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Isolation and characterization of a novel CXC chemokine in common carp (Cyprinus carpio L.)
Molecular Immunology 39 (2003) 829–834
Isolation and characterization of a novel CXC chemokine in common carp (Cyprinus carpio L.) Ram Savan a , Tomoya Kono a , Azumi Aman b , Masahiro Sakai b,∗ a
United Graduate School of Agricultural Sciences, Kagoshima University, Korimoto 1-21-24, Kagoshima 890-0065, Japan b Faculty of Agriculture, Miyazaki University, Gakuen kibanadai nishi 1-1, Miyazaki 889-2192, Japan Received 23 July 2002; received in revised form 8 October 2002; accepted 25 October 2002
Abstract A novel CXC chemokine was identified for the first time in fish from common carp (Cyprinus carpio L.). The gene was obtained from the head kidney (HK) stimulated with LPS and Con A. The cDNA consists of 619 bp with a 37 bp 5 UTR and a 287 bp 3 UTR. An open reading frame of 368 bp encodes a 97 amino acid peptide, with a putative signal peptide of 20 aa. The gene has four cysteines residues, which are conserved, with first two cysteines separated with phenylalanine. By homology and phylogenetic analysis, the chemokine was found to be closer to human IP-10. Identities were significantly low to the CXC chemokines cloned from lamprey (Lampetra fluviatilis), flounder (Paralichthys olivaceus), rainbow trout (Onchorhynchus mykiss) and zebrafish (Danio rerio). The carp CXC chemokine contains three exons interrupted by two introns. The gene was transcribed from an early time point by stimulation with LPS and Con A. Organs in resting phase as well as stimulated expressed the gene. © 2002 Elsevier Science Ltd. All rights reserved. Keywords: Cytokines; Innate immunity; Inflammation; Mitogens; Fish; Expression analysis; Head kidney
1. Introduction Cytokines play significant role in initiating and regulating the inflammatory process, which is one of the important defenses in innate immunity. These are a group of molecules, which is subdivided into families like interleukins, lymphokines, growth factors, interferon’s and chemokines. Chemokines are a super family of small related protein molecules that are secreted by a variety of cells and that have, among their diverse biological properties, the ability to recruit a wide range of immune cells to the sites of infection and disease. These molecules act as chemo-attractants causing an influx of neutrophils, monocytes, T cells and basophils in humans. The functions like Abbreviations: Mig, monokine induced by interferon ␥; I-TAC, interferon-inducible T-cell chemoattractant; IP-10, interferon inducible protein-10; PF-4, platelet factor 4; GCP, granulocyte chemotactic protein; BLC, B-lymphocyte chemoattractant; BRAK, breast and kidney; NAP, neutrophil activating protein; SDF-1, stromal cell derived factor; GRO, growth related oncogene; LPS, lipopolysaccharide; Con A, concanavalin A; EST, expressed sequence tags ∗ Corresponding author. Tel.: +81-985-587219; fax: +81-985-587219. E-mail address: [email protected] (M. Sakai).
integrin activation, chemotaxis, lipid mediator biosynthesis, superoxide radical production, and granule enzyme release have been reported (Oppenheim et al., 1991; Schall and Bacon, 1994; Baggiolini and Dahinden, 1994; Bacon and Schall, 1996). Chemokines are classified into four groups: CXC(␣), CC(), C(␥) and CX3 C(␦) based on the arrangement of first two cysteine residues within the protein (Yoshie et al., 2000). Phylogenetic position that fish occupy makes them an attractive model for studying the evolution of immunity. In fish, only a few cytokines and chemokines have been known. As innate immunity is known to be important effectors in defense from the pathogens, isolation and characterization of cytokines in fish is of prime importance. The cytokines cloned in fish are either by EST or PCR mediated homology cloning. Apart from zebrafish chemokine (Long et al., 2000), which resembles to mammalian BRAK CXC chemokine, IL-8 like molecules have been recently cloned in rainbow trout (Laing et al., 2002), flounder (Lee et al., 2001) and lamprey (Najakshin et al., 1999). Phylogenetically, the CXC chemokines cloned, in fish, resemble closely to vertebrate IL-8 molecule and are represented in a single clade. The present report describes the isolation and characterization
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R. Savan et al. / Molecular Immunology 39 (2003) 829–834
of a cDNA transcript encoding a novel CXC chemokine in teleost.
2. Materials and methods 2.1. Stimulation of carp head kidney (HK) by mitogens Common carp, Cyprinus carpio L., (mean weight = 100 g) was obtained from Sunaso fisheries farm, Miyazaki, Japan. The fish were acclimated in an aerated fresh water tank at 20 ◦ C under natural photoperiod and fed for 2 weeks. HK cells of 12-fish were stimulated by treatment with 5 g/ml Con-A (Wako, Japan) and 10 g/ml LPS (E. coli 055:B5; Difco, USA) for 1, 4, 8, 12, 24 and 48 h in RPMI 1640 (Nissui, Japan) medium supplemented with 5% carp serum and 1% Streptomycin/Penicillin (Gibco, USA). 2.2. cDNA library construction Total RNA was isolated from 0.2 g of HK cells stimulated with LPS and Con-A using ISOGEN (Nippon Gene, Japan) according to the manufacturer’s instructions. Poly (A) RNA was purified using a quick prep micro mRNA kit (Amersham Pharmacia Biotech, Sweden). cDNA synthesis was performed using a cDNA synthesis kit (Invitrogen, USA), in a pSPORT cloning vector.
using primers CXC Fw 1, CXC Fw 2, CXC Fw 3, CXC Rv 1, CXC Rv 2 and CXC Rv 2a as shown in the Fig. 1. After an initial denaturation at 94 ◦ C for 5 min, 10 cycles were carried at 94 ◦ C, 10 s; 57 ◦ C, 30 s and 72 ◦ C for 2 min. Amplification was carried out for another 20 cycles with the same temperature profile but with increasing the holding time of elongation (3 s/cycle). A final delay was allowed for 7 min at 72 ◦ C. The products obtained were cloned into PGEM-T Easy vector (Promega, UK) and transformed into JM109 by electroporation (BTX 399; Genetronics, USA). 2.6. Accession numbers The nucleotide sequence data from this article have been retrieved from DDBJ/GenBank data libraries under the given accession numbers: common carp CXC, AB082985; European river lamprey, AJ231072; Japanese flounder, AF216646; rainbow trout, AJ300835; zebrafish, AF279919. Chemokines from human are under the accession numbers: PF4 (CXCL4), NM 002619; NAP2 (CXCL7), NM 002704; Mig (CXCL9), NM 002416; I-TAC (CXCL11), NM 005409; IP10 (CXCL10), NM 001565; IL8 (CXCL8), M17017; Gro α (CXCL1), NM 001511; Gro (CXCL2), NM 002089; Gro γ (CXCL3), NM 002090; BLC (CXCL13), NM 006419; CXCL16, AF301016; GCP2 (CXCL6), NM 002993; ENA78 (CXCL5), NM 002994; BRAK (CXCL14), NM 004887; SDF-1 (CXCL12), L36034.
2.3. Plasmid preparation and sequencing Plasmid DNA was extracted by alkaline lysis (Sambrook et al., 2001). The cDNA clones were subjected to pre-sequencing reaction using ThermoSequenase (Amersham, UK) with T7 or Sp6 (Nisshinbo, Japan) primers and sequenced on an automated DNA sequencer LIC-4200L (Li-Cor, USA). 2.4. Sequence and phylogenetic analysis The sequences were compared with those in the database using the BlastX algorithm (Altschul et al., 1990). Assignment of putative identities was set to a minimum P-value of 10−5 . Protein alignment and percentage identities were calculated by clustal W using Bioedit software (Hall, 1999). Phylogenetic analysis was carried out for the deduced amino acid sequences of carp and other vertebrate CXC chemokines. The phylogenetic tree was obtained by NJ (Neighbor Joining) method using PAUP software (Swofford, 1998). A detailed EST analysis of carp HK stimulated by LPS and Con A has been reported (Savan and Sakai, 2002). 2.5. PCR amplification and sequencing of the genomic DNA Genomic DNA was isolated from carp liver using formamide method as described in Sambrook et al. (2001). PCR was performed with 2 l (500 ng) template genomic DNA
2.7. Reverse transcription PCR analysis of CXC chemokine expression HK cells of carp were stimulated by treatment with 5 g/ml Con-A and 10 g/ml LPS for 1, 4, 8, 12, 24 and 48 h, individually, in RPMI 1640 medium supplemented with 5% carp serum and 1% Streptomycin/Penicillin. After determining the time course expression of chemokine gene in HK by LPS stimulation, organs like spleen, kidney, liver, heart, and brain were stimulated by LPS for 1 h. Unstimulated organs were also isolated. Total RNA extracted, from these treatments, were used for cDNA synthesis by ReverTra Dash (Toyobo, Japan). Gene specific primers for chemokine amplification were designed using highly conserved regions (CXC Fw 1 & CXC Rv 1), and amplified product gave a specific product of 293 bp. A set of -actin primers (Fw 5 -ACTACCTCATGAAGATCCTG-3 and Rv 5 -TTGCTGACCAC ATCTGCTG-3 ) served as an internal control of amount and quality of cDNA. All PCR reactions were performed according to the following protocol: 1 l of cDNA was mixed with 5 l dNTPs (10 M of each dNTP), 0.5 l Taq polymerase (5 units/l), 5 l of each gene specific primer and 27.5 l of water. The PCR was performed in a PCR apparatus (MJ Research, USA) with 30 reaction cycles of 0.5 min at 94 ◦ C, 0.5 min at 58 ◦ C and 3 min at 72 ◦ C. PCR products were electrophoresed on a 2% agarose gel to detect the specific bands.
Fig. 1. Genomic sequence structure of carp CXC chemokine. Coding sequences are shown in uppercase whereas UTR and introns are shown in lower case. Intron splice sites (gt. . . ag) are shown as underlined italics. The deduced aminoacid sequence is given below the nucleotides. The motifs associated to mRNA instability are shown by an over score and polyadenylation signal with double underscore. The stop codon is represented with an asterix. The primers used for gene expression and genomic sequences are indicated as arrows.
Fig. 2. Alignment of deduced amino acid sequence of carp CXC chemokine with other known genes in vertebrates. Identical amino acid residues are indicated by underlining. Dots indicate gaps that are introduced for optimal alignment. Triangles indicate the CXC motif. The percentages in parentheses indicate the overall and mature amino acid identities. The amino acid residues in bold fonts are the signal sequences of the respective chemokines.
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3. Results The carp CXC chemokine cDNA consists of 619 bp with a 37 bp 5 UTR, a 368 bp open reading frame encoding an 97 amino acid peptide and a 287 bp 3 UTR (Fig. 1). The 3 UTR contains a single typical polyadenylation signal (AATAAA) between nucleotides 567 and 571 and two instability motifs (ATTTA) responsible for the rapid degradation of mRNA. The predicted cleavage site of the signal sequence to the mature protein is between Glycine20 and Glutamine21 . The mature peptide has four cysteines residues, which are conserved, with first two cysteines separated with phenylalanine. One conserved sequence motif in both CC as well as CXC involves two adjacent hydrophobic residues (Isoleucine49 –Isoleucine50 ) and this motif is also conserved in carp chemokine.
The ELR motif, which is associated with specificity to neutrophil, is absent in the cloned carp CXC chemokine. Interestingly, the IL-8 molecules cloned so far do not harbor ELR motif, instead DLR motif is seen in trout IL-8 sequence. Carp CXC chemokines shares higher identities to mammalian non-ELR chemokines. Identities to both whole and mature protein were calculated. The aa sequence of carp chemokine draws highest identity (Figs. 2 and 3) to human IP-10 (38.0%), with similar levels of identities to human I-TAC (37.3%) and human Mig (33.1%). Carp chemokine shared identities of 29.1–30.7% to IL-8 genes of lamprey, rainbow trout, Japanese flounder. When only mature protein was compared, the highest identity was to human IP-10 (39.7%) followed by I-TAC (33.7%) and Mig (25.7%). Furthermore, the phylogenetic analysis revealed that, carp chemokine shares the same cluster with human
Fig. 3. Schematic representation of CXC chemokine intron/exon organization along with human IP 10 and trout IL-8 genomic structure. The alignment was performed using Bioedit software. Boxes are exons and horizontal lines are introns, with their nucleotide lengths. Shaded areas represent 5 and 3 UTRs.
Fig. 4. An unrooted phylogenetic tree constructed by PAUP software from the amino acid sequences of CXC chemokine genes from human and fish.
R. Savan et al. / Molecular Immunology 39 (2003) 829–834
Fig. 5. Expression patterns of common carp CXC chemokine gene studied by RT-PCR. (a) RT-PCR of chemokine expressed from head kidney cells stimulated by LPS at different time intervals. (b) Transcripts of CXC chemokine from different organs (Hk, head kidney, Br, brain; S, spleen; L, liver; G, gill) stimulated for 1 h by LPS. (c) Expression of CXC chemokine in organs at resting phase. β-Actin was used as a control of the amount and quality of cDNA for the above studies (h; hours).
IP-10, I-TAC and Mig (Fig. 4). Chemokines from other fishes share the same clade with human IL-8, except zebrafish CXC chemokine, which is closer to human BRAK chemokine. The carp chemokine harbors two introns in the sequence encoding the gene. The first and second introns are 143 and 109 bp, respectively. Typical intron splice motifs were observed at the 5 (GT) and 3 (AG) ends of each intron. The two introns are positioned exactly to the introns of mammalian chemokines. Interestingly, the third intron was absent in the carp chemokine, the mammalian non-ELR chemokines as well as IL-8 like chemokine from rainbow trout harbors three introns. The carp chemokine transcripts were expressed after 1 h of LPS and Con A stimulation (Fig. 5). All hematopoietic organs expressed the gene in resting phase.
4. Discussion Chemokines are key components in the process of leukocyte recruitment in inflammatory sites. The interaction of various chemokines with their receptors on leukocytes allows activation and chemotaxis of neutrophils, eosinophils, lymphocytes, monocytes necessary for migration to the sites of evolving inflammation. We have identified a novel CXC chemokine from the HK cells cDNA library stimulated by LPS and Con A. The identification and cloning of carp chemokine was based on the EST analysis. LPS is
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known to trigger a wide range of cellular responses, including the production of cytokines and chemokines, release of arachidonic acid, production of oxygen and nitrogen intermediaries responsible for pathophysiological reactions. LPS stimulation up regulated chemokines in human monocytes in SAGE analysis (Suzuki et al., 2000). The carp chemokine is 97 aa in length, similar to trout chemokine. The lengths of human I-TAC, Mig and PF 4 is 94, 125 and 101 aa, respectively. The carp homologue carries two ATTTA motifs, in 3 UTR region, which are instability motifs. These motifs are typical to inflammatory genes. Carp chemokine is shorter than the human non-ELR type chemokines. Studies have identified that three critical aa residues immediately preceding the first N-terminal cystine are important for neutrophil binding. Non-ELR chemokines like Mig, I-TAC and IP-10 the major targets are B and T cells. Carp chemokine has all the four cysteines and in conserved positions as found in mammalian and fish CXC chemokines. It is interesting to note that phenylalanine separates the first two cysteines forming the CXC motif. A molecular phylogenetic tree was constructed to further analyze the evolutionary relationship with known CXC chemokines. In unison to the percentage identity, the carp gene occupied a different clad in relation to the known CXC chemokines of fish and was closest to human IP-10. According to Najakshin et al. (1999) early divergence of CXC type chemokine lead to three basic types: SDF, IL-8 and I-TAC. Till recently, most of the CXC type chemokines cloned in fish were of IL-8 type. This study reports a novel CXC chemokine isolated for the first time in teleosts. Carp chemokine is composed of three exons and two introns. The typical ag–gt rule was followed at the acceptor and donor sites. The genomic structure of human IP-10 has three introns, however, the third intron was absent in carp and the first two introns were smaller in fish than its mammalian counterparts. The homologue of carp chemokine in zebrafish was examined by in silico approach from the genomic database of zebrafish. The intron/exon structure was similar to that of carp chemokine gene with only two introns. The conserved cystine residues spanned in the second and third introns. Although, trout IL-8 chemokine has three introns, the fourth exon is made up of only two aminoacids in the coding region. The CXC chemokines so far isolated in fish are homologues of human IL-8 like chemokines (Secombes et al., 2001). However, only zebrafish chemokine resembles BRAK like chemokine. This chemokine is implicated in developmental processes such as neural development. Our carp chemokine shows higher homology to I-TAC and IP-10 than to chemokines isolated from fish. IL-2 and interferons up regulate the non-ELR CXC chemokines like IP-10, Mig, and SDF-1. This suggests that, the interferon and IL-2 genes may be present in fish but not yet cloned in fish. IP-10 and Mig have selectivity to T-cells that have been stimulated by IL-2 and interferon. This results in the recruitment of activated/effector T cells,
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thereby, initiating the effector T cell immunity (Mackay, 1996). Furthermore, chemokines are known to exert their biological activities through G-protein coupled receptors of the target cells. In mammals, receptors of for CXC and CC have been identified. The IP-10, Mig and I-TAC have specificity to CXCR3, which is expressed on activated T cells and is important in selective recruitment of T cells during inflammation (Loetscher et al., 1996; Mackay, 1996; Cole et al., 1998). The fact that the carp CXC chemokine shares high homology with IP-10, Mig and I-TAC, it might have an important role to play during inflammation. However, functional studies have to be carried out to know the receptor specificity of carp CXC chemokine and its role during inflammation. Carp CXC chemokine transcripts could be detected in all the hematopoietic tissues of unchallenged carp, using RT-PCR. After LPS induction, the gene was transcribed in tissues at an higher level. The LPS and Con-A challenge at various time intervals indicated that the gene is induced at an early time point and the message is strong in the early period of induction. The chemokine tissues demonstrate protracted expression kinetics. The expression of the gene may reflect the importance in acute and chronic stresses in fish. At tissue level, the expression in all organs may be due to tissue macrophages present in the organs. We have cloned a novel non-ELR chemokine in fish. The genomic and expression data will form a solid basis for conducting further functional studies of this gene.
Acknowledgements This study was supported in part by a grant from the “Research for the Future (JSPS-RFTF 97L00902)” program from the Japan Society for the Promotion of Science. References Altschul, S.F., Gish, W., Miller, W., Myers, E.W., Lipman, E.W., 1990. Basic local alignment search tool. J. Mol. Biol. 215, 403. Bacon, K.B., Schall, T.J., 1996. Chemokines as mediators of allergic inflammation. Int. Arch. Aller. Immunol. 109, 97. Baggiolini, M., Dahinden, C.A., 1994. CC chemokines in allergic inflammation. Immunol. Today 15, 127.
Cole, K.E., Strick, C.A., Paradis, T.J., Ogborne, K.T., Loetscher, M., Gladue, R.P., Lin, W., Boyd, J.G., Moser, B., Wood, D.E., Sahagan, B.G., Neote, K., 1998. Interferon-inducible T cell alpha chemoattractant (I-TAC): a novel non-ELR CXC chemokine with potent activity on activated T cells through selective high affinity binding to CXCR3. J. Exp. Med. 187, 2009. Hall, T.A., 1999. BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucl. Acids. Symp. Ser. 41, 95. Laing, K.J., Zou, J.J., Wang, T., Bols, N., Hirono, I., Aoki, T., Secombes, C.J.., 2002. Identification and analysis of an interleukin 8-like molecule in rainbow trout Onchorynchus mykiss. Dev. Comp. Immunol. 26, 433–444. Lee, E.Y., Park, H.H., Kim, Y.T., Choi, T.J., 2001. Cloning and sequence analysis of the interleukin-8 gene from flounder (Paralichthys olivaceus). Gene 274, 237. Loetscher, M., Gerber, B., Loetscher, P., Jones, S.A., Piali, L., Clark-Lewis, I., Baggiolini, M., Moser, B., 1996. Chemokine receptor specific for IP10 and Mig: structure, function, and expression in activated T-lymphocytes. J. Exp. Med. 84, 963. Long, Q., Quint, E., Lin, S., Ekker, M., 2000. The zebrafish scyba gene encodes a novel CXC-type chemokine with distinctive expression patterns in the vestibulo-acoustic system during embryogenesis. Mech. Dev. 97, 183. Mackay, C.R., 1996. Chemokine receptors and T cell chemotaxis. J. Exp. Med. 184, 799. Najakshin, A.M., Mechetina, L.V., Alabyev, B.Y., Taranin, A.V., 1999. Identification of an IL-8 homolog in lamprey (Lampetra fluviatilis): early evolutionary divergence of chemokines. Eur. J. Immunol. 29, 375. Oppenheim, J.J., Zachariae, C.O., Mukaida, N., Matsushima, K., 1991. Properties of the novel proinflammatory supergene intercrine cytokine family. Ann. Rev. Immunol. 9, 617. Sambrook, J., Fritsch, E.F., Maniatis, T., 2001. Molecular Cloning: A Laboratory Manual, 3rd ed. Cold Spring Harbor Laboratory Press, New York. Savan, R., Sakai, M., 2002. Cyprinus carpio L., head kidney cells after stimulation by two mitogens, lipopolysaccharide and concanavalin-A. Comp. Biochem. Physiol. (Biochem. Mol. Biol.) 131, 71. Schall, T.J., Bacon, K.B., 1994. Chemokines, leukocyte trafficking and inflammation. Curr. Opin. Immunol. 6, 865. Secombes, C.J., Wang, T., Hong, S., Peddie, S., Crampe, M., Laing, K.J., Cunningham, C., Zou, J., 2001. Cytokines and innate immunity in fish. Dev. Comp. Immunol. 25, 713. Suzuki, T., Hashimoto, S., Toyoda, N., Nagai, S., Yamazaki, N., Dong, H.Y., Sakai, J., Yamashita, T., Nukiwa, T., Matsushima, K., 2000. Comprehensive gene expression profile of LPS-stimulated human monocytes by SAGE. Blood 96, 2584. Swofford, D.L., 1998. PAUP∗ . Phylogenetic Analysis Using Parsimony (∗ and Other Methods), Version 4. Sinauer Associates, Sunderland, MA. Yoshie, O., Nomiyama, N., Miyasaka, M., 2000. Chemokine Handbook: Cell Technology. Shujunsha Co. Ltd., Tokyo.
Isolation and characterization of a novel CXC chemokine in common carp (Cyprinus carpio L.) Ram Savan a , Tomoya Kono a , Azumi Aman b , Masahiro Sakai b,∗ a
United Graduate School of Agricultural Sciences, Kagoshima University, Korimoto 1-21-24, Kagoshima 890-0065, Japan b Faculty of Agriculture, Miyazaki University, Gakuen kibanadai nishi 1-1, Miyazaki 889-2192, Japan Received 23 July 2002; received in revised form 8 October 2002; accepted 25 October 2002
Abstract A novel CXC chemokine was identified for the first time in fish from common carp (Cyprinus carpio L.). The gene was obtained from the head kidney (HK) stimulated with LPS and Con A. The cDNA consists of 619 bp with a 37 bp 5 UTR and a 287 bp 3 UTR. An open reading frame of 368 bp encodes a 97 amino acid peptide, with a putative signal peptide of 20 aa. The gene has four cysteines residues, which are conserved, with first two cysteines separated with phenylalanine. By homology and phylogenetic analysis, the chemokine was found to be closer to human IP-10. Identities were significantly low to the CXC chemokines cloned from lamprey (Lampetra fluviatilis), flounder (Paralichthys olivaceus), rainbow trout (Onchorhynchus mykiss) and zebrafish (Danio rerio). The carp CXC chemokine contains three exons interrupted by two introns. The gene was transcribed from an early time point by stimulation with LPS and Con A. Organs in resting phase as well as stimulated expressed the gene. © 2002 Elsevier Science Ltd. All rights reserved. Keywords: Cytokines; Innate immunity; Inflammation; Mitogens; Fish; Expression analysis; Head kidney
1. Introduction Cytokines play significant role in initiating and regulating the inflammatory process, which is one of the important defenses in innate immunity. These are a group of molecules, which is subdivided into families like interleukins, lymphokines, growth factors, interferon’s and chemokines. Chemokines are a super family of small related protein molecules that are secreted by a variety of cells and that have, among their diverse biological properties, the ability to recruit a wide range of immune cells to the sites of infection and disease. These molecules act as chemo-attractants causing an influx of neutrophils, monocytes, T cells and basophils in humans. The functions like Abbreviations: Mig, monokine induced by interferon ␥; I-TAC, interferon-inducible T-cell chemoattractant; IP-10, interferon inducible protein-10; PF-4, platelet factor 4; GCP, granulocyte chemotactic protein; BLC, B-lymphocyte chemoattractant; BRAK, breast and kidney; NAP, neutrophil activating protein; SDF-1, stromal cell derived factor; GRO, growth related oncogene; LPS, lipopolysaccharide; Con A, concanavalin A; EST, expressed sequence tags ∗ Corresponding author. Tel.: +81-985-587219; fax: +81-985-587219. E-mail address: [email protected] (M. Sakai).
integrin activation, chemotaxis, lipid mediator biosynthesis, superoxide radical production, and granule enzyme release have been reported (Oppenheim et al., 1991; Schall and Bacon, 1994; Baggiolini and Dahinden, 1994; Bacon and Schall, 1996). Chemokines are classified into four groups: CXC(␣), CC(), C(␥) and CX3 C(␦) based on the arrangement of first two cysteine residues within the protein (Yoshie et al., 2000). Phylogenetic position that fish occupy makes them an attractive model for studying the evolution of immunity. In fish, only a few cytokines and chemokines have been known. As innate immunity is known to be important effectors in defense from the pathogens, isolation and characterization of cytokines in fish is of prime importance. The cytokines cloned in fish are either by EST or PCR mediated homology cloning. Apart from zebrafish chemokine (Long et al., 2000), which resembles to mammalian BRAK CXC chemokine, IL-8 like molecules have been recently cloned in rainbow trout (Laing et al., 2002), flounder (Lee et al., 2001) and lamprey (Najakshin et al., 1999). Phylogenetically, the CXC chemokines cloned, in fish, resemble closely to vertebrate IL-8 molecule and are represented in a single clade. The present report describes the isolation and characterization
0161-5890/02/$ – see front matter © 2002 Elsevier Science Ltd. All rights reserved. PII: S 0 1 6 1 - 5 8 9 0 ( 0 2 ) 0 0 2 5 7 - 2
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R. Savan et al. / Molecular Immunology 39 (2003) 829–834
of a cDNA transcript encoding a novel CXC chemokine in teleost.
2. Materials and methods 2.1. Stimulation of carp head kidney (HK) by mitogens Common carp, Cyprinus carpio L., (mean weight = 100 g) was obtained from Sunaso fisheries farm, Miyazaki, Japan. The fish were acclimated in an aerated fresh water tank at 20 ◦ C under natural photoperiod and fed for 2 weeks. HK cells of 12-fish were stimulated by treatment with 5 g/ml Con-A (Wako, Japan) and 10 g/ml LPS (E. coli 055:B5; Difco, USA) for 1, 4, 8, 12, 24 and 48 h in RPMI 1640 (Nissui, Japan) medium supplemented with 5% carp serum and 1% Streptomycin/Penicillin (Gibco, USA). 2.2. cDNA library construction Total RNA was isolated from 0.2 g of HK cells stimulated with LPS and Con-A using ISOGEN (Nippon Gene, Japan) according to the manufacturer’s instructions. Poly (A) RNA was purified using a quick prep micro mRNA kit (Amersham Pharmacia Biotech, Sweden). cDNA synthesis was performed using a cDNA synthesis kit (Invitrogen, USA), in a pSPORT cloning vector.
using primers CXC Fw 1, CXC Fw 2, CXC Fw 3, CXC Rv 1, CXC Rv 2 and CXC Rv 2a as shown in the Fig. 1. After an initial denaturation at 94 ◦ C for 5 min, 10 cycles were carried at 94 ◦ C, 10 s; 57 ◦ C, 30 s and 72 ◦ C for 2 min. Amplification was carried out for another 20 cycles with the same temperature profile but with increasing the holding time of elongation (3 s/cycle). A final delay was allowed for 7 min at 72 ◦ C. The products obtained were cloned into PGEM-T Easy vector (Promega, UK) and transformed into JM109 by electroporation (BTX 399; Genetronics, USA). 2.6. Accession numbers The nucleotide sequence data from this article have been retrieved from DDBJ/GenBank data libraries under the given accession numbers: common carp CXC, AB082985; European river lamprey, AJ231072; Japanese flounder, AF216646; rainbow trout, AJ300835; zebrafish, AF279919. Chemokines from human are under the accession numbers: PF4 (CXCL4), NM 002619; NAP2 (CXCL7), NM 002704; Mig (CXCL9), NM 002416; I-TAC (CXCL11), NM 005409; IP10 (CXCL10), NM 001565; IL8 (CXCL8), M17017; Gro α (CXCL1), NM 001511; Gro (CXCL2), NM 002089; Gro γ (CXCL3), NM 002090; BLC (CXCL13), NM 006419; CXCL16, AF301016; GCP2 (CXCL6), NM 002993; ENA78 (CXCL5), NM 002994; BRAK (CXCL14), NM 004887; SDF-1 (CXCL12), L36034.
2.3. Plasmid preparation and sequencing Plasmid DNA was extracted by alkaline lysis (Sambrook et al., 2001). The cDNA clones were subjected to pre-sequencing reaction using ThermoSequenase (Amersham, UK) with T7 or Sp6 (Nisshinbo, Japan) primers and sequenced on an automated DNA sequencer LIC-4200L (Li-Cor, USA). 2.4. Sequence and phylogenetic analysis The sequences were compared with those in the database using the BlastX algorithm (Altschul et al., 1990). Assignment of putative identities was set to a minimum P-value of 10−5 . Protein alignment and percentage identities were calculated by clustal W using Bioedit software (Hall, 1999). Phylogenetic analysis was carried out for the deduced amino acid sequences of carp and other vertebrate CXC chemokines. The phylogenetic tree was obtained by NJ (Neighbor Joining) method using PAUP software (Swofford, 1998). A detailed EST analysis of carp HK stimulated by LPS and Con A has been reported (Savan and Sakai, 2002). 2.5. PCR amplification and sequencing of the genomic DNA Genomic DNA was isolated from carp liver using formamide method as described in Sambrook et al. (2001). PCR was performed with 2 l (500 ng) template genomic DNA
2.7. Reverse transcription PCR analysis of CXC chemokine expression HK cells of carp were stimulated by treatment with 5 g/ml Con-A and 10 g/ml LPS for 1, 4, 8, 12, 24 and 48 h, individually, in RPMI 1640 medium supplemented with 5% carp serum and 1% Streptomycin/Penicillin. After determining the time course expression of chemokine gene in HK by LPS stimulation, organs like spleen, kidney, liver, heart, and brain were stimulated by LPS for 1 h. Unstimulated organs were also isolated. Total RNA extracted, from these treatments, were used for cDNA synthesis by ReverTra Dash (Toyobo, Japan). Gene specific primers for chemokine amplification were designed using highly conserved regions (CXC Fw 1 & CXC Rv 1), and amplified product gave a specific product of 293 bp. A set of -actin primers (Fw 5 -ACTACCTCATGAAGATCCTG-3 and Rv 5 -TTGCTGACCAC ATCTGCTG-3 ) served as an internal control of amount and quality of cDNA. All PCR reactions were performed according to the following protocol: 1 l of cDNA was mixed with 5 l dNTPs (10 M of each dNTP), 0.5 l Taq polymerase (5 units/l), 5 l of each gene specific primer and 27.5 l of water. The PCR was performed in a PCR apparatus (MJ Research, USA) with 30 reaction cycles of 0.5 min at 94 ◦ C, 0.5 min at 58 ◦ C and 3 min at 72 ◦ C. PCR products were electrophoresed on a 2% agarose gel to detect the specific bands.
Fig. 1. Genomic sequence structure of carp CXC chemokine. Coding sequences are shown in uppercase whereas UTR and introns are shown in lower case. Intron splice sites (gt. . . ag) are shown as underlined italics. The deduced aminoacid sequence is given below the nucleotides. The motifs associated to mRNA instability are shown by an over score and polyadenylation signal with double underscore. The stop codon is represented with an asterix. The primers used for gene expression and genomic sequences are indicated as arrows.
Fig. 2. Alignment of deduced amino acid sequence of carp CXC chemokine with other known genes in vertebrates. Identical amino acid residues are indicated by underlining. Dots indicate gaps that are introduced for optimal alignment. Triangles indicate the CXC motif. The percentages in parentheses indicate the overall and mature amino acid identities. The amino acid residues in bold fonts are the signal sequences of the respective chemokines.
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3. Results The carp CXC chemokine cDNA consists of 619 bp with a 37 bp 5 UTR, a 368 bp open reading frame encoding an 97 amino acid peptide and a 287 bp 3 UTR (Fig. 1). The 3 UTR contains a single typical polyadenylation signal (AATAAA) between nucleotides 567 and 571 and two instability motifs (ATTTA) responsible for the rapid degradation of mRNA. The predicted cleavage site of the signal sequence to the mature protein is between Glycine20 and Glutamine21 . The mature peptide has four cysteines residues, which are conserved, with first two cysteines separated with phenylalanine. One conserved sequence motif in both CC as well as CXC involves two adjacent hydrophobic residues (Isoleucine49 –Isoleucine50 ) and this motif is also conserved in carp chemokine.
The ELR motif, which is associated with specificity to neutrophil, is absent in the cloned carp CXC chemokine. Interestingly, the IL-8 molecules cloned so far do not harbor ELR motif, instead DLR motif is seen in trout IL-8 sequence. Carp CXC chemokines shares higher identities to mammalian non-ELR chemokines. Identities to both whole and mature protein were calculated. The aa sequence of carp chemokine draws highest identity (Figs. 2 and 3) to human IP-10 (38.0%), with similar levels of identities to human I-TAC (37.3%) and human Mig (33.1%). Carp chemokine shared identities of 29.1–30.7% to IL-8 genes of lamprey, rainbow trout, Japanese flounder. When only mature protein was compared, the highest identity was to human IP-10 (39.7%) followed by I-TAC (33.7%) and Mig (25.7%). Furthermore, the phylogenetic analysis revealed that, carp chemokine shares the same cluster with human
Fig. 3. Schematic representation of CXC chemokine intron/exon organization along with human IP 10 and trout IL-8 genomic structure. The alignment was performed using Bioedit software. Boxes are exons and horizontal lines are introns, with their nucleotide lengths. Shaded areas represent 5 and 3 UTRs.
Fig. 4. An unrooted phylogenetic tree constructed by PAUP software from the amino acid sequences of CXC chemokine genes from human and fish.
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Fig. 5. Expression patterns of common carp CXC chemokine gene studied by RT-PCR. (a) RT-PCR of chemokine expressed from head kidney cells stimulated by LPS at different time intervals. (b) Transcripts of CXC chemokine from different organs (Hk, head kidney, Br, brain; S, spleen; L, liver; G, gill) stimulated for 1 h by LPS. (c) Expression of CXC chemokine in organs at resting phase. β-Actin was used as a control of the amount and quality of cDNA for the above studies (h; hours).
IP-10, I-TAC and Mig (Fig. 4). Chemokines from other fishes share the same clade with human IL-8, except zebrafish CXC chemokine, which is closer to human BRAK chemokine. The carp chemokine harbors two introns in the sequence encoding the gene. The first and second introns are 143 and 109 bp, respectively. Typical intron splice motifs were observed at the 5 (GT) and 3 (AG) ends of each intron. The two introns are positioned exactly to the introns of mammalian chemokines. Interestingly, the third intron was absent in the carp chemokine, the mammalian non-ELR chemokines as well as IL-8 like chemokine from rainbow trout harbors three introns. The carp chemokine transcripts were expressed after 1 h of LPS and Con A stimulation (Fig. 5). All hematopoietic organs expressed the gene in resting phase.
4. Discussion Chemokines are key components in the process of leukocyte recruitment in inflammatory sites. The interaction of various chemokines with their receptors on leukocytes allows activation and chemotaxis of neutrophils, eosinophils, lymphocytes, monocytes necessary for migration to the sites of evolving inflammation. We have identified a novel CXC chemokine from the HK cells cDNA library stimulated by LPS and Con A. The identification and cloning of carp chemokine was based on the EST analysis. LPS is
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known to trigger a wide range of cellular responses, including the production of cytokines and chemokines, release of arachidonic acid, production of oxygen and nitrogen intermediaries responsible for pathophysiological reactions. LPS stimulation up regulated chemokines in human monocytes in SAGE analysis (Suzuki et al., 2000). The carp chemokine is 97 aa in length, similar to trout chemokine. The lengths of human I-TAC, Mig and PF 4 is 94, 125 and 101 aa, respectively. The carp homologue carries two ATTTA motifs, in 3 UTR region, which are instability motifs. These motifs are typical to inflammatory genes. Carp chemokine is shorter than the human non-ELR type chemokines. Studies have identified that three critical aa residues immediately preceding the first N-terminal cystine are important for neutrophil binding. Non-ELR chemokines like Mig, I-TAC and IP-10 the major targets are B and T cells. Carp chemokine has all the four cysteines and in conserved positions as found in mammalian and fish CXC chemokines. It is interesting to note that phenylalanine separates the first two cysteines forming the CXC motif. A molecular phylogenetic tree was constructed to further analyze the evolutionary relationship with known CXC chemokines. In unison to the percentage identity, the carp gene occupied a different clad in relation to the known CXC chemokines of fish and was closest to human IP-10. According to Najakshin et al. (1999) early divergence of CXC type chemokine lead to three basic types: SDF, IL-8 and I-TAC. Till recently, most of the CXC type chemokines cloned in fish were of IL-8 type. This study reports a novel CXC chemokine isolated for the first time in teleosts. Carp chemokine is composed of three exons and two introns. The typical ag–gt rule was followed at the acceptor and donor sites. The genomic structure of human IP-10 has three introns, however, the third intron was absent in carp and the first two introns were smaller in fish than its mammalian counterparts. The homologue of carp chemokine in zebrafish was examined by in silico approach from the genomic database of zebrafish. The intron/exon structure was similar to that of carp chemokine gene with only two introns. The conserved cystine residues spanned in the second and third introns. Although, trout IL-8 chemokine has three introns, the fourth exon is made up of only two aminoacids in the coding region. The CXC chemokines so far isolated in fish are homologues of human IL-8 like chemokines (Secombes et al., 2001). However, only zebrafish chemokine resembles BRAK like chemokine. This chemokine is implicated in developmental processes such as neural development. Our carp chemokine shows higher homology to I-TAC and IP-10 than to chemokines isolated from fish. IL-2 and interferons up regulate the non-ELR CXC chemokines like IP-10, Mig, and SDF-1. This suggests that, the interferon and IL-2 genes may be present in fish but not yet cloned in fish. IP-10 and Mig have selectivity to T-cells that have been stimulated by IL-2 and interferon. This results in the recruitment of activated/effector T cells,
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thereby, initiating the effector T cell immunity (Mackay, 1996). Furthermore, chemokines are known to exert their biological activities through G-protein coupled receptors of the target cells. In mammals, receptors of for CXC and CC have been identified. The IP-10, Mig and I-TAC have specificity to CXCR3, which is expressed on activated T cells and is important in selective recruitment of T cells during inflammation (Loetscher et al., 1996; Mackay, 1996; Cole et al., 1998). The fact that the carp CXC chemokine shares high homology with IP-10, Mig and I-TAC, it might have an important role to play during inflammation. However, functional studies have to be carried out to know the receptor specificity of carp CXC chemokine and its role during inflammation. Carp CXC chemokine transcripts could be detected in all the hematopoietic tissues of unchallenged carp, using RT-PCR. After LPS induction, the gene was transcribed in tissues at an higher level. The LPS and Con-A challenge at various time intervals indicated that the gene is induced at an early time point and the message is strong in the early period of induction. The chemokine tissues demonstrate protracted expression kinetics. The expression of the gene may reflect the importance in acute and chronic stresses in fish. At tissue level, the expression in all organs may be due to tissue macrophages present in the organs. We have cloned a novel non-ELR chemokine in fish. The genomic and expression data will form a solid basis for conducting further functional studies of this gene.
Acknowledgements This study was supported in part by a grant from the “Research for the Future (JSPS-RFTF 97L00902)” program from the Japan Society for the Promotion of Science. References Altschul, S.F., Gish, W., Miller, W., Myers, E.W., Lipman, E.W., 1990. Basic local alignment search tool. J. Mol. Biol. 215, 403. Bacon, K.B., Schall, T.J., 1996. Chemokines as mediators of allergic inflammation. Int. Arch. Aller. Immunol. 109, 97. Baggiolini, M., Dahinden, C.A., 1994. CC chemokines in allergic inflammation. Immunol. Today 15, 127.
Cole, K.E., Strick, C.A., Paradis, T.J., Ogborne, K.T., Loetscher, M., Gladue, R.P., Lin, W., Boyd, J.G., Moser, B., Wood, D.E., Sahagan, B.G., Neote, K., 1998. Interferon-inducible T cell alpha chemoattractant (I-TAC): a novel non-ELR CXC chemokine with potent activity on activated T cells through selective high affinity binding to CXCR3. J. Exp. Med. 187, 2009. Hall, T.A., 1999. BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucl. Acids. Symp. Ser. 41, 95. Laing, K.J., Zou, J.J., Wang, T., Bols, N., Hirono, I., Aoki, T., Secombes, C.J.., 2002. Identification and analysis of an interleukin 8-like molecule in rainbow trout Onchorynchus mykiss. Dev. Comp. Immunol. 26, 433–444. Lee, E.Y., Park, H.H., Kim, Y.T., Choi, T.J., 2001. Cloning and sequence analysis of the interleukin-8 gene from flounder (Paralichthys olivaceus). Gene 274, 237. Loetscher, M., Gerber, B., Loetscher, P., Jones, S.A., Piali, L., Clark-Lewis, I., Baggiolini, M., Moser, B., 1996. Chemokine receptor specific for IP10 and Mig: structure, function, and expression in activated T-lymphocytes. J. Exp. Med. 84, 963. Long, Q., Quint, E., Lin, S., Ekker, M., 2000. The zebrafish scyba gene encodes a novel CXC-type chemokine with distinctive expression patterns in the vestibulo-acoustic system during embryogenesis. Mech. Dev. 97, 183. Mackay, C.R., 1996. Chemokine receptors and T cell chemotaxis. J. Exp. Med. 184, 799. Najakshin, A.M., Mechetina, L.V., Alabyev, B.Y., Taranin, A.V., 1999. Identification of an IL-8 homolog in lamprey (Lampetra fluviatilis): early evolutionary divergence of chemokines. Eur. J. Immunol. 29, 375. Oppenheim, J.J., Zachariae, C.O., Mukaida, N., Matsushima, K., 1991. Properties of the novel proinflammatory supergene intercrine cytokine family. Ann. Rev. Immunol. 9, 617. Sambrook, J., Fritsch, E.F., Maniatis, T., 2001. Molecular Cloning: A Laboratory Manual, 3rd ed. Cold Spring Harbor Laboratory Press, New York. Savan, R., Sakai, M., 2002. Cyprinus carpio L., head kidney cells after stimulation by two mitogens, lipopolysaccharide and concanavalin-A. Comp. Biochem. Physiol. (Biochem. Mol. Biol.) 131, 71. Schall, T.J., Bacon, K.B., 1994. Chemokines, leukocyte trafficking and inflammation. Curr. Opin. Immunol. 6, 865. Secombes, C.J., Wang, T., Hong, S., Peddie, S., Crampe, M., Laing, K.J., Cunningham, C., Zou, J., 2001. Cytokines and innate immunity in fish. Dev. Comp. Immunol. 25, 713. Suzuki, T., Hashimoto, S., Toyoda, N., Nagai, S., Yamazaki, N., Dong, H.Y., Sakai, J., Yamashita, T., Nukiwa, T., Matsushima, K., 2000. Comprehensive gene expression profile of LPS-stimulated human monocytes by SAGE. Blood 96, 2584. Swofford, D.L., 1998. PAUP∗ . Phylogenetic Analysis Using Parsimony (∗ and Other Methods), Version 4. Sinauer Associates, Sunderland, MA. Yoshie, O., Nomiyama, N., Miyasaka, M., 2000. Chemokine Handbook: Cell Technology. Shujunsha Co. Ltd., Tokyo.