Cystatins in human tear fluid

Abstract

The activities of cysteine proteinases which include several lysosomal cathepsins are controlled by naturally occuring inhibitory proteins termed cystatins. Cystatins occur both intracellularly and extracellularly in various tissue fluids including tears. Tears were collected by the Schirmer paper strip method from healthy volunteers who had no history or signs of external ocular disease. The tear components were extracted from the filter papers, and used to determine the apparent free cystatin activity and cystatin levels of tears, and for immunoblots. Tears were also collected using capillary tubes for the measurements of cystatins. By titrating papain, a cysteine proteinase, of known specific activity with tear fluid, relatively high levels of apparent free cystatin activity were demonstrated in tears: 28.8±3.47 (S.E.M.) pmols papain inhibited per mg tear protein (n=9). The concentrations of cystatins in tear samples were measured by an indirect enzymelinked iummunosorbent assay (ELISA) using antibodies against human salivary cystatin S and purified cystatin S as standard. The ELISAs revealed that tears contain high levels of cystatin-like immunoreactive material, amounting to about 10% of tear proteins. In μg cystatin S/mg protein the values were: right eye: 94.7±9.9; left eye: 115.5±14.8; n=12. Cystatin levels of tears collected using capillary tubes were comparable: 120.7±19 μg/mg protein (n=10). Immunoblots of tear fluids revealed a protein of about 14,000 molecular weight which reacted with antihuman cystatin SN monoclonal antibodies. Protein(s) of similar molecular weight were visualized using antibodies against human cystatins S and C. Less abundant additional cystatin-like immuno-reactive proteins were detected by using the two latter antibodies. Thus, tears contain a number of cystatins capable of inhibiting a typical cysteine proteinase papain added to the tear samples. Cystatins in tears presumably have a defensive role in protecting corneal and conjunctival epithelia from the harmful effects of some of the proteolytic enzymes under physiologic and pathologic conditions.