Immunohistochemical Detection of Protein Adducts of 2,4

Chem. Res. Toxicol. 2001, 14, 1209-1217

1209

Immunohistochemical Detection of Protein Adducts of 2,4-Dinitrochlorobenzene in Antigen Presenting Cells and Lymphocytes after Oral Administration to Mice: Lack of a Role of Kupffer Cells in Oral Tolerance Cynthia Ju* and Lance R. Pohl Molecular and Cellular Toxicology Section, Laboratory of Molecular Immunology, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland Received March 13, 2001

Although current studies suggest that most drug-induced allergic reactions (DIARS) are caused by immunogenic conjugates formed from the reaction of a reactive metabolite of a drug with cellular proteins, it is not clear why these reactions are relatively rare. One possible pathway that may explain the low incidence of DIARS in many cases is oral tolerance, an antigen-specific immunological hyporesponsiveness induced by oral administration of antigens. The mechanism of oral tolerance, however, is not clearly understood and is difficult to study directly with drugs, because animal models of DIARS have been elusive. We chose 2,4dinitrochlorobenzene (DNCB) as a model compound to circumvent this problem because animal models of allergic reactions have been established for this compound. DNCB forms immunogenic 2,4-dinitrophenylated (DNP) protein conjugates that can induce immune reactions and it causes oral tolerance when it is fed to animals prior to sensitization. We hypothesized that DNPprotein conjugates may have a role in oral tolerance. To test this idea, we have begun to identify cells bearing these conjugates after the oral administration of DNCB. Female C57BL/6J mice were fed DNCB and tissues were examined after 6 and 24 h. Immunohistochemical analysis indicated the presence of DNP-protein conjugates in enterocytes of the small intestine, in macrophages and lymphocytes of the mesenteric lymph nodes, in dendritic cells and lymphocytes of the spleen, and in Kupffer cells and other sinusoidal cells of the liver. It was found that Kupffer cell depletion did not affect oral tolerance to DNCB. The findings suggest that the cells bearing DNP-protein conjugates, other than Kupffer cells, in the liver and other tissues may be important in the induction of oral tolerance against DNCB. Protein adducts of drugs administered orally may also be present in these cells, and they may have a role in the downregulation of DIARS in many individuals.

Introduction (DIARS)1

Drug-induced allergic reactions account for approximately 6-10% of all adverse drug reactions (1). These reactions can be life threatening and result in the removal of useful drugs from the market, such as practolol, benoxaprofen, ticrynafen, and nomefensine (2). Unfortunately, little is known about the mechanism(s) involved in DIARS, and it is almost impossible to predict who will develop DIARS to a given drug. It is believed that DIARS are caused by immunogenic protein adducts formed from the reaction of a reactive metabolite of a drug with cellular proteins (3, 4). The reason only a small proportion of patients develop immune reactions against drug protein adducts remains unclear. One possibility for the relatively low incidence of DIARS may be due to * To whom correspondence should be addressed. Phone: (301) 4027323. Fax (301) 480-4852. E-mail: [email protected]. 1 Abbreviations: DIARS, drug-induced allergic reactions; DNCB, 2,4-dinitrochlorobenzene; DNP, 2,4-dinitrophenyl; DTH, delayed type hypersensitivity; DNP-lysine, N-2,4-DNP-L-lysine; FCS, fetal calf serum; IgG, immunoglobulin G; DTT, dithiothreitol; PBS, phosphatebuffered saline; ABC, avidin-biotin complex; PE, phycoerythrin; FITC, fluorescein isothiocyanate; H/E, hematoxylin/eosin; APCs, antigenpresenting cells; MHC, major histocompatibility complex; Th1, T helper cell 1; NK T cells, natural killer T cells; FACS, fluorescence activated cell sorting.

10.1021/tx0100587

protein adducts causing oral tolerance instead of DIARS in most individuals (5). However, it is difficult to study this hypothesis directly with drugs because animal models for most DIARS have not been developed. In contrast, several animal models of allergic reactions have been established for chemically reactive haptens. One of the best-studied models involves the hapten 2,4-dinitrochlorobenzene (DNCB). This chemical reacts with lysine and cysteine residues of proteins to form immunogenic 2,4-dinitrophenylated (DNP)-protein adducts (6) and causes T-cell mediated delayed hypersensitivity (DTH) reactions in the skin. Moreover, when DNCB is administered orally prior to skin sensitization, the DTH reaction is blocked, presumably by DNP-protein adducts causing oral tolerance (7, 8). Therefore, it seems reasonable that understanding the mechanism of oral tolerance caused by DNCB may provide clues to how protein adducts of drugs may cause oral tolerance. Although the mechanism(s) of tolerance caused by the oral feeding of DNCB and other reactive haptens as well as protein antigens has been extensively studied, many of the details of this process remain to be explained (9, 10). For example, several studies suggest that cells of the gut-associated lymphoid tissues and the draining me-

This article not subject to U.S. Copyright. Published 2001 by the American Chemical Society Published on Web 08/24/2001

1210

Chem. Res. Toxicol., Vol. 14, No. 9, 2001

senteric lymph nodes are important in the induction of oral tolerance (8, 11), while other evidence supports the role of splenocytes (12, 13) and liver cells (14-17) in oral tolerance. Consequently, the cellular basis of oral tolerance remains to be clearly defined. To help resolve this problem, particularly as it relates to haptens, we report here an immunochemical method for identifying cells that become DNP-labeled after the feeding of DNCB to mice.

Experimental Procedures Chemicals and Reagents. The following chemicals and reagents were purchased commercially: cholesterol, dichloromethylene diphosphonate (clodronate), DNCB, rabbit anti-DNP sera, Gill No. 1 hematoxylin solution, N-2,4-DNP-L-lysine (DNP-lysine), phosphatidylcholine, trypsin tablets (containing 1 mg of porcine trypsin), fetal calf serum (FCS), and purified mouse immunoglobulin G (IgG) (Sigma, St. Louis, MO); dithiothreitol (DTT, ICN Biomedicals, Aurora, OH); xylene and 10% neutral buffered formalin (Fisher, Fairlawn, NJ); India Ink (VisualSystems, Rockville, MD); paraformaldehyde (Polysciences Inc, Warrington, PA); Ficoll-paque (Amersham Pharmacia Biotech AB, Uppsala, Sweden); calcium- and magnesium-free phosphate-buffered saline (PBS, Biofluids, Rockville, MD); Vectastain anti-rabbit IgG (peroxidase-conjugated) avidinbiotin complex (ABC) kit (Vector Laboratories Inc., Burlingame, CA); rat IgG2b, phycoerythrin (PE)-conjugated hamster IgG1κ anti-mouse CD3, PE-conjugated hamster IgG1κ, fluorescein isothiocyanate (FITC)-conjugated rat IgG2a, and rat anti-mouse FcγIII/II receptor (PharMingen, San Diego, CA); FITC-conjugated rat IgG2a anti-DNP (Accurate Chemical & Scientific Co., Westbury, NY); and rat IgG2b anti-F4/80 antibody (Serotec, Raleiph, NC). Animal Treatment. Female C57BL/6J mice (6-8 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. The animals were used approximately 1 week after arrival. DNCB was dissolved in acetone:olive oil (1:9, v/v), and administered by gastric gavage (250 mg/kg, 200 µL total volume). Immunohistochemical Detection of DNP-Protein Adducts. Mice were killed at 6 and 24 h after the oral administration of DNCB (250 mg/kg). The liver, spleen, mesenteric lymph nodes and small intestine were removed. The luminal contents of the small intestine were flushed out with PBS containing 1 mM DTT. The small intestine was then segmented into five equal sections (15-18 cm/section). A 2 mm thick section from the liver, spleen, and each segment of the small intestine was fixed in neutral buffered formalin (3.7%, v/v, formaldehyde in 0.029 M NaH2PO4‚H2O and 0.046 M NaHPO4‚7H2O, pH 7.0), embedded in paraffin, and sections (5 µm) were mounted on poly (L-lysine)-treated glass slides by American Histolabs (Gaithersburg, MD). The mesenteric lymph nodes were fixed in neutral buffered formalin without segmentation. All of the following steps were done at room temperature. Poly (L-lysine)-treated slides were placed in a staining dish, and paraffin was removed from the sections by washes with xylene (two incubations of 5 min each), followed by washes for 2 min each with absolute ethanol, 70% ethanol, 35% ethanol, and PBS. To improve the intensity of immunohistochemical staining, the tissue sections on the slides were subjected to trypsin digestion (1 trypsin tablet/mL of dionized water) for 30 min at 37 °C. After washing in PBS for 5 min, endogenous peroxidase activity was inhibited by incubating the sections with 0.3% (v/v) H2O2 in methanol for 30 min. After blocking nonspecific binding sites with normal goat serum for 20 min, the sections were treated for 30 min with rabbit polyclonal anti-DNP antibodies. The antibodies were diluted 1 to 2000 for the liver, spleen, and small intestine. For staining of the mesenteric lymph nodes, the antibodies were

Ju and Pohl diluted 1 to 1000. Similarly diluted rabbit preimmune sera were used for negative controls. Labeled proteins were visualized using the Vectastain immunoperoxidase ABC kit, following manufacture’s instructions. The tissue was counterstained with Gill No. 1 hematoxylin solution. Flow Cytometry. Animals treated with DNCB or vehicle were killed 6 h after dosing. Spleen cell suspension from each animal was prepared by teasing the tissue apart between two frosted microscope slides. Mononuclear cells were purified with the use of a Ficoll-Paque density gradient following manufacture’s instruction. The cells were aliquoted to a total of 1 × 106 cells per tube for immunofluorescence staining. Before staining, the cells were incubated with anti-FcγIII/II receptor (1 to 100 dilution) and mouse IgG (1 to 100 dilution) in 100 µL of PBS containing 5% FCS to block nonspecific binding of fluorescentlabeled antibodies. After 30 min incubation on ice, the cells were washed in PBS and incubated with PE-conjugated hamster IgG1κ anti-CD3 (1:100) and FITC-conjugated rat IgG2a antiDNP (1:100) diluted in 100 µL of PBS containing 5% FCS. Cells stained with isotype-matched Ig were used as negative controls. After 45 min incubation on ice, the cells were washed twice in PBS and fixed in 4% (v/v) paraformaldehyde dissolved in PBS. Staining was then measured by flow cytometry on a FACSort flow cytometer using the CellQuest analysis program (Becton Dickinson, Mountain View, CA). Experiments were repeated on four independent occasions. Depletion of Kupffer Cells with Clodronate-Containing Liposomes. Clodronate-containing liposomes were prepared as described by Van Rooijen et al. (18). Briefly, phosphatidylcholine (86 mg) and 8 mg cholesterol were dissolved in 10 mL of chloroform in a 500 mL round-bottom flask. Chloroform was removed by low vacuum rotary evaporation at 37 °C. The thin film that formed on the walls of the flask was dispersed by gentle shaking for 10 min in 10 mL of PBS or 0.6 M clodronate dissolved in PBS. The suspensions were kept under argon for 2 h at room temperature, sonicated for 3 min in a water bath sonicator (Sonicor Instrument Co., Copiague, NY), and kept under argon for another 2h. The nonencapsulated clodronate was removed by centrifugation (10000g for 15 min) of the liposomes. The white band at the top of the suspension, which contained clodronate-containing liposomes, was retrieved and washed twice with PBS (sterilized) by centrifugation (25000g for 30 min). Finally, the pellet was resuspended in 4 mL of sterilized PBS and it can be stored at 4 °C for up to one month before use. Mice were injected intravenously with clodronatecontaining liposomes (100 µL). Various times after liposome administration, the livers were removed and PLL slides of the liver sections were prepared as described earlier. The depletion of Kupffer cells was determined immunohistochemically using a murine macrophage marker, F4/80 (19), following the procedure used for the detection of DNP-protein adducts. The phagocytic activity in the liver was determined by administering intravenously India Ink (100 µL) to mice (19). After 20 min, the livers were removed and sections were fixed in neutral buffered formalin and then embedded in paraffin and used to prepare hematoxylin/eosin (H/E) stained slides (American Histolabs). The uptake of carbon particles was detected by light microscopy. The Effect of Kupffer Cell Depletion on DNCB-Induced Oral Tolerance. Animals were divided into four groups: group A was injected intravenously with 100 µL of empty liposomes 2 days prior to the oral administration of the vehicle without DNCB; group B was injected intravenously with 100 µL of empty liposomes 2 days prior to the oral administration of DNCB; group C was injected intravenously with 100 µL of clodronatecontaining liposomes 2 days prior to the oral administration of the vehicle without DNCB; group D was injected intravenously with 100 µL of clodronate-containing liposomes 2 days prior to the oral administration of DNCB. This treatment was carried out once per week for 3 weeks. The DTH responses to subsequent DNCB sensitization were assessed by ear swelling test as described (20). Briefly, 5 weeks after the final DNCB feeding,

Study of the Mechanism of Oral Tolerance to Haptens

Chem. Res. Toxicol., Vol. 14, No. 9, 2001 1211

Figure 1. Immunohistochemical detection of DNP-protein adducts in the small intestine of mice 6 h after the oral administration of DNCB. Anti-DNP sera were diluted 1-2000. (A) Detection of DNP-protein adducts in a section of the small intestine from an animal treated with DNCB (75×); (B) detection of DNP-protein adducts in the villus enterocytes (arrowhead) and in cells of the lamina propria (arrow) (300×); panel C, no DNP-protein adducts were detected in a section of the small intestine from an animal treated with vehicle (75×); VT, villus tip, and LP, lamina propria. 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 (v/v, 1:9) 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 the net increase in ear thickness. Statistical Analysis. All comparisons between control animals and animals fed with DNCB were made by Student’s t test (unpaired) and judged significantly different at P < 0.05.

Results Immunohistochemical Detection of DNP-Protein Adducts in Vivo after oral Administration of DNCB. DNP-protein adducts was detected immunohistochemically, with the use of rabbit anti-DNP sera, in cells of several tissues 6 h after the oral administration of DNCB. In the small intestine, DNP-protein adducts were observed predominantly in the villus enterocytes, especially in the villus-tip enterocytes (Figure 1, panels A and B). Immunohistochemical staining was also detected in cells of the lamina propria (Figure 1B), and low levels were found in cells of the dome region of the Peyer’s patches (data not shown). No immunochemical staining was observed when it was done with sections from control animals, which were fed with vehicle only (Figure 1C). DNP-protein binding was detected in cells of the mesenteric lymph nodes and the spleen (Figure 2). Cells with the morphology and size of macrophages and lymphocytes appeared to be labeled in the paracortical zones of the mesenteric lymph nodes (Figure 2A). In the spleen, a number of DNP-labeled cells were detected in the white pulp (Figure 2B). Some of these cells had the morphology and size of lymphocytes (Figure 2C). Other DNP-labeled cells appeared to be dendritic cells that were located not only in the marginal zone but also in the T-cell area of the white pulp (Figure 2D), and many of these cells formed clusters (Figure 2E).

DNP-protein adducts were also detected in the liver. Staining was observed in epithelial cells of bile ducts, in endothelial cells of the portal veins, and in a number of sinusoid cells throughout the liver (Figure 3A). Some of these cells appeared to have the morphology and size of Kupffer cells (Figure 3, panels B and C). However, most of the DNP-labeled sinusoid cells did not appear to be depleted by pretreatment with clodronate-containing liposomes (Figure 3D). The number of DNP-labeled sinusoidal cells was 194 and 221 in the liver sections shown in panels C and D of Figure 3, respectively. The specificity of the immunochemical detection of DNPprotein adducts was confirmed by the finding that preincubation of anti-DNP sera with DNP-lysine conjugate blocked the staining in the liver (Figure 3E). Very little DNP-protein binding was observed in cells of the small intestine, spleen and liver 24 h after the oral administration of DNCB. In contrast, the number of DNP-labeled cells in the mesenteric lymph nodes remained constant for at least 24 h after DNCB was administered to the mice (data not shown). Detection of DNP-Labeled Splenic T Cells by Flow Cytometry after the Oral Administration of DNCB. Immunohistochemical analysis suggested that certain DNP-labeled cells in the white pulp of the spleen may be T lymphocytes. To confirm this finding, two-color flow cytometry analysis was performed, with the use of anti-CD3 and anti-DNP antibodies, on mononuclear cells isolated from animals 6 h after the oral administration of vehicle or DNCB. A typical flow cytometry analysis is shown in Figure 4, where approximately 1.9% of splenic CD3+ T cells from DNCB treated mice were found to stain positively for both CD3 and DNP (Figure 4A). Only 0.2% of cells isolated from vehicle control animals stained double positive for these markers (Figure 4B). Statistical analysis, with the use of 15 DNCB treated mice and 9 vehicle treated mice, confirmed this finding and showed

1212


Ju and Pohl

Figure 2. Immunohistochemical detection of DNP-protein adducts in the mesenteric lymph nodes and the spleen of mice 6h after the oral administration of DNCB. Anti-DNP sera were diluted 1-1000 for immunohistochemical staining of cells in the mesenteric lymph nodes and 1-2000 for cells in the spleen. (A) Detection of DNP-protein adducts in cells of the paracortical zones of mesenteric lymph nodes (300×). DNP-labeled cells with morphology and size of macrophages and lymphocytes are indicated with arrowheads and arrows, respectively. (B-E) Detection of DNP-protein adducts in cells of the white pulp of the spleen. (B) 150×; (C) cells with morphology and size of lymphocytes are indicated with arrows (300×); (D) cells with morphology of dendritic cells are indicated with arrowheads (300×); (E) a cluster of dendritic cells (300×).

that the number of DNP-labeled CD3+ T-cells was significantly higher in DNCB treated animals than those of vehicle treated animals (Figure 4C). Role of Kupffer Cells in DNCB-Induced Oral Tolerance. Immunohistochemcial analysis using antiF4/80 antibody showed that Kupffer cells were nearly eliminated from the liver 24 h after the intravenous injection of clodronate-containing liposomes as reported by others (18) (Figure 5, panels A and B). The depletion of Kupffer cells was confirmed by the substantial inhibition of uptake of carbon particles in the liver by this treatment (Figure 5, panels C and D). Kupffer cells stayed depleted for 2 days and began to reappear in the

liver on day 3 (data not shown). In addition, the number of macrophages in the spleen was also diminished after the intravenous injection of clodronate-containing liposomes, and as in the liver, the amount of DNP-protein adducts detected in the spleen did not appear to be affected by this treatment (data not shown). The effect of the depletion of Kupffer cells on DNCBinduced oral tolerance was investigated. Mice sensitized with 5% DNCB developed a DTH response that was inhibited by prior oral administration of DNCB (Figure 6, panels A and B). Kupffer cell depletion, however, did not prevent DNCB-induced oral tolerance (Figure 6, panels C and D).



Figure 3. Immunohistochemical detection of DNP-protein conjugates in the liver of mice 6 h after the oral administration of DNCB. Anti-DNP sera were diluted 1-2000. (A) Detection of DNP-protein adducts in epithelial cells of the bile ducts (BD), in endothelial cells of a portal vein (PV) and in sinusoid cells (arrows) (300×); panels B and C show two DNP-labeled sinusoid cells with different morphology (arrows, 750×); (D) depletion of Kupffer cells by intravenous injection of clodronate-containing liposomes did not appear to decrease the number of DNP-labeled cells in the sinusoids (arrows, 300×); (E) immunochemical staining was inhibited by preincubation of anti-DNP sera with 20 µM DNP-lysine conjugate (150×).

Discussion Because most drugs are administered orally, the relatively low incidence of DIARS may be due to the induction of oral tolerance in many cases. Most studies on the mechanism of oral tolerance have been done with protein antigens. When proteins are administered orally at low doses, suppressor cells are thought to have a major role in mediating tolerance against subsequent immunization with the protein antigens (10, 21). Indeed, when cells from the gut associated lymphoid tissues, the mesenteric lymph nodes and spleen of orally tolerized animals are adoptively transferred to naive animals, antigen specific tolerance is produced (12, 22-24). How-

ever, the identities of the cells and the mechanism by which they cause tolerance are not well defined. Evidence suggests that inappropriate presentation of protein antigens by antigen-presenting cells (APCs) with low or no costimulatory activities may induce T cell tolerance (25, 26). Considerably less is known about the mechanism of hapten-induced oral tolerance. The relatively slow progress in this research is mainly due to the limited knowledge of the cells involved in hapten-induced oral tolerance. In an earlier report, DNCB was tracked in the gastrointestinal tract and the peripheral lymphoid organs by immunofluorescence microscopy after its oral administration to guinea pigs (27). However, the resolution of the

1214


Figure 4. Detection of DNP-labeled T-cells by flow cytometry analysis in the spleen of mice treated with DNCB orally. Splenic mononuclear cells were isolated from mice 6 h after treatment with DNCB (A) or vehicle (B). The cells were stained with monoclonal antibodies specific for CD3 (diluted 1-100) and DNP (diluted 1-100). The numbers in the upper-left corner indicate the percentage of CD3+DNP- cells, and the numbers in the upper-right corner indicate the percentage of CD3+DNP+ cells. The ratios of the percentage of CD3+DNP+ cells to that of total CD3+ cells were compared between vehicle- and DNCB-treated animals (panel C). The data are expressed as the mean value ( standard deviation for 9 controls (black bar) and 15 DNCB treated animals (white bar). (*) p < 0.05 relative to vehicletreated controls.

technique was relatively low and did not result in the identification of the cells that carried the DNP-protein adducts. In the present study, our immunohistochemical findings are more revealing due to the use of more modern techniques. The findings of high levels of DNP-protein adducts in villus enterocytes relative to other parts of the small intestine suggest that these cells may have a role in inducing oral tolerance to haptens. In this regard, it has been shown that enterocytes from DNCB-fed mice induced T cell anergy in vitro, possibly by inappropriate antigen presentation with low or no costimulatory signals (8). Because protein adducts of diclofenac, acetaminophen, and halothane have also been detected in the villus enterocytes in vivo, enterocytes may also be important in the downregulation of DIARS in many individuals (5). The detection of specific DNP-labeled dendritic cells and lymphocytes in the spleen (Figures 2 and 4) has important implications. It suggests that adduct formation was not likely due to the direct interaction of DNCB with cellular protein constituents of these cells as it was with enterocytes. Instead, DNP-protein adducts released from injured parenchymal cells may have been taken up by dendritic cells in the peripheral tissues before they migrated to the spleen through the blood circulation. It is also possible that DNP-protein adducts formed in the blood or released into the blood from damaged cells were taken up by dendritic cells that resided in the spleen. The detection of DNP-protein adducts in the cluster of dendritic cells (Figure 2E), which are in close proximity to numerous T cells in the white pulp of the spleen,

Ju and Pohl

supports the idea that dendritic cells are highly efficient APCs (28). It also suggests that DNP-labeled dendritic cells may have transferred the DNP-protein adducts in the form of peptide derivatives bound to major histocompatibility complex (MHC) class II molecules, to DNPspecific T cells (29, 30), which could conceivably differentiate into suppressor T cells (31, 32). In this regard, it has been shown that splenic T cells, from animals orally tolerized to protein antigens, can transfer protein tolerance to naı¨ve animals (12, 13). DNP-labeled cells in the mesenteric lymph nodes, which appeared to be lymphocytes (Figure 2A), likely acquired the antigen from dendritic cells or other APCs in the mesenteric lymph nodes or those that had migrated to the mesenteric lymph nodes from the gut associated lymphoid tissues. These DNP-labeled T cells, like those in the spleen, may be suppressor T cells (12). Evidence suggests that the liver may also have a role in oral tolerance. First of all, most of the blood draining from the intestine goes directly to the liver via the portal vein. Second, the liver is an immune privileged site in the sense that liver allografts are accepted across MHC barriers in mice and rats without immunosuppressive therapy (33, 34). Third, systemic tolerance to proteins or haptens can be induced by prior exposure via the portal vein (14, 15, 35, 36). Fourth, results from some studies have shown that a portacaval shunt can block oral tolerance to protein antigens or haptens (15, 37). In our study, DNP-protein adducts were detected in the sinusoid cells throughout the liver and some of these cells were initially thought to be Kupffer cells based on their size and morphology (Figure 3). This idea seemed reasonable because a number of studies have provided evidence for a role of Kupffer cells in tolerance induction. For example, it has been shown that depletion of Kupffer cells by gadolinium chloride prevents portal vein tolerance (38) and inhibits allograft survival induced by portal vein infusion of donor cells (16, 17, 39). Moreover, although Kupffer cells can act as APCs (40-43), they are poor stimulators of allogeneic T-cells (44) and produce immunosuppressive mediators such as IL-10 (45) and prostaglandin E2 (46). The role of Kupffer cells in oral tolerance to DNCB was studied by investigating the effect of their depletion on tolerance induction. It has been shown that liposomeincorporated clodronate can selectively deplete macrophages without affecting other cells (47). The mechanism is due to apoptosis of macrophages induced by intracellularly released clodronate after liposomes are phagocytosed (18). We showed that Kupffer cells were virtually depleted 24 h after intravenous injection of clodronatecontaining liposomes (Figure 5). However, the depletion of Kupffer cells did not abrogate oral tolerance to DNCB (Figure 6), nor did it appear to reduce the number of DNP-labeled sinusoidal cells (Figure 3, panels A and D), suggesting that other DNP-labeled cells in the sinusoids may be involved in the mechanism of DNCB-induced tolerance. These cells may include a number of different resident lymphocytes in the liver, such as natural killer (NK) T cells and γδ T cells. It has been shown that liver NK T cells are crucial for the induction of oral tolerance in an experimental model of trinitrochlorobenzeneinduced colitis (48), while γδ T cells in the liver are responsible for the prolonged skin allografts survival induced by portal vein injection of donor spleen cells (49).



Figure 5. Depletion of Kupffer cells by clodronate-containing liposome treatment. Animals were injected intravenously with either empty liposomes (panels A and C, 290×) or clodronate-containing liposomes (panels B and D, 290×). After 24 h, some animals were killed and the liver sections were stained with F4/80 antibody (diluted 1-50) for Kupffer cells (panels A and B), while other animals were killed 20 min after injection of IndiaInk for the determination of carbon particle uptake by Kupffer cells in H/E stained sections (panels C and D). Kupffer cells (labeled with arrows) were nearly eliminated by the treatment with clodronate-containing liposomes.

Figure 6. Effect of Kupffer cell depletion on DNCB-induced oral tolerance. Mice were injected intravenously with empty liposomes (A and B) or clodronate-containing liposomes (C and D). After 2 days, they were gavaged with either vehicle (A and C, black bars) or DNCB (B and D, white bars). This treatment was repeated once a week for 3 weeks. Five weeks after the last treatment, mice were sensitized with 5% DNCB and challenged 5 days later by applying 2.5% DNCB to the ear. The DTH response was measured 24 h after challenge by measuring the ear swelling. The results are expressed as the mean value ( standard deviation (n ) 10 for each group). (*) p < 0.05 relative to vehicle-treated controls.

The intense immunopositive staining in the endothelial cells of the portal veins (Figure 3) is likely due to DNCB reaching the liver mainly through the portal veins following the oral administration. The high levels of DNP-protein adducts detected in the epithelial lining of the bile duct was an unexpected finding, but may be explainable (Figure 3A). It is known that the major metabolic pathway of DNCB is to form a glutathione (GSH) conjugate (DNP-SG) (6), and DNP-SG is secreted into the bile. We found that DNP-protein binding in the

epithelial cells of the bile duct was diminished when DNCB was administered after GSH depletion by buthionine sulfoximine treatment (data not shown). These results suggest that the DNP moiety on DNP-SG may be transferred to bile epithelial proteins, possibly after it is metabolically activated by oxidation of the sulfur atom (50, 51). In summary, immunohistochemical analysis indicated the presence of DNP-protein conjugates in enterocytes of the small intestine, in macrophages and lymphocytes of the mesenteric lymph nodes, in dendritic cells and lymphocytes of the spleen, and in Kupffer cells and other sinusoidal cells of the liver. The involvement of Kupffer cells in DNCB-induced oral tolerance was eliminated because the depletion of these cells with clodronatecontaining liposomes prior to the oral administration of DNCB did not block oral tolerance. However, other DNPlabeled sinusoidal cells in the liver and those detected in the gut associated lymphoid tissues, the mesenteric lymph nodes and spleen, may be important in DNCBinduced oral tolerance. Nevertheless, Kupffer cells may have a role in immunological tolerance when protein adducts of drugs are formed in the liver. Indeed, protein adducts of halothane and acetaminophen have been detected in vivo in Kupffer cells (52, 53) where they may have a role in suppressing immune reactions against these protein adducts. In the future, the DNP-labeled cells may be purified by fluorescence activated cell sorting (FACS) (54) or cell isolation techniques with the use of antibody-labeled magnetic beads (55). Subsequently, the role of these DNP-labeled cells in tolerance can be determined by adoptive transfer experiments. This in-

1216


formation should lead to a better understanding of not only the mechanism of hapten-induced oral tolerance, but also the role of oral tolerance in preventing DIARS.

References (1) Goldstein, R.A., and Patterson, R. (1984) Summary. J. Allergy Clin. Immunol. 74, 643-644. (2) Uetrecht, J. P. (1990) Drug metabolism by leukocytes and its role in drug-induced lupus and other idiosyncratic drug reactions. CRC Crit. Rev. Toxicol. 20, 213-235. (3) Cohen, S. D., Pumford, N., Khairallah, E. A., Boehelheide, K., Pohl, L. R., Amouzadeh, H. R., and Hinson, J. A. (1997) Contemporary issues in toxicology: selective protein covalent binding and target organ toxicity. Toxicol. Appl. Pharmacol. 143, 1-12. (4) 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. (5) Ware, J. A., Graf, M. L. M., Martin, B. M., Lustberg, L. R., and Pohl, L. R. (1998) Immunochemical detection and identification of protein adducts of diclofenac in the small intestine of rats: possible role in allergic reactions. Chem. Res. Toxicol. 11, 164171. (6) Tingle, M. D., Clarke, J. B., Kitteringham, N. R., and Park, B. K. (1990) Influence of glutathione conjugation on the immunogenicity of dinitrophenyl derivatives in the rat. Int. Arch. Allergy Appl. Immunol. 91, 160-165. (7) Chase, M. W. (1946) Inhibition of experimental drug allergy by prior feeding of the sensitizing agent. Proc. Soc. Exp. Biol. Med. 61, 257. (8) Galliaerde, V., Desvignes, C., Peyron, E., and Kaiserlian, D. (1995) Oral tolerance to haptens: intestinal epithelial cells from 2,4dinitrochlorobenzene-fed mice inhibit hapten-specific T cell activation in vitro. Eur. J. Immunol. 25, 1385-1390. (9) Gautam, S. C., and Battisto, J. R. (1983) Suppression of contact sensitivity and cell-mediated lympholysis by oral administration of hapten is caused by different mechanisms. Cell. Immunol. 78, 295-304. (10) Weiner, H. L. (1997) Oral tolerance: immune mechanisms and treatment of autoimmune diseases. Immunol. Today 18, 335343. (11) Weiner, H. L. (1994) Oral tolerance. Science 91, 10762-10765. (12) Lider, O., Santos, L. M., Lee, C. S., Higgins, P. J., and Weiner, H. L. (1989) Suppression of experimental autoimmune encephalomyelitis by oral administration of myelin basic protein. II. Suppression of disease and in vitro immune responses is mediated by antigen-specific CD8+ T lymphocytes. J. Immunol. 142, 748752. (13) Yoshida, H., Hachimura, S., Hirahara, K., Hisatsune, T., Nishijima, K., Shiraishi, A., and Kaminogawa, S. (1998) Induction of oral tolerance in splenocyte-reconstituted SCID mice. Clin. Immunol. Immunopathol. 87, 282-291. (14) Battisto, J. R., and Miller, J. (1962) Immunologic unresponsiveness produced in adult guinea pigs by parenteral introduction of minute quantities of hapten or protein antigen. Proc. Soc. Exp. Biol. Med. 111, 111-115. (15) Cantor, H. M., and Dumont, A. E. (1967) Hepatic suppression of sensitization to antigen absorbed into the portal system. Nature 215, 744-745. (16) Kamei, T., Callery, M. P., and Flye, M. W. (1990) Kupffer cell blockade prevents induction of portal venous tolerance in rat cardiac allograft transplantation. J. Surg. Res. 48, 393-396. (17) Squiers, E., Salomon, D., Pickard, L., Howard, R., and Pfaff, W. (1990) Abrogation of the induction of portal venous tolerance in cardiac transplant model resulting from Kupffer cell inhibition by gadolinium. Transplantation 50, 171-173. (18) Van Rooijen, N., and Sanders, A. (1994) Liposome mediated depletion of macrophages: mechanism of action, preparation of liposomes and applications. J. Immunol. Methods 174, 83-93. (19) Witmer-Pack, M. D., Crowley, M. T., Inaba, K., and Steinman, R. M. (1993) Macrophages, but not dendritic cells, accumulate colloidal carbon following administration in situ. J. Cell Sci. 105, 965-973. (20) 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 Appl. Immunol. 116, 318-324. (21) Strobel, S., and Mowat, A. M. (1998) Immune responses to dietary antigens: Oral tolerance. Immunol. Today 19, 173-181.

Ju and Pohl (22) Chen, Y., Kuchroo, V. K., Inobe, J. I., Hafler, D. A., and Weiner, H. L. (1994) Regulatory T-cell clones induced by oral tolerance: suppression of autoimmune encephalomyelitis. Science 265, 1237-1240. (23) MacDonald, T. T. (1983) Immunosuppression caused by antigen feeding II. Suppressor T cells mask Peyer’s patch B cell priming to orally administered antigen. Eur. J. Immunol. 13, 138-142. (24) Ngan, J., and Kind, L. S. (1978) Suppressor T cells for IgE and IgG in peyer’s patches of mice made tolerant by the oral administration of ovalbumin. J. Immunol. 120, 861-865. (25) Bertolino, P. T.-B. M., and Rabourdin-Combe, C. (1998) Hepatocytes induce functional activation of naive CD8+ T lymphocytes but fail to promote survival. Eur. J. Immunol. 28, 221-236. (26) Knolle, P., Schmitt, E., Jin, S., Germann, T., Duchmann, R., Hegenbarth, S., Gerken, G., and Lohse, A. W. (1999) Induction of cytokine production in naive CD4+ T cells by antigenpresenting murine liver sinusoidal endothelial cells but failure to induce differentiation toward Th1 cells. Gastroenterology 116, 1428-1440. (27) Takei, Y., Nakagawa, S., Oka, D., and Ueki, H. (1985) The distribution of 2,4-dinitrophenyl groups in guinea pigs following feeding of 2,4-dinitrochlorobenzene. Arch. Dermatol. Res. 278, 97101. (28) Unternaehrer, J. J., Inaba, K., Turley, S., Steinman, R., and Mellman, I. (2000) Dendritic cells retain antigen presenting molecules in a clustered distribution at the plasma membrane, improving their efficiency as T cell activators. Mol. Biol. Cell 11, 598-598. (29) Arnold, P. Y., and Mannie, M. D. (1999) Vesicles bearing MHC class II molecules mediate transfer of antigen from antigenpresenting cells to CD4+ T cells. Eur. J. Immunol. 29, 13631373. (30) Patel, D. M., Arnold, P. Y., White, G. A., Nardella, J. P., and Mannie, M. D. (1999) Class II MHC/peptide complexes are released from APC and are acquired by T cell responders during specific antigen recognition. J. Immunol. 163, 5201-5210. (31) Dhodapkar, M. V., Steinman, R. M., Krasovsky, J., Munz, C., and Bhardwaj, N. (2001) Antigen-specific inhibition of effector T cell function in humans after injection of immature dendritic cells. J. Exp. Med. 193, 233-238. (32) Jonuleit, H., Schmitt, E., Schuler, G., Knop, J., and Enk, A. H. (2000) Induction of interleukin 10-producing, nonproliferating CD4+ T cells with regulatory properties by repetitive stimulation with allogeneic immature human dendritic cells. J. Exp. Med. 192, 1213-1222. (33) Qian, S., Demetris, A. J., Murase, N., Rao, A. S., Fung, J. J., and Starzl, T. E. (1994) Murine liver allograft transplantation: tolerance and donor cell chimerism. Hepatology 19, 916-924. (34) Zimmermann, F. A., Butcher, G. W., Davies, H. S., Brons, G., Kamada, N., and Turel, O. (1979) Techniques for orthotopic liver transplantation in the rat and some studies of the immunologic responses to fully allogeneic liver grafts. Transplant. Proc. 11, 571-577. (35) Ghobrial, R. R., Hamashima, T., Wang, M. E., Wang, M., Stepkowski, S. M., and Kahan, B. D. (1996) Induction of transplantation tolerance by chimeric donor/recipient class I RT1.Aa molecules. Transplantation 62, 1002-1010. (36) Triger, D. R., Cynamon, M. H., and Wright, R. (1973) Studies on the hepatic uptake of antigen, I. Comparison of inferior vena cava and portal vein routes of immunization. Immunology 25, 941946. (37) Yang, R., Liu, Q., Grosfeld, J. L., and Pescovitz, M. D. (1994) Intestinal venous drainage through the liver is a prerequisite for oral tolerance induction. J. Pediatr. Surg. 29, 1145-1148. (38) Cuison, A., Tasaka, K., Chuang, C. K., Minai, M., Yoshikawa, H., and Nakajima, Y. (1995) Schistosome eggs in the portal vein can induce tolerance. Int. J. Parasitol. 25, 993-998. (39) Callery, M., Kamei, T., and Flye, M. W. (1989) Kupffer cell blockade inhibits induction of tolerance by the portal venous route. Transplantation 47, 1092-1094. (40) Lohse, A. W., Knolle, P. A., Bilo, K., Uhrig, A., Waldmann, C., Ibe, M., Schmitt, E., Gerken, G., and Meyer Zum Buschenfelde, K. H. (1996) Antigen-presenting function and B7 expression of murine sinusoidal endothelial cells and Kupffer cells. Gastroenterology 110, 1175-1181. (41) Roland, C. R., Mangino, M. J., Duffy, B. F., and Flye, M. W. (1993) Lymphocyte suppression by Kupffer cells prevents portal venous tolerance induction: A study of macrophage function after intravenous gadolinium. Transplantation 55, 1151-1158. (42) Roland, C. R., Walp, L., Stack, R. M., and Flye, M. W. (1994) Outcome of Kupffer cell antigen presentation to a cloned murine Th1 lymphocyte depends on the inducibility of nitric oxide synthase by IFN-g. J. Immunol. 153, 5453-5464.

Study of the Mechanism of Oral Tolerance to Haptens (43) Squiers, E. C., Brunson, M. E., and Salomon, D. R. (1993) Kupffer cells can present alloantigen in vitro: An effect abrogated by gadolinium. J. Surg. Res. 55, 551-554. (44) Rubenstein, D., Roska, A. K., and Lipsky, P. E. (1986) Liver lining sinusoidal cells express class II major histocompatibility antigens but are poor stimulators of free allogeneic T lymphocytes. J. Immunol. 137, 1803-1810. (45) Knolle, P. A., Uhrig, A., Hegenbarth, S., Loser, E., Schmitt, E., Gerken, G., and Lohse, A. W. (1998) IL-10 down-regulates T cell activation by antigen-presenting liver sinusoidal endothelial cells through decreased antigen uptake via the mannose receptor and lowered surface expression of accessory molecules. Clin. Exp. Immunol. 114, 427-433. (46) Karck, U., Peters, T., and Decker, K. (1988) The release of tumor necrosis factor from endotoxin-stimulated rat Kupffer cells is regulated by prostaglandin E2 and dexamethasone. J. Hepatol. 7, 352-361. (47) Van Rooijen, N. (1989) The liposome-mediated macrophage ‘suicide’ technique. J. Immunol. Methods 124, 1-6. (48) Trop, S., Samsonov, D., Gotsman, I., Alper, R., Diment, J., and Ilan, Y. (1999) Liver-associated lymphocytes expressing NK1.1 are essential for oral immune tolerance induction in a murine model. Hepatology 29, 746-755. (49) Gorczynski, R. M. (1994) Adoptive transfer of unresponsiveness to allogeneic skin grafts with hepatic gamma delta T cells. Immunology 81, 27-35. (50) Ripp, S. L., Overby, L. H., Philpot, R. M., and Elfarra, A. A. (1997) Oxidation of cysteine S-conjugates by rabbit liver microsomes and


(51)

(52)

(53)

(54)

(55)

cDNA-expressed flavin-containing monooxygenases: studies with S-(1,2-dichlorovinyl)-L-cysteine, S-(1,2,2-trichlorovinyl)-L-cysteine, S-allyl-L-cysteine, and S-benzyl-L-cysteine. J. Pharmacol. Exp. Ther. 51, 507-515. Werner, M., Birner, G., and Dekant, W. (1996) Sulfoxidation of mercapturic acids derived from tri- and tetrachloroethene by cytochromes P450 3A: a bioactivation reaction in addition to deacetylation and cysteine conjugate β-lyase mediated cleavage. Chem. Res. Toxicol. 9, 41-49. Furst, S. M., Luedke, D., and Gandolfi, A. J. (1997) Kupffer cells from halothane-exposed guinea pigs carry trifluoroacetylated protein adducts. Toxicology 120, 119-132. Roberts, D. W., Bucci, T. J., Benson, R. W., Warbritton, A. R., McRae, T. A., Pumford, N. R., and Hinson, J. A. (1991) Immunohistochemical localization and quantification of the 3-(cysteins-yl)-acetaminophen protein adduct in acetaminophen hepatoxicity. Am. J. Phathol. 138, 359-371. Shimizu, J., Yamazaki, S., and Sakaguchi, S. (1999) Induction of tumor immunity by removing CD25+CD4+ T cells: A common basis between tumor immunity and autoimmunity. J. Immunol. 163, 5211-5218. Thornton, A. M., and Shevach, E. M. (1998) CD4+CD25+ Immunoregulatory T cells suppress polyclonal T cell activation in vitro by inhibiting interleukin 2 production. J. Exp. Med. 188, 287-296.

TX0100587

Immunohistochemical Detection of Protein Adducts of 2,4

Recommend Documents