Purinergic Regulation of Degranulation by Human LAD2 Mast Cells

Abstract

Purpose: : Mast-cell degranulation releases histamine and other products crucial in allergic conjunctivitis. Adenosine receptors (ARs) have been reported to modify degranulation, but observations vary with species, tissue and stimulus. This study tested the roles of ARs in degranulation of human LAD2 mast cells activated immunologically or by C3a complement component.

Methods: : LAD2 cells were gifts from NIAID. Gene expression of the ARs was quantified by real-time PCR. Degranulation was triggered by cross-linking FcεRI anchored IgE or by adding 0.2 µM C3a, and monitored as percentage β-hexosaminidase release. ATP concentrations were measured by the luciferin-luciferase method.

Results: : Relative expression of A_2A, A_2B and A₃ ARs was 1.00, 0.24 and 0.32, respectively, and A₁ was undetectable (n=3). The non-selective AR agonist NECA (1 µM) enhanced both C3a-(63 ± 4%; n=76) and immunologically-triggered (54 ± 4%; n=78) degranulation. The selective A_2A antagonist SCH 442416 blocked enhancement of C3a- and immunologically-triggered degranulation by ~60% and ~70%, respectively. The A_2B antagonist MRS 1754 blocked only enhancement of C3a-degranulation by ~60%. The A₃ antagonist MRS 1191 did not significantly reduce either enhancement. The antagonists little affected C3a-stimulated degranulation alone. Addition of all 3 AR antagonists nearly abolished the NECA-enhanced effect of degranulation by both stimuli. Mast cells themselves were not a significant source of adenosine delivery since baseline bath concentration of ATP (29 ±4 nM; n=36) was not increased after stimulation with C3a (n=12), cross-linking FcεRI (n=24) or 50% hypotonicity (n=18).

Conclusions: : LAD2 express ARs in the order A_2A>A_2B, A₃, but A₁ was undetectable. Non-selective activation of ARs with NECA enhances degranulation stimulated immunologically or by C3a. Selective AR inhibition reduced the enhancements, with A_2A≥A_2B>A₃. Ecto-enzymatic metabolism of autocrine-released ATP is not a significant source of adenosine. We conclude that mast cell degranulation can be modulated by ecto-enzymatic dephosphorylation of ATP found in inflammatory milieux, largely through A_2AR.

Keywords: adenosine • receptors: pharmacology/physiology • conjunctivitis