Feline interleukin-8 expression in peripheral blood mononuclear cells induced by egg white derivatives

Abstract

Feline peripheral blood mononuclear cells (PBMC)-derived chemotactic factor induced by egg white derivatives (EWD) treatment was analyzed at the protein and messenger ribonucleic acid (mRNA) level. EWD itself was not active chemotactic for feline peripheral blood polymorphonuclear cells (PMN). But chemotaxis of PMN was enhanced by either culture supernatant from PBMC treated with EWD or human recombinant (hr) interleukin (IL)-8. Both hr IL-8 and the culture supernatant from PBMC treated with EWD yielded a distinct band, molecular weight of 6–8 kDa, in sodium-dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with 15% loading gel. Therefore, to identify this chemotactic factor, culture supernatant from PBMC treated with EWD was partially purified by anion exchange chromatography on diethylaminoethyl (DEAE)–Sepharose CL-6B and concentrated by ultrafiltration. Only the fraction, which was eluted with 0.3 M NaCl, showed a high concentration of total protein and also enhanced the chemotactic activity of PMN. This activity was thereafter designated as eluate. The chemotactic activity of eluate was inhibited by anti-hr IL-8 polyclonal antibody (pAb). A single protein band with 6–8 kDa was shown in both the eluate and hr IL-8 when analyzed by SDS-PAGE and Western blotting using anti-hr IL-8 pAb, suggesting that the chemotactic factor for feline PMN is IL-8, 6–8 kDa, produced by PBMC treated with EWD. The physicochemical characteristics of eluate were stable in heated (60–100 °C), acid (pH 3.0), and alkaline (pH 9.0) conditions. The eluate under these conditions also showed a distinct band in molecular weight of 6–8 kDa in SDS-PAGE and Western blotting and was very active in chemotactic activity of PMN. IL-8 mRNA gene expression on feline PBMC was analyzed by reverse transcriptase-polymerase chain reaction (RT-PCR) assay using a series of oligonucleotides, each 22 mer, derived from feline IL-8. Feline IL-8 mRNA showed low level in 3-h incubation without EWD, but it was increased in a dose-dependent manner by addition of EWD. Following EWD (10 μg/ml) treatment, IL-8 mRNA expression was rapidly increased up to 6 h and decreased by 12 h although it was not expressed in freshly prepared PBMC. This study strongly suggested that immunoenhancing effect of EWD on chemotactic response of PMN is mediated by feline IL-8, 6–8 kDa, produced by PBMC stimulated with EWD. In addition, the expression of feline IL-8 mRNA on PBMC is increased when stimulated with EWD.