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.
Fujishima
S
S
Department of Medicine, School of Medicine, Keio University, Tokyo, Japan.
Nakamura
H
H
Waki
Y
Y
Soejima
K
K
Takeuchi
Y
Y
Ogawa
M
M
Shiozawa
M
M
Hiraoka
Y
Y
Kawashiro
T
T
Aiso
S
S
Kanazawa
M
M
D000911:Antibodies, Monoclonal; D002478:Cells, Cultured; D005434:Flow Cytometry / Q000379:methods*; D006801:Humans; D007150:Immunohistochemistry; D016209:Interleukin-8 / Q000032:analysis* / Q000276:immunology; D007424:Intracellular Fluid / Q000737:chemistry / Q000276:immunology; D008070:Lipopolysaccharides / Q000494:pharmacology; D009000:Monocytes / Q000187:drug effects / Q000378:metabolism*; D012503:Saponins / Q000494:pharmacology; D013997:Time Factors
D000911:Antibodies, Monoclonal; D016209:Interleukin-8; D008070:Lipopolysaccharides; D012503:Saponins