Lymphocyte Loss and Immunosuppression Following Acetaminophen

Acetaminophen-Induced Hepatotoxicity in Mice as a Potential ... a hepatotoxic dose of acetaminophen in C57Bl/6 mice, lymphocyte loss that appeared to ...
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Chem. Res. Toxicol. 2007, 20, 20-26

Lymphocyte Loss and Immunosuppression Following Acetaminophen-Induced Hepatotoxicity in Mice as a Potential Mechanism of Tolerance Mary Jane Masson,*,† Richard A. Peterson,‡ Christine J. Chung,† Mary L. Graf,† Leah D. Carpenter,† Jeffrey L. Ambroso,‡ David L. Krull,‡ Janeice Sciarrotta,‡ and Lance R. Pohl† Molecular and Cellular Toxicology Section, Laboratory of Molecular Immunology, National Heart, Lung, and Blood Institute, National Institutes of Health, Department of Health and Human SerVices, Bethesda, Maryland, and Safety Assessment, GlaxoSmithKline, Research Triangle Park, North Carolina ReceiVed August 14, 2006

Current evidence suggests that drug-induced liver disease can be caused by an allergic response (druginduced allergic hepatitis, DIAH) induced by hepatic drug-protein adducts. The relatively low incidence of these reactions has led us to hypothesize that tolerogenic mechanisms prevent DIAH from occurring in most people. Here, we present evidence for the existence of one of these regulatory pathways. Following a hepatotoxic dose of acetaminophen in C57Bl/6 mice, lymphocyte loss that appeared to be due at least in part to apoptosis was noted in the spleen, thymus, and draining lymph nodes of the liver. There was no observable lymphocyte loss in the absence of hepatotoxicity. Acetaminophen-induced liver injury (AILI) also led to a functional suppression of the immune system as determined by the inhibition of a delayed-type hypersensitivity response to dinitrochlorobenzene. Further studies with adrenalectomized mice suggested a role for corticosterone in the depletion of lymphocytes following APAP-induced liver injury. In conclusion, these findings suggest that lymphocyte loss and immunosuppression following AILI may prevent subsequent occurrences of allergic hepatitis and possibly other forms of APAP-induced allergies induced by hepatic drug-protein adducts. Similar regulatory pathways may inhibit other hepatotoxic drugs from causing allergic reactions. Introduction Drug-induced liver disease (DILD) accounts for one-half of all cases of acute hepatic failure in the United States resulting in significant morbidity and mortality (1). This presents a major problem for both the patients relying on the drugs and the pharmaceutical industry producing them. One type of DILD, drug-induced allergic hepatitis (DIAH), is believed to be initiated in many cases by immunogenic drug-protein adducts formed by the covalent interaction of reactive metabolites with endogenous target proteins (2, 3). Subsequent immunopathologic reactions may be directed against drug-protein adducts and/or the unaltered target proteins. Before drug-protein adducts can induce immunopathologic reactions, they must come into contact with cells of the immune system either by being secreted from hepatocytes or, more likely, by being released from dying hepatocytes. The latter possibility could also lead to the release of endogenous adjuvants known as danger signals that are probably required to initiate autoreactive adaptive immune responses (4-7). However, recent evidence suggests that the dominant immune response against drug-protein adducts may be tolerance (8, 9). We now describe another potential tolerogenic mechanism that may play a role in preventing drug-protein adducts from causing DIAH. Following acetaminophen (APAP)-induced hepatotoxicity, it was discovered that lymphocyte depletion * Corresponding author. Phone: 301-451-2888. Fax: 301-480-4852. E-mail: [email protected]. † National Institutes of Health. ‡ GlaxoSmithKline.

occurred in the spleen, thymus, and hepatic draining lymph nodes, resulting in immunosuppression. It appeared that this tolerance mechanism was mediated by the hypothalamicpituitary-adrenal axis.

Experimental Procedures Chemicals and Reagents. The following chemicals and reagents were purchased commercially: APAP, N-acetyl-L-cysteine (NAC), and 2,4-dinitrochlorobenzene (DNCB) (Sigma, St. Louis, MO); EZprep, CC1 antigen retrieval system, avidin-biotin block, and diaminobenzidine (DAB) (Ventana Medical Systems, Inc., Tucson, AZ). Animal Treatment. Male C57BL/6J mice (8 weeks of age; Jackson Laboratory, Bar Harbor, ME) were maintained in a humidity- and temperature-controlled environment in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. For experiments involving adrenalectomized (Adx) mice, the mice were allowed to drink sterile saline (0.9%) instead of water following the surgical removal of the adrenal glands (performed by Jackson Laboratory). The animals were acclimatized for one week prior to experiments. In experiments involving APAP, animals were fasted overnight (16 h) before treatment with APAP in PBS or with PBS by intraperitoneal injection. Where indicated, NAC (450 mg/kg in saline adjusted to pH 7.4 with NaOH) was administered by intraperitoneal injection at the same time as APAP. At various times thereafter, mice were euthanized in a carbon dioxide chamber and blood was collected through the inferior vena cava followed by the removal of spleens and thymuses and the draining hepatic lymph nodes. Assessment of Hepatotoxicity. Liver injury was determined by measuring serum levels of alanine transaminase (ALT) with a diagnostic kit (Teco Diagnostics, Anaheim, CA).

10.1021/tx060190c CCC: $37.00 © 2007 American Chemical Society Published on Web 12/09/2006

APAP-Induced LiVer Injury Results in Immunosuppression Assessment of Lymphocytolysis. Lymphoid organs were fixed in 10% buffered formalin, embedded in paraffin, dewaxed, mounted on glass slides and stained with hematoxylin/eosin. A board-certified pathologist (RAP) assessed the extent of lymphocytolysis following APAP treatment in a blind fashion. Detection of Apoptotic Cells by Cleaved-Caspase 3 Immunochemical Staining. Formalin-fixed, paraffin-embedded tissues (American HistoLabs, Gaithersburg, MD) were de-waxed and hydrated using EZprep. Antigen retrieval was followed by avidinand biotin-blocking. Rabbit polyclonal anti-cleaved caspase-3 (ASP175, 1:100) (Cell Signaling Technology, Beverly, MA) and goat anti-rabbit (8 µg/mL) (Vector Laboratories, Burlingame, CA) were used as the primary and secondary antibodies, respectively. Following the detection of positive cells with DAB, slides were counterstained with hematoxylin. Flow Cytometry. Single cell suspensions of spleen, thymus, and draining hepatic lymph nodes (LN) were made by passing organs through 40 µm mesh cell strainers (BD Falcon, Bedford, MA) into 10% FCS in PBS. Following red blood cell lysis with ACK lysis buffer, cells were washed and counted by trypan blue exclusion. Prior to adding primary antibodies, Fc receptors were blocked with the anti-FcγIII/II receptor (BD Pharmingen, San Diego, CA) for 10 min. The primary antibodies (BD Pharmingen) used were allophycocyanin (APC) anti-mouse CD8R (53-6.7), fluorescein isothiocyanate (FITC) anti-mouse CD4 (L3T4), phycoerythrin (PE) anti-mouse CD3 (145-2C11), PE anti-mouse 1-Ab (MHCII; AF6120.1), and FITC anti-mouse CD19 (1D3). Cells (5 × 105) were incubated with primary antibody (1:100 dilution in 100 µL of 10% FCS in PBS) for 20 min at 4 °C and washed twice. The viability stain 7-AAD (5 µL) was added 20 min prior to data acquisition to exclude dead cells from analysis. Data was acquired using a CyAnTM LX Flow Cytometer (DakoCytomation, Carpintaria, CA) and later analyzed using FlowJo Software (Tree Star, Ashland, OR). DNCB-Induced Delayed-Type Hypersensitivity (DTH) Response. Twenty-four hours after treatment with PBS (control), 80 or 250 mg/kg APAP, the DTH response to DCNB sensitization was assessed by the ear swelling test as described (10). Briefly, mice were sensitized by applying 25 µL of 5% (w/v) DNCB in acetone/olive oil (4:1, v/v) to shaved abdominal skin and challenged 5 days later by applying 10 µL of 2.5% (w/v) DNCB in acetone/ olive oil (9:1, v/v) to each side of one ear. Ear thickness was measured prior to challenge and 24 h afterward with a caliper micrometer (Dyer, Lancaster, PA), and the results were expressed as a net increase in ear thickness. Assay for Serum Corticosterone. Serum corticosterone levels were measured by MP Biomedicals, LLC (Solon, OH; www.mpdiagnostics.com) using a radioimmunoassay. Statistical Analysis. ANOVA analysis was used to compare the means. The results shown represent the means ( SD. Correlation analysis was used to determine the correlation coefficients (r). Differences were considered significant when p < 0.05.

Results APAP Hepatotoxicity Induces Lymphocyte Depletion in Lymphoid Organs. Twenty-hours after mice were administered hepatotoxic doses of APAP (200 or 300 mg/kg) (Figure 1), cell counts in the spleen, thymus, and draining hepatic LN were significantly reduced (Figure 2). In contrast, lymphoid counts were not reduced in mice treated with a non-hepatotoxic dose of APAP (80 mg/kg) (Figures 1 and 2) or in mice protected against the hepatotoxic effects of 300 mg/kg APAP by NAC treatment (Figure 3). There was also a trend toward increased lymphocyte loss in mice treated with 300 mg/kg APAP compared to that in mice treated with 200 mg/kg APAP (Figures 1 and 2). When mice were followed for 5 days after treatment with 300 mg/kg of APAP (Figure 4A), cell counts in the spleen recovered within 48 h (Figure 4B), whereas thymic cells remained depressed over the 5-day period (Figure 4C).

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Figure 1. Dose-dependent APAP-induced liver injury in C57Bl/6 mice. Twenty-four hours following the treatment of mice with PBS (control) or APAP (80, 200, or 300 mg/kg), liver injury was assessed by measuring serum ALT activity. The results shown represent the means ( SD of 7 to 8 mice per group. (*) p < 0.05 relative to controls and mice treated with 80 mg/kg of APAP. (γ) p < 0.05 relative to mice treated with 200 mg/kg APAP. No mice died during this 24 h period.

Figure 2. Dose-dependent decreases in lymphoid cell counts following APAP treatment. The total cell counts were determined in the spleen (A), thymus (B), and hepatic LN (C) of mice 24 h after treatment with PBS (control) or APAP (80, 200, or 300 mg/kg). The results shown represent the means ( SD of 7 to 8 mice per group. Because of low cell counts, the hepatic LN cells were pooled in C, and statistics could not be applied. (*) p < 0.05 relative to controls and mice treated with 80 mg/kg of APAP. No mice died during this 24 h period.

Flow cytometric analysis of lymphocyte subsets demonstrated that 24 h following a hepatotoxic dose of 200 mg/kg APAP, the total number of CD4+ T cells (CD4+CD3+), CD8+ T cells (CD8+CD3+), and B cells (CD19+MHCII+) in the spleen decreased by 19, 20, and 27%, respectively. A trend toward even greater loss of these splenocyte subsets was seen following treatment with 300 mg/kg APAP (Figure 5A-C). In the thymus, 200 and 300 mg/kg APAP resulted in a 32 and 51% decrease, respectively, in the number of immature CD4+CD8+ thymocytes (Figure 5D), although these values were not significantly different. In contrast, treatment with 80 mg/kg APAP had no effect on lymphocyte counts in these organs.

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Figure 3. NAC prevents APAP-induced liver injury and immune cell loss. (A) Twenty-four hours following the treatment of mice with APAP only (300 mg/kg), APAP (300 mg/kg) and NAC (450 mg/kg), NAC only (450 mg/kg), or PBS (control), serum ALT activity was measured, and the total numbers of plenocytes (B), thymocytes (C), and hepatic LN cells (D) were determined. The results shown represent the means ( SD of 7 mice per group. Because of low cell counts, the hepatic LN cells were pooled in (D), and statistics could not be applied. (*) p < 0.05 relative to controls. No mice died during this 24 h period.

significantly inhibited, whereas no inhibition was noted following the non-hepatotoxic dose of APAP (Figure 6). Possible Role of Corticosterone in APAP-Induced Lymphocyte Depletion. Histological analysis of tissue sections of the spleen and thymus 24 h after the treatment of mice with a hepatotoxic dose of APAP revealed lymphocytolysis in the white pulp region of the spleen and to a greater extent in the outer subcapsular and cortex zones of the thymus (data not shown). There were more cells immunohistochemically stained for cleaved caspase-3, a specific marker of apoptosis (11), in the thymus (Figure 7B) than in the spleen (Figure 7A), whereas in contrast, very few cells stained positively for this marker in the spleen and thymus of mice treated with PBS (Figure 7C and D, respectively) or 80 mg/kg APAP (data not shown). Because glucocorticoids are known to induce apoptosis in lymphocytes (12), we studied the potential role of corticosterone in APAP-induced lymphocyte depletion. A significant correlation was found between corticosterone levels and the degree of liver injury (r2 ) 0.750, p < 0.01)(data not shown), suggesting that this hormone was involved in the loss of lymphocytes following APAP-induced liver injury. In support of this idea, adrenalectomized mice treated with a hepatotoxic dose of APAP (Figure 8A) were resistant to lymphocyte depletion in the thymus, spleen, and hepatic LN (Figure 8B-D, respectively).

Discussion Figure 4. Time course of APAP-induced liver injury and lymphoid cell loss and recovery in the spleen and thymus. Liver injury (A) and total cell counts in the spleen (B) and thymus (C) of mice were measured at 0 (control; 0/6 lethality), 24 (0/6 lethality), 48 (3/7 lethality), 72 (3/8 lethality), and 120 h (3/6 lethality) following treatment with 300 mg/kg APAP. The results shown represent the means ( SD of 3-6 mice per group. (*) p < 0.05 relative to controls.

The changes in lymphocyte counts were also associated with a suppression of immune function. Following a hepatotoxic dose of 250 mg/kg APAP, the DNCB-induced DTH response was

Despite evidence suggesting that DIAH is mediated by drugprotein adducts (2, 3), it remains unclear why these reactions are uncommon. One possibility is that the dominant immune response against drug-protein adducts released from injured hepatocytes is tolerance. In this regard, it is clear that the liver is a tolerogenic organ having multiple mechanisms to prevent adaptive immune reactions. Its unique environment consists largely of NK, NKT, and γδ T cells of the innate immune system and contains far fewer lymphocytes of the adaptive immune system than other lymphoid tissues (13, 14). Antigen

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Figure 5. Reduced lymphocyte subsets in the spleen and thymus following APAP treatment. The total number of splenic CD4+ T-cells (A), CD8+ T-cells (B), B cells (C), and immature CD4+CD8+ thymocytes (D) were measured by flow cytometry in mice 24 h after treatment with PBS (control) or APAP (80, 200, or 300 mg/kg). The results shown represent the means ( SD of 6 mice per group. (*) p < 0.05 relative to controls and mice treated with 80 mg/kg APAP. No mice died during this 24 h period.

Figure 6. Inhibition of DTH response induced by DNCB following APAP-induced liver injury. Twenty-four hours after treatment with PBS (control) or 80 or 250 mg/kg APAP, (A) liver injury was assessed by determining ALT activity, and mice were sensitized with 5% DNCB and challenged 5 days later by applying 2.5% DNCB to the ear. (B) The DTH response was assessed 24 h after the challenge by measuring ear swelling. The results shown represent the means ( SD of 10-12 mice per group. (γ) p < 0.05 relative to controls and mice treated with 80 mg/kg APAP. No mice died during this experiment.

presentation by Kupffer cells, the major antigen-presenting cells of the liver, may favor the induction of tolerance (8, 15) because these cells produce immunosuppressive factors, such as IL-10 and prostaglandin E2, upon activation (16, 17). Similarly, antigen presentation by other cells of the liver, namely, liver sinusoidal endothelial cells and hepatocytes, can also lead to tolerance (18, 19). Consequently, conventional T cells activated in the liver are less likely to initiate an adaptive immune response than T cells activated elsewhere. CD8+ T cells undergoing primary activation in the liver exhibit a shortened half-life and defective cytotoxic function compared to those of CD8+ T cells activated in LN (20, 21). In addition, the proliferation of hepatic CD4+ T cells in response to allogeneic APCs is suppressed in the presence of regulatory NKT and γδ T cells (22). We provide evidence here for another mechanism of tolerance that may prevent drug-protein adducts and immune-activating danger signals released from injured liver cells (4-7) from inducing allergic hepatitis and possibly other types of allergic reactions. Twenty-four hours after a hepatotoxic dose of APAP, the number of cells in the spleen, hepatic draining LN, and particularly the thymus were significantly reduced (Figure 2).

Consistent with this finding, immature CD4+CD8+ thymocytes were depleted most extensively (Figure 5D) by APAP relative to the amount of depletion observed in splenic CD4+ T, CD8+ T, and B cells (Figure 5A-C, respectively), which were all similarly affected by APAP treatment. Other findings indicate that liver injury is a prerequisite for lymphoid cell depletion. First, a non-hepatotoxic dose of APAP had no effect on lymphocyte cell counts in lymphoid organs (Figure 2). Second, NAC, a known inhibitor and antidote of AILN (23, 24) inhibited 300 mg/kg APAP from causing liver injury and blocked the loss of lymphocytes from the spleen, thymus, and draining hepatic LN (Figure 3). Lymphoid cell depletion caused by AILI also resulted in T cell immunosuppression, as measured by the DTH response to DNCB (Figure 6). Other investigators have previously reported thymocyte depletion and suppression of humoral and cellular immunity following APAP-induced liver injury (26). In accordance with our results, a relationship between the degree of liver injury and the extent of immunosuppression was suggested by the findings. However, a later report by the same group concluded that the immunosuppressive effects of APAP were not a result of liver injury but a direct effect of APAP (27). Although it is

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Figure 7. Hepatotoxic dose of APAP induces apoptosis of cells in the spleen and thymus. Immunohistochemical detection of cleaved-caspase 3 positive cells in the spleen (A) and thymus (B) 24 h after the treatment of mice with 300 mg/kg APAP. Very few cells stained positively for this marker in the spleen (C) and thymus (D) of mice treated with PBS. The results shown are representative of 5 other mice in each group (original magnification 400×).

Figure 8. APAP-induced lymphocyte loss does not occur in adrenalectomized mice. (A) Following treatment with 300 mg/kg APAP or PBS (control), serum ALT activity was measured at 24 h, and the total numbers of thymocytes (B), splenocytes (C), and hepatic LN cells (D) were determined. The results shown represent the means ( SD of 5 mice in the control group and 12 mice in the treated group. Because of low cell counts, the control hepatic LN cells were pooled in D, and statistics could not be applied. (*) p < 0.05 relative to controls. Following treatment with 300 mg//kg APAP, 43% of mice died within this 24 h period.

not clear why our findings differ from this report, a recent study indicates that concanavalin-A (Con-A)-induced liver injury also leads to thymic atrophy and splenocyte depletion (28). An immunohistochemical examination of tissue sections from the spleen and thymus revealed increased numbers of cleavedcaspase 3 positive cells after a hepatotoxic dose of APAP, particularly in the thymus (Figure 7), suggesting that one mechanism of AILI-induced lymphoid cell depletion was apoptotic-mediated cell death (29). Similar apoptotic-mediated lymphocyte depletion in these organs was reported after ConA-induced liver injury (28). Glucocorticoids are known to induce lymphocyte death through an apoptotic mechanism (30-32), with immature CD4+CD8+ thymocytes being most sensitive to

these effects (33, 34) as found in both our study (Figure 5D) and in the Con-A investigation (28). In the case of AILI, it appears that corticosterone may be involved in lymphocyte death because we found that not only a significant correlation existed between corticosterone levels and the degree of AILI (r2 ) 0.750, p < 0.01) (data not shown) but also that adrenalectomized mice were resistant to lymphoid cell depletion following AILI (Figure 8). Further support for this pathway of lymphocyte cell death is based on the findings that AILI is associated with increased serum levels of several inflammatory cytokines, including IL-1, TNFR, IL-6, and IFNγ (35-38), which have all been shown to induce the release of glucocorticoids by stimulating the hypothalamic-pituitary-adrenocortical axis (39,

APAP-Induced LiVer Injury Results in Immunosuppression

40)). Although we cannot exclude the effects of epinephrine and aldosterone on AILI-induced lymphocyte depletion, these adrenal hormones, unlike glucocorticoids, cause only mild thymic atrophy when injected into mice (41, 42). In contrast to these results, the lymphocyte depletion caused by Con-Ainduced liver injury did not appear to be mediated by corticosterone (28), indicating that other mechanisms could contribute to lymphoid cell depletion following liver injury. Although AILI is a major cause of acute liver failure (43) with concomitant release of potentially immunogenic APAPprotein adducts into the blood (44), there are very few reports of APAP-induced allergic hepatitis (45, 46) or other allergic reactions attributed to APAP (47-49). Our findings here suggest that one reason for the low incidence of allergic reactions associated with AILI and possibly other drugs that cause severe liver injury is that in many cases DILI may cause the depletion of lymphocytes leading to immunologic tolerance against drugprotein adducts. Acknowledgment. This research was supported in part by the Intramural Research Program of the National Institutes of Health, National Heart, Lung, and Blood Institute and by a Cooperative Research and Development Agreement (CRADA) with GlaxoSmithKline.

References (1) Ostapowicz, G., Fontana, R. J., Schiodt, F. V., Larson, A., Davern, T. J., Han, S. H., McCashland, T. M., Shakil, A. O., Hay, J. E., Hynan, L., Crippin, J. S., Blei, A. T., Samuel, G., Reisch, J., and Lee, W. M. (2002) Results of a prospective study of acute liver failure at 17 tertiary care centers in the United States. Ann. Intern. Med. 137, 947-954. (2) Pohl, L. R. (1990) Drug-induced allergic hepatitis. Semin. LiVer Dis. 10, 305-315. (3) Liu, Z. X., and Kaplowitz, N. (2002) Immune-mediated drug-induced liver disease. Clin. LiVer Dis. 6, 755-774. (4) Matzinger, P. (2002) The danger model: a renewed sense of self. Science 296, 301-305. (5) Uetrecht, J. P. (1999) New concepts in immunology relevant to idiosyncratic drug reactions: the ‘danger hypothesis’ and innate immune system. Chem. Res. Toxicol. 12, 387-395. (6) Rock, K. L., Hearn, A., Chen, C. J., and Shi, Y. (2005) Natural endogenous adjuvants. Springer Semin. Immunopathol. 26, 231-246. (7) Tsung, A., Sahai, R., Tanaka, H., Nakao, A., Fink, M. P., Lotze, M. T., Yang, H., Li, J., Tracey, K. J., Geller, D. A., and Billiar, T. R. (2005) The nuclear factor HMGB1 mediates hepatic injury after murine liver ischemia-reperfusion. J. Exp. Med. 201, 1135-1143. (8) Ju, C., McCoy, J. P., Chung, C. J., Graf, M. L., and Pohl, L. R. (2003) Tolerogenic role of Kupffer cells in allergic reactions. Chem. Res. Toxicol. 16, 1514-1519. (9) Masson, M. J., and Uetrecht, J. P. (2004) Tolerance induced by low dose D-penicillamine in the brown Norway rat model of drug-induced autoimmunity is immune-mediated. Chem. Res. Toxicol. 17, 82-94. (10) Oliver, A. R., and Silbart, L. K. (1998) Local and systemic tolerance to orally administered dinitrochlorobenzene is not broken by cholera toxin. Int. Arch. Allergy Immunol. 116, 318-324. (11) Fernandes-Alnemri, T., Litwack, G., and Alnemri, E. S. (1994) CPP32, a novel human apoptotic protein with homology to Caenorhabditis elegans cell death protein Ced-3 and mammalian interleukin-1 betaconverting enzyme. J. Biol. Chem. 269, 30761-30764. (12) Compton, M. M., and Cidlowski, J. A. (1986) Rapid in vivo effects of glucocorticoids on the integrity of rat lymphocyte genomic deoxyribonucleic acid. Endocrinology 118, 38-45. (13) Kita, H., Mackay, I. R., Van De Water, J., and Gershwin, M. E. (2001) The lymphoid liver: considerations on pathways to autoimmune injury. Gastroenterology 120, 1485-1501. (14) Racanelli, V., and Rehermann, B. (2006) The liver as an immunological organ. Hepatology 43, S54-S62. (15) Ju, C., and Pohl, L. R. (2005) Tolerogenic role of Kupffer cells in immune-mediated adverse drug reactions. Toxicology 209, 109-112. (16) Knolle, P. A., Uhrig, A., Protzer, U., Trippler, M., Duchmann, R., Meyer, zum Buschenfelde, K. H., and Gerken, G. (1998) Interleukin10 expression is autoregulated at the transcriptional level in human and murine Kupffer cells. Hepatology 27, 93-99.

Chem. Res. Toxicol., Vol. 20, No. 1, 2007 25 (17) Ogle, C. K., Wu, J. Z., Wood, S., Ogle, J. D., Alexander, J. W., and Warden, G. D. (1990) The increased release of prostaglandin E2 by Kupffer cells from burned guinea pigs. J. Burn Care Rehabil. 11, 287294. (18) Limmer, A., Ohl, J., Kurts, C., Ljunggren, H. G., Reiss, Y., Groettrup, M., Momburg, F., Arnold, B., and Knolle, P. A. (2000) Efficient presentation of exogenous antigen by liver endothelial cells to CD8+ T cells results in antigen-specific T-cell tolerance. Nat. Med. 6, 13481354. (19) Bertolino, P., Trescol-Biemont, M. C., and Rabourdin-Combe, C. (1998) Hepatocytes induce functional activation of naive CD8+ T lymphocytes but fail to promote survival. Eur. J. Immunol. 28, 221236. (20) Bowen, D. G., McCaughan, G. W., and Bertolino, P. (2005) Intrahepatic immunity: a tale of two sites? Trends Immunol. 26, 512-517. (21) Bowen, D. G., Zen, M., Holz, L., Davis, T., McCaughan, G. W., and Bertolino, P. (2004) The site of primary T cell activation is a determinant of the balance between intrahepatic tolerance and immunity. J. Clin. InVest. 114, 701-712. (22) Katz, S. C., Pillarisetty, V. G., Bleier, J. I., Kingham, T. P., Chaudhry, U. I., Shah, A. B., and DeMatteo, R. P. (2005) Conventional liver CD4 T cells are functionally distinct and suppressed by environmental factors. Hepatology 42, 293-300. (23) Jollow, D. J., Thorgeirsson, S. S., Potter, W. Z., Hashimoto, M., and Mitchell, J. R. (1974) Acetaminophen-induced hepatic necrosis. VI. Metabolic disposition of toxic and nontoxic doses of acetaminophen. Pharmacology 12, 251-271. (24) Piperno, E., and Berssenbruegge, D. A. (1976) Reversal of experimental paracetamol toxicosis with N-acetylcysteine. Lancet 2, 738739. (25) Kaplowitz, N. (2005) Idiosyncratic drug hepatotoxicity. Nat. ReV. Drug. DiscoVery 4, 489-499. (26) Ueno, K., Yamaura, K., Nakamura, T., Satoh, T., and Yano, S. (2000) Acetaminophen-induced immunosuppression associated with hepatotoxicity in mice. Res. Commun. Mol. Pathol. Pharmacol. 108, 237251. (27) Yamaura, K., Ogawa, K., Yonekawa, T., Nakamura, T., Yano, S., and Ueno, K. (2002) Inhibition of the antibody production by acetaminophen independent of liver injury in mice. Biol. Pharm. Bull. 25, 201-205. (28) Fayad, R., Sennello, J. A., Kim, S. H., Pini, M., Dinarello, C. A., and Fantuzzi, G. (2005) Induction of thymocyte apoptosis by systemic administration of concanavalin A in mice: role of TNF-alpha, IFNgamma and glucocorticoids. Eur. J. Immunol. 35, 2304-2312. (29) Krajewska, M., Wang, H. G., Krajewski, S., Zapata, J. M., Shabaik, A., Gascoyne, R., and Reed, J. C. (1997) Immunohistochemical analysis of in vivo patterns of expression of CPP32 (Caspase-3), a cell death protease. Cancer Res. 57, 1605-1613. (30) Hechter, O., and Johnson, S. (1949) In vitro effect of adrenal cortical extract upon lymphocytolysis. Endocrinology 45, 351-369. (31) Turnell, R. W., Clarke, L. H., and Burton, A. F. (1973) Studies on the mechanism of corticosteroid-induced lymphocytolysis. Cancer Res. 33, 203-212. (32) Ma, D. D., Ho, A. H., and Hoffbrand, A. V. (1984) Effect of thymosin on glucocorticoid receptor activity and glucocorticoid sensitivity of human thymocytes. Clin. Exp. Immunol. 55, 273-280. (33) Cohen, J. J. (1991) Programmed cell death in the immune system. AdV. Immunol. 50, 55-85. (34) Umansky, S. R., Korol, B. A., and Nelipovich, P. A. (1981) In vivo DNA degradation in thymocytes of gamma-irradiated or hydrocortisone-treated rats. Biochim. Biophys. Acta 655, 9-17. (35) 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. (36) Ishida, Y., Kondo, T., Ohshima, T., Fujiwara, H., Iwakura, Y., and Mukaida, N. (2002) A pivotal involvement of IFN-gamma in the pathogenesis of acetaminophen-induced acute liver injury. FASEB J. 16, 1227-1236. (37) Masubuchi, Y., Bourdi, M., Reilly, T. P., Graf, M. L., George, J. W., and Pohl, L. R. (2003) Role of interleukin-6 in hepatic heat shock protein expression and protection against acetaminophen-induced liver disease. Biochem. Biophys. Res. Commun. 304, 207-212. (38) Hogaboam, C. M., Bone-Larson, C. L., Steinhauser, M. L., Matsukawa, A., Gosling, J., Boring, L., Charo, I. F., Simpson, K. J., Lukacs, N. W., and Kunkel, S. L. (2000) Exaggerated hepatic injury due to acetaminophen challenge in mice lacking C-C chemokine receptor 2. Am. J. Pathol. 156, 1245-1252. (39) Sapolsky, R. M., Romero, L. M., and Munck, A. U. (2000) How do glucocorticoids influence stress responses? Integrating permissive, suppressive, stimulatory, and preparative actions. Endocr. ReV. 21, 55-89. (40) Haeryfar, S. M., and Berczi, I. (2001) The thymus and the acute phase response. Cell. Mol. Biol. (Paris, Fr., Print) 47, 145-156.

26 Chem. Res. Toxicol., Vol. 20, No. 1, 2007 (41) Sagiyama, K., Tsuchida, M., Kawamura, H., Wang, S., Li, C., Bai, X., Nagura, T., Nozoe, S., and Abo, T. (2004) Age-related bias in function of natural killer T cells and granulocytes after stress: reciprocal association of steroid hormones and sympathetic nerves. Clin. Exp. Immunol. 135, 56-63. (42) Ben, Rhouma, K., Schimchowitsch, S., Stoeckel, M. E., Felix, J. M., and Sakly, M. (1997) Implication of type II glucocorticoid receptors in aldosterone induced apoptosis of rat thymocytes. Arch. Physiol. Biochem. 105, 216-224. (43) Larson, A. M., Polson, J., Fontana, R. J., Davern, T. J., Lalani, E., Hynan, L. S., Reisch, J. S., Schiodt, F. V., Ostapowicz, G., Shakil, A. O., and Lee, W. M. (2005) Acetaminophen-induced acute liver failure: results of a United States multicenter, prospective study. Hepatology 42, 1364-1372. (44) Davern, T. J., 2nd, James, L. P., Hinson, J. A., Polson, J., Larson, A. M., Fontana, R. J., Lalani, E., Munoz, S., Shakil, A. O., and Lee, W. M. (2006) Measurement of serum acetaminophen-protein adducts in patients with acute liver failure. Gastroenterology 130, 687-694. (45) Shinzawa, H., Togashi, H., Sugahara, K., Ishibashi, M., Terui, Y., Aoki, M., Mitsuhashi, H., Matsuo, T., Watanabe, H., Abe, T., Ohno,

Masson et al.

(46) (47)

(48)

(49)

S., Saito, K., Saito, T., Yamada, N., Takahashi, T., and Horiuchi, R. (2001) Acute cholestatic hepatitis caused by a probable allergic reaction to paracetamol in an adolescent. Tohoku J. Exp. Med. 193, 255-258. Guerin, C., Casez, J. P., Vital-Durand, D., and Levrat, R. (1984) Allergy to paracetamol. A case of hepatic and cutaneous involvement. Therapie 39, 47-49. Liccardi, G., Cazzola, M., De, Giglio, C., Manfredi, D., Piscitelli, E., D’Amato, M., and D’Amato, G. (2005) Safety of celecoxib in patients with adverse skin reactions to acetaminophen (paracetamol) and other non-steroidal anti-inflammatory drugs. J. InVest. Allergol. Clin. Immunol. 15, 249-253. Titchen, T., Cranswick, N., and Beggs, S. (2005) Adverse drug reactions to nonsteroidal anti-inflammatory drugs, COX-2 inhibitors and paracetamol in a paediatric hospital. Br. J. Clin. Pharmacol. 59, 718-723. Boussetta, K., Ponvert, C., Karila, C., Bourgeois, M. L., Blic, J., and Scheinmann, P. (2005) Hypersensitivity reactions to paracetamol in children: a study of 25 cases. Allergy 60, 1174-1177.

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