A flow cytometric method for determination of the interferon receptor IFNAR2 subunit in peripheral blood leukocyte subsets.
BACKGROUND
It is expected that expression levels in the peripheral blood mononuclear cells (PBMC) of IFNAR2, a subunit of the interferon (IFN) receptor, may be a marker for predicting IFN response. In the present study, we have established a rapid and convenient method for assaying IFNAR2, using flow cytometry.


METHODS
Fifty microliters of whole blood from healthy volunteers was treated with an anti-IFNAR2 antibody and stained with a Fluorescein isothiocyanate (FITC)-conjugated secondary antibody. In addition, the cells were stained with subset-specific antibodies conjugated with phycoerythrin (PE) and PE covalently linked to cyanin 5 at the same time. The mean FITC-fluorescence intensities were analyzed separately by gating on subset-specific regions.


RESULTS
IFNAR2 was detected in most lymphocytes, monocytes, and granulocytes, although IFNAR2 expression was higher in the monocytes and granulocytes than in the lymphocytes. The intra- and interdaily variations of IFNAR2 in lymphocytes, monocytes, and granulocytes were small. Among the lymphocyte subsets, IFNAR2 showed high expression in natural killer (NK) cells and low expression in T lymphocytes. The effect of IFN-alpha on IFNAR2 expression was examined in vitro. A down-regulation of IFNAR2 was observed by IFN-alpha above 100 IU/ml.


CONCLUSIONS
This assay may be useful for examining IFNAR2 in various leukocyte subsets, separately, as well as providing a rapid and easy method for monitoring expression of type I IFN receptors.
Tochizawa
Shiro
S
Research Institute of Pharmacological and Therapeutical Development, Otsuka Pharmaceutical Co., Ltd., 463-10 Kagasuno Kawauchi-cho Tokushima 771-0192, Japan.
Akamatsu
Seiji
S
Sugiyama
Yoshihiro
Y
Muraguchi
Masahiro
M
Ohmoto
Yasukazu
Y
Ono
Yukihisa
Y
Ishikawa
Hiroshi
H
Tanigami
Akira
A
Sumida
Takumi
T
Mori
Toyoki
T
D005434:Flow Cytometry / Q000379:methods*; D016650:Fluorescein-5-isothiocyanate; D006801:Humans; D016898:Interferon-alpha / Q000494:pharmacology; D015640:Ion Channel Gating; D007962:Leukocytes / Q000378:metabolism*; D016131:Lymphocyte Subsets; D008565:Membrane Proteins; D010799:Phycoerythrin; D053633:Receptor, Interferon alpha-beta; D017471:Receptors, Interferon / Q000378:metabolism*
C508033:IFNAR2 protein, human; D016898:Interferon-alpha; D008565:Membrane Proteins; D017471:Receptors, Interferon; D010799:Phycoerythrin; D053633:Receptor, Interferon alpha-beta; D016650:Fluorescein-5-isothiocyanate