Clara cell secretory protein decreases lung inflammation after acute virus infection

Kevin S. Harrod1, Amber D. Mounday1, Barry R. Stripp2, and Jeffrey A. Whitsett1

1 Division of Pulmonary Biology, Children's Hospital Medical Center, Cincinnati, Ohio 45229; and 2 Department of Environmental Medicine, University of Rochester, Rochester, New York 14642

    ABSTRACT
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Clara cell secretory protein (CCSP) is an abundant 10-kDa polypeptide synthesized and secreted primarily by nonciliated bronchiolar epithelial cells in the mammalian lung. To determine the potential role of CCSP in pulmonary inflammation after acute viral infection, CCSP gene-targeted {CCSP-deficient [CCSP(-/-)]} mice were exposed to a recombinant E1- and E3-deficient adenoviral vector, Av1Luc1, intratracheally. Lung inflammation was markedly increased in CCSP(-/-) mice compared with wild-type control mice and was associated with an increased number of polymorphonuclear cell infiltrates and epithelial cell injury in both conducting airways and alveolar regions. Histological evidence of pulmonary inflammation in CCSP(-/-) mice was associated with increased production of cytokine (interleukin-1beta and -6 and tumor necrosis factor-alpha ) mRNA and protein, as well as chemokine (macrophage inflammatory protein-1alpha and -2 and monocyte chemoattractant protein-1) mRNA expression within the lung in response to adenoviral infection. Adenoviral-mediated gene transfer was decreased in CCSP(-/-) mice relative to wild-type mice as measured by luciferase enzyme activity in lung homogenates. The present study suggests that CCSP is involved in modulating lung inflammation during viral infection and supports a role for CCSP in lung host defense.

lung epithelium; pulmonary infection; viral pathogenesis

    INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

CLARA CELL SECRETORY PROTEIN (CCSP) is a 10-kDa dimeric protein synthesized primarily by nonciliated bronchiolar epithelial cells (11). CCSP is one of the most abundant soluble proteins within the epithelial lining fluid of the lung (11). Nonciliated bronchiolar epithelial cells (also termed Clara cells) lining the conducting airways synthesize and secrete CCSP and a variety of proteins implicated in host defense, including surfactant protein (SP) A, SP-B, and SP-D (17). CCSP is structurally similar to the rabbit uteroglobin (UG) protein, which is known to bind polychlorinated biphenyls (12, 13) and inhibit secretory phospholipases A2 (6) and chemotaxis of inflammatory cells in uterine tissues (15). The physiological function of CCSP in vivo as well as the role of CCSP in lung infection and injury is not well understood. To delineate the function of CCSP, CCSP gene-targeted {CCSP-deficient [CCSP(-/-)]} mice were generated (14). Pulmonary accumulation of polychlorinated biphenyls after intraperitoneal administration was markedly decreased in CCSP(-/-) mice, suggesting that CCSP is a determinant of lipophilic toxin accumulation within the lung (14). After hyperoxic lung injury, pulmonary edema was exacerbated and proinflammatory cytokine gene expression was increased in CCSP(-/-) mice, providing support for the concept that CCSP plays a role in host responses to lung injury (5).

Lung inflammation after viral infection with adenoviruses and adenoviral vectors has been well characterized. Adenoviruses are ubiquitous viral pathogens that cause respiratory, gastrointestinal, and genitourinary infections (16). Adenoviruses usually cause acute respiratory pathology; however, adenoviruses can also persist as asymptomatic infections of the respiratory tract (16). Acutely, adenoviral infection causes lung infiltration of macrophages and neutrophils in the alveolar air spaces, generally observed 2-3 days after infection (2). Concentrations of the cytokines tumor necrosis factor (TNF)-alpha , interleukin (IL)-1, and IL-6 are also increased in pulmonary tissues after adenoviral infection, coinciding with the appearance of macrophages in alveolar regions (2). Because of their tropism for airway epithelial cells, adenoviruses have been utilized as vectors for gene transfer to the lung. The efficiency and duration of gene expression with recombinant adenoviruses, however, are limited by host inflammatory and immune responses (1, 18, 19, 22). Thus lung inflammation after infection with adenoviral vectors has been well characterized and provides a useful model for studying viral-induced inflammation in the lung.

Although the role of CCSP in infectious injury in the lung has not been addressed, this report tests whether CCSP modulates host responses to viral infection in the lung. CCSP(-/-) mice were infected with a replication-deficient adenoviral vector, and lung inflammation and acute immune responses were measured. The deficiency of CCSP in mice increases lung inflammation after intratracheal administration of an adenovirus.

    MATERIALS AND METHODS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Mice. CCSP(-/-) (129J Ola/129J hybrid) and wild-type 129J (Taconic Farms, Germantown, NY) mice were housed under pathogen-free conditions in the Children's Hospital (Cincinnati, OH) Research Foundation vivarium as required by American Association for Accreditation of Laboratory Animal Care guidelines.

Intratracheal administration of adenovirus. Eight- to twelve-week-old CCSP(-/-) and 129J wild-type control mice (n = 6-12 mice/group) were used. The procedure for intratracheal administration of adenoviral vectors was previously described by Zsengeller et al. (22). Briefly, mice were anesthetized with methoxyflurane vapor, and a ventral midline incision was made to expose the trachea. Intratracheal inoculation of 1 × 109 plaque-forming units of Av1Luc1, an E1- and E3-deleted adenoviral vector expressing firefly luciferase from the Rous sarcoma virus promoter, in 100 µl of delivery vehicle (10 mM Tris, 1 mM MgCl2, and 10% glycerol, pH 7.4) was performed with a bent, 27-gauge tuberculin syringe (Monoject, St. Louis, MO). The incision was closed with one drop of Nexaband liquid, and the mice were allowed to recover. Mice recover rapidly and remain active after the procedure. At a predetermined time of biological analysis, the mice were killed by a lethal injection of pentobarbital sodium. A midline incision was made in the abdomen. Exsanguination was accomplished by transection of the inferior vena cava to reduce hemorrhage in the lung. For histological studies, RNA and protein analyses, the right upper, middle, and lower lobes of the lung were clamped with a hemostat and removed for measurement of luciferase activity, RNA, and protein. The left lung lobe was inflated with 4% paraformaldehyde (Electron Microscopy Sciences, Ft. Washington, PA) and fixed overnight for histological examination.

Evaluation of acute inflammatory cell infiltrates. Inflammatory cell numbers and percentages were evaluated at 4 and 24 h after adenoviral vector administration. Bronchoalveolar lavage (BAL; n = 6 mice/group) fluid was obtained by intratracheal instillation of 1 ml of PBS into the lung while it was maintained within the thoracic cavity. The lavage was reinfused into the lung two times before final collection. BAL cells were isolated by centrifugation at 500 g and resuspended in 500 µl of PBS, 100 µl of the cell suspension were mixed in 100 µl of 0.4% trypan blue (GIBCO BRL, Grand Island, NY), and the cells were counted with a hemocytometer. To determine inflammatory cell types in BAL, 5 × 104 cells were mounted on slides by cytospin centrifugation in 100 µl of PBS at 600 rpm for 3 min. Cell types were identified and counted by differential staining microscopy with Diff-Quik (Baxter Healthcare, Miami, FL). Inflammatory cell populations were determined by counting 100 cells, and a percentage was calculated based on five sample sets from three animals per group.

Cytokine analysis. Cytokine mRNA abundance was determined by RT-PCR analysis of whole lung total RNA. Briefly, whole lung total RNA was isolated by phenol-chloroform extraction and precipitation with isopropanol with the Phase-Lock protocol (5 Prime right-arrow 3 Prime, Boulder, CO). Total RNA quantitation was confirmed by gel electrophoresis. Total RNA was converted to cDNA by the RT reaction (GIBCO BRL, Gaithersburg, MD). PCR for cytokine cDNA was performed with the following primer tandems: beta -actin primer 1, 5'-GTGGGCCGCTCTAGGCACCAA-3' and primer 2, 5'-CTCTTTGATGTCACGCACGATTTC-3'; IL-6 primer 1, 5'-TTGCCTTCTTGGGACTGATGCT-3' and primer 2, 5'-GTATCTCTCTGAAGGACTCTGG-3'; TNF-alpha primer 1, 5'-CCAGACCTCACACTCAGAT-3' and primer 2, 5'-AACACCCATTCCCTTCACAG-3'; macrophage inflammatory protein (MIP)-1alpha primer 1, 5'-ACTGCCCTTGCTGTTCTTCTCT-3' and primer 2, 5'-AGGCATTCAGTTCCAGGTCAGT-3'; MIP-2 primer 1, 5'-ATGGCCCCTCCCACCTGC-3' and primer 2, 5'-TCAGTTAGCCTTGCCTTTGTT-3'; and monocyte chemoattractant protein (MCP)-1 primer 1, 5'-ATGCAGGTCCCTGTCATGCTT-3' and primer 2, 5'-CTAGTTCACTGTCACACTGGT-3'. PCR with the OptiPrime reagents (Stratagene, La Jolla, CA) was performed for 25 cycles on a Perkin-Elmer 2400 Gene Amp System thermal cycler by the following parameters: initiation at 94°C for 30 s, annealing temperature at 59°C for 30 s, and elongation temperature of 72°C for 30 s. Ethidium bromide staining of 2% agarose gel electrophoresis was used to visualize PCR products.

Cytokine concentrations were assessed in lung homogenates by ELISA according to the manufacturer's recommendations (Endogen, Woburn, MA). Standard curves were calculated for known standards to verify linearity of analysis and for calculation of cytokine concentration.

Adenoviral-mediated transgene expression. To detect adenoviral-mediated luciferase activity, the right upper lobe was removed and immediately homogenized in 800 µl of lysis buffer as previously described (22). The luciferase reaction was initiated by injection of 100 µl of 1 mM luciferin, and data were collected for 10 s at 25°C with a Monolight 2010 luminometer (Analytical Luminescence Laboratories, San Diego, CA). Light units were normalized to total lung homogenate protein as measured by the Lowry assay. Luciferase activity in the mouse lung is expressed as relative light units per microgram of lung protein.

Pulmonary histopathology. Histopathological changes were evaluated at 7 and 14 days after Av1Luc1 administration in the inflation-fixed mouse lung. Inflation-fixed lungs were washed in PBS three times and divided in half for preparation of paraffin embedding. Paraffin-embedded lungs were sectioned at 5 µm and stained with hematoxylin and eosin for morphological analysis. Pathological assessment of lung inflammation was graded blindly on a scale of 0-4, and a score was determined from the mean (±SE) of six animals.

Statistical analysis. Statistical analysis for multiple groups was determined by ANOVA with StatWorks computer software. All data are presented as means ± SE. Differences were considered significant at P <=  0.05.

    RESULTS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

BAL cell counts are increased in CCSP(-/-) mice. To assess the role of CCSP in modulating lung inflammatory cell infiltrates after adenoviral infection, adult CCSP(-/-) mice (in 129J strain background) and 129J wild-type mice (7-15 wk of age) were intratracheally injected with a recombinant, replication-deficient (E1- and E3-deleted) adenoviral vector, Av1Luc1. Inflammatory cells in BAL fluid were assessed at 4 and 24 h postinfection for cell counts in BAL fluid. Both wild-type (129J) and CCSP(-/-) mice tolerated 109 plaque-forming units of adenovirus and were responsive and active within 1 h of the surgical procedure. No mortality was observed after administration of the virus to either group of mice. At 4 h after adenoviral infection, cell counts in BAL fluid from control 129J mice were not different from cell counts in uninfected mice (Fig. 1A). Four hours after Av1Luc1 administration, cell counts in BAL fluid from CCSP(-/-) mice were increased threefold compared with those in wild-type mice. Twenty-four hours after adenoviral infection, cell counts in BAL fluid from wild-type mice were increased compared with cell counts in BAL fluid from uninfected wild-type mice. Cell counts in BAL fluid from infected CCSP(-/-) mice at 24 h were increased 3.5-fold compared with those in infected wild-type mice. Cell counts in BAL fluid from uninfected mice did not vary in either group. The number of cells obtained from BAL in CCSP(-/-) and wild-type mice were similar before infection.


View larger version (21K):
[in this window]
[in a new window]
 


View larger version (52K):
[in this window]
[in a new window]
 
Fig. 1.   Inflammatory cell number (A) and identification of inflammatory cell type (B) in bronchoalveolar lavage (BAL) fluid from wild-type and Clara cell secretory protein-deficient [CCSP(-/-)] mice. A: wild-type and CCSP(-/-) mice were intratracheally administered 109 plaque-forming units of Av1Luc1, and BAL fluid was recovered at 4 and 24 h after infection. Inflammatory cell counts were determined by trypan blue exclusion and counted with a hemocytometer. Normal cell counts in BAL fluid were acquired from uninfected wild-type mice. * P <=  0.05 compared with wild-type mice at similar time points. B: determination of inflammatory cell types in BAL fluid at 4 h after adenoviral infection in CCSP(-/-) and wild-type (wt) mice. Inflammatory cells in BAL were recovered by cytospin centrifugation and identified by differential staining. Arrowheads, appearance of neutrophils in BAL fluid of CCSP(-/-) mice; arrows, appearance of macrophages.

To determine whether individual inflammatory cell populations were altered in BAL fluid from CCSP(-/-) mice, BAL fluid was obtained from adenoviral-infected wild-type and CCSP(-/-) mice and examined after cytospin centrifugation and differential cell staining. Four hours after adenoviral infection, inflammatory cells in BAL fluid from wild-type mice were predominantly macrophages and were not different from those in BAL fluid from uninfected mice (Fig. 1B, Table 1). Four hours after adenoviral infection of CCSP(-/-) mice, BAL fluid contained primarily macrophages, with the notable appearance of neutrophils. Twenty-four hours after adenoviral infection, numerous macrophages and increased numbers of neutrophils were noted in wild-type mice. Increased macrophages and neutrophils were detected in BAL fluid from CCSP(-/-) mice 24 h after adenoviral infection compared with those from wild-type control mice.

                              
View this table:
[in this window]
[in a new window]
 
Table 1.   Differential cell counts in BAL from wild-type and CCSP(-/-) mice after adenoviral infection

Increased cytokine and chemokine responses after adenoviral infection in CCSP(-/-) mice. Cytokine and chemokine mRNAs were assessed from lung homogenates of CCSP(-/-) and control mice 4 and 24 h after adenoviral infection. The proinflammatory cytokine TNF-alpha and IL-6 as well as the neutrophilic chemokine MIP-2 and MIP-1alpha mRNAs were unchanged 4 h after infection in either wild-type or CCSP(-/-) mice (Fig. 2). After 4 h, the monocytic chemokine MCP-1 was increased in the lungs of CCSP(-/-) mice but was not detected in wild-type mice. At 24 h after adenoviral administration, proinflammatory cytokine TNF-alpha and IL-6 mRNAs as well as chemokine MIP-2 and MIP-1alpha mRNAs were increased in the lungs of CCSP(-/-) mice compared with wild-type mice. MCP-1 mRNA was also increased in the lungs of CCSP(-/-) mice at 24 h after infection compared with wild-type mice.


View larger version (55K):
[in this window]
[in a new window]
 


View larger version (51K):
[in this window]
[in a new window]
 
Fig. 2.   Increased cytokine and chemokine mRNAs in CCSP(-/-) mice at 4 (A) and 24 h (B) after adenoviral infection in replicates (n = 4 experiments) of CCSP(-/-) and wt [CCSP(+/+)] mice. beta -Actin, control; MCP-1, monocyte chemoattractant protein-1; IL-6, interleukin-6, TNF-alpha , tumor necrosis factor-alpha ; MIP-1alpha and MIP-2, macrophage inflammatory protein-1alpha and macrophage inflammatory protein-2, respectively. Total RNA was isolated from lung homogenates of CCSP(-/-) and wt mice. mRNA was converted to cDNA by RT reaction. Amplification of cytokine and chemokine cDNA species was performed by PCR and visualized by ethidium bromide-stained agarose gel electrophoresis.

Concentrations of the proinflammatory cytokines TNF-alpha , IL-6, and IL-1beta were measured in lung homogenates from CCSP(-/-) and wild-type mice by ELISA. Four hours after infection, IL-6, IL-1beta , and TNF-alpha were unchanged in either wild-type or CCSP(-/-) mice (data not shown). However, 24 h after infection, the concentration of IL-6 was increased in wild-type mice compared with uninfected control mice (Fig. 3). In CCSP(-/-) mice 24 h after infection, the concentration of IL-6 was increased threefold compared with that in wild-type mice. Concentrations of IL-1beta and TNF-alpha were also increased to a greater extent in CCSP(-/-) mice compared with wild-type mice 24 h after adenoviral infection.


View larger version (14K):
[in this window]
[in a new window]
 
Fig. 3.   Increased concentrations of cytokines in lungs of CCSP(-/-) mice. Normal, untreated wt mice. Cytokine concentrations for IL-6 (A), IL-1beta (B), and TNF-alpha (C) were increased in lung homogenates from CCSP(-/-) vs. wt mice (n = 6/group). * Significant difference from wt mice, P <=  0.05.

Adenoviral gene expression is reduced in the lungs of CCSP(-/-) mice. The adenoviral vector Av1Luc1 used in these studies encodes the luciferase reporter gene under the control of the Rous sarcoma virus promoter region. To determine whether viral gene expression is altered in the lungs of CCSP(-/-) mice after adenoviral infection, luciferase activity was measured in lung homogenates of control and CCSP(-/-) mice 7 and 14 days after adenoviral infection. Luciferase activity in control mice was higher 7 days after adenoviral infection and was decreased 14 days after infection. Luciferase activity in the lungs of CCSP(-/-) mice was significantly decreased compared with that in the lungs of wild-type mice 7 and 14 days after adenoviral administration (Fig. 4). Luciferase activity in the lungs of CCSP(-/-) mice was higher at 7 days after infection and decreased at 14 days after adenoviral administration.


View larger version (20K):
[in this window]
[in a new window]
 
Fig. 4.   Luciferase activity in lungs of adenoviral-infected CCSP(-/-) and wt mice. Luciferase activity was determined in lung homogenates from individual animals (n = 12/group) at 7 and 14 days after infection. Protein concentration was determined by Lowry assay. * Significant difference from wt mice, P <=  0.05.

Increased lung inflammation in CCSP(-/-) mice after adenoviral infection. To determine whether adenoviral-mediated lung inflammation was influenced by CCSP, lung histology was assessed after administration of the adenovirus. Pulmonary infiltrates were observed in the lungs of all mice receiving adenovirus at 7 and 14 days after infection. In wild-type mice 7 days after infection, lung inflammation consisted of focal alveolar infiltrates composed primarily of mononuclear cells, with occasional neutrophils (Fig. 5). In CCSP(-/-) mice 7 days after infection, alveolar inflammation consisted of large areas of consolidation. Lung inflammation in CCSP(-/-) mice was more extensive and involved more regions of the lung. Lung inflammation was increased 14 days after infection in both CCSP(-/-) and wild-type mice. Severe lung inflammation persisted in CCSP(-/-) mice and included alveolar septal thickening, with extensive regions of consolidation noted in the lung parenchyma. Lung inflammation in the CCSP(-/-) mice was increased compared with that in wild-type mice 7 and 14 days after administration of the virus.


View larger version (114K):
[in this window]
[in a new window]
 
Fig. 5.   Pulmonary histopathology of CCSP(-/-) and wt mice at 7 (7d) and 14 (14d) days after adenoviral infection. Lung sections from wt (A and C) and CCSP(-/-) (B and D) mice at 7 (A and B) and 14 days (C and D) are shown. A, airway; v, vessel. Parenchymal inflammation, alveolitis, and areas of cellular consolidation were observed in CCSP(-/-) mice at 7 and 14 days after infection. Original magnification, ×245.

    DISCUSSION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

The present study demonstrates increased inflammatory responses in CCSP gene-targeted mice after intratracheal administration of Av1Luc1, an E1- and E3-deleted recombinant adenoviral vector. Inflammatory cells in BAL fluid were increased in CCSP(-/-) mice, and neutrophils appeared earlier in the course of infection. Expression of the proinflammatory cytokines IL-6, IL-1beta , and TNF-alpha as well as the neutrophilic chemokines MIP-1alpha and MIP-2 and the monocytic chemokine MCP-1 were increased in the lungs of CCSP(-/-) mice after infection. Lung inflammation was increased and luciferase activity, as a measure of viral gene expression, was decreased in the lungs of CCSP(-/-) mice. These results indicate that CCSP deficiency exacerbates the early host responses and inflammation to adenoviral infection in the lung.

In a previous study (2) using the mouse model of adenoviral pneumonia, lung infiltrates appeared in the lung parenchyma 2 days after infection and were monocytic or neutrophilic in appearance. In the present study, total inflammatory cells in BAL fluid and neutrophilic infiltration increased earlier in CCSP(-/-) mice than in wild-type mice and were associated with earlier expression of cytokines and chemokines in the CCSP(-/-) mice. The induction of inflammatory responses and cytokine production after adenoviral exposure in wild-type mice is consistent with previous findings regarding host inflammatory responses after adenoviral infection (2). The findings in the present study that inflammatory cell influx and proinflammatory cytokines and chemokines are increased in the lungs of CCSP(-/-) mice are consistent with the concept that CCSP plays a role in limiting alveolar influx of macrophages and neutrophils and cytokine responses early during the course of adenoviral infection.

Adenoviral infection is associated with acute and chronic cytopathic effects that disrupt the respiratory epithelium (2, 22). After adenoviral vector administration in mice, respiratory epithelial cell proliferation is increased in both normal and immunodeficient mice, suggesting that adenoviral infection per se has cytopathic effects independent of viral clearance by immune cells (22). Surfactant protein homeostasis was also disrupted in both immunocompetent and immunodeficient mice after adenoviral vector infection (21). In the present study, the increase in lung inflammation observed in CCSP(-/-) mice may reflect a possible role for CCSP in cytoprotection of the lung epithelium after injury. In support of this concept, CCSP(-/-) mice succumbed to lung injury earlier than wild-type mice during oxygen exposure (5).

In the present study, monocytic and neutrophilic infiltration was more extensive in the lung parenchyma of CCSP(-/-) mice after adenoviral infection. The early enhancement of cytokine expression in CCSP(-/-) mice likely contributes to the increase in lung inflammation seen later in the course of infection. The proinflammatory cytokines IL-6 and TNF-alpha initiate both acute and chronic inflammatory events, including the activation of adhesion molecules and chemokines (10). TNF-alpha also exerts important cytopathic effects on virally infected cells (8, 9). Increased expression of the chemokines MIP-1alpha , MIP-2, and MCP-1 in adenoviral-infected CCSP(-/-) mice may contribute to increased lung inflammation by induction of inflammatory cell chemotaxis. The findings in the present study suggest that CCSP deficiency increases the expression of important inflammatory mediators after adenoviral infection and may influence later lung inflammatory events.

Host immune responses to adenoviral vectors limit the efficiency and duration of viral gene expression. Adenoviral vector administration to immunodeficient mouse models (1, 18, 19, 22) or the use of immunomodulatory therapies (3, 22) limits host immune responses and extends the duration and level of adenoviral-mediated gene expression. The loss of adenoviral transgene expression has been attributed, at least in part, to the rapid loss of viral DNA in infected cells (22). A number of mechanisms may explain the loss of adenoviral DNA, including the cytopathic effects of virus infection, the uptake and depletion of viral particles by macrophages and other inflammatory cells, and T cell-mediated cytolytic killing during the later phase (7-14 days) of infection. In the present study, adenoviral gene expression in CCSP(-/-) mice was decreased and associated with increased pulmonary inflammation. Thus the present study is consistent with previous findings that host immune and inflammatory responses to adenoviral infection limit adenoviral vector gene expression in vivo.

Although the function of CCSP has not been clearly defined, there is increasing evidence that CCSP plays an important role in the modulation of various inflammatory responses (4, 5, 7, 11, 20). In a hyperoxic lung injury model, survival of CCSP(-/-) mice was reduced compared with control mice (5). Likewise, the onset of lung edema occurred earlier in CCSP(-/-) mice. Expression of the proinflammatory cytokines IL-3, IL-6, and IL-1beta was increased in the lungs of CCSP(-/-) mice, and in the case of IL-1beta , increased expression was localized to the lung parenchyma (5). Lung inflammation and injury in hyperoxic CCSP(-/-) mice were not limited to the bronchiolar epithelium but also involved the alveolar epithelium, suggesting that CCSP plays a role in limiting lung injury and inflammation in both the alveolar and bronchiolar regions of the lung. In the present study, lung inflammation in CCSP(-/-) mice was increased markedly in the lung parenchyma compared with that in wild-type mice. Whether CCSP produced by conducting airway cells traffics to alveolar regions of the lung is not known at present. It is also possible that CCSP itself modulates inflammation in the lung parenchyma by events mediated by its action in the conducting airways.

The findings in the present study suggest that the lack of CCSP increases the host response to viral infection in the lung. Alternately, altered Clara cell function may also contribute to the observed increase in lung inflammatory responses in CCSP(-/-) mice. Ultrastructural analysis demonstrated that secretory granules in Clara cells of CCSP(-/-) mice were abnormal or absent (14). Thus it is possible that altered inflammatory responses to adenoviral infection in the present study resulted from disrupted Clara cell function rather than the lack of CCSP. Clara cells secrete a number of host defense molecules, including SP-A and SP-D (13). Both SP-A and SP-D are likely important in host defense after lung infection (17). Thus the production and secretion of important immunomodulatory factors by the lung epithelium may play an important role in lung injury after infection.

Despite the relative abundance of CCSP in the BAL fluid, the physiological function of CCSP is not understood. CCSP (also termed CC10 and UG) has been shown in vitro to act as an immunosuppressant mediated, in part, by its ability to inhibit secretory phospholipases A2. Recently, another gene-targeted mouse model of CCSP called the UG gene-targeted [UG(-/-)] mouse model was described (20). UG(-/-) mice spontaneously developed severe renal fibrosis, with extensive deposition of fibronectin and collagen. As in the present study involving the CCSP(-/-) mice, no lung pathology was reported in the UG(-/-) mouse model. The CCSP(-/-) mice used in the present study have no apparent renal pathology. The discrepancy in phenotype between the two CCSP(-/-) mice may be related to differences in vivarium conditions or genetic strains.

The present findings support the concept that CCSP functions to modulate the host responses during viral-induced lung inflammation. CCSP deficiency exacerbates early inflammatory responses to viral infection, suggesting that CCSP plays a role in innate immunity to infectious agents. Cytokines and chemokines likely play a role in the transition of early nonspecific immune responses to more specific adaptive immune mechanisms. In the present study, the enhanced induction of cytokines and chemokines in the CCSP(-/-) mice after adenoviral infection may explain the increased lung inflammation later during the course of infection and supports the concept that secretory products of lung epithelial cells are important modulators of lung inflammation and injury. The precise molecular mechanisms by which CCSP limits lung inflammation in vivo remain to be discerned. However, the present findings support the potential utility of CCSP as a therapeutic strategy to influence inflammation after lung injury and infection.

    ACKNOWLEDGEMENTS

We thank Shilpa Jain-Vora and Michael Jones for preparation of the cytokine primers, Nannette Mittereder and Bruce Trapnell for preparation of the adenoviral vector, and Anne Marie Levine and Thomas Korfhagen for thoughtful discussion.

    FOOTNOTES

This work was supported by the Cystic Fibrosis Foundation; National Heart, Lung, and Blood Institute Grants HL-41496 (to J. A. Whitsett) and HL-51376 (to B. R. Stripp); and the Parker B. Francis Foundation (K. S. Harrod).

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. §1734 solely to indicate this fact.

Address for reprint requests: J. A. Whitsett, Children's Hospital Medical Center, Div. of Neonatology and Pulmonary Biology, 3333 Burnet Ave., Cincinnati, OH 45229-3039.

Received 20 March 1998; accepted in final form 14 August 1998.

    REFERENCES
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

1.   Dai, Y., E. M. Schwarz, D. Gu, W. Zhang, N. Sarvetnick, and I. M. Verma. Cellular and humoral immune responses to adenoviral vectors containing factor IX gene: tolerization of factor IX and vector antigens allows for long-term expression. Proc. Natl. Acad. Sci. USA 92: 1401-1405, 1995[Abstract].

2.   Ginsberg, H. S., L. L. Moldawer, P. B. Sehgal, M. Redington, P. L. Kilian, R. M. Chanock, and G. A. Prince. A mouse model for investigating the molecular pathogenesis of adenoviral pneumonia. Proc. Natl. Acad. Sci. USA 88: 1651-1655, 1991[Abstract].

3.   Guerette, B., J. T. Vilquin, M. Gingras, C. Gravel, K. J. Wood, and J. P. Tremblay. Prevention of immune reactions triggered by first-generation adenoviral vectors by monoclonal antibodies and CTLA4Ig. Hum. Gene Ther. 7: 1455-1463, 1996[Medline].

4.   Hay, J. G., C. Danel, C. S. Chu, and R. G. Crystal. Human CC10 gene expression in airway epithelium and subchromosomal locus suggest linkage to airway disease. Am. J. Physiol. 268 (Lung Cell. Mol. Physiol. 12): L565-L575, 1995[Abstract/Free Full Text].

5.   Johnston, C. J., G. W. Mango, J. N. Finkelstein, and B. R. Stripp. Altered pulmonary response to hyperoxia in Clara cell secretory protein deficient mice. Am. J. Respir. Cell Mol. Biol. 17: 147-155, 1997[Abstract/Free Full Text].

6.   Levin, S. W., J. D. Butler, U. K. Schumacher, P. D. Wightman, and A. B. Mukherjee. Uteroglobin inhibits phospholipase A2 activity. Life Sci. 38: 1813-1819, 1986[Medline].

7.   Magdaleno, S. M., G. Wang, K. J. Jackson, M. K. Ray, S. Welty, R. H. Costa, and F. J. DeMayo. Interferon-gamma regulation of Clara cell gene expression: in vivo and in vitro. Am. J. Physiol. 272 (Lung Cell. Mol. Physiol. 16): L1142-L1151, 1997[Abstract/Free Full Text].

8.   Mestan, J., W. Digel, S. Mittnacht, H. Hillen, D. Blohm, A. Moller, H. Jacobsen, and H. Kirchner. Antiviral effects of recombinant tumour necrosis factor in vitro. Nature 323: 816-819, 1986[Medline].

9.   Paya, C. V., N. Kenmotsu, R. A. Schoon, and P. J. Leibson. Tumor necrosis factor and lymphotoxin secretion by human natural killer cells leads to antiviral cytotoxicity. J. Immunol. 141: 1989-1995, 1988[Abstract/Free Full Text].

10.   Ramsay, A. J., J. Ruby, and I. A. Ramshaw. A case for cytokines as effector molecules in the resolution of virus infection. Immunol. Today 143: 2031-2037, 1993.

11.   Singh, G., and S. L. Katyal. Clara cells and Clara cell 10 kD protein (CC10). Am. J. Respir. Cell Mol. Biol. 17: 141-143, 1997[Free Full Text].

12.   Singh, G., S. L. Katyal, W. E. Brown, and A. L. Kennedy. Mouse Clara cell 10-kDa (CC10) protein: cDNA nucleotide sequence and molecular basis for the variation in progesterone binding of CC10 from different species. Exp. Lung Res. 19: 67-75, 1993[Medline].

13.   Singh, G., S. L. Katyal, W. E. Brown, A. L. Kennedy, U. Singh, and M. L. Wong-Chong. Clara cell 10 kDa protein (CC10): comparison of structure and function to uteroglobin. Biochim. Biophys. Acta 1039: 348-355, 1990[Medline].

14.   Stripp, B. R., J. Lund, G. W. Mango, K. C. Doyen, C. Johnston, K. Hultenby, M. Nord, and J. A. Whitsett. Clara cell secretory protein: a determinant of PCB bioaccumulation in mammals. Am. J. Physiol. 271 (Lung Cell. Mol. Physiol. 15): L656-L664, 1996[Abstract/Free Full Text].

15.   Vasanthakumar, G., R. Manjunath, A. B. Mukherjee, H. Warabi, and E. Schiffmann. Inhibition of phagocyte chemotaxis by uteroglobin, an inhibitor of blastocyst rejection. Biochem. Pharmacol. 37: 389-394, 1988[Medline].

16.   Vitalis, T. Z., N. Keicho, S. Itabashi, S. Hayashi, and J. C. Hogg. A model of latent adenovirus 5 infection in the guinea pig (Cavia porcellus). Am. J. Respir. Cell Mol. Biol. 14: 225-231, 1996[Abstract].

17.   Wright, J. R. Immunomodulatory functions of surfactant. Physiol. Rev. 77: 931-962, 1997[Abstract/Free Full Text].

18.   Yang, Y., Q. Li, H. C. J. Ertl, and J. M. Wilson. Cellular and humoral immune responses to viral antigens create barriers to lung-directed gene therapy with recombinant adenoviruses. J. Virol. 69: 2004-2015, 1995[Abstract].

19.   Yang, Y., F. A. Nunes, K. Berensci, E. E. Furth, E. Gonczol, and J. M. Wilson. Cellular immunity to viral antigens limits E1-deleted adenoviruses for gene therapy. Proc. Natl. Acad. Sci. USA 91: 4407-4411, 1994[Abstract].

20.   Zhang, Z., G. C. Kundu, C.-J. Yuan, J. M. Ward, E. J. Lee, F. DeMayo, H. Westphal, and A. B. Mukherjee. Severe fibronectin-deposit renal glomerular disease in mice lacking uteroglobin. Science 276: 1408-1412, 1997[Abstract/Free Full Text].

21.   Zsengeller, Z. K., S. E. Wert, C. J. Bachurski, K. L. Kirwan, B. C. Trapnell, and J. A. Whitsett. Recombinant adenoviral vector disrupts surfactant homeostasis in mouse lung. Hum. Gene Ther. 8: 1331-1344, 1997[Medline].

22.   Zsengeller, Z. K., S. E. Wert, W. M. Hull, X. Hu, S. Yei, B. C. Trapnell, and J. A. Whitsett. Persistence of replication-deficient adenovirus-mediated gene transfer in lungs of immune-deficient (nu/nu) mice. Hum. Gene Ther. 6: 457-467, 1995[Medline].


Am J Physiol Lung Cell Mol Physiol 275(5):L924-L930
0002-9513/98 $5.00 Copyright © 1998 the American Physiological Society