DISCUSSION

Several animal models of allergic asthma, including those which utilized parasite, OVA, and plant extracts as allergens, have been developed.(Mapp et al., 1985; Pritchard et al., 1983) Among these models, OVA asthma model with aluminum hydroxide as adjuvant is the standard animal model and contributed greatly to the understanding of many important mechanisms of asthma and basic processes that occur during allergic airway responses. Nevertheless, the OVA model is somewhat different from those of human asthma: Allergic diseases in human have a chronic nature resulting from intermittent or continuous exposure to allergen, and structural changes such as airway remodeling are characteristics. On the other hand, however, animal models using OVA are inadequate to evaluate some of the processes involved in airway remodeling.(Blyth et al., 1996; Leigh et al., 2002)

Therefore, many investigators attempted to develop more physiologic asthma model resembling that of human. CR is not only important human allergen, but also potent allergen in mouse. Exposure to CR is closely correlated to asthma severity, and it can sensitize host via respiratory system without any adjuvant.(Arruda et al., 2001; Hong et al., 2004) In the present study, we used CR allergen only with intranasal sensitization, and our results showed that CR allergen dose dependently induced airway hyper-reactivity, eosinophilic and neutrophilic inflammation, and goblet cell hyperplasia of respiratory epithelium. Such presentations resemble those that have been observed in human asthmatic patients.

Although some asthma animal models using CR have been introduced, there are some critical problems in these models.(McKinley et al., 2004; Zhou et al., 1998) These studies did

not explain the function of endotoxin in CR allergen: Crude CR contains a relatively large much amount of endotoxin, (Hong et al., 2004) and the role of endotoxin contaminated in CR allergen is controversial. Some investigators have attempted to explain the function of endotoxin by challenging mouse with endotoxin, however, the clinical relevance of this model is not clear. It has been suggested that exposure to low levels of endotoxin may exacerbate asthmatic responses, whereas exposure to high levels might be protective. This is based on two animal studies with opposite findings: Wan et al. showed that mouse exposed to 40 ng/ml aerosolized endotoxin prior to OVA challenge resulted in higher specific IgE levels and inhibited tolerance to OVA compared to the mouse exposed to saline prior to OVA challenge.(Wan et al., 2000) On the other hand, Tulic et al. found that exposure to 50 mg/ml concentration endotoxin prior to OVA challenge or up to 6 days after OVA challenge was protective and prevented the IgE response in rats.(Tulic et al., 2002)

The lack of a good animal model of endotoxin has so far made it difficult for investigators to explain the relationship between endotoxin, allergen and asthmatic response. However, we developed a novel mouse model of asthma induced by CR allergen that is characterized by airway hyper-responsiveness and pulmonary inflammation. The results, obtained with this model showed that the endotoxin added (15,810 EU/mL) did not improve AHR induced by CR allergen, however rather decreased in eosinophilic inflammation in BAL fluid, IL-13, and goblet cell hyperplasia compared with those induced by crude CR endotoxin (5,810 EU/mL). These data are in good agreement with previous studies, and indicate that high dose of endotoxin is protective in allergic disease progression. However, AHR, Th2 cytokine (IL-13), and goblet cells in airway were not changed in endotoxin-depletion experiment.

14

-In the present study, we also used TLR-4 mutant (C3H/HeJ) and TLR-4 wild type (C3H/HeJ) mouse to evaluate the function of endotoxin in the induction of CR animal model of asthma. TLR-4 functions as the signal transducing receptor for endotoxin in the cells of immune systems including macrophages and dendritic cells.(Medzhitov, 2001) Our results showed that AHR, eosinophil count in BAL fluid, IL-13 in lung homogenate, and goblet cell hyperplasia induced in C3H/HeJ mouse by crude CR were more severe than in wild type mouse (C3H/HeN).

These results agree with those of added endotoxin BALB/C mouse: Endotoxin added to crude CR had protective effects on the features of allergic asthma, allergic inflammation, goblet hyperplasia and Th2 responses in BALB/C mouse. These features were aggravated also in TLR-4 non-functioning mouse, suggesting that endotoxin in CR may have a protective role in the development of allergic asthma. These results are also concordant with human epidemiologic study, which showed that endotoxin levels in samples of dust from the child'smattress were inversely related to the occurrence of hay fever, atopic asthma, and atopic sensitization, and that cytokine production by leukocytes was inverselyrelated to the endotoxin level in the bedding.(Braun-Fahrlander et al., 2002)

In this study, endotoxin in CR was found to have limited roles in the development of CR asthma, nevertheless, CR itself can induce allergic asthma. CR allergen contains several important allergens, including Bla g 1, Bla g 2, Bla g 4, and Bla g 5. Among these allergens, Bla g 2 is a potent allergen that elicits hypersensitivity response in 60~80% of CR allergic patients, and is a 36-kD protein that shows primary sequence homology to aspartic protease.(Arruda et al., 2001; Arruda et al., 1995) However, the strong capacity of Bla g 2 to stimulate IgE production is unrelated to aspartic protease activity: Pomes et al. reported that aspartic protease activity of the

allergen is not a prerequisite of allergenicity, and other enzyme such as serine protease of CR could contribute to inflammation although aspartic protease is not required for proteins to elicit IgE response.(Pomes et al., 2002) Kheradmand et al. reported that proteases may be generally required to overcome the allergen tolerance and develope allergic inflammation.(Kheradmand et al., 2002) Especially, serine protease has a specific receptor system that is protease activating receptor-2 (PAR-2), on a variety cell type including macrophage. PAR-2 activation in the airways concurrently with exposure to inhaled OVA induces allergic sensitization via dendritic cell activation, whereas exposure to OVA alone induces allergen tolerance.(Ebeling et al., 2007) Therefore, CR allergen sensitizes host without adjuvant and causes chronic inflammation.

In present study, we studied the characteristics of a mouse model of asthma in response to CR and the roles of endotoxin. CR exposure can elicit asthma-like pulmonary inflammation characterized by AHR, eosinophilia, elevation of Th2 cytokine and goblet cell hyperplasia, and these features are related to the amount of exposed CR during sensitization. Higher dose of endotoxin inhibited eosinophilic inflammation, the elevation of Th2 cytokine level and goblet cell hyperplasia induced by CR allergen. Endotoxin contaminated in CR allergen may have limited roles in the development of CR asthma model and even have protective roles in the development of asthma. This novel mouse model of asthma induced by CR represents a valuable tool for further study and may allow dissecting of the allergen-endotoxin interactions which seem to be important in the pathogenesis of asthma.

16

-V. CONCLUSION

CR can induce an allergic asthma presenting increased AHR, eosinophilic and neutrophilic inflammation, and goblet cell hyperplasia of respiratory epithelium. Endotoxin contaminated in CR has limited roles or protective in the development of CR asthma and does not affect the development of AHR in CR asthma model.

Fig. 1 Protocols of cockroach sensitization: CR allergen dose dependent experiment (A), CR allergen and addition of endotoxin experiment (B), CR allergen and endotoxin-depletion experiment (C), TLR-4 dependent experiment (D)

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-Fig. 2 Cockroach induced asthma model in BALB/C mouse. Developments of AHR (A), eosinophilic inflammation (B), IL-13 secretion (C) and goblet cell hyperplasia of respiratory epithelium (D) are dose-dependent on the CR extracts administered.

Fig. 3 Effect of endotoxin added to CR-induced asthma model in BALB/C mouse.

Addition of endotoxin suppressed eosinophilic inflammation (B) and IL-13 productions (C), but not the development of AHR (A).

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-Fig. 4 Endotoxin-depleted CR induced asthma model in BALB/C mouse. Depletion of endotoxin in CR extracts suppressed eosinophilic inflammation (B), but not AHR (A).

Fig. 5 PAS staining of cockroach induced asthma model in BALB/C mouse. Sham (A), CR (B), endotoxin added to CR (C) and endotoxin depleted CR (D). The results of PAS staining were quantified in E.

22

-Fig. 6 Trichrome staining of cockroach-induced asthma model in BALB/C mouse.

Sham (A), CR (B), endotoxin added to CR (C) and endotoxin-depleted CR (D). The results of Trichrome staining were quantified in E.

Fig. 7 AHR and differential cell count in BAL fluid in TLR-4 mutant mouse exposed to cockroach. Change of AHR (A) and inflammatory cells in BAL fluid (B) of TLR-4 wild type (C3HeN) and TLR-4 receptor mutant (C3HeJ) mouse in CR asthma model. *; p<0.01,

#;p<0.05

24

-Fig. 8 The level of cytokines in TLR-4 mutant mouse exposed to cockroach.

Expression of IL-13 (A) and IFN-γ (B) in TLR-4 wild type (C3HeN) and mutant mouse (C3HeJ). The cytokines in the lung homogenates were measured.

Fig. 9 PAS staining of TLR-4 mutant mouse exposed to cockroach. Wild type (sham - A, CR - B) and mutant (sham - C, CR - D) mouse. Goblet hyperplasia of respiratory epithelium was quantified in E.

26

-Fig. 10 Trichrome staining of TLR-4 mutant mouse exposed to cockroach. Wild type (sham - A, CR - B) and mutant (sham - C, CR - D) mouse. Fibrosis area was quantified in E.

Table 1. The level of endotoxin concentrations in the applied CR extract

28

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- 국문요약 -

34

-염증반응, 기도과민성, 점액 샘의 증식을 일으켰다. 바퀴 알레르기항원에 추가로 내독소를 주입한 마우스에서는 기관지의 호산구성 염증과 기관지폐포액 속의 인터루킨-13, 술잔 세포의 증식이 감소되는 소견을 보였으나 기도과민성에는 영향을 미치지 않았다. 또한 내독소가 없는 바퀴 알레르기항원도 일반적인 바퀴항원에 비하여 기관지폐포액 속의 호산구성 염증과 임파구를 감소시켰으나 기도과민성과 기관지폐포액 속의 인터루킨-13에는 영향을 미치지 않았다. TLR-4가 없는 마우스에서도 바퀴 알레르기항원은 호산구성 염증, 술잔 세포의 증식과 기도과민성의 증가를 나타냈다.

결론: 바퀴는 마우스에서 호산구성 염증, 호중구성 염증, 기도과민성과 술잔 세포 증식이 있는 천식을 유발한다. 비록 바퀴 알레르기항원내의 내독소가 호산구성 염증에 대하여 양면성이 있지만, 바퀴 천식 모델의 발생에 영향은 미미하며, 바퀴 천식 모델의 기도과민성 발생에는 영향을 미치지 않는다.

핵심되는 말: 바퀴, 내독소, Toll like receptor-4