Toxicology and Molecular Biology Branch, Health Effects Laboratory Division, National Institute for Occupational Safety and Health, Morgantown, West Virginia 26505
1 To whom correspondence should be addressed at Toxicology and Molecular Biology Branch, Health Effects Laboratory Division, National Institute for Occupational Safety and Health, 1095 Willowdale Road, Morgantown, WV 26505. Fax: (304) 285-6038. E-mail: mluster{at}cdc.gov.
Received July 8, 2004; accepted December 4, 2004
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ABSTRACT |
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Key Words: isocyanate-induced asthma; toluene diisocyanate; airway hyperreactivity; cytokines; occupational asthma.
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INTRODUCTION |
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As recently reviewed by Johnson et al. (2004), studies in murine models of occupational asthma using epicutaneous sensitization protocols have supported an immune etiology in diisocyanate-induced asthma. For example, Dearman et al. (1996)
demonstrated that topical application of TDI induced the production of specific IgE antibodies with both CD4+ and CD8+ T cells participating as response effectors. Scheerens et al. (1996)
, using epicutaneous sensitization and intranasal challenge, showed that TDI led to increased airway hyperreactivity (AHR) to carbachol, and the response could be adoptively transferred with lymphocytes from sensitized mice, suggesting an immunological etiology. Herrick et al. (2002)
, using a similar exposure design with hexamethylene diisocyanate (HDI), detected airway eosinophilia, mucous hypersecretion, and induction of both Th1 and Th2 cytokines. Subsequent studies from this group (Herrick and Bottomly, 2003
) showed that CD4+ T cells were critical for airway eosinophilia, while CD8+ T cells were the major effector cells in contact hypersensitivity. Previous studies in our laboratory demonstrated that low-level subchronic and, to a lesser extent, acute high-dose inhalation to TDI effectively sensitized mice, resulting in challenge-induced increases in AHR, airway eosinophilia, mucous hypersecretion, TDI-specific serum antibodies, and elevated pulmonary Th1 and Th2 cytokines (Matheson et al., 2005
). Furthermore, in extending observations by Scheerens et al. (1996)
, adoptive and passive transfer experiments indicated that both antibody and lymphocytes (T and B cells) participated in this response. The present studies were conducted to determine whether TDI-induced asthma, produced by low-level (20 ppb) inhalation exposure, is associated with a predominant CD4+ T-cell/Th2 cytokine response, as occurs with most common large molecular weight respiratory allergens.
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MATERIALS AND METHODS |
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Experimental design. Mice were exposed by inhalation to 20 ppb of TDI (Mondur TD80; 80:20 molar mixture of 2,4:2,6 isomers provided by Bayer, USA, Pittsburgh, PA) for 6 weeks, 5 days per week, 4 h per day in a 10-l inhalation chamber, with only the heads of the animals extended into the chamber. TDI vapors in the chamber were generated by passing dried air through an impinger that contained 3 ml TDI. A computer interfaced mass flow controller (Model GFC-37, 020 LPM, Aalborg Instruments, Orangeburg, NY) regulated the TDI concentration in the chamber while a similar mass flow controller (Model GGC-47, 0100 1/min, Aalborg Instruments) regulated the diluent air. Temperature (76°C) and relative humidity (
4050%) were monitored by a Type HP-233 transmitter (Vaisala, Helsinki, Finland) interfacing with the TDI and diluent air controllers in a National Instruments (Austin, TX) data acquisition/control system. The generation system produces TDI vapor, free of TDI aerosol. Real-time monitoring of the chamber atmosphere was performed using an AutostepTM continuous toxic gas analyzer (Bacharach, Inc, Pittsburgh, PA) with concentrations never varying more than 0.5 ppb. Challenge by inhalation (1 h, 20 ppb TDI) was performed following a rest period of 14 days during which there was no exposure to TDI. The 6-week exposure period is the time during which sensitization to TDI develops in the current model (Matheson et al., 2005
). Therefore mice that were exposed to TDI during this 6-week period followed by challenge are, henceforth, referred to as "sensitized/challenged" groups. Two controls groups were examined, including an air-sensitized/air-challenged and air-sensitized/TDI-challenged treatment group. As both control groups responded similarly, for convenience, only results from the air-sensitized/TDI-challenge control treatment are shown and are, henceforth, referred to as "controls." Administration of cytokine-specific neutralizing antibodies to the control mice did not influence any of the parameters tested (data not shown). In addition, CD4 KO, CD8 KO, and IL-4 KO mice exposed to air for 6 weeks followed by challenge with TDI were not different from concurrent wild-type mice for the parameters examined (data not shown). Thus, for convenience only wild-type control values are presented.
Tissue collection. Forty-eight hours after airway challenge, mice were sacrificed by CO2 asphyxia, and lungs were collected. Lungs were inflated with 1.0 ml of 10% neutral buffered formalin (NBF), and immersed in 10% NBF for 24 h. The tissues were embedded in paraffin, serially sectioned, and stained with hematoxylin and eosin for histopathological assessment. PAS staining was performed to detect goblet metaplasia, and Chromatrope 2R/Mayer's Hematoxylin staining for eosinophil identification. The histopathological grading system was performed blinded and expressed on a 05 scale for each animal, with 0 representing no changes, 1 equal to minimal change, 2 equal to slight/mild changes, 3 equal to moderate changes, 4 equal to moderate/severe changes, and 5 equal to severe changes. Additional groups of mice were utilized for bronchoalveolar lavage fluid (BALF) and blood collection 24 h after challenge. To obtain BALF, mice were anesthetized, exsanguinated, and intubated with a 20-gauge cannula positioned at the tracheal bifurcation. Each mouse lung was lavaged three times with 1.0 ml of sterile HBSS and the fluid pooled. Samples of BALF (104 cells in 0.1-ml volumes) from individual mice were used for cytospin preparations. The slides were fixed and stained with Diff-Quick (VWR, Pittsburgh, PA), and differential cell counts were obtained using light microscopic evaluation of 300 cells/slide. Total cell counts were performed with a hemocytometer. Nonlavaged lungs were collected 24 h after challenge, frozen in RNAlater (Qiagen, Valencia, CA) or liquid nitrogen and stored at 80°C for reverse transcription-polymerase chain reaction (RT-PCR) analysis. Tissues frozen in liquid nitrogen were incubated with RNAlaterICE (Ambion, Austin, TX) at 20°C for 24 h prior to RNA isolation.
Antibody detection. Total serum IgE levels were measured using a modified (Matheson et al., 2001) sandwich ELISA (Satoh et al., 1995
) employing rat monoclonal anti-mouse IgE (BD-PharMingen) as the capture antibody. Serial two-fold dilutions of test sera, starting at 1:5, were added and incubated with peroxidase-goat anti-mouse IgE (1:1000, Nordic Immunological Laboratories, Capistrano Beach, CA) and developed with ABTS substrate (2,2'-azino-bis{3-ethylbenzthiazoline-6-sulfonic acid}). TDI-specific IgG antibodies were detected by ELISA using a TDI-mouse serum albumin conjugate, using a previously described procedure (Satoh et al., 1995
), kindly provided by Dr. Meryl Karol (University of Pittsburgh PA), and modified in our lab (Matheson et al., 2001
).
Eosinophil peroxidase activity (EPO). EPO activity was measured in BALF supernatants of mice according to the method of Bell et al. (1996), with slight modifications. Briefly, 0.1 ml of peroxidase substrate solution consisting of o-phenylenediamine dihydrochloride (OPD), urea hydrogen peroxide, and phosphate-citrate buffer (Sigma Fast Tablets®, Sigma, St. Louis, MO) was added to 0.1 ml of the BAL supernatant. The mixture was incubated at 37°C for 30 min before stopping the reaction with 50 µl of 2 N hydrochloric acid. Optical densities were measured at 490 nm (OD490). Nonspecific reactions (always <10%) were corrected by treating duplicate sample sets with the EPO inhibitor, 2 mM 3-amino-1,2,4-triazole (Sigma). The results were expressed as OD490 corrected for background and volume of supernatant retrieved.
Airway hyperreactivity (AHR). Airway responsiveness was assessed 24 h following TDI challenge in response to increasing concentrations of methacholine, using a single chamber whole body plethysmograph (Buxco, Troy, NY). A spontaneously breathing mouse was placed into the main chamber of the plethysmograph, and pressure differences between the main chamber and a reference chamber were recorded. AHR was expressed as enhanced pause (Penh), which correlates with measurement of airway resistance, impedance, and intrapleural pressure, and is derived from the formula: Penh = ([Te Tr] / Tr) x Pef / Pif; where Te = expiration time, Tr = relaxation time, Pef = peak expiratory flow, and Pif = peak inspiratory flow (Schwarze et al., 1999). Mice were placed into the plethysmograph and exposed for 3 min to nebulized PBS followed by 5 min of data collection to establish baseline values. This was followed by increasing concentrations of nebulized methacholine (050 mg contained in 1.0 ml of PBS) for 3 min per dose using an AeroSonic ultrasonic nebulizer (DeVilbiss, Somerset, PA). Recordings were taken for 5 min after each nebulization. For convenience, only the 50 mg/ml methacholine concentration data are presented. The Penh values during each 5-min sequence were averaged and expressed as percent change from values obtained in control animals. Control animals for each cytokine deficiency were examined, and no major discrepancies in base line AHR were detected between experimental groups (data not shown).
BAL cell phenotypic characterization. Phenotypic characterization of BALF cell constituents of TDI-sensitized/challenged wild-type and control mice was conducted as previously described with only antibody modifications (Matheson et al., 2002), using two-color flow cytometric analysis with combinations of monoclonal antibodies from BD-Pharmingen including fluorescein isothiocyanate (FITC)-conjugated anti-CD3, phycoerythrin (PE)-conjugated anti-CD4, and PE-conjugated anti-CD8. Data were acquired on a FACS Calibur (BD-Pharmingen) with 10,000 events assessed.
Real-time RT-PCR. Tissues were homogenized, and total cellular RNA was extracted using the Qiagen RNeasy kit® (Qiagen, Valencia, CA) according to the manufacturer's instructions. One microgram of RNA was reverse-transcribed using random hexamers and 60 U of Superscript II (Life Technologies, Grand Island, NY). Real-time PCR primer/probe sets for murine 18S, IFN, IL-4, IL-5, and IL-13 were purchased from Applied Biosystems (Foster City, CA), and real-time PCR was performed with an iCycler (Bio-Rad, Hercules, CA) according to the manufacturer's instructions, with initial incubation at 50°C for 2 min, 95°C for 10 min, and then 60 cycles at 95°C for 15 sec and 60°C for 1 min. The fold difference in mRNA expression between treatment groups was determined using the relative quantification method utilizing real-time PCR efficiencies (Pfaffl, 2001
) and normalized to the housekeeping gene, 18S/rRNA, thus comparing CT changes between controls and experimental samples. Statistical analysis was performed comparing the CT values of the control group to the CT values of the sensitized/challenged treatment groups, n = 4. Prior to conducting statistical analyses, the fold change from the challenge-only control was calculated for each individual sample (including individual control samples to assess variability in this group).
Statistical analysis. All studies were conducted in duplicate or triplicate with representative data shown. For statistical analysis, standard one-way analysis of variance (ANOVA) followed by Student-Newman-Keuls (SNK) test was used for multiple group comparisons. Student's two-tailed unpaired t-test was used to determine the level of difference between two experimental groups, and p < 0.05 was considered a statistically significant difference.
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RESULTS |
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DISCUSSION |
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Allergic asthma, which is associated with IgE antibodies and eosinophilia, is considered a Th2 predominant response and can be adoptively transferred with Th2 cells in experimental animal models (Cohn et al., 1998; Li et al., 1999
). In this respect, animal studies have demonstrated both the requirement and interdependence for Th2 cytokines in many manifestations of allergic asthma (Foster et al., 1996
; Grunig et al., 1998
; Hamelmann et al., 1997
; Kumar et al., 2002
; Webb et al., 2000
; Wills-Karp et al., 1998
). Our studies also indicate that Th2 cytokines play a primary role in TDI-induced asthma. For example, IL-4 depletion, although only moderately inhibiting AHR, markedly attenuated total serum IgE levels, as well as TDI-specific antibody levels and histopathological changes in the lung, including airway remodeling. IL-4 promotes mucus secretion and goblet cell hyperplasia, in addition to the immunoglobulin switch to IgE (Gelfand, 1998
; Grunewald et al., 1998
; Pauwels et al., 1997
). IL-13 deficiency had moderate attenuating effects on most endpoints measured, except in the case of goblet metaplasia, which was markedly attenuated. Combined IL-4/IL-13 deficiency had a profound effect, as almost all endpoints were essentially abrogated. IL-4 and IL-13 have many overlapping functions as a result of sharing a common receptor subunit, IL-4Ra. IL-13 also has distinct functions and, like IL-4, is important in the development of AHR and mucus production (Hershey, 2003
). Some unique capabilities of IL-13 include its ability to suppress NF-
B, as well as to promote eosinophil recruitment through an IL-5 and eotaxin-dependent mechanism (Mattes et al., 2002
; Pope et al., 2001
) and its dominant role in airway remodeling (Elias et al., 2003
).
Our data also demonstrate dissociation between AHR and tissue manifestations of TDI-asthma, since IFN depletion diminished AHR to TDI, while only slightly affecting antibody and inflammatory responses. Consistent with these observations, IFN
expression in the lungs was observed following TDI sensitization. While Th1 cytokines are normally considered to negatively influence allergic responses, increasing data indicate that there is a cooperative interaction between Th1 and Th2 cytokines in the pathogenesis of asthma. For example, it was demonstrated that cooperation between Th1 and Th2 cells is necessary for a robust eosinophil inflammatory response and pulmonary recruitment of antigen-specific T cells (Randolph et al., 1999
). In an ovalbumin-mediated asthma model, significant increases in Th1 chemokines, such as IP-10, were observed, and overexpression of IP-10 augmented AHR, eosinophilia, CD8+ cell numbers, and IL-4 expression (Medoff et al., 2002
). Adoptive-transfer of ovalbumin-specific Th1 cells not only failed to reverse Th2-mediated airway inflammation, but also caused severe airway inflammation (Hansen et al., 1999
) and enhanced AHR (Takaoka et al., 2001
). Studies in humans have suggested that the number of IFN
-producing cells found in asthmatic lungs correlate with asthma severity, bronchial hyperresponsiveness, and blood eosinophilia (Krug et al., 1996
; Magnan et al., 2000
; van Rijt and Lambrecht, 2001
) and endogenous IFN
potentiates IL-13 induced lung inflammation (Ford et al., 2001
; Hofstra et al., 1998
).
In conclusion, these studies indicate that occupational asthma, induced by low-molecular-weight chemicals, represented in these studies by TDI, evokes similar immune mechanisms as allergic asthma caused by large-molecular-weight antigens. Activated CD4+ T cells play a predominant role in the pathogenesis of TDI-induced asthma. Furthermore, it would appear that Th2 cytokines are decisive in the initial phase of occupational asthma, in the priming and development of Th2 cells, and in the permeation of eosinophils into the airway lumen. However, as has been suggested previously for allergic asthma (Akdis et al., 1999; Hershey, 2003
; Jung et al., 1996
; Webb et al., 2000
), a cooperative interaction with CD8+ T cells and Th1 cytokines in the pathogenesis of asthma lesions clearly exists. This was particularly evident with IFN
and the development of AHR and the reduction of AHR, inflammation, and Th1/Th2 cytokine production in CD8 knockout mice.
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ACKNOWLEDGMENTS |
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