Expression of tumor cell properties in synovial cells in culture

The tumorigenic properties of human rheumatoid arthritis synovial cells in culture were investigated. The synovial cells developed good colonies and secreted plasminogen activator (PA) and collagenase in the cell cultures, as do Hela cells. Since PA and progesterone receptor (PgR) are considered to be end products of estradiol action in breast cancer cells, the estrogen receptor (ER) and PgR content in these cells was also assayed. Large amounts of ER and PgR were detected in the synovial cells in culture, even though these cells are not targets for sex steroids. Study of the cytomorphologic changes in the synovial cells in culture revealed many characteristics generally observed in neoplastic cells. Whether any or all of these observations have any implication in prognosis or therapy in this disease remains to be studied.     

TWEAK and Fn14 expression in the pathogenesis of joint inflammation and bone erosion in rheumatoid arthritis

Introduction

TNF-like weak inducer of apoptosis (TWEAK) has been proposed as a mediator of inflammation and bone erosion in rheumatoid arthritis (RA). This study aimed to investigate TWEAK and TWEAK receptor (Fn14) expression in synovial tissue from patients with active and inactive rheumatoid arthritis (RA), osteoarthritis (OA) and normal controls and assess soluble (s)TWEAK levels in the synovial fluids from patients with active RA and OA. Effects of sTWEAK on osteoclasts and osteoblasts were investigated in vitro.

Methods

TWEAK and Fn14 expression were detected in synovial tissues by immunohistochemistry (IHC). Selected tissues were dual labelled with antibodies specific for TWEAK and lineage-selective cell surface markers CD68, Tryptase G, CD22 and CD38. TWEAK mRNA expression was examined in human peripheral blood mononuclear cells (PBMC) sorted on the basis of their expression of CD22. sTWEAK was detected in synovial fluid from OA and RA patients by ELISA. The effect of sTWEAK on PBMC and RAW 264.7 osteoclastogenesis was examined. The effect of sTWEAK on cell surface receptor activator of NF Kappa B Ligand (RANKL) expression by human osteoblasts was determined by flow cytometry.

Results

TWEAK and Fn14 expression were significantly higher in synovial tissue from all patient groups compared to the synovial tissue from control subjects (P < 0.05). TWEAK was significantly higher in active compared with inactive RA tissues (P < 0.05). TWEAK expression co-localised with a subset of CD38⁺ plasma cells and with CD22⁺ B-lymphocytes in RA tissues. Abundant TWEAK mRNA expression was detected in normal human CD22⁺ B cells. Higher levels of sTWEAK were observed in synovial fluids isolated from active RA compared with OA patients. sTWEAK did not stimulate osteoclast formation directly from PBMC, however, sTWEAK induced the surface expression of RANKL by human immature, STRO-1⁺ osteoblasts.

Conclusions

The expression of TWEAK by CD22⁺ B cells and CD38⁺ plasma cells in RA synovium represents a novel potential pathogenic pathway. High levels of sTWEAK in active RA synovial fluid and of TWEAK and Fn14 in active RA tissue, together with the effect of TWEAK to induce osteoblastic RANKL expression, is consistent with TWEAK/Fn14 signalling being important in the pathogenesis of inflammation and bone erosion in RA.     

Synovial Fluid Progenitors Expressing CD90+ from Normal but Not Osteoarthritic Joints Undergo Chondrogenic Differentiation without Micro-Mass Culture

Objective

Mesenchymal progenitor cells (MPCs) can differentiate into osteoblasts, adipocytes, and chondrocytes, and are in part responsible for maintaining tissue integrity. Recently, a progenitor cell population has been found within the synovial fluid that shares many similarities with bone marrow MPCs. These synovial fluid MPCs (sfMPCs) share the ability to differentiate into bone and fat, with a bias for cartilage differentiation. In this study, sfMPCs were isolated from human and canine synovial fluid collected from normal individuals and those with osteoarthritis (human: clinician-diagnosed, canine: experimental) to compare the differentiation potential of CD90+ vs. CD90− sfMPCs, and to determine if CD90 (Thy-1) is a predictive marker of synovial fluid progenitors with chondrogenic capacity in vitro.

Methods

sfMPCs were derived from synovial fluid from normal and OA knee joints. These cells were induced to differentiate into chondrocytes and analyzed using quantitative PCR, immunofluorescence, and electron microscopy.

Results

The CD90+ subpopulation of sfMPCs had increased chondrogenic potential compared to the CD90− population. Furthermore, sfMPCs derived from healthy joints did not require a micro-mass step for efficient chondrogenesis. Whereas sfMPCs from OA synovial fluid retain the ability to undergo chondrogenic differentiation, they require micro-mass culture conditions.

Conclusions

Overall, this study has demonstrated an increased chondrogenic potential within the CD90+ fraction of human and canine sfMPCs and that this population of cells derived from healthy normal joints do not require a micro-mass step for efficient chondrogenesis, while sfMPCs obtained from OA knee joints do not differentiate efficiently into chondrocytes without the micro-mass procedure. These results reveal a fundamental shift in the chondrogenic ability of cells isolated from arthritic joint fluids, and we speculate that the mechanism behind this change of cell behavior is exposure to the altered milieu of the OA joint fluid, which will be examined in further studies.     

Medication persistence over 2 years of follow-up in a cohort of early rheumatoid arthritis patients: associated factors and relationship with disease activity and with disability

Introduction

Tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) is a tumour necrosis factor (TNF) family member capable of inducing apoptosis in many cell types.

Methods

Using immunohistochemistry, terminal deoxynucleotidyl transferase biotin-dUTP nick end labelling (TUNEL) and real-time PCR we investigated the expression of TRAIL, TRAIL receptors and several key molecules of the intracellular apoptotic pathway in human synovial tissues from various types of arthritis and normal controls. Synovial tissues from patients with active rheumatoid arthritis (RA), inactive RA, osteoarthritis (OA) or spondyloarthritis (SpA) and normal individuals were studied.

Results

Significantly higher levels of TRAIL, TRAIL R1, TRAIL R2 and TRAIL R4 were observed in synovial tissues from patients with active RA compared with normal controls (p < 0.05). TRAIL, TRAIL R1 and TRAIL R4 were expressed by many of the cells expressing CD68 (macrophages). Lower levels of TUNEL but higher levels of cleaved caspase-3 staining were detected in tissue from active RA compared with inactive RA patients (p < 0.05). Higher levels of survivin and x-linked inhibitor of apoptosis protein (xIAP) were expressed in active RA synovial tissues compared with inactive RA observed at both the protein and mRNA levels.

Conclusions

This study indicates that the induction of apoptosis in active RA synovial tissues is inhibited despite stimulation of the intracellular pathway(s) that lead to apoptosis. This inhibition of apoptosis was observed downstream of caspase-3 and may involve the caspase-3 inhibitors, survivin and xIAP.     

Neutral amino acid transport in human synovial cells: substrate specificity of adaptative regulation and transinhibition

Neutral amino acid transport was characterized in human synovial cells. The amino acids tested are transported by all three major neutral amino acid transport systems, that is, A, L, and ASC. The model amino acid 2-aminoisobutyric acid (AIB) was found to be a strong specific substrate for system A in synovial cells. When cells were starved of amino acids, the activity of AIB transport increased, reaching a maximum within 1 h. The stimulation of transport activity was not blocked by cycloheximide and would thus appear to be related to a release from transinhibition. Similarly, the decrease in the activity of AIB transport observed after the addition of alpha-methyl-aminoisobutyric acid (meAIB) appeared to be related to transinhibition. However, using a different approach, that is, amino acid starvation followed by incubation with 10 mM meAIB and transfer to an amino acid-free medium with or without cycloheximide supplementation, a clear increase in AIB uptake, due both to derepression and a release from transinhibition, was observed. Unlike human fibroblasts, the depression of system A in these synovial cells was not serum-dependent. The process of derepression was observed only after preloading with meAIB. Neither AIB nor alanine produced this phenomenon. Moreover, alanine preloading led to a large increase in AIB transport activity due to a release from transinhibition. These observations indicate that the process of derepression and release from transinhibition are specific to the substrates present in the culture medium prior to amino acid starvation.     

Effect of Interleukin-1beta and Dehydroepiandrosterone on the Expression of Lumican and Fibromodulin in Fibroblast-Like Synovial Cells of the Human Temporomandibular Joint

Several epidemiological studies have reported that temporomandibular disorders (TMDs) are more prevalent in women than in men. It has recently been proposed that sex hormones such as estrogen, testosterone and dehydroepiandrosterone (DHEA) are involved with the pathogenesis of TMDs. Although studies have investigated the relationship between estrogen and testosterone and the restoration of TMDs, the relationship between DHEA and TMDs is unknown. The synovial tissue of the temporomandibular joint (TMJ) is made up of connective tissue with an extracellular matrix (ECM) composed of collagen and proteoglycan. One proteoglycan family, comprised of small leucine-rich repeat proteoglycans (SLRPs), was found to be involved in collagen fibril formation and interaction. In recent years, the participation of SLRPs such as lumican and fibromodulin in the internal derangement of TMJ has been suggested. Although these SLRPs may contribute to the restoration of the synovium, their effect is still unclear. The purpose of this study was to investigate the effect of DHEA, a sex hormone, on the expression of lumican and fibromodulin in human temporomandibular specimens and in cultured human TMJ fibroblast-like synovial cells in the presence or absence of the pro-inflammatory cytokine interleukin-1beta (IL-1beta). In the in vivo study, both normal and osteoarthritic (OA) human temporomandibular synovial tissues were immunohistochemically examined. In the in vitro study, five fibroblast-like synoviocyte (FLS) cell lines were established from human TMJ synovial tissue of patients with osteoarthritis. The subcultured cells were then incubated for 3, 6, 12 or 24 h with/without IL-1beta (1 ng/mL) in the presence or absence of DHEA (10 μM). The gene expression of lumican and fibromodulin was examined using the real-time polymerase chain reaction (PCR) and their protein expression was examined using immunofluorescent staining. We demonstrated that the expression of lumican differs from that of fibromodulin in synovial tissue and furthermore, that IL-1beta induced a significant increase in lumican mRNA and immunofluorescent staining in FLS compared to cells without IL-1beta. DHEA plus IL-1beta induced a significant increase in fibromodulin, but not in lumican mRNA, compared to DHEA alone, IL-1beta alone and in the absence of DHEA and IL-1beta. In immunofluorescent staining, weaker fibromodulin staining of FLS cells was observed in cells cultured in the absence of both DHEA and IL-1beta compared to fibromodulin staining of cells cultured with DHEA alone, with DHEA plus IL-1beta, or with IL-1beta alone. These results indicate that DHEA may have a protective effect on synovial tissue in TMJ by enhancing fibromodulin formation after IL-1beta induced inflammation. DHEA enhancement of fibromodulin expression may also exert a protective effect against the hyperplasia of fibrous tissue that TGF-beta1 induces. In addition lumican and fibromodulin are differentially expressed under different cell stimulation conditions and lumican and fibromodulin may promote regeneration of the TMJ after degeneration and deformation induced by IL-1beta.Key words: Temporomandibular joint, dehydroepiandrosterone, lumican, fibromodulin, small leucine rich repeat proteoglycan     

Synovial mesenchymal progenitor derived aggrecan regulates cartilage homeostasis and endogenous repair capacity

Aggrecan is a critical component of the extracellular matrix of all cartilages. One of the early hallmarks of osteoarthritis (OA) is the loss of aggrecan from articular cartilage followed by degeneration of the tissue. Mesenchymal progenitor cell (MPC) populations in joints, including those in the synovium, have been hypothesized to play a role in the maintenance and/or repair of cartilage, however, the mechanism by which this may occur is unknown. In the current study, we have uncovered that aggrecan is secreted by synovial MPCs from healthy joints yet accumulates inside synovial MPCs within OA joints. Using human synovial biopsies and a rat model of OA, we established that this observation in aggrecan metabolism also occurs in vivo. Moreover, the loss of the “anti-proteinase” molecule alpha-2 macroglobulin (A2M) inhibits aggrecan secretion in OA synovial MPCs, whereas overexpressing A2M rescues the normal secretion of aggrecan. Using mice models of OA and cartilage repair, we have demonstrated that intra-articular injection of aggrecan into OA joints inhibits cartilage degeneration and stimulates cartilage repair respectively. Furthermore, when synovial MPCs overexpressing aggrecan were transplanted into injured joints, increased cartilage regeneration was observed vs. wild-type MPCs or MPCs with diminished aggrecan expression. Overall, these results suggest that aggrecan secreted from joint-associated MPCs may play a role in tissue homeostasis and repair of synovial joints.Subject terms: Mesenchymal stem cells, Cartilage, Experimental models of disease     

Borrelia burgdorferi downregulates ICAM-1 on human synovial cells in vitro.

Lyme arthritis following infection with Borrelia burgdorferi (B. burgdorferi) is associated with the presence of bacteria in the joint, but the mechanism of persistent infection in the presence of specific antibodies and lymphocytes remains unknown. To investigate how an infection with B. burgdorferi might influence the local immune response in the joint, we examined the expression of cell adhesion molecules, human leucocyte antigens and inducible nitric oxide synthase (iNOS)-1 and -2 in human synovial cells after infection with B. burgdorferi in vitro. Synovial cells are known to influence the function of local immunologic effector cells and play a key role in the pannus formation of erosive arthritis. It has been shown previously that B. burgdorferi can persist in the cytosol of human synovial cells. The expression of the surface molecules ICAM-1, VCAM-1, HLA-class-I and -class-II and the cytosolic production of iNOS-1 and -2 in synovial cells was measured by flow cytometry for up to 5 days after infection with B. burgdorferi. A significant, lasting downregulation of surface ICAM-1 could be demonstrated on synovial cells, whereas no significant changes were seen in the expression of VCAM-1, HLA-class-I and -II, and of iNOS-1 and -2. To determine the biological significance of this downregulation an in vitro adhesion assay using peripheral blood mononuclear cells was developed. After infection with B. burgdorferi a significantly smaller number of mononuclear cells was adhering to the synovial cell monolayer. Adhesion of peripheral mononuclear cells was shown to be in part mediated by ICAM-1 by using a blocking mononuclear antibody against ICAM-1. Downregulation of ICAM-1 on synovial cells due to infection with B. burgdorferi might suppress the local immunosurveillance and might help the bacteria to persist in joint cells in vivo.     

Prolonged, granulocyte–macrophage colony-stimulating factor-dependent, neutrophil survival following rheumatoid synovial fibroblast activation by IL-17 and TNFalpha

Introduction

A surprising feature of the inflammatory infiltrate in rheumatoid arthritis is the accumulation of neutrophils within synovial fluid and at the pannus cartilage boundary. Recent findings suggest that a distinct subset of IL-17-secreting T-helper cells (T_H17 cells) plays a key role in connecting the adaptive and innate arms of the immune response and in regulating neutrophil homeostasis. We therefore tested the hypothesis that synovial fibroblasts bridge the biological responses that connect T_H17 cells to neutrophils by producing neutrophil survival factors following their activation with IL-17.

Methods

IL-17-expressing cells in the rheumatoid synovium, and IL-17-expressing cells in the peripheral blood, and synovial fluid were examined by confocal microscopy and flow cytometry, respectively. Peripheral blood neutrophils were cocultured either with rheumatoid arthritis synovial fibroblasts (RASF) or with conditioned medium from RASF that had been pre-exposed to recombinant human IL-17, TNFα or a combination of the two cytokines. Neutrophils were harvested and stained with the vital mitochondrial dye 3,3'-dihexyloxacarbocyanine iodide before being enumerated by flow cytometry.

Results

T_H17-expressing CD4⁺ cells were found to accumulate within rheumatoid synovial tissue and in rheumatoid arthritis synovial fluid. RASF treated with IL-17 and TNFα (RASF_IL-17/TNF) effectively doubled the functional lifespan of neutrophils in coculture. This was entirely due to soluble factors secreted from the fibroblasts. Specific depletion of granulocyte–macrophage colony-stimulating factor from RASF_IL-17/TNF-conditioned medium demonstrated that this cytokine accounted for approximately one-half of the neutrophil survival activity. Inhibition of phosphatidylinositol-3-kinase and NF-κB pathways showed a requirement for both signalling pathways in RASF_IL-17/TNF-mediated neutrophil rescue.

Conclusion

The increased number of neutrophils with an extended lifespan found in the rheumatoid synovial microenvironment is partly accounted for by IL-17 and TNFα activation of synovial fibroblasts. T_H17-expressing T cells within the rheumatoid synovium are likely to contribute significantly to this effect.     

Histopathology and molecular pathology of synovial B-lymphocytes in rheumatoid arthritis

B-cells of the rheumatoid synovial tissue are a constant part of and, in some histopathological subtypes, the dominant population of the inflammatory infiltrate, located in the region of tissue destruction. The pattern of B-cell distribution and the relationship to the corresponding antigen-presenting cells (follicular dendritic reticulum cells: FDCs) show a great variety. B-cells may exhibit (i) a follicular organization forming secondary follicles; (ii) follicle-like patterns with irregularly formed FDC networks, and (iii) a diffuse pattern of isolated FDCs. Molecular analysis of immunoglobulin VH and VL genes from human synovial B-cell hybridomas and synovial tissue demonstrates somatic mutations due to antigen activation. The FDC formations in the synovial tissue may therefore serve as an environment for B-cell maturation, which is involved in the generation of autoantibodies. An autoantibody is defined as "pathogenic" if it fulfills the Witebsky-Rose-Koch criteria for classical autoimmune diseases: definition of the autoantibody; induction of the disease by transfer of the autoantibody; and isolation of the autoantibody from the disease-specific lesion. B-cells from rheumatoid synovial tissue show specificity for FcIgG, type II collagen, COMP, sDNA, tetanus toxoid, mitochondrial antigens (M2), filaggrin and bacterial HSPs. The contributions of these antigens to the pathogenesis of RA are still hypothetical. A possible contribution could derive from crossreactivity and epitope mimicry: due to crossreaction, an antibody directed originally against a foreign infectious agent could react with epitopes from articular tissues, perpetuating the local inflammatory process. The characteristic distribution pattern, the localisation within the area of tissue destruction, the hypermutated IgVH and IgVL genes, and their exclusive function to recognize conformation-dependent antigens suggest a central role for B-cells in the inflammatory process of rheumatoid arthritis. Therefore, the analysis of synovial B-cell hybridomas and experimental expression of synovial IgVH and IgVL genes will help to characterise the antigens responsible for the pathogenesis of rheumatoid arthritis.     

Immunolocalization of lamins in the thick nuclear lamina of human synovial cells

While in the great majority of cells the nuclear lamina is not resolved as a distinct structure separating the chromatin from the nuclear envelope, a demonstrable nuclear lamina ("fibrous lamina") of 30 to 300 nm thickness, interposed between the inner nuclear membrane and the peripheral chromatin, is characteristic for certain types of cells of vertebrates and invertebrates. We have examined whether the thick (50-70 nm) fibrous lamina of human synovial cells from patients suffering from rheumatoid arthritis indeed contains the lamins found in the indiscernible lamina structures present in most normal cells. We have observed, by electron microscopic immunolocalization, that both the A and the B type lamins occur throughout the entire nuclear lamina of these cells and that this structure is also resistant to treatments with nucleases and high salt buffers. This shows that the thick fibrous lamina only seen in certain vertebrate cells is compositionally related to the "masked" nuclear lamina of most other cells which usually is identified only upon removal of the adjacent nuclear structures.     

"Matrigenin" activity from bovine bone--I. Partial purification of activity

1. Bovine bone contains an extractable activity which stimulated the synthesis of glycosaminoglycans by bovine synovial, human synovial and mouse 3T3 fibroblastic cells in culture. Human cells were used to develop an assay for purification of the stimulatory activity ("matrigenin" activity) from bovine bone. 2. Partial purification of "matrigenin" activity was achieved by precipitation of the EDTA extract at pH 3.5 and Sepharose CL-6B chromatography in 4 M guanidinium HCl. Dissociative conditions were necessary to prevent aggregation. 3. On SDS-polyacrylamide gel electrophoresis the activity ran with a mobility equivalent to a Mr = 27,500 and could be recovered from the SDS gels.     

Gap junctions in human synovial cells and tissue

Our objective was to establish the existence of intercellular communication through gap junctions in synovial lining cells and in primary and passaged cultures of human synovial cells. Communication between cells was assessed using the nystatin perforated-patch method, fluorescent dye transfer, immunochemistry, transmission electron microscopy, and immunoblotting. Functional gap junctions were observed in primary and passaged cultures and were based on measurements of the transient current response to a step voltage. The average resistance between cells in small aggregates was 300 +/- 150 MOmega. Gap junctions were also observed between synovial lining cells in tissue explants; the size of the cell network in synovial tissue was estimated to be greater than 40 cells. Intercellular communication between cultured cells and between synovial lining cells was confirmed by dye injection. Punctate fluorescent regions were seen along intercellular contacts between cultured cells and in synovial membranes in cells and tissue immunostained for connexin43. The presence of the protein was verified in immunoblots. Regular 2-nm intermembrane gap separations characteristic of gap junctions were seen in transmission electron micrographs of synovial biopsies. The results showed that formation of gap-junction channels capable of mediating ionic and molecular communication was a regular feature of synovial cells, both in tissue and in cultured cells. The gap junctions contained connexin43 protein and perhaps other proteins. The physiological purpose of gap junctions in synovial cells is unknown, but it is reasonable to anticipate that intercellular communication serves some presently unrecognized function.     

The expression of matrix metalloproteinases and their inhibitors by pig synovial cells and their regulation by combinations of cytokines and growth factors

• 1.1. Pig synovial fibroblasts in culture were studied to determine if they were an easily reproducible model system for studying the actions of cytokines and growth factors on human synovial cells. The biochemical analyses were conducted by activity assays, enzymography and Northern blot.
• 2.2. Human recombinant interleukin-1α, basic fibroblast growth factor and transforming growth factor-β₁ were studied in combinations because of their known involvement in controlling tissue remodelling.
• 3.3. The response of pig fibroblasts to these agents, in terms of the expression of matrix metalloproteinases (collagenase, gelatinase and stromelysin) and their inhibitors (TIMPs), show that they behave similarly enough to human cells for use when supplies of human primary cells are unavailable.

     

Effects of retinol and dexamethasone on cytokine-mediated control of metalloproteinases and their inhibitors by human articular chondrocytes and synovial cells in culture

Human articular chondrocytes in culture produced large amounts of specific mammalian collagenase, gelatinase and proteoglycanase when exposed to dialysed supernatant medium derived from cultured human blood mononuclear cells (mononuclear cell factor) or to conditioned medium, partially purified by fractionation with ammonium sulphate (60–90% fraction), from cultures of human synovial tissue (synovial factor). Human chondrocytes and synovial cells also released into culture medium an inhibitor of collagenase of apparent molecular weight about 30 000, which appeared to be similar to the tissue inhibitor of metalloproteinases synthesised by tissues in culture. The amounts of free collagenase inhibitor were reduced in culture media from chondrocytes or synovial cells exposed to mononuclear cell factor or synovial factor. While retinol inhibited the production of collagenase brought about by mononuclear cell factor or synovial factor, it restored the levels of inhibitor, which were reduced in the presence of mononuclear cell factor or synovial factor. Dexamethasone markedly reduced the production of collagenase by synovial cells, while only partially inhibiting factor-stimulated collagenase production by chondrocytes. Addition of puromycin as an inhibitor of protein synthesis reduced the amounts of both collagenase and inhibitor to control or undetectable levels.     

The effect of solubilized bone matrix fractions from different mammalian species on glycosaminoglycan synthesis by cultured fibroblasts

Human and bovine bone matrices were extracted with salt solutions of different composition and the extracts tested for stimulation of incorporation of radioactivity from [3H]glucosamine and [35S]SO4 into the hyaluronic acid and chondroitin sulfate of the cell pellet, the cell surface and the medium fractions of human synovial cells in culture. Stimulatory activity was extracted with a solution of 0.3 M EDTA in 2.5 M NaCl from bovine but not human bone. Subsequent extraction of the residues with 4 M guanidinium hydrochloride yielded activity from both matrices. A major stimulation of incorporation of radioactivity was observed in the cell surface fractions. Human synovial cells constitute a more sensitive assay system for the stimulatory activity than rabbit synovial cells.     

Prolonged, granulocyte-macrophage colony-stimulating factor-dependent, neutrophil survival following rheumatoid synovial fibroblast activation by IL-17 and TNFalpha

Introduction

A surprising feature of the inflammatory infiltrate in rheumatoid arthritis is the accumulation of neutrophils within synovial fluid and at the pannus cartilage boundary. Recent findings suggest that a distinct subset of IL-17-secreting T-helper cells (T_H17 cells) plays a key role in connecting the adaptive and innate arms of the immune response and in regulating neutrophil homeostasis. We therefore tested the hypothesis that synovial fibroblasts bridge the biological responses that connect T_H17 cells to neutrophils by producing neutrophil survival factors following their activation with IL-17.

Methods

IL-17-expressing cells in the rheumatoid synovium, and IL-17-expressing cells in the peripheral blood, and synovial fluid were examined by confocal microscopy and flow cytometry, respectively. Peripheral blood neutrophils were cocultured either with rheumatoid arthritis synovial fibroblasts (RASF) or with conditioned medium from RASF that had been pre-exposed to recombinant human IL-17, TNFα or a combination of the two cytokines. Neutrophils were harvested and stained with the vital mitochondrial dye 3,3''-dihexyloxacarbocyanine iodide before being enumerated by flow cytometry.

Results

T_H17-expressing CD4⁺ cells were found to accumulate within rheumatoid synovial tissue and in rheumatoid arthritis synovial fluid. RASF treated with IL-17 and TNFα (RASF_IL-17/TNF) effectively doubled the functional lifespan of neutrophils in coculture. This was entirely due to soluble factors secreted from the fibroblasts. Specific depletion of granulocyte–macrophage colony-stimulating factor from RASF_IL-17/TNF-conditioned medium demonstrated that this cytokine accounted for approximately one-half of the neutrophil survival activity. Inhibition of phosphatidylinositol-3-kinase and NF-κB pathways showed a requirement for both signalling pathways in RASF_IL-17/TNF-mediated neutrophil rescue.

Conclusion

The increased number of neutrophils with an extended lifespan found in the rheumatoid synovial microenvironment is partly accounted for by IL-17 and TNFα activation of synovial fibroblasts. T_H17-expressing T cells within the rheumatoid synovium are likely to contribute significantly to this effect.