Carolyn J. Kelly

The peroxisome proliferator-activated receptor-γ is a negative regulator of macrophage activation

The peroxisome proliferator-activated receptor-γ (PPAR-γ) is a member of the nuclear receptor superfamily of ligand-dependent transcription factors that is predominantly expressed in adipose tissue, adrenal gland and spleen. PPAR-γ has been demonstrated to regulate adipocyte differentiation and glucose homeostasis in response to several structurally distinct compounds, including thiazolidinediones and fibrates. Naturally occurring compounds such as fatty acids and the prostaglandin D2 metabolite 15-deoxy-Δ12,14prostaglandin J2 (15d-PGJ2) bind to PPAR-γ and stimulate transcription of target genes. Prostaglandin D2metabolites have not yet been identified in adipose tissue, butaremajor products of arachidonic-acid metabolism in macrophages, raising the possibility that they might serve as endogenous PPAR-γ ligands in this cell type. Here we show that PPAR-γ is markedly upregulated in activated macrophages and inhibits the expression of the inducible nitric oxide synthase, gelatinase B and scavenger receptor A genes in response to 15d-PGJ2 and synthetic PPAR-γ ligands. PPAR-γ inhibits gene expression in part by antagonizing the activities of the transcription factors AP-1, STAT and NF-κB. These observations suggest that PPAR-γ and locally produced prostaglandin D2 metabolites are involved in the regulation of inflammatory responses, and raise the possibility that synthetic PPAR-γ ligands may be of therapeutic value in human diseases such as atherosclerosis and rheumatoid arthritis in which activated macrophages exert pathogenic effects.

Interleukin-4-dependent production of PPAR-γ ligands in macrophages by 12/15-lipoxygenase

The peroxisome proliferator-activated receptor-γ (PPAR-γ) is a ligand-dependent nuclear receptor that has been implicated in the modulation of critical aspects of development and homeostasis, including adipocyte differentiation, glucose metabolism, and macrophage development and function. PPAR-γ is activated by a range of synthetic and naturally occurring substances, including antidiabetic thiazolidinediones,, polyunsaturated fatty acids, 15-deoxy-Δ12,14prostaglandin J2 (refs 8, 9) and components of oxidized low-density lipoprotein, such as 13-hydroxyoctadecadienoic acid (13-HODE) and 15-hydroxyeicosatetraenoic acid (15-HETE). However, the identities of endogenous ligands for PPAR-γ and their means of production in vivo have not been established. In monocytes and macrophages, 13-HODE and 15-HETE can be generated from linoleic and arachidonic acids, respectively, by a 12/15-lipoxygenase that is upregulated by the TH2-derived cytokine interleukin-4 (ref. 11). Here we show that interleukin-4 also induces the expression of PPAR-γ and provide evidence that the coordinate induction of PPAR-γ and 12/15-lipoxygenase mediates interleukin-4-dependent transcription of the CD36 gene in macrophages. These findings reveal a physiological role of 12/15-lipoxygenase in the generation of endogenous ligands for PPAR-γ, and suggest a paradigm for the regulation of nuclear receptor function by cytokines.

Regulation of cytokine expression by ligands of peroxisome proliferator activated receptors

Peroxisome proliferator activated receptors (PPARs) are ligand-activated transcription factors with diverse actions including adipocyte differentiation and lipid metabolism. Recent studies have revealed anti-inflammatory activities, but the majority of these studies have been performed in monocyte/macrophages. In these studies, we investigate the effects of PPAR ligands in murine mitogen-activated splenocytes. Ciglitazone, a PPARγ ligand, consistently decreased IFN-γ and IL-2 production by mitogen-activated splenocytes and had modest effects on splenocyte proliferation. The effects of WY14,643, a representative of the fibrate class of PPARα ligands, on splenocyte proliferation and IL-2 levels are less marked than those observed with the PPARγ ligand. In addition, treatment with WY14,643 and other fibrates led to marked increases in supernatant concentrations of IL-4. However, treatment with a potent and specific PPARα ligand (GW7,647) did not augment IL-4. Also, WY14,643 induced IL-4 expression in splenocytes from PPARα knockout mice, suggesting that the fibrate effect on IL-4 was largely through a PPARα-independent mechanism. This increase in IL-4 was associated with and causatively related to augmented expression of CD23 by CD45R/B220+ cells. We also demonstrate that PPARγ gene expression is up-regulated in T cells by mitogen activation, that it is positively regulated by IL-4 and WY14,643, and that it is blocked by anti-IL-4. Finally, we demonstrate that WY14,643 can modestly augment IL-4 promoter activity in a PPARα-independent manner. In concert, these findings support the roles of PPAR ligands in modulating inflammatory responses involving lymphocytes but also establish potent effects of the fibrate class of PPARα ligands on IL-4 expression that are receptor independent.

Distinct Domains of CD98hc Regulate Integrins and Amino Acid Transport

CD98 is a cell surface heterodimer formed by the covalent linkage of CD98 heavy chain (CD98hc) with several different light chains to form amino acid transporters. CD98hc also binds specifically to the integrin β1A cytoplasmic domain and regulates integrin function. In this study, we examined the relationship between the ability of CD98hc to stimulate amino acid transport and to affect integrin function. By constructing chimeras with CD98hc and a type II transmembrane protein (CD69), we found that the cytoplasmic and transmembrane domains of CD98hc are required for its effects on integrin function, while the extracellular domain is required for stimulation of isoleucine transport. Consequently, the capacity to promote amino acid transport is not required for CD98hcs effect on integrin function. Furthermore, a mutant of CD98hc that lacks its integrin binding site can still promote increased isoleucine transport. Thus, these two functions of CD98hc are separable and require distinct domains of the protein.

WY14,643, a PPARα Ligand, Has Profound Effects on Immune Responses In Vivo

Peroxisome proliferator-activated receptors (PPARs) are ligand-activated transcription factors with diverse actions. PPARα and PPARγ are expressed in different lymphocyte subpopulations. Recently, we have observed that PPARα ligands elicit augmented IL-4 expression in cultures of mitogen-activated splenocytes. The following studies were undertaken to characterize the in vivo effects of WY14,643, a PPARα ligand. Our studies demonstrate that oral administration of WY14,643 markedly reduces splenocyte number in immunized and nonimmunized C57BL/6 mice. Mice fed WY14,643 display impaired IgG responses to myelin oligodendrocyte glycoprotein peptide 35–55 (pMOG35–55), following immunization with pMOG35–55/CFA. Following in vitro restimulation with pMOG35–55, splenocytes harvested from WY14,643-fed mice demonstrate impaired production of IFN-γ, IL-6, and TNF-α despite similar proliferative responses. We also demonstrate higher expression of PPARα in B than T cells. Finally, to obtain an understanding of the cause of splenocyte depletion with fibrate therapy, we studied the effect of WY14,643 on apoptosis of activated splenocytes. WY14,643 in vitro induces apoptosis in lymphocytes and this effect appears to occur in a PPARα-independent manner. Thus WY14,643, a fibrate, is a profound immunosuppressive agent.

Suppression of inducible nitric oxide generation by agmatine aldehyde: Beneficial effects in sepsis

The induction of inducible nitric oxide synthase (iNOS) serves an important immuno‐protective function in inflammatory states, but ungoverned nitric oxide (NO) generation can contribute to a number of pathologic consequences. Delineation of the mechanisms that can downregulate iNOS‐generated NO in inflammation could have therapeutic relevance. Here we show that agmatine, a metabolite of arginine, inhibits iNOS mediated nitric oxide generation in cytokine stimulated cell culture preparations. This effect was not cell type specific. Increased diamine oxidase (DAO) and decreased aldehyde dehydrogenase (AldDH) activities are also representative of inflammatory settings. Increasing the conversion of agmatine to an aldehyde form by addition of purified DAO or suppression of aldehyde breakdown by inhibition of AldDH activity increases the inhibitory effects of agmatine in an additive fashion. Inhibitors of DAO, but not monoamine oxidase (MAO), decreased the inhibitory effects of agmatine, as did the addition of AldDH or reacting aldehydes with phenylhydrazine. We examined rats given lipopolysaccharide (LPS) to evaluate the potential effects of agmatine in vivo. Endotoxic rats administered agmatine prevented the decreases in blood pressure and renal function normally associated with sepsis. Agmatine treatment also increased the survival of LPS treated mice. Our data demonstrate the capacity of agmatine aldehyde to suppress iNOS mediated NO generation, and indicate a protective function of agmatine in a model of endotoxic shock. How agmatine may aid in coordinating the early NO phase and the later repair phase responses in models of inflammation is discussed.

Repression of IFN-γ Expression by Peroxisome Proliferator-Activated Receptor γ

Peroxisome proliferator-activated receptors (PPARs) are ligand-activated transcription factors expressed in a wide variety of cells. Our studies and others have demonstrated that both human and murine T cells express PPARγ and that expression can be augmented over time in mitogen-activated splenocytes. PPARγ ligands decrease proliferation and IL-2 production, and induce apoptosis in both B and T cells. PPARγ ligands have also been shown to be anti-inflammatory in multiple models of inflammatory disease. In the following study, we demonstrate for the first time that PPARγ is expressed in both murine CD4 and CD8 cells and that PPARγ ligands directly decrease IFN-γ expression by murine and transformed T cell lines. Unexpectedly, GW9662, a PPARγ antagonist, increases lymphocyte IFN-γ expression. Transient transfection studies reveal that PPARγ ligands, in a PPARγ-dependent manner, potently repress an IFN-γ promoter construct. Repression localizes to the distal conserved sequence of the IFN-γ promoter. Our studies also demonstrate that PPARγ acts on the IFN-γ promoter by interfering with c-Jun activation. These studies suggest that many of the observed anti-inflammatory effects of PPARγ ligands may be related to direct inhibition of IFN-γ by PPARγ.

T Cells and Minimal Change Disease

It is one of the ironies of medical practice that as physicians we can competently and confidently treat diseases of whose pathogenesis we remain woefully ignorant. Such is the case with minimal change disease, the most common diagnosis associated with the nephrotic syndrome in children (MCNS). The

Regulation of intracellular polyamine biosynthesis and transport by NO and cytokines TNF-α and IFN-γ

Nitric oxide (NO) has been described to exert cytostatic effects on cellular proliferation; however the mechanisms responsible for these effects have yet to be fully resolved. Polyamines, conversely, are required components of cellular proliferation. In experimental models of inflammation, a relationship between these two pathways has been suggested by the temporal regulation of a common precursor, arginine. This study was undertaken to determine the effects NO and the NO synthase (NOS)-inducing cytokines, tumor necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma), exert on polyamine regulation. The transformed kidney proximal tubule cell line, MCT, maintains high constitutive levels of the first polyamine biosynthetic enzyme, ornithine decarboxylase (ODC). NO donors markedly suppressed ODC activity in MCT and all other cell lines examined. TNF-alpha and IFN-gamma induction of NO generation resulted in suppressed ODC activity, an effect prevented by the inducible NOS inhibitor L-N6-(1-iminoethyl)lysine (L-NIL). Dithiothreitol reversal of NO-mediated ODC suppression supports nitrosylation as the mechanism of inactivation. We also evaluated polyamine uptake, inasmuch as inhibition of ODC can result in a compensatory induction of polyamine transporters. Administration of NO donors, or TNF-alpha and IFN-gamma, suppressed [3H]putrescine uptake, thereby preventing transport-mediated reestablishment of intracellular polyamine levels. This study demonstrates the capacity of NO and inflammatory cytokines to regulate both polyamine biosynthesis and transport.

T Cell Function in Chronic Renal Failure and Dialysis

This article briefly summarizes the literature regarding possible defects in cell-mediated immunity in the setting of chronic renal failure. It is difficult to precisely determine the proximate cause or level of such defects from most studies. Confounding variables include reports on mixed patient populations (predialytic chronic renal failure, hemodialysis patients, and peritoneal dialysis patients) and studies before and after the introduction of erythropoietin for end-stage renal disease patients. While it seems clear that lymphopenia, suboptimal responses to mitogens, abnormal cytokine gene expression, and abnormal IL-2R expression are seen in a number of dialysis patients, the role of uremia versus dialysis in producing these abnormalities is unclear. In addition, it is difficult to determine whether T cell abnormalities are primary or secondary to impaired function of other interacting immune cells, such as macrophages. Clinical implications of defects in cell-mediated immunity are additionally discussed.