Room air entrainment during beta-agonist delivery with heliox.

Studies of the efficacy of heliox in patients with severe asthma have shown mixed results. Among the factors that are responsible for variable outcomes, the failure of heliox delivery systems to prevent room air entrainment (RAE) during beta-agonist delivery is probably the most critical. While keeping the rotameter flow rate (FR) of heliox mixed 70:30 to a nebulizer at 10 L/min, the FR of heliox from a second gas source to a T-connector (TC) was increased during the delivery of the beta-agonist with a conventional T-nebulizer delivery system (TNDS). A negative peak inspiratory flow (pneumotachometer reading) or a helium concentration of < 70% (quadralizer reading) were indicators of RAE. RAE was tested during spontaneous tidal breathing and acute asthma. A rotameter FR of 10 L/m to the nebulizer with no flow from a second gas source to a TC (conventional TNDS) resulted in a significant drop in helium concentration during tidal breathing (46.2%) and acute asthma (27.5%) due to RAE. This degree of helium dilution can negate the beneficial effects of heliox to lung mechanics almost completely. A rotameter FR of 10 L/m each to a nebulizer and a TC resulted in a helium concentration 69.8% during tidal breathing (no RAE), but 49% (significant RAE) during asthma events. A rotameter FR of 15 L/m (pressure regulator setting, 100 lbs per square inch) to a TC, while maintaining a rotameter FR of 10 L/m to a nebulizer prevented RAE during asthma (helium concentration, 69.9%). Conventional TNDS may be used to deliver the beta-agonist with heliox during asthma without RAE.

TGF-beta down-regulates IL-6 signaling in intestinal epithelial cells: critical role of SMAD-2.

Interleukin-6 (IL-6) is a pro-inflammatory cytokine that plays an important role in the pathogenesis of inflammatory bowel disease. TGF-beta, a multifunctional cytokine, is a potent negative regulator of mucosal inflammation in the intestine. The aim of the present study is to determine possible cross-talk between IL-6 and TGF-beta signaling pathways. Model intestinal epithelial cell lines, Caco2-BBE were used. We show that TGF-beta receptor Type II is predominantly present in the basolateral membrane of intestinal epithelial cells. TGF-beta1 induces a time-dependent phosphorylation of Smad2 and co-immunoprecipitation of SMAD-2 with Smad-4 and its subsequently translocation to the nucleus. We show that pretreatment of cells with TGF-beta1 is associated with a down-regulation of IL-6 induced tyrosine phosphorylation of STAT1 and STAT3 and suppression of ICAM-1 expression. Furthermore, TGF-beta1 pretreatment resulted in a significant inhibition of IL-6 induced ICAM-1 promoter activity. TGF-beta mediated inhibition of IL-6 induced ICAM-1 expression was reversed by transfection with dominant negative Smad2 constructs. In conclusion, we show that: 1) TGF-beta receptor Type II is predominantly located on basolateral membrane and receptor stimulation activates Smad pathway; 2) TGF-beta1 down-regulates IL-6-induced tyrosine phoshorylation of STAT1 and STAT3 and ICAM-1 expression; and 3) Smad2 is required for the down-regulation of IL-6 signaling by TGF-beta. Collectively, our data demonstrate a cross-talk between TGF-beta and IL-6, and TGF-beta may play a role in the negative regulation of IL-6 signaling in intestinal epithelial cells.

Polarized fibronectin secretion induced by adenosine regulates bacterial-epithelial interaction in human intestinal epithelial cells.

Fibronectin (FN) is a multifunctional protein that plays important roles in many biological processes including cell adhesion and migration, wound healing and inflammation. Cellular FNs are produced by a wide variety of cell types including epithelial cells, which secrete them and often organize them into extensive extracellular matrices at their basal surface. However, regulation of FN synthesis and the polarity of FN secretion by intestinal epithelial cells have not been investigated. In the present study we investigated the role of adenosine, whose levels are up-regulated during inflammation, in modulating FN synthesis, the polarity of FN secretion and the downstream effects of the secreted FN. Polarized monolayers of T84 cells were used as an intestinal epithelial model. Adenosine added to either the apical or basolateral aspect of the cells led to a time- and dose-dependent accumulation of FN in the culture supernatants, polarized to the apical compartment and reached maximal levels 24 h after apical or basolateral addition of adenosine. Confocal microscopy confirmed that FN localized to the apical domain of model intestinal epithelial cells stimulated with apical or basolateral adenosine. The induction of FN was significantly down-regulated in response to the adenosine receptor antagonist alloxazine and was inhibited by cycloheximide. Moreover, adenosine increased FN promoter activity (3.5-fold compared with unstimulated controls) indicating that FN induction is, in part, transcriptionally regulated. Interestingly, we demonstrated that adenosine, as well as apical FN, significantly enhanced the adherence and invasion of Salmonella typhimurium into cultured epithelial cells. In summary, we have shown for the first time that FN, a classic extracellular matrix protein, is secreted into the apical compartment of epithelial cells in response to adenosine. FN may be a critical host factor that modulates adherence and invasion of bacteria, thus playing a key role in mucosal immune responses during inflammation.

Targeted deletion of metalloproteinase 9 attenuates experimental colitis in mice: central role of epithelial-derived MMP.

BACKGROUND & AIMS: There is mounting evidence that matrix metalloproteinases are the predominant proteinases expressed in the gut mucosa during active inflammatory bowel disease. We investigated the role of metalloproteinase 9 (MMP-9), a secreted gelatinase that is consistently up-regulated in both animal models and human inflammatory bowel disease and is associated with disease severity, in the pathogenesis of colitis by using mice containing a targeted deletion of the MMP-9 gene. METHODS: Dextran sodium sulfate-induced colitis and Salmonella typhimurium-induced enterocolitis were used as animal models to study colitis. RESULTS: MMP-9 activity and protein expression were absent from normal colonic mucosa but were up-regulated during experimental colitis. MMP-9-/- mice exposed to dextran sodium sulfate or salmonella had a significantly reduced extent and severity of colitis. Immunohistochemical studies showed that MMP-9 was localized to epithelial cells and granulocytes during active colitis. The immune response to systemic administration of Salmonella typhimurium was not affected in MMP-9-/- mice. Neutrophil transmigration studies and bone marrow chimeras showed that neutrophil MMP-9 is neither required for its migration nor sufficient to induce tissue damage during colitis and that epithelial MMP-9 is important for tissue damage. MMP-9 inhibited cell attachment and wound healing in the model intestinal epithelial cell line, Caco2-BBE. CONCLUSIONS: Taken together, our data suggest that MMP-9 expressed by epithelial cells may play an important role in the development of colitis by modulating cell-matrix interaction and wound healing. Thus, strategies to inhibit MMP-9 may be of potential therapeutic benefit.

IL-6 induces NF-kappa B activation in the intestinal epithelia.

IL-6 is a potent proinflammatory cytokine that has been shown to play an important role in the pathogenesis of inflammatory bowel disease (IBD). It is classically known to activate gene expression via the STAT-3 pathway. Given the crucial role of IL-6 in the pathogenesis of chronic intestinal inflammation, it is not known whether IL-6 activates NF-kappaB, a central mediator of intestinal inflammation. The model intestinal epithelial cell line, Caco2-BBE, was used to study IL-6 signaling and to analyze whether suppressor of cytokine signaling 3 (SOCS-3) proteins play a role in the negative regulation of IL-6 signaling. We show that IL-6 receptors are present in intestinal epithelia in a polarized fashion. Basolateral IL-6 and, to a lesser extent, apical IL-6 induces the activation of the NF-kappaB pathway. Basolateral IL-6 stimulation results in a maximal induction of NF-kappaB activation and NF-kappaB nuclear translocation at 2 h. IL-6 induces polarized expression of ICAM-1, an adhesion molecule shown to be important in the neutrophil-epithelial interactions in IBD. Using various deletion constructs of ICAM-1 promoter, we show that ICAM-1 induction by IL-6 requires the activation of NF-kappaB. We also demonstrate that overexpression of SOCS-3, a protein known to inhibit STAT activation in response to IL-6, down-regulates IL-6-induced NF-kappaB activation and ICAM-1 expression. In summary, we demonstrate the activation of NF-kappaB by IL-6 in intestinal epithelia and the down-regulation of NF-kappaB induction by SOCS-3. These data may have mechanistic and therapeutic implications in diseases such as IBD and rheumatoid arthritis in which IL-6 plays an important role in the pathogenesis.

Agonist-induced polarized trafficking and surface expression of the adenosine 2b receptor in intestinal epithelial cells: role of SNARE proteins.

Adenosine, acting through the A2b receptor, induces vectorial chloride and IL-6 secretion in intestinal epithelia and may play an important role in intestinal inflammation. We have previously shown that apical or basolateral adenosine receptor stimulation results in the recruitment of the A2b receptor to the plasma membrane. In this study, we examined domain specificity of recruitment and the role of soluble N-ethylmaleimide (NEM) attachment receptor (SNARE) proteins in the agonist-mediated recruitment of the A2b receptor to the membrane. The colonic epithelial cell line T84 was used because it only expresses the A2b-subtype adenosine receptor. Cell fractionation, biotinylation, and electron microscopic studies showed that the A2b receptor is intracellular at rest and that apical or basolateral adenosine stimulation resulted in the recruitment of the receptor to the apical membrane. Upon agonist stimulation, the A2b receptor is enriched in the vesicle fraction containing vesicle-associated membrane protein (VAMP)-2. Furthermore, in cells stimulated with apical or basolateral adenosine, we demonstrate a complex consisting of VAMP-2, soluble NEM-sensitive factor attachment protein (SNAP)-23, and A2b receptor that is coimmunoprecipitated in cells stimulated with adenosine within 5 min and is no longer detected within 15 min. Inhibition of trafficking with NEM or nocodazole inhibits cAMP synthesis induced by apical or basolateral adenosine by 98 and 90%, respectively. cAMP synthesis induced by foskolin was not affected, suggesting that generalized signaling is not affected under these conditions. Collectively, our data suggest that 1) the A2b receptor is intracellular at rest; 2) apical or basolateral agonist stimulation induces recruitment of the A2b receptor to the apical membrane; 3) the SNARE proteins, VAMP-2 and SNAP-23, participate in the recruitment of the A2b receptor; and 4) the SNARE-mediated recruitment of the A2b receptor may be required for its signaling.