Genomic organization and biological characterization of the novel human CC chemokine DC-CK-1/PARC/MIP-4/SCYA18

The chemokines are a group of chemotactic molecules that appear to regulate the directed movement of white blood cells in vitro and in vivo and may therefore play important roles in inflammation and immunity. The genes encoding the chemokines are clustered in close physical proximity to each other. A large cluster of human CC chemokine genes resides on chromosome 17. We have used this information in a positional cloning approach to identify novel chemokine genes within this cluster. We constructed a YAC contig encompassing the MIP-1alpha (HGMW-approved symbol SCYA3) gene region and used exon trapping and sequence analysis to isolate novel chemokine genes. Using this approach, a gene encoding a chemokine named MIP-4, based on its homology with MIP-1alpha (49.5% identity at the nucleotide level and 59.6% at the predicted amino acid level), was found. The MIP-4 gene (HGMW-approved symbol SCYA18) consists of three exons spread over 7.1 kb and is separated from the MIP-1alpha gene by 16 kb. The MIP-4 gene encodes a 750-bp mRNA that is expressed in lung and macrophages but not in brain or muscle. The mRNA encodes an 89-amino-acid protein and includes a predicted signal peptide of 21 amino acids. Recombinant or synthetic MIP-4 induced calcium mobilization in naive and activated T lymphocyte subpopulations in vitro. Injection of synthetic MIP-4 into the peritoneal cavity of mice led to the accumulation of both CD4(+) and CD8(+) T lymphocytes, but not monocytes or granulocytes. These observations provide new information concerning the arrangement of the CC chemokine gene cluster on human chromosome 17 and indicate that the MIP-4 gene product is chemotactic in vivo for both CD4(+) and CD8(+) T lymphocytes and may therefore be implicated in both humoral and cell-mediated immunity.     

Novel chicken CXC and CC chemokines

Upon stimulation with lipopolysaccharide (LPS) the chicken macrophage cell line HD-11 secretes factors with cytokine activity. To characterize these molecules, representational difference analysis with RNA of LPS-induced and uninduced HD-11 cells was performed. Two cDNA clones were isolated that code for polypeptides with structural features of chemokines. cDNA K60 codes for a novel CXC chemokine of 104 residues including a putative signal peptide of 20 amino acids at the N-terminus. It is 67% identical to the previously cloned chicken chemokine 9E3/CEF4. K60 exhibits a similar degree of sequence identity to human interleukin 8 and other related CXC chemokines (about 50%), rendering straight-forward predictions of its biological properties difficult. cDNA K203 codes for a novel CC chemokine of 89 amino acids including a putative N-terminal signal peptide of 21 residues. It is 43% identical to a previously characterized chicken protein with homology to mammalian macrophage inflammatory protein 1beta (MIP-1beta). K203 exhibits about 50% sequence identity to human MIP-1beta and other related CC chemokines.     

Identification of chemokines and a chemokine receptor in cichlid fish,shark, and lamprey

Chemokines are small, inducible, structurally related proteins that guide cells expressing the right chemokine receptors to sites of immune response. They have been identified and studied extensively in mammals, but little is known about their presence in other vertebrate groups. Here we describe seven new chemokines in bony fish and one in a cartilaginous fish, as well as one chemokine receptor in a jawless vertebrate. All eight chemokines belong to the SCYA (CC) subfamily characterized by four conserved cysteine residues of which the first two are adjacent. The chemokine receptor is of the CXCR4 type. Phylogenetic analysis does not reveal any clear evidence of orthology of fish and human chemokines. Although the divergence of the subfamilies began before the fish-tetrapod split, much of the divergence within the subfamilies took place separately in the two vertebrate groups. The existence of a chemokine receptor in the lamprey indicates that chemokines are apparently also present in the Agnatha.     

Molecular Cloning and Characterization of a Novel Human CC Chemokine, SCYA26

By searching the Expressed Sequence Tag database, a full-length cDNA for a novel human CC chemokine was cloned. This cDNA encoded a 94-amino-acid protein with a putative signal peptide of 26 amino acids. The deduced mature protein had the four conserved cysteine residues characteristic of CC chemokines and showed 44% identity with MIP-1beta and 40% identity with MIP-1alpha, RANTES, and MCP-4. mRNA for this chemokine was expressed constitutively in human heart and liver and with lesser but detectable levels in skeletal muscle, kidney, and small intestine. To investigate its biological activity, the protein was expressed in mammalian cells and purified by affinity chromatography. The recombinant protein demonstrated chemotactic activity in vitro for T cells and monocytes but not for neutrophils. The gene was mapped to chromosome 7q11.2 by fluorescence in situ hybridization. Based on its structural identity with other CC chemokines and the chemotactic activity and chromosomal location of this chemokine, we designate this chemokine small inducible cytokine subfamily A, member 26 (SCYA26). This gene symbol has been approved by the HUGO Gene Nomenclature Committee.     

Chemokine PARC gene (SCYA18) generated by fusion of two MIP-1alpha/LD78alpha-like genes

Two loci in the human genome, chromosomes 4q12-q21 and 17q11.2, contain clusters of CXC and CC chemokine subfamily genes, respectively. Since mice appear to contain fewer chemokine genes than humans, numerous gene duplications might have occurred in each locus of the human genome. Here we describe the genomic organization of the human pulmonary and activation-regulated CC chemokine (PARC), also known as DC-CK1 and AMAC-1. Despite high sequence similarity to a CC chemokine macrophage inflammatory protein-1alpha (MIP-1alpha)/LD78alpha, PARC is chemotactic for lymphocytes and not for monocytes and does not share its receptor with MIP-1alpha. Analyses of the BAC clones containing the human PARC gene indicated that the gene is located most closely to MIP-1alpha (HGMW-approved symbol SCYA3) and MIP-1beta (HGMW-approved symbol SCYA4) on chromosome 17q11.2. Dot-plot comparison suggested that the PARC gene had been generated by fusion of two MIP-1alpha-like genes with deletion and selective usage of exons. Base changes accumulated before and after the fusion might have adapted the gene to a new function. Since there are variably duplicated copies of the MIP-1alpha gene called LD78beta (HGMW-approved symbol SCYA3L) in the vicinity of the MIP-1alpha gene, the locus surrounding the MIP-1alpha gene seems to be a "hot spring" that continuously produces new family genes. This evidence provides a new model, duplication and fusion, of the molecular basis for diversity within a gene family.     

Molecular cloning of a novel human CC chemokine (Eotaxin-3) that is a functional ligand of CC chemokine receptor 3.

Previously, we mapped the novel CC chemokine myeloid progenitor inhibitory factor 2 (MPIF-2)/eotaxin-2 to chromosome 7q11.23 (Nomiyama, H., Osborne, L. R., Imai, T., Kusuda, J., Miura, R., Tsui, L.-C., and Yoshie, O. (1998) Genomics 49, 339-340). Since chemokine genes tend to be clustered, unknown chemokines may be present in the vicinity of those mapped to new chromosomal loci. Prompted by this hypothesis, we analyzed the genomic region containing the gene for MPIF-2/eotaxin-2 (SCYA24) and have identified a novel CC chemokine termed eotaxin-3. The genes for MPIF-2/eotaxin-2 (SCYA24) and eotaxin-3 (SCYA26) are localized within a region of approximately 40 kilobases. By Northern blot analysis, eotaxin-3 mRNA was constitutively expressed in the heart and ovary. We have generated recombinant eotaxin-3 in a baculovirus expression system. Eotaxin-3 induced transient calcium mobilization specifically in CC chemokine receptor 3 (CCR3)-expressing L1.2 cells with an EC(50) of 3 nM. Eotaxin-3 competed the binding of (125)I-eotaxin to CCR3-expressing L1.2 cells with an IC(50) of 13 nM. Eotaxin-3 was chemotactic for normal peripheral blood eosinophils and basophils at high concentrations. Collectively, eotaxin-3 is yet another functional ligand for CCR3. The potency of eotaxin-3 as a CCR3 ligand seems, however, to be approximately 10-fold less than that of eotaxin. Identification of eotaxin-3 will further promote our understanding of the control of eosinophil trafficking and other CCR3-mediated biological phenomena. The strategy used in this study may also be applicable to identification of other unknown chemokine genes.     

Evolution of CC chemokines in teleost fish: a case study in gene duplication and implications for immune diversity

Chemokines are a superfamily of cytokines responsible for regulating cell migration under both inflammatory and physiological conditions. CC chemokines are the largest subfamily of chemokines, with 28 members in humans. A subject of intense study in mammalian species, the known functional roles of CC chemokines ligands in both developmental and disease conditions continue to expand. They are also an important family for the study of gene copy number variation and tandem duplication in mammalian species. However, little is known regarding the evolutionary origin and status of these ligands in primitive vertebrates such as teleost fish. In this paper, we review the evolution of the teleost fish CC chemokine gene family, noting evidence of widespread tandem gene duplications and examining the implications of this phenomenon on immune diversity. Through extensive phylogenetic analysis of the CC chemokine sets of four teleost species, zebrafish, catfish, rainbow trout, and Atlantic salmon, we identified seven large groups of CC chemokines. It appeared that several major groups of CC chemokines are highly related including the CCL19/21/25 group, the CCL20 group, CCL27/28 group, and the fish-specific group. In the three remaining groups that contained the largest number of members, the CCL17/22 group, the MIP group, and the MCP group, similarities among species members were obscured by rapid, tandem duplications that may contribute to immune diversity.     

Isolation and characterization of two alleles of the chicken cytochrome c gene.

Analysis of total chicken DNA by genomic blot hybridization indicates that only one cytochrome c gene exists in the chicken genome. The two alleles of this single cytochrome c gene have been isolated from a Charon 4A-chicken genomic library. This isolation made use of the yeast CYC1 cytochrome c gene as a specific hybridization probe. The 2 chicken alleles, CC9 and CC10, have been sequenced. The amino acid sequence predicted by these 2 alleles is identical, and agrees with the published chicken cytochrome c protein sequence. The flanking regions of these 2 alleles exhibit approximately 1% divergence, indicating a very limited polymorphism. Comparative sequence analysis with the flanking regions of previously isolated cytochrome c genes (yeast and rat) indicate no significant regions of homology. The presence of only one cytochrome c-like sequence in the chicken genome is in striking contrast with mammalian genomes, which contain as many as 20-30 cytochrome c-like sequences.     

Cloning of a novel rainbow trout (Oncorhynchus mykiss) CC chemokine with a fractalkine-like stalk and a TNF decoy receptor using cDNA fragments containing AU-rich elements

An activation-specific cDNA library was made from phytohaemagglutinin (PHA)-activated haematopoietic cells of the rainbow trout (Oncorhynchus mykiss) using the technique of suppression subtractive hybridization. Several immune system genes were identified, including an interleukin (IL)1 receptor related protein and two invariant chain-like proteins. Many clones showed no similarity by BLAST search, but had AU-rich elements. These fragments were labelled and used for hybridization with a PHA-activated head kidney cDNA library. Several immune system genes were isolated by this technique, including a tumour necrosis factor (TNF) decoy receptor and a novel chemokine, designated trout chemokine 2. The TNF receptor is 285 amino acids in length and is 32-36% identical to a brook trout and human homologue. The CC chemokine is 44% identical at the amino acid level to a carp CC chemokine and approximately 20% identical to several mammalian CC chemokines. However, it has a 91 amino acid stalk-like structure at its COOH end, which is similar to the glycosylated stalk of fractalkine, a mammalian CX(3)C chemokine. In summary, AU-rich fragments obtained from an activation-specific library proved useful as hybridization probes for isolating trout immune system genes.     

In silico identification and expression analysis of 12 novel CC chemokines in catfish

Chemokines, a superfamily of chemotactic cytokines involved in recruitment, activation, and adhesion of a variety of leukocyte types to inflammatory foci, are a crucial component of the immune system of Sarcopterygiian vertebrates. Although all mammalian chemokines are believed to have been found, the status of these molecules in Actinopterygii was unknown until recently. The identification of chemokines in fish species has been complicated by low sequence conservation and confusion over expected numbers. Earlier discoveries of single fish chemokines coupled with rapidly expanding genetic resources in these species have recently provided a foundation for large-scale in silico discoveries of these important immune regulators. We report here the identification and expression analysis of 12 new CC chemokine sequences from catfish. When added to our previous report of 14 catfish CC chemokines, the number of CC chemokines in catfish now stands at 26, two more than known from humans. Establishing orthologous relationships among the majority of catfish CC chemokines, a newly available set of chicken CC chemokines, and their mammalian counterparts remain difficult, suggesting high levels of duplication and divergence within individual species.     

Cloning, characterization and genomic organization of LCC-1 (scya16), a novel human CC chemokine expressed in liver

By homology search of expressed sequence tags (EST) in GenBank a novel member of the CC chemokine family was identified. The full-length sequence of this liver-specific CC chemokine (LCC-1) predicted a mature protein of 97 amino acids with 31-48% identity to other CC chemokines. There was a characteristic amino acid C-term extension when aligned with other chemokines. Northern blot analysis from a panel of human tissues revealed that LCC-1 mRNA expression is restricted to adult and fetal liver. Different polyadenylation results in two mRNA species of 1.5 kb and 0.5 kb in size. LCC-1 is constitutively expressed in human HepG2 hepatoma cells and is induced by hypoxic exposure. The promoter region of the LCC-1 gene contains potential HIF-1 binding sites. The EST for LCC-1 has been previously mapped to the CC chemokine cluster on human chromosome 17q11.2. The organization of the LCC-1 gene (scya16) into three exons interrupted by two introns is identical to that found for other members of the CC chemokine family.     

Chemokine PARC Gene (SCYA18) Generated by Fusion of Two MIP-1α/LD78α-like Genes

Two loci in the human genome, chromosomes 4q12–q21 and 17q11.2, contain clusters of CXC and CC chemokine subfamily genes, respectively. Since mice appear to contain fewer chemokine genes than humans, numerous gene duplications might have occurred in each locus of the human genome. Here we describe the genomic organization of the human pulmonary and activation-regulated CC chemokine (PARC), also known as DC-CK1 and AMAC-1. Despite high sequence similarity to a CC chemokine macrophage inflammatory protein-1α (MIP-1α)/LD78α, PARC is chemotactic for lymphocytes and not for monocytes and does not share its receptor with MIP-1α. Analyses of the BAC clones containing the humanPARCgene indicated that the gene is located most closely toMIP-1α(HGMW-approved symbolSCYA3) andMIP-1β(HGMW-approved symbolSCYA4) on chromosome 17q11.2. Dot-plot comparison suggested that thePARCgene had been generated by fusion of twoMIP-1α-like genes with deletion and selective usage of exons. Base changes accumulated before and after the fusion might have adapted the gene to a new function. Since there are variably duplicated copies of theMIP-1αgene calledLD78β(HGMW-approved symbolSCYA3L) in the vicinity of theMIP-1αgene, the locus surrounding theMIP-1αgene seems to be a “hot spring” that continuously produces new family genes. This evidence provides a new model, duplication and fusion, of the molecular basis for diversity within a gene family.     

Constitutive inositol phosphate formation in cytomegalovirus-infected human fibroblasts is due to expression of the chemokine receptor homologue pUS28

An open reading frame (ORF), US28, with homology to mammalian chemokine receptors has been identified in the genome of human cytomegalovirus (HCMV). Its protein product, pUS28, has been shown to bind several human CC chemokines, including RANTES, MCP-1, and MIP-1 alpha, and the CX(3)C chemokine fractalkine with high affinity. Addition of CC chemokines to cells expressing pUS28 was reported to cause a pertussis toxin-sensitive increase in the concentration of cytosolic free Ca(2+). Recently, pUS28 was shown to mediate constitutive, ligand-independent, and pertussis toxin-insensitive activation of phospholipase C via G(q/11)-dependent signaling pathways in transiently transfected COS-7 cells. Since these findings are not easily reconciled with the former observations, we analyzed the role of pUS28 in mediating CC chemokine activation of pertussis toxin-sensitive G proteins in cell membranes and phospholipase C in intact cells. The transmembrane signaling functions of pUS28 were studied in HCMV-infected cells rather than in cDNA-transfected cells. Since DNA sequence analysis of ORF US28 of different laboratory and clinical strains had revealed amino acid sequence differences in the amino-terminal portion of pUS28, we compared two laboratory HCMV strains, AD169 and Toledo, and one clinical strain, TB40/E. The results showed that infection of human fibroblasts with all three HCMV strains led to a vigorous, constitutively enhanced formation of inositol phosphates which was insensitive to pertussis toxin. This effect was critically dependent on the presence of the US28 ORF in the HCMV genome but was independent of the amino acid sequence divergence of the three HCMV strains investigated. The constitutive activity of pUS28 is not explained by expression of pUS28 at high density in HCMV-infected cells. The pUS28 ligands RANTES and MCP-1 failed to stimulate binding of guanosine 5'-O-(3-[(35)S]thiotriphosphate to membranes of HCMV-infected cells and did not enhance constitutive activation of phospholipase C in intact HCMV-infected cells. These findings raise the possibility that the effects of CC chemokines and pertussis toxin on G protein-mediated transmembrane signaling previously observed in HCMV-infected cells are either independent of or not directly mediated by the protein product of ORF US28.     

Identification, sequence characterization, and analysis of expression profiles of three novel CC chemokines from domestic duck (Anas platyrhynchos)

Chemokines are low-molecular weight-chemotactic cytokines, which are involved in lymphocyte trafficking and migration of leucocytes to sites of injury, in immune surveillance and in healing process. They also play a role in pathogenesis of inflammatory diseases. Three novel CC chemokines were identified from domestic duck (Anas platyrhynchos) by screening of an enriched cDNA library constructed from mitogen-stimulated splenic mononuclear cells. Two of the clones (AB163 and AB330) had a very high nucleotide (both about 81%) and predicted amino acid level (71 and 76%, respectively) identity to the reported chicken macrophage inflammatory protein 1- (MIP-1; SCYA4) and regulated upon activation of normal T-cell expressed and secreted (RANTES; SCYA5) sequences. In phylogenetic analysis, these molecules clustered together with corresponding chemokines reported from other vertebrates. The third clone (AB187) had highest homology to chicken MIP-1 (36% amino acid identity) and showed closer relation to a number of chemokines belonging to monocyte chemoattractant proteins and MIP-1 chemokines. Expression of these molecules was upregulated upon mitogen stimulation of splenocytes as detected by semiquantitative RT-PCR. AB187 showed several fold increases (about 8.5 times) in the mRNA expression. Basal level expression of some of these chemokines was detected in both lymphoid and nonlymphoid tissues, including spleen, liver, lung, and bone marrow. Considering the importance of this animal species as a model for diseases such as chronic human hepatitis B, further studies will offer valuable insights into the role of these molecules in immunopathology of such diseases.The nucleotide sequences that are reported in this paper have been submitted to the NCBI Genbank database. Accession nos. AB163 (AY641435), AB187 (AY641436), and AB330 (AY641437).     

Identification of a gammaherpesvirus selective chemokine binding protein that inhibits chemokine action

Chemokines are involved in recruitment and activation of hematopoietic cells at sites of infection and inflammation. The M3 gene of gammaHV68, a gamma-2 herpesvirus that infects and establishes a lifelong latent infection and chronic vasculitis in mice, encodes an abundant secreted protein during productive infection. The M3 gene is located in a region of the genome that is transcribed during latency. We report here that the M3 protein is a high-affinity broad-spectrum chemokine scavenger. The M3 protein bound the CC chemokines human regulated upon activation of normal T-cell expressed and secreted (RANTES), murine macrophage inflammatory protein 1alpha (MIP-1alpha), and murine monocyte chemoattractant protein 1 (MCP-1), as well as the human CXC chemokine interleukin-8, the murine C chemokine lymphotactin, and the murine CX(3)C chemokine fractalkine with high affinity (K(d) = 1. 6 to 18.7 nM). M3 protein chemokine binding was selective, since the protein did not bind seven other CXC chemokines (K(d) > 1 microM). Furthermore, the M3 protein abolished calcium signaling in response to murine MIP-1alpha and murine MCP-1 and not to murine KC or human stromal cell-derived factor 1 (SDF-1), consistent with the binding data. The M3 protein was also capable of blocking the function of human CC and CXC chemokines, indicating the potential for therapeutic applications. Since the M3 protein lacks homology to known chemokines, chemokine receptors, or chemokine binding proteins, these studies suggest a novel herpesvirus mechanism of immune evasion.     

Cutting edge: identification of a novel chemokine receptor that binds dendritic cell- and T cell-active chemokines including ELC, SLC, and TECK

Searching for new receptors of dendritic cell- and T cell-active chemokines, we used a combination of techniques to interrogate orphan chemokine receptors. We report here on human CCX CKR, previously represented only by noncontiguous expressed sequence tags homologous to bovine PPR1, a putative gustatory receptor. We employed a two-tiered process of ligand assignment, where immobilized chemokines constructed on stalks (stalkokines) were used as bait for adhesion of cells expressing CCX CKR. These cells adhered to stalkokines representing ELC, a chemokine previously thought to bind only CCR7. Adhesion was abolished in the presence of soluble ELC, SLC (CCR7 ligands), and TECK (a CCR9 ligand). Complete ligand profiles were further determined by radiolabeled ligand binding and competition with >80 chemokines. ELC, SLC, and TECK comprised high affinity ligands (IC50 <15 nM); lower affinity ligands include BLC and vMIP-II (IC50 <150 nM). With its high affinity for CC chemokines and homology to CC receptors, we provisionally designate this new receptor CCR10.     

Modulation of immunity against herpes simplex virus infection via mucosal genetic transfer of plasmid DNA encoding chemokines

In this study, we examined the effects of murine chemokine DNA, as genetic adjuvants given mucosally, on the systemic and distal mucosal immune responses to plasmid DNA encoding gB of herpes simplex virus (HSV) by using the mouse model. The CC chemokines macrophage inflammatory protein 1beta (MIP-1beta) and monocyte chemotactic protein 1 (MCP-1) biased the immunity to the Th2-type pattern as judged by the ratio of immunoglobulin isotypes and interleukin-4 cytokine levels produced by CD4(+) T cells. The CXC chemokine MIP-2 and the CC chemokine MIP-1alpha, however, mounted immune responses of the Th1-type pattern, and such a response rendered recipients more resistant to HSV vaginal infection. In addition, MIP-1alpha appeared to act via the upregulation of antigen-presenting cell (APC) function and the expression of costimulatory molecules (B7-1 and B7-2), whereas MIP-2 enhanced Th1-type CD4(+) T-cell-mediated adaptive immunity by increasing gamma interferon secretion from activated NK cells. Our results emphasize the value of using the mucosal route to administer DNA modulators such as chemokines that function as adjuvants by regulating the activity of innate immunity. Our findings provide new insight into the value of CXC and CC chemokines, which act on different innate cellular components as the linkage signals between innate and adaptive immunity in mucosal DNA vaccination.