RAPID COMMUNICATION
Altered lung gene expression in CCSP-null mice suggests immunoregulatory roles for Clara cells

T. M. Watson1, S. D. Reynolds2, G. W. Mango1, I.-M. Boe3, J. Lund3, and B. R. Stripp2

1 Division of Respiratory Biology and Toxicology, Department of Environmental Medicine, University of Rochester School of Medicine and Dentistry, Rochester, New York 14642; 2 Department of Environmental and Occupational Health, University of Pittsburgh, Pittsburgh, Pennsylvania 15260; and 3 Department of Cell Biology and Anatomy, University of Bergen, Bergen, N5009 Norway


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Clara cell secretory protein (CCSP) is one of the most abundant proteins present in airway lining fluid of mammals. In an effort to elucidate the function of CCSP, we established CCSP-null [CCSP(-/-)] mice and demonstrated altered sensitivity to various environmental agents including oxidant pollutants and microorganisms. Although CCSP deficiency itself may be central to the observed changes in environmental susceptibility, altered lung gene expression associated with CCSP deficiency may contribute to the observed phenotype. To determine whether CCSP deficiency results in altered lung gene expression, high-density cDNA microarrays were used to profile gene expression in the total lung RNA of wild-type and CCSP(-/-) mice. Genes that were differentially expressed between wild-type and CCSP(-/-) mice included a previously nonannotated expressed sequence tag (EST W82219) and immunoglobulin A (IgA), both of which were elevated with CCSP deficiency. mRNA expression of EST W82219 and IgA was localized in the lungs of wild-type and CCSP(-/-) mice to airway Clara cells and peribronchial lymphoid tissues, respectively. We conclude that CCSP deficiency is associated with 1) altered gene expression in Clara cells of the conducting airway epithelium and 2) alterations to peribronchial B lymphocytes. These findings identify new roles for Clara cells and their secretions in airway homeostasis.

10-kDa Clara cell secretory protein; secretoglobin; uteroglobin; hyperoxia; oxidant injury


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

CLARA CELL SECRETORY PROTEIN (CCSP), otherwise commonly known as uteroglobin, is the prototypical member of a family of secretory proteins that have recently been termed secretoglobins (16). The predominant site of CCSP expression is in the nonciliated airway epithelial (Clara) cell of the conducting airway (3, 17). However, CCSP expression has been observed among other secretory cell types in the epithelia of both the respiratory and genitourinary tracts in certain species (6, 17). Sequence polymorphisms associated with the human CCSP gene are associated with variability in the levels of CCSP expression and have been associated with susceptibility to asthma and glomerulonephropathy (18, 42). Moreover, extensive studies of human patients have shown a relationship between chronic lung disease, such as that observed among asthmatic patients and individuals with chronic obstructive pulmonary disease (COPD), and reductions in lung and serum CCSP levels (reviewed in Ref. 10).

Many potential functions have been ascribed to CCSP, with a role as an anti-inflammatory mediator the most frequently cited (25, 27, 28). This assertion is based on a combination of in vitro and in vivo studies, including the findings that CCSP is a potential regulator of secretory phospholipase A2, that it has anticytokine activities, and that peptides derived from the CCSP sequence exhibit anti-inflammatory properties (21, 26, 29). However, despite the numerous properties described for CCSP, many remain controversial (1, 36). Stripp et al. (39) and others (48) have established CCSP-null [CCSP(-/-)] mice to define in vivo functions for CCSP. The pulmonary phenotype of CCSP(-/-) mice includes alterations in Clara cell ultrastructure (39, 41), susceptibility to oxidants (13, 23) and resistance to microorganisms (9), all of which are associated with changes in inflammatory responses after challenge. However, the finding that CCSP(-/-) mice show identical inflammatory responses to agents such as endotoxin (12) argues against a direct role for CCSP in the regulation of pulmonary inflammation per se.

Interestingly, the virtually undetectable steady-state phenotype of CCSP(-/-) mice generated by Stripp et al. (39) and further characterized by Reynolds et al. (32) is in stark contrast to the severe phenotype, including wasting, multiorgan damage, and premature mortality, observed in an independently generated line of CCSP(-/-) mice (48). The phenotype of CCSP(-/-) mice generated by Zhang et al. (48) was of variable penetrance, yet kidney defects observed in these mice could be reproduced with an antisense strategy to reduce levels of CCSP expression (49). Variable phenotypes observed among different mouse models of CCSP deficiency highlight the importance of other genetic and epigenetic factors in dictating the phenotypic outcome.

Phenotypic differences between mouse models of CCSP deficiency, coupled with loose associations between CCSP deficiency and human disease, have led us to hypothesize that CCSP deficiency among CCSP(-/-) mice is associated with the altered expression of other genes in the lung. If correct, understanding the identity and localization of differentially expressed genes would provide new insights into cellular and molecular changes resulting from CCSP deficiency and the impact of these genes on susceptibility to environmental agents. This hypothesis was tested in the present study through screening high-density cDNA microarrays to profile and compare gene expression between steady-state wild-type (WT) and CCSP(-/-) mice. We demonstrate that CCSP deficiency is associated with changes in the abundance of mRNA species in the lung. In this study, we demonstrate that CCSP deficiency is associated with altered gene expression in Clara cells and bronchus-associated lymphoid tissues (BALT).


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Animals. Strain 129 CCSP(-/-) mice (32, 39) and strain-matched WT control mice were maintained as specific pathogen-free in-house colonies. All animals were housed in humidity- and temperature-controlled rooms on a 12:12-h light-dark cycle and were allowed food and water ad libitum. Representative mice were screened quarterly for pathogens represented on a 16-agent serological panel (Microbiological Associates, Rockville, MD). Animals used in this study were 8-12 wk of age.

RNA isolation and gene microarray. Six WT and six CCSP(-/-) mice were killed with 100 mg/kg of pentobarbital sodium. The animals were perfused with saline, and the lungs were removed and homogenized in 4 M guanidinium. RNA was extracted as previously described (5). Polyadenylated RNA was selected from 100 µg of total lung RNA from six WT and six CCSP(-/-) mice and used to probe a mouse GEM1 cDNA microarray representing 8,700 cDNAs (Incyte Pharmaceuticals, Palo Alto, CA). Standard labeling and hybridization conditions established by the manufacturer were employed. Differences in probe-labeling efficiency were accounted for through derivation of balanced coefficients, and the WT fluorescence intensity and balanced CCSP(-/-) fluorescence intensity were reported. These values were used to calculate the ratio of signal intensities between CCSP(-/-) and WT samples.

Northern hybridization. Two criteria were used to select clones for validation by Northern blot analysis. Clones with a balanced differential expression equal to or greater than 1.9 or equal to or less than -1.9 were identified, and the 20 clones with the highest P1 or balanced P2 intensities were selected for further analysis. RNA was prepared as described for the gene microarray except that the tissue was not perfused. Five micrograms of total lung RNA from one WT or CCSP(-/-) mouse were separated by electrophoresis through formaldehyde gels (34) and transferred to Durolon membranes (Stratagene, La Jolla, CA). Plasmids were prepared by standard methodology, and inserts were isolated after double digestion with NotI and EcoR1. Inserts were labeled with [32P]dCTP with a random-primer labeling kit (GIBCO BRL, Rockville, MD). Hybridization was carried out at the calculated melting temperature (Tm) of -25°C, and blots were washed at Tm -10°C, as previously described (32). Hybridized membranes were exposed to film (Kodak, Rochester, NY), and band intensities were analyzed with a phosphorimager and ImageQuant software (Molecular Dynamics, Sunnyvale, CA). Blots were rehybridized with beta -actin or L32 for normalization. Differential expression of two clones, expressed sequence tag (EST) W82219 and immunoglobulin A (IgA) heavy chain constant region was further evaluated in six WT and CCSP(-/-) mice by Northern blot hybridization as described above. Expression of EST W82219 in WT and CCSP(-/-) heart, intestine, kidney, liver, lung, salivary gland, spleen, testis, and thymus was assessed by Northern blot hybridization as described above.

In situ hybridization. Mice were killed with 100 mg/kg of pentobarbital sodium and exsanguinated. The lungs were inflation fixed with neutral buffered formalin through a tracheal cannula for 10 min at 10 cmH2O pressure. Lungs were then immersed in formalin for 16 h, immersed in PBS for 24 h, dehydrated, and paraffin embedded. Paraffin-embedded lung tissue was cut at 5 µm and placed onto Superfrost slides. 35S-labeled sense and antisense riboprobes (6 × 108 dpm/µg) were generated from rat CCSP (38) or mouse cytochrome P-450 2F2 (CYP2F2) cDNA (40) cloned into pGEM3Z and from EST W82219 and IgA cDNA cloned into pT3T7 with a riboprobe transcription kit (Promega, Madison, WI). Conditions and solutions for hybridization were essentially as previously described (43). Hybridization was carried out overnight at 54°C, and the slides were washed under high-stringency conditions. The slides were dipped in Kodak NTB2 emulsion, exposed for 16 h, and developed with Kodak D19 developer following the manufacturer's protocol. Images from WT and CCSP(-/-) tissue were captured under identical conditions, and pseudocolored silver grains (red) were superimposed over the bright-field image in Adobe Photoshop. Sense probes were used as negative controls and demonstrated no nonspecific hybridization under the conditions used.

Dual in situ hybridization and immunohistochemistry. WT tissue was hybridized as described in In situ hybridization with the exception that 3H-labeled riboprobes were used. After RNase treatment, the sections were blocked and incubated with rabbit anti-rat CCSP (1:16,000) overnight at 4°C. Antigen-antibody complexes were detected as previously described (32) with biotinylated goat anti-rabbit Ig secondary antibody, streptavidin-horseradish peroxidase, and diaminobenzidine substrate. The slides were then washed sequentially at 65°C for 30 min in 50% formamide-2× SSC-10 mM dithiothreitol (DTT), at room temperature for 15 min in 2× SSC-10 mM DTT, and at room temperature for 15 min in 0.1× SSC-10 mM DTT. Autoradiography and imaging were done as described in In situ hybridization.

IgA ELISA. Six WT and six CCSP(-/-) mice were lavaged twice with 1 ml of saline. Cells were pelleted at 300 g, and the supernatants were concentrated two- to fourfold with Centricon 10 microconcentrators (Amicon, Beverly, MA). Serum was obtained from a similar number of WT and CCSP(-/-) mice. Protein concentrations for lavage fluid and serum were determined by bicinchoninic acid assay (Pierce, Rockford, IL). Lavage fluid and serum IgA concentrations were determined with an IgA ELISA kit as described by the manufacturer (Bethyl Laboratories, Montgomery, TX). All samples were assayed in triplicate, and IgA levels were normalized to protein concentration. Significance was determined by Student's t-test.


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Altered lung gene expression associated with CCSP deficiency. Our laboratory and others have previously demonstrated that CCSP deficiency among CCSP(-/-) mice is associated with ultrastructural changes to Clara cells (39, 41) and susceptibility to inhaled oxidant gases (23, 48). Experiments performed in the present study were designed to test the hypothesis that CCSP deficiency is associated with changes in lung gene expression and that the identity of differentially expressed genes would provide insight into functions for Clara cells and/or CCSP in lung homeostasis. Differences in steady-state lung expression of 8,700 genes were determined for pools of six adult male strain 129 CCSP(-/-) mice and six age-, sex-, and strain-matched WT control mice with Incyte mouse GEM1 cDNA microarrays (Fig. 1). Differentially expressed genes were those whose estimated mRNA abundance was increased by at least 190% or decreased by at least 53% in total lung RNA from CCSP(-/-) mice relative to that in WT control mice as suggested by Incyte Pharmaceuticals (24). To validate these findings, five downregulated genes and five upregulated genes were selected for Northern blot analysis based on their abundance in total lung RNA of CCSP(-/-) and WT mice. Fluorescence intensity (Table 1) was used as an index of abundance for the corresponding mRNA. Northern hybridization indicated that only 2 of the 10 genes identified as differentially expressed through microarray analysis could be confirmed with a standard quantitative methodology (Table 1). No further analysis of quantitative gene expression data from microarray screening was undertaken without prior validation with alternative approaches to confirm differential gene expression.


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Fig. 1.   Differential gene expression in wild-type (WT) and Clara cell secretory protein-null [CCSP(-/-)] mice. Gene expression in pools of total lung RNA from 6 WT and 6 CCSP(-/-) mice were compared by hybridization to a GEM1 cDNA microarray. Balanced CCSP(-/-) fluorescence intensities were compared with WT fluorescence intensities for all 8,700 genes analyzed. EST, expressed sequence tag.


                              
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Table 1.   Validation of select differentially expressed genes

Consistent with the genotype of mice used for this analysis was the finding that CCSP mRNA showed a 97% decline in abundance in total lung RNA from CCSP(-/-) mice relative to that in WT control mice. Low levels of detectable mRNA observed with the microarray approach are similar to the low levels of a nonfunctional truncated CCSP transcript detected at <1% of control levels by RNase protection analysis (32). Other differentially expressed genes included IgA heavy chain constant region and an unknown gene identified by EST W82219. These genes were overexpressed in total lung RNA of CCSP(-/-) mice as determined by Northern blot analysis (Table 1, Fig. 2). To further define the tissue specificity of altered EST W82219 mRNA expression, Northern analysis was performed with total RNA isolated from various mouse tissues of WT and CCSP(-/-) mice. Among the tissues evaluated, mRNA for EST W82219 was detected only in total lung RNA of WT and CCSP(-/-) mice (Fig. 2B).


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Fig. 2.   EST W82219 and IgA mRNA abundance in WT and CCSP(-/-) mice. A: EST W82219 and IgA transcript levels in 6 WT and 6 CCSP(-/-) [knockout (KO)] total lung RNA samples were determined by Northern hybridization. beta -Actin and L32 hybridization demonstrated equal loading of RNA samples. B: hybridization of EST W82219 to total RNA from WT (+) and CCSP(-/-) (-) heart, intestine, kidney, liver, lung, salivary gland, spleen, testis, and thymus. A digital image of an ethidium bromide (EtBr)-stained gel is included as a measure of RNA loading.

CCSP-dependent changes in airway epithelial and peribronchial lymphoid tissues. To precisely localize sites of altered gene expression in lungs of CCSP(-/-) mice, the distribution of IgA and EST mRNAs were compared in lung tissue sections from WT and CCSP(-/-) mice (Fig. 3). Consistent with a previous study by Stripp et al. (39), hybridization of an antisense riboprobe for the Clara cell-specific CYP2F2 mRNA revealed no apparent differences between strains (Fig. 3, A and B). Antisense riboprobes generated toward EST W82219 hybridized to transcripts localized exclusively in conducting airways of both WT and CCSP(-/-) mice, with an intensity that decreased in a proximal to distal gradient (Fig. 3, C, D, G, and H). Longer (1 mo) exposures to photographic emulsion failed to detect EST W82219 transcript in non-airway epithelial cells. Despite similarities in the distribution of EST W82219 and Clara cell-specific CYP2F2 mRNAs, the extent of EST W82219 mRNA hybridization was noticeably higher in airways of CCSP(-/-) mice relative to that in WT control mice. Cellular colocalization of EST W82219 mRNA with CCSP by dual in situ hybridization with a 3H-labeled EST W82219 antisense riboprobe coupled with immunohistochemical detection of CCSP revealed a Clara cell-specific pattern of EST W82219 mRNA expression (Fig. 3J) in the bronchiolar epithelium. These data, coupled with previous observations by Stripp et al. (39, 41) of ultrastructural changes to Clara cells of CCSP(-/-) mice, demonstrate that CCSP deficiency results in phenotypic changes to Clara cells.


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Fig. 3.   Localization of EST and IgA transcripts in lung tissue of WT and CCSP(-/-) mice. The spatial distribution of cytochrome P-450 2F2 (A and B), EST W82219 (C, D, G, and H), and IgA (E, F, and I) was determined by in situ hybridization in WT (A, C, E, and G) and CCSP(-/-) (B, D, F, H, and I) tissues with 35S-labeled antisense riboprobes. Silver grains detected by dark-field microscopy were pseudocolored red and superimposed over bright-field images. Images are oriented with the proximal portion of the airway to the right. G-I, magnification of boxes in C, D, and F, respectively. Original magnifications: ×10 in A-F; ×40 in G and H; ×100 in I. J: colocalization of EST W82219 transcript and CCSP protein by dual in situ hybridization with tritiated riboprobe and immunohistochemistry, respectively. Silver grains detected by dark-field microscopy were pseudocolored black and superimposed on the bright-field image. Light brown staining was indicative of CCSP immunoreactivity. Original magnification, ×400.

In contrast to the airway epithelial cell-specific pattern of EST W82219 mRNA expression, the pattern of IgA gene expression in CCSP(-/-) mice is suggestive of altered BALT. Clusters of IgA message-positive cells were rarely detected in WT tissue (Fig. 3E). In contrast, IgA mRNA-positive cells were readily detected in the lung tissue of CCSP(-/-) mice and exhibited a peribronchial distribution consistent with the diffuse localization of BALT (Fig. 3, F and I). These results suggest a role for Clara cells and/or their secretions in regulation of local lymphoid populations.

CCSP-dependent alterations in airway and systemic IgA production. ELISA was used to determine whether changes in IgA mRNA expression in cells of the BALT resulted in either local or systemic alterations in the abundance of IgA protein (Fig. 4). Bronchoalveolar lavage fluid and plasma were recovered from healthy adult male WT and CCSP(-/-) mice with no history of infection or other environmental challenges. Levels of IgA were elevated ~2.5-fold in the bronchoalveolar lavage fluid and 2.7-fold in the plasma of CCSP(-/-) mice relative to those in WT control mice. Collectively, changes in IgA expression in the lungs of CCSP(-/-) mice indicate a potential role for Clara cells and/or CCSP in the modulation of local lymphoid populations.


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Fig. 4.   IgA protein concentration in bronchoalveolar (BAL) fluid (A) and plasma (B). A: BAL fluid was obtained from WT (n = 6) and CCSP(-/-) (KO) (n = 5) mice, and IgA protein levels were determined by ELISA. Values were normalized to total protein concentration and are reported as the ratio of IgA to total protein. * P < 0.001. B: IgA protein content in peripheral blood. Plasma was collected from peripheral blood of WT (n = 6) and CCSP(-/-) (n = 6) mice, and IgA protein levels were determined with ELISA. Values were normalized to total protein concentration and are reported as the ratio of IgA to total protein. * P < 0.05.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

We demonstrate that CCSP deficiency results in changes in lung gene expression, indicating perturbations in lung homeostasis. A novel gene was identified whose expression was confined to Clara cells and upregulated in airways of CCSP(-/-) mice. However, changes in lung gene expression in CCSP(-/-) mice were not confined to Clara cells. Local production of IgA was significantly elevated at both the mRNA and protein levels in addition to a significant elevation in serum IgA levels. Increased production of lung IgA involved a dramatic expansion of peribronchial IgA-expressing lymphocytes, consistent with expansion of BALT.

CCSP mRNA and protein abundance have been used as biomarkers to follow changes in airways associated with either acute or chronic lung disease (11, 19, 39, 44). Even though these studies have revealed relationships between the abundance of CCSP and the type or magnitude of lung pathology, functional consequences of these changes are likely to be complex, particularly as they relate to chronic alterations in airway homeostasis and perpetuation of airway disease. CCSP(-/-) mice have the potential to reveal functional roles for Clara cells and their secretions in airway homeostasis. A number of related phenotypes have been described for CCSP(-/-) mice, including increased susceptibility to inhaled oxidant gases (13, 23) and altered responses to pathogens (8, 9). A common finding of these studies is the induction of a more profound inflammatory reaction among CCSP(-/-) mice. However, a previous demonstration by Johnston et al. (13) that CCSP(-/-) and WT mice show identical inflammatory responses when challenged with low doses of inhaled endotoxin argues against a direct role for CCSP in mediating these differential responses. Moreover, findings of the present study reinforce the notion that CCSP(-/-) mice have more complex changes to the lung than simple CCSP deficiency.

Altered expression of a novel lung and Clara cell-specific genes in total lung RNA of CCSP(-/-) mice clearly indicates the potential for functional changes to Clara cells. The many possible explanations for altered Clara cell gene expression include compensatory upregulation of related genes, nonspecific alterations in Clara cell gene expression that result from the dramatic reduction in secretory product expression, or differences in the accumulation and/or sequestration of endogenous ligands. A study by McDowell et al. (24) highlights the possibility of similar CCSP and EST W82219 regulatory mechanisms by demonstrating a similar decrease in their mRNA levels after murine lung injury induced by nickel exposure (24). Preliminary analysis of the predicted protein product of EST W82219 indicates that it shares distant homology with the secretoglobin family of proteins for which CCSP is the founding member (16, 30; Reynolds, P, Reynolds S, and Stripp B, unpublished observations). This raises the possibility that elevated expression of EST W82219 in Clara cells of CCSP(-/-) mice represents a mechanism to compensate for CCSP deficiency. Importantly, functional changes to Clara cells indicated by altered expression of EST W82219 are consistent with earlier observations by Stripp et al. (39, 41) of ultrastructural perturbations observed in Clara cells of CCSP(-/-) mice. Prominent ultrastructural changes to Clara cells of CCSP(-/-) mice include the absence of secretory granules and the frequent appearance of multilamellar inclusions surrounding cytoplasmic organelle components. The basis for these ultrastructural changes in Clara cells of CCSP(-/-) mice is not known. However, similar ultrastructural alterations have been observed in Clara cells in association with airway pathology such as equine COPD (14). Similarities in Clara cell alterations occurring between clinical chronic airway disease and CCSP(-/-) mice are further supported by studies (4, 20, 35) demonstrating that CCSP levels are significantly decreased in the airways and serum of asthmatic patients, cigarette smokers, and individuals with COPD. As such, functional changes to Clara cells of CCSP(-/-) mice and the associated changes in airway homeostasis represent a model of a primary Clara cell defect that may yield important new insights into the pathobiology of chronic airway disease.

Alterations in IgA expression highlight a potentially important role for Clara cells and/or CCSP in local immunoregulation. Increased IgA mRNA abundance observed in peribronchial lymphoid tissue of CCSP(-/-) mice is associated with a significant elevation in IgA protein levels in both the airway lining fluid and serum. IgA serves important functions in innate mucosal defense against pathogens through its ability to nonspecifically opsonize bacteria and contribute to their clearance (33). Elevated steady-state levels of IgA observed in the airway lining fluid of CCSP(-/-) mice may therefore contribute to an increased clearance rate and resistance to Pseudomonas infection (9). However, mechanisms contributing to elevated IgA production in the lung are unknown. CCSP has been proposed to act as an anticytokine as a result of its ability to modulate inflammation and the activity of interferon (IFN)-gamma (7, 29). Moreover, in vitro studies (22, 45, 46) indicated that CCSP may be directly regulated at the transcriptional level by IFN-gamma and tumor necrosis factor (TNF)-alpha . Even though a study (12) in CCSP(-/-) mice showed no evidence for altered inflammatory responses to stimuli that do not cause concomitant cellular injury, it is possible that altered local production or activity of IFN-gamma or other cytokines such as TNF-alpha could contribute to altered immunoregulation. Both IFN-gamma and members of the TNF family have been shown to function as potent regulators of the immune response (15, 37, 47).

Interestingly, even though CCSP(-/-) mice show no signs of autoimmune disease (32), an independently generated line of mice harboring a different knockout of the same gene show systemic disease including IgA glomerulonephropathy and focal pancreatic necrosis (48, 49). Based on observations presented herein and those of Zheng et al. (49), we propose that immunoregulatory changes and increases in local production of IgA that are associated with CCSP deficiency may contribute to systemic organ dysfunction. The potential contribution of altered Clara cell function to systemic disease is suggested from epidemiological studies demonstrating that smoking, which is associated with a significant decline in CCSP abundance (2, 4), represents a significant risk factor for progression to renal failure (31). Additional studies are necessary to determine the mechanisms whereby CCSP deficiency leads to altered IgA production and to determine whether these changes are associated with increased risk of systemic disease.

In summary, our results demonstrate that CCSP deficiency is associated with changes in gene expression in both Clara cells and other cells in the peribronchial region. Changes in the expression of IgA mRNA are associated with local increases in IgA protein, suggesting that CCSP deficiency leads to altered regulation of the immune response in the lung. Additional studies are necessary to determine whether immunoregulatory alterations observed among CCSP(-/-) mice are a direct function of CCSP deficiency or whether other changes to Clara cells contribute to this phenotype.


    ACKNOWLEDGEMENTS

This study was supported by National Institute of Environmental Health Sciences Grants ES-08964 and ES-01247.


    FOOTNOTES

T. M. Watson was supported by National Institute of Environmental Health Sciences Toxicology Training Grant T32-ES-07026.

Address for reprint requests and other correspondence: B. R. Stripp, Univ. of Pittsburgh, Dept. of Environmental and Occupational Health, 3343 Forbes Ave, Rm. 314, Pittsburgh, PA 15260 (E-mail: bstripp{at}server.ceoh.pitt.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.

Received 26 May 2001; accepted in final form 10 September 2001.


    REFERENCES
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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