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.
- Publikační typ
- časopisecké články MeSH
The aim of this study was to determine if cytokeratins (CKs) 8 and 18--typical epithelial cell markers--are constitutively expressed in adult human corneal endothelium. Cryosections, paraffin-embedded sections and corneal endothelial imprints obtained from eleven adult human corneal discs not suitable for transplantation were used. Different fixative solutions were applied before indirect immunofluorescent or enzymatic staining was performed with antibodies against CK8 (Chemicon), CK18 (Dako and Sigma) and CK8/18 (Novocastra). Semi-quantitative RT-PCR and Western blotting (mRNA or proteins were isolated from Millicell membranes) were used to determine cytokeratin mRNA and protein levels. Approximately 50% of the corneal endothelial cells were positive for CK8 (Chemicon), CK18 (Sigma) and the CK pair 8/18 (Novocastra) in the endothelium when acetone was used for fixation. Four and 52% CK18-positive cells were observed using immunofluorescent and enzymatic immunohistochemistry, respectively, when the CK18 antibody from Dako was used. No signal was detected when 4% formalin or 10% paraformaldehyde was used as a fixative, irrespective of the antibody used. CK8 and CK18 proteins and mRNAs were detected in the endothelium of all tested corneas by Western blotting or semi-quantitative RT-PCR, respectively. We detected both CK8 and CK18 in the endothelium of all specimens at both the protein and mRNA levels. These results clearly demonstrate that cells of the corneal endothelium express CKs 8 and 18 and share some features with simple epithelia.
- MeSH
- exprese genu MeSH
- fluorescenční protilátková technika nepřímá metody MeSH
- keratin-18 genetika metabolismus MeSH
- keratin-8 genetika metabolismus MeSH
- lidé středního věku MeSH
- lidé MeSH
- messenger RNA genetika MeSH
- oční proteiny genetika metabolismus MeSH
- polymerázová řetězová reakce s reverzní transkripcí metody MeSH
- rohovkový endotel metabolismus MeSH
- senioři MeSH
- Check Tag
- lidé středního věku MeSH
- lidé MeSH
- senioři MeSH
- Publikační typ
- práce podpořená grantem MeSH
Posterior polymorphous corneal dystrophy (PPCD) is a hereditary bilateral disorder affecting primarily the endothelium and Descemet's membrane (DM). The aim of this study was to determine the changes in the presence and localization of the alpha1-alpha6 collagen IV chains and alpha1, alpha2 collagen VIII chains in Czech patients with PPCD. Twelve corneal buttons from ten PPCD patients who underwent corneal grafting, as well as eight unaffected corneas, were used. Enzymatic indirect immunohistochemistry was performed on cryosections using antibodies against the alpha1-alpha6 collagen IV chains and alpha1, alpha2 collagen VIII chains. The intensity of the signal was examined separately in the basal membrane of the epithelium (BME), stroma and DM. More than 50% of PPCD specimens exhibited positivity for alpha1 and alpha2 collagen IV chains in the BME and in the posterior stroma, while no staining was detected in these areas in control specimens. The signal for the alpha1 and alpha2 collagen IV chains was more intense in DM of PPCD corneas compared to controls and it was shifted from the stromal side (in control tissue) to the endothelial side of DM (in the patients). A less intensive signal in PPCD corneas for the alpha3 and alpha5 chains in DM and an accumulation of alpha3-alpha5 in the posterior stroma in diseased corneas were the only differences in staining for the alpha3-alpha6 collagen IV chains. The alpha1 collagen VIII chain was detected on both the endothelial and the stromal sides of DM in 90% of patients with PPCD, compared with the prevailing localization on the stromal side of DM in control corneas. A change in the localization of the alpha2 collagen VIII chain in DM from vertically striated features in control specimens to double line positivity in the DM of PPCD corneas and positive staining in the posterior collagenous layer of four patients were also detected. In three PPCD patients a fibrous pannus located under the BME, positive for alpha1-alpha3, alpha5 collagen IV chains and alpha1 collagen VIII chain, was observed. The increased expression of the alpha1, alpha2 collagen IV and alpha1 collagen VIII chains and the change in their localization in DM may contribute to the increased endothelial proliferative capacity observed in PPCD patients.
- MeSH
- bazální membrána metabolismus patologie MeSH
- Bowmanova membrána metabolismus patologie MeSH
- dědičné dystrofie rohovky metabolismus patologie MeSH
- dospělí MeSH
- imunoenzymatické techniky MeSH
- kolagen typ VIII metabolismus MeSH
- kolagen typu IV metabolismus MeSH
- lidé středního věku MeSH
- lidé MeSH
- mladiství MeSH
- mladý dospělý MeSH
- rohovka metabolismus MeSH
- senioři MeSH
- Check Tag
- dospělí MeSH
- lidé středního věku MeSH
- lidé MeSH
- mladiství MeSH
- mladý dospělý MeSH
- mužské pohlaví MeSH
- senioři MeSH
- ženské pohlaví MeSH
- Publikační typ
- práce podpořená grantem MeSH
Cieľ: Cieľom tejto práce bolo zistiť výskyt jednotlivých reťazcov kolagénu IV (?1, ?2, ?3, ?4, ?5 a ?6) v kontrolných rohovkách a v rohovkách pacientov so zadnou polymorfnou dystrofiou rohovky (ZPD). Metodika: Pre experimenty bolo použitých 7 kontrolných rohoviek a 7 rohovkových terčov získaných pri keratoplastike pacientov so ZPD. Jednotlivé reťazce kolagénu IV boli detekované v kryorezoch o hrúbke 7 Km pomocou nepriamej fluorescenčnej imunohistochémie. Výsledky: V kontrolných rohovkách sa v bazálnej membráne epitelu nachádzali reťazce ?3, ?5 a ?6, v strome reťazce ?3 a ?5. V Descemetovej membráne boli prítomné všetky reťazce kolagénu IV s výnimkou reťazca ?1, ktorý bol pozitívny len u jednej kontrolnej rohovky zo siedmych. V bunkách epitelu a endotelu kontrolných rohoviek nebol kolagén IV detekovaný. U rohoviek pacientov so ZPD sa v bazálnej membráne epitelu okrem reťazcov ?3, ?5 a ?6 vyskytoval aj reťazec ?2, v strome boli vedľa reťazcov ?3 a ?5 detekované i reťazce ?1, ?2 a ?4. V Descemetovej membráne rohoviek pacientov so ZPD boli vo zvýšenej (?1, ?2, ?3), zníženej (?4, ?6), alebo nezmenenej (?5) miere oproti kontrolám prítomné všetky reťazce kolagénu IV.V bunkách epitelu a endotelu rohoviek pacientov nebol kolagén IV detekovaný. Záver: Ľudská rohovka vykazuje značnú heterogenitu vo výskyte reťazcov ? kolagénu IV, a to predovšetkým v bazálnej membráne epitelu a v Descemetovej membráne, pre ktoré je prítomnosť kolagénu IV jednou z charakteristických čŕt. U pacientov so ZPD dochádza k zmenám expresie jednotlivých reťazcov kolagénu IV na úrovni Descemetovej membrány a stromy.
The expression of all six chains of collagen IV was studied using the indirect fluorescent immunohistochemistry in seven control corneas and seven corneas obtained from patients suffering from the posterior polymorphous corneal dystrophy. Heterogeneous staining, especially in the epithelial basement membrane and Descemet's membrane, was observed in the control corneas. An increase of the staining intensity for the ?1 and ?2 chains was observed, especially in the Descemet's membrane and the corneal stroma in samples obtained from the patients compared to the control tissues.
