Lan Jornot

Modulation of the DNA binding activity of transcription factors CREP, NFκB and HSF by H2O2 and TNFα. Differences between in vivo and in vitro effects

Human endothelial cells exposed to H2O2 showed reduced CREP DNA binding activity, enhanced HSF activation, and no induction of NFκB binding activity. Interestingly, H2O2 was able to induce NFκB subunit p65 translocation in the nucleus. In contrast, cells exposed to TNFα showed enhanced CREP binding activity, activation of NFκB and no induction of HSE‐HSF complex. Addition of H2O2, diamide and iodoacetic acid to the binding reaction mixture markedly reduced the DNA binding ability of the three transcription factors. Thus free sulfhydryls were important in DNA binding activity of CREP, NFκB and HSF, and the lack of induction of NFκB by H2O2 in intact cells was likely caused by oxidation on a thiol, and not by a deficiency in the activation pathway.

N-acetylcysteine inhibits Na+ absorption across human nasal epithelial cells

N‐acetylcysteine (NAC) is a widely used mucolytic drug in patients with a variety of respiratory disorders. The mechanism of action is based on rupture of the disulfide bridges of the high molecular glycoproteins present in the mucus, resulting in smaller subunits of the glycoproteins and reduced viscosity of the mucus. Because Na+ absorption regulates airway surface liquid volume and thus the efficiency of mucociliary clearance, we asked whether NAC affects the bioelectric properties of human nasal epithelial cells. A 24‐h basolateral treatment with 10 mM of NAC decreased the transepithelial potential difference and short‐circuit current (ISC) by 40%, and reduced the amiloride‐sensitive current by 50%, without affecting the transepithelial resistance. After permeabilization of the basolateral membranes of cells with amphotericin B in the presence of a mucosal‐to‐serosal Na+ gradient (135:25 mM), NAC inhibited 45% of the amiloride‐sensitive current. The Na+‐K+‐ATPase pump activity and the basolateral K+ conductance were not affected by NAC treatment. NAC did not alter total cell mRNA and protein levels of α‐epithelial Na+ channel (EnaC) subunit, but reduced abundance of α‐ENaC subunits in the apical cell membrane as quantified by biotinylation. This effect can be ascribed to the sulphydryl (SH) group of NAC, since N‐acetylserine and S‐carboxymethyl‐l‐cysteine were ineffective. Given the importance of epithelial Na+ channels in controlling the thin layer of fluid that covers the surface of the airways, the increase in the fluidity of the airway mucus following NAC treatment in vivo might be in part related to downregulation of Na+ absorption and consequently water transport. J. Cell. Physiol. 201: 106–116, 2004.

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)-α 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-α 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-α, 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.

Effects of amphotericin B on ion transport proteins in airway epithelial cells

Topical intranasal application of the antifungal Amphotericin B (AmphoB) has been shown as an effective medical treatment of chronic rhinosinusitis. Because this antibiotic forms channels in lipid membranes, we considered the possibility that it affects the properties and/or cell surface expression of ion channels/pumps, and consequently transepithelial ion transport. Human nasal epithelial cells were exposed apically to AmphoB (50 μM) for 4 h, 5 days (4 h daily), and 4 weeks (4 h daily, 5 days weekly) and allowed to recover for 18–48 h. AmphoB significantly reduced transepithelial potential difference, short‐circuit current, and the amiloride‐sensitive current. This was not due to generalized cellular toxicity as judged from normal transepithelial resistance and mitochondrial activity, but was related to inhibitory effects of AmphoB on ion transport proteins. Thus, cells exposed to AmphoB for 4 h showed decreased apical epithelial sodium channels (ENaC) activity with no change in basolateral Na+K+‐ATPase activity and K+ conductance, and reduced amount of αENaC, α1‐Na+K+‐ATPase, and NKCC1 proteins at the cell membrane, but no change in mRNA levels. After a 5‐day treatment, there was a significant decrease in Na+K+‐ATPase activity. After a 4‐week treatment, a decrease in basolateral K+ conductance and in αENaC and α1‐Na+K+‐ATPase mRNA levels was also observed. These findings may reflect a feedback mechanism aimed to limit cellular Na+ overload and K+ depletion subsequently to formation of AmphoB pores in the cell membrane. Thus, the decreased Na+ absorption induced by AmphoB resulted from reduced cell surface expression of the ENaC, Na+K+‐ATPase pump and NKCC1 and not from direct inhibition of their activities.

Neuroendocrine cells of nasal mucosa are a cellular source of brain-derived neurotrophic factor

There is growing evidence for extensive interaction between sensory neurons, immune and mucosal epithelial cells during airway inflammation and hyperreactivity. This neuro-immune cross-talk (neurogenic inflammation) involves different groups of mediators, which include the neurotrophin family (nerve growth factor, brain-derived neurotrophic factor (BDNF) and neurotrophin-3 and -4). Neurotrophins modulate airway inflammation by enhancing sensory nerve excitability and production of neuropeptides, and by interaction with different immune cell types. In the present study, it was questioned whether airway epithelial cells express BDNF, and if proinflammatory cytokines (tumour necrosis factor-α, interleukin-1β and interferon-γ) and a glucocorticoid (budesonide) affect this expression. Primary cultures of nasal epithelial cells were used. It was found that BDNF was stored in chromogranin A-containing secretory granules of specialised epithelial cells, i.e. neuroendocrine cells, and was secreted in a polarised manner. Apical secretion appears to be constitutive, whereas basolateral secretion is markedly enhanced upon stimulation with cytokines. This enhanced basolateral secretion was not due to enhanced synthesis and was not affected by inhibitors of the processing enzymes, such as furin and the metalloproteinases involved in the maturation of BDNF, but was considerably diminished by budesonide. Therefore, airway mucosa might contribute to neurogenic inflammation through increased secretion of brain-derived neurotrophic factor by neuroendocrine cells under inflammatory conditions.

N-acetylcysteine augments adenovirus-mediated gene expression in human endothelial cells by enhancing transgene transcription and virus entry

It has previously been shown that oxidants reduce the efficiency of adenoviral transduction in human umbilical vein endothelial cells (HUVECs). In this study, the effect of the antioxidant N‐acetylcysteine (NAC) in adenovirus‐mediated gene transfer has been investigated.

Differential effects of hyperoxia and hydrogen peroxide on thymidine kinase and adenosine kinase activities of cultured endothelial cells

To compare the respective sensitivity of two nucleoside kinases, adenosine kinase and thymidine kinase, to oxidative stress, we measured these enzyme activities in cultured aortic endothelial cells exposed for 48 h to various O2 concentrations, and in cell extracts treated with H2O2 or the enzyme system hypoxanthine-xanthine oxidase. Adenosine kinase activity was not significantly influenced by the exposure to hyperoxia, nor by treatment with the enzyme system hypoxanthine-xanthine oxidase or with H2O2. On the other hand, there was a dose-dependent inhibitory effect on thymidine kinase activity resulting from exposure to various O2 concentrations or from treatment with various amounts of xanthine oxidase. Incubation of cell extracts in the presence of H2O2 also resulted in a significant reduction of thymidine kinase activity. These results indicate that thymidine kinase exhibits a selective sensitivity to the toxic effect of O2 concentrations and of O2 intermediates such as H2O2.

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

Hypothesis T cells modulate the antiviral and inflammatory responses of airway epithelial cells to human rhinoviruses (HRV). Methods Differentiated primary human nasal epithelial cells (HNEC) grown on collagen-coated filters were exposed apically to HRV14 for 6 h, washed thoroughly and co-cultured with anti-CD3/CD28 activated T cells added in the basolateral compartment for 40 h. Results HRV14 did not induce IFNγ, NOS2, CXCL8 and IL-6 in HNEC, but enhanced expression of the T cell attractant CXCL10. On the other hand, HNEC co-cultured with activated T cells produced CXCL10 at a level several orders of magnitude higher than that induced by HRV14. Albeit to a much lower degree, activated T cells also induced CXCL8, IL-6 and NOS2. Anti-IFNγ antibodies and TNF soluble receptor completely blocked CXCL10 upregulation. Furthermore, a significant correlation was observed between epithelial CXCL10 mRNA expression and the amounts of IFNγ and TNF secreted by T cells. Likewise, increasing numbers of T cells to a constant number of HNEC in co-cultures resulted in increasing epithelial CXCL10 production, attaining a plateau at high IFNγ and TNF levels. Hence, HNEC activation by T cells is induced mainly by IFNγ and/or TNF. Activated T cells also markedly inhibited viral replication in HNEC, partially through activation of the nitric oxide pathway. Conclusion Cross-talk between T cells and HNEC results in activation of the latter and increases their contribution to airway inflammation and virus clearance.