Non-typeable Haemophilus influenzae decreases cilia beating via protein kinase Cε.

BACKGROUND: Haemophilus influenzae infection of the nasal epithelium has long been associated with observations of decreased nasal ciliary beat frequency (CBF) and injury to the ciliated epithelium. Previously, we have reported that several agents that slow CBF also have the effect of activating protein kinase C epsilon (PKCε) activity in bronchial epithelial cells. The subsequent auto-downregulation of PKCε or the direct inhibition of PKCε leads to the specific detachment of the ciliated cells. METHODS: Primary cultures of ciliated bovine bronchial epithelial cells were exposed to filtered conditioned media supernatants from non-typeable H. influenzae (NTHi) cultures. CBF and motile points were measured and PKCε activity assayed. RESULTS: NTHi supernatant exposure significantly and rapidly decreased CBF in a dose-dependent manner within 10 minutes of exposure. After 3 hours of exposure, the number of motile ciliated cells significantly decreased. Direct measurement of PKCε activity revealed a dose-dependent activation of PKCε in response to NTHi supernatant exposure. Both CBF and PKCε activity changes were only observed in fresh NTHi culture supernatant and not observed in exposures to heat-inactivated or frozen supernatants. CONCLUSIONS: Our results suggest that CBF slowing observed in response to NTHi is consistent with the stimulated activation of PKCε. Ciliated cell detachment is associated with PKCε autodownregulation.

Alcohol increases the permeability of airway epithelial tight junctions in Beas-2B and NHBE cells.

BACKGROUND: Tight junctions form a continuous belt-like structure between cells and act to regulate paracellular signaling. Protein kinase C (PKC) has been shown to regulate tight junction assembly and disassembly and is activated by alcohol. Previous research has shown that alcohol increases the permeability of tight junctions in lung alveolar cells. However, little is known about alcohol's effect on tight junctions in epithelium of the conducting airways. We hypothesized that long-term alcohol exposure reduces zonula occluden-1 (ZO-1) and claudin-1 localization at the cell membrane and increases permeability through a PKC-dependent mechanism. METHODS: To test this hypothesis, we exposed normal human bronchial epithelial (NHBE) cells, cells from a human bronchial epithelial transformed cell line (Beas-2B), and Beas-2B expressing a PKCα dominant negative (DN) to alcohol (20, 50, and 100 mM) for up to 48 hours. Immunofluorescence was used to assess changes in ZO-1, claudin-1, claudin-5, and claudin-7 localization. Electric cell-substrate impedance sensing was used to measure the permeability of tight junctions between monolayers of NHBE, Beas-2B, and DN cells. RESULTS: Alcohol increased tight junction permeability in a concentration-dependent manner and decreased ZO-1, claudin-1, claudin-5, and claudin-7 localization at the cell membrane. To determine a possible signaling mechanism, we measured the activity of PKC isoforms (alpha, delta, epsilon, and zeta). PKCα activity significantly increased in Beas-2B cells from 1 to 6 hours of 100 mM alcohol exposure, while PKCζ activity significantly decreased at 1 hour and increased at 3 hours. Inhibiting PKCα with Gö-6976 prevented the alcohol-induced protein changes in both ZO-1 and claudin-1 at the cell membrane. PKCα DN Beas-2B cells were resistant to alcohol-induced protein alterations. CONCLUSIONS: These results suggest that alcohol disrupts ZO-1, claudin-1, claudin-5, and claudin-7 through the activation of PKCα, leading to an alcohol-induced "leakiness" in bronchial epithelial cells. Such alcohol-induced airway-leak state likely contributes to the impaired airway host defenses associated with acute and chronic alcohol ingestion.

Adenosine activation of A(2B) receptor(s) is essential for stimulated epithelial ciliary motility and clearance.

Mucociliary clearance, vital to lung clearance, is dependent on cilia beat frequency (CBF), coordination of cilia, and the maintenance of periciliary fluid. Adenosine, the metabolic breakdown product of ATP, is an important modulator of ciliary motility. However, the contributions of specific adenosine receptors to key airway ciliary motility processes are unclear. We hypothesized that adenosine modulates ciliary motility via activation of its cell surface receptors (A(1), A(2A), A(2B), or A(3)). To test this hypothesis, mouse tracheal rings (MTRs) excised from wild-type and adenosine receptor knockout mice (A(1), A(2A), A(2B), or A(3), respectively), and bovine ciliated bronchial epithelial cells (BBECs) were stimulated with known cilia activators, isoproterenol (ISO; 10 µM) and/or procaterol (10 µM), in the presence or absence of 5'-(N-ethylcarboxamido) adenosine (NECA), a nonselective adenosine receptor agonist [100 nM (A(1), A(2A), A(3)); 10 µM (A(2B))], and CBF was measured. Cells and MTRs were also stimulated with NECA (100 nM or 10 µM) in the presence and absence of adenosine deaminase inhibitor, erythro-9- (2-hydroxy-3-nonyl) adenine hydrochloride (10 µM). Both ISO and procaterol stimulated CBF in untreated cells and/or MTRs from both wild-type and adenosine knockout mice by ~3 Hz. Likewise, CBF significantly increased ~2-3 Hz in BBECs and wild-type MTRs stimulated with NECA. MTRs from A(1), A(2A), and A(3) knockout mice stimulated with NECA also demonstrated an increase in CBF. However, NECA failed to stimulate CBF in MTRs from A(2B) knockout mice. To confirm the mechanism by which adenosine modulates CBF, protein kinase activity assays were conducted. The data revealed that NECA-stimulated CBF is mediated by the activation of cAMP-dependent PKA. Collectively, these data indicate that purinergic stimulation of CBF requires A(2B) adenosine receptor activation, likely via a PKA-dependent pathway.

Malondialdehyde-acetaldehyde-adducted protein inhalation causes lung injury.

In addition to cigarette smoking, alcohol exposure is also associated with increased lung infections and decreased mucociliary clearance. However, little research has been conducted on the combination effects of alcohol and cigarette smoke on lungs. Previously, we have demonstrated in a mouse model that the combination of cigarette smoke and alcohol exposure results in the formation of a very stable hybrid malondialdehyde-acetaldehyde (MAA)-adducted protein in the lung. In in vitro studies, MAA-adducted protein stimulates bronchial epithelial cell interleukin-8 (IL-8) via the activation of protein kinase C epsilon (PKCɛ). We hypothesized that direct MAA-adducted protein exposure in the lungs would mimic such a combination of smoke and alcohol exposure leading to airway inflammation. To test this hypothesis, C57BL/6J female mice were intranasally instilled with either saline, 30µL of 50µg/mL bovine serum albumin (BSA)-MAA, or unadducted BSA for up to 3 weeks. Likewise, human lung surfactant proteins A and D (SPA and SPD) were purified from human pulmonary proteinosis lung lavage fluid and successfully MAA-adducted in vitro. Similar to BSA-MAA, SPD-MAA was instilled into mouse lungs. Lungs were necropsied and assayed for histopathology, PKCɛ activation, and lung lavage chemokines. In control mice instilled with saline, normal lungs had few inflammatory cells. No significant effects were observed in unadducted BSA- or SPD-instilled mice. However, when mice were instilled with BSA-MAA or SPD-MAA for 3 weeks, a significant peribronchiolar localization of inflammatory cells was observed. Both BSA-MAA and SPD-MAA stimulated increased lung lavage neutrophils and caused a significant elevation in the chemokine, keratinocyte chemokine, which is a functional homologue to human IL-8. Likewise, MAA-adducted protein stimulated the activation of airway and lung slice PKCɛ. These data support that the MAA-adducted protein induces a proinflammatory response in the lungs and that the lung surfactant protein is a biologically relevant target for malondialdehyde and acetaldehyde adduction. These data further implicate MAA-adduct formation as a potential mechanism for smoke- and alcohol-induced lung injury.