Essential role of MHC II-independent CD4+ T cells, IL-4 and STAT6 in contact hypersensitivity induced by fluorescein isothiocyanate in the mouse

Keisuke Takeshita, Tsugiko Yamasaki, Shizuo Akira, Florian Gantner and Kevin B. Bacon

Bayer Yakuhin, Ltd., Research Center Kyoto, Department of Respiratory Diseases Research, Japan

Correspondence to: K. B. Bacon; E-mail: kevin.bacon.kb{at}bayer.co.jp
Transmitting editor: K. Inaba


    Abstract
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Contact hypersensitivity (CHS) induced by a hapten is thought to be mediated by T helper type 1 (Th1) cells. However, FITC can induce contact allergy in vivo, and in vitro studies suggest that this response is Th2-type driven. We compared CHS reactions induced by FITC or dinitrofluorobenzene (DNFB), a well-known Th1 inducing hapten, in Balb/c mice, C57/B6 mice, and several gene knock-out mice, and investigated the role of Th1/Th2 cytokines, T cell populations, eosinophils, and mast cells. Balb/c mice (Th2 dominant strain) had a stronger response to FITC than C57/B6 mice (Th1 dominant strain). The skin inflammation was characterized by edema and eosinophilia, and serum IgE levels were elevated following FITC challenge. All responses were enhanced by a second round of sensitization. Anti-TNF-{alpha} or anti-very late antigen-4 (VLA-4) antibody partly inhibited both FITC- and DNFB-induced CHS. Pretreatment of mice with anti-IL-4 antibody, anti-IL-5 antibody, recombinant INF-{gamma}, or the mast-cell depleting agent 48/80 significantly diminished edema formation, and Stat6–/– mice were fully protected from FITC-induced CHS, while DNFB-induced CHS was enhanced (Stat6–/–, mast cell depletion) or not affected (anti-IL-5 antibody). Further, mice lacking CD4+ T cells and mice lacking both CD8 and MHC II showed very little reaction at all to FITC, while the absence of CD8 T cells alone or MHC II alone conferred partial protection only. These findings indicate a contribution of MHC II-independent CD4+ T cells and/or CD4+ NKT cells to the Th2 response triggered by FITC in vivo, and makes FITC-induced CHS a suitable animal model for atopic dermatitis.

Keywords: allergy, eosinophils, mast cells, skin, Th1/2 cells


    Introduction
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Murine CD4 T helper clones have been categorized into two subsets based on the differences in their profiles of cytokine production. The Th1 subset secretes INF-{gamma} and IL-2, while Th2 cells produce IL-4, IL-5, IL-6, IL-10 and IL-13. IL-3, GM-CSF and TNF-{alpha} are produced by both subsets (1). Contact hypersensitivity (CHS) is an inflammatory, T cell-mediated skin reaction to a hapten such as dinitrofluorobenzene (DNFB) which is thought to be associated with the activation of type 1 helper T cells. During sensitization, Langerhans cells (LC), the professional APC in the skin, can process the hapten–carrier protein complex, present it in context with their surface-expressed MHC II molecules and migrate to the skin-draining lymph nodes. In these secondary lymphoid organs DNFB-specific T cells are primed to the Ag, and become activated and polarized toward type 1 helper T cells (afferent phase). Upon challenging the skin with the same hapten, the hapten–protein complex is presented by LC and/or other APC to recruit DNFB-specific T cells, which in turn produce the type 1 cytokines INF-{gamma} and IL-2 between 12–24 h after challenge (efferent phase). The pattern of cell infiltration observed in this murine CHS response has been well characterized: 24 h after challenge the edema is severe and the cell infiltration is moderate. The ratio of mononuclear cells to polymorphonuclear cells (PMN) is similar. At 48 h and 72 h after challenge, however, a predominance of mononuclear cells over PMNs becomes obvious. Additional studies have shown that the majority of these lymphocytes are of the helper/inducer phenotype, although some killer/suppressor cells are also present (24). These data support the concept that Th1 cells are important in the CHS response. Since the importance of Th2-derived cytokines has also been illustrated in other studies (5), the specificity of the mechanism is still open to question. IL-4 inhibited the efferent phase of CHS, but not the afferent phase, by inhibiting TNF-{alpha}-induced migration of LC (6,7). Contrary to these reports, it has been demonstrated that the CHS response is diminished in IL-4-deficient mice at a late stage of the efferent phase (8). No alteration in the CHS response in IL-4-deficient mice however was reported for another hapten, 4-ethoxyl methlene-2-oxazolin-5-one [Oxa (9)]. Recently, it was shown that topical exposure of mice to FITC results in the selective development of activated lymph node cells (LNC) expressing a preferential type 2 cytokine-secretion profile with high levels of IL-4 and IL-10, but low levels of IFN-{gamma}, when restimulated by FITC in vitro (10). Negative selection by complement depletion identified CD4+ Th 2-type cells as the primary source of IL-4 and IL-10 among activated LNC, whereas the low IFN-{gamma} levels were exclusively derived from CD8+ T cytotoxic (Tc) 1-type cells (11). These data indicate that the skin lesions provoked in mice by FITC are primarily a result of the activation of Th2-type cells. However, direct evidence of Th2 cells being crucial for FITC-induced CHS responses in vivo is still lacking. In the present study, we characterized the features of FITC-induced CHS reactions in mice. Our findings indicate that FITC but not DNFB can induce a Th2-type response in vivo which depends on CD4+ cells, and that the ensuing tissue pathology reflects that observed in human atopic dermatitis.


    Methods
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Chemicals and reagents
DNFB (98% pure) and FITC were obtained from Sigma Chemical Co. (St Louis, MO). DNFB was dissolved 4:1 in acetone:olive oil (AOO), and FITC was prepared as a 1:1 solution of dibutylphthalate:acetone (DBP). Hapten solutions were always prepared freshly prior to dosing.

Monoclonal antibody against IL-4 (11B11, rat IgG1), IL-5 (TRFK5, rat IgG1), TNF-{alpha} (MP6-XT-3, rat IgG1) and control Ig were obtained from PharMingen (San Diego, CA). Monoclonal antibody against very late antigen-4 (VLA-4) (PS/2, rat IgG2b) was obtained from Southern Biotechnology Associate, Inc. (San Diego, CA). Recombinant INF-{gamma} was purchased from Pepro Tech, Inc. (Rocky Hill, NJ). Compound 48/80 (mast cell depleting drug) was obtained from Sigma Chemical Co.

Mice
Female Balb/c or C57/B6 mice were obtained from JCR (Charles River, Kanagawa, Japan), maintained under specific pathogen-free conditions, and used at 7–8 weeks of age. CD4 knockout (k.o.) (CD4–/–), MHC II I-A k.o. (MHC II–/–), CD8 T cell lacking ß2-microglobulin (ß2m) k.o. (CD8–/–), and CD8–/–/MHC II–/– double k.o. mice on a C57/B6 genetic background were purchased from IBL (Immuno-Biological Laboratories, Gunma, Japan). Signal transducer and activator of transcription 6 (STAT6) k.o. mice (Stat6–/–, C57/B6 background) were generated in the laboratory of Professor S. Akira [Osaka University (12)]. Animals were kept under standard conditions in a 12 h day/night rhythm with free access to food and water ad libitum. All animals received humane care and the studies have been approved by the internal ethics committee in accordance with the guidelines recommended by JALAS (Japanese Association of Laboratory Animal Science).

Protocol A for sensitization and challenge (1 week model)
Four hundred microliters of 0.5% FITC dissolved in DBP (1:1) or 30 µl of 0.5% DNFB dissolved in AOO (4:1) was painted onto the shaved abdominal skin on day 0 and 1, respectively. Six days later (day 6), mice were challenged by applying 20 µl of 0.5% FITC or 20 µl of 0.3% DNFB solution onto both sides of the right ear.

Protocol B for sensitization and challenge (3 week model)
Four hundred microliters of 0.5% FITC dissolved in DBP (1:1) or 30 µl of 0.5% DNFB dissolved in AOO (4:1) was painted onto the shaved abdominal skin on day 0, 1, 14 and 15. On day 20, mice were challenged by applying 20 µl of 0.5% FITC or 20 µl of 0.3% DNFB onto both sides of the right ear.

Measurement of the ear thickness
In all models, the corresponding volume of vehicle was applied to the left ear as control. Ear thickness was measured at 24, 48, or 72 h after FITC or DNFB challenge using a calibrated thickness gauge (Mitsutoyo, Tokyo, Japan) under anesthetization with ether. Ear edema was expressed as (R – L) – (R0 – L0), where R0 and L0 represent the thickness of the right and left ear, respectively, at the beginning of the experiment (0 h), and R and L stand for the thickness values obtained at the respective time points. Signficant differences between experimental groups were analyzed using Student’s t-test.

Measurement of serum concentrations of IgE
Blood was drawn from the sinus cavernous, and serum was obtained by centrifugation at 1500 g for 10 min. Immunoglobulin E levels were determined by a commercially available ELISA kit (Yamasa, Japan) according to the manufacturer’s instructions.

Measurement of EPO activity
Skin specimens for measurement of eosinophil peroxidase (EPO) activity were homogenized in 1 ml of ice-cold buffer (0.05 M Tris–HCl pH 8.0 containing 0.1% Triton X-100) using a tissue homogenizer. The homogenized samples were centrifuged at 1500 g for 20 min at 4°C and the supernatants were subjected to measurement of EPO activity. Substrate solution (100 µl) consisting of 10 mM O-phenylenediamine in 0.05 M Tris–HCl (pH 8.0) and 4 mM hydrogen peroxide was added to 20-fold diluted supernatant samples in homogenization buffer (100 µl) in 96-well microplate. The reaction mixture was incubated at room temperature for 60 min before the reaction was stopped by the addition of 100 µl of 2 M sulfuric acid. The absorbance at 490 nm was measured by a microplate reader.

Histological examination
The ear skin specimens were excised and fixed in 10% formalin, then processed and stained with hematoxylin and eosin. The sections were examined at a magnification of 10 or 40x. At least 10 fields were examined for each lobe.

Immunopharmacological studies
For antibody treatment, mice received an intravenous injection of 500 µg per mouse of anti-IL-5 (13), anti-TNF-{alpha} (14), anti-VLA-4 antibody (15) or the corresponding control Ig in a total volume of 0.5 ml 30 min prior to challenge. Anti-IL-4 antibody (500 µg per mouse) or the corresponding control antibody was injected subcutaneously (s.c.) 30 min prior to sensitization (16). Recombinant INF-{gamma} (30 µg/mouse) was administered s.c. at day 0 and 1 after sensitization (17), compound 48/80 (mast cell depleting drug) was injected intradermally into the ear at the site of challenge 4 days before challenge at a dose of 200 µg/mouse (18).

Statistics
If not otherwise stated, data are expressed as mean values ± SEM. Statistical differences of data sets were analyzed using one-way ANOVA and differences between groups were assessed by Dunnett’s method or, if applicable, by Student’s t-test using commercially available statistics software (GraphPad Software, Inc., San Diego, CA). P-values <0.05 were considered statistically significant. Details are given in the figure legends.


    Results
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Induction of CHS by FITC or DNFB in Balb/C and C57/B6 mice
We investigated whether a Th1 dominant strain, C57/B6, would also respond to FITC. FITC has been reported to induce a CHS response in Balb/c mice (10,11,19). A single topical application of FITC to the ears induced a CHS response as assessed by an increase of ear thickness in both strains. However, 24 h after FITC challenge (protocol A) the response was more pronounced and showed a peak in Balb/c mice (0.276 ± 0.0255 mm compared to 0.147 ± 0.008 mm in C57/B6; Figure 1A) before dropping to the level of the reaction seen in C57/B6. In general, the response to FITC 24 h after challenge was stronger in both strains when CHS was induced over 3 weeks using a second round of sensitization (protocol B; Balb/c: 0.41 ± 0.0135 mm; C57/B6: 0.162 ± 0.0454 mm; n = 5; Figure 1B).



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Fig. 1. Comparison of the CHS response induced by FITC or DNFB in Balb/c and C57/B6 mice. Balb/c (open circles) or C57/B6 mice (filled circles) were sensitized by FITC (A and B) or DNFB (C and D) according to protocol A (1 week model; A and C) or protocol B (3 week model; B and D) (see Methods for details). Mice were challenged by the application of FITC or DNFB solution onto both sides of the right ear. Measurements of ear thickness were performed using a micrometer at 24, 48 and 72 h after challenge and CHS is expressed as the increase in ear thickness. Data represent mean values ± SEM of five animals at each given time point.

 
In contrast to the different sensitivity of Th1- and Th2-dominant strains to FITC, a very similar response pattern was observed over 72 h after challenge when CHS was induced by DNFB. Moreover, no significant enhancement was seen in the 3 week model after a second round of sensitization (protocol A, Figure 1C; protocol B, Figure 1D).

Elevation of serum IgE in BALB/c mice sensitized and challenged with FITC
We next investigated whether FITC-sensitized mice would develop a typical Th2 response in vivo. As IgE is thought to be a reliable marker of Th2 activity, we measured total serum IgE levels 24 h after FITC challenge. FITC-sensitized Balb/c mice produced slightly higher amounts of serum IgE (138.3 ng/ml) compared to non-sensitized mice (78.68 ng/ml) in the 1 week model (protocol A) as shown in Figure 2A. In line with the aggravated CHS response, however, total IgE production was significantly enhanced 24 h after challenge in the 3 week model (protocol B) as shown in Figure 2B.



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Fig. 2. Serum IgE levels and skin EPO activity in Balb/c mice challenged by FITC. Balb/c mice received vehicle (open bars, non-sensitized controls) or were sensitized by FITC (solid bars) according to protocol A (A, 1 week model) or protocol B (B, 3 week model). All mice were challenged by the application of FITC solution to both sides of the right ear. Twenty-four hours after challenge, mice were euthanized, serum and ear samples collected, and total IgE levels and EPO activity determined. Each column indicates the mean ± SEM of five animals. Note differences in the scale of y-axes. Statistical differences were analyzed using Student’s t-test (*P < 0.05, **P < 0.01, ***P < 0.001).

 
Increase in EPO activity in challenged ear of FITC-sensitized Balb/c mice
Since eosinophils are typically present in Th2-mediated allergic diseases, we next measured EPO activity as a specific index of eosinophil infiltration into the challenged skin. EPO activity in ear samples taken from FITC-sensitized and -challenged mice was significantly increased compared to non-sensitized, challenged animals and again, a second boost of treatment resulted in further enhanced EPO activity (Figure 2A and B, respectively). These data clearly indicate that eosinophils migrate into the skin of FITC-sensitized mice upon challenge.

Histopathology of FITC-induced CHS
To further corroborate our biochemical findings, we performed histopathological analyses on skin specimens from FITC-challenged mouse ears over a time period of 72 h after challenge. Sensitized, vehicle-challenged controls did not significantly differ from non-sensitized, non-challenged controls. No significant vascular enlargement, dermal edema or cell infiltrates were detectable (Figure 3A). One hour after FITC challenge, however, mast cell degranulation was observed (Figure 3B), and significant thickening of dermis and epidermis became obvious after 8 h, paralleled by eosinophil-containing cell infiltrates (Figure 3C). Further, eosinophils were found in the areas of epidermal intercellular edema between 8 (not shown) and 24 h after FITC challenge (Figure 3D and E). All responses reached a maximum at 24 h followed by a decrease after 48–72 h in the 1 week model (protocol A, Figure 3F) but remained high until the end of the experiment in the 3 week model (protocol B, Figure 3G and data not shown). All histological changes were observed in both Balb/c (Figure 3) and C57/B6 (not shown) strains.



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Fig. 3. Histopathological findings in CHS skin lesions induced by FITC. Balb/c mice were left untreated or sensitized and challenged with FITC according to protocol A and B, respectively. Skin specimens were taken at day 0 (normal, untreated controls; A), or FITC challenge (B: 1 h; C: 8 h; D, E: 24 h; F: 72 h; all protocol A). Histological changes in the 3 week model (protocol B) 24 h after FITC challenge is exemplified in G. Arrows indicate examples of a mast cell (B). Magnifications: A, C, F, G: 10x; B, D, E: 40x.

 
These findings confirm the induction of an experimental hypersensitivity reaction by FITC which causes lesions, in some cases pustular, containing activated mast cells and eosinophilic infiltrates as hallmark features.

Effect of anti-TNF-{alpha} or anti-VLA-4 mAb on the CHS response to FITC or DNFB
TNF-{alpha} is a proinflammatory cytokine involved in LC migration and it has been reported that 2 h following FITC painting, mRNA for TNF-{alpha} was up-regulated in normal mice (20). Further, the migration of epidermal LC induced by hapten or LPS is known to be sensitive to anti-TNF-{alpha} antibody treatment (21). We thus set out to examine the role of TNF-{alpha} for the establishment of CHS induced by FITC or DNFB. Indeed, anti-TNF-{alpha} antibody-pretreated mice showed significantly reduced ear swelling upon challenge in both models at any time point investigated in the 1 week model (protocol A; Figure 4A and B), and similar results were obtained in the 3 week model (not shown).



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Fig. 4. Effect of TNF-{alpha} or VLA-4 neutralization on the CHS response induced by FITC or DNFB in Balb/c mice. Balb/c mice were sensitized for FITC (A and C) or DNFB (B and D) according to protocol A. Anti-mouse TNF-{alpha}, anti-mouse VLA-4 or control antibody were injected s.c. at a dose of 500 µg/mouse 1 h before challenge by FITC or DNFB, respectively. Control animals received the same volume of PBS. All experiments were performed according to protocol A (1 week model). Measurements of ear thickness were performed using a micrometer at 24, 48 and 72 h after challenge as described in the Methods. All data represent mean values ± SEM of n = 5 animals at each given time point. Statistical differences were analyzed using Student’s t-test (*P < 0.05, **P < 0.01, ***P < 0.001).

 
VLA-4 is expressed on T cells, monocytes and eosinophils, and regulates the adhesion cascade of leukocytes including tethering, rolling and firm arrest. VLA-4+ cells are increased and correlate with the eosinophils and disease severity in Th2 diseases such an asthma (22). Since in our FITC-induced CHS model T cell-dependency was shown and eosinophilic infiltrates in skin were noted (compare Figures 24) we tested the effect of anti-VLA-4 antibody treatment. A 30–40% inhibition of ear swelling was observed in mice that received anti-VLA-4 antibody compared to control animals. Similar effects were seen in the DNFB model, i.e. anti-VLA-4 antibody pretreatment resulted in a 50% reduction of the CHS response, a finding which is in agreement with previously published data [Figure 4C and D; (15)].

These antibody studies show that both TNF-{alpha} and VLA-4 play a significant role in the establishment of hapten-induced CHS, regardless of whether a Th1 or Th2 response dominates.

Treatment regimens directed against Th2 cells
The biochemical and histological evidence for a Th2-driven reaction prompted us to investigate the effect of cytokine modulation on the outcome of the CHS reaction. In line with our hypothesis, pretreatment of the animals with a neutralizing anti-IL-4 antibody reduced ear thickness triggered by FITC challenge by over 60% at all time points investigated while a control antibody had no effect. The administration of rmuINF-{gamma}, a typical Th1 cytokine, significantly diminished the response to FITC by ~35% (Figure 5A and B).



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Fig. 5. Protection from FITC-induced CHS by anti-IL-4 antibody, INF-{gamma} or STAT6 gene disruption. Balb/c mice were left untreated (non-sensitized), received a control antibody or a neutralizing anti-IL-4 antibody (500 µg/mouse s.c., A) 1 h before sensitization and FITC challenge (protocol A). (B) mice were pretreated with rmuINF-{gamma} (30 µg/mouse s.c.) or the corresponding volume of PBS at day 0 and 1, respectively. Stat6–/– mice or their wt controls were sensitized and challenged by FITC (C) or DNFB (D) according to protocol B. Measurements of ear thickness were performed using a micrometer at 24 (D), 48 and 72 h (A–C) after challenge as described in the Methods. All data represent mean values ± SEM of n = 5 animals at each given time point. Statistical differences were analyzed using Student’s t-test (**P < 0.01).

 
To evaluate the role of STAT6, a crucial mediator of IL-4 receptor signaling, in the development of a CHS response, we compared mice with targeted disruption of the STAT6 gene (Stat6–/–) to their WT counterparts (wt). As expected, and in agreement with the protective effect of anti-IL-4 antibody pretreatment, the CHS response to FITC was dramatically diminished and ear thickness did not differ from non- sensitized mice both in the 1 week (not shown) and in the 3 week model (Figure 5C). In contrast, the CHS response to DNFB was significantly enhanced in mice lacking STAT6 compared to wt animals (0.326 ± 0.029 mm vs 0.208 ± 0.021 mm, respectively) revealing basic mechanistic differences induced by those two contact allergens (Figure 5D).

Blocked CHS response to FITC but not to DNFB by anti-IL-5 antibody pretreatment
Tissue eosinophilia invariably depends on the presence of the Th2-cytokine IL-5, as shown in numerous studies using neutralization approaches or gene knockout strategies (23). We thus investigated whether the neutralization of this eosinophil survival factor would affect FITC-induced ear swelling which paralleled eosinophil infiltration and increased EPO activity (see Figures 3 and 2, respectively). First, we injected anti-IL-5 antibody into Balb/c mice. Both in the one week (Figure 6A) and in the three week model (data not shown), IL-5 neutralization resulted in a diminished inflammatory response to FITC, and ear swelling was reduced by 70% compared to PBS or antibody controls. In addition, anti-IL-5 antibody pretreatment also showed efficacy in C57/B6 mice (Figure 6B). In contrast, the anti-IL-5 antibody failed to modulate the CHS response induced by DNFB, supporting the view that DNFB induces a Th1-response with no major role for eosinophils (Figure 6C).



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Fig. 6. Effect of anti-IL-5 antibody on the CHS response induced by FITC or DNFB. BALB/c mice (A and C) or C57/B6 mice (B) were sensitized by FITC (A and B) or DNFB (C) according to protocol A. Anti-mouse IL-5 antibody or control antibody were injected at a dose of 500 µg/mouse s.c., control animals received the corresponding volume of PBS 1 h before challenge. Measurements of ear thickness were performed using a micrometer at 24, 48 and 72 h after challenge as described in the Methods. All data represent mean values ± SEM of n = 5 animals at each given time point. Statistical differences were analyzed using Student’s t-test (*P < 0.05, **P < 0.01).

 
Mast cells contribute to CHS induced by FITC
The fundamental differences between CHS induced by FITC and DNFB prompted us to further investigate the role of mast cells, a population of inflammatory cells found in the inflamed skin of FITC-challenged animals (see Figure 3) and known to be capable of producing IL-5. Compound 48/80 is a well-established tool to experimentally deplete mast cells in vivo. Unlike IgE, this drug can induce activation, degranulation and death of mast cells via non-immunologic stimulation, and it was demonstrated that pretreatment of mice with compound 48/80 reduces the number of intact mast cells by 95% (24). To validate this tool, we performed preliminary experiments and observed a clearly diminished passive cutaneous anaphylaxis response induced by anti-dinitrophenyl (DNP) IgE in compound 48/80 pretreated mice (data not shown). Indeed, such mast cell-depleted mice showed a clearly diminished reaction to FITC-induced CHS. A 60% reduction was seen in the one week model (Figure 7A) and the effect of compound 48/80 pretreatment was even more pronounced in the 3 week model (Figure 7C). In mast cell-depleted animals, however, the reaction to DNFB was even aggravated and an increase in ear swelling compared to control mice of 40% was noted (Figure 7B).



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Fig. 7. Effect of mast cell depletion by compound 48/80 on the CHS response induced by FITC or DNFB. BALB/c mice were sensitized by FITC (A and C) or DNFB (B) according to protocol A (A and B) or to protocol B (C). Compound 48/80 (200 µg/mouse) or the corresponding volume of PBS was injected i.d. into the ear 4 days before challenge. Measurements of ear thickness were performed using a micrometer at 24, 48 and 72 h after challenge as described in the Methods. All data represent mean values ± SEM of n = 5 animals at each given time point. Statistical differences were analyzed using Student’s t-test (*P < 0.05, **P < 0.01)

 
Diminished CHS responses to FITC in CD4–/– mice
In order to gain better insight into the T cell subsets that orchestrate the responses to FITC and DNFB, respectively, we made use of gene-targeted mice that show defined T cell defects (see Table 1 for genotypes and phenotypes). CD4-deficient mice (CD4–/–) mice exert normal development of CD8+ T cells and myeloid components, and full cytotoxic T cell activity against viruses, but have markedly decreased helper activity for antibody production (25). Mice lacking ß2 microglobulin (ß2m) show a complex phenotype characterized by a normal distribution of {gamma}{delta} T cells and CD4+ T cells, an inability to express CD1 and class I MHC molecules which require ß2m for assembly, a decreased number of mature NK cells, a defect in NKT function and, importantly, a defect in T cell-mediated cytotoxicity due to the complete lack of mature {alpha}ß+CD8+ T cells [CD8–/–; (26)]. Thus, ß2m k.o. mice show a CD8–/–/MHC I–/– double k.o. phenotype and are here referred to as CD8–/– mice.


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Table 1. Overview of the gene-targeted mouse strains used (all of the C57/B6 background)
 
The CHS response to FITC was significantly diminished in such CD8–/– mice. CD4–/– mice, however, hardly responded to FITC at all, and only a very small increase in ear thickness was detectable upon FITC challenge. When CHS was induced by DNFB, partial protection was seen in CD4–/– and CD8–/– mice 24 h after challenge compared to wt animals. The diminished response was more pronounced in CD8–/– mice (Figure 8A).



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Fig. 8. Effect of CD4, ß2m, I-A or ß2m/I-A double gene depletion on the CHS response induced by FITC or DNFB. CD4/–, ß2m k.o. (CD8/–), I-A k.o. (MHC II/–), double k.o. (CD8/–/MHC II/–) or C57/B6 wt mice were sensitized and challenged by FITC or DNFB solution according to protocol A. Measurements of ear thickness were performed using a micrometer at 24 h after challenge as described in the Methods. All data represent mean values ± SEM of n = 5 animals at each given time point. Statistical differences compared to the respective wt control were analyzed using Student’s t-test (**P < 0.01, ***P < 0.001).

 
MHC II-independent CD4+ cells are critical for the CHS response induced by FITC, and the response to DNFB is strongly diminished by MHC I and II depletion
I-A gene disrupted mice are deficient in the cell surface expression of MHC II molecules (MHC II–/–). The number of single positive CD4+ cells in the periphery of these MHC II-deficient mice is reduced by ~97% (27,28). Although the nature of this population is unclear, it is speculated that these cells represent either class I-restricted CD4+ T cells or CD4+ NKT cells (29). Mice completely lacking MHC molecules have been generated by mating ß2m k.o. mice (CD8–/–/MHC I–/–) with I-A k.o. (MHC II–/–) mice. Phenotypically, these mice harbor neither CD4+ nor CD8+ T cells in their peripheral lymphoid organs. MHC II–/– mice showed a significantly diminished response to FITC and DNFB, respectively (Figure 8B), compared to their wt counterparts. However, in comparison to CD4–/– mice the protection from FITC-induced CHS was relatively low (see Figure 8A). MHC-deficient mice, i.e. ß2m/I-A double k.o. mice (CD8–/–/MHC II–/–) were fully resistant to FITC, and showed a strongly reduced response to DNFB (Figure 8B).

Collectively, these investigations performed in k.o. mice demonstrate the absolute necessity for cells that are either class I-restricted CD4+ T cells or CD4+ NKT cells in the establishment of a full response to FITC. Further, the contribution of CD4+ and CD8+ cells to the DNFB response is approximately equal, and only the complete lack of MHC I and MHC II molecules confers full protection from DNFB-induced CHS.


    Discussion
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
CHS is a T cell-dependent response to skin sensitization and challenge with lipid-soluble sensitizers (haptens) that are able to bind directly to soluble, or cell (keratinocytes)-associated protein. The lipophilic compound fluorescein isothiocyanate (FITC) was shown to be a strong sensitizer for the CHS response (30), rapidly entering the circulation by way of local lymph nodes, and the regional lymph nodes, spleen and distant lymph nodes (31).

Chemical contact allergy induced by numerous haptens [DNFB, trinitrofluorobenzene (TNFB) and Oxa], is regarded as a cell-mediated immune response mediated by INF-{gamma}- producing Th/Tc1 cells (32). However Tang et al. (10) have indicated that the FITC-induced CHS response is Th2- dominant, while the DNFB response was Th1-dominant. When FITC was used as the immunogen, a Th2-like response was observed with an IL-4/INF-{gamma} ratio of 25. In contrast, more INF-{gamma} than IL-4-secreting cells were found in draining lymph nodes from DNFB-sensitized mice with an IL-4/INF-{gamma} ratio of 0.026 (10). These results suggest that the CHS response induced by FITC in Balb/c mice is primarily a result of the activation of Th2-type cells. However, there is no evidence for strain dependency in the FITC-induced CHS response. To clarify this question, we compared the CHS response induced by FITC in Balb/c and C57/B6 mice. A single topical application of FITC to the ears could induce a CHS response in both the Th2 dominant strain BALB/c mice and the Th1 dominant strain, C57/B6, but this response was stronger in BALB/c mice than in C57/B6 mice.

Epidermal cell-derived proinflammatory cytokines, including IL-1 and TNF-{alpha}, play an important role in promoting LC migration during the induction phase of CHS. Wang et al. demonstrated that IL-10 KO mice had greater numbers of hapten-bearing cells in the LN following FITC, and that pretreatment with either neutralizing TNF-{alpha} antibody or rhIL-1Ra significantly diminished the enhanced LC migration in IL-10 k.o. mice (20). We could confirm that the neutralizing TNF-{alpha} antibody significantly inhibited CHS responses induced by FITC in normal mice, suggesting that TNF-{alpha} can contribute to promoting LC migration during the induction phase of CHS induced by FITC.

Lymphocyte–endothelial cell recognition is an active multistep process central to the pathophysiology of inflammation. Chisholm et al. demonstrated that the intravenous administration of 75 µg of the anti-VLA-4 antibody 4–6 h prior to challenge significantly inhibited the efferent response of DNFB-sensitized mice. This inhibition of CHS responses using anti-VLA-4 antibody treatment was evident as a 60% reduction in FITC similarly to DNFB. However the inhibitory effect was likely not only due to the inhibition of the migration of effector lymphocytes, but also eosinophils, since VLA-4 is suggested to play a critical roll in eosinophil infiltration in many models of inflammation (22,33).

Mice challenged by peptide antigen, parasite or anti-IgD show a polarization to Th2 responses and an enhanced secretion of IL-4 that promotes IgE production. FITC-sensitized mice produced significantly higher amounts of serum IgE, thus it appears that FITC can induce Th2 dependent responses in vivo. Anti-IL-4 or rIHF-{gamma} could suppress CHS induced by FITC in Balb/c mice. Since Th2 differentiation is IL-4-dependent, IL-4 neutralization may inhibit the development of Th2 cells and the subsequent events that lead to allergic inflammation such as IL-5-dependent eosinophilia or IgE-mediated, mast cell-dependent inflammation. Furthermore, because IL-4 up-regulates collagen and fibronectin synthesis in subepithelial fibroblasts (leading to remodelling), inhibiting IL-4 may also prevent hyperplasia (34). INF-{gamma} suppression of IL-4-mediated IgE production may shift the T cell population toward the Th1 subset, accounting for the reduced FITC-induced CHS responses. In addition, with the CHS response model induced by FITC on the Balb/C background, anti-IL-4 antibody treated mice had reduced CHS responses and STAT6 KO C57BL/6 mice did not develop a CHS response. These phenomena indicate that CHS responses to FITC in both strains (Balb/c and C57/B6) mice can be used as typical Th2 type response model.

Our histological data indicated that large numbers of eosinophils were observed at the site of inflammation in Balb/c or C57/B6 mice, and EPO activity was increased. However, the contribution of eosinophils to the CHS response induced by FITC is unknown. To better understand this, we investigated whether in vivo administration of anti-IL-5 suppresses CHS induced by FITC in Balb/c or C57/B6 mice. The inhibition induced by anti-IL-5 antibody treatment was ~50–60% reduction in both FITC models, but not in either DNFB model. These data indicate that the inhibitory effect was only due to the inhibition of the migration of eosinophils and not to inhibition of the migration of other cells (neutrophils and mononuclear cells).

Various groups that have examined the CHS response in normal and mast cell-deficient mice have reported different results. Some found equivalent ear swelling responses in mast cell-deficient and WT mice, whereas others found strongly reduced ear swelling responses in mast cell–deficient animals (3538). This difference is influenced by various additional factors such as the concentration of the allergen/hapten, or the adjuvant used for sensitization and elicitation of the immune response. Importantly, the deficiency in ear swelling responses in W/W-v mice can be due to factors that are independent of mast cells. We therefore used compound 48/80 for the depletion of mast cells. Pretreatment of mice with compound 48/80 reduced the number of intact mast cells by 95% and the PCA response (data not shown) and partly inhibited the ear swelling response to FITC challenge compared with PBS. However, the CHS response to DNFB was significantly enhanced in compound 48/80 treated mice compared with PBS treated mice. The reason for this effect is at present unclear and is currently under investigation.

While it has been considered that contact hypersensitivity is a T cell-mediated cutaneous immune/inflammatory reaction to haptens, the relative contribution of CD4+ vs CD8+ T cells in contact hypersensitivity (CHS) remains unclear. First, in vivo depletion of CD4+, but not CD8+, T cells significantly inhibited CHS responses to DNFB (39). In vitro depletion of CD4+, but not CD8+, T cells from LN cells (LNCs) was shown to prevent the ability to adoptively transfer CHS, and CD4+ T cells have been shown to be capable of adoptively transferring CHS to naive mice (40). Furthermore, CD4 knockout mice mounted a reduced CHS response (41). However, some studies suggest that CHS responses are mediated by CD8+ T cells and down-regulated by CD4+ T cells. For example, in vivo depletion of CD8+ or CD4+ T cells resulted in reduction or exaggeration of the CHS responses to DNFB, respectively. MHC class I-deficient mice were unable to mount a CHS response to DNFB, whereas MHC class II-deficient mice induced an exaggerated CHS response (42). The enhanced CHS responses in MHC class II-deficient mice were decreased by treatment with anti-CD8 antibody (43), or by transfer of WT CD4+ T cells. These studies demonstrated that type 1 cytotoxic T (Tc1) cells or helper T (Th1) cells contribute to the CHS response but not type 2 cells (44). Dearman and Kimber indicated that Th2-type CD4+ cells were responsible for the delayed-type component of the dermal hypersensitivity reaction by adoptive transfer experiments (11). However, there is no direct evidence for the contribution of CD4+ cells to CHS induced by FITC, so we studied CD4 and ß2-m k.o. mice CHS models induced by FITC or DNFB. The CHS response to FITC was significantly diminished in CD4 k.o. mice compared with wild type mice, while depletion of CD8+ T cells mice partly inhibited the ear swelling response to FITC. These and Stat6 k.o. studies indicate that CD4+ T cells shifted to a type 2 response are essential for CHS responses induced by FITC. Our results suggest that both CD4+ Th1 and CD8+ type 1 cytotoxic T cells are crucial effector cells in CHS responses to DNFB in C57BL/6 mice, using the protocols listed above.

Epitope recognition of most CD4+ T cells is MHC II-dependent. A small subset of MHC II-independent CD4+ T cells is present in normal and MHC II-deficient mice (27,28). MHC II-independent CD4+ T cells in I-A –/– mice are thymically derived, appear early in ontogeny, localize preferentially to the B rather than to the T cell areas in peripheral lymphoid organs, exhibit the phenotype of resting or activated memory T cells, and have a diverse TCR repertoire that is potentially functional (45). Only one subset within the MHC II-independent CD4+ T cells, i.e. CD1d-restricted NK1+ T cells expressing the NK1 marker (NKT) cells, is well defined. MHC II-independent, CD1d-restricted NKT cells are absent from ß2-m k.o. mice (46). The strong capacity to produce IL-4 has led to speculation that NKT cells might drive the differentiation of Th2 responses (47). But the presence and function of MHC II-independent CD4+ T cells in the CHS response is not clear. To be clear whether it was class I-restricted CD4+ T cells or CD4+ NKT cells whose contribution to the CHS response was most relevant, we studied MHC-II or MHC-II/ß2-m k.o. mice on the CHS response induced by FITC or DNFB. Although the CHS response to FITC was significantly diminished in CD4 k.o. mice, resistance to the CHS was observed in class II-deficient mice, and significantly diminished in MHC-II/ß2-m k.o. mice to a similar level as the CD4 k.o. mice. There was no difference in the level of inhibition in the CHS response induced by DNFB between ß2-m k.o. and CD4 k.o. mice. These data indicate that CD 4+ T cells shifted to a type 2 response by Stat6 are essential for the CHS response induced by FITC, and that not only CD 4+ T cells but potentially also CD4+ NKT cells can induce type 2 CHS in an MHC II-dependent or MHC II-independent manner (MHC I or CD1d) (48,49). In addition, it is suggested that this shift is important during the induction phase of the response (50). In our experiments, given that the anti-IL-4 antibody was capable of inhibiting the response prior to challenge with FITC, we can also conclude that the induction phase of the response is affected by the shift to a Th2 phenotype.

Acute atopic dermatitis skin lesions are characterized by marked epidermal intercellular edema. Antigen-presenting cells (Langerhans cells, dendritic cells and macrophages) in acute lesions express IgE molecules. Chronic lesions are characterized by an acanthotic epidermis, parakeratosis and only minimum spongiosis. Chronic lichenified lesions have an increased number of IgE-bearing Langerhans cells and inflammatory dendritic epidermal cells in the epidermis, and macrophages dominate the dermal mononuclear cell infiltrate. These lesions also contain eosinophils, which are thought to contribute to inflammation and tissue injury through production of reactive oxygen intermediates and proinflammatory cytokines and release of toxic granule proteins (51). We have demonstrated that in an FITC-induced CHS model, an inflammatory skin pathology reproducing all the key features of human atopic dermatitis develops, including what appears to be a Th2-driven immune response involving mast cells, macrophage-like mononuclear cells and eosinophil infiltration, and elevation of total serum IgE. This may therefore represent a suitable mouse model with which to analyze therapeutics developed for the human disease.


    Acknowledgements
 
The authors thank K. Nagao for stimulating discussions and experimental help. Special thanks also go to Dr S. Akira for kindly providing Stat6–/– mice.


    Abbreviations
 
AOO—acetone:olive oil

ß2m—ß2 microglobulin

CHS—contact hypersensitivity

DBP—dibutylphthalate:acetone

DTH—delayed-type hypersensitivity

DNFB—dinitrofluorobenzene

DNP—dinitrophenyl

EPO—eosinophil peroxidase

k.o.—knockout

LC—Langerhans cell

Oxa—oxazolone

PMN—polymorphonuclear cell

STAT—signal transducer and activator of transcription

TNFB—trinitrofluorobenzene

VLA-4—very late antigen-4


    References
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

  1. Mosmann, T. R. and Coffman, R. L. 1989. Th1 and Th2 cells: different patterns of lymphokine secretion lead to different functional properties. Annu Rev Immunol 7:145.[CrossRef][ISI][Medline]
  2. Dvorak, H. F., Galli, S. J. and Dvorak, A. M. 1986. Cellular and vascular manifestations of cell-mediated immunity. Hum Pathol 17:122.[ISI][Medline]
  3. Crowle, A. J. 1975. Delayed hypersensitivity in the mouse. Adv Immunol 20:197.[Medline]
  4. Chapman, J. R., Ruben, Z. and Butchko, G. M. 1986. Histology of and quantitative assays for oxazolone-induced allergic contact dermatitis in mice. Am J Dermatopathol 8:130.[ISI][Medline]
  5. Thomson, J. A., Troutt, A. B. and Kelso, A. 1993. Contact sensitization to oxazolone: involvement of both interferon-gamma and interleukin-4 in oxazolone-specific Ig and T-cell responses. Immunology 78:185.[ISI][Medline]
  6. Biedermann, T., Mailhammer, R., Mai, A., Sander, C., Ogilvie, A., Brombacher, F., Maier, K., Levine, A. D. and Rocken, M. 2001. Reversal of established delayed type hypersensitivity reactions following therapy with IL-4 or antigen-specific Th2 cells. Eur J Immunol 31:1582.[CrossRef][ISI][Medline]
  7. Wang, B., Fujisawa, H., Zhuang, L., Freed, I., Howell, B. G., Shahid, S., Shivji, G. M., Mak, T. W. and Sauder, D. N. 2000. CD4+ Th1 and CD8+ type 1 cytotoxic T cells both play a crucial role in the full development of contact hypersensitivity. J Immunol 165:6783.[Abstract/Free Full Text]
  8. Weigmann, B., Schwing, J., Huber, H., Ross, R., Mossmann, H., Knop, J. and Reske-Kunz, A. B. 1997. Diminished contact hypersensitivity response in IL-4 deficient mice at a late phase of the elicitation reaction. Scand J Immunol 45:308.[CrossRef][ISI][Medline]
  9. Dieli, F., Sireci, G., Scire, E., Salerno, A. and Bellavia, A. 1999. Impaired contact hypersensitivity to trinitrochlorobenzene in interleukin-4-deficient mice. Immunology 98:71.[CrossRef][ISI][Medline]
  10. Tang, A., Judge, T. A., Nickoloff, B. J. and Turka, L. A. 1996. Suppression of murine allergic contact dermatitis by CTLA4Ig. Tolerance induction of Th2 responses requires additional blockade of CD40-ligand. J Immunol 157:117.[Abstract]
  11. Dearman, R. J. and Kimber, I. 2000. Role of CD4(+) T helper 2-type cells in cutaneous inflammatory responses induced by fluorescein isothiocyanate. Immunology 101:442.[CrossRef][ISI][Medline]
  12. Takeda, K., Tanaka, T., Shi, W., Matsumoto, M., Minami, M., Kashiwamura, S., Nakanishi, K., Yoshida, N., Kishimoto, T. and Akira, S. 1996. Essential role of Stat6 in IL-4 signalling. Nature 380:627.[CrossRef][ISI][Medline]
  13. Proust, B., Nahori, M. A., Ruffie, C., Lefort, J. and Vargaftig, B. B. 2003. Persistence of bronchopulmonary hyper-reactivity and eosinophilic lung inflammation after anti-IL-5 or -IL-13 treatment in allergic BALB/c and IL-4Ralpha knockout mice. Clin Exp Allergy 33:119.[CrossRef][ISI][Medline]
  14. Macari, D. M., Teixeira, M. M. and Hellewell, P. G. 1996. Priming of eosinophil recruitment in vivo by LPS pretreatment. J Immunol 157:1684.[Abstract]
  15. Catalina, M. D., Estess, P. and Siegelman, M. H. 1999. Selective requirements for leukocyte adhesion molecules in models of acute and chronic cutaneous inflammation: participation of E- and P- but not L-selectin. Blood 93:580.[Abstract/Free Full Text]
  16. To, Y., Dohi, M., Tanaka, R., Sato, A., Nakagome, K. and Yamamoto, K. 2001. Early interleukin 4-dependent response can induce airway hyperreactivity before development of airway inflammation in a mouse model of asthma. Lab Invest 81:1385.[ISI][Medline]
  17. Iwamoto, I., Nakajima, H., Endo, H. and Yoshida, S. 1993. Interferon gamma regulates antigen-induced eosinophil recruitment into the mouse airways by inhibiting the infiltration of CD4+ T cells. J Exp Med 177:573.[Abstract]
  18. Stankiewicz, E., Wypasek, E. and Plytycz, B. 2001. Opposite effects of mast cell degranulation by compound 48/80 on peritoneal inflammation in Swiss and CBA mice. Pol J Pharmacol 53:149.[ISI][Medline]
  19. Nuriya, S., Enomoto, S. and Azuma, M. 2001. The role of CTLA-4 in murine contact hypersensitivity. J Invest Dermatol 116:764.[Abstract/Free Full Text]
  20. Wang, B., Zhuang, L., Fujisawa, H., Shinder, G. A., Feliciani, C., Shivji, G. M., Suzuki, H., Amerio, P., Toto, P. and Sauder, D. N. 1999. Enhanced epidermal Langerhans cell migration in IL-10 knockout mice. J Immunol 162:277.[Abstract/Free Full Text]
  21. Roake, J. A., Rao, A. S., Morris, P. J., Larsen, C. P., Hankins, D. F. and Austyn, J. M. 1995. Dendritic cell loss from nonlymphoid tissues after systemic administration of lipopolysaccharide, tumor necrosis factor, and interleukin 1. J Exp Med 181:2237.[Abstract]
  22. Bocchino, V., Bertorelli, G., D’Ippolito, R., Castagnaro, A., Zhuo, X., Grima, P., Di Comite, V., Damia, R. and Olivieri, D. 2000. The increased number of very late activation antigen-4-positive cells correlates with eosinophils and severity of disease in the induced sputum of asthmatic patients. J Allergy Clin Immunol 105:65.[ISI][Medline]
  23. Foster, P. S., Hogan, S. P., Ramsay, A. J., Matthaei, K. I. and Young, I. G. 1996. Interleukin 5 deficiency abolishes eosinophilia, airways hyperreactivity, and lung damage in a mouse asthma model. J Exp Med 183:195.[Abstract]
  24. Ajuebor, M. N., Das, A. M., Virag, L., Flower, R. J., Szabo, C. and Perretti, M. 1999. Role of resident peritoneal macrophages and mast cells in chemokine production and neutrophil migration in acute inflammation: evidence for an inhibitory loop involving endogenous IL-10. J Immunol 162:1685.[Abstract/Free Full Text]
  25. Rahemtulla, A., Kundig, T. M., Narendran, A., Bachmann, M. F., Julius, M., Paige, C. J., Ohashi, P. S., Zinkernagel, R. M. and Mak, T. W. 1994. Class II major histocompatibility complex-restricted T cell function in CD4-deficient mice. Eur J Immunol 24:2213.[ISI][Medline]
  26. Koller, B. H., Marrack, P., Kappler, J. W. and Smithies, O. 1990. Normal development of mice deficient in beta 2M, MHC class I proteins, and CD8+ T cells. Science 248:1227.[ISI][Medline]
  27. Grusby, M. J., Johnson, R. S., Papaioannou, V. E. and Glimcher, L. H. 1991. Depletion of CD4+ T cells in major histocompatibility complex class II-deficient mice. Science 253:1417.[ISI][Medline]
  28. Grusby, M. J., Auchincloss, H. Jr, Lee, R., Johnson, R. S., Spencer, J. P., Zijlstra, M., Jaenisch, R., Papaioannou, V. E. and Glimcher, L. H. 1993. Mice lacking major histocompatibility complex class I and class II molecules. Proc Natl Acad Sci USA 90:3913.[Abstract]
  29. Trobonjaca, Z., Leithauser, F., Moller, P., Bluethmann, H., Koezuka, Y., MacDonald, H. R. and Reimann, J. 2001. MHC-II-independent CD4+ T cells induce colitis in immunodeficient RAG–/– hosts. J Immunol 166:3804.[Abstract/Free Full Text]
  30. Thomas, W. R., Edwards, A. J., Watkins, M. C. and Asherson, G. L. 1980. Distribution of immunogenic cells after painting with the contact sensitizers fluorescein isothiocyanate and oxazolone. Different sensitizers form immunogenic complexes with different cell populations. Immunology 39:21.[ISI][Medline]
  31. Dearman, R. J., Cumberbatch, M., Hilton, J., Clowes, H. M., Fielding, I., Heylings, J. R. and Kimber, I. 1996. Influence of dibutyl phthalate on dermal sensitization to fluorescein isothiocyanate. Fundam Appl Toxicol 33:24.[CrossRef][ISI][Medline]
  32. Watanabe, H., Unger, M., Tuvel, B., Wang, B. and Sauder, D. N. 2002. Contact hypersensitivity: the mechanism of immune responses and T cell balance. J Interferon Cytokine Res 22:407.[CrossRef][ISI][Medline]
  33. Kanehiro, A., Takeda, K., Joetham, A., Tomkinson, A., Ikemura, T., Irvin, C. G. and Gelfand, E. W. 2000. Timing of administration of anti-VLA-4 differentiates airway hyperresponsiveness in the central and peripheral airways in mice. Am J Respir Crit Care Med 162:1132.[Abstract/Free Full Text]
  34. Lewis, D. B. 2002. Allergy immunotherapy and inhibition of Th2 immune responses: a sufficient strategy? Curr Opin Immunol 14:644.[CrossRef][ISI][Medline]
  35. Askenase, P. W., Van Loveren, H., Kraeuter-Kops, S., Ron, Y., Meade, R., Theoharides, T. C., Nordlund, J. J., Scovern, H., Gerhson, M. D. and Ptak, W. 1983. Defective elicitation of delayed-type hypersensitivity in W/Wv and SI/SId mast cell-deficient mice. J Immunol 131:2687.[Abstract/Free Full Text]
  36. Webb, E. F., Tzimas, M. N., Newsholme, S. J. and Griswold, D. E. 1998. Intralesional cytokines in chronic oxazolone-induced contact sensitivity suggest roles for tumor necrosis factor alpha and interleukin-4. J Invest Dermatol 111:86.[Abstract]
  37. Galli, S. J. and Hammel, I. 1984. Unequivocal delayed hypersensitivity in mast cell-deficient and beige mice. Science 226:710.[ISI][Medline]
  38. Asada, H., Linton, J. and Katz, S. I. 1997. Cytokine gene expression during the elicitation phase of contact sensitivity: regulation by endogenous IL-4. J Invest Dermatol 108:406.[Abstract]
  39. Anderson, C., Hehr, A., Robbins, R., Hasan, R., Athar, M., Mukhtar, H. and Elmets, C. A. 1995. Metabolic requirements for induction of contact hypersensitivity to immunotoxic polyaromatic hydrocarbons. J Immunol 155:3530.[Abstract]
  40. Gautam, S. C., Matriano, J. A., Chikkala, N. F., Edinger, M. G. and Tubbs, R. R. 1991. L3T4 (CD4+) cells that mediate contact sensitivity to trinitrochlorobenzene express I-A determinants. Cell Immunol 135:27.[ISI][Medline]
  41. Kondo, S., Beissert, S., Wang, B., Fujisawa, H., Kooshesh, F., Stratigos, A., Granstein, R. D., Mak, T. W. and Sauder, D. N. 1996. Hyporesponsiveness in contact hypersensitivity and irritant contact dermatitis in CD4 gene targeted mouse. J Invest Dermatol 106:993.[Abstract]
  42. Bour, H., Peyron, E., Gaucherand, M., Garrigue, J. L., Desvignes, C., Kaiserlian, D., Revillard, J. P. and Nicolas, J. F. 1995. Major histocompatibility complex class I-restricted CD8+ T cells and class II-restricted CD4+ T cells, respectively, mediate and regulate contact sensitivity to dinitrofluorobenzene. Eur J Immunol 25:3006.[ISI][Medline]
  43. Bouloc, A., Cavani, A. and Katz, S. I. 1998. Contact hypersensitivity in MHC class II-deficient mice depends on CD8 T lymphocytes primed by immunostimulating Langerhans cells. J Invest Dermatol 111:44.[Abstract]
  44. Xu, H., DiIulio, N. A. and Fairchild, R. L. 1996. T cell populations primed by hapten sensitization in contact sensitivity are distinguished by polarized patterns of cytokine production: interferon gamma-producing (Tc1) effector CD8+ T cells and interleukin (Il) 4/Il-10-producing (Th2) negative regulatory CD4+ T cells. J Exp Med 183:1001.[Abstract]
  45. Kontgen, F., Suss, G., Stewart, C., Steinmetz, M. and Bluethmann, H. 1993. Targeted disruption of the MHC class II Aa gene in C57BL/6 mice. Int Immunol 5:957.[Abstract]
  46. Bendelac, A., Rivera, M. N., Park, S. H. and Roark, J. H. 1997. Mouse CD1-specific NK1 T cells: development, specificity, and function. Annu Rev Immunol 15:535.[CrossRef][ISI][Medline]
  47. Yoshimoto, T., Bendelac, A., Watson, C., Hu-Li, J. and Paul, W. E. 1995. Role of NK1.1+ T cells in a TH2 response and in immunoglobulin E production. Science 270:1845.[Abstract]
  48. Mendiratta, S. K., Martin, W. D., Hong, S., Boesteanu, A., Joyce, S. and Van Kaer, L. 1997. CD1d1 mutant mice are deficient in natural T cells that promptly produce IL-4. Immunity 6:469.[ISI][Medline]
  49. Chen, Y. H., Chiu, N. M., Mandal, M., Wang, N. and Wang, C. R. 1997. Impaired NK1+ T cell development and early IL-4 production in CD1-deficient mice. Immunity 6:459.[ISI][Medline]
  50. Yokozeki, H., Ghoreishi, M., Takagawa, S., Takayama, K., Satoh, T., Katayama, I., Takeda, K., Akira, S. and Nishioka, K. 2000. Signal transducer and activator of transcription 6 is essential in the induction of contact hypersensitivity. J Exp Med 191:995.[Abstract/Free Full Text]
  51. Leung, D. Y. and Bieber, T. 2003. Atopic dermatitis. Lancet 361:151.[CrossRef][ISI][Medline]
  52. Zijlstra, M., Bix, M., Simister, N. E., Loring, J. M., Raulet, D. H. and Jaenisch, R. 1990. Beta 2-microglobulin deficient mice lack CD4-8+ cytolytic T cells. Nature 344:742.[CrossRef][ISI][Medline]