ARTICLE |
Correspondence to: Matthias F. Seidel, Medizinische Poliklinik der Universität Bonn, Wilhelmstr. 35-37, D-53111 Bonn, Germany.
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Summary |
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Collagen-induced arthritis in rats is a widely used model of rheumatoid arthritis (RA). However, the joint immunohistopathology is less well characterized. The objective of this study was therefore to analyze whole ankle joints for markers known to mediate inflammatory mechanisms in RA. Indirect immunohistochemistry was performed on undecalcified cryostat sections for intercellular adhesion molecule-1 (ICAM-1, clone 1A 29) and leukocyte function-associated antigen-1 (LFA-1, clone WT.1) expression, for CD4+ lymphocytes (clone W3/25), B-cells (clone HIS 14), and macrophages (clone ED2). Acute, osteodestructive arthritis (n = 8) induced with bovine collagen Type II was verified by clinical and radiological measures. LFA-1 expression was found almost exclusively at sites associated with cartilage erosion or osteodestruction. ICAM-1 was similarly expressed in the vicinity of tissue degradation but also by blood vessels in peripheral areas of joint swelling. CD4+ lymphocytes and macrophages were more ubiquitous. B-cells were infrequent. In control animals (n = 4) ICAM-1 was expressed by synovial blood vessels. Macrophages were identified at the synovial lining. The results suggest that LFA-1 and ICAM-1 mediate important inflammatory events in this model. Similar findings in human RA synovium provide further arguments that collagen-induced arthritis in rats might be regarded as a comparable disease. (J Histochem Cytochem 45:1247-1253, 1997)
Key Words: rheumatoid, collagen, rat, arthritis, osteodestruction, immunohistopathology, ICAM-1, LFA-1, CD4
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Introduction |
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Type II collagen-induced arthritis (
Human RA synovial tissue typically demonstrates putatively autoreactive lymphocytes and macrophages infiltrating and destroying the joint tissues; B-cells are less commonly encountered (
The objective of this study was to determine if ICAM-1 and LFA-1 expression, along with other cell markers typical of RA, occurs in the collagen-induced arthritis model. We studied the early chronic inflammatory joint lesion. Indirect immunohistology was performed on undecalcified, native cryostat sections of ankle joints using a modified protocol of a previously described tissue sectioning technique (
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Materials and Methods |
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Reagents
All reagents were purchased from Sigma (St Louis, MO) except where otherwise indicated.
Induction of Arthritis
The animal experiments were conducted with permission of the Regierungspräsidium Köln (German animal welfare act AZ 23.203.2 BN 40, 10/94). Specific pathogen-free-reared 9-10-week-old inbred male Dark Agouti rats were obtained from Harlan-Winkelman (Borchen, Germany). The rats were kept in cages with filtertops (Techniplast, Buguggiate, Italy; Becker, Castrop-Rauxel, Germany) and separate from other laboratory animals. Pathogen-free processed laboratory chow (Altromin; Lage, Germany) and water were given ad libitum. Bovine collagen Type II was obtained from Dr. Mary Griffiths and was prepared by pepsin digestion as previously described (
Monitoring and Development of Arthritis
The animals were weighed two times per week and were monitored for the onset of clinical symptoms of arthritis. To quantify the intensity of the arthritis, a clinical score for each animal was calculated 6 days after the onset of arthritis. This score was composed of the sum of involvement in each limb and an element for weight loss. The scale for each limb: 0 (no swelling), 1 (faint erythema), 2 (massive erythema/faint edema), 3 (massive erythema/extensive edema). Weight loss was scored as 0 (normal weight gain), 1 (no weight gain), 2 (5% weight loss), and 3 (5-10% weight loss).
Radiography
Five to six days after the onset of arthritis, the animals were anesthetized with ether. Lateral radiographs of both hindpaws were obtained using a Phillips Rotaldex apparatus with 43 kV, 75 mAs on a standard Agfa Curix HT 1000 G film.
Processing for Whole-joint Frozen Sections of Undecalcified Tissue
After radiography, the animals were sacrificed under ether anesthesia by cervical dislocation. From some animals blood was obtained by intracardial puncture and serum aliquots were stored at -80C. For tissue embedding, the skin was removed from the hindpaws and the joints immersed in 8% gelatin. Because rapid freezing causes disruption of tissue integrity, the specimens were frozen slowly in liquid nitrogen and then stored at -80C. As control tissue for immunohistochemical staining, the spleens of some animals were removed and processed similarly. For in toto joint analysis, cryostat sections were obtained by a modification of a previously described method for unfixed joints of small animals (
Immunohistochemistry
Immunohistochemical staining was performed with mouse monoclonal antibodies (prepared against rat tissue) as specified in Table 1 and using an avidin-biotin-peroxidase staining kit (Vectastain Elite ABC Kit; Vector, Burlingame, CA). All incubations were carried out at 37C in a humidified chamber. The washes were done in PBS at RT for 5 min each. Antibodies were titered using spleen sections from diseased animals (Table 1). The tissue was fixed in acetone for 1 min or in 95% ethanol for 10 min at RT (see Table 1). The sections were rinsed and then blocked in 5% horse serum for 10 min. The primary antibodies (Table 1) were diluted in a mixture of PBS, 0.1% bovine serum albumin (BSA, globin-free), and 0.02% sodium azide, and were applied for 15 min. Negative controls consisted of PBS with 1% normal mouse serum or PBS alone. To efficiently quench endogenous peroxidases, the samples were then incubated in methanol containing 0.3% H2O2 for 20 min at RT (
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Quantification and Statistical Analysis of Immunostained Cells in Joints
One whole ankle joint from each animal was analyzed. Staining was scored for three locations in the inflamed joint. Areas in proximity to the joint cartilage and bone destruction (central osteodestructive lesions) were scored separately from peripheral areas of joint edema (peripheral synovial pannus areas) and from bone marrow. In the normal joint, only synovium and bone marrow were distinguished. Because individual cells could not always be delineated as positively or negatively stained, a semiquantitative score (0-4) was assigned for each joint and antigen according to percentages of stained cells: <0.5%, 0.5-5%, 5-10%, 10-25%, >25%, respectively. The mean scores derived from all joints evaluated were used to compute an average percent of positive cells in these areas. The data were analyzed with Student's t-test. Differences between the individual anatomic compartments analyzed were considered significant at p<0.05.
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Results |
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Clinical, Radiographic, and Histological Characterization Confirmed the Early Nature of Arthritic Joints
All animals immunized with collagen Type II developed severe arthritis. The first symptoms of the disease were observed at the distal hindpaws between Days 14 and 17 after immunization. The interdigital erythema of distal joints spread to more proximal areas and was followed by increased swelling. In one animal, significant inflammation was observed only at the left hindpaw. By Day 20, massive erythema and edema indicated a profound inflammatory process. The manifestation of the disease at the forepaws was less constant. Other joints did not appear to be affected, as judged by clinical examination. Clinical scores ranged from 13 to 19 (16.5 ± 1.96). Compared to control animals, lateral radiographs (not shown) indicated intensive swelling of the entire surrounding joint tissue. In 15 inflamed ankle joints, bony destruction was observed radiologically (14/15 at the talocalcaneal articulation). These findings corresponded to previously described radiological changes in collagen-induced arthritis in rats (
Prevalence and Distribution of Immunological Phenotypes in Control Animals and in Collagen-induced Arthritis
Representative peripheral synovial pannus areas and central osteodestructive lesion areas of each inflamed joint were scored for the prevalence of each phenotype. We also scored normal synovium and both normal and immunized bone marrow for comparative purposes (the mean percentages for eight immunized and four control joints are shown in Figure 1). Endogenous peroxidase was not detected in control sections treated with PBS or normal mouse serum. Healthy synovium contained virtually no B-cells, whereas this cell type was readily identified in the bone marrow compartment. Vascular endothelium strongly expressed ICAM-1 (Figure 2A). LFA-1 was absent from normal synovium; approximately 7% of the bone marrow cell population was stained. No definitive CD4 staining was present in the normal synovium (Figure 3A), with subtle infiltration of the bone marrow. In contrast, many synovial cells expressed the macrophage marker and were concentrated at the synovial lining (Figure 3C), but were not significantly different from bone marrow densities. In collagen-induced arthritic joints, CD4+ cells were the predominant cell population in peripheral synovial pannus areas, mostly scattered as individual cells and infrequently as perivascular cell infiltrations. CD4+ lymphocytes were also widely distributed in central osteodestructive lesions (Figure 3B) but were less closely associated with bony destruction. Bone marrow infiltration was not significantly different from synovial infiltration, suggesting that the CD4 lymphocytes can be regarded as a general and less specific cellular response. Staining with the ED2 antibody revealed a ubiquitous distribution of macrophages in the peripheral synovial pannus area and central osteodestructive lesion (Figure 3D). Macrophages were also detected in close contact with the joint cartilage erosion. Staining for B-cells with the HIS 14 monoclonal antibody was rare in peripheral synovial pannus areas; groups of 50-100 cells were found in the vicinity of the central osteodestructive lesions (Figure 3E). Bone marrow staining demonstrated expected increases in CD4 and ED2 (macrophage)-positive cells.
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Expression of ICAM-1 and LFA-1 Staining Correlates with Intensity of Arthritic Inflammation
In collagen-induced arthritic joints, ICAM-1 stained small vascular structures in peripheral synovial pannus areas (Figure 2C), consistent with ongoing neovascularization. The same vascular staining patterns were rarely observed in central osteodestructive lesions at the cartilage surface (Figure 2B). Instead, ICAM-1 staining there was confined mostly to cells surrounding the eroded cartilage surface. The infiltration close to these bony lesions was not significantly different from the bone marrow, suggesting a more unspecific cellular response for this marker. LFA-1 staining was not observed in the peripheral synovial pannus area but was found at the cartilage surface, frequently associated with bony destruction (Figure 2D). Not all areas of osteodestruction manifested LFA-1 staining, presumably because the small groups of LFA-1+ cell infiltration were not sectioned on every plane of the slide. In two animals, intensive LFA-1 staining was observed in circular lymphoid aggregates near the joint but not in direct contact with the cartilage. Compared to control animals, the LFA-1 phenotype was greatly increased in the bone marrow in collagen-induced arthritic animals (Figure 1).
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Discussion |
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Although collagen-induced arthritis in rodents is considered as perhaps the most important model of RA in humans, the joint immunopathology is not well known. Joint inflammatory mechanisms in collagen-induced arthritis may be distinct from human pathology because the autoantigen in RA is still speculative and is probably not related to collagen Type II. In this study, we therefore aimed to characterize inflammatory infiltrates in separate joint compartments for markers typically found in RA. Acute experimental arthritis was verified by clinical and radiological assessment. In toto joint analysis was accomplished by our special cryostat sectioning protocol. The novel polypropylene tape enabled us to use various tissue fixatives. Furthermore, with this procedure we did not have to consider immunohistochemical requirements for conventional bone processing, such as demineralization and paraffin-embedding. Rapid processing time (less than 6 hr for sectioning and immunostaining) and convenience can be considered as additional and substantial advantages.
Our analysis was semiquantitative because not all antibodies yielded the same quality of immunostaining. To ensure comparison to other studies, we therefore estimated the numbers of stained cells. The absence of HIS-14 (for B-cells) or LFA-1 staining in the synovium of control animals indicated a high degree of tissue specificity (although these markers were detected in the bone marrow of the same sections). Furthermore, CD4 staining in the central osteodestructive lesion but also in the peripheral synovial pannus area corresponded to previously published results in collagen-induced arthritis (
LFA-1+ cells were the most sensitive indicators of osteodestructive lesions in our study. However, not all areas of osteodestruction were always associated with this phenotype, possibly owing to the small size of LFA-1-associated inflammatory infiltrates. LFA-1 expression may also change over time within the same articular surface. The important role of this molecule was demonstrated when an anti-LFA-1 antibody treatment suppressed the development of collagen-induced arthritis in DBA/1 mice (
ICAM-1 was found to be expressed mostly by small blood vessels in the peripheral synovial pannus area. In central osteodestructive lesions, ICAM-1 was predominantly expressed by a cell population other than the endothelium, suggesting that this adhesion molecule may not only mediate lymphocyte trafficking. Stimulated human synovial fibroblasts co-expressing HLA class II antigens and ICAM-1 have been reported to serve as potent antigen-presenting cells for staphylococcal enterotoxin B to T-lymphocytes (
In summary, our study shows that LFA-1 and ICAM-1 expressions appear to be closely associated with osteodestruction and may therefore play an important role in tissue degradation and the chronicity of the disease. In addition, our results support further similarity to human rheumatoid arthritis (
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Acknowledgments |
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We thank Dr Samuel Hunter and Dr Mary Griffiths for critically reviewing the manuscript, Dr Jörg Kriegsmann and Dr Thomas Muche for technical advice, and Ms D. Menrath for excellent assistance with taking the radiographs.
Received for publication February 24, 1997; accepted March 7, 1997.
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