IDENTIFICATION, ORIGINS AND THE DIURNAL ROLE OF THE PRINCIPAL SERINE-PROTEASE INHIBITORS IN HUMAN TEAR FLUID

Purpose. Previous work identified polymorphonuclear leukocyte (PMN) el astase as the major caseinolytic entity in tears collected after overn ight eye closure. This study was designed to identify the principal se rine protease inhibitors (serpins) in tears and to determine their fun ction in the regulation of PMN cell proteases on eye closure. Methods. Reflex and closed eye tear samples were collected by microcapillary t ube and centrifuged. After reflex and closed rye supernatants (R and C ) were fractionated by HPLC, samples were subjected to casein zymograp hy and reverse zymography. Western blots were utilized to screen tears and HPLC fractions for elastase, cathepsin G and proteinase-3 and to obtain semi-quantitative data on alpha I-protease inhibitor (alp1), al pha I-antichymotrypsin (al-Achy), secretory leukocyte protease inhibit or (SLPI), elafin and alpha2-macroglobulin (alpha 2-M) as well as asso ciated complexes and products. To confirm specificity of reactivity, s amples were immunoprecipitated for a given protease or serpin and scre ened for the coprecipitation of interacting species. Results. Although R fluid contains no caseinolytic activity, it contains low levels of serpin-like activity principally in the form of SLPI (5-10 ng/mu l). L esser amounts of alpha 2-M, alpha 1-Achy and alp1 (similar to<1-3 ng/m u l) are also evident. C fluid is associated with very high levels of PMN cell proteases along with a similar to 5-2O-fold increase in the c oncentrations of all of the above inhibitors. Trace levels of elafin w ere also detected. The concentrations of rapid reacting inhibitors exc eeded that of proteases, with SLPI, alpha 1-Achy and alp1 being the pr incipal functional entities. In atypical samples, complexes of elastas e and alpha 2-M were also encountered. Conclusions. SLPI, a known anti microbial agent and an elastase and cathepsin G inhibitor, is the prin cipal serpin in R fluid. C fluid is associated with a marked increase in the concentrations of an array of rapid reacting serpins capable of inhibiting all known PMN cell serine proteases. In the normal closed eye, the concentration of rapid reacting inhibitors always exceeds tha t of proteases with C fluid also containing a functional reserve of th e slow reacting inhibitor alpha 2-M.