CFTR chloride channel regulation by an interdomain interaction.

The cystic fibrosis gene encodes a chloride channel, CFTR (cystic fibrosis transmembrane conductance regulator), that regulates salt and water transport across epithelial tissues. Phosphorylation of the cytoplasmic regulatory (R) domain by protein kinase A activates CFTR by an unknown mechanism. The amino-terminal cytoplasmic tail of CFTR was found to control protein kinase A-dependent channel gating through a physical interaction with the R domain. This regulatory activity mapped to a cluster of acidic residues in the NH(2)-terminal tail; mutating these residues proportionately inhibited R domain binding and CFTR channel function. CFTR activity appears to be governed by an interdomain interaction involving the amino-terminal tail, which is a potential target for physiologic and pharmacologic modulators of this ion channel.

Syntaxin 1A is expressed in airway epithelial cells, where it modulates CFTR Cl(-) currents.

The CFTR Cl(-) channel controls salt and water transport across epithelial tissues. Previously, we showed that CFTR-mediated Cl(-) currents in the Xenopus oocyte expression system are inhibited by syntaxin 1A, a component of the membrane trafficking machinery. This negative modulation of CFTR function can be reversed by soluble syntaxin 1A peptides and by the syntaxin 1A binding protein, Munc-18. In the present study, we determined whether syntaxin 1A is expressed in native epithelial tissues that normally express CFTR and whether it modulates CFTR currents in these tissues. Using immunoblotting and immunofluorescence, we observed syntaxin 1A in native gut and airway epithelial tissues and showed that epithelial cells from these tissues express syntaxin 1A at >10-fold molar excess over CFTR. Syntaxin 1A is seen near the apical cell surfaces of human bronchial airway epithelium. Reagents that disrupt the CFTR-syntaxin 1A interaction, including soluble syntaxin 1A cytosolic domain and recombinant Munc-18, augmented cAMP-dependent CFTR Cl(-) currents by more than 2- to 4-fold in mouse tracheal epithelial cells and cells derived from human nasal polyps, but these reagents did not affect CaMK II-activated Cl(-) currents in these cells.

Neutrophils negatively regulate induction of mucosal IgA responses after sublingual immunization.

Induction of mucosal immunoglobulin-A (IgA) capable of providing a first line of defense against bacterial and viral pathogens remains a major goal of needle-free vaccines given via mucosal routes. Innate immune cells are known to play a central role in induction of IgA responses by mucosal vaccines, but the relative contribution of myeloid cell subsets to these responses has not firmly been established. Using an in vivo model of sublingual vaccination with Bacillus anthracis edema toxin (EdTx) as adjuvant, we examined the role of myeloid cell subsets for mucosal secretory IgA responses. Sublingual immunization of wild-type mice resulted in a transient increase of neutrophils in sublingual tissues and cervical lymph nodes. These mice later developed Ag-specific serum IgG responses, but not serum or mucosal IgA. Interestingly, EdTx failed to increase neutrophils in sublingual tissues and cervical lymph nodes of IKKß(ΔMye) mice, and these mice developed IgA responses. Partial depletion of neutrophils before immunization of wild-type mice allowed the development of both mucosal and serum IgA responses. Finally, co-culture of B cells with neutrophils from either wild-type or IKKß(ΔMye) mice suppressed secretion of IgA, but not IgM or IgG. These results identify a new role for neutrophils as negative regulators of IgA responses.

IKKß in intestinal epithelial cells regulates allergen-specific IgA and allergic inflammation at distant mucosal sites.

Regulation of allergic responses by intestinal epithelial cells (IECs) remains poorly understood. Using a model of oral allergen sensitization in the presence of cholera toxin as adjuvant and mice with cell-specific deletion of inhibitor-κB kinase (IKKß) in IECs (IKKß(ΔIEC)), we addressed the contribution of IECs to allergic sensitization to ingested antigens and allergic manifestations at distant mucosal site of the airways. Cholera toxin induced higher pro-inflammatory responses and altered the profile of the gut microbiota in IKKß(ΔIEC) mice. Antigen-specific immunoglobulin E (IgE) responses were unaltered in IKKß(ΔIEC) mice, but their IgA antibodies (Abs), T helper type 1 (Th1) and Th17 responses were enhanced. Upon nasal antigen challenge, these mice developed lower levels of allergic lung inflammation, which correlated with higher levels of IgA Abs in the airways. The IKKß(ΔIEC) mice also recruited a higher number of gut-sensitized T cells in the airways after nasal antigen challenge and developed airway hyper-responsiveness, which were suppressed by treatment with anti-interleukin-17A. Fecal microbiota transplant during allergic sensitization reduced Th17 responses in IKKß(ΔIEC) mice, but did not affect IgA Ab responses. In summary, we show that IKKß in IECs shapes the gut microbiota and immune responses to ingested antigens and influences allergic responses in the airways via regulation of IgA Ab responses.

EAAT1 is involved in transport of L-glutamate during differentiation of the Caco-2 cell line.

Little is known concerning the expression of amino acid transporters during intestinal epithelial cell differentiation. The transport mechanism of L-glutamate and its regulation during the differentiation process were investigated using the human intestinal Caco-2 cell line. Kinetic studies demonstrated the presence of a single, high-affinity, D-aspartate-sensitive L-glutamate transport system in both confluent and fully differentiated Caco-2 cells. This transport was clearly Na(+) dependent, with a Hill coefficient of 2. 9 +/- 0.3, suggesting a 3 Na(+)-to-1 glutamate stoichiometry and corresponding to the well-characterized X(A,G)(-) system. The excitatory amino acid transporter (EAAT)1 transcript was consistently expressed in the Caco-2 cell line, whereas the epithelial and neuronal EAAT3 transporter was barely detected. In contrast with systems B(0) and y(+), which have previously been reported to be downregulated when Caco-2 cells stop proliferating, L-glutamate transport capacity was found to increase steadily between day 8 and day 17. This increase was correlated with the level of EAAT1 mRNA, which might reflect an increase in EAAT1 gene transcription and/or stabilization of the EAAT1 transcript.

Secretion of sparfloxacin from the human intestinal Caco-2 cell line is altered by P-glycoprotein inhibitors.

The mechanism of intestinal secretion of the difluorinated quinolone sparfloxacin was investigated with the epithelial cell line Caco-2 and was compared to that of the P-glycoprotein (P-gp) substrate vinblastine. The P-gp inhibitors verapamil and progesterone significantly increased the epithelial cell accumulation of both vinblastine and sparfloxacin. This increase is likely to result from an inhibition of drug secretion since both vinblastine uptake and sparfloxacin uptake are known to proceed through a passive transmembrane diffusion. The unidirectional fluxes across cell monlayers grown on permeable filters indicated that a net secretion of sparfloxacin and vinblastine occurred across Caco-2 cells. These secretions were significantly inhibited by the MDR-reversing agent verapamil. We conclude that the P-gp is likely to be involved in the intestinal elimination of the difluorinated quinolone sparfloxacin.

Oral QS-21 requires early IL-4 help for induction of mucosal and systemic immunity.

The highly purified saponin derivative, QS-21, from the Quillaja saponaria Molina tree has been proved to be safe for parenteral administration and represents a potential alternative to bacterial enterotoxin derivatives as a mucosal adjuvant. Here we report that p.o. administration of QS-21 with the vaccine protein tetanus toxoid elicited strong serum IgM and IgG Ab responses, which were only slightly enhanced by further oral immunization. The IgG Ab subclass responses were predominantly IgG1 followed by IgG2b for the 50-microg p.o. dose of QS-21, whereas the 250-microg p.o. dose also induced IgG2a and IgG3 Abs. Low oral QS-21 doses induced transient IgE Ab responses 7 days after the primary immunization, whereas no IgE Ab responses were seen in mice given the higher QS-21 dose. Further, low but not high p.o. QS-21 doses triggered Ag-specific secretory IgA (S-IgA) Ab responses. Th cell responses showed higher IFN-gamma (Th1-type) and lower IL-5, IL-6, and IL-10 (Th2-type) secretion after the high QS-21 p.o. dose than after low doses. Interestingly, the mucosal adjuvant activity of low oral QS-21 doses was diminished in IL-4(-/-) mice, suggesting a role for this cytokine in the initiation of mucosal immunity by oral QS-21. In summary, our results show that oral QS-21 enhances immunity to coadministered Ag and that different doses of QS-21 lead to distinct patterns of cytokine and serum Ab responses. We also show that an early IL-4 response is required for the induction of mucosal immunity by oral QS-21 as adjuvant.