Studies of isolated synovial lining cells of rheumatoid and nonrheumatoid synovial membranes

Synovial membranes from patients with rheumatoid arthritis and other arthritides were digested with tryps.n to produce suspensions of synovial cells. The cells were stained by the direct fluorescent antibody technique for IgG, IgM and the β_1c (C3) component of complement, using fluorescein and rhodamine isothiocyanate-labeled antisera. A characteristic diffuse staining pattern for IgG and β_1c was observed in the cytoplasm of the phagocytic lining cells of rheumatoid synovium. In seropositive patients, inclusions containing IgG, IgM, and β_1c were also stained in these cells. The role of these immunoglobulins, localized in the lining cells, is discussed in relation to rheumatoid inflammation.     

Resistin in rheumatoid arthritis synovial tissue, synovial fluid and serum

BACKGROUND: Resistin is a newly identified adipocytokine which has demonstrated links between obesity and insulin resistance in rodents. In humans, proinflammatory properties of resistin are superior to its insulin resistance-inducing effects. OBJECTIVES: To assess resistin expression in synovial tissues, serum and synovial fluid from patients with rheumatoid arthritis, osteoarthritis and spondylarthropathies (SpA), and to study its relationship with inflammatory status and rheumatoid arthritis disease activity. METHODS: Resistin expression and localisation in synovial tissue was determined by immunohistochemistry and confocal microscopy. Serum and synovial fluid resistin, leptin, interleukin (IL)1beta, IL6, IL8, tumour necrosis factor alpha, and monocyte chemoattractant protein-1 levels were measured. The clinical activity of patients with rheumatoid arthritis was assessed according to the 28 joint count Disease Activity Score (DAS28). RESULTS: Resistin was detected in the synovium in both rheumatoid arthritis and osteoarthritis. Staining in the sublining layer was more intensive in patients with rheumatoid arthritis compared with those with osteoarthritis. In rheumatoid arthritis, macrophages (CD68), B lymphocytes (CD20) and plasma cells (CD138) but not T lymphocytes (CD3) showed colocalisation with resistin. Synovial fluid resistin was higher in patients with rheumatoid arthritis than in those with SpA or osteoarthritis (both p<0.001). In patients with rheumatoid arthritis and SpA, serum resistin levels were higher than those with osteoarthritis (p<0.01). Increased serum resistin in patients with rheumatoid arthritis correlated with both CRP (r=0.53, p<0.02), and DAS28 (r=0.44, p<0.05), but not with selected (adipo) cytokines. CONCLUSION: The upregulated resistin at local sites of inflammation and the link between serum resistin, inflammation and disease activity suggest a role for resistin in the pathogenesis of rheumatoid arthritis.     

Fibroblast-like synovial cells derived from synovial fluid

OBJECTIVE: To obtain fibroblast-like synovial cells (FLS) from synovial fluid (SF). METHODS: SF aspirated from joints of patients with rheumatoid arthritis (RA), other types of inflammatory arthritis, and osteoarthritis (OA) was centrifuged and the resulting cell pellet resuspended in growth medium. After 2 days, nonadherent cells were removed. FLS were also cultured from surgical specimens of synovial tissue (td-FLS). Phenotype characterization of fluid derived FLS (fd-FLS) was accomplished by flow cytometry and immunohistochemistry staining. Tumor necrosis factor-alpha (TNF-alpha) induced interleukin 6 (IL-6), IL-8, and cyclooxygenase 2 (COX-2) mRNA levels were assessed. RESULTS: Second and later passage fd-FLS exhibited uniform fibroblast-like morphology. Fd-FLS and td-FLS expressed a similar profile of cell surface antigens including the fibroblast marker Thy-1. Less than 2% of either cell type expressed surface markers characteristic of dendritic cells, phagocytic cells, T cells, or leukocytes. Immunohistochemistry staining revealed the presence of fibroblast products prolyl-4 hydroxylase, procollagen I, and procollagen III in both culture types. TNF-a induced increases in IL-6, IL-8, and COX-2 mRNA were suppressed by dexamethasone in both fd-FLS and td-FLS. CONCLUSION: FLS can be cultured from SF. The fibroblast phenotype was confirmed by analysis of surface antigens and intracellular proteins. Inflammatory mediators produced after stimulation of both fd-FLS and td-FLS were suppressed by dexamethasone. In addition to providing a more accessible source of FLS, fd-FLS may also facilitate study of synovial cells in early RA when tissue specimens are not readily available.     

Osteochondral repair in synovial joints

PURPOSE OF REVIEW: One of the major challenges in rheumatology remains the induction of osteochondral repair in synovial joints. Remarkable progress has been made in controlling the inflammatory pathways of chronic synovitis and tissue damage in rheumatoid arthritis and spondyloarthropathy. Here, we provide an overview of the current knowledge on the mechanisms involved in osteochondral repair in degenerative joint diseases, as well as in immune mediated inflammatory arthritides, with special emphasis on tumor necrosis factor alpha and IL-1. RECENT FINDINGS: Homeostasis of articular cartilage and subchondral bone are essential for maintaining the integrity of osteochondral structures within synovial joints. This is achieved by the regulation of a delicate balance between anabolic and catabolic signals. In articular cartilage one cell type, the chondrocyte, is responsible for regulation of homeostasis. In bone, however, two distinct cell types, osteoblasts and osteoclasts, are responsible for anabolic and catabolic pathways, respectively. In inflammatory joint disorders, this tight regulation is profoundly dysregulated, with tumor necrosis factor alpha acting as an important catalyst of a disturbed homeostasis, together with IL-1. Targeting these cytokines may restore the intrinsic repair capacity of osteochondral structures. SUMMARY: To restore catabolic cytokine balances appears to be a suitable strategy to promote osteochondral repair.     

Inhibitors of interleukin 1 activity in synovial fluids and in cultured synovial fluid mononuclear cells.

Measurement of interleukin 1 (IL-1) in synovial fluids (SF) yielded variable results and implied the presence of an inhibitory activity. As peripheral blood monocytes produce an IL-1 receptor antagonist (IL-1ra), we investigated whether SF mononuclear cells (SFMC) also secreted such inhibitory activity. MC isolated from inflammatory SF produced, in addition to variable levels of IL-1, a specific IL-1 inhibitor of approximately 23 kDa which blocked both IL-1 biological activity and binding to its receptor. Western blot, using a polyclonal antibody to rhIL-1ra, indicated that SFMC secreted material that shared immunological crossreactivity with the cloned IL-1ra. IL-1 inhibitory activity was also detected in SF but not formally demonstrated to be related to IL-1ra. In conclusion, SFMC could produce IL-1ra and an imbalance between IL-1 and its specific antagonist may be relevant to the severity of joint destruction.     

Chondroprogenitor cells of synovial tissue

OBJECTIVE: To assess the chondrogenic potential of cells within the synovium. METHODS: Explants of synovium taken from various sites in the joint were embedded in agarose and cultured with transforming growth factor beta1 (TGFbeta1) to assess their chondrogenic potential. Isolated synovial cells were also tested for their chondrogenic potential by culturing them as aggregates in a chemically defined medium with TGFbeta1. Cartilage formation was determined with histologic staining and immunohistochemistry. The osteochondral potential of the isolated cells was also assessed after subcutaneous implantation of the cells, loaded into porous calcium phosphate ceramic cubes, in athymic mice. RESULTS: A total of 48 synovial explants were cultured in agarose with TGFbeta1. The formation of cartilage was observed in the outer region of 21 explants, and type II collagen was localized in that region by immunohistochemistry. A larger percentage of TGFbeta1+ explants from the inner synovium sites formed cartilage compared with those from the outer synovium sites. Chondrogenesis occurred in aggregates incubated with TGFbeta1 as early as day 7, and by day 14, all TGFbeta1+ aggregates demonstrated chondrogenesis. In contrast with the results of the in vitro aggregate assay for chondrogenesis, no formation of cartilage or bone was evident in any section containing synovial cell-loaded ceramic cubes that were harvested at either 3 or 6 weeks after implantation subcutaneously in athymic mice. CONCLUSION: Synovial explants and isolated synovial cells will undergo chondrogenesis when cultured in the presence of TGFbeta1. The data indicate a possible synovial origin for the chondrocytic cells found in rheumatoid pannus. Furthermore, these data are consistent with the clinical findings of synovial chondrogenesis leading to synovial chondromatosis.     

DNA hypomethylation in rheumatoid arthritis synovial fibroblasts

Objective

Rheumatoid arthritis synovial fibroblasts (RASFs) are phenotypically activated and aggressive. We undertook this study to investigate whether the intrinsic activation of RASFs is due to global genomic hypomethylation, an epigenetic modification.

Methods

Global genomic hypomethylation was assessed by immunohistochemistry, flow cytometry, and L1 promoter bisulfite sequencing. The levels of Dnmt1 were determined in synovial tissue and cultured SFs by Western blotting before and after treatment with cytokines and growth factors. Normal SFs were treated for 3 months with a nontoxic dose of the DNA hypomethylation drug 5‐azacytidine (5‐azaC), and changes in gene expression were revealed using complementary DNA arrays. The phenotypic changes were confirmed by flow cytometry.

Results

In situ and in vitro, RASF DNA had fewer 5‐methylcytosine and methylated CG sites upstream of an L1 open‐reading frame than did DNA of osteoarthritis SFs, and proliferating RASFs were deficient in Dnmt1. Using 5‐azaC, we reproduced the activated phenotype of RASFs in normal SFs. One hundred eighty‐six genes were up‐regulated >2‐fold by hypomethylation, with enhanced protein expression. These included growth factors and receptors, extracellular matrix proteins, adhesion molecules, and matrix‐degrading enzymes. The hypomethylating milieu induced irreversible phenotypic changes in normal SFs, which resembled those of the activated phenotype of RASFs.

Conclusion

DNA hypomethylation contributes to the chronicity of RA and could be responsible for the limitation of current therapies.

     

Expression of laminins and their integrin receptors in different conditions of synovial membrane and synovial membrane-like interface tissue

OBJECTIVE: To demonstrate the expression of laminins (Lns) and their integrin (Int) receptors in different synovial samples and synovial membrane-like interface tissues from well fixed and aseptically loosened total hip replacement (THR), and the potential role of Ln-Int interaction in the production of collagenases and cytokines. METHODS: Immunohistochemical staining was done to detect the distribution of EHS Ln, Ln alpha2, alpha3, alpha5, beta1, beta2 chains and Int alpha1, alpha2, alpha3, alpha6, beta1, beta4 subunits in different samples. Double immunofluorescence labelling was used to find colocalisation of Int alpha6 subunit and collagenase-1/collagenase-3/TNFalpha/IL6. RESULTS: General Ln immunoreactivity was detected in all specimens. Ln alpha5, beta1 and beta2, but not alpha2 and alpha3 chains were seen in the synovial lining and the basement membrane of blood vessels with the intensity/extent of labelling in the following rank order: rheumatoid arthritis (RA) loosened prostheses, osteoarthritis, well fixed prostheses, traumatic knees. Among Int subunits, staining for beta1 was usually the strongest, followed by staining for Int alpha6, alpha1, alpha3, and alpha2 subunits, with the same rank order for overall expression of Lns. Int beta4 subunit was not detectable in most of the specimens. Double labelling focused on Int alpha6 subunit disclosed its frequent colocalisation with collagenases 1 and 3 and with tumour necrosis factor alpha and interleukin 6 in synovial lining. CONCLUSION: Synovial lining contains Ln-10, Ln-11, and Int alpha6beta1 and alpha1beta1 receptors. In aseptic loosening of THR, interface tissue has a similar Ln subtype and Int receptor composition as RA synovium, which confirms its "lining-like" phenotype. Synovial lining does not contain Ln-5 (alpha3beta3gamma2) or Int alpha6beta4, which are components of epithelial hemidesmosomes. The expression of Lns and their Int receptors is upregulated in inflammation. The close spatial relation between Ln and its Int receptors in synovial lining cells containing proteinases and cytokines suggests a potential role in joint destruction and prosthetic loosening.     

Lubrication of synovial membrane.

(1) An apparatus has been constructed to measure the coefficient of friction of synovial membrane against a glass slider. (2) Provision was made to vary the load on the specimens. (3) The coefficient of friction varied from 0-006 to 0-07. (4) A presentation similar to that used in the Sommerfeld analysis of journal bearings indicated that in these experiments synovial membrane was lubricated in the hydrodynamic regimen.     

Acidosis of synovial fluid correlates with synovial fluid leukocytosis.

The antibacterial activity of aminoglycoside antibiotics is significantly reduced by lowering the pH of the incubation medium. Since gram-negative septic arthritis responds poorly to aminoglycoside antibiotic therapy, we sought to determine whether synovial fluid acidosis contributes to this poor outcome. Synovial fluid samples from 22 patients with various forms of acute and chronic arthritis were examined for white blood cell count and pH. A close correlation (r = -0.92, p is less than 0.001) between an increasing white blood cell count and a decreasing pH was demonstrated. Since septic arthritis is associated with high white blood cell counts, in synovial fluid, the resultant low pH may contribute to the poor response to gram-negative septic arthritis treated with aminoglycoside antibiotics.     

The influence of synovial fluid on adenovirus‐mediated gene transfer to the synovial tissue

Objective

To determine the effect of synovial fluid (SF) from rheumatoid arthritis (RA) patients on adenovirus type 5 (Ad5)–mediated gene transfer to synoviocytes, and to explore new strategies for vector development based on the neutralization data obtained.

Methods

SF was derived from 63 randomly selected RA patients. Ten samples were used to study the effect of SF on Ad5‐mediated gene transfer in synoviocytes. IgG and <100‐kd fractions were purified from these 10 SF, and their effect on gene transfer was determined. Neutralizing activity against wild‐type Ad5 (wt‐Ad5), wt‐Ad26, wt‐Ad34, wt‐Ad35, and wt‐Ad48 was tested in the SF from the remaining 53 patients.

Results

Seven of 10 SF samples inhibited Ad5‐mediated gene transfer. Purified antibodies exhibited inhibition patterns similar to those seen with unfractionated SF. In 5 of 10 SF samples, low molecular weight fractions inhibited gene transfer at low dilutions. Neutralization of wt‐Ad35 by SF from RA patients was less frequent than neutralization of other wt‐Ad tested (4% versus 42–72%; n = 53).

Conclusion

SF from 70% of the RA patients contained neutralizing antibodies that hamper Ad5‐mediated gene transfer to synoviocytes. The activity of neutralizing antibodies may be circumvented in the majority of RA patients when vectors based on an Ad35 backbone are used.

     

The effects of synovial fluid macrophages and interleukin-1 on hyaluronic acid synthesis by normal synovial fibroblasts

Summary The effects of peripheral blood monocyte and rheumatoid synovial fluid macrophage conditioned media were studied on hyaluronic acid (HA) metabolism of normal synovial fibroblasts. Both media stimulated HA synthesis about two-fold compared to controls (1% fetal calf serum). The activated mononuclear phagocyte conditioned media did not contain HA-degrading activity in these experiments. The effects of various concentrations of interleukin-1 (IL-1) on HA synthesis and proliferation of synovial fibroblasts were studied. Even at very low concentrations (0.1 IU IL-1/ml) HA synthesis was stimulated. With increasing concentrations HA synthesis did not increase but proliferation was stimulated. Stimulated fibroblasts synthesized mainly high molecular weight HA. Thus with IL-1-activation, normal synovial fibroblasts could not produce increased amounts of abnormal HA with decreased molecular weight.     

Identification of hydroxyapatite crystals in synovial fluid

A semiquantitative technique employing (¹⁴C) ethane-1-hydroxy 1, -1-diphosphonate (EHDP) binding has been used to detect crystals, presumably hydroxyapatite, in human synovial fluid samples which were handled to prevent the formation of artifactual mineral phase. Binding material was found in 29% of noninflammatory and in none of inflammatory joint fluids. Nuclide binding material was strongly correlated with the presence of CPPD crystals and with radiographic evidence of cartilaginous degeneration.     

Effects of pulse methylprednisolone on inflammatory mediators in peripheral blood,synovial fluid,and synovial membrane in rheumatoid arthritis

Objective. To establish whether the clinical efficacy of pulse methylprednisolone (MP; 1,000 mg intravenously) is related to the modulation of proinflammatory cytokines within the peripheral blood, synovial membrane, or synovial fluid compartments. Methods. Eighteen patients with active rheumatoid arthritis (RA) were studied. Peripheral blood (11 patients) and knee synovial fluid (9 patients, 10 knees) were obtained before and at 4 and 24 hours after MP therapy. Interleukin-1β (IL-1β), IL-8, and tumor necrosis factor α (TNFα) were measured by enzyme-linked immunosorbent assay and biologic assays; prostaglandin E₂ (PGE₂) was measured by competitive radioimmunoassay. In 10 patients, arthroscopically directed synovial biopsies were obtained before and at 24 hours after treatment, at disease relapse (4 patients), and after retreatment (1 patient). Membranes were stained by immunohistochemical techniques with monoclonal antibodies against TNFα, IL-8, IL-1β, and the IL-1 receptor antagonist protein (IL-1Ra). Results. MP therapy was associated with a rapid (within 24 hours) and substantial decrease in the expression of TNFα in the lining and sublining regions of the synovial membrane, as well as substantial decreases in the levels of TNFα in serum and synovial fluid. There was also reduced IL-8 expression in the synovial lining, as well as reduced synovial fluid IL-8 levels. No effect on synovial membrane IL-1β and IL-1Ra or synovial fluid IL-1β and PGE₂ was found. Conclusion. MP therapy rapidly reduces IL-8 and TNFα levels in the synovial compartment, with cytokine changes in the serum and synovial fluid reflecting the changes in the synovial membrane. Alterations in TNFα expression in the synovial membrane correlated with clinical response to, and subsequent relapse after, MP therapy.     

Suppression of human synovial cell proliferation by dihomo-gamma-linolenic acid

Prostaglandin E1 (PGE1) and oils enriched in its precursor fatty acids suppress inflammation and joint tissue injury in several animal models. Since synovial cell proliferation is a hallmark of rheumatoid arthritis, we studied the effect of dihomo-gamma-linolenic acid (DGLA), an immediate precursor of PGE1, on the growth of human adherent synovial cells (ASC) in tissue culture. When stimulated by appropriate concentrations of recombinant interleukin-1 beta (rIL-1 beta), ASC proliferate and produce PGE. DGLA-enriched medium suppressed both baseline and rIL-1 beta-stimulated ASC growth fivefold, compared with medium supplemented with arachidonic acid. Indomethacin reduced the effect of the DGLA. Synovial cells incorporated the DGLA, and rIL-1 beta-stimulated cells that were incubated with DGLA exhibited a 14-fold increase in PGE1 (to 25.2 +/- 6.0 ng/ml, mean +/- SD) and a 70% decrease in PGE2 (to 25.2 +/- 4.2 ng/ml) compared with cells in control medium. At equivalent concentrations (5 x 10(-7) M), PGE1 increased the level of cellular cAMP to a greater extent than did PGE2 (16.8 +/- 2.0 pmoles versus 4.3 +/- 1.9 pmoles, mean +/- SEM). Exogenous PGE1 was also a more effective inhibitor of cell growth. Similarly, cAMP concentrations in cells exposed to DGLA for 6 hours were greater than concentrations in arachidonic acid-enriched cultures (17.8 +/- 3.3 pmoles versus 2.1 +/- 2.0 pmoles). These observations suggest that DGLA can restrain ASC growth, an effect which may be due to its capacity to increase PGE1 production and subsequent cellular cAMP concentration.     

Sequential synovial fluid sampling suggests plasma and synovial fluid IL-6 vary independently in rheumatoid arthritis

SIR, Inflammatory cytokines, including interleukin-6 (IL-6),are raised in rheumatoid arthritis (RA). Single paired daytimesamples show that the IL-6 and TNF- concentrations in synovialfluid exceed blood concentrations during the day [1–3],and these and other data suggest that joints are the source.Recent data have shown that plasma IL-6 in RA follows a circadianpattern with a peak at 6 a.m. [4]. If IL-6 is generated insidejoints and diffuses into plasma, it might be expected that thesynovial fluid IL-6 concentration     

Mast cell numbers in rheumatoid synovial tissues

Synovial biopsy specimens from 20 patients with rheumatoid arthritis were subjected to quantitative analysis for several parameters of inflammation and for enumeration of synovial tissue mast cells. Strong positive correlations were found between numbers of mast cells per cubic millimeter of synovial tissue and the following synovial tissue parameters: inflammatory index (a quantification of lymphocytic infiltration), Leu-3a grade (T helper/inducer lymphocytes), Leu-1 grade (T lymphocyte), and plasma cell grade. A strong negative correlation was found between the synovial mast cell count and the extent of sublining layer fibrin deposition. Correlations between synovial mast cell count and Leu-2a grade, ratio of Leu-3a grade:Leu-2a grade, OKM1 grade, HLA–DR grade, and lining layer thickness grade did not reach statistical significance. In addition, we obtained synovial specimens from 6 of the patients both before and after long-term therapy with oral methotrexate and from 3 of the patients before, and 1 week after, an intraarticular injection of steroid. The 3 patients who had an intraarticular steroid injection showed a 67–96% decrease in the number of synovial tissue mast cells; there was no significant change in the number of synovial mast cells in the tissues of the 6 patients who received oral methotrexate. These observations are the first documentation of a quantitative relationship between the number of mast cells and the number and phenotypic profile of infiltrating lymphocytes in an inflamed tissue, which in this case, is human synovium. Our findings suggest that mast cells are involved in the pathologic interactions in rheumatoid arthritis and might play a role in the early phases of exacerbations of disease activity.