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J. Xie, D. Stevenson, F.A. Yarber, S.F. Hamm–Alvarez, M.D. Trousdale; Transduced Viral Il–10 Is Exocytosed From Lacrimal Acinar Secretory Vesicles In Response To Carbachol . Invest. Ophthalmol. Vis. Sci. 2006;47(13):1964.
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© ARVO (1962-2015); The Authors (2016-present)
To determine the intracellular trafficking and release pathways of viral IL–10 (vIL–10) from transduced acinar epithelial cells from rabbit lacrimal gland.
Rabbit lacrimal gland acinar cells (LGAC) were isolated and maintained for 2 days in a laminin–based primary culture system. Reconstituted acini were transduced with an adenoviral vector carrying the Epstein Barr virus IL–10 gene (AdvIL–10) at a MOI of 5 for 24 hours. The distribution of vIL–10 and that of other compartment markers was assessed by confocal fluorescence microscopy using appropriate primary and fluorescent secondary antibodies. To determine how vIL–10 secretion from LGAC responded to secretagogue stimulation, basal and carbachol (CCH, 100 µM)–stimulated release of vIL–10 and the secretory product, ß–hexosaminidase, were measured from LGAC at time intervals from 0–180 minutes using ELISA and biochemical assays, respectively, and values normalized to pellet protein.
Confocal fluorescence microscopy revealed that vIL–10 immunoreactivity was detected in a punctate vesicular pattern in resting acini, accumulating primarily in large (0.5–1 µm) subapical vesicles although intracellular labeling could also be detected closer to the basolateral plasma membrane. Preliminary colocalization studies indicated that vIL–10 was partially co–localized with the trans–Golgi network marker, γ–adaptin, and was co–localized to a lesser extent with the Golgi marker, Rab6. vIL–10 was not co–localized with the dynein–associated cofactor, p150Glued. Secretion experiments showed that transduced LGAC secreted vIL–10 at a low but detectable rate in the absence of CCH; CCH stimulation significantly (p≤0.05, n=3) increased the secretory rate at all times measured (0–180 minutes). The curve appeared biphasic, with a robust and linear increase from 0–90 minutes, and tapering off from 90–180 minutes.
These studies suggest that vIL–10 transgene product is likely to be targeted to mature secretory vesicles localized beneath the apical membrane, which are released in response to CCH stimulation.
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