inducible nitric oxide synthase
monocyte chemoattractant protein
macrophage inflammatory protein
Neutrophils and macrophages infiltrate after acetaminophen (APAP)-induced liver injury starts to develop. However, their precise roles still remain elusive. In untreated and control IgG-treated wild-type (WT) mice, intraperitoneal APAP administration (750 mg/kg) caused liver injury including centrilobular hepatic necrosis and infiltration of neutrophils and macrophages, with about 50% mortality within 48 h after the injection. APAP injection markedly augmented intrahepatic gene expression of inducible nitric oxide synthase (iNOS) and heme oxygenase (HO)-1. Moreover, neutrophils expressed iNOS, which is presumed to be an aggravating molecule for APAP-induced liver injury, while HO-1 was mainly expressed by macrophages. All anti-granulocyte antibody-treated neutropenic WT and most CXC chemokine receptor 2 (CXCR2)-deficient mice survived the same dose of APAP, with reduced neutrophil infiltration and iNOS expression, indicating the pathogenic roles of neutrophils in APAP-induced liver injury. However, APAP caused more exaggerated liver injury in CXCR2-deficient mice with reduced macrophage infiltration and HO-1 gene expression, compared with neutropenic WT mice. An HO-1 inhibitor, tin-protoporphyrin-IX, significantly increased APAP-induced mortality, implicating HO-1 as a protective molecule for APAP-induced liver injury. Thus, CXCR2 may regulate the infiltration of both iNOS-expressing neutrophils and HO-1-expressing macrophages, and the balance between these two molecules may determine the outcome of APAP-induced liver injury.
Acetaminophen (APAP) is widely prescribed as an analgesic and antipyretic drug in clinics and is also sold as numerous over-counter preparations as a single compound or in combination with other medications 1, 2. Although it is generally safe, an overdose of APAP can cause severe liver failure with a significant morbidity and mortality. Thus, its wide availability and severe toxicities has placed APAP overdose as the leading cause for calls to Poison Control Centers in the United States (>100 000/year). Moreover, APAP overdose accounts for more than 56 000 emergency room visits, 2600 hospitalizations, and an estimated 458 deaths due to acute liver failure each year in the United States alone 3.
The toxic response is initiated by the metabolism of APAP to a toxic metabolite, N-acetyl-p-benzoquinone imine. Because N-acetyl-p-benzoquinone imine can react rapidly with sulfhydryl groups, it first depletes glutathione in hepatocytes and then reacts with a number of intracellular proteins, thereby causing their dysfuntions 4, 5. APAP-induced liver injury is histopathologically characterized by centrilobular hepatic necrosis with a massive infiltration of leukocytes, particularly neutrophils. The recruitment of leukocytes in the damaged tissue is a hallmark of the inflammatory process, which may contribute to the development of APAP-induced liver injury 6, 7. In line with this assumption, accumulating evidence has implicated neutrophils as the leukocyte type essentially involved in the pathogenesis of various liver diseases 8–12. However, we previously observed that the increases in serum alanine transferase (ALT) levels preceded neutrophil infiltration into liver in APAP-induced liver injury 13, 14. Inactivation of neutrophils by anti-CD18 Ab had no effects on APAP-induced liver injury 15. In contrast, Smith and his colleagues 16 demonstrated that rats treated by anti-neutrophil serum exhibited an enhanced resistance to APAP-induced hepatotoxicity, although they did not explore its mechanisms in detail. Thus, there still remain controversies on the pathogenic roles of neutrophils in APAP-induced liver injury.
Neutrophil recruitment is regulated by CXC chemokine ligands (CXCL), murine KC/CXCL1, and macrophage inflammatory protein (MIP)-2/CXCL2 17, 18. Several lines of evidence demonstrated that the recruitment of neutrophils was significantly reduced in some disease models using mice lacking CXC chemokine receptor 2 (CXCR2), a receptor for CXCL1 and CXCL2 19–26. Our previous study demonstrated that the magnitude of liver injury induced by APAP well correlated with intrahepatic neutrophil number and intrahepatic myeloperoxidase activity, suggesting that neutrophil recruitment was involved in the pathogenesis of APAP-induced liver injury 13, 14.
Here, in order to address this point, we caused APAP-induced liver injury in anti-granulocyte Ab (Ly-6G)-treated neutropenic WT and CXCR2-KO mice. Both Ly-6G-treated neutropenic WT mice and CXCR2-KO mice showed attenuated APAP-induced liver injury, with reduced neutrophil infiltration. Moreover, we demonstrated that neutrophils expressed CXCR2 and inducible NO synthase (iNOS), which can produce NO, a potent injurious mediator for APAP-induced liver injury 13. When compared between neutropenic WT and CXCR2-KO mice, the latter were less resistant to APAP-induced hepatotoxicity, with less macrophage infiltration. We further showed that a substantial portion of macrophages expressed CXCR2 and that macrophages were a major source of heme oxygenase (HO)-1, which exhibits protective activities for various liver injuries including APAP-induced liver injury 27–30. Thus, the present study implied that CXCR2 has double-edged roles in APAP-induced liver injury, by regulating the recruitment of both iNOS-expressing neutrophils and HO-1-expressing macrophages.
Alleviation of APAP-induced liver injury in neutropenic WT and CXCR2-KO mice
We previously observed that the increases in serum ALT levels preceded neutrophil infiltration into liver in APAP-induced acute hepatotoxicity 13, 14. These observations raised questions on the pathogenic roles of neutrophil infiltration in APAP-induced liver dysfunction. Hence, in order to address this point, we injected the same dose of APAP into untreated WT, control IgG-treated WT, anti-Ly-6G Ab-treated neutropenic WT mice, and mice deficient in CXCR2, a murine unique receptor for CXC chemokines with an ELR motif and neutrophil chemotactic activities.
Within 48 h after APAP administration (750 mg/kg), about 50% of IgG-treated WT and WT mice succumbed to acute liver failure (IgG-treated WT, ten deaths/22 mice; WT, nine deaths/19 mice), while all neutropenic WT (no deaths/14 mice) and most of CXCR2-KO mice (four deaths/26 mice) survived at this time point (Fig. 1a). Before APAP challenge, there were no discernible differences in serum ALT levels and liver morphology among the four groups (Fig. 1b, c). Both untreated and control IgG-treated WT mice exhibited centrilobular hepatic necrosis with severe hemorrhage and leukocyte infiltration to similar extents at 6 and 24 h after APAP treatment, whereas these morphological alterations were apparently diminished in neutropenic WT mice and, to a lesser degree, CXCR2-KO mice (Fig. 1b). Consistent with these morphological changes, the increases in serum ALT levels were depressed in neutronpenic WT mice and, to a lesser degree, CXCR2-KO mice, compared with untreated and control IgG-treated WT mice (Fig. 1c). Taken collectively, these observations would imply that neutrophil infiltration contributed to the development of APAP-induced liver dysfunction.
Leukocyte recruitment in the liver after APAP challenge
The attenuation of APAP-induced liver injury was less prominent in CXCR2-KO mice than in neutropenic WT mice, suggesting that CXCR2 was only partially responsible for neutrophil infiltration. In order to explore this possibility, we next examined the effects of peripheral blood neutrophil depletion or CXCR2 deficiency on leukocyte infiltration in the liver after APAP injection.
Few neutrophils and discernible numbers of macrophages were present in the liver among the four groups before APAP treatment. APAP treatment increased the number of neutrophils and macrophages in both untreated and control IgG-treated WT mice progressively until 24 h (Fig. 2). In contrast, neutrophil infiltration was significantly reduced in neutropenic WT and CXCR2-KO mice to a similar extent, compared with untreated and control IgG-treated WT mice (Fig. 2a, c). The numbers of intrahepatic macrophages were increased in neutropenic WT mice to the same extent as in untreated and control IgG-treated WT mice at any time intervals examined (Fig. 2b, d). On the contrary, the infiltration of macrophages was significantly reduced in CXCR2-KO mice, compared with the other three groups (Fig. 2b, d). These observations suggest that CXCR2 could regulate the infiltration of macrophages as well as neutrophils after APAP challenge
The roles of CXCR2-mediated signals in macrophage infiltration in the liver after APAP challenge
A double-color immunofluorescence analysis was performed to determine the types of CXCR2-expressing cells. Most Ly-6G-positive neutrophils expressed CXCR2 in the liver of mice after APAP challenge (Fig. 3a). Of interest is that a small but substantial proportion of F4/80-positive macrophages also expressed CXCR2 (Fig. 3b). Thus, CXCR2 and its ligands may be able to regulate the infiltration of F4/80-positive macrophages as well as neutrophils.
In order to address this possibility, we administered Ab to mouse CXCL1 and CXCL2 before APAP treatment. Either anti-CXCL1 or anti-CXCL2 Ab significantly reduced the infiltration of F4/80-positive cells induced by APAP treatment (Fig. 4a). Moreover, the combined administration of anti-CXCL1 and anti-CXCL2 Ab reduced F4/80-positive cell infiltration to a similar extent as in CXCR2-KO mice and anti-CXCR2 Ab-treated WT mice (Fig. 2a; 4). These observations suggest that CXCR2 and its ligands can regulate F4/80 cell infiltration induced by APAP treatment. However, CXCL1 and CXCL2 exhibited marginal in vitro chemotactic activities even at 100 ng/mL (Table 1). Thus, the absence of CXCR2 may impair macrophage infiltration rather indirectly by affecting the production of chemokines with macrophage chemotactic activities, such as monocyte chemoattractant protein (MCP)-1/CCL2 and MIP-1α/CCL3
|Control||CXCL1 (20 ng/mL)||CXCL1 (100 ng/mL)||CXCL2 (20 ng/mL)||CXCL2 (100 ng/mL)|
|Migrated macrophages||6.5 ± 0.9||15.5 ± 2.0||16.8 ± 2.2||14.8 ± 1.2||15.3 ± 1.3|
Under the employed experimental conditions, the gene expression of CCL2 and CCL3 was faintly detected in the liver of all four groups. APAP induced the intrahepatic gene expression of CCL2 and CCL3 to similar extents in untreated, control IgG-treated, and neutropenic WT mice (Fig. 5). On the contrary, APAP-induced enhancement in CCL2 and CCL3 gene expression was significantly attenuated in CXCR2-KO mice, compared with the other groups (Fig. 5). Thus, the absence of CXCR2 may diminish expression of these chemokines, thereby reducing macrophage infiltration into the liver.
The roles of iNOS after APAP challenge
To clarify the differences in the sensitivities to APAP between neutropenic WT and CXCR2-KO mice, we next examined the intrahepatic gene expression of iNOS, because iNOS-mediated NO generation can be detrimental to APAP-induced liver injury 12, 13. Even before APAP treatment, the gene expression of iNOS was detected at weak but similar levels in the four groups (Fig. 6a). At 24 h after APAP treatment, iNOS mRNA expression was remarkably enhanced in untreated and control IgG-treated WT mice. However, iNOS expression was not augmented in both neutropenic WT and CXCR2-KO mice (Fig. 6a, b).
Consistent with iNOS mRNA levels, nitrotyrosine, a marker of peroxynitrite-induced tissue injury, was detected immunohistochemically in centrilobular regions in the liver of untreated and control IgG-treated WT mice but not neutropenic WT or CXCR2-KO mice, at 24 h after APAP challenge (Fig. 7). Thus, different sensitivities between neutropenic WT and CXCR2-KO mice cannot be due to differences in iNOS expression and eventual peroxynitrite-induced tissue injury. Moreover, a double-color immunofluorescence analysis demonstrated that iNOS was expressed mainly by Ly-6G-positive neutrophils (Fig. 6d). Collectively, Ly-6G treatment and the lack of CXCR2 reduced the infiltration of neutrophils, a main cellular source of iNOS, to a similar extent.
The roles of HO-1 in APAP-induced liver injury
Because several lines of evidence suggest protective roles of HO-1 in experimental liver injury 27–30, we next examined the intrahepatic HO-1 gene expression. Before APAP injection, HO-1 mRNA was detected faintly but to a similar extent in the liver of all four groups. APAP treatment augmented the intrahepatic HO-1 gene expression markedly and to similar extents in untreated, control IgG-treated, and neutropenic WT mice, but not CXCR2-KO mice (Fig. 6a, c). Moreover, HO-1 proteins were detected in most F4/80-positive macrophages (Fig. 6e) and a few neutrophils (data not shown).
In order to define the role of HO-1 in APAP-induced liver injury, WT and neutropenic WT mice were administered with an HO-1 inhibitor, tin-protoporphyrin-IX (SnPP; 25 mg/kg), or vehicle at 24 h before APAP challenge. In vehicle-treated groups, survival rates were 50% at 48 h after APAP challenge (six deaths/12 mice), whereas the administration of SnPP significantly increased mortality rates, reaching more than 80% (ten deaths/12 mice) (Fig. 8a). Moreover, SnPP treatment aggravated APAP-induced liver injury in neutropneic WT mice as evidenced by an increase in serum ALT levels (Fig. 8b), although all these mice survived the challenge. Thus, these observations implied that HO-1 had a protective role in APAP-induced liver injury and that the reduction in HO-1-expressing macrophages may account for incomplete attenuation in APAP-induced liver injury in CXCR2-KO mice, compared with neutropenic WT mice.
Several lines of evidence indicate that neutrophils contribute to the development of various types of liver injury 8–12, 31–35. However, our previous observations that liver dysfunction preceded neutrophil infiltration in APAP-induced liver injury 13, 14, prompted us to investigate the pathogenic roles of neutrophils, using the anti-granulocyte Ab. Here, we demonstrated that depletion of neutrophils from peripheral blood prevented the development of APAP-induced liver injury as well as neutrophil infiltration almost completely. Thus, the direct hepatotoxicities of APAP may induce liver injury in the early phase, but neutrophils subsequently infiltrate and aggravate the precedent liver injury probably by producing injurious factors.
NO is mainly produced by iNOS and contributes to tissue damages in various kinds of liver injury 36, 37. Consistently, we provided definitive evidence on the essential involvement of NO and iNOS also in APAP-induced liver injury 13. Here, we further proved that neutrophils were a main cellular source of iNOS and that depletion of neutrophils attenuated APAP-induced iNOS expression as well as neutrophil infiltration almost to a basal level. Moreover, immunohistochemical analysis demonstrated that depletion of neutrophils diminished the generation of nitrotyrosine, a representative marker of NO-mediated tissue damage. Thus, infiltrating neutrophil-derived iNOS could generate NO, thereby causing tissue damages.
Hogaboam and his colleagues 38, 39 claimed that CXC chemokines with an ELR motif such as IL-8/CXCL8, MIP-2, CXCL1, and IFN-γ-inducible protein-10/CXCL10 were protective for APAP-induced liver injury when administered intravenously, although there exist inconsistencies on the effects of IFN-γ-inducible protein-10/CXCL10 in their studies. They further asserted that the protective effects of these chemokines could be ascribed to mitogenic activity of CXCR2-mediated signals on hepatocytes 40.
On the contrary, we observed that APAP-induced liver injury was markedly attenuated in CXCR2-KO mice with reduced neutrophil infiltration, compared with WT mice, in line with previous reports that CXCR2-KO mice exhibited attenuated neutrophil infiltration in various disease models 16–27. Indeed, Hogaboam and colleagues 38 observed that only a low level of CXCR2 was expressed on untreated hepatocytes and that APAP could enhance CXCR2 protein expression only transiently with a time lag. Moreover, pharmacological doses of CXCL2 could significantly accelerate hepatocyte proliferation after a partial hepatectomy, later than 36 h after the injection 40. Thus, it is probable that hepatoprotective effects of CXCR2-mediated signals may be apparent in the later phase of APAP-induced liver injury. On the contrary, until 48 h after APAP injection as in our experimental conditions, CXCR2-mediated signals may be more responsible for neutrophil-mediated liver injury. This assumption may be supported by the similar observation on myocardial reperfusion injury, where CXCR2 exerted both direct cardioprotective effects and indirect damaging effects by recruiting neutrophils 41.
APAP-induced F4/80-positive macrophage infiltration was attenuated in CXCR2-KO but not neutropenic WT mice. In line with our previous observation that CXCR2 was expressed by murine peripheral blood monocytes 42, we observed that CXCR2 was expressed by a portion of F4/80-positive macrophages in the present study. The genetic disruption of CXCR2 or the administration of neutralizing Ab to CXCR2 or its ligands significantly attenuated macrophage recruitment into the liver after APAP challenge, indicating that CXCR2-mediated signals can regulate macrophage recruitment. Contrary to this assumption, CXCL1 or CXCL2 exhibited in vitro marginal macrophage chemotactic activities. These discrepancies may be explained by the previous report that a CXCR2 ligand, human CXCL8, can cause the adherence of human monocytes to the endothelium under shear stress, an essential step for monocyte transendothelial migration 43. Alternatively, infiltrating neutrophils can regulate subsequent mononuclear leukocyte infiltration as previously reported 44–48. Consistently, the gene expression of CCL2 and CCL3, potent monocyte/macrophage chemoattractant chemokines, was reduced in CXCR2-KO mice. Thus, CXCR2 may regulate the expression of these chemokines, thereby modulating intrahepatic monocyte/macrophage trafficking.
CXCR2-KO mice were more susceptible to APAP challenge than neutropenic WT mice, as judged by serum ALT levels and pathological changes. One prominent difference between neutropenic WT and CXCR2-KO mice was the degree of macrophage infiltration. The protective role of macrophages in APAP-induced liver injury was suggested by the observations that APAP-induced liver injury was augmented in CCR2-KO mice 49, 50, which exhibited impaired monocyte/macrophage trafficking, due to the absence of CCR2, a specific receptor for potent monocyte/macrophage chemotactic chemokines, MCP-1/CCL2 and MCP-2/CCL8. Moreover, the depletion of intrahepatic macrophages/Kupffer cells exaggerated the susceptibility to APAP-induced liver injury 51. These observations suggest that macrophages could exert protective effects against APAP-induced liver injury probably by expressing a protective mediator(s).
HO-1, also known as heat shock protein 32, is a rate-limiting enzyme in the degradation of heme into biliverdin, carbon monoxide, and iron 52, 53. Several lines of evidence suggest that HO-1 can attenuate tissue injury caused by various inflammatory stimuli 54–56. Moreover, the pretreatment of rats with hemin, a potent inducer of HO-1, prevented APAP-induced hepatotoxicity 29.
Consistently, we observed that an HO-1 inhibitor, SnPP, aggravated APAP-induced liver injury as evidenced by increased mortality rates in WT mice and elevated serum ALT levels in neutropenic WT mice. Moreover, our double-color immunofluorescence analysis revealed that macrophages were a main cellular source of HO-1 as reported previously 57. Furthermore, macrophage infiltration and enhanced HO-1 gene expression were observed to similar extents in the liver of untreated and neutropenic WT mice, but not CXCR2-KO mice, after APAP challenge. Thus, different sensitivities to APAP-induced liver injury could be ascribed to the differences in macrophage trafficking between neutropenic WT and CXCR2-KO mice. Collectively, CXCR2 can regulate the infiltration of iNOS-expressing neutrophils and HO-1-expressing macrophages, and the balance between these two gaseous mediators may modulate the outcome of APAP-induced liver dysfunction. Thus, the modulation of these two enzymes may be a good therapeutic target for APAP-induced liver injury.
Materials and methods
Reagents and antibodies
APAP was purchased from Sigma Chemical Co. (St. Louis, MO). A hybridoma clone, RB6-8C5, which secretes a rat anti-mouse Ly-6G mAb 58, 59, was a kind gift from Dr. Robert Coffman (DNAX Research Institute of Molecular and Cellular Biology, Palo Alto, CA). The anti-Ly-6G Ab was purified as described previously and used to deplete peripheral blood granulocytes 58, 59.
The following monoclonal Ab (mAb) and polyclonal Ab (pAb) were used for immunohistochemical or double-color immunofluorescence analysis: rat anti-mouse Ly-6G mAb, rat anti-mouse F4/80 mAb (Dainippon Pharmaceutical Co., Osaka, Japan), rabbit anti-nitrotyrosine pAb (Upstate Biotechnology, Lake Placid, NY), rabbit anti-CXCR2 pAb 60, rat anti-CXCL1 mAb, rat anti-CXCL2 mAb (R&D Systems, Inc., Minneapolis, MN), rabbit anti-mouse iNOS pAb, rabbit anti-mouse HO-1 pAb (Stressgen Biotechnologies, Victoria, Canada), cyanine dye 3-conjugated donkey anti-rat IgG pAb, and FITC-conjugated donkey anti-rabbit IgG pAb (Jackson Immunoresearch Laboratories, West Grove, PA). SnPP, a selective inhibitor of HO-1 activity 61, was obtained from Frontier Scientific (Logan, UT). Recombinant murine CXCL1 and CXCL2 were purchased from PeproTech (London, UK).
Pathogen-free 8-wk-old male BALB/c mice were obtained from Sankyo Laboratories (Tokyo, Japan) and were designated as WT mice in the present experiments. CXCR2-deficient (CXCR2-KO) mice on a BALB/c genetic background were purchased from Jackson Laboratories (Bar Harbor, ME). All these mice were kept under specific pathogen-free conditions at the Animal Research Center of Kanazawa University. Age- and sex-matched mice were used for the present experiments. All animal experiments in this study complied with the Guidelines for the Care and Use of Laboratory Animals on the Takara-machi Campus of Kanazawa University.
APAP-induced liver injury
APAP-induced liver injury was established as described previously 13, 14. Briefly, APAP solution was made immediately before each experiment by dissolving the compound in phosphate-buffered saline (PBS; pH 7.2) and was warmed to 37°C. In all experiments, mice were allowed free access to water but not food for 10 h before APAP challenge, and were administered intraperitoneally with 750 mg/kg of APAP. Preliminary experiments demonstrated that intraperitoneal injection of 250 µg anti-mouse Ly-6G reduced the peripheral blood neutrophil numbers below 5% of control IgG-treated animals until 2 days after the treatment (our unpublished data). Thus, in some experiments, WT mice were injected intraperitoneally with 250 µg anti-mouse Ly-6G or isotype-matched control IgG 1 day before APAP administration 58, 59. In another series of experiments, WT or neutropenic WT mice were injected intraperitoneally with SnPP (25 mg/kg) 24 h before APAP challenge. In some experiments, WT mice received intraperitoneally 250 µg neutralizing anti-CXCR2, anti-CXCL1, anti-CXCL2, or control Ab 1 h before the administration of APAP (750 mg/kg).
Determination of serum alanine aminotransferase levels
Whole-blood samples were collected at the indicated time intervals after APAP injection to determine serum ALT levels with a Fuji DRI-CHEM 3500V as instructed by the manufacturer (Fuji Medical System, Tokyo, Japan).
Liver tissues were obtained from the mice at the indicated time intervals after APAP challenge, and were fixed in 4% formaldehyde buffered with PBS (pH 7.2), and then embedded with paraffin. Sections (6 µm thick) were stained with hematoxylin and eosin. Histopathological findings of the livers were scored as described previously 62, 63. Briefly, histologically normal sections were graded 0; minimal centrilobular necrosis was graded 1+; more extensive necrosis confined to centrilobular regions was graded 2+; necrosis extending from central zone to portal triads was graded 3+; and massive necrosis of most of the liver was graded 4+. All evaluations were performed by an examiner without prior knowledge of the experimental procedures.
An immunohistochemical analysis was performed as described previously 13, 14. Briefly, deparaffinized sections were immersed in 0.3% H2O2 in methanol for 30 min to eliminate endogenous peroxide activities. The sections were further incubated with PBS containing 1% normal serum derived from the same species as that used for the preparation of the secondary Ab and 1% BSA to reduce nonspecific reactions. The sections were incubated with anti-Ly-6G, anti-F4/80, or anti-nitrotyrosine Ab at a concentration of 1 μg/mL at 4°C overnight. After the incubation with biotinylated secondary Ab (2 μg/mL) at room temperature for 30 min, immune complexes were visualized using a Catalyzed Signal Amplification system (DAKO, Kyoto, Japan) according to the manufacturer's instructions. The number of infiltrating neutrophils and macrophages in the liver were enumerated on ten randomly chosen visual fields at ×400 magnification and the average of the ten selected microscopic fields was calculated 13, 14. All measurements were performed by an examiner without prior knowledge of the experimental procedures.
Double-color immunofluorescence analysis
A double-color immunofluorescence analysis was also performed to identify the types of CXCR2-expressing cells in the liver of mice as described previously 14. Briefly, deparaffinized sections were incubated with PBS containing 1% normal donkey serum and 1% BSA to reduce nonspecific reactions. Thereafter, the sections were further incubated with the combination of anti-CXCR2 and anti-Ly-6G, or anti-CXCR2 and anti-F4/80 Ab at 4°C overnight. All Ab were used at a concentration of 1 μg/mL. After the incubation with fluorochrome-conjugated secondary Ab (15 μg/mL) at room temperature for 30 min, the sections were observed under a fluorescence microscope. A double-color immunofluorescence analysis was also performed using anti-Ly-6G or anti-F4/80 mAb, together with anti-iNOS or anti-HO-1 Ab at 4°C overnight. All Ab were used at a concentration of 1 μg/mL. Fluorescence images were observed as mentioned above.
Extraction of total RNA and RT-PCR
Semi-quantitative RT-PCR analysis was performed as described previously 13, 14. Briefly, total RNA were extracted from the removed liver tissues using ISOGEN (Nippon Gene, Toyama, Japan) according to the manufacturer's instructions. Five micrograms of total RNA was reverse-transcribed into cDNA using the Superscript II Reverse Transcriptase (Invitrogen Life Technologies, Tokyo, Japan) with oligo(dT) Primers (Invitrogen Life Technologies). Thereafter, the resultant cDNA was amplified together with Taq polymerase (Nippon Gene, Tokyo, Japan) using the specific sets of primers shown in Table 2. The generated PCR products did not reach a saturable level with the determined optimal cycle numbers. The amplified PCR products were fractionated on a 2% agarose gel and visualized by ethidium bromide staining. The band intensities were measured using NIH Image Analysis software version 1.63 (National Institutes of Health, Bethesda, MD), and the ratio of each band to β-actin was calculated.
|Transcript||Sequence||Annealing temperature (°C)||Cycles||Product size (bp)|
(R) 5′- GGCAATCAGTTCCAGGTCAGT-3′
Mouse peritoneal macrophages were collected 24 h after intraperitoneal injection of thioglycollate and were suspended at a concentration of 0.8×105/mL in chemotaxis buffer consisting of Hepes/Hanks balanced salt solution (Teijin Bio, Tokyo Japan). After recombinant murine CXCL1 or CXCL2 were added to lower wells of 5-μm-pore-size chemotaxis chambers (Chemotaxicells; Kurabou, Osaka, Japan) at a concentration of 20 or 100 ng/mL, 200 µL of the cell suspension was added to the upper wells. After incubation for 4 h at 37°C in a 5% CO2 atmosphere, the side of the polycarbonate membrane in contact with the cell suspension was scraped and washed to remove any cells. After fixation in ethanol for 15 min, the migrated cells adhering to the underside of the membrane facing the chemoattractant were stained with hematoxylin. The number of cells that had migrated through the filter was determined by counting the cells in five high-power fields under a microscope. Four replicate filters were used for each treatment. As a negative control, chemotaxis buffer without any chemokine was added to the lower chamber. These experiments were repeatedly carried out four times.
The means and SEM were calculated for all parameters determined in this study. Statistical significance was evaluated using one-way ANOVA with post-hoc testing with the Scheffé's F multiple comparisons test or Mann–Whitney's U-test. The survival curves by the Kaplan–Meier procedure were analyzed by a log-rank test. p<0.05 was accepted as statistically significant.
The work was financially supported in part by Grants-in-Aids from the Ministry of Education, Culture, Sports, Science, and Technology of the Japanese Government.