645 Gene expression profile of T cell-specific chemokines in hepatitis C virus-transfected human hepatocyte-derived cell lines

HEPATOLOGY, Vol. 34, No. 4, Suppl. 1, 2003

AASLD ABSTRACTS

471A

Among F3-4 patients, genes involved in carbohydrate metabolism (e.g. glucosidase) tended to be highly expressed in cases with HCC. Conclusions: We identified genes possibly associated with fibrosis progression and hepatocarcinogenesis. This information can be used to detect increased carcinogenic potential of the liver in patients with HCV infection. Disclosures: Yujin Hoshida - No relationships to disclose Naoya Kato - No relationships to disclose Takao Kawabe - No relationships to disclose Masaru Moriyama - No relationships to disclose Shuntaro Obi - No relationships to disclose Masao Omata - No relationships to disclose Motoyuki Otsuka - No relationships to disclose Run-Xuan Shao - No relationships to disclose Shuichiro Shiina - No relationships to disclose Hiroyoshi Taniguchi - No relationships to disclose Ryosuke Tateishi - No relationships to disclose

Ca2+-independent manner, of which mechanisms differ from those in other pseudotype VSV-sensitive cell line. Disclosures: Norio Hayashi - No relationships to disclose Michiyo Inoue - No relationships to disclose Tatsuya Kanto - No relationships to disclose Yasumasa Komoda - No relationships to disclose Chang K Limn - No relationships to disclose Yoshiharu Matsuura - No relationships to disclose Hideki Miyatake - No relationships to disclose Chika Oki - No relationships to disclose Mitsuru Sakakibara - No relationships to disclose Aki Sato - No relationships to disclose Tetsuo Takehara - No relationships to disclose Takayuki Yakushijin - No relationships to disclose

644 PSEUDOTYPE HEPATITIS C VIRUS INFECTS IMMATURE MYELOID DENDRITIC CELLS THROUGH THE INTERACTION WITH LECTIN. Aki Sato, Tatsuya Kanto, Osaka

Apolinario, Pedro L Majano, Raquel Lorente, Hospital Universitario Santa Cristina, Madrid, Spain; Gerardo Clemente, Hospital Universitario Gregorio Mara~on, Madrid, Spain; Carmelo GarciaMonzon, Hospital Universitario Santa Crist~'na,Madrid, Spain

University Graduate School of Medicine, Suita, Osaka, Japan; Chang K Limn, Yasumasa Komoda, Research Instffute for Microbial Diseases, Osaka University, Osaka, Japan; Chika Oki, Michiyo Inoue, Hideki Miyatake, Mitsuru Sakakibara, Takayuki Yakushijin, Tetsuo Takehara, Osaka University Graduate School of Medicine, Suita, Osaka, Japan; Yoshiharu Matsuura, Research Instffute for Microbial Diseases, Osaka University, Osaka, Japan; Norio Hayashi, Osaka University Graduate School of Medicine, Suita, Osaka, Japan Background and Aim Hepatitis C virus (HCV) causes persistent infection that leads to liver cirrhosis and hepatocellular carcinoma. One of the mechanisms for HCV persistence is the ability of HCV to suppress the dendritic cell (DC) function (Kanto T. et al. J Immuno11998). DC is the most potent APC that regulates various immune responses. Blood DC mainly consists of two subsets, i. e, myeloid and plasmacytoid DC. Myeloid DC (MDC) are characterized by their potent immunostimulatory properties for both primary and secondary T-cell responses against virus. Plasmacytoid DC (PDC), previously known as IFN-producing cells, produces a large amount of type I IFN upon vorus infection. Direct HCV infection to blood DC has b e e n implicated for DC dysfunction, however, it is yet to be determined which DC subsets are susceptible to HCV. The aim of this study is to clarify the susceptibility of each DC subset to HCV and to establish the methods to protect DC from HCV infection. Subjects and Methods Monocytes, MDC and PDC were magnetically isolated from PBMC obtained from healthy volunteers. Isolated monocytes or MDC were cultured in the presence of GM-CSF with or without IL-4 for 4-7 days. PDC were cultured in the presence of IL-3. To these separated cell and cultured cells, we inoculated pseudotype vesicular stomatitis virus (VSV) coated with chimeric HCV envelope glycoproteins, which enables us to identify infected cells by expression of green fluorescent protein (GFP). The infected cells (GFP+ cells) were observed under fluorescence microscopy, and their positive percentages were determined by FACS analysis. Results We demonstrate that pseudotype VSV enters MDC ((% of GFP+ cells, median (range) 23.3% (12,3-27.0%), n - 3 ) ) and monocyte-derived DC (MoDC) ((5.89% (4.11-6.43%), n - 3 ) ) but not PDC ((0.02% ( 0-0.03%), n - 3 ) ) recovered from HCV-negative donors. Hepatoblastoma cell line, HepG2, also showed susceptibility to pseudotype VSV ((% of GFP+ cells, 84.6%)). The highest efficiency of pseudotype VSV or authentic HCV entry to MDC was observed w h e n they were cultured with GM-CSF. Such efficiency decreased with the treatment of IL-4 or CpG oligodeoxynucleotide, showing that MDC lose susceptibility to pseudotype VSV as they mature. Mannan inhibited pseudotype VSV entry to MDC in a dosedependent manner; however, it did not inhibit the virus entry to HepG2. In addition, Ca2+ chelators did not influence pseudotype VSV entry to MDC. Conclusion These results show that HCV infects immature MDC through the interaction with lectins in a

645

GENE EXPRESSION PROFILE OF T CELL-SPECIFIC C H E M O K I N E S IN HEPATITIS C VIRUS-TRANSFECTED H U M A N HEPATOCYTE-DERIVED CELL LINES. Arantxa

INTRODUCTION: An enhanced intrahepatic expression of T cellspecific chemokines (CK), such as RANTES and IP-10, has been shown in patients with chronic hepatitis C virus (HCV) infection. There is an increasing experimental evidence that certain HCV proteins, such as the structural core and the nonstructural 5A (NS5A), can fimcfion as a transcripcional trans-activator modifying gene expression through activation of transcription factors as NF-KB. OBJECTIVE: To determine whether these HCV proteins might regulate T cellspecific CK gene expression in cultured human hepatocyte-derived cell lines. METHODS: We assessed 1) RANTES and IP-10 mRNA levels in different clones of core and NS5A stably-transfected HepG2 and CCL13 cell lines, stimulated or not with a cytokine mixture (TNF-cY, IL-1/3, IFN-T) (CM) by RT-PCR and 2) ELISA measurements of RANTES andIP-10 concentrations in the supernatants of HCVtransfected and untransfected cells studied. RESULTS: Although RANTES mRNA was expressed in resting untransfected HepG2 and CCL13 cells, induction of RANTES transcripts in both resting HCVtransfected cell lines was dearly observed, being higher in resting core-transfected cells (3-fold) and NS5A-transfected cells (4-fold) than in resting mock-transfected (control) cells, as well as to a similar extent in CM-stimulated core- and NS5A-transfected cells. Induction of RANTES secretion followed the same pattern observed for mRNA expression. Interestingly, the highest mRNA and protein levels were always observed in CM-stimulated HCV-transfected cells, with no significant diffrences between core and NS5A. On the other h a n g IP-10 mRNA and protein levels were undetectable in resting HCVtransfected and unstransfected cells, whereas a vigorous mRNA expression and protein synthesis appeared only when these cells were stimulated with cytokines. Noticeable, a higher mRNA upregulation was observed in CM-stimulated core-transfected cells (10-fold) and in NS5A-transfected cells (7-fold) than in control cells. Furthermore, induction of IP-10 production was higher in CM-stimulated NS5Atransfected cells (2.4 fold) and in core-transfected cells (1.6-fold) than in control cells. Finally, no significant differences in mRNA and protein expression levels were found between distincts core- and NS5Atransfected cell clones assessed. CONCLUSIONS: These results demonstrate that core and NS5A proteins, alone or by the synergistic effect of cytokines, are able to upregulate RANTES and IP-10 gene expression in human hepatocytes, thus potentially contributing to hepatocellular necroinflammatory damage f e a s t i n g chronic hepatitis C by promoting the recruitment and migration of effector T cells to HCV-infected hepatocytes. Disclosures: Arantxa Apolinario - No relationships to disclose Gerardo Clemente - No relationships to disclose Carmelo Garcia-Monzon - No relationships to disclose Raquel Lorente - No relationships to disclose Pedro L Majano - No relationships to disclose