Interleukin-4 in the Generation of the AERD Phenotype: Implications for Molecular Mechanisms Driving Therapeutic Benefit of Aspirin Desensitization

Abstract

Aspirin-exacerbated respiratory disease (AERD) is explained in part by over-expression of 5-lipoxygenase, leukotriene C4 synthase (LTC(4)S) and the cysteinyl leukotriene (CysLT) receptors (CysLT1 and 2), resulting in constitutive over-production of CysLTs and the hyperresponsiveness to CysLTs that occurs with aspirin ingestion. Increased levels of IL-4 have been found in the sinus mucosa and nasal polyps of AERD subjects. Previous studies demonstrated that IL-4 is primarily responsible for the upregulation of LTC4S by mast cells and the upregulation of CysLT1 and 2 receptors on many immune cell types. Prostaglandin E(2) (PGE(2)) acts to prevent CysLT secretion by inhibiting mast cell and eosinophil activation. PGE(2) concentrations are reduced in AERD reflecting diminished expression of cyclooxygenase (COX)-2. IL-4 can inhibit basal and stimulated expression of COX-2 and microsomal PGE synthase 1 leading to decreased capacity for PGE(2) secretion. Thus, IL-4 plays an important pathogenic role in generating the phenotype of AERD. This review will examine the evidence supporting this hypothesis and describe a model of how aspirin desensitization provides therapeutic benefit for AERD patients.

Figure 1

Cytokine modulation of cysteinyl leukotriene receptor protein expression on T cells. T cells were separated from blood using magnetic bead affinity chromatography and stimulated with 20 ng/mL IL-4 for 16 hrs before cells were collected for analysis. Cell surface expression of the CysLTR1 receptor was evaluated using rabbit polyclonal anti-CysLTR1 followed by labeling with FITC-conjugated goat anti-rabbit IgG. Isotype control is shown in white, unstimulated in black, and IL-4 stimulated in gray. Reprinted with permission of the American Thoracic Society [31].

Figure 2

COX-2 protein expression in resting and LPS-stimulated monocytes. Monocytes were isolated from blood by magnetic bead purification. Cells were treated with IL-4 (10 ng/mL), IL-13 (10 ng/mL), or LPS (1 μg/mL) for 24 hrs and whole cell lysates collected. Proteins were separated on a 10% SDS acrylamide gel and transferred to nitrocellulose. The membrane was probed with anti-COX-2 and then stripped and reprobed with anti-β-actin.

Figure 3

IL-4 inhibits monocyte PGE₂ secretion. IL-4 was added to the cells (10 ng/mL) alone or either with LPS (1 μg/mL) or IL-1β (10 ng/mL). Cells were incubated for 24 hrs before supernatants were collected. PGE₂ levels were measured by ELISA and reported as pg/mL [43].

Figure 4

EMSA for STAT6. (a) EMSAs were performed using ³²P-labeled oligomers comprising the STAT6 site within the CysLT1R promoter. Nuclear extracts were purified from THP-1 mononuclear cell lines in the resting state, IL-4 stimulated (10 ng/mL), and IL-4 stimulated in the additional presence of aspirin (10 mM). STAT6 binding was evaluated by performing EMSAs in the presence of 100–300-fold molar excess unlabeled STAT6 consensus sequence (comprising the ε heavy chain promoter) or a mutated STAT6 consensus sequence. EMSAs were also performed using STAT6, phosphoSTAT6, and, as a control, STAT4 antibodies. (b) Relevance to normal tissue was evaluated using nuclear extracts prepared as above, derived from enriched peripheral blood-derived mononuclear phagocytes [43].

Figure 5

Western hybridization of nuclear extracts. Nuclear extracts obtained as described for Figure 4 were electrophoresed on a 10% SDS polyacrylamide gel and transferred to a nitrocellulose membrane. Presence of phosphoSTAT6 was determined via probing with anti-phosphoSTAT6 antibodies and a secondary peroxidase-labeled antibody [43].

Figure 6

Summary of IL-4 activity on the leukotriene and prostaglandin synthesis pathways. Activation of gene synthesis by IL-4 is shown in pink while inhibition of gene synthesis by IL-4 is shown in red.