* Department of Pharmacology and Toxicology, and Department of Pathobiology and Diagnostic Investigation, Center for Integrative Toxicology, Michigan State University, East Lansing, Michigan 48824
Received October 15, 2003; accepted February 2, 2004
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ABSTRACT |
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Key Words: murine; allergic rhinitis; intranasal instillation; trimellitic anhydride; dinitrochlorobenzene; oxazolone.
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INTRODUCTION |
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One of the difficulties associated with the intranasal instillation of LMW chemicals is the selection of a suitable, nontoxic vehicle in which to deliver these chemicals to the airways. Most LMW chemicals linked to occupational asthma and allergic rhinitis are lipophilic and hydrolyze in aqueous solutions, which precludes the use of aqueous vehicles (Ebino et al., 1999). Also, many organic solvents such as ethyl acetate and acetone are very irritating to the airway mucosa. Ideally, a vehicle should allow complete dissolution of the chemical and should not induce cytotoxicity and inflammation in the respiratory tract. In the present study, we used a vehicle consisting of ethyl acetate and olive oil in a 1:4 combination that allowed effective dissolution of the LMW chemicals and caused no detectable nasal or pulmonary pathology.
Th2 cytokines have been implicated in LMW chemical-induced allergic airway disease both in humans and in experimental animal models. For example, the production of IL-4 and IL-5 proteins was increased in bronchial biopsies of patients with TDI-induced asthma (Maestrelli et al., 1997). In addition, the selective enhancement of Th2 cytokines was detected in several murine models of TMA-induced asthma (Betts et al., 2002
; Dearman et al., 2002
; Vandebriel et al., 2000
). The methods by which cytokine measurements are made vary in murine models. They include measuring cytokine secretion from stimulated local draining lymph node cells that drain the site of dermal application or by analyzing the cells from BALF for cytokine protein or mRNA expression. In the present study, we examined the local mRNA expression of Th2 cytokines in right lung lobe tissue after intranasal administration of TMA, DNCB, and OXA and micro-dissected nasal airway tissue after intranasal administration of TMA.
The primary objective of this study was to test the hypothesis that intranasal sensitization and challenge with the known chemical respiratory allergen TMA, but not with the nonrespiratory sensitizers DNCB and OXA, will induce the characteristic features of LMW chemical-induced allergic airway disease in the nasal and pulmonary airways. DNCB and OXA are known LMW chemical contact allergens that elicit contact hypersensitivity in the skin mediated by Th1 cytokines such as IFN- and do not cause occupational asthma or allergic rhinitis (Dearman and Kimber, 2001
). The histopathology in the nasal and pulmonary airways of mice after intranasal sensitization and challenge with TMA was compared to that of mice intranasally sensitized and challenged to DNCB or OXA. In addition, the right lung lobes were assessed for any changes in Th2 and Th1 cytokine mRNA expression. The nasal airways of TMA sensitized and challenged mice were also assessed for changes in Th2 and Th1 cytokine mRNA expression. This was done to examine the utility of measuring local cytokine gene expression as a biomarker for the respiratory allergenicity of low molecular weight chemicals.
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MATERIALS AND METHODS |
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For each chemical, there were five different treatment groups: (1) untreated naïve group, (2) mice sensitized and challenged with the vehicle alone, (3) mice sensitized with the vehicle alone and then challenged with the chemical in vehicle, (4) mice sensitized with the chemical in vehicle and then challenged with the vehicle alone, and (5) mice sensitized and challenged with the chemical in vehicle. Mice were sacrificed 24 or 96 h (Day 28 or Day 31) after the final instillation.
An additional group of mice was subsequently added to specifically assess cytokine mRNA expression in the nasal airways of mice sensitized and challenged with TMA alone. In this study, the mice were sensitized and challenged with TMA as described above, but were sacrificed 48 h after the final instillation.
Necropsy, blood sample collection, and tissue preparation.
At the designated sacrifice times, mice were deeply anesthetized via ip injection of 0.1 ml of 12% pentobarbital in saline. Blood samples (0.1 to 0.5 ml) were taken from the abdominal aorta and collected in Beckton Dickinson vacutainers. Serum samples were collected and stored at
20°C after the blood samples were centrifuged to remove cells. The abdominal aorta and renal artery were then severed to exsanguinate the animals. Immediately after death, the trachea was cannulated and the heart/lung block removed. A syringe was then attached to the cannula and bronchoalveolar lavage fluid was collected from the whole lung with 2 ml saline. Differential cell counts were determined by cytocentrifuging cells onto glass slides and then staining with Diff-Quik (DADE Behring, Newark, DE). Neutrophils, macrophages, eosinophils, and other cell types were microscopically identified on the slides. The total number of each cell type per ml of lavage fluid was determined by multiplying the percentage of each cell type from a total of 200 cells by the total number of all cells per ml of lavage fluid. The right bronchus was then clamped using suture thread. All four right lung lobes were placed in 2 ml Tri reagent (Molecular Research Center, Cincinnati, OH), homogenized and stored at
80°C.
After removal of the right lung lobes, the left lung lobe was intratracheally perfused with 10% neutral buffered formalin at a constant intra-airway pressure of 30 cm of fixative. After 1 h, the trachea was ligated, and the inflated left lung lobe was immersed in a large volume of the same fixative for 24 h. After fixation, the left lung lobe was microdissected along the axial airways, and sections were then excised at the level of the fifth (proximal airway) and eleventh (distal airway) airway generation (Fig. 2), as has been described previously in detail (Steiger et al., 1995). Paraffin sections (5 µ;m) were stained with Alcian Blue (pH 2.5) and Periodic Acid Schiffs (AB/PAS) reagent, which stains both neutral and acidic mucosubstances within airway mucous (goblet) cells.
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For the analysis of cytokine mRNA expression in the nasal mucosa, the heads of mice were split in a sagittal plane adjacent to the midline exposing the mucosal surfaces lining the nasal lateral wall and septum. The nasoturbinate and maxilloturbinate from each nasal airway and the proximal septum were micro-dissected. The excised tissues from each animal were placed in 1 ml Tri Reagent, homogenized, and stored at 80°C.
Morphometry of stored intraepithelial mucosubstances (IM) in nasal airways.
The amount of stored mucosubstances in the respiratory epithelium lining the mid-septum in the most proximal nasal section was estimated by determining the volume density of AB/PAS-stained mucosubstances using computerized image analysis and standard morphometric techniques. The area of AB/PAS-stained mucosubstances was calculated by circumscribing the perimeter of the stained material using the Scion Image program (Scion Corporation, Frederick, MD). The length of the basal lamina underlying the surface epithelium was calculated from the contour length of the digitized image of the basal lamina. The volume of stored mucosubstances (volume density, Vs) per unit of surface area was estimated using the method described in detail by Harkema et al. (1987) and was expressed as nanoliters of intraepithelial mucosubstances (IM) per square millimeter of basal lamina.
ELISA for total serum IgE.
Total serum IgE was measured using a 96-well Immulon ELISA plate (Dynex, Technologies, Chantilly, VA) coated with 2 µ;g/ml anti-mouse IgE (Purified Rat Anti-Mouse IgE Monoclonal Ab, Pharmingen, San Diego, CA) and incubated overnight at 4°C. After washing, the plates were incubated in 3% bovine serum albumin (3% BSA) at 37°C for 1 h (BSA, CALBIOCHEM, La Jolla, CA). Serum samples at 1:10 dilution were then added followed by incubation at 37°C for 1 h. After washing, biotinylated anti-mouse IgE (Biotin-conjugated Rat Anti-Mouse IgE Monoclonal Ab, Pharmingen, San Diego, CA) was then added at 2 µ;g/ml and allowed to incubate at 25°C for 1 h. After washing, 1.5 µ;g/ml of streptavidin peroxidase was added followed by incubation at 25°C for 1 h. After washing, TMB substrate (12.5 ml citric-phosphate buffer + 200 µ;l of tetramethylbenzidine [TMB] stock solution [6 mg/ml in DMSO] + 100 µ;l 1 % H2O2 [Fluka Chemical Co., Ronkonkoma, NY]) was added to produce a color reaction. The reaction was terminated by the addition of 6 N H2SO4. Optical density was determined at 450 nm using an EL-808 microplate reader (Bio-Tek Instruments, Winooski, VT).
Real-time RT-PCR.
Total RNA was isolated from lung, nasal airway tissue, small intestine, or spleen using the TRI REAGENT method (Molecular Research Center, Cincinnati, OH) following the manufacturers protocol. The evaluation of the relative expression levels of the cytokines IL-4, IL-5, IL-10, IL-13, and IFN- mRNA was determined using the TaqMan one-step real-time multiplex RT-PCR with TaqMan pre-developed primers and probe using the manufacturers recommended protocol (Applied Biosystems, Foster City, CA). Briefly, aliquots of isolated tissue RNA (100 ng total RNA) were added to the RT-PCR reaction mixture, which included the target gene (IL-4, IL-5, IL-10, IL-13, or IFN-
) primers and probe, endogenous reference primers and probe (18S ribosomal RNA), AmpliTaq DNA polymerase and Multiscribe reverse transcriptase (MuLV). The probes are designed to exclude detection of genomic DNA. RNA samples were first reverse transcribed and then immediately amplified by PCR. Following the PCR, amplification plots (change in dye fluorescence versus cycle number) were examined and a dye fluorescence threshold within the exponential phase of the reaction was set separately for the target gene and the endogenous reference (18S). The cycle number at which each amplified product crosses the set threshold represents the CT value. The amount of target gene normalized to its endogenous reference was calculated by subtracting the endogenous reference CT from the target gene CT (
CT). Relative mRNA expression was calculated by subtracting the mean
CT of the control samples from the
CT of the treated samples (
CT). The amount of target mRNA, normalized to the endogenous reference and relative to the calibrator (i.e., RNA from control) is calculated by using the formula 2
CT.
Statistics.
The data obtained from each experimental group were expressed as a mean group value ± the standard error of the mean (SEM). The differences among groups were determined by one way or two-way analysis of variance (ANOVA) and an All Pairwise Comparison Test (Tukey), using SigmaStat software from Jandel Scientific (San Rafael, CA).
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RESULTS |
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In mice similarly instilled with TMA but sacrificed 96 h after the last instillation, the nasal inflammatory response was similar in character and distribution but considerably attenuated from that in the mice that were sacrificed earlier at 24 h post-instillation. These mice also had mild lymphoid hyperplasia of the NALT. In addition, mucous cell hyperplasia was present in the respiratory epithelium lining the mid-septum in T1 (Fig. 4). No exposure-related alterations were microscopically evident in the nasal or pulmonary airways of DNCB or OXA-instilled mice (data not shown). In addition, there were no morphologic alterations in the gut or spleen of all treated mice relative to the naïve controls.
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DISCUSSION |
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Th2 cells produce IL-4, IL-5, IL-10, and IL-13 (Yssel and Groux, 2000). IL-4 promotes T cell activation and differentiation into the Th2 subtype while both IL-4 and IL-13 promote IgE production in B cells and mucus production in the airways (Frew, 1996
; Shim et al., 2001
). The mechanism of nonoccupational allergic rhinitis is dependent on Th2 cytokines (Andersson et al., 2000
). Th2 cytokines have also been linked to the pathogenesis of LMW chemical-induced asthma. Several groups have shown that lymph node cells draining the site of dermal application in murine models of TMA-induced asthma preferentially secrete Th2 cytokines including IL-4, IL-10, and IL-13 (Dearman et al., 2003
; Plitnick et al., 2002
). Limited evidence exists, however, that demonstrates a link between LMW chemical-induced allergic rhinitis and the up-regulation of Th2 cytokines. Nevertheless, the similarity in pathogenesis between LMW chemical-induced allergic rhinitis and nonoccupational allergic rhinitis and the evidence that Th2 cytokines are up-regulated in TMA-induced asthma suggests a critical role for Th2 cytokines in LMW chemical-induced allergic rhinitis. Only TMA sensitized and challenged mice had an increase in the nasal airway-derived mRNA levels of the Th2 cytokines IL-4, IL-5, and IL-13 and no change in the levels of the Th1 cytokine IFN-
. This is the first report to our knowledge that demonstrates, in a murine model, a link between TMA-induced allergic rhinitis and the selective enhancement of Th2 cytokine mRNA levels within the nasal airway. The nasal airways of DNCB or OXA treated mice were not assessed for Th2 cytokine transcripts. The rationale for not doing so was the absence of any DNCB- or OXA-induced nasal histopathology. However, studies specifically designed to assess the expression of Th2 and Th1 cytokines in mice treated with DNCB or OXA should be conducted in the future.
In the present study, no airway inflammation or epithelial lesions were found in any lungs instilled with TMA, DNCB, or OXA. There was only a minor increase in the number of alveolar macrophages lavaged from the lungs of TMA-instilled mice. The lack of significant airway pathology suggests that the amount of chemical that entered the lung may not have been sufficient to elicit an irritant/adaptive immune response in the lung. This may have been due to the viscosity of the ethyl acetate and olive oil vehicle, which may have prevented effective distribution of the chemicals into the pulmonary airways. In addition, the inherent high reactivity of these chemicals may have limited the distribution of these chemicals to the upper airways. It is likely that a large portion of this chemical reacted with proteins in the nasal airways preventing distribution to the lungs of these mice.
Greenberg et al. (1994) have reported that after aerosolized toluene diisocyanate exposure in guinea pigs, the vast majority of the chemical remains in the nasal airway. The small increase in macrophages in the BALF and the increase in lung-derived IL-5 mRNA after sensitization and challenge with TMA, however, suggests that there was some distribution of the chemical into the lung. The increase in macrophages in the BALF was not an immune-mediated effect as it also took place after just a single instillation of the chemical and may be due to the inherent irritating properties of the chemical.
Despite the lack of any significant immune-mediated pathology in the lungs of mice sensitized and challenged to TMA, TMA sensitized and challenged mice had a 30-fold increase in lung-derived IL-5 mRNA levels 24 h after the final TMA instillation. There was no increase in the transcripts for any of the other Th2 cytokines or the Th1 cytokine IFN-. In contrast, intranasal sensitization and challenge with DNCB or OXA did not result in a significant change in mRNA levels for any of the measured cytokines including IFN-
. These findings support other reports that link LMW chemical-induced occupational asthma with the selective expression of Th2 cytokines unlike known nonsensitizers of the respiratory tract. Several possibilities exist that may explain the TMA-induced increase in lung-derived IL-5 transcripts in the absence of pulmonary airway inflammation. One possibility is that this increase originated from infiltrating lymphocytes in areas of the lung not examined histologically such as the right lung lobes or more proximal regions of the left lung lobe. Another possibility is that the source of the cytokine transcripts was the bronchial-associated lymphoid tissue or the airway epithelium. Or perhaps a systemic immune response was elicited that originated in some extra-pulmonary organ that caused an increase in lymphocytes in the circulation and likewise the pulmonary vasculature. Further studies are required to determine the exact source of the increased expression of Th2 cytokines induced by TMA.
Although a pathologic response was not elicited in the pulmonary airways after intranasal instillation, this model of allergen-induced effects in the nasal airway may be used to assess the potential of a chemical agent to be a respiratory allergen. Allergic rhinitis and asthma were at one time thought of as separate disease entities, but there is increasing support for the concept of "one airway, one disease," i.e., that both these diseases are really a "continuum of inflammation within one airway" (Grossman, 1997, pg. 115). Several lines of evidence support this theory and the use of this model of nasal airway changes as a concurrent model to assess the allergenic effects of chemicals in the lower airways. Epidemiologic data suggests that allergic rhinitis and asthma coexist. In one study, it was found that 92% of subjects with occupational asthma experienced symptoms of rhinitis (Leynaert et al., 2000
). In addition to their association, allergic rhinitis and asthma share pathologic characteristics including the fact that they are both immediate type hypersensitivity reactions characterized by the infiltration of mast cells, eosinophils, and lymphocytes into the airways and mucus hypersecretion and both are elicited by the same inflammatory mediators such as histamine (WHO, 1999
). In addition, the airway epithelia lining the nasal and pulmonary airways are similar. Both the nasal and pulmonary airways contain respiratory epithelium that includes ciliated cells and mucous cells that respond similarly to allergen exposure (Andersson et al., 2000
). Also, a common therapeutic approach is used in the treatment of both diseases. For example, inhaled corticosteroids are used to ameliorate symptoms of both allergic rhinitis and asthma (Grossman, 1997
). Despite the limited distribution of the chemicals into the pulmonary airways, the effects of such chemicals in the upper airways may be used as a surrogate to predict their effects in the pulmonary airways.
The role of IgE antibodies in the pathogenesis of LMW chemical-induced occupational asthma and allergic rhinitis is unclear and controversial. Only 10 to 30% of all cases of toluene diisocyanate (TDI)-induced occupational asthma, for example, exhibit increases in IgE antibodies (Weissman and Lewis, 2000). There is a stronger, yet still inconsistent, link between IgE antibodies and TMA-induced allergic airway disease (Dearman, 2002
). Some investigators have suggested that the failure to establish a consistent link between high circulating IgE levels and LMW chemical-induced allergic airway disease is due to the deficiencies in IgE detection methods (Kimber and Dearman, 2002
). In most of the assays used to detect serum IgE against specific chemical allergens in humans or in animal models, the antigen used is in the form of a hapten-protein conjugate. The most prevalent protein used to prepare such conjugates has been albumin (Kimber and Dearman, 2002
). Tests for specific IgE that make use of hapten-protein conjugates are likely to identify only those patients in which binding of the chemical sensitizer to albumin has stimulated IgE production, not those in which IgE may have been induced by other protein-hapten conjugates (Griffin et al., 2001
). In such instances IgE levels would be underestimated possibly resulting in a false diagnosis. Other investigators have demonstrated that non-IgE-dependent mechanisms of LMW chemical-induced allergic airway disease may exist (Bernstein et al., 2002
; Herrick et al., 2002
; Larsen et al., 2001
). Nevertheless, we found that mice intranasally sensitized and challenged with TMA exhibited increases in total serum IgE. The increase in serum IgE, however, was not restricted to mice exposed to chemical respiratory allergens as DNCB sensitized and challenged mice exhibited a similar increase in total serum IgE. This was despite the fact that DNCB, at a nonirritating concentration, failed to elicit any pathologic response in the nasal and pulmonary airways and did not elicit a cytokine response in the lung. It is possible that DNCB at higher concentrations is irritating to the airway and may elicit a cell-mediated mononuclear cellular inflammation in the airways that is not mediated by Th2 cytokines as others have shown (van Houwelingen et al., 2002
). The present study, however, was dependent on repeated intranasal instillation to achieve sensitization; thus, it was important that the irritant effects of the LMW chemicals in the airways were minimal to distinguish between an immunological response and overt toxicity. Others have shown that DNCB elicits an increase in serum IgE while failing to sensitize the respiratory tract (Ban and Hettich, 2001
).
The IgE data from the present study has several putative implications. One is that serum IgE levels are not a good biomarker of LMW chemical-induced allergic airway disease as DNCB, a chemical not known to cause IgE-mediated allergic airway disease of the respiratory tract, induced increases in serum IgE. There is data that supports this possibility. One group showed that IgE levels are inversely correlated with LMW chemical-induced airway inflammation in a murine model of LMW chemical-induced asthma (Herrick et al., 2002). Another implication is that DNCB, unlike most non-sensitizers of the respiratory tract, has a unique capacity to elicit an IgE response despite its inability to induce airway inflammation. This possibility is supported by the finding that intranasal sensitization and challenge with another chemical not known to cause IgE-mediated allergic airway disease of the respiratory tract, OXA, failed to induce an increase in serum IgE. Again it is possible that OXA at higher, irritating concentrations in this model, may elicit a cell-mediated mononuclear cellular inflammation. The exact role of IgE antibodies in the present study is unclear. Further studies with other LMW chemical respiratory sensitizers and nonsensitizers need to be conducted in order to determine the relevance of enhanced circulating IgE levels in LMW chemical-induced allergic airway disease.
The present study illustrated that intranasal sensitization and challenge with TMA in an ethyl acetate and olive oil vehicle may not result in sufficient pulmonary deposition of the allergen resulting in the absence of an allergic response in the lung. Intranasal sensitization and challenge with TMA, but not DNCB or OXA, was effective in generating some of the hallmark histopathologic features of allergic airway disease in the nasal airways of mice. In addition, the TMA-induced allergic rhinitis was accompanied by local increases in Th2-specific cytokine mRNA within the nasal airway. The analysis of local cytokine mRNA levels in the nasal airway after intranasal exposure may prove useful in the identification of other chemical respiratory allergens.
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ACKNOWLEDGMENTS |
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NOTES |
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1 To whom correspondence should be addressed at Department of Pharmacology and Toxicology, Michigan State University, 315 National Food Safety and Toxicology Center, East Lansing, MI 48824. Fax: (517) 432-3218. E-mail: kamins11{at}msu.edu
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