Heidi Kokkonen

Up-regulation of cytokines and chemokines predates the onset of rheumatoid arthritis

OBJECTIVE To identify whether cytokines, cytokine-related factors, and chemokines are up-regulated prior to the development of rheumatoid arthritis (RA). METHODS A nested case-control study was performed in 86 individuals who had donated blood samples before experiencing any symptoms of disease (pre-patients) and 256 matched control subjects (1:3 ratio). In 69 of the pre-patients, blood samples were also obtained at the time of the diagnosis of RA. The plasma levels of 30 cytokines, related factors, and chemokines were measured using a multiplex system. RESULTS The levels of several of the cytokines, cytokine receptors, and chemokines were significantly increased in individuals before disease onset compared with the levels in control subjects; i.e., those representing signs of general immune activation (interleukin-1beta [IL-1beta], IL-2, IL-6, IL-1 receptor antagonist, and tumor necrosis factor), activation of Th1 cells (interferon-gamma, IL-12), Th2 cells (IL-4, eotaxin), Treg cells (IL-10), bone marrow-derived factors (IL-7, granulocyte-macrophage colony-stimulating factor, and granulocyte colony-stimulating factor), as well as chemokines (monocyte chemotactic protein 1 and macrophage inflammatory protein 1alpha). The levels were particularly increased in anti-cyclic citrullinated peptide antibody- and rheumatoid factor-positive individuals, and the concentration of most of these increased further after disease onset. The concentration of IL-17 in individuals before disease onset was significantly higher than that in patients after disease onset. Individuals in whom RA subsequently developed were discriminated from control subjects mainly by the presence of Th1 cells, Th2 cells, and Treg cell-related cytokines, while chemokines, stromal cell-derived cytokines, and angiogenic-related markers separated patients after the development of RA from individuals before the onset of RA. CONCLUSION Individuals in whom RA later developed had significantly increased levels of several cytokines, cytokine-related factors, and chemokines representing the adaptive immune system (Th1, Th2, and Treg cell-related factors); after disease onset, the involvement and activation of the immune system was more general and widespread.

Antibodies of IgG, IgA and IgM isotypes against cyclic citrullinated peptide precede the development of rheumatoid arthritis.

IntroductionWe and others have previously shown that antibodies against cyclic citrullinated proteins (anti-CCP) precede the development of rheumatoid arthritis (RA) and in a more recent study we reported that individuals who subsequently developed RA had increased concentrations of several cytokines and chemokines years before the onset of symptoms of joint disease. Here we aimed to evaluate the prevalence and predictive values of anti-CCP antibodies of IgG, IgM and IgA isotype in individuals who subsequently developed RA and also to relate these to cytokines and chemokines, smoking, genetic factors and radiographic score.MethodsA case-control study (1:4 ratio) was nested within the Medical Biobank and the Maternity cohorts of Northern Sweden. Patients with RA were identified from blood donors predating the onset of disease by years. Matched controls were selected randomly from the same registers. IgG, IgA and IgM anti-CCP2 antibodies were determined using EliA anti-CCP assay on ImmunoCAP 250 (Phadia AB, Uppsala, Sweden).ResultsOf 86 patients with RA identified as blood donors prior to the onset of symptoms, samples were available from 71 for analyses. The median (Q1 to Q3) predating time was 2.5 years (1.1 to 5.9 years). The sensitivity of anti-CCP antibodies in the pre-patient samples was 35.2% for IgG, 23.9% for IgA, and 11.8% for IgM. The presence of IgG and IgA anti-CCP antibodies was highly significant compared with controls. IgG and IgA anti-CCP2 predicted RA significantly in conditional logistic regression models odds ratio (OR) = 94.1, 95% confidence interval (CI) 12.7 to 695.4 and OR = 11.1, 95% CI 4.4 to 28.1, respectively, the IgM anti-CCP showed borderline significance OR = 2.5 95% CI 0.9 to 6.3. Concentrations of all anti-CCP isotypes increased the closer to the onset of symptoms the samples were collected with an earlier and higher increase for IgG and IgA compared with IgM anti-CCP. IgA and IgG anti-CCP positive individuals had different patterns of up-regulated chemokines and also, smoking brought forward the appearance of IgA anti-CCP antibodies in pre-RA individuals.ConclusionsAnti-CCP2 antibodies of both the IgG and IgA isotypes pre-dated the onset of RA by years; also, both IgG and IgA anti-CCP2 antibodies predicted the development of RA, with the highest predictive value for IgG anti-CCP2 antibodies.

Autoantibodies predate the onset of systemic lupus erythematosus in northern Sweden

IntroductionAutoantibodies have a central role in systemic lupus erythematosus (SLE). The presence of autoantibodies preceding disease onset by years has been reported both in patients with SLE and in those with rheumatoid arthritis, suggesting a gradual development of these diseases. Therefore, we sought to identify autoantibodies in a northern European population predating the onset of symptoms of SLE and their relationship to presenting symptoms.MethodsThe register of patients fulfilling the American College of Rheumatology criteria for SLE and with a given date of the onset of symptoms was coanalysed with the register of the Medical Biobank, Umeå, Sweden. Thirty-eight patients were identified as having donated blood samples prior to symptom onset. A nested case-control study (1:4) was performed with 152 age- and sex-matched controls identified from within the Medical Biobank register (Umeå, Sweden). Antibodies against anti-Sjögrens syndrome antigen A (Ro/SSA; 52 and 60 kDa), anti-Sjögrens syndrome antigen B, anti-Smith antibody, ribonucleoprotein, scleroderma, anti-histidyl-tRNA synthetase antibody, double-stranded DNA (dsDNA), centromere protein B and histones were analysed using the AtheNA Multi-Lyte ANA II Plus Test System on a Bio-Plex Array Reader (Luminex200). Antinuclear antibodies test II (ANA II) results were analysed using indirect immunofluorescence on human epidermal 2 cells at a sample dilution of 1:100.ResultsAutoantibodies against nuclear antigens were detected a mean (±SD) of 5.6 ± 4.7 years before the onset of symptoms and 8.7 ± 5.6 years before diagnosis in 63% of the individuals who subsequently developed SLE. The sensitivity (45.7%) was highest for ANA II, with a specificity of 95%, followed by anti-dsDNA and anti-Ro/SSA antibodies, both with sensitivities of 20.0% at specificities of 98.7% and 97.4%, respectively. The odds ratios (ORs) for predicting disease were 18.13 for anti-dsDNA (95% confidence interval (95% CI), 3.58 to 91.84) and 11.5 (95% CI, 4.54 to 28.87) for ANA. Anti-Ro/SSA antibodies appeared first at a mean of 6.6 ± 2.5 years prior to symptom onset. The mean number of autoantibodies in prediseased individuals was 1.4, and after disease onset it was 3.1 (P < 0.0005). The time predating disease was shorter and the number of autoantibodies was greater in those individuals with serositis as a presenting symptom in comparison to those with arthritis and skin manifestations as the presenting symptoms.ConclusionsAutoantibodies against nuclear antigens were detected in individuals who developed SLE several years before the onset of symptoms and diagnosis. The most sensitive autoantibodies were ANA, Ro/SSA and dsDNA, with the highest predictive OR being for anti-dsDNA antibodies. The first autoantibodies detected were anti-Ro/SSA.

Influence of female hormonal factors, in relation to autoantibodies and genetic markers, on the development of rheumatoid arthritis in northern Sweden: a case–control study

Objectives: To study the influence of female hormonal factors on the development of rheumatoid arthritis (RA) in relation to the human leucocyte antigen (HLA)-DRB1 shared epitope (SE), the protein tyrosine phosphatase (PTPN22) 1858T variant, anti-citrullinated protein antibodies (ACPAs), and immunoglobulin (Ig)M-rheumatoid factor (IgM-RF). Methods: A case–control study (1:4) was nested within the Medical Biobank of northern Sweden. Females who had subsequently developed RA (n = 70), median of 2.7 years before the onset of symptoms, and matched controls (n = 280) were identified from among the blood donors. A questionnaire concerning previous exposures until disease onset, including hormonal and reproductive factors, and smoking habits was distributed. Results: Breastfeeding was significantly associated with the development of RA [odds ratio (OR) 4.8, 95% confidence interval (CI) 1.43–15.8]. Increasing time of breastfeeding increased the risk of RA (OR 5.7, 95% CI 1.83–17.95) for breastfeeding ≥ 17 months. In a multiple logistic regression analysis, increasing time of breastfeeding (OR 9.5, 95% CI 2.14–42.43 for ≥ 17 months), seropositivity for ACPAs (OR 19.5, 95% CI 4.47–84.81), and carriage of the PTPN22 1858T variant (OR 3.2, 95% CI 1.36–7.54) remained significant predictors of RA. Users of oral contraceptives (OC) for ≥ 7 years had a decreased risk for development of RA (OR 0.37, 95% CI 0.15–0.93). Conclusions: A longer duration of breastfeeding increased the risk of developing RA, especially among individuals seropositive for ACPA or IgM-RF or carrying the PTPN22 1858T variant. Use of OC for ≥ 7 years was associated with a decreased risk.

Associations of antibodies against citrullinated peptides with human leukocyte antigen-shared epitope and smoking prior to the development of rheumatoid arthritis

IntroductionIt has previously been shown that an increased number of antibodies against citrullinated peptides/proteins (ACPA) predate the onset of rheumatoid arthritis (RA). Over time antibody positivity expands, involving more specific responses when approaching the onset of symptoms. We investigated the impact of human leukocyte antigen-shared epitope (HLA-SE) alleles and smoking on the development of ACPA, as well as in combination with ACPA during the state of quiescent autoimmunity (before the onset of symptoms), on the development of RA.MethodsBlood samples donated to the Medical Biobank of Northern Sweden from individuals prior to the onset of symptoms of RA (n = 370) and after onset (n = 203) and from population-based controls (n = 585) were used. Antibodies against 10 citrullinated peptides, fibrinogen (Fibα561-583, α580-600, ß62-81a, ß62-81b, ß36-52), vimentin (Vim2-17, 60-75), filaggrin (CCP-1/Fil307-324), α-enolase (CEP-1/Eno5-21), collagen type II (citC1359-369), and anti-cyclic citrullinated peptide (CCP)2 antibodies were analysed.ResultsHLA-SE-positive individuals were more frequently positive for ACPA compared with HLA-SE-negative individuals prior to the onset of symptoms of RA, particularly for antibodies against CEP-1 and Fibß62-81a (72). Smoking was associated with antibodies against Vim2-17 and citC1359-369. HLA-SE and smoking showed increasing association to the presence of the antibodies closer to disease onset. The highest odds ratio (OR) for development of RA was for the combination of HLA-SE alleles and ACPA positivity, especially for antibodies against Fibß62-81b, CCP-1/Fil307-324, and Fibβ36-52. A gene-environment additive interaction between smoking and HLA-SE alleles for the risk of disease development was found, with the highest OR for individuals positive for antibodies against Fibβ36-52, CEP-1, and Fibα580-600.ConclusionsThe relationships between antibodies against the different ACPA specificities, HLA-SE, and smoking showed a variable pattern in individuals prior to the onset of RA. The combination of smoking and HLA-SE alleles was significantly associated with the development of some of the antibody specificities closer to onset of symptoms, and these associations remained significant at diagnosis. An additive gene-environment interaction was found for several of the antibodies for the development of RA.

Plasma phospholipid fatty acid content is related to disease activity in ankylosing spondylitis.

Objective. To investigate fatty acid composition in the diet, plasma phospholipids, and adipose tissue in a cohort of patients with ankylosing spondylitis (AS), and to determine their correlations to disease activity and blood lipids in a cross-sectional study. Methods. Diet was assessed using a food frequency questionnaire in 66 patients with AS. Polyunsaturated fatty acids in plasma phospholipids and gluteal adipose tissue were measured using gas chromatography. Disease status was quantified using the Bath Ankylosing Spondylitis Disease Activity Index (BASDAI), erythrocyte sedimentation rate (ESR), high sensitivity C-reactive protein, and proinflammatory cytokines. Results. Diet did not correlate with disease activity assessed by the BASDAI, but there were negative correlations between the dietary intake of long-chain omega-3 fatty acids and ESR (rs = –0.27, p < 0.05). The plasma phospholipid content of arachidonic acid correlated significantly with the BASDAI score (rs = 0.39, p < 0.01). There were correlations between the intake of long-chain omega-3 fatty acids and high-density lipoproteins and serum triglycerides (rs = 0.26 and rs = –0.25, respectively, p < 0.05). Conclusion. There was a positive correlation between levels of arachidonic acid in plasma phospholipids and disease activity assessed by BASDAI in patients with AS. A Western diet does not appear to influence this correlation, but seems to affect blood lipids involved in atherogenic processes.

Preferential Association of Interferon Regulatory Factor 5 Gene Variants with Seronegative Rheumatoid Arthritis in 2 Swedish Case-Control Studies

Objective. Two interferon regulatory factor 5 (IRF5) gene variants were examined for association with rheumatoid arthritis (RA). Methods. A total of 2300 patients with RA and 1836 controls were recruited from 2 independent RA studies in Sweden. One insertion-deletion polymorphism (CGGGG indel) and one single-nucleotide polymorphism (rs10488631) in the IRF5 gene were genotyped and analyzed within RA subgroups stratified by rheumatoid factor (RF) and anticitrullinated peptide antibodies (ACPA). Results. The CGGGG indel was preferentially associated with the RF-negative (OR 1.29, p = 7.9 × 10−5) and ACPA-negative (OR 1.27, p = 7.3 × 10−5) RA subgroups compared to the seropositive counterparts. rs10488631 was exclusively associated within the seronegative RA subgroups (RF-negative: OR 1.24, p = 0.016; ACPA-negative: OR 1.27, p = 4.1 × 10−3). Conclusion. Both the CGGGG indel and rs10488631 are relevant for RA susceptibility, especially for seronegative RA.

Marginal jawbone loss is associated with onset of rheumatoid arthritis and is related to plasma level of receptor activator of nuclear factor kappa-B ligand (RANKL)

To investigate whether periodontitis, characterized by marginal jawbone loss, precedes the onset of symptoms of rheumatoid arthritis (RA), and to analyze plasma levels of RANKL (a cytokine that is crucial for bone resorption) and anti–citrullinated peptide antibodies (ACPAs) in presymptomatic individuals compared with matched referent controls.Objectives We investigated whether periodontitis, displayed as marginal jawbone loss, preceded onset of symptoms of RA. Furthermore, we analysed plasma levels of receptor activator of nuclear factor kappa-B (RANKL), a cytokine crucial for bone resorption and of anti-citrullinated peptide antibodies (ACPA) in pre-symptomatic individuals compared with controls. Methods Marginal jawbone levels were measured on dental radiographs from the premolar/molar regions of the jaws of 176 subjects of whom 93 had developed RA. Of these, 46 had documented radiographs predating symptom onset and sex, age and smoking status referents could be matched for 45 of them. The plasma RANKL concentrations were analysed using ELISA. The receiver operating characteristic curve was used to define the cut-off value. Results Compared with matched referents, bone loss was significantly higher in never-smoking, pre-symptomatic subjects and increasing levels of bone loss was associated with higher risk to develop subsequent RA (hazard ratio=1.06, 95%CI 1.01, 1.11). No association was found in smokers. In the pre-symptomatic RANKL-positive individuals a significantly higher extent of marginal jawbone loss was detected, and those who were both RANKL- and ACPA positive displayed an even more pronounced jawbone loss. Conclusions Marginal jawbone loss preceded clinical onset of symptoms of RA but the difference was only manifested in non-smokers. Moreover, pre-symptomatic RA-individuals, who were RANKL positive, displayed a significantly higher degree of marginal jawbone loss, particularly in ACPA positive individuals. This article is protected by copyright. All rights reserved.

An increased concentration of receptor activator of nuclear factor kappa-B ligand pre-dates the onset of rheumatoid arthritis

Objectives RANK ligand (RANKL) is involved in destruction and osteoporosis in RA. In this study, the relationships between RANKL and ACPA, anti-carbamylated protein antibodies (anti-CarP), cytokines and chemokines were analysed in individuals before the onset of RA symptoms, and their associations with radiological findings at disease onset were assessed. Methods This was a case-control study performed within the Medical Biobank of Northern Sweden that included 470 pre-symptomatic individuals [334 women and 136 men; mean (s.d.) age 52.3 (9.4) years] using blood samples donated before symptom onset (pre-dating time; 5.0 years) and 96 controls (60 women and 36 men). Plasma was analysed for RANKL (BioVendor, Karasek, Brno, Czech Republic), anti-CCP2 antibodies (Eurodiagnostics, Malmö, Sweden), anti-CarP antibodies (in-house ELISA), ACPA specificities (ISAC-platform, Phadia AB, Uppsala, Sweden) and cytokines/chemokines (Meso Scale Discovery methods, Rockville, MD, USA). Radiographs of hands and feet were graded using the Larsen score. Results The concentration of RANKL was higher in the pre-symptomatic individuals compared with controls; mean (s.e.m.): 0.50 (0.03) vs 0.22 (0.02) nmol/l (P < 0.001). The concentration increased gradually over time until symptom onset but appeared later than ACPA/RF/anti-CarP antibodies. Positivity for these antibodies yielded higher levels of RANKL compared with seronegativity (P < 0.001). RANKL concentrations were significantly associated with IL-6 and IL-10 concentrations. The combination of positivity for RANKL and anti-CarP antibodies resulted in a higher Larsen score at diagnosis β = 6.18 (95% CI: 0.93, 11.43; P = 0.022). Conclusion RANKL concentrations were increased several years before symptom onset for RA, particularly in ACPA/RF/anti-CarP-positive individuals, all detectable earlier than RANKL. Positivity for RANKL and anti-CarP antibodies yielded the highest Larsen score at disease onset.

OP0256 Presence of Risk Factors for Cardiovascular Disease Predates The Onset of Symptoms of Rheumatoid Arthritis

Background Patients with rheumatoid arthritis (RA) are at increased risk of developing cardiovascular (CV) comorbidity compared with the general population. Contradictory results concerning CV disease prior to onset of RA have been reported. Of the known CV risk factors, a more atherogenic lipid profile and smoking have been presented prior to RA onset. Objectives In this study, lifestyle factors, lipid levels, presence of hypertension, and diabetes were evaluated in individuals prior to onset of symptoms of RA and matched population controls from northern Sweden. Methods A nested case-control study was based on information from The Västerbotten Intervention Programme (VIP) and the WHO Multinational Monitoring of Trends and Determinants in Cardiovascular Disease (MONICA). Data were collected by questionnaire (socioeconomic and lifestyle factors), assessments by a nurse (body mass index;BMI, waist and blood pressure), and blood sampling (lipids and glucose loading). The registers of patients with RA (ARA criteria) attending the Department of Rheumatology, Umeå was co-analysed with the registers from VIP and MONICA. This study included 547 pre-symptomatic individuals (mean age 51.5years; 372f/175m, median (IQR) predating time 5.0 (7.0) years), and 1654 controls (mean age 51.6years; 1123f/531m). CV risk factors were defined as: hypertension (systolic≥140 mmHg, diastolic≥90 mmHg), ApoB/ApoA1 ratio (females ≥0.7, males ≥0.8), BMI≥25, diabetes, and ever smoker. Results Lower levels of ApoA1 (OR=2.0 (95%CI1.25, 3.25) and increased ApoB/ApoA1 ratio (OR=2.25 95%CI1.25, 4.06) were associated with RA development, particularly in females besides smoking, BMI and waist range. Plasma glucose at baseline was related to disease development, particularly in males (OR=1.18 95%CI1.01, 1.37). Blood pressure did not differ between the groups. Conditional logistic regression models identified elevated ApoB/ApoA1 ratio (OR=1.25 95%CI1.01, 1.56), being ever smoker (OR=2.00 95%CI1.63, 2.47), BMI≥25 (OR=1.33 95%CI1.09, 1.63) and diabetes (OR=1.98 95%CI1.06, 3.68) but not hypertension (OR=0.89 95%CI 0.72–1.11) to be associated with RA development. The pre-symptomatic individuals had significantly higher frequency of these risk factors, 32.3% of these individuals had ≥3 of these factors compared with 23.2% of the matched controls (OR=2.52 95%CI1.67, 3.83). Conclusions Several of the CV risk factors are present in individuals already years before onset of symptoms of RA. One third of the pre-symptomatic individuals had at least 3 of these factors present. These results urge early CV prevention in patients with RA. Acknowledgement The Medical Biobank of northern Sweden is acknowledged. Disclosure of Interest None declared