Peripheral blood monocytes of rheumatoid arthritis patients do not express elevated TNF alpha, IL-1beta, and IL-8 mRNA levels. A comparison of monocyte isolation procedures.
We have compared three monocyte isolation procedures for their suitability in the analysis of cytokine mRNA expression in circulating monocytes. Monocytes were isolated from peripheral blood (1) using antiCD14 coated magnetic beads, (2) by Ficoll centrifugation, or (3) by Ficoll centrifugation followed by plastic adherence. The effect of the isolation procedure on the cytokine mRNA expression levels of the isolated monocytes was evaluated by RT-PCR. Results show that the expression of cytokine mRNAs determined in monocytes isolated using antiCD14 coated magnetized beads reflects best the cytokine mRNA levels in circulating monocytes. We subsequently applied this method to the analysis of cytokine mRNA expression levels in rheumatoid arthritis and control monocytes, which revealed that RA and control monocytes isolated by antiCD14 beads produce similar, very low TNF alpha, IL-1beta, and IL-8 mRNA levels.
Rodenburg
R J
RJ
Department of Biochemistry, University of Nijmegen, The Netherlands. r.rodenburg@bioch.kun.nl
van den Hoogen
F H
FH
van de Putte
L B
LB
van Venrooij
W J
WJ
D001172:Arthritis, Rheumatoid / Q000097:blood*; D005260:Female; D006801:Humans; D007375:Interleukin-1 / Q000096:biosynthesis*; D016209:Interleukin-8 / Q000096:biosynthesis*; D007963:Leukocytes, Mononuclear / Q000166:cytology* / Q000378:metabolism*; D008297:Male; D012333:RNA, Messenger / Q000097:blood*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014409:Tumor Necrosis Factor-alpha / Q000096:biosynthesis*
D007375:Interleukin-1; D016209:Interleukin-8; D012333:RNA, Messenger; D014409:Tumor Necrosis Factor-alpha