[Expression of nuclear factor-kappaB and its inhibitor in alveolar macrophages of patients with neonatal hyaline membrane disease].
OBJECTIVE
Inflammatory reaction and injury in immature lungs are associated with activation of nuclear factor-kappa B (NF-kappaB) to trigger proinflammatory cytokine release, but the mechanism thereof is not fully understood. The present study was conducted to understand possible relationship between expression of NF-kappaB and its inhibitor and severity and outcome of neonates with hyaline membrane disease (HMD).


METHODS
Serial samples of bronchoalveolar lavage fluid (BALF) were obtained during mechanical ventilation from 31 preterm infants with HMD. These infants were divided into two groups: survivors group [n = 22, birth weight (1500 +/- 320) g and gestational age (31.2 +/- 1.8) weeks] and nonsurvivors group [birth weight (1340 +/- 280) g, gestational age (30.8 +/- 2.1) weeks]. Nineteen preterm infants [birth weight (1470 +/- 280) g, gestational age (30.6 +/- 1.9) weeks] without respiratory disorders were enrolled as control subjects. Alveolar macrophages (AM) were isolated by differential adherence. AM was cultured and treated with lipopolysaccharide (LPS) for 1 hr. Then, nuclear extracts of AM were analyzed by electrophoretic mobility shift assay (EMSA) for NF-kappaB expression. NF-kappaB inhibitor (IkappaB-alpha protein) in cytoplasmic extracts was detected by using Western blotting and IL-1beta and IL-8 in BALF by enzyme-linked immunosorbent assay (ELISA).


RESULTS
NF-kappaB complexes were observed by EMSA, they were characterized by competition with cold oligonucleotide and p65-specific antibodies. The addition of an excess of cold oligonucleotide, corresponding to the NF-kappaB binding site, turned off the signal of the band, showing that the band was specific. An excess of an irrelevant oligonucleotide (corresponding to the SP-1) did not show any effect. The addition of an anti-p65 antibody caused the supershift of the two upper bands. After EMSA, the NF-kappaB complexes were quantified by using a ImageQuant software. NF-kappaB expression in AM at 24 hrs was higher in all the patients with HMD as compared with control subjects (survives/control, 34.1 vs 11.4 RDU, P < 0.01; nonsurvivors/control, 55.2 vs 11.4 RDU, P < 0.01). The NF-kappaB expression in AM at 72 hrs was higher than that in control subjects but not for nonsurvivors (survivors/control, 47.8 vs 25.6 RDU, P < 0.01; nonsurvivors/control, 21.8 vs 25.6, P > 0.05). The NF-kappaB expression in AM from nonsurvivors was depressed at 72 hrs as compared to 24 hrs (21.8 vs 55.2, P < 0.01), whereas the NF-kappaB expression in AM from survivors was still higher at 72 hrs than that at 24 hrs (47.8 vs 34.1, t = 4.43, P < 0.01).


CONCLUSIONS
Altered NF-kappaB activation in AM of BALF of neonates with HMD was observed, and it may be mediated by decreased IkappaB synthesis, increased IkappaB degradation, or both. In HMD nonsurvivors NF-kappaB translocation was hampered upon LPS activation.
Liu
Cui-qing
CQ
Department of Neonatology, Childrens' Hospital of Hebei Provence, Shijiazhuang 050031, China.
Cao
Lei
L
Zheng
Hua-cheng
HC
Jia
Xi-qun
XQ
Kang
Li-min
LM
Li
Lan-feng
LF
Liu
Su-zhe
SZ
D001724:Birth Weight; D015153:Blotting, Western; D001992:Bronchoalveolar Lavage Fluid / Q000166:cytology; D018929:Cell Culture Techniques; D002467:Cell Nucleus / Q000187:drug effects / Q000378:metabolism; D003593:Cytoplasm / Q000187:drug effects / Q000378:metabolism; D024202:Electrophoretic Mobility Shift Assay; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D005865:Gestational Age; D006801:Humans; D006819:Hyaline Membrane Disease / Q000276:immunology* / Q000628:therapy; D020672:I-kappa B Proteins / Q000276:immunology*; D007231:Infant, Newborn; D007234:Infant, Premature / Q000276:immunology; D053583:Interleukin-1beta / Q000276:immunology; D016209:Interleukin-8 / Q000276:immunology; D008070:Lipopolysaccharides / Q000494:pharmacology; D016676:Macrophages, Alveolar / Q000187:drug effects / Q000276:immunology*; D008297:Male; D000072000:NF-KappaB Inhibitor alpha; D016328:NF-kappa B / Q000276:immunology*; D012121:Respiration, Artificial; D012720:Severity of Illness Index; D013997:Time Factors
D020672:I-kappa B Proteins; D053583:Interleukin-1beta; D016209:Interleukin-8; D008070:Lipopolysaccharides; D016328:NF-kappa B; C000606204:NFKBIA protein, human; D000072000:NF-KappaB Inhibitor alpha