Elsevier

International Immunopharmacology

Volume 4, Issue 9, September 2004, Pages 1171-1185
International Immunopharmacology

Ciglitizone and 15d PGJ2 induce apoptosis in Jurkat and Raji cells

https://doi.org/10.1016/j.intimp.2004.05.007Get rights and content

Abstract

Several studies have shown that PPARγ agonists play a role in the regulation of lymphocytes function and apoptosis. However, the molecular mechanism(s) underlying the immunomodulatory effects of PPARγ agonists are not defined yet. In this study, the effects of PPARγ (15d PGJ2 and ciglitizone) ligands on proliferation, cytokine production and apoptosis of Jurkat and Raji cells (human T and B lymphocytes, respectively) were examined. Ciglitizone and 15d PGJ2 presented antiproliferative and cytotoxic effects on Jurkat and Raji cells as shown by [14C]-thymidine incorporation and cell viability assay. In addition, 15d PGJ2 inhibited cytokine production (IL-2 in Jurkat cells and IL-10 in Raji cells). The mechanism whereby PPARγ agonists induced cytotoxicity is via apoptosis as shown by DNA fragmentation, nuclear condensation and phosphatidylserine externalization. The induction of apoptosis by ciglitizone and 15d PGJ2 on Jurkat and Raji cells may explain the suppression of cytokine production and the decrease in proliferation observed in both cell types. The apoptotic process was associated with a decrease in mitochondrial membrane potential and a marked down-regulation of the c-myc expression. These findings might play a key role in the apoptosis of T and B lymphocytes induced by PPARγ agonists.

Introduction

Peroxisome proliferator activated receptors (PPARs) are members of the nuclear hormone receptor superfamily that includes receptors for steroids, retinoids, and thyroid hormones [1]. To date, three different PPAR subtypes have been identified: PPARα, PPARβ (PPARδ) and PPARγ [2]. Agonists of both PPARα (fibrates, e.g. gemfibrosil, Wy 14643 and clofibrate) [3], [4], [5] and PPARγ (TZDs, e.g. troglitazone, pioglitazone and rosiglitazone) [5], [6] have been therapeutically used for the treatment of hyperlipidaemia, atherosclerosis, diabetes, obesity and X syndrome.

PPARγ has also been reported to affect leukocyte function [7], [8], [9], [10], [11], [12], [13], [14], [15], [16], [17], [18]. PPARγ expression is augmented in activated peritoneal murine macrophages and PPARγ ligands inhibit the expression of inducible nitric oxide synthase, gelatinase B and scavenger receptor A genes, in part by antagonizing the activities of transcription factors such as AP-1, STAT and NF-κB [10], [11]. Activation of PPARγ in monocytes and macrophages inhibits the expression of inflammatory cytokines such as TNFα, IL-1α and IL-6 [10], [11], [12]. In addition, 15d PGJ2 is a negative regulator of macrophage functions such as phagocytosis, superoxide anion and hydrogen peroxide production [13]. Several groups have demonstrated that T and B lymphocytes express PPARγ [14], [15], [16], [17], [18]. The PPARγ expressed in T lymphocytes is of functional significance and ligands such as 15d PGJ2 and ciglitizone cause marked inhibition of proliferative response [17], [18] and IL-2 secretion [14]. The exposure to ligands of PPARγ (15d PGJ2 and troglitazone) leads to significant decrease in T cell viability probably due to apoptosis as indicated by DNA fragmentation (TUNEL assay) and phosphatidylserine externalization [17], [18]. In human peripheral T lymphocytes, 15d PGJ2 and troglitazone, but not Wy14643 (PPARα ligand), inhibit IL-2 production and PHA-induced proliferation [14]. Normal mouse and human B lymphocytes and neoplastic B cells also express PPARγ [15], [16]. When B cells are exposed to 15d PGJ2, their proliferation is inhibited in a dose-dependent manner [15], [16]. PPARγ agonists are toxic for B lymphocytes via apoptosis as also indicated by DNA fragmentation and phosphatidylserine externalization [15], [16]. PPARγ and its endogenous ligands have been indicated as participating factors for type 1/type 2 T and NK (natural killer) cells differentiation and development [8].

In spite of the information above, the molecular mechanism(s) underlying the effects of PPARγ agonists are still undefined. Also, a systematic study comparing the apoptotic effects of activators of PPARγ and PPARα in both B and T lymphocytes remains to be carried out.

The purpose of the present study was then to determine the effects of PPARγ (15d PGJ2 and ciglitizone) ligands on proliferation, cytokine production and apoptosis of Jurkat and Raji cells (human T and B lymphocytes cell lines, respectively). The following morphological and biochemical assays were performed to characterize the process of apoptosis: chromatin condensation, DNA fragmentation, phosphatidylserine externalization, mitochondrial despolarization and the expression of pro-apoptotic (bcl-xS, p53, c-myc, bax) and anti-apoptotic (bcl-xL, bcl-2) genes.

Section snippets

Reagents

15 Deoxy Δ12,14 prostaglandin J2 (15d PGJ2), 4-chloro-6-(2,3-xylidino)-2 pyrimidinylthioacetic acid (Wy 14463) and clofibrate were obtained from Bio Mol (Plymouth Meeting, Pennsylvania, USA). Ciglitizone and 9-cis retinoic acid (RA) were obtained from Sigma (St. Louis, MO). Ciglitizone, 15d PGJ2, 9-cis retinoic acid, clofibrate and WY 14643 were dissolved in DMSO and added to the culture medium. Controls received the same amount of DMSO, which was always lower than 0.5% (v/v) of the culture

Evidence that Jurkat and Raji cells express PPARγ and PPARα

The expression of the transcripts for PPARγ and PPARα in Jurkat and Raji cells was examined by RT-PCR. The product size for PPARγ was 474 pb, for PPARα was 276 pb, and for β-actin (control) 538 pb. Both cells expressed mRNA for PPARγ and PPARα (Fig. 1). However, Jurkat cells express a relatively larger amount of PPARγ than Raji cells under similar conditions. The immunocytochemistry revealed the cytoplasmic and perinuclear localization of PPARα and PPARγ in both Jurkat and Raji cells (Fig. 2).

Discussion

Evidence is presented herein that Jurkat and Raji cells express PPARγ and PPARα (Fig. 1). As proposed by Akiyama et al. [26], in the case of the PPAR receptors, localization is dependent on the context of the cell type. Perhaps, variations in PPAR localization between cell types may reflect differences in the exposure to endogenous ligands, nuclear translocation proteins, or other proteins that may interact with and affect PPAR localization. In our study, PPARα and PPARγ are predominantly

Acknowledgements

The authors are indebted to the technical assistance of J.R. Mendonça, G. de Souza, E.P.P. Silva, and to the discussion and suggestion of Dr. C.K. Miyasaka and Dr S. Bordin. This research is supported by FAPESP, CNPq, CAPES and PRONEX.

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