Tue Wenzel Kragstrup

A simple set of validation steps identifies and removes false results in a sandwich enzyme-linked immunosorbent assay caused by anti-animal IgG antibodies in plasma from arthritis patients

Rheumatoid arthritis (RA) and spondyloarthritis (SpA) are chronic diseases characterized by activation of the immune system and production of antibodies. Thus, rheumatoid factor, anti-animal IgG antibodies and heterophilic antibodies in plasma samples from arthritis patients can interfere with immunoassays such as sandwich enzyme-linked immunosorbent assay (ELISA) systems often used in arthritis research. However, standard methodologies on how to test for false results caused by these antibodies are lacking. The objective of this study was to design a simple set of steps to validate a sandwich ELISA before using it for measuring analytes in plasma from arthritis patients. An interleukin-24 (IL-24) sandwich ELISA system was prepared with a monoclonal mouse capture antibody and a polyclonal goat detection antibody and tested for interference by rheumatoid factor, anti-animal IgG antibodies and heterophilic antibodies. Plasma samples from 23 patients with RA and SpA were used. No differences were found between plasma samples measured in wells coated with anti-IL-24 specific antibody and in wells coated with isotype control antibody (false positive results), and recombinant human IL-24 was not recovered in spiked samples (false negative results). This interference was removed after preincubating the plasma samples from patients with arthritis with goat or bovine IgG, suggesting that anti-animal IgG antibodies found in the plasma of the arthritis patients caused the false results. Additional testing showed that the signal-to-noise ratio could be increased by titration of the capture and detection antibodies and by using the ELAST amplification system. Finally, the calculated concentration of IL-24 was increased in ethylenediaminetetraacetic acid (EDTA) plasma compared to heparin plasma and serum and decreased with repetitive freeze/thaw cycles of the samples illustrating how sample handling could additionally contribute to the variations reported by different laboratories in measurement of the same analyte. This study proposes a simple set of validation steps to evaluate and optimize a sandwich ELISA before using it for measuring analytes in plasma from arthritis patients. Anti-animal IgG antibodies are also present in healthy individuals, suggesting that validation of ELISA systems for measuring non-arthritis samples could also be improved by this simple set of validation steps.

The p38 MAPK regulates IL-24 expression by stabilization of the 3' UTR of IL-24 mRNA.

Background IL-24 (melanoma differentiation-associated gene-7 (mda-7)), a member of the IL-10 cytokine family, possesses the properties of a classical cytokine as well as tumor suppressor effects. The exact role of IL-24 in the immune system has not been defined but studies have indicated a role for IL-24 in inflammatory conditions such as psoriasis. The tumor suppressor effects of IL-24 include inhibition of angiogenesis, sensitization to chemotherapy, and p38 mitogen-activated protein kinase (MAPK)-mediated apoptosis. Current knowledge on the regulation of IL-24 expression is sparse. Previous studies have suggested that mRNA stabilization is of major importance to IL-24 expression. Yet, the mechanisms responsible for the regulation of IL-24 mRNA stability remain unidentified. As p38 MAPK is known to regulate gene expression by interfering with mRNA degradation we examined the role of p38 MAPK in the regulation of IL-24 gene expression in cultured normal human keratinocytes. Methodology/Principal Findings In the present study we show that anisomycin- and IL-1β- induced IL-24 expression is strongly dependent on p38 MAPK activation. Studies of IL-24 mRNA stability in anisomycin-treated keratinocytes reveal that the p38 MAPK inhibitor SB 202190 accelerates IL-24 mRNA decay suggesting p38 MAPK to regulate IL-24 expression by mRNA-stabilizing mechanisms. The insertion of the 3′ untranslated region (UTR) of IL-24 mRNA in a tet-off reporter construct induces degradation of the reporter mRNA. The observed mRNA degradation is markedly reduced when a constitutively active mutant of MAPK kinase 6 (MKK6), which selectively activates p38 MAPK, is co-expressed. Conclusions/Significance Taken together, we here report p38 MAPK as a regulator of IL-24 expression and determine interference with destabilization mediated by the 3′ UTR of IL-24 mRNA as mode of action. As discussed in the present work these findings have important implications for our understanding of IL-24 as a tumor suppressor protein as well as an immune modulating cytokine.

Shedding of Large Functionally Active CD11/CD18 Integrin Complexes from Leukocyte Membranes during Synovial Inflammation Distinguishes Three Types of Arthritis through Differential Epitope Exposure

CD18 integrins are adhesion molecules expressed on the cell surface of leukocytes and play a central role in the molecular mechanisms supporting leukocyte migration to zones of inflammation. Recently, it was discovered that CD11a/CD18 is shed from the leukocyte surface in models of inflammation. In this study, we show that shedding of human CD11/CD18 complexes is a part of synovial inflammation in rheumatoid arthritis and spondyloarthritis but not in osteoarthritis. In vivo and in vitro data suggest that the shedding is driven by TNF-α, which links the process to central events in the inflammatory response. The shed complexes contain multiple heterodimers of CD11/CD18, are variable in size, and differ according to the type of synovial inflammation. Furthermore, the differential structures determine the avidity of binding of the complexes to the ICAM-1. With the estimated concentrations of CD11/CD18 in plasma and synovial fluid a significant coverage of binding sites in ICAM-1 for CD18 integrins is expected. Based on cell adhesion experiments in vitro, we hypothesize that the large soluble complexes of CD11/CD18 act in vivo to buffer leukocyte adhesion by competing with the membrane-bound receptors for ICAM-1 binding sites. As reported here for synovial inflammation changes in the concentration or structure of these complexes should be considered as likely contributors to disease activity.

Infections as risk factor for autoimmune diseases - A nationwide study.

Viruses, bacteria and other infectious pathogens are the major postulated environmental triggers of autoimmunity. In the present nation-wide study we describe the association between infections and 29 autoimmune diseases. We used the Danish Civil Registration System to identify 4.5 million persons born between 1945 and 2000. Information on infections and autoimmune diseases was obtained from the Danish Hospital Register. The cohort was followed from 1977 to 2012. Incidence rate ratios for developing an autoimmune disease were estimated using poisson regression. We found an association between hospital admission for an infection and 29 autoimmune diseases. This study shows that infections are risk factors for a broad spectrum of autoimmune diseases in a dose-response and temporal manner, in agreement with the hypothesis that infections are an environmental risk factor contributing to the etiology of autoimmune diseases together with genetic factors.

Elimination of erroneous results in flow cytometry caused by antibody binding to Fc receptors on human monocytes and macrophages

Nonspecific binding of monoclonal antibodies (mAbs) to Fc‐receptors on leukocytes is an important cause of background fluorescence in flow cytometry, and failing to block such nonspecific binding can lead to erroneous results. A major part of previous studies on blocking reagents for flow cytometry have been done in mice, and published results are not completely in agreement. In humans, Fc‐receptors are found on most leukocytes, with highest abundance on monocytes/macrophages. Therefore, in the present study our aim was to thoroughly investigate the efficiency of different commonly used blocking reagents regarding inhibition of nonspecific binding of mouse mAbs to human peripheral blood mononuclear cells (MNCs) and monocyte‐derived macrophages (MDMs). Monocytes and MDMs showed strong nonspecific binding of IgG1 and IgG2a isotypes, but not IgG2b. In contrast, B‐cells, T‐cells, and NK‐cells did not substantially bind any of the mouse isotype control antibodies evaluated (IgG1, IgG2a, and IgG2b). Importantly, we show that binding of IgG1 and IgG2a to monocytes and MDMs can be eliminated by blocking, either with a commercial Fc‐blocking reagent, with mouse or human serum, or with mouse or human IgG in high concentration. Previously, isotype controls have been widely used in flow cytometry assays. However, we show that such controls may be highly unreliable, and we believe they should not be used as gating controls, or to determine background signal. Based on these results, as well as considerations of price and applicability, our recommendation is not to use isotypes as gating controls in flow cytometry, but instead to use 100 μg/mL of purified human IgG as blocking reagent for elimination of nonspecific binding of mouse mAbs to human MNCs and MDMs.

Toll‐like receptor 2 and 4 induced interleukin‐19 dampens immune reactions and associates inversely with spondyloarthritis disease activity

Spondyloarthritis (SpA) is a group of immune mediated inflammatory diseases affecting joints, gut, skin and entheses. The inflammatory process involves activation of Toll‐like receptor (TLR)‐2 and TLR‐4 and production of cytokines and chemokines such as monocyte chemoattractant protein 1 (CCL2/MCP‐1). This proinflammatory chemokine recruits monocytes to sites of inflammation and is central in the development of several immune‐mediated inflammatory diseases. Interleukin (IL)‐19 is a member of the IL‐10 family of cytokines. IL‐19‐deficient mice are more susceptible to innate‐mediated colitis and develop more severe inflammation in response to injury. In this work, we studied inducers of IL‐19 production and effect of IL‐19 on the production of CCL2/MCP‐1 and proinflammatory cytokines in peripheral blood mononuclear cells (PBMCs) from healthy controls (HCs) and in PBMCs and synovial fluid mononuclear cells (SFMCs) from SpA patients. Further, we measured IL‐19 in plasma from HCs and in plasma and synovial fluid from SpA patients. Constitutive IL‐19 expression was present in both PBMCs and SFMCs and the secretion of IL‐19 was increased by TLR‐2 and TLR‐4 ligands. Neutralizing IL‐19 in HC PBMCs and SpA SFMCs resulted in increased production of CCL‐2/MCP‐1. IL‐19 concentrations were decreased in synovial fluid compared with plasma and associated inversely with disease activity in SpA. SpA SFMCs produced less IL‐19 in response to LPS compared with HC PBMCs. These findings indicate that IL‐19 production is diminished in SpA. Taken together, impaired IL‐19 control of the innate immune system might be involved in the pathogenesis of SpA.

Increased interleukin (IL)-20 and IL-24 target osteoblasts and synovial monocytes in spondyloarthritis: IL-20 and IL-24 in SpA

The pathogenesis of spondyloarthritis (SpA) involves activation of the innate immune system, inflammation and new bone formation. The two cytokines interleukin (IL)‐20 and IL‐24 have been shown to link innate immune activation and tissue homeostasis. We hypothesized that these two cytokines are secreted as part of activation of the innate immune system and affect bone homeostasis in SpA. IL‐20 and IL‐24 were measured in plasma from axial SpA patients (n = 83). Peripheral SpA patients (n = 16) were included for in‐vitro cell culture studies. The plasma IL‐20 and IL‐24 levels were increased in SpA patients compared with healthy controls (HCs) by 57 and 83%, respectively (both P < 0·0001). The Toll‐like receptor 4‐induced secretion of the two cytokines was greater in SpA peripheral blood mononuclear cells (PBMCs) compared with HC PBMCs. IL‐20 and IL‐24 increased the production of monocyte chemoattractant protein‐1 by activated SpA synovial fluid monocytes, decreased the production of Dickkopf‐1 by SpA fibroblast‐like synovial cells and induced mineralization in human osteoblasts. Taken together, our findings indicate disease‐aggravating functions of IL‐20 and IL‐24 in SpA.

Increased IL‐20 and IL‐24 target osteoblasts and synovial monocytes in spondyloarthritis

The pathogenesis of spondyloarthritis (SpA) involves activation of the innate immune system, inflammation and new bone formation. The two cytokines interleukin (IL)‐20 and IL‐24 have been shown to link innate immune activation and tissue homeostasis. We hypothesized that these two cytokines are secreted as part of activation of the innate immune system and affect bone homeostasis in SpA. IL‐20 and IL‐24 were measured in plasma from axial SpA patients (n = 83). Peripheral SpA patients (n = 16) were included for in‐vitro cell culture studies. The plasma IL‐20 and IL‐24 levels were increased in SpA patients compared with healthy controls (HCs) by 57 and 83%, respectively (both P < 0·0001). The Toll‐like receptor 4‐induced secretion of the two cytokines was greater in SpA peripheral blood mononuclear cells (PBMCs) compared with HC PBMCs. IL‐20 and IL‐24 increased the production of monocyte chemoattractant protein‐1 by activated SpA synovial fluid monocytes, decreased the production of Dickkopf‐1 by SpA fibroblast‐like synovial cells and induced mineralization in human osteoblasts. Taken together, our findings indicate disease‐aggravating functions of IL‐20 and IL‐24 in SpA.

The IL-20 receptor axis in immune-mediated inflammatory arthritis: novel links between innate immune recognition and bone homeostasis.

The treatment of rheumatoid arthritis (RA) and spondyloarthritis (SpA) was transformed a little over a decade ago by the introduction of agents neutralizing the pro-inflammatory cytokine tumour necrosis factor (TNF)-α. Nevertheless, some patients do not achieve remission and the inhibition of the normal immune system with current drugs increases the risk of infection. The interleukin (IL)-20 receptor (IL-20R) axis is pivotal for tissue homeostasis. By contrast, this axis does not seem to directly activate cells of the immune system. Thus, modulation of the IL-20R axis might not result in increased risk of infection. The IL-20R axis consists of the three cytokines IL-19, IL-20, and IL-24 (termed the IL-20R cytokines) and their shared receptors. All three cytokines bind the receptor complex of IL-20R2/IL-20R1 whereas only IL-20 and IL-24 also bind the receptor complex of IL-20R2/IL-22R1. This short review describes how the IL-20R axis could be a novel link between innate immune recognition and bone homeostasis. The IL-20R cytokines are produced in response to both danger-associated molecular patterns and immune complexes formed by RA-associated autoantibodies. This could be of importance because these mediators can thus be present even in situations without inflammation. IL-19 shows anti-inflammatory properties in arthritis through IL-20R1. IL-20 and IL-24 through IL-22R seem to participate in the recruitment of mononuclear cells to the synovial joint and to sites of bone erosion in particular. Our results indicate that dual inhibition of IL-20 and IL-24 or attenuation of the shared IL-22R subunit could have a beneficial effect on radiographic progression, especially in seropositive RA.

A disintegrin and metalloprotease-17 and galectin-9 are important regulators of local 4-1BB activity and disease outcome in rheumatoid arthritis

OBJECTIVE Co-stimulatory T cell cytokines are important in the progression of RA. This study investigates the interplay between 4-1BB, a disintegrin and metalloprotease-17 (ADAM17) and galectin-9 (Gal-9) in RA. METHODS Stimulated mononuclear cells from patients with chronic RA (n = 12) were co-incubated with tissue inhibitor of metalloproteinase, 4-1BB ligand and Gal-9. Plasma samples were examined for soluble 4-1BB (s4-1BB) in newly diagnosed, treatment-naïve patients with RA (n = 97). The 28-joint DAS with CRP (28DAS-CRP), total Sharp score, erosion score and joint space narrowing were used to evaluate treatment outcome serially over a 2-year period. RESULTS RA CD4(+) and CD8(+) synovial T cells express high levels of 4-1BB. The addition of TNF-α to cultured synovial mononuclear cells increased shedding of 4-1BB. 4-1BB ligand only increased TNF-α shedding in combination with Gal-9. RNA interference-mediated knockdown of ADAM17 or the addition of an ADAM17 inhibitor reduced the 4-1BB shedding. Shedding of 4-1BB was not influenced by Gal-9. Plasma levels of s4-1BB were increased in early RA and correlated with the number of swollen joints at baseline. After 3 months of treatment, the plasma levels of s4-1BB were equal to those of the controls. Baseline plasma levels of s4-1BB were inversely correlated with DAS28-CRP after 2 years of treatment, but not with total Sharp score, erosion score or joint space narrowing. CONCLUSION ADAM17 induces 4-1BB shedding in RA. Gal-9 is pivotal for the function of 4-1BB and induction of TNF-α. Furthermore, high plasma levels of s4-1BB were associated with the number of swollen joints, but also with a low DAS28-CRP after 2 years treatment in early RA.