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

Arthritis Rheum. 1998 Sep;41(9):1669-76. doi: 10.1002/1529-0131(199809)41:9<1669::AID-ART19>3.0.CO;2-G.


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.

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 IFNgamma and IL-10.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Aged
  • Aged, 80 and over
  • Anti-Bacterial Agents / pharmacology
  • Antigens, Surface / metabolism
  • Arthritis, Rheumatoid / immunology
  • Arthritis, Rheumatoid / metabolism*
  • Brefeldin A
  • CD28 Antigens / immunology
  • CD3 Complex / immunology
  • CD4-Positive T-Lymphocytes / immunology
  • CD4-Positive T-Lymphocytes / metabolism*
  • CD8-Positive T-Lymphocytes / immunology
  • CD8-Positive T-Lymphocytes / metabolism
  • Calcimycin / pharmacology
  • Cell Separation
  • Cyclopentanes / pharmacology
  • Cytokines / biosynthesis*
  • Flow Cytometry
  • Humans
  • Immunophenotyping
  • Ionophores / pharmacology
  • Lymphocyte Activation
  • Macrolides
  • Middle Aged
  • Protein Synthesis Inhibitors / pharmacology
  • Synovial Membrane / cytology
  • Synovial Membrane / immunology
  • Synovial Membrane / metabolism*
  • T-Lymphocytes, Helper-Inducer / metabolism


  • Anti-Bacterial Agents
  • Antigens, Surface
  • CD28 Antigens
  • CD3 Complex
  • Cyclopentanes
  • Cytokines
  • Ionophores
  • Macrolides
  • Protein Synthesis Inhibitors
  • Brefeldin A
  • Calcimycin