IL-13 desensitizes β2-adrenergic receptors in human airway epithelial cells through a 15-lipoxygenase/G protein receptor kinase 2 mechanism

Abstract

Background Beta-2 adrenergic receptor (β2AR) agonists are critical treatments for asthma. However, receptor desensitization can lead to loss of therapeutic effects. While desensitization to repeated use of β2 agonists is well studied, Type-2 inflammation could also impact β2AR function. Objective To evaluate the impact of the Type-2 cytokine, IL-13, on β2AR desensitization in human airway epithelial cells (HAECs) and determine whether 15-Lipoxygenase-1 (15LO1) binding with phosphatidylethanolamine binding protein-1 (PEBP1) contributes to desensitization through release of G-protein Receptor Kinase-2 (GRK2). Methods HAECs in air-liquid-interface (ALI) culture with/without IL-13 (48 hrs) or isoproterenol (ISO) (30 min) pretreatment were stimulated with ISO (10min). cAMP was measured by ELISA and β2AR and GRK2 phosphorylation by Western Blot. siRNA was utilized for 15LO1 knockdown. Interactions of GRK2, PEBP1 and 15LO1 were detected by Immunoprecipitation/Western Blot and immunofluorescence. HAECs and airway tissue from controls and asthmatics were evaluated for I5LO1, PEBP1 and GRK2. Results Pretreatment with ISO or IL-13 decreased ISO-induced cAMP generation compared to ISO for 10 min alone, paralleled by increases in β2AR and GRK2 phosphorylation. GRK2 associated with PEBP1 after 10 min of ISO in association with low pGRK2 levels. In contrast, in the presence of IL-13+ISO (10 min), binding of GRK2 to PEBP1 decreased, while 15LO1 binding and pGRK2 increased. 15LO1 knockdown restored ISO-induced cAMP generation. These findings were recapitulated in freshly brushed HAEC from asthmatic cells and tissue. Conclusion IL-13 treatment of HAECs leads to β2AR desensitization which involves 15LO1/PEBP1 interactions to free GRK2 and allow it to phosphorylate (and desensitize) β2ARs, suggesting beneficial effects of β2 agonists could be blunted in Type-2 associated asthma.

Inhibition of PI3K promotes dilation of human small airways in a rho kinase‐dependent manner

“…We previously reported that IL‐13 augments carbachol‐induced airway narrowing and attenuates β 2 ‐adrenoceptor agonist‐induced airway dilation in human PCLS (Cooper et al , ; Robinett et al , ). Although IL‐13 attenuates β 2 ‐adrenoceptor agonist‐induced airway (Laporte et al , ; Grunstein et al , ; Duan et al , ; Cooper et al , ; Robinett et al , ; Albano et al , ), IL‐13 has little effect on PI3K p110 inhibitor‐induced airway dilation as shown in Figure . We and others have shown that HASM cells derived from fatal asthma donors manifest distinct phenotypes compared to cells derived from non‐asthmatic patients, including cell proliferation (Roth et al , ), enzymatic function (Chambers et al , ; Trian et al , ), receptor signalling and sustained calcium transients consistent with contractility of the muscle (Ma et al , ; Leguillette and Lauzon, ; Chin et al , ).…”

“…The inflammatory milieu in asthma engenders AHR to contractile agonists and attenuates β 2 agonist‐induced bronchodilation (Cooper et al , ; Albano et al , ; Gupta et al , ). Previously, we showed that exposure to IL‐13 renders airways hyposensitive to β 2 ‐adrenoceptor agonist‐mediated airway dilation but not TAS2 receptor‐mediated airway dilation (Robinett et al , ).…”

Serum IL‐5 and IL‐13 consistently serve as the best predictors for the blood eosinophilia phenotype in adult asthmatics

“…A mouse model in which asthmatic features persisted for 6 months after cessation of allergen exposure demonstrated that a critical network of feedback and feed forward interactions between epithelial cells and ILC2s is taking place, in which IL‐13 is central and involved in maintaining chronic asthma . In addition, it was shown that IL‐13 desensitizes β2‐adrenergic receptors in human airway epithelial cells , thus offering a potential explanation for the increased AHR.…”

15-Lipoxygenase 1 in nasal polyps promotes CCL26/eotaxin 3 expression through extracellular signal-regulated kinase activation

“…Cell lysates were run on 4-12% gradient SDS-PAGE gels under reducing conditions, as previously described. 29 Western blotting (WB) was performed with primary antibodies against 15LO1 (1:1000 dilution; a gift of Dr Doug Conrad, University of California, San Diego, Calif), CCL26 (1:100 dilution; R&D Systems, Minneapolis, Minn), phosphorylated ERK (pERK; 1:5000 dilution; Sigma, St Louis, Mo), total ERK (tERK; 1:5000 dilution; Sigma), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH; 1:1000 dilution; Novus Biologicals, Littleton, Colo). Densitometric analysis was performed with ImageJ software (National Institutes of Health, Bethesda, Md).…”