Autophagy of mucin granules contributes to resolution of airway mucous metaplasia.

Exacerbations of muco-obstructive airway diseases such as COPD and asthma are associated with epithelial changes termed mucous metaplasia (MM). Many molecular pathways triggering MM have been identified; however, the factors that regulate resolution are less well understood. We hypothesized that the autophagy pathway is required for resolution of MM by eliminating excess non-secreted intracellular mucin granules. We found increased intracellular levels of mucins Muc5ac and Muc5b in mice deficient in autophagy regulatory protein, Atg16L1, and that this difference was not due to defects in the known baseline or stimulated mucin secretion pathways. Instead, we found that, in mucous secretory cells, Lc3/Lamp1 vesicles colocalized with mucin granules particularly adjacent to the nucleus, suggesting that some granules were being eliminated in the autophagy pathway rather than secreted. Using a mouse model of MM resolution, we found increased lysosomal proteolytic activity that peaked in the days after mucin production began to decline. In purified lysosomal fractions, Atg16L1-deficient mice had reduced proteolytic degradation of Lc3 and Sqstm1 and persistent accumulation of mucin granules associated with impaired resolution of mucous metaplasia. In normal and COPD derived human airway epithelial cells (AECs), activation of autophagy by mTOR inhibition led to a reduction of intracellular mucin granules in AECs. Our findings indicate that during peak and resolution phases of MM, autophagy activity rather than secretion is required for elimination of some remaining mucin granules. Manipulation of autophagy activation offers a therapeutic target to speed resolution of MM in airway disease exacerbations.

Noninvasive Imaging of CCR2+ Cells in Ischemia-Reperfusion Injury After Lung Transplantation.

Ischemia-reperfusion injury-mediated primary graft dysfunction substantially hampers short- and long-term outcomes after lung transplantation. This condition continues to be diagnosed based on oxygen exchange parameters as well as radiological appearance, and therapeutic strategies are mostly supportive in nature. Identifying patients who may benefit from targeted therapy would therefore be highly desirable. Here, we show that C-C chemokine receptor type 2 (CCR2) expression in murine lung transplant recipients promotes monocyte infiltration into pulmonary grafts and mediates graft dysfunction. We have developed new positron emission tomography imaging agents using a CCR2 binding peptide, ECLi1, that can be used to monitor inflammatory responses after organ transplantation. Both 64 Cu-radiolabeled ECL1i peptide radiotracer (64 Cu-DOTA-ECL1i) and ECL1i-conjugated gold nanoclusters doped with 64 Cu (64 CuAuNCs-ECL1i) showed specific detection of CCR2, which is upregulated during ischemia-reperfusion injury after lung transplantation. Due to its fast pharmacokinetics, 64 Cu-DOTA-ECL1i functioned efficiently for rapid and serial imaging of CCR2. The multivalent 64 CuAuNCs-ECL1i with extended pharmacokinetics is favored for long-term CCR2 detection and potential targeted theranostics. This imaging may be applicable for diagnostic and therapeutic purposes for many immune-mediated diseases.

Lung transplant acceptance is facilitated by early events in the graft and is associated with lymphoid neogenesis.

Early immune responses are important in shaping long-term outcomes of human lung transplants. To examine the role of early immune responses in lung rejection and acceptance, we developed a method to retransplant mouse lungs. Retransplantation into T-cell-deficient hosts showed that for lungs and hearts alloimmune responses occurring within 72 h of transplantation are reversible. In contrast to hearts, a 72-h period of immunosuppression with costimulation blockade in primary allogeneic recipients suffices to prevent rejection of lungs upon retransplantation into untreated allogeneic hosts. Long-term lung acceptance is associated with induction of bronchus-associated lymphoid tissue, where Foxp3(+) cells accumulate and recipient T cells interact with CD11c(+) dendritic cells. Acceptance of retransplanted lung allografts is abrogated by treatment of immunosuppressed primary recipients with anti-CD25 antibodies. Thus, events contributing to lung transplant acceptance are established early in the graft and induction of bronchus-associated lymphoid tissue can be associated with an immune quiescent state.

Role of foxj1 and estrogen receptor alpha in ciliated epithelial cell differentiation of the neonatal oviduct.

Estrogen regulates proliferation and differentiation of epithelial cells in the mammalian oviduct, but pathways for cell-specific differentiation are not well understood. In the epithelial cells of the developing rat oviduct, we found estrogen receptor (ER) alpha is expressed at birth and persists in all cells through neonatal day (ND) 7 when ciliated cells appear. To determine a specific function of ER and foxj1, a transcription factor known to have fundamental roles in ciliogenesis in the lung, in differentiation of the ciliated epithelial cells, we treated newborn rats from ND 0 to 5 with estradiol-17beta (E2) with and without a selective ER antagonist. E2 enhanced the number of proliferating cells and accelerated the process of epithelial cell differentiation resulting in ciliogenesis by ND 5, and co-treatment with an ER antagonist inhibited these changes. Foxj1 was expressed only in the infundibulum and ampulla (INF/AMP). That expression preceded the appearance of cilia and was induced by E2. Cilia were absent in oviducts of foxj1-deficient mice, indicating that foxj1 plays a critical role in oviductal ciliogenesis. However, we found the presence of cilia in the ERalpha-deficient mouse oviduct. The widespread expression of ERalpha in oviductal epithelium, but restriction of cilia to the INF/AMP regions, and importantly, the presence of cilia in the ERalpha-deficient mice, suggested ER signaling is not essential for ciliated epithelial cell differentiation. These observations demonstrate that, although E2 stimulates the differentiation process of ciliated epithelial cells, foxj1 is directly required for epithelial cell ciliogenesis of the neonatal oviduct.

Effects of paramyxoviral infection on airway epithelial cell Foxj1 expression, ciliogenesis, and mucociliary function.

To elucidate molecular mechanisms underlying the association between respiratory viral infection and predisposition to subsequent bacterial infection, we used in vivo and in vitro models and human samples to characterize respiratory virus-induced changes in airway epithelial cell morphology, gene expression, and mucociliary function. Mouse paramyxoviral bronchitis resulted in airway epithelial cell infection and a distinct pattern of epithelial cell morphology changes and altered expression of the differentiation markers beta-tubulin-IV, Clara cell secretory protein, and Foxj1. Furthermore, changes in gene expression were recapitulated using an in vitro epithelial cell culture system and progressed independent of the host inflammatory response. Restoration of mature airway epithelium occurred in a pattern similar to epithelial cell differentiation and ciliogenesis in embryonic lung development characterized by sequential proliferation of undifferentiated cells, basal body production, Foxj1 expression, and beta-tubulin-IV expression. The effects of virus-induced alterations in morphology and gene expression on epithelial cell function were illustrated by decreased airway mucociliary velocity and impaired bacterial clearance. Similar changes in epithelial cell Foxj1 expression were also observed in human paramyxoviral respiratory infection. Taken together, these model systems of paramyxoviral respiratory infection mimic human pathology and identify epithelial cell Foxj1 expression as an early marker of epithelial cell differentiation, recovery, and function.

Vesicular stomatitis virus G-pseudotyped lentivirus vectors mediate efficient apical transduction of polarized quiescent primary alveolar epithelial cells.

We investigated the use of lentivirus vectors for gene transfer to quiescent alveolar epithelial cells. Primary rat alveolar epithelial cells (AEC) grown on plastic or as polarized monolayers on tissue culture-treated polycarbonate semipermeable supports were transduced with a replication-defective human immunodeficiency virus-based lentivirus vector pseudotyped with the vesicular stomatitis virus G (VSV-G) protein and encoding an enhanced green fluorescent protein reporter gene. Transduction efficiency, evaluated by confocal microscopy and quantified by fluorescence-activated cell sorting, was dependent on the dose of vector, ranging from 4% at a multiplicity of infection (MOI) of 0.1 to 99% at an MOI of 50 for AEC grown on plastic. At a comparable titer and MOI, transduction of these cells by a similarly pseudotyped murine leukemia virus vector was approximately 30-fold less than by the lentivirus vector. Importantly, comparison of lentivirus-mediated gene transfer from the apical or basolateral surface of confluent AEC monolayers (R(t) > 2 kOmega. cm(2); MOI = 10) revealed efficient transduction only when VSV-G-pseudotyped lentivirus was applied apically. Furthermore, treatment with EGTA to increase access to the basolateral surface did not increase transduction of apically applied virus, indicating that transduction was primarily via the apical membrane domain. In contrast, differentiated tracheal epithelial cells were transduced by apically applied lentivirus only in the presence of EGTA and at a much lower overall efficiency (approximately 15-fold) than was observed for AEC. Efficient transduction of AEC from the apical cell surface supports the feasibility of using VSV-G-pseudotyped lentivirus vectors for gene transfer to the alveolar epithelium and suggests that differences exist between upper and lower airways in the polarity of available receptors for the VSV-G protein.

Induction, distribution and modulation of upper airway allergic inflammation in mice.

BACKGROUND: To further elucidate mechanisms of human allergic rhinosinusitis, we studied the induction, distribution and modulation of allergen-induced upper airway inflammation in a BALB/c mouse model. METHODS: Allergic inflammation induced with ovalbumin (OVA) by intraperitoneal (IP) injection in alum was compared to repeated intranasal instillation. The type and distribution of inflammatory cells was compared in the respiratory and olfactory epithelial compartments. Eosinophil distribution was assessed using Scarlet Red stain and a polyclonal antibody recognizing eosinophil major basic protein (MBP). The role of interleukin (IL)-5 in upper airway inflammation was tested by administration of polyclonal anti-IL-5 antibody during the sensitization protocol. RESULTS: Unsensitized control mice receiving saline failed to develop upper airway eosinophil infiltration. IP OVA-sensitized mice developed marked upper airway mucosal eosinophil infiltration after aerosol OVA challenge, whereas repeated intranasal instillation of OVA produced qualitatively similar, but less intense eosinophil infiltration. Using either sensitization protocol, eosinophil infiltration was seen in areas of the lower portion of the nasal septum, the floor and the lower lateral walls of the mid-caudal region of the nasal cavity. Immunofluorescence staining for MBP confirmed this distribution of eosinophils but also demonstrated some eosinophils in the maxillary sinuses and in circumscribed regions of the ethmoturbinates. All areas of eosinophil infiltration were lined by respiratory epithelium. The selective infiltration of respiratory but not olfactory epithelium by eosinophils was unassociated with a measurable induction of epithelial ICAM-1 or eotaxin expression. OVA-induced upper airway eosinophil infiltration was found to be IL-5 dependent, since administration of a polyclonal anti-IL-5 antibody (TRFK-5) during OVA sensitization resulted in a marked modulation (80% decrease) in eosinophil infiltration in response to subsequent OVA challenge. CONCLUSION: The mouse upper airway, specifically in areas containing respiratory epithelium, is a target for OVA-induced allergic inflammation. This selective infiltration of respiratory, but not olfactory, epithelium is, in part, dependent upon IL-5. This model is useful for further dissection of the inflammatory response with genetic manipulations and targeted immunological approaches.

Proximal promoter of the surfactant protein D gene: regulatory roles of AP-1, forkhead box, and GT box binding proteins.

Surfactant protein D (SP-D) plays roles in pulmonary host defense and surfactant homeostasis and is increased following lung injury. Because AP-1 proteins regulate cellular responses to diverse environmental stimuli, we hypothesized that the conserved AP-1 motif (at -109) and flanking sequences in the human SP-D promoter contribute to the regulation of SP-D expression. The AP-1 sequence specifically bound to fra-1, junD, and junB in H441 lung adenocarcinoma nuclear extracts. Mutagenesis of the AP-1 motif in a chloramphenicol acetyltransferase reporter construct containing 285 base pairs of upstream sequence nearly abolished promoter activity, and co-transfection of junD significantly increased wild type but not mutant promoter activity. The sequence immediately downstream of the AP-1 element contained a binding site for HNF-3 (FOXA), and simultaneous mutation of this site (fox-d) and an upstream FoxA binding site (-277, fox-u) caused a 4-fold reduction in chloramphenicol acetyltransferase activity. Immediately upstream of the AP-1-binding site, we identified a GT box-containing positive regulatory element. Despite finding regions of limited homology to the thyroid transcription factor 1-binding site, SP-D promoter activity did not require thyroid transcription factor 1. Thus, transcriptional regulation of SP-D gene expression involves complex interactions with ubiquitous and lineage-dependent factors consistent with more generalized roles in innate immunity.

Ciliogenesis and left-right axis defects in forkhead factor HFH-4-null mice.

Cilia have been classified as sensory or motile types on the basis of functional and structural characteristics; however, factors important for regulation of assembly of different cilia types are not well understood. Hepatocyte nuclear factor-3/forkhead homologue 4 (HFH-4) is a winged helix/forkhead transcription factor expressed in ciliated cells of the respiratory tract, oviduct, and ependyma in late development through adulthood. Targeted deletion of the Hfh4 gene resulted in defective ciliogenesis in airway epithelial cells and randomized left-right asymmetry so that half the mice had situs inversus. In HFH-4-null mice, classic motile type cilia with a 9 + 2 microtubule ultrastructure were absent in epithelial cells, including those in the airways. In other organs, sensory cilia with a 9 + 0 microtubule pattern, such as those on olfactory neuroepithelial cells, were present. Ultrastructural analysis of mutant cells with absent 9 + 2 cilia demonstrated that defective ciliogenesis was due to abnormal centriole migration and/or apical membrane docking, suggesting that HFH-4 functions to direct basal body positioning or anchoring. Evaluation of wild-type embryos at gestational days 7.0 to 7.5 revealed Hfh4 expression in embryonic node cells that have monocilium, consistent with a function for this factor at the node in early determination of left- right axis. Analysis of the node of HFH-4 mutant embryos revealed that, in contrast to absent airway cilia, node cilia were present. These observations indicate that there are independent regulatory pathways for node ciliogenesis compared with 9 + 2 type ciliogenesis in airways, and support a central role for HFH-4 in ciliogenesis and left-right axis formation.

Forkhead transcription factor HFH-4 expression is temporally related to ciliogenesis.

Members of the forkhead/winged-helix family of transcription factors are expressed in tissue-specific patterns and play critical roles in development and cell differentiation. The expression of forkhead family member hepatocyte nuclear factor-3/forkhead homologue 4 (HFH-4) has been localized by RNA-blot analysis and in situ hybridization to the proximal airway of the lung (trachea, bronchi, and bronchioles) with onset at mouse embryonic day (E) 14.5 and is present in the choroid plexus, ependymal cells, oviduct, and testis. We hypothesized that the restricted expression of HFH-4 messenger RNA suggests a function common to these tissues and therefore a cell-specific role for HFH-4. Accordingly, an anti-HFH-4 antibody was generated and used for cell-specific localization of protein expression to begin to identify the functions of HFH-4. We found HFH-4 expression in proximal airway ciliated epithelial cells, but not Clara cells or alveolar epithelial cells. HFH-4 was also expressed in ciliated epithelial cells of the nose and paranasal sinuses, choroid plexus, ependyma, and oviduct. In developing mouse lung, HFH-4 expression was initially detected in airway epithelial cells at E15.5, before the appearance of cilia, and at later stages was localized to epithelial cells with cilia. In the testis, HFH-4 expression in spermatids was coincident with stage-specific generation of flagella. The temporal relationship of HFH-4 expression to the development of cilia and flagella, and the restricted expression in ciliated epithelial cells, suggest that this transcription factor has a role in regulation and maintenance of the ciliated cell phenotype in epithelial cells.

A human forkhead/winged-helix transcription factor expressed in developing pulmonary and renal epithelium.

Members of the forkhead/winged-helix transcription factor family play crucial roles during vertebrate development. A human hepatocyte nuclear factor/forkhead homolog (HFH)-4 cDNA encoding a 421-amino acid protein was isolated from a human fetal lung cDNA library. By Southern blot analysis of human-rodent somatic cell hybrid genomic DNA, the human HFH-4 gene localizes to chromosome 17q23-qter. This is the locus of another forkhead/winged-helix gene, the interleukin enhancer binding factor gene. RNA blot analysis revealed a 2.5-kilobase human HFH-4 transcript in fetal lung, kidney, and brain as well as in adult reproductive tissues, lung, and brain. By in situ hybridization, HFH-4 expression is associated with differentiation of the proximal pulmonary epithelium, starting during the pseudoglandular stage of human lung development. During human renal morphogenesis, HFH-4 is expressed in the developing epithelial cells of the ureteric duct, glomerulus, and epithelial vesicles. The unique pattern of HFH-4 expression during human fetal development suggests a role for this forkhead/winged-helix factor during pulmonary and renal epithelial development.

Structural characterization of the mouse Hfh4 gene, a developmentally regulated forkhead family member.

Hepatocyte nuclear factor-3/forkhead homologue 4 (HFH-4) is a forkhead/winged-helix transcription factor family member that has a unique temporal and spatial pattern of gene expression in the developing and adult lung, choroid plexus, testis, and oviduct. To characterize HFH-4 further, mouse genomic clones were isolated and analyzed. The Hfh4 gene is encoded on a 5.5-kb region located on the distal end of mouse chromosome 11 and consists of two exons and one intron. Unlike most forkhead genes, the DNA binding domain is divided between two exons, and the intron position corresponds precisely to the site of gene translocations involving two known human forkhead homologues. Multiple putative transcription start sites are identified in a G+C-rich sequence that does not contain TATA or CAAT boxes. Within 2.1 kb of 5' flanking sequence are three identical E boxes and multiple putative transcription factor binding sites. Transfection of plasmids containing Hfh4 5' flanking sequence linked to a reporter gene results in promoter activity in lung epithelial cells but not in epithelial-like fibrosarcoma cells, suggesting that this 5' flanking sequence can function as a promoter with the proper cell-type specificity.

Primary structure of hepatocyte nuclear factor/forkhead homologue 4 and characterization of gene expression in the developing respiratory and reproductive epithelium.

Members of the winged helix/forkhead family of transcription factors are believed to play a role in cell-specific gene expression. A cDNA encoding a member of this family of proteins, termed hepatocyte nuclear factor/forkhead homologue 4 (HFH-4), has been isolated from rat lung and rat testis cDNA libraries. This cDNA contains an open reading frame of 421 amino acids with a conserved DNA binding domain and several potential transactivating regions. During murine lung development, a single species of HFH-4-specific transcript (2.4 kb long) is first detected precisely at the start of the late pseudoglandular stage (embryonic day 14.5) and, by in situ hybridization, is specifically localized to the proximal pulmonary epithelium. The unique temporal and spatial pattern of HFH-4 gene expression in the developing lung defines this protein as a marker for the initiation of bronchial epithelial cell differentiation and suggests that it may play an important role in cell fate determination during lung development. In addition to expression in the pulmonary epithelium, RNA blot analysis reveals 2.4-kb HFH-4 transcripts in the testis and oviduct. By using mice with genetic defects in spermatogenesis, HFH-4 expression in the testis is found to be associated with the appearance of haploid germ cells and in situ hybridization studies indicate that HFH-4 expression is confined to stages I-VII of spermatogenesis. This pattern of HFH-4 gene expression during the early stages of differentiation of haploid germ cells suggests that HFH-4 may play a role in regulating stage-specific gene expression and cell-fate determination during lung development and in spermatogenesis.

Gene therapy for the respiratory manifestations of cystic fibrosis.

Cystic fibrosis (CF) is caused by mutations of the cystic fibrosis transmembrane conductance regulator (CFTR) gene. The major manifestations are on the airway epithelial surface, with purulent mucus, recurrent infections, chronic inflammation, and loss of lung function. Consequent to mutations in both parental genes, airway epithelial cells have insufficient CFTR function. Because this can be corrected in vitro by transfer of the normal CFTR gene into airway epithelial cells, it is reasonable to hypothesize that the respiratory manifestations of CF could be prevented by transfer of the normal human CFTR cDNA to the airway epithelium in vivo. Over the past 6 years, our laboratory has developed a strategy to accomplish this goal using a replication deficient E1-E3- recombinant adenovirus (Ad) serotype 5 vector containing the normal human CFTR cDNA (AdCFTR). Studies with experimental animals demonstrate that with administration of such a vector to the airways, the human CFTR cDNA could be transferred to the airway epithelium, with expression of the human CFTR cDNA for at least 6 weeks. Extensive preclinical studies in vitro and in vivo demonstrated that the risks to humans were sufficiently low to initiate a Phase I trial using the AdCFTR vector to treat the respiratory manifestations of CF in humans. Following approval by the National Heart, Lung, and Blood Institute Institutional Review Board, the National Institutes of Health Biosafety Committee, the National Institutes of Health Recombinant DNA Advisory Committee, and the Food and Drug Administration, we initiated the first human trial of gene therapy for CF on April 17, 1993. The clinical study is still ongoing, with safety and efficacy data being evaluated, but there is clear evidence that it is feasible to transfer and express the normal CFTR cDNA to the airway epithelium in vivo in individuals with CF.

Clinical supervision: a three-way mirror.

The principles and process of a model of clinical supervision for psychotherapy are presented and described through two cases. The model draws heavily from Bowen's family systems theory and Peplau's theory of interpersonal relations. The relationships in the client-family, client-therapist, and therapist-supervisor systems mirror each other, and changes in self-definition, and in the management of anxiety of persons in one system catalyzes changes in parallel systems. Two cases illustrate the model. In the first case, anxiety needed to be raised to levels optimal for learning. In the second case, no learning occurred until levels of anxiety were reduced.

In vivo suppression of injury-induced vascular smooth muscle cell accumulation using adenovirus-mediated transfer of the herpes simplex virus thymidine kinase gene.

Restenosis, a process characterized in part by excessive smooth muscle cell (SMC) proliferation in areas of vascular injury, occurs in up to 50% of patients undergoing balloon angioplasty. In an effort to develop a treatment strategy for restenosis, we constructed a replication-deficient recombinant adenovirus (AdMLP.HSTK) containing the herpes simplex virus thymidine kinase gene (HSV tk). This viral gene product phosphorylates the prodrug ganciclovir to form a nucleoside analog that inhibits DNA synthesis. Cultured primary rat SMCs infected with AdMLP.HSTK were completely growth-inhibited by incubation in ganciclovir-containing medium. In addition, when only a portion of the SMC population received the HSV tk transgene, an inhibitory effect on neighboring SMCs was evident. Evaluation of this strategy in vivo using a rat carotid balloon injury model demonstrated that local infection of injured arteries with AdMLP.-HSTK followed by 2 weeks of systemic ganciclovir treatment significantly (P < 0.01) reduced injury-induced SMC accumulation. In contrast, there was no suppression of injury-induced SMC accumulation in animals infected with AdMLP.HSTK but not receiving ganciclovir or in those animals infected with a control adenovirus and either treated or not treated with ganciclovir. These results demonstrate the potential utility of adenovirus-mediated gene transfer for treatment of restenosis after balloon injury.

Suppression of in vivo tumorigenicity of human lung cancer cells by retrovirus-mediated transfer of the human tumor necrosis factor-alpha cDNA.

The clinical use of tumor necrosis factor-alpha (TNF) is constrained by tumor cell resistance and systemic toxicity. Based on observations with murine tumors, we hypothesized that induction of local TNF production by the tumor may suppress growth of human cancer cells. To evaluate this, a human TNF cDNA was transferred to human lung cancer cell lines in vitro using a retrovirus vector to produce TNF cDNA-modified cell lines secreting TNF. In vitro cell growth was similar for parental and modified cells. All cells were resistant to TNF. The in vivo tumorigenicity of parental and modified cells was compared in nude mice. Animals injected subcutaneously with parental cells uniformly developed tumors. Tumor growth was markedly less for all modified cells, and this suppression of tumor development was reversed by anti-TNF antibody administration. Animals injected with a mixture of 50% modified and 50% parental cells or parental cell tumors injected with modified cells had decreased tumor growth, demonstrating that modified cells could suppress tumorigenicity. These data suggest that TNF can induce antitumor defenses to suppress in vivo human tumor cell growth and provide a rationale for transferring the human TNF cDNA directly to malignant cells for the therapy of human lung cancer.

Administration of an adenovirus containing the human CFTR cDNA to the respiratory tract of individuals with cystic fibrosis.

We have administered a recombinant adenovirus vector (AdCFTR) containing the normal human CFTR cDNA to the nasal and bronchial epithelium of four individuals with cystic fibrosis (CF). We show that this vector can express the CFTR cDNA in the CF respiratory epithelium in vivo. With doses up to 2 x 10(9) pfu, there was no recombination/complementation or shedding of the vector or rise of neutralizing antibody titres. At 2 x 10(9) pfu, a transient systemic and pulmonary syndrome was observed, possibly mediated by interleukin-6. Follow-up at 6-12 months demonstrated no long term adverse effects. Thus, it is feasible to use an adenovirus vector to transfer and express the CFTR cDNA in the respiratory epithelium of individuals with CF. Correction of the CF phenotype of the airway epithelium might be achieved with this strategy.