- Email: [email protected]
Jic female mice
Exp Toxic Pathol 2000; 52: 235-240 URBAN & FISCHER http://www.urbanfischer.de/joumals/exptoxpath I Department of Veterinary Pathology, Faculty of Agriculture, The University of Tokyo, Japan 2National Institute of Industrial Health, Japan
Picryl chloride-induced allergic dermatitis in IQIIJic female mice MIHO IKEDA I , KOJI KUROKI I , HODAKA SUZUKI I , HIROYUKI NAKAYAMA I , JUNZO SAEGUSA 2, and KUNIO DOli With 4 figures and 1 table Received: February 8, 1999; Accepted: June 14, 1999 Address for correspondence: Dr. MIHO IKEDA, Department of Veterinary Pathology, Faculty of Agriculture, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan; Tel.lFax: +81-3-5841-8185. Key words: Allergic dermatitis; IQI/Jic female mice; Picryl chloride; Dermatitis, allergic.
Summary IQIIJic (IQI) mice are an ICR-derived inbred strain developed in Japan, and it is known that aged females of this strain develop allergic dermatitis of spontaneous nature. In the present study, young IQI female mice which were sensitized with picryl chloride (PCL) to the shaved skin of abdomen and then topically applied with PCL to the ear at 4, II, 18 and 25 days after the sensitization were examined. The ear swelling response increased rapidly after the 1st application, peaked after the 2nd one, and then gradually decreased. Histopathologically, edema with inflammatory cell infiltration developed after the 1st application and progressed after the 2nd one. The number of mast cells, CD4positive cells and MHC class II-positive cells became prominent accompanied with epidermal thickening and dermal fibroplasia after the 4th application when clear elevation of total serum IgE levels was observed in many mice. Compared with the dermatitis induced in the same way in BALB/c female mice, the nature was similar with each other but the degree was obviously severer in IQI female mice. IQI female mice are considered to be a useful laboratory animal for the investigation of allergic dermatitis.
Introduction The number of patients with immune-mediated dermatitis such as atopic dermatitis is increasing year by year. However, the exact etiology and pathogenesis of such dermatitis have not yet been clarified, resulting in the delay in developing new drugs for such dermatitis. To find a clue to solve these problems, comparative studies using various animal models are necessary. IQIIJic (lQI) mice are an ICR-derived inbred strain established in Japan. It is well known that IQI mice induce
a high level of antinuclear autoantibody following mercuric chloride-treatment (SAEGUSA et al. 1990), have thymic B cells (SAEGUSA et al. 1996), and show an agerelated infiltration of lymphocytes with parenchymal destruction in both salivary and lacrimal glands (SAEGUSA et al. 1997). It is also reported that IQI mice develop an age-related spindle cell hyperplasia with mast cell infiltration in adrenal cortex (KIM et al. 1997). The authors suggest that certain abnormalities in the immune system might underlie the above-mentioned phenomena observed in IQI mice. Recently, the prevalence of skin lesions of spontaneous nature was noticed in IQI mice of aged group. The dermatitis was observed only in females, and its incidence and severity increased with age. The dermatitis was considered to be due to Type I allergy judging from the histopathological findings and serum IgE levels, of which details will be published elsewhere. In this study, we tried to induce experimental dermatitis in young IQI female mice which have not developed spontaneous dermatitis yet, and compared its characteristics with those in BALB/c female mice, which are most often used for the induction of allergic dermatitis.
Material and methods Animals: Twenty-three 8-week-old IQI female mice were transported from National Institute of Industrial Health (Kawasaki, Japan) to our animal facility. In addition, 23 7-week-old BALB/c female mice were purchased from Japan SLC Inc. (Hamamatsu, Japan). The animals were individually kept in polycarbonate cages and fed standard laboratory pellets (MF, Oriental Yeast CO., Tokyo, Japan) and tap water ad libitum throughout the experimental period. The animal room was air-conditioned 0940-2993/00/52/03-235 $ 12.00/0
235
(temperature: 23 ± 2°C; relative humidity: 55 ± 5%) with a 14-hr light and lO-hr dark cycle. The animals were used after acclimation for 3 or 4 weeks. Treatments: Eighteen 12-to 13-week-old IQI mice and 18 lO-week-old BALB/c mice were sensitized by topical application with 150 III of 5% picryl chloride (PCL) (Nakalai Tesque Inc., Kyoto, Japan) to the shaved skin of abdomen. At 4, 11, 18 and 25 days after the sensitization, the ear was topically applied with 20 III of 0.8% PCL. PCL was dissolved in acetone and olive oil (4: 1). Five control mice of each strain were applied with vehicle alone in the same way. Measurement of ear swelling response: Ear thickness of each mouse was measured before the first application and at 24 h after the Ist, 2nd, 3rd and the 4th application with PCL or vehicle, respectively, using a digimatic micrometer (Mitutoyo Co., Kawasaki, Japan) under light ether anesthesia. The ear swelling response was judged from the difference between the ear thickness before the 1st application and that at 24 h after each application. Data were expressed as mean ± SD (10- 3 cm) of 5 (vehicle-applied) and 6 (PCLapplied) mice at each point of measurement. Measurement of total serum IgE levels: Total serum IgE levels were measured on 6 mice of each strain at 24 h after the 1st, 2nd and 4th application with PCL, and on 5 control mice of each strain at 24 h after the 4th application with vehicle, respectively, by sandwich ELISA method using mouse IgE measurement kit "YAMASA" EIA (Yamasa Shoyu Co., Ltd., Choshi, Japan). Histopathology: At 24 h after the 1st, 2nd and 4th application, 6 mice of each strain were killed by heart puncture under ether anesthesia, respectively. In addition, 5 control mice were killed in the same way at 24 h after the 4th application. For histopathological examination, the ears applied with PCL or vehicle were obtained from each mouse and fixed in 10% neutral buffered formalin. Paraffin sections (211m) were stained with hematoxylin and eosin (HE) or toluidine blue (TB). Immunohistochemistry: Pieces of the above-mentioned ear samples were embedded in O.c.T. compound a)
(Sakura Finetechnical Co., Ltd., Tokyo, Japan), rapidly frozen in dry ice-acetone, and then stored at -80°C until used. Cryosections (6 11m) were fixed in acetone for 10 minutes before immunohistochemical staining according to the avidin-biotin-peroxidase complex (ABC) method using Vectastain ABC kit (Vector Laboratories, California, USA). As the primary antibodies, rat monoclonal antibodies against mouse B220 (clone RA3-6B2), Mac-l (CDII b) (clone NInO) and CD4 (clone RM4-5), and hamster monoclonal antibody against mouse CD3e (clone 145-2Cll) were purchased from Pharmingen (California, USA). Rat monoclonal antibody against mouse MHC class II (I-Ab·d.q and I-Ed. k) (clone M5/l14.15.2) was supplied by Dr. S. KYUWA (Institute of Medical Science, the University of Tokyo), and rat monoclonal antibody against mouse CD8 (clone 2.43) was supplied by Dr. S. YAMAMOTO (Institute of Public Health, Tokyo). After ABC reaction, sections were visualized in 3,3'-diaminobenzidine tetrahydrochloride (DAB; Sigma, St. Louis MO, USA) solution. Sections were counterstained with methyl green.
Results Ear swelling response: Figure 1 shows the time course of ear swelling response in both strains. In IQI mice, the ear swelling response increased rapidly after the 1st application, peaked after the 2nd one, and then decreased gradually. On the other hand, the ear swelling response increased more slowly and peaked after the 3rd application in BALB/c mice. In addition, it was always weaker in BALB/c mice than in IQI mice at each point examined. Total serum IgE levels: Clear elevation of total serum IgE levels was recorded after the 4th application in both strains, and the level was much higher in BALB/c mice than in IQI mice (fig. 2). Histopathological findings: In the dermis of the ear of IQI mice treated with PCL, edema developed after the 1st application (fig. 3a) and became more prominent after the 2nd one, followed by prominent fibroplasia
b)
(X10·3 cm)
60 50 40
T
T
T
30
1
1
20
T
1
T
10 "'T
0
.1
1st
236
2nd
3rd 4th application
Exp Toxic Pathol 52 (2000) 3
1st
2nd
3rd application
4th
Fig. 1. Ear swelling response in IQI/Jic (a) and BALB/c (b) female mice after repeated application with PCL (G) or vehicle (A) following the sensitization with PCL. Data are shown as mean ± SD.
b)
a)
ng/ml
700 o
600 500
o
o
400
'0
8
300
Fig. 2. Total serum IgE levels after repeated PCL-application following the sensitization with PCL in IQI/Jic (a) and BALB/c (b) female mice. - = average value.
200 detection limit
o .".
O.l.-----r---.,.-----,r--~-
2nd 4th application
1st
after the 4th one (fig. 3b). Inflammatory cell infiltration was slight after the I st application (fig. 3a), but it progressed thereafter (fig. 3b). Infiltrated cells were mainly composed of neutrophils, eosinophils and mononuclear cells. As to mast cells, they showed no detectable change after the I st application. Mild increase in number of mast cells was observed in the superficial dermis after
. L
.'
• . " ..
~-
•
•
.
•.
'.....
•
,
,. ,
•
o
8
+-O~)()--o-----(lO--O
.
Control
1st
2nd 4th application
Control
the 2nd application, and the number of mast cells prominently increased after the 4th one (fig. 3c). In the epidermis, epidermal thickening due to keratinocyte hyperplasia and hyperkeratosis was observed after the I st application, and it progressed thereafter accompanied with inflammatory cell infiltration (fig. 3a, b).
- .. •
Fig. 3. Histopathological findings of the ear after repeated PCL-application following the sensitization with PCL in IQIIJic (a, b, c) and BALB/c (d) female mice. a) Moderate edema with mild inflammatory cell infiltration in the dermis after the 1st application. HE, x240. b) Marked epidermal thickening with prominent inflammatory cell infiltration and fibroplasia in the dermis after the 4th application. HE, x240. c) Many mast cells are seen in the dermis after the 4th application. TB, x480. d) Similar but less severe changes than those in b) are seen after the 4th application. HE, x240. Exp Toxic Pathol 52 (2000) 3
237
Table 1. Immunohistochemistry of component cells in the ear of IQI/Jic (a) and BALB/c (b) female mice after repeated PCL-application following the sensitization with PCL. B220
Application CD3
a) 1st 2nd 4th Control b) 1st 2nd 4th Control
MHC class II
Mac-I
Epidermis
Dermis
Epidermis
Dermis
Epidermis
Dermis
Epidermis
Dermis
± + +
+ ++
± ±
± ±
± + ++
+ ++ +++
± + ++ ±
± ++ ++++ ±
± ±
± + ++
± ±
± ±
± + +
+ ++ ++
± ++ +++ ±
± + ++ ±
* Degree of the increase in number of positive cells (-: none, ±: minimal, +: mild, +++: moderate, +++: marked) In the ear of BALB/c mice, although apparently less severe, changes with the same histopathological nature to that in the ear of IQI mice were observed (fig. 3d). There were no histopathological changes in the ear applied with vehicle.
Immunohistochemical findings: Table I shows the outline of immunohistochemical findings. In the ear of IQI mice, the increase in number of CD3e-positive cells became apparent after the 4th application of PCL. The kinetics of CD4-positive cells corresponded with that of
Fig. 4. Immunohistochemical staining of the ear after the 4th application of PCL in IQlIJic (a, b) and BALB/c (c) female mice. a) Many CD-4 positive cells are seen in the dermis. x240. b) Marked increase in number of MHC class II-positive cells are seen especially in the dermis. x240. c) Many MHC class II-positive keratinocytes are seen in the epidermal basal layer. x240. 238
Exp Toxic Pathol 52 (2000) 3
CD3e-positive cells, and only a few CD8-positive cells were noticed throughout the observation period. Therefore, almost all of the CD33-positive cells were thought to be CD4-positive ones (fig. 4a). B220-positive cells were rarely seen throughout the observation period. Mac-I-positive cells appeared after the 1st application, and their number increased with time. Similar but somewhat less severe changes were also seen in the ear of BALB/c mice. As to MHC class II-positive cells, their number increased with time in both strains. In addition, the number was larger in the dermis than in the epidermis in IQI mice while that was larger in the epidermis than in the dermis in BALB/c mice (fig. 4b, c).
Discussion Pathological studies were carried out on IQI female mice which were first sensitized with PCL to the shaved skin of abdomen and then topically applied with PCL to the ear repeatedly. BALB/c female mice were also treated in the same way and served as controls. The ear swelling response was apparently more intense and increased more rapidly in IQI mice than in BALB/c mice. This strain difference in the ear swelling response corresponded well with the strain difference in the severity of inflammatory exudation at the early phase and of fibroplasia at the late phase, respectively. In the ear of IQI mice, intradermal edema with infiltration of neutrophils, eosinophils and mononuclear cells was observed after the Ist application as reported by ROUPE and RIDELL (1979) in the skin of CBA mice after a single PCL application following the sensitization with PCL. Cellular infiltration increased with time, but edema subsided and was followed by fibroplasia after the 4th application. In the immunohistochemical examination, the numbers of Mac-l-, CD4- and MHC class II-positive cells also increased with time, especially after the 4th application. The increase in numbers of these cells was more prominent in IQI mice than in BALB/c mice. In addition, epidermal changes composed of epidermal thickening due to keratinocyte hyperplasia and hyperkeratosis and inflammatory cell infiltration increased with time and were also more prominent in IQI mice than in BALB/c mice. Up to the present time, there are several reports suggesting an important role of keratinocytes in the induction of contact dermatitis through expression of MHC class II (ROBERTS et al. 1985; GAWKROGER et al. 1987). In the present study, as mentioned above, the number of keratinocytes expressing MHC class II increased with time accompanied with the increment of inflammatory cell infiltration in both strains. However, the number of MHC class II-positive keratinocytes was larger in BALB/c mice while that of dermal positive cells, probably macrophages, was larger in IQI mice.
This suggests that there may be a strain difference in the mode of MHC class II expression in the skin after repeated PCL-application following the sensitization with PCL. Total serum IgE levels elevated after the 4th application when the increase in numbers of mast cells and CD4-positive T lymphocytes became prominent in the dermis. Mast cell response was larger in IQI mice than in BALB/c mice, whereas the total serum IgE level was apparently higher in BALB/c mice than in IQI mice. In this regard, it is generally said that BALB/c mice are high IgE-responders (HOLT et al. 1981; AZUMA et al. 1987). There are several reports of allergic contact dermatitis induced in mice by topical exposure to chemicals. The characteristics of dermatitis differs by sensitizing agents used. For example, trimellic anhydride (TMA) and diphenylmethane-4,4'-diisocyanate (MOl) induced dermatitis with increase in total serum IgE after a single application, but dermatitis induced by 2,4-dinitrochlorobenzen (DNCB), dicyclohgexylmethane-4,4'-diisocyanate (HMDI) or isophorone diisocyanate (IPDI) did not accompany the production ofIgE (DEARMAN et al. 1991, 1992). Therefore, they discussed that TMA and MOl selectively activated Th2 cells, and DNCB, HMDI and IPDI activated Thl cells. Moreover, KITAGAKI et al. (1995) reported that immediate-type hypersensitivity response with increase of antigen-specific IgE levels was induced by repeated epicutaneous application of PCL but it had never been observed after a single application of PCL in BALB/c mice. NAGI et al. (1997) also reported the similar findings in mice treated with 2-4-dinitrofluorobenzene (DNFB). They suggested that the shift from Thl-dominant response to Th2 dominant one might occur following the repeated application. Recent in vivo studies have also demonstrated that many responses started as Th 1dominant type and then shifted to Th2-dominant one (CLERICI and SHEARER 1993; ROOK et al. 1994; ELENKOV et al. 1998). Putting those findings and the present data together, there seems to be a possibility that the shift from Th 1dominant response to Th2-dominant one may also occur in the skin of the ear of both strains following the repeated PCL-application, although the analysis of subsets of CD4-positive lymphocytes had not been done in the present study. In conclusion, the dermatitis with characteristics of type I allergy could be induced in the ear of IQI mice after the 4th peL-application following the sensitization with PCL. The nature of dermatitis was similar between IQI and BALBIc female mice, but the degree of the dermatitis was obviously severer in IQI female mice than in BALB/c ones, suggesting that IQI female mice have higher susceptibility to topical application of contact sensitizing agents. Therefore, IQI female mice seem to be a useful laboratory animal for the investigation of allergic dermatitis. Exp Toxic Pathol 52 (2000) 3
239
References AZUMA M, HIRANO T, MIYAJIMA H, et al.: Regulation of murine IgE production in SJA/9 and nude mice. Potentiation of IgE production by recombinant interleukin 4. J Immunol 1987; 139: 2538-2544. CLERICI M, SHEARER GM: A Thl~Th2 switch is a critical step in the etiology of HIV infection. Immunol Today 1993; 14: 107-111. DEARMAN RJ, KIMBER I: Differential stimulation of immune function by respiratory and contact chemical allergens. Immunology 1991; 72: 563-570. DEARMAN RJ, SPENCE LM, KIMBER I: Characterization of murine immune responses to allergic diisocyanates. Toxicol Appl Pharmacol 1992; 112: 190-197. ELENKOV n, WEBSTER E, PAPANICOLAOU DA, et al.: Histamine potently suppresses human IL-12 and stimulates IL-IO production via H2 recepters. J Immunol 1998; 161: 2586-2593. GAWKRODGER DJ, CARR MM, MCVITTIE E, et al.: Keratinocyte expression of MHC class II antigens in allergic sensitization and challenge reactions and in irritant contact dermatitis. J Invest Dermatol 1987; 88: 11-16. HOLT PG, ROSE AH, BATTY JE, et al.: Induction of adjuvant-independent IgE responses in inbred mice: primary, secondary, and persistent IgE responses to ovalbumin and ovomucoid. Int Arch Allergy Appl Immunol 1981; 65: 42-50. KIM JS, KUBOTA H, DOl K, et al.: Correlation of mast cells
240
Exp Toxic Pathol 52 (2000) 3
with spindle cell hyperplasia in the adrenal cortex of IQIIJic mice. Exp Anim 1997; 46: 103-109. KITAGAKI H, FUJISAWA S, WATANABE K, et al.: lmmidiatetype hypersensitivity response followed by a late reaction is induced by repeated epicutaneous application of contact sensitizing agents in mice. J Invest Dermatol 1995;105:749-755. NAGAI H, MATSUO A, HIYAMA H, et al.: Immunoglobulin E production in mice by means of contact sensitization with a simple chemical, hapten. J Allergy Clin Immunol 1997; 100: 39-44. ROBERTS LK, SPANGRUDE GJ, DAYNES RA, et al.: Correlation between keratinocyte expression of la and the intensity and duration of contact hyper sensitivity responses in mice. J Immunol1985; 135: 2929-2936. ROOK GAW, HERNANDES-PANDO R, LIGHTMAN SL: Hormones, peripherally activated prohormones and regulation of the ThlfTh2 balance. Immunol Today 1994; 15: 301-303. ROUPE G and RIDELL B: The cellular infiltrate in contact hypersensitivity to picryl chloride on the mouse. Acta Dermatovener (Stockholm) 1979; 59: 191-195. SAEGUSA J, KIUCHI Y, ITOH T: Antinucleolar autoantibody induced in mice by mercuric chloride. - strain difference in susceptibility - Exp Anim 1990; 39: 597-599. SAEGUSA J, YASUDA A, KUBOTA H: IQIIJic mice have thymic B cells. Exp Anim 1996; 45: 353-360. SAEGUSA J, KUBOTA H: Sialadenitis in IQIIJic mice: a new animal model of Sjogren's syndrome. J Vet Med Sci 1997; 59: 897-903.
Picryl chloride-induced allergic dermatitis in IQIIJic female mice MIHO IKEDA I , KOJI KUROKI I , HODAKA SUZUKI I , HIROYUKI NAKAYAMA I , JUNZO SAEGUSA 2, and KUNIO DOli With 4 figures and 1 table Received: February 8, 1999; Accepted: June 14, 1999 Address for correspondence: Dr. MIHO IKEDA, Department of Veterinary Pathology, Faculty of Agriculture, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan; Tel.lFax: +81-3-5841-8185. Key words: Allergic dermatitis; IQI/Jic female mice; Picryl chloride; Dermatitis, allergic.
Summary IQIIJic (IQI) mice are an ICR-derived inbred strain developed in Japan, and it is known that aged females of this strain develop allergic dermatitis of spontaneous nature. In the present study, young IQI female mice which were sensitized with picryl chloride (PCL) to the shaved skin of abdomen and then topically applied with PCL to the ear at 4, II, 18 and 25 days after the sensitization were examined. The ear swelling response increased rapidly after the 1st application, peaked after the 2nd one, and then gradually decreased. Histopathologically, edema with inflammatory cell infiltration developed after the 1st application and progressed after the 2nd one. The number of mast cells, CD4positive cells and MHC class II-positive cells became prominent accompanied with epidermal thickening and dermal fibroplasia after the 4th application when clear elevation of total serum IgE levels was observed in many mice. Compared with the dermatitis induced in the same way in BALB/c female mice, the nature was similar with each other but the degree was obviously severer in IQI female mice. IQI female mice are considered to be a useful laboratory animal for the investigation of allergic dermatitis.
Introduction The number of patients with immune-mediated dermatitis such as atopic dermatitis is increasing year by year. However, the exact etiology and pathogenesis of such dermatitis have not yet been clarified, resulting in the delay in developing new drugs for such dermatitis. To find a clue to solve these problems, comparative studies using various animal models are necessary. IQIIJic (lQI) mice are an ICR-derived inbred strain established in Japan. It is well known that IQI mice induce
a high level of antinuclear autoantibody following mercuric chloride-treatment (SAEGUSA et al. 1990), have thymic B cells (SAEGUSA et al. 1996), and show an agerelated infiltration of lymphocytes with parenchymal destruction in both salivary and lacrimal glands (SAEGUSA et al. 1997). It is also reported that IQI mice develop an age-related spindle cell hyperplasia with mast cell infiltration in adrenal cortex (KIM et al. 1997). The authors suggest that certain abnormalities in the immune system might underlie the above-mentioned phenomena observed in IQI mice. Recently, the prevalence of skin lesions of spontaneous nature was noticed in IQI mice of aged group. The dermatitis was observed only in females, and its incidence and severity increased with age. The dermatitis was considered to be due to Type I allergy judging from the histopathological findings and serum IgE levels, of which details will be published elsewhere. In this study, we tried to induce experimental dermatitis in young IQI female mice which have not developed spontaneous dermatitis yet, and compared its characteristics with those in BALB/c female mice, which are most often used for the induction of allergic dermatitis.
Material and methods Animals: Twenty-three 8-week-old IQI female mice were transported from National Institute of Industrial Health (Kawasaki, Japan) to our animal facility. In addition, 23 7-week-old BALB/c female mice were purchased from Japan SLC Inc. (Hamamatsu, Japan). The animals were individually kept in polycarbonate cages and fed standard laboratory pellets (MF, Oriental Yeast CO., Tokyo, Japan) and tap water ad libitum throughout the experimental period. The animal room was air-conditioned 0940-2993/00/52/03-235 $ 12.00/0
235
(temperature: 23 ± 2°C; relative humidity: 55 ± 5%) with a 14-hr light and lO-hr dark cycle. The animals were used after acclimation for 3 or 4 weeks. Treatments: Eighteen 12-to 13-week-old IQI mice and 18 lO-week-old BALB/c mice were sensitized by topical application with 150 III of 5% picryl chloride (PCL) (Nakalai Tesque Inc., Kyoto, Japan) to the shaved skin of abdomen. At 4, 11, 18 and 25 days after the sensitization, the ear was topically applied with 20 III of 0.8% PCL. PCL was dissolved in acetone and olive oil (4: 1). Five control mice of each strain were applied with vehicle alone in the same way. Measurement of ear swelling response: Ear thickness of each mouse was measured before the first application and at 24 h after the Ist, 2nd, 3rd and the 4th application with PCL or vehicle, respectively, using a digimatic micrometer (Mitutoyo Co., Kawasaki, Japan) under light ether anesthesia. The ear swelling response was judged from the difference between the ear thickness before the 1st application and that at 24 h after each application. Data were expressed as mean ± SD (10- 3 cm) of 5 (vehicle-applied) and 6 (PCLapplied) mice at each point of measurement. Measurement of total serum IgE levels: Total serum IgE levels were measured on 6 mice of each strain at 24 h after the 1st, 2nd and 4th application with PCL, and on 5 control mice of each strain at 24 h after the 4th application with vehicle, respectively, by sandwich ELISA method using mouse IgE measurement kit "YAMASA" EIA (Yamasa Shoyu Co., Ltd., Choshi, Japan). Histopathology: At 24 h after the 1st, 2nd and 4th application, 6 mice of each strain were killed by heart puncture under ether anesthesia, respectively. In addition, 5 control mice were killed in the same way at 24 h after the 4th application. For histopathological examination, the ears applied with PCL or vehicle were obtained from each mouse and fixed in 10% neutral buffered formalin. Paraffin sections (211m) were stained with hematoxylin and eosin (HE) or toluidine blue (TB). Immunohistochemistry: Pieces of the above-mentioned ear samples were embedded in O.c.T. compound a)
(Sakura Finetechnical Co., Ltd., Tokyo, Japan), rapidly frozen in dry ice-acetone, and then stored at -80°C until used. Cryosections (6 11m) were fixed in acetone for 10 minutes before immunohistochemical staining according to the avidin-biotin-peroxidase complex (ABC) method using Vectastain ABC kit (Vector Laboratories, California, USA). As the primary antibodies, rat monoclonal antibodies against mouse B220 (clone RA3-6B2), Mac-l (CDII b) (clone NInO) and CD4 (clone RM4-5), and hamster monoclonal antibody against mouse CD3e (clone 145-2Cll) were purchased from Pharmingen (California, USA). Rat monoclonal antibody against mouse MHC class II (I-Ab·d.q and I-Ed. k) (clone M5/l14.15.2) was supplied by Dr. S. KYUWA (Institute of Medical Science, the University of Tokyo), and rat monoclonal antibody against mouse CD8 (clone 2.43) was supplied by Dr. S. YAMAMOTO (Institute of Public Health, Tokyo). After ABC reaction, sections were visualized in 3,3'-diaminobenzidine tetrahydrochloride (DAB; Sigma, St. Louis MO, USA) solution. Sections were counterstained with methyl green.
Results Ear swelling response: Figure 1 shows the time course of ear swelling response in both strains. In IQI mice, the ear swelling response increased rapidly after the 1st application, peaked after the 2nd one, and then decreased gradually. On the other hand, the ear swelling response increased more slowly and peaked after the 3rd application in BALB/c mice. In addition, it was always weaker in BALB/c mice than in IQI mice at each point examined. Total serum IgE levels: Clear elevation of total serum IgE levels was recorded after the 4th application in both strains, and the level was much higher in BALB/c mice than in IQI mice (fig. 2). Histopathological findings: In the dermis of the ear of IQI mice treated with PCL, edema developed after the 1st application (fig. 3a) and became more prominent after the 2nd one, followed by prominent fibroplasia
b)
(X10·3 cm)
60 50 40
T
T
T
30
1
1
20
T
1
T
10 "'T
0
.1
1st
236
2nd
3rd 4th application
Exp Toxic Pathol 52 (2000) 3
1st
2nd
3rd application
4th
Fig. 1. Ear swelling response in IQI/Jic (a) and BALB/c (b) female mice after repeated application with PCL (G) or vehicle (A) following the sensitization with PCL. Data are shown as mean ± SD.
b)
a)
ng/ml
700 o
600 500
o
o
400
'0
8
300
Fig. 2. Total serum IgE levels after repeated PCL-application following the sensitization with PCL in IQI/Jic (a) and BALB/c (b) female mice. - = average value.
200 detection limit
o .".
O.l.-----r---.,.-----,r--~-
2nd 4th application
1st
after the 4th one (fig. 3b). Inflammatory cell infiltration was slight after the I st application (fig. 3a), but it progressed thereafter (fig. 3b). Infiltrated cells were mainly composed of neutrophils, eosinophils and mononuclear cells. As to mast cells, they showed no detectable change after the I st application. Mild increase in number of mast cells was observed in the superficial dermis after
. L
.'
• . " ..
~-
•
•
.
•.
'.....
•
,
,. ,
•
o
8
+-O~)()--o
.
Control
1st
2nd 4th application
Control
the 2nd application, and the number of mast cells prominently increased after the 4th one (fig. 3c). In the epidermis, epidermal thickening due to keratinocyte hyperplasia and hyperkeratosis was observed after the I st application, and it progressed thereafter accompanied with inflammatory cell infiltration (fig. 3a, b).
- .. •
Fig. 3. Histopathological findings of the ear after repeated PCL-application following the sensitization with PCL in IQIIJic (a, b, c) and BALB/c (d) female mice. a) Moderate edema with mild inflammatory cell infiltration in the dermis after the 1st application. HE, x240. b) Marked epidermal thickening with prominent inflammatory cell infiltration and fibroplasia in the dermis after the 4th application. HE, x240. c) Many mast cells are seen in the dermis after the 4th application. TB, x480. d) Similar but less severe changes than those in b) are seen after the 4th application. HE, x240. Exp Toxic Pathol 52 (2000) 3
237
Table 1. Immunohistochemistry of component cells in the ear of IQI/Jic (a) and BALB/c (b) female mice after repeated PCL-application following the sensitization with PCL. B220
Application CD3
a) 1st 2nd 4th Control b) 1st 2nd 4th Control
MHC class II
Mac-I
Epidermis
Dermis
Epidermis
Dermis
Epidermis
Dermis
Epidermis
Dermis
± + +
+ ++
± ±
± ±
± + ++
+ ++ +++
± + ++ ±
± ++ ++++ ±
± ±
± + ++
± ±
± ±
± + +
+ ++ ++
± ++ +++ ±
± + ++ ±
* Degree of the increase in number of positive cells (-: none, ±: minimal, +: mild, +++: moderate, +++: marked) In the ear of BALB/c mice, although apparently less severe, changes with the same histopathological nature to that in the ear of IQI mice were observed (fig. 3d). There were no histopathological changes in the ear applied with vehicle.
Immunohistochemical findings: Table I shows the outline of immunohistochemical findings. In the ear of IQI mice, the increase in number of CD3e-positive cells became apparent after the 4th application of PCL. The kinetics of CD4-positive cells corresponded with that of
Fig. 4. Immunohistochemical staining of the ear after the 4th application of PCL in IQlIJic (a, b) and BALB/c (c) female mice. a) Many CD-4 positive cells are seen in the dermis. x240. b) Marked increase in number of MHC class II-positive cells are seen especially in the dermis. x240. c) Many MHC class II-positive keratinocytes are seen in the epidermal basal layer. x240. 238
Exp Toxic Pathol 52 (2000) 3
CD3e-positive cells, and only a few CD8-positive cells were noticed throughout the observation period. Therefore, almost all of the CD33-positive cells were thought to be CD4-positive ones (fig. 4a). B220-positive cells were rarely seen throughout the observation period. Mac-I-positive cells appeared after the 1st application, and their number increased with time. Similar but somewhat less severe changes were also seen in the ear of BALB/c mice. As to MHC class II-positive cells, their number increased with time in both strains. In addition, the number was larger in the dermis than in the epidermis in IQI mice while that was larger in the epidermis than in the dermis in BALB/c mice (fig. 4b, c).
Discussion Pathological studies were carried out on IQI female mice which were first sensitized with PCL to the shaved skin of abdomen and then topically applied with PCL to the ear repeatedly. BALB/c female mice were also treated in the same way and served as controls. The ear swelling response was apparently more intense and increased more rapidly in IQI mice than in BALB/c mice. This strain difference in the ear swelling response corresponded well with the strain difference in the severity of inflammatory exudation at the early phase and of fibroplasia at the late phase, respectively. In the ear of IQI mice, intradermal edema with infiltration of neutrophils, eosinophils and mononuclear cells was observed after the Ist application as reported by ROUPE and RIDELL (1979) in the skin of CBA mice after a single PCL application following the sensitization with PCL. Cellular infiltration increased with time, but edema subsided and was followed by fibroplasia after the 4th application. In the immunohistochemical examination, the numbers of Mac-l-, CD4- and MHC class II-positive cells also increased with time, especially after the 4th application. The increase in numbers of these cells was more prominent in IQI mice than in BALB/c mice. In addition, epidermal changes composed of epidermal thickening due to keratinocyte hyperplasia and hyperkeratosis and inflammatory cell infiltration increased with time and were also more prominent in IQI mice than in BALB/c mice. Up to the present time, there are several reports suggesting an important role of keratinocytes in the induction of contact dermatitis through expression of MHC class II (ROBERTS et al. 1985; GAWKROGER et al. 1987). In the present study, as mentioned above, the number of keratinocytes expressing MHC class II increased with time accompanied with the increment of inflammatory cell infiltration in both strains. However, the number of MHC class II-positive keratinocytes was larger in BALB/c mice while that of dermal positive cells, probably macrophages, was larger in IQI mice.
This suggests that there may be a strain difference in the mode of MHC class II expression in the skin after repeated PCL-application following the sensitization with PCL. Total serum IgE levels elevated after the 4th application when the increase in numbers of mast cells and CD4-positive T lymphocytes became prominent in the dermis. Mast cell response was larger in IQI mice than in BALB/c mice, whereas the total serum IgE level was apparently higher in BALB/c mice than in IQI mice. In this regard, it is generally said that BALB/c mice are high IgE-responders (HOLT et al. 1981; AZUMA et al. 1987). There are several reports of allergic contact dermatitis induced in mice by topical exposure to chemicals. The characteristics of dermatitis differs by sensitizing agents used. For example, trimellic anhydride (TMA) and diphenylmethane-4,4'-diisocyanate (MOl) induced dermatitis with increase in total serum IgE after a single application, but dermatitis induced by 2,4-dinitrochlorobenzen (DNCB), dicyclohgexylmethane-4,4'-diisocyanate (HMDI) or isophorone diisocyanate (IPDI) did not accompany the production ofIgE (DEARMAN et al. 1991, 1992). Therefore, they discussed that TMA and MOl selectively activated Th2 cells, and DNCB, HMDI and IPDI activated Thl cells. Moreover, KITAGAKI et al. (1995) reported that immediate-type hypersensitivity response with increase of antigen-specific IgE levels was induced by repeated epicutaneous application of PCL but it had never been observed after a single application of PCL in BALB/c mice. NAGI et al. (1997) also reported the similar findings in mice treated with 2-4-dinitrofluorobenzene (DNFB). They suggested that the shift from Thl-dominant response to Th2 dominant one might occur following the repeated application. Recent in vivo studies have also demonstrated that many responses started as Th 1dominant type and then shifted to Th2-dominant one (CLERICI and SHEARER 1993; ROOK et al. 1994; ELENKOV et al. 1998). Putting those findings and the present data together, there seems to be a possibility that the shift from Th 1dominant response to Th2-dominant one may also occur in the skin of the ear of both strains following the repeated PCL-application, although the analysis of subsets of CD4-positive lymphocytes had not been done in the present study. In conclusion, the dermatitis with characteristics of type I allergy could be induced in the ear of IQI mice after the 4th peL-application following the sensitization with PCL. The nature of dermatitis was similar between IQI and BALBIc female mice, but the degree of the dermatitis was obviously severer in IQI female mice than in BALB/c ones, suggesting that IQI female mice have higher susceptibility to topical application of contact sensitizing agents. Therefore, IQI female mice seem to be a useful laboratory animal for the investigation of allergic dermatitis. Exp Toxic Pathol 52 (2000) 3
239
References AZUMA M, HIRANO T, MIYAJIMA H, et al.: Regulation of murine IgE production in SJA/9 and nude mice. Potentiation of IgE production by recombinant interleukin 4. J Immunol 1987; 139: 2538-2544. CLERICI M, SHEARER GM: A Thl~Th2 switch is a critical step in the etiology of HIV infection. Immunol Today 1993; 14: 107-111. DEARMAN RJ, KIMBER I: Differential stimulation of immune function by respiratory and contact chemical allergens. Immunology 1991; 72: 563-570. DEARMAN RJ, SPENCE LM, KIMBER I: Characterization of murine immune responses to allergic diisocyanates. Toxicol Appl Pharmacol 1992; 112: 190-197. ELENKOV n, WEBSTER E, PAPANICOLAOU DA, et al.: Histamine potently suppresses human IL-12 and stimulates IL-IO production via H2 recepters. J Immunol 1998; 161: 2586-2593. GAWKRODGER DJ, CARR MM, MCVITTIE E, et al.: Keratinocyte expression of MHC class II antigens in allergic sensitization and challenge reactions and in irritant contact dermatitis. J Invest Dermatol 1987; 88: 11-16. HOLT PG, ROSE AH, BATTY JE, et al.: Induction of adjuvant-independent IgE responses in inbred mice: primary, secondary, and persistent IgE responses to ovalbumin and ovomucoid. Int Arch Allergy Appl Immunol 1981; 65: 42-50. KIM JS, KUBOTA H, DOl K, et al.: Correlation of mast cells
240
Exp Toxic Pathol 52 (2000) 3
with spindle cell hyperplasia in the adrenal cortex of IQIIJic mice. Exp Anim 1997; 46: 103-109. KITAGAKI H, FUJISAWA S, WATANABE K, et al.: lmmidiatetype hypersensitivity response followed by a late reaction is induced by repeated epicutaneous application of contact sensitizing agents in mice. J Invest Dermatol 1995;105:749-755. NAGAI H, MATSUO A, HIYAMA H, et al.: Immunoglobulin E production in mice by means of contact sensitization with a simple chemical, hapten. J Allergy Clin Immunol 1997; 100: 39-44. ROBERTS LK, SPANGRUDE GJ, DAYNES RA, et al.: Correlation between keratinocyte expression of la and the intensity and duration of contact hyper sensitivity responses in mice. J Immunol1985; 135: 2929-2936. ROOK GAW, HERNANDES-PANDO R, LIGHTMAN SL: Hormones, peripherally activated prohormones and regulation of the ThlfTh2 balance. Immunol Today 1994; 15: 301-303. ROUPE G and RIDELL B: The cellular infiltrate in contact hypersensitivity to picryl chloride on the mouse. Acta Dermatovener (Stockholm) 1979; 59: 191-195. SAEGUSA J, KIUCHI Y, ITOH T: Antinucleolar autoantibody induced in mice by mercuric chloride. - strain difference in susceptibility - Exp Anim 1990; 39: 597-599. SAEGUSA J, YASUDA A, KUBOTA H: IQIIJic mice have thymic B cells. Exp Anim 1996; 45: 353-360. SAEGUSA J, KUBOTA H: Sialadenitis in IQIIJic mice: a new animal model of Sjogren's syndrome. J Vet Med Sci 1997; 59: 897-903.