Evaluation of the Use of Reporter Antigens in an Auricular Lymph Node Assay to Assess the Immunosensitizing Potential of Drugs

Stefan Nierkens1, Leonie Nieuwenhuijsen, Mieke Thomas and Raymond Pieters

Institute for Risk Assessment Sciences, Immunotoxicology, Utrecht University, Utrecht, Netherlands

Received October 16, 2003; accepted January 9, 2004


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Immune-mediated idiosyncratic drug reactions are a major problem for susceptible patients, physicians, and the pharmaceutical industry. Validated screening tools to assess the immunosensitizing capacity of orally or intravenously administered pharmaceuticals are currently not available. To date, the popliteal lymph node assay (PLNA) seems the most promising tool for this purpose. The PLNA has recently been extended with the use of reporter antigens (RA) that are coinjected together with the drug of interest. The measurement of isotypes of RA-specific antibody-secreting cells (ASC) enables the distinction of sensitizing chemicals and (nonsensitizing) irritants without radio-isotopic end points. However, the use of footpad injections raises ethical concerns. Therefore, we examined the use of RA after intradermal injection into the ear of BALB/c mice and measured RA-specific ASC in the draining auricular lymph node (ALN). We show that RA-specific IgG isotype ASC numbers are very useful and sensitive parameters to identify drug-induced hypersensitivity in both PLN and ALN. However, the type 1–associated parameters (CD8+ cells, macrophages, IFN-{gamma}, TNF-{alpha}, and IL-1ß) that are induced in the PLN by streptozotocin were less pronounced in the ALN. Thus, the PLNA may provide more immunologically relevant information on the mechanisms of certain chemical-induced hypersensitivity reactions. The RA-ALN assay may provide an alternative for the RA-PLNA; both assays can be used to distinguish sensitizing compounds from nonsensitizing ones.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
It is estimated that immune-mediated drug hypersensitivity reactions (IDHR) account for 6–10% of all adverse drug reactions (Adkinson et al., 2002Go) and that 5–20% of patients receiving certain drugs can suffer from some form of drug-related allergic or autoimmune-like disease (Luster et al., 1999Go). Also, drug hypersensitivity has been reported as the most frequent cause (64% of cases) of drug failure during clinical development in humans, which was not predicted from standard preclinical animal studies (Dean, 2000Go). The idiosyncratic nature and multifactorial etiology of IDHR complicate the development of predictive tests and, to date, no validated assays are available to identify a compound's ability to induce manifest hypersensitivity or autoimmune derangements in humans. However, the popliteal lymph node assay (PLNA), being a simple, straightforward animal test, has been recognized as a very useful tool. It focuses on the initiation of lymphocyte activation by low molecular weight chemicals and thus lacks the complex immunoregulatory interference with the outcome of the responses that may occur in general toxicity studies.

Originally, the PLNA was used to assess graft-versus-host reactivity (GVHR), but since Gleichmann and Gleichmann (1976)Go proposed that chemicals could alter structures of the major MHC complex in such a way that autoreactive T cells might be activated in a GVHR-like manner, the PLNA came into view as a test to screen for allergenic potential of pharmaceuticals. So far, more than 130 compounds have been tested (Gutting et al., 2002Go; Kammuller et al., 1989Go; Nierkens et al., 2002Go), and the results of these tests showed close correlation with documented immunological side effects of these compounds. However, animal welfare committees often raise objections to the use of the PLNA, because chemicals may cause more or less severe paw inflammation and subsequent hindrance in moving activity in test animals.

Therefore, we set out to search for an alternative injection site, which causes less animal stress but enables the use of local, immunologically relevant read-out parameters in a selective draining lymph node, i.e., similar to the PLNA. The present study describes an evaluation of intradermal injection of the ears of mice and the use of cells from the draining auricular lymph node (ALN) to assess immunosensitization. The idea to use the ALN is derived from the validated local lymph node assay (LLNA; reviewed in Kimber et al., 2002Go), which is used to distinguish skin sensitizers by their capacity to induce proliferation of ALN cells after topical application of compounds on the ears of mice. To achieve a model for the assessment of allergic or autoimmunogenic potency of a wide variety of (non–skin-sensitizing) drugs, which are generally administered orally or intravenously, we decided to inject the selected compounds intradermally into the ear. Using this approach, we also circumvented the fact that some drugs, such as streptozotocin (STZ), lack the physicochemical properties to effectively pass the stratum corneum of the skin (Ashby et al., 1995Go).

In their most simple form, both the PLNA and the LLNA use lymphocyte proliferation in the draining local lymph node as objective measurement. Alternatively, we have previously introduced the use of reporter antigens (RA) in the PLNA, referred to as RA-PLNA (Albers et al., 1997Go; Pieters and Albers, 1999Go). In the RA-PLNA, RA-specific antibodies are analyzed after simultaneous injection of the chemical of interest and either trinitrophenyl-ovalbumin (TNP-OVA; T-dependent RA) or TNP-Ficoll (T-independent RA). The RA are injected in such a small amount (10 µg) that no TNP-specific, antibody-secreting cells (ASC) are measured. Efficient immune responses to TNP-OVA require cognate T-B cell interactions and costimulatory adjuvant signals (Nierkens et al., 2002Go). Therefore, any reactive drug that induces inflammatory mediators, including nonsensitizing irritants or adjuvants, will induce the formation of TNP-specific ASC. On the other hand, TNP-Ficoll alone does not elicit TNP-specific ASC with Ig-isotypes other than IgM. Therefore, a TNP-specific IgG response can only occur in the presence of T cell help. Importantly, naive T cells are incapable of specifically responding to TNP-Ficoll. Hence, the formation of TNP-specific IgG ASC in responses that are induced upon coinjection of TNP-Ficoll together with a drug indicates soluble help from hapten- or neo-antigen-specific T cells. Thus, the use of these two RA in the PLNA provides evidence for chemical-induced sensitization to either the compound itself (allergy) or neo-epitopes (autoimmune hypersensitivity) and discerns sensitizing drugs from inflammatory (but nonsensitizing) compounds and innocent (noninflammatory, nonsensitizing) drugs (Albers et al., 1997Go). In this approach, the response to well-defined bystander antigens enables the use of a single, non–radio-isotopic, generalized, and specific, T cell–dependent read-out parameter to assess the immunosensitizing potential of a wide variety of drugs.

In the present study, we have investigated whether the RA approach could be used to detect immunogenic drugs when injecting the drugs and TNP-Ficoll intradermally in the pina of mice and with subsequent assessment of the read-out parameters in the ALN, referred to as the RA-ALN assay (RA-ALNA). Of note, we used the RA-PLNA as a positive control assay. Compounds that were selected as being positive are ofloxacin (OFLX), D-penicillamine (D-Pen), diphenylhydantoin (DPH), and streptozotocin (STZ), and chemicals expected to be negative are procainamide (PA), sulphamethoxazole (SMX), which are both known to require metabolism, phenobarbital (PhB), and metformin (MF). The drugs that were tested were mostly selected from a US-FDA database (Weaver et al., manuscript in preparation), and the decision to test D-Pen was based on previous experience with this compound (Nierkens et al., 2002Go). Apart from the numbers of TNP- and isotype-specific ASC, we measured cytokine production by in vitro restimulated lymph node cells, determined the relative and absolute numbers of T cells, B cells, and macrophages in the lymph nodes, and used immunohistology to confirm lymph node activation.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 


Mice.
Female, specific pathogen–free BALB/c mice (6 weeks old) were obtained from Harlan (Horst, Netherlands). They were maintained under barrier conditions in filter-topped macrolon cages with wood-chip bedding, at a mean temperature of 23 ± 2°C, 50–55% relative humidity, and 12 h light/dark cycle. Drinking water and standard laboratory food pellets were provided ad libitum. Mice were allowed to settle for a week before random assignment to specific treatment groups. The experiments were conducted according to the guidelines of the Animal Experiments Committee of the Faculty of Veterinary Medicine, Utrecht University (Utrecht, Netherlands).

Chemicals.
Chemicals were obtained from Sigma-Aldrich Co. (St. Louis, MO) unless stated otherwise. All test compounds were diluted in 0.9% saline (B. Braun, Melsungen, Germany). DPH, OFLX, and SMX were injected as suspensions due to limited solubility in saline. These samples were left in an ultrasound bath for 15 min prior to further dilution, resulting in homogenous suspensions. TNP-Ficoll was prepared as previously described (Albers et al., 1996Go).

RA-PLNA and RA-ALNA.
For the RA-PLNA, we used the standard protocol as previously described (Albers et al., 1997Go). Accordingly, mice were subcutaneously (sc) injected into the right hind footpad with a freshly prepared mixture (50 µl) of the compound of interest diluted in saline together with a predefined, subsensitizing dose (10 µg) of TNP-Ficoll. Injection was performed with a 25-gauge needle in toe-to-heel direction. Chemicals were injected in quantities that have been demonstrated to be stimulatory in the PLNA before (D-Pen and STZ; Nierkens et al., 2002Go), were equimolar to a related compound (PhB), or were obtained by performing dose-response experiments in the RA-PLNA (DPH, OFLX, SMX, MF, and PA). To gain the optimal doses in final experiments, both the effectivity (based on TNP-specific ASC as read-out parameter) and the extent of paw inflammation were considered. For instance, 3 mg DPH or 3–4 mg OFLX per mouse induced higher TNP-specific antibody production compared to lower doses but resulted in such severe inflammation of the paw that tests were not repeated. Thus, 1 mg of STZ or D-Pen, 2 mg of DPH, OFLX, SMX, MF, or PhB, or 1.5 mg of PA was injected. Control animals were injected with 10 µg TNP-Ficoll in vehicle.

Since our goal was to evaluate the capacity of the RA-ALNA to identify immunosensitizing chemicals rather than a quantitative comparison of the RA-ALNA with the RA-PLNA, we decided to use the same dose instead of the same concentration. For the RA-PLNA, we used the standard injection volume of 50 µl according to our standard protocol (Albers et al., 1997Go). So, in the RA-ALNA we used 20 µl instead of 50 µl as injection volume, resulting in higher concentrations of TNP-Ficoll and test compounds than in the RA-PLNA. In the RA-ALNA, mice were injected with the compound/TNP-Ficoll mixture diluted in saline in similar doses/kg body weight as in the RA-PLNA. The injection was performed with a 25-gauge needle in the middle and at the dorsum of the ear, resulting in a circular swelling in the middle of the auricle. Mice were briefly sedated with 20 µl ketamine (50 mg/ml)/xylazine (10 mg/ml), intramuscularly, prior to the injection of DPH, OFLX, and SMX. Although intradermal ear injections can be performed without sedation, injection of the ear is more convenient when mice are immobilized, particularly when relatively insoluble or highly viscous solutions are injected. The effect of this sedation on the measured parameters (total cell counts, TNP-specific Ig formation, and shifts in lymphocyte subtypes) was elucidated in the cases of OFLX and DPH. Because there were no differences in these parameters, we used sedation for further studies with OFLX, DPH, and SMX.

At day 7, mice were sacrificed and the ALN, located at the bifurcation of the jugular vein, was removed or the PLN was excised, as described previously (Bloksma et al., 1995Go). The lymph node was separated from adherent fatty tissue and isolated in ice-cold PBS/1% BSA in which single-cell suspensions were prepared. Cells were resuspended in 1 ml PBS/1% BSA, counted using a Coulter Counter (Coulter Electronics, Luton, UK), and adjusted to 1 x 106 cells/ml for ELISPOT and flow cytometry and to 2.5 x 106 cells/ml for ex vivo restimulation. For immunohistochemistry, lymph nodes were snap-frozen in liquid nitrogen and stored at –20°C until the preparation of sections.

ELISPOT assay.
The ELISPOT assay was performed based on the operating procedure previously described (Schielen et al., 1995Go). Immobilon-P membranes (Immobilon PVDF Transfer, Millipore, Billerica, MA) were coated overnight at 4°C with PBS-Tween (0.05%)/TNP-BSA (10 µg/ml) and blocked for 1 h at room temperature with PBS-Tween/1% BSA. Membranes were washed and clamped in (in-house-made) spot blocks, and 5 x 105 cells were centrifuged onto the membranes and incubated for 4 h at 37°C. Membranes were vigorously washed with PBS and PBS-Tween to remove cells and debris and incubated overnight at 4°C with alkalic phosphatase-conjugated goat antimouse, human-adsorbed IgG1, IgG2a, and IgM antibodies (Southern Biotechnology Associates, Inc., Birmingham, AL) diluted in PBS-Tween. Membranes were washed and incubated with fresh stock solutions of para-nitroblue tetrazolium and 5-bromo-4-chloro-3-indolylphosphate toluidine salt in dimethylformamide (BDH, Poole, England) diluted in Tris (100 mM Trizma base, 100 mM NaCl, 5 mM MgCl2 · 6H2O, pH 9.5) to visualize development of TNP-specific antibody spots. Spots were counted by two independent observers using a stereomicroscope.

Flow cytometry.
Cell subtypes were analyzed using flow cytometry. Cells (1 x 105) were centrifuged in 96-well plates and resuspended in 50 µl PBS/0.05% BSA/0.1% NaN3/3% normal mouse serum with FcBlock (BD Pharmingen, Hamburg, Germany) (CD16/CD32, Fc{gamma}III/II receptor, 2.4G2) for 30 min in darkness at 4°C. Cells were washed and incubated with fluorescein isothiocyanate–(FITC), phycoerythrin- (PE), and cy-chrome–(CY) conjugated monoclonal antibodies (30 min in darkness, 4°C). Cells were characterized based on the following monoclonal antibodies: CD3{epsilon} CY (145-2C11), CD4 FITC (RM4-5), CD8a PE (53-6.7), CD19 PE (1D3), CD11c FITC (HL3), streptavidin CY, rat-antimouse MHC-II biotin (NIMR-4; BD Pharmingen, Hamburg, Germany), and F4/80 PE (Caltag, Burlingame, CA). Samples incubated with biotin-conjugated monoclonal antibodies were washed and incubated with streptavidin-CY (BD Pharmingen). Cells were stored in formalin (0.1%) and analyzed within 18 h on a FACScan with standard FACSflow using CELLQuest software (Becton Dickinson, Franklin Lakes, NJ).

Immunohistology.
Cryostat sections (6 µm) were fixed in acetone and incubated with predetermined dilutions of naphthol AS-BI phosphate in acetate/1% dimethylformamide and red-violet LB salt for 1 h at 37°C to visualize macrophages.

Cell culture and cytokine measurement.
Cell suspensions (3.75 x 106 cells) in complete RPMI 1640 with Glutamax-I (Invitrogen, Life Technologies, Paisley, Scotland) supplemented with 10% FBS (ICN Pharmaceuticals, Costa Mesa, CA) and 2% penicillin-streptomycin (Invitrogen) were incubated with medium, Con A (5 µg/ml), or LPS (2 µg/ml) in 96-well plates (Highbond 3590; Costar, Cambridge, MA) overnight at 37°C and 5% CO2. Supernatant was collected and stored at –20°C until analysis. IFN-{gamma}, IL-1ß, and IL-4 were determined by sandwich ELISA. IL-4 and IFN-{gamma} capture and detecting antibodies were obtained from BD Pharmingen and IL-1ß from R&D Systems (Abingdon, UK). Plates were coated overnight at 4°C with 1 µg/ml rat-antimouse IFN-{gamma}, 2 µg/ml rat-antimouse IL-1ß, or 1 µg/ml rat-antimouse IL-4 in 0.05 M carbonate buffer; pH 9.6, washed with PBS-Tween, and blocked with PBS-Tween/casein for 4 h at room temperature. Samples and IL-4, IL-1ß, and IFN-{gamma} standards were added in several dilutions and incubated overnight at 4°C. After washing, plates were incubated with 0.25 µg/ml rat-antimouse IFN-{gamma},IL-1ß, or IL-4 conjugate diluted in PBS-Tween/casein for 1 h at room temperature. Plates were incubated with streptavidine-conjugated horseradish peroxidase (0.3 µg/ml; Sanquin, Amsterdam, Netherlands) diluted in PBS-Tween/casein for 45 min at room temperature. After the final washes, 3,3',5,5'-tetramethylbenzidine-substrate (0.1 mg/ml) was added and the color reaction was stopped after 10 min with 2 M H2SO4. Absorbance was measured at 450 nm using an ELISA reader ELX800 (BIO-TEK Instruments Inc., Winooski, VT). TNF-{alpha}ELISA (BioSource, Camarillo, CA) was performed in accordance with the manufacturer's instructions. Lowest detection levels were 1.0 pg/ml for IL-4 and IL-1ß and 4.0–10.0 pg/ml for IFN-{gamma} and TNF-{alpha}.

Statistics.
Statistical analyses were performed using independent-samples t-test procedure, and p < 0.05 was considered statistically significant.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

Determination of Cell Count
Figure 1 shows the total cell numbers of the PLN (Fig. 1A) or ALN (Fig. 1B) after sc injection of the compound of interest together with a low dose of TNP-Ficoll. Levels of control animals that were treated with TNP-Ficoll alone were not different from levels in untreated mice or mice that were injected with saline alone (not shown). In the RA-PLNA, total lymph node cell numbers increased in mice upon treatment with STZ, DPH, D-Pen, OFLX, and MF, whereas cellularity was similar to controls with PA, PhB, and SMX. In the RA-ALNA, all chemicals, including presumed negative ones, significantly increased cellularity compared to controls.



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FIG. 1. Total cell numbers present in the draining lymph nodes. Mice were injected with TNP-Ficoll alone (Control) or together with the indicated compound in the footpad or in the ear. At day 7, draining lymph nodes (PLN for footpad injections and ALN for intradermal ear injections) were isolated and cells were counted using a Coulter Counter. Bars represent group means ± SEM of 4–6 mice per group. *Significant differences (p < 0.05) refer to the corresponding control.

 
Exposures to D-Pen, OFLX, and DPH were associated with the induction of inflammation in footpad or ear after 5–7 days. Ears or paws of mice that were treated with STZ, MF, and PhB were not different from control mice. SMX induced slight reddening of the skin in both the paw and the ear.

TNP-Specific ASC Numbers
In control animals in the RA-ALNA, IgM and IgG1 ASC were around 25 and 10 ASC/106 cells, respectively. In the RA-PLNA, these control levels were 5 and 1 ASC/106 cells, respectively.

The IgM and IgG1 (or IgG2a in the case of STZ) ASC numbers were significantly increased in the RA-PLNA as well as in the RA-ALNA for OFLX, D-Pen, DPH, and STZ, whereas ASC numbers in mice treated with PA, PhB, SMX, and MF were all similar to controls (Fig. 2).



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FIG. 2. TNP-specific IgM and IgG ASC in the draining lymph nodes. Mice were injected with TNP-Ficoll alone (Control) or together with the indicated compound in the footpad or in the ear. At day 7, draining lymph nodes (PLN for footpad injections [black bars] and ALN for intradermal ear injections [white bars]) were isolated and TNP-specific IgM and IgG1 (or IgG2a for STZ) were measured using the ELISPOT assay. Bars represent mean TNP-specific ASC levels ± SEM of 4–6 mice per group and are expressed as ASC numbers per 1 x 106 cells. *Significant differences (p < 0.05) refer to the corresponding control.

 
The Effects on Proportions of Cell Subtypes
Previously, the use of the B cell marker B220 was found useful in the differentiation between allergen and irritant responses in the LLNA (Gerberick et al., 2002Go; Manetz and Meade, 1999Go). In the present study, we evaluated the changes in percentages of T cell subpopulations, B cells, and macrophages in the PLN and ALN in response to different chemicals. Our results showed an increase in B cell percentages for the strong sensitizers DPH and D-Pen and a decrease for STZ in the RA-PLNA (Table 1). This effect was accompanied by a decrease in Th cell percentages for D-Pen and DPH, whereas STZ increased CD8+ cell and macrophage proportions and decreased Th cell percentages.


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TABLE 1 Cell Substypes in the PLN or ALN7 Days After Exposure to the Respective Compound Together with TNP-Ficoll

 
In the RA-ALNA, D-Pen was the only compound that caused a significant increase in B cell percentages and a subsequent decrease in Th cell proportions. The increased proportions of CD8+ cells and macrophages that were visible in the PLN after STZ exposure were not observed in the ALN. These flow cytometric results were confirmed by immunohistology, which showed the presence and absence of macrophages in the PLN and ALN, respectively (Fig. 3).



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FIG. 3. Immunohistochemistry. Naphtol AS-BI phosphate-stained cryostat sections from ALN or PLN. Lymph nodes were dissected 7 days after the initial injection with saline or STZ. Mice were injected intradermally in the pina (ALN) or in the hind foodpad (PLN); (A) PLN control, (B) PLN STZ, (C) ALN control, and (D) ALN STZ. Macrophages are clearly present in the PLN but not in the ALN in STZ-treated mice.

 
Cytokine Measurements
The cytokines IFN-{gamma} and IL-4 are generally used to broadly identify immune responses as being type 1 or type 2, respectively, and the balance between IFN-{gamma} and IL-4 is generally considered to be indicative of type 1/type 2 differentiation. We measured these cytokines in cultures of Con A–restimulated lymph node cells isolated from D-Pen–or STZ-treated mice. The choice for these two chemicals is based on the knowledge that they may serve as typical type 2 or type 1 skewing compounds (Nierkens et al., 2002Go). Because STZ is an effective macrophage-activating chemical in the PLNA, we also decided to measure the proinflammatory cytokines TNF-{alpha} and IL1-ß in LPS-restimulated cultures of lymph node cells.

Remarkably, production of IFN-{gamma} and IL-4 in controls (TNP-Ficoll in vehicle) was much higher in ALN cultures than in PLN cultures but was not significantly different from mice that were injected with saline alone. STZ significantly increased the levels of IL-4 and induced high levels of IFN-{gamma} in both ALN and PLN cultures. However, the increase of IFN-{gamma} levels was more pronounced in PLN cultures than in ALN cultures (Fig. 4). D-Pen, on the other hand, significantly increased levels of IL-4 and IFN-{gamma} in PLN cultures only, but the levels of IFN-{gamma} were much lower than those observed with STZ (note the log-scale for IFN-{gamma} in Fig. 4). Together, the Th1-Th2 balance is shifted to Th1 for STZ and more to Th2 for D-Pen.



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FIG. 4. Cytokine measurements after in vitro restimulation of draining lymph node cells of mice exposed to D-Pen or STZ. Mice were injected with TNP-Ficoll alone (Control) or together with D-Pen as a reference of type 2 immune responses or STZ as a reference of type 1 immune responses. At day 7, draining lymph nodes (PLN for footpad injections [black bars] and ALN for intradermal ear injections [white bars]) were isolated and restimulated with Con A (IL-4 and IFN-{gamma}) or LPS (TNF-{alpha} or IL-1ß) for 24 h. Bars represent mean cytokine levels ± SEM of 4–6 mice per group. *Significant differences (p < 0.05) refer to the corresponding control.

 
STZ induced TNF-{alpha} and IL-1ß production exclusively in the PLN and not in the ALN cultures. Interestingly, this difference concurs with the absence of an increase in macrophages in the ALN in STZ-induced responses.


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The PLNA has been recognized as a promising tool for its potency to distinguish allergenic pharmaceuticals, which are normally taken orally or intravenously. We have previously introduced the use of RA in the PLNA to enable discrimination between immunosensitizing chemicals and mere irritants or innocent compounds (Pieters and Albers, 1999Go) and provide more insight into the mechanisms of immunosensitization (Nierkens et al., 2002Go). However, the PLNA may give cause for ethical concerns because footpad injections with certain irritant compounds might cause paw inflammation and animal stress. Therefore, we decided to explore the use of the RA TNP-Ficoll in a model with intradermal injection of the pina and determination of relevant immunological parameters in the draining ALN.

Our data show that, with the use of TNP-specific ASC numbers in both the RA-PLNA and RA-ALNA, we could clearly identify compounds that are known to provoke hypersensitivity reactions in mice (STZ, diabetogen in mice [Rossini et al., 1977Go]) or the human population (DPH [Alarcón-Segovia and Alarcón-Riquelme, 1989Go], D-Pen [Emery and Panayi, 1989Go], and OFLX [Melde, 2001Go]). The compounds that are not known for immune-mediated adverse effects in humans (MF) or require metabolism of the original compound (SMX and PA; Pirmohamed et al., 2002Go; Uetrecht, 1990Go) are tested negative. Thus, based on these results, intradermal pina injections seem to be an effective alternative for footpad injections with less animal stress. The use of RA was successful in both the PLNA and ALNA and might provide a test system for the identification of immunosensitizing drugs.

Apart from the antibody response to TNP-Ficoll, we have also determined changes in cell composition for all chemicals and cytokine production for two chemicals. Evidently, in both the RA-PLNA and RA-ALNA, specific antibody formation is a more robust immunological parameter for detecting the immunosensitizing potential of chemicals than changes in cell numbers and is functionally more relevant than changes in B cell numbers or cytokine production. Linked to the robustness, control B cells are not secreting specific antibodies in the PLN, resulting in low background levels and high sensitivity.

Additionally, as shown with exposure to STZ, the use of RA and specific staining for IgG1 or IgG2a provides information about the type of immune response that is elicited by the drug. Taken together, detection of RA-specific ASC clearly improves sensitivity and possibly selectivity of both the PLNA and the ALNA.

Phenobarbital has been reported to have the potential to elicit adverse reactions in some humans (Hashizume et al., 2002Go), but the drug was tested negative in our assays. The mechanisms of PhB-induced hypersensitivity are not yet clear. One possibility might be that PhB has to be metabolized before it can be immunologically active, similar to PA and SMX. The lack of metabolic activity in the paw is a well-recognized drawback of the PLNA but apparently also applies to the ALNA. However, skin tissue might be capable of drug metabolism as indicated in studies with eugenol and isoeugenol or SMX (reviewed in Baron and Merk, 2001Go). This indicates that the intradermal exposure route might bypass metabolically competent cells, such as keratinocytes. In those cases where hypersensitivity reactions are dependent on metabolism of the parent drug, it can be considered to inject the reactive intermediates instead of the original compound. Alternatively, the compound of interest can be preincubated with phagocytes (Kubicka-Muranyi et al., 1993Go; Wulferink et al., 2001Go) or injected together with metabolizing systems, such as a S9 mix (Katsutani and Shionoya, 1992Go).

A marked difference between the RA-PLNA and RA-ALNA became visible with STZ. As previously documented and in line with its diabetogenic effect, STZ induced characteristic type 1 phenomena in the RA-PLNA in the present study. Apart from IgG2a production, these phenomena included an increase in the numbers of CD8+ cells and macrophages and enhanced production of cytokines like IFN-{gamma}, IL-1ß, and TNF-{alpha}. The typical type 1 effects were less pronounced or absent in the ALNA. Most remarkably, macrophages were less attracted to the draining lymph node when STZ was injected in the ear region compared to injection in the footpad. The reason for this difference between the PLNA and ALNA is not known, but tissue-specific cell populations and processes may differently influence the local immune response and the type of effector responses (Matzinger, 2002Go). For instance, IL-4, which has been shown to inhibit the migration of Langerhans cells from the ear to the draining lymph node (Takayama et al., 1999Go), might be involved. Importantly, results with STZ from the two assays together indicate a clear, site-specific difference in immune response to an identical compound. Intriguingly, responses in RA-PLNA appear more in line with the effects that are expected for STZ in animal models for diabetes (Lukic et al., 1998Go), and, hence, the RA-PLNA seems more suitable for studying the initiating mechanisms of STZ-induced diabetes.

Although both the RA-PLNA and the RA-ALNA can be used to distinguish sensitizers from nonsensitizers, the RA-ALNA might be less convenient for the injection of relatively insoluble compounds or highly viscous solutions. Injection of compounds in the footpad is fairly simple and is performed without sedation of the animal in any circumstance, whereas injection of suspensions or viscous exposure samples is more convenient when animals are slightly sedated before intradermal injection. Moreover, the injection volume used in the RA-PLNA is larger than in the RA-ALNA, which enables higher exposure amounts and less difficulty with relatively insoluble compounds or viscous solutions. Using organic solvents, such as dimethyl sulphoxide (DMSO), might solve the lack of aqueous solubility. However, 10 µl DMSO was shown to cause moderate PLN enlargement upon injection into the footpad (De Bakker et al., 1990Go). Therefore, we decided to inject all chemicals using one and the same vehicle and to prepare homogenous suspensions using an ultrasound bath. Our data show that injection of the poorly soluble compounds OFLX and DPH resulted in positive responses, whereas SMX, also poorly soluble, tested negative in both assays. So, limited aqueous solubility seems not necessarily related to the induction of inflammation. For instance, D-Pen (soluble in saline) resulted in paw and ear inflammation, whereas SMX only caused a minor reddening of the skin. Therefore, these assays are able to discriminate immunosensitizing and nonsensitizing compounds even when they are injected as suspensions. Also, the use of one vehicle minimizes the use of additional control groups.

In conclusion, we show that both the RA-PLNA and RA-ALNA can distinguish sensitizers from nonsensitizers or prohaptenic drugs provided that a functional immunological parameter, such as RA-specific ASC, is used for detection. As such, intradermal injections might provide an alternative to footpad injections to diminish animal stress. However, for STZ, results of the RA-PLNA seem more reflective of the immune mechanisms of STZ-induced autoimmunity, and, therefore, the RA-PLNA may be more useful when investigating the mechanisms of drug-induced sensitization. Overall, we found that the RA approach may be incorporated in the search for a first screen to determine the immunosensitizing capacity of orally or intravenously taken drugs.


    ACKNOWLEDGMENTS
 
We thank Professor W. Seinen (Institute for Risk Assessment Sciences, Utrecht University) for critical reading of the manuscript.


    NOTES
 

1 To whom correspondence should be addressed at IRAS-IT, Utrecht University, P.O. Box 80176, 3508 TD Utrecht, Netherlands. Fax: +31 30 2535077. E-mail: s.nierkens{at}iras.uu.nl


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
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