Epithelial Neutrophil Activating Peptide 78 (ENA-78) is a member of the CXC subfamily of chemokines. Full-length ENA-78 is 114 amino acids (aa) in length with a predicted molecular weight of 12 kDa. ENA-78 is expressed by many immune cells, such as macrophages, eosinophils, as well as non-immune cells including mesothelial cells, and fibroblasts. Soluble ENA-78 potently chemoattracts T cells and monocytes, while the cell-bound chemokine promotes strong adhesion of leukocytes to activated endothelial cells, where it is primarily expressed. Thus, chemotaxis assay used 24-well microchemotaxis system was undertaken to detect the chemotactic effect of ENA-78 on THP-1 the human monocytic cell line. Briefly, THP-1 cells were seeded into the upper chambers (150 ul cell suspension,106 cells/ml in RPMI 1640 with FBS free) and different concentrations of recombinant mouse ENA-78 diluted with serum free RPMI 1640 was added in lower chamber with a polycarbonate filter (8 um pore size) used to separate the two compartments. After incubation at 37 ℃ with 5% CO2 for 1h, the filter was removed, then cells in low chamber were observed by inverted microscope at low magnification (×10) and the number of migrated cells were counted using Fluorescence Activating Cell Sorter. Result shows ENA-78 is able to induce migration of THP-1 cells. The migrated THP-1 cells in low chamber at low magnification (×10) were shown in Figure 1. Statistical results of FACS were shown in Figure 2. The optimum chemotaxis of ENA-78 occurs at 1 ng/ml.

Figure 2. The chemotactic effect of ENA-78 on THP-1 cells