Prostaglandin E2 resistance in granulocytes from patients with aspirin-exacerbated respiratory disease

Abstract

Background: Aspirin-exacerbated respiratory disease (AERD) is an inflammatory condition of the respiratory tract and is characterized by overproduction of leukotrienes (LT) and large numbers of circulating granulocyte-platelet complexes. LT production can be suppressed by prostaglandin E(2) (PGE(2)) and the cyclic AMP-dependent protein kinase A (PKA).

Objective: To determine if PGE(2)-dependent control of LT production by granulocytes is dysregulated in AERD.

Methods: Granulocytes from well-characterized patients with and without AERD were activated ex vivo and subjected to a range of functional and biochemical analyses.

Results: Granulocytes from subjects with AERD generated more LTB4 and cysteinyl LTs than did granulocytes from controls with aspirin-tolerant asthma and controls without asthma. When compared with controls, granulocytes from subjects with AERD had comparable levels of EP(2) protein expression and PGE(2)-mediated cAMP accumulation, yet were resistant to PGE(2)-mediated suppression of LT generation. Percentages of platelet-adherent neutrophils correlated positively with LTB4 generation and inversely with responsiveness to PGE(2)-mediated suppression of LTB(4). The PKA inhibitor H89 potentiated LTB4 generation by control granulocytes but was inactive in granulocytes from individuals with AERD and had no effect on platelet P-selectin induction. Both tonic PKA activity and levels of PKA catalytic gamma subunit protein were significantly lower in granulocytes from individuals with AERD relative to those from controls.

Conclusions: Impaired granulocyte PKA function in AERD may lead to dysregulated control of 5-lipoxygenase activity by PGE(2), whereas adherent platelets lead to increased production of LTs, which contributes to the features of persistent respiratory tract inflammation and LT overproduction.

Keywords: AERD; Samter's triad; aspirin triad; aspirin-exacerbated respiratory disease; asthma; cyclic AMP; leukotriene; nonsteroidal anti-inflammatory drug; prostaglandin E(2); protein kinase A.

FIG 1

LT production by fMLP-stimulated granulocytes and suppression by PGE₂ and EP agonists. LTB₄ (A) and cysLT (B) production by 0.5 × 10⁶ granulocytes stimulated with 1 μM fMLP are shown from controls without asthma (white columns), controls with ATA (gray columns), and subjects with AERD (black columns). Percentage suppression of LTB₄ (C) and cysLT (D) production by fMLP-stimulated granulocytes pretreated with the listed agonists is shown for cells from controls without asthma, controls with ATA, and subjects with AERD. Data are expressed as mean (±SEM) (*P < .05; **P < .01).

FIG 2

Platelet-adherent neutrophils, LTB₄ generation, and suppression of 5-LO activity in AERD. A, Percentages of platelet-adherent neutrophils in whole blood from 8 subjects with AERD are plotted against quantity of LTB₄ generated by fMLP-stimulated granulocytes from the same individuals. Percentage suppression of fMLP-induced LTB₄ by pretreatment with PGE₂ (B) or the EP₂ receptor-specific agonist (C) plotted against percentages of platelet-adherent neutrophils in the blood of each subject. Effect size (Pearson correlation) is denoted as an r value.

FIG 3

EP₂ protein expression, cAMP accumulation, and inhibition of platelet CD62P. A, Representative flow cytometric histogram of intracellular EP₂ is shown for neutrophils from a subject with AERD. Mean intra-cellular EP₂ levels of neutrophils and eosinophils (B) and of platelets (D) are shownfor controls without asthma (n = 6), controls with ATA (n = 5), and subjects with AERD (n = 16). Intracellular cAMP accumulation within granulocytes (C) or platelets (E) stimulated with listed agonists is shown. F, Inhibition of ADPβS-stimulated CD62P expression on platelets in platelet-rich plasma is shown. Data are expressed as mean (±SEM). There were no significant differences between patient groups for any comparisons in panels B–F.

FIG 4

Effect of H89 on LTB₄ production from blood granulocytes. The effect of pretreatment of granulocytes with H89 on fMLP-induced LTB₄ production is shown for controls without asthma (A) (n = 6), controls with ATA (B) (n = 6), and subjects with AERD (C) (n = 8). Data are expressed as mean (±SEM) (*P < .05; **P < .01).

FIG 5

Granulocyte PKA activity and PKACγ subunit expression. A, Basal PKA activity measured by ELISA is shown. B, The net effect of H89 on granulocyte production of LTB₄ is shown plotted again the basal PKA activity for each patient. Effect size is denoted as an r value. C, Representative Western blot of granulocytes for PKACγ protein, with GAPDH shown below. Of note, the 2 control subjects with ATA with the lowest PKACγ protein levels of that patient group are the 2 shown on this blot. D, Mean PKACγ protein levels normalized to GAPDH are shown. *P < .05.