Is the cystatin-like domain of TSL functionally active in external ocular infections and during the normal diurnal cycle?
OBJECTIVE
To test whether the cystatin-like functional domain in tear specific lipocalin (TSL) is functionally active in tears during the normal diurnal cycle and during external ocular infections.


METHODS
Capillary tube collected reflex (RTF), open (OTF) and closed (CTF) eye tear samples were recovered from six normals and semi-quantitatively western blot assayed for cystatin C and TSL. CTF samples were immunoprecipitated with antibodies raised against TSL, cystatin C and other antiproteases and screened for the co-precipitation of proteases by casein and gelatin zymography. OTF samples recovered from individuals with viral, fungal and bacterial keratitis were similarly screened for TSL-bound proteases. Human tissue was subjected to immunohistochemical study.


RESULTS
Western blot analysis reveals a progressive increase in cystatin C in going from RTF to OTF to CTF samples (approximately 3, 7 and 30 ng microl(-1), respectively). In contrast, the concentration of TSL remains constant (approximately 1500 ng microl(-1)). Immunocytochemistry data show staining of the apical surface of the human conjunctiva and some intra-cellular staining for cystatin C, but not for cystatin A. Zymography confirms earlier data that CTF contains exceptionally high levels of proteases bound to a wide range of specific inhibitors. However, only trace amounts of proteases are complexed with cystatin C and no protease can be detected bound to TSL in either the pathological or CTF samples.


CONCLUSIONS
Although TSL contains a functional cystatin-like domain, it is not physiologically active during the normal diurnal cycle or during external ocular infections. Reactive proteases in CTF are most likely controlled by the presence of excess levels of more reactive cystatins, especially cystatin C, which accumulates during prolonged eye closure. Immunohistochemical data suggest that the apical conjunctiva may be a contributing source for the accumulating cystatin C.
Sack
Robert A
RA
State University of New York State College of Optometry, 33 West 42nd Street at Bryant Park, Manhattan, NY 10036, USA. rsack@suny.opt.edu
Sathe
Sonal
S
Beaton
Ann
A
Kozinski
Margaret
M
Bogart
Bruce
B
Lew
George
G
Sharma
Savitri
S
Upponi
Anjali
A
D002352:Carrier Proteins / Q000502:physiology*; D002940:Circadian Rhythm / Q000502:physiology; D055316:Cystatin C; D015891:Cystatins / Q000502:physiology*; D015817:Eye Infections / Q000378:metabolism*; D005136:Eye Proteins / Q000502:physiology; D019084:Fluorescent Antibody Technique, Indirect; D006801:Humans; D007634:Keratitis / Q000378:metabolism; D054835:Lipocalin 1; D013666:Tears / Q000378:metabolism*
C525030:CST3 protein, human; D002352:Carrier Proteins; D055316:Cystatin C; D015891:Cystatins; D005136:Eye Proteins; C518443:LCN1 protein, human; D054835:Lipocalin 1