Published ahead of print on April 17, 2008, doi:10.1165/rcmb.2008-0044OC
© 2008 American Thoracic Society DOI: 10.1165/rcmb.2008-0044OC Pulmonary Mastocytosis and Enhanced Lung Inflammation in Mice Heterozygous Null for the Foxf1 Gene1 Division of Pulmonary Biology, Cincinnati Children's Hospital Research Foundation, Cincinnati, Ohio; and 2 Department of Medicine, University of Chicago, Chicago, Illinois Correspondence and requests for reprints should be addressed to Vladimir V. Kalinichenko, Division of Pulmonary Biology, Cincinnati Children's Hospital Research Foundation, 3333 Burnet Ave., MLC 7009, Cincinnati, OH 45229. E-mail: Vladimir.Kalinichenko{at}cchmc.org
The Forkhead Box f1 (Foxf1) transcriptional factor (previously known as HFH-8 or Freac-1) is expressed in endothelial and smooth muscle cells in the embryonic and adult lung. To assess effects of Foxf1 during lung injury, we used CCl4 and butylated hydroxytoluene (BHT) injury models. Foxf1+/– mice developed severe airway obstruction and bronchial edema, associated with increased numbers of pulmonary mast cells and increased mast cell degranulation after injury. Pulmonary inflammation in Foxf1+/– mice was associated with diminished expression of Foxf1, increased mast cell tryptase, and increased expression of CXCL12, the latter being essential for mast cell migration and chemotaxis. After ovalbumin (OVA) sensitization and OVA challenge, increased lung inflammation, airway hyperresponsiveness to methacholine, and elevated expression of CXCL12 were observed in Foxf1+/– mice. During lung development, Foxf1+/– embryos displayed a marked increase in pulmonary mast cells before birth, and this was associated with increased CXCL12 levels in the lung. Expression of a doxycycline-inducible Foxf1 dominant-negative transgene in primary cultures of lung endothelial cells increased CXCL12 expression in vitro. Foxf1 haploinsufficiency caused pulmonary mastocytosis and enhanced pulmonary inflammation after chemically induced or allergen-mediated lung injury, indicating an important role for Foxf1 in the pathogenesis of pulmonary inflammatory responses.
Key Words: Foxf1 • CXCL12 • SDF-1 • mast cells • lung injury
Mast cells are highly specialized cells that play important roles in adaptive and innate immunity, immunoglobulin E–mediated allergy, and inflammatory responses in the lung (1, 2). Immediately after exposure to allergens, mast cells release a variety of inflammatory mediators, including histamine, heparin, prostaglandins, and mast cell proteases, causing airway constriction, blood vessel dilatation and permeation, tissue swelling, hyperoxia, and eosinophil infiltration, contributing to pathogenesis of bronchial asthma (1, 2). Mast cells store tumor necrosis factor (TNF) and are thus primed to trigger TNF-mediated inflammatory responses (3). Mast cells also produce IL-4, IL-6, and IL-8, which play important roles in acute and chronic lung inflammation (2, 4). Mast cell recruitment is mediated by various factors, including platelet-derived growth factor (PDGF), vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), transforming growth factor β (TGF-β), and stromal cell–derived factor-1 (SDF-1 or CXCL12) (4–6). CXCL12 is produced by fibroblasts and endothelial cells and binds to its receptor, CXCR4, present on the surface of mast cells. CXCL12 stimulates mast cell migration and IL-8 production (4, 7). Interestingly, activated mast cells are found in excessive numbers in tissues in heterogeneous clinical disorders associated with mastocytosis (8). Although a subset of patients with mastocytosis displayed activating mutations in c-KIT gene, little is known about transcriptional and signaling pathways contributing to molecular pathogenesis of this disease (8). The Forkhead Box (Fox) family of transcription factors shares homology in the winged helix DNA-binding domain (9) and its members play important roles in cellular proliferation, differentiation, and metabolic homeostasis (10, 11). Forkhead Box f1 (Foxf1) is expressed in capillary endothelial cells, fibroblasts, and peribronchial smooth muscle cells of the embryonic and adult lung and other organs (12, 13). We and others have previously generated mice with targeted disruption of the Foxf1 gene and demonstrated that Foxf1–/– embryos die by 8.5 days post coitum (dpc) due to defective vasculogenesis in the yolk sac and allantois (13, 14). Haploinsufficiency of the Foxf1 gene in Foxf1+/– mice causes perinatal pulmonary hemorrhage, and severe defects in alveolarization and vascularization, as well as fusion of lung lobes and arteries (13, 15, 16). Perinatal lethality from pulmonary hemorrhage was observed in half of newborn Foxf1+/– mice that displayed the most severe reduction in pulmonary Foxf1 levels (13). Interestingly, the other half of the newborn Foxf1+/– mice displayed compensatory increases of Foxf1 mRNA levels in the lung, exhibited diminished alveolar septation without pulmonary hemorrhage, and survived postnatally (13). Adult Foxf1+/– mice had normal life spans and exhibited normal lung morphology in adulthood, suggesting that these mice compensated for the alveolar septation defect (17). However, in response to butylated hydroxytoluene (BHT)-mediated lung injury, the Foxf1+/– mice exhibited severe defects in lung repair and died from pulmonary hemorrhage (17).
Our previous studies demonstrated that Foxf1 is expressed in hepatic stellate and endothelial cells (18, 19). After carbon tetrachloride (CCl4) injury, Foxf1+/– livers exhibited diminished activation of the hepatic stellate cells, associated with a significant reduction in In this article, we have demonstrated increased numbers of pulmonary mast cells and susceptibility of Foxf1+/– mice to pulmonary injury and inflammation. Increased numbers of pulmonary mast cells, increased mast cell degranulation, and bronchial edema caused airway obstruction in CCl4-treated Foxf1+/– mice. Increased pulmonary inflammation in Foxf1+/– mice was associated with increased pulmonary expression of mast cell tryptase and CXCL12, the latter of which is essential for mast cell migration and chemotaxis. After ovalbumin (OVA) sensitization and OVA challenge, Foxf1+/– mice displayed increased lung inflammation, airway hyperresponsiveness to methacholine, and elevated expression of tryptase and CXCL12. Furthermore, increased numbers of mast cells and elevated CXCL12 levels were found in lungs of Foxf1+/– embryos, suggesting that Foxf1 haploinsufficiency causes a genetic predisposition to inflammatory lung diseases. Finally, using primary endothelial cells containing a doxycycline-inducible Foxf1 dominant-negative transgene, we demonstrated that Foxf1 deficiency increased CXCL12 expression in vitro. Foxf1 haploinsufficiency caused abnormal accumulation of mast cells during lung development and lung injury, contributing to genetic predisposition to chemically induced and antigen-mediated lung inflammation.
Foxf1+/– Mice Foxf1+/– mice in which the Foxf1 allele was disrupted by an in-frame insertion of a nuclear localizing β-galactosidase (β-Gal) gene were bred for 10 generations into the Black Swiss mouse genetic background (13). Carbon tetrachloride (CCl4; Sigma, St. Louis, MO) was dissolved in mineral oil at a 1:20 ratio vol/vol, and a single intraperitoneal injection of CCl4 (0.5 µl of CCl4/1 g of body weight) was administered to male Foxf1+/– mice or their wild-type (WT) littermates as described (18, 20). In separate experiments, Foxf1+/– and WT mice were injected intraperitoneally with BHT (300 mg/kg body weight) to induce lung injury as described (12, 17, 21). To determine statistical significance of any observed differences, we used five Foxf1+/– and WT mice in each group. Mice were killed by CO2 asphyxiation, and the left lung lobe was used to prepare total RNA. Right lung lobes were fixed overnight in 4% paraformaldehyde (PFA) at 4°C and then paraffin embedded.
Immunization and Airway Challenge with OVA
Collection and Analysis of Bronchoalveolar Lavage Fluid
Measurement of Cytokine Levels
Measurements of Airway Responsiveness to Methacholine
Immunohistochemical Staining
RNase Protection Assay
Analysis of Applied Biosystems Expression Arrays
Quantitative Real-Time RT-PCR
Cotransfection Studies and Infection of Primary Endothelial Cells with Recombinant Adenoviruses The TetO-Foxf1 dominant-negative (DN) transgene contains the CMV-Tet operator (TetO) promoter, which drives the expression of a T7-tagged Foxf1 DNA binding domain fused to engrailed transcriptional repression domain (29). Cultured ECs were transiently transfected with 6x Foxf1-TATA-luciferase (LUC) reporter construct (30) and CMV-Foxf1 expression plasmid using Fugene 6 reagent (Roche, Indianapolis, IN) as described previously (29, 30). A CMV-Renilla construct was used as an internal control to normalize transfection efficiency. Two hours after transfection, ECs were infected at a multiplicity of infection (MOI) of 100 ifu per cell with adenovirus containing Tetracycline activator (Ad-TA, Tet-off system) or with control LacZ adenovirus (Ad-LacZ) as described (19, 29). Dual luciferase assays (Promega) were performed 48 h after the adenoviral infection as described previously (19, 30). In separate experiments, WT and transgenic ECs were infected with either Ad-TA or Ad-LacZ and then used for preparation of total RNA or for immunofluorescent staining. ECs were fixed with 10% paraformaldehyde and then stained with mouse monoclonal antibodies against T7 followed by anti-mouse antibody conjugated with TRITC as described (29).
Statistical Analysis
CCl4 Treatment Causes Bronchial Obstruction in Foxf1+/– Mice To assess whether Foxf1 insufficiency influenced lung injury, we used CCl4 and BHT injury models. In studies of CCl4 toxicity in Foxf1+/– mice, approximately 60% of the Foxf1+/– mice died within the initial 24 hours after CCl4 treatment (Figure 1A). While CCl4 is hepatotoxic, serum levels of liver aminotransferases, alkaline phosphatase, bilirubin, and albumin were similar in CCl4-treated Foxf1+/– and WT mice (data not shown), indicating that the increased mortality of Foxf1+/– mice during the initial 24 hours of CCl4 treatment did not result from liver failure. Since Foxf1 is essential for formation of pulmonary capillaries (13), we considered the possibility that capillary insufficiency and lung hemorrhage associated with partial loss of Foxf1 function may contribute to the observed increased early mortality in Foxf1+/– mice. However, at the light microscopic level, no detectable changes were observed in the vascular bed of CCl4-treated Foxf1+/– lungs compared with WT lungs (data not shown).
In contrast to WT mice, CCl4-treated Foxf1+/– lungs displayed severe narrowing of large airways (Figures 1B-1I), leading to airway occlusion (Figures 1J and 1K). The average diameter of pulmonary bronchioles in CCl4-treated Foxf1+/– mice was significantly decreased compared with pulmonary bronchioles in either WT mice or vehicle-treated Foxf1+/– mice (Figure 1N). Furthermore, the airway obstruction in CCl4-treated Foxf1+/– mice was associated with increased airway resistance as demonstrated by airway responsiveness to methacholine (Figure 1O). These results suggested that CCl4-induced bronchial obstruction contributed to the increased mortality of Foxf1+/– mice.
CCl4 Treatment Causes Abnormal Lung Inflammation in Foxf1+/– Mice
Increased Numbers of Mast Cells in Foxf1+/– Lungs Morphologic examination of HE-stained lung sections and immunostaining with tryptase antibody revealed increased numbers of mast cells in CCl4-treated Foxf1+/– lungs (Figures 3A–3F). Tryptase-positive mast cells were observed in alveolar, peribronchial, and perivascular regions (Figures 3G–3I). Increased tryptase immunoreactivity was also observed in the lumen of Foxf1+/– pulmonary airways (Figure 3F), a finding consistent with activation of mast cells (31, 32). Consistent with increased tryptase staining in CCl4-injured Foxf1+/– lungs, tryptase mRNA levels were increased 7-fold (Figure 3J). Furthermore, numbers of tryptase-positive mast cells in liver, kidney, and intestine of CCl4-treated Foxf1+/– mice remained unaltered (data not shown), indicating that the CCl4-mediated inflammation was restricted to the Foxf1+/– lung. Thus, haploinsufficiency of the Foxf1 gene caused bronchial obstruction and increased numbers of activated mast cells after CC14-induced injury, perhaps contributing to bronchial edema and mortality of CCl4-treated Foxf1+/– mice. Interestingly, numbers of tryptase-positive mast cells were increased in lungs of Foxf1+/– mice before injury (Figure 3D). Mast cells were not detected in normal lung tissue from untreated WT mice (Figure 3A). Thus, Foxf1 influences mast cell accumulation in the lung that may in turn influence susceptibility to lung injury.
CCl4 Treatment Causes Increased Expression of CXCL12 in the Foxf1+/– Lung To identify genes influenced by Foxf1 during lung injury, we performed hybridization analysis of Applied Biosystems mouse expression arrays with cDNA probes generated from total lung RNA prepared from CCl4-treated Foxf1+/– and CCl4-treated WT mice (see GEO database for a complete list of genes with altered expression levels in CCl4-treated Foxf1+/– lungs; www.ncbi.nim.nih.gov/geo; accession # GSE11112). This analysis revealed that CCl4-treated Foxf1+/– lungs exhibited a 2.5-fold reduction in Foxf1 mRNA levels (data not shown), consistent with diminished Foxf1 expression during BHT-mediated lung injury (17). Expression of several inflammation-associated genes was increased in Foxf1+/– mice after CCl4 treatment (data not shown), a finding consistent with increased inflammation in CCl4-treated Foxf1+/– mice. The up-regulated genes included endomucin and stromal cell–derived factor 1 (SDF-1 or CXCL12), the latter of which is known to regulate mast cell migration and chemotaxis (4). Quantitative real-time RT-PCR (qRT-PCR) analysis was used to confirm that the CCl4-treated Foxf1+/– lungs exhibited diminished expression of Foxf1, whereas CXCL12 and endomucin mRNAs were increased in Foxf1+/– lungs (Figure 3K). Increased levels of CXCL12 protein were also found in BAL fluid of CCl4-treated Foxf1+/– mice (Figure 3L). CXCL12 expression was not altered in the liver (Figure 3K) or blood serum of Foxf1+/– mice (Figure 3L). Furthermore, CXCL12 mRNA and protein levels were similar in vehicle-treated WT and Foxf1+/– lungs (Figures 3K and 3L), indicating that the CXCL12 expression was induced by CCl4-mediated pulmonary inflammation.
BHT Injury Causes Increased Tryptase and CXCL12 Levels
Persistent Pulmonary Inflammation and Airway Hyperresponsiveness in Foxf1+/– Mice after OVA-Mediated Lung Injury Our studies demonstrated that haploinsufficiency of the Foxf1 gene causes an increase in the number of pulmonary mast cells, and renders the mice sensitive to bronchial inflammation and airway obstruction after CCl4 and BHT injury. Since increased numbers of mast cells were found in lungs of untreated Foxf1+/– mice, we considered that Foxf1 haploinsufficiency might contribute to antigen-mediated lung inflammation similar to that occurring in asthma. To test this, Foxf1+/– and WT mice were sensitized with three injections of OVA and then challenged with either aerosolized OVA or saline (22). Consistent with previous studies (22), OVA challenges induced inflammation in interstitial tissue surrounding airways and pulmonary blood vessels in control and Foxf1+/– mice (Figure 5A). Inflammation was more severe in Foxf1+/– mice, as demonstrated by an increased number of inflammatory cells in the lung tissue (Figure 5A) and BAL fluid (Figure 5B). BAL fluid of OVA-treated Foxf1+/– mice contained a significant increase in numbers of eosinophils, whereas numbers of macrophages, lymphocytes, and neutrophils remained unaltered compared with OVA-treated WT mice (Figure 5B). OVA-treated Foxf1+/– mice also displayed increased concentrations of IL-4 and IL-5 proteins in BAL fluid compared with OVA-treated WT mice (Figure 5C). Consistent with the increased pulmonary inflammation in Foxf1+/– lungs, airway resistance during methacholine treatment was significantly increased (Figures 5D and 5E). Increased inflammation in Foxf1+/– lungs was associated with reduced Foxf1 mRNA and increased expression of mast cell tryptase (Figure 5G), as well as with increased CXCL12 mRNA (Figure 5G) and protein levels (Figure 5F). These results are consistent with BHT and CCl4-mediated lung injury in Foxf1+/– mice. Taken together, these lung injury studies demonstrate that Foxf1 haploinsufficiency causes an increased susceptibility to either antigen-mediated or chemically-induced lung inflammation.
Increased Numbers of Pulmonary Mast Cells and Elevated CXCL12 Levels in Foxf1+/– Embryos Next we determined whether increased CXCL12 expression and increased mast cell numbers occurred in Foxf1+/– embryonic lungs before birth. Paraffin lung sections from WT and Foxf1+/– E18.5 embryos were used for immunohistochemical staining with antibodies specific to mast cell protease 7 (tryptase), a known marker for mast cells (33). Although tryptase staining was not detected in WT lungs, increased numbers of tryptase-positive mast cells were observed in lungs from Foxf1+/– embryos at E18.5 (Figure 6C). Likewise, tryptase and mast cell–specific protease 5 mRNAs were increased in RNase protection assays (Figure 6A) and RT-PCR analysis (Figure 6B), confirming increased numbers of mast cells in Foxf1+/– lungs. Increased tryptase was observed in Foxf1+/– mice as early as E15.5 (Figure 6A). Furthermore, CXCL12 mRNA was increased in lungs from Foxf1+/– embryos at E15.5 and E18.5 (Figure 6D) and was correlated with elevated tryptase expression (Figure 6A) and increased numbers of pulmonary mast cells (Figure 6C). Since CXCL12 is essential for migration of mast cells into lung tissue (4), our results suggest that increased CXCL12 expression may contribute to the accumulation of mast cells seen in Foxf1+/– embryonic lungs. Interestingly, accumulation of mast cells was specific for Foxf1+/– lung tissue because normal mast cell numbers and tryptase mRNA levels were observed in embryonic Foxf1+/– livers and intestine (data not shown). These results suggest that Foxf1 haploinsufficiency causes pulmonary mastocytosis and increased CXCL12 expression during lung development.
Foxf1 Regulates CXCL12 Expression In Vitro The Foxf1 transcription factor is not expressed in pulmonary mast cells or their precursors (18, 34), but is co-expressed with CXCL12 in pulmonary endothelial cells (18, 34), supporting the concept that pulmonary mastocytosis is related to migration/ engraftment of mast cell precursors into the lung rather then changes in differentiation or growth of hematopoetic precursors per se. To determine whether Foxf1 directly affects CXCL12 expression in endothelial cells, we used primary lung ECs prepared from either WT or TetO-Foxf1 DN transgenic mice. The TetO-Foxf1 DN mouse line uses the CMV-Tet operator (TetO) promoter to drive expression of a T7-tagged Foxf1 dominant-negative (Foxf1 DN) fusion protein (29). Purity of pulmonary ECs was 95.1 ± 2.7%, as demonstrated by cytochemical staining with FITC-conjugated isolectin B4 (Figures 7A and 7B), a specific marker of endothelial cells (13).
To activate the TetO-Foxf1 DN transgene in cell culture, transgenic ECs were infected for 48 hours with either CMV-Tetracycline activator adenovirus (Ad-TA) or LacZ control adenovirus (AdLacZ). Immunofluorescent staining with T7 antibody demonstrated that the Foxf1 DN protein was induced by Ad-TA infection (Tet-off system) in transgenic TetO-Foxf1 DN ECs, but was not in Ad-TA–infected wild type ECs (Figure 7E) or in transgenic TetO-Foxf1 DN ECs infected with AdLacZ adenovirus (Figure 7D). Cotransfection studies with the CMV-Foxf1 expression vector and the 6x Foxf1-TATA-luciferase reporter plasmid showed that Foxf1 DN protein inhibited Foxf1 transcriptional activity in transgenic TetO-Foxf1 DN ECs when they were infected with Ad-TA adenovirus (Figure 7G). In contrast, both AdLacZ-infected transgenic ECs and Ad-TA–infected WT ECs displayed normal Foxf1 transcriptional activity compared with uninfected ECs (Figure 7G). Thus, conditional expression of the Foxf1 DN transgene in ECs reduced the Foxf1 transcriptional activity. Decreased Foxf1 activity in TetO-Foxf1 DN ECs was associated with increased CXCL12 mRNA as demonstrated by qRT-PCR (Figure 7H). Thus CXCL12 expression is enhanced by inhibition of Foxf1 in cultured pulmonary endothelial cells.
Foxf1 is an important transcriptional regulator expressed in the developing mesenchyme and its derivatives: fibroblasts, endothelial cells, and smooth muscle cells (13, 14, 18). Although previous studies demonstrated that Foxf1 was critical for vascular integrity during lung development and lung injury (13, 17), molecular mechanisms underlying the Foxf1 function in the lung remain largely unknown. In this study, we demonstrated that lungs of Foxf1+/– embryos exhibited increased numbers of mast cells and increased expression of tryptase, mast cell protease-5, and CXCL12 chemokine, the latter of which is known to promote mast cell migration and chemotaxis (4). These results suggest that haploinsufficiency of the Foxf1 gene increases pulmonary mast cell numbers during embryonic lung development, perhaps through a CXCL12-dependent mechanism. Activation of mast cells causes blood vessel dilatation and inhibits blood coagulation due to release of histamine and heparin, respectively (1, 2). Because a majority of Foxf1+/– mice exhibited a perinatal lethal phenotype due to pulmonary hemorrhage (13), it is tempting to speculate that mast cell–derived mediators contribute to the pulmonary hemorrhage seen in newborn Foxf1+/– mice (13).
In the present study, we demonstrated an increased susceptibility of Foxf1+/– mice to both chemical and allergen-mediated lung inflammation. In studies of CCl4 toxicity, severe airway obstruction and bronchial edema in Foxf1+/– mice preceded the onset of severe hepatic injury (18), suggesting that the liver injury does not cause mortality in Foxf1+/– mice. Pulmonary inflammation was associated with elevated tryptase and increased numbers of mast cells. Since degranulation of mast cells is known to cause the release of tryptase and histamine into the airways enhancing inflammation and leading to bronchial edema and bronchoconstriction (31, 32, 35, 36), our results suggest that CCl4 injury caused degranulation of pulmonary mast cells, contributing to bronchial edema and lethality of the Foxf1+/– mice from airway obstruction. Although CCl4-treated Foxf1+/– mice developed respiratory symptoms before death, it is not sufficient to conclude that the lethality occurred solely due to mast cell degranulation and pulmonary inflammation. Liver injury can also contribute, at least in part, to the lethality observed in CCl4-treated Foxf1+/– mice. In fact, abnormal degranulation of mast cells in Foxf1+/– lungs may be triggered by systemic release of cytokines, chemokines, and/or enzymes from injured liver. We previously demonstrated that hepatic expression of IFN-β, IFN- Our previous studies demonstrated that Foxf1+/– mice died of pulmonary hemorrhage after BHT lung injury (17). This mortality with Foxf1+/– lung injury was associated with a significant reduction in pulmonary Foxf1 levels and diminished expression of genes critical for lung repair (17). In present study, we also found that BHT-treated Foxf1+/– lungs displayed elevated numbers of pulmonary mast cells and increased tryptase levels, causing airway obstruction. These results suggest that mast cell degranulation and bronchial edema contributes to the lethality in BHT-treated Foxf1+/– mice. Another important finding of our studies is that Foxf1+/– mice displayed increased lung inflammation and airway hyperresponsiveness to methacholine after OVA sensitization and OVA challenge. Increased tryptase levels were also observed in OVA-challenged Foxf1+/– lungs, suggesting that antigen-mediated response can trigger mast cell degranulation in Foxf1+/– lungs. This may enhance pulmonary inflammation and airway hyperresponsiveness in Foxf1+/– lungs. Because increased numbers of mast cells were observed in untreated Foxf1+/– lungs, Foxf1 haploinsufficiency may contribute to a genetic predisposition to antigen-mediated lung inflammation and asthma. Interestingly, an increased number of activated mast cells is also characteristic of systemic mastocytosis, a heterogeneous group of human disorders characterized by myeloproliferative syndrome and elevated tryptase levels in multiple organs (8). Since ablation of a single allele of the Foxf1 gene caused increased numbers of mast cells, it will be of interest to assess status of the Foxf1 gene in human mastocytosis. Although mechanisms underlying increased numbers of mast cells in Foxf1+/– lungs remain unclear, our results suggest that Foxf1 is involved in regulation of mast cell homeostasis or migration. The Foxf1 protein is a transcription factor, which is expressed in pulmonary fibroblasts, ECs, and smooth muscle cells, but not in hematopoetic cells (12, 13). Using either Foxf1 antibody or LacZ staining detecting a β-galactosidase reporter knocked into endogenous Foxf1 gene locus (13), we were unable to detect the Foxf1 in mast cells, basophils, or their precursors in the bone marrow or embryonic liver (18, 34, and data not shown). Therefore, Foxf1 regulates mast cell homeostasis or recruitment by an indirect mechanism, possibly involving pulmonary endothelial cells, a major cell type expressing Foxf1 protein in the lung (12, 13). In the present study, we used an inducible Foxf1 dominant-negative transgene (Foxf1 DN) (29) to inhibit Foxf1 function in primary lung ECs prepared from either WT or TetO-Foxf1 DN transgenic mice. Our results demonstrated that Foxf1 depletion in cultured ECs induces the expression of CXCL12, a chemokine known to promote mast cell migration and chemotaxis (4). Thus, Foxf1 negatively regulates CXCL12 expression in cultured endothelial cells in vitro. Consistent with this hypothesis, increased expression of CXCL12 was found in Foxf1+/– lungs after either chemically induced (CCl4 and BHT) or antigen-mediated (OVA) lung injury, correlating with increased number of mast cells and elevated tryptase levels in Foxf1+/– lungs. Interestingly, five potential forkhead-binding sites are present in the –2 kb promoter region of the mouse CXCL12 gene (–936, –1,219, –1,335, –1,419, and –1,807), raising the possibility that Foxf1 may bind to this promoter region and act as a repressor of CXCL12 gene transcription. Alternatively, since a repressor function has not previously been reported for the Foxf1 transcription factor, the Foxf1 protein may indirectly regulate the CXCL12 gene. It is also possible that the absence of Foxf1 allows other transcriptional activators, more potent then Foxf1, to bind the promoter region, thus inducing CXCL12 gene expression. In summary, Foxf1+/– mice displayed severe airway obstruction, elevated numbers of pulmonary mast cells, and increased tryptase levels after various forms of lung injury. Pulmonary inflammation in Foxf1+/– mice was associated with diminished expression of Foxf1, and increased expression of CXCL12, the latter of which is essential for mast cell migration and chemotaxis. Increased numbers of mast cells, and elevated CXCL12 levels, were found in lungs of Foxf1+/– embryos. Foxf1 depletion in cultured endothelial cells led to a significant increase in CXCL12 expression. Our results suggest that Foxf1 haploinsufficiency causes genetic predisposition to inflammatory lung diseases in mice due, at least in part, to increased recruitment of mast cells to the lung and increased CXCL12 production by pulmonary endothelial cells.
The authors thank S. Ramakrishna, D. Malin, and I. M. Kim for technical assistance and Lloyd Graf Jr. for useful comments. The authors dedicate this manuscript to the memory of Robert H. Costa, a pioneer in discovery and characterization of Forkhead transcription factors.
This work was supported by American Heart Association Grant 0335036N (to V.V.K.), Research Grant 6-FY2005–325 from the March of Dimes Birth Defects Foundation (to V.V.K.), and US Public Health Service Grant HL 84151 (to V. V. K.). Originally Published in Press as DOI: 10.1165/rcmb.2008-0044OC on April 17, 2008 Conflict of Interest Statement: None of the authors has a financial relationship with a commercial entity that has an interest in the subject of this manuscript. Received in original form January 23, 2008 Accepted in final form April 16, 2008
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Abstract
CLINICAL RELEVANCE
-smooth muscle actin, type I collagen, notch-2, and IP-10 proteins, indicating an important role for Foxf1 in liver repair and function (
in BAL fluid were measured using a Mouse Cytokine CBA kit (BD Biosciences, San Diego, CA) according to the manufacturer's recommendations. The concentrations of CXCL12 in BAL fluid and serum was determined by a quantitative immunoassay from R&D Systems (Minneapolis, MN). 
0.05 are identified with asterisks. (H) CXCL12 expression is induced in transgenic TetO-Foxf1 DN ECs after infection with AdTA. Total RNAs were prepared from transgenic TetO-Foxf1 DN and WT endothelial cells that were infected with either AdTA adenovirus or control AdLacZ adenovirus. CXCL12 expression was determined by qRT-PCR. CXCL12 mRNA levels were normalized to the corresponding cyclophilin levels and presented as a mean ± SD in four different EC cultures.