Although there have been post mortem
studies of the airway pathology of CF (10
), we believe ours is the first study to quantify mucous cells, mucins, and inflammatory changes in CF using tissue obtained by bronchoscopy and methods of design-based stereology. We found that the volume fraction of submucosal glands was fourfold higher than normal in CF, a finding that is much more striking than the changes in goblet cells, which were more subtle. These subtleties included a modest increase in the volume of stored mucin in the epithelium, which was the result of increased goblet cell size rather than increase in goblet cell number. Mucin stores in goblet cells may be relatively low in CF because of ongoing degranulation signals. Nevertheless, we were surprised that we did not find more evidence of goblet cell hyperplasia in the large airways in CF.
Using immunohistochemistry, we found that MUC5AC was the predominant mucin expressed by goblet cells, and MUC5B was the predominant mucin in gland mucous cells. This pattern was not different in subjects with CF compared with healthy subjects. Thus, although the large inflammatory stimuli in CF may up-regulate mucin gene expression in mucous cells in the epithelium and submucosa, they do not alter basic patterns of expression of gel-forming mucins. The increased mucin stores in the submucosal glands represent a reservoir of mucins that could contribute to mechanisms of mucin hypersecretion in CF. Although mechanisms of mucous cell degranulation are distinct from those of intracellular mucin accumulation, it is reasonable to assume that increases in mucin stores in the submucosa lead to increases in mucins in secretions.
Neutrophilic airway inflammation is a known pathologic feature of CF, but our goal was to examine neutrophils in relation to goblet cells in the epithelium and gland cells in the submucosa. We found that neutrophil elastase immunostaining was largely absent around glands in the submucosa, so that neutrophil-mediated degranulation of gland mucous cells is very unlikely. Other mucin secretagogues must regulate gland mucin secretion in CF. In contrast, in the epithelium, we found intense neutrophil elastase immunostaining. It is unclear what causes neutrophils to accumulate in the epithelium. Recent data suggest that there are epithelial cell changes related to CFTR deficiency that cause neutrophil accumulation in the epithelial layer (28
). For example, epithelial cells in CF have an augmented production of interleukin 8 (30
) and increased intercellular adhesion molecule-1 expression (28
). Whatever the mechanism of neutrophil accumulation in the epithelial layer, the consequences could include degranulation of goblet cells, or stimulation of goblet cell hypertrophy.
The increased volume of glands in the submucosa was the most striking aspect of the pathologic phenotype that we observed in the subjects with CF. Our data showing an increase in the volume fraction of glands in the submucosa could result from the numbers of gland units being increased, or because individual glands become hypertrophied. Our methods do not allow us to distinguish between these possibilities. We are able to state that the gland volume increases with proportionate increases in the serous and mucous acini. Published data do not shed light on whether gland enlargement in CF results from growth of new submucosal glands, the enlargement of existing glands, or both. There are data to support the concept of new gland growth in adult airways. For example, in health, submucosal glands are confined to large cartilaginous airways. However, in autopsy studies of CF lungs, submucosal glands are found in more distal, noncartilaginous airways (33
). In addition, using animal models, it is possible to induce gland growth in the airways (34
), suggesting that new gland growth is possible in adult lungs. Any such development of glands from surface epithelial cells would require epithelial progenitor cells and recapitulation of the epithelial tubulogenesis that occurs during lung development. During fetal lung development, buds form from the surface epithelium followed by lateral morphogenesis into the mesenchyme (36
), a sequence that can be reproduced in cell culture. Normal human tracheobronchial epithelial cells cocultured with human fetal lung fibroblasts penetrate collagen matrices (39
), form tubular structures, and undergo dichotomous branching (40
). Polarized noninvasive epithelial cells may transform into nonpolar invasive mesenchymal cells (“epithelial mesenchymal transition”), break through the basement membrane, and invade surrounding extracellular matrix (41
Our study design did not address the mechanism of gland enlargement in CF, but we hypothesize that neutrophil-directed epithelial cell activation could lead to epithelial mesenchymal transition, epithelial tubulogenesis, and new glands. Our findings for the epithelial-restricted nature of neutrophilic inflammation in CF and the positive correlation between epithelial neutrophils and gland volume in the submucosa provide some support for this hypothesis as does the finding of thinning of the basal lamina zone in CF. This latter result could occur because of the actions of neutrophil proteases and thinning of the basal lamina, which could, in turn, facilitate tubulogenesis. Interactions between epithelial cells and extracellular matrix have been particularly well studied in the kidney. For example, locally produced growth factors, such as EGFR ligands, have roles in nephrogenesis and in renal regeneration after acute tubular injury (42
). Both the number and extent of tyrosine phosphorylation of EGF receptors increase in the developing kidney during late fetal development, coincident with the timing of tubulogenesis and glomerulogenesis (26
). TGF-α has been shown to be important for branching tubulogenesis of renal epithelial cells in matrigel, and in complete metanephric organ culture, in which induction of the mesenchyme by ureteric budding occurs. In this context, our finding that TGF-α expression in the submucosa in CF is restricted to submucosal glands suggests that this particular ligand for EGFR may play a role in the development and maintenance of gland structures in CF.