Rheumatoid Arthritis Synovial Mononuclear Cells Research Articles

Programmed death ligand 2 – A link between inflammation and bone loss in rheumatoid arthritis

ObjectiveActive rheumatoid arthritis (RA) is accompanied by increased appendicular and axial bone loss, closely associated to the degree of inflammation. The programmed death-1 (PD-1) pathway is important for maintaining peripheral tolerance, and its ligand PD-L2 has recently been associated with bone morphogenetic protein activity. Here, we report that PD-L2 plays a central role in RA osteoimmunology. MethodsFemoral bone mineral density (BMD) and trabecular bone microstructure were evaluated by micro-CT in wild type (WT) and PD-L2−/− mice. Osteoclasts were generated from RA synovial fluid mononuclear cells and peripheral blood monocytes. The effects of recombinant PD-L2, was evaluated by tartrate-resistant acid phosphatase (TRAP) activity and the development of bone erosions in the presence of anti-citrullinated protein antibodies (ACPA). Plasma soluble (s)PD-L2 levels were measured in patients with early (e)RA (n ​= ​103) treated with methotrexate alone or in combination with the TNF inhibitor Adalimumab. ResultsPD-L2−/− mice had a decreased BMD and deteriorated trabecular bone microstructure that was not related to the RANKL/OPG pathway. PD-L2 decreased TRAP activity in osteoclasts and decreased ACPA-induced erosions. In the RA synovial membrane PD-L2 was highly expressed especially in the lining layer and plasma sPD-L2 levels were increased in eRA patients and decreased with treatment. One-year sPD-L2 correlated inversely with erosive progression two years after treatment initiation with methotrexate and placebo. ConclusionPD-L2 regulates bone homeostasis in RA. Our findings provide new insight into the relationship between the immune system and bone homeostasis, and suggest a potential therapeutic target for limiting inflammatory bone loss in RA.

Extracellular Vesicles Transfer the Receptor Programmed Death-1 in Rheumatoid Arthritis.

Extracellular vesicles (EVs) have been recognized as route of communication in the microenvironment. They transfer proteins and microRNAs (miRNAs) between cells, and possess immunoregulatory properties. However, their role in immune-mediated diseases remains to be elucidated. We hypothesized a role for EVs in the rheumatoid arthritis (RA) joint, potentially involving the development of T cell exhaustion and transfer of the co-inhibitory receptor programmed death 1 (PD-1). Synovial fluid mononuclear cells (SFMCs) and peripheral blood mononuclear cells (PBMCs) from RA patients were investigated for PD-1 and other markers of T cell inhibition. EVs were isolated from RA plasma and synovial fluid. In addition, healthy control (HC) and RA PBMCs and SFMCs were cultured to produce EVs. These were isolated and investigated by immunogold electron microscopy (EM) and also co-cultured with lymphocytes and PD-1 negative cells to investigate their functions. Finally, the miRNA expression profiles were assessed in EVs isolated from RA and HC cell cultures. Cells from the RA joint expressed several T cell co-inhibitory receptors, including PD-1, TIM-3, and Tigit. ELISA demonstrated the presence of PD-1 in EVs from RA plasma and synovial fluid. Immunogold EM visualized PD-1 expression by EVs. Co-culturing lymphocytes and the PD-1 negative cell line, U937 with EVs resulted in an induction of PD-1 on these cells. Moreover, EVs from RA PBMCs increased proliferation in lymphocytes when co-cultured with these. All EVs contained miRNAs associated with PD-1 and other markers of T cell inhibition and the content was significantly lower in EVs from RA PBMCs than HC PBMCs. Stimulation of the cells increased the miRNA expression. However, EVs isolated from stimulated RA SFMCs did not change their miRNA expression profile to the same extend. EVs carrying both the PD-1 receptor and miRNAs associated with T cell inhibition were present in RA cell cultures. Upon stimulation, these miRNAs failed to be upregulated in EVs from RA SFMCs. This was in line with increased expression of T cell co-inhibitory markers on SFMCs. In conclusion, we suggest EVs to play a significant role in the RA microenvironment, potentially favoring the progression of T cell exhaustion.

Changes in Soluble CD18 in Murine Autoimmune Arthritis and Rheumatoid Arthritis Reflect Disease Establishment and Treatment Response.

IntroductionIn rheumatoid arthritis (RA) immune activation and presence of autoantibodies may precede clinical onset of disease, and joint destruction can progress despite remission. However, the underlying temporal changes of such immune system abnormalities in the inflammatory response during treat-to-target strategies remain poorly understood. We have previously reported low levels of the soluble form of CD18 (sCD18) in plasma from patients with chronic RA and spondyloarthritis. Here, we study the changes of sCD18 before and during treatment of early RA and following arthritis induction in murine models of rheumatoid arthritis.MethodsThe level of sCD18 was analyzed with a time-resolved immunoflourometric assay in 1) plasma from early treatment naïve RA patients during a treat-to-target strategy (the OPERA cohort), 2) plasma from chronic RA patients, 3) serum from SKG and CIA mice following arthritis induction, and 4) supernatants from synovial fluid mononuclear cells (SFMCs) and peripheral blood mononuclear cells (PBMCs) from 6 RA patients cultured with TNFα or adalimumab.ResultsPlasma levels of sCD18 were decreased in chronic RA patients compared with early RA patients and in early RA patients compared with healthy controls. After 12 months of treatment the levels in early RA patients were similar to healthy controls. This normalization of plasma sCD18 levels was more pronounced in patients with very early disease who achieved an early ACR response. Plasma sCD18 levels were associated with radiographic progression. Correspondingly, the serum level of sCD18 was decreased in SKG mice 6 weeks after arthritis induction compared with healthy littermates. The sCD18 levels in both SKG and CIA mice exhibited a biphasic course after arthritis induction with an initial increase above baseline followed by a decline. Shedding of CD18 from RA SFMC and RA PBMC cultures was increased by TNFα and decreased by adalimumab.ConclusionsThe plasma sCD18 levels were altered in patients with RA, in mice with autoimmune arthritis and in cell cultures treated with TNFα and adalimumab. Decreased levels of plasma sCD18 could reflect autoimmunity in transition from early to chronic disease and normalization in response to treatment could reflect autoimmunity in remission.

THU0550 Changes in Soluble CD18 Reflect Latency in Restoration of the Immune System after Early Treatment of Rheumatoid Arthritis

BackgroundIn rheumatoid arthritis (RA) immune activation and leukocyte migration to the joints precede clinical onset of disease. Early inhibition of inflammation is the main aim of treat-to-target strategy, but the...

THU0548 Interleukin-7 Accelerates Osteoclast Formation from Monocytes in Synovial Fluid from Patients from Rheumatoid Arthritis

BackgroundInterleukin-7 (IL-7), a member of the common gamma-chain cytokine family, plays a crucial role in T cell development, which induces T cell survival, proliferation, differentiation in mature naive and memory...

FRI0056 Antibody-based proximity ligation assay combined with flow cytometry is a powerful tool to detect active IL-7 receptors in arthritis

BackgroundIL-7 is a leading growth factor for memory T cells and is believed to play a central role in the sustained inflammation observed in many chronic inflammatory diseases, including rheumatoid...

Blockade of Toll-like receptor 2 prevents spontaneous cytokine release from rheumatoid arthritis ex vivo synovial explant cultures

IntroductionThe aim of this study was to examine the effect of blocking Toll-like receptor 2 (TLR2) in rheumatoid arthritis (RA) synovial cells.MethodsRA synovial tissue biopsies, obtained under direct visualization at arthroscopy, were established as synovial explant cultures ex vivo or snap frozen for immunohistology. Mononuclear cell cultures were isolated from peripheral blood and synovial fluid of RA patients. Cultures were incubated with the TLR1/2 ligand, Pam3CSK4 (200 ng, 1 and 10 μg/ml), an anti-TLR2 antibody (OPN301, 1 μg/ml) or an immunoglobulin G (IgG) (1 μg/ml) matched control. The comparative effect of OPN301 and adalimumab (anti-tumour necrosis factor alpha) on spontaneous release of proinflammatory cytokines from RA synovial explants was determined using quantitative cytokine MSD multiplex assays or ELISA. OPN301 penetration into RA synovial tissue explants cultures was assessed by immunohistology.ResultsPam3CSK4 significantly upregulated interleukin (IL)-6 and IL-8 in RA peripheral blood mononuclear cells (PBMCs), RA synovial fluid mononuclear cells (SFMCs) and RA synovial explant cultures (P < 0.05). OPN301 significantly decreased Pam3CSK4-induced cytokine production of tumour necrosis factor alpha (TNF-α), IL-1β, IL-6, interferon (IFN)-γ and IL-8 compared to IgG control in RA PBMCs and SFMCs cultures (all P < 0.05). OPN301 penetration of RA synovial tissue cultures was detected in the lining layer and perivascular regions. OPN301 significantly decreased spontaneous cytokine production of TNF-α, IL-1β, IFN-γ and IL-8 from RA synovial tissue explant cultures (all P < 0.05). Importantly, the inhibitory effect of OPN on spontaneous cytokine secretion was comparable to inhibition by anti-TNFα monoclonal antibody adalimumab.ConclusionsThese findings further support targeting TLR2 as a potential therapeutic agent for the treatment of RA.

Expansion of a unique macrophage subset in rheumatoid arthritis synovial lining layer

The Z39Ig protein (complement receptor for C3b and iC3b) is expressed on resident tissue macrophages in various tissues. This study was undertaken to examine the distribution of Z39Ig+cells and their phenotypic features in rheumatoid arthritis (RA) synovium, in comparison with those of osteoarthritis (OA) and psoriatic arthritis (PsA) synovium. Monoclonal anti-Z39Ig antibody was produced by immunizing Z39Ig transfected murine pre B cells and used for the identification of Z39Ig+cells. Z39Ig+cells were further stained with antibodies to macrophages, fibroblast-like synoviocytes, complement receptors and dendritic cells by using the double immunostaining method in normal, RA, OA and PsA synovium. RA synovial mononuclear cells were double-stained using anti-Z39Ig and anti-CD11c antibodies and sorted into Z39Ig+CD11c+cells and Z39Ig+CD11c-cells. These cell populations were then analysed by electron microscopy. The expression of the Z39Ig protein was limited to intimal macrophages in normal, RA, OA and PsA synovium. The numbers of Z39Ig+CD11c+cells and the ratios of Z39Ig+CD11c+cells to Z39Ig+cells were increased in the synovial lining layer of RA as compared with those of OA and PsA. The ultrastructural analysis of Z39Ig+CD11c+cells showed the character of macrophages with many secondary lysosomes and swelling of mitochondria. Z39Ig+ cells appeared to be useful for identification of resident tissue macrophages in normal synovium and the corresponding macrophages in the synovial lining layer of inflammatory arthritis. Expansion of Z39Ig+CD11c+cells was characteristic of RA synovial lining layer.

Expression of the chemokine superfamily in rheumatoid arthritis.

The infiltration of leucocytes into the joint of rheumatoid arthritis (RA) is believed to be mediated by chemotactic factors released by activated cells. In this study, examination was made of the gene expression and production of the chemokine superfamily in RA patients by reverse transcriptase-polymerase chain reaction (RT-PCR) and immunoprecipitation. Cultured synovial fibroblasts were found capable of expressing and producing IL-8, GRO, monocyte chemotactic and activating factor (MCAF), macrophage inflammatory protein-1 alpha (MIP-1 alpha), MIP-1 beta and RANTES in response to IL-1 alpha. The expression of IL-8, GRO, MCAF, MIP-1 alpha, and MIP-1 beta was clearly shown to increase in freshly isolated synovial fluid mononuclear cells (SFMC) of RA patients, in contrast to peripheral blood mononuclear cells (PBMC) of RA patients and normal subjects. The gene expression of RANTES appeared to be the same for RA SFMC, RA PBMC, and normal PBMC. Thus, the over-expression of various chemokines may promote the recruitment of inflammatory cells into rheumatoid inflamed joints.

IL-23 P19 Expression Induced by IL-17 and IL-1β in Rheumatoid Arthritis Synovial Mononuclear Cells

Interleukin-23 (IL-23) is a novel pro-inflammatory cytokine which has been implicated to play a pathogenic role in rheumatoid arthritis (RA). This study was undertaken to investigate the IL-23 inductive activity of the proinflammatory cytokine IL-17, IL-1β and tumor necrosis factor (TNF-α) in RA synovial fluid mononuclear cells (SFMC). Expression of IL-23p19, IL-17, IL-1β and TNF-α in joint was examined by immunohistochemistry (IHC) of patients with RA and osteoarthritis (OA). The effects of IL-17 and IL-1β on expression of IL-23p19 in human SFMC from RA patients were determined by reverse transcriptase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA). IL-23p19 was expressed in the RA fibroblast like synoviocyte (FLS), but not from OA FLS. Similar to the protein expression, IL-23p19 mRNA could be detected by RT-PCR in RA SFMC. IL-17 and IL-1β could induce RA SFMC to produce the IL-23p19. The effects of IL-17 were much stronger than IL-1β or TNF-α. These responses were observed in a doseresponsive manner. In addition, IL-17 or IL-1β neutralizing antibody down-regulated the expression of IL-23p19 induced by LPS in RA-SFMC. Our results demonstrate that IL-23p19 is overexpressed in RA synovium and IL-17 and IL-1β appears to upregulate the expression of IL-23p19 in RA-SFMC.

Interleukin (IL)-23 p19 expression induced by IL-1 in human fibroblast-like synoviocytes with rheumatoid arthritis via active nuclear factor- B and AP-1 dependent pathway

To explore the source of the p19 subunit of interleukin-23 (IL-23) in joints with rheumatoid arthritis (RA), the effects of IL-1beta and tumour necrosis factor (TNF)-alpha on IL-23 gene expression in RA fibroblast-like synoviocytes and the effect of IL-23 on proinflammatory cytokines. Expression of IL-23 p19 in joints was examined by immunohistochemical analysis of patients with RA and osteoarthritis (OA). The effects of IL-1beta and TNF-alpha on the expression, of IL-23 p19 and IL-12 p35 subunits in human fibroblast-like synoviocytes from RA patients (HFLS-RA) were determined by reverse transcriptase polymerase chain reaction (RT-PCR), quantitative PCR and western blotting assay. Blockade of nuclear factor kappaB (NF-kappaB) or AP-1 activation was used to verify the involvement of intracellular signal pathways of the induction of p19. IL-23-induced IL-8 and IL-6 productions were determined in HFLS-RA by RT-PCR and enzyme-linked immunosorbent assay. IL-23 p19 was expressed in the synovium from RA, but not from OA patients. Similar to the protein expression, IL-23 p19 mRNA could be detected by RT-PCR in four of five RA synovial fluid mononuclear cells (SFMC). IL-1beta and TNF-alpha could induce RA fibroblast-like synoviocytes to produce the IL-23 p19 subunit. The effects of IL-1beta were much stronger than TNF-alpha. These responses were observed in both a dose-responsive and time-dependent manner. IL-1beta produced weakly enhanced gene expression of the p35 subunits of IL-12. IL-1beta also promotes the p35 expression, a subunit of IL-12, but weakly. In addition, the NF-kappaB and the AP-1 inhibitors down-regulated the expression of IL-23 p19 mRNA induced by IL-1beta. IL-23 receptor (IL-23R) was of constitutive expression in HFLS-RA. Moreover, IL-23 up-regulated the IL-8 and IL-6 mRNA and protein levels in a dose-dependent manner in HFLS-RA. Our results demonstrate that IL-23, produced by mononuclear cells in synovial fluid with RA and HFLS-RA, promotes inflammatory responses in RA by inducing IL-8 and IL-6 production from HFLS. IL-1beta regulates IL-23 p19 expression via NF-kappaB and AP-1 pathways. This report also demonstrates that IL-23 could promote inflammatory responses in HFLS-RA by stimulating IL-8 and IL-6 production.

Expression of interferon-gamma (IFN-gamma), IL-10, IL-12 and transforming growth factor-beta (TGF-beta) mRNA in synovial fluid cells from patients in the early and late phases of rheumatoid arthritis (RA).

The expression of immunoregulatory cytokines was investigated in freshly isolated synovial fluid mononuclear cells (SFMC) and peripheral blood mononuclear cells (PBMC) from patients with RA, using a quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) assay. IFN-gamma, TGF-beta, IL-10 and IL-12 (p40) transcripts were detected in SFMC of patients with early disease (<1 year duration) as well as in patients with long standing arthritis (>1 year). The expression of IFN-gamma, IL-10 and IL-12 mRNA was increased in SFMC compared with RA PBMC. In addition, the expression was higher in RA SFMC than in PBMC from health control individuals. Immunoassay analysis of the secreted IL-12 heterodimer demonstrated increased levels in RA SF compared with levels found in serum from RA patients and control individuals. High levels of TGF-beta mRNA were found in SFMC, but a significantly decreased TGF-beta/beta2-microglobulin (beta2-M) ratio was found compared with PBMC from both patients and control individuals. IL-4mRNA could not be detected, either in SFMC or in PBMC. Cytokine expression in RA PBMC did not differ from control PBMC, with the exception of a decreased TGF-beta/beta2-M ratio in RA patients with early disease. Our findings of IFN-gamma mRNA and IL-12, but undetectable levels of IL-4 mRNA, suggest that the synovitis is characterized by a type 1 immune response. The presence of TGF-beta and IL-10 mRNA indicates that immunosuppressive cytokines may also operate in the inflamed joint, although their level of expression may not be sufficient for down-modulation of immune activation.

Effects of antirheumatic treatments on the prostaglandin E2 biosynthetic pathway

Microsomal prostaglandin E synthase 1 (mPGES-1) is up-regulated in experimental arthritis and markedly expressed in synovial tissue biopsy samples from patients with rheumatoid arthritis (RA). This study was carried out to determine the effects of tumor necrosis factor (TNF) blockers and glucocorticoids on mPGES-1 and cyclooxygenase (COX) expression, as well as biosynthesis of PGE(2) in rheumatoid joints. In vitro effects of TNF blockers and dexamethasone on the PGE(2) biosynthetic pathway were examined in RA synovial fluid mononuclear cells (SFMCs) by flow cytometry. PGE(2) levels in culture supernatants were measured by enzyme immunoassay. Expression of enzymes responsible for PGE(2) synthesis ex vivo was evaluated by immunohistochemistry in synovial biopsy samples obtained from 18 patients before and after treatment with TNF blockers and from 16 patients before and after intraarticular treatment with glucocorticoids. Double immunofluorescence was performed using antibodies against mPGES-1, COX-1, COX-2, and CD163. Double immunofluorescence revealed that mPGES-1 and COX-2 were colocalized in SFMCs as well as in RA synovial tissue cells. The addition of either TNF blockers or dexamethasone suppressed lipopolysaccharide-induced mPGES-1 and COX-2 expression in synovial fluid monocyte/macrophages in vitro and decreased the production of PGE(2). Intraarticular treatment with glucocorticoids significantly reduced both mPGES-1 and COX-2 expression in arthritic synovial tissue ex vivo. The number of COX-1-expressing cells in synovial tissue was also significantly decreased by glucocorticoid treatment. In contrast, neither mPGES-1 nor COX-2 expression in synovial tissue was significantly suppressed by anti-TNF therapy. These data are the first to demonstrate the effects of antirheumatic treatments on mPGES-1 expression in RA and suggest that the inhibition of PGE(2) biosynthesis, preferably by targeting mPGES-1, might complement anti-TNF treatment for optimal antiinflammatory results in RA.

Rheumatoid arthritis synovial T cells regulate transcription of several genes associated with antigen-induced anergy.

Rheumatoid arthritis (RA) is a chronic, inflammatory synovitis whose pathogenesis may involve autoimmune mechanisms. Anergy is a state of T-cell nonresponsiveness characterized by downregulated IL-2 production. Paradoxically, RA T cells are hyporesponsive and proliferate poorly to antigens and mitogens, thus sharing some characteristics with anergic T cells. We analyzed the molecular basis of anergy in cloned human CD4+ T cells using differential display RT-PCR and subsequently examined the levels of differentially expressed transcripts in RA and, as control, reactive arthritis (ReA) synovium. Several transcriptional events were common to anergic T cells and RA synovium. These included downregulation of CALMODULIN:, which is critical to T-cell activation, and of cellular apoptosis susceptibility protein, which may mediate resistance to apoptosis in RA. Transcription of CALMODULIN: in RA synovium was less than 1% of that in ReA and was lower in RA synovial fluid mononuclear cells than in paired PBMCs. Following anti-TNF-alpha therapy in vivo, RA PBMC CALMODULIN: transcripts increased five- to tenfold. Pharmacological calmodulin blockade in vitro impaired antigen-specific proliferation. These data provide a link between reduced CALMODULIN: transcription and impaired T-cell responsiveness in RA. The identification of transcriptional changes common to anergic and RA synovial T cells should help interpret some of the characteristic RA cellular defects.

Selective expression of folate receptor beta and its possible role in methotrexate transport in synovial macrophages from patients with rheumatoid arthritis.

To investigate the expression of folate receptors (FR) and reduced folate carrier (RFC) and determine their relevance to methotrexate (MTX) transport in synovial mononuclear cells (SMC) from patients with rheumatoid arthritis (RA). Levels of FR and RFC messenger RNA (mRNA) were examined by reverse transcriptase-polymerase chain reaction (RT-PCR) in SMC from RA patients and peripheral blood mononuclear cells from healthy donors. Expression of FR-beta mRNA and protein was determined by Northern blot and Western blot analyses in RA SMC and monocyte/macrophage-lineage cells. FR-beta expression and folic acid binding capacity on the cell surface were examined by flow cytometric analysis and 3H-folic acid binding analysis. Studies of the inhibition of 3H-MTX uptake in the presence of unlabeled folic acid were performed to investigate the uptake of MTX through FR in RA SMC. RT-PCR, Northern blot, and Western blot analyses showed that FR-beta mRNA and protein were expressed selectively in activated monocytes and CD14+ RA SMC. These cells exhibited folic acid binding capacity. Furthermore, the FR-beta protein was shown to have folic acid binding capacity. Uptake of 3H-MTX through RA SMC was significantly inhibited in the presence of unlabeled folic acid. These results demonstrate that FR-beta expression is selectively elevated in RA synovial macrophages and suggest that MTX is transported through FR-beta in RA synovial macrophages. The findings suggest that folate antagonists with higher affinity for FR-beta would be useful in the treatment of RA.

The CD69 activation pathway in rheumatoid arthritis synovial fluid t cells

To study the CD69 activation pathway in synovial fluid (SF) T lymphocytes from patients with rheumatoid arthritis (RA). Peripheral blood mononuclear cells (PBMC) or SF mononuclear cells (SFMC) were used in proliferation assays with anti-CD69, anti-CD28, anti-CD3, phorbol myristate acetate (PMA), and/or recombinant interleukin-2 (IL-2). CD69+, CD69-, and resting SF T cells were also proliferated. CD25 expression and production of IL-2 after CD69 activation were assessed by flow cytometry and in a bioassay with the IL-2-dependent cell line CTLL-2. RA SFMC did not proliferate either in the presence of anti-CD69 monoclonal antibodies alone or with concomitant PMA activation, when compared with paired or control PBMC. Similar low proliferative responses via the CD3 or CD28 pathway with PMA were observed. This defective proliferation of RA SFMC after stimulation through the CD69 molecule was explained in part by a failure to express CD25 and to produce IL-2. SF CD69- T cells and resting SF T cells had higher rates of proliferation through the alternative costimulatory pathway CD28 than did SF CD69+ T cells or freshly isolated SF T cells. Freshly isolated SF T cells present a profound state of hyporesponsiveness through the CD69 and CD28 costimulatory pathways. This state appears to be dependent on the activation status of SF T cells, since CD69- and resting SF T cells showed recovery of the ability to proliferate through the CD28 activation pathway.

Epithelial neutrophil activating peptide-78: a novel chemotactic cytokine for neutrophils in arthritis.

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.

The biased Vγ gene usage in the synovial fluid of patients with rheumatoid arthritis

Taking advantage of the reverse transcriptase-polymerase chain reaction (RT-PCR), we have analyzed T cell receptor gamma-chain mRNA of synovial fluid gamma/delta T cells from patients with rheumatoid arthritis (RA) in comparison with those of peripheral blood mononuclear cells (PBMC) from RA patients and healthy individuals. The quantitative RT-PCR method in conjunction with nucleotide sequencing revealed the frequent usage of the V gamma 3 gene segment in RA synovial fluid mononuclear cells (SFMC) (p < 0.01) which in PBMC of healthy individuals occurred rarely. PBMC of most healthy individuals expressed the V gamma 9 gene predominantly (p < 0.01) as expected. However, only half of RA patients showed elevated levels of the V gamma 9 gene expression in their PBMC. The gamma-chain mRNA containing the V gamma 3 gene in RA SFMC showed no conserved junctional sequence (complementarity-determining region 3). To investigate the nature of ligands recognized by the V gamma 3-bearing T cells, we analyzed V gamma gene usage of RA SFMC, RA PBMC, and normal PBMC stimulated with Mycobacterium tuberculosis (MT) or MT plus interleukin-2 since there is mounting evidence of high reactivity of RA SFMC to MT and mycobacterial heat-shock protein 65. However, the V gamma usage appeared to be mostly V gamma 9 in RA SFMC, RA PBMC and normal PBMC. Taken together these results suggest that an as yet unknown antigen(s) (other than MT) might select gamma/delta T cells expressing the V gamma 3 gene in RA SFMC.

Production of soluble ICAM-1 by mononuclear cells from patients with rheumatoid arthritis patients.

The present study was designed to quantify the level of the soluble form of ICAM-1 (sICAM-1) produced by mononuclear cells (MNC) of rheumatoid arthritis (RA) patients, and to correlate these levels with the disease activity and with the amounts of cytokines or rheumatoid factors (RF) produced by MNC. Unstimulated synovial fluid (SF) MNC produced higher amounts of sICAM-1 than peripheral blood (PB) MNC in RA patients (P < 0.01). sICAM-1 production by PHA-stimulated MNC was higher in RA SF MNC than RA or normal PB MNC (P < 0.01). The amounts of SICAM-1 produced correlated with the amounts of soluble IL-2 receptor produced (P < 0.02) but not with IL-1B or the Lansbury activity index in RA PB MNC. sICAM-1 correlated with the amounts of soluble CD23 and IL-4 produced by normal PB MNC (P < 0.01). The amounts of sICAM-1 correlated with IgG-RF (P < 0.02) and IgM-RF (P < 0.01) produced by unstimulated MNC obtained from the bone marrow (BM) of RA patients. ICAM-1 expression of T-lymphocyte subsets, B lymphocytes, and monocytes obtained from RA PB and RA BM assayed by two-color flow cytometry ranged from 0.1 to 6%, which was not appreciably different from that of normal controls. The monocyte fraction of RA PB MNC produced significantly higher amounts of sICAM-1 than lymphocyte fraction. These results suggest that sICAM-1 produced by MNC may be a marker of cell activation in T and B lymphocytes, in contrast to the transient increase of ICAM-1 expression.

Beta 1 integrin-mediated interaction with extracellular matrix proteins regulates cytokine gene expression in synovial fluid cells of rheumatoid arthritis patients.

Inflammatory cytokines have been implicated in the pathogenesis of rheumatoid arthritis (RA), whereas the mechanisms for constitutive production of inflammatory cytokines in affected joints are largely unknown. Recently, integrin-mediated interaction with extracellular matrix (ECM) proteins has been demonstrated to play a role in regulating cytokine production in T cells and monocytes. In this study, we investigated the contribution of the beta 1 integrin-mediated interaction with ECM proteins to the persistent cytokine gene expression in RA synovial fluid mononuclear cells (SFMNC). We examined mRNA expression of 14 cytokines in the SFMNC of three RA patients, which were either fresh or cultured overnight in serum-free medium on ECM-coated plates, by polymerase chain reaction with a panel of oligonucleotide primers specific for each cytokine. The persistent expression of various cytokine mRNA found in fresh SFMNC was maintained after overnight culture in serum-free medium on ECM proteins, especially on laminin (LM), but not on serum albumin. This effect of LM was inhibited by an anti-integrin beta 1 chain (CD29) mAb, as well as by an anti-CD3 mAb, indicating an important role of the beta 1 integrin-mediated interaction with ECM proteins in regulating persistent cytokine gene expression in RA SFMNC, and a key role of T cells in regulating inflammatory monokine production.