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J. R. Sparrow, B. Westlund, Y. P. Jang, S. Kim, J. Zhou; Complement Activation by Photooxidation Products of A2E. Invest. Ophthalmol. Vis. Sci. 2007;48(13):2357.
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Studies implicate local inflammation and activation of complement amongst the processes involved in the pathogenesis of age-related macular degeneration (AMD). A question of importance pertains to the trigger(s) responsible for the complement-associated events. We previously reported that photooxidation products of A2E can activate complement (PNAS 103:16185, 2006). Here we test the participation of the alternative pathway.
We developed an in vitro assay using normal human serum (NHS) as a source of complement. Complement activation was monitored using enzyme immunoassay to measure the C3 split product iC3b in undiluted serum incubated with ARPE-19 cells that had accumulated A2E and were irradiated (430 nm) to generate photooxo-A2E products. Alternatively serum was incubated in wells precoated with A2E or two species of oxidized A2E, peroxy-A2E and furano-A2E. Serum incubated in empty wells served as background subtraction. iC3b generation in NHS was compared to that in factor B-depleted human serum.
iC3b levels were elevated in NHS overlying irradiated A2E-laden RPE as compared to cells irradiated in the absence of A2E, unirradiated A2E-laden RPE or serum irradiated in the absence of cells. iC3b was also increased in serum incubated with solid-form peroxy-A2E and furano-A2E but was not elevated in serum incubated with A2E. Substitution of NHS with factor B-depleted human sera containing 20 mM calcium and magnesium abrogated the increase in iC3b. The inhibition was reversed by the addition of factor B protein.
We suggest that cleavage products of photooxidized A2E redistribute inside and outside the cell with some of these fragments serving to activate complement. These events could predispose the macula to disease and, over time, contribute to the chronic inflammation considered to be associated with drusen formation. Our results also indicate that complement activation by this mechanism is dependent on the alternative pathway. Further studies will test for involvement of the classical pathway.
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