The cyclopentone prostaglandin 15-deoxy-Δ12,14 prostaglandin J2 represses nitric oxide, TNF-α, and IL-12 production by microglial cells

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Abstract

Prostaglandins are generally considered pro-inflammatory molecules that contribute to the pathology associated with a variety of immune-mediated diseases including multiple sclerosis. However, recently it has been demonstrated that specific cyclopentone prostaglandin metabolites including 15-deoxy-Δ12,14 prostaglandin J2 (15d-PGJ2) are capable of repressing the production of pro-inflammatory molecules by cells of the monocyte/macrophage lineage. Activated microglia produce nitric oxide (NO) and TNF-α, molecules which can be toxic to cells including oligodendrocytes, thus potentially contributing to the pathology associated with multiple sclerosis. The current study demonstrates that 15d-PGJ2 inhibits lipopolysachharide (LPS) induction of NO and TNF-α production by rat primary microglia and mouse N9 microglial cells. 15d-PGJ2 also inhibits NO production by microglial cells activated in response to IFN-γ and TNF-α, cytokines believed to be important modulators of multiple sclerosis. IL-12 plays a critical role in stimulating the production of Th1 cells, which are believed to contribute to the pathology associated with multiple sclerosis. The current studies demonstrate that 15d-PGJ2 represses the production of IL-12 by microglial cells. Collectively, these studies demonstrate that the prostaglandin metabolite 15d-PGJ2 represses microglial production of potentially cytotoxic molecules, as well as molecules capable of altering T-cell phenotype. These in vitro studies suggest the possibility that the prostaglandin 15d-PGJ2 may modulate inflammatory diseases including multiple sclerosis.

Introduction

Prostaglandins have been implicated in a variety of inflammatory disorders, including those associated with the central nervous system Lipsky, 1999, O'Banion, 1999. Paradoxically, it has been demonstrated that specific prostaglandin metabolites including 15-deoxy-Δ12,14 prostaglandin J2 (15d-PGJ2) may inhibit the production of pro-inflammatory molecules by macrophages Jiang et al., 1998, Ricote et al., 1998. A potential therapeutic role for 15d-PGJ2 in inflammatory disorders is suggested by in vivo studies which demonstrated that this prostaglandin ameliorates inflammatory bowel disease in mice (Su et al., 1999). In addition, elevated serum 15d-PGJ2 levels correlate temporally with the resolution of inflammation in a carrageenin-induced pleurisy model in rats (Gilroy et al., 1999). 15d-PGJ2 inhibits the production of pro-inflammatory molecules from monocyte/macrophages Jiang et al., 1998, Ricote et al., 1998, suggesting a potential mechanisms of action of this prostaglandin in mediating inflammation.

Microglia are resident CNS cells that may serve as antigen presenting cells, and can be phagocytic. A wide variety of neuroimmunological and neurodegenerative diseases including multiple sclerosis are characterized by the presence of activated microglia Sriram and Rodriguez, 1997, Gonzalez-Scarano and Baltuch, 1999, McGeer and McGeer, 1999. Upon activation, microglia proliferate, change morphology, and exhibit increased production of a variety of cytokines including TNF-α, as well as increased synthesis of MHC class II and NO (Benveniste, 1997). Although molecules including NO and TNF-α are toxic to pathogens, these agents can also be toxic to CNS cells including myelin-producing oligodendrocytes (reviewed in Benveniste, 1997), which are compromised in the course of multiple sclerosis (reviewed in Raine, 1997). These molecules also may be toxic to neurons, and thus may contribute to axonal degeneration characteristic of multiple sclerosis (Trapp et al., 1998). Interestingly, NO and TNF-α have alternatively been reported to protect CNS cells (reviewed in Mattson et al., 1997, Munoz-Fernandez and Fresno, 1998). However, the basis for these disparate experimental results have not been elucidated. Experimental autoimmune encephalomyelitis (EAE), like multiple sclerosis, is an autoimmune disorder characterized by central nervous system (CNS) demyelination, and remittent paralysis (Martin et al., 1992). The levels of NO and TNF-α are elevated in EAE and multiple sclerosis, and inhibition of the synthesis of these molecules blocks development of EAE Ruddle et al., 1990, Selmaj et al., 1991, Zhou et al., 1996, Ding et al., 1998. Thus, theoretically, agents which inhibit NO and TNF-α synthesis may be effective in the treatment of multiple sclerosis.

EAE can be induced in susceptible rodent strains by adoptive transfer of CD4+ T-cells specific for myelin-antigens into naive, syngeneic recipients (Pettinelli and McFarlin, 1981). CD4+ T-cells exhibit two distinct patterns of cytokine production and are designated T helper 1 (Th1) and T helper 2 (Th2) cells, both of which are believed to be derived from a common precursor. In EAE, Th1 cells are believed to be encephalitogenic, while Th2 cells may be protective Ando et al., 1989, Powell et al., 1990, Olsson, 1995, Liblau et al., 1995. Numerous studies have indicated that IL-12 is critical in Th1 cell differentiation (reviewed in Adorini, 1999). IL-12 is a 75-kDa heterodimer consisting of two covalently linked p35 and p40 proteins, encoded by distinct genes Gubler et al., 1991, Wolf et al., 1991. This cytokine is principally produced in monocytes and dendritic cells (Cassatella et al., 1995). In the brain, IL-12 is produced in microglia (Lodge and Sriram, 1996). IL-12 is an important mediator of EAE and multiple sclerosis. Administration of the cytokine increases the severity of EAE (Samtambrogio et al., 1995), and IL-12 antibodies inhibit the development of EAE (Leonard et al., 1995). IL-12-deficient mice also do not develop EAE (Segal et al., 1998). The potential role of IL-12 in the initiation of multiple sclerosis is suggested by the presence of IL-12 p40 RNA early in disease (Windhagen et al., 1995), and the observation that IL-12 levels appear to correlate with multiple sclerosis disease progression (Balashov et al., 1997). Thus, agents that repress IL-12 production in the brain could inhibit the development of multiple sclerosis, through effects on T-cell phenotype.

The current study demonstrates that the prostaglandin 15d-PGJ2 inhibits microglial production of NO and TNF-α, molecules that are potentially toxic to cells including myelin producing oligodendrocytes. In addition, this prostaglandin inhibits microglial production of IL-12, a cytokine critical for the differentiation of Th1 cells, which are believed to contribute to the development of multiple sclerosis. Significantly, 15d-PGJ2 represses the production of these molecules by microglia stimulated with IFN-γ and TNF-α, molecules that contribute to the pathology associated with multiple sclerosis. These in vitro studies suggest the possibility that 15d-PGJ2 may modulate inflammatory diseases including multiple sclerosis.

Section snippets

Cell culture

Pure rat microglia cultures were obtained through a modification of the McCarthy and deVellis (1980) protocol. Briefly, cerebral cortices from 1–2-day-old rats were excised, meninges removed, and cortices minced into small pieces. Cells were separated by trypsinization followed by trituration of cortical tissue. Cells were plated into tissue culture flasks and allowed to grow to confluence (∼1 week) in DMEM media containing 10% FBS and 1.4 mM glutamine. Flasks were shaken overnight (200 rpm at

Effect of prostaglandin 15d-PGJ2 upon NO production by microglial cells

Activated microglia produce NO that may be toxic to oligodendrocytes, cells which are compromised in multiple sclerosis. The prostaglandin 15d-PGJ2 was previously demonstrated to inhibit NO production by monocyte/macrophages Jiang et al., 1998, Ricote et al., 1998, and more recently by microglial cells (Petrova et al., 1999). In the present studies, we demonstrated that LPS increased the production of nitrite in N9 microglial cells (Fig. 1). 15d-PGJ2 significantly repressed LPS-induced nitrite

Discussion

The role of prostaglandins as proinflammatory molecules is well documented. Paradoxically, cyclopentone prostaglandins have been demonstrated to repress the activation of cells of the monocyte/macrophage lineage. For example, the cyclopentone prostaglandin 15d-PGJ2 has been shown to repress the production of NO, TNF-α, IL-1, IL-6, matrix metalloproteinase-9, and scavenger receptor A, by these cells Jiang et al., 1998, Ricote et al., 1998. This prostaglandin also inhibits NO production by

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