In vivo imaging of complement activation in mouse choroidal neovascularization using a novel monocolonal antibody against the C3 activation fragment C3d

Abstract

Purpose: Uncontrolled activation of the alternative complement pathway is thought to be associated with age-related macular degeneration. During activation of the complement cascade complement C3 protein is hydrolyzed resulting in the generation and fixation of C3 activation fragments on affected tissues. Previously, we have shown that in mouse laser-induced choroidal neovascularization (CNV), C3 fragments are present in the CNV lesions and can be used as addressable ligands for targeted therapeutics to reduce CNV. Here we examined the potential use of novel anti-C3d antibodies to detect complement activation in vivo in mouse CNV.

Methods: C3-deficient mice were immunized with human C3d protein. Monoclonal antibodies (mAb) were screened by ELISA, and their ability to bind to epitopes on C3d that are not present or exposed on full-length C3 was assayed by immunoprecipitation and Western blotting. Binding to tissue-bound C3 activation products was examined in CNV lesions in vitro (flatmounts of RPE/choroid) and in vivo (after tailvein injection of FITC-labeled mAb).

Results: (1) Three mAbs preferentially bound to the iC3b, C3dg, and C3d fragments, without binding to or interacting with C3 or C3b. (2) One of the mAbs (C3d29) identified tissue-bound C3 activation products in CNV lesions by immunohistochemistry in wildtype (WT) mice. (3) Staining was abolished or attenuated in CNV of fB-deficient mice or WT mice treated with an inhibitor of the complement alternative pathway. (4) FITC-C3d29, bound to CNV lesions could be imaged in vivo 24 hours after the injection by Micron III retinoscopy, when compared to a nonspecific FITC-labeled mAb.

Conclusions: Antibodies specific to tissue-bound C3 activation fragments can be used to visualize sites of complement activation. This technique may be employed in the future for diagnostic purposes and to monitor effects of therapeutic agents.

Keywords: 412 age-related macular degeneration • 551 imaging/image analysis: non-clinical • 557 inflammation