Effects of paclitaxel on cultured synovial cells from patients with rheumatoid arthritis

BACKGROUND: Proliferation of synovial cells is considered to play a key role in rheumatoid arthritis (RA). Using paclitaxel, a unique antineoplastic agent known to suppress collagen-induced arthritis, we conducted an in vitro study of cell kinetics on cultured synovial cells from patients with RA. METHODS: Alterations of the cell cycle of cultured fibroblast-like synovial cells (FLSs) from patients with RA were studied using flow cytometry and laser scanning cytometry. Apoptosis and accumulation of cyclin concerning effects of paclitaxel were detected. RESULTS: Paclitaxel induced arrest of the cell cycle at G2/M phase and apoptosis in FLSs. The late stage of apoptosis was determined by the positivity of terminal deoxynucleotidyl transferase assay. Morphological observation by combined usage of both annexin V and propidium iodide on FLSs on a slide glass showed early apoptotic changes in detail. FLSs arrested at G2/M phase showed marked accumulation of cyclin B1. The effects of paclitaxel decreased on FLSs, which diminished proliferative activity. CONCLUSIONS: These data indicate that paclitaxel induces cell arrest at G2/M phase followed by apoptosis in human FLSs, which have high proliferative activity, and possible therapeutic effects of paclitaxel on RA.     

VLA-4-dependent and -independent pathways in cell contact-induced proinflammatory cytokine production by synovial nurse-like cells from rheumatoid arthritis patients

Nurse-like stromal cell lines from the synovial tissue of patients with rheumatoid arthritis (RA-SNC) produce, on coculture with lymphocytes, large amounts of proinflammatory cytokines. In the present paper, we analyze the molecular events necessary for the induction of cytokine release from RA-SNC cells, and particularly the roles played by cell adhesion and the transmigration (also known as pseudoemperipolesis) of lymphocytes. For this purpose, the effects of various mAbs on the binding and transmigration of a human B-cell line, MC/car, were examined using a cloned RA-SNC line, RA-SNC77. To analyze the role of lymphocyte binding and transmigration on upregulated cytokine production by the RA-SNC77 cells, we used C3 exoenzyme-treated MC/car cells, which could bind to RA-SNC77 cells but could not transmigrate. Treatment with anti-CD29 or anti-CD49d mAb significantly reduced binding and transmigration of the MC/car cells. In contrast, the neutralizing anti-CD106/vascular cell adhesion molecule 1 mAb did not show any inhibitory effect. Likewise, none of the neutralizing mAbs against CD11a, CD18, CD44, CD49e, or CD54 showed significant effects. Binding of C3-treated or untreated MC/car cells to RA-SNC77 cells induced comparable levels of IL-6 and IL-8 production. In addition, the enhanced cytokine production by RA-SNC77 cells required direct lymphocyte contact via a very late antigen-4 (VLA-4)-independent adhesion pathway. These results indicate that, although both the VLA-4-dependent/vascular cell adhesion molecule 1-independent and the VLA4-independent adhesion pathways are involved in MC/car binding and subsequent transmigration, only the VLA4-independent adhesion pathway is necessary and sufficient for the enhanced proinflammatory cytokine production by RA-SNC77 cells. The transmigration process, which is dependent on Rho-GTPase, is not a prerequisite for this phenomenon.     

Interleukin-29 modulates proinflammatory cytokine production in synovial inflammation of rheumatoid arthritis

Introduction

The association between rheumatoid arthritis (RA) and periodontitis is suggested to be linked to the periodontal pathogen Porphyromonas gingivalis. Colonization of P. gingivalis in the oral cavity of RA patients has been scarcely considered. To further explore whether the association between periodontitis and RA is dependent on P. gingivalis, we compared host immune responses in RA patients with and without periodontitis in relation to presence of cultivable P. gingivalis in subgingival plaque.

Methods

In 95 RA patients, the periodontal condition was examined using the Dutch Periodontal Screening Index for treatment needs. Subgingival plaque samples were tested for presence of P. gingivalis by anaerobic culture technique. IgA, IgG and IgM antibody titers to P. gingivalis were measured by ELISA. Serum and subgingival plaque measures were compared to a matched control group of non-RA subjects.

Results

A higher prevalence of severe periodontitis was observed in RA patients in comparison to matched non-RA controls (27% versus 12%, p < 0.001). RA patients with severe periodontitis had higher DAS28 scores than RA patients with no or moderate periodontitis (p < 0.001), while no differences were seen in IgM-RF or ACPA reactivity. Furthermore, RA patients with severe periodontitis had higher IgG- and IgM-anti P. gingivalis titers than non-RA controls with severe periodontitis (p < 0.01 resp. p < 0.05), although subgingival occurrence of P. gingivalis was not different.

Conclusions

Severity of periodontitis is related to severity of RA. RA patients with severe periodontitis have a more robust antibody response against P. gingivalis than non-RA controls, but not all RA patients have cultivable P. gingivalis.     

RANTES and chemotactic activity in synovial fluids from patients with rheumatoid arthritis and osteoarthritis

A massive accumulation of inflammatory cells in synovial tissues is a major pathological feature of rheumatoid arthritis (RA). Neutrophiles dominate synovial fluid while rheumatoid synovium is infiltrated with mononuclear cells. Mechanisms regulating influx of particular subpopulations of leukocytes into articular cavity and synovium compartment are not completely defined. An increasing amount of data supports a crucial role of a C-C chemokine RANTES in the RA pathogenesis. Our objective is to evaluate chemotactic activity for neutrophils (NCA), lymphocytes (LCA), and monocytes (MoCA) in SFs obtained from patients with RA and osteoarthritis (OA). We also aimed to characterise the relation between chemotactic activity, RANTES, and percentage distribution of leukocytes in SF. SFs from 11 patients with RA and 6 with OA were included in the study. Modified microchamber Boyden method was employed to assess chemotactic activity. Cytological and biochemical analysis of SF was performed. RANTES was measured with ELISA. Rheumatoid SFs were rich in cells with predominance of neutrophiles while osteoarthritic fluids were lymphocytic. RA SFs were also characterised by increased lactoferrin level. Both NCA and LCA were higher in SF from patients with RA (62 +/- 12 and 24 +/- 6 cells/HPF, resp) as compared to patients with OA (23 +/- 6; P < .05 and 6 +/- 2 cells/HPF; P < 0.05). The chemoattractive effect of RA SF was more pronounced on neutrophiles than on lymphocytes. RA SF expressed high RANTES levels (145+/- 36 pg/mL), while OA SF was characterised by only trace amount of this chemokine (2 +/- 1 pg/mL). We found positive correlation of RANTES with chemotactic activity for mononuclear cells (LCA + MoCA; R = 0.61; P < .05). Surprisingly, RANTES correlated also positively with neutrophiles number (R = 0.77; P < 0.001). Rheumatoid SF possesses strong chemotactic potency for leukocytes. RANTES is overexpressed in RA SF and is a potential mediator influencing intensity and composition of cellular infiltration in joints affected with inflammatory arthritis.     

Immunological detection of myeloperoxidase in synovial fluid from patients with rheumatoid arthritis.

We have used rocket immunoelectrophoresis and immunoblotting to detect myeloperoxidase in synovial fluid from patients with rheumatoid arthritis. This protein was enzymatically inactive but its identity as myeloperoxidase was confirmed by comparing its subunit structure with that of the purified enzyme. When neutrophils were stimulated to secrete myeloperoxidase in vitro, a polypeptide with an apparent molecular mass of 62 kDa was detected extracellularly by immunoblotting. Neutrophils isolated from synovial fluid showed a reduced level of this 62 kDa polypeptide but it was detected extracellularly in synovial fluid by immunoblotting. Thus, we conclude that neutrophils in synovial fluid from patients with rheumatoid arthritis have been activated in vivo to secrete myeloperoxidase and propose that the products of this enzyme system can contribute to the tissue damage associated with this disease.     

The loss of sympathetic nerve fibers in the synovial tissue of patients with rheumatoid arthritis is accompanied by increased norepinephrine release from synovial macrophages.

Our objective was to investigate sympathetic and sensory nerve fibers in synovial tissue in rheumatoid arthritis (RA) and osteoarthritis (OA) in relation to histological inflammation and synovial cytokine and norepinephrine (NE) secretion. Immunohistochemistry was used to detect nerve fibers and inflammatory parameters. A superfusion technique of synovial tissue pieces was used to investigate cytokine and NE secretion. In RA, we detected 0.2 +/- 0.04 tyrosine hydroxylase-positive (TH-positive=sympathetic) nerve fibers/mm2 as compared to 4.4 +/- 0. 8 nerve fibers/mm2 in OA (P<0.001). In RA, there was a negative correlation between the number of TH-positive nerve fibers and inflammation index (RRank=-0.705, P=0.002) and synovial IL-6 secretion (RRank=-0.630, P=0.009), which was not found in OA. Substance P-positive (=sensory) nerve fibers were increased in RA as compared to OA (3.5+/-0.2 vs. 2.3+/-0.3/mm2, P=0.009). Despite lower numbers of sympathetic nerve fibers in RA than in OA, NE release was similar at baseline (RA vs. OA: 152+/-36 vs. 106+/-21 pg/ml, n.s.). Basal synovial NE secretions correlate with the number of TH-positive CD 163+ synovial macrophages (RA: RRank=0.622, P=0.031; OA: RRank=0.299, n.s.), and synovial macrophages have been shown to produce NE in vitro. Whereas sympathetic innervation is reduced, sensory innervation is increased in the synovium from patients with longstanding RA when compared to the synovium from OA patients. The differential patterns of innervation are dependent on the severity of the inflammation. However, NE secretion from the synovial tissue is maintained by synovial macrophages. This demonstrates a loss of the influence of the sympathetic nervous system on the inflammation, accompanied by an up-regulation of the sensory inputs into the joint, which may contribute to the maintenance of the disease.     

Do synovial fluid acute phase proteins from patients with rheumatoid arthritis originate from serum?

This study was performed in order to gain insight into the occurrence, glycosylation and the possible origin of the acute-phase proteins α1-acid glycoprotein (AGP) and α1-protease inhibitor (PI) in sera and synovial fluid from patients with rheumatoid arthritis (RA). Therefore paired sera and synovial fluid samples from patients with RA, and paired synovial fluid samples from right and left knees of patients with varying degrees of arthritis were studied. Crossed affinity immunoelectrophoresis (CAIE) was used with concanavalin A and Aleuria aurantia lectin for the detection of the degree of branching and fucosylation, respectively, and the monoclonal CSLEX-1 for the detection of Sialyl Lewisx (SLex) groups on AGP. For PI, not only CAIE, but also high-pressure-anion-exchange chromatography with pulsed amperometric detection was used to study the glycosylation. It was established that the concentrations of AGP and PI were increased in the serum of RA patients compared to normal healthy controls, but that the concentration of both proteins, as well as albumin, was significantly lower in synovial fluid than in serum. Furthermore, the type of glycosylation of both AGP and PI found in RA was significantly different from that found in normals, with increased fucosylation, but there were no major differences in the degree of branching of AGP- or PI-glycans in RA, compared to normals. No differences in glycosylation could be established between serum and synovial fluid in RA. For PI an increased fucosylation was found, both in serum and synovial fluid, using both methods of detection, and it could be established that only the α1→3- and not the α1→6-fucosylation of PI was affected by RA. The increased fucosylation of AGP resulted in an increased expression of SLex on AGP-glycans. Since the α1→3- fucosylation of AGP was significantly increased in both serum and synovial fluid from RA patients, and this correlated with systemic but not with local disease parameters, it can be suggested that acute phase proteins in synovial fluid are most probably of hepatic origin. Abbreviations: AGP, α1-acid glycoprotein; AAL, Aleuria Aurantia Lectin; Con A, concanavalin A; PI, α1-protease inhibitor; CAIE, crossed affino-immunoelectrophoresis; SLex, sialyl Lewis X; IL-6, interleukin-6; RA, rheumatoid arthritis; PMN, polymorphonuclear cells; HPAEC, high pressure anion exchange chromatography This revised version was published online in November 2006 with corrections to the Cover Date.     

Acute-phase serum amyloid A production by rheumatoid arthritis synovial tissue

Acute-phase serum amyloid A (A-SAA) is a major component of the acute-phase response. A sustained acute-phase response in rheumatoid arthritis (RA) is associated with increased joint damage. A-SAA mRNA expression was confirmed in all samples obtained from patients with RA, but not in normal synovium. A-SAA mRNA expression was also demonstrated in cultured RA synoviocytes. A-SAA protein was identified in the supernatants of primary synoviocyte cultures, and its expression colocalized with sites of macrophage accumulation and with some vascular endothelial cells. It is concluded that A-SAA is produced by inflamed RA synovial tissue. The known association between the acute-phase response and progressive joint damage may be the direct result of synovial A-SAA-induced effects on cartilage degradation.     

Kallikrein in synovial fluid with rheumatoid arthritis

The levels of kallikrein and collagenase in synovial fluid from rheumatoid arthritis (RA) patients were examined and the role of kallikrein in procollagenase activation is discussed. Both prekallikrein and active kallikrein in synovial fluid from patients with RA were significantly elevated when compared to synovial fluid from patients with osteoarthritis (OA). In RA synovial fluid, the ratio of the active form to total kallikrein was also higher than that in OA synovial fluid. Both active collagenase and the alpha 2-macroglobulin (alpha 2M)-collagenase complex in RA synovial fluid were higher than in OA synovial fluid. A partial correlation (r = 0.58) between active kallikrein and total collagenase (active and alpha 2M-collagenase complex) was observed in RA synovial fluid. These observations indicate that both kallikrein and collagenase are associated with the destruction of cartilage, but the role of kallikrein in procollagenase activation was not fully clarified.     

IL-29 enhances Toll-like receptor-mediated IL-6 and IL-8 production by the synovial fibroblasts from rheumatoid arthritis patients

Introduction

We previously reported that IL-29, a newly described member of interferon (IFN) family, was overexpressed in blood and synovium of rheumatoid arthritis (RA) patients and triggered proinflammatory cytokine IL-6 and IL-8 mRNA expression in RA synovial fibroblasts (RA-FLS). This suggests that IL-29 has an important role in synovial inflammation. Toll-like receptors (TLRs) also activate RA-FLS to produce inflammatory mediators including tumor necrosis factor α (TNF-α) and IL-1β in RA-FLS. Since the TLR family plays an early role in the innate immune response and the subsequent induction of the adaptive immune response, we hypothesize that IL-29 interacts with TLRs in RA inflammation. This study aimed to investigate the effect of IL-29 on TLR-mediated proinflammatory cytokine production in RA-FLS.

Methods

The mRNA level of IL-29 receptors (IL-28Rα and IL-10R2) in RA-FLS was determined by semi-quantitative RT- PCR. IL-6 and IL-8 mRNA expressions in RA-FLS were evaluated by real-time PCR after pre-incubation with IL-29 and subsequent stimulation with peptidoglycan (PGN, TLR2 ligand), or polycytidylic acid (poly(I:C), TLR3 ligand), or lipopolysaccharide (LPS, TLR4 ligand) . The production of TLR2, 3, and 4 in RA-FLS after IL-29 stimulation was also assessed by real-time PCR and flow cytometry. IL-29 mRNA and protein expression in RA-FLS after stimulation with PGN, poly(I:C), or LPS were measured by real-time PCR and enzyme-linked immunosorbent assay (ELISA), respectively.

Results

The IL-29 receptor complex (IL-28Rα and IL-10R2) was identified in RA-FLS. IL-29 enhanced TLR-mediated IL-6 and IL-8 expression in RA-FLS. IL-29 upregulated expression of TLR2, 3 and 4 in RA-FLS. Exposure to PGN, poly(I:C) or LPS triggered IL-29 production by RA-FLS.

Conclusions

We show for the first time that IL-29 enhances TLR-induced proinflammatory cytokine production in RA-FLS via upregulation of TLRs.     

Rubella and rheumatoid arthritis: hyaluronic acid and susceptibility of cultured rheumatoid synovial cells to viruses.

Synovial cell lines were established from patients with rheumatoid arthritis (RA) and from normal human embryos. High levels of hyaluronic acid (HA) were produced by some RA cell lines, some of which were partially or completely resistant to infection with Newcastle disease virus (NDV), vesicular stomatitis virus (VSV), and rubella virus (RV). Normal fetal synovial cells lines were susceptible to NDV, VSV, and RV. Infection with virus became possible after treatment of RA cells with hyaluronidase to depolymerize HA, and HA prevented infection of normal synovial cells with VSV. These results provide evidence that HA and not chronic or latent viral infection is responsible for the lack of susceptibility of RA synovial cells to certain viruses.     

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

Introduction

The aim of this study was to examine the effect of blocking Toll-like receptor 2 (TLR2) in rheumatoid arthritis (RA) synovial cells.

Methods

RA 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.

Results

Pam3CSK4 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.

Conclusions

These findings further support targeting TLR2 as a potential therapeutic agent for the treatment of RA.     

Prorenin-renin axis in synovial fluid in patients with rheumatoid arthritis and osteoarthritis.

This study was undertaken 1) to determine whether or not renin is present in synovial fluid in patients with rheumatoid arthritis and osteoarthritis, and, if present, 2) to investigate whether it is synthesized in synovial fluid, or it is only transported from the circulation into the synovial cavity. The active renin concentration (indirect) was measured with angiotensin I radioimmunoassay kits. Inactive renin was converted into active renin with Sepharose-bound trypsin. Both active and inactive forms of renin were found in synovial fluid. They were significantly higher in patients with rheumatoid arthritis (n = 9) than in those with osteoarthritis (n = 16). In plasma, the concentration of inactive renin was significantly higher (P less than 0.001) in the former. Albumin, transferrin, alpha 2-macroglobulin, ceruloplasmin and immunoglobulins G and M were also found in synovial fluid. In each disease, a plot of the log ratio of synovial fluid to the serum concentration against the log molecular weight of each protein gave an approximately straight line curve, suggesting that these proteins are derived from the circulation and are transported into the synovial cavity. In contrast, the ratio of synovial fluid to plasma concentrations of active renin was significantly higher than that predicted on the basis of the above-mentioned interrelationships in both diseases, whereas the ratio of inactive renin was significantly lower. These findings suggest that 1) inactive and active renin are filtered into the synovial fluid from the circulation, and that 2) inactive renin is converted into the active form in the fluid.     

Evidence for oxidised low density lipoprotein in synovial fluid from rheumatoid arthritis patients

The oxidative modification of human LDL has been implicated in atherosclerosis, but the mechanisms by which such modification occurs in vivo are not fully understood. In the present study, we have isolated LDL from knee-joint synovial fluid of patients with rheumatoid arthritis. We demonstrate that such LDL is oxidatively modified as evidenced by an increased negative charge, distorted particulate nature and more rapid degradation by cultured macrophages. These results indicate that formation of oxidised LDL is associated with the local inflammatory response. Because the cellular interactions in rheumatoid arthritis have analogies with those in atherogenesis, we suggest that the rheumatoid joint is a useful model of atherosclerosis in which the in vivo process of LDL oxidation may be readily studied.     

Evidence for oxidised low density lipoprotein in synovial fluid from rheumatoid arthritis patients

The oxidative modification of human LDL has been implicated in atherosclerosis, but the mechanisms by which such modification occurs in vivo are not fully understood. In the present study, we have isolated LDL from knee-joint synovial fluid of patients with rheumatoid arthritis. We demonstrate that such LDL is oxidatively modified as evidenced by an increased negative charge, distorted particulate nature and more rapid degradation by cultured macrophages. These results indicate that formation of oxidised LDL is associated with the local inflammatory response. Because the cellular interactions in rheumatoid arthritis have analogies with those in atherogenesis, we suggest that the rheumatoid joint is a useful model of atherosclerosis in which the in vivo process of LDL oxidation may be readily studied.     

Increased native chemiluminescence in granulocytes isolated from synovial fluid and peripheral blood of patients with rheumatoid arthritis

Polymorphonuclear leukocytes (PMNs) isolated from peripheral blood and synovial fluid of patients with rheumatoid arthritis and from peripheral blood of volunteers were stimulated with 12-Phorbol-13-myristate acetate (PMA). No significant differences in luminol-amplified chemiluminescence were found between different patients and control groups. However, two distinct patterns of native chemiluminescence were observed. Type I showed no, or only a small, increase in native chemiluminescence with integral counts over 30 min less than 3 × 10⁵ cpm, and the majority of samples from volunteers were of this type. Type II was characterized by a burst of native chemiluminescence starting 8 to 15 min after cell stimulation. It was found in most PMN samples from patients with rheumatoid arthritis. Integral counts over 30 min were always higher than 10⁶ cpm and as high as 10⁸ cpm in some cases. A strong inhibition of the Type II native chemiluminescence was caused by desferal, catalase, thiourea, and glutathione. However, the luminol-amplified chemiluminescence remained unchanged or was only slightly decreased under the same experimental conditions. Sodium azide strongly inhibited both kinds of luminscence. Hydroxyl radicals, formed in a Fenton reaction, may be important intermediates in the Type II native chemiluminescence.