Influence of beta2-adrenoceptor gene polymorphisms on beta2-adrenoceptor-mediated responses in human lung mast cells

Abstract

Background and purpose: Previous studies have shown that beta(2)-adrenoceptor-mediated responses in human lung mast cells are highly variable. The aims of the present study were to establish whether polymorphisms of the beta (2)-adrenoceptor gene (ADRB2) influence this variability in (a) beta(2)-adrenoceptor-mediated inhibition and (b) desensitization of beta(2)-adrenoceptor-mediated responses in human lung mast cells.

Experimental approach: Mast cells were isolated from human lung tissue. The inhibitory effects of the beta-adrenoceptor agonist, isoprenaline (10(-10)-10(-5) M), on IgE-mediated histamine release from mast cells were determined (n=92). Moreover, the inhibitory effects of isoprenaline were evaluated following a desensitizing treatment involving long-term (24 h) incubation of mast cells with isoprenaline (10(-6) M) (n=65). A potential influence of polymorphisms on these functional responses was determined by genotyping 11 positions, in the promoter and coding regions, of ADRB2 previously reported as polymorphic.

Key results: There was no influence of any of the polymorphic positions of ADRB2 on the potency of isoprenaline to inhibit histamine release from mast cells with the exception of position 491C>T (Thr164Ile). There was no influence of any of the polymorphic positions of ADRB2 on the extent of desensitization of the isoprenaline-mediated response following a desensitizing treatment except for position 46G>A (Gly16Arg). Analyses at the haplotype level indicated that there was no influence of haplotype on beta (2)-adrenoceptor-mediated responses in mast cells.

Conclusions and implications: These data indicate that certain polymorphisms in ADRB2 influence beta(2)-adrenoceptor-mediated responses in human lung mast cells.

Figure 1

Effect of isoprenaline on histamine release from human lung mast cells. Cells were incubated for 10 min with or without isoprenaline, before challenge for 25 min with a maximal releasing concentration of anti-IgE (1:300). (a) Results are expressed as the % inhibition of the unblocked histamine release, which was 36±1% and data points are means±s.e.mean, n=92. (b) Twelve individual concentration–response curves, representative of the 92 experiments performed, are shown.

Figure 2

Influence of SNPs on inhibition. Cells were incubated for 10 min with or without isoprenaline, before challenge for 25 min with a maximal releasing concentration of anti-IgE (1:300). The figure shows the influence of (a) position 46G>A (amino acid 16, Gly>Arg) and (b) position 491C>T (amino acid 164, Thr>Ile) on the isoprenaline inhibition. Results are expressed as the % inhibition of the unblocked histamine releases, which were for (a) 34±2 (Gly16Gly), 38±2 (Gly16Arg) and 34±4% (Arg16Arg) and for (b) 37±1 (Thr164Thr) and 24±7% (Thr164Ile). Values are means±s.e.mean, for (a) 33 (Gly16Gly), 38 (Gly16Arg), and 19 (Arg16Arg) experiments and for (b) 88 (Thr164Thr) and four (Thr16Iile) experiments. SNP, single nucleotide polymorphism.

Figure 3

Effect of desensitizing treatment on the isoprenaline inhibition. (a) Mast cells were incubated for 24 h with (desensitized) or without (control) isoprenaline (10⁻⁶ M), after which the cells were washed extensively. Cells were then incubated for 10 min with or without isoprenaline, before challenge for 25 min with a maximal releasing concentration of anti-IgE (1:300). Results are expressed as the % inhibition of the unblocked histamine releases, which were 38±2 (control) and 34±2% (desensitized). Values are means±s.e.mean, n=65. (b) The figure shows the variable effect that desensitizing treatment (24 h with 10⁻⁶ M isoprenaline) has on E_max values for isoprenaline. Each line represents one of 65 experiments, although, for reasons of clarity, data from 32 experiments are shown in the figure.

Figure 4

Influence of 46G>A (amino acid 16) on functional desensitization. Data generated from preparations expressing (a) the more common allele (Gly16Gly), (b) both alleles (Gly16Arg) and (c) the less common allele (Arg16Arg) are shown. Mast cells were incubated for 24 h with (desensitized) or without (control) isoprenaline (10⁻⁶ M), after which the cells were washed extensively. Cells were then incubated for 10 min with or without isoprenaline, before challenge for 25 min with a maximal releasing concentration of anti-IgE (1:300). Results are expressed as the % inhibition of the unblocked histamine releases, which were for (a) 36±2 (control) and 33±2% (desensitized), for (b) 40±2 (control) and 36±3% (desensitized), and for (c) 37±5 (control) and 30±4% (desensitized). Values are means±s.e.mean, for (a) 22, (b) 29 and (c) 14 experiments.

Figure 5

Influence of haplotypes on inhibition. Cells were incubated for 10 min with or without isoprenaline, before challenge for 25 min with a maximal releasing concentration of anti-IgE (1:300). The figure shows the influence of (1) haplotype A, (2) haplotype B and (3) haplotype C on the isoprenaline inhibition. Results are expressed as the % inhibition of the unblocked histamine releases which were 39±3, 36±5 and 40±4% for (1), (2) and (3), respectively. Values are means±s.e.mean, for (1) 13, (2) 14 and (3) five experiments.

Figure 6

Influence of haplotypes on functional desensitization. Mast cells were incubated for 24 h with (desensitized) or without (control) isoprenaline (10⁻⁶ M) after which the cells were extensively washed. Cells were then incubated for 10 min with or without isoprenaline (10⁻¹⁰–10⁻⁵ M) before challenge for 25 min with a maximal releasing concentration of anti-IgE (1:300). E_max values for the isoprenaline inhibition were determined in control and desensitized sets and % desensitization values were calculated as follows: (1−[E_max of isoprenaline after desensitizing treatment/E_max of isoprenaline]) × 100. Each point reflects data from an individual experiment and horizontal bars represent mean % desensitization values, which were 46±7 (n=10), 65±9 (n=9) and 52±8% (n=4) for haplotypes A, B and C, respectively.

Figure 7

Haplotype designation using an inclusive or a simplified approach. Haplotypes were established by considering 8 (−654, −468, −367, −47^(BUP19), −20, 46⁽¹⁶⁾, 79⁽²⁷⁾, 491⁽¹⁶⁴⁾), 5 positions (−654, −468, −367, −47, −20), 4 (−367, −47, 46, 79), 3 (−47, 46, 79), 2 (46, 79) or 2^* (−47, 46) polymorphic positions of the β₂-adrenoceptor gene. Data show how the complexion of groups does not change (haplotype A), or changes slightly (haplotypes B and C) if all eight SNPs across the breadth of ADRB2 or fewer SNPs are considered. This analysis was performed in a population of 92 individuals. SNP, single nucleotide polymorphism.