Activation of invariant natural killer T (iNKT) cells and signaling through receptor for advanced glycation end products (RAGE) are known to independently mediate lung ischemia–reperfusion (IR) ...injury. This study tests the hypothesis that activation of RAGE specifically on iNKT cells via alveolar macrophage‐produced high mobility group box 1 (HMGB1) is critical for the initiation of lung IR injury. A murine in vivo hilar clamp model was utilized, which demonstrated that RAGE−/− mice were significantly protected from IR injury. Treatment of WT mice with soluble RAGE (a decoy receptor), or anti‐HMGB1 antibody, attenuated lung IR injury and inflammation, whereas treatment with recombinant HMGB1 enhanced IR injury in WT mice but not RAGE−/− mice. Importantly, lung dysfunction, cytokine production and neutrophil infiltration were significantly attenuated after IR in Jα18−/− mice reconstituted with RAGE−/− iNKT cells (versus WT iNKT cells). In vitro studies demonstrated that, after hypoxia‐reoxygenation, alveolar macrophage‐derived HMGB1 augmented IL‐17 production from iNKT cells in a RAGE‐dependent manner. These results suggest that HMGB1‐mediated RAGE activation on iNKT cells is critical for initiation of lung IR injury and that a crosstalk between macrophages and iNKT cells via the HMGB1/RAGE axis mediates IL‐17 production by iNKT cells causing neutrophil infiltration and lung IR injury.
This study uses a murine in vivo lung ischemia reperfusion model and in vitro experiments to demonstrate that macrophage‐produced HMGB1 mediates RAGE activation on iNKT cells to induce IL‐17 production, which is critical for neutrophil infiltration and initiation of lung ischemia reperfusion injury. See editorial by Gelman and Scozzi on page 2237.
Background and purpose:
Prenatal patency of ductus arteriosus is maintained by prostaglandin (PG) E2 in concert with nitric oxide (NO) and carbon monoxide (CO). Accordingly, we have previously found ...that NO activity increases upon deletion of either COX. Here, we have examined whether COX inhibition by indomethacin mimics COX deletion in promoting NO.
Experimental approach:
Experiments were performed in vitro and in vivo with wild‐type (WT) and eNOS−/−, near‐term mouse foetuses. Indomethacin was given p.o. to the mother as single (acute treatment) or repeated (daily for 3 days; chronic treatment) doses within a therapeutic range (2 mg kg−1).
Key results:
Indomethacin promoted eNOS mRNA expression in the WT ductus. Coincidentally, the drug enhanced the contraction of the isolated ductus to the NOS inhibitor, NG‐nitro‐L‐arginine methyl ester, and its effect augmented with the length of treatment. No such enhancement was seen with the eNOS−/− ductus. Chronic indomethacin also increased, albeit marginally, the contraction of the WT ductus to the CO synthesis inhibitor, zinc protoporphyrin. Whether given acutely or chronically, indomethacin induced a little narrowing of the ductus antenatally and had no effect on postnatal closure of the vessel.
Conclusions and implications:
We conclude that activation of NO and, to a much lesser degree, CO mechanisms is an integral part of the indomethacin effect on the ductus. This relaxing influence may oppose the contraction from PGE2 suppression and could explain the failures of indomethacin therapy in premature infants with persistent duct.
British Journal of Pharmacology (2008) 153, 1631–1640; doi:10.1038/bjp.2008.36; published online 25 February 2008
Nitric oxide produced by cytokine-inducible nitric oxide synthase (iNOS) is thought to be important in the pathogenesis of septic shock. To further our understanding of the role of iNOS in normal ...biology and in a variety of inflammatory disorders, including septic shock, we have used gene targeting to generate a mouse strain that lacks iNOS. Mice lacking iNOS were indistinguishable from wild-type mice in appearance and histology. Upon treatment with lipopolysaccharide and interferon γ, peritoneal macrophages from the mutant mice did not produce nitric oxide measured as nitrite in the culture medium. In addition, lysates of these cells did not contain iNOS protein by immunoblot analysis or iNOS enzyme activity. In a Northern analysis of total RNA, no iNOS transcript of the correct size was detected. No increases in serum nitrite plus nitrate levels were observed in homozygous mutant mice treated with a lethal dose of lipopolysaccharide, but the mutant mice exhibited no significant survival advantage over wild-type mice. These results show that lack of iNOS activity does not prevent mortality in this murine model for septic shock.
Nitric oxide produced in endothelial cells affects vascular tone. To investigate the role of endothelial nitrix oxide synthase (eNOS) in blood pressure regulation, we have generated mice heterozygous ...(+/-) or homozygous (-/-) for disruption of the eNOS gene. Immunohistochemical staining with anti-eNOS antibodies showed reduced amounts of eNOS protein in +/- mice and absence of eNOS protein in -/- mutant mice. Male or female mice of all three eNOS genotypes were indistinguishable in general appearance and histology, except that -/- mice had lower body weights than +/+ or +/- mice. Blood pressures tended to be increased (by approximately 4 mmHg) in +/- mice compared with +/+, while -/- mice had a significant increase in pressure compared with +/+ mice (≈ 18 mmHg) or +/- mice (≈ 14 mmHg). Plasma renin concentration in the -/- mice was nearly twice that of +/+ mice, although kidney renin mRNA was modestly decreased in the -/- mice. Heart rates in the -/- mice were significantly lower than in +/- or +/+ mice. Appropriate genetic controls show that these phenotypes in F2mice are due to the eNOS mutation and are not due to sequences that might differ between the two parental strains (129 and C57BL/6J) and are linked either to the eNOS locus or to an unlinked chromosomal region containing the renin locus. Thus eNOS is essential for maintenance of normal blood pressures and heart rates. Comparisons between the current eNOS mutant mice and previously generated inducible nitric oxide synthase mutants showed that homozygous mutants for the latter differ in having unaltered blood pressures and heart rates; both are susceptible to lipopolysaccharide-induced death.
Delivery of soluble CD39, or CD39‐like molecules, may offer a clinical therapeutic strategy for combating ischemia reperfusion injury. See article by Crikis et al on page 2586.
The goal of this study was to interrogate the role of inducible NO synthase (iNOS) in the late phase of ischemic preconditioning (PC) in vivo. A total of 321 mice were used. Wild-type mice ...preconditioned 24 h earlier with six cycles of 4-min coronary occlusion/4-min reperfusion exhibited a significant$(P<0.05)$increase in myocardial iNOS protein content, iNOS activity (assessed as calcium-independent L-citrulline formation), and nitrite + nitrate tissue levels. In contrast, endothelial NOS protein content and calcium-dependent NOS activity remained unchanged. No immunoreactive neuronal NOS was detected. When wild-type mice were preconditioned 24 h earlier with six 4-min occlusion/4-min reperfusion cycles, the size of the infarcts produced by a 30-min coronary occlusion followed by 24 h of reperfusion was reduced markedly (by 67%;$P<0.05)$compared with sham-preconditioned controls, indicating a late PC effect. In contrast, when mice homozygous for a null iNOS allele were preconditioned 24 h earlier with the same protocol, infarct size was not reduced. Disruption of the iNOS gene had no effect on early PC or on infarct size in the absence of PC. These results demonstrate that (i) the late phase of ischemic PC is associated with selective up-regulation of iNOS, and (ii) targeted disruption of the iNOS gene completely abrogates the infarct-sparing effect of late PC (but not of early PC), providing unequivocal molecular genetic evidence for an obligatory role of iNOS in the cardioprotection afforded by the late phase of ischemic PC. Thus, this study identifies a specific protein that mediates late PC in vivo.
Since the lung is repeatedly subjected to injury by pathogens and toxicants, maintenance of pulmonary homeostasis requires rapid repair of its epithelial surfaces. Ciliated bronchiolar epithelial ...cells, previously considered as terminally differentiated, underwent squamous cell metaplasia within hours after bronchiolar injury with naphthalene. Expression of transcription factors active in morphogenesis and differentiation of the embryonic lung, including beta-catenin, Foxa2, Foxj1, and Sox family members (Sox17 and Sox2), was dynamically regulated during repair and redifferentiation of the bronchiolar epithelium after naphthalene injury. Squamous cells derived from ciliated cells spread beneath injured Clara cells within 6-12 h after injury, maintaining the integrity of the epithelium. Dynamic changes in cell shape and gene expression, indicating cell plasticity, accompanied the transition from squamous to cuboidal to columnar cell types as differentiation-specific cell markers typical of the mature airway were restored. Similar dynamic changes in the expression of these transcription factors occurred in ciliated and Clara cells during regeneration of the lung after unilateral pneumonectomy. Taken together, these findings demonstrate that ciliated epithelial cells spread and transdifferentiate into distinct epithelial cell types to repair the airway epithelium.
Demyelination is often associated with acute inflammatory events involving the recruitment-activation of microglia/macrophage, astrocytes, and leukocytes. The ultimate role of inflammatory products ...in demyelinating disease and in the survival of oligodendrocytes, the myelin forming cells, is unresolved. The current study examines the role of inducible NO synthase (iNOS)-derived NO in a neurotoxicant-induced model of demyelination. NO levels were greatly elevated in the midline corpus callosum during demyelination in genetically intact C57BL/6 mice, and this NO was due solely to the induction of iNOS, as the correlates of NO were not found in mice lacking iNOS. C57BL/6 mice lacking iNOS exhibited more demyelination, but did not display an increased overall cellularity in the corpus callosum, attributable to an unimpeded microglia/macrophage presence. An enhanced course of pathology was noted in mice lacking iNOS. This was associated with a greater depletion of mature oligodendrocytes, most likely due to apoptosis of oligodendrocytes. Microglia and astrocytes did not undergo apoptosis during treatment. Our results suggest a moderately protective role for NO during acute inflammation-association demyelination.
Background and purpose:
Prenatal patency of ductus arteriosus is maintained by prostaglandin (PG) E2, possibly along with nitric oxide (NO) and carbon monoxide (CO), and cyclooxygenase (COX) deletion ...upregulates NO. Here, we have examined enzyme source and action of NO for ductus patency and whether NO and CO are upregulated by deletion of, respectively, heme oxygenase 2 (HO‐2) and COX1 or COX2.
Experimental approach:
Experiments were performed in vitro and in vivo with wild‐type and gene‐deleted, near‐term mouse fetuses.
Key results:
NG‐nitro‐L‐arginine methyl ester (L‐NAME) contracted the isolated ductus and its effect was reduced by eNOS, but not iNOS, deletion. L‐NAME contraction was not modified by HO‐2 deletion. Zinc protoporphyrin (ZnPP) also contracted the ductus, an action unaffected by deletion of either COX isoform. Bradykinin (BK) relaxed indomethacin‐contracted ductus similarly in wild‐type and eNOS−/− or iNOS−/−. BK relaxation was suppressed by either L‐NAME or ZnPP. However, it reappeared with combined L‐NAME and ZnPP to subside again with K+ increase or K+ channel inhibition. In vivo, the ductus was patent in wild‐type and NOS‐deleted fetuses. Likewise, no genotype–related difference was noted in postnatal closure.
Conclusions and implications:
NO, formed mainly via eNOS, regulates ductal tone. NO and CO cooperatively mediate BK‐induced relaxation in the absence of PGE2. However, in the absence of PGE2, NO and CO, BK induces a relaxant substance behaving as an endothelium‐derived hyperpolarizing factor. Ductus patency is, therefore, sustained by a cohort of agents with PGE2 and NO being preferentially coupled for reciprocal compensation.
British Journal of Pharmacology (2007) 151, 54–62. doi:10.1038/sj.bjp.0707211
To determine the contribution of the inducible nitric oxide synthase (iNOS) to hepatic injury following warm ischemia-reperfusion, we developed a model of partial hepatic ischemia-reperfusion in mice ...and studied the injury response in iNOS knockout (KO) mice. Compared with wild types, iNOS KO animals exhibited lower plasma transaminase levels after 1 and 6 h of reperfusion following 1 h of ischemia. At the 3-h time point, enzyme levels were not different between the two groups. iNOS mRNA was not detectable in the ischemic hepatic lobes of wild-type mice until 3 h of reperfusion; however, perfusion studies identified a significant delay in reperfusion of the ischemic lobe in the iNOS KO mice at the 1-h time point with similar perfusion rates at 3 and 6 h compared with wild type. By way of comparison, mice deficient in the endothelial NOS (eNOS) were also assessed for the degree of hepatic damage 3 h post-reperfusion. Plasma transaminase levels were significantly increased in eNOS KO animals compared with wild-type controls. These data suggest that systemic as well as local sources of iNOS regulate reperfusion, and local iNOS contributes to hepatic injury, while eNOS is protective in warm hepatic ischemia-reperfusion.
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