MOLECULAR-CLONING OF PORCINE ALVEOLAR MACROPHAGE-DERIVED NEUTROPHIL CHEMOTACTIC FACTOR-I AND FACTOR-II - IDENTIFICATION OF PORCINE IL-8 AND ANOTHER INTERCRINE-ALPHA PROTEIN

Alveolar macrophages (AM) mediate lung inflammation by producing lipid and peptide molecules that attract neutrophils (PMN) to the lung. Recently we described two porcine proteins called alveolar macrophage-derived chemotactic factors, AMCF-I and -II, that are potent, efficacious, and specific PMN chemoattractants both in vitro and in vivo. We report here the cloning of the full-length cDNAs which code for each protein. Porcine AM were stimulated for 4 h in vitro with Escherichia coli endotoxin (LPS), and a cDNA library was created from poly(A)+-selected mRNA. Specific oligonucleotide probes for AMCF-I and AMCF-II were amplified from the porcine AM cDNA library by the polymerase chain reaction using degenerate oligonucleotide primer pairs derived from the N-terminal amino acid sequences of the proteins. These probes were used to isolate 2 full-length cDNAs of 1466 (AMCF-I) and 1515 (AMCF-II) base pairs. Both cDNAs code for proteins with four cysteine residues containing the C-X-C sequence characteristic of the intercrine-alpha family of neutrophil chemoattractants. AMCF-I shares 74% identity with human IL-8 and 84% identity with rabbit IL-8, and likely represents the porcine homologue of IL-8. By contrast, AMCF-II has no obvious human homologue. AMCF-II shares 53% identity with human neutrophil activating peptide 2. Its shared identity with the GRO-related proteins is as high as 61% (rat CINC/GRO), and its shared identity with the 78 amino acid epithelial cell-derived neutrophil activator (ENA-78) is 67%. AMCF-II may represent a new member of the intercine-alpha family of neutrophil chemoattractants. Steady-state mRNA levels for AMCF-I following LPS stimulation are detectable at 4 h, peak at 16-24 h, and persist for more than 72 h. Steady-state mRNA levels for AMCF-II are detectable immediately after adherence in vitro, peak at 8-16 h, and persist for greater than 72 h. Since both of these proteins are produced by porcine AM, strategies designed at limiting AM-mediated inflammation in the human lung should be aimed at the human homologues of AMCF-I and AMCF-II.