Age-Associated Changes in the Respiratory Epithelial Response to Influenza Infection.

Older adults suffer a disproportionate burden of influenza-related morbidity and mortality typically attributed to defects in the aging immune system collectively known as immunosenescence. While the age-related decline in the adaptive immune system has been well characterized, little is known about how aging affects the principal site of influenza infection-the nasal epithelium. In human nasal epithelial cell cultures (hNECs) from older adults, we found similar or increased levels of cytokines during influenza infection compared with hNECs from younger individuals. However, hNECs from older individuals demonstrated decreased mRNA expression for several key proteins that affect clearance of infected cells, including MHC-I and transporter associated with antigen presentation (TAP). These findings were confirmed at the level of protein expression. In vivo studies corroborated the in vitro differences in MHC-I and TAP gene expression and also revealed important decreases in the expression of key influenza-specific antiviral mediators MX1 and IFITM1. Furthermore, epithelial cell-cytotoxic T lymphocyte co-cultures demonstrate that CTL cytotoxic activity is dose-dependent on MHC-I antigen presentation. Taken together, these results indicate that aging is associated with important changes in the nasal epithelium, including antigen presentation and antiviral pathways, which may contribute to increased severity of disease in older adults through impaired clearance of infected cells.

Ion transport and barrier function in a telomerase-immortalized human nondysplastic, Barrett's cell line (BAR-T).

Barrett's specialized columnar epithelium (SCE) replaces reflux-damaged squamous epithelium. The benefits of SCE lie in its superior protection of the esophagus against further reflux damage. It was shown that this protection is dependent on ion transport and barrier function of SCE. The risks of SCE lie in its higher predisposition to malignant transformation. An understanding of underlying mechanisms of both processes would benefit considerably from greater knowledge of the structure and function of native SCE - the latter recently advanced by the availability of a telomerase-immortalized, nonneoplastic, human Barrett's cell line (BAR-T). Some of BAR-T characteristics for growth and differentiation have been described recently, but not its capacity to serve as a model for ion transport and barrier function of SCE. To determine the latter, BAR-T cells were grown in enriched media, seeded on permeable supports, and subjected to electrical, biochemical, and morphologic study. HET-1A (esophageal epithelial cell line), a nonneoplastic, human esophageal squamous cell line, was also studied for comparison. BAR-T, but not HET-1A cells in HEPES Ringer solution behaved as polarized monolayers with the capacity for ion transport and barrier function. This was evident electrically with a volt-ohm meter (EVOM),which recorded in BAR-T a resting potential difference of 2.0 +/- 0.2 mV, Isc of 17.4 +/- 3.3 microAmps/cm2 and resistance of 103 +/- 12 ohms x cm2. Further, Isc in BAR-T was inhibitable by exposure to Na-free solution, serosal ouabain, and luminal 4-acetamido4'-isothiocyano-2,2'-stilbenedisulfonic acid. Expression of tight junction genes were determined in BAR-T and HET-1A cells using quantitative reverse transcriptase-polymerase chain reaction, with expression of zonula occludens-1 (ZO-1) set at 1 as reference. Claudins 1, 4, and 12 were prominently expressed in BAR-T (0.2-0.6 of ZO-1), while claudins 1, 11, and 12 were prominently expressed in HET-1A(0.1-0.8 of ZO-1). BAR-T, but not HET-1A, expressed claudins 4, 8, 16, 18, and 23, and HET-1A, but not BAR-T, expressed claudins 11, 15, and 20. Protein expression of prominently expressed claudins in BAR-T correlated with mRNA expression. Immunofluorescence and confocal microscopy localized claudins 1 and 4 in BAR-T to cell membranes and claudin 18, specifically to the tight junction. Membrane polarization was also documented in BAR-T by immunolocalization of NaK,ATPase to the basolateral membrane. BAR-T, but not HET-1A cells grown on permeable supports form a polarized monolayer with both ion transport and barrier function. Since a number of features of BAR-T are similar to Barrett's SCE and distinct from HET-1A, the BAR-T cell line represents a valuable resource for the study of ion transport and barrier function of nondysplastic SCE.