T Lymphocytes Promote the Antiviral and Inflammatory Responses of Airway Epithelial Cells

T cells modulate the antiviral and inflammatory responses of airway epithelial cells to human rhinoviruses (HRV)

Src signaling links mediators of inflammation to Cx43 gap junction channels in primary and transformed CFTR-expressing airway cells

Dysfunction of the cystic fibrosis transmembrane conductance regulator (CFTR) is associated with recurrent pulmonary infections and inflammation. We previously reported that tumor necrosis factor (TNF)-alpha decreases gap junction connectivity in cell lines derived from the airway epithelium of non-cystic fibrosis (non-CF) subjects, a mechanism that was defective in cells derived from CF patients, and identified the tyrosine kinase c-Src as a possible bridge between TNF-alpha and Cx43. To examine whether this modulation also takes place in primary epithelial cells, the functional expression of Cx43 was studied in non-CF and CF airway cells, obtained from surgical polypectomies and turbinectomies, which were grown either on culture dishes or permeable filters. Expression of Cx43 was detected by immunofluorescence on cells grown under both culture conditions. Non-CF and CF airway cells also showed intercellular diffusion of Lucifer Yellow. Dye coupling was rapidly abolished in non-CF cells in the presence of TNF-alpha, lipopolysaccharide and lysophosphatidic acid, and could be prevented by tyrphostin47, an inhibitor of Src tyrosine kinases. This down-regulation, however, was not detected in CF airway cells. These data indicate that CFTR dysfunction is associated with altered Src signaling, resulting in the persistence of gap junction connectivity in primary and transformed CF airway cells

Long-term cultures of polarized airway epithelial cells from patients with cystic fibrosis

The poor ability of respiratory epithelial cells to proliferate and differentiate in vitro into a pseudostratified mucociliated epithelium limits the general use of primary airway epithelial cell (AEC) cultures generated from patients with rare diseases, such as cystic fibrosis (CF). Here, we describe a procedure to amplify AEC isolated from nasal polyps and generate long-term cultures of the respiratory epithelium. AEC were seeded onto microporous permeable supports that carried on their undersurface a preformed feeder layer of primary human airway fibroblasts. The use of fibroblast feeder layers strongly stimulated the proliferation of epithelial cells, allowing the expansion of the cell pool with successive passages. AEC at increasing passage were seeded onto supports undercoated with airway fibroblasts and exposed to air. Either freshly isolated or amplified AEC could differentiate into a pseudostratified mucociliated epithelium for at least 10 mo. Thus, CF epithelia cultures showed elevated Na+ transport, drastic hyperabsorption of surface liquid, and absence of cAMP-induced Cl- secretion as compared with non-CF cultures. They were also characterized by thick apical secretion that hampered the movement of cell surface debris by cilia. However, CF respiratory epithelia did not show increased production of mucins or IL-8. The method described here is now routinely used in our laboratory to establish long-term cultures of well differentiated respiratory epithelia from human airway biopsies

Epithelial CXCL10 protein secreted into the basolateral compartment increases with increasing T cells numbers and increasing IFNγ and TNF amounts, reaching a plateau at high concentrations.

<p>A constant number of HNEC (0.5×10⁶ cells) was co-cultured with increasing numbers of activated T cells (0.1, 0.5, 2.5, 5×10⁶ cells). CXCL10 protein amounts secreted by HNEC into the basolateral media are expressed as a function of the numbers of T cells in co-cultures (A) and the amounts of IFNγ (B) and TNF (C) secreted by increasing numbers of T cells.</p

Activated T cells inhibit virus replication in infected HNEC partially through the NO pathway.

<p>HNEC were exposed apically to HRV14, washed thoroughly then co-cultured with activated T cells in the presence or absence of FP and L-NIL, inhibitors of NOS2 mRNA and activity, respectively. (A) NO secretion in the basolateral compartment and (B) viral RNA levels. NO is expressed as nitrite, the stable oxidation product of NO (µM). Viral RNAs was measured by real-time PCR and expressed as fold change relative to those observed in infected HNEC cultured in the absence of activated T cells. *p<0.05</p