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Victoria E. McGilligan, Meredith S. Gregory-Ksander, Tara C. Moore, Johnny E. Moore, Robin R. Hodges, Darlene A. Dartt; Expression Of Nalp3 Inflammasome Components In Human And Rat Conjunctival Goblet Cells. Invest. Ophthalmol. Vis. Sci. 2011;52(14):4788. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
To investigate the expression of NALP3 inflammasome components in human and rat conjunctival goblet cells as the NALP3 inflammasome is a widely reported mediator of innate immunity in mucosal surfaces.
Conjunctiva was removed from sacrificed rats, fixed, and sectioned. Primary cultures of human and rat conjunctival goblet cells from conjunctval explants were grown and propagated in RPMI medium supplemented with 10% fetal bovine serum. Goblet cells were characterized by immunofluorescence microscopy for the goblet cell specific markers cytokeratin (CK)-7, Helix pomatia agglutinin (HPA), UEA-I and MUC5AC. Conjunctival sections and cultured goblet cells were analysed by immunofluorescence microscopy or western blotting for NALP3 and its constituent components, ASC, Caspase 1 and pro IL-1 beta. Cells and sections were also analysed for the presence of the bacterial receptors Toll Like Receptor (TLR) 2, TLR4 and CD14 and the purinergic receptors P2X4 and P2X7.
Human and rat conjunctival goblet cells were positive for CK-7, HPA, UEA1 and MUC5AC. Primary human and rat goblet cell cultures and rat conjunctiva from sections, constitutively expressed NALP3 with cytoplasmic and perinuclear staining observed by immunofluorescence microscopy. Two NALP3 protein bands were demonstrated by western blotting at 50kDa and 150kDa. Similar staining patterns and protein detection by western blotting were observed for ASC (24kDa), Caspase 1 (45kDa and 20kDa) and pro-IL-1 beta (31kDa), indicating constitutive expression of these inflammasome components in human and rat and conjunctival goblet cells. The presence of all three bacterial receptors TLR2 (100kDa), TLR4 (95kDa) and CD14 (55kDa) was confirmed as was the presence of the purinergic receptors P2X4 (50kDa) and P2X7 (75kDa). All receptors demonstrated punctate and perinuclear staining patterns and P2X4r was of filamentous appearance.
Multiple components of the NALP3 inflammasome as well as the receptors used by bacteria are present in rat and human goblet cells. These results suggest a potential role of the NALP3 inflammasome in goblet cell regulation of innate immunity in the conjunctiva.
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