Time-Dependent Airway Epithelial and Inflammatory Cell Responses Induced by Influenza Virus A/PR/8/34 in C57BL/6 Mice

Abstract

The present study examines the kinetics of airway epithelial remodeling and inflammation in the airways of C57BL/6J mice infected with influenza virus A/PR/8/34 (PR8). Mice were intranasally instilled with 50 plaque forming units (pfu) of virus or its respective vehicle, saline, and then were sacrificed at 3, 7, 10, 15, or 21 days postinfection (dpi). PR8 treatment resulted in airway epithelial cell regeneration as suggested by proliferating cell nuclear antigen (PCNA) positive staining at 7 and 10 dpi and mucous cell metaplasia (MCM) evident at 10, 15, and 21 dpi. PR8 treatment resulted in a classic pattern of inflammation observed in bronchoalveolar lavage fluid (BALF), in which neutrophils peaked at 3 and 7 dpi and monocytes, lymphocytes, and eosinophils peaked at 10 dpi before returning to background levels of detection. Chemokine (MCP-1) and cytokine (IL-6, TNF-α, IFN-γ, IL-5, IL-4, and IL-9) levels peaked at 7 dpi in BALF. IL-13 levels were unaffected by PR8 treatment. Concurrent with inflammation, MUC5AC gene expression was markedly increased by PR8 treatment at 7 dpi. Collectively, the results of this study indicate that the onset of MCM in airway epithelium occurs during the remodeling process and persists after the inflammatory response has diminished.

Introduction

Influenza is a highly contagious respiratory disease associated with considerable morbidity and mortality in the global community each year. Virus transmission readily occurs by the airborne route or by direct contact. In individuals that have not been vaccinated, the virus multiplies rapidly in epithelial cells along the entire respiratory tract (Van Reeth, 2000). Studies exploring the response of airway epithelium to influenza have focused primarily on determinants of viral entry (Zambon, 1999) and mechanisms by which the host-immune system defends (Van Reeth, 2000; Tamura and Kurata, 2004) against influenza infection resulting in viral clearance and, consequently, epithelial desquamation. However, subsequent airway epithelial remodeling with mucous cell metaplasia has received no attention.

The airway epithelium is under constant insult by environmental chemicals and airborne pathogens. Inhalation of these xenobiotics can impair mucociliary clearance by killing epithelial cells responsible for the production of mucus (e.g., mucous goblet cells) or involved in the movement of mucus (e.g. ciliated cells). Moreover, these xenobiotics can impair alveolar macrophage function (e.g., phagocytosis and production of chemical mediators) and alveolar type II cell function (e.g., production of surfactant). The loss of any of these lung defenses renders the host more susceptible to infection by a variety of pathogens. With repeated exposures, remodeling of the epithelium lining the conducting airways that ensues may lead to mucus hypersecretion and plugging, which can be detrimental to the host by compromising gas exchange.

Mucins are goblet cell-derived proteins containing sialic acid residues that are recognized for their ability to aggregate and inhibit hemagglutinin activity of influenza virus (White et al., 2005a). In addition, mucins also assist in reducing the oxidant response of neutrophils caused by viral infection (White et al., 2005b). Therefore mucins are a component of the host-derived protective response against viral infection and subsequent pathologic sequelae.

MCM has been well characterized in models of airway remodeling after allergen exposure (Tesfaigzi, 2002; Reader et al., 2003b; Kumar et al., 2004) as well as after toxicant exposure (Harkema and Hotchkiss, 1993; Fanucchi et al., 1998; Tesfaigzi, 2002). The pathogenesis of MCM in these models appears to be intimately linked with soluble mediators derived from pro-inflammatory neutrophils and monocytes as well as T helper type 2 (T_H2) cells during the adaptive immune response. For instance, neutrophil-derived elastase (Jamil et al., 1997), tumor necrosis factor–alpha (TNF-α) (Kawano et al., 2002) and the T_H2 cytokines IL-4 (Dabbagh et al., 1999), IL-5 (Justice et al., 2002), IL-9 (Reader et al., 2003a), and IL-13 (Shim et al., 2001) have been implicated in the etiology of MCM. More recently, there has been an increasing body of evidence that MCM also occurs as a component of the progressive changes in respiratory pathology in models of viral infections (Holtzman et al., 2006), including adenovirus (Kajon et al., 2003), respiratory syncytial virus (RSV) (Harrod et al., 2003) and influenza virus (Wohlleben et al., 2003) infection.

To initiate an investigation of influenza-induced MCM, a model of influenza infection of the lower airways was implemented (Wiley et al., 2001). The present study was conducted to provide a temporal morphometric characterization of the airway epithelial remodeling and MCM occurring at generation 5 along the main axial airway of the left lung lobe following a primary influenza infection. Furthermore, this study examined the biochemical and cellular composition of BALF as an indicator of the status of the concurrent inflammatory response associated with infection. This study served as the foundation for which the effects of immunotoxic chemical agents can be examined in the context of host-resistance to a common respiratory pathogen.

Methods

Animals

Seventy female C57BL/6 mice (8–10 weeks old) were purchased from Charles River (Portage, MI). On arrival, mice were randomly assigned to the experimental groups, transferred to plastic cages containing sawdust bedding (6 animals per cage), and quarantined for 1 week. Mice were free of pathogens and respiratory disease, and used in accordance with guidelines set forth by the All-University Committee on Animal Use and Care at Michigan State University. Mice were given food (Purina Certified Laboratory Chow) and water ad libitum and not used for experimentation until their body weight was 17–20 g. Animal holding rooms were maintained at 21–24°C and 40–60% relative humidity with a 12-hour light/dark cycle.

Experimental Design

Two separate studies were conducted for the characterization of PR8-induced inflammation and MCM. In the first study, 60 female mice were randomly assigned to 10 treatment groups (n = 6/group). Mice received either an intranasal instillation of SAL or PR8 and were terminated at either 3, 7, 10, 15, or 21 days postinfection (dpi). The lungs from these mice were first lavaged with SAL to retrieve inflammatory cells and biochemical markers of inflammatory cell activation, then formalin-fixed for immunohistochemical analysis. In the second study, 10 mice were randomly assigned to 2 treatment groups (n = 5/group). Mice received either an intranasal instillation of SAL or PR8 and were terminated at 7 dpi. RNA was isolated from whole lung homogenates obtained from these mice.

Influenza A/PR/8/34 Instillation

Influenza A/PR/8/34 (PR8) was generously supplied by the laboratory of Dr. Alan Harmsen (Montana State University, MT). Mice were anesthetized with 4% isoflurane in oxygen, and 50μl of PR8 in pyrogen-free saline was instilled as 25 μl per nare at a total dose of 50 plaque forming units (pfu).

Necropsy, Lavage Collection, and Tissue Preparation

Mice were sacrificed at 3, 7, 10, 15, and 21 days postinfection (dpi). On each of the aforementioned days, mice were anesthetized by an intraperitoneal injection of 0.1 mL of 12% pentobarbital solution, a midline laparotomy was performed, and animals exsanguinated by cutting the abdominal aorta. Immediately after death, the trachea was exposed and cannulated; the heart and lung were excised en bloc. One milliliter of sterile saline was instilled through the tracheal cannula and withdrawn to recover bronchoalveolar lavage fluid (BALF). A second saline lavage was performed and combined with the first.

After lavage, the lung was processed for histological analysis as follows. The left and right lung lobes were inflated under constant pressure (30 cm H₂O) with 10% neutral-buffered formalin (Sigma Chemical Co., St. Louis, MO) for 1 hour. The tracheal airway was then ligated and the inflated lobes were stored in the same fixative for at least 24 hours until further processing.

The intrapulmonary airways of the fixed left lung lobe from each rodent was microdissected according to a modified version of the technique of Plopper et al. (1983), fully described in a previous publication (Harkema and Hotchkiss, 1992). Beginning at the lobar bronchus, airways are split down the long axis of the largest daughter branches (i.e., main axial airway; large diameter conducting airway) through the 12th airway generation. Tissue blocks that transverse the entire lung lobe at the level of the 5th and 11th airway generation of the main axial airway were excised. Each lobe was sectioned in half, perpendicular to the primary bronchus. The tissue blocks were embedded in paraffin, and 5–6 μm thick sections were cut from the anterior surface. Lung sections were stained with hematoxylin and eosin (H&E) for routine histopathology or with Alcian Blue (pH 2.5)/periodic acid-Schiff (AB/PAS) to detect intraepithelial mucosubstances.

Immunocytochemistry

Hydrated paraffin sections (5–6 μm thick) from formalin-fixed lung tissues were treated with 0.05% proteinase K for 2 minutes and washed with 1 N HCI for 1 hour. Sections were then treated with 3% H₂0₂ (in methanol) to block endogenous peroxide and were incubated with a monoclonal antibody (PC10) cocktail to PCNA (Biogenex, San Ramon, CA) consisting of the primary antibody to PCNA 1:50, a secondary antibody to Immunoglobulin 1:500 and mouse serum 1:50 for 1 hour. Immunoreactive PCNA was visualized with the Vectastain Elite ABC kit (Vectastain Laboratories Inc., Burlingame, CA) using 3′,3′-diaminobenzidine (DAB) tetrahydrochloride (Sigma Chemical Co., St. Louis, MO) as a chromagen.

Total and PCNA Positive Epithelial Numeric Cell Density and Labeling Index

The total number of epithelia lining the luminal surface of the main axial airway at generation 5 were enumerated per length of basal lamina. Likewise, cells with nuclei staining positive for PCNA were also enumerated per length of basal lamina. A labeling index for PCNA was determined by dividing the number of PCNA positive cells per unit length of basal lamina by the total number of epithelial cells per unit length of basal lamina.

Morphometry of Stored Intraepithelial Mucosubstances

The appearance of MCM in a model of Influenza A infection has only been reported once previously at a fixed time point (Wohlleben et al., 2003). Therefore, to begin to characterize the pathogenesis of MCM in this particular model, it was important to first establish the temporal relationship between a primary PR8 infection and the time-to-onset of MCM. This was accomplished by employing standard morphometric techniques on the epithelial lining of the main axial airway at generation 5 (G5) from left lung lobe sections stained with AB/PAS. G5 was chosen for analysis for two reasons: (1) the clinical correlate of influenza infections in humans begins in the nose and subsequent viral progeny make their way down the trachea and infect the lower airways in a proximal to distal manner with respect to the branching of the bronchi; (2) variability in the lesions at this site were minimal in this model; therefore, allowing reproducible events to be quantified.

The volume density (Vs) of AB/PAS-stained mucosubstances in the respiratory epithelium lining the main axial airway at 3, 7, 10, 15, and 21 dpi was quantified using computerized image analysis and standard morphometric techniques. The area of AB/PAS stained mucosubstance was calculated from the automatically circumscribed perimeter of stained material using a Power Macintosh 7100/66 computer and the public domain NIH Image program (written by Wayne Rasband, U.S. National Institutes of Health and available on the Internet at 〈http://rsb.info.nih.gov/nih-image/〉). The length of the basal lamina underlying the surface epithelium was calculated from the contour length of the digitized image of the basal lamina. The volume of stored mucosubstances per unit of surface area of epithelial basal lamina was estimated using the method described in detail by Harkema et al. (1987). The Vs of intraepithelial mucosubstances is expressed as nanoliters of intraepithelial mucosubstances per mm² of basal lamina.

Bronchoalveolar Lavage Cellularity

Total leukocytes in BALF were enumerated using a hemocytometer, and fractions of eosinophils, lymphocytes, monocytes/macrophages, and neutrophils were determined by counting 200 cells in a cytospin sample stained with Diff-Quick (Dade Behring, Newark, DE).

Total Protein

Total protein was quantified in BALF using the BCA (bicinchoninic acid) protein assay (Pierce, Rockford, IL) and following the manufacturer’s instructions.

Neutrophil Elastase

Airway elastase recovered in BALF was determined by an ELISA for elastase using a rabbit monoclonal antibody to the human elastase (Calbiochem, La Jolla, CA). Fifty-microliter aliquots of BALF were applied to a 96-well microtiter plate (Microfluor 2 Black, Dynex Technologies, Chantilly, VA) and dried overnight at 40°C. Plates were blocked with a solution of 1.5% goat serum in Automation Buffer Solution (ABS, pH 7.5; Biomeda Corp., Foster City, CA) for 30 minutes at 37°C. Plates were then incubated with anti-elastase antibody (1:400 in ABS containing 1.5% goat serum) for 1 hour at 37°C and then washed 3 times with ABS. Bound primary antibody was detected with a biotinylated goat anti-rabbit secondary antibody and quantified using horseradish-peroxidase-conjugated avidin/biotin complex (ABC Reagent; Vector Laboratories, Burlingame, CA) and a fluorescent substrate (QuantaBlue; Pierce Chemical, Rockford, IL) using a fluorescence microplate reader (SpectraMax Gemini; Molecular Devices; 318 nm excitation/410 nm emission). Readings were taken at 3-minute intervals for 24 minutes. Duplicate samples were averaged, and the group data is represented as mean Vmax units/s.

Inflammatory Cytokines

Inflammatory cytokines retrieved in the BALF were analyzed by flow cytometry using cytometric bead array technology by Becton Dickinson Biosciences (San Diego, CA) on a BD FACSCalibur flow cytometer. In brief, 50 μl of BALF from each sample was incubated individually with a mixture of beads coated with antibodies to (IL-6, IL-10, MCP-1, IFN-γ, TNF-α, and IL-12p70) from the mouse inflammation kit or a mixture of beads coated with antibodies to (IFN-γ, TNF-α, IL-2, IL-4, and IL-5) from the mouse T_H1/T_H2 kit. Similarly, the cytokines IL-9 and IL-13 were analyzed using BD flex-sets. Samples and standards were analyzed according to manufacturer-based instructions.

RNA Isolation

From a second study employing a similar design, total RNA was isolated from the lung in the 7 dpi group using the TRI reagent RNA isolation method. The evaluation of the relative expression levels of MUC5AC mRNA were determined using the TaqMan real-time multiplex RT-PCR with custom designed TaqMan primers and probe to the target gene and the manufacturer’s predeveloped primers and probe to 18S (Applied Biosystems, Foster City, CA). The primers and probe to both the target gene and endogenous reference gene were specifically designed to exclude detection of genomic DNA. Aliquots of isolated tissue RNA (1 μg total RNA) were converted to cDNA using random primers. The resultant cDNA (2 μl) was added to a reaction mixture that consisted of the target gene primers and probe, endogenous reference primers and probe (18S ribosomal RNA), and Taqman universal master mix to a final volume of 30 μl.

Following PCR, amplification plots (change in dye fluorescence versus cycle number) were examined and a dye fluorescence threshold within the exponential phase of the reaction was set separately for the target gene and the endogenous reference (18S). The cycle number at which each amplified product crosses the set threshold represents the C_T value. The amount of target gene normalized to its endogenous reference was calculated by subtracting the endogenous reference C_T from the target gene C_T (ΔC_T). Relative mRNA expression was calculated by subtracting the mean ΔC_T of the control samples from the ΔC_T of the treated samples (ΔΔC_T). The amount of target mRNA, normalized to the endogenous reference and relative to the calibrator (i.e., RNA from control) is calculated by using the formula $2_{\text{T}}^{-\mathrm{\Delta }\mathrm{\Delta }\text{C}}$ .

Statistical Analysis

Data are expressed as mean ± standard error of the mean (SEM). Outliers were removed from sample sets by using a Grubb’s test. The differences among groups were determined by either a standard t-test or 2-way analysis of variance (ANOVA) with multiple comparisons made by the Student–Newman–Keuls post hoc test using SigmaStat software from Jandel Scientific (San Rafael, CA). The criterion for significance was taken to be p < 0.05.

Results

Pulmonary Histopathology

No microscopic alterations were present in the examined proximal (at G5 of the main axial airway) and distal (at G11 of the main axial airway) tissue sections from the left lung lobe of mice that were intranasally instilled with vehicle alone (controls) (Figure 1A–1B, 1E–1F, and 1I–1J).

The principal pulmonary alteration in mice intranasally instilled with influenza virus and sacrificed 3 dpi was an acute necrotizing bronchiolitis characterized by multiple focal areas of necrosis and luminal shedding (exfoliation) of the surface epithelial cells lining the main axial airway (Figure 1C–1D) and smaller diameter preterminal and occasional terminal bronchioles. Similar but much less severe lesions were observed in the distal tissue section of some of these exposed mice. Airway epithelial lesions were accompanied by a mild intramural inflammatory cell infiltrate (Figure 1C) composed principally of mononuclear cells (lymphocytes and monocytes) and lesser numbers of neutrophils. A similar inflammatory cell infiltrate was present in adjacent peribronchiolar and perivascular regions.

Virus-instilled mice that were sacrificed 7 dpi had a marked bronchiolitis and alveolitis again restricted mainly to the hilar region of the lung lobe (proximal tissue section). Necrosis and exfoliation of the bronchiolar ciliated epithelium observed at 3 dpi was replaced by a hyperplastic/hypertrophic, nonciliated, cuboidal and basophilic epithelium accompanied by a marked lymphocytic inflammatory cell infiltrate in the affected bronchioles and adjacent alveolar septa (interstitial pneumonia). Lesser, but conspicuous, numbers of eosinophils were also intermixed with the mononuclear inflammatory cells. There was also mild-to-moderate alveolar type II cell hyperplasia and hypertrophy in these affected pulmonary regions along with numerous large highly vacuolated alveolar macrophages, smaller monocytes, and lymphocytes within alveolar air spaces. Various amounts of proteinacious material were also present in some of the alveolar lumens in the affected regions of the lung.

Mice that were instilled with virus and sacrificed at 10 dpi (Figure 1G–1H) had a chronic bronchiolitis and alveolitis, again restricted mainly to the proximal tissue section (hilar aspect of the lung lobe). At this time postinfection, the affected bronchiolar epithelium was composed of tall cuboidal to columnar ciliated and nonciliated cells (Figure 1G). Many of the nonciliated epithelial cells were mucous cells with conspicuous amounts of AB/PAS stained, intracytoplasmic mucosubstances (i.e., MCM) (Figure 1H, arrow).

The associated inflammatory cell infiltrate (Figure 1G, asterisk) in and around the bronchiolar walls, adjacent blood vessels and alveolar septa was similar in composition to that observed in mice sacrificed at 7 dpi. However, the most conspicuous change in the affected alveolar regions at 10 dpi compared to that at 7 dpi was the addition of coalescing regions of interstitial fibrosis accompanying the type II cell hyperplasia and the mainly lymphocytic inflammatory cell infiltrate (chronic alveolitis). Mice instilled with virus and sacrificed at 15 and 21 dpi (Figure 1K–1L) had similar but less severe airway and alveolar lesions as compared to those lungs of the mice sacrificed at 10 dpi.

Temporal Analysis of Stored Intraepithelial Mucosubstances

The Vs of intracytoplasmic acidic and neutral mucosubstances (Figure 2) in the airway epithelium was increased by approximately 4-fold in lung lobe sections obtained from PR8-treated mice as compared to respective time-matched SAL controls starting as early as 10 dpi. This marked elevation in mucosubstance Vs was maintained through 21 dpi.

Expression of MUC5AC

Recognizing that the time to onset of MCM was established at 10 dpi, RNA from whole lung homogenates at 7 dpi was analyzed for MUC5AC mRNA levels (Figure 3). MUC5AC levels were nearly 3-fold greater in lung homogenates from mice treated with PR8 than those treated with saline.

Numeric Cell Denisities and Cellular Remodeling

By numeric cell density counts, the total airway epithelial cell counts (Figure 4A) observed at 10 dpi were mildly elevated with respect to counts enumerated in SAL controls. In addition to total cell counts, cells with nuclei staining positive for PCNA (Figure 4B) were also enumerated. PCNA numeric cell density and labeling index (Figure 4C) were significantly elevated as compared to time-matched SAL controls by four to six-fold in the PR8-treated group at 7 and 10 dpi, respectively.

Bronchoalveolar Lavage Fluid (BALF)

Protein

The presence of protein (Figure 5) in BALF was significantly increased in samples collected from mice treated with influenza. This was observed between 7 and 21 dpi with an apparent apex at 10 dpi.

Total and Differential Inflammatory Cell Counts

To further characterize the inflammatory cell milieu that is present in the airways during these epithelial changes, differential cell counts were performed. A significant rise in the total number of cells (Figure 6A) retrieved in the BALF of influenza-treated mice was observed as compared to that enumerated in the respective saline control as early as 3 dpi and lasting through 15 dpi with an apparent apex at 10 dpi. The early innate immune response was marked by significant increases in neutrophils (Figure 6B) by 3 dpi and tapering by 10 dpi. Macrophages and other monocytic cells (Figure 6C) were significantly elevated between 7 and 15 dpi. The adaptive immune response was characterized by marked increases in lymphocytes (Figure 6D) between 7 and 21 dpi with peak numbers observed at 10 dpi. Eosinophils (Figure 6E) were also abundant between 7 and 15 dpi.

Inflammatory Chemokines and Cytokines

With ongoing inflammation there are a host of chemokines and cytokines being released by these activated inflammatory cells. To characterize these chemical mediators found in BALF, a mouse inflammation cytometric bead array analysis was employed. Concentrations of TNF-α (Figure 7A), IFN-γ (Figure 7B), IL-6 (Figure 7C), and MCP-1 (Figure 7D), were significantly elevated in influenza-instilled mice compared to controls at 7 dpi, and concentrations of IL-10 (Figure 7E) were significantly decreased in influenza instilled mice compared to controls at 21 dpi. Levels of TNF-α and IL-6 remained significantly elevated through 10 dpi with similar trends observed with MCP-1. There were no changes in concentrations for IL-12p70 (Figure 7F) between PR8 treatment and saline.

T_H 2 Cytokines IL-4, IL-5, IL-9, and IL-13

The T_H2 cytokines IL-4 (Figure 8A), IL-5 (Figure 8B), IL-9 (Figure 8C) and IL-13 (Figure 8D) have been implicated in the development of MCM. By employing cytometric bead array kits and flex sets for these cytokines, BALF analysis yielded marked increases in the levels of IL-5 detected at 7 dpi. Furthermore, there were significant, albeit mild increases in the levels of IL-4 and IL-9 at 7 dpi as well. The amounts of IL-13 in BALF were low in all treatment groups with no detectable differences between virus- and saline-instilled mice.

Elastase

In addition to factors derived from lymphocytes, neutrophil-derived elastase has also been implicated as a factor known to induce MCM. Accordingly, neutrophil-derived elastase (Figure 9) was significantly elevated between 7 and 15 dpi in mice instilled with influenza compared to control mice instilled with saline.

Discussion

MCM has been well characterized in models of airway remodeling after allergen exposure (Tesfaigzi, 2002; Reader et al., 2003b; Kumar et al., 2004) as well as after nonallergenic toxicant exposure (Harkema and Hotchkiss, 1993; Fanucchi et al., 1998; Tesfaigzi, 2002). A common thread between these models is that the inhaled xenobiotic incites an initial inflammatory response in the pulmonary airways followed by a remodeling of the airway epithelium with MCM. Similarly, many viral respiratory pathogens target the epithelium and incite a robust and diverse (innate, humoral and cell-mediated) immune response that results in the release of soluble-mediators, epithelial injury, and subsequent regeneration. Again, MCM is an adaptive response, or consequence, of cellular signaling by mediators released during the inflammatory response to the invading pathogen.

Indeed, MCM has been observed in models of human adenovirus (Kajon et al., 2003) and RSV (Harrod et al., 2003; Miller et al., 2003). In these models, underlying factors involved in the onset of MCM are only partially understood. Likewise, there have been no efforts prior to this study to begin to characterize MCM using the common respiratory pathogen influenza. The impetus of this study was to characterize the temporal relationship between PR8 infection and the onset of MCM with respect to epithelial apoptosis and remodeling in the context of the host-immune response to the virus.

This study also provides the foundation upon which subsequent studies investigating the relationship between immune modulating chemical agents and influenza host-resistance and their effects upon airway epithelial cell death and regeneration can be initiated. Some studies have already been conducted to address the effects of chemical exposure on host-resistance to influenza virus infectivity (Burleson et al., 1996; Jaspers et al., 2005) and the role these agents play in modulating host-immunity. In addition to the examination of influenza virus, other studies have embarked upon characterizing chemical-induced decreases in host-immunity toward other airborne pathogens such as Legionella pneumophila (Klein et al., 1993).

The host relies upon the innate defenses of the lung (e.g., mucociliary clearance, phagocytosis by resident alveolar macrophages, and neutralization by surfactants) to provide protection against foreign pathogens. Exposure of the host to environmental chemicals (e.g., ozone, diesel exhaust, chemical components of smoke, etc.) can injure epithelium lining the conducting airways of the lung and other cell types contributing to the innate defense of the lung. Moreover, some of these chemicals (e.g., TCDD and cannabinoids) act systemically on cells involved with the adaptive host-immune response.

Regeneration of an epithelium that has been compromised by either chemical agents or viral pathogens is an important step in reestablishing mucociliary clearance and preventing secondary infection. MCM that results from the host immune response to chemical and/or pathogen-induced epithelial injury may lead to a hypersecretory condition. Therefore, characterization of the MCM associated with influenza virus and its temporal relationship to infection and the immune mediators involved is an important first step toward understanding when and how concurrent exposure to immunotoxicants could enhance or attenuate hypersecretory conditions associated with MCM.

In the present study, C57BL/6 mice were utilized. This strain does not have a T_H1 or T_H2-skewed immune response to pathogens like other mouse strains (e.g. BALB/c or AJ mice). In addition, we chose a PR8 exposure that utilized a low concentration (50 pfu) of virus, to ensure survival of the mice for 21 days, in a large volume of saline (50 μl), that facilitated delivery of the virus to the lower airways. This paradigm is unlike most studies, wherein immune responses to the virus are examined within the first 6–10 days following a high concentration exposure.

As stated previously, influenza targets the epithelium lining the respiratory tract to gain entry and replicate. More specifically, influenza targets the viral receptor on host cells, a cell surface carbohydrate sialic acid moiety, and gains entry through N-linked glycoprotein (Chu and Whittaker, 2004). Once the virus has gained entry, replication is highly dependent on the host-cells’ replicative machinery. As a result, signaling cascades that allow for the replication of the virus may also promote host-cell death (Ludwig et al., 2006).

In addition, competent immune responses support the induction of cytotoxic T cell responses that are required for host-dependent clearance of virally infected cells. Exposure to virus in this model yielded an observed airway epithelial degeneration and necrosis by 3 dpi with more extensive epithelial regeneration by 7 and 10 dpi, evidenced by the basophilic nature of the epithelium observed upon H&E staining and the positive staining of cellular nuclei with antibodies directed against PCNA. Regeneration peaked at 10 dpi, and had resolved by 15 dpi. Coincident with the regenerative process, was an increase in the total number of epithelial cells enumerated in the PR8 treatment group.

While epithelial regeneration is still active at 10 dpi, we observe MCM that does not resolve by 21 dpi. As expected, MCM occurs coincident with peaks of retrievable inflammatory cells in the BALF. As stated previously, the pathogenesis of MCM is complex, and appears to be linked to soluble mediators released during an inflammatory response following epithelial injury. Accordingly, this study took into consideration the kinetics of inflammatory cells entering the airways and examined the profile of soluble mediators that were being actively secreted.

Inflammatory cells entering the airways followed a classic pattern of inflammation, in which an early pro-inflammatory response, represented by neutrophilic influx followed by monocytes and macrophages, was observed early at 3 and 7 dpi, respectively, and a delayed host-immune response, characterized predominantly by an influx of lymphocytes and to a lesser extent eosinophils appeared to peak by 10 dpi. Each of these cell types has been implicated in the development of MCM via their release of soluble mediators (e.g., neutrophil-derived elastase and lymphocyte/eosinophil-derived cytokines). Therefore, we also investigated the chemokine and cytokine composition of the BALF to begin to establish a role for these cellular and chemical mediators.

In addition to replicating in the epithelial cells of the respiratory tract, influenza also infects monocytes/macrophages and other leukocytes by binding to sialic acid receptor moieties on these cell types (Ronni et al., 1995). Virus infection activates several transcription factors within these cells that are involved in the induction of chemokine and cytokine gene expression. These include nuclear factor kappa B (NF-κB), interferon regulatory factors (IRFs), activating protein (AP)-1, signal transducers and activators of transcription (STATs) and nuclear factor-interleukin 6 (NF-IL-6 or C/EBPβ) (Julkunen et al., 2001).

Therefore, influenza plays a direct role in influencing the chemokine and cytokine makeup of the inflammatory response (reviewed by Julkunen et al., 2000, 2001). In the current study, concentrations of MCP-1, TNF-α, IL-6, and IFN-γ within the BALF of PR8-treated mice are provided. MCP-1 is a chemokine that can be derived from either influenza-infected epithelia or infected monocytes/macrophages. IL-6 and TNF-α are proinflammatory cytokines that are produced predominantly by monocytes/macrophages.

Instead of participating in an antiviral capacity, TNF-α has been suggested to be a driving force for MCP-1 expression. IL-6 serves as a differentiation factor for lymphocytes and stimulates immunoglobulin production by B cells. Interferon-γ, produced by NK cells and/or T_H1 cells, enhances the over-all development of cell-mediated immunity, macrophage activation, antigen presentation, and chemokine gene expression. In the present study, each of these cytokines was significantly elevated at 7 dpi. Because these cytokines are present during epithelial regeneration and immediately precede MCM, we also considered the potential role of these mediators in mucin gene expression.

Mucins are associated with normal mucociliary clearance in the lungs. Xenobiotics and infectious pathogens can incite inflammatory/immune-mediators that function as secretagogues to activate mucin secretion. These mediators can up-regulate MUC gene expression as well. MUC genes are differentially expressed in goblet cells in diverse epithelial tissues. MUC5AC is the predominant mucin normally expressed in goblet cells in the lung (Rose and Voynow, 2006). In our study, MUC5AC expression is enhanced at 7 dpi. This is also the same day that a number of cytokines and chemokines in the BALF have peaked. Two of these cytokines, TNF-α and IL-6, have been implicated as being key regulators of MUC5AC gene expression. TNF-α has been shown to regulate expression of MUC5AC at the transcriptional level in human airway epithelial cells (Song et al., 2003). Likewise, IL-6 has also been shown to increase MUC5AC mRNA steady-state expression in differentiated cultures of primary human tracheobronchial epithelial (TBE) cells (Chen et al., 2003).

Another soluble mediator examined in our study was neutrophil elastase. Neutrophil elastase is a serine protease, which has been shown to increase expression of MUC5AC (Voynow et al., 1999). Neutrophil elastase regulates MUC5AC through either the induction of oxidant stress (Fischer and Voynow 2002) or via its proteolytic activation of an EGFR signaling cascade involving TGF-α (Kohri et al., 2002). Our studies indicate that neutrophil elastase is also elevated in the BALF as early as 7 dpi and is maintained through 15 dpi.

T_H2 cytokines are important contributors to MCM in models where there is sound immunological evidence for an essential role of effector T_H2 type CD4⁺ T cells. In the current model, a MCM response of the epithelium was elicited 10 days after a primary infection with PR8. Clearance of a primary influenza infection is predominantly mediated by CD8⁺ T cells (Topham et al., 1997). However, there is growing support for a role for CD4⁺ T cells as contributing immune effectors in the protection against influenza (Brown et al., 2004, 2006; Swain et al. 2006). Regardless, it was not too surprising that T_H2 cytokines from CD4⁺ T cells were not a prevailing feature in this model.

Although a potentially important role for these cytokines, in particular IL-9 and IL-13, has been emphasized (Holtzman et al., 2006), levels of these two cytokines in the current study were at the level of detection in BALF. Likewise, IL-4 levels were consistent with IL-9 and IL-13. Whether the levels of these cytokines observed in BALF were sufficient to promote MCM in a regenerative epithelium is questionable. Certainly, signaling between CD4⁺ T cells and the epithelium in the context of the microenvironment and cell-cell contact cannot be excluded. Concentrations of IL-5, on the other hand, were markedly enhanced at 7 dpi. IL-5 is chemoattractive for eosinophils. Indeed, the BALF differentials provided evidence in this study that eosinophils were recruited to the lungs as early as 7 dpi. IL-5 has been implicated for participating indirectly with CD4⁺ T cells for inducing MCM, presumptively through IL-4 (Justice et al., 2002). However, there is no direct evidence to support this in the current model.

Collectively, this paper provides an initial characterization of the effects of a common respiratory virus on the development of MCM in the pulmonary airways. There is a clear progression of epithelial changes in response to the injurious actions of influenza and its associated immune response. Despite the complexity of the immune response, many cellular and chemical mediators recognized for their involvement in MCM are present. Therefore, this is a relevant model for elucidating the mechanisms by which viruses, like influenza, alter the epithelial composition, in particular mucus-secreting cells, of the respiratory tract.

In this model, the appearance of MCM after primary infection may support the notion of a beneficial effect, in which the respiratory tract is increasing the secretion of mucus to assist in mucocilliary clearance, thereby, further protecting against secondary infections or subsequent reinfection. Conversely, unwarranted hypersecretory conditions could result in the obstruction of airflow that is detrimental to the host. Hence, this is also a relevant model for investigating how concurrent exposure to immunomodulatory xenobiotics can influence viral clearance, epithelial desquamation, and subsequent remodeling of the airway epithelium.