Introduction

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It has become well established that allergic asthma is characterized by chronic airway inflammation associated with goblet cell hyperplasia and mucus plugging of airways, subepithelial fibrosis, focal desquamation of bronchial epithelium, and airway smooth muscle cell hypertrophy (1). Inflammatory and immune cells are thought to contribute to this process by producing a variety of inflammatory mediators, toxic oxygen radicals, and cytokines (2, 3). Histologically, eosinophils often predominate, and a large body of evidence points to activated eosinophils as key effector cells in mediating tissue damage (4). The role of other inflammatory cells, such as neutrophils, is less clear (5). Although increased numbers of neutrophils in lung biopsies of patients with asthma have not generally been observed, recruitment of neutrophils to the lungs on allergen challenge has been demonstrated by many investigators. In addition, massive infiltration of the lungs by neutrophils has been found in cases of sudden-onset fatal asthma (6).

Animal models have been valuable for investigation of the underlying pathology of allergic pulmonary diseases. Rat models are becoming more useful as many immunological reagents including monoclonal antibodies to cell adhesion molecules and cytokines have become available. The brown Norway (BN) rat model mimics human allergic asthma in several aspects. This strain exhibits a helper T cell type 2 (Th2)-driven response to allergic sensitization (7) with high levels of allergen-specific IgE (8, 9). Following aeroallergen challenge of sensitized animals, early- and late-phase bronchoconstrictions occur (10), associated with pulmonary inflammation and bronchial hyperresponsiveness to methacholine (11). However, airway responses in the BN model are relatively weak and somewhat inconsistent (12). Fischer rats have been reported to have hyperreactive airways compared to other strains (13), but it has not been described whether they develop airway inflammation on allergic sensitization and allergen challenge.

To study the inflammatory component during disease development, methods are needed to quantitate inflammatory cells in the lung tissue. Techniques commonly used to quantitate inflammatory cells in the lungs include: (1) histology of perfused and fixed lungs, (2) ex vivo radiolabeling of leukocytes and quantitation of their accumulation in the lung by -counting, and (3) enzymatic dispersion by tissue mincing and digestion with collagenase followed by cell counting. However, histology and enzymatic dispersion techniques are time and labor intensive and neutrophils may not be easily identified in tissue sections by standard histologic techniques. Furthermore, heterogeneous involvement of lung tissue might complicate histologic analysis and nonspecific cell loss, especially of adherent cells such as granulocytes, may be difficult to avoid with dispersion techniques. Studies with radiolabeled eosinophils have been hampered by difficulties in purifying rat or guinea pig blood eosinophils. Exudate eosinophils have been used for migration studies in rat (14) and guinea pig (15), but they may be primed or activated and the expression of adhesion molecules may differ from blood eosinophils (16). Furthermore, in vitro handling of cells may also cause cell activation (17, 18).

To overcome these limitations, we have developed a technique for quantitating lung tissue eosinophils and neutrophils by specific assays for eosinophil peroxidase (EPO) and the neutrophil myeloperoxidase (MPO) in lung extracts (19). Here, we characterize the time course of the inflammatory response after allergen challenge of sensitized BN rats, using specific peroxidase enzyme assays in conjunction with bronchoalveolar lavage (BAL) and histology.

Functional activity of the granulocytes, either in the airways directly surrounding tissues or within the parenchymal septa, may to some extent be modulated by degranulation. This may, under certain circumstances, become of considerable pathophysiologic importance and influence quantitation of the cells. Thus, we considered it also of importance to assess the status of eosinophil and neutrophil cell integrity.

	Materials and Methods

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Animals

Male, inbred brown Norway (BN/SSN), Lewis, and Fischer 344 rats, weighing 125-149 g, were purchased from Harlan-Sprague Dawley, Inc., (Indianapolis, IN). The animal experiments were performed in accordance with protocols approved by our University Committee on Laboratory Animals.

Sensitization and Allergen Challenge of BN Rats

Ovalbumin (OA, grade V; Sigma Chemical Co., St. Louis, MO) was prepared at 2 mg/ml in 0.9% sterile, pyrogen-free NaCl and precipitated at a 1:1 ratio with Al(OH)₃ (45 mg/ml, Imject^TM Alum; Pierce, Rockford, IL), following the instructions of the manufacturer. Rats were immunized with 1 mg of OA (1 ml OA-Al[OH]₃ suspension) given subcutaneously at two sites on the back of the neck and 10¹⁰ heat-killed Bordetella pertussis bacilli (gift from S. Halperin, Halifax, Nova Scotia, Canada) in 0.5 ml of saline given intraperitoneally as an adjuvant, following the sensitization procedure of Renzi and coworkers (20). Sham immunizations were done without OA but in the same manner with injection of B. pertussis intraperitoneally and saline-Al(OH)₃ subcutaneously. Fourteen days later, the rats were placed in a Plexiglas chamber (21 liters) and challenged for 1 h with an aerosol of 0.5% OA in saline, delivered at 3 liters/min by an ultrasonic nebulizer (Monaghan 670; Monaghan Co., Littleton, CO) at setting 7. 

Dissection and Lung Perfusion

Rats were investigated at various time points (3 h, 14 h, 24 h, 48 h, 72 h, 6 d, and 13 d) after allergen challenge. To prevent laryngospasms, which can lead to lung hemorrhages, the rats were premedicated intraperitoneally with 0.2 mg/kg atropine sulfate 5 min before being anesthetized by intraperitoneal administration of ketamine (50 mg/kg) and xylazine (10 mg/kg, Rompun; Chemagro, Ltd., Etobicoke, Ontario, Canada). The abdominal cavity was opened, a 25-gauge butterfly needle was inserted into the inferior vena cava, and 100 U of heparin in 1 ml of saline was injected. The abdominal aorta was severed and at the same time, Tyrode's solution (pH 7.4, 37°C) was infused into the inferior vena cava. After the administration of 25 ml of Tyrode's solution, the chest was opened and another 25 ml of Tyrode's solution was infused via the vena cava above the diaphragm, followed by 5 ml of phosphate-buffered saline (PBS, pH 7.4) containing 0.1% EDTA, followed by 5 ml more through the pulmonary artery. This protocol consistently cleared the lung vasculature of blood cells.

Bronchoalveolar Lavage and BAL Cell Determination

The upper trachea was cannulated and the lungs were lavaged four times (7 ml/lavage) with cold (4°C), Ca²⁺- and Mg²⁺-free PBS, 0.1% EDTA. Cells in the bronchoalveolar lavage fluid (BALF) were sedimented by centrifugation (10 min at 200 × g, 4°C) and resuspended in PBS. Total leukocytes were determined by hemacytometer counting using crystal violet stain, and eosinophils were counted using 0.05% phloxine B in 50% propylene glycol (all from Sigma Chemical Co.) in water (21). Cytocentrifuge preparations of the BALF leukocytes were stained with Diff-Quik^TM (Baxter Healthcare Corp., Miami, FL) and at least 200 cells were differentiated according to standard morphologic criteria. Total BAL eosinophils are reported on the basis of the phloxine B stain and were in good agreement with counts calculated from the differential on the stained cytocentrifuge preparations and the total leukocyte count. BALF protein content was estimated spectrophotometrically by absorbance (OD) at 280 nm, and 1 OD unit was converted to 1 mg/ml total protein.

Quantitation of Lung Tissue Eosinophils and Neutrophils

Quantitation of lung tissue eosinophils and neutrophils was done as described previously (19). Briefly, after BAL the lungs were removed, separated into the individual lobes, and weighed, after trimming away all extrapulmonary airway tissue. Samples of parenchyma from each lobe, approximating 20% of the total lung wet weight, were pooled, stored at 70°C, and later freeze-dried. For enzyme extraction, lyophilized samples were homogenized in 50 mM Hepes, pH 8.0, at 0.5% (dry wt/vol) with a pestle homogenizer (Talboys Engineering Corp., Emerson, NJ), centrifuged at 10,000 × g for 30 min at 4°C, and the supernatant was discarded. The pellet was resuspended in 0.5% cetyltrimethylammonium chloride (CTAC) in distilled water to the original volume, rehomogenized, and centrifuged again as described previously. An aliquot of the supernatant was taken for analysis of EPO and MPO activity.

Lung extracts were diluted 1:10 in 50 mM Hepes, pH 8.0 (EPO dilution buffer) or 10 mM citrate buffer, pH 5.0 (MPO dilution buffer). Aliquots of 75 µl of each sample were pipetted into 4 wells of a 96-well tissue culture plate. Cold stop solution (4 N H₂SO₄, containing also 2 mM resorcinol for the EPO assay) was added to two wells (150 µl/well) to stop the reaction at t = 0 s (background OD). The EPO substrate solution consisted of 50 mM Hepes (pH 8.0), 6 mM KBr, 3 mM o-phenylenediamine (OPD), 8.8 mM H₂O₂. The MPO substrate solution was 3 mM 3',5,5'-tetramethylbenzidene dihydrochloride (TMB), 120 µM resorcinol, and 2.2 mM H₂O₂ in distilled water. Substrate solution (75 µl) was added to each well, and the reaction was stopped after 30 s (EPO) or 2 min (MPO) with 150 µl of cold stop solution. The OD_{490 nm} (EPO) or OD_450 nm (MPO) was determined. As an additional control, 75 µl of dilution buffer (without lung extract) was placed into four wells, 75 µl of substrate buffer was added, followed by 150 µl of stop solution after 30 s or 2 min. No color reaction was observed in these control wells. The reaction was carried out at room temperature (21-22°C). The enzyme activities of the lung samples were calculated by subtracting the mean background OD and are expressed as change in optical density per minute.

Standard curves for calculating the number of eosinophils and neutrophils in the lungs, based on the enzyme activities, were developed as previously described (19). Briefly, 10 × 10⁶ BAL eosinophils were injected into a piece of noninflamed control lung and this tissue was frozen, lyophilized, and extracted as described previously. The EPO activity of the resulting extract was tested at different dilutions and the activity plotted against the theoretical eosinophil equivalents in the dilution of lung extract. This standard curve was used to estimate the eosinophil number in the test lung extracts and, on the basis of weight, in the lungs. In the extract from the eosinophil-injected control lung, the MPO activity was also measured and correlated with the EPO activity. The resulting linear regression was used as a correction of EPO spillover (approximately 5-6%) to the MPO assay (activity) in lung extracts, as shown previously (19). The neutrophil content of the lungs was estimated again as previously described, i.e., by injection of purified neutrophils (in this study, 53 × 10⁶) into control lung tissue, and then this tissue was lyophilized and extracted. Different dilutions of this extract were analyzed for MPO activity and this standard curve was used for estimation of the neutrophil content of the lungs. There was no MPO activity detected using the EPO assay conditions (19).

Histology, Immunohistochemistry, Transmission Electron Microscopy, and Degranulation Status Assessment

Freshly removed lung tissue was fixed for at least 24 h in 10% phosphate-buffered neutral formalin. After fixation, samples were cut from the hilus to the periphery and embedded in paraffin, using an automated tissue processor. Sections were cut at 5-µm thickness and stained with hematoxylin and eosin.

For preparation of semithin sections and transmission electron microscopy (TEM), lungs were first perfused via the pulmonary artery and BAL performed as usual and then fixed in situ by slow perfusion for 10-15 min of whole lungs with 1% glutaraldehyde in cacodylate buffer (0.1 M, pH 7.3). Representative samples of lung tissue were taken and processed for TEM after embedding in Epon (TAAB resin; Marivac Ltd., Halifax, Nova Scotia, Canada) and osmium tetroxide postfixation. Semithin sections were prepared and stained with toluidine blue to allow for assessment of compartmentalization of granulated cells over vasculature, parenchymal interstitium, and airways. TEM sections (300 nm) were counterstained with uranyl acetate and lead citrate. Sections were viewed on a Philips 300 TEM instrument (Philips, Eindhoven, the Netherlands) at various magnifications for assessment of granulocyte degranulation.

For immunohistochemistry, lungs were inflated in situ with O.C.T. compound (Tissue-Tek; Miles Inc., Elkhart, IN) via a tracheal cannula. After removal, single lobes were embedded in O.C.T. and snap-frozen immediately to 80°C in isopentane, immersed in a liquid nitrogen bath. Cryostat sections (6 µm) were cut from 1-cm³ blocks, air-dried, fixed in ice-cold acetone (10 min), and stored at 20°C until stained. For staining, slides were rehydrated in PBS and endogenous peroxidase was blocked by incubation for 20 min in methanol-3% hydrogen peroxide (4:1, vol/vol), and then blocked by incubation for 20 min with 1% bovine serum albumin (BSA), 10% goat serum in PBS. After a PBS wash, sections were stained with BMK-13, a monoclonal mouse IgG₁ antibody, directed against human eosinophil major basic protein (MBP), cross-reactive for rat MBP (22). An isotype-matched control antibody against an irrelevant epitope was used as a negative control. Slides were incubated for 60 min with BMK-13 hybridoma culture supernatant at a 1:20 dilution at room temperature and washed for 10 min with PBS. The slides were then incubated for 45 min with biotinylated goat anti-mouse Ig, adsorbed with rat serum proteins (Sigma Chemical Co.), diluted 1:50 in PBS, containing also 1% BSA and 0.5% rat serum, followed by a 10-min PBS wash and 45-min incubation with streptavidin-horseradish peroxidase (HRP) (Amersham Life Science, Oakville, Ontario, Canada), diluted 1:200 in PBS, 1% BSA. Bound enzyme was visualized with 3-amino-9-ethylcarbazole (Sigma Chemical Co.). Slides were counterstained with Mayer's hematoxylin and a coverslip was mounted with glycerin-gelatin.

Measurement of OA-specific IgE and IgG

From a group of BN and the Fischer and Lewis rats, EDTA (0.2%) blood was collected at the time of sacrifice (48 h after challenge) and plasma samples were stored at 20°C until analyzed. OA-specific IgE and IgG were determined by ELISA on 96-well plates (Costar 3690; Costar Corp., Cambridge, MA).

Data Analysis

All data are reported as arithmetic means. Error bars represent 1 SEM. Because the results are not normally distributed, the sensitized group was compared with the corresponding sham-sensitized group by the nonparametric Mann-Whitney U test (25). Correlations between different measured parameters were analyzed by Spearman's rank correlation (25). The OA-specific IgG and IgE ELISAs were analyzed by Kruskal-Wallis test (25). When this test showed significant differences, the Mann-Whitney U test was used to compare individual groups. All analyses were performed with the StatView data analysis system version 4.5 (Abacus Concepts, Inc., Berkeley, CA). A P value < 0.05 was considered to be significant.

	Results

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BALF Inflammatory Cell Profile

There was a vigorous influx of neutrophils and eosinophils into the BALF of sensitized BN rats, which was not observed in sham-sensitized, but OA-challenged animals as shown in Figure 1A. Neutrophils preceded the eosinophils and were observed as early as 3 h after allergen challenge. The increase in neutrophil counts became statistically significant at 14 h after challenge and they peaked at 24 h with a mean of 2.9 × 10⁶. Thereafter, the neutrophil counts declined rapidly, returning to baseline by day 6. The increase in BALF eosinophils first became significant at 14 h after challenge, and at 24 h reached a mean of 2.3 × 10⁶, i.e., slightly less than the neutrophils. In contrast to the neutrophils, eosinophils continued to accumulate and reached their peak at 3 d after challenge with a mean of 10 × 10⁶, comprising up to 85% of lavage cells at this time. Eosinophil counts were still significantly elevated at 6 d after challenge but had returned to baseline by day 13. 

View larger version (21K): [in this window] [in a new window]   View larger version (22K): [in this window] [in a new window]   Figure 1.   Time course of inflammatory cell accumulation in the bronchoalveolar compartment. BAL was performed in sensitized (n = 4-11, except for 48-h time point, when n was 25) or sham-sensitized (n = 3-9) BN rats at 3 h, 14 h, 24 h, 48 h, 72 h, 6 d, and 13 d after OA challenge and cells enumerated by hemacytometer counting and differentiation of cytocentrifuge preparations. (A) Recruitment of neutrophils and eosinophils. (B) Accumulation of macrophages and lymphocytes in BALF. Lymphocyte counts increased slightly and reached maxima at 2 d (mean, 4.4 × 105) and 6 d (mean, 5.5 × 105) after challenge. The nonparametric Mann- Whitney U test was used to compare the sensitized group of rats with the sham-sensitized group for each corresponding time point. **,P < 0.01, *,P < 0.05.

The number of BALF macrophages and lymphocytes increased in sensitized, OA-challenged rats, as shown in Figure 1B. In sensitized rats, the macrophage count increased about fivefold with the most rapid increase within 48 h after challenge, from a baseline of 0.85 × 10⁶ to a peak mean of 4.3 × 10⁶ at 6 d after challenge. The macrophage count was still significantly elevated on day 13 after challenge. In sensitized rats, many of the macrophages at 2-6 d after challenge had the phenotypic appearance of newly recruited monocytes (slightly indented nucleus and relatively sparse cytoplasmic seam), and overall, many macrophages showed morphologic signs of cell activation such as increased vacuolation and cytoplasmic projections. Multinucleated giant cells were also found at 3 d and later after challenge (not shown). There was a relatively small increase in lymphocyte counts between 2 and 6 d after challenge (Figure 1B), which roughly paralleled the time course of the macrophage accumulation. No significant rise in the lymphocyte count of sham-sensitized rats was observed. Mast cells and epithelial cells were observed only occasionally and were not enumerated.

Quantitation and Kinetics of Lung Tissue Eosinophils and Neutrophils

The time course of neutrophil and eosinophil migration to the lung tissue is shown in Figure 2. There was a rapid influx of neutrophils into the lung tissue, which was already significant at 3 h after challenge. Neutrophils peaked at 24 h with a mean of 98 × 10⁶ and declined rapidly thereafter, returning to baseline within 6 d. The influx of eosinophils into the lung tissue was much slower and almost linear until day 2 after challenge, when they peaked with a mean of 128 × 10⁶. Lung tissue eosinophilia persisted for at least 6 d but declined to the level in the sham-sensitized control group by day 13 after challenge. In general, the baseline lung tissue eosinophil count was quite variable in the sham-sensitized group with a range of 4- 22 × 10⁶ eosinophils, the highest counts observed 13 d after challenge.

View larger version (24K): [in this window] [in a new window]   Figure 2.   Time course of neutrophil and eosinophil accumulation in lung parenchyma following allergen challenge of sensitized or sham-sensitized BN rats (n as in Figure 1). The tissue content was quantitated by specific MPO and EPO assays of lung extracts as described under MATERIALS AND METHODS. **,P < 0.01, *,P < 0.05 by Mann-Whitney U test for sensitized versus sham-sensitized rats at corresponding time points.

Increased BALF Protein Content and Lung Wet Weight after Challenge

The BALF protein content increased by 3 h and continued to rise in a nearly linear fashion until day 2 or 3, when it peaked at nearly fivefold that in the sham-sensitized group (Figure 3). Lung wet weights increased markedly (by more than 50%, P < 0.05) within 3 h after OA challenge of sensitized rats but not significantly in sham-sensitized rats and continued to increase slightly until day 3, followed by a slow decline (not shown).

View larger version (16K): [in this window] [in a new window]   Figure 3.   Time course of BALF protein content following OA challenge of sensitized or sham-sensitized BN rats. BAL was performed at the indicated time points after challenge, cells were sedimented by centrifugation, and the BALF protein content in the supernatant was determined by measuring the absorbance at 280 nm. A value of 1 OD was converted to 1 mg/ml protein. *P < 0.05; **P < 0.01, by Mann-Whitney U test for sensitized versus sham-sensitized rats at corresponding time points. n as in Figure 1.

Histology, Immunohistochemistry, and TEM of OA-challenged Lungs

At 48 h postchallenge, there was a prominent edematous reaction in the adventitia of venules and arterioles and in the submucosa of adjacent bronchi and bronchioli, as shown in Figure 4B, but not in sham-sensitized control rats (Figure 4A). Accumulations of eosinophils as well as aggregates of mononuclear cells and a few multinucleated giant cells were found mainly in perivascular and peribronchial sites. Specific staining of the eosinophils on cryosections with an antibody directed to MBP (Figure 4D), revealed a heavy accumulation of eosinophils, most prominent in the adventitia of medium-size vessels and the adjacent submucosa of respiratory and terminal bronchioli. However, many eosinophils were also found in the interstitial space of the septa (not shown). At 72 h, most clusters of eosinophils were found in the bronchial submucosa, as shown in Figure 4E, and the bronchial epithelium itself showed signs of cell activation such as enlargement of nuclei with prominent nucleoli and dissolution of chromatin. At 6 d after challenge, many eosinophils were still found in the bronchial submucosa and some airways were occluded by mucus plugs (Figure 4F). The inflammatory response had largely resolved by day 13 after challenge (Figure 4G), when there was restoration to normal lung morphology except for some focal mononuclear and eosinophilic aggregates. Specific staining of collagen fibers with a trichrome stain did not show significant increase in collagen at day 13 (not shown).

View larger version (158K): [in this window] [in a new window]   Figure 4.   Histopathologic investigation of lung tissue from OA-challenged BN rats. (A) Hematoxylin-eosin (H&E)-stained paraffin section of lung tissue from a sham-sensitized BN rat at 48 h after OA challenge. This section shows a normal lung histology with no inflammatory signs. Original magnification: ×50. (B) H&E-stained paraffin section of lung tissue from a sensitized BN rat at 48 h after challenge. The adventitial space of vessels appears widened due to edema (arrows) and inflammatory foci are found throughout the lungs. Original magnification: ×50. (C and D) Cryosection of lung tissue from a sensitized BN rat at 48 h after challenge: Staining with a negative-control monoclonal antibody (C) or specific detection of eosinophils with an antibody against eosinophil MBP (D). Eosinophil accumulation was most prominent in the adventitia of medium-size vessels. Original magnification: ×50 (C) and ×125 (D). (E) H&E-stained paraffin section of lung tissue from a sensitized rat at 72 h after challenge, showing accumulation of eosinophils in the submucosa of a bronchus. The bronchial epithelium shows signs of cell activation such as enlargement of nuclei with prominent nucleoli (arrow). Original magnification: ×250. (F) H&E-stained paraffin section of lung tissue from a sensitized rat at 6 d after challenge, showing submucosal and interstitial inflammation with eosinophils and mononuclear cells. Some airways contained mucus plugs with eosinophilic and mononuclear aggregates. Original magnification: ×125. (G) Inflammation had largely resolved at 13 d postchallenge as shown by this H&E-stained paraffin section of lung tissue from a sensitized rat. Original magnification: ×50.

Assessment of semithin sections confirmed the virtual absence of granulated cells in blood vessels or capillaries, confirming the efficacy of the perfusion vascular washout procedure (Figure 5A).

View larger version (57K): [in this window] [in a new window]   Figure 5.   (A) Microphotograph of section of rat lung 24 h after antigen challenge at time of tissue peak of eosinophil and neutrophil infiltration. Note the absence of erythrocytes and white blood cells in the septal capillaries and arterioles/venules. (Epon section, 1 µm, stained with toluidine blue. Bar: 40 µm.) (B) TEM photograph of peribronchial area of rat lung 24 h after antigen challenge at time of tissue peak of eosinophil and neutrophil infiltration. Note plentiful, typical crystal-containing granules in the cytoplasm of eosinophilic cells (E) and granules in the cytoplasm of a neutrophil (N) with continuous intact cell membrane. Note the absence of granular structures of eosinophilic or degenerated type in the remaining stromal compartment. (Epon section, 300 nm. Bar: 5 µm).

Analysis of TEM of representative samples confirmed that degranulation was minimal with eosinophilic granules well contained within intact cell cytoplasm confined by intact and continuous cell membrane contours (Figure 5B). There were no intrastromal granules found, either of eosinophilic or indeterminate morphology (Figure 5B).

Correlation Analysis

Using the nonparametric Spearman's rank correlation analysis, we investigated for each of the time points whether a significant correlation existed between the different parameters. These results are summarized in Table 1. No significant correlations were found at 3 h, 14 h, 3 d, and later after challenge. At 24 h after challenge, there was a significant correlation between the number of lung neutrophils and BAL neutrophils, between BAL eosinophils and BAL neutrophils, and between lung eosinophils and BAL eosinophils. There was also a significant correlation between the number of BAL macrophages and BALF protein content. At 48 h, significant correlations were found between lung tissue eosinophils and BAL eosinophils, BAL eosinophils and BALF protein, BAL macrophages and BAL eosinophils and neutrophils, and between BALF protein content and lung tissue eosinophils and lung tissue neutrophils (Table ).

Pulmonary Inflammation in Fischer and Lewis Rats

In view of the severe eosinophilic pulmonary inflammation elicited in the BN rats by the immunization and prolonged aerosol challenge used here, we decided to investigate if similar responses might also occur in other rat strains that tend to have a more Th1-type biased immune response, such as Lewis and Fischer rats. Fischer rats have been described to have hyperreactive airways (13) and may therefore be a suitable rat strain for studying allergic pulmonary responses. A group of Fischer (n = 5) and Lewis (n = 5) rats was investigated at 48 h after challenge. Only a few inflammatory cells were found in the BALF of Fischer ([2.0 ± 0.7] × 10⁵ neutrophils, [4.0 ± 1.3] × 10⁴ eosinophils) and Lewis ([1.4 ± 0.86] × 10⁵ neutrophils, [1.2 ± 1.2] × 10⁴ eosinophils) rats. Quantitation of lung tissue eosinophils (Fischer, [2.7 ± 0.3] million; Lewis, [2.5 ± 0.2] million) and neutrophils (Fischer, [7 ± 0.4] million; Lewis, [10.6 ± 1.9] million) by enzyme assays revealed that there was only a very weak inflammatory cell response compared with BN rats (Figure 2) in either rat strain. Histology of lungs at 48 h postchallenge in both Lewis and Fischer rats showed a minimal inflammatory infiltrate with a very patchy distribution (not shown).

Measurements of OA-specific IgE and IgG

As shown in Figure 6, BN rats had the highest titers of OA-specific IgE of the three rat strains investigated. The specific IgE was very low in Fischer rats (P < 0.01 versus BN), and almost undetectable in Lewis rats (P < 0.01 versus BN and P < 0.01 versus Fischer). BN rats had also the highest specific IgG (P < 0.01 versus Lewis), Fischer rats had the second highest titer, and Lewis rats had again the lowest titer of the three strains.The differences between BN and Fischer and between Fischer and Lewis rats did not, however, reach significance (P = 0.17 for both comparisons).

View larger version (22K): [in this window] [in a new window]   Figure 6.   OA-specific IgE and IgG in BN, Fischer, and Lewis rats following immunization with alum-precipitated OA and B. pertussis vaccine as adjuvant. Blood samples were obtained 16 d after immunization (48 h after challenge). *P < 0.01 BN compared with Fischer, #P < 0.01 BN or Fischer compared with Lewis. Number of animals n = 12 (BN), 5 (Fischer), and 5 (Lewis).

	Discussion

Top Abstract Introduction Materials & Methods Results Discussion References

Although BN rats are widely used for studies of allergic pulmonary responses, the kinetics and magnitude of neutrophil and eosinophil recruitment into lung parenchyma as well as BALF in a BN rat model has, to our knowledge, not hereto been described in detail. In this study, we employed MPO and EPO assay methods for quantitating lung tissue neutrophils and eosinophils, which we have shown to be highly specific for each enzyme in the rat (19), to analyze the pulmonary inflammatory response following allergen challenge. By combining this method with the technique of BAL, we could analyze the kinetics and magnitude of the inflammatory response independently in the bronchoalveolar compartment and the lung parenchyma itself. The absence of any intravascular component to the granulocytic cell presence in the lungs as confirmed by analysis of semithin sections (Figure 5A) suggests that the perfusion technique employed here effectively removed blood cells from the pulmonary vasculature and that the measured granulocyte numbers reflect cell accumulation in the lung parenchyma itself.

Somewhat unexpected was the large number of neutrophils, which accumulated in the lung parenchyma by 24 h (ca. 100 × 10⁶) and exceeded the number of eosinophils at this time (ca. 60 × 10⁶; see Figure 2). Renzi and coworkers retrieved 25 × 10⁶ and 10 × 10⁶ neutrophils at 8 and 32 h, respectively, and 6 × 10⁶ eosinophils at 32 h from the lung tissue of BN rats sensitized in a similar manner as here, but challenged with OA only for 5 min (20). Similarly, Laberge and coworkers recovered 21 × 10⁶ neutrophils and 18 × 10⁶ eosinophils at 32 h postchallenge (26). Thus, the neutrophil and eosinophil tissue accumulation observed by us was much greater compared to other studies in the BN rat. Several differences between these experiments and our's may account for this. First, in these studies, anesthetized rats were challenged for 5 min by intratracheal delivery of the allergen, whereas in our experiments, awake animals were exposed for 60 min, which possibly resulted in a larger total dose of allergen. Second, in both studies mentioned previously, enzymatic dispersion of lung followed by leukocyte enumeration by hemacytometer counting was used to quantitate parenchymal leukocytes. The latter technique might lead to an underestimation of adherent leukocytes such as granulocytes owing to incomplete recovery during the isolation procedure and the percentage recovery of lung leukocytes is not readily ascertainable.

The role of neutrophils in allergic asthma is controversial (5). However, neutrophils are capable of releasing potent agents such as proteases, reactive oxygen species, and lipid mediators, and there is evidence in animal models that this can contribute to tissue damage and airway responses (5, 27). Although recruitment of neutrophils after allergen challenge to the lungs has been described by many investigators in humans (28, 29) and in animals (30- 32), including BN rats (33), it has been argued that this response might be related to traces of lipopolysaccharide (LPS) in the aerosol or may be due to an unspecific response to the challenge itself. In our experiments, no increase in neutrophil counts in Al(OH)₃ and B. pertussis sham-sensitized rats, challenged with OA aerosol, or in sensitized animals, challenged with saline (not shown), was observed. Hence, the neutrophil recruitment was allergen induced and not due to the introduction of LPS or foreign protein into the airways. Interestingly, there were two phases of neutrophil accumulation in both BALF and parenchyma: a first wave within 3 h, a plateau to 14 h, and a second rapid increase between 14 and 24 h. It is tempting to speculate that the first wave was in response to preformed mediator release, whereas the second wave may be the result of induced cytokines or mediator production by the first wave of neutrophils. The significant correlation between BAL neutrophils and eosinophils at 24 h post challenge (Table ) suggests that, in the early phase of the allergic response, both cell types may respond to the same stimuli or that the neutrophil influx may be a priming factor for the eosinophil recruitment. The techniques used here could be applied to test these possibilities.

In contrast to neutrophils, eosinophils continued to accumulate in tissue and BALF and persisted for many more days. Our data provide some insights into the relative contribution of neutrophils and eosinophils in the response in the two different compartments investigated. Whereas the maximum number of neutrophils in BALF was only one-quarter the number of eosinophils in BALF, the peak tissue content was similar (100 × 10⁶ neutrophils at 24 h versus 130 × 10⁶ eosinophils at 48 h), suggesting that analysis of BAL cells alone might lead to an underestimation of the neutrophil participation. Interestingly, the time course of neutrophils in BALF and tissue was almost superimposable (Figures 1A and 2), perhaps indicating that there might be differences in the migration routes into the bronchoalveolar space for neutrophils and eosinophils. It has been shown that, in the lungs, in contrast to the systemic circulation, neutrophils emigrate mainly through the capillary bed in various inflammatory states (34), whereas little is known about eosinophil migration pathways to the lungs. In the model used here, leukocyte recruitment appeared to occur at all levels of the bronchoalveolar tree (Figures 4 and 5). Interestingly, morphometric analysis in human asthma also demonstrates that in patients with asthma, leukocyte accumulation (especially T cells and eosinophils) occurs at all levels including small airways and parenchyma (35).

Neutrophils are not consistently found in lung biopsy specimens from patients with asthma, except in cases of sudden-onset fatal asthma (6). However, we show here that neutrophils are a large part of the allergen-induced leukocyte infiltrate into the lung tissue. In marked contrast to eosinophils they do not persist, likely because of their relatively short survival and rapid clearance, and presumably because of the cessation in the production of neutrophil-recruiting mediators. This might explain why neutrophils are usually sparse in biopsies. The frequency of contact with allergen and the temporal relationship of biopsy to acute exacerbations in human asthma may be important determining factors for observing neutrophils in the tissue.

The recognition that eosinophilic and neutrophilic cells as a whole were not degranulated (Figure 5B) in our study requires discussion in the light of the common description of this change in clinical and experimental models of asthma. One possible explanation may be found in the fact that the model presented here consists of a single allergen challenge. The decreasing or later absence of sufficient quantities of antigen may be the reason for the lack of degranulation of the influxed populations of eosinophils and neutrophils. In addition, the TEM findings confirm that the termination procedure and organ/tissue handling in this model maintain the physiologic status of eosinophils sufficiently for valid conclusions to be drawn from analyses such as presented in our studies.

In addition to the BALF granulocytes, we observed a significant increase in alveolar macrophages and lymphocytes after challenge. Whereas increase in lymphocytes in BN rats after allergen challenge has been described previously (22, 36), this is, to our knowledge, the first report showing an allergen-induced increase in the number of alveolar macrophages (Figure 1B). This may reflect the recruitment of blood monocytes to the lungs, and this view is supported by the histologic appearance, as described under RESULTS. We also observed increased numbers of giant cells in the lung tissue and the BALF, especially at 48 h or later after allergen challenge. This might be an indication of macrophage activation, which can lead to cell fusion (37). Macrophages are potent cytokine producers and can be activated by interaction with allergen through IgE pathways (38, 39). They have been shown in a BN rat model to be an important source for the synthesis of cysteinyl-leukotrienes, powerful bronchoconstrictor agents (40). Increase in tissue macrophages with an immature phenotype has also been found in human asthma (41). The significant correlations between BALF macrophage count and BALF protein content at 24 h as well as between BAL macrophage and BAL eosinophil and also neutrophil counts at 48 h (Table ) suggest that this cell type plays an immunomodulatory role in pulmonary allergic responses, and the model described here may be of value for studying the role of this cell type.

The rapid increase in BALF protein (Figure 3) and lung wet weights (not shown) in sensitized rats on allergen exposure likely resulted from an increase in vascular and bronchoalveolar permeability, leading to plasma exudation into the interstitial space and eventually into the airways. Accordingly, widening of the adventitial space was prominent in histologic sections of allergen-challenged rats at 3 h (not shown) and later after challenge (e.g., at 48 h; Figure 4B). Tissue edema and plasma exudation into interstitium and airways are characteristic features of allergic asthma (42) and airway edema can potentiate airway reactivity (43). Plasma leakage is thought to be caused by numerous inflammatory mediators (42). The BALF protein content doubled by 3 h after challenge, at the time when the greatest increase in lung wet weight occurred. However, protein continued to increase and then decreased by day 6 in parallel with the eosinophil response (Figures 2 and 3). Thus, the leakage of protein due to increased permeability may be in part acute, associated with early mediator release and/or neutrophil influx but then also sustained, perhaps because of eosinophil-derived mediators or toxic products. This is supported by the observation that there were significant correlations between BALF protein and BAL eosinophils, lung eosinophils, and lung neutrophils at 48 h and between BALF protein and BAL macrophage counts at 24 h postchallenge (Table ).

The lung inflammation largely resolved by day 13 after challenge as assessed by BALF analysis, EPO and MPO assays of lung parenchyma (Figures 1-3), and histology (Figure 4G). Despite the apparent severity of the inflammatory response, no permanent changes, e.g., fibrosis, were observed. This model might therefore also be suitable for the study of mechanisms of inflammatory cell clearance and tissue repair. Interestingly, virtually no inflammatory reaction was observed in Fischer and Lewis rats with the same sensitization and allergen challenge protocol used for the BN rats. A possible explanation for this is the lack of a specific IgE response (Figure 6) following immunization with alum-precipitated OA and B. pertussis vaccine as adjuvant, a regimen that is known to trigger IgE production (44). Surprisingly, specific IgG titers were also lower in Fischer and particularly in Lewis rats compared to BN rats. Thus, the immunization protocol used here might trigger a strong Th2-type response in BN rats and Lewis rats may not mount this type of immune response, at least not with the immunization regimen used here, and the Fischer rats have a relatively weak response compared to the BN strain. Furthermore, it is likely that not only the IgE responses, but also the cytokine pattern in the lung before and after allergen challenge, is important in determining the inflammatory response. BN rats have been shown to express predominantly the Th2 cytokines IL-4 and -5 in their lungs but not the Th1-specific cytokines IL-2 and interferon  (IFN-) (7). In contrast, Sprague-Dawley rats, which are also low IgE producers, as are Lewis rats, and do not develop pulmonary responses after allergen challenge, expressed predominantly the Th1 cytokines after challenge (7). Although cytokine expression was not investigated in our study, we speculate that the lack of Th2 cytokine expression in Fischer and Lewis rats might be one reason for the absence of an eosinophilic inflammatory response in these strains.

In summary, the severe allergic lung inflammation in the BN model described here is complex and involves multiple cell types such as eosinophils, neutrophils, lymphocytes, and macrophages, and probably stromal cells such as the bronchial epithelium. It should be noted that the correlations found in this study (Table ) do not imply that the relationship is linear or even direct. Further investigation is necessary to address this point. The techniques described here for quantitating lung tissue eosinophils and neutrophils should be useful to elucidate the mechanisms of inflammatory cell recruitment, persistence, clearance, and relationship to tissue damage and pulmonary function in experimental allergic inflammation.