Exacerbated inflammation is now recognized as an important component of cystic fibrosis (CF) airway disease. Whether inflammation is part of the basic defect in CF or a response to persistent infection remains controversial. We addressed this question using human fetal tracheal grafts in severe combined immunodeficient mice. This model yields histologically mature, and most importantly, naive CF and non-CF surrogate airways. Significant inflammatory imbalance was found in naive CF airway grafts, including a highly increased intraluminal interleukin 8 content (CF: 10.1 +/- 2.2 ng/ml; non-CF: 1.2 +/- 0.6 ng/ml; P < 0.05) and consistent accumulation of leukocytes in the subepithelial region (P < 0.001). CF airway grafts were not histologically affected until challenged with Pseudomonas aeruginosa, which provoked: (1) early (before 3 h) and massive leukocyte transepithelial migration, (2) intense epithelial exfoliation, and (3) rapid progression of bacteria toward the lamina propria. In non-CF grafts, these three sets of events were not observed before 6 h. Using a model of naive human airways, we thus demonstrate that before any infection, CF airways are in a proinflammatory state. After infection, the basal inflammatory imbalance contributes to exert severe damage to the mucosa, paving the way for bacterial colonization and subsequent steps of CF airway disease.

Accumulating evidence suggests that the early pulmonary inflammation pathogenesis in cystic fibrosis (CF) may be associated with an abnormal increase in the production of pro-inflammatory cytokines in the CF lung, even in the absence of infectious stimuli. We have postulated that if baseline abnormalities in airway epithelial cell production of cytokines occur in CF, they should be manifested in the CF bronchial submucosal glands, which are known to express high levels of CFTR (cystic fibrosis transmembrane conductance regulator) protein, the gene product mutated in CF disease. Immunohistochemical analyses showed that CF bronchial submucosal glands in patients homozygous for the deltaF508 deletion expressed elevated levels of the endogenous chemokine interleukin (IL)-8 but not the pro-inflammatory cytokines IL-1beta and IL-6, compared with non-CF bronchial glands. Moreover, basal protein and mRNA expression of IL-8 were constitutively up-regulated in cultured deltaF508 homozygous CF human bronchial gland cells, in an unstimulated state, compared with non-CF bronchial gland cells. Furthermore, the exposure of CF and non-CF bronchial gland cells to an elevated extracellular Cl- concentration markedly increased the release of IL-8, which can be corrected in CF gland cells by reducing the extracellular Cl- concentration. We also found that, in contrast to non-CF gland cells, dexamethasone did not inhibit the release of IL-8 by cultured CF gland cells. The selective up-regulation of bronchial submucosal gland IL-8 could represent a primary event that initiates early airway submucosal inflammation in CF patients. These findings are relevant to the pathogenesis of CF and suggest a novel pathophysiological concept for the early and sustained airway inflammation in CF patients.

Embryonic stem (ES) cell lines can be expanded indefinitely in culture while maintaining their potential to differentiate into any cell type. During embryonic development, the skin forms as a result of reciprocal interactions between mesoderm and ectoderm. Here, we report the in vitro differentiation and enrichment of keratinocytes from murine ES cells seeded on extracellular matrix (ECM) in the presence of Bone Morphogenic Protein-4 (BMP-4) or ascorbate. The enriched preparation of keratinocytes was able to form an epidermal equivalent composed of a stratified epithelium when cultured at the air-liquid interface on a collagen-coated acellular substratum. Interestingly, an underlying cellular compartment that belongs to the fibroblast lineage was systematically formed between the reconstituted epidermis and the inert membrane. The resulting tissue displayed morphological patterns similar to normal embryonic skin, as evidenced by light and transmission electron microscopy. Immunohistochemical studies revealed expression patterns of cytokeratins, basement membrane (BM) proteins and late differentiation markers of epidermis, as well as fibroblast markers, similar to native skin. The results demonstrate the capacity of ES cells to reconstitute in vitro a fully differentiated skin. This ES-derived bioengineered skin provides a powerful tool for studying the molecular mechanisms controlling epidermal and dermal commitments.

Embryonic stem (ES) cells are self-renewable and pluripotent cells derived from the inner cell mass of a blastocyst-stage embryo. ES cell pluripotency is being investigated increasingly to obtain specific cell lineages for therapeutic treatments and tissue engineering. Type II alveolar epithelial cells have been derived from murine ES cells, but the capacity of the latter to generate differentiated airway epithelial tissue has never been reported. Herein, we show by RT-PCR and immunocytochemistry that murine ES cells are able to differentiate into nonciliated secretory Clara cells, and that type I collagen induces this commitment. Moreover, when cultured at the air-liquid interface, ES cells give rise to a fully differentiated airway epithelium. By quantitative histologic examination, immunohistochemistry, and scanning electron microscopy, we show that the bioengineered epithelium is composed of basal, ciliated, intermediate, and Clara cells, similar to those of native tracheobronchial airway epithelium. Transmission electron microscopy and Western blotting reveal that the generated epithelium also exhibits the ultrastructural features and secretory functions characteristic of airway epithelial tissue. These results open new perspectives for cell therapy of injured epithelium in airway diseases, such as bronchopulmonary dysplasia, cystic fibrosis, or bronchiolitis obliterans.

Deletion of the amino acid residue Phe 508 of the cystic fibrosis transmembrane conductance regulator (CFTR) protein represents the most common mutation identified in cystic fibrosis (CF) patients. A monoclonal and a polyclonal antibody directed against different regions of CFTR were used to localize the CFTR protein in normal and CF airway epithelium derived from polyps of non-CF and CF subjects homozygous for the delta Phe 508 CFTR mutation. To identify the cellular and subcellular localization of CFTR, immunofluorescent light microscopy, confocal scanning microscopy, and immunogold transmission electron microscopy were performed on cryofixed tissue. A markedly different subcellular distribution was identified between normal and CF airway epithelial cells. In normal epithelium, labeling was restricted to the surface apical compartment of the ciliated cells. In contrast, in the epithelium from homozygous delta Phe 508 CF patients, CFTR markedly accumulated in the cytosol of all the epithelial cells. These findings are consistent with the concept that the CFTR delta Phe 508 mutation modifies the intracellular maturation and trafficking of the protein, leading to an altered subcellular distribution of the delta Phe 508 mutant CFTR.

The cell migration that occurs during wound repair is dependent on modifications of the cell-matrix interaction in which extracellular matrix proteins and their receptors, the integrins, are involved. To study the interactions between airway epithelial cells and the extracellular matrix during the process of wound repair, we developed an in vitro wound model of human epithelial cells. Surface epithelial cells were dissociated from human nasal polyps and cultured on a type I collagen matrix. At confluency, a wound was made by the addition of 2 microliters of NaOH (1 N) to the cell culture. After the cell culture was washed, the wound area was recorded every 12 h for 96 h by a videomicroscopic technique. We calculated the wound-repair index that represents the decrease in the wound area per hour. Using immunofluorescence techniques, we first examined the localization, during wound repair, of fibronectin and of the beta 1-, alpha v-, alpha 2-, alpha 3-, and alpha 5-integrin subunits. Secondly, we carried out a series of wound-repair blocking experiments with the use of anti-integrin or anti-fibronectin antibodies diluted in the culture medium. We observed that fibronectin and the alpha 5- integrin subunit were exclusively expressed by the migratory cells in the wounded area. No difference in the localization of the alpha v-, alpha 2-, and alpha 3-integrin subunits was observed between the nonrepairing and repairing cells. The blocking experiments showed a significant decrease in the wound-repair index in the presence of either the anti-beta 1, -alpha 3, alpha 5, or the anti-fibronectin antibodies. Furthermore, the addition of fibronectin to the culture medium induced a significant increase in the wound repair index. These results suggest that fibronectin and the corresponding alpha 5 beta 1-integrin play an important role in the process of airway epithelium wound repair.

SUMMARY:To investigate the role of P. aeruginosa virulence factors in the repair of human airway epithelial cells (HAEC) in culture, we evaluated the effect of stationary-phase supernatants from the wild-type strain PAO1 on cell migration, actin cytoskeleton distribution, epithelial integrity during and after repair of induced wounds, and the balance between matrix metalloproteinases (MMP) and their tissue inhibitors (TIMP). PAO1 supernatant altered wound repair by slowing the migration velocity in association with altered actin cytoskeleton polymerization in the lamellipodia of migrating airway epithelial cells and delaying or inhibiting the restoration of epithelial integrity after wound closure. PAO1 virulence factors overactivated two of the gelatinolytic enzymes, MMP-2 and MMP-9, produced by HAEC during repair. During HAEC repair in the presence of PAO1 virulence factors, enhanced MMP-2 activation was associated with decreased rates of its specific inhibitor TIMP-2, whereas enhanced MMP-9 activation was independent of changes of its specific inhibitor TIMP-1. These inhibitory effects were specific to P. aeruginosa elastase-producing strains (PAO1 and lipopolysaccharide-deficient AK43 strain); supernatants from P. aeruginosa strain elastase-deficient PDO240 and Escherichia coli strain DH5 ␣ had no inhibitory effect. To mimic the effects of P. aeruginosa, we further analyzed HAEC wound closure in the presence of increasing concentrations of activated MMP-9 or MMP-2. Whereas increasing concentrations of active MMP-9 accelerated repair, excess activated MMP-2 generated a lower migration velocity. All these data demonstrate that P. aeruginosa virulence factors, especially elastase, may impede airway epithelial wound closure by altering cell motility and causing an imbalance between pro-and activated forms of MMP-2. (Lab Invest 2000, 80:209-219).

Human nasal polyps from non-CF and AF 508 homozygous CF patients were used to compare the expression of CFTR and markers of epithelial differentiation, such as cytokeratins (CK) and desmoplakins (DP), at the transcriptional and translational levels. mRNA expression was assessed by semiquantitative RT/PCR kinetic assays while the expression and distribution of proteins were evaluated by immunofluorescence analysis. In parallel, for each nasal tissue specimen, the importance of surface epithelium remodeling and inflammation was estimated after histological observations. Our results show that the steady-state levels of CFTR, CK13, CK18, CK14, or DP 1 mRNA transcripts in AF 508 CF nasal polyps were not significantly different from those of non-CF tissues. A variability in the CFTR mRNA transcript level and in the pattern of CFTR immunolabeling has been observed between the different tissue samples. However, no relationship was found between the level of CFTR mRNA transcripts and the CFTR protein expression and distribution, either in the non-CF or in the CF group. The histological observations of non-CF and CF nasal polyp tissue indicated that the huge variations in the expression and distribution of the CFTR protein were associated with the variations in the degree of surface epithelium remodeling and inflammation in the lamina propria. A surface epithelium, showing a slight basal cell hyperplasia phenotype associated with diffuse inflammation, was mainly characterized by a CFTR protein distribution at the apex of ciliated cells in both non-CF and CF specimens. In contrast, in a remodeled surface epithelium associated with severe inflammation, CFTR protein presented either a diffuse distribution in the cytoplasm of ciliated cells, or was absent. These results suggest that abnormal expression and distribution of the CFTR protein in CF airways is not only caused by CFTR mutations. Airway surface epithelium remodeling and inflammation could play a critical role in the posttranscriptional and/ or the posttranslational regulation of the CFIR protein expression in non-CF and CF airways. (J. Clin. Invest. 1995Invest. . 95:1601Invest. -1611