Dysregulation of IL-2 and IL-8 production in circulating T lymphocytes from young cystic fibrosis patients.

It is well documented that patients with cystic fibrosis (CF) are unable to clear persistent airway infections in spite of strong local inflammation, suggesting a dysregulation of immunity in CF. We and others have reported previously that T lymphocytes may play a prominent role in this immune imbalance. In the present work, we compared the reactivity of CD3+ T cells obtained from young CF patients in stable clinical conditions (n = 10, aged 9-16.5 years) to age-matched healthy subjects (n = 6, aged 9-13.5 years). Intracellular levels of interferon (IFN)-gamma, interleukin (IL)-2, IL-8 and IL-10 were determined by flow cytometry after whole blood culture. The data identified T lymphocyte subsets producing either low levels (M1) or high levels (M2) of cytokine under steady-state conditions. We found that the production of IFN-gamma and IL-10 by T lymphocytes was similar between young CF patients and healthy subjects. In contrast, after 4 h of activation with PMA and ionomycin, the percentage of T cells producing high levels of IL-2 (M2) was greater in CF patients (P = 0.02). Moreover, T cells from CF patients produced lower levels of IL-8, before and after activation (P = 0.007). We conclude that a systemic immune imbalance is present in young CF patients, even when clinically stable. This disorder is characterized by the capability of circulating T lymphocytes to produce low levels of IL-8 and by the emergence of more numerous T cells producing high levels of IL-2. This imbalance may contribute to immune dysregulation in CF.

Inflammation and infection in naive human cystic fibrosis airway grafts.

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.

Cell wall-associated protein A as a tool for immunolocalization of Staphylococcus aureus in infected human airway epithelium.

Staphylococcus aureus is a common human pathogen involved in non-bronchial diseases and in genetic and acquired bronchial diseases. In this study, we applied an immunolabeling approach for in vivo and in vitro detection of S. aureus, based on the affinity of staphylococcal protein A (SpA) for the Fc region of immunoglobulins, especially IgG. Most strains of S. aureus, including clinical strains, can be detected with this labeling technique. The approach can be used for detection and localization with transmission electron microscopy or light-fluorescence microscopy of S. aureus in infected tissues such as human bronchial tissue from cystic fibrosis (CF) patients. The methodology can also be applied to cell culture models with the aim of characterizing bacterial adherence to epithelial cells in backscattered electron imaging with scanning electron microscopy. Application to the study of S. aureus adherence to airway epithelium showed that the bacteria did not adhere in vivo to intact airway epithelium. In contrast, bacteria adhered to the basolateral plasma membrane of columnar cells, to basal cells, to the basement membrane and were identified beneath the lamina propria when the epithelium was injured and remodeled, or in vitro when the epithelial cells were dedifferentiated.

Pseudomonas aeruginosa virulence factors delay airway epithelial wound repair by altering the actin cytoskeleton and inducing overactivation of epithelial matrix metalloproteinase-2.

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 DH5alpha 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.

Differentiated and functional human airway epithelium regeneration in tracheal xenografts.

To investigate the regeneration process of a well-differentiated and functional human airway epithelium, we adapted an in vivo xenograft model in which adult human nasal epithelial cells adhere and progressively repopulate denuded rat tracheae grafted in nude mice. The proliferating activity, the degree of differentiation, and the barrier integrity of the repopulated epithelium were studied during the regeneration process at optical and ultrastructural levels with immunocytochemistry and a permeability tracer. Three days after implantation in nude mice, tracheal xenografts were partially repopulated with a flattened nonciliated and poorly differentiated leaky epithelium. By the end of the first week after the graft, cell proliferation produced on the entire surface of the rat trachea an epithelium that was stratified into multiple layers and tightly sealed. During successive weeks, cell proliferation dramatically decreased. Moreover, the epithelium became progressively columnar, secretory, ciliated, and transiently leaky. At 4-5 wk, a fully differentiated pseudostratified functional epithelial barrier impermeable to a low-molecular-weight tracer was reconstituted. The regeneration of a well-differentiated and functional human airway epithelium in rat tracheae grafted in nude mice includes several steps that mimic the regeneration dynamics of airway epithelium after injury.

Early expression of beta- and gamma-subunits of epithelial sodium channel during human airway development.

The amiloride-sensitive epithelial Na(+) channel (ENaC) is an apical membrane protein complex involved in active Na(+) absorption and in control of fluid composition in airways. There are no data reporting the distribution of its pore-forming alpha-, beta-, and gamma-subunits in the developing human lung. With use of two different rabbit polyclonal antisera raised against beta- and gamma-ENaC, immunohistochemical localization of the channel was performed in fetal (10-35 wk) and in adult human airways. Both subunits were detected after 17 wk of gestation on the apical domain of bronchial ciliated cells, in glandular ducts, and in bronchiolar ciliated and Clara cells. After 30 wk, the distribution of beta- and gamma-subunits was similar in fetal and adult airways. In large airways, the two subunits were detected in ciliated cells, in cells lining glandular ducts, and in the serous gland cells. In the distal bronchioles, beta- and gamma-subunits were identified in ciliated and Clara cells. Ultrastructural immunogold labeling confirmed the identification of beta- and gamma-ENaC proteins in submucosal serous cells and bronchiolar Clara cells. Early expression of ENaC proteins in human fetal airways suggests that Na(+) absorption might begin significantly before birth, even if secretion is still dominant.

Selective up-regulation of chemokine IL-8 expression in cystic fibrosis bronchial gland cells in vivo and in vitro.

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.

Distribution of integrins during human fetal lung development.

Interactions between epithelial cells and the extracellular matrix through integrins play a key role in the development of the lung by modulating branching morphogenesis, epithelial cell polarization, and differentiation. To determine the role of integrins during the different stages of lung development, we investigated the distribution of eight integrin subunits in the trachea and lung from human fetuses. In distal airways, during the early pseudoglandular stage of development, the alpha2-, alpha5-, alpha6-, alphav-, and beta1-subunits were detected in all epithelial cell plasma membranes, and polarized but undifferentiated tracheal epithelial cells expressed alpha3-, alpha6-, and beta1-subunits in the plasma membrane of the cells facing the basement membrane. The alpha6- and beta4-chains were detected along the basal plasma membrane of the basal cells in differentiated tracheal epithelia. The alpha4-subunit was detected in all respiratory cells throughout fetal development. In the submucosal glands, myoepithelial cells expressed the integrin subunits found in the undifferentiated cells of the developing airways, whereas the secretory cells expressed only alpha2-, alpha3-, alpha4-, alpha6-, and beta1-subunits. These results demonstrate differential expression of integrins during lung development and suggest that integrins may play multiple roles in organogenesis and maturation of respiratory surface epithelium and glands.

ATP depletion induces a loss of respiratory epithelium functional integrity and down-regulates CFTR (cystic fibrosis transmembrane conductance regulator) expression.

To mimic the effect of ischemia on the integrity of airway epithelium and expression of cystic fibrosis transmembrane conductance regulator (CFTR), we induced an ATP depletion of the respiratory epithelium from upper airway cells (nasal tissue) and human bronchial epithelial 16HBE14o- cell line. Histological analysis showed that 2 h of ATP depletion led to a loss of the epithelium integrity at the interface between basal cells and columnar cells. The expression of connexin 43 (Cx43, subunit of the gap junctions) and desmoplakins 1 and 2 (DPs 1 and 2, major components of the desmosomes) proteins was inhibited. After 90 min of ATP depletion, a significant decrease of the transepithelial resistance (25%) was observed but was reversible. Similar results were obtained with the 16HBE14o- human bronchial epithelial cell line. ATP depletion led to actin filaments depolymerization. The expression of the mature CFTR (170 kDa) and fodrin proteins at the apical domain of the ciliated cells was down-regulated. The steady-state levels of CFTR, Cx43, DPs 1 and 2 mRNAs, semiquantified by RT-polymerase chain reaction kinetics, remained constant throughout ATP depletion in nasal tissue as in the homogeneous cell population of 16HBE14o- human bronchial epithelial cell line. This suggests that the down-regulation of these proteins might be posttranscriptional. The intercellular diffusion through gap junctions of Lucifer dye was completely inhibited after 90 min of ATP depletion but was reversible. The volume-dependent and the cAMP-dependent chloride secretion were inhibited in a nonreversible way. Taken together, these results suggest that an ATP depletion in human airway epithelium, mimicking ischemia, may induce a marked alteration in the junctional complexes and cytoskeleton structure concomitantly with a loss of apical CFTR expression and chloride secretion function.

Induction of a cAMP-stimulated chloride secretion in regenerating poorly differentiated airway epithelial cells by adenovirus-mediated CFTR gene transfer.

In cystic fibrosis (CF), the airway epithelium is in the process of injury and regeneration. In the context of the CF gene therapy, we previously reported that regenerating poorly differentiated (PD) cells of human airway epithelium represent preferential cell targets for recombinant adenoviral gene vectors. To define whether PD non-CF and CF epithelial cells possess a functional cystic fibrosis transmembrane conductance regulator protein (CFTR) chloride channel, we analyzed the CFTR expression and the regulation of chloride secretion under cyclic (c)AMP stimulation in these regenerating PD epithelial cells of non-CF and CF airway tissue. Moreover, we studied the effects of CFTR gene transfer mediated by a replication-defective adenovirus containing the wild-type CFTR gene (AdCFTR) on CFTR expression and on cAMP-stimulated chloride secretion. Distribution of the CFTR protein was evaluated in regenerating PD airway cells by light fluorescence microscopy and scanning laser confocal microscopy. The cAMP-mediated regulation of cell membrane chloride secretion was investigated using the whole-cell patch clamp and SPQ (6-methoxy-N-[3-sulfopropyl]quinolinium) techniques. Compared with the absence of CFTR expression and cAMP-regulated chloride secretion in nontransduced regenerating PD cells of either non-CF or CF origin, transduction with AdCFTR induces a CFTR expression and a cAMP-regulated stimulation of the cell membrane chloride secretion in the regenerating PD cells. These results suggest that, out of the context of CF, remodeled and poorly differentiated airway epithelium may present abnormalities in ion transport. Moreover, our data suggest that, in the context of CF gene therapy, adenoviral vectors can be efficient in correcting, at least partially, the chloride secretion defect in the remodeled CF airway epithelium.

Decreased expression of the cystic fibrosis transmembrane conductance regulator protein in remodeled airway epithelium from lung transplanted patients.

The absence or mislocalization of cystic fibrosis transmembrane conductance regulator (CFTR) is regarded as being specific for cystic fibrosis (CF). In principle, the supply of a non-CF lung transplant to a CF patient should bring up normal CFTR expression in the lower airways. Immunolocalization of CFTR and of epithelial differentiation markers (ie, cytokeratins 13, 14, and 18, and desmoplakins 1 and 2) was carried out on 21 mucosal biopsies from the upper lobe of grafts in non-CF (n = 12) and CF patients (n = 9) retrieved between days 23 and 1,608 after lung transplantation. Biopsy specimens from seven non-CF and four CF patients presented either a pseudostratified respiratory epithelium or slight basal cell hyperplasia. CFTR was distributed at the apical membrane of the ciliated cells. In remodeled epithelia with basal cell hyperplasia or squamous metaplasia, CFTR was either weakly expressed in the cytoplasm of the superficial epithelial cells or was undetectable. The extent of epithelium remodeling was significantly correlated with an impairment of lung function. The results suggest that posttransplant airway epithelium dedifferentiation of the graft leads to the loss of properly targeted CFTR irrespective of the underlying disease of the recipient.

Early alterations in airway mucociliary clearance and inflammation of the lamina propria in CF mice.

In cystic fibrosis (CF), whether cystic fibrosis transmembrane conductance regulator (CFTR) dysfunction leads to decreased mucociliary clearance and mucus hypersecretion, before bacterial infection, remains an open question. To answer this question, we quantified in a blind trial the mucociliary transport velocity, the histological state, and the degree of inflammation of the tracheal mucosa in 23 cftr(m1HGU/cftr(m1HGU) transgenic mice (Dorin, J. R., P. Dickinson, E. W. F. W. Alton, S. N. Smith, D. M. Geddes, B. J. Stevenson, W. L. Kimber, S. Fleming, A. R. Clark, M. L. Hooper, L. Anderson, R. S. P. Beddington, and D. J. Porteous. Nature Lond. 359: 211-215, 1992) and in 30 control littermates housed in pathogen-free conditions. The nasal and tracheal transepithelial potential difference (PD) measured in basal conditions was significantly more negative in the cftr(m1HGU) mutant mice as compared with the control mice (nasal PD: -7.1 +/- 0.6 and -4.6 +/- 0.5 mV, respectively, P < 0.01; tracheal PD: -30.8 +/- 2.1 and -21.4 +/- 1.8 mV, respectively, P < 0.04). In the cftr(m1HGU)/cftr(m1HGU) mice, the mucociliary transport velocity was significantly lower (14.2 +/- 4.4 microm/mm, P < 0.04) compared with the control mice (30.6 +/- 5.9 microm/mm). The number of inflammatory cells in the lamina propria was significantly higher in the cftr(m1HGU)/cftr(m1HGU) mice (1048.7 +/- 124.7 cells/mm2, P < 0.03) compared with the control mice (640.5 +/- 58.2 cells/mm2). These results suggest that in CF, decreased airway mucociliary clearance and airway submucosal inflammation represent early alterations, before any airway infection.

Epithelial barrier integrity during in vitro wound repair of the airway epithelium.

The surface epithelium of the airway mucosa forms a continuous barrier to a wide number of noxious substances present in the lumen. The restoration of the barrier integrity after injury represents a key issue in the defense capacity of the airway epithelium. Using an in vitro wound repair model of the airway epithelium, we investigated the dynamic of the restoration of the epithelial barrier integrity during the wound repair process. Airway epithelial cells in culture were chemically wounded by sodium hydroxide. The immunolocalization of zonula occludens 1 (ZO-1), a cytoplasmic protein associated with the tight junctions, was examined during the wound repair process. Junctional integrity was examined by analyzing the transepithelial resistance (TER) and the permeability to [3H]mannitol and by visualizing the permeability to lanthanum nitrate during 5 days after injury. Immediately after injury, we simultaneously observed a 36.7% decrease in the TER and a 74.9% rise in the permeability to [3H]mannitol. In addition, lanthanum nitrate penetrated in the intercellular spaces in the repairing areas, which was also characterized by the absence of ZO-1 staining, as opposed to nonrepairing cells. TER and [3H]mannitol flux values as well as lanthanum nitrate and ZO-1 localizations were found to be similar to those observed in confluent cultures only 1 to 2 days after complete wound closure. This study demonstrates that using our culture model, confluent airway epithelial cells form a continuous and efficient barrier with tight junctions. Epithelial integrity is affected immediately after injury and is completely restored within 1 to 2 days after wound closure. During such a period of time, the airway epithelium may remain exposed to the noxious effect of environment in vivo, which can prevent the epithelial barrier restoration by modifying tight junction formation.

Fibronectin and its alpha 5 beta 1-integrin receptor are involved in the wound-repair process of airway epithelium.

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.

CFTR is involved in membrane endocytosis but not in fluid-phase and receptor-mediated endocytosis in human respiratory epithelial cells.

Cystic fibrosis transmembrane conductance regulator (CFTR) protein has been reported to be a cAMP-regulator of plasma membrane recycling in epithelial cells overexpressing CFTR. To assess its role in the different endocytic processes in human respiratory epithelial cells, the rates of internalization of membrane, fluid-phase and receptor-mediator tracers were compared, under control conditions and after treatment with the cAMP agonist forskolin in normal and cystic fibrosis (CF) cells. In both control and treated-cells, CFTR was only present in the plasma membrane of normal but not in CF cells. Similarly, activation of Cl- efflux only occurred in normal and not in CF-treated cells. The rate of membrane endocytosis was significantly decreased by 35% in normal treated-cells, whereas it was not significantly decreased (12%) in CF-treated cells. Upon forskolin treatment, the decrease of the rate of both fluid-phase and receptor-mediated endocytosis was not significantly different between normal and CF cells. These results demonstrate that CFTR is involved in membrane endocytosis but not in fluid-phase and receptor-mediated endocytosis in human respiratory epithelial cells.

CFTR and differentiation markers expression in non-CF and delta F 508 homozygous CF nasal epithelium.

Human nasal polyps from non-CF and delta F 508 homozygous CF patients were used to compare the expression of CFTR and markers 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, CK18, CK14, or DP 1 mRNA transcripts in delta F 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 of 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 CFTR protein expression in non-CF and CF airways.

A change in gating mode leading to increased intrinsic Cl- channel activity compensates for defective processing in a cystic fibrosis mutant corresponding to a mild form of the disease.

The effects of the mild cystic fibrosis (CF) mutation P574H were analysed and compared with those of three severe ones (delta I507, delta F508 and R560T). Immunochemical and functional analyses indicate that the rank order of CFTR expression at the cell surface is: wild type CFTR > P574H >> delta F508 >> R560T approximately 0. Patch-clamp analysis indicates that the open probability of P574H Cl- channels is almost twice as high as that of the wild type CFTR-Cl- channel. This increased intrinsic activity of individual P574H CFTR-Cl- channels compensates for the lower number of P574H CFTR-Cl- channels reaching the cell surface, and probably explains the milder form of CF associated with the P574H mutation. NS004, a recently described activator, restores near normal CFTR activity in cells expressing the P574H-CFTR channel. The P574H mutation modifies the gating mode of the channel with a large increase (approximately x 7) in the mean channel open time. Proline 574 might play an important role in the process connecting ATP hydrolysis at the nucleotide binding domain and opening and closing events of the CFTR-Cl- channel.

Expression and localization of CFTR in the rhesus monkey surface airway epithelium.

The Rhesus monkey has been used as a model for evaluating the possibility of introducing the CFTR gene into the airway epithelium in vivo. We addressed the question of whether the simian airway surface epithelium exhibits a CFTR distribution and functional activity (ciliary beating frequency) similar to that of human airway surface epithelium. Expression of CFTR mRNA was demonstrated on Rhesus monkey tracheobronchial tissue by reverse transcription polymerase chain reaction (RT-PCR) analysis. By immunofluorescent light microscopy, CFTR was localized on the apical plasma membrane of ciliated cells as we previously described for human tracheobronchial surface epithelium. The ciliary beat frequency (CBF) measured on the explant of Rhesus monkey tracheobronchial tissue appeared to be similar to that of the CBF of human tracheobronchial ciliated cells. To compare the Rhesus monkey CFTR gene with that of the human, we sequenced parts of exon 13 (encoding the R domain) and exon 24 (encoding the C terminal part of the protein) of the Rhesus monkey CFTR gene. The nucleotide sequence identity with the human counterpart was found to be 98% and 94% respectively, although restriction enzyme differences allow discrimination between Rhesus monkey and human CFTR cDNA. Taken together, these results suggest that the airway epithelium of the Rhesus monkey is a suitable model of human respiratory epithelium for analyzing the effect of human CFTR gene transfer.

Decreased expression of the CFTR protein in remodeled human nasal epithelium from non-cystic fibrosis patients.

BACKGROUND: In normal adult pseudostratified human nasal surface epithelium, the cystic fibrosis transmembrane conductance regulator (CFTR) is localized to the apical domain of the ciliated cells, whereas in cystic fibrosis (CF), the mutated delta F 508 CFTR exhibits an abnormal cytoplasmic localization. Frequent airway injuries either in CF or non-CF patients may induce a remodeling of the surface epithelium characterized by a change in the morphological structure from normal columnar pseudostratified epithelium to either basal cell hyperplasia, mucous cell hyperplasia, or squamous metaplasia. EXPERIMENTAL DESIGN: The localization of CFTR parallel to markers of cell differentiation, such as cytokeratin 14 (CK14, a marker of basal cells), cytokeratin 18 (CK 18, a marker of ciliated and mucous cells), cytokeratin 13 (CK13, a marker of squamous metaplasia cells), and desmoplakins (DP) 1 and 2 (markers of desmosomes) was analyzed by indirect immunofluorescence. RESULTS: In normal pseudostratified epithelium, CFTR was detected at the apical plasma membrane of the ciliated cells, CK14 was identified in basal cells of focal areas, CK18 was localized in both ciliated and mucous cells, CK 13 was detected in all basal cells, and DP 1 and 2 were preferentially detected at the interface between columnar and basal cells. In basal cell hyperplasia, CFTR was poorly expressed in the cytoplasm of the more superficial cells, CK14 and CK13 were localized in basal cell multilayers, CK18 labeling was present in the more superficial cell layers, and DP 1 and 2 were preferentially detected at the interface between the more basal cells. In squamous metaplasia, CFTR labeling was either very low or even undetectable, CK14 was found in focal areas of the more basal cell layers, CK18 labeling was either very low or undetectable, CK13 expression was restricted to the flattened cells toward the epithelial surface, and DP 1&2 were intensively present between all the epithelial cells. CONCLUSIONS: These results suggest that the localization of CFTR in human nasal surface epithelium is related to the differentiation state of this epithelium. Abnormally low expression of the CFTR protein may not only be caused by CFTR gene mutations but can also be associated with airway surface epithelium dedifferentiation and remodeling.

Expression of gelatinase A, a mediator of extracellular matrix remodeling, by tracheal gland serous cells in culture and in vivo.

Tracheal gland morphogenesis and gland hypertrophy in disease involve the penetration of epithelial cells into the submucosa, a process that requires digestion of the basal lamina and the surrounding extracellular matrix. We observed that bovine tracheal gland cells invaded collagen substrates and were inhibited from doing so in the presence of a metalloproteinase inhibitor GM6001. The gland cells, but not bovine tracheal surface epithelial cells, secreted a 72-kDa metalloproteinase. The purified enzyme could be activated with 4-aminophenylmercuric acetate and converted to an active 65-kDa form that was far more effective in degrading denatured collagen (gelatin) than nondenatured type I and IV collagens and was ineffective in degrading intact interstitial collagen fibers. At 25 degrees C, the initial rate of degradation of acid-solubilized type I collagen was approximately 50 mg of type I collagen cleaved per min per mg of enzyme, whereas acid-solubilized type IV collagen was degraded at approximately 250 mg cleaved per min per mg of enzyme. In contrast, at the same temperature, heat-denatured type I collagen was degraded 1000-fold more rapidly, while heat-denatured type IV collagen was cleaved 50-fold more rapidly. The activity of the enzyme was maximal at pH 7-8 and was completely abolished by the metalloproteinase inhibitors EDTA and 1,10-phenanthroline. In immunoblots, the enzyme was recognized by an antibody directed against human gelatinase A, the 72-kDa gelatinase. The purified enzyme disrupted the distribution pattern of type IV collagen in the gland basal lamina, as well as of interstitial collagen in the underlying stromal tissue, as shown in tissue sections by immunocytochemistry. Using an antibody directed against the purified enzyme, we also showed by immunocytochemistry that the gelatinase was present in tracheal tissue and was specifically located at the periphery of some tracheal gland acini. Northern blots showed higher concentrations of gelatinase A mRNA in glands than in epithelium microdissected from adult cow tracheas. These data indicate that gelatinase A is a specialized product of the tracheal gland epithelial cell, a cell type normally invasive as part of its developmental program; the enzyme may play an important role in normal gland development and disease-associated hypertrophy.