Tolerogenic Role of Kupffer Cells in Allergic Reactions - American

Nov 8, 2003 - Laboratory of Molecular Immunology, NHLBI, Department of Health and Human Services, ... University of Colorado Health Sciences Center...
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Chem. Res. Toxicol. 2003, 16, 1514-1519

Tolerogenic Role of Kupffer Cells in Allergic Reactions Cynthia Ju,*,† J. Philip McCoy,‡ Christine J. Chung,§ Mary Louise M. Graf,§ and Lance R. Pohl§ Department of Pharmaceutical Sciences, University of Colorado Health Sciences Center, Denver, Colorado, Flow Cytometry Core Facility, and Molecular and Cellular Toxicology Section, Laboratory of Molecular Immunology, NHLBI, Department of Health and Human Services, National Institutes of Health, Bethesda, Maryland Received August 26, 2003

Drug-induced allergic reactions (DIARs), including allergic hepatitis, cutaneous reactions, and blood dyscrasias, are unpredictable and can be life threatening. Although current studies suggest that DIARs are caused by immunogenic drug-protein adducts, it remains unclear what factors determine the susceptibility to DIARs. We hypothesized that most individuals may be resistant to DIARs in part because they become immunologically tolerant to drugprotein adducts in the liver, an organ with tolerogenic properties. Because animal models of DIARs are elusive, we tested this hypothesis using a murine model of 2,4-dinitrochlorobenzene (DNCB)-induced delayed type hypersensitivity reaction that is mediated by immunogenic 2,4dinitrophenylated (DNP)-protein adducts. Intravenous pretreatment of mice with DNP-BSA led to its accumulation in hepatic Kupffer cells (KC) and induced immunological tolerance to subsequent DNCB sensitization. Tolerance could be abrogated by prior depletion of KC or induced in naı¨ve mice by transferring a T cell-depleted, KC-enriched fraction of liver nonparenchymal cells from mice tolerized 1 month earlier by DNP-BSA pretreatment. These findings implicate KC as a primary and sustained inducer of tolerance against DNP-protein adducts and suggest a similar role in modulating allergic reactions against drug-protein adducts. Perhaps genetic and/or environmental factors affecting the activities of these cells may play a role in determining individual susceptibility to DIARs.

Introduction Adverse drug reactions represent a major cause of death in the United States and a significant detriment to new drug development (1, 2). Approximately 10% of adverse drug reactions, such as allergic hepatitis, lupus, cutaneous reactions, and blood dyscrasias, appear to have an allergic etiology (3). Unfortunately, it remains impossible to predict the occurrences of DIARs1 due to our limited knowledge of the underlying mechanisms. Current studies suggest that most DIARs are caused by immunogenic protein adducts formed from covalent interactions of cellular proteins with a reactive metabolite of a drug (4, 5), although it is not clear why DIARs are restricted to a susceptible group of patients. One possible reason may be that most individuals develop immunological tolerance to drug-protein adducts instead of DIARs. The liver is the predominant site in which drugprotein adducts are formed, but little work has been done to explore the role of the liver in inducing tolerance to these protein adducts even though this organ is known * To whom correspondence should be addressed. Tel: 303-315-2180. Fax: 303-315-6281. E-mail: [email protected]. † University of Colorado Health Sciences Center. ‡ Flow Cytometry Core Facility, National Institutes of Health. § Molecular and Cellular Toxicology Section, National Institutes of Health. 1 Abbreviations: DIAR, drug-induced allergic reactions; DTH, delayed type hypersensitivity; DNCB, 2,4-dinitrochlorobenzene; DNP, 2,4-dinitrophenylated; DNP-BSA, DNP-bovine serum albumin conjugate; KC, Kupffer cell(s); FCS, fetal calf serum; APC, allophycocyanin; PerCP, peridinin chlorophyll protein; PE, phycoerythrin; FITC, fluorescein isothiocyanate; liposome/clodronate, clodronate-containing liposomes; NPC, nonparenchymal cells.

to have immunosuppressive properties (6, 7). For example, it has been demonstrated that allografts across MHC barriers of different mouse and rat strains are rendered resistant to rejection by preexposure of the recipients to donor cells through the portal vein (6, 7). Similarly, antigen-specific DTH reactions can be suppressed by preexposure of the antigens via the portal vein (8, 9). Accordingly, we hypothesized that the liver might also play an important role in inducing immunological tolerance to the protein adducts of drugs. It is difficult to test this hypothesis directly with drugs because animal models for most DIARs have been elusive. In contrast, animal models of allergic reactions have been established for chemically reactive haptens, including DNCB. DNCB causes a T cell-mediated DTH reaction when applied onto the skin. This reaction is mediated by immunogenic DNP-protein adducts formed by the reaction of DNCB with lysine and cysteine residues of proteins in the skin (10). Therefore, a murine model of DNCB-induced DTH response was used to investigate the role of the liver in inducing immunological tolerance to DNP-protein adducts. We found that pretreatment of mice intravenously with DNP-BSA conjugate induced tolerance against subsequent DNCB sensitization and that KCs (hepatic macrophages) appeared to play an important role in both initiating and sustaining the tolerance.

Materials and Methods Reagents and Antibodies. The following chemicals and reagents were purchased commercially: BSA (ICN Biomedical

10.1021/tx0341761 CCC: $25.00 © 2003 American Chemical Society Published on Web 11/08/2003

Tolerogenic Kupffer Cells in Xenobiotic Allergy Inc., Aurora, OH); DNP-BSA (Biosearch Technologies, Novato, CA); DNCB, dichloromethylene diphosphonate (clodronate), collagenase (type IV), Percoll, and rabbit anti-DNP sera (Sigma, St. Louis, MO); Ficoll-paque (Amersham, Piscataway, NJ); FCS (Invitrogen, Carlsbad, CA); 10% neutral buffered formalin (Fisher, Fairlawn, NJ); and peroxidase-conjugated anti-rat IgG avidin-biotin complex (ABC) kit (Vector Laboratories Inc., Burlingame, CA). The following anti-mouse mAbs were obtained from BD PharMingen (San Diego, CA): purified rat anti-FcγRII/ III (2.4G2), APC-conjugated rat anti-CD90.2 (53-2.1) and rat IgG2a (isotype control), PerCP-conjugated rat anti-CD45 (30-F11) and rat IgG2b (isotype control), PE-conjugated hamster antiCD11c (HL3), rat anti-CD19 (1D3), and the respective isotype control antibodies, including hamster IgG1 and rat IgG2a. FITCconjugated rat anti-mouse F4/80 (CI:A3-1) and rat IgG2b (isotype control) were purchased from Serotec (Raleigh, NC). Animals. Female C57BL/6J mice (6-8 weeks of age; Jackson Laboratory, Bar Harbor, ME) were acclimated to a 12 h lightdark cycle for approximately 1 week before use. All animals were maintained in autoclaved microisolator cages in a humidity- and temperature-controlled specific pathogen-free environment in accordance with the National Institutes of Health standards and the Guide for the Care and Use of Laboratory Animals. Immunohistochemical Detection of DNP-Labeled Cells in the Liver After Intravenous Injection of DNP-BSA. Female C57BL/6J mice were injected intravenously with 5 mg of DNP-BSA dissolved in saline. After 2 h, the livers were removed, and 2 mm thick sections were fixed in 10% neutral buffered formalin. Paraffin sections (5 µm) were mounted on poly(L-lysine)-treated glass slides by Histoserve Inc. (Gaithersburg, MD). The liver sections were stained with rabbit antiDNP sera (1:1000 dilution), and DNP-labeled cells were visualized using the Vectastain immunoperoxidase ABC kit, following the manufacturer’s instructions. Induction of Immunological Tolerance Against DNCBInduced DTH Reaction by Intravenous Pretreatment with DNP-BSA. Mice were injected intravenously through the lateral tail vein with 5 mg of DNP-BSA or BSA dissolved in 100 µL of saline. One week later, the mice were sensitized by applying 25 µL of 5% (w/v) DNCB in acetone/olive oil (4/1, v/v) to shaved abdominal skin. After 5 days, mice were challenged by applying 10 µL of 2.5% (w/v) DNCB in acetone/olive oil (1/9, 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). The results were expressed as the net increase in ear thickness. Effect of KC Depletion on the Induction of Immunological Tolerance Against DNCB-Induced DTH Reaction. To determine the role of KCs in the mechanism of tolerance, these cells were depleted by intravenous injection with liposome/ clodronates, which were prepared as previously described (11, 12). KC depletion was confirmed immunohistochemically with the use of a murine macrophage marker, F4/80, following an established procedure (11). Two days after KC depletion, the mice were injected intravenously with DNP-BSA to render them tolerant against DNCB-induced DTH reaction. Isolation of Liver NPCs. Liver NPCs were isolated following a previously described method (13). Mice were anesthetized, and the peritoneal cavity was widely exposed. The inferior vena cava was ligated, a 20 G catheter was inserted into the superior vena cava, and the portal vein was cut so that the perfusion was confined to the liver. The liver was perfused in situ at 37 °C with Hank’s balanced salt solution (HBSS) for 4 min followed by a 0.1% collagenase solution (in HBSS) for 10 min at a rate of 6 mL/min. Livers from 10 mice were pooled, and after complete dispersion of the cells, the suspension was filtered through nylon mesh to remove large aggregates and centrifuged at 50g for 2 min at 4 °C. The supernatant was then centrifuged at 500g for 10 min, and the pellet was resuspended in 100 mL of PBS containing 5% FCS (10 mL/liver). Ten milliliters of this suspension was layered on a discontinuous density gradient consisting of 15 and 20 mL of Percoll at 1.066 and 1.037 g/mL,

Chem. Res. Toxicol., Vol. 16, No. 12, 2003 1515 respectively, and centrifuged at 800g for 15 min at 4 °C. NPCs were obtained by collecting the fraction extending from the interface throughout the lower Percoll layer. The cells were washed twice in PBS containing 5% FCS before quantitation. One hundred to 150 million NPCs were obtained from 10 livers. Isolation of Splenic Mononuclear Cells. Spleens from two mice were placed in a 60 mm × 15 mm Petri dish containing a 5 mL solution of PBS and 5% FCS. Splenocytes were dispersed from the splenic capsule by smearing the tissues with frosted glass slides. The cells were then filtered through nylon mesh to remove large aggregates, and the filtrate was centrifuged at 1000 rpm for 8 min. The pellet was resuspended in 10 mL of PBS containing 5% FCS and layered on 4 mL of Ficoll-paque. The final suspension was centrifuged at 1000 rpm for 20 min. Mononuclear cells were obtained by collecting the interface, and the cells were washed twice in PBS before quantitation. Flow Cytometry. To characterize freshly isolated liver NPCs, cells were stained (at 1:100 dilution) with monoclonal antibodies to identify T cells (APC anti-CD90.2), leukocytes (PerCP anti-CD45), KCs (FITC anti-F4/80), dendritic cells (PE anti-CD11c), and B cells (PE anti-CD19). To prevent nonspecific binding, all samples were preincubated with blocking antiFcγRII/III antibody (2.4G2, 1:50 dilution) plus rat serum (1:10 dilution) for 5 min on ice. Samples were analyzed on a FACSCalibur using CellQuest software (BD Biosciences). To obtain purified subpopulations of liver NPCs, cells were stained with PE anti-CD90.2 and APC anti-CD45 antibodies. The T cell (CD45+CD90.2+) and non-T cell (CD45+CD90.2-) subpopulations were sorted with a Moflo cell sorter (Cytomation, Fort Collins, CO), yielding a purity of each subset of >99%. Adoptive Transfer of Tolerance against DNCB-Induced DTH Reaction to Naı1ve Mice. Donor mice were injected intravenously with 5 mg of DNP-BSA or BSA in 100 µL of saline, and 4 weeks later, the liver NPCs and splenic mononuclear cells were isolated. Splenic mononuclear cells (37 × 106), liver NPCs (3 × 106), as well as purified T cell (6 × 105) or non-T cell subpopulations (8 × 105) of liver NPCs were transferred to separate groups of naı¨ve mice by intravenous injection (100 µL in PBS), followed by DNCB sensitization 2 h later. Subsequent challenge and evaluation of DTH response were carried out as described above. Statistical Analysis. Data are presented as means ( SEM. Statistical comparisons between two groups were made using a Student’s t-test and between various groups using ANOVA with a post-hoc test of significance between individual groups. Differences were considered significant when p < 0.05.

Results Uptake of DNP-BSA by KC After Intravenous Administration. DNP-labeled cells were detected immunohistochemically in the liver sinusoids of mice between 2 and 6 h after the intravenous injection of DNPBSA but were undetectable after this time (Figure 1). To determine whether the labeled cells were KCs, the experiment was repeated by pretreating the animals with liposome/clodronate 2 days prior to the intravenous injection with DNP-BSA. This reagent has been shown previously to deplete KCs within 24 h and maintain their depletion for approximately 1 week (11, 12, 14). Indeed, it was found that DNP staining of sinusoidal cells was markedly decreased when KCs were depleted by liposome/clodronate pretreatment (Figure 1B). In contrast, very little staining of the sinusoidal endothelial cells was observed. Role of KCs in Tolerance against DNCB-Induced DTH Reactions. In preliminary studies, it was found that the intravenous pretreatment of mice with 5 mg of DNP-BSA 1 week prior to DNCB skin sensitization resulted in an inhibition of the subsequent DTH response

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Figure 3. Effect of KC depletion on tolerization of DNCB sensitization by DNP-BSA pretreatment. C57BL/6J female mice were injected intravenously with empty liposomes (Lip/ pbs) or liposome/clodronate (Lip/cld). After 2 days, mice were injected intravenously with 5 mg of DNP-BSA (filled bars) or BSA (open bars). One week later, mice were sensitized with 5% DNCB and challenged 5 days later by applying 2.5% DNCB to the ear. The DTH responses were determined 24 h after challenge by measuring ear swelling. The results are expressed as the mean ( SEM of 10 mice per group. (*) p < 0.05 relative to BSA-treated controls.

Figure 1. Immunohistochemical detection of DNP-labeled cells in the liver of KC-depleted and nondepleted mice. C57BL/6J female mice were injected intravenously with either empty liposomes (A) or liposome/clodronate (B). After 2 days, the mice were injected intravenously with DNP-BSA (5 mg), and 2 h later, the animals were sacrificed, and liver sections were stained for DNP-labeled cells with rabbit anti-DNP sera (11000 dilution; original magnification, 400×).

Figure 2. Induction of tolerance against DNCB sensitization by intravenous pretreatment with DNP-BSA. C57BL/6J female mice were injected intravenously with DNP-BSA, BSA, or saline 1 week prior to sensitization with 5% DNCB. After 5 days, the mice were challenged by applying 2.5% DNCB to the ear, and DTH responses were determined 24 h postchallenge by measuring ear swelling using a caliper micrometer. The results are expressed as the mean ( SEM of 10 mice per group. (*) p < 0.05 relative to BSA or saline-treated controls.

after DNCB challenge (Figure 2). In contrast, intravenous pretreatment with 5 mg of BSA did not inhibit the DNCB-induced DTH responses (Figure 2). To determine whether KCs had a role in induction of the tolerance, the

Figure 4. Adoptive transfer of tolerance against DNCB sensitization in naı¨ve mice with splenic mononuclear cells (MNC) or liver NPC obtained from mice tolerized with DNPBSA. Donor mice were injected intravenously with 5 mg of DNP-BSA or BSA. Four weeks later, spleen MNC (37 × 106) or liver NPC (3 × 106) were isolated from both groups and then injected directly to recipient mice. Two hours later, recipient mice were sensitized with 5% DNCB. After 6 days, the DTH response was determined by measuring ear swelling 24 h after challenge with 2.5% DNCB. The results are expressed as the mean ( SEM of 10 mice per group. (*) p < 0.05 relative to recipients of spleen MNC or liver NPC obtained from BSAtreated donor mice.

experiment was repeated when KCs were depleted from the liver by liposome/clodronate treatment 2 days prior to the DNP-BSA pretreatment. The results showed that KC depletion led to an inhibition of the tolerance induced by DNP-BSA (Figure 3). Because it has been shown that intravenous injection of liposome/clodronate also depletes macrophages in the spleen (12), adoptive transfer studies were performed to delineate the role of macrophages in the liver and/or the spleen in DNP-BSA-induced tolerance against DNCB sensitization. Liver NPC or splenic mononuclear cells, obtained from donor mice 4 weeks after the intravenous pretreatment with DNP-BSA, were transferred to naı¨ve mice prior to sensitization with DNCB. Tolerance against DNCB sensitization was induced significantly in naı¨ve mice by the adoptive transfer of liver NPC but not by the transfer of splenic mononuclear cells even though a 10-fold higher number of cells were transferred as compared to the liver NPCs (Figure 4). Flow cytometric analysis revealed that CD45+CD90.2+ T cells and CD45+CD90.2- non-T cells accounted for 10

Tolerogenic Kupffer Cells in Xenobiotic Allergy

Figure 5. Flow cytometric analysis of liver NPC. C57BL/6J female mice were injected intravenously with 5 mg of DNPBSA. Four weeks later, the liver NPCs were isolated and the cells were stained with APC anti-CD90.2, PerCP anti-CD45, FITC anti-F4/80, and PE anti-CD11c or anti-CD19. The percentages of T cells (CD45+CD90.2+) and non-T cells (CD45+CD90.2-) are shown in (A). (B-D) Percentages of various types of cells (gated on the non-T cell subset), including (B) F4/80+ KC, (C) CD11c+ dendritic cells (DC), and (D) CD19+ B cells (B). The nonfilled histograms with fluorescence intensities between 101 and 102 represent positive staining with F4/80, CD11c, and CD19 antibodies and the filled histograms with fluorescence intensities below 101 represent background staining with respective isotype-matched control antibodies.

and 28% of freshly isolated liver NPCs, respectively (Figure 5A). The remaining CD45-CD90.2- cells were primarily endothelial cells, which account for approximately 50% of liver NPCs (15). The CD45+CD90.2- non-T cell subpopulation was composed of approximately 72% F4/80+ KC, 7% CD11c+ dendritic cells, and 3% CD19+ B cells (Figure 5B-D, respectively). To further investigate which type(s) of cells within the mixture of liver NPCs played an important role in inducing tolerance in naı¨ve mice, CD45+CD90.2+ T cells (6 × 105) and CD45+CD90.2- non-T cells (8 × 105) were purified by flow cytometric sorting and adoptively transferred to separate groups of naı¨ve mice. Both CD45+CD90.2+ T cells and CD45+CD90.2- non-T cells were able to transfer tolerance (Figure 6). Moreover, when adoptive transfer was carried out with liver NPC that had been depleted of KCs, tolerance was blocked (Figure 7). It is likely that the number of T cells (approximately 3 × 105) in the KC-depleted fraction of liver NPCs was insufficient to transfer tolerance.

Discussion Evidence suggests that DIARs are induced in most cases by immunogenic neoantigens formed by the reaction of endogenous proteins with reactive metabolites of drugs that act as haptens (4, 5). However, the low incidence of DIARs suggests that most individuals may develop immunological tolerance rather than deleterious immune reactions to drug-protein adducts (11). Because the liver is the site where drug-protein adducts are predominantly formed and is known to possess tolerogenic properties (6, 7), it seems reasonable that this organ may be a major site of tolerance induction against DIARs.

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Figure 6. Adoptive transfer of DNP-BSA-induced tolerance against DNCB sensitization to naı¨ve mice with nonfractionated, purified hepatic T cells or T cell-depleted subpopulation of liver NPCs. Donor mice were injected intravenously with 5 mg of DNP-BSA or BSA. Four weeks later, liver NPCs were isolated from both groups and then either injected directly to recipient naı¨ve mice (3 million cells/mouse) or used for the purification by cell sorting and injection into naı¨ve mice of CD45+CD90.2+ hepatic T cells (T, 600 000 cells/mouse, >99% pure) or T celldepleted CD45+CD90.2- (non-T) subpopulation of liver NPCs (800 000 cells/mouse, >99% pure). Two hours later, recipient mice were sensitized with 5% DNCB. After 6 days, the DTH response was determined by measuring ear swelling 24 h after challenge with 2.5% DNCB. The results are expressed as the mean ( SEM of 10 mice per group. (*) p < 0.05 relative to recipients of liver NPCs obtained from BSA-treated donor mice.

Figure 7. Adoptive transfer of DNP-BSA-induced tolerance against DNCB sensitization to naı¨ve mice with liver NPC obtained from KC-depleted or KC-nondepleted mice. Donor mice were injected intravenously with 5 mg of DNP-BSA or BSA, and after 4 weeks, they were treated with liposome/clodronate to deplete KC or with empty liposomes as a control. After 2 days, liver NPCs were isolated and injected into three groups of recipient mice (3 million cells/mouse). Two groups of naı¨ve recipient mice were injected with liver NPCs isolated from KCnondepleted mice that were pretreated with BSA or DNP-BSA. The third group was injected with liver NPCs isolated from KCdepleted mice that were pretreated with DNP-BSA (NPC-KC). Two hours later, mice were sensitized with 5% DNCB, and after 6 days, the DTH response was determined by measuring ear swelling 24 h after challenge with 2.5% DNCB. The results are expressed as the mean ( SEM of 10 mice per group. (*) p < 0.05 relative to recipients of liver NPCs from BSA-treated donor mice.

The tolerogenicity of the liver has been attributed to the unique cell populations within this organ. Natural killer T cells make up nearly half of the T cell population in the liver but are found in much lower proportions elsewhere, such as in the spleen, blood, lymph nodes, and thymus (16). These cells appear to have a role in preventing autoimmunity (17) and inducing oral tolerance in an experimental model of trinitrochlorobenzene-

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induced colitis (18). The liver also contains a higher percentage of γδ T cells as compared with that in the blood (19), and these cells have been shown to have a role in the delayed rejection of skin allografts induced by portal vein injection of allogeneic donor cells (20). Data from the present study indicate that hepatic T cells also play a role in tolerizing DTH reactions induced by hapten-protein adducts. We showed that adoptive transfer of highly purified (>99%) hepatic T cells obtained from DNP-BSA-tolerized mice could induce tolerance against DNCB sensitization in naı¨ve mice (Figure 6). These cells may include natural killer T cells, γδ T cells, and/or a subset of regulatory/suppressor T cells, which may be rendered tolerogenic through their interaction with KCs (21-23). Other work suggests that liver sinusoidal endothelial cells may act as antigen presenting cells and induce T cells to undergo apoptosis or to become regulatory T cells (24, 25). KCs have also been linked to the tolerogenicity of the liver. For example, studies of organ transplantation in animals indicated that inhibition of KC activity abrogates the prolonged survival of allografts induced by portal vein infusion of allogeneic donor cells (6, 26). However, the role of KCs in inducing tolerance against antigens other than allografts remains unclear even though these cells represent a primary population of antigen presenting cells in the liver and are known to scavenge both particulate and soluble materials in the circulation (27). In this regard, protein adducts of the drugs halothane and acetaminophen, which are produced primarily in the liver, have been detected in vivo in KCs (28, 29), suggesting that these cells may be important in modulating T cell responses against drug-protein adducts. Previous studies have shown that tolerance against the skin sensitizing haptens, DNCB and trinitrochlorobenzene, could be induced by prior intravenous injection with the hapten covalently bound to mouse immunoglobulin G or serum (30, 31) as well as various types of syngeneic cells (32), but the underlying mechanism was not thoroughly investigated. The present study, however, provides the first evidence for a direct role of KC in the induction of tolerance to hapten-protein adducts. We found that pretreatment of mice intravenously with DNP-BSA induced tolerance against subsequent DNCB sensitization and that the tolerance was preceded by an accumulation of DNP-BSA predominantly in KCs rather than sinusoidal endothelial cells (Figure 1). The induction of tolerance could be inhibited when KCs were depleted with liposome/clodronate prior to DNP-BSA treatment (Figure 3). It could also be induced in naı¨ve mice by adoptive transfer of a KC-enriched fraction of liver NPCs that were depleted of sinusoidal endothelial cells and T cells and obtained from mice tolerized by DNP-BSA pretreatment 1 month earlier (Figure 6). In contrast, splenocytes did not appear to have a role in the development of tolerance (Figure 4). It is possible, however, that hepatic dendritic cells may also contribute in part to the induction of the tolerance against DNCB-induced DTH reactions despite the paucity of these cells in the liver (Figure 5). In this regard, it has been demonstrated that administration of liver dendritic cell progenitors may increase allografts survival (33). Although it is not clear how hepatic dendritic cells become tolerogenic, their interaction with KCs may be a contributing factor (34, 35).

Ju et al.

The tolerogenic property of KCs appears to be related at least in part to their low expression level of costimulatory molecules (36) and secretion of tolerogenic factors, such as interleukin-10, transforming growth factor-β, and prostaglandin-E2 (37-39). Our findings further indicate for the first time that the tolerogenicity of KCs is longlived. We were able to transfer tolerance to naı¨ve mice with a KC-enriched fraction of liver NPCs obtained from donor mice 1 month (Figure 6) or 10 weeks (data not shown) after the induction of tolerance by DNP-BSA treatment. This sustained tolerogenicity may be associated with the persistent presentation of low levels of DNP-BSA that were below immunohistochemical detection 6 h after DNP-BSA treatment. In this regard, it is known that KCs survive for weeks to months in the liver (40, 41). Moreover, a link between persistent antigen presentation and T cell tolerance has been suggested in studies of donor cell chimerism (42, 43). The long-term tolerogenic effect of KCs may also be associated with the continuous production of immunosuppressive cytokines. Nevertheless, our results indicate that the tolerogenicity of KC is antigen-specific because KC-enriched fractions of cells from DNP-BSA-treated, but not BSA-treated mice, could transfer tolerance against DNCB sensitization to naı¨ve mice (Figures 3, 4, and 6), whereas they could not transfer tolerance to naı¨ve mice against skin sensitization caused by oxazolone (data not shown). In summary, our data indicate that KCs are a primary and sustained inducer of immunological tolerance against a hapten-induced DTH response. This finding suggests that these cells may play an important role in modulating immune responses against drug-protein adducts and preventing DIARs in most individuals. Perhaps genetic polymorphisms (44) and environmental factors, such as drugs (45, 46), alcohol (47), and infectious pathogens (48), that affect the activities of KCs may contribute as risk factors in determining individual susceptibility to DIARs. A better understanding of the immunosuppressive function of KCs in the future may lead to new strategies of minimizing allograft rejection, as well as predicting and preventing DIARs.

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