Definition of human interleukin-4 receptor alpha chain haplotypes and allelic association with atopy markers

Abstract

Cumulative evidence indicates that the human interleukin-4 receptor α chain gene (IL-4Rα, CD124) is highly polymorphic in contrast to other cytokine receptor genes. Our group recently identified the IL-4Rα variant R551 as being strongly associated with decreased kidney allograft survival. Due to the key immunoregulatory role of IL-4 and controversial reports on the association of IL-4Rα variants with atopy, we present here the development of polymerase chain reaction-primer sets for sequence-specific amplification of all seven hitherto described amino acid polymorphisms, and we investigated 158 blood donors prospectively. By using an Expectation-Maximization algorithm, we calculated the presence of 11 putative human IL-4Rα haplotypes and identified 4 putative IL-4Rα haplotypes with a cumulative frequency of >90%. None of the polymorphisms showed a significant association with the phenotype atopy. All mutant alleles showed a trend toward decreased total IgE levels. This association was only significant (p < 0.05; Mann-Whitney U-test) for the A375, R406, and P478 variants in non-atopic blood-donors (n = 90), presumably due to the high variance of IgE levels among the smaller group of atopic individuals. We postulate that IL-4Rα mutations are associated to different extents with a decrease in function of the receptor but do not present a major atopy locus.

Introduction

Interleukin-4 (IL-4) is an immunoregulatory cytokine produced by activated T cells, mast cells and basophils that plays a central role in the regulation of B-cell and T-cell mediated immune responses 1, 2. IL-4 is a growth factor for pre-activated B cells and T cells, is crucial for the development of T-helper 2 (Th₂)-cells, stimulates immunoglobulin G1 (IgG1) and immunoglobulin E (IgE) production, and enhances cell surface expression of CD23 and major histocompatibility complex (MHC) class II molecules on B cells [3]. Since IL-4 exerts its biological effects through binding to the IL-4 receptor complex, the IL-4 receptor itself is an essential component of the IL-4 pathway. The functional IL-4 receptor is a heterodimer composed of the IL-4 receptor α chain (IL-4Rα, CD124) 4, 5 and the common cytokine receptor γ chain (γc chain, CD 132) 6, 7. Numerous studies have confirmed the key regulatory role of IL-4 in the pathogenesis of atopy [8], as well as in animal models of experimental allergic encephalomyelitis [9], lupus erythematodes [10], and allogeneic tolerance 11, 12. Intriguing data obtained in the murine model of leishmaniasis indicate that the observed T-helper 1 (Th₁/Th₂ immune deviation of C57BL/6 vs. Balb/c mice may be due to different IL-4Rα allotypes. Schulte et al. reported the presence of a IL-4Rα allotype in Balb/c mice that differ in three extracellular and five cytoplasmic amino acids from C57BL/6 mice [13]. They further showed that this IL-4Rα allotype leads to decreased IL-4 neutralizing activity and an increased dissociation rate of the soluble IL-4Rα, causing IL-4 hyperresponsiveness in Balb/c mice. Concerning solid organ transplantation, our group recently reported that the IL-4Rα variant R551 is strongly associated with decreased kidney allograft survival in humans [14].

As a result of the identification of polymorphisms in the human IL-4Rα gene [15], several controversial reports on the association of different IL-4Rα polymorphisms with atopy markers have been published. Hershey et al. reported that the R551 mutation in the IL-4Rα gene is strongly associated with atopy, hyper-IgE syndrome, and enhanced expression of CD23 in response to IL-4 [16]. However, these findings could not be reproduced by other investigators. Grimbacher et al. reported that the R551 variant is not associated with the hyper-IgE syndrom [17], and Noguchi et al. did not find either an association of atopy with the R551 variant in atopic adults or a preferential transmission of the R551 variant to atopy- or asthma-affected children in a Japanese population [18]. Furthermore, in direct contrast to the results of Hershey et al., Kruse et al. reported that the R551 and P478 polymorphisms of the IL-4Rα gene are associated with lowered total IgE levels [19]. Finally, Mitsuyasu et al. investigated an extracellular polymorphism in the IL-4Rα gene and reported that the I50 variant is strongly associated with atopy and elevated total IgE levels [20].

Despite the intriguing results reported, these studies were hampered by the fact that they were focused on different mutations of the human IL-4Rα gene, which may segregate with other mutations in the same gene that may also influence signal transduction via the IL-4 receptor. In an attempt to improve this situation, we present in this study the development of seven polymerase chain reaction (PCR) primer sets for sequence-specific amplification (PCR-SSP) of all hitherto described amino acid polymorphisms in the human IL-4Rα gene, and we investigated 158 prospectively recruited healthy adult blood donors. We present in this study data on the linkage disequilibibrium of all IL-4Rα variants that lead to amino acid substitutions and calculated putative IL-4Rα haplotypes by using an Expectation-Maximization algorithm. We investigated also the relationship between IL-4Rα mutations, as well as putative haplotypes, and clinical symptoms of atopy, a family history of atopy, total IgE serum levels, and specific sensitization against aeroallergens. Results indicate that IL-4Rα mutants affect IgE serum levels, presumably by impaired IL-4 signaling. Due to the central role of IL-4 in the regulation of adaptive immune responses, our findings may have important implications for the research in different clinical settings, for example, autoimmunity, tolerance induction, and infectious diseases.