Abstract 280 Poster Session IV, Tuesday, 5/4 (poster 280)

The inflammation that occurs during acute hyperoxic lung injury is mediated in part by leukocyte chemoattractants and activators known as chemokines. Although a large part of the neutrophil attractant activity in human lung diseases is due to the chemokine interleukin-8 (IL-8), other closely related chemokines have also been implicated. We used reverse transcriptase polymerase chain reaction (RT-PCR) to measure levels of the neutrophil attractant chemokine Groβ during oxygen stress in both isolated, cultured rabbit alveolar macrophages (AM) and in lung tissue from newborn rabbits. AM isolated from adult rabbits were cultured in DME/F12, adherence purified for 24 hours, and then exposed for a further 24 hours to room air/5% CO2, 95% O2/5% CO2 or bacterial lipopolysaccharide (LPS) 1 µg/ml before having RNA extracted. A 303 base pair fragment of Groβ was detectable after 30 cycles of RT-PCR under all conditions (n=4 experiments). Groβ was detectable after 20 cycles of RT-PCR in RNA from 0/4 normoxic cultures, 1/4 hyperoxic cultures and 3/5 cultures with added LPS, suggesting that its expression was upregulated by LPS but not hyperoxia in isolated AM. By contrast, Groβ was detectable after 30 cycles of RT-PCR in 7/8 lungs of newborn rabbits exposed to 100% O2 for 8-11 days, but in 1/8 age-matched, air-exposed controls (p=0.01). The specificity of the RT-PCR result was confirmed by DNA sequencing and by Southern hybridization of a Groβ cDNA probe to blots containing the RT-PCR fragments. Groβ mRNA abundance was so low as to be undetectable by RNase protection assay under any of the above conditions. As previously described for IL-8, Groβ mRNA is upregulated in rabbit lung during hyperoxia exposure, but not similarly stimulated by oxygen stress in the isolated rabbit AM. Further, the very low abundance of Groβ mRNA makes it likely that other neutrophil attractants play more significant roles in hyperoxic lung injury.