Introduction

Hepatitis B virus (HBV) causes acute and chronic hepatitis and leads to the development of life-threatening liver diseases, including cirrhosis and hepatocellular carcinoma (HCC).¹ It is estimated that 350 million people worldwide are chronically infected with HBV.² Therefore, chronic HBV infection remains one of the most prevalent chronic viral infections and causes a serious global health problem.

Woodchuck hepatitis virus (WHV) is a member of the family Hepadnaviridae and resembles a human HBV in having a similar genetic organization, identical replication mode and common tropism for hepatocytes.³ WHV infection of its natural host woodchuck (Marmota monax) resembles HBV infection in humans regarding the major virological and immunological aspects as well as associated diseases, including chronic hepatitis and HCC.^{3, 4, 5, 6, 7} The chronically WHV-infected woodchuck model thus represents an informative animal model to study the immunopathogenesis of HBV infection and develop therapeutic strategies combating HBV-related diseases. Therefore, basic information about and the availability of woodchuck-specific cytokines/chemokines are critical. Recently, several woodchuck cytokines have been characterized by molecular cloning, including the genes for woodchuck tumor necrosis factor, lymphotoxin-, -, interferon (IFN)- and cDNA sequences for IFN-, interleukin (IL)-2, IL-4, IL-6,⁸ IL-15⁹ and GM-CSF.¹⁰ In addition, cDNA sequence information for IL-10 and IL-12 can be found in the GeneBank database.⁸ This sequence information and resultant reagents have greatly facilitated studies of the immunopathogenesis of and immunotherapy for chronic hepatitis B in this model.

Interleukin-8 (IL-8) is a pleiotropic CXC chemokine with neutrophil chemotactic activity and a wide range of physiologic and pathophysiological activities.^{11, 12} It is produced by macrophages and a wide variety of cells, including hepatocytes, upon exposure to inflammatory stimulants.¹³ IL-8 is an important inflammatory chemokine involved in enhancing early host defense responses. Serum IL-8 levels were found to be increased significantly in patients with chronic viral hepatitis and have been associated with hepatic flares.^{14, 15} Furthermore, the expression of IL-8 has been found in various human cancers,¹⁶ including HCC.^{17, 18} Therefore, it is worthwhile to use an HBV infection-relevant animal model, the WHV-infected woodchucks, to further investigate the roles of IL-8 in the immunopathogenesis of chronic hepatitis B and the development of HCC. In this study, we describe the cloning of woodchuck IL-8 (wk-IL-8) cDNA and its genomic DNA. The biological activities of the cloned wk-IL-8 cDNA were demonstrated and the expression of wk-IL-8 in liver tissues from woodchucks with chronic hepatitis and HCC was characterized. These data provide a basis for further studies of IL-8 in the woodchuck model.

Results

Cloning and sequence analysis of wk-IL-8 cDNA

A PCR-based strategy with primers designed to areas of high homology observed in multiple alignments of known mammalian IL-8 gene sequences was used to clone the wk-IL-8 cDNA. Using cDNA from mitogen-stimulated woodchuck splenocytes as the template, a specific DNA fragment of 369 bp was amplified by PCR using primers WK-IL8-F and WK-IL8-R (Supplementary Table 1). Sequence analysis identified an open reading frame of 303 bp that represents the complete coding sequence of wk-IL-8. The 5'-untranslated sequence of wk-IL-8 was further obtained by 5'-RACE. The nucleotide sequence and deduced amino acid sequence are shown in Figure 1.

Figure 1.

Cloning of woodchuck IL-8 cDNA and its corresponding genomic DNA. Nucleotide and encoded amino acid sequences of wk-IL-8 are shown. The predicted amino acid sequence is indicated under the nucleotide sequence. The asterisk marks the stop codon. The nucleotides for the initiation codon in the first exon and the stop codon in the fourth exon are boxed and in bold. The conserved GT/AG sequences in the exon–intron boundaries are also marked in bold. The nucleotide sequences of wk-IL-8 cDNA and its corresponding genomic DNA have been submitted to GenBank under accession number EU332349.

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The wk-IL-8 cDNA codes for a 101-amino acid protein containing a CXC family signature sequence, which is conserved in mammals and other vertebrates. Figure 2a represents a comparison between the deduced amino acid sequence of the IL-8 protein and those of other mammals, including humans. The complete coding sequence of wk-IL-8 shares high identity with the IL-8 sequences of other species, ranging from 74 to 86% at the nucleotide level and from 67 to 87% at the amino acid level (Supplementary Table 2). A phylogenetic tree of these species was generated based on the amino acid sequences (Figure 2b). The amino acid sequence of wk-IL-8 has the highest identity to rabbit IL-8, which is also apparent in the phylogenetic analysis, in that wk-IL-8 clusters with rabbit IL-8.

Figure 2.

(a) Alignment of the encoded wk-IL-8 amino acid sequences with those of IL-8 from other species, including rabbit, cat, dog, sheep, bovine, human and guinea pig. Dashes indicate gaps introduced for maximal alignment. Conservation of amino acid identity is indicated in the consensus line with an asterisk (^*) and (:) and (.) indicate conservative and semi-conservative substitutions, respectively. Conserved ELRCXC and GPH motifs are boxed. The four highly conserved cysteine residues and predicted cleavage site for the signal peptide are indicated by arrowheads and an arrow, respectively. The amino acid sequences of human, bovine, sheep, dog, cat, guinea pig and rabbit IL-8 correspond to GenBank entries NP_000575, AAI03311, NP_001009401, NP_001003200, NP_001009281, AAA37049 and AAA31422, respectively. Multiple sequence alignment was generated by using the program Clustal W. (b) Phylogenetic analysis of IL-8 from different species on the amino acid level. The phylogenetic tree calculation was based on a sequence distance method and used the Neighbor Joining (NJ) algorithm.¹⁹

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According to the homology alignment and SignalP 3.0 analysis,²⁰ it was predicted that wk-IL-8 has a putative signal peptide of 22 aa and the start of the mature wk-IL-8 protein is at Ala₂₃ (the first amino acid of wk-IL-8 precursor is assigned No. 1). Comparison of wk-IL-8 with human IL-8 (hu-IL-8) also revealed that many amino acid residues significant for IL-8 structure and functions are conserved. These residues include four conserved cysteine residues, the CXC motif, the ELR motif, the GPH motif in the 57–61 turn and other conserved residues (Figure 2a). Four conserved cysteine residues are present in the wk-IL-8 precursor peptide at positions 34, 36, 61 and 77 (the numbers of amino acid residues are based on the amino acid sequence of the wk-IL-8 precursor). These cysteines have been shown to be essential for the formation of intramolecular disulfide bridges and subsequent tertiary structure in hu-IL-8.²¹ The first two cysteine residues, at positions 34 and 36, and the single intervening amino acid between them (Q in wk-IL-8) comprise the CXC motif, a signature sequence of the CXC chemokine family. Immediately preceding the CXC motif is the ELR motif (Glu₃₁-Leu₃₂-Arg₃₃), which confers chemokines with the ability to bind and activate PMNs (polymorphonuclear leukocytes) and to mediate angiogenesis.^{22, 23} Residues 57–61 (Ser₅₇, Gly₅₈, Pro₅₉, His₆₀ and Cys₆₁) form an atypical turn and, together with the disulfide bridges, provide a structural scaffold for the NH₂ terminal region²⁴ and play significant roles in ligand–receptor interactions. ELR and GPH motif (Gly₅₈, Pro₅₉, His₆₀) in the 57–61 turn have also been suggested to be important for receptor-binding affinity and specificity.^{22, 25, 26} The conservation of these significant residues and motifs in wk-IL-8 indicates that wk-IL8 may have structure and functions similar to hu-IL-8, including chemotactic effects on neutrophils and angiostatic effects.

Cloning and sequence analysis of wk-IL-8 genomic DNA

Cloning of the wk-IL-8 gene was performed by amplification of woodchuck liver genomic DNA with primers wk-IL-8F and wk-IL-8+491R (Supplementary Table 1). The overall arrangement and structure of the wk-IL-8 gene was determined by comparing the genomic sequence with the wk-IL-8 cDNA sequence, as shown in Figure 1. It has a four-exon and three-intron organization that is highly homologous with human IL-8 gene structures.²⁷ Exon 1 contains the 5' UTR and 64 bp of coding sequences, exons 2 and 3 contain 136 and 84 nucleotides, respectively, and exon 4 contains 22 bp of coding sequence (including the stop codon) as well as the 3' UTR. The nucleotide sequence of every exon is in complete agreement with that of the wk-IL-8 cDNA. The lengths of the three introns are 889, 246 and 432 bp, respectively, and they interrupt the codons for the amino acids as indicated in Figure 1. The exon–intron boundaries of all three introns in the wk-IL-8 gene are in accordance with the AG/GT rule,²⁸ with a consensus AG at all 3' acceptor sites and a GT at all 5' donor sites of the introns.

Neutrophil chemotactic activity of wk-IL-8

To test whether the cloned wk-IL-8 cDNA encoded a biologically active protein, we subcloned wk-IL-8 cDNA into a eukaryotic expression vector. The ability of the expressed protein to attract neutrophils was then assayed using a chemotaxis assay, a representative system for evaluating IL-8 functional activity. The wk-IL-8 expressing vector, pCMV-Tag-4A/wk-IL-8, encodes a full-length recombinant wk-IL-8 with a C-terminal Flag tag. After transfecting pCMV-Tag-4A/wk-IL-8 into 293FT cells, the expression of wk-IL-8 in transfected cells was confirmed by RT-PCR and immunoprecipitation–western blot analysis (Figures 3a and b). Culture supernatants from pCMV-Tag-4A/wk-IL-8 or vector-transfected 293FT cells were used to assay chemotactic activity for woodchuck neutrophils. Figure 3b shows that migration of woodchuck neutrophils exposed to culture supernatants from pCMV-Tag-4A/wk-IL-8-transfected cells was significantly greater than that of neutrophils exposed to supernatant from vector-transfected cells. These chemotaxis results demonstrated that the wk-IL-8 cDNA we cloned encodes a functionally active protein that possesses neutrophil chemotaxis activity. We also used the culture supernatants from pCMV-Tag-4A/wk-IL-8-transfected cells to test whether wk-IL-8 can attract human neutrophils. Our result showed that wk-IL-8 could induce the chemotaxis of human neutrophils across the species barrier (data not shown).

Figure 3.

Detection of wk-IL-8 expression in pCMV-Tag4A/wk-IL-8-transfected cells by RT-PCR. (a) and immunoprecipitation–western blot analysis (b). (a) Total RNA was isolated from cells transfected with vector (designated as V) or pCMV-Tag4A/wk-IL-8 (designated as IL-8). RT-PCRs were performed in the presence (+RT) or absence (-RT) of reverse transcriptase with primers WK-IL8+114F and WK-IL8+294R. The PCR products were then analyzed by agarose gel electrophoresis and visualized by ethidium bromide staining. M represents a 100 bp DNA size marker. (b) Culture supernatants from cells transfected with vector (V) or pCMV-Tag4A/wk-IL-8 (IL-8) were subjected to immunoprecipitation with anti-FLAG M2. Bound proteins were separated on a 4–12% Tris-Bis gel and detected by chemoluminescence with anti-FLAG M2 antibody followed by HRP-conjugated anti-mouse antibody. H and L indicate heavy chain and light chain of antibodies eluted from anti-FLAG beads after boiling the beads in sample buffer. (c) Chemotaxis activity of wk-IL-8 for woodchuck neutrophils. Woodchuck neutrophils were purified and loaded onto inserts in wells containing different dilutions of culture supernatants from pCMV-Tag-4A/wk-IL-8 (designated IL-8) or vector (designated 4A)-transfected 293FT cells and incubated for 30 min at 37 °C. To minimize the interference of fetal bovine serum (FBS) on the chemotaxis, the supernatants were diluted in FBS-containing DMEM to give a final concentration of FCS of 5% in the upper and lower chambers in each experimental group. Medium (DMEM) without chemokine and medium containing 10^–6 M FMLP were tested as negative and positive controls, respectively. After incubation, neutrophils migrating to the lower chambers were collected by Cytospin centrifugation, stained with Leu's stain and then enumerated. Cell numbers in at least five fields at a magnification of 200 were counted and the mean number was calculated. The extent of migration is expressed as a chemotactic index by dividing the number of cells that had migrated in the presence of chemokine by the number of cells that had migrated in the absence of chemokine (DMEM alone). The experiment was repeated two times and s.d. are shown with bars.

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Expression of wk-IL-8 in woodchuck liver tissues

It has been suggested that the liver may participate in inflammatory processes through the production of IL-8.²⁹ IL-8 has also been reported to be commonly and constitutively produced in human HCC specimens and HCC cell lines.¹⁸ Therefore, we investigated the expression of IL-8 in the livers of woodchucks with chronic hepatitis or HCC by real-time RT-PCR with wk-IL-8-specific primers based on wk-IL8 cDNA sequence that we cloned. First, using the woodchuck hepatoma cell line, WCH-17³⁰ we first studied whether the expression of wk-IL-8 could be induced in hepatocytes by lipopolysaccharides (LPS). We found that wk-IL-8 could be easily detected by conventional RT-PCR without any stimulation (data not shown), indicating that wk-IL-8 was constitutively expressed in WCH-17. The level of wk-IL-8 mRNA in this cell line was significantly increased by LPS treatment (10 g ml^-1) to more than 200 fold above unstimulated control as early as 1 h after stimulation, and then decreased to about 30-fold above unstimulated control at 3 h post-stimulation (Figure 4).

Figure 4.

Induction of IL-8 expression in the woodchuck hepatoma cell line WCH-17 by LPS stimulation. WCH-17 cells were seeded in 6-well plates at the density of 3 10⁵ cells per well 16 h before LPS stimulation. The culture media for the cells were then changed to medium with (LPS, 10 g ml^-1) or without (M) lipopolysaccharide and cultured for another 1 or 3 h. Total RNA was extracted and wk-IL-8 levels in cells were determined by real-time RT-PCR with primers WK-IL8+114F and WK-IL8+294R using the LightCycler system. Wk-IL-8 mRNA levels were normalized to the levels of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and the fold induction was obtained by taking the levels of wk-IL-8 in cells without LPS stimulation (M) as 1. Representative results from two experiments are shown as fold inductions.d.

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We then studied the expression levels of wk-IL-8 in clinical hepatoma specimens and adjacent non-tumor liver tissues from four chronic WHV carriers. The result showed that wk-IL-8 was expressed in all liver tissues examined, although the levels varied greatly in the different samples. The levels of wk-IL-8 were higher in the non-tumor tissues than in the tumor tissues from these woodchucks (Figure 5a). We further addressed the question of whether the levels of wk-IL-8 in liver tissues are correlated to the levels of viral load or viral RNA. As shown in Figure 5b, all woodchucks displayed medium to high levels of viral DNA in the serum and viral RNAs in the liver tissues. In all four WHV carrier woodchucks, viral RNA expression was more active in non-tumor tissues than in tumor tissues. Nevertheless, there was no obvious correlation between the levels of wk-IL-8 in the liver tissues and the levels of WHV RNA in the same tissues or the viral load in the corresponding serum samples.

Figure 5.

(a) IL-8 expression in liver tissues (tumor tissues vs non-tumor tissues) from chronically WHV-infected woodchucks. Total RNA was extracted from tumor and non-tumor tissues from livers obtained at autopsy. Wk-IL-8 mRNA levels were determined by real-time RT-PCR as described in Figure 4. (b) WHV RNA levels in liver tissues subjected to wk-IL-8 expression analysis in panel a. WHV RNA levels were determined by real-time RT-PCR with primers WHV+2117 and WHV-2303. Virus titers in the sera of these woodchucks before autopsy were also determined by real-time PCR.

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Discussion

In this study, we cloned the cDNA of wk-IL-8 and its corresponding genomic DNA to establish a basis for investigation of IL-8 in the woodchuck immune system and the pathogenesis of hepadnavirus-related diseases. The biological activity of the wk-IL-8 encoded by the cDNA that we cloned was assessed by neutrophil chemotaxis assay. Many mammalian IL-8 sequences have now been published,^{27, 31, 32, 33, 34} and the cDNA sequence and gene organization of the wk-IL-8 showed high similarity to the IL-8 sequences of other mammalian species, indicating a high degree of evolutionary conservation. Comparing the deduced amino acid sequence of wk-IL-8 with that of IL-8 proteins from humans and other species indicated that the critical structural features of IL-8 protein are well conserved in wk-IL-8, including the four cysteine residues involved in disulfide formation and ELR motif involved in receptor-binding activity and neutrophil-activating functions. The conservation of these structural features implies the conservation of functions. The demonstration that the wk-IL-8 we cloned, like the IL-8 proteins from other species, also possesses neutrophil chemotaxis activity further supports this notion.

A number of functional domains have been identified as essential for the binding of human IL-8 (hu-IL-8) to its receptors. ELR motifs, disulfide bridges and the SGPHCA turn (GPH motif was boxed in Figure 2a) have been shown to be critical for IL-8 receptor binding.^{24, 35} As all these residues are conserved in wk-IL-8, it is reasonable to predict that wk-IL-8, like rabbit IL-8,³⁶ can bind to cognate human receptors and stimulate them. The demonstration that culture supernatants containing wk-IL-8 could induce the chemotaxis of human neutrophils in this study supports this prediction. There are two receptors for hu-IL-8: CXCR1 and CXCR2. CXCR1 is highly selective for IL-8, whereas CXCR2 is relatively non-selective for IL-8 and can be activated by all other CXC chemokines with an ELR motif, including CXCL1-3 (growth-related oncogene-, , ), CXCL5 and CXCL7 (neutrophil-activating protein-2).^{12, 37} It has been shown that Tyr₁₃ and Lys₁₅ in mature hu-IL-8 (hu-IL-8) (corresponding to residues His₄₀ and Thr₄₂ in the wk-IL-8 precursor) are major determinants modulating human IL8 CXCR1-binding affinity. Rabbit IL-8 with His₁₃ and Thr₁₅ at these two positions can bind to both hu-IL-8 receptors but has 200-fold lower binding affinity for human CXCR1 than its hu-IL-8 homolog.³⁶ Replacement of rabbit His₁₃ and Thr₁₅ (corresponding to His₄₀ and Thr₄₂ in the wk-IL-8 precursor) with Tyr₁₃ and Lys₁₅ of the human molecules converted the low-affinity binding of the rabbit IL-8 to high-affinity binding of human CXCR1.³⁶ As residues 40 and 42 in the wk-IL-8 precursor are the same to those in rabbit IL-8, we predict that the binding properties of wk-IL-8 to human CXCL1 and CXCL2 should be very similar to those of rabbit IL-8, that is, wk-IL-8 can bind to human CXCR1 but with much lower affinity than its human homolog.

Although the woodchuck is classified as a rodent and is phylogenetically close to mouse species, the distance between the woodchuck and human may be closer than that between the woodchuck and mouse at the genetic level. It has been noted that the sequences of most woodchuck cytokine/chemokine genes available have higher homology to those of humans than to those of other rodents.^{10, 38} This trend also holds true for wk-IL-8 as shown in this study. A homolog of hu-IL-8 is missing in specific rodents, for example, mice and rats.³⁹ Hence, murine or rat models have not been considered fully representative of human disease(s) mediated by IL-8. The presence of a hu-IL-8 homolog with similar structure and functions in woodchucks has further strengthened the appropriateness of using chronic WHV carrier woodchucks to study the immunopathogenesis of chronic hepatitis B in humans.

During the preparation of this paper, a partial sequence of wk-IL-8 cDNA was deposited in the NCBI GenBank (accession no. EF216348). Comparison of this registered nucleotide sequence and our cDNA sequence revealed a one nucleotide mismatch that results in an amino acid residue change from a cysteine (our wk-IL-8) to an arginine at residue 20. As shown by multiple amino acid alignments, the cysteine residue is conserved at this position among various species. However, an amino acid difference at this position should not affect the biological properties of mature wk-IL-8 because this residue is located in the signal peptide.

Cloning of wk-IL-8 cDNA allowed us to develop a wk-IL-8-specific real-time RT-PCR assay to study the expression of IL-8 in woodchucks chronically infected with WHV. We paid attention to the liver tissues with chronic WHV-related hepatitis (non-tumor tissues) and HCC (tumor tissues). We first used a woodchuck hepatoma cell line, WCH-17, to demonstrate that woodchuck hepatocyte-derived cells could constitutively express IL-8 and that the expression of wk-IL-8 could be induced to much higher levels in the presence of LPS. This result is consistent with that found in human hepatocyte-derived cells, for example, HepG2 cells.^{40, 41} We then studied the expression of wk-IL-8 in surgically resected liver specimens from WHV-carrier woodchucks. It has been reported that IL-8 plays a role in hepatic injury in patients with chronic viral hepatitis.¹⁴ In line with this notion, wk-IL-8 RNA was readily detected in the livers (non-tumor parts) of woodchucks with chronic hepatitis. It was noted that there was no obvious correlation between the levels of wk-IL-8 in the liver tissues and the levels of WHV RNA in the same tissues or viral load in the corresponding serum samples. The expression of IL-8 in HCC was also found not to significantly differ with viral infection in human studies.^{42, 43} These results suggested that hepadnavirus itself may not directly contribute to the induction of wk-IL-8 in infected livers.

IL-8 has also been reported to contribute to human cancer progression through potential mitogenic, angiogenic and motogenic functions.⁴² It has been demonstrated in humans that HCC cells were the major producer of IL-8 in these tissues.^{18, 44} The incidence of portal and venous invasion was significantly higher in patients expressing more IL-8 at tumor sites than in the normal liver, implying that IL-8 might play an important role in metastasis.^{18, 42} In woodchucks, we could also detect wk-IL-8 expression in liver tumors; however, unlike in human HCC samples, the levels of wk-IL-8 in the tumor tissues were usually lower than those in non-tumor liver tissues. Although most of the clinical course of HCC in woodchucks is similar to that observed in humans, metastases, which commonly occur in human HCCs, are rare in woodchucks.⁴⁵ Lower levels of wk-IL-8 expression might therefore be one of the explanations for the rarity of HCC metastases in woodchucks. In human studies, it has been found that higher IL-8 expression is associated with HCCs having advanced pathologic stage, venous invasion and no tumor capsule.^{43, 44} Because most hepatic neoplasms associated with WHV infection are characteristically well differentiated trabecular HCCs,⁴⁶ this may partly explain why we could not detect higher wk-IL-8 expression in woodchuck HCCs. One study investigating the differential expression of IL-8 in breast cancer cells of various invasiveness revealed that transcriptional regulation could be a general mechanism of IL-8 overexpression. It was suggested that complex cooperation between NF-B, AP-1 and C/EBP transcription factors is required for greater expression of IL-8 in invasive cancer cells.⁴⁷ We thus speculated that lower expression of wk-IL-8 in woodchuck HCCs may also be attributed to the absence of activation of these transcriptional factors. Under this circumstance, cells in the non-tumor tissues would be the one predominantly expressing IL-8 due to the ongoing chronic hepatitis in these chronic WHV carriers. It will be interesting to test the speculation and investigate the underlying mechanisms for the differential regulation of IL-8 expression in woodchucks and humans. As the sample number and pathological information for the woodchucks in this study were quite limited, further investigation will be required to confirm this hypothesis.

As IL-8 is an important chemokine in many diseases, the cloning of wk-IL-8 cDNA enables us to study the role(s) of IL-8 in chronic hepatitis and HCC in the context of hepadnavirus infection in WHV carrier woodchucks. Results from this animal model will facilitate our understanding on the pathogenesis of HBV-related diseases in humans and the development of rational therapeutic strategies.

Materials and methods

Woodchucks and tissue samples

Woodchucks (Marmota monax) were purchased from North Eastern Wildlife (Ithaca, NY, USA) and maintained at the laboratory animal center, National Taiwan University College of Medicine. Experiments were conducted in accordance with the protocols approved by the Animal Care and Use committee of National Taiwan University College of Medicine. Spleen was taken out after euthanasia and put in RPMI medium for further purification of splenocytes. Autopsy liver tissues of woodchucks were snap frozen in liquid and stored at -80 °C until DNA or RNA extraction.

RT-PCR amplification and cloning of woodchuck IL-8 cDNA

Woodchuck splenocytes were purified using Ficoll/Paque (Amersham Pharmacia Biotech, Uppsala, Sweden). Splenocytes were cultured in 6-well dishes at 4 10⁶ cells per well and stimulated with 1 g ml^-1 ConA for 24 h. Total RNA was extracted from stimulated splenocytes using Trizol (Invitrogen, San Diego, CA, USA). cDNA was synthesized from 1 g of total RNA by Moloney murine leukemia virus reverse transcriptase (M-MLV RT) with oligo(dT) primer following the manufacturer's instructions (Invitrogen Life Technologies, CA, USA) and used as a template for PCR. Synthesis and amplification of cDNA encoding for wk-IL-8 was performed by polymerase chain reaction (PCR) with primers that were designed based on the highly conserved regions of the human (accession No. NM_000584), rabbit (M57439), sheep (NM_001009401), bovine (BC103310), cat (NM_001009281), dog (NM_001003200) and guinea pig (L04986) IL-8 genes. The primers used to amplify the woodchuck IL-8 were wk-IL-8 F and wk-IL-8 R (Supplementary Table 1). PCR was performed over 35 cycles of 95 °C, 30 s; 50 °C, 45 s; 72 °C, 45 s, followed by an extension step at 72 °C for 7 min. PCR products were cloned into the pyT&A vector according to the manufacturer's instruction (Yeastern Biotech, Taipei, Taiwan). Cloned cDNA was sequenced by a commercial service (Genomics, Taipei, Taiwan) using an ABI automated DNA 3730 sequencer (Applied Biosystems). Generated sequences were analyzed for similarity with other known IL-8 sequences using the BLAST program from the NCBI (National Center for Biotechnology Information). Having the cDNA clone covering the coding region of woodchuck IL-8, 5' sequences of wk-IL-8 cDNA were further determined by 5' RACE (CapFishingTM Full-length cDNA Premix Kit, Seegene, Rockville, MD, USA) with the primer WK-IL8+294R (Supplementary Table 1), according to the manufacturer's protocol.

Cloning of genomic DNA

Woodchuck genomic DNA was extracted from woodchuck liver tissues by Blood & Tissue Genomic DNA extraction kit (Viogene, Taiwan). To amplify the genomic DNA of woodchuck IL-8, 1 g of genomic DNA was used as a template for PCR with primers WK-IL-8+1F and WK-IL-8+491R (Supplementary Table 1). An amplified DNA fragment was cloned into pyT&A vector (Yeastern Biotech, Taipei, Taiwan) and verified by sequencing. The genomic DNA sequence of wk-IL-8 was submitted to GenBank data base under the NCBI accession number EU332349.

Construction of a mammalian expression plasmid for wk-IL-8

To express woodchuck IL-8 in mammalian cells and assay its biological activities, an expression plasmid for wk-IL-8 (pCMV-Tag-4A/wk-IL-8) was constructed. Briefly, a cDNA fragment encoding full-length wk-IL-8 was amplified from the plasmid pyT&A/wk-IL-8 by PCR using sense (WK-IL-8+1F) and anti-sense (WK-IL-8+303R) primers (Supplementary Table 1). Restriction sites for EcoRI and EcoRV endonucleases were introduced into the primers for construction of the expression plasmid. The amplified fragment was digested with EcoRI and EcoRV and ligated into EcoRI and EcoRV-digested vector pCMV-Tag 4A (Stratagene, Cedar Creek, TX, USA), resulting in the plasmid pCMV-Tag-4A/wk-IL-8.

Cell culture and transfection

Cells (293FT) were grown at 37 °C under 5% CO₂ in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal calf serum (FCS) (Biological Industries, Israel). 293FT was purchased from Invitrogen and is a cell line derived from 293, a human embryonal kidney-derived cell line,⁴⁸ and stably expresses adenovirus E1A protein and SV40 large T antigen. The cells were transfected with pCMV-Tag-4A/wk-IL-8 or vector (Invitrogen Life Technologies, CA, USA) at a cell density of 4 10⁵ cells per well in 12-well plates using lipofectamine 2000 following the user guidelines. Culture supernatants were collected on the second day post-transfection to assay for chemotactic activity for neutrophils.

Detection of wk-IL-8 by immunoprecipitation–western blotting

Culture supernatants from pCMV-Tag-4A/wk-IL-8- or vector-transfected cells were pre-cleared by centrifugation at 15 000 r.p.m. for 1 min to remove cell debris. Immunoprecipitation was done at 4 °C overnight with anti-FLAG M2 agarose beads (Sigma-Aldrich, St Louis, MO, USA). Immunoprecipitates were washed three times in RIPA buffer . Bound proteins were eluted by boiling in Laemmli sample buffer for 5 min and subjected to NuPage electrophoresis on a 4–12% Tris-Bis Gel (Invitrogen Life Technologies, CA, USA). For immunoblotting, the proteins were electrotransferred to a nitrocellulose membrane (Amersham, Arlington Heights, IL, USA) at 90 V for 40 min in the cold. The proteins on the membrane were then probed with anti-Flag antibody at a 1:5000 dilution (Sigma-Aldrich, St Louis, MO, USA) and then visualized by chemiluminescence using the Immobilon Western reagents according to the manufacturer's protocol (Millipore, Billerica, MA, USA).

Chemotaxis assay

Woodchuck peripheral blood neutrophils were freshly isolated from heparinized woodchuck blood according to methods described earlier.⁴⁹ Briefly, dextran (4.5% (wt/vol); T500 (Pharmacia, Uppsala, Sweden)) was added to blood at a ratio of 1:5, and incubated at room temperature for 40 min. The upper fraction of leukocyte-enriched plasma was then layered onto Ficoll/Paque (Pharmacia) at a ratio of 4:3 and centrifuged at 2000 r.p.m. for 30 min. Pellets at the bottom of the centrifuge tubes were washed, and residual erythrocytes were lysed by distilled water treatment for 30 s. An equal volume of 1.8% sodium chloride solution was added to stop this hypotonic shock. Cells were finally suspended at 5 10⁶ cells per ml in DMEM without fetal calf serum.

Chemotaxis assays were performed by a method described earlier,⁵⁰ with slight modification. Transwell inserts with a 5-M pore size in 24-well plates (Corning Costar Crop., NY, USA) were used for the chemotaxis assay. Peripheral blood leukocytes (5 10⁵) in 100 l were loaded into the insert above the well containing 600 l of culture supernatant collected from vector or pCMV-Tag-4A/wk-IL-8-transfected 293FT cells. Various dilutions of culture supernatants were tested for neutrophil chemotactic activity. Serum-free culture medium (DMEM) was used as a negative control. N-formylmethionylleucyl phenylalanine (FMLP, Sigma) was used as a positive control. The plates were incubated at 37 °C in a CO₂ incubator for 30 min. After incubation, cells attached to the bottom of the insert were gently washed off and combined with the cells in the lower chamber. The cells were mounted onto slides using a Cytospin centrifuge at 500 r.p.m. for 5 min at room temperature. The slides were air dried and stained with Liu's stain.⁵¹ The cells in five randomly selected fields were counted under a microscope at 200-fold magnification and the mean of the five fields in each well was calculated. The extent of cell migration was expressed as a chemotactic index determined by dividing the number of cells that had migrated in the presence of chemokine by the number of cells that had migrated in the absence of chemokine (DMEM alone).

RT-PCR analysis of wk-IL-8 expression

Total RNAs from cultured cells or liver tissues were extracted by Trizol (Invitrogen Life Technologies) according to the manufacturer's instructions. The wk-IL-8 mRNA expression levels were quantitatively analyzed by real-time RT-PCR. Briefly, 1 g of total RNA was reverse transcribed to cDNA by M-MLV RT (Invitrogen Life Technologies) with oligo(dT) primer at 50 °C for 60 min. Quantitative real-time PCR was carried out using a LightCycler System (Roche Applied Science, Mannheim, Germany) with primers WK-IL8+114F and WK-IL8+294R by a Lightcycler FastStart DNA master SYBR I kit. The thermal profile for real-time PCR was: 40 cycles of 10 s at 95 °C, 10 s at 60 °C and 10 s at 72 °C. Melting curve analysis was performed to ensure the specificity of the PCR product. PCR products were also run on agarose gels to validate the expected product length. A standard curve was constructed using dilutions with defined copy numbers of a plasmid containing the wk-IL-8 cDNA fragment. Standard PCR was performed in a Biometra thermal cycler (Biometra, Gottingen, Germany) with primers WK-IL8+114F and WK-IL8+294R using the following temperature profile: denaturation at 94 °C for 30 s, primer annealing at 60 °C for 45 s, and primer extension at 72 °C for 30 s, for a total of 30 cycles.

Quantitation of serum viral load and liver WHV pregenomic RNA

A Syber green I-based real-time PCR was used to quantitate WHV DNA in the sera and WHV pregenomic RNA in the liver tissues using a LightCycler System (Roche Applied Science). WHV DNA was extracted from 100 l woodchuck serum samples and eluted into 200 l double-distilled water using the QIAamp DNA Blood kit (Qiagen) according to the manufacturer's protocol. One microliter of extracted DNA solution was used for real-time PCR with primers WHV+2117 and WHV-2303 and a Lightcycler FastStart DNA master SYBR I kit. The amplification conditions included initial denaturation at 95 °C for 10 min, followed by 40 cycles of denaturation at 95 °C for 10 s, annealing at 55 °C for 15 s and extension at 72 °C for 14 s. The WHV DNA standard used as a control was BamHI-linearized whole WHV genomic DNA cloned at BamHI site of pGem4Z (Promega). The amount of WHV pregenomic RNA in woodchuck liver tissue was also determined by the real-time PCR described above except that cDNAs reverse-transcribed from total liver RNAs were used as templates in the reaction.

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Acknowledgements

We are grateful to Drs Betty A Wu-Hsieh, Chung-Yi Hu and Fang Liao for helpful suggestions and discussions on neutrophil chemotaxis assay. We also thank Miss Hui-Chu Tu and Miss Hsiu-Li Chou for their excellent technical assistance.

This research was supported by funds from the National Research Program for Genomic Medicine of National Science Council (Grant NSC 96-3112-B-002-009), National Taiwan University (Grant 95R0066-BM02-04) and National Taiwan University Hospital (Grant NTUH.97A10).

Supplementary Information accompanies the paper on Genes and Immunity website (http://www.nature.com/gene)