The immunogenicity of seasonal and pandemic influenza vaccination in autoimmune inflammatory rheumatic patients-a 6-month follow-up prospective study.

INTRODUCTION: Influenza may cause severe complications in patients with autoimmune inflammatory rheumatic disease (AIRD), to whom vaccinations are especially recommended. However, AIRD patients require cautious scrutiny of immunogenicity as they might exhibit poor antibody response to vaccination, especially when taking immunomodulatory medications. AIM: The aim was to determine immunogenicity of seasonal and pandemic influenza vaccine in AIRD patients, its timeline/persistence, and influence of medications on immune response. METHODS: One hundred and thirty-seven AIRD and 54 healthy controls were vaccinated with trivalent seasonal influenza. After 3-5 weeks, 15 healthy controls and 93 AIRD were vaccinated with pandemic influenza vaccine, and 63 of patients were vaccinated a second time after 3-5 weeks. Sera were collected before vaccination, 18-90 days after each vaccination, and more than 180 days after the last vaccination. The immune response was measured using hemagglutination inhibition (HI) assay and IgG/IgA antibodies against influenza A/B with ELISA. RESULTS: Our findings indicate that following vaccination with seasonal influenza vaccine, seroprotection, seroresponse, and change in geometric mean titers (GMT) in AIRD patients was not compromised compared to healthy. Similarly, we report for pandemic influenza vaccination little added benefit of the second dose. We confirm lowest increase in HI titer in rituximab-treated AIRD compared to other medications. Vaccination largely tilts the balance from negative ELISA A IgG and IgA titers to positive titers in seasonal H1N1 seroresponsive AIRD patients and controls. A significant decrease in HI GMT and seroprotection was observed only in AIRD at > 180 days after vaccination highlighting an absent persistence of immunogenic response in AIRD patients. Due to high initial HI titers for influenza vaccine, we foresee their benefit in personalized medicine in the future. CONCLUSION: Influenza vaccination is immunologically active for AIRD, with little value of the second dose of the pandemic vaccine and further scrutiny on persistence of immune response to vaccine in AIRD is needed.

Autoantibodies against dsDNA measured with nonradioactive Farr assay-an alternative for routine laboratories.

Autoantibodies against dsDNA are utilized for the diagnosis and prognosis of SLE as they are highly specific and correlate with disease activity/renal involvement. However, different detection methods are used in routine diagnostic laboratories. Farr radioimmunoassay (Farr-RIA) has been designated as the preferred method, since it provides very specific and at the same time quantitative results, enabling follow-up of level variations over time. Using intercalating fluorescent dsDNA dye would enable all the benefits of Farr-RIA without the radioactive material and organic solvents. To develop a modified fluorescent Farr method (Farr-FIA) and compare it to the classical Farr-RIA in regard to laboratory parameters, as well as clinical utility. Assays were tested on sera of 70 SLE patients, 78 other autoimmune patients, and 145 healthy blood donors. DNA for Farr-FIA was isolated from healthy donor, for Farr-RIA, 14C-labeled dsDNA from E. coli was used and mixed with sera in borate-buffered saline, followed by precipitation with saturated ammonium sulfate solution and centrifugation. The supernatant (S) was separated from the precipitate (P), and content of dsDNA was measured with PicoGreen (Invitrogen) in Farr-FIA or radioactive isotope in scintillation solution in Farr-RIA. The results were calculated as a ratio (P-S)/(P+S). Farr-FIA has a diagnostic sensitivity of 53% and diagnostic specificity of 100% (ROC AUC 0.781). Good correlation and agreement were shown between Farr-RIA and Farr-FIA. Also, there is good correlation between Farr-FIA and SLEDAI, comparable to that of Farr-RIA. Farr-FIA differs from Farr-RIA in the changed detection system yielding comparable results and thus could represent a nonradioactive replacement for Farr-RIA.

Evaluating the utility of autoantibodies for disease activity and relapse in giant cell arteritis.

In patients with giant cell arteritis (GCA), autoantibodies against cytoskeletal elements, cardiolipin, neutrophil cytoplasmic antigens, ferritin, endothelial and smooth muscle cells have been reported, however no updated reviews are available evaluating their clinical utility. Methodology of detection is important, especially for quantitative assays, e.g. enzyme-linked immunoassays and multiplex beadbased immunoassays, while semiquantitative assays contribute valuable data on isoforms, epitope mapping and cellular localization. Most studies to date reporting on antiphospholipid antibodies in GCA have focused on anti-cardiolipin antibodies (aCL), while the highest prevalence of autoantibodies in GCA patients was reported for the anti-N-terminal peptides of the ferritin heavy chain (92%). Antineutrophil cytoplasmic antibodies were shown to be present in only a small percentage of GCA patients, decreasing after therapy, however in combination with aCL and antibodies against peptides of N-terminal ferritin heavy chain, they could represent an added value in detecting relapse in GCA patients.

Analysis of Drug Effects on Primary Human Coronary Artery Endothelial Cells Activated by Serum Amyloid A.

BACKGROUND: RA patients have a higher incidence of cardiovascular diseases compared to the general population. Serum amyloid A (SAA) is an acute-phase protein, upregulated in sera of RA patients. AIM: To determine the effects of medications on SAA-stimulated human coronary artery endothelial cells (HCAEC). METHODS: HCAEC were preincubated for 2 h with medications from sterile ampules (dexamethasone, methotrexate, certolizumab pegol, and etanercept), dissolved in medium (captopril) or DMSO (etoricoxib, rosiglitazone, meloxicam, fluvastatin, and diclofenac). Human recombinant apo-SAA was used to stimulate HCAEC at a final 1000 nM concentration for 24 hours. IL-6, IL-8, sVCAM-1, and PAI-1 were measured by ELISA. The number of viable cells was determined colorimetrically. RESULTS: SAA-stimulated levels of released IL-6, IL-8, and sVCAM-1 from HCAEC were significantly attenuated by methotrexate, fluvastatin, and etoricoxib. Both certolizumab pegol and etanercept significantly decreased PAI-1 by an average of 43%. Rosiglitazone significantly inhibited sVCAM-1 by 58%. CONCLUSION: We observed marked influence of fluvastatin on lowering cytokine production in SAA-activated HCAEC. Methotrexate showed strong beneficial effects for lowering released Il-6, IL-8, and sVCAM-1. Interesting duality was observed for NSAIDs, with meloxicam exhibiting opposite-trend effects from diclofenac and etoricoxib. This represents unique insight into specific responsiveness of inflammatory-driven HCAEC relevant to atherosclerosis.

A concise review of significantly modified serological biomarkers in giant cell arteritis, as detected by different methods.

Giant cell arteritis (GCA) is a primary systemic vasculitis present in subjects older than 50years with involvement of large- and medium-sized arteries. Early diagnosis for GCA is essential to prevent serious complications, such as permanent vision loss and/or cerebrovascular events. Elevated inflammatory cytokines, with acute phase and other proteins dominate large- and medium-sized arteries leading to stenosis or occlusion of arterial lumen. To date, there are no reliable serological markers for monitoring GCA. The review aims to provide concise overview of published GCA studies in order to: a) identify significantly changed serological biomarkers in GCA and compare the influences of techniques for marker evaluation and b) investigate most promising markers in GCA using analyte frequency and meta-analysis.

A study of extracellular vesicle concentration in active diabetic Charcot neuroarthropathy.

It was the aim of this work to determine whether the plasma concentration of extracellular vesicles (EVs) in active diabetic Charcot neuroarthropathy (CN) is connected to the inflammatory markers, temperature elevation in the affected foot and concentration of soluble receptor for advanced glycation end products (RAGE). EVs were isolated from peripheral blood of 35 patients with active CN. EVs were counted after repetitive centrifugation and washing of samples, by flow cytometry. Foot temperature was measured by infrared thermometer. Concentration of soluble receptor for advanced glycation end products (RAGE) was determined by enzyme-linked immunosorbent assay (ELISA). We found statistically significant correlations of EV concentration (but not soluble RAGE concentration) with C-Reactive Protein (CRP) and with temperature difference between the affected and the contralateral foot (r=0.40, p=0.032; r=0.89, p<10-8, respectively). We provide evidence that the concentration of EVs is related to elevation of markers of inflammation (CRP and foot temperature difference) in acute Diabetic CN. EV-based markers could be considered as a potential aid in early diagnosis of CN.

Methotrexate reduces HbA1c concentration but does not produce chronic accumulation of ZMP in patients with rheumatoid or psoriatic arthritis.

OBJECTIVES: The mechanism by which methotrexate (MTX) improves glucose homeostasis in patients with rheumatoid (RA) and psoriatic arthritis (PsA) remains undetermined. Animal studies indicate a role for intracellular accumulation of 5-aminoimidazole-4-carboxamide-1-ß-d-ribofuranosyl 5'-monophosphate (ZMP) but this has not been directly demonstrated in humans. We explored whether accumulation of ZMP is associated with improvements in glucose homeostasis during MTX therapy. METHOD: MTX-naïve, non-diabetic RA (n = 16) and PsA (n = 10) patients received uninterrupted MTX treatment for 6 months. To evaluate whether ZMP accumulated during MTX therapy, we measured the concentration of ZMP in erythrocytes and the concentration of its dephosphorylated derivative 5-aminoimidazole-4-carboxamide-1-ß-d-ribofuranoside (AICAR) in urine using liquid chromatography mass spectrometry (LC-MS/MS). To assess glucose homeostasis, we determined the concentration of glycated haemoglobin (HbA1c) and homeostasis model assessment of insulin resistance [HOMA-IR: fasting glucose (mmol/L) × fasting insulin (µU/mL)/22.5]. RESULTS: Erythrocyte ZMP and urinary AICAR concentrations did not increase during 6 months of MTX therapy. HbA1c concentration was reduced from 5.80 ± 0.29% at baseline to 5.51 ± 0.32% at 6 months (p < 0.001), while HOMA-IR remained unaltered. Reduction in HbA1c concentration was not associated with increased ZMP or AICAR concentrations. CONCLUSIONS: MTX therapy probably does not produce a chronic increase in erythrocyte ZMP or urinary AICAR concentrations. Collectively, our data do not support the hypothesis that MTX improves glucose homeostasis through chronic accumulation of ZMP.

Oxidatively altered IgG with increased immunoreactivity to ß2-glycoprotein I and its peptide clusters influence human coronary artery endothelial cells.

Oxidative stress has been shown to play a role in modifying antibodies in favor of higher auto-immunoreactivity. We studied the immunoreactivity of oxidized IgG (oxIgG) to ß2-glycoprotein I (ß2GPI), six peptide sequences corresponding to amino acid clusters on its different domains, to determine their effects on human coronary artery endothelial cells (HCAEC). Human IgG was purified from seven donors, electro-oxidized and checked for immunoreactivity and avidity to ß2GPI and to peptides by ELISA. Conformational stability and antibody-antigen complex formation of oxIgG was analyzed by fluorescence spectroscopy and dynamic light scattering. Resting and activated sub-confluent HCAEC were stimulated with oxIgG or IgG. Secreted cytokines were measured by ELISA. Immunoreactivity of seven oxIgG samples increased to 7.5-fold against ß2GPI and to 3.8-fold against six peptides as compared to IgG. oxIgG showed low avidity "properties." Conformational changes and exposure of protein hydrophobic regions were confirmed by an elevation in fluorescence (2.4- to 5.0-fold) on bis-ANS dye binding to oxIgG. oxIgG significantly elevated the release of GROα and IL-8 in resting and activated states of HCAEC. Oxidation alters IgG in favor of autoreactivity toward whole ß2GPI and corresponding peptides on different domains of ß2GPI and could lead to dysfunction of arterial endothelium by upregulation of chemokines.

Immunoreactivity and avidity of IgG anti-ß2-glycoprotein I antibodies from patients with autoimmune diseases to different peptide clusters of ß2-glycoprotein I.

The pathogenicity of antibodies against ß2-glycoprotein I (anti-ß2GPI) depends on multiple factors such as subclass type, epitope binding and avidity. Due to their large heterogeneity, their impact on antiphospholipid syndrome (APS) onset is still not fully clarified. We studied the binding characteristics of IgG anti-ß2GPI with known avidity from sera of 201 autoimmune patients (87 with APS, 67 with APS associated with systemic lupus erythematosus (SLE), 47 with only SLE) to six ß2GPI peptides corresponding to amino acid clusters on domains I-II, II, III and III-IV by indirect ELISA and evaluated their association with clinical features of APS. Peptides A (LKTPRV; domain I-II), B (KDKATF; domain IV) and C (TLRVYK; domain III) were derived from a hexapeptide phage display library previously shown to react with pathogenic monoclonal anti-ß2GPI. Peptides D (NGPANSK; domain III), E (YNPLWFV; domain II) and F (KMDGNHP; domain III-IV) represent surface amino acid clusters on ß2GPI. The percentage of patients positive for peptides were observed as follows: 30.3% for peptide D, 28.90% for B, 25.9% for C, 24.9% for E, 24.4% for F and 10.0% for A. The anti-peptide antibodies in studied serum samples were predominantly of heterogeneous avidity, followed by law avidity anti-peptide antibodies, whereas only a few were of high avidity. Positive and negative correlations were found between several anti-peptide antibodies and the rate of thrombosis. Our results indicated diverse reactivity of IgG anti-ß2GPI to different epitopes on ß2GPI. Classification of IgG anti-ß2GPI into subgroups regarding epitope specificity and avidity could represent an additional tool in understanding their pathogenicity in APS.

Antibodies to phosphatidylserine/prothrombin complex as an additional diagnostic marker of APS?

Antiprothrombin antibodies can be measured by ELISA using either a prothrombin/phosphatidylserine complex (aPS/PT) or prothrombin alone (aPT) as antigen. We aimed to compare the clinical features of autoimmune patients with avidity of aPS/PT and determine the diagnostic efficiency of aPS/PT and aPT for assessing antiphospholipid syndrome (APS). aPS/PT were of low (n = 9), heterogeneous (n = 31) and high (n = 8) avidity out of 48 cases. None of the samples with low avidity were positive in aPT ELISA. Among patients with heterogeneous or high avidity aPS/PT, there was a significantly greater number of patients with APS as compared to patients with low avidity (38/39 vs. 7/9; p < 0.05). No SLE patients had high avidity antiprothrombin antibodies.

Autoimmune response following influenza vaccination in patients with autoimmune inflammatory rheumatic disease.

Vaccines have undoubtedly brought overwhelming benefits to mankind and are considered safe and effective. Nevertheless, they can occasionally stimulate autoantibody production or even a recently defined syndrome known as autoimmune/inflammatory syndrome induced by adjuvants (ASIA). There is scarce data regarding autoimmune response after seasonal/influenza A (H1N1) vaccine in patients with autoimmune inflammatory rheumatic disease (AIRD). The objective of our study was therefore to determine autoimmune response in a large group of AIRD patients vaccinated against seasonal and/or H1N1 influenza. We conducted a prospective cohort study with a 6-month follow-up. Two-hundred and eighteen patients with AIRD (50 vaccinated against seasonal influenza, six against H1N1, 104 against both, 58 non-vaccinated controls) and 41 apparently healthy controls (nine vaccinated against seasonal influenza, three against H1N1, 18 against both, 11 non-vaccinated controls) were included. Blood samples were taken and screened for autoantibodies [antinuclear antibody (ANA), anti-extractable nuclear antigen (anti-ENA), anticardiolipin (aCL) IgG/IgM antibodies, anti-beta 2-glycoprotein I (anti-ß2GPI)] at inclusion in the study, before each vaccination, 1 month after the last vaccination and 6 months after inclusion. For non-vaccinated participants (patients and healthy controls) blood samples were taken at the time of inclusion in the study and 6 months later. We report that after the administration of seasonal/H1N1 vaccine there were mostly transient changes in autoantibody production in AIRD patients and in healthy participants. However, a small subset of patients, especially ANA-positive patients, had a tendency towards anti-ENA development. Although no convincing differences between the seasonal and H1N1 vaccines were observed, our results imply that there might be a slight tendency of the H1N1 vaccine towards aCL induction. Although seasonal and H1N1 vaccines are safe and effective, they also have the potential to induce autoantibodies in selected AIRD patients and healthy adults. Follow-up of such individuals is proposed and further research is needed.

Serum amyloid A: an acute-phase protein involved in tumour pathogenesis.

The synthesis of acute-phase protein serum amyloid A (SAA) is largely regulated by inflammation- associated cytokines and a high concentration of circulating SAA may represent an ideal marker for acute and chronic inflammatory diseases. However, SAA is also synthesized in extrahepatic tissues, e.g. human carcinoma metastases and cancer cell lines. An increasing body of in vitro data supports the concept of involvement of SAA in carcinogenesis and neoplastic diseases. Accumulating evidence suggests that SAA might be included in a group of biomarkers to detect a pattern of physiological events that reflect the growth of malignancy and host response. This review is meant to provide a broad overview of the many ways that SAA could contribute to tumour development, and accelerate tumour progression and metastasis, and to gain a better understanding of this acute-phase reactant as a possible link between chronic inflammation and neoplasia.

Autoimmune and proinflammatory activity of oxidized immunoglobulins.

AIMS: Oxidation reactions can modify protein activity or specificity. Recently, a novel redox-reactive family of autoantibodies was described, which indicated involvement of altered antibodies (beside altered antigens) into autoimmune reactions. The aim of our study was to determine the binding capacity alterations of electro-oxidized blood donors' IgGs, and to evaluate their effects on released proinflammatory interleukin 6 in HUVEC. RESULTS: We found out that 1.) Isolated blood donor IgGs bound after electro-oxidation to beta2-glycoprotein I, cardiolipin, citrullinated cyclic peptide and protein 3 by enzyme-linked immunosorbent assay, extractable nuclear antigens by counterimmuno-electrophoresis, and cell antigens by indirect immunofluorescence; 2.) Alterations in immunoreactivity of IgGs due to oxidation highly depend on electric current, time of exposure and the presence of antioxidants, 3.) Treatment of HUVEC with oxidized IgGs resulted in changed cell morphology, accompanied by an increase in released interleukin-6. CONCLUSIONS: Our data suggest repeatable transformation of antibodies present in the blood of healthy persons and patients. Inter-individual differences in chemical stability of antibodies, patient's antioxidant status, and the microenvironmental changes at the cellular level may influence the range of antibody alterations and their involvement in pathophysiological autoimmune processes.

Prevalence and clinical associations of anti-Ku antibodies in patients with systemic sclerosis: a European EUSTAR-initiated multi-centre case-control study.

OBJECTIVES: To determine the prevalence of anti-Ku antibodies in 625 patients with systemic sclerosis (SSc) from six European rheumatological centres and to evaluate their clinical and serological characteristics. METHODS: Sera of 625 consecutive patients with either limited cutaneous or diffuse cutaneous SSc were tested for antibodies to Ku antigen together with other extractable nuclear antigens by counterimmunoelectrophoresis. A case-control design with calculation of bootstrap 95% confidence intervals derived from anti-Ku negative control patients was used to evaluate clinical associations of anti-Ku antibodies. Sera from anti-Ku positive patients with SSc and a control group were additionally tested by immunofluorescence on Hep-2 cell substrates and line immunoassay. RESULTS: Anti-Ku antibodies were found in the sera of 14/625 (2.2%) patients with SSc. Of 14 anti-Ku positive patients with SSc, 10 had no other anti-extractable nuclear antigen (ENA) antibodies detected by counterimmunoelectrophoresis. Using a case-control study design, anti-Ku antibodies were significantly associated with musculoskeletal manifestations such as clinical markers of myositis, arthritis and joint contractures. In addition, a significant negative correlation of anti-Ku antibodies was found with vascular manifestation such as fingertip ulcers and teleangiectasias. There was a striking absence of anti-centromere antibodies as well as anti- polymyositis (PM)/scleroderma (Scl) antibodies in patients that were anti-Ku positive. As expected, anti-Scl70 and punctate nucleolar immunofluorescence patterns were present only in single cases. CONCLUSION: This is the largest cohort to date focusing on the prevalence of anti-Ku antibodies in patients with SSc. The case-control approach was able to demonstrate a clinically distinct subset of anti-Ku positive patients with SSc with only relative clinical differences in skeletal features. However, the notable exceptions were signs of myositis. This shows the importance of anti-Ku antibody detection for the prediction of this specific clinical subset.

Beta2-glycoprotein I and its clinical significance: from gene sequence to protein levels.

In order to elucidate beta2-GPI at the DNA level and characterize its polymorphisms, mRNA expression, protein levels and clinical significance at each of these steps, a molecular review of beta2-GPI literature was performed. The human beta2-GPI complete nucleotide sequence has been reported and it consists of 8 exons separated by large introns. The beta2-GPI gene is polymorphic with four alleles. The distribution of point mutations can be significantly different between various racial populations. DNA variation studies of the beta2-GPI gene identified a total of 151 single-nucleotide polymorphisms, 26 of which are within regions with potential clinical significance. Southern blot analysis indicated the presence of one gene product only. An atypical TATA box and a hepatic nuclear factor-1 element are both essential for beta2-GPI promoter activity. Transcription factor binding sites for STAT, CREB, C/EBPbeta, NF-1, AP-1, NFAT, HNF-3beta and HNF-1 have been identified in the promoter region of the beta2-GPI gene by computer analysis. The beta2-GPI transcriptional signal of 1.5 kb was detected in Northern blot analysis and its 326-amino-acid sequence was found to be one of the most proline-rich eukaryotic proteins. Amino acid substitutions have been shown to be associated with loss of phospholipid binding, development and recognition of antiphospholipid antibodies.

Serum amyloid A in autoimmune thrombosis.

The objectives of this study were (1) to determine how levels of serum amyloid A (SAA), high sensitivity C-reactive protein (CRP) and interleukin-6 (IL-6) correlate to autoimmune diseases in patients with or without thrombosis, and (2) to discuss the parameters that influence the relative SAA values. SAA, CRP and IL-6 concentrations were determined by enzyme linked immunosorbent assay (ELISA). 84 patients with secondary antiphospholipid syndrome (SAPS), primary antiphospholipid syndrome (PAPS), systemic lupus erythematosus with antiphospholipid antibodies (SLE+aPL), SLE, venous thrombosis (VT), arterial thrombosis (AT) were compared to healthy donors (n=60). The percentages of patients above cut-off were highest in the SAPS, SLE and SLE+aPL groups. Significant differences were observed between healthy donors and inflammatory groups of patients (SAPS and SLE+aPL) in all three measured parameters. SAA and CRP were shown to be correlated to a greater extent in SAPS patients than SLE+aPL patients. In summary, this cross-sectional, retrospective, small study and accompanying clinical considerations limit the ability to make definite conclusions. SAA would not serve as a useful marker for venous, arterial thrombosis or PAPS (pro-coagulant events). It could however, be a good predictor of progression from a non-inflammatory thrombotic condition to an inflammatory one.

Opposing regulation of interleukin-8 and NF-kappaB responses by lipoxin A4 and serum amyloid A via the common lipoxin A receptor.

Lipoxin A4 (LXA4) is a potent eicosanoid that inhibits IL-1beta-induced activation of human fibroblast like synoviocytes (FLS) via the ALX4 receptor. Serum amyloid A (SAA) is an acute phase reactant with cytokine-like properties. SAA has been shown to bind the same seven transmembrane G protein-coupled receptor ligated by LXA4. Here we compared the inflammatory responses of lipid (LXA4) and peptide (SAA) ligands in human FLS via the shared ALX, and characterized their downstream signaling. LXA4 induced stimulation of tissue inhibitors of metalloproteinase-2, whereas SAA induced interleukin-8 and matrix metalloproteinase-3 production. SAA up-regulated NF-kappaB and AP-1 DNA binding activity, while LXA4 markedly inhibited these responses after IL-1beta stimulation. A human IL-8 promoter luciferase construct was transfected into CHO cells stably expressing ALXR in order to determine the role of NF-kappaB and/or AP-1 in the regulation of IL-8 gene expression. The NF-kappaB pathway proved to be the preeminent for the biological responses elicited by both ligands. These findings suggest that two endogenous molecules, targeting a common receptor, could participate in the pathogenesis of inflammatory arthritis by differentially regulating inflammatory responses in tissues expressing the ALXR.

Lipoxin A4 counteracts synergistic activation of human fibroblast-like synoviocytes.

Excessive production of interleukin-6 (IL-6) and metalloproteinases (MMPs) have been implicated in the pathogenesis of rheumatoid arthritis. Lipoxin A4 (LXA4) and transforming growth factor beta 2 (TGF-beta 2), mediators with potential anti-inflammatory activities, were tested to determine how they affect IL-1 beta-dependent release of IL-6 and MMPs in human fibroblast like synoviocytes. The results showed dramatic differences between the mediators: TGF-beta 2 acted synergistically with IL-1 beta to stimulate IL-6 protein levels, whereas LXA4 inhibited IL-6 expression in dose- and time-dependent manner. Inhibition, by LXA4 was abrogated when cells were pre-incubated with antibody against the LXA4R whereas TGF-beta 2 by itself had no significant effect on IL-6 or MMP levels. LXA4, at nanomolar concentrations, altered the MMP-1 and MMP-3 expression levels of IL-1 beta and TGF-beta 2 stimulated fibroblast like synoviocytes at 5 days. Furthermore, IL-1 beta and TGF-beta 2 up-regulated LXA4R mRNA. These results demonstrate, for the first time, that LXA4Rs mediate the effects of LXA4 on inflammatory responses after combined stimulation of human fibroblast like synoviocytes with IL-1 beta and TGF-beta 2. These activities might constitute an important mechanism by which LXA4 regulates human synovial fibroblast activation.