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Keywords:

  • Concanavalin A;
  • Hepatic inflammation;
  • CCL3/MIP-1α;
  • CD4+ T cells;
  • IFN-γ

Abstract

  1. Top of page
  2. Abstract
  3. 1 Introduction
  4. 2 Results
  5. 3 Discussion
  6. 4 Materials and methods
  7. Acknowledgements

T cell-mediated hepatitis is associated with significant morbidity and mortality worldwide. Levels of C-C chemokine ligand 3/macrophage inflammatory protein-1α (CCL3/MIP-1α) are elevated in the serum of patients with T cell-mediated liver diseases, but its role is not fully understood. Con A-induced hepatitis is a murine liver-specific inflammation mediated by activated T cells and is driven by an up-regulation of the hepatic expression of IFN-γ. In this study, we have used CCL3/MIP-1α gene-deficient mice to examine the role of CCL3/MIP-1α in the pathogenesis of Con A-induced hepatitis. We demonstrate a novel pro-inflammatory role for CCL3/MIP-1α since CCL3/MIP-1α deficiency significantly attenuated hepatic injury, both biochemically and histologically. Moreover, the recruitment of CCR1-expressing CD4+ T cells to the liver after Con A treatment was strikingly attenuated by CCL3/MIP-1α deficiency. Correspondingly, hepatic IFN-γ produced by the recruited CD4+ T cells was significantly reduced by CCL3/MIP-1α deficiency during Con A-induced hepatitis. Furthermore, treatment of mice with a dual CCR1/CCR5 peptide antagonist, methionylated RANTES, also markedly reduced hepatic injury and decreased the numbers of CD4+ T cells within the liver producing IFN-γ during Con A-induced hepatitis. These findings demonstrate that blockade of the CCL3/MIP-1α-CCR1 pathway may represent a novel therapeutic target for treating T cell-mediated liver diseases.

Abbreviations:
CCL3:

C-C chemokine ligand 3

MIP-1α:

Macrophage inflammatory protein-1α

ALT:

Alanine transaminase

Met-RANTES:

Methionylated-RANTES

KO:

Knockout

WT:

Wild type

1 Introduction

  1. Top of page
  2. Abstract
  3. 1 Introduction
  4. 2 Results
  5. 3 Discussion
  6. 4 Materials and methods
  7. Acknowledgements

T cells are known to play a crucial role in the pathogenesis of infectious (hepatitis B and C) and immune-mediated (autoimmune) hepatitis 13. Specifically, a number of clinical studies have reported that CD4+ T cells are the predominant T cells infiltrating into the liver in autoimmune hepatitis 2. Furthermore, in chronic hepatitis B or C, both CD4+ and CD8+ T cells have been implicated in the progression of liver injury 3. However, despite tremendous progress in our understanding of the pathophysiology of T cell-mediated liver diseases, several key questions remain unresolved. First and foremost, the mechanisms governing T cell migration from the blood into tissues during T cell-mediated hepatitis remain incompletely understood. Furthermore, the endogenous mediators that promote the recruitment of T cells to the liver during T cell-mediated liver diseases have been poorly characterized. There is now a growing realization that chemokines may facilitate T cell recruitment and activation during T cell-mediated liver damage 1, 4, 5.

C-C chemokine ligand 3/macrophage inflammatory protein-1α (CCL3/MIP-1α) is a CC chemokine that promotes the recruitment of various leukocyte subtypes, including T cells 6. CCL3/MIP-1α is a ligand for the chemokine receptors CCR1and CCR5 6, 7, and increases in its expression have been linked to the progression of several inflammatory/autoimmune diseases including experimental enteritis, influenza-induced pneumonitis and multiple sclerosis (reviewed in 6).

Concanavalin (Con) A-induced hepatitis is mediated mainly by activated CD4+ T cells recruited to the liver and serves as a prototypic model mimicking many aspects of human T cell-mediated liver diseases and fulminant hepatic failure 814. Previous studies using this model have documented an important pro-inflammatory role for hepatic IFN-γ during Con A-induced hepatitis in mice 10, 13. In addition, several reports have revealed that IFN-γ is produced by CD4+ T cells recruited to the liver during Con A-induced hepatitis 15, 16. Although, CCL3/MIP-1α mRNA expression is elevated during Con A-induced hepatitis 5, the contribution of CCL3/MIP-1α to the progression of hepatic inflammation and injury associated with murine T cell-mediated hepatitis induced by Con A injection into mice remains unknown. Of particular relevance to the present study are clinical reports of elevated serum levels of CCL3/MIP-1α and increased hepatic CCL3/MIP-1α mRNA expression in patients with T cell-mediated liver disease 4, 5.

In the present study, we have assessed the contribution of CCL3/MIP-1α and its receptor CCR1 to the pathogenesis of murine T cell-mediated hepatitis induced by Con A injection.

2 Results

  1. Top of page
  2. Abstract
  3. 1 Introduction
  4. 2 Results
  5. 3 Discussion
  6. 4 Materials and methods
  7. Acknowledgements

2.1 CCL3/MIP-1α expression in Con A-induced hepatitis

In agreement with previous reports 814, intravenous administration of a single dose of Con A (13.5 mg/kg) caused significant hepatitis, as demonstrated by a marked increase in plasma alanine transaminase (ALT) levels at 8 h (154-fold increase above control PBS; p<0.01) and 24 h (100-fold increase above control PBS; p<0.01) post Con A injection (Fig. 1). We next determined hepatic CCL3/MIP-1α mRNA expression and levels at 30 min, 90 min, 8 h and 24 h following Con A administration. By RNase protection assay, we observed significant increases in hepatic CCL3/MIP-1α mRNA expression at 90 min and 8 h (>2-fold increase above control PBS at both time points; p<0.01) following Con A treatment (Fig. 2A). However, by 24 h post Con A treatment, hepatic CCL3/MIP-1α mRNA expression had returned to control levels (Fig. 2A). In parallel, a serial and significant increase in hepatic levels of CCL3/MIP-1α was observed at 90 min (4.5-fold increase above control; p<0.05) and 8 h (6.2-fold increase above control; p<0.01) after Con A injection (Fig. 2B). The chemokine receptors CCR1 and CCR5 are the primary receptors through which CCL3/MIP-1α mediates its biological effects 7. We observed by flow cytometry that CCR1-positive CD4+ T cells were maximally recruited to the liver 8 h after Con A administration compared to controls (Fig. 2C). However, the recruitment of CCR1-bearing CD4+ T cells to the liver at 8 h following Con A injection was not paralleled by a reduction in the number of CCR1-positive CD4+ T cells circulating in the blood (Table 1). On the contrary, we observed an enrichment of CCR1-positive CD4+ T cells in the blood after Con A administration (Table 1). We speculate that the release of CCR1-expressing CD4+ T cells from the bone marrow or spleen could explain the enrichment of CCR1-bearing CD4+ T cells in the blood after Con A treatment. In contrast to CCR1, there was no significant increase in the number of CCR5-expressing CD4+ T cells recruited to the liver at any time point after Con A injection when compared to PBS-treated controls (Fig. 2C). In addition, Con A treatment did not increase the recruitment of monocytes/macrophages, or CCR1- and CCR5-expressing monocytes/macrophages, to the liver at 8 h post Con A injection relative to naive mice (M. N. Ajuebor and M. G. Swain, unpublished observations; Table 2).

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Figure 1. Time course of plasma ALT levels during hepatitis induced by Con A (13.5 mg/kg in PBS) or vehicle. Results are expressed as mean ± SD (n=4–7 mice per group). **p<0.01 versus control (PBS-treated group).

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Figure 2. Hepatic CCL3/MIP-1α, CCR1 and CCR5 expression during Con A-induced hepatitis. (A) Densitometric analysis of hepatic CCL3/MIP-1α mRNA expression (as a ratio of GAPDH mRNA expression; n=4–5 per group). (B) Hepatic CCL3/MIP-1α levels (n=6–16 per group). (C) Mean absolute number of CCR1/CCR5 chemokine receptor-positive CD4+ T cells/liver ± SD (n=3–6 per group). *p<0.05, **p<0.01 versus PBS control.

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Table 1. Absolute numbers of circulating CD4+ T cells, IFN-γ-producing CD4+ T cells and CCR1-bearing CD4+ T cells during Con A-induced hepatitis
Treatment

CD4+ T cells

(×105)

IFN-γ-producing CD4+ T cells (×105)CCR1-bearing CD4+ T cells (×105)
  1. Mice were given Con A (13.5 mg/kg) for 8 h or left untreated (naive). Peripheral blood lymphocytes were co-stained with either fluorochrome-conjugated IFN-γ and CD4+ T cell mAb or CCR1 and CD4+ T cell mAb as described in Sect. 4. Data are from four to six mice per group. *p<0.05 versus naive mice.

Naive6.40±2.404.20±1.97
Con A 2.80±0.6*04.20±2.00
Table 2. Percentage of hepatic NK, CD8+ T cells and monocytes/macrophages during Con A-induced hepatitis
TreatmentNK cellsCD8+ T cellsMonocytes/macrophages
  1. Mice were given Con A (13.5 mg/kg) for 8 h or left untreated (naive). Hepatic leukocytes were isolated from mice and then stained for NK cells with fluorochrome-conjugated NK1.1+ mAb (PharMingen), for CD8+ T cells with fluorochrome-conjugated CD8+ T cell mAb (PharMingen) and for monocytes/macrophages with fluorochrome-conjugated F4/80 mAb (Serotec). Data are from three to six mice per group. *p<0.05 versus naive mice.

Naive22.68±5.269.88±2.6726.7 ±3.73
Con A16.9 ±5.288.13±1.72 2.53±0.14*

2.2 CCL3/M1P-1α gene deletion impairs the development of Con A-induced hepatitis

CCL3/MIP-1α knockout (KO) mice exhibited significantly less hepatic injury 8 h following Con A injection relative to wild-type (WT) controls, as demonstrated biochemically by a striking reduction in ALT levels (WT mice: 7785±1180 U/ml; CCL3/MIP-1α gene-deficient mice: 1672±1050 U/ml; p<0.001; n=8 per group; Fig. 3A). Moreover, livers from CCL3/MIP-1α KO mice exhibited improved hepatic histology with very patchy hepatocellular necrosis and mild lymphocytic infiltration 8 h after Con A treatment (Fig. 3B), whereas livers obtained from WT mice exhibited widespread/confluent hepatocellular necrosis and marked lymphocytic infiltration following Con A treatment (Fig. 3B). Our observation of residual elevations of serum ALT levels in CCL3/MIP-1α KO mice during Con A-induced hepatitis, despite the striking improvement in hepatic histology, would imply that other mediators also contribute to the pathology of Con A-induced hepatitis. Nonetheless, our results suggest a crucial pro-inflammatory role for CCL3/MIP-1α in the setting of T cell-mediated hepatic injury induced by Con A injection in mice.

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Figure 3. CCL3/MIP-1α deficiency impairs the development of Con A-induced hepatic injury. WT or CCL3/MIP-1α KO mice were injected with Con A (13.5 mg/kg), and all mice were sacrificed 8 h after Con A injection for determination of plasma ALT levels (A). Results are presented as mean ± SD (n=5–8 mice per group). ##p<0.01 versus PBS-treated WT; ***p<0.001 versus Con A-treated WT. (B) Representative H&E staining of liver sections of normal (non-inflamed) mice. In addition, a representative H&E staining of liver sections obtained from WT mice (middle panel) or CCL3/MIP-1α KO mice (right panel) 8 h after Con A administration. In WT mice, widespread necrosis is evident in the hepatocytes with substantial lymphocyte infiltration (middle panel), whereas the degree of necrosis and lymphocytic infiltrates is strikingly reduced in CCL3/MIP-1α KO mice (right panel) after Con A treatment.

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2.3 CCL3/M1P-1α deficiency reduces the recruitment of hepatic CCR1-positive CD4+ T cells

In preliminary experiments, we used standard immunofluorescence imaging 17 to confirm that an anti-CCR1 polyclonal antibody recognizing the cytoplasmic tail of murine CCR1 was detecting CCR1 expression on the cell surface membrane of lymphocytes. Indeed, we observed CCR1 expression confined mainly to the cell surface membrane (Fig. 4A), an indication that this anti-CCR1 antibody recognizes functional cell surface-expressed CCR1. In addition, Shang et al. 18 have used this same anti-CCR1 antibody to determine the contribution of functional CCR1 to the inflammatory response in the lung. In this study, the authors observed that CCR1 was up-regulated on NK cells infiltrating into lung granulomata and mediated their recruitment to the lung granulomata. In addition, antibody binding was absent on NK cells from CCR1 knockout mice.

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Figure 4. Effects of CCL3/MIP-1α deficiency on the recruitment of CCR1-positive CD4+ T cells, IFN-γ-producing CD4+ T cells and neutrophils to the liver during Con A-induced hepatitis. (A) Representative immunofluorescence image depicting CCR1 expression on the cell surface membrane of lymphocytes. (B) Percentage of CCR1-expressing CD4+ T cells/liver at the 8-h time point post Con A treatment (n=3–4). *p<0.05 versus naive WT or naive KO mice; #p<0.05 versus Con A-treated WT mice. (C) Mean absolute number of CCR1-expressing CD4+ T cells/liver after 8 h of Con A administration (n=3–4). *p<0.05 versus naive WT or naive KO mice; #p<0.05 versus Con A-treated WT mice. (D) Percentage of IFN-γ-positive CD4+ T cells/liver at the 8-h time point after Con A treatment (n=3–4). *p<0.05 versus naive WT or naive KO mice; #p<0.05 versus Con A-treated WT mice. (E) Mean absolute number of IFN-γ-positive CD4+ T cells/liver after 8 h of Con A treatment (n=3–4). *p<0.05 versus naive WT or naive KO mice; #p<0.05 versus Con A-treated WT mice. (F) Number of hepatic neutrophil counts in WT (n=5) and CCL3/MIP-1α KO mice (n=5) at 8 h post Con A treatment. *p<0.05 versus naive WT or naive KO mice.

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In our study, peak increases in the number of CCR1-positive CD4+ T cells infiltrating into the liver were observed at 8 h post Con A injection relative to controls (Fig. 2C). Moreover, infiltrating CD4+ T cells (but not monocytes/macrophages, NK or CD8+ T cells) are the predominant cells in the liver during Con A-induced hepatitis 11, 15, 16. Therefore, we specifically examined the effect of CCL3/MIP-1α deficiency on the number of CCR1-expressing CD4+ T cells infiltrating into the liver during Con A-induced hepatitis. By flow cytometry, we observed a significant reduction in the percentage and absolute number of CCR1-expressing CD4+ T cells in the liver of CCL3/MIP-1α gene-deficient mice at 8 h after Con A treatment relative to WT (Fig. 4B, C). Similarly, significant reductions in both the percentage and absolute number of isolated hepatic IFN-γ-producing CD4+ T cells were seen in CCL3/MIP-1α KO mice relative to WT controls 8 h after Con A treatment (Fig. 4D, E). Of note, the total number of CD4+ T cells isolated from the livers of CCL3/MIP-1α KO mice 8 h after Con A treatment was also significantly less than that observed in Con A-treated WT mice (WT mice: 1.44±0.26×105 total cells/liver; CCL3/MIP-1α KO mice: 0.44±0.32×105 total cells/liver; p<0.05; n=3–4 per group). Similarly, the percentage of CD4+ T cells in the liver after Con A treatment was significantly reduced by CCL3/MIP-1α deficiency at the 8-h time point (WT mice: 39.9±5.4%; CCL3/MIP-1α KO mice: 29.6±4.2%; p<0.05; n=3–4 per group). CCL3/MIP-1α is also a potent neutrophil chemoattractant 19. Moreover, it has been postulated that neutrophils contribute to Con A-induced hepatitis 20. However, the numbers of hepatic neutrophils at 8 h after Con A injection were identical in both CCL3/MIP-1α KO and WT mice, as determined by esterase staining (Fig. 4F). Our findings suggest an important role for liver-infiltrating CCR1-positive IFN-γ-producing CD4+ T cells in the pro-inflammatory effects of CCL3/MIP-1α during T cell-mediated hepatic injury induced by Con A injection.

2.4 Effect of CCL3/MIP-1α deficiency on peripheral CD4+ T cells

To exclude the possibility that an inherent deficiency in peripheral CD4+ T cell numbers in CCL3/MIP-1α KO mice may underlie the decreased accumulation of CCR1-positive CD4+ T cells in the liver of CCL3/MIP-1α KO mice after Con A injection, we compared the number of peripheral blood CD4+ T cells in naive CCL3/MIP-1α KO versus naive WT mice. Peripheral blood CD4+ T cell counts were similar in CCL3/MIP-1α KO mice compared to WT mice (WT mice: 6.06±2.66×105 CD4+ T cells/ml; CCL3/MIP-1α KO mice: 5.36±1.4×105 CD4+ T cells/ml; n=3 per group).

2.5 Effect of CCL3/M1P-1α deficiency on hepatic IFN-γ levels during Con A-induced hepatitis

CD4+ T cell counts in peripheral blood were significantly reduced at the 8-h time point after Con A injection relative to naive mice (Table 1). However, we did not observe IFN-γ-producing CD4+ T cells in peripheral blood of naive or Con A-treated mice (Table 1). Since hepatic IFN-γ is significantly elevated at 8 h after Con A injection 14, we propose that CD4+ T cells are recruited to the liver from the circulation after Con A administration and that these recruited CD4+ T cells subsequently produce IFN-γ in the liver after Con A injection. Therefore, we next determined the effect of CCL3/MIP-1α deficiency on hepatic IFN-γ levels at 8 h after Con A treatment. We observed that the improvement in Con A-induced hepatitis in CCL3/MIP-1α KO mice was paralleled by a striking reduction (>40%) in hepatic levels of the pro-inflammatory cytokine IFN-γ (Fig. 5) 8 h after Con A treatment, relative to WT controls. The reason for basal expression of IFN-γ in the liver of naive mice is unknown. It likely reflects the fact that the liver is continuously exposed to gut-derived endotoxins and other bacterial products, which would certainly be expected to result in a continuous state of low-grade immune activation. It is noteworthy that hepatic levels of TNF-α in CCL3/MIP-1α KO mice were significantly less than that seen in WT controls at the 8-h time point following Con A administration (WT mice: 225±96 pg/mg total protein; CCL3/MIP-1α KO mice: 116±42 pg/mg total protein; p<0.05; n=5 per group).

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Figure 5. Effect of CCL3/MIP-1α deficiency on hepatic IFN-γ levels during Con A-induced hepatitis. In all cases, results are presented as means ± SD of 3–5 mice per group. #p<0.05 versus PBS-treated (no Con A) WT mice; *p<0.05 versus Con A-treated WT mice.

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2.6 Met-RANTES treatment ameliorates Con A-induced hepatic injury

As shown above (Fig. 4), CCR1-positive IFN-γ-producing CD4+ T cells are important for the pro-inflammatory effects of CCL3/MIP-1α during Con A-induced hepatitis. To further characterize the role of CCR1 in Con A-induced hepatic injury, methionylated RANTES (Met-RANTES), a dual CCR1/CCR5 antagonist was used. Treatment of mice with Met-RANTES significantly reduced hepatic injury at 8 h post Con A treatment as observed biochemically by reduced ALT levels (PBS + Con A: 5238±2379 U/ml; Met-RANTES + Con A: 502±405 U/ml; p<0.001; n=4–6; Fig. 6A) and by histology (Fig. 6B). Extensive confluent necrosis was observed in PBS/Con A-treated mice, whereas only patchy necrosis was observed after Met-RANTES/Con A treatment. Although Met-RANTES is reported to have possible weak agonistic effects, we found that mice pretreated only with Met-RANTES (i.e. no Con A treatment) did not develop any evidence of liver injury at 8 h compared to PBS-treated (i.e. no Con A treatment) mice, as demonstrated biochemically (ALT levels; PBS: 12±9U/ml; Met-RANTES: 14±10 U/ml; n=4 per group; Fig. 6A) and histologically (data not shown).

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Figure 6. Effects of Met-RANTES on Con A-induced hepatic injury. (A) ALT levels. Results are presented as mean ± SD (n=4–6 mice per group). ##p<0.01 versus PBS-treated (no Con A) group; ***p<0.001 versus Con A/PBS-treated group. (B) H&E staining of liver sections of PBS-treated mice (left panel) or Met-RANTES-treated mice (right panel) 8 h after Con A administration. Widespread necrosis is evident in the hepatocytes with marked lymphocyte infiltrates in PBS-treated mice (left panel); in contrast, mild necrosis and a decrease in hepatic lymphocyte accumulation is observed in Met-RANTES-treated mice (right panel).

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2.7 Effect of Met-RANTES treatment on hepatic IFN-γ levels during Con A-induced hepatitis

We observed a significant reduction (>40%) in hepatic levels of IFN-γ (Fig. 7) at 8 h after Con A treatment in the Met-RANTES-treated group when compared with controls. In addition, treatment of mice with Met-RANTES alone (i.e. no Con A treatment) did not change hepatic levels of IFN-γ at 8 h relative to PBS-treated controls (i.e. no Con A or Met-RANTES treatment; Fig. 7). Furthermore, hepatic levels of TNF-α after Met-RANTES treatment were significantly lower than levels observed in PBS-treated mice at the 8-h time point following Con A administration (PBS: 234±99 pg/mg total protein; Met-RANTES: 79.3±10 pg/mg total protein; p<0.01; n=4–5 per group).

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Figure 7. Met-RANTES treatment reduces hepatic IFN-γ levels during Con A-induced hepatitis. Results are presented as mean ± SD of n=4–6 mice per group. #p<0.05 versus PBS (no Con A) control; *p<0.05 versus Con A/PBS-treated controls.

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2.8 Met-RANTES treatment inhibits the recruitment of CCR1-positive IFN-γ-producing CD4+ T cells

By flow cytometry, we observed a significant reduction in the number of CD4+ T cells recruited to the liver at 8 h after Con A treatment in the Met-RANTES-treated group relative to the PBS-treated group (Fig. 8A). Moreover, the absolute number of CCR1-positive IFN-γ-producing CD4+ T cells recruited to the liver at 8 h after Con A injection was also markedly reduced by Met-RANTES treatment when compared to the PBS-treated group (Fig. 8B). Given that neutrophils in mice also express the CCR1 receptor 6, 7, we also determined the effect of Met-RANTES treatment on hepatic neutrophil accumulation after Con A injection. However, the number of neutrophils in the liver at 8 h after Con A treatment was unaltered by Met-RANTES treatment (relative to controls), as determined by esterase staining (Fig. 8C).

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Figure 8. Met-RANTES inhibits the recruitment of CCR1-positive IFN-γ-producing CD4+ T cells into the liver during Con A-induced hepatitis. (A) Mean absolute number of CD4+ T cells/liver ± SD of PBS-treated (n=3) and Met-RANTES-treated (n=4) mice 8 h after Con A treatment. (B) Mean absolute number of CCR1-positive IFN-γ-producing CD4+ T cells/liver ± SD of PBS-treated (n=3) and Met-RANTES-treated (n=4) mice at 8 h after Con A injection. (C) Hepatic neutrophil counts from PBS-treated mice (n=5) and Met-RANTES-treated mice (n=4) at 8 h post Con A treatment. Dotted line denotes mean number of positively stained neutrophil cells ± SD in naive mice. Data are presented as the mean number of positively stained neutrophil cells ± SD. In all cases, *p<0.05 versus PBS-treated group.

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3 Discussion

  1. Top of page
  2. Abstract
  3. 1 Introduction
  4. 2 Results
  5. 3 Discussion
  6. 4 Materials and methods
  7. Acknowledgements

The last decade has seen the accumulation of a large volume of evidence supporting an important role for CCL3/MIP-1α in the pathophysiology of several diseases including multiple sclerosis and enteritis (reviewed by 6). However, the role played by CCL3/MIP-1α in modulating hepatic inflammation and the mechanisms underlying this modulation in T cell-mediated liver diseases remain incompletely understood 5. The present study delineates a significant role and mechanism of action of CCL3/MIP-1α in contributing to the pathogenesis of T cell-mediated hepatitis induced by Con A treatment in the mouse. In this study, we have identified a striking increase in hepatic CCL3/MIP-1α mRNA expression and levels within 90 min of Con A injection, before hepatic injury is evident. More importantly, we demonstrate that disruption of the CCL3/MIP-1α gene significantly attenuates Con A-induced hepatitis, both biochemically and histologically. Our data strongly suggest that hepatic CCL3/MIP-1α plays an important role in the progression of hepatic inflammation and injury associated with Con A-induced hepatitis. As previous reports have documented hepatic macrophages (Kupffer cells) to be a major source of CCL3/MIP-1α during hepatic inflammation 5, 21, we speculate that Kupffer cells are the major hepatic cell type producing CCL3/MIP-1α following Con A injection.

CD4+ T cells are the predominant T lymphocytes recruited to the liver following Con A injection 11, 15, 16. Evidence that recruited CD4+ T cells mediate Con A-induced hepatitis in mice stems from a series of studies in which athymic nude or SCID mice were found to be resistant to Con A-induced liver damage 8, 11. Furthermore, mice treated with anti-CD4 mAb or with FK506, a T cell-specific immunosuppressant, did not develop hepatitis following Con A administration 8, 11. CCL3/MIP-1α has been shown to contribute to inflammatory tissue injury by virtue of its chemotactic effects on leukocytes, including T cells, via CCR1 and CCR5 chemokine receptors 6. The recruitment of CCR1-positve CD4+ T cells to the liver peaked at 8 h following Con A injection. Therefore, we determined the role of CCL3/MIP-1α in the recruitment of CCR1-expressing CD4+ T cells to the liver during Con A-induced hepatitis. Interestingly, the improvement in hepatic injury in CCL3/MIP-1α gene-deficient mice after Con A injection was paralleled by a reduction in the number of CCR1-expressing CD4+ T cells in the liver of Con A-treated CCL3/MIP-1α KO mice relative to Con A-treated WT controls. Our data suggest that CCL3/MIP-1α plays a critical role in the recruitment of CCR1-bearing CD4+ T cells to the liver during Con A-induced hepatitis and that recruited hepatic CCR1-positive CD4+ T cells are of central importance in the pathogenic effects of CCL3/MIP-1α during murine T cell-mediated hepatitis induced by Con A injection. Interestingly, our findings are in agreement with clinical observations of Lichterfeld et al. 22 who documented that elevated serum levels of CCL3/MIP-1α and increased liver-infiltrating CCR1-expressing CD4+ T cells correlated with disease activity in patients with T cell-mediated liver disease. Furthermore, a recent study by Leifeld et al. 5 reported increased serum levels of CCL3/MIP-1α in patients with fulminant hepatic failure. CCL3/MIP-1α has been suggested to promote the recruitment of neutrophils during an inflammatory response 19, which could contribute to the pathogenesis of Con A-induced hepatitis 20. Our current findings do not support this possibility since neutrophil accumulation in the liver was not defective in CCL3/MIP-1α KO mice after Con A injection relative to WT controls.

The association of reduced hepatic injury in Con A-treated CCL3/MIP-1α KO mice with the reduced recruitment of CCR1-positive CD4+ T cells to the liver implies that CCR1-bearing CD4+ T cells are important to the pro-inflammatory effects of CCL3/MIP-1α during Con A-induced hepatitis. Therefore, we evaluated the effect of CCR1 blockade on hepatic injury during Con A-induced hepatitis. Treatment with Met-RANTES, an antagonist of the CCR1 and CCR5 receptors, resulted in a significant reduction in the severity of hepatic injury and a significant reduction in the numbers of CD4+ T cells in the liver after Con A administration. Although CCR1 is also expressed on neutrophils in mice 6, 7, we found that hepatic neutrophil recruitment after Met-RANTES treatment was similar to that observed in controls after Con A injection. Nevertheless, the effectiveness of Met-RANTES in ameliorating hepatitis suggests that CCR1 and possibly CCR5 are key receptors in mediating the effects of CCL3/MIP-1α in this model. Moreover, we did not observe increased CCR5-bearing CD4+ T cell recruitment to the liver in Con A-treated mice relative to controls, whereas the recruitment of CCR1-expressing CD4+ T cells to the liver was maximally increased at 8 h after Con A injection relative to controls. Taken together, we speculate that the lack of effect of CCL3/MIP-1α deficiency and Met-RANTES treatment on hepatic neutrophil recruitment during Con A-induced hepatitis establishes the crucial role of CCR1-bearing CD4+ T cells rather than neutrophils in the pro-inflammatory effects of CCL3/MIP-1α in the setting of murine T cell-mediated hepatitis induced by Con A injection.

IFN-γ is a critical cytokine contributing to the pathogenesis of Con A-induced hepatitis, since mice pretreated with anti-IFN-γ antibody or IFN-γ-deficient mice are resistant to Con A-mediated liver damage 10, 13. In recent years, it has become apparent that in Con A-treated mice, IFN-γ is produced mainly by activated CD4+ T cells recruited to the liver 15, 16. In agreement, we found that hepatic IFN-γ was produced by CD4+ T cells after recruitment to the liver, since circulating CD4+ T cells did not produce IFN-γ after Con A treatment (as documented in Table 1). Therefore, a decrease in the recruitment of CCR1-positive CD4+ T cells to the liver of CCL3/MIP-1α KO mice, and after Met-RANTES treatment during Con A-mediated hepatitis, is consistent with our current observations of (i) reduced hepatic IFN-γ-producing CD4+ T cells as well as hepatic levels of IFN-γ and (ii) improved hepatic injury in CCL3/MIP-1α gene-deficient mice and in Met-RANTES-treated mice after Con A treatment. Our observations using the Con A-induced hepatitis model are in contrast to the observations of Salazar-Mather et al.21, 23 using the murine cytomegalovirus infection-induced hepatitis model in which type 1 interferons (IFN-α/β) induced the synthesis of CCL3/MIP-1α, which in turn was found to promote liver inflammation by recruiting IFN-γ-producing NK cells. We did not observe NK cell or CD8+ T cell recruitment to the liver in Con A-treated mice relative to naive mice (see Table 2). Moreover, in recombination-activating gene-deficient mice which bear only NK cells, IFN-γ levels did not increase after Con A treatment 15. These observations suggest that CCL3/MIP-1α exerts its pro-inflammatory effects within the liver by a different mechanism in these different models of hepatitis.

In recent years, several studies have used anti-TNF-α neutralizing antibodies or TNF-α knockout mice to demonstrate the crucial pro-inflammatory effect of TNF-α in Con A-mediated liver damage 11, 13. Specifically, depletion of Kupffer cells (i.e. resident hepatic macrophages) ameliorates Con A-induced hepatic injury 24. Furthermore, TNF-α produced by Kupffer cells is reported to promote liver damage after Con A administration via interaction with two distinct surface receptors, TNFR1 and TNFR2 9, 12. Therefore, our observation of reduced hepatic levels of TNF-α during Con A-induced hepatitis after Met-RANTES treatment or in CCL3/MIP-1α KO mice could indicate that the CCL3/MIP-1α-CCR1 interaction could also contribute to the pathology of Con A-induced hepatitis by regulating hepatic TNF-α levels.

In summary, we propose that in response to Con A injection (i) CD4+ T cells are recruited into the liver from the circulation; (ii) recruited hepatic CD4+ T cells produce IFN-γ 15, 16; (iii) the direct physical contact between activated macrophages and recruited CD4+ T cells during Con A-mediated liver damage 8 may trigger IFN-γ to activate Kupffer cells, thereby producing TNF-α 24; (iv) both cytokines then act synergistically to mediate Con A-induced hepatic injury 13; an effect which is attenuated by CCL3/MIP-1α deficiency. Support for this link between hepatic IFN-γ-producing CD4+ T cells, TNF-α and CCL3/MIP-1α derives from our observation that the recruitment of IFN-γ-producing CD4+ T cells to the liver, as well as hepatic TNF-α levels, were severely impaired in CCL3/MIP-1α KO mice during Con A-induced hepatitis. We believe that our findings identify a crucial pro-inflammatory role for CCL3/MIP-1α in Con A-induced hepatitis, a model of T cell-mediated hepatitis, which is mediated mainly through the hepatic recruitment of IFN-γ-producing CD4+ T cells. Therefore, manipulation of the hepatic CCL3/MIP-1α-CCR1 pathway may be a novel prospective target for the treatment of T cell-mediated liver diseases.

4 Materials and methods

  1. Top of page
  2. Abstract
  3. 1 Introduction
  4. 2 Results
  5. 3 Discussion
  6. 4 Materials and methods
  7. Acknowledgements

4.1 Con A-induced hepatitis

Specific pathogen-free male C57BL/6J mice (5–7 weeks old) were obtained from The Jackson Laboratories (Bar Harbor, Maine). Mice were injected intravenously with freshly prepared Con A (13.5 mg/kg; Sigma Chem. Co., St Louis, MO) 814. At 30 min, 90 min, 8 h and 24 h after Con A administration and under halothane anesthesia, blood was collected for measurement of plasma ALT levels (commercial kit, Sigma). Livers were perfused with ice-cold sterile PBS to remove blood elements and then used for the determination of hepatic chemokine and cytokine (TNF-α, IFN-γ and CCL3/MIP-1α) levels as well as for the determination of chemokine receptor (CCR1 and CCR5) expression by FACS and for histological evaluation of liver injury. PBS-treated mice served as controls. In a separate set of experiments, a group of specific pathogen-free male CCL3/MIP-1α KO mice (on a C57BL/6J background; The Jackson Laboratories) and corresponding WT mice were injected with Con A (13.5 mg/kg). All mice were killed at 8 h after Con A injection. All procedures in this study were approved by the Animal Care Committee of the University of Calgary and conformed to the guidelines established by the Canadian Council on Animal Care.

4.2 Histology and cytochemistry

Tissues were processed and stained with hematoxylin/eosin according to standard protocols and analyzed by light microscopy by an observer unaware of the treatments 14. Another set of liver sections were stained with a chloroacetate esterase stain (Leder Stain; Sigma) to quantitate hepatic neutrophil infiltration 20. Data are presented as the mean number of positively stained neutrophils over ten high-power fields (hpf).

4.3 RNase protection assay

Total RNA was extracted from liver tissue using Trizol reagent (Invitrogen Canada, Burlington, Canada). The mouse template set mCK-5 (PharMingen) was used for the detection of CCL3/M1P-1α mRNA expression 14.

4.4 CCL3/MIP-1α, IFN-γ and TNF-α ELISA

CCL3/MIP-1α, IFN-γ and TNF-α levels in liver homogenates were determined by specific ELISA, as described 14, 25.

4.5 Isolation of liver-infiltrating lymphocytes and flow cytometry analysis

The isolation of liver-infiltrating lymphocytes was conducted as described 14. Isolated hepatic lymphocytes (1×106 cells) were first incubated with a FITC-conjugated anti-mouse CD4 mAb or isotype control (Serotec Inc, Raleigh, NC), then fixed and permeabilized using a cytofix-cytoperm plus kit (PharMingen). Following permeabilization, cells were incubated with a PE-labeled goat IgG isotype control (Southern Biotech Inc., Birmingham, AL) or a polyclonal antibody against the cytoplasmic tail of murine CCR1 (Santa Cruz Biotechnology, Santa Cruz, CA) 14, 18 for 30 min at 4°C. Cells were then washed and stained in the dark with a PE-labeled anti-goat IgG (Biomeda Corp, Foster City, CA) and analyzed by FACS using CellQuest software (Becton Dickinson, Mountain View, CA). The number of CCR5-positive CD4+ T cells recruited to the liver after Con A injection was determined as described above for CCR1, using a PE-conjugated CCR5 mAb (PharMingen).

4.6 Immunofluorescence cellular localization of CCR1 expression

Mononuclear cells were isolated from peripheral blood leukocytes of naive C57BL/6 mice. Cells were fixed and then permeabilized as described above. Permeabilized cells were stained with an alexa 488-fluoro-labeled goat IgG isotype control (Molecular Probes Inc., Eugene, OR) or a polyclonal antibody against the cytoplasmic tail of murine CCR1 (Santa Cruz Biotechnology) 14, 18 for 30 min at 4°C. Cells were then washed and stained in the dark with an alexa 488®-fluoro-labeled rabbit anti-goat secondary antibody (Molecular Probes Inc.) and analyzed by immunofluorescence imaging 17; the cells were visualized at 100× magnification using a 1.35NA objective on an Olympus 1X70 microscope (Rockleigh, NJ) attached to a 14-bit cooled CCD camera (Princeton Instruments, Monomouth Junction, NJ), as described 17. Lymphocytes were identified by morphology and size.

4.7 Detection of intracellular IFN-γ by flow cytometry

Isolated hepatic lymphocytes (1×106 cells/tube) were initially incubated with a PerCP-conjugated anti-mouse CD4 mAb or isotype control (PharMingen) for 30 min at 4°C. Stained cells were fixed and permeabilized using cytofix-cytoperm plus kit (PharMingen). After permeabilization, cells were incubated with a PE-conjugated anti-mouse IFN-γ mAb (clone XMG1.2; PharMingen) or/and an anti-mouse CCR1 polyclonal Ab/FITC-labeled anti-goat IgG (as described above) or their respective isotype controls for 45 min at 4°C. Cell viability was greater than 95%, as determined by trypan blue. Gates were set using appropriate isotype control antibodies. The cells were acquired with the FACScan flow cytometer.

4.8 Effect of Met-RANTES

Met-RANTES (30 µg/mouse; i.v.), a functional antagonist of the chemokine receptors CCR1 and CCR5 26, or PBS was injected into mice 1 h prior to Con A administration. All mice were sacrificed 8 h after Con A administration for assessment of hepatic injury, hepatic cytokine levels and mononuclear leukocyte recruitment, as described above. In a separate set of experiments, mice were given Met-RANTES or PBS only (but no Con A treatment) and killed 8 h later. The dose of Met-RANTES used in this study has been shown to effectively reduce renal inflammation 27.

4.9 Statistical analysis

All data are shown as mean ± SD. For comparison of means between two experimental groups, the Student's unpaired t-test was used. Comparison among three or more experimental groups was performed using the one-way ANOVA followed by either Dunnett's multiple comparison test or Newman-Kuels comparison test. A p value of ⩽0.05 was considered significant. All statistical analyses were performed using GraphPad Instat (version 3.00) software.

Acknowledgements

  1. Top of page
  2. Abstract
  3. 1 Introduction
  4. 2 Results
  5. 3 Discussion
  6. 4 Materials and methods
  7. Acknowledgements

The authors are grateful to Laurie Robertson for her invaluable assistance with the flow cytometry studies. We are also grateful to Dr. Stefan Urbanski (Dept. of Pathology, University of Calgary) for his critical evaluation of the histological data. The authors are highly indebted to Dr. Kamala Patel (Live Cell Imaging Core of the Immunology Research Group, University of Calgary) for kindly providing her expertise on the immunofluorescence imaging experiments. This study was supported by a grant from the Canadian Institutes of Health Research (CIHR)/Health Canada Hepatitis C Initiative. Maureen N. Ajuebor was supported by a Canadian Association of Gastroenterology/Schering/CIHR post-doctoral fellowship. Mark G. Swain is an Alberta Heritage Foundation for Medical Research Senior Scholar and a CIHR/Health Canada Hepatitis C Initiative Investigator.

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