Ligation of Human CD46 with Purified Complement C3b or F(ab)of Monoclonal Antibodies Enhances Isoform-Specific Interferon Gamma-Dependent Nitric Oxide Production in Macrophages
Department of Microbiology, University of Washington School of Medicine, Seattle, Washington, WA 98195, USA. Journal of Biochemistry
(Impact Factor: 2.58).
07/2002; 132(1):83-91. DOI: 10.1093/oxfordjournals.jbchem.a003203
CD46, a complement regulatory protein widely expressed on human cells, serves as an entry receptor for measles virus (MV). We have previously shown that the expression of human CD46 in mouse macrophages restricts MV replication in these cells and enhances the production of nitric oxide (NO) in the presence of gamma interferon (IFN-gamma). In this study, we show that crosslinking human CD46 expressed on the mouse macrophage-like cell line RAW264.7 with purified C3b multimer but not monomer enhances NO production. The enhanced production of NO in response to IFN-gamma was observed again with C3b multimer but not monomer. The augmentation of NO production is human CD46-dependent with a CYT1>CYT2 profile. Thus, the reported MV-mediated NO production, irrespective of whether it is IFN-gamma-dependent or -independent, should be largely attributable to CD46 signaling but not to MV replication. Similar CYT1-dependent augmentation of NO production was reproducible with two CD46 ligating reagents, CD46-specific monoclonal antibodies (mAb) or their F(ab')(2) and MV hemagglutinin (H) and fusion (F) glycoproteins. Co-cultivation of mouse macrophages bearing human CD46 with Chinese hamster ovary (CHO) cells expressing MV H and F enhanced IFN-gamma-induced NO production. Yet, the NO levels induced by F(ab')(2) against CD46 or MV H/F on CHO cells were much lower than those induced by CD46-crosslinking mAb with Fc or MV infection. Removing the cytoplasmic tails of CD46 abrogated the augmentation of NO production triggered by all three stimulators. Thus, the CD46 CYT1 and CYT2 isoforms functionally diverge to elicit innate immune responses, which can be modulated by purified C3b multimer or anti-CD46 mAbs.
Available from: Tsukasa Seya
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ABSTRACT: Mouse cells ubiquitously express CRRY, which is a functional orthologue of human decay-accelerating factor (DAF; CD55) and membrane cofactor protein (MCP; CD46), and thus protects cells from homologous complement. NIH3T3 cells expressed minute levels of mouse CD46 (mCD46) mRNA but barely produced mCD46 protein. mCD46 message and protein levels were markedly increased during mouse cytomegalovirus (mCMV) infection. Consistently, mCD46-expressing cells became resistant to mouse complement; primary-cultured fibroblasts from mCD46 gene-disrupted mice showed no increase in protection, resulting in complement-dependent cytolysis. Thus, the marked up-regulation of mCD46 in mouse fibroblast cells/cell lines by mCMV infection participates in host cell protection from complement. By mCD46 promoter deletion assay, the region necessary for induction of the promoter activity by mCMV infection was shown to be restricted to a sequence of 19 bp, which was homologous to the corresponding portion in human CD46, and the promoter regions of early-inducible human CMV UL36 and human herpesvirus 6 UL29. The results were confirmed by mutation analysis of this 19-bp region. We designated this sequence as the CMV-responsive element (CMVRE). Electrophoretic mobility shift assay demonstrated the existence of a CMVRE-binding factor, expression of which was significantly increased after mCMV infection. Thus, mCMV up-regulates the gene expression of mCD46 via CMVRE and CMVRE-binding factor, resulting in mCD46 protein expression on mCMV-infected cells. Since both the membrane and soluble mCD46retained complement regulatory activity, mCD46 induced by mCMV infection may act as a regulator of systemic complement activation. This represents a unique strategy of mCMV survival in host cells with sufficient replication by circumventing host complement attack.
European Journal of Immunology 10/2002; 32(10):2954-64. DOI:10.1002/1521-4141(2002010)32:10<2954::AID-IMMU2954>3.0.CO;2-2 · 4.03 Impact Factor
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ABSTRACT: Lipopolysaccharide (LPS) is an agonist for Toll-like receptor (TLR) 4 and expresses many genes including NF-kappaB- and interferon regulatory factor (IRF)-3/IFN-inducible genes in macrophages and dendritic cells (DCs). TICAM-1/TRIF was identified as an adapter that facilitates activation of IRF-3 followed by expression of interferon (IFN)-beta genes in TLR3 signaling, but TICAM-1 does not directly bind TLR4. Although MyD88 and Mal/TIRAP adapters functions downstream of TLR4, DC maturation and IFN-beta induction are independent of MyD88 and Mal/TIRAP. In this investigation, we report the identification of a novel adapter, TICAM-2, that physically bridges TLR4 and TICAM-1 and functionally transmits LPS-TLR4 signaling to TICAM-1, which in turn activates IRF-3. In its structural features, TICAM-2 resembled Mal/TIRAP, an adapter that links TLR2/4 and MyD88. However, TICAM-2 per se exhibited minimal ability to activate NF-kappaB and the IFN-beta promoter. Hence, in LPS signaling TLR4 recruits two types of adapters, TIRAP and TICAM-2, to its cytoplasmic domain that are indirectly connected to two effective adapters, MyD88 and TICAM-1, respectively. We conclude that for LPS-TLR4-mediated activation of IFN-beta, the adapter complex of TICAM-2 and TICAM-1 plays a crucial role. This results in the construction of MyD88-dependent and -independent pathways separately downstream of the two distinct adapters.
Journal of Biological Chemistry 01/2004; 278(50):49751-62. DOI:10.1074/jbc.M305820200 · 4.57 Impact Factor
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ABSTRACT: During the 1980s CD46 was discovered in a search for C3b binding proteins of human peripheral blood cells. Its role as an inactivator of C3b and C4b deposited on self-tissue is highlighted by the observation that partial deficiency of CD46 is a predisposing factor to hemolytic uremic syndrome. This discovery has an impact on the treatment options for these patients. Other new findings have expanded the role of CD46 in immunity and disease. For example, signaling through CD46 on human T lymphocytes drives them to become regulatory cells, indicating a novel link between the complement system and cellular immunity. Also, CD46 interacts with at least seven human pathogens and participates in reproduction/fertilization, further suggesting that dissecting its multi-faceted activities will have important clinical implications.
Trends in Immunology 10/2004; 25(9):496-503. DOI:10.1016/j.it.2004.07.004 · 10.40 Impact Factor
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