Accumulating evidences support that CD4 + CD25 high T regulatory (Treg) cells play an essential role in controlling and preventing autoimmunity. Paradoxically, RA patients have elevated numbers of circulating CD4 + CD25 high T cells, however, the inflammation is still ongoing. Further identification of these CD4 + CD25 high T cells may contribute to a better understanding of underlying mechanisms. We show here that these CD4 + CD25 high T cells were composed of CD4 + CD25 high FoxP3 + Treg cells and activated CD4 + CD25 high FoxP3 − effector cells. Moreover, there were significantly more Treg cells and effector T cells expressing GITR, and more monocytes expressing GITR-L. Thus, although RA patients have elevated numbers of CD4 + CD25 high T cells, the suppressive function is not increased, because of the increased number of activated effector T cells. In addition, the GITR-GITR-L system was activated in RA patients, which might lead to diminish suppressive activity of Treg cells and/or lead to resist to suppression of Treg cells by activated effector T cells, thus, contributing to the ongoing inflammation in RA patients.

Intracellular cytokine staining is an increasingly popular analytical tool that can be used to define the profile of cytokines in various disease states. One important requirement for this assay is the inclusion of a protein transport inhibitor in stimulated cell cultures to trap the cytokine, thus allowing a brighter signal. Two compounds commonly used for this purpose are brefeldin A (BFA) and monensin (MN). Flow cytometry was used to assess the differential effects of BFA and MN on surface CD3, -4, -8, and -69 expression and the intracellular expression of gamma interferon (IFN-␥) and tumor necrosis factor alpha (TNF-␣) following stimulation with phorbol myristate acetate and ionomycin. We found that BFA blocked the majority of CD3 Methods for intracellular cytokine staining are becoming widely accepted as an analytical tool in the immunology laboratory. This staining technique allows the delineation of distinct cytokine-producing leukocyte subsets within a mixed cell population. The methodology involves the fixation and permeabilization of the target cells for detection of intracellular cytokines by flow cytometry. This method was first proposed by Jung et al. (9) as a modification of a protocol that was originally designed for analysis using a fluorescence microscope (21). Further publications on this subject have resulted in reports addressing the kinetic aspects of cytokine expression (11) and the development of a whole-blood method (23). Using this methodology, a number of groups have detected imbalances in cytokine production in different disease states (4,6, 26,27).A key aspect of intracellular cytokine detection is trapping the cytokine within the cell. Generally, unstimulated cells produce undetectable amounts of cytokine. Therefore, the cells must be stimulated; a popular choice for stimulation is the combination of phorbol myristate acetate (PMA) and ionomycin (ION), which induces rapid induction of many cytokines. Thus, early quantification (i.e., at 2 to 4 h) of the number of cells expressing a cytokine and determination of the relative amount of cytokine per cell can be made. A protein transport inhibitor is added to the cultures to prevent the release of cytokines from the cells. Two commonly used compounds are monensin (MN) and brefeldin A (BFA). MN is derived from Streptomyces cinnamonensis and is an Na ϩ ionophore that disrupts intracellular Na ϩ and H ϩ gradients, exerting its greatest effects on the regions of the Golgi apparatus that are associated with the final stages of secretory vesicle maturation (13; E. Chu, J. Elia, D. Sehy, D. Ernst, and C. Shih, Hotlines [Pharmingen] 3:9-10, 1997). BFA is a macrocyclic lactone that is produced by a variety of fungi and is synthesized from palmitate. BFA was originally isolated from Penicillium brefeldianum as described by Dinter and Berger (7) and appears to inhibit protein secretion early in a pre-Golgi compartment (between the endoplasmic reticulum and Golgi). The mechanism of this action is complicated and is best explained by Dinter and Berge...