Barrier dysfunction of the corneal endothelium in response to TNF-α: role of p38 MAP kinase.

The corneal endothelium maintains stromal deturgescence, which is required for corneal transparency. The glycosaminoglycans of the stroma pose a threat to stromal deturgescence because of their tendency to imbibe water across the endothelium. This fluid leak into the stroma is restricted by the putative barrier function associated with the corneal endothelium. The barrier integrity of the endothelium, in conjunction with its active fluid pump mechanism,1,2 is responsible for the stromal deturgescence. Furthermore, tight junctions (TJs) of the endothelium, in addition to their innate role in the maintenance of barrier integrity, are also intrinsically coupled to the fluid pump activity. This is because the TJs also maintain polarity of the transmembrane proteins, which are involved in active fluid transport. Barrier integrity is also critical to sustain local osmotic gradients, which elicit active fluid movement.3,4 Hence, barrier integrity of the endothelium is indispensable for the maintenance of stromal hydration control; therefore, it is crucial for corneal transparency. Despite a continuous age-related loss of endothelial cells (∼0.5% per year), stromal hydration is maintained as long as the cell density is greater than 700 cells/mm2.1,5 When the corneal endothelium is subjected to inflammation, disease, or surgical trauma, a loss of stromal hydration control is induced concomitant with a rapid decline in cell density.1,6 Loss of corneal transparency because of decompensated endothelium is a major indication for corneal transplantation, of which there are approximately 40,000 annually in the United States.7 Even after transplantation, survival of the endothelium is a major concern6 because the proinflammatory mediators, which are released secondary to immune response, are known to affect gene expression8 and barrier integrity in other cell types. A basic understanding of the mechanisms involved in barrier dysfunction is, therefore, essential to develop therapeutic strategies that could be used to rescue transplanted corneas from endothelial failure. TNF-α, a 17-kDa proinflammatory cytokine, is thought to play a major role in corneal endothelial dysfunction during allograft rejection9,10 and anterior uveitis.11 Its levels are elevated in the aqueous humor of rabbits undergoing allograft rejection.10,12 Prolongation of endothelial graft survival was observed by interfering with the activities of TNF-α through the administration of TNFR-Ig, a recombinant TNF receptor.10 In general, TNF-α is known to have pathophysiological influence in many cell types through mechanisms leading to apoptosis,13 loss of barrier integrity,14,15 and prolongation of the immune response through enhanced expression of cellular adhesion molecules.16 In a study involving rabbit corneal endothelium, Watsky et al.17 showed that TNF-α increases the permeability to carboxyfluorescein, concomitant with disruption of the actin cytoskeleton. Although they found that 8 Br-cAMP (a membrane-permeable analog of cAMP) opposed the response to TNF-α, the molecular mechanisms underlying the barrier dysfunction and mechanism of rescue by elevated cAMP remain unknown. In several studies involving vascular endothelium, TNF-α is known to induce barrier dysfunction concomitant with the disruption of actin cytoskeleton14,18 and microtubule disassembly15 through mechanisms involving the activation of reactive oxygen species (ROS),19 RhoA GTPase,20 MAP kinases,15 transcriptional activation of myosin light chain kinase,18,21 and heat shock protein 27 (Hsp27).22 However, a preponderance of evidence collected to date seems to indicate a major role for p38 MAP kinase because it is also implicated in the activation of ROS production through components of the NADPH complex,23 Hsp27,22 and microtubule disruption.15,24 Therefore, in the present study, for the first time, we have investigated the involvement of p38 MAP kinase in the TNF-α–induced breakdown of the barrier integrity of corneal endothelium. Our results demonstrate that the TNF-α–induced activation of p38 MAP kinase is a crucial event that results not only in the loss of barrier integrity but also in the disruption of cytoskeleton and the remodeling of apical junctional complex (AJC).