Linda Mathsson

Antibodies against citrullinated vimentin in rheumatoid arthritis: Higher sensitivity and extended prognostic value concerning future radiographic progression as compared with antibodies against cyclic citrullinated peptides

OBJECTIVE The Sa autoantigen can be found in inflamed synovium of patients with rheumatoid arthritis (RA), and at least part of the humoral RA-specific anti-Sa response is directed against citrullinated vimentin. This study was undertaken to evaluate the sensitivity, specificity, and prognostic value of determination of levels of antibodies against modified citrullinated vimentin (anti-MCV) as compared with antibodies against cyclic citrullinated peptides (anti-CCP) in an inception cohort of patients with early RA. METHODS Clinical data, radiographs, and measurements of levels of anti-MCV and anti-CCP antibodies were obtained in 273 patients with early RA at baseline, after 3 months, and after 1, 2, 3, and 5 years. Autoantibodies were also analyzed in 100 healthy controls. RESULTS Of the 273 patients, 193 (70.7%) were anti-MCV positive and 158 (57.9%) were anti-CCP positive at the time of diagnosis, with nearly equal specificities (95% and 96%, respectively). Forty (14.7%) were anti-MCV positive only, and 5 (1.8%) were anti-CCP positive only. Anti-MCV-positive and anti-MCV-negative patients had similar disease activity at baseline, but presence of anti-MCV was predictive of subsequent high disease activity and continued radiographic progression. Changes in anti-MCV level showed stronger correlation with changes in clinical parameters than did changes in anti-CCP level. The subgroup of patients who were anti-MCV positive and anti-CCP negative showed a higher rate of radiographic destruction than did patients who were negative for both anti-MCV and anti-CCP. CONCLUSION These findings show that when patients with early RA are compared with healthy controls, analysis of anti-MCV yields greater sensitivity and unchanged specificity as compared with analysis of anti-CCP. Anti-MCV also appears to perform better than anti-CCP in identifying poor radiographic prognosis in patients with early RA.

Multiplex analyses of antibodies against citrullinated peptides in individuals prior to development of rheumatoid arthritis.

OBJECTIVE The presence of antibodies against cyclic citrullinated peptides has been demonstrated to precede the onset of symptoms of rheumatoid arthritis (RA) by several years. The aim of this study was to analyze antibodies against 10 citrullinated autoantigen-derived peptides for reactivity before the onset of RA symptoms. METHODS A case-control study was conducted within the Medical Biobank of Northern Sweden. The study was performed in 409 individuals, 386 of whom donated 717 blood samples before the onset of symptoms of RA (pre-patients). The median period of time predating the onset of RA was 7.4 years. A total of 1,305 population-based control subjects were also studied. Antibodies to 10 citrullinated peptides, fibrinogen α573 (Fibα573), Fibα591, Fibβ36-52, Fibβ72, Fibβ74, α-enolase (citrullinated α-enolase peptide 1 [CEP-1]), triple-helical type II collagen peptide C1 (citC1III), filaggrin, vimentin 2-17 (Vim2-17), and Vim60-75, were analyzed using a microarray system. RESULTS The fluorescence intensity of antibodies against Fibβ36-52, Fibβ74, CEP-1, citC1III, and filaggrin was significantly increased in pre-patients compared with controls (P<0.001). The levels of the earliest-detectable antibodies (Fibα591 and Vim60-75) fluctuated over time, with only a slight increase after the onset of disease. The frequency of antibodies against Fibβ36-52, CEP-1, and filaggrin increased gradually, reaching the highest levels before symptom onset. The frequency of a cluster of antibodies, citC1III, Fibα573, and Fibβ74, increased only slightly before the onset of symptoms but increased prominently after disease onset. The odds ratio for the development of RA in individuals expressing both CEP-1 and Fibβ36-52 antibodies (using data from samples obtained <3.35 years predating symptom onset) was 40.4 (95% confidence interval 19.8-82.3) compared with having either antibody alone. CONCLUSION Development of an immune response toward citrullinated peptides is initially restricted but expands with time to induce a more specific response, with levels, particularly those of antibodies against CEP-1, Fibβ36-52, and filaggrin, increasing during the predating time period closer to the onset of symptoms.

Immune complexes from SLE sera induce IL10 production from normal peripheral blood mononuclear cells by an FcγRII dependent mechanism: implications for a possible vicious cycle maintaining B cell hyperactivity in SLE

Background: Raised interleukin (IL)6 and IL10 levels are thought to contribute to the pathogenesis of systemic lupus erythematosus (SLE) by enhancing autoantibody production and immune complex (IC) formation. These immune complexes can then stimulate cellular reactions through Fc and complement receptors. Objective: To investigate whether circulating SLE ICs stimulate type 2 cytokine production. Methods: Twenty serum samples from patients with active SLE were compared with sera from 18 healthy controls. Sera and polyethylene glycol (PEG) precipitates from sera were added to peripheral blood mononuclear cell (PBMC) cultures, and the production of IL10 and IL6 was investigated by enzyme linked immunospot assay (ELISPOT) and enzyme linked immunosorbent assay (ELISA). Fc gamma receptor (FcγR) antibodies were used in blocking experiments, and flow cytometry was used to assess the correlation between monocyte FcγR expression and IC-induced cytokine production. Results: Ten per cent dilutions of the SLE sera induced a significantly increased number of IL10-producing cells in comparison with control sera (median, 11.75 v 1.25 spot forming cells/50 000 PBMC; p<0.0001). PEG precipitates from SLE sera also induced significantly increased levels of IL10 (p=0.016) and IL6 (p=0.042) in comparison with control PEG precipitates. IL10 production induced by SLE PEG precipitates or by artificial ICs could be blocked by anti-FcγRII antibodies, and the FcγRII expression on CD14+ monocytes correlated with the IC-induced production of IL10 and IL6. Conclusions: SLE sera stimulate IL10 and IL6 production from PBMC, and this effect is at least partly explained by precipitable ICs acting through FcγRII. This effect provides a possible mechanism for the enhanced production of IL10 in SLE, whereby B cell activation, antibody production, IC stimulated monocytes/macrophages, and type 2 cytokines create a vicious cycle that may help to maintain B cell hyperactivity in SLE.

Regulation of the interferon-alpha production induced by RNA-containing immune complexes in plasmacytoid dendritic cells.

OBJECTIVE Interferon-alpha (IFNalpha) is produced in several autoimmune diseases, including systemic lupus erythematosus (SLE), and may be important in their pathogenesis. We undertook this study to investigate how IFNalpha production induced by RNA-containing immune complexes (ICs) in plasmacytoid dendritic cells (PDCs) is regulated. METHODS Normal PDCs purified from peripheral blood mononuclear cells (PBMCs) were cocultivated with other cell populations isolated from healthy individuals or SLE patients. IFNalpha production was induced by RNA-containing ICs, which consisted of anti-RNP autoantibodies and U1 small nuclear RNP particles, and the effects of prostaglandin E2 (PGE2), reactive oxygen species (ROS), or the cytokines IFNalpha2b, granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin-10 (IL-10), or tumor necrosis factor alpha (TNFalpha) were explored. RESULTS Monocytes inhibited IFNalpha production by PDCs in PBMC cultures, while natural killer (NK) cells were stimulatory. The monocytes had little effect on IFNalpha production by pure PDCs but inhibited its stimulation by NK cells. Monocytes from SLE patients were less inhibitory. Exposure of PBMCs or PDCs to IFNalpha2b/GM-CSF increased their IFNalpha production. RNA-containing ICs caused production of ROS, PGE2, and TNFalpha, especially in monocytes. These mediators and IL-10 suppressed IFNalpha production in PBMC cultures, with ROS and PGE2 also inhibiting IFNalpha production by purified PDCs. Inhibition by all of these agents, except for ROS, was abolished by IFNalpha2b/GM-CSF. The inhibitory effect of monocytes was significantly counteracted by the ROS scavengers serotonin and catalase. CONCLUSION IFNalpha production induced by RNA-containing ICs in PDCs is regulated by a network of interactions between monocytes, NK cells, and PDCs, involving several pro- and antiinflammatory molecules. This should be considered when designing and applying new therapies.

Inflammatory cytokines, behaviour and age as determinants of self-rated health in women

Self-rated health is a powerful and independent predictor of long-term health, but its biological basis is unknown. We have shown previously that self-rated health is associated with increased levels of circulating cytokines in women. The main aim of the present study was to increase the understanding of the association between markers of wellbeing, such as self-rated health, and cytokines and to investigate the impact of age on these associations. In 174 female consecutive primary health care patients divided into three age groups, we examined subjective ratings of health and aspects of wellbeing and circulating levels of IL (interleukin)-1beta, IL-1ra (IL-1 receptor antagonist), IL-6 and TNF-alpha (tumour necrosis factor-alpha). Poor self-rated health was significantly associated with higher levels of TNF-alpha in all of the age groups. For IL-1beta and IL-1ra, the correlations with self-rated health were significant only in the oldest age group. Lower ratings of other measurements of health and wellbeing were related to higher levels of cytokines, most pronounced for TNF-alpha and IL-1beta, and in the middle and olderst age groups. More symptoms resembling a sickness response induced by inflammation were implicated to be associated with lower self-rated health. The strength of the association between inflammatory cytokines and poor health perception increased with advanced age, indicating an increased vulnerability for inflammatory activity during aging. It is suggested that higher levels of TNF-alpha are connected to a sickness response that, in turn, is connected to self-rated health. The results provide a possible psychobiological basis to understand better diffuse subjective symptoms and poor subjective health in women.

High anti-collagen type-II antibody levels and induction of proinflammatory cytokines by anti-collagen antibody-containing immune complexes in vitro characterise a distinct rheumatoid arthritis phenotype associated with acute inflammation at the time of disease onset

Objective: To investigate whether the cytokine-inducing properties of surface-bound collagen type II (CII)-containing immune complexes (IC), which were reported earlier, have any clinical impact. Methods: Anti-CII serology was analysed in 274 patients with early rheumatoid arthritis (RA). Patients with increased levels of anti-CII were followed serially for 1–5 years with regard to anti-CII IC-induced levels of tumour necrosis factor (TNF)α, interleukin (IL)1β and IL8. Levels of antibodies and IC-induced cytokines were compared with clinical indices over 5 years of follow-up. Results: 5/100 healthy controls and 24/274 (8.8%) patients with RA exhibited increased levels (>29 arbitrary units (AU)/ml) of anti-native CII antibodies, a non-significant difference. 9/274 (3.3%) patients with RA and no controls comprised a discrete group with high anti-CII levels >450 AU/ml. These high anti-CII level sera were associated with induction of pro-inflammatory cytokines by anti-CII-containing IC formed in vitro. 8/9 patients with high baseline anti-CII levels exhibited a parallel decline in antibody levels, IC-induced cytokines, C reactive protein (CRP) and erythrocyte sedimentation rate (ESR). Anti-CII-positive patients had significantly increased levels of CRP and ESR at baseline, but not later during the follow-up. Conclusions: Anti-native CII-positive patients with RA have a distinct clinical phenotype characterised by an early acute phase response that might be driven by anti-CII-containing IC in joint cartilage.

The inhibitory Fc gamma IIb receptor dampens TLR4-mediated immune responses and is selectively up-regulated on dendritic cells from rheumatoid arthritis patients with quiescent disease.

Rheumatoid arthritis (RA) is a common autoimmune disease leading to profound disability and premature death. Although a role for FcγRs and TLRs is accepted, their precise involvement remains to be elucidated. FcγRIIb is an inhibitory FcR important in the maintenance of tolerance. We hypothesized that the inhibitory FcγRIIb inhibits TLR responses on monocyte-derived dendritic cells (DC) and serves as a counterregulatory mechanism to dampen inflammation, and we surmised that this mechanism might be defective in RA. The expression of the inhibitory FcγRIIb was found to be significantly higher on DCs from RA patients having low RA disease activity in the absence of treatment with antirheumatic drugs. The expression of activating FcγRs was similarly distributed among all RA patients and healthy controls. Intriguingly, only DCs with a high expression of FcγRIIb were able to inhibit TLR4-mediated secretion of proinflammatory cytokines when stimulated with immune complexes. In addition, when these DCs were coincubated with the combination of a TLR4 agonist and immune complexes, a markedly inhibited T cell proliferation was apparent, regulatory T cell development was promoted, and T cells were primed to produce high levels of IL-13 compared with stimulation of the DCs with the TLR4 agonist alone. Blocking FcγRIIb with specific Abs fully abrogated these effects demonstrating the full dependence on the inhibitory FcγRIIb in the induction of these phenomena. This TLR4-FcγRIIb interaction was shown to dependent on the PI3K and Akt pathway.

Validation of a multiplex chip-based assay for the detection of autoantibodies against citrullinated peptides

IntroductionAutoantibodies directed against citrullinated proteins/peptides (ACPAs) are highly specific and predictive for the development of rheumatoid arthritis (RA). Different subgroups of RA patients, which have different prognoses and may require different treatments, are characterized by different autoantibody profiles. The objective of this study was to develop a microarray for the detection of multiple RA-associated autoantibodies, initially focusing on responses against citrullinated epitopes on candidate autoantigens in RA.MethodsThe microarray is based on Phadias ImmunoCAP ISAC system, with which reactivity to more than 100 antigens can be analyzed simultaneously, by using minute serum volumes (< 10 μl). Twelve citrullinated peptides, and the corresponding native arginine-containing control peptides, were immobilized in an arrayed fashion onto a chemically modified glass slide, allowing a three-dimensional layer with high binding capacity. The assay was optimized concerning serum dilution and glass surface, whereas each individual antigen was optimized concerning coupling chemistry, antigen concentration, and selection of spotting buffer. The performance of each peptide in the ImmunoCAP ISAC system was compared with the performance in enzyme-linked immunosorbent assays (ELISAs). Serum from 927 RA patients and 461 healthy controls from a matched case-control study were applied onto reaction sites on glass slides, followed by fluorescent-labeled anti-human immunoglobulin G (IgG) antibody. Fluorescence intensities were detected with a laser scanner, and the results analyzed by using image-analysis software.ResultsStrong correlations between the ImmunoCAP ISAC system and ELISA results were found for individual citrullinated peptides (Spearman ρ typically between 0.75 and 0.90). Reactivity of RA sera with the peptides was seen mainly in the anticyclic citrullinated peptide 2 (CCP2)-positive subset, but some additional reactivity with single citrullinated peptides was seen in the anti-CCP2-negative subset. Adjusting for reactivity against arginine-containing control peptides did not uniformly change the diagnostic performance for antibodies against the individual citrullinated peptides.ConclusionsThe multiplexed array, for detection of autoantibodies against multiple citrullinated epitopes on candidate RA autoantigens, will be of benefit in studies of RA pathogenesis, diagnosis, and potentially as a guide to individualized treatment.

Antibodies to citrullinated α-enolase peptide 1 and clinical and radiological outcomes in rheumatoid arthritis

Introduction The anticyclic citrullinated peptide 2 (anti-CCP2) assay is a generic test for antibodies to citrullinated proteins, among which there is a subset of about 50% with antibodies to citrullinated enolase peptide 1 (CEP-1). The anti-CEP-1 positive subset is strongly associated with the HLA-DRB1 shared epitope and its interaction with smoking. Objective To investigate whether anti-CEP-1 antibodies may be helpful in predicting outcome. Methods Anti-CEP-1 and anti-CCP2 antibodies were measured in two prospective cohorts of patients (Karolinska n=272, Norfolk Arthritis Register (NOAR) n=408) with early rheumatoid arthritis (RA). Outcomes measured were C-reactive protein, erythrocyte sedimentation rate, visual analogue scales for pain and global assessment of disease activity, Health Assessment Questionnaire, physicians assessment, swollen and tender joint counts and radiological progression. Results Anti-CCP2 antibodies were present in 57% and 50%, and anti-CEP-1 in 27% and 24% of the Karolinska and NOAR cohorts, respectively. Importantly, no statistically significant differences in clinical outcomes were demonstrated between the anti-CEP-1−/CCP2+ and the anti-CEP-1+/CCP2+ subsets in either cohort, or in radiological outcomes in the Karolinska cohort. Conclusion Although antibodies to specific citrullinated proteins may have distinct genetic and environmental risk factors, the similarity in clinical phenotype suggests that they share common pathways in the pathogenesis of joint disease in RA.

Cytokine induction by circulating immune complexes and signs of in-vivo complement activation in systemic lupus erythematosus are associated with the occurrence of anti-Sjögren's syndrome A antibodies

Circulating immune complexes (IC) and levels of IC‐induced cytokines have been correlated with complement activation and autoantibody profiles in systemic lupus erythematosus (SLE). SLE sera were analysed concerning levels of immune complexes (IC), classical complement function and different antinuclear and anti‐C‐reactive protein (CRP) autoantibodies. Blood mononuclear cells from healthy donors were stimulated with isolated IC and production of interleukin (IL)‐10, IL‐6 and IL‐12p40 was measured. Functional experiments revealed that increased levels of IC‐induced cytokines were associated with both increased classical complement activation and the occurrence of anti‐Sjögrens syndrome A (SSA) and anti‐SSB but not other autoantibodies. Biochemical measurement of circulating IC showed that the degree of complement activation and the occurrence of anti‐SSA were synergistically associated with levels of circulating IC in SLE sera, as complement activation was a prerequisite for the enhancing effect of anti‐SSA. Anti‐CRP was associated with complement activation, but not with other autoantibodies. Our results indicate that anti‐SSA and possibly anti‐SSB antibodies influence IC formation and subsequent IC‐induced cytokine induction, and that they thereby participate in the inflammatory process in active SLE.