PPAR activators as antiinflammatory mediators in human T lymphocytes: implications for atherosclerosis and transplantation-associated arteriosclerosis

Abstract

Activation of T lymphocytes and their ensuing elaboration of proinflammatory cytokines, such as interferon (IFN)-gamma, represent a critical step in atherogenesis and arteriosclerosis. IFNgamma pathways also appear integral to the development of transplantation-associated arteriosclerosis (Tx-AA), limiting long-term cardiac allograft survival. Although disruption of these IFNgamma signaling pathways limits atherosclerosis and Tx-AA in animals, little is known about inhibitory regulation of proinflammatory cytokine production in humans. The present study investigated whether activators of peroxisome proliferator-activated receptor (PPAR)alpha and PPARgamma, with their known antiinflammatory effects, might regulate the expression of proinflammatory cytokines in human CD4-positive T cells. Isolated human CD4-positive T cells express PPARalpha and PPARgamma mRNA and protein. Activation of CD4-positive T cells by anti-CD3 monoclonal antibodies significantly increased IFNgamma protein secretion from 0 to 504+/-168 pg/mL, as determined by ELISA. Pretreatment of cells with well-established PPARalpha (WY14643 or fenofibrate) or PPARgamma (BRL49653/rosiglitazone or pioglitazone) activators reduced anti-CD3-induced IFNgamma secretion in a concentration-dependent manner. PPAR activators also inhibited TNFalpha and interleukin-2 protein expression. In addition, PPAR activators markedly reduced cytokine mRNA expression in these cells. Such antiinflammatory actions were also evident in cell-cell interactions with medium conditioned by PPAR activator-treated T cells attenuating human monocyte CD64 expression and human endothelial cell major histocompatibility complex class II induction. Thus, activation of PPARalpha and PPARgamma in human CD4-positive T cells limits the expression of proinflammatory cytokines, such as IFNgamma, yielding potential therapeutic benefits in pathological processes, such as atherosclerosis and Tx-AA.

Figure 1

CD4-positive human T cells express PPARα and PPARγ. A, RT-PCR reaction of PPARα and PPARγ RNA in freshly isolated human CD4-positive T cells (T4) reveals a cDNA fragment of the expected size. Also shown are a DNA ladder (MW), RT-PCR product from macrophage RNA as a positive control (MØ), and a negative control consisting of RT-PCR reactions lacking RT (Co). B, Western blot analysis on nucleic (Nucl) and cytosolic (Cyto) fractions of human CD4-positive T cells with use of an anti-human PPARα antibody or an anti-human PPARγ antibody.

Figure 2

PPAR activators inhibit IFNγ expression in human CD4-positive T cells. A, Isolated CD4-positive T cells were pretreated with PPARα activators (WY14643 or fenofibrate) 2 hours before stimulation with anti-CD3 antibodies. After 48 hours, cytokine protein content in cell-free supernatants was measured by ELISA. Results are expressed as percentage of CD3-activated cells (% control). Bars represent mean±SEM (n=6). **P<0.01. B, Isolated CD4-positive T cells were pretreated with PPARγ activators (BRL or pioglitazone) 2 hours before stimulation with anti-CD3 antibodies. After 48 hours, cytokine protein content in cell-free supernatants was measured by ELISA. Results are expressed as percentage of CD3-activated cells (% control). Bars represent mean±SEM (n=6). *P<0.05; **P<0.01.

Figure 3

PPAR activators reduce TNFα and IL-2 protein secretion from CD4-positive human T cells. A and B, Isolated CD4-positive T cells were pretreated with PPARα-activating WY14643 (A) or PPARγ-activating BRL 2 hours before stimulation with anti-CD3 antibodies (B). After 48 hours, cytokine protein content in cell-free supernatants was measured by ELISA. Results are expressed as percentage of CD3-activated cells (% control). Bars represent mean±SEM (n=3). *P<0.05; **P<0.01. C, PPAR activators do not induce expression of IL-4 in CD4-positive human T cells. Isolated CD4-positive T cells were treated with PPARα activators (250 μ mol/L WY14643 or 100 μ mol/L fenofibrate) or PPARγ activators (10 μ mol/L BRL or 10 μ mol/L pioglita-zone) for 24 hours before IL-4 protein content in cell-free supernatant was measured by ELISA. PMA/ionomycin (100 ng/mL/1 μ mol/L)–treated cells served as a positive control. Results are expressed as percentage of PMA/ionomycin-activated cells (% control). Bars represent mean±SEM (n=3). **P<0.01 compared with unstimulated cells. D, PPAR activators inhibit PMA/ionomycin-induced IFNγ expression. Isolated CD4-positive T cells were stimulated and treated with PMA/ ionomycin (10 ng/mL/0.5 μ mol/L) in the absence or presence of the PPARα activator WY14643 or the PPARγ activator BRL. After 6 hours, cytokine protein contents in cell-free supernatants were measured by ELISA. Results are expressed as percentage of PMA/ionomycin-treated cells (% control). Bars represent mean±SEM (n=3). *P<0.05; **P<0.01.

Figure 4

PPAR activators inhibit proinflammatory cytokine mRNA expression in human CD4-positive cells. A, Representative Northern blot analysis for cytokine expression of human CD4-positive T cells pretreated with WY14643 (250 μ mol/L) or BRL (10 μ mol/L) before 24-hour stimulation with anti-CD3 antibodies. Three independent experiments yielded similar results. B, Representative Northern blot analysis for cytokine expression of human CD4-positive T cells pretreated with WY14643 (top) or BRL (bottom) at concentrations indicated before incubation with anti-CD3 antibodies for 24 hours. Three independent experiments yielded similar results. C, Representative Northern blot analysis for cytokine expression of human CD4-positive T cells pretreated with BRL or pioglitazone (both at 10 μ mol/L) before incubation with anti-CD3 antibodies for 24 hours (top). At the bottom is a densitometric analysis of IFNγ mRNA expression normalized to housekeeping gene B41 of 3 independent experiments. D, Representative Northern blot analysis for cytokine mRNA expression of human CD4-positive T cells pretreated with WY14643 (250 μ mol/L) or BRL (10 μ mol/L) before 2-hour stimulation with PMA/ionomycin. Three independent experiments yielded similar results.

Figure 5

PPAR activators reduce proinflammatory activity of T-cell supernatants on human monocytes in the absence of any direct effects on monocyte CD64 response. A, Freshly isolated human monocytes were incubated for 18 hours with conditioned media from CD4-positive T cells stimulated with anti-CD3 monoclonal antibodies in the presence or absence of PPAR agonists, and mean fluorescence intensity of monocyte CD64 expression was measured by flow cytometry (right). Results are expressed as percentage of control (monocytes incubated with supernatants from activated T cells). Bars represent mean±SEM (n=4). *P<0.05; **P<0.01. B, Human monocytes were incubated with conditioned media from CD3-activated T cells to induce CD64 expression and then directly stimulated with PPAR activators for 18 hours before CD64 expression was assessed by flow cytometry (right). Results are expressed as percentage of control (monocytes incubated with supernatants from activated T cells). Bars represent mean±SEM (n=4). No significant difference was seen, except for comparison with unstimulated cells. **P<0.01. C, Human monocytes were incubated with IFNγ (200 U/L) in the presence or absence of PPAR activators. After 18 hours, CD64 expression was measured by flow cytometry (right). Results are expressed as percentage of control (monocytes stimulated with IFNγ). Bars represent mean±SEM (n=5). No significant difference was seen, except for comparison with unstimulated cells. **P<0.01.

Figure 6

PPAR activators reduce proinflammatory activity of T-cell supernatants on human ECs. A, Human ECs were incubated for 72 hours with conditioned media from CD4-positive T cells stimulated with or without PPAR activators, and endothelial MHC II surface expression was measured by flow cytometry (mean fluorescence intensity) (right). Results are expressed as percentage of ECs incubated with supernatants from activated T cells. Bars represent mean±SEM (n=3). *P<0.05. B, Human ECs were incubated with conditioned media from CD3-activated T cells to induce MHC II expression and then directly stimulated with PPAR activators for 72 hours before MHC II surface expression was assessed by flow cytometry (right). Results are expressed as percentage of control (ECs incubated with supernatants from activated T cells). Bars represent mean±SEM (n<4). No significant difference was seen, except for comparison with unstimulated cells. **P<0.01. C, Human ECs were incubated with IFNγ (1000 U/L) in the presence or absence of PPAR activators. After 72 hours, MHC II expression was measured by flow cytometry (right). Results are expressed as percentage of control (ECs stimulated with IFNγ). Bars represent mean±SEM (n=4). No significant difference was seen, except for comparison with unstimulated cells. *P<0.05.