Flow cytometric single-cell analysis of cytokine production by CD4+ T cells in synovial tissue and peripheral blood from patients with rheumatoid arthritis.
OBJECTIVE
To determine the cytokine profile of CD4+ T cells in the synovial tissue (ST) of rheumatoid arthritis (RA) patients at the single-cell level.


METHODS
Unseparated ST cells and paired CD4+ T cells separated from the peripheral blood (PB) and ST of RA patients were stimulated for 4 hours with phorbol myristate acetate (PMA) plus calcium ionophore A23187, or for 6 hours with immobilized anti-CD3 plus anti-CD28, in the presence of brefeldin A. Cells were stained for intracellular cytokines such as interferon-gamma (IFNgamma), interleukin-2 (IL-2), IL-4, IL-10, and IL-13, in combination with cell surface markers. The percentages of cytokine-producing T cells were analyzed by flow cytometry.


RESULTS
When ST cells were stimulated with PMA plus A23187 in bulk culture, IFNgamma-producing T cells were more frequently detected in the CD8+ subset, but cells producing other cytokines were found in the CD4+ subset. Purified ST CD4+ T cells, after stimulation with PMA plus A23187, were able to produce higher levels of IFNgamma but lower levels of IL-4 and IL-13, by analysis at the single-cell level, as compared with the PB CD4+, CD45RO+ T cells. The majority of IL-4- or IL-13-producing ST CD4+ cells produced IFNgamma, although PB CD4+ T cells rarely showed this cytokine pattern. IL-10-producing CD4+ T cells were more frequently found in the ST than in the PB. Of interest, most of the IL-10-producing ST CD4+ T cells were able to produce IFNgamma. IL-2-producing CD4+ T cells were similarly present in both compartments. Similar intracellular cytokine patterns were observed with anti-CD3 plus anti-CD28 stimulation, although the number of detected cells was lower.


CONCLUSIONS
These data indicate that CD4+ T cells present within the inflamed synovium have apparently distinct cytokine profiles from those of memory CD4+ T cells in the PB, as typified by their ability to secrete both IFNgamma and IL-10.
Morita
Y
Y
Okayama University Medical School, Japan.
Yamamura
M
M
Kawashima
M
M
Harada
S
S
Tsuji
K
K
Shibuya
K
K
Maruyama
K
K
Makino
H
H
D000368:Aged; D000369:Aged, 80 and over; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D000954:Antigens, Surface / Q000378:metabolism; D001172:Arthritis, Rheumatoid / Q000276:immunology / Q000378:metabolism*; D020126:Brefeldin A; D018106:CD28 Antigens / Q000276:immunology; D017252:CD3 Complex / Q000276:immunology; D015496:CD4-Positive T-Lymphocytes / Q000276:immunology / Q000378:metabolism*; D018414:CD8-Positive T-Lymphocytes / Q000276:immunology / Q000378:metabolism; D000001:Calcimycin / Q000494:pharmacology; D002469:Cell Separation; D003517:Cyclopentanes / Q000494:pharmacology; D016207:Cytokines / Q000096:biosynthesis*; D005434:Flow Cytometry; D006801:Humans; D016130:Immunophenotyping; D007476:Ionophores / Q000494:pharmacology; D008213:Lymphocyte Activation; D018942:Macrolides; D008875:Middle Aged; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D013583:Synovial Membrane / Q000166:cytology / Q000276:immunology / Q000378:metabolism*; D006377:T-Lymphocytes, Helper-Inducer / Q000378:metabolism
D000900:Anti-Bacterial Agents; D000954:Antigens, Surface; D018106:CD28 Antigens; D017252:CD3 Complex; D003517:Cyclopentanes; D016207:Cytokines; D007476:Ionophores; D018942:Macrolides; D011500:Protein Synthesis Inhibitors; D020126:Brefeldin A; D000001:Calcimycin