SUMMARY Tartrate-resistant acid phosphatase (TRAP) is a histochemical marker of the osteoclast. It is also characteristic of monohistiocytes, particularly alveolar macrophages, and is associated with diverse pathological conditions, including hairy cell leukemia and AIDS encephalopathy. To study the biology of this enzyme, we investigated its expression and activity in mouse tissues. Confocal fluorescence studies showed that TRAP is localized to the lysosomal compartment of macrophages. In adult mice, high activities of the enzyme were demonstrated in bone, spleen, liver, thymus, and colon, with lower amounts in lung, stomach, skin, brain, and kidney. Trace amounts were detected in testis, muscle, and heart. Expression of TRAP mRNA was investigated in tissue sections by in situ hybridization and protein expression was monitored by histochemical staining or immunohistochemically. TRAP is widely expressed in many tissues, where it is associated with cells principally originating from the bone marrow, including those of osteoclast/macrophage lineage. The cellular distribution of TRAP mRNA and enzyme antigen in the tissues corresponds closely to that of cells staining with an antibody directed to the CD80 (B7) antigen. Therefore, to confirm its putative localization in dendritic cells, isolated bone marrow dendritic cells were matured in culture. These co-stained strongly for TRAP protein and the CD80 antigen. These studies demonstrate that TRAP is a lysosomal enzyme that is found in diverse murine tissues, where it is expressed in dendritic cells as well as osteoclasts and macrophages, as previously shown.
SUMMARYTartrate-resistant acid phosphatase (TRAP) is a lysosomal di-iron protein of mononuclear phagocytes and osteoclasts. Hitherto, no role for the enzyme in immunity has been identi®ed; however, knockout mice lacking TRAP have a skeletal phenotype caused by an intrinsic osteoclast defect. To investigate a putative function for TRAP in macrophages (Mw), we investigated proin¯ammatory responses and systemic microbial clearance in knockout mice compared with age-and gendermatched congenic wild-type mice. Phorbol 12-myristate 13-acetate (PMA)-stimulated and interferon-c (IFN-c)-induced superoxide formation was enhanced in peritoneal Mw lacking TRAP; nitrite production in response to stimulation with lipopolysaccharide (LPS) and IFN-c was also increased. In addition, secretion of the proin¯ammatory cytokines, tumour necrosis factor-a (TNF-a), interleukin (IL)-1b and IL-12, was signi®cantly greater in TRAP-de®cient Mw when stimulated with LPS, with or without addition of either TNF-a or IFN-c. The activity of tartratesensitive (lysosomal) acid phosphatase was increased in Mw from the knockout mice but activities of the lysosomal hydrolases N-acetyl b-glucosaminidase and acid b-glucuronidase were unchanged, indicating selective activation of compensatory acid phosphatase activity. Evidence of impaired Mw function in vivo was obtained in TRAP knockout mice, which showed delayed clearance of the microbial pathogen, Staphylococcus aureus, after sublethal intraperitoneal inoculation. After microbial challenge, peritoneal exudates obtained from TRAP knockout mice had a reduced population of Mw. As peritoneal Mw and neutrophils lacking TRAP were able to phagocytose and kill S. aureus normally in vitro, TRAP may directly or indirectly in¯uence recruitment of Mw to sites of microbial invasion. Our study shows that TRAP participates in the in¯ammatory response of the Mw and in¯uences effector signalling pathways in innate immunity.
1. Endotoxin induces a shock-like syndrome with increased nitric oxide synthesis. To clarify the cellular source of NO in endotoxic shock we used immunohistochemistry and in situ hybridization to localize inducible NO synthase in rats given lipopolysaccharide or Corynebacterium parvum and lipopolysaccharide. Immunohistochemistry was carried out with an antibody raised against a synthetic peptide of mouse macrophage NO synthase. In situ hybridization was performed with 35S-labelled oligonucleotide probes corresponding to cDNA sequences common to mouse macrophage inducible NO synthase and rat vascular smooth inducible NO synthase. Monocytes and macrophages were identified by immunohistochemistry with the mouse monoclonal antibody ED1. 2. After lipopolysaccharide alone, the major site of NO synthase induction was monocytes and macrophages in multiple organs, principally liver and spleen. Bronchial, bile duct, intestinal and bladder epithelium and some hepatocytes also expressed inducible NO synthase. Expression peaked at 5 h and had returned to normal by 12 h except in spleen. 3. After priming with C. parvum, lipopolysaccharide led to a similar distribution of inducible NO synthase as lipopolysaccharide alone, but in addition there was more prominent hepatocyte staining, staining in macrophage granulomas in the liver and inducible NO synthase was present in some endothelial cells in the aorta. 4. These findings provide a direct demonstration of the cellular localization of inducible NO synthase after lipopolysaccharide.
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