Maturational differences in lung NF-kappaB activation and their role in tolerance to hyperoxia

Abstract

Neonatal rodents are more tolerant to hyperoxia than adults. We determined whether maturational differences in lung NF-kappaB activation could account for the differences. After hyperoxic exposure (O2 > 95%), neonatal (<12 hours old) lung NF-kappaB binding was increased and reached a maximum between 8 and 16 hours, whereas in adults no changes were observed. Additionally, neonatal NF-kappaB/luciferase transgenic mice (incorporating 2 NF-kappaB consensus sequences driving luciferase gene expression) demonstrated enhanced in vivo NF-kappaB activation after hyperoxia in real time. In the lungs of neonates, there was a propensity toward NF-kappaB activation as evidenced by increased lung I-kappaB kinase protein levels, I-kappaBalpha phosphorylation, beta-transducin repeat-containing protein levels, and total I-kappaBalpha degradation. Increased lung p-JNK immunoreactive protein was observed only in the adult lung. Inhibition of pI-kappaBalpha by BAY 11-7085 resulted in decreased Bcl-2 protein levels in neonatal lung homogenates and decreased cell viability in lung primary cultures after hyperoxic exposure. Furthermore, neonatal p50-null mutant (p50(-/-)) mice showed increased lung DNA degradation and decreased survival in hyperoxia compared with WT mice. These data demonstrate that there are maturational differences in lung NF-kappaB activation and that enhanced NF-kappaB may serve to protect the neonatal lung from acute hyperoxic injury via inhibition of apoptosis.

Figure 1

Demonstration of hyperoxia-mediated neonatal (Neo) lung NF-κB activation in C57BL/6 mice. (A) Representative of 3 EMSA blots. F, free probe; 0–72 h, duration of hyperoxia; C, competition with 100-fold excess of unlabeled oligonucleotide probe; M, competition with 100-fold excess of unlabeled mutated oligonucleotide probe; S, supershift with p50 antibody in lung extract collected at 8 hours of hyperoxia; SS, supershift retardation band. (B) Densitometric evaluation of the EMSA blots. Densitometric units were expressed as a ratio of air-exposed values. The data are the mean ± SE of 3 separate experiments. *P < 0.05 vs. similarly exposed adults. (C) NF-κB activation in the neonatal LPS-injected lung. LPS (20 mg/kg) was injected intraperitoneally into 5-day-old C57BL/6 mice. Ctl, mice injected with PBS as vehicle control; LPS, 1 hour after injection with LPS; p65, sample as in LPS lane incubated with p65 antibody. Note: The decreased NF-κB complex and the supershift gel retardation band demonstrating the binding complex are specific to p65 in this model.

Figure 2

Visualization of NF-κB activation in NF-κB/luc Tg mice. (A) In the pseudoimages, the blue to red coloring represents the lowest to the highest light intensity. 0–72 hours, duration of hyperoxia. (B) Immunohistochemical staining of luciferase protein in the neonatal lung after 24 hours of hyperoxia. Luciferase protein was localized to bronchiolar and alveolar epithelial cells (lower left and middle panels, white arrows) as well as endothelial cells (lower left panel, arrowhead). Colocalization of the luciferase (red) and alveolar macrophage CD68 (green) is demonstrated by yellow-colored cells (lower right panel, yellow arrow). Note that the luciferase protein is not exclusively localized to the macrophages.

Figure 3

Maturational differences in pI-κBα, total I-κBα, and β-TrCP in hyperoxia. (A) Expression of pI-κBα at early time points. 0–4 hours, duration of hyperoxia. (B) Expression of I-κBα from 0 to 72 hours of hyperoxia. (C) Densitometric evaluation of I-κBα protein levels from the blot shown in B. (D) Relative mobility of the I-κBα bands after hyperoxia as compared with that in cell extracts with (T) or without (U) TNF-α treatment. Note that the lower band of the I-κBα represents pI-κBα. (E) β-TrCP protein levels after hyperoxia in the nuclear fractions of the mouse lung.

Figure 4

Maturational differences in total I-κBα, IKK core subunit, p-JNK, and JNK protein levels in hyperoxia. Mice aged 0 days, 7 days, 14 days, 21 days, and 60 days (adult) were exposed to air or hyperoxia (O₂) for 72 hours. A representative of 3 Western analyses is shown.

Figure 5

Levels of pro- and antiinflammatory cytokine mRNA in the lungs of mice exposed to air or 72 hours of hyperoxia. Densitometric values were expressed as a ratio to GAPDH. Values represent the mean ± SE of 3 separate experiments. *P < 0.05 vs. air-exposed mice. Black bars, TNF-α; dark gray bars, IL-1β; light gray bars, TGF-β; white bars, GM-CSF.

Figure 6

Bcl-2 expression and TUNEL staining in lungs of WT mice exposed to 72 hours of hyperoxia. (A) Bcl-2 mRNA levels were evaluated by RT-PCR. Densitometric values are expressed as a ratio to GAPDH. Values are the mean ± SE of 3 separate experiments. *P < 0.05 vs. air-exposed mice. ^† P < 0.05 vs similarly exposed neonates. (B) Immunoreactive Bcl-2 and Bax protein levels. (C) Immunohistochemical staining of Bcl-2 protein. Note that increased Bcl-2 was localized predominantly in the alveolar epithelial cells (arrowhead). (D) TUNEL staining in mouse lung tissues after hyperoxia. Note the relatively low number of TUNEL-positive cells in the neonatal lung but the high signal intensity in the similarly exposed adult lung (yellow).

Figure 7

Effect of inhibition of I-κΒα phosphorylation on primary lung cells exposed to hyperoxia. (A) Visualization of NF-κB activation in primary lung cells cultured from NF-κB/luc Tg mice. (B) Effect of inhibition of I-κΒα phosphorylation on NF-κB activation. Cells were incubated with 1 mM BAY 11-7085 or BAY 11-7082 and then exposed to 24 hours of hyperoxia. Controls were incubated with 0.1% ethanol, the vehicle for BAY. Note the decreased light intensity after BAY treatment. (C) Cell viability was evaluated using trypan blue exclusion. The number of surviving cells in each group was assessed after 24 hours of hyperoxic exposure.

Figure 8

Inhibition of NF-κB activation in neonates injected with BAY. After 24 hours of hyperoxia, BAY-injected mice were injected with luciferin and anesthetized to allow for visualization (see Methods). (A) Photon emission was quantified over the lung area (arrow) and expressed as a ratio to pre-O₂ for each animal. Pre-O₂ lane: images taken prior to injection and hyperoxia; O₂/PEG lane: mice injected with PEG (vehicle for BAY compounds); O₂/BAY lane: mice injected with BAY 11-7085. (B) Values represent the mean ± SE of 3 experiments. *P < 0.05 vs. polyethylglycol 400 (PEG) and uninjected. (C) pI-κBα and Bcl-2 protein levels in C57BL/6 neonatal lung homogenates after 24 hours of hyperoxia and BAY injection.

Figure 9

Lung injury in neonatal p50^–/– mice after hyperoxia. (A) Upper panels: Representative H&E-stained lung tissue slide from neonatal WT and p50^–/– mice exposed to hyperoxia for 144 hours (O₂). The slides were viewed at ×200. Lower left panel: Inflammatory cells per power field. Values are the mean ± SE of 3 animals in each group. *P < 0.05 vs. oxygen-exposed WT mice. Lower right panels: Evaluation of lung injury after 144 hours of hyperoxia. Images were obtained at high magnification (×400); left, increased inflammatory cells in the bronchiolar ephithelium; right, bronchiolar epithelia necrosis. (B) Lung DNA fragmentation in WT and p50^–/– mice exposed to 72 hours of hyperoxia. Genomic DNA was isolated, and DNA fragments were amplified by PCR. Left: Visualization of lung DNA fragments in ethidium bromide–stained 0.8% agarose gel. Right: Densitometric evaluation of the agarose gel .Values are the mean ± SE of 3 experiments. *P < 0.05 vs. air-exposed p50^–/–; ^† P < 0.05 vs. O₂-exposed WT mice.

Figure 10

Lung Bcl-2 and TUNEL staining in neonatal p50^–/– mice after hyperoxia. (A) Immunohistochemical staining of Bcl-2 protein in neonatal p50^–/– mice exposed to 72 hours of air or hyperoxia. Note the unchanged Bcl-2 protein levels after hyperoxia. (B) TUNEL staining in neonatal WT and p50^–/– mice after 144 hours of hyperoxia. Note the visibly increased amount of TUNEL-positive staining in the lungs of neonatal p50^–/– mice as compared with the similarly exposed WT neonatal mice.

Figure 11

Survival of neonatal WT (A) and p50^–/– (B) mice after chronic hyperoxia. Values are expressed as the percentage of surviving animals.