Depletion of neutrophils blocks the recruitment of antigen-nonspecific cells into the liver without affecting the antiviral activity of hepatitis B virus-specific cytotoxic T lymphocytes

Abstract

Using transgenic mice that replicate hepatitis B virus (HBV) in their livers, we previously showed that passively transferred HBV-specific cytotoxic T cells (CTLs) recruit antigen-nonspecific lymphomononuclear and polymorphonuclear inflammatory cells that contribute to the pathogenesis of liver disease. This process is chemokine-dependent, because we recently showed that blocking the chemokines CXCL9 and CXCL10 reduces the recruitment of antigen-nonspecific lymphomononuclear cells and the severity of liver disease after CTL injection. In the current study we show that the severity of the CTL-initiated liver disease is also ameliorated by the depletion of neutrophils. Interestingly, depletion of neutrophils does not affect the intrahepatic migration or antiviral activity of CTLs, but it profoundly inhibits the recruitment of all antigen-nonspecific cells into the liver. This effect occurs in face of high intrahepatic levels of chemokine gene expression, suggesting that neutrophil-dependent functions other than chemokine induction are necessary for the recruitment process to occur. The notion that depletion of neutrophils 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

Anti-Gr-1 treatment quantitatively depletes PMNs in vivo. Nine age- and serum HBeAg-matched female transgenic mice from lineage 1.3.32 (three mice per group) were injected with anti-Gr-1 Abs and 1 × 10⁷ HBsAg-specific CTLs (clone 6C2). Mice were bled and killed, and livers were harvested at days 1, 2, and 5 after CTL transfer. 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 Gr-1⁺/CD11b⁺ cells (PMNs) represent the numbers detected in the whole liver. The results were compared with additional groups of transgenic mice (three mice per group) that were injected with either saline (NaCl) alone or CTL and Irr control Abs.

Figure 2

Depletion of PMNs does not affect the intrahepatic recruitment of HBV-specific CTLs. The recruitment of the passively transferred clone 6C2 (originally produced in male mice) was measured in the same livers described in the legend to Fig. 1 by quantifying the amount of Sry- and CCKAR-specific sequences by real-time PCR. The indicated numbers represent the average copy numbers of Sry-specific amplicon per 10,000 liver-cell genomes.

Figure 3

Effect of PMN depletion on HBV replication, liver disease, and expression of cytokines and chemokines in the liver of CTL-injected HBV transgenic mice. Age- and serum HBeAg-matched female transgenic mice (three mice per group) described in the legend to Fig. 1 were killed at the indicated time points, and total hepatic DNA was analyzed for HBV replication by Southern blot analysis. 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 (± standard deviation), measured at the time of autopsy, is indicated for each group and is expressed in units/liter. Total hepatic RNA from the same mice was analyzed also by RNase protection assay for the expression of various cytokines and chemokines as indicated. The housekeeping mRNAs encoding GAPDH and the ribosomal protein L32 were used to normalize the amount of RNA loaded in each lane. Results were compared with those observed in livers pooled from two age-, sex-, and serum HBeAg-matched transgenic littermates injected with saline (NaCl).

Figure 4

Depletion of PMNs blocks the recruitment of antigen-nonspecific cells into the liver: histological features. (A and B) Histological analysis of the necroinflammatory foci (arrowheads) detected in livers from animals treated either with control Irr Abs (A) or anti-Gr-1 Abs (α-Gr-1, B) that were killed 1 day after CTL transfer. (C and D) Histological analysis of the same livers at higher magnification. Cells displaying the histological features of apoptotic hepatocytes (asterisks), lymphocytes (long arrows), macrophages (arrowheads), and PMNs (short arrows) are indicated. Note that much fewer inflammatory cells (lymphocytes and macrophages) were detected along with apoptotic hepatocytes in the foci of anti-Gr-1-treated mice. Original magnification, ×100 (A and B) and ×600 (C and D).

Figure 5

Depletion of PMNs blocks the recruitment of antigen-nonspecific cells into the liver: IHL analysis. IHL analysis in the same animals described in the legend to Fig. 4. 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.