C4b-binding Protein Protects ß-Cells from Islet Amyloid Polypeptide-induced Cytotoxicity.

C4BP (C4b-binding protein) is a polymer of seven identical α chains and one unique ß chain synthesized in liver and pancreas. We showed previously that C4BP enhances islet amyloid polypeptide (IAPP) fibril formation in vitro Now we report that polymeric C4BP strongly inhibited lysis of human erythrocytes incubated with monomeric IAPP, whereas no lysis was observed after incubation with preformed IAPP fibrils. In contrast, incubation with the monomeric α-chain of C4BP was less effective. These data indicate that polymeric C4BP with multiple binding sites for IAPP neutralizes lytic activity of IAPP. Furthermore, addition of monomeric IAPP to a rat insulinoma cell line (INS-1) resulted in decreased cell viability, which was restored in the presence of physiological concentrations of C4BP. Treatment of INS-1 cells and primary rat islets with IAPP also diminished their ability to secrete insulin upon stimulation with glucose, which was reversed in the presence of C4BP. Further, C4BP was internalized together with IAPP into INS-1 cells. Pathway analyses of mRNA expression microarray data indicated that cells exposed to C4BP and IAPP in comparison with IAPP alone increased expression of genes involved in cholesterol synthesis. Depletion of cholesterol through methyl-ß-cyclodextrin or cholesterol oxidase abolished the protective effect of C4BP on IAPP cytotoxicity of INS-1 cells. Also, inhibition of phosphoinositide 3-kinase but not NF-κB had a similar effect. Taken together, C4BP protects ß-cells from IAPP cytotoxicity by modulating IAPP fibril formation extracellularly and also, after uptake by the cells, by enhancing cholesterol synthesis.

The complement inhibitor CD59 regulates insulin secretion by modulating exocytotic events.

Type 2 diabetes is triggered by reduced insulin production, caused by genetic and environmental factors such as inflammation originating from the innate immune system. Complement proteins are a component of innate immunity and kill non-self cells by perforating the plasma membrane, a reaction prevented by CD59. Human pancreatic islets express CD59 at very high levels. CD59 is primarily known as a plasma membrane protein in membrane rafts, but most CD59 protein in pancreatic ß cells is intracellular. Removing extracellular CD59 disrupts membrane rafts and moderately stimulates insulin secretion, whereas silencing intracellular CD59 markedly suppresses regulated secretion by exocytosis, as demonstrated by TIRF imaging. CD59 interacts with the exocytotic proteins VAMP2 and Syntaxin-1. CD59 expression is reduced by glucose and in rodent diabetes models but upregulated in human diabetic islets, potentially reflecting compensatory reactions. This unconventional action of CD59 broadens the established view of innate immunity in type 2 diabetes.

Heavy chains of inter alpha inhibitor (IαI) inhibit the human complement system at early stages of the cascade.

Inter alpha inhibitor (IαI) is an abundant serum protein consisting of three polypeptides: two heavy chains (HC1 and HC2) and bikunin, a broad-specificity Kunitz-type proteinase inhibitor. The complex is covalently held together by chondroitin sulfate but during inflammation IαI may interact with TNF-stimulated gene 6 protein (TSG-6), which supports transesterification of heavy chains to hyaluronan. Recently, IαI was shown to inhibit mouse complement in vivo and to protect from complement-mediated lung injury but the mechanism of such activity was not elucidated. Using human serum depleted from IαI, we found that IαI is not an essential human complement inhibitor as was reported for mice and that such serum has unaltered hemolytic activity. However, purified human IαI inhibited classical, lectin and alternative complement pathways in vitro when added in excess to human serum. The inhibitory activity was dependent on heavy chains but not bikunin and detected at the level of initiating molecules (MBL, properdin) in the lectin/alternative pathways or C4b in the classical pathway. Furthermore, IαI affected formation and assembly of the C1 complex and prevented assembly of the classical pathway C3-convertase. Presence and putative interactions with TSG-6 did not affect the ability of IαI to inhibit complement thus implicating IαI as a potentially important complement inhibitor once enriched onto hyaluronan moieties in the course of local inflammatory processes. In support of this, we found a correlation between IαI/HC-containing proteins and hemolytic activity of synovial fluid from patients suffering from rheumatoid arthritis.

Islet amyloid polypeptide triggers limited complement activation and binds complement inhibitor C4b-binding protein, which enhances fibril formation.

Islet amyloid polypeptide (IAPP) is synthesized in pancreatic ß-cells and co-secreted with insulin. Aggregation and formation of IAPP-amyloid play a critical role in ß-cell death in type 2 diabetic patients. Because Aß-fibrils in Alzheimer disease activate the complement system, we have here investigated specific interactions between IAPP and complement factors. IAPP fibrils triggered limited activation of complement in vitro, involving both the classical and the alternative pathways. Direct binding assays confirmed that IAPP fibrils interact with globular head domains of complement initiator C1q. Furthermore, IAPP also bound complement inhibitors factor H and C4b-binding protein (C4BP). Recombinant C4BP mutants were used to show that complement control protein (CCP) domains 8 and 2 of the α-chain were responsible for the strong, hydrophobic binding of C4BP to IAPP. Immunostaining of pancreatic sections from type 2 diabetic patients revealed the presence of complement factors in the islets and varying degree of co-localization between IAPP fibrils and C1q, C3d, as well as C4BP and factor H but not membrane attack complex. Furthermore, C4BP enhanced formation of IAPP fibrils in vitro. We conclude that C4BP binds to IAPP thereby limiting complement activation and may be enhancing formation of IAPP fibrils from cytotoxic oligomers.

The factor H variant associated with age-related macular degeneration (His-384) and the non-disease-associated form bind differentially to C-reactive protein, fibromodulin, DNA, and necrotic cells.

Recently, a polymorphism in the complement regulator factor H (FH) gene has been associated with age-related macular degeneration. When histidine instead of tyrosine is present at position 384 in the seventh complement control protein (CCP) domain of FH, the risk for age-related macular degeneration is increased. It was recently shown that these allotypic variants of FH, in the context of a recombinant construct corresponding to CCPs 6-8, recognize polyanionic structures differently, which may lead to altered regulation of the alternative pathway of complement. We show now that His-384, corresponding to the risk allele, binds C-reactive protein (CRP) poorly compared with the Tyr-384 form. We also found that C1q and phosphorylcholine do not compete with FH for binding to C-reactive protein. The interaction with extracellular matrix protein fibromodulin, which we now show to be mediated, at least in part, by CCP6-8 of FH, occurs via the polypeptide of fibromodulin and not through its glycosaminoglycan modifications. The Tyr-384 variant of FH bound fibromodulin better than the His-384 form. Furthermore, we find that CCP6-8 is able to interact with DNA and necrotic cells, but in contrast the His-384 allotype binds these ligands more strongly than the Tyr-384 variant. The variations in binding affinity of the two alleles indicate that complement activation and local inflammation in response to different targets will differ between His/His and Tyr/Tyr homozygotes.