Abstract
Purpose: :
To investigate the effects of TNF-α, a pro-inflammatory cytokine implicated in corneal allograft rejection, on barrier integrity of the corneal endothelium.
Methods: :
Experiments were performed with cultured bovine corneal endothelial cells. Barrier integrity was monitored continuously by TER (Transendothelial electrical resistance). Peri-junctional actomyosin ring (PAMR), which forms a dense band of actin cytoskeleton at the apical junctional complex, was visualized by staining with Texas-Red conjugated phalloidin. MLC (myosin light chain) phosphorylation, essential for actomyosin contraction, was assessed by Western blotting.
Results: :
Exposure to TNF-α (20 ng/mL) resulted in a sustained loss of TER, formation of inter-endothelial gaps, increase in MLC phosphorylation, and significant thinning of PAMR. Co-treatment with forskolin (10 µM) + rolipram (PDE4 inhibitor; 50 µM) opposed the TNF-α-induced loss of TER by more than 50% and thinning of PAMR. Similar co-treatment with the nonselective A2B agonist NECA (20 µM) also opposed the decrease in TER but was less efficacious compared to forskolin + rolipram.
Conclusions: :
Depolymerization of PAMR, in addition to its contraction (based on studies with thrombin and histamine), contributes to dynamic regulation of the barrier integrity of corneal endothelial cells. Thus, the effects of elevated cAMP on TNF-α-induced loss of barrier integrity may involve opposition to actomyosin contraction and/or enhancement in actin polymerization at the apical junctional complex.
Keywords: cornea: endothelium • cell adhesions/cell junctions • inflammation