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We introduce a novel in vivo model of human mucosal immunity, based on the implantation of human fetal bronchial mucosa and autologous peribronchial lymph node (PLN) in the severe combined immunodeficiency (SCID) mouse. In the SCID host, human fetal bronchi implanted alone retain macrophages and mast cells but lose T cells. In contrast, fetal bronchi co-implanted with PLN contain, in addition to macrophages and mast cells, numerous T cells and B cells, often clustered in intramucosal bronchus-associated lymphoid tissue (BALT). Functionally, bronchus-PLN cografts are able to mount robust alphabeta and gammadelta T-cell-mediated immune responses to Pseudomonas aeruginosa and 3,4-epoxy-3-methyl-1-butyl-diphosphate challenges. No other autologous lymphoid organ (bone marrow, thymus, liver) allows for BALT development in co-implanted bronchi, which suggests special ontogenetic and functional relations between extramucosal PLN and intramucosal BALT. Overall, the bronchus-PLN cograft appears as a promising model for human bronchial immune development and function. Our study is the first to document long-term ex vivo maintenance of functional human lymph nodes as native appendices to mucosal tissue. Our results, therefore, suggest a simple strategy for developing similar experimental models of human immune function in other mucosae.

To examine the effects of the uPA/plasmin system on cell migration in relation to the activation of MMP-9, we used ex vivo and in vitro wound-repair models of human bronchial epithelial cells and videomicroscopy techniques that make possible cell tracking and quantification of cell migration speeds. We observed that uPA was only detected in migrating cells at the wound edges and located at crucial sites for cell/extracellular matrix interactions. The implication of uPA in human bronchial epithelial cell migration was studied by incubating cultures with a monoclonal antibody raised against uPA and these experiments led to a 70% reduction in cell velocity. To examine the effects of the plasmin system on cell migration, we incubated cultures with increasing concentrations of plasmin or activated MMP-9. We observed a significant dose-dependent increase in cell migration velocity with plasmin (P < 0.001) and MMP-9 (P < 0.001). Moreover, addition of exogenous plasmin led to a twofold increase of activated MMP-9 in migrating cells. We also demonstrated that the addition of anti-uPA IgG led to an inhibition of 43% of activated MMP-9. In conclusion, these results show that uPA is involved in human bronchial epithelial cells migration. This action is mediated by the generation of plasmin, which in turn activates MMP-9, thus making possible cell migration., (Copyright 2001 Academic Press.)

Initial infection of the airway by Pseudomonas aeruginosa may occur through a variety of bacterial strategies including binding to epithelial receptors present at the surface of the respiratory epithelium. In order to characterize the adherence sites for P. aeruginosa in damaged and repairing bronchial tissue, an ex vivo model of airway epithelial injury and repair was developed using primary cell cultures of nasal cells from 14 subjects with polyposis. P. aeruginosa strongly adhered to flattened dedifferentiated (FD) bronchial and nasal cytokeratin 13-positive epithelial cells in the process of migration for repair. In in vitro experiments, competitive binding inhibition assays demonstrated that alpha5beta1 integrins and cellular fibronectin, in particular the RGD sequence, are receptors involved in P. aeruginosa adherence to FD nasal epithelial cells. Fluorescent cell sorting analysis and immunofluorescence techniques revealed that the alpha5beta1 integrins are overexpressed and apically exposed in FD nasal epithelial cells. One 50 kDa outer membrane protein was identified in piliated and nonpiliated strains of P. aeruginosa that was involved in binding to cellular fibronectin and alpha5beta1 epithelial integrins. These results demonstrate that Pseudomonas aeruginosa adherence is related to the dedifferentiation of airway epithelium during the repair process which unmasks and upregulates the alpha5beta1 integrin expression and induces active synthesis of cellular fibronectin. These epithelial receptors are then used by a Pseudomonas aeruginosa 50 kDa outer membrane protein as sites of bacterial adherence.

Internalization of Pseudomonas aeruginosa by epithelial respiratory cell lines has been suggested to be dependent on the cystic fibrosis transmembrane conductance regulator (CFTR) protein. Because we have observed intracellular (IC) P. aeruginosa only in cells that do not express apical CFTR, we addressed the question of whether bacterial internalization by epithelial cells depends on the degree of cell differentiation and polarity. Internalization of piliated P. aeruginosa PAO-1 and PAK by human epithelial respiratory cells in primary culture and by the 16 human bronchial epithelial 14o- cell line cultured either on thick collagen gels or on thin collagen films was evaluated by the gentamicin exclusion assay. Cells cultured on thick gels were differentiated, polarized, and tight. They exhibited CFTR at their apical membranes, expressed beta1 integrins at their basal membranes, excluded lanthanum nitrate, and uniformly expressed ZO-1 protein. In contrast, in cells cultured on thin films, CFTR was present mainly in the cytoplasm, whereas beta1 integrins were detected at apical membranes. Most cells cultured on thin films did not exclude lanthanum nitrate and rarely expressed ZO-1 protein. Cells grown on thick and thin collagen substrates differed markedly in bacterial internalization: no IC bacteria could be detected in cells cultured on gels, whereas high IC bacterial concentrations were isolated from cells cultured on thin films. Treatment of cells cultured on thin films with ethylenediaminetetraacetic acid, to disrupt intercellular junctions further, significantly enhanced P. aeruginosa internalization. Our results suggest that P. aeruginosa internalization by epithelial respiratory cells does not depend on CFTR protein expression at the epithelial cell surface but rather on cell polarity and junctional complex integrity.

The tracheobronchial tree begins to form during the fourth week of development through a series of dichotomic divisions of an entoblastic evagination. The morphogenesis and maturation of the respiratory tract depend both on the nature of the extracellular matrix which facilitates cell migration and on epithelial-mesenchymal interactions which induce the proliferation and differentiation of epithelial cells. The study of the plasticity and the phenotypic modifications of secretory cells during both development and inflammatory remodeling of the tracheobronchial mucosa suggests an important role for secretory cells during ciliogenesis and repair.