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. 2022 May 23;11(5):793.
doi: 10.3390/biology11050793.

Presence of Protease Inhibitor 9 and Granzyme B in Healthy and Pathological Human Corneas

Stanislava Reinstein Merjava  1 , Jan Kossl  2 , Ales Neuwirth  3 , Pavlina Skalicka  4 , Zuzana Hlinomazova  5 , Vladimir Holan  2 , Katerina Jirsova  1
Affiliations

Presence of Protease Inhibitor 9 and Granzyme B in Healthy and Pathological Human Corneas

Stanislava Reinstein Merjava et al. Biology (Basel). .

Abstract

The aim of this study was to find out whether protease inhibitor 9 (PI-9) and granzyme B (GrB) molecules that contribute to immune response and the immunological privilege of various tissues are expressed in healthy and pathological human corneas. Using cryosections, cell imprints of control corneoscleral discs, we showed that PI-9 was expressed particularly in the endothelium, the superficial and suprabasal epithelium of healthy corneas, limbus, and conjunctiva. GrB was localized in healthy corneal and conjunctival epithelium, while the endothelium showed weak immunostaining. The expression of PI-6 and GrB was confirmed by qRT-PCR. Increased expression levels of the PI-9 and GrB genes were determined when the corneas were cultured with proinflammatory cytokines. Fluorescent and enzymatic immunohistochemistry of pathological corneal explants (corneal melting and herpes virus keratitis) showed pronounced PI-9, GrB, human leucocyte antigen (HLA)-DR, and leukocyte-common antigen (CD45) signals localized in multicellular stromal infiltrates and inflammatory cells scattered in the corneal stroma. We conclude that increased expression of the PI-9 and GrB proteins under pathological conditions and their upregulation in an inflammatory environment indicate their participation in immune response of the cornea during the inflammatory process.

Keywords: cornea; corneal endothelium; corneal epithelium; granzyme B; protease inhibitor 9.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Detection of PI-9 and GrB in the control human cornea, limbus, and conjunctiva. (A) Enzymatic immunohistochemical detection of PI-9 and GrB in control human cornea (Ep: epithelium and En: endothelium), limbus, and conjunctiva. Human tonsil and testes were used as positive controls for PI-9 and GrB, respectively. (B) Detection of PI-9 and GrB in the control human cornea using indirect fluorescent immunocytochemistry. Corneal endothelium, epithelium, and conjunctival epithelium show positive intracytoplasmic dots. Nuclei were stained with propidium iodide. Scale bars = 10 μm.
Figure 2
Figure 2
Expression of the PI-9 and GrB genes in human corneas. (A) PI-9 and GrB gene expression in the corneal epithelium, endothelium, and conjunctiva. (B) Enhanced PI-9 and GrB gene expression in corneas cultured in medium (control) or in medium with proinflammatory cytokines (Stimul.). Bars represent ± SD from five individual corneas. * p ≤ 0.05 and *** p ≤ 0.001 indicate statistical significance as compared with the control.
Figure 3
Figure 3
Enzymatic immunohistochemical detection of PI-9, GrB, HLA-DR, CD45, and CD34 in melted corneas (M3 and M5) and in the cornea with herpes virus keratitis (K1). Some examples of infiltrates are indicated by asterisks. (*). Scale bar = 50 μm.
See this image and copyright information in PMC

References

    1. Clarke E.P., Cates G.A., Ball E.H., Sanwal B.D. A collagen-binding protein in the endoplasmic reticulum of myoblasts exhibits relationship with serine protease inhibitors. J. Biol. Chem. 1991;266:17230–17235. doi: 10.1016/S0021-9258(19)47363-8. - DOI - PubMed
    1. Gettins P.G. Serpin structure, mechanism, and function. Chem. Rev. 2002;102:4751–4804. doi: 10.1021/cr010170+. - DOI - PubMed
    1. Maas C., de Maat S. Therapeutic SERPINs: Improving on Nature. Front. Cardiovasc. Med. 2021;8:648349. doi: 10.3389/fcvm.2021.648349. - DOI - PMC - PubMed
    1. Bird C.H., Blink E.J., Hirst C.E., Buzza M.S., Steele P.M., Sun J., Jans D.A., Bird P.I. Nucleocytoplasmic distribution of the ovalbumin serpin PI-9 requires a nonconventional nuclear import pathway and the export factor Crm1. Mol. Cell Biol. 2001;21:5396–5407. doi: 10.1128/MCB.21.16.5396-5407.2001. - DOI - PMC - PubMed
    1. Sun J., Bird C.H., Sutton V., McDonald L., Coughlin P.B., De Jong T.A., Trapani J.A., Bird P.I. A cytosolic granzyme B inhibitor related to the viral apoptotic regulator cytokine response modifier A is present in cytotoxic lymphocytes. J. Biol. Chem. 1996;271:27802–27809. doi: 10.1074/jbc.271.44.27802. - DOI - PubMed