Laminin γ2 knockout mice rescued with the human protein exhibit enamel maturation defects.

The epithelial ameloblasts are separated from the maturing enamel by an atypical basement membrane (BM) that is enriched in laminin 332 (LM-332). This heterotrimeric protein (α3, ß3 and γ2 chains) provides structural integrity to BMs and influences various epithelial cell processes including cell adhesion and differentiation. Mouse models that lack expression of individual LM-332 chains die shortly after birth. The lethal phenotype of laminin γ2 knockout mice can be rescued by human laminin γ2 (LAMC2) expressed using a doxycycline-inducible (Tet-on) cytokeratin 14 promoter-rtTA. These otherwise normal-looking rescued mice exhibit white spot lesions on incisors. We therefore investigated the effect of rescue with human LAMC2 on enamel maturation and structuring of the atypical BM. The maturation stage enamel organ in transgenic mice was severely altered as compared to wild type controls, a structured BM was no longer discernible, dystrophic matrix appeared in the maturing enamel layer, and there was residual enamel matrix late into the maturation stage. Microtomographic scans revealed excessive wear of occlusal surfaces on molars, chipping of enamel on incisor tips, and hypomineralization of the enamel layer. No structural alterations were observed at other epithelial sites, such as skin, palate and tongue. These results indicate that while this humanized mouse model is capable of rescue in various epithelial tissues, it is unable to sustain structuring of a proper BM at the interface between ameloblasts and maturing enamel. This failure may be related to the atypical composition of the BM in the maturation stage and reaffirms that the atypical BM is essential for enamel maturation.

The anatomical distribution of genetic associations.

Deeper understanding of the anatomical intermediaries for disease and other complex genetic traits is essential to understanding mechanisms and developing new interventions. Existing ontology tools provide functional, curated annotations for many genes and can be used to develop mechanistic hypotheses; yet information about the spatial expression of genes may be equally useful in interpreting results and forming novel hypotheses for a trait. Therefore, we developed an approach for statistically testing the relationship between gene expression across the body and sets of candidate genes from across the genome. We validated this tool and tested its utility on three applications. First, we show that the expression of genes in associated loci from GWA studies implicates specific tissues for 57 out of 98 traits. Second, we tested the ability of the tool to identify novel relationships between gene expression and phenotypes. Specifically, we experimentally confirmed an underappreciated prediction highlighted by our tool: that white blood cell count--a quantitative trait of the immune system--is genetically modulated by genes expressed in the skin. Finally, using gene lists derived from exome sequencing data, we show that human genes under selective constraint are disproportionately expressed in nervous system tissues.

Myb permits multilineage airway epithelial cell differentiation.

The epithelium of the pulmonary airway is specially differentiated to provide defense against environmental insults, but also subject to dysregulated differentiation that results in lung disease. The current paradigm for airway epithelial differentiation is a one-step program whereby a p63(+) basal epithelial progenitor cell generates a ciliated or secretory cell lineage, but the cue for this transition and whether there are intermediate steps are poorly defined. Here, we identify transcription factor Myb as a key regulator that permits early multilineage differentiation of airway epithelial cells. Myb(+) cells were identified as p63(-) and therefore distinct from basal progenitor cells, but were still negative for markers of differentiation. Myb RNAi treatment of primary-culture airway epithelial cells and Myb gene deletion in mice resulted in a p63(-) population with failed maturation of Foxj1(+) ciliated cells as well as Scbg1a1(+) and Muc5ac(+) secretory cells. Consistent with these findings, analysis of whole genome expression of Myb-deficient cells identified Myb-dependent programs for ciliated and secretory cell differentiation. Myb(+) cells were rare in human airways but were increased in regions of ciliated cells and mucous cell hyperplasia in samples from subjects with chronic obstructive pulmonary disease. Together, the results show that a p63(-) Myb(+) population of airway epithelial cells represents a distinct intermediate stage of differentiation that is required under normal conditions and may be heightened in airway disease.

Increased iron sequestration in alveolar macrophages in chronic obstructive pulmonary disease.

Free iron in lung can cause the generation of reactive oxygen species, an important factor in chronic obstructive pulmonary disease (COPD) pathogenesis. Iron accumulation has been implicated in oxidative stress in other diseases, such as Alzheimer's and Parkinson's diseases, but little is known about iron accumulation in COPD. We sought to determine if iron content and the expression of iron transport and/or storage genes in lung differ between controls and COPD subjects, and whether changes in these correlate with airway obstruction. Explanted lung tissue was obtained from transplant donors, GOLD 2-3 COPD subjects, and GOLD 4 lung transplant recipients, and bronchoalveolar lavage (BAL) cells were obtained from non-smokers, healthy smokers, and GOLD 1-3 COPD subjects. Iron-positive cells were quantified histologically, and the expression of iron uptake (transferrin and transferrin receptor), storage (ferritin) and export (ferroportin) genes was examined by real-time RT-PCR assay. Percentage of iron-positive cells and expression levels of iron metabolism genes were examined for correlations with airflow limitation indices (forced expiratory volume in the first second (FEV1) and the ratio between FEV1 and forced vital capacity (FEV1/FVC)). The alveolar macrophage was identified as the predominant iron-positive cell type in lung tissues. Furthermore, the quantity of iron deposit and the percentage of iron positive macrophages were increased with COPD and emphysema severity. The mRNA expression of iron uptake and storage genes transferrin and ferritin were significantly increased in GOLD 4 COPD lungs compared to donors (6.9 and 3.22 fold increase, respectively). In BAL cells, the mRNA expression of transferrin, transferrin receptor and ferritin correlated with airway obstruction. These results support activation of an iron sequestration mechanism by alveolar macrophages in COPD, which we postulate is a protective mechanism against iron induced oxidative stress.

Keratinocyte-targeted expression of human laminin γ2 rescues skin blistering and early lethality of laminin γ2 deficient mice.

Laminin-332 is a heterotrimeric basement membrane component comprised of the α3, ß3, and γ2 laminin chains. Laminin-332 modulates epithelial cell processes, such as adhesion, migration, and differentiation and is prominent in many embryonic and adult tissues. In skin, laminin-332 is secreted by keratinocytes and is a key component of hemidesmosomes connecting the keratinocytes to the underlying dermis. In mice, lack of expression of any of the three Laminin-332 chains result in impaired anchorage and detachment of the epidermis, similar to that seen in human junctional epidermolysis bullosa, and death occurs within a few days after birth. To bypass the early lethality of laminin-332 deficiency caused by the knockout of the mouse laminin γ2 chain, we expressed a dox-controllable human laminin γ2 transgene under a keratinocyte-specific promoter on the laminin γ2 (Lamc2) knockout background. These mice appear similar to their wild-type littermates, do not develop skin blisters, are fertile, and survive >1.5 years. Immunofluorescence analyses of the skin showed that human laminin γ2 colocalized with mouse laminin α3 and ß3 in the basement membrane zone underlying the epidermis. Furthermore, the presence of "humanized" laminin-332 in the epidermal basement membrane zone rescued the alterations in the deposition of hemidesmosomal components, such as plectin, collagen type XVII/BP180, and integrin α6 and ß4 chains, seen in conventional Lamc2 knockout mice, leading to restored formation of hemidesmosomes. These mice will be a valuable tool for studies of organs deficient in laminin-332 and the role of laminin-332 in skin, including wound healing.

Functional consequences of cell type-restricted expression of laminin α5 in mouse placental labyrinth and kidney glomerular capillaries.

The labyrinth is the highly vascularized part of the rodent placenta that allows efficient transfer of gases, nutrients, wastes, and other molecules between the maternal and embryonic circulations. These two blood compartments are separated by blastocyst-derived trophoblasts and endothelial cells with an intervening basement membrane that contains laminin and other typical basement membrane components. Previously we reported that the labyrinth of laminin α5 knockout (LMα5-/-) embryos exhibits reduced vascularization and detachment of endothelial cells from the basement membrane, which normally contains LMα5. As very little is known about the origin of this vascular basement membrane, we investigated the cellular requirements for LMα5 expression in the mouse placental labyrinth. By fluorescence-activated cell sorting and RT-PCR we confirmed that both endothelial cells and trophoblasts normally express LMα5. Using Cre-loxP technology and doxycycline-mediated gene expression, we generated genetically mosaic placentas in which either the trophoblasts or the endothelial cells, but not both, expressed LMα5. We found that the overall architecture of the labyrinth was normal as long as one of these two cell types expressed LMα5, even if it was transgene-derived human laminin α5. These results suggest that laminin trimers containing α5 that are synthesized and secreted by endothelium or by trophoblasts are capable of integrating into the basement membrane and promoting normal vascularization of the placenta. Additional studies showed that endothelium-expressed human LMα5 can support vascularization of the kidney glomerulus, consistent with previous studies using a tissue grafting approach.

Oxidative modifications of the C-terminal domain of tropoelastin prevent cell binding.

Tropoelastin (TE), the soluble monomer of elastin, is synthesized by elastogenic cells, such as chondrocytes, fibroblasts, and smooth muscle cells (SMCs). The C-terminal domain of TE interacts with cell receptors, and these interactions play critical roles in elastic fiber assembly. We recently found that oxidation of TE prevents elastic fiber assembly. Here, we examined the effects of oxidation of TE on cell interactions. We found that SMCs bind to TE through heparan sulfate (HS), whereas fetal lung fibroblasts (WI-38 cells) bind through integrin α(v)ß(3) and HS. In addition, we found that oxidation of TE by peroxynitrite (ONOO(-)) prevented binding of SMCs and WI-38 cells and other elastogenic cells, human dermal fibroblasts and fetal bovine chondrocytes. Because the C-terminal domain of TE has binding sites for both HS and integrin, we examined the effects of oxidation of a synthetic peptide derived from the C-terminal 25 amino acids of TE (CT-25) on cell binding. The CT-25 peptide contains the only two Cys residues in TE juxtaposed to a cluster of positively charged residues (RKRK) that are important for cell binding. ONOO(-) treatment of the CT-25 peptide prevented cell binding, whereas reduction of the CT-25 peptide had no effect. Mass spectrometric and circular dichroism spectroscopic analyses showed that ONOO(-) treatment modified both Cys residues in the CT-25 peptide to sulfonic acid derivatives, without altering the secondary structure. These data suggest that the mechanism by which ONOO(-) prevents cell binding to TE is by introducing negatively charged sulfonic acid residues near the positively charged cluster.

Oxidative and nitrosative modifications of tropoelastin prevent elastic fiber assembly in vitro.

Elastic fibers are extracellular structures that provide stretch and recoil properties of tissues, such as lungs, arteries, and skin. Elastin is the predominant component of elastic fibers. Tropoelastin (TE), the precursor of elastin, is synthesized mainly during late fetal and early postnatal stages. The turnover of elastin in normal adult tissues is minimal. However, in several pathological conditions often associated with inflammation and oxidative stress, elastogenesis is re-initiated, but newly synthesized elastic fibers appear abnormal. We sought to determine the effects of reactive oxygen and nitrogen species (ROS/RNS) on the assembly of TE into elastic fibers. Immunoblot analyses showed that TE is oxidatively and nitrosatively modified by peroxynitrite (ONOO(-)) and hypochlorous acid (HOCl) and by activated monocytes and macrophages via release of ONOO(-) and HOCl. In an in vitro elastic fiber assembly model, oxidatively modified TE was unable to form elastic fibers. Oxidation of TE enhanced coacervation, an early step in elastic fiber assembly, but reduced cross-linking and interactions with other proteins required for elastic fiber assembly, including fibulin-4, fibulin-5, and fibrillin-2. These findings establish that ROS/RNS can modify TE and that these modifications affect the assembly of elastic fibers. Thus, we speculate that oxidative stress may contribute to the abnormal structure and function of elastic fibers in pathological conditions.

Maintenance of glomerular filtration barrier integrity requires laminin alpha5.

Mutation of the mouse laminin alpha5 gene results in a variety of developmental defects, including defects in kidney structure and function. Whereas the total absence of laminin alpha5 results in breakdown of the glomerular basement membrane (GBM) and failed glomerular vascularization, a hypomorphic Lama5 mutation (the Lama5(neo) allele) results in proteinuria, hematuria, polycystic kidney disease (PKD), and death 3 to 4 weeks after birth. Here, we examined the role of podocyte-derived laminin alpha5 via podocyte-specific inactivation of Lama5 and podocyte-specific rescue of the Lama5(neo) mutation. Podocyte-specific inactivation of Lama5 resulted in varying degrees of proteinuria and rates of progression to nephrotic syndrome. The GBM of proteinuric mice appeared thickened and "moth-eaten," and podocyte foot processes became effaced. Podocyte-specific restoration of laminin alpha5 production using two distinct strategies in Lama5(neo/neo) mice resulted in the resolution of proteinuria, hematuria, and PKD. These results suggest that the development of normal GBM structure and function requires podocyte-derived laminin alpha5 during and after glomerulogenesis and present a unique mechanism for the pathogenesis of PKD in these mice.

Cigarette smoke induces nucleic-acid oxidation in lung fibroblasts.

Oxidative stress is widely proposed as a pathogenic mechanism for chronic obstructive pulmonary disease (COPD), but the molecular pathway connecting oxidative damage to tissue destruction remains to be fully defined. We suggest that reactive oxygen species (ROS) oxidatively damage nucleic acids, and this effect requires multiple repair mechanisms, particularly base excision pathway components 8-oxoguanine-DNA glycosylase (OGG1), endonuclease III homologue 1 (NTH1), and single-strand-selective monofunctional uracil-DNA glycosylase 1 (SMUG1), as well as the nucleic acid-binding protein, Y-box binding protein 1 (YB1). This study was therefore designed to define the levels of nucleic-acid oxidation and expression of genes involved in the repair of COPD and in corresponding models of this disease. We found significant oxidation of nucleic acids localized to alveolar lung fibroblasts, increased levels of OGG1 mRNA expression, and decreased concentrations of NTH1, SMUG1, and YB1 mRNA in lung samples from subjects with very severe COPD compared with little or no COPD. Mice exposed to cigarette smoke exhibited a time-dependent accumulation of nucleic-acid oxidation in alveolar fibroblasts, which was associated with an increase in OGG1 and YB1 mRNA concentrations. Similarly, human lung fibroblasts exposed to cigarette smoke extract exhibited ROS-dependent nucleic-acid oxidation. The short interfering RNA (siRNA)-dependent knockdown of OGG1 and YB1 expression increased nucleic-acid oxidation at the basal state and after exposure to cigarette smoke. Together, our results demonstrate ROS-dependent, cigarette smoke-induced nucleic-acid oxidation in alveolar fibroblasts, which may play a role in the pathogenesis of emphysema.

Neutrophil elastase and neurovascular injury following focal stroke and reperfusion.

Neutrophil elastase (NE) degrades basal lamina and extracellular matrix molecules, and recruits leukocytes during inflammation; however, a basic understanding of the role of NE in stroke pathology is lacking. We measured an increased number of extravascular NE-positive cells, as well as increased levels of tissue elastase protein and activity, following transient middle cerebral artery occlusion (tMCAo). Both pharmacologic inhibition of NE with ZN200355 (ZN), and genetic deletion of NE, significantly reduced infarct volume, blood-brain barrier disruption, vasogenic edema, and leukocyte-endothelial adherence 24 h after tMCAo. ZN also reduced infarct volume in MMP9-null mice following tMCAo. There were, however, no reductions in infarct volume or vasogenic edema in NE-null mice in two models of permanent middle cerebral artery occlusion. Our findings confirm the involvement of NE in neurovascular stroke pathology, when reperfusion allows neutrophils access to vulnerable brain, with pharmacologic or genetic inhibition of NE being both neuro- and vasculo-protective in this setting.

Oxidative damage to nucleic acids in severe emphysema.

BACKGROUND: Oxidative stress is a key element in the pathogenesis of emphysema, but oxidation of nucleic acids has been largely overlooked. The aim of this study was to investigate oxidative damage to nucleic acids in severe emphysematous lungs. METHODS: Thirteen human severe emphysematous lungs, including five with alpha(1)-antitrypsin deficiency (AATD), were obtained from patients receiving lung transplantation. Control lung tissue was obtained from non-COPD lungs (n = 8) and donor lungs (n = 8). DNA and RNA oxidation were investigated by immunochemistry. Morphometry (mean linear intercept [Lm] and CT scan) and immunostaining for CD68 and neutrophil elastase also were performed. RESULTS: Nucleic acid oxidation was increased in alveolar wall cells in emphysematous lungs compared to non-COPD and donor lungs (p < 0.01). In emphysematous lungs, oxidative damage to nucleic acids in alveolar wall cells was increased in the more severe emphysematous areas assessed by histology (Lm, > 0.5 mm; p < 0.05) and CT scan (< -950 Hounsfield units; p < 0.05). Compared to classic emphysema, AATD lungs exhibited higher levels of nucleic acid oxidation in macrophages (p < 0.05) and airway epithelial cells (p < 0.01). Pretreatments with DNase and RNase demonstrated that RNA oxidation was more prevalent than DNA oxidation in alveolar wall cells. CONCLUSIONS: We demonstrated for the first time that nucleic acids, especially RNA, are oxidized in human emphysematous lungs. The correlation between the levels of oxidative damage to nucleic acids in alveolar wall cells and the severity of emphysema suggest a potential role in the pathogenesis of emphysema.

Distal airways in mice exposed to cigarette smoke: Nrf2-regulated genes are increased in Clara cells.

Cigarette smoke (CS) is the main risk factor for chronic obstructive pulmonary disease (COPD). Terminal bronchioles are critical zones in the pathophysiology of COPD, but little is known about the cellular and molecular changes that occur in cells lining terminal bronchioles in response to CS. We subjected C57BL/6 mice to CS (6 d/wk, up to 6 mo), looked for morphologic changes lining the terminal bronchioles, and used laser capture microdissection to selectively isolate cells in terminal bronchioles to study gene expression. Morphologic and immunohistochemical analyses showed that Clara cell predominance remained despite 6 months of CS exposure. Since Clara cells have a role in protection against oxidative stress, we focused on the expression of antioxidant/detoxification genes using microarray analysis. Of the 35 antioxidant/detoxification genes with at least 2.5-fold increased expression in response to 6 months of CS exposure, 21 were NF-E2-related factor 2 (Nrf2)-regulated genes. Among these were cytochrome P450 1b1, glutathione reductase, thioredoxin reductase, and members of the glutathione S-transferase family, as well as Nrf2 itself. In vitro studies using immortalized murine Clara cells (C22) showed that CS induced the stabilization and nuclear translocation of Nrf2, which correlated with the induction of antioxidant and detoxification genes. Furthermore, decreasing Nrf2 expression by siRNA resulted in a corresponding decrease in CS-induced expression of several antioxidant and detoxification genes by C22 cells. These data suggest that the protective response by Clara cells to CS exposure is predominantly regulated by the transcription factor Nrf2.

Fragments of extracellular matrix as mediators of inflammation.

Classically, the extracellular matrix (ECM) was viewed as a supporting structure for stabilizing the location of cells in tissues and for preserving the architecture of tissues. This conception has changed dramatically over the past few decades with discoveries that ECM has profound influences on the structure, viability, and functions of cells. Much of the data supporting this new paradigm has been obtained from studies of normal and pathological structural cells such as fibroblasts, smooth muscle cells, and malignant cells, as, for example, breast cancer epithelial cells. However, there has also been recognition that effects of ECM on cells extend to inflammatory cells. In this context, attention has been drawn to fragments of ECM components. In this review, we present information supporting the concept that proteolytic fragments of ECM affect multiple functions and properties of inflammatory and immune cells. Our focus is particularly upon neutrophils, monocytes, and macrophages and fragments derived from collagens, elastin, and laminins. Hyaluronan fragments, although they are not products of proteolysis, are also discussed, as they are a notable example of ECM fragments that exhibit important effects on inflammatory cells. Further, we summarize some exciting recent developments in this field as a result of mouse models in which defined ECM fragments and their receptors are clearly implicated in inflammation in vivo. Thus, this review underscores the idea that proteolysis of ECM may well have implications that go beyond modifying the structural environment of cells and tissues.

Neutrophil elastase cleaves laminin-332 (laminin-5) generating peptides that are chemotactic for neutrophils.

Proteolytic processing of laminin-332 by matrix metalloproteinase (MMP)-2 and MMP-14 has been shown to yield fragments that are promigratory for epithelial cells. During acute and chronic inflammation, proteases are elaborated by neutrophils and macrophages that can degrade basement membranes. We investigated the susceptibility of laminin-332 to degradation by the following neutrophil and macrophage proteases: neutrophil elastase (NE), cathepsin G, proteinase-3, and MMPs-2, -8, -9, and -12. Protease-specific differences were seen in the capacity to cleave the individual chains of laminin-332. NE and MMP-12 showed the greatest activity toward the gamma2 chain, generating a fragment similar in size to the gamma2x fragment generated by MMP-2. The digestion pattern of laminin-332 by degranulated neutrophils was nearly identical to that generated with NE alone. Digestion by supernatants of degranulated neutrophils was blocked by an inhibitor of NE, and NE-deficient neutrophils were essentially unable to digest laminin-332, suggesting that NE is the major neutrophil-derived protease that degrades laminin-332. In vivo, laminin gamma2 fragments were found in the bronchoalveolar lavage fluid of wild-type mice treated with lipopolysaccharide, whereas that obtained from NE-deficient mice showed a different cleavage pattern. In addition, NE cleaved a synthetic peptide derived from the region of human laminin gamma2 containing the MMP-2 cleavage site, suggesting that NE may generate laminin-332 fragments that are also promigratory. Both laminin-332 fragments generated by NE digestion and NE-digested laminin gamma2 peptide were found to be chemotactic for neutrophils. Collectively, these data suggest that degradation of laminin-332 by NE generates fragments with important biological activities.

Clara cell adhesion and migration to extracellular matrix.

BACKGROUND: Clara cells are the epithelial progenitor cell of the small airways, a location known to be important in many lung disorders. Although migration of alveolar type II and bronchiolar ciliated epithelial cells has been examined, the migratory response of Clara cells has received little attention. METHODS: Using a modification of existing procedures for Clara cell isolation, we examined mouse Clara cells and a mouse Clara-like cell line (C22) for adhesion to and migration toward matrix substrate gradients, to establish the nature and integrin dependence of migration in Clara cells. RESULTS: We observed that Clara cells adhere preferentially to fibronectin (Fn) and type I collagen (Col I) similar to previous reports. Migration of Clara cells can be directed by a fixed gradient of matrix substrates (haptotaxis). Migration of the C22 cell line was similar to the Clara cells so integrin dependence of migration was evaluated with this cell line. As determined by competition with an RGD containing-peptide, migration of C22 cells toward Fn and laminin (Lm) 511 (formerly laminin 10) was significantly RGD integrin dependent, but migration toward Col I was RGD integrin independent, suggesting that Clara cells utilize different receptors for these different matrices. CONCLUSION: Thus, Clara cells resemble alveolar type II and bronchiolar ciliated epithelial cells by showing integrin mediated pro-migratory changes to extracellular matrix components that are present in tissues after injury.

Bronchiolar chemokine expression is different after single versus repeated cigarette smoke exposure.

BACKGROUND: Bronchioles are critical zones in cigarette smoke (CS)-induced lung inflammation. However, there have been few studies on the in vivo dynamics of cytokine gene expression in bronchiolar epithelial cells in response to CS. METHODS: We subjected C57BL/6J mice to CS (whole body exposure, 90 min/day) for various periods, and used laser capture microdissection to isolate bronchiolar epithelial cells for analysis of mRNA by quantitative reverse transcription-polymerase chain reaction. RESULTS: We detected enhanced expression of keratinocyte-derived chemokine (KC), macrophage inflammatory protein-2 (MIP-2), tumor necrosis factor-alpha (TNF-alpha), and interleukin-1beta (IL-1beta) by bronchial epithelial cells after 10 consecutive days of CS exposure. This was mirrored by increases in neutrophils and KC, MIP-2, TNF-alpha, and IL-1beta proteins in the bronchoalveolar lavage (BAL) fluid. The initial inhalation of CS resulted in rapid and robust upregulation of KC and MIP-2 with concomitant DNA oxidation within 1 hr, followed by a return to control values within 3 hrs. In contrast, after CS exposure for 10 days, this initial surge was not observed. As the CS exposure was extended to 4, 12, 18 and 24 weeks, the bronchiolar KC and MIP-2 expression and their levels in BAL fluid were relatively dampened compared to those at 10 days. However, neutrophils in BAL fluid continuously increased up to 24 weeks, suggesting that neutrophil accumulation as a result of long-term CS exposure became independent of KC and MIP-2. CONCLUSION: These findings indicate variable patterns of bronchiolar epithelial cytokine expression depending on the duration of CS exposure, and that complex mechanisms govern bronchiolar molecular dynamics in vivo.

Tumor necrosis factor-alpha from macrophages enhances LPS-induced clara cell expression of keratinocyte-derived chemokine.

Tumor necrosis factor (TNF)-alpha is a cytokine produced by alveolar macrophages in response to LPS in the lung. Clara cells are bronchiolar epithelial cells that produce a variety of proinflammatory cytokines in response to LPS but not to TNF-alpha. In this study, we examined whether TNF-alpha affects Clara cell cytokine production in the setting of LPS stimulation. Using a transformed murine Clara cell line (C22), we observed that both LPS and TNF-alpha induced production of keratinocyte-derived chemokine (KC) and monocyte chemoattractant protein (MCP)-1. We also found that simultaneous LPS and TNF-alpha stimulation is synergistic for KC production, but additive for MCP-1 production. By using a Transwell coculture system of RAW264.7 macrophages and Clara cells isolated from C57Bl/6 mice, we found that macrophages produce a soluble factor that enhances Clara cell KC production in response to LPS. Cocultures of Clara cells from mice deficient in TNF-alpha receptors with RAW264.7 macrophages demonstrated that the effect of macrophages on Clara cells is mediated primarily via TNF-alpha. To determine whether these findings occur in vivo, we treated wild-type and TNF receptor-deficient mice intratracheally with LPS and examined the expression of KC. LPS-treated, TNF receptor-deficient mice showed much less KC mRNA in airway epithelial cells compared with wild-type mice. In contrast, a similar number of KC-expressing cells was seen in the lung periphery. Thus, upregulation of KC by Clara cells in the setting of LPS stimulation is largely dependent on TNF-alpha originating from alveolar macrophages. These findings shed light on macrophage-Clara cell interactions in regulating the pulmonary inflammatory response to LPS.

Clara cells impact the pulmonary innate immune response to LPS.

Airway epithelial cells secrete proinflammatory mediators in response to LPS, but cytokine production by a prominent nonciliated bronchiolar epithelial cell, the Clara cell, specifically, is unknown. To investigate Clara cell cytokine production in response to LPS, we used a transformed murine Clara cell line, C22, and isolated Clara cells from C57Bl/6 mice. Stimulation of both cell types with LPS resulted in significant upregulation of keratinocyte-derived chemokine (KC) and monocyte chemoattractant protein-1, but did not induce TNF-alpha production. To determine whether LPS induces cytokine production by Clara cells in vivo, LPS was instilled intratracheally into mice. KC was expressed by Clara cells, alveolar type 2 cells, and alveolar macrophages, 2 h after LPS administration, as determined by in situ hybridization. TNF-alpha, although not expressed in airway epithelial cells, was expressed primarily in alveolar macrophages in response to LPS. To assess the impact of Clara cells on KC and TNF-alpha production in the lung in the early response to LPS, mice were treated with naphthalene to selectively induce Clara cell injury before LPS stimulation. KC expression in the airways and the lung periphery, and KC and TNF-alpha levels in the bronchoalveolar lavage fluid, were significantly reduced in naphthalene-treated vs. vehicle-treated mice after LPS stimulation. Furthermore, transwell cocultures of C22 cells and RAW264.7 macrophages indicated that C22 cells released a soluble factor(s) in response to LPS that enhanced macrophage production of TNF-alpha. These results indicate that Clara cells elaborate cytokines and modulate the lung innate immune response to LPS.