Detection of TNF alpha and Fas ligand mRNA within synovial mononuclear cells by fluorescence in-cell labeling PCR (FICL-PCR).
T cells that infiltrate the synovial lesions of rheumatoid arthritis may play a key role in its pathogenesis. To learn more about their functional nature, we determined the frequency of synovial T cells that harbored the TNF alpha and Fas ligand transcript by a technique, called Fluorescence In-Cell Labeling Polymerase Chain Reaction (FICL-PCR). The mRNA of interest was detected in fixed cells by the incorporation during PCR of a fluorescein-12-dUTP label following an initial reverse transcription PCR step. Using this technique the CD3 transcript was detected in the T leukemic cell line, MOLT-4, with calculated sensitivity and specificity values of 91% and 100%, respectively. The percentage mean (+/-S.D.) of TNF alpha mRNA positive cells and Fas ligand mRNA positive cells in peripheral blood mononuclear cells from 12 rheumatoid arthritis patients were 5.1+/-2.3% and 4.8+/-3.1%, respectively. The percentage mean (+/-S.D.) of TNF alpha mRNA positive cells and Fas ligand mRNA positive cells among synovial mononuclear cells from six rheumatoid arthritis patients was 16.8+/-8.3% and 10.8+/-1.8%, respectively. This result indicates that the cytotoxic T cells expressing TNF alpha accumulate in rheumatoid arthritic lesions where they may play a pathogenic role.
Okubo
M
M
Department of Internal Medicine II, Fukushima Medical College, Fukushima City, Japan.
Brown
M P
MP
Chiba
K
K
Kasukawa
R
R
Nishimaki
T
T
D001172:Arthritis, Rheumatoid / Q000276:immunology; D017252:CD3 Complex / Q000378:metabolism; D053222:Fas Ligand Protein; D005453:Fluorescence; D006801:Humans; D008024:Ligands; D008562:Membrane Glycoproteins / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D013582:Synovial Fluid / Q000166:cytology* / Q000276:immunology; D013601:T-Lymphocytes / Q000276:immunology / Q000378:metabolism*; D014409:Tumor Necrosis Factor-alpha / Q000235:genetics / Q000378:metabolism*
D017252:CD3 Complex; C505661:FASLG protein, human; D053222:Fas Ligand Protein; D008024:Ligands; D008562:Membrane Glycoproteins; D012333:RNA, Messenger; D014409:Tumor Necrosis Factor-alpha