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1.  Calcium-Activated Potassium Channel KCa3.1 in Lung Dendritic Cell Migration 
Migration to draining lymph nodes is a critical requirement for dendritic cells (DCs) to control T-cell–mediated immunity. The calcium-activated potassium channel KCa3.1 has been shown to be involved in regulating cell migration in multiple cell types. In this study, KCa3.1 expression and its functional role in lung DC migration were examined. Fluorescence-labeled antigen was intranasally delivered into mouse lungs to label lung Ag-carrying DCs. Lung CD11chighCD11blow and CD11clowCD11bhigh DCs from PBS-treated and ovalbumin (OVA)-sensitized mice were sorted using MACS and FACS. Indo-1 and DiBAC4(3) were used to measure intracellular Ca2+ and membrane potential, respectively. The mRNA expression of KCa3.1 was examined using real-time PCR. Expression of KCa3.1 protein and CCR7 was measured using flow cytometry. Migration of two lung DC subsets to lymphatic chemokines was examined using TransWell in the absence or presence of the KCa3.1 blocker TRAM-34. OVA sensitization up-regulated mRNA and protein expression of KCa3.1 in lung DCs, with a greater response by the CD11chighCD11blow than CD11clowCD11bhigh DCs. Although KCa3.1 expression in Ag-carrying DCs was higher than that in non–Ag-carrying DCs in OVA-sensitized mice, the difference was not as prominent. However, Ag-carrying lung DCs expressed significantly higher CCR7 than non–Ag-carrying DCs. CCL19, CCL21, and KCa3.1 activator 1-EBIO induced an increase in intracellular calcium in both DC subsets. In addition, 1-EBIO–induced calcium increase was suppressed by TRAM-34. In vitro blockade of KCa3.1 with TRAM-34 impaired CCL19/CCL21-induced transmigration. In conclusion, KCa3.1 expression in lung DCs is up-regulated by OVA sensitization in both lung DC subsets, and KCa3.1 is involved in lung DC migration to lymphatic chemokines.
doi:10.1165/rcmb.2010-0514OC
PMCID: PMC3262686  PMID: 21493782
allergic airway inflammation; antigen uptake; asthma; calcium-activated potassium channel; dendritic cell
2.  Increased Expression of Angiopoietins and Tie2 in the Lungs of Chronic Asthmatic Mice 
Angiopoietin (Ang)1 and Ang2 are ligands for Tie2 tyrosine kinase receptor (Tie2). Elevated levels of Ang1 and Ang2 in induced sputum of patients with asthma have been reported, with a positive correlation of Ang2 levels with the severity of airway occlusion. Although studies have shown Tie2-mediated regulation of nonvascular cells in some pathological conditions, current knowledge on Tie2 signaling in asthma is limited to the vasculature. We examined the expression pattern of Ang1, Ang2, vascular endothelial growth factor (VEGF), and Tie2 and their correlation with the degree of airway remodeling in the lung of ovalbumin (OVA)-sensitized and OVA-challenged mice with airway hyperresponsiveness. Lung tissues were isolated from Balb/c mice after OVA sensitization and challenge. Hematoxylin and eosin, periodic acid-Schiff, and trichrome staining were used to show the lung pathology. The expression of Ang1, Ang2, VEGF, and Tie2 was examined using immunofluorescence, Western blot, ELISA, and real-time PCR. In the lung of normal mice, Tie2 expression was detected only in the blood vessels. However, in the lung of OVA-sensitized and OVA-challenged mice, Tie2 was abundantly expressed in airway epithelial cells and in a subset of macrophages in addition to constitutive expression in pulmonary vessels. The increase in Tie2 expression correlated with the severity of airway remodeling. Macrophages and airway epithelial cells express Ang2 and VEGF only in allergic models. Ang1 was constitutively expressed, with a decrease in mRNA level in allergic models. In conclusion, increased expression of Tie2 and Ang2 in allergic airway epithelium and alveolar macrophages correlates with the severity of airway remodeling.
doi:10.1165/rcmb.2009-0330OC
PMCID: PMC3095938  PMID: 20463289
airway epithelial cells; airway remodeling; angiopoietins; Tie2 tyrosine kinase receptor; vascular endothelial growth factor
3.  Fms-Like Tyrosine Kinase 3 Ligand Decreases T Helper Type 17 Cells and Suppressors of Cytokine Signaling Proteins in the Lung of House Dust Mite–Sensitized and –Challenged Mice 
We previously reported that Fms-like tyrosine kinase 3 ligand (Flt3-L) reversed airway hyperresponsiveness (AHR) and airway inflammation, and increased the number of regulatory CD11chighCD8αhighCD11blow dendritic cells and CD4+CD25+ICOS+Foxp3+IL-10+ T-regulatory cells in the lung of allergen-sensitized and -challenged mice. In this study, we evaluated the effect of Flt3-L on Th17 cells and expression of suppressors of cytokine signaling (SOCS) proteins in the lungs of house dust mite (HDM)–sensitized and –challenged mice. BALB/c mice were sensitized and challenged with HDM, and AHR to methacholine was established. Mice were treated with Flt3-L (5 μg, intraperitoneal) daily for 10 days. Levels of IL-4, -5, -6, -8, and -13, and transforming growth factor (TGF)–β in the bronchoalveolar lavage fluid (BALF) were examined by ELISA. Flt3-L treatment reversed existing AHR to methacholine and substantially decreased eosinophils, neutrophils, IL-5, -6, -8, and IL-13, and TGF-β levels in the BALF. HDM-sensitized and -challenged mice showed a significant increase in lung CD4+IL-17+IL-23R+CD25− T cells with high expression of retinoic acid–related orphan receptor (ROR)–γt transcripts. However, administration of Flt3-L substantially decreased the number of lung CD4+IL-17+IL-23R+CD25− T cells, with significantly decreased expression of ROR-γt mRNA in these cells. HDM sensitization caused a significant increase in the expression of SOCS-1, -3, and -5 in the lung. Flt3-L treatment abolished the increase in SOCS-1 and SOCS-3 proteins, whereas SOCS-5 expression was significantly reduced. These data suggest that the therapeutic effect of Flt3-L in reversing the hallmarks of allergic asthma in a mouse model is mediated by decreasing IL-6 and TGF-β levels in the BALF, which, in turn, decrease CD4+IL-17+IL-23R+ROR-γt+CD25− T cells and the expression of SOCS-1 and SOCS-3 in the lung of HDM-sensitized and -challenged mice.
doi:10.1165/rcmb.2009-0241OC
PMCID: PMC2970852  PMID: 19933379
airway hyperresponsiveness; house dust mite; retinoic acid–related orphan receptor–γt; suppressors of cytokine signaling; T helper cell type 17
4.  Programmed Death-1 Antibody Blocks Therapeutic Effects of T-Regulatory Cells in Cockroach Antigen-Induced Allergic Asthma 
We recently reported that the adoptive transfer of T-regulatory cells (Tregs) isolated from lung and spleen tissue of green fluorescent protein–transgenic mice reversed airway hyperresponsiveness and airway inflammation. Because Programmed Death-1 (PD-1) is a pivotal receptor regulating effector T-cell activation by Tregs, we evaluated whether PD-1 is involved in the therapeutic effect of naturally occurring Tregs (NTregs) and inducible Tregs (iTregs) in cockroach (CRA)-sensitized and challenged mice. The CD4+CD25+ NTregs and CD4+CD25− iTregs isolated from the lungs and spleens of BALB/c mice were adoptively transferred into CRA-sensitized and CRA-challenged mice with and without anti–PD-1 antibody (100 μg/mice). The CD4+CD25+ T cells in the lung were phenotyped after adoptive transfer. Concentrations of IL-4, IL-5, IL-10, IFN-γ, and IL-13 in bronchoalveolar lavage fluid (BALF) were measured using ELISA. The NTregs and iTregs from either lung or spleen tissue reversed airway hyperresponsiveness for at least 4 wk. However, the therapeutic effect was blocked by administering the anti–PD-1 antibody. The administration of Tregs-recipient mice with anti–PD-1 antibody significantly decreased cytotoxic T-lymphocyte antigen-4 expression, with low concentrations of Forkhead-winged transcriptional factor box 3 (Foxp3) mRNA transcripts in lung CD4+CD25+ T cells. These mice had substantially higher concentrations of BALF IL-4, IL-5, and IL-13, but significantly decreased levels of BALF IL-10. Adoptive therapy recipients without the anti–PD-1 antibody exhibited high levels of CTLA-4 expression and Foxp3 transcripts in lung CD4+CD25+ T cells, with a significant decrease in BALF IL-4, IL-5, and IL-13 concentrations and a substantial increase in BALF IL-10 concentrations. These data suggest that the reversal of airway hyperresponsiveness and airway inflammation by Tregs is mediated in part by PD-1, because other costimulatory molecules (e.g., inducible costimulatory molecule [ICOS] or CTLA-4) have been shown to play a role in Treg-mediated suppression.
doi:10.1165/rcmb.2009-0258OC
PMCID: PMC2951873  PMID: 19901343
airway hyperresponsiveness; airway inflammation; anti–PD-1 antibody; cockroach antigen; Forkhead-winged transcriptional factor box P3
5.  Flt3-L Increases CD4+CD25+Foxp3+ICOS+ Cells in the Lungs of Cockroach-Sensitized and -Challenged Mice 
We previously reported in an ovalbumin-induced model of allergic asthma that Fms-like tyrosine kinase 3 ligand (Flt3-L) reversed airway hyperresponsiveness (AHR) and airway inflammation, and increased the number of regulatory CD11chighCD8αhighCD11blow dendritic cells in the lung. In this study, we investigated the effect of Flt3-L in a clinically relevant aeroallergen-induced asthma on the phenotypic expression of lung T cells. Balb/c mice were sensitized and challenged with cockroach antigen (CRA), and AHR to methacholine was established. These mice received three intraperitoneal injections of anti-CD25 antibody (PC61; 250 μg) and Flt3-L (3 μg) daily for 10 days. Cytokines and Ig levels in the serum were measured and differential bronchoalveolar lavage fluid (BALF) cell counts were examined. Flt3-L reversed AHR to methacholine to the control level. Flt3-L significantly decreased levels of BALF IL-5, IFN-γ, eosinophilia and substantially increased IL-10 and the number of CD4+CD25+ Forkhead winged helix transcription factor box P3 (Foxp3+) IL-10+ T cells in the lung. Administration of PC61 antibody blocked the effect of Flt3-L and substantially increased AHR, eosinophilia, and BALF IL-5 and IFN-γ levels, and decreased BALF IL-10 levels and the number of CD4+CD25+Foxp3+IL-10+ T cells. Flt3-L significantly decreased CD62-L, but increased inducible costimulatory molecule and Foxp3 mRNA expression in the CD4+CD25+ T cells isolated from lungs of Flt3-L–treated, CRA-sensitized mice compared to CRA-sensitized mice without Flt3-L treatment and PBS control group. Flt3-L significantly inhibited the effect of CRA sensitization and challenge to increase GATA3 expression in lung CD4+CD25+ T cells. Collectively, these data suggest that the therapeutic effect of Flt3-L is mediated by increased density of naturally occurring CD4+CD25+Foxp3+IL-10+ICOS+ T-regulatory cells in the lung. Flt3-L could be a therapeutic strategy for the management and prevention of allergic asthma.
doi:10.1165/rcmb.2008-0397OC
PMCID: PMC2830405  PMID: 19448155
airway hyperresponsiveness; anti-CD25 antibody; Fms-like tyrosine kinase 3 ligand; Forkhead winged helix transcription factor box P3; naturally occurring CD4+CD25+ T-regulatory cells

Results 1-5 (5)