Excessive complement activation can result in extreme tissue damage and systemic inflammatory responses, similar to innate immune responses rapidly elicited after systemic adenovirus (Ad) injections. To determine if Ad interactions with the complement system impact upon Ad-induced innate immune responses, we injected Ad into complement-deficient, C3-knockout mice (C3-KO) or wild-type mice (WT) and quantitatively compared multiple anti-Ad innate immune responses in both strains of mice. In Ad-treated WT mice, we noted rapid increases in plasma KC levels (1 h post injection), followed by increases in IL-6, IFN-gamma, RANTES, IL-12(p40), IL-5, G-CSF, and GM-CSF and subsequently thrombocytopenia. Conversely, in Ad-treated C3-KO mice, many of these inflammatory responses were significantly blunted, including the avoidance of Ad-induced thrombocytopenia. Global liver transcriptome responses in Ad-treated WT mice were assessed by RT-PCR-validated gene array analysis and were found to be also significantly affected by the lack of complement activity in Ad-treated C3-KO mice. Finally, our results confirmed the ability of high dose Ads to transduce hepatocytes despite a lack of complement activity. In summary, Ad interactions with the mammalian complement system are significant and likely initiate and/or exacerbate many of the inflammatory responses noted after systemic Ad injections.
We studied complement 1 inhibitor (C1-INH) as an inhibitor of the alternative complement pathway. C1-INH prevented lysis, induced by the alternative complement pathway, of paroxysmal nocturnal hemoglobinuria (PNH) erythrocytes in human serum. It inhibited the binding of both factors B and C3 to PNH and rabbit erythrocytes and blocked the ability of factor B to restore alternative-pathway function in factor B–depleted serum. C1-INH did not bind to factors B or D but did bind to immobilized C3b and cobra venom factor (CVF), a C3b analogue. C1-INH prevented factor B from binding to CVF-coated beads and dissociated bound factor B from such beads. Factor B and C1-INH showed cross competition in binding to CVF-coated beads. Factor D cleaved factor B into Bb and Ba in the presence of C3b. Cleavage was markedly inhibited when C3b was preincubated with C1-INH. C1-INH inhibited the formation of CVFBb and decreased the C3 cleavage. Removal of C1-INH from serum, in the presence of Mg-EGTA with an anti–C1-INH immunoabsorbant, markedly increased alternative-pathway lysis. C1-INH interacts with C3b to inhibit binding of factor B to C3b. At physiologic concentrations, it is a downregulator of the alternative pathway convertase.
Complement factor H (CFH) is an important regulatory protein in the alternative pathway of the complement system, and CFH polymorphisms increase the genetic risk of age-related macular degeneration dramatically. These same human CFH variants have also been associated with dense deposit disease. To mechanistically study the function of CFH in the pathogenesis of these diseases, we created transgenic mouse lines using human CFH bacterial artificial chromosomes expressing full-length human CFH variants and crossed these to Cfh knockout (Cfh(-/-)) mice. Human CFH protein inhibited cleavage of mouse complement component 3 and factor B in plasma and in retinal pigment epithelium/choroid/sclera, establishing that human CFH regulates activation of the mouse alternative pathway. One of the mouse lines, which express relatively higher levels of CFH, demonstrated functional and structural protection of the retina owing to the Cfh deletion. Impaired visual function, detected as a deficit in the scotopic electroretinographic response, was improved in this transgenic mouse line compared with Cfh(-/-) mice, and transgenics had a thicker outer nuclear layer and less sub-retinal pigment epithelium deposit accumulation. In addition, expression of human CFH also completely protected the mice from developing kidney abnormalities associated with loss of CFH. These humanized CFH mice present a valuable model for study of the molecular mechanisms of age-related macular degeneration and dense deposit disease and for testing therapeutic targets.
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