Blocking chemokine responsive to gamma-2/interferon (IFN)-gamma inducible protein and monokine induced by IFN-gamma activity in vivo reduces the pathogenetic but not the antiviral potential of hepatitis B virus-specific cytotoxic T lymphocytes

Abstract

Using transgenic mice that replicate hepatitis B virus (HBV) at high levels in the liver as recipients of HBV-specific cytotoxic T lymphocytes (CTLs), we showed that the chemokines responsive to gamma-2/IFN-gamma inducible protein ([Crg2]IP-10) and monokine induced by interferon-gamma (Mig) are rapidly and strongly induced in the liver after CTL transfer. The transferred CTLs produce neither chemokine; rather, they activate (via the secretion of IFN-gamma) hepatocytes and nonparenchymal cells of the liver to produce (Crg2)IP-10 and Mig. Importantly, blocking these chemokines in vivo reduces the recruitment of host-derived lymphomononuclear cells into the liver and the severity of the liver disease without affecting the IFN-gamma-dependent antiviral potential of the CTLs. The finding that neutralization of these chemokines is associated with maintenance of antiviral effects but diminished tissue damage may be significant for the development of immunotherapeutic approaches for the treatment of chronic HBV infection.

Figure 1.

In vitro characteristics of HBsAg-specific CTLs. Total hepatic RNA from clone 6C2 before and after a 4-h stimulation in vitro with plate-bound anti-CD3 mAbs was analyzed by RNase protection assay (RPA) for the expression of various cytokines, (Crg2)IP-10, Mig, and CXCR3 as indicated. The RNA encoding the ribosomal protein L32 was used to normalize the amount of RNA loaded in each lane.

Figure 2.

Kinetics of HBV replication, disease, and expression of cytokine, T cell and macrophage markers and chemokines in the liver of CTL-injected HBV transgenic mice. Age- and serum HBeAg–matched male transgenic mice (three mice per group) from lineage 1.3.32 were injected intravenously with 10⁷ CTLs (clone 6C2), killed at the indicated time points, and total hepatic DNA was analyzed for HBV replication by Southern blot analyses. Bands corresponding to the integrated transgene, relaxed-circular (RC) and single-stranded (SS) linear HBV DNA replicative forms are indicated. The integrated transgene can be used to normalize the amount of DNA bound to the membrane. The mean sALT activity, measured at the time of autopsy, is indicated for each group and is expressed in U/l. Total hepatic RNA from the same mice was also analyzed by RPA for the expression of T cell (CD3, CD4, and CD8) and macrophage (F480) markers, various cytokines, chemokines, and CXCR3 as indicated. The RNA encoding the ribosomal protein L32 was used to normalize the amount of RNA loaded in each lane. Results were compared with those observed in livers pooled from 10 age-, sex-, and serum HBeAg–matched transgenic littermates injected with saline (NaCl).

Figure 3.

Intrahepatic distribution of (Crg2)IP-10 and Mig mRNA in NaCl- and CTL-injected livers. Livers from HBV transgenic mice (lineage 1.3.32) that were injected 4 h earlier either with saline (NaCl) or HBV-specific CTLs were analyzed for the expression of Mig (A and B) or (Crg2)IP-10 (C and D) mRNA by in situ hybridization using specific ³³[P]-labeled riboprobes. Note that most Mig- or (Crg2)IP-10 RNA is contained in the hepatocytes of CTL-injected livers (B and D). Mig- or (Crg2)IP-10–positive nonparenchymal cells (arrowheads, B and D) and Mig- or (Crg2)IP-10–negative apoptotic hepatocytes (asterisks, B and D) and inflammatory cells (arrows, A) are also indicated (H&E; original magnification: ×400).

Figure 4.

The intrahepatic induction of (Crg2)IP-10 and Mig is mediated by IFN-γ produced by the transferred CTLs. (Left panel) Age- and serum HBeAg–matched male transgenic mice (lineage 1.3.32) were intraperitoneally injected with 250 μg of hamster mAbs to IFN-γ and killed 24 h after CTL administration. Control mice (Irr Ab) were simultaneously injected with 250 μg of irrelevant hamster IgG before CTL transfer and killed at the same time after CTL administration. (right panel). Age- and serum HBeAg–matched male transgenic mice (lineage 1.3.46) that were either heterozygous (+/−) or homozygous (−/−) for the IFN-γ null mutation were injected with 10⁷ CTLs (clone 6C2) and killed 24 h later. Total hepatic RNA was analyzed for (Crg2)IP-10, Mig, and CXCR3 as indicated. The RNA encoding the ribosomal protein L32 was used to normalize the amount of RNA loaded in each lane. Results were compared with those observed in livers pooled from 10 age-, sex-, and serum HBeAg–matched transgenic littermates injected with saline (NaCl).

Figure 5.

(Crg2)IP-10 and Mig activity do not mediate the antiviral potential of HBV-specific CTLs but mediate part of the accompanying liver disease. 1 ml of either a cocktail of anti–(Crg2)IP-10 and anti-Mig neutralizing rabbit Ig or NRS was administered intraperitoneally into six groups (three mice per group) of age- (8–10 wk), sex-, and serum HBeAg–matched transgenic mice (lineage 1.3.32) twice, first 16 h before and then simultaneously with the transfer of HBsAg-specific CTLs (clone 6C2). Mice were bled and killed, and livers were harvested 4, 24, or 48 h later. Total hepatic DNA was analyzed for HBV replication by Southern blot analyses. Bands corresponding to the integrated transgene, relaxed-circular (RC) and single-stranded (SS) linear HBV DNA replicative forms are indicated. The integrated transgene can be used to normalize the amount of DNA bound to the membrane. The mean sALT activity, measured at the time of autopsy, is indicated for each group and is expressed in U/l. Total hepatic RNA from the same mice was also analyzed by RPA for the expression of various cytokines as indicated. The RNA encoding the ribosomal protein L32 was used to normalize the amount of RNA loaded in each lane. Results were compared with those observed in livers pooled from 10 age-, sex-, and serum HBeAg–matched transgenic littermates injected with saline (NaCl).

Figure 6.

Passive neutralization of (Crg2)IP-10 and Mig reduced the recruitment of lymphomononuclear cells and the size of the intrahepatic inflammatory foci. (Top) Histological analysis of the necroinflammatory foci detected in the livers from animals treated either with control Abs (NRS, left) or antichemokine Abs (αMig + αIP-10, right) that were killed 48 h after CTL transfer. Cells displaying the histological features of apoptotic hepatocytes (asterisks), lymphocytes (arrows), and macrophages (arrowheads) are indicated. Cells displaying the histological features of polymorphonuclear cells are not indicated. Note that fewer apoptotic hepatocytes and lymphomononuclear cells were detectable in the foci of mice treated with antichemokine Abs (right). (Bottom) Histological analysis of the same livers at lower magnification displaying several necroinflammatory foci (arrowheads). Original magnifications: ×1,000 (top) and ×100 (bottom).

Figure 7.

Passive neutralization of (Crg2)IP-10 and Mig reduced the recruitment of most cell subsets and particularly lymphomononuclear cells such as NK cells, myeloid dendritic cells, and CTLs. IHLs analysis in the same animals described in the legend to Fig. 5. Livers were weighed at the time of autopsy. IHLs were isolated from two liver lobes of a known weight and analyzed by flow cytometry. The indicated numbers of total IHLs and different cell subsets represent the numbers detected in the whole liver.