Alpha-1 antitrypsin limits neutrophil extracellular trap disruption of airway epithelial barrier function.

Neutrophil extracellular traps contribute to lung injury in cystic fibrosis and asthma, but the mechanisms are poorly understood. We sought to understand the impact of human NETs on barrier function in primary human bronchial epithelial and a human airway epithelial cell line. We demonstrate that NETs disrupt airway epithelial barrier function by decreasing transepithelial electrical resistance and increasing paracellular flux, partially by NET-induced airway cell apoptosis. NETs selectively impact the expression of tight junction genes claudins 4, 8 and 11. Bronchial epithelia exposed to NETs demonstrate visible gaps in E-cadherin staining, a decrease in full-length E-cadherin protein and the appearance of cleaved E-cadherin peptides. Pretreatment of NETs with alpha-1 antitrypsin (A1AT) inhibits NET serine protease activity, limits E-cadherin cleavage, decreases bronchial cell apoptosis and preserves epithelial integrity. In conclusion, NETs disrupt human airway epithelial barrier function through bronchial cell death and degradation of E-cadherin, which are limited by exogenous A1AT.

Altered stability of pulmonary surfactant in SP-C-deficient mice.

The surfactant protein C (SP-C) gene encodes an extremely hydrophobic, 4-kDa peptide produced by alveolar epithelial cells in the lung. To discern the role of SP-C in lung function, SP-C-deficient (-/-) mice were produced. The SP-C (-/-) mice were viable at birth and grew normally to adulthood without apparent pulmonary abnormalities. SP-C mRNA was not detected in the lungs of SP-C (-/-) mice, nor was mature SP-C protein detected by Western blot of alveolar lavage from SP-C (-/-) mice. The levels of the other surfactant proteins (A, B, D) in alveolar lavage were comparable to those in wild-type mice. Surfactant pool sizes, surfactant synthesis, and lung morphology were similar in SP-C (-/-) and SP-C (+/+) mice. Lamellar bodies were present in SP-C (-/-) type II cells, and tubular myelin was present in the alveolar lumen. Lung mechanics studies demonstrated abnormalities in lung hysteresivity (a term used to reflect the mechanical coupling between energy dissipative forces and tissue-elastic properties) at low, positive-end, expiratory pressures. The stability of captive bubbles with surfactant from the SP-C (-/-) mice was decreased significantly, indicating that SP-C plays a role in the stabilization of surfactant at low lung volumes, a condition that may accompany respiratory distress syndrome in infants and adults.

Secretion of surfactant protein C, an integral membrane protein, requires the N-terminal propeptide.

Proteolytic processing of surfactant protein C (SP-C) proprotein in multivesicular bodies of alveolar type II cells results in a 35-residue mature peptide, consisting of a transmembrane domain and a 10-residue extramembrane domain. SP-C mature peptide is stored in lamellar bodies (a lysosomal-like organelle) and secreted with surfactant phospholipids into the alveolar space. This study was designed to identify the peptide domain of SP-C required for sorting and secretion of this integral membrane peptide. Deletion analyses in transiently transfected PC12 cells and isolated mouse type II cells suggested the extramembrane domain of mature SP-C was cytosolic and sufficient for sorting to the regulated secretory pathway. Intratracheal injection of adenovirus encoding SP-C mature peptide resulted in secretion into the alveolar space of wild type mice but not SP-C (-/-) mice. SP-C secretion in null mice was restored by the addition of the N-terminal propeptide. The cytosolic domain, consisting of the N- terminal propeptide and extramembrane domain of mature SP-C peptide, supported secretion of the transmembrane domain of platelet-derived growth factor receptor. Collectively, these studies indicate that the N-terminal propeptide of SP-C is required for intracellular sorting and secretion of SP-C.

The role of homodimers in surfactant protein B function in vivo.

Surfactant protein B (SP-B) is detected in the airways as a sulfhydryl-dependent dimer (M(r) approximately 16,000). To test the hypothesis that formation of homodimers is critical for SP-B function, the cysteine residue reported to be involved in SP-B dimerization was mutated to serine (Cys(248) --> Ser) and the mutated protein was targeted to the distal respiratory epithelium of transgenic mice. Transgenic lines which demonstrated appropriate processing, sorting, and secretion of human SP-B monomer were crossed with SP-B +/- mice to achieve expression of human monomer in the absence of endogenous SP-B dimer (hSP-B(mon), mSP-B-/-). In two of three transgenic lines, hSP-B(mon), mSP-B-/- mice had normal lung structure, complete processing of SP-C proprotein, well formed lamellar bodies, and normal longevity. Pulmonary function studies revealed an altered hysteresis curve for hSP-B(mon), mSP-B-/- mice relative to wild type mice. Large aggregate surfactant fractions from hSP-B(mon), mSP-B-/- mice resulted in higher minimum surface tension in vitro compared with surfactant from wild type mice. Surfactant lipids supplemented with 2% hSP-B monomer resulted in slower adsorption and higher surface tension than surfactant with 2% hSP-B dimer. Taken together, these data indicate a role for SP-B dimer in surface tension reduction in the alveolus.

Ablation of a critical surfactant protein B intramolecular disulfide bond in transgenic mice.

The 79-amino acid, mature SP-B peptide contains three intramolecular disulfide bonds shared by all saposin-like proteins. This study tested the hypothesis that the disulfide bond formed between cysteine residues 35 and 46 (residues 235 and 246 of the SP-B proprotein) is essential for proper function of SP-B. To test the role of this bridge in SP-B function in vivo, a construct was generated in which cysteine residues 235 and 246 of the human SP-B proprotein were mutated to serine and cloned under the control of the 3.7-kilobase hSP-C promoter (hSP-B(C235S/C246S)). In two transgenic mouse lines, expression of the mutant peptide in the wild-type murine SP-B background was invariably lethal in the neonatal period. In four additional lines, survival was inversely related to the level of transgene expression. To test the ability of the mutant peptide to functionally replace the wild-type protein, transgenic mice were crossed into the SP-B null background. No animals that expressed hSP-B(C235S/C246S) in the murine SP-B-/- background survived the neonatal period. hSP-B(C235S/C246S) proprotein accumulated in the endoplasmic reticulum and was not processed to the mature, biologically active peptide. The results of these studies demonstrate that the intramolecular bridge between residues 235 and 246 is critical for intracellular trafficking of SP-B and suggest that overexpression of mutant SP-B in the wild-type background may be lethal.

Surfactant protein B (SP-B) -/- mice are rescued by restoration of SP-B expression in alveolar type II cells but not Clara cells.

Surfactant protein B (SP-B) mRNA and protein are restricted to alveolar Type II and Clara cells in the respiratory epithelium. In order to investigate the function of SP-B in these distinct cell types, transgenic mice were generated in which SP-B expression was selectively restored in Type II cells or Clara cells of SP-B -/- mice. The 4.8-kilobase murine SP-C promoter was used to generate 3 transgenic lines which expressed human SP-B in Type II cells (mSP-C/hSP-B). Likewise, the 2.3-kilobase murine CCSP promoter was used to generate two transgenic lines which expressed human SP-B in Clara cells (mCCSP/hSP-B). mSP-C/hSP-B and mCCSP/hSP-B transgenic mice were subsequently bred to SP-B +/- mice in order to selectively express SP-B in Type II cells or Clara cells of SP-B -/- mice. Selective restoration of SP-B expression in Type II cells completely rescued the neonatal lethal phenotype in SP-B -/- mice. Expression of SP-B in some, but not all Type II cells of SP-B -/- mice, allowed postnatal survival, but resulted in significantly altered lung architecture and function. Selective restoration of SP-B expression in Clara cells of SP-B -/- mice resulted in respiratory dysfunction and invariable neonatal death, related to the complete absence of mature SP-B peptide in these mice. These results indicate that expression and processing of the SP-B proprotein to the mature peptide in Type II cells is absolutely required for lung function in vivo and that SP-B expression in Clara cells cannot substitute for this function.