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Mite Allergen Induces Allergic Dermatitis with Concomitant Neurogenic Inflammation in Mouse

2003; Elsevier BV; Volume: 121; Issue: 2 Linguagem: Inglês

10.1046/j.1523-1747.2003.12356.x

1523-1747

Chiung Hui Huang, I-Chun Kuo, Hui Xu, Kaw Yan Chua, Yoke Sun Lee,

Allergic Rhinitis and Sensitization

Pathogenesis of atopic dermatitis involved the interactions of immune and neuroendocrine systems. Here we describe a mouse model for atopic dermatitis with concomitant neurogenic inflammation, by epicutaneous sensitization with a dust mite allergen. Allergen patching resulted in localized dermatitis characterized by pronounced epidermal hyperplasia and spongiosis, which was associated with infiltration of eosinophils and neutrophils, degranulated mast cells, CD4+ and CD8+ T cells, and dendritic cells. There was increased innervation of calcium gene related peptides and substance P in inflamed skins, interactions between nerve fibers and mast cells were seen, indicating the coexistence of neurogenic inflammation. Splenic T cells produced T helper 2-polarized cytokines in response to allergen stimulation in vitro, indicating systemic allergen sensitization. This is the first report of a mouse model of eczema, accompanied by neurogenic inflammation, which shows close resemblance to human allergic diseases. This work supports the notion that the skin is an important site for the initiation of primary allergen sensitization. Besides, this model may also be useful for study of other stress-associated neuroinflammatory skin disorders such as neurogenic pruritus and psoriasis. Pathogenesis of atopic dermatitis involved the interactions of immune and neuroendocrine systems. Here we describe a mouse model for atopic dermatitis with concomitant neurogenic inflammation, by epicutaneous sensitization with a dust mite allergen. Allergen patching resulted in localized dermatitis characterized by pronounced epidermal hyperplasia and spongiosis, which was associated with infiltration of eosinophils and neutrophils, degranulated mast cells, CD4+ and CD8+ T cells, and dendritic cells. There was increased innervation of calcium gene related peptides and substance P in inflamed skins, interactions between nerve fibers and mast cells were seen, indicating the coexistence of neurogenic inflammation. Splenic T cells produced T helper 2-polarized cytokines in response to allergen stimulation in vitro, indicating systemic allergen sensitization. This is the first report of a mouse model of eczema, accompanied by neurogenic inflammation, which shows close resemblance to human allergic diseases. This work supports the notion that the skin is an important site for the initiation of primary allergen sensitization. Besides, this model may also be useful for study of other stress-associated neuroinflammatory skin disorders such as neurogenic pruritus and psoriasis. atopic dermatitis dermal dendritic cell Atopic dermatitis (AD) is a common chronic relapsing inflammatory skin disease afflicting up to 10 to 15% of the population in industrialized countries (Rudikoff and Lebwohl, 1998Rudikoff D. Lebwohl M. Atopic dermatitis.Lancet. 1998; 351: 1715-1721Abstract Full Text Full Text PDF PubMed Scopus (242) Google Scholar). Both clinical and epidemiologic studies indicate that AD is closely associated with respiratory allergies, also known as the atopic triad (atopic eczema, rhinitis, and asthma), usually manifesting with eczema in early life followed by the development of respiratory allergies later on (Hanifin and Rajka, 1980Hanifin J.M. Rajka G. Diagnostic feature of atopic dermatitis.Acta Derm Venereol. 1980; 92: 44-47Google Scholar). The pathogenesis of AD is multifactorial, and genetically determined immunologic aberrations are thought to play a central part in the pathogenesis of chronic AD. The mechanisms involved in disease development are not well understood. In addition, there is some evidence that T cell responses to inhalant allergens, such as house dust mite allergens and pollens or food allergens are crucial to the propagation of AD (Werfel and Kapp, 1998Werfel T. Kapp A. Environmental and other major provocation factors in atopic dermatitis.Allergy. 1998; 53: 731-739Crossref PubMed Scopus (141) Google Scholar). Recent studies indicate that both T helper (Th)2 (interleukin (IL)-4, IL-5, IL-13) and Th1 (interferon (IFN)-γ) type cytokines contribute to the pathogenesis of skin inflammation in AD and the biphasic cytokine expression pattern was therefore proposed (Thepen et al., 1996Thepen T. Langeveld-Wildschut E.G. Bihari I.C. van Wichen D.F. van Reijsen F.C. Mudde G.C. Bruijnzeel-Koomen C.A. Biphasic response against aeroallergen in atopic dermatitis showing a switch from an initial Th2 response to a Th1 response in situ: An immunocytochemical study.J Allergy Clin Immunol. 1996; 97: 828-837Abstract Full Text PDF PubMed Scopus (364) Google Scholar;Spergel et al., 1999Spergel J.M. Mizoguchi E. Oettgen H. Bhan A.K. Geha R.S. Roles of Th1 and Th2 cytokines in a murine model of allergic dermatitis.J Clin Invest. 1999; 103: 1103-1111Crossref PubMed Scopus (305) Google Scholar). It is known that exacerbation of AD can be provoked by stress-associated and abnormal local expression of neuropeptides (Anand et al., 1991Anand P. Springall D.R. Blank M.A. Sellu D. Polak J.M. Bloom S.R. Neuropeptides in skin diseases: Increased VIP in eczema and psoriasis but not axillary hyperhidrosis.Br J Dermatol. 1991; 124: 547-549Crossref PubMed Scopus (94) Google Scholar). Inflammatory changes mediated by sensory nerves have been reported in the skin and other tissues (Baluk, 1997Baluk P. Neurogenic inflammation in skin and airways.J Invest Dermatol Symp Proc. 1997; 2: 76-81Abstract Full Text PDF PubMed Scopus (74) Google Scholar). Increasing evidence indicated that the neuropeptides released by sensory nerves such as c-fibers in the skin are capable of activating specific cutaneous target cells located in close proximity of the activated nerve fibers to induce a range of inflammatory activities (Baraniuk et al., 1990Baraniuk J. Marek L. Kowalski L. Kaliner M. Neuropeptides in the skin.in: Bos J. Skin Immunology. CRC Press, Boca Raton, FL1990: 307-326Google Scholar;Ansel et al., 1996Ansel J.C. Kaynard A.H. Armstrong C.A. Olerud J. Bunnett N. Payan D. Skin–nervous system interactions.J Invest Dermatol. 1996; 106: 198-204Crossref PubMed Scopus (184) Google Scholar). For example, mast cell degranulation can be induced by neuropeptides such as substance P (SP) released by cutaneous nerve (Ebertz et al., 1987Ebertz J.M. Hirshman C.A. Kettelkamp N.S. Uno H. Hanifin J.M. Substance P-induced histamine release in human cutaneous mast cells.J Invest Dermatol. 1987; 88: 682-685Abstract Full Text PDF PubMed Google Scholar;Sugiura et al., 1992Sugiura H. Maeda T. Uehara M. Mast cell invasion of peripheral nerve in skin lesions of atopic dermatitis.Acta Derm Venereol Suppl. 1992; 176: 74-76Google Scholar). In addition, it has been shown that SP can induce keratinocytes and mast cells to produce IL-1 and tumor necrosis factor-α, respectively (Brown et al., 1990Brown J. Perry P. Hefeneider S. Ansel J. Neuropeptide modulation of keratinocyte cytokine production.Molecular and Cellular Biology of Cytokines. Wiley-Liss, Inc., New York1990: 451-456Google Scholar;Ansel et al., 1993Ansel J.C. Brown J.R. Payan D.G. Brown M.A. Substance P selectively activates TNF-alpha gene expression in murine mast cells.J Immunol. 1993; 150: 4478-4485PubMed Google Scholar). Given the complexity of the immunopathogenesis of AD, a good animal model would provide important insights into the potential mechanisms contributing to disease studies. A murine model is a good research tool as the murine immune system is well characterized and the research reagents for murine works are readily available. To date several murine models have been reported for human AD, most of them are not induced by specific protein antigens, and therefore not useful for the study of disease mechanisms. There have been two reports of murine AD models that involved the use of protein antigens (Hsu et al., 1996Hsu C.H. Chua K.Y. Huang S.K. Chiang I.P. Hsieh K.H. Glutathione S-transferase induces murine dermatitis that resembles human atopic dermatitis.Clin Exp Allergy. 1996; 26: 1329-1337Crossref PubMed Scopus (8) Google Scholar;Spergel et al., 1998Spergel J.M. Mizoguchi E. Brewer J.P. Martin T.R. Bhan A.K. Geha R.S. Epicutaneous sensitization with protein antigen induces localized allergic dermatitis and hyperresponsiveness to methacholine after single exposure to aerosolised antigen in mice.J Clin Invest. 1998; 101: 1614-1622Crossref PubMed Scopus (499) Google Scholar). The main drawback of these models, however, is that the protein antigens used are not relevant to human AD. As numerous recent studies have suggested that house dust mite allergens play a direct pathophysiologic role in AD (Maeda et al., 1992Maeda K. Yamamoto K. Tanaka Y. Anan S. Yoshida H. House dust mite (HDM) antigen in naturally occurring lesions of atopic dermatitis (AD): The relationship between HDM antigen in the skin and HDM antigen-specific IgE antibody.J Dermatol Sci. 1992; 3: 73-77Abstract Full Text PDF PubMed Scopus (45) Google Scholar;Kimura et al., 1998Kimura M. Tsuruta S. Yoshida T. Correlation of house dust mite-specific lymphocyte proliferation with IL-5 production, eosinophilia, and the severity of symptoms in infants with atopic dermatitis.J Allergy Clin Immunol. 1998; 101: 84-89Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar), the use of house dust mite allergen to establish a murine model for AD is a logical and more desirable choice. In this study, we report the establishment of a murine model for allergic dermatitis with AD-like symptoms using an house dust mite allergen from the mite species Dermatophagoides pteronyssinus (Der p) through epicutaneous patching. This allergen, designated as Der p 8, is a glutathione-S-transferase of Der p mites and shown to have a 40 to 50% frequency of IgE reactivity with mite allergic patients (O'Neill et al., 1994O'Neill G.M. Donovan G.R. Baldo B.A. Cloning and characterization of a major allergen of the house dust mite, Dermatophagoides pteronyssinus, homologous with glutathione S-transferase.Biochim Biophys Acta. 1994; 1219: 521-528Crossref PubMed Scopus (69) Google Scholar). This is a first report of the induction of strong systemic Th2-skewed immune responses, as well as allergic and neurogenic inflammation of the skin of sensitized mice through epicutaneous sensitization with a mite allergen of clinical importance. Six to eight week old female BALB/C mice from the Sembawang Laboratory Animals Center, National University of Singapore, were used in the experiments. Recombinant Der p 8 expressed in Pichia pastoris was used as a sensitizing antigen. Animal experiments were performed according to Institutional guidelines for Animal Care and Handling, National University of Singapore. Rat and hamster monoclonal antibodies used for immunofluorescence, FACScan analysis, and magnetic cell sorting (MACS) were anti-DEC205 (NLDC-145, Serotec Ltd, Oxford, UK), biotin-conjugated anti-CD3ε (145–2C11), anti-CD8α (53–6.7), fluorescein isothiocyanate-conjugated anti-CD4 (RM4.5), anti-IFN-γ (XMG-2.1), phycoerythrin-conjugated anti-CD4 (RM4.5), anti-CD8α (53–6.7), and anti-IL-4 (BVD4–1D11). Monoclonal antibodies used in enzyme-linked immunosorbent assay (ELISA) were rat antibodies to mouse IL-4 (BVD4–1D11), IL-5 (TRFK5), IFN-γ (R4–6A2), and biotin-conjugated rat antibodies to mouse IL-4 (BVD6–24G2), IL-5 (TRFK4), and IFN-γ (XMG1.2). The recombinant mouse IL-4, IL-5, and IFN-γ were used as standards in sandwich ELISA. All monoclonal antibodies and reagents were purchased from PharMingen (San Diego, California) unless otherwise specified. Rabbit polyclonal anti-SP and anti-calcitonin gene related peptides were purchased from DiaSorin Inc. (Stillwater, Minnesota). Cy-chrome-conjugated streptavidin, fluorescein isothiocyanate-conjugated streptavidin, and streptavidin microbeads were used for fluorescence studies and cell sorting. Epicutaneous sensitization of mice was performed as described byWang et al., 1996Wang L.F. Lin J.Y. Hsieh K.H. Lin R.H. Epicutaneous exposure of protein antigen induces a predominant Th2-like response with high IgE production.J Immunol. 1996; 156: 4079-4082Google Scholar andSpergel et al., 1998Spergel J.M. Mizoguchi E. Brewer J.P. Martin T.R. Bhan A.K. Geha R.S. Epicutaneous sensitization with protein antigen induces localized allergic dermatitis and hyperresponsiveness to methacholine after single exposure to aerosolised antigen in mice.J Clin Invest. 1998; 101: 1614-1622Crossref PubMed Scopus (499) Google Scholar. Briefly, 50 μg of Der p 8 in 100 μL of phosphate-buffered saline (PBS) or PBS alone was applied to 1 cm2 gauze, which was patched to the skin with a transparent dressing and further secured with an elastic bandage. The patch was applied for 4 d and then removed. Seventeen days later, an identical patch was reapplied to the same skin site. This procedure was repeated twice over a period of 50 d. A group of mice received two patches of PBS and were challenged with Der p 8 at the last patch. Cells were culture with RPMI-1640 supple-mented with 10% heat-inactivated bovine calf serum, 2 mM L-glutamine, antibiotics (100 U per mL penicillin and 100 μg per mL streptomycin) (Hyclone Laboratories, Logan, Utah), 1 mM sodium pyruvate, and 5.5 × 10−2 mM 2-mercaptoethanol (Life Technology, Grand Island, New York). The spleen cells were cultured in 24-well plates in the presence of Der p 8 (10 μg per mL) for 3 d. Recombinant IL-2 was added to the cultured cell on days 3, 5, and 7. On day 10, the cells were harvested. For characterizing the cytokine profile of T cell subsets, cells were separated into two populations by AutoMACS (Miltenyi Biotec GmbH Bergisch Gladback, Germany) and the purified CD4+ or CD8+ T cells (1 × 105 cells per well) were stimulated with immobilized anti-CD3 (10 μg per mL) and anti-CD28 (2 μg per mL) in 96 well U-bottom plates for 24 h. All the antibodies used for detection of cytokines in the cultured supernatant were purchased from Pharmingen (Pharmagen, San Diego, CA). The ELISA was performed according to the manufacturer's instructions. Cells were stimulated with immobilized anti-CD3 and soluble anti-CD28 in the presence of 3 μM of monensin (Sigma, St Louis, Missouri) for 10 h. After staining of anti-CD4 or anti-CD8 monoclonal antibodies, cells were fixed and permeabilized with 0.1% saponin (Sigma) and further stained with IL-4 and IFN-γ. For histologic examination, specimens were obtained from the patched skins at day 50 after sensitization and fixed in 10% buffered neutral formalin immediately. After embedding in the paraffin, sections of 4 μm were cut and stained with hematoxylin and eosin. For immunofluorescence staining, the skins obtained from the patched area were embedded in Tissue-Tek O.C.T. compound, snap-frozen in liquid nitrogen and 10 μm sections were prepared. These sections were further incubated with 5% normal mouse serum before the first antibodies were applied. The fluorescence was viewed under the Zeiss laser scanning confocal microscope. For staining of neuropeptides, mice were perfused with 4% paraformaldehyde, embedded in (Tissue-Tek) O.C.T. compound (Sakura Finetek USA, Inc., Torrance, California) and 20 μm sections were prepared. Sections were stained by an avidin–biotin complex method (Vector Laboratories, Inc., Burlingame, California) and counterstained by methylene green. All data were represented as mean±SEM. Mast cells in the dermatitis layers were quantified from fields of 400 × magnification. All the results were analyzed by two-tailed Student's t test. Histologic examina-tion showed features of dermatitis with epidermal hyperplasia and spongiosis in the area of skin patched with Der p 8 (Figure 1a). A significant lichenification and excoriation could be observed in the thickened skin (Figure 1b). Eosinophilia and neutrophilia were seen in the dermis and subcutaneous layers (Figure 1c). To rule out the irritant effect of Der p 8, PBS patched mice were challenged with Der p 8 at the last patch (Figure 1d). It showed similar histology as PBS patched mice. An inflammatory T cell infiltrate is one of the main histologic characteristics of AD and increasing evidence suggests that T cells and the cytokines they release play a pathologic role in AD (Yamaguchi et al., 1991Yamaguchi Y. Suda T. Ohta S. Tominaga K. Miora Y. Kashara T. Analysis of the survival of mature human eosinophils: Interleukin 5 prevents apoptosis in mature human eosinophils.Blood. 1991; 78: 2542-2547Crossref PubMed Google Scholar;Hamid et al., 1996Hamid Q. Naseer T. Minshall E.M. Song Y.L. Boguniewicz M. Leung D.Y. In vivo expression of IL-12 and IL-13 in atopic dermatitis.J Allergy Clin Immunol. 1996; 98: 225-231Abstract Full Text Full Text PDF PubMed Scopus (328) Google Scholar;Akdis et al., 1999Akdis M. Simon H.U. Weigl L. Kreyden O. Blaser K. Akdis C.A. Skin homing (cutaneous lymphocyte-associated antigen-positive) CD8+ T cells respond to superantigen and contribute to eosinophilia and IgE production in atopic dermatitis.J Immunol. 1999; 163: 466-475PubMed Google Scholar;Herz et al., 1998Herz U. Bunikowski R. Renz H. Role of T cells in atopic dermatitis.Int Arch Allergy Immunol. 1998; 115: 179-190Crossref PubMed Scopus (100) Google Scholar). In our data, the most dominant infiltrating cell type in the epidermis and dermis layers of the affected skin was the mononuclear cell, and most of these were CD4+ and CD8+ T lymphocytes (Figure 2). Our preliminary results (data not shown) showed that the cultured cutaneous T cells from the inflamed skin produced significant levels of IL-4 and IL-10, as well as high levels of IL-5 and IL-13. The presence of these Th2-like cytokines as well as the eosinophilia of the inflamed skin strongly suggest that a Th2-skewed immune response has been elicited and is associated with the pathophysiology of AD seen in our model.Figure 2Der p 8 sensitization induced infiltration of T cells and dendritic cells. Skin sections were stained with fluorescein isothiocyanate labeled α-CD3, phycoerythrin labeled α-CD4, phycoerythrin labeled α-CD8, and biotinylated α-DEC205 plus Streptavidin phycoerythrin as indicated. The green color indicated CD3+ or DEC205+ cells. The single positive CD4+ or CD8+ cells are indicated by red color. The double positive cells are indicated by orange to yellow color (as indicated by arrows). Scale bar: 50 μm.View Large Image Figure ViewerDownload (PPT) Langerhans cells and dermal dendritic cells (DDC) are potent antigen-capturing and presenting cells, and play an important role in the pathogenesis of AD (Mudde et al., 1990Mudde G.C. Van Reijsen G.C. Boland G.J. DeGast G.C. Bruijnzeel P.L.B. Bruijnzeel-Koomen C.A.F.M. Allergen presentation by epidermal Langerhans' cells from patients with atopic dermatitis is mediated by IgE.Immunology. 1990; 69: 335-341PubMed Google Scholar). The Der p 8 patched skin showed a prominent increase in the number of Langerhans cells/dendritic cells at the junctions of dermis and epidermis of the skin (Figure 2; as indicated by DEC205+/DEC205+CD8+ cells). It has been reported that DDC are rapidly recruited from blood into the inflamed skin. These DDC could serve as an immediate source of antigen-presenting cells to CLA+ T cells in the inflamed skin. Although the precise role of these DDC remains speculative, it is tempting to hypothesize that DDC play a part in the development of systemic sensitization. To determine the cytokine profiles of the T cell induced by epicutaneous patching of Der p 8, splenocytes from patched mice were cultured with Der p 8 in vitro. As shown in Figure 3(a), splenocytes of Der p 8 patched mice produced higher IL-4 (p<0.001) and IL-5 (p<0.001) as well as lower IFN-γ than the control mice. The data indicated that patching with Der p 8 allergen induced a systemic and predominantly Th2 skewed cytokine profile. To characterize further the cytokine profile of T cell subsets, we cultured the T cells with Der p 8 in vitro for 10 d and analyzed the cells by flow cytometry. As revealed by intracellular cytokine staining, there was a significant increase in the percentages of IL-4 (4.3%±2.2% vs 0.8%±0.3%, p<0.01), and IFN-γ (2.4%±0.2% vs 1.9%±0.8%, p<0.05) producing cells in the CD4+ subset of Der p 8 patched mice (Figure 3b). These results were further confirmed by cytokine ELISA. As shown in Figure 3(c), CD4+ T cells of Der p 8 patched mice produced a significant amount of IL-4 and IL-5. The amount of IFN-γ produced was 2-fold lower than that of IL-4. A significant amount of IL-13 was also detected by reverse transcription–polymerase chain reaction in these CD4+ T cells (data not shown). After 10 d of in vitro culture with Der p 8, there was a slight increase in the ratio of CD8+/CD4+ T cells in the Der p 8 patched mice (0.24 and 0.18 for Der p 8 and PBS patched mice, respectively) (data not shown). Further studies using purified CD8+ T cells revealed that CD8+ cells of Der p 8 patched mice produced a significant amount of IL-4 (p<0.05) and IL-5 (p<0.05) than control mice (Figure 4). These data indicated that a subset of CD8+ T cells producing Th2-like cytokines could be induced in Der p 8 patched mice. The induction of IL-4/IL-5 producing CD8+ T cells by patching is a novel observation that has not been reported in other animal models for AD. In humans, a pathologic role for CD8+ T cells in AD has been suggested (Ishii et al., 1998Ishii N. Takahashi K. Sugita Y. Nakajima H. Atopic dermatitis apparently caused by type 2 CD8+ T cells in an AIDS patient.Clin Exp Dermatol. 1998; 23: 121-122Crossref PubMed Scopus (4) Google Scholar). The frequency of IL-4+CD8+ T cells correlated with the level of serum IgE in allergic patients. These CD8+ T cells enhanced IgE production in vitro (Meissner et al., 1997Meissner N. Kussebi F. Jung T. Ratti H. Baumgarten C. Werfel T. Heusser C. Renz H. A subset of CD8+ T cells from allergic patients produce IL-4 and stimulate IgE production in vitro.Clin Exp Allergy. 1997; 27: 1380-1382Crossref PubMed Scopus (33) Google Scholar). Furthermore, CD8+CLA+ T cells induced IgE production by B cells, mainly through IL-13, and secreted IL-5 that enhanced eosinophil survival by delaying apoptosis of eosinophil (Yamaguchi et al., 1991Yamaguchi Y. Suda T. Ohta S. Tominaga K. Miora Y. Kashara T. Analysis of the survival of mature human eosinophils: Interleukin 5 prevents apoptosis in mature human eosinophils.Blood. 1991; 78: 2542-2547Crossref PubMed Google Scholar;Akdis et al., 1999Akdis M. Simon H.U. Weigl L. Kreyden O. Blaser K. Akdis C.A. Skin homing (cutaneous lymphocyte-associated antigen-positive) CD8+ T cells respond to superantigen and contribute to eosinophilia and IgE production in atopic dermatitis.J Immunol. 1999; 163: 466-475PubMed Google Scholar). In comparison with PBS patched mice, Der p 8 patched mice showed a significant increase in the number of mast cells (203.7±11.4 vs 157.4±10.5, p<0.01, n=9) and degranulated mast cells (37.8±5.5 vs 11.4±2.1, p<0.01) in the dermis layer (Figure 5a, b). Degranulating mast cells in AD skin release a number of mediators that have profound effects on the pathogenesis of atopic skin (Klein et al., 1989Klein L.M. Lavker R.M. Matis W.L. Murphy G.F. Degranulation of human mast cells induces an endothelial antigen central to leukocyte adhesion.Proc Natl Acad Sci USA. 1989; 86: 8972-8976Crossref PubMed Scopus (204) Google Scholar;Katayama et al., 1992Katayama I. Yokozeki H. Nishioka K. Mast-cell-derived mediators induce epidermal cell proliferation: Clue for lichenified skin lesion formation in atopic dermatitis.Int Arch Allergy Immunol. 1992; 98: 410-414Crossref PubMed Scopus (29) Google Scholar). Furthermore, mast cell degranulation can be induced by neuropeptides such as SP released by cutaneous nerves (Ebertz et al., 1987Ebertz J.M. Hirshman C.A. Kettelkamp N.S. Uno H. Hanifin J.M. Substance P-induced histamine release in human cutaneous mast cells.J Invest Dermatol. 1987; 88: 682-685Abstract Full Text PDF PubMed Google Scholar). This interaction between cutaneous target cells and neuropeptides contributes to the neurogenic inflammation seen in AD. We therefore investigated the interaction between the nervous and immune systems in the dermatitis lesions. The presence of neuropeptides, calcium gene related peptide and SP was revealed by immunohistochemical staining of the inflamed skin in mice treated with Der p 8 and undetectable in the control mice (Figure 5c). Numerous nerve fibers ramified into the dermis and appeared to be in close apposition with a number of granular cells that are most likely the mast cells in the dermis (Figure 5d). To our knowledge, this is one of the first animal models that shows the phenomenon of neurogenic inflammation associated with AD. An understanding of these interactions can facilitate the development of more specific treatments for a wide range of chronic neuroinflammatory skin diseases, and the current animal model will serve as an excellent research tool for such studies. It has been speculated that some genetically determined elements causing an epidermal barrier dysfunction could predispose an atopic individual to allergen sensitization very early in life (Ogawa and Yoshiike, 1993Ogawa H. Yoshiike T. A speculative view of atopic dermatitis: Barrier dysfunction in pathogenesis.J Dermatol Sci. 1993; 5: 197-204Abstract Full Text PDF PubMed Scopus (93) Google Scholar;Taieb, 1999Taieb A. Hypothesis: from epidermal barrier dysfunction to atopic disorders.Contact Dermatitis. 1999; 31: 177-180Crossref Scopus (127) Google Scholar). The synergy of such epidermal barrier breakdown and other immunologic abnormalities could lead to the propagation and enhancement of allergic inflammation associated with the pathogenesis of AD and allergic asthma. Incidentally, the Der p 8-patched mice developed lung allergic inflammation and airways hyperreactivity upon subsequent intratracheal allergen challenge (data not shown). Taken together, the data derived from this animal model support the notion that skin is not only a target site for atopic disease, but it is also an important site for the initiation of primary allergic sensitization to environmental irritants or allergens. Our findings also support the notion that primary prevention of allergic diseases should start very early in life and the problems associated with skin permeability should be addressed for high-risk atopic babies. We would like to thank Dr Patrick Holt, Dr Wayne Thomas, and Dr Claudia Wolfowicz for helpful discussions. This work was supported by a National Medical Research Council grant (0225/1997, 0442/2000) and the National University of Singapore Academic Research Fund (R-178-000-010-112).