Atypical development of the tracheal basement membrane zone of infant rhesus monkeys exposed to ozone and allergen

Michael J. Evans,1 Michelle V. Fanucchi,1 Gregory L. Baker,1 Laura S. Van Winkle,1 Lorraine M. Pantle,1 Susan J. Nishio,1 Edward S. Schelegle,1 Laurel J. Gershwin,1 Lisa A. Miller,1 Dallas M. Hyde,1 Philip L. Sannes,2 and Charles G. Plopper1

1Department of Anatomy, Physiology, and Cell Biology, School of Veterinary Medicine, Center for Comparative Respiratory Biology and Medicine, University of California, Davis, California 95616; and 2Department of Molecular Biomedical Sciences, College of Veterinary Medicine, North Carolina State University, Raleigh, North Carolina 27606

Submitted 30 May 2003 ; accepted in final form 19 June 2003


    ABSTRACT
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 DISCLOSURES
 REFERENCES
 
Development of the basement membrane zone (BMZ) occurs postnatally in the rhesus monkey. The purpose of this study was to determine whether house dust mite allergen (HDMA) plus ozone altered this process. Rhesus monkeys were exposed to a regimen of HDMA and/or ozone or filtered air for 6 mo. To detect structural changes in the BMZ, we measured immunoreactivity of collagen I. To detect functional changes in the BMZ, we measured perlecan and fibroblast growth factor-2 (FGF-2). We also measured components of the FGF-2 ternary signaling complex [fibroblast growth factor receptor-1 (FGFR-1) and syndecan-4]. The width of the BMZ was irregular in the ozone groups, suggesting atypical development of the BMZ. Perlecan was also absent from the BMZ. In the absence of perlecan, FGF-2 was not bound to the BMZ. However, FGF-2 immunoreactivity was present in basal cells, the lateral intercellular space (LIS), and attenuated fibroblasts. FGFR-1 immunoreactivity was downregulated, and syndecan-4 immunoreactivity was upregulated in the basal cells. This suggests that FGF-2 in basal cells and LIS may be bound to the syndecan-4. We conclude that ozone and HDMA plus ozone effected incorporation of perlecan into the BMZ, resulting in atypical development of the BMZ. These changes are associated with specific alterations in the regulation of FGF-2, FGFR-1, and syndecan-4 in the airway epithelial-mesenchymal trophic unit, which may be associated with the developmental problems of lungs associated with exposure to ozone.

perlecan; fibroblast growth factor-2; fibroblast growth factor receptor-1; syndecan-4; epithelial-mesenchymal trophic unit


THE BASEMENT MEMBRANE ZONE (BMZ) is the central structure of the epithelial-mesenchymal trophic unit. The epithelial-mesenchymal trophic unit consists of opposing layers of epithelial and mesenchymal cells separated by the BMZ (11, 13, 17). The primary trophic unit consists of a layer of basal cells, the BMZ, and the attenuated fibroblast sheath. Recognition of the attenuated fibroblast sheath as a distinct layer of resident fibroblasts is key to the concept of an epithelial-mesenchymal trophic unit. The BMZ has a number of functions in the epithelial-mesenchymal trophic unit. It is specialized for attachment of epithelium with the extracellular matrix; it also serves as a barrier, binds specific growth factors, hormones, and ions, and is involved with electrical charge and cell-cell communication (1, 8, 40). Binding and storage of growth factors is an important function of the BMZ. The exchange of information between the epithelium and fibroblasts in the epithelial-mesenchymal trophic unit occurs via the BMZ.

With transmission electron microscopy, the BMZ appears as three component layers: the lamina lucida, the lamina densa, and the lamina reticularis (LR). Together they make up the basal lamina. The LR is especially pronounced under the respiratory epithelium of large conducting airways, where it may be several micrometers thick. Collagen type I, III, and V form heterogeneous fibers that account for the thickness of the LR. The collagen fibers are arranged as a mat of large fibers oriented along the longitudinal axis of the airway. Smaller fibers are cross-linked with the larger fibers to complete this structure (14). The BMZ also has numerous pores (14, 19, 20). Heparan sulfate proteoglycans (perlecan) and chondroitin sulfate proteoglycans (bamacan) are an intrinsic part of the BMZ that are involved with most of its functions (37-39). Attenuated fibroblasts beneath the BMZ are thought to synthesize the collagen type I, III, and V components of the BMZ (11, 13, 17).

In previous studies, we found that development of the epithelial BMZ occurred postnatally in the rhesus monkey (10). The collagen BMZ increased in width from 1 to 6 mo, and perlecan was localized in the BMZ at all stages of development. Fibroblast growth factor-2 (FGF-2) was strongly expressed in basal cells at 1-3 mo but not in the BMZ. However, by 6 mo the distribution of FGF-2 had changed; FGF-2 was now expressed throughout the BMZ and weakly in basal cells. Fibroblast growth factor receptor-1 (FGFR-1) was present in basal cells and the nuclei of columnar cells throughout this time period. In rhesus monkeys treated with house dust mite allergen (HDMA) during this period of development, we found significant thickening of the tracheal BMZ (12). Both perlecan and FGF-2 were evenly distributed throughout the thickened BMZ. We also found that all HDMA tracheal samples expressed thin focal areas of the BMZ (<2.0 µm) associated with leukocyte trafficking. In these areas, the collagen BMZ was damaged and depleted of perlecan and FGF-2. However, in the adjacent basal cells, there was increased FGF-2 immunoreactivity. We concluded that basal cells and FGF-2 are involved with significant remodeling of the BMZ in the developing trachea of infant rhesus monkeys exposed to HDMA.

Remodeling of the epithelial BMZ involves increased deposition of subepithelial collagen, resulting in thickening of the BMZ (4, 5, 12, 36). Thickening of the BMZ is thought to protect against airway narrowing and air trapping (26). It is not known how thickening affects the various functions of the BMZ, such as its role in FGF-2 signaling. Ozone is an oxidant gas thought to act synergistically with allergens in airway remodeling. Two fundamental characteristics have been defined for ozone affects on the lung. First, postnatal animals, before weaning, are less susceptible to acute pulmonary injury than are adults. Second, chronic exposure to oxidant gases retards postnatal maturation of the lung. The purpose of the present study was to determine whether combined cyclic inhalation of ozone plus HDMA affected development of the BMZ in infant rhesus monkeys during the first postnatal 6 mo. From our previous study, we know this is a time when the BMZ is undergoing active development (10) and that HDMA alters the normal pattern of development (12). To detect structural changes in BMZ development, we measured collagen I. To detect functional changes in the BMZ, we measured the distribution of perlecan and FGF-2. To detect functional changes in the adjacent basal cells and attenuated fibroblasts, we measured the expression of FGFR-1 and the cell surface proteoglycan syndecan-4, both components of the FGF-2 ternary signaling complex (2, 30, 48). We found that ozone depleted BMZ perlecan and caused structural changes in the BMZ collagen. These changes altered the expression of FGF-2, FGFR-1, and syndecan-4 in the epithelial-mesenchymal trophic unit.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 DISCLOSURES
 REFERENCES
 
Animals and experimental protocol. All monkeys selected for these studies were California Regional Primate Research Center colony-born rhesus macaques (Macaca mulatta). Care and housing of animals complied with the provisions of the Institute of Laboratory Animal Resources and conforms to practices established by the American Association for Accreditation of Laboratory Animal Care.

Twenty-four infant rhesus monkeys (30 days old) were exposed to 11 episodes of either filtered air, HDMA aerosol, ozone, or HDMA plus ozone (5 days each followed by 9 days of filtered air). Ozone was delivered for 8 h/day at 0.5 parts per million. Twelve of the monkeys (HDMA and HDMA + ozone groups) were sensitized to HDMA (Dermatophagoides farinae) at age 14 and 28 days by subcutaneous inoculation (SQ) of HDMA in alum and intraperitoneal injection of heat-killed Bordetella pertussis cells. HDMA sensitization was confirmed via skin testing with SQ HDMA on day 38 of the exposure protocol. Sensitized monkeys were exposed to HDMA aerosol for 2 h/day on days 3-5 of either filtered air (HDMA, n = 6) or ozone (HDMA + ozone, n = 6) exposure. Nonsensitized monkeys were exposed to either filtered air (n = 6) or ozone (n = 6). Details of the ozone and allergen exposure procedures are given in another paper (42). Immunoreactivity for collagen I, perlecan, and FGF-2 in the filtered air and HDMA groups were presented in a previous paper concerning remodeling of the BMZ and leukocyte trafficking (12). Data from this paper are included here for comparison with the ozone treatment groups of this study.

Preparation of animals. After the exposure protocol, monkeys were killed with an overdose of pentobarbital sodium after being sedated with Telazol (8 mg/kg intramuscularly) and anesthetized with Diprivan (0.1-0.2 mg · kg-1 · min-1 intravenously). Monkeys were necropsied after exsanguination through the abdominal aorta, and the lungs were prepared for analysis as previously described (41). Tracheal samples were sliced perpendicular to the long axis of the airway into rings fixed in either 1.0 or 4.0% paraformaldehyde for 1 h and embedded in paraffin.

Immunohistochemistry. For routine histology, 5-µm sections were stained with hematoxylin and eosin. For immunohistochemistry, 5-µm sections were deparaffinized in xylene, hydrated in ethanol, and washed in PBS. For collagen, sections fixed in 1.0% paraformaldehyde (PFA) were treated with pepsin (1.0 mg pepsin/ml 3.0% acetic acid) at 37°C for 2 h, blocked with bovine serum albumin, and then treated with antibody to collagen I (1:250; rabbit anti-human polyclonal antibody; Biogenesis, Kingston, NH) overnight at 4°C. For perlecan, sections fixed in 1.0% PFA were treated with 0.1% Pronase in PBS for 30 min, rinsed in nanopure water followed by PBS, blocked with bovine serum albumin for 30 min, and incubated with an antibody to perlecan (1:2,000; mouse anti-human monoclonal antibody, clone 7B5; Zymed, San Francisco, CA) overnight at 4°C. For syndecan-4, sections fixed in 4.0% PFA were placed in citrate buffer and heated in a microwave oven at 95°C for 5 min and then placed in fresh buffer for 10 min at room temperature. Nonspecific binding was blocked with bovine serum albumin. The sections were treated with antibody to syndecan-4 (1:200; Santa Cruz Biotechnology) overnight at 4°C. For FGF-2, sections fixed in 1.0% PFA were treated with 0.1% H202 in methanol for 60 min, followed by 50 mg/ml of bovine testicular hyaluronidase in 0.05 M Tris buffer (pH 7.6) for 30 min. The sections were blocked with 5.0% horse serum for 30 min and incubated with an antibody to FGF-2 (1:750; mouse anti-human monoclonal antibody, clone bFM-2; Upstate Biotechnology, Lake Placid, NY) overnight at 4°C. For FGFR-1, sections fixed in 1.0% PFA were treated with 0.02% trypsin in PBS at room temperature for 30 min, washed, blocked with 25 µg of purified goat IgG in PBS for 60 min, and incubated with an antibody to FGFR-1 (1:500; rabbit anti-chicken polyclonal antibody; Upstate Biotechnology) overnight at 4°C. After immunohistochemistry, the sections were washed in PBS, treated with the secondary antibody (1: 1,000; Alexa Fluor 568; Molecular Probes, Eugene, OR) for 30 min, washed in PBS, and the coverslip was mounted in enzyme-linked fluorescence, fluorescent safe media (Molecular Probes). Fluorescence was visualized on an Olympus BH-2 fluorescent microscope.

Antibody specificity. The antibodies for collagen type I and V and FGFR-1 had negligible cross-reactivity with other collagens or noncollagen matrix proteins (per supplier). The antibody for collagen type III has no cross-reactivity with other collagens (16). The antibody for FGF-2 has no cross-reactivity with FGF-1 or heparin-binding growth factor-1 (25). The antibody for perlecan may cross-react with the short arm of laminin A and B chains (28). The antibody for syndecan-4 is specific for a peptide mapping within an internal region of syndecan-4.

Semiquantitation. The intensity of immunoreactivity for perlecan, FGF-2, FGFR-1, and syndecan-4 expression was graded on a scale of 0-3 for each animal (23). A scale of 0 indicates no staining beyond background, 1 indicates isolated areas of staining in the BMZ or cells, 2 indicates staining in 25-75% of the BMZ or cells, and 3 indicates strong staining in most of the BMZ or cells. Analysis of the tissues was performed in a blinded fashion. The mean and SD of perlecan, FGF-2, FGFR-1, and syndecan-4 expression for each group of animals were then determined.

Quantitation of BMZ width. The width of the BMZ was measured morphometrically to quantitate the immunohistochemical results. In human biopsy samples, it was demonstrated that 31-45 measurements, at least 20 µm apart and covering 1,000 µm of BMZ, are necessary to give an accuracy of ±15.0% (45). In this study, eight micrographs were taken equidistant apart around the circumference of the tracheas. The width of the BMZ was measured at four points, 50 µm apart, on each micrograph. A total of 1,600 µm of BMZ were sampled in each tracheal ring in this manner. The average width of the BMZ was determined from these measurements for each animal. To estimate the proportion of the BMZ that was thin, the percentage of measurements that was <2.0 µm was determined for each animal. The mean and SD of BMZ width and the percentage of the measurements <2.0 µm were then determined for each group of animals (10).

Statistics. The differences between treatment groups were compared with Mann-Whitney's rank sum test, with significance set at P < 0.05.


    RESULTS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 DISCLOSURES
 REFERENCES
 
Collagen I. The width of the BMZ in the filtered air group was 4.3 ± 0.7 µm. It increased in the HDMA group to 6.3 ± 0.8 µm. The width of the BMZ in the ozone group was 5.7 ± 1.2 µm and in the HDMA plus ozone group was 4.7 ± 1.1 µm. The width of the BMZ was irregular in each group but more so in the ozone and HDMA plus ozone groups (Fig. 1, A and B). Thin regions of the BMZ (<2.0 µm) made up 4.7 ± 3.3 and 4.7 ± 4.3% of the measurements in the filtered air and HDMA groups. In the ozone and HDMA plus ozone groups, thin regions of the BMZ made up 7.8 ± 3.8 and 16.7 ± 6.1% of the measurements (Fig. 2). It is not clear whether these thin areas represent regions of the BMZ damaged by leukocyte trafficking (12) or regions of the BMZ that had developed abnormally (10).



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Fig. 1. A: collagen I immunoreactivity in the basement membrane zone (BMZ; arrowheads) of a house dust mite allergen (HDMA)-treated monkey. Both of the surfaces are irregular. The mean width of the BMZ was 6.3 ± 0.8 µm. The percentage of the BMZ <2.0 µm in width was 4.7 ± 3.3. B: collagen I immunoreactivity of an HDMA + O3-treated monkey. Some areas of the BMZ appear normal (arrowheads) and other areas are thin (<2.0 µm) and incomplete (arrows). It is not clear whether these irregular areas represent regions of the BMZ damaged by leukocyte trafficking (12) or regions of the BMZ that had developed abnormally (10). The mean width of the BMZ was 4.7 ± 1.1 µm. The percentage of the BMZ <2.0 µm was 16.7 ± 6.1. Bar = 10 µm.

 


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Fig. 2. Graphic representation of the percentage of the BMZ that was <2.0 µm in width. There was a significant increase in the number of animals in the HDMA + O3 group with BMZ measurements that were <2.0 µm in width. *P < 0.05 vs. the filtered air (FA) group.

 

Perlecan. Immunoreactivity was evenly distributed throughout the epithelial BMZ, the walls of blood vessels, and smooth muscle bundles in both the filtered air and HDMA groups (Fig. 3A). The relative immunoreactivity was strong in the BMZ. In contrast, perlecan immunoreactivity was uniformly weak or absent from the epithelial BMZ of the ozone and HDMA plus ozone groups (Fig 3B). However, in the walls of blood vessels and smooth muscle bundles, it remained high. The decrease in the relative intensity of perlecan immunoreactivity in the epithelial BMZ of the O3 treatment groups, but not in the walls of blood vessels, suggests that perlecan was not being incorporated into the epithelial BMZ during development.



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Fig. 3. A: perlecan immunoreactivity in the BMZ of an HDMA-treated monkey. There is strong immunoreactivity in the epithelial BMZ (arrowheads) and in the walls of blood vessels (arrows). B: in the epithelial BMZ of an HDMA + O3-treated monkey (arrowheads), there is very little perlecan immunoreactivity; however, in the walls of blood vessels (arrows), it remained strong. Bar = 10 µm.

 

FGF-2. Immunoreactivity for FGF-2 in the epithelial BMZ mirrored that of perlecan in each treatment group. It was evenly distributed throughout the epithelial BMZ and the walls of blood vessels but not in smooth muscle bundles in the filtered air and HDMA groups (Fig. 4A). The relative immunoreactivity was strong in the epithelial BMZ but was less in the walls of blood vessels and smooth muscle bundles. In contrast, FGF-2 immunoreactivity was uniformly weak or absent from the epithelial BMZ of the ozone and HDMA plus ozone groups (Fig. 4B), whereas in the walls of blood vessels and smooth muscle bundles, it remained the same. The decrease in the relative intensity of FGF-2 immunoreactivity in the epithelial BMZ of the ozone and HDMA plus ozone groups suggests that FGF-2 was not being stored in the epithelial BMZ during development due to a lack of perlecan (Fig. 5). However, FGF-2 immunoreactivity was present in basal cells and the lateral intercellular space on the epithelial side of the BMZ. On the mesenchymal side of the BMZ, it was present in attenuated fibroblasts and other cells in the extracellular matrix of both ozone treatment groups. This is in contrast to the filtered air and HDMA groups in which FGF-2 immunoreactivity was relatively weak on both sides of the BMZ (Fig. 6).



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Fig. 4. A: fibroblast growth factor-2 (FGF-2) immunoreactivity in the BMZ of an FA-treated monkey. There is strong immunoreactivity in the epithelial BMZ (arrowheads). In the walls of blood vessels (arrow), it is less, and in the lateral intercellular space between basal cells and columnar cells (*), it is faint. B: in contrast, FGF-2 immunoreactivity was uniformly weak or absent from the epithelial BMZ (arrowheads) of O3-treated monkeys, whereas in the walls of blood vessels, it remained the same. Strong FGF-2 immunoreactivity was now present in basal cells (*) and the lateral intercellular space of the epithelium. On the mesenchymal side of the BMZ, strong immunoreactivity was present in attenuated fibroblasts (arrows) and other cells in the extracellular matrix. Bar = 10 µm.

 


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Fig. 5. Graphic representation of perlecan and FGF-2 immunoreactivity expression scores (means ± SD). In the ozone treatment groups, there was a significant decrease in the perlecan and FGF-2 scores. These findings suggest that perlecan was not being incorporated into the BMZ during development in the ozone groups. In the absence of perlecan, FGF-2 was not stored in the BMZ. *P < 0.05 vs. the FA group.

 


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Fig. 6. Graphic representation of FGF-2 immunoreactivity expression scores in the basal cells and attenuated fibroblast compartments (means ± SD). In the ozone treatment groups, there was a significant increase in expression scores in both the basal cell and attenuated fibroblast compartments. *P < 0.05 vs. the FA group.

 

Syndecan-4. Weak to moderate syndecan-4 immunoreactivity was expressed on the surface and cytoplasm of basal cells and occasional ciliated cells on the epithelial side of the BMZ in the filtered air and HDMA groups (Fig. 7A). Weak immunoreactivity was present in some attenuated fibroblasts on the mesenchymal side of the BMZ. However, in the ozone and HDMA plus ozone groups, syndecan-4 immunoreactivity was strong in the basal cells and lateral intercellular space on the epithelial side of the BMZ (Fig. 7B). Immunoreactivity in attenuated fibroblasts on the mesenchymal side remained the same. The relative intensity of basal cell syndecan-4 immunoreactivity was significantly more in the ozone and ozone plus HDMA groups than the filtered air or HDMA groups, suggesting that it had been upregulated in the ozone exposure groups. Syndecan-4 immunoreactivity mirrored that of FGF-2 in the epithelium, suggesting that FGF-2 normally bound to BMZ perlecan is now bound to basal cell-associated syndecan-4.



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Fig. 7. A: weak syndecan-4 immunoreactivity was expressed on the surface and cytoplasm of basal cells and lateral intercellular space (*) of FA-treated monkeys. B: in contrast, syndecan-4 immunoreactivity was strong in the basal cells and lateral intercellular space (*) of HDMA + O3-treated monkeys. Weak immunoreactivity was expressed by attenuated fibroblasts in all treatment groups. Bar = 10 µm.

 

FGFR-1. In both the filtered air and HDMA groups, FGFR-1 immunoreactivity was expressed on the surface and cytoplasm of basal cells (Fig. 8A). It was also expressed in ciliated and goblet cell nuclei, cilia, and in some basal cell nuclei similar to that seen in developing BMZ (10). In contrast, FGFR-1 immunoreactivity was reduced in the basal cells and columnar cell nuclei in the ozone and HDMA plus ozone groups (Fig. 8B). However, it was still strong in the cilia. The relative intensity of FGFR-1 immunoreactivity was less in the ozone and ozone plus HDMA groups compared with the filtered air and HDMA groups, suggesting that it had been downregulated in the ozone exposure groups. Immunoreactivity for FGFR-1 was inversely related to that of syndecan-4 in the epithelium (Fig. 9). Immunoreactivity was not present in attenuated fibroblasts in any of the treatment groups.



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Fig. 8. A: strong fibroblast growth factor receptor-1 (FGFR-1) immunoreactivity was expressed on the surface and cytoplasm of basal cells (*) and cilia in FA-treated monkeys. Weak immunoreactivity was expressed in ciliated and goblet cell nuclei and in some basal cell nuclei. B: in contrast, FGFR-1 immunoreactivity was reduced to isolated groups of basal cells (*) in HDMA + O3-treated monkeys. Bar = 10 µm.

 


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Fig. 9. Graphic representation syndecan-4 and FGFR-1 immunoreactivity expression scores in basal cells. Immunoreactivity for syndecan-4 was inversely related to that of FGFR-1 in the epithelium. There was a significant increase in syndecan-4 scores in the ozone treatment groups. Conversely, there was a significant decrease in FGFR-1 scores. *P < 0.05 vs. the FA group.

 


    DISCUSSION
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 DISCLOSURES
 REFERENCES
 
In contrast to the previous study (12), the tracheal BMZ was significantly different in monkeys exposed to either ozone or HDMA plus ozone during this same developmental period. The collagen BMZ was irregular and thin in many areas, and perlecan was depleted or severely reduced in BMZ. These changes were present throughout the entire BMZ, and not only with areas of leukocyte trafficking, as seen in animals exposed to HDMA (12). Only the epithelial BMZ was depleted of perlecan; the BMZ around blood vessels and smooth muscle cells was not affected. This suggests that cells associated with synthesis of the epithelial BMZ perlecan are the main cells affected by the ozone. Examples of perlecan depletion in the BMZ have been reported in perlecan knockout mice (6), lung tumors (squamous cell carcinoma and adenocarcinoma) (29), and atheroslerotic blood vessels (32). Also, in a previous study, total proteoglycans were shown to be decreased in rats chronically exposed to ozone (34). In perlecan knockouts, there is BMZ deterioration in blood vessels in areas of mechanical stress (such as the heart), which leads to bleeding and death of the animal (6). These studies demonstrate that perlecan depletion is associated with abnormal development of the BMZ, similar to what we found in monkeys exposed to either ozone or HDMA plus ozone.

FGF-2 is the main growth factor stored in the BMZ (10, 15, 40). FGF-2 is stored in the BMZ through binding with perlecan, a heparan sulfate proteoglycan that is an intrinsic constituent of the BMZ. FGF-2 is stored in the BMZ of airway and alveolar epithelium, endothelium, and smooth muscle cells in the lungs of developing and adult rats (10, 33, 38). Presumably, it is stored in the BMZ for rapid cellular responses to changes in local environmental conditions, such as leukocyte trafficking or sloughing of columnar epithelium (3, 22, 30, 46). It can be released from perlecan in response to various conditions and become an important cytokine within the local microenvironment of the epithelial-mesenchymal trophic unit (3, 9, 27, 47). The significance of FGF-2 signaling in airway epithelium has not been determined. However, it may be associated with regulation of a number of molecules associated with airway remodeling, e.g., FGFs, epidermal growth factor, endothelin-1, and transforming growth factor-{beta} (17).

In the BMZ depleted of perlecan, FGF-2 was not stored but instead was present around basal cells and lateral intercellular space in the epithelium and attenuated fibroblasts in the extracellular matrix. Here, it may be bound to cell surface proteoglycans, such as glypican or syndecan or FGF receptors. Basal cells express the cell surface proteoglycan, syndecan-4 (29), the most widespread member of the syndecan family (7, 48). Syndecan-4 expression is rapidly upregulated in injured tissues, and shedding of the syndecan-4 ectodomain into the surrounding extracellular matrix is stimulated. These ectodomains in the extracellular matrix exist as soluble regulatory macromolecules in the tissue. Syndecan-4 ectodomains in wound fluids bind to growth factors, proteases, and protease inhibitors (2, 31, 35). The results of the present study suggest that syndecan-4 was upregulated in ozone and HDMA plus ozone groups and possibly shed into the lateral intercellular space. In contrast, FGFR-1 was down-regulated. Immunoreactivity for FGF-2 was colocalized with syndecan-4 immunoreactivity and basal cells. These findings suggest that syndecan-4 could act as a regulatory molecule to sequester and control FGF-2 signaling in the absence of perlecan.

In addition to atypical development of the BMZ, lack of BMZ perlecan probably also affected other developmental processes in the airway due to its regulatory role in FGF-2 signaling. FGF-2, an important cytokine in development that is stored in the BMZ through binding with perlecan, can be released from perlecan in response to various conditions and function as an important regulatory cytokine in the epithelial-mesenchymal trophic unit. FGF signal through a ternary complex that consists of FGF plus FGFR plus heparan sulfate proteoglycan (30). When the FGF ternary complex is formed, it initiates tyrosine kinase signaling associated with cell proliferation, migration, and differentiation. In airway epithelium, basal cells are the main cell type involved with FGF-2 ternary complex formation and signaling (Fig. 10). Basal cells express the cell surface receptors FGFR-1 (10, 21, 33) and syndecan-4. Syndecan-4, in combination with FGFR-1, has been shown to selectively regulate FGF-2 signaling (18, 43, 50). In the absence of perlecan, syndecan-4 was upregulated and FGFR-1 downregulated. The inverse relationship between these two molecules and the abundance of FGF-2 in the epithelium suggests that signaling through the FGF-2 ternary complex may have been negatively influenced by a lack of BMZ perlecan.



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Fig. 10. Illustration of BMZ-bound FGF-2, extracellular signaling via diffusion or FGF-binding protein (FGF-BP), and formation of the FGF-2 ternary complex with basal cells of airway epithelium. [Adapted from Nugent and Iozzo (30).]

 

Altered regulation of FGF-2 signaling in animals exposed to ozone may be associated with the abnormal development of airways that has been reported. For example, ozone exposure reduces the postnatal morphogenesis of the gas exchange area (44), impairs bronchiolar formation (10, 49), and retards the differentiation of the mucociliary apparatus of proximal airways (24). Recently, we showed that the combined cyclic inhalation of ozone and HDMA, by HDMA-sensitized infant monkeys, resulted in a marked increase in serum IgE and histamine and airway eosinophilia (42). Furthermore, combined cyclic inhalation of ozone and HDMA resulted in alteration of airway structure that was associated with a significant increase in baseline airway resistance and reactivity. These results indicate that ozone can amplify the allergic and structural remodeling effects of HDMA sensitization and inhalation.

In conclusion, we found that exposure to ozone and HDMA plus ozone depleted the BMZ of perlecan and caused atypical development of the epithelial BMZ. This resulted in altered regulation of FGF-2, FGFR-1, and syndecan-4 in the airway epithelial-mesenchymal trophic unit. We suggest that these alterations in FGF-2 regulation may be associated with the atypical development of the lung observed in the rhesus monkey after exposure to O3 and may constitute an important mechanism that modulates responses to injury.


    DISCLOSURES
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 DISCLOSURES
 REFERENCES
 
This work was supported by National Institutes of Health Grants P01-ES-00628, P01-ES-11617, ES-04311, ES-06700, ES-05707, HL-44497, and RR-000169 and American Lung Association and Environmental Protection Agency Grant R82744 [GenBank] 2-01-0.


    FOOTNOTES
 

Address for reprint requests and other correspondence: M. J. Evans, Dept. of Anatomy, Physiology, and Cell Biology, School of Veterinary Medicine, Univ. of California, Davis, CA 95616 (E-mail: mevans{at}ucdavis.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


    REFERENCES
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 DISCLOSURES
 REFERENCES
 

  1. Adachi E, Hopkinson I, and Hayashi T. Basement-membrane stromal relationships: interactions between collagen fibrils and the lamina densa. Int Rev Cytol 173: 73-156, 1997.[ISI][Medline]
  2. Bass MD and Humphries MJ. Cytoplasmic interactions of syndecan-4 orchestrate adhesion receptor and growth factor receptor signalling. Biochem J 368: 1-15, 2002.[ISI][Medline]
  3. Bikfalvi A, Klein S, Pintucci G, and Rifkin DB. Biological roles of fibroblast growth factor-2. Endocr Rev 18: 26-45, 1997.[Abstract/Free Full Text]
  4. Bousquet J, Jeffery PK, Busse WW, Johnson M, and Vignola AM. Asthma. From bronchoconstriction to airway inflammation and remodeling. Am J Respir Crit Care Med 161: 1720-1745, 2000.[Free Full Text]
  5. Brewster CE, Howarth PH, Djukanovic R, Wilson J, Holgate ST, and Roche WR. Myofibroblasts and subepithelial fibrosis in bronchial asthma. Am J Respir Cell Mol Biol 3: 507-511, 1990.[ISI][Medline]
  6. Costell M, Gustafsson E, Aszodi A, Morgelin M, Bloch W, Hunziker E, Addicks K, Timpl R, and Fassler R. Perlecan maintains the integrity of cartilage and some basement membranes. J Cell Biol 147: 1109-1122, 1999.[Abstract/Free Full Text]
  7. Couchman JR, Chen L, and Woods A. Syndecans and cell adhesion. Int Rev Cytol 207: 113-150, 2001.[ISI][Medline]
  8. Crouch EC, and Martin GR, Brody JS, and Laurie GW. Basement membranes. In: Lung, edited by Crystal RG, West JB, Wiebel ER, and Barnes PJ. Philadelphia, PA: Lippencott Raven, 1997, p. 769-791.
  9. Dowd CJ, Cooney CL, and Nugent MA. Heparan sulfate mediates bFGF transport through basement membrane by diffusion with rapid reversible binding. J Biol Chem 274: 5236-5244, 1999.[Abstract/Free Full Text]
  10. Evans MJ, Fanucchi MV, Van Winkle LS, Baker GL, Murphy AE, Nishio SJ, Sannes PL, and Plopper CG. Fibroblast growth factor-2 during postnatal development of the tracheal basement membrane zone. Am J Physiol Lung Cell Mol Physiol 283: L1263-L1270, 2002.[Abstract/Free Full Text]
  11. Evans MJ, Guha SC, Cox RA, and Moller PC. Attenuated fibroblast sheath around the basement membrane zone in the trachea. Am J Respir Cell Mol Biol 8: 188-192, 1993.[ISI][Medline]
  12. Evans MJ, Van Winkle LS, Fanucchi MV, Baker GL, Murphy AE, Nishio SJ, Schelegle ES, Gershwin LJ, Sannes PL, and Plopper CG. Fibroblast growth factor-2 in remodeling of the developing basement membrane zone in the trachea of infant rhesus monkeys sensitized and challenged with allergen. Lab Invest 82: 1747-1754, 2002.[ISI][Medline]
  13. Evans MJ, Van Winkle LS, Fanucchi MV, and Plopper CG. The attenuated fibroblast sheath of the respiratory tract epithelial-mesenchymal trophic unit. Am J Respir Cell Mol Biol 21: 655-657, 1999.[Free Full Text]
  14. Evans MJ, Van Winkle LS, Fanucchi MV, Toskala E, Luck EC, Sannes PL, and Plopper CG. Three-dimensional organization of the lamina reticularis in the rat tracheal basement membrane zone. Am J Respir Cell Mol Biol 22: 393-397, 2000.[Abstract/Free Full Text]
  15. Folkman J, Klagsbrun M, Sasse J, Wadzinski M, Ingber D, and Vlodavsky I. A heparin-binding angiogenic protein-basic fibroblast growth factor-is stored within basement membrane. Am J Pathol 130: 393-400, 1988.[Abstract]
  16. Godfrey M, Keene DR, Blank E, Hori H, Sakai LY, Sherwin LA, and Hollister DW. Type II achondrogenesis-hypochondrogenesis: morphologic and immunohistopathologic studies. Am J Hum Genet 43: 894-903, 1988.[ISI][Medline]
  17. Holgate ST, Davies DE, Lackie PM, Wilson SJ, Puddicombe SM, and Lordan JL. Epithelial-mesenchymal interactions in the pathogenesis of asthma. J Allergy Clin Immunol 105: 193-204, 2000.[ISI][Medline]
  18. Horowitz A, Tkachenko E, and Simons M. Fibroblast growth factor-specific modulation of cellular response by syndecan-4. J Cell Biol 157: 715-725, 2002.[Abstract/Free Full Text]
  19. Howat WJ, Barabas T, Holmes JA, Holgate ST, and Lackie PM. Distribution of basement membrane pores in bronchus revealed by microscopy following epithelial removal. J Struct Biol 139: 137-145, 2002.[ISI][Medline]
  20. Howat WJ, Holmes JA, Holgate ST, and Lackie PM. Basement membrane pores in human bronchial epithelium: a conduit for infiltrating cells? Am J Pathol 158: 673-680, 2001.[Abstract/Free Full Text]
  21. Hughes SE and Hall PA. Immunolocalization of fibroblast growth factor receptor 1 and its ligands in human tissues. Lab Invest 69: 173-182, 1993.[ISI][Medline]
  22. Iozzo RV. Matrix proteoglycans: from molecular design to cellular function. Annu Rev Biochem 67: 609-652, 1998.[ISI][Medline]
  23. Kranenburg AR, De Boer WI, Van Krieken JH, Mooi WJ, Walters JE, Saxena PR, Sterk PJ, and Sharma HS. Enhanced expression of fibroblast growth factors and receptor FGFR-1 during vascular remodeling in chronic obstructive pulmonary disease. Am J Respir Cell Mol Biol 27: 517-525, 2002.[Abstract/Free Full Text]
  24. Mariassy AT, Sielczak MW, McCray MN, Abraham WM, and Wanner A. Effects of ozone on lamb tracheal mucosa. Quantitative glycoconjugate histochemistry. Am J Pathol 135: 871-879, 1989.[Abstract]
  25. Matsuzaki K, Yoshitake Y, Matuo Y, Sasaki H, and Nishikawa K. Monoclonal antibodies against heparin-binding growth factor II/basic fibroblast growth factor that block its biological activity: invalidity of the antibodies for tumor angiogenesis. Proc Natl Acad Sci USA 86: 9911-9915, 1989.[Abstract]
  26. Milanese M, Crimi E, Scordamaglia A, Riccio A, Pellegrino R, Canonica GW, and Brusasco V. On the functional consequences of bronchial basement membrane thickening. J Appl Physiol 91: 1035-1040, 2001.[Abstract/Free Full Text]
  27. Mongiat M, Otto J, Oldershaw R, Ferrer F, Sato JD, and Iozzo RV. Fibroblast growth factor-binding protein is a novel partner for perlecan protein core. J Biol Chem 276: 10263-10271, 2001.[Abstract/Free Full Text]
  28. Murdoch AD, Liu B, Schwarting R, Tuan RS, and Iozzo RV. Widespread expression of perlecan proteoglycan in basement membranes and extracellular matrices of human tissues as detected by a novel monoclonal antibody against domain III and by in situ hybridization. J Histochem Cytochem 42: 239-249, 1994.[Abstract/Free Full Text]
  29. Nackaerts K, Verbeken E, Deneffe G, Vanderschueren B, Demedts M, and David G. Heparan sulfate proteoglycan expression in human lung cancer cells. Int J Cancer 74: 335-345, 1997.[ISI][Medline]
  30. Nugent MA and Iozzo RV. Fibroblast growth factor-2. Int J Biochem Cell Biol 32: 115-120, 2000.[ISI][Medline]
  31. Park PW, Reizes O, and Bernfield M. Cell surface heparan sulfate proteoglycans: selective regulators of ligand-receptor encounters. J Biol Chem 275: 29923-29926, 2000.[Free Full Text]
  32. Pillarisetti S. Lipoprotein modulation of subendothelial heparan sulfate proteoglycans (perlecan) and atherogenicity. Trends Cardiovasc Med 10: 60-65, 2000.[ISI][Medline]
  33. Powell PP, Wang CC, Horinouchi H, Shepherd K, Jacobson M, Lipson M, and Jones R. Differential expression of fibroblast growth factor receptors 1 to 4 and ligand genes in late fetal and early postnatal rat lung. Am J Respir Cell Mol Biol 19: 563-572, 1998.[Abstract/Free Full Text]
  34. Radhakrishnamurthy B. Consequences of prolonged inhalation of ozone on F344/N rats: collaborative studies. Part III: effects on complex carbohydrates of lung connective tissue. Res Rep Health Eff Inst: 3-14; discussion 15-23, 1994.
  35. Rapraeger AC. Molecular interactions of syndecans during development. Semin Cell Dev Biol 12: 107-116, 2001.[ISI][Medline]
  36. Redington AE. Fibrosis and airway remodelling. Clin Exp Allergy 30, Suppl 1: 42-45, 2000.[ISI][Medline]
  37. Roberts CR, Wright TN, and Hascall VC. Proteoglycans. In: Lung, edited by Crystal RG, West JB, Wiebel ER, and Barnes PJ. Philadelphia, PA: Lippincott Raven, 1997, p. 757-767.
  38. Sannes PL, Burch KK, and Khosla J. Immunohistochemical localization of epidermal growth factor and acidic and basic fibroblast growth factors in postnatal developing and adult rat lungs. Am J Respir Cell Mol Biol 7: 230-237, 1992.[ISI][Medline]
  39. Sannes PL, Burch KK, Khosla J, McCarthy KJ, and Couchman JR. Immunohistochemical localization of chondroitin sulfate, chondroitin sulfate proteoglycan, heparan sulfate proteoglycan, entactin, and laminin in basement membranes of postnatal developing and adult rat lungs. Am J Respir Cell Mol Biol 8: 245-251, 1993.[ISI][Medline]
  40. Sannes PL and Wang J. Basement membranes and pulmonary development. Exp Lung Res 23: 101-108, 1997.[ISI][Medline]
  41. Schelegle ES, Gershwin LJ, Miller LA, Fanucchi MV, Van Winkle LS, Gerriets JP, Walby WF, Omlor AM, Buckpitt AR, Tarkington BK, Wong VJ, Joad JP, Pinkerton KB, Wu R, Evans MJ, Hyde DM, and Plopper CG. Allergic asthma induced in rhesus monkeys by house dust mite (Dermatophagoides farinae). Am J Pathol 158: 333-341, 2001.[Abstract/Free Full Text]
  42. Schelegle ES, Miller LA, Gershwin LJ, Fanucchi MV, Van Winkle LS, Gerriets JP, Walby WF, Omlor AM, Tarkington BK, Wong VJ, Baker GL, Pantle L, Joad JP, Pinkerton KB, Wu R, Evans M, Hyde D, and Plopper CG. Repeated episodes of ozone inhalation amplifies the effects of allergen sensitization and inhalation on airway immune and structural developments in rhesus monkeys. Toxicol Appl Pharmacol. In press.
  43. Simons M and Horowitz A. Syndecan-4-mediated signalling. Cell Signal 13: 855-862, 2001.[ISI][Medline]
  44. Stiles J and Tyler WS. Age-related morphometric differences in responses of rat lungs to ozone. Toxicol Appl Pharmacol 92: 274-285, 1988.[ISI][Medline]
  45. Sullivan P, Stephens D, Ansari T, Costello J, and Jeffery P. Variation in the measurements of basement membrane thickness and inflammatory cell number in bronchial biopsies. Eur Respir J 12: 811-815, 1998.[Abstract/Free Full Text]
  46. Taipale J and Keski-Oja J. Growth factors in the extracellular matrix. FASEB J 11: 51-59, 1997.[Abstract/Free Full Text]
  47. Tassi E, Al-Attar A, Aigner A, Swift MR, McDonnell K, Karavanov A, and Wellstein A. Enhancement of fibroblast growth factor (FGF) activity by an FGF-binding protein. J Biol Chem 276: 40247-40253, 2001.[Abstract/Free Full Text]
  48. Tumova S, Woods A, and Couchman JR. Heparan sulfate proteoglycans on the cell surface: versatile coordinators of cellular functions. Int J Biochem Cell Biol 32: 269-288, 2000.[ISI][Medline]
  49. Tyler WS, Tyler NK, Last JA, Gillespie MJ, and Barstow TJ. Comparison of daily and seasonal exposures of young monkeys to ozone. Toxicology 50: 131-144, 1988.[ISI][Medline]
  50. Volk R, Schwartz JJ, Li J, Rosenberg RD, and Simons M. The role of syndecan cytoplasmic domain in basic fibroblast growth factor-dependent signal transduction. J Biol Chem 274: 24417-24424, 1999.[Abstract/Free Full Text]