Tartrate-resistant acid phosphatase in bone and cartilage following decalcification and cold-embedding in plastic.

Tartrate-resistant acid phosphatase (TRAP) has been proposed as a cytochemical marker for osteoclasts. We have developed an improved technique for the localization of TRAP in rat and mouse bone and cartilage. This procedure employs JB-4 plastic as the embedding medium, permits decalcification, and results in improved morphology compared with frozen sections. Peritoneal lavage cells were used to determine the appropriate isomer and concentration of tartrate necessary for inhibition of tartrate-sensitive acid phosphatase. After incubation in medium containing 50 mM L(+)-tartaric acid, osteoclasts and chondroclasts were heavily stained with reaction product. On the basis of their relative sensitivity to tartrate inhibition, three populations of mononuclear cells could also be distinguished. These three populations may represent: heavily stained osteoclast/chondroclast precursors; sparsely stained osteoblast-like cells lining the bone surface; and unstained cells of monocyte-macrophage lineage. Our results are consistent with the use of TRAP as a histochemical marker for study of the osteoclast.

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Demonstration of tartrate-resistant acid phosphatase in un-decalcified, glycolmethacrylate-embedded mouse bone: a possible marker for (pre)osteoclast identification.

Journal of Histochemistry & Cytochemistry ◽

10.1177/34.10.3745910 ◽

1986 ◽

Vol 34 (10) ◽

pp. 1317-1323 ◽

Cited By ~ 144

Author(s):

F P van de Wijngaert ◽

E H Burger

Keyword(s):

Bone Marrow ◽

Acid Phosphatase ◽

Phosphatase Activity ◽

Acid Phosphatase Activity ◽

Mononuclear Cells ◽

Histochemical Demonstration ◽

Splenic Tissue ◽

Tartrate Resistant Acid Phosphatase ◽

Excellent Preservation ◽

Histochemical Marker

Fixed, undecalcified mouse long bones were embedded in glycol methacrylate (GMA), sectioned, and incubated for acid phosphatase in the presence or absence of tartrate, to investigate the feasibility of tartrate-resistant acid phosphatase as a histochemical marker for osteoclast identification. Naphthol AS-BI phosphate was used as the substrate and hexazonium pararosanaline as coupler. Cytocentrifuge preparations of mouse, rat, and quail bone marrow or frozen and GMA sections of mouse splenic tissue were used as controls to specify acid phosphatase activity. After adequate fixation, acid phosphatase activity sensitive to tartrate inhibition (TS-AP) was demonstrated in macrophages from spleen, bone marrow, and loose connective tissue surrounding bone rudiments. Acid phosphatase activity resistant to tartrate inhibition (TR-AP), was detected in multi-nuclear osteoclasts and in some mononuclear cells from bone marrow and periosteum. In cytocentrifuge preparations and frozen sections of mouse spleen, TR-AP was demonstrated after simultaneous incubation with substrate and tartrate. In GMA sections, however, TR-AP could only be demonstrated after pre-incubation with tartrate before application of substrate. We suggest that histochemical demonstration of TR-AP versus TS-AP on GMA-embedded bone sections by means of a pre-incubation method can be used as an identification marker of (pre)osteoclasts. Plastic embedding is recommended for its excellent preservation of morphology and enzyme activity.

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Tartrate-resistant acid phosphatase in mononuclear and multinuclear cells during the bone resorption of tooth eruption.

Journal of Histochemistry & Cytochemistry ◽

10.1177/35.11.3655324 ◽

1987 ◽

Vol 35 (11) ◽

pp. 1227-1230 ◽

Cited By ~ 31

Author(s):

S C Marks ◽

M L Grolman

Keyword(s):

Acid Phosphatase ◽

Bone Resorption ◽

Connective Tissue ◽

Mononuclear Cells ◽

Alveolar Bone ◽

Tooth Eruption ◽

Tartrate Resistant Acid Phosphatase ◽

Dental Follicle ◽

Multinuclear Cells ◽

Cytochemical Marker

Tartrate-resistant acid phosphatase (TRAP) has been used as a cytochemical marker for the cell mediators of bone resorption, osteoclasts and their mononuclear precursors. We have applied a cytochemical method for TRAP to study the dependence of the osteoclast-mediated bone resorption of tooth eruption on the dental follicle, a connective tissue investment of the developing tooth, by analyzing the TRAP activity of mononuclear cells in the dental follicle before and during pre-molar eruption in dogs. The percentage of TRAP-positive monocyte cells increases until mid-eruption, slightly preceding a previously demonstrated rise in numbers of osteoclasts on adjacent bone surfaces. These data suggest an ontogenetic relationship between follicular mononuclear cells and osteoclasts on adjacent alveolar bone surfaces during tooth eruption. However, because TRAP occurs in other tissues and is not an exclusive indicator of pre-osteoclasts, proof of their relationship will have to await application of more definitive techniques.

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Giant Cell Tumors: Inquiry Into Immunohistochemical Expression of CD117 (c-Kit), Microphthalmia Transcription Factor, Tartrate-Resistant Acid Phosphatase, and HAM-56

Archives of Pathology & Laboratory Medicine ◽

10.5858/2005-129-360-gctiii ◽

2005 ◽

Vol 129 (3) ◽

pp. 360-365

Author(s):

Rolando Y. Ramos ◽

Helen M. Haupt ◽

Peter A. Kanetsky ◽

Rakesh Donthineni-Rao ◽

Carmen Arenas-Elliott ◽

...

Keyword(s):

Transcription Factor ◽

Acid Phosphatase ◽

Giant Cell ◽

Stromal Cells ◽

Mononuclear Cells ◽

Osteoclast Differentiation ◽

Tartrate Resistant Acid Phosphatase ◽

Immunohistochemical Expression ◽

Giant Cell Tumors ◽

Microphthalmia Transcription Factor

Abstract Context.—Osteoclast-like giant cells (GCs) in giant cell tumors (GCTs) are thought to derive from a monocyte-macrophage lineage. Microphthalmia transcription factor (MITF) is necessary for osteoclast gene expression and tartrate-resistant acid phosphatase (TRAP) activation; c-Kit plays a role in regulation of MITF. Objective.—To gain insight into the differentiation of GCTs of bone (GCTBs) and GCTs tendon sheath (GCTTSs) by investigating immunohistochemical staining for c-Kit, MITF, TRAP, and HAM-56 in the GCs and stroma. Design.—Immunoreactivity for CD117 (c-Kit), MITF, TRAP, and HAM-56 was studied in 35 GCTBs, 15 GCTTSs, and 5 foreign-body GC controls. Results.—Across tumors, MITF and TRAP but not c-Kit were generally expressed in GCs; TRAP was variably expressed in stromal cells. The MITF was expressed more consistently in stromal cells of GCTTSs than GCTBs (P < .001). The GCTBs showed more intense MITF stromal (P < .001) and TRAP GC staining (P = .04) than GCTTSs. HAM-56 staining by stromal cells was associated with MITF stromal staining (r2 = 0.6, P < .001). Conclusions.—Results suggest that MITF and TRAP are expressed during osteoclast differentiation and that a proportion of mononuclear cells in GCTs express the macrophage marker HAM-56. Both GCTBs and GCTTSs show similar patterns of immunohistochemical expression.

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Human osteoclast and giant cell differentiation: the apparent switch from nonspecific esterase to tartrate resistant acid phosphatase activity coincides with the in situ expression of osteopontin mRNA.

Journal of Histochemistry & Cytochemistry ◽

10.1177/43.12.8537635 ◽

1995 ◽

Vol 43 (12) ◽

pp. 1193-1201 ◽

Cited By ~ 40

Author(s):

J R Connor ◽

R A Dodds ◽

I E James ◽

M Gowen

Keyword(s):

Acid Phosphatase ◽

Mononuclear Cells ◽

Giant Cells ◽

Mononuclear Phagocyte ◽

Osteoclast Precursor ◽

Tartrate Resistant Acid Phosphatase ◽

Nonspecific Esterase ◽

Connective Tissues ◽

Human Osteoclast

Animal model and in vitro cultures suggest that osteoclasts and cells of the mononuclear phagocyte system share a common precursor. However, the human osteoclast precursor has not been positively identified. We attempted to identify the precursor in situ by using a number of osteoclast- and macrophage-selective markers, together with the expression of osteopontin mRNA, previously shown to be abundant in human osteoclasts. Sections of osteophytic bone and a panel of inflammatory connective tissues were processed for in situ hybridization; serial sections were analyzed for tartrate-resistant acid phosphatase (TRAP) and nonspecific esterase (NSE) activity, selective cytochemical markers for the osteoclast and cells of the macrophage/monocyte lineage, respectively. The murine anti-human osteoclast monoclonal antibodies 23C6 (vitronectin receptor) and C35 (osteoclast-selective) were used to further identify the osteoclast phenotype. We compared osteoclasts, giant cells, and their respective putative mononuclear precursors. At resorption sites within osteophytic bone, osteopontin mRNA was expressed in osteoclasts and a distinct population of TRAP+, NSE- mononuclear cells. Adjacent clusters of mononuclear cells were TRAP- and NSE+ or were active for both enzymes; these cells demonstrated variable expression of osteopontin mRNA. In the inflammatory connective tissues, abundant macrophage-like cells (NSE+/TRAP-) did not express osteopontin mRNA. However, TRAP+ mononuclear cells observed among clusters of NSE+ cells did express osteopontin mRNA. At these sites, clusters of putative macrophage polykaryons removing fragments of bone debris were observed. These giant cells and associated mononuclear cells were NSE- and distinctly TRAP+, and expressed osteopontin mRNA, C35, and 23C6 (human osteoclast) reactivity. Therefore, cells involved in the remodeling (resorption) of bone or the removal of bone debris, together with their immediate precursors, switch from being NSE+/TRAP- to NSE-/TRAP+ cells that express osteopontin mRNA. We propose that the clusters of NSE+/TRAP- mononuclear cells represent the immature osteoclast precursor. In support of this, TRAP+/NSE+ cells were occasionally observed in both tissues, representing an intermediate stage in differentiation. These results further suggest that cells of the mononuclear phagocyte lineage within bone and inflammatory connective tissue have the potential to differentiate into osteoclasts.

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Changes in the Tartrate-resistant Acid Phosphatase Cell Population in Dental Follicles and Bony Crypts of Rat Molars during Tooth Eruption

Journal of Dental Research ◽

10.1177/00220345890680021001 ◽

1989 ◽

Vol 68 (2) ◽

pp. 150-156 ◽

Cited By ~ 95

Author(s):

G.E. Wise ◽

W. Fan

Keyword(s):

Acid Phosphatase ◽

Mononuclear Cells ◽

Osteoclast Formation ◽

Tooth Eruption ◽

Enamel Organ ◽

Tartrate Resistant Acid Phosphatase ◽

Dental Follicle ◽

Cellular Events ◽

Large Numbers ◽

Rat Molars

It was the aim of this study to determine the cellular changes that occur in the enamel organ, dental follicle, and surrounding bony crypt of the rat molar prior to and during tooth eruption. By use of light microscope histochemistry to detect cells containing tartrate-resistant acid phosphatase (TRAP), it was seen that TRAP-positive mononuclear cells were present in the dental follicle prior to the onset of eruption (e.g., three days postnatal age) and then declined in number during eruption. Concurrently, TRAP-positive osteoclasts were initially present in large numbers on the surface of the bony crypt surrounding the molars (three days postnatal age) and then declined in number as eruption progressed. Electron microscopy confirmed that these were mononuclear cells and osteoclasts. The results suggest that the mononuclear cells are either precursors of the osteoclasts or perhaps release cytokines that affect osteoclast formation or activity. Staining for alkaline phosphatase (ALP) activity indicated that at an early postnatal age (secretory stage of amelogenesis), ALP was detected only in the stratum intermedium of the enamel organ, whereas at a later age (maturation phase of amelogenesis), it was present only in the ameloblasts. These results, combined with a survey of the literature, strongly suggest that ALP moves from the base of the enamel organ to the enamel itself over a period of time ranging from pre- to post-eruption. Rat molars are teeth of limited eruption, and the cellular events that occur in eruption appear comparable with what is seen in dog and human dentition, especially in terms of the cellular events seen in the dental follicle prior to and during eruption. Thus, because rat molars are often more amenable to experimental protocol, they may be a suitable choice for answering questions pertaining to tooth eruption in the dog and in humans.

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Acid phosphatase activity is detected preferentially in the osteoclastic lineage by pre-treatment with cyanuric chloride.

Journal of Histochemistry & Cytochemistry ◽

10.1177/39.10.1940313 ◽

1991 ◽

Vol 39 (10) ◽

pp. 1415-1420 ◽

Cited By ~ 15

Author(s):

Y Nakamura ◽

A Yamaguchi ◽

T Ikeda ◽

S Yoshiki

Keyword(s):

Acid Phosphatase ◽

Soft Tissues ◽

Mononuclear Cells ◽

Cell Types ◽

Cyanuric Chloride ◽

Tartrate Resistant Acid Phosphatase ◽

Simple Method ◽

Splenic Macrophages ◽

Trap Staining ◽

Pre Treatment

We previously reported a simple method to detect osteoid matrices in decalcified bone sections by pre-treatment with cyanuric chloride. We have applied this technique to identify osteoclasts and their precursors in rats. In JB-4 sections prepared from untreated bone tissues with cyanuric chloride, both acid phosphatase (ACP) and tartrate-resistant acid phosphatase (TRAP) were found not only in osteoclasts and bone marrow mononuclear cells but also in osteoblasts. In contrast, treatment of bones with cyanuric chloride resulted in staining ACP preferentially in osteoclasts and mononuclear cells adjacent to the bone surface. In the osteoclasts and most of the ACP-positive mononuclear cells, autoradiography showed calcitonin binding. Decalcification with EDTA did not affect the staining for ACP activity in bones treated with cyanuric chloride. It was possible to simultaneously identify ACP and osteoid matrix in a decalcified section. In soft tissues without treatment with cyanuric chloride, both ACP and TRAP were detected in splenic macrophages, alveolar macrophages, and proximal convoluted ducts in kidney. Neither ACP nor TRAP was found in these cell types in the tissues treated with cyanuric chloride. This procedure provides a new, simple method to identify a more restricted population in the osteoclastic lineage than that detected by TRAP staining.

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Inhibition of tartrate-resistant acid phosphatase gene expression by hemin and protoporphyrin IX. Identification of a hemin-responsive inhibitor of transcription

Blood ◽

10.1182/blood.v88.6.2288.bloodjournal8862288 ◽

1996 ◽

Vol 88 (6) ◽

pp. 2288-2297 ◽

Cited By ~ 21

Author(s):

SV Reddy ◽

O Alcantara ◽

GD Roodman ◽

DH Boldt

Keyword(s):

Acid Phosphatase ◽

Mammalian Cells ◽

Mononuclear Cells ◽

Binding Protein ◽

Protoporphyrin Ix ◽

Tartrate Resistant Acid Phosphatase ◽

Sv40 Promoter ◽

Response Element ◽

Nuclear Extracts ◽

Flanking Region

Tartrate-resistant acid phosphatase (TRAP) is an iron-containing protein encoded by the same gene that codes for uteroferrin, a placental iron transport protein. In human peripheral mononuclear cells, TRAP expression is inhibited by both hemin (ferric protoporphyrin IX) and protoporphyrin IX. Nuclear run-on assays confirmed that this inhibition occurs at the level of gene transcription. Previous studies with mTRAP deletion mutants showed that the hemin effect was dependent on repressor activity in the mTRAP 5′- flanking region at -1846 bp to -1240 bp relative to ATG (Reddy et al, J Bone Mineral Res 10:601, 1995). We now report that gel shift assays showed a DNA binding protein in nuclear extracts of hemin-treated cells termed hemin response element binding protein (HREBP). Additional studies have localized the HREBP binding region in the mTRAP 5′- flanking DNA to a 27-bp sequence at -1815 to -1789 bp relative to ATG. A tandem repeat sequence, GAGGC;GAGGC, contained within this DNA segment, was shown to be involved in binding of HREBP. Highly homologous sequences are present in the 5′-flanking region of the hTRAP gene. Binding of HREBP to the mTRAP DNA sequence was inhibited by anti- HAP1 antibodies, indicating homology between the hemin-responsive factor and the yeast heme-dependent transcription factor, HAP1. A 607- bp segment of the mTRAP 5′-flanking region containing the candidate hemin response element and surrounding sequences conferred hemin regulation on the viral SV40 promoter. Southwestern blotting experiments probing nuclear extracts of hemin-treated U937 cells with the 27-bp binding sequence showed two protein bands at 37 and 133 kD representing candidate HREBPs. A GENINFO search showed several other mammalian genes with tandem GAGGC motifs in noncoding regions, providing the possibility that additional genes may also be regulated by hemin at the level of transcription. These studies provide the first description of a novel iron/hemin-responsive transcriptional regulatory mechanism in mammalian cells.

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Tartrate-resistant acid ATPase as a cytochemical marker for osteoclasts.

Journal of Histochemistry & Cytochemistry ◽

10.1177/37.1.2461980 ◽

1989 ◽

Vol 37 (1) ◽

pp. 115-117 ◽

Cited By ~ 60

Author(s):

G N Andersson ◽

S C Marks

Keyword(s):

Acid Phosphatase ◽

Substrate Specificity ◽

Light Microscopy ◽

Mononuclear Cells ◽

Histochemical Method ◽

Bone Surface ◽

Specific Staining ◽

Surface Staining ◽

And Function ◽

Cytochemical Marker

We present a modified histochemical method for staining osteoclasts and adjacent mononuclear cells which takes advantage of the recently described substrate specificity for ATP of osteoclastic acid phosphatase. Staining of osteoclasts using ATP as substrate exhibits by light microscopy the same tartrate resistance as conventional acidic phosphatases, without the bone surface staining seen with other substrates. This feature, coupled with specific staining of fewer vicinal mononuclear cells, makes this method potentially useful for studying osteoclast ontogeny and function.

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