The nature and designation of osteoarthritis determined by its histopathology

Specimens of synovial tissue and articular cartilage removed at operations of patients with primary degenerative joint disease were scrutinized for defined histopathological parameters. The findings in the synovial membrane were subsequently compared as to frequency and intensity with identical parameters ascertained in specimens removed from patients with definite rheumatoid arthritis. These examinations disclosed the presence of synovial inflammatory features, although at lesser incidence and intensity than in rheumatoid arthritis; therefore the designation of arthritis is indicated. On the basis of the concurrently examined tissue specimens of the articular cartilage, and in consensus with the generally acknowledged start of the degenerative joint disease by disintegrating changes in the articular cartilage, the prefix of chondrosis is appropriate.     

Biochemical and histological changes in osteoarthritic synovial membrane.

In order to investigate the relationship between the synovial inflammatory response and lysosomal enzyme activity in osteoarthritis, synovial specimens obtained from 19 osteoarthritic patients and control specimens from 10 normal joints were analysed for cathepsin D and acid phosphatase enzyme levels. In estimating enzyme activities methods previously developed for quantitative enzyme determination in cartilage were modified and applied to synovial tissues for the first time. In addition, samples of osteoarthritic synovium were histologically graded according to their degree of inflammation. It was found that in osteoarthritic synovium cathepsin D and acid phosphatase, which is a general marker for lysosomal enzyme activity, were significantly increased compared with normal control synovium. No significant relationship was found between the degree of synovial tissue inflammation and lysosomal enzyme activity.     

Immunolocalization of inducible nitric oxide synthase in synovium and cartilage in rheumatoid arthritis and osteoarthritis

Nitric oxide has been implicated as a mediator of inflammatory arthritis, and recent work has shown that pro-inflammatory cytokines stimulate NO production in vitro by activation of the inducible nitric oxide synthase (iNOS) pathway. In order to identify the cellular sources of NO production within the joint, we have used immunohistochemical techniques to study the distribution of iNOS in synovium and cartilage from normal and diseased joints. iNOS was most strongly expressed in the synovial lining layer, subsynovium, vascular smooth muscle and chondrocytes from patients with rheumatoid arthritis (RA). Analysis of serial sections, coupled with double immunofluorescent staining, showed that the CD68+ macrophages in the synovial lining layer and, to a lesser extent, fibroblasts were the predominant source of iNOS within synovium, whereas T cells, B cells and neutrophils were negative. A similar pattern of iNOS staining was seen in osteoarthritis, but fewer cells were iNOS positive and the intensity of staining, particularly in cartilage, was much weaker than in RA. In contrast, no evidence of iNOS was detected in non- inflammatory synovium or in cartilage derived from normal joints (fractured neck of femur). In conclusion, these data support the hypothesis that synovium and cartilage are important sources of increased NO production in patients with inflammatory arthritis. Localization of iNOS at these sites within the inflamed joint raises the possibility that increased local production of NO may contribute to the pathogenesis of inflammatory arthritis by increasing synovial blood flow and by modulating cellular function within synovium and articular cartilage.      

Nitric oxide is a mediator of apoptosis in the rheumatoid joint

OBJECTIVE: To study the role of nitric oxide (NO) derived from the inducible nitric oxide synthase (iNOS) pathway in the induction of apoptosis in the rheumatoid joint. METHODS: Joint tissue was obtained from four rheumatoid arthritis (RA) patients, three osteoarthritis patients and two patients with a fractured neck of the femur (NOF#), and apoptotic cells were identified in cryosections using the TUNEL (terminal dUTP nick end labelling) assay. Expression of iNOS was determined using immunohistochemistry. NO synthesis and the effect of NOS inhibitors on apoptosis levels were studied in explant cultures of RA cartilage and synovium. RESULTS: Numbers of apoptotic cells were greatly increased in rheumatoid synovium and articular cartilage compared with NOF# and osteoarthritic synovium. Immunohistochemistry showed co-localization of iNOS staining and apoptosis in the synovial lining layer and articular cartilage. The NOS inhibitor L-NMMA (L-N(G)-monomethylarginine) strongly inhibited apoptosis in explant cultures of synovium and cartilage, and this was reversed by the NO donor S-nitroso-acetyl-penicillamine. CONCLUSION: This study indicates that NO acts as a mediator of apoptosis in RA and suggests that NOS inhibitors reverse this process.     

Collagenase, cathepsin B and cathepsin L gene expression in the synovial membrane of patients with early inflammatory arthritis.

OBJECTIVE: To examine the expression of the matrix metalloproteinase, MMP-1, and the cysteine proteases, cathepsin B (CB) and cathepsin L (CL), in the synovial membrane (SM) of patients with early inflammatory arthritis. METHODS: Samples of SM were obtained by blind needle biopsy or needle arthroscopy from inflamed knees of 28 patients with early inflammatory arthritis (mean disease duration 10.2 months, range 2 weeks-18 months). Sixteen patients had rheumatoid arthritis (RA), nine psoriatic arthritis and there was one each with ankylosing spondylitis, gout and an undifferentiated arthritis. Comparison was made with tissue from two patients with established erosive RA and three normal synovial tissue samples. In situ hybridization was performed using digoxigenin-labelled RNA probes. RESULTS: MMP-1, CB and CL were expressed in all patients with early arthritis and in established erosive RA, whereas normal synovium showed only scanty expression. The three proteases were prominent in perivascular infiltrates and endothelial cells of early arthritis tissue. MMP-1 was observed primarily in the lining layer, but was also evident in the sublining area. CB and CL were expressed to a lesser extent in the lining layer, and were present mainly in the subintima. The three proteases were not found in lymphoid aggregrates. No differences were observed between the disease categories. CONCLUSIONS: The detection of MMP-1, CB and CL in the synovium shortly after symptom onset implies that the potential for joint destruction exists at a very early stage in the disease. In addition, the perivascular and endothelial cell expression suggests a role for these proteases in mononuclear cell influx to the inflamed synovium and in angiogenesis.     

YKL-39 (chitinase 3-like protein 2), but not YKL-40 (chitinase 3-like protein 1), is up regulated in osteoarthritic chondrocytes

OBJECTIVE: To investigate quantitatively the mRNA expression levels of YKL-40, an established marker of rheumatoid and osteoarthritic cartilage degeneration in synovial fluid and serum, and a closely related molecule YKL-39, in articular chondrocytes. METHODS: cDNA array and online quantitative polymerase chain reaction (PCR) were used to measure mRNA expression levels of YKL-39 and YKL-40 in chondrocytes in normal, early degenerative, and late stage osteoarthritic cartilage samples. RESULTS: Expression analysis showed high levels of both proteins in normal articular chondrocytes, with lower levels of YKL-39 than YKL-40. Whereas YKL-40 was significantly down regulated in late stage osteoarthritic chondrocytes, YKL-39 was significantly up regulated. In vitro both YKLs were down regulated by interleukin 1beta. CONCLUSIONS: The up regulation of YKL-39 in osteoarthritic cartilage suggests that YKL-39 may be a more accurate marker of chondrocyte activation than YKL-40, although it has yet to be established as a suitable marker in synovial fluid and serum. The decreased expression of YKL-40 by osteoarthritic chondrocytes is surprising as increased levels have been reported in rheumatoid and osteoarthritic synovial fluid, where it may derive from activated synovial cells or osteophytic tissue or by increased matrix destruction in the osteoarthritic joint. YKL-39 and YKL-40 are potentially interesting marker molecules for arthritic joint disease because they are abundantly expressed by both normal and osteoarthritic chondrocytes.     

Detailed analysis of the cell infiltrate and the expression of mediators of synovial inflammation and joint destruction in the synovium of patients with psoriatic arthritis: implications for treatment

BACKGROUND: The synovial tissue is a primary target of many inflammatory arthropathies, including psoriatic arthritis (PsA). Identification of proinflammatory molecules in the synovium may help to identify potentially therapeutic targets. OBJECTIVE: To investigate extensively the features of cell infiltration and expression of mediators of inflammation and joint destruction in the synovium of patients with PsA compared with patients with rheumatoid arthritis matched for disease duration and use of drugs. METHODS: Multiple synovial tissue biopsy specimens were obtained by arthroscopy from an inflamed joint in 19 patients with PsA (eight oligoarthritis, 11 polyarthritis) and 24 patients with rheumatoid arthritis. Biopsy specimens were analysed by immunohistochemistry to detect T cells, plasma cells, fibroblast-like synoviocytes, macrophages, proinflammatory cytokines, matrix metalloproteinases and tissue inhibitor metalloproteinase-1, adhesion molecules and vascular markers. Stained sections were evaluated by digital image analysis. RESULTS: The synovial infiltrate of patients with PsA and rheumatoid arthritis was comparable with regard to numbers of fibroblast-like synoviocytes and macrophages. T cell numbers were considerably lower in the synovium of patients with PsA. The number of plasma cells also tended to be lower in PsA. The expression of tumour necrosis factor alpha (TNFalpha), interleukin (IL) 1beta, IL6 and IL18 was as high in PsA as in rheumatoid arthritis. The expression of matrix metalloproteinases, adhesion molecules and vascular markers was comparable for PsA and rheumatoid arthritis. CONCLUSION: These data show increased proinflammatory cytokine expression in PsA synovium, comparable to results obtained for rheumatoid arthritis, and support the notion that, in addition to TNFalpha blockade, there may be a rationale for treatments directed at IL1beta, IL6 and IL18.     

The role of vascular cell adhesion molecule 1/ very late activation antigen 4 in endothelial progenitor cell recruitment to rheumatoid arthritis synovium

OBJECTIVE: Marrow-derived endothelial progenitor cells (EPCs) are important in the neovascularization that occurs in diverse conditions such as cardiovascular disorders, inflammatory diseases, and neoplasms. In rheumatoid arthritis (RA), synovial neovascularization propels disease by nourishing the inflamed and hyperproliferative synovium. This study was undertaken to investigate the hypothesis that EPCs selectively home to inflamed joint tissue and may perpetuate synovial neovascularization. METHODS: In a collagen-induced arthritis (CIA) model, neovascularization and EPC accumulation in mouse ankle synovium was measured. In an antibody-induced arthritis model, EPC recruitment to inflamed synovium was evaluated. In a chimeric SCID mouse/human synovial tissue (ST) model, mice were engrafted subcutaneously with human ST, and EPC homing to grafts was assessed 2 days later. EPC adhesion to RA fibroblasts and RA ST was evaluated in vitro. RESULTS: In mice with CIA, cells bearing EPC markers were significantly increased in peripheral blood and accumulated in inflamed synovial pannus. EPCs were 4-fold more numerous in inflamed synovium from mice with anti-type II collagen antibody-induced arthritis versus controls. In SCID mice, EPC homing to RA ST was 3-fold greater than to normal synovium. Antibody neutralization of vascular cell adhesion molecule 1 (VCAM-1) and its ligand component alpha4 integrin potently inhibited EPC adhesion to RA fibroblasts and RA ST cryosections. CONCLUSION: These data demonstrate the selective recruitment of EPCs to inflamed joint tissue. The VCAM-1/very late activation antigen 4 adhesive system critically mediates EPC adhesion to cultured RA fibroblasts and to RA ST cryosections. These findings provide evidence of a possible role of EPCs in the synovial neovascularization that is critical to RA pathogenesis.     

Arthroscopic and immunohistologic characterization of knee joint synovitis in osteoarthritis

We studied 10 patients who had arthritis of the knee joint, but no other signs of rheumatic disease. The clinical diagnosis of osteoarthritis was corroborated by arthroscopic evidence of characteristic cartilage degeneration. Signs of inflammation were confined to areas of the synovial membrane that lay near the cartilage; thus, the major part of the joint cavity was not affected. The intensity of the synovial inflammation varied within the areas involved, but was always most pronounced in regions rimming the cartilage. Biopsy samples selected from regions of intensely inflamed synovium contained foci of T lymphocytes, which were bordered by immunoglobulin-carrying B lymphocytes and plasma cells, as well as strongly HLA-DR positive dendritic-like cells adjoined to alpha Leu-3a+ T helper lymphocytes. In tissue samples taken from macroscopically noninflamed areas, only a few infiltrating lymphocytes were seen. Thus, the inflammatory synovial changes found in osteoarthritis appear to be anatomically restricted and of varied intensity but, when present, are microscopically indistinguishable from the changes that have been previously described as indicative of rheumatoid arthritis.     

Sequential changes of parathyroid hormone related protein (PTHrP) in articular cartilage during progression of inflammatory and degenerative arthritis

OBJECTIVE: To investigate immunolocalisation of parathyroid hormone related protein (PTHrP) in two sequential models of experimental cartilage damage (inflammatory and degenerative) in order to elucidate differences in chondrocyte response to the disease. METHODS: Immunohistochemistry with a polyclonal rabbit antiserum to the N-terminal domain of PTHrP was used to detect this protein in two different rabbit models sharing progressive cartilage damage: antigen induced arthritis (AIA) and osteoarthritis (OA) secondary to partial medial meniscectomy. Cartilage specimens from early (2 days in AIA; 8 weeks in experimental OA) and late (3 weeks in AIA; 52 weeks in OA) disease were compared. RESULTS: Cell and matrix PTHrP staining in early AIA and OA was similar to that in controls. Late AIA cartilage showed a significant decrease in PTHrP positive cells and in the cartilage matrix. In contrast, at late OA stages, distinct PTHrP positivity was detected in proliferating cell clones, as assessed by proliferating cell nuclear antigen staining around cartilage damaged areas. CONCLUSION: PTHrP staining of hyaline articular cartilage shows a different pattern during progression of each type of arthritis: an overall decrease associated with the inflammatory disease, and an increase in the proliferating chondrocyte clones with degenerative arthritis.     

TENASCIN IS INCREASED IN CARTILAGE AND SYNOVIUM FROM ARTHRITIC KNEES

Tenascin is a major extracellular glycoprotein known to haveimportant functions in processes such as wound repair and embryogenesisincluding bone and cartilage formation. The expression of thismolecule in articular cartilage and synovium from normal andabnormal (OA and inflammatory joint disease including RA) humanknee joints was studied by an immunohistological technique usingparaffin embedded tissue and a specific anti-human tenascinmonoclonal antibody (BC4). The results show that in normal articularcartilage tenascin is expressed in small amounts in the surfacezone and in synovium is present in significant levels in thewalls of blood vessels only. In diseased joints expression isgreatly increased both in articular cartilage and synovium.Increased production of tenascin is likely to be part of a reparativeresponse in injured joints the understanding of which may suggestnovel mechanisms to modify disease progression in degenerativeand inflammatory joint disease. KEY WORDS: Extracellular matrix, Osteoarthritis, Rheumatoid arthritis     

The different stages of synovitis: acute vs chronic, early vs late and non-erosive vs erosive

Rheumatoid arthritis is a systemic inflammatory disease that, by definition, can affect all parts of the human body, including severe complications such as uveitis/episcleritis and vasculitis of the heart, lungs, kidneys and the central and peripheral nervous systems. Its primary and by far the most common manifestations, however, affect the joints and are characterised by inflammatory reactions and activation of the synovial lining tissue and associated structures, the latter resulting in tenovaginitis and rheumatoid nodules. As all pathophysiological mechanisms are based on pathways that are inherent in the different components of a joint, it is necessary to examine the unique features of normal synovium prior to analysing disease-specific pathways. This chapter will therefore describe the physiological structure of the synovium and the inflammatory pathology of rheumatoid synovitis in early and chronic stages of the disease.     

Pathobiology of the synovium in osteoarthritis

The synovium is a vascularized soft tissue lining the capsule and intra‐articular structures of diarthrodial joints. The synovial intima consists of a discontinuous layer of synovial lining cells supported by a cell‐containing scaffolding of loose extracellular matrix (ECM) or interstitium, composed of collagen (types III, IV, V, VI), proteoglycans and other extracellular proteins such as fibronectin, tenascin, entactin and laminin. The synovium has several functions critical for maintaining normal joint homeostasis. Synovial fluid (SF) and its constituents are produced by this tissue, which also mediates blood/synovial fluid exchanges, such as some nutrition of the surface cartilage chondrocytes and absorption of foreign particles from the synovial cavity. The synovial ECM also provides hydraulic resistance, preventing rapid seepage of SF out of the joint cavity and modifying the exchange of macromolecules and other solutes between the subintimal capillaries and SF. Two main types of synovial cell have been identified. The most common synovial lining cell is the type B or fibroblast‐like synoviocyte (FLS). FLS are characterised by abundant rough endoplasmic reticulum and dendritic processes which form a network over the intimal surface but are also present in the subintima. The highly proliferative FLS are involved in the production of specialised matrix constituents, including hyaluronan, collagens and fibronectin for the intimal interstitium and high molecular weight hyaluronan for the SF, essential for the preservation of high viscosity and low coefficient of friction between the surfaces of articular cartilage of the joint. The type A lining cell or macrophage‐like synoviocytes (MLS) are round, nonfixed, nonproliferative, immunoreactive cells which express MHC II molecules and cathepsins B, D and L. MLS absorb and degrade extracellular matrix constituents, cell debris, microorganisms and antigens in the synovial fluid and intimal matrix and use a system of capillaries and lymphatics to clear the joint of particles. Activated MLS are an abundant source of pro‐inflammatory mediators which can promote resorption of articular cartilage. Changes which occur in the osteoarthritic (OA) synovium are not as visually striking as those which occur in RA joints, however, this does not mean that OA synovium can be legitimately used as a control tissue for RA studies. In early OA, the synovium can appear grossly normal. A degree of synovial villous hypertrophy, fibrosis and a significant low grade inflammation are usually present in this tissue from subjects with OA, with a mild infiltrate composed primarily of lymphocytes and mononuclear cells. The cellularity is generally much less than that seen in RA tissue, however, natural killer cell activity and mast cell levels are increased in OA synovium, often greater than in RA tissue. Advanced OA is often characterised by cartilaginous fragments embedded in the synovium, resulting in more pronounced synovitis. Corresponding increases in several molecular markers have been described, including cytokines (interleukin‐1β, IL‐1β receptor antagonist) and other molecules implicated in cartilage destruction (stromelysin, aggrecanase‐2, inducible nitric oxide synthase, urokinase‐type plasminogen activator). Most of these changes in the pathology of the synovium have been demonstrated in recent years by immunohistological studies on both human synovial tissues and synovia from animals with surgically induced OA.     

Pathogenesis of haemophilic synovitis: experimental studies on blood-induced joint damage

Summary.  Hemarthrosis is a common manifestation of haemophilia, and joint arthropathy remains a frequent complication. Even though the exact mechanisms related to blood-induced joint disease have not yet been fully elucidated, it is likely that iron deposition in the synovium induces an inflammatory response that causes not only immune system activation but also stimulates angiogenesis. This process ultimately results in cartilage and bone destruction. Investigating the processes that occur in the early stages of blood-induced joint disease in humans has been very limited. Therefore, the use of haemophilic animal models is critical to augment the understanding of this phenomenon. This article discusses three cellular regulators (p53, p21 and TRAIL) induced in synovial tissue that are important for iron metabolism. A cartilage remodelling programme induced by the release of cytokines and growth factors that result in articular damage is also discussed. Full elucidation of the pathogenesis of haemophilic joint disease is required to identify new avenues for prevention and therapy.     

Identification of collagen subtypes in synovial fluid sediments from arthritic patients

Collagen fibers in synovial fluid sediment were described a decade ago. Since then, tissue-specific collagen molecules (types) have been characterized. Techniques were devised to identify the collagen types in joint fluid sediment. Collagens were found in 12 of 17 pellets prepared from fluid aspirates from 17 knee joints of patients with various forms of arthritis. Collagen types I and III and polypeptide chains A and B (basement membrane collagen) were specifically identified in four of seven fluids from patients with active systemic lupus erythematosus (SLE) and in a single fluid from a patient with severe septic arthritis. This “collagen profile” was identical to that of rheumatoid synovium. Type II collagen, characteristic of hyaline articular cartilage, was found in two of six fluids from osteoarthritic joints. The presence of sufficient collagen (about 5 μg) to permit typing was correlated with roentgenpgraphic evidence of joint space narrowing; the presence of the “synovial” collagen profile was correlated with decreased joint fluid pH.     

Appearance of calpain correlates with arthritis and cartilage destruction in collagen induced arthritic knee joints of mice.

OBJECTIVES--To determine the relevance of calpain in murine collagen induced arthritis (CIA) and to correlate the presence of m-calpain with the appearance of arthritis and cartilage destruction. METHODS--The immunohistochemical appearance and localisation of m-calpain at different stages of arthritis were analysed and compared with the histological changes occurring during type II CIA. The arthritic knee joint lavage was also examined for m-calpain by immunoelectrophoretic blotting. RESULTS--Immunohistochemical staining demonstrated a clear positive correlation between the appearance of m-calpain and both a histological grade of arthritis and an acute phase of cartilage destruction. Further development of the disease showed continual presence of m-calpain but with reduced intensity. Intra-articular inflammatory cells (mainly polymorphonuclear leucocytes, synovial lining cells, and sublining fibroblasts) were found to be the most positively stained, but extracellular localisation of m-calpain on the surface of cartilage and synovium, and in the articular cartilage matrix and chondrocyte lacunae, was also observed. In the knee joint lavage obtained at the most intensive stage of acute arthritis, m-calpain was detectable by immunoelectrophoretic blotting. CONCLUSIONS--The findings suggest that m-calpain may act at an early phase of CIA as a matrix proteinase and take part in the destruction of articular cartilage or activate other destructive enzymes.     

CRYSTAL SHEDDING IN SEPTIC ARTHRITIS: CASE REPORTS AND IN VIVO EVIDENCE IN AN ANIMAL MODEL

Abstract Two cases of coexisting septic and crystalline joint disease are reported. In one patient polyarticular septic artaritis occurred simultaneously with gout and pseudogout. In a second patient septic arthritis preceded the appearance of calcium pyrophosphate dihydrate (CPPD) crystals in the joint fluid, supporting an earlier postulate that lysosomal enzymes released during sepsis lead to shedding of crystals from cartilage and synovium into the joint space. This sequence was demonstrated in a rat air pouch model of synovium, in which CPPD crystals embedded in facsimile synovial tissue were released after injection of pyogenic bacteria. Coexisting septic arthritis should always be considered when crystals are identified in inflamed joints, particularly in elderly patients with concurrent infections.     

Immunolocalization of matrix metalloproteinase 3 (stromelysin) in rheumatoid synovioblasts (B cells): correlation with rheumatoid arthritis.

Metalloproteinases produced by connective tissue cells may play a key part in the destruction of joints in rheumatoid arthritis. Matrix metalloproteinase 3 (MMP-3; stromelysin) capable of degrading cartilage proteoglycans and type IX collagen and of activating procollagenase was immunolocalised in hyperplastic synovial lining cells in rheumatoid synovium, but not in the cells of normal synovium. Cells responsible for synthesis of MMP-3 have the phenotype of synovioblasts (B cells) by immunoelectron microscopy, but not of phagocytic synovial macrophages (A cells). Cultured monolayer of rheumatoid synovial cells synthesises MMP-3 only under treatment with macrophage conditioned medium. Immunolocalisation of MMP-3 in rheumatoid synovium and cultured synovial cells was possible when the specimens were treated with a monovalent ionophore, monensin. These results suggest that MMP-3 is synthesised and secreted continuously without storage from hyperplastic synovioblasts stimulated by factor(s) derived from activated macrophages present in the synovium.