Toxicological Sciences 54, 509-516 (2000)
Copyright © 2000 by the Society of Toxicology
The Hepatic Inflammatory Response after Acetaminophen Overdose: Role of Neutrophils
>,1
* Department of Pharmacology, Pharmacia & Upjohn, Inc., Kalamazoo, Michigan 49007;
Department of Pathology, University of Texas Health Science Center, Houston, Texas 77030; and
Department of Pharmacology and Toxicology, University of Arkansas for Medical Sciences, Little Rock, Arkansas 72205
Received September 16, 1999; accepted December 8, 1999
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Acetaminophen overdose induces severe liver injury and hepatic failure. There is evidence that inflammatory cells may be involved in the pathophysiology. Thus, the aim of this investigation was to characterize the neutrophilic inflammatory response after treatment of C3Heb/FeJ mice with 300 mg/kg acetaminophen. A time course study showed that neutrophils accumulate in the liver parallel to or slightly after the development of liver injury. The number of neutrophils in the liver was substantial (209 ± 64 PMN/50 high-power fields at 12 h) compared to baseline levels (7 ± 1). Serum levels of TNF-
and the C-X-C chemokines KC and MIP-2 increased by 28-, 14-, and 295-fold, respectively, over levels found in controls during the injury process. In addition, mRNA expression of MIP-2 and KC were upregulated in livers of acetaminophen-treated animals as determined by ribonuclease protection assay. However, none of these mediators were generated in large enough quantities to account for neutrophil sequestration in the liver. There was no upregulation of Mac-1 (CD11b/CD18) or shedding of L-selectin on circulating neutrophils. Moreover, an anti-CD18 antibody had no protective effect against acetaminophen overdose during the first 24 h. These results indicate that there is a local inflammatory response after acetaminophen overdose, including a substantial accumulation of neutrophils in the liver. Because of the critical importance of ß2 integrins for neutrophil cytotoxicity, these results suggest that neutrophils do not contribute to the initiation or progression of AAP-induced liver. The inflammation observed after acetaminophen overdose may be characteristic for a response sufficient to recruit neutrophils for the purpose of removing necrotic cells but is not severe enough to cause additional damage. Key Words: cytokines; chemokines; adhesion molecules; inflammation, liver necrosis; Mac-1 (CD11b/CD18); L-selectin (CD62L).
|
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
An overdose of the analgesic acetaminophen (AAP) causes liver injury and potential hepatic failure in experimental animals and humans (Thomas, 1993
). The toxic response is initiated by the metabolism of AAP to the electrophilic metabolite N-acetyl-p-benzoquinone imine (NAPQI) (Dahlin et al., 1984). Because of the high reactivity of NAPQI with sulfhydryl groups, it first depletes glutathione in hepatocytes (Mitchell et al., 1973b) and then reacts with a number of intracellular proteins (Cohen and Khairallah, 1997; Pumford et al., 1997). Subsequent events, which may be important for the pathophysiology, are an increase in cytosolic and nuclear Ca2+ (Shen et al., 1991; Tsokos-Kuhn et al., 1988), resulting in DNA fragmentation (Shen et al., 1991; Ray et al., 1993), mitochondrial dysfunction (Esterline et al., 1989; Meyers et al., 1988) and a mitochondrial oxidant stress (Jaeschke, 1990). However, it is controversial whether or not some of these effects are exclusively the results of NAPQI protein binding or if reactive metabolites generated by inflammatory cells also contribute to the toxicity. The first description of hepatic neutrophil accumulation (Mitchell et al., 1973a) and Kupffer cell activation (Laskin and Pilaro, 1986) after AAP overdose was published some time ago. However, only recently was clear evidence provided for a contribution of Kupffer cells to the injury process (Laskin et al., 1995; Michael et al., 1999). In contrast, the role of neutrophils in the pathophysiology of AAP overdose remains unclear.
Neutrophils have been shown to be involved in a number of liver disease processes including hepatic ischemia-reperfusion injury (Jaeschke, et al., 1990), endotoxemia (Hewett et al., 1992; Jaeschke et al., 1991), sepsis (Molnar et al., 1997), alcoholic hepatitis (Bautista, 1997) and after exposure to certain chemicals such as -naphthylisothiocyanate (Dahm et al. 1991). Neutrophil-induced liver injury is a multistep process that includes the activation and recruitment of these inflammatory cells into the liver vasculature, transendothelial migration, and adherence to parenchymal cells (Jaeschke et al., 1996; Jaeschke and Smith, 1997). Members of the C-X-C chemokine family are potent chemoattractants for neutrophils and have been shown to contribute to hepatic neutrophil recruitment and injury during endotoxemia and ischemia-reperfusion (Colletti et al., 1996; Maher et al., 1997; Zhang et al., 1995). In addition, a number of adhesion molecules, particularly members of the ß2 integrin family (CD11/CD18), are required for neutrophil-induced cell killing (Jaeschke, 1997). One member, Mac-1 (CD11b/CD18), proved to be essential for the adherence-dependent oxidant burst and cell injury (Shappell et al., 1990). Consequently, antibodies against CD11b and CD18 (Jaeschke et al., 1991, 1993b; Liu et al., 1995), which functionally inactivated neutrophils in vivo, effectively protected against a neutrophil-mediated injury. Therefore, the aims of our study were to characterize and quantify hepatic neutrophil sequestration during AAP overdose in mice and to test whether liver injury can be attenuated by a monoclonal antibody against CD18. In addition, we determined the formation of C-X-C chemokines after AAP to evaluate if these chemoattractants could be responsible for the recruitment of neutrophils into the liver.
|
MATERIALS AND METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Animals.
Male C3Heb/FeJ mice (20–25 g body weight) were purchased from Jackson Laboratories (Bar Harbor, ME). The animals had free access to food (certified rodent diet 5002C; PMI Feeds, Inc., Richmond, IN) and water. The experimental protocols followed the criteria of Pharmacia & Upjohn, Inc. and the National Research Council for the care and use of laboratory animals in research. Animals were fasted overnight and then treated intraperitoneally with 300 mg/kg acetaminophen (Sigma Chemical Co, St. Louis, MO) dissolved in warm (37°C) saline (16 mg/ml). Some animals were treated with 2 mg/kg (iv) of the anti-mouse CD18 monoclonal antibody 2E6 (Endogen Inc., Woburn, MA). The first dose was injected 2 h after AAP treatment and a second dose at 8 h. This treatment regimen with IgG antibodies proved to be successful for 24-h experiments (Farhood et al., 1995
; Jaeschke et al., 1990; Liu et al., 1995). Control animals received vehicle (10 ml/kg PBS containing 0.1% BSA) at the same time. Some animals were intravenously injected with recombinant mouse MIP-2 or KC (Endogen Inc.) (8 µg/kg).
Experimental protocol.
Groups of animals (n = 5–6) were treated with AAP. At different time points (0–24 h), animals were anesthetized with pentobarbital (60 mg/kg). Blood was collected from the vena cava into a syringe; 0.1 ml of the blood was heparinized and the rest was allowed to coagulate on ice. All samples were centrifuged and the plasma was used for determination of alanine aminotransferase (ALT) activity with Sigma test kit DG 159-UV. The serum samples were aliquoted and stored at –80°C until analysis. Livers were sectioned transversely across the midportion of each lobe; parts were fixed in phosphate-buffered formalin for histologic analysis, and the rest was frozen in liquid nitrogen for hemoglobin analysis and RNA isolation.
Histology.
Formalin-fixed portions of the liver were paraffin-embedded and 5-µm thick sections were cut. Cell damage was evaluated in parallel sections stained with hematoxylin-eosin. The percentage of necrosis was estimated by evaluating the number of medium power microscopic fields (x 10) with necrosis compared to the entire histologic section. Parallel sections were stained with naphthol AS-D chloroacetate esterase as described in detail (Jaeschke et al., 1996). Neutrophils were counted in 50 high-power fields (x 400) using a Nikon Labophot Microscope. The pathologist (AF) performing the histologic evaluation was blinded as to the treatment of animals.
Biochemical and immunologic analyses.
Serum samples were analyzed for TNF-, KC, and MIP-2 with the respective ELISA kits, i.e., Factor-TestTM Mouse TNF ELISA Kit (Genzyme, Inc., Cambridge, MA), Quantikine Mouse KC (R&D Systems, Inc., Minneapolis, MN). and Quantikine Mouse MIP-2 (R&D Systems, Inc.). Tissue hemoglobin as indicator for hemorrhage was determined with the Total Hemoglobin Kit (Sigma Diagnostics, St. Louis, MO). Briefly, a 20% liver homogenate was prepared in 50 mM Na-phosphate buffer (120 mM NaCl, 10 mM EDTA). After centrifugation at 16,000 x g for 10 min at 4°C, the supernatant was diluted in Drabkin's reagent and the absorbance measured at 550 nm. To account for different background absorbance, the absorbance at 550 nm was obtained from a spectrum (400–700 nm). The hemoglobin (Hb) concentration was determined with a calibration curve and calculated as milligrams hemoglobin/gram liver tissue.
Ribonuclease protection assay (RPA).
Total cellular RNA was isolated from mouse liver tissue according to the method of Chomczynski and Sacchi (1987) as described in detail (Essani et al., 1997). For the RPAs, all protocols followed the instructions of the RiboQuant Multi-Probe RNase Protection Assay System (PharMingen, San Diego, CA). Using the In Vitro Transcription Kit and a customized template set containing mouse MIP-2, mouse KC, and L32, a radiolabeled probe set was synthesized using [-32P]UTP. These probes were hybridized with total RNA isolated from liver tissue for 16 h. After digestion of nonhybridized RNA with RNase, the protected probes were separated on a denaturing acrylamide gel. The gel was dried and then exposed to X-ray film (Kodak X-OMatTM, Fisher Scientific, Pittsburgh, PA) for 12 h at –80°C. The developed X-ray films were scanned using a Bio-Rad GS-710 Calibrated Imaging Densitometer (Bio-Rad Laboratories, Hercules, CA). The images were labeled and printed using Adobe Photoshop 4.0.
Flow cytometric analysis (Essani et al., 1997).
Peripheral blood neutrophils were stained using Coulter Immunology's (Hialeah, FL) whole blood lysis kit according to the manufacturer's instructions. Briefly, 100 µl of whole blood was washed three times with phosphate-buffered saline containing 0.1% bovine serum albumin (PBS/BSA). Cells were resuspended with 100 µl of PBS/BSA containing 1 µg of fluorescein isothiocyanate (FITC)-conjugated RB6-8C5 (anti-GR-1) and phycoerythrin (PE)-conjugated M1/70 (anti-CD11b), or PE-conjugated Mel-14 (anti-L-selectin) antibodies (Pharmingen, San Diego, CA). Following incubation for 30 min on ice, cells were pelleted by centrifugation and washed twice with PBS. Cells were lysed with 1 ml Immuno-Lyse (Coulter) for 2 min at room temperature and fixed with 250 µl of Coulter Clone fixative. Cells were then pelleted by centrifugation, washed twice with PBS, and resuspended in PBS. Two-color analysis of antibody binding to cells was conducted by flow cytometry using a FACScan flow cytometer (Becton Dickinson, San Diego, CA). Peripheral blood neutrophils were gated by the forward and light angle scatter and Gr-1 FITC fluorescence. Nonspecific fluorescence was determined on cells incubated with isotype and cytochrome-matched control antibodies.
Statistics.
Data are given as mean ± SE. Comparisons between multiple groups were performed with one-way ANOVA followed by Bonferroni t test. p < 0.05 was considered significant.
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Administration of 300 mg/kg AAP induced liver injury, which was first evident at 2 h with a moderate increase of plasma ALT values, and then rapidly progressed to very high levels between 4 and 24 h (Fig. 1A
). Histologic evaluation showed the known pattern of centrilobular necrosis (data not shown). The number of necrotic hepatocytes was estimated with 54 ± 2% at 6 h and 60 ± 6% at 24 h. Parallel to the development of severe parenchymal cell damage, there was hemorrhage, as indicated by the substantial accumulation of hemoglobin in liver tissue (Fig. 1A). The liver hemoglobin content, which was low in controls (0.4 ± 0.1 mg Hb/g liver tissue), did not change at 2 h, but increased by 73-fold at 4 h. Peak values were measured between 6 and 24 h (86- to 103-fold increase). In control livers, relatively few neutrophils were observed in sinusoids (7 ± 1 PMN/50 HPF). This did not change at the 2-h time point, but with the development of liver injury, the number of neutrophils increased gradually to reach their peak levels between 12 and 24 h (Fig. 1B). Thus, neutrophil accumulation in the liver occurred parallel to or slightly trailed the development of hemorrhage and parenchymal cell injury.
|
To investigate whether AAP toxicity caused a systemic activation of neutrophils, the expression of Mac-1 (CD11b/CD18) and L-selectin (CD62L) was determined on circulating neutrophils by flow cytometry. Upregulation of Mac-1 and shedding of L-selectin are markers of neutrophil activation (Jutila et al., 1989
). As shown in Figure 2, mouse neutrophils have a baseline expression of Mac-1 and L-selectin. Evaluation of the adhesion molecule expression 6 h after AAP administration showed no change in the expression of either Mac-1 or L-selectin (Fig. 2). The same results were obtained 4 h after AAP (data not shown). These findings indicate that there is no systemic neutrophil activation during AAP-induced liver injury.
|
To test whether the accumulated neutrophils in the liver contribute to the injury, animals were treated with the anti-CD18 monoclonal antibody 2E6 2 h after AAP administration. A second dose was administered at 8 h. This dosing regimen proved successful in a number of in vivo studies using IgG antibodies (Farhood et al., 1995
; Jaeschke et al., 1990; Liu et al., 1995). At 24 h, AAP caused severe liver injury, as indicated by high levels of plasma ALT, hemorrhage (tissue Hb content), and necrosis in vehicle-treated animals (Table 1). The anti-CD18 antibody had no significant effect on any of these parameters during the first 24 h. In addition, blocking CD18 had no significant effect on hepatic neutrophil accumulation (Table 1).
|
Measurement of serum levels of the proinflammatory cytokine TNF-
(Fig. 3A) showed an increase parallel to the initial injury. Although the levels remained elevated, no significant further increase beyond the 4-h value was observed. In contrast, serum levels of the C-X-C chemokines KC and MIP-2 increased moderately between 4 and 6 h, peaked at 12 h, and declined at 24 h (Figs. 3B and 3C). To demonstrate that at least part of the C-X-C chemokines are generated by the liver during AAP toxicity, mRNA formation of MIP-2 and KC were analyzed by RNase Protection Assay. In the liver of untreated animals, no mRNA for either chemokines was detectable (Fig. 4). Only minor levels of KC mRNA were found in vehicle-treated animals. However, 6 h after AAP administration, both KC and MIP-2 mRNA levels were increased (Fig. 4). These results suggest that C-X-C chemokines are generated in the liver after a toxic dose of AAP.
|
|
To test whether the presence of high levels of MIP-2 and KC in the blood can cause significant neutrophil accumulation in the liver, 8 µg/kg of the recombinant proteins were injected intravenously. Compared to vehicle-treated animals (14 ± 2 PMN/50 HPF), KC had no effect on hepatic neutrophils (10 ± 1 PMN/50 HPF; n = 4) at 2 h. In contrast, MIP-2 had a very moderate effect (32 ± 4 PMN/50 HPF; n = 4; p < 0.05 compared to controls). Similar results were obtained after 4 h (KC: 10 ± 1 PMN/50 HPF; MIP-2: 25 ± 2 PMN/50 HPF, p < 0.05). These data indicate that mouse rMIP-2, but not KC, are able to cause neutrophil sequestration in the liver vasculature. However, when compared to the number of neutrophils accumulated after AAP administration, the effect of MIP-2 is very limited.
|
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
The objective of this investigation was to characterize and quantify hepatic neutrophil accumulation during AAP toxicity and to test if these inflammatory cells aggravate the injury. There is increasing evidence that neutrophils can contribute to a number of liver pathophysiologies, including ischemia-reperfusion injury (Jaeschke et al., 1990
), endotoxemia (Hewett et al., 1992; Jaeschke et al., 1991), sepsis (Molnar et al., 1997), alcoholic hepatitis (Bautista, 1997), and -naphthylisothionate toxicity (Dahm et al., 1991). In these cases, neutrophils are activated and accumulate in the liver vasculature several hours before an involvement in the injury process is detectable (Chosay et al., 1997; Jaeschke et al., 1990 Jaeschke et al., 1991). Although the sequestration in sinusoids is independent of adhesion molecules (Jaeschke et al., 1996), the subsequent transmigration step and the adherence to hepatocytes requires ß2 integrins (CD11/CD18) and intercellular adhesion molecule-1 (ICAM-1) on endothelial cells and hepatocytes (Jaeschke and Smith, 1997). Thus, antibodies against CD11b or CD18 (Jaeschke et al., 1991, 1993b; Liu et al., 1995) and their counterreceptor ICAM-1 (Essani et al., 1995; Farhood et al., 1995) effectively protected against the neutrophil-mediated injury in the liver. In contrast to these findings, neutrophils started to accumulate in the liver shortly after the initiation of the injury and followed with some delay the progression of AAP-induced liver damage. Furthermore, an IgG anti-CD18 monoclonal antibody had no effect on cell damage during the 24-h time period after AAP administration, suggesting that interference with ß2 integrins on neutrophils did not prevent injury. Because of the critical importance of ß2 integrins for neutrophil cytotoxicity (Shappell et al., 1990), these results suggest that neutrophils do not contribute to the initiation or progression of AAP-induced liver toxicity. This conclusion is justified for several reasons, even if the antibody treatment had no effect on the number of neutrophils in the tissue. First, it was shown that neutrophils isolated from the liver after treatment with an anti-CD11b antibody were functionally inactivated, i.e., their capacity to generate reactive oxygen was reduced by > 50% (Jaeschke et al., 1993b). Consistent with these results, anti-CD11b and anti-CD18 antibodies prevented a neutrophil-induced oxidant stress and injury in vivo (Jaeschke et al., 1991). Second, reducing hepatic neutrophils by 60% with a neutropenia-inducing monoclonal antibody had no effect on reperfusion injury; a reduction by 90% was necessary to protect (Jaeschke et al., 1990). These results are consistent with the fact that only 30–35% of accumulating neutrophils actually transmigrate and attack hepatocytes (Essani et al., 1995, 1997). Third, we showed protective effects against neutrophil-mediated injury with antiadhesion antibodies or by interference with chemotaxis without affecting neutrophil counts in the liver (Essani et al., 1995, 1997; Farhood et al., 1995; Jaeschke et al., 1998; Lawson et al., 1998). Thus, overall neutrophil counts do not correlate with effectiveness of protective interventions in these models.
Further support for the conclusion that neutrophils may not be sufficiently activated to cause additional injury during AAP toxicity comes from the observation that there was neither Mac-1 (CD11b/CD18) upregulation nor L-selectin (CD62L) shedding on circulating neutrophils after AAP administration. Enhanced Mac-1 expression and L-selectin shedding are characteristic for activation of neutrophils (Jutila et al., 1989). In all models where neutrophils accumulated in the liver and contributed to the injury process, upregulation of Mac-1 was observed on circulating neutrophils. This includes hepatic ischemia-reperfusion injury (Jaeschke et al., 1993b), galactosamine (Gal)/endotoxin (Essani et al., 1995), Gal/TNF (Essani et al., 1995), and high doses of endotoxin (Spitzer et al., 1994; Witthaut et al., 1994). In contrast, hepatic neutrophil accumulation without Mac-1 upregulation, as seen after Gal/IL-1 administration, did not result in injury (Essani et al., 1995). The activation status of circulating neutrophils closely reflects those accumulated in the liver vasculature (Spitzer et al., 1994). Therefore, it can be concluded that there was no relevant systemic activation of neutrophils during AAP intoxication. A potential local activation in the liver, which may be not reflected on circulating cells, appears less likely, because the anti-CD18 antibody had no effect. Previous experience showed that in all models with Mac-1 upregulation on neutrophils, antibodies against CD11b and/or CD18 were highly protective (Jaeschke et al., 1991, 1993b; Liu et al., 1995). Moreover, transcriptional upregulation of ICAM-1 was observed and ICAM-1 antibodies were also beneficial (Essani et al., 1995; Farhood et al., 1995). However, toxic doses of AAP did not induce ICAM-1 mRNA expression in the liver (Welty et al., 1993) suggesting that this important counterreceptor for ß2 integrins is not upregulated on liver cells. Although we can not exclude an involvement of neutrophils at a later stage, the delay between accumulation and the injury was never longer than 5–7 h. The fact that neutrophils were present in the liver for more than 20 h without involvement in the injury process suggests that neutrophils may not contribute to AAP-induced liver injury unless additional chemotactic factors are generated at later times.
A recent manuscript appears to contradict the findings of our study. Smith et al. (1998) showed a protective effect of an antineutrophil polyclonal antiserum against AAP toxicity in the rat. A limitation of this study is the use of a neutropenia-inducing antiserum. Previous experiments with monoclonal antibodies and antisera, which cause neutropenia, showed that the majority of neutrophils will be accumulating in the liver after treatment (Bautista et al., 1994). These neutrophils are functionally inactivated and are phagocytosed by Kupffer cells (Bautista et al., 1994). This results in the initial activation and priming of Kupffer cells, as demonstrated by enhanced release of superoxide (Bautista et al., 1994) and TNF- (Hewett et al., 1993). Consequently, prolonged pretreatment, as done in the AAP study (Smith et al., 1998), may cause inactivation of Kupffer cells. Recent studies showed that inactivation of Kupffer cells has a profound protective effect in the AAP model (Laskin et al., 1995; Michael et al., 1999). Thus, without further clarification it is unclear if the protective effect of a neutropenia-inducing antiserum was due to neutrophil or Kupffer cell inactivation. Because of these problems, we used a monoclonal antibody that does not cause neutropenia.
Quantitatively, the number of neutrophils accumulating in the liver after AAP (200–250 PMNs/50 HPF) is approximately 50–70% of the cell numbers seen after endotoxemia (Chosay et al., 1997), which involves massive formation of proinflammatory cytokines and chemokines (Colletti et al., 1996; Schlayer et al., 1988; Zhang et al., 1995). Consistent with previous observations of TNF- and IL-1 mRNA and protein formation after AAP (Blazka et al., 1995), we observed low levels of TNF- in blood at the time of injury between 4 and 24 h. However, peak plasma levels of 100–150 pg/ml were less than 5% of the concentrations measured after endotoxemia (Essani et al., 1995; Schlayer et al., 1988). TNF- and IL-1 are the major cytokines responsible for hepatic neutrophil sequestration after low doses of endotoxin (Essani et al., 1995; Schlayer et al., 1988). In addition, TNF- is responsible for neutrophil activation, as indicated by increased Mac-1 expression (Essani et al., 1995; Witthaut et al., 1994). In these experiments, endogenously generated TNF resulted in peak serum levels of 3–5 ng/ml (Witthaut et al., 1994). Consistent with these in vivo observations, TNF-mediated upregulation of Mac-1 on neutrophils in vitro requires 
1 ng/ml for any effect and 5–10 ng/ml for maximal expression of Mac-1 (Thompson and Matsushima, 1992; von Asmuth et al., 1991). Thus, the minor increase of TNF- formation after AAP treatment is consistent with the lack of Mac-1 upregulation (Fig. 2
) and ICAM-1 mRNA expression (Welty et al., 1993). Consequently, it appears unlikely that this very limited cytokine formation could be responsible for neutrophil recruitment into the liver during AAP-induced injury.
Other potential mediators of hepatic neutrophil sequestration could be C-X-C chemokines. Members of this family are potent neutrophil chemoattractants and have been shown to be involved in the neutrophil pathophysiology during ischemia-reperfusion (Colletti et al., 1996; Lentsch et al., 1998). In addition, transgenic mice that overexpress the human IL-8 gene (Simonet et al., 1994) or rats transfected with an adenovirus containing the rat chemokine gene CINC (Maher et al., 1997) have increased numbers of neutrophils in the liver. These observations suggest that a general or local overproduction of C-X-C chemokines can cause neutrophil accumulation in the liver. If this chemokine formation occurs selectively in hepatocytes, neutrophils can transmigrate and attack parenchymal cells, leading to liver damage (Maher et al., 1997). Our data with AAP showed that both KC and MIP-2 are generated during the injury phase. The fact that mRNA levels of these chemokines increased in the liver indicates that at least some of these peptides were generated in the liver. However, when KC and MIP-2 levels obtained with AAP treatment are compared to those during endotoxemia (KC: 60,000–70,000 pg/ml; MIP-2: 12,000–15,000 pg/ml) (Lawson and Jaeschke, unpublished), the increase is only less than 5%. Although there is evidence that chemokines may contribute to some degree to hepatic neutrophil recruitment during endotoxemia in a rat model (Zhang et al., 1995), it seems unlikely that the relative moderate formation of KC and MIP-2 could have a major effect on AAP-induced neutrophil accumulation. This conclusion is supported by our results that intravenous injection of KC and MIP-2 had very little or no effect on neutrophil sequestration in the hepatic vasculature. Thus, neither cytokines nor C-X-C chemokines formed after AAP overdose appear to be generated in sufficient quantities to recruit neutrophils into the liver. This leaves complement factors as potential mediators of hepatic neutrophil sequestration. Cell injury and the substantial release of cell contents can activate complement. Complement factors, e.g., C5a, are potent activators and chemoattractants for neutrophils (Perez, 1984) and have been shown to be at least partially responsible for hepatic neutrophil accumulation during ischemia-reperfusion (Jaeschke et al., 1993a) and endotoxemia (Witthaut et al., 1994). However, more detailed studies are necessary to confirm this hypothesis.
In summary, our study showed an inflammatory response in the liver after AAP overdose with cytokine/chemokine formation and sequestration of neutrophils in the hepatic vasculature. However, neither TNF- nor the C-X-C chemokines KC and MIP-2 are generated in large enough quantities to be able to account for the substantial neutrophil accumulation in the liver. In addition, neutrophils appear to be only moderately activated and interference with ß2 integrins on neutrophils did not prevent injury. Because of the critical importance of ß2 integrins for neutrophil cytotoxicity, these results suggest that neutrophils do not contribute to the initiation or progression of AAP-induced liver during the first 24 h. The moderate inflammatory response after AAP-induced liver injury may be sufficient to remove necrotic cell debris but is not severe enough to cause additional damage.
|
ACKNOWLEDGMENTS |
|---|
This investigation was supported in part by a National Institute of Environmental Health Sciences grant ES-06091.
|
NOTES |
|---|
1 To whom correspondence should be addressed at Department of Pharmacology and Toxicology, University of Arkansas for Medical Sciences, 4301 W. Markham St. (Mailslot 638), Little Rock, AR 72205-7199. Fax: (501) 686-8970. E-mail: JaeschkeHartmutW{at}exchange.uams.edu.
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Bautista, A.P. (1997). Chronic alcohol intoxication induces hepatic injury through enhanced macrophage inflammatory protein-2 production and intercellular adhesion molecule-1 expression in the liver. Hepatology 25, 335–342.[ISI][Medline]
Bautista, A. P., Spolarics, Z., Jaeschke, H., Smith, C. W., and Spitzer, J. J. (1994). Antineutrophil monoclonal antibody (1F12) alters superoxide anion release by neutrophils and Kupffer cells. J. Leukoc. Biol. 55, 328–335.[Abstract]
Blazka, M. E., Wilmer, J. L., Holladay, S. D., Wilson, R. E., and Luster, M. I. (1995). Role of proinflammatory cytokines in acetaminophen hepatotoxicity. Toxicol. Appl. Pharmacol. 133, 43–52.[ISI][Medline]
Chomczynski, P., and Sacchi, N. (1987). Single step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal. Biochem. 162, 156–162.[ISI][Medline]
Chosay, J. G., Essani, N. A., Dunn, C. J., and Jaeschke, H. (1997). Neutrophil margination and extravasation in sinusoids and venules of the liver during endotoxin-induced injury. Am. J. Physiol. 272, G1195–G1200.
Cohen, S. D., and Khairallah, E. A. (1997). Selective protein arylation and acetaminophen-induced hepatotoxicity. Drug Metab. Rev. 29, 59–77.[ISI][Medline]
Colletti, L. M., Kunkel, S. L., Walz, A., Burdick, M. D., Kunkel, R. G., Wilke, C. A., and Strieter, R. M. (1996). The role of cytokine networks in the local liver injury following hepatic ischemia/reperfusion in the rat. Hepatology 23, 506–514.[ISI][Medline]
Dahlin, D. C., Miwa, G. T., Lu, A. Y. H., and Nelson, S. D. (1984). N-Acetyl-p-benzoquinone imine: a cytochrome P-450-mediated oxidation product of acetaminophen. Proc. Natl. Acad. Sci. U S A 81, 1327–1331.
Dahm, L. J., Schultze, A. E., and Roth, R. A. (1991). An antibody to neutrophils attenuates alpha-naphthylisothiocyanate-induced liver injury. J. Pharmacol. Exp. Ther. 256, 412–420.
Essani, N. A., Bajt, M. L., Farhood, A., Vonderfecht, S. L., and Jaeschke, H. (1997). Transcriptional activation of vascular cell adhesion molecule-1 (VCAM-1) gene in vivo and its role in the pathophysiology of neutrophil-induced liver injury in murine endotoxin shock. J. Immunol. 158, 5941–5948.[Abstract]
Essani, N. A, Fisher, M. A., Farhood, A., Manning, A. M., Smith, C. W., and Jaeschke, H. (1995). Cytokine-induced upregulation of hepatic intercellular adhesion molecule-1 (ICAM-1) mRNA expression and its role in the pathophysiology of murine endotoxin shock and acute liver failure. Hepatology 21, 1632–1639.[ISI][Medline]
Esterline, R. L., Ray, S. D., and Ji, S. (1989). Reversible and irreversible inhibition of hepatic mitochondrial respiration by acetaminophen and its toxic metabolite, N-acetyl-p-benzoquinone imine (NAPQI). Biochem. Pharmacol. 38, 2387–2390.[ISI][Medline]
Farhood, A., McGuire, G. M., Manning, A. M., Miyasaka. M., Smith, C. W., and Jaeschke, H. (1995). Intercellular adhesion molecule-1 (ICAM-1) gene expression and its role in neutrophil-induced ischemia-reperfusion injury in the liver. J. Leukoc. Biol. 57, 368–374.[Abstract]
Hewett, J. A., Jean, P. A., Kunkel, S. L., and Roth, R. A. (1993). Relationship between tumor necrosis factor- and neutrophils in endotoxin-induced liver injury. Am. J. Physiol. 265, G1011–G1015.
Hewett, J. A., Schultze, A. E., VanCise, S., and Roth, R. A. (1992). Neutrophil depletion protects against liver injury from bacterial endotoxin. Lab Invest. 66, 347–361.[ISI][Medline]
Jaeschke, H. (1990). Glutathione disulfide formation and oxidant stress during acetaminophen-induced hepatotoxicity in mice in vivo: the protective effect of allopurinol. J. Pharmacol. Exp. Ther. 255, 935–941.
Jaeschke, H. (1997). Cellular adhesion molecules: regulation and functional significance in the pathogenesis of liver diseases. Am. J. Physiol. 273, G602–G611.
Jaeschke, H., Farhood, A., Bautista, A. P., Spolarics, Z., and Spitzer, J. J. (1993a). Complement activates Kupffer cells and neutrophils during reperfusion after hepatic ischemia. Am. J. Physiol. 264, G801–G809.
Jaeschke, H., Farhood, A., Bautista, A. P., Spolarics, Z., Spitzer, J. J., and Smith, C. W. (1993b). Functional inactivation of neutrophils with a Mac-1 (CD11b/CD18) monoclonal antibody protects against ischemia-reperfusion injury in rat liver. Hepatology 17, 915–923.[ISI][Medline]
Jaeschke, H., Farhood, A., Fisher, M. A., and Smith, C. W. (1996). Sequestration of neutrophils in the hepatic vasculature during endotoxemia is independent of ß2 integrins and intercellular adhesion molecule-1. Shock 6, 351–356.[ISI][Medline]
Jaeschke, H., Farhood, A., and Smith, C. W. (1990). Neutrophils contribute to ischemia/reperfusion injury in rat liver in vivo. FASEB J. 4, 3355–3359.[Abstract]
Jaeschke, H., Farhood, A., and Smith, C. W. (1991) Neutrophil-induced liver cell injury in endotoxin shock is a CD11b/CD18-dependent mechanism. Am. J. Physiol. 261, G1051–G1056.
Jaeschke, H., Fisher, M. A., Lawson, J. A., Simmons, C. A., Farhood, A., and Jones, D. A. (1998). Activation of caspase-3 (CPP32)-like proteases is essential for TNF--induced hepatic parenchymal cell apoptosis and neutrophil-mediated necrosis in a murine endotoxin shock model. J. Immunol. 160, 3480–3486.
Jaeschke, H. and Smith, C. W. (1997). Mechanisms of neutrophil-induced parenchymal cell injury. J. Leukoc. Biol. 61, 647–653.[Abstract]
Jaeschke, H., Smith, C. W., Clemens, M. G., Ganey, P. E., and Roth, R. A. (1996). Mechanisms of inflammatory liver injury: adhesion molecules and cytotoxicity of neutrophils. Toxicol. Appl. Pharmacol. 139, 213–226.[ISI][Medline]
Jutila, M. A., Rott, L., Berg, E. L., and Butcher, E. C. (1989). Function and regulation of the neutrophil MEL-14 antigen in vivo: comparison with LFA-1 and MAC-1. J. Immunol. 143, 3318–3324.[Abstract]
Laskin, D. L., Gardner, C. R., Price, V. F., and Jollow, D. J. (1995). Modulation of macrophages functioning abrogates the acute hepatotoxicity of acetaminophen. Hepatology 21, 1045–1050.[ISI][Medline]
Laskin, D. L., and Pilaro, A. M. (1986). Potential role of activated macrophages in acetaminophen hepatotoxicity: I. Isolation and characterization of activated macrophages from rat liver. Toxicol. Appl. Pharmacol. 86, 204–215.[ISI][Medline]
Lawson, J. A., Fisher, M. A., Simmons, C. A., Farhood, A., and Jaeschke, H. (1998). Parenchymal cell apoptosis as a signal for sinusoidal sequestration and transendothelial migration of neutrophils in murine models of endotoxin and Fas-antibody-induced liver injury. Hepatology 28, 761–767.[ISI][Medline]
Lentsch, A. B., Yoshidome, H., Cheadle, W. G., Miller, F. N., and Edwards, M. J. (1998). Chemokine involvement in hepatic ischemia/reperfusion injury in mice: roles for macrophage inflammatory protein-2 and KC. Hepatology 27, 1172–1177.[ISI][Medline]
Liu, P., McGuire, G. M., Fisher, M. A., Farhood, A., Smith, C. W., and Jaeschke, H. (1995) Activation of Kupffer cells and neutrophils for reactive oxygen formation is responsible for endotoxin-enhanced liver injury after hepatic ischemia. Shock 3, 56–62.[ISI][Medline]
Maher, J. J., Scott, M. K., Saito, J. M., and Burton, M. C. (1997). Adenovirus-mediated expression of cytokine-induced neutrophil chemoattractant in rat liver induces a neutrophilic hepatitis. Hepatology 25, 624–630.[ISI][Medline]
Meyers, L. L., Beierschmitt, W. P., Khairallah, E. A., and Cohen, S. D. (1988). Acetaminophen-induced inhibition of mitochondrial respiration in mice. Toxicol. Appl. Pharmacol. 93, 378–387.[ISI][Medline]
Michael, S. L., Pumford, N. R., Mayeux, P. R., Niesman, M. R., and Hinson, J. A. (1999). Pretreatment of mice with macrophage inactivators decreases acetaminophen hepatotoxicity and the formation of reactive oxygen and nitrogen species. Hepatology 30, 186–195.[ISI][Medline]
Mitchell, J. R., Jollow, D. J., Potter, W. Z., Davis, D. C., Gillette, J. R., and Brodie, B. B. (1973a). Acetaminophen-induced hepatic necrosis. I. Role of drug metabolism. J. Pharmacol. Exp. Ther. 187, 185–194.
Mitchell, J. R., Jollow, D. J., Potter, W. Z., Gillette, J. R., and Brodie, B. B. (1973b). Acetaminophen-induced hepatic necrosis. IV. Protective role of glutathione. J. Pharmacol. Exp. Ther. 187, 211–217.
Molnar, R. G., Wang, P., Ayala, A., Ganey, P. E., Roth, R. A., and Chaudry, I. H. (1997). The role of neutrophils in producing hepatocellular dysfunction during the hyperdynamic stage of sepsis in rats. J. Surg. Res. 73, 117–122.[ISI][Medline]
Perez, H. D. (1984). Biologically active complement (C5)-derived peptides and their relevance to disease. Crit. Rev. Oncol. Hematol. 1, 199–225.[Medline]
Pumford, N. R., Halmes, N. C., and Hinson, J. A. (1997). Covalent binding of xenobiotics to specific proteins in the liver. Drug Metab. Rev. 29, 39–57.[ISI][Medline]
Ray, S. D., Kamendulis, L. M., Gurule, M. W., Yorkin, R. D., and Corcoran, G. B. (1993). Ca2+ antagonists inhibit DNA fragmentation and toxic cell death induced by acetaminophen. FASEB J. 106, 453–463.
Schlayer, H. J., Laaff, H., Peters, T., Woort-Menker, M., Estler, H. C., Karck, U., Schaefer, H. E., and Decker, K. (1988). Involvement of tumor necrosis factor in endotoxin-triggered neutrophil adherence to sinusoidal endothelial cells of mouse liver and its modulation in acute phase. J. Hepatol. 7, 239–249.[ISI][Medline]
Shappell, S. B., Toman, C., Anderson, D. C., Taylor, A. A., Entman, M. L., and Smith, C. W. (1990). Mac-1 (CD11b/CD18) mediates adherence-dependent hydrogen peroxide production by human and canine neutrophils. J. Immunol. 144, 2702–2711.[Abstract]
Shen, W., Kamendulis, L. M., Ray, S. D., and Corcoran, G. B. (1991). Acetaminophen-induced cytotoxicity in cultured mouse hepatocytes: correlation of nuclear Ca2+ accumulation and early DNA fragmentation with cell death. Toxicol. Appl. Pharmacol. 111, 242–254.[ISI][Medline]
Simonet, W. S, Hughes, T. M., Nguyen, H. Q., Trebasky, L. D., Danilenko, D. M., and Medlock, E. S. (1994). Long-term impaired neutrophil migration in mice overexpressing human interleukin-8. J. Clin. Invest. 94, 1310–1319.
Smith, G. S., Nadig, D. E., Kokoska, E. R., Solomon, H., Tiniakos, D. G., and Miller, T. A. (1998). Role of neutrophils in hepatotoxicity induced by oral acetaminophen administration in rats. J. Surg. Res. 80, 252–258.[ISI][Medline]
Spitzer, J. A., Zhang, P, and Mayer, A. M. (1994). Functional characterization of peripheral circulating and liver recruited neutrophils in endotoxic rats. J. Leukoc. Biol. 56, 166–173.[Abstract]
Thomas, S. H. L. (1993). Paracetamol (acetaminophen) poisoning. Pharmacol. Ther. 60, 91–120.[ISI][Medline]
Thompson, H. L., and Matsushima, K. (1992). Human polymorphonuclear leukocytes stimulated by tumour necrosis factor-alpha show increased adherence to extracellular matrix proteins which is mediated via the CD11b/CD18 complex. Clin. Exp. Immunol. 90, 280–285.[ISI][Medline]
Tsokos-Kuhn, J. O., Hughes, H., Smith, C. V., and Mitchell, J. R. (1988). Alkylation of the liver plasma membrane and inhibition of the Ca2+-ATPase by acetaminophen. Biochem. Pharmacol. 37, 2125–2131.[ISI][Medline]
Von Asmuth, E. J., van der Linden, C. J., Leeuwenberg, J. F., and Buurman, W. A. (1991). Involvement of the CD11b.CD18 integrin, but not of the endothelial cell adhesion molecules ELAM-1 and ICAM-1 in tumor necrosis factor-alpha-induced neutrophil toxicity. J. Immunol. 147, 3869–3875.[Abstract]
Welty, S. E., Smith, C. V., Benzick, A. E., Montgomery, C. A., and Hansen, T. N. (1993). Investigation of possible mechanisms of hepatic swelling and necrosis caused by acetaminophen in mice. Biochem. Pharmacol. 45, 449–458.[ISI][Medline]
Witthaut, R., Farhood, A., Smith, C. W., and Jaeschke, H. (1994). Complement and tumor necrosis factor- contribute to Mac-1 (CD11b/CD18) upregulation and systemic neutrophil activation during endotoxemia in vivo. J. Leukoc. Biol. 55, 105–111.[Abstract]
Zhang, P., Xie, M., Zagorski, J., and Spitzer, J. A.(1995). Attenuation of hepatic neutrophil sequestration by anti-CINC antibody in endotoxic rats. Shock 4, 262–268.[ISI][Medline]
CiteULike 
Connotea 
Del.icio.us What's this?
This article has been cited by other articles:
|
|
 |
|
  S. K. Ramaiah and H. Jaeschke Role of Neutrophils in the Pathogenesis of Acute Inflammatory Liver Injury Toxicol Pathol, October 1, 2007; 35(6): 757 - 766. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 B. A. Merrick, M. E. Bruno, J. H. Madenspacher, B. A. Wetmore, J. Foley, R. Pieper, M. Zhao, A. J. Makusky, A. M. McGrath, J. X. Zhou, et al. Alterations in the Rat Serum Proteome during Liver Injury from Acetaminophen Exposure J. Pharmacol. Exp. Ther., August 1, 2006; 318(2): 792 - 802. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Y. Ito, E. R. Abril, N. W. Bethea, M. K. McCuskey, C. Cover, H. Jaeschke, and R. S. McCuskey Mechanisms and pathophysiological implications of sinusoidal endothelial cell gap formation following treatment with galactosamine/endotoxin in mice Am J Physiol Gastrointest Liver Physiol, August 1, 2006; 291(2): G211 - G218. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P. Kodali, P. Wu, P. A. Lahiji, E. J. Brown, and J. J. Maher ANIT toxicity toward mouse hepatocytes in vivo is mediated primarily by neutrophils via CD18 Am J Physiol Gastrointest Liver Physiol, August 1, 2006; 291(2): G355 - G363. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
B. Donahower, S. S. McCullough, R. Kurten, L. W. Lamps, P. Simpson, J. A. Hinson, and L. P. James Vascular endothelial growth factor and hepatocyte regeneration in acetaminophen toxicity Am J Physiol Gastrointest Liver Physiol, July 1, 2006; 291(1): G102 - G109. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 L. P. James, P. M. Simpson, H. C. Farrar, G. L. Kearns, G. S. Wasserman, J. L. Blumer, M. D. Reed, J. E. Sullivan, J. A. Hinson, and The Pediatric Pharmacology Research Unit Network Cytokines and Toxicity in Acetaminophen Overdose J. Clin. Pharmacol., October 1, 2005; 45(10): 1165 - 1171. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. Cover, P. Fickert, T. R. Knight, A. Fuchsbichler, A. Farhood, M. Trauner, and H. Jaeschke Pathophysiological Role of Poly(ADP-Ribose) Polymerase (PARP) Activation during Acetaminophen-Induced Liver Cell Necrosis in Mice Toxicol. Sci., March 1, 2005; 84(1): 201 - 208. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 O. Teufelhofer, W. Parzefall, E. Kainzbauer, F. Ferk, C. Freiler, S. Knasmuller, L. Elbling, R. Thurman, and R. Schulte-Hermann Superoxide generation from Kupffer cells contributes to hepatocarcinogenesis: studies on NADPH oxidase knockout mice Carcinogenesis, February 1, 2005; 26(2): 319 - 329. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
|