Meconium induces expression of inducible NO synthase and activation of NF-kappaB in rat alveolar macrophages.
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.
Li
Y H
YH
Neonatal Unit, Astrid Lindgren Children's Hospital, Karolinska Institute, Stockholm, Sweden.
Yan
Z Q
ZQ
Brauner
A
A
Tullus
K
K
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
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