Meconium Induces Expression of Inducible NO Synthase and Activation of NF-κB 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-␥. 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 Ͻ 0.05) and time-(4 -48 h, p Ͻ 0.05) dependent manner. This capability of meconium could be further enhanced in the presence of interferon-␥ (100 IU/mL, p Ͻ 0.05). Budesonide (10 Ϫ4 -10 Ϫ10 M) or dexamethasone (10 Ϫ4 -10 Ϫ6 M) effectively inhibited the meconiuminduced NO production (p Ͻ 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. Meconium-stained amniotic fluid is frequently encountered during both term and postterm deliveries, and 1-3% of affected infants consequently develop MAS. This disorder is characterized by respiratory distress complicated with persistent pulmonary hypertension caused by airway obstruction and pneumonitis. MAS is a major cause of neonatal morbidity and mortality (1).NO is an important molecule active in a number of biologic reactions (2, 3), especially with implications in inflammation (4). It is generated from L-arginine by three different NO synthases; of these, two are constitutive isoforms. The third is an inducible and Ca 2ϩ -independent NO synthase (iNOS), normally produced only after transcriptional activation of its gene (5, 6). High levels of NO produced by iNOS can mediate lung injury (7). Laboratory studies have suggested that NO can potentiate the lung injury by promoting oxidative or nitrosative stress (8), inactivating surfactant, and stimulating inflammation (9).The expression of iNOS is mediated by differential signaling transduction pathways. Among them, the NF-B signaling pathway has been suggested as the determinant mechanism, for example, for cytokine production, regulation of adhesion mol-

Dexamethasone Alleviates Meconium‐Induced Airway Hyperresponsiveness and Lung Inflammation in Rabbits

“…Administration of dexamethasone recently inhibited the meconium-stimulated expression of cyclooxygenase-2 and iNO-synthase in respiratory epithelium, macrophages, and endothelial cells. 25,26 Interruption of the cytokine cascade and inhibition of chemotactic factors by corticosteroids may result in a reduction of neutrophil margination and influx into the lungs, with a simultaneous increase of the blood leukocyte count. 13,15 Similarly, in this study, dexamethasone decreased neutrophils in the lungs, and increased circulating neutrophils.…”

Meconium-induced release of nitric oxide in rabbit alveolar cells

“…Steroids have been used to inhibit the inflammatory response and can downregulate the pro-inflammatory cytokine production in vitro. Li et al 9 have shown that steroids also inhibits meconium-stimulated NO production and suggested that steroids could be used in the treatment of MAS.…”

“…8 Nitric oxide is an important inflammatory mediator. In MAS, an intense inflammatory reaction occurs and Li et al (2001) reported an increase in rat alveolar macrophage cell line (ATCC8383) when exposed to meconium. They also found iNOS expression leading to increased production of NO.…”

“…They also found iNOS expression leading to increased production of NO. 9,10 The purpose of the study is to measure the NO production in meconium-induced injury in rabbit alveolar cells.…”

The role of complement in meconium aspiration syndrome

“…20 The transcription factor NF-kB, known to induce a number of proinflammatory cytokines, was shown to be activated by meconium in rat alveolar macrophages. 21 Human meconium is shown to contain phospholipase A 2 , thereby capable of directly damaging alveolar cells. 22 Meconium was found to increase the production of arachidonic acid metabolites by phospholipase A 2 23 and elevated catalytic activity of phospholipase A 2 was found both in tissue and lavage fluid.…”