LaboratoryComparative Medicine Science Vol 50, No 2 Copyright 2000 April 2000 by the American Association for Laboratory Animal Science

Atopic Dermatitis in NC/Jic Mice Associated with Infestation

Osamu T. Iijima,1,2 Hiroshi Takeda,2 Yasuhiro Komatsu,1 Teruhiko Matsumiya,2 and Hisahide Takahashi3

The NC (NC/Nga) mouse strain was established as an inbred NC/Jic mice also were purchased from CLEA Japan. The SPF strain by Kondo in 1957 on the basis on Japanese fancy mice (1, male BALB/cA Jcl mice also were obtained from CLEA Japan as 2). The NC/Nga mice were originally recognized as an autoim- the reference strain. These SPF mice were monitored and cer- mune disease model mice and were known to manifest signs of tificated as free from the aforementioned pathogens and any a number of disorders. Dermatitis with extremely high inci- ectoparasites. The mice were housed in groups of 4 to 6 in 31 x dence was the most evident disorder in NC/Nga mice (1, 3, 4). 23 x 15.5-cm plastic shoebox cages (M-55-TG, Okazaki Sangyo In 1997, Matsuda et al. documented that NC/Nga mouse derma- Co., Saitama, Japan). They were maintained in a laminar flow titis is a model of human atopic dermatitis (AD) (5). According rack (LFR-A-2, Tokiwa Kagaku Kikai Co., Tokyo, Japan) in the to Matsuda, NC/Nga mice develop skin lesions that are clini- animal facility (room temperature: 23 Ϯ 2ЊC, relative humidity: cally and histologically similar to human AD, spontaneously 55 Ϯ 10%, all fresh air ventilation: 15 to 20 times/h, 12 hours’ appearing on the face, neck, ears, and dorsal skin of the mice. light and 12 hours’ dark). Feed (MF, Oriental Yeast Co., Tokyo, Plasma concentration of total immunoglobulin E (IgE) in the Japan) and water were available ad libitum, and the bedding mice is markedly high from eight weeks of age, correlating with (Paper Clean, Japan SLC, Shizuoka, Japan) was changed once a the clinical severity of dermatitis. One important aspect of the week. NC/Nga strain as a human AD model is that the lesions of the Diagnosis of M. musculi: Cellophane tape (14 x 40 mm) mice appear when they are raised under non-sterile (conven- was pressed against areas of the pelt of the head, neck, and tional) circumstances, but not under specific-pathogen-free shoulders of mice then was affixed to a slide. All fields of the (SPF) conditions. Thus, NC/Nga mice suffer from dermatitis tape were examined microscopically (100x) for the presence of similar to human AD, with IgE hyperproduction, which may be M. musculi. triggered by some environmental factor(s). Although the possi- Eradication of M. musculi: Eradication was performed ac- bility of an allergic mechanism in the cause of the dermatitis of cording to a method described elsewhere (7), with slight modi- NC/Nga mice was reported (6), others denied the immunologic fication. A 1:100 dilution of ivermectin (0.1 mg/ml; IvomecTM, mechanism in the course of the development of the dermatitis Merial London, London, UK) in distilled water was applied as four (4). The cause of this dermatitis remained unclear. squirts per cage (2.6 ml of solution or 0.26 mg of ivermectin) once In a study of NC/Jic mice, which were derived from Kondo’s a week just after the change of bedding. After confirmation of colony of NC/Nga mice after establishment of the strain in 1957 the eradication of M. musculi, we applied additional squirts of and have been sustained in the Central Institute for Experi- ivermectin for two weeks, on the basis of the life cycle of this mental , we observed that the mice with dermatitis (8). were infested with the common mouse mite, Myobia musculi. Clinical severity score: Clinical skin conditions in the mice We designed the present study to clarify the relation between were examined and were scored by two persons unaware of the dermatitis and M. musculi infestation. which mice were from which group, according to described crite- Animals: Male NC/Jic mice purchased from CLEA Japan ria (5), with slight modification. A clinical severity score for the (Tokyo, Japan) were transferred to Tsumura Central Laborato- skin lesions was defined as the sum of individual scores graded ries from Fujita Health University and were sustained in a con- as 0 (none), 1 (mild), 2 (moderate), and 3 (severe) for each of five ventional animal facility. Although these mice were originally signs (itch, edema, erythema/hemorrhage, excoriation/erosion, SPF (monitored and certificated as free of Escherichia. coli O115a, and scaling/dryness). Before skin conditions were scored, indi- c : K(B), Pasteurella pneumotropica, Pseudomonas aeruginosa, Sal- vidual scratching behavior was observed for 2 minutes. Itching monella typhimurium, Corynebacterium kutscheri, Mycoplasma was evaluated by observing scratching behavior. Lack of pulmonis, Tyzzer’s organism, Ectromelia virus, mouse adenovi- scratching behavior was scored as grade 0, 1 to 2 times was rus, mouse hepatitis virus, Sendai virus, Giardia muris, scored as grade 1, 3 to 5 times was scored as grade 2, and more Spironucleus muris, and Syphacia spp. throughout the experi- than 5 times was scored as grade 3. Edema was evaluated by mental period), they had been infested with M. musculi. We de- scaling the thickness of both ears by use of a thickness gauge scribe these mice as “conventional” in this study. The SPF male (Dial thickness gauge G, Ozaki Mfg. Co., Tokyo, Japan). Mean thickness < 0.3 mm was scored as grade 0, 0.3 to 0.39 mm was 1Kampo & Pharmacognosy Laboratory, Tsumura & Co., 3586 Ami-machi, grade 1, 0.4 to 0.59 mm was grade 2, and > 0.59 mm was grade Yoshiwara, Inashiki- gun, Ibaraki, 300-1192; 2Department of Pharmacology and 3. Other 3 signs are graded as area of lesions. No lesion was Intractable Disease Research Center (Division of Drug Research and Develop- ment), Tokyo Medical University, 6-1-1 Shinjuku Shinjuku-ku, Tokyo 160-8402; scored as grade 0, around 1 mm of diameter of the lesion was and 3Laboratory Animal Center, School of Medicine, Fujita Health University, grade 1, around 3 mm was grade 2 and Ն 5 mm was grade 3. 1-98 Kutsukake, Toyoake, Aichi, 470-1101 Japan. Differences in evaluation between two persons were averaged. 225 Vol 50, No 2 Comparative Medicine April 2000

Detection of plasma IgE: Plasma IgE was quantified, using a sandwich enzyme-linked immunosorbent assay (ELISA). First, 96-well immunoplates were coated with purified mouse IgE monoclonal antibody (2 ␮g/ml; Yamasa Corp., Chiba, Japan) diluted in coating buffer (0.1M NaHCO3/0.1M Na2CO3). After overnight incubation at 4ЊC, the wells were blocked for 1 hour at room temperature. The plates were washed three times with wash buffer (0.1% Tween-20 in phosphate-buffered saline), and the standard or samples were added at 50 ␮l/well, respectively. The plates were incubated for 2.5 hours at room temperature, the wells were washed four times with wash buffer, biotinylated mouse IgE monoclonal antibody (100 ␮l/ml; Yamasa Corp.) was added, and incubation proceeded for 1 hour at room tempera- ture. The wells were washed six times, followed by addition of 1:500 dilution of a streptavidin-peroxidase complex (100 ␮l/well; Sigma Chemical Co., St. Louis, MO) for 30 minutes at room tem- perature. Wells were washed eight times, and 2,2-azino-bis (3- ethylbenz-thiazoline-6-sulfonic acid) (ABTS; 100 ␮l/well) substrate solution (10.3 mg of ABTS/ml of 0.1M citric acid buffer, pH 4.35) was added. On color development, the plate was analyzed by use of an ELISA plate reader at optical density (OD) of 405 nm. Experimental design: Three experiments were conducted: control, experimental eradication, and experimental infestation. In study 1 (control), six conventional male NC/Jic mice and eight SPF male NC/Jic mice were used. Plasma IgE values, clinical severity score, and numbers of mice with were evaluated at the ages of 4, 8, 12, 16, 20, and 24 weeks. In study 2, eight conventional male NC/Jic mice that had de- veloped severe dermatitis and had extremely high plasma IgE values, were used. The experimental eradication study was be- gun at the age of 16 weeks. Before mite eradication, all mice were confirmed to have been infested with M. musculi. Plasma IgE values, clinical severity score, and numbers of mice with mites were evaluated at 4 and 8 weeks after the onset of eradi- cation. In study 3, four SPF male NC/Jic and four SPF male BALB/ cA mice of four-week-old (mite-free) and two conventional male NC/Jic mice of 17-month-old (with mites) were used. Total plasma IgE values and clinical severity scores for each conven- tional NC/Jic mouse were 140 and 134 ␮g/ml and 12 and 11, re- spectively, at the onset of the experiment. One conventional NC/ Jic mouse and two SPF NC/Jic and SPF BALB/cA mice each were housed together for four weeks in a cage. We used two cages for experimentally induced infestation. Plasma IgE val- ues, clinical severity score, and numbers of mice with mites were evaluated at 4, 8, and 12 weeks after the onset of experi- mentally induced infestation. All experimental procedures were conducted according to the guidelines of The Animal Care and Use Committee of Tsumura Figure 1. Total plasma IgE concentrations in conventional and spe- cific-pathogen-free (SPF) male NC/Jic mice of study 1. All conven- Central Laboratories. tional mice were infested with Myobia musculi throughout the ex- Results are summarized in Figure 1 and Table 1. In study 1, perimental period. : Conventional NC/Jic mice; : SPF NC/Jic mice. the conventional NC/Jic mice had high IgE values at 8 weeks of Data are expressed as the mean Ϯ SD of 6 (conventional) or 8 (SPF) age, when mild dermatitis was clinically evident. The IgE val- mice. Total plasma IgE concentrations in conventional male NC/Jic ues then increased markedly and reached a plateau at 16 mice of study 2 (experimental eradication of mites). Experimental eradication was started at the age of 16 week. Ivermectin (0.26 mg/ weeks. Severe dermatitis was apparent at 12 weeks of age, and cage) was applied once a week for 6 times. : Conventional NC/Jic continued to increase age. Existence of M. musculi was proved mice. Data are expressed as the mean Ϯ SD of 8 mice. Total plasma throughout the experimental period. On the other hand, SPF IgE concentrations in SPF male NC/Jic mice and SPF male BALB/cA NC/Jic mice did not show these findings. mice of study 3 (experimentally induced infestation). Two strains of

SPF mice (mite-free) were caged together with conventional male NC/ In study 2, mean clinical severity score of the conventional Jic mice (with mites) for 4 weeks. : SPF NC/Jic mice;∇ : SPF BALB/ NC/Jic mice was 8.1 at the onset of eradication, decreased cA mice. Data are expressed as the mean Ϯ SD of four mice. 226 Mouse Atopic Dermatitis Model by Mite Infestation

Table 1. Clinical severity score of male NC/Jic mice with or without Myobia musculi Age (wk) No. of 4 8 12 16 20 24 Group Mice Nc Scored N Score N Score N Score N Score N Score Conventional NC/Jic mice 6 6 0 Ϯ 060.5Ϯ 0.5 6 5.0 Ϯ 1.6 6 6.0 Ϯ 2.9 6 7.3 Ϯ 2.2 6 7.8 Ϯ 3.6 SPF NC/Jic mice 8 0 0 Ϯ 00 0Ϯ 00 0Ϯ 00 0Ϯ 00 0Ϯ 00 0Ϯ 0 Conventional NC/Jic mice 8 8 0 Ϯ 081.1Ϯ 0.4 8 6.5 Ϯ 3.2 8 8.1 Ϯ 3.6 0 4.5 Ϯ 4.9 0 3.2 Ϯ 3.2 treated with ivermectina SPF NC/Jic mice 4 0 0 Ϯ 041.3Ϯ 0.5 4 5.8 Ϯ 3.0 4 10.0 Ϯ 2.9 - - - - infested with M. musculib SPF BALB/cA mice 4 0 0 Ϯ 040Ϯ 04 0Ϯ 04 0Ϯ 0 - - - - infested with M. musculib aIvermectin was given at 16, 17, 18, 19, 20, and 21 weeks of age. bMice were caged together with conventional NC/Jic mice (with mites) from 4 to 8 weeks of age. cNumber infested. dClinical severity score (mean Ϯ SD). SPF = Specific-pathogen-free status. gradually, and reached 3.2, indicating healing of the dermatitis. neck, and shoulder region of the host and feeds on skin secre- Although plasma IgE values did not change by 4 weeks after tions. This mite is a purely surface dweller and does not pen- the onset of eradication, all mites were eradicated at that time. etrate the deeper layers of the skin at any stage of its life. Plasma IgE values were decreased at 8 weeks after the onset of Transmission requires close direct contact between animals eradication. (15). Although M. musculi is a latently pathogenic ectoparasite In study 3, the SPF NC/Jic mice began to develop dermatitis (16, 17); it is not on the list of microbes selected for monitoring 4 weeks after the onset of experimentally induced infestation. by the U.S. National Institutes of Health (18). It may go unrec- After 12 weeks, severe dermatitis and markedly high plasma ognized by routine microbiological monitoring of laboratory ro- IgE values were observed. The SPF BALB/cA mice caged to- dents. It is thought to be difficult, if not impossible, to control M. gether did not show these findings. All SPF mice caged together musculi by use of good sanitation alone (19). were infested with mites 4 weeks after the onset of experimen- Dawson et al. (10) studied variable susceptibility to dermati- tally induced infestation. tis attributable to M. musculi infestation in breeding stocks of There is controversy concerning the existence of a sex differ- 16 inbred strains of mice and observed significant differences in ence in susceptibility to dermatitis associated with mite infesta- the frequencies of the lesion. According to Dawson et al., strains tion (9, 10). In the control experiment of the study reported of mice vary significantly with regard to susceptibility to M. here, male NC/Jic mice had high plasma IgE concentration and musculi infestation, and the C57BL strain is thought to be a high clinical severity scores (female data not shown). We, there- high responder, as reported by others (13, 14). Development of fore, decided to conduct the subsequent experiments using male dermatitis is controlled by at least two gene loci: some non-H-2- NC/Jic mice. We found that eradication of M. musculi led to linked gene(s) shared by all C57BL background strains, and a healing and that experimentally induced infestation led to the gene or genes within the H-2 complex (10). Jungmann et al. re- onset of dermatitis and increase in plasma IgE concentration. ported that BALB/c mice, a strain previously classified as resis- Although possible role of trauma from fighting, which is antici- tant to this type of lesion, developed dermatitis after infestation pated in group-housed mice, might be considered, the lesions of with Myocoptes musculinus, a fur mite closely related to M. NC/Jic mice appear on the face, neck, ears, and dorsal skin, sites musculi (20). Jungmann et al. (21) also reported that infestation that differ from those of trauma from fighting. The results of with M. musculinus in BALB/c mice leads to a substantial im- our study clearly indicated that the dermatitis in NC/Jic mice munologic disorder, resulting in a Th-2 type response, with was developed by infestation with M. musculi. marked systemic consequences. They described this dermatitis The NC/Jic mice were derived from the NC/Nga strain after it as mite-associated ulcerative dermatitis (MAUD). Clinical and was established at Nagoya University and transferred to the histologic skin lesion findings, immunologic responses, and Central Institute for Experimental Animals. Both strains have other characteristics of MAUD seem similar to those of the der- been sustained by strict sib mating, and a difference has not matitis in NC mice. Compared with the MAUD data, the inci- been found by use of routine genetic monitoring (11). We did not dence of lesions in NC mice is higher than that in C57BL mice find significant differences in clinical and histologic characteris- (10) and the age of onset of the dermatitis in NC mice is tics of dermatitis (histologic data not shown) between NC/Nga younger than that in BALB/c mice (20). These spontaneous dis- and NC/Jic mice used in this study (5). Kondo reported that: eases seem to be suitable natural models for studying allergic age of onset and the severity of dermatitis in NC/Nga mice de- reactions, including AD in human beings. Although the cause of pend considerably on conditions of the animal facility, clinical human AD remains controversial, the role of the mites has been signs of disease wax and wane, and reduction in reproduction thought to be most important (22–24). efficiency appears in connection with development of the der- In conclusion, we revealed one important cause of the derma- matitis (1). These characteristics are in accord with that of the titis of NC strain mice that had been thought to be induced by dermatitis induced by infestation with M. musculi (12–14). We an unknown environmental factor when the mice were raised propose that it is necessary to consider the possibility of an ec- under conventional circumstances. To clarify the validity of this toparasite, such as M. musculi infestation, in the case of derma- animal model of human AD, NC strain mice should be studied titis in NC/Nga mice. from the viewpoint of immunologic responses caused by mite Myobia musculi is known as a common parasite of laboratory infestation or MAUD. The findings from studies of this model with- mice in conventional circumstances. It is found on the head, out consideration of mite infestation may be unduly influenced. 227 Vol 50, No 2 Comparative Medicine April 2000

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