Paracellular Permeability Restricts Airway Epithelial Responses to Selectively Allow Activation by Mediators at the Basolateral Surface

Abstract

Respiratory pathogens and toxins often assault the lung from the airway lumen. Airway epithelia may initiate and amplify inflammation in response to these attacks, but under certain conditions confinement of inflammation to the airway lumen may be beneficial to the host. Accordingly, we hypothesized that airway epithelial polarity allows different responses to basolateral vs apical stimuli that may modulate inflammation. Using primary human airway epithelial cells differentiated at an air-liquid interface in culture, we found that responses to several cytokines required basolateral mediator application. In contrast, responses to Haemophilus influenzae occurred after either basolateral or apical interaction with airway epithelia. Experiments focused on IFN-γ receptor polarity confirmed its predominant basolateral location in cultured airway epithelia as well as in normal human airway tissue. Furthermore, physical and pharmacologic disruption of barrier function in airway epithelia allowed responses to apical application of IFN-γ and other cytokines. These in vitro studies directly correlated with experiments in mice in which an airway epithelial response to IFN-γ injected into the airway lumen was seen only after disruption of barrier function. The results indicate that airway epithelia with intact barrier function restrict inflammatory responses by limitation of cell activation through requiring interaction of selected mediators with the basolateral surface. However, loss of barrier integrity allows epithelial responses to these mediators if located in the airway lumen to amplify airway defenses.

Inhibition by Cigarette Smoke of Nuclear Factor-κB–Dependent Response to Bacteria in the Airway

“…NF-kB-dependent gene activation was determined using a recombinant adenoviral vector that expresses a luciferase gene driven by four tandem NF-kB enhancer sequences, as described previously (11,18). Photinus pyralis luciferase activity was determined using a commercial luciferase reporter assay kit (Promega) and a Lumat LB 9501 luminometer (Berthold, Bad Wildbad, Germany).…”

“…Human IL-8 and IL-6 and mouse keratinocyte chemoattractant (KC) protein concentrations were determined using commercial sandwich enzyme-linked immunoassay kits (R&D Systems, Minneapolis, MN), as described previously (11,(16)(17)(18). The sensitivity of this assay system for IL-8 is greater than 37.5 pg/ml, its sensitivity for IL-6 is greater than 9.4 pg/ml, and its sensitivity for KC is greater than 2.0 pg/ml.…”

“…Whole-cell and nuclear protein extract preparation and immunoblot analysis were performed as described previously (15,18). Primary antibodies used to detect specific cellular and nuclear proteins included rabbit polyclonal IgG 4915 against human ICAM-1, mouse IgG1 monoclonal antibody (mAb) clone 3G2 against human caspase- 3, rabbit IgG mAb clone 5A1E against human cleaved caspase-3, rabbit IgG mAb clone C22B4 against human RelA/p65, rabbit IgG mAb clone C84E11 against human IKKa phosphorylated on Ser-176 and IKKb phosphorylated on Ser-177, rabbit IgG mAb clone 14D4 against human IkBa phosphorylated on Ser-32 (which also binds mouse phosphorylated IkBa), and mouse IgG1 mAb clone L35A5 against human and mouse IkBa from Cell Signaling Technology (Beverly, MA); mouse IgG2a mAb clone AC-74 against human b-actin (which also binds mouse b-actin) from Sigma-Aldrich (St. Louis, MO); and rabbit polyclonal antiserum against human heat shock protein (HSP)-90 from Assay Designs (Ann Arbor, MI).…”

Local Blockade of Epithelial PDL-1 in the Airways Enhances T Cell Function and Viral Clearance during Influenza Virus Infection

“…The airway epithelium lining the respiratory tract not only represents the first barrier of defense against respiratory pathogens but is often the primary target of infection (1). Consequently, apart from a critical role in gas exchange function, airway epithelial cells additionally participate in host defense mechanisms by producing cytokines and chemokines (2)(3)(4)(5)(6)(7)(8). Since exacerbated responses by airway epithelial cells in response to innocuous antigens or pathogens can prove detrimental to lung function, airway epithelial cells have also evolved to regulate pulmonary homeostasis by multiple mechanisms, including, among others, expression of CD200 (9), MUC-1 (10), and surfactant proteins (11) and reduced expression of adaptor molecules (12)(13)(14).…”

Plasticity and Virus Specificity of the Airway Epithelial Cell Immune Response during Respiratory Virus Infection

“…The airway epithelium is a ciliated, pseudostratified, columnar epithelium consisting of ciliated cells, basal cells, and secretory goblet cells that, together with locally produced IgA, provide mechanisms for mucociliary clearance of inhaled microorganisms (19). In response to antigenic insults, AECs participate in host defense mechanisms by producing cytokines and chemokines, such as interleukin 6 (IL-6), IL-8, IL-1␣, RANTES, MIP-1␣, granulocyte-macrophage colony-stimulating factor (GM-CSF), and granulocyte colony-stimulating factor (G-CSF) (1,8,21,26,44,47,48). Regulation of the intensity and duration of inflammation in the airways is critical for maintaining respiratory function, and thus, epithelial cells also mediate a plethora of processes with the goal of limiting airway inflammation (22).…”