Comparative studies of complement responses to ICs

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The complement systems of humans and laboratory animals (mice and rats) show striking species differences in function, particularly with regards to the classical pathway of complement activation. Indeed, studies since the 1950’s have documented markedly reduced classical pathway function in mice as compared to rats and humans. In preliminary data, we confirm and extend these findings using model immune complexes (ICs) formed by monoclonal antibodies to platelet factor 4 (PF4) and heparin (KKO ICs) or polyclonal rabbit anti-dinitrophenol (DNP) antibodies to DNP conjugated with keyhole limpet hemocyanin (DNP ICs). Specifically, we show: 1) robust C3 activation by KKO and DNP ICs in whole blood and plasma from mice, rats and humans 2) classical pathway requirements for complement activation by KKO ICs in rats and human, but alternative pathway requirements in mice 3) incorporation of alternative pathway proteins into KKO ICs in mice, but not rats or humans 4) comparable requirements for the alternative pathway among four murine strains and 5) differential expression of alternative pathway proteins by proteomics in mice as compared to rats and humans. Based on these preliminary data and published observations, we will test the overall hypothesis that impaired classical pathway activity in mice is counterbalanced by the alternative pathway C3b feedback cycle. To test this hypothesis, we propose the following two aims: 1) Comparative studies of human, mouse, and rat complement systems. In this aim, we will test that hypothesis that genetic variation in murine classical pathway proteins contributes to impaired classical pathway function. We will apply molecular approaches to compare genetic sequences and perform structural homologies of classical pathway proteins in mice v rats and humans. In other studies, we will investigate the plasma proteome of mice, rats, and humans to examine protein/immune complex interactions, co-expression of complement and complement-associated proteins and correlate complement protein levels with functional responses to KKO and DNP ICs. 2) Functional studies of murine and human complement systems. In this aim, we will test the hypothesis that mice utilize the C3b feedback cycle of the alternative pathway as a compensatory response to low classical pathway activity. To test this hypothesis, we will use purified human and mouse complement proteins to demonstrate reduced impaired classical C3 convertase activity and normal/heightened activity of the C3b feedback cycle in mice, examine the effects of interchanging human and mouse complement proteins, and identify potential murine inhibitors of classical pathway activation from mouse serum. Together, these studies are expected to delineate the molecular and functional basis of divergent complement pathway responses in mice and humans. We expect these studies to generate preliminary data for an expanded application to elucidate the molecular and structural divergence of murine complement responses with the long term goal of developing translationally relevant animal models.