Synovial Neutrophils Research Articles

Role of C3a receptors, C5a receptors, and complement protein C6 deficiency in collagen antibody-induced arthritis in mice.

The complement system, especially the alternative pathway, plays essential roles in the induction of injury in collagen Ab-induced arthritis (CAIA) in mice. The goal of the current study was to directly compare the roles of receptors for C3a and C5a, as well as the membrane attack complex, as effector mechanisms in the pathogenesis of CAIA. Clinical disease activity in C3aR(-/-), C5aR(-/-), and C6-deficient (C6-def) mice was decreased by 52, 94, and 65%, respectively, as compared with wild-type mice. Decreases in histopathologic injury as well as in IgG and C3 deposition paralleled the clinical disease activity. A decrease in the percentage of synovial neutrophils was observed in C3aR(-/-), C5aR(-/-), and C6-def mice, and a decrease in macrophages was observed in C3aR(-/-) and C5aR(-/-), but not in C6-def, mice. Synovial mRNA obtained by laser capture microdissection exhibited a decrease in TNF-α in C5aR(-/-) mice and in IL-1β in both C5aR(-/-) and C6-def mice, whereas C3aR(-/-) mice demonstrated no change in either cytokine. Our findings show that absent C3aR-, C5aR-, or membrane attack complex-initiated effector mechanisms each decrease susceptibility to CAIA, with clinical effects most pronounced in C5aR-deficient mice. Although the absence of C3aR, C5aR, or C6 led to differential deficiencies in effector mechanisms, decreased proximal joint IgG and C3 deposition was common to all three genotypes in comparison with wild-type mice. These data suggest the existence of positive-feedback amplification pathways downstream of all three effectors that promote additional IgG deposition and C3 activation in the joint.

The effect of blood contamination on equine synovial fluid analysis

Based on a systemic complete blood count and a synovial fluid sample, to develop a mathematical model enabling the approximation of corrected values for synovial fluid white blood cell (WBC) count, neutrophil percentage, and total protein concentration in samples of synovial fluid that were contaminated by blood. Peripheral venous blood and synovial fluid samples were obtained from ten horses. A pooled synovial fluid sample from each horse was separated into 2 mL aliquots, which were subsequently contaminated with a known percentage of autogenous blood (0 to 50% of the synovial fluid volume). A complete blood count, packed cell volume, total protein (TP) concentration, and differential cytological examination were performed in all the synovial fluid and venous blood samples. Regression analysis was used to generate a model to calculate non-contaminated synovial WBC count, TP concentration and synovial neutrophil percentage. Using a further five horses these models were applied in blinded fashion to contaminated synovial fluid samples. Calculated values were compared to non-contaminated measured values. Model results for synovial WBC count and TP concentration were not significantly different from measured values. Calculated neutrophil percentage of synovial fluid WBC was a mean of 6.6% higher than measured values and was significantly different. There was no effect of the severity of contamination (as a percentage of volume) on the ability of the models to predict any of the outcome variables. It is possible to calculate non-contaminated synovial fluid WBC and TP values, but not neutrophil percentage, from heavily contaminated samples. Further study would allow for improved prediction, validation and extrapolation to a wider horse population.

Morphological study of synovial changes in two-stage reconstructions of the infected hip and knee arthroplasties

ObjectivesTo study the morphological changes of the regenerating synovium in two-stage revision arthroplasty, which is the gold standard for treatment of periprosthetic joint infection.DesignThe authors analysed a series of synovial...

Neutrophil Extracellular Trap Formation Is Associated with IL-1β and Autophagy-Related Signaling in Gout

BackgroundGout is a prevalent inflammatory arthritis affecting 1–2% of adults characterized by activation of innate immune cells by monosodium urate (MSU) crystals resulting in the secretion of interleukin-1β (IL-1β). Since neutrophils play a major role in gout we sought to determine whether their activation may involve the formation of proinflammatory neutrophil extracellular traps (NETs) in relation to autophagy and IL-1β.Methodology/Principal FindingsSynovial fluid neutrophils from six patients with gout crisis and peripheral blood neutrophils from six patients with acute gout and six control subjects were isolated. MSU crystals, as well as synovial fluid or serum obtained from patients with acute gout, were used for the treatment of control neutrophils. NET formation was assessed using immunofluorescence microscopy. MSU crystals or synovial fluid or serum from patients induced NET formation in control neutrophils. Importantly, NET production was observed in neutrophils isolated from synovial fluid or peripheral blood from patients with acute gout. NETs contained the alarmin high mobility group box 1 (HMGB1) supporting their pro-inflammatory potential. Inhibition of phosphatidylinositol 3-kinase signaling or phagolysosomal fusion prevented NET formation, implicating autophagy in this process. NET formation was driven at least in part by IL-1β as demonstrated by experiments involving IL-1β and its inhibitor anakinra.Conclusions/SignificanceThese findings document for the first time that activation of neutrophils in gout is associated with the formation of proinflammatory NETs and links this process to both autophagy and IL-1β. Modulation of the autophagic machinery may represent an additional therapeutic study in crystalline arthritides.

Evaluation of the Bcl-2 family antagonist ABT-737 in collagen-induced arthritis

Therapeutic manipulation of cellular apoptosis holds great promise for malignant and potentially nonmalignant diseases. A relative resistance to apoptosis in RA synovium is associated with increased expression of prosurvival Bcl-2 family members. In this study, we demonstrate that treatment of DBA/1 mice, prior to the onset of CIA with ABT-737, a BH3 mimetic targeting Bcl-2, Bcl-w, and Bcl-x(L), ameliorated disease development. In contrast, treatment of mice with ABT-737 in established CIA did not alter the course of disease. ABT-737 induced lymphopenia, however pathogenic lymphoid populations in CIA mice were less affected, as shown by relatively normal T and B cell responses to CII. Naïve lymphocytes were highly sensitive to apoptosis after culture with ABT-737, but synovial macrophages and neutrophils were not. Mcl-1 was detected in synovial monocyte/macrophages and neutrophils and strikingly, its expression, rather than Bcl-2 and Bcl-x(L), increased in the affected paws and lymphoid organs of mice with CIA. These observations implicate Mcl-1, which is not targeted by ABT-737, in the survival of inflammatory cells in established CIA and suggest that antagonism of Mcl-1 may be more effective in diseases such as RA.

Levels of lectin pathway proteins in plasma and synovial fluid of rheumatoid arthritis and osteoarthritis

The innate immune system contributes to the development of rheumatoid arthritis (RA). A potent contributor to such processes is the complement system. The complement system is known to be activated in the inflammatory phases of osteoarthritis (OA). The lectin pathway of the complement system is activated through the recognition of pathogens or altered self-structures by mannan-binding lectin (MBL) or one of the three ficolins in collaboration with MBL-associated serine proteases (MASPs). We assessed the lectin pathway in plasma and synovial fluid (SF) of 27 RA patients and 30 OA patients by measuring MBL, MASP-2, MASP-3, M-ficolin, and H-ficolin. The concentration for all 5 proteins was significantly higher in plasma than in SF (P < 0.001) and the concentration in paired plasma and SF samples correlated in both RA and OA (significance levels between <0.001 and 0.02). The ratio of SF/plasma concentration was for all proteins significantly elevated in RA compared with OA patients (all P < 0.001). The M-ficolin concentration correlated with the neutrophils in both plasma (P = 0.01) and SF (P < 0.001) of RA, and in plasma of 78 controls (P = 0.03). To our knowledge, this is the first report on these proteins in SF, except for MBL where our results are in contrast to the one previous publication. The results support an important physiological role of the neutrophils in determining the M-ficolin levels in both RA and healthy adults. We suggest that quantifications of white blood cells should be included in future clinical investigations of M-ficolin.

Synovial fluid cells in proteoglycan-induced arthritis contain a myeloid-derived suppressor population (167.21)

Abstract Joint damage in rheumatoid arthritis (RA) is largely mediated by synovial fluid (SF) neutrophils. In proteoglycan (PG)-induced arthritis (PGIA), an autoimmune mouse model of RA, SF cells show high expression of the myeloid lineage markers CD11b and Gr-1. The effects of this neutrophil-like SF cell population on the activation of dendritic cells (DCs) or T cells have not been studied in RA or its animal models. We co-cultured SF cells of PGIA mice with bone marrow-derived DCs, and found that levels of MHC II and CD86 expression by DCs decreased significantly in the presences of SF cells. To determine if SF cells also reduced the antigen (Ag)-presenting capacity of the DCs, we added naïve PG-specific TCR transgenic T cells and cognate Ag to the co-cultures. T cells cultured in the presence of SF cells and Ag-loaded DCs showed significant decreases in CD69 expression and proliferation compared to T cells cultured with Ag-loaded DCs only. Arginase-1, a marker of myeloid-derived suppressor cells (MDSCs) was upregulated at both transcriptional and protein levels in SF cells. Expression and activity of arginase-1 were also elevated in spleen cells of arthritic mice as compared to spleen cells of naïve non-arthritic mice. Our results suggest that SF cells in the arthritic joints and spleens of mice with PGIA contain a population of MDSCs, which might have the capacity to suppress autoreactive T cell responses and thus accelerate the resolution of joint inflammation.

Macrophage migration inhibitory factor regulates neutrophil chemotactic responses in inflammatory arthritis in mice

Macrophage migration inhibitory factor (MIF) facilitates multiple aspects of inflammatory arthritis, the pathogenesis of which has been significantly linked to the activity of neutrophils. The effects of MIF on neutrophil recruitment are unknown. This study was undertaken to investigate the contribution of MIF to the regulation of neutrophil chemotactic responses. K/BxN serum-transfer arthritis was induced in wild-type (WT), MIF(-/-) , and monocyte chemotactic protein 1 (MCP-1; CCL2)-deficient mice as well as in WT mice treated with monoclonal antibodies to cytokine-induced neutrophil chemoattractant (anti-KC). Leukocyte trafficking in vivo was examined using intravital microscopy, and neutrophil function in vitro was examined using migration chambers and assessment of MAP kinase activation. K/BxN serum-transfer arthritis was markedly attenuated in MIF(-/-) mice, with reductions in the clinical and histologic severity of arthritis and the synovial expression of KC and interleukin-1. Arthritis was also reduced by anti-KC antibody treatment, but not in MCP-1-deficient mice. In vivo, neutrophil recruitment responses to KC were reduced in MIF(-/-) mice. Similarly, MIF(-/-) mouse neutrophils exhibited reduced chemotactic responses to KC in vitro, despite displaying unaltered chemokine receptor expression. Reduced chemotactic responses of MIF(-/-) mouse neutrophils were associated with reduced phosphorylation of p38 and ERK MAP kinases. These findings suggest that MIF promotes neutrophil trafficking in inflammatory arthritis via facilitation of chemokine-induced migratory responses and MAP kinase activation. Therapeutic MIF inhibition could limit synovial neutrophil recruitment.

Viability and cell death of synovial fluid neutrophils as diagnostic biomarkers in equine infectious joint disease: A pilot study

Synovial fluid samples from culture-confirmed infected joints ( n = 13), joints with pronounced non-infectious synovitis ( n = 11) and healthy joints ( n = 14) were collected from 24 equine patients and seven slaughterhouse horses. The samples from the joints with non-infectious synovitis and healthy joints served as negative controls. After isolation, counting and identification of neutrophils, the percentage viability, and the proportion apoptotic and necrotic neutrophils were determined by flow cytometry. Viability was significantly higher in infected samples compared to the controls. A significant difference in cell death type was observed, with apoptosis predominating in infected joints, and necrosis being more present in joints with pronounced non-infectious synovitis and healthy joints. The results of this pilot study suggest that flow cytometric analysis of neutrophil viability and cell death dynamics may assist the discrimination between infected and non-infected joints.

Analysis of Synovial Fluid in Culture-Negative Samples of Suspicious Periprosthetic Infections

Synovial fluid analysis can help to rule out a periprosthetic infection. The goal of this study was to evaluate the prognostic significance of synovial fluid suspicious for a periprosthetic infection given the synovial leukocyte count and percent of neutrophils. From August 2006 to November 2008, patients who had synovial fluid aspirated for painful knees and elevated C-reactive protein levels after total knee arthroplasty but revealed no growth of any microorganism were retrospectively evaluated by medical record review. Mean follow-up period was 827.7±250.6 days from the date of joint aspiration results. The optimal cut-off values for synovial leukocyte counts and percent neutrophils were determined using receiver operating characteristic curves. From a total of 56 patients, 14 were classified as having poor results. The remaining 42 patients had an uneventful clinical course with a stable prosthesis on radiographs without specific therapy during the follow-up period. The receiver operating characteristic curve showed that a synovial leukocyte count of 3600/uL had an 86% sensitivity and 79% specificity; with 89% synovial neutrophils, the sensitivity was 72% and specificity 62%. The synovial fluid leukocyte count and percent neutrophils were significant predictors of outcome in patients with cultures that were negative but suspicious for periprosthetic infections.

Prevention of the progression of adjuvant induced arthritis by oral supplementation of Indian fresh water mussel (Lamellidens marginalis) aqueous extract in experimental rats

Mussel is well accepted as food all over India. Beside for its nutritive value, people residing in Kosi river basin, Bihar, India, consume a preparation of soup, made from the footpad of molluscan species, with the belief that it gives relief from signs and symptoms of joint pain and related problems. This study was designed to explore the preventive activity of Indian fresh water mussel (Lamellidens marginalis) aqueous extract oral supplementation in experimental arthritis model. Arthritis was induced in male albino rats by intradermal injection of Freund's complete adjuvant in right hind footpad. Lamellidens marginalis extract (LME1, 500 mg/kg/day and LME2, 1 g/kg/day) peroral supplementation started from the 1st day after adjuvant injection and was continued for the subsequent 13 days. Severity of arthritis was evaluated from paw diameter, ankle diameter, paw weight, urinary hydroxyproline, glucosamine level, serum interleukin-1β, IL6, IL10, CINC1, TNFα level, lysosomal enzyme levels and from histopathological assessment. Lamellidens marginalis extract supplementation significantly (p<0.05) decreased paw diameter, ankle diameter, and paw weight in treated groups (LME1, 500 mg/kg/day and LME2, 1 g/kg/day) as compared with arthritic group. Urinary hydroxyproline, glucosamine level, serum IL1β, IL6, CINC1, TNFα, IL10 and lysosomal enzyme levels were restored significantly (p<0.05) in treated groups (LME1, 500 mg/kg/day and LME2, 1 g/kg/day) as compared to arthritic group. Synovial membrane damage and neutrophil infiltration in histopathological examination was restored significantly by LME supplementation as compared to arthritic group. Thus, it might be concluded that experimental animals supplemented with Lamellidens marginalis extract were protected against the severity of disease progression in adjuvant induced arthritis.

Possible Protective Role of Chloramphenicol in TSST-1 and Coagulase-Positive Staphylococcus aureus-Induced Septic Arthritis with Altered Levels of Inflammatory Mediators

Chloramphenicol is mostly used against coagulase-negative Staphylococcus aureus, and its protective role against coagulase-positive S. aureus is not well studied. In our study, arthritis was induced in mice by S. aureus (Apollo Gleneagles 33 (AG-33) or American Type Culture Collection 25923 (ATCC-25923)) infection. Chloramphenicol was administered after 2 h of infection. Mice were killed at 1, 3, 5 days post-infection. Mice inoculated with pathogenic Staphylococci (AG-33) expressing coagulase and Toxic shock syndrome toxin-1 (TSST-1), displayed severe arthritis with enhanced bacterial burden in the spleen, cytokine production in serum and synovial tissue, neutrophil recruitment, and cyclooxegenase-2 expression in synovial tissue compared with ATCC-25923-infected groups. Severity of arthritis was regulated by chloramphenicol treatment. Our study suggests that alteration in the inflammatory cytokine levels and pronounced production of cyclooxygenase-2 play important roles in progression of arthritis which is regulated by application of chloramphenicol.

Deficiency of CXCR2, but not other chemokine receptors, attenuates autoantibody-mediated arthritis in a murine model.

Chemokines coordinate leukocyte trafficking in homeostasis and during immune responses. Prior studies of their role in arthritis have used animal models with both an initial adaptive immune response and an inflammatory effector phase. We undertook analysis of chemokines and their receptors in the effector phase of arthritis using the K/BxN mouse serum-transfer model. A time-course microarray analysis of serum-transferred arthritis was performed, examining ankle tissue, synovial fluid, and peripheral blood leukocytes. Up-regulation of chemokines was confirmed by quantitative reverse transcriptase-polymerase chain reaction. The functional relevance of chemokine induction was assessed by transferring serum into mice deficient in CCR1-7, CCR9, CXCR2, CXCR3, CXCR5, CX(3)CR1, CCL2, or CCL3. Further mechanistic analysis of CXCR2 involved treatment of arthritic mice with a CXCR2 antagonist, bone marrow (BM) cell transfers with CXCR2(+/-) and CXCR2(-/-) donors and recipients, flow cytometry of synovial cells, and competition experiments measuring enrichment of CXCR2-expressing neutrophils in arthritic joints of mice with mixed CXCR2(+/+) and CXCR2(-/-) BM cells. Gene expression profiling revealed up-regulation of the CXCR2 ligands CXCL1, CXCL2, and CXCL5 in the joint in parallel with disease activity. CXCR2(-/-) mice had attenuated disease relative to CXCR2(+/-) littermates, as did mice receiving the CXCR2 inhibitor, while deficiency of other chemokine receptors did not affect arthritis severity. CXCR2 was required only on hematopoietic cells and was widely expressed on synovial neutrophils. CXCR2-expressing neutrophils were preferentially recruited to arthritic joints in the presence of CXCR2-deficient neutrophils. CXCR2 (but not other chemokine receptors) is critical for the development of autoantibody-mediated arthritis, exhibiting a cell-autonomous role in neutrophil recruitment to inflamed joints.

Lack of evidence for association of two functional SNPs of CHI3L1 gene (HC-gp39) with rheumatoid arthritis

CHI3L1 gene encodes for a glycoprotein (HC-gp39 or YKL40) secreted by synovial fibroblasts, macrophages, neutrophil granulocytes and chondrocytes. Its expression is under the control of NF-kB. It is regarded as an acute phase protein, and its levels are significantly elevated in rheumatic diseases. Furthermore, HC-gp39 has been shown to be recognized by autoreactive T cells in rheumatoid arthritis. In the present study, we have examined two functional variants of the promoter region of CHI3L1 gene (CHI3L1-1 (rs4950928) and CHI3L1-2 (rs10399931) that have been reported earlier to be associated with schizophrenia and sarcoidosis. We used TaqMan allelic discrimination assays to study the genotypes of Hungarian patients with rheumatoid arthritis (n=182) and of healthy controls (n=194). No significant association of the investigated SNPs with the disease was found. Here we report that CHI3L1 SNPs, shown to be involved in the predisposition of schizophrenia, are not associated with rheumatoid arthritis.

Immunologic rheumatic disorders

We provide the basics for clinicians who might be called on to consider the diagnosis of diseases such as systemic lupus erythematosus (SLE) or rheumatoid arthritis (RA) in their practice. We will emphasize clinical recognition and first-line laboratory testing. Only characteristics of the classic rheumatic inflammatory diseases (ie, RA, seronegative spondyloarthropathy, SLE, antiphospholipid syndrome, Sjögren syndrome, scleroderma, and polymyositis/dermatomyositis) will be covered. In the past decade, treatment for RA and seronegative spondyloarthropathy has substantially improved. Their treatment has been revolutionized by the use of methotrexate and, more recently, TNF inhibitors, T-cell costimulation modulators, and B-cell depletion. The goal of RA treatment today is to induce a complete remission as early as possible in the disease process, with the mantra being "elimination of synovitis equals elimination of joint destruction." The hope is that if the major mediators of Sjögren syndrome, SLE, or scleroderma can be identified and then blocked, as in the example of TNF inhibitors in patients with RA, more specific treatments will become available. Thus RA has become an excellent model of this evolving paradigm. Through the identification of major mediators in its pathogenesis, novel and highly efficacious therapeutic agents have been developed.

Release of the Soluble Urokinase-Type Plasminogen Activator Receptor (suPAR) by Activated Neutrophils in Rheumatoid Arthritis

Soluble form of the urokinase-type plasminogen activator receptor (suPAR) is markedly increased in biological fluids during different inflammatory conditions. It has previously been observed that the highest suPAR concentrations in inflammatory exudates tend to be associated with the presence of high number of neutrophils. Guided by this observation and our recent finding that activated neutrophils release suPAR we investigated whether neutrophils can be a source of suPAR during the inflammatory response in vivo. To address this question we conducted the comparative analysis of neutrophils isolated from the paired samples of synovial fluid (SF) and peripheral blood (PB) of rheumatoid arthritis patients. Freshly isolated SF neutrophils released significantly (p < 0.01) higher amounts of suPAR compared with PB neutrophils. We demonstrated that neutrophils from both sources release predominantly the truncated D2D3 form of suPAR. Migration of formyl peptide receptor-like 1 (FPRL1)-transfected human embryonic kidney (HEK) 293 cells toward the supernatants harvested from in vitro cultured SF neutrophils was significantly diminished when D2D3 form of suPAR was immunodepleted from the supernatants. Taken together, these data demonstrate that neutrophils, first, contribute to or are responsible for the generation of the increased suPAR levels during the inflammatory response and, second, release the chemotactically active form of suPAR that might be involved in the recruitment of formyl peptide receptors-expressing leukocytes into the inflamed tissues.

Surface RANKL of Toll-like receptor 4–stimulated human neutrophils activates osteoclastic bone resorption

AbstractInflammatory bone loss in septic and inflammatory conditions is due to increased activity of osteoclasts that requires receptor activator of NF-kappa B-ligand (RANKL). Neutrophils are the predominant infiltrating cells in these conditions. Although disease severity is linked to neutrophils, their role in evolution of bony lesions is not clear. We show that lipopolysaccharide (LPS), a toll-like receptor 4 ligand, up-regulated the expression of membrane RANKL in human blood neutrophils and murine air pouch–derived neutrophils. LPS-activated human and murine neutrophils, cocultured with human monocyte-derived osteoclasts and RAW 264.7 cells, respectively, stimulated bone resorption. Transfection of PLB-985 neutrophil-like cells with RANKL antisense RNA reduced osteoclastogenesis. Synovial fluid neutrophils of patients with exacerbation of rheumatoid arthritis strongly expressed RANKL and activated osteoclastogenesis in coculture systems. Osteoprotegerin, the RANKL decoy receptor, suppressed osteoclast activation by neutrophils from these different sources. Moreover, direct cell-cell contact between neutrophils and osteoclasts was visualized by confocal laser microscopy. Activation of neutrophil membrane-bound RANKL was linked to tyrosine phosphorylation of Src-homology domain–containing cytosolic phosphatase 1 with concomitant down-regulation of cytokine production. The demonstration of these novel functions of neutrophils highlights their potential role in osteoimmunology and in therapeutics of inflammatory bone disease.

Effect of Repeated Arthrocentesis on Cytologic Analysis of Synovial Fluid in Dogs

Serial arthrocentesis and synovial fluid examination can be used to monitor treatment efficacy in immune-mediated polyarthritis (IMPA), but whether this procedure induces inflammation that interferes with test result interpretation is unknown. The aim of this study was to determine the effect of repeated arthrocentesis on synovial fluid cytology in healthy dogs. Nine healthy client-owned dogs. Prospective study. Arthrocentesis was performed under sedation on 4 joints (both carpi, 1 tarsus, 1 stifle) on each dog every 3 weeks, a total of 4 times. Automated cell counts were done on stifle fluid, smears were made, and differential cell counts done on smears from all joints. Slides were evaluated microscopically for erythrocyte numbers, total nucleated cell count, differential cell count, and cell morphology. Data were analyzed by 2-way analysis of variance. A total of 144 synovial fluid samples were examined. Repeated arthrocentesis was not associated with increases in synovial fluid neutrophil numbers. Mild mononuclear inflammation was detected in 13 samples from 6 dogs. Serial arthrocentesis at 3-week intervals can rarely be associated with mild mononuclear joint inflammation, but does not appear to induce neutrophilic inflammation, at least in healthy dogs, and can be useful to monitor treatment response in canine IMPA.

Proteinase activated receptor (PAR) involvement in mediating arthritis pain and inflammation

Proteinase activated receptors (PARs) are a newly identified family of G-protein-coupled receptors that are activated by proteinases released into tissues during inflammation. Evidence has accumulated which shows that PARs can exhibit both anti- and pro-inflammatory properties in different organ systems. During arthritis, proteinases are known to be released into the joint where they orchestrate tissue remodelling and degeneration. By activating PARs, these proteinases have the potential to modulate joint inflammation and pain by a highly targeted and selective receptor pathway. The recent identification of PARs on synovial fibroblasts, neutrophils, macrophages and mast cells is further evidence that this intriguing family of receptors could play a role in the pathogenesis and symptoms of arthritis. This review article provides an overview of the current knowledge regarding PARs and their emerging role in arthritis.

Kallikreins, kininogens and kinin receptors on circulating and synovial fluid neutrophils: role in kinin generation in rheumatoid arthritis

Neutrophils traffic into and have the capacity to generate kinins in SF of RA patients. The aim of this study was to assess the expression of kallikreins, kininogens and kinin receptors in circulating and SF neutrophils, as well as synovial tissue of RA patients, and to assess kinin generation in SF. Neutrophils were isolated from blood and SF of RA patients and blood of healthy volunteers. Expression of kallikreins, kininogens and kinin receptors in neutrophils and synovial tissue was assessed by immunocytochemistry using specific antibodies, with visualization by brightfield and confocal microscopy. Levels of basal and generated kinins in SF of RA patients were measured by ELISA. Kinin labelling was significantly reduced, indicating the loss of the kinin moiety from kininogen on circulating (P < 0.001) and SF neutrophils (P < 0.05) of RA patients. Immunolabelling of tissue kallikrein was also decreased, whereas kinin B(1) and B(2) receptor expression was increased in circulating and SF neutrophils of RA patients. Immunolabelling of kallikreins and kinin receptor proteins was similar in RA and normal synovial tissues. The basal kinin level in SF of RA patients was 5.7 +/- 6.1 ng/ml and the mean concentration of kinins generated in vitro was 80.6 +/- 56.3 ng/ml. The capacity for kinin generation was positively correlated with measures of disease activity. Kallikrein-kinin proteins on neutrophils play an important role in kinin generation and the pathophysiology of RA. Specific kallikrein and kinin receptor antagonists may be useful as IA therapies for inflamed joints.