Sarah G. Harris

Prostaglandins as modulators of immunity

Prostaglandins are potent lipid molecules that affect key aspects of immunity. The original view of prostaglandins was that they were simply immunoinhibitory. This review focuses on recent findings concerning prostaglandin E2 (PGE2) and the PGD2 metabolite 15-deoxy-Delta(12,14)-PGJ2, and their divergent roles in immune regulation. We will highlight how these two seminal prostaglandins regulate immunity and inflammation, and play an emerging role in cancer progression. Understanding the diverse activities of these prostaglandins is crucial for the development of new therapies aimed at immune modulation.

The nuclear receptor PPAR gamma is expressed by mouse T lymphocytes and PPAR gamma agonists induce apoptosis.

Peroxisome proliferator‐activated receptor (PPAR)‐γ is a nuclear hormone receptor that serves as a trans factor to regulate lipid metabolism. Intense interest is focused on PPAR‐γ and its ligands owing to its putative role in adipocyte differentiation. Little is known, however, about the functions of PPAR‐γ in the immune system, especially in T lymphocytes. We demonstrate that both naive and activated ovalbumin‐specific T cells from DO11.10‐transgenic mice express PPAR‐γ mRNA and protein. In order to determine the function of PPAR‐γ, T cells were stimulated withphorbol 12‐myristate 13‐acetate and ionomycin or antigen and antigen‐presenting cells. Simultaneous exposure to PPAR‐γ ligands (e. g. 15‐deoxy‐Δ12, 14‐prostaglandin J2, troglitazone) showed drastic inhibition of proliferation and significant decreases in cell viability. The decrease in cell viability was due to apoptosis of the T lymphocytes, and occurred only when cells were treated with PPAR‐γ, and not PPAR‐α agonists, revealing specificity of this response for PPAR‐γ. These observations suggest that PPAR‐γ agonists play an important role in regulating T cell‐mediated immune responses by inducing apoptosis. T cell death via PPAR‐γ ligation may act as a potent anti‐inflammatory signal in the immune system, and ligands could possibly be used to control disorders in which excessive inflammation occurs.

15-Deoxy-Δ12,1412,14-PGJ2 Induces IL-8 Production in Human T Cells by a Mitogen-Activated Protein Kinase Pathway

Mast cells, platelets, and some macrophages are abundant sources of PGD2 and its active metabolite 15-deoxy-Δ12,14-PGJ2 (15-d-PGJ2). The lipid mediator 15-d-PGJ2 regulates numerous processes, including adipogenesis, apoptosis, and inflammation. The 15-d-PGJ2 has been shown to both inhibit as well as induce the production of inflammatory mediators such as TNF-α, IL-1β, and cyclooxygenase, mostly occurring via a nuclear receptor called peroxisome proliferator-activated receptor-γ (PPAR-γ). Data concerning the effects of 15-d-PGJ2 on human T cells and immune regulation are sparse. IL-8, a cytokine with both chemotactic and angiogenic effects, is produced by T lymphocytes following activation. Whether 15-d-PGJ2 can regulate the production of IL-8 in T cells in unknown. Interestingly, 15-d-PGJ2 treatment of unstimulated T cells induces cell death. In contrast, in activated human T lymphocytes, 15-d-PGJ2 does not kill them, but induces the synthesis of IL-8. In this study, we report that 15-d-PGJ2 induced a significant increase in both IL-8 mRNA and protein from activated human T lymphocytes. The induction of IL-8 by 15-d-PGJ2 did not occur through the nuclear receptor PPAR-γ, as synthetic PPAR-γ agonists did not mimic the IL-8-inducing effects of 15-d-PGJ2. The mechanism of IL-8 induction was through a mitogen-activated protein kinase and NF-κB pathway, as inhibitors of both systems abrogated IL-8 protein induction. Therefore, 15-d-PGJ2 can act as a potent proinflammatory mediator in activated T cells by inducing the production of IL-8. These findings show the complexity with which 15-d-PGJ2 regulates T cells by possessing both pro- and anti-inflammatory properties depending on the activation state of the cell. The implications of this research also include that caution is warranted in assigning a solely anti-inflammatory role for 15-d-PGJ2.

Prostaglandin D2, its metabolite 15-d-PGJ2, and peroxisome proliferator activated receptor-γ agonists induce apoptosis in transformed, but not normal, human T lineage cells

Prostaglandin D2 (PGD2) is abundantly produced by mast cells, platelets, and alveolar macrophages and has been proposed as a key immunoregulatory lipid mediator. 15‐Deoxy‐Δ12,14‐PGJ2 (15‐d‐PGJ2), a key PGD2 metabolite, is under intense study as a potential anti‐inflammatory mediator. Little is known about PGD2 or the role of 15‐d‐PGJ2, if any, in regulating the activities of human T lineage cells. In this report we demonstrate that both PGD2 and 15‐d‐PGJ2 have potent antiproliferative effects, and in fact kill human T lymphocyte lines derived from malignant cells by an apoptotic mechanism. Interestingly, normal human T cells were not similarly affected. Although the T lymphocyte lines express mRNA for the PGD2 receptor (DP‐R), a potent DP receptor agonist, BW245C, did not inhibit the proliferation or viability of the cells, suggesting an alternative mechanism of action. PGD2 and 15‐d‐PGJ2 can bind to the peroxisome proliferator activated receptor‐γ (PPAR‐γ) which is implicated in lipid metabolism and apoptosis. Exposure to synthetic PPAR‐γ ligands (e.g. ciglitazone, troglitazone) mimicked the inhibitory responses of PGD2 and 15‐d‐PGJ2, and induced apoptosis in the transformed T cells consistent with a PPAR‐γ‐dependent mechanism. These observations suggest that PPAR‐γ ligands (which may include PGD2) provide strong apoptotic signals to transformed, but not normal T lymphocytes. Thus, the efficacy of utilizing PPAR‐γ and its ligands as therapeutics for human T cell cancers needs to be further evaluated.

PPAR‐γ‐Mediated Regulation of Normal and Malignant B Lineage Cells

Abstract: Prostaglandins of the E‐series stimulate B lymphocytes by enhancing immunoglobulin‐class switching and antibody production. Little is known about whether or not other prostaglandins affect B lineage cells and perhaps counterbalance the stimulatory effects of PGE2. PGD2 is a major product of cyclooxygenase in bone marrow and in macrophages, suggesting a role for this lipid product in immunological responses. PGD2 undergoes dehydration to the biologically active prostaglandin 15‐deoxy‐Δ12,14‐PGJ2 (15d‐PGJ2) that binds to the nuclear receptor known as peroxisome proliferator‐activated receptor gamma (PPAR‐γ). We found that normal mouse B cells and a Wvariety of B lymphoma cells (e.g., 70Z/3, WEHI‐231, CH12, and J558) express PPAR‐γmRNA and the 67‐kDa PPAR‐γ protein. 15d‐PGJ2 had a dose‐dependent antiproliferative/cytotoxic effect on normal and malignant B cells, as shown by 3H‐thymidine and MTT assays. Only PPAR‐γ agonists (i.e., thiazolidinediones) mimicked the effect of 15d‐PGJ2 on B lineage cells, indicating that the mechanism by which 15d‐PGJ2 negatively affects B lineage cells involves PPAR‐γ. The mechanism whereby PPAR‐γ agonists induced cytotoxicity is via apoptosis, as shown by Annexin V assays. PPAR‐γ agonists may serve as a counterbalance to the stimulating effects of PGE2, which promotes B‐cell differentiation. The use of prostaglandins, such as 15d‐PGJ2, and synthetic PPAR‐γ agonists to induce apoptosis in B lineage cells may lead to the development of therapies for fatal PGE2‐resistant B lymphomas.

induction of apoptosis in mouse t cells upon peroxisome proliferator-activated receptor gamma (ppar-γ) binding

Peroxisome proliferator-activated receptors (PPARs) are a family of ligand-activated nuclear receptor transcription factors. PPARs heterodimerize with the retinoic acid X receptor upon ligand binding. This complex then binds to PPAR responsive elements (PPREs) located in the promoter regions of various genes and acts to regulate their expression (Westin, et al., 1998). Although there are several PPARs, much attention has recently focused on PPAR-γ. Two isoforms of PPAR-γ have been found in mice - PPARγl and PPAR-γ2. PPAR-γ2 is found exclusively in adipose tissue and regulates adipogenesis. PPAR-γl, however, is expressed at low levels in many cells such as smooth muscle cells, macrophages, and epithelial cells (Ma, et al., 1998). Ligand binding of PPAR-γl has many functions including inhibition of migration of vascular smooth muscle cells, inhibition of inducible nitric oxide synthase in murine macrophages, and apoptosis of some colonic tumor cell lines (Kitamura, et al., 1999, Spiegelman, et al., 1997, Vedin, et al., 1996). Metabolites of prostaglandin D2 (PGD2) have been suggested to be natural ligands for PPAR-γ. For example, 15-deoxy-Δ12,14-PGJ2(herein referred to as 15-d-PGJ2) as well as other prostaglandins of the J series can bind and activate PPARγin vitro.Synthetic ligands for PPAR-yhave also been identified, and include the thiazoldinediones (i.e. ciglitazone, troglitazone) (Schoonjans, et al., 1997, Willson, et al., 1996). These compounds are anti-hyperglycemic drugs currently used to treat non-insulin dependent diabetes.

Prostaglandin E2 as a modulator of lymphocyte mediated inflammatory and humoral responses.

Prostaglandins are a family of structurally related small lipid molecules that can regulate cellular growth, differentiation, and homeostasis. Prostaglandins are primarily derived from arachidonic acid, which is released from the membrane by phospholipases in response to a variety of extrinsic stimuli. Constitutive and inducible forms of cyclooxygenase in conceit with isomerses convert arachidonic acid into different prostaglandins including prostaglandin E2 (PGE2). PGE2 was previously known as an immunosuppressive prostaglandin as it inhibits T cell production of IL-2 and B lymphocyte production of IgM. Recently a new concept emerged that PGE2 is a critical modulator of B and T cell function and is not necessarily immunosuppressive. The focus of this paper is to present new developments that indicate PGE2 functions by promoting antibody driven responses at the expense of inflammatory responses.

Peroxisome Proliferator‐Activated Receptor Gamma (PPAR‐γ) Activation in Naive Mouse T Cells Induces Cell Death

Peroxisome proliferator–activated receptor gamma (PPAR-γ) is a nuclear hormone receptor found mainly in adipocytes, but has also recently been found in fibroblasts, myocytes, macrophages, and epithelial cells.1 In mice, there are two isoforms of PPAR-γ—PPAR-γ1 and PPAR-γ2. While PPAR-γ1 is found at low levels in many cell types, the PPAR-γ2 isoform seems restricted to adipocytes. The functional significance of the two isoforms has not yet been determined.2 Synthetic ligands for PPAR-γ include the antidiabetic drug, troglitazone, as well as other thiazolidinediones and LY,171883. Natural ligands include linoleic acid and 15deoxy∆PGJ2 (a metabolite of prostaglandin D2). Upon ligand binding, PPARs heterodimerize with the retinoic X receptor. This complex regulates gene expression by acting as a transcription factor, binding to PPAR-responsive elements (PPREs) in promoter regions. Binding of the receptors by ligand can induce a variety of responses including fat cell differentiation, regulation of colonic epithelial cell differentiation, and TNF-α stimulation in macrophages.5–7 To date, there is very little information concerning the effects of PPAR-γ activation on the immune system. This topic is extremely important and timely due to the recent use of synthetic PPAR-γ agonists (i.e., the thiazolidinediones) in the treatment of non-insulin-dependent diabetes. Therefore, we have studied the expression and function of this receptor in T lymphocyte development. Interestingly, naive mouse T cells isolated from DO11.10 T cell receptor transgenic mice express mRNA for the PPAR-γ1 isoform, as well as PPAR-γ protein (FIG. 1). Also, when naive mouse T cells are cultured with PPAR-γ (but not PPAR-α) agonists in the presence of either antigen and antigen-presenting cells or PMA and ionomycin, the proliferation of the cells is inhibited in a dose-dependent fashion. The inhibition of proliferation is statistically significant (p < 0.05) at concentrations greater than 3 μM (FIG. 2). To determine if the observed decrease in proliferation was due to a decrease in cell viability, MTT assays were performed. FIGURE 2 shows that PPAR-γ agonists do, in fact, induce cell death in mouse T lymphocytes. Significant inhibition of viability was observed at concentrations ranging from 3 to 100 μM. The PPAR-α agonist WY,14643 did not induce cell death, suggesting that the response is specific for PPAR-γ.

Prostaglandin Receptors of the EP2 and EP4 Subtypes Regulate B Lymphocyte Activation and Differentiation to IGE-Secreting Cells

Immunoglobulin E (IgE) and T-helper type 2 (Th2) cytokines initiate physiologic cascades which cause allergic disease and asthma. Understanding how these cascades are triggered is critical for comprehending disease pathogenesis and for developing effective therapies. Novel therapies are needed to curb the increasing global incidence of allergy and asthma. Numerous mechanisms regulate IgE and Th2 cytokine production, however, an emerging concept is that E-series prostaglandins (PGEs), shift the immune response towards allergy by promoting production of IgE and a Th2 profile of cytokines (1). PGEs are lipid molecules which regulate diverse processes throughout the body. Prostaglandins are produced in most tissues (including lymphoid) by “constitutive” cyclooxygenases (COX-1), and in response to inflammatory stimuli, production is increased by expression of “inducible” cyclooxygenases (COX-2), (2). PGEs bind receptors (EP receptors) expressed by hematopoietic cells and promote Th2 and IgE responses at multiple levels. Firstly, PGEs induce development of Th2 cells via modulating cytokine production by antigen presenting cells. Secondly, they inhibit Th1 and promote Th2 profiles of cytokine production from mature differentiated T cells. Lastly, PGEs directly target B lymphocytes and enhance cytokine-directed immunoglobulin isotype class switching.