Lipopolysaccharide-induced cytokine production in peripheral blood mononuclear cells: intracellular localization of tumor necrosis factor alpha and interleukin 1 beta detected with a three-color immunofluorescence technique.
Lipopolysaccharide (LPS) can induce monocytes to produce various cytokines such as tumor necrosis factor alpha (TNF alpha) and interleukin 1 beta (IL-1 beta). In the present study, the kinetics of both intracellular and extracellular accumulation of TNF alpha and IL-1 beta in LPS stimulated mononuclear cell (MNC) cultures has been determined. A three-color-immunofluorescence technique was used to detect intracellular accumulation of cytokines. Intracellular accumulation of TNF alpha in monocytes starts shortly after initiation of the culture; i.e., TNF alpha is detectable after 1 h, reaching a peak level after 3-4 hours with 50-65% of monocytes staining positive. In parallel with its increased intracellular presence, TNF alpha was also found in the culture supernatant. The intracellular accumulation of IL-1 beta in monocytes became detectable after 2 h of culture in the presence of LPS. After 4 h, a plateau was reached, with 90% of the monocytes being positive. In parallel, but with a little delay, IL-1 beta could be detected in the culture supernatant. TNF alpha and IL-1 beta can be produced simultaneously in the same monocytes as was shown by a three-color-immunofluorescence technique. It is concluded that TNF alpha and IL-1 beta are good parameters for the early measurement of monocyte activation and that both the intracellular accumulation in monocytes and the amount of secreted cytokines can be used for such a purpose. The intracellular accumulation in monocytes can be measured by the three-color-immunofluorescence technique described.
de Bont
E S
ES
Department of Pediatric Oncology, Beatrix Children Hospital, University Hospital Groningen, The Netherlands.
Niemarkt
A E
AE
Tamminga
R Y
RY
Kimpen
J L
JL
Kamps
W A
WA
de Leij
L H
LH
D000328:Adult; D002478:Cells, Cultured / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism; D003116:Color; D016207:Cytokines / Q000096:biosynthesis* / Q000187:drug effects; D005455:Fluorescent Antibody Technique / Q000379:methods*; D006801:Humans; D007375:Interleukin-1 / Q000032:analysis*; D007700:Kinetics; D007963:Leukocytes, Mononuclear / Q000737:chemistry / Q000166:cytology / Q000378:metabolism*; D008070:Lipopolysaccharides / Q000494:pharmacology; D014409:Tumor Necrosis Factor-alpha / Q000032:analysis*
D016207:Cytokines; D007375:Interleukin-1; D008070:Lipopolysaccharides; D014409:Tumor Necrosis Factor-alpha