Flow cytometric single-cell analysis of cytokine production by CD4+ T cells in synovial tissue and peripheral blood from patients with rheumatoid arthritis

Yoshitaka Morita, Masahiro Yamamura, Masanori Kawashima, Seishi Harada, Kazuhide Tsuji, Kazuko Shibuya, Keisuke Maruyama, Hirofumi Makino

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Abstract

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-γ (IFNγ), 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, IFNγ-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 IFNγ 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 IFNγ, 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 IFNγ. 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. Conclusion. 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 IFNγ and IL-10.

Original languageEnglish
Pages (from-to)1669-1676
Number of pages8
JournalArthritis and Rheumatism
Volume41
Issue number9
DOIs
Publication statusPublished - Sep 1 1998

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Single-Cell Analysis
Rheumatoid Arthritis
Cytokines
T-Lymphocytes
Interferons
Interleukin-10
Interleukin-13
Calcimycin
Tetradecanoylphorbol Acetate
Interleukin-4
Interleukin-2
Brefeldin A
Calcium Ionophores
Synovial Membrane

ASJC Scopus subject areas

  • Immunology and Allergy
  • Rheumatology
  • Immunology
  • Pharmacology (medical)

Cite this

Flow cytometric single-cell analysis of cytokine production by CD4+ T cells in synovial tissue and peripheral blood from patients with rheumatoid arthritis. / Morita, Yoshitaka; Yamamura, Masahiro; Kawashima, Masanori; Harada, Seishi; Tsuji, Kazuhide; Shibuya, Kazuko; Maruyama, Keisuke; Makino, Hirofumi.

In: Arthritis and Rheumatism, Vol. 41, No. 9, 01.09.1998, p. 1669-1676.

Research output: Contribution to journalArticle

Morita, Yoshitaka ; Yamamura, Masahiro ; Kawashima, Masanori ; Harada, Seishi ; Tsuji, Kazuhide ; Shibuya, Kazuko ; Maruyama, Keisuke ; Makino, Hirofumi. / Flow cytometric single-cell analysis of cytokine production by CD4+ T cells in synovial tissue and peripheral blood from patients with rheumatoid arthritis. In: Arthritis and Rheumatism. 1998 ; Vol. 41, No. 9. pp. 1669-1676.
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abstract = "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-γ (IFNγ), 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, IFNγ-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 IFNγ 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 IFNγ, 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 IFNγ. 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. Conclusion. 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 IFNγ and IL-10.",
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AU - Yamamura, Masahiro

AU - Kawashima, Masanori

AU - Harada, Seishi

AU - Tsuji, Kazuhide

AU - Shibuya, Kazuko

AU - Maruyama, Keisuke

AU - Makino, Hirofumi

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N2 - 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-γ (IFNγ), 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, IFNγ-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 IFNγ 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 IFNγ, 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 IFNγ. 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. Conclusion. 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 IFNγ and IL-10.

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