RESUMO
In this study, we hypothesized that the granulomatous disorder sarcoidosis is not caused by a single pathogen, but rather results from abnormal responses of Toll-like receptors (TLRs) to conserved bacterial elements. Unsorted bronchoalveolar lavage (BAL) cells from patients with suspected pulmonary sarcoidosis and healthy non-smoking control subjects were stimulated with representative ligands of TLR-2 (in both TLR-2/1 and TLR-2/6 heterodimers) and TLR-4. Responses were determined by assessing resulting production of tumour necrosis factor (TNF)-α and interleukin (IL)-6. BAL cells from patients in whom sarcoidosis was confirmed displayed increased cytokine responses to the TLR-2/1 ligand 19-kDa lipoprotein of Mycobacterium tuberculosis (LpqH) and decreased responses to the TLR-2/6 agonist fibroblast stimulating ligand-1 (FSL)-1. Subsequently, we evaluated the impact of TLR-2 gene deletion in a recently described murine model of T helper type 1 (Th1)-associated lung disease induced by heat-killed Propionibacterium acnes. As quantified by blinded scoring of lung pathology, P. acnes-induced granulomatous pulmonary inflammation was markedly attenuated in TLR-2(-/-) mice compared to wild-type C57BL/6 animals. The findings support a potential role for disordered TLR-2 responses in the pathogenesis of pulmonary sarcoidosis.
Assuntos
Sarcoidose Pulmonar/metabolismo , Receptor 2 Toll-Like/metabolismo , Adolescente , Adulto , Idoso , Animais , Líquido da Lavagem Broncoalveolar/citologia , Líquido da Lavagem Broncoalveolar/imunologia , Estudos de Casos e Controles , Citocinas/biossíntese , Modelos Animais de Doenças , Feminino , Expressão Gênica , Humanos , Ligantes , Masculino , Camundongos , Camundongos Knockout , Pessoa de Meia-Idade , Pneumonia/genética , Pneumonia/imunologia , Propionibacterium acnes/imunologia , Multimerização Proteica , Sarcoidose Pulmonar/genética , Sarcoidose Pulmonar/imunologia , Receptor 2 Toll-Like/antagonistas & inibidores , Receptor 2 Toll-Like/genética , Receptor 4 Toll-Like/agonistas , Receptor 4 Toll-Like/metabolismo , Receptores Toll-Like/química , Receptores Toll-Like/genética , Receptores Toll-Like/metabolismo , Adulto JovemRESUMO
SETTING: Previous studies have shown that isolates from cases in IS6110 restriction fragment length polymorphism (RFLP) clusters that have persisted over several years and are widely distributed grow significantly faster in macrophages than isolates from cases with unique RFLP patterns. As members of the Beijing family of Mycobacterium tuberculosis are widely distributed and have been responsible for several large outbreaks, it has been suggested that this genotype may have a selective advantage over other strains. OBJECTIVE: To determine whether rapid growth in macrophages is a common characteristic of Beijing family strains. DESIGN: T-helper precursor-1 human macrophages were infected with various Beijing family strains, and intracellular growth and tumor necrosis factor alpha (TNF-alpha) secretion were assessed. Strains differed in their genotype, with IS6110 copy number ranging from 9 to 22. RESULTS: Strains demonstrated a range of growth phenotypes over the 7-day infection period. Three grew significantly more slowly than the other strains, whereas the fastest growth was observed consistently with isolates of strain 210. CONCLUSION: Rapid growth in macrophages is not a common characteristic of all Beijing strains. Few Beijing strains are as virulent as strain 210. The growth advantage is consistent with strain 210 having persisted many years in different locations and having caused many outbreaks.
Assuntos
DNA Bacteriano/genética , Macrófagos/microbiologia , Mycobacterium tuberculosis/crescimento & desenvolvimento , Células Th1/microbiologia , Técnicas de Tipagem Bacteriana , Surtos de Doenças , Ensaio de Imunoadsorção Enzimática , Humanos , Macrófagos/metabolismo , Macrófagos/patologia , Mycobacterium tuberculosis/genética , Polimorfismo Genético , Análise de Sequência de DNA , Células Th1/metabolismo , Células Th1/patologia , Tuberculose/epidemiologia , Tuberculose/microbiologia , Tuberculose/patologia , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Cytotoxic effector phenotype and function of MHC-restricted Mycobacterium tuberculosis (MTB)-reactive CD4(+) and CD8(+) T lymphocytes were analyzed from healthy tuberculin skin test-positive persons. After stimulation in vitro with MTB, both CD4(+) and CD8(+) T cells up-regulated mRNA expression for granzyme A and B, granulysin, perforin, and CD95L (Fas ligand). mRNA levels for these molecules were greater for resting CD8(+) than CD4(+) T cells. After MTB stimulation, mRNA levels were similar for both T cell subsets. Increased perforin and granulysin protein expression was confirmed in both in CD4(+) and CD8(+) T cells by flow cytometry. Both T cell subsets lysed MTB-infected monocytes. Biochemical inhibition of the granule exocytosis pathway in CD4(+) and CD8(+) T cells decreased cytolytic function by >90% in both T cell subsets. Ab blockade of the CD95-CD95L interaction decreased cytolytic function for both T cell populations by 25%. CD4(+) and CD8(+) T cells inhibited growth of intracellular MTB in autologous monocytes by 74% and 84%, respectively. However, inhibition of perforin activity, the CD95-CD95L interaction, or both CTL mechanisms did not affect CD4(+) and CD8(+) T cell mediated restriction of MTB growth. Thus, perforin and CD95-CD95L were not involved in CD4(+) and CD8(+) T cell mediated restriction of MTB growth.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/microbiologia , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/microbiologia , Mycobacterium tuberculosis/imunologia , Antígenos de Diferenciação de Linfócitos T/genética , Antígenos de Diferenciação de Linfócitos T/metabolismo , Atividade Bactericida do Sangue/imunologia , Citotoxicidade Imunológica , Proteína Ligante Fas , Granzimas , Humanos , Técnicas In Vitro , Complexo Principal de Histocompatibilidade , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/imunologia , Mycobacterium tuberculosis/crescimento & desenvolvimento , Perforina , Proteínas Citotóxicas Formadoras de Poros , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Serina Endopeptidases/genética , Serina Endopeptidases/metabolismo , Regulação para Cima , Receptor fas/genética , Receptor fas/imunologiaRESUMO
Despite the continued importance of tuberculosis as a world-wide threat to public health, little is known about the mechanisms used by human lymphocytes to contain and kill the intracellular pathogen Mycobacterium tuberculosis. We previously described an in vitro model of infection of human monocytes (MN) with virulent M. tuberculosis strain H37Rv in which the ability of peripheral blood lymphocytes to limit intracellular growth of the organism could be measured. In the current study, we determined that lymphocyte-mediated killing of intracellular M. tuberculosis occurs within the first 24 h of coculture with infected MN. Natural killer (NK) cells isolated from both purified protein derivative (PPD)-positive and PPD-negative subjects were capable of mediating this early killing of intracellular H37Rv. NK cell-mediated killing of intracellular M. tuberculosis was not associated with the production of gamma interferon. Transferred supernatants of cocultured NK cells and M. tuberculosis-infected MN could not mediate the killing of intracellular M. tuberculosis, and Transwell studies indicated that direct cell-to-cell contact was required for NK cells to mediate the killing of the organism. Killing was not dependent upon exocytosis of NK cell cytotoxic granules. NK cells induced apoptosis of mycobacterium-infected MN, but neither killing of intracellular M. tuberculosis by NK cells nor NK cell-induced apoptosis of infected MN was inhibited by blocking the interaction of FasL and Fas. Thus, human NK cells may mediate killing of intracellular M. tuberculosis via alternative apoptotic pathways.
Assuntos
Apoptose , Grânulos Citoplasmáticos/metabolismo , Células Matadoras Naturais/imunologia , Mycobacterium tuberculosis/imunologia , Adulto , Comunicação Celular , Técnicas de Cocultura , Proteína Ligante Fas , Humanos , Interferon gama/metabolismo , Células Matadoras Naturais/microbiologia , Linfócitos/imunologia , Glicoproteínas de Membrana/metabolismo , Pessoa de Meia-Idade , Mycobacterium bovis/imunologia , Receptor fas/metabolismoRESUMO
The alternate RNA polymerase sigma factor gene, sigF, which is expressed in stationary phase and under stress conditions in vitro, has been deleted in the virulent CDC1551 strain of Mycobacterium tuberculosis. The growth rate of the DeltasigF mutant was identical to that of the isogenic wild-type strain in exponential phase, although in stationary phase the mutant achieved a higher density than the wild type. The mutant showed increased susceptibility to rifampin and rifapentine. Additionally, the DeltasigF mutant displayed diminished uptake of chenodeoxycholate, and this effect was reversed by complementation with a wild-type sigF gene. No differences in short-term intracellular growth between mutant and wild-type organisms within human monocytes were observed. Similarly, the organisms did not differ in their susceptibilities to lymphocyte-mediated inhibition of intracellular growth. However, mice infected with the DeltasigF mutant showed a median time to death of 246 days compared with 161 days for wild-type strain-infected animals (P < 0.001). These data indicate that M. tuberculosis sigF is a nonessential alternate sigma factor both in axenic culture and for survival in macrophages in vitro. While the DeltasigF mutant produces a lethal infection of mice, it is less virulent than its wild-type counterpart by time-to-death analysis.
Assuntos
Proteínas de Bactérias/genética , Deleção de Genes , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/patogenicidade , Fator sigma/genética , Tuberculose/microbiologia , Animais , Células Cultivadas , Modelos Animais de Doenças , Feminino , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Monócitos/microbiologia , Mycobacterium tuberculosis/crescimento & desenvolvimento , Mycobacterium tuberculosis/metabolismo , Fenótipo , Tuberculose/mortalidade , VirulênciaRESUMO
We assessed the applicability of an in vitro model of low-level infection of human monocytes to the characterization of the virulence of strains of the Mycobacterium tuberculosis family. Peripheral blood monocytes were infected at a 1:1 ratio with the virulent M. tuberculosis strain H37Rv, the avirulent M. tuberculosis strain H37Ra, and the attenuated M. bovis strain BCG. Both the percentages of cells infected by the three strains and the initial numbers of intracellular organisms were equivalent, as were levels of monocyte viability up to 7 days following infection. Intracellular growth reflected virulence, as H37Rv replicated in logarithmic fashion throughout the assay, BCG growth reached a plateau at 4 days, and H37Ra did not grow at all. The same patterns of growth were observed following infection of human alveolar macrophages with H37Rv and H37Ra. Monocyte production of tumor necrosis factor alpha was significantly higher following infection with virulent H37Rv than with either BCG or H37Ra. In contrast, there was no clear correlation of interleukin 10 production with virulence. Nonadherent cells of purified-protein-derivative-positive donors mediated equivalent degrees of reduction of the intracellular growth of H37Rv, BCG, and H37Ra. Low-level infection of human monocytes with H37Rv, BCG, and H37Ra thus provides an in vitro model for assessment of the virulence of these M. tuberculosis family strains. Furthermore, it is suggested that the virulence of these strains is expressed primarily by their differing abilities to adapt to the intracellular environment of the mononuclear phagocyte.
Assuntos
Linfócitos/fisiologia , Monócitos/microbiologia , Mycobacterium tuberculosis/patogenicidade , Fator de Necrose Tumoral alfa/biossíntese , Adulto , Humanos , Interleucina-10/biossíntese , Pessoa de Meia-Idade , Monócitos/fisiologia , Mycobacterium tuberculosis/crescimento & desenvolvimento , Mycobacterium tuberculosis/imunologia , Fagocitose , VirulênciaRESUMO
Protective human immunity to Mycobacterium tuberculosis (M. tb) has proven difficult to characterize, in part because of technical obstacles to in vitro infection of human cells with virulent M. tb. We established a reproducible method of infecting human monocytes (MN) with the virulent M. tb strain H37Rv that did not reduce MN viability. TNF-alpha had no effect on replication of H37Rv within MN, and IFN-gamma mediated only a 1.9-fold reduction in bacterial growth. In contrast, nonadherent cells (NAC) from purified protein derivative (PPD)-positive and PPD-negative subjects reduced intracellular growth of H37Rv by 6- and 10.6-fold, respectively (p = 0.007 and p = 0.005). CD4+ T cells were essential to growth inhibition mediated by NAC of PPD-positive subjects, whereas containment of M. tb by NAC of PPD-negative subjects did not require CD4+ cells. CD8+ T cells did not contribute to protection mediated by NAC of either group. Supernatants of cocultured H37Rv-infected MN and NAC only partially reduced intracellular growth of M. tb despite containing nanogram concentrations of TNF-alpha and IFN-gamma. Neutralizing antibodies to TNF-alpha, IFN-gamma, and IL-12 failed to affect the NAC-mediated growth limitation. NAC treated with emetine retained approximately 40% of their capacity to contain intracellular H37Rv, however. These studies indicate that protective human recall responses to M. tb are mediated primarily by CD4+ T cells, whereas CD4-CD8- lymphocytes may contribute to innate immunity to M. tb. The ability of NAC to activate M. tb-infected MN is only partly attributable to soluble mediators and may also involve contact-mediated mechanisms.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Monócitos/imunologia , Monócitos/microbiologia , Mycobacterium tuberculosis/crescimento & desenvolvimento , Mycobacterium tuberculosis/imunologia , Tuberculina/imunologia , Adulto , Anticorpos Bloqueadores/farmacologia , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/microbiologia , Adesão Celular/imunologia , Comunicação Celular/imunologia , Separação Celular , Sobrevivência Celular/imunologia , Sistema Livre de Células/imunologia , Células Cultivadas , Humanos , Imunidade Celular , Interferon gama/biossíntese , Interferon gama/imunologia , Interferon gama/farmacologia , Interleucina-12/imunologia , Líquido Intracelular/imunologia , Líquido Intracelular/microbiologia , Pessoa de Meia-Idade , Mycobacterium tuberculosis/efeitos dos fármacos , Mycobacterium tuberculosis/patogenicidade , Reprodutibilidade dos Testes , Fator de Necrose Tumoral alfa/biossíntese , Fator de Necrose Tumoral alfa/imunologia , Fator de Necrose Tumoral alfa/farmacologia , VirulênciaRESUMO
Sarcoidosis is a multisystem disorder characterized by non-caseating granulomas and the accumulation of CD4+ T cells in involved tissues such as the lung. To evaluate the diversity of the CD4+ T-cell repertoire in this disorder, a detailed clonal analysis was performed in five individuals with active sarcoidosis who demonstrated preferential accumulation of T cells expressing the T-cell receptor variable gene family V beta 8 in either the lung or blood. In three individuals, analysis of unselected samples of nucleotide sequences derived from V beta 8+ lung T cells demonstrated degrees of clonality ranging from 11% to 46%, indicating the expansion of limited numbers of V beta 8+ T-cell clones in the lung. Analysis of the corresponding deduced amino acid sequences demonstrated common VDJ junctional amino acid residues in the dominant V beta 8+ T-cell clones derived from two oligoclonal V beta 8+ lung T-cell populations, consistent with an antigen-specific T-cell response. In contrast, analysis of V beta 8+ CD4+ T cells from the blood of an individual with a marked bias for peripheral blood V beta 8+ T cells demonstrated no evidence of oligoclonality, suggesting that the stimulus for circulating biased V beta-specific T cells in sarcoidosis may derive from a different, perhaps superantigenic, origin. Clinical improvement in the disease either in response to treatment with corticosteroids or as a result of spontaneous resolution was associated with a decrease in the proportion of V beta 8-specific T cells in the biased lung and/or blood T-cell compartments. Together, these observations are consistent with a role for this T-cell subset in the clinical manifestations of active granulomatous disease.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Pulmão/imunologia , Receptores de Antígenos de Linfócitos T alfa-beta/genética , Sarcoidose Pulmonar/imunologia , Adulto , Sequência de Aminoácidos , Sequência de Bases , Líquido da Lavagem Broncoalveolar/imunologia , Feminino , Humanos , Masculino , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , Receptores de Antígenos de Linfócitos T alfa-beta/metabolismo , Sarcoidose Pulmonar/genéticaRESUMO
The fibronectin-binding 30-kDa alpha Ag is a major secretory protein of growing mycobacteria that stimulates in vitro lymphocyte blastogenesis in most healthy purified protein derivative-positive individuals, but only a minority of patients with active tuberculosis. T cell epitopes of the alpha Ag were assessed using blastogenic responses of PBMC from 12 healthy purified protein derivative-positive subjects to a set of synthetic peptides based on the 325-amino acid sequence of the alpha Ag of Mycobacterium bovis BCG. Because epitope-specific precursor cells are infrequent and randomly distributed, we used Poisson analysis to determine positive responses to 10 micrograms/ml of each peptide in 12 replicate culture wells. Seven immunodominant regions of the alpha Ag were identified. Each subjects responded to at least one of the two most dominant epitopes, which correspond to amino acids 131-155 and 233-257 (from N terminus). Peptides of these two epitopes induced production of IFN-gamma by sorted CD4+ T cells. The immunodominant peptides may have use a components of a vaccine and as tools to study the evolution of the immune response to M. tuberculosis. The two most dominant epitopes both occur in regions of the alpha Ag that differ from those of the atypical pathogens M. avium and M. kansasii. In addition, the M. bovis epitope of amino acids 133-155 differs from that of M. tuberculosis by a single amino acid. It may be possible to exploit the sequence differences for development of diagnostic tests with increased specificity.
Assuntos
Antígenos de Bactérias/imunologia , Linfócitos T CD4-Positivos/imunologia , Mapeamento de Epitopos/métodos , Mycobacterium bovis/imunologia , Adulto , Sequência de Aminoácidos , Células Cultivadas , Feminino , Humanos , Epitopos Imunodominantes/imunologia , Interferon gama/biossíntese , Ativação Linfocitária/imunologia , Masculino , Pessoa de Meia-Idade , Dados de Sequência Molecular , Teste TuberculínicoRESUMO
Sarcoidosis is a multiorgan granulomatous disorder of unknown etiology characterized by noncaseating granulomas in involved tissues. A positive Kveim-Siltzbach reaction is a granulomatous response to an intradermal injection of a suspension of sarcoid tissue extract in individuals with sarcoidosis. The protracted time course and granulomatous features of this reaction have a striking resemblance to the Mitsuda reaction in tuberculous leprosy, which suggests that the Kveim-Siltzbach reaction is a response to an unknown Ag(s). To evaluate whether this reaction is Ag-driven, an analysis of the TCR V beta repertoire in 15 Kveim-Siltzbach reaction sites was performed using a PCR technique and primers specific for 20 V beta gene families. Results of this analysis demonstrated a pattern of V beta expression dominated by expression of V beta 2, V beta 3, V beta 6, or V beta 8 to levels > 20% of total V beta gene expression in nine of 15 individuals. Analysis of paired biopsy and blood specimens revealed a preferential expression of specific V beta genes, such as V beta 3, V beta 5, and V beta 8, at sites of Kveim-Siltzbach reactions to levels four to seven times that of the corresponding peripheral blood. Sequence analysis demonstrated that preferential expression of specific V beta genes at Kveim-Siltzbach reaction sites is oligoclonal. Furthermore, the dominant V beta 8 sequence present at one of the reaction sites contained a sequence motif in the variable-diversity-joining junctional region previously identified in sarcoid lung and blood T cell populations. These results suggest that the Kveim-Siltzbach reaction is characterized by a limited TCR beta-chain repertoire consistent with an Ag-driven T cell immune response.
Assuntos
Teste de Kveim/métodos , Receptores de Antígenos de Linfócitos T alfa-beta/genética , Sarcoidose/imunologia , Sequência de Aminoácidos , Sequência de Bases , Humanos , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , Receptores de Antígenos de Linfócitos T alfa-beta/imunologia , Sarcoidose/genética , Linfócitos T/imunologiaRESUMO
Sarcoidosis is a granulomatous disease in which activated T cells, responding to an unidentified stimulus, accumulate at sites of disease such as the lung. To evaluate the hypothesis that active sarcoidosis is characterized by a selective activation and expansion of a limited repertoire of T cell receptor (TCR) specific T cells, we analyzed TCR V beta gene expression in lung and blood T cells of patients with active sarcoidosis and, for comparison, normal individuals using polymerase chain reaction amplification of 20 V beta gene families. Analysis of normal bronchoalveolar lavage T cells revealed TCR V beta distributions similar to that of normal blood, providing evidence for a lack of generalized skewing of the T cell repertoire in the normal, noninfected lung. Compared to normal lung and blood, subgroups of individuals with sarcoidosis demonstrated biased expression of one or more V beta genes in either the lung or blood. Five V beta gene families (V beta 5, V beta 8, V beta 15, V beta 16, and V beta 18) were most frequently utilized in a biased fashion by sarcoid lung or blood T cells. Furthermore, dramatic skewing of the T cell repertoire was apparent when sarcoid lung and blood T cells were expanded by short-term culture with IL-2. Sequence analysis demonstrated a bias in V beta gene expression was usually due to expansion of select V beta-specific clones, some of which contained a similar V(D)J junctional region motif. These observations provide evidence for a selective activation and accumulation of antigen-specific V beta-expressing T cells in sarcoidosis.