Abstract
Clara cell 10 kDa protein (CC10) is the major secretory protein of Clara cells and is thought to play a protective role in the lung owing to its anti-inflammatory properties. There is little information on the anatomical distribution of CC10-positive cells in rat lung following lipopolysaccharide (LPS) challenge. We have determined the expression of CC10 along the tracheobronchial tree in saline-treated and LPS-treated rats. Saline-treated rats showed sporadic CC10 staining in central airways and abundant staining in bronchioles. In transitional airways, most cells were positive except for squamous cells. Following LPS challenge, there was a reduction in staining in the upper airways but little change within bronchioles. Squamous epithelia within the transitional airways now showed positive staining. These cells also co-stained for pancytokeratin and appeared to co-localize with surfactant D- and Ki67-positive cells, indicating the presence of a dedifferentiated cell type with both epithelial and pneumocyte phenotypes. These data show that diffuse inflammatory injury results in generalized loss of CC10 in central airways. Conversely, the transitional airways showed evidence of a dedifferentiated population of squamous cells that now stained for CC10. We hypothesize that this is an attempt by peripheral lung to maintain alveolar sac integrity during an inflammatory episode.
Introduction
Clara cell 10 kDa protein (CC10) is the major secretory protein of the Clara cell, a nonciliated secretory epithelial cell present throughout the tracheobronchial tree. It has a variety of names, including CCSP, CC16, uteroglobin, and SCGB1A1, and is part of the family of secretoglobins (Klug et al. 2000). CC10 is known to be steroid inducible and to exhibit anti-inflammatory and immunomodulatory functions, which have been documented in both humans and animals. It is now recognized that CC10 is a sensitive marker of lung injury, and it has been proposed as a peripheral biomarker of airway disease.
It is now well established that CC10-positive cells are found throughout the tracheobronchial tree, including the bronchi, in a variety of species including humans, monkeys, and rodents (Barth et al. 2000; Boers et al. 1999; Coppens et al. 2007; Dodge et al. 1993). It has been documented that “classic” CC10-positive Clara cells (i.e., nonciliated, dome-shaped cells), are the predominant cell type in the lower respiratory tree (Jensen et al. 1994; Boers et al., 1999), and there is speculation that the CC10-positive cells seen in the human and rodent bronchi are not true Clara cells but may represent a subset of Clara cells (Broers et al. 1992; Dodge et al. 1993). Clara cells secrete proteins into the epithelial lining fluid of the airways, including CC10 and surfactant proteins A (and D), which are essential for maintaining airway patency in the parenchyma. In addition to their secretory function, Clara cells are thought to be progenitor cells of terminally differentiated bronchiolar epithelium, and it has been shown that the location of the Clara cell can influence this role (Ji et al. 1995; Boers et al. 1999). Clara cells in the bronchi and bronchioles of human airways are not proliferative (Boers et al. 1999; Barth et al. 2000), whereas in terminal bronchioles, Clara cells contributed 15% of the proliferative cells and this fraction rose to 44% in the respiratory bronchioles (Boers et al. 1999). In hyperproliferation of the conducting airways, such as hyperplasia and metaplasia, there is a decrease in CC10 in humans (Jensen et al. 1994; Barth et al. 2000) mice (Hicks et al. 2003) and rats (S. Bolton, unpublished observations). In contrast, peripheral tumors induced in mice under control of the surfactant promoter did not show a reduction in CC10 (Wikenheiser and Whitsett 1997). This finding would suggest that CC10 may have different functions at different anatomical locations. Clara cells are not found in the alveolar bed, but the resident Type II pneumocytes share several key functions with Clara cells, such as proliferative capacity (regarded as stem cell population) and secretion of surfactant (Castranova et al. 1988).
CC10 protein is also thought to play a protective role in the lung owing to its anti-inflammatory properties. Studies have shown modulation of CC10 levels in bronchoalveolar lavage fluid (BALF) and plasma during lung inflammation in both human and animal studies, which may represent a common sequela of airway damage. The levels of CC10 were reduced in both serum and BALF of healthy smokers compared to controls (Shijubo et al. 1997; Shijubo et al. 1999), and this finding was reflected in a reduction in the numbers of CC10-positive cells in the airways (Shijubo et al. 1997). In contrast to the BALF data, a recent study showed that there was no alteration in levels of CC10 in sputum from asthmatics (de Burbure et al. 2007), although this result could possibly be attributed to a difference in the areas of lung sampled compared to BALF. Similar reductions in CC10 have been reported in various human lung diseases (reviewed in Shijubo et al. 2003), including non-small cell lung carcinoma (Linnoila et al. 1992; Jensen et al. 1994), asthma (Laing et al. 2000; Shijubo et al. 1999), and sarcoidosis (Ohchi et al., 2004). It has also been demonstrated that a polymorphism in the CC10 gene led to decreased levels of CC10 and was associated with development of the airway disease (Laing et al. 2000; Mansur et al. 2002; Ohchi et al. 2004).
Animal studies have shown that CC10 exerts its anti-inflammatory effects by inhibition of phospholipases A and C (Mantile et al. 1993) and PGD2 (Mandal et al. 2004), which are known to be involved in animal models of allergic and inflammatory disease. In the CC10 knock-out (KO) mouse, there was an exaggerated inflammatory response to ovalbumin (OVA) (Chen et al. 2001; Hung et al. 2004; Mandal et al. 2004; Wang et al. 2001) that could be inhibited by treatment with recombinant CC10 (Hung et al. 2004; Mandal et al. 2004), consistent with the proposed anti-inflammatory protective role for CC10. These studies also showed that CC10 could directly modulate cytokine responses (Chen et al. 2001) specifically from Th2 cells (Hung et al. 2004), which putatively drive the OVA-induced pathology. In contrast to the chronic allergic models, acute inflammation induced by lipopolysaccharide (LPS) instillation into rats led to a decrease in CC10-positive epithelial cells in bronchioles (Arsalane et al. 2000; Ooi et al. 1994), which was accompanied by a decrease in CC10 levels in BALF and an increase in serum, owing to leakage across the alveolocapillary barrier (Arsalane et al. 2000). Presence of CC10 in plasma has also been shown in humans following a single inhaled challenge of LPS (Michel et al. 2005). Changes in the levels of CC10, as an indicator of epithelial barrier integrity, can therefore be detected and monitored in blood and lung fluids.
Clara cells are also target cells for toxicants such as naphthalene owing to high levels of the cytochrome P450 required for their metabolism (Devereux et al. 1989). In mice exposed to either intraperitoneal or inhaled naphthalene, there was epithelial vacuolation and necrosis accompanied by epithelial sloughing (Plopper et al. 1992; West et al. 2001). Effects on Clara cells and CC10 have been seen in other models of acute lung injury where animals are exposed to other toxicants such as tobacco smoke (Van Miert et al. 2005), ipomeanol (Hermans et al. 1999), ozone (Pinkerton et al. 1993), and LPS (Michel et al. 2005).
The Clara cell is a known target for LPS injury following a single exposure (Ooi et al. 1994; Arsalane et al. 2000; Elder et al. 2000; Elizur et al. 2007). Repeat dosing of LPS in rodents is known to induce a level of adaptation or tolerance in the airways that is typified by a reduction in the infiltrating neutrophils, with an increase in macrophages with each subsequent exposure (Elder et al. 2000; Elizur et al. 2007). There are no published reports on the effect of repeat LPS challenge on the resident cells such as Clara cells or expression of CC10 throughout the tracheobronchial tree. Previous studies in rats have described changes in only the bronchioles after a single challenge of LPS (Ooi et al. 1994; Arsalane et al. 2000). We have examined, using an immunohistochemical approach, the phenotype and distribution of CC10-positive cells throughout the tracheobronchial tree in rats exposed to aerosolized LPS for five days. These cells were further characterized for expression of cytokeratin (epithelial marker), surfactant D (SP-D, type II pneumocyte marker), and Ki67 (a proliferative marker) in an attempt to determine the origins and proliferative capacity of these CC10-positive cells.
Materials and Methods
Reagents and Antibodies
LPS, serotype 026:B6, was purchased from Sigma (Poole, Dorset, UK). Antibodies were purchased as follows; rabbit anti-CC10 from Upstate Cell Signalling (Millipore, Chandlers Ford, Hants, UK); mouse anti-pan cytokeratin (pan-CK as marker for epithelial cells; clone C11, cytokeratins 4, 5, 6, 8, 10, 13, 18) from Chemicon (Millipore); mouse anti-rat SP-D (clone SPDE, marker for Type II pneumocytes) from Abcam (Cambridge, Cambs, UK); and mouse anti-Ki67 (clone MM1, proliferation marker) from Vector Labs (Peterborough, Cambs, UK). Isotype control mouse antibodies were purchased from Dako (Ely, Cambs, UK), and rabbit antibodies were purchased from Serotec (Kidlington, Oxon, UK). Alexa-conjugated antibodies were purchased from Molecular Probes (Invitrogen, Paisley, Scotland, UK).
Exposure of Rats to Aerosolized LPS
All procedures were conducted in accordance with the UK Animals (Scientific Procedures) Act, 1986. Male CRL/CD rats (Charles River, Margate, Kent, UK), eight to ten weeks old and weighing approximately 300–400 g, were placed into open-fronted holding cones that were attached to a cylindrical metal aerosol chamber. Their noses were exposed to an aerosol of saline or LPS (0.1 mg/mL) for 30 minutes. Aerosols were generated using two disposable side-stream nebulizers (Respironics UK Ltd, Bognor Regis, West Sussex, UK) per flow through inhalation column (ADG Developments, Codicote, Hertfordshire, UK) with airflow of 121.min−1 (61.min−1 per nebulizer). At the beginning of the aerosol challenge, 10 mL of agent was placed into each nebulizer, and the solution was removed and replaced with fresh half way through the aerosol challenge period. The challenge was repeated daily for five days, and rats were euthanized four hours following the final aerosol challenge, with ip injection of Euthatal™ (pentobarbitone sodium, 200 mg/mL−1).
Tissue Preparation
At necropsy, the trachea was cannulated and the lungs instilled with 10% neutral buffered formalin at a set height of 25 cm H2O pressure. Once the lungs were fully distended, the trachea was tied off and the lungs removed from the thoracic cavity and post-fixed in 10% neutral buffered formalin for forty-eight hours. Individual lobes were then trimmed, and sagittal sections approxiately 5 mm thick were taken from the lung hilum to the base and embedded in paraffin.
Immunohistochemistry and Histology
Sections 4 μm thick were cut on a microtome and picked up on Superfrost + slides and dried overnight at 37°C. Sections were dewaxed in xylene, taken through graded alcohols into water. For H&E staining, sections were stained with Gills II Haematoxylin (Pioneer Research Chemicals, Colchester, Essex, UK) and Eosin Y (Acros Organics, Fisher Scientific, Loughborough, Leicestershire, UK) on a Leica ST5020 Autostainer (Leica Microsystems, Milton Keynes, Buckinghamshire, UK). Antigen retrieval was performed using heat treatment (98°C, five minutes, RHS-2 rapid Microwave Histoprocessor, Milestone Srl, Sorisole, Italy) in Vector Unmasking Fluid (Vector Labs), and all steps were carried out using a LabVision Autostainer except for incubation in the chromogen DAB. Sections were incubated for sixty minutes at room temperature with antibodies against CC10 (1/2000, ~1μg/mL), SP-D (8μg/mL), pan-CK (1/400, ~5μg/mL), Ki67 (0.28μg/mL), or isotype control antibody at appropriate concentration. Bound antibody was detected using either standard streptavidinbiotin complex protocols (Duet StreptABComplex, Dako, pan-CK, and CC10) or TSA amplification kit (Perkin Elmer, SP-D and Ki67) according to manufacturers’ instructions. Sections were then counterstained in Gills II Haematoxylin, dehydrated, and mounted in DPX mounting medium (Merck, Lutterworth, Leicestershire, UK). Immunofluorescent double staining was performed by incubating the sections sequentially with anti-CC10 antibody followed by Alexa594 antirabbit antibody, and then antipan CK antibody followed by Alexa488 antimouse antibody. Sections were counterstained and mounted in VectaShield antifade medium plus DAPI (Vector Labs). Neither the SP-D mAb nor the Ki67 mAb was suitable for immunofluorescent double staining owing to a lack of signal (S. Bolton, unpublished observations).
Morphometry
Standard DAB-stained sections were assessed using a Zeiss Axiophot 2 microscope and images were captured using a Leica DFC360 camera. Morphometry measurements were made using Leica Qwin software (ver. 3.1.0, Leica Microsystems Imaging Solutions Ltd., Cambridge, UK, 2004). To determine area of staining, CC10-stained sections were subject to a threshold analysis, with the levels determined by staining in control sections. In central airways, a first area was selected at random close to the hilar region of the central airway, and then subsequent areas were selected by moving the slide along at intervals of approximately 2 mm. Five areas were sampled from either side of the airway. All bronchioles with diameters ranging from 150 to 300 μm were sampled, up to a maximum of ten bronchioles per rat. Cell counts were determined (manual counts), and the length of the airway sampled was then determined (Qwin); the results were expressed as number of cells/mm airway length or area of staining/mm airway length.
Statistical Analysis
All data were analyzed using GraphPad Prism (ver. 4.01, GraphPad Software Inc., San Diego, CA, USA, 2004), and differences were considered significant where the p value was less than .05 using an unpaired two-tailed t-test.
Results
CC10 Expression in Normal Airways
CC10 immunostaining was carried out on saline-treated control rats and assessed using standard light microscopy. In the central airways, there was sporadic staining of nonciliated, dome-shaped columnar epithelial cells—typical of the classical Clara cell morphology (Figure 1A, CC). Occasional ciliated columnar epithelial cells were also stained (Figure 1A, CC10 + EC). Within the smaller bronchioles, the staining was more frequent, and the cells showed a typical Clara cell phenotype (Figure 1B), with small, dome-shaped apical caps of CC10-positive material. In transitional airways, typical Clara cells were frequently stained positive (Figure 1C, CC, arrow). At the transitional zone, occasional cuboidal cells were positively stained (Figure 1C, arrow), but not the squamous cells that run into the alveolar bed (Figure 1C, arrowhead). Staining was observed only in epithelial cells, and there was no other positive staining within the alveolar bed apart from the transitional zone. CC10 immunostaining was also carried out on lung samples from naïve rats, with identical results (data not shown). Sections incubated with isotype control rabbit IgG showed no staining (data not shown). Also of note, Ki67 staining (proliferation marker) in the central airways was sparse, with predominantly the basal cell staining positive with no Clara cells showing positive staining (data not shown). Ki67-positive cells in the smaller airways appeared to be a mixture of both Clara cells and basal cells (data not shown).
CC10 Expression Following Repeat LPS Challenge
CC10 expression was examined in rats treated once per day for five days with LPS. The aerosolized LPS induced a peribronchial (Figure 2A) and peribronchiolar (Figure 2B) infiltrate of inflammatory cells, which consisted of a mixture of neutrophils and macrophages (Figures 2A and 2B, arrows). Within the transitional airways, the inflammatory cells were seen in the submucosa and within the terminal bifurcation, where there was also a cluster of squamous cells, which were not apparent in control rats (Figure 2C, arrowhead, SqC). In the central airways, there appeared to be a change in the phenotype of some of the CC10-positive cells. Typical Clara cells were still apparent (Figure 2D, CC, arrow), but there were also some CC10-positive epithelial cells without a domed top, which appeared much thinner (Figure 2D, arrowhead). This appearance would suggest that the thin CC10-positive cells represent a subpopulation of CC10-positive cells that have secreted a portion of their CC10 into the airway lumen. Within the bronchioles, there were a few sporadic thin cells, which were not seen in control rats (Figure 2E, arrow; compare to Figure 1B), but the predominant cell type had a Clara cell morphology. Within the transitional airways, the squamous cells at the terminal bifurcation showed positive staining for CC10 (Figure 2F, arrow, SqC). In addition, in some of the cells, the CC10 staining appeared as a ring around the edge of the cell with a clearer zone in the center, suggestive of a secretory morphology (Figure 2F, arrowhead).
Cell counts of all airway compartments were carried out, and cells were classified as either CC10-positive, CC10-positive thin (thin without a domed top), or negative. Within the central airways, there was an increase in the numbers of thin CC10 cells after LPS challenge (p = .0428) with a concomitant decrease in normal CC10-positive cells (p = .0056) without any significant change in the overall cell numbers (Figure 3A). In support of this observation, the overall area of CC10 staining was also reduced in the central airways of LPS-treated rats (Figure 3B). In the bronchioles, there were very few thin CC10-positive cells (Figure 3C). However, this number was significantly increased over saline-treated rats, where there were none, although it was not sufficient to affect the overall area of CC10 staining (Figure 3D). The results from the bronchioles were re-analyzed depending on whether there was an inflammatory infiltrate around the epithelial cells, but there was no difference in saline-treated compared to LPS-treated rats (data not shown). No morphometry was done on the transitional airways owing to the presence of the squamous cells, as there was no equivalent cell type in the saline-treated rats.
Characterization of CC10-positive Squamous Cells in Transitional Airways of LPS-treated Rats
In many of the transitional airways of LPS-treated rats, there was a cluster of squamous cells at the terminal bifurcation (compare Figure 1C with Figure 2C). We further characterized these cells using double immunofluorescent staining for CC10 and pan-CK. In larger airways, all CC10-positive cells were shown to represent a subset of pan-CK–positive cells (Figure 4A). The squamous cells at the terminal bifurcation of the transitional airways were also CC10-positive and pan-CK–positive (Figure 4B and 4C, F). Staining of sequential sections showed that these cells were also SP-D–positive (Figure 4D) and that some but not all of the cells were also Ki67-positive (Figure 4E).
Discussion
CC10 Expression in Normal Bronchi
We have demonstrated that CC10-positive cells are present throughout the tracheobronchial tree of rats. Staining was sporadic in the central airways, and cells had both a Clara and a non-Clara cell morphology, whereas the cells in the smaller airways appeared to be all Clara cells.
Our observations described in this study confirm earlier reports that there exists a population of bronchial epithelial cells that are CC10-positive in both rats (Dodge et al. 1993) and humans (Broers et al. 1992; Barth et al. 2000). However, whether they are classical Clara cells or represent a subset of Clara cells, or indeed whether they are a completely distinct cell type, is unclear. There are several studies, including our own, that lead us to suggest that the bronchial CC10-positive cells are clearly distinct from their bronchiolar counterparts. First, Dodge et al. (1993) demonstrated that rat bronchial CC10-positive cells have a different cellular distribution of CC10 compared to bronchiolar Clara cells. Second, in our study, some of these bronchial CC10-positive cells appeared to have Clara cell morphology and some were ciliated, which would suggest a mixed population of two distinct cell types of Clara and non-Clara cells. Third, human bronchial CC10-positive cells do not show any proliferative capability, unlike their bronchiolar Clara cell counterparts (Barth et al. 2000). Similarly, we also found that Ki67-positive cells in the central airways were restricted mainly to basal cells, indicating that the bronchial CC10-positive cells do not serve a proliferative stem cell role like true Clara cells. Fourth, modulation of CC10-positive cells in the bronchi as opposed to bronchioles or alveolar bed has also been shown in human lung cancer samples (Jensen et al. 1994) and in the current rat study described here, where we found no effect in the bronchioles. Of course, rather than considering them as distinct cell types, it still remains feasible that they are all Clara cells but with slightly different functions, depending on the anatomical location. With regard to the CC10-positive ciliated cells, it is of course feasible that the cilia seen associated with the cells are from adjacent cells. However, the cilia do not appear to have a tangential cut and are arranged perpendicularly to the stained cell, suggesting that they are directly attached.
CC10 Expression Following Repeated LPS Challenge: Central Airways and Bronchioles
CC10-positive cells appeared to have two phenotypes following LPS challenge: normal Clara cells with a dome-shaped, apical protrusion and cells that appeared thinner, with no domed top. This change was most apparent within the central airways, but there was little difference in the bronchiole or transitional airways. In central airways, this transition to a thin cell phenotype was also reflected in a decrease in the total area of epithelial CC10 staining that was not a result of a decrease in cell numbers in the airways. Similar differences in expression of CC10 in different compartments of the lung have also been demonstrated in human pulmonary carcinoma samples with, again, the least change in bronchioles (Jensen et al. 1994). We hypothesize that these thin cells have secreted their CC10 protein into the airway lumen in response to the LPS and the associated inflammation. Similar morphological effects have been reported for Clara cells in the bronchioles of rats exposed to NO2 (Evans et al. 1978) and LPS (Ooi et al. 1994). We did not find any differences in the expression of CC10 in the bronchioles. It is interesting to speculate that it may be owing to a protective effect of the high numbers of Clara cells within the bronchioles. This finding is in contrast to a previous publication (Arsalane et al. 2000), but in that study the route of administration (intratracheal vs. whole-body aerosol) and dosing regime of LPS (single dose vs. five consecutive doses) were different than the current study.
It has been documented in many human studies that chronic pulmonary inflammation, as a result of either cigarette smoke exposure or disease such as asthma, results in a decrease in CC10 levels in BALF and serum (Laing et al. 2000; Mutti et al. 2006; Shijubo et al. 1997; Shijubo et al. 1999). A study by Shijubo (Shijubo et al. 1999) found that the actual numbers of CC10-positive cells were decreased in asthmatics. Unfortunately, as the current study was a retrospective immunohistochemical study on archival material, there was no opportunity to assess the CC10 levels in BALF or blood from these rats. Nevertheless, our study provides evidence that the Clara cell is acutely sensitive to epithelial insult and is reduced in chronic inflammatory conditions, which may help to exacerbate and perpetuate the inflammatory cycle.
CC10 Expression Following Repeated LPS Challenge: Transitional Airways
The inflammatory infiltrate in the submucosa of the transitional airways consisted of neutrophils and macrophages. This finding would be consistent with findings from previous studies, which described the adaptive response to repeat LPS challenge where a mixture of both neutrophils and macrophages are present in BALF (Elder et al. 2000; Shimada et al. 2000). Our study also showed that in the transitional airways of LPS-treated rats, clusters of squamous cells that stained for CC10 were seen. These cells are morphologically consistent with a reparative phenotype, which can repair and repopulate the tissue following injury (Puchelle et al. 2006), and given the rapid turnover of the rodent lung, this lesion would most likely resolve over time. The positive staining is perhaps surprising, as previous reports have shown that squamous metaplastic cells do not stain for CC10 (Barth et al. 2000). However, that study was in human bronchi, and the cells showed a classic squamous tessellating metaplasia, whereas our study in rats showed small clusters of squamous-like epithelial cells in transition airways. A study by Wikenheiser et al. (1992) showed that in a mouse model of pulmonary adenocarcinoma, tumors in the alveolar bed stained positive for CC10. The levels of CC10 in tumors may also be indicative of the state of maturity of the carcinoma, as larger, more mature carcinomas showed consistently less staining (Wikenheiser and Whitsett 1997). We have also found that a classic squamous metaplasia in the central airways of spontaneously hypertensive rats exposed to tobacco smoke did not stain for CC10 or showed a gradation of staining (S. Bolton, manuscript in preparation). Our study provides evidence that early squamous cell changes in the peripheral lung are associated with CC10 expression and that the changes are focal depending on the lung compartment.
Characterization of CC10-positive Squamous Cells in Transitional Airways of LPS-treated Rats
Although CC10 is absent from the normal alveolar bed, it can be associated with pathological and phenotypical changes to the alveolar bed. The CC10-positive squamous cells seen in the transitional airways were confirmed as epithelial cells owing to their CK-positive co-staining. However, many of these squamous cells were also SP-D–positive, indicating a Type II cell origin rather than an epithelial origin. Increased levels of CC10 are seen in regions of the alveolar bed associated with Type II cell hyperplasia and bronchiolization in humans (Jensen et al. 1994), and the authors conclude that there are two distinct cell types that are capable of repopulating the alveolar bed during hyperplastic and metaplastic growth. Similarly, in mice with experimentally induced pulmonary tumors, there was a mutually exclusive nonoverlapping staining pattern for either CC10 or surfactant C (SP-C), indicating that these tumors arose from either epithelial cells (CC10-positive, SP-C–negative) or Type II pneumocytes (SP-C–positive, CC10-negative) (Wikenheiser and Whitsett 1997). Our results show that the squamous cells can exhibit both an epithelial and a Type II pneumocyte phenotype, suggestive of a dedifferentiated state, which can proliferate and attempt to repair injury in the periphery. Unfortunately, attempts at double staining for CC10 and SP-D were unsuccessful, as the SP-D antibody did not show any staining when used with fluorescent secondary antibodies. However, the staining of sequential sections gave sufficient evidence to show that at least a subset of the CC10 cells also stains for SP-D. The presence of CC10 in these squamous cells may provide some level of protection for the surfactant against PLA2-mediated hydrolysis (Guy et al. 1991).
We have shown the appearance of CC10-positive cells within the transitional airways of rats following repeated LPS challenge. An increase in levels or appearance of CC10 within a cell population may be indicative of either a change in cell maturity or a change in cell phenotype. CC10 appears to be a sensitive indicator of epithelial cell function, and it is conceivable that the increased expression in the peripheral airways is a mechanism to compensate for the loss seen in the central airways after injury. As well as a possible role in inflammation, this finding could also be interpreted as an attempt to maintain alveolar sac patency during an acute inflammatory episode.