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Association between RANTES promoter polymorphism −401A and enhanced RANTES production in atopic dermatitis patients
Journal of Dermatological Science (2005) 39, 189—191
www.intl.elsevierhealth.com/journals/jods
LETTER TO THE EDITOR Association between RANTES promoter polymorphism 401A and enhanced RANTES production in atopic dermatitis patients Dear Editor Chemokine regulated on activation, normal T-cellexpressed and secreted (RANTES) is located on 17q, where is reported to be genetic linkage with atopic dermatitis (AD). Enhanced RANTES production has been found in serum and skin lesions of AD patients, while treatment with steroids has the important effect of lowering the expression level of RANTES in atopic patients [1—3]. These reports suggest a potential role of RANTES in AD, and that the alteration of RANTES gene expression might be involved in the development and progression of AD. Recently, a functional mutation in the RANTES gene promoter 401G/A was identified to be associated with AD in a German cohort [4]. Using transient transfection of human cell lines, the RANTES 401A polymorphism was also shown to exert higher constitutive transcription activities, indicating that it may up-regulate RANTES production in vivo. However, it remains unclear whether this polymorphism is correlated with the expression of RANTES mRNA and protein in AD patients. In this study, we investigate the association of the 401A polymorphism with RANTES mRNA as well as protein expression in AD patients. The subjects were 62 Japanese patients with AD and 14 healthy individuals. All of the patients fulfilled the diagnostic criteria of AD [5], and on the basis of serum IgE levels, they were classified as high IgE AD (IgE 500 IU/mL) (n = 39) or low IgE AD (IgE < 500 IU/mL) (n = 23). According to the severity-scoring index [6], 31 AD patients had severe disease (scoring 50). None of the patients was being treated with systemic steroids, though some received topical corticosteroids infrequently. Genomic DNA was extracted from peripheral leukocytes and the 401 genotypes were determined according to a method previously described [7].
PBMC were isolated from peripheral venous blood. Total RNA was then extracted from the PBMC with Trizol reagent and 1 mg of total RNA was subjected to reverse-transcription. Real-time PCR for RANTES and GAPDH was performed with an initial denaturation at 95 8C for 10 min, followed by 40 cycles at 95 8C for 10 s, 68 8C for 10 s and 72 8C for 16 s. Serum was taken from 58 AD patients and 10 healthy controls, and RANTES protein levels were measured using ELISA. Data are expressed as mean S.E. mRNA values are expressed as relative to the inter-assay control measured in the same reaction, and RANTES mRNA values are normalized against those of GAPDH, which allowed the results from different reactions to be compared and ensured that original cDNA copies were not interfering factors. Statistical Analysis was performed using SPSS for Windows. We found no significant differences in mRNA expression between patients and normal controls with RANTES 401 genotypes (Table 1). There was a significant difference in RANTES levels between AD patients and normal controls ( p = 0.016) (Fig. 1a). In AD patients, 401A carriers showed significantly higher protein levels than normal controls or normal controls who carry the 401A allele ( p = 0.008 and 0.02), whereas 401G carriers did not show this difference ( p = 0.524 and 0.458). In addition, AD 401A carriers had higher protein levels than AD 401G carriers; however, the difference was not significant ( p = 0.068). We also found that AD 401A carriers with a high IgE concentration showed higher RANTES protein levels than normal controls or normal controls who carry the 401A allele ( p = 0.025 and 0.046). Further, 401A carriers with severe AD showed significantly higher RANTES levels than those with moderate or mild AD ( p = 0.008) and normal controls ( p = 0.006) (Fig. 1b). In contrast, 401G carriers with severe AD had similar protein levels as those with moderate or mild AD (data not shown). The present study clarifies that compared with normal controls; the RANTES 401A polymorphism is associated with enhanced RANTES production in AD patients. This result suggests that RANTES 401A
0923-1811/$30.00 # 2005 Published by Elsevier Ireland Ltd on behalf of Japanese Society for Investigative Dermatology. doi:10.1016/j.jdermsci.2005.06.003
190
Letter to the Editor
Table 1 Association of RANTES 401 genotypes and the expression of mRNA and protein mRNA (relative units)
p-Value
Control
AD
Total (n)
84.57 + 25.32 (14)
39.30 10.55 (62)
0.074 a
AG + AA (n) IgE > 500 IgE < 500
88.63 42.57 (8)
34.25 13.40 (37) 29.03 14.46 (25) 45.12 29.10 (12)
0.256 b 0.219 b 0.393 b
GG (n)
79.16 22.28 (6)
54.28 18.33 (25)
0.532 b
a b *
Protein (ng/mL) Control
p-Value AD 43.78 5.25 (56)
0.016a, *
29.93 4.29 (6)
50.92 7.44 (34) 52.41 9.84 (23) 47.79 10.87 (11)
0.008a, * 0.025a, * 0.096 a
0.020b, * 0.046b, * 0.151 b
21.12 9.38 (4)
32.76 6.29 (22)
0.524 a
0.458 b
26.4 4.46 (10)
AD vs. control (total). AD vs. control with the same genotype. Statistically significant.
polymorphism is involved in AD by up-regulating RANTES protein expression. The RANTES 401A promoter polymorphism results in a binding element for GATA transcription factors which play roles in upregulating RANTES promoter activity [4]. It has been reported that levels of GATA-3 mRNA in PBMC in AD patients were elevated as compared with normal controls [8]. It is therefore reasonable to expect that the 401A polymorphism binds with the elevated GATA transcription factor, and thus at least
Fig. 1 (a) The RANTES levels in normal controls and AD patients. There was a significant difference in RANTES levels between AD patients and normal controls. AD 401A carriers showed significantly higher protein levels than normal controls or normal controls who carry the 401A allele, whereas 401G carriers did not show this difference. (b) Comparison of RANTES serum levels according to the disease severity with the 401A genotype. Mean RANTES serum levels were significantly higher in AD 401A carriers with severe disease as compared to those with mild or moderated disease and normal controls. *p < 0.05.
partly, contribute to the high RANTES levels in AD patients. Furthermore, in this study, AD 401A polymorphism carriers with a high IgE concentration also showed enhanced RANTES production as compared to normal controls, which might provide further support to the hypothesis that AD patients are genetically heterogeneous. We also demonstrated that AD 401A polymorphism carriers with severe disease showed significantly increased RANTES levels, as compared to those with moderate or mild disease and normal controls. This result is in accordance with the well-documented role of RANTES in the pathophysiology of chronic inflammatory disease and suggests that enhanced RANTES production by the 401A polymorphism result in the recruitment of more inflammatory cells, which may attribute to the disease activity. In this study, no differences in RANTES mRNA levels were found between AD patients and controls with the 401 genotype. The true reason for the discrepancy between RANTES protein levels and mRNA expression is unclear. A post-transcriptional regulation might exist on RANTES expression. It was shown that certain stimulated eosinophils expressed increased RANTES protein, but not RANTES mRNA in cell culture [9]. However, it is also possible that from quantitative mRNA data, RNA levels may be insufficient to reflect the levels of protein produced by cells [10]. The present results indicate that the RANTES promoter 401A polymorphism plays a role in the development of AD by up-regulating serum levels of RANTES in Japanese patients, thus providing an explanation for the association of this polymorphism with AD.
References [1] Morita E, Kameyoshi Y, Hiragun T, Mihara S, Yamamoto S. The C—C chemokines, RANTES and eotaxin, in atopic dermatitis. Allergy 2001;6:194—5. [2] Kaburagi Y, Shimada Y, Nagaoka T, Hasegawa M, Takehara K, Sato S. Enhanced production of C—C chemokines (RANTES,
Letter to the Editor
[3]
[4]
[5] [6]
[7]
[8]
MCP-1, MIP-1alpha MIP-beta and eotaxin) in patients with atopic dermatitis. Arch Dermatol Res 2001;293:350—5. Di Gioacchino M, Verna N, Cavallucci E, Paolini F, Caruso R, Grana M, et al. Steroid and antihistamines modulate RANTES release in cultured peripheral blood mononuclear cells of atopic patients. Int J Immunopathol Pharmacol 2002;15:27— 34. Nickel RG, Casolaro V, Wahn U, Beyer K, Barnes KC, Plunkett BS, et al. Atopic dermatitis is associated with a functional mutation in the promoter of the C—C chemokine RANTES. J Immunol 2000;164:1612—6. Hanifin JM, Rajka G. Diagnostic features of atopic dermatitis. Acta Derm Venereol 1982;92(Suppl.):44—7. Consensus Report of The European Task Force on Atopic Dermatitis. Severity scoring of atopic dermatitis: the SCORAD index. Dermatology 1993;186:23—31. Fryer AA, Spiteri MA, Bianco A, Hepple M, Jones PW, Strange RC, et al. The 403G ! A promoter polymorphism in the RANTES gene is associated with atopy and asthma. Genes Immun 2000;1:509—14. Arakawa S, Hatano Y, Katagiri K. Differential expression of mRNA for Th1 and Th2 cytokine-associated transcription factors and suppressors of cytokine signalling in peripheral blood mononuclear cells of patients with atopic dermatitis. Clin Exp Immunol 2004;135(3):505—10.
191
[9] Ying S, Meng Q, Taborda-Barata L, Corrigan CJ, Barkans J, Assoufi B, et al. Human eosinophils express messenger RNA encoding RANTES and store and release biologically active RANTES protein. Eur J Immunol 1996;26:70—6. [10] Gygi SP, Rochon Y, Franza BR, Aebersold R. Correlation between protein and mRNA abundance in yeast. Mol Cell Biol 1999;19:1720—30.
Bingxue Bai Keiko Tanaka Takahiro Tazawa Naoko Yamamoto Hisashi Sugiura* Japan *Corresponding author E-mail address: [email protected] (H. Sugiura)
1 March 2005
www.intl.elsevierhealth.com/journals/jods
LETTER TO THE EDITOR Association between RANTES promoter polymorphism 401A and enhanced RANTES production in atopic dermatitis patients Dear Editor Chemokine regulated on activation, normal T-cellexpressed and secreted (RANTES) is located on 17q, where is reported to be genetic linkage with atopic dermatitis (AD). Enhanced RANTES production has been found in serum and skin lesions of AD patients, while treatment with steroids has the important effect of lowering the expression level of RANTES in atopic patients [1—3]. These reports suggest a potential role of RANTES in AD, and that the alteration of RANTES gene expression might be involved in the development and progression of AD. Recently, a functional mutation in the RANTES gene promoter 401G/A was identified to be associated with AD in a German cohort [4]. Using transient transfection of human cell lines, the RANTES 401A polymorphism was also shown to exert higher constitutive transcription activities, indicating that it may up-regulate RANTES production in vivo. However, it remains unclear whether this polymorphism is correlated with the expression of RANTES mRNA and protein in AD patients. In this study, we investigate the association of the 401A polymorphism with RANTES mRNA as well as protein expression in AD patients. The subjects were 62 Japanese patients with AD and 14 healthy individuals. All of the patients fulfilled the diagnostic criteria of AD [5], and on the basis of serum IgE levels, they were classified as high IgE AD (IgE 500 IU/mL) (n = 39) or low IgE AD (IgE < 500 IU/mL) (n = 23). According to the severity-scoring index [6], 31 AD patients had severe disease (scoring 50). None of the patients was being treated with systemic steroids, though some received topical corticosteroids infrequently. Genomic DNA was extracted from peripheral leukocytes and the 401 genotypes were determined according to a method previously described [7].
PBMC were isolated from peripheral venous blood. Total RNA was then extracted from the PBMC with Trizol reagent and 1 mg of total RNA was subjected to reverse-transcription. Real-time PCR for RANTES and GAPDH was performed with an initial denaturation at 95 8C for 10 min, followed by 40 cycles at 95 8C for 10 s, 68 8C for 10 s and 72 8C for 16 s. Serum was taken from 58 AD patients and 10 healthy controls, and RANTES protein levels were measured using ELISA. Data are expressed as mean S.E. mRNA values are expressed as relative to the inter-assay control measured in the same reaction, and RANTES mRNA values are normalized against those of GAPDH, which allowed the results from different reactions to be compared and ensured that original cDNA copies were not interfering factors. Statistical Analysis was performed using SPSS for Windows. We found no significant differences in mRNA expression between patients and normal controls with RANTES 401 genotypes (Table 1). There was a significant difference in RANTES levels between AD patients and normal controls ( p = 0.016) (Fig. 1a). In AD patients, 401A carriers showed significantly higher protein levels than normal controls or normal controls who carry the 401A allele ( p = 0.008 and 0.02), whereas 401G carriers did not show this difference ( p = 0.524 and 0.458). In addition, AD 401A carriers had higher protein levels than AD 401G carriers; however, the difference was not significant ( p = 0.068). We also found that AD 401A carriers with a high IgE concentration showed higher RANTES protein levels than normal controls or normal controls who carry the 401A allele ( p = 0.025 and 0.046). Further, 401A carriers with severe AD showed significantly higher RANTES levels than those with moderate or mild AD ( p = 0.008) and normal controls ( p = 0.006) (Fig. 1b). In contrast, 401G carriers with severe AD had similar protein levels as those with moderate or mild AD (data not shown). The present study clarifies that compared with normal controls; the RANTES 401A polymorphism is associated with enhanced RANTES production in AD patients. This result suggests that RANTES 401A
0923-1811/$30.00 # 2005 Published by Elsevier Ireland Ltd on behalf of Japanese Society for Investigative Dermatology. doi:10.1016/j.jdermsci.2005.06.003
190
Letter to the Editor
Table 1 Association of RANTES 401 genotypes and the expression of mRNA and protein mRNA (relative units)
p-Value
Control
AD
Total (n)
84.57 + 25.32 (14)
39.30 10.55 (62)
0.074 a
AG + AA (n) IgE > 500 IgE < 500
88.63 42.57 (8)
34.25 13.40 (37) 29.03 14.46 (25) 45.12 29.10 (12)
0.256 b 0.219 b 0.393 b
GG (n)
79.16 22.28 (6)
54.28 18.33 (25)
0.532 b
a b *
Protein (ng/mL) Control
p-Value AD 43.78 5.25 (56)
0.016a, *
29.93 4.29 (6)
50.92 7.44 (34) 52.41 9.84 (23) 47.79 10.87 (11)
0.008a, * 0.025a, * 0.096 a
0.020b, * 0.046b, * 0.151 b
21.12 9.38 (4)
32.76 6.29 (22)
0.524 a
0.458 b
26.4 4.46 (10)
AD vs. control (total). AD vs. control with the same genotype. Statistically significant.
polymorphism is involved in AD by up-regulating RANTES protein expression. The RANTES 401A promoter polymorphism results in a binding element for GATA transcription factors which play roles in upregulating RANTES promoter activity [4]. It has been reported that levels of GATA-3 mRNA in PBMC in AD patients were elevated as compared with normal controls [8]. It is therefore reasonable to expect that the 401A polymorphism binds with the elevated GATA transcription factor, and thus at least
Fig. 1 (a) The RANTES levels in normal controls and AD patients. There was a significant difference in RANTES levels between AD patients and normal controls. AD 401A carriers showed significantly higher protein levels than normal controls or normal controls who carry the 401A allele, whereas 401G carriers did not show this difference. (b) Comparison of RANTES serum levels according to the disease severity with the 401A genotype. Mean RANTES serum levels were significantly higher in AD 401A carriers with severe disease as compared to those with mild or moderated disease and normal controls. *p < 0.05.
partly, contribute to the high RANTES levels in AD patients. Furthermore, in this study, AD 401A polymorphism carriers with a high IgE concentration also showed enhanced RANTES production as compared to normal controls, which might provide further support to the hypothesis that AD patients are genetically heterogeneous. We also demonstrated that AD 401A polymorphism carriers with severe disease showed significantly increased RANTES levels, as compared to those with moderate or mild disease and normal controls. This result is in accordance with the well-documented role of RANTES in the pathophysiology of chronic inflammatory disease and suggests that enhanced RANTES production by the 401A polymorphism result in the recruitment of more inflammatory cells, which may attribute to the disease activity. In this study, no differences in RANTES mRNA levels were found between AD patients and controls with the 401 genotype. The true reason for the discrepancy between RANTES protein levels and mRNA expression is unclear. A post-transcriptional regulation might exist on RANTES expression. It was shown that certain stimulated eosinophils expressed increased RANTES protein, but not RANTES mRNA in cell culture [9]. However, it is also possible that from quantitative mRNA data, RNA levels may be insufficient to reflect the levels of protein produced by cells [10]. The present results indicate that the RANTES promoter 401A polymorphism plays a role in the development of AD by up-regulating serum levels of RANTES in Japanese patients, thus providing an explanation for the association of this polymorphism with AD.
References [1] Morita E, Kameyoshi Y, Hiragun T, Mihara S, Yamamoto S. The C—C chemokines, RANTES and eotaxin, in atopic dermatitis. Allergy 2001;6:194—5. [2] Kaburagi Y, Shimada Y, Nagaoka T, Hasegawa M, Takehara K, Sato S. Enhanced production of C—C chemokines (RANTES,
Letter to the Editor
[3]
[4]
[5] [6]
[7]
[8]
MCP-1, MIP-1alpha MIP-beta and eotaxin) in patients with atopic dermatitis. Arch Dermatol Res 2001;293:350—5. Di Gioacchino M, Verna N, Cavallucci E, Paolini F, Caruso R, Grana M, et al. Steroid and antihistamines modulate RANTES release in cultured peripheral blood mononuclear cells of atopic patients. Int J Immunopathol Pharmacol 2002;15:27— 34. Nickel RG, Casolaro V, Wahn U, Beyer K, Barnes KC, Plunkett BS, et al. Atopic dermatitis is associated with a functional mutation in the promoter of the C—C chemokine RANTES. J Immunol 2000;164:1612—6. Hanifin JM, Rajka G. Diagnostic features of atopic dermatitis. Acta Derm Venereol 1982;92(Suppl.):44—7. Consensus Report of The European Task Force on Atopic Dermatitis. Severity scoring of atopic dermatitis: the SCORAD index. Dermatology 1993;186:23—31. Fryer AA, Spiteri MA, Bianco A, Hepple M, Jones PW, Strange RC, et al. The 403G ! A promoter polymorphism in the RANTES gene is associated with atopy and asthma. Genes Immun 2000;1:509—14. Arakawa S, Hatano Y, Katagiri K. Differential expression of mRNA for Th1 and Th2 cytokine-associated transcription factors and suppressors of cytokine signalling in peripheral blood mononuclear cells of patients with atopic dermatitis. Clin Exp Immunol 2004;135(3):505—10.
191
[9] Ying S, Meng Q, Taborda-Barata L, Corrigan CJ, Barkans J, Assoufi B, et al. Human eosinophils express messenger RNA encoding RANTES and store and release biologically active RANTES protein. Eur J Immunol 1996;26:70—6. [10] Gygi SP, Rochon Y, Franza BR, Aebersold R. Correlation between protein and mRNA abundance in yeast. Mol Cell Biol 1999;19:1720—30.
Bingxue Bai Keiko Tanaka Takahiro Tazawa Naoko Yamamoto Hisashi Sugiura* Japan *Corresponding author E-mail address: [email protected] (H. Sugiura)
1 March 2005