Li Y, Zhang T, Ho C, Douglas SD, Orange JS, Ho WZ; Conference on Retroviruses and Opportunistic Infections (11th : 2004 : San Francisco, Calif.).
Program Abstr Conf Retrovir Oppor Infect 11th 2004 San Franc Calif. 2004 Feb 8-11; 11: abstract no. 789.
Children's Hosp. of Philadelphia, Univ. of Pennsylvania Sch. of Med., USA
BACKGROUND: Natural killer (NK) cells are critical in host innate immune defense against certain viruses. We investigated whether NK cells release anti-viral factor(s) that inhibits HCV replicon expression in human hepatic cells (FCA-1, Huh.2, and Huh.8).METHODS: Primary NK cells (>95% of purity) were obtained either by negative selection with NK isolation kit from Miltenyi Biotec or by sterile 2-color flow cytometric cell sorting. Trans-well cell culture plates were employed for co-culture experiments to determine whether NK cells produce soluble factor(s) capable of suppressing HCV RNA expression in HCV replicon-containing hepatic cell lines. The supernatants collected from NK cell lines (NK-92 and YTS) and primary NK cell cultures were filtered through 0.22-mm-pore-size filters before being added to HCV replicon cell cultures. The amounts of interferon-g (IFN-g) produced by NK cells were measured by ELISA. HCV RNA copy numbers in HCV replicon cell lines were quantified by real-time RT-PCR. HCV NS5 protein expression was determined by Western blot and immunoblot assays.RESULTS: NK cells, when co-cultured (in a trans-well) with the HCV replicon-containing hepatic cells, have the ability to release soluble factor(s) that markedly suppressed HCV RNA expression. The inhibitory effect of NK cell-released factor(s) on HCV replicon correlates with the numbers of NK cells added into the transwells in the co-cultures. Supernatants collected from NK cell lines (NK-92 and YTS) as well as from primary NK cells isolated from healthy donors effectively inhibit HCV replicon expression at both RNA and protein levels in a concentration-dependent fashion. Investigations of the factor(s) involved in NK cell-mediated non-cytolytic anti-HCV activity showed that IFN-g is a primary agent, since antibodies to IFN-g or IFN-g receptors abolished the anti-HCV activity of NK cell-released factor(s). The role of IFN-g in NK cell-mediated anti-HCV activity is further supported by the evidence that NK cell supernatants enhance expression of signal transducer and activator of transcription-1 (STAT1), a critical nuclear factor that is essential in IFN-g-mediated antiviral pathway.CONCLUSIONS: Our data provide direct in vitro evidence that NK cell-mediated innate immunity may play a vital role in controlling HCV replication in human hepatic cells.
Publication Types:
Keywords:
- Antiviral Agents
- Cell Line
- Gene Expression
- Hepacivirus
- Hepatitis C Antibodies
- Hepatocytes
- Humans
- In Vitro
- Interferon Type II
- Interferon-gamma, Recombinant
- Interferons
- Killer Cells, Natural
- Protein Binding
- Replicon
- Trans-Activators
- Transcription Factors
- genetics
Other ID:
UI: 102270978
From Meeting Abstracts