Hyperoxic Increases in Lung ICAM-1 mRNA are Independent of TNF-α and IL-1β mRNA

Hyperoxia-exposure results in neutrophil accumulation and edema in the exposed lung. Intercellular adhesion molecule-1 (ICAM-1), a ligand for neutrophil β2 integrins, is upregulated in hyperoxia-exposed lungs and enhances neutrophil-mediated injury. Because tumor necrosis factor-α (TNF-α) and interleukin-1 (IL-1) are potent inducers of ICAM-1, we investigated whether TNF-α and IL-1β mRNA increase prior to the increase in ICAM-1 mRNA in hyperoxia-exposed mouse lungs. We exposed mice to >95% oxygen for up to 96 h, isolated lung RNA, and assessed ICAM-1, TNF-α, and IL-1β mRNA by Northern blotting. We found that neither TNF-α nor IL-1β mRNA was detectable prior to 96 h, while ICAM-1 mRNA increased by 48 h. To further assess TNF-α and IL-1β mRNA, we employed quantitative reverse-transcriptase polymerase chain reaction (RTPCR) using a mimic DNA (mimic) species as an internal control for PCR. Mimic DNA was identical to reverse-transcribed cDNA (wild type), except for 147 bp of irrelevant DNA ligated into the original cDNA. For each lung RNA sample we reverse transcribed total lung RNA and coamplified the resulting wild-type cDNA with serial dilutions of mimic DNA in a PCR containing [32P] dCTP. After PCR, we electrophoresed the samples and determined the concentration of TNF-α and IL-1β wild-type cDNAs by the ratios of wild type to mimic counts. We found no increase in TNF-α or IL-1β mRNA through 72 h of hyperoxia exposure, while there was an approximately 10-fold increase in TNF-α mRNA and a 35-fold increase in IL-1β mRNA within 2 h in the lungs of animals exposed to endotoxin. In conclusion, our data suggest that TNF-α and IL-1β are not responsible for the upregulation of ICAM-1 in hyperoxia-exposed mouse lungs.