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Cytokines in relation to autoantibodies before onset of symptoms for systemic lupus erythematosus C Eriksson and S Rantapää-Dahlqvist Lupus 2014 23: 691 originally published online 14 February 2014 DOI: 10.1177/0961203314523869 The online version of this article can be found at: http://lup.sagepub.com/content/23/7/691
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CONCISE REPORT
Cytokines in relation to autoantibodies before onset of symptoms for systemic lupus erythematosus C Eriksson1 and S Rantapa¨a¨-Dahlqvist2 1
Department of Clinical Immunology/Clinical Microbiology, Umea˚ University, Umea˚, Sweden; and 2Department of Public Health and Clinical Medicine/Rheumatology, Umea˚ University, Umea˚, Sweden
Objectives: A number of cytokines and chemokines were analysed and related to autoantibodies in blood samples pre-dating the onset of symptoms of systemic lupus erythematosus. Methods: Thirty-five patients with systemic lupus erythematosus (American College of Rheumatology criteria) were identified as having donated blood samples, prior to symptom onset, to the Biobank of northern Sweden. Altogether, 140 age- and sex-matched controls were also identified. The concentrations of interferon-a, interleukin-4, interleukin-9, interleukin-10, interferon inducible protein-10 and monocyte chemotactic protein-1 were analysed using multiplex technology and related to autoantibodies (ANA, ENA, anti-dsDNA and anti-histone antibodies) analysed from the same blood sample. Results: The interferon-g inducible protein-10 levels were higher in the pre-symptomatic individuals than in controls (p < 0.05) and correlated with interferon-a (p < 0.01). The interferon-g inducible protein-10 and interferon-a concentrations were significantly increased in individuals positive for autoantibodies: interferon-g inducible protein-10 for ANA; anti-SSA/Ro and anti-Jo-1 antibodies; interferon-a with anti-SSB/La antibodies. The levels of interleukin-10, interferon-g inducible protein-10 and monocyte chemotactic protein-1 increased significantly from the pre-symptomatic individuals to after onset of systemic lupus erythematosus. Conclusions: An increased concentration of interferon-g inducible protein-10 pre-dated the onset of systemic lupus erythematosus and was related to autoantibodies before the onset of disease. The levels of interferon-g inducible protein-10 and interferon-a were correlated. These findings support the proposal that the interferon system is important early in the pathogenesis of systemic lupus erythematosus and autoantibody formation. Lupus (2014) 23, 691–696. Key words: Systemic lupus erythematosus; cytokines; autoantibodies; pathogenesis
Introduction Systemic lupus erythematosus (SLE) is a heterogeneous disease presenting with diverse clinical manifestations and exhibiting variable severity in individual patients and between different ethnic populations.1 A typical pathophysiological indicator in patients with SLE is the production of autoantibodies directed against nuclear antigens (ANA). The generation of these autoantibodies has been shown to precede the development of clinical manifestations with a predictable course in a time-related progression.1,2 We have previously Correspondence to: Catharina Eriksson Department of Clinical Immunology/Clinical Microbiology, Umea˚ University, S-901 87 Umea˚, Sweden. Email: [email protected] Received 1 July 2013; accepted 23 January 2014
reported that ANA specificities were detectable a mean time-scale of 5.6 years before the onset of symptoms in 63% of individuals who subsequently developed SLE.3 The sensitivity was highest for ANA, at 45.7%, with a specificity of 95%, followed by anti-dsDNA and anti-Ro/SSA antibodies, both with a sensitivity of 20.0% and specificities of 98.7% and 97.4%, respectively. Anti-Ro/SSA was the earliest detectable autoantibody, occurring a mean of 6.6 years before the onset of symptoms.3 Interferon (IFN)-a has been shown to be an important factor involved in the pathogenesis of SLE.4 An up-regulation of IFN-a-regulated genes in blood cells is present in lupus patients and has been termed ‘the interferon signature’. IFN-a has both direct and indirect effects on B-cells and on the production of pathogenic autoantibodies in patients with SLE.5 IFN-related chemokines, such as CXCL10/IFN-g inducible protein 10 (IP-10) and
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10.1177/0961203314523869
Cytokines in relation to autoantibodies before onset of symptoms for SLE C Eriksson and S Rantapa¨¨a-Dahlqvist
692
CCL2/monocyte chemotactic protein-1 (MCP-1), have also been associated with disease activity and organ damage in patients with SLE, which indirectly supports a proposed role for type I IFN in disease outcome.6,7 Interleukin (IL)-4 has been shown to have a critical role by promoting autoantibody production and development of lupus nephritis in an animal model.8 In another study, it was shown that the expression of mRNA for IL-4 in blood mononuclear cells was decreased in lupus patients, while expression of that for IL-10 and IFN-g was increased.9 IL-10 is predominantly an anti-inflammatory cytokine but has also been shown to be elevated in SLE patients and to be associated with both anti-dsDNA antibodies and disease activity measured using the SLE Disease Activity Index (SLEDAI).10 IL-9, produced by follicular T helper subset, or Th-9 cells, stimulates the production of immunoglobulin in B-cells and could subsequently be of relevance in the pathogenesis of SLE.11 The aim of this study was to analyse a selected range of cytokines and chemokines and their relationships to previously detected autoantibodies in samples collected prior to the onset of symptoms of SLE in a population from northern Europe.
A nested case–control study, designed as 1:4, was undertaken based on the 35 identified individuals, referred to as ‘pre-symptomatic ‘individuals and randomly selected controls (n ¼ 140) from the same population based cohorts as the cases, matched for sex, age and date of blood sampling. Samples from three controls were no longer available. Following a diagnosis of SLE, samples were available for analysis from 28 of the 35 presymptomatic individuals. The mean age (range) at the time of blood sampling before onset of symptoms was 37.7 (16.8–60.2) years, for the matched controls 36.2 (17.8–62.3) years and at diagnosis of SLE 47.5 (23–67) years. At the time of blood sampling after SLE was diagnosed, the median value for SLEDAI was 3.5 (range 0–26) and Systemic Lupus International Collaborative Clinics (SLICC)/ACR Damage Index was 1 (range 0–8).13,14 Twenty-one of the 28 patients, were prescribed active medication at the time of the sampling (corticosteroids (n ¼ 17), Disease Modifying Anti Rheumatic Drugs (DMARDs); azathioprine, methotrexate or mycophenolate mofetil (n ¼ 11), hydroxychloroquine (n ¼ 11), cyclophosphamide (n ¼ 1) and rituximab (n ¼ 1). The study was approved by the Regional Ethics Committee of the University in Umea˚ and all participants gave their written informed consent.
Materials and methods
Analysis of cytokines and antibodies
Patients and controls
IFN-a, IL-4, IL-9, IL-10 and the chemokines CXCL10/IP-10 and CCL2/MCP-1 were detected using the multiplex detection kit Milliplex map (Millipore Corporation, Billerica, MA, USA) and analysed on a Bio-Plex Array Reader (Luminex200, LabmapTM system; Luminex Corporation, Austin, TX, USA). IL-9 was subsequently excluded from statistical analyses because all of the measured values were similar for all individuals irrespective of whether a patient or control. Analyses of autoantibodies from the same blood samples had been performed earlier using the multiplex detection kit AtheNA Multi-Lyte ANA-II Plus Test (ZEUS Scientific, Raritan, NJ, USA) on a Bio-Plex Array Reader (Luminex200, LabmapTM system), while anti-nuclear antibodies (ANA) were detected by indirect immunofluorescence on HEp2-slides (Immunoconcepts) using samples diluted 1:100.3
The register of patients with SLE attending the Department of Rheumatology, University Hospital, Umea˚, with a known date for the onset of symptoms was co-analysed with those of the Medical Biobank and of the maternity cohort (i.e. the record of samples obtained for screening pregnant women for anti-rubella antibodies) of northern Sweden as previously described.3 Full details for the patient recruitment criteria and the collection and storage of blood samples have been described previously.3 A total of 35 patients (three male and 32 female), of whom 34 fulfilled four and one fulfilling only three, of the American College of Rheumatology (ACR) criteria for SLE,12 were identified as having donated blood samples before the onset of any symptoms of SLE. The median (interquartile range (IQR)) time before the first symptom was 6.7 (7.7) years and 8.9 (9.3) years before SLE was diagnosed. Nineteen of the patients were identified from the Medical Biobank (plasma samples) and the other 16 from the maternity cohort collection (sera).
Statistics Statistical calculations were performed using SPSS for Windows version 18.0 (SPSS, Chicago, IL, USA). Continuous data were compared by nonparametric analyses with Wilcoxon’s signed rank
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Cytokines in relation to autoantibodies before onset of symptoms for SLE C Eriksson and S Rantapa¨¨a-Dahlqvist
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test for matched pairs (pre-patients versus SLE patients), Mann-Whitney’s U-test (pre-patients versus control subjects) and Spearman’s signed rank test for correlations. Relationships between categorical data (positive versus negative) were compared using Chi-square analysis or Fisher’s exact test when appropriate. All p-values are two sided and p-value 0.05 were considered statistically significant.
Results The concentrations of cytokines in pre-symptomatic individuals, SLE patients and controls are presented in Table 1. The levels of IP-10 were significantly higher in the pre-symptomatic individuals than in the controls (median value (IQR): 361 pg/ mL (298) versus 256 pg/mL (182), p ¼ 0.02). The concentrations of IL-10, IP-10 and MCP-1 were significantly increased following diagnosis compared with the levels in the pre-symptomatic individuals (p < 0.05, p < 0.0001 and p < 0.0001, respectively; Figure 1(a) to (c)). The concentrations of the other cytokines did not differ significantly between pre-symptomatic individuals and controls or between pre-symptomatic individuals and SLE patients.
In those pre-symptomatic individuals positive for any autoantibody the concentrations of IFN-a and IP-10 were significantly higher compared with seronegative individuals (p < 0.05 and p < 0.001, respectively; Table 1). Stratification for the separate autoantibodies yielded different patterns of cytokine/chemokine concentrations for the various antibodies. IP-10 was significantly higher in presymptomatic individuals positive for ANA, antiSSA/Ro and anti-Jo1 antibodies (p < 0.01, p < 0.05 and p < 0.05, respectively), while IFN-a was significantly higher in pre-symptomatic individuals positive for anti-SSB/La (p < 0.05). IL-10 was present at significantly higher concentrations in anti-RNP antibody positive individuals (p < 0.05). The concentration of MCP-1 was significantly lower in antibody positive individuals overall and specifically in those with anti-SSA/Ro and anti-SSB/La antibodies (p < 0.05, p < 0.01 and p < 0.05, respectively). The concentration of IL-4 was significantly lower in anti-dsDNA positive individuals (p ¼ 0.05). There were no identifiable relationships between cytokine/chemokines concentrations and the presence of any of the autoantibodies in the control group or patients following a diagnosis of SLE. The number of autoantibodies in presymptomatic individuals correlated with the concentrations of IFN-a (rs ¼ 0.32, p ¼ 0.066) and
Table 1 Median values (IQR) of cytokines in relation to autoantibodies. The pre-symptomatic individuals and SLE patients were compared with the controls, respectively Antibody positive and antibody negative pre-symptomatic individuals for each autoantibody were compared.
Controls SLE patients All pre-symptomatic Individuals Any antibody No antibody ANA pos neg dsDNA pos neg histone pos neg SSA pos neg SSB pos neg RNP pos neg Jo-1 pos neg
n
IFN- pg/mL
IL-4 pg/mL
IL-10 pg/mL
IP-10 pg/mL
MCP-1 pg/mL
137 28
24.09 (32.3) 15.4 (27.18)
8.25 (12.6) 15.20 (18.47)
4.20 (5.8) 13.79 (25.70)*a
256.1 (182.4) 693.8 (709.6)*a
236.4 (265.2) 1122 (764.8)*a
27.91 39.2 15.40 33.48 23.27 22.90 29.71 41.06 27.72 43.08 27.82 58.91 27.72 48.89 25.72 27.91 28.7
15.16 15.20 6.07 13.61 15.18 4.00 15.20 5.12 15.20 15.2 11.42 15.20 11.41 10.85 15.16 4.07 15.18
7.93 8.39 3.30 8.50 3.39 8.32 7.44 8.46 5.99 8.46 7.44 8.46 7.44 15.95 5.03 16.30 7.44
361.4 479.4 203.5 409.5 201.9 489.4 347.5 489.4 347.5 598.4 336.8 506.8 347.5 626.5 384.0 610.1 347.5
35 22 13 16 19 7 28 5 30 7 28 3 32 4 31 3 32
(39.55) (11.9)*b (21.4) (36.4) (28.5) (26.5) (41.1) (27.4) (40.0) (45.3) (36.1) (14.8)*b (27.6) (35.5) (34.6) (28.3) (31.1)
(12.5) (12.5) (12.6) (12.5) (12.5) (2.6)*b (10.7) (9.6) (12.5) (12.6) (12.5) (3.9) (12.5) (19.0) (12.5) (7.3) (12.5)
(8.98) (11.6) (8.6) (9.9) (8.1) (13.3) (8.6) (30.8) (8.8) (12.3) (8.9) (8.7) (8.9) (6.4)*b (8.1) (12.4) (8.6)
(298.3)*a (306.4)**b (136.7) (362.7)*b (336.4) (332.3) (285.8) (199.7) (325.2) (434.4)*b (257.7) (493.1) (284.0) (853.2) (284.6) (243.7)*b (278.6)
286.9(561.5) 221.9 (174.9)*b 391.3 (615.3) 237.1 (432.5) 381.4 (567.3) 219.1 (122.9) 340.1 (565.5) 210.0 (67.0) 340.1 (570.1) 192.8 (123.1)*b 340.1 (557.6) 152.2 (49.7)*b 328.7 (560.8) 208.3 (181.5) 325.2 (565.5) 254.4 (55.9) 328.7 (565.7)
a
Compared with controls. Positive compared with antibody negative. *p < 0.05; **p < 0.001. b
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Cytokines in relation to autoantibodies before onset of symptoms for SLE C Eriksson and S Rantapa¨¨a-Dahlqvist
694
Figure 1 Levels of cytokines (pg/mL) in controls, pre-symptomatic individuals and SLE patients, i.e. before the onset of symptoms and after SLE diagnosis stratified for: (a) IP-10; (b) IL-10; (c) MCP-1. The three groups were compared using Kruskal–Wallis test. The pre-symptomatic individuals/SLE patients were compared with the controls using Mann–Whitney’s U-test and the presymptomatic individuals with the SLE patients using Wilcoxon signed ranks test.
significantly with, IP-10 (rs ¼ 0.65, p < 0.001), whilst MCP-1 was inversely correlated (rs ¼ 0.41, p ¼ 0.015; Figures 2 and 3). The concentration of IFN-a correlated significantly in the presymptomatic individuals with the concentrations of IP-10 (rs ¼ 0.45, p < 0.01), IL-4, (rs ¼ 0.35, p < 0.05), IL-10 (rs ¼ 0.36, p < 0.05) and inversely with MCP-1 (rs ¼ –0.45, p < 0.01). The concentration of IP-10 correlated non-significantly with the level of IL-10 (rs ¼ 0.33, p ¼ 0.053) and inversely with MCP-1 (rs ¼ 0.52, p ¼ 0.001). There were no time-dependent relationships between the time before onset of symptoms and the levels of the analytes (data not shown).
Discussion This analysis of cytokine and chemokine levels in individuals before the onset of symptom of SLE is, to the best of our knowledge, the first reported study; consequently, there are no other studies to compare our results with. The concentration of IP-10 was found to be significantly higher in the pre-symptomatic individuals than in the controls. The level of IFN-a was not elevated before the onset of symptoms compared with that of the matched controls, but because the concentrations of IP-10 and IFN-a were correlated this finding could suggest IFN-a to be of relevance
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Cytokines in relation to autoantibodies before onset of symptoms for SLE C Eriksson and S Rantapa¨¨a-Dahlqvist
695
Figure 2 Correlation between IP-10 concentration (pg/mL) and the number of antibodies in individuals before symptom onset.
Figure 3 Correlation between MCP-1 concentration (pg/mL) and number of antibodies in individuals before symptom onset.
in the early development of SLE. We have previously shown an increasing number of autoantibody specificities in samples collected prior to diagnosis the closer to the onset of symptoms,3 but no corresponding increase in cytokine levels was found in this study. However, the levels of IFN-a and IP-10 were related to the presence of autoantibodies in the pre-symptomatic individuals. These observations could, perhaps, be explained by the limited sample size. IFN-a has been found, in patients with SLE and in incomplete lupus syndrome, to correlate inversely with IgM autoantibodies and to correlate positively with IgG autoantibodies suggesting that IFN-a promotes a class switch from IgM to IgG for autoantibodies in early lupus.15 High levels of
IFN gene expression in both groups correlated with IgG class of autoantibodies, suggesting that the IFN gene expression signature may identify individuals at risk for disease development. Our findings of increased levels in pre-symptomatic individuals, but not in SLE patients, could be considered as supporting the importance of IFN and IFN-associated molecules in the early pathogenic process of disease development of SLE. Additionally, the genes for IFN-inducible chemokines have been reported to be up-regulated in leucocytes from SLE-patients and were associated with disease activity as well as organ damage and disease outcome.16 In the study conducted by Fu et al.16 there was an association between the expression of these chemokine genes – the so called ‘interferon signature’ – and the presence of anti-Sm and anti-RNP autoantibodies. The concentrations of CCL2/MCP-1 and IL-4 were negatively correlated with the presence of certain autoantibodies before the onset of disease. However, we are unable to fully explain the negative association between MCP-1 and IL-4 respectively and antibodies and comparative studies in SLE patients are lacking. The level of MCP-1 has been shown to exhibit a negative relationship with the number of antibodies in patients with type-I diabetes.17 One study in man showed that the expression of IL-4 at the mRNA level in blood mononuclear cells was decreased in lupus patients.9 In the present study, there were no differences between either controls or pre-symptomatic individuals and SLE patients concerning IL-4 except for a significant negative correlation between IL-4 and the presence of anti-dsDNA antibodies in the pre-symptomatic individuals. Significantly higher concentrations of IL-10 were found in anti-RNP positive pre-symptomatic individuals, albeit IL-10 has been shown by others to be associated with anti-dsDNA, anti-SSA/Ro and anti-SSB/La antibodies in patients with SLE.18 In the SLE patient group, our findings were in agreement with previous reports by showing elevated blood levels of IP10, MCP-1 and IL-10.6,7,19 The limitations of this study are the small number of pre-symptomatic individuals included, particularly after stratification, and in some cases a long sample storage time that could possibly influence the levels of cytokine levels. However, the controls were identified from the same sample bank registers as the cases and, consequently, were matched for sampling and storage conditions. After the diagnosis of SLE there are many factors that may influence cytokine levels, such as on-going Lupus
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Cytokines in relation to autoantibodies before onset of symptoms for SLE C Eriksson and S Rantapa¨¨a-Dahlqvist
696
medication and the inflammatory response activity at the time of blood sampling. In conclusion, IFN-a and the IFN-associated chemokines CXCL10/IP-10 and CCL2/MCP-1 have the highest relationship with autoantibody formation and to the number of autoantibodies per individual before the onset of SLE disease. These findings support previous theories that the IFN-system is important in the early phase of pathogenesis and autoantibody formation in patients with SLE, even years before clinical symptoms are apparent.
Funding This project was supported by funding from ‘VISARE NORR FUND’, Umea˚, Sweden.
Conflict of interest None declared.
Acknowledgements Professor Go¨ran Wadell and Professor Go¨ran Hallmans are gratefully acknowledged for making samples from the ‘Maternity cohort’ and the ‘Medical Biobank’ available. Mrs A˚sa A˚gren is gratefully acknowledged for computer service with the registers.
References 1 Arbuckle MR, McClain MT, Rubertone MV, et al. Development of autoantibodies before the clinical onset of systemic lupus erythematosus. N Engl J Med 2003; 349: 1526–1533. 2 Jacobsen S, Petersen J, Ullman S, et al. A multicentre study of 513 Danish patients with systemic lupus erythematosus. I. Disease manifestations and analyses of clinical subsets. Clin Rheumatol 1998; 17: 468–477.
3 Eriksson C, Kokkonen H, Johansson M, et al. Autoantibodies predate the onset of systemic lupus erythematosus in northern Sweden. Arthritis Res Ther 2011; 13: R30. 4 Bennett L, Palucka AK, Arce E, et al. Interferon and granulopoiesis signatures in systemic lupus erythematosus blood. J Exp Med 2003; 197: 711–723. 5 Ronnblom L, Alm GV, Eloranta ML. Type I interferon and lupus. Curr Opin Rheumatol 2009; 21: 471–477. 6 Eriksson C, Eneslatt K, Ivanoff J, et al. Abnormal expression of chemokine receptors on T-cells from patients with systemic lupus erythematosus. Lupus 2003; 12: 766–774. 7 Bauer JW, Baechler EC, Petri M, et al. Elevated serum levels of interferon-regulated chemokines are biomarkers for active human systemic lupus erythematosus. PLoS Med 2006; 3: e491. 8 Peng SL, Moslehi J, Craft J. Roles of interferon-gamma and interleukin-4 in murine lupus. J Clin Invest 1997; 99: 1936–1946. 9 Csiszar A, Nagy G, Gergely P, et al. Increased interferon-gamma (IFN-gamma), IL-10 and decreased IL-4 mRNA expression in peripheral blood mononuclear cells (PBMC) from patients with systemic lupus erythematosus (SLE). Clin Exp Immunol 2000; 122: 464–470. 10 Park YB, Lee SK, Kim DS, et al. Elevated interleukin-10 levels correlated with disease activity in systemic lupus erythematosus. Clin Exp Rheumatol 1998; 16: 283–288. 11 Li H, Rostami A. IL-9: basic biology, signaling pathways in CD4þ T cells and implications for autoimmunity. J Neuroimmune Pharmacol 2010; 5: 198–209. 12 Hochberg MC. Updating the American College of Rheumatology revised criteria for the classification of systemic lupus erythematosus. Arthritis Rheum 1997; 40: 1725. 13 Bombardier C, Gladman DD, Urowitz MB, et al. Derivation of the SLEDAI. A disease activity index for lupus patients. The Committee on Prognosis Studies in SLE. Arthritis Rheum 1992; 35: 630–640. 14 Gladman D, Ginzler E, Goldsmith C, et al. Systemic lupus international collaborative clinics: development of a damage index in systemic lupus erythematosus. J Rheumatol 1992; 19: 1820–1821. 15 Li QZ, Zhou J, Lian Y, et al. Interferon signature gene expression is correlated with autoantibody profiles in patients with incomplete lupus syndromes. Clin Exp Immunol 2010; 159: 281–291. 16 Fu Q, Chen X, Cui H, et al. Association of elevated transcript levels of interferon-inducible chemokines with disease activity and organ damage in systemic lupus erythematosus patients. Arthritis Res Ther 2008; 10: R112. 17 Hanifi-Moghaddam P, Schloot NC, Kappler S, et al. An association of autoantibody status and serum cytokine levels in type 1 diabetes. Diabetes 2003; 52: 1137–1142. 18 Gomez D, Correa PA, Gomez LM, et al. Th1/Th2 cytokines in patients with systemic lupus erythematosus: is tumor necrosis factor alpha protective? Semin Arthritis Rheum 2004; 33: 404–413. 19 Willeke P, Schotte H, Erren M, et al. Concomitant reduction of disease activity and IL-10 secreting peripheral blood mononuclear cells during immunoadsorption in patients with active systenic lupus erythematosus. Cell Mol Biol 2002; 48: 323–329.
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Cytokines in relation to autoantibodies before onset of symptoms for systemic lupus erythematosus C Eriksson and S Rantapää-Dahlqvist Lupus 2014 23: 691 originally published online 14 February 2014 DOI: 10.1177/0961203314523869 The online version of this article can be found at: http://lup.sagepub.com/content/23/7/691
Published by: http://www.sagepublications.com
Additional services and information for Lupus can be found at: Email Alerts: http://lup.sagepub.com/cgi/alerts Subscriptions: http://lup.sagepub.com/subscriptions Reprints: http://www.sagepub.com/journalsReprints.nav Permissions: http://www.sagepub.com/journalsPermissions.nav
>> Version of Record - May 30, 2014 OnlineFirst Version of Record - Feb 14, 2014 What is This?
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Lupus (2014) 23, 691–696 http://lup.sagepub.com
CONCISE REPORT
Cytokines in relation to autoantibodies before onset of symptoms for systemic lupus erythematosus C Eriksson1 and S Rantapa¨a¨-Dahlqvist2 1
Department of Clinical Immunology/Clinical Microbiology, Umea˚ University, Umea˚, Sweden; and 2Department of Public Health and Clinical Medicine/Rheumatology, Umea˚ University, Umea˚, Sweden
Objectives: A number of cytokines and chemokines were analysed and related to autoantibodies in blood samples pre-dating the onset of symptoms of systemic lupus erythematosus. Methods: Thirty-five patients with systemic lupus erythematosus (American College of Rheumatology criteria) were identified as having donated blood samples, prior to symptom onset, to the Biobank of northern Sweden. Altogether, 140 age- and sex-matched controls were also identified. The concentrations of interferon-a, interleukin-4, interleukin-9, interleukin-10, interferon inducible protein-10 and monocyte chemotactic protein-1 were analysed using multiplex technology and related to autoantibodies (ANA, ENA, anti-dsDNA and anti-histone antibodies) analysed from the same blood sample. Results: The interferon-g inducible protein-10 levels were higher in the pre-symptomatic individuals than in controls (p < 0.05) and correlated with interferon-a (p < 0.01). The interferon-g inducible protein-10 and interferon-a concentrations were significantly increased in individuals positive for autoantibodies: interferon-g inducible protein-10 for ANA; anti-SSA/Ro and anti-Jo-1 antibodies; interferon-a with anti-SSB/La antibodies. The levels of interleukin-10, interferon-g inducible protein-10 and monocyte chemotactic protein-1 increased significantly from the pre-symptomatic individuals to after onset of systemic lupus erythematosus. Conclusions: An increased concentration of interferon-g inducible protein-10 pre-dated the onset of systemic lupus erythematosus and was related to autoantibodies before the onset of disease. The levels of interferon-g inducible protein-10 and interferon-a were correlated. These findings support the proposal that the interferon system is important early in the pathogenesis of systemic lupus erythematosus and autoantibody formation. Lupus (2014) 23, 691–696. Key words: Systemic lupus erythematosus; cytokines; autoantibodies; pathogenesis
Introduction Systemic lupus erythematosus (SLE) is a heterogeneous disease presenting with diverse clinical manifestations and exhibiting variable severity in individual patients and between different ethnic populations.1 A typical pathophysiological indicator in patients with SLE is the production of autoantibodies directed against nuclear antigens (ANA). The generation of these autoantibodies has been shown to precede the development of clinical manifestations with a predictable course in a time-related progression.1,2 We have previously Correspondence to: Catharina Eriksson Department of Clinical Immunology/Clinical Microbiology, Umea˚ University, S-901 87 Umea˚, Sweden. Email: [email protected] Received 1 July 2013; accepted 23 January 2014
reported that ANA specificities were detectable a mean time-scale of 5.6 years before the onset of symptoms in 63% of individuals who subsequently developed SLE.3 The sensitivity was highest for ANA, at 45.7%, with a specificity of 95%, followed by anti-dsDNA and anti-Ro/SSA antibodies, both with a sensitivity of 20.0% and specificities of 98.7% and 97.4%, respectively. Anti-Ro/SSA was the earliest detectable autoantibody, occurring a mean of 6.6 years before the onset of symptoms.3 Interferon (IFN)-a has been shown to be an important factor involved in the pathogenesis of SLE.4 An up-regulation of IFN-a-regulated genes in blood cells is present in lupus patients and has been termed ‘the interferon signature’. IFN-a has both direct and indirect effects on B-cells and on the production of pathogenic autoantibodies in patients with SLE.5 IFN-related chemokines, such as CXCL10/IFN-g inducible protein 10 (IP-10) and
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Cytokines in relation to autoantibodies before onset of symptoms for SLE C Eriksson and S Rantapa¨¨a-Dahlqvist
692
CCL2/monocyte chemotactic protein-1 (MCP-1), have also been associated with disease activity and organ damage in patients with SLE, which indirectly supports a proposed role for type I IFN in disease outcome.6,7 Interleukin (IL)-4 has been shown to have a critical role by promoting autoantibody production and development of lupus nephritis in an animal model.8 In another study, it was shown that the expression of mRNA for IL-4 in blood mononuclear cells was decreased in lupus patients, while expression of that for IL-10 and IFN-g was increased.9 IL-10 is predominantly an anti-inflammatory cytokine but has also been shown to be elevated in SLE patients and to be associated with both anti-dsDNA antibodies and disease activity measured using the SLE Disease Activity Index (SLEDAI).10 IL-9, produced by follicular T helper subset, or Th-9 cells, stimulates the production of immunoglobulin in B-cells and could subsequently be of relevance in the pathogenesis of SLE.11 The aim of this study was to analyse a selected range of cytokines and chemokines and their relationships to previously detected autoantibodies in samples collected prior to the onset of symptoms of SLE in a population from northern Europe.
A nested case–control study, designed as 1:4, was undertaken based on the 35 identified individuals, referred to as ‘pre-symptomatic ‘individuals and randomly selected controls (n ¼ 140) from the same population based cohorts as the cases, matched for sex, age and date of blood sampling. Samples from three controls were no longer available. Following a diagnosis of SLE, samples were available for analysis from 28 of the 35 presymptomatic individuals. The mean age (range) at the time of blood sampling before onset of symptoms was 37.7 (16.8–60.2) years, for the matched controls 36.2 (17.8–62.3) years and at diagnosis of SLE 47.5 (23–67) years. At the time of blood sampling after SLE was diagnosed, the median value for SLEDAI was 3.5 (range 0–26) and Systemic Lupus International Collaborative Clinics (SLICC)/ACR Damage Index was 1 (range 0–8).13,14 Twenty-one of the 28 patients, were prescribed active medication at the time of the sampling (corticosteroids (n ¼ 17), Disease Modifying Anti Rheumatic Drugs (DMARDs); azathioprine, methotrexate or mycophenolate mofetil (n ¼ 11), hydroxychloroquine (n ¼ 11), cyclophosphamide (n ¼ 1) and rituximab (n ¼ 1). The study was approved by the Regional Ethics Committee of the University in Umea˚ and all participants gave their written informed consent.
Materials and methods
Analysis of cytokines and antibodies
Patients and controls
IFN-a, IL-4, IL-9, IL-10 and the chemokines CXCL10/IP-10 and CCL2/MCP-1 were detected using the multiplex detection kit Milliplex map (Millipore Corporation, Billerica, MA, USA) and analysed on a Bio-Plex Array Reader (Luminex200, LabmapTM system; Luminex Corporation, Austin, TX, USA). IL-9 was subsequently excluded from statistical analyses because all of the measured values were similar for all individuals irrespective of whether a patient or control. Analyses of autoantibodies from the same blood samples had been performed earlier using the multiplex detection kit AtheNA Multi-Lyte ANA-II Plus Test (ZEUS Scientific, Raritan, NJ, USA) on a Bio-Plex Array Reader (Luminex200, LabmapTM system), while anti-nuclear antibodies (ANA) were detected by indirect immunofluorescence on HEp2-slides (Immunoconcepts) using samples diluted 1:100.3
The register of patients with SLE attending the Department of Rheumatology, University Hospital, Umea˚, with a known date for the onset of symptoms was co-analysed with those of the Medical Biobank and of the maternity cohort (i.e. the record of samples obtained for screening pregnant women for anti-rubella antibodies) of northern Sweden as previously described.3 Full details for the patient recruitment criteria and the collection and storage of blood samples have been described previously.3 A total of 35 patients (three male and 32 female), of whom 34 fulfilled four and one fulfilling only three, of the American College of Rheumatology (ACR) criteria for SLE,12 were identified as having donated blood samples before the onset of any symptoms of SLE. The median (interquartile range (IQR)) time before the first symptom was 6.7 (7.7) years and 8.9 (9.3) years before SLE was diagnosed. Nineteen of the patients were identified from the Medical Biobank (plasma samples) and the other 16 from the maternity cohort collection (sera).
Statistics Statistical calculations were performed using SPSS for Windows version 18.0 (SPSS, Chicago, IL, USA). Continuous data were compared by nonparametric analyses with Wilcoxon’s signed rank
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test for matched pairs (pre-patients versus SLE patients), Mann-Whitney’s U-test (pre-patients versus control subjects) and Spearman’s signed rank test for correlations. Relationships between categorical data (positive versus negative) were compared using Chi-square analysis or Fisher’s exact test when appropriate. All p-values are two sided and p-value 0.05 were considered statistically significant.
Results The concentrations of cytokines in pre-symptomatic individuals, SLE patients and controls are presented in Table 1. The levels of IP-10 were significantly higher in the pre-symptomatic individuals than in the controls (median value (IQR): 361 pg/ mL (298) versus 256 pg/mL (182), p ¼ 0.02). The concentrations of IL-10, IP-10 and MCP-1 were significantly increased following diagnosis compared with the levels in the pre-symptomatic individuals (p < 0.05, p < 0.0001 and p < 0.0001, respectively; Figure 1(a) to (c)). The concentrations of the other cytokines did not differ significantly between pre-symptomatic individuals and controls or between pre-symptomatic individuals and SLE patients.
In those pre-symptomatic individuals positive for any autoantibody the concentrations of IFN-a and IP-10 were significantly higher compared with seronegative individuals (p < 0.05 and p < 0.001, respectively; Table 1). Stratification for the separate autoantibodies yielded different patterns of cytokine/chemokine concentrations for the various antibodies. IP-10 was significantly higher in presymptomatic individuals positive for ANA, antiSSA/Ro and anti-Jo1 antibodies (p < 0.01, p < 0.05 and p < 0.05, respectively), while IFN-a was significantly higher in pre-symptomatic individuals positive for anti-SSB/La (p < 0.05). IL-10 was present at significantly higher concentrations in anti-RNP antibody positive individuals (p < 0.05). The concentration of MCP-1 was significantly lower in antibody positive individuals overall and specifically in those with anti-SSA/Ro and anti-SSB/La antibodies (p < 0.05, p < 0.01 and p < 0.05, respectively). The concentration of IL-4 was significantly lower in anti-dsDNA positive individuals (p ¼ 0.05). There were no identifiable relationships between cytokine/chemokines concentrations and the presence of any of the autoantibodies in the control group or patients following a diagnosis of SLE. The number of autoantibodies in presymptomatic individuals correlated with the concentrations of IFN-a (rs ¼ 0.32, p ¼ 0.066) and
Table 1 Median values (IQR) of cytokines in relation to autoantibodies. The pre-symptomatic individuals and SLE patients were compared with the controls, respectively Antibody positive and antibody negative pre-symptomatic individuals for each autoantibody were compared.
Controls SLE patients All pre-symptomatic Individuals Any antibody No antibody ANA pos neg dsDNA pos neg histone pos neg SSA pos neg SSB pos neg RNP pos neg Jo-1 pos neg
n
IFN- pg/mL
IL-4 pg/mL
IL-10 pg/mL
IP-10 pg/mL
MCP-1 pg/mL
137 28
24.09 (32.3) 15.4 (27.18)
8.25 (12.6) 15.20 (18.47)
4.20 (5.8) 13.79 (25.70)*a
256.1 (182.4) 693.8 (709.6)*a
236.4 (265.2) 1122 (764.8)*a
27.91 39.2 15.40 33.48 23.27 22.90 29.71 41.06 27.72 43.08 27.82 58.91 27.72 48.89 25.72 27.91 28.7
15.16 15.20 6.07 13.61 15.18 4.00 15.20 5.12 15.20 15.2 11.42 15.20 11.41 10.85 15.16 4.07 15.18
7.93 8.39 3.30 8.50 3.39 8.32 7.44 8.46 5.99 8.46 7.44 8.46 7.44 15.95 5.03 16.30 7.44
361.4 479.4 203.5 409.5 201.9 489.4 347.5 489.4 347.5 598.4 336.8 506.8 347.5 626.5 384.0 610.1 347.5
35 22 13 16 19 7 28 5 30 7 28 3 32 4 31 3 32
(39.55) (11.9)*b (21.4) (36.4) (28.5) (26.5) (41.1) (27.4) (40.0) (45.3) (36.1) (14.8)*b (27.6) (35.5) (34.6) (28.3) (31.1)
(12.5) (12.5) (12.6) (12.5) (12.5) (2.6)*b (10.7) (9.6) (12.5) (12.6) (12.5) (3.9) (12.5) (19.0) (12.5) (7.3) (12.5)
(8.98) (11.6) (8.6) (9.9) (8.1) (13.3) (8.6) (30.8) (8.8) (12.3) (8.9) (8.7) (8.9) (6.4)*b (8.1) (12.4) (8.6)
(298.3)*a (306.4)**b (136.7) (362.7)*b (336.4) (332.3) (285.8) (199.7) (325.2) (434.4)*b (257.7) (493.1) (284.0) (853.2) (284.6) (243.7)*b (278.6)
286.9(561.5) 221.9 (174.9)*b 391.3 (615.3) 237.1 (432.5) 381.4 (567.3) 219.1 (122.9) 340.1 (565.5) 210.0 (67.0) 340.1 (570.1) 192.8 (123.1)*b 340.1 (557.6) 152.2 (49.7)*b 328.7 (560.8) 208.3 (181.5) 325.2 (565.5) 254.4 (55.9) 328.7 (565.7)
a
Compared with controls. Positive compared with antibody negative. *p < 0.05; **p < 0.001. b
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Figure 1 Levels of cytokines (pg/mL) in controls, pre-symptomatic individuals and SLE patients, i.e. before the onset of symptoms and after SLE diagnosis stratified for: (a) IP-10; (b) IL-10; (c) MCP-1. The three groups were compared using Kruskal–Wallis test. The pre-symptomatic individuals/SLE patients were compared with the controls using Mann–Whitney’s U-test and the presymptomatic individuals with the SLE patients using Wilcoxon signed ranks test.
significantly with, IP-10 (rs ¼ 0.65, p < 0.001), whilst MCP-1 was inversely correlated (rs ¼ 0.41, p ¼ 0.015; Figures 2 and 3). The concentration of IFN-a correlated significantly in the presymptomatic individuals with the concentrations of IP-10 (rs ¼ 0.45, p < 0.01), IL-4, (rs ¼ 0.35, p < 0.05), IL-10 (rs ¼ 0.36, p < 0.05) and inversely with MCP-1 (rs ¼ –0.45, p < 0.01). The concentration of IP-10 correlated non-significantly with the level of IL-10 (rs ¼ 0.33, p ¼ 0.053) and inversely with MCP-1 (rs ¼ 0.52, p ¼ 0.001). There were no time-dependent relationships between the time before onset of symptoms and the levels of the analytes (data not shown).
Discussion This analysis of cytokine and chemokine levels in individuals before the onset of symptom of SLE is, to the best of our knowledge, the first reported study; consequently, there are no other studies to compare our results with. The concentration of IP-10 was found to be significantly higher in the pre-symptomatic individuals than in the controls. The level of IFN-a was not elevated before the onset of symptoms compared with that of the matched controls, but because the concentrations of IP-10 and IFN-a were correlated this finding could suggest IFN-a to be of relevance
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Figure 2 Correlation between IP-10 concentration (pg/mL) and the number of antibodies in individuals before symptom onset.
Figure 3 Correlation between MCP-1 concentration (pg/mL) and number of antibodies in individuals before symptom onset.
in the early development of SLE. We have previously shown an increasing number of autoantibody specificities in samples collected prior to diagnosis the closer to the onset of symptoms,3 but no corresponding increase in cytokine levels was found in this study. However, the levels of IFN-a and IP-10 were related to the presence of autoantibodies in the pre-symptomatic individuals. These observations could, perhaps, be explained by the limited sample size. IFN-a has been found, in patients with SLE and in incomplete lupus syndrome, to correlate inversely with IgM autoantibodies and to correlate positively with IgG autoantibodies suggesting that IFN-a promotes a class switch from IgM to IgG for autoantibodies in early lupus.15 High levels of
IFN gene expression in both groups correlated with IgG class of autoantibodies, suggesting that the IFN gene expression signature may identify individuals at risk for disease development. Our findings of increased levels in pre-symptomatic individuals, but not in SLE patients, could be considered as supporting the importance of IFN and IFN-associated molecules in the early pathogenic process of disease development of SLE. Additionally, the genes for IFN-inducible chemokines have been reported to be up-regulated in leucocytes from SLE-patients and were associated with disease activity as well as organ damage and disease outcome.16 In the study conducted by Fu et al.16 there was an association between the expression of these chemokine genes – the so called ‘interferon signature’ – and the presence of anti-Sm and anti-RNP autoantibodies. The concentrations of CCL2/MCP-1 and IL-4 were negatively correlated with the presence of certain autoantibodies before the onset of disease. However, we are unable to fully explain the negative association between MCP-1 and IL-4 respectively and antibodies and comparative studies in SLE patients are lacking. The level of MCP-1 has been shown to exhibit a negative relationship with the number of antibodies in patients with type-I diabetes.17 One study in man showed that the expression of IL-4 at the mRNA level in blood mononuclear cells was decreased in lupus patients.9 In the present study, there were no differences between either controls or pre-symptomatic individuals and SLE patients concerning IL-4 except for a significant negative correlation between IL-4 and the presence of anti-dsDNA antibodies in the pre-symptomatic individuals. Significantly higher concentrations of IL-10 were found in anti-RNP positive pre-symptomatic individuals, albeit IL-10 has been shown by others to be associated with anti-dsDNA, anti-SSA/Ro and anti-SSB/La antibodies in patients with SLE.18 In the SLE patient group, our findings were in agreement with previous reports by showing elevated blood levels of IP10, MCP-1 and IL-10.6,7,19 The limitations of this study are the small number of pre-symptomatic individuals included, particularly after stratification, and in some cases a long sample storage time that could possibly influence the levels of cytokine levels. However, the controls were identified from the same sample bank registers as the cases and, consequently, were matched for sampling and storage conditions. After the diagnosis of SLE there are many factors that may influence cytokine levels, such as on-going Lupus
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medication and the inflammatory response activity at the time of blood sampling. In conclusion, IFN-a and the IFN-associated chemokines CXCL10/IP-10 and CCL2/MCP-1 have the highest relationship with autoantibody formation and to the number of autoantibodies per individual before the onset of SLE disease. These findings support previous theories that the IFN-system is important in the early phase of pathogenesis and autoantibody formation in patients with SLE, even years before clinical symptoms are apparent.
Funding This project was supported by funding from ‘VISARE NORR FUND’, Umea˚, Sweden.
Conflict of interest None declared.
Acknowledgements Professor Go¨ran Wadell and Professor Go¨ran Hallmans are gratefully acknowledged for making samples from the ‘Maternity cohort’ and the ‘Medical Biobank’ available. Mrs A˚sa A˚gren is gratefully acknowledged for computer service with the registers.
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