Priming of phospholipases A2 of human neutrophils by tumor necrosis factor.

99S macrophage represented at least one important source of BAL IL-8 in the patients who subsequently developed ARDS. In established ARDS, both neutrophil chemotaxins were elevated together with MCP-1, but not MIP-ia. These data suggest that different chemotactic cytokines are employed at different stages of the evolution of ARDS. BAL IL-8 may represent a very early marker of the inflammatory response leading to ARDS which is of potential predictive value in at-risk groups. I nterleukin-8 (IL-8) is a potent inflammatory mediator in the hung. Alveolar macrophages (AM) are the primary lung cells producing IL-8 in response to endo-toxin. However, IL-8 only accounts for a portion of the polymorphonuclear (PM N) chemotactic activity produced by LPS-stimulated AM. Recently, we purified to homo-geneity the PMN chemotactic activity produced by LPS-stimulated porcine AM and identified two proteins. The full-length cDNAs identify the first protein, AM-derived chemotactic factor-I (AMCF-I), as the porcine homo-logue of IL-8. The identity of the second protein, AMCF-II, remains unknown, but shares its highest homologies with the 78-amino acid epithehial cell-derived neutrophil activator, ENA-78, and the CR0 subfamily of chemoat-tractants. Thus, we sought to determine whether LPS-stimulated human AM transcribe the mRNAs for ENA-78 and CR0, as well as IL-8. Human AM were incubated in the presence or absence of 1 mg/mI LPS for 2, 4, and 8 h. Total RNA was isolated at each time point. Detection of the mRNAs for IL-S. ENA-78, and CR0 was performed by two methods: polymerase chain reaction (PCR) and Northern analysis. For PCR detection, aliquots of total RNA at each time point were treated in the presence or absence of reverse transcriptase. PCR was performed using primers for IL-8, ENA-78, and CR0. The signals for each chemokine are LPS-inducible and depend on reverse transcriptase, indicating that we are detecting their mRNAs rather than their genomic DNA. We confirmed the identities of ENA-78 and GRO by eluting their PCR bands from agarose, ligating them into a plasmid vector, expanding them in Escherichia coli, and sequencing them. This further identified the CR0 PCR product as CR0-beta. Using the PCR-cloned cDNAs for IL-8, ENA-78, and CR0 as templates, we labelled antisense polynucleotide probes by PCR for use in Northern analysis. We observed a time-dependent and LPS-inducibie accumulation of each of the mRNAs for IL-8, ENA-78, and CR0. Thus, human AMs transcribe the mRNAs for ENA-78 and CR0-beta as well as IL-8. CR0 and ENA-78 may account …