Secreted phospholipase A2 group X overexpression in asthma and bronchial hyperresponsiveness

Abstract

Rationale: Secreted phospholipase A(2) enzymes (sPLA(2)s) play key regulatory roles in the biosynthesis of eicosanoids, such as the cysteinyl leukotrienes, but the role of these enzymes in the pathogenesis of asthma is not known.

Objectives: To establish if sPLA(2)s are overexpressed in the airways of patients with asthma, and to determine if these enzymes may play a role in the generation of eicosanoids in exercise-induced bronchoconstriction.

Methods: Induced sputum samples were obtained from subjects with asthma with exercise-induced bronchoconstriction and nonasthmatic control subjects at baseline, and on a separate day 30 minutes after exercise challenge. The expression of the PLA(2)s in induced sputum cells and supernatant was determined by quantitative polymerase chain reaction, immunocytochemistry, and Western blot.

Measurements and main results: The sPLA(2)s expressed at the highest levels in airway cells of subjects with asthma were groups X and XIIA. Group X sPLA(2) (sPLA(2)-X) was differentially overexpressed in asthma and localized to airway epithelial cells and bronchial macrophages. The gene expression, immunostaining in airway epithelial cells and bronchial macrophages, and the level of the extracellular sPLA(2)-X protein in the airways increased in response to exercise challenge in the asthma group, whereas the levels were lower and unchanged after challenge in nonasthmatic control subjects.

Conclusions: Increased expression of sPLA(2)-X may play a key role in the dysregulated eicosanoid synthesis in asthma.

Figure 1.

Expression of secreted phospholipase A₂ enzymes (sPLA₂s) in induced sputum cells from subjects with asthma and exercise-induced bronchoconstriction. Semiquantitative polymerase chain reaction was used to assess the expression of the secreted (A) and cytosolic PLA₂s (cPLA₂) (B) relative to the expression of glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The bars represent the mean ± SD.

Figure 2.

Baseline differences in secreted phospholipase A₂ (sPLA₂) groups V, X, and XIIA between subjects with asthma and exercise-induced bronchoconstriction and nonasthmatic control subjects. The gene expression of sPLA₂-X was increased relative to control (A), whereas there was no difference in the expression of sPLA₂-XIIA (B). The expression of sPLA₂-V was below the level of detection. No difference was detected for immunostaining in induced sputum cells for sPLA₂-V (C). The percentage of cells immunostaining for sPLA₂-X (D) and sPLA₂-XIIA (E) was increased in subjects with asthma relative to control subjects.

Figure 3.

Immunostaining for secreted phospholipase A₂ (sPLA₂) groups V, X, and XIIA in induced sputum cells. Representative photomicrographs of immunocytochemistry for sPLA₂-V (A, B), sPLA₂-X (C, D), and sPLA₂-XIIA (E, F) demonstrate immunostaining predominantly in columnar epithelial cells (arrows) and macrophages (arrowheads). The representative slides were from subjects with asthma after exercise challenge.

Figure 4.

Effects of exercise challenge on group X secreted phospholipase A₂ (sPLA₂-X) in subjects with asthma with exercise-induced bronchoconstriction and nonasthmatic control subjects. The gene expression (A) and percentage of cells in induced sputum immunostaining (B) for sPLA₂-X increased in subjects with asthma after challenge, but not in control subjects. Immunostaining for sPLA₂-X in subjects with asthma increased in bronchial macrophages (Mac) and columnar epithelial cells (Epi), but not in eosinophils (EOS) (C), whereas no changes were observed in the control group (D). Western blots of induced sputum supernatant revealed an increase in sPLA₂-X protein in subjects with asthma (E), but not in control subjects (F), resulting in a higher level of sPLA₂-X in subjects with asthma than in control subjects after exercise (G). BL = baseline; post = postexercise.

Figure 5.

Effects of exercise challenge on group XIIA secreted phospholipase A₂ (sPLA₂-XIIA) in subjects with asthma and exercise-induced bronchoconstriction and nonasthmatic control subjects. The gene expression (A) and percentage of cells in induced sputum immunostaining (B) for sPLA₂-XIIA increased in subjects with asthma after challenge, but not in control subjects. BL = baseline; post = postexercise.

Figure 6.

Effects of exercise challenge on group V secreted phospholipase A₂ (sPLA₂-V) in subjects with asthma and exercise-induced bronchoconstriction and nonasthmatic control subjects. The percentage of cells in induced sputum immunostaining for sPLA₂-V was no different postexercise compared with baseline in either the subjects with asthma or control subjects (A). Western blot of induced sputum supernatant had no increase in sPLA₂-V protein in subjects with asthma (B), and could not be detected in control subjects. BL = baseline; post = postexercise.