Phagocytosis of apoptotic eosinophils but not neutrophils by bronchial epithelial cells

BACKGROUND: We have previously demonstrated that human bronchial epithelial cells engulf apoptotic eosinophils. OBJECTIVES: To compare and contrast the phagocytic capabilities of monocyte-derived macrophage and primary airway epithelial cells for apoptotic granulocytes. RESULTS: Here we compared phagocytosis of human apoptotic eosinophils and neutrophils by small and large airway epithelial cells (SAEC and LAEC) and monocyte-derived macrophages. Confocal microscopy of F-actin staining and scanning and transmission electron microscopy revealed phagocytic cup formation around apoptotic eosinophils by airway epithelial cells (AEC) membranes with evidence of their digestion. Resting and cytokine-stimulated AEC did not recognize and ingest apoptotic neutrophils. The latter were phagocytosed by macrophages that exhibited greater ingestion of and higher capacity for, apoptotic eosinophils over apoptotic neutrophils. Cytochalasin D completely abolished uptake of apoptotic eosinophils by SAEC, LAEC or macrophage monolayers. Ligation of epithelial cell CD44 receptors for 24 h increased phagocytosis of apoptotic eosinophils by SAEC and LAEC with a potency comparable with that of IL-1. Phagocytosis was a specific receptor-mediated process involving integrin- (alphavbeta3, alphavbeta5, CD36), phosphatidylserine receptor- and lectin-dependent mechanisms. No significant differences were observed in avarice for apoptotic eosinophils by SAEC or LAEC either resting, CD44 monoclonal antibodies- or cytokine- stimulated, or in their usage and expression of recognition receptors. CONCLUSION: These findings further suggest and define an important role for the bronchial epithelium in the selective removal of apoptotic eosinophils from the airways in asthma.     

C3 synthesis by A549 alveolar epithelial cells is increased by interferon-gamma and dexamethasone.

The third component of complement, C3, is produced in the lung by several cell types including alveolar epithelial cells. Since interferon-gamma (IFN-gamma) and dexamethasone regulate C3 gene expression in non-pulmonary cells, and because IFN-gamma and dexamethasone interact to regulate the functional activity of alveolar epithelial cells, we investigated the effects of IFN-gamma and dexamethasone on C3 production by A549 human alveolar epithelial cells. Treatment of A549 cells with IFN-gamma alone increased C3 production in a time-and dose-dependent manner. Maximal increase in C3 production occurred after stimulation of A549 cells with 500 IU/ml IFN-gamma for 3 days and was 3.4-fold greater than control. Dexamethasone (0.1 microM) stimulation of A549 cells increased C3 production 6.7-fold over controls on day 3. Treatment of A549 cells with IFN-gamma plus dexamethasone resulted in an 11-to 13-fold increase in C3 synthesis. C3 mRNA levels were increased in A549 cells treated with IFN-gamma and dexamethasone individually and in combination suggesting that IFN-gamma and dexamethasone increase C3 synthesis by a pre-translational mechanism. IFN-gamma and dexamethasone did not alter the two-chain structure of the C3 molecule produced by A549 cells, as assessed by Western blotting. We speculate that IFN-gamma and glucocorticoids may be important in the local regulation of C3 synthesis in the lung.     

Synergic production of neutrophil chemotactic activity by colonic epithelial cells and eosinophils

The presence of eosinophils in the lumen and mucosa of the intestine is characteristic of both ulcerative colitis (UC) and Crohn's disease (CD). There is evidence of eosinophil activation in the intestine during acute inflammatory episodes of these diseases; these episodes are also characterized by an influx of neutrophils, which have the potential to cause extensive tissue damage. We undertook a study to determine whether eosinophils in contact with colonic epithelial cells produce factors that may attract neutrophils in response to immunological stimulation. Neutrophil chemotactic activity (NCA) and concentrations of three neutrophil-attracting CXC chemokines – CXCL1 (Groα), CXCL5 (Ena78) and CXCL8 (IL8) – were measured in supernatants of T84 colonic epithelial cells and blood eosinophils or eosinophil-like myeloid leukaemia cells (AML14.3D10), alone or in combination. Cells were stimulated with serum-opsonized zymosan (OZ) particles. NCA (P < 0.005) and CXCL5 levels (P < 0.05) in the supernatants of OZ-stimulated epithelial/eosinophil co-cultures were significantly higher than in the supernatants of either cell type alone. Release of CXCL1 (P < 0.05) and CXCL8 (P < 0.01) from OZ-stimulated co-culture supernatants was significantly higher than from OZ-stimulated eosinophils but not higher than from OZ-stimulated epithelial cells. Eosinophils and colonic epithelial cells exhibit synergy in production of neutrophil chemoattractants in response to immunological stimulation. This may represent a mechanism for exaggerated recruitment of neutrophils to the intestine in response to acute infection in conditions that are characterized by the presence of eosinophils in the bowel.     

Neutrophil defensins stimulate the release of cytokines by airway epithelial cells: modulation by dexamethasone

OBJECTIVE AND DESIGN: Neutrophils may contribute to recruiting other cells to sites of inflammation by generating chemotactic signals themselves, or by stimulating other cell types to release chemoattractants such as interleukin-8 (IL-8). Recently, we demonstrated that neutrophil-derived alpha-defensins are able to increase IL-8 expression in airway epithelial cells. In addition, it has previously been reported that neutrophil elastase-induced IL-8 synthesis was insensitive to inhibition by the glucocorticoid dexamethasone. The aim of the present study was to investigate the effect of defensins on the expression of various cytokines in cultured airway epithelial cells and to examine the effect of dexamethasone on defensin-induced cytokine synthesis in these cells. METHODS: Cultures of A549 cells and primary bronchial epithelial cells (PBEC) were stimulated with defensins either alone or in the presence of dexamethasone. Supernatants were analyzed for IL-8, ENA-78, IL-6, MCP-1 and GM-CSF by ELISA. In addition, IL-8 and ENA-78 mRNA was detected by Northern blot analysis. RESULTS: Defensins increased IL-8 expression, ENA-78, MCP-1 and GM-CSF release from A549 cells, whereas in PBEC only IL-8 and IL-6 were increased. Pre-treatment with dexamethasone significantly reduced defensin-induced IL-6, IL-8 and ENA-78 synthesis in airway epithelial cells. In addition, dexamethasone also reduced the neutrophil chemotactic activity in supernatants of these cells. CONCLUSIONS: The results from the present study indicate that defensins differentially induce cytokine secretion by A549 cells and PBEC. Glucocorticoids may interfere with the defensin-induced inflammatory process by reducing defensin-induced cytokine secretion in lung epithelial cells.     

Internalization of a non-pathogenic mycobacteria by macropinocytosis in human alveolar epithelial A549 cells

Mycobacterium smegmatis (MSM) a non-pathogenic mycobacterium is often employed as a tool to understand many aspects of the mycobacterial infections. However, its own biology and particularly its mechanism of entry into non-phagocytic cells are not well known. Previously, we demonstrated that Mycobacterium tuberculosis (MTB) invades epithelial cells by macropinocytosis. In the present study, we investigated whether MSM also invades human epithelial type II pneumocytes (A549) by macropinocytosis. Infection of A549 cells with MSM elicited actin filaments redistribution, lamellipodia formation and increased fluid phase uptake, suggesting macropinocytosis. Furthermore, macropinocytosis inhibitors like cytochalasin D and amiloride caused inhibition of fluid phase and bacterial uptake. We can conclude that MSM, like MTB, takes advantage of macropinocytosis for entry into epithelial cells, however, unlike MTB, internalized MSM are killed by host cells. These findings suggest that induction of macropinocytosis and cell invasion is not an exclusive feature of pathogenic organisms.     

Human eosinophils and mast cells: Birds of a feather flock together

While the origin of the phrase “birds of a feather flock together” is unclear, it has been in use for centuries and is typically employed to describe the phenomenon that people with similar tastes or interests tend to seek each other out and congregate together. In this review, we have co-opted this phrase to compare innate immune cells of related origin, the eosinophil and mast cell, because they very often accumulate together in tissue sites under both homeostatic and inflammatory conditions. To highlight overlapping yet distinct features, their hematopoietic development, cell surface phenotype, mediator release profiles and roles in diseases have been compared and contrasted. What emerges is a sense that these two cell types often interact with each other and their tissue environment to provide synergistic contributions to a variety of normal and pathologic immune responses.     

低氧对人肺泡上皮细胞糖皮质激素受体表达及对地塞米松抗炎效应的影响

目的 研究低氧条件下人肺泡上皮细胞糖皮质激素受体(GR)的两种亚型GRα和GRβ水平的表达及在地塞米松抗炎效应的变化.方法 以人A549肺泡上皮细胞系为模型,细胞分别在常氧(37℃,950 mL/L空气、50 mL/L CO2)和低氧(37℃,950 mL/L N2、250 mL/L CO2)条件下培养24、48、72 h,Western blot法检测各组细胞GRα和GRβ的蛋白表达水平;脂多糖(LPS 10μg/mL)和不同浓度的地塞米松(DEX 10-8 mol/L～ 10-6 mol/L)一起加入A549细胞培养液中,分别常氧和低氧培养48 h,放射免疫法检测细胞培养上清中的IL-8水平.结果 24h低氧组的GRα水平与24h常氧组相比无明显差别(P＞0.05),48 h和72 h低氧组的GRα水平与相应时间点的常氧组相比明显下降(P＜0.05,P＜0.01),24、48、72 h低氧组GRα水平呈进行性下降(P＜0.05),而各相应时间点的常氧组GRα水平无明显差别(P＞0.05);低氧组和常氧组各时间点GRβ的表达无明显差别.在10-7 mol/L和10-6 mol/L DEX浓度时,低氧培养48 h的A549细胞上清的IL-8均高于相应的常氧组的IL-8水平(P＜0.05).结论 低氧可以时间依赖性的导致人肺泡上皮细胞A549 GRα表达水平下降并减弱地塞米松的抗炎效应.     

Integrin alphavbeta3-mediated endocytosis of immobilized fibrinogen by A549 lung alveolar epithelial cells

Fibrinogen (FBG), together with its polymerized form fibrin, modulates cellular responses during wound repair and tissue remodeling. Thus, we sought to determine whether A549 lung epithelial type II-like cells would endocytose insoluble, surface-bound FBG as a potential mechanism of alveolar matrix remodeling. Surface-bound FBG was endocytosed into either lysosomes or late endosomes by A549 cells through arg-gly-asp-dependent binding to alphavbeta3 but not alpha5beta1 integrin receptors. Soluble FBG added to confluent monolayers of A549 cells was not endocytosed. Unlike the uptake of the extracellular matrix glycoproteins vitronectin and thrombospondin by other cell types, endocytosis of FBG by A549 cells was neither inhibited by heparin nor dependent on binding to cell-surface heparan sulfate proteoglycans. FBG did not colocalize with endocytosed transferrin, whereas dextran showed partial colocalization with FBG in endocytic vesicles, suggesting nonclathrin-mediated endocytosis. Inhibition of actin filament polymerization blocked endocytosis of both dextran and FBG but not transferrin, providing further support that FBG is endocytosed via a nonclathrin pathway. Disruption of actin polymerization inhibited integrin-mediated cell spreading, which contributed to an overall reduction in FBG clearance that was most likely due to reduced cell migration and associated pericellular proteolysis. Trasylol inhibition of extracellular plasmin activity did not inhibit endocytosis of FBG. The endocytosed FBG was degraded to trichloroacetic acid-soluble fragments that showed an electrophoretic pattern distinctly different from plasmin-degraded FBG. Together, these results suggest that endocytosis of matrix-associated FBG by alveolar epithelial cells may be involved in the processes of alveolar tissue repair and matrix remodeling.     

The milieu of damaged alveolar epithelial type 2 cells stimulates alveolar wound repair by endogenous and exogenous progenitors

Alveolar epithelial integrity is dependent upon the alveolar milieu, yet the milieu of the damaged alveolar epithelial cell type 2 (AEC2) has been little studied. Characterization of its components may offer the potential for ex vivo manipulation of stem cells to optimize their therapeutic potential. We examined the cytokine profile of AEC2 damage milieu, hypothesizing that it would promote endogenous epithelial repair while recruiting cells from other locations and instructing their engraftment and differentiation. Bronchoalveolar lavage and lung extract from hyperoxic rats represented AEC2 in vivo damage milieu, and medium from a scratch-damaged AEC2 monolayer represented in vitro damage. CINC-2 and ICAM, the major cytokines detected by proteomic cytokine array in AEC2 damage milieu, were chemoattractive to normoxic AECs and expedited in vitro wound healing, which was blocked by their respective neutralizing antibodies. The AEC2 damage milieu was also chemotactic for exogenous uncommitted human amniotic fluid stem cells (hAFSCs), increasing migration greater than 20-fold. hAFSCs attached within an in vitro AEC2 wound and expedited wound repair by contributing cytokines migration inhibitory factor and plasminogen activator inhibitor 1 to the AEC2 damage milieu, which promoted wound healing. The AEC2 damage milieu also promoted differentiation of a subpopulation of hAFSCs to express SPC, TTF-1, and ABCA3, phenotypic markers of distal alveolar epithelium. Thus, the microenvironment created by AEC2 damage not only promotes autocrine repair but also can attract uncommitted stem cells, which further augment healing through cytokine secretion and differentiation.     

S100P expression in human esophageal epithelial cells: Human esophageal epithelial cells sequentially produce different S100 proteins in the process of differentiation

Extracellular calcium ions (Ca(2+)) are important in regulating the differentiation of keratinocytes and squamous epithelial cells. To clarify the mechanisms involved in the differentiation of human esophageal epithelial cells (EECs), we used the primary culture of human EECs, which can be differentiated by increasing the concentration of extracellular Ca(2+), and tried to reveal the extracellular Ca(2+) inducible genes using a differential display (DD) method. We found that the calcium-binding protein S100P showed a Ca(2+)-inducible expression in the EECs. Our immunohistochemical study demonstrated that differentiated large EECs expressing S100P overlie immature proliferating cells which lack S100P immunoreactivity. S100P was detected in vivo in the suprabasal layers of the epithelium. These findings indicate that S100P expression is closely associated with differentiation of human EECs. We also investigated the expression of other S100 proteins, including S100A2, S100A6, and CAAF1 (S100A12), in human EECs. Most of the immature EECs were positive for S100A2 and S100A6, whereas the S100A12-producing cells were similar to the S100P-producing cells. In vivo, S100A12 was strongly detected on all epithelial cells except for basal and proliferating cells. S100A2 was detected on all of the epithelial cells. S100A6 was preferentially seen in the cells of basal layers. These findings suggest that within EECs S100 proteins might play important roles in cell differentiation during specific stages. Among them, S100P expression is unique in that this protein is transiently expressed during the early stage of differentiation.     

A morphometric examination of type II alveolar epithelial cells in normal and isolated-perfused dog lungs

The volume densities of type II alveolar cell cytoplasmic organelles and alveolar surface densities were estimated by established stereologic procedures. The morphometric measurements were obtained from normal dog lungs (in situ) and isolated dog lungs perfused for 30-minute, 1-hour, and 2-hour periods. The type II cell lamellar body volume densities and the alveolar surface densities progressively decreased as the times of perfusion were increased. The volume densities of the granular and agranular endoplasmic reticulum progressively increased during the periods of perfusion. These morphometric parameters from lungs in situ and isolated lungs suggest that changes occur in pulmonary surfactant synthesis and activity during perfusion. It is further postulated that progressive increases in the rates of surfactant removal and/or inactivation during perfusion may contribute to spontaneous edema in lungs isolated for periods exceeding two hours. The morphologic and physiologic integrity of isolated perfused lung preparations, widely used as models of lungs in vivo, in situ requires further evaluation.     

Modulation of cadherin and catenins expression by tumor necrosis factor-alpha and dexamethasone in human bronchial epithelial cells

Asthma is an inflammatory disease, and the epithelial mesenchymal unit appears to be of importance in regulating the disease mechanisms. Cell-cell adhesion plays an important role in tissue morphogenesis and homeostasis and is commonly mediated by cadherins, a family of Ca(2+)-dependent transmembrane adhesion receptors. The cadherin family is involved in control of the cellular architecture. Proinflammatory cytokines such as tumor necrosis factor (TNF)-alpha are involved in asthma and may interfere with epithelial integrity. In the present study, we investigated the role of TNF-alpha and dexamethasone on the expression of E-cadherin, beta-catenin, and gamma-catenin. We used two bronchial epithelial cell models: primary small airway epithelial cell cultures and primary culture obtained from human bronchial tubes. After 48 h of TNF-alpha stimulation with or without dexamethasone expression of E-cadherin, beta-catenin and gamma-catenin were analyzed using Western blot analysis and immunofluorescence. This study showed a decrease in the expression of adhesion molecules in both epithelial cell cultures after stimulation. Dexamethasone and anti-TNF-alpha inhibited this effect. In unstimulated cells, E-cadherin and beta- and gamma-catenin expression was membranous, expressed only on the lateral cell wall with minimal cytoplasmic expression. Immunoreactivity was cytoplasmic in stimulated cells. We demonstrated, using Western blot analysis and immunofluorescence, that proinflammatory cytokines could be responsible for structural damage to the epithelium and that this process was potentially reversed by steroids.