Differential desensitization of homozygous haplotypes of the beta2-adrenergic receptor in lymphocytes

Abstract

Single-nucleotide polymorphisms of the beta(2)-adrenergic receptor gene and its 5' promoter have been associated with differences in receptor function and desensitization. Linkage disequilibrium may account for inconsistencies in reported effects of isolated polymorphisms. Therefore, we have investigated the three most common homozygous haplotypes of the beta(2)-adrenergic receptor (position 19 [Cys/Arg] of the 5' leader cistron and positions 16 [Arg/Gly] and 27 [Gln/Glu] of the receptor) for putative differences in agonist-induced desensitization. Lymphocytes of well defined nonasthmatic, nonallergic subjects homozygous for the haplotype CysGlyGln, ArgGlyGlu, or CysArgGln were isolated. Desensitization of (-)-isoproterenol-induced cyclic adenosine monophosphate (cAMP) accumulation and beta(2)-adrenergic receptor sequestration and downregulation were measured in relation to beta(2)-adrenergic receptor-mediated inhibition of IFN-gamma and interleukin-5 production. We observed that lymphocytes of individuals bearing the CysGlyGln haplotype were more susceptible to desensitization of the beta-agonist-induced cAMP response than those of individuals with the ArgGlyGlu or CysArgGln haplotype. The haplotype-dependent desensitization of beta-agonist-induced cAMP response was not associated with haplotype-dependent beta(2)-adrenergic receptor sequestration or downregulation. In addition, our data suggest reduced inhibition, in lymphocytes of subjects with the CysGlyGln haplotype, of interleukin-5 production induced by treatment with antibodies to the T-cell receptor-CD3 complex and to costimulatory molecule CD28 (alphaCD3/alphaCD28). This is the first study demonstrating haplotype-related differences in agonist-induced beta(2)-adrenergic receptor desensitization in primary human cells. This haplotype-related desensitization of the beta(2)-adrenergic receptor in lymphocytes might have consequences regarding the regulation of helper T-cell type 2 inflammatory responses.

Figures

Figure 1.

(−)-Isoproterenol–induced desensitization of β2-adrenergic receptor (β2AR)–mediated cyclic adenosine monophosphate (cAMP) accumulation in lymphocytes. (A) Effects of 30 minutes and 2 hours of pretreatment with vehicle (veh) or (−)-isoproterenol (iso) on β2AR-mediated cAMP accumulation induced by (−)-isoproterenol (1 μM). (B) Percentage (−)-isoproterenol–induced desensitization of cAMP accumulation after 30 minutes and 2 hours of pretreatment. *p < 0.01; **p < 0.0001. Data were obtained in lymphocytes from 22 subjects.

Figure 2.

(−)-Isoproterenol–induced desensitization of cAMP accumulation after 30 minutes and 2 hours of pretreatment, stratified for β2AR haplotype. (A) Effects of 30 minutes (top) and 2 hours (bottom) of pretreatment with vehicle (veh) or (−)-isoproterenol (iso) on β2AR-mediated cAMP accumulation induced by (−)-isoproterenol (1 μM), stratified for β2AR haplotype. (B) Percentage of (−)-isoproterenol–induced desensitization of cAMP accumulation after 30 minutes (shaded boxes) and 2 hours (open boxes) of pretreatment, stratified for β2AR haplotype. Data were obtained in lymphocytes from five subjects with the CysGlyGln haplotype, 10 subjects with the ArgGlyGlu haplotype, and seven subjects with the CysArgGln β2AR haplotype. *p < 0.05 for differences between pretreatment conditions; #p = 0.03 and ##p = 0.01 for differences between haplotypes.

Figure 3.

(−)-Isoproterenol–induced β2AR downregulation. (A) (−)-Isoproterenol (1 μM)–induced β2AR downregulation after 30 minutes, 2 hours, and 6 hours of treatment. *p < 0.02 compared with 30 minutes and 2 hours of pretreatment. Data were obtained in lymphocytes from 21 subjects. (B) β2AR downregulation after 6 hours of treatment with (−)-isoproterenol, stratified for β2AR haplotype: five subjects with the CysGlyGln haplotype, nine subjects with the ArgGlyGlu haplotype, and seven subjects with the CysArgGln β2AR haplotype.

Figure 4.

(−)-Isoproterenol (1 μM)–induced inhibition of αCD3/αCD28-induced IFN-γ and interleukin (IL)-5 production. Effects of 30 minutes and 2 hours of pretreatment with vehicle (shaded boxes) or (−)-isoproterenol (1 μM; open boxes) on (−)-isoproterenol–induced inhibition of IFN-γ (A) and IL-5 production (B). (−)-Isoproterenol significantly inhibited αCD3/αCD28-induced IFN-γ and IL-5 production under all treatment conditions (p < 0.005). *p = 0.005 and **p < 0.0001 compared with vehicle. Data for IL-5 were obtained in lymphocytes from 24 subjects. Data for IFN-γ were obtained from 25 subjects.

Figure 5.

(−)-Isoproterenol (1 μM)–induced inhibition of IFN-γ and IL-5 production after pretreatment with the same agonist concentration (open boxes) or vehicle (shaded boxes) for 30 minutes and 2 hours, stratified for β2AR haplotype. The numbers 1, 2, and 3 at the abscissa represent haplotypes CysGlyGln, ArgGlyGlu, and CysArgGln, respectively. (A) (−)-Isoproterenol significantly inhibited IFN-γ production in every haplotype under all treatment conditions (p < 0.05). No haplotype-related differences were found. Data were obtained in lymphocytes from five subjects with the CysGlyGln haplotype, 11 subjects with the ArgGlyGlu haplotype, and nine subjects with the CysArgGln β2AR haplotype. (B) (−)-Isoproterenol significantly inhibited IL-5 production for every haplotype at the indicated time points (p < 0.05), except for those indicated by the following: +p = 0.08 and ++p = 0.5. *p < 0.05 and §0.05 < p ⩽ 0.1 for differences between haplotypes. Data were obtained in lymphocytes from five subjects with the CysGlyGln β2AR haplotype, 11 subjects with the ArgGlyGlu β2AR haplotype, and eight subjects with the CysArgGln β2AR haplotype.