Clinically acute exacerbation of skin manifestations is

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1 Stress Response, Tachykinin, and Cutaneous In ammation Ichiro Katayama, Sang-Jae Bae, Yo-ichiro Hamasaki, Ken Igawa,* Yasuhiro Miyazaki,* Hiroo Yokozeki,* and Kiyoshi Nishioka* Department of Dermatology, Nagasaki University School of Medicine, Nagasaki, Japan; *Department of Dermatology, Tokyo Medical and Dental University, School of Medicine, Tokyo, Japan In the last decade, several new aspects of glucocorticoid (GC)-actions on immune cells have been recognized. This recognition has been largely obtained through clinical observations of stress-induced exacerbations of certain dermatologic diseases. To clarify whether GC modulates cutaneous in ammatory reactions besides its known anti-in ammatory effect, rst we examined the effect of long-term application of topical GC on several kinds of in ammatory responses induced in the murine model and demonstrated that these regimens signi cantly augmented the classical contact sensitivity reaction, the croton oil-induced irritant reaction, and the IgEmediated biphasic cutaneous reaction. In addition, large dose topical steroid and its withdrawal enhanced scratching behavior in hapten-challenged mice. This augmented scratching behavior correlated with the induction of preprotachykinin mrna expression in the challenged skin. In an in vitro experiment, a low-dose, stress-induced level of glucocorticoid signi cantly upregulated hapten-induced proin ammatory cytokine (IL1a) production by murine keratinocyte cell line Pam 212 and induced substance P peptide production from cultured human keratinocytes. Our results suggest that unsuitable use of GC in addition to stress-induced GC may modulate immune function in the skin through aberrant production of tachykinin, such as substance P or other epidermal cell derived cytokines. Key words: allergic reactions/glucocorticoid/stress/tachykinin. Journal of Investigative Dermatology Symposium Proceedings 6:81± 86, 2001 Clinically acute exacerbation of skin manifestations is occasionally observed in patients with atopic dermatitis after withdrawal of topical corticosteroid or under emotional stress. Itching is another predominant symptom among allergic dermatoses, i.e., atopic dermatitis, urticaria, pruriginous diseases, and certain patients with psoriasis vulgaris. All of those frequently are exacerbated under emotional stress; however, its mechanism is not clearly understood. It was well known that the mediators causing itching in in amed skin are histamine, prostaglandin, and tachykinins. Among these, substance P (SP) plays a central role in mediating the sensation of peripheral itch because intradermal injection of SP causes itching through histamine release from mast cells, and capsaicin depletes cutaneous nerve endings of SP resulting in inhibition of the itch± scratch cycle in certain pruritic dermatoses. This paper summarizes the kinetics of glucocorticoid (GC)- induced augmentation of cutaneous in ammatory reactions and the interaction of GC and tachykinin in the skin. Our ndings provide new clues to better understand the neuroendocrineimmune circuit in the skin and management of refractory skin diseases such as atopic dermatitis, psoriasis, or urticaria, all of those are easily aggravated under physiologic and/or psychiatric stress. Manuscript received June 14, 2001; accepted for publication June 14, Reprint requests to: Dr. I. Katayama, Department of Dermatology, Nagasaki University School of Medicine, 1-7-1, Sakamoto, Nagasaki, Japan. GC AND RODENT-CONTACT SENSITIVITY GC augments hapten-speci c contact sensitivity reaction To evaluate the effect of long-term topical GC on cutaneous in ammatory response, we applied GC on BALB/C mouse skin before and after contact sensitization (Fig 1). As is shown in Fig 2(A), nine applications of 0.1% di ucotolone on alternate days (seven applications before and two applications after sensitization) on a sensitized site clearly augmented a DNFBspeci c contact sensitivity reaction induced on the pinna of BALB/ C mice in a concentration-dependent fashion. Prednisolone butylacetate or triamcinolone acetate showed a rather weak enhancing effect (Fig 2B). This regimen also augmented TNCB or oxazolone-speci c contact sensitivity. Similarly, however, a relatively weak enhancing effect was obtained when C3H mice were used. Application of GC four times (two times before and two times after sensitization) showed a rather reduced augmentative effect compared with that in the nine-times group. Vehicle control (ethanol) did not show any modulatory activity. In contrast to epicutaneous application of GC, no enhancing effect was observed when the same doses of GC were applied intraperitoneally. In addition to sensitizers, nonspeci c reactions induced by croton oil or a suboptimal concentration of DNFB (0.15%) were also augmented by GC pretreatment on the ank skin before elicitation of nonspeci c skin reactions on the pinna. GC inhibited contact sensitivity reaction when applied on the challenged sites It is generally accepted that GC downregulates contact sensitivity reaction through a reduction of the number of epidermal Langerhans cells (Belsito et al, 1982; Aberer et al, 1984). Therefore, we applied GC to a challenged site on the right pinna twice before and twice after sensitization on the /01/$15.00 Copyright # 2001 by The Society for Investigative Dermatology, Inc. 81

2 82 KATAYAMA ET AL JID SYMPOSIUM PROCEEDINGS Figure 1. Experimental protocol. BALB/C mice received nine epicutaneous applications of 0.1% di ucotolone on alternate days on the ank skin. After seven applications, each mouse was sensitized with DNFB, TNCB. or oxazolone. Twenty-four hours after the last applications of 0.1% di ucotolone, each mouse was challenged with corresponding hapten on the right pinna skin. The left pinna was used as vehicle control. ank skin. Treatment at the challenged site with GC four times clearly inhibited the contact sensitivity reaction, which contrasts with the results reported by Grabbe et al (1995). The number of Langerhans cells, thy 1.2 (+) epidermal dendritic cells, or gd (+) epidermal dendritic cells of the GC-treated ank skin was reduced after topical application of GC (Table I). Similar results were obtained at the challenged pinna skin from the mice with GC pretreatment on the ank skin. Paradoxically, contact sensitivity reaction was augmented by GC pretreatment, although local Langerhans cells were reduced in number at the sensitized site. To clarify the sensitizing activity of GC-treated Langerhans cells, we sensitized mice by haptenated epidermal cells from GC-treated or ethanol-treated skin. An early reaction was induced by haptenated epidermal cells from GC-pretreated ank skin, whereas a 24 h reaction was slightly weak compared with that of ethanolpretreated ank skin. No augmentative effect was observed when a large concentration of epidermal cells was used for sensitization. This result might suggest that Langerhans cells are not involved in the GC-induced augmentation of contact sensitivity reaction. Local skin environment of GC-pretreated mice would play some role in exacerbated the skin reaction through an elevated production of proin ammatory cytokines (Miyazaki et al, 2000). GC augments biphasic cutaneous reactions induced by monoclonal IgE anti-dnp antibody To evaluate the effect of long-term topical GC on IgE-mediated in ammatory response, we applied GC on the mouse skin before induction of biphasic cutaneous reactions by intravenous injection of monoclonal IgE anti-dnp antibody and a challenge test. A similar clinical state is occasionally observed in atopic dermatitis. Sensitization and elicitation to various allergens might occur in the patient's skin with chronic dermatitis, and long-standing use of topical steroid might modulate these reactions. As has already been reported (Katayama et al, 1990, 1996, 1997), intravenous application of monoclonal IgE anti-dnp antibody induced hapten-speci c biphasic cutaneous reactions. When mice received seven applications of 0.1% di ucortolone on the ank skin before IgE anti-dnp antibody injection, signi cantly augmented early (4 h) and late phase (24±72 h) cutaneous reactions were observed in BALB/C mice (Fig 3). There have been several reports that mast cell de cient mice WBB6F1 vol/vol lack both early and late phase cutaneous reactions after IgE antibody application and a challenge test in a different experimental system (Wershil et al, 1991). In contrast, previous applications of GC induced an augmented late phase but not an early phase cutaneous reaction in WBB6F1 mice. Histopathologic analysis demonstrated that in ammatory cells such as lymphocytes, eosinophils, and some neutrophils in ltrated Figure 2. Augmentation of contact sensitivity skin reaction by GC. Igawa et al (1997). 0.1% di ucotolone valerate was applied on the ank skin nine times on alternate days (seven applications before and two applications after sensitization). Mice were sensitized with 0.5% DNFB on day 12 (seventh application of di ucotolone valerate) on the same site of ank skin that mice received 0.1% di ucotolone seven times, and then skin was tested on the right pinna on day 17 (one day after the nine applications of di ucotolone valerate). (A) Dose response of di ucotolone valerate on DNFB contact sensitivity reaction; (B) effect of a different kind of GC on DNFB contact sensitivity reaction. to the skin in the late phase reaction (72 h). Mast cells were not demonstrated in all specimens of WBB6F1 vol/vol mice after GC application, in contrast to the presence of mast cells in WBB6F1+/ + mice. These results might suggest that long-term application of topical steroid and its withdrawal augment cutaneous in ammatory reactions through induction of cytokine or cytokine receptors (Almawi et al, 1996) by the resident cells of the skin, without affecting Langerhans cell function. Our results support this possibility (Miyazaki et al, 2000). LOW-DOSE GC INDUCES IL1a FROM CULTURED MOUSE KERATINOCYTES (KC) THROUGH ACTIVATION OF NFkB AND AP-1 In an in vitro experiment, low-dose GC signi cantly upregulated hapten-induced proin ammatory cytokine (IL1a) production (Fig 4A) from a cultured murine KC cell line Pam 212 through activation of NFkB (Fig 4B) and AP-1 (Miyazaki et al, 2000). Low-dose GC moderately upregulated hapten-induced TNFa production from Pam 212, whereas it did not affect IL10 production. These results also suggest that GC modulates cytokine production by epidermal KC resulting in augmented cutaneous in ammation.

3 VOL. 6, NO. 1 NOVEMBER 2001 NEUROENDOCRINE-IMMUNE CIRCUIT IN THE SKIN 83 Table I. The number of Langerhans cells, Thy I (+) cells, and gd (+) T cells per mm 2 in GC-treated and untreated skin a Ia (+) Thy 1 (+) gd (+) Ear Trunk Ear Trunk Ear Trunk Normal Di ucortolone ** * ** ** ** ** a Number of cells of pinna skin were counted on day 17 after nine applications of di ucortolone valerate on the ank skin. FITC labeled antibodies were used for cell staining and number of cells per mm 2 in three randomly selected elds were counted. Results were expressed as mean SE. *p < 0.05; **p < 0.01; NS, not signi cant compared with normal skin. Figure 3. Augmentation of IgE anti-dnp antibody induced passive cutaneous anaphylaxis elicited on the pinna by longterm topical GC applied on distant ank skin. Fifty microliters of 0.1% di ucortolone valerate or 1a±24 (OH)2 D3 (0.05 mg per ml) in ethanol were applied on the ank skin seven times on alternate days. Mice then received intravenous application of monoclonal IgE anti-dnp antibody 1 h after the last application ( nal PCA titer > 32560). One hour after injection, mice were challenged with 0.15% DNFB in acetone:olive oil (4:1) on the right pinna. The left pinna was painted with vehicle as a control. GC AND PRURITIS GC affect scratching behaviors in a contact sensitivity reaction To clarify the effect of GC on the scratching behavior in a DNFB contact sensitivity reaction, each mice was challenged at 1.5, 4, and 24 h after the ninth GC application, and then scratching behavior was recorded using a video camera for 80 min after the challenge test with DNFB. Scratching behavior was signi cantly increased when mice were challenged at 24 h, but not at 1 h or 4 h after the last GC application (data not shown). Scratching behaviors were observed shortly after the challenge test in experimental, vehicle control, and nonsensitized groups. The degree of scratching was not very different among each group in the rst 50 min, although increased scratching behavior was observed in GC-pretreated mice in contrast to decreased behavior in other groups from 50 to 80 min after the challenge test (manuscript submitted for publication). Effect of cytokines (IL1a, IL-2, IL-3, IL-4, and TNFa) on scratching behavior Next, we examined whether prurigenic cytokines such as IL-la, IL-2, IL-3, IL-4, or TNFa affect the scratching behavior in DNFB-sensitized mice before and after the challenge test to the pinna skin. Signi cantly augmented scratching behaviors were observed in the mice who had IL-3 and TNFa ± but not other cytokines ±preinjected into the ear lobe after the challenge test (manuscript submitted for publication). In contrast, same cytokines did not induce signi cant scratching behavior in mice when they were injected before the challenge test, which suggests that these cytokines could not elicit scratching but augment DNFB-induced scratching behavior possibly mediated by initiators of DNFB contact sensitivity. Expression of preprotachykinin A (PPT-A) and inos mrna in GC-pretreated mice We analyzed the expression of mrna of SP precursor and inos, both of which are possible mediators causing itch, in GC-pretreated and DNFB-sensitized mouse challenged with DNFB at 1 and 24 h after the last GC application. Analysis by RT-PCR demonstrated that PPT-A and inos mrna were only expressed in the challenged skin elicited at 24 h, but not at 1 h after the last GC application (manuscript submitted for publication). PPT-A mrna was expressed in the challenged skin of sensitized but not GC-pretreated mice preinjected with IL-3 before the challenge test with DNFB. Another cytokine such as IL-1a, IL-2, IL-4, or TNFa did not cause itching, which suggests that GC pretreatment induces IL-3 and inos in the early phase of DNFB contact sensitivity reaction, resulting in itch sensation in the mouse. Becherel et al recently reported that inos is expressed by human KC during acute urticaria, which induces mast cell degranulation resulting in itch sensation (Becherel et al, 1997). Hermes et al reported that IL-3 and TNFa are expressed by dermal microvasculature endothelial cells in various forms of urticaria (Hermes et al, 1999). These observations suggest that inos and/or IL-3 cause an itching sensation in GC-pretreated and DNFB-challenged mice. Induction of SP by cultured normal human KC After we demonstrated PPT-A mrna in the challenged skin of GCpretreated mice, we then analyzed whether SP, major prurigenic mediators of the skin, can be generated by epidermal KC. Among the various stimuli, including cytokines, chemical mediators, or various neuropeptides, SP itself stimulated KC to induce SP (Bae et al, 1999).

4 84 KATAYAMA ET AL JID SYMPOSIUM PROCEEDINGS Figure 4. Effect of hydrocortisone on IL1a production by Pam 212 cells through NFkB activation. Miyazaki et al (2000). (A) Various concentrations of hydrocortisone were added to the culture medium of Pam 212 KC cell lines and IL1a concentration was measured by ELISA 24 h after stimulation. (B) Effect of PDTC on TNBS and hydrocortisone-induced NFkB expression and IL1a production by Pam 212 cells. As shown in Fig 5(A, B), autocrine induction of SP in normal KC was observed in a concentration- and culture period-dependent fashion. SP concentration without KC was decreased during the culture period, possibly due to the degradation at 37 C. The degradation is temperature dependent with a much larger loss of activity at 37 C than at 0 C, and the half-life in plasma is thought to be 12±15 min. To exclude the possibility that cell-associated SP is responsible for SP secretion into the medium during the apoptotic process, cell-associated SP was measured after SP stimulation. Cell-associated SP in SP-stimulated KC after several freeze and thaw treatments was unchanged at 3 and 18 h after stimulation. SP gene expression in SP-stimulated normal KC To clarify the SP gene expression in normal KC, we performed RT-PCR and Southern blot analysis. In nonstimulated conditions, normal KC express a moderate amount of PPT-A mrna expression and addition of exogenous SP signi cantly augmented PPT-A mrna expression in a concentration-dependent fashion in normal KC (Fig 6). This basal expression of PPT-A mrna may be due to a nonspeci c process such as the effect of medium change or change of incubation temperature on mrna induction. Figure 6 shows the time course of expression of PPT-A mrna in normal KC stimulated with exogenous SP. Peak time of expression of PPT-A mrna is 10 h after stimulation with SP. Southern blot analysis con rmed the expression of PPT-A mrna in SP-treated KC. Effect of various agents on SP production in normal KC In addition to SP, several biologic active substances modulate KC functions in both in vitro and in vivo. We next Figure 5. Time course and concentration-dependent production of SP by cultured KC. Bae et al (1999). Normal human KC were cultured in KGM CC-3111 medium (complete medium) supplemented with BPE, hegf, insulin, antibiotics, and hydrocortisone as described above. At the semicon uent stage, the cells were washed with ice-chilled DMEM and replaced with KGM medium without hydocortisone, for 24 h. At the start of the experiment, the medium was replaced with fresh KGM medium containing variously diluted SP and then culture supernatants were collected at various times and donated for ELISA assay. (A) The dose±response effect of SP on autocrine production of SP in cultured normal human KC (NHK). (B) Time course of SP-induced SP production in cultured normal human KC (NHK). Results are expressed as the means 6 SD. screened several agents on SP production by normal KC. Relatively low but signi cant SP was induced by high concentration of acetylcholine, epinephrine, histamine, and CGRP. Glucocorticoid also induced a signi cant amount of SP by cultured human KC (submitted for publication). We could not identify IL-3 or IL-3 mrna from cultured KC, which suggests that IL-3 is not involved in GC-induced SP production by cultured KC. GC, TACHYKININ, AND CUTANEOUS INFLAMMATORY REACTIONS GC plays an important role in the maintenance of homeostasis, vital functions, or primary host defense and acts to modulate stressinduced immune responses. GC has been widely used in modern medicine for its established anti-in ammatory properties; however, undesired topical side-effects have also been recognized in dermatologic elds, including atrophy of the skin, increased risk of cutaneous infectious diseases, or topical corticosteroid withdrawal syndrome. Clinically it has also been reported that some patients show GC resistance or GC-induced exacerbation of allergic disease such as bronchial asthma (Sher et al, 1994; Adcock et al, 1995) or atopic dermatitis (Clayton et al, 1995). Recent reports suggest that inappropriate or long-standing use of topical GC induce refractory eyelid dermatitis (Rapaport and Rapaport, 1999).

5 VOL. 6, NO. 1 NOVEMBER 2001 NEUROENDOCRINE-IMMUNE CIRCUIT IN THE SKIN 85 Figure 6. SP gene expression in SP-stimulated normal KC. Bae et al (1999). To clarify the SP gene expression in normal KC, we performed RT-PCR and Southern blot analysis. For speci city of mrna expression of PPT-A, we performed Southern blot analysis as described in the Materials and Methods. The values were determined by scanning densitometer and expressed in relation to actin scanning density. In addition to clinical observations, topical GC also augment cutaneous immune reactions in the mouse model (Igawa et al, 1997). This augmentative effect of GC is not fully understood. GC induces apoptosis in mouse T cells and immature rat thymocytes and regulates leukocyte adhesion to endothelial cells; however, Dhabhar et al suggested that the GC determines the suppression at high concentrations or the augmentation at low concentrations of the immune response (Dhabhar and McEwen, 1996). Recently, Kuraishi et al (1995) established a mouse model to evaluate and analyze the mediators mediating the itching sensation by observing the scratching behavior when mice were injected with prurigenic agents. In these studies, we also demonstrated that scratching behavior in GC-pretreated mice was more severe than that in the vehicle-pretreated group (ethanol applicated group and control group) after a challenge test with simple chemicals. This enhancing effect of GC was more evident when mice were challenged at 24 h after the last GC application than when they were challenged at 1 h, which might re ect one aspect of steroid withdrawal observed in clinical practice. In a previous study, we have demonstrated that cultured KC express PPT-A mrna and generate signi cant amounts of SP peptide (Bae et al, 1999). It was reported that repeated applications of hapten induce an augmented skin reaction in the mouse contact sensitivity reaction, possibly due to overproduction of Th2 cytokines by in ltrating or resident cells of the skin (Kitagaki et al, 1995). The results of our studies suggest that GC signi cantly affects scratching behavior by inducing local cytokines and neuropeptide such as IL-1a, TNFa, and SP by resident cells of the skin. Furthermore, RT-PCR analysis demonstrated that mrna of SP, which is a known mediator causing itch (Andoh et al, 1998), and inos, which is expressed by KC in urticaria (Becherel et al, 1997), were only expressed in the DNFB-challenged skin from the mice with repeated GC application and from IL3-preinjected mice without GC application. Therefore, in our mouse model, absorbed GC augments hapten-speci c contact sensitivity reaction, IgEmediated biphasic cutaneous reaction, and scratching behavior by modulation of proin ammatory cytokines, SP, or inos synthesis by the resident skin KC. In addition, SP mrna was only expressed in mice preinjected with IL-3 but not with any other cytokines, which suggests that IL- 3 also augments scratching by upregulation of SP synthesis in DNFB contact sensitivity. Figure 7. Stress-induced augmentation of cutaneous in ammation. Stress-induced GC or long-term topical GC and its withdrawal might induce epidermal cytokine and SP, resulting in augmented cutaneous in ammations. Th1-derived IFNg or Th1/Th2- derived IL-3 might modulate these circuits. IL-3 has been reported as a trophic factor for central cholinergic neurons in vitro and in vivo (Kamegai et al, 1990). The source of IL- 3 in this augmented contact sensitivity reaction has not been identi ed immunohistochemically at present. IL-3 might be generated by Th2 cells, mast cells, or endothelial cells in a certain allergic in ammation under stressful conditions, resulting in possible induction of SP by skin resident cells. Further studies are needed to clarify the role of IL-3 in neuroendocrine-immune system in this mouse model and human allergic diseases. Dhabhar et al (2000) reported that stress-induced enhancement of skin immune function is mediated by IFNg. The role of IFNg should be also clari ed in our system. Figure 7 summarizes the role and interaction of GC, tachykinin, and cytokines in stress-induced augmentation of cutaneous in ammation. Topical steroid and its withdrawal might modulate these circuits resulting in unexpected local and/or systemic reactions in human allergic skin diseases. 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