Vincent et al. Arthritis Research & Therapy 2013, 15:R97 http://arthritis-research.com/content/15/4/R97 RESEARCHARTICLE Open Access Clinical associations of serum interleukin-17 in systemic lupus erythematosus Fabien B Vincent1*, Melissa Northcott2, Alberta Hoi2, Fabienne Mackay1 and Eric F Morand2 Abstract Introduction: Serum interleukin (IL)-17 concentrations have been reported to be increased in systemic lupus erythematosus (SLE), but associations with clinical characteristics are not well understood. We characterized clinical associations of serum IL-17 in SLE. Methods: We quantified IL-17 in serum samples from 98 SLE patients studied cross-sectionally, and in 246 samples from 75 of these patients followed longitudinally over two years. Disease activity was recorded using the SLE Disease Activity Index (SLEDAI)-2k. Serum IL-6, migration inhibitory factor (MIF), and B cell activating factor of the tumour necrosis factor family (BAFF) were also measured in these samples. Results: Serum IL-17 levels were significantly higher in SLE patients compared to healthy donors (P <0.0001). No correlation was observed between serum IL-17 and SLEDAI-2k, at baseline or during longitudinal follow-up. However, we observed that SLEDAI-2k was positively correlated with IL-17/IL-6 ratio. Serum IL-17 was significantly increased in SLE patients with central nervous system (CNS) disease (P = 0.0298). A strong correlation was observed between serum IL-17 and IL-6 (r = 0.62, P <0.0001), and this relationship was observed regardless of disease activity and persisted when integrating cytokine levels over the period observed (r = 0.66, P <0.0001). A strong correlation of serum IL-17 was also observed with serum BAFF (r = 0.64, P <0.0001), and MIF (r = 0.36, P = 0.0016). Conclusions: Serum IL-17 concentration correlates poorly with SLE disease activity but is significantly elevated in patients with CNS disease. IL-17/IL-6 ratio may be more useful than IL-17 or IL-6 alone to characterize Th17-driven disease, such as SLE. The association of other cytokines with serum IL-17 suggests that IL-17 may drive activation of diverse immune pathways in SLE. Keywords: Autoimmunity, B cell activating factor of the tumour necrosis factor (TNF) family (BAFF), interleukin-6, interleukin-17, macrophage migration inhibitory factor (MIF), systemic lupus erythematosus (SLE), T helper 17 (Th17) Introduction effector cells, [3,4] there has been much interest regard- Systemic lupus erythematosus (SLE) is a chronic idio- ing the role of the Th17 axis in the pathogenesis of pathic systemic autoimmune disorder characterized by autoimmune disease. IL-17 has been associated with the multiple organ damage, particularly the joints, skin, pathogenesis of a range of autoimmune diseases, includ- brain and kidneys [1]. This condition is characterized by ing rheumatoid arthritis (RA) [5], systemic sclerosis [6], both innate and adaptive immune dysregulation multiple sclerosis (MS) [7], and SLE [8]. IL-17 is recog- (reviewed in Vincent et al. [2]), but the relative impor- nized to have a central role in lupus-prone mouse mod- tance of cytokines operative in these systems is els [9,10] and therefore presumed to be involved in unknown. Interleukin (IL)-17 is the prototypic T helper human SLE pathogenesis (reviewed in Nalbandian et al. 17 (Th17) cell pro-inflammatory cytokine. Since the [8]). Although not observed in all studies [6], serum IL- identification of Th17 cells as a third subset of CD4+ T 17 levels have been found to be elevated in SLE patients compared to controls [11-13]. Correlations between * Correspondence: [email protected] serum IL-17 levels and SLE disease activity and anti- 1 Department of Immunology, Monash University, Central Clinical School, double stranded DNA (anti-dsDNA) antibody levels Alfred Medical Research and Education Precinct (AMREP), 89 Commercial Road, Melbourne, VIC, 3004, Australia have previously been reported in some studies [11,13]. Full list of author information is available at the end of the article © 2013 Vincent et al.; licensee BioMed Central Ltd. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Vincent et al. Arthritis Research & Therapy 2013, 15:R97 Page 2 of 9 http://arthritis-research.com/content/15/4/R97 However, research into the potential relationships -20°C until use. Measurements of C-reactive protein between IL-17 and the clinical expression of SLE has (CRP), erythrocyte sedimentation rate (ESR), creatinine been limited, and data from longitudinally followed and anti-dsDNA antibodies (Abs) titres (normal <7; Farr cohorts is lacking. Cytokines with diverse effects on the assay, Anti-dsDNA kit, Trinity Biotech, Wicklow, Ire- immune system such as IL-6 [14-17], B cell activating land), were performed. Healthy control samples were factor of the tumour necrosis factor family (BAFF), and obtains from the Australian Red Cross Blood Service. macrophage migration inhibitory factor (MIF) have each been shown to have some interplay with IL-17 [18,19], Serum cytokine quantification and to have potential roles in the pathogenesis or clini- Sandwich enzyme-linked immunosorbent assay (ELISA) cal expression of SLE [2,20]. However, it is not clear was used to determine serum concentrations of IL-16, whether IL-17 regulates the expression of other cyto- IL-17, MIF, and BAFF. ELISA was performed using kines in patients with SLE. R&D human IL-17 DuoSet ELISA development kit In this study, we aimed to examine the associations (R&D Systems, Minneapolis, MN, USA). Ninety-six-well between serum IL-17 levels and disease expression in ELISA plates (Immunoplates, Nunc, Roskilde, Denmark) SLE in terms of disease activity and organ involvement. were coated with monoclonal mouse anti-human IL-17 We also analysed the relationship between serum IL-17 antibody. Plates were blocked with 1% bovine serum levels and serum levels of IL-6, BAFF, and MIF. The albumin in phosphate-buffered saline after overnight results indicate that although serum IL-17 levels do not incubation at 4°C. After washing, samples and standards correlate with standard measures of disease activity, it is of recombinant human IL-17 were added to the plate increased in patients with CNS disease, and is strongly and incubated overnight. Biotinylated goat anti-human associated with serum concentrations of IL-6, BAFF, IL-17 antibodies and streptavidin conjugated to horse- and MIF. Finally, our findings suggest that IL-17/IL-6 radish peroxidase were used for detection, followed by ratio may be a novel biomarker for disease activity in 3,3’,5,5’- tetramethylbenzidine (Sigma-Aldrich, Sydney, SLE. NSW, Australia) to develop colour. Plates were read at 450nm against standard curves for IL-17. The detection Material and methods limit of the assay was 16 pg/mL. Patients and clinical assessments The above ELISA protocol was similarly completed Patients aged over 18 fulfilling the 1982 American Col- using the human IL-6 DuoSet ELISA development kit lege of Rheumatology (ACR) criteria [21] for the classifi- (R&D Systems, Minneapolis, MN, USA). Similarly, MIF cation of SLE were recruited between May 2007 and concentrations were measured using mouse monoclonal June 2009 from the Lupus Clinic of Monash Medical anti-human MIF antibodies (R&D Systems, Minneapolis, Centre, Melbourne, Australia. Written informed consent MN, USA) for capture and goat biotinylated anti-human was obtained from each individual. Disease-related MIF antibodies (R&D Systems, Minneapolis, MN, USA) damage was recorded at baseline using the Systemic for detection. Recombinant human MIF was used as a Lupus International Collaborating Clinics (SLICC) SLE standard (R&D Systems, Minneapolis, MN, USA). 1% Damage Index (SDI) [22]. Other baseline variables col- bovine serum albumin in 5% sucrose-phosphate-buffered lected included patient age, gender, ethnicity and disease saline was used as a blocking agent. For BAFF, a similar duration. Disease activity was recorded at each clinic ELISA protocol was performed, using purified mouse visit using the SLE Disease Activity Index-2000 (SLE- anti-human BAFF antibodies (BD Biosciences, San Jose, DAI-2k) [23]. Patients were recorded as having renal or CA, USA) for coating the plates, and standards of central nervous system (CNS) involvement if they had recombinant human BAFF (R&D Systems, Minneapolis, evidence of involvement in ACR renal or CNS criteria, MN, USA), and biotinylated goat anti-human BAFF or on any renal or CNS domains of the SLEDAI-2k or antibodies (R&D Systems, Minneapolis, MN, USA). The SLICC-SDI at any time during the study period. Active detection limit of the assay was 1.5ng/ml. Each sample renal disease was defined as a non-zero score in any of was tested in duplicate. the renal components of the SLEDAI-2k. Patient follow- up was as scheduled by the treating physician. Patients Statistical analysis received standard-of-care therapy. The Human Research Data are expressed as median and interquartile range Ethics Committee, Southern Health, approved the study (IQR), except where otherwise indicated. As serum cyto- protocol and materials. kine concentrations were not normally distributed, non- parametric analysis was used in this study. Spearman’s Samples rank test was used to analyze correlations between vari- Paired sera and SLEDAI-2k scores were collected from ables. Mann-Whitney U tests and chi-square tests were patients at routine medical visits. Sera were stored at used to compare groups of continuous and categorical Vincent et al. Arthritis Research & Therapy 2013, 15:R97 Page 3 of 9 http://arthritis-research.com/content/15/4/R97 data respectively. A P value of <0.05 was considered sta- tistically significant. All tests were two-tailed unless otherwise stated. Statistical analyses were performed using GraphPad software (Prism Version 5.0D, 2010, San Diego, CA, USA).