* Toxicology and Molecular Biology Branch and Pathology and Physiology Research 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, 1095 Willowdale Road, Morgantown, WV 26505. Fax: (304) 285-6038. E-mail: mluster{at}cdc.gov.
Received June 3, 2004; accepted November 2, 2004
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ABSTRACT |
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Key Words: toluene diisocyanate; isocyanate-induced asthma; lymphocytes.
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INTRODUCTION |
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As with high-molecular-weight molecules, such as house dust mite allergens and detergent enzymes, immunologic mechanisms are considered the most likely cause, although inflammatory, pharmacologic, and neurogenic mechanisms have been implicated (Mapp et al., 1994; Raulf-Heimsoth and Baur, 1998
; Scheerens et al., 1996
). Clinical features of isocyanate-induced asthma can include the onset of the asthmatic symptoms within 1 h following challenge, persistent airway hyperresponsiveness (AHR) to chemical-specific and nonspecific (e.g., methacholine) challenge, and airway inflammation, involving the presence of activated T cells, eosinophils, neutrophils, and mast cells. However, specific IgE antibodies are detected in only 530% of individuals with isocyanate-induced asthma (Tee et al., 1998
), and a similar percentage of exposed workers have detectable IgG antibodies but no asthmatic symptoms, suggesting that IgG antibodies may represent an indicator of exposure rather than disease (Bernstein et al., 1997
). Furthermore, atopy is not considered a risk factor for diisocyanate asthma (Tee et al., 1998
). A lack of association with IgE-mediated immunity, however, does not exclude immunological mechanisms, as clinical evidence suggests involvement of CD8+ lymphocytes in occupational asthma induced by exposure to isocyanates (Finotto et al., 1991
; Maestrelli et al., 1994
).
The present studies were conducted to determine the role of the immune system in the asthmatic phenotype induced by TDI and to determine the influence of exposure dose on disease phenotype. To accomplish this goal, we established and characterized a mouse model for TDI-induced asthma following exposure conditions relevant to the workplace (i.e., via inhalation route). Mice were exposed to aerosol-free TDI vapor in a nose-only chamber using low-level (20 ppb) subchronic (6 weeks) inhalation or high-dose (500 ppb) acute inhalation, as might occur following an accidental spill. The 20-ppb dose of TDI was selected since this is the Occupational Safety and Health Administration's (OSHA) current 15-minute Permissible Exposure Limit, expressed as a Ceiling Limit (PEL-C). Workplace concentrations in excess of 20 ppb TDI are still being reported, although less frequently in recent years (Ott et al., 2003).
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MATERIALS AND METHODS |
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Exposure. TDI (80:20 molar mixture of 2,4:2,6 isomers provided by Bayer, USA, Pittsburgh, PA) vapors were generated by passing dried air through an impinger that contained 3 ml TDI. A computer-interfaced mass flow controller (Aalborg Instruments, Orangeburg, NY, model GFC-37, 020 LPM) regulated the TDI concentration in the chamber, while a similar mass flow controller (model GGC-47, 0100 LPM) regulated the diluent air. Temperature and relative humidity were monitored by a Vaisala transmitter (Vaisala Inc., Woburn MA, type HP-233) 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 TDI concentrations never varying more than 10% in the study. Mice were exposed to TDI by inhalation either of 20 ppb of TDI for 6 weeks, 5 days per week, 4 h per day (subchronic exposure), or of 500 ppb TDI for 2 h (acute exposure), in a 10 L inhalation chamber with only the heads of the animals extended into the chamber. Challenge (1 h, 20 ppb TDI) was performed on all groups 14 days following the last day of subchronic or acute exposure. The 6-week exposure period is the time during which sensitization to TDI develops in the current models. Therefore, mice that were exposed to TDI during this 6-week period followed by challenged are, henceforth, referred to as "sensitized/challenged" groups. Three control groups were examined, including an air sensitized/air challenged, TDI sensitized/air challenged, and air sensitized/TDI challenged treatment group. As all control groups responded similarly, for convenience, only results from the air sensitized/TDI challenged control treatment are shown and are, henceforth, referred to as "controls" except in AHR studies, where values for all groups are reported.
Tissue collection. Groups of mice from each treatment group were sacrificed 48 h after airway challenge, using a CO2 atmosphere, and lungs and nares were collected. Lungs were inflated with 10% neutral buffered formalin (NBF), and tissues were immersed in 10% NBF for 24 h, after which the nares were decalcified. The tissues were embedded in paraffin, serially sectioned, and stained with hematoxylin and eosin for histopathological assessment. PAS staining was performed to identify 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 change, 1 = minimal, 2 = slight/mild, 3 = moderate,4 = moderate/severe, and 5 = severe. Additional groups of mice were sacrificed 24 h after challenge and utilized for bronchoalveolar lavage fluid (BALF) and blood collection. To obtain BALF, mice were anesthetized with 50 mg/kg of pentobarbital, 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 pooled. BALF recovery was 80 ± 5% for all animals. The BALF samples were centrifuged, and the supernatant frozen at 80°C until enzyme analysis. The cells were resuspended at 105 cells/ml of HBSS, and 0.1 ml was 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. In replicate experiments, lungs were collected 24 h following challenge, and tissues were frozen in RNAlater®(Qiagen, Valencia, CA) 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.
Transfer experiments. Adoptive and passive transfer experiments were conducted to assess the role of specific immunity in the asthma response. For adoptive transfer experiments, single cell suspensions were prepared from groups of mice exposed to TDI for 6 weeks or air sham controls by gently pressing pooled lymph nodes (mediastinal and auricular) and spleens through a stainless steel screen. The cell suspensions were washed with HBSS (Gibco, Grand Island, New York), the cell number adjusted to 2 x 107 cells/ml, and aliquots layered onto Lympholyte-M (Accurate Chemical, Westbury, NY). After centrifugation at 2,500 rpm, the lymphocyte interface was collected and washed, and 5.0 x 107 cells in 0.5 ml volumes were injected intravenously into naive recipients. B or T cell depletion was conducted by incubating isolated lymphoid cells with either panT or panB Dynabeads® (Dynal Biotech Inc., Lake Success, NY) at a 7:1 cell:bead ratio, according to the manufacturer's instructions. The respective T and B cell populations were >98% pure, as assessed by FACS analysis on a FACS Calibur® (BD Biosciences, Palo Alto, CA) utilizing anti-CD3 and anti-B220 FITC conjugated monoclonal antibodies (PharMingen, San Diego, CA). The resulting T and B lymphocyte populations were injected intravenously into naive recipients at a concentration of 2.9 x 107 cells and 2.5 x 107 cells, respectively, in 0.5-ml volumes. To measure TDI-specific serum activity, naive mice received an intradermal injection of 30 µl heat-inactivated (56°C, 4 h) or nonheated pooled serum into the dorsum of the right ear from either TDI sensitized/challenged mice or control mice. Animals were challenged 24 h later with 1% TDI (in acetone:olive oil, 4:1) on the dorsum of the same ear, and the change in ear thickness was compared to the thickness prechallenge. Additional groups of mice received an intravenous injection of 200 µl of either heated or unheated pooled sera from TDI sensitized/challenged or control mice.
Twenty-four hours after intravenous lymphocyte or serum transfer, mice were challenged either by inhalation with 20 ppb TDI for 1 h or by a single application of 25 µl of 1% TDI (in acetone:olive oil, 4:1) onto the dorsum of the right ear, as previously described (Ebino et al., 1999). Respiratory responses including pathology (as outlined above) and airway responsiveness to methacholine (see below) were determined 48 and 24 h following challenge, respectively. The ear challenge response was determined by measuring the change in ear thickness from baseline prechallenge ear thickness 24 h following TDI application. Cell proliferation in the draining lymph node was determined in an additional group of recipient mice using a modification of the local lymph node assay, as originally described by Dearman and Kimber (2000)
. Twenty-four hours after challenge, the mice were injected intravenously with 200 µl of 3H-thymidine (specific activity 0.1 µCi/ml; Amersham, Piscataway, NJ), and incorporation of 3H-thymidine into DNA in the draining auricular lymph nodes was measured.
Antibody detection. Total serum IgE was measured using a sandwich enzyme-linked immunosorbent assay (ELISA) as previously described (Satoh et al., 1995). Briefly, plates were coated with 5 µg/ml of rat monoclonal anti-mouse IgE (PharMingen). Serial two-fold dilutions of test sera, starting at a 1:5 dilution, 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)]. Total serum IgE concentrations were derived from a standard curve obtained using murine monoclonal anti-DNP IgE (Sigma, St. Louis, MO).
TDI-specific antibodies were detected by ELISA using a TDI-mouse serum albumin conjugate, kindly provided by Dr. Meryl Karol (University of Pittsburgh, Pittsburgh, PA), as previously described (Satoh et al., 1995). Serial two-fold dilutions of test sera, starting at a 1:5 dilution, were added to individual wells and incubated with peroxidase-conjugated, goat anti-mouse antibodies against either total IgG (1:400, Sigma, St. Louis, MO), IgG1, or IgG2a (both at 1:400, The Binding Site, Birmingham, UK) and developed with ABTS substrate. Antibody titers were determined by plotting the serial dilution curve for each sample individually versus the OD for each dilution of that sample. A cutoff OD of 0.2 (average OD of challenge only mouse serum was 0.06 ± 0.005) was used to determine the titer.
Eosinophil peroxidase activity (EPO). Measurement of EPO activity was performed on BALF supernatants 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 BALF supernatant. The mixture was incubated at 37°C for 30 min before stopping the reaction with 50 M of 2 N hydrochloric acid. Optical densities were measured at 490 nm (OD490). Nonspecific activity was determined by treating duplicate sample sets with the EPO inhibitor, 3-amino-1,2,4-triazole (2 mM, Sigma), and was always less than 10% of the nontreated samples. Results are expressed as OD490 corrected for background and volume of BALF supernatant retrieved (BALF recovery was 80 ± 5%).
Airway hyperresponsiveness (AHR). AHR to methacholine challenge was assessed, 24 h following TDI challenge, 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. Only data from the 50 mg/ml methacholine exposure are presented, as similar but less robust changes were observed between treatment and control groups at the 10 and 25 mg/ml methacholine concentrations (data not shown). The PenH values during each 5-min sequence were averaged and expressed as percentage increase over baseline values following PBS exposure for each methacholine concentration.
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 TNF
were purchased as predeveloped kits from Applied Biosystems (Foster City, CA). Real-time PCR was performed using Taqman Universal Master mix with Amperase in an iCycler (Bio-Rad, Hercules, CA) for 1 cycle at 50°C for 2 min (degrade carry over using Amperase), and 95°C for 10 min, followed by 60 cycles at 95°C for 15 sec and 60°C for 1 min. The differences in mRNA expression between control and treatment groups were determined by the relative quantification method developed by Pfaffl (2001)
utilizing the threshold cycle (CT) method and real-time PCR efficiencies of the target gene normalized to the housekeeping gene 18S/rRNA.
Statistical analysis. All studies were replicated with representative data shown. For statistical analysis, standard one-way analysis of variance (ANOVA) followed by the Student-Newman-Keuls 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. For the analyses of RT-PCR data, the fold change from the mean of the control group was calculated for each individual sample (including individual control samples to assess variability in this group centered around one) prior to ANOVA and SNK.
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RESULTS |
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DISCUSSION |
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These data also lend mechanistic understanding to the often-controversial finding in humans, where both IgE and non-IgE etiologies have been suggested. For example, previous clinical studies have shown that specific antibodies do not correlate well with disease activity, although TDI-specific IgG antibodies are often associated with exposure (Bernstein et al., 1997; Lushniak et al., 1998
), while in guinea pigs, antibody titers correspond only to exposure concentrations (Karol, 1983
). Mice exposed to TDI by inhalation for 6 weeks (20 ppb, 4 h/day, 5 days/week) demonstrated a marked allergic response evidenced by increases in airway inflammation, eosinophilia, goblet cell metaplasia, epithelial cell alterations, airway hyperreponsiveness (AHR), TH1/TH2 cytokine expression in the lung, elevated levels of serum total IgE, and TDI-specific IgG antibodies, as well as the ability to transfer many of these responses to naïve mice with lymphocytes and serum from mice subchronically exposed to TDI. Increased AHR as well as TDI-specific IgG antibodies and asthma-associated lung pathology were also observed in mice following acute high-dose (500 ppb) exposure. Although AHR could be transferred in animals that received acute TDI exposure, mice exposed using the acute exposure design failed to demonstrate elevated serum IgE levels, lung eosinophilia, or elevated TH1/TH2 lung cytokine expression. Thus, unlike that which occurred following subchronic TDI exposure, the pathologies observed following acute exposure lack definitive evidence that TH2 or IgE responses are involved. In this respect, the immunopathogenesis of allergic asthma is usually associated with a TH2 phenotype and can be adoptively transferred with TH2 cells (Cohn et al., 1998
; Li et al., 1999
; Scheerens et al., 1996
). The TH2 cytokines, IL-4 and IL-5 are associated with isotype switching to IgE and eosinophilic responses, both of which are hallmarks of allergic asthma. Although adoptive transfer experiments using cells collected from acute TDI exposed mice resulted in increased AHR in recipients, it has recently been suggested that unrestrained plethysmography, while a sensitive indicator for altered lung function, may not always be a reliable measure of airway responsiveness (Adler et al., 2004
) and, without other supporting clinical data, does not indicate that the pathology is of immunological origin. A number of non-IgE mechanisms can be postulated to be responsible for the pulmonary response following acute TDI exposure. For example, the response may involve specific T-cell immunity or IgG1 antibodies. Alternatively, nonspecific immune mechanisms may be evoked, such as the so-called "by-stander" response (Curtsinger et al., 1999
). This mechanism would imply that inflammation, induced by TDI acting as an irritant, activates macrophages and neutrophils and the release of mediators such as TNF
and IL-1ß, which stimulates T-cell responses independently of MHC-T cell receptor interactions.
The importance of exposure duration and dose in the pathogenic mechanisms that lead to TDI-induced asthma have been addressed previously in animal studies (Scheerens et al., 1999). In these investigations, dermal sensitization for 6 weeks on days 0, 7, 14, 21, 28, and 35, followed by intranasal challenge, resulted in significant respiratory involvement that was not evident following a 2-day sensitization period. Our results, at least using acute exposure, are also consistent with clinical studies which have suggested that isocyanate-induced airway reactivity can result from upper airway irritation without evoking TDI-specific immune mediators (Bernstein, 1982
; Leroyer et al., 1998
; Luo et al., 1990
).
To help clarify the events evoked in TDI-asthma, adoptive and passive transfer experiments were performed. We observed that transfer of lymphocytes from either the acute or subchronic TDI exposed mice to naïve recipients allowed for increased AHR in response to methacholine. Additional adoptive transfer experiments indicated that both T cells and B cells contributed to TDI-asthma following subchronic TDI exposure, as evidenced by both increased AHR and TDI-specific dermal responses in recipient mice of both lymphocyte populations following TDI challenge. These observations are consistent with those of Scheerens et al. (1996), in which TDI asthma was adoptively transferred using lymphocytes from mice epicutaneously sensitized to TDI. A role for antibody was also suggested to be involved in the response following subchronic TDI exposure, as naïve mice administered serum from subchronically exposed mice responded to specific TDI challenge. Reaginic antibodies, particular those of the IgE class, may be involved, since serum was rendered ineffective by heating and FcErIg transgenic mice, which lack both IgE and IgG Fc receptors, failed to become sensitized to TDI. It has been suggested that the lack of a strong association between the presence of specific IgE antibodies and human disease in isocyanate-induced asthma may be due to phenotyping methods (Bernstein and Jolly, 1999
), as the usage of polymeric isocyanate conjugates, rather than monomeric isocyanate-human serum albumin test antigens, provides greater assay sensitivity (Aul et al., 1999
; Park et al., 2001
).
Our data are also consistent with the idea that subchronic TDI sensitization involves a mixed TH1/TH2 response. This was evidenced by increased expression of the TH2 cyokines, IL-4 and IL-5, as well as the TH1 cytokine, IFN, expression in lungs of mice exposed to TDI using the subchronic paradigm. While allergic asthma is usually considered a TH2-mediated disease, a large body of evidence suggests that TH1 cells participate in the response. For example, cooperation between TH1 and TH2 cells is necessary for a robust eosinophil response, as well as TH2 cell recruitment into the lung in ovalbumin-specific cell transfer experiments (Randolph et al., 1999
). In an ovalbumin murine asthma model, significant increases in the TH1 chemokines, such as IP-10, have been observed after challenge, and overexpression of IP-10 augmented AHR, eosinophilia, CD8+ cell numbers, and IL-4 expression while airway hyperresponsiveness in IP-10 knockout mice was ablated (Medoff et al., 2002
). Studies in humans have found both CD4+ and IFN
-secreting CD8+ T cells are present in the airways of asthmatics (Krug et al., 1996
; Magnan et al., 2000
; van Rijt and Lambrecht, 2001
), and that the number of IFN
producing cells relates to asthma severity, bronchial hyperresponsiveness, and blood eosinophilia (Magnan et al., 2000
). In this respect, endogenous IFN
may be necessary for optimal IgE production (Hofstra et al., 1998
) and potentiation of IL-13-induced lung inflammation (Ford et al., 2001
). Patients with occupational asthma also show both TH1 and TH2 cytokines following in vitro lymphocyte stimulation (Del Prete et al., 1993
; Lee et al., 1998
; Lummus et al., 1998
; Maestrelli et al., 1997
; Sumi et al., 2003
). Furthermore, TH2 cells comprise only a minor portion of the T-cell population in the airways of TDI-allergic asthmatics, as most cells present a TH1 phenotype (Bernstein and Jolly, 1999
). In fact, the majority of T-cell clones derived from bronchial mucosa of patients with isocyanate-induced asthma present a CD8+ phenotype capable of secreting IL-5 (Maestrelli et al., 1994
) and IFN
(Wisnewski et al., 2003
).
In conclusion, a mouse model is described that demonstrates low-level subchronic TDI inhalation induces pathology, consistent with allergic asthma, manifested by airway inflammation, lung eosinophilia, increased AHR, asthma associated histopathology, Th cytokine expression, elevated serum IgE, and TDI-specific antibodies. Asthmatic symptoms also occur following high-dose, acute exposure, but the response is less robust, failing to demonstrate eosinophilia, elevated serum IgE levels, or Th cytokines. Evidence is also presented that, like allergic asthma, TDI asthma following subchronic exposure, while associated with a TH2 response involving IgE antibodies, also involves TH1 responses. Establishing the relative contribution of TH1 and TH2 mediators is addressed in a subsequent manuscript.
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ACKNOWLEDGMENTS |
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