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Originally published online as doi:10.1189/jlb.0905527 on February 3, 2006

Published online before print February 3, 2006
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(Journal of Leukocyte Biology. 2006;79:696-705.)
© 2006 by Society for Leukocyte Biology

L-selectin and intercellular adhesion molecule-1 regulate the development of Concanavalin A-induced liver injury

Ayako Kawasuji*, Minoru Hasegawa*,1, Mayuka Horikawa*, Tomoyuki Fujita*, Yukiyo Matsushita*, Takashi Matsushita*, Manabu Fujimoto*, Douglas A. Steeber{dagger}, Thomas F. Tedder{ddagger}, Kazuhiko Takehara* and Shinichi Sato§

* Department of Dermatology, Kanazawa University Graduate School of Medical Science, Kanazawa, Ishikawa, Japan;
{dagger} Department of Biological Sciences, University of Wisconsin-Milwaukee, Milwaukee, Wisconsin;
{ddagger} Department of Immunology, Duke University Medical Center, Durham, North Carolina; and
§ Department of Dermatology, Nagasaki University Graduate School of Biomedical Sciences, Nagasaki, Japan

1Correspondence: Department of Dermatology, Kanazawa University Graduate School of Medical Science, 13-1, Takara-machi, Kanazawa, 920-8641, Japan. E-mail: minoruha{at}derma.m.kanazawa-u.ac.jp

ABSTRACT

Concanavalin A (Con A)-induced hepatitis is a model for human T cell-mediated hepatitis. We evaluated the role of L-selectin and intercellular adhesion molecule-1 (ICAM-1) in this model by injecting Con A intravenously in mice lacking L-selectin (L-selectin–/–), ICAM-1 (ICAM-1–/–), or both (L-selectin/ICAM-1–/–). Blood and liver samples were collected 0, 8, 24, and 48 h after Con A treatment. Increases in plasma transaminase levels, which peaked 8 h after injection, were reduced significantly in L-selectin–/–, ICAM-1–/–, and L-selectin/ICAM-1–/– mice compared with wild-type mice. Liver necrosis was more strongly inhibited in ICAM-1–/– mice than in L-selectin–/– mice but was most prominently reduced in L-selectin/ICAM-1–/– mice, in parallel with decreased plasma transaminase levels. The reduced severity of hepatitis in the mutant mice correlated with decreases in numbers of liver CD4+ T cells but not numbers of CD8+ T cells or neutrophils. Following Con A treatment, L-selectin deficiency reduced liver mRNA expression of tumor necrosis factor-{alpha}, and ICAM-1 deficiency reduced expression of interleukin-4. By contrast, reductions in liver macrophage inhibitor protein-1{alpha} mRNA occurred in all mutant mice. These results indicate that L-selectin and ICAM-1 contribute cooperatively to the development of Con A-induced hepatitis by regulating leukocyte infiltration and subsequent cytokine production.

Key Words: hepatitis • cytokine • animal model

INTRODUCTION

Hepatitis induced by Concanavalin A (Con A) is commonly used as an experimental model of human T cell-mediated hepatitis [1 ]. Con A is a T cell mitogenic lectin, inducing polyclonal T cell activation in vitro, and causes severe and acute liver injury in mice, which is dependent on the presence of T cells [1 ]. Following Con A administration, T cells and macrophages release various cytokines, such as tumor necrosis factor {alpha} (TNF-{alpha}), interferon-{gamma} (IFN-{gamma}), and interleukin (IL)-4, which have been linked to the development of hepatic lesions [2 3 4 5 ]. In this model, following Con A attachment to endothelial cells, T cells promptly migrate out of vessels and accumulate in the perivascular areas, such as the space of Disse, perivenular interstitial tissue, and the portal tract. Thus, hepatocyte injury is associated with massive CD4+ T cell activation and infiltration into the liver parenchyma, leading to secretion of various cytokines [1 2 3 ]. However, leukocytes other than CD4+ T cells have also been suggested to play a role in this model [6 7 8 ].

Leukocyte recruitment is a multistep process regulated by multiple adhesion molecules [9 10 11 ]. Leukocytes first tether and roll on vascular endothelial cells before they are activated to adhere firmly and subsequently emigrate into the extravascular space. The selectin family (L-, E-, and P-selectin) mediates tethering and rolling of leukocytes, and immunoglobulin (Ig) superfamily members, including intercellular adhesion molecule-1 (ICAM-1; CD54) and their integrin ligands, are critical for the firm adhesion [12 , 13 ]. L-selectin (CD62L), which primarily mediates leukocyte capture and rolling on the endothelium, is constitutively expressed by most leukocytes [12 ]. In vitro, L-selectin binds to several glycosylated mucin-like proteins expressed by high endothelial venules [12 ]. L-selectin-deficient (L-selectin–/–) mice demonstrate decreased trauma- and TNF-{alpha}-induced rolling of leukocytes, decreased leukocyte recruitment into an inflamed peritoneum, decreased delayed-type hypersensitivity (DTH) responses, delayed rejection of allogeneic skin transplants, and resistance to lipopolysaccharide (LPS)-induced septic shock [14 15 16 17 18 19 ]. ICAM-1 is constitutively expressed at low levels by endothelial cells and is up-regulated rapidly during inflammation, resulting in increased leukocyte-endothelial cell adhesion [20 ]. Leukocytes express ß2 integrins, including lymphocyte function-associated antigen-1 (LFA-1; CD11a/CD18), which interact with ICAM-1, and ICAM-1/ß2 integrin interactions promote leukocyte rolling but also mediate firm adhesion and the transmigration of leukocytes at sites of inflammation [11 , 21 ]. ICAM-1–/– mice have significantly reduced numbers of infiltrating neutrophils during peritonitis, reduced susceptibility to LPS-induced septic shock, delayed skin wound repair, and impaired DTH reactions, although allogeneic skin graft rejection is normal [15 , 22 23 24 ]. Recent studies demonstrate that the loss of L-selectin and ICAM-1 expression reduces leukocyte recruitment into sites of inflammation beyond what is observed with loss of either receptor alone [21 , 24 25 26 ]. Therefore, L-selectin and ICAM-1 mediate optimal leukocyte accumulation during inflammation through overlapping as well as synergistic functions.

Despite the crucial role of adhesion molecules in inflammatory cell infiltration, their role in Con A-induced hepatitis remains unclear. One study has reported inhibitory effects on leukocyte recruitment using anti-E-selectin and anti-vascular cell adhesion molecule-1 (VCAM-1) monoclonal antibodies (mAb) [27 ]. Another study showed that genetic deficiency of P-selectin or P-selectin-blocking mAb impaired lymphocyte adhesion to liver venules and sinusoids and prevented liver injury after Con A treatment [28 ]. Furthermore, Con A-induced cytotoxicity was inhibited by treatment with anti-ICAM-1/LFA-1 mAb but not by anti-VCAM-1 mAb in vitro and in vivo [29 ]. By contrast, other groups found that pretreatment with blocking mAb or genetic deletion of ICAM-1 did not affect development of Con A-induced liver injury [30 , 31 ]. Thus, the contribution of ICAM-1 to the development of Con A-induced liver injury remains controversial. Furthermore, to our knowledge, the role of L-selectin in generation of Con-A induced hepatitis has not been assessed.

In this study, the role of L-selectin and ICAM-1 in Con A-induced hepatitis was assessed directly using mice lacking L-selectin, ICAM-1, or both adhesion molecules. The results of this study suggest that ICAM-1 and L-selectin contribute cooperatively to Con A-induced hepatitis by regulating the influx of CD4+ T cells and their subsequent cytokine production.

MATERIALS AND METHODS

Animals
L-selectin–/– mice were produced as described [14 ]. ICAM-1–/– mice [22 ], expressing residual amounts of ICAM-1 splice variants in the thymus and spleen but not in other organs including liver [32 ], were obtained from the Jackson Laboratory (Bar Harbor, ME). Mice lacking L-selectin and ICAM-1 (L-selectin/ICAM-1–/–) were generated as described [21 ]. All mice were healthy, fertile, and did not display evidence of infection or disease. All mice were back-crossed between five and 10 generations onto the C57BL/6 genetic background. Mice used for experiments were 7–10 weeks old. Wild-type littermates were used as controls. All mice were housed in a specific pathogen-free barrier facility and screened regularly for pathogens. All studies and procedures were approved by the Committee on Animal Experimentation of Kanazawa University Graduate School of Medical Science (Japan).

Con A administration
Con A was purchased from Sigma Chemical Co. (St. Louis, MO). Con A was dissolved in sterile, pyrogen-free phosphate-buffered saline (PBS) and injected intravenously via the tail vein in a dose of 25 mg/kg body weight.

Transaminase measurements
Blood samples were obtained by retro-orbital venous plexus puncture of anesthetized mice at 0, 8, 24, and 48 h after Con A injection. All plasma samples were stored at –80°C until use. Plasma asparate aminotransferase (AST) and alanine transferase (ALT) levels were determined with the aid of Fuji-Dri Chem FDC5500 (Fuji Film, Tokyo, Japan), which is based on a dry chemistry method, according to the manufacturer’s instructions.

Histological examination and immunohistochemistry
Livers were fixed in 3.5% paraformaldehyde and then paraffin-embedded. Sections (6 µm) were stained with hematoxylin and eosin (H&E) for general histological evaluation. For immunohistochemistry, frozen sections of liver were acetone-fixed and then incubated with 10% normal rabbit serum in PBS (10 min, 37°C) to block nonspecific staining. Sections were then incubated with rat mAb specific for mouse ICAM-1 (MCA818, Serotec, Sapporo, Japan), CD4 (Clone RM4-5, BD PharMingen, San Diego, CA), and CD8 (Clone 53-6.7, BD PharMingen). Rat IgG (Southern Biotechnology Associates Inc., Birmingham, AL) was used as a control for nonspecific staining. Sections were incubated sequentially (20 min, 37°C) with a biotinylated rabbit anti-rat IgG antibody (Vectastain ABC kit, Vector Laboratories, Burlingame, CA) and then horseradish peroxidase-conjugated avidin-biotin complexes (Vector Laboratories). Sections were developed with 3,3'-diaminobenzidine tetrahydrochloride and hydrogen peroxide and counterstained with methyl green.

Assessment of leukocyte infiltration
The number of infiltrating neutrophils in the liver parenchyma was counted around hepatic and portal venules and sinusoids in H&E-stained sections at 0, 8, and 24 h after Con A injection. Neutrophils were distinguished from other cells by nuclear morphology. The number of lymphocytes adhering to sinusoidal endothelium and hepatic and portal venules was determined in each section stained with anti-CD4 or anti-CD8 mAb at 0, 8, and 24 h after Con A treatment. Five sections of the liver were chosen randomly from each mouse. Ten microscopic fields (x400) were taken at random from each sample, and all the leukocytes included in the fields were assessed. Ten mice of each genotype were examined. Three investigators independently examined each section in a blinded manner.

Histological determination of hepatic necrosis
Hepatic necrosis was assessed in each section as the percentage of liver parenchyma with necrotic change. Eight microscopic fields (x25) were taken from each sample. Images were processed with the Scion image analysis system. The total hepatic parenchyma area and the necrotic area were measured for each sample, and the percentage of necrotic area was calculated for each mouse strain.

Reverse transcriptase-polymerase chain reaction (RT-PCR) in Con A-induced hepatitis
Livers were collected 8 h after Con A administration. All liver samples were snap-frozen in liquid nitrogen and stored at –80°C until use. Total RNA from the liver was extracted using Qiagen RNeasy spin columns (Qiagen Ltd., Crawley, UK) and digested with DNaseI (Qiagen Ltd.) to remove chromosomal DNA in accordance with the manufacturer’s protocols. Total RNA was reverse-transcribed to cDNA using a RT system with random hexamers (Promega, Madison, WI). Real-time quantitative RT-PCR was performed using the TaqMan® system (Applied Biosystems, Foster City, CA). Each cytokine’s probe and primers were obtained from TaqMan® gene expression assays (Applied Biosystems). Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used to normalize mRNA, and probe and primers were from predeveloped TaqMan® assay reagents (Applied Biosystems). Real-time PCR was performed using an ABI Prism 7000 sequence detector (Applied Biosystems) according to the manufacturer’s instructions. Relative expression of real-time PCR products was determined using the {Delta}{Delta}CT technique as described previously [33 ]. Briefly, we normalized each set of samples using the difference in threshold cycle (CT) between the target gene and housekeeping gene (GAPDH): {Delta}CT = (CT target gene–CT GAPDH). Relative mRNA levels were calculated by the expression 2–{Delta}{Delta}CT, where {Delta}{Delta}CT = {Delta}CT sample (n) – {Delta}CT calibrator (n). At the minimum, each reaction was done in triplicate.

Statistical analysis
The Mann-Whitney U-test was used for determining the level of significance of differences in sample means, and Bonferroni’s test was used for multiple comparisons.

RESULTS

Liver injury is inhibited in the absence of L-selectin and/or ICAM-1 expression
AST and ALT plasma levels were measured at 0, 8, 24, and 48 h after Con A injection in wild-type, L-selectin–/–, ICAM-1–/–, and L-selectin/ICAM-1–/– mice to assess hepatic injury. Con A treatment increased AST levels, especially in wild-type mice, with a peak increase at 8 h, which returned close to baseline levels by 48 h after injection. AST levels at the 8-h time-point were reduced significantly by 70% in L-selectin–/– mice compared with wild-type mice (P<0.001; Fig. 1 ). The AST level at 8 h was reduced by 79% in ICAM-1–/– mice relative to wild-type mice (P<0.001), and this level was significantly lower than that found in the L-selectin–/– mice (P<0.05). Furthermore, L-selectin/ICAM-1–/– mice exhibited a 92% decrease in AST levels compared with wild-type mice (P<0.001) at 8 h after Con A injection, and these levels were significantly lower relative to L-selectin–/– or ICAM-1–/– mice (P<0.001 and P<0.01, respectively).


Figure 1
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  		Figure 1. Time course of plasma transaminase levels after Con A injection (25 mg/kg) in wild-type, L-selectin–/–, ICAM-1–/–, and L-selectin/ICAM-1–/– mice. Plasma AST and ALT levels were assessed 0, 8, 24, and 48 h after Con A injection. All values represent the means ± SEM of results obtained using 10 mice in each group. Statistical analysis is provided in Results.

 
Con A treatment also raised plasma ALT levels at 8 and 24 h after treatment in wild-type mice (Fig. 1) . The ALT levels at 8 h were reduced by 45% in L-selectin–/– mice (P<0.001), by 85% in ICAM-1–/– mice (P<0.001), and by 93% in L-selectin/ICAM-1–/– mice (P<0.001) relative to wild-type mice. The ALT levels were also significantly lower in ICAM-1–/– and L-selectin/ICAM-1–/– mice than in L-selectin–/– mice (P<0.001). Furthermore, L-selectin/ICAM-1–/– mice showed significantly decreased ALT levels compared with ICAM-1–/– mice (P<0.05). Thus, L-selectin or ICAM-1 loss alone reduced liver injury, and ICAM-1 loss inhibited it more strongly than L-selectin loss. Furthermore, Con A-induced liver injury was nearly eliminated in the absence of both ICAM-1 and L-selectin expression.

Liver necrosis is inhibited in the absence of L-selectin and/or ICAM-1 expression
Liver damage was also assessed histologically at 0, 8, and 24 h after Con A injection in wild-type, L-selectin–/–, ICAM-1–/–, and L-selectin/ICAM-1–/– mice. It has been demonstrated that DNA fragmentation starts after 4 h, and maximum levels are found 8 h after Con A treatment, consistent with the rise in aminotransferases [34 ]. Nonetheless, only slight degenerative changes and necrosis were observed in the histological section of the liver from wild-type and mutant mice after 8 h of Con A treatment (Fig. 2A ). Although mutant mice showed decreased necrosis compared with wild-type mice, no significant difference was found among each strain at this time-point. After 24 h of Con A treatment, hepatocytes in wild-type mice showed large, bridging necroses (Fig. 2) , as demonstrated in previous studies [2 , 35 ]. Loss of L-selectin or ICAM-1 alone significantly reduced liver necrosis, and ICAM-1–/– mice had more modest necrosis than L-selectin–/– mice. Furthermore, liver necrosis was nearly absent in L-selectin/ICAM-1–/– mice. Similar findings were found until 48 h after Con A injection, by which time, the progress of hepatic injury had ceased (Fig. 2A) . Thus, the concurrent loss of L-selectin and ICAM-1 expression resulted in almost complete inhibition of liver necrosis.


Figure 2
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  		Figure 2. Evaluation of Con A-induced liver necrosis in wild-type, L-selectin–/–, ICAM-1–/–, and L-selectin/ICAM-1–/– mice. (A) The percentage of liver parenchyma with necrotic injury was measured in liver sections at 8, 24, and 48 h after Con A treatment. All values represent the mean ± SEM of results obtained from 10 mice in each group. *, P < 0.05, versus wild-type; **, P < 0.01, versus wild-type. (B) Representative H&E stained sections of livers from wild-type, L-selectin–/–, ICAM-1–/–, and L-selectin/ICAM-1–/– mice 24 h following Con A administration. Original magnification, x200. Arrows indicate necrotic area.

 
Effect of L-selectin and/or ICAM-1 loss on liver leukocyte infiltration
The suppressed liver injury observed in mutant mice could have resulted from impaired leukocyte infiltration into the liver. Therefore, to assess this possibility, the number of CD4+ T cells, CD8+ T cells, and neutrophils around hepatic and portal venules and sinusoids was measured at 0, 8, and 24 h after Con A injection (Fig. 3 ). Before Con A treatment, only a few infiltrating CD4+ T cells, CD8+ T cells, and neutrophils were observed around hepatic and portal venules and sinusoids in each section (data not shown). By contrast, at 8 h after Con A injection, large numbers of infiltrating CD4+ T cells were found in the sinusoids of wild-type mice (Figs. 3A and 4A ). Numbers of infiltrating CD4+ T cell were significantly reduced in L-selectin–/– (32% decrease, P<0.001), ICAM-1–/– (73%, P<0.001), and L-selectin/ICAM-1–/– (78%, P<0.001) mice compared with wild-type mice (Figs. 3A and 4A) . Although the numbers of CD8+ T cells and neutrophils within the sinusoids were elevated following Con A treatment, no significant differences were observed between any of the genotypes of mice (Fig. 3B and 3C) . It is interesting that in wild-type and L-selectin–/– mice, numbers of infiltrating CD4+ T cells in the sinusoids were decreased at 24 h relative to 8 h after Con A treatment, and the opposite was found in the ICAM-1–/– and L-selectin/ICAM-1–/– mice (Fig. 3A) . By contrast, numbers of CD4+ T cells were increased around the portal venules at 24 h relative to 8 h after Con A injection (Fig. 3A) . However, the number of CD4+ T cells adhering around the portal venules after 24 h was reduced significantly in L-selectin–/– (32% decrease, P<0.05), ICAM-1–/– (61%, P<0.001), and L-selectin/ICAM-1–/– (73%, P<0.001) mice relative to wild-type mice (Figs. 3A and 4B) . Thereby, the suppressed CD4+ T cell accumulation around the portal area at 24 h was in parallel with the reduced cell attachment observed in the sinusoids at 8 h after Con A treatment in each of the mutant mice (Fig. 3A) . Regardless, the number of CD4+ T cells adhering to hepatic venules in each mutant mouse was comparable with that in wild-type mice at 8 h and 24 h after Con A injection (Fig. 3A) . No significant differences in the number of CD8+ T cells or neutrophils were observed among wild-type, L-selectin–/–, ICAM-1–/–, and L-selectin/ICAM-1–/– mice in the hepatic and portal veins or sinusoids (Fig. 3B and 3C) . Thus, L-selectin and ICAM-1 contributed to CD4+ T cell-specific infiltration into the liver during Con A-induced liver hepatotoxicity.


Figure 3
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  		Figure 3. Con A-induced recruitment of CD4+ T cells, CD8+ T cells, and neutrophils around portal and hepatic venules and sinusoids in wild-type, L-selectin–/–, ICAM-1–/–, and L-selectin/ICAM-1–/– mice. Numbers of neutrophils were determined by counting in H&E-stained liver sections. Serial sections were stained with anti-CD4 mAb and anti-CD8 mAb to assess the number of CD4+ T cells and CD8+ T cells, respectively. The number of CD4+ T cells (A), CD8+ T cells (B), and neutrophils (C) was evaluated by averaging the numbers of leukocytes present in 10 successive microscopic fields from 10 mice in each group (original, x400). Statistical analysis is provided in Results.

 

Figure 4
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  		Figure 4. Representative immunohistochemical sections of sinusoids at 8 h (A) and portal area at 24 h (B) after Con A administration. Sections were stained with anti-CD4 mAb. Arrows indicate CD4+ T cells. Original magnification, x200.

 
ICAM-1 expression in the liver
ICAM-1 expression on various types of cells, including sinusoidal endothelial cells, is induced by stimulation with proinflammatory cytokines in vitro [20 ]. To assess a role for ICAM-1 in Con A-induced hepatitis, ICAM-1 expression was examined immunohistochemically in the liver of wild-type mice (Fig. 5 ). Weak constitutive expression of ICAM-1 was observed on vascular and sinusoidal endothelial cells, and hepatocytes showed no expression of ICAM-1. A time-course analysis revealed uniformly augmented ICAM-1 expression on vascular and sinusoidal endothelial cells by 8 h after Con A treatment, which remained up-regulated after 24 h. Thus, ICAM-1 expression was increased on vascular and sinusoidal endothelial cells during Con A-induced inflammation.


Figure 5
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  		Figure 5. ICAM-1 expression in the liver at 0, 8, and 24 h after Con A administration in wild-type mice. ICAM-1 expression was assessed by immunohistochemistry using anti-ICAM-1 mAb. Arrows indicate ICAM-1 expression on vascular and sinusoidal endothelial cells. Original magnification, x200.

 
Effect of L-selectin and/or ICAM-1 loss on cytokine and growth factor production
A variety of cytokines and growth factors, including TNF-{alpha}, IFN-{gamma}, and IL-4, has been implicated in the development of Con A-induced liver injury [2 , 5 , 36 ]. These cytokines are generated and released, in part, by inflammatory cells infiltrating into the liver. To assess the effects of L-selectin and/or ICAM-1 deficiency on production of cytokines, mRNA levels of TNF-{alpha}, IFN-{gamma}, IL-4, IL-6, macrophage inflammatory protein-1{alpha} (MIP-1{alpha}), monocyte chemoattractant protein-1 (MCP-1), and transforming growth factor-ß (TGF-ß) in the liver were examined 8 h after Con A challenge by RT-PCR (Fig. 6 ). TNF-{alpha} mRNA levels were decreased equally in L-selectin–/– and L-selectin/ICAM-1–/– mice compared with ICAM-1–/– and wild-type mice (P<0.05). Conversely, ICAM-1–/– and L-selectin/ICAM-1–/– mice exhibited significantly decreased IL-4 mRNA expression relative to L-selectin–/– and wild-type mice (P<0.05). MIP-1{alpha} mRNA expression levels were reduced significantly in L-selectin–/–, ICAM-1–/–, and L-selectin/ICAM-1–/– mice compared with wild-type mice (P<0.05, P<0.01, and P<0.01, respectively). By contrast, no significant differences in levels of IFN-{gamma}, IL-6, MCP-1, or TGF-ß were observed among groups of mice. Thus, loss of L-selectin and ICAM-1 differentially affected cytokine production in the liver during Con A-induced hepatitis.


Figure 6
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  		Figure 6. Relative mRNA expression of TNF-{alpha}, IFN-{gamma}, IL-4, IL-6, MIP-1{alpha}, MCP-1, and TGF-ß in the livers of wild-type, L-selectin–/–, ICAM-1–/–, and L-selectin/ICAM-1–/– mice 8 h after Con A injection. Relative mRNA expression was quantified by real-time RT-PCR. All values represent the mean ± SEM of results obtained from 10 mice in each group.

 
DISCUSSION

The present study demonstrates a critical in vivo role for L-selectin and ICAM-1 in development of Con A-induced hepatitis using gene-targeted mice. Deficiency of L-selectin or ICAM-1 inhibited Con A-induced liver injury (Figs. 1 and 2) , and less damage occurred in ICAM-1–/– mice than in L-selectin–/– mice. It is remarkable that Con A-induced liver injury was almost completely eliminated in the absence of L-selectin and ICAM-1 expression. Therefore, the present study reveals critical and cooperative roles of L-selectin and ICAM-1 in the development of Con A-induced hepatitis. Reduced severity of hepatitis in mutant mice was associated with reduced numbers of liver CD4+ T cells but not numbers of CD8+ T cells or neutrophils (Fig. 3) . In addition, liver IL-4 expression was reduced in ICAM-1–/– and L-selectin/ICAM-1–/– mice, and TNF-{alpha} expression was decreased in L-selectin–/– and L-selectin/ICAM-1–/– mice. Furthermore, MIP-1{alpha} mRNA levels were inhibited by deficiency of L-selectin and/or ICAM-1. These findings suggest that L-selectin and ICAM-1 function are necessary for the development of Con A-induced hepatitis through regulating the selective recruitment of CD4+ T cell subsets and cytokine production in the liver.

An important finding of this study is that ICAM-1 deficiency significantly inhibited liver injury and CD4+ T cell recruitment in Con A-induced hepatitis (Figs. 1 and 2) . Early findings are controversial concerning the effect of pretreatment with anti-ICAM-1 mAb in Con A-induced hepatitis [29 30 31 ]. Although the administration of anti-ICAM-1/LFA-1 mAb almost completely inhibited development of Con A-induced liver injury [29 ], anti-ICAM-1 mAb neutralization did not affect Con A-induced hepatitis in other reports [30 , 31 ]. Nonetheless, our findings using ICAM-1–/– mice, which circumvented the complexity of the mAb-blocking approach, may reveal more reliable roles of ICAM-1. However, in contrast to our findings, a previous report demonstrated that Con A-induced hepatitis was not suppressed in ICAM-1–/– mice [30 ]. As a striking strain difference has been demonstrated in Con A-induced hepatitis [37 ], a subtle difference of genetic background may influence the results. Although the previous study used ICAM-1–/– mice of mixed genetic backgrounds (C57BL/6x129), our mutant mice were back-crossed into C57BL/6 mice more than five generations, and then only littermates were used for the experiments. Therefore, our results are likely less affected by genetic background compared with the previous report.

In the present study, protection from development of Con A-induced hepatitis observed in adhesion molecule-deficient mice correlated with decreased accumulation of CD4+ T cells but not with CD8+ T cells and neutrophils (Figs. 3 and 4) . After Con A administration, the number of CD4+ T cells adhering to the sinusoidal endothelium increased at 8 h and then reduced significantly by 24 h in wild-type mice (Fig. 3A) . By contrast, CD4+ T cells adhering to the portal venules showed the largest number 24 h after Con A administration in wild-type mice (Fig. 3A) . The cellular accumulation in the portal tracts results from the translocation of extrasinusoidally migrating lymphocytes, as the space of Disse is a primary site for hepatic lymph formation and has an anatomical continuity with the portal tract [27 ]. As the majority of lymphocytes adheres in liver sinusoids rather than in the hepatic veins after Con A treatment (Fig. 3) , the adhesion molecule, which is required for firm adhesion to the sinusoidal endothelium and migration into the space of Disse, may play a crucial role in the pathogenesis of Con A hepatitis. It is interesting that ICAM-1 is expressed at higher levels in the liver than in other tissues [38 ], and the sinusoidal endothelial cells constitutively express ICAM-1 (Fig. 5) [30 , 39 ]. Furthermore, ICAM-1 expression on vascular and sinusoidal endothelial cells was up-regulated during 8–24 h after Con A injection (Fig. 5) , as previously reported [30 ]. ICAM-1 has been shown to mediate firm adhesion and to support transendothelial migration of lymphocytes in the liver [40 41 42 ]. Taken together, ICAM-1 expression on sinusoidal endothelial cells, as well as vascular endothelial cells, may contribute to accumulation of CD4+ T cells and subsequent liver injury in the Con A-induced hepatitis model.

It is remarkable that L-selectin deficiency showed a significant inhibitory effect on development of liver damage and CD4+ T cell accumulation in Con A-induced hepatitis (Figs. 1 and 2) . In the liver, the large cross-sectional area of the sinusoids results in a low blood flow rate, which can eliminate the requirement for selectin-mediated rolling and tethering of the leukocytes [43 , 44 ]. Consistent with this, pretreatment with blocking antibodies against E-selectin, P-selectin, and VCAM-1 failed to prevent Con A-induced hepatitis [30 ]. The unimpaired trapping of cells in the livers of E- and P-selectin-deficient mice provides experimental evidence for the minimal role of selectins in this process [19 , 45 ]. A previous paper reported that P-selectin expression plays a significant role in the development of Con A-induced hepatitis [28 ]. However, it has been speculated that P-selectin expression on platelets but not endothelial cells is important for CD4+ T cell infiltration in the liver, as P-selectin was not detected on sinusoids after Con A treatment [28 ]. Thus, previous studies indicate no significant roles of selectins in the sinusoidal endothelium. However, our findings indicate that L-selectin expressed on leukocytes is necessary for optimal CD4+ T cell adhesion to sinusoids, which do not express other selectins. By contrast, neutrophil recruitment was not influenced by the deficiency of ICAM-1 or L-selectin (Fig. 3C) . This is consistent with a previous report demonstrating that no typical adhesion molecules likely affect the neutrophil recruitment in liver sinusoids [45 , 46 ]. Therefore, neutrophil recruitment may be mediated by unidentified adhesion molecules or physical trapping rather than adhesive forces. Thus, L-selectin in addition to ICAM-1 may be important for CD4+ T cell infiltration into the liver in Con A-induced hepatitis.

Deficiencies in L-selectin and ICAM-1 were also found to influence expression of cytokines in the Con A-induced hepatitis model (Fig. 6) . It is important that the TNF-{alpha} mRNA levels in the liver were inhibited specifically by the loss of L-selectin expression. Following the destruction of sinusoidal endothelial cells by T cells, TNF-{alpha} seems to play a central role in the induction of hepatocyte injury [1 2 3 , 47 ]. Therefore, a possible explanation of reduced liver injury by L-selectin deficiency is decreased TNF-{alpha} expression in the liver (Fig. 6) . By contrast, IL-4 expression was reduced by ICAM-1 deficiency (Fig. 6) . Several recent reports have revealed that IL-4, a T helper cell type 2 (Th2) cytokine, which is elevated in the liver following Con A injection, exhibits proinflammatory effects during Con A-induced hepatitis, as demonstrated by anti-IL-4 mAb or IL-4 gene-deficient mouse studies [5 , 7 , 48 , 49 ]. Although it is unclear how these adhesion molecules influenced each cytokine expression, this is likely a result of the selective accumulation of lymphocyte subsets by each adhesion molecule. A recent investigation demonstrated that Th1 cells use {alpha}4-integrin to adhere within liver sinusoids, whereas Th2 cells exclusively use vascular adhesion protein-1 in inflamed liver [46 ]. Like this, it is becoming clear that each organ has its own leukocyte recruitment paradigms, dependent on each inflammatory setting. Future studies will be needed to clarify the mechanism of L-selectin or ICAM-1-dependent lymphocyte subset recruitment in Con A-induced hepatitis.

The accumulation of leukocytes during inflammation is regulated by adhesion molecules and chemokines. MIP-1{alpha} may be involved in the development of Con A-induced hepatitis, as liver MIP-1{alpha} expression was decreased by the loss of L-selectin and/or ICAM-1 (Fig. 6) . MIP-1{alpha} is a CC chemokine that promotes the recruitment of various leukocyte subsets including T cells. Hepatic macrophages are major sources of MIP-1{alpha} during hepatic inflammation [50 , 51 ]. The findings using MIP-1{alpha}-deficient mice have demonstrated that hepatic MIP-1{alpha} plays an important role in the progression of hepatic inflammation and injury associated with Con A-induced hepatitis [52 ]. It is interesting that our results showed that mice deficient for adhesion molecule or both exhibited reduced hepatic MIP-1{alpha} expression, which was parallel with the decreases in CD4+ T cell infiltration and liver injury. Therefore, it is possible that decreased leukocyte infiltration or cytokine production resulting from the loss of L-selectin and ICAM-1 suppresses hepatic MIP-1{alpha} production by Kupffer cells, leading to further impairment in leukocyte recruitment. Thus, interaction between adhesion molecules and chemokines may be a key step in the selective recruitment of effecter cells from the circulation to sites of Con A-induced liver injury.

The unique circulatory system of the liver may diminish the requirement for adhesion molecules, especially selectins. Nonetheless, our findings indicate that adhesion molecules have critical functions in the development of Con A-induced hepatitis by regulating selective leukocyte subset infiltration. The finding that liver injury was virtually eliminated by the loss of L-selectin and ICAM-1 expression indicates that these adhesion molecules are potential therapeutic targets for human T cell-mediated hepatitis.

ACKNOWLEDGEMENTS

This work was supported by a grant-in-aid from the Ministry of Education, Science, and Culture of Japan (to M. Hasegawa and S. S.) and National Institutes of Health, USA, Grants CA54464 and CA81776 (to T. F. T.). We are grateful to Dr. Naofumi Mukaida for use of the Fuji-Dri Chem FDC5500. We also thank Ms. M. Matsubara and Y. Yamada for technical assistance.

Received September 19, 2005; revised November 6, 2005; accepted November 29, 2005.

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P. F. LALOR, C. TUNCER, C. WESTON, A. MARTIN-SANTOS, D. J. SMITH, and D. H. ADAMS
Vascular Adhesion Protein-1 as a Potential Therapeutic Target in Liver Disease
Ann. N.Y. Acad. Sci., September 1, 2007; 1110(1): 485 - 496.
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