Effect of neutralizing transforming growth factor beta1 on the immune response against Mycobacterium tuberculosis in guinea pigs

Transforming growth factor beta (TGF-beta) is a cytokine which has been shown to suppress the antimycobacterial immune responses of humans and experimental animals. In this study, the contributions of TGF-beta to cytokine production in vivo were investigated by using the established guinea pig model of tuberculous pleurisy. Mycobacterium bovis BCG-vaccinated guinea pigs were injected intrapleurally with heat-killed virulent Mycobacterium tuberculosis. Eight days following induction of an antigen-specific pleural effusion, guinea pigs were injected intrapleurally with anti-TGF-beta1 or isotype control antibody. The following day, pleural exudates were removed, and the fluid volume and characteristics of the infiltrating cells were determined. Pleural fluid was analyzed for total interferon (IFN) and tumor necrosis factor (TNF) protein levels by using appropriate bioassays. RNA from pleural effusion cells was examined to determine TGF-beta1, TNF-alpha, IFN-gamma, and interleukin-8 mRNA levels by using real-time PCR. Proliferative responses of pleural effusion lymphocytes were examined in response to concanavalin A and purified protein derivative (PPD) in vitro. Treatment with anti-TGF-beta1 resulted in decreased pleural fluid volume and decreased cell numbers in the pleural space along with an increased percentage of lymphocytes and a decreased percentage of neutrophils. The bioactive TNF protein levels in pleural fluid were increased in guinea pigs treated with anti-TGF-beta1, while the bioactive IFN protein concentrations were not altered. Expression of TGF-beta1 and TNF-alpha mRNA was significantly increased following TGF-beta1 neutralization. Finally, PPD-induced proliferative responses of pleural cells from anti-TGF-beta1-treated animals were significantly enhanced. Thus, TGF-beta1 may be involved in the resolution of this local, mycobacterial antigen-specific inflammatory response.     

Interleukin-8 production in tuberculous pleurisy: role of mesothelial cells stimulated by cytokine network involving tumour necrosis factor-alpha and interleukin-1 beta

Interleukin-8 (IL-8) plays an important role in the host immune response to Mycobacterium tuberculosis by recruiting inflammatory cells to the site of infection. Here, we investigated the role of pleural macrophages and mesothelial cells in the production of IL-8 in tuberculous pleurisy. Large concentrations of IL-8 were detected in tuberculous pleural effusions, but not in pleural effusions associated with congestive heart failure (CHF). Tuberculous pleural macrophages and M. tuberculosis-infected CHF pleural macrophages produced large concentrations of IL-8. When immunohistochemistry was performed on pleural tissues, antigenic IL-8 was detected in the mesothelial cells lining the tuberculous pleura. Direct stimulation of cultured CHF pleural mesothelial cells with M. tuberculosis induced IL-8 secretion. However, conditioned media from M. tuberculosis-infected pleural macrophages (CoMTB) induced greater mesothelial cell IL-8 secretion. Tumour necrosis factor-alpha (TNF-alpha) and IL-1beta induced mesothelial cell IL-8 mRNA expression, and neutralizing anti-TNF-alpha antibody and IL-1 receptor antagonist nearly completely obliterated CoMTB-induced mesothelial cell IL-8 mRNA expression and protein secretion. These findings demonstrate that both pleural macrophages and mesothelial cells produce IL-8 in tuberculous pleurisy, and cytokines produced by M. tuberculosis-infected macrophages mediate mesothelial cell IL-8 production.     

Local production and localization of transforming growth factor-beta in tuberculous pleurisy.

Transforming growth factor-beta (TGF-beta) is one of the cytokines which play an immunosuppressive role in an inflammatory process. To investigate the local production of TGF-beta, we evaluated the levels of TGF-beta in tuberculous pleural effusions (TBPE) and non-tuberculous benign pleural effusions (non-TBPE) by the growth inhibition assay with Mv1Lu mink lung epithelial cells. The mean level of TGF-beta in TBPE (46.1 +/- 31.5 pM; mean +/- s.d.) was higher than in non-TBPE (21.7 +/- 12.3 pM) (P < 0.05). Although the level of interferon-gamma (IFN-gamma) in TBPE measured by ELISA was significantly higher than in non-TBPE, there was no significant difference in the levels of tumour necrosis factor-alpha (TNF-alpha) measured by ELISA between these two groups. Moreover, to elucidate localization of TGF-beta in tuberculous pleurisy, immunohistochemical studies of pleura, using the rabbit polyclonal antibody Ab39 against latent TGF-beta 1 binding protein (LTBP) were performed. Results revealed that LTBP was localized in immature fibrotic areas where infiltrations of T lymphocytes and macrophages were absent. Importantly, the major sources of LTBP in these areas were thought to be mesothelial cells and fibroblasts. LTBP was not found in granulomas and mature fibrotic areas. Our data suggest that TGF-beta in tuberculous pleurisy may play important roles for regression of granulomatous inflammation and pleural fibrosis for tissue repair.     

非小细胞性肺癌局部免疫微环境中细胞因子mRNA表达的分析

目的:以非小细胞肺癌(non-small cell lung cancer,NSCLC)为对象,探讨肿瘤局部微环境中免疫反应的特点及其对抗肿瘤免疫的影响。方法:以5例结核性胸膜炎患者作为对照,采用地高辛末端标记的寡核苷酸探针,以原位杂交技术检测了23例非小细胞性肺癌(NSCLC)患者的新鲜胸腔积液和/或手术切除标本中的淋巴细胞和肿瘤细胞中,IL-2、INF-γ、IL-12(p40)、IL-18、IL-4、IL-10、TGF-β1、IL-1、IL-3、IL-8、GM-CSF、TNF-α及TGF-αmRNA的表达。结果:NSCLC患者胸腔积液的单个核细胞及肿瘤组织中,IL-4、IL-10、TGF-α和TGF-β1mRNA的表达水平,明显高于IL-2、IL-12、IL-18和INF-γmRNA的表达;而结核性胸腔积液的单个核细胞中上述细胞因子mRNA的水平均降低,并且各细胞因子mRNA的表达水平之间没有明显的差异。结论:NSCLC患者胸腔积液的单个核细胞及肿瘤组织中,II型细胞因子和免疫抑制细胞因子mRNA的表达占主导地位,反映了肿瘤局部的免疫微环境处于抑制状态。上述结果有助于了解肿瘤逃逸的机制,并为制定NSCLC免疫治疗的方案提供了重要的实验依据。     

Differential regulation of Th1-type and Th2-type cytokine profiles in pancreatic islets of C57BL/6 and BALB/c mice by multiple low doses of streptozotocin

In the nonobese diabetic (NOD) mouse, the T helper (Th)1-type inflammatory cytokines interferon (IFN)-gamma and tumor necrosis factor (TNF)-alpha play a critical role in the development of type 1 diabetes, whereas the Th2-type anti-inflammatory cytokines interleukin (IL)-4 and IL-10 operate counterregulatory. There are no comprehensive analyses on cytokine profiles in the mouse model of diabetes induced with multiple low doses of streptozotocin (MLD-STZ). Therefore, we used islets to study ex vivo effects of MLD-STZ and in vitro effects of STZ on IFN-gamma, TNF-alpha, IL-4, and IL-10 on both levels of protein-producing cells and the mRNA expression, as well as the mRNA expression of the Th3-type cytokine transforming growth factor TGF-beta1. C57BL/6 and BALB/c mice of both genders were injected intraperitoneally with 40 mg/kg body wt STZ on five consecutive days and islets were isolated on day I and 3 after the fifth STZ-injection. Control mice received the solvent of STZ. In islets of C57BL/6 mice of both genders MLD-STZ similarly stimulated production of IFN-gamma and TNF-alpha, but significantly reduced IL-4 and IL-10 levels in male mice only. Opposite results were obtained in islets of BALB/c mice of both genders. Here, MLD-STZ markedly decreased the levels of IFN-gamma and TNF-alpha, but significantly increased the levels of IL-4 and IL-10. The functional results were in line with MLD-STZ effects on the mRNA expression of the cytokines. Moreover, MLD-STZ effects on the TGF-beta1 mRNA expression were reversed to the effects on IFN-gamma and TNF-alpha. The in vitro effects of STZ in islets, in general, were similar to those exerted by MLD-STZ. Apparently, reduction and upregulation of Th2-type cytokines was more associated with susceptibility and resistance, respectively, to MLD-STZ-induced diabetes than upregulation of Th1-type cytokine levels.     

Immunohistochemical analysis of cytokines and apoptosis in tuberculous lymphadenitis

Relatively little is known about the effector mechanisms whereby the human immune system controls Mycobacterium tuberculosis infection. In this study we elaborate on the immune response and mechanisms of persistence of mycobacteria in lesions by analysing, using immunohistochemistry, the expression of cytokines [tumour necrosis factor-alpha (TNF-alpha), interleukin-10 (IL-10), transforming growth factor-beta (TGF-beta) and interferon-gamma (IFN-gamma)], apoptotic cells and apoptosis-related proteins [Bcl2, Bax, Fas ligand (FasL) and Fas] in the human tuberculous lymphadenitis lesions. The expression of apoptosis-related proteins has been shown to be exploited by mycobacteria to evade the immune response and persist in the host. Foreign body (FB) granulomas were used as controls. In tuberculosis (TB) granulomas, epithelioid cells and multinucleated giant cells expressed cytokines differently. In epithelioid cells, the numbers of TNF-alpha-, IL-10- and TGF-beta-stained cells were higher than IFN-gamma-stained cells (P < 0.01). TGF-beta and FasL were strongly expressed in the necrotic centres as compared with other cytokines. More giant cells expressed IL-10 and TGF-beta than expressed TNF-alpha and IFN-gamma (P < 0.01). Staining of consecutive sections revealed that some giant cells expressed IL-10 but not TNF-alpha. Apoptotic TB giant cells correlated positively with the expression of TNF-alpha, IFN-gamma and TGF-beta, but not with the expression of IL-10. The percentage of giant cells expressing Bax was lower than those expressing Fas, unlike the epithelioid cells, suggesting that TB giant cells are less susceptible to apoptosis. Compared with FB giant cells, there were fewer TB giant cells showing TNF-alpha, IFN-gamma, FasL, Fas expression or undergoing apoptosis (P < 0.05). Taken together, these observations show that the cellular microenvironment of TB granulomas down-regulates microbicidal functions, favouring bacillary survival and persistence. TGF-beta and FasL may be responsible for tissue destruction. The giant cells, being less susceptible to apoptosis, may remain a continuous source of pro-inflammatory cytokines, causing immune pathology.     

Recombinant guinea pig tumor necrosis factor alpha stimulates the expression of interleukin-12 and the inhibition of Mycobacterium tuberculosis growth in macrophages

Tumor necrosis factor alpha (TNF-alpha) plays an important role in the host immune response to infection with the intracellular pathogen Mycobacterium tuberculosis. It is essential for the formation of protective tuberculous granulomas and regulates the expression of other cytokines which contribute to a protective immune response. Interleukin-12 (IL-12) is known to promote a Th1 response, which is essential for antimycobacterial resistance. Recombinant guinea pig TNF-alpha (rgpTNF-alpha) protein (17 kDa) was purified, and its bioactivity was confirmed by its cytotoxicity for L929 fibroblasts. High titers of polyclonal anti-gpTNF-alpha antibody were obtained by immunization of rabbits. Resident alveolar and peritoneal macrophages were isolated from guinea pigs and infected with either the H37Ra or H37Rv strain of M. tuberculosis. The mRNA levels for TNF-alpha and IL-12 p40 were measured using real-time PCR. IL-12 p40 mRNA was up-regulated in a dose-dependent manner by rgpTNF-alpha alone. In infected macrophages, a lower dose of rgpTNF-alpha intensified the mRNA levels of TNF-alpha and IL-12 p40. However, higher doses of rgpTNF-alpha suppressed TNF-alpha and IL-12 p40 mRNA. The antimycobacterial activity of macrophages was assessed by metabolic labeling of M. tuberculosis with [3H]uracil. Resident alveolar and peritoneal macrophages treated with anti-gpTNF-alpha antibody to block endogenous TNF-alpha exhibited increased intracellular mycobacterial growth. These data suggest that the dose of TNF-alpha is crucial to the stimulation of optimal expression of protective cytokines and that TNF-alpha contributes to the control of mycobacterial replication to promote host resistance against M. tuberculosis.     

Transforming growth factor-beta 1 primes macrophages for enhanced expression of the nitric oxide synthase gene for nitric oxide-dependent cytotoxicity against Entamoeba histolytica.

Nitric oxide (NO) produced by activated macrophages is the major cytotoxic molecule for in vitro cytotoxicity against Entamoeba histolytica trophozoites. Transforming growth factor-beta 1 (TGF-beta 1) is a potent negative regulator of several macrophage functions, including NO production. In this study, we investigated the effect of TGF-beta 1 on macrophage nitric oxide synthase (mac-NOS) mRNA expression and NO production for macrophage cytotoxicity against E. histolytica trophozoites. TGF-beta 1 by itself was incapable of inducing mouse bone marrow-derived macrophage (BMM) amoebicidal activity and NO production (as measured by nitrite). In contrast, TGF-beta 1 pretreatment (4 hr) primed BMM for an enhanced amoebicidal activity of 15% and 23% in response to (interferon-gamma) IFN-gamma+tumour necrosis factor-alpha (TNF-alpha) or IFN-gamma+lipopolysaccharide LPS, concomitant with increased NO production of 85% and 27%, respectively. TGF-beta 1 pretreatment increased NO production in response to IFN-gamma+TNF-alpha/LPS stimulation in a time- and dose-dependent manner. By Northern blot analysis, the increased NO production of TGF-beta 1-pretreated BMM was preceded by markedly enhanced expression of mac-NOS mRNA. The priming effect of TGF-beta 1 on NO production was critically dependent on both a TNF-alpha (> or = 100 U) and a LPS (> or = 100 ng) triggering dose in the presence of IFN-gamma. TGF-beta 1 pretreatment enhanced TNF-alpha mRNA expression, but had no effect on TNF-alpha production in culture supernatants after 4 hr of stimulation with IFN-gamma+TNF-alpha/LPS; however, at a later time-point (16-48 hr), even though the levels of TNF-alpha mRNA expression were unaffected, TNF-alpha production was reduced. These data demonstrate that TGF-beta 1 priming for increased mac-NOS mRNA expression for NO-dependent cytotoxicity against E. histolytica in response to IFN-gamma+TNF-alpha/LPS stimulation may be involved in the modulation of a TNF-alpha triggering signal by TGF-beta 1.     

Circulating TNF-alpha, TGF-beta, and IL-10 in tuberculosis patients and healthy contacts

Levels of tumour necrosis factor (TNF)-alpha, transforming growth factor (TGF)-beta, and interleukin (IL)-10 in plasma of pulmonary tuberculosis (TB) patients and healthy contacts and plasma and pleural fluid of patients with tuberculous pleuritis were examined by enzyme immunoassay. Plasma TNF-alpha and IL-10 were elevated to significant levels in healthy contacts. High levels of TGF-beta and IL-10 were also detected in plasma from TB patients and healthy contacts. Pleural fluid contained all three cytokines with the level of IL-10 being highest followed by TGF-beta and TNF-alpha. Plasma of tuberculous pleuritis patients also had detectable levels of the three cytokines. Increased levels of TNF-alpha in plasma of contacts and to some extent pleural fluid of pleuritis patients, is perhaps to limit the infection, while elevated IL-10 in plasma of TB patients and contacts and pleural fluid would perhaps modulate excess proinflammation. Elevated TGF-beta in TB patients suggests its role in the immunopathogenesis.     

Pulmonary tuberculosis in BALB/c mice with non-functional IL-4 genes: changes in the inflammatory effects of TNF-alpha and in the regulation of fibrosis

In BALB/c mice, as in man, progressive pulmonary tuberculosis is accompanied by increasing expression of IL-4. Therefore we have used BALB/c mice with disrupted IL-4 genes (IL-4(-/-)) to investigate the role of IL-4 in pulmonary tuberculosis, with particular emphasis on the toxicity of TNF-alpha and on fibrosis, both of which are neglected aspects of human tuberculosis. Delayed-type hypersensitivity (DTH) sites in IL-4(+/+) mice were sensitive to the toxicity of locally injected TNF-alpha, whereas DTH sites in IL-4(-/-) mice were not. However, intravenous administration of IL-4 to IL-4(-/-) mice restored the sensitivity of the DTH sites to pro-inflammatory effects of TNF-alpha. In late disease, the lungs of IL-4(+/+) mice expressed low IFN-gamma, but high TGF-beta and IL-4, correlating with fibrosis, detected as a high hydroxyproline content. In contrast, TGF-beta peaked 7 days after infection in the lungs of the IL-4(-/-) mice, and then fell to very low levels in the late disease, while IFN-gamma remained high. Accordingly, hydroxyproline content was reduced in infected IL-4(-/-) mice compared to IL-4(+/+) controls. In conclusion, the findings suggest that IL-4 has modestly detrimental effects on the antibacterial efficacy of the Th1 response, and larger effects on the toxicity of TNF-alpha, and on fibrosis.     

IL-18 production in human pulmonary and pleural tuberculosis

Interleukin-18 (IL-18) has multiple important pro-inflammatory effects, including the induction of interferon-gamma (IFN-gamma) in various diseases. In this study, we investigated the IL-18-producing activities in human pulmonary and pleural tuberculosis (TB) in response to purified protein derivative (PPD) antigen (Ag) from Mycobacterium tuberculosis. The most significant IL-18 production was found in chronic refractory TB (CRTB) patients. However, IFN-gamma production in CRTB patients was significantly less than that in healthy tuberculin reactors or in patients with tuberculous pleurisy (TBP). Elevated levels of both IL-18 and IFN-gamma were found in pleural fluids from TBP patients. In vitro production of IL-18 was dramatically decreased following an 18 h stimulation with PPD. However, IFN-gamma was markedly increased in pleural mononuclear cells from TBP patients after in vitro stimulation with PPD. The mesothelial cell type was the main source of pro-IL-18 in pleural cells from TBP patients, suggesting an important role for these cells in TBP. Taken together, these data indicate that IL-18 is elevated in peripheral blood mononuclear cells from CRTB patients, as well as at the site of TBP, indicating a possible role for IL-18 in both protective immunity and pathologic responses in human TB.     

Cytokine profiles in primary and secondary pulmonary granulomas of Guinea pigs with tuberculosis

The cytokine mRNA profiles of primary (arising from inhaled bacilli) and secondary (arising from hematogenous reseeding of the lung) granulomas from the lung lobes of bacillus Calmette-Guérin (BCG)-vaccinated and unimmunized guinea pigs challenged with virulent Mycobacterium tuberculosis by the pulmonary route were assessed in situ using laser capture microdissection (LCM) at 6 weeks after infection. The challenge dose chosen was so low that some lung lobes did not receive an implant from the airway. In unimmunized guinea pigs, some lobes contained either large, necrotic primary lesions or small, non-necrotic secondary lesions, or both. The lobes of BCG-vaccinated animals contained only non-necrotic primary tubercles, and no secondary lesions were visible. Real-time PCR analysis of the acquired RNA clearly demonstrated that primary tubercles from BCG-vaccinated guinea pigs were overwhelmed with mRNA from the anti-inflammatory cytokine, transforming growth factor (TGF)-beta, with some IFN-gamma and IL-12p40 mRNA. In contrast, primary lesions from unimmunized animals were dominated by proinflammatory TNF-alpha mRNA. The cytokine mRNA profile of secondary lesions from unimmunized animals was strikingly similar to the profile of primary lesions from BCG-vaccinated guinea pigs (i.e., a predominance of TGF-beta mRNA with some IL-12p40 and IFN-gamma mRNA), indicating that the lung lobes from which these lesions were retrieved had been naturally "vaccinated" by the time the bloodborne bacilli returned to the lung at 3 to 4 weeks after infection. Furthermore, cytokine mRNA analysis of splenic granulomas from nonvaccinated and vaccinated animals showed close resemblance to primary granulomas recovered from the lungs of the same animal, that is, high levels of TNF-alpha mRNA in unimmunized animals, and mostly TGF-beta mRNA in BCG-vaccinated guinea pigs. Taken together, these data indicate that mycobacteria returning to the lungs of unimmunized guinea pigs 3 to 4 weeks after infection induce a local cytokine response that is fundamentally different from the response to inhaled bacilli and is reminiscent of the primary response in a vaccinated animal.     

ESAT-6- and CFP-10-specific Th1, Th22 and Th17 cells in tuberculous pleurisy may contribute to the local immune response against Mycobacterium tuberculosis infection

Th1 cell-mediated adaptive immune response is very important but may not be sufficient to control Mycobacterium tuberculosis (M. tuberculosis) infection. The roles of the various T cell subsets and cytokines in the inflammatory processes are not clearly elucidated. We investigated whether Th1, Th22 and Th17 cells mediated cellular immunity at the local site of M. tuberculosis infection in patients with tuberculous pleurisy (TBP). The results showed that the cytokines IFN-γ and IL-22 but not IL-17 were elevated in tubercular pleural fluid. Following stimulation with immune-dominant peptides of early secreted antigenic target-6 (ESAT-6), culture filtrate protein-10 (CFP-10) or Bacille Calmette-Guerin, pleural fluid mononuclear cells expressed high levels of cytokines IFN-γ, IL-22 and IL-17 as revealed by mRNA and protein measurements. In addition, we showed that cytokines IFN-γ, IL-22 and IL-17 were produced in M. tuberculosis-specific immune response by distinct subsets of CD4+ T cells with the phenotype of CD45RA-CD62L-CCR7+CD27+ . Our results demonstrated for the first time that ESAT-6- and CFP-10-specific Th1, Th22 and Th17 cells existed in the patients with TBP and might play an essential role against M. tuberculosis infection. The findings of this study raised the possibility of unravelling the critical targets for therapeutic intervention in chronic inflammatory diseases such as TBP.     

联合检测IFN-γ、VEGF-C、CRP及ADA对结核性与恶性胸腔积液鉴别诊断的价值

目的探讨干扰素-γ(IFN-γ)、血管内皮生长因子(VEGF-C)、C-反应蛋白(CRP)及腺苷脱氨酶(ADA)在结核性与恶性胸腔积液鉴别诊断中的应用价值。方法检测122例临床确诊的胸腔积液患者(恶性胸腔积液56例,结核性胸膜炎48例,其他类型18例)胸水和血清中的IFN-γ、VEGF-C、CRP及ADA含量。结果结核组的IFN-γ、CRP浓度及ADA活性明显高于恶性肿瘤组,差异有统计学意义(P0.01),根据受试者工作特征(ROC)曲线结果判断,以100ng/L为临界值,IFN-γ对结核性胸腔积液诊断的灵敏度、特异性分别为83.1%、92.3%;以45U/L为临界值,ADA对结核性胸腔积液诊断的灵敏度、特异性分别为85.6%、96.3%;以110mg/L为临界值,CRP对结核性胸腔积液诊断的灵敏度、特异性分别为79.1%、84.2%;三项指标联合检测,其灵敏度、特异性分别达到87.8%和86.0%。恶性胸腔积液中VEGF-C高于结核性及其他类型胸腔积液(P0.01);VEGF-C/ADA≥8对恶性胸腔积液诊断的灵敏度、特异性分别为86.3%、82.6%;VEGF-C/ADA≤3对结核性胸腔积液诊断的灵敏度、特性度分别为85.1%、87.1%。结论联合检测IFN-γ、VEGF-C、CRP及ADA可以提高结核性胸膜炎诊断的灵敏度及特异性,VEGF-C与ADA浓度比值对胸腔积液的鉴别诊断具有较好的临床价值。     

Reduced apoptosis and increased inflammatory cytokines in granulomas caused by tuberculous compared to non-tuberculous mycobacteria: role of MPT64 antigen in apoptosis and immune response

Inhibition of apoptosis of infected macrophages by pathogenic mycobacteria is suggested to be an important virulence mechanism, but little is known about the mycobacterial proteins involved in the inhibition of apoptosis. In this study we investigated differences in apoptosis and immune response and their correlation with the expression of Mycobacterium tuberculosis complex-specific secretory protein MPT64 in lesions caused by tuberculous or non-tuberculous mycobacteria by analysing the in situ expression of apoptosis-related proteins (FasL, Fas, Bax, Bcl-2), apoptotic cells, inflammatory cytokines [tumour necrosis factor (TNF)-alpha, interleukin (IL)-10, transforming growth factor (TGF)-beta, interferon (IFN)-gamma] and MPT64 antigen. The discrimination of mycobacteria was made by nested polymerase chain reaction (PCR) amplification of IS6110, which is specific for M. tuberculosis complex organisms. Forty-seven cases of lymphadenitis with necrotic granulomas were evaluated. With nested PCR, 30/47 cases were positive for M. tuberculosis. MPT64 antigen was detected specifically in the PCR-positive cases. Granulomas caused by tuberculous mycobacteria had fewer apoptotic cells, higher numbers of cells expressing TNF-alpha and TGF-beta and less extensive necrosis than granulomas caused by non-tuberculous mycobacteria. There was a significant negative correlation between apoptotic cells and the number of cells expressing MPT64 antigens, suggesting a role for MPT64 protein in the inhibition of apoptosis. Granulomas with higher amounts of MPT64 also showed a greater number of cells expressing TGF-beta than those with lower amounts of MPT64. In conclusion, this study supports the hypothesis that inhibition of apoptosis is a virulence mechanism for tuberculous mycobacteria. Correlation of MPT64 antigen with expression of macrophage deactivating cytokines and reduced apoptosis suggests its role in pathogenesis and bacillary persistence.     

Mycobacterium tuberculosis-induced gamma interferon production by natural killer cells requires cross talk with antigen-presenting cells involving Toll-like receptors 2 and 4 and the mannose receptor in tuberculous pleurisy

Tuberculous pleurisy allows the study of human cells at the site of active Mycobacterium tuberculosis infection. In this study, we found that among pleural fluid (PF) lymphocytes, natural killer (NK) cells are a major source of early gamma interferon (IFN-gamma) upon M. tuberculosis stimulation, leading us to investigate the mechanisms and molecules involved in this process. We show that the whole bacterium is the best inducer of IFN-gamma, although a high-molecular-weight fraction of culture filtrate proteins from M. tuberculosis H37Rv and the whole-cell lysate also induce its expression. The mannose receptor seems to mediate the inhibitory effect of mannosylated lipoarabinomannan, and Toll-like receptor 2 and 4 agonists activate NK cells but do not induce IFN-gamma like M. tuberculosis does. Antigen-presenting cells (APC) and NK cells bind M. tuberculosis, and although interleukin-12 is required, it is not sufficient to induce IFN-gamma expression, indicating that NK cell-APC contact takes place. Indeed, major histocompatibility complex class I, adhesion, and costimulatory molecules as well as NK receptors regulate IFN-gamma induction. The signaling pathway is partially inhibited by dexamethasone and sensitive to Ca2+ flux and cyclosporine. Inhibition of p38 and extracellular-regulated kinase mitogen-activated protein kinase pathways reduces the number of IFN-gamma+ NK cells. Phosphorylated p38 (p-p38) is detected in ex vivo PF-NK cells, and M. tuberculosis triggers p-p38 in PF-NK cells at the same time that binding between NK and M. tuberculosis reaches its maximum value. Thus, interplay between M. tuberculosis and NK cells/APC triggering IFN-gamma would be expected to play a beneficial role in tuberculous pleurisy by helping to maintain a type 1 profile.     

Elevated IL-5 levels in pleural fluid of patients with paragonimiasis westermani

To investigate the pathogenic mechanisms of eosinophilic pleural effusion in patients with paragonimiasis, we measured the levels of IL-5, granulocyte-macrophage colony-stimulating factor (GM-CSF) and interferon-gamma (IFN-gamma) in pleural effusions. Samples were obtained from 11 patients with Paragonimus westermani infection. In addition, samples from 12 patients with pleural transudates, 16 with tuberculous pleurisy, seven with empyema and 20 with lung cancer were also examined. Eosinophilia was remarkable in peripheral blood (range 4-34%, median 23.4%) and pleural fluid (range 0-95%, median 71%) of paragonimiasis patients. IL-5 concentrations in pleural effusions of paragonimiasis were markedly higher than those in other groups. Although marked elevation of GM-CSF and IFN-gamma levels was observed in pleural effusion of empyema and tuberculosis patients, it was marginal in the pleural effusion of paragonimiasis patients. In paragonimiasis patients, IL-5 levels in the pleural effusion correlated well with the percentage of eosinophils in peripheral blood and pleural fluid. Such a correlation was not observed between GM-CSF levels in pleural effusion and percentages of eosinophils in pleural fluid or peripheral blood. Our findings suggest that in paragonimiasis IL-5 in the local inflammatory site is particularly important in mediating eosinophilia in peripheral blood and pleural effusion.     

Differential expression of gamma interferon mRNA induced by attenuated and virulent Mycobacterium tuberculosis in guinea pig cells after Mycobacterium bovis BCG vaccination

To determine whether Mycobacterium bovis BCG vaccination would alter gamma interferon (IFN-gamma) mRNA expression in guinea pig cells exposed to Mycobacterium tuberculosis, we cloned a cDNA encoding guinea pig IFN-gamma from a spleen cell cDNA library. The cDNA is composed of 1,110 bp, with an open reading frame encoding a 166-amino-acid protein which shows 56 and 41% amino acid sequence homology to human and mouse IFN-gamma, respectively. Spleen or lymph node cells from na?ve and BCG-vaccinated guinea pigs were stimulated with purified protein derivative (PPD) or M. tuberculosis H37Ra or H37Rv, and the total RNA was subjected to Northern blot analysis with a (32)P-labeled probe derived from the cDNA clone. Compared to the IFN-gamma mRNA expression in cells of na?ve animals, that in spleen and lymph node cells exposed to various stimuli was enhanced after BCG vaccination. However, there was a significant reduction in IFN-gamma mRNA levels when cells were stimulated with a multiplicity of infection of greater than 1 virulent M. tuberculosis bacterium per 10 cells. The enhanced IFN-gamma mRNA response in BCG-vaccinated animals was associated with an increase in the proportions of CD4(+) T cells in the spleens, as determined by fluorescence-activated cell sorter analysis. Furthermore, the nonadherent population in the spleens enriched either by panning with anti-guinea pig immunoglobulin G-coated plates or by purification on nylon wool columns produced more IFN-gamma mRNA than whole spleen cells following stimulation with concanavalin A or PPD. This indicates that T cells are principally responsible for the upregulation of IFN-gamma mRNA expression following BCG vaccination. The mechanism by which virulent mycobacteria suppress IFN-gamma mRNA accumulation is currently under investigation.     

Transforming growth factor-beta mRNA and protein in hypertrophic scar tissues and fibroblasts: antagonism by IFN-alpha and IFN-gamma in vitro and in vivo.

Hypertrophic scarring (HSc) following burn injury is a common, disfiguring, and functionally limiting form of dermal fibrosis, compromising recovery. Previously, elevated levels of transforming growth factor-beta1 (TGF-beta1), a fibrogenic cytokine, were found in wounds and serum of severely injured patients, antagonized in part by treatment with systemic interferon-alpha2b (IFN-alpha2b) both in vitro and in vivo. It is hypothesized that in wound healing after injury, platelets are an initial source of TGF-beta, but wound fibroblasts may be capable, after activation, of autoamplification of the initial response to injury by increasing TGF-beta mRNA and protein that may subsequently be responsive to IFN therapy with IFN-alpha or IFN-gamma or both. Using three pairs of site-matched HSc and normal fibroblasts from the same individuals, nonconfluent and near confluent fibroblasts were treated with TGF-beta, and cell proliferation and collagen production were assayed using cell counting and 18O2 isotopic uptake into hydroxyproline before analysis by gas chromatography-mass spectrometry (GC-MS). HSc and normal fibroblasts were assayed for the production of TGF-beta protein secretion using ELISA for TGF-beta1, TGF-beta2, and TGF-beta3 after acidification of medium samples from 96-h cultures. HSc and normal fibroblasts were treated with IFN-alpha2b or IFN-gamma or both for 96 h. Quantitative RT-PCR and Northern analysis were performed using newly synthesized internal standards for human TGF-beta1. TGF-beta stimulates both HSc and normal fibroblast proliferation. Collagen synthesis is greater in HSc than in normal fibroblasts and is maximally stimulated at 75 pM TGF-beta. TGF-beta stimulated collagen metabolism is antagonized by IFN-alpha or IFN-gamma or both in an additive fashion. HSc and normal fibroblasts not only possess the mRNA for TGF-beta1 but also secrete mature TGF-beta protein. Treatment of HSc and normal fibroblasts with IFN-alpha2b or IFN-gamma antagonizes TGF-beta protein production, and additive effects occur. RT-PCR demonstrates that after IFN treatment, downregulation of TGF-beta1 mRNA accounts in part for the reduction in protein secretion in HSc fibroblasts. Elevations of systemic TGF-beta may be due to wound fibroblasts. TGF-beta synthesis and antagonism of fibroblast TGF-beta protein secretion occurs with either IFN-alpha or IFN-gamma, in part by downregulation of TGF-beta1 mRNA levels.