CCL26-targeted siRNA treatment of alveolar type II cells decreases expression of CCR3-binding chemokines and reduces eosinophil migration: implications in asthma therapy.

The underlying inflammation present in chronic airway diseases is orchestrated by increased expression of CC chemokines that selectively recruit leukocyte populations into the pulmonary system. Human CCL26 signals through CC chemokine receptor 3 (CCR3), is dramatically upregulated in challenged asthmatics, and stimulates recruitment of eosinophils (EOSs) and other leukocytes. CCL26 participates in regulation of its receptor CCR3 and modulates expression of a variety of chemokines in alveolar type II cells. Utilizing the A549 alveolar type II epithelial cell culture model, we carried out studies to test the hypothesis that CCL26-siRNA treatment of these cells would ameliorate Th2-driven release of the eotaxins and other CCR3 ligands that would, in turn, decrease recruitment and activation of EOSs. Results demonstrate that CCL26-siRNA treatments decreased interleukin-4-induced CCL26 and CCL24 expression by >70%. CCL26-directed small-interfering RNA (siRNA) treatments significantly decreased release of CCL5 (RANTES), CCL15 (MIP-1δ), CCL8 (MCP-2), and CCL13 (MCP-4). In bioactivity assays it was shown that EOS migration and activation were reduced up to 80% and 90%, respectively, when exposed to supernatants of CCL26-siRNA-treated cells. These results provide evidence that CCL26 may be an appropriate target for development of new therapeutic agents designed to alleviate the underlying inflammation associated with chronic diseases of the airways.

Post-transcriptional silencing of CCR3 downregulates IL-4 stimulated release of eotaxin-3 (CCL26) and other CCR3 ligands in alveolar type II cells.

Trafficking and inflammation in airway diseases are, in part, modulated by members of the CC chemokine family, eotaxin-1 (CCL11), eotaxin-2 (CCL24), and eotaxin-3 (CCL26), which transduce signals through their CCR3 receptor. In this context, we hypothesized that transfecting alveolar type II epithelial cells with CCR3-targeted siRNA or antisense (AS-ODN) sequences will downregulate cellular synthesis and release of the primary CCR3 ligands CCL26 and CCL24 and will modulate other CCR3 ligands. The human A549 alveolar type II epithelium-like cell culture model was used for transfection and subsequent effects on CCR3 agonists. siRNAs were particularly effective. PCR showed a 60-80% decrease in mRNA and immunoblots showed up to 75-84% reduction of CCR3 in siRNA treated cells. CCR3-siRNA treatments reduced IL-4 stimulated CCL26 release and constitutive CCL24 release by 65% and 80%, respectively. Release of four additional CCR3 agonists RANTES, MCP-2, MCP-3 and MCP-4 was also significantly reduced by CCR3-siRNA treatments of the alveolar type II cells. Activation of eosinophils, assessed as superoxide anion generation, was reduced when eosinophils were treated with supernatants of A549 cells pretreated with CCR3-targeted siRNAs or AS-ODNs. Collectively, the data suggest that post-transcriptional regulation of CCR3 receptors may be a potential therapeutic approach for interrupting proinflammatory signaling.

Modulation of eotaxin-3 (CCL26) in alveolar type II epithelial cells.

Airway epithelial inflammation associated with emphysema, chronic bronchitis, chronic obstructive pulmonary disease (COPD) and asthma is regulated in part by alveolar type II cell chemokine signaling. Data suggest that resident lung cells use CCR3, CCR5 and CCR2 chemokine receptor/ligand systems to regulate the profile of leukocytes recruited in disease-associated inflammatory conditions. Thus studies were designed to test whether alveolar type II cells possess a Th1-activated CCR5-ligand system that modulates the Th2-activated CCR3/eotaxin-2 (CCL24), eotaxin-3 (CCL26) chemokine systems. The A549 alveolar type II epithelial-like cell culture model was used to demonstrate that alveolar type II cells constitutively express CCR5 which may be upregulated by MIP-1alpha (CCL3) whose expression was induced by the Th1 cytokines IL-1beta and IFN-gamma. Selective down-regulation of CCL26, but not CCL24, was observed in CCL3 and IL-4/CCL3 stimulated cells. Down-regulation was reversed by anti-CCR5 neutralizing antibody treatment. Thus, one mechanism through which Th1-activated CCCR5/ligand pathways modulate Th2-activated CCR3/ligand pathways is the differential down-regulation of CCL26 expression. Results suggest that the CCR3 and CCR5 receptor/ligand signaling pathways may be important targets for development of novel mechanism-based adjunctive therapies designed to abrogate the chronic inflammation associated with airway diseases.

Cytokine-stimulated human lung alveolar epithelial cells release eotaxin-2 (CCL24) and eotaxin-3 (CCL26).

Asthma is a complex inflammatory disease characterized by a prolonged underlying airway inflammation resulting from cytokine-orchestrated signaling between many types of cells, including airway epithelial cells. Trafficking, recruitment, and activation of cells in airway disease are, in part, modulated by the newly discovered CC subfamily of chemokines, eotaxin (CCL11), eotaxin-2 (CCL24) and eotaxin-3 (CCL26), which transduce signals by acting as agonists for the CCR3 receptor. The specific cytokine stimuli that modulate CCL24 and CCL26 release in airway epithelial cells remain poorly defined. Thus, human 549 alveolar type II epithelium-like cells were stimulated singly and with combinations of 1-100 ng/ml tumor necrosis-factor-alpha (TNF-alpha), interleukin-1beta (IL-1beta), and IL-4, cytokines known to be elevated in the airways of asthmatics. Release of CCL11, CCL24, and CCL26 was quantified by ELISA, and CCR3 receptors monitored by immunocytochemistry and FACS analysis. Results suggest that epithelial cells release CCL11 during the first 24 h of stimulation, in contrast to a significant increase in CCL24 and CCL26 release after 24-48 h of stimulation. Differential release of the eotaxins in response to cytokine combinations was noted. The alveolar type II epithelial cells were found to possess constitutive CCR3 receptors, which increased after proinflammatory cytokine stimulation. The airway epithelium CCR3 receptor/eotaxin ligand signal transduction system may be an important target for development of novel mechanism-based adjunctive therapies designed to interrupt the underlying chronic inflammation in allergic and inflammatory disorders.

Syntaxin 2 and SNAP-23 are required for regulated surfactant secretion.

The secretion of lung surfactant in alveolar type II cells is a complex process involving the fusion of lamellar bodies with the plasma membrane. This process is somewhat different from the exocytosis of hormones and neurotransmitters. For example, it is a relatively slower process, and lamellar bodies are very large vesicles with a diameter of approximately 1 microm. SNARE proteins are the conserved molecular machinery of exocytosis in the majority of secretory cells. However, their involvement in surfactant secretion has not been reported. Here, we showed that syntaxin 2 and SNAP-23 are expressed in alveolar type II cells. Both proteins are associated with the plasma membrane, and to some degree with lamellar bodies. An antisense oligonucleotide complementary to syntaxin 2 decreased its mRNA and protein levels. The same oligonucleotide also inhibited surfactant secretion, independent of secretagogues. A peptide derived from the N-terminus of syntaxin 2 or the C-terminus of SNAP-23 significantly inhibited Ca(2+)- and GTPgammaS-stimulated surfactant secretion from permeabilized type II cells in a dose-dependent manner. Furthermore, introduction of anti-syntaxin 2 or anti-SNAP-23 antibodies into permeabilized type II cells also inhibited surfactant release. Our results suggest that syntaxin 2 and SNAP-23 are required for regulated surfactant secretion.

Reorganization of cytoskeleton during surfactant secretion in lung type II cells: a role of annexin II.

The secretion of lung surfactant requires the movement of lamellar bodies to the plasma membrane through cytoskeletal barrier at the cell cortex. We hypothesized that the cortical cytoskeleton undergoes a transient disassembly/reassembly in the stimulated type II cells, therefore allowing lamellar bodies access to the plasma membrane. Stabilization of cytoskeleton with Jasplakinolinde (JAS), a cell permeable actin microfilament stabilizer, caused a dose-dependent inhibition of lung surfactant secretion stimulated by terbutaline. This inhibition was also observed in ATP-, phorbol 12-myristate 13-acetate (PMA)- or Ca(2+) ionophore A23187-stimulated surfactant secretion. Stimulation of type II cells with terbutaline exhibited a transient disassembly of filamentous actin (F-actin) as determined by staining with Oregon Green 488 Phalloidin. The protein kinase A inhibitor, H89, abolished the terbutaline-induced F-actin disassembly. Western blot analysis using anti-actin and anti-annexin II antibodies showed a transient increase of G-actin and annexin II in the Triton X-100 soluble fraction of terbutaline-stimulated type II cells. Furthermore, introduction of exogenous annexin II tetramer (AIIt) into permeabilized type II cells caused a disruption in the cortical actin. Treatment of type II cells with N-ethylmaleimide (NEM) resulted in a disruption of the cortical actin. NEM also inhibited annexin II's abilities to bundle F-actin. The results suggest that cytoskeleton undergoes reorganization in the stimulated type II cells, and annexin II tetramer plays a role in this process.

Fusion of lamellar body with plasma membrane is driven by the dual action of annexin II tetramer and arachidonic acid.

Annexin II has been implicated in membrane fusion during the exocytosis of lamellar bodies from alveolar epithelial type II cells. Most previous studies were based on the fusion assays by using model membranes. In the present study, we investigated annexin II-mediated membrane fusion by using isolated lamellar bodies and plasma membrane as determined by the relief of octadecyl rhodamine B (R18) self-quenching. Immunodepletion of annexin II from type II cell cytosol reduced its fusion activity. Purified annexin II tetramer (AIIt) induced the fusion of lamellar bodies with the plasma membrane in a dose-dependent manner. This fusion is Ca2+-dependent and is highly specific to AIIt because other annexins (I and II monomer, III, IV, V, and VI) were unable to induce the fusion. Modification of the different functional residues of AIIt by N-ethylmaleimide, nitric oxide, or peroxynitrite abolished AIIt-mediated fusion. Arachidonic acid enhanced AIIt-mediated fusion and reduced its Ca2+ requirement to an intracellularly achievable level. This effect is due to membrane-bound arachidonic acid, not free arachidonic acid. Other fatty acids including linolenic acid, palmitoleic acid, myristoleic acid, stearic acid, palmitic acid, and myristic acid had little effect. AIIt-mediated fusion was suppressed by the removal of arachidonic acid from lamellar body and plasma membrane using bovine serum albumin. The addition of arachidonic acid back to the arachidonic acid-depleted membranes restored its fusion activity. Our results suggest that the fusion between lamellar bodies with the plasma membrane is driven by the synergistic action of AIIt and arachidonic acid.

Characterization of alpha-soluble N-ethylmaleimide-sensitive fusion attachment protein in alveolar type II cells: implications in lung surfactant secretion.

N-ethylmaleimide-sensitive fusion protein (NSF) and soluble NSF attachment protein (alpha-SNAP) are thought to be soluble factors that transiently bind and disassemble SNAP receptor complex during exocytosis in neuronal and endocrine cells. Lung surfactant is secreted via exocytosis of lamellar bodies from alveolar epithelial type II cells. However, the secretion of lung surfactant is a relatively slow process, and involvement of SNAP receptor and its cofactors (NSF and alpha-SNAP) in this process has not been demonstrated. In this study, we investigated a possible role of alpha-SNAP in surfactant secretion. alpha-SNAP was predominantly associated with the membranes in alveolar type II cells as determined by Western blot and immunocytochemical analysis using confocal microscope. Membrane-associated alpha-SNAP was not released from the membrane fraction when the cells were lyzed in the presence of Ca2+ or Mg2+ATP. The alkaline condition (0.1 M Na2CO3, pH 12), known to extract peripheral membrane proteins also failed to release it from the membrane. Phase separation using Triton X-114 showed that alpha-SNAP partitioned into both aqueous and detergent phases. NSF had membrane-bound characteristics similar to alpha-SNAP in type II cells. Permeabilization of type II cells with beta-escin resulted in a partial loss of alpha-SNAP from the cells, but cellular NSF was relatively unchanged. Addition of exogenous alpha-SNAP to the permeabilized cells increased surfactant secretion in a dose-dependent manner, whereas exogenous NSF has much less effects. An alpha-SNAP antisense oligonucleotide decreased its protein level and inhibited surfactant secretion. Our results suggest a role of alpha-SNAP in lung surfactant secretion.