Comparison of effects of Y-27632 and Isoproterenol on release of cytokines from human peripheral T cells
Introduction
T cells play an important role in inflammatory responses through secreting T helper type 1 (Th-1) cytokines such as interleukin (IL)-2 and interferon (IFN)-gamma, and Th-2 cytokines such as IL-4 and IL-5 upon stimulation through T cell receptor (TCR). RhoA, a Rho family GTPase, is involved in the activation and function of T cells via TCR [1].
Y-27632, {(+)-(R)-trans-4-(1-aminoethyl)-N-(4-pyridyl) cyclohexanecarboxamide dihydrochloride, monohydrate}, selectively inhibits the effector of RhoA p21, which exists as two isoforms of Rho-associated protein serine/threonine kinase; Rho-associated coiled-coil forming kinase (ROCK) I and ROCK II (correspondent with ROK alpha or Rho kinase) [2], [3]. Y-27632 inhibited inflammatory reactions of human peripheral blood cells including lymphocytes [4], [5], besides relaxing contracted human bronchial smooth muscle [6]. These in vitro studies were generally performed in 0.01 to 100 μM of Y-27632, showing that its maximum effects appeared at 10 μM.
Recent studies have indicated that the beta agonists and other agents elevating intracellular levels of cyclic adenosine monophosphate (cAMP) mimicked effects of Y-27632 on human cells [7]. Moreover, dibutyryl cAMP inactivated RhoA in human cytotoxic lymphocytes [8]. Salbutamol, a beta agonist, was similar to but superior than Y-27632 in relaxant effect on contracted trachea [9]. We previously reported that Isoproterenol (Iso) (0.01–10 μM) inhibited production of IL-2, IL-4, and IL-5, and increased levels of cAMP in human peripheral T cells stimulated with Concanavalin A (Con A) [10]. Thus, we examined effect of both Y-27632 and Iso on release of Th-1 cytokines (IL-2 and IFN-gamma) and Th-2 cytokines (IL-4 and IL-5) from Con A-activated T cells obtained from peripheral blood of normal subjects, and compared the effects in the range of 0.1 to 10 μM.
Section snippets
Lymphocyte isolation and preparation
Heparinized venous blood was obtained from seven healthy volunteers. Lymphocytes and other mononuclear cells were isolated by standard density gradient centrifugation in lymphocyte separation medium (the density is 1.0770–1.0800 g/ml) (Organon Teknika, Durham, NC, USA). Cells recovered from the interface were washed three times with RPMI 1640 culture medium (GIBCO BRL, Gaithersburg, MD, USA), and suspended at 1.0×107/ml in culture medium containing 10% fetal bovine serum (FBS) (Equitech-Bio.,
Effect of Con A on T cells
We initially examined the effect of Con A on release of cytokines. Con A at 50 μg/ml optimally induced the secretion of IL-2, IFN-gamma, IL-4, and IL-5 from T cells (data not shown). When we stimulated T cells with 50 μg/ml Con A, we found that an average (±S.D.) of release of IL-2, IFN-gamma, IL-4, and IL-5 in the seven subjects was 1942.4±499.8, 1024.7±786.8, 273.4±117.4, and 346.5±227.9 pg/ml, respectively (Fig. 1a). We also found that the basal secretion of each cytokine from T cells
Discussion
Our data showed that Y-27632 suppressed the Con A-induced release of Th-1 cytokines (IL-2 and IFN-gamma), weakly decreased the release of Th-2 cytokines (IL-4 and IL-5), and reduced the ratio of Th-1/Th-2 cytokine release from human peripheral T cells. We confirmed that Y-27632 did not directly affect viability of T cells (data not shown). Recent studies have suggested that RhoA activated by Vav, a GDP/GTP exchange factor for Rho GTPase, following stimulation of TCR was involved in activation
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