Showing papers by "Joseph M. Pilewski published in 2008"

IL-22 mediates mucosal host defense against Gram-negative bacterial pneumonia

Abstract: Emerging evidence supports the concept that T helper type 17 (TH17) cells, in addition to mediating autoimmunity, have key roles in mucosal immunity against extracellular pathogens. Interleukin-22 (IL-22) and IL-17A are both effector cytokines produced by the TH17 lineage, and both were crucial for maintaining local control of the Gram-negative pulmonary pathogen, Klebsiella pneumoniae. Although both cytokines regulated CXC chemokines and granulocyte colony–stimulating factor production in the lung, only IL-22 increased lung epithelial cell proliferation and increased transepithelial resistance to injury. These data support the concept that the TH17 cell lineage and its effector molecules have evolved to effect host defense against extracellular pathogens at mucosal sites.

Egr-1 Regulates Autophagy in Cigarette Smoke-Induced Chronic Obstructive Pulmonary Disease

Abstract: Background: Chronic obstructive pulmonary disease (COPD) is a progressive lung disease characterized by abnormal cellular responses to cigarette smoke, resulting in tissue destruction and airflow limitation. Autophagy is a degradative process involving lysosomal turnover of cellular components, though its role in human diseases remains unclear. Methodology and Principal Findings: Increased autophagy was observed in lung tissue from COPD patients, as indicated by electron microscopic analysis, as well as by increased activation of autophagic proteins (microtubule-associated protein-1 light chain-3B, LC3B, Atg4, Atg5/12, Atg7). Cigarette smoke extract (CSE) is an established model for studying the effects of cigarette smoke exposure in vitro. In human pulmonary epithelial cells, exposure to CSE or histone deacetylase (HDAC) inhibitor rapidly induced autophagy. CSE decreased HDAC activity, resulting in increased binding of early growth response-1 (Egr-1) and E2F factors to the autophagy gene LC3B promoter, and increased LC3B expression. Knockdown of E2F-4 or Egr-1 inhibited CSEinduced LC3B expression. Knockdown of Egr-1 also inhibited the expression of Atg4B, a critical factor for LC3B conversion. Inhibition of autophagy by LC3B-knockdown protected epithelial cells from CSE-induced apoptosis. Egr-1 2/2 mice, which displayed basal airspace enlargement, resisted cigarette-smoke induced autophagy, apoptosis, and emphysema. Conclusions: We demonstrate a critical role for Egr-1 in promoting autophagy and apoptosis in response to cigarette smoke exposure in vitro and in vivo. The induction of autophagy at early stages of COPD progression suggests novel therapeutic targets for the treatment of cigarette smoke induced lung injury.

Autoantibodies in patients with chronic obstructive pulmonary disease.

Abstract: Rationale: Adaptive immune responses are present in patients with chronic obstructive pulmonary disease (COPD), and it has been postulated that these processes could be autoreactive. Objectives: To ascertain if humoral autoimmunity could play a role in COPD pathogenesis. Methods: Circulating IgG autoantibodies were detected by immunofluorescence and immunoprecipitation. Immunohistochemistry and immunofluorescence were used to evaluate intrapulmonary IgG and complement (C3) deposition in human lung explants. Autoantibody pathogenicity was also investigated with an antibody-dependent cell-mediated cytotoxicity assay. Measurements and Main Results: The prevalence of anti–HEp-2 epithelial cell autoantibodies in 47 smokers/former smokers with COPD (GOLD stages 1–4) was greater than among 8 subjects with a smoking history but normal spirometry and 21 healthy control subjects who had never smoked (68 vs. 13 vs. 10%, respectively; P < 0.0001). Antibodies against primary pulmonary epithelial cells were found in 12 of 12 patients with COPD versus 3 of 12 never-smoked control subjects (P < 0.001). Self-antigens immunoprecipitated from 34 of 35 (97%) of COPD plasmas (vs. 0/12 never-smoked controls). Antibodies against a particular 130-kD autoantigen (n = 7) were associated with decreased body mass index (23.2 ± 2.1 vs. 29.5 ± 1.0 kg/m2, P = 0.007). Intrapulmonary immune complexes were present in six of six and C3 was seen in five of six COPD lung explants, unlike zero of six and one of six normals, respectively. Cytotoxicity of pulmonary epithelial cells by allogeneic mononuclear cells also increased 46% after incubation with COPD plasmas (n = 10), compared with identical treatments with eight normal specimens (P = 0.03). Conclusions: IgG autoantibodies with avidity for pulmonary epithelium, and the potential to mediate cytotoxicity, are prevalent in patients with COPD. Autoreactive adaptive immune responses may be important in the etiology of this disease.

Defining an inhibitory domain in the gamma subunit of the epithelial sodium channel.

Abstract: Proteases activate the epithelial sodium channel (ENaC) by cleaving the large extracellular domains of the α- and γ-subunits and releasing peptides with inhibitory properties. Furin and prostasin activate mouse ENaC by cleaving the γ-subunit at sites flanking a 43 residue inhibitory tract (γE144-K186). To determine whether there is a minimal inhibitory region within this 43 residue tract, we generated serial deletions in the inhibitory tract of the γ-subunit in channels resistant to cleavage by furin and prostasin. We found that partial or complete deletion of a short segment in the γ-subunit, R158-N171, enhanced channel activity. Synthetic peptides overlapping this segment in the γ-subunit further identified a key 11-mer tract, R158-F168 (RFLNLIPLLVF), which inhibited wild-type ENaC expressed in Xenopus laevis oocytes, and endogenous channels in mpkCCD cells and human airway epithelia. Further studies with amino acid-substituted peptides defined residues that are required for inhibition in this key 11-mer tract. The presence of the native γ inhibitory tract in ENaC weakened the intrinsic binding constant of the 11-mer peptide inhibitor, suggesting that the γ inhibitory tract and the 11-mer peptide interact at overlapping sites within the channel.

CX3CL1 up-regulation is associated with recruitment of CX3CR1+ mononuclear phagocytes and T lymphocytes in the lungs during cigarette smoke-induced emphysema.

Abstract: CX3CR1 is expressed on monocytes, dendritic cells, macrophages, subsets of T lymphocytes, and natural killer cells and functions in diverse capacities such as leukocyte adhesion, migration, and cell survival on ligand binding. Expression of the CX3CL1 gene, whose expression product is the sole ligand for CX3CR1, is up-regulated in human lungs with chronic cigarette smoke-induced obstructive lung disease. At present, it is unknown whether CX3CL1 up-regulation is associated with the recruitment and accumulation of immune cells that express CX3CR1. We show that mice chronically exposed to cigarette smoke up-regulate CX3CL1 gene expression, which is associated with an influx of CX3CR1+ cells in the lungs. The increase in CX3CR1+ cells is primarily comprised of macrophages and T lymphocytes and is associated with the development of emphysema. In alveolar macrophages, cigarette smoke exposure increased the expression of both CX3CR1 and CX3CL1 genes. The inducibility of CX3CR1 expression was not solely dependent on a chronic stimulus because lipopolysaccharide up-regulated CX3CR1 in RAW264.7 cells in vitro and in mononuclear phagocytes in vivo. Our findings suggest a mechanism by which macrophages amplify and promote CX3CR1+ cell accumulation within the lungs during both acute and chronic inflammatory stress. We suggest that one function of the CX3CR1-CX3CL1 pathway is to recruit and sustain divergent immune cell populations implicated in the pathogenesis of cigarette smoke-induced emphysema.

Prostasin expression is regulated by airway surface liquid volume and is increased in cystic fibrosis

Abstract: Airway surface liquid (ASL) absorption is initiated by Na+ entry via epithelial Na+ channels (ENaC), which establishes an osmotic gradient that drives fluid from the luminal to serosal airway surfa...

Chaperone displacement from mutant cystic fibrosis transmembrane conductance regulator restores its function in human airway epithelia

Abstract: Mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene cause cystic fibrosis (CF). The most common mutation, DeltaF508, omits the phenylalanine residue at position 508 in the first nucleotide binding domain (NBD1) of CFTR. The mutant protein is retained in the endoplasmic reticulum and degraded by the ubiquitin-proteasome system. We demonstrate that expression of NBD1 plus the regulatory domain (RD) of DeltaF508 CFTR (DeltaFRD) restores the biogenesis of mature DeltaF508 CFTR protein. In addition, DeltaFRD elicited a cAMP-stimulated anion conductance response in primary human bronchial epithelial (HBE) cells isolated from homozygous DeltaF508 CF patients. A protein transduction domain (PTD) could efficiently transduce (approximately 90%) airway epithelial cells. When fused to a PTD, direct addition of the DeltaFRD peptide conferred a dose-dependent, cAMP-stimulated anion efflux to DeltaF508 HBE cells. Hsp70 and Hsp90 associated equally with WT and DeltaF508 CFTR, whereas nearly twice as much of the Hsp90 cochaperone, Aha1, associated with DeltaF508 CFTR. Expression of DeltaFRD produced a dose-dependent removal of Aha1 from DeltaF508 CFTR that correlated with its functional rescue. These findings indicate that disruption of the excessive association of the cochaperone, Aha1, with DeltaF508 CFTR is associated with the correction of its maturation, trafficking and regulated anion channel activity in human airway epithelial cells. Thus, PTD-mediated DeltaFRD fragment delivery may provide a therapy for CF.

Post-transplant Colonization With Non-Aspergillus Molds and Risk of Development of Invasive Fungal Disease in Lung Transplant Recipients

Abstract: Background: The clinical significance and risk of progression to invasive disease of the non-Aspergillus molds from the bronchoalveolar lavage (BAL) of lung transplant (LTx) recipients are not known. Methods: We reviewed the medical records from March 1996 to March 2006 of all LTx recipients whose BAL culture grew non-Aspergillus mold. The clinical characteristics, administration of prophylaxis, and outcomes were recorded. Results: Eighty-five non-Aspergillus molds were isolated from the BAL of 75 patients. Of these LTx recipients, 14.5% had a BAL with non-Aspergillus mold. Emphysema was the most common underlying diagnosis for transplantation (41.3%) and the most common isolate was Cladosporium. Isolation of a non-Aspergillus mold occurred at a median of 415 days after LTx. Prophylaxis with an anti-mold agent was noted in 44.7% (38 of 85) of the isolates. Median follow-up was 765 days. There were no cases of proven invasive fungal infection up to 1 year after isolation of the mold; and only 1 case of probable zygomycosis. Conclusions: Isolation of non-Aspergillus molds in the BAL of LTx recipients may not be associated with the development of invasive disease, irrespective of anti-fungal prophylaxis. These results suggest that initiation of targeted anti-fungal prophylaxis after isolation of non-Aspergillus molds from BAL may not be warranted.

Postdeposition Dispersion of Aerosol Medications Using Surfactant Carriers

Abstract: Inhaled aerosol drugs provide a means of directly treating the lungs; however, aerosol deposition and drug distribution can be nonuniform, especially in obstructive lung disease. We hypothesize that surfactant-based aerosol carriers will disperse medications over airway surfaces after deposition through surface tension driven flows, increasing dose uniformity and improving drug distribution into underventilated regions. We considered saline and surfactant aerosol delivery via cannula onto several model airway surfaces including porcine gastric mucus (PGM) and both cystic fibrosis (CF) and non-CF human bronchial epithelial cells (HBEs). Fluorescent dye and microspheres (d = 100 nm, 1 μm) were used to qualitatively and quantitatively assess postdeposition dispersion. Aerosol volume median diameters were in the 1–4 μm range. The tested surfactants included sodium dodecyl sulfate (SDS), cetyl trimethyl ammonium bromide (CTAB), tyloxapol, and calfactant. All surfactants tested on PGM (tyloxapol, calfactant, SD...

Anti-human leukocyte antigen antibodies, vascular C4d deposition and increased soluble c4d in broncho-alveolar lavage of lung allografts.

Abstract: BACKGROUND The hallmark of humoral rejection is the presence of subendothelial C4d in the allograft. A simultaneous determination of vascular C4d with soluble C4d in broncho-alveolar lavage fluid (BAL) and circulating anti-human leukocyte antigen (HLA) antibodies (HLA-Ab) has not been reported in lung transplantation. METHODS Forty-two consecutive lung-transplant patients were included in this cross-sectional study. The presence and specificity of HLA-Ab was determined at the same frequency with transbronchial biopsies. Soluble C4d levels were measured by enzyme-linked immunosorbent assay in all 42 patients. In a subgroup of 32 patients with available timely matched paraffin-embedded tissue sections, the vascular C4d deposition was also assessed. RESULTS The presence of HLA-Ab in 16 patients was associated with biopsy-proven acute rejection (10/16 vs. 3/16, P 0.5 microg/mL) elevated in patients with HLA-Ab and vascular C4d and was moderately (0.2 microg/mL) increased in patients with antibodies but C4d-negative. In contrast, only a slight elevation of soluble C4d (<0.1 microg/mL) was detected in patients without HLA-specific antibodies. CONCLUSIONS The association of HLA-specific antibodies with vascular C4d deposition and soluble C4d in BAL, in addition to the reduced pulmonary function, might constitute a diagnostic triad for antibody-mediated rejection in lung transplant patients.

Surfactant-based antimicrobial solution for inhalation

Abstract: A surfactant can be added, safely and effectively, to a drug solution containing any antimicrobial agent, such as an antibiotic like tobramycin, that is suitable for administration to the lungs via inhalation. Thus, when an aerosolized drug solution includes surfactant, Marangoni flows cause the drug particles, once deposited in the lungs, to spread over a wider surface area, thereby ensuring greater antimicrobial efficacy. A solution that contains, for example, an antibiotic and tyloxapol or another surfactant providing a similar surface tension to the composition is optimally delivered by the functional combination of a breath-actuated nebulizer and a high-flow compressor.