ARTICLE |
Correspondence to: Jürgen Mollenhauer, Dept. of Biochemistry, Rush Medical College at Rush-PresbyterianSt. Luke’s Medical Center, 1653 W. Congress Parkway, Chicago, IL 60612.
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Summary |
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In its tissue-specific function as a collagen receptor of chondrocytes, cartilage annexin V (anchorin CII) occupies a key position in the organization of the cellextracellular matrix (ECM) junction for the tissue. The general role of annexin V (Anx V) in other tissues suggests involvement in cellular secretory processes and in regulation of apoptosis. Immunohistochemical analysis of Anx V in growth plate cartilage, confirmed by in situ hybridization, suggests that Anx V is prominently expressed and forms a major constituent of growth plate chondrocytes. Anx V epitopes are also located in the pericellular matrix of hypertrophic cartilage. In adult articular cartilage the expression is downregulated, with the highest levels of immunostaining found in the upper third of the articular cartilage layers and almost no antigen found in the deep layers. Osteoarthritic (OA) cartilage is characterized by a significant upregulation of message and protein throughout the entire depth of the tissue, an accumulation of cytoplasmic annexin V epitopes, and a release of epitopes into the pericellular and interterritorial matrix, in part co-localized with granular structures. Therefore, Anx V expression and tissue distribution may serve as a histological marker for metabolic alterations and for changes in the cellular phenotype associated with OA. (J Histochem Cytochem 47:209220, 1999)
Key Words: annexin V, immunohistochemistry, in situ hybridization, articular cartilage, growth plate
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Introduction |
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The annexins (Anx) are a family of calcium and phospholipid binding proteins (for review see
One member, Anx V, Mr 3436 kD (depending on species), contains four conserved 70-amino-acid repeat domains found in all annexins (
Anx V has been used as a marker for apoptosis, or controlled cell death (
In cartilage, annexins II, V, and VI have been detected (
Fetal chicken chondrocytes have high expression levels of Anx V mRNA (
Adult articular cartilage, a bradytrophic (slowly metabolizing) tissue, is characterized by a significantly downregulated metabolism. Cellular turnover of molecules from the ECM, in particular collagen Type II, is minimal. Osteoarthritis (OA) is characterized by an increased turnover of molecules from the ECM. An imbalance of synthesis and degradation of aggrecan and collagen Type II, the major matrix molecules, results in progressive destruction of articular cartilage. The tissue gradually degenerates with the development of fibrillation, fissures, ulceration, and full-thickness defects. In a number of patients, secondary ossification takes place with the formation of osteophytes. Mineralizing matrix vesicles are deposited in the ECM of OA cartilage and may serve as initiation sites for pathological mineral deposits (
In this study we compared the expression of Anx V in cartilage obtained from OA patients at the time of knee arthroplasty with normal cartilage obtained from adult donors (3993 years) with no known history of joint disease. Two samples from newborn donors and one fetal sample were included to obtain information on growing articular (epiphyseal) and growth plate cartilage. The expression of Anx V was upregulated in the diseased cartilage samples to a degree comparable to those of the fetal and newborn growing cartilage, which displayed very intense signals. This was true both for protein, as detected by immunohistology, and for message, as detected by in situ hybridization.
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Materials and Methods |
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Reagents
Chemicals, either reagent or molecular biology grade, were purchased from Sigma Chemical (St Louis, MO) unless otherwise noted. Chondroitin ABC lyase (EC 4.2.2.2), keratanase (EC 3.2.1.103), and keratanase II were obtained from Seikagaku America (Rockville, MD).
Tissue Acquisition
Normal human cartilage samples were obtained through the Regional Organ Bank of Illinois from 21 donors within 24 h of their death and from six OA patients through the Department of Orthopaedic Surgery, Rush-PresbyterianSt. Luke's Medical Center, Chicago, Illinois, according to the institutions' protocols and with institutional approval. Of the 21 donors, 18 were adults (8 women and 10 men aged 3993 years). In addition, the growth plate of two fetuses and the epiphyseal cartilage of one newborn were obtained for analysis.
Recombinant Human Annexin V (rhanxV)
RhanxV was produced from a bacterial strain transfected with an expression vector including the cDNA from human placental Anx V (purchased from American Type Culture Collection, Rockville, MD; ATCC 67916) as described elsewhere (
Anx V Antisera
Polyclonal serum #9757 was raised against a 20-amino-acid synthetic peptide specific for the N-terminal sequence of human Anx V (NH2-M A Q V L R G T V T D F P G F D E R A D-COOH). The sequence was tested for specificity by the BLASTP program (
Histological Processing
Full-thickness articular cartilage without the calcified cartilage or subchondral bone was sliced into approximately 3 x 3 mm2 pieces. The cartilage was fixed in 4% paraformaldehyde, dehydrated, and embedded in paraffin under RNase-free conditions. Sections of 6 µm were cut and processed for histology, immunohistochemistry, or in situ hybridization. For histological grading, the sections were stained with Safranin O and Fast Green and then graded using a modification of the scale of Mankin (
Immunohistochemistry
Rabbit anti-human Anx V peptide antiserum (#9757) was used as the source of the primary antibodies for indirect immunostaining. To increase the penetration of antibodies into cartilage tissue, deparaffinized sections were digested with keratanase (10 mU/ml), keratanase II (0.1 mU/ml), and chondroitinase ABC (10 mU/ml) in 100 mM Tris/50 mM sodium acetate buffer (pH 6.5) at 37C for 90 min. Sections were then incubated with nonimmune goat serum in PBS (1:100 dilution) for 30 min to block nonspecific binding sites. Primary antibody was applied at a dilution of 1:500 in 1% bovine serum albumin (BSA) in PBS overnight at 4C. The bound antibody was detected by biotinylated second antibody and an avidinbiotinalkaline phosphatase reaction kit according to the manufacturer's protocols, including the inhibition of tissue-endogenous alkaline phosphatase with levamisole (Pierce). Alternatively, bound #9757 antibodies were detected with rhodamine-conjugated goat anti-rabbit IgG (Jackson ImmunoResearch; West Grove, PA) in a 1:100 dilution and a 1-hr incubation at room temperature (RT). Control reactions were performed by incubating sections with (a) no primary antibodies, (b) normal rabbit serum (1:250 dilution) followed by second antibody, or (c) #9757 antiserum (1:250 dilution) preabsorbed with synthetic peptide (M A Q V L R G T V T D F P G F D E R A D) conjugated to BSA and followed with alkaline phosphatase-labeled goat anti-rabbit IgG.
Western Blotting
Following the methods of
Isolation of Chicken and Human Chondrocyte Membranes
Chicken chondrocyte membranes were isolated from adult chicken sternal cartilage as described (
In Situ Hybridization
A 29-mer anti-sense oligonucleotide probe was designed with a sequence (5'-CCAGGGAAGTCAGTCACAGTGCCT-CTGAG-3') complementary to BP 106134 of human Anx V mRNA, exon 3 (accession number U05761). The specificities and sequence homologies of this probe were compared with sequence data available from the EMBL/GenBank DDB database. Gel-purified oligonucleotide probes were 3'-end-labeled with 5'-[-thiol-35S]-dCTP using terminal deoxynucleotidyl transferase (New England Nuclear; Wilmington, DE). The radiolabeled probe was hybridized to cartilage sections as previously described (
Amplification of Annexin V mRNA
Polymerase chain reaction (PCR) was performed to validate the signal obtained by in situ hybridization, as described previously by
Digestion of Chicken Chondrocyte Membranes with Matrix Metalloproteinases (MMPs)
MMP-1 (fibroblast collagenase) and MMP-3 (stromelysin) were gifts from Dr. Elizabeth Arner (DuPontMerck, Wilmington). MMP-8 (neutrophil collagenase) (
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Results |
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Specificity of Antiserum #9757
The antiserum was tested by Western blotting using rhanxV and the peptideBSA conjugate as target antigens (Figure 1). In addition, an aliquot of the antiserum was blocked before the incubation with the blotted proteins by incubation with the BSA-conjugated peptide. As shown in Figure 1, the antibodies recognized exclusively the recombinant Anx V in the bacterial extract, the BSA-conjugated peptide, and in the chondrocyte membrane extract, the Anx V polypeptide at about 34 kD. Pretreatment of the antiserum with the conjugated peptide to block the specific immune reactions completely eliminated the antibody reaction against the blotted antigens.
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Anx V Immunohistology in Human Growing Cartilage
The epiphyseal and growth plate cartilages that were used for the immunohistochemistry were obtained from two fetuses and one newborn. The cartilage sections were incubated with primary antibody #9757 followed by a secondary antibody conjugated to either avidinbiotinalkaline phosphatase (Figure 2AE) or rhodamine (Figure 2FK). To more precisely define the location of chondrocytes within the epiphyses and growth plates, a low-power photomicrograph of a section stained with hematoxylin and eosin is included (Figure 2L). Four regions (IIV) are marked to indicate the region of the cartilage from which the higher-power photomicrographs were taken. In this section from a fetal metatarsus, the epiphysis contains cartilage canals but no secondary center. Across the young cartilage there was a gradient of staining, with the least intense at the developing articular surface (Figure 2A, Zone I) and the most intense in the hypertrophic region of the growth plate (Figure 2D and Figure 2I, Zone IV). Cells immediately adjacent to the joint space had no detectable staining for Anx V; cells located just beneath the articular surface varied in the intensity of staining. Some cells were intensely stained, whereas others were either very lightly stained or were negative. Cells located more centrally in the epiphysis and in the proliferative zone of the growth plate were more uniform in staining. In the hypertrophic chondrocytes there was intense intracellular staining as well as strong granular staining in the ECM surrounding the hypertrophic chondrocytes. A similar pattern was seen in the hypertrophic zones in the early secondary centers of ossification of the tissues (not shown), except that the cells were in a circular array around the invading blood capillaries. These staining patterns were detectable with both fluorescence and alkaline phosphatase detection kits and were abolished by blocking the antibody with the BSA-conjugated peptide.
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Chondrocytes from normal adult tissue stained most strongly in the areas of the plasma membranes and the lacunar walls. This staining pattern was persistent throughout the entire age range tested (3993 years) in the normal joint cartilage (Figure 3A and Figure 4AG). The staining patterns could be suppressed by preincubating the antiserum (#9757) with the peptide-conjugated BSA (Figure 5). The overall intensity of staining declined from the middle to the deep zone, although all the tissues were treated with chondroitinase ABC and keratanases to unblock epitopes. Staining was always present in the plasma membranes and the lacunar wall, indicating maintenance of the Anx V expression on the plasma membranes. Although the cells of the deeper zones of the adult articular cartilage were not always visibly stained in the plasma membranes after alkaline phosphatase staining, the presence of some plasma membrane/lacunar wall epitopes in these cells was revealed by staining with the fluorescent (rhodamine-conjugated) second antibody (not shown). In the deepest layers, in the vicinity of or just underneath the tidemark, occasional weak staining of the ECM in the pericellular matrix was seen (Figure 4G).
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Anx V Immunohistology in Normal and Osteoarthritic Cartilage
In normal adult articular cartilage (Mankin grade 0), Anx V was localized in the upper layers of the cartilage, with no detectable staining in the deeper layers (Figure 3A). In cartilage (Mankin grade 5) with a damaged superficial layer, staining was intense along the outer surface and some chondrocytes within the deep zone were now positive.
The cartilage received from OA patients showed various degrees of damage, with fibrillation and/or fissures penetrating into the deep layers of the joint cartilage and clusters of cells (Mankin grades >5). In this cartilage, the intensity of stain was enhanced (Figure 3CE) even in areas that appeared undamaged (Figure 3E) compared to normal cartilage (Figure 3A).
The increased intensity of staining was more apparent at higher magnification (Figure 4). In the normal cartilage (Figure 3AG) there was cytoplasmic staining of the chondrocytes and the lacunar wall that was less intense than that in the OA cartilage (Figure 4HM, Figure 5A, and Figure 5C). The cells in clusters in the OA cartilage were always heavily stained (Figure 4K). As a particular feature of the OA cartilage, staining in the ECM beyond the lacunar wall was observed (Figure 4LO and Figure 5C), quite similar to that in the zone of hypertrophic chondrocytes in the growth plates (Figure 2D and Figure 2I). Frequently, the staining had a granular pattern, with the granules displaying very different sizes (Figure 4N and Figure 4O). Both the cytoplasmic and matrix staining could be blocked with the peptide-conjugated BSA (Figure 5B and Figure 5D), thus indicating a binding of the antibodies to Anx V-specific epitopes both inside and outside of the cells.
In situ Hybridization of Anx V Message
To compare relative differences in Anx V mRNA between growth plate, normal adult, and OA cartilage, each in situ hybridization was performed using tissues for all three from multiple donors. The tissues were processed using standardized protocols to minimize differences in mRNA preservation. For each hybridization, all three types of cartilage were handled simultaneously with a single probe preparation, the same length of exposure, and the same conditions for developing the emulsion (Figure 6). In the young cartilage, Anx V mRNA was detectable at low levels in chondrocytes throughout the epiphysis. In the growth plates, chondrocytes in the resting and proliferative zones had levels of mRNA similar to those in the epiphyses. However, in the hypertrophic zone the expression of Anx V mRNA was higher. Overall detectable mRNA for Anx V was lower in the normal articular cartilage, with expression highest in the superficial layer and some chondrocytes in the middle layer. Chondrocytes in the deep layer were only weakly positive or negative. Expression was elevated in the chondrocytes from the OA cartilage, especially in those cells located in clusters. The signals could be competed with the same but nonradioactive oligonucleotide probe, indicating specificity of the probe. The specificity of the probe was further tested by using PCR with the probe being one of the two necessary primers and the other primer designed according to a site 545 BP downstream. As shown in Figure 7, only one species of cDNA was amplified, with a molecular weight matching the expected size of about 500 BP.
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Susceptibility of Anx V to Metalloproteinase Activity
The observation of Anx V-specific epitopes in the ECM of the OA cartilage prompted us to explore the basis for this staining pattern because annexins are not described as part of the ECM, with the exception of Anx V-containing mineralizing matrix vesicles released by chondrocytes of growth plates (
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Discussion |
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The purpose of this study was to determine the pattern of expression of Anx V in human fetal and adult normal cartilage and to compare these findings with those obtained from OA cartilage. The study was motivated by two particular functional properties of Anx V in cartilage: to serve as a collagen "receptor" on chondrocytes (
Although Anx V is a constitutive component of the cytoplasm of many different cell types, its preferential location in chondrocytes is the outer surface of the plasma membranes (
This strong staining of the cytoplasm of hypertrophic chondrocytes of the calcifying portion of the growth plate suggests a profound difference in the metabolic processing and function of Anx V in these cells. A gradient of increasing mRNA expression of Anx V from the resting zone through the proliferating zone into the hypertrophic zone has also been recently described in chicken growth plates (
As mentioned earlier, some investigators found parallels in the expression pattern of matrix proteins in growth plate cartilage and in OA (
The pericellular staining of the ECM is another feature of hypertrophic growth plate cartilage (
Alternatively, the detection of Anx V epitopes by the antibodies could be the result of extracellular proteolytic cleavage of the surface-associated Anx V and the release of proteolytic fragments into the ECM. Employing recombinant MMP-1, MMP-3, and MMP-8, enzymes that are upregulated in damaged human cartilage (
Whether due to the production of mineralizing vesicles or to the proteolytic release, a depletion of Anx V from the chondrocyte membranes might place additional demands on the biosynthetic capacity of chondrocytes and might explain the upregulation in message levels and cytoplasmic protein observed here. Because these features are also seen in hypertrophic fetal growth plate chondrocytes, they may indicate a switch to some embryogenetic differentiation patterns in OA cartilage. In that, our findings would coincide with those made for other proteins of the growth plates: the expression of the growth plate-specific collagen Type X (
Finally, the wide variation in size of the deposited granules suggests that at least some of the deposits are the remnants of dead chondrocytes, apoptotic bodies (
It is possible that apoptosis and hypertrophic differentiation are linked in growth plate cartilage to allow the controlled transition of the fetal cartilage to bone (
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Footnotes |
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1 Present address: School of Biological Sciences, National University of Singapore, Singapore.
2 Present address: Department of Cell and Molecular Biology, Lund University, Lund, Sweden.
3 Present address: Abbott Laboratories, North Chicago, Illinois.
4 Present address: Department of Orthopaedic Surgery, University of Ulm, Ulm, Germany.
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Acknowledgments |
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Supported in part by the NIH/NIAMS SCOR grant 2-P50-AR-39239 and by a local chapter grant from the Arthritis Foundation. HK is the recipient of a postdoctoral fellowship from the Merck Foundation.
We would like to acknowledge the skillful technical support of Larry Madsen and by Michele Healey. The collaboration of Dr. Allan Valdellon and his staff from the Regional Organ Bank of Illinois is gratefully acknowledged.
Received for publication September 23, 1998; accepted September 29, 1998.
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