The assessment of intracellular cytokines at the single-cell level by flow cytometry has recently become a potent tool in many areas of cell biology and in defining the role of cytokines in various human diseases. Three-color flow cytometry for detection of intracellular cytokines combined with simultaneous determination of lymphocytes (CD3+ and CD4+) or monocytes (CD33+ and CD14+) was used for comparison of phytohemagglutinin (PHA)-and phorbol myristate acetate (PMA)-ionomycin-induced production of intracellular cytokines in peripheral blood mononuclear cells (PBMCs) of healthy donors. We found that the number of PBMCs stained for tumor necrosis factor alpha and gamma interferon after 6 h of activation was higher when PMA-ionomycin was used for stimulation, while the frequencies of cells positive for interleukin 4 (IL-4) were similar for both stimulators. However, PMA-ionomycin stimulation caused prominent alterations of cell morphology and membrane expression of CD4 and CD14. In contrast, PHA did not cause downregulation of surface markers and resulted in less pronounced alterations in both forward and side scatter signals during flow cytometry analysis. Moreover, during 48 h of culture PHA stimulated tumor necrosis factor beta and IL-10 production, which was not observed when PMA-ionomycin was used. We conclude that the use of PHA for cell activation may limit in vitro artifacts and allow more precise analysis of intracellular cytokine production in various disease states.
Power Doppler ultrasound (PDUS) is increasingly used to assess synovitis in Rheumatoid Arthritis (RA). Prior studies have shown correlations between PDUS scores and vessel counts, but relationships with T cell immunopathology have not been described.
PBMC were isolated from healthy controls (HC) or RA patients and stimulated ex vivo with PMA and ionomycin for 3 hours in the presence of Golgistop. Paired synovial fluid (SF) or synovial tissue (ST) were analysed where available. Intracellular expression of IL-17, IFNγ, and TNFα by CD4+ T cells was determined by flow cytometry. Synovial blood flow was evaluated by PDUS signal at the knees, wrists and metacarpophalangeal joints of RA patients. Serum, SF and fibroblast culture supernatant levels of vascular endothelial growth factor-A (VEGF-A) were measured by ELISA. The frequency of IL17+IFNγ-CD4+ T cells (Th17 cells) was significantly elevated in peripheral blood (PB) from RA patients vs. HC (median (IQR) 0.5 (0.28–1.59)% vs. 0.32 (0.21–0.54)%, p = 0.005). Th17 cells were further enriched (mean 6.6-fold increase) in RA SF relative to RA PB. Patients with active disease had a higher percentage of IL-17+ T cells in ST than patients in remission, suggesting a possible role for Th17 cells in active synovitis in RA. Indeed, the percentage of Th17 cells, but not Th1, in SF positively correlated with CRP (r = 0.51, p = 0.04) and local PDUS-defined synovitis (r = 0.61, p = 0.002). Furthermore, patients with high levels of IL-17+CD4+ T cells in SF had increased levels of the angiogenic factor VEGF-A in SF. Finally, IL-17, but not IFNγ, increased VEGF-A production by RA synovial fibroblasts in vitro.
Our data demonstrate a link between the presence of pro-inflammatory Th17 cells in SF and local PDUS scores, and offer a novel immunological explanation for the observation that rapid joint damage progression occurs in patients with persistent positive PDUS signal.
OBJECTIVE—To test the hypothesis that ankylosing spondylitis (AS) is a T helper cell type 2 polarised disease by quantifying the T cell cytokines interferon γ (IFNγ), interleukin 4 (IL4), tumour necrosis factor α (TNFα), and IL10 at the single cell level in patients with AS in comparison with healthy HLA-B27 negative and HLA-B27 positive controls.
METHODS—Peripheral blood mononuclear cells from 65 subjects (25 HLA-B27 positive patients with active AS, 18 healthy HLA-B27 positive controls, and 22 healthy HLA-B27 negative controls) were stimulated with phorbol myristate acetate/ionomycin for six hours, surface stained for CD3 and CD8, intracellularly stained for the cytokines IFNγ, TNFα, IL4, and IL10, and analysed by flow cytometry. TNFα production was related to the genotype of the TNFα promoter at the -308 and -238 polymorphisms.
RESULTS—In peripheral blood the percentage of TNFα+ T cells was significantly lower in HLA-B27 positive patients with AS (median 5.1% for CD4+ T cells) than in healthy HLA-B27 negative controls (median 9.5%; p=0.008). Surprisingly, the percentage of TNFα+ T cells was also significantly lower in healthy HLA-B27 positive controls (median 7.48%) than in healthy HLA-B27 negative controls (p=0.034). Furthermore, the percentage of IFNγ+ T cells was lower in patients with AS and in healthy HLA-B27 positive controls than in healthy HLA-B27 negative controls (p=0.005 and p=0.003, respectively). The percentage of IL10+/CD8+ T cells was higher in patients with AS than in both control groups. In HLA-B27 positive subjects, TNF1/2 heterozygosity at -308 (n=6) was associated with a higher percentage of TNFα+ T cells than TNF1/1 homozygosity (n=25; median 9.97% v 5.11% for CD4+ T cells; p=0.017). In contrast, in HLA-B27 negative controls (n=18) there was no such genotype/phenotype correlation (median 9.4% v 10.6%).
CONCLUSIONS—The lower T cell production of TNFα and IFNγ shown at the single cell level in HLA-B27 positive patients with AS and healthy HLA-B27 positive controls may contribute to the increased susceptibility of HLA-B27 positive subjects to develop AS. Preliminary genotype-phenotype correlations suggest that in HLA-B27 positive subjects TNF2 at -308 or a linked gene results in higher TNFα production and, therefore, might be a marker for a protective haplotype.
Interleukin (IL)-17 is a proinflammatory cytokine that is produced largely by a unique CD4+ T-helper (Th) subset called Th17 cells. The development of Th17 cells is suppressed by interferon (IFN)-γ produced by Th1 cells, suggesting cross-regulation between Th17 and Th1 cells. Thus, this study analyzed the balance of CD4+ Th17 and Th1 cell responses in peripheral blood from patients with systemic lupus erythematosus (SLE) and healthy subjects.
Twenty-five adult patients with SLE and 26 healthy subjects matched for gender and age (± 2 years) were recruited. Peripheral blood mononuclear cells (PBMCs) from patients and healthy subjects were stimulated for 4 h ex vivo with phorbol myristate acetate (PMA) and ionomycin. The frequency of CD4+ T cells producing IL-17 and/or IFN-γ was measured by using flow cytometry. Expression of Th17-associated chemokine receptors CCR4 and CCR6 on CD4+ T cells as well as plasma levels of Th17-polarizing cytokines were assessed. Disease activity was evaluated by the SLE disease activity index score (SLEDAI). Unpaired t test and Pearson correlation were used for statistical analyses.
Patients with SLE had an increased frequency of CD4+IL-17+ T cells compared with healthy subjects. However, the frequency of CD4+IFN-γ+ T cells was similar between the two groups, indicating an altered balance of Th17 and Th1 cell responses in SLE. Patients with SLE also had an increased frequency of CD4+CCR4+CCR6+ T cells that are known to produce IL-17. The frequency of CD4+IL-17+ T cells and CD4+CCR4+CCR6+ T cells correlated with disease activity. In measuring plasma levels of the Th17-polarizing cytokines, levels of IL-6 were higher in patients with SLE than in healthy subjects, although levels of IL-1β, IL-21, IL-23, and transforming growth factor (TGF)-β were not different between the two groups.
We demonstrate an enhanced Th17 cell response that correlates with disease activity in patients with SLE, suggesting a role for IL-17 in the pathogenesis of lupus. Our data indicate that the mechanisms involved in balancing Th1 and Th17 regulation, as well as in producing IL-6, are aberrant in SLE, leading to an increased Th17 response. We suggest that CCR4 and CCR6 expression on CD4+ T cells should be considered as markers of disease activity, and that IL-17 blocking may offer a therapeutic target in SLE.
The aim was to characterize the expression of CCL19 and CCL21 in rheumatoid arthritis (RA) synovial tissue and to examine their regulation and pathogenic role in macrophages and RA synovial tissue fibroblasts.
Expression of CCL19 and CCL21 was demonstrated in RA and normal (NL) synovial tissues employing immunohistochemistry. CCL19 and CCL21 levels were quantified in fluids from osteoarthritis (OA), juvenile idiopathic arthritis (JIA), psoriatic arthritis (PsA) and RA using ELISA. Regulation of CCL19 and CCL21 expression was determined in RA peripheral blood in vitro differentiated macrophages as well as RA synovial tissue fibroblasts by real-time RT-PCR. CCL19 and CCL21 activated peripheral blood in vitro differentiated macrophages and RA synovial tissue fibroblasts were examined for proangiogenic factor production employing ELISA.
CCL19 and CCL21 were elevated in RA synovial tissue compared to NL controls. Levels of CCL19 and CCL21 were greatly increased in RA and PsA synovial fluid versus OA synovial fluid. In RA macrophages and fibroblasts, expression of CCL19 was increased by LPS, TNF-α and IL-1β stimulation. However, CCL21 expression was modulated by IL-1β in RA fibroblasts as well as TNF-α and RA synovial fluid in RA macrophages. CCL19 and CCL21 activation induced VEGF and Ang-1 production from RA synovial tissue fibroblasts and secretion of IL-8 and Ang-1 from macrophages.
We identify, for the first time, regulators of CCL19 and CCL21 in RA fibroblasts and RA peripheral blood in vitro differentiated macrophages and we document a novel role of CCL19/21 in RA angiogenesis.
CCL19; CCL21; RA synovial tissue fibroblast; macrophages and proangiogenic factors
OBJECTIVE—The aim of this study was to investigate in situ the expression of the classic vitronectin (VN) receptor consisting of the αv and β3 subunits in synovial lining cells (SLC) of chronic synovitis occurring in osteoarthritis (OA) and in rheumatoid arthritis (RA). The expression and function of αv and β3 as VN receptor in cultured fibroblast-like synoviocytes (FBS) derived from patients with OA and RA was also compared.
METHODS—Expression of αv and β3 was examined immunohistochemically in normal synovial tissue and in synovial tisssue from patients with OA and RA. The effect of proinflammatory cytokines and of a synovial fluid of a patient with RA on the expression of the αv and β3 subunits of cultured FBS was determined by flow cytometry. Binding of OA and RA-FBS to VN was quantified using adhesion assays and the effect of interleukin 1β (IL1β) and tumour necrosis factor α (TNFα) on adhesion was measured. The specifity of the adhesion was tested by inhibition studies using monoclonal antibodies to integrin subunits.
RESULTS—In in situ studies normal SLC showed a parallel distribution of αv and β3 subunits. OA-SLC strongly and uniformly expressed αv whereas RA-SLC showed heterogeneous expression of αv. In situ both OA-SLC and RA-SLC lacked the expression of the integrin subunit β3. In in vitro studies, OA-FBS and RA-FBS did not differ as regards expression of αv and β3, and VN attachment. Binding of RA-FBS to VN was partially blocked by antibodies against αv, β1, and β3 subunits, whereas only antibodies against αv and β3 inhibited the binding of OA-FBS to VN. The proinflammatory cytokines TNFα and IL1β increased the expression of αv and β3, and the VN binding of OA-FBS, whereas αv and β3 expression, and VN binding were downregulated in RA-FBS. Similar effects were found when the synovial fluid of an RA patient was used.
CONCLUSION—The integrin subunit β3 seems to be one partner but not the major one with which the subunit αv forms functional vitronectin receptors in OA-FBS and RA-FBS. The interaction between synovial cells and inflammatory cytokines seems to be different for OA and RA; the basis for this difference, however, remains to be established.
Regulatory T cells (Tregs) exert their anti‐inflammatory activity predominantly by cell contact‐dependent mechanisms. A study was undertaken to investigate the regulatory capacity of autologous peripheral blood Tregs in contact with synovial tissue cell cultures, and to evaluate their presence in peripheral blood, synovial tissue and synovial fluid of patients with rheumatoid arthritis (RA).
44 patients with RA and 5 with osteoarthritis were included in the study. The frequency of interferon (IFN)γ‐secreting cells was quantified in synovial tissue cell cultures, CD3‐depleted synovial tissue cell cultures, synovial tissue cultures co‐cultured with autologous CD4+ and with CD4+CD25+ peripheral blood T cells by ELISPOT. Total CD3+, Th1 polarised and Tregs were quantified by real‐time PCR for CD3ε, T‐bet and FoxP3 mRNA, and by immunohistochemistry for FoxP3 protein.
RA synovial tissue cell cultures exhibited spontaneous expression of IFNγ which was abrogated by depletion of CD3+ T cells and specifically reduced by co‐culture with autologous peripheral blood Treg. The presence of Treg in RA synovitis was indicated by FoxP3 mRNA expression and confirmed by immunohistochemistry. The amount of FoxP3 transcripts, however, was lower in the synovial membrane than in peripheral blood or synovial fluid. The T‐bet/FoxP3 ratio correlated with both a higher grade of synovial tissue lymphocyte infiltration and higher disease activity.
This study has shown, for the first time in human RA, the efficacy of autologous Tregs in reducing the inflammatory activity of synovial tissue cell cultures ex vivo, while in the synovium FoxP3+ Tregs of patients with RA are reduced compared with peripheral blood and synovial fluid. This local imbalance of Th1 and Treg may be responsible for repeated rheumatic flares and thus will be of interest as a target for future treatments.
In this study, we analysed the number of IL-17+ cells in facet joints, in the peripheral blood (PB) and synovial fluid (SF) of spondyloarthritis (SpA) patients and compared these results with those of patients with other rheumatic diseases and controls.
Immunohistochemical analysis of IL-17+ cells was performed in facet joints of 33 ankylosing spondylitis (AS) patients and compared with data from 20 osteoarthritis (OA) patients. The frequency of IL-17+CD4+ T cells in PB and SF of SpA patients (PB n = 30, SF n = 11), rheumatoid arthritis (RA) patients (PB n = 14, SF n = 7), OA patients (PB n = 10) and healthy controls (PB n = 12) was analysed after stimulation with Staphylococcus aureus Enterotoxin B and phorbol 12-myristate 13-acetate/ionomycin and quantified by flow cytometry.
In AS facet joints, the frequency of IL-17-secreting cells was significantly higher than in samples obtained from OA patients (P < 0.001), with a slight predominance of IL-17+ cells among the mononuclear cells (61.5% ± 14.9%) compared to cells with polysegmental nuclei. Immunofluorescence microscopy revealed that the majority of IL-17+ cells were myeloperoxidase-positive (35.84 ± 13.06/high-power field (HPF) and CD15+ neutrophils (24.25 ± 10.36/HPF), while CD3+ T cells (0.51 ± 0.49/HPF) and AA-1+ mast cells (2.28 ± 1.96/HPF) were less often IL-17-positive. The frequency of IL-17+CD4+ T cells in the PB and SF of SpA patients did not differ significantly compared to RA patients, OA patients or healthy controls.
Our data suggest an important role for IL-17 in the inflammatory processes in AS. However, the innate immune pathway might be of greater relevance than the Th17-mediated adaptive immune response.
The metastasis‐associated protein S100A4 promotes the progression of cancer by regulating the remodelling of the extracellular matrix. The expression of S100A4 in vivo is shown and the functional role of S100A4 in the pathogenesis of osteoarthritis and rheumatoid arthritisis is explored. The expression of S100A4 in rheumatoid arthritis, osteoarthritis and normal synovial tissues was determined by immunohistochemistry. The expression of matrix metalloproteinase (MMP) mRNA was measured in rheumatoid arthritis and osteoarthritis synovial fibroblasts treated and untreated with S100A4 oligomer by real‐time polymerase chain reaction. Levels of released MMPs were confirmed by ELISA in cell culture supernatants. S100A4 protein was expressed in rheumatoid arthritis and osteoarthritis synovial tissues, in contrast with normal synovium. S100A4 up regulated MMP‐3 mRNA in rheumatoid arthritis synovial fluid, with a peak after 6 h. This resulted in release of MMP‐3 protein. MMP‐1, MMP‐9 and MMP‐13 mRNA were also up regulated in synovial fluid, but with different kinetics. MMP‐14 mRNA showed no change. Thus, S100A4 protein is expressed in synovial tissues of patients with rheumatoid arthritis and osteoarthritis in contrast with healthy people. It induces the expression and release of MMP‐3 and other MMPs from synovial fluid. The data suggest that S100A4‐producing cells could be involved in the pathogenesis of osteoarthritis and rheumatoid arthritis, including pannus formation and joint destruction.
Interleukin-17 (IL-17)-producing T helper (Th) 17 cells are considered as a new subset of cells critical to the development of rheumatoid arthritis (RA). We aimed to investigate the distribution of Th1 and Th17 cells and their association with disease activity, and determine the Th17-related cytokine levels in the peripheral blood of RA patients.
Peripheral blood mononuclear cells from 55 RA and 20 osteoarthritis (OA) patients were stimulated with mitogen, and the distributions of CD4+Interferon (INF)+IL-17- (Th1 cells) and CD4+INF-IL-17+ (Th17 cells) were examined by flow cytometry. Serum levels of IL-6, IL-17, IL-21, IL-23, and tumor necrosis factor (TNF)-α were measured by ELISA. Erythrocyte sedimentation rate (ESR) and C-reactive protein (CRP) were recorded. The 28-joint disease activity score (DAS28) was also assessed.
The median percentage of Th17 cells was higher in RA patients than in OA patients (P=0.04), and in active than in inactive RA (P=0.03), whereas that of Th1 cells was similar in both groups. Similarly, the levels of IL-17, IL-21, and IL-23 were detected in a significantly higher proportion of RA patients than OA patients and the frequencies of detectable IL-6, IL-17, and IL-21 were higher in active RA than in inactive RA group. The percentage of Th17 cells positively correlated with the DAS28, ESR, and CRP levels.
These observations suggest that Th17 cells and Th17-related cytokines play an important role in RA pathogenesis and that the level of Th17 cells in peripheral blood is associated with disease activity in RA.
Rheumatoid arthritis; T helper 1 cell; T helper 17 cell; Cytokines
OBJECTIVES—To evaluate the effect of anti-TNFα on the Th1 and Th2 cytokines in patients with spondyloarthropathy (SpA).
METHODS—Peripheral blood mononuclear cells (PBMC) were obtained from 20 patients with active SpA treated with infliximab (5 mg/kg). For comparison, PBMC were also obtained from 15 healthy controls and 19 patients with active rheumatoid arthritis (RA). After stimulation with PMA/ionomycin, the intracellular cytokines interleukin (IL)2, IL4, IL10, and interferon (IFN)γ were determined in CD3+ T cells and in CD3+/CD56+ natural killer (NK) T cells by flow cytometry.
RESULTS—At baseline the percentage of T cells positive for IFNγ (p=0.020) and IL2 (p=0.046) was decreased in patients with SpA compared with healthy controls, while IL10 (p=0.001) was increased. This cytokine profile, confirmed by the mean fluorescence intensities (MFI), was more pronounced in CD3+/CD8- cells and contrasted with higher IL2 production in RA. NK T cells, characterised by high IL4 and IL10 numbers, were also increased in patients with SpA (p=0.017). Treatment with infliximab induced a significant and persistent increase in IFNγ and IL2 in patients with SpA. Moreover, there was a transient decrease in IL10 and NK T cells in patients with high baseline values, resulting in values comparable with those of healthy controls. This switch in cytokine profile was seen in both the CD3+/CD8- and CD3+/CD8+ subsets.
CONCLUSIONS—Before treatment patients with SpA had an impaired Th1 cytokine profile compared with healthy controls and patients with RA. TNFα blockade induced restoration of the Th1 cytokines, resulting in a normal cytokine balance. These data confirm the effect of anti-TNFα on the immune changes in SpA, and provide insights into the mechanisms involved in TNFα blockade.
The oxidative metabolism of neutrophils isolated from the bloodstream and synovial fluid of 16 patients with rheumatoid arthritis was compared by measuring the ability of neutrophils to generate luminol dependent chemiluminescence and to secrete O2-. Measurements of receptor mediated--that is, N-formyl-methionyl-leucyl-phenylalanine stimulated--activation or receptor and second message independent--that is phorbol myristate acetate stimulated--activation showed that synovial fluid neutrophils had biochemical characteristics to suggest that they had been either up-regulated (primed) or down-regulated (activated) in vivo. These conclusions were confirmed by comparison of these responses with the changes in oxidative metabolism observed during in vitro priming and activation of control neutrophils: synovial fluid neutrophils possessed lower levels of myeloperoxidase than paired bloodstream cells, and unlike bloodstream cells could not be primed in vitro. These data thus suggest that synovial fluid neutrophils have been exposed to both priming and activating agents within rheumatoid joints.
The immunoregulatory function of interleukin (IL)-29 has recently been recognized. However, little is known about the involvement of IL-29 in the pathogenesis of rheumatoid arthritis (RA). This study aimed to examine the expression profiles of IL-29 in blood, synovial fluid (SF) and synovium in RA patients and investigate the effect of IL-29 on cytokines production in RA synovial fibroblasts.
The transcript levels of IL-29 and its specific receptor IL-28Rα in peripheral blood mononuclear cells (PBMC) and synovium were determined by real-time reverse transcription-polymerase chain reaction (real-time PCR). The concentrations of IL-29 in serum and synovial fluid (SF) were quantified by enzyme-linked immunoassay (ELISA), and the correlation of serum IL-29 levels with disease activity in RA patients was investigated. Furthermore, the expression of IL-29 in RA synovium was examined by immunohistochemistry and double immunofluorescence analysis. Finally, the expression of IL-6, IL-8, IL-10, IL-17 and matrix metalloproteinase-3 (MMP-3) in synovial fibroblasts upon IL-29 stimulation was determined by real-time PCR.
IL-29 and IL-28Rα mRNA expression in PBMC was significantly increased in patients with RA compared with healthy controls (HC). The serum levels of circulating IL-29 were higher in RA than those in HC. Increased IL-29 levels were detected in RA SF when compared with osteoarthritis (OA) SF. However, serum IL-29 levels showed no significant correlation with RA disease activity. IL-29 was mostly expressed in the lining region of RA synovium. Moreover, IL-29 was expressed predominately in synovial macrophages and fibroblasts. RA synovial fibroblasts exposed to IL-29 specifically upregulated IL-6, -8 and MMP-3 but downregulated IL-10.
The findings in the present study indicate, for the first time, that IL-29 is dysregulated in patients with RA, which may contribute to the RA pathogenesis via inducing the production of proinflammatory cytokines, chemokines or matrix metalloproteinases in synovial fibroblasts.
OBJECTIVE: To compare expression of the p75 chain of the interleukin-2 receptor (IL-2Rp75, CD122) on peripheral and synovial mononuclear cells in rheumatoid and non-rheumatoid inflammatory arthritis. METHODS: Peripheral blood (PBMC) and synovial (SFMC) mononuclear cells were isolated from subjects with rheumatoid arthritis (n = 16) and non-rheumatoid inflammatory arthritis (n = 12). PBMC were isolated from six healthy controls. Expression of CD122 was examined using indirect immunofluorescence and quantitative flow cytometry. RESULTS: There was no difference in IL-2Rp75 expression on PBMC from rheumatoid arthritis patients, non-rheumatoid arthritis patients, and controls. In subjects with rheumatoid arthritis there was no difference in IL-2Rp75 expression on PBMC and SFMC. However, in the non-rheumatoid arthritis group there was an increase in IL-2Rp75 expression on SFMC compared with PBMC (P = 0.0032). On SFMC there was a greater expression of IL-2Rp75 in non-rheumatoid arthritis than in rheumatoid arthritis (P = 0.0007). Expression was greater on CD8 positive cells and in subjects with shorter duration of disease. CONCLUSIONS: The p75 chain of the IL-2 receptor, an important T cell activation antigen, is not upregulated in synovial fluid. This appears to be a disease specific defect and provides further support for the concept of "frustrated" or incomplete T cell activation in this disease.
CD4+ memory T cells (Tm) from rheumatoid arthritis peripheral blood (RAPB) or peripheral blood from normal donors produced IL-2, whereas fewer cells secreted IFN-γ or IL-4 after a brief stimulation. RAPB Tm contained significantly more IFN-γ producers than normal cells. Many rheumatoid arthritis (RA) synovial Tm produced IFN-γ alone (40%) and fewer cells produced IL-2 or IL-4. An in vitro model was employed to generate polarized T-helper (Th) effectors. Normal and RAPB Tm differentiated into both IFN-γ- and IL-4-producing effectors. RA synovial fluid (RASF) Tm demonstrated defective responsiveness, exhibiting diminished differentiation of IL-4 effectors, whereas RA synovial tissue (RAST) Tm exhibited defective generation of IFN-γ and IL-4 producers.
CD4+ T-helper cells; cytokines; rheumatoid arthritis
Steroid hormone receptors such as glucocorticoid receptors, androgen receptors, and oestrogen receptors α (ERα) and β (ERβ) have been identified in synovial cells of patients with rheumatoid arthritis and osteoarthritis.
To find a quantitative relationship between the number of receptor positive cells and markers of inflammation, and to compare the two groups of patients with rheumatoid arthritis and osteoarthritis.
A total of 36 patients with rheumatoid arthritis (n = 17) and osteoarthritis (n = 19) were included, and receptor positive cells and cellular markers of synovial inflammation were quantified by immunohistochemistry and ELISA (interleukin 6 (IL6) and IL8).
Patients with rheumatoid arthritis showed a higher degree of histologically determined inflammation compared with those with osteoarthritis. However, synovial density of gluco‐corticoid receptor positive (GR+), androgen receptor positive (AR+), ERα+ and ERβ+ cells were not different among patients with rheumatoid arthritis and osteoarthritis. In patients with osteoarthritis, the density of GR+ cells positively correlated with the density of AR+, ERα+ and ERβ+ cells (p = 0.007), which was not observed in patients with rheumatoid arthritis. This indicates positively coupled steroid hormone receptor expression in patients with osteoarthritis but not in those with rheumatoid arthritis. In patients with rheumatoid arthritis, secretion of synovial IL6 and IL8 positively correlated with the density of ERα+ and ERβ+ cells (not with gluco‐corticoid receptor and androgen receptor), which was not found in the synovium of patients with osteoarthritis. This indicates that inflammatory factors might up regulate the expression of oestrogen receptors in patients with rheumatoid arthritis, or vice versa.
In patients with osteoarthritis, expression of different steroid receptors is positively coupled, which was not observed in the synovium of patients with rheumatoid arthritis. This uncoupling phenomenon in rheumatoid arthritis might lead to an imbalance of the normal synovial homeostasis.
The purpose of this study was to investigate the profile of histone deacetylase (HDAC) expression in the synovial tissue of rheumatoid arthritis (RA) compared with that of normal control and osteoarthritis (OA), and to examine whether there is a link between HDAC activity and synovial inflammation.
HDAC activity and histone acetyltransferase (HAT) activity were determined in nuclear extracts of total synovial tissue surgically obtained from normal, OA and RA joints. The level of cytoplasmic tumor necrosis factor a (TNFα) fraction was measured by ELISA. Total RNA of synovial tissue was used for RT-PCR of HDAC1-8. In synovial fibroblasts from RA (RASFs), the effects of TNFα on nuclear HDAC activity and class I HDACs (1, 2, 3, 8) mRNA expressions were examined by quantitative real-time PCR. The protein expression and distribution of class I HDACs were examined by Western blotting.
Nuclear HDAC activity was significantly higher in RA than in OA and normal controls and correlated with the amount of cytoplasmic TNFα. The mRNA expression of HDAC1 in RA synovial tissue was higher than in OA and normal controls, and showed positive correlation with TNFα mRNA expression. The protein level of nuclear HDAC1 was higher in RA synovial tissue compared with OA synovial tissue. Stimulation with TNFα significantly increased the nuclear HDAC activity and HDAC1 mRNA expression at 24 hours and HDAC1 protein expression at 48 hours in RASFs.
Our results showed nuclear HDAC activity and expression of HDAC1 were significantly higher in RA than in OA synovial tissues, and they were upregulated by TNFα stimulation in RASFs. These data might provide important clues for the development of specific small molecule HDAC inhibitors.
Macrophage migration inhibitory factor (MIF) is one of key regulators in acute and chronic immune-inflammatory conditions including rheumatoid arthritis (RA). We examined the effect of MIF on osteoclastogenesis, which is known to play a crucial role in bone destruction in RA.
The concentration of MIF and receptor activator of nuclear factor-κB ligand (RANKL) in the synovial fluid was measured by ELISA. MIF-induced RANKL expression of RA synovial fibroblasts was determined by real-time PCR and western blot. Osteoclastogenesis was analyzed in culture of human peripheral blood mononuclear cells (PBMC) with MIF. Osteoclastogenesis was also determined after co-cultures of rhMIF-stimulated RA synovial fibroblasts with human PBMC.
Synovial fluid MIF concentration in RA patients was significantly higher than in osteoarthritis (OA) patients. The concentration of RANKL correlated with that of MIF in RA synovial fluids (r = 0.6, P < 0.001). MIF stimulated the expression of RANKL mRNA and protein in RA synovial fibroblasts, which was partially reduced by blocking of interleukin (IL)-1β. Osteoclasts were differentiated from PBMC cultures with MIF and M-CSF, even without RANKL. Osteoclastogenesis was increased after co-culture of MIF-stimulated RA synovial fibroblasts with PBMC and this effect was diminished by RANKL neutralization. Blocking of PI3 kinase, p38 MAP kinase, JAK-2, NF-κB, and AP-1 also led to a marked reduction in RANKL expression and osteoclastogenesis.
The interactions among MIF, synovial fibroblasts, osteoclasts, RANKL, and IL-1β have a close connection in osteoclastogenesis and they could be a potential gateway leading to new therapeutic approaches in treating bone destruction in RA.
The phenotypic characterization of enzymatically dissociated mononuclear cells in synovial membrane samples from multiple sites in two patients with rheumatoid arthritis (RA) were examined by fluorescence activated flow cytometry. In synovial membrane samples from each patient there was a consistent increase in the proportion of CD8+ cells (suppressor/cytotoxic), CD14+ cells (monocytes/macrophages), and HLA-DR+ cells compared with paired peripheral blood mononuclear cells. The proportion of CD4+ cells (helper/inducer) in synovial membrane was variable. Studies of in vitro production of IgM and IgM rheumatoid factor in one patient showed strikingly similar values for synovial membrane rheumatoid factor production at the two sites, which was enhanced compared with production in peripheral blood. These results suggest that in individual patients with RA the intra-articular immune response is comparable at multiple anatomical sites and that it is distinct from that in peripheral blood.
OBJECTIVES--To investigate the role of T helper-1 cell (Th1) activation in the induction of proinflammatory cytokine production and cartilage damage by rheumatoid arthritis (RA) synovial fluid mononuclear cells (SFMNC) and the subsequent possible beneficial role of the T helper-2 cell (Th2) cytokine interleukin-4 (IL-4) in the inhibition of this process. METHODS--SFMNC were stimulated with bacterial antigen (hsp60) to activate Th1 cells. Th1 and Th2 specific cytokine profiles (interferon gamma (IFN gamma) and IL-4) and proinflammatory cytokines interleukin-1 (IL-1) and tumour necrosis factor alpha (TNF alpha) in the conditioned media were analysed. In addition, the conditioned media were tested for their ability to induce cartilage damage. The same parameters were measured in the presence of IL-4. RESULTS--Stimulation of SFMNC with bacterial antigen resulted in an increase in IFN gamma, IL-1, and TNF alpha production which was accompanied by the induction of cartilage damage. Th1 activation could be inhibited by IL-4 as shown by a reduction of IFN gamma. This was accompanied by a decrease in IL-1 and TNF alpha production and inhibition of cartilage damage. CONCLUSIONS--Th1 activation is a possible mechanism by which inflammation in RA joints is enhanced. The Th2 cytokine IL-4 inhibits this Th1 activity and may diminish inflammation and induction of cartilage damage in RA joints.
We and others have shown that cells obtained from inflamed joints of rheumatoid arthritis (RA) patients produce interleukin-8, a potent chemotactic cytokine for neutrophils (PMNs). However, IL-8 accounted for only 40% of the chemotactic activity for PMNs found in these synovial fluids. Currently, we have examined the production of the novel PMN chemotactic cytokine, epithelial neutrophil activating peptide-78 (ENA-78), using peripheral blood, synovial fluid, and synovial tissue from 70 arthritic patients. RA ENA-78 levels were greater in RA synovial fluid (239 +/- 63 ng/ml) compared with synovial fluid from other forms of arthritis (130 +/- 118 ng/ml) or osteoarthritis (2.6 +/- 1.8 ng/ml) (P < 0.05). RA peripheral blood ENA-78 levels (70 +/- 26 ng/ml) were greater than normal peripheral blood levels (0.12 +/- 0.04 ng/ml) (P < 0.05). Anti-ENA-78 antibodies neutralized 42 +/- 9% (mean +/- SE) of the chemotactic activity for PMNs found in RA synovial fluids. Isolated RA synovial tissue fibroblasts in vitro constitutively produced significant levels of ENA-78, and this production was further augmented when stimulated with tumor necrosis factor-alpha (TNF-alpha). In addition RA and osteoarthritis synovial tissue fibroblasts as well as RA synovial tissue macrophages were found to constitutively produce ENA-78. RA synovial fluid mononuclear cells spontaneously produced ENA-78, which was augmented in the presence of lipopolysaccharide. Immunohistochemical localization of ENA-78 from the synovial tissue of patients with arthritis or normal subjects showed that the predominant cellular source of this chemokine was synovial lining cells, followed by macrophages, endothelial cells, and fibroblasts. Synovial tissue macrophages and fibroblasts were more ENA-78 immunopositive in RA than in normal synovial tissue (P < 0.05). These results, which are the first demonstration of ENA-78 in a human disease state, suggest that ENA-78 may play an important role in the recruitment of PMNs in the milieu of the inflamed joint of RA patients.
Background: Interferon (IFN) ß displays anti-inflammatory and immunosuppressive activity and has been considered for the treatment of rheumatoid arthritis (RA). Information about the effects of this molecule on joint cells is scarce, however.
Objective: To investigate the effects of IFNß on the production of interleukin-1 receptor antagonist (IL1Ra) in human articular chondrocytes and synovial fibroblasts.
Methods: Chondrocytes and synovial fibroblasts were stimulated with IFNß alone or in combination with interleukin (IL) 1ß. IL1Ra concentrations in culture supernatants and cell lysates were determined by ELISA. Expression of mRNA encoding the secreted sIL1Ra or the intracellular icIL1Ra1 isoforms was quantified by real time reverse transcriptase-polymerase chain reaction.
Results: In chondrocytes, IFNß alone had no effect, but dose dependently enhanced the secretion of IL1Ra induced by IL1ß. Chondrocyte cell lysates contained undetectable or low levels of IL1Ra, even after stimulation with IL1ß and IFNß. Consistently, IL1ß and IFNß induced sIL1Ra mRNA expression in chondrocytes, while expression of icIL1Ra1 was not detectable. Human articular chondrocytes thus mainly produce secreted IL1Ra. In synovial fibroblasts, IFNß alone dose dependently increased IL1Ra secretion. In addition, IFNß enhanced the stimulatory effect of IL1ß on IL1Ra production. In synovial cell lysates, IFNß and IL1ß also increased IL1Ra levels. Consistently, IFNß and IL1ß induced the expression of both sIL1Ra and icIL1Ra1 mRNA in synovial fibroblasts.
Conclusion: IFNß increases IL1Ra production in joint cells, which may be beneficial in cartilage damaging diseases such as RA or osteoarthritis.
Several microRNA, which are ~22-nucleotide noncoding RNAs, exhibit tissue-specific or developmental stage–specific expression patterns and are associated with human diseases. The objective of this study was to identify the expression pattern of microRNA-146 (miR-146) in synovial tissue from patients with rheumatoid arthritis (RA).
The expression of miR-146 in synovial tissue from 5 patients with RA, 5 patients with osteoarthritis (OA), and 1 normal subject was analyzed by quantitative reverse transcription–polymerase chain reaction (RT-PCR) and by in situ hybridization and immunohistochemistry of tissue sections. Induction of miR-146 following stimulation with tumor necrosis factor α (TNFα) and interleukin-1β (IL-1β) of cultures of human rheumatoid arthritis synovial fibroblasts (RASFs) was examined by quantitative PCR and RT-PCR.
Mature miR-146a and primary miR-146a/b were highly expressed in RA synovial tissue, which also expressed TNFα, but the 2 microRNA were less highly expressed in OA and normal synovial tissue. In situ hybridization showed primary miR-146a expression in cells of the superficial and sublining layers in synovial tissue from RA patients. Cells positive for miR-146a were primarily CD68+ macrophages, but included several CD3+ T cell subsets and CD79a+ B cells. Expression of miR-146a/b was markedly up-regulated in RASFs after stimulation with TNFα and IL-1β.
This study shows that miR-146 is expressed in RA synovial tissue and that its expression is induced by stimulation with TNFα and IL-1β. Further studies are required to elucidate the function of miR-146 in these tissues.
OBJECTIVES--The aim of this study was to determine if neutrophils isolated from the blood and synovial fluid of patients with rheumatoid arthritis had patterns of receptor expression resembling those of blood neutrophils from controls which had been activated and primed in vitro. METHODS--Fluorescence activated cell sorting was used to measure receptor expression in paired blood and synovial fluid neutrophils from patients and in control neutrophils exposed to phorbol myristate acetate and granulocyte-macrophage colony stimulating factor. RESULTS--There was no significant difference in the patterns of receptor expression in blood neutrophils from patients and healthy controls, but neutrophils in the synovial fluid had been primed and activated within the joint. About 50% of rheumatoid synovial fluid neutrophil samples expressed Fc gamma RI, a high affinity receptor for monomeric IgG, which is only expressed in neutrophils exposed to cytokines. CONCLUSIONS--Synovial fluid neutrophils are activated and primed within the inflamed joint and hence their ability to respond to activating factors such as immune complexes will be modulated. As the expression of Fc gamma RI requires active biosynthesis, this work indicates that selective gene activation occurs when neutrophils are recruited into rheumatoid joints.
Background: An imbalance of T cell subsets in asthma with a predominance of Th2 type cells has been proposed. The aim of this study was simultaneously to detect surface markers and intracellular production of cytokines in T cells from the airways of children with and without asthma.
Methods: Bronchoalveolar lavage (BAL) fluid was obtained by wedging a suction catheter into the distal airway immediately before elective surgery. Cells were stimulated with phorbol 12-myristrate 13-acetate (PMA) and ionomycin and intracytoplasmic cytokine retention was achieved using monensin. The cells were stained with the relevant antibodies and analysed by flow cytometry.
Results: No statistical difference was observed between children with atopic asthma, atopic non-asthmatic subjects, and normal controls in the percentage of CD3+ cells producing interleukin (IL)-2 or IL-4. Interferon (IFN)γ+ T cells were, however, present in a much higher percentage than either IL-2 or IL-4 positive cells. The percentage of IFNγ+ T cells was significantly increased in subjects with atopic asthma (median 71.3%, interquartile range (IQR) 65.1–82.2, n=13) compared with both atopic non-asthmatic subjects (51.9%, IQR 37.2–70.3, n=12), p<0.05 and normal controls (58.1%, IQR 36.1–66.1, n=23), p<0.01.
Conclusions: These findings indicate that IFNγ producing T cells are more abundant in the airways of children with atopic asthma than in atopic non-asthmatic subjects and controls. The proinflammatory activities of IFNγ may play an important role in the pathogenesis of childhood asthma and may suggest that asthma is not simply a Th2 driven response.