The effect of taurine chloramine on pro-inflammatory cytokine production by peripheral blood mononuclear cells isolated from rheumatoid arthritis and osteoarthritis patients

OBJECTIVE: Pro-inflammatory cytokines play a critical role in the pathogenesis of RA. A natural oxidant, TauCl exerts anti-inflammatory activities. Here, the effects of Tau and TauCl on key pro-inflammatory cytokines--IL-1beta, IL-6 and TNF-alpha production by LPS-triggered peripheral blood mononuclear cells (PBMCs) isolated from RA and OA patients and healthy blood donors--were examined. METHODS: PBMCs were stimulated with LPS (24 h) in the presence of Tau or TauCl (200-400 microM). Cytokine production was measured in culture supernatants (secreted) and cells lysates (cell-associated) using specific ELISAs. RESULTS: Production of the secretedforms of IL-1beta and IL-6 was inhibited by TauCl with IC50 approximately equal to 250 microM and 300-400 microM respectively, in all investigated groups. In all cultures of PBMCs TauCl raised the TNF-alpha production at the low concentration (200 mM), while at the higher concentration (400 microM) either reduced it (55% of RA, 70% of OA patients and 55% of healthy donors) or exerted no effect (remainder of patients). Interestingly, Tau did not significantly affect any cytokine production. CONCLUSION: TauCl at high concentrations down-regulates pro-inflammatory cytokine production. However, the impact of TauCl on TNF-alpha production by PBMCs from RA is more limited than in cells isolated from OA patients.     

Reduced chemokine and matrix metalloproteinase expression in patients with rheumatoid arthritis achieving remission

OBJECTIVE: To quantify the changes in synovial expression of mediators of macrophage chemotaxis, matrix degradation, and macrophage infiltration in the synovial membrane of patients with rheumatoid arthritis (RA) achieving American College of Rheumatology (ACR) defined remission and radiological arrest. METHODS: Knee synovial biopsies were taken from a selected group of 18 patients with RA before and after treatment and immunostained with antibodies specific for CD68; the chemokines macrophage inflammatory protein (MIP)-1a and monocyte chemoattractant protein (MCP)-1; matrix metalloproteinases (MMP-1 and 3) and their inhibitors, the tissue inhibitors of metalloproteinases (TIMP-1 and 2); as well as isotype-specific negative controls. Immunostaining was quantified using a computer assisted color video image analysis system. Radiographs were performed before and after treatment and the Larsen score determined. Patients were arbitrarily divided into 2 groups: the radiological arrest group (defined as change in Larsen score pound 5 from baseline) and radiological progressors (defined as change in Larsen score > 5). Patients were classified according to ACR response criteria. RESULTS: In the 8 patients who achieved ACR defined remission, there were tendencies toward reductions in the synovial lining layer (LL) expression of MIP-1a by 36% (p = 0.1) and MCP-1 by 48% (p = 0.1). Significant reductions occurred in the expression of MMP-1, by 53% in the LL (p = 0.008) and 59% in synovial sublining layer (SL) (p = 0.02) and MMP-3, by 76% in LL (p = 0.02), and 72% in SL (p = 0.008), but not in TIMP expression. In this group of patients there were reductions in MMP:TIMP ratios, in particular the MMP-1:TIMP-1 ratio in the LL (p = 0.05), MMP-3:TIMP-1 ratio in the LL (p = 0.05) and SL (p = 0.008), and MMP-3:TIMP-2 ratio in the LL (p = 0.04) and SL (p = 0.08). In this group of patients CD68+ macrophage infiltration was significantly reduced in the LL by 59% (p = 0.008) and in the SL by 52% (p = 0.008), which corresponded with the reductions in chemokine expression. In the remaining 10 patients who did not achieve full remission there were no significant changes in the variables studied. In the group achieving ACR 50% or 70% response there was a reduction in CD68 expression that approached significance (p = 0.06 in LL and SL), but there was no significant change in the other variables. There were no significant changes in the patients with an ACR 20% response. In the radiological arrest group (12 patients) there was a 41% reduction in LL expression of MIP-1a (p = 0.05) and MMP-1 (p = 0.06). Reductions in MMP:TIMP expression were also noted, in particular in MMP-1:TIMP-1 expression in the LL (p = 0.04) and MMP-3:TIMP-1 in the SL (p = 0.01). There were corresponding reductions in CD68 expression by 49% (p = 0.009) in LL and by 42% (p = 0.0005) in SL. In the radiological progressors (6 patients) there were no significant reductions in mediator expression. CONCLUSION: In RA, ACR defined remission is associated with reductions in MMP-1 and 3 expression, with a corresponding reduction in macrophage infiltration and a tendency to reduction in MIP-1a expression. Radiological arrest is associated with reductions in MMP-1 expression, and significant reductions in macrophage infiltration, MIP-1 expression, and MMP:TIMP ratio.     

Serum cytokines in patients with rheumatoid arthritis

Serum cytokines such as interleukin 1 (IL-1), interferon (IFN-), and tumor necrosis factor (TNF) were measured in 40 patients with rheumatoid arthritis (RA). In the 40 patients studied, serum IL-1 was detected in 5 patients, IFN- in 10 patients, and TNF in 20 patients. The IL-1-positive group showed increased values of activity indices compared to the IL-1-negative group. Values of serum IFN- correlated well with the number of peripheral blood lymphocytes and CD3⁺ cells and with the percentage of CD3⁺ CD26⁺ cells. Values of serum TNF correlated positively with the number of peripheral blood monocytes and the percentage of CD3⁺ HLA-DR⁺ and CD3⁺ CD25⁺ cells. These results indicated that serum IL-1 in RA patients reflects the activity of RA, while the serum IFN- and TNF in RA patients may be related to circulating activated lymphocytes and monocytes, respectively.     

Cytokine expression in synovial membranes of patients with rheumatoid arthritis and osteoarthritis.

OBJECTIVES--To compare, by immunohistochemistry, the cellular and cytokine profile in rheumatoid arthritis (RA) and osteoarthritis (OA) synovial membranes (SMs). Synovium was obtained at knee arthroplasty from 10 patients with RA and 10 with OA. METHODS--Synovial membranes were stained with a panel of monoclonal antibodies (MAb) to assess cytokine expression (IL-1 alpha, IL-1 beta, IL-6, GM-CSF, TNF-alpha and EGF) and the intensity of the mononuclear cellular infiltrate (MNC). RESULTS--Significantly greater percentages of IL-1 alpha, IL-1 beta, IL-6, TNF-alpha, GM-CSF and EGF cells were detected in all areas of the rheumatoid SMs when compared with osteoarthritic SMs. Five RA but only one OA SM demonstrated focal lymphoid aggregates. Lining layer thickening was noted in RA SMs only. The intensity of the MNC and number of blood vessels were greater in the RA group. CONCLUSION--The results suggest that the differences in cytokine production by RA and OA SMs are quantitative but that the greater thickness of the synovial lining layer and higher vascularity may be specific to RA.     

Chemokine and chemokine receptor expression in paired peripheral blood mononuclear cells and synovial tissue of patients with rheumatoid arthritis, osteoarthritis, and reactive arthritis

BACKGROUND: Chemokine receptors and chemokines have a crucial role in leucocyte recruitment into inflamed tissue. OBJECTIVE: To examine the expression of an extensive number of chemokines and receptors in a unique bank of paired samples of synovial tissue (ST) and peripheral blood (PB) from patients with different forms of arthritis to assist in identifying suitable targets for therapeutic intervention. METHODS: Synovial biopsy specimens were obtained from 23 patients with rheumatoid arthritis (RA), 16 with osteoarthritis, and 8 with reactive arthritis. ST chemokine (CCL2/MCP-1, CCL5/RANTES, CCL7/MCP-3, CCL8/MCP-2, CCL14/HCC-1, CCL15/HCC-2, CCL16/HCC-4), chemokine receptor (CCR1, CCR2b, CCR5, CXCR4), and CD13 expression was analysed by immunohistochemistry and two colour immunofluorescence. Chemokine receptor expression (CCR1, CCR3, CCR5, CCR6, CCR7) on PB cells was studied by flow cytometry. Non-parametric tests were used for statistical analysis. RESULTS: Abundant expression of CCR1, CXCR4, and CCR5 was found in all forms of arthritis, with a specific increase of CCL5 and CCL15 in RA. CCL7, CCL8, CCL14, CCL15, and CCL16 were detected for the first time in ST. The results for PB analysis were comparable among different arthritides. Interestingly, compared with healthy controls, significantly lower expression of CCR1 (p<0.005) and CCR5 (p<0.05) by PB monocytes in the patient groups was seen. DISCUSSION: A variety of chemokines and receptors might have an important role in several inflammatory joint disorders. Although other receptors are involved as well, migration of CCR1(+) and CCR5(+) cells towards the synovial compartment may play a part in the effector phase of various forms of arthritis.     

Elevated circulating T cell subsets and cytokines expression in patients with rheumatoid arthritis

Clinical Rheumatology - This study aimed to assess the role of different subsets of circulating follicular helper T cells (Tfh), central memory (TCM), effector memory (TEM), Naïve T,...     

CYP7B expression and activity in fibroblast-like synoviocytes from patients with rheumatoid arthritis: regulation by proinflammatory cytokines

OBJECTIVE: The cytochrome P450 enzyme CYP7B catalyzes the conversion of dehydroepiandrosterone (DHEA) into 7alpha-hydroxy-DHEA (7alpha-OH-DHEA). This metabolite can stimulate the immune response. We previously reported that the severity of murine collagen-induced arthritis is correlated with CYP7B messenger RNA (mRNA) expression and activity in the arthritic joint. The purpose of this study was to investigate the presence of 7alpha-OH-DHEA in synovial samples and the cytokine regulation of CYP7B activity in fibroblast-like synoviocytes (FLS) from patients with rheumatoid arthritis (RA). METHODS: The presence of 7alpha-OH-DHEA was examined in synovial biopsy tissues, synovial fluid, and serum by radioimmunoassay. The effect of cytokines on CYP7B mRNA expression and CYP7B activity in FLS was examined by determining the formation of the CYP7B enzyme product 7alpha-OH-DHEA with the use of high-performance liquid chromatography. RESULTS: The CYP7B enzyme product 7alpha-OH-DHEA was found in synovial biopsy tissues, synovial fluid, and serum from RA patients. The proinflammatory cytokines tumor necrosis factor alpha (TNFalpha), interleukin-1alpha (IL-1alpha), IL-1beta, and IL-17 up-regulated CYP7B activity in an FLS cell line 2-10-fold. Enhanced CYP7B activity was correlated with an increase in CYP7B mRNA. The cytokine transforming growth factor beta inhibited CYP7B activity. Moreover, CYP7B activity was detected in 10 of 13 unstimulated synovial fibroblast cell lines. Stimulation with TNFalpha increased CYP7B activity in all cell lines tested. CONCLUSION: Exposure to the proinflammatory cytokines TNFalpha, IL-1alpha, IL-1beta, and IL-17 increases CYP7B activity in synovial tissue. Increased CYP7B activity leads to higher levels of the DHEA metabolite 7alpha-OH-DHEA in synovial fluid, which may contribute to the maintenance of the chronic inflammation observed in RA patients.     

Interleukin-1 receptor antagonist production in cultured synovial cells from patients with rheumatoid arthritis and osteoarthritis.

OBJECTIVE--To measure the amounts of interleukin-1 receptor antagonist (IL-1ra) protein produced by cultured synovial cells obtained from patients with rheumatoid arthritis (RA) and osteoarthritis (OA). METHODS--Synovial cells obtained from patients with either RA or OA were cultured and the supernatants were measured for IL-1ra by enzyme linked immunosorbent assay. RESULTS--The synovial cells obtained from patients with RA produced significantly smaller amounts of IL-1ra than did those obtained from patients with OA, in a late passage (third to fifth) without stimulation and a first passage both with and without stimulation (p < 0.025, respectively). In addition, when the patients with RA were divided into two groups according to the maximum number of lining cell layers, the amounts of IL-1ra produced by the proliferative type were smaller than those produced by the less proliferative type (p < 0.025). CONCLUSIONS--The above findings suggest that IL-1ra production in RA synovial cells is suppressed, and that reduced IL-1ra protein production is one of the causes which leads to the proliferation of lining cells and persistent joint inflammation.     

Osteoprotegerin expression in synovial tissue from patients with rheumatoid arthritis,spondyloarthropathies and osteoarthritis and normal controls

OBJECTIVES: To demonstrate the expression of osteoprotegerin (OPG) and receptor activator of nuclear factor kappaB ligand (RANKL) in synovial tissue from rheumatoid arthritis (RA) patients, establish the cell lineage expressing OPG and compare the expression of OPG in RA, spondyloarthropathies, osteoarthritis and normal synovial tissue. METHODS: Synovial biopsy specimens were obtained at arthroscopy from 16 RA and 12 spondyloarthropathy patients with active synovitis of a knee joint, six RA patients with no evidence of active synovitis, 10 patients with osteoarthritis and 18 normal subjects. Immunohistological analysis was performed using monoclonal antibodies (mAb) to detect OPG and RANKL expression. In addition, dual immunohistochemical evaluation was performed with lineage-specific monoclonal antibodies (macrophages, fibroblasts and endothelial cells) and OPG to determine the cell lineages expressing OPG. The sections were evaluated by computer-assisted image analysis and semiquantitative analysis. RESULTS: Two patterns of OPG expression were seen, one exclusively in endothelial cells and one expressed predominantly in macrophages in the synovial lining layer. Both patterns of OPG staining could be blocked with excess recombinant OPG. Endothelial and synovial lining expression of OPG was seen in all synovial tissues except those from patients with active RA. In contrast, RANKL expression was seen predominantly in synovial tissue from patients with active disease, mainly in sublining regions, particularly within areas of lymphocyte infiltration. CONCLUSIONS: OPG expression on macrophage type synovial lining cells as well as endothelial cells is deficient in RA patients with active synovitis, in contrast to that seen in spondyloarthropathy patients with active synovitis. This deficiency in OPG expression in the inflamed joint of RA patients may be important in the development of radiologically defined joint erosions.     

Phospholipase activity in synovial fluid from patients with rheumatoid arthritis, osteoarthritis and crystal-associated arthritis

Phospholipase activity was assayed in cell-free synovial fluid (SF) from patients with rheumatoid arthritis (RA, n = 28), osteoarthritis (OA, n = 10), and crystal-associated arthritis (C, n = 7) by measuring the release of either [14C]oleic acid or [3H]arachidonic acid from radiolabeled E. coli phospholipids. Activity measured by oleic acid release was not significantly different between the three groups of patients (RA = 571 +/- 43.3, OA = 460 +/- 54.7 and C = 718 +/- 162.6 pmol/min/mg). Arachidonic acid release was significantly (p less than 0.005) less in OA (31 +/- 7.3) than RA (61 +/- 4.7) which was similar to C (58 +/- 17.6 pmol/min/mg). Arachidonic acid release correlated significantly with the SF white blood cell count (r = 0.483, p less than 0.01). This study shows the importance of the type of substrate used to measure phospholipase activity and indicates that differences in the capacity to release arachidonic acid may exist between RA and OA disease states.     

The chemokine receptor CCR5 genetic polymorphism and expression in rheumatoid arthritis patients

OBJECTIVES: To identify the genetic polymorphism of the chemokine receptor CCR5 (the Delta32 allelic variant) in patients with rheumatoid arthritis (RA) and compare the findings with healthy controls. To compare the CCR5 phenotypic expression in T cells and monocytes isolated from the peripheral blood and synovial fluid in a subgroup of RA patients. METHODS: CCR5 genes of 92 RA patients and 160 healthy controls were genotyped using specific primers flanking the region of deletion. The ethnic distribution was similar between the groups. Flow cytometric analysis was used for immunophenotyping the T cells and monocytes isolated from the peripheral blood and synovial fluid of eight RA patients. The isolated cells were triple stained with CD4 or CD8, CD25 and CCR5 monoclonal antibodies. RESULTS: There was no difference in the CCR5Delta32 genotypic frequency between the RA patients and the control group (0.055 and 0.063, respectively, p = 0.989). No homozygote for the CCR5Delta32 allele was seen in either group. Five heterozygotes were identified in the RA patient group, whose disease was shown to be aggressive. A significant enrichment of activated CCR5+ monocytes was seen in the synovial fluid of the RA patients subjected to arthrocentesis, who were all homozygotes for the CCR5 wild-type genotype. CONCLUSION: A protective role for the CCR5 allelic variant in RA development was not observed. Disease severity in the heterozygotes suggests that other proinflammatory mechanisms might overcome this mutation in vivo. The activated CCR5+ monocyte enrichment in the rheumatoid synovial fluid might indicate that this cell population has an important role in the pathogenesis of the disease.     

The impact of rheumatoid arthritis and osteoarthritis: the activities of patients with rheumatoid arthritis and osteoarthritis compared to controls

We measured the impact of rheumatoid arthritis (RA) and osteoarthritis (OA) by comparing the activities of patients with these illnesses to controls matched for age, sex, and community of residence. Our results indicate that patients with RA experience more losses than controls in every domain of human activity and that patients with OA experience more losses in the performance of household chores, shopping and errands, and leisure activities. The methods described here provide a simple, reliable way to assess the impacts of illness in the same terms for all dimensions of human activity.     

The SHAP-HA complex in sera from patients with rheumatoid arthritis and osteoarthritis.

OBJECTIVE: To investigate serum derived hyaluronan (HA) associated protein-hyaluronan (SHAP-HA) complex in sera from patients with rheumatoid arthritis (RA) and osteoarthritis (OA) and determine levels of the complex in comparison with those of hyaluronan (HA), in order to assess the role of the complex as an indicator of joint inflammation. METHODS: ELISA and HA binding assays were used for quantitation of the SHAP-HA complex and HA levels in serum samples from 142 patients (114 with RA, 28 with OA) and 31 healthy controls. Clinical evaluations were also performed. RESULTS: In some RA sera, SHAP-HA complex levels were extremely high compared to control levels, but in OA sera no marked increase was observed compared to controls. This was also the case with the HA levels compared between RA and OA sera. However, in RA the levels of the SHAP-HA complex appear to be more related to clinical variables than are levels of HA, and the most significant correlations between levels of SHAP-HA complex and HA were found in the RA group. CONCLUSION: Quantitation of the SHAP-HA complex in sera may be useful as a joint marker that directly correlates to the degree of joint inflammation in RA, and offers new insight into the pathogenesis of arthritis. It may also serve as an independent criterion in the subsequent classification of RA and OA.     

Autoantibodies to osteopontin in patients with osteoarthritis and rheumatoid arthritis

OBJECTIVE: Osteopontin (OPN), secreted mainly from chondrocytes, is suggested to be involved in the ossification and remodeling of bone and also in regulation of cytokine profiles. We investigated whether patients with osteoarthritis (OA) and rheumatoid arthritis (RA) display autoimmunity against OPN. METHODS: Recombinant human OPN (rhOPN) was prepared as a fusion protein with beta-galactosidase using E. coli. Serum samples from patients with OA or RA and from age matched healthy donors were tested for autoantibodies to rhOPN using ELISA and Western blotting. Reactivity of the same samples to purified native human OPN (nhOPN) was investigated by ELISA separately, to evaluate conformational epitopes. RESULTS: By ELISA, autoantibodies to rhOPN were found in one (0.95%) of 105 patients with OA and 2 (2.3%) of 88 patients with RA. These autoantibodies to rhOPN were confirmed by Western blotting. In contrast, 11 (9.5%) of 105 OA serum and 13 (15%) of 88 RA serum samples reacted to nhOPN. The anti-OPN positive RA patients showed high serum levels of rheumatoid factor and C-reactive protein and accelerated erythrocyte sedimentation rate compared to the anti-OPN negative group, although the differences did not achieve statistical significance. CONCLUSION: Our data showed that OPN is one of the autoantigens in OA and RA. Preferential recognition of nhOPN to rhOPN indicates that major epitope(s) of OPN would be conformational. Clinically, existence of the anti-OPN antibodies may be linked to disease severity in RA.     

Neuropeptides in synovium of patients with rheumatoid arthritis and osteoarthritis

The presence of neural elements in synovial tissue proper has earlier been suggested on the basis of nonspecific silver impregnation techniques and is now confirmed in a study based on specific demonstration of cytoskeletal neurofilaments and various neuropeptides. With both the neurofilament and neuropeptide antisera, nerves were seen predominantly in a perivascular location, there being fewer nerves freely in the stromal tissue. In the synovium of patients with rheumatoid arthritis (RA), free stromal nerves stained with neurofilament antiserum often lacked neuropeptide immunoreactivity, while this was not the case in normal synovium or synovial samples from patients with osteoarthritis (OA). Furthermore, the intensity of staining of neurotransmitter peptides was weaker in RA than in OA or normal synovial tissue. It is suggested that neurogenic inflammation may play a role in RA and that neuropeptide nerves possibly release their mediators in RA.     

Distribution of mast cells in human synovial tissue of patients with osteoarthritis and rheumatoid arthritis

Mast cells are demonstrated in synovial membranes of patients with osteo-arthritis and rheumatoid arthritis using a new staining principle based on interaction of heparin in mast cell granules with peroxidase labeled avidin. It was found that mast cell numbers in the subsynovial layer of patients with rheumatoid arthritis were significantly lower than those in patients suffering from osteo-arthritis. This decrease can be mainly attributed to patients with rheumatoid arthritis whose synovitis was characterized by a distinct histomorphological pattern consisting of lining cell ulcers and granulation tissue. However, when mast cell numbers in rheumatoid arthritis and osteo-arthritis patients were compared without respect to mast cell distribution in the subsynovial layer or the stratum fibrosum, no statistical differences between the diseases could be observed.