Dres Damgaard

Peptidylarginine deiminase 2 is required for tumor necrosis factor alpha-induced citrullination and arthritis, but not neutrophil extracellular trap formation

Citrullination, the post-translational conversion of arginines to citrullines, may contribute to rheumatoid arthritis development given the generation of anti-citrullinated protein antibodies (ACPAs). However, it is not known which peptidylarginine deiminase (PAD) catalyzes the citrullination seen in inflammation. PAD4 exacerbates inflammatory arthritis and is critical for neutrophil extracellular traps (NETs). NETs display citrullinated antigens targeted by ACPAs and thus may be a source of citrullinated protein. However, PAD4 is not required for citrullination in inflamed lungs. PAD2 is important for citrullination in healthy tissues and is present in NETs, but its role in citrullination in the inflamed joint, NETosis and inflammatory arthritis is unknown. Here we use mice with TNFα-induced inflammatory arthritis, a model of rheumatoid arthritis, to identify the roles of PAD2 and PAD4 in citrullination, NETosis, and arthritis. In mice with TNFα-induced arthritis, citrullination in the inflamed ankle was increased as determined by western blot. This increase was unchanged in the ankles of mice that lack PAD4. In contrast, citrullination was nearly absent in the ankles of PAD2-deficient mice. Interestingly, PAD2 was not required for NET formation as assessed by immunofluorescence or for killing of Candida albicans as determined by viability assay. Finally, plasma cell numbers as assessed by flow cytometry, IgG levels quantified by ELISA, and inflammatory arthritis as determined by clinical and pathological scoring were all reduced in the absence of PAD2. Thus, PAD2 contributes to TNFα-induced citrullination and arthritis, but is not required for NETosis. In contrast, PAD4, which is critical for NETosis, is dispensable for generalized citrullination supporting the possibility that NETs may not be a major source of citrullinated protein in arthritis.

Reduced glutathione as a physiological co-activator in the activation of peptidylarginine deiminase

BackgroundCitrullination catalysed by peptidylarginine deiminases (PADs) plays an important pathogenic role in anti-citrullinated protein antibody (ACPA)-positive rheumatoid arthritis (RA) and, possibly, several other inflammatory diseases. Non-physiological reducing agents such as dithiothreitol (DTT) are normally added to the reaction buffer when determining PAD activity in vitro. We investigated the ability of reduced glutathione (GSH), the most abundant intracellular small-molecule thiol in vivo, to activate PADs.MethodsActivity of recombinant human (rh) PAD2 and PAD4, PADs contained in synovial fluid (SF) samples from RA patients and PADs released from phorbol 12-myristate 13-acetate (PMA)-stimulated cells was measured using an in-house PAD activity assay detecting citrullination of fibrinogen.ResultsNo activity of rhPAD2, rhPAD4 or PADs within SF was observed without addition of an exogenous reducing agent. Activity of both recombinant and SF PAD was observed in the presence of 1 mM DTT or 10–15 mM GSH. Following stimulation with PMA, human isolated leucocytes, but not mononuclear cells, released enzymatically active PAD, the activity of which was abolished upon pre-incubation of the cells with the glutathione reductase inhibitor 2-AAPA. No PAD activity was observed in the corresponding supernatants, but addition of exogenous GSH restored activity.ConclusionsCatalytic activity of PAD requires reducing conditions. GSH meets this requirement at concentrations comparable with those found within cells. Active PAD, reduced by GSH, is released from PMA-stimulated granulocytes, but becomes inactivated in the extracellular space.

Increased levels of peptidylarginine deiminase 2 in synovial fluid from anti-CCP-positive rheumatoid arthritis patients: Association with disease activity and inflammatory markers

Abstract Objective. To quantify peptidylarginine deiminase 2 (PAD2) in SF of RA patients and OA patients, and to determine the association between PAD2 levels, disease activity and inflammatory markers in RA. Methods. Blood and SF samples were obtained from 39 RA patients and 40 patients with OA. PAD2 content and PAD activity were measured by means of in-house assays. TNF-α, IL-1β, IL-6, IL-8, IL-10 and IL-12 were measured using flow cytometry. Results. PAD2 levels and PAD activity were higher in SF from RA than OA patients (P < 0.0001 and P = 0.03, respectively), as were all cytokine levels (P < 0.0001–0.05). SF PAD2 levels were higher among anti-CCP-positive patients than among anti-CCP-negative patients (P = 0.005). PAD2 levels in SF from RA patients correlated with disease activity, as assessed by DAS28 (P < 0.005). Moreover, SF PAD2 levels correlated with circulating CRP and anti-CCP levels (P < 0.0006), as well as with leucocyte count, IL-6, IL-8 and IL-10 levels in SF (P < 0.0001–0.02). PAD activity in SF was higher in RA patients than in OA patients, and correlated with PAD2 concentration. Conclusion. Extracellular PAD2 levels in SF correlate with disease activity in RA patients. Anti-CCP-positive RA patients have higher PAD2 levels in SF than anti-CCP-negative RA patients and OA patients. Since we could demonstrate enzymatically active PADs in SF, we propose that free, extracellular PAD is of pathogenic relevance.

Generation of monoclonal antibodies against peptidylarginine deiminase 2 (PAD2) and development of a PAD2-specific enzyme-linked immunosorbent assay

The enzyme peptidylarginine deiminase 2 (PAD2) has been associated with inflammatory diseases, such as rheumatoid arthritis and neurodegenerative diseases including multiple sclerosis. To investigate the association of various diseases with extracellular PAD2, we raised monoclonal antibodies (mAbs) against rabbit PAD2 and evaluated their cross-reactivity with human PAD2 by indirect enzyme-linked immunosorbent assay (ELISA), western blotting and immunohistological staining of inflamed synovial tissue. Moreover, we established a sandwich ELISA detecting human PAD2, based on two different monoclonal antibodies, mAbs DN2 and DN6. The assay had a lower detection limit of 200pg/mL in serum and plasma samples, and showed dilution linearity and recovery ranging from 95 to 106%. The mAbs and the ELISA showed isotype specificity for PAD2. Circulating PAD2 was found in 8/28 (29%) serum samples from healthy donors. In conclusion, several of our mAbs proved useful in western blotting and immunohistochemistry, and the ELISA described here reliably measures PAD2 levels in blood. This allows investigation of PAD2 as a possible biomarker and further investigation of PAD2s involvement in various inflammatory diseases.

Reactive oxygen species inhibit catalytic activity of peptidylarginine deiminase

Abstract Protein citrullination catalysed by peptidylarginine deiminase (PAD) may play an important pathogenic role in several chronic inflammatory diseases and malignancies. PAD2, PAD4, and citrullinated proteins are found in the synovium of rheumatoid arthritis patients. PAD activity is dependent on calcium and reducing conditions. However, reactive oxygen species (ROS) have been shown to induce citrullination of histones in granulocytes. Here we examine the ability of H2O2 and leukocyte-derived ROS to regulate PAD activity using citrullination of fibrinogen as read-out. H2O2 at concentrations above 40 µM inhibited the catalytic activity of PAD2 and PAD4 in a dose-dependent manner. PMA-stimulated leukocytes citrullinated fibrinogen and this citrullination was markedly enhanced when ROS formation was inhibited by the NADPH oxidase inhibitor diphenyleneiodonium (DPI). In contrast, PAD released from stimulated leukocytes was unaffected by exogenously added H2O2 at concentrations up to 1000 µM. The role of ROS in regulating PAD activity may play an important part in preventing hypercitrullination of proteins.

Calgizzarin (S100A11): a novel inflammatory mediator associated with disease activity of rheumatoid arthritis

BackgroundCalgizzarin (S100A11) is a member of the S100 protein family that acts in different tumors by regulating a number of biologic functions. Recent data suggest its association with low-grade inflammation in osteoarthritis (OA). The aim of our study is to compare S100A11 expression in the synovial tissues, synovial fluid and serum of patients with rheumatoid arthritis (RA) and osteoarthritis (OA) and to characterize the potential association between S100A11 and disease activity.MethodsS100A11 protein expression was detected in synovial tissue from patients with RA (n = 6) and patients with OA (n = 6) by immunohistochemistry and immunofluorescence. Serum and synovial fluid S100A11 levels were measured by ELISA in patients with RA (n = 40) and patients with OA (n = 34). Disease activity scores in 28 joints based on C-reactive protein (DAS28-CRP) were used to assess disease activity. Cytokine content in peripheral blood mononuclear cells (PBMCs), synovial fibroblasts (SFs) and synovial fluid was analysed by ELISA, western blotting or cytometric bead array.ResultsS100A11 expression was significantly up-regulated in the synovial lining and sublining layers (p < 0.01) and vessels (p < 0.05) of patients with RA compared to patients with OA, and was associated with fibroblasts and T cells. S100A11 was significantly increased in synovial fluid (p < 0.0001) but not in serum (p = 0.158) from patients with RA compared to patients with OA when adjusted for age and sex. Synovial fluid S100A11 correlated with DAS28 (r = 0.350, p = 0.027), serum CRP (r = 0.463, p = 0.003), synovial fluid leukocyte count (r = 0.677, p < 0.001), anti-cyclic citrullinated peptide antibodies (anti-CCP) (r = 0.424, p = 0.006) and IL-6 (r = 0.578, p = 0.002) and IL-8 (r = 0.740, p < 0.001) in synovial fluid from patients with RA. PBMCs and SFs isolated from patients with RA synthesized and spontaneously secreted higher levels of S100A11 in comparison with PBMCs and SFs from patients with OA (p = 0.011 and 0.03, respectively). S100A11 stimulated the production of the pro-inflammatory cytokine IL-6 by PBMCs (p < 0.05) and SFs (p < 0.01).ConclusionsOur data provide the first evidence of S100A11 up-regulation and its association with inflammation and disease activity in patients with RA.

Dietary exposure to benzoxazinoids enhances bacteria-induced monokine responses by peripheral blood mononuclear cells

SCOPE To examine potentially immunomodulating effects of dietary benzoxazinoids (BXs), present in cereal grains. METHODS AND RESULTS Nineteen healthy volunteers were randomly distributed into two groups, who received diets with high or low content of BXs for 3 wk. After a weeks wash-out, the groups switched diets. Peripheral blood mononuclear cells (PBMCs) were stimulated with Porphyromonas gingivalis, Escherichia coli lipopolysaccharide (LPS), or tetanus toxoid (TT). PBMCs from a healthy donor received the same stimuli in presence of serum from each participant receiving BXs. The production of monokines, T-cell cytokines and T-helper cell proliferation were assessed. A 3-wk diet with high BX content enhanced IL-1β responses against LPS and P. gingivalis, as well as TNF-α response against P. gingivalis, after 24 h of stimulation. Moreover, IL-6 was found to be increased after 7 days of stimulation with LPS. No effect was observed on T-cell cytokines or proliferation. BX levels in serum after a single meal did not modify cytokine responses. CONCLUSION High dietary intake of BXs enhances bacteria-induced production of pro-inflammatory monokines by PBMCs, but not T-cell responses; presumably due to intrinsic changes within PBMCs, built up over 3 wk of BX-rich diet, rather than to an immediate effects of BXs contained in serum.

Relative efficiencies of peptidylarginine deiminase 2 and 4 in generating target sites for anti-citrullinated protein antibodies in fibrinogen, alpha-enolase and histone H3

Objective Peptidylarginine deiminase 2 (PAD2) and PAD4 are expressed in the synovium of rheumatoid arthritis (RA) patients and catalyze citrullination of arginine residues in proteins targeted by anti-citrullinated protein antibodies (ACPAs). Little is known about the relative importance of PAD2 and PAD4 in generating citrullinated self-antigens. Here we investigate the ability of PAD2 and PAD4 to generate citrullinated targets for ACPAs in four human proteins. Methods Synovial fluid (SF) and plasma were collected from 42 RA patients. Human fibrinogen, human alpha-enolase (ENO1), human histone H3, and human serum albumin (HSA) were citrullinated in vitro by PAD2 or PAD4. The total degree of citrullination was determined using the anti-modified citrulline approach. Antibody binding to native and citrullinated proteins was measured by ELISA. Results ACPAs within pooled SF from multiple RA patients reacted equally well with, and cross-reacted with, PAD2- and PAD4-citrullinated fibrinogen. ACPAs from most individual patient SF and plasma samples bound equally well to PAD2- and PAD4-citrullinated fibrinogen or ENO1. When histone H3 was used as target, PAD4 was generally superior in generating epitopes recognized by ACPAs. No binding to citrullinated HSA was observed. Conclusion In most patients, PAD2 and PAD4 are equally efficient in generating citrullinated target sites for ACPAs in fibrinogen and ENO1. The binding of autoantibodies to histone H3 was generally higher after citrullination with PAD4 than with PAD2. Citrullinated HSA is not a target for ACPAs.

PAD Activation in Arthritis

The findings that citrullinated proteins are targeted by the most specific immune response in rheumatoid arthritis, and that citrullination also plays an important role in neurodegenerative diseases and certain cancers, have triggered many researchers to study various aspects of this form of posttranslational modification. This chapter is focused on the conditions that are needed for peptidylarginine deiminases to become active citrullinating enzymes, in particular in relation to joint inflammation as observed in rheumatoid arthritis. After briefly discussing the available methods to study the activity of peptidylarginine deiminases and their substrate specificity, the isoforms that are most relevant for citrullination during inflammation and the factors that are mediating their activation are addressed. Citrullination is crucial for one of the processes that are tightly associated with inflammation, NETosis, which is more extensively discussed in Chap. 8. Finally, peptidylarginine deiminase activation and the resulting citrullination in the context of the inflamed joints in rheumatoid arthritis are described.