Geri L. Traver

The air-liquid interface and use of primary cell cultures are important to recapitulate the transcriptional profile of in vivo airway epithelia

Organotypic cultures of primary human airway epithelial cells have been used to investigate the morphology, ion and fluid transport, innate immunity, transcytosis, infection, inflammation, signaling, cilia, and repair functions of this complex tissue. However, we do not know how closely these cultures resemble the airway surface epithelium in vivo. In this study, we examined the genome-wide expression profile of tracheal and bronchial human airway epithelia in vivo and compared it with the expression profile of primary cultures of human airway epithelia grown at the air-liquid interface. For comparison, we also investigated the expression profile of Calu-3 cells grown at the air-liquid interface and primary cultures of human airway epithelia submerged in nutrient media. We found that the transcriptional profile of differentiated primary cultures grown at the air-liquid interface most closely resembles that of in vivo airway epithelia, suggesting that the use of primary cultures and the presence of an air-liquid interface are important to recapitulate airway epithelia biology. We describe a high level of similarity between cells of tracheal and bronchial origin within and between different human donors, which suggests a very robust expression profile that is specific to airway cells.

IL-4-induced transcription factor NFIL3/E4BP4 controls IgE class switching

IL-4 signaling promotes IgE class switching through STAT6 activation and the induction of Ig germ-line ε (GLε) transcription. Previously, we and others identified a transcription factor, Nfil3, as a gene induced by IL-4 stimulation in B cells. However, the precise roles of nuclear factor, IL-3-regulated (NFIL3) in IL-4 signaling are unknown. Here, we report that NFIL3 is important for IgE class switching. NFIL3-deficient mice show impaired IgE class switching, and this defect is B-cell intrinsic. The induction of GLε transcripts after LPS and IL-4 stimulation is significantly reduced in NFIL3-deficient B cells. Expression of NFIL3 in NFIL3-deficient B cells restores the impairment of IgE production, and overexpression of NFIL3 in the presence of cycloheximide induces GLε transcripts. Moreover, NFIL3 binds to Iε promoter in vivo. Together, these results identify NFIL3 as a key regulator of IL-4-induced GLε transcription in response to IL-4 and subsequent IgE class switching.

A role for the PDZ-binding domain of the coxsackie B virus and adenovirus receptor (CAR) in cell adhesion and growth.

The coxsackie and adenovirus receptor (CAR) plays a role in viral infection, maintenance of the junction adhesion complex in polarized epithelia, and modulation of cellular growth properties. As a viral receptor, the C-terminus appears to play no role indicating that the major function of CAR is to tether the virus to the cell. By contrast, the C-terminus is known to play a role in cellular localization and probably has a significant function in CAR-mediated adhesion and cell growth properties. We hypothesized that the CAR PDZ (PSD-95/Disc-large/ZO-1) binding motif interacts with PDZ-domain-containing proteins to modulate the cellular phenotype. CAR was modified by deleting the last four amino acids (CARΔGSIV) and evaluated for cell-cell adhesion in polarized primary human airway epithelia and growth characteristics in stably transfected L-cells. Although ablation of the CAR PDZ-binding motif did not affect adenoviral infection, it did have a significant effect both on cell-cell adhesion and on cell growth. Expression of CARΔGSIV failed to increase the transepithelial resistance in polarized epithelia to the same degree as wild-type CAR and failed to act as a growth modulator in L-cells. Furthermore, we provide evidence for three new CAR interacting partners, including MAGI-1b, PICK1 and PSD-95. CAR appears to interact with several distinct PDZ-domain-containing proteins and may exert its biological function through these interactions.

Interdependency of β-Adrenergic Receptors and CFTR in Regulation of Alveolar Active Na+ Transport

&bgr;-Adrenergic receptors (&bgr;AR) regulate active Na+ transport in the alveolar epithelium and accelerate clearance of excess airspace fluid. Accumulating data indicates that the cystic fibrosis transmembrane conductance regulator (CFTR) is important for upregulation of the active ion transport that is needed to maintain alveolar fluid homeostasis during pulmonary edema. We hypothesized that &bgr;AR regulation of alveolar active transport may be mediated via a CFTR dependent pathway. To test this hypothesis we used a recombinant adenovirus that expresses a human CFTR cDNA (adCFTR) to increase CFTR function in the alveolar epithelium of normal rats and mice. Alveolar fluid clearance (AFC), an index of alveolar active Na+ transport, was 92% greater in CFTR overexpressing lungs than controls. Addition of the Cl− channel blockers NPPB, glibenclamide, or bumetanide and experiments using Cl− free alveolar instillate solutions indicate that the accelerated AFC in this model is due to increased Cl− channel function. Conversely, CFTR overexpression in mice with no &bgr;1- or &bgr;2-adrenergic receptors had no effect on AFC. Overexpression of a human &bgr;2AR in the alveolar epithelium significantly increased AFC in normal mice but had no effect in mice with a non-functional human CFTR gene (&Dgr;&phgr;508 mutation). These studies indicate that upregulation of alveolar CFTR function speeds clearance of excess fluid from the airspace and that CFTRs effect on active Na+ transport requires the &bgr;AR. These studies reveal a previously undetected interdependency between CFTR and &bgr;AR that is essential for upregulation of active Na+ transport and fluid clearance in the alveolus.

Tim-3 Directly Enhances CD8 T Cell Responses to Acute Listeria monocytogenes Infection

T cell Ig and mucin domain (Tim) 3 is a surface molecule expressed throughout the immune system that can mediate both stimulatory and inhibitory effects. Previous studies have provided evidence that Tim-3 functions to enforce CD8 T cell exhaustion, a dysfunctional state associated with chronic stimulation. In contrast, the role of Tim-3 in the regulation of CD8 T cell responses to acute and transient stimulation remains undefined. To address this knowledge gap, we examined how Tim-3 affects CD8 T cell responses to acute Listeria monocytogenes infection. Analysis of wild-type (WT) mice infected with L. monocytogenes revealed that Tim-3 was transiently expressed by activated CD8 T cells and was associated primarily with acquisition of an effector phenotype. Comparison of responses to L. monocytogenes by WT and Tim-3 knockout (KO) mice showed that the absence of Tim-3 significantly reduced the magnitudes of both primary and secondary CD8 T cell responses, which correlated with decreased IFN-γ production and degranulation by Tim-3 KO cells stimulated with peptide Ag ex vivo. To address the T cell–intrinsic role of Tim-3, we analyzed responses to L. monocytogenes infection by WT and Tim-3 KO TCR-transgenic CD8 T cells following adoptive transfer into a shared WT host. In this setting, the accumulation of CD8 T cells and the generation of cytokine-producing cells were significantly reduced by the lack of Tim-3, demonstrating that this molecule has a direct effect on CD8 T cell function. Combined, our results suggest that Tim-3 can mediate a stimulatory effect on CD8 T cell responses to an acute infection.

Functional Effects of Coxsackievirus and Adenovirus Receptor Glycosylation on Homophilic Adhesion and Adenoviral Infection

ABSTRACT The coxsackievirus and adenovirus receptor (CAR) is both a viral receptor and homophilic adhesion protein. The extracellular portion of CAR consists of two immunoglobulin (Ig)-like domains, each with a consensus sequence for N-glycosylation. We used chemical, genetic, and biochemical studies to show that both sites are glycosylated and contribute to the function of CAR. Although the glycosylation of CAR does not alter cell surface levels or junctional localization, it affects both adhesion and adenovirus infection in unique ways. CAR-mediated adhesion appears to require at least one site of glycosylation since cells expressing CAR without glycosylation do not cluster with each other. In contrast, glycosylation of the Ig-like domain proximal to the membrane is key to the cooperative behavior of adenovirus binding and infection. Contrary to the hypothesis that cooperativity improves viral infection, our data show that although glycosylation of the D2 domain is required for adenovirus cooperative binding, it has a negative consequence upon infection. This is the first report dissecting the adhesion and receptor activities of CAR, revealing that factors other than the binding interface play a significant role in the function of CAR. These data have important implications for both cancers with altered glycosylation states and cancer treatments using oncolytic adenovirus.

768. Activity of the Extracellular Domain of the Coxsackie and Adenovirus Receptor

The Coxsackie virus and Adenovirus Receptor (CAR) plays a dual role as a viral receptor and a homotypic junctional adhesion protein. CAR is a transmembrane protein and a member of the Immunoglobulin superfamily with two extracellular Ig-like domains. According to x-ray crystallographic data, the most distal Ig-like domain (D1) mediates the homophilic interaction. This same domain is also responsible for the high affinity binding of the adenovirus (Ad) fiber protein. Currently no activity has been ascribed to the proximal Ig-like domain (D2). We asked whether the D2 domain plays a role in the function of CAR as either a viral receptor or an adhesion protein.