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. 2001 Nov;85(11):1336–1340. doi: 10.1136/bjo.85.11.1336

Detection of galectin-3 in tear fluid at disease states and immunohistochemical and lectin histochemical analysis in human corneal and conjunctival epithelium

E Hrdlickova-Cela 1, J Plzak 1, K Smetana 1, Z Melkova 1, H Kaltner 1, M Filipec 1, F Liu 1, H Gabius 1
PMCID: PMC1723761  PMID: 11673302

Abstract

BACKGROUND/AIM—Components of the tear fluid contribute to the biochemical defence system of the eye. To reveal whether the immune mediator and lipopolysaccharide binding galectin-3 is present in tears, tear samples were collected from eyes in healthy and pathological states. Investigation of expression of galectin-3 and galectin-3 reactive glycoligands in normal human conjunctival and corneal epithelia was also initiated as a step to understand the role of galectin-3 in ocular surface pathology.
METHODS—Immunoblot analysis using either a rabbit polyclonal or a mouse monoclonal antibody against galectin-3 was employed to detect galectin-3 in tear fluid. Galectin-3 expression in tissue specimens was detected by immunocytochemistry employing A1D6 mouse monoclonal antibody, and galectin-3 reactive glycoligands were visualised by lectin histochemistry using labelled galectin-3.
RESULTS—Galectin-3 was found only in tears from patients with ocular surface disorders. It was expressed in normal corneal and conjunctival epithelia but not in lacrimal glands. Inflammatory leucocytes and goblet cells found in galectin-3 containing tear fluid also expressed galectin-3. Galectin-3 binding sites were detected on the surface of conjunctival and corneal epithelial cells co-localising with desmoglein.
CONCLUSIONS—This study revealed expression of galectin-3 in tear fluid obtained from patients with eye diseases. The role of this endogenous lectin (produced by inflammatory as well as epithelial cells) in antimicrobial action and inflammation modulation could be expected.



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Figure 1  .

Figure 1  

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Western blotting for detection of galectin-3 using polyclonal antibody. MW: molecular weight (demonstrated according to position in acrylamide gel, because it is not visible in western analysis). Lanes 1, 2: tear fluid samples from patients with sarcoidosis, lane 3: tear fluid sample from patient with corneal degeneration, lane 4: tear sample from patient with adenovirus conjunctivitis, lane M: extract from cultured human macrophage cell line producing galectin-3 used as a positive control. Only the tear samples from a few of the investigated patients are shown.

Figure 2  .

Figure 2  

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Positivity of inflammatory and goblet cells for galectin-3 detected by A1D6 antibody. Inflammatory cells in conjunctival stroma from a patient with Stevens-Johnson syndrome (A) expressed galectin-3 similarly to leucocytes (B) harvested from another patient with galectin-3 in tear fluid. The inflammatory cells infiltrating the cornea express the galectin-3 in cytoplasm in contrast with absence of galectin-3 in conjunctival epithelium. Immunofluorescence and immunoperoxidase detection of galectin-3, scale 50 µm (A) and 10 µm (B).

Figure 3  .

Figure 3  

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Galectin-3 expression in conjunctiva and cornea. Conjunctival (A) and corneal (B) areas are positive in the immunohistochemical analysis. The galectin-3 was expressed predominantly on the cell surface. However, the expression of galectin-3 in the superficial part of conjunctiva was non-uniform; some areas were galectin-3 negative. The surface of corneal and conjunctival epithelium is marked by arrows. Detection of galectin-3 using A1D6 antibody, scale 25 µm (A) and 50 µm (B).

Figure 4  .

Figure 4  

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Co-localisation of galectin-3 binding sites with desmoglein in conjunctiva (A) and cornea (B). The binding sites for galectin-3 (red signal) co-localised with expression of the desmosomal protein desmoglein (green signal). Desmoglein was detected by immunohistochemistry and galectin-3 reactive glycloligands by lectin histochemistry, scale 25 µm (A, B).

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Selected References

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