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1.
Curr Biol ; 22(24): 2375-82, 2012 Dec 18.
Article in English | MEDLINE | ID: mdl-23219724

ABSTRACT

Chemokines are essential in many cell migration processes, including the recruitment of leukocytes to sites of infection. In the latter context, chemokines promote leukocyte extravasation into the relevant tissue through a well-studied cascade of events. It is widely believed that chemokines further guide leukocytes within tissues via chemotaxis, the directed migration along gradients of soluble ligands. However, the basic mechanism of chemokine action within tissues has yet to be formally addressed in vivo. We identified a chemokine (zCxcl8) that recruits zebrafish neutrophils to infection loci and analyzed its function directly within interstitial tissues of living larvae. Using noninvasive imaging and a controlled cellular source of zCxcl8, we found that zCxcl8 guides neutrophils in a 2-fold manner: by biasing cell speed according to direction (orthotaxis) and by restricting cell motility near the source. We further show that zCxcl8 establishes tissue-bound gradients in vivo by binding to heparan sulfate proteoglycans (HSPGs). Inhibition of this interaction compromised both directional guidance and restriction of neutrophil motility. Thus, by interacting with extracellular HSPGs, chemokines establish robust surface-bound (haptotactic) gradients that mediate both recruitment and retention of leukocytes at sites of infection.


Subject(s)
Cell Movement , Chemokines/physiology , Inflammation Mediators/physiology , Leukocytes/cytology , Neutrophils/metabolism , Polysaccharides/metabolism , Humans
2.
Dev Comp Immunol ; 38(2): 243-53, 2012 Oct.
Article in English | MEDLINE | ID: mdl-22705555

ABSTRACT

We earlier identified two CXCL8-like lineages in cyprinid fish, which are functional homologues of the mammalian CXCL8, but with diverged functions. We here investigated whether the carp IFN-γ-inducible CXCb gene, related to the mammalian CXCL9, -10 and -11 chemokines, was subject to a similar diversification. On the zebrafish genome, a cluster of seven CXCb genes was found on chromosome five. Analysis of the promoter of the zebrafish CXCb genes suggests a partially shared, but differential induction. A second CXCb gene, CXCb2, was identified in common carp by homology cloning. CXCb2 is constitutively expressed in immune-related tissues, predominantly in head kidney lymphocytes/monocytes. Interestingly, an induction of CXCb2 gene expression with recombinant carp IFN-γ2 and LPS was observed in macrophages and granulocytes. Finally, difference in sensitivity to LPS, and kinetics of CXCb1 and CXCb2 gene expression during zymosan-induced peritonitis, was observed. These results indicate a functional diversification for cyprinid CXCb chemokines, with functional homology to mammalian CXCL9-11.


Subject(s)
Carps/immunology , Chemokines, CXC/genetics , Interferon-gamma/metabolism , Animals , Carps/metabolism , Cloning, Molecular , Evolution, Molecular , Interferon-gamma/immunology , Leukocytes/immunology , Leukocytes/metabolism , Lipopolysaccharides/metabolism , Peritoneum/cytology , Peritoneum/immunology , Peritonitis/immunology , Promoter Regions, Genetic , Transcriptome , Zebrafish/genetics , Zebrafish/immunology , Zymosan
3.
Dev Comp Immunol ; 36(4): 741-50, 2012 Apr.
Article in English | MEDLINE | ID: mdl-22182503

ABSTRACT

Numerous CXC chemokines have been identified in fish, however, their role in inflammation is not well established. Here, CXC chemokines of the CXCL8-like (CXCa_L1 and CXCL8_L2) and CXCL9/10/11-like (CXCb) subset were investigated in carp. Recombinant CXCa_L1, CXCL8_L2 and CXCb all stimulated chemotaxis of macrophages and granulocytes in vitro. CXCb also attracted lymphocytes. Distinct effects on phagocyte activation were observed: the CXCL8-like chemokines increase respiratory burst activity, but not nitrite production. The three chemokines differentially induced a moderate increase in IL-1ß, CXCa_L1 and CXCL8_L2 gene expression. Intracellular calcium mobilization in granulocytes upon CXCa_L1 stimulation implies signal transduction through G-protein coupled CXC receptors. Notably, upon intraperitoneal administration, carp CXCL8-like chemokines strongly induced in vivo leukocyte recruitment, including neutrophils and monocytes/macrophages, in contrast to CXCb, for which the number of recruited leukocytes was low. The results indicate functional homology for carp CXCL8-like and CXCb chemokines with mammalian CXCL8 and CXCL9-11, respectively.


Subject(s)
Carps/immunology , Chemokines, CXC/immunology , Fish Proteins/immunology , Animals , Granulocytes/immunology , Head Kidney/cytology , Macrophages/immunology
4.
Dev Comp Immunol ; 36(2): 433-41, 2012 Feb.
Article in English | MEDLINE | ID: mdl-21907235

ABSTRACT

TRIM proteins have recently emerged as novel players in antiviral defense. TRIM proteins contain a tri-partite motif, composed of a RING zinc finger, one or two B-boxes and a coiled-coil domain. Many members of this large protein family of E3 ubiquitin ligases catalyze the attachment of ubiquitin to a substrate protein, an activity dependent on the RING domain. We earlier made a full description of the TRIM gene family in zebrafish and pufferfish and identified three multigene TRIM subsets, a feature unique to fish. To determine their biological role, we further characterized members of the finTRIM subset. FinTRIM gene expression was studied during development and in multiple tissues in adult rainbow trout. Upregulation of a large number of finTRIM upon viral stimulation suggests they are involved in antiviral immunity. We also demonstrate that two finTRIM members display E3 ubiquitin ligase activity, indicating that finTRIMs could regulate antiviral signaling through ubiquitination.


Subject(s)
Fish Proteins/metabolism , Novirhabdovirus/metabolism , Oncorhynchus mykiss/metabolism , Ubiquitin-Protein Ligases/metabolism , Animals , Cell Line , Cloning, Molecular/methods , Cluster Analysis , Fish Proteins/genetics , Fish Proteins/immunology , Gene Expression Regulation, Developmental , Gene Expression Regulation, Enzymologic , Immunohistochemistry/veterinary , Novirhabdovirus/immunology , Oncorhynchus mykiss/immunology , Oncorhynchus mykiss/virology , RNA/chemistry , RNA/genetics , Real-Time Polymerase Chain Reaction/veterinary , Reverse Transcriptase Polymerase Chain Reaction/veterinary , Ubiquitin-Protein Ligases/genetics , Ubiquitin-Protein Ligases/immunology , Ubiquitination
5.
PLoS One ; 6(7): e22022, 2011.
Article in English | MEDLINE | ID: mdl-21789205

ABSTRACT

Tripartite motif proteins (TRIM) constitute a large family of proteins containing a RING-Bbox-Coiled Coil motif followed by different C-terminal domains. Involved in ubiquitination, TRIM proteins participate in many cellular processes including antiviral immunity. The TRIM family is ancient and has been greatly diversified in vertebrates and especially in fish. We analyzed the complete sets of trim genes of the large zebrafish genome and of the compact pufferfish genome. Both contain three large multigene subsets--adding the hsl5/trim35-like genes (hltr) to the ftr and the btr that we previously described--all containing a B30.2 domain that evolved under positive selection. These subsets are conserved among teleosts. By contrast, most human trim genes of the other classes have only one or two orthologues in fish. Loss or gain of C-terminal exons generated proteins with different domain organizations; either by the deletion of the ancestral domain or, remarkably, by the acquisition of a new C-terminal domain. Our survey of fish trim genes in fish identifies subsets with different evolutionary dynamics. trims encoding RBCC-B30.2 proteins show the same evolutionary trends in fish and tetrapods: they evolve fast, often under positive selection, and they duplicate to create multigenic families. We could identify new combinations of domains, which epitomize how new trim classes appear by domain insertion or exon shuffling. Notably, we found that a cyclophilin-A domain replaces the B30.2 domain of a zebrafish fintrim gene, as reported in the macaque and owl monkey antiretroviral TRIM5α. Finally, trim genes encoding RBCC-B30.2 proteins are preferentially located in the vicinity of MHC or MHC gene paralogues, which suggests that such trim genes may have been part of the ancestral MHC.


Subject(s)
Evolution, Molecular , Fish Proteins/genetics , Tetraodontiformes/genetics , Zebrafish/genetics , Amino Acid Sequence , Animals , Conserved Sequence/genetics , Fish Proteins/chemistry , Fish Proteins/metabolism , Gene Duplication , Gene Expression Profiling , Gene Expression Regulation , Genome/genetics , Humans , Major Histocompatibility Complex/genetics , Molecular Sequence Data , Multigene Family/genetics , Phylogeny , Protein Structure, Tertiary , Selection, Genetic , Sequence Homology, Amino Acid , Synteny/genetics
6.
PLoS One ; 5(8): e12384, 2010 Aug 26.
Article in English | MEDLINE | ID: mdl-20865040

ABSTRACT

BACKGROUND: During the inflammatory process, chemokine CXCL8 plays a pivotal role in recruitment of human neutrophilic granulocytes. A diversity of sequences similar to CXCL8 was reported in fish, but their evolutionary relationships and functional homology with their human homolog remain unclear. PRINCIPAL FINDINGS: We screened fish genomes to seek for sequences related to CXCL8. A first lineage was retrieved in all teleosts, while a second CXCL8 lineage was found in zebrafish and carp only. An early inflammatory function for both lineages was indicated by several lines of evidence. The induction of carp CXCL8s, CXCb, and CXC receptor-1 and -2 was analyzed after in vitro stimulation of leukocyte subpopulations and in two in vivo inflammation models. Recombinant proteins of carp CXCL8 proteins were produced and showed significant chemotactic activity for carp leukocytes. CONCLUSIONS: While both carp CXCL8s appear to be functional homologs of mammalian CXCL8, their different induction requirements and kinetics evoke a gene-specific sub-functionalization.


Subject(s)
Carps/genetics , Fish Proteins/genetics , Fish Proteins/immunology , Gene Expression Profiling , Interleukin-8/genetics , Interleukin-8/immunology , Zebrafish/genetics , Amino Acid Sequence , Animals , Base Sequence , Carps/classification , Carps/immunology , Cells, Cultured , Fish Proteins/chemistry , Fishes/classification , Fishes/genetics , Fishes/immunology , Interleukin-8/chemistry , Leukocytes/immunology , Molecular Sequence Data , Phylogeny , Sequence Alignment , Zebrafish/immunology
7.
BMC Biol ; 7: 7, 2009 Feb 05.
Article in English | MEDLINE | ID: mdl-19196451

ABSTRACT

BACKGROUND: In mammals, the members of the tripartite motif (TRIM) protein family are involved in various cellular processes including innate immunity against viral infection. Viruses exert strong selective pressures on the defense system. Accordingly, antiviral TRIMs have diversified highly through gene expansion, positive selection and alternative splicing. Characterizing immune TRIMs in other vertebrates may enlighten their complex evolution. RESULTS: We describe here a large new subfamily of TRIMs in teleosts, called finTRIMs, identified in rainbow trout as virus-induced transcripts. FinTRIMs are formed of nearly identical RING/B-box regions and C-termini of variable length; the long variants include a B30.2 domain. The zebrafish genome harbors a striking diversity of finTRIMs, with 84 genes distributed in clusters on different chromosomes. A phylogenetic analysis revealed different subsets suggesting lineage-specific diversification events. Accordingly, the number of fintrim genes varies greatly among fish species. Conserved syntenies were observed only for the oldest fintrims. The closest mammalian relatives are trim16 and trim25, but they are not true orthologs. The B30.2 domain of zebrafish finTRIMs evolved under strong positive selection. The positions under positive selection are remarkably congruent in finTRIMs and in mammalian antiviral TRIM5alpha, concentrated within a viral recognition motif in mammals. The B30.2 domains most closely related to finTRIM are found among NOD-like receptors (NLR), indicating that the evolution of TRIMs and NLRs was intertwined by exon shuffling. CONCLUSION: The diversity, evolution, and features of finTRIMs suggest an important role in fish innate immunity; this would make them the first TRIMs involved in immunity identified outside mammals.


Subject(s)
Amino Acid Motifs/genetics , Gene Duplication , Genetic Variation/genetics , Multigene Family , Selection, Genetic , Amino Acid Sequence , Animals , COS Cells , Chlorocebus aethiops , Evolution, Molecular , Molecular Sequence Data , Oncorhynchus mykiss , Phylogeny , RING Finger Domains/genetics , Zebrafish
8.
J Endocrinol ; 193(3): 349-57, 2007 Jun.
Article in English | MEDLINE | ID: mdl-17535873

ABSTRACT

Corticotropin-releasing factor (CRF) plays a central role in the regulation of the stress axis. In mammals, CRF as well as its receptors and its CRF-binding protein (CRF-BP) are expressed in a variety of organs and tissues outside the central nervous system. One of these extrahypothalamic sites is the adrenal gland, where the paracrine actions of adrenal CRF influence cortical steroidogenesis and adrenal blood flow. Although the central role of CRF signaling in the initiation and regulation of the stress response has now been established throughout vertebrates, information about the possible peripheral presence of CRF in earlier vertebrate lineages is scant. We established the expression of CRF, CRF-BP, and the CRF receptor 1 in a panel of peripheral organs of common carp (Cyprinus carpio). Out of all the peripheral organs tested, CRF and CRF-BP are most abundantly expressed in the carp head kidney, the fish equivalent of the mammalian adrenal gland. This expression localizes to chromaffin cells. Furthermore, detectable quantities of CRF are released from the intact head kidney following in vitro stimulation with 8-bromo-cAMP in a superfusion setup. The presence of CRF and CRF-BP within the chromaffin compartment of the head kidney suggests that a pathway homologous to the mammalian intra-adrenal CRF system is present in the head kidney of fish. It follows that such a system to locally fine-tune the outcome of the centrally initiated stress response has been an integral part of the vertebrate endocrine system since the common ancestor of teleostean fishes and mammals.


Subject(s)
Adrenal Glands/chemistry , Biological Evolution , Carps/metabolism , Carrier Proteins/analysis , Corticotropin-Releasing Hormone/analysis , 8-Bromo Cyclic Adenosine Monophosphate/pharmacology , Adrenal Glands/drug effects , Adrenal Glands/metabolism , Animals , Base Sequence , Carrier Proteins/metabolism , Chromaffin Cells/chemistry , Chromaffin Cells/drug effects , Chromaffin Cells/metabolism , Corticotropin-Releasing Hormone/genetics , Corticotropin-Releasing Hormone/metabolism , DNA Primers/genetics , Gene Expression , Immunohistochemistry , Microscopy, Confocal , Molecular Sequence Data , Organ Culture Techniques , RNA, Messenger/analysis , Radioimmunoassay , Receptors, Corticotropin-Releasing Hormone/analysis , Receptors, Corticotropin-Releasing Hormone/genetics , Stimulation, Chemical
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