Cysteinyl leukotrienes acting via granule membrane-expressed receptors elicit secretion from within cell-free human eosinophil granules

Abstract

Background: Cysteinyl leukotrienes (cysLTs) are recognized to act via receptors (cysLTRs) expressed on cell surface plasma membranes. Agents that block cysLT(1) receptor (cysLT(1)R) are therapeutics for allergic disorders. Eosinophils contain multiple preformed proteins stored within their intracellular granules. Cell-free eosinophil granules are present extracellularly as intact membrane-bound organelles in sites associated with eosinophil infiltration, including asthma, rhinitis, and urticaria, but have unknown functional capabilities.

Objective: We evaluated the expression of cysLTRs on eosinophil granule membranes and their functional roles in eliciting protein secretion from within eosinophil granules.

Methods: We studied secretory responses of human eosinophil granules isolated by subcellular fractionation. Granules were stimulated with cysLTs, and eosinophil cationic protein and cytokines were measured in the supernatants. Receptor expression on granule membranes and eosinophils was evaluated by flow cytometry and Western blot.

Results: We report that receptors for cysLTs, cysLT(1)R, cysLT(2) receptor, and the purinergic P2Y12 receptor, are expressed on eosinophil granule membranes. Leukotriene (LT) C(4) and extracellularly generated LTD(4) and LTE(4) stimulated isolated eosinophil granules to secrete eosinophil cationic protein. MRS 2395, a P2Y12 receptor antagonist, inhibited cysLT-induced eosinophil cationic protein release. Montelukast, likely not solely as an inhibitor of cysLT(1)R, inhibited eosinophil cationic protein release elicited by LTC(4) and LTD(4) as well as by LTE(4).

Conclusion: These studies identify previously unrecognized sites of localization, the membranes of intracellular eosinophil granule organelles, and function for cysLT-responsive receptors that mediate cysteinyl leukotriene-stimulated secretion from within eosinophil granules, including those present extracellularly.

FIG 1

Isolated granules expressed extracellular domains of the A, CysLT₁ receptor (CysLT₁R) and B, CysLT₂ receptor (CysLT₂R), but not C, the carboxy-terminal intracellular domain of CysLT₁R. Shaded histograms represent staining with control antibody (Ab). Solid and dashed lines represent staining with polyclonal specific antibodies (pAb) and anti-CysLTR pAbs neutralized by absorption with their respective immunogen peptide, respectively. Data are from one experiment, representative of three.

FIG 2

Isolated granules were stimulated with different concentrations (0.03 – 3,000 nM) of A, LTC₄, B, LTD₄ and C, LTE₄ and eosinophil cationic protein (ECP) levels were measured in the supernatants. Data is shown for one experiment representative of three. + represents significantly increased ECP release (P < 0.05) compared with non-stimulated granules.

FIG 3

MRS 2395, a selective P2Y12 receptor (P2Y12R) antagonist, dose-dependently inhibited the eosinophil cationic protein (ECP) release induced by A, LTC₄ 30 nM, B, LTD₄ 0.3 nM and C, 300 nM and D, LTE₄ 30 nM. + and * represent P < 0.05 for ECP released compared with non-stimulated and leukotriene-stimulated granules, respectively. E, Eosinophil and F, isolated granule surface membranes expressed the P2Y12R. Shaded histograms and solid lines represent staining with control Ab and with an anti- P2Y12R specific polyclonal Ab, respectively. G, The expression of the P2Y12R was confirmed on eosinophil and isolated granules by Western blots. Specificity of immunodetection was confirmed by neutralization with the anti-P2Y12R pAb by absorption with its respective immunogen peptide. All data is shown for one experiment representative of three. Gran, granules; Eos, eosinophils.

FIG 4

Montelukast dose-dependently inhibited eosinophil cationic protein (ECP) secretion induced by A, LTC₄ 30 nM, B, LTD₄ 0.3 nM and C, 300 nM and D, LTE₄ 30 nM. Data is shown for one experiment representative of three. + and * represent P < 0.05 for ECP released compared with non-stimulated granules and leukotriene-stimulated granules, respectively.