* Institute of Toxicology, Bayer AG, Building No. 514, 42096 Wuppertal, Germany;
Institute of Experimental Pathology, Medical School, 30625 Hanover, Germany
Received November 2, 1999; accepted March 12, 2000
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ABSTRACT |
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Key Words: respiratory sensitization; allergy; airway hypersensitivity; respiratory rate; airway eosinophils; confounding factors.
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INTRODUCTION |
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Despite the guinea pigs' sensitivity for the identification of the allergenic potential of proteins, in many instances sensitized guinea pigs did not fully display surrogate immediate onset type asthmatic responses from challenge with diphenylmethane-4,4'-diisocyanate (MDI), but did when challenged with the low molecular weight chemical trimellitic anhydride (TMA) (Pauluhn et al., 1999a). Though difficult to reconcile, one source of potential variability appears to be related to the particle size of MDI aerosol used for inhalation induction and elicitation of sensitization responses. To clarify as to whether particle size is an important variable in this bioassay, the impact of MDI aerosol particle size on the outcome on both the induction and elicitation of sensitization response was examined. Attempts were made to induce and challenge guinea pigs to two-size distributions of MDI aerosol. One was preferentially deposited in the lower airways in the absence of appreciable deposition in the nasopharyngeal airways (small aerosol: targeted MMAD
1.5 µm), whereas the large aerosol was targeted to be deposited preferentially in the extrathoracic, nasopharyngeal airways (large aerosol: targeted MMAD
5 µm). Though it can be assumed that the total deposition of the large aerosol exceeds that of the small one by a factor of approximately 2 (Martonen and Yang, 1994
), no attempt was made to exactly qualify the most likely regional deposition because of the polydisperse nature of the aerosol generated.
Guinea pigs sensitized by inhalation to either small or large aerosol were compared with both naive animals and those sensitized by an extrapulmonary (intradermal) route. To minimize the occurrence of potential confounding factors upon challenge due to pre-existing, induction-related airway inflammation, inhalation sensitization was made using a brief, high-level exposure of 135 mg/m3 polymeric MDI without any additional booster inhalation exposures.
Two endpoints were examined to characterize positive response. One focused on the identification of immediate-onset response by measurements of respiratory rate during challenge exposure; the other focused on the identification of an influx of eosinophils in bronchial airways and lung-associated lymph nodes. This allowed the identification of a late-phase inflammatory response. In this context, increasing evidence suggests that eosinophilic granulocytes play a critical role in the pathogenesis of asthma and of other hyperresponsive airway diseases and differentiates asthma from other inflammatory conditions of the airways (Barnes et al., 1991a,b
; Davis et al., 1984
; Frigas and Gleich, 1986
; Kay et al., 1991
; Lapa e Silva et al., 1993
, 1997
; Obata et al., 1992
). No attempts were made to demonstrate an associated late-phase bronchoconstriction by measurements of respiratory function because they were often described to be unsuccessful (Pauluhn and Eben, 1991
; Pauluhn, 1997
; Richards et al., 1992
).
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MATERIALS AND METHODS |
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Animals, diet, and housing conditions.
Female Dunkin-Hartley guinea pigs (Crl:[HA]BR) with an initial weight range of 300350 g were obtained from Charles River, Sulzfeld, Germany. Animals were acclimatized for approximately 1 week, randomized, and housed four per cage. Guinea pigs were allowed food and tap water ad libitum except during exposures. A 12-h on/off light cycle was maintained in the animal housing room. Temperature was 23 ± 2°C and relative humidity was in the range of 4070%. All experiments and procedures described were performed in compliance with GLP requirements (OECD, 1983), taking into account the EU animal welfare regulations (EC Directive 86/609/EC, 1986).
Sensitization to and challenge with MDI.
All inhalation exposures were nose-only, as described in the section on inhalation exposure techniques.
Intradermal sensitization.
One group of guinea pigs (n = 16) received a single id injection on days 0, 2, and 4 (injection volume 100 µl, 0.3% MDI [w/v]) MDI in dehydrated corn oil as vehicle. The relative locations on the flanks used for repeated injection were cranial, thoracic, and caudal. Control animals (n = 16) received vehicle alone under otherwise identical conditions. Prior to each injection, the MDI content of the solution was verified analytically.
Inhalation sensitization.
Two groups of guinea pigs, each of 16 animals, were sensitized with 135 mg/m3 of respirable MDI aerosol (actual gravimetric concentration) by a single 15-min nose-only inhalation exposure. The aerosol atmospheres generated in the first and second group differed in particle size. In the first group, guinea pigs were exposed to a small aerosol and in the second group to a large aerosol. The small and large aerosol were targeted at MMADs of 1.5 and
5 µm, respectively.
Challenge
MDI.
After a postexposure period of approximately 3 weeks (range: days 1923), guinea pigs were challenged with MDI aerosol using a ramped exposure regimen (target concentrations 15 and 45 mg/m3 air; each challenge duration was 15 min, subsequent exposure). For the first and second challenge periods, the mean actual concentrations (± standard deviation) obtained by filter analyses were 16.0 ± 2.2 and 49.4 ± 12.7 mg MDI/m3 air, respectively. Half of the animals in each group were challenged with a small aerosol; the other half of the animals were challenged with a large-size aerosol.
Exposure technique, aerosol generation, and characterization.
Atmospheres of MDI were generated under dynamic conditions using a digitally controlled Hamilton Microlab M pump and a modified Schlick-nozzle Type 970, form-S 3 (Schlick GmbH, Coburg, Germany). Temperature control of the nozzle was made by a water jacket connected to a digitally controlled thermostat. MDI was nebulized using conditioned (dry, oil-free) compressed air. The aerosol for induction (I) and challenge (C) exposures was generated as follows: (I) the liquid polymeric MDI was nebulized using compressed, conditioned (dry, oil-free) air. The small aerosol of MDI was generated by nebulization of 25 µl x min1 and a dispersion pressure of approximately 600 kPa. All parts of the nozzle coming into contact with MDI were maintained at approximately 40°C using a water jacket connected to a digitally controlled thermostat. The large aerosol was generated under similar conditions but using 150 kPa dispersion pressure and a nozzle temperature of 25°C. (C) The aerosol was generated as described above using the following modifications: the small aerosol of MDI was generated by nebulization of 10 µl x min1, a dispersion pressure of approximately 600 kPa, and a temperature of approximately 40°C. The large aerosol was generated under similar conditions but using a dispersion pressure of 100 kPa, a supply rate of 50 µl MDI x min1, and a nozzle temperature of 25°C. The total airflow to the nose-only inhalation chamber (internal volume of chamber 7.2 L) was 30 L x min1. The increase of temperature within the nozzle resulted in a marked decrease in viscosity and hence increased reproducibly the output of aerosol of polymeric MDI. The respective target concentration was attained by using an extraction/dilution cascade as depicted in Figure 1
. With respect to the generation of this aerosol as well as to this modular directed-flow nose-only inhalation chamber have been described elsewhere (Pauluhn, 1994b
; Pauluhn et al., 1999b
). The generation of the monomeric aerosol used in the pilot study has been published previously (Pauluhn et al., 1999a
). The exposure atmospheres were characterized using filter analyses (Sartorius glass fiber filters). Chamber air was sampled from the vicinity of the breathing zone of the guinea pigs. For particle size analyses, a low-pressure critical orifice AERAS stainless steel cascade impactor (HAUKE, 4810 Gmunden, Austria) was used. The mass median aerodynamic diameter (MMAD) and the geometric standard deviation (GSD) were calculated as described previously (Pauluhn, 1994b
).
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Necropsy and histopathology.
In all groups, animals were sacrificed 1 day after challenge, and the nasal cavities, lung, trachea, and lung-associated lymph nodes (LALN) were preserved for histopathological examinations. Following complete exsanguination, lungs were weighed and fixed by instillation. The fixation of tissue was in 4% neutral buffered formaldehyde. All tissues were routinely processed, dehydrated, and embedded in Paraplast-plus, and sections approximately 5 µm thick were cut from each tissue block. Tissue sections were stained with hematoxylin and eosin according to Lillie & Mayer (Lillie and Fullmer, 1976) hematoxylin-eosin-azure (Tan and Bethel, 1992
) to improve the identification of eosinophilic granulocytes. All examinations were made by light microscopy. For grading, the number of eosinophilic granulocytes in a rectangular unit area of 0.1722 mm2 (0.82 x 0.21 mm) was counted and graded as follows: grade 0, no eosinophils; grade 1 (slight), up to 30 eosinophils/unit area; grade 2 (moderate), 3180 eosinophils/unit area; grade 3 (severe), more than 81 eosinophils/unit area. Three distinct locations were examined: the perivascular, the submucosal, and the muscularis mucosae areas. These areas were examined and scored independently in the five lung lobes, i.e., lobus dexter accessorius, lobus sinister caudalis, lobus sinister cranialis, lobus dexter mediatus, and lobus dexter caudalis. The examination of influx of eosinophilic granulocytes at these topographical locations was made because they were also described for the occurrence of dendritic cell (DC) in the guinea pig airways (Lapa e Silva et al., 1993
; Lawrence et al., 1997
; Maarten et al., 1994
). Lawrence et al. (1997) have observed a large influx of eosinophils into the epithelium of the trachea and bronchi 18 h after antigen challenge as a characteristic late-phase inflammatory response in guinea pigs. Therefore, animals were sacrificed 1 day after the inhalation challenge exposure.
Enzyme-linked immunosorbent assay (ELISA).
For serological analyses, blood was drawn from guinea pigs by cardiac puncture approximately 3 weeks following inhalation exposure or initiation of id injection. Serum was prepared and stored at 20°C until analyzed. Plastic microtiter plates (Nunc Immunoplate type II, Nunc, Copenhagen, Denmark) were coated with 50 µl per well of 5 µg/ml MDI-guinea pig serum albumin conjugate (MDI-GPSA) in 0.5 M sodium carbonate/bicarbonate buffer (pH 9.6) by overnight incubation at 4°C. Various dilutions of guinea pig serum in phosphate-buffered saline containing 0.05% Tween 20 (PBS-Tween) were added in duplicate (100-µl aliquots) and plates were incubated for 30 min at 37 °C. Plates were washed three times in PBS-Tween and 100 µl of rabbit anti-guinea pig IgG1 antibody (Miles Scientific, Slough, UK), diluted 1:2500 in PBS-Tween, was added to each well. Plates were again incubated for 30 min at 37°C and washed prior to addition of a peroxidase-labeled goat anti-rabbit IgG antibody (Miles Scientific), diluted 1:5000 in PBS-Tween. Following a further 30-min incubation at 37°C, the plates were washed again and substrate (o-phenylenediamine and urea hydrogen peroxide) was added. Reactions were terminated after approximately 10 min by addition of 50 µl 0.5M citric acid per well. Absorbance was measured at 450 nm using an automatic reader (Multiskan, Flow Laboratories, Irvine, Ayrshire, UK). Criterion for positivity of individual serum samples was as follows: mean optical density for the reagent blank wells was calculated for each plate, as were the means of each duplicate serum sample. A reading was regarded as positive if it was higher than twice the reagent blank for the particular plate. The titer of each serum was the highest dilution of serum that gave a positive reading.
Preparation and characterization of conjugate for ELISA.
MDI-GPSA was prepared as described previously (Rattray et al, 1994). Briefly, approximately 200 mg GPSA was dissolved in 20 ml 0.05 M sodium borate buffer (pH 9.4). Approximately 60 mg monomeric MDI was added and the solution was stirred at 4°C for 30 min. Glass vessels were used throughout, as MDI reacts with plastic. The solution was dialyzed successively against phosphate-buffered saline (pH 7.2) and distilled water for a period of approximately 48 h at 4°C. The lyophilized conjugate was stored at 20°C until use. The degree of substitution of the MDI conjugate was assessed using a method based upon the determination of free amino groups by reaction with 2,4,6-trinitrobenzene sulphonic acid (TNBS), as described previously (Rattray et al., 1994
). Briefly, approximately 1 mg MDI-GPSA conjugate and 1 mg GPSA were each dissolved in approximately 1 ml 0.1 M sodium borate buffer (pH 9.3); 25 µl of a stock solution of 1.2 M TNBS in 0.1 M sodium borate buffer (pH 9.3) was added to each sample, and the samples were incubated for approximately 20 min at room temperature. The optical density at 420 nm (OD) was measured using a Philips spectrophotometer (PU 8880uv/vis). GPSA had approximately 30 readily available hapten-binding sites per molecule. The degree of substitution was approximately 15:1 moles hapten:moles protein.
Pilot study for the evaluation of anti-MDI antibody formation.
In a separate pilot study, groups of guinea pigs (approximately 810 animals/group) were sensitized with respirable aerosol of polymeric MDI by single high-level inhalation exposure of 15-min duration. Actual concentrations of MDI aerosol ranged from 5 to 835 mg/m3 air, i.e., from virtually nonirritant to strongly irritant. In this pilot study, the MMAD of polymeric MDI was approximately 1.6 µm (range 1.41.9), and the GSD was approximately 1.5. In one additional group ( 135 mg/m3), 16 guinea pigs were exposed to monomeric MDI aerosol (MMAD
1.1 µm, GSD 1.4). Exposure concentrations up to a concentration of
135 mg/m3 were tolerated without specific clinical signs, whereas guinea pigs exposed to 835 mg/m3 experienced a labored breathing pattern and respiratory distress up to the fourth postexposure day. One out of eight guinea pigs succumbed on the first postexposure day. Following id induction, inflammatory skin reactions were observed at the injection sites.
Statistical evaluation of data.
Quantitative histopathological data were analyzed by one-way analysis of variance and Tukey-Kramer post hoc test. Data addressing the respiratory response was analyzed by a Mann-Whitney Rank Sum Test (SPSS). Quantal histopathological findings (incidence of airway eosinophilia) were compared with the concurrent control using the pairwise Fisher test with R x C chi-square test (Gad and Weil, 1982).
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RESULTS |
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Analysis of Respiratory Rate
For elicitation of specific respiratory hypersensitivity, guinea pigs of all groups were exposed subsequently to gravimetrically determined concentrations of 16 and 49 mg MDI/m3 air, each concentration for 15 min. The first eight guinea pigs of each group were challenged with the small, the second half with the large aerosol. The results of measurements of respiratory rate made upon challenge with MDI aerosol are summarized in Figure 4.
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Histopathology
Findings in the nasal cavities and trachea were unremarkable (Table 1). As no appreciable or systematic differences of influx of eosinophilic granulocytes in airways of the lobus dexter accessorius, lobus sinister caudalis, lobus sinister cranialis, lobus dexter mediatus, and lobus dexter caudalis of the lung were observed, all five locations were merged into one. As illustrated in Figure 5
, a characteristic influx of eosinophils in the bronchial submucosa and muscularis mucosae existed in MDI-sensitized guinea pigs. There was an apparent shift towards a more pronounced influx in groups induced with the large MDI aerosol.
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Thus, in relation to animals sensitized by inhalation induction to a large size aerosol, it appears that a greater influx of eosinophilic granulocytes was produced than a similar induction regimen using a small aerosol (Fig. 6, Table 2
). Overall, for the elicitation of respiratory hypersensitivity, as characterized by an increased influx of eosinophilic granulocytes into the mucosa and submucosa of the airways, a more clear tendency of pronounced response was observed when challenge exposure was with the small rather than the large size aerosol. In either case, however, this response was statistically significantly different from the response seen in the control.
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DISCUSSION |
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Although of different magnitude, in all MDI-sensitized groups the degree of influx of eosinophils in the muscularis mucosae and submucosa was clearly different from similarly challenged, naive controls. The most pronounced difference in response to the small-size and large(r)-size challenge aerosol was observed in the LALN of id-sensitized guinea pigs. When challenge was with the small MDI aerosol, most animals displayed an influx of eosinophils. On the other hand, when challenge was with the large aerosol of MDI, no response was observed. Intradermal induction appears to favor an influx of eosinophils in LALN, whereas induction by the inhalation route (large aerosol) promotes an influx at the perivascular location of the bronchi.
The overall scoring of results obtained by measurement of respiratory rate during or following MDI challenge and the examination of influx of eosinophilic granulocytes into the bronchial airways and LALN are summarized in Table 2. From this comparison it appears that guinea pigs, when challenged with the small aerosol, tended to display a greater respiratory response as well as a more consistent influx of eosinophils within the muscularis mucosae, perivascular area, and LALN than animals challenged with the large aerosol. However, responses at the submucosal location were indistinguishable. Thus, the id route of induction and the single high-level inhalation induction regimen using the large aerosol appeared to demonstrate a greater sensitization efficiency than the single high-level inhalation induction regimen using the small aerosol. Though speculative, this may be related to the greater deposition efficiency of large particles and, accordingly, a greater total deposited dose within the respiratory tract than a preferential deposition at any specific region within the respiratory tract. Moreover, the influx of eosinophils at the perivascular location and LALN appears to depend significantly on the route of induction. Overall, it may be concluded that challenge exposures with this type of irritant aerosol appear to evoke more consistent effects when the MMAD is in the range of
2 rather than
5 µm. For inhalation induction exposures, exposure intensity (and accordingly, exposure dose) may be more important than particle size.
Likewise, combined experimental evidence suggests that in the guinea pig respiratory allergy model, MDI appears to evoke a late-phase rather than any vigorous immediate-onset response as is observed under similar conditions with TMA (Pauluhn et al., 1999a). This interpretation of findings appears to be supported by other authors using this animal model (Karol and Thorne, 1988
). Using an aerosol of MDI, only late-onset responses were obtained. In contrast, inhalation challenge with an MDI-protein conjugate yielded both type of responses.
In summary, with respect to the inhalation challenge to irritant concentrations, guinea pigs appear to respond differently when challenged with small and large aerosols of the pulmonary irritant MDI. It appears that changes in breathing patterns are more likely to be misconstrued as a positive change of breathing pattern when challenge is made with a large rather than small aerosol, ostensibly due to upper respiratory tract irritation. Especially for agents likely to evoke a late-onset response, the analysis of influx of eosinophils into bronchial airways is considered to be an important adjunct to respiratory function measurements, which commonly focus on respiratory responses occurring during or shortly after challenge exposures. Furthermore, findings appear to demonstrate that particle size is an important variable in inhalation sensitization studies with irritant aerosols, which potentially contributes to interlaboratory variability. However, these data suggest that for the induction of respiratory allergy, the magnitude of the exposure used for induction is apparently more important than particle size, whereas for elicitation of sensitization response more consistent effects were obtained when challenge exposures were made with a small-size aerosol (MMAD 2).
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ACKNOWLEDGMENTS |
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NOTES |
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