Progressive Changes In Inflammatory And Matrix Adherence Of Human Bronchial Epithelial Cells With Respiratory Syncytial Virus Infection

ObjectivesThe functions of airway epithelial cells (AECs) have attracted more and more attentions of respiratory researchers and physicians. The airway epithelium is a protective barrier from inhaled particulates and aerosols from the external environment and forms the mucosal immune barrier, the first structural cell defense against environmental insults such as virus and particulate matter. Generally, the airway epithelium is mainly for keeping the airway open and constitutive integrity, and maintaining the homeostasis of the local microenvironment under normal condition. However, when stressed or injured, epithelial cells can function as both "target" and "effector" cells. As target cells, they are influenced by exogenous inflammatory agents. As effector cells, they produce and release adhesion molecules as well as soluble factors such as chemokines. It has become increasingly clear that airway epithelial cells play a critical role in innate and adaptive immune responses as well as mucosal inflammation.The balanced expression of adhesive molecules in AECs should be important for keeping airway homeostasis. Intercellular adhesion molecule-1(ICAM-1)[belonging to the immunoglobulin supergene family] is for epithelial cell regulation of the airway response to pathogens. ICAM-1binds with β2integrins expressed on leukocytes and participates in neutrophil recruitment into the airway. In addition to attracting diapedesis, ICAM-1can also function as a signaling molecule transmitting outside-inside signaling, resulting in transcription factor and protein kinases activation through phosphorylation of intracellular proteins. More and more evidence suggests that adhesion molecules play central roles in maintaining the integrity of the epithelium. There are many evidences that integrity of the tight junctions between airway epithelial cells could be influenced by E-cadherin, a transmembrane glycoprotein for calcium-dependent, homophilic connection between adjacent cells. The cytoplasmic domain of E-cadherin associates with a-, β-and y-catenin which link E-cadherin to the actin cytoskeleton. Cell-cell adhesion is dependent on the levels of expression of E-cadherin.Respiratory syncytial virus (RSV) is the leading causes of bronchiolitis and pneumonia worldwide, especially in infants, the elderly, and the immunocompromised. RSV normally is highly restricted to the airway epithelial cells and it can replicate in these cells. RSV infection of airway epithelial cells is an important initial event and can cause the airway epithelial cell necrosis, loss and decay of mucosa, and a large number of the release of inflammatory mediators, which initiate the immune and pathological reaction. The airway epithelial cells respond to RSV infection with specific alterations in gene expression, including production of ICAM-1, cytokines, chemokines and other inflammatory mediators. Moreover, RSV can cause disruption of the epithelial apical junction complex and remodeling of the apical actin cytoskeleton. On the other hand, RSV possesses a lot of expressed genes which antagonize the effect of type I interferons and other related host factor pathways that inhibit viral replication efficiency. RSV infection alters host gene transcription and transcripts translation through specific antagonism of the function of host proteins, induction of RNA stress granules and altered patterns of host gene expression. Thus, the interplay between epithelial cells and RSV infection involves a dynamic process whose course of progression and persistence depends upon how efficiently RSV is cleared by the epithelial cells.In addition to the acute manifestations induced by respiratory syncytial virus infection, persistent infection may be associated with long-term complications in the development of chronic respiratory diseases. The mechanism for long-term airway insufficiency after RSV infection is not yet understood. To understand the mechanisms underlying RSV-induced long-term consequences, we established an in vitro RSV (strain A2) infection model using human bronchial epithelial cells (16HBECs) that persists over several generations and dynamic analyses were used to investigate the function of bronchial epithelial cell inflammatory and matrix adherence to further the understanding of the development of airway dysfunction following acute RSV infection.Therefore, this study focused on:(1) To establish an in vitro RSV (strain A2) infection model using16HBECs that persists over several generations, and analyze the viral replication in cells and the susceptibility to RSV infection.(2) To observe the dynamic changes of the function of bronchial epithelial cell inflammatory and matrix adherence using RSV-infected16HBECs that persist over four generations.(3) To analyze the effects of ICAM-1and E-cadherin on the function of bronchial epithelial cell inflammatory and matrix adherence and explore its molecular mechanism.Methods and results1. To establish an in vitro RSV (strain A2) infection model using16HBE cells that persists over several generations60%-70%confluent monolayer cultures of16HBECs were infected with RSV at multiplicity of infections (MOIs) of0.00025,0.0005,0.0010,0.0067,0.0134,0.0268and0.0536according to experiments in advance. During passages, RSV persistence was verified and monitored using a Respiratory Syncytial Virus Real-time PCR Kit and the number of syncytial cells in each generation was analyzed using a Leica DC200digital camera system.The results showed that when RSV at MOIs≤0.0010was used, samples were tested clearly positive for RSV in the first generation (G), but lost their positivity in G2to G4in most cases. Absence of positivity remained up to the twentieth generation. Very little apparent changes in cell morphologic characteristics were observed. However, when RSV at MOI=0.0067was used to infect16HBECs, RSV was progressively cleared by16HBECs (or the16HBECs were destroyed) in G2or G3during successive passages. In most cases (about80%), the16HBECs survived to G4. Surviving16HBECs in G2showed similar healthy cell monolayer morphology as cultures of uninfected cells, while in G3some small syncytia and irregularly shaped cells began to form and in G4large syncytia were observed with decreased numbers of cells. G5cultures were found to contain predominantly lysed cells, large amounts of syncytia, and a residue of scattered islands of cells adherent to the substratum that died shortly afterwards. Though it was effective at promoting syncytia formation at G3, higher MOIs of RSV infection (MOIs≥0.0134) led to minimal levels of survival. Therefore, MOI0.0067was used to analyze progressive changes in inflammatory and matrix adherence at G1to G4.These data demonstrate the susceptibility of16HBECs to RSV strain A2and their capacity to produce long-term progressive RSV infection. Therefore, we establish an in vitro RSV (strain A2) infection model using16HBECs that persists over four generations. 2. The dynamic changes of the function of bronchial epithelial cell inflammatory and matrix adherence after RSV-infected16HBECs that persist over four generationsConcentrations of interleukin (IL)-1β, IL-6, IL-8, tumor necrosis factor (TNF)-α, interferon-γ-induced protein (IP)-10, and macrophage inflammatory protein (MIP)-1α in the culture supernatant were determined using enzyme-linked immunosorbent assay (ELISA) kits according to the manufacturer’s protocol. It was found that RSV infection up-regulated the release of an array of inflammatory molecules in16HBECs. IL-6levels progressively increased in G2through G4; IL-1β, IL-8, TNF-α, and MIP-1α increased in G3and G4, and IP-10increased only in G4.Leukocyte-16HBEC adherence was analyzed using Wright-Giemsa staining. The results show that the adherence of leukocytes to16HBECs was low in control group. After infection with RSV, leukocyte adherence of16HBECs remained low in G1, but significantly and gradually increased17.8-to43.0-fold in G2to G4(p<0.001compared to control). Parallel experiments using fluorescence-activated cell sorting analysis yielded similar results. There was no significant difference between the G1and control group. However, G2to G416HBECs showed progressively enhanced binding to leukocytes.To assess the effects of RSV infection on adherence of16HBECs to matrix, we coated plates with rat tail tendon collagen type I prior to plating cells and assessed numbers of adherent cells after RSV infection. Results showed that similar numbers of16HBECs remained adherent in G1and G2, while progressively fewer cells adhered to matrix in G3and G4.These results suggested that leukocyte and matrix adherence of RSV-infected16HBECs did not change in G1or G2, but subsequently, cytokine/chemokine secretion and leukocyte adherence increased drastically while matrix adherence decreased until the cells died.3. The effects of ICAM-1and E-cadherin on the function of bronchial epithelial cell inflammatory and matrix adherenceTo further examine the role of adhesion molecules in the progression of RSV infection, we examined expression and secretion of ICAM-1and E-cadherin throughout the successive generations of infection. With the exception of G1, we found that the levels of ICAM-1mRNA in RSV-infected16HBECs gradually increased with each progressive generation and peaked in G4(about6.2-fold increase compared to control). Western blot analysis verified that ICAM-1protein was significantly upregulated in G2(about4.8-fold), G3(about5.9-fold), and G4(about6.7-fold) compared with the control. Similar results were obtained using either a whole-cell ELISA for ICAM-1level or an ELISA of soluble ICAM-1(sICAM-1) in conditioned medium (4.4-fold increase in G3and25.2-fold increase in G4).We also assessed the expression of the CADHERIN1(CDH1), the gene for E-cadherin. Real-time PCR results showed that CDH1mRNA expression in RSV-infected16HBECs was up-regulated compared to control until G4. However, it was dramatically decreased in G4relative to G3. Western blot assay results supported this finding:while E-cadherin protein expression was increased in G3relative to the control, it was almost undetectable in G4.To determine whether adhesive molecules may be involved in RSV-infected16HBECs adherence, we subjected cells in G3to neutralizing antibodies against ICAM-1and E-cadherin. Neutralization of ICAM-1but not E-cadherin resulted in significantly decreased TNF-a levels and leukocyte adherence to16HBECs compared with G3group, while the levels of other cytokines and chemokines remained unchanged. Furthermore, the adherence of16HBECs to matrix was significantly reduced by anti-E-cadherin antibody, but not anti-ICAM-1antibody.These findings suggest that the changes of ICAM-1and E-cadherin expression in progressive generations of RSV-infection16HBECs may contribute to inflammation mobilization and epithelial shedding.Conclusion This study established an in vitro RSV (strain A2) infection model using16HBECs that persists over four generations. These data demonstrate the susceptibility of16HBECs to RSV and their capacity to produce long-term progressive RSV infection, which may contribute to inflammation mobilization and epithelial shedding by the changes of ICAM-1and E-cadherin expression. Understanding of the progression of RSV infection may provide new insight into the cellular mechanisms of RSV-induced airway dysfunction following acute RSV infection. We therefore propose that adhesive molecules could be effective therapeutic target for developing drugs against RSV-induced chronic airway dysfunction.