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(The FASEB Journal. 2005;19:1245-1252.)
© 2005 FASEB

The role of histamine H1 receptor and H2 receptor in LPS-induced liver injury

Takayuki Masaki, Seiichi Chiba, Hiroshi Tatsukawa, Hitoshi Noguchi, Tetsuya Kakuma, Mizuki Endo, Masataka Seike, Takeshi Watanabe* and Hironobu Yoshimatsu1

Department of Internal Medicine, School of Medicine, Oita University, Oita, Japan; and
* Laboratory for Immune Surveillance RIKEN Research Center for Allergy and Immunology, Yokohama, Japan

1Correspondence: Department of Internal Medicine, School of Medicine, Oita University, Hasama, Oita, 879-5593, Japan. E-mail: MASAKI{at}med.oita-u.ac.jp


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
To examine the role of histamine H1 and H2 receptors in the regulation of lipopolysaccharide (LPS)-induced liver injury, a combination of D-galactosamine and LPS (GalN/LPS) was administered to histamine H1 receptor knockout (H1-R KO) and H2 receptor knockout (H2-R KO) mice. The numbers of necrotic and apoptotic hepatocytes in the liver, as well as the levels of serum aspartate transaminase (AST) and alanine transaminase (ALT), were increased significantly by GalN/LPS treatment compared to the appropriate controls. Pretreatment with histamine ameliorated the GalN/LPS-induced necrotic and apoptotic changes in the hepatocytes and inhibited the elevation of serum AST and ALT levels. Histamine attenuated the GalN/LPS-induced increases in the levels of TNF-{alpha}, but augmented those of IL-10 both in the liver and serum. Histamine inhibited the GalN/LPS-induced caspase-3 activity in the liver. Furthermore, these effects of histamine were completely or partially attenuated in H2-R KO mice, but not in H1-R KO mice. Peritoneal macrophages from H2-R KO mice exhibited blunted changes in the effects of histamine on LPS-induced TNF-{alpha} and IL-10 production in vitro compared to the wild-type (WT) controls. In summary, the present findings suggest that the histamine H2-R-TNF-{alpha} and -IL-10 pathways play protective roles in endotoxin-induced hepatic injury.—Masaki, T., Chiba, S., Tatsukawa, H., Noguchi, H., Kakuma, T., Endo, M., Seike, M., Watanabe, T., Yoshimatsu, H. The role of histamine H1 receptor and H2 receptor in LPS-induced liver injury.


Key Words: lipopolysaccharide • histamine receptor • tumor necrosis factor-{alpha} • IL-10


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
ACUTE HEPATIC FAILURE and hepatitis are associated with high patient mortality, and are often refractory to clinical treatment (1 , 2) . Bacterial lipopolysaccharide (LPS), the release of which is responsible for systemic reactions in patients with severe infections, is implicated in the pathogenesis of liver injury (3) . LPS exerts its effects by stimulating inflammatory cells, such as monocytes in the circulation and macrophages that have infiltrated the liver (4 5 6) . Kupffer cells in the liver also play a crucial role in determining the development of immune responses to LPS (7 8 9) . Cytokines, such as tumor necrosis factor-{alpha} (TNF-{alpha}), interferon-{gamma} (INF-{gamma}), interleukin-1ß (IL-1ß), and interleukin-10 (IL-10), all of which are released from inflammatory cells and hepatic Kupffer cells, have been shown to be involved in LPS-induced liver injury (7 , 10 11 12 13) .

Administration of LPS to animals is used as an experimental model to analyze the mechanism underlying endotoxin-induced acute liver injury, since it induces the infiltration of inflammatory cells into the liver and causes acute liver injury (14) . Furthermore, the administration of LPS concomitant with, or subsequent to, a subtoxic dose of D-galactosamine (GalN) produces more severe hepatic damage in this animal model, with consequent apoptotic and necrotic changes in the liver (14 , 15) . In these cases, proinflammatory cytokines induce hepatocyte apoptosis (11 , 16) , and treatment with anti-inflammatory cytokines prevents GalN/LPS-induced fulminant hepatic failure in experimental animals (17 , 18) .

Histamine, a well-known bioactive monoamine in inflammatory cells, plays an important role in inflammatory and allergic responses (19 20 21 22) . Studies have demonstrated that the interactions between histamine and its specific receptors promote inflammatory reactions by modulating levels of inflammatory cytokines (23 24 25 26 27 28 29 30) . Recent studies have demonstrated that histamine regulates T cell and antibody responses by promoting the differential expression of the histamine H1 and H2 receptors (31) . Histamine and the histamine receptors have been shown to influence immune responses by regulating the production of LPS-induced cytokines (26 27 28 29 30) .

The present study aims to clarify the functional roles of histamine and its receptor in the development of acute fulminant hepatic injury in mice that are deficient for histamine H1-R and H2-R. First, we analyzed the effects of exogenous histamine on GalN/LPS-induced changes on serum levels of AST and ALT and on levels of hepatocyte apoptosis and necrosis. Second, we examined the effects of histamine on changes in the levels of cytokines in the liver induced by GalN/LPS treatment. Third, to understand the receptor mechanism, we investigated the effects of histamine on those parameters that are related to hepatic injury in mice deficient for histamine H1-R and H2-R.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Animals
Eight-wk-old male C57Bl/6 mice, weighing ~22–25 g, were purchased from Seac Yoshitomi (Yoshitomi, Fukuoka, Japan). Histamine H1-R knockout (H1-R KO) (32 33 34) and H2-R knockout (H2-R KO) mice (35) were obtained from Kyushu University (Kyushu University, Fukuoka, Japan). Backcrossing H1R–/– or H2R–/– homozygous mice to the C57Bl/6 strain for six generations resulted in the incipient congenic N5 mice. Southern and/or Northern blot confirmed the genotypes. Mice were housed in a bacteria-free room at a temperature of 21 ± 1°C and with 55 ± 5% humidity and daily illumination between 07:00 and 19:00 (12 h light: 12 h dark). All animals were treated in accordance with Oita University Guidelines for the Care and Use of Laboratory Animals.

Reagents
LPS (Sigma, St Louis, MO, USA) or histamine phosphate (Sigma) were dissolved in phosphate-buffered saline (PBS). These solutions were freshly prepared on the day of administration.

Experimental protocols
For preparation of mice with GalN/LPS-induced fulminant hepatic failure, C57Bl/6, H1-R KO, and H2-R KO mice were given an intraperitoneal (i.p.) injection of GalN (800 µg/g body weight; Sigma), immediately followed by an i.p. injection of LPS (30 ng/g body weight). To determine the effects of histamine on mice with fulminant hepatic failure, histamine (10 (µg/g body weight) was injected s.c. at 0.5 and 12 h before GalN/LPS administration. The doses of GalN, LPS, and histamine were determined in previous studies (36–38) and in our study (39) . In addition, the above doses of histamine or GalN alone did not induce liver injury as determined by evaluating liver enzymes and liver histology. Serum and liver samples for cytokines analysis or histology were obtained at 0, 1.5, 4, 6, 12, or 24 h after the administration of GalN/LPS (n=4–6 for each group).

AST, ALT, TNF-{alpha}, INF-{gamma}, and IL-10 levels
Serum levels of liver enzymes, including AST and ALT, were determined using an automatic analyzer (SRL, Tokyo, Japan). Liver, serum, and medium TNF-{alpha}, INF-{gamma}, and IL-10 were determined with an enzyme-linked immunosorbent assay (ELISA) kit (BioSource) and an OD reader according to the manufacturer’s instructions. Liver samples (100 mg) that had been frozen at –80°C were homogenized in 1 mL of PBS and centrifuged at 1500 rpm. The supernatant was collected and stored at –80°C. Protein concentrations of the liver solutions were analyzed using the method of Bradford (Bio-Rad Lab, CA, USA); OD readings of samples were converted to pg/mL using standard curves generated with the recombinant cytokine supplied with the kit.

Histopathological analysis
Small pieces of liver were removed and rinsed with saline. Tissue sections were cut at a thickness of 5 µm and stained with hematoxylin and eosin (HE). To examine hepatocytes, HE-stained liver sections were analyzed with an image analysis system (Olympus, Tokyo, Japan).

Preparation of peritoneal macrophages, peritoneal fluid, and culture medium
Mice were maintained free of specific pathogens in autoclaved cages in a laminar flow hood to minimize the spontaneous activation of macrophages. To produce responsive macrophages, 4 mL of sterile PBS was injected i.p and PBS was collected. The fluid was centrifuged at 300 x g for 15 min at 4°C, and the supernatant was decanted and analyzed. The resultant peritoneal cells were obtained and plated at 5 x 105 cells/well in 96-well plates with RPM1 medium and 10% heat-inactivated PBS (Invitrogen Corp., Tokyo, Japan). After incubation for 30 min at 37°C in a 5% CO2 atmosphere, nonadherent cells were removed by washing with ice-cold saline; adherent macrophages were used for the experiments. Adherent cells were >95% macrophages. Resident peritoneal macrophages were prepared from untreated control mice in an LPS-free system. Attached macrophages were cultured in RPM1 and used for experiments. All reagents to which macrophages were exposed in culture were free of detectable LPS. Peritoneal macrophages cultured in RPM1 medium with histamine (10–5 M) in 96-well plates were stimulated with LPS (0–5 ng/mL); the medium was replaced with 0.2 mL of fresh medium at 0, 2, or 4 h after the addition of LPS. After incubation for 0, 2, or 4 h, the replaced medium was collected into microcentrifuge tubes and centrifuged at 900 x g for 5 min at 4°C. The supernatant was concentrated by centrifugal filtration using filter tubes (Millipore, Bedford, MA, USA) and the cytokines released from macrophages were analyzed by ELISA.

Determination of apoptosis
Apoptotic hepatocytes were detected with the in situ terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) method using an apoptosis kit (Medical Biological Lab, Nagoya, Japan). Liver sections (5 µm) were treated with proteinase K, and endogenous peroxidase activity was blocked by treatment with 0.02% hydrogen peroxide. Tissue sections were treated with a mixture of terminal deoxynucleotidyl transferase, digoxigenin-labeled dUTP, and dATP at 37°C for 1 h, followed by incubation with peroxidase-labeled anti-digoxigenin antibody solution for 30 min. As a negative control, PBS was substituted for the mixture of deoxynucleotidyl transferase, digoxigenin-labeled dUTP, and dATP. In addition, apoptosis in liver was analyzed by nuclear staining with 500 ng/mL of 4',6-diamidino-2-phenylindole (DAPI) for 10 min at room temperature. Furthermore, DNA fragmentation was also quantified by using an apoptosis ELISA kit (Funakoshi Co., Ltd., Tokyo, Japan) as in other studies (40 , 41) .

Immunohistochemistry
Liver sections were transferred without rinsing to a solution that contained the primary antibody (anti-histamine H2 receptor; Cosmo Bio Co., Ltd., Tokyo, Japan). Sections were incubated on ice for 24 h, rinsed in PBS, then processed with an ABC kit (Vector Laboratories, Burlingame, CA, USA). Sections were transferred to a solution that contained biotinylated antibody for 1 h, rinsed, transferred to avidin-biotinylated peroxidase for 1 h, rinsed, and finally developed using diaminobenzidine as a substrate.

Determination of caspase-3 and -8 activity
To determine the activation of caspase-3 and -8 in the liver tissue of mice, liver homogenates were prepared in lysis buffer and analyzed using a caspase-3 activity assay kit (Medical Biological Laboratories Co., Ltd., Nagoya, Japan) and a caspase-8 activity assay kit (B-Bridge Japan, Tokyo, Japan) according to the manufacturer's instruction. Briefly, liver homogenates were centrifuged at 10,000 g for 1 min at 4°C and their protein content determined (Bio-Rad Lab, CA, USA). Liver homogenates were incubated with DEVD or IETD-pNA substrate at 37°C for 1 h, and a wavelength of 405 nm was measured in a plate reader (Multiscan Multisoft, LabSystems, Tokyo, Japan).

Real-time quantitative reverse transcription polymerase chain reaction
Liver TNF-{alpha}, INF-{gamma}, IL-10, and H2-R mRNAs were determined by polymerase chain reaction (PCR) amplification and quantified by real-time quantitative PCR. Total cellular RNA was prepared from selected mouse tissues using TRIzol (Lifetech, Tokyo, Japan) according to the manufacturer’s protocol. Total RNA (20 µg) was electrophoresed on 1.2% formaldehyde-agarose gels. RNA quality and quantity were assessed by EtBr-agarose gel electrophoresis and by measuring the relative absorbance at 260 nm vs. that at 280 nm. cDNA was synthesized from 150 ng of total RNA in a volume of 20 µL with a ReverTra-Dash reverse transcriptase kit (Toyobo, Tokyo, Japan) using random hexamer primers. Reactions were diluted to 50 µL with sterile distilled H2O and stored at –20°C. Primers for mouse TNF-{alpha}, INF-{gamma}, IL-10, and H2-R were designed, synthesized, optimized, and provided as preoptimized kits: TNF-{alpha} (Cat. no. Mm 00443258m1), INF-{gamma} (Cat. no. 00801778m1), IL-10 (Cat. no. Mm 00439616m1), and H2-R (Cat. no. Mm 00434009m1). Primers for rRNA as internal controls were provided as a preoptimized kit (Cat. no. Hs99999901). Using an ABI PRISM 7000 sequence detector, PCR amplifications were performed in volumes of 50 µL containing 100 ng cDNA template in PCR Master Mix (Roche, NJ, USA) according to the following program: 50°C for 2 min; 95°C for 10 min; 40 cycles at 95°C for 15 s; and 60°C for 1 min. Samples were analyzed in duplicate. Results were analyzed with Sequence Detection Software (ABI) and expression levels of TNF-{alpha}, INF-{gamma}, IL-10, and H2-R mRNAs were normalized to rRNA, as outlined in Perkin-Elmer’s User Bulletin No. 2.

Statistical analysis
All the data were expressed as the mean ± standard error of the mean (SE). The statistical analysis of difference was assessed by ANOVA for multiple comparisons or the unpaired t test was used where appropriate. The relationship between apoptosis in the liver and the level of TNF-{alpha} was examined by the Pearson's correlation coefficient.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Effects of histamine on GalN/LPS-induced serum AST and ALT levels in C57Bl/6 mice
Figure 1 A–C demonstrates the effects of histamine on GalN/LPS-induced liver injury in C57Bl/6 mice. The administration of GalN/LPS increased serum levels of AST and ALT 12 h post-treatment compared with PBS-treated controls (P<0.01 for each) (Fig. 1C) . Pretreatment with histamine abrogated the GalN/LPS-induced increases in serum AST and ALT compared with pretreatment with PBS 12 h after GalN/LPS treatment (P<0.01 for each) (Fig. 1C ). The administered dosages of histamine did not induce liver injury when given alone, as assessed by the serum levels of AST and ALT (Fig. 1C ).



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  		Figure 1. Effects of histamine (HA) on GalN/LPS-induced changes. A) Histology, hematoxylin and eosin (HE). B) Histology, TUNEL and DAPI. C) Serum aspartate transaminase (AST) and alanine transaminase (ALT) levels. D) Apoptotic changes in the liver. E) caspase-3 activity in liver. Mice were injected i.p. with GalN and LPS. *P < 0.05, **P < 0.01 vs. PBS-treated controls, +P < 0.05, ++P < 0.01, vs. LPS-treated controls.

Effects of histamine on GalN/LPS-induced hepatic apoptosis and necrosis in C57Bl/6 mice
Administration of GalN/LPS induced apoptosis and necrosis of hepatocytes (Fig. 1) . TUNEL staining, DAPI staining and a DNA fragmentation ELISA revealed significant hepatic apoptosis 6 h after GalN/LPS treatment (control vs. LPS; P<0.01) (Fig. 1B, D ). Pretreatment with histamine reduced the apoptotic changes examined by staining with TUNEL, DAPI, and a DNA fragmentation ELISA (LPS vs. LPS-HA; P<0.05) in the liver (Fig. 1B, D ). Focal necrosis induced by accumulated macrophages and neutrophils and extensive necrosis were observed in HE-stained liver sections 24 h after GalN/LPS treatment (Fig. 1A ).

Relationship between GalN/LPS-induced liver apoptosis and TNF-{alpha}
We analyzed the relationship between GalN/LPS-influenced value of DNA fragmentation in the liver and the serum level of TNF-{alpha}. The value of DNA fragmentation was positively correlated with serum TNF-{alpha} (P<0.01).

Effects of histamine on GalN/LPS-induced caspase-3 and -8 activity in the liver
GalN/LPS-induced activation of caspase-3 in PBS pretreated mice 6 h after administration, whereas activation was inhibited in histamine pretreated mice (LPS, 0.248±0.041; LPS+HA, 0.081±0.019; P<0.01) (Fig. 1E ). In addition, activation of caspase-8 was increased by GalN/LPS, which was also inhibited by histamine pretreatment (LPS, 0.067±0.007; LPS+HA, 0.036± 0.005; P<0.05).

Effects of histamine on GalN/LPS-induced changes in the levels of TNF-{alpha}, INF-{gamma}, and IL-10
As shown in Fig. 2 , the administration of GalN/LPS-induced increases in the serum levels of TNF-{alpha}, INF-{gamma}, and IL-10. Similarly, administration of GalN/LPS-induced increases in the levels of TNF-{alpha}, INF-{gamma}, and IL-10 mRNA in the liver compared with PBS-treated controls after 6 h of treatment (P<0.01 for each) (Fig. 2A ). Pretreatment with histamine attenuated the GalN/LPS-induced increases in serum TNF-{alpha} (1.5 h after treatment) and INF-{gamma} (4 h after treatment) (P<0.05 or P<0.01) (Fig. 2B ). In contrast, pretreatment with histamine exacerbated the GalN/LPS-induced increases in serum and hepatic IL-10 compared with the effects of PBS pretreatment 4 h after treatment (P<0.05 or P<0.01) (Fig. 2A, B ). Similar results were obtained for the protein concentrations of TNF-{alpha} , INF-{gamma}, and IL-10 in the liver (Fig. 2C ). Histamine alone did not significantly regulate serum TNF-{alpha} (control, 11.2±1.9; HA, 8.2±1.4; P>0.1), INF-{gamma} (control, 5.8±1.0; HA, 5.2±0.5; P>0.1), and IL-10 (control, 6.2±0.7; HA, 9.7±2.0; P>0.1) (Fig. 2B ).



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  		Figure 2. Effects of HA on LPS-induced changes in TNF-{alpha}, INF-{gamma}, and IL-10. A) Hepatic TNF-{alpha}, INF-{gamma}, and IL-10 mRNA expression levels in vivo. B) Serum TNF-{alpha}, INF-{gamma}, and IL-10 in vivo. C) Hepatic TNF-{alpha}, INF-{gamma}, and IL-10 protein levels. D) Supernatant TNF-{alpha} levels in vitro. E) Supernatant IL-10 levels in vitro. **P < 0.01 vs. PBS-treated controls. +P < 0.05, ++P < 0.01, vs. LPS-treated controls.

Effects of histamine on the LPS-induced TNF-{alpha} and IL-10 levels of peritoneal macrophages in vitro
Administration of LPS to peritoneal macrophages in vitro induced increases in the supernatant levels of TNF-{alpha} compared with untreated macrophages (P<0.01) (Fig. 2D ). Exogenous histamine treatment for 2 h attenuated the LPS-induced increases in the peritoneal macrophage culture supernatant levels of TNF-{alpha} compared with control levels (P<0.01) (Fig. 2D ). In contrast, histamine treatment for 2 h augmented the LPS-induced increases in the macrophage culture supernatant levels of IL-10 compared with the control levels (P<0.05) (Fig. 2E ).

Effect of histamine on GalN/LPS-induced liver injury in histamine H1-R and H2-R KO mice
Detection of histamine H2-Rs around the hepatic vein was enhanced by GalN/LPS treatment (Fig. 3 A). In addition, the expression level of histamine H2-R mRNA in the liver was increased by GalN/LPS treatment (control, 100.0±9.9; GalN/LPS, 204.2±34.2; P<0.05) (Fig. 3B ). As described above, pretreatment with histamine antagonized the effects of GalN/LPS on serum AST and ALT levels, as well as on the levels of hepatic apoptosis and necrosis (Fig. 1) . These effects of histamine on GalN/LPS-induced changes in the AST and ALT levels were partially attenuated in histamine H2-R KO mice, but not in histamine H1-R KO mice compared with the WT controls after 12 h treatment (P<0.01) (Fig. 4 C). The effects of histamine on liver tissues were attenuated in histamine H2-R KO mice compared with WT controls when gauged by effects on TUNEL staining, DAPI staining, and DNA fragmentation ELISA (GalN/LPS-WT vs. GalN/LPS-H2-R; P<0.05) (Fig. 4B, D ). Focal necrosis induced by accumulated macrophages and neutrophils and extensive necrosis were exacerbated in the histamine H2-R KO mice compared with the WT controls after 24 h treatment (Fig. 4A ). The effect of histamine on hepatic caspase-3 activity was attenuated in histamine H2-R KO mice compared with the WT controls after 6 h treatment (GalN/LPS-WT, 0.065±0.005; GalN/LPS-H2-R, 0.111± 0.017; P<0.05) (Fig. 4E ). In addition, activation of hepatic caspase-8 was exaggerated in histamine H2-R KO mice (GalN/LPS-WT, 0.047±0.006; GalN/LPS-H2-R, 0.069±0.003; P<0.05).



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  		Figure 3. GalN/LPS-induced changes in histamine H2-R in the liver. A) Hepatic histamine H2-R immunohistochemistry (x400) B) Hepatic histamine H2-R mRNA expression levels; *P < 0.05 vs. PBS-treated controls.



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  		Figure 4. Effects of HA on GalN/LPS-induced changes in histamine H1-R knockout (H1R-KO) and histamine H2-R knockout (H2R-KO) mice. A) Histology, hematoxylin and eosin (HE). B) Histology,TUNEL and DAPI; C)Serum aspartate transaminase (AST) and alanine transaminase (ALT) levels. D) Apoptotic changes in the liver. E) Caspase-3 activity in liver. Mice were injected i.p. with GalN and LPS. +P < 0.05, ++P < 0.01 vs. WT.

Effects of histamine on GalN/LPS-induced changes in the serum and hepatic cytokine levels of both histamine H1-R and H2-R KO mice
The treatment of WT mice with histamine attenuated the GalN/LPS-induced increases in the levels of serum and hepatic TNF-{alpha} and INF-{gamma}, but augmented the increases in serum and hepatic IL-10 levels compared with pretreatment with PBS after 1.5 or 4 h treatment (P<0.05 or P<0.01) (Fig. 2A, B ). The effects of histamine on GalN/LPS-induced changes in the serum or hepatic levels of TNF-{alpha} and INF-{gamma}, and IL-10 were blunted in histamine H2-R KO mice, but not in H1-R KO mice compared with WT mice (P<0.05 or P<0.01) (Fig. 5 A–C).



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  		Figure 5. Effects of HA on LPS-induced changes in TNF-{alpha}, INF-{gamma}, and IL-10 changes in H1R-KO and H2R-KO mice. A) Hepatic TNF-{alpha}, INF-{gamma}, and IL-10 mRNA expression levels in vivo. B) Serum TNF-{alpha}, INF-{gamma}, and IL-10 in vivo. C) Hepatic TNF-{alpha}, INF-{gamma}, and IL-10 protein levels. D) Supernatant TNF-{alpha} levels in vitro. E) Supernatant IL-10 levels in vitro. +P < 0.05, ++P < 0.01, vs. WT.

Effects of histamine on LPS-induced TNF-{alpha} and IL-10 levels in peritoneal macrophages of histamine H1-R and H2-R KO mice in vitro
In vitro, the effects of histamine on LPS-induced increases in TNF-{alpha} levels were blunted in histamine H2-R KO mice compared with WT mice (P<0.01) (Fig. 5D) . In addition, the effects of histamine on the levels of LPS-induced IL-10 were blunted in histamine H2-R KO mice compared with WT mice in vitro (P<0.05) (Fig. 5E ).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In agreement with previous reports (11 , 14 , 16 17 18 , 36) , we found that injection of LPS induced increases in the serum levels of AST and ALT, as well as apoptotic and necrotic changes in hepatocytes, which are biochemical and histological parameters of liver damage, respectively. LPS increased levels of TNF-{alpha}, INF-{gamma}, and IL-10 in both the liver and serum (36 , 38) . Previous studies have demonstrated that histamine influences LPS-induced inflammatory reactions by modulating the levels of proinflammatory cytokines (23 24 25 26 27 28 29 30) . The results of the present study show that pretreatment with histamine attenuates acute hepatic injury that is induced by LPS. The elevation of serum levels of AST and ALT, increased rates of hepatocyte apoptosis and necrosis, and changes in the levels of TNF-{alpha}, INF-{gamma}, and IL-10 were all recovered partially by histamine treatment in vivo. In addition, peritoneal macrophages pretreated with histamine in vitro exhibited significantly decreased LPS-induced TNF-{alpha} production and increased IL-10 production compared with controls. These results indicate that histamine regulates LPS-induced liver injury and cytokine production, both in vivo and in vitro.

It is important to elucidate how histamine rescues the liver from GalN/LPS-induced injury. Liver apoptosis and necrosis develop in a stepwise fashion. The first step is apoptosis, which is caused by various humoral factors, including proinflammatory cytokines (14 15 16) . The second step is focal necrosis, which is induced by the accumulated polymorphonuclear cells and lymphomononuclear cells (14 15 16) . Finally, massive necrosis appears to be caused by intra-hepatic macrophages and neutrophils (14 15 16) . Since histamine is involved in lymphocyte proliferation and neutrophil chemotaxis and infiltration, the possibility exists that histamine prevents liver injury by affecting the late stage of hepatic cell damage, which includes necrosis, induced by inflammatory cells. However, the present study demonstrates that histamine attenuates GalN/LPS-induced apoptosis of hepatocytes, which implicates histamine activity in the early stage of hepatic cell damage. TNF-{alpha} has been shown to be the key cytokine in the induction of extensive apoptosis and necrosis of hepatocytes in GalN/LPS-induced fulminant hepatic failure (11 , 15 , 16) . Histamine treatment blocks the expression of TNF-{alpha}. Taken together, our results suggest that histamine prevents the development of liver injury at an early stage of cell damage by inhibiting the production and/or release of TNF-{alpha}, which are regulators of apoptosis.

Several inflammatory cytokines, including TNF-{alpha}, INF-{gamma}, and IL-10, have been shown to be involved in liver injury induced by GalN/LPS (7 , 11 12 13) . Among these factors, it has been reported that TNF-{alpha} INF-{gamma} accelerate, and IL-10 decelerates, the development of liver injury (7 , 11 12 13) . In the present study, the levels of all of these proinflammatory cytokines were increased in the liver by GalN/LPS treatment. Pretreatment with histamine attenuated the increases in the levels of hepatic TNF-{alpha} and INF-{gamma} and augmented the increase in the level of IL-10, thereby suppressing liver injury. These results indicate that histamine protects against LPS-induced liver injury by regulating the levels of TNF-{alpha}, INF-{gamma}, and IL-10. Especially, TNF-{alpha} binds death receptor, leading to activation of caspase-8 and caspase-3 (42 43 44) . The present study demonstrated histamine inhibited LPS/GalN-induced TNF-{alpha} level, caspase-8 and caspase-3 activity. In addition, GalN/LPS-induced value of DNA fragmentation was positively correlated with the level of TNF-{alpha}. These results indicated that TNF-{alpha}-caspase-8 and -3-mediated pathways might contribute to the effect of histamine on GalN/LPS-induced liver apoptosis.

We analyzed the receptors involved in histamine protection against liver injury. Our results show that in histamine H2-R KO mice, but not in histamine H1-R KO mice, histamine inhibition of the development of liver injury was attenuated either completely or partially. Thus, it appears that histamine H2-R can rescue GalN/LPS-induced liver injury by regulating the levels of proinflammatory cytokines. Our results agree with previous reports using histamine receptor agonists and/or antagonists (26 27 28 29) , which have shown that histamine and the H2 receptor modulate immune responses by inhibiting the production of TNF-{alpha} and INF-{gamma}, and by stimulating the release of IL-10 in H2-R KO mice. In addition, peritoneal macrophages from histamine H2-R KO mice, but not from H1-R KO mice, exhibited increased LPS-induced TNF-{alpha} and IL-10 regulation in vitro compared with the controls. These results indicate the direct effects of histamine H2-R on LPS-induced toxicity and that H2-R is more important than H1-R in suppressing LPS-induced proinflammatory cytokine production. Previous studies demonstrated that the effect of histamine in the heart, lung, and the hepatoma-derived cell line is based on H2-R-mediated activation of adenylate cyclase and cyclic AMP (45 46 47) . H2-R-mediated activation of adenylate cyclase and cyclic AMP might be involved in LPS-induced liver injury.

In summary, we have shown that appropriate histamine preadministration can inhibit GalN/LPS-induced liver injury. The ability of histamine to inhibit the production, release, or action of proinflammatory cytokines partially accounts for the protective effect of histamine on LPS toxicity in mice. The present findings suggest that the histamine H2-R pathway play a crucial role in endotoxin-induced hepatic injury by regulating the production of proinflammatory cytokines in the liver. Therefore, the manipulation of the histamine H2-R pathway may be useful for treatment of endotoxin-induced liver injury and related inflammatory disorders.


   ACKNOWLEDGMENTS
 
This work was supported by Grants-in-Aid 136770067 from the Japanese Ministry of Education, Science and Culture and by Research Grants for Intractable Diseases from the Japanese Ministry of Health and Welfare, 2001–2002. This work was also supported in part by a grant from the Smoking Research Foundation, venture business laboratory in Oita University, and Grants 16922171 from Japan Society for the Promotion of Science.

Received for publication October 3, 2004. Accepted for publication April 8, 2005.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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