Limited Applicability of GW9662 to Elucidate PPARγ-Mediated Fatty Acid Effects in Primary Human T-Helper Cells

Synthetic antagonists of the nuclear receptor PPARγ such as GW9662 are widely used to elucidate receptor-mediated ligand effects. In addition and complementary to recent work, we examined whether GW9662 is suitable to serve for mechanistic investigation in T-helper cells. 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 PPARγ-activating fatty acids with immunomodulatory properties. Corresponding cultures were incubated with GW9662 in the absence of the fatty acid. After 19 h, cells were mitogen stimulated for further 5 h. Subsequently, intracellular IL-2 was measured in CD3+CD4+ lymphocytes by means of flow cytometry. 100 μmol/L c9,t11-CLA reduced the number of T-helper cells expressing IL-2 by 68%. 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. Moreover, GW9662 dose-dependently induced cell death in human leukocytes. These results suggest that application of GW9662 is not conducive in this experimental setting.


Introduction
During the last decades, the scientific knowledge about the role of peroxisome proliferator-activated receptors (PPARs) in controlling metabolic and inflammatory processes has increased steadily. Among the three isoforms of the PPAR family, designated PPAR (NR1C1), PPAR / (NR1C2; NUC1), and PPAR (NR1C3), the latter has been specifically implicated in the regulation of immune cell function, for example, in macrophages [1] and T-helper cells [2]. In Thelper cells, the predominately expressed splice variant 1 is inducible by agonist ligation [3]. Its activation by ligand binding antagonizes the proinflammatory capability of several transcription factors such as nuclear factor-B (NF-B), signal transducer and activator of transcription (STAT) [4,5], and nuclear factor of activated T cells (NFAT) to control the expression of immunostimulatory cytokines such as IL-2 and IL-4 [6,7].
Due to their ability to activate PPAR with micromolar affinity [8], conjugated linoleic acids (CLA), naturally occurring fatty acids in ruminant fats, aroused scientific interest as potentially anti-inflammatory agents. 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 [9], inhibits IL-2 and TNF-in Thelper cells [10], and prevents experimentally induced airway inflammation in mice at least in part via a PPAR -dependent mechanism [11].
GW9662 is widely used to elucidate PPAR -dependent anti-inflammatory mechanisms in vitro [12,13] and in vivo [14][15][16]. This molecule covalently modifies the ligand-binding domain by arylation on the cysteine residue Cys 285 [17] and 2 International Journal of Inflammation thereby inhibits irreversibly ligand binding to and activation of PPAR .
In the present study, which is complementary to previously published work of our group [10], we examined whether GW9662 is suitable to explain PPAR -mediated effects of c9,t11-CLA in primary human T-helper cells.

Purification of PBMC.
Mononuclear cells were isolated from buffy coats obtained from peripheral blood of healthy volunteers who gave their written consent for blood donation. Buffy coat blood was 1 : 1 diluted with PBS (PAA, Cölbe, Germany), layered onto Lymphocyte Separation Medium (LSM) 1077 (1.077 g/mL; PAA; ratio 1 : 1), and centrifuged at 700 ×g for 20 min at 20 ∘ C. The PBMC interphase was collected, washed three times with PBS, and resuspended in RPMI 1640 medium supplemented with 10% FBS Gold (PAA).

Cell Viability.
To assess the impact of GW9662 on cell viability, PBMC (1 × 10 6 /mL) were incubated without or with 0.4, 2, and 10 mol/L of this compound for 19 h, followed by 5 h stimulation with PMA (2.5 ng/mL) and ionomycin (0.5 g/mL) in the presence of brefeldin A (5 g/mL). Control cultures contained the according volume of DMSO. Cell viability was analyzed by annexin-V (Immunotech, Marseille, France) and propidium iodide (PI; Sigma-Aldrich, Munich, Germany) exclusion double staining as previously described [10].

Statistics.
Differences in the percentages of IL-2 positive cells were evaluated using a linear mixed model with the fixed factors "fatty acid treatment" (c9,t11-CLA and DMSO) and "PPAR antagonist treatment" (GW9662 and control) and the interaction of these two factors. The assumption of normality and homoscedasticity was justified by visual inspection of QQ-plots and predicted versus residual plots. A random intercept specific for each subject was included to control for interindividual differences. Tukey-Kramer was conducted as posthoc test and values were adjusted for multiple comparisons. For evaluation of data obtained in the absence of c9,t11-CLA, the concentration of GW9662 was entered into the model as fixed factor while IL-2 positive cells, MFI, and viability were defined as dependent variables, respectively. Because the distribution of viability was skewed, a log-transform was applied. For the latter outcome, differences between concentrations 0 mol/L and 0.4 mol/L were additionally evaluated by defining posthoc contrasts between these two concentration levels. Significance of difference was set at < 0.05. All calculations were carried out using SAS 9.3 (PROC MIXED).

GW9662 Fails to Abrogate the Inhibitory Effect of c9,t11-CLA on IL-2 Expression in T-Helper Cells.
In stimulated control cultures, 15 ± 2% of the T cells (CD3 + ) were identified as IL-2 positive T-helper cells (CD3 + CD4 + ; Figure 1(a)). Incubation with 100 mol/L c9,t11-CLA for 24 h significantly reduced the intracellular content of IL-2 in stimulated Thelper cells by 68% to 5 ± 1%. Preincubation with 0.4 mol/L GW9662 did not result in reexpansion of the IL-2 positive Thelper cell population. This was unexpected as preincubation with 0.4 mol/L of the PPAR antagonist T0070907, a compound with similar molecular structure to GW9662 except for one single N atom, did so in the aforementioned similar approach [10].
We further tested in a range of fivefold increases of the concentration of GW9662 whether a reversal of the fatty acid effect, in terms of blocked PPAR , was achieved. Interestingly, pretreatment with increasing concentrations of GW9662 did not lead to increased IL-2 production but even to a reduction. At 10 mol/L and in the presence of c9,t11-CLA, GW9662 caused a drop in the percentage of IL-2 positive T-helper cells even stronger than did the c9,t11-CLA treatment alone (Figure 1(b)).

GW9662 Dose-Dependently Downregulates IL-2 Expression in T-Helper Cells.
We next examined whether the PPAR antagonist exerted a fatty acid independent effect itself. Indeed, with increasing concentrations of GW9662 we found a continuous reduction in the IL-2 expressing T-helper cell population. Simultaneously, mean fluorescence intensity (MFI) reflecting the cytokine levels on a per-cell basis dosedependently decreased (Figure 2).

GW9662 Dose-Dependently Induces Cell Death of Human
Primary Leukocytes. We further assessed whether putative cytotoxic effects underlie the failure of GW9662 to restore the cytokine production inhibited by c9,t11-CLA. As revealed by annexin-V and PI exclusion double staining, GW9662 dose-dependently caused cell death in PBMC (Figures 3(a)  and 3(b)). After 24 h in the presence of GW9662, viability decreased by up to 35 ± 8% at 10 mol/L. However, at 0.4 mol/L GW9662 did not affect cell viability significantly (>95% of the control, = 0.531).

Discussion
In line with previous work of our group [10], we demonstrated at first that c9,t11-CLA reduces the expression of the immunostimulatory cytokine IL-2 in T-helper cells. We have previously shown that c9,t11-CLA acts at least in part via a PPAR -mediated pathway, since low-dose cotreatment with the PPAR inhibitor T0070907 largely reverted this fatty acid effect [10]. Though intended to be likewise applicable, GW9662 failed to abrogate the fatty acid effect at all tested concentrations in the present approach. This outcome was unexpected, as a large body of evidence exists that indicates suitability of GW9662 to elucidate PPAR -dependent mechanisms when used at concentrations within the single-to double-digit micromolar range, including own results from in vitro studies in human epithelial cells [9]. However, we have indications that GW9662 acts differently from T0070907 not only in primary lymphocytes but also in other cells International Journal of Inflammation 5 such as macrophages (unpublished findings). Nevertheless, in agreement with the literature, in a similar designed study like the one herein, GW9662 completely negated the modulating effects of t10,c12-CLA, a synthetic CLA isomer, on TNFexpression in stimulated porcine PBMC [18]. However, corroborating our findings, Raman et al. recently reported in the Jurkat T-cell line that not only PPAR agonists but also its antagonists decreased the mitogen stimulated elevation in intracellular Ca 2+ , which could lead to IL-2 suppression via decreased transcriptional activity of NFAT [19].
In order to justify our data, we repeated the experiments with GW9662 purchased from different manufacturers (not shown). Since the results were comparable we can exclude that false-negative data have been produced. Besides PPAR , PPAR , and PPAR / are also expressed by PBMC [20,21] and are bound and activated by CLA [22,23]. However, it is not plausible that the fatty acid effects have been mediated through either of these isoforms, as Cys 285 , the modified residue in PPAR , is conserved among all three PPARs. Moreover, significantly higher concentrations of GW9662 are required for inhibition of ligand binding to PPAR (factor ∼10 over PPAR ) and PPAR / (factor ∼600 over PPAR ), respectively [17]. We clearly found that GW9662 dose-dependently exerts an own fatty acid independent diminishing effect on IL-2 production in primary T-helper cells. This finding is new and of significance since effects of the antagonist by its own might mask those which should be actually explained by its usage. Moreover, GW9662 is cell toxic in PBMC with increasing concentrations. GW9662 has previously been shown to cause apoptotic cell death in a concentration-dependent manner in oral squamous cells [24] and colon cells [25]. However, in these studies cancer cell lines were used and these cells underwent apoptosis also after treatment with T0070907 at concentrations higher than 10 mol/L. The cell death inducing effect of high doses of PPAR antagonists led to discuss them as potential therapeutic agents in the treatment of cancer [25,26] but must be considered undesired in primary cells. However, as cell viability was not affected at 0.4 mol/L in our experiments, other effects than cytotoxic underlie the failure of GW9662 to serve for mechanistic exploration of the fatty acid effect that remains elusive.
In summary, and with the restriction that concentrations below 0.4 mol/L have not been tested, our data suggest that GW9662 is not valuable for determining the specific PPARmediated mode of fatty acid action in primary T-helper cells due to own regulatory and cytotoxic effects.