Two colour flow cytometry was used to analyse in situ
cytokine expression by human monocytes. Whole blood was cultured in siliconised glass bottles, with or without E. coli
lipopolysaccharide (LPS), for various times, and the mononuclear cells (MNCs) then exposed to a variety of permeabilisation procedures prior to flow cytometric analysis.
Paraformaldehyde (PF)/
saponin fixation preserved cellular morphology, and caused a reproducible degree of permeabilisation (estimated by
propidium iodide inclusion: mean 94%, range 86-99% (n = 33)). After fixation with 4% PF and permeabilisation with 1%
saponin at 0 degrees C in PBS containing 20% human serum, MNCs were incubated with
phycoerythrin(PE)-conjugated mouse anti-CD14 (monocyte phenotype) and polyclonal rabbit anti-human
interleukin-1 alpha (IL-1 alpha),
IL-1 beta, tumour
necrosis factor alpha (
TNF-alpha), or control rabbit
IgG. Binding of rabbit
antibodies was detected using goat anti-rabbit
IgG fluorescein isothiocyanate (
FITC).
FITC fluorescence was increased in CD14 PE positive cells with the three anti-
cytokine antibodies following LPS stimulation, compared with controls. There was a reproducible dose related response in monocyte
IL-1 beta and
TNF-alpha expression following LPS stimulation, with early peaks in
TNF-alpha (2 h), compared with
IL-1 beta (4 h), and
IL-1 alpha (12 h). Specificity of this
cytokine detection system was confirmed by inhibition studies using the corresponding recombinant human
cytokines, by an absence of staining in CD14 negative or unpermeabilised MNCs, and by the characteristic cytoplasmic localisation of the different
cytokines visualised with UV immunochemistry. Hence, the methods described here provide a reproducible, semiquantitative and specific assay for the detection of cell associated
monokines. The technique may be applicable to the analysis of a variety of different
cytokines in other phenotypically defined cell populations.