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International Journal of Experimental Pathology logoLink to International Journal of Experimental Pathology
. 2008 Jun;89(3):188–200. doi: 10.1111/j.1365-2613.2008.00577.x

Acute uriticaria-like lesions in allergen-unexposed cutaneous tissues in a mouse model of late allergic rhinitis

Toshiharu Hayashi 1, Taeko Fujii 1
PMCID: PMC2525774  PMID: 18460071

Abstract

The mechanisms of distant manifestation after a local allergic reaction are largely unknown. This study examined the development of cutaneous lesions in a mouse model of late allergic rhinitis (LAR). BALB/c mice were sensitized by ovalbumin (OVA) intraperitoneally two times (on days 0 and 10) and challenged by OVA intranasally on day 14. Four days after OVA challenge, nasal and cutaneous lesions including helper T (Th) responses, expression of adhesion molecules and presence of OVA and IgE were examined, and compared with unsensitized and unchallenged (control) mice. Compared with the control group, the LAR group developed LAR characterized by infiltration of lymphocytes and eosinophils, increased IgE values and increased productions of IL-4 and IL-5, but not IFN-γ. A dominant infiltration of eosinophils and increase in mast cells, attachment of eosinophils to endothelium, intense expression of VCAM-1 on endothelium in venules and VLA-4 expression on eosinophils and mast cells were recognized in the cutaneous tissues. There were no differences in the expression of ICAM-1 on vascular endothelium and LFA-1 on infiltrated leucocytes between the two groups. CLA expression on lymphocytes was not detected, and the binding of OVA and IgE on mast cells and eosinophils was found in the cutaneous lesions in the LAR group, but not in the control group. This study suggests that acute uriticaria-like lesions in OVA-unexposed cutaneous tissues may be induced by immediate allergic reaction due to the systemic development of Th2-type response in a mouse model of LAR.

Keywords: cutaneous lesion, eosinophil, IgE, immediate, late, mast cell, rhinitis, Th2, uriticaria, VCAM-1, VLA-4

Allergic diseases are multifactorial diseases influenced by genetic predispositions and environmental factors and characterized by a predominant helper T (Th)2 response (Gelfand 2004). Allergic airway diseases are largely divided into two phases: early- and late-phase reactions (O’byrne et al. 1987; Lei et al. 1989; Yu et al. 1996). For example, in allergic rhinitis early-phase reactions occur within minutes of exposure to the allergen and are induced by mainly IgE and mast cells, leading to sneezing, itching and clear rhinorrhoea. On the other hand, late-phase reactions occur 6–24 h after local allergen exposure and are characterized by congestion, fatigue, malaise irritability (Skoner 2001) induced by persistent tissue oedema and infiltration of eosinophils and CD4+ Th cells into the nasal mucosa (Gelfand 2004).

Allergic reactions are not limited to the area they originated, and there has been a lot of speculation on the relation between dermatitis and sensitization to aero-allergens and food allergens in humans (Tupker et al. 1996; Brinkman et al. 1997; Burks et al. 1998; Schafer et al. 1999; Sicherer & Sampson 1999; Beyer et al. 2002; Kyllönen et al. 2006). However, the mechanisms which lead to the development of systemic element, and particularly the mechanisms of distant manifestations after a local allergic reaction have not been elucidated (Togias 2004). Possibilities of the development of cutaneous lesions in humans with food and milk allergy include locally and systemically produced cytokines, chemokines and several chemical mediators (Prescott et al. 2006). Especially, interactions between inflammatory cells and endothelial cells via adhesion molecules may play important roles in various inflammatory and immune responses (Nakamura et al. 1993; Cotran & Pober 1989; Hayashi et al. 2005). For example, milk-induced urticaria is associated with the expansion of T cells expressing cutaneous lymphocyte antigen (CLA) on lymphocytes in the cutaneous tissues (Beyer et al. 2002), leading to the development of cutaneous lesion. On the other hand, very late antigen-4 (VLA-4), a counter receptor of VCAM-1 on endothelium, plays an important role in the migration of mainly eosinophils and lymphocytes to the sites of inflammation (Okigami et al. 2007). In addition, lymphocyte function-associated antigen (LFA)-1 on neutrophils and monocytes interacts with intercellular-associated molecule (ICAM)-1, a counter receptor for LFA-1, on endothelium and those cells may play a role in inflamed areas (Nakamura et al. 1993).

It has been suggested that late-phase reactions are manifestations of the systemic inflammatory events, leading to pathologic response in other parts of body (Togias 2004). In addition, it has never been reported that allergen inhalation challenge causes cutaneous changes in subjects who do not have atopic skin disease (Togias 2004). Thus, we focused on the cutaneous reaction with special reference to the expression of adhesion molecules (CLA, ICAM-1/LFA-1 and VCAM-1/VLA-4) and distribution of OVA and IgE in a mouse model of late allergic rhinitis (LAR) (Hayashi et al. 2007).

Materials and methods

Mice

Specific pathogen-free, 8-week-old female BALB/c mice (Japan Charles River Co., Kanagawa, Japan) were used (total number of mice; n = 27). They were kept at 25 ± 2 °C (room temperature), 55 ± 10% humidity with a 12-h/12-h light/dark cycle (lightening time 08:00–20:00) in metal cages sterilized by dry heating (180 °C, 30 min) and proved autoclaved (120 °C, 30 min) pellets (CA-1, Clea Japan Inc., Tokyo, Japan) and water ad libitum. The number of mice used in each set of experiments is given in parenthesis. The animal experiments were approved by the Research Ethics Board of the Faculty of Agriculture, Yamaguchi University.

Experimental protocols

The mice were divided into following two groups. The sensitization and challenge procedures (Figure 1a) were carried out by the methods described previously (Hayashi et al. 2007) with a simple modification. In brief, the animals (n = 22; LAR group) were sensitized intraperitoneally (i.p.) by the injection of 10 μg of ovalubumin (OVA; Grade V; Sigma, St Louis, MO, USA) in 1.2 mg of aluminium hydroxide gel (Alum; SERVA, Heidelberg, Germany) adjuvant (OVA/Alum), which was suspended in 100 μl of phosphate-buffered saline (PBS; pH 7.4), on day 0 and boosted i.p. with 10 μg of OVA/Alum on day 10. Four days after the second sensitization, the mice were challenged intranasally (i.n.) with 500 μg of OVA in 50 μl of PBS (both right and left nasal cavities by pipettes alternately). The control group (n = 5) was injected with 100 μl of PBS i.p. on days 0 and 10, and challenged with 50 μl of PBS i.n. on day 14.

Figure 1.

Figure 1

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Experimental protocol in the induction of late allergic rhinitis (LAR). On days 0 and 10, mice (n = 22) were sensitized intraperitoneally (i.p.) with 10 μg of OVA with Alum adjuvant (OVA/Alum). Four days after the second sensitization, mice were challenged intranasally (i.n.) with 500 μg of OVA (LAR group). Control group (n = 5) was injected with 100 μl of PBS i.p. on days 0 and 10, and 50 μl of PBS i.n. on day 14. Plasma was obtained on days 0 and 18 to count the number of eosinophils and measure IgE concentrations in both groups. On day 18, the whole blood, spleens, skins and the head were obtained for histopathology, immunohistochemistry and cytokine assays. Nasal (b, c, d, e and f) and cutaneous tissues (b) were sampled and examined for histopathology and immunohistochemistry (See details in Materials and methods).

Sampling

Plasma was obtained from the angular vein of the eye on days 0 and 18 by heparinized pipes (Terumo, Tokyo, Japan) to count the number of blood eosinophils and measure IgE concentrations. On day 18, whole blood, spleens, cutaneous tissues (four different parts; the nose, anterior and hind limbs and back; Figure 1b) and whole heads (Figure 1c) were obtained under anaesthesia by diethylether (Sigma-Aldrich, St Louis, MO, USA).

Tissue processing for histopathology and immunohistochemistry

Cutaneous tissues (n = 5, control group; n = 22, LAR group) were fixed in 10% neutral-buffered formalin (pH 7.0) and embedded in paraffin blocks. On the other hand, heads (n = 5; control group, n = 22; LAR group) were fixed in 10% neutral-buffered formalin for 2 days, and those were decalcified in 1 mM Tris–HCl (pH 7.2) with 20% EDTA–2Na for 2 weeks. Nasal lesion was sliced at the anterior margin of orbit transversely (Figure 1b). Sections of 4-μm thickness were stained with haematoxylin and eosin (HE), toluidine blue (TB), congo red or periodic acid Schiff.

For immunohistochemistry, the cutaneous tissues of the hind limb (n = 5, control group; n = 22, LAR group) were embedded in OCT and rapidly frozen in n-hexane (Sigma-Aldrich) at −80 °C and 4-μm frozen sections were made by a cryostat (Bright Instrument Co., Limited, Huntingdon, UK) and fixed in acetone and air dried.

Evaluation of histopathology

For a semiquantative analysis, score index (severity of inflammation; degree of infiltration of inflammatory cells) per mouse in left septal respiratory areas (200 μm2; dotted rectangle area, Figure 1d) was scored on 0–4 scale by light microscopy as follows: 0 = none, 1 = weak and partial, 2 = weak and diffuse, 3 = moderate and partial and 4 = moderate and diffuse. In addition, the total amount of respiratory epithelium and the number of goblet and non-goblet cells were counted, and the total number of epithelium (200 μm wide; Figure 1d) and the ratio of goblet/non-goblet cells were calculated. In addition, the number of inflammatory cells (200 μm2; rectangle areas; Figure 1e) in each of four different parts of both lateral and septum areas was counted and the distance from the tip of respiratory epithelium to the edge of nasal septum cartilage on the left-hand side (five different parts; Figure 1e) was measured by an eyepiece micrometer. The number of infiltrating cells in the cutaneous tissues was determined by four different parts (200 μm2; Figure 1b) in each left cutaneous site. Each result was averaged and totalled for each mouse, and thereafter the mean ± standard error (SEM) of each experiment was calculated. Each cell type calculated by two authors was averaged and the number of inflammatory cells was expressed per mm2.

Total number of eosinophils in blood

The absolute number of leucocytes per microlitre per mouse (n = 5; control group, n = 22; LAR group) was counted, and then the percentage of eosinophils was calculated after blood smear was stained with Giemsa solution. Then, the total number of eosinophils was determined.

Immunohistochemistry

For a direct or an indirect immunoperoxidase, after washing with PBS to remove mounting media, sections were treated with 5% bovine serum albumin (BSA; Sigma-Aldrich Co.) in PBS for 10 min at room temperature to block non-specific binding of antibody. Sections were incubated overnight at 4 °C with peroxidase-conjugated rabbit anti-OVA polyclonal antibody (2 μg/ml; Rockland Immunochemicals Inc., Gilbertsville, PA, USA), rat anti-mouse IgE monoclonal antibody (mAb) (0.01 mg/ml; PhaMingen, San Diego, CA, USA), rat anti-mouse VCAM-1 mAb (0.05 mg/ml; Antigenix America Inc., Huntington Station, NY, USA), rat anti-mouse VLA-4 mAb (0.05 mg/ml; Antigenix America Inc.) or rat anti-human CLA mAb (0.0125 mg/ml; Biosciences, Camarillo, CA, USA), which reacts with mouse CLA. After washing with 0.2% Tween 80 (Kanto chemical Co., Tokyo, Japan) in PBS, endogenous peroxidase activity was inactivated in 0.3% H2O2 in methanol for 30 min at room temperature. After washing with distilled water, sections were incubated with peroxidase-labelled goat anti-rat IgG (or IgM) (0.025 mg/dl; ICN Pharmaceutical Inc., Aurora, OH, USA) overnight at 4 °C. After washing with PBS, 3,3′-diaminobenzidine substrate (Roche Diagnostics GmbH, Penzberg, Germany) was added for 10 min at room temperature. Sections were counter-stained with 0.2% methyl green solution or haematoxylin.

For indirect immunofluorescent technique, sections were incubated overnight at 4 °C with rat anti-mouse ICAM-1 mAb (0.0125 mg/ml, clone KAT-1; Seikagaku, Tokyo, Japan) or rat anti-mouse LFA-1 mAb (0.25 mg/ml, CD11a, clone KBA; Seikagaku). After washing with 0.2% Tween 80 in PBS, sections were incubated with fluorescein isothiocyanate (FITC)-labelled goat anti-rat IgG (0.0025 mg/ml; ICN Pharmaceuticals) overnight.

For indirect immunoperoxidase or indirect immunofluorescence assays, the reaction without primary antibody served as a negative control. Mesenteric lymph node and spleen, respectively, from 8-week-old female BALB/c (normal) mice (n = 1) were used as a positive control of each antibody to adhesion molecules. In addition, as a positive control to detect OVA or IgE, OVA (10 μg/0.05 ml, Grade V; Sigma) or IgE (3.2 ng/0.05 ml; Morinaga, Kanagawa, Japan) were injected into the right and left ears of a normal mouse (n = 1) intradermally and 10 min after the injection the presence of OVA and IgE in cutaneous tissues was confirmed by the method described above.

Isolation of splenocytes

Spleens were aseptically removed and placed in a phosphate-buffered balanced salt solution (PBBS; pH 7.4). Splenic cells were obtained by the method described previously (Sasaki et al. 2007). In brief, single-cell suspensions were collected in sterile conical tubes (Assist Trading Co., Tokyo, Japan) after red blood cells were haemolysed by Tris-buffered NH4Cl solution, and washed in PBBS containing 0.1% heat-inactivated foetal calf serum (FCS; Gibco, Grand Island, NY, USA) followed by centrifugation at 800 g for 10 min at 4 °C. Cells were counted using a haemocytometer and diluted in minimum essential medium (MEM; Nissui Pharmaceutical Co., Tokyo, Japan) with kanamicin (20 μg/200 μl) to a density of 1 × 106 cells/ml. Viability of cells was more than 81% as determined by trypan blue dye exclusion test. Splenocytes (2 × 105/200 μl MEM with 5% FCS) were cultured for 48 h at 37 °C in 5% CO2 in the presence of 1.1 μg/200 μg of concanavalin A (Con A; ICN Biomedicals Inc.) in 96-well plates (Coster, Cambridge, MA, USA) and supernatants were obtained.

Measurements of IFN-γ, IL-4 and IL-5 in supernatants from cultured cells and IgE in plasma by ELISA

The concentration of IFN-γ, IL-4 (eBioscience, San Diego, CA, USA) and IL-5 (R&D system, Inc., Minneapolis, MN, USA) in supernatants from cultured splenocytes and IgE (Morinaga) in plasma was measured by mouse ELISA kits according to the manufacturer's instructions. Detectable amount for IgE is 500 pg/ml, for IFN-γ is 0.7 pg/ml and for IL-4 and IL-5 is 4 pg/ml. If cytokines could not be detected in the supernatants, their concentration was estimated as 0.

Statistical analysis

The data are expressed as the mean ± SEM of examined mice. Mann–Whitney U-test and Student's t-test (two-tailed) were used to evaluate the differences (comparison between the two groups) and a value P < 0.05 was considered statistically significant.

Results

The number of eosinophils and values of IgE in blood

As shown in Figure 2a, the number of eosinophils in peripheral blood in the LAR group (421.38 ± 44.33/μl) on day 18, but not in the control group, significantly increased compared with that on day 0 (233.35 ± 36.78/μl) (P < 0.05). The total IgE concentration in the LAR group was significantly higher than that in the control group (81.55 ± 3.07 vs. 10.27 ± 1.07 ng/ml) (P < 0.05). In addition, the concentration of IgE (81.55 ± 3.07 ng/ml) on day 18 increased compared with that on day 0 (10.45 ± 0.84 ng/ml) (P < 0.05) in the LAR group.

Figure 2.

Figure 2

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Number of eosinophils (a) and IgE values (b) in blood on days 0 and 18 respectively. Each value represents the mean ± SEM. *P < 0.05.

Production of cytokines in supernatants from cultured splenocytes

γ-Interferon production (Figure 3a) decreased in the LAR group (59.85 ± 6.39 pg/ml) compared with that in the control group (66.97 ± 3.06 pg/ml), although there was no statistical difference. On the other hand, IL-4 production (Figure 3b) in the LAR group (67.16 ± 8.09 pg/ml) increased compared with the control group (30.72 ± 3.59 pg/ml). IL-5 (Figure 3c) was detected in the LAR group (5.69 ± 2.66 pg/ml), whereas there was no IL-5 production in the control group (Figure 3c).

Figure 3.

Figure 3

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Production of cytokines in supernatants from cultured splenocytes stimulated with Con A on day 18. Concentration of IFN-γ (a), IL-4 (b) and IL-5. Each value represents the mean ± SEM. *P < 0.05.

Histopathology of rhinitis

The thickness of nasal septum (Figure 4a, 88.07 ± 2.14 μm), the total number of epithelial cells (Figure 4b, 62.69 ± 2.39), the ratio of goblet cells/non-goblet cells (Figure 4c) and score index (Figure 4d, 2.43 ± 0.20) in the LAR group were increased compared with the control group (Figure 4a, 70.16 ± 0.30 μm; Figure 4b, 53.60 ± 3.64; Figure 4c, 0.05 ± 0.03 or Figure 4d, 1.20 ± 0.15) (P < 0.05, except for the total number of epithelium). In addition, the number of eosinophils (260.23 ± 83.64 × 103 vs. 7.50 ± 5.26 × 103/mm2), lymphocytes (87.50 ± 15.03 × 103 vs. 28.75 ± 18.24 × 103/mm2), plasma cells (13.64 ± 5.82 × 103 vs. 3.75 ± 4.10 × 103/mm2) and neutrophils (59.09 ± 17.71 × 103 vs. 28.75 ± 13.71 × 103/mm2) was more in the LAR group than in the control group (P < 0.05 both).

Figure 4.

Figure 4

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Evaluation of late allergic rhinitis. The thickness of nasal septum (a), number of epithelium (b), ratio of the number of goblet cell and non-goblet cell (c), score index (d; severity of inflammation) and the number of each leucocyte (e) in the respiratory region. Each value represents the mean ± SEM. *P < 0.05.

In the LAR group, inflammatory lesions were located mainly in nasal mucosa near the nasal-associated lymphoid tissues, lateral, septum and floor of respiratory epithelium, and no inflammatory lesions were observed in the nasal mucosa lining olfactory epithelium. Rhinitis was characterized by the accumulation of inflammatory cells predominantly by eosinophils and lymphocytes accompanied by some neutrophils and a few plasma cells (Figure 4e). In addition, there was moderate-to-severe congestion in whole respiratory areas of nasal mucosa, and hypertrophic and hyperplastic respiratory epithelial cells with elongated and disorganized cilia showing vacuolar degeneration and necrosis in inflamed areas. Moreover, goblet cell hyperplasia and/or metaplasia with much mucus were observed (Figure 5b). On the other hand, histology revealed normal appearance except for a few inflammatory cell infiltrates in the control group (Figure 5a).

Figure 5.

Figure 5

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A control mouse shows relatively normal appearance with a few cell infiltration in nasal mucosa (a). Severely infiltrated eosinophils and lymphocytes with hyperplastic and degenerative respiratory epithelium are seen in a LAR mouse (b). HE ×400 (original magnification).

Histopathology of cutaneous tissues

The cutaneous tissues in the LAR group revealed mild-to-moderate inflammatory cells in the dermis and subcutaneous tissue, including eosinophils, lymphocytes, mast cells and neutrophils but not macrophages (Figure 6a–d). The number of inflammatory cells especially of eosinophils and mast cells in the LAR group was increased compared with that in the control group. However, the kinds of the infiltrating cell and the degree of lesion in cutaneous tissues varied with their location. Compared with the control group, the DAR group significantly increased inflammatory cell number as follows: the control vs. the LAR; mast cells (204.76 ± 23.28 × 103 vs. 88.75 ± 29.57 × 103/mm2) in the nose (P < 0.05; Figure 6a), eosinophils (132.95 ± 28.95 × 103 vs. 56.25 ± 46.01 × 103/mm2) and mast cells (183.81 ± 26.80 × 103 vs. 72.50 ± 50.12 × 103/mm2) in the anterior limbs (P < 0.05; Figure 6b) and eosinophils (210.80 ± 40.98 × 103 vs. 81.25 ± 103.35 × 103/mm2), lymphocyte (75.85 ± 15.00 × 103 vs. 17.50 ± 10.94 × 103/mm2), plasma cells (8.81 ± 4.57 × 103 vs. 1.25 ± 2.50 × 103/mm2) and neutrophils (34.66 ± 9.26 × 103 vs. 16.25 ± 8.3 3 × 103/mm2) in the hind limbs (P < 0.05; Figure 6c). On the other hand, there was no difference in the number of cells of the back cutaneous tissues between the two groups (Figure 6d).

Figure 6.

Figure 6

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Number of inflammatory cells in the cutaneous tissues of the nose (a), anterior limb (b), hind limb (c) and back (d). The kinds and degrees of the infiltrating cells are variable with their location. Each value represents the mean ± SEM. *P < 0.05.

Compared with the mice of the control group (Figure 7a), those of the LAR group showed hyperplasia of epidermis with epidermal spongyosis in the hind limb (Figure 7b), although, in general, no prominent epidermal changes at different parts of the skin were observed in the two groups. There were oedema and hyperaemia in the dermis and subcutaneous tissues where mainly mast cells and eosinophils with some neutrophils and plasma cells were recognized, although cutaneous lesions varied in the site (Figure 7c). Perivascular lymphocytic infiltration in the hind limb was rarely found. Increased number of eosinophils with some neutrophils and lymphocytes within the venular lumen and destruction of venular walls with haemorrhage was sometimes observed where eosinophils adhered to venular endothelium (eosinophilic venulitis) (Figure 7e,f). Degranulation of eosinophilic granules was often observed in the lumen walls and outside venules. These degranulated eosinophils increased their size with degeneration. These vascular changes were not observed in the control group (Figure 7d). In addition, degranulation from enlarged mast cells in the dermis and subcutaneous tissues was often observed and invasion of mast cells with degranulation into the peripheral nerve was often observed in the LAR group (Figure 7g) but not in the control group.

Figure 7.

Figure 7

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Histopathology of cutaneous tissues of the hind limb. A control mouse shows normal appearance in (a) and an arrow indicates venules (d; enlargement of a) without infiltration and destruction of vessel wall. A LAR mouse shows spongiosis with epidermal thickening (b), and infiltration of eosinophils and increase in mast cells with oedema in dermis and subcutaneous tissues (c). Details of a thin rectangle and a bold rectangle in (c) were shown in (e) and (f) respectively. (e) shows attachment of eosinophils to endothelial cells of venules (small arrows), destruction of venular wall (a bold arrow) and degranulation of mast cells (a large arrow) and (f) shows aggregation of eosinophils with degeneration and extravasation, and destruction of venular wall can be seen (a bold arrow). (g) shows peripheral nerve (an asterisk mark) with infiltration of mast cells (arrows). HE ×100 (a, b and c), ×200 (d, e, f and g) (original magnification).

Characterization of inflammatory cell and detection of adhesion molecules in cutaneous tissue

In the control group, mast cells (Figure 8a) and eosinophils (Figure 8c) were distributed sparsely, whereas those cells increased diffusely (Figure 8b,d) in the LAR group in the cutaneous tissues of the hind limb. As a positive control of CLA-positive lymphocytes, endothelial cells of the postcapillary venules in the lymph node were stained intensely (Figure 8e), whereas the CLA-positive cells were not recognized in both groups (Figure 8f). Although no VLA-4-positive cells were observed in the control mice (Figure 8g), numerous VLA-4-positive eosinophils and mast cells were observed in the LAR group (Figure 8h). No VCAM-1-positive vascular endothelial cells in the control group were recognized (Figure 8i), whereas intense VCAM-1 expression on endothelial cells was observed in the DAR group (Figure 8j). On the other hand, although ICAM-1 expression on vascular endothelial cells (Figure 8k,l) and LFA-1-positive leucocytes (data not shown) were observed in both groups, there was no difference in their expression between the two groups. There was neither IgE (Figure 8m) nor OVA (Figure 8o) in the control group, whereas the binding of IgE (Figure 8n) and OVA (Figure 8p) on mast cells and eosinophils and leak of those from venules were observed in the LAR group.

Figure 8.

Figure 8

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Characterization of inflammatory cells and detection of adhesion molecules in cutaneous tissues of the hind limb. Inflammatory cell types, which bind to OVA and IgE, were determined by cell morphology (e.g. cell size and nuclear morphology) and by comparison with serial sections stained by HE. Representative photographs showing mast cells (a and b), eosinophils (c and d), CLA (e and f), VLA-4 (g and h), VCAM-1(i and j), ICAM-1 (k and l), OVA (m and n) and IgE (o and p) in the control (a, c, e, g, i, k, m and o) and LAR group (b, d, f, h, j, l, n and p). Degranulation of enlarged mast cell (b; insert) and degenerated eosinophils with degranulation (d; left-hand side of insert, right-hand side of insert shows node granulation of eosinophil) are visible, whereas no eosinophils are visible (c). Endothelial cells of the high endothelial venules of a lymph node (e; positive control for CLA on cutaneous lymphocytes) can be identified (an arrow), whereas no CLA-positive lymphocytes were seen (f). (h), but not (g), shows VLA-4 expression (arrows) on mast cells (h; left-hand side of insert) and eosinophils (h; right-hand side of insert). (j), but not (i), shows VCAM-1 expressions on venules (arrows). (k) and (l) show ICAM-1 expression on endothelial cells of venules (arrows), and right half of (k) and(l), which were stained without a primary antibody, shows no fluorescence. (n), but not (m), shows binding of OVA (arrows) on mast cells (n; left-hand side of insert) and eosinophils (n; right-hand side of insert). (p), but not (o), shows binding of IgE (arrows) on mast cells (p; left-hand side of insert) and eosinophils (p; right-hand side of insert). (a, b) TB; (c, d) congo red; (e–j and m–p) immunoperoxidase; (k and l) immunofluorescence. (a–j) ×100, (m–p) ×400, (k and l) ×500; inserts (b, d, h, n and p) (original magnification). Arrows indicate inflammatory cells (a-d, h, n and p) and endothelium (j, and left half of K and I).

Discussion

This study demonstrated that mild cutaneous lesions developed in the mouse model of LAR. In addition, compared with the control group, eosinophilia, increased blood IgE values and increased productions of IL-4 and IL-5, but not IFN-γ, from splenocytes in the LAR group suggest the development of systemic Th2 responses, which may be shifted from Th1 to Th2 response by mutual Thl/Th2 inhibitory effect (Mosmann & Coffman 1989) in the LAR group. This is supported by the evidence that less number of blood eosinophils, a low blood IgE value, a high IFN-γ production and a low IL-4 and no IL-5 productions may represent Th1-dominant response in the control group compared with that in the LAR group.

IL-4 and IL-13, both share a number of biological properties, in the circulation may activate not only VCAM-1 expression on endothelium but also mast cells (Fukushi et al. 2000). Then, VLA-4, a counter receptor of VCAM-1, on eosinophils and mast cells may contribute to a firm attachment of eosinophilic to endothelium (Okigami et al. 2007). Additionally, VLA-4 expression on mast cells indicates their activation (Yasuda et al. 1995). On the other hand, ICAM-1 expression on venular endothelium and LFA-1 on neutrophils may contribute their infiltration in the cutaneous tissues (Franson et al. 2004). However, similar events such as ICAM-1 expression on endothelium and LFA-1-positive neutrophils were recognized in the control mice. Thus, neutrophil reaction may play a minor role in the development of cutaneous lesions. In addition, compared with the control group, infiltration of lymphocytes increased in cutaneous tissues of the hind limb, although, in general, there was no difference in the number of infiltrated lymphocytes in several cutaneous tissues between the two groups. Moreover, infiltration of mast cells into peripheral nerve and degranulation were often observed, suggesting that this may play a role in cutaneous inflammation. Taken together, adhesion molecules are to allow for eosinophil cells to attach to the vascular endothelium in dermis and subcutaneous tissues, and transmigration and infiltration may occur by the release of a series of preformed and newly synthesized mediators as a result of the interaction of IgE-sensitized mast cells with OVA. In addition, the binding of OVA and IgE was detected on eosinophils. Interaction of allergen with surface-bound IgE on eosinophils (Ying et al. 1998) may result in the release of inflammatory mediators (Gounni et al. 1994). Moreover, it has been reported that IL-4 upregulates FcεRI alpha in eosinophils (Terada et al. 1995). Thus, after extravasation, IgE-bound eosinophils with OVA may also play a role in the further development of cutaneous lesions in this way.

The presence of IgE and OVA outside blood vessels in the dermis and subcutaneous tissues suggests that those in the circulation may be derived from blood vessels. Origin of OVA may be slowly absorbed from the nasal lesions of OVA-contacted nasal mucosa into the circulation (Togias 2004). Thereafter, leak of IgE and OVA may occur by increased permeability of venules. There are several possibilities of increased permeability as follows. First, venular wall destruction will lead to increased permeability. It is probable as vascular damage by eosinophilic venulitis was observed at the site of eosinophils adhered to endothelium of venules. Then, release of cytotoxic substances (e.g. eosinophil peroxidase and major basic protein) from eosinophils may cause damages to endothelium, resulting in an increased vascular permeability (Rihoux 1990). Second, increased production of IL-4 in the LAR group may contribute to an increase in vascular permeability (Kotowicz et al. 2004). As a result, circulating OVA and IgE will leak to the cutaneous tissues. One might suppose that some of the inflammatory mediators especially histamine produced by the original nasal lesions and circulated in the peripheral blood may contribute to the increased permeability of venules in the dermis and subcutaneous tissues. However, there was no evidence of the increase in the number of mast cells of nasal mucosa under our experimental conditions including our previous studies (Hayashi et al. 2007; Sasaki et al. 2007), suggesting that this possibility is less likely. Locally produced IgE by plasma cells may be another possibility rather than leak of IgE. However, a few plasma cells were detected. Thus, also this possibility is less likely.

Leaked IgE cross-linked on mast cell with OVA may result in the release of histamine, eosinophil chemotactic factor (ECF)-A (Burks et al. 1988), LTB4, LTC4, LTD4, PGD2 as well as cytokines, such as IL-5 and IL-13, which directly induce allergic symptoms (Galli 1993). Thereafter, especially ECF-A may attract eosinophils from blood vessels to the cutaneous tissues. On the other hand, the number of mast cells increased in cutaneous tissues. It may be due to stem cell factor (SCF) produced by a number of tissue-resident cells including fibroblats and endothelial cells, which regulates mast cell growth and survival, differentiation and function in physical condition after interaction of SCF and c-kit on mast cells (Grabbe et al. 1994). Further studies are needed to clarify this point, since several growth factors have been suggested in the regulation of cutaneous mast cell growth and in the differentiation other than SCF (Grabbe et al. 1994).

The quality of inflammatory cell infiltration between rhinitis and dermatitis was quite different. First, the nasal lesion was characterized by the infiltration of mainly eosinophils and lymphocytes with some destructive change in respiratory epithelium (Hayashi et al. 2007). In addition, we have reported previously in the same experimental protocol as the present study that LAR may be induced by local and systemic production of IL-13 (Hayashi et al. 2007). In addition, Miyahara et al. (2006) have pointed out that IL-13 is a major contributor to the development of the late nasal response. These suggest that the allergic response in the nasal lesions may be induced by a late Th2 response (O’byrne et al. 1987; Kosgren et al. 1997; Takeda et al. 1997; Miyahara et al. 2005, 2006; Hayashi et al. 2007; Sasaki et al. 2007). On the other hand, in general, cutaneous lesions showed a predominant infiltration of eosinophils and an increase in mast cells in different cutaneous tissues. Moreover, the binding of IgE and OVA on mast cells and eosinophils was detected outside blood vessels. However, there was no infiltration of CLA+ lymphocytes, which were characteristically seen in late hypersensitivity reaction and CD4+ T lymphocytes are thought to regulate eosinophil recruitment to the site of allergic skin reaction through the activity of Th2 cytokines (Prescott et al. 2006). Thus, this is in contrast to the previous report that the underlying mechanisms of the reaction in dermatitis of food allergy are a combination of immediate and delayed hypersensitivity reactions (Würthrich 1998). This discrepancy may be due to the different mechanisms by different types of allergens and route of challenge.

There were two forms of allergic skin reaction. In acute urticaria, mast cells are generally viewed as key elicitors and the reaction is due to allergen-induced cross-linkage of mast cell membrane-bound IgE, which results in the release of vasoactive and chemotactic mediators, leading to localized oedema and eosinophil infiltration (Henz & Zuberbier 2000). On the other hand, atopic dermatitis was induced by Th2 cells (Hamid et al. 1994) and memory T cells migrating into the skin were highly enriched for CLA expressing memory-effector T cells (Picker 1994; Picker et al. 1994) other than IgE-mediated mast cell reaction. Thus, the development of cutaneous lesions of the present study seems probably the same as that of acute urticaria. In addition, the quality of the cutaneous lesions was variable, suggesting that the mechanisms may differ in different sites of the skin. Perhaps, the concentration of OVA in the circulation in each cutaneous tissue is different. Most likely is due to the number of mast cells existed in each cutaneous site as shown in the control mice.

Finally, systemically absorbed allergen would be taken up by every mast cell in every tissue, on the basis of the assumption that every one of these cells carries specific IgE-recognizing allergens against which allergic sensitization has occurred (Togias 2004). If so, an allergic reaction induced in the nasal mucosa should simultaneously result in manifestations (e.g. gastrointestinal tract) other than the cutaneous tissues as reported here. Thus, we are now investigating this point.

In conclusion, the present study demonstrated the systemic development of Th2-type reaction in LAR associated with the development of cutaneous lesions and suggests that acute uriticaria-like lesions may be induced by immediate allergic reactions. To the best of our knowledge, this is the first report on the development of inflammation at the distal site from the site of allergen exposure in a mouse model of LAR.

Acknowledgments

This study was supported in part by a grant-in-aid of the Ministry of Education, Science, Sports and Culture of Japan (No. 17658142).

References

  1. Beyer K, Castro R, Feidel C, Feidel C, Sampson HA. Milk-induced urticaria is associated with the expansion of T cells expressing cutaneous lymphocyte antigen. J. Allergy Clin. Immunol. 2002;109:688–693. doi: 10.1067/mai.2002.123235. [DOI] [PubMed] [Google Scholar]
  2. Brinkman L, Aslander MM, Raaijmakersb JA, Lammers JW, Koenderman L, Bruijnzeel-Koomen CA. Bronchial and cutaneous responses in atopic dermatitis patients after allergen inhalation challenge. Clin. Exp. Allergy. 1997;27:1043–1051. doi: 10.1111/j.1365-2222.1997.tb01256.x. [DOI] [PubMed] [Google Scholar]
  3. Burks AW, Mallory SB, Williams LW, Shirrell MA. Atopic dermatitis: clinical relevance of food hypersensitivity reactions. J. Pediatr. 1998;113:447–451. doi: 10.1016/s0022-3476(88)80626-7. [DOI] [PubMed] [Google Scholar]
  4. Cotran RS, Pober JS. Effects of cytokines on vascular endothelium: their role in vascular and immune injury. Kidney Int. 1989;35:969–975. doi: 10.1038/ki.1989.80. [DOI] [PubMed] [Google Scholar]
  5. Franson M, Benson M, Wennergren G, Gardell LO. A role for neutrophils in intermittent allergic rhinitis. Acta Otolaryngol. 2004;124:616–620. doi: 10.1080/00016480310015173. [DOI] [PubMed] [Google Scholar]
  6. Fukushi J, Ono M, Morikawa W, Iwamoto Y, Kuwano M. The activity of soluble VCAM-1 in angiogenesis stimulated by IL-4 and IL-13. J. Immunol. 2000;165:2818–2823. doi: 10.4049/jimmunol.165.5.2818. [DOI] [PubMed] [Google Scholar]
  7. Galli SJ. New concepts about the mast cell. N. Engl. J. Med. 1993;328:257–265. doi: 10.1056/NEJM199301283280408. [DOI] [PubMed] [Google Scholar]
  8. Gelfand EW. Inflammatory mediators in allergic rhinitis. J. Allergy Clin. Immunol. 2004;114:S135–S138. doi: 10.1016/j.jaci.2004.08.043. [DOI] [PubMed] [Google Scholar]
  9. Gounni AS, Lamkhioued B, Ochiai K, et al. High-affinity IgE receptor on eosinophils is involved in defence against parasites. Nature. 1994;367:183–186. doi: 10.1038/367183a0. [DOI] [PubMed] [Google Scholar]
  10. Grabbe J, Welker P, Dippel E, Czarnetzki BM. Stem cell factor, a novel cutaneous growth factor for mast cells and melanocytes. Arch. Dermatol. Res. 1994;287:78–84. doi: 10.1007/BF00370723. [DOI] [PubMed] [Google Scholar]
  11. Hamid Q, Boguniewicz M, leung DYM. Differential in situ cytokine gene expression in acute versus chronic atopic dermatitis. J. Clin. Invest. 1994;94:870–876. doi: 10.1172/JCI117408. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Hayashi T, Hasegawa K, Ichinohe N. ICAM-1 expression on endothelium and systemic cytokine production in cutaneous neutrophilic leukocytoclastic vasculitis in NZB × NZWF1 mice. Histol. Histopathol. 2005;20:45–52. doi: 10.14670/HH-20.45. [DOI] [PubMed] [Google Scholar]
  13. Hayashi T, Hasegawa K, Sasaki Y. Systemic administration of olygodeoxynucleotides with CpG motifs at priming phase reduces local Th2 response and late allergic rhinitis in BALB/c mice. Inflammation. 2008;31:47–56. doi: 10.1007/s10753-007-9048-9. [DOI] [PubMed] [Google Scholar]
  14. Henz BM, Zuberbier T. Uriticaria: new development and perspectives. Hautatarzt. 2000;51:302–308. doi: 10.1007/s001050051039. [DOI] [PubMed] [Google Scholar]
  15. Kosgren M, Erjefalt JS, Korsgren F, Sundler F, Persson CG. Allergic eosinophil-rich inflammation develops in lungs and airways of B cell-deficient mice. J. Exp. Med. 1997;185:885–893. doi: 10.1084/jem.185.5.885. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Kotowicz K, Callard RE, Klein NJ, Jacobs MG. Interleukin-4 increases the permeability of human cells in culture. Clin. Exp. Allergy. 2004;34:445–449. doi: 10.1111/j.1365-2222.2004.01902.x. [DOI] [PubMed] [Google Scholar]
  17. Kyllönen H, Malmberg P, Remitz A, Rytilä P, Metso T, Helenius I, Haähtela T. Respiratory symptoms, bronchial hyper-responsiveness, and eosinophilic airway inflammation in patients with moderate-to-severe atopic dermatitis. Clin. Exp. Allergy. 2006;36:192–197. doi: 10.1111/j.1365-2222.2006.02419.x. [DOI] [PubMed] [Google Scholar]
  18. Lei HY, Huang KJ, Shen CL, Huang JL. An antigen-specific hypersensitivity which does not fit into traditional classification of hypersensitivity. J. Immunol. 1989;143:432–438. [PubMed] [Google Scholar]
  19. Miyahara S, Miyahara N, Takeda K, Joetam A, Gelfand EW. Physiologic assessment of allergic rhinitis in mice: role of the high-affinity IgE receptor (FcεRI) J. Allergy Clin. Immunol. 2005;116:1020–1027. doi: 10.1016/j.jaci.2005.08.020. [DOI] [PubMed] [Google Scholar]
  20. Miyahara S, Miyahara N, Takeda K, et al. IL-13 is essential to the late-phase response in allergic rhinitis. J. Allergy Clin. Immunol. 2006;118:1110–1116. doi: 10.1016/j.jaci.2006.06.014. [DOI] [PubMed] [Google Scholar]
  21. Mosmann TR, Coffman R. TH1 and Th2 cells: different patterns of lymphokine secretion lead to different functional properties. Annu. Rev. Immunol. 1989;7:145–173. doi: 10.1146/annurev.iy.07.040189.001045. [DOI] [PubMed] [Google Scholar]
  22. Nakamura S, Ohtani H, Watanabe Y, et al. In situ expression of the cell adhesion molecules in inflammatory bowel disease. Lab. Invest. 1993;69:77–85. [PubMed] [Google Scholar]
  23. O’byrne PM, Dolovich J, Hargreave FE. Late asthmatic responses. Am. Rev. Respir. Dis. 1987;136:740–751. doi: 10.1164/ajrccm/136.3.740. [DOI] [PubMed] [Google Scholar]
  24. Okigami H, Takesita K, Tajimi M, et al. Inhibition of eosinophilia in vivo by a small molecule inhibitor of very late antigen (VLA)-4. Eur. J. Pharmacol. 2007;559:202–209. doi: 10.1016/j.ejphar.2006.11.065. [DOI] [PubMed] [Google Scholar]
  25. Picker LJ. Control of lymphocyte homing. Curr. Opin. Immunol. 1994;6:394–406. doi: 10.1016/0952-7915(94)90118-x. [DOI] [PubMed] [Google Scholar]
  26. Picker LJ, Martin RJ, Trumble A, et al. Differential expression of lymphocyte homing receptors by human memory/effector T cells in pulmonary versus cutaneous immune effector sites. Eur. J. Immunol. 1994;24:1269–1277. doi: 10.1002/eji.1830240605. [DOI] [PubMed] [Google Scholar]
  27. Prescott V, Forbes E, Foster PS, Matthaei K, Hogan SP. Mechanistic analysis of experimental food allergen-induced cutaneous reaction. J. Leukoc. Biol. 2006;80:258–266. doi: 10.1189/jlb.1105637. [DOI] [PubMed] [Google Scholar]
  28. Rihoux JP. Allergic reaction and vascular permeability. Allerg. Immunol. 1990;22:428–433. [PubMed] [Google Scholar]
  29. Sasaki Y, Hayashi T, Hasagawa K. Lactate dehydrogenase-elevation virus infection at the sensitization and challenge phases reduces the development of delayed eosinophilic allergic rhinitis in BALB/c mice. Scand. J. Immunol. 2007;66:628–635. doi: 10.1111/j.1365-3083.2007.02014.x. [DOI] [PubMed] [Google Scholar]
  30. Schafer T, Heinrich J, Wjst M, Adam H, Ring J, Wichmann H-E. Association between severity of atopic eczema and degree of sensitization to aeroallergens in school children. J. Allergy Clin. Immunol. 1999;104:1280–1284. doi: 10.1016/s0091-6749(99)70025-4. [DOI] [PubMed] [Google Scholar]
  31. Sicherer S, Sampson H. Food hypersensitivity and atopic dermatitis: pathophysiology, epidemiology, diagnosis, and management. J. Allergy Clin. Immunol. 1999;104:S114–S122. doi: 10.1016/s0091-6749(99)70053-9. [DOI] [PubMed] [Google Scholar]
  32. Skoner DP. Allergic rhinitis: definition, epidemiology, pathophysiology, detection, and diagnosis. J. Allergy Clin. Immunol. 2001;108:S2–S8. doi: 10.1067/mai.2001.115569. [DOI] [PubMed] [Google Scholar]
  33. Takeda K, Hamelmann E, Joetham A, et al. Development of eosinophilic airway inflammation and airway hyperresponsiveness in mast cell-deficient mice. J. Exp. Med. 1997;186:449–454. doi: 10.1084/jem.186.3.449. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Terada N, Konno A, Terada Y, et al. IL-4 upregulates Fc epsilon RI alpha-chain messenger RNA in eosinophils. J. Allergy Clin. Immunol. 1995;96:1161–1169. doi: 10.1016/s0091-6749(95)70201-6. [DOI] [PubMed] [Google Scholar]
  35. Togias A. Systemic effect of local allergic disease. J. Allergy Clin. Immunol. 2004;113:S8–S14. doi: 10.1016/j.jaci.2003.09.051. [DOI] [PubMed] [Google Scholar]
  36. Tupker R, De Monchy J, Coenraads P, Homan A, van Der Meer J. Induction of atopic dermatitis by inhalation of house dust mite. J. Allergy Clin. Immunol. 1996;97:1064–1070. doi: 10.1016/s0091-6749(96)70259-2. [DOI] [PubMed] [Google Scholar]
  37. Würthrich B. Food-induced cutaneous adverse reaction. Allergy. 1998;53:131–135. doi: 10.1111/j.1398-9995.1998.tb04983.x. [DOI] [PubMed] [Google Scholar]
  38. Yasuda M, Hasunuma Y, Adachi H, et al. Expression and function of fibronectin binding on rat mast cells. Int. Immunol. 1995;7:251–258. doi: 10.1093/intimm/7.2.251. [DOI] [PubMed] [Google Scholar]
  39. Ying S, Barata LT, Meng Q, et al. High-affinity immunoglobulin E receptor (FcεRI)-bearing eosinophils, mast cells, macrophages and Langerhans's cell in allergen-induced late-phase cutaneous reactions in atopic subjects. Immunology. 1998;93:281–288. doi: 10.1046/j.1365-2567.1998.00418.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Yu CK, Lee SC, Wang JY, Hsiue TR, Lei HY. Early-type hypersensitivity-associated airway inflammation and eosinophilia induced by dermatophagoides farinae in sensitized mice. J. Immunol. 1996;156:1923–1930. [PubMed] [Google Scholar]

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