Identification, origins and the diurnal role of the principal serine protease inhibitors in human tear fluid.
OBJECTIVE
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 (alpha 1-Achy), secretory leukocyte protease inhibitor (SLPI), elafin and alpha 2-macroglobulin (alpha 2-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 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/microliter). Lesser amounts of alpha 2-M, alpha 1-Achy and alp1 (approximately < 1-3 ng/microliter) are also evident. C fluid is associated with very high levels of PMN cell proteases along with a approximately 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, alpha 1-Achy and alp1 being the principal functional entities. In atypical samples, complexes of elastase and alpha 2-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 alpha 2-M.
Sathe
S
S
SUNY, College of Optometry, NY, USA.
Sakata
M
M
Beaton
A R
AR
Sack
R A
RA
D015153:Blotting, Western; D056649:Cathepsin G; D002403:Cathepsins / Q000037:antagonists & inhibitors / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D055316:Cystatin C; D015891:Cystatins / Q000032:analysis; D015853:Cysteine Proteinase Inhibitors / Q000032:analysis; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D053146:Myeloblastin; D009504:Neutrophils / Q000201:enzymology; D010196:Pancreatic Elastase / Q000037:antagonists & inhibitors / Q000378:metabolism; D053491:Proteinase Inhibitory Proteins, Secretory; D011506:Proteins / Q000378:metabolism; D053481:Secretory Leukocyte Peptidase Inhibitor; D012697:Serine Endopeptidases / Q000378:metabolism; D015842:Serine Proteinase Inhibitors / Q000032:analysis* / Q000502:physiology; D015843:Serpins / Q000032:analysis* / Q000502:physiology; D013666:Tears / Q000737:chemistry*; D000514:alpha 1-Antichymotrypsin / Q000378:metabolism; D000511:alpha-Macroglobulins / Q000378:metabolism
C525030:CST3 protein, human; D055316:Cystatin C; D015891:Cystatins; D015853:Cysteine Proteinase Inhibitors; D053491:Proteinase Inhibitory Proteins, Secretory; D011506:Proteins; C506814:SLPI protein, human; D053481:Secretory Leukocyte Peptidase Inhibitor; D015842:Serine Proteinase Inhibitors; D015843:Serpins; D000514:alpha 1-Antichymotrypsin; D000511:alpha-Macroglobulins; D002403:Cathepsins; D012697:Serine Endopeptidases; C534094:CTSG protein, human; D056649:Cathepsin G; D010196:Pancreatic Elastase; D053146:Myeloblastin