vided by Dr P Brennan, Fort Collins, Colorado,. Division of Pulmonary ..... 17 Birkedal-Hansen H, Moore WGI, Bodden MK, Windsor. LJ, Birkedal-Hansen B, ...
Thorax 1996;51:306-311
306
Effect of Mycobacterium tuberculosis and its components on macrophages and the release of matrix metalloproteinases Jui C Chang,* Annette Wysocki, Kam-Meng Tchou-Wong, Naomi Moskowitz, Yihong Zhang, William N Rom Background - Pulmonary tuberculosis is associated with caseating necrosis, parenchymal lung destruction, and cavity formation. It was hypothesised that tuberculous lung destruction is mediated, at least in part, by the participation ofmatrix metalloproteinases released by mononuclear phagocytes. Methods - Cells of the myelomonocytic leukaemia cell line THP-1 were incubated with lipoarabinomannan (LAM), the major antigenic cell wall component, and with Mycobacterium tuberculosis and analysed by Northern blot analysis. Two patients with active cavitary tuberculosis also underwent bronchoalveolar lavage and the cells were analysed by Northern blotting. Results - Incubation of THP-1 cells with LAM resulted in the stimulated release of matrix metalloproteinase-9 (MMP-9), a 92 kDa gelatinase, by 24 hours in a dosedependent fashion. In addition, Northern analysis revealed that LAM upregulated the gene for MMP-9 by 24 hours, but not the gene for the 72 kDa gelatinase MMP2. Heat killed M tuberculosis H37Ra also upregulated the MMP-9 gene. Bronchoalveolar lavage of the two patients with active cavitary tuberculosis showed striking upregulation of the MMP-9 gene comDivision of Pulmonary pared with a normal control using Northern analysis. LAM also upregulated and Critical Care Medicine, the type I interstitial collagenase (MMPDepartments of 1) gene by 24 hours in both THP-1 cells Medicine, and peripheral blood monocytes. Environmental Medicine and Surgery, Conclusions - These data suggest that M and Bellevue Hospital tuberculosis and its major cell antigenic Chest Service, New component, LAM, stimulate the release of York University Medical Center, New MMP-9 and upregulate the expression of Yori, NY 10016, USA genes for MMP-1 and MMP-9. It is posJ C Chang sible that M tuberculosis and its comA Wysocki K-M Tchou-Wong ponents contribute directly to cavity N Moskowitz formation by their ability to stimulate Y Zhang macrophages to release matrix metalloW N Rom proteinases that digest collagens I-IV, and *Current address: indirecdy by stimulating the release of the Department of Veterans cytokines interleukin 1, and tumour necAffairs Medical Center, rosis factor a that induce fibroblasts to Mount Sinai School of Medicine, Bronx, New amplify the release of matrix metalloYork, USA. proteinases. Correspondence to: (Thorax 1996;51:306-311) Dr W N Rom. Received 24 January 1994 Returned to authors 6 May 1994 Revised version received 25 September 1995 Accepted for publication 23 October 1995
Keywords: collagenases, matrix metalloproteinases, tuberculosis, cavity.
to destruction of lung tissue with cavity formation. The granulomatous lesion is tyically a cell mediated response involving macrophages and lymphocytes as the immunoresponsive cells. Fibroblasts, one of the major connective tissue cells, are also involved in the course of the acute and chronic phase of granulomatous inflammation as evidenced by the deposition of collagen fibres in the granulomatous lesion.' Advanced pulmonary tuberculosis is associated with a locally destructive process of cavitary lesions which plays an important part in transmission of disease. The pathogenesis of the destructive lung disease caused by Mycobacterium tuberculosis is still poorly understood. Dannenberg' suggested that the tissue damage such as liquefaction, caseation, and cavity formation was the result of a hypersensitivity reaction - that is, excess bacillary antigen causing host tissue or cells to produce cytotoxic cytokines, oxygen radicals, hydrolytic enzymes and eventual cell death.3 Since the degradation of connective tissue matrix involves an initial extracellular cleavage of insoluble proteins mediated by neutral proteinases such as elastases and collagenases,4 it would be important to assess neutral matrix metalloproteinase activity in host cells during tuberculosis infection.5 To test the hypothesis that M tuberculosis or its components modulate the synthesis of matrix metalloproteinases, especially collagenases, we have studied the effects of M tuberculosis and lipoarabinomannan (LAM), the major antigenic component of the mycobacterial cell wall,67 on the stimulation of matrix metalloproteinases in human peripheral blood monocytes and a myelomonocytic leukaemia cell line (THP-1).7 Bronchoalveolar lavage (BAL) was also performed in two patients with active cavitary tuberculosis to obtain BAL cells to assess expression of the matrix metalloproteinase-9 gene. Since we8 and others9-" have shown that LAM can stimulate the release of tumor necrosis factor oa (TNF-cx) and interleukin 1 ,B (IL1 I) from mononuclear phagocytes, and since these two cytokines are known to stimulate collagenase production in synovial cells,4 15 we evaluated gene expression of the type I interstitial collagenase following stimulation of human lung fibroblasts.
Methods CELL CULTURE AND STIMULATION
Lipoarabinomannan (LAM) from laboratory Central caseating necrosis is the hallmark of attenuated Mycobacterium spp was kindly protuberculous granulomas. Tissue caseation leads vided by Dr P Brennan, Fort Collins, Colorado,
Effect of M tuberculosis on macrophage matrix metalloproteinases
USA. Evaluation of tuberculosis reagents for the presence of Gram negative bacterial endotoxin was performed with the amoebocyte lysate assay (E-Toxate Kit, Sigma). A lipopolysaccharide (LPS) standard curve was generated with an assay limit of 1 pg/ml LPS. Six different batches of LAM from Mycobacterium spp were tested and all contained
