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1.  Three-Color Flow Cytometry Detection of Intracellular Cytokines in Peripheral Blood Mononuclear Cells: Comparative Analysis of Phorbol Myristate Acetate-Ionomycin and Phytohemagglutinin Stimulation 
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.
PMCID: PMC96054  PMID: 11238213
2.  Linking Power Doppler Ultrasound to the Presence of Th17 Cells in the Rheumatoid Arthritis Joint 
PLoS ONE  2010;5(9):e12516.
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.
Methodology/Principal Findings
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.
PMCID: PMC2931706  PMID: 20824142
3.  Characterization of CCL19 and CCL21 in Rheumatoid Arthritis 
Arthritis and rheumatism  2011;63(4):914-922.
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.
PMCID: PMC3079365  PMID: 21225692
CCL19; CCL21; RA synovial tissue fibroblast; macrophages and proangiogenic factors
4.  S100A4 is expressed at site of invasion in rheumatoid arthritis synovium and modulates production of matrix metalloproteinases 
Annals of the Rheumatic Diseases  2006;65(12):1645-1648.
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.
PMCID: PMC1798462  PMID: 17105852
5.  Differential expression and functional behaviour of the αv and β3 integrin subunits in cytokine stimulated fibroblast-like cells derived from synovial tissue of rheumatoid arthritis and osteoarthritis in vitro 
Annals of the Rheumatic Diseases  1997;56(12):729-736.
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.

PMCID: PMC1752301  PMID: 9496152
6.  Low T cell production of TNFα and IFNγ in ankylosing spondylitis: its relation to HLA-B27 and influence of the TNF-308 gene polymorphism 
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.

PMCID: PMC1753353  PMID: 11114280
7.  Imbalance in distribution of functional autologous regulatory T cells in rheumatoid arthritis 
Annals of the Rheumatic Diseases  2007;66(9):1151-1156.
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.
PMCID: PMC1955165  PMID: 17392348
8.  Analysis of IL-17+ cells in facet joints of patients with spondyloarthritis suggests that the innate immune pathway might be of greater relevance than the Th17-mediated adaptive immune response 
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.
PMCID: PMC3218910  PMID: 21689402
9.  Dysregulated balance of Th17 and Th1 cells in systemic lupus erythematosus 
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.
PMCID: PMC2888202  PMID: 20334681
10.  Macrophage migration inhibitory factor enhances osteoclastogenesis through upregulation of RANKL expression from fibroblast-like synoviocytes in patients with rheumatoid arthritis 
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.
PMCID: PMC3132025  PMID: 21401926
11.  Increased activity and expression of histone deacetylase 1 in relation to tumor necrosis factor-alpha in synovial tissue of rheumatoid arthritis 
Arthritis Research & Therapy  2010;12(4):R133.
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.
PMCID: PMC2945023  PMID: 20609223
12.  Characterization of IL-7 and IL-7R in the pathogenesis of Rheumatoid Arthritis 
Arthritis and rheumatism  2011;63(10):2884-2893.
The aim of the study was to characterize the expression of IL-7 and IL-7R in rheumatoid arthritis (RA) synovial tissue and to examine their regulation and pathogenic role in macrophages, endothelial cells and RA synovial tissue fibroblasts.
Expression of IL-7 and IL-7R was demonstrated in RA and normal synovial tissues employing immunohistochemistry. Expression and regulation of IL-7 and IL-7R was determined in RA peripheral blood in vitro differentiated macrophages, RA synovial tissue fibroblasts and human microvascular endothelial cells (HMVECs) by real-time RT-PCR and/or flow cytometry. Next, IL-7 activated macrophages, RA fibroblasts and endothelial cells were examined for production of proangiogenic factors employing ELISA.
IL-7 and IL-7R were coexpressed on RA synovial tissue lining and sublining macrophages and endothelial cells. Consistently, expression of IL-7 and its receptor were significantly elevated in RA synovial fluid and peripheral blood macrophages as well as RA fibroblasts compared to normal cells. TLR4 ligation and stimulation with TNF-α modulated expression of IL-7 and IL-7R on RA macrophages and HMVECs. However, in RA fibroblasts only expression of IL-7R was increased by LPS and TNF-α activation. IL-7 also mediated RA pathogenesis by inducing production of potent proangiogenic factors from macrophages and endothelial cells.
We identify, for the first time, regulators of IL-7 and IL-7R expression in RA fibroblasts, RA peripheral blood in vitro differentiated macrophages and endothelial cells and we document a novel role of IL-7 in RA angiogenesis.
PMCID: PMC3614067  PMID: 21647866
IL-7; IL-7R; RA synovial tissue fibroblast; macrophages and proangiogenic factors
13.  Receptor expression in synovial fluid neutrophils from patients with rheumatoid arthritis. 
Annals of the Rheumatic Diseases  1993;52(5):354-359.
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.
PMCID: PMC1005049  PMID: 8100700
14.  Neutrophils isolated from the synovial fluid of patients with rheumatoid arthritis: priming and activation in vivo. 
Annals of the Rheumatic Diseases  1991;50(3):147-153.
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.
PMCID: PMC1004363  PMID: 1849716
15.  Impaired Th1 cytokine production in spondyloarthropathy is restored by anti-TNFα 
Annals of the Rheumatic Diseases  2001;60(8):750-755.
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.

PMCID: PMC1753790  PMID: 11454638
16.  Elevated Levels of T Helper 17 Cells Are Associated with Disease Activity in Patients with Rheumatoid Arthritis 
Annals of Laboratory Medicine  2012;33(1):52-59.
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.
PMCID: PMC3535197  PMID: 23301223
Rheumatoid arthritis; T helper 1 cell; T helper 17 cell; Cytokines
17.  Rheumatoid synovial CD4+ T cells exhibit a reduced capacity to differentiate into IL-4-producing T-helper-2 effector cells 
Arthritis Research  2000;3(1):54-64.
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.
PMCID: PMC17825  PMID: 11178127
CD4+ T-helper cells; cytokines; rheumatoid arthritis
18.  Reactive oxygen species induce expression of vascular endothelial growth factor in chondrocytes and human articular cartilage explants 
Vascular endothelial growth factor (VEGF) promotes cartilage-degrading pathways, and there is evidence for the involvement of reactive oxygen species (ROS) in cartilage degeneration. However, a relationship between ROS and VEGF has not been reported. Here, we investigate whether the expression of VEGF is modulated by ROS.
Aspirates of synovial fluid from patients with osteoarthritis (OA) were examined for intra-articular VEGF using ELISA. Immortalized C28/I2 chondrocytes and human knee cartilage explants were exposed to phorbol myristate acetate (PMA; 0–20 μg/ml), which is a ROS inducer, or 3-morpholino-sydnonimine hydrochloride (SIN-1; 0–20 μM), which is a ROS donor. The levels of VEGF protein and nitric oxide (NO) production were determined in the medium supernatant, using ELISA and Griess reagent, respectively. Gene expression of VEGF-121 and VEGF-165 was determined by splice variant RT-PCR. Expression of VEGF and VEGF receptors (VEGFR-1 and VEGFR-2) was quantified by real-time RT-PCR.
Synovial fluid from OA patients revealed markedly elevated levels of VEGF. Common RT-PCR revealed that the splice variants were present in both immortalized chondrocytes and cartilage discs. In immortalized chondrocytes, stimulation with PMA or SIN-1 caused increases in the levels of VEGF, VEGFR-1 and VEGFR-2 mRNA expression. Cartilage explants produced similar results, but VEGFR-1 was only detectable after stimulation with SIN-1. Stimulation with PMA or SIN-1 resulted in a dose-dependent upregulation of the VEGF protein (as determined using ELISA) and an increase in the level of NO in the medium.
Our findings indicate ROS-mediated induction of VEGF and VEGF receptors in chondrocytes and cartilage explants. These results demonstrate a relationship between ROS and VEGF as multiplex mediators in articular cartilage degeneration.
PMCID: PMC1794535  PMID: 17187682
19.  Interleukin-29 modulates proinflammatory cytokine production in synovial inflammation of rheumatoid arthritis 
Arthritis Research & Therapy  2012;14(5):R228.
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.
PMCID: PMC3580539  PMID: 23078630
20.  Angiopoietin-1 is expressed in the synovium of patients with rheumatoid arthritis and is induced by tumour necrosis factor α 
Annals of the Rheumatic Diseases  2003;62(2):100-107.
Objectives: To examine the potential role of the angiogenic growth factor angiopoietin-1 (Ang-1) in inflammatory arthritis.
Methods: Eighteen synovial tissue samples were obtained from 17 patients with a clinical diagnosis of rheumatoid arthritis (RA) and compared with six synovial tissue samples from six patients with osteoarthritis (OA). Ang-1 expression in synovial tissues was determined by immunohistochemistry and in situ hybridisation. Ang-1 mRNA and protein expression were also examined by northern blot analysis and enzyme linked immunosorbent assay (ELISA) in cultured synovial fibroblasts and human umbilical vein endothelial cells (HUVECs) before and after treatment with tumour necrosis factor (TNF)α.
Results: Ang-1 protein expression was detected by immunohistochemistry in 16/18 RA synovial tissue samples. Ang-1 protein was frequently observed in the synovial lining layer and in cells within the sublining synovial tissue, in both perivascular areas and in areas remote from vessels. In contrast, Ang-1 was only weakly detected in these sites in OA samples. Ang-1 mRNA and protein were also expressed in cultured synovial fibroblasts derived from patients with RA. In addition, induction of Ang-1 mRNA and protein was observed by northern blot analysis and ELISA after stimulation of RA synovial fibroblasts, but not HUVECs, with the proinflammatory cytokine TNFα.
Conclusions: Ang-1 mRNA and protein are expressed in the synovium of patients with RA, and are up regulated in synovial fibroblasts by TNFα. Ang-1 may therefore be an important regulator of angiogenesis in inflammatory arthritis.
PMCID: PMC1754433  PMID: 12525377
21.  Quantitative determination of steroid hormone receptor positive cells in the synovium of patients with rheumatoid arthritis and osteoarthritis: is there a link to inflammation? 
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.
PMCID: PMC1798416  PMID: 16815861
22.  FMS-related tyrosine kinase 3 ligand (Flt3L)/CD135 axis in rheumatoid arthritis 
Arthritis Research & Therapy  2013;15(6):R209.
The FMS-related tyrosine kinase 3 ligand (Flt3L)/CD135 axis plays a fundamental role in proliferation and differentiation of dendritic cells (DCs). As DCs play an important role in rheumatoid arthritis (RA) immunopathology we studied in detail the Flt3L/CD135 axis in RA patients.
The levels of Flt3L in (paired) serum and synovial fluid (SF) were quantified by enzyme-link immunosorbent assay (ELISA). Expression of Flt3L and CD135 in paired peripheral blood mononuclear cells (PBMCs) and synovial fluid mononuclear cells (SFMCs) was quantified by fluorescence-activated cell sorting (FACS). The expression of Flt3L, CD135 and TNF-Converting Enzyme (TACE) in synovial tissues (STs) and in vitro polarized macrophages and monocyte-derived DCs (Mo-DCs) was assessed by quantitative PCR (qPCR). CD135 ST expression was evaluated by immunohistochemistry and TACE ST expression was assessed by immunofluorescence. Flt3L serum levels were assessed in RA patients treated with oral prednisolone or adalimumab.
Flt3L levels in RA serum, SF and ST were significantly elevated compared to gout patients and healthy individuals (HI). RA SF monocytes, natural killer cells and DCs expressed high levels of Flt3L and CD135 compared to HI. RA ST CD68+ and CD163+ macrophages, CD55+ fibroblast-like synoviocytes (FLS), CD31+ endothelial cells or infiltrating monocytes and CD19+ B cells co-expressed TACE. IFN-γ-differentiated macrophages expressed higher levels of Flt3L compared to other polarized macrophages. Importantly, Flt3L serum levels were reduced by effective therapy.
The Flt3L/CD135 axis is active in RA patients and is responsive to both prednisolone and adalimumab treatment. Conceivably, this ligand receptor pair represents a novel therapeutic target.
PMCID: PMC3978611  PMID: 24314260
23.  Differential expression of the urokinase receptor (CD87) in arthritic and normal synovial tissues. 
Journal of Clinical Pathology  1997;50(4):314-319.
AIM: To determine whether the urokinase plasminogen activator receptor (u-PAR; CD87) exhibits a possible pathogenic role in rheumatoid and osteoarthritis. METHODS: A semiquantitative, indirect immunoperoxidase histochemical analysis was performed on frozen synovial tissue sections. The recently characterised monoclonal antibody 10G7 recognising transfectants bearing u-PAR was used. Synovial tissue was obtained from 10 patients with rheumatoid arthritis, 10 patients with osteoarthritis, and four normal subjects. RESULTS: u-PAR was expressed on 70-90% of synovial tissue lining cells and subsynovial, interstitial macrophages from the arthritis patients, but only on a few myeloid cells from the normal subjects. It was also present on more endothelial cells from the rheumatoid and osteoarthritis patients, than from normal synovial tissue. CONCLUSIONS: Plasminogen activators are important in joint destruction underlying arthritis. The up-regulated expression of u-PAR in diseased versus normal synovial tissue suggests a role for this antigen in the inflammatory and angiogenic mechanisms underlying rheumatoid and osteoarthritis.
PMCID: PMC499882  PMID: 9215148
24.  Characteristics of immunocompetent cells in synovial membranes from multiple sites in patients with rheumatoid arthritis. 
Annals of the Rheumatic Diseases  1992;51(9):1066-1068.
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.
PMCID: PMC1004839  PMID: 1384441
25.  Differential expression of the angiogenic Tie receptor family in arthritic and normal synovial tissue 
Arthritis Research  2002;4(3):201-208.
Angiopoietins (Ang) are vascular endothelial cell-specific growth factors that play important roles principally during the later stages of angiogenesis. We have compared the distribution of the receptor tyrosine kinase (Tie) and the Ang ligands in synovial tissues from normal subjects and those with rheumatoid arthritis (RA) and osteoarthritis (OA).
Immunohistochemical analysis was used to determine the expression of Ang-1, Ang-2, Tie1 and Tie2 in synovial tissue of normal subjects and those with RA and OA. Ang-1, Ang-2, Tie1 and Tie2 mRNA and protein expression were quantified in synovial tissues and RA synovial tissue fibroblasts with real-time reverse transcription polymerase chain reaction and western blot analysis.
In RA, Ang-1 positive immunostaining on lining cells, macrophages and endothelial cells was significantly higher than in OA and normal synovial tissue. The expression pattern of Ang-2 in synovial tissue was similar in RA and OA, whereas the Ang-2 expression was low in normal tissue. Synovial tissue from subjects with RA and OA showed a significant upregulation of Tie1 on lining cells, macrophages and endothelial cells compared to that from normal subjects. Tie2 was significantly upregulated in the RA and OA synovial tissue lining cells, macrophages and smooth muscle cells compared to normal synovial tissue. Generally Ang-1, Ang-2, Tie1 and Tie2 mRNA levels were higher in RA synovial tissue compared to normal and OA synovial tissues, and RA synovial tissue fibroblasts. Western blot analysis also demonstrated greater Tie1 and Tie2 protein expression in RA and OA synovial tissue compared to RA synovial tissue fibroblasts. In conclusion, the dominance of Ang-1 mRNA and protein expression over Ang-2 is in agreement with an active neovascularization in RA synovial tissue.
PMCID: PMC111023  PMID: 12010571
angiopoietin; mRNA; rheumatoid arthritis; Tie receptor expression

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