: x400). In (D), the percentages of nNOS expression in the different regions for the four control lungs and eight tuberculous lungs (see legend to Figure 4 for abbreviations). ns = not significant with p > 0.05.

 
The chemical reaction by which NO combines with O2- to form peroxynitrite (ONOO-) is one of the most rapid and complete reactions recorded. Because ONOO- has the capacity to nitrate tyrosine residues, the presence of nitrotyrosine in tissues accurately reflects the presence of NO. Thus, to determine whether the increased NOS expression in the TB lungs correlated with increased NO production and to assess its immunolocalization, the lung tissues were stained for nitrotyrosine. Compared with normal lungs (Figure 7A), there was increased nitrotyrosine immunostaining of the tuberculous lungs, principally localized to the macrophages and multinucleated giant cells in both the granulomatous (Figure 7B) and pneumonitis (Figure 7C) areas. The increased expression of nitrotyrosine in the inflammatory regions of the granulomas and the pneumonitis areas was significant (Figure 7D).



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  		Figure 7. Nitrotyrosine expression. In (A), a representative control lung stained for nitrotyrosine (original magnification: x150). This particular field displayed normal cells but atelectatic lung tissues. Shown in (B) is a granuloma with necrotic (n), inflammatory (i), and fibrotic ( f ) zones identified (original magnification: x100). The inset (x400) reveals a multinucleated giant cell with positive staining for nitrotyrosine. Shown in (C) is a pneumonitis zone (original magnification: x400). Note the giant cell (arrowhead) and the alveolar macrophages (arrows) with intense nitrotyrosine staining. In (D), the percentages of nitrotyrosine expression for the four control lungs, and the different granulomatous and pneumonitis regions for the eight tuberculous lungs are shown (see legend to Figure 4 for abbreviations). *p < 0.05 compared with control lungs.

 
Localization of TNF-{alpha}
Because there are convincing data on the importance of TNF-{alpha} in tuberculous granuloma formation and effective immune responses against M. tuberculosis (2, 3, 20, 21), we determined if TNF-{alpha} colocalized to the same areas of the tuberculous lungs in which the NOS isoforms and nitrotyrosine were found. As shown in Figure 8A, there was little TNF-{alpha} expression in control lungs. In contrast, there was a significant increase in TNF-{alpha} expression in all three zones of granulomas of the tuberculous lungs, especially in the necrotic and inflammatory zones and in the pneumonitis area (Figures 8B–8D).



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  		Figure 8. TNF-{alpha} expression. In (A), a representative control lung stained for TNF-{alpha} (original magnification: x150). Shown in (B) is a granuloma immunostained for TNF-{alpha}. Note that the necrotic zone (n) is well-delineated, but in this particular granuloma, the inflammatory (i) and fibrotic (f ) zones were not well-demarcated (original magnification: x100). The inset (x400) shows a giant cell with positive TNF-{alpha} staining. Shown in (C) is a pneumonitis area with abundant TNF-{alpha}–positive interstitial macrophages (original magnification: x400). In (D), the percentages of TNF-{alpha} expression in the different regions for the four control lungs and eight tuberculous lungs. *p < 0.05, **p < 0.01 compared with control lungs. See legend to Figure 4 for abbreviations.

 
Localization of Macrophages, T Cells, and B Cells
To localize macrophages, T cells, and B cells in the three granulomatous zones and pneumonitis area, lung tissue sections in both the control and TB patient groups were immunostained for CD68, CD3, and CD20, respectively. Immunostaining with anti-CD68 antibody revealed numerous macrophages (Figure 9A) and multinucleated giant cells in the inflammatory areas of the granuloma, although macrophage markers were significantly increased in the pneumonitis areas and all three granulomatous zones (Figures 9A and 9D). Intriguingly, there was an abundance of CD68 in the necrotic area, although there were minimal distinct cells identifiable in this region. Significantly increased numbers of T cells were also found in the inflammatory and fibrotic areas of granulomas (Figures 9B and 9D). B cell accumulation was only significantly increased in the inflammatory region of the granulomas (Figures 9C and 9D).



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  		Figure 9. Localization of macrophages (CD68), T cells (CD3), and B cells (CD20) in the granulomatous and pneumonitis areas. Shown in (A) is the immunolocalization of CD68 in the pneumonitis area of a tuberculous lung (original magnification: x400). In (B), a granuloma with the necrotic (n), inflammatory (i), and fibrotic ( f ) zones identified is immunostained for CD3 (original magnification: x100). Shown in (C) is a granuloma with the necrotic (n) and inflammatory (i) zones identified, immunostained for CD20 (original magnification: x100). Shown in (D) is the quantitation of each of the three cells types in the control lungs and in the four microanatomical zones of the tuberculous granulomas (see legend to Figure 4 for abbreviations). *p < 0.05, **p < 0.01 compared with their respective control lungs.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The potential mechanisms by which NO and other reactive nitrogen intermediates affect antimicrobial activity are protean, including (1) modification of bacterial proteins and lipids at both microbial surfaces and intracellularly, (2) deamination of bacterial DNA, generating abasic sites and strand breaks, (3) direct interaction with accessory protein targets, resulting in enzymatic inactivation or other protein malfunctions, and (4) induction of macrophage apoptosis, considered by many to be necessary to initiate intracellular mycobacterial killing (7, 22, 23). Despite these potential effects of NO, distinctive M. tuberculosis strains have variable susceptibility to different species of reactive nitrogen intermediates; the reason for these differences is not known (24–26).

The present investigation demonstrates that human tuberculous lungs have increased expression of iNOS, eNOS, and nitrotyrosine in the inflammatory zone of granulomas and in pneumonitis areas. The principal cell in which NOS isoforms and nitrotyrosine were found was the alveolar macrophage and its derivatives, epithelioid macrophages and multinucleated giant cells. The eNOS isoform showed abundant expression in both macrophages and bronchial epithelial cells of TB patients. This finding is consistent with reports that all the NOS isoforms may be expressed in monocytes and macrophages (27) and that eNOS is also expressed by epithelial cells (28). Because eNOS is activated by increased intracellular calcium or by AKT signaling (29, 30), it is of considerable interest that TB granulomas may increase calcium absorption via ectopic vitamin D production and that the AKT pathway may be activated in macrophages by whole Mycobacterium avium (31) or by lipoarabinomannan of M. tuberculosis (32). Furthermore, it is becoming increasingly clearer that constitutive eNOS may also be transcriptionally regulated by extracellular stimuli, in addition to activation by increases in intracellular calcium and by posttranslational modifications (27, 33, 34). Thus, the relative contribution of eNOS to total NO expression in vivo is not known.

Although the findings from this descriptive study would lend credence to the notion that NO may play a role in tuberculous granuloma formation, what is the evidence in the literature linking NO to such a role? The precise factors necessary for granuloma formation are not fully understood and are likely to vary with different granuloma types. Recruitment of intravascular mononuclear cells is one obvious requirement for granuloma formation. For example, mice with a truncated intercellular adhesion molecule-1, a molecule necessary for diapedesis, were deficient in granuloma formation (35). In addition, chemokine and chemokine receptor expression likely contribute to the formation and maintenance of granulomas in chronic infections such as TB. It has been hypothesized that the importance of TNF-{alpha} in granuloma formation and maintenance is, in part, due to its regulation of chemokines and their receptors (36). In addition, mycobacterial lipoarabinomannan, a cell wall lipoglycan, may induce expression of the chemokines interleukin-8, macrophage chemotactic protein-1, and macrophage inhibitory protein-1ß from human peripheral blood mononuclear cells by a TNF-{alpha}–dependent mechanism (37).

NO has been implicated in granuloma formation due to either noninfectious stimuli (17, 38) or infectious agents (11–16), including mycobacterial infections. The precise mechanism(s) for this effect of NO is not known. One possibility is that NO may activate signaling pathways such as the mitogen-activated protein kinases (39), which regulate the expression of inflammatory cytokines and chemokines (40). Indeed, Kuo and colleagues (10) showed that alveolar macrophages isolated from TB patients expressed increased NO and that the generated NO amplified the synthesis of TNF-{alpha} and interleukin-1ß, linking NO to granuloma-associated cytokines. iNOS and NO have also been directly implicated in granuloma formation in response to various microbes such as Salmonella species (16), Cryptococcus neoformans (12), Leishmania donovani (14), Listeria monocytogenes (11), Schistosoma species (15), and M. bovis–bacillus Calmette-Guérin (13). Facchetti and colleagues (18) examined iNOS expression in abnormal human lymph nodes due to nonspecific, infectious, or noninfectious causes and found abundant catalytically active iNOS expression in epithelioid macrophages and multinucleated giant cells of infectious granulomas and sarcoidosis (18). Bacillus Calmette-Guérin–infected mice treated with an NOS inhibitor had granulomas that had greater necrosis, disruption, and bacillary load (13). Others have also shown that cytokines and iNOS were required for granuloma formation and control of tuberculous infection in C57BL/6 mice (1). Although less direct in its association, both the TNF-{alpha} p55 receptor knockout mice and the IFN{gamma} knockout mice had little NO production and showed poorly formed or absent granuloma formation with mycobacterial or paracoccidioidomycosis infection (41, 42). Treatment of Salmonella typhimurium–infected BALB/c mice with anti–IL-12 resulted in reduced IFN{gamma} and iNOS expression, and disruption of granuloma formation (43). Cooper and colleagues (5) demonstrated that iNOS-/- mice infected with M. tuberculosis by inhalation had increased neutrophil infiltration in the lungs, with heightened necrosis and disruption of the pulmonary granulomas. In contrast to these evidences that a lack of NO production may deleteriously alter the morphology of the granulomas, MacMicking and coworkers (4) found that lung granulomas appeared equally well-formed in either iNOS-/- or wild-type mice intravenously infected with M. tuberculosis. Perhaps differences in the route of infection explain these disparate reports via differential expression of relevant cytokines and chemokines. In addition, redundant roles may be played by NO, cytokines, and chemokines in the trafficking of cells that constituted granulomas. The role of NO in mycobacteria-induced granuloma formation may also be mycobacterial species–specific, because in BALB/c mice infected with M. avium, administration of an iNOS inhibitor in drinking water led to markedly increased numbers, cellularity, and sizes of granulomatous lesions in the absence of any effect on bacterial load (44).

As alluded to previously, perhaps NO serves as a chemotactic agent in the context of granuloma formation. Consistent with this possibility is the observation that NO may be a direct or indirect chemoattractant for neutrophils, monocytes, and lymphocytes (45–48). However, increased neutrophil accumulation in the granulomas of iNOS-/- mice infected with aerosolized M. tuberculosis would argue against NO being a neutrophil chemotactic factor (5, 49). In a rat model of zymosan- or silica-induced pulmonary disease, NO and ONOO- mediated the migration of monocytes and macrophages to the granuloma sites and induced macrophage chemotactic protein-1 in the lesions (48). In addition to its potential role as a chemoattractant, NO is required for epithelioid macrophage differentiation and activation in M. bovis infection (50). Furthermore, Garbe and colleagues (51) showed that reactive nitrogen intermediates may promote M. tuberculosis to a stationary phase of growth, a finding that is particularly relevant to the tuberculous granuloma, a site containing latent mycobacteria. The findings from the present study that eNOS, iNOS, and nitrotyrosine expression are increased in the pulmonary lesions of human TB would help support these experimental models.


   Acknowledgments
 
The authors are very grateful to Dr. Michael D. Iseman for his helpful discussions and support, and to Cally Duncan and Lynn Cunningham for their expert technical assistance. They also thank Dr. Matt Strand for help with the statistical analysis and Dr. James Fisher for helpful comments and critical review of the manuscript. The authors are also indebted to Mr. and Mrs. Paul Lowerre for their support of the Lowerre Foundation Mycobacteriology Research Award.

Supported by the Parke-Davis Atorvastatin Research Award, American Lung Association Career Investigator Award, and NIH-HL-66-112 (E. D. C.).

Received in original form January 11, 2002; accepted in final form April 23, 2002


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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