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The -2518A>G promoter polymorphism in the CCL2 gene is not associated with systemic sclerosis susceptibility or phenotype: Results from a multicenter study of European Caucasian patients
Human Immunology 70 (2009) 130-133
Contents lists available at ScienceDirect
Human Immunology journal homepage: www.elsevier.com/locate/humimm
The -2518A⬎G promoter polymorphism in the CCL2 gene is not associated with systemic sclerosis susceptibility or phenotype: Results from a multicenter study of European Caucasian patients Timothy R.D.J. Radstake a,*, Madelon C. Vonk a, Marieke Dekkers a, Mascha M.V.A.P. Schijvenaars b, William L. Treppichio c, Robert Lafyatis d, Gabriela Riemekasten e, Frank van den Hoogen f, Marieke J.H. Coenen b a
Department of Rheumatology Radboud University Nijmegen Medical Centre, The Netherlands Department of Human Genetics, Radboud University Nijmegen Medical Centre, The Netherlands Millenium Pharmaceuticals, Cambridge, Massachusetts, USA d The Arthritis Center, Boston Medical Center, Boston, Massachusetts, USA e Department of Rheumatology and Clinical Immunology, Charitè—University Medicine Berlin, Berlin, Germany f Department of Rheumatology, St. Maartenkliniek, Nijmegen, The Netherlands b c
A R T I C L E
I N F O
Article history: Received 5 September 2008 Accepted 22 October 2008 Available online 24 November 2008
Keywords: CCL2 Systemic sclerosis Single nucleotide polymorphism
A B S T R A C T
A single nucleotide polymorphism (SNP) of the gene encoding monocyte chemoattractant protein–1 (MCP-1, CCL2) has previously been suggested to be involved in the susceptibility of systemic sclerosis (SSc). Here we have tested whether the -2518A⬎G CCL2 variant is associated with SSc susceptibility and/or phenotype using a cohort of SSc patients (n ⫽ 345). Clinical data from SSc patients attending rheumatology clinics in the Netherlands and Germany was collected DNA was obtained after informed consent. The control group used (n ⫽ 272) was randomly recruited from comparable geographic regions. The -2518A⬎G SNP in CCL2 (rs1024611) was determined using a Taqman SNP Genotyping assay. The genotype distribution was found to be similarly distributed among SSc patients and healthy controls. In addition, no association could be detected between the genotype and the presence of antinuclear antibodies, anticentromere antibodies, and antitopoisomerase antibodies or pulmonary involvement. Our results demonstrate that the functional variant -2518A⬎G of CCL2 is not implicated in the susceptibility or phenotype of SSc. 䉷 2009 American Society for Histocompatibility and Immunogenetics. Published by Elsevier Inc. All rights reserved.
1. Introduction Systemic sclerosis (SSc) is a connective tissue disease of unknown etiology, which is characterized by excessive deposition of collagen in the skin and various other organs and is clinically known as fibrosis. One of the first characteristic histologic features observed in the skin from SSc patients is the presence of infiltrating macrophages, T cells, B cells and fibroblasts implying the implication the local secretion of chemokines. Chemokines comprise a family of small secreted proteins that act as chemotactic ligands through interaction with seven membrane spanning chemokine receptors. Monocyte chemoattractant protein–1 (MCP-1, CCL2) is a multifunctional inflammatory chemokine belonging to the C-C chemokines. CCL2 is predominantly produced by monocytes but also macrophages, fibroblasts, endothelial cells and keratinocytes are T.R.D.J. Radstake is currently at the Department of Rheumatology, Boston Medical Center & The Arthritis Center, Boston, Massachusetts. * Corresponding author. E-mail address: [email protected] (T.R.D.J. Radstake).
also able to secrete CCL2. The roles of CCL2 have been thoroughly investigated and it has found that its biologic activities extend beyond that of chemo-attraction as it can also directly induce cell activation and fibrosis. In this light, CCL2 has been demonstrated to be upregulated in a variety of fibrotic conditions including idiopathic pulmonary fibrosis, systemic sclerosis, and bleomycineinduced experimental scleroderma [1– 4]. In addition, the direct role of CCL2 in the fibrotic process has been further substantiated by the observations that CCL2 is able to directly increase type I collagen gene expression by the modulation of TGF expression [5], and that mice lacking CCL2 receptor (CCR2) are protected from fluorescein isothiocynate- and bleomycine-induced lung fibrosis [6]. More recently, SKL-2841, a small-molecule antagonist of CCL2 and MIP-1 was shown to significantly suppress the inflammation of inflammatory cells in bleomycin-induced scleroderma, further attesting to the pivotal role of CCL2 in fibrosis [7]. In SSc patients, increased serum CCL2 levels were found to correlate with clinical symptoms such as pulmonary fibrosis [2,8,9]. More recently, CCL2 protein levels were suggested as a useful tool for risk stratification in early-stage disease [10]. Simi-
0198-8859/09/$32.00 - see front matter 䉷 2009 American Society for Histocompatibility and Immunogenetics. Published by Elsevier Inc. All rights reserved. doi:10.1016/j.humimm.2008.10.012
T.R.D.J. Radstake et al. / Human Immunology 70 (2009) 130-133
larly, supernatants collected from peripheral blood mononuclear cells (PBMCs) from SSc patients had significantly higher spontaneous production of CCL2 and the protein was highly expressed in SSc skin and isolated skin fibroblasts [2,11,12]. Interestingly, and in contrast to mice, a recent report from Distler et al. suggested a role for IL-4 in the pro-fibrotic effect of CCL2; however no direct effect on fibroblast behaviour could be observed [13]. Various single nucleotide polymorphisms (SNPs) have been described in the CCL2 gene located on chromosome 17q11.2– q12. One of these SNPs in the promoter of the gene changing an A to G at position -2518 (-2518A⬎G, rs1024611) has been functionally related to CCL2 levels. So far, this SNP has been associated with CCL2 expression in chronic hepatitis C infection [14], pulmonary tuberculosis [15], reduced insulin sensitivity in nondiabetic individuals, and systemic lupus erythematosus [16]. So far, three studies have studied the potential role of the CCL2 gene in SSc leading to much controversy. Karerr et al. were the first to describe a positive association between CCl2 functional variant and SSc susceptibility using an exceptional small number (n ⫽ 18) of SSc patients [17]. After this study, two recent reports showed an absence of association between SSc susceptibility using small cohorts of UK and Slovak patients (n ⫽ 94 and 48, respectively) [18,19]. Because numerous reports suggest for a role for CCL2 in SSc, we investigated whether the CCL2 SNP is associated with susceptibility to and/or the phenotype of SSc. In contrast to previous findings on the protein level, we could not prove an association between the -2518A⬎G CCL2 variant and SSc or disease phenotype using a large cohort of patients with well-documented SSc. 2. Subjects and methods 2.1. Study subjects Patients at European League Against Rheumatism Scleroderma Trials and Research Group centers (Radboud University Nijmegen Medical Centre, Nijmegen, the Netherlands; St. Maartens Kliniek Nijmegen, Nijmegen, the Netherlands; and CharitÊ—University Medicine Berlin, Berlin, Germany) were included in this study (n ⫽ 345). The control group (n ⫽ 272) was randomly recruited from a comparable population. SSc was classified as either a limited subtype or a diffuse subtype according to the extent of the skin involved, as proposed by Leroy et al. [20]. The following clinical data were collected for ascertainment of clinical phenotype: age, gender, disease duration, and presence of autoantibodies (antitopoisomerase and anticentromere). Involvement of the lungs was assessed according to international guidelines [21]. Pulmonary fibrosis was investigated by a computed tomographic scan. Restrictive syndrome and diffusion capacity of the lungs was defined as a forced vital capacity ⬍75% of the predicted value and a diffusion capacity for carbon monoxide of ⬍75% of predicted (based on age, gender, height, and ethnic origin). The control population consisted of unrelated healthy individuals recruited in the same geographic region as SSc patients and matched by sex and ethnicity with the SSc patients groups. From these individuals, DNA was extracted from blood using salt extraction [22]. All patients gave written informed consent, and the ethics committee of each hospital approved the study. 2.2. Polymerase chain reaction The -2518A⬎G SNP in CCL2 (rs1024611) was determined using a Taqman SNP Genotyping assay (assay ID, C_2590362_10, Applied Biosystems, Nieuwekerk aan de Ijssel, the Netherlands). Reactions were performed according to the manufacturer’s protocol using 10 ng of DNA. Genotypes were generated automatically using the 7500 Fast Real-Time PCR Instrument (Applied Biosystems, Nieuwekerk aan de Ijssel, the Netherlands).
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2.3. Statistical analysis Test for deviation from Hardy Weinberg equilibrium was performed by means of Chi-square tests in controls only. A 2 test was performed using SPSS, version 14.0 statistical package (SPSS Inc., Chicago, IL) to test for association of the genotype with SSc by comparing the genotype frequencies of cases and controls. To test for association with a specific disease phenotype, 2 tests were performed to compare the frequencies of the genotypes between patients with a limited and diffuse phenotypes, and tests were conducted as to whether lung fibroses was associated with a specific genotype. 3. Results In the current study we genotyped 345 SSc patients and 272 healthy controls for the -2518 A⬎G CCL2 variant. Table 1 summarizes characteristics of the SSc patients and healthy controls. The mean age (⫾SD) of the SSc patients was 56 ⫾ 13 years, the percentage of female individuals was 81.7%, the percentage of patients having limited phenotype of SSc was 62.6%, and the presence of anti nuclear antibodies was 94.5%, which is in line with previous genetic studies in European populations [23,24]. The genotype and allele distribution among the SSc samples from the Netherlands and Germany was similar, and no deviation from Hardy-Weinberg equilibrium was observed in the healthy control group (data not shown). The genotype and allele distribution was found to be similarly distributed among SSc patients and healthy controls and also within clinical phenotypes between German and Dutch patients (Table 2). Moreover, the -2518 GG genotype that was previously suggested to be associated with SSc susceptibility and increased CCL2 levels in fibroblasts was represented equally among patients having diffuse or limited disease, between patients with or without computed tomography–proven lung fibrosis, or patients with or without decreased lung capacity. Also no association could be detected between the genotype and the presence of antinuclear antibodies, anticentromere antibodies, and antitopoisomerase antibodies. 4. Discussion Systemic sclerosis is characterized by the involvement of inflammatory cells implying the role of chemokines. Currently, accumulating evidence suggests a role for CCL2 proteins in SSc. A recent report by Carulli et al. demonstrated that CCL2 protein levels were consistently and significantly elevated in the circulation of SSc patients [10]. Moreover, high CCL2 levels tended to be associated with internal organ involvement in early SSc, particularly pulmonary hypertension and cardiac involvement. In addition, Antonelli et al. also observed increased levels of CCL2 protein in the serum of SSc patients early after its diagnosis [9]. Interestingly, here it was found that CCL2 levels were quite stable throughout disease Table 1 Demographic variables of patients and healthy controls Characteristic
Healthy controls
SSc patients
N Age, years, mean (SD) Female (%) Limited phenotype (%) Disease duration, months, mean (SD) Positivity ANA antibodies Positivity anticentromere antibodies Positivity antitopoisomerase antibodies Pulmonary fibrosis on CT scan Low FVC (⬍75% predicted) Low DLCO (⬍75% predicted)
272 42 (12) 43.8%
345 56 (13) 81.7% 62.6% 112 (92) 94.5% 34.8% 22.3% 34.8% 20.9% 61.7%
— — — — — —
ANA, anti-nuclear antibodies; DLCO, diffuse capacity of the lung for carbon monoxide; FVC, forced vital capacity; SSc, systemic sclerosis.
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T.R.D.J. Radstake et al. / Human Immunology 70 (2009) 130-133
Table 2 Genotype distributions of the -2518A⬎G CCL2 polymorphism within different disease variables from systemic sclerosis (SSc) patients ⫺2518 CCL2 genotype
Patients with disease vs. controls SSc patients Healthy controls Diffuse vs. limited Limited Diffuse ANA positive vs. negative Positive Negative Antitopoisomerase abs Positive Negative Anticentromere abs Positive Negative Pulmonary fibrosis Present Absent Predicted FVC ⬍75% ⬎75% Predicted DLCO ⬍75% ⬎75%
A/A
A/G
G/G
Total (n)
p Value
201 (58.3) 139 (51.1)
122 (35.4) 112 (41.2)
22 (6.4) 21 (7.7)
345 272
0.209
127 (58.8) 74 (57.4)
73 (33.8) 49 (38.0)
16 (7.4) 6 (4.7)
216 129
0.497
188 (57.7) 8 (57.1)
117 (35.9) 5 (35.7)
21 (6.4) 1 (7.1)
326 14
1.0
49 (63.6) 144 (56.0)
23 (29.9) 96 (37.4)
5 (6.5) 17 (6.6)
77 257
0.462
71 (59.2) 125 (57.1)
41 (34.2) 80 (36.5)
8 (6.7) 14 (6.4)
120 219
0.916
69 (57.5) 119 (58.6)
39 (32.5) 75 (36.9)
12 (10) 9 (4.4)
120 203
0.135
41 (56.9) 154 (58.3)
26 (36.1) 93 (35.2)
5 (6.9) 17 (6.4)
72 264
0.977
125 (58.7) 73 (56.6)
73 (34.3) 49 (38.0)
15 (7) 7 (5.4)
213 129
0.725
Abs, antibodies; ANA, antinuclear antibodies; DLCO, diffuse capacity of the lung for carbon monoxide; FVC, forced vital capacity.
p Values were calculated using 2 tests.
whereas CXCL10 levels dropped significantly after 5 years. Altogether, these observations underscore the potential role in SSc, either in its pathogenesis and/or as a useful marker for follow-up. Here we focused on determining a potential association between the -2518A⬎G CCL2 variant and SSc susceptibility and/or phenotype. We demonstrate that the functional variant of CCL2 is not implicated in susceptibility to SSc as previously suggested. In addition, we were not able to detect an association between the -2518A⬎G CCL2 variant and any clinical parameter of SSc phenotype. To our knowledge, this is the first study attempting to replicate previous observations of CCL2 involvement using a large and well-documented cohort of SSc patients and allowing robust conclusions. The observation that the genotype distribution is almost completely overlapping validates the combined analysis of the samples. Several explanations might be responsible for the lack of association with the genetic variant. In our view, the most likely explanation lies within the fact that other triggers than the currently investigated genetic variant drive the increased CCL2 protein levels. There are several SNPs described in the CCL2 gene. Although the -2518A⬎G variant has been suggested to be functional, it is likely that this is true for others as well. In addition, activated immune cells mainly secrete CCL2 protein. The underlying pathway responsible for the activation of these immune cells in SSc is still unknown and might involve a direct relationship with SSc. In conclusion, although CCL2 protein may be involved in the pathogenesis of SSc, our results indicate a lack of association between the genetic variant and SSc. Therefore, we believe that the -2518A⬎G CCL2 functional variant cannot be used as a marker for SSc susceptibility and or phenotype. Acknowledgments We are greatly thankful to all of the rheumatologists and research nurses who were involved in the collection of blood. We also thank Christel Brouwer and Jasper Broen for their invaluable work
regarding the isolation, measurement, and storage of DNA samples. This study was funded by Millenium Inc, a personal grant from the Netherlands Organization for Scientific Research (NWO, VENI grant T.R.), and the Young Investigators grant from EULAR (T.R.). This publication reflects solely the authors’ views and not those of the supporting organizations. Conflict of interest statement: Timothy R.D.J. Radstake received a grant from Millenium that covered the analysis of the CCL2 polymorphism. No other authors have any conflict of interest. M. Vonk and M. Coenen, solely, had access to the data and are fully responsible for the reliability of the data. References [1] Distler O, Pap T, Kowal-Bielecka O, Meyringer R, Guiducci S, Landthaler M, et al. Overexpression of monocyte chemoattractant protein 1 in systemic sclerosis: Role of platelet-derived growth factor and effects on monocyte chemotaxis and collagen synthesis. Arthritis Rheum 2001;44:2665–78. [2] Hasegawa M, Sato S, Takehara K. Augmented production of chemokines (monocyte chemotactic protein-1 (MCP-1), macrophage inflammatory protein-1alpha (MIP-1alpha) and MIP-1beta) in patients with systemic sclerosis: MCP-1 and MIP-1alpha may be involved in the development of pulmonary fibrosis. Clin Exp Immunol 1999;117:159 – 65. [3] Loyd JE, Slovis B, Phillips JA, 3rd, Butler MG, Foroud TM, Conneally PM, Newman JH. The presence of genetic anticipation suggests that the molecular basis of familial primary pulmonary hypertension may be trinucleotide repeat expansion. Chest 1997;111(6 Suppl):82S– 83S. [4] Rollins BJ. Monocyte chemoattractant protein 1: A potential regulator of monocyte recruitment in inflammatory disease. Mol Med Today 1996;2: 198 –204. [5] Gharaee-Kermani M, McCullumsmith RE, Charo IF, Kunkel SL, Phan SH. CCchemokine receptor 2 required for bleomycin-induced pulmonary fibrosis. Cytokine 2003;24:266 –76. [6] Moore BB, Paine R, 3rd, Christensen PJ, Moore TA, Sitterding S, Ngan R, et al. Protection from pulmonary fibrosis in the absence of CCR2 signaling. J Immunol 2001;167:4368 –77. [7] Kimura M, Kawahito Y, Hamaguchi M, Nakamura T, Okamoto M, Matsumoto Y, et al. SKL-2841, a dual antagonist of MCP-1 and MIP-1 beta, prevents bleomycininduced skin sclerosis in mice. Biomed Pharmacother 2007;61:222– 8. [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. [9] Antonelli A, Ferri C, Fallahi P, Ferrari SM, Giuggioli D, Colaci M, et al. CXCL10 (alpha) and CCL2 (beta) chemokines in systemic sclerosis—a longitudinal study. Rheumatology (Oxford) 2008;47:45–9. [10] Carulli MT, Handler C, Coghlan JG, Black CM, Denton CP. Can CCL2 serum levels be used in risk stratification or to monitor treatment response in systemic sclerosis? Ann Rheum Dis 2008;67:105–9. [11] Yamamoto T, Eckes B, Hartmann K, Krieg T. Expression of monocyte chemoattractant protein-1 in the lesional skin of systemic sclerosis. J Dermatol Sci 2001;26:133–9. [12] Yamamoto T, Eckes B, Krieg T. High expression and autoinduction of monocyte chemoattractant protein-1 in scleroderma fibroblasts. Eur J Immunol 2001;31: 2936 – 41. [13] Distler JH, Jungel A, Caretto D, Schulze-Horsel U, Kowal-Bielecka O, Gay RE, et al. Monocyte chemoattractant protein 1 released from glycosaminoglycans mediates its profibrotic effects in systemic sclerosis via the release of interleukin-4 from T cells. Arthritis Rheum 2006;54:214 –25. [14] Muhlbauer M, Bosserhoff AK, Hartmann A, Thasler WE, Weiss TS, Herfarth H, et al. A novel MCP-1 gene polymorphism is associated with hepatic MCP-1 expression and severity of HCV-related liver disease. Gastroenterology 2003; 125:1085–93. [15] Flores-Villanueva PO, Ruiz-Morales JA, Song CH, Flores LM, Jo EK, Montano M, et al. A functional promoter polymorphism in monocyte chemoattractant protein-1 is associated with increased susceptibility to pulmonary tuberculosis. J Exp Med 2005;202:1649 –58. [16] Maruyama-Furuta N, Yano Y, Gabazza EC, Suematsu M, Matsumoto K, Akatsuka H, et al. Monocyte chemoattractant protein-1 promoter -2518 polymorphism is associated with post-challenge insulin and glucose levels in nondiabetic Japanese subjects. Diabetes Res Clin Pract 2007;78:208 –10. [17] Karrer S, Bosserhoff AK, Weiderer P, Distler O, Landthaler M, Szeimies RM, et al. The -2518 promotor polymorphism in the MCP-1 gene is associated with systemic sclerosis. J Invest Dermatol 2005;124:92– 8. [18] Carulli MT, Spagnolo P, Fonseca C, Welsh KI, duBois RM, Black CM, Denton CP. Single-nucleotide polymorphisms in CCL2 gene are not associated with susceptibility to systemic sclerosis. J Rheumatol 2008;35:839 – 44. [19] Navratilova Z, Lukac J, Mrazek F, Kriegova E, Bucova M, Bosak V, Petrek M. MCP-1 -2518 A/G single nucleotide polymorphism in Slovak patients with systemic sclerosis. Mediators Inflamm 2008:204063.
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[20] LeRoy EC, Black C, Fleischmajer R, Jablonska S, Krieg T, Medsger TA, Jr., et al. Scleroderma (systemic sclerosis): Classification, subsets and pathogenesis. J Rheumatol 1988;15:202–5. [21] Matucci-Cerinic M, D’Angelo S, Denton CP, Vlachoyiannopoulos P, Silver R. Assessment of lung involvement. Clin Exp Rheumatol 2003;21(3 Suppl 29):S19 –S23. [22] Miller SA, Dykes DD, Polesky HF. A simple salting out procedure for extracting DNA from human nucleated cells. Nucleic Acids Res 1988;16:1215.
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[23] Allanore Y, Borderie D, Airo P, Guiducci S, Czirjak L, Nasonov EL, et al. Lack of association between three vascular endothelial growth factor gene polymorphisms and systemic sclerosis: Results from a multicenter EUSTAR study of European Caucasian patients. Ann Rheum Dis 2007;66:257–9. [24] Wipff J, Giraud M, Sibilia J, Mouthon L, Meyer O, Tiev K, et al. Polymorphic markers of the fibrillin-1 gene and systemic sclerosis in European Caucasian patients. J Rheumatol 2008;35:643–9.
Contents lists available at ScienceDirect
Human Immunology journal homepage: www.elsevier.com/locate/humimm
The -2518A⬎G promoter polymorphism in the CCL2 gene is not associated with systemic sclerosis susceptibility or phenotype: Results from a multicenter study of European Caucasian patients Timothy R.D.J. Radstake a,*, Madelon C. Vonk a, Marieke Dekkers a, Mascha M.V.A.P. Schijvenaars b, William L. Treppichio c, Robert Lafyatis d, Gabriela Riemekasten e, Frank van den Hoogen f, Marieke J.H. Coenen b a
Department of Rheumatology Radboud University Nijmegen Medical Centre, The Netherlands Department of Human Genetics, Radboud University Nijmegen Medical Centre, The Netherlands Millenium Pharmaceuticals, Cambridge, Massachusetts, USA d The Arthritis Center, Boston Medical Center, Boston, Massachusetts, USA e Department of Rheumatology and Clinical Immunology, Charitè—University Medicine Berlin, Berlin, Germany f Department of Rheumatology, St. Maartenkliniek, Nijmegen, The Netherlands b c
A R T I C L E
I N F O
Article history: Received 5 September 2008 Accepted 22 October 2008 Available online 24 November 2008
Keywords: CCL2 Systemic sclerosis Single nucleotide polymorphism
A B S T R A C T
A single nucleotide polymorphism (SNP) of the gene encoding monocyte chemoattractant protein–1 (MCP-1, CCL2) has previously been suggested to be involved in the susceptibility of systemic sclerosis (SSc). Here we have tested whether the -2518A⬎G CCL2 variant is associated with SSc susceptibility and/or phenotype using a cohort of SSc patients (n ⫽ 345). Clinical data from SSc patients attending rheumatology clinics in the Netherlands and Germany was collected DNA was obtained after informed consent. The control group used (n ⫽ 272) was randomly recruited from comparable geographic regions. The -2518A⬎G SNP in CCL2 (rs1024611) was determined using a Taqman SNP Genotyping assay. The genotype distribution was found to be similarly distributed among SSc patients and healthy controls. In addition, no association could be detected between the genotype and the presence of antinuclear antibodies, anticentromere antibodies, and antitopoisomerase antibodies or pulmonary involvement. Our results demonstrate that the functional variant -2518A⬎G of CCL2 is not implicated in the susceptibility or phenotype of SSc. 䉷 2009 American Society for Histocompatibility and Immunogenetics. Published by Elsevier Inc. All rights reserved.
1. Introduction Systemic sclerosis (SSc) is a connective tissue disease of unknown etiology, which is characterized by excessive deposition of collagen in the skin and various other organs and is clinically known as fibrosis. One of the first characteristic histologic features observed in the skin from SSc patients is the presence of infiltrating macrophages, T cells, B cells and fibroblasts implying the implication the local secretion of chemokines. Chemokines comprise a family of small secreted proteins that act as chemotactic ligands through interaction with seven membrane spanning chemokine receptors. Monocyte chemoattractant protein–1 (MCP-1, CCL2) is a multifunctional inflammatory chemokine belonging to the C-C chemokines. CCL2 is predominantly produced by monocytes but also macrophages, fibroblasts, endothelial cells and keratinocytes are T.R.D.J. Radstake is currently at the Department of Rheumatology, Boston Medical Center & The Arthritis Center, Boston, Massachusetts. * Corresponding author. E-mail address: [email protected] (T.R.D.J. Radstake).
also able to secrete CCL2. The roles of CCL2 have been thoroughly investigated and it has found that its biologic activities extend beyond that of chemo-attraction as it can also directly induce cell activation and fibrosis. In this light, CCL2 has been demonstrated to be upregulated in a variety of fibrotic conditions including idiopathic pulmonary fibrosis, systemic sclerosis, and bleomycineinduced experimental scleroderma [1– 4]. In addition, the direct role of CCL2 in the fibrotic process has been further substantiated by the observations that CCL2 is able to directly increase type I collagen gene expression by the modulation of TGF expression [5], and that mice lacking CCL2 receptor (CCR2) are protected from fluorescein isothiocynate- and bleomycine-induced lung fibrosis [6]. More recently, SKL-2841, a small-molecule antagonist of CCL2 and MIP-1 was shown to significantly suppress the inflammation of inflammatory cells in bleomycin-induced scleroderma, further attesting to the pivotal role of CCL2 in fibrosis [7]. In SSc patients, increased serum CCL2 levels were found to correlate with clinical symptoms such as pulmonary fibrosis [2,8,9]. More recently, CCL2 protein levels were suggested as a useful tool for risk stratification in early-stage disease [10]. Simi-
0198-8859/09/$32.00 - see front matter 䉷 2009 American Society for Histocompatibility and Immunogenetics. Published by Elsevier Inc. All rights reserved. doi:10.1016/j.humimm.2008.10.012
T.R.D.J. Radstake et al. / Human Immunology 70 (2009) 130-133
larly, supernatants collected from peripheral blood mononuclear cells (PBMCs) from SSc patients had significantly higher spontaneous production of CCL2 and the protein was highly expressed in SSc skin and isolated skin fibroblasts [2,11,12]. Interestingly, and in contrast to mice, a recent report from Distler et al. suggested a role for IL-4 in the pro-fibrotic effect of CCL2; however no direct effect on fibroblast behaviour could be observed [13]. Various single nucleotide polymorphisms (SNPs) have been described in the CCL2 gene located on chromosome 17q11.2– q12. One of these SNPs in the promoter of the gene changing an A to G at position -2518 (-2518A⬎G, rs1024611) has been functionally related to CCL2 levels. So far, this SNP has been associated with CCL2 expression in chronic hepatitis C infection [14], pulmonary tuberculosis [15], reduced insulin sensitivity in nondiabetic individuals, and systemic lupus erythematosus [16]. So far, three studies have studied the potential role of the CCL2 gene in SSc leading to much controversy. Karerr et al. were the first to describe a positive association between CCl2 functional variant and SSc susceptibility using an exceptional small number (n ⫽ 18) of SSc patients [17]. After this study, two recent reports showed an absence of association between SSc susceptibility using small cohorts of UK and Slovak patients (n ⫽ 94 and 48, respectively) [18,19]. Because numerous reports suggest for a role for CCL2 in SSc, we investigated whether the CCL2 SNP is associated with susceptibility to and/or the phenotype of SSc. In contrast to previous findings on the protein level, we could not prove an association between the -2518A⬎G CCL2 variant and SSc or disease phenotype using a large cohort of patients with well-documented SSc. 2. Subjects and methods 2.1. Study subjects Patients at European League Against Rheumatism Scleroderma Trials and Research Group centers (Radboud University Nijmegen Medical Centre, Nijmegen, the Netherlands; St. Maartens Kliniek Nijmegen, Nijmegen, the Netherlands; and CharitÊ—University Medicine Berlin, Berlin, Germany) were included in this study (n ⫽ 345). The control group (n ⫽ 272) was randomly recruited from a comparable population. SSc was classified as either a limited subtype or a diffuse subtype according to the extent of the skin involved, as proposed by Leroy et al. [20]. The following clinical data were collected for ascertainment of clinical phenotype: age, gender, disease duration, and presence of autoantibodies (antitopoisomerase and anticentromere). Involvement of the lungs was assessed according to international guidelines [21]. Pulmonary fibrosis was investigated by a computed tomographic scan. Restrictive syndrome and diffusion capacity of the lungs was defined as a forced vital capacity ⬍75% of the predicted value and a diffusion capacity for carbon monoxide of ⬍75% of predicted (based on age, gender, height, and ethnic origin). The control population consisted of unrelated healthy individuals recruited in the same geographic region as SSc patients and matched by sex and ethnicity with the SSc patients groups. From these individuals, DNA was extracted from blood using salt extraction [22]. All patients gave written informed consent, and the ethics committee of each hospital approved the study. 2.2. Polymerase chain reaction The -2518A⬎G SNP in CCL2 (rs1024611) was determined using a Taqman SNP Genotyping assay (assay ID, C_2590362_10, Applied Biosystems, Nieuwekerk aan de Ijssel, the Netherlands). Reactions were performed according to the manufacturer’s protocol using 10 ng of DNA. Genotypes were generated automatically using the 7500 Fast Real-Time PCR Instrument (Applied Biosystems, Nieuwekerk aan de Ijssel, the Netherlands).
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2.3. Statistical analysis Test for deviation from Hardy Weinberg equilibrium was performed by means of Chi-square tests in controls only. A 2 test was performed using SPSS, version 14.0 statistical package (SPSS Inc., Chicago, IL) to test for association of the genotype with SSc by comparing the genotype frequencies of cases and controls. To test for association with a specific disease phenotype, 2 tests were performed to compare the frequencies of the genotypes between patients with a limited and diffuse phenotypes, and tests were conducted as to whether lung fibroses was associated with a specific genotype. 3. Results In the current study we genotyped 345 SSc patients and 272 healthy controls for the -2518 A⬎G CCL2 variant. Table 1 summarizes characteristics of the SSc patients and healthy controls. The mean age (⫾SD) of the SSc patients was 56 ⫾ 13 years, the percentage of female individuals was 81.7%, the percentage of patients having limited phenotype of SSc was 62.6%, and the presence of anti nuclear antibodies was 94.5%, which is in line with previous genetic studies in European populations [23,24]. The genotype and allele distribution among the SSc samples from the Netherlands and Germany was similar, and no deviation from Hardy-Weinberg equilibrium was observed in the healthy control group (data not shown). The genotype and allele distribution was found to be similarly distributed among SSc patients and healthy controls and also within clinical phenotypes between German and Dutch patients (Table 2). Moreover, the -2518 GG genotype that was previously suggested to be associated with SSc susceptibility and increased CCL2 levels in fibroblasts was represented equally among patients having diffuse or limited disease, between patients with or without computed tomography–proven lung fibrosis, or patients with or without decreased lung capacity. Also no association could be detected between the genotype and the presence of antinuclear antibodies, anticentromere antibodies, and antitopoisomerase antibodies. 4. Discussion Systemic sclerosis is characterized by the involvement of inflammatory cells implying the role of chemokines. Currently, accumulating evidence suggests a role for CCL2 proteins in SSc. A recent report by Carulli et al. demonstrated that CCL2 protein levels were consistently and significantly elevated in the circulation of SSc patients [10]. Moreover, high CCL2 levels tended to be associated with internal organ involvement in early SSc, particularly pulmonary hypertension and cardiac involvement. In addition, Antonelli et al. also observed increased levels of CCL2 protein in the serum of SSc patients early after its diagnosis [9]. Interestingly, here it was found that CCL2 levels were quite stable throughout disease Table 1 Demographic variables of patients and healthy controls Characteristic
Healthy controls
SSc patients
N Age, years, mean (SD) Female (%) Limited phenotype (%) Disease duration, months, mean (SD) Positivity ANA antibodies Positivity anticentromere antibodies Positivity antitopoisomerase antibodies Pulmonary fibrosis on CT scan Low FVC (⬍75% predicted) Low DLCO (⬍75% predicted)
272 42 (12) 43.8%
345 56 (13) 81.7% 62.6% 112 (92) 94.5% 34.8% 22.3% 34.8% 20.9% 61.7%
— — — — — —
ANA, anti-nuclear antibodies; DLCO, diffuse capacity of the lung for carbon monoxide; FVC, forced vital capacity; SSc, systemic sclerosis.
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Table 2 Genotype distributions of the -2518A⬎G CCL2 polymorphism within different disease variables from systemic sclerosis (SSc) patients ⫺2518 CCL2 genotype
Patients with disease vs. controls SSc patients Healthy controls Diffuse vs. limited Limited Diffuse ANA positive vs. negative Positive Negative Antitopoisomerase abs Positive Negative Anticentromere abs Positive Negative Pulmonary fibrosis Present Absent Predicted FVC ⬍75% ⬎75% Predicted DLCO ⬍75% ⬎75%
A/A
A/G
G/G
Total (n)
p Value
201 (58.3) 139 (51.1)
122 (35.4) 112 (41.2)
22 (6.4) 21 (7.7)
345 272
0.209
127 (58.8) 74 (57.4)
73 (33.8) 49 (38.0)
16 (7.4) 6 (4.7)
216 129
0.497
188 (57.7) 8 (57.1)
117 (35.9) 5 (35.7)
21 (6.4) 1 (7.1)
326 14
1.0
49 (63.6) 144 (56.0)
23 (29.9) 96 (37.4)
5 (6.5) 17 (6.6)
77 257
0.462
71 (59.2) 125 (57.1)
41 (34.2) 80 (36.5)
8 (6.7) 14 (6.4)
120 219
0.916
69 (57.5) 119 (58.6)
39 (32.5) 75 (36.9)
12 (10) 9 (4.4)
120 203
0.135
41 (56.9) 154 (58.3)
26 (36.1) 93 (35.2)
5 (6.9) 17 (6.4)
72 264
0.977
125 (58.7) 73 (56.6)
73 (34.3) 49 (38.0)
15 (7) 7 (5.4)
213 129
0.725
Abs, antibodies; ANA, antinuclear antibodies; DLCO, diffuse capacity of the lung for carbon monoxide; FVC, forced vital capacity.
p Values were calculated using 2 tests.
whereas CXCL10 levels dropped significantly after 5 years. Altogether, these observations underscore the potential role in SSc, either in its pathogenesis and/or as a useful marker for follow-up. Here we focused on determining a potential association between the -2518A⬎G CCL2 variant and SSc susceptibility and/or phenotype. We demonstrate that the functional variant of CCL2 is not implicated in susceptibility to SSc as previously suggested. In addition, we were not able to detect an association between the -2518A⬎G CCL2 variant and any clinical parameter of SSc phenotype. To our knowledge, this is the first study attempting to replicate previous observations of CCL2 involvement using a large and well-documented cohort of SSc patients and allowing robust conclusions. The observation that the genotype distribution is almost completely overlapping validates the combined analysis of the samples. Several explanations might be responsible for the lack of association with the genetic variant. In our view, the most likely explanation lies within the fact that other triggers than the currently investigated genetic variant drive the increased CCL2 protein levels. There are several SNPs described in the CCL2 gene. Although the -2518A⬎G variant has been suggested to be functional, it is likely that this is true for others as well. In addition, activated immune cells mainly secrete CCL2 protein. The underlying pathway responsible for the activation of these immune cells in SSc is still unknown and might involve a direct relationship with SSc. In conclusion, although CCL2 protein may be involved in the pathogenesis of SSc, our results indicate a lack of association between the genetic variant and SSc. Therefore, we believe that the -2518A⬎G CCL2 functional variant cannot be used as a marker for SSc susceptibility and or phenotype. Acknowledgments We are greatly thankful to all of the rheumatologists and research nurses who were involved in the collection of blood. We also thank Christel Brouwer and Jasper Broen for their invaluable work
regarding the isolation, measurement, and storage of DNA samples. This study was funded by Millenium Inc, a personal grant from the Netherlands Organization for Scientific Research (NWO, VENI grant T.R.), and the Young Investigators grant from EULAR (T.R.). This publication reflects solely the authors’ views and not those of the supporting organizations. Conflict of interest statement: Timothy R.D.J. Radstake received a grant from Millenium that covered the analysis of the CCL2 polymorphism. No other authors have any conflict of interest. M. Vonk and M. Coenen, solely, had access to the data and are fully responsible for the reliability of the data. References [1] Distler O, Pap T, Kowal-Bielecka O, Meyringer R, Guiducci S, Landthaler M, et al. Overexpression of monocyte chemoattractant protein 1 in systemic sclerosis: Role of platelet-derived growth factor and effects on monocyte chemotaxis and collagen synthesis. Arthritis Rheum 2001;44:2665–78. [2] Hasegawa M, Sato S, Takehara K. Augmented production of chemokines (monocyte chemotactic protein-1 (MCP-1), macrophage inflammatory protein-1alpha (MIP-1alpha) and MIP-1beta) in patients with systemic sclerosis: MCP-1 and MIP-1alpha may be involved in the development of pulmonary fibrosis. Clin Exp Immunol 1999;117:159 – 65. [3] Loyd JE, Slovis B, Phillips JA, 3rd, Butler MG, Foroud TM, Conneally PM, Newman JH. The presence of genetic anticipation suggests that the molecular basis of familial primary pulmonary hypertension may be trinucleotide repeat expansion. Chest 1997;111(6 Suppl):82S– 83S. [4] Rollins BJ. Monocyte chemoattractant protein 1: A potential regulator of monocyte recruitment in inflammatory disease. Mol Med Today 1996;2: 198 –204. [5] Gharaee-Kermani M, McCullumsmith RE, Charo IF, Kunkel SL, Phan SH. CCchemokine receptor 2 required for bleomycin-induced pulmonary fibrosis. Cytokine 2003;24:266 –76. [6] Moore BB, Paine R, 3rd, Christensen PJ, Moore TA, Sitterding S, Ngan R, et al. Protection from pulmonary fibrosis in the absence of CCR2 signaling. J Immunol 2001;167:4368 –77. [7] Kimura M, Kawahito Y, Hamaguchi M, Nakamura T, Okamoto M, Matsumoto Y, et al. SKL-2841, a dual antagonist of MCP-1 and MIP-1 beta, prevents bleomycininduced skin sclerosis in mice. Biomed Pharmacother 2007;61:222– 8. [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. [9] Antonelli A, Ferri C, Fallahi P, Ferrari SM, Giuggioli D, Colaci M, et al. CXCL10 (alpha) and CCL2 (beta) chemokines in systemic sclerosis—a longitudinal study. Rheumatology (Oxford) 2008;47:45–9. [10] Carulli MT, Handler C, Coghlan JG, Black CM, Denton CP. Can CCL2 serum levels be used in risk stratification or to monitor treatment response in systemic sclerosis? Ann Rheum Dis 2008;67:105–9. [11] Yamamoto T, Eckes B, Hartmann K, Krieg T. Expression of monocyte chemoattractant protein-1 in the lesional skin of systemic sclerosis. J Dermatol Sci 2001;26:133–9. [12] Yamamoto T, Eckes B, Krieg T. High expression and autoinduction of monocyte chemoattractant protein-1 in scleroderma fibroblasts. Eur J Immunol 2001;31: 2936 – 41. [13] Distler JH, Jungel A, Caretto D, Schulze-Horsel U, Kowal-Bielecka O, Gay RE, et al. Monocyte chemoattractant protein 1 released from glycosaminoglycans mediates its profibrotic effects in systemic sclerosis via the release of interleukin-4 from T cells. Arthritis Rheum 2006;54:214 –25. [14] Muhlbauer M, Bosserhoff AK, Hartmann A, Thasler WE, Weiss TS, Herfarth H, et al. A novel MCP-1 gene polymorphism is associated with hepatic MCP-1 expression and severity of HCV-related liver disease. Gastroenterology 2003; 125:1085–93. [15] Flores-Villanueva PO, Ruiz-Morales JA, Song CH, Flores LM, Jo EK, Montano M, et al. A functional promoter polymorphism in monocyte chemoattractant protein-1 is associated with increased susceptibility to pulmonary tuberculosis. J Exp Med 2005;202:1649 –58. [16] Maruyama-Furuta N, Yano Y, Gabazza EC, Suematsu M, Matsumoto K, Akatsuka H, et al. Monocyte chemoattractant protein-1 promoter -2518 polymorphism is associated with post-challenge insulin and glucose levels in nondiabetic Japanese subjects. Diabetes Res Clin Pract 2007;78:208 –10. [17] Karrer S, Bosserhoff AK, Weiderer P, Distler O, Landthaler M, Szeimies RM, et al. The -2518 promotor polymorphism in the MCP-1 gene is associated with systemic sclerosis. J Invest Dermatol 2005;124:92– 8. [18] Carulli MT, Spagnolo P, Fonseca C, Welsh KI, duBois RM, Black CM, Denton CP. Single-nucleotide polymorphisms in CCL2 gene are not associated with susceptibility to systemic sclerosis. J Rheumatol 2008;35:839 – 44. [19] Navratilova Z, Lukac J, Mrazek F, Kriegova E, Bucova M, Bosak V, Petrek M. MCP-1 -2518 A/G single nucleotide polymorphism in Slovak patients with systemic sclerosis. Mediators Inflamm 2008:204063.
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