Cell-associated IL-8 in human blood monocytes: analysis by flow cytometry

Several cell-associated cytokines, such as interleukin-1 (IL-1) and tumor necrosis factor, exist on the cell surface and are biologically active. Although extracellular IL-8, a potent chemotactic factor for primarily neutrophils, has been studied extensively, cell-associated IL-8 has barely been studied. In this study, we analyzed the intracellular and cell-surface IL-8 in human blood monocytes in vitro by using flow cytometry and predicted the biological activity of the cell-associated IL-8 in vivo. After fixation with paraformaldehyde, mononuclear cells were divided into two subgroups. One subgroup was left untreated to study cell-associated antigens, and the other subgroup was permeabilized with saponin to detect intracellular antigens. In lipopolysaccharide (LPS)-stimulated monocytes, IL-8 was detected solely intracellularly, whereas both the intracellular and cell-surface IL-1 beta was detectable. In a time-course study, the intracellular IL-8 increased in response to LPS stimulation, but the cell-surface IL-8 was undetectable throughout the course. In an LPS-stimulated monocytic cell line, both ELISA and flow cytometry detected the quantitative change of the intracellular IL-8. The dissimilar localization between IL-8 and IL-1 beta within cells was confirmed by the immunohistochemical analysis. In summary, LPS stimulation induced a time-dependent increase in intracellular but not cell-surface IL-8 in monocytes. Thus, it is unlikely that the cell-associated IL-8 is functioning physiologically. The semiquantitative flow cytometric procedure may be useful for simultaneous examination for cell-surface and intracellular cytokines.