Biology and Biomedical Sciences: Immunology
Date of Award
Doctor of Philosophy (PhD)
Chair and Committee
The complement system is an essential branch of the innate immune system and acts as a bridge to the adaptive immune system. It serves as the first line of defense against pathogens, as well as in the clearance of immune complexes and apoptotic cells. Deficiencies in many of the complement components lead to an increase in bacterial infections while others predispose to autoimmune conditions, especially systemic lupus erythematosus: SLE). Regulation of the complement system is important for protection of the host tissues against inappropriate activation. In the absence of appropriate regulation, commonly due to mutations in regulatory proteins, complement can attack self-cells. This thesis work will examine two situations in which the central component of complement, C3, is involved in a human disease state. In the first case, there is a loss of appropriate regulation of C3 and in the second an autoantibody to the C3 convertase leads to a secondary C3 deficiency. Atypical hemolytic uremic syndrome: aHUS) is a thrombotic microangiopathy that primarily affects the kidneys. This disease is characterized by microangiopathic hemolytic anemia, thrombocytopenia, and acute renal failure. Heterozygous mutations in the regulatory proteins Factor H, CD46 and Factor I have been shown to predispose to the disease. This is due to a lack of proper control of complement activation on endothelial cells in the renal microvasculature. The first goal of this thesis work was to characterize the functional consequences of heterozygous mutations in C3 that have been identified in patients with aHUS. These mutant C3 proteins were expressed recombinantly in mammalian cells and characterized for functional changes. In ELISA binding studies, fluid phase cofactor assays and surface plasmon resonance, the majority of these proteins: 11/17) had a defect in their interaction with CD46 or Factor H, both critical C3 regulatory proteins. Additionally, one mutation, R139W, led to increased binding of Factor B, resulting in formation of an inappropriately stabilized alternative pathway C3 convertase and, secondarily, to an undesirable increase in C3 activation. Complete C3 deficiency predisposes to multiple pyogenic bacterial pathogens and was lethal in early childhood prior to the advent of antibiotics. Additionally, C3 deficiency impairs the antibody response to vaccinations and causes a defect in dendritic cell maturation. In the second part of this thesis, I will describe a case of C3 deficiency in an 18 year-old patient who presented with a life-threatening Neisseria meningitides infection. He had a zero whole complement titer: CH50) and C3 antigen was not detected in the standard hospital laboratory tests. Sequencing of his C3 DNA did not reveal any mutations or truncations. Further analysis of his serum demonstrated quite low levels of C3 degradation products. Mixing the patient's serum with normal human serum led to an accelerated C3 turnover followed by the expected degradation of the C3 activation products. In hemolytic assays performed to assess the decay of the classical pathway C3 convertase, his serum stabilized the convertase, preventing its decay. Finally, it was determined that the factor stabilizing the C3 convertase was in the IgG fraction of the patient's serum. This factor, presumably an acquired autoantibody to the convertase, appears analogous to a previously reported C4 nephritic factor observed in a few patients with glomerulonephritis. However, this patient's presentation is unique and two years later he does not have renal disease. The studies presented here establish that mutations in C3 predispose to the development of aHUS due to inadequately regulated activation of the complement cascade. These data are the first described example of C3 mutations leading to this disease. The second part of this work focuses on a novel case in which stabilization of the classical pathway C3 convertase by an autoantibody led to complete loss of functionally intact C3. Together, this work emphasizes the requirement for precise regulation of complement activation at the key C3 step.
Miller, Elizabeth, "Characterization of Complement C3 Dysregulation Predisposing to Two Human Disease States" (2012). All Theses and Dissertations (ETDs). Paper 719.