Persistent secretion of IL-18 in the skin contributes to IgE response in mice

Hiroki Nakano1,2, Hiroko Tsutsui1,5, Makoto Terada1,3, Koubun Yasuda1,5, Kiyoshi Matsui3, Shizue Yumikura-Futatsugi1,5, Kei-ichi Yamanaka4,5, Hitoshi Mizutani4,5, Takehira Yamamura2 and Kenji Nakanishi1,5

1 Department of Immunology & Medical Zoology, 2 Second Department of Surgery and 3 Department of Internal Medicine, Hyogo College of Medicine, Nishinomiya 663-8501, Japan 4 Department of Dermatology, Mie University School of Medicine, Tsu 514-8507, Japan 5 Core Research for Evolutional Science and Technology, Japan Science and Technology Corp., Tokyo 101-0062, Japan

Correspondence to: K. Nakanishi, Department of Immunology & Medical Zoology, Hyogo College of Medicine, 1-1 Mukogawa-cho, Nishinomiya 663-8501, Japan. E-mail: nakaken{at}hyo-med.ac.jp
Transmitting editor: T. Kurosaki


    Abstract
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
After exposure of the skin to microbes, the host develops skin-specific inflammation and an acquired immune response, in which keratinocytes (KC) and Langerhans cells play critical roles respectively. We established two animal models. (i) We examined the importance of KC-derived IL-18 for the systemic IgE response by using a skin transplantation model. As previously reported, transgenic mice (KCASP1Tg), that over-express caspase-1 in their KC, display high serum levels of IgE, and spontaneously develop chronic dermatitis by production of IL-18 and IL-1ß. We examined the capacity of transplantation of cutaneous lesions from KCASP1Tg to induce IgE production in wild-type or mutant mice with a syngeneic background. Transplantation of active cutaneous lesions, that expressed high levels of IL-18 and IL-1ß, induced long-lasting IgE production in wild-type mice without elevation of circulating IL-18 and IL-1ß. Furthermore, IL-18R-, CD4- or stat6-deficient mice transplanted with the lesions did not produce IgE, indicating that this IgE response is initiated by IL-18, and dependent on host-derived CD4+ T cells and stat6. (ii) We investigated IL-18 secretion from KC upon stimulation with microbe products. Freshly isolated KC from wild-type mice secreted IL-18 in response to Protein A purified from Cowan 1 strain of Staphylococcus aureus (SpA), which often exacerbates human skin diseases, including atopic dermatitis. Cutaneous application of SpA increased serum levels of IL-18 and IgE. These results indicate that local accumulation of IL-18 triggers systemic IgE responses without exposure to antigen.

Keywords: atopic disease, caspase-1, keratinocyte, skin transplantation, Staphylococcus aureus protein A


    Introduction
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Skin is the largest organ in the body and the front line of the host defense. Epidermis consists of keratinocytes (KC), melanocytes, epidermal Langerhans cells (LC) and intraepithelial T cells (15). LC are immature dendritic cells (DC), whose function is to capture and transport locally exposed protein antigen to draining lymph nodes, in which systemic adaptive immune responses are generally accomplished. During their migration to lymph node, LC develop into mature DC with professional antigen-presenting capacity, which trigger development of systemic immunity specific for the antigen the LC/DC carry (13). Inversely, the antigen-specific immune response in the skin is closely associated with the systemic response to the same antigen. Therefore, skin and immune organs appear to be tightly connected with each other via the circulatory trooping of LC/DC and antigen-specific immune cells. In contrast, KC and melanocytes reside in the skin, and do not seem to principally participate in the development of adaptive immune responses. However, KC might contribute to development of local innate immunity and local inflammation, and might potentially modify adaptive immunity by influencing LC, presumably based on their unique property of producing various cytokines upon stimulation with microbes or chemical reagents (4,5). Therefore, it is important to determine whether KC-induced cutaneous inflammation can also affect the systemic immune response.

We have established caspase-1-transgenic mice (KCASP1Tg) that KC-specifically express caspase-1 and develop atopic dermatitis (AD)-like skin lesions in an IL-18- and IL-1ß-dependent manner (6,7). These cytokines are produced as biologically inactive precursors and released as the active form after being cleaved by the appropriate intracellular enzymes such as caspase-1 (812). IL-18 has diverse biological actions depending on its immunological environment (1320). In the presence of IL-12, IL-18 promotes inflammatory responses via induction of IFN-{gamma}, a potent pro-inflammatory cytokine (1316,20). In contrast, in the absence of IL-12, IL-18 initiates atopic responses via induction of production of Th2-related cytokines including IL-4, an essential cytokine for induction of IgE response (1720). We also showed that IL-1ß enhances the capacity of IL-18 to induce an atopic response (7). These results suggested that KC may also contribute to the systemic immune response by the production of several cytokines including IL-18 and IL-1ß.

In this study, we investigated whether skin having activated KC solely has the potential to induce systemic immune responses. To test this we examined the capacity of lesion skin of KCASP1Tg to induce Th2 response by its transplantation onto syngeneic wild-type mice. We found that the active cutaneous lesion skin of KCASP1Tg has the potential to induce elevation of serum levels of IgE in the host without apparent elevation of IL-18 in their circulation. However, mutant mice deficient in CD4 or stat6, that is essential to transduce IL-4 signaling (21,22), failed to produce IgE upon challenge with the skin graft. Furthermore, IL-18 receptor (IL-18R) {alpha}-deficient mice lacking responsiveness to IL-18 (23) did not show elevation of IgE after the skin transplantation. These results clearly indicate that IL-18 in the skin causes high serum levels of IgE, depending on host-originated CD4+ T cells, stat6- and IL-18R-expressing cells. Furthermore, we examined the ability of IL-18 from activated KC to induce the IgE response in mice. Here, we demonstrated that cutaneous application of Protein A purified from Staphylococcus aureus Cowan 1 (SpA) (24) induced IgE production through release of IL-18, but not IL-12, from KC. Thus, local release of IL-18 in the skin might be sufficient for IgE induction in mice. These results show the importance of IL-18 production from KC upon stimulation with some microbe products, providing new insights into the mechanisms that may be relevant to the pathogenesis of allergic disorders with unidentified antigens.


    Methods
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Mice
Female C57BL/6 (B6) wild-type mice (6–10 weeks old) were purchased from CLEA Japan (Osaka, Japan). CD4-deficient mice on a B6 background (female, 6–10 weeks old) were kindly provided by Dr Taniguchi (University of Tokyo, Tokyo, Japan). Stat6-deficient mice generated on a B6 background (female, 6–10 weeks old) were a kind gift from Dr Takeda (Osaka University, Osaka, Japan) (22). IL-18R{alpha}-deficient mice were backcrossed with B6 mice and F10 mice (female, 6–10 weeks old) were kindly provided by Dr Hoshino (Osaka University), and used for this study (23). KCASP1Tg were described elsewhere (6,7,18), and female mice suffering from chronic dermatitis with high serum levels of IgE (10–12 µg/ml) and IL-18 (5 to 7 ng/ml) were selected, and used as donors of skin grafts. Caspase-1-deficient mice were crossed with B6 wild-type mice and F6 female mice (6–10 weeks old) were used for this study (25). Myeloid differentiation factor 88 (MyD88)-deficient mice on a B6/129 background and F4 mice of Toll-like receptor (TLR) 2-deficient mice backcrossed with B6 wild-type mice were kindly provided by Dr Akira (Osaka University), and female mice (6–10 weeks old) were used for this study (25). All mice were maintained under specific pathogen-free conditions.

Reagents
SpA purified from S. aureus Cowan 1 was purchased from Calbiochem (La Jolla, CA). Lipopolysaccharide (LPS) from Escherichia coli (O55:B5) was purchased from Difco (Detroit, IL). Murine Fas ligand transfectant (mFasL) was described elsewhere (9). z-VAD-FMK (ZVAD), a broad caspase inhibitor, and Ac-YCAD-CMK (YVAD), a caspase-1 inhibitor, were purchased from Peptide Institute (Osaka, Japan). The culture medium generally used in this study was RPMI 1640 containing 10% FCS, 100 U/ml penicillin, 100 µg/ml streptomycin, 50 µM 2-mercaptoethanol and 2 mM L-glutamine. PAM212, a mouse KC cell line, was kindly provided by Dr Tamaki (University of Tokyo).

Skin transplantation
Skin specimens (1 cm2) were prepared from wild-type B6 mice, or from lesion or non-lesion skin of KCASP1Tg and were transplanted onto the back of wild-type B6, CD4-deficient, stat6-deficient or IL-18R{alpha}-deficient B6 mice. Two pieces of skin specimens were prepared from lesion and non-lesion skin of one KCASP1Tg for grafts. After skin transplantation, the recipients were fed with drinking water supplemented with 1 mg/ml neomycin sulfate (Sigma, St Louis, MO) and 1000 U/ml polymixin B sulfate (Sigma) to prevent any possible infection. At the indicated time points, serum was sampled for determining the IgE concentration. Graft survival in each host transplanted was monitored for up to 48 days. For histological study, we stained skin specimens with hematoxylin & eosin as described previously (6,7).

Skin lysate
Skin specimens (1 cm2) were prepared from wild-type B6 mice or KCASP1Tg. The epidermal sheets prepared from the skin specimens were homogenized in PBS at 4°C and filtered. The concentrations of various cytokines and protein in each lysate were measured by ELISA kits for cytokines (described below) and by protein assay reagent (Pierce, Rockford, IL) respectively.

Th1/Th2 differentiation
Splenic CD4+ T cells were isolated from variously treated mice by using AutoMACS (Miltenyi Biotec, Auburn, CA) following the incubation of freshly isolated spleen cells with anti-CD4 beads (Miltenyi Biotec). The purity of CD4+ T cells was >98%. The cells (1 x 106/ml) were incubated with immobilized anti-CD3{epsilon} (PharMingen, San Diego, CA) for 48 h, and the concentrations of IFN-{gamma} and IL-4 in each supernatant were determined by ELISA.

Assay for cytokines and IgE
IL-18 concentration was determined by an ELISA kit (MBL, Nagoya, Japan). IL-4, IFN-{gamma} and IL-1ß were measured by an ELISA kit (Genzyme TECHNE, Minneapolis, MN). The IL-5 ELISA kit was purchased from Endogen (Woburn, MA). The IgE serum level was determined by ELISA (PharMingen) according to the manufacturer’s instructions. IFN-{gamma}-inducing activity of IL-18 was determined by bioassay using IL-18-responsive mouse NK cell clones, designated as LNK series, as shown previously (9,26,27). Briefly, LNK5E6 cells, having higher responsiveness to IL-18 in terms of IFN-{gamma} production than LNK5E3 cells (9), were incubated with various samples plus 100 pg/ml rIL-12 in the presence or absence of anti-IL-18 antibody (50 µg/ml) for 48 h. IL-18 activity was defined as concentration of IFN-{gamma} produced by the cells in response to IL-18 as follows (9): IL-18 activity = (IFN-{gamma} in the supernatant without anti-IL-18 antibody) – (IFN-{gamma} in the supernatant with anti-IL-18 antibody).

Application of SpA
Daily, we applied various doses of SpA in 5 µl vehicle, 50% glycerol in PBS, to wild-type ear skin for 14 days. For the control study, 5 µl vehicle without SpA was used. We kept one mouse in a separate cage to prevent the effects from other mice. The serum levels of IL-18 and IgE were measured by ELISA.

Preparation of KC
KC were prepared from various genotypes of mice according to the method described by Tamaki et al. (28,29) and were incubated with the medium overnight for normal recovery of their surface molecular expression. To delete DC, CD11c+ cells were depleted by using AutoMACS after incubation of the KC preparation with CD11c–micro-beads (Miltenyi Biotec). KC (5 x 105/ml) or PAM212 cells (2 x 105/ml) were incubated with various doses of SpA and 1 µg/ml LPS or mFasL (1 x 106/ml) for 24 h. In some experiments wild-type KC were incubated with 100 µg/ml SpA in the presence of 20 µM ZVAD or YVAD for 24 h. IL-18 concentration and its activity in each supernatant were determined by ELISA and bioassay, respectively. In some experiments, KC were incubated with 500 µg/ml SpA for 4 h and total RNA was extracted, followed by RT-PCR (27). Primers for IL-18, IL-12p35, IL-12p40 and ß-actin, and PCR conditions for individual cytokines are shown elsewhere (27).

Preparation of Kupffer cells
Kupffer cells were prepared as previously reported (9). Kupffer cells (1 x 106/ml) were incubated with 1 µg/ml LPS for 4 h, and their IL-18, IL-12p35, IL-12p40 and ß-actin expression as mRNA levels determined by RT-PCR.

FACS analysis
Proportions of DC, CD4+ T cells and CD8+ T cells were determined by two-color flow cytometric analysis after incubation of KC preparations with phycoerythrin (PE)-conjugated anti-CD11c (PharMingen) and FITC-conjugated anti-I-Ab (PharMingen) or PE-conjugated anti-CD4 (PharMingen) and FITC-conjugated anti-CD8 (PharMingen) as previously described (9).

Statistics
All data are shown as the mean ± SD of triplicate samples. Significance between the control group and a treated group was examined with the unpaired Student’s t-test. P < 0.05 was considered significant.


    Results
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Induction of IgE by transplantation of skin graft from KCASP1Tg
We first investigated whether lesion skin of KCASP1Tg has the potential to induce elevation of serum levels of IgE in syngeneic normal wild-type mice. To normalize the conditions of skin grafts in each transplantation, we selected donor KCASP1Tg that had suffered from chronic dermatitis in their ears, face and trunk, and had certain levels of IL-18 and IgE in their sera (described in Methods). We transplanted the grafts of lesion or non-lesion skin of KCASP1Tg onto normal B6 mice and measured host serum levels of IgE. As shown in Fig. 1, serum levels of IgE in the host increased promptly after transplantation with the lesion skin of KCASP1Tg, while non-lesion skin-grafted B6 mice showed delayed and poor IgE responses. Control B6 mice, that had been transplanted with wild-type skin grafts, exhibited no elevation of IgE (Fig. 1). Lesion skin-grafted mice showed high IgE levels in their sera, but decreased levels at the time of graft detachment, while non-lesion skin-grafted mice maintained low IgE levels (Fig. 1). However, to our surprise, lesion skin-grafted mice started to increase their serum IgE levels even after rejection of active skin lesion grafts (Fig. 1). Serum levels of IL-18 were not elevated after challenge with any type of skin grafts (data not shown). Serum IL-4 or IL-6 were undetectable by commercially available ELISA kits (data not shown). These results indicate that lesion skin of KCASP1Tg is capable of inducing long-lasting systemic IgE elevation when transplanted onto normal B6 mice, contrasting with exogenous IL-18-dependent IgE production that ceased immediately after stopping administration of IL-18 (our unpublished data).



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Fig. 1. IgE induction by transplantation of lesion skin from KCASP1Tg. Normal B6 mice were transplanted with lesion (closed circles and bar, upper photo) or non-lesion (hatched circles and bar, middle photo) skin from KCASP1Tg, or with normal skin from wild-type mice (open squares and bar, lower photo). At the indicated time points, sera were sampled for measurement of IgE by ELISA. Graft survival was observed. The data represent mean + SD of three mice in each experimental group. The graft survival is shown in the upper panel. The similar results were obtained by three independent experiments.

 
Accumulation of IL-18- and Th2-related cytokines in lesion skin of KCASP1Tg
To understand the mechanism by which only transplantation of the skin lesion can efficiently induce IgE responses in the host, we compared the concentration of IL-18 between the skin grafts with and without lesions. Lesion skin of KCASP1Tg displayed high levels of IL-18 measured by ELISA, which can detect both precursor and mature forms of IL-18. In contrast, the IL-18 concentration was low in the non-lesion skin lysate from KCASP1Tg and lowest in the skin lysate from wild-type (Fig. 2A). As previously reported, immunoblotting analyses for IL-18 revealed that cutaneous lesions of KCSP1Tg express both bioactive IL-18 of 18 kDa and precursor IL-18 of 24 kDa, and that wild-type skin and non-lesion skin of KCASP1Tg express only 24 kDa IL-18 (6). To confirm this we performed bioassay for IL-18 (9,26,27). Lesion skin of KCASP1Tg showed a high titer of IFN-{gamma}-inducing activity of IL-18, while non-lesion skin showed little activity (Fig. 2B). As expected, normal skin of B6 mice had no such biologically active IL-18 (Fig. 2B). In addition to IL-18, IL-1ß, another product of caspase-1 in the KC (6,8,12) was also condensed only in lesion skin (Fig. 2C). Thus, the IgE concentration in the hosts paralleled that of IL-18 and IL-1ß levels in the graft transplanted.



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Fig. 2. Predominant accumulation of IL-18 in lesion skin of KCASP1Tg. The lesion (closed bars) or non-lesion (hatched bars) skin specimens were sampled from two KCASP1Tg or control specimens were from three wild-type mice (open bars). Epidermal sheet lysate was prepared from each specimen. IL-18 (A), IL-1ß (C), IFN-{gamma} (D), IL-4 (E) or IL-5 (F) concentrations in each lysate were determined by ELISA. IL-18 activity in various samples was determined by IFN-{gamma} production by IL-18-responsive LNK5E6 cells (described in Methods). IFN-{gamma} levels were determined by ELISA (B). The data indicate mean ± SD of triplicates in each sample. Similar results were obtained from three independent experiments. ND, not detected.

 
Next, we measured concentrations of IFN-{gamma}, IL-4 and IL-5 in the lysate of lesion skin of KCASP1Tg. IFN-{gamma}, IL-4 and IL-5 levels were all increased in the lesion skin of KCASP1Tg as compared with wild-type or KCASP1Tg non-lesion skin lysate (Fig. 2D–F). Thus, the lesion skin graft stored large amounts of IL-18, IL-1ß, IFN-{gamma}, IL-5 and IL-4.

To investigate which types of cells accumulated in the lesion skin of KCASP1Tg, we measured the proportions of CD4+ T cells, a possible cell source of Th1- and Th2-related cytokines, and CD8+ T cells, potent IFN-{gamma}-producing cells, in the KC preparation by FACS. As shown in Fig. 3, the CD4+ T cell number was substantially elevated in the lesion skin, but not in the non-lesion skin, as compared with wild-type mice. There is no difference in the CD8+ T cell or DC proportions among these three types of recipients (Fig. 3). Consistent with our previous report (7), mast cell number was remarkably increased in the lesion skin, but not in the non-lesion skin, of KCASP1Tg as compared with wild-type mice (data not shown). Thus, CD4+ T cells and mast cells are preferentially accumulated in the lesion skin of KCASP1Tg.



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Fig. 3. Accumulation of CD4+ T cells in lesion skin of KCASP1Tg. KC preparations were prepared from normal skin of wild-type mice (wild-type) or non-lesion (Tg/non-lesion) and lesion (Tg/lesion) skin of KCASP1Tg mice. Individual KC preparations were incubated with PE-conjugated anti-CD4 and FITC-conjugated anti-CD8 (upper panels) or PE-conjugated anti-CD11c and FITC-conjugated anti-I-Ab (lower panels). Proportions of CD4+ T cells or CD8+ T cells, or CD11c+ I-Ab+ DC are shown in the upper and lower panels respectively. Data represent one of three mice in each experimental group. Similar results were obtained from three independent experiments.

 
Induction of Th1 and Th2 cells in active skin lesion-grafted mice
Because IgE response usually requires activation of Th2 cells (21,30,31), we next tested whether CD4+ T cells in the host develop into Th2 cells after lesion skin transplantation. As shown in Fig. 4, splenic CD4+ T cells from hosts at day 21 after the transplantation of lesion skin of KCASP1Tg produced higher amounts of both IL-4 and IFN-{gamma} in response to immobilized anti-CD3 compared with hosts transplanted with normal B6 skin. In contrast, splenic CD4+ T cells from recipients transplanted with non-lesion skin produced comparable amounts of IL-4 and IFN-{gamma} in response to plate-bound anti-CD3 as those from recipients with B6 wild-type skin (Fig. 4). At day 7, however, splenic CD4+ T cells from the recipients transplanted with lesion skin or non-lesion skin of KCASP1Tg secreted comparable amounts of IL-4 and IFN-{gamma} as in control recipients transplanted with normal skin from wild-type mice (data not shown). These results indicate that it takes 2–3 weeks to develop Th1/Th2 cells after challenge with the lesion graft. Thus, accumulation of IgE in vivo does not entirely require selective Th2 cell development like the mutant mice over-releasing IL-18 (6,7,18,19).



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Fig. 4. Absence of selective Th2 cell development in hosts transplanted with lesion skin. Normal B6 mice were transplanted with lesion skin of KCASP1Tg or normal skin of wild-type mice. At day 21, splenic CD4+ T cells were incubated with immobilized anti-CD3 for 48 h. IL-4 (closed bars) and IFN-{gamma} levels (open bars) in each supernatant were determined by ELISA. The data indicate mean ± SD of triplicates. Similar results were obtained from three independent experiments. NS; not significant.

 
IL-18-, CD4- and stat6-dependent IgE induction
We analyzed cellular and molecular mechanisms underlying KCASP1Tg lesion-induced IgE. As CD4+ T cell-depleted mice did not show any elevation of serum levels of IgE after treatment with IL-18 (18,19), we tested whether host-derived CD4+ T cells are essentially required or whether lesion-infiltrating IL-4-producing donor T cells (Fig. 2E) are equipped with the complete IgE-inducing machinery. CD4-deficient mice did not produce IgE even after transplantation with lesion skin of KCASP1Tg (Fig. 5A). These results indicate that infiltrates in the skin graft of KCASP1Tg are incapable of inducing systemic elevation of IgE without host CD4+ T cells. As stat6 is essential for signal transduction of IL-4 (22), we next analyzed whether host-derived stat6 is critical for the IgE induction. As shown in Fig. 5(A), stat6-deficient mice did not show IgE elevation after lesion skin transplantation, suggesting that the skin graft-mediated IgE response is host-derived CD4+ T cell and stat6 dependent. Transplantation with normal B6 skin also did not induce IgE production in CD4- or stat6-deficient hosts (data not shown). Therefore, IgE induction by skin transplantation occurs only when the host is equipped with intact CD4+ T cells and stat6.



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Fig. 5. Requirement of host CD4+ T cells, stat6 and IL-18 responsiveness for induction of IgE, and Th1/Th2 development. (A) Wild-type, CD4-deficient (CD4KO), stat6-deficient (stat6KO) or IL-18R{alpha}-deficient (IL-18RKO) mice were transplanted with the lesion skin of KCASP1Tg. At day 0 (pre, open bars) or day 21 (post, closed bars), serum was sampled for measurement of IgE. The data indicate mean ± SD of three mice in each experimental group. (B) IL-18R{alpha}-deficient mice were transplanted with B6 wild-type normal skin or lesion from KCASP1Tg (Tg/lesion). At day 21 splenic CD4+ T cells were isolated from these recipient mice and stimulated with immobilized anti-CD3 for measuring IL-4 and IFN-{gamma} production by ELISA. The data indicate mean ± SD of one of three mice in each experimental group. Similar results were obtained from three independent experiments. ND, not detected. NS, not significant.

 
Next, we investigated whether IL-18 in the graft causes induction of IgE in the host or not. For this purpose, we used IL-18R{alpha}-deficient mice that cannot respond to IL-18 (23) as hosts. IL-18R{alpha}-deficient mice failed to increase IgE (Fig. 5A). IL-18R{alpha}-deficient mice transplanted with normal B6 skin showed no elevation of IgE (data not shown). Taken together, these results indicate that persistent accumulation of a small amount of IL-18 in the skin lesion induces systemic elevation of IgE depending on host-derived CD4+ T cells and stat6.

As wild-type mice transplanted with lesion skin showed an IgE response accompanied by the deviation of CD4+ T cells into Th1 and Th2 cells (Fig. 4), we investigated whether this Th1/Th2 cell development also depends on endogenous IL-18 in the lesion graft. As shown in Fig. 5(B), CD4+ T cells from IL-18R{alpha}-deficient mice transplanted with the inflammatory skin graft produced comparable amounts of IL-4 or IFN-{gamma} as in those from mutant mice transplanted with wild-type normal skin. These results suggest that lesion graft-mediated Th1/Th2 cell development might depend on IL-18 released from the graft.

SpA induction of IL-18 and IgE in vivo
It is crucial to identify natural stimuli that induce IL-18 release without induction of IL-12 production. Because S. aureus infection occasionally exacerbates cutaneous inflammatory changes in patients with AD (32, 33) and some patients with AD showed elevated serum levels of IL-18 (34), we next investigated whether S. aureus products can cause systemic elevation of IL-18 in normal B6 mice. To test this, we daily applied SpA to ear skin of normal wild-type B6 mice for 2 weeks. Serum levels of IL-18 were increased in mice treated with SpA, but not in mice with vehicle, in a dose-dependent manner (Fig. 6A). We did not detect IL-12p40 or IL-12p70 in the sera of SpA- or vehicle-treated mice by ELISA (data not shown). IgE levels were also elevated dose dependently (Fig. 6B). In addition, SpA-treated mice did not display preferential development of Th2 cells (Fig. 6C). These results indicate that, like IL-18 treatment (18,19), SpA has the potential to induce systemic IgE without preferential development of Th1 or Th2 cells.



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Fig. 6. Induction of IL-18 and IgE by in vivo treatment with SpA. Ear skin of normal B6 mice was treated daily with various doses of SpA or vehicle alone (Veh). At day 14, sera and the spleen were sampled for determination of the concentration of IL-18 (A) or IgE (B). Splenic CD4+ T cells from vehicle- or SpA (100 µg/day)-treated mice were incubated with plate-bound anti-CD3 for 48 h, and IFN-{gamma} (open bars) and IL-4 levels (closed bars) in each supernatant were determined by ELISA (C). The data (A and B) indicate mean ± SD of three mice in each experimental group. The data (C) indicate mean ± SD of triplicate samples and represent the results from one of the three mice in each experimental group. The similar results were obtained from three independent experiments. ND, not detected. NS, not significant.

 
KC secrete IL-18, but not IL-12, in response to SpA
IL-18 without IL-12 is capable of inducing IgE, while IL-18 with IL-12 inversely inhibits IgE induction (16,17). To date, the stimuli including LPS that induce IL-18 secretion always cause IL-12 production (9,10,27). Next, we investigated what cell types secrete IL-18 and whether these cells release IL-18 without the production of IL-12 in response to SpA. After stimulation with SpA, PAM212 cells, a mouse KC line, secreted IL-18 that has the capacity to induce IFN-{gamma} production (Fig. 7A). To substantiate further whether freshly isolated KC secrete IL-18 in response to SpA, we incubated KC from wild-type B6 mice with various doses of SpA for 24 h. As shown in Fig. 7(B, left), freshly isolated KC could dose dependently release IL-18 in response to SpA. As LC/DC have the capacity to release IL-18 (20), we depleted CD11c+ cells from the KC preparation and incubated them with SpA. Even after removal of CD11c+ cells, KC showed secretion of IL-18 following stimulation with SpA (Fig. 7B, right).



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Fig. 7. Release of IL-18, but not IL-12, from SpA-stimulated KC. (A) PAM212 cells were incubated with or without SpA for 24 h. The IL-18 concentration (left panel) and IFN-{gamma}-inducing activity of IL-18 (right panel) in each supernatant were determined by ELISA and bioassay respectively. (B) KC (unfractionated, closed bars) or CD11c+ cell-depleted KC (CD11c-depleted, open bars) prepared from the skin of wild-type B6 mice were analyzed for their expression of CD11c and MHC class II (I-Ab) by flow cytometry. These cells were incubated with or without SpA for 24 h and IL-18 levels in the resulting supernatants were measured by ELISA. (C) KC were prepared from wild-type, caspase-1-deficient (ICE) (open column), TLR2-deficient (TLR2) (dotted column) or MyD88-deficient (MyD) (hatched column) mice. Wild-type KC were incubated with 500 µg/ml SpA in the presence of 20 µM ZVAD, 20 µM YVAD or the same volume of DMSO (Veh) for 24 h. Simultaneously, KC from various mutant mice were incubated with 500 µg/ml SpA for 24 h. IL-18 in each supernatant was determined by ELISA. Wild-type KC incubated with 500 µg/ml SpA without DMSO produced 28.4 ± 3.5 pg/ml IL-18. There are no significant differences in SpA-stimulated IL-18 concentrations among variously treated wild-type KC and among KC with various genotypes. (D) KC and Kupffer cells from wild-type B6 mice were incubated with or without (–) 500 µg/ml SpA or 1 µg/ml LPS respectively for 4 h. IL-18, IL-12p40, IL-12p35 and ß-actin mRNA expression levels in individual total RNA extracted were determined by RT-PCR. The data (A–C) indicate mean ± SD of triplicates. The data (D) represent three RT-PCR results of one experiment. The similar results were obtained from three independent experiments. ND, not detected.

 
Next, we investigated the molecular mechanisms underlying SpA-induced IL-18 secretion from KC. As caspase-1 is required for LPS-induced IL-18 secretion, we examined IL-18 secretion from SpA-stimulated caspase-1-deficient KC. As shown in Fig. 7(C), caspase-1-deficient KC secreted comparable levels of IL-18 as wild-type KC, indicating caspase-1-independent IL-18 secretion from KC upon stimulation with SpA. As a caspase-1 inhibitor, YVAD, and a broad caspase inhibitor, ZVAD, potently inhibit IL-18 secretion from Kupffer cells, tissue macrophages in the liver, upon LPS or membrane-associated FasL (9), we examined the effects of ZVAD or YVAD on IL-18 secretion from SpA-stimulated KC. As shown in Fig. 7(C), these two types of caspase inhibitors did not inhibit IL-18 secretion from SpA-stimulated wild-type KC, indicating a dispensable role of caspase in SpA-induced IL-18 secretion from KC. This was also the case for caspase-1-deficient KC (data not shown). As many microbe products stimulate TLR/MyD88 signaling pathways (35,36), we investigated whether KC secrete IL-18 depending on TLR2, a signaling receptor for Gram-positive bacterial products, or on MyD88, an essential adaptor molecule shared by all TLR members. As shown in Fig. 7(C), both TLR2 and MyD88 are dispensable for SpA-induced IL-18 secretion from KC, indicating TLR-independent IL-18 secretion. As Kupffer cells secrete IL-18 upon stimulation with LPS or membrane-associated FasL (9), we next examined whether these stimuli induce IL-18 release from KC. In contrast to Kupffer cells, KC did not secrete IL-18 after being stimulated with LPS or mFasL (Table 1).


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Table 1. KC do not release IL-18 upon stimulation with LPS or mFasL
 
Finally, we analyzed whether SpA-stimulated KC simultaneously produce IL-12 or not. To identify a trace amount of IL-12, we performed RT-PCR using total RNA from KC that had been incubated with SpA for 4 h. As shown in Fig. 7(D), neither IL-12p35 nor IL-12p40 mRNA was detectable in SpA-stimulated KC, whereas Kupffer cells stimulated with LPS expressed both of them, as is consistent with our previous reports (9,10,27). We performed RT-PCR for IL-12 using total RNA from KC having been incubated with SpA for 8 or 16 h and did not find either components of IL-12 in them (data not shown). We also carried out ELISA for IL-12. We did not find IL-12p40 or IL-12p70 in the resulting supernatants of SpA-stimulated KC after 24 h incubation (data not shown). In contrast, like Kupffer cells (9,10,27), KC constitutively express IL-18 and did not change the level of expression after stimulation with SpA (Fig. 7D). Kupffer cells did not secrete IL-12 or IL-18 in response to SpA (data not shown). Collectively, the present results indicate that SpA applied to skin stimulates the local KC to secrete IL-18, but not IL-12, leading to induction of IgE.


    Discussion
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Skin is a potent, front-line organ that principally protects the host from pathogens. The skin consists of various unique immune cells including LC, and also cytokine-producing non-immune cells, including KC, that are a major constituent of the epidermis. The present study demonstrated that local accumulation of IL-18 in the skin can affect the systemic immune conditions. Transplantation of an IL-18-releasing skin graft causes systemic IgE elevation in a host-derived CD4+ T cell-, stat6- and IL-18-responsiveness-dependent manner with mild Th1/Th2 cell development (Figs 1, 4 and 5). Skin application with SpA, that induces secretion of a small amount of IL-18 from KC, triggers elevation of the serum levels of IgE without development of Th2 cells (Figs 6 and 7). Various types of epithelium, including KC, respiratory epithelial cells and intestinal epithelial cells, have been shown to express IL-18 and to have potential to release IL-18, although its pathophysiological roles are unclear (37,38). Interestingly, these epithelial cells also have aspects as the target cells of various atopic diseases, such as AD, bronchial asthma and food allergy. Our present results led us to assume that accidental or incidental release of IL-18 from these epithelial cells might induce systemic IgE responses and might presumably result in the development of atopic responses. This is the first report of an allergen-independent, bacterial product-dependent IgE response, opening a new concept of atopic disorders.

Genetically engineered mice releasing high levels of IL-18 show spontaneous elevation of serum levels of IgE under specific pathogen-free conditions (6,7,18,19). IL-18 transgenic mice that over-express biologically active IL-18 in their lymphocytes increase their serum levels of IgE (39). KCASP1Tg show elevation of serum levels of IgE and spontaneous development of progressive skin inflammatory changes resembling those in AD (6,7). This was also the case for KC-specific IL-18 transgenic mice (KIL-18Tg) that over-express biologically active IL-18 selectively in their KC (7). Although both types of KC-specific transgenic mice provided us with an indication that excessive release of IL-18 causes IgE secretion, it is still to be elucidated whether IL-18 locally and persistently released in the skin simply causes systemic IgE production. In this study, we analyzed whether cutaneous lesions of KCASP1Tg have the potential to solely transfer high-level IgE-inducing conditions in normal mice. After transplantation with the cutaneous lesion, B6 mice revealed IgE production without any apparent elevation of IL-18 in their circulation. However, mutant mice deficient in CD4 or stat6, that is essential to transduce IL-4 signaling (21,22), failed to produce IgE upon challenge with the skin graft. Furthermore, IL-18 R{alpha}-deficient mice lacking responsiveness to IL-18 (23) did not show elevated IgE after skin transplantation. These results clearly indicate that IL-18 in the skin causes systemic elevation of serum levels of IgE, depending on host-originated CD4+ T cells, stat6- and IL-18R- expressing cells. Thus, local release of IL-18 in the skin might be sufficient for IgE induction in mice. These results strongly suggest the importance of activation of KC for induction of a systemic IgE response.

In spite of the possible equilibrium of distribution of transgenes in the skin of KCASP1Tg (6), IL-18 and IL-1ß were preferentially secreted in lesion skin compared with non-lesion skin (Fig. 2B). This might be, in part, because the KC extensively accumulated in the lesion skin of KCASP1Tg as compared with non-lesion skin (Fig. 1), perhaps explaining much higher concentrations of IL-18 in the former (Fig. 2A–C). Alternatively, this may be explained by the presence of scratching-induced possible activation of endogenous caspase-1 in the lesion skin (6,40). Indeed, the skin alterations in mice were limited in the area to which their limbs can reach (6), although we could not observe activated components of mouse caspase-1 in the lesions by immunoblotting because of the technical restrictions. Alternatively, cell death might be involved in IL-18 release in the limited part of the skin. To address this, we compared apoptotic cell numbers between lesion and non-lesion skin of KCASP1Tg. As previously reported, many apoptotic cells are observed in lesion skin, but not in non-lesion skin, suggesting a possible contribution of apoptotic cells to local IL-18 accumulation, although KC apoptosis preferentially observed in the lesion might be induced by the product of the transgene, human caspase-1 itself. Furthermore, IL-18 itself may enhance Fas-mediated IL-18 release by enhancing FasL expression on lymphocytes (9). Furthermore, SpA induces IL-18 release from KC without induction of apoptotic cell death (Fig. 7 and data not shown), indicating the presence of apoptosis-independent IL-18 accumulation in the skin. Thus, cell death might be involved in, but not solely relevant for, IL-18 release in the lesion skin of KCASP1Tg.

Skin grafts prepared from lesions of KCASP1Tg were detached, although the relation between host and donor mice was syngeneic. This may be partly due to the infiltration of host-derived inflammatory cells that potently produce pro-inflammatory cytokines in response to the skin graft with activated KC. Alternatively, IL-18 released from the graft might inhibit the angiogenesis (41) that is primarily required for the engraftment. Reportedly, vascular endothelial cells have the potential to express various kinds of cytokines/cytokine receptors, including tumor necrosis factor receptor, in response to various types of stress (42,43). This led us to propose the following scenario. These vascular endothelial cells in the graft might have been endogenously activated to induce recruitment of host-derived effector cells via chemokine and/or chemokine receptor interaction. The host-derived effector cells might participate in the amputation of the graft from the host. Alternatively, the activated endothelial cells might accelerate intravascular clot formation via synthesis of coagulation factors (44).

Daily administration of IL-18 causes elevation of serum levels of IgE in an IL-4-, CD40 ligand- and CD4+ T cell-dependent manner (18,19). However, IL-18-stimulated T cells cannot produce IL-4 upon TCR engagement, suggesting that treatment with IL-18 without antigen stimulation did not induce the development of Th2 cells in vivo. In contrast, T cells stimulated with both antigen and IL-18, with or without IL-12, can develop into Th1 cells and Th2 cells respectively (20). Indeed, exogenous IL-18 enhances Th2 cell and IgE responses in helminth-inoculated mice, indicating that IL-18 plus antigen induces and accelerates both Th2 cell and IgE responses (16). In the case of transplantation with lesion skin onto normal B6 mice, human caspase-1, a foreign protein for the recipients, together with a high level of IL-18 in the graft enhances both Th2 and IgE responses in the recipients (Figs 1, 4 and 5), strongly indicating that the levels of IL-18 determine the degree of IgE and Th2 response under the conditions of exposure to antigen in vivo, too. This is also supported by the fact that IL-18R{alpha}-deficient mice did not show Th2 or IgE responses even after transplantation with lesion skin (Fig. 5B). Therefore, it is expected that exogenous IL-18 enhances IgE and Th2 responses in the host transplanted with non-lesion skin. In fact, daily injection of IL-18 induces IgE response in hosts transplanted with non-lesion skin, in line with our previous study (18), although the level of development of Th1/Th2 cells was moderate as compared with the mice transplanted with lesion skin (Fig. 4 and data not shown). Collectively, these results suggest that transplantation of IL-18- plus IL-1ß-producing inflammatory skin lesions induces acquired immune responses in normal mice. LC/DC might be important for the activation of host adaptive immune responses. LC/DC may be activated by ingestion of apoptotic KC or apoptotic infiltrates in the inflammatory skin lesion and then recruit into immune organs to exert their role as antigen-presenting cells. A recent study demonstrated the importance of ingestion of apoptotic cells by DC for their presentation of the antigen that the apoptotic cells produce. CD8+ DC have the capacity to capture dying cells and present antigens derived from the dying cells to T cells (45). Lesion skin, but not non-lesion skin, of KCASP1Tg actually contains large numbers of apoptotic KC (6). The apoptotic KC in the lesion skin may activate LC/DC to induce Th1/Th2 cell differentiation, although we do not know whether host- and/or graft-derived DC are involved in this event. We are now crossing green fluorescence protein (GFP) transgenic mice and KCASP1Tg, and investigating whether GFP-expressing DC can migrate into the host immune organs to interact with host T cells after transplantation of the inflammatory skin lesion of GFP-expressing KCASP1Tg. Our results suggest that local release of IL-18 in the inflammatory skin might activate host adaptive immunity to induce long-lasting IgE response in mice. Concerning the mechanism underlying Th1/Th2 cell development in the host without exposure to apparent foreign antigen, we proposed the following possibilities. First, as described above, the product of transgene, which would not initiate adaptive immune responses in the mutant mice because of the induction of self-tolerance, might trigger adaptive immune response in wild-type host mice. Second, the lesion skin of KCASP1Tg might have potent adjuvant activity, presumably due to accumulation of various cytokines (Fig. 2). Finally, commensal bacteria in the flora might contribute to this development. We need further study.

As previously reported, caspase-1-dependent IL-18 secretion occurs after stimulation of TLR, innate immune receptors, with corresponding pathogen-associated molecular patterns (PAMP) (10,35,36). Upon stimulation with LPS, cytoplasmic domains of TLR4 are associated with MyD88, an intracellular adaptor molecule, followed by a relay of signals and activation of NF-{kappa}B. After stimulation of their TLR4 with LPS, macrophages produce IL-12 in a MyD88-dependent manner and simultaneously secrete IL-18 in a caspase-1-dependent, but MyD88-independent, manner (10). This is also the case for IL-12 and IL-18 release after infection with pathogens, such as Listeria monocytogenes, a Gram-positive intracellular bacterium (10) and Plasmodium berghei, a pathogenic protozoa (46). In contrast, SpA selectively induces IL-18 secretion from KC independently of caspase-1 or other caspases (Fig. 7C). It is intriguing to speculate that despite its potential analogy to PAMP, SpA does not require TLR2/MyD88 for its signaling (Fig. 7C) and lacks the capacity to induce IL-12 production from KC (Fig. 7D). TLR/MyD88-dependent signal pathways are potently involved in the activation of pathogen-induced Th1 cell development (35). In contrast, the SpA-triggered Th2-relating biological events (Figs 6 and 7) may imply that TLR/MyD88-independent cell activation might dominantly contribute to caspase-1-independent IL-18 secretion without production of IL-12, which might result in the activation of Th2-deviated acquired immune responses. However, it is to be elucidated whether DC can secrete both IL-12 and IL-18 upon SpA stimulation. Further study is eagerly required for the identification of the accurate molecular mechanism involved in SpA-stimulated IL-18 secretion from KC. Therefore, KC may be the key target cells for the treatment of hyper IgE conditions and provide new insights into the mechanism underlying the pathogenesis of atopic diseases that may be primarily initiated by the activation of KC.

In contrast to the spontaneous development of Th2 cells and inflammatory cutaneous changes in KCASP1Tg (6,7), mice treated with SpA did not display these changes (Fig. 6C and data not shown). This may be in part attributable to the period and dose of application of SpA. In fact, it takes 8 weeks and longer for KCASP1Tg to develop the skin alterations (6,7) and the mice contained >10-fold levels of serum IL-18 than SpA-treated mice (Fig. 6A). Additionally, some other promoting factors such as physical stress might be required for the development of actual skin changes.

Although we need further study, this study suggested the importance of non-immune cells in induction of host immune response. Here, we demonstrated that local skin inflammation induces systemic immune response, suggesting prompt treatment of local inflammatory response might inhibit development of a systemic harmful immune response.


    Acknowledgements
 
This work was supported in part by a Hitec Research Center Grant from the Ministry of Education, Culture, Sports, Science and Technology of Japan (MEXT), and Grant-in-Aid for Scientific Research on Priority Areas from MEXT.


    Abbreviations
 
AD—atopic dermatitis

B6—C57BL/6

DC—dendritic cells

FasL—Fas ligand

IL-18R—IL-18 receptor

KCASP1Tg—keratinocyte-specific caspase-1 transgenic mice

KC—keratinocyte

KIL-18Tg—keratinocyte-specific IL-18 transgenic mice

LC—Langerhans cell

MyD88—myeloid differentiation factor 88

PAMP—pathogen-associated molecular pattern

PE—phycoerythrin

SpA—protein A from Staphylococcus aureus Cowan 1

TLR—Toll-like receptor

YVAD—Ac-YCAD-CMK

ZVAD—z-VAD-FMK


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 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

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