Detection of the Chemotactic Factor for Canine Peripheral Blood Mononuclear Cells and Polymorphonuclear Cells in the Culture Supernatant of Cos7 Cells Transfected with Canine Interleukin-8 cDNA

Abstract

Chemotactic activities in the culture supernatants of Cos7 cells transfected with a cloned canine IL-8 cDNA (pcIL-8SRα14)were evaluated by using mononuclear cells (MNC) and polymorphonuclear cells (PMN) from the peripheral blood of dogs. The culture supernatants of Cos7 cells were collected 66 hr after the transfection of pcIL-8SRα14 (Cos7/cIL-8). Chemotactic activities in the culture supernatants for MNC and PMN were determined as migration distances in Millipore membrane filters in a modified Boyden's chamber method. Peroxidase staining for MNC was effective not only for cells in cytospun smears but also for cells migrated in the filters. PMN in cytospun smears were well stained by peroxidase staining, whereas migrated PMN in the filters were stained weakly. Chemotactic activities in the culture supernatant of Cos7/cIL-8 cells for both MNC and PMN were significantly higher than those of control Cos7 cells. In addition, the culture supernatant of Cos7/cIL-8 cells was chemotactic for peroxidase negative nonadherent MNC (lymphocytes), but not for peroxidase positive adherent MNC (monocytes). This Cos7/cIL-8 supernatant also showed chemotactic activities for neutrophils in a dose dependent manner. These results suggest that the culture supernatant of Cos7/cIL-8 is chemotactic for lymphocytes as well as for neutrophils.