Characterization of a C3a Receptor in Rainbow Trout and Xenopus: The First Identification of C3a Receptors in Nonmammalian Species

Department of Pathobiology, University of Pennsylvania School of Veterinary Medicine, Philadelphia, PA 19104, USA.
The Journal of Immunology (Impact Factor: 5.36). 09/2005; 175(4):2427-37. DOI: 10.4049/jimmunol.175.4.2427
Source: PubMed

ABSTRACT Virtually nothing is known about the structure, function, and evolutionary origins of the C3aR in nonmammalian species. Because C3aR and C5aR are thought to have arisen from the same common ancestor, the recent characterization of a C5aR in teleost fish implied the presence of a C3aR in this animal group. In this study we report the cloning of a trout cDNA encoding a 364-aa molecule (TC3aR) that shows a high degree of sequence homology and a strong phylogenetic relationship with mammalian C3aRs. Northern blotting demonstrated that TC3aR was expressed primarily in blood leukocytes. Flow cytometric analysis and immunofluorescence microscopy showed that Abs raised against TC3aR stained to a high degree all blood B lymphocytes and, to a lesser extent, all granulocytes. More importantly, these Abs inhibited trout C3a-mediated intracellular calcium mobilization in trout leukocytes. A fascinating structural feature of TC3aR is the lack of a significant portion of the second extracellular loop (ECL2). In all C3aR molecules characterized to date, the ECL2 is exceptionally large when compared with the same region of C5aR. However, the exact function of the extra portion of ECL2 is unknown. The lack of this segment in TC3aR suggests that the extra piece of ECL2 was not necessary for the interaction of the ancestral C3aR with its ligand. Our findings represent the first C3aR characterized in nonmammalian species and support the hypothesis that if C3aR and C5aR diverged from a common ancestor, this event occurred before the emergence of teleost fish.

Download full-text


Available from: Hani Boshra, Jul 01, 2015
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Interleukin-6 (IL-6) is a pleiotropic cytokine that regulates hematopoiesis, inflammation, immune responses and bone homeostasis in mammals. Fish IL-6 has been cloned in recent years but to date no functional studies have been reported. Thus, in this paper we present for the first time in fish the functional characterisation of IL-6, using rainbow trout (Oncorhynchus mykiss) as the fish model and with a focus on macrophage effects. Trout IL-6 (tIL-6) expression in macrophages could be induced by proinflammatory agents (LPS, polyI:C, and IL-1β) and recombinant tIL-6 (rtIL-6) rapidly induced STAT3 phosphorylation and expression of SOCS-1 to -3, CISH and IRF-1, as seen in mammals. However, three findings in this study suggest a novel role of tIL-6 in fish. Firstly, macrophage growth was enhanced by rtIL-6 in vitro, suggesting that IL-6 produced during inflammatory events may promote macrophage proliferation locally. Secondly, rtIL-6 induced the expression of cathelicidin-2, an antimicrobial peptide with immune-modulatory function, but down-regulated the expression of IL-1β and TNF-α, indicating a role of IL-6 in host defence and also in limiting inflammation. Thirdly, rtIL-6 induced the expression of hepcidin in macrophages. In mammals hepcidin is antimicrobial but also regulates iron homeostasis by inhibiting iron absorption, and its expression is induced by IL-6 only in hepatocytes but not macrophages. Thus, in fish if IL-6 is induced in patrolling macrophages during sepsis this may act to reduce iron availability by induction of hepcidin expression and lead to iron deficiency, as a means to limit the spread of infection.
    Molecular Immunology 06/2011; 48(15-16):1903-16. DOI:10.1016/j.molimm.2011.05.027 · 3.00 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: In mammals, the bioactive fragment C3a, released from C3 during complement activation, is a potent mediator of inflammatory reactions and exerts its functional activity through the specific binding to cell surface G protein-coupled seven-transmembrane receptors. Recently, we demonstrated a Ciona intestinalis C3a (CiC3a)-mediated chemotaxis of hemocytes in the deuterostome invertebrate Ciona intestinalis and suggested an important role for this molecule in inflammatory processes. In the present work, we have cloned and characterized the receptor molecule involved in the CiC3a-mediated chemotaxis and studied its expression profile. The sequence, encoding a 95,394 Da seven-transmembrane domain protein, shows the highest sequence homology with mammalian C3aRs. Northern blot analysis revealed that the CiC3aR is expressed abundantly in the heart and neural complex and to a lesser extent in the ovaries, hemocytes, and larvae. Three polyclonal Abs raised in rabbits against peptides corresponding to CiC3aR regions of the first and second extracellular loop and of the third intracellular loop react specifically in Western blotting with a single band of 98-102 kDa in hemocyte protein extracts. Immunostaining performed on circulating hemocytes with the three specific Abs revealed that CiC3aR is constitutively expressed only in hyaline and granular amoebocytes. In chemotaxis experiments, the Abs against the first and second extracellular loop inhibited directional migration of hemocytes toward the synthetic peptide reproducing the CiC3a C-terminal sequence, thus providing the compelling evidence that C. intestinalis expresses a functional C3aR homologous to the mammalian receptor. These findings further elucidate the evolutionary origin of the vertebrate complement-mediated proinflammatory process.
    The Journal of Immunology 10/2006; 177(6):4132-40. DOI:10.1016/j.molimm.2006.07.215 · 5.36 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: The recent accumulation of genomic information of many representative animals has made it possible to trace the evolution of the complement system based on the presence or absence of each complement gene in the analyzed genomes. Genome information from a few mammals, chicken, clawed frog, a few bony fish, sea squirt, fruit fly, nematoda and sea anemone indicate that bony fish and higher vertebrates share practically the same set of complement genes. This suggests that most of the gene duplications that played an essential role in establishing the mammalian complement system had occurred by the time of the teleost/mammalian divergence around 500 million years ago (MYA). Members of most complement gene families are also present in ascidians, although they do not show a one-to-one correspondence to their counterparts in higher vertebrates, indicating that the gene duplications of each gene family occurred independently in vertebrates and ascidians. The C3 and factor B genes, but probably not the other complement genes, are present in the genome of the cnidaria and some protostomes, indicating that the origin of the central part of the complement system was established more than 1,000 MYA.
    Immunogenetics 10/2006; 58(9):701-13. DOI:10.1007/s00251-006-0142-1 · 2.49 Impact Factor