In vitro evidence of involvement of the epithelial y+ transporter in β-defensin production on the ocular surface.

To analyse the hypothesis as to whether there is a functional relationship between human cationic amino acid transporters (hCATs, y(+) transporter, the main transporter of L-arginine and L-lysine) and human β-defensin (important components of immune function) production on the ocular surface, arginase and nitrate monoxide synthase (NOS), enzymes that compete for L-arginine, were inhibited by norNOHA (N(omega)-hydroxy-nor-L-arginine) and/or L-NAME (NG-nitro-L-arginine methyl ester) in cultured human corneal epithelial cells. In addition, the transport activity of hCAT proteins was inhibited or activated through α-tocopherol or PMA (phorbol myristate acetate), respectively. Concentrations of the human inducible β-defensins (hBD) 2 and 3 were determined by ELISA experiments. The basic expression of hBD3 in non-stimulated HCE cells significantly exceeded that of hBD2. Both β-defensins also differed as to how readily their excretion could be stimulated. HBD2 excretion rate was 3.5 time more by L-NAME, whereas norNOHA had no effect. In contrast, hBD3 excretion was increased by norNOHA by a factor of 1.5 but L-NAME alone had no effect. The excretion of both β-defensins was increased 3- and 6-fold by combined administration of L-NAME, norNOHA and interleukin (IL)-1β. Administration of α-tocopherol increased hBD2 excretion twofold. No effect was observed for hBD3. With PMA, on the other hand, a reduction in secretion for both β-defensins was observed. These in vitro findings provide evidence of a functional association between CAT proteins and β-defensins 2 and 3 opening up a new field of research with pharmacological perspectives for treatment of inflammatory diseases such as keratitis or dry eye disease.