Tartrate-resistant acid phosphatase (TRAP), once considered to be just a histochemical marker of osteoclasts is now recognised to be a molecule of widespread occurrence with functions in both the skeleton and the immune system. TRAP is expressed by osteoclasts, macrophages, dendritic cells and a number of other cell types. It has a critical role in many biological processes including skeletal development, collagen synthesis and degradation, the mineralisation of bone, cytokine production by macrophages and dendritic cells, macrophage recruitment, dendritic cell maturation and a role in the development of Th1 responses. TRAP is able to degrade skeletal phosphoproteins including osteopontin (OPN), identical to the T-cell cytokine, Eta-1. In this review, we discuss the role of TRAP in bone and immune cells and suggest that TRAP may be implicated in autoimmune disorders regulated by Th1 inflammatory responses as well as certain cancers.

We studied ten individuals from eight families showing features consistent with the immuno-osseus dysplasia spondyloenchondrodysplasia (SPENCD). Of particular note was the diverse spectrum of autoimmune phenotypes observed in these patients, including systemic lupus erythematosus (SLE), Sjögren's syndrome, haemolytic anemia, thrombocytopenia, hypothyroidism, inflammatory myositis, Raynaud's disease, and vitiligo. Haplotype data indicated the disease gene to be on chromosome 19p13 and linkage analysis yielded a combined multipoint lod score of 3.6. Sequencing of the ACP5 gene, encoding tartrate resistant acid phosphatase (TRAP), identified biallelic mutations in each of the patients studied, and in vivo testing confirmed a loss of expressed protein. All eight patients assayed demonstrated elevated serum interferon alpha activity, and gene expression profiling in whole blood defined a type I interferon signature. Our findings reveal a previously unrecognised link between TRAP activity and interferon metabolism, and highlight the importance of type I interferon in the genesis of autoimmunity.

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.

Histochemical demonstration of tartrate-resistant acid phosphatase (TRAP) is used for the specific identification of osteoclasts. The enzyme, which we have shown to be critical for normal bone development in mice, is also characteristic of monohistiocytes, including alveolar macrophages, and is associated with diverse pathological conditions such as Gaucher's disease and hairy cell leukemia. TRAP activity is enhanced in serum when bone resorption is increased, and the activity is used routinely to monitor treatment responses in Gaucher's disease. We have lately shown widespread expression of the enzyme in murine tissues with particular reference to the skin, thymus, gut epithelia, and isolated dendritic cells, suggesting a possible role in immunity. To further clarify the significance of TRAP in human physiology, we have examined its distribution in non-skeletal human tissues and in CD34+-derived human dendritic cells. TRAP mRNA determined by Northern blotting analysis was expressed abundantly in spleen, liver, colon, lung, small intestine, kidney, stomach, testis, placenta, lymph node, thymus, peripheral blood leukocyte, bone marrow, and fetal liver. Expression of TRAP protein was investigated by immunohistochemistry, with which the enzyme was identified in multiple tissues. Histochemical staining detected enzymatically active protein in spleen, lung, skin, colon, stomach, and ileum. Active TRAP was identified in CD34+-derived immature dendritic cells and co-localized to intracellular CD63 positive organelles. When these cells were matured by induction with LPS, the TRAP activity increased fivefold and remained within the cell during the phase associated with CD63 surface expression. Our findings demonstrate widespread expression of TRAP in human tissues. Its abundant expression in epithelia and dendritic cells suggests a potential role in antigen processing and in immune responses.

Tartrate-resistant acid phosphatase is one of the major enzymes produced and secreted by osteoclasts. To obtain sufficient enzyme for biochemical characterization, we have purified this enzyme from human osteoclastomas by sequential chromatography on SP-Sephadex, CM-Sephadex, hydroxylapatite, Sephadex G-150 and concanavalin A-Sepharose. The purification over the original tumour extract was about 2000-fold, with a yield of 10%. The enzyme appeared to be homogeneous when assessed by SDS/polyacrylamide-gel electrophoresis. Both gel filtration and SDS/polyacrylamide-gel electrophoresis indicated an Mr of about 30,000. The reduced and alkylated enzyme consists of two subunits with Mrs of 15,000 and 17,500. The N-terminal amino acid sequence of both subunits indicates that there is a high degree of identity between the osteoclastoma enzyme and similar enzymes purified from spleen and uterus. Using 4-methylumbelliferyl phosphate as substrate, the specific activity of the purified enzyme was 387 units.mg-1, and the Km was 284 microns. The pH optimum was 5.7. Unlike similar enzymes purified from human and bovine bone, osteoclastoma acid phosphatase is not activated by reducing agents (2-mercaptoethanol or ascorbic acid). The enzyme contains 4.8 mol of Fe2+/3+, 0.3 mol of Mn2+ and 1.7 mol of Mg2+ per mol of enzyme. Although the enzyme loses 50% of its activity in the presence of EDTA, it is not inhibited by the iron chelator 1,10-phenanthroline. However, the enzyme is activated to a small extent by Mn2+ and Mg2+. Using a variety of substrates and inhibitors, we demonstrate that there are differences between the osteoclastoma acid phosphatase and the enzyme purified from other sources.

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Highlights► We examined the role of the TRAP enzyme during endochondral bone formation in mice. ► TRAP does not impair vascularization as well as ossification of the long bone epiphysis. ► TRAP−/− mice show an expansion of the cartilaginous growth plate. ► This malformation is first detected at week three and during further development. ► TRAP appears to be critical for the formation of the metaphysis but not of the epiphysis.