We recently described a model of Th1 recall responses based on segmental antigen challenge with purified protein derivative of Mycobacterium tuberculosis (PPD). Bronchoscopic instillation of 0.5 tuberculin units of PPD resulted in localized lymphocytic inflammation in PPD-positive subjects only. Recruited lymphocytes were predominantly CD4ϩ and were enriched for cells capable of PPDspecific interferon (IFN)-␥ production. In the current study, we investigated the mechanisms by which this localized recall response is mobilized. Bronchoscopic PPD challenge of skin test-positive subjects resulted in the production of CXCR3 ligands IFN-␥-inducible protein (IP)-10 and monokine induced by IFN-␥ (Mig), but not of CCR5 ligands macrophage inflammatory protein-1␣ and regulatedupon activation, normal T-cell expressed and secreted, whereas skin test-negative subjects produced none of these chemokines. The great majority of individuals infected with Mycobacterium tuberculosis do not develop active disease, but instead develop specific cell-mediated immunity that is manifest clinically by the development of positive skin-test responses to purified protein derivative of M. tuberculosis (PPD) (1). Skin-test responsiveness is associated with relative protection against subsequent infection with M. tuberculosis (2), although the mechanisms by which this protection is mediated remain poorly understood. We previously sought to establish a model of M. tuberculosis-specific recall responses in the human lung to provide a means to investigate the local responses of immune individuals upon re-exposure to the organism. To do so, we adapted the technique of bronchoscopic segmental antigen challenge that had previously been used extensively to characterize the local Th2-mediated immune responses in the lungs of individuals with atopic asthma using challenge with allergens such as ragweed (3-5). We instead used PPD as the challenge antigen, and compared responses of naturally infected skin test-positive subjects with those of healthy PPD-negative control subjects. A baseline bronchoalveolar lavage (BAL) was performed, followed by administration of 0.5 tuberculin units (TU) of PPD (i.e., 1/10th of the standard skintest dose) into the challenged segment and a control instillation of 10 cc of normal saline into a corresponding segment of the contralateral lung. Repeat BAL of control and challenged segments was performed 48 h later.In our initial studies, we demonstrated that bronchoscopic challenge with PPD resulted in a localized inflammatory response in challenged lung segments of PPD-positive subjects only. Compared with BAL of both baseline and control lung segments of these subjects, PPD-challenged segments displayed a 2.7-fold increase in the total BAL cells and an increase in the percentage of BAL lymphocytes from 10% to 19%. PPDchallenged segments of skin test-positive subjects also displayed an increased percentage of CD4ϩ T cells, and were enriched for cells capable of antigen-specific production of interferon (IFN)-␥ in response to ...

H37Rv and H37Ra have been widely used as models of virulent and avirulent strains, respectively, of Mycobacterium tuberculosis. Since the sequencing of H37Rv, microarrays have been used to investigate gene expression of M. tuberculosis strains under various conditions, and to compare gene expression of specific isolates of the organism. Because differences in the virulence of these organisms could also be manifest via their differential induction of host genes, we used Affymetrix Human Genome Arrays U133A and U133B to evaluate human alveolar macrophage (AM) responses to infection with H37Rv and H37Ra. H37Rv altered expression of far more genes than did H37Ra. Moreover, the genes induced by H37Rv to a greater extent than by H37Ra were predominantly associated with the development of effective immunity. H37Rv markedly increased expression of IL-23 p19, whereas neither organism significantly induced IL-12 p35 expression. Quantitative PCR confirmed that H37Rv induced significantly more AM p19 expression than did H37Ra. After low-level infection of both AM and peripheral blood monocytes (MN) with H37Rv, neither cell type produced IL-12 (by ELISA). In contrast, AM displayed significant IL-23 production in response to H37Rv, whereas MN did not. Our findings thus suggest an important role for IL-23 in human host responses to pulmonary infection with M. tuberculosis, and are consistent with epidemiologic and genetic studies that imply that H37Rv may not have unusual capacity to cause human disease.

SummaryIn 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.