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Regulation and function of epithelial secreted phospholipase A2 group X in asthma - PubMed

  • ️Tue Jan 01 2013

Regulation and function of epithelial secreted phospholipase A2 group X in asthma

Teal S Hallstrand et al. Am J Respir Crit Care Med. 2013.

Abstract

Rationale: Indirect airway hyperresponsiveness (AHR) is a fundamental feature of asthma that is manifest as exercise-induced bronchoconstriction (EIB). Secreted phospholipase A2 group X (sPLA2-X) plays a key role in regulating eicosanoid formation and the development of inflammation and AHR in murine models.

Objectives: We sought to examine sPLA2-X in the airway epithelium and airway wall of patients with asthma, the relationship to AHR in humans, and the regulation and function of sPLA2-X within the epithelium.

Methods: We precisely phenotyped 34 patients with asthma (19 with and 15 without EIB) and 10 normal control subjects to examine in vivo differences in epithelial gene expression, quantitative morphometry of endobronchial biopsies, and levels of secreted protein. The regulation of sPLA2-X gene (PLA2G10) expression was examined in primary airway epithelial cell cultures. The function of epithelial sPLA2-X in eicosanoid formation was examined using PLA2 inhibitors and murine tracheal epithelial cells with Pla2g10 deletion.

Measurements and main results: We found that sPLA2-X protein is increased in the airways of patients with asthma and that epithelial-derived sPLA2-X may be increased in association with indirect AHR. The expression of sPLA2-X increases during in vitro epithelial differentiation; is regulated by inflammatory signals including tumor necrosis factor, IL-13, and IL-17; and is both secreted from the epithelium and directly participates in the release of arachidonic acid by epithelial cells.

Conclusions: These data reveal a relationship between epithelial-derived sPLA2-X and indirect AHR in asthma and that sPLA2-X serves as an epithelial regulator of inflammatory eicosanoid formation. Therapies targeting epithelial sPLA2-X may be useful in asthma.

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Figures

<i>Figure 1.</i>
Figure 1.

Expression of PLA2G10 in airway epithelial brushings and induced sputum cells. (A) Copy number analysis of PLA2G10 expression in all groups combined demonstrates higher expression in airway epithelial cells than in airway cells derived from induced sputum. (B) There was no difference between the PLA2G10 expression in airway epithelial brushings between subjects with and without asthma and between subjects with and without exercise-induced bronchoconstriction (EIB). (C) There was also no difference in the PLA2G10 expression in induced sputum cells between each of the groups.

<i>Figure 2.</i>
Figure 2.

Quantification of secreted phospholipase A2 group X (sPLA2-X) protein in the airway lining fluid and airway cells. The concentration of sPLA2-X protein in induced sputum supernatant (A) as well as the amount of sPLA2-X protein relative to total protein (B) was increased in subjects with asthma relative to subjects without asthma. The differences were greater for subjects with exercise-induced bronchoconstriction (EIB) relative to subjects without EIB. (C) The amount of sPLA2-X protein relative to total protein in lysates from epithelial cells obtained by bronchoscopic brushings of the airway epithelium was increased in the group of subjects with asthma with EIB. (D) The concentration of sPLA2-X in cell lysates from the induced sputum cell pellet was increased in subjects with EIB. Ctrl = control.

<i>Figure 3.</i>
Figure 3.

Quantification of epithelial secreted phospholipase A2 group X (sPLA2-X) immunostaining by design-based stereology. (A) The volume (Vs) of the epithelium (epi) relative to the surface area of the basal lamina (bala) was similar between the asthma and control groups. (B) The volume of sPLA2-X immunostaining relative to the area of the basal lamina had more variation in the asthma groups and tended to be higher in the exercise-induced bronchoconstriction (EIB [+]) group, but these differences did not reach statistical significance. (C) Within the asthma group, the volume of sPLA2-X immunostaining in the epithelium tended to be associated with severity of airway hyperresponsiveness (AHR). Epithelial sPLA2-X immunostaining could be identified through the epithelium in many sections (D), but was also prominent in basal cells (BC) in some regions (E), as well as staining at the terminal bar (arrow) of ciliated cells (F). Goblet cells (GC) do not appear to be a significant source of sPLA2-X staining (F).

<i>Figure 4.</i>
Figure 4.

PLA2G10 expression during differentiation and repair in primary airway epithelial cell organotypic culture. (A) The expression of PLA2G10 increased over multiple time points during in vitro epithelial differentiation in organotypic culture in epithelial cells derived from subjects with asthma with exercise-induced bronchoconstriction (EIB [+]), EIB (−) subjects with asthma, and control subjects without asthma. The change in gene expression over time differed by phenotype. (B) Treatment with retinoic acid (RA, 50 nM) in primary airway epithelial cells cultured in basal medium increased the PLA2G10 expression change over time. (C) In fully differentiated epithelial cells, the secreted phospholipase A2 group X (sPLA2-X) protein accumulated in the apical fluid relative to the intracellular protein found in the lysate (P = 0.01), but there was no difference by phenotype. (D) After an in vitro scratch wound in fully differentiated cells the PLA2G10 expression changed over time but was similar between the two groups studied.

<i>Figure 5.</i>
Figure 5.

Effects of serum replacements and asthma-related cytokines on PLA2G10 expression in cultured primary epithelial cells. (A) The addition of growth factors and serum supplements in defined bronchial epithelial growth medium had reduced PLA2G10 expression over time relative to primary airway epithelial cells cultured in basal media. (B) The addition of epinephrine (Epi) or insulin, or to a lesser extent epidermal growth factor (EGF) or hydrocortisone (Hydrocort) to basal medium reduced the expression of epithelial PLA2G10. (C) The histone deacetylase inhibitor sodium butyrate markedly increased the expression of PLA2G10 in epithelial cells after 18 hours in culture. (D) Treatment of primary epithelial cells in basal media with tumor necrosis factor (TNF) and IL-1β increased the expression of PLA2G10 relative to untreated epithelial cells. (E) Treatment of epithelial cells with IL-17 and to a lesser extent IL-13 increased the expression of PLA2G10, whereas IL-4 and to a lesser extent IL-25 and thymic stromal lymphopoietin decreased PLA2G10 expression. (F) Treatment of epithelial cells from each of the study groups with SB, TNF/IL-1β, RA, IL-13, and IL-17 did not reveal significant differences in the increase in PLA2G10 expression between the groups. *P < 0.05, †P < 0.01, ‡P < 0.001, §P < 0.0001 RA = retinoic acid.

<i>Figure 6.</i>
Figure 6.

Respective roles of secreted phospholipase A2 group X (sPLA2-X) and cytosolic phospholipase A2 alpha (cPLA2α) in epithelial release of arachidonic acid (AA) and prostaglandin E2 (PGE2). (A) The release of radiolabeled AA into the culture medium increased over time in the BEAS2B human airway epithelial cell line, and treatment with tumor necrosis factor (TNF) in combination with IL-1β increased the release of AA. (B) The spontaneous release of AA was reduced at 18 and 24 hours in murine tracheal epithelial cells (mTECs) from PLa2g10−/− mice compared with wild-type (WT) mice, whereas the cPLA2 inhibitor pyrrophenone (Pyr-2) inhibited AA release to a lesser extent. (C) Treatment with TNF plus IL-1β increased AA release in a manner that was partially inhibited by Pyr-2 in PLa2g10−/− mice. (D) The generation of PGE2 by mTECs was increased by the addition of TNF plus IL-1β and decreased by the cPLA2 inhibitor Pyr-2, but the synthesis of PGE2 was not altered in PLa2g10−/− mice.

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References

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