#ELUCIDATE SY FREE#
c9, t11-CLA (Matreya LLC, Pleasant Gap, USA) in free fatty acid form, phorbol 12-myristate 13-acetate (PMA), ionomycin, and brefeldin A (all Enzo) were likewise dissolved in DMSO, aliquoted, and stored at −20☌. Lyophilized 2-chloro-5-nitrobenzanilide (GW9662) was solubilized in sterile dimethylsulfoxide (DMSO) according to the manufacturer’s instruction (Enzo, Lörrach, Germany, and Sigma, Taufkirchen, Germany) and stored in aliquots at −20☌.
In the present study, which is complementary to previously published work of our group, we examined whether GW9662 is suitable to explain PPAR γ-mediated effects of c9, t11-CLA in primary human T-helper cells. This molecule covalently modifies the ligand-binding domain by arylation on the cysteine residue Cys 285 and thereby inhibits irreversibly ligand binding to and activation of PPAR γ. GW9662 is widely used to elucidate PPAR γ-dependent anti-inflammatory mechanisms in vitro and in vivo. For instance, we have previously shown that the predominant natural isomer c9, t11-CLA reduces expression of the chemokine IL-8 in airway epithelial cells, inhibits IL-2 and TNF- α in T-helper cells, and prevents experimentally induced airway inflammation in mice at least in part via a PPAR γ-dependent mechanism. ĭue to their ability to activate PPAR γ with micromolar affinity, conjugated linoleic acids (CLA), naturally occurring fatty acids in ruminant fats, aroused scientific interest as potentially anti-inflammatory agents.
#ELUCIDATE SY ACTIVATOR#
Its activation by ligand binding antagonizes the proinflammatory capability of several transcription factors such as nuclear factor- κB (NF- κB), signal transducer and activator of transcription (STAT), and nuclear factor of activated T cells (NFAT) to control the expression of immunostimulatory cytokines such as IL-2 and IL-4. In T-helper cells, the predominately expressed splice variant γ1 is inducible by agonist ligation. Among the three isoforms of the PPAR family, designated PPAR α (NR1C1), PPAR β/ δ (NR1C2 NUC1), and PPAR γ (NR1C3), the latter has been specifically implicated in the regulation of immune cell function, for example, in macrophages and T-helper cells. Introductionĭuring the last decades, the scientific knowledge about the role of peroxisome proliferator-activated receptors (PPARs) in controlling metabolic and inflammatory processes has increased steadily.
These results suggest that application of GW9662 is not conducive in this experimental setting. Moreover, GW9662 dose-dependently induced cell death in human leukocytes. GW9662 failed to abrogate this fatty acid effect, likely due to the fact that the compound exerted an own inhibitory effect on IL-2 production. 100 μmol/L c9, t11-CLA reduced the number of T-helper cells expressing IL-2 by 68%. Subsequently, intracellular IL-2 was measured in CD3 +CD4 + lymphocytes by means of flow cytometry. After 19 h, cells were mitogen stimulated for further 5 h. Corresponding cultures were incubated with GW9662 in the absence of the fatty acid. Human peripheral blood mononuclear cells (PBMC) were preincubated with increasing concentrations of GW9662 (0, 0.4, 2, and 10 μmol/L) 30 min before adding the c9, t11-isomer of conjugated linoleic acid ( c9, t11-CLA) as representative of PPAR γ-activating fatty acids with immunomodulatory properties. In addition and complementary to recent work, we examined whether GW9662 is suitable to serve for mechanistic investigation in T-helper cells.
Synthetic antagonists of the nuclear receptor PPAR γ such as GW9662 are widely used to elucidate receptor-mediated ligand effects.
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