Chronic obstructive pulmonary disease is associated with the - Nature

Genes and Immunity (2002) 3, 436–439 & 2002 Nature Publishing Group All rights reserved 1466-4879/02 $25.00 www.nature.com/gene

BRIEF COMMUNICATION

Chronic obstructive pulmonary disease is associated with the -1055 IL-13 promoter polymorphism TCTM van der Pouw Kraan1, M Ku¨c, u¨kaycan2, AM Bakker3, JMC Baggen1, JS van der Zee4, MA Dentener2, EFM Wouters2 and CL Verweij1 1 Department of Molecular Cell Biology, Free University, Amsterdam, The Netherlands; 2Department of Pulmonology, Maastricht University, Nutrition and Toxicology Research Institute Maastricht (Nutrim), Maastricht, The Netherlands; 3Department of Rheumatology, Leiden University Medical Center, Leiden, The Netherlands; 4Department of Pulmonology, Academic Medical Center, Amsterdam, The Netherlands

IL-13 is strongly implicated in the development of asthma and chronic obstructive polymonary disease (COPD). We previously identified an IL-13 promoter polymorphism (1055 C to T) that is associated with allergic asthma. We now report an increased frequency of the 1055 T allele in COPD patients compared to healthy controls (P ¼ 0.002) and compared to a second control group consisting of smoking individuals with normal lung function (P ¼ 0.01). A closely linked IL-13 exon polymorphism is present at normal allelic frequencies (P ¼ 0.3 and 0.4, respectively). In addition, we observed a normal distribution of two IL-4 polymorphisms at positions 590 and +33 (P ¼ 0.2 and 0.9, respectively). These results could implicate a functional role for the IL-13 promoter polymorphism in the enhanced risk to develop COPD. Genes and Immunity (2002) 3, 436–439. doi:10.1038/sj.gene.6363896 Keywords: IL-13; COPD; polymorphism; immunology; genetics; human

Chronic obstructive pulmonary disease (COPD) is a genetically and environmentally determined disease characterized by airflow limitation that is not fully reversible (reviewed by Barnes).1 The most important risk factor for development of COPD is smoking, although only 15% of all smokers develop COPD, indicating that genetic factors may determine in which smokers COPD will develop. COPD is characterized by airway inflammation with infiltrating leucocytes as observed in biopsies, induced sputum and bronchoalveolar lavage (BAL) fluids.1–3 Similar features are observed in asthma. Infiltrating T cells are predominantly CD4+ in asthma, in contrast to a predominance of CD8+ T cells in COPD. In COPD patients, neutrophils are highly increased in BAL fluid and induced sputum, whereas eosinophils are more increased in asthma. It has been hypothesized that asthma and COPD are not absolute distinct disease entities and have common underlying mechanisms leading to the pathology of both disorders, later referred to as the Dutch Hypothesis.4 Indeed airway hyper-responsiveness, a characteristic of asthma, turned out to be a strong predictor of progression of airway obstruction in early COPD patients.5 Mortality from COPD is also increased with more severe

Correspondence: Tineke C.T.M. van der Pouw Kraan, VUMC, Department of Molecular Cell Biology J283, Van Boechorststr. 7, 1081 BT Amsterdam, The Netherlands. E-mail: [email protected]

airway hyper-responsiveness.6 Other risk factors for COPD include the combination of blood eosinophilia and reported asthma attacks,7 although the single parameters are not important risk factors. Over the past few years it has become clear that IL-13 is a critical and specific effector molecule in experimental asthma as well as in COPD. The human IL-13 gene is together with the IL-4 gene located within 15 kb on chromosome 5q31, a region associated with airway hyper-responsiveness, asthma and IgE levels.8–11 IL-4 and IL-13 share many (but not all) functions, such as the induction of a class switch to IgE in B cells, which can be explained by shared receptor usage of the IL-4Ra chain mediating stat 6 activation. IL-4 mediates its activity through the IL-4Ra chain and either the IL-13Ra chain or the common gamma chain, while IL-13 utilizes the IL-4Ra chain in combination with the IL-13Ra chain.12 Inhibition of IL-13 activity in the lungs of sensitized mice prevents several characteristics of asthma, such as airway hyper-responsiveness, pulmonary eosinophilia and mucus production,13,14 whereas neutralization of IL-4 fails to do so. Pulmonary expression of transgenic IL-13 in adult lungs results in a COPD phenotype with inflammation, mucus metaplasia and matrix-metalloproteinase- and cathepsin-dependent emphysema.15 These results clearly indicate the prominent and unique role of IL-13 in asthma as well as COPD. Recently, we reported an IL-13 promoter polymorphism (1055 C to T),16 which is associated with allergic asthma, altered regulation of IL-13 production and increased binding of nuclear proteins. The frequency of the T allele in the Dutch population is 0.149 and is

COPD is associated with the IL-13-1055 T allele TCTM Van der Pouw Kraan et al

comparable to the frequency found in Great Britain (0.135).17 Recently, six novel polymorphisms have been described for the IL-13 locus,18 including a polymorphism in exon 4 (+2044 G/A), leading to an amino acid substitution at position 130 (Arg130Gln). The Gln form is strongly associated with increased serum IgE levels in American and German populations.18 In addition to the association with high IgE levels, the Gln130 form is also associated with atopic dermatitis in another German population.19 However, in Japanese and British populations,20 the Gln130 is associated with asthma rather than high IgE levels. For IL-4 a promoter polymorphism has been identified (590 C to T)21 that is 100% linked with a polymorphism in the 50 UTR region (+33 C to T),22 and moderately associated with enhanced serum IgE levels in Japanese asthmatics.23 Due to the overlapping activities of IL-4 and IL-13, their close chromosomal location and the IL13-induced COPD phenotype in mice, we determined the polymorphic genotypes of both cytokines in COPD patients in comparison to normal controls. We selected 151 COPD patients (see Table 1) with poor lung function as indicated by a very low FEV1, forced expiratory volume in 1 s, ie, the volume of air exhaled in the first second of a forced vital capacity expiratory manoeuvre (mean 37% of the predicted value according to age, body length and gender). Screening of these patients for the IL-13 and IL-4 polymorphisms revealed a higher frequency of the IL-13 1055 T allele in COPD patients (26.3%) compared to healthy controls (14.6%) and to a second healthy control group consisting of smoking individuals (16.0%, P ¼ 0.002 and 0.01, respectively, w2). Since the distribution of the IL-13 1055 alleles was not different between males and females within the COPD group (P ¼ 0.6) nor between all males and females from the three groups (P ¼ 0.5), we can exclude sex as a confounding factor. The mean age in the COPD patient group was higher than in the healthy control groups. It can be anticipated that some of the

Table 1 Patient characteristics COPD patients (n ¼ 151)

Age (years) Male/female FEV1 (%) Pack years

65 (10) 100/51 37 (13) 33 (19)

Controls (n ¼ 99) Mean (s.d.) 41 (13) 57/42 Nd Nd

Smoking controls (n ¼ 78)

43 (8.9) 35/43 104 (14) 34 (16)

Note: Caucasian COPD patients were selected from the pulmonary rehabilitation center Hornerheide (Horn, The Netherlands) and the Academic Medical Center (Amsterdam, The Netherlands). COPD was defined according to the criteria of the American Thoracic Society.24 For all patients the percentage of predicted forced expiratory volume in 1 s (FEV1) was o70% and FEV1 reversibility o10% of the predicted value after inhalation of a beta2-agonist. Patients with alpha1-antitrypsin deficiency and patients with bronchial asthma were excluded from the study. Healthy blood donors from the Netherlands were selected as controls. The smoking control group consisted of 78 Caucasian smokers who took part in a smoking cessation program and were living in the same region of the Netherlands as the COPD patients. They had no clinical history of COPD and an FEV1% predicted within normal limits.

control individuals, especially those within the smoking group, may develop COPD later in life. Nevertheless we observed a higher frequency of the IL-13 1055 T allele in COPD patients, which may become more significant when patients and controls are completely age matched. The frequency of the IL-13 +2044 G/A polymorphism was not different between COPD patients and controls, indicating the specificity of the association of COPD with the IL-13 1055 T allele. Also for the IL-4 polymorphisms at positions –590 and +33, we did not observe a difference between patients and controls. In addition, the data confirmed that the 590 C/T polymorphism in the IL-4 promoter is 100% linked to the +33 C/T polymorphism at the 50 UTR region of IL-4 (see Table 2). Interestingly, we observed a significant association of the IL-13 –1055 TT genotype with IL-13 production capacity.16 These findings suggest a functional role for IL-13 in COPD in accordance with animal models in which pulmonary expression of IL-13 leads to several characteristics of emphysema. Because COPD is not associated with an allergic Th2 biased phenotype, it can be anticipated that characteristics of IL-13 not related to its Th2-associated functions may contribute to pulmonary disease with inflammatory cell influx, mucus overproduction and induction of cathepsins and matrix metalloproteinases. Further studies are required to establish how all these features are regulated by IL-13. In mice, pulmonary mucus production is mediated via the shared IL-4 Ra chain even in the absence of IL-4,25 indicating a role for IL-13. Moreover, in gene-targeted mice infected with the gastrointestinal nematode N. Brasiliensis, induction of mucus hypersecretion is clearly a unique characteristic of IL-13 and independent of a Th2 immune response. Mice deficient for IL-13, unlike wildtype and IL-4 deficient mice, do not show intestinal goblet cell hyperplasia and as a consequence fail to expel N. Brasiliensis, despite a robust Th2 response.26 Overproduction of pulmonary mucus secretion is a key feature of IL-13-induced COPD in mice15 and in COPD patients.1 Chronic mucus hypersecretion is associated with declined FEV1, morbidity, pulmonary infection and death in COPD patients,27,28 signifying that IL-13 is an important target for intervention in the pathological process of chronic pulmonary disease. Graves et al18 found a strong association of increased serum IgE levels with the IL-13 +2044 A allele and to a lesser extent with the IL-13 1055 T allele. When we compared IgE levels in the normal controls, we did not observe a significant difference between serum IgE levels in the different IL-13 genotypes (data not shown, ANOVA). This could be due to the lower number of individuals tested, but indicates that the association is not very strong, as was also observed in a population consisting of asthma patients and normal controls.20 The fact that the human IL-13 gene is located on a chromosomal region associated with AHR, a strong risk factor for COPD, and the capacity of IL-13 to induce the entire COPD phenotype in mice combined with our finding fact that COPD is specifically associated with the IL-13 1055 T allele, while we found no association with the +2044 IL-13 exon polymorphism, suggests that the 1055 polymorphism itself may be responsible for the increased risk to develop COPD.

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Table 2 IL-13 and IL-4 genotypes and allelic frequencies in Dutch COPD patients and normal Dutch individuals* Genotype

n

(%)

IL-13-1055 CC CT TT Total

80 62 9 151

(53.0) (41.0) (6.0)

Controls

CC CT TT Total

72 25 2 99

Smoking controls

CC CT TT Total

COPD patients

COPD patients

IL-4-590 CC CT TT Total

Allele

n

(%)

Genotype

n

C T

222 80

(73.5) (26.5)*

(72.7) (25.3) (2.0)

C T

169 29

56 19 3 78

(71.8) (24.4) (3.8)

C T

107 33 0 140

(76.4) (23.6) (0)

IL-13 +2044 GG GA AA Total

90 45 8 143

(85.4) (14.6)

GG GA AA Total

131 25

(84.0) (16.0)

GG GA AA Total

C T

247 33

(88.2) (11.8)

IL-4+33 CC CT TT Total

Allele

n

(62.9) (31.5) (5.6)

G A

225 61

(78.7) (21.3)

62 29 2 93

(66.7) (31.2) (2.1)

G A

153 33

(82.3) (17.7)

52 24 2 78

(66.7%) (30.8) (2.6)

G A

128 28

(82.1) (17.9)

107 33 0 140

(76.4) (23.6) (0)

C T

247 33

(88.2) (11.8)

(%)

(%)

Controls

CC CT TT Total

65 27 1 93

(69.9) (29.0) (1.1)

C T

157 29

(84.4) (15.6)

CC CT TT Total

65 27 1 93

(69.9) (29.0) (1.1)

C T

157 29

(84.4) (15.6)

Smoking controls

CC CT TT Total

40 12 0 52

(76.9) (23.1) (0)

C T

92 12

(88.5) (11.5)

CC CT TT Total

40 12 0 52

(76.9) (23.1) (0)

C T

92 12

(88.5) (11.5)

*P ¼ 0.002 compared to controls and P ¼ 0.01 compared to smoking controls. Note: Genomic DNA was used as a template for amplification by PCR. Standard PCR reactions were carried out in 40 ml volumes, containing the appropriate buffer, I U Taq polymerase, 1.5–2.5 mm MgCl2, 250 mm dNTP’s, and 300 mM of relevant primers. The following primer sets were used. For 1055 C/T: sense 50 -ACTTCTGGGAGTCAGAGCCA-30 and antisense 50 -TACAGCCATGTCGCCTTTTCCTGCTCTTCCGTC-30 ; for +2044 G/A: sense 50 -ACGTGGCCTTCGGGATTTAC-30 and antisense: 50 -GCAAATAATGATGCTTTCGAAGTTTCAGTGGA-30 . For determination of the IL-4 polymorphisms we used: for 590 C/T: sense 50 -AAATAAAAATAAAAATGAGC-30 and antisense 50 -TTAACCTGGCTTCTTCCAAG-30 ; for IL-4 +33 C/T: sense 50 -GTAAACTCATTTTCGCTCGG-30 and antisense 50 CAGAGCGGGAAGCAGTTGGGACGTG-30 . Restriction sites were introduced by the underlined sequences. The pcr products were digested by Hpy99 I (IL-13 1055), Nla IV (IL-13 +2044), BsmFI (IL-4 590) or MnlI (IL-4 +33) and visualized by UV light on ethidium bromide stained agarose gels.

Acknowledgements This study was financially supported by the Dutch League against Rheumatism and GlaxoSmithKline.

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