ABSTRACT
Chronic mucus hypersecretion (CMH) contributes to the morbidity and mortality of asthma, and remains uncontrolled by current therapies in the subset of patients with severe, steroid-resistant disease. Altered cross-talk between airway epithelium and airway smooth muscle cells (ASMCs), driven by pro-inflammatory cytokines such as interleukin (IL)-1ß, provides a potential mechanism that influences CMH. This study investigated mechanisms underlying CMH by comparing IL-1ß-induced gene expression profiles between asthma and control-derived ASMCs and the subsequent paracrine influence on airway epithelial mucus production in vitroIL-1ß-treated ASMCs from asthmatic patients and healthy donors were profiled using microarray analysis and ELISA. Air-liquid interface (ALI)-cultured CALU-3 and primary airway epithelial cells were treated with identified candidates and mucus production assessed.The IL-1ß-induced CCL20 expression and protein release was increased in ASMCs from moderate compared with mild asthmatic patients and healthy controls. IL-1ß induced lower MIR146A expression in asthma-derived ASMCs compared with controls. Decreased MIR146A expression was validated in vivo in bronchial biopsies from 16 asthmatic patients versus 39 healthy donors. miR-146a-5p overexpression abrogated CCL20 release in ASMCs. CCL20 treatment of ALI-cultured CALU-3 and primary airway epithelial cells induced mucus production, while CCL20 levels in sputum were associated with increased levels of CMH in asthmatic patients.Elevated CCL20 production by ASMCs, possibly resulting from dysregulated expression of the anti-inflammatory miR-146a-5p, may contribute to enhanced mucus production in asthma.
Subject(s)
Asthma/metabolism , Chemokine CCL20/metabolism , Interleukin-1beta/pharmacology , MicroRNAs/metabolism , Myocytes, Smooth Muscle/metabolism , Adolescent , Adult , Aged , Asthma/drug therapy , Case-Control Studies , Cells, Cultured , Epithelial Cells/drug effects , Epithelial Cells/metabolism , Female , Gene Expression , Humans , Male , Middle Aged , Mucus/metabolism , Myocytes, Smooth Muscle/drug effects , Sputum/metabolism , Young AdultABSTRACT
Exposure to urban particulate matter (UPM) exacerbates asthmatic lung inflammation. Lung dendritic cells (DCs) are critical for stimulating T cell immunity and in maintaining airway tolerance, but they also react to airway UPM. The adjuvant role of UPM in enhancing primary immune responses by naive cells to allergen has been reported, but the direct effects of UPM-activated DCs on the functionality of human memory CD4 T cells (Tms), which constitute the majority of T cells in the lung, has not been investigated. Blood CD1c(+) DCs were purified and activated with UPM in the presence or absence of house dust mite or tetanus toxoid control antigen. 5-(and -6)-Carboxyfluorescein diacetate succinimidyl ester-labeled blood Tms were cocultured with autologous DCs, T cell proliferation and effector function were assessed using flow cytometry, and secreted cytokines were measured by combined bead array. UPM-DCs elicited IFN-γ and IL-13 secretion and induced proliferation in Tms isolated from both allergic patients with asthma and healthy control subjects, whereas only IL-13 was produced by Tms from patients with atopic asthma stimulated by house dust mite-loaded DCs. UPM-DCs drove the expansion and differentiation of a mixed population of Th1, Th2, and Th17 cell effectors through a mechanism that was dependent on major histocompatibility class II but not on cytokine-driven expansion. The data suggest that UPM not only has adjuvant properties but is also a source of antigen that stimulates the generation of Th2, Th1, and Th17 effector phenotypes, which have been implicated in both exacerbations of asthma and chronic inflammatory diseases.
Subject(s)
Cell Proliferation/drug effects , Dendritic Cells/drug effects , Lymphocyte Activation/drug effects , Particulate Matter/toxicity , Th1 Cells/drug effects , Th17 Cells/drug effects , Th2 Cells/drug effects , Urban Health , Adult , Allergens/immunology , Animals , Asthma/immunology , Asthma/metabolism , Case-Control Studies , Cells, Cultured , Coculture Techniques , Cytokines/immunology , Cytokines/metabolism , Dendritic Cells/immunology , Dendritic Cells/metabolism , Female , Histocompatibility Antigens Class II/immunology , Histocompatibility Antigens Class II/metabolism , Humans , Immunologic Memory , Inflammation Mediators/immunology , Inflammation Mediators/metabolism , Male , Middle Aged , Particulate Matter/immunology , Phenotype , Pyroglyphidae/immunology , Receptors, Antigen, T-Cell/immunology , Receptors, Antigen, T-Cell/metabolism , Tetanus Toxin/immunology , Th1 Cells/immunology , Th1 Cells/metabolism , Th17 Cells/immunology , Th17 Cells/metabolism , Th2 Cells/immunology , Th2 Cells/metabolism , Young AdultABSTRACT
Airway smooth muscle (ASM) cells are thought to contribute to the pathogenesis of allergic asthma by orchestrating and perpetuating airway inflammation and remodeling responses. In this study, we evaluated the IL-17RA signal transduction and gene expression profile in ASM cells from subjects with mild asthma and healthy individuals. Human primary ASM cells were treated with IL-17A and probed by the Affymetrix GeneChip array, and gene targets were validated by real-time quantitative RT-PCR. Genomic analysis underlined the proinflammatory nature of IL-17A, as multiple NF-κB regulatory factors and chemokines were induced in ASM cells. Transcriptional regulators consisting of primary response genes were overrepresented and displayed dynamic expression profiles. IL-17A poorly enhanced IL-1ß or IL-22 gene responses in ASM cells from both subjects with mild asthma and healthy donors. Interestingly, protein modifications to the NF-κB regulatory network were not observed after IL-17A stimulation, although oscillations in IκBε expression were detected. ASM cells from subjects with mild asthma up-regulated more genes with greater overall variability in response to IL-17A than from healthy donors. Finally, in response to IL-17A, ASM cells displayed rapid activation of the extracellular signal-regulated kinase/ribosomal S6 kinase signaling pathway and increased nuclear levels of phosphorylated extracellular signal-regulated kinase. Taken together, our results suggest that IL-17A mediated modest gene expression response, which, in cooperation with the NF-κB signaling network, may regulate the gene expression profile in ASM cells.
Subject(s)
Asthma/genetics , Asthma/metabolism , Interleukin-17/genetics , Interleukin-17/metabolism , Myocytes, Smooth Muscle/metabolism , Adult , Asthma/pathology , Cell Line , Extracellular Signal-Regulated MAP Kinases/genetics , Extracellular Signal-Regulated MAP Kinases/metabolism , Female , Humans , Interleukin-1beta/genetics , Interleukin-1beta/metabolism , Interleukins/genetics , Interleukins/metabolism , Male , Myocytes, Smooth Muscle/pathology , NF-kappa B/genetics , NF-kappa B/metabolism , Phosphorylation , Signal Transduction , Transcriptome , Up-Regulation , Interleukin-22ABSTRACT
Urban particulate matter (UPM) exacerbates asthmatic lung inflammation and depresses lung immunity. Lung dendritic cells (DCs) react to airway particulates, and have a critical role in linking innate and adaptive immunity, but the direct effects of UPM on DCs, that have been activated by granulocyte/macrophage colony-stimulating factor (GM-CSF), a product of stimulated normal human bronchial epithelial cells, has not been investigated. Human blood CD1c(+) DCs were purified and activated with UPM in the presence or absence of GM-CSF with and without LPS, and DC maturation was assessed by flow cytometry. DC stimulatory capacity and priming of 5-(and -6)-carboxyfluorescein diacetate succinimidyl ester-labeled naive CD4 T cells was investigated using the allogeneic mixed lymphocyte reaction. T cell proliferation and effector function were assessed using flow cytometry and secreted cytokines were measured by combined bead array. UPM enhanced DC maturation in an LPS-independent manner. DCs activated by UPM plus GM-CSF (UPM + GM-CSF DCs) induced higher naive CD4 T cell proliferation in the allogeneic mixed lymphocyte reaction than DCs pretreated by GM-CSF alone (GM-CSF DCs), and elicited both substantially lower levels of IFN-γ, IL-13, and IL-5 secretion, and lower frequencies of alloantigen-specific T helper (Th) type 1 effector cells than naive CD4 T cells primed by GM-CSF DCs. UPM-stimulated DCs produced IL-6 and TNF-α. Neutralization of IL-6 decreased naive CD4 T cell proliferation stimulated by UPM + GM-CSF DCs, and significantly increased the frequency of alloantigen-specific Th1 effector cells, but did not reverse UPM-induced inhibition of IFN-γ secretion. We conclude that UPM enhances GM-CSF-induced DC maturation and stimulatory capacity, but inhibits the generation of Th1 cells. Thus, UPM exposure may impair Th1 responses to pulmonary pathogens.
Subject(s)
Dendritic Cells/drug effects , Granulocyte-Macrophage Colony-Stimulating Factor/pharmacology , Macrophage Colony-Stimulating Factor/pharmacology , Particulate Matter/pharmacology , Th1 Cells/immunology , Cell Differentiation/immunology , Dendritic Cells/immunology , Dendritic Cells/metabolism , Granulocyte-Macrophage Colony-Stimulating Factor/immunology , Humans , Interleukin-13/immunology , Interleukin-5/immunology , Interleukin-6/immunology , Macrophage Colony-Stimulating Factor/immunology , Particulate Matter/immunology , Th1 Cells/metabolism , Tumor Necrosis Factor-alpha/metabolismABSTRACT
IL-25 (IL-17E) is a T-helper cell type 2 (Th2) cytokine best described as a potentiator of Th2 memory responses. Reports of expression of its receptor, IL-25R, on airways structural cells suggest a wider role for IL-25 in remodeling. We hypothesized that IL-25 stimulates local angiogenesis in the asthmatic bronchial mucosa. Immunoreactive IL-25(+), IL-25R(+), and CD31(+) (endothelial) cells in sections of bronchial biopsies from asthmatics and controls were detected by immunohistochemistry. The effect of IL-25 on angiogenesis was examined using an in vitro assay. Real-time PCR was used to detect expression of IL-25R and VEGF mRNA in cultured human vascular endothelial cells (HUVEC), and a cell proliferation kit (WST-8) was used to measure the effect of IL-25 on HUVEC proliferation. Immunostaining showed that IL-25(+), IL-25R(+), and CD31(+)/IL-25R(+) cells were significantly elevated in the bronchial mucosa of asthmatics compared with controls (P < 0.003). In asthmatics, the numbers of IL-25(+) cells correlated inversely with the forced expiratory volume in 1 s (r = -0.639; P = 0.01). In vitro, HUVEC constitutively expressed IL-25R, which was up-regulated further by TNF-α. IL-25 and TNF-α also increased expression of VEGF and VEGF receptors. IL-25 increased HUVEC proliferation and the number, length, and area of microvessel structures in a concentration-dependent manner in vitro. VEGF blockade, the PI3K-specific inhibitor LY294002, and the MAPK/ERK1/2 (MEK1/2)-specific inhibitor U0126 all markedly attenuated IL-25-induced angiogenesis, and the inhibitors also reduced IL-25-induced proliferation and VEGF expression. Our findings suggest that IL-25 is elevated in asthma and contributes to angiogenesis, at least partly by increasing endothelial cell VEGF/VEGF receptor expression through PI3K/Akt and Erk/MAPK pathways.
Subject(s)
Asthma/immunology , Bronchi/blood supply , Interleukin-17/immunology , Th2 Cells/immunology , Adult , Asthma/metabolism , Asthma/physiopathology , Blotting, Western , Bronchi/drug effects , Bronchi/pathology , Cell Proliferation/drug effects , Cells, Cultured , Endothelial Cells/drug effects , Endothelial Cells/metabolism , Extracellular Signal-Regulated MAP Kinases/metabolism , Humans , Immunohistochemistry , Immunologic Memory/immunology , Interleukin-17/metabolism , Interleukin-17/pharmacology , Middle Aged , Phosphatidylinositol 3-Kinases/metabolism , Phosphorylation/drug effects , Platelet Endothelial Cell Adhesion Molecule-1/genetics , Platelet Endothelial Cell Adhesion Molecule-1/metabolism , Proto-Oncogene Proteins c-akt/metabolism , Receptors, Interleukin/genetics , Receptors, Interleukin/metabolism , Reverse Transcriptase Polymerase Chain Reaction , STAT6 Transcription Factor/metabolism , Th2 Cells/metabolism , Vascular Endothelial Growth Factor A/genetics , Vascular Endothelial Growth Factor A/metabolism , Young AdultABSTRACT
The increase in airway smooth muscle (ASM) mass is a major structural change in asthma. This increase has been attributed to ASM cell (ASMC) hyperplasia and hypertrophy. The distance between ASMC and the epithelium is reduced, suggesting migration of smooth muscle cells toward the epithelium. Recent studies have suggested a role of chemokines in ASMC migration toward the epithelium; however, chemokines have other biological effects. The objective of the current study is to test the hypothesis that chemokines (eotaxin, RANTES, IL-8, and MIP-1α) can directly influence ASMC mass by increasing the rate of proliferation or enhancing the survival of these cells. Human ASMCs were exposed to different concentrations of eotaxin, RANTES, IL-8, or MIP-1α. To test for proliferation, matched control and stimulated ASMC were pulsed with [(3)H]thymidine, or ASMCs were stained with BrdU and then analyzed with flow cytometry. Apoptosis was measured using Annexin V staining and flow cytometry. Expression of phosphorylated p42/p44 and MAPKs was assessed by Western blot. In a concentration-dependent manner, chemokines including eotaxin, RANTES, IL-8, and MIP-1α increased ASMC's [(3)H]thymidine incorporation and DNA synthesis. IL-8, eotaxin, and MIP-1α decreased the rate of apoptosis of ASMCs compared with the matched controls. A significant increase in phosphorylated p42/p44 MAPKs was seen after treating ASMCs with RANTES and eotaxin. Moreover, inhibition of p42/p44 MAPK phosphorylation reduced the level of chemokine-induced ASM proliferation. We conclude that chemokines might contribute to airway remodeling seen in asthma by enhancing the number and survival of ASMCs.
Subject(s)
Airway Remodeling/immunology , Cell Proliferation , Chemokine CCL11/physiology , Chemokine CCL3/physiology , Chemokine CCL5/physiology , Interleukin-8/physiology , Myocytes, Smooth Muscle/immunology , Up-Regulation/immunology , Apoptosis/immunology , Asthma/immunology , Asthma/metabolism , Asthma/pathology , Bronchi/immunology , Bronchi/metabolism , Bronchi/pathology , Cell Survival/immunology , Cells, Cultured , Chemokine CCL11/biosynthesis , Chemokine CCL3/biosynthesis , Chemokine CCL5/biosynthesis , DNA Replication/immunology , Down-Regulation/immunology , Humans , Interleukin-8/biosynthesis , Mitogen-Activated Protein Kinase 1/metabolism , Mitogen-Activated Protein Kinase 1/physiology , Mitogen-Activated Protein Kinase 3/metabolism , Mitogen-Activated Protein Kinase 3/physiology , Myocytes, Smooth Muscle/metabolism , Myocytes, Smooth Muscle/pathology , Phosphorylation/immunology , Thymidine/biosynthesis , Thymidine/metabolismABSTRACT
BACKGROUND: Allergic asthma is characterized by reversible airway obstruction and bronchial hyperresponsiveness associated with T(H)2 cell-mediated inflammation. Cysteinyl leukotrienes (CysLTs) are potent lipid mediators involved in bronchoconstriction, mucus secretion, and cell trafficking in asthmatic patients. Recent data have implicated CysLTs in the establishment and amplification of T(H)2 responses in murine models, although the precise mechanisms are unresolved. OBJECTIVES: Preliminary microarray studies suggested that human T(H)2 cells might selectively express cysteinyl leukotriene receptor 1 (CYSLTR1) mRNA. We sought to establish whether human T(H)2 cells are indeed a CysLT target cell type. METHODS: We examined the expression of CYSLTR1 using real-time PCR in human T(H)1 and T(H)2 cells. We functionally assessed cysteinyl leukotriene receptor 1 protein (CysLT(1)) expression using calcium flux, cyclic AMP, and chemotaxis assays. RESULTS: We show that human T(H)2 cells selectively express CYSLTR1 mRNA at high levels compared with T(H)1 cells after in vitro differentiation from naive precursors. Human T(H)2 cells are selectively responsive to CysLTs in a calcium flux assay when compared with T(H)1 cells with a rank order of potency similar to that described for CysLT(1) (leukotriene [LT] D(4) > LTC(4) > LTE(4)). We also show that LTD(4)-induced signaling in T(H)2 cells is mediated through CysLT(1) coupled to G(α)q and G(α)i proteins, and both pathways can be completely inhibited by selective CysLT(1) antagonists. LTD(4) is also found to possess potent chemotactic activity for T(H)2 cells at low nanomolar concentrations. CONCLUSIONS: These findings suggest a novel mechanism of action for CysLTs in the pathogenesis of asthma and provide a potential explanation for the anti-inflammatory effects of CysLT(1) antagonists.
Subject(s)
Cysteine/pharmacology , Immunologic Factors/pharmacology , Leukotrienes/pharmacology , Receptors, Leukotriene/genetics , Th2 Cells/immunology , Calcium Signaling/immunology , Chemotaxis/drug effects , Chemotaxis/immunology , Cysteine/metabolism , GTP-Binding Protein alpha Subunits/metabolism , Gene Expression Profiling , Gene Expression Regulation , Humans , Immunologic Factors/metabolism , Leukotriene D4/pharmacology , Leukotrienes/metabolism , Protein Binding/drug effects , Receptors, Leukotriene/metabolism , Th2 Cells/drug effects , Th2 Cells/metabolismABSTRACT
BACKGROUND: Prostaglandin E(2) (PGE(2)) is thought to play a role in the pathogenesis of aspirin-sensitive asthma (ASA). OBJECTIVE: We sought to extend our previous observations implicating impaired inflammatory cell responsiveness to PGE(2) as a pathogenetic mechanism in patients with aspirin-sensitive rhinosinusitis to the bronchial mucosa in patients with ASA. METHODS: Immunohistochemistry was used to enumerate inflammatory cells and their expression of cysteinyl leukotriene receptors 1 and 2 (CysLT(1) and CysLT(2)) and the PGE(2) receptors E-prostanoid 1 to 4 (EP(1)-EP(4)) in bronchial biopsy specimens from patients with ASA, patients with aspirin-tolerant asthma, and control subjects (n= 15 in each group). Concentrations of PGE(2) in bronchoalveolar lavage fluid were measured by using ELISA. The effects of PGE(2) and EP receptor agonists on CD3/CD28-stimulated cytokine production by PBMCs were measured by using ELISA. Airways responsiveness to LTD(4)in vivo was measured in asthmatic patients by means of bronchial challenge. RESULTS: Compared with patients with aspirin-tolerant asthma, patients with ASA had increased bronchial mucosal neutrophil and eosinophil numbers but reduced percentages of T cells, macrophages, mast cells, and neutrophils expressing EP(2). Both groups showed increased bronchial sensitivity to inhaled LTD(4), but this did not correlate with mucosal expression of CysLT(1) or CysLT(2). Bronchoalveolar lavage fluid PGE(2) concentrations were comparable in all groups. In vitro PGE(2) inhibited cytokine production by PBMCs through EP(2) but not other PGE(2) receptors. CONCLUSION: Our data are consistent with the hypothesis that impaired inhibition of inflammatory leukocytes by PGE(2) acting through the EP(2) receptor has a role in the pathogenesis of ASA.
Subject(s)
Asthma, Aspirin-Induced/metabolism , Bronchi/metabolism , Leukocytes/metabolism , Receptors, Prostaglandin E, EP2 Subtype/metabolism , Respiratory Mucosa/metabolism , Adult , Asthma, Aspirin-Induced/immunology , Bronchi/pathology , Bronchoalveolar Lavage Fluid/chemistry , Cytokines/biosynthesis , Female , Humans , Leukocytes, Mononuclear/metabolism , Male , Middle Aged , Receptors, Leukotriene/metabolism , Receptors, Prostaglandin E, EP2 Subtype/agonists , Young AdultABSTRACT
Aspirin-sensitive respiratory disease (ASRD) is a condition characterized by persistent and often severe inflammation of the upper and lower respiratory tracts. Patients develop chronic eosinophilic rhinosinusitis, nasal polyposis, and asthma. The ingestion of aspirin and other cyclooxygenase-1 (COX-1) inhibitors induces exacerbations of airway disease that may be life-threatening. Thus, aspirin sensitivity is a phenotypic marker for the syndrome, yet nearly all affected individuals can be desensitized by the administration of graded doses of aspirin, leading to long-term clinical benefits. Patients with aspirin sensitivity are often able to tolerate selective COX-2 inhibitors. The pathogenesis of ASRD is underpinned by abnormalities in eicosanoid biosynthesis and eicosanoid receptor expression coupled with intense mast cell and eosinophilic infiltration of the entire respiratory tract. This review focuses on the molecular, cellular, and biochemical abnormalities characterizing ASRD and highlights unanswered questions in the literature and potential future areas of investigation.
Subject(s)
Aspirin/adverse effects , Cyclooxygenase Inhibitors/adverse effects , Lung Diseases/chemically induced , Dose-Response Relationship, Drug , Drug Tolerance/physiology , Eicosanoids/metabolism , Humans , Lung Diseases/metabolism , Lung Diseases/physiopathologyABSTRACT
Eicosanoids are lipid-signaling mediators released by many cells in response to various stimuli. Increasing evidence suggests that eicosanoids such as leukotrienes and prostaglandins (PGs) may directly mediate remodeling. In this study, we assessed whether these substances could alter extracellular matrix (ECM) proteins and the inflammatory profiles of primary human airway smooth muscle cells (ASM) and fibroblasts. PGE(2) decreased both fibronectin and tenascin C in fibroblasts but only fibronectin in ASM. PGD(2) decreased both fibronectin and tenascin C in both ASM and fibroblasts, whereas PGF(2α) had no effect on ECM deposition. The selective PGI(2) analog, MRE-269, decreased fibronectin but not tenascin C in both cell types. All the PGs increased IL-6 and IL-8 release in a dose-dependent manner in ASM and fibroblasts. Changes in ECM deposition and cytokine release induced by prostaglandins in both ASM and fibroblasts were independent of an effect on cell number. Neither the acute nor repeated stimulation with leukotrienes had an effect on the deposition of ECM proteins or cytokine release from ASM or fibroblasts. We concluded that, collectively, these results provide evidence that PGs may contribute to ECM remodeling to a greater extent than leukotrienes in airway cells.
Subject(s)
Bronchi/metabolism , Extracellular Matrix Proteins/metabolism , Fibroblasts/metabolism , Interleukin-6/metabolism , Interleukin-8/metabolism , Leukotrienes/metabolism , Myocytes, Smooth Muscle/metabolism , Prostaglandins/metabolism , Adolescent , Adult , Aged , Aged, 80 and over , Brefeldin A/pharmacology , Bronchi/cytology , Bronchi/drug effects , Cell Proliferation/drug effects , Enzyme-Linked Immunosorbent Assay , Extracellular Matrix/metabolism , Female , Fibroblasts/cytology , Fibroblasts/drug effects , Fibronectins/metabolism , Humans , Male , Middle Aged , Myocytes, Smooth Muscle/cytology , Myocytes, Smooth Muscle/drug effects , Protein Synthesis Inhibitors/pharmacology , Tenascin/metabolism , Transforming Growth Factor beta/pharmacology , Young AdultABSTRACT
Phenotypic modulation of airway smooth muscle (ASM) is an important feature of airway remodeling in asthma that is characterized by enhanced proliferation and secretion of pro-inflammatory chemokines. These activities are regulated by the concentration of free Ca(2+) in the cytosol ([Ca(2+)](i)). A rise in [Ca(2+)](i) is normalized by rapid reuptake of Ca(2+) into sarcoplasmic reticulum (SR) stores by the sarco/endoplasmic reticulum Ca(2+) (SERCA) pump. We examined whether increased proliferative and secretory responses of ASM from asthmatics result from reduced SERCA expression. ASM cells were cultured from subjects with and without asthma. SERCA expression was evaluated by western blot, immunohistochemistry and real-time PCR. Changes in [Ca(2+)](i), cell spreading, cellular proliferation, and eotaxin-1 release were measured. Compared with control cells from healthy subjects, SERCA2 mRNA and protein expression was reduced in ASM cells from subjects with moderately severe asthma. SERCA2 expression was similarly reduced in ASM in vivo in subjects with moderate/severe asthma. Rises in [Ca(2+)](i) following cell surface receptor-induced SR activation, or inhibition of SERCA-mediated Ca(2+) re-uptake, were attenuated in ASM cells from asthmatics. Likewise, the return to baseline of [Ca](i) after stimulation by bradykinin was delayed by approximately 50% in ASM cells from asthmatics. siRNA-mediated knockdown of SERCA2 in ASM from healthy subjects increased cell spreading, eotaxin-1 release and proliferation. Our findings implicate a deficiency in SERCA2 in ASM in asthma that contributes to its secretory and hyperproliferative phenotype in asthma, and which may play a key role in mechanisms of airway remodeling.
Subject(s)
Asthma/metabolism , Bronchi/metabolism , Sarcoplasmic Reticulum/enzymology , Asthma/pathology , Asthma/physiopathology , Blotting, Western , Bronchi/pathology , Bronchi/physiopathology , Calcium/metabolism , Cell Movement , Cell Proliferation , Cells, Cultured , Chemokine CCL11/metabolism , Female , Gene Expression Regulation, Enzymologic , Homeostasis , Humans , Immunohistochemistry , Interleukin-13/pharmacology , Male , Myocytes, Smooth Muscle/drug effects , Myocytes, Smooth Muscle/metabolism , RNA Interference , Reverse Transcriptase Polymerase Chain Reaction , Sarcoplasmic Reticulum Calcium-Transporting ATPasesABSTRACT
BACKGROUND: IL-25 is thought to participate in allergic inflammation by propagating T(h)2-type responses. OBJECTIVE: To address the hypothesis that allergen provocation increases expression of IL-25 and its receptor IL-25R in the asthmatic bronchial mucosa and skin dermis of atopic subjects. METHODS: Sequential single and double immunostaining was used to evaluate the numbers and phenotypes of IL-25 and IL-25R immunoreactive cells in bronchial biopsies from mild atopic subjects with asthma (n = 10) before and 24 hours after allergen inhalation challenge and skin biopsies from atopic subjects (n = 10) up to 72 hours after allergen subepidermal injection. RESULTS: IL-25 immunoreactivity was expressed by a majority of epidermal cells in both organs at baseline and was not further augmented by challenge. IL-25R immunoreactive cells were rare in the epidermis before or after challenge. Allergen challenge was associated with significantly (P < .01) increased expression of IL-25 and IL-25R immunoreactivity in the submucosa of both organs. IL-25 immunoreactivity colocalized with eosinophils, mast cells, and endothelial cells, whereas IL-25R immunoreactivity colocalized with eosinophils, mast cells, endothelial cells, and T lymphocytes. In both organs, correlations were observed between increases in IL-25 expression and the magnitudes of the late-phase allergen-induced clinical responses. CONCLUSION: Allergen provocation induces functionally relevant, increased expression of IL-25 and its receptor in the asthmatic bronchial mucosa and dermis of sensitized atopic subjects. In addition to T cells, eosinophils, mast cells, and endothelial cells are potential sources and targets of IL-25 in the course of allergic inflammation.
Subject(s)
Asthma/immunology , Hypersensitivity, Immediate/immunology , Interleukin-17/biosynthesis , Receptors, Interleukin-17/biosynthesis , Receptors, Interleukin/biosynthesis , Respiratory Mucosa/immunology , Skin/immunology , Adult , Allergens/immunology , Asthma/metabolism , Bronchi/immunology , Bronchi/metabolism , Female , Humans , Hypersensitivity, Immediate/metabolism , Immunohistochemistry , Interleukin-17/immunology , Male , Receptors, Interleukin/immunology , Respiratory Mucosa/metabolism , Skin/metabolism , Young AdultABSTRACT
Asthma is characterised by airway hyper-responsiveness and remodelling, and there is mounting evidence that alterations in the phenotype of airway smooth muscle (ASM) play a central role in these processes. Although the concept that dysregulation of ASM Ca(2+) homeostasis may underlie at least part of these alterations has been around for many years, it is only relatively recently that the availability of ASM biopsies from subjects with mild and moderate asthma has allowed it to be properly investigated. In this article, critical components of the pathobiology of asthmatic ASM, including contractile function, proliferation, cell migration and secretion of proinflammatory cytokines and chemokines, are reviewed and related to associated changes in ASM Ca(2+) homeostasis. Based on this evidence, it is proposed that a unifying mechanism for the abnormal asthmatic phenotype is dysregulation of Ca(2+) homeostasis caused at least in part by a downregulation in expression and function of sarcoendoplasmic Ca(2+) ATPases (SERCAs).
Subject(s)
Airway Remodeling/physiology , Asthma/physiopathology , Calcium/metabolism , Muscle, Smooth/physiopathology , Cytokines/metabolism , Homeostasis/physiology , Humans , Muscle Contraction/physiology , Sarcoplasmic Reticulum Calcium-Transporting ATPases/physiologyABSTRACT
Asthma and chronic obstructive pulmonary disease (COPD) are associated with Th2 and Th1 differentiated T cells. The cytokine thymic stromal lymphopoietin (TSLP) promotes differentiation of Th2 T cells and secretion of chemokines which preferentially attract them. We hypothesized that there is distinct airways expression of TSLP and chemokines which preferentially attract Th1- and Th2-type T cells, and influx of T cells bearing their receptors in asthma and COPD. In situ hybridization, immunohistochemistry, and ELISA were used to examine the expression and cellular provenance of TSLP, Th2-attracting (TARC/CCL17, MDC/CCL22, I-309/CCL1), and Th1-attracting (IP-10/CXCL10, I-TAC/CXCL11) chemokines in the bronchial mucosa and bronchoalveolar lavage fluid of subjects with moderate/severe asthma, COPD, and controls. Cells expressing mRNA encoding TSLP, TARC/CCL17, MDC/CCL22, and IP-10/CXCL10, but not I-TAC/CXCL11 and I-309/CCL1, were significantly increased in severe asthma and COPD as compared with non-smoker controls (p < 0.02). This pattern was reflected in bronchoalveolar lavage fluid protein concentrations. Expression of the same chemokines was also increased in ex- and current smokers. The cellular sources of TSLP and chemokines were strikingly similar in severe asthma and COPD. The numbers of total bronchial mucosal T cells expressing the chemokine receptors CCR4, CCR8, and CXCR3 did not significantly differ in asthma, COPD, and controls. Both asthma and COPD are associated with elevated bronchial mucosal expression of TSLP and the same Th1- and Th2-attracting chemokines. Increased expression of these chemokines is not, however, associated with selective accumulation of T cells bearing their receptors.
Subject(s)
Asthma/immunology , Asthma/metabolism , Chemokines/biosynthesis , Chemotaxis, Leukocyte/immunology , Cytokines/biosynthesis , Pulmonary Disease, Chronic Obstructive/immunology , Pulmonary Disease, Chronic Obstructive/metabolism , Thymus Gland/immunology , ADAM Proteins/biosynthesis , Adult , Aged , Asthma/pathology , Chemokine CCL1/biosynthesis , Chemokine CCL17/biosynthesis , Chemokine CXCL10/biosynthesis , Chemokine CXCL11/biosynthesis , Chemokines/genetics , Cytokines/physiology , Female , Humans , Male , Middle Aged , Pulmonary Disease, Chronic Obstructive/pathology , Respiratory Mucosa/immunology , Respiratory Mucosa/metabolism , Respiratory Mucosa/pathology , Severity of Illness Index , Stromal Cells/immunology , Stromal Cells/metabolism , Stromal Cells/pathology , T-Lymphocyte Subsets/immunology , T-Lymphocyte Subsets/metabolism , T-Lymphocyte Subsets/pathology , Th1 Cells/immunology , Th1 Cells/metabolism , Th1 Cells/pathology , Th2 Cells/immunology , Th2 Cells/metabolism , Th2 Cells/pathology , Thymus Gland/metabolism , Thymus Gland/pathology , Tumor Suppressor Proteins/biosynthesis , Thymic Stromal LymphopoietinABSTRACT
Leukotriene (LT) E(4) mediates many of the principal features of bronchial asthma, such as bronchial constriction, hyperresponsiveness, eosinophilia, and increased vascular permeability. Furthermore, it is the most stable of the cysteinyl leukotrienes (CysLTs) and can be active at the site of release for a prolonged time after its synthesis. There might be several reasons why LTE(4) has been forgotten. LTE(4) demonstrated low affinity for CysLT(1) and CysLT(2) receptors in equilibrium competition assays. It was less potent than other CysLTs in functional assays, such as calcium flux, in cells transfected with CysLT(1) and CysLT(2). The introduction of CysLT(1) antagonists into clinical practice diverted interest into CysLT(1)-related mechanisms, which were mediated mainly by LTD(4). However, experiments with animal models and human studies have revealed that LTE(4) has unique characteristics that cannot be explained by the current knowledge of CysLT(1) and CysLT(2). These activities include its potency relative to other CysLTs to increase airway responsiveness to histamine, to enhance eosinophilic recruitment, and to increase vascular permeability. Asthmatic airways also demonstrate marked in vivo relative hyperresponsiveness to LTE(4), especially in patients with aspirin-sensitive respiratory disease. This has stimulated a search for additional LT receptors that would respond preferentially to LTE(4) stimulation.
Subject(s)
Asthma/immunology , Bronchial Hyperreactivity/immunology , Drug Hypersensitivity/immunology , Leukotriene E4/immunology , Animals , Aspirin/adverse effects , Asthma/drug therapy , Bronchial Hyperreactivity/drug therapy , Drug Hypersensitivity/drug therapy , Histamine , Humans , Leukotriene C4/immunology , Leukotriene D4/immunology , Methacholine Chloride , Receptors, Leukotriene/immunology , Skin/drug effects , Skin/immunology , Skin/pathologyABSTRACT
BACKGROUND: T(H)1 cell-mediated immunity is essential for host defense against a variety of intracellular pathogens, such as mycobacteria, salmonella, and Leishmania species. A major T(H)1-mediated effector mechanism involves the IFN-gamma-induced killing of the pathogen by infected macrophages. OBJECTIVES: The range of known T(H)1-specific effector molecules is limited, especially in human subjects. We sought to identify novel effector molecules that might be involved in T(H)1-mediated pathogen clearance. METHODS: We performed microarray-based analysis of human T(H)1 and T(H)2 cells to identify T(H)1-specific molecules. These analyses identified the extracellular matrix molecule fibronectin as a highly expressed T(H)1-specific molecule. We examined the expression of fibronectin in a variety of human cell types by using real-time RT-PCR, ELISA, and Western blotting. We also studied the role of fibronectin in modulating monocyte phenotype using in vitro culture. RESULTS: We show that human T(H)1 cells constitutively express and secrete fibronectin after in vitro differentiation from naive precursors. Furthermore, we demonstrate that ex vivo human T(H)1 cells selectively express fibronectin when compared with T(H)2 cells. The predominant isoform of fibronectin expressed by T(H)1 cells contains additional domains of the protein responsible for alpha4beta1 integrin binding and activation of Toll-like receptor 4. We show that treatment of monocytes with T(H)1 cell-derived fibronectin induces expression of the proinflammatory cytokine IL-6 while inhibiting IL-10 expression. CONCLUSIONS: Because fibronectin also plays a major role in the attachment and opsonization of numerous intracellular pathogens, we propose that it might be a critical molecule produced by T(H)1 cells involved in pathogen eradication.
Subject(s)
Fibronectins/metabolism , Monocytes/immunology , Th1 Cells/immunology , Th2 Cells/immunology , Cell Differentiation/immunology , Cells, Cultured , Fibronectins/genetics , Fibronectins/pharmacology , Host-Pathogen Interactions/immunology , Humans , Integrin alpha4beta1/immunology , Integrin alpha4beta1/metabolism , Interleukin-10/antagonists & inhibitors , Interleukin-10/immunology , Interleukin-10/metabolism , Interleukin-6/agonists , Interleukin-6/immunology , Interleukin-6/metabolism , Monocytes/drug effects , Monocytes/metabolism , Oligonucleotide Array Sequence Analysis , Protein Isoforms/immunology , Protein Isoforms/metabolism , Th1 Cells/metabolism , Th2 Cells/metabolism , Toll-Like Receptor 4/immunology , Toll-Like Receptor 4/metabolismABSTRACT
We previously reported that human CD4+ Tregs secrete high levels of IL-10 when stimulated in the presence of dexamethasone and calcitriol (vitamin D3). We now show that following stimulation by allergen, IL-10-secreting Tregs inhibit cytokine secretion by allergen-specific Th2 cells in an IL-10-dependent manner. A proportion of patients with severe asthma fail to demonstrate clinical improvement upon glucocorticoid therapy, and their asthma is characterized as glucocorticoid resistant (SR, abbreviation derived from "steroid resistant"). Dexamethasone does not enhance secretion of IL-10 by their CD4+ T cells. Addition of vitamin D3 with dexamethasone to cultures of SR CD4+ T cells enhanced IL-10 synthesis to levels observed in cells from glucocorticoid-sensitive patients cultured with dexamethasone alone. Furthermore, pretreatment with IL-10 fully restored IL-10 synthesis in these cells in response to dexamethasone. Vitamin D3 significantly overcame the inhibition of glucocorticoid-receptor expression by dexamethasone while IL-10 upregulated glucocorticoid-receptor expression by CD4+ T cells, suggesting potential mechanisms whereby these treatments may overcome poor glucocorticoid responsiveness. We show here that administration of vitamin D3 to healthy individuals and SR asthmatic patients enhanced subsequent responsiveness to dexamethasone for induction of IL-10. This strongly suggests that vitamin D3 could potentially increase the therapeutic response to glucocorticoids in SR patients.
Subject(s)
Asthma/immunology , CD4-Positive T-Lymphocytes/immunology , Dexamethasone/therapeutic use , Drug Resistance/immunology , Glucocorticoids/therapeutic use , Interleukin-10/metabolism , Anti-Inflammatory Agents/therapeutic use , Asthma/drug therapy , Cholecalciferol/therapeutic use , Humans , Reference ValuesABSTRACT
RATIONALE: Airway remodeling in asthma involves accumulation of airway smooth muscle (ASM) and increased vascularity due to angiogenesis. Bronchial blood vessels and ASM are found in close proximity, and ASM releases multiple proinflammatory mediators, including vascular endothelial growth factor (VEGF). OBJECTIVES: We examined whether release of proangiogenic mediators is increased in ASM from subjects with asthma and whether this is translated to induction of angiogenesis. METHODS: Biopsy-derived ASM cells were cultured from 12 subjects with mild asthma, 8 with moderate asthma, and 9 healthy control subjects. Angiogenesis induced by cell-conditioned medium (CM) from ASM was evaluated in a tubule formation assay. Anti-CD31-labeled tubules were quantified by image analysis. Angiogenic factors in CM were quantified by antibody arrays and by enzyme-linked immunosorbent assay. MEASUREMENTS AND MAIN RESULTS: Induction of angiogenesis by CM from unstimulated ASM was increased in subjects with mild asthma (twofold) and moderate asthma (threefold), compared with healthy CM (P < 0.001). Levels of angiogenic factors (VEGF, angiopoietin [Ang]-1, angiogenin) were similarly elevated in CM from subjects with asthma compared with that from healthy subjects (P < 0.05), whereas antiangiogenic factors (endostatin, Ang-2) were unchanged. VEGF, Ang-1, and angiogenin in combination increased vascularity (twofold, P < 0.01) in cultured intact biopsies. Selective VEGF immunodepletion abolished enhanced tubule formation by CM from asthmatic ASM (P < 0.01), but CM depletion of Ang-1 or angiogenin had no effect. CONCLUSIONS: ASM cultured from subjects with mild or moderate asthma, but not from healthy control subjects, promotes angiogenesis in vitro. This proangiogenic capacity resides in elevated VEGF release and suggests that ASM regulates airway neovascularization in asthma.
Subject(s)
Angiogenesis Inducing Agents/metabolism , Angiogenic Proteins/metabolism , Asthma/physiopathology , Muscle, Smooth/metabolism , Neovascularization, Pathologic , Adult , Angiopoietin-1 , Bronchi/blood supply , Bronchi/pathology , Case-Control Studies , Cells, Cultured , Female , Humans , Male , Myocytes, Smooth Muscle/metabolism , Protein Array Analysis , Vascular Endothelial Growth Factor A/metabolismABSTRACT
The inflammatory response observed in allergic disease involves multiple cell types but is orchestrated in part by the T(H)2 cytokines IL-4, IL-5, and IL-13. In recent years, the transcription factors that control the expression and function of these cytokines have been elucidated, including signal transducer and activator of transcription 6, GATA3, nuclear factor of activated T cells, and nuclear factor kappaB. These molecules are attractive targets for therapeutic intervention because they regulate the expression of numerous effector molecules and functions simultaneously. For instance, the immunosuppressive agents glucocorticoids and cyclosporin A both function by repressing the activity of transcription factors through a variety of mechanisms. In this review we examine the role of each transcription factor in allergic disease and discuss approaches that have been taken to therapeutically interfere with transcription factor function in allergic disease.
Subject(s)
Hypersensitivity/pathology , Hypersensitivity/prevention & control , Inflammation Mediators/antagonists & inhibitors , Inflammation Mediators/physiology , Transcription Factors/antagonists & inhibitors , Transcription Factors/physiology , Animals , Drug Delivery Systems , Humans , Hypersensitivity/drug therapy , Hypersensitivity/immunology , Immunosuppressive Agents/adverse effects , Immunosuppressive Agents/therapeutic use , Inflammation Mediators/adverse effects , Signal Transduction/drug effects , Signal Transduction/immunology , Th2 Cells/immunology , Th2 Cells/metabolism , Th2 Cells/pathology , Transcription Factors/adverse effectsABSTRACT
BACKGROUND: Low emission zones (LEZ) are an increasingly common, but unevaluated, intervention aimed at improving urban air quality and public health. We investigated the impact of London's LEZ on air quality and children's respiratory health. METHODS: We did a sequential annual cross-sectional study of 2164 children aged 8-9 years attending primary schools between 2009-10 and 2013-14 in central London, UK, following the introduction of London's LEZ in February, 2008. We examined the association between modelled pollutant exposures of nitrogen oxides (including nitrogen dioxide [NO2]) and particulate matter with a diameter of less than 2·5 µm (PM2·5) and less than 10 µm (PM10) and lung function: postbronchodilator forced expiratory volume in 1 s (FEV1, primary outcome), forced vital capacity (FVC), and respiratory or allergic symptoms. We assigned annual exposures by each child's home and school address, as well as spatially resolved estimates for the 3 h (0600-0900 h), 24 h, and 7 days before each child's assessment, to isolate long-term from short-term effects. FINDINGS: The percentage of children living at addresses exceeding the EU limit value for annual NO2 (40 µg/m3) fell from 99% (444/450) in 2009 to 34% (150/441) in 2013. Over this period, we identified a reduction in NO2 at both roadside (median -1·35 µg/m3 per year; 95% CI -2·09 to -0·61; p=0·0004) and background locations (-0·97; -1·56 to -0·38; p=0·0013), but not for PM10. The effect on PM2·5 was equivocal. We found no association between postbronchodilator FEV1 and annual residential pollutant attributions. By contrast, FVC was inversely correlated with annual NO2 (-0·0023 L/µg per m3; -0·0044 to -0·0002; p=0·033) and PM10 (-0·0090 L/µg per m3; -0·0175 to -0·0005; p=0·038). INTERPRETATION: Within London's LEZ, a smaller lung volume in children was associated with higher annual air pollutant exposures. We found no evidence of a reduction in the proportion of children with small lungs over this period, despite small improvements in air quality in highly polluted urban areas during the implementation of London's LEZ. Interventions that deliver larger reductions in emissions might yield improvements in children's health. FUNDING: National Institute for Health Research Biomedical Research Centre at Guy's and St Thomas' National Health Service (NHS) Foundation Trust and King's College London, NHS Hackney, Lee Him donation, and Felicity Wilde Charitable Trust.