ARTICLE |
Correspondence to: Timothy M. Cox, Dept. of Medicine, Box 157, Addenbrooke's Hospital, Cambridge CB2 2QQ, UK.
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Summary |
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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.
(J Histochem Cytochem 49:675683, 2001)
Key Words: tartrate-resistant acid phosphatase, dendritic cells, localization, expression, tissues
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Introduction |
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OSTEOCLASTS and other cells of monohistiocytic lineage, including the macrophage, express the band 5 isozyme of tartrate-resistant acid phosphatase, TRAP (EC 3.1.3.2) (
TRAP has long been used as a histochemical marker for the osteoclast (2-macroglobulin (
Previous studies of acid phosphatase function lacked specificity because of overlapping enzymatic activities of multiple phosphatase isozymes with the capacity to hydrolyze common substrates. The development of specific antibodies as well as gene probes for the Acp 5 isozyme of TRAP has greatly facilitated the systematic study of its enzyme expression in relation to tissue distribution and physiology.
The human TRAP gene maps to chromosome 19 and contains a putative N-terminal lysosomal leader sequence (
The enzyme has been purified from a number of sources, including human osteoclastomas, in which it is composed of two subunits initially translated as a single polypeptide (
Mice deficient in TRAP generated by selective gene targeting have demonstrated that TRAP is essential for the normal mineralization of cartilage in developing bones and for maintenance of the adult skeleton (
In the mouse, TRAP is expressed in many tissues in addition to bone (
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Materials and Methods |
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Northern Blotting
Multiple-choice Northern blots were purchased from Cambridge Biosciences (Cambridge, UK) and Clontech Laboratories (Basingstoke, UK). Blots were made with 2 µg poly A+ RNA per lane from human tissues. The RNA samples were obtained from healthy adult tissues and from more than a single donor. The membranes from Cambridge Biosciences were wetted in 4 x SSC for 20 min and incubated for 2 hr at 65C in Rapid-hyb buffer (Amersham Pharmacia Biotech; Little Chalfont, UK). Human TRAP cDNA (25 ng) was labelled with [32P]-dCTP using the Prime-a-Gene Labeling System from Promega (Southampton, UK). The probe was purified down a column of Sephadex G-50 and added to the prehybridization solution with the blot. Hybridization was carried out for 2 hr at 65C using 2 x 106 cpm/100 µl. Blots were washed at the same temperature: three times for 5 min with 2 x SSC0.1% SDS and subsequently twice in 0.25 x SSC0.1% SDS for 30 min. Blots from Clontech were prehybridized in ExpressHyb at 68C for 30 min. This solution was replaced with radiolabeled probe as previously described in fresh ExpressHyb and incubated for 1 hr at 68C. Washes were for 3040 min at room temperature (RT) in 2 x SSC0.05% SDS, followed by twice for 20 min at 50C in 0.1 x SSC0.1%SDS. All membranes were exposed to Hyperfilm TM-ßmax (Amersham Pharmacia Biotech) at -80C for 4 days.
Histochemical Staining for TRAP
Unfixed, undecalcified cryostat sections were stained for TRAP activity using the standard naphthol AS-BI phosphate postcoupling method, using Fast Red as the coupler. Tissue samples were obtained during the course of surgical biopsy for unrelated disorders in separate adult subjects and were found to be disease-free after histological examination. The incubation was carried out for 10 min at RT in 0.4 M sodium acetate buffer, pH 5.6, containing 2 mM naphthol AS-BI phosphate and 100 mM sodium tartrate. The reaction was stopped in distilled water and postcoupled in the same buffer containing 2 mM Fast Red for 2 min or until color developed, followed by washing in distilled water (
Immunohistochemistry
Immunohistochemistry was performed using a Vectastain Elite ABC kit and peroxidase staining from Vector Laboratories (Peterborough, UK). Frozen sections obtained from disease-free tissue of adult individuals after surgical biopsy were fixed in acetone at 20C for 20 min. Formalin-fixed, paraffin-embedded sections were treated with two changes of xylene for 10 min each with constant stirring. Sections were rehydrated through a series of ethanol concentrations and washed with water. All sections, irrespective of their method of preparation, were washed in PBS for 5 min and incubated for 20 min in diluted normal blocking serum prepared from the species in which the secondary antibody was made. Excess serum was blotted from sections and primary antibody diluted in PBS was applied. The primary antibodies were polyclonals: rabbit anti uteroferrin (
Harvesting and Culture of Human Dendritic Cells
CD34+ stem cells were immunomagnetically purified from leukapheresis products obtained from disease-free adult donors for hematopoietic stem cell storage and transplantation with a midiMACs system (Miltenyi Biotec; Bisley, Surrey, UK). The cells were cultured in 24-well plates in serum-free medium containing GM-CSF, TNF, TGFß-1, stem cell factor, and F1t3 ligand (Peprotech) (
Confocal Microscopy of Human Dendritic Cells
Dendritic cells were cultured on alcian blue-coated coverslips and fixed in methanol/acetone (1:1) at -20C overnight. Cells were washed three times with PBS0.1% BSA and incubated with mouse antibody directed to the CD63 antigen (Bio-design; Kennebunk, ME) and rabbit antibody to porcine uteroferrin (
Assay of TRAP Activity in Dendritic Cells and Tissues
Dendritic cells and human spleen were homogenized in 0.5 m1 0.4 M sodium acetate, pH 5.6, containing 1% w/v Triton X-100. To assay specifically for TRAP (band 5), immunoabsorption with immobilized rabbit antibodies to porcine uteroferrin was carried out (
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Results |
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Northern Blotting Analysis
An investigation of TRAP mRNA expression in 17 human tissues was carried out by Northern blotting analysis (Fig 1). Abundant expression of TRAP transcripts of the expected size of 1.5 kb was identified after hybridization of radiolabeled full-length human TRAP cDNA in spleen, liver, lung, kidney, stomach, lymph node, bone marrow, and fetal liver, with smaller amounts in colon, small intestine, placenta, and thymus, and a trace in testis and peripheral blood leukocyte. Transcripts were absent in muscle, brain, and heart. The isozyme-specific activities of TRAP in immature human dendritic cells and mature dendritic cells were respectively 0.053 and 0.245 µmols/mg protein/minute, indicating a substantial increase on maturation in vitro. Therefore, the activity of the enzyme is eightfold greater in dendritic cells than in human spleen but increases further on maturation to a value approximately 40-fold greater in mature dendritic cells.
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Enzymatic Activity of TRAP in Tissues
An extract of human spleen exhibited a TRAP activity of 0.006 µmols/mg protein/min. Histochemical examination of tissues for TRAP activity revealed a positive enzymatic reaction in several tissues, especially lung, where high activity was widespread (Fig 2A). Activity was present in the skin, with higher activity concentrated around the hair follicles than in the epidermis (Fig 2B and Fig 2C). Here enzyme activity was detected only after prolonged incubation in vitro (see arrows). Weak enzymatic activity was also demonstrated in colon, ileum, stomach, liver, kidney, and spleen (not shown).
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Immunohistochemistry
An investigation of TRAP by immunohistochemistry identified protein in several tissues (Fig 3 and Fig 4). In the lung, staining was in cells surrounding the alveolar spaces, comparable with expression in pulmonary alveolar macrophages as well as sparse dendritic cells (Fig 3A). Abundant protein immunostaining was detected in sinusoidal cells surrounding the blood spaces around the vessels in the spleen (Fig 3B). In the liver (Fig 3C), immunohistochemical staining gave a positive reaction in hepatocytes surrounding the hepatic venous radicals. In the stomach, colon, and ileum (Fig 4A4C), TRAP protein was concentrated in the muscular layer of the submucosal epithelium. In the uterus (Fig 4D), TRAP immunostaining was prominent around the secretory ducts and endometrial glands.
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In the skin, as in the mouse, a dual localization of TRAP protein was demonstrated by immunostaining. TRAP protein was identified with strong staining reactions in cells of the stratum basale, in regions of the developing papillae, and in a subepithelial layer to which Langerhans cells migrate (Fig 5).
TRAP Expression in Dendritic Cells
The presence of TRAP in CD34+-derived human dendritic cells was investigated. Human dendritic cells were cultured from CD34+ stem cells obtained at leukapheresis after stem cell mobilization with GM-CSF. Both immature and activated dendritic cells stained positively for TRAP (Fig 6). In immature dendritic cells, as defined by low cell surface expression of MHC class II molecules CD83 and CD86, the TRAP protein co-localized to intracellular CD63-positive organelles. After activation with LPS, the dendritic cells mature into an MHC class II CD83 and CD86 high-expressing population. In these cells, the TRAP protein remained intracellular, whereas CD63 was expressed at the cell surface. Isolated dendritic cells were assayed for TRAP activity after lysis. Activity that could be removed by immobilized antibody was fivefold higher in the activated dendritic cell population compared with the immature cells.
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Discussion |
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Because dendritic cells have been recognized as the professional antigen-presenting cells of the immune system and because of our earlier demonstration that Acp 5 shows widespread expression in murine tissues and specifically can be demonstrated in isolated dendritic cells, a comparative study using human tissues was undertaken to determine the potential involvement of Acp 5 in the human immune system, including tissues with epithelia that interface with the external environment.
We have observed that TRAP expression is widely distributed in human tissues, despite having long been employed as a familiar marker of the osteoclast. Osteoclasts indeed show intense staining for TRAP in normal bone (
In view of these findings and of the importance of dendritic cells in human immunity, we report here multi-tissue expression of Acp 5 in human organs at sites corresponding to cells of hematopoietic lineage and, specifically, in cells of dendritic phenotype invested with antigen-presenting properties. A striking finding was the extent to which TRAP expression localized to tissues and organs with environmentally exposed surfaces, including the skin, lung, gastrointestinal tract, and endometrium. These are clearly sites at which continual interactions occur between dendritic phagocytes and microbial and other antigens. TRAP expression, as expected, was also detected in visceral tissues that contain macrophages and also contribute component cells of the mononuclear phagocyte system, including the spleen and liver. The skin revealed a particularly interesting dual localization, especially around the hair follicle and appendages.
In skin and in tissues lining the gastrointestinal tract, TRAP activity was unexpectedly low compared with the amount of protein present, as detected by the strong immunostaining reactions and confirmed by the studies of steady-state transcript abundance. This may be in readiness for activation when triggered by appropriate conditions. The cysteine proteinases are a family of proteases of which members are present in all sites at which TRAP has been identified. In vitro studies show that TRAP enzyme is greatly activated on cleavage of the single-chain pro-enzyme into the disulfide-linked heterodimer by cysteine proteinases (
Here for the first time we have identified abundant TRAP expression in cultured human dendritic cells. Located in most tissues, DCs capture and process antigens. They upregulate their co-stimulatory molecules and migrate to the spleen and the lymph nodes, where they activate antigen-specific T-cells (
Acp 5 expression in dendritic cells within the muscular layer of the submucosa of the gastrointestinal tract is also at a site where a putative role in cells that mediate antigen capture and processing is likely. In the skin, non-keratinocyte epidermal cells, especially migratory Langerhans cells, are involved in presentation of processed foreign antigens to the T-lymphocytes. Pulmonary alveolar macrophage cells are activated by continual exposure to environmental antigens.
In summary, these studies in humans and previous studies carried out in mice and rats (
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Footnotes |
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1 Present address: Division of Molecular and Cellular Biology, School of Clinical Veterinary Medicine, University of Bristol, UK.
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Acknowledgments |
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ARH is in receipt of a Research Fellowship from the Arthritis Research Campaign. PM and PJL are supported by The Wellcome Trust.
Joan Grantham and Philip Ball kindly prepared the manuscript and figures.
Received for publication August 15, 2000; accepted January 24, 2001.
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