129
Views
59
CrossRef citations to date
0
Altmetric
Original Article

Identification, origins and the diurnal role of the principal serine protease inhibitors in human tear fluid

, , &
Pages 348-362 | Received 02 Jun 1997, Accepted 22 Dec 1997, Published online: 02 Jul 2009
 

Abstract

Purpose. Previous work identified polymorphonuclear leukocyte (PMN) elastase as the major caseinolytic entity in tears collected after overnight eye closure. This study was designed to identify the principal serine protease inhibitors (serpins) in tears and to determine their function in the regulation of PMN cell proteases on eye closure.

Methods. Reflex and closed eye tear samples were collected by microcapillary tube and centrifuged. After reflex and closed eye supernatants (R and C) were fractionated by HPLC, samples were subjected to casein zymography 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 1-protease inhibitor (alp1), alpha 1-antichymotrypsin (α1-Achy), secretory leukocyte protease inhibitor (SLPI), elafin and alpha2-macroglobulin (α2-M) as well as associated complexes and products. To confirm specificity of reactivity, samples were immunoprecipitated for a given protease or serpin and screened for the co-precipitation 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/μl). Lesser amounts of α2-M, α1-Achy and alp1 (∼<1–3 ng/μl) are also evident. C fluid is associated with very high levels of PMN cell proteases along with a ∼5–20-fold increase in the concentrations of all of the above inhibitors. Trace levels of elafin were also detected. The concentrations of rapid reacting inhibitors exceeded that of proteases, with SLPI, α1-Achy and alp1 being the principal functional entities. In atypical samples, complexes of elastase and α2-M were also encountered.

Conclusions. SLPI, a known antimicrobial agent and an elastase and cathepsin G inhibitor, is the principal 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 that of proteases with C fluid also containing a functional reserve of the slow reacting inhibitor α2-M. Curr. Eye Res. 17: 348–362, 1998.

Reprints and Corporate Permissions

Please note: Selecting permissions does not provide access to the full text of the article, please see our help page How do I view content?

To request a reprint or corporate permissions for this article, please click on the relevant link below:

Academic Permissions

Please note: Selecting permissions does not provide access to the full text of the article, please see our help page How do I view content?

Obtain permissions instantly via Rightslink by clicking on the button below:

If you are unable to obtain permissions via Rightslink, please complete and submit this Permissions form. For more information, please visit our Permissions help page.