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The Journal of Immunology, 2006, 176: 401-415.
Copyright © 2006 by The American Association of Immunologists

Defining the Origins and Evolution of the Chemokine/Chemokine Receptor System1

Mark E. DeVries*, Alyson A. Kelvin*, Luoling Xu*, Longsi Ran*, John Robinson{dagger} and David J. Kelvin2,*

* Division of Experimental Therapeutics and Department of Immunology, University of Toronto, Toronto General Research Institute, Toronto General Hospital, University Health Network, Toronto, Ontario, Canada; and {dagger} Robarts Research Institute, London, Ontario, Canada


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The chemokine system has a critical role in mammalian immunity, but the evolutionary history of chemokines and chemokine receptors are ill-defined. We used comparative whole genome analysis of fruit fly, sea urchin, sea squirt, pufferfish, zebrafish, frog, and chicken to identify chemokines and chemokine receptors in each species. We report 127 chemokine and 70 chemokine receptor genes in the 7 species, with zebrafish having the most chemokines, 63, and chemokine receptors, 24. Fruit fly, sea urchin, and sea squirt have no identifiable chemokines or chemokine receptors. This study represents the most comprehensive analysis of the chemokine system to date and the only complete characterization of chemokine systems outside of mouse and human. We establish a clear evolutionary model of the chemokine system and trace the origin of the chemokine system to ~650 million years ago, identifying critical steps in their evolution and demonstrating a more extensive chemokine system in fish than previously thought.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The modern mammalian immune system is composed of innate and adaptive systems that function cooperatively to control invading pathogens recognized as nonself. The origins of mammalian adaptive and innate immunity are ancient. For example, RAG-based adaptive immunity appears to have been present at least as far back as the last common ancestor of sharks and mammals, somewhere around 400 million years ago (mya)3 (1, 2). Mammalian innate immune mechanisms appear to have originated even earlier with elements of systems such as complement and TLRs found in mammalian and invertebrate species (3, 4, 5, 6). This suggests that the last common ancestor of these two lineages (964 mya) used similar systems for host immune responses.

The coordinated movement of leukocytes is critical to both innate and adaptive immune systems and is mediated primarily by the chemokine system. Chemokines are low m.w. cytokines that are released at the sites of infection, inflammation, and injury and chemoattract leukocytes bearing chemokine receptors. Constitutively expressed chemokines in lymphoid organs also regulate leukocyte homing and maturation. Chemokines have demonstrated roles in angiogenesis, neurological development and function, organogenesis, and germ cell migration (7, 8, 9, 10, 11). The human genome has 18 identified chemokine receptors and 42 chemokines (12, 13, 14). Chemokine molecules can be divided into four subgroups (C, CC, CXC, and CX3C) based upon the presence and positioning of the first two of four conserved cysteine residues. Chemokine receptors are {gamma} subfamily rhodopsin G protein-coupled receptors (GPCRs) (15). This family includes receptors such as somatostatin, angiotensin, bradykinin, fMLP, and adrenomedullin receptors (ADMRs) (15). Chemokine receptors are classified based upon the class of chemokines they bind. In many cases, receptors are promiscuous, capable of binding multiple ligands, just as certain ligands can bind multiple receptors (12). Nevertheless, certain members, such as CXCL12 and its receptor CXCR4, appear to be exclusive binding partners. Knocking out either CXCL12 or CXCR4 is lethal in the mouse model (9, 16). In humans, the majority of chemokine receptors are present on chromosome 3 (CCR1, 2, 3, 4, 5, 8, 9, CCRL1, CCRL2, CXCR6, CX3CR1), with the remaining of the receptors distributed across chromosome 2 (CXCR1, 2, 4), chromosome 17 (CCR7, 10), chromosome 6 (CCR6), chromosome 11 (CXCR5), and chromosome X (CXCR3). Chemokines tend to be located in large clusters with 12 of 16 CXC chemokines on chromosome 4, 15 CC chemokines on chromosome 17, 3 CC chemokines on chromosome 9, 2 CC chemokines on chromosome 7, 2 chemokines on chromosome 5, 3 chemokines on chromosome 16, the 2 C chemokines on chromosome 1, and the remainder located singly on their respective chromosomes. Chemokine and chemokine receptor repertoires between human and mouse species are highly conserved, both in content and in genomic location.

To date, the chemokine system has only been completely characterized in mouse and human, although there is fragmentary information from several non mammalian species, including ray-finned fish, suggesting that the chemokine system arose early in vertebrate evolution (12). The availability of draft genomes for many nonmammalian vertebrate and invertebrate species now allows us to systematically identify immune-related genes across species and infer the evolutionary origins of various immune systems (17). Here we used the draft genomes of chicken (Gallus gallus), frog (Xenopus tropicalis), zebrafish (Danio rerio), pufferfish (Fugu rubripes), sea squirt (Ciona intestinalis), and sea urchin (Strongylocentrotus purpuratus) to systematically identify all chemokines and chemokine receptors in each species and infer the evolutionary origins and history of chemokines and chemokine receptors.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Identification of putative chemokines and chemokine receptors within the draft genomes of F. rubripes, D. rerio, X. tropicalis, G. gallus, and C. intestinalis

All chemokines and chemokine receptors were identified from current assemblies within the Ensembl Database (<www.ensembl.org>) for D. rerio, F. rubripes, G. gallus, and X. tropicalis, the Department of Energy Joint Genome Institute database for C. intestinalis (<http://genome.jqi-psf.org/ciona4/ciona4.home.html>), and the Baylor College of Medicine database for S. purpuratus (<www.hgsc.bcm.tmc.edu/projects/seaurchin>). All known chemokines and chemokine receptors were compared with predicted and known protein sequences from the databases by basic local alignment search tool search (expected score: 0.0001). Only hits that, when compared back to the genpept database (National Center for Biotechnology Information <www.ncbi.nlm.nih.gov>), returned known chemokines or chemokine receptors as their top scoring hit were retained. In addition to examining automated gene predictions, we examined genomic sequence direction by comparing known chemokines and chemokine receptors to draft genomes by tblastn (expected score: 0.1), and putative peptide fragments were identified. Putative peptide fragments that returned a known chemokine or chemokine receptor when compared with the genpept database were retained. Appropriate positioning of conserved cysteine residues and conserved exon boundaries were also required for chemokine identification. Only putative chemokines where at least two exons were identified are included in this study. Sequences were then compared with the National Center for Biotechnology Information dbEST database (<www.ncbi.nlm.nih.gov/dbEST/index.html>) and any matches were used to refine predictions. Only putative receptors that placed phylogenetically within the chemokine receptor subgroup of {gamma} rhodopsin GPCRs (as determined by protein parsimony and nearest neighbor analysis with all human {gamma} rhodopsin GPCRs) were considered putative chemokine receptors for this study. All putative chemokines and chemokines receptors and their genomic locations are listed in Tables I and II.


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  		Table I. Identified chemokine receptors, chemokine binding proteins, RDC1, and adrenomedullin receptors from zebrafish (dr), pufferfish (fr), frog (xt), and chicken (gg)

 

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  		Table IA. (Continued)

 

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  		Table II. Identified chemokines from zebrafish (dr), pufferfish (fr), frog (xt), and chicken (gg)

 

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  		Table IIA. (Continued)

 

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  		Table IIB. (Continued)

 
Pairwise homology of putative chemokines and chemokine receptors

Human chemokines and chemokine receptors were compared individually to all putative chemokines and chemokine receptors from the examined species (Tables I and II) by Needlemen-Wunsch pairwise alignment using the needle program from the EMBOSS package with default parameters and the percent identity of the top-scoring pair listed (18). To avoid penalizing incomplete sequences, terminal 3' and 5' gaps were not scored.

Phylogenetic analysis of chemokines and chemokine receptors

All known and putative chemokines and chemokine receptors and the GPCRs RDC1 and the ADMR were aligned using ClustalW (19), and phylogenetic analysis was performed using both protein parsimony and nearest neighbor methods with a bootstrap score of 100 using the PHYLIP software package (Phylogeny Inference Package, version 3.5c; J. Felsenstein, University of Washington, Seattle, WA). Predicted chemokine sequences are detailed in the supplemental material. 4 Known sequences used were: hsADMR, Ensembl ID: ENSP00000300098; hsCCBP2, Ensembl ID: ENSP00000273145; hsRDC1, Ensembl ID: ENSP00000272928; hsCCR1-CCR10, Ensembl IDs: ENSP00000296140, ENSP00000344867, ENSP00000329591, ENSP00000332659, ENSP00000343985, ENSP00000339393, ENSP00000246657, ENSP00000326432, ENSP00000308707, ENSP00000332504; hsCCRL1, hsCCRL2, Ensembl IDs: ENSP00000249887, ENSP00000346872; hsCX3CR1, Ensembl ID: ENSP00000304320; hsCXCR1-CXCR6, Ensembl IDs: ENSP00000295683, ENSP00000319635, ENSP00000335095, ENSP00000241393, ENSP00000292174, ENSP00000304414, ENSP00000310405; mmCXCR2-CXCR6, GenBank gis: 547718, 10719927, 46577576, 416719, 13507658; mmCCR1-CCR10, GenBank gis: 1705891, 2506483, 1705893, 1705895, 2851566, 8134362, 1352336, 3334152, 8134364, 12643802; mmCCRL1-CCRL2, GenBank gis: 21746187, 31980870; mmCCBP2, GenBank gi: 14547939; mmXCR1, GenBank gi: 12585214; mmCX3CR1, GenBank gi: 8134357; mmADMR, GenBank gi: 1169785; mmRDC1, GenBank gi: 47117863; Cyprinus carpio chemokines, GenBank gis: 19912841, 3298342, 33945321, 33945571, 42661784; human chemokines, ENSEMBL IDs: ENSP00000225842, ENSP00000302234, ENSP00000225844, ENSP00000334867, NP_004158, ENSP00000293275, ENSP00000219244, ENSP00000004921, ENSP00000308815, ENSP00000225831, ENSP00000351671, ENSP00000259607, ENSP00000219235, ENSP00000293280, ENSP00000222902, ENSP00000253451, ENSP00000005180, ENSP00000259631, ENSP00000281629, ENSP00000225245, ENSP00000250151, ENSP00000293272, ENSP00000200307, ENSP00000225840, ENSP00000006053, ENSP00000296031, ENSP00000305651, ENSP00000306884, ENSP00000339913, ENSP00000286758, ENSP00000337065, ENSP00000293778, ENSP00000264492, ENSP00000296026, ENSP00000296029, ENSP00000296027, ENSP00000226317, ENSP00000296028, ENSP00000306512, ENSP00000264492, ENSP00000271401, ENSP00000271402; Ictalurus furcatus chemokines, GenBank gis: 33285946, 49175627, 49175631, 49175633, 49175637, 49175639, 49175641, 49175643, 49175645, 49175653; Ictalurus punctatus chemokines, GenBank gis: 23194396, 33285944, 49175629, 49175635, 49175647, 49175649, 49175651, 49175655, 49175657; Melanochromis auratus chemokines, 27802625, 27802627; Oncorhynchus mykiss chemokines, GenBank gis: 15029046, 20065775, 31087938, 40644207; Paralabidochromis chilotes chemokines, GenBank gis: 27802629, 27802631, 27802633, 27802635, 27802637; Paralichthys olivaceus chemokines, GenBank gis: 22531647, 24024792, 39104470; Scyliorhinus canicula SCYA107, GenBank gi: 27802641; Triakis scyllium chemokines, GenBank gi: 52546152, 52546154, 52546156; and mouse chemokines, Ensembl IDs: ENSMUSP00000031319, ENSMUSP00000034232, GenBank gis: 121623, 127100, 24638135, 1708848, 127092, 124494, 13124561, 3914966, 7673945, 7674363, 7674364, 37088434, 134512, 126844, 127079, 1346534, 548926, 115197, 417192, 13431906, 1709026, 1352377, 2493663, 6094382, 6094383, 6094384, 6175078, 6094388, 13431902, 3122923, 27151761, 12229756. Both methods yielded consistent trees. Genes were considered orthologous to a human chemokine or chemokine receptor if they placed phylogenetically with that chemokine or chemokine receptor protein (bootstrap score of 85 or higher) and shared higher homology to an individual chemokine or chemokine receptor (by amino acid identity) than any other known human protein or if they displayed characteristics unique to a particular human gene (such as the CX3C motif and membrane domain associated with human CX3CL1).

Identification of parologous groups in mouse and human

Mouse genomic regions homologous to the syntenic regions on human chromosomes 2, 3, 7, 12, and 17 were obtained by blasting the human genes in these regions (Draft Human Genome, <http://genome.cse.ucsc.edu>: chromosome 2, 131244135–253119605; chromosome 3, 41708408–59905815; chromosome 7, 20163390–88809276; chromosome 12, 50081050–68181262; chromosome 17, 30013700–58946198) against the mouse genome database of the Celera Discovery System (Celera Genomics). Complete gene lists were compiled from the syntenic regions obtained from mouse (chromosome 1 (49997463–93050906), chromosome 1 (117961192–129903758), chromosome 2 (50673337–83082136), chromosome 6 (48949722–59042650), chr9 (115948450–122035962), chr10 (113151679–129585868), chromosome 11 (84990369–119253191), chromosome 15 (99035058–102695874), numbers are Celera Discovery System chromosomal positions). Mouse genes that had a homolog within the human syntenic regions were kept, and homolog pairs were blasted individually against a database containing all the syntenic human and mouse genes with an minimum expected score of 0.00001. Those gene pairs that returned at least two other paralogous genes were used to form initial paralogy groups, which were then merged together based upon common genes.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
To reconstruct the evolutionary history of the chemokine system, as well as to provide a catalog of the chemokine system in important model species, we sought to systematically identify all chemokines and chemokine receptors within the draft genomes of the species chicken (G. gallus), frog (X. tropicalis), zebrafish (D. rerio), pufferfish (F. rubripes), sea squirt (C. intestinalis), and sea urchin (S. purpuratus) (Fig. 1). We performed de novo chemokine and chemokine receptor gene prediction on the draft genomes of each species and merged our gene predictions with any available gene information characterized in the literature, as well as any automated gene prediction information from public databases and expressed sequence tag information. Due to their small size and gene structure of three or four exons, chemokines were often not identified by automated gene prediction programs and were only identified when detailed analysis was performed paying particular attention to the positioning of conserved residues and exon boundaries. Orthology to human chemokines and chemokine receptors was determined by phylogenetic and discriminating feature analysis as described in Materials and Methods.



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  		FIGURE 1. The timing of last common ancestors with human beings. Phylogeny showing the dates of the last common ancestors of represented classes with human beings. Dates are given as molecular clock estimates (5 6 ) and commonly used model species are enclosed in parentheses. The emergence of various physiological and immunological features is shown at the top of the figure.

 
Chemokines and chemokine receptors in chicken (G. gallus)

Within the vertebrate subphylum, Aves (birds) are the most closely related class to Mammalia (mammals) with a last common ancestor ~310 mya (5). In addition to being an important commercial resource, chickens are commonly used in immune models and are important hosts for multiple human diseases such as influenza. We identified and annotated all putative chemokines and chemokine receptor genes in the draft genome of the chicken (G. gallus) (Figs. 2–4 and Tables I and II). Based on homology and phylogenetic placement, 14 putative chemokine receptor and 23 putative chemokine genes are present in chicken, collectively representing all four classes of chemokines (CXC, CC, XC, CX3C) (Figs. 2–4 and Tables I and II). Where clear orthology could be established, genes were named based upon their human ortholog. For example, ggCXCL14 is the chicken ortholog of human CXCL14. Otherwise genes were named as follows: species abbreviation-location-closest human-percent identity-public protein id. For example gg-scaf86-hsCCR8–56.4%-EK5496, indicates a gene from G. gallus located on scaffold 86 with 56.4% identity to human CCR8 and listed in the Ensembl database with ID 000005496 (EK, Ensembl known; EP, Ensembl predicted; GB, GenBank ID; DN, de novo prediction). Complete gene information including base position and predicted peptides are available in Tables I and II, and in the supplemental material. The chicken chemokine receptor repertoire consists of orthologs to most human receptors including CCR4, 6, 7, 8, 9, CCRL1, CXCR4, 5, XCR1, and CX3CR1 as well as a receptor that places phylogenetically with CXCR1 and CXCR2 (gg-unplaced-CXCR2–41.8%-GB11320935) (Fig. 3). Our exploration of the chicken genome failed to yield orthologs of CCR1, 2, 3, 5, CCR10, or CXCR6. However, there are two receptors that formed a branch with mammalian CCR2 and CCR5 in the phylogenetic tree (gg-chr2-CCR2–57.2%-EK19142 and gg-chr2-CCR5–62.6%-EK19141), although we could not establish orthology (Fig. 3). Chicken possesses three CXC chemokines with the "ELR" motif. In mammals, the ELR motif is common to CXC chemokines with ligand specificity for CXCR1 and CXCR2 and is commonly associated with neutrophil chemotaxis. In addition, the ELR motif has implicated in angiogenesis with mutation of this motif leading to an angiostatic effect (20). Chicken also possesses orthologs of human CCL17, CXCL12 (2 genes), CXCL14, CX3CL1, and an XC motif chemokine (gg-chr1-XCL1–33.0%-EK24539). CCL17 is a ligand for CCR4 and has been implicated in Th2 responses and T cell maturation. Although we identified a CX3C chemokine, it should be noted that the mucin stalk of mammalian CX3CL1 is absent in chicken CX3CL1 although it possesses an elongated C terminus akin to human CXCL16. CX3CL1 is highly expressed in neurological tissue and in mounting Th1 responses as well as being implicated in atherosclerosis (21, 22, 23). Of all the chemokines identified, ggCXCL14, ggCXCL12a, and ggCXCL12b showed the highest homology to human with 60, 78.5, and 73% identity, respectively. In mice, CXCL12 and its receptor CXCR4 are critical to neural development, organogenesis, T cell homing, germ cell migration, and hemopoiesis with deletion of either gene yielding a lethal phenotype (9, 16, 24, 25). CXCL14, in contrast, is poorly characterized with no known receptor although it has been implicated in monocyte and dendritic cell chemotaxis and is an angiostatic factor (26, 27). Although many chicken chemokines do not have definitive orthologs, this is hardly surprising. Shields (28) previously noted that the amino acid sequences of chemokines are too short to provide a large enough sample size to determine accurate branching except in cases of very high conservation. As such, the receptor repertoire is likely a better indication of the nature of the chemokine system in a given species.



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  		FIGURE 2. Systematic characterization of chemokines and chemokine receptors. Putative chemokines and chemokine receptors were identified from the draft genomes of chicken (G. gallus), frog (X. tropicalis), zebrafish (D. rerio), sea squirt (C. intestinalis), and sea urchin (S. purpuratus). The number of putative chemokines (A) and chemokine receptors (B) are shown along with the numbers of previously characterized receptors from human and mouse. Human chemokines (C) and chemokine receptors (D) were compared individually by Needlemen-Wunsch pairwise alignment to all putative identified chemokines and chemokine receptors from the indicated species, and the percent identity of the top-scoring pair was plotted. As expected, percent identity tends to increase as the length of time since the last common ancestor decreases. Interestingly, certain proteins such as CXCR4, CXCL12, and CXCL14 exhibit sharp peaks, demonstrating a high degree of evolutionary conservation.

 


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  		FIGURE 3. Phylogeny of chemokine receptors. A, Putative chemokine receptors identified in chicken (gg), frog (xt), zebrafish (dr), and known chemokine receptors from mouse (mm) and human (hs) were aligned, and nearest neighbor analysis was performed. Bootstrap values (of 100) are shown.

 


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  		FIGURE 4. Phylogeny of chemokines. Putative chemokines identified in chicken (gg), frog (xt), zebrafish (dr), and known chemokines from mouse (mm) and human (hs) were aligned, and protein parsimony analysis was performed. Branches with bootstrap values of 85 or greater (of 100) are shown. Chemokines with putative membrane domains are marked with an asterisk.

 
One chicken chemokine that also bears mentioning (gg-chr4-CXCL3–36.4%-EK16811) has striking homology (70% similarity, 54% identity) to a viral chemokine (vIL-8) from Gallid herpesvirus 2 that causes Marek’s disease (29, 30). Marek’s disease is a contagious, lymphoproliferative disease of domestic chickens in which mononuclear infiltration, demyelination of peripheral nerves, and T cell lymphomas are common features. However, this virokine has much lower homology to chicken IL-8 (CXCL8) (46% similarity, 31% identity vs chicken CXCL8) and has been implicated in monocyte recruitment rather than the neutrophil recruitment, for which CXCL8 is best known. Our data suggests that this viral chemokine in fact is mimicking—and is likely a viral copy of—the novel chicken chemokine we have identified gg-chr4-CXCL3–36.4%-EK16811, rather than CXCL8 as has been previously thought, and that research into the function of this novel chemokine may aid in understanding this disease.

Chemokines and chemokine receptors in frog (X. tropicalis)

Class Amphibia shares a last common ancestor with mammals living ~360 mya (5). Within vertebrate evolution, amphibians represent the evolutionary shift to a terrestrial environment and mark the emergence of tetrapods. We identified and annotated all putative chemokines and chemokine receptor genes in the draft genome of the frog (X. tropicalis) (Figs. 2–4 and Tables I and II). As with chicken, the chemokine receptor system in frog is highly represented with 15 chemokine receptors and 25 chemokines including orthologs to CCR6, 7, 9, CCRL1, CXCR3, 4, 5, 6, and XCR1. There is also a receptor that places phylogenetically with human CCR2 and CCR5 (xt-sc26-CCR5–40.5%-DN) and two receptors that resemble human CCR3 (xt-sc219-CCR3–37.1%-DN, xt-sc26-CCR3–32.9%-DN) although orthology could not be clearly established (Figs. 2 and 3, Table I). It should be noted that the CXCR6 receptor is present in frog, suggesting this receptor may have been lost in chicken. No CXCR1, CXCR2, CCR8, CCR10, or CX3CR1 receptors were evident in frog. Frog possesses three ELR-containing CXC chemokines, a CXCL12 ortholog, and a CXCL14 ortholog. Phylogenetic analysis was unable to identify clear orthologs for the remaining chemokines, although they appear to represent a variety of subfamilies (Fig. 4). We were unable to identify a CX3C chemokine or any chemokines with putative membrane-associated domains. As with chicken, the frog chemokines CXCL12 and CXCL14 showed striking homology (72.4 and 66.3% identity, respectively) to their human counterparts.

Chemokines and chemokine receptors in zebrafish

The ray-finned fish (class Osteichthyes) are the most abundant vertebrate class and shared a last common ancestor with mammals some 450 mya (5). Zebrafish has rapidly become an important model species for studying immune responses due to its ease of manipulation during development (for example, gene inactivation by morpholino oligonucleotides) and its prolific fecundity (31). We identified and annotated all putative chemokines and chemokine receptor genes in the draft genome of the zebrafish (D. rerio) (Figs. 2–4 and Tables I and II). There are 24 putative chemokine receptor and 63 putative chemokine genes within zebrafish, far more than other species examined (Figs. 2–4 and Tables I and II). This is a startling result as all previous studies predicted that the number of fish chemokines would be substantially less then those in mammals (32, 33, 34). These include orthologs of CCR6, 7, CCRL1, CXCR1/2, 3, 4, 5, and to the chemokines CXCL12 (three genes) and CXCL14 (two genes). Even though they possess a larger number of chemokines and chemokine receptors than either human or mouse, zebrafish lacks clear orthologs to CCR1, 2, 3, 5, 8, and CX3CR1 and have no chemokines that are obvious ligands of CCR1, 2, 3, 5, 8, and CX3CR1 (Figs. 3 and 4). Examination of the loci corresponding to human chromosome 3 in different species highlights the evolution of this group of chemokine receptors (Fig. 5). None of the zebrafish CXC chemokines possess an ELR motif, although we did identify six chemokines with possible membrane motifs. Many of the expansions observed among zebrafish chemokines appear to be duplications within single select lineages, such as a cluster of closely related chemokines on chromosome 25 (Fig. 4). The absence of these chemokines in mammals may indicate that they serve functions specific to an aquatic environment. A combination of tandem and en bloc duplications is likely responsible for the observed increase in chemokine and chemokine receptor repertoires above those in mammals (see Discussion). The same analysis on the pufferfish genome reveals a similar representation of chemokines and chemokine receptors, although fewer in number. Pufferfish does possess a CX3CR1 ortholog that we did not identify in zebrafish. The lower number of genes may be related to the uniqueness of the pufferfish genome, namely that it has undergone compaction so that it is a fraction of the size of other fish and mammalian genomes (35).



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  		FIGURE 5. Comparison of primary CCR loci between species. In humans, the majority of CCRs reside in a single locus on chromosome 3. The equivalent loci are shown from mouse, human, chicken, frog, and zebrafish. The CCR1, 2, 3, 5, CCRL2 minicluster is only completely represented in human and mouse.

 
Comparative genomic analysis of invertebrates failed to identify chemokines or chemokine receptors

The sea squirt (C. intestinalis) is the only extant invertebrate belonging to the chordate phylum to have its genome completed (36). The last common ancestor of sea squirt and mammals (650 mya) represents an important stage in the evolution of vertebrates (6, 37). Sea squirt possess a dorsal notochord (in the larval) stage as well as the rudiments of multiple organ systems typically characterized as vertebrate. It is commonly used to examine the evolution of the nervous and immune systems. Purple sea urchin (S. purpuratus) is an echinoderm that represents a separate branch of deuterostomes (the other two being chordates and hemichordates). Sea urchin is commonly used for studying developmental gene networks and embryogenesis (38). The draft genomes of sea squirt and sea urchin, as well as that of fruit fly (an invertebrate protostome) allow us to study the origins of chemokines and chemokine receptors. These species have last common ancestors with mammals ranging from 650 to 964 mya (5, 6). In all species, we failed to identify any putative chemokine or chemokine receptors. Furthermore, we also failed to identify any orthologs of closely related GPCR subfamilies. Chemokine receptors are {gamma} rhodopsin GPCRs and have been previously classified into a subgroup that includes angiotensin, bradykinin fMLP, C5a, leukotriene, and an ADMR (15). As part of our systematic identification of chemokine receptors, we identified all GPCRs from sea urchin and sea squirt. We were unable to identify clear orthologs of chemokine receptor-related subfamilies (angiotensin, bradykinin fMLP, C5a, leukotriene, and ADMRs) in sea squirt, sea urchin, or fruit fly, suggesting that this subfamily of GPCRs that include chemokine receptors had not evolved by the last common ancestor of sea squirt and vertebrates. Indeed, even among other {gamma} rhodopsin GPCRs, only orthologs of the galanin receptor and the relaxin-3 receptor (GPR135) as well as a receptor of the somatostain/opioid lineage were identifiable, suggesting most of the GPCR {gamma} subfamily arose later in evolution and that chemokine receptors were not present at the last common ancestor of sea squirt and vertebrates.

En bloc duplication of chemokines and chemokine receptors

En bloc duplication is the simultaneous duplication of multiple genes via the copying of a segment of chromosome. The resulting duplicate regions are said to be syntenic. Examination of the genomic loci harboring chemokine receptors in human and mouse genomes revealed five syntenic regions (human chromosomes 2, 3, 7, 12, and 17), with many genes in common in addition to chemokine receptors (Fig. 6A). This suggests that these loci were the result of en bloc duplications and not the result of individual gene duplication. Four of these loci possess the highly characterized homeobox (HOX) clusters. The locus on chromosome 3, which does not possess a HOX cluster, was likely joined with the HOX containing locus from human chromosome 7 in the ancestral state. This is evident by examining the corresponding loci in chicken, which form a single locus (chicken chromosome 2), suggesting that the last common ancestor of birds and mammals had four syntenic regions that contained HOX clusters, chemokines, and chemokine receptors (Fig. 6B).



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  		FIGURE 6. Chemokines and chemokine receptors were propagated by en bloc duplication. A, Examination of the genomic loci containing chemokines and chemokine receptors demonstrated that four of these loci (chromosome 2, 3, 7, 12, 17) are syntenic, possessing 116 paralogous gene groups, a subset of which is shown. In particular, HOX clusters are present in four of these loci. B, The five human syntenic regions were originally four. The chromosome 3 chemokine receptor locus in human does not possess a HOX cluster. However, the orthologous locus in chicken is associated with the HOX cluster of chicken chromosome 2. The genes of chicken chromosome 2 (20–55 Mb) were blasted individually against the human proteome and the human chromosome containing the closest blast hit plotted. This clearly demonstrates that both the human chromosome 3 and chromosome 7 loci comprise a single locus within chicken chromosome 2.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
By systematically identifying chemokine and chemokine receptor genes across model species from multiple taxonomic classes, we can infer the origins and model the evolutionary history of the chemokine/chemokine receptor system. Chemokine receptors form a monophyletic group within the {gamma} rhodopsin GPCR family and likely arose through repeated duplication of a single ancestral gene (15). As we will demonstrate later in Discussion, expansion of the chemokine receptor gene family likely occurred through two mechanisms: en bloc duplication and tandem duplication.

En bloc duplication led to initial radiation of the chemokine system during vertebrate evolution

We demonstrate that the majority of chemokines and chemokine receptors exist as part of five syntenic regions (human chromosomes 2, 3, 7, 12, and 17) likely resulting from en bloc duplication (Fig. 6A). These syntenic regions were also identified by McLysaght et al. (39). We also show that the ancestral state of the syntenic regions on chromosomes 3 and 7 was most likely a single locus, as is the case in chicken (Fig. 6B). All four of these ancestral loci contain HOX gene clusters.

HOX genes are highly conserved genes critical to body plan development, and the clusters containing these genes have been sequenced from a large number of species (36, 40, 41, 42, 43). Many have suggested that the en bloc duplication of these gene clusters was actually part of two whole genome duplications early in the vertebrate lineage, whereas others suggest the duplications were of a smaller scale (39, 40, 44). Regardless of whether the duplication events encompassed the whole genome, the tight association of the majority of chemokine receptors with HOX clusters provides us with an invaluable tool for tracking the origin of the chemokine system. The only extant invertebrate chordates are the sea squirt (model species: C. intestinalis) and amphioxus (model species: Branchiostoma floridae), which are thought to share a common ancestor with vertebrates ~650 mya (6, 37). The class Agnatha (jawless fish lamprey and hagfish) contains the most primitive vertebrates, sharing a last common ancestor with jawed vertebrates ~564 mya (the hagfish thought to be the more primitive of the two) (5, 45, 46). Both amphioxus and sea squirt (as well as insect species and sea urchin) have only a single HOX cluster, whereas lamprey has four (36, 43, 45, 46). From this we can conclude two things: 1) the duplication of HOX clusters was accompanied by the duplication of chemokine receptors and thus species possessing multiple HOX clusters (such as lamprey and hagfish) should possess multiple HOX-associated chemokine receptors; and 2) because chemokine receptor duplication accompanied HOX cluster duplication, the chemokine system existed more than 564 mya when the first HOX cluster duplication occurred and before the last common ancestor with jawed vertebrates.

The HOX locus on human chromosome 12 has no associated chemokine receptors, but does have a very closely related receptor, the ADMR. ADMR is vital to cardiac development (47). Other studies of GPCRs have placed ADMR and the very closely related RDC1 receptor—located in the HOX-associated locus of chromosome 2 along with CXCR1, 2, and 4—as the closest relatives of the chemokine receptor family (15). By considering the genomic context, it seems evident that ADMR and chemokine receptors are more closely related to each other than to any other GPCR subclass and share their most immediate ancestor. Previous studies of the HOX clusters in teleost fish demonstrated seven clusters in zebrafish and six clusters in pufferfish, with their composition suggestive of a single fish-specific genome duplication followed by loss of one or two clusters, respectively (35, 48). The duplication seems a likely explanation for some of the additional zebrafish chemokines and chemokine receptors that we observed. For example, the zebrafish CC chemokine clusters on chromosome 20 and 25, the CXC chemokine clusters on chromosomes 1 and 5, and the additional copies of the chemokine receptors CCR9 and CCRL1 may be the result of this duplication event.

Tandem duplications as a means of selectively expanding the chemokine and chemokine receptor repertoire

Chemokine and chemokine receptor genes tend to be organized in tandem arrays. Tandem duplication, the copying of a chromosomal segment within a locus to yield two adjacent copies) is a common source of gene duplication and likely played an important role in the formation of chemokine and chemokine receptor repertoires. For example, in human, chromosome 3 has 11 receptors, chromosome 4 has 16 CXC chemokines, and chromosome 17 has 2 receptors and 15 chemokines in close proximity. In fact, examining individual genomes in different vertebrate classes demonstrates that tandem duplication is an ongoing process, with each species exhibiting chemokines and chemokine receptors apparently duplicated in a lineage-specific fashion. For example, three of the putative membrane-associated chemokines of zebrafish occur in a single cluster on chromosome 25 and form their own phylogenetic group (Fig. 4). Indeed, a number of recently reported catfish chemokines (32) did not have clear orthologs in zebrafish, suggesting that there may be substantial variation in the chemokine system between fish species due to gene duplication and loss. In humans, multiple receptors from the chromosome 3 locus (and corresponding chemokine ligands from chromosome 17) are not present in chicken, frog, or fish genomes. Although we cannot discount the possible role of gene deletion in all nonmammalian species, the comparative genomic analysis of multiple species suggests that a number of these genes are specific to the mammalian lineage.

Based on sequence homology, genomic position, and identification of paralogous regions, we have created a model of the evolutionary history of the chemokine receptor system as it arose in mammals (Fig. 7). Before the first en bloc duplication, the ancestral locus contained a chemokine, a chemokine receptor, and ADMR (as the result of a tandem duplication), along with the HOX cluster and several other genes, a subset of which is shown including a pair of STATs that are essential to cytokine signaling (49). Two rounds of en bloc duplication led to the classical four HOX clusters in tetrapods (human chromosomes 2, 12, 17, and 3/7). Both the CC and CXC arms of the chemokine system would have been established before the second en bloc duplication. Although some tandem duplication of receptors occurred in between en bloc duplications, the majority of tandem duplications of receptors likely occurred afterward. The rudimentary chromosome 3 cluster (likely resembling that seen in fish) underwent continual tandem duplications, which we have tracked by examining frog and chicken, culminating in the expansion of the CCR1, 2, 3, and 5 receptors that are only clearly defined in mammals.



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  		FIGURE 7. Model for chemokine receptor evolution. The majority of chemokine receptors are present in syntenic regions of chromosomes 2, 3, 7, 12, and 17. After the first en bloc duplication, the HOXCD protocluster would contain two chemokine receptors that would ultimately become CXCR1 and CXCR4 on chromosome 2 after the second genome duplication. The chemokine receptors present in the HOXAB protocluster, in contrast, would possess the CCR7/9 ancestor and the CCR10/CCRL1 ancestor after tandem duplication. After the second genome duplication, chromosome 17 would retain CCR7 and CCR10. Chromosome 7 originally would have possessed the primary CCR cluster that arose through repeated tandem duplication and was translocated to human chromosome 3.

 
The role of the chemokine system in vertebrate evolution

Although previous studies have provided piecemeal information on the chemokine system in nonmammalian vertebrates, we have provided a comprehensive comparative genomics approach to understand the origins and evolutionary history of chemokines and chemokine receptors. It has been proposed previously that fish species possess a small number of chemokines relative to mammals with little promiscuity (32, 33, 34). Our data clearly demonstrate that zebrafish has an extensive chemokine system (with more members than mammals) and that the CC family was well established by the time of the last common ancestor of fish and mammals, and, based upon the evidence of en bloc duplications, we can conclude that both CC and CXC families were present during the initial radiation of chemokine and chemokine receptor genes, somewhere around 564 mya.

We have placed the origins of ancestral chemokine receptors between 650 and 564 mya at the emergence of vertebrates (5, 6). This period is marked by the evolution of neural crest tissue, a skull, gill bars for respiration, muscularized pharynx and gut, a chambered heart, closed circulatory system, blood-based oxygen transport, and a hemopoietic system. It is interesting to speculate that the diverse role of the chemokine system was established early in their ontogeny and that they may have had integral roles in the evolution of these vertebrate systems.

Chemokines are most clearly recognized for their role as leukocyte chemoattractants in both innate and adaptive immunity (10, 50). Many chemokines are closely associated with RAG-based adaptive immunity. Although some have speculated that many cytokines and chemokines arose only with the acquisition of RAG genes by vertebrates, recent evidence of alternate forms of adaptive immunity suggest that much of the genetic basis for adaptive immunity was in place before the acquisition of RAG genes (51). Indeed, the jawless vertebrates, hagfish and lamprey, do not possess RAG genes, but lamprey CXCR4 was previously shown to be expressed on a lymphocyte-like cell set (52). This suggests there were chemokines and chemokine receptors that may have served an adaptive immune role before classical RAG-based adaptive immunity. Furthermore, many genes (particularly CC chemokines from human chromosome 17, ELR-CXC chemokines from chromosome 4, and CC receptors from chromosome 3) that are apparently absent from zebrafish suggest that they are not critical for RAG-based adaptive immunity—because zebrafish possesses a RAG-based adaptive immune system—although they may represent a refinement of the system.

Chemokines and chemokine receptors continue to undergo en bloc and tandem duplications, giving rise to duplicate genes that may acquire novel functions. Zebrafish has two orthologs of human CXCR4 that apparently have acquired some independent functions (53). Likewise, catfish appears to have a number of closely related chemokines not present in zebrafish (32). A closer examination of distantly related species of a single class will give better information regarding environment-specific adaptations of the chemokine system. For example, the frog species Xenopus laevis has undergone a genome duplication not found in X. tropicalis and, therefore, may have several novel members of the chemokine system (54). Similarly, it is unclear whether other fish species, such a lobe-finned fish or lungfish, underwent the genome duplication responsible for seven HOX clusters in zebrafish and examination of these species will more clearly establish the state of the chemokine system at the divergence of the mammal and fish lineages. Also clear is that the interspecies variability of chemokines and chemokine receptors can differ significantly. CXCR4 and its ligand, CXCL12, are both highly conserved across all chemokine-containing species examined and this likely reflects the critical role of these genes as is evidenced by the lethality of their respective knockouts in mice (9, 16). The chemokine CXCL14 is more highly conserved through evolution than CXCL12 but has no known receptor and has minimal functional information available. This suggests that this molecule also serves a critical function and that knockout of this gene may also be lethal. The CCRL1 receptor has no known ligand but is also well conserved in the species examined and may represent a candidate receptor for CXCL14.

Concluding remarks

Our study represents the most comprehensive comparative genomic analysis of the chemokine system known to date and the only complete characterization of chemokine systems outside of mouse and human. We have demonstrated that taking a whole-genome approach, using phylogenetic analysis coupled with their genomic context across multiple species can be a powerful tool in dissecting complicated systems that are the result of many duplication events. Through this method, we have established a clear evolutionary model for the development of the loci and functions of the chemokine system, not only tracing the origin of the chemokine system to ~650 mya, but also identifying critical steps in the evolution of specific chemokines and chemokine receptors as well as demonstrating a more extensive chemokine system in fish than previously thought.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by grants from the Canadian Institutes of Health Research, Canvac, and Genome Canada. M.E.D. is a Canadian Institutes of Health Research scholar. Back

2 Address correspondence and reprint requests to Dr. David J. Kelvin, Division of Experimental Therapeutics, Toronto General Research Institute, 200 Elizabeth Street, MBRC5R425, Toronto, Ontario M5G 2C4, Canada. E-mail address: dkelvin{at}uhnres.utoronto.ca Back

3 Abbreviations used in this paper: mya, million years ago; GPCR, G protein-coupled receptor; ADMR, adrenomedullin receptor; HOX, homeobox. Back

4 The online version of this article contains supplemental material. Back

Received for publication June 28, 2005. Accepted for publication October 12, 2005.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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