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J H Lass, E I Walter, T E Burris, H E Grossniklaus, M I Roat, D L Skelnik, L Needham, M Singer, M E Medof; Expression of two molecular forms of the complement decay-accelerating factor in the eye and lacrimal gland.. Invest. Ophthalmol. Vis. Sci. 1990;31(6):1136-1148.
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Complement is present in ocular fluids, but the molecular mechanism(s) restricting its activation to exogenous targets and not to autologous ocular cells are currently unknown. To clarify how this control is achieved, monoclonal antibody (mAb)-based techniques were used to examine the eye, the lacrimal gland, and ocular fluids for the decay-accelerating factor (DAF), a membrane regulatory protein which protects blood cells from autologous complement activation on their surfaces. Immunohistochemical staining of tissue sections revealed DAF antigen on corneal and conjunctival epithelia, corneal endothelium, trabecular meshwork, and retina, as well as on lacrimal gland acinar cells and in adjacent lumens. By flow cytometry, cultures of conjunctival epithelium exhibited the highest DAF levels and levels on corneal epithelium greater than corneal endothelium greater than conjunctival fibroblasts. Biosynthetic labeling of corneal endothelium yielded de novo DAF protein with an apparent molecular weight (Mr) of 75 kD, approximating that of blood cell DAF protein, and digestions of conjunctival epithelium with phosphatidylinositol-specific phospholipase C (PI-PLC), an enzyme which cleaves glycoinositolphospholipid membrane anchors, released approximately 70% of the ocular surface DAF protein similar to leukocyte surface DAF protein. Quantitations of DAF by radioimmunometric assay employing mAbs against two DAF epitopes revealed 325 ng/ml (n = 12), 4.8 ng/ml (n = 10), and 22.0 ng/ml (n = 8) of soluble DAF antigen in tears, aqueous humor, and vitreous humor, respectively. Western blot analyses of the tear DAF antigen revealed two DAF forms, one with an apparent Mr of 72 kD resembling membrane DAF forms in other sites, and a second with an apparent Mr of 100 kD, which is previously undescribed. Since DAF activity is essential physiologically in protecting blood cells from autologous complement attack, the identification of DAF on the ocular surface, intraocularly, in the lacrimal gland, and in tears suggests that DAF-mediated control of complement activation is also required in these locations.
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