title: Meconium induces expression of inducible NO synthase and activation of NF-kappaB in rat alveolar macrophages.
abstract: Meconium aspiration causes intensive inflammatory reactions in the lungs, and may lead to neonatal respiratory disorder. Infiltrated inflammatory cells, particularly macrophages, play an important role in such an inflammation. A rat alveolar macrophage cell line (ATCC8383) was exposed to meconium alone or in combination with dexamethasone, budesonide, or interferon-gamma. Nitric oxide (NO) accumulation in the supernatant of the cell culture was detected by Griess reaction, and mRNA of inducible NO synthase (iNOS) expression was detected by reverse transcriptase-PCR. Nuclear factor-kappa B was analyzed by electrophoretic mobility shift assay, and iNOS location and nuclear factor-kappa B transactivation were determined by immunostaining. Our results showed that meconium was capable of inducing production of NO and expression of iNOS in alveolar macrophages in a dose- (1-25 mg/mL, p < 0.05) and time- (4-48 h, p < 0.05) dependent manner. This capability of meconium could be further enhanced in the presence of interferon-gamma (100 IU/mL, p < 0.05). Budesonide (10(-4)-10(-10) M) or dexamethasone (10(-4)-10(-6) M) effectively inhibited the meconium-induced NO production (p < 0.05). Using the protein synthesis inhibitor cycloheximide, we demonstrated that meconium directly induced iNOS in macrophages. Furthermore, meconium also triggered nuclear factor-kappa B activation, a mechanism possibly responsible for the iNOS expression. Our findings suggest that meconium is a potent inflammatory stimulus, resulting in iNOS expression, leading to overproduction of NO from the macrophages, which may be of pathogenic importance in meconium aspiration syndrome. In vitro steroids down-regulated the iNOS expression, thus suggesting a potential to down-regulate NO-mediated inflammation in neonates with meconium aspiration syndrome.
authors.affiliation: Neonatal Unit, Astrid Lindgren Children's Hospital, Karolinska Institute, Stockholm, Sweden.
authors.forename: Y H
authors.initials: YH
authors.lastname: Li
authors.forename: Z Q
authors.initials: ZQ
authors.lastname: Yan
authors.forename: A
authors.initials: A
authors.lastname: Brauner
authors.forename: K
authors.initials: K
authors.lastname: Tullus
mesh_terms: D000818:Animals; D000893:Anti-Inflammatory Agents / Q000494:pharmacology; D001483:Base Sequence; D019819:Budesonide / Q000494:pharmacology; D002460:Cell Line; D017931:DNA Primers / Q000235:genetics; D003907:Dexamethasone / Q000494:pharmacology; D015870:Gene Expression; D005938:Glucocorticoids / Q000494:pharmacology; D006801:Humans; D007231:Infant, Newborn; D016676:Macrophages, Alveolar / Q000187:drug effects / Q000378:metabolism*; D008470:Meconium / Q000378:metabolism*; D008471:Meconium Aspiration Syndrome / Q000235:genetics / Q000378:metabolism; D016328:NF-kappa B / Q000378:metabolism*; D009569:Nitric Oxide / Q000096:biosynthesis; D019001:Nitric Oxide Synthase / Q000235:genetics* / Q000378:metabolism*; D052247:Nitric Oxide Synthase Type II; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D051381:Rats
chemical_list: D000893:Anti-Inflammatory Agents; D017931:DNA Primers; D005938:Glucocorticoids; D016328:NF-kappa B; D012333:RNA, Messenger; D009569:Nitric Oxide; D019819:Budesonide; D003907:Dexamethasone; C496319:NOS2 protein, human; D019001:Nitric Oxide Synthase; D052247:Nitric Oxide Synthase Type II; C496318:Nos2 protein, rat