Abstract
Purpose: :
To determine if leukotriene (LT) D4 produced during allergic inflammation stimulates conjunctival goblet cell mucin secretion and whether this response can be terminated by the pro-resolution mediators resolvin (Rv) RvE1 and RvD1.
Methods: :
Rat goblet cells were cultured from conjunctival pieces in RPMI media and passaged once. The presence of the receptors for LTD4, CysLT1 and CysLT2, was investigated in cultured cells by immunofluorescence microscopy and western blotting analysis using antibodies specific to the receptors. Mucin secretion was measured from cultured goblet cells that were: 1) incubated with increasing concentrations (10-15-10-8 M) or for increasing time (0.5-8 hr) with LTD4; 2) preincubated with the LTD4 receptor antagonists monteleukast or MK571 each at 10-7-10-5 M and then stimulated with LTD4 at 10-10 and 10-9 M; and 3) preincubated with RvE1 and RvD1 each at 10-10-10-8 M. Secretion was measured by a enzyme-linked lectin assay using the biotinylated lectin UEA-1 and streptavadin/alkaline phosphatase conjugate for detection. This assay measures high molecular weight glycoconjugates including mucin. Values for secretion were standardized to total protein.
Results: :
Both LTD4 receptors were detected in rat conjunctival goblet cells. LTD4 significantly stimulated goblet cell secretion in a concentration-dependent manner with a maximum 4.5±2.4 -fold increase occurring at 10-11 M and a maximum 2.2±0.5-fold increase at 0.5 hr. Montelukast or MK571 (each at 10-5 M) significantly inhibited LTD4-stimulated secretion a maximum of 84±14 % and 78±15 %, respectively. RvE1 and RvD1 each completely inhibited LTD4-stimulated secretion in a concentration-dependent manner with 100% inhibition of secretion at 10-9 M RvE1 and 10-10 M RvD1.
Conclusions: :
Conjunctival goblet cell mucin secretion is stimulated by the pro-allergic inflammatory mediator LTD4 and this response is actively terminated by the resolvins RvE1 and RvD1.
Keywords: conjunctiva • inflammation