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Airway inflammatory and immunological events in a rat model exposed to toluene diisocyanate
Food and Chemical Toxicology 43 (2005) 1281–1288 www.elsevier.com/locate/foodchemtox
Airway inflammatory and immunological events in a rat model exposed to toluene diisocyanate Kouame Kouadio *, Kui-Cheng Zheng, Mallet K.-N. Tuekpe, Hidemi Todoriki, Makoto Ariizumi Department of Environmental and Preventive Medicine, School of Medicine, University of the Ryukyus, 207 Uehara, Nishihara, Okinawa 903-0215, Japan Received 9 August 2004; accepted 7 March 2005
Abstract To investigate the inflammatory and immunological events in the airway induced by a short period of repeated exposure to toluene diisocyanate (TDI), an animal model was established, which resembled the industrial field exposure. After whole body exposure of Wistar rats to 0.38 and 1.20 ppm TDI vapor 4 h a day for five consecutive days in a glass chamber, bronchoalveolar lavage (BAL) was performed. BAL fluid cellular and cytokine contents were then determined. Histopathological examinations were also carried out on the lungs. The TDI vapor exposure resulted in airway symptoms similar to those in occupational asthma. BAL fluid cellular analysis and lung histophathological examination revealed that inflammatory response was characterized by marked eosinophil infiltration of the airways. The cytokine assay revealed significant production of IL-4 in the airways of the TDI exposed rats as compared to the control rats. These findings indicated that a short period of repeated exposure to TDI vapor may cause respiratory hypersensitivity in which airway inflammatory and immunological events represented by eosinophil infiltration and Th2 cytokines may play an important role. Also, this animal model may be suitable for exploring the mechanism underlying TDI-induced occupational asthma. 2005 Elsevier Ltd. All rights reserved. Keywords: TDI; Eosinophils; Cytokines; Occupational asthma
1. Introduction Allergic contact dermatitis and asthma are two important types of allergic diseases at the workplace caused by the exposure to exogenous substances. Occupational asthma is the most frequently diagnosed form of acute occupational respiratory disease in the industrialized countries (Meredith et al., 1991; Ross et al., 1995). Toluene diisocyanate (TDI), a low molecular weight
* Corresponding author. Tel.: +81 98 895 3331x2321; fax: +81 98 895 1412. E-mail addresses: [email protected], kouadiokouame @hotmail.com (K. Kouadio).
0278-6915/$ - see front matter 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.fct.2005.03.005
chemical widely used in industries as a polymerizing agent in the production of polyurethane, has been reported to be one of the most important causes of occupational asthma (Bernstein, 1982). NIOSH (1977) has proposed that the threshold level value (TLV) for industrial diisocyanate vapor be lowered to 0.005 ppm as a time weighted average (TWA) and 0.02 ppm as a ceiling concentration. Although the clinical aspects of TDI induced occupational asthma have been well defined, the mechanisms involved in its pathogenesis are still not fully understood. In order to better understand these mechanisms, several animal models have been developed based on cutaneous, intradermal, intranasal, or head only inhalation exposure to TDI (Karol et al.,
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1981; Karol, 1983; Patterson et al., 1983; Abe et al., 1993; Dearman et al., 1996; Niimi et al., 1996). However, all these methods of exposure do not resemble the exposure conditions observed in industrial fields since individuals are usually whole body exposed to a TDI atmosphere at the workplace. Also, it is obscure whether the airway hypersensitivity induced by repeated short-period exposure to TDI is due to immunological and inflammatory response or simply to TDIÕs chemical irritation. To establish an in vivo animal model resembling industrial exposure and to elucidate the mechanisms involved in TDI induced airway hypersensitivity, Wistar rats were sensitized with repeated short period whole body exposure to TDI vapor, after which the inflammatory and immunological events in the airways were investigated. Occupational asthma has been reported to be a complex pathophysiological event involving the interaction of many cell types and cytokines. It has been shown that activation of selected T cells with subsequent eosinophil recruitment and secretion of eosinophil derived mediators may contribute to both epithelial cell damage and airway hyperresponsiveness (Bentley et al., 1992). Furthermore, studies have suggested that T cell derived cytokines may play a role in the initiation of eosinophilic inflammation (Mattoli et al., 1991; Maestrelli et al., 1995, 1997). T cell-mediated inflammatory reaction has been hypothesized to be involved in the pathogenesis of TDI induced asthma through an array of cytokines. Therefore, the present study was conducted to further investigate the status and the role of infiltration of inflammatory cells, particularly eosinophils, the secretion and the role of Th2 cytokines in airway hyperresponsiveness induced by TDI in a rat model exposed to TDI atmosphere.
2. Materials and methods 2.1. Animals Thirty-eight-week old female Wistar rats, weighing an average of 240 ± 30 g were obtained from a Kyudo breeding laboratory (Kumamoto, Japan). Upon arrival, the rats were housed in pathogen free steel mesh cages under environmentally controlled conditions in compliance with the Ryukyus University Policy on Animal Care and Use. They were kept at a constant room temperature (25 ± 2 C) and humidity (50–70%) and at a 12 h light dark cycle. Food and water were provided ad libitum through out the experiment except during the exposure periods. The animals were allowed to acclimatize to our laboratory for a week, and then 10 rats were randomly selected for the control group. The remaining 20 rats were randomly divided into two expo-
sure groups (1 and 2) with each exposure group contained 10 rats. 2.2. Exposure procedures and quantification of TDI concentrations The rats were placed in a 22 l glass chamber with a dynamic adjustable laminar airflow and exposed to TDI vapor, 4 h per day for five consecutive days. The TDI atmosphere in the chamber was generated by bubbling air at a rate of 22 l per minute through a flask containing 15 ml of 2–4 toluene diisocyanate (TDI, Wako Chemical Co., Japan) into the chamber. By varying the airflow rate through the flask, different TDI concentrations were obtained. A constant concentration was maintained in the chamber by maintaining a constant airflow meter rate during the whole exposure period. This generation system produced TDI vapor free of aerosol. Temperature and humidity were monitored during the exposure period. The air was conditioned to a temperature of 24 ± 1 C and 25 ± !1% relative humidity as reported by Gagnaire et al. (1996). The concentration was checked each hour in order to maintain a stable concentration for each exposure group. The concentrations of TDI atmospheres in the chamber were determined according to Marcali (1957), which was modified by NIOSH (1977). Briefly, the air in the chamber was sampled with an impinger containing an absorber medium made from acetic and hydrochloric acids. Several solutions, diazotization solution containing sodium nitrite and sodium bromide, sulfamic acid solution, N-1-napthylethylenediamine and sodium carbonate solution, were then added to the absorber medium. When a reddish-blue colored solution was finally obtained, its transmittance was measured with a spectrophotometer at 550 nm. A calibration curve of a series of standardized TDI solutions was prepared by plotting transmittance versus TDI concentrations. This curve was then used to evaluate TDI concentrations. The chamber concentrations were 0.31, 0.45, 0.33, 0.38, and 0.45 ppm for exposure group 1, and 0.99, 1.44, 1.20, 0.98 and 1.41 ppm for exposure group 2. The mean TDI concentrations were 0.38 ± 0.07 ppm and 1.20 ± 0.22 ppm for exposure groups 1 and 2, respectively. The control group was placed in the same chamber and treated under the same condition with physiological saline in the flask instead of TDI. Since the effect of TDI vapor on the breathing pattern and frequency before, during and after sensitization have been well recorded, our focus in this study was mainly on the inflammatory and immunological events in the airway after a repeated short-period exposure to TDI vapor. We did not therefore record breathing pattern and frequency in this study.
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2.3. Bronchoalveolar lavage (BAL)
2.6. Statistical analysis
Twenty-four hours after a 5-day exposure, the rats in both the exposure and control groups were weighed and anesthetized by intraperitoneal injection of 50 mg/kg of sodium pentobarbital. A cervical incision was made and the trachea was isolated. A catheter was then inserted into the trachea and bronchoalveolar lavage (BAL) was performed. The trachea was slowly infused with 5 ml sterile phosphate buffered saline (PBS, 37 C), and the effluent (BAL fluid) was gently aspirated. The recovered aliquot (approximately 3 ml per rat) was centrifuged at 500 g (4 C). The supernatant was collected and stored at 80 C until cytokines determination. To collect the BAL cells, the trachea was further infused 2 times with 2 ml each time of PBS (37 C), and all the recovered aliquot (2.5 ml) was pooled and centrifuged at 500 g (4 C). The cell pellet was resuspended in RPMI 1640 medium (Gibco, Life Technologies, NY, USA) and the total number of cells and their viability were determined using 0.2% trypan blue exclusion method. To perform the differential leukocyte cell count, 0.1 ml of the cell suspension was smeared on a glass slide and stained with Wright–Giemsa. Three hundred nucleated cells were then examined under a microscope. The cells were classified either as macrophages, neutrophils, eosinophils or lymphocytes. The first washing was used for cytokine determination and the second for leucocyte differential count because the cytokine concentration in the second washing would have been reduced while the proportions of the various leucocytes would have been maintained in both the first and second BAL washings.
The cellular contents and cytokine productions were presented as means ± SD. Two-tailed StudentÕs t-test was performed to reveal differences between the means of each exposure group and the control group. Values of p < 0.05 were considered to be statistically significant.
2.4. Cytokine assay Cytokines IL-2, IL-4 and IL-6 productions in the BAL fluid were quantified respectively with rat IL-2, IL-4 and IL-6 ELISA kits (Endogen, Inc, MA, USA) according to the protocols recommended by the manufacturer. The inter-assay and intra-assay coefficient of variation, CV, were <10%. The sensitivities of the assays were <5 pg/ml, <2 pg/ml and <8 pg/ml for IL-2, IL-4 and IL-6, respectively. 2.5. Lung histopathology Immediately after BAL was performed, 1 ml formaldehyde solution was injected through the catheter into the trachea. The lungs of each rat were resected, weighed and then fixed in buffered formalin. A section encompassing the maximum cross-sectional area of the right lung was taken perpendicular to the major bronchi, dehydrated through a series of ethanol solutions, and then embedded in paraffin. Two micrometer thick sections were then sliced and stained with hematoxylin eosin for examination.
3. Results 3.1. Hypersensitivity symptoms in the airways Exposure to both 0.38 and 1.20 ppm TDI resulted in airway hypersensitivity in the rats. When the rats were exposed to 0.38 ppm TDI, airway symptoms characterized by hyperrhinorrhea and sneezing occurred within two hours. On the third day of exposure, the animals started coughing and showed moderate prolongation of the expiratory phase. On the last day of the exposure period, all the rats were wheezing and gasping for air. In comparison with 0.38 ppm TDI, 1.20 ppm TDI induced airway symptoms in the rats within one hour. The symptoms, particularly exertional breathing (wheezing, gasping for air and prolongation of the expiratory rate) were also more severe. This implies that airway hypersensitivity symptoms were aggravated along with the increase in TDI concentration. The rats in the control group, however, showed no obvious respiratory symptoms. 3.2. Analysis of BAL fluid BAL fluid cellular analysis demonstrated that exposure to both 0.38 ppm and 1.20 ppm TDI vapors resulted in inflammatory responses in the airways of the rats. As shown in Table 1, a five-day exposure to TDI resulted in a significant increase in the numbers of total cells and each differential leukocyte as compared to the control rats. Eosinophils and neutrophils in particular were increased 17.20 and 4.62 times, respectively, in the rats exposed to 0.38 ppm. When the rats were exposed to 1.20 ppm, the total number of cells, as well as each leukocyte were further increased significantly as compared to the control rats. The number of eosinophils and neutrophils in the rats exposed to 1.20 ppm were increased 30.2 and 6.26 times, respectively. 3.3. Cytokines in the BAL fluid The cytokine content in the BAL fluid was determined by an ELISA test. As shown in Fig. 1 , the productions of IL-2 in exposure groups 1 and 2 were not significantly different from those in the control group (p = 0.53 and p = 0.12, respectively). Similarly, there was no difference in the IL-6 productions between each of the exposure groups and the control group (p = 0.23
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Table 1 Total cell and differential cell counts in the BAL fluid (·105 cells/ml) Treatment
Total cells
Differential leucocytes Eosinophils
Neutrophils
Lymphocytes
Macrophages
Control group Exposure group 1 Exposure group 2
15.93 ± 1.24 25.78 ± 4.62* 34.18 ± 6.58#
0.05 ± 0.01 0.86 ± 0.29# 1.51 ± 0.49#
0.26 ± 0.06 1.20 ± 0.39* 1.63 ± 0.24#
1.51 ± 0.41 2.18 ± 0.75 3.16 ± 0.91*
14.05 ± 1.13 21.40 ± 3.51# 27.68 ± 5.24#
Values are means ± SD. Each group contains 10 rats. * Significantly different from the control group values at p < 0.05. # Significantly different from the control group values at p < 0.01.
Fig. 1. Cytokine production in the BAL fluid. Values are means ± SD. Each group contains 10 rats. * Significantly different from the control group values at p < 0.05. ** Significantly different from the control group values at p < 0.01.
and p = 0.16, respectively). However, IL-4 was significantly increased in exposure group 1 (p = 0.041) and exposure group 2 (p = 0.0092) as compared to the control group. 3.4. Lung histopathological analysis The histopathological examination showed that inflammatory events occurred in the lungs of the rats exposed to TDI (Fig. 2). A prominent infiltration of eosinophils in the bronchi and a massive goblet cells metaplasia of the epithelium of the central airways were observed. These pathological changes were more severe in the rats exposed to 1.20 ppm than in those exposed to 0.38 ppm TDI vapor. In contrast, no pathological changes were found in the lungs of the control rats.
4. Discussion Animal models are extremely important for research and /or testing purposes in the study of industrial chemicals. For occupational asthma, animal models are particularly important because of our current incomplete
understanding of the mechanisms involved in chemical sensitization (Karol, 1988, 1994). Several animal models have been developed to understand the mechanisms underlying TDI induced asthma (Karol et al., 1981; Karol, 1983; Patterson et al., 1983; Abe et al., 1993; Dearman et al., 1996; Niimi et al., 1996). In contrast to other animal models previously reported, a Wistar rat model characterized by whole body exposure to TDI vapor in a glass chamber was established in this study. It is considered that the whole body exposure method has an advantage over the other methods in that it closely resembles the individualÕs exposure to TDI vapor at the workplace. It simulates potential human exposure to environmental chemicals or pesticides and it is possible to expose a large number of animals at the same time and therefore reduce the cost of the experiment. Also the animals are not fixed in a specific place and therefore are not under much stress. Feces and urine can easily escape and thermoregulation via the tail is also not compromised. We exposed 10 rats for each exposure group in a 22 l inhalation chamber. There could have been overcrowding in the chamber. However we tried to duplicate exposure in industrial fields where it is not always that procedures regarding space are followed, especially in developing countries where sometimes proper precautions are not taken. As compare to a head only exposure to TDI (Karol, 1983), the animals were not under much stress despite the number of rats in the chamber. It has been reported that not only the exposure method but also the exposure concentrations are important for the development of asthmatic symptoms in animal models exposed to TDI (Karol, 1983). A previous study by Karol (1983) has revealed that exposing guinea pigs to 0.12 ppm of TDI vapor did not show any respiratory hypersensitivity. In contrast, 0.36 ppm or greater resulted in airways hypersensitivity while a concentration of 2 ppm was pneumotoxic and few pulmonary hypersensitivity reactions were observed (Karol, 1983). Considering KarolÕs experiment, our preliminary ones in rats (data not shown) and that of Zheng et al. (2004), we used two different concentrations of TDI, 0.38 ppm and 1.20 ppm in the present study. The Wistar
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Fig. 2. Light microscopic images of the lungs of the control group rats (A), TDI exposure group 1 (B) and TDI exposure group 2 (C) after hematoxylin eosin staining. A, B and C are the bronchi of the rats. Goblet cells (black arrows) and eosinophils (white arrows) are present in the bronchi of TDI exposure groups: Magnification 200· (A,C) and 400· (B).
rats were sensitized and the results show that these concentrations also induced airway hypersensitivity in the exposed rats.
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A report by Tanaka et al. (1983) has shown that guinea pigs sensitized and subsequently challenged repeatedly with nasal applications of TDI showed asthma-like symptoms such as exertional breathing, prolonged expiration, and cough immediately after the challenge. In the present study, although the rats were repeatedly exposed to TDI vapor for short periods without challenge, the rats showed airway hypersensitivity symptoms, which were asthma-like. The pathological findings in these animals were not completely in agreement with reported histopathological findings for asthma (Fabbri et al., 1991) because the exposure period was short. Studies have shown that in vivo airway hyperresponsiveness was not observed at any point in time in a mouse model exposed to TDI according to the short-term protocol (Scheerens et al., 1999). It has been shown that by altering the exposure time and/or cumulative dosage of TDI, different biological reactions can be elicited in exposed mice. This important finding therefore might be a reflection of the diversity of symptoms found in patients suffering from TDI-induced asthma. Both the short-exposure and the long-exposure models could be useful for further investigation into the mechanisms involved in the action of TDI (Scheerens et al., 1999). Zheng et al. (2004) found that concentrations of 0.34 ppm and above sensitized all the rats by the second day after exposing for 4 h a day. Further exposure on the other days resulted in hypersensitivity. The results of airway inflammatory cell dynamics by means of BAL fluid and histopathological examination of the lung as well as cytokine productions revealed that repeated exposure to a low concentration (0.38 ppm) or a high concentration (1.20 ppm) of TDI results in airway hypersensitivity in which inflammatory and immunological inter-response may be involved. These data are consistent with those in a guinea pig model repeatedly exposed to a series of TDI vapor concentrations reported by Karol (1983). In KarolÕs experiment, the guinea pig model was established using the method of head inhalation exposure to TDI vapors, 3 h per day for five consecutive days, the immunologic response to TDI exposure (TDI specific IgE) occurred and was exposure concentration dependent. In the present study, although only two concentrations (0.38 ppm and 1.20 ppm) of TDI vapor were used to sensitize the rats, the data also revealed that exposure to a high concentration (1.20 ppm) of TDI vapor resulted in a severer immunologic and inflammatory responses characterized with a higher productions of cytokines and a more prominent infiltration of eosinophils in the airways as compared with exposure to a lower concentration (0.38 ppm) of TDI. This suggests a concentration dependent relationship between TDI exposure and immunoinflammatory responses. It has been reported that in TDI induced asthma patients, only 10–30% have detectable IgE (Karol, 1981). This suggests that IgE may not
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be specific to occupational asthma and therefore was not measured in this study. A previous report has shown that, in general the pathological features in TDI induced occupational asthmatics are similar to those of status asthmaticus seen in non-occupational asthma (Fabbri et al., 1988). In occupational asthmatics, the airways were plugged with mucus containing abundant exudates; the airway epithelium was extensively desquamated, and the mucosa and the lamina propria were edematous and markedly infiltrated with eosinophils (Fabbri et al., 1991). Airway inflammation has been reported to play a significant role in the pathogenesis of asthma, and eosinophil infiltration in the airway is a known hallmark histopathological character of asthma (Djukanovic et al., 1990; Saetta et al., 1992). Late asthmatic response (LAR) in humans after exposure to TDI have been shown to be associated with the increase of airway hyperresponsiveness and airway eosinophilia (Fabbri et al., 1987). Furthermore, studies have demonstrated a strong correlation between asthma severity and eosinophil numbers in biopsies (Moqbel et al., 1992), sputum (Frigas et al., 1981) and blood (Durham and Kay, 1985). Eosinophils are potent proinflammatory cells which release toxic granule proteins such as major basic protein (MBP) and eosinophil cationic protein (ECP), generate lipid mediators such as platelet-activating factor (PAF), leukotriene C4 and oxygen metabolites which have profound potential to injure the airway epithelium, promote bronchial responsiveness, exacerbate inflammatory response and contract airway smooth muscle (Gleich, 1990; Weller, 1991). Eosinophils therefore possess properties that can directly and indirectly cause airway obstruction and promote bronchial hyperresponsiveness. The pathological changes in the TDI exposed rats in this study were in board agreement with those in occupational asthmatics and other previously reported animal models (Tanaka et al., 1983; Fabbri et al., 1991; Mapp et al., 1996). It has also been revealed that asthmatic bronchial inflammation is a specialized form of cell mediated immunity in which immunological mediators such as lymphokines and cytokines, secreted principally by activated CD4+ T lymphocytes, orchestrate the accumulation and activation of specific granulocyte effector cells, particularly eosinophils (Kay, 1991). In atopic asthma, activated T helper lymphocytes are present in bronchial biopsy specimens and bronchoalveolar lavage (BAL) fluid. The production of cytokines may be important in the pathogenesis of this disorder. Bronchial inflammation in asthma may depend in part on the activation of T helper lymphocytes that elaborate proinflammatory cytokines (Azzawi et al., 1990; Walker et al., 1991). Some studies have shown that the Th2 type cytokines interleukin (IL)-4 and IL-5 have an important role in atopic disease and more specifically in atopic
asthma (Seder et al., 1992; Bjorksten et al., 1995; Daher et al., 1995). IL-4 is involved in the switch of B cells to IgE production (Le gros et al., 1990) and IL-5 has been shown to cause eosinophil infiltration into the airway wall (Campbell et al., 1987). Both IL-4 and IL-5 have been implicated in the airway hypersensitivity associated with TDI-induced occupational asthma (Maestrelli et al., 1997). Animal models exposed to TDI have also revealed the important role of IL-4 and IL-5 in airway hypersensitivity (Dearman et al., 1996). The significant increase of IL-4 production (but not IL-2 and IL-6) in the airways of TDI-exposed rats in this study is partly consistent with previous studies on TDI induced occupational asthma (Dearman et al., 1996; Maestrelli et al., 1997), further supporting the important role of Th2 cytokines in airway hypersensitivity. Possible oral ingestion and dermal absorption of TDI may have occurred in this study. All these routes have the potential of sensitizing. However, apart from the fact that the vapor form of TDI is more likely to affect the airways than other body parts and systems, Karol et al. (1981) have shown that dermal contact of TDI results in pulmonary sensitization. The shortcoming of the present study is that IL-5 production was not determined due to unavailability of commercial rat IL-5 kit at the time the experiment was carried out. Lacking data on IL-5 productions hamper the explanation of the regulation of eosinophil infiltration and the conclusion of the role of Th2 cytokines in this animal model. From the results of this study, it can be seen that repeated whole body exposure to TDI may result in inflammatory and immunological events in the airway of rats in which, an inflammation characterized by eosinophil infiltration and Th2 type cytokines may play a key role. Also the animal model used in this study can be used to further unravel the complex mechanisms underlying TDI induced occupational asthma.
Acknowledgement The authors would like to thank Dr. Takamitsu Morioka of the first Department of Pathology, Faculty of Medicine, University of the Ryukyus, Okinawa, Japan, for his technical support. This animal experiment was done according to the regulation of Japanese laws related to the use of animal for research.
References Abe, Y., Ogino, S., Irifune, M., Imamura, I., Liu, Y.Q., Fukui, H., Matsunaga, T., 1993. Histamine content, synthesis and degrada-
K. Kouadio et al. / Food and Chemical Toxicology 43 (2005) 1281–1288 tion in nasal mucosa and lung of guinea pigs treated with toluene diisocyanate (TDI). Clinical and Experimental Allergy 23, 512– 517. Azzawi, M., Bradley, B., Jeffery, P.K., Frew, A.J, Wardlaw, A.J., Knowles, G., Assoufi, B., Collins, J.V., Durham, S., Kay, A.B., 1990. Identification of activated T lymphocytes and eosinophils in bronchial biopsies in stable atopic asthma. The American Review of Respiratory Disease 142, 1407–1413. Bentley, A.M., Maestrelli, P., Saetta, M., Fabbri, L.M., Robinson, D.S., Bradley, B.L., Jeffery, P.K., Durham, S.R., Kay, A.B., 1992. Activated T lymphocytes and eosinophils in the bronchial mucosa in isocyanate induced asthma. Journal of Allergy and Clinical Immunology 89, 821–829. Bernstein, I.L., 1982. Isocyanate induced pulmonary diseases: a current perspective. Journal of Allergy and Clinical Immunology 70, 24–31. Bjorksten, B., Borres, M.P., Einarsson, R., 1995. Interleukin-4, soluble CD23 and interferon-gamma levels in serum during the first 18 months of life. International Archives of Allergy and Immunology 107, 34–36. Campbell, H.D., Tucker, W.Q., Hort, Y., Martinson, M.E., Mayo, G., Clutterbuck, E.J., Sanderson, C.J., Young, I.G., 1987. Molecular cloning, nucleotide sequence, and expression of the gene encoding human eosinophil differentiation factor (interleukin-5). Proceedings of the National Academy of Science of United States of America 84, 6629–6633. Daher, S., Santos, L.M., Sole, D., De Lima, M.G., Naspitz, C.K., Musatti, C.C., 1995. Interleukin-4 and soluble CD23 serum levels in asthmatic atopic children. Journal of Investigational Allergology and Clinical Immunology 5, 251–254. Dearman, R.J., Basketter, D.A., Kimber, I., 1996. Characterization of chemical allergens as a function of divergent cytokine secretion profiles induced in mice. Toxicology and Applied Pharmacology 138, 308–316. Djukanovic, R., Wilson, J.W., Britten, K.M., Wilson, S.J., Walls, A.F., Roche, W.R., Howarth, P.H., Holgate, S.T., 1990. Quantification of mast cells and eosinophils in the bronchial mucosa of symptomatic atopic asthmatics and healthy control subjects using immunohistochemistry. American Review of Respiratory Disease 142, 863–871. Durham, S.R., Kay, A.B., 1985. Eosinophil bronchial hyperreactivity and late asthmatic reactions. Clinical Allergy 15, 411–418. Fabbri, L.M., Boschetto, P., Zocca, E., 1987. Bronchoalveolar neutrophilia during late asthmatic reactions induced by toluene diisocyanate. American Review of Respiratory Disease 136, 36– 42. Fabbri, L.M., Danieli, D., Crescioli, S., 1988. Fatal asthma in a toluene diisocyanate sensitized subject. American Review of Respiratory Disease 137, 1494–1498. Fabbri, L.M., Maestrelli, P., Saetta, M., Mapp, C.E., 1991. Airway inflammation during late asthmatic reactions induced by toluene diisocyanate. American Review of Respiratory Disease 143, S37– S38. Frigas, E., Loegering, D.A., Solley, G.O., Farrow, G.M., Gleich, G.J., 1981. Elevated levels of the eosinophil granule major basic protein in the sputum of patients with bronchial asthma. Mayo Clinic Proceedings 56, 345–353. Gagnaire, F., Ban, M., Micillino, M., Lemonier, M., Bonnet, P., 1996. Bronchial responsiveness and inflammation in guinea pigs exposed to toluene diisocyanate: a study on a single and repeated exposure. Toxicology 114, 91–100. Gleich, G.J., 1990. The eosinophil and bronchial asthma: current understanding. Journal of Allergy and Clinical Immunology 85, 422–436. Karol, M.H., 1981. Survey of industrial workers for antibodies to industrial workers for antibodies to toluene diisocyanate. Journal of Occupational Medicine 23, 741–747.
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Karol, M.H., 1983. Concentration dependent immunologic response to toluene diisocyanate (TDI) following inhalation exposure. Toxicology and Applied Pharmacology 68, 229–241. Karol, M.H., 1988. The development of an animal model for TDI asthma. Bulletin Europeen de Physiopathologie Respiratoire 23, 571–576. Karol, M.H., 1994. Animal models of occupational asthma. European Respiratory Journal 7, 555–568. Karol, M.H., Hauth, B.A., Riley, E.J., Magreni, C.M., 1981. Dermal contact with toluene diisocyanate (TDI) produces respiratory tract hypersensitivity in guinea pigs. Toxicology and Applied Pharmacology 58, 221–230. Kay, A.B., 1991. Asthma and inflammation. Journal of Allergy and Clinical Immunology 87, 893–910. Le gros, G., Ben-Sasson, S.Z., Seder, R., Finkelman, F.D., Paul, W.E., 1990. Generation of interleukin-4 (IL-4) producing cells in vivo and in vitro: IL-2 and IL-4 are regulated for in vitro generation of IL-4 producing cells. Journal of Experimental Medicine 172, 921–929. Maestrelli, P., Di Stefano, A., Occari, P., Turato, G., Milani, G., Pivirotto, F., Mapp, C.E., Fabbri, L.M., Saetta, M., 1995. Cytokines in the airway mucosa of subjects with asthma induced by toluene diisocyanate. American Journal of Respiratory and Critical Care Medicine 151, 607–612. Maestrelli, P., Occari, P., Turato, G., Papiris, S.A., Di Stefano, A., Mapp, C.E., Milani, G.F., Fabbri, L.M., Saetta, M., 1997. Expression of IL-4 and IL-5 proteins in asthma induced by toluene diisocyanate (TDI). Clinical and Experimental Allergy 27, 1292– 1298. Mapp, C.E., Lapa E Silva, J.R., Lucchini, R.E., Chitano, P., Rado, V., Saetta, M., Pretolani, M., Karol, M.H., Maestrelli, P., Fabbri, L.M., 1996. Inflammatory events in the blood and airways of guinea pigs immunized to toluene diisocyanate. American Journal of Respiratory and Critical Care Medicine 154, 201–208. Marcali, K., 1957. Microdetermination of toluene diisocyanates in atmosphere. Annals of Chemistry 29, 552–558. Mattoli, S., Colotta, F., Fincato, G., Mezzetti, M., Mantovani, A., Patalano, F., Fasoli, A., 1991. Time course of IL1 and IL6 synthesis and release in human bronchial epithelial cell cultures exposed to toluene diisocyanate. Journal of Cellular Physiology 149, 260–268. Meredith, S.K., Taylor, V.M., McDonald, J.C., 1991. Occupational respiratory disease in the United Kingdom 1989: a report to the British Thoracic Society and Society of Occupational Medicine by the SWORD project group. British Journal of Industrial Medicine 48, 292–298. Moqbel, R., Barkans, J.R., Bradley, B., Durham, S.R., Kay, A.B., 1992. Application of monoclonal antibodies against major basic protein (BMK-13) and eosinophil cationic protein (EG1 and EG2) for quantifying eosinophils in bronchial biopsies from atopic asthma. Clinical and Experimental Allergy 22, 265–273. Niimi, A., Amitani, R., Yamada, K., Tanaka, K.I., Kuze, F., 1996. Late respiratory response and associated eosinophilic inflammation induced by repeated exposure to toluene diisocyanate in guinea pigs. Journal of Allergy and Clinical Immunology 97, 1308–1319. NIOSH, US Department of Health, Education and Welfare, Washington, DC, 1977. 2-4-toluene diisocyanate (TDI) in air. In: Manual of Analytical Methods, 2nd ed., vol 1, pp. 1411–1418. Patterson, R., Zeiss, C.R., Harris, K.E., 1983. Immunologic and respiratory responses to airway challenges of dogs with toluene diisocyanate. Journal of Allergy and Clinical Immunology 71, 604– 611. Ross, D.J., Sallie, B.A., McDonald, J.C., 1995. SWORDÕs 1994 surveillance of work related and occupational respiratory disease in the United Kingdom. Occupational Medicine 45, 175–178. Saetta, M., Di Stefano, A., Maestrelli, P., De Marzo, N., Milani, G.F., Pivirotto, F., Mapp, C.E., Fabri, L.M., 1992. Airway mucosal inflammation in occupational asthma induced by toluene
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diisocyanate. American Review of Respiratory Disease 145, 160– 168. Scheerens, H., Buckley, T.L., Muis, T.L., Garssen, J., Dormans, J, Nijkamp, F.P., Van Loveren, H., 1999. Dormans. Long-term topical exposure to toluene diisocyanate in mice leads to antibody production and in vivo airway hyperresponsiveness three hours after intranasal challenge. American Journal of Respiratory Care Medicine 159, 1074–1080. Seder, R.A., Paul, W.E., Davis, M.M., Fazekas de St Groth, B., 1992. The presence of IL4 during in vitro priming determines the lymphokine producing potential of CD4+ T cells from T cell receptor transgenic mice. Journal of Experimental Medicine 176, 1091–1098.
Tanaka, K., Kawai, M., Maekawa, N., 1983. Experimental model of asthma by toluene diisocyanate (TDI). Bulletin of Chest Disease and Respiratory Institute of Kyoto University 16, 1–9. Walker, C., Virchow Jr., J.C., Bruijnzeel, P.L.B., Blaser, K., 1991. T cell subsets and their soluble products regulate eosinophilia in allergic and non-allergic asthma. Journal of Immunology 146, 1829–1835. Weller, P.F., 1991. The immunobiology of eosinophils. New England Journal of Medicine 324, 1110–1118. Zheng, K.C., Kouadio, K., Tuekpe, M.K.N., Makoto, A., 2004. Eosinophil infiltration in airways of mice exposed to toluene diisocyanate. Journal of Occupational Health 46, 299–302.
Airway inflammatory and immunological events in a rat model exposed to toluene diisocyanate Kouame Kouadio *, Kui-Cheng Zheng, Mallet K.-N. Tuekpe, Hidemi Todoriki, Makoto Ariizumi Department of Environmental and Preventive Medicine, School of Medicine, University of the Ryukyus, 207 Uehara, Nishihara, Okinawa 903-0215, Japan Received 9 August 2004; accepted 7 March 2005
Abstract To investigate the inflammatory and immunological events in the airway induced by a short period of repeated exposure to toluene diisocyanate (TDI), an animal model was established, which resembled the industrial field exposure. After whole body exposure of Wistar rats to 0.38 and 1.20 ppm TDI vapor 4 h a day for five consecutive days in a glass chamber, bronchoalveolar lavage (BAL) was performed. BAL fluid cellular and cytokine contents were then determined. Histopathological examinations were also carried out on the lungs. The TDI vapor exposure resulted in airway symptoms similar to those in occupational asthma. BAL fluid cellular analysis and lung histophathological examination revealed that inflammatory response was characterized by marked eosinophil infiltration of the airways. The cytokine assay revealed significant production of IL-4 in the airways of the TDI exposed rats as compared to the control rats. These findings indicated that a short period of repeated exposure to TDI vapor may cause respiratory hypersensitivity in which airway inflammatory and immunological events represented by eosinophil infiltration and Th2 cytokines may play an important role. Also, this animal model may be suitable for exploring the mechanism underlying TDI-induced occupational asthma. 2005 Elsevier Ltd. All rights reserved. Keywords: TDI; Eosinophils; Cytokines; Occupational asthma
1. Introduction Allergic contact dermatitis and asthma are two important types of allergic diseases at the workplace caused by the exposure to exogenous substances. Occupational asthma is the most frequently diagnosed form of acute occupational respiratory disease in the industrialized countries (Meredith et al., 1991; Ross et al., 1995). Toluene diisocyanate (TDI), a low molecular weight
* Corresponding author. Tel.: +81 98 895 3331x2321; fax: +81 98 895 1412. E-mail addresses: [email protected], kouadiokouame @hotmail.com (K. Kouadio).
0278-6915/$ - see front matter 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.fct.2005.03.005
chemical widely used in industries as a polymerizing agent in the production of polyurethane, has been reported to be one of the most important causes of occupational asthma (Bernstein, 1982). NIOSH (1977) has proposed that the threshold level value (TLV) for industrial diisocyanate vapor be lowered to 0.005 ppm as a time weighted average (TWA) and 0.02 ppm as a ceiling concentration. Although the clinical aspects of TDI induced occupational asthma have been well defined, the mechanisms involved in its pathogenesis are still not fully understood. In order to better understand these mechanisms, several animal models have been developed based on cutaneous, intradermal, intranasal, or head only inhalation exposure to TDI (Karol et al.,
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1981; Karol, 1983; Patterson et al., 1983; Abe et al., 1993; Dearman et al., 1996; Niimi et al., 1996). However, all these methods of exposure do not resemble the exposure conditions observed in industrial fields since individuals are usually whole body exposed to a TDI atmosphere at the workplace. Also, it is obscure whether the airway hypersensitivity induced by repeated short-period exposure to TDI is due to immunological and inflammatory response or simply to TDIÕs chemical irritation. To establish an in vivo animal model resembling industrial exposure and to elucidate the mechanisms involved in TDI induced airway hypersensitivity, Wistar rats were sensitized with repeated short period whole body exposure to TDI vapor, after which the inflammatory and immunological events in the airways were investigated. Occupational asthma has been reported to be a complex pathophysiological event involving the interaction of many cell types and cytokines. It has been shown that activation of selected T cells with subsequent eosinophil recruitment and secretion of eosinophil derived mediators may contribute to both epithelial cell damage and airway hyperresponsiveness (Bentley et al., 1992). Furthermore, studies have suggested that T cell derived cytokines may play a role in the initiation of eosinophilic inflammation (Mattoli et al., 1991; Maestrelli et al., 1995, 1997). T cell-mediated inflammatory reaction has been hypothesized to be involved in the pathogenesis of TDI induced asthma through an array of cytokines. Therefore, the present study was conducted to further investigate the status and the role of infiltration of inflammatory cells, particularly eosinophils, the secretion and the role of Th2 cytokines in airway hyperresponsiveness induced by TDI in a rat model exposed to TDI atmosphere.
2. Materials and methods 2.1. Animals Thirty-eight-week old female Wistar rats, weighing an average of 240 ± 30 g were obtained from a Kyudo breeding laboratory (Kumamoto, Japan). Upon arrival, the rats were housed in pathogen free steel mesh cages under environmentally controlled conditions in compliance with the Ryukyus University Policy on Animal Care and Use. They were kept at a constant room temperature (25 ± 2 C) and humidity (50–70%) and at a 12 h light dark cycle. Food and water were provided ad libitum through out the experiment except during the exposure periods. The animals were allowed to acclimatize to our laboratory for a week, and then 10 rats were randomly selected for the control group. The remaining 20 rats were randomly divided into two expo-
sure groups (1 and 2) with each exposure group contained 10 rats. 2.2. Exposure procedures and quantification of TDI concentrations The rats were placed in a 22 l glass chamber with a dynamic adjustable laminar airflow and exposed to TDI vapor, 4 h per day for five consecutive days. The TDI atmosphere in the chamber was generated by bubbling air at a rate of 22 l per minute through a flask containing 15 ml of 2–4 toluene diisocyanate (TDI, Wako Chemical Co., Japan) into the chamber. By varying the airflow rate through the flask, different TDI concentrations were obtained. A constant concentration was maintained in the chamber by maintaining a constant airflow meter rate during the whole exposure period. This generation system produced TDI vapor free of aerosol. Temperature and humidity were monitored during the exposure period. The air was conditioned to a temperature of 24 ± 1 C and 25 ± !1% relative humidity as reported by Gagnaire et al. (1996). The concentration was checked each hour in order to maintain a stable concentration for each exposure group. The concentrations of TDI atmospheres in the chamber were determined according to Marcali (1957), which was modified by NIOSH (1977). Briefly, the air in the chamber was sampled with an impinger containing an absorber medium made from acetic and hydrochloric acids. Several solutions, diazotization solution containing sodium nitrite and sodium bromide, sulfamic acid solution, N-1-napthylethylenediamine and sodium carbonate solution, were then added to the absorber medium. When a reddish-blue colored solution was finally obtained, its transmittance was measured with a spectrophotometer at 550 nm. A calibration curve of a series of standardized TDI solutions was prepared by plotting transmittance versus TDI concentrations. This curve was then used to evaluate TDI concentrations. The chamber concentrations were 0.31, 0.45, 0.33, 0.38, and 0.45 ppm for exposure group 1, and 0.99, 1.44, 1.20, 0.98 and 1.41 ppm for exposure group 2. The mean TDI concentrations were 0.38 ± 0.07 ppm and 1.20 ± 0.22 ppm for exposure groups 1 and 2, respectively. The control group was placed in the same chamber and treated under the same condition with physiological saline in the flask instead of TDI. Since the effect of TDI vapor on the breathing pattern and frequency before, during and after sensitization have been well recorded, our focus in this study was mainly on the inflammatory and immunological events in the airway after a repeated short-period exposure to TDI vapor. We did not therefore record breathing pattern and frequency in this study.
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2.3. Bronchoalveolar lavage (BAL)
2.6. Statistical analysis
Twenty-four hours after a 5-day exposure, the rats in both the exposure and control groups were weighed and anesthetized by intraperitoneal injection of 50 mg/kg of sodium pentobarbital. A cervical incision was made and the trachea was isolated. A catheter was then inserted into the trachea and bronchoalveolar lavage (BAL) was performed. The trachea was slowly infused with 5 ml sterile phosphate buffered saline (PBS, 37 C), and the effluent (BAL fluid) was gently aspirated. The recovered aliquot (approximately 3 ml per rat) was centrifuged at 500 g (4 C). The supernatant was collected and stored at 80 C until cytokines determination. To collect the BAL cells, the trachea was further infused 2 times with 2 ml each time of PBS (37 C), and all the recovered aliquot (2.5 ml) was pooled and centrifuged at 500 g (4 C). The cell pellet was resuspended in RPMI 1640 medium (Gibco, Life Technologies, NY, USA) and the total number of cells and their viability were determined using 0.2% trypan blue exclusion method. To perform the differential leukocyte cell count, 0.1 ml of the cell suspension was smeared on a glass slide and stained with Wright–Giemsa. Three hundred nucleated cells were then examined under a microscope. The cells were classified either as macrophages, neutrophils, eosinophils or lymphocytes. The first washing was used for cytokine determination and the second for leucocyte differential count because the cytokine concentration in the second washing would have been reduced while the proportions of the various leucocytes would have been maintained in both the first and second BAL washings.
The cellular contents and cytokine productions were presented as means ± SD. Two-tailed StudentÕs t-test was performed to reveal differences between the means of each exposure group and the control group. Values of p < 0.05 were considered to be statistically significant.
2.4. Cytokine assay Cytokines IL-2, IL-4 and IL-6 productions in the BAL fluid were quantified respectively with rat IL-2, IL-4 and IL-6 ELISA kits (Endogen, Inc, MA, USA) according to the protocols recommended by the manufacturer. The inter-assay and intra-assay coefficient of variation, CV, were <10%. The sensitivities of the assays were <5 pg/ml, <2 pg/ml and <8 pg/ml for IL-2, IL-4 and IL-6, respectively. 2.5. Lung histopathology Immediately after BAL was performed, 1 ml formaldehyde solution was injected through the catheter into the trachea. The lungs of each rat were resected, weighed and then fixed in buffered formalin. A section encompassing the maximum cross-sectional area of the right lung was taken perpendicular to the major bronchi, dehydrated through a series of ethanol solutions, and then embedded in paraffin. Two micrometer thick sections were then sliced and stained with hematoxylin eosin for examination.
3. Results 3.1. Hypersensitivity symptoms in the airways Exposure to both 0.38 and 1.20 ppm TDI resulted in airway hypersensitivity in the rats. When the rats were exposed to 0.38 ppm TDI, airway symptoms characterized by hyperrhinorrhea and sneezing occurred within two hours. On the third day of exposure, the animals started coughing and showed moderate prolongation of the expiratory phase. On the last day of the exposure period, all the rats were wheezing and gasping for air. In comparison with 0.38 ppm TDI, 1.20 ppm TDI induced airway symptoms in the rats within one hour. The symptoms, particularly exertional breathing (wheezing, gasping for air and prolongation of the expiratory rate) were also more severe. This implies that airway hypersensitivity symptoms were aggravated along with the increase in TDI concentration. The rats in the control group, however, showed no obvious respiratory symptoms. 3.2. Analysis of BAL fluid BAL fluid cellular analysis demonstrated that exposure to both 0.38 ppm and 1.20 ppm TDI vapors resulted in inflammatory responses in the airways of the rats. As shown in Table 1, a five-day exposure to TDI resulted in a significant increase in the numbers of total cells and each differential leukocyte as compared to the control rats. Eosinophils and neutrophils in particular were increased 17.20 and 4.62 times, respectively, in the rats exposed to 0.38 ppm. When the rats were exposed to 1.20 ppm, the total number of cells, as well as each leukocyte were further increased significantly as compared to the control rats. The number of eosinophils and neutrophils in the rats exposed to 1.20 ppm were increased 30.2 and 6.26 times, respectively. 3.3. Cytokines in the BAL fluid The cytokine content in the BAL fluid was determined by an ELISA test. As shown in Fig. 1 , the productions of IL-2 in exposure groups 1 and 2 were not significantly different from those in the control group (p = 0.53 and p = 0.12, respectively). Similarly, there was no difference in the IL-6 productions between each of the exposure groups and the control group (p = 0.23
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Table 1 Total cell and differential cell counts in the BAL fluid (·105 cells/ml) Treatment
Total cells
Differential leucocytes Eosinophils
Neutrophils
Lymphocytes
Macrophages
Control group Exposure group 1 Exposure group 2
15.93 ± 1.24 25.78 ± 4.62* 34.18 ± 6.58#
0.05 ± 0.01 0.86 ± 0.29# 1.51 ± 0.49#
0.26 ± 0.06 1.20 ± 0.39* 1.63 ± 0.24#
1.51 ± 0.41 2.18 ± 0.75 3.16 ± 0.91*
14.05 ± 1.13 21.40 ± 3.51# 27.68 ± 5.24#
Values are means ± SD. Each group contains 10 rats. * Significantly different from the control group values at p < 0.05. # Significantly different from the control group values at p < 0.01.
Fig. 1. Cytokine production in the BAL fluid. Values are means ± SD. Each group contains 10 rats. * Significantly different from the control group values at p < 0.05. ** Significantly different from the control group values at p < 0.01.
and p = 0.16, respectively). However, IL-4 was significantly increased in exposure group 1 (p = 0.041) and exposure group 2 (p = 0.0092) as compared to the control group. 3.4. Lung histopathological analysis The histopathological examination showed that inflammatory events occurred in the lungs of the rats exposed to TDI (Fig. 2). A prominent infiltration of eosinophils in the bronchi and a massive goblet cells metaplasia of the epithelium of the central airways were observed. These pathological changes were more severe in the rats exposed to 1.20 ppm than in those exposed to 0.38 ppm TDI vapor. In contrast, no pathological changes were found in the lungs of the control rats.
4. Discussion Animal models are extremely important for research and /or testing purposes in the study of industrial chemicals. For occupational asthma, animal models are particularly important because of our current incomplete
understanding of the mechanisms involved in chemical sensitization (Karol, 1988, 1994). Several animal models have been developed to understand the mechanisms underlying TDI induced asthma (Karol et al., 1981; Karol, 1983; Patterson et al., 1983; Abe et al., 1993; Dearman et al., 1996; Niimi et al., 1996). In contrast to other animal models previously reported, a Wistar rat model characterized by whole body exposure to TDI vapor in a glass chamber was established in this study. It is considered that the whole body exposure method has an advantage over the other methods in that it closely resembles the individualÕs exposure to TDI vapor at the workplace. It simulates potential human exposure to environmental chemicals or pesticides and it is possible to expose a large number of animals at the same time and therefore reduce the cost of the experiment. Also the animals are not fixed in a specific place and therefore are not under much stress. Feces and urine can easily escape and thermoregulation via the tail is also not compromised. We exposed 10 rats for each exposure group in a 22 l inhalation chamber. There could have been overcrowding in the chamber. However we tried to duplicate exposure in industrial fields where it is not always that procedures regarding space are followed, especially in developing countries where sometimes proper precautions are not taken. As compare to a head only exposure to TDI (Karol, 1983), the animals were not under much stress despite the number of rats in the chamber. It has been reported that not only the exposure method but also the exposure concentrations are important for the development of asthmatic symptoms in animal models exposed to TDI (Karol, 1983). A previous study by Karol (1983) has revealed that exposing guinea pigs to 0.12 ppm of TDI vapor did not show any respiratory hypersensitivity. In contrast, 0.36 ppm or greater resulted in airways hypersensitivity while a concentration of 2 ppm was pneumotoxic and few pulmonary hypersensitivity reactions were observed (Karol, 1983). Considering KarolÕs experiment, our preliminary ones in rats (data not shown) and that of Zheng et al. (2004), we used two different concentrations of TDI, 0.38 ppm and 1.20 ppm in the present study. The Wistar
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Fig. 2. Light microscopic images of the lungs of the control group rats (A), TDI exposure group 1 (B) and TDI exposure group 2 (C) after hematoxylin eosin staining. A, B and C are the bronchi of the rats. Goblet cells (black arrows) and eosinophils (white arrows) are present in the bronchi of TDI exposure groups: Magnification 200· (A,C) and 400· (B).
rats were sensitized and the results show that these concentrations also induced airway hypersensitivity in the exposed rats.
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A report by Tanaka et al. (1983) has shown that guinea pigs sensitized and subsequently challenged repeatedly with nasal applications of TDI showed asthma-like symptoms such as exertional breathing, prolonged expiration, and cough immediately after the challenge. In the present study, although the rats were repeatedly exposed to TDI vapor for short periods without challenge, the rats showed airway hypersensitivity symptoms, which were asthma-like. The pathological findings in these animals were not completely in agreement with reported histopathological findings for asthma (Fabbri et al., 1991) because the exposure period was short. Studies have shown that in vivo airway hyperresponsiveness was not observed at any point in time in a mouse model exposed to TDI according to the short-term protocol (Scheerens et al., 1999). It has been shown that by altering the exposure time and/or cumulative dosage of TDI, different biological reactions can be elicited in exposed mice. This important finding therefore might be a reflection of the diversity of symptoms found in patients suffering from TDI-induced asthma. Both the short-exposure and the long-exposure models could be useful for further investigation into the mechanisms involved in the action of TDI (Scheerens et al., 1999). Zheng et al. (2004) found that concentrations of 0.34 ppm and above sensitized all the rats by the second day after exposing for 4 h a day. Further exposure on the other days resulted in hypersensitivity. The results of airway inflammatory cell dynamics by means of BAL fluid and histopathological examination of the lung as well as cytokine productions revealed that repeated exposure to a low concentration (0.38 ppm) or a high concentration (1.20 ppm) of TDI results in airway hypersensitivity in which inflammatory and immunological inter-response may be involved. These data are consistent with those in a guinea pig model repeatedly exposed to a series of TDI vapor concentrations reported by Karol (1983). In KarolÕs experiment, the guinea pig model was established using the method of head inhalation exposure to TDI vapors, 3 h per day for five consecutive days, the immunologic response to TDI exposure (TDI specific IgE) occurred and was exposure concentration dependent. In the present study, although only two concentrations (0.38 ppm and 1.20 ppm) of TDI vapor were used to sensitize the rats, the data also revealed that exposure to a high concentration (1.20 ppm) of TDI vapor resulted in a severer immunologic and inflammatory responses characterized with a higher productions of cytokines and a more prominent infiltration of eosinophils in the airways as compared with exposure to a lower concentration (0.38 ppm) of TDI. This suggests a concentration dependent relationship between TDI exposure and immunoinflammatory responses. It has been reported that in TDI induced asthma patients, only 10–30% have detectable IgE (Karol, 1981). This suggests that IgE may not
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be specific to occupational asthma and therefore was not measured in this study. A previous report has shown that, in general the pathological features in TDI induced occupational asthmatics are similar to those of status asthmaticus seen in non-occupational asthma (Fabbri et al., 1988). In occupational asthmatics, the airways were plugged with mucus containing abundant exudates; the airway epithelium was extensively desquamated, and the mucosa and the lamina propria were edematous and markedly infiltrated with eosinophils (Fabbri et al., 1991). Airway inflammation has been reported to play a significant role in the pathogenesis of asthma, and eosinophil infiltration in the airway is a known hallmark histopathological character of asthma (Djukanovic et al., 1990; Saetta et al., 1992). Late asthmatic response (LAR) in humans after exposure to TDI have been shown to be associated with the increase of airway hyperresponsiveness and airway eosinophilia (Fabbri et al., 1987). Furthermore, studies have demonstrated a strong correlation between asthma severity and eosinophil numbers in biopsies (Moqbel et al., 1992), sputum (Frigas et al., 1981) and blood (Durham and Kay, 1985). Eosinophils are potent proinflammatory cells which release toxic granule proteins such as major basic protein (MBP) and eosinophil cationic protein (ECP), generate lipid mediators such as platelet-activating factor (PAF), leukotriene C4 and oxygen metabolites which have profound potential to injure the airway epithelium, promote bronchial responsiveness, exacerbate inflammatory response and contract airway smooth muscle (Gleich, 1990; Weller, 1991). Eosinophils therefore possess properties that can directly and indirectly cause airway obstruction and promote bronchial hyperresponsiveness. The pathological changes in the TDI exposed rats in this study were in board agreement with those in occupational asthmatics and other previously reported animal models (Tanaka et al., 1983; Fabbri et al., 1991; Mapp et al., 1996). It has also been revealed that asthmatic bronchial inflammation is a specialized form of cell mediated immunity in which immunological mediators such as lymphokines and cytokines, secreted principally by activated CD4+ T lymphocytes, orchestrate the accumulation and activation of specific granulocyte effector cells, particularly eosinophils (Kay, 1991). In atopic asthma, activated T helper lymphocytes are present in bronchial biopsy specimens and bronchoalveolar lavage (BAL) fluid. The production of cytokines may be important in the pathogenesis of this disorder. Bronchial inflammation in asthma may depend in part on the activation of T helper lymphocytes that elaborate proinflammatory cytokines (Azzawi et al., 1990; Walker et al., 1991). Some studies have shown that the Th2 type cytokines interleukin (IL)-4 and IL-5 have an important role in atopic disease and more specifically in atopic
asthma (Seder et al., 1992; Bjorksten et al., 1995; Daher et al., 1995). IL-4 is involved in the switch of B cells to IgE production (Le gros et al., 1990) and IL-5 has been shown to cause eosinophil infiltration into the airway wall (Campbell et al., 1987). Both IL-4 and IL-5 have been implicated in the airway hypersensitivity associated with TDI-induced occupational asthma (Maestrelli et al., 1997). Animal models exposed to TDI have also revealed the important role of IL-4 and IL-5 in airway hypersensitivity (Dearman et al., 1996). The significant increase of IL-4 production (but not IL-2 and IL-6) in the airways of TDI-exposed rats in this study is partly consistent with previous studies on TDI induced occupational asthma (Dearman et al., 1996; Maestrelli et al., 1997), further supporting the important role of Th2 cytokines in airway hypersensitivity. Possible oral ingestion and dermal absorption of TDI may have occurred in this study. All these routes have the potential of sensitizing. However, apart from the fact that the vapor form of TDI is more likely to affect the airways than other body parts and systems, Karol et al. (1981) have shown that dermal contact of TDI results in pulmonary sensitization. The shortcoming of the present study is that IL-5 production was not determined due to unavailability of commercial rat IL-5 kit at the time the experiment was carried out. Lacking data on IL-5 productions hamper the explanation of the regulation of eosinophil infiltration and the conclusion of the role of Th2 cytokines in this animal model. From the results of this study, it can be seen that repeated whole body exposure to TDI may result in inflammatory and immunological events in the airway of rats in which, an inflammation characterized by eosinophil infiltration and Th2 type cytokines may play a key role. Also the animal model used in this study can be used to further unravel the complex mechanisms underlying TDI induced occupational asthma.
Acknowledgement The authors would like to thank Dr. Takamitsu Morioka of the first Department of Pathology, Faculty of Medicine, University of the Ryukyus, Okinawa, Japan, for his technical support. This animal experiment was done according to the regulation of Japanese laws related to the use of animal for research.
References Abe, Y., Ogino, S., Irifune, M., Imamura, I., Liu, Y.Q., Fukui, H., Matsunaga, T., 1993. Histamine content, synthesis and degrada-
K. Kouadio et al. / Food and Chemical Toxicology 43 (2005) 1281–1288 tion in nasal mucosa and lung of guinea pigs treated with toluene diisocyanate (TDI). Clinical and Experimental Allergy 23, 512– 517. Azzawi, M., Bradley, B., Jeffery, P.K., Frew, A.J, Wardlaw, A.J., Knowles, G., Assoufi, B., Collins, J.V., Durham, S., Kay, A.B., 1990. Identification of activated T lymphocytes and eosinophils in bronchial biopsies in stable atopic asthma. The American Review of Respiratory Disease 142, 1407–1413. Bentley, A.M., Maestrelli, P., Saetta, M., Fabbri, L.M., Robinson, D.S., Bradley, B.L., Jeffery, P.K., Durham, S.R., Kay, A.B., 1992. Activated T lymphocytes and eosinophils in the bronchial mucosa in isocyanate induced asthma. Journal of Allergy and Clinical Immunology 89, 821–829. Bernstein, I.L., 1982. Isocyanate induced pulmonary diseases: a current perspective. Journal of Allergy and Clinical Immunology 70, 24–31. Bjorksten, B., Borres, M.P., Einarsson, R., 1995. Interleukin-4, soluble CD23 and interferon-gamma levels in serum during the first 18 months of life. International Archives of Allergy and Immunology 107, 34–36. Campbell, H.D., Tucker, W.Q., Hort, Y., Martinson, M.E., Mayo, G., Clutterbuck, E.J., Sanderson, C.J., Young, I.G., 1987. Molecular cloning, nucleotide sequence, and expression of the gene encoding human eosinophil differentiation factor (interleukin-5). Proceedings of the National Academy of Science of United States of America 84, 6629–6633. Daher, S., Santos, L.M., Sole, D., De Lima, M.G., Naspitz, C.K., Musatti, C.C., 1995. Interleukin-4 and soluble CD23 serum levels in asthmatic atopic children. Journal of Investigational Allergology and Clinical Immunology 5, 251–254. Dearman, R.J., Basketter, D.A., Kimber, I., 1996. Characterization of chemical allergens as a function of divergent cytokine secretion profiles induced in mice. Toxicology and Applied Pharmacology 138, 308–316. Djukanovic, R., Wilson, J.W., Britten, K.M., Wilson, S.J., Walls, A.F., Roche, W.R., Howarth, P.H., Holgate, S.T., 1990. Quantification of mast cells and eosinophils in the bronchial mucosa of symptomatic atopic asthmatics and healthy control subjects using immunohistochemistry. American Review of Respiratory Disease 142, 863–871. Durham, S.R., Kay, A.B., 1985. Eosinophil bronchial hyperreactivity and late asthmatic reactions. Clinical Allergy 15, 411–418. Fabbri, L.M., Boschetto, P., Zocca, E., 1987. Bronchoalveolar neutrophilia during late asthmatic reactions induced by toluene diisocyanate. American Review of Respiratory Disease 136, 36– 42. Fabbri, L.M., Danieli, D., Crescioli, S., 1988. Fatal asthma in a toluene diisocyanate sensitized subject. American Review of Respiratory Disease 137, 1494–1498. Fabbri, L.M., Maestrelli, P., Saetta, M., Mapp, C.E., 1991. Airway inflammation during late asthmatic reactions induced by toluene diisocyanate. American Review of Respiratory Disease 143, S37– S38. Frigas, E., Loegering, D.A., Solley, G.O., Farrow, G.M., Gleich, G.J., 1981. Elevated levels of the eosinophil granule major basic protein in the sputum of patients with bronchial asthma. Mayo Clinic Proceedings 56, 345–353. Gagnaire, F., Ban, M., Micillino, M., Lemonier, M., Bonnet, P., 1996. Bronchial responsiveness and inflammation in guinea pigs exposed to toluene diisocyanate: a study on a single and repeated exposure. Toxicology 114, 91–100. Gleich, G.J., 1990. The eosinophil and bronchial asthma: current understanding. Journal of Allergy and Clinical Immunology 85, 422–436. Karol, M.H., 1981. Survey of industrial workers for antibodies to industrial workers for antibodies to toluene diisocyanate. Journal of Occupational Medicine 23, 741–747.
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Karol, M.H., 1983. Concentration dependent immunologic response to toluene diisocyanate (TDI) following inhalation exposure. Toxicology and Applied Pharmacology 68, 229–241. Karol, M.H., 1988. The development of an animal model for TDI asthma. Bulletin Europeen de Physiopathologie Respiratoire 23, 571–576. Karol, M.H., 1994. Animal models of occupational asthma. European Respiratory Journal 7, 555–568. Karol, M.H., Hauth, B.A., Riley, E.J., Magreni, C.M., 1981. Dermal contact with toluene diisocyanate (TDI) produces respiratory tract hypersensitivity in guinea pigs. Toxicology and Applied Pharmacology 58, 221–230. Kay, A.B., 1991. Asthma and inflammation. Journal of Allergy and Clinical Immunology 87, 893–910. Le gros, G., Ben-Sasson, S.Z., Seder, R., Finkelman, F.D., Paul, W.E., 1990. Generation of interleukin-4 (IL-4) producing cells in vivo and in vitro: IL-2 and IL-4 are regulated for in vitro generation of IL-4 producing cells. Journal of Experimental Medicine 172, 921–929. Maestrelli, P., Di Stefano, A., Occari, P., Turato, G., Milani, G., Pivirotto, F., Mapp, C.E., Fabbri, L.M., Saetta, M., 1995. Cytokines in the airway mucosa of subjects with asthma induced by toluene diisocyanate. American Journal of Respiratory and Critical Care Medicine 151, 607–612. Maestrelli, P., Occari, P., Turato, G., Papiris, S.A., Di Stefano, A., Mapp, C.E., Milani, G.F., Fabbri, L.M., Saetta, M., 1997. Expression of IL-4 and IL-5 proteins in asthma induced by toluene diisocyanate (TDI). Clinical and Experimental Allergy 27, 1292– 1298. Mapp, C.E., Lapa E Silva, J.R., Lucchini, R.E., Chitano, P., Rado, V., Saetta, M., Pretolani, M., Karol, M.H., Maestrelli, P., Fabbri, L.M., 1996. Inflammatory events in the blood and airways of guinea pigs immunized to toluene diisocyanate. American Journal of Respiratory and Critical Care Medicine 154, 201–208. Marcali, K., 1957. Microdetermination of toluene diisocyanates in atmosphere. Annals of Chemistry 29, 552–558. Mattoli, S., Colotta, F., Fincato, G., Mezzetti, M., Mantovani, A., Patalano, F., Fasoli, A., 1991. Time course of IL1 and IL6 synthesis and release in human bronchial epithelial cell cultures exposed to toluene diisocyanate. Journal of Cellular Physiology 149, 260–268. Meredith, S.K., Taylor, V.M., McDonald, J.C., 1991. Occupational respiratory disease in the United Kingdom 1989: a report to the British Thoracic Society and Society of Occupational Medicine by the SWORD project group. British Journal of Industrial Medicine 48, 292–298. Moqbel, R., Barkans, J.R., Bradley, B., Durham, S.R., Kay, A.B., 1992. Application of monoclonal antibodies against major basic protein (BMK-13) and eosinophil cationic protein (EG1 and EG2) for quantifying eosinophils in bronchial biopsies from atopic asthma. Clinical and Experimental Allergy 22, 265–273. Niimi, A., Amitani, R., Yamada, K., Tanaka, K.I., Kuze, F., 1996. Late respiratory response and associated eosinophilic inflammation induced by repeated exposure to toluene diisocyanate in guinea pigs. Journal of Allergy and Clinical Immunology 97, 1308–1319. NIOSH, US Department of Health, Education and Welfare, Washington, DC, 1977. 2-4-toluene diisocyanate (TDI) in air. In: Manual of Analytical Methods, 2nd ed., vol 1, pp. 1411–1418. Patterson, R., Zeiss, C.R., Harris, K.E., 1983. Immunologic and respiratory responses to airway challenges of dogs with toluene diisocyanate. Journal of Allergy and Clinical Immunology 71, 604– 611. Ross, D.J., Sallie, B.A., McDonald, J.C., 1995. SWORDÕs 1994 surveillance of work related and occupational respiratory disease in the United Kingdom. Occupational Medicine 45, 175–178. Saetta, M., Di Stefano, A., Maestrelli, P., De Marzo, N., Milani, G.F., Pivirotto, F., Mapp, C.E., Fabri, L.M., 1992. Airway mucosal inflammation in occupational asthma induced by toluene
1288
K. Kouadio et al. / Food and Chemical Toxicology 43 (2005) 1281–1288
diisocyanate. American Review of Respiratory Disease 145, 160– 168. Scheerens, H., Buckley, T.L., Muis, T.L., Garssen, J., Dormans, J, Nijkamp, F.P., Van Loveren, H., 1999. Dormans. Long-term topical exposure to toluene diisocyanate in mice leads to antibody production and in vivo airway hyperresponsiveness three hours after intranasal challenge. American Journal of Respiratory Care Medicine 159, 1074–1080. Seder, R.A., Paul, W.E., Davis, M.M., Fazekas de St Groth, B., 1992. The presence of IL4 during in vitro priming determines the lymphokine producing potential of CD4+ T cells from T cell receptor transgenic mice. Journal of Experimental Medicine 176, 1091–1098.
Tanaka, K., Kawai, M., Maekawa, N., 1983. Experimental model of asthma by toluene diisocyanate (TDI). Bulletin of Chest Disease and Respiratory Institute of Kyoto University 16, 1–9. Walker, C., Virchow Jr., J.C., Bruijnzeel, P.L.B., Blaser, K., 1991. T cell subsets and their soluble products regulate eosinophilia in allergic and non-allergic asthma. Journal of Immunology 146, 1829–1835. Weller, P.F., 1991. The immunobiology of eosinophils. New England Journal of Medicine 324, 1110–1118. Zheng, K.C., Kouadio, K., Tuekpe, M.K.N., Makoto, A., 2004. Eosinophil infiltration in airways of mice exposed to toluene diisocyanate. Journal of Occupational Health 46, 299–302.