POLYMORPHONUCLEAR LEUKOCYTE CELLS AND ELASTASE IN TEARS

Purpose. To characterize the effects that mode of sampling and overnig ht eye closure have on the nature of caseinolytic activity recovered i n tear fluid. Methods. Reflex, open smd closed (R, O and C) eye tear f luids were collected by microcapillary tubes or from the inferior forn ix by Schirmer strip. Microcapillary collected samples were centrifuge d and recovered cells cytochemically characterized and probed by immun ofluorescence microscopy, or alternatively extracted in acidic PBS, Te ar supernatants, pellets and Schirmer strip extracts were subjected to casein zymography or SDS-PAGE and immunoprobed for plasmin/plasminoge n. To identify caseinolytic activity samples were immunoprecipitated w ith antibodies to plasmin/plasminogen or to elastase, and the immunopr ecipitated materials were subjected to zymographic analysis. Results. Immunoblot assays revealed R and O samples contained low levels of pla sminogen (similar to 1.1 mu g/ml) and only trace levels of plasmin (<0 .1 ng/ml), Insufficient levels of caseinolytic activity were present t o allow zymographic detection. Cytochemical analysis revealed that R a nd O pellets consisted almost exclusively of desquamated epithelium. I mmunoblot analysis revealed that C fluid was associated with an increa se in plasminogen and its partial conversion to plasmin (similar to 3. 2 ng/mu l), high molecular weight covalent complexes and degradative p roducts. Zymographic analysis disclosed much greater caseinolytic acti vity than could be attributed to plasmin or its cleavage products. Thi s consisted primarily of three bands (30-26 kDa) which were identified as polymorphonuclear leukocyte (PMN) cell elastase based on size and antigenicity, This is derived from PMNs recovered from the C pellet. E lastase could also be recovered from Schirmer strips from 90% of donor s, provided that the strips were extracted in sample loading buffer. T he activity was restricted to the portion of the strip that had been i n contact with the ocular tissue. Conclusions. The main source of case inolytic activity in C fluid is elastase. This arises from PMNs that u ndergo recruitment, activation and degranulation in the C environment. In contrast, the elastase recovered in Schiner strip extracts is deri ved from intact PMNs that adhere to the strip during sample collection . This would suggest char PMN cells undergo a low level of recruitment into the open eye environment.