Key words

H

D

, M

H

, A

B

,

A

W

−H

, A

C

−D

Association of Soluble IL−4R Serum Levels

and IL−4R

αα

Chain Gene Polymorphisms*

Stężenie rozpuszczalnej formy receptora IL−4

w odniesieniu do polimorfizmu genu IL−4R

αα

1_{First Department and Clinic of Pediatrics, Allergology, and Cardiology, Wroclaw Medical University, Poland} 2_{Chair and Department of Clinical Pharmacology, Wroclaw Medical University, Poland}

Adv Clin Exp Med 2009, 18, 6, 559–565 ISSN 1230−025X

ORIGINAL PAPERS

© Copyright by Wroclaw Medical University

Abstract

Background.The IL−4 receptor plays a key role in IL−4 signal transmission, immunoglobulin isotype switching,

and Th2 differentiation. These processes are directly associated in the response to allergens and chronic allergic in− flammation. Soluble sIL−4R is part of the homeostatic mechanism for IL−4. With an appropriate sIL−4R/IL−4 ratio, it can act as an IL−4 inhibitor; this mechanism was implemented in innovative asthma therapy. Polymorphisms in the IL−4Rαgene, among others C−3223T and I50V, have been reported to be associated with atopy. They may po− tentially affect the function of the gene and the decoded protein.

Objectives. The aim of the study was to analyze the serum levels of sIL−4R in children and adults in relation to

atopy and C−3223T and I50V genotype.

Material and Methods.Serum levels of sIL−4R were determined by ELISA in children and adults (n = 106) pre−

viously genotyped for both polymorphisms.

Results.The study found statistically significant differences in the level of sIL−4R in relation to genotype in the

group of children (p< 0.05, Whitney−Mann Utest). The atopic children had lower levels of sIL−4R than controls, although the results were not statistically significant. In the adults there were no statistical significant differences in serum sIL−4R levels.

Conclusions.The findings point to a potentially significant role of C−3223T and possibly of I50V in the negative

regulation of the IL−4 pathway (Adv Clin Exp Med 2009, 18, 6, 559–565).

Key words: sIL−4R, IL−4Rαgene promoter, genetic polymorphism.

Streszczenie

Wprowadzenie. Receptor dla IL−4 pełni kluczową rolę w przekazywaniu sygnału związanego z IL−4, a tym samym

w procesie przełączania klas immunoglobulin oraz przekierowaniu odpowiedzi układu immunologicznego w kie− runku Th2. Procesy te są związane bezpośrednio zarówno z reakcją organizmu na alergeny, jak i utrzymywaniem się stanu przewlekłego zapalenia alergicznego. Rozpuszczalna forma receptora dla IL−4 (sIL−4R) jest elementem układu homeostatycznego dla IL−4. Przy odpowiednim stosunku stężeń obu czynników może pełnić funkcję hamu− jącą działanie IL−4, który to mechanizm wykorzystano w innowacyjnych formach terapii astmy. Polimorfizmy w re− gionie genu dla łańcucha alfa receptora IL−4, między innymi C−3223T i I50V, zostały opisane jako czynniki zwią− zane z występowaniem atopii. Potencjalnie mogą wpływać na funkcje genu oraz kodowanego białka.

Cel pracy.Analiza surowiczych stężeń sIL−4R u dzieci oraz osób dorosłych w odniesieniu do cech atopii i wymie−

nionych genotypów.

Materiał i metody. Surowicze stężenia sIL−4R oznaczono z wykorzystaniem techniki ELISA w grupie dzieci

i osób dorosłych (n = 106), u których pierwotnie oznaczono genotyp dla obu polimorfizmów.

Wyniki. Wykazano różnice w stężeniach sIL−4R w grupie dzieci w odniesieniu do genotypów (p < 0,05, Whitney−

−Mann Urank test). U dzieci z atopią obserwowano mniejsze stężenia sIL−4R w porównaniu z grupą kontrolną,

The significant role of IL−4 in processes with an allergic background has been unquestionable since the 1990s. The first Polish report on the role of this cytokine in children was published as early as 1993 [1]. Swedish authors first demonstrated a predictive character of increased serum IL−4 level in infants as a factor indicating a later develop− ment of atopic diseases [2]. Many other studies confirmed the correlation between atopy or asthma and elevated levels of IL−4 in serum, bronchial biopsy specimens, and bronchoalveolar lavage fluid [3–5] as well as increased production of IL−4 by peripheral blood mononuclear cells in relation to the effect of allergens [6]. Moreover, it was con− firmed that the production of IL−4 by T lympho− cytes is increased in atopic patients [7]. IL−4 exerts its function by binding to IL−4R. Increased expres− sion of the IL−4 receptor alpha subunit in epitheli− um and subepithelium was demonstrated in bronchial epithelium biopsy specimens from patients diagnosed with atopic asthma in compari− son with controls, i.e. atopic patients without asth− ma [8]. Moreover, patients diagnosed with asthma who underwent specific immunotherapy devel− oped decreased IL−4R expression in peripheral blood monocytes [9].

The IL−4 soluble receptor (sIL−4R) is a natu− rally occurring protein formed by alternative splic− ing of exon 8 of the IL−4Rα gene (IL−4 receptor alpha chain). sIL−4R contains only the extracellular part of IL−4R. It plays an ambivalent role in the organism which depends on the sIL−4R/IL−4 ratio. There are only a few reports regarding sIL−4R serum levels in healthy subjects. An age−dependent decrease in this factor in serum has been reported by Japanese authors [10] and decreased levels were observed in atopic patients [11]. On the other hand, an increase in this factor may express activation of the immune system under the effect of allergens, as it is one of the components of the homeostatic mechanisms. Increased levels of sIL−4R were demonstrated in bronchoalveolar lavage fluid from asthmatic patients [10] as well as patients with allergic rhinitis [11]. Another study found de− creased serum levels of sIL−4R in atopic patients compared with controls after allergen provocation [12]. Moreover, fluctuations in sIL−4R level were observed during specific immunotherapy [13].

As IL−4Rα is an important element in the pathway of the signal induced by both IL−4 and

IL−13, the search for polymorphisms in the gene’s functional regions enjoys significant interest among geneticists. These polymorphisms may affect the stability and function of the IL−4Rα membrane form or sIL−4R. A report by Hackstein [14] suggests an association of the C−3223T poly− morphism within this gene with decreased sIL−4R level in healthy subjects. C−3223T has been shown to be in linkage disequilibrium with I50V, so it is possible that I50V has some effect on sIL−4R serum level as well. The I50V polymorphism has been extensively studied and associated with IgE levels and atopic asthma; however, these results have not been replicated in different populations. In the present authors’ own study, a relationship between C−3223T and atopy or asthma status was found; however, no linkage disequilibrium with I50V was observed [15]. Thus it could be suspect− ed that C−3222T correlates with sIL−4R serum level in a study population. The aim of the present study was to evaluate serum levels of sIL−4R in relation to atopy status and C−3223T and I50V genotype.

Materials and Methods

Subjects

Determination of Soluble IL−4

Receptor Level

The serum levels of sIL−4R were determined by Quantikine quantitative sandwich ELISA kits using the method recommended by the manufac− turer (R&D Systems). The intensity of staining was read with a Dynatech MR 5000 plate reader at 450 nm and expressed in pg/ml. Individual sam− ples were measured twice and the mean values were further analyzed. The sensitivity of the method was < 5 pg/ml and the range 25–1000 pg/ml.

Statistical Analysis

Statistical analysis was performed by means of the Man−Whitney Utest, ANOVA Kruskal−Wallis rank test, multiple regression test, and Spearman’s correlation test using the Statistica 8 statistical package. Significance was defined as a pvalue of less than 0.05.

Results

Analysis of sIL−4R Levels

The levels of sIL−4R in the serum samples from the children are summarized in Table 1. The levels were lower in the atopic group than in the control group. However, the difference was not statistically significant (Mann−Whitney U test,

multiple regression p > 0.05). The effects of age and sex were also not statistically significant. Regarding the C−3223T genotype, the highest serum levels of sIL−4R were found in the children with the CC genotype. A statistically significant difference was demonstrated when comparing the combined CT + TT genotype group vs. CC geno− types (p < 0.05). Higher sIL−4R levels were demonstrated in the control group with CC geno− type than in the atopic subjects with CC genotype. Atopic subjects with CT + TT genotype had lower levels than the control subjects with the same genotype. For the I50V genotype, significantly higher levels were demonstrated for the II geno− type vs. the IV + VV genotype. They were higher in the control group than in the atopic subjects. The scatter plots of serum sIL−4R depending on C−3223T and I50V genotype are shown in Figu− res 1 and 2, respectively. Moreover, the effect of both polymorphisms on serum sIL−4R level was investigated. The highest serum sIL−4R levels were found in the subgroup with wild alleles for both SNPs (single−nucleotide polymorphism) (homo−zygotes) and were higher in the control group. The lowest mean serum levels of sIL−4R were in the subgroup of children with mutations for both polymorphisms. However, the results were not statistically significant for that kind of analysis (ANOVA Kruskal−Wallis test, data not shown).

The levels of sIL−4R in the serum samples from the adults are summarized in Table 2. The

Table 1.Serum sIL−4R levels of the children according to atopy status and C−3223T and I50V genotype. Significance of differences was determined by the Mann−Whitney Utest. Apvalue < 0.05 was considered statistically significant

Tabela 1.Surowicze stężenie sIL−4R w grupie dzieci w odniesieniu do statusu atopii oraz genotypu C−3223T i I50V. W ce− lu wykazania statystycznie istotnych różnic wykorzystano test U Manna−Whitneya, wartość p mniejszą niż 0,05 uznano za statystycznie istotną

sIL−4R (pg/ml) Mean ± SD CI−95% CI+95% Median p

atopic, n = 34 35.91 ± 5.92 33.85 37.98 35.94 ns

control, n = 36 38.82 ± 8.85 35.83 41.82 37.23

CC, n = 28 39.53 ± 9.13 35.99 43.07 38.22 < 0.05

CT + TT, n = 42 35.99 ± 6.22 34.06 37.93 35.83

II, n = 39 39.05 ± 9.04 36.11 41.99 37.99 < 0.05

IV + VV, n = 31 (nVV = 1) 35.34 ± 4.82 33.57 37.11 35.31

Analysis in the subgroups

atopic CC, n = 6 38.06 ± 4.79 33.05 43.08 37.3 ns

atopic CT + TT, n = 28 35.45 ± 6.11 33.08 37.82 35.94

control CC, n = 22 39.93 ± 10.05 35.47 44.39 38.22 ns

control CT + TT, n = 14 37.07 ± 6.51 33.31 40.84 35.66

atopic II, n = 19 37.67 ± 6.5 34.53 40.8 37.01 ns

atopic IV + VV, n = 15 33.68 ± 4.33 31.29 36.09 35.06

control II, n = 20 40.37 ± 10.95 35.24 45.50 38.57 < 0.05

control IV + VV, n = 16 36.89 ± 4.87 34.30 39.48 35.61

analysis in relation to allergic status gave similar results to those of the children. Parents with a pos− itive history for allergic diseases had lower serum levels of sIL−4R than those with a negative history. As far as genotype was concerned, higher levels of sIL−4R were found in the parents with the homozy− gote genotype for wild CC compared with the CT + TT genotype. As far as I50V polymorphism was concerned, higher sIL−4R levels were observed in the subgroup with the IV genotype than in that with the II genotype. In all the analyses concerning the parents, the results were not statis− tically significant; thus only a trend was observed. No age−dependent effect on sIL−4R serum level was observed in the study population (multiple regression analysis, Spearman’s correlation test).

Discussion

According to current knowledge of the patho− genesis of atopic diseases, immunological factors, especially the cytokine network, are the most important components. The predominance of Th2 lymphocytes seems to play the key role in the ear− liest months of life by conditioning the process of allergic sensitization. IL−4 is the main component of the Th2−type response due to its stimulation of IgE production and lymphocyte differentiation. IL−4 also promotes allergic inflammation by inducing the expression of VCAM−1 on vascular endothelium, which stimulates the migration of T lymphocytes, monocytes, basophils, and eosino− phils to the site of allergic inflammation. IL−4 also

sI

L

-4

R

(p

g

/m

mean± 0.95 CI

individual data

CT + TT CC

20 30 40 50

60 _{tusu atopii oraz genotypu C−3223T}

i I50V. W celu wykazania statystycz− nie istotnych różnic wykorzystano test U Manna−Whitneya. Wartość p mniejszą niż 0,05 uznano za staty− stycznie istotną

sI

L

-4

R

(p

g

/m

l)

mean± 0.95 CI

individual data

IV + VV II

20 30 40 50 60 70 80 90

p < 0,05

Fig. 2.Serum sIL−4R levels of the

children in relation to I50V genotype. Significance of differences was deter− mined by the Mann−Whitney Utest. Apvalue < 0.05 was considered sta− tistically significant

Ryc. 2.Surowicze stężenie sIL−4R

inhibits apoptosis and prolongs eosinophilic sur− vival. Another mechanism which is important in the pathogenesis of asthma depends on the induc− tion of mucin gene expression and mucus produc− tion. IL−13, which binds to the same receptor, exerts a different effect. It plays a role in the later stages of the immunological response, especially in allergic inflammation and airway remodeling. Both IL−13 and IL−4 have been identified in inflammatory tissues. Mice with gene hyperex− pression for IL−13 develop eosinophilic inflamma− tion, hypersecretion of mucus, fibrosis of the res− piratory tract, and nonspecific hyperreactivity of the respiratory tract [16].

Both cytokines act through the agency of a common component, the IL−4 receptor α chain. Modifications of the gene for this factor may play a significant role in the actions of both cytokines. Whether the relationship is direct or indirect remains an open question. The functional signifi− cance of at least three IL−4Rα polymorphisms have been confirmed for the actions mediated by IL−4; however, for IL−13 the relation has the char− acter of a gene−gene interaction [17]. Ober [18] remarked that genetic defects involving IL−4Rα may affect the incidence of bronchial asthma through their effect on IL−13, while those which are primarily associated with IL−4 response condi− tion atopy. This theory is further supported by observations on the function of IL−13, which seems to be associated with the inflammatory pathomechanism in the bronchi of asthmatic patients on a pathway partially independent of IL−4 [16]. Many studies have been published on the 16p11.2−p12.1 region and its associations with asthma/ /atopy, many of them contradictory. Two reports demonstrated an association between sIL−4R and C−3223T polymorphism in the gene promoter region for IL−4Rα, another confirmed the partici− pation of I50V polymorphism (for TVR haplo−

type) in this correlation, while still another study excluded such correlation [13–19]. Func−tional studies have not demonstrated that the polymor− phic variant of the promoter has different promot− er activity in vitro. However, it is still possible that this polymorphism may alter crucial function in vivo[20].

The present study found lower levels of sIL−4R in atopic patients in relation to controls, although the difference was not statistically significant. Statistical significance was achieved when the mean levels of sIL−4R were compared in the groups corresponding to the genotypes C−3223T and I50V. Taking into account genotype coinci− dence, the lowest levels of sIL−4 were observed in the children with mutations for both polymor− phisms. This suggests a genotype effect in the reg− ulation of sIL−4R level. The results from the group of parents were statistically insignificant; howev− er, a trend for C−3223T was comparable to that observed in the children’s group.

The results confirm the hypothesis of an inad− equate negative regulation of the Th2 (IL−4) sys− tem in atopic patients with a genetic “defect” con− cerning this type of interaction. Providing evi− dence for such a mechanism is crucial for targeted therapy. Apart from the classical methods of ther− apy using a recombinant form of sIL−4R (sIL−4R) in the form of aerosol [21], initial trials have been conducted to use IL−4Rαantagonists in gene ther− apy. A trial to introduce to a model a vector con− taining a gene for an IL−4R antagonist (a mutated form of IL−4 which, binding the receptor, blocks its function) was described in which expression of the gene was obtained in 20 weeks. The therapy result− ed in a decrease in IL−4− and IL−13−induced bronchial hypersensitivity and respiratory eosino− philia. The introduced gene, which underwent sys− temic and local expression, caused inhibition of eosinophilic inflammation induced by allergic

Table 2.Serum sIL−4R levels of adults in relation to allergy status and C−3223T and I50V genotype. Significance of diffe− rences was determined by the Mann−Whitney Utest. Apvalue < 0.05 was considered statistically significant

Tabela 2.Surowicze stężenie sIL−4R w grupie dorosłych w odniesieniu do statusu alergii oraz genotypu C−3223T i I50V. W celu wykazania statystycznie istotnych różnic wykorzystano test U Manna−Whitneya. Wartość p mniejszą niż 0,05 uzna− no za statystycznie istotną

sIL−4R (pg/ml) Mean ± SD CI−95% CI+95% Median p

allergy positive, n = 12 37.67 ± 4.27 34.96 40.39 37.36 ns.

allergy negative history, n = 24 38.51 ± 7.12 35.51 41.52 35.73

CC, n = 6 41.48 ± 8.75 32.30 50.66 39.31 ns.

CT + TT, n = 30 37.58 ± 5.61 35.49 39.68 36.53

II, n = 17 38.05 ± 7.02 34.44 41.66 35.71 ns.

IV + II, n = 18 38.60 ± 5.78 35.73 41.48 37.92

The Th2−type response is important in view of pro− tection of the organism against parasites. Mice which produce excessive amounts of sIL−4R do not show any visible physiological or developmen− tal abnormalities. Mice which do not produce IL−4 are able to protect themselves against parasites of the Nippostrongylus brasyliensistype, which means that inhibition of IL−4 function alone does not interfere with the immunological response to para− sites. On the other hand, organisms deprived of IL− 4 receptor are unable to respond to N. brasyliensi, which probably results from the inhibition of IL−13. In such cases the organism’s defense on rsIL−4R administration may be significantly dis− turbed if we assume that the drug affects the func−

important in alerting IL−13 function and thus the remodeling process in asthmatic lungs. It was reported that hsIL−4R has the ability to stabilize the binding of IL−13 to its receptor to augment IL−13− mediated responses [26].

The authors concluded that the C−3223T poly− morphism potentially indicates a tendency to pro− duce lower sIL−4R levels, regardless of atopy sta− tus. It seems that a similar role may be played by the I50V polymorphism, although its significance is ambiguous. Subjects with the T−3223 allele who have decreased levels of sIL−4R potentially consti− tute the most suitable group for therapy with the recombinant form of sIL−4R.

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Address for correspondence:

First Department and Clinic of Pediatrics, Allergology, and Cardiology Wroclaw Medical University

Hoene−Wrońskiego 13c 50−379 Wrocław Poland

Tel.: +48 71 328 12 06 E−mail: [email protected]

Conflict of interest: None declared