Potential Interaction between CCR1 and Its Ligand, CCL3, Induced by Endogenously Produced Interleukin-1 in Human Hepatomas
2003; Elsevier BV; Volume: 162; Issue: 4 Linguagem: Inglês
10.1016/s0002-9440(10)63921-1
ISSN1525-2191
AutoresPeirong Lu, Yasunari Nakamoto, Yoko Nemoto‐Sasaki, Chifumi Fujii, Hui Wang, Minako Hashii, Yasukazu Ohmoto, Shuichi Kaneko, Kenichi Kobayashi, Naofumi Mukaida,
Tópico(s)Immunotherapy and Immune Responses
ResumoHepatoma cell lines can produce a massive amount of chemokines in response to various stimuli including hepatitis viruses and their products. However, it remains elusive on the types of chemokine receptor(s) expressed in the hepatoma tissues and its roles in hepatoma development. To clarify these points, we examined the chemokine receptor expression in six human hepatoma cell lines. All of the hepatoma cell lines constitutively and exclusively expressed CCR1 mRNA and its protein on their cell surface. CCR1 expression was also detected on hepatoma cells and to a lesser degree, on endothelial cells in hepatoma tissues but not in normal liver tissues. Furthermore, CCL3 expression was detected in hepatoma cells, endothelial cells, and to a lesser degree, fibroblast-like cells in hepatoma tissue, whereas only occasional vascular endothelial cells and inflammatory cells in normal liver tissues were weakly positive for CCL3. Moreover, the forskolin-mediated increases in intracellular cAMP concentrations were inhibited by the ligands for CCR1, CCL3, CCL4, and CCL5, suggesting that the expressed CCR1 was functional. Four hepatoma cell lines produced CCL3 only in response to interleukin (IL)-1α and IL-1β. Finally, IL-1α and IL-1β were detected abundantly in hepatoma tissues but not in normal liver tissues. Thus, IL-1 may enhance the local production of CCL3, which may interact with CCR1 expressed on hepatoma cells, in an autocrine and/or paracrine manner. Hepatoma cell lines can produce a massive amount of chemokines in response to various stimuli including hepatitis viruses and their products. However, it remains elusive on the types of chemokine receptor(s) expressed in the hepatoma tissues and its roles in hepatoma development. To clarify these points, we examined the chemokine receptor expression in six human hepatoma cell lines. All of the hepatoma cell lines constitutively and exclusively expressed CCR1 mRNA and its protein on their cell surface. CCR1 expression was also detected on hepatoma cells and to a lesser degree, on endothelial cells in hepatoma tissues but not in normal liver tissues. Furthermore, CCL3 expression was detected in hepatoma cells, endothelial cells, and to a lesser degree, fibroblast-like cells in hepatoma tissue, whereas only occasional vascular endothelial cells and inflammatory cells in normal liver tissues were weakly positive for CCL3. Moreover, the forskolin-mediated increases in intracellular cAMP concentrations were inhibited by the ligands for CCR1, CCL3, CCL4, and CCL5, suggesting that the expressed CCR1 was functional. Four hepatoma cell lines produced CCL3 only in response to interleukin (IL)-1α and IL-1β. Finally, IL-1α and IL-1β were detected abundantly in hepatoma tissues but not in normal liver tissues. Thus, IL-1 may enhance the local production of CCL3, which may interact with CCR1 expressed on hepatoma cells, in an autocrine and/or paracrine manner. Chemokines are a family of chemoattractant cytokines that can be classified into four groups based on the position of the first two cysteines adjacent to the amino-terminus, CXC, CC, C, and CX3C.1Premack BA Schall TJ Chemokine receptors: gateways to inflammation and infection.Nat Med. 1996; 2: 1174-1178Crossref PubMed Scopus (571) Google Scholar, 2Rollins BJ Chemokines.Blood. 1997; 90: 909-928Crossref PubMed Google Scholar, 3Rossi D Zlotnik A The biology of chemokines and their receptors.Annu Rev Immunol. 2000; 18: 217-242Crossref PubMed Scopus (2083) Google Scholar, 4Mellado M Rodriguez-Frade JM Manes S Martinez AC Chemokine signaling and functional responses: the role of receptor dimerization and TK pathway activation.Annu Rev Immunol. 2001; 19: 397-421Crossref PubMed Scopus (292) Google Scholar The biological effects of chemokines seem to be mediated through their interaction with a family of specific G protein-coupled seven-transmembrane domain receptors.1Premack BA Schall TJ Chemokine receptors: gateways to inflammation and infection.Nat Med. 1996; 2: 1174-1178Crossref PubMed Scopus (571) Google Scholar, 2Rollins BJ Chemokines.Blood. 1997; 90: 909-928Crossref PubMed Google Scholar, 3Rossi D Zlotnik A The biology of chemokines and their receptors.Annu Rev Immunol. 2000; 18: 217-242Crossref PubMed Scopus (2083) Google Scholar, 4Mellado M Rodriguez-Frade JM Manes S Martinez AC Chemokine signaling and functional responses: the role of receptor dimerization and TK pathway activation.Annu Rev Immunol. 2001; 19: 397-421Crossref PubMed Scopus (292) Google Scholar Based on their specific chemokine ligands, the receptor proteins are categorized as CC chemokine receptors (CCR), CXC chemokine receptors (CXCR), C chemokine receptor (CR), and CX3C chemokine receptor (CX3CR).1Premack BA Schall TJ Chemokine receptors: gateways to inflammation and infection.Nat Med. 1996; 2: 1174-1178Crossref PubMed Scopus (571) Google Scholar, 2Rollins BJ Chemokines.Blood. 1997; 90: 909-928Crossref PubMed Google Scholar, 3Rossi D Zlotnik A The biology of chemokines and their receptors.Annu Rev Immunol. 2000; 18: 217-242Crossref PubMed Scopus (2083) Google Scholar, 4Mellado M Rodriguez-Frade JM Manes S Martinez AC Chemokine signaling and functional responses: the role of receptor dimerization and TK pathway activation.Annu Rev Immunol. 2001; 19: 397-421Crossref PubMed Scopus (292) Google Scholar Until the present, nearly 20 chemokine receptors have been identified and each receptor mediates the functions of a corresponding chemokine(s) (http://cytokine.medic.kumamoto-u.ac.jp). A wide variety of tumor cells and normal cells can produce various chemokines under various conditions. Moreover, several lines of evidence suggest that produced chemokines have a crucial role in the progression of tumor cells by recruiting leukocyte infiltration and modulating tumor cell motility.5Gerard C Rollins BJ Chemokines and disease.Nat Immunol. 2001; 2: 108-115Crossref PubMed Scopus (1191) Google Scholar, 6Desbaillets I Diserens AC Tribolet N Hamou MF Van Meir EG Upregulation of interleukin 8 by oxygen-deprived cells in glioblastoma suggests a role in leukocyte activation, chemotaxis, and angiogenesis.J Exp Med. 1997; 186: 1201-1212Crossref PubMed Scopus (218) Google Scholar, 7Kunz M Hartmann A Flory E Toksoy A Koczan D Thiesen HJ Mukaida N Neumann M Rapp UR Brocker EB Gillitzer R Anoxia-induced up-regulation of interleukin-8 in human malignant melanoma. A potential mechanism for high tumor aggressiveness.Am J Pathol. 1999; 155: 753-763Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar, 8Mukaida N Interleukin-8: an expanding universe beyond neutrophil chemotaxis and activation.Int J Hematol. 2000; 72: 391-398PubMed Google Scholar Persistent chronic infection with hepatitis B virus or hepatitis C virus frequently resulted in the development of hepatocellular carcinoma, which ranks as the eighth cause of death among human cancers and is endemic in Asia, Africa, and southern Europe.9Schafer DF Sorrell MF Hepatocellular carcinoma.Lancet. 1999; 353: 1253-1257Abstract Full Text Full Text PDF PubMed Scopus (515) Google Scholar We previously observed that the X protein of hepatitis B virus and the hepatitis C virus NS5A gene product can induce in vitro gene expression and protein production of interleukin (IL)-8/CXCL8,10Mahe Y Mukaida N Kuno K Akiyama M Ikeda N Matsushima K Murakami S Hepatitis B virus X protein transactivates human interleukin-8 gene through acting on nuclear factor kB and CCAAT/enhancer-binding protein-like cis-elements.J Biol Chem. 1991; 266: 13759-13763Abstract Full Text PDF PubMed Google Scholar, 11Polyak SJ Khabar KS Paschal DM Ezelle HJ Duverlie G Barber GN Levy DE Mukaida N Gretch DR Hepatitis C virus nonstructural 5A protein induces interleukin-8, leading to partial inhibition of the interferon-induced antiviral response.J Virol. 2001; 75: 6095-6106Crossref PubMed Scopus (281) Google Scholar one of CXC chemokines. Moreover, IL-8/CXCL8 protein was detected in hepatoma cells in some hepatoma tissues.12Iguchi A Kitajima I Yamakuchi M Ueno S Aikou T Kubo T Matsushima K Mukaida N Maruyama I PEA3 and AP-1 are required for constitutive IL-8 gene expression in hepatoma cells.Biochem Biophys Res Commun. 2000; 279: 166-171Crossref PubMed Scopus (43) Google Scholar However, it still remains elusive whether receptors for IL-8/CXCL8 are expressed in hepatoma tissues. Moreover, little is currently known about the expression of other chemokine receptors in hepatoma tissues. Hence, we examined chemokine receptor expression in hepatoma cell lines. CCR1 was the only chemokine receptor expressed consistently in all hepatoma cell lines that we investigated. Moreover, CCR1 and its ligand, CCL3/macrophage inflammatory protein-1α, were expressed abundantly in hepatoma cells in human hepatoma tissues. Furthermore, we provided evidence that a proinflammatory cytokine, IL-1, may regulate the expression of CCL3 in hepatoma tissues. Human recombinant CCL3/macrophage inflammatory protein-1α and an anti-human CCL3 monoclonal antibody (clone ANOC801) were generously provided by Ohtsuka Pharmaceutical Co., Ltd. (Tokushima, Japan). Human IL-1α and tumor necrosis factor (TNF)-α were kindly provided by Dainippon Pharmaceutical Company (Osaka, Japan). Rabbit anti-human CCR1 antibodies were prepared as described previously.13Su SB Mukaida N Wang J Nomura H Matsushima K Preparation of specific polyclonal antibodies to a C-C chemokine receptor, CCR1, and determination of CCR1 expression on various types of leukocytes.J Leukoc Biol. 1996; 60: 658-666PubMed Google Scholar, 14Furuichi K Wada T Sakai N Iwata Y Yoshimoto K Shimizu M Kobayashi K Takasawa K Kida H Takeda SI Mukaida N Matsushima K Yokoyama H Distinct expression of CCR1 and CCR5 in glomerular and interstitial lesions of human glomerular diseases.Am J Nephrol. 2000; 20: 291-299Crossref PubMed Scopus (74) Google Scholar, 15de Wynter EA Heyworth CM Mukaida N Jaworska E Weffort-Santos A Matushima K Testa NG CCR1 chemokine receptor expression isolates erythroid from granulocyte-macrophage progenitors.J Leukoc Biol. 2001; 70: 455-460PubMed Google Scholar, 16Su S Mukaida N Wang J Zhang Y Takami A Nakao Inhibition of immature erythroid progenitor cell proliferation by macrophage inflammatory protein-1alpha by interacting mainly with a C-C chemokine receptor, CCR1.Blood. 1997; 90: 605-611PubMed Google Scholar Human recombinant CCL4/macrophage inflammatory protein-1β, CCL5/regulated on activation normal T lymphocyte expressed and secreted (RANTES), CCL11/eotaxin, and IL-1β were purchased from Pepro Tech EC (London, UK). Monoclonal mouse anti-human IL-1α and IL-1β antibodies were purchased from Genzyme (Cambridge, MA) and R&D Systems, Inc. (Minneapolis, MN), respectively. Rabbit anti-human CCL4 antibodies were prepared by immunizing rabbits with recombinant human CCL4. Six human hepatoma-derived cell lines including HuH7 (differentiated hepatoma cell line), HepG2 (epithelial-like cell line), HLE and HLF (nondifferentiated epithelial cell lines), SK-Hep1 (endothelial cell origin), and Hep3B (hepatitis B virus-positive hepatocellular carcinoma cell line) were used in the experiments.17Pugh JC Yaginuma K Koike K Summers J Duck hepatitis B virus (DHBV) particles produced by transient expression of DHBV DNA in a human hepatoma cell line are infectious in vitro.J Virol. 1988; 62: 3513-3516Crossref PubMed Google Scholar, 18Diamond L Kruszewski F Aden DP Knowles BB Baird WM Metabolic activation of benzo[a]pyrene by a human hepatoma cell line.Carcinogenesis. 1980; 1: 871-875Crossref PubMed Scopus (100) Google Scholar, 19Dor I Namba M Sato J Establishment and some biological characteristics of human hepatoma cell lines.Gann. 1975; 66: 385-392PubMed Google Scholar, 20Heffelfinger SC Hawkins HH Barrish J Taylor L Darlington GJ SK HEP-1: a human cell line of endothelial origin.In Vitro Cell Dev Biol. 1992; 28A: 136-142Crossref PubMed Scopus (130) Google Scholar, 21Knowles BB Howe CC Aden DP Human hepatocellular carcinoma cell lines secrete the major plasma proteins and hepatitis B surface antigen.Science. 1980; 209: 497-499Crossref PubMed Scopus (1491) Google Scholar These cell lines were maintained in Dulbecco's modified essential medium (DMEM; Sigma Chemical Co., St. Louis, MO) supplemented with 10% heat-inactivated fetal bovine serum (Atlanta Biologicals, Norcross, GA) in a humidified incubator at 37°C in 5% CO2. Liver specimens were surgically obtained from 15 individuals with their informed consent. Twelve of them were confirmed to be positive for antibodies against hepatitis C virus and presented with histological diagnosis of cirrhosis with hepatocellular carcinoma. The remaining three specimens were obtained from the surrounding hepatic parenchyma of metastatic liver cancers, in which no evidence of metastasis or chronic liver disease was diagnosed by at least two pathologists specializing in liver disease. These three specimens were also enrolled as controls. Study protocols were approved by the Ethical Committee of Kanazawa University, Graduate School of Medicine. Total RNAs were extracted from cultured human hepatoma cell lines at a subconfluence with the use of RNAzol (Tel-Test Inc., TX) according to the manufacturer's instructions. Then the RNA preparations were treated with ribonuclease-free deoxyribonuclease (DNase) I (Life Technologies, Inc., Gaithersburg, MD) to remove residual genomic DNA. Two μg of total RNA were reverse-transcribed at 42°C for 1 hour in 20 μl of reaction mixture containing mouse Moloney leukemia virus reverse transcriptase (Toyobo, Osaka, Japan) and hexanucleotide random primers (Amersham Bioscience, Tokyo, Japan). Serially twofold diluted cDNA products were amplified for β-actin using the specific set of the primers (Table 1) with 27 cycles of 94°C for 30 seconds, 55°C for 1 minute, and 72°C for 1 minute, in 20 μl of reaction mixture containing Taq polymerase (Takara Shuzo, Kyoto, Japan) to evaluate the amount of the transcribed cDNA. Thereafter, equal amounts of cDNA products were amplified for chemokine and chemokine receptor genes using the specific sets of primers based on the reported sequences (Table 1). As a negative control, PCRs were performed without reverse transcriptase treatment (no-RT-PCR). Total RNAs extracted from human peripheral blood mononuclear cells were used as a positive control for chemokine receptor mRNA expression. After the amplification, the resultant PCR products were fractionated on a 1.2% agarose gel and visualized by ethidium bromide staining under ultraviolet light transillumination.Table 1Specific Sets of Primers and Conditions of PCRPrimersNucleotide sequence (5′ → 3′) sense/anti-senseAnnealing temperature, °PCR Cyclesβ-actinCACTGTGTTGGCGTACAGGT5527TCATCACCATTGGCAATGAGCXCR1CATGTCAAATATTACAGATCCA5535CCAGCAGCCAAGACAAACAAACCXCR2CATGGAGAGTGACAGCTTTG5835GCTGGGCTAACATTGGATGACXCR3CCACCCACTGCCAATACAAC6035CGGAACTTGACCCCTACAAACXCR4GGTGGTCTATGTTGGCGTCT5735TCGATGCTGATCCCAATGTACXCR5GAGAGAACCTCACGCACCTC5735TGCTTGGTCAAGATGACTGCCCR1CTCTTCCTGTTCACGCTTCC5735GCCTGAAACAGCTTCCACTCCCR2ATGCTGTCCACATCTCGTTCTC5535GGACAGAAGCAAACACAGCCACCCR3GTCATCACCAGCATCGTCAC5735TCATGCAGCAGTGGGAGTAGCCR5GGGATAGCACTGAGCAAAGC5735GTTTTAGCCATCCCCCAAATCCL3/MIP-1αCAGGTCTCCACTGCTGCC5935CACTCAGCTCCAGGTCACTCCL4/MIP-1βCCAAACCAAAAGAAGCAAGC5735ACAGTGGACCATCCCCATAGCCL5/RANTESACCCAGCAGTCGTCTTTGTC5735CTCCCAAAGTGCTGGGATTAC Open table in a new tab For an immunocytochemical analysis of hepatoma cell lines, cultured hepatoma cells were seeded into the wells of a Lab-Tec chamber slide with eight wells (Nalge Nunc International Corp., Naperville, IL) at 2 × 104 cells/well. The cells were cultured for 1 to 2 days in a 37°C incubator with 5% CO2 and then subjected to immunocytochemical study. The cells in the wells were fixed by sequential treatment with 4% paraformaldehyde for 15 minutes at room temperature and with a freshly prepared mixture of methanol/acetone (1:1) for 15 minutes. For an immunohistochemical analysis of tissue samples, paraffin-embedded sections were deparaffinized with xylene and graded concentrations of ethanol. After treatment with 0.3% (v/v) hydrogen peroxide in methanol, sections were incubated sequentially with an avidin/biotin blocking kit (Vector Laboratories Inc., Burlingame, CA) and with goat normal serum. For the detection of CCR1 protein expression, the slides were incubated sequentially with primary rabbit anti-human CCR1 antibody (3 μg/ml) at 4°C overnight and with goat anti-rabbit IgG (Vector Laboratories, Inc.) for 30 minutes at room temperature. Immune complexes were detected with the use of the Vectastain Elite ABC kit (Vector Laboratories, Inc.) according to the manufacturer's instructions. For the detection of CCL3, IL-1α, and IL-1β proteins, the slides were sequentially incubated with mouse anti-human CCL3 monoclonal antibody (0.5 μg/ml) for 1 hour at room temperature, mouse anti-human IL-1α (3 μg/ml), and mouse anti-human IL-1β (2 μg/ml) antibodies overnight, respectively. Then, the slides were incubated with Envision (goat anti-mouse IgG; DAKO, Tokyo, Japan) for 30 minutes at room temperature. The resultant immune complexes were detected with the use of DAB kit (DAKO). Before being mounted, the slides were counterstained with hematoxylin. Negative and isotype controls were also performed in the same condition. An observer without a prior knowledge on the histological diagnosis examined at least randomly chosen five fields of the tissue sections at ×400 magnification and scored the immune reactions as follows: −, less than 10% cells were positive; +, 10 to 20% cells exhibited positive reactions; ++, 20 to 50% cells showed positive reactions; +++, more than 50% showed positive reactions. Cultured hepatoma cells were harvested and washed two times in washing buffer (phosphate-buffered saline, pH 7.4, 0.1% w/v bovine serum albumin, 0.1% w/v sodium azide) and were first incubated in a washing buffer containing 5% normal goat serum to block nonspecific binding. For antibody staining, 5 to 10 × 105 cells were incubated with 20 μg/ml of rabbit anti-human CCR1 or the same concentration of normal rabbit IgG as a control. After being washed twice with washing buffer, the cells were incubated with fluorescein isothiocyanate-conjugated F(ab′)2 goat anti-rabbit IgG (5 μg/ml; Caltag Laboratories, Burlingame, CA). The cells were filtered thorough nylon mesh after being washed twice. All incubations were done for 20 minutes on ice followed by two washings in a washing buffer. Cells were gated based on forward and side scatter and propidium-iodine-positive dead cells were excluded from the analysis. A total of 1 × 104-gated events was collected from each flow cytometry sample. Hepatoma cell lines were suspended in DMEM supplemented with 10% fetal bovine serum at a cell concentration of 1 × 105 cells/ml. One ml of cell suspension was incubated in the absence or presence of the designated concentrations of human IL-1α, IL-1β, TNF-α, and lipopolysaccharide for 24 hours. Supernatants were collected after centrifugation at 1,500 × g to remove particles, and aliquots were stored frozen at −70°C until use in enzyme-linked immunosorbent assay. CCL3 concentrations in the supernatants were determined with the use of human CCL3 assay kit (Genzyme) according to the manufacturer's instructions. Total RNAs were extracted from hepatoma cell lines cultured for 6 hours in the presence or the absence of either IL-1α or IL-1β (100 ng/ml) and subjected to RT-PCR as described above. Approximately 1 × 105 Hep3B cells were suspended in 1 ml of DMEM supplemented with 10% fetal bovine serum and seeded into each well of 24-multiwell plates. After incubation overnight, the medium was changed to serum-free DMEM. After the incubation for additional 24 hours, the medium was again removed. Then, 0.5 ml of serum-free DMEM was added to each well in the presence or the absence of 10 μmol of forskolin (Wako Pure Chemical Industries, Osaka, Japan) in the presence of the indicated concentrations of CCR1 ligands or CCR1 ligands with 10 μg/ml of anti-CCR1. The effects of CCL11/eotaxin were also examined in parallel. After incubation at 37°C for 20 minutes, the cells were lysed and intracellular cAMP levels were determined with the use of Biotrak cAMP enzyme immunoassay system (Amersham Bioscience) according to the manufacturer's instructions. All data were expressed as mean ± SD and were analyzed statistically with analysis of variance. P < 0.05 was considered to be significant. Several lines of evidence suggest the potential involvement of chemokines and their receptors in the invasion and metastatic processes of tumors. Hence, we first examined the gene expression of chemokine receptors in several hepatoma cell lines with the use of RT-PCR. Among the chemokine receptor genes that we examined, CCR1 mRNA was detected in all hepatoma cell lines (Figure 1). In addition, two hepatoma cell lines (HuH7 and Hep3B) expressed CXCR4 mRNA, while HuH7 and HLF cells expressed CCR5 mRNA. To exclude the possibility that contaminated genomic DNA gave rise to the generation of the amplified bands, we used total RNA samples that were treated with DNase. Moreover, we performed RT-PCR under the same conditions except that RT was omitted from the reaction and did not detect any bands at all (Figure 1, bottom). Thus, although the coding region of most chemokine receptor genes consists of one exon, the amplified bands derived from the expressed transcript but not the contaminated genomic DNA. We next examined by immunocytochemical analysis whether hepatoma cell lines expressed CCR1 protein. We detected positive immune reactions in all of the cell lines that we examined (Figure 2A). The use of control antibodies did not give rise to positive reactions at all (Figure 2A; a, b, and c). Moreover, positive reactions were abolished completely when the primary antibodies were absorbed with the peptide used for the immunization (Figure 2A; d, e, and f). These results implied that the observed immunoreactivities were specific. Furthermore, flow cytometric analysis also detected CCR1 expression on all hepatoma cell lines (Figure 2B), indicating that CCR1 was expressed on the cell surface of these hepatoma cell lines. Because most chemokine receptors are coupled with the Gα subunit and adenylyl cyclase, we examined the effects of chemokines on the forskolin-induced increase in intracellular cAMP levels. Forskolin induced a marked increase in intracellular cAMP levels in Hep3B cells (Figure 3A). All three ligands for CCR1, CCL3, CCL4, and CCL5, reduced the forskolin-induced increases in intracellular cAMP levels (Figure 3A). Because CCL5 can bind CCR3, we also examined the effect of CCL11/eotaxin, another specific ligand for CCR3. However, CCL11/eotaxin failed to decrease forskolin-induced cAMP accumulation (Figure 3B). Furthermore, anti-CCR1 antibodies abrogated the effects of CCL3 (Figure 3B). Similar results were obtained when HuH7 cells were used instead of Hep3B cells (data not shown). Because CCR1 but not CCR5 mRNA was detected in Hep3B cells, these results would indicate that Hep3B cells responded to CCL3, CCL4, and CCL5 through functional CCR1 on their surface. We next examined CCR1 protein expression in hepatoma tissues by an immunohistochemical analysis. We could not detect any CCR1 immunoreactivities in normal liver tissues (Figure 4D and Table 2). On the contrary, CCR1 immunoreactivities were detected on hepatoma tissues (Figure 4, A to C; and Table 2). The observation at a higher magnification demonstrated the immunoreactivities in hepatoma cells and to a lesser degree, vascular endothelial cells and occasionally small bile duct epithelial cells (Figure 4, B and C; and Table 2). However, only in a small number of cases (3 of 12), fibroblast-like cells were also weakly positive for CCR1.Table 2CCR1 and CCL3 Expression in Normal and Hepatoma Tissue Samples by Immunohistochemical AnalysisCCR1CCL3Sample no.HistologyHepatocyte or hepatomaEndotheliumFibroblastHepatocyte or hepatomaEndotheliumFibroblast1Normal−−−−−−2Normal−−−−−−3Normal−−−−+−4HCC+++−+++−5HCC+++++++++−6HCC+++−+++++7HCC++++++8HCC++−+++9HCC++++−+++++10HCC++++++++++11HCC+−−++−12HCC−−−+−−13HCC+++−+++++14HCC++−++−15HCC++−++−HCC, Hepatocellular carcinoma.−, 50% of the cells are positive. Open table in a new tab HCC, Hepatocellular carcinoma. −, 50% of the cells are positive. We next examined whether hepatoma cell lines expressed ligands for CCR1. However, we detected neither CCL3 (Figure 5, A and B) nor CCL4 immunoreactivities in any hepatoma cell lines (data not shown). In normal liver tissues, CCL3 immunoreactivities were weakly detected in a few inflammatory cells and endothelium cells (Figure 5F and Table 2). Unexpectedly, CCL3 immunoreactivities were detected in hepatoma cells, vascular endothelial cells, and small bile duct epithelial cells, and to a lesser degree, fibroblast-like cells in hepatoma tissues (Figure 5, C to E; and Table 2). The immunoreactivities were not detected when control antibodies or the antibodies preabsorbed with the corresponding antigens were used as a primary antibody (data not shown), indicating the specificity of the reactions. However, we did not detect immunoreactive CCL4 expression in hepatoma tissues (data not shown). Hepatoma cells were positive for CCL3 although hepatoma cell lines did not express CCL3 at all without any stimulation. These observations prompted us to assume that hepatoma cells in tissues produce CCL3 in response to various stimuli such as proinflammatory cytokines and lipopolysaccharide, which may be present in hepatoma tissues. To explore this possibility, we measured CCL3 production by hepatoma cell lines stimulated with IL-1α, IL-1β, TNF-α, and lipopolysaccharide. The treatment with human IL-1α or IL-1β enhanced markedly CCL3 secretion into the supernatants in a dose-dependent manner in four cell lines among six cell lines that we investigated (Figure 6). SK-Hep1 cell line but not other cell lines secreted CCL3 in a dose-dependent manner in the stimuli of TNF-α and lipopolysaccharide (data not shown). Only the HuH7 cell line secreted CCL4 in response to IL-1α in a dose-dependent manner (data not shown). The induction of CCL3 production was at pretranslational levels because CCL3 mRNA expression was induced in HuH7 and HLE cells when they were stimulated with either IL-1α or IL-1β (Figure 7).Figure 7Total RNAs were extracted from HuH7 or HLE cell lines, which were incubated in the presence or the absence of IL-1α (100 ng/ml) or IL-1β (100 ng/ml) for 6 hours. RT-PCR was performed on the obtained total RNAs to detect CCL3 as described in Materials and Methods. Amplification of β-actin is shown to ensure the same amount of used cDNA. A representative result from three independent experiments is shown.View Large Image Figure ViewerDownload Hi-res image Download (PPT) To further support our hypothesis that hepatoma cells in tissue produce CCL3 in response to proinflammatory cytokines, we examined IL-1α and IL-1β expression in hepatoma and normal liver tissue by an immunohistochemical analysis. In normal liver tissue, IL-1α (Figure 8D) and IL-1β (data not shown) were scarcely detected in hepatocytes. In hepatoma tissues, IL-1α was detected abundantly in hepatoma cells, vascular endothelial cells, and small bile duct epithelial cells (Figure 8, A to C) whereas IL-1β was detected in hepatoma cells and infiltrating cells (data not shown). The immunoreactivities were not detected when the primary antibody was omitted or the isotype-matched control antibody was used as the primary one (data not shown), indicating the specificity of the reactions. These results suggest that endogenously produced IL-1 induces CCL3 expression in hepatoma tissues that express the specific receptor for CCL3, CCR1. Chemokines were originally discovered as chemotactic factors for specific types of leukocytes. Subsequent studies have demonstrated that chemokines exert a wide variety of actions also on nonleukocytic cells. Of interest is that some CXC chemokines such as IL-8/CXCL8 is presumed to be involved in neovascularization in some types of tumors through their potent angiogenic activities.7Kunz M Hartmann A Flory E Toksoy A Koczan D Thiesen HJ Mukaida N Neumann M Rapp UR Brocker EB Gillitzer R Anoxia-induced up-regulation of interleukin-8 in human malignant melanoma. A potential mechanism for high tumor aggressiveness.Am J Pathol. 1999; 155: 753-763Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar, 22Huang S Mills L Mian B Tellez C McCarty M Yang XD Gudas JM Bar-Eli M Fully humanized neutralizing antibodies to interleukin-8 (ABX-IL8) inhibit angiogenesis, tumor growth, and metastasis of human melanoma.Am J Pathol. 2002; 161: 125-134Abstract Full Text Full Text PDF PubMed Scopus (311) Google Scholar, 23Yoshizaki T Horikawa T Qing-Chun R Wakisaka N Takeshita H Sheen TS Lee SY Sato H Furukawa M Induction of interleukin-8 by Epstein-Barr virus latent membrane protein-1 and its correlation to angiogenesis in nasopharyngeal carcinoma.Clin Cancer Res. 2001; 7: 1946-1951PubMed Google Scholar, 24Addison CL Daniel TO Burdick MD Liu H Ehlert JE Xue YY Buechi L Walz A Richmond A Strieter RM The CXC chemokine receptor 2, CXCR2, is the putative receptor for ELR+ CXC chemokine-induced angiogenic activity.J Immunol. 2000; 165: 5269-5277PubMed Google Scholar, 25Inoue K Slaton JW
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