|Year : 2018 | Volume
| Issue : 2 | Page : 107-112
Serum Interleukin-6, Interleukin-17A, and transforming growth factor beta are raised in systemic sclerosis with interstitial lung disease
Anupam Wakhlu1, Rasmi Ranjan Sahoo1, Jyoti Ranjan Parida2, Mohit K Rai2, Durga P Misra2, Vinita Agrawal3, Vikas Agarwal2
1 Department of Rheumatology, King George's Medical University, Lucknow, Uttar Pradesh, India
2 Department of Clinical Immunology, Sanjay Gandhi Postgraduate Institute of Medical Sciences, Lucknow, Uttar Pradesh, India
3 Department of Pathology, Sanjay Gandhi Postgraduate Institute of Medical Sciences, Lucknow, Uttar Pradesh, India
|Date of Web Publication||24-May-2018|
Prof. Anupam Wakhlu
Department of Rheumatology, King George's Medical University, Lucknow - 226 003, Uttar Pradesh
Source of Support: None, Conflict of Interest: None
Background: Dysregulation in cytokines like interleukin-6 (IL-6), interleukin-17A (IL-17A) and transforming growth factor-beta (TGF β1) has been pathogenically implicated in systemic sclerosis (SSc). The present study aimed to assess their serum levels in patients with SSc and correlate with clinical manifestations.
Methods: This cross-sectional, observational study included 93 patients fulfilling the 2013 revised ACR/EULAR SSc classification criteria and 33 age-and sex-matched healthy controls. Antinuclear antibody (ANA), extractable nuclear antigen (ENA) profile, chest radiograph, pulmonary function tests and electrocardiography were done. HRCT of thorax and echocardiography were done wherever indicated. Modified Rodnan skin score (MRSS) was calculated. Serum IL-6, IL-17A and TGF β1 levels were assayed using ELISA kit and compared among disease subtypes and clinical parameters. Spearman coefficient was used to test correlation between continuous variables. P value of <0.05 was considered significant.
Results: The mean age of patients was 37.8+10.3 years (Female:Male: 30:1) with median duration of disease of 3 years. Serum IL-6, IL-17A and TGF β1 levels were significantly higher in patients as compared to controls (IL-6: 19.4±11 vs 6.7±3.9 pg/ml (P < 0.0001); IL-17A: 39.1±14.8 vs 16.4±2.1 pg/ml (P < 0.0001); TGF β1: 862.2±443 vs 377.2±208.8 pg/ml (P < 0.0001). Higher levels of these cytokines were also observed in patients of diffuse cutaneous SSc, those with lung fibrosis and anti-topoisomerase positivity.
Conclusion: Serum IL-6, IL-17A and TGF β1 levels were significantly higher in SSc patients and higher levels were associated with ILD and skin fibrosis.
Keywords: Cytokine, interleukin-6, interleukin-17A, interstitial lung disease, scleroderma, systemic sclerosis, transforming growth factor β1
|How to cite this article:|
Wakhlu A, Sahoo RR, Parida JR, Rai MK, Misra DP, Agrawal V, Agarwal V. Serum Interleukin-6, Interleukin-17A, and transforming growth factor beta are raised in systemic sclerosis with interstitial lung disease. Indian J Rheumatol 2018;13:107-12
|How to cite this URL:|
Wakhlu A, Sahoo RR, Parida JR, Rai MK, Misra DP, Agrawal V, Agarwal V. Serum Interleukin-6, Interleukin-17A, and transforming growth factor beta are raised in systemic sclerosis with interstitial lung disease. Indian J Rheumatol [serial online] 2018 [cited 2019 Aug 17];13:107-12. Available from: http://www.indianjrheumatol.com/text.asp?2018/13/2/107/222108
| Introduction|| |
Systemic sclerosis (SSc) is a heterogeneous disorder of unknown etiology, characterized by vasculopathy, autoimmunity, and fibrosis of the skin and internal organs. The pathogenesis of SSc is incompletely understood, and treatment is mainly symptomatic with the use of immunosuppressive and vasodilator therapy for specific indications. Immune dysregulation has long been demonstrated in SSc, implying that immune cells, by secreting cytokines, amplify the disease process. The current trend in defining the role of cytokines in disease process has led to the successful use of monoclonal antibodies targeting these pathogenic cytokines.
De Lauretis et al. studied eight different cytokines including interleukin-6 (IL-6), IL-8 and IL-10 in SSc and reported IL-6 to be an independent predictor of diffusing capacity of lungs for carbon monoxide (DLCO) decline in SSc patients with interstitial lung disease (ILD). Other studies have also reported the high levels of serum IL-6 in SSc. There are case reports of anti-IL-6 treatment (tocilizumab) in SSc with variable results.
The role of IL-17 in SSc has not been extensively studied. High levels of IL-17 have been reported in SSc patients, indicating that it may have a role in SSc pathogenesis. One study demonstrated increased IL-17 production by T cells from the peripheral blood and fibrotic lesions of the skin and lungs in SSc patients. However, the association between IL-17 and SSc-ILD is not well defined as of now.
Transforming growth factor beta (TGF β), a potent stimulator of extracellular matrix production, has been extensively studied in SSc. In early disease, TGF β1 levels have been reported to be reduced in the sera of SSc patients compared to controls and inversely correlate with disease activity in some studies. Other studies have reported increased TGF β1 immunoreactivity in skin during early disease course which gradually decreased as the disease progressed.
The present study aims to assess serum IL-6, IL-17A, and TGF β1 levels in a North Indian SSc cohort and correlate with clinical manifestations with special reference to ILD.
| Methods|| |
This was a cross-sectional, observational study was jointly conducted at the Department of Rheumatology, King George's Medical University and Department of Clinical Immunology, Sanjay Gandhi Postgraduate Institute of Medical Sciences, Lucknow, India.
Ninty-three patients fulfilling the 2013 revised ACR/EULAR systemic sclerosis classification criteria, were included in the study. Thirty-three age and sex-matched healthy controls were also enrolled. All patients with overlap syndrome were excluded from the study.
Details were collected regarding duration of symptoms, other comorbidities, and relevant previous investigations. Depending on the extent of skin thickening, patients were grouped into two major subtypes: limited cutaneous (lc-SSc; thickening distal to the elbows or knees, including face) and diffuse cutaneous (dc-SSc; thickening proximal to the elbows or knees).
The severity of skin thickening was determined by modified Rodnan skin score (MRSS). Total MRSS is the sum of 17 body areas with score in each area ranges from 0 to 3: 0-normal skin; 1-mild thickening; 2-with a greater degree of skin tautness; and 3-hide-bound skin.
Laboratory investigations included routine hematologic and biochemical tests along with autoantibody profile (antinuclear antibody [ANA], extractable nuclear antigen [ENA] profile (where possible). Chest radiograph, pulmonary function test, and electrocardiography were done for all patients. High-resolution computed tomography CT scan of thorax (HRCT) and 2D echocardiography were done wherever indicated.
Venous blood samples (2 ml) were obtained from all individuals in plain vacutainers. Serum was separated immediately by centrifugation and stored at −80°C for future analysis. Serum IL-6, IL-17A and TGF β1 levels were measured by enzyme-linked immunosorbent assay kit according to the manufacturer's instructions (human IL-6 and TGF β1 kit: BD Bioscience, USA; IL-17A kit: R&D, USA).
Statistical analysis was done by GraphPad prism 5.01 (GraphPad Software, San Diego, CA, USA). Quantitative data were expressed as mean with standard deviation or median with interquartile range according to the distribution of data whereas qualitative data expressed as frequency and percentage. Student's unpaired t-test was used to compare quantitative variables, and Spearman's rank correlation was used to analyze relationships between cytokine levels and clinical parameters. P < 0.05 was considered statistically significant.
The study was approved by the ethics committee of both the institutes. Informed written consent was obtained from all patients prior to their enrollment in this study.
| Results|| |
The study included 93 SSc patients and 33 age- and sex-matched healthy controls. The mean age of the patients was 37.8 ± 10.3 years, and that of controls was 34.5 ± 8 years. The median duration of disease was 36 months (range: 2–360). Among patients, 90 were female (96.8%), and 3 were male (3.2%). 58 patients were of dc (62.4%), and 35 were of lc subtype (37.6%).
There were 55 patients with ILD (59.1%) and 38 patients without ILD (40.9%). The demographic and clinical characteristics of patients with and without ILD are shown in [Table 1].
|Table 1: Demographic and clinical characteristics of patients with and without interstitial lung disease|
Click here to view
HRCT thorax in ILD patients revealed nonspecific interstitial pneumonia (NSIP) in 51.9% and usual interstitial pneumonia (UIP) pattern in 48.9%.
ANA was positive 96.8% patients, with speckled pattern seen in 52.7% and homogenous pattern in 47.3%. ENA profile could be done in 61 patients. Antitopoisomerase antibody was positive in 67.3%, anti-Ro in 23.6%, anti-La in 5.5%, and anti-RNP in 18.2% of patients.
Serum IL-6, IL-17A, and TGF β1 levels (mean ± SD) were significantly high in patients as compared to controls [Figure 1]a and [Figure 1]b (IL-6: 19.4 ± 11 vs. 6.7 ± 3.9 pg/ml (P < 0.0001); IL-17A: 39.1 ± 14.8 vs. 16.4 ± 2.1 pg/ml (P < 0.0001); TGF-β1: 862.2 ± 443 vs. 377.2 ± 208.8 pg/ml (P < 0.0001). There were significantly higher levels of serum IL-6, IL-17A and TGF β1 levels in patients with ILD compared to those without ILD (IL-6: 23.9 ± 12.4 vs. 13 ± 2.9 pg/ml (P < 0.0001); IL-17A: 46.4 ± 14.8 vs. 28.6 ± 6.2 pg/ml (P < 0.0001); TGF β1: 1157 ± 296.6 vs. 443.6 ± 220.2 pg/ml (P < 0.0001) [Table 2]. The predicted forced vital capacity (FVC%), total lung capacity (TLC), and DLCO did not correlate with the cytokine levels in our study. Higher levels of serum IL-6, IL-17A and TGF β1 were also observed in patients with dc scleroderma and those with antitopoisomerase positivity [Table 2]. There was no difference in cytokine levels among patients with and without digital ulcer [Table 2]. Serum levels of IL-6, IL-17A, and TGF β1 correlated positively with MRSS [Figure 2]. There was no statistically significant difference in the levels of cytokines among patients with disease duration <5 years and more than 5 years.
|Figure 1: (a) Comparison of serum interleukin-6 and interleukin-17A levels among patients and controls: Serum interleukin-6 and interleukin-17A levels were significantly higher in the patient group. (b) Comparison of serum Transforming growth factor beta 1 levels among patients and controls: Serum Transforming growth factor beta 1 was significantly higher in the patient group|
Click here to view
|Table 2: Comparison of serum transforming growth factor beta 1, interleukin-6, and interleukin-17A levels (mean±standard deviation) among different subgroups of systemic sclerosis patients (manifestations and serology)|
Click here to view
|Figure 2: Correlation of serum Transforming growth factor beta 1, interleukin-6, and interleukin-17A levels with modified Rodnan skin score: The levels correlated positively with modified Rodnan skin score|
Click here to view
| Discussion|| |
The present study found significantly higher serum IL-6, IL-17A, and TGF β1 levels in SSc patients as compared to age- and sex-matched healthy controls. Patients with ILD had higher serum levels of IL-6, IL-17A, and TGF β1 as compared to non-ILD scleroderma patients.
IL-6, a pleiotropic cytokine is critical to the pathogenesis of SSc. It is secreted by a wide variety of cells and stimulates SSc fibroblasts to differentiate, proliferate, and produce collagen leading to fibrosis. In our patients, serum IL-6 levels were significantly high and were associated with diffuse skin involvement and lung fibrosis. Elevated levels of IL-6 in SSc have been observed in dc subtype. One study reported high serum IL-6 levels in SSc patients with alveolitis and also demonstrated a positive correlation between IL-6 and HRCT score. Abdel-Magied et al. observed a positive correlation between IL-6 and severity of ILD measured by 6-min walk distance, FVC%, TLC%, DLCO%, and forced expiratory volume 1%, which was not observed in the present study. Antitopoisomerase antibody was associated with high serum IL-6 levels in our study. Other studies have also demonstrated similar association indicating IL-6 may have a role in severe disease.
IL-17A is secreted by T helper 17 cells under the influence of IL-6 and TGF β and is involved in fibroblast differentiation and collagen production. We observed high serum IL-17A levels in our patients with diffuse skin involvement, ILD, and anti-topoisomerase antibody positive patients. IL-17A levels also correlated positively with MRSS. High levels of IL-17A have been reported in the sera and skin of SSc patients. One study demonstrated increased expression of IL-17A and its receptor in early SSc skin lesions. IL-17A levels are increased early in the course of SSc and reduced with time. An association between IL-17A levels and pulmonary fibrosis has been reported in SSc. Earlier, IL-17A has been reported to significantly enhance pulmonary fibroblast proliferation and their type I collagen, TGF-β and IL-6, in vitro. Th17 cells have been reported to play an important role in skin and lung fibrosis by enhancing fibroblast proliferation and cytokine production in a mouse bleomycin model of SSc. Moreover, IL-17A derived from patients with SSc has been reported to induce the proliferation, collagen synthesis and secretion, and migration of dermal vascular smooth muscle cells through ERK1/2 signaling pathway. Both IL-17A and ERK1/2 have been proposed to be promising therapeutic targets for the treatment of SSc-related vasculopathy. In an earlier study, SSc patients were reported to have significantly higher serum IL-17A levels, but not IL-17F, and that patients with high serum IL-17A levels tend to have pitting scars and tend to have less severe skin sclerosis. The authors proposed that increased IL-17A levels in the sera and involved skin of SSc may be due to negative feedback. On the other hand, IL-17A has been reported to contribute to the pathogenesis of renal fibrosis by regulating RANTES-mediated inflammatory cell infiltration.
Elevated TGF β1 levels were observed in our patients and were associated with dc subtype, pulmonary involvement, and anti-topoisomerase antibody. There was a positive correlation between serum TGF β1 and MRSS. A recent study by Dantas et al. revealed raised serum active TGF β1 levels in patients of SSc as compared to healthy controls. They found a positive correlation between levels of active TGF β1 and diffuse subset, digital ulcers, lung fibrosis, skin involvement, and positive anti topoisomerase I. Although serum TGF β1 levels have been reported to be high in both dc-and lc-SSc, higher levels were observed in dc subtype. TGF β1 plays an important role in the pathogenesis of skin and lung fibrosis in SSc by increased production of collagen and extracellular matrix. Increased expression of TGF β1 has been observed in skin and lungs in SSc. Serum TGF β1 levels have been shown to be low in SSc during early disease. Decrease in serum TGF β may be due to increased expression of receptors in the tissues which sequester circulating TGF β.
Disease activity in scleroderma reduces with time, and this may have a bearing on serum cytokine levels. Cytokine levels could reduce as disease activity abates. In early disease, paradoxical results have been described in some studies, showing increased serum cytokine levels  while others had reported reduced levels. This has been attributed to the mopping up of cytokines by increased receptor expression in skin and other tissues. Although the cytokine levels did not differ significantly with respect to the duration of disease in the present study, patients with disease duration more than 5 years had lower levels of cytokines when compared to patients with disease duration <5 years.
In the present study, the percentage of NSIP and UIP patterns of ILD were almost equal. Literature describes NSIP to be the predominant pattern seen in scleroderma. UIP pattern is associated with more fibrosis and honey-combing reflective of aggressive and advanced lung disease. The high levels of circulating cytokines seen may explain the aggressive nature of ILD seen in our patients.
In the present study, ANA was positive in 96.8% patients, with speckled pattern seen in 52.7% and homogenous pattern in 47.3%. ENA could be done in 61 patients only, mostly dc-SSc. The relative absence of anticentromere antibodies and nucleolar pattern could be because of limited ENA data available in lc-SSc patients or because of ethnic differences. Pradhan et al. from Mumbai, Western India reported anti-centromere antibodies in 22.7% and anti-scl70 antibodies in 62.7% of a cohort of 110 patients. ANA positivity was seen in 85.5% patients of which 63.9% had speckled, 18.1% had nucleolar, 7.4% had centromere, and 2.1% had rim or peripheral pattern. They did not report a homogenous pattern. They recognized a geographic variation in autoantibody profile in patients of scleroderma.
The present study did not demonstrate an association between IL-6, IL-17A, TGF β1 levels, and digital ulcers. This variation from the reported literature may be due to vasodilator therapy, the presence of healed ulcers and disease duration when cytokines were assessed.
This study demonstrates the possible pathogenic importance of profibrotic cytokines IL-6, IL-17A and TGF β1 in SSc as evidenced by higher levels in SSc patients. In addition, the serum levels of these cytokines correlated significantly with severity of lung and skin involvement.
The duration of disease and treatment could have important effects on the level of cytokines and their correlation with clinical manifestations. Serial sampling and long-term follow-up may better elucidate relationship to duration of disease, disease activity, and various manifestations of disease. To mitigate the effects of treatment, sampling in a treatment-naïve inception cohort is warranted.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
| References|| |
Fleischmajer R, Perlish JS, Reeves JR. Cellular infiltrates in scleroderma skin. Arthritis Rheum 1977;20:975-84.
De Lauretis A, Sestini P, Pantelidis P, Hoyles R, Hansell DM, Goh NS, et al.
Serum interleukin 6 is predictive of early functional decline and mortality in interstitial lung disease associated with systemic sclerosis. J Rheumatol 2013;40:435-46.
Abdel-Magied RA, Kamel SR, Said AF, Ali HM, Abdel Gawad EA, Moussa MM, et al.
Serum interleukin-6 in systemic sclerosis and its correlation with disease parameters and cardiopulmonary involvement. Sarcoidosis Vasc Diffuse Lung Dis 2016;33:321-30.
Shima Y, Kuwahara Y, Murota H, Kitaba S, Kawai M, Hirano T, et al.
The skin of patients with systemic sclerosis softened during the treatment with anti-IL-6 receptor antibody tocilizumab. Rheumatology (Oxford) 2010;49:2408-12.
Murata M, Fujimoto M, Matsushita T, Hamaguchi Y, Hasegawa M, Takehara K, et al.
Clinical association of serum interleukin-17 levels in systemic sclerosis: Is systemic sclerosis a Th17 disease? J Dermatol Sci 2008;50:240-2.
Kurasawa K, Hirose K, Sano H, Endo H, Shinkai H, Nawata Y, et al.
Increased interleukin-17 production in patients with systemic sclerosis. Arthritis Rheum 2000;43:2455-63.
Olewicz-Gawlik A, Danczak-Pazdrowska A, Kuznar-Kaminska B, Gornowicz-Porowska J, Katulska K, Trzybulska D, et al.
Interleukin-17 and interleukin-23: Importance in the pathogenesis of lung impairment in patients with systemic sclerosis. Int J Rheum Dis 2014;17:664-70.
Dziadzio M, Smith RE, Abraham DJ, Black CM, Denton CP. Circulating levels of active transforming growth factor beta1 are reduced in diffuse cutaneous systemic sclerosis and correlate inversely with the modified Rodnan skin score. Rheumatology (Oxford) 2005;44:1518-24.
Higley H, Persichitte K, Chu S, Waegell W, Vancheeswaran R, Black C, et al.
Immunocytochemical localization and serologic detection of transforming growth factor beta 1. Association with type I procollagen and inflammatory cell markers in diffuse and limited systemic sclerosis, morphea, and Raynaud's phenomenon. Arthritis Rheum 1994;37:278-88.
van den Hoogen F, Khanna D, Fransen J, Johnson SR, Baron M, Tyndall A, et al
. 2013 classification criteria for systemic sclerosis: An American college of rheumatology/European league against rheumatism collaborative initiative. Arthritis Rheum 2013;65:2737-47.
Khanna D, Furst DE, Clements PJ, Allanore Y, Baron M, Czirjak L, et al.
Standardization of the modified rodnan skin score for use in clinical trials of systemic sclerosis. J Scleroderma Relat Disord 2017;2:11-8.
Scala E, Pallotta S, Frezzolini A, Abeni D, Barbieri C, Sampogna F, et al.
Cytokine and chemokine levels in systemic sclerosis: Relationship with cutaneous and internal organ involvement. Clin Exp Immunol 2004;138:540-6.
De Santis M, Bosello S, La Torre G, Capuano A, Tolusso B, Pagliari G, et al.
Functional, radiological and biological markers of alveolitis and infections of the lower respiratory tract in patients with systemic sclerosis. Respir Res 2005;6:96.
Dantas AT, Gonçalves SM, de Almeida AR, Gonçalves RS, Sampaio MC, Vilar KM, et al.
Reassessing the role of the active TGF-β
1 as a biomarker in systemic sclerosis: Association of serum levels with clinical manifestations. Dis Markers 2016;2016:6064830.
Nakashima T, Jinnin M, Yamane K, Honda N, Kajihara I, Makino T, et al.
Impaired IL-17 signaling pathway contributes to the increased collagen expression in scleroderma fibroblasts. J Immunol 2012;188:3573-83.
Zhou Y, Hou W, Xu K, Han D, Jiang C, Mou K, et al.
The elevated expression of th17-related cytokines and receptors is associated with skin lesion severity in early systemic sclerosis. Hum Immunol 2015;76:22-9.
Rolla G, Fusaro E, Nicola S, Bucca C, Peroni C, Parisi S, et al.
Th-17 cytokines and interstitial lung involvement in systemic sclerosis. J Breath Res 2016;10:046013.
Lei L, Zhao C, Qin F, He ZY, Wang X, Zhong XN, et al.
Th17 cells and IL-17 promote the skin and lung inflammation and fibrosis process in a bleomycin-induced murine model of systemic sclerosis. Clin Exp Rheumatol 2016;34 Suppl 100:14-22.
Liu M, Yang J, Xing X, Cui X, Li M. Interleukin-17A promotes functional activation of systemic sclerosis patient-derived dermal vascular smooth muscle cells by extracellular-regulated protein kinases signalling pathway. Arthritis Res Ther 2014;16:4223.
Peng X, Xiao Z, Zhang J, Li Y, Dong Y, Du J, et al.
IL-17A produced by both γδ T and Th17 cells promotes renal fibrosis via RANTES-mediated leukocyte infiltration after renal obstruction. J Pathol 2015;235:79-89.
Christmann RB, Sampaio-Barros P, Stifano G, Borges CL, de Carvalho CR, Kairalla R, et al.
Association of interferon- and transforming growth factor β-regulated genes and macrophage activation with systemic sclerosis-related progressive lung fibrosis. Arthritis Rheumatol 2014;66:714-25.
Sato S, Hasegawa M, Takehara K. Serum levels of interleukin-6 and interleukin-10 correlate with total skin thickness score in patients with systemic sclerosis. J Dermatol Sci 2001;27:140-6.
Lafyatis R. Transforming growth factor β – At the centre of systemic sclerosis. Nat Rev Rheumatol 2014;10:706-19.
Pradhan V, Rajadhyaksha A, Nadkar M, Pandit P, Surve P, Lecerf M, et al.
Clinical and autoimmune profile of scleroderma patients from Western India. Int J Rheumatol 2014;2014:983781.
[Figure 1], [Figure 2]
[Table 1], [Table 2]