Modulation of the epithelial barrier by dexamethasone and prolactin in cultured Madin-Darby canine kidney (MDCK) cells

Glucocorticoids and prolactin (PRL) have a direct effect on the formation and maintenance of tight junctions (TJs) in cultured endothelial and mammary gland epithelial cells. In this work, we investigated the effect of a synthetic glucocorticoid dexamethasone (DEX) and PRL on the paracellular barrier function in MDCK renal epithelial cells. DEX (4 microM)+PRL (2 microg/ml) and DEX alone increased significantly the transepithelial electrical resistance after chronic treatment (4 days) of confluent MDCK monolayers or after 24 h treatment of subconfluent monolayers. Immunoblotting and immunocytochemistry revealed no changes in the expression and distribution of TJ-associated proteins occludin, ZO-1 and claudin-1 in confluent monolayers after hormone addition. However, a marked increase in junctional content for occludin and ZO-1 with no changes in their total expression was observed in subconfluent MDCK monolayers 24 h exposed to DEX or DEX+PRL. No change in cell proliferation/growth was detected at subconfluent conditions following hormone treatment. An increase in the total number of viable cells was observed only in confluent MDCK monolayers after exposure to DEX+PRL suggesting that the main effect of these hormones on already established barrier may be associated with the inhibition of cell death. In conclusion, our data suggest that these hormones (specially dexamethasone) have an effect on TJ structure and function only during the formation of MDCK epithelial barrier by probably modulating the localization, stability or assembly of TJ proteins to membrane sites of intercellular contact.     

Relative contribution of hemopoietic and pulmonary parenchymal cells to lung inducible nitric oxide synthase (inos) activity in murine endotoxemia

Acute lung injury is an important feature of sepsis and increased iNOS expression and NO production contribute to the pathogenesis of this syndrome. We generated bone marrow-transplanted chimeric mice with iNOS expression limited to either inflammatory or pulmonary parenchymal cells, and assessed pulmonary iNOS activity and systemic levels of NO metabolites in an endotoxemic model of sepsis. We found that while both pulmonary parenchymal cells and inflammatory cells contribute to the increased lung iNOS activity in endotoxemia, pulmonary parenchymal cells contribute to a significantly greater degree. Using measurement of plasma NO(-)(x), whole body NO production was assessed in this model. We found that the main source of NO(-)(x) was again, parenchymal cells and not inflammatory cells. This is the first study to demonstrate that most of the increased NO production in this model of endotoxemic sepsis derives from parenchymal cells rather than inflammatory cells.     

Somatostatin ameliorates lipopolysaccharide-induced tight junction damage via the ERK–MAPK pathway in Caco2 cells

Dysfunction of the epithelial barrier is an important pathogenic factor of inflammatory bowel disease and other inflammatory conditions of the gut. Somatostatin (SST) has been demonstrated to reduce local and systemic inflammation reactions and maintain the integrity of the blood–brain barrier (BBB). To determine the beneficial effect of SST on lipopolysaccharide (LPS)-induced damage of the tight junction (TJ) and its mechanisms, Caco2 cells pretreated with SST (1 nM) or MEK inhibitor U0126 (10 μM) were exposed to LPS. LPS significantly reduced the expression of TJ proteins in a dose-dependent way. LPS (100 μg/ml) greatly induced Caco2 monolayer barrier dysfunction by decreasing transepithelial resistance and increasing epithelial permeability. Pretreatment with SST effectively improved the barrier dysfunction of Caco2 cells. SST significantly increased the expression of TJ proteins occludin and ZO-1 and inhibited the redistribution of TJ proteins due to LPS stimulation. Furthermore, SST decreased the LPS-induced phosphorylation of ERK1/2, and a selective MEK inhibitor markedly protected the barrier function against LPS disturbance by blocking the activation of the ERK–MAPK pathway in Caco2 cells. Besides, LPS significantly increased the mRNA level of SSTR5, which was partly inhibited by pretreatment with SST. In conclusion, the present study indicates that SST protects the Caco2 monolayer barrier against LPS-induced tight junction breakdown by down-regulating the activation of the ERK–MAPK pathway and suppression the activation of SSTR5.     

Resolution of experimental lung injury by monocyte-derived inducible nitric oxide synthase

Although early events in the pathogenesis of acute lung injury (ALI) have been defined, little is known about the mechanisms mediating resolution. To search for determinants of resolution, we exposed wild type (WT) mice to intratracheal LPS and assessed the response at intervals to day 10, when injury had resolved. Inducible NO synthase (iNOS) was significantly upregulated in the lung at day 4 after LPS. When iNOS(-/-) mice were exposed to intratracheal LPS, early lung injury was attenuated; however, recovery was markedly impaired compared with WT mice. iNOS(-/-) mice had increased mortality and sustained increases in markers of lung injury. Adoptive transfer of WT (iNOS(+/+)) bone marrow-derived monocytes or direct adenoviral gene delivery of iNOS into injured iNOS(-/-) mice restored resolution of ALI. Irradiated bone marrow chimeras confirmed the protective effects of myeloid-derived iNOS but not of epithelial iNOS. Alveolar macrophages exhibited sustained expression of cosignaling molecule CD86 in iNOS(-/-) mice compared with WT mice. Ab-mediated blockade of CD86 in iNOS(-/-) mice improved survival and enhanced resolution of lung inflammation. Our findings show that monocyte-derived iNOS plays a pivotal role in mediating resolution of ALI by modulating lung immune responses, thus facilitating clearance of alveolar inflammation and promoting lung repair.     

A pyrrolidinone derivative inhibits cytokine-induced iNOS expression and NF-kappaB activation by preventing phosphorylation and degradation of IkappaB-alpha

We previously showed that 1-[3-(3-pyridyl)-acryloyl]-2-pyrrolidinone hydrochloride (N2733) inhibits lipopolysaccharide (LPS)-induced tumour necrosis factor (TNF)-alpha secretion and improves the survival of endotoxemic mice. Since overproduction of nitric oxide (NO) by inducible NO synthase (iNOS) in vascular smooth muscle cells (VSMCs) is largely responsible for the development of endotoxemic shock, and iNOS gene expression is mainly regulated by LPS and inflammatory cytokines, we studied whether or not N2733 affects interleukin (IL)-1beta-induced iNOS gene expression, NF-kappaB activation, and NF-kappaB inhibitor (IkappaB)-alpha degradation in cultured rat VSMCs. N2733 dose-dependently (10-100 microM) inhibited IL-1beta-stimulated NO production, and decreased IL-1beta-induced iNOS mRNA and protein expression, as found on Northern and Western blot analyses, respectively. Gel shift assay and an immunocytochemical study showed that N2733 inhibited IL-1beta-induced NF-kappaB activation and its nuclear translocation. Western blot analyses involving anti-IkappaB-alpha and anti-phospho IkappaB-alpha antibodies showed that IL-1beta induced transient degradation of IkappaB-alpha preceded by the rapid appearance of phosphorylated IkappaB-alpha, both of which were markedly blocked by N2733. N2733 blocked IL-1beta-induced phosphorylated IkappaB-alpha even in the presence of a proteasome inhibitor (MG115). Immunoblot analysis involving anti-IkappaB kinase (IKK)-alpha and anti-phosphoserine antibodies revealed that N2733 inhibited IL-1beta-induced IKK-alpha phosphorylation, whereas N2733 had no inhibitory effect on IL-1beta-stimulated p42/p44 MAP kinase or p38 MAP kinase activity. Our results suggest that the inhibitory action of N2733 toward IL-1beta-induced NF-kappaB activation and iNOS expression is due to its blockade of the upstream signal(s) leading to IKK-alpha activation, and subsequent phosphorylation and degradation of IkappaB-alpha in rat VSMCs.     

Diethylmaleate and buthionine sulfoximine, glutathione-depleting agents, differentially inhibit expression of inducible nitric oxide synthase in endotoxemic mice.

Diethylmaleate (DEM) and buthionine sulfoximine (BSO), glutathione (GSH)-depleting agents, reduced the metabolic activity and the protein level of iNOS in both macrophages and hepatocytes activated by lipopolysaccharide (LPS). In this study, we examined the effects of DEM and BSO on iNOS expression in LPS-treated mice under the assumption that the level of GSH may alter the expression of nitric oxide synthase. Serum levels of interleukin-1beta (IL-1beta) and tumor necrosis factor-alpha (TNF-alpha) were also determined. DEM markedly decreased the levels of hepatic GSH in response to LPS. Treatment of mice with DEM significantly reduced serum nitrite/nitrate levels and hepatic iNOS protein and mRNA induction by LPS. Although BSO inhibited the level of hepatic GSH in LPS-treated mice, the agent did not alter serum nitrite/nitrate levels and hepatic iNOS expression. DEM completely inhibited an increase of serum IL-1beta level by LPS, whereas BSO failed to inhibit it. Neither DEM nor BSO significantly affected the induction of serum TNF-alpha level by LPS. These results showed that DEM and BSO differentially affect the expression of iNOS in endotoxemic mice, suggesting the possibility that suppression of iNOS expression by DEM may be associated with the inhibition of IL-1beta but not of TNF-alpha.     

Hyperoxia disrupts pulmonary epithelial barrier in newborn rats via the deterioration of occludin and ZO-1

Background

Prolonged exposure to hyperoxia in neonates can cause hyperoxic acute lung injury (HALI), which is characterized by increased pulmonary permeability and diffuse infiltration of various inflammatory cells. Disruption of the epithelial barrier may lead to altered pulmonary permeability and maintenance of barrier properties requires intact epithelial tight junctions (TJs). However, in neonatal animals, relatively little is known about how the TJ proteins are expressed in the pulmonary epithelium, including whether expression of TJ proteins is regulated in response to hyperoxia exposure. This study determines whether changes in tight junctions play an important role in disruption of the pulmonary epithelial barrier during hyperoxic acute lung injury.

Methods

Newborn rats, randomly divided into two groups, were exposed to hyperoxia (95% oxygen) or normoxia for 1–7 days, and the severity of lung injury was assessed; location and expression of key tight junction protein occludin and ZO-1 were examined by immunofluorescence staining and immunobloting; messenger RNA in lung tissue was studied by RT-PCR; transmission electron microscopy study was performed for the detection of tight junction morphology.

Results

We found that different durations of hyperoxia exposure caused different degrees of lung injury in newborn rats. Treatment with hyperoxia for prolonged duration contributed to more serious lung injury, which was characterized by increased wet-to-dry ratio, extravascular lung water content, and bronchoalveolar lavage fluid (BALF):serum FD4 ratio. Transmission electron microscopy study demonstrated that hyperoxia destroyed the structure of tight junctions and prolonged hyperoxia exposure, enhancing the structure destruction. The results were compatible with pathohistologic findings. We found that hyperoxia markedly disrupted the membrane localization and downregulated the cytoplasm expression of the key tight junction proteins occludin and ZO-1 in the alveolar epithelium by immunofluorescence. The changes of messenger RNA and protein expression of occludin and ZO-1 in lung tissue detected by RT-PCR and immunoblotting were consistent with the degree of lung injury.

Conclusions

These data suggest that the disruption of the pulmonary epithelial barrier induced by hyperoxia is, at least in part, due to massive deterioration in the expression and localization of key TJ proteins.     

Interleukin-1 is not essential for expression of inducible NOS in hepatocytes induced by lipopolysaccharide in vivo

Interleukin (IL)-1 and tumor necrotic factor alpha (TNFalpha) are pivotal in the pathogenesis of endotoxemia. In spite of the in vitro finding that IL-1beta, but not TNFalpha, can induce iNOS mRNA and NO production as a single stimulus in hepatocytes in primary culture, the involvement of IL-1 in iNOS induction in the liver has been less clear in vivo. To address this, we challenged IL-1alpha/beta double-knockout (IL-1alpha/beta(-/-)) and TNFalpha(-/-) mice with lipopolysaccharide (LPS). As compared with wild-type mice, the increases in the plasma NO level measured as nitrite and nitrate and hepatic iNOS were significantly reduced in IL-1alpha/beta(-/-) and TNFalpha(-/-) mice 8 and 12h after the LPS challenge. In the wild-type mice, iNOS protein was first detected in Kupffer cells around the portal vein 2h after LPS challenge; and then it spread to hepatocytes throughout the intralobular region of the liver by 8h. Although the expression of iNOS protein was detected in Kupffer cells of both IL-1alpha/beta(-/-) and TNFalpha(-/-) mice, its level was moderate in hepatocytes of IL-1alpha/beta(-/-) mice, but negligible in those of TNFalpha(-/-) mice, 8h after LPS challenge. Concomitant with the expression of iNOS protein in the liver, Toll-like receptor 4, the signaling receptor for LPS, was expressed in hepatocytes of wild-type and IL-1alpha/beta(-/-) mice, but not of TNFalpha(-/-) mice. These results demonstrate that the expression of Toll-like receptor 4 is well correlated with that of iNOS protein in hepatocytes in vivo after LPS challenge and that IL-1 is not essential for the induction of iNOS in hepatocytes in vivo.     

Role of free radicals and poly(ADP-ribose) synthetase in intestinal tight junction permeability

BACKGROUND: Small intestine permeability is frequently altered in inflammatory bowel disease and may be caused by the translocation of intestinal toxins through leaky small intestine tight junctions (TJ) and adherence (1,2). The role of hydrogen peroxide (H2O2), and nitric oxide (NO) and PARS in the permeability and structure of small intestine TJ is not clearly understood. MATERIALS AND METHODS: In vitro study, MDCK (Madin-Darby Canine Kidney) cells were exposed to H2O2 (100 microM for 2h), or zymosan (200 microl of stock solution 1 mg/ml for 4h), in the presence or absence of a treatment with poly(ADP-ribose) synthetase (PARS) inhibitor 3-aminobenzamide (3-AB: 3 mM) or with n-acetylcysteine (NAC 10 mM). In vivo study, wild-type mice (WT) and mice lacking (KO) of the inducible (or type 2) nitric oxide synthase (iNOS) were treated with zymosan (500 mg/kg, suspended in saline solution, i.p.). In addition INOSWT mice were treated with 3-AB (10 mg/kg, i.p.) or with NAC (40 mg/kg, i.p.) 1 hour and 6 h after zymosan administration. RESULTS: Exposure of MDCK cells to hydrogen peroxide caused a significant impairment in mitochondrial respiration that was associated with a reduction of cells adherence as well as derangement of the junctional proteins. A significant increase of nitrate and nitrite levels, stable metabolites of nitric oxide (NO), were found in MDCK supernatant after zymosan incubation. NO production was associated with a significant reduction of cell adherence and impairment of occludin protein. Pre-treatment of the cells with 3-AB or with NAC caused a significant prevention of H2O2-mediated occludin junctional damage as well as reduced the NO-induced occludin damage. In addition, H2O2 and NO are able to induce a significant derangement of beta-catenin and Zonula Ocludence-1 (ZO-1). We found an increase of tight junctional permeability to lanthanum nitrate (molecular weight, 433) in the terminal ileal TJs in zymosan-treated iNOSWT mice compared with permeable TJ in the control animals. Zymosan-treated iNOSKO mice showed a significant increase of tight junctional permselectivity. There were no differences in strand count or strand depth in the ilea from control or treated animals. In addition, a significant disrupted immunofluorescence signal for occludin, ZO-1 and beta-catenin was observed in the terminal ilea of zymosan-treated iNOSWT mice. In ileal fragments from zymosan-treated iNOSKO mice, we found less irregular distribution patterns of occludin, ZO-1 and beta-catenin. Similarly NAC or 3-AB treatments were able to prevent zymosan-induced damage of junctional proteins in iNOSWT mice. CONCLUSION: In conclusion, this study demonstrates that the alteration of permselectivity is most likely induced by ROS and PARS activation.     

Role of nitric oxide on diaphragmatic contractile failure in Escherichia coli endotoxemic rats

Contractile dysfunction of the respiratory muscles plays an important role in the genesis of respiratory failure during sepsis. Nitric oxide (NO), a free radical that is cytotoxic and negatively inotropic in the heart and skeletal muscle, is produced in large amounts during sepsis by a NO synthase inducible (iNOS) by LPS and/or cytokines. The aim of this study was to investigate whether iNOS was induced in the diaphragm of Escherichia coli endotoxemic rats and whether inhibition of iNOS induction or of NOS synthesis attenuated diaphragmatic contractile dysfunction. Rats were inoculated intravenously (IV) with 10 mg/kg of E. coli endotoxin (LPS animals) or saline (C animals). Six hours after LPS inoculation animals showed a significant increase in diaphragmatic NOS activity (L-citrulline production, P < 0.005). Inducible NOS protein was detected by Western-Blot in the diaphragms of LPS animals, while it was absent in C animals. LPS animals had a significant decrease in diaphragmatic force (P < 0.0001) measured in vitro. In LPS animals, inhibition of iNOS induction with dexamethasone (4 mg/kg IV 45 min before LPS) or inhibition of NOS activity with N(G)-methyl-L-arginine (8 mg/kg IV 90 min after LPS) prevented LPS-induced diaphragmatic contractile dysfunction. We conclude that increased NOS activity due to iNOS was involved in the genesis of diaphragmatic dysfunction observed in E. coli endotoxemic rats.     

ZO-2 silencing in epithelial cells perturbs the gate and fence function of tight junctions and leads to an atypical monolayer architecture

ZO-2 is a tight junction (TJ) protein that shuttles between the plasma membrane and the nucleus. ZO-2 contains several protein binding sites that allow it to function as a scaffold that clusters integral, adaptor and signaling proteins. To gain insight into the role of ZO-2 in epithelial cells, ZO-2 was silenced in MDCK cells with small interference RNA (siRNA). ZO-2 silencing triggered: (A) changes in the gate function of the TJ, determined by an increase in dextran flow through the paracellular route of mature monolayers and achievement of lower transepithelial electrical resistance values upon TJ de novo formation; (B) changes in the fence function of the TJ manifested by a non-polarized distribution of E-cadherin on the plasma membrane; (C) altered expression of TJ and adherens junction proteins, determined by a decreased amount of occludin and E-cadherin in mature monolayers and a delayed arrival to the plasma membrane of ZO-1, occludin and E-cadherin during a calcium switch assay; and (D) an atypical monolayer architecture characterized by the appearance of widened intercellular spaces, multistratification of regions in the culture and an altered pattern of actin at the cellular borders.     

Penehyclidine hydrochloride attenuates LPS-induced iNOS production by inhibiting p38 MAPK activation in endothelial cells

The aim of this study was to investigate the inhibitory effect of penehyclidine hydrochloride (PHC) on lipopolysaccharide (LPS)-induced nitric oxide (NO) and inducible nitric oxide synthase (iNOS) production in human endothelial cell. Cultured endothelial cells were pretreated with PHC, followed by LPS treatment. NO activity were determined. iNOS expression and p38 mitogen-activated protein kinase (p38 MAPK) protein expression were measured by Western blot analysis. LPS treatment significantly induced p38 MAPK activation, iNOS expression, and NO production, which could be attenuated by 2 μg/ml PHC pretreatment. Furthermore, our study showed LPS-induced NO production and iNOS expression were suppressed by p38 MAPK inhibitor SB203580 pretreatment. We concluded that PHC attenuates NO production and iNOS expression by suppressing the activation of p38 MAPK pathway, thereby implicating a mechanism by which PHC may exert its protective effects against LPS-induced endothelial cell injury.     

autoregulatory role of endothelium-derived nitric oxide (NO) on Lipopolysaccharide-induced vascular inducible NO synthase expression and function

Nitric oxide (NO) produced by inducible nitric oxide synthase (iNOS) is responsible for sepsis-induced hypotension and plays a major contributory role in the ensuing multiorgan failure. The present study aimed to elucidate the role of endothelial NO in lipopolysaccharide (LPS)-induced iNOS expression, in isolated rat aortic rings. Exposure to LPS (1 mug/ml, 5 h) resulted in a reversal of phenylephrine precontracted tone in aortic rings (70.7 +/- 3.2%). This relaxation was associated with iNOS expression and NF-kappaB activation. Positive immunoreactivity for iNOS protein was localized in medial and adventitial layers of LPS-treated aortic rings. Removal of the endothelium rendered aortic rings resistant to LPS-induced relaxation (8.9 +/- 4.5%). Western blotting of these rings demonstrated an absence of iNOS expression. However, treatment of endothelium-denuded rings with the NO donor, diethylamine-NONOate (0.1 mum), restored LPS-induced relaxation (61.6 +/- 6.6%) and iNOS expression to levels comparable with arteries with intact endothelium. Blockade of endothelial NOS (eNOS) activation using geldanamycin and radicicol, inhibitors of heat shock protein 90, in endothelium-intact arteries suppressed both LPS-induced relaxation and LPS-induced iNOS expression (9.0 +/- 8.0% and 2.0 +/- 6.2%, respectively). Moreover, LPS treatment (12.5 mg/kg, intravenous, 15 h) of wild-type mice resulted in profound elevation of plasma [NO(x)] measurements that were reduced by approximately 50% in eNOS knock-out animals. Furthermore, LPS-induced changes in vascular reactivity and iNOS expression evident in wild-type tissues were profoundly suppressed in tissues taken from eNOS knockout animals. Together, these data suggest that eNOS-derived NO, in part via activation of NF-kappaB, regulates iNOS-induction by LPS. This study provides the first demonstration of a proinflammatory role of vascular eNOS in sepsis.     

Inhibitory mechanisms of highly purified vitamin B2 on the productions of proinflammatory cytokine and NO in endotoxin-induced shock in mice

Inhibitory effects of highly purified vitamin B2 (riboflavin-5'-sodium phosphate, >97%) on the interleukin (IL)-6, macrophage inflammatory protein (MIP)-2 and nitric oxide (NO) in LPS-induced shock mice were evaluated. Vitamin B2 at 20 mg/kg (protective effect on mice mortality induced by LPS), intravenously administered 6 h after LPS injection, significantly decreased the plasma elevated levels of IL-6 and MIP-2 at 9 and 12 h. In addition, vitamin B2 lowered the tissue concentration and the mRNA expression of IL-6 in lung and those of MIP-2 in liver at 9 h. Vitamin B2 also reduced concentration of MIP-2 concentration in lung, and inhibited mRNA expression in kidney, respectively. Vitamin B2 decreased the plasma elevated NO levels in accordance with a reduction in expression of inducible NO synthase (iNOS) both at 21 and 24 h. Accordingly, the reduction in elevated plasma cytokine levels and NO based on the inhibitory effect on local cytokine mRNA expression and iNOS would be responsible for the anti-septic effect of vitamin B2.     

EP2 and EP4 receptors on muscularis resident macrophages mediate LPS-induced intestinal dysmotility via iNOS upregulation through cAMP/ERK signals

Intestinal resident macrophages play an important role in gastrointestinal dysmotility by producing prostaglandins (PGs) and nitric oxide (NO) in inflammatory conditions. The causal correlation between PGs and NO in gastrointestinal inflammation has not been elucidated. In this study, we examined the possible role of PGE(2) in the LPS-inducible inducible NO synthase (iNOS) gene expression in murine distal ileal tissue and macrophages. Treatment of ileal tissue with LPS increased the iNOS and cyclooxygenase (COX)-2 gene expression, which lead to intestinal dysmotility. However, LPS did not induce the expression of iNOS and COX-2 in tissue from macrophage colony-stimulating factor-deficient op/op mice, indicating that these genes are expressed in intestinal resident macrophages. iNOS and COX-2 protein were also expressed in dextran-phagocytized macrophages in the muscle layer. CAY10404, a COX-2 inhibitor, diminished LPS-dependent iNOS gene upregulation in wild-type mouse ileal tissue and also in RAW264.7 macrophages, indicating that PGs upregulate iNOS gene expression. EP(2) and EP(4) agonists upregulated iNOS gene expression in ileal tissue and isolated resident macrophages. iNOS mRNA induction mediated by LPS was decreased in the ileum isolated from EP(2) or EP(4) knockout mice. In addition, LPS failed to decrease the motility of EP(2) and EP(4) knockout mice ileum. EP(2)- or EP(4)-mediated iNOS expression was attenuated by KT-5720, a PKA inhibitor and PD-98059, an ERK inhibitor. Forskolin or dibutyryl-cAMP mimics upregulation of iNOS gene expression in macrophages. In conclusion, COX-2-derived PGE(2) induces iNOS expression through cAMP/ERK pathways by activating EP(2) and EP(4) receptors in muscularis macrophages. NO produced in muscularis macrophages induces dysmotility during gastrointestinal inflammation.     

MKP-1 switches arginine metabolism from nitric oxide synthase to arginase following endotoxin challenge

L-Arginine (L-arg) is metabolized to nitric oxide (NO) by inducible NO synthase (iNOS) or to urea and L-ornithine (L-orn) by arginase. NO is involved in the inflammatory response, whereas arginase is the first step in polyamine and proline synthesis necessary for tissue repair and wound healing. Mitogen-activated protein kinases (MAPK) mediate LPS-induced iNOS expression, and MAPK phosphatase-1 (MKP-1) plays a crucial role in limiting MAPK signaling in macrophages. We hypothesized that MKP-1, by attenuating iNOS expression, acts as a switch changing L-arg metabolism from NO production to L-orn production after endotoxin administration. To test this hypothesis, we performed studies in RAW264.7 macrophages stably transfected with an MKP-1 expression vector in thioglyollate-elicited peritoneal macrophages harvested from wild-type and Mkp-1^–/– mice, as well as in vivo in wild-type and Mkp-1^–/– mice. We found that overexpression of MKP-1 resulted in lower iNOS expression and NO production but greater urea production in response to LPS. Although deficiency of MKP-1 resulted in greater iNOS expression and NO production and lower urea production in response to LPS, neither the overexpression nor the deficiency of MKP-1 had any substantial effect on the expression of the arginases. lung injury; macrophage; ornithine; mitogen-activated protein kinases     

Knockdown of lung phosphodiesterase 2A attenuates alveolar inflammation and protein leak in a two-hit mouse model of acute lung injury

Phosphodiesterase 2A (PDE2A) is stimulated by cGMP to hydrolyze cAMP, a potent endothelial barrier-protective molecule. We previously found that lung PDE2A contributed to a mouse model of ventilator-induced lung injury (VILI). The purpose of the present study was to determine the contribution of PDE2A in a two-hit mouse model of 1-day intratracheal (IT) LPS followed by 4 h of 20 ml/kg tidal volume ventilation. Compared with IT water controls, LPS alone (3.75 μg/g body wt) increased lung PDE2A mRNA and protein expression by 6 h with a persistent increase in protein through day 4 before decreasing to control levels on days 6 and 10. Similar to the PDE2A time course, the peak in bronchoalveolar lavage (BAL) neutrophils, lactate dehydrogenase (LDH), and protein concentration also occurred on day 4 post-LPS. IT LPS (1 day) and VILI caused a threefold increase in lung PDE2A and inducible nitric oxide synthase (iNOS) and a 24-fold increase in BAL neutrophilia. Compared with a control adenovirus, PDE2A knockdown with an adenovirus expressing a short hairpin RNA administered IT 3 days before LPS/VILI effectively decreased lung PDE2A expression and significantly attenuated BAL neutrophilia, LDH, protein, and chemokine levels. PDE2A knockdown also reduced lung iNOS expression by 53%, increased lung cAMP by nearly twofold, and improved survival from 47 to 100%. We conclude that in a mouse model of LPS/VILI, a synergistic increase in lung PDE2A expression increased lung iNOS and alveolar inflammation and contributed significantly to the ensuing acute lung injury.