Objective The most prominent alteration in the immune system of patients with systemic lupus erythematosus (SLE) and antiphospholipid syndrome (APS) is the increased expression of type I interferon (IFN) inducible genes, known as the IFN signature. This signature is related to disease activity, vascular disease and predicts future flares and the response to anifrolumab. There is an urgent need for easily measurable biomarkers to detect an IFN signature. Galectin-9 may steer T-cell responses and is a potential biomarker for autoimmune diseases. In the present study we investigated the performance of galectin-9 as a biomarker to detect the IFN signature in SLE and APS and its production by dendritic cell (DC) subsets. Methods Serum levels of galectin-9 and CXCL10 were measured by Luminex in an identification cohort (n=43) and replication cohort (n=148) of patients with SLE, SLE +APS and primary APS (PAPS) and healthy controls (HC). Isolated monocytes were used to quantify IFN scores by measuring the expression of 4 type I IFN inducible genes. The performance of potential biomarkers for the IFN signature were assessed by Receiver Operating Characteristics curves. RNA sequencing on plasmacytoid and myeloid DC isolated from patients with SLE, SLE +APS and PAPS (n=54) were analysed to identify potential sources of galectin-9 in SLE and APS. Results In both identification and replication cohort, serum levels of galectin-9 and CXCL10 were elevated in SLE, SLE +APS and PAPS patients as compared with HC (all p<0.05) and both galectin-9 and CXCL10 correlated with the IFN signature (r=0.66, p<0.001 and r=0.46, p<0.001 respectively). ROC-curve analysis revealed a better performance of galectin-9 (AUC 0.86) than CXCL10 (AUC 0.78) or traditional serological biomarkers for SLE (AUC <0.75) to detect an IFN signature. The expression of galectin-9 was increased in both pDC and mDC in SLE and APS, in particular in IFN-high patients. In vitro, IFNα upregulated galectin-9 expression in pDC and mDC. Conclusion Galectin-9 is produced by dendritic cells in SLE and APS upon activation by IFNα and serves as an easily measurable biomarker that outclasses CXCL10 or traditional measures of disease activity to detect an IFN signature in patients with SLE and APS.

Background/purpose Dendritic cells (DC) are key cells in the pathogenesis of autoimmune diseases by potently activating T-cells. Systemic lupus erythematosus (SLE) and antiphospholipid syndrome (APS) are characterised by an IFN signature, caused by elevated levels of IFNα. Plasmacytoid DC (pDC) are held responsible for the increased levels of IFNα in SLE and APS. The molecular mechanisms underlying the increased activation of pDC in SLE and APS are unknown. Using RNA sequencing (RNAseq) and further in vitro validation experiments on plasmacytoid and myeloid DC (mDC) obtained from patients with SLE and APS we assessed the causes and consequences of increased type I IFN signalling on the dysregulation of both pDC and mDC in patients with SLE and APS. Methods RNAseq was performed on pDC and mDC isolated from peripheral blood of patients with SLE, SLE +APS and primary APS (PAPS) and healthy controls (n=54). Weighted gene correlation network analysis (WGCNA) was used to identify pDC- and mDC-specific gene modules and to stratify patients into those with (IFN-high) or without (IFN-low) an IFN signature. The response of pDC and mDC (in co-culture with T-cells) to IFNα and TLR agonists were analysed by RT-qPCR and flow cytometry to functionally validate RNAseq data. Results WGCNA identified IFN modules in pDC and mDC that perfectly stratified patients from HC. Comparing the IFN modules of pDC and mDC revealed cell specific alterations related to the IFN signature in pDC/mDC. Increased expression of TLR7 and its downstream intermediates was confined to IFN-high patients in pDC. In contrast, genes involved in the activation of T-cells were related to the IFN module in mDC. Both pDC and mDC showed increased expression of BAFF. In vitro, IFNα upregulated TLR7 in pDC and augmented TLR7-mediated IFNα production. In contrast to pDC, IFNα primed mDC for enhanced T-cell proliferation via the upregulation of co-stimulatory molecules. Furthermore, in SLE/APS, pDC and mDC produced BAFF and expressed chemokine receptors. Conclusion pDC and mDC are differentially affected by IFNα in SLE and APS. IFNα primes pDC for enhanced IFNα production which potentiates T-cell activation by mDC, thereby sustaining the IFN signature in SLE and APS.

Background and objectives Osteoarthritis (OA) is a degenerative joint disease, clinically characterised by joint pain and disability. Underlying structural changes are degeneration of articular cartilage, intra-articular inflammation with synovitis and changes in peri-articular and subchondral bone. The ideal drug to treat OA should have analgesic, anti-inflammatory and cartilage protective effects. To date, no such drug has been described. In human and animal in and ex-vivo models, the immunoregulatory cytokines interleukin 4 (IL-4) and interleukin 10 (IL-10), especially in combination therapy, can prevent joint degeneration effectively. To use the combined activities of both IL-4 and IL-10, we developed IL4–10 synerkine, preserving the functional activity of each cytokine. In several well-established animal models, we have shown the potential of IL4–10 synerkine to inhibit inflammatory pain in animal models. In this study, we evaluated the potential of the synerkine to reduce inflammation and to provide cartilage protection in an ex vivo model for human OA. Materials and methods Osteoarthritic cartilage (n = 8) and synovium (n = 6) was obtained at joint replacement surgery and was cultured for 72 h in conditioned medium, in the presence or absence of equimolar concentrations of IL-4 (10 ng/ml), IL-10 (10 ng/ml), the combination of IL-4 and IL-10 (10 ng/ml + 10 ng/ml) and IL4-10 synerkine (20 ng/ml). After culture, cartilage was selected to study changes in proteoglycan (PG) synthesis and release. Supernatants of both cartilage and synovium cultures were collected for measurement of a broad spectrum of inflammatory mediators. Results In both synovium and cartilage cultures, elevated levels of pro-inflammatory mediators like IL-6 (resp. 7.5 µg/mg and 531 µg/mg) and IL-8 (resp. 2.6 µg/mg and 142 µg/mg) were measured. These levels were strongly inhibited by the IL4-10 synerkine (about 80%; all p < 0.01). IL4-10 synerkine also showed a reducing effect on the release of other important mediators, like IL-1, MMP-1 and MMP-3, whereas levels of interleukin-1 receptor antagonist (IL-1RA) and tissue inhibitor of metalloproteinase-1 (TIMP-1) remained unchanged. Additionally, the synerkine had a beneficial effect on proteoglycan metabolism, inducing proteoglycan synthesis with 32%, while the proteoglycan release was slightly reduced (-1.3%). Conclusions IL4-10 synerkine directly affects proteoglycan turnover and cytokine production of OA cartilage, combined with reduced secretion of inflammatory cytokines and proteases by OA synovial tissue. The immunoregulatory and cartilage protective effects in combination with it’s analgesic effects, suggest that the synerkine is an effective disease modifying drug in the treatment of osteoarthritis.

Background A considerable percentage of patients shows a limited response to biologics targeting one specific inflammatory mediator, largely because of redundancy of these mediators. An attractive alternative is inhibition of multiple proinflammatory mediators and induction of immunoregulatory activity by regulatory cytokines such as IL4 and IL10. Many studies demonstrated the strong capacity of IL4 and IL10 as stand-alone drugs to inhibit inflammation and tissue-destructive responses in animal and human in vitro models. Clinical results of these cytokines, however, have been modest, possibly because of poor bioavailability that is mainly due to low molecular weight and rapid renal clearance. Objectives To execute a feasibility study to develop IL4 and IL10 as one biologic (called IL4–10 synerkine), preserving distinct characteristics of each molecule plus improving bioavailablity by increasing the molecular size. Materials and methods Biochemical properties of IL4–10 synerkine were determined by western blot, size exclusion chromatography, and ELISA. Functional properties were studied by measuring the capacity of IL4–10 synerkine to regulate production of pro-inflammatory cytokines and their inhibitors, as well as proinflammatory and regulatory T-cell activity, and expression of Fc receptors. In addition, blockade of IL4 and IL10 receptor was performed to confirm the specific activities of IL4 and IL10. Results IL4–10 synerkine appeared as a glycosylated dimeric protein with a molecular size of ˜70 kDa, consisted of intact IL10 and IL4 subunits. In whole blood assays IL4–10 synerkine robustly and dose-dependently inhibited multiple pro-inflammatory cytokines, which was almost complete at 20 ng/ml (IL1β, TNFα, IL6 and IL8, all p < 0.001). This effect was dependent on interaction with IL10R and IL4R. Oppositely, the synerkine significantly induced production of IL1RA and preserved sTNFR levels. IL4–10 synerkine strongly inhibited Th1 and Th17 cytokine secretion (P < 0.01), while maintaining FoxP3 expression. Finally, while IL4 upregulated FcεR expression and IL10 upregulated expression of activating FcγRs on monocytes (all p < 0.001), both Fcε and FcγR expression were largely preserved at control levels in the presence of IL4–10 synerkine (all at least p < 0.01). Conclusions IL4–10 synerkine is a novel anti-inflammatory drug that shifts multiple proinflammatory pathways towards immunoregulation. The increased molecular mass predicts better bioavailabilty in humans than the wild-type molecules, which potentially enhances its clinical efficacy. The strong and improved inhibitory activities of IL4–10 synerkine (as compared to IL4 and IL-10 monotherapy) that we observed in several animal models for inflammatory pain underscores the potential for treatment of inflammatory and possibly degenerative rheumatic diseases.

OBJECTIVE:To determine the effect of methotrexate (MTX) on expression levels of activating receptors for IgG (FcgammaRs) on monocytes of rheumatoid arthritis (RA) patients in relation to changes in disease activity.METHODS:The effect of MTX on FcgammaRs on monocytes of RA patients was evaluated ex vivo as well as in vitro. Recently diagnosed, disease-modifying antirheumatic drug (DMARD)-naive RA patients were treated with low-dose MTX. At baseline and 16 weeks after the start of MTX treatment, changes in FcgammaR expression levels on peripheral blood monocytes were evaluated by fluorescence-activated cell sorting analysis and were correlated to changes in disease parameters. To study the direct effects of MTX on monocytes, these cells were isolated from peripheral blood monocytes of healthy controls and cultured with MTX. Other monocyte surface molecules (CD40, CD80, CD86, MHC class II) were also determined to test the specificity of the effect on FcgammaR expression levels.RESULTS:Eleven out of 15 patients improved clinically (mean disease activity score before 6.2 +/- 0.8 vs 4.3 +/- 1.7 after). Sixteen weeks after the start of MTX therapy, the expression levels of FcgammaRI and IIa on monocytes were significantly decreased, whereas the decreases in FcgammaRIIIa expression levels on monocytes were less marked. The percentage decrease in FcgammaRI expression correlated with the percentage decrease in CRP and well-being. In vitro MTX selectively decreased FcgammaRI and FcgammaRIIa expression levels of isolated monocytes, in contrast to other surface molecules.CONCLUSION:The disease-modifying effect of MTX in the treatment of RA is accompanied by down-regulation of activating FcgammaRI and IIa on monocytes, which could be a direct effect of MTX on monocytes. This down-regulation represents a new mode of action of MTX which should be considered in RA patients, especially during conditions that could give rise to monocyte activation by IgG-containing immune complexes, e.g. during antibody-based therapy of RA.

Objectives: To investigate whether differences in T helper (Th) 1 and Th2 cell activity in salivary glands ("local'') or ("peripheral'') blood can discriminate between Sjogren's syndrome (SS) and non-Sjogren's sicca syndrome (nSS-sicca). Additionally, to study relationships of local and peripheral Th cell activities with each other and with disease activity measures.Methods: 62 sicca patients ( 32 with SS, 30 with nSS-sicca) were studied. Local Th1 ( interferon gamma ( IFN gamma)) and Th2 ( interleukin (IL) 4) activity were determined using immunohistochemistry. T cell production of IFN gamma and IL4 in peripheral blood (PB) was determined by ELISA. Erythrocyte sedimentation rate (ESR) and serum IgG were considered disease activity measures.Results: ESR and serum IgG were higher in patients with SS than in patients with nSS-sicca. Local Th1 cell activity was higher and PB Th1 activity lower in patients with SS than in those with nSS-sicca. Th2 cell activity did not differ significantly between the patient groups. The ratio IFN gamma/IL4 was higher in salivary glands and lower in PB in patients with SS than in patients with nSS-sicca. Local and peripheral Th1 and Th2 cell activities correlated with ESR and serum IgG levels. ESR, serum IgG, and local or peripheral Th1 or Th2 cell activity did not discriminate between patients with SS and nSS-sicca.Conclusions: An imbalance between Th1 and Th2 activity in sicca patients is clearly related to the severity of disease, but cannot be used to distinguish between patients with SS and those with nSS-sicca.

Background: Monocytes/macrophages have an important and versatile role in joint inflammation and destruction in rheumatoid arthritis (RA). Objective: To determine the efficiency of monocyte/macrophage elimination by a new drug conjugated antibody (CD64-calicheamicin (CD64-CaMi)) directed to the high affinity receptor for IgG (FcγRI). Methods: Mononuclear cells from peripheral blood and synovial fluid of patients with RA were cultured in the presence of CD64-CaMi. Cell death of monocytes/macrophages was measured by analysis of phenotypic changes (light scatter patterns, CD14 expression, and FcγRI expression) and nuclear DNA fragmentation. The selectivity of CD64-CaMi was checked by using FcγRI deficient and FcγRI transfected cell lines. In addition, the indirect effect of CD64-CaMi-induced macrophage cell death on arthritogenic T(h1) cell activity was determined. Results: Inflammatory macrophages from RA synovial fluid, expressing increased FcγRI levels, were efficiently killed by CD64-CaMi through induction of DNA fragmentation. CD64-CaMi-induced cell death of monocytes/macrophages from peripheral blood of patients with RA proved less efficient. Induction of synovial macrophage death by CD64-CaMi was accompanied by efficient inhibition of proinflammatory T(h1) cytokine production. Conclusion: Together, the presented data suggest that elimination of macrophages through a new FcγRI directed CD64-CaMi is feasible. Because monocytes from peripheral blood are also eliminated by this immunoconjugate, additional experimental studies should validate its potential for local (intra-articular) application in the treatment of RA.

A guanine to adenine point mutation results in an arginine (R) to histidine (H) substitution in FcgammaRIIa at residue 131 that strongly impacts receptor function. This FcgammaRIIa polymorphism is mostly typed by allele-specific polymerase chain reactions (PCR) or in functional assays, dependent on ligand binding. Both types of methods are laborious, time consuming, and not readily available in routine laboratories.We generated a panel of human antibodies against FcgammaRII, and one of them, MDE-9, selectively recognized the FcgammaRIIa-H131 allotype. MDE-9 was applicable to detect FcgammaRIIa-H131 in both flow cytometry and immunohistochemistry. MDE-9 was used to develop an FcgammaRIIa allotyping method based on flow cytometry. In a "single-tube assay", FITC-labeled MDE-9 (specific for FcgammaRIIa-H131) and Cy3-labeled mAb41H16 (specific for FcgammaRIIa-R131) were added to 50 mul samples of whole blood. The results of flow cytometric FcgammaRIIa allotyping correlated completely with PCR genotyping. This novel allotyping assay should facilitate the screening of patients in a routine diagnostic setting. In addition, a combination of MDE-9 and 41H16 can be used in FcgammaRIIa-H/H131 homozygous individuals to detect FcgammaRIIa and FcgammaRIIb surface expression on monocytes. This is an important application of these antibodies because, to this day, no antibodies were available to specifically study the surface expression of FcgammaRIIb. (C) 2004 Elsevier B.V. All rights reserved.

Objectives. Levels of immunoglobulin G (IgG) Fc receptors (FcgammaRs) affect the activity and function of monocytes/macrophages when binding IgG-containing immune complexes. Hence, the expression level of FcgammaRs on monocytic cells may influence inflammation in patients with rheumatoid arthritis (RA). In this study the expression levels of FcgammaRI, IIa and IIIa on peripheral blood monocytes of RA patients were compared with those of healthy controls and related to patient and disease characteristics and the use of disease-modifying anti-rheumatic drugs (DMARDs). In addition, FcgammaR expression levels were determined on RA synovial fluid macrophages and compared with those in RA peripheral blood.Methods. Mononuclear cells from peripheral blood and synovial fluid were isolated and FcgammaR expression levels on CD14-positive cells were analysed by flow cytometry. The effects of patient and disease characteristics and the use of DMARDs were assessed.Results. A high expression level of FcgammaRIIa and high percentages of FcgammaRIIIa-expressing monocytes were found in RA patients with a high erythrocyte sedimentation rate. DMARD-naive early RA patients had higher FcgammaRIIa expression levels but a similar amount of FcgammaRIIIa-positive monocytes compared with RA patients using DMARDs. In synovial fluid, FcgammaRIIa expression levels were lower than in RA peripheral blood, whereas the percentage of FcgammaRIIIa-positive monocytic cells was higher in synovial fluid than in peripheral blood.Conclusions. These data point to the involvement of FcgammaRs, specifically FcgammaRIIa and IIIa, in the immune response of RA and suggest that FcgammaR expression levels are susceptible to modulation by DMARD therapy.

Objective. Several clinical studies performed with human recombinant interleukin 10 (IL-10) in patients with rheumatoid arthritis (RA) have shown little efficacy. We investigated potentially proinflammatory in vivo effects of IL-10 in humans. We evaluated the upregulation of Fcgamma receptor (FcgammaR) expression on monocytes/macrophages (and granulocytes) in patients with RA receiving different dosages of IL-10.Methods. Together with changes in disease activity and several cell markers, the expression of FcgammaRI, FcgammaRIIa, and FcgammaRIII was determined on granulocytes and monocytes/macrophages from the peripheral blood of 6 patients with active RA before and after treatment with recombinant human IL-10. In addition, the in vitro effect of IL-10 on FcgammaR expression on monocytes/macrophages in combination with their susceptibility to immune complex induced production of tumor necrosis factor-alpha (TNF-alpha) was assessed.Results. Clinical improvement was not observed in the IL-10 treated patients (based on ACR20 criteria). Significant decreases in thrombocyte numbers were observed in patients receiving IL-10. No changes in cell markers such as CD14 were found. On the other hand, expression of FcgammaRI and FcgammaRIIa on monocytes/macrophages was increased upon high dose IL-10 treatment. Interestingly, increases in expression of FcgammaRI and FcgammaRIIa correlated with a decrease in thrombocyte numbers. In vitro, IL-10 similarly upregulated FcgammaRI and FcgammaRIIa expression on monocytes/macrophages from RA patients. This was accompanied by increased TNF-alpha production after immune complex stimulation.Conclusion. These findings indicate that upregulation of FcgammaR expression in RA with IL-10 treatment may counteract the otherwise antiinflammatory effects of IL-10 by potentiating immune complex mediated proinflammatory responses.

Background: A large number of activated T cells are found in the joints of patients with rheumatoid arthritis (RA). Interleukin 7 (IL7), a T cell growth factor and a regulator of Th1 and Th2 cytokine production, is produced by synoviocytes from patients with RA.Objective: To investigate the effect on proinflammatory cytokine production of synovial fluid mononuclear cells (SFMC) and the mechanism by which IL7 influences CD4+ T cell activity in patients with RA.Methods: In a cross sectional group of patients with RA, IL7 levels were compared with those of healthy controls and related to disease activity. The effect of IL7 on cytokine production was tested by RA SFMC and on SF CD4+ T cells in the presence of mononuclear cells (MC). Production of tumour necrosis factor alpha (TNFalpha), IL1beta, interferon gamma (IFNgamma), and IL4 was measured by enzyme linked immuno-sorbent assay (ELISA) and by single cell FACS analysis. Expression of the IL7 receptor alpha chain on CD4+ T cells (essential for IL7 signalling) was assessed. Direct effects of IL7 on isolated synovial fluid (SF) CD4+ T cells were studied by cytokine analysis. By neutralisation of IL12 in MC cultures, indirect effects of IL7 on T cells through accessory cells were studied.Results: IL7 serum levels were higher in patients with RA than in healthy controls and correlated positively with C reactive protein levels. IL7 stimulated TNFalpha production by SFMC and very potently stimulated IFNgamma and TNFalpha production by SF CD4+ T cells. These effects were probably mediated through the IL7 receptor alpha chain, which was abundantly expressed on SF CD4+ T cells. Besides the direct stimulation of T cell cytokine production by IL7, its action was partly dependent on IL12, indicating that IL7 also stimulates accessory cell function, leading to T cell activation.Conclusion: IL7 stimulates proinflammatory cytokine production of intra-articular CD4+ T cells and accessory cells from patients with RA. The correlation with measures of disease activity indicates that IL7 might substantially contribute to the perpetuation of Th1 and TNFalpha mediated proinflammatory responses in patients with RA.

Can atopy help to clarify the role of Th2 mediated regulation in these diseases? Amongst the heterogeneity of human immune responses T helper (Th) lymphocyte subsets have been shown to have an important role.1 Of these different subsets, Th1 cells mediate cellular immunity, including cytotoxicity and delayed-type hypersensitivity responses through the specific production of interferon γ (IFNγ) and interleukin (IL) 2. Th2 cells, characterised by IL4, IL5, and IL13 production, favour humoral immunity and down regulate Th1 mediated cellular immunity. Th2 responses are associated with IL4/IL13 mediated IgE production and IL5 mediated eosinophilia. Th1 activity in its turn inhibits these responses and results in effective immune responses against several infectious agents such as bacteria and viruses. Also, in several autoimmune diseases Th1 cells contribute to the induction and persistence of inflammation and inflammation-induced tissue damage. Numerous studies have shown that Th1-induced immunity is inhibited by suppressive Th cells other than IL4+ Th2 cells. These suppressive cells are also distinguished by their particular cytokine secretion and/or function: transforming growth factor β (TGFβ)+ Th3 cells, IL10+ T regulatory 1 (Tr1) cells, and CD4+CD25+ anergic/suppressive cells.2,3 Although all these subsets may contribute to suppression of Th1 activity, the balance between Th1 and Th2 cells has been shown to strongly influence many inflammatory responses. Owing to the mutually antagonising abilities of Th1 and Th2 cells in many experimental animal and human in vitro studies, the Th1/Th2 balance in rheumatoid arthritis (RA) has been extensively studied. “Balance between Th1 and Th2 cells strongly influences inflammatory responses” In RA synovial tissue, synovial fluid, and serum, analysis of IFNγ and IL4 production to indicate Th1 and Th2 activity, showed that Th1 activity was clearly predominant and Th2 activity was absent compared with control subjects (table 1).4–6 …