Susan H. Guttentag

Cxcr2 and Cxcl5 regulate the IL-17/G-CSF axis and neutrophil homeostasis in mice

Neutrophils are essential for maintaining innate immune surveillance under normal conditions, but also represent a major contributor to tissue damage during inflammation. Neutrophil homeostasis is therefore tightly regulated. Cxcr2 plays a critical role in neutrophil homeostasis, as Cxcr2(-/-) mice demonstrate mild neutrophilia and severe neutrophil hyperplasia in the bone marrow. The mechanisms underlying these phenotypes, however, are unclear. We report here that Cxcr2 on murine neutrophils inhibits the IL-17A/G-CSF axis that regulates neutrophil homeostasis. Furthermore, enterocyte-derived Cxcl5 in the gut regulates IL-17/G-CSF levels and contributes to Cxcr2-dependent neutrophil homeostasis. Conversely, G-CSF was required for Cxcl5-dependent regulation of neutrophil homeostasis, and inhibition of IL-17A reduced plasma G-CSF concentrations and marrow neutrophil numbers in both Cxcl5(-/-) and Cxcr2(-/-) mice. Cxcr2(-/-) mice constitutively expressed IL-17A and showed increased numbers of IL-17A-producing cells in the lung, terminal ileum, and spleen. Most IL-17-producing splenocytes were responsive to IL-1β plus IL-23 in vitro. Depletion of commensal microbes by antibiotic treatment in Cxcr2(-/-) mice markedly decreased IL-17A and G-CSF expression, neutrophilia, and marrow myeloid hyperplasia. These data suggest a critical role for Cxcr2, Cxcl5, and commensal bacteria in regulation of the IL-17/G-CSF axis and neutrophil homeostasis at mucosal sites and have implications for the development of treatments for pathologies resulting from either excessive or ineffective neutrophil responses.

IL-17A and TNF-α exert synergistic effects on expression of CXCL5 by alveolar type II cells in vivo and in vitro.

CXCL5, a member of the CXC family of chemokines, contributes to neutrophil recruitment during lung inflammation, but its regulation is poorly understood. Because the T cell-derived cytokine IL-17A enhances host defense by triggering production of chemokines, particularly in combination with TNF-α, we hypothesized that IL-17A would enhance TNF-α–induced expression of CXCL5. Intratracheal coadministration of IL-17A and TNF-α in mice induced production of CXCL1, CXCL2, and CXCL5, which was associated with increased neutrophil influx in the lung at 8 and 24 h. The synergistic effects of TNF-α and IL17A were greatly attenuated in Cxcl5−/− mice at 24 h, but not 8 h, after exposure, a time when CXCL5 expression was at its peak in wild-type mice. Bone marrow chimeras produced using Cxcl5−/− donors and recipients demonstrated that lung-resident cells were the source of CXCL5. Using differentiated alveolar epithelial type II (ATII) cells derived from human fetal lung, we found that IL-17A enhanced TNF-α–induced CXCL5 transcription and stabilized TNF-α–induced CXCL5 transcripts. Whereas expression of CXCL5 required activation of NF-κB, IL-17A did not increase TNF-α–induced NF-κB activation. Apical costimulation of IL-17A and TNF-α provoked apical secretion of CXCL5 by human ATII cells in a transwell system, whereas basolateral costimulation led to both apical and basolateral secretion of CXCL5. The observation that human ATII cells secrete CXCL5 in a polarized fashion may represent a mechanism to recruit neutrophils in host defense in a fashion that discriminates the site of initial injury.

Prolonged Survival in Hereditary Surfactant Protein B (SP-B) Deficiency Associated with a Novel Splicing Mutation

Hereditary surfactant protein B (SP-B) deficiency has been lethal in the first year of life without lung transplantation. We tested the hypothesis that SP-B gene mutations may result in milder phenotypes by investigating the mechanisms for lung disease in two children with less severe symptoms than have been previously observed in SP-B deficiency. Immunostaining patterns for pulmonary surfactant proteins were consistent with SP-B deficiency in both children. DNA sequence analysis indicated that both children were homozygous for a mutation in exon 5 that created an alternative splice site. Reverse transcriptase PCR and sequence analysis confirmed use of this splice site, which resulted in a frameshift and a premature termination codon in exon 7. The predominant reverse transcriptase PCR product, however, lacked exon 7, which restored the reading frame but would not allow translation of the exons that encode mature SP-B. Western blot analysis detected reduced amounts of mature SP-B as well as an aberrant SP-B proprotein that corresponded to the size expected from translation of the abnormal transcript. We conclude that a novel splicing mutation was the cause of lung disease in these children and that hereditary SP-B deficiency can be the cause of lung disease in older children.

Surfactant protein B processing in human fetal lung.

Surfactant protein B (SP-B8), an 8-kDa hydrophobic protein essential for surfactant and normal lung function, is produced from the intracellular processing of preproSP-B. To characterize SP-B processing in human type 2 cells, we used human fetal lung in explant culture and polyclonal antibodies to human SP-B8(Phe201-Met279) and to specific epitopes within the NH2- and COOH-terminal propeptide domains (Ser145-Leu160, Gln186-Gln200, and Gly284-Ser304). Western blot analysis revealed a novel intermediate at ∼9 kDa, representing mature SP-B8, with a residual NH2-terminal peptide of ∼10 amino acids. Pulse-chase studies showed a precursor-product relationship between the 9- and 8-kDa forms. During differentiation of type 2 cells in explant culture, the rate of proSP-B conversion to 25-kDa intermediate remained constant, whereas the rate of 25-kDa intermediate conversion to SP-B8increased, resulting in a net increase in tissue SP-B8. Dexamethasone did not affect the rate of proSP-B processing but markedly enhanced the rate of SP-B8 accumulation. We conclude that NH2-terminal propeptide cleavage of proSP-B is a multistep process and that more distal processing events are rate limiting and both developmentally and hormonally regulated.Surfactant protein B (SP-B8), an 8-kDa hydrophobic protein essential for surfactant and normal lung function, is produced from the intracellular processing of preproSP-B. To characterize SP-B processing in human type 2 cells, we used human fetal lung in explant culture and polyclonal antibodies to human SP-B8 (Phe201-Met279) and to specific epitopes within the NH2- and COOH-terminal propeptide domains (Ser145-Leu160, Gln186-Gln200, and Gly284-Ser304). Western blot analysis revealed a novel intermediate at approximately 9 kDa, representing mature SP-B8, with a residual NH2-terminal peptide of approximately 10 amino acids. Pulse-chase studies showed a precursor-product relationship between the 9- and 8-kDa forms. During differentiation of type 2 cells in explant culture, the rate of proSP-B conversion to 25-kDa intermediate remained constant, whereas the rate of 25-kDa intermediate conversion to SP-B8 increased, resulting in a net increase in tissue SP-B8. Dexamethasone did not affect the rate of proSP-B processing but markedly enhanced the rate of SP-B8 accumulation. We conclude that NH2-terminal propeptide cleavage of proSP-B is a multistep process and that more distal processing events are rate limiting and both developmentally and hormonally regulated.

Notch1 and Jagged1 expression by the developing pulmonary vasculature

The molecular mechanisms of pulmonary vascular development are poorly understood. Cell‐specific developmental pathways are influenced by cell–cell signaling. Notch signaling molecules are highly conserved receptors active in many cell‐fate determination systems. Recent observations of Notch molecules and a Notch ligand, Jagged1, suggest their importance in vascular morphogenesis, and particularly pulmonary vascular development. We performed a systematic evaluation of Notch1/Jagged1 gene and protein expression in the developing mouse lung from embryonic day 11 until adulthood by using quantitative PCR, immunofluorescence, and electron microscopic analysis. mRNA transcripts for Notch1‐4 and Jagged1 increased progressively from early to later lung development, accompanied by a simultaneous rise in endothelial cell‐specific gene expression, a pattern not seen in other organs. Notch1 mRNA was identified on both epithelial and mesenchymal structures of the embryonic lung. Immunofluorescence staining revealed the progressive acquisition of Notch1 and Jagged1 proteins by the emerging endothelium. Notch1 and Jagged1 were seen initially on well‐formed, larger vessels within the embryonic lung bud and progressively on finer vascular networks. Each was also expressed on surrounding nonvascular structures. The localization of Notch1 and Jagged1 on endothelial cell surface membranes within the alveolar microvasculature was confirmed by immuno‐electron microscopy. These temporal and spatial patterns in Notch1/Jagged1 gene and protein expression suggest multiple potential paths of cell–cell signaling during lung development and vascular morphogenesis.

Surfactant proteins in pulmonary alveolar proteinosis in adults

Pulmonary alveolar proteinosis (PAP) is a rare disorder characterised histologically by an intra-alveolar accumulation of fine granular eosinophilic and periodic acid-Schiff positive material. In a retrospective study, the composition of the intra-alveolarly accumulated material of adult patients with PAP was analysed by means of immunohistochemistry and Western blotting. In patients with PAP, the current authors found an intra-alveolar accumulation of surfactant protein (SP)-A, precursors of SP-B, SP-B, variable amounts of mono-, di-, and oligomeric SP-C forms, as well as SP-D. Only in one patient was a precursor of SP-C detected. By means of immuno-electron microscopy, the current authors identified not only transport vesicles labelled for precursors of SP-B and SP-C, but also transport vesicles containing either precursors of SP-B or SP-C in type-II pneumocytes in normal human lungs. It is concluded that pulmonary alveolar proteinosis in adults is characterised by an intra-alveolar accumulation of surfactant protein A, precursors of surfactant protein B, and surfactant proteins B, C and D. The current data provide evidence that not only an impairment of surfactant clearance by alveolar macrophages, but also an abnormal secretion of transport vesicles containing precursors of surfactant protein B (but not surfactant protein C) and an insufficient palmitoylation of surfactant protein C, which may lead to the formation of di- and oligomeric surfactant protein C forms, play a role in the pathogenesis of pulmonary alveolar proteinosis.

TGF-β1 inhibits surfactant component expression and epithelial cell maturation in cultured human fetal lung

Transforming growth factor-β1 (TGF-β1) is a multifunctional cytokine shown to play a critical role in organ morphogenesis, development, growth regulation, cellular differentiation, gene expression, and tissue remodeling after injury. We examined the effect of exogenously administered TGF-β1 on the expression of surfactant proteins (SPs) and lipids, fatty acid synthetase, and ultrastructural morphology in human fetal lung cultured for 5 days with and without dexamethasone (10 nM). Expression of the type II cell-specific marker surfactant proprotein C (proSP-C), studied by [35S]Met incorporation and immunoprecipitation, increased sevenfold with dexamethasone treatment. TGF-β1 (0.1-100 ng/ml) in the presence of dexamethasone inhibited 21-kDa proSP-C expression in a dose-dependent manner (maximal inhibition 31% of control level at 100 ng/ml). There was no change in [35S]Met incorporation into total protein in any of the treatment groups vs. the control group. In immunoblotting experiments, TGF-β1 blocked culture-induced accumulation of SP-A and SP-B. Under the same conditions, TGF-β1 reduced mRNA content for SP-A, SP-B, and SP-C to 20, 38, and 41%, respectively, of matched control groups but did not affect levels of β-actin mRNA. SP transcription rates after 24 h of exposure to TGF-β1 were reduced to a similar extent (20-50% of control level). In both control and dexamethasone-treated explants, TGF-β1 (10 ng/ml) also decreased fatty acid synthetase mRNA, protein, and enzyme activity and the rate of [3H]choline incorporation into phosphatidylcholine. By electron microscopy, well-differentiated type II cells lining potential air spaces were present in explants cultured with dexamethasone, whereas exposure to TGF-β1 with or without dexamethasone resulted in epithelial cells lacking lamellar bodies. We conclude that exogenous TGF-β1 disrupts culture-induced maturation of fetal lung epithelial cells and inhibits expression of surfactant components through effects on gene transcription.

Expression and Glucocorticoid Regulation of Surfactant Protein C in Human Fetal Lung

The hydrophobic surfactant protein C (SP-C) is known to modulate the biophysical properties of surfactant phospholipid. Although SP-C mRNA has been demonstrated in human fetal lung, there is limited information regarding developmental expression and processing of proSP-C protein. Two epitopespecific human proSP-C antisera, anti-hCPROSP-C(His59-Ser72) and anti-hCTERMSP-C (Gly162-Gly175), were generated to complement previously produced anti-NPROSP-C(Met10-Gln23) for the study of proSP-C expression in human fetal lung. Western blotting and immunocytochemistry detected expression of proSP-C protein by 12-16 wk of gestation. ProSP-C immunoreactivity of preculture lung, limited to expression of proSP-C21 in airway epithelial cells, was markedly enhanced by culture of lung explants in dexamethasone. To examine synthesis of proSP-C, homogenates from explants were labeled with35 S-Met/Cys for 0.5-4 h. Immunoprecipitation with anti-NPROSP-C detected 35S-proSP-C21 by 30 min and, after 2 h of labeling, there was a 15-fold increase in 35S-proSP-C21 in dexamethasone-treated lungs versus controls. Synthesis of proSP-C21 was followed by the appearance of a 24-kD form and smaller processing intermediates including 6-10-kD forms. Posttranslational processing of proSP-C21 was not observed in control explants. SP-C6-10 were not recognized by either anti-CPROSP-C or anti-hCTERMSP-C. These results indicate that low level expression of proSP-C protein first occurs in epithelial cells early in the second trimester and that expression can be enhanced by dexamethasone. Initial posttranslational processing of human proSP-C involves modification of proSP-C21 to SP-C24 and subsequent proteolysis of C-terminal propeptide domains. We speculate that absence of low Mr intermediates in unstimulated second trimester fetal lung tissue reflects developmental and glucocorticoid dependent regulation of proSP-C21 synthesis and posttranslational processing.

Differences in N-linked glycosylation between human surfactant protein-B variants of the C or T allele at the single-nucleotide polymorphism at position 1580: implications for disease

Human surfactant protein-B (SP-B), a hydrophobic protein, is essential for normal lung function. SP-B is expressed and secreted by specific lung cell types, i.e. alveolar type II and Clara cells, of the respiratory epithelium. The SP-B precursor (42 kDa) undergoes post-translational processing to generate an 8 kDa mature SP-B. A single-nucleotide polymorphism (SNP) at nucleotide 1580 (C/T) in exon 4 of SP-B that changes amino acid 131 from threonine to isoleucine (Thr131-->Ile) is associated with several pulmonary diseases. The Thr131-->Ile substitution can eliminate a potential N-linked glycosylation site, Asn129-Gln-Thr131, which is present in the SP-B variant of the C allele (ACT/Thr) but not in that of the T allele (ATT/Ile). To determine whether the C allele SP-B variant is indeed glycosylated at Asn(129)-Gln-Thr131, we first generated stably transfected Chinese hamster ovary cell lines that expressed each version of SP-B, and developed specific SP-B polyclonal anti-peptide antibodies. Using both the stably transfected cell lines and fetal lung explants, we observed that the C allele variant is indeed glycosylated at the Asn129-Gln-Thr131 site, whereas the T allele variant, which served as a control, is not. In addition, we also confirmed that both SP-B variants contain another N-linked glycosylation site, Asn311-Ser-Ser313. Given its association with several pulmonary diseases, this finding provides useful information for future studies in disease systems associated with this SNP. Further, we speculate that, given the fact that this SNP is found frequently in the general population, N-linked glycosylation at residue Asn129 interferes with SP-B processing, secretion and folding under certain disease conditions.

MAINTENANCE OF DIFFERENTIATED FUNCTION OF THE SURFACTANT SYSTEM IN HUMAN FETAL LUNG TYPE II EPITHELIAL CELLS CULTURED ON PLASTIC

We report a simplified culture system for human fetal lung type II cells that maintains surfactant expression. Type II cells isolated from explant cultures of hormone-treated lungs (18–22 wk gestation) by collagenase + trypsin digestion were cultured on plastic for 4 days in serum-free medium containing dexamethasone (Dex, 10 nM) + 8-bromo-cAMP (0.1 mM) + isobutylmethylxanthine (0.1 mM) or were untreated (control). Surfactant protein (SP) mRNAs decreased markedly in control cells between days 1 and 4 of culture, but mRNA levels were high in treated cells on day 4 (SP-A, SP-B, SP-C, SP-D; 600%, 100%, 85%, 130% of day 0 content, respectively). Dex or cAMP alone increased SP-B, SP-C, and SP-D mRNAs and together had additive effects. The greatest increase in SP-A mRNA occurred with cAMP alone. Treated cells processed pro-SP-B and pro-SP-C proteins to mature forms and had a higher rate of phosphatidylcholine (PG) synthesis (2-fold) and higher saturation of PC (∼34% versus 27%) than controls. Only treated cells maintained secretagogue-responsive phospholipid synthesis. By electron microscopy, the treated cells retained lamellar bodies and extensive microvilli. We conclude that Dex and cAMP additively stimulate expression of surfactant components in isolated fetal type II cells, providing a simplified culture system for investigation of surfactant-related, and perhaps other, type II cell functions.