Interleukin (IL)-18 was originally discovered in the Proprionibacterium acnes-induced toxic shock model as interferon (IFN)-
inducing factor (Okamura et al, 1995). IL-18 is intracellularly produced from a biologically inactivated precursor, pro-IL-18, and mature IL-18 is secreted after the cleavage of pro-IL-18 by caspase-1, originally identified as IL-1
converting enzyme (ICE) (Dinarello, 1999). It has been reported that pro-IL-18 mRNA is expressed in a wide range of cells, including Kupffer cells, macrophages, T cells, B cells, osteoblasts, keratinocytes, dendritic cells, astrocytes, and microglia, whereas mature IL-18 is only weakly detected in the sera or tissues (Dinarello, 1999). IL-18 is biologically similar, although not structurally related, to IL-12, a strong T helper (Th) 1 T cell inducer. IL-18 is thought to be a strong cofactor in Th1 cell development (Dinarello, 1999); however, we demonstrated that IL-18 and IL-2 in combination synergistically induced expression of IL-13, a Th2 cytokine, in T cells and a unique IL-13 producing natural killer cell population (Hoshino et al, 1999). We and other investigators have reported that IL-18 not only induces production of the Th1 cytokine IFN-, but also that of Th2 cytokines, including IL-4, IL-5, IL-10, and IL-13 and IgE and IgG1, in murine model systems (Yoshimoto et al, 1999,2000; Hoshino et al, 2000; Wild et al, 2000; Xu et al, 2000; Nakanishi et al, 2001). Recently, we described the generation of IL-18 transgenic (TG) mice in which mature murine IL-18 cDNA was expressed under the control of a mouse immunoglobulin promoter (Hoshino et al, 2001). Serum IgE, IgG1, IL-4, and IFN- levels were significantly higher in these TG mice than their wild-type (WT) counterparts and splenic T cells from these TG mice produced more IFN-, IL-4, IL-5, and IL-13 than those from control WT mice. Moreover, recent studies suggest that IL-18 plays an important part in the pathogenesis of asthma, which is a chronic inflammatory airways disease characterized by infiltration of antigen-specific Th2, or Th2-like, cells (Nakanishi et al, 2001). These observations indicate that not only can IL-18 act as a cofactor in both Th1 and Th2 cell development, but it may also contribute to allergic disorders.
Murine contact hypersensitivity (CH) induced by application of a hapten such as 2,4-dinitrofluorobenzene (Thomas et al, 1999), trinitrochlorobenzene (TNCB) (Kitagaki et al, 1999), or oxazolone (Dieli et al, 1999) is a well-established experimental model of human contact dermatitis. A previous study found that both CD4+ and CD8+ T cells play a part in inducing CH (Gocinski and Tigelaar, 1990). CH was thought to be associated with the activation of Th1 type T cells. It has been demonstrated that in IL-4/IL-10-producing CD4+ T cells, negatively regulates CH response (Xu et al, 1996). Recent studies suggest that both Th1 and Th2 cytokines such as IFN-, IL-4, IL-5, and IL-13 expressed at the site of inflammation or the draining lymph node play important parts in TNCB-induced cutaneous inflammation in mice (Dieli et al, 1999;Yokozeki et al, 2000). Repetitive elicitation of CH response induced by TNCB results in a shift in cutaneous cytokine expression from a Th1 to a Th2 phenotype (Kitagaki et al, 1999). Moreover, the requirement of cytokines in CH might depend on the kind of hapten because IL-4 is required for the CH reaction to TNCB but not oxazolone (Dieli et al, 1999). The increased expression of pro-IL-18 mRNA and IL-18 protein by keratinocytes was reported following oxazolone and dinitrochlorobenzene stimulation in mice (Xu et al, 1998) and humans (Naik et al, 1999), respectively. Furthermore, a recent report revealed that Langerhans cell-derived IL-18 might contribute to the oxazolone-elicited CH reaction (Wang et al, 2002). Thus, these results suggest that IL-18 produced at the site of cutaneous inflammation is likely to be involved in the manifestation of these CH reactions.
To elucidate a role of IL-18 in cutaneous inflammation, we generated keratin (K) 5/IL-18 TG mice in which keratinocytes express mouse mature IL-18 fused with the signal peptide (SP) of the mouse immunoglobulin 
-chain under the control of the human keratinocyte K5 promoter (Sano et al, 1999). The K5/IL-18 TG mice showed not only an aggravated TNCB-elicited CH reaction but also exacerbated and prolonged croton oil-induced irritant contact dermatitis compared with WT littermate or immunoglobulin/IL-18 TG mice in which mature IL-18 was expressed by B and T cells (Hoshino et al, 2001). Furthermore, the repeated elicitation of a CH reaction to TNCB resulted in cutaneous inflammation with more severe histologic changes. These observations imply that IL-18 plays a crucial part in the aggravation and prolongation of allergic and nonallergic cutaneous inflammation.
Materials and Methods
Mice
Male and female C57BL/6N (B6) mice were purchased from Charles River Japan (Yokohama, Japan) and bred in our laboratory. Immunoglobulin/IL-18 TG mice and their WT littermates were generated as described previously (Hoshino et al, 2001). They were housed in plastic cages in an air-conditioned room at a temperature of 24
1°C, a humidity of 55%5% and a 12 h light/dark cycle. All procedures were approved by the Committee on the Ethics of Animal Experiments, Kurume University (approval no. 570, 2002). Animal care was provided in accordance with the procedures outlined in the Guide for the Care and Use of Laboratory Animals (National Institutes of Health Publication No. 86-23, 1985).
Generation of TG mice expressing mouse mature IL-18
The IL-18 transgene for TG mice was generated as described previously (Hoshino et al, 2001). Briefly, mature mouse IL-18 cDNA fused with the SP from the V-J2-C region of the mouse immunoglobulin -chain (IL-18SP) were generated by polymerase chain reaction (PCR) using mouse pro-IL-18 cDNA (provided by Dr Kiyoshi Takeda, Osaka University, Osaka, Japan). The amplified IL-18SP DNA was subcloned into the pCR2.1 vector (Invitrogen, Carlsbad, California), and sequenced. Then, IL-18SP was subcloned into a pcdEF3 vector containing the human elongation factor 1
promoter and bovine poly(A) signal, (obtained from Dr Jerome Langer, Robert Wood Johnson Medical School, New Jersey), and was designated as pEF-IL-18SP. The KpnI/BbsI-digested pEF-IL-18SP DNA fragment containing IL-18SP and the bovine poly(A) signal was subcloned into the NotI site of a pBSK vector containing the human K5 promoter (Sano et al, 1999). The BssHII-digested linear DNA fragment (K5/IL-18SP/poly(A); Figure 1a) was injected into fertilized eggs of B6 mice at Oriental Bio Service (Kyoto, Japan). Hemizygous TG mice were generated by mating founder mice with B6 mice and the offspring were screened by the PCR using genomic DNA prepared from their tails, an enzyme-linked immunosorbent assay for serum mature mouse IL-18 (mIL-18), and western blotting for skin mature mIL-18 as previously reported (Hoshino et al, 2001).
Figure 1.
(A) Transgene structure. Mature mouse IL-18 cDNA fused with the SP from the mouse immunoglobulin -chain was linked to the human K5 promoter to achieve keratinocyte-specific expression. The transgene construct also contained the K5 polyadenylation sequence to aid in processing of the transcript and secretion of the protein. pA: bovine poly(A) signal. (B) TG IL-18 mRNA expression in the ear tissue (left), and serum IL-18 level (right) from 13 wk old male WT and K5/IL-18 TG mice (n=6, each group).
Induction of CH by an application of TNCB
Male K5/IL-18 TG, male immunoglobulin/IL-18 TG and their age-matched male WT mice were sensitized by topical application of 100 
L of acetone/olive oil (4:1 v/v) (vehicle) or 100 L of 3% (w/v) TNCB (Nacalai Tesque, Kyoto, Japan) on the shaved abdomen. Six days after the sensitization, an aliquot (10 L) of 1% (w/v) TNCB was applied to each site on the surface of the right ear and the vehicle was applied to the left ear. The thickness of each ear was measured using an engineering micrometer (SM-112, Teclock, Nagano, Japan) on blind basis before, and 24 h after TNCB application on the ear.
Induction of croton oil-induced irritant contact dermatitis
Male K5/IL-18 TG (11 wk old), immunoglobulin/IL-18 TG (10–12 wk old) and their age-matched WT mice were topically applied by 10 L of 2% (v/v) croton oil (Sigma, St Louis, Missouri) to each site on the surface of the right. The vehicle (acetone/olive oil, 4:1 v/v) was applied on the left ear as well. The thickness of each ear was measured using an engineering micrometer on blind basis before, and 6, 24, and 48 h and 7 d after application.
Induction of contact dermatitis by repeated application of TNCB
Male K5/IL-18 TG (8–11 wk old), immunoglobulin/IL-18 TG (14 wk old) and their age-matched WT mice were sensitized by topical application of 10 L of TNCB (0.25% w/v) or vehicle (acetone/olive oil, 4:1 v/v) to the external and internal surfaces of each ear. TNCB was applied six times at 1 wk intervals. The thickness of each ear was measured using an engineering micrometer on blind basis before, and 1, 4, and 24 h after TNCB application. Twenty-four hours after the sixth TNCB application, blood was drawn, and the mice were sacrificed. A 6 mm diameter punch biopsy specimen was taken from each left ear, homogenized in 1 mL 50 mM potassium phosphate buffer (pH 6.0) containing 0.5% hexadecyltrimethylammonium bromide (Sigma), centrifuged (40,000
g, 10 min), and the supernatant was stored at -80°C until analysis. The remainder of each left ear was fixed with 10% formalin-buffered saline (Nacalai Tesque) for histopathologic analysis, the right ears were excised for mRNA quantification and the cervical lymph nodes were removed for flow cytometric analysis.
Myeloperoxidase (MPO) and eosinophil peroxidase (EPO) activities
For measuring MPO activity, aliquots (25 L) of the ear tissue supernatants and 225 L of 50 mM potassium phosphate buffer (pH 6.0) containing 167 g per mL o-dianisidine dihydrochloride (Sigma) and 0.0005% hydrogen peroxide were incubated for 20 min at room temperature, and their absorbance at 450 nm was measured using a microplate reader (Spectra Max 250, Molecular Device, Sunnyvale, California). For measuring EPO activity, aliquots (50 L) of the supernatants and 100 L of 50 mM Tris–HCl buffer (pH 8.0) containing 0.1% Triton X-100, 1 mM o-phenylenediamine (Sigma) and 500 M hydrogen peroxide were incubated for 30 min at room temperature. The reaction was stopped by adding 2 M sulfuric acid (50 L) and the absorbance at 490 nm was measured using a microplate reader.
Quantification of cytokines and immunoglobulins
Cytokine and immunoglobulin concentrations were measured using specific enzyme-linked immunosorbent assay kits; for mouseIL-18 (MBL, Nagoya, Japan), mIL-4 (R&D Systems, Minneapolis, Minnesota), mouseIFN- (R&D Systems), mouseIL-1 (R&D Systems), mouseIgE (Yamasa, Choshi, Japan), and mouseIgG1 (Bethyl Laboratories, Montgomery, Texas) according to the manufacturers' instructions.
Histologic analysis
Formalin-fixed ear tissues were embedded in paraffin, cut into 2 m thick sections, and stained with hematoxylin and eosin. Alternatively, mast cell granules were identified by metachromasia with toluidine blue stain. The number of mast cells in a 2.6 mm epidermal distance on cross section of skin tissue was microscopically counted.
Flow cytometric analysis
The tissues were digested with Hanks balanced salt solution containing 1 mg per mL collagenase D (Roche, Mannheim, Germany) for 25 min at 37°C, the reaction was stopped by adding 500 mM of ethylenediamine tetraacetic acid-2Na to produce a final concentration of 10 mM, the cells were placed on ice for 5 min and then washed with RPMI-1640 medium containing 10% heat-inactivated fetal calf serum (Invitrogen, Tokyo, Japan). Flow cytometric analysis was performed using an EPICS Flow Cytometer (Beckman Coulter, Fullerton, California). B lineage cells were detected using a phycoerythrin-conjugated anti-mouseCD45/B220 monoclonal antibody (RA3-6B2, rat IgG2a, PharMingen, San Diego, California) and fluorescein isothiocyanate-conjugated affinity-purified goat anti-mouse IgM antibodies (-heavy chain-specific, LO-MM-9, Zymed, San Francisco, California). T cell subsets were detected with fluorescein isothiocyanate-anti-mouseCD4 (GK1.5, PharMingen), phycoerythrin-anti-mouseCD8a (53-6.7, PharMingen), and CyChrome-anti-mouseCD3 (2C11, PharMingen) monoclonal antibodies. fluorescein isothiocyanate-, phycoerythrin-, CyChrome-conjugated isotype-matched immuno-globulin (PharMingen) were used for flow cytometric analysis. An anti-mouseCD16/CD32 monoclonal antibody (2.4G2, PharMingen) was used to block non-specific binding.
Quantitative real-time reverse transcriptase–PCR isolated of total RNA from mouse ear
Each ear tissue was excised, immediately frozen in liquid nitrogen, pulverized using Cryo-press (Microtech, Funabashi, Japan), lysed in 1 mL Isogen (Nippon Gene, Tokyo, Japan) at room temperature, and the total RNA was isolated, according to manufacturer's instructions. First strand cDNA were synthesized from total RNA (2 g) using a Super Script Preamplification System for First-Strand cDNA Synthesis kit (Invitrogen, Tokyo, Japan). The synthesized cDNA (2 L) in a total reaction volume of 50 L comprising TaqMan 1000 reaction PCR core reagents (Applied Biosystems Japan, Tokyo, Japan), 300 nM primers and 200 nM probes was amplified using GeneAmp 5700 (Applied Biosynthesis). The mixture solution were incubated for 2 min at 50°C, denatured for 10 min at 95°C and subjected to 45 two-step amplification cycles each comprising annealing/extension at 60°C for 1 min followed by denaturation at 95°C for 15 s. Sequence-specific amplification was detected as an increased fluorescent signal of 6-carboxy-fluorescein during the amplification cycle. The primers and probes were designed using Primer Express software (Applied Biosynthesis) and synthesized by Invitrogen Japan and Applied Biosynthesis, respectively. The primer and probe sequences were as follows; mIL-18 transgene sense primer: 5'-TGGGTACTGCTGCTCTGGGT-3'; mIL-18 transgene anti-sense primer: 5'-ATTCCGTATTACTGCGGTTGTACAG-3'; mIL-18 transgene probe: 5'-CCACTGGTGACAACTTTGGCCGACTTC-3'; pro-mIL-18 sense primer: 5'-TCAGGACAAAGAAAGCCGCC-3'; pro-mIL-18 anti-sense primer: 5'-TCTGACATGGCAGCCATTGT-3'; pro-mIL-18 probe: 5'-ACCTTCCAAATCACTTCCTCTTGGCCC-3'; mIL-12 sense primer: 5'-AAATGAAGCTCTGCATCCTGC-3'; mIL-12 anti-sense primer: 5'-TCACCCTGTTGATGGTCACG-3'; mouseIL-12 probe: 5'-CACGCCTTCAGCACCCGCG-3'; m tumor necrosis factor (mTNF)- sense primer: 5'-TCTCTTCAAGGGACAAGGCTG; mouseTNF- anti-sense primer: 5'-ATAGCAAATCGGCTGACGGT; mTNF- probe: 5'-CCCG ACTACGTGCTCCTCACCCA; mIFN- sense primer: 5'-AGCTCATCC GAGTGGTCCAC-3'; mIFN- anti-sense primer: 5'-AGCAGCGACTC CTTTTCCG-3'; mIFN- probe: 5'-TGTTGCCGGAATCCAGCCTCA GG-3'; mIL-4 sense primer: 5'-GGCATTTTGAACGAGGTCACA-3'; mIL-4 anti-sense primer: 5'-AGGACGTTTGGCACATCCA-3'; mIL-4 probe: 5'-CTCCGTGCATGGCGTCCCTTCT-3'; mouseCCL20 sense primer: 5'-GGTGGCAAGCGTCTGCTC-3'; mCCL20 anti-sense primer: 5'-GCCTGGCTGCAGAGGTGA-3'; mCCL20 probe: 5'-TCCTTGCTTT GGCATGGGTACTGCTG-3'. The sense and anti-sense primers and probe for mouseGAPDH were purchased from Applied Biosystems. Amplification of the genes for mouseGAPDH was performed on all samples tested to control for variations in RNA contents and all transcript values were normalized with reference to mGAPDH mRNA levels. A no-template control was included in each amplification reaction to control for contaminating templates. For valid sample analysis, the fluorescence intensity of the no-template control was required to be zero.
Statistical analysis
The results were expressed as meanSE. Statistical analyses were carried out using SAS software (SAS Institute Inc., Cary, North Carolina). Student's t-test and repeated measure ANOVA were applied for the statistical analyses. Differences at p-values of less than 0.05 were considered to be significant.
Results
TG mice overexpressing mature IL-18 targeted to keratinocytes
Transfection experiments were carried out to confirm that the transgene construct could induce optimal secretion of mature IL-18 in vitro. Mature mouse IL-18 cDNA fused to the SP was subcloned into a pcdEF3 vector containing the human elongation factor 1 promoter and bovine poly(A) signal (pEF-IL-18SP) and aliquots (0.5–2 g) of this construct were transiently transfected into 293 cells. Mature mouse IL-18 was detected in the supernatants of the pEF-IL-18SP-transfected 293 cells by enzyme-linked immunosorbent assay analysis (data not shown). The construct design for an IL-18 TG mouse under the control of the human K5 promoter is shown in Figure 1(a). One female and three male founders that showed transgene integration were obtained. K5/IL-18 TG mice were mated with B6 mice and K5/IL-18 TG and WT offspring were generated at a male/female ratio of approximately 1:1. These K5/IL-18 TG mice were healthy at birth and grew normally. For most studies shown below, we used hemizygous mice from the no. 8 founder. The mRNA of soluble mature IL-18 (transgene) was highly expressed in the skin tissue of K5/IL-18 TG but not WT mice. The mean serum IL-18 level of 13 wk old male K5/IL-18 TG mice was 4.50.7 ng per mL (n=6), in contrast to less than the detection limit (0.26 ng per mL) in WT mice (Figure 1b). IL-1, IL-4, IFN-, and IgE were undetectable in the sera of these K5/IL-18 TG mice, whereas their serum IgG1 levels were increased with aging (data not shown). The other three founder lines of K5/IL-18 TG mice showed similar serum IL-18 levels, IL-18 transgene expressions in the skin tissues and responses for repetitive TNCB application, when compared with no. 8 founder line (data not shown).
CH reaction to TNCB
In order to determine the role of IL-18 in the CH response, we examined responsiveness in male K5/IL-18 TG, immunoglobulin/IL-18 TG and their WT mice. These mice have been sensitized with a vehicle or TNCB on the shaved abdomen. Six days after the sensitization, the vehicle and TNCB was challenged to each site on the surface of the left and right ear, respectively. No significant ear swelling was found in the TG and WT mice that have been sensitized and challenged with the vehicle. Slight ear swelling was found in the K5/IL-18 TG, immunoglobulin/IL-18 TG and WT mice that have been sensitized with the vehicle and challenged with TNCB. The mean increase of ear thickness in these mice was 62.5 m, 44.0 m, and 44.0 m, respectively. Compared with WT mice, the extent of ear thickness was slightly but significantly (p<0.05) increased in K5/IL-18 TG but not in immunoglobulin/IL-18 TG mice. The mice sensitized with TNCB showed antigen-specific ear swelling 24 h after challenge with TNCB but not the vehicle. Compared with WT mice, the extent of ear swelling was significantly increased in K5/IL-18 TG mice but not in immunoglobulin/IL-18 TG mice (Figure 2).
Figure 2.
Ear thickness of K5/IL-18 TG, immunoglobulin/IL-18 TG and their littermate WT mice after TNCB-elicited CH reaction. K5/IL-18 TG and their littermate WT, or immunoglobulin/IL-18 TG and their littermate WT mice were sensitized by topical application of 100 L of 3% (w/v) TNCB on the shaved abdomen. Six days after the sensitization, 10 L of 1% (w/v) TNCB was applied to each site on the surface of the right ear and the vehicle (acetone/olive oil, 4:1 v/v) was applied to the left ear. The ear thickness was determined 24 h after elicitation. Repeated experiments were performed, and the representative data is shown. Data represent the meanSE for six animals. **p<0.01, significantly different from the corresponding vehicle (V)-treated mice (Student's t test), ##p<0.01, significantly different from the corresponding WT mice (Student's t test).
Irritation response to a topical application of croton oil
We examined the cutaneous response after challenge of croton oil in K5/IL-18 TG and immunoglobulin/IL-18 TG mice to determine a role of IL-18 in the skin (Figure 3). After topical application of 2% croton oil, significant ear swelling was detected at 6 and 24 h, and disappeared at 48 h in WT mice. In K5/IL-18 TG mice, the significant ear swelling was sustained until 48 h after the challenge with the croton oil. There was no significant difference in the croton oil-induced ear swelling between immunoglobulin/IL-18 TG and the WT mice.
Figure 3.
Ear thickness of K5/IL-18 TG, immunoglobulin/IL-18 TG and WT mice after topical application of an irritant croton oil. The mice were topically applied by 10 L of 2% croton oil to each site on the surface of the right ear and 10 L of vehicle (acetone/olive oil, 4:1 v/v) on the left ear as well. The ear thickness was determined 6 h, 24 h, 48 h, and 7 d after application. Repeated experiments were performed, and the representative data is shown. Data represent the meanSE of five animals. ##p<0.01, significantly different from the corresponding WT mice (Student's t test).
Dermatitis caused by repetitive topical application of TNCB
As described above, CH reaction caused by TNCB and ear swelling caused by croton oil in K5/IL-18 TG mice was stronger than those in immunoglobulin/IL-18 TG and WT mice. Next, we examined the responsiveness by repetitive exposure of TNCB in the ears of K5/IL-18 TG mice to evaluate the role of IL-18 in chronic skin inflammation. The ear thickness was measured 1, 4, and 24 h after the second to sixth TNCB applications. Six repeated topical applications of TNCB to the ears induced CH responses and two peaks of ear thickness were observed 1 and 24 h after the second to sixth TNCB treatments in K5/IL-18 TG, immunoglobulin/IL-18 TG and WT mice (Figure 4); however, the extent and the duration of the ear swelling after each TNCB application was much greater in K5/IL-18 TG than observed in WT mice. In addition, the ear swelling in K5/IL-18 TG mice was cumulatively increased as it had not subsided by the next elicitation with TNCB in contrast to the results observed in WT mice. There was no significant difference in ear swelling between immunoglobulin/IL-18 TG and WT mice in the repetitive TNCB-induced chronic ear swelling analysis.
Figure 4.
Increase (%) in ear thickness following repeated application of TNCB to the ears of K5/IL-18 TG, immunoglobulin/IL-18 TG and their WT mice. TNCB (0.25%) or vehicle was applied topically to both ears of each mouse once a week for 6 wk. The ear thickness was determined 1, 4, and 24 h after the second to sixth TNCB applications. Repeated experiments were performed twice, and the representative data are shown. Data represent the meanSE for six mice for K5/IL-18 TG and four mice for immunoglobulin/IL-18 TG mice. Two group comparison was performed by repeated measures ANOVA. In K5/IL-18 TG mice (upper panel), the group effect was statistically different in TG TNCB versus WT TNCB (p=0.0089), TG TNCB versus TG vehicle (p=0.001), and WT TNCB versus WT vehicle (p
0.0001), but not in TG vehicle versus WT vehicle (p=0.2996). In immunoglobulin/IL-18 TG mice (lower panel), the group effect was statistically different in TG TNCB versus TG vehicle (p=0.0045) and WT TNCB versus WT vehicle (p=0.0007), but not in TG TNCB versus WT TNCB (p=0.0531); TG vehicle versus WT vehicle (p=0.6036).
In order to quantify inflammatory cell infiltration of the skin tissue, the MPO and EPO activities were determined and the numbers of mast cells in toluidine blue-stained specimens were counted (Figure 5). In TNCB-treated K5/IL-18 TG and WT mice, the MPO and EPO activities and numbers of mast cells were significantly higher than those in the corresponding vehicle-treated mice. Moreover, the MPO and EPO activities and numbers of mast cells were significantly higher in the TNCB-treated K5/IL-18 TG than the corresponding WT mice; however, there were no differences between the MPO or EPO activities or the numbers of mast cells in vehicle-treated WT and K5/IL-18 TG mice.
Figure 5.
MPO and EPO activities and mast cell infiltration of the inflamed lesions caused by repeated application of TNCB in K5/IL-18 TG and WT mice. The skin tissue was excised 24 h after the final application of TNCB or vehicle, the MPO and EPO activities of the tissue samples were measured to obtain indices of the numbers of neutrophils and eosinophils, respectively, and the mast cells in a 2.6 mm epidermal length were counted on toluidine blue-stained skin specimens. Data represent the meanSE value for five or six mice. *p<0.05, **p<0.01, significantly different from the corresponding vehicle-treated mice (Student's t test), #p<0.05, ##p<0.01, significantly different from the corresponding WT mice (Student's t test).
As described above, serum levels of IL-18 were higher in K5/IL-18 TG mice than those in WT mice. After the sixth TNCB treatment, the serum IL-18 levels showed a tendency to increase in K5/IL-18 TG mice (data not shown). Although the serum level of IgE was undetectable (0.25 g per mL) in both vehicle-treated K5/IL-18 TG and WT mice, the repetitive treatment with TNCB increased serum IgE levels to 0.70.3 g per mL (n=6) in K5/IL-18 TG but undetectable in WT mice. The serum levels of IL-1, IL-4, and IFN- were undetectable in both K5/IL-18 TG and WT mice treated with vehicle or TNCB (data not shown).
Histopathologic changes in the skin after repetitive treatment with TNCB
The skin tissues were harvested 24 h after the sixth applications of vehicle or TNCB, and hematoxylin and eosin sections were then microscopically examined. Whereas vehicle treatment did not induce histologic skin damages in WT mice (Figure 6aa and e, Figure 6ba), TNCB treatment induced mild acanthosis and thickness of the dermis with edema and infiltration of mononuclear cells (Figure 6ab and f, Figure 6bb). In TNCB-treated K5/IL-18 TG mice, more severe hyperkeratosis, acanthosis, migration of inflammatory cells into the epidermis (exocytosis), and swelling of the dermis were observed than seen in WT mice (Figure 6ad and h, Figure 6bd). Consistent with these histologic results, we found higher MPO and EPO activities in TNCB-treated K5/IL-18 TG mice than the corresponding WT mice (Figure 5). It is worth to note that vehicle-treated K5/IL-18 TG but not WT mice showed only but significant epidermal hyperplasia with nuclear swelling of keratinocytes and infiltration of mononuclear cells and eosinophils in the dermis (Figure 6ac and g, Figure 6bc).
Figure 6.
Histopathology of ear skin lesions caused by repeated application of TNCB in K5/IL-18 TG and WT mice. The skin tissues were excised 24 h after the final application of vehicle or TNCB and formalin-fixed/paraffin embedded sections were stained with hematoxylin and eosin (a). Toluidine blue staining was also performed for the detection of mast cells (arrow) (b). (a) A and E: vehicle-treated WT; B and F: TNCB-treated WT. C and G: Vehicle-treated TG. D and H: TNCB-treated TG. (b) A: Vehicle-treated WT. B: TNCB-treated WT. C: Vehicle-treated TG. D: TNCB-treated TG. Original magnification: (a) A–D100, E–H400; (b) A–D400. Bar=50 m.
Altered cell populations in the lymph nodes after repetitive treatment with TNCB
As shown in Table I, the percentage and absolute number of B lineage cells in lymph nodes from vehicle-treated K5/IL-18 TG mice were significantly higher than those of vehicle-treated WT mice. Following the repetitive TNCB treatment, the percentage and absolute number of B lineage cells in lymph nodes were significantly increased in WT mice; however, the number of B lineage cells was increased more significantly in K5/IL-18 TG mice compared with those in WT mice. Also the absolute number of CD4+ and CD8+ T cells was increased in K5/IL-18 TG mice after the repetitive TNCB treatment.
Table I - Lymphoid cells were isolated from the cervical lymph nodes of K5/IL-18 TG and their WT mice treated with TNCB or vehicle, as described in Materials and Methods. The cells were subjected to flow cytometric analysis with fluorescein isothiocyanate-conjugated anti-mCD4, PE-anti-mCD8, PE-anti-mCD45/B220 monoclonal antibodies, and fluorescein isothiocyanate-anti-mIgM antibody. Each value represents the meanSE
for three mice.
![Table I - Lymphoid cells were isolated from the cervical lymph nodes of K5/IL-18 TG and their WT mice treated with TNCB or vehicle, as described in Materials and Methods. The cells were subjected to flow cytometric analysis with fluorescein isothiocyanate-conjugated anti-mCD4, PE-anti-mCD8, PE-anti-mCD45/B220 monoclonal antibodies, and fluorescein isothiocyanate-anti-mIgM antibody. Each value represents the mean[plusmn]SE[mu] for three mice - Unfortunately we are unable to provide accessible alternative text for this. If you require assistance to access this image, please contact help@nature.com or the author](/common/images/table_thumb.gif)
Full table Expression of mRNA for cytokines and chemokines in skin tissue after repetitive treatment with TNCB
In order to investigate the involvement of cytokines and chemokines in the TNCB-induced chronic skin inflammation, the expression levels of mRNA for pro-IL-18, IL-12, IFN-, IL-4, TNF-, and CCL20 in ear tissues collected 24 h after the sixth TNCB application were measured using quantitative real-time reverse transcriptase–PCR. As shown in Figure 7, the repetitive treatment with TNCB increased the expression of mRNA for pro-IL-18 (not transgene-derived mature IL-18 mRNA), IFN-, IL-4, and CCL20 mRNA in both WT and K5/IL-18 TG mice. IL-12 and TNF- mRNA expressions were significantly increased by the repetitive treatment with TNCB in K5/IL-18 TG mice as compared with corresponding vehicle-treated mice. The magnitudes of the increased expression of mRNA for IFN-, IL-4, and CCL20 were 2-fold or higher in K5/IL-18 TG mice as compared with WT mice, although the increases in IFN-, IL-4, and CCL20 observed in the TG mice were not statistically significant. There were no differences between the cytokine/chemokine mRNA expression levels in vehicle-treated K5/IL-18 TG and WT mice.
Figure 7.
Expression of mRNA for cytokines and chemokines in inflamed skin of K5/IL-18 TG and WT mice. The ear tissues were excised from the mice 24 h after the sixth application of TNCB or vehicle. The total RNA was extracted from the tissues and the cytokine and chemokine expression levels were determined by semi quantitative real-time reverse transcriptase–PCR methods. All transcript values (expression ratios) were normalized according to the expression levels of a housekeeping gene GAPDH. Data represent the meanSE for three mice (each in duplicate). *p<0.05, **p<0.01, significantly different from the corresponding vehicle-treated mice (Student's t test). ND: not detectable.
Discussion
Keratinocytes are major producers of IL-18 and constitutively produce it as an inactive precursor (Companjen et al, 2000;Mee et al, 2000). Pro-IL-18 is cleaved by caspase-1 to produce biologically active IL-18, which is secreted after treatment with pro-inflammatory mediators by keratinocytes (Naik et al, 1999. In psoriasis, the immunoreactive IL-18 in extracts of psoriatic scales contained the mature form of IL-18. Thus, in chronic skin inflammation, pro-IL-18 may be rapidly processed, followed by the release of active mature IL-18 by keratinocytes.
In this study, we generated IL-18 TG mice under the control of the human K5 promoter. These TG mice exhibited high serum levels of IL-18 but not IFN-, IL-4, and IgE. The serum IL-18 concentration of the K5/IL-18 TG mice at 13 wk old, was 4.50.7 ng per mL (n=6), whereas that of age-matched WT mice was undetectable. This observation indicates that implanted mature IL-18 with immunoglobulin SP was secreted by K5-expressing keratinocytes (Sano et al, 1999) and entered the systemic circulation. In a recent study on immunoglobulin/IL-18 TG mice, B and mature T cells of which could express mature IL-18 under the control of an IgH promoter (Hoshino et al, 2001), the immunoglobulin/IL-18 TG mice exhibited high serum levels of IFN-, IL-4, and IgE. Thus, K5/IL-18 TG mice has weaker effects on aberrant production of both of Th1 and Th2 cytokines compared with immunoglobulin/IL-18 TG mice. Although the precise reason remains unclear, the lower serum IL-18 level (7.60.9 ng per mL, n=3 for immunoglobulin/IL-18 TG mice) and the differences in cell types where IL-18 is overexpressed might be associated with less aberrant cytokine production in K5/IL-18 TG mice.
K5/IL-18 TG mice used in this study exhibited no spontaneous skin abnormalities until they were about 16 wk old. In contrast, elicitation of skin abnormalities due to ICE (Yamanaka et al, 2000), IL-4 (Tepper et al, 1990), and IFN- (Carroll et al, 1997) overexpression has been demonstrated by studies on TG mice. When skin-specific ICE TG mice reached 8 wk of age, they exhibit high serum IL-18 and IL-1 levels and severe dermatitis characterized by apoptosis of keratinocytes. In the ICE TG mouse, ICE-induced apoptotic tissue injury and elevated systemic proinflammatory cytokine levels are thought to contribute to the serious dermatitis (Yamanaka et al, 2000). In IL-4 TG mice, facial atopy-like dermatitis accompanied by eosinophilic infiltration and high serum IgE production have been reported (Tepper et al, 1990). Three week old TG mice that overproduced IFN- in their skins have also been reported to exhibit skin abnormalities accompanied by accumulation of T cells, monocytic cells, and Langerhans cells (Carroll et al, 1997). Thus, the apparent skin condition of K5/IL-18 TG mice seems to differ from that of ICE TG or IL-4 TG or IFN- TG mice unless the mouse skin was subjected to any stimulation.
Compared with immunoglobulin/IL-18 TG or WT mice, the ear swelling resulting from CH reaction to TNCB was significantly enhanced 24 h after the challenge in K5/IL-18 TG mice. As a recent study (Wang et al, 2002) implicated IL-18 in the pathogenesis of CH response to oxazolone, our present data indicated a role for IL-18 in the CH response to TNCB. Furthermore, the ear swelling after application of croton oil significantly increased and was prolonged in K5/IL-18 TG but not immunoglobulin/IL-18 TG or WT mice. These observations indicate that IL-18 may act as an aggravating factor in not only nonallergic but also allergic cutaneous inflammation, such as CH. Also, IL-18 locally produced from the skin tissue but not from lymphocytes likely contributes to the aggravation of these cutaneous inflammations. The ability of IL-18 to prolong cutaneous inflammation prompts us to examine the potential role of IL-18 on chronic allergic contact dermatitis.
As was expected, the repetitive application of TNCB to the ears of K5/IL-18 TG mice elicited more severe dermatitis than that of immunoglobulin/IL-18 or WT mice. Enhanced infiltration of neutrophils, eosinophils, and mast cells was observed in the TNCB-treated K5/IL-18 TG mice in comparison with that in WT mice. Histopathologic examination revealed that TNCB treatment to the ears induced more severe epidermal hyperplasia and extravasation of inflammatory cells and edema in the dermis in K5/IL-18 TG than in WT mice (Figure 6). Besides an ability of IL-18 to induce IFN- production by T cells, natural killer cells, and natural killer T and B cells (Dinarello, 1999), direct effects of IL-18 on neutrophils (Leung et al, 2001), eosinophils (Wang et al, 2001), and basophils (Yoshimoto et al, 1999) were reported. IL-18 not only acts as a chemoattractant to neutrophils but also activates expression of TNF- mRNA by neutrophils (Leung et al, 2001). Induction of eosinophil-derived IL-8 production by IL-18 (Wang et al, 2001) may be responsible for neutrophilic migration to inflamed sites. IL-18 has also been reported to stimulate histamine and IL-4 release by basophils (Yoshimoto et al, 1999). Thus in K5/IL-18 TG mice, a continuous recruitment of inflammatory cells and production of cytokines or growth factors at the inflamed site might underlie the exacerbated inflamed cutaneous reaction, which is a result of the continuous production of IL-18 in the skin.
We investigated the possible involvement of cytokines and chemokines in TNCB-induced CH by measuring expression levels of mRNA for pro-IL-18, IL-12, IFN-, IL-4, TNF-, and CCL20 in ear skin tissues using a quantitative real-time reverse transcriptase–PCR (Figure 7). The repetitive TNCB application elicited higher expression of mRNA for Th1 (IFN-) and Th2 (IL-4) cytokines and chemokines (CCL20) in the skin tissue in K5/IL-18 TG than in WT mice. IFN- is a strong inducer of production of adhesion molecules and chemokines, including CCL20 (Homey et al, 2000). The expression of CCL20, which can be secondarily induced by IL-12 and IL-18 via IFN- production, from keratinocytes, dermal fibroblasts, dermal microvascular endothelial cells, and dendritic cells is reported to attract the memory subset of T cells and dendritic cells, especially epithelial Langerhans-type dendritic cells (Homey et al, 2000). As antigen-presenting cells and lymphocytes have been suggested to make a significant contribution to the CH response, IL-12, IFN-, and CCL20 produced at inflammatory sites may play a part in the exaggerated inflammatory reaction observed in K5/IL-18 TG mice. IL-4 is thought to be one of the factors that aggravate TNCB-induced CH (Kitagaki et al, 1999) because of a reduced severity of the response to TNCB in IL-4-deficient mice (Dieli et al, 1999) and STAT (signal transducer and activator of transcription) 6-deficient mice (Yokozeki et al, 2000). In addition, IL-4 is known to be a strong activator of mast cells. Histamine, a major chemical mediator released by activated mast cells/basophils, was reported to induce IL-18 production by human peripheral mononuclear cells (Kohka et al, 2000). Thus, in addition to the anticipated direct effect of IL-18 on the inflammatory cells, the upregulation of Th1 and Th2 cytokines and chemokines may be involved in the exacerbation of CH responses through positive feedback.
In addition to the pathologic changes in the skin, TNCB treatment also affected serum cytokine and immunoglobulin levels, and the cell numbers and populations in draining lymph nodes. Although elevated serum IgE levels have been reported in models of 2,4-dinitrofluorobenzene-induced CH (Nagai et al, 2000) and TNCB-induced CH (Yokozeki et al, 2000), the serum IgE level was not detectable in WT mice in this study. A high serum IgE level was detected only in TNCB-treated K5/IL-18 TG mice but not vehicle-treated K5/IL-18 TG mice. Thus, the repetitive TNCB-elicited CH response in this study might be a relatively weak immunologic reaction compared with other reports (Yokoze ki et al, 2000; Nagai et al, 2000). A synergistic effect of the transgene-derived IL-18, such as enhanced cutaneous IL-4 production, may have resulted in the elevated serum IgE levels in this model. As reported previously, mice exposed to contact allergens such as TNCB showed a significant increase in B lineage cells compared with irritants such as benzalkonium chloride or sodium lauryl sulfate (Gerberick et al, 1999). In this repeated TNCB-induced CH model, the percentage of B lineage cells was highest in the TNCB-treated K5/IL-18 TG mice where the strongest cutaneous inflammation was elicited (Table I). Thus the increased IgE production and B lineage cell population in draining lymph nodes implies that IL-18 enhances the immune response in this repeated TNCB-induced CH model as well as inflammation itself.
In this study, we showed that excessive production of IL-18 by keratinocytes aggravated the CH response. Recent studies showed that serum IL-18 levels in patients with atopic dermatitis and in a murine atopic dermatitis model were higher than the respective control levels (Tanaka et al, 2001). More recently,Konishi et al (2002) reported that keratinocyte-specific overexpressed IL-18 TG mice under the control of human K14 promoter developed atopic dermatitis-like skin lesions at about 24 wk under the specific pathogen free conditions (Konishi et al, 2002). Our female K5/IL-18 TG mice also show hair-loss and erosion of the back skin at about 18 wk after birth (manuscript in preparation); however, we did not observe spontaneous skin disorder in the 7 to 17 wk old K5/IL-18 TG mice that we used in this study. These findings suggest that IL-18 acts as a factor that causes deterioration rather than induction, of allergic and nonallergic skin orders. Further analysis should be needed to clarify this issue.
In conclusion, K5/IL-18 TG mice exhibited more severe TNCB-induced CH than immunoglobulin/IL-18 TG or WT mice. Application of an irritant (croton oil) induced stronger and more sustained ear swelling in K5/IL-18 TG mice than in immunoglobulin/IL-18 TG or WT mice. Repetitive topical application of TNCB to their ears elicited a stronger cutaneous inflammation in K5/IL-18 TG mice than seen in immunoglobulin/IL-18 TG or WT mice. In addition to a direct effect of IL-18 on inflammatory cells, upregulation of various cytokines and chemokines by IL-18 at inflamed sites may contribute to the exacerbation of cutaneous inflammation in these TG mice. Our results suggest that overexpression of IL-18 in the skin is a critically important therapeutic target in both allergic and nonallergic dermatitis.
References
| 1. | Carroll JM, Crompton T, Seery JP & Watt FM. Transgenic mice expressing IFN-gamma in the epidermis have eczema, hair hypopigmentation, and hair loss. J Invest Dermatol (1997) 180: 412–422. |
| 2. | Companjen AR, van der Velden VH, Vooys A, Debets R, Benner R & Prens EP. Human keratinocytes are major producers of IL-18: Predominant expression of the unprocessed form. Eur Cytokine Netw (2000) 11: 383–390. | PubMed | ISI | ChemPort | |
| 3. | Dieli F, Sireci G, Scire E, Salerno A & Bellavia A. Impaired contact hypersensitivity to trinitrochlorobenzene in interleukin-4-deficient mice. Immunology (1999) 98: 71–79. | Article | PubMed | ISI | ChemPort | |
| 4. | Dinarello CA. IL-18: A TH1-inducing, proinflammatory cytokine and new member of the IL-1 family. J Allergy Clin Immunol (1999) 103: 11–24. | Article | PubMed | ISI | ChemPort | |
| 5. | Gerberick GF, Cruse LW & Ryan CA. Local lymph node assay: Differentiating allergic and irritant responses using flow cytometry. Methods (1999) 19: 48–55. | Article | PubMed | ISI | ChemPort | |
| 6. | Gocinski BL & Tigelaar RE. Roles of CD4+ and CD8+ T cells in murine contact sensitivity revealed by in vivo monoclonal antibody depletion. J Immunol (1990) 144: 4121–4128. | PubMed | ISI | ChemPort | |
| 7. | Homey B, Dieu-Nosjean MC & Wiesenborn A et al. Up-regulation of macrophage inflammatory protein-3 alpha/CCL20 and CC chemokine receptor 6 in psoriasis. J Immunol (2000) 164: 6621–6632. | PubMed | ISI | ChemPort | |
| 8. | Hoshino T, Wiltrout RH & Young HA. IL-18 is a potent coinducer of IL-13 in NK and T cells: A new potential role for IL-18 in modulating the immune response. J Immunol (1999) 162: 5070–5077. | PubMed | ISI | ChemPort | |
| 9. | Hoshino T, Yagita H, Ortaldo JR, Wiltrout RH & Young HA. In vivo administration of IL-18 can induce IgE production through Th2 cytokine induction and up-regulation of CD40 ligand (CD154) expression on CD4+ T cells. Eur J Immunol (2000) 30: 1998–2006. | Article | PubMed | ISI | ChemPort | |
| 10. | Hoshino T, Kawase Y & Okamoto M et al. Cutting edge: IL-18-transgenic mice. In vivo evidence of a broad role for IL-18 in modulating immune function. J Immunol (2001) 166: 7014–7018. | PubMed | ISI | ChemPort | |
| 11. | Kitagaki H, Kimishima M, Teraki Y, Hayakawa J, Hayakawa K, Fujisawa S & Shiohara T. Distinct in vivo and in vitro cytokine profiles of draining lymph node cells in acute and chronic phases of contact hypersensitivity: Importance of a type 2 cytokine-rich cutaneous milieu for the development of an early-type response in the chronic phase. J Immunol (1999) 163: 1265–1273. | PubMed | ISI | ChemPort | |
| 12. | Kohka H, Nishibori M & Iwagaki H et al. Histamine is a potent inducer of IL-18 and IFN-gamma in human peripheral blood mononuclear cells. J Immunol (2000) 164: 6640–6646. | PubMed | ISI | ChemPort | |
| 13. | Konishi H, Tsutsui H & Murakami T et al. IL-18 contributes to the spontaneous development of atopic dermatitis-like inflammatory skin lesion independently of IgE/stat6 under specific pathogen-free conditions. Proc Natl Acad Sci USA (2002) 99: 11340–11345. | Article | PubMed | ChemPort | |
| 14. | Leung BP, Culshaw S & Gracie JA et al. A role for IL-18 in neutrophil activation. J Immunol (2001) 167: 2879–2886. | PubMed | ISI | ChemPort | |
| 15. | Mee JB, Alam Y & Groves RW. Human keratinocytes constitutively produce but do not process interleukin-18. Br J Dermatol (2000) 143: 330–336. | Article | PubMed | ISI | ChemPort | |
| 16. | Nagai H, Ueda Y, Ochi T, Hirano Y, Tanaka H, Inagaki N & Kawada K. Different role of IL-4 in the onset of hapten-induced contact hypersensitivity in BALB/c and C57BL/6 mice. Br J Pharmacol (2000) 129: 299–306. | Article | PubMed | ISI | ChemPort | |
| 17. | Naik SM, Cannon G & Burbach GJ et al. Human keratinocytes constitutively express interleukin-18 and secrete biologically active interleukin-18 after treatment with pro-inflammatory mediators and dinitrochlorobenzene. J Invest Dermatol (1999) 113: 766–772. | Article | PubMed | ISI | ChemPort | |
| 18. | Nakanishi K, Yoshimoto T, Tsutsui H & Okamura H. Interleukin-18 regulates both Th1 and Th2 responses. Annu Rev Immunol (2001) 19: 423–474. | Article | PubMed | ISI | ChemPort | |
| 19. | Okamura H, Tsutsi H & Komatsu T et al. Cloning of a new cytokine that induces IFN-gamma production by T cells. Nature (1995) 378: 88–91. | Article | PubMed | ISI | ChemPort | |
| 20. | Sano S, Itami S & Takeda K et al. Keratinocyte-specific ablation of Stat3 exhibits impaired skin remodeling, but does not affect skin morphogenesis. EMBO J (1999) 18: 4657–4668. | Article | PubMed | ISI | ChemPort | |
| 21. | Tanaka T, Tsutsui H & Yoshimoto T et al. Interleukin-18 is elevated in the sera from patients with atopic dermatitis and from atopic dermatitis model mice, NC/Nga. Int Arch Allergy Immunol (2001) 125: 236–240. | Article | PubMed | ISI | ChemPort | |
| 22. | Tepper RI, Levinson DA, Stanger BZ, Campos-Torres J, Abbas AK & Leder P. IL-4 induces allergic-like inflammatory disease and alters T cell development in transgenic mice. Cell (1990) 62: 457–467. | Article | PubMed | ISI | ChemPort | |
| 23. | Thomas JA, Allen JL & Tsen M et al. Impaired cytokine signaling in mice lacking the IL-1 receptor-associated kinase. J Immunol (1999) 163: 978–984. | PubMed | ISI | ChemPort | |
| 24. | Wang B, Feliciani C, Howell BG, Freed I, Cai Q, Watanabe H & Sauder DN. Contribution of Langerhans cell-derived IL-18 to contact hypersensitivity. J Immunol (2002) 168: 3303–3308. | PubMed | ISI | ChemPort | |
| 25. | Wang W, Tanaka T, Okamura H, Sugita M, Higa S, Kishimoto T & Suemura M. Interleukin-18 enhances the production of interleukin-8 by eosinophils. Eur J Immunol (2001) 31: 1010–1016. | Article | PubMed | ISI | ChemPort | |
| 26. | Wild JS, Sigounas A, Sur N, Siddiqui MS, Alam R, Kurimoto M & Sur S. IFN-gamma-inducing factor (IL-18) increases allergic sensitization, serum IgE, Th2 cytokines, and airway eosinophilia in a mouse model of allergic asthma. J Immunol (2000) 164: 2701–2710. | PubMed | ISI | ChemPort | |
| 27. | Xu B, Aoyama K & Yu S et al. Expression of interleukin-18 in murine contact hypersensitivity. J Interferon Cytokine Res (1998) 18: 653–659. | PubMed | ISI | ChemPort | |
| 28. | Xu D, Trajkovic V & Hunter D et al. IL-18 induces the differentiation of Th1 or Th2 cells depending upon cytokine milieu and genetic background. Eur J Immunol (2000) 30: 3147–3156. | Article | PubMed | ISI | ChemPort | |
| 29. | Xu H, DiIulio NA & Fairchild RL. 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 (1996) 183: 1001–1012. | Article | PubMed | ISI | ChemPort | |
| 30. | Yamanaka K, Tanaka M & Tsutsui H et al. Skin-specific caspase-1-transgenic mice show cutaneous apoptosis and pre-endotoxin shock condition with a high serum level of IL-18. J Immunol (2000) 165: 997–1003. | PubMed | ISI | ChemPort | |
| 31. | Yokozeki H, Ghoreishi M & Takagawa S et al. Signal transducer and activator of transcription 6 is essential in the induction of contact hypersensitivity. J Exp Med (2000) 191: 995–1004. | Article | PubMed | ISI | ChemPort | |
| 32. | Yoshimoto T, Tsutsui H & Tominaga K et al. IL-18, although antiallergic when administered with IL-12, stimulates IL-4 and histamine release by basophils. Proc Natl Acad Sci USA (1999) 96: 13962–13966. | Article | PubMed | ChemPort | |
| 33. | Yoshimoto T, Mizutani H & Tsutsui H et al. IL-18 induction of IgE: Dependence on CD4+ T cells, IL-4 and STAT6. Nature Immunol (2000) 1: 132–137. | Article | PubMed | ISI | ChemPort | |
Acknowledgments
We thank Dr Howard A. Young (NCI-Frederick, Frederick, MD) and Dr Hideo Yagita (Juntendo University, Tokyo, Japan) for helpful discussions regarding this manuscript, and Masanori Nakamura and Akiko Shibata for expert technical assistance. Dr T. Hoshino is supported by the Japan Chemical Industry Association (Tokyo, Japan), Uehara Memorial (Tokyo, Japan), Kanae (Osaka, Japan), Nagao Memorial (Tokyo, Japan), Kaibara Morikazu Medical Science Promotion (Fukuoka, Japan), Ishibashi (Tokyo, Japan) and Mitsui Medical Science Promotion (Tokyo, Japan) Foundations, and a Grant-in-Aid for Scientific Research on Priority Areas (C) "Medical Genome Science" from the Ministry of Education, Science, Sports and Culture of Japan.
