Rheumatology Advance Access published April 4, 2014

RHEUMATOLOGY

Original article

54

doi:10.1093/rheumatology/ket428

The association of cytokines with disease activity and damage scores in systemic lupus erythematosus patients Eoghan M. McCarthy1,2, Siobha´n Smith2, Ruth Z. Lee1, Gaye Cunnane3, Michele F. Doran3, Suzanne Donnelly4, Donough Howard1, Paul O’Connell1, Grainne Kearns1, Joan Nı´ Gabhann2 and Caroline A. Jefferies2

Methods. Serum levels of the following cytokines were determined by ELISA in SLE patients (diagnosed as per ACR diagnostic criteria): IL-1b, IL-10, IL-12p70 and TNF-a. Demographic data, disease activity as per the SLEDAI and damage scores (SLICC) at the 5-year follow-up were calculated. Results. Enhanced production of TNF-a, IL-1 and IL-10 were observed in SLE patients compared with controls. A strong positive correlation was seen between levels of IL-12p70 and IL-10. In addition, IL-10, TNF-a and IL-1 demonstrated a significant relationship with disease activity. Interestingly, elevated levels of IL-10 were observed in SLE patients with CNS involvement while patients with elevated levels of TNF-a were more likely to have renal involvement and sustain damage over the follow-up period. Additionally, the ratio of all cytokines assayed to IL-12p70 levels were significantly higher in SLE patients when compared with controls, with an association seen between damage accrual and the IL-1b/IL-12p70 ratio (r = 0.431, P = 0.003), IL-10/IL-12p70 ratio (r = 0.351, P = 0.018) and TNF-a/IL-12p70 ratio (r = 0.33, P = 0.028). When the respective ratios were analysed for organ-specific disease, significant differences were observed for the IL-1b/IL-12p70 ratio (0.79 vs 0.47, P = 0.036), IL-10/IL-12p70 ratio (4.29 vs 1.87, P = 0.018) and TNF-a/IL-12p70 ratio (7.49 vs 5.21, P = 0.018) with respect to renal involvement. Conclusion. Increased levels of a number of immunomodulatory cytokines relative to IL-12p70 in this Caucasian SLE patient population are seen in patients with renal involvement and are associated with increased accrual of damage at the 5-year follow-up. Key words: systemic lupus erythematosus, disease activity, damage accrual, cytokines.

Introduction SLE is a complex autoimmune disease characterized by enhanced autoantibody formation, excessive proinflammatory cytokine production and damage to multiple 1 Department of Rheumatology, Beaumont Hospital, 2Molecular and Cellular Therapeutics, Royal College of Surgeons in Ireland, 3 Department of Rheumatology, St James Hospital and 4Department of Rheumatology, Mater Misericordiae University Hospital, Dublin, Ireland.

Submitted 7 April 2013; revised version accepted 4 November 2013. Correspondence to: Caroline A. Jefferies, Molecular and Cellular Therapeutics and RCSI Research Institute, Royal College of Surgeons in Ireland, 123 St Stephens Green, Dublin 2, Ireland. E-mail: [email protected]

organ systems. While the role of autoantibodies and immune complexes in the initiation of the disease is well characterized, the significance of aberrant cytokine production in disease pathogenesis is becoming increasingly apparent. Cytokines are soluble factors that play a vital role in the differentiation, maturation and activation of immune cells. In addition to their role in the systemic immune deregulation observed in SLE, they are also involved in the local inflammatory response that ultimately leads to tissue injury and end organ damage. The precise relationship of such abnormalities to disease pathogenesis and progression remains unclear [1]. For example, cytokines that regulate CD4+ Th cell differentiation have been implicated in the pathogenesis of SLE.

! The Author 2014. Published by Oxford University Press on behalf of the British Society for Rheumatology. All rights reserved. For Permissions, please email: [email protected]

1

CLINICAL SCIENCE

Objective. The aim of this study was to explore the role of cytokines in the pathogenesis of SLE in a genetically homogeneous Caucasian SLE patient population.

Downloaded from http://rheumatology.oxfordjournals.org/ at Anadolu University on May 19, 2014

Abstract

Eoghan M. McCarthy et al.

2

Given the marked heterogeneity of cytokine profiles, disease course and patient outcomes among lupus patients from different genetic backgrounds, studies are warranted in SLE patients from genetically homogeneous populations in order to enable us to better understand the differences underpinning these variations. Detailed analysis of cytokine levels, their interrelationships and how these relate to disease activity and damage accumulation may enable us to better predict the course of disease. Thus the primary objective of this study is to explore the relationship between pro- and anti-inflammatory cytokines involved in the aetiology and pathogenesis of SLE in a homogeneous Caucasian population. We sought to identify differences in cytokine profiles in Caucasian SLE patients and elucidate the role cytokines play in both disease activity and damage accrual.

Patients and methods Study population The study was performed in accordance with the Declaration of Helsinki and approved by the medical ethics committees of both Beaumont and St James Hospital. Study participants provided written informed consent. Forty-five patients who met at least four of the ACR classification criteria for SLE were included [24]. As a control population, 20 age-matched females (±5 years) were recruited from the Royal College of Surgeons in Ireland staff, all of whom had no history of autoimmune disease. Consent for this arm of the study was obtained from the RCSI institutional ethics committee. Patients were eligible for enrolment if they could confirm they were of Irish descent for three generations. Only patients for whom all clinical data were available over the entire 5-year follow-up period were included in the study.

Determination of cytokines in sera A baseline venous blood sample was obtained from each participant. Serum was isolated and stored immediately at 80 C until analysis. Serum levels of the following cytokines (IFN-g, IL-1b, IL-10, IL-12p70 and TNF-a) were quantitatively determined by a multiplex immunoassay system that enables the measurement of biomarkers using electrochemiluminescence (Human Pro-inflammatory Multiplex Array, Meso Scale Discovery, Gaithersburg, MD, USA). B lymphocyte stimulator (BLyS) levels were also evaluated to assess the relationship of these cytokines responses with B cell function. Serum BLyS levels were determined by ELISA (R&D Systems, Minneapolis, MN, USA). Experiments were performed in accordance with manufacturers’ instructions, all samples were analysed in duplicate and the mean coefficient of variation between duplicate wells was 6.2%. The analytic sensitivity for the cytokines determined by multiplex array was IFN-g, 0.8 pg/ml; IL-1b, 0.58 pg/ml; IL-10, 0.57 pg/ml; IL-12p70, 0.77 pg/ml and TNF-a, 0.28 pg/ml. The lower detection limit of the assay for BLyS was 3.39 pg/ml. ELISAs were also used to evaluate sera for antibodies to dsDNA, Sm, RNP, Ro, La and cardiolipin.

www.rheumatology.oxfordjournals.org

Downloaded from http://rheumatology.oxfordjournals.org/ at Anadolu University on May 19, 2014

IL-12, a key cytokine regulating Th1 differentiation, is produced by SLE monocytes and has been shown to trigger lupus nephritis and promote renal damage [2]. In addition, an abnormally activated IL-23/IL-17 axis has been demonstrated in SLE patients, resulting in high levels of IL-23, a cytokine important for proliferation of inflammatory Th17 cells [3]. Other cytokines exhibit more diverse effects. IL-10 inhibits the antigen-presenting capacity of monocytes through the down-regulation of MHC class II and co-stimulatory molecules such as CD86 [4]. IL-10 has also been implicated in inhibiting the proliferation of CD4+ T cells and production of cytokines such as TNF-a [5]. However, IL-10 also functions as a potent B cell stimulator, enhancing activation and differentiation of these cells, known to play a key role in the pathogenesis of SLE [6]. Therefore, while the molecular mechanisms underpinning cytokine production in SLE are increasingly well understood, the association of the resultant cytokine expression with disease characteristics and manifestations is less well defined. For example, IL-10 and IL-12p70 levels have been demonstrated to both positively and negatively correlate with disease activity [7–10]. In addition, while several studies have revealed increased serum levels of IFN-g and TNF-a in patients with SLE [11–14], others have reported no significant changes in these cytokines [15, 16]. Some studies have suggested that an imbalance in the Th1/Th2 ratio plays a role in the aetiopathogenesis of SLE [17], a finding that has not been replicated, potentially due to the different genetic backgrounds of the patients in the respective studies. The prevalence of SLE varies between ethnic groups and it is well established that patients from different genetic backgrounds have different disease severity and clinical phenotypes [18]. Family studies have suggested that 75% of the variation in IL-10 production is genetically determined [19]. Genetic polymorphisms at the promoter regions on IL-10 and TNF-a genes are associated with different cytokine production in SLE patients [20]. Furthermore, IL-10 gene polymorphisms have been demonstrated to predict damage accumulation in Asian patients but not African American or Caucasian patients [21]. Accordingly, genetic heterogeneity may largely explain the contrasting results seen in the literature with regards to the role of serum cytokines in SLE. Disease activity plays a key role in potentiating irreversible organ damage in SLE patients [22]. Damage accumulation is associated with significant morbidity and mortality. While the role of cytokines in promoting disease activity in SLE is well studied, differences in cytokine levels and their individual ratios in those that sustain irreversible damage and those that remain damage free over long-term follow-up are less well defined. An imbalance of the traditionally defined peripheral Th1/Th2 ratio as well as an IL-12-driven Th1 polarization have previously been proposed as promoters of irreversible renal damage in patients with lupus nephritis, but studies have not extrapolated these findings to all organ damage scores [23].

Cytokine imbalance promotes damage accrual in SLE

Data collection

Statistical analysis Categorical variables were analysed using Fisher’s exact test. Normally distributed continuous variables were analysed using an unpaired t-test. Differences in cytokine levels between patients and controls were examined using the non-parametric Mann–Whitney test and are presented as median [interquartile range (IQR) (Q1–Q3)]. Likewise the Mann–Whitney test was used to examine differences in cytokine ratios between patients and controls as well the differences in ratios with regard to damage accrual and organ involvement in the SLE group. Spearman’s rank correlation was used to assess the interrelationship between cytokine levels as well as their relationship with disease activity and disease duration at baseline. The strength of the correlation was graded using Cohen’s criteria as follows: 0.3–0.5 = weak, 0.5–0.7 = moderate and >0.7 = strong [29]. Similarly, the relationship between baseline cytokine expression and the subsequent 5-year damage scores was analysed using Spearman’s correlation. All analyses were performed using GraphPad Prism version 5.04 for Windows (GraphPad Software, La Jolla, CA, USA). Statistical significance was defined as a P-value 95th percentile for TNF-a in our healthy control population), a significant association was seen with renal involvement [odds ratio (OR) 12.7 (95% CI 1.3, 71.2), P = 0.017]. Of note, no significant

4

difference was noted in renal function between patients with elevated and normal TNF-a levels. Regarding autoantibody profiles, higher levels of TNF-a were observed in patients who were Sm antigen positive as compared with those who were Sm antigen negative [17 pg/ml (IQR 13.8–201.4) vs 9.7 pg/ml (IQR 6.67–18.3), P = 0.035]. A similar profile was seen for IFN-g and the Ro antigen. Ro-positive patients had higher levels of IFN-g than those who were Ro negative [3.3 pg/ml (IQR 2.43–4.63) vs 2.6 pg/ml (IQR 1.95–3.16), P = 0.027]. Furthermore levels of TNF-a, BLyS and IL-1b were higher in patients who were dsDNA positive when compared with those who were dsDNA negative [IL-1b: 1.601 pg/ml (IQR 1.19–2.62) vs 1.234 pg/ml (IQR 0.64–1.57); TNF-a: 14.91 pg/ml (IQR 7.61–51.92) vs 8.67 pg/ml (IQR 6.09–13.48); BLyS: 869.1 pg/ml (IQR 618.3–1229) vs 642.6 pg/ml (IQR 567–742.1) (P < 0.05)] (supplementary Fig. S1, available at Rheumatology Online).

The relationship of cytokine levels with disease activity, damage scores, age at diagnosis and disease duration in Caucasian SLE patients The levels of IL-1b, IL-10 and TNF-a demonstrated weak correlation with the SLEDAI in SLE patients (Table 3). In addition, patients with an elevated level of TNF-a at baseline accrued significantly more damage over the 5-year follow-up period [median SDI 1 (IQR 0–1.25) vs 0 (IQR 0–1), P = 0.03]. Given the previous studies indicating that the ratio of TNF-a, IL-1b and IFN-g to IL-10 may play a role in damage accrual, we next examined the relationship of these various ratios to damage accrual. Although no association was found between the various ratios and damage accrual at the 5-year follow-up, patients with a shorter duration of disease had higher levels of BLyS and IL-10 (data not shown). Current steroid therapy, HCQ use or additional immunosuppression (AZA, MMF, etc.) was

www.rheumatology.oxfordjournals.org

Downloaded from http://rheumatology.oxfordjournals.org/ at Anadolu University on May 19, 2014

Cytokine analysis of serum in SLE patients (n = 45) and healthy controls (n = 20). Serum was isolated and stored immediately at 80 C until analysis was performed. Serum cytokine levels were quantitatively determined by a multiplex immunoassay system. All samples were analysed in duplicate. Data are presented as median values (Q1–Q3). P-values denote differences using Mann–Whitney two-sample statistics. SELENA-SLEDAI: Safety of Estrogens in Lupus Erythematosus National Assessment version of the SLE Disease Activity Index; SDI: SLICC/ACR Damage Index.

Cytokine imbalance promotes damage accrual in SLE

FIG. 1 Correlation coefficients of IL-12p70 with pro- and anti-inflammatory cytokines in patients with SLE

not observed to influence cytokine levels on univarate analysis (supplementary Fig. S2, available at Rheumatology Online).

IL-12p70 levels play a role in damage accrual in SLE patients As IL-12p70 has been suggested to play a key role in promoting irreversible renal damage, we examined the role of IL-12p70 in potentiating all organ damage in our patient population. Although no difference in IL-12p70 levels were observed in our patient population, the ratio of all cytokines assayed to IL-12 was significantly higher in SLE patients compared with controls (P < 0.001) (Fig. 3A). As highlighted above, although none of the individual cytokines measured demonstrated a relationship with damage accrual, the IL-1b/IL-12p70 ratio was significantly higher in those patients who suffered composite damage as assessed by the SLICC score during follow-up compared with those who remained damage free (Fig. 3B). In addition, over the 5-year follow-up period an association was seen between damage accrual and the baseline IL1b/IL-12p70 ratio [r = 0.431 (95% CI 0.15, 0.65), P = 0.003],

www.rheumatology.oxfordjournals.org

IL-10/IL-12p70 ratio [r = 0.351 (95% CI 0.06, 0.59), P = 0.018] and TNF-a/IL-12p70 ratio [r = 0.33 (95% CI 0.03, 0.57), P = 0.028]. In addition, comparing patients with biopsy-proven renal involvement and those with no renal disease revealed significantly higher IL-1b/IL-12p70, IL-10/IL-12p70 and TNF-a/IL-12p70 ratios for those with renal disease vs those without [IL-1b/IL-12p70: 0.79 (IQR 0.37–2.58) vs 0.47 (IQR 0.31–0.7), P = 0.036; IL-10/IL12p70: 4.29 (IQR 1.94–4.98) vs 1.87 (IQR 1.33–2.33), P = 0.018; TNF-a/IL-12p70: 7.49 (IQR 4.83–53.65) vs 5.21 (IQR 2.77–7.34), P = 0.018] (Fig. 3C). No differences were observed for other organ-specific domains. These results indicate that the relative balance of circulating cytokines plays a key role in both renal involvement and damage accrual in Caucasian SLE patients.

Discussion SLE is a heterogeneous disease and it is becoming clear that individual cytokine patterns are important in the stratification of disease. In this study we investigated a number of cytokines involved in SLE pathogenesis in a Caucasian

5

Downloaded from http://rheumatology.oxfordjournals.org/ at Anadolu University on May 19, 2014

Serum was isolated and stored at 80 C until analysis was performed. Serum cytokine levels were quantitatively determined by multiplex array. All samples were analysed in duplicate. Data are presented as non-parametric Spearman’s correlation coefficients. Correlations are indicated as per Cohen’s criteria: 0.3–0.5, weak; 0.5–0.7, moderate; >0.7, strong.

Eoghan M. McCarthy et al.

FIG. 2 Ratio of proinflammatory cytokines to IL-10 in SLE patients

TABLE 3 Correlation coefficients of cytokines with disease activity, damage accrual and disease duration in Irish SLE patients SLEDAI (95%CI) IFN- g IL-1b IL-10 IL-12p70 TNF-a

New damage (95% CI)

0.088 ( 0.38, 0.22) 0.394* (0.11, 0.62) 0.324* (0.03, 0.57) 0.129 ( 0.18, 0.41) 0.330** (0.03, 0.57)

0.111 0.192 0.072 0.123 0.165

( ( ( ( (

0.39, 0.12, 0.23, 0.41, 0.14,

0.19) 0.47) 0.37) 0.19) 0.45)

Disease duration (95% CI) 0.19 ( 0.12, 0.47) 0.14 ( 0.42, 0.17) 0.326* ( 0.57, 0.03) 0.253 ( 0.52, 0.06) 0.016 ( 0.29, 0.32)

*P < 0.001; **P < 0.05. Serum cytokine levels were quantitatively determined by multiplex assay. Data presented are nonparametric Spearman’s correlation coefficients. Correlations are indicated as per Cohen’s criteria: 0.3–0.5, weak; 0.5–0.7, moderate; >0.7, strong.

population. Elevated levels of cytokines (IL-1b, TNF-a, IFN-g and IL-10) are demonstrated in patients compared with controls, with a weak association seen between most cytokines and disease activity. In contrast, individual cytokine levels do not appear to drive damage accrual. Importantly our results demonstrate an association of increased cytokine levels relative to IL-12p70 with renal involvement as well as an increased accrual of damage at the 5-year follow-up. The results highlight the importance of conducting research in homogeneous patient populations to better understand the underlying mechanisms of disease. It is well established that cytokines play a key role in SLE, but the exact contribution of individual cytokines and their relationships to disease activity and damage accrual in SLE remains undefined, with specific/particular differences being observed among patients from different genetic backgrounds. In support of this, previous work by our group has highlighted BLyS as playing a significant role in damage accrual in Caucasian SLE patients [30].

6

Our current study further identifies a clear role for TNF-a in promoting damage in Caucasian patients. This is in contrast to some studies that have suggested TNF-a may be protective in Hispanic SLE patients [12]. Our findings indicate that TNF-a levels correlate with disease activity, and while TNF-a levels did not predict damage accrual in our entire population, those patients with elevated levels of TNF-a at baseline were more likely to suffer damage over the follow-up period. In addition, we identified patients who are Smith positive as having higher levels of TNF-a. Hence a signature of elevated TNF-a with Smith positivity may help treating physicians better identify patients at risk of poorer outcomes early in the disease course and adjust treatment protocols accordingly, particularly since these patients are also more likely to have renal involvement. It is likely that the increased TNF-a levels observed in our patients are a direct manifestation of SLE rather than being attributable to renal failure. In support of this, no significant difference in renal function was seen between those with elevated

www.rheumatology.oxfordjournals.org

Downloaded from http://rheumatology.oxfordjournals.org/ at Anadolu University on May 19, 2014

The ratio of IFN-g to IL-10 is not higher in Irish SLE patients when compared with controls, nor were any differences observed in the ratios of either IL-1b/IL-10 or TNF-a/IL-10 between patients and controls. Serum was isolated and stored immediately at 80 C. Serum cytokine levels were quantitatively determined by multiplex array. All samples were analysed in duplicate. The ratios of the various cytokines to IL-10 were calculated by dividing the various serum cytokine levels by those of IL-10 in both SLE patients and healthy controls. Data are presented as median values. P-values denote differences using Mann–Whitney two-sample statistics.

Cytokine imbalance promotes damage accrual in SLE

FIG. 3 The balance of serum cytokines is associated with renal involvement and damage accrual

TNF-a and those without. This is in keeping with studies in the nephrology literature that have demonstrated no significant difference in TNF-a levels between healthy controls and patients with chronic renal failure or those requiring dialysis [31]. Cytokines have been proposed as a key driver of disease activity and damage in SLE. We have demonstrated that a number of individual cytokines are associated with disease activity in Caucasian SLE patients. Despite a clear association of cytokines with disease activity, none of the cytokines assayed appear to promote damage accrual. There was no difference in the ratio of the proinflammatory cytokines assayed in our study to IL-10 between patients and controls, suggesting that excess IL-10 production may balance out the effects of these proinflammatory cytokines. The exact contribution of IL-10 to the pathogenesis of SLE is undefined. IL-10 is pleiotropic in its abilities to stimulate B lymphocyte proliferation and immunoglobulin secretion, inhibit Th1 responses and

www.rheumatology.oxfordjournals.org

promote Th2 responses. IL-10 has been shown to down-regulate murine lupus through it affect on pathogenic Th1 cytokine responses [32]. In addition, recent studies have identified certain regulatory B cells whose effects are mediated by IL-10, suggesting that IL-10 may have a protective effect during lupus progression [33]. IL-10 has also been demonstrated to play a key role in down-regulating Th17/Th1 responses in collageninduced arthritis [34]. Of note, IL-10 was the only cytokine in our study to demonstrate correlation with BLyS, a key mediator of B cell function and antibody production. BLyS and IL-10 levels were the only cytokines higher in our patient group early in the course of disease. Thus in Caucasian patients, the predominant role for IL-10 may be in promoting B cell proliferation early in the disease course, explaining its correlation with the SLEDAI while, as the disease progresses, IL-10 may potentially be protective, explaining the lack of association between damage scores.

7

Downloaded from http://rheumatology.oxfordjournals.org/ at Anadolu University on May 19, 2014

(A) The ratio of pro- and anti inflammatory cytokines relative to IL-12p70 was significantly higher in SLE patients when compared with controls. (B) Patients with higher IL-1b/IL-12p70, TNF-a/IL-12p70 and IL-10/IL-12p70 ratios accrued more damage over the 5-year follow-up period. (C) Patients with higher IL-1b/IL-12p70, TNF-a/IL-12p70 and IL-10/IL12p70 ratios were more likely to have renal involvement. Serum was isolated and stored at 80 C until use. Serum cytokine levels were quantitatively determined by multiplex array. All samples were analysed in duplicate. The ratios of the various cytokines to IL-12p70 were calculated by dividing the various serum cytokine levels by those of IL-12p70. Data are presented as median values. P-values denote differences using Mann–Whitney two-sample statistics.

Eoghan M. McCarthy et al.

8

Rheumatology key messages Increased cytokine levels relative to IL-12p70 in SLE patients are associated with renal involvement. . In SLE patients it is the balance of cytokines that appears to promote damage accrual. .

Funding: This work was supported by the Science Foundation Ireland (grant 08/IN.1/B2091), the Health Research Board Ireland (grant RP 2001 26) and the Royal College of Physicians in Ireland. The funders had no role in study design, data collection and analysis, decision to publish or preparation of the manuscript. Disclosure statement: The authors have declared no conflicts of interest.

Supplementary data Supplementary data are available at Rheumatology Online.

References 1 Doria A, Ghirardello A, Iaccarino L et al. Pregnancy, cytokines, and disease activity in systemic lupus erythematosus. Arthritis Rheum 2004;51:989–95. 2 Liu TF, Jones BM. Impaired production of IL-12 in systemic lupus erythematosus. I. Excessive production of IL-10 suppresses production of IL-12 by monocytes. Cytokine 1998;10:140–7. 3 Wong CK, Lit LC, Tam LS et al. Hyperproduction of IL-23 and IL-17 in patients with systemic lupus erythematosus: implications for Th17-mediated inflammation in autoimmunity. Clin Immunol 2008;127:385–93. 4 Ding L, Linsley PS, Huang LY et al. IL-10 inhibits macrophage costimulatory activity by selectively inhibiting the up-regulation of B7 expression. J Immunol 1993;151: 1224–34. 5 Joss A, Akdis M, Faith A et al. IL-10 directly acts on T cells by specifically altering the CD28 co-stimulation pathway. Eur J Immunol 2000;30:1683–90. 6 Llorente L, Zou W, Levy Y et al. Role of interleukin 10 in the B lymphocyte hyperactivity and autoantibody production of human systemic lupus erythematosus. J Exp Med 1995; 181:839–44. 7 Arora V, Verma J, Marwah V et al. Cytokine imbalance in systemic lupus erythematosus: a study on northern Indian subjects. Lupus 2012. 8 Llorente L, Richaud-Patin Y, Fior R et al. In vivo production of interleukin-10 by non-T cells in rheumatoid arthritis, Sjogren’s syndrome, and systemic lupus erythematosus. A potential mechanism of B lymphocyte hyperactivity and autoimmunity. Arthritis Rheum 1994;37:1647–55. 9 Houssiau FA, Lefebvre C, Vanden Berghe M et al. Serum interleukin 10 titers in systemic lupus erythematosus reflect disease activity. Lupus 1995;4:393–5. 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;16:283–8.

www.rheumatology.oxfordjournals.org

Downloaded from http://rheumatology.oxfordjournals.org/ at Anadolu University on May 19, 2014

In contrast to the potentially protective effect of IL-10 seen on damage accrual, we found a significant association in the ratio of a number of cytokines to IL-12p70 and damage accrual. Previous studies have suggested IL-12p70 is a pathogenic mediator of renal damage in SLE [17]. However, others have demonstrated levels of IL-12 to be unrelated to both disease activity and injury in SLE patients [14, 35], although these studies lacked racial and genetic homogeneity. In our study, conducted among a homogeneous SLE population, IL-12p70 levels reflected neither disease activity nor damage accrual. However, our study suggests the relative balance between a number of cytokines, and IL-12p70 rather than absolute cytokine levels plays a significant role in damage accrual in SLE patients. Higher TNF-a/IL12p70, IL-1b/IL-12p70 and IL-10/1L-12p70 ratios were associated with increased damage accrual. Regarding how the relative levels of IL-1b, IL-10 and TNF-a to IL12p70 may be driving damage accrual, it is interesting to note that relatively low levels of the Th1-inducing cytokine IL-12p70 and higher levels of the Th17-inducing cytokine IL-1 could contribute to increased numbers of autoreactive IL-17-producing Th cells. In addition, the imbalance in cytokine levels may also contribute to the decreased function of autoimmunity-suppressing Tregs documented in patients with SLE [36, 37], in turn promoting damage. When cytokine levels are measured in serum it is not possible to determine their origin. In-depth studies at the cellular level are therefore needed to explain our data. Nonetheless, the recent introduction of anti-BLyS therapy highlights the potential benefits of better understanding the role of circulating cytokines in SLE patient populations, with a view to identifying patients at risk of poorer outcomes as well as potential targeted interventions. The number of patients enrolled in this study was small due to all participants having to confirm they were of Irish descent for three generations prior to enrolment. Given the limited number of patients meeting the inclusion criteria and the fact that this was an exploratory study, we did not adjust for multiple comparisons when analysing the data, a limitation of this study that must be highlighted. Therefore further studies involving larger numbers of patients from different genetic backgrounds are warranted to facilitate correction for multiple comparisons and to confirm our findings. Nonetheless, the strength of our study lies in the genetic homogeneity of our lupus population. Absolute cytokine levels are associated with disease activity in Caucasian SLE patients. In contrast, a relative imbalance of circulating cytokine levels was associated with damage accrual. Our results highlight that increased cytokine levels relative to IL-12p70 in this SLE patient population are seen in those with renal involvement and are associated with increased accrual of damage at the 5-year follow-up and indicate that future investigations into the role of IL12p70 in SLE may benefit from examining its role in the context of other circulating cytokines rather than in isolation.

Cytokine imbalance promotes damage accrual in SLE

11 Chun HY, Chung JW, Kim HA et al. Cytokine IL-6 and IL-10 as biomarkers in systemic lupus erythematosus. J Clin Immunol 2007;27:461–6. 12 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;33:404–13. 13 Kim T, Kanayama Y, Negoro N et al. Serum levels of interferons in patients with systemic lupus erythematosus. Clin Exp Immunol 1987;70:562–9. 14 Tokano Y, Morimoto S, Kaneko H et al. Levels of IL-12 in the sera of patients with systemic lupus erythematosus (SLE)—relation to Th1- and Th2-derived cytokines. Clin Exp Immunol 1999;116:169–73.

16 Viallard JF, Pellegrin JL, Ranchin V et al. Th1 (IL-2, interferon-gamma (IFN-gamma)) and Th2 (IL-10, IL-4) cytokine production by peripheral blood mononuclear cells (PBMC) from patients with systemic lupus erythematosus (SLE). Clin Exp Immunol 1999;115:189–95. 17 Tucci M, Lombardi L, Richards HB et al. Overexpression of interleukin-12 and T helper 1 predominance in lupus nephritis. Clin Exp Immunol 2008;154:247–54. 18 Dean GS, Tyrrell-Price J, Crawley E et al. Cytokines and systemic lupus erythematosus. Ann Rheum Dis 2000;59: 243–51. 19 Westendorp RG, Langermans JA, Huizinga TW et al. Genetic influence on cytokine production and fatal meningococcal disease. Lancet 1997;349:170–3. 20 Gibson AW, Edberg JC, Wu J et al. Novel single nucleotide polymorphisms in the distal IL-10 promoter affect IL-10 production and enhance the risk of systemic lupus erythematosus. J Immunol 2001;166: 3915–22. 21 Sung YK, Park BL, Shin HD et al. Interleukin-10 gene polymorphisms are associated with the SLICC/ACR Damage Index in systemic lupus erythematosus. Rheumatology 2006;45:400–4.

26 FitzGerald JD, Grossman JM. Validity and reliability of retrospective assessment of disease activity and flare in observational cohorts of lupus patients. Lupus 1999;8: 638–44. 27 Gladman D, Ginzler E, Goldsmith C et al. The development and initial validation of the Systemic Lupus International Collaborating Clinics/American College of Rheumatology damage index for systemic lupus erythematosus. Arthritis Rheum 1996;39:363–9. 28 Thumboo J, Lee HY, Fong KY et al. Accuracy of medical record scoring of the SLICC/ACR damage index for systemic lupus erythematosus. Lupus 2000;9:358–62. 29 Cohen J. Statistical Power Analysis for the Behavioral Sciences. 2nd edn. Hillsdale, NJ: Lawrence Erlbaum Associates, 1988. 30 McCarthy EM, Lee RZ, Ni Gabhann J et al. Elevated B lymphocyte stimulator levels are associated with increased damage in an Irish systemic lupus erythematosus cohort. Rheumatology 2013;52:1279–84. 31 Pereira BJ, Shapiro L, King AJ et al. Plasma levels of IL-1 beta, TNF alpha and their specific inhibitors in undialyzed chronic renal failure, CAPD and hemodialysis patients. Kidney Int 1994;45:890–6. 32 Watanabe R, Ishiura N, Nakashima H et al. Regulatory B cells (B10 cells) have a suppressive role in murine lupus: CD19 and B10 cell deficiency exacerbates systemic autoimmunity. J Immunol 2010;184:4801–9. 33 Yanaba K, Bouaziz JD, Haas KM et al. A regulatory B cell subset with a unique CD1dhiCD5+ phenotype controls T cell-dependent inflammatory responses. Immunity 2008; 28:639–50. 34 Carter NA, Rosser EC, Mauri C. Interleukin-10 produced by B cells is crucial for the suppression of Th17/Th1 responses, induction of T regulatory type 1 cells and reduction of collagen-induced arthritis. Arthritis Res Ther 2012;14:R32.

22 Gladman DD, Urowitz MB, Rahman P et al. Accrual of organ damage over time in patients with systemic lupus erythematosus. J Rheumatol 2003;30:1955–9.

35 Wong CK, Ho CY, Li EK et al. Elevation of proinflammatory cytokine (IL-18, IL-17, IL-12) and Th2 cytokine (IL-4) concentrations in patients with systemic lupus erythematosus. Lupus 2000;9:589–93.

23 Uhm WS, Na K, Song GW et al. Cytokine balance in kidney tissue from lupus nephritis patients. Rheumatology 2003; 42:935–8.

36 Miyara M, Amoura Z, Parizot C et al. Global natural regulatory T cell depletion in active systemic lupus erythematosus. J Immunol 2005;175:8392–400.

24 Hochberg MC. Updating the American College of Rheumatology revised criteria for the classification of systemic lupus erythematosus. Arthritis Rheum 1997; 40:1725.

37 Valencia X, Yarboro C, Illei G et al. Deficient CD4+CD25high T regulatory cell function in patients with active systemic lupus erythematosus. J Immunol 2007; 178:2579–88.

www.rheumatology.oxfordjournals.org

9

Downloaded from http://rheumatology.oxfordjournals.org/ at Anadolu University on May 19, 2014

15 Lacki JK, Leszczynski P, Kelemen J et al. Cytokine concentration in serum of lupus erythematosus patients: the effect on acute phase response. J Med 1997;28: 99–107.

25 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;35:630–40.