ABSTRACT
Costimulatory signals are critical to T cell activation, but how their effects are mediated remains incompletely characterized. Here, we demonstrate that locally produced C5a and C3a anaphylatoxins interacting with their G protein-coupled receptors (GPCRs), C5aR and C3aR, on APCs and T cells both upstream and downstream of CD28 and CD40L signaling are integrally involved in T cell proliferation and differentiation. Disabling these interactions reduced MHC class II and costimulatory-molecule expression and dramatically diminished T cell responses. Importantly, impaired T cell activation by Cd80-/-Cd86-/- and Cd40-/- APCs was reconstituted by added C5a or C3a. C5aR and C3aR mediated their effects via PI-3 kinase-gamma-dependent AKT phosphorylation, providing a link between GPCR signaling, CD28 costimulation, and T cell survival. These local paracrine and autocrine interactions thus operate constitutively in naive T cells to maintain viability, and their amplification by cognate APC partners thus is critical to T cell costimulation.
Subject(s)
CD4-Positive T-Lymphocytes/immunology , Cell Survival/immunology , Complement C3a/immunology , Complement C5a/immunology , Lymphocyte Activation/immunology , Animals , Antigen Presentation/immunology , Antigen-Presenting Cells/immunology , B7-1 Antigen/immunology , B7-1 Antigen/metabolism , CD28 Antigens/immunology , CD28 Antigens/metabolism , CD4-Positive T-Lymphocytes/cytology , CD4-Positive T-Lymphocytes/metabolism , CD40 Antigens/immunology , CD40 Antigens/metabolism , CD40 Ligand/immunology , CD40 Ligand/metabolism , Cell Differentiation/immunology , Complement C3a/metabolism , Complement C5a/metabolism , Flow Cytometry , Immunoblotting , Immunoprecipitation , Mice , Mice, Transgenic , Polymerase Chain Reaction , RNA, Messenger/analysis , Receptor, Anaphylatoxin C5a/immunology , Receptor, Anaphylatoxin C5a/metabolism , Signal Transduction/immunologyABSTRACT
Decay-accelerating factor (Daf) dissociates C3/C5 convertases that assemble on host cells and thereby prevents complement activation on their surfaces. We demonstrate that during primary T cell activation, the absence of Daf on antigen-presenting cells (APCs) and on T cells enhances T cell proliferation and augments the induced frequency of effector cells. The effect is factor D- and, at least in part, C5-dependent, indicating that local alternative pathway activation is essential. We show that cognate T cell-APC interactions are accompanied by rapid production of alternative pathway components and down-regulation of Daf expression. The findings argue that local alternative pathway activation and surface Daf protein function respectively as a costimulator and a negative modulator of T cell immunity and explain previously reported observations linking complement to T cell function. The results could have broad therapeutic implications for disorders in which T cell immunity is important.
Subject(s)
CD55 Antigens/immunology , Cell Communication/immunology , Complement C3-C5 Convertases/immunology , Dendritic Cells/immunology , Lymphocyte Activation/immunology , T-Lymphocytes/immunology , Animals , Cell Proliferation , Complement System Proteins/immunology , Down-Regulation/immunology , Enzyme Activation/immunology , Female , Immunity, Cellular , Interleukin-6/immunology , Leukemia Inhibitory Factor , Male , Mice , Mice, Knockout , Signal Transduction/immunologyABSTRACT
Focused complement activation on foreign targets depends on regulatory proteins that decay the bimolecular C3 convertases. Although this process is central to complement control, how the convertases engage and disassemble is not established. The second and third complement control protein (CCP) modules of the cell surface regulator, decay-accelerating factor (DAF, CD55), comprise the simplest structure mediating this activity. Positioning the functional effects of 31 substitution mutants of DAF CCP2 to -4 on partial structures was previously reported. In light of the high resolution crystal structure of the DAF four-CCP functional region, we now reexamine the effects of these and 40 additional mutations. Moreover, we map six monoclonal antibody epitopes and overlap their effects with those of the amino acid substitutions. The data indicate that the interaction of DAF with the convertases is mediated predominantly by two patches approximately 13 A apart, one centered around Arg69 and Arg96 on CCP2 and the other around Phe148 and Leu171 on CCP3. These patches on the same face of the adjacent modules bracket an intermodular linker of critical length (16 A.) Although the key DAF residues in these patches are present or there are conservative substitutions in all other C3 convertase regulators that mediate decay acceleration and/or provide factor I-cofactor activity, the linker region is highly conserved only in the former. Intra-CCP regions also differ. Linker region comparisons suggest that the active CCPs of the decay accelerators are extended, whereas those of the cofactors are tilted. Intra-CCP comparisons suggest that the two classes of regulators bind different regions on their respective ligands.
Subject(s)
CD55 Antigens/chemistry , Complement C3-C5 Convertases/chemistry , Amino Acid Sequence , Animals , Binding Sites , CD55 Antigens/metabolism , Complement C3-C5 Convertases/metabolism , Crystallography, X-Ray , Epitopes/chemistry , Humans , Leucine/chemistry , Models, Molecular , Molecular Sequence Data , Mutation , Phenylalanine/chemistry , Protein Binding , Sequence Homology, Amino AcidABSTRACT
We describe the first complete mitochondrial genome sequence from a representative of the insect order Coleoptera, the flour beetle Tribolium castaneum. The 15,881 bp long Tribolium mitochondrial genome encodes 13 putative proteins, two ribosomal RNAs and 22 tRNAs canonical for animal mitochondrial genomes. Their arrangement is identical to that in Drosophila melanogaster, which is considered ancestral for insects and crustaceans (Boore et al., 1998; Hwang, et al., 2001a). Nucleotide composition, amino acid composition, and codon usage fall within the range of values observed in other insect mitochondrial genomes. Most notable features are the use of TCT as tRNA(Ser(AGN)) anticodon instead of GCT, which is used in most other arthropod species, and the relative scarcity of special sequence motifs in the 1431 bp long control region. Phylogenetic analysis confirmed resolving power in the conserved regions of the mitochondrial proteome regarding diversification events, which predate the emergence of pterygote insects, while little resolution was obtained at the level of basal perygote diversification. The partition of faster evolving amino acid sites harbored strong support for joining Lepidoptera with Diptera, which is consistent with a monophyletic Mecopterida.
Subject(s)
DNA, Mitochondrial/genetics , Phylogeny , Tribolium/classification , Tribolium/genetics , Animals , Base Composition , Base Sequence , Codon , Evolution, Molecular , Gene Order , Likelihood Functions , Molecular Sequence DataABSTRACT
The cleavage of C3 by the C3 convertases (C3bBb and C4b2a) determines whether complement activation proceeds. Dissociation (decay acceleration) of these central enzymes by the regulators decay-accelerating factor (DAF), complement receptor 1 (CR1), factor H, and C4-binding protein (C4BP) controls their function. In a previous investigation, we obtained evidence implicating the alpha4/5 region of the type A domain of Bb (especially Tyr338) in decay acceleration of C3bBb and proposed this site as a potential interaction point with DAF and long homologous repeat A of CR1. Because portions of only two DAF complement control protein domains (CCPs), CCP2 and CCP3, are necessary to mediate its decay of the CP C3 convertase (as opposed to portions of at least three CCPs in all other cases, e.g. CCPs 1-3 of CR1), DAF/C4b2a provides the simplest structural model for this reaction. Therefore, we examined the importance of the C2 alpha4/5 site on decay acceleration of C4b2a. Functional C4b2a complexes made with the C2 Y327A mutant, the C2 homolog to factor B Y338A, were highly resistant to DAF, C4BP, and long homologous repeat A of CR1, whereas C2 substitutions in two nearby residues (N324A and L328A) resulted in partial resistance. Our new findings indicate that the alpha4/5 region of C2a is critical to decay acceleration mediated by DAF, C4BP, and CR1 and suggest that decay acceleration of C4b2a and C3bBb requires interaction of the convertase alpha4/5 region with a CCP2/CCP3 site of DAF or structurally homologous sites of CR1 and C4BP.