Introduction

Top Abstract Introduction Materials and Methods Results and Discussion References

The alveoli and distal airways are lined by pulmonary surfactant, a mixture of phospholipid and protein, that functions to reduce surface tension at the air-liquid interface and to participate in protective mechanisms against inhaled toxic, infectious, or allergenic substances (1, 2). Impairment of the intricately regulated synthesis, release, and reuptake of four unique surfactant proteins (SPs)SP-A, SP-B, SP-C, and SP-Dby type II pneumocytes results in surfactant dysfunction (3) and is believed to play a major role in disorders of the lung parenchyma, such as respiratory distress syndrome (6). Although potentially significant, the role of SPs in inflammatory diseases that affect the airways, such as asthma, remains unknown.

Allergen-induced asthmatic responses include activation of T cells leading to a T helper (Th) 2-type inflammation with high serum immunoglobulin (Ig) E, eosinophilia, reversible airway obstruction, and airway hyperresponsiveness (AHR) (7). Pulmonary surfactant changes in this process may be important for two reasons. First, the hydrophobic SPs SP-B and SP-C shown to prevent collapse of alveoli at low lung volumes (8, 9) have also been implicated in maintaining patency of narrow conducting airways (10, 11). Thus, dysfunction of SP-B and SP-C may contribute to the enhanced resistance of the asthmatic airways (12). Second, the hydrophilic SPs (lung collectins) SP-A and SP-D are part of the innate immune system, and as such, they have potent effects on its cellular components and a binding ability to complex antigens, including microorganisms (13) and allergens (14). Collectins, therefore, may play a significant role in development and modulation of allergic responses in the airways. Notably, SP-A and SP-D were shown to specifically bind particles of Aspergillus fumigatus (Af ), indicating a direct involvement of these molecules in the processing of Af antigens and in the resulting inflammation (15, 16).

	Materials and Methods

Top Abstract Introduction Materials and Methods Results and Discussion References

Mice, Sensitization, and Intranasal Challenge with Af Extract

To study the role of SPs, a murine model of Af-induced allergic sensitization was characterized. Female BALB/c mice were housed under pathogen-free conditions. Experiments were performed between 8 and 12 wk of age. All experimental animals used in this study were under a protocol approved by the Institutional Animal Care and Use Committee of the University of Pennsylvania. Two groups of mice were compared: "Naive" mice received intranasal vehicle challenges only, with 21% glycerol in phosphate-buffered saline (PBS). "Sensitized" mice were injected intraperitoneally with 20 µg Af (Bayer Pharmaceuticals, Elkhart, IN) together with 20 mg alum (Imject Alum; Pierce, Rockford, IL) in 100 µl on Days 1 and 14, followed by intranasal challenge on Days 25, 26, and 27 with 25 µl of Af extract in PBS (12.5 mg in 21% glycerol, PBS). Intranasal treatment was carried out essentially as described previously (17). Briefly, sensitized and control mice were anesthetized by isoflurane inhalation, and 25 µl of Af extract or vehicle, respectively, was applied to the left naris. The studies were performed and all mice were killed 24 h after their last intranasal treatment, when the peaks of eosinophil infiltration and airway responses were assumed to occur. Naive mice that received intranasal glycerol treatment alone showed no difference from nonsensitized, nonexposed normal BALB/c mice in any of the study parameters that we investigated, including lung histology, bronchoalveolar lavage (BAL) fluid (BALF) cellular content, Ig and cytokine profile, and airway responses to acetylcholine (ACh) (not shown).

In Vivo Measurement of Airway Responsiveness to ACh

Airway function measurements were carried out as previously described (20), modified as follows: Mice were anesthetized, cannulated, and ventilated (140 breaths/min; 0.2 ml tidal volume) after administration of pancuronium bromide (1.0 mg/kg). Transduced alveolar pressure and airflow rate (DP45 and DP103; Validyne, Northridge, CA) was used to calculate lung resistance (RL) and dynamic compliance (C_dyn) by a computer (Buxco Electronics, Inc., Troy, NY). Baseline RL and C_dyn values were established and after administration of saline, ACh was given intravenously at concentrations ranging from 80 to 1,280 µg/kg in five increments.

BALF Analysis for Differential Cell Count and Cytokine and Surfactant Content

Lungs were lavaged with either a small volume (1 ml) of sterile PBS or a large volume (5 ml) of sterile saline, and total and differential cell counts were performed as described previously (20, 21). Cytokine levels were determined from cell-free supernate of the small-volume BAL by enzyme-linked immunosorbent assay (ELISA) using antibodies and recombinant cytokines from PharMingen (San Diego, CA). To remove cells the large-volume BALF samples were centrifuged at 400 × g for 10 min at 4°C. The cell-free supernate was separated into two fractions by a second centrifugation at 20,000 × g for 60 min at 4°C. The pellet contained the large-aggregate (LA) surfactant fraction and the supernate contained soluble proteins as well as the small-aggregate (SA) fraction. For consistency with previous work (22, 23) we termed the supernate the SA fraction.

Total protein and phospholipid contents of the LA and SA fractions were determined using standard methods. Sodium dodecyl sulfate polyacrylamide gel electrophoresis of LA and SA surfactant samples was carried out using NuPAGE 10% Bis-Tris gels (Novex, San Diego, CA) according to instructions of the manufacturer.

Rabbit polyclonal antisera against SP-A, SP-B, and SP-D were obtained and Western blots were performed as previously described (22). Antimature SP-C antibody was obtained from Byk Goulden Pharmaceuticals (Constance, Germany). Each lane was loaded with 5 µg of total protein.

Serum ELISA for Igs

Serum samples were collected as described elsewhere (20, 21). Antibodies and recombinant IgE were purchased from PharMingen and antibody levels were determined according to instructions of the manufacturer. For Af-specific antibody levels, plates (Dynatech, Chantilly, VA) were coated with Af (50 µg/ml in PBS, pH 7.1) and incubated overnight at 4°C. Samples were diluted 1:5 for Af-specific IgE and IgG2a, and 1:25 for IgG1 and total IgE. Data were analyzed with the Microplate Manager software program for the PC version (Bio-Rad, Hercules, CA).

Lung Tissue Histology and Analysis of Messenger RNA Expression

After lavage, lungs were inflated and fixed in paraformaldehyde (4% wt/vol in sodium cacodylate, 0.1 M, pH 7.3) for histologic analysis. Paraffin sections prepared from the lungs of naive and sensitized mice were stained with hematoxylin and eosin (H&E) for evaluation of airway inflammation.

In a different set of mice, total RNA was isolated from lungs after BAL (23). Specific messenger RNA (mRNA) content was determined by Northern blot analysis. Nitrocellulose blots loaded with 10 µg total RNA per lane were hybridized under high stringency with [-³²P]complementary DNA (cDNA) probes for rat SP-A, SP-B (23), SP-C, and SP-D (22) prepared from purified plasmid inserts by labeling with [-³²P]deoxycytidine triphosphate (Ready-to-Go Kit; Pharmacia, Piscataway, NJ) to specific activities of 6 to 8 × 10⁶ counts per min/µg DNA as previously described (22, 23). The specific signals were normalized for loading by hybridization of each blot with a ³²P end-labeled ([-³²P]adenosine triphosphate) 28S ribosomal RNA oligonucleotide probe. Specific mRNA bands were quantified by Phosphoimager (Bio-Rad).

Data Analysis

Data are expressed as means ± standard error of the mean (SEM). Student's t test assuming equal variances was performed to test differences between groups and correlation was investigated by regression analysis. P < 0.05 was considered significant. Data were analyzed with the Sigmastat standard statistical package (Jandel Scientific, San Rafael, CA).

	Results and Discussion

Top Abstract Introduction Materials and Methods Results and Discussion References

Characterization of Af-Induced Acute Airway Inflammation and Hyperresponsiveness

Af is a major airborne fungus responsible for a number of allergic disorders, including allergic bronchopulmonary aspergillosis (ABPA) and IgE-mediated asthma. In contrast to ABPA, in asthma the particles of Af sensitize the airways without fungal colonization (24). Our study aimed to reproduce this poorly characterized disease process in an animal model and to investigate whether pulmonary surfactants play a role.

To determine whether intranasally instilled Af would elicit an eosinophilic inflammatory response, a hallmark of Af-induced asthma, H&E sections of the lung tissue and the cellular content of the BALF were analyzed. Figure 1A shows that Af induced a predominantly perivascular (right upper panel) and peribronchial (right lower panel) inflammatory infiltrate that contained mainly mononuclear cells and eosinophils (Figure 1B, upper panel), with the lung parenchyma and the alveolar spaces being relatively preserved. The columnar epithelium of the airways remained intact, indicating that our model of acute allergic responses does not induce denudation or other damage to the epithelium. This is somewhat different from previously published models of Af-induced allergic sensitization in which mice received repeated intranasal Af exposure over a period of 3 wk and demonstrated more pronounced inflammatory changes with epithelial damage (18, 19). The BALF of sensitized and challenged mice (Figure 1B, lower panel, and Figure 1C) showed significant eosinophilia (P < 0.001), with approximately 40% of all the recovered cells being eosinophils.

View larger version (87K): [in this window] [in a new window]   View larger version (13K): [in this window] [in a new window]   View larger version (10K): [in this window] [in a new window]   Figure 1.   Sensitization and challenge with Af resulted in airway eosinophilic inflammation and elevated serum Ig production. (A) Representative paraffin-embedded, H&E-stained lung sections prepared from the left lung showing blood vessel (upper panels) and airway (lower panels) of naive mice (left panels) and mice sensitized and challenged with Af (right panels). Sections were evaluated under light microscopy. Original magnification: ×20. (B) High-power micrographs showing eosinophil cells in the subepithelial airway tissue (arrows, upper panel, H&E) and in the BALF (lower panel, Giemsa). (60× oil immersion.) (C) Absolute numbers of BALF eosinophil cells were derived from counts in Giemsa-stained cytospin preparations and the total cell number in each sample as described. Data are expressed as means ± SEM of n = 16 in the naive (open bar) and n = 22 in the sensitized ( filled bar) groups. **P < 0.01. (D) Total serum IgE and Af-specific Ig profile (IgE, IgG1, and IgG2a) were analyzed by ELISA. Total serum IgE (left panel): expressed as ng/ml. Af-specific Igs IgE (middle panel) and IgG1 and IgG2a (right panel) expressed as optical density (O.D.). Data are expressed as means ± SEM of n = 16 in the naive (open bar) and n = 22 in the sensitized ( filled bar) group. *P < 0.05, **P < 0.01.

Because elevated serum IgE and IgG to Af is a diagnostic feature of allergic asthma induced by Af and may be partly responsible for pathologic changes of the human lung (24), we analyzed total serum IgE together with the Af-specific Ig profile (IgE, IgG1, and IgG2a). There was an order of magnitude increase in the total serum IgE content after sensitization and challenge with Af (P < 0.001; Figure 1D, left panel) in addition to significantly elevated Af-specific IgE levels (P < 0.05; Figure 1D, middle panel). There were also robust increases in Af-specific IgG1 (P < 0.001, sensitized versus naive; Figure 1D, right panel). Levels of Af-specific IgG2a were slightly but also significantly elevated (P < 0.05). Our data indicate that sensitization and challenge with Af resulted in a characteristic Th2-type (interleukin [IL]-4-mediated) antibody profile in which total IgE and Af-specific IgG1 predominated.

Airway function measurements of mice sensitized and exposed to Af showed significant increases in RL and decreases in C_dyn in response to ACh when compared with naive animals (analysis of variance P < 0.001). Regression analysis revealed a significant correlation between the numbers of eosinophils and the extent of RL given at an ACh dose of 320 µg/kg (r = 0.75; P < 0.05), confirming a common base for Af-induced allergic airway inflammation and AHR (Figure 2A, right panel).

View larger version (29K): [in this window] [in a new window]   Figure 2.   Sensitization and challenge with Af resulted in Th2-type cytokine-driven AHR. (A) Mice were sensitized and exposed to Af and their lung function was assessed by measuring RL (left panel) and Cdyn (middle panel) by a Buxco online system as described. Data are expressed as % changes from baseline. The naive group (open squares, n = 8) received intranasal glycerol treatment alone. The sensitized group (filled squares, n = 8) received intraperitoneal sensitization and intranasal treatment with Af extract. Baseline RL values in the naive and in the sensitized groups were 0.84 ± 0.04 and 1.25 ± 0.11 Hgmm/cmH2O/ min, respectively. Regression analysis (right panel) was carried out between number of BALF eosinophils (x axis) and RL given at an ACh dose of 320 µg/kg (y axis) (r = 0.75; P < 0.05). (B) Cytokine levels in BALF were determined by ELISA. Regression analysis was carried out between number of BALF eosinophils (absolute number of cells, x axis) and IL-5 (pg/ml, y axis) (r = 0.764; P < 0.01; left panel), log converted values of the IL-4 levels (x axis) and IL-5 (r = 0.883; P < 0.001; middle panel), and IL-4 (x axis) and IFN- (y axis) (r = 0.669; P < 0.01; right panel).

Because IL-4 and IL-5 are central in the development of a Th2-type immune response, particularly in IgE production and airway eosinophilia, we characterized the local release of these cytokines by analyzing the BALF. IL-5 levels in sensitized mice increased significantly (132 ± 32 versus 24 ± 6 pg/ml in naives; P < 0.01) and showed a significant correlation with the numbers of eosinophils recovered from the BALF of Af-sensitized mice (r = 0.764; P < 0.01; Figure 2B, lower left panel). In addition, sensitization markedly enhanced IL-4 release (60 ± 11 versus 1 ± 0 pg/ml; P < 0.01). IL-4 positively correlated with IL-5 (r = 0.883; P < 0.001) and negatively correlated with interferon (IFN)- (r = 0.669; P < 0.01), indicating that predominance of Th2-type cytokines may exert an inhibitory effect on local IFN- after Af-induced allergic sensitization. Taken together, similar to our previously characterized ovalbumin-induced models of allergic AHR (20, 21), intraperitoneal sensitization and intranasal challenges with Af induced a predominantly Th2-like inflammatory response manifested by increases in serum IgE, IgG1, and an IL-5-driven eosinophil accumulation in the lungs with altered airway function.

Sensitization and Challenge with Af Inhibited Hydrophobic SP-B and SP-C and mRNA Expression

LA and SA surfactant fractions of the BALF are morphologically and functionally different (25). Tubular myelin, large multilamellar vesicles, and lamellar bodies are observed in the LA fraction that is also responsible for the biophysical properties of surfactant. SP-A, SP-B, and SP-C are found mainly in this fraction. The SA fractions contain only small unilamellar vesicles and show poor surface activity both in vitro and in vivo. Centrifugation of cell-free BALF at 20,000 × g for 60 min results in a supernatant that contains the majority of SP-D and, also, the SA fraction is isolated with this portion.

In human respiratory distress syndrome and in models of experimental lung injury, changes in total protein and phospholipid levels are prominent (1). To study whether allergic inflammation in our model altered lung protein and phospholipid content, we analyzed the LA and SA pools as well as the cellular fraction of BALF samples in sensitized and control mice. Protein levels in the SA fraction of BALF were significantly higher than in the LA of the cellular fractions. Allergic sensitization resulted in increased protein levels in the SA fraction, which, however, did not attain statistical significance in this study (Table 1). The phospholipid levels were markedly higher in the LA than in the SA fraction in both animal groups, as expected; however, we observed no significant differences between sensitized and naive mice (Table 1), indicating no significant damage of type II epithelial cell function during allergic inflammation in this model.

Western blot analysis (Figures 3A and 3B) of the LA aggregate fraction of sensitized mice demonstrated significant reduction of the SP-B and SP-C levels when compared with naive mice (P < 0.05). No detectable levels of either SP-B or SP-C were found in the SA fraction. In addition, Northern blot analysis and densitometric evaluation of the 2-kb SP-B and 0.9-kb SP-C bands revealed marked decreases in normalized mRNA content in mice sensitized and challenged with Af when compared with nonsensitized control mice (P < 0.05 and P < 0.005, respectively). Thus, the alterations in hydrophobic SP levels (SP-B and SP-C) induced by allergic airway inflammation were reflected in commensurate changes in mRNA expression (Figures 3C and 3D). Cell activation during inflammation, as well as an influx of inflammatory cells, may result in decreased specific mRNA through increasing the total RNA levels in the lung. Although we took extra precautions to avoid such bias by normalizing mRNA levels to the 28S RNA content in each sample and by quantifying the amount of total RNA extracted from each lung that showed no significant differences between naive and sensitized groups in this study, we cannot entirely exclude the possibility that a dilutional effect contributed to the reduction of SP-B and SP-C mRNA we observed. Nevertheless, the proportionate decreases we found in the SP-B and SP-C levels were not the result of any such dilution by total protein because in the LA fraction the protein levels were virtually identical between sensitized and naive mice (Table 1).

View larger version (53K): [in this window] [in a new window]   Figure 3.   SP-B and SP-C content is decreased after allergic sensitization and challenge with Af. (A) Representative SP-B and SP-C Western blot in the LA fraction of BALF. Nitrocellulose blots with samples of SA surfactant from naive and sensitized mice were probed with polyclonal anti-SP-B and anti-SP-C antibody as described. (B) Quantification of SP-B and SP-C content. The relative content of mature SP-B or SP-C in each sample was determined by densitometric scanning of the 8- or 3.7-kD bands from multiple blots. Open bars: naive mice; filled bars: sensitized mice. Data are expressed as % of control levels. n = 7-8 samples in each group. Means ± SEM were calculated after deriving the average of the results from two independent experiments. Each lane contains 5 µg total protein. (C) Representative autoradiographs of the 0.9-kB SP-C and 2-kB SP-B mRNA bands. Total RNA for Northern blot analysis was prepared from the lungs of naive and sensitized mice as described. After transfer to nylon membranes, blots were probed with full-length rat SP-B and SP-C cDNA probe labeled with -P32 and exposed to film. (D) Quantification of SP-B and SP-C mRNA expression. Intensity was quantified by densitometric scanning and values were normalized to 28S mRNA levels. SP mRNA contents are expressed as % of control values. Open bars: naive mice; filled bars: sensitized mice. n = 7-8 samples in each group. Means ± SEM were calculated after deriving the average of the results from two independent experiments. Each lane contains 10 µg total RNA. *P < 0.05 versus naive control.

The mechanism and physiologic significance of reduction in SP-B and SP-C during asthmatic inflammation are unknown and require further investigation. Although a series of studies appears to corroborate the hypothesis that surfactant dysfunction may be an important factor in obstruction of the asthmatic airways (10, 26) a role for dysregulation of hydrophobic SPs has not been raised. Previously proposed mechanisms involve infiltration of airways with plasma proteins resulting in altered surfactant phospholipid composition (26) and inhibition of surface activity (10, 11). Our findings extend this hypothesis by demonstrating that the hydrophobic SPs responsible for maintenance of surfactant activity (SP-B and SP-C) were inhibited during asthmatic inflammation at both the protein and mRNA levels. Thus, we suggest that the regulatory pathways of SP production are also affected after sensitization and challenge with the allergen. This mechanism is not unique for allergic airway inflammation, inasmuch as inhibition of hydrophobic SPs were demonstrated in other inflammatory diseases such as respiratory distress syndrome (27) and Pneumocistis carinii pneumonia (22, 23) and are attributed to the regulatory effects of proinflammatory cytokines, particularly of tumor necrosis factor- (28, 29). The hydrophobic SPs may play an important role in maintaining surface tension of the alveoli and presumably patency of the small conducting airways (10, 26). The beneficial effects of exogenous surfactant therapy have been indicated in a guinea-pig model (30) as well as in asthmatic patients (31). Thus, surfactant replacement may provide a useful adjunctive therapy in asthma.

Af-Induced Allergic Inflammation Is Associated with Increased SP-D Protein Expression

SP-D is a member of a novel, growing family of proteins that are believed to play a role in non-antibody-mediated innate immune response (2, 4). This family consists of SP-A and SP-D in the lung as well as mannose binding protein in the blood. Termed collectins, for collagen-like lectin, the primary function of these proteins appears to be modulation of host defense and inflammation. Previously, we have published reports that SP-A and SP-D expression are altered in response to P. carinii-induced pulmonary inflammation, supporting the concept that these lung collectins may play an active role in the inflammatory process (22, 23). After sensitization and exposure to Af we analyzed BALF for SP-A and SP-D levels and found that lung collectins were detectable in both LA and SA fractions of naive and sensitized mice by Western blot analysis. In the SA fraction, expression of the 43-kD SP-D protein was markedly increased in sensitized mice (P < 0.001, Figures 4A and 4B) with levels of the 35-kD SP-A remaining unaffected. In the LA surfactant fraction, levels of SP-A and SP-D were maintained during allergic inflammation (111 ± 9 and 148 ± 28% of control level, n = 10, respectively).

View larger version (15K): [in this window] [in a new window]   Figure 4.   Effect of allergic challenge on lung collectin protein expression. (A) Representative Western blots performed sequentially with polyclonal antisera against SP-A and SP-D visualized using enhanced chemiluminescence. Samples of SA surfactants were prepared from BALF of naive and sensitized mice as described; 29- to 35-kD SP-A doublet and 43-kD SP-D bands in LA and SA fractions. Each lane contains 5 µg total protein. (B) Quantification of SP-A (26 to 35 kD) and SP-D (43 kD) content. Densitometric scanning was performed from multiple blots and quantified as described. Open bars: naive mice; filled bars: sensitized mice. Data are expressed as % of control levels. n = 7-8 samples in each group. Means ± SEM were calculated after deriving the average of the results from two independent experiments. *P < 0.05. Regression analysis (C) was carried out between total IgE (x axis) and SP-D levels (y axis) (r = 0.851; P < 0.001).

The mechanism of SP-D upregulation by type II cells in allergic inflammation is unclear. In contrast to SP-B and SP-C mRNA expression, levels of the 0.9-kb SP-A and 1.2-kb SP-D mRNA were not significantly affected by allergic changes (115 ± 19 and 143 ± 27% of control level, n = 5, respectively). These results suggest that increases in SP-D protein release into the airways may be due largely to altered post-translational processes, augmented release, and/ or reduced reuptake by epithelial cells (32) during allergic inflammation. Although it has been reported that regulators of SP expression include a variety of mediators and cytokines that also play important roles in the asthmatic inflammation (33), the exact mechanism of heightened SP-D protein production in our model awaits further clarification. Interestingly, in transgenic mice overexpressing IL-4, the cytokine central to the development of allergic changes and IgE synthesis, levels of SP-D protein were also disproportionately elevated when compared with SP-D mRNA (35). Further, analysis of the associations between SP-D and components of allergic airway inflammation revealed a significant positive correlation with serum total IgE (r = 0.85; P < 0.001), indicating a close relationship and a possible common regulatory pathway (Figure 4C). Taken together, these may suggest a regulatory function for IL-4 in SP-D production. The selective upregulation of SP-D during allergic AHR, coupled with its ability to bind allergens (14), implies a possible role for this lung collectin in development of allergic airway inflammation.

This report is the first detailed characterization of altered SP production in a model of allergic inflammation and AHR. Further studies are needed to clarify the role of SPs in the pathogenesis of asthma.