The Journal of Immunology
Clara Cell Secretory Protein Modulates Lung Inflammatory and Immune Responses to Respiratory Syncytial Virus Infection1 Shan-Ze Wang,* Cynthia L. Rosenberger,* Yi-Xiao Bao,* James M. Stark,† and Kevin S. Harrod2* Clara cell secretory protein (CCSP) has been shown to have anti-inflammatory and immunomodulatory functions in the lung. Respiratory syncytial virus (RSV) is the most common cause of respiratory infection in infants and young children. RSV usually infects small airways and likely interacts with the Clara cells of bronchioles. To determine a possible role for CCSP during acute RSV infection, CCSP-deficient (CCSPⴚ/ⴚ) and wild-type (WT) mice were intratracheally infected with RSV and the lung inflammatory and immune responses to RSV infection were assessed. RSV-F gene expression was increased in the lungs of CCSPⴚ/ⴚ mice as compared with WT mice following RSV infection, consistent with increased viral persistence. Lung inflammation was significantly increased in CCSPⴚ/ⴚ mice as compared with WT mice after infection. Moreover, although the levels of Th1 cytokines were similar, the levels of Th2 cytokines and neutrophil chemokines were increased in the lungs of CCSPⴚ/ⴚ mice following infection. Physiologic endpoints of exacerbated lung disease, specifically airway reactivity and mucus production, were increased in CCSPⴚ/ⴚ mice after RSV infection. Importantly, restoration of CCSP in the airways of CCSPⴚ/ⴚ mice abrogated the increased viral persistence, lung inflammation, and airway reactivity. These findings suggest a role for CCSP and Clara cells in regulating lung inflammatory and immune responses to RSV infection. The Journal of Immunology, 2003, 171: 1051–1060.
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lara cell secretory protein (CCSP,3 also known as CC10, CC16, or uteroglobin) is an abundant protein secreted from nonciliated bronchial epithelial cells (Clara cells) of the conducting airways and distal bronchioles (1, 2). It is the prototypic member of a new subfamily of secretoglobins with putative anti-inflammatory functions (3). Although the precise molecular function of CCSP has not been defined, studies in CCSP genetargeted (CCSP⫺/⫺) mice strongly support its role in modulating lung inflammatory responses to infection or injury (4 – 6). In addition, clinical studies indicate that CCSP expression and production are decreased in chronic asthmatic patients (7–9). The decreased production and expression may in turn contribute to the pathophysiology of asthma. Recent studies from this and other laboratories indicate that OVA-immunized CCSP null mice have increased airway hyperreactivity (AHR), lung inflammatory responses, mucus production, and pulmonary Th2 cytokine responses following OVA challenge (10, 11). Taken together, these
*Asthma and Pulmonary Immunology Program, Lovelace Respiratory Research Institute, Albuquerque, NM 87108; and †Department of Molecular Microbiology and Immunology, University of Missouri, Columbia, MO 65211 Received for publication March 7, 2003. Accepted for publication May 21, 2003. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 This study was supported in part by grants from the American Lung AssociationAsthma Research Center (to S.-Z.W.) and by National Heart, Lung, and Blood Institute Grant HL-66994 (to K.S.H.). 2 Address correspondence and reprint requests to Dr. Kevin S. Harrod, Asthma and Pulmonary Immunology Program, Lovelace Respiratory Research Institute, 2425 Ridgecrest Drive SE, Albuquerque, NM 87108. E-mail address:
[email protected] 3 Abbreviations used in this paper: CCSP, Clara cell secretory protein; AHR, airway hyperactivity; SP, surfactant protein; RSV, respiratory syncytial virus; WT, wild type; rh, recombinant human; AR, airway reactivity; BALF, bronchoalveolar lavage fluid; MIP, macrophage inflammatory protein; AB-PAS, Alcian blue-periodic acid Schiff; Penh, enhanced pause.
Copyright © 2003 by The American Association of Immunologists, Inc.
findings indicate that CCSP modulates lung inflammatory and immune responses to infection, injury, and allergen challenge. CCSP gene-targeted mice are healthy and fertile, with no gross physiological or pathological abnormalities, (12), suggesting that CCSP does not mediate de novo critical lung function. Clara cell morphology is altered by CCSP deficiency, with markedly attenuated secretory granules present in ultrastructural studies of the airway epithelium of CCSP⫺/⫺ mice (13). Although Clara cells are known to produce surfactant proteins (SP), such as SP-A, -B, and -D, with critical lung homeostatic functions, the regulation of these proteins appears not to be altered by CCSP deficiency (12, 14). Recent evidence indicates that CCSP deficiency may alter the composition of the airway surface fluid (13). Although the implications of this are not entirely clear, numerous host defense compounds are produced and secreted by the airway epithelium (2). Furthermore, it has also been shown that CCSP⫺/⫺ mice have increased IgA mRNA expression in peribronchial B lymphocytes (6), indicating a heightened ability to induce or regulate innate immune and inflammatory mechanisms consistent with the previous findings from multiple laboratories (6, 10, 11). Respiratory syncytial virus (RSV) is the most common pathogen of the respiratory tract in infants and young children worldwide, and RSV infection in early childhood is strongly associated with the subsequent development of allergy and asthma (15, 16). Currently, there is no effective therapy for RSV infection, in part due to the poorly understood pathogenesis of RSV-induced airway disease. Although RSV infection can occur throughout the tracheobronchial tree, infection of small airways is more commonly associated with severe clinical outcomes (17). Thus, RSV likely interacts with Clara cells and CCSP in the lung during RSV infection. However, the role of CCSP in modulating lung inflammatory responses to RSV infection is unknown. To define a role for CCSP in regulating the host response to RSV infection, CCSP⫺/⫺ mice were infected with human RSV, 0022-1767/03/$02.00
1052 and lung disease was assessed for distinct markers of lung epithelial and immunological endpoints important in RSV-induced lung disease. The present study indicates that CCSP deficiency increases the pathogenesis of RSV infection. Furthermore, restoration of CCSP in the airways of CCSP⫺/⫺ mice abrogated the increase in viral persistence, lung inflammation, and AHR induced by RSV infection. Thus, CCSP and Clara cells likely play an important role in regulating inflammatory and immune responses to RSV infection.
Materials and Methods Animals CCSP⫺/⫺ mice (129J Ola/129J hybrid, kindly provided by Dr. J. A. Whitsett, Children’s Hospital, Cincinnati, OH) (12) and wild-type (WT) mice (129J; The Jackson Laboratory, Bar Harbor, ME) were housed under pathogen-free conditions in the Lovelace Respiratory Research Institute vivarium following Association for the Assessment and Accreditation of Laboratory Animal Care International guidelines.
Respiratory syncytial virus A stock of RSV A2 was generated in HEp-2 cells and viral titration performed as described (18, 19). Briefly, purified RSV titers were determined in triplicate for each culture dilution using standard plaque assay procedures. Plaque formation was counted manually by visualization following hematoxylin staining of cell monolayers.
Mouse model of RSV infection Intratracheal inoculation of RSV (107 PFU in 100 l of culture medium) was performed by insertion of a 27-gauge tube through the oral cavity of mice under anesthesia. Intratracheal insertion was confirmed by the lack of negative pressure applied by retraction of a syringe plunger. A syringe containing the RSV preparation was expelled into the trachea followed by multiple injections of air to facilitate dispersion of the inoculant into the lung periphery. Mice usually recovered within 5 min and resumed normal eating and grooming activities.
CCSP MODULATES RSV INFECTION Doc documentation system and Quantity One software (Bio-Rad, Hercules, CA).
Cytokine production Cytokine protein levels were measured as described (11). Briefly, the apical and intermediate lobes of the right lungs were homogenized in 1 ml of PBS and centrifuged at 2000 rpm for 7 min at 4°C. The supernatants were collected for cytokine protein production analysis. The levels of neutrophil chemokine macrophage inflammatory protein (MIP)-2 and CXC chemokine KC, and Th1/Th2 cytokines IFN-␥, IL-4, IL-5, IL-12, and IL-13 were analyzed using ELISA (R&D Systems, Minneapolis, MN). According to the manufacturer’s recommendations, data were normalized by volume of homogenate supernatants. Standard curves were calculated from known standards for calculation of cytokine concentrations.
Pulmonary histopathology Lung inflammation was assessed by histological staining of lung sections as described (11). Left lungs were inflated via a tracheal cannula at 20 cm of pressure with 4% paraformaldehyde and removed en bloc from the thorax. For paraffin embedding, inflation-fixed lungs were washed in PBS three times and bisected transversely in the dorsoventral direction just caudal to the entry of the mainstream bronchus. Lung sections (5 m) were taken starting 100 m from the designated reference point and collected at 100-m intervals. Two sections of the left lung for each animal were stained with H&E, and graded on a minimal, mild, moderate, and marked scale corresponding to numbers 1 (minimal) to 4 (marked) for the following criteria: septal, perivascular, and peribronchiolar infiltrates; epithelial cell hyperplasia/hypertrophy. The score for each animal is the average of the above four criteria. All slides were scored blindly, and a score was determined for the mean (⫾SEM) of six to eight animals per group.
Alcian blue-periodic acid Schiff (AB-PAS) staining
Purified recombinant human CCSP (rhCCSP, also known as rhCC10) was kindly provided by Claragen (College Park, MD). Previous studies have determined that 50 g of recombinant rat CCSP are sufficient to block LPS-mediated neutrophil migration in the lung (20). For the current studies, 50 g of rhCCSP diluted in 100 l of sterile water was intratracheally instilled into each mouse. RSV was intratracheally instilled (107 PFU/ mouse) 2 h later following rhCCSP restoration. Purified human albumin (Sigma-Aldrich, St. Louis, MO) was used as a control protein.
Lung sections (5 m) of the left lung lobe (six to eight mice per group) at 100 –200 m from the reference point were stained by AB-PAS to identify mucus-secreting cells as described (11). Briefly, the lung sections were deparaffinized in xylene and hydrated in decreasing concentrations of ethanol. The slides were then stained in AB for 30 min, washed in running water for 5 min, oxidated in 1% periodic acid for 10 min, and washed in running water for another 5 min. After staining in Schiff’s reagent for 10 min, slides were rinsed three times in sodium metabisulfite, washed in running water for 10 min, and mounted following dehydration in ethanol and xylene. Photomicrographs (at ⫻400) show the mucus-producing cells with the distinctive color for PAS positivity (pink) and AB-PAS positivity (purple). In all mice, only the mucous cells in epithelia lining the main intrapulmonary bronchus were analyzed. The length of basal lamina lining the large airways of WT and CCSP⫺/⫺ mice and the volume density of the AB-PAS-stained mucosubstances in the surface epithelium were determined using Scion Image Software (NIH, Bethesda, MD); mucosubstance calculations were normalized to mm2 of basal lamina as described (22).
Sample preparation and analysis
Measurement of airway reactivity
Mice (8 –10 per group) were used for measuring airway reactivity (AR), and bronchoalveolar lavage fluid (BALF) was then collected from these mice. In parallel, six to eight mice per group were used for pathological and molecular studies. The left lung was inflation-fixed, paraffin-embedded, and sectioned for histopathological and immunohistochemical studies (11). Two lobes of the right lung were homogenized for protein studies, and RNA was extracted from the other two lobes of the right lung.
AR was measured with a whole-body plethysmograph (Buxco Electronics, Sharon, CT) (11). As previously described (23), AR is expressed as enhanced pause (Penh), a calculated value showing a strong correlation with airway resistance measured using standard procedures (24, 25). Individual mice were placed in parallel chambers connected to an automatic electric nebulizer and a recording system. Baseline AR of mice was recorded for 5 min. Subsequently, the mice were challenged for 1 min to nebulized saline and increasing concentrations (3, 6, 12, and 25 mg/ml) of nebulized methacholine (ICN Biomedicals, Aurora, OH). After each nebulization, a recording was taken for 10 min. The Penh values measured during the first 5 min were averaged and expressed as mean ⫾ SEM (n ⫽ 8 –10 mice/group).
Restoration of CCSP in the airways of CCSP⫺/⫺ mice
RNA isolation and RT-PCR Total RNA was isolated from the cardiac and diaphragmatic lobes of the right lungs using Tri-reagents (Molecular Research Center, Cincinnati, OH) following the manufacturer’s protocol. RT-PCR analysis was designed to detect nascent viral mRNA transcripts, but not genomic or progeny RSV RNA, as a measure of viral transcriptional activity as described (21). The virus-specific mRNA transcript for RSV gene F (designated RSV-F) was converted to cDNA by the reverse transcriptase reaction using the following virus-specific primer sequence: RSV-F, 5⬘-CAACTCCAT TGTTATTTGCC-3⬘. The RSV-specific sequence was amplified by PCR using the following primer set: RSV-F upper primer, 5⬘-CCAGCAAAGT GTTAGACCTCAAAA-3⬘; RSV-F lower primer, 5⬘-AATCGCACCCGT TAGAAAATG-3⬘. Primer sequences were identified using Lasergene software (DNAstar, Madison, WI). The RSV-specific sequence was amplified for 30 cycles, and RT-PCR amplification products were visualized by ethidium bromide-stained gel electrophoresis under UV light. Gel images were captured and densitometric analysis was performed using the Gel-
BALF and cell counting BALF was obtained by three serial intratracheal instillations of 1 ml of PBS into the lung, and the samples were pooled for each animal. Cells in the BALF were isolated by centrifugation at 1500 rpm for 10 min and resuspended in 200 l of PBS. Viable cells were counted by hemacytometer in a 1:5 solution of BALF suspension to 0.4% trypan blue (Life Technologies, Grand Island, NY). To determine inflammatory cell types in BALF, 5 ⫻ 104 cells were mounted onto slides by cytospin centrifugation (400 rpm for 4 min) in 100 l of PBS. Lung cytology was identified and counted by differential staining microscopy with Hema-Tek Stain Pak, a modified Wright-Giemsa stain (Bayer, Elkhart, IN). Inflammatory cell populations were determined by counting 100 cells, and a percentage was calculated based on two sample sets from 8 –10 animals per group. The
The Journal of Immunology
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absolute cell numbers for each cell type were calculated according to the total leukocyte counts in BALF and the percentage of each cell type for every sample.
Statistical analysis Results from 6 –10 different mice in each group were expressed as mean ⫾ SEM unless otherwise indicated. Differences between groups were assessed for significance by Student’s t test, when data were available in only two groups. When data were available in more than two groups, ANOVA was used to perform pairwise comparisons, followed by Fisher’s least significant difference test. Differences were considered significant at p ⬍ 0.05.
Results
⫺/⫺
RSV clearance is decreased in the lungs of CCSP
mice
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CCSP and WT mice (8 –9 wk of age) were intratracheally infected with RSV (107 PFU/mouse) for 2, 4, 7, and 10 days, respectively. To assess viral clearance in the lungs of CCSP⫺/⫺ mice, RT-PCR analysis of the viral-specific gene expression of RSV-F was performed from total lung RNA. RT-PCR analysis of endogenous -actin mRNA steady state levels was used as loading and assay controls. After 2 days of RSV infection, the RSV-F gene was expressed readily in the lungs of all CCSP⫺/⫺ and four of six WT mice. However, the relative level of RSV-F gene expression was increased in the lungs of CCSP⫺/⫺ mice as compared with WT mice (Fig. 1). After 4 days of RSV infection, RSV-F gene expression was still readily detectable in the lungs of all CCSP⫺/⫺ mice. In contrast, the RSV-F gene was only weakly expressed in the lungs from three of six WT mice. Densitometric analysis of RSV-F mRNA levels indicated a significant increase of RSV-F in the lungs of CCSP⫺/⫺ mice as compared with WT mice (Fig. 1B). On day 7 postinfection, the RSV-F gene was only detected at a very
FIGURE 1. RSV gene expression is increased in CCSP-deficient (⫺/⫺) mice following RSV infection. WT and CCSP⫺/⫺ mice were treated with RSV (1 ⫻ 107 PFU/mouse) for 2, 4, 7, or 10 days (n ⫽ 6 mice/group). Total RNA was isolated from the right lungs of the mice. RSV-F steady state, positive strand RNA levels were assessed by RT-PCR as described in Materials and Methods. RSV-F-amplified cDNA was visualized by ethidium bromide-stained gel electrophoresis, and -actin was used as a loading control. A, Representative RT-PCR analysis of RSV-F and -actin in the lung homogenates of WT and CCSP⫺/⫺ mice after RSV infection for 2, 4, 7, and 10 days. B, Densitometric analysis of RSV-F mRNA levels. The intensity of the RSV-F PCR product was normalized to that of -actin for each sample after subtraction of background. The numerator indicates the number of animals in which RSV-F gene expression was detected, and the denominator indicates the total number of animals in each group of the experiment. The OD ratio of RSV-F over -actin is significantly increased in CCSP⫺/⫺ mice as compared with WT mice at 2, 4, and 7 days following RSV infection (ⴱ, p ⬍ 0.05).
low level in the lungs of one of six WT mouse. However, RSV-F gene expression was still weak in five of six CCSP⫺/⫺ mice. On day 10 postinfection, the RSV-F gene was not detectable in any WT mice and only at a very low level in one of six CCSP⫺/⫺ mouse. -actin mRNA steady state levels were not changed in the lungs of CCSP⫺/⫺ mice as compared with WT mice at any time following RSV infection. Lung inflammation is increased in CCSP⫺/⫺ mice after RSV infection To determine the effect of CCSP deficiency on lung inflammation, BALF cellularity from CCSP⫺/⫺ and WT mice was assessed following RSV inoculation. The total cell numbers, and the numbers of neutrophils and lymphocytes were significantly increased in the BALF from CCSP⫺/⫺ mice as compared with those from WT mice 2 days after RSV infection (Fig. 2A). The total inflammatory cell numbers were further increased in the BALF from CCSP⫺/⫺ as compared with those from either WT mice infected with RSV for 7 days or CCSP⫺/⫺ mice infected with RSV for 2 days. The increase in inflammatory cells in the lungs of CCSP⫺/⫺ mice at 7 days postinfection comprised mainly macrophages, although increased neutrophils and eosinophils were noted (Fig. 2B). To further determine the role of CCSP in modulating lung inflammatory responses to RSV infection, lung inflammation and epithelial morphology were analyzed by histological staining of lung sections. The sections were scored blindly according to septal infiltrates, perivascular infiltrates, peribronchiolar infiltrates, and epithelial hyperplasia/hypertrophy in CCSP⫺/⫺ and WT mice at 2 and 7 days following RSV infection. Increased inflammatory cell infiltration was noted in both proximal and distal airways of CCSP⫺/⫺ mice as compared with WT mice after RSV infection for
FIGURE 2. Total and differential cell counts are increased in BALF from CCSP⫺/⫺ mice after RSV infection. Total cells were counted by a hemacytometer following trypan blue exclusion staining. Differential staining was used to identify cell types based upon nuclear and cytoplasmic morphology and staining patterns. Total and differential cells were counted in BALF from WT and CCSP⫺/⫺ mice treated with either vehicle (Veh) or RSV (1 ⫻ 107 PFU/mouse) for 2 (A) and 7 days (B) (n ⫽ 8 –10 mice/ group). Data are expressed as mean cell counts ⫾ SEM. Total, total leukocytes; Mac, macrophage; Lymph, lymphocyte; Neut, neutrophil; Eosin, eosinophil. ⴱ, p ⬍ 0.05, as compared with respective vehicle control group; ⴱⴱ, p ⬍ 0.05 as compared with both vehicle control group and WT RSV group.
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CCSP MODULATES RSV INFECTION
FIGURE 3. Lung histopathological scores are increased in the lungs of CCSP⫺/⫺ mice after RSV infection. A, Photomicrographs were taken from representative slides of WT and CCSP⫺/⫺ mice infected with RSV for 7 days (original magnification, ⫻200). Leukocyte infiltration in the lung parenchyma and lung epithelial hypertrophy were increased in both proximal and distal airways of CCSP⫺/⫺ mice as compared with those in WT mice after RSV infection. Arrows point to the thickened myofibril layer in the basal compartments of the large airways in CCSP⫺/⫺ mice. B, Pathological scoring of lung inflammation and injury was assessed in WT and CCSP⫺/⫺ mice treated with either vehicle or RSV (1 ⫻ 107 PFU/mouse) for 2 and 7 days (n ⫽ 6 – 8 mice/group). Vehicle control mice were assessed and scored as zero. Data are expressed as mean scores ⫾ SEM. The histopathological scores were significantly increased in CCSP⫺/⫺ mice as compared with those in WT mice after RSV infection for either 2 or 7 days (ⴱ, p ⬍ 0.05; ⴱⴱ, p ⬍ 0.01).
2 or 7 days (Fig. 3A, representative photomicrographs from mice infected with RSV for 7 days). Areas of consolidated inflammatory cell infiltrates were distinct in CCSP⫺/⫺ mice. Furthermore, morphologic changes in airway epithelial cells were readily observable in both large and small airways of CCSP⫺/⫺ mice following RSV infection, with loss of the normal cuboidal appearance of nonciliated bronchiolar epithelial (Clara) cells, the increased appearance of columnar and pseudostratified epithelial cell morphology, and airway epithelial cell sloughing. Increased thickening of the myofibril layer in the basal compartments of the large airways was apparent in the CCSP⫺/⫺ mice following RSV infection. Accordingly, the mean pathological score was significantly increased in the lungs of CCSP⫺/⫺ mice as compared with WT mice following RSV infection for both 2 and 7 days (Fig. 3B). No significant difference of epithelial morphology was noted between WT and CCSP⫺/⫺ mice treated with vehicle. Collectively, leukocyte recruitment in the BALF and lung histopathological scores were increased in CCSP⫺/⫺ mice following RSV infection. Increased neutrophil chemokines in the lungs of CCSP⫺/⫺ mice infected with RSV To determine a possible mechanism by which CCSP deficiency causes increased neutrophil recruitment in BALF and increased lung infiltration, the neutrophil chemokines MIP-2 and KC were assessed in the lung homogenates of CCSP⫺/⫺ and WT mice at either 2 or 7 days after RSV inoculation. The levels of MIP-2 (Fig. 4A) and KC (Fig. 4B) were significantly increased in WT mice infected with RSV as compared with vehicle control WT mice after either 2 or 7 days. MIP-2 and KC levels were further increased in CCSP⫺/⫺ mice as compared with those in WT mice following RSV infection at 2 and 7 days. Overall, the production of both MIP-2 and KC was increased in the lungs of CCSP⫺/⫺ mice after RSV infection for either 2 or 7 days, in accordance with the observed increase in lung neutrophil numbers.
FIGURE 4. The neutrophil chemokines MIP-2 and KC are increased in the lungs of CCSP⫺/⫺ mice following RSV infection. MIP-2 and KC levels were determined by ELISA in lung homogenates from WT and CCSP⫺/⫺ mice treated with either vehicle or RSV (1 ⫻ 107 PFU/mouse) for 2 or 7 days (n ⫽ 6 – 8/group). Data are expressed as mean levels ⫾ SEM. The levels of MIP-2 (A) and KC (B) were significantly increased in WT mice infected with RSV compared with vehicle control WT mice (ⴱ, p ⬍ 0.05) after either 2 or 7 days of treatment. The MIP-2 and KC levels were further increased in CCSP⫺/⫺ mice as compared with those in WT mice following RSV infection for 2 and 7 days, respectively (ⴱⴱ, p ⬍ 0.05).
The Journal of Immunology Increased Th2 cytokines in the lungs of CCSP⫺/⫺ mice following RSV infection To the determine the role of CCSP in modulating immune responses to RSV infection, the levels of IFN-␥, IL-4, IL-5, IL-12, and IL-13 were assessed in the lung homogenates of CCSP⫺/⫺ and WT mice at 2 or 7 days following either vehicle treatment or RSV infection. The levels of IL-13 (Fig. 5A) and IL-5 (Fig. 5B) were only slightly changed in the lungs of either WT or CCSP⫺/⫺ mice at 2 days following RSV infection as compared with vehicle control treatment. However, IL-13 (Fig. 5A) and IL-5 (Fig. 5B) levels were markedly increased in the lungs of CCSP⫺/⫺ mice infected with RSV for 7 days as compared with vehicle control CCSP⫺/⫺ mice, CCSP⫺/⫺ mice infected with RSV for 2 days, or WT mice infected with RSV for 7 days. The levels of IFN-␥ were increased in the lungs of both WT and CCSP⫺/⫺ mice at 7 days following RSV infection as compared with vehicle control mice, respectively. However, there was no significant change of IFN-␥ levels between the WT and CCSP⫺/⫺ mice following RSV infection (data not shown). The IL-4 and IL-12 protein levels were similar in the lungs of WT and CCSP⫺/⫺ mice following RSV infection for either 2 or 7 days (data not shown).
1055 2 days following RSV infection, respectively. Furthermore, both the numbers of AB-PAS-positive cells and intensity of the mucous staining were increased in the airways of CCSP⫺/⫺ mice as compared with WT at 7 days following RSV infection (Fig. 6A, representative micrographs). The volume density of AB-PAS-positive material was also increased in the airways of CCSP⫺/⫺ mice as compared with WT mice at 7 days after RSV infection (Fig. 6B). This increased AB-PAS-positive mucosubstance is consistent with the increased IL-5 and IL-13 levels in the lungs of CCSP⫺/⫺ mice at 7 days following RSV infection. AR is increased in CCSP⫺/⫺ mice following RSV infection To assess the role of CCSP in modulating the development of AHR induced by RSV infection, the AR of CCSP⫺/⫺ and WT mice was measured at 2 and 7 days following either RSV or vehicle treatment. Both WT and CCSP⫺/⫺ vehicle control mice responded poorly to methacholine challenges, and the AR of WT and CCSP⫺/⫺ mice was similar on days 2 and 7 after vehicle treatment (data not shown). The AR was only slightly increased in WT mice infected with RSV (Fig. 7) as compared with vehicle treatment for 2 and 7 days, respectively. However, the AR was significantly increased in CCSP⫺/⫺ mice as compared with WT mice infected
AB-PAS-positive mucosubstances are increased in CCSP⫺/⫺ mice following RSV infection The role of CCSP in modulating RSV-induced mucous cell metaplasia was examined by AB-PAS staining of the airways in CCSP⫺/⫺ and WT mice at 2 and 7 days following RSV infection or vehicle treatment. Mucus staining was not apparent in the airway epithelium of vehicle-treated WT and CCSP⫺/⫺ mice (data not shown). AB-PAS-positive cells were visible in the airways of WT and CCSP⫺/⫺ mice after RSV infection for 2 days (data not shown). The intensity and number of AB-PAS-stained cells were increased in the airways of both WT and CCSP⫺/⫺ mice infected with RSV for 7 days as compared with WT and CCSP⫺/⫺ mice at
FIGURE 5. The Th2 cytokines IL-13 and IL-5 are increased in the lungs of CCSP⫺/⫺ mice after RSV infection. IL-13 and IL-5 protein levels were determined by ELISA in lung homogenates from WT and CCSP⫺/⫺ mice treated with either vehicle or RSV (1 ⫻ 107 PFU/mouse) at 2 or 7 days following RSV infection (n ⫽ 6 – 8/group). The data are expressed as mean levels ⫾ SEM. The levels of IL-13 (A) and IL-5 (B) were significantly increased in CCSP⫺/⫺ mice infected with RSV for 7 days as compared with either vehicle control CCSP⫺/⫺ mice, or CCSP⫺/⫺ mice infected with RSV for 2 days, or WT mice infected with RSV for either 2 or 7 days (ⴱ, p ⬍ 0.05; ⴱⴱ, p ⬍ 0.01).
FIGURE 6. Mucus staining in the airways is increased in CCSP⫺/⫺ mice after RSV infection. Mucus-producing cells in the airways were identified by AB-PAS staining. A, Photomicrographs were taken from representative slides of WT and CCSP⫺/⫺ mice infected with RSV (1 ⫻ 107 PFU/mouse) for 7 days (original magnification, ⫻400). AB-PAS staining was increased in the airway epithelia of CCSP⫺/⫺ mice compared with that in WT mice at 7 days after RSV infection. B, Volume of stored AB-PASpositive material in airway epithelia was significantly increased in CCSP⫺/⫺ mice compared with WT mice after RSV infection for 7 days (n ⫽ 6 – 8 mice/group) (ⴱ, p ⬍ 0.05).
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FIGURE 7. AR is increased in CCSP⫺/⫺ mice following RSV infection. AR was recorded in WT and CCSP⫺/⫺ mice treated with either vehicle or RSV (1 ⫻ 107 PFU/mouse) for 2 or 7 days. The data are presented as mean Penh ⫾ SEM (n ⫽ 8 –10 mice/group). The AR of CCSP⫺/⫺ mice was significantly increased as compared with that of either vehicle-treated CCSP⫺/⫺ mice or RSV-infected WT mice (ⴱ, p ⬍ 0.05; ⴱⴱ, p ⬍ 0.01). The AR of WT mice infected with RSV for either 2 or 7 days was not significantly increased as compared with that in vehicle-treated WT mice. There was no significant difference between the AR of vehicle-treated WT mice and vehicle-treated CCSP⫺/⫺ mice (data not shown).
with RSV for either 2 or 7 days (Fig. 7). The increased AR was consistent with increased lung inflammation and mucus staining in the lungs of CCSP⫺/⫺ following RSV infection. Restoration of CCSP in the airways of CCSP⫺/⫺ mice abrogates decreased viral clearance and reduces lung inflammation and AR To further confirm the role of CCSP in modulating viral persistence, lung inflammation, and the development of AHR in RSV infection, CCSP⫺/⫺ mice were instilled intratracheally with 50 g of rhCCSP. In a control group of CCSP⫺/⫺ mice, 50 g of human albumin was instilled. RSV (107 PFU/mouse) was intratracheally instilled 2 h following either rhCCSP restoration or albumin treatment. RSV-F gene expression, AR, and lung inflammation were assessed 2 days postinfection. RSV-F gene expression was decreased in the lungs of CCSP⫺/⫺ mice pretreated with rhCCSP compared with pretreatment with albumin or no pretreatment followed by RSV infection (Fig. 8). In contrast, pretreatment with albumin did not significantly affect RSV-F gene expression in the lungs of CCSP⫺/⫺ mice infected with RSV as compared with no pretreatment. These data indicate that restoration of CCSP in the airways of CCSP⫺/⫺ mice abrogates the increase of viral persistence. Similarly, restoration of CCSP in the airways of CCSP⫺/⫺ mice modulated overall lung inflammation (Fig. 9A) and lung histopathological scores (Fig. 9B), as well as the total cell and neutrophil numbers in the BALF (Fig. 9C), compared with those in CCSP⫺/⫺ mice pretreated with albumin or CCSP⫺/⫺ mice without pretreatment followed by RSV infection. Consistent with the decreased viral persistence and lung inflammation, AR was also significantly decreased in CCSP⫺/⫺ mice with CCSP restoration followed by RSV infection for 2 days as compared with that in CCSP⫺/⫺ mice treated with RSV or albumin and RSV for 2 days (Fig. 10). Comparison of the abrogated host response in the lungs of CCSP⫺/⫺ mice with that of CCSP⫹/⫹ mice following RSV infection indicates that restoration of CCSP in the lungs of CCSP⫺/⫺ is markedly similar to that of CCSP⫹/⫹ at 2 days following infection.
CCSP MODULATES RSV INFECTION
FIGURE 8. Restoration of CCSP in the airways of CCSP⫺/⫺ mice decreases RSV gene expression. CCSP⫺/⫺ mice were treated with RSV alone, albumin and RSV, or rhCCSP and RSV (1 ⫻ 107 PFU/mouse), respectively, for 2 days (n ⫽ 7 mice/group), as described in Materials and Methods. RSV-F steady state, positive strand RNA levels were assessed by RT-PCR of lung total RNA. RSV-F-amplified cDNA was visualized by ethidium bromide-stained gel electrophoresis, and -actin was used as a loading control. A, Representative RT-PCR analysis of RSV-F and -actin in the lung homogenates of CCSP⫺/⫺ mice treated with RSV alone, albumin and RSV, and rhCCSP and RSV, respectively. B, Densitometric analysis of RSV-F mRNA levels. The intensity of the RSV-F PCR product was normalized to that of -actin for each sample after background subtraction. The OD ratio of RSV-F over -actin is significantly decreased in CSSP (⫺/⫺) mice pretreated with rhCCSP followed by RSV infection as compared with CCSP⫺/⫺ mice treated only with RSV (ⴱ, p ⬍ 0.05).
Discussion RSV lung infection accounts for significant morbidity in both young children and the elderly (26). The role of lung epithelium in regulating host immune responses to RSV infection is not well understood. Clara cells are abundant in distal and terminal airways of the human respiratory tract (1, 2), which are important sites for asthma or RSV infection (17). Although a definitive molecular function has been not been elucidated, previous studies suggest that CCSP, an abundant Clara cell secretory product, may have important anti-inflammatory functions in the lung (4 – 6, 10, 11, 27). The present study indicates that CCSP deficiency increases viral persistence and lung inflammation. Furthermore, increased Th2 cytokines, mucus production, and AR, hallmarks of asthma or bronchiolitis, were observed following RSV infection. Restoration of CCSP in the airways abrogated the increased viral persistence and pulmonary inflammatory responses of CCSP⫺/⫺ mice to RSV infection. These studies suggest that CCSP may play an important role in modulating lung inflammatory and immune responses to RSV infection. Viral persistence is a critical element of virus-induced disease, reflecting a balance between viral replication and viral clearance that can alter the severity or duration of disease. RSV is a singlestranded, negative sense RNA virus which requires transcription of a nascent positive strand RNA from the parental-negative RNA as an initial step in RSV replication. Herein, RSV-F gene expression was increased in the lungs of CCSP⫺/⫺ mice, consistent with increased viral persistence in CCSP-deficient mice. Our previous studies indicate that exacerbation of lung disease by RSV infection
The Journal of Immunology
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FIGURE 10. Restoration of CCSP in the airways of CCSP⫺/⫺ mice abrogates the increase in AR by RSV infection. CCSP⫺/⫺ mice were pretreated with albumin or rhCCSP, or were untreated, and subsequently infected with RSV (1 ⫻ 107 PFU/mouse) for 2 days (n ⫽ 6 – 8 mice/group). AR is expressed as mean Penh ⫾ SEM. AR of CCSP⫺/⫺ mice was significantly decreased by rhCCSP pretreatment as compared with those of CCSP⫺/⫺ mice pretreated with albumin followed by RSV infection and in CCSP⫺/⫺ mice infected with RSV without pretreatment. Albumin pretreatment did not significantly affect the increased AR induced by RSV infection as compared with the RSV-infected mice without pretreatment (n ⫽ 6 – 8/mice group). ⴱ, p ⬍ 0.05 denotes statistical difference from RSV alone group; ⴱⴱ, p ⬍ 0.05 indicates statistical difference from both RSV alone group and albumin plus RSV group.
FIGURE 9. Lung histopathological scores are abrogated by reconstitution of CCSP in the airways of CCSP⫺/⫺ mice. A, Photomicrographs were taken from representative slides of CCSP⫺/⫺ mice treated with RSV alone, albumin and RSV, or rhCCSP and RSV (1 ⫻ 107 PFU/mouse) for 2 days (n ⫽ 6 – 8 mice/group). The original magnification was ⫻200. Leukocyte infiltration in the lung parenchyma and lung epithelial proliferation were decreased in the airways of CCSP⫺/⫺ mice treated with rhCCSP and RSV as compared with treatment of RSV alone or albumin and RSV. B, Lung histopathology and inflammation were scored in CCSP⫺/⫺ mice treated with RSV alone, albumin and RSV, or rhCCSP and RSV infection (1 ⫻ 107 PFU/mouse) for 2 days (n ⫽ 6 – 8 mice/group). Vehicle control mice were assessed and scored as zero. Data are expressed as mean scores ⫾ SEM. The histopathological scores were significantly decreased in CCSP⫺/⫺ mice pretreated with rhCCSP followed by RSV infection as compared with CCSP⫺/⫺ mice untreated before RSV infection or pretreated with albumin before RSV (ⴱ, p ⬍ 0.05). C, The inflammatory cells in BALF from these mice were assessed 2 days following RSV infection. The total inflammatory cell numbers and neutrophil cell numbers in BALF from CCSP⫺/⫺ mice were decreased by rCCSP pretreatment as compared with those of CCSP⫺/⫺ mice pretreated with albumin followed by RSV infection and in the CCSP⫺/⫺ mice infected with RSV alone (n ⫽ 6 – 8 mice/ group). Albumin pretreatment did not affect lung inflammation to RSV infection as compared with untreated, RSV-infected animals. ⴱⴱ, p ⬍ 0.05, as compared with both the RSV alone group and albumin plus RSV group.
can induce expression of other viral genes, suggesting that increased viral gene expression is not confined to a single viral gene (21). Furthermore, increased viral persistence has been associated
with decreased CCSP levels (4). The increased viral persistence concurrent with the observed increase in lung inflammation in CCSP⫺/⫺ mice is surprising, as increased inflammation is associated with increased clearance of pathogens. The findings presented herein are consistent with other models in which a constitutive host defense molecule secreted by the lung epithelium has been genetically or functionally attenuated (4, 28 –31). Mice deficient in SP-A, a host defense molecule secreted by Clara and alveolar type II cells, have increased inflammation to RSV infection, presumably due to inadequate clearance of RSV through viral clearance mechanisms specific to the lung epithelium (30). The role of CCSP in viral clearance and the mechanisms by which CCSP deficiency increases RSV persistence are unknown. However, similarities from this study with studies of other models of deficient host defense suggest the importance of epithelial-derived pathogen clearance mechanisms, as well as regulation of innate and adaptive immune response by epithelial-derived mediators (4, 28 –31). Consistent with the increase in RSV persistence in the lungs of CCSP⫺/⫺ mice, lung inflammation was also increased in CCSP⫺/⫺ mice after RSV infection. Early during the course of infection, inflammatory cells were comprised predominantly of neutrophils in the lungs of CCSP⫺/⫺ mice as compared with WT mice, while increased macrophages, eosinophils, and neutrophils were observed at later time points. The neutrophil chemokines MIP-2 and KC were also increased in the lungs of CCSP⫺/⫺ mice, concomitant with the increased neutrophil migration into the lungs of CCSP⫺/⫺ mice at early time points following infection. These findings are consistent with previous studies suggesting that CCSP may modulate neutrophil responses to infection (4) (20), injury (32), and allergen challenge (11) by regulating the production of neutrophil chemokines. It has also been shown that Th2 cytokine IL-13 can activate neutrophils and induce neutrophil recruitment to the airways in a rat model (33). Therefore, the increased IL-13 levels in CCSP⫺/⫺ mice may contribute to the increased neutrophil numbers in the airways on day 7 postinfection. There is currently
1058 no evidence for a direct effect of CCSP on neutrophil recruitment. Neutrophils are the predominant leukocytes in the airways of infants with RSV-induced bronchiolitis (34 –36). Neutrophils and their secreted products likely damage the airway epithelium during RSV infection (37–39) and other lung diseases (40). Furthermore, neutrophil elastase can induce mucin MUC5AC gene expression in airway epithelium (41) and induce mucus production (42, 43). Neutrophil infiltration into the lungs of CCSP⫺/⫺ mice and subsequent release of neutrophil elastase into the airways may contribute to the observed increased mucus production after RSV infection. Pretreatment with selective neutrophil elastase inhibitors can prevent OVA-induced goblet cell degranulation (42), and inhibit goblet cell hypersecretion induced by ozone exposure (44) or endotoxin treatment (45). However, these experimental approaches have not been studied to RSV infection. Therefore, inhibition of neutrophil elastase or blockade of neutrophil recruitment may ameliorate mucus production in asthma, RSV infection, and other chronic airway diseases. AHR is a hallmark of airway disease associated with asthma and bronchiolitis, both of which have been linked to RSV infection (15, 16). AR was markedly increased in CCSP⫺/⫺ mice following RSV infection, concurrent with increased lung inflammation and RSV lung burdens. The role of CCSP in regulating airway responses during RSV infection has not been previously investigated. Clinical studies indicate that CCSP-positive epithelial cells were reduced in small airways of patients with chronic asthma, and CCSP-positive epithelial cell proportions were inversely correlated with numbers of T cells and mast cells in the small airways of these patients (7). These findings are consistent with the notion that CCSP deficiency may lead to increased inflammatory or immune responses, which may contribute to the development of AHR and asthma (46). Likewise, CCSP levels in BALF (9) and serum (8) are also decreased in asthmatic patients. Recent studies indicate that CCSP deficiency increases lung inflammation (10) and AHR in a mouse model of asthma (11). The studies herein support the concept that CCSP may diminish the development of lung inflammation and subsequently AHR in the pathogenesis of RSV-induced airway disease. Human studies indicate that severe RSV infection in early life is associated with increased IL-5 and IL-13 production (47). In the present study, the Th2 cytokines IL-5 and IL-13 were increased in the lungs of CCSP⫺/⫺ mice following RSV infection. Th2 immune responses have been increased in an experimental allergen-induced asthma model using CCSP⫺/⫺ mice (10). Taken together, these studies suggest a role for CCSP in modulating Th2 immune responses in asthma and RSV infection. The present study also showed that mucus production by the airway epithelium of CCSP⫺/⫺ mice was increased after RSV infection, and may contribute to the development of AHR, although the mucus production observed herein is small as compared with those in OVA-challenge studies (11) or in other studies with RSV infection (48), using a different strain of mice. Both IL-5 and IL-13 are important mediators for mucus production, lung inflammation, and the development of AHR. IL-13 can directly induce mucus overproduction and AHR in asthma (49, 50), and pulmonary expression of IL-5 alone can induce goblet cell metaplasia and mucus production (51). IL-5 and IL-13 may also induce the development of airway inflammation and AHR in RSV infection in mice (52–54). IL-5 and IL-13 have eosinophil activities, and the increase of these two cytokines is consistent with the increased eosinophil numbers observed. Furthermore, there is evidence indicate that, besides the classical pathway IFN-␥, Th2 cytokines IL-4 and IL-13 can also activate macrophages (55). Therefore, the increased macrophages in the lungs of CCSP⫺/⫺ mice after 7 days of RSV infection may
CCSP MODULATES RSV INFECTION be due to the increased levels of IL-13. The current study does not define the cellular sources of the cytokines measured herein, however multiple cellular sources of these cytokines are present in the lung, including both immune and epithelial cell lineages. The present findings suggest that CCSP modulates Th2 immune responses and mucus production during RSV infection. In the current study, restoration of CCSP in the airways of CCSP⫺/⫺ mice abrogated the increased lung pathophysiology of RSV infection. These findings are consistent with the notion that the exacerbated inflammatory responses to RSV infection in CCSP⫺/⫺ mice are likely due to the deficiency in CCSP per se (13). Additional phenotypic changes have been identified in the CCSP⫺/⫺ mouse model, including attenuated Clara cell secretory granules, altered protein composition of the airway surface fluid, and increased IgA expression in peribronchial regions (6, 13). The importance of CCSP per se as compared with the phenotypic changes in the CCSP⫺/⫺ mouse model has not been addressed. Studies have shown that CCSP replacement with rhCCSP in the lungs of CCSP⫺/⫺ mice can attenuate lung inflammation and cytokine production following LPS challenge (20), confirming the anti-inflammatory effect of CCSP restored in the airways. Most recently, it has been reported that synthetic peptides of the conserved regions of CCSP and lipocortin can reverse the inflammation of allergic conjunctivitis induced by ragweed, suggesting that novel recombinant peptides from CCSP may be safe and effective agents for the treatment of inflammatory or allergic diseases (56). The findings presented herein suggest that CCSP in the airways may be an important regulator of the host inflammatory responses to a number of infectious or allergic stimuli. The molecular function of CCSP is not fully understood. Previous studies indicate that CCSP possesses varied biochemical and biological properties including inhibition of phospholipase A2 (57) and phospholipase C activities (58), and immunomodulatory (59) or anti-inflammatory activity (27, 60). CCSP may play a direct role in reducing IL-13 production of T cells in response to Ag challenge (61). CCSP is a prototypic member of a subfamily of secretoglobins, proteins with putative inflammatory or immune modulatory functions (62). Secretory cells of the conducting airway epithelium express distinct members of the secretoglobin family in a partially overlapping fashion, and altered expression of secretoglobins in airway disease may contribute to immunoregulatory perturbations commonly seen in airway disease (62). The findings presented herein suggest CCSP as an important modulator of inflammatory and immune responses in the lung, but do not rule out the importance of other airway-specific proteins with immunomodulatory function. Clara cells are mainly distributed in the conducting airways and distal bronchioles of the mammalian lung (1). Ultrastructural studies indicate an abundance of secretory granules in Clara cells, suggesting that they may play an important role in producing critical components of distal airway surface fluid, many of which have immunosuppressive properties (63). Indeed, Clara cells likely produce a number of important host defense proteins involved in maintaining airway homeostasis against injury or infection (2, 64). As Clara cells may be progenitor cells during development and remodeling (65), injury to Clara cells may inhibit bronchiolar epithelial differentiation and affect epithelial repair (66). Similarly, epithelial injury by RSV infection in infants and young children, whose lungs are still developing, may have long-lasting consequences of pulmonary disease (67). Furthermore, stem cell populations expressing Clara cell markers have been identified in terminal airways, and Clara cells may function to maintain epithelial diversity after injury (68). These studies indicate that Clara cells and their products may play an important role in maintaining airway homeostasis and regulating lung immune
The Journal of Immunology responses; injury detrimental to Clara cell function may lead to an inappropriate inflammatory and immune response resulting in airway dysfunction. Recent studies have indicated that gene targeting of the CCSP gene loci can alter the expression of a subset of genes in the lungs of CCSP⫺/⫺ mice (6, 13). One of these genes includes another member of the secretoglobin gene family, of which CCSP is the prototypic member (3, 62). The functional importance of altered gene expression in the lungs of CCSP⫺/⫺ mice has not been fully defined. Furthermore, the genetic background of mice strongly influences the host response and degree of lung disease associated with RSV infection (19). The 129J background mouse strain used in the current study has been shown to be relatively susceptible to RSV infection as compared with other mouse strains, such as C57BL/6 strains (19). The findings herein that CCSP restoration in the airways of CCSP⫺/⫺ mice was able to abrogate the increased lung disease to RSV is consistent with the notion that CCSP per se is important in regulating the host response to RSV infection. Further studies are required to elucidate the molecular function of CCSP in the context of RSV-induced disease. Clara cells secrete a number of important immunomodulatory and host defense molecules in the lining of the airway epithelium (2). Of these potential regulators, the surfactant proteins SP-A and SP-D have received increasing attention for their role in host defense to bacterial and viral infection (63, 69). SP-A and SP-D function as novel pattern recognition receptors for pathogen-associated surface molecules, with the ability to modulate adaptive immune responses to infection. The breadth of pathogens to which SP-A and SP-D have been shown to mediate host defense indicates the importance of this novel class of pattern recognition receptors (70). The role of SP-A and SP-D secreted by the Clara cells has not been distinguished from that of SP-A and SP-D secreted from alveolar type II cells of the distal lung. Gene targeting of CCSP does not appear to alter SP-A gene expression or protein levels in the lung (12) (K. S. Harrod, unpublished results). However, given the alterations in the airway surface fluid composition in the lungs (13), posttranslational or secretory regulation of surfactant proteins may still regulate the function of these host defense proteins. In summary, CCSP deficiency increases viral persistence, lung inflammation, Th2 immune responses, and AR in acute RSV infection. All are abrogated upon restoration of CCSP in the airways, indicating an immunomodulatory function for CCSP during RSV infection. Clinical studies have shown that CCSP is diminished in the airways of patients with asthma and bronchiolitis, both associated with RSV infection. The findings from this study suggest a role for CCSP in coordinating the host inflammatory and immune response to RSV infection in the lung. The results from this and other studies also suggest that airway epithelium and the secretory products of lung epithelial cells are important modulators of lung inflammatory and immune responses to infection, allergen, or injury. The molecular functions of CCSP and the precise mechanisms by which CCSP modulates lung inflammatory and immune responses in vivo remain to be determined.
Acknowledgments We are grateful to Dr. Barney S. Graham for providing RSV and Hep-2 cells, and Drs. Aprile L. Pilon and Richard Welch for providing rhCCSP. We also thank Dee Esparza for assistance in RSV instillation, Yoneko Knighton for help in preparing and staining slides, and Roger Henson for technical support in measuring airway reactivity.
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