Blockade of neutrophil elastase attenuates severe liver injury in hepatitis B transgenic mice

Abstract

Serine proteinases produced by polymorphonuclear neutrophils play important roles in neutrophil-mediated tissue injury at inflammatory sites. Although neutrophil recruitment to the liver has been shown to be involved in the exacerbation of liver inflammation, the function of neutrophil elastase (NE) in liver injury remains unclear. Here, we found that administration of an NE inhibitor (NEI) reduced serum alanine aminotransferase (sALT) activity and inflammatory cell infiltration into the liver from 8 to 24 h after injection of antigen-specific cytotoxic T lymphocytes (CTLs) into hepatitis B virus transgenic mice. Furthermore, the NEI treatment reduced the expressions of inflammatory cytokines and chemokines in the liver and tumor necrosis factor alpha production by macrophages. In addition, the NEI treatment suppressed the mRNA expressions of CC chemokine ligand 3 (CCL-3), CCL-4, and macrophage inflammatory protein 2 (MIP-2) in neutrophils in the liver at 8 h after the CTL injection. In support of these results, we confirmed that administration of anti-CCL-3, anti-CCL-4, and anti-MIP-2 monoclonal antibodies suppressed sALT activity and leukocyte migration into the liver. In conclusion, the present results suggest that NE contributes to the early step of the inflammatory cascade in acute viral hepatitis and that NEIs may have potential as therapeutic drugs against acute severe viral hepatitis.

FIG. 1.

NEI treatment suppresses CTL-induced liver injury and cell recruitment. (A) Three HBV transgenic mice per group were injected intravenously with 4 × 10⁶ 6C2 cells or NaCl. The mean sALT activity measured at the time of autopsy is indicated for each group and expressed as international units per liter (mean ± SD). *, P < 0.05 compared with NaCl injection. (B and C) IHLs and PBCs were isolated from the animals studied for panel A, and the effects of the NEI treatment were analyzed. The numbers in each subset of cells were calculated by multiplying the total number of IHLs or PBCs by the frequency of each subset in the IHL or PBC population by FACS analysis (mean ± SD). *, P < 0.05 compared between the presence and absence of NEI treatment. (D) To calculate the number of HBs28-39-specific CTLs in the liver, IHLs were isolated from three mice per group at 24 h after the CTL injection and stained with Ld-restricted tetramer HBs28-39-PE and CD8-FITC. The numbers were calculated as described above and are expressed as means ± SD.

FIG.2.

Histological analysis. (A to D) Liver sections were obtained from mice sacrificed at 24 h after the injection of CTLs and stained with hematoxylin and eosin. Note that in the CTL-injected mice at 24 h, small inflammatory foci containing mostly lymph mononuclear cells are present and apoptotic hepatocytes are detected in the liver (arrows). In CTL-injected mice with NEI treatment, lymph mononuclear cells and apoptotic hepatocytes are reduced in the parenchyma (arrows). (E to H) To evaluate the neutrophil distribution in the liver after the CTL injection with or without NEI treatment, liver sections were stained with an anti-mouse Gr-1 MAb. NEI-treated livers show a marked decrease in the number of Gr-1⁺ cells after the CTL injection (F and H). (I to L) To evaluate the induction of apoptosis, liver sections were stained by the in situ TUNEL assay. The CTL-treated livers demonstrate a marked increase in the number of TUNEL-positive cells after the injection (I and K). In contrast, TUNEL-positive hepatocytes are decreased after NEI treatment (J and L) compared with NaCl treatment. (M) The relative percentages of apoptotic hepatocytes among the total hepatocytes were determined by TUNEL staining. Data are expressed as means ± SD for three mice. *, P < 0.01 compared with NaCl injection. Original magnification, ×100 (A, B, E, F, I, and J) and ×400 (C, D, G, H, K, and L).

FIG. 3.

Inflammatory cytokine and chemokine expressions and intracellular cytokine expressions in macrophages in the liver. (A) Three HBV transgenic mice per group were injected intravenously with 4 × 10⁶ 6C2 cells and the NEI or NaCl. (Left) Total hepatic RNA (20 μg) was isolated from the livers at various time points and analyzed for cytokine and chemokine expressions by RPA. (Right) To quantify the differences in the mRNA expressions at various time points, the mRNA expression levels were calculated as the relative percentage values of the L32 housekeeping gene expression. *, P < 0.05 compared with NaCl treatment. (B) The intracellular cytokine expressions in IHLs were examined at 4 and 8 h after injection of either the NEI or NaCl into CTL-injected mice. IHLs were isolated from the liver as described in Materials and Methods and stained with anti-CD11b-allophycocyanin or anti-Gr-1-FITC and anti-mouse TNF-α-PE or anti-rat IgG₁-PE. The expression levels of TNF-α (open histograms) and the isotype control (filled histograms) are shown at the indicated time points. Representative results of three independent experiments are shown.

FIG. 4.

Chemokine expressions by neutrophils after the CTL injection. (A) Gr-1⁺ cells from IHLs were purified by positive selection using BD IMag anti-mouse Gr-1 Particles-MSC. (B) After separation of the Gr-1⁺ cells, total RNA was isolated, and the chemokine mRNA expression levels were analyzed by RPA. (C) To quantify the differences in the mRNA expressions at 4 and 8 h, the mRNA expression levels were calculated as the relative percentage values of the L32 housekeeping gene expression.

FIG. 5.

Roles of CCL3, CCL4, and MIP-2 in the liver injury. Three HBV transgenic mice per group were injected with anti-CCL3, anti-CCL4, and anti-MIP-2 MAb or rat IgG as a control prior to the injection of CTLs. (A) Mean sALT activity was measured at 8 h after the CTL injection. (B) IHLs were isolated, and the effects of the antibodies and the NEI were analyzed. The numbers in each subset of cells in the liver were calculated by multiplying the total number of IHLs by the frequency of each subset in the IHL population by FACS analysis (mean ± SD). *, P < 0.05 compared with IgG injection. (C) Total hepatic RNA was analyzed for cytokine mRNA expressions by RPA as described in the legend for Fig. 3A. (D) The mRNA expression levels were calculated as the relative percentage values of L32 housekeeping gene expression. *, P < 0.05 compared with 6C2 plus rat IgG treatment.