Novel role for peroxisome proliferator-activated receptor alpha in T cell physioloogy

Lipids and lipid metabolism have a number of regulatory effects on immune and inflammatory responses. The nuclear hormone receptor PPAR?, a transcription factor that can be activated by various lipid species, is important in regulating various aspects of inflammation. This dissertation demonstrates that PPAR? also plays an active role in regulating host adaptive immune responses. PPAR? is expressed by a number of cells in the immune system, including T and B lymphocytes. The PPAR? that is expressed in lymphocytes is transactivation and transrepression competent, both processes being important for its regulation of lymphocyte function. Expression of PPAR? in CD4+ T cells regulates the expression and production of IL-2 and IFN-gamma following activation of these cells. The regulation of cytokine production by PPAR? is mediated, in part, through its ability to regulate the expression of T-bet. PPAR? regulates T-bet through a novel IFN-? independent signaling mechanism that does not require DNA binding or ligand activation of the nuclear hormone receptor. PPAR? was subsequently found to suppress T-bet expression in activated CD4+ T cells through its ability to inhibit the activation of p38 MAP kinase. Additional studies in this dissertation demonstrate that the temporal regulation of activated CD4+ T cells by PPAR? is important for the development of normal humoral immune responses. PPAR?-/- mice immunized with foreign protein antigens produced markedly lower levels of antigen-specific antibodies when compared to identically immunized PPAR?;+/+ mice. Polyclonal activation of B cells <italic>in vitro

A NOVEL ROLE FOR PEROXISOME PROLIFERATOR-ACTIVATED RECEPTOR ALPHA IN T CELL PHYSIOLOGY by Dallas C. Jones A dissertation subnlitted to the faculty of The University of Utah in partial fulfillment of the requirements for the degree of Doctor of Philosophy 111 Experinlental Patho logy Department of Pathology The University of Utah August 2003 Copyright © Dallas C. Jones 2003 All Rights Reserved THE UNIVERSITY OF UTAH GRADUATE SCHOOL SlTPERVISORY COMMITTEE APPROVAL of a dissertation submitted by Dallas C. Jones This dissertation has been read by each member of the following supervisory committee and by majority vote has been found to be satisfactory. S -I cl3 Gerald J. Spa~grude THE UNIVERSITY OF UTAH GRADUATE SCHOOL FINAL READING APPROVAL To the Graduate Council of the University of Utah: I have read the dissertation in its final forn1 and have found that (1) its format, citations, and bibliographic style are consistent and acceptable; (2) its illustrative materials including figures, tables, and charts are in place; and (3) the final manuscript is satisfactory to the supervisory conlmittee and is ready for submission to The Graduate School. Date ' .~tnmg~~ Rayl ond A. Dayncs Chair: Supervisory Committee Approved for the Major Department CaFrR. Kjeldsberg Chair Approved for the Graduate Council ABSTRACT Lipids and lipid metabolism have a number of regulatory effects on imnlune and int1ammatory responses. The nuclear hormone receptor PPARa, a transcription factor that can be activated by various lipid species, is important in regulating various aspects of inf1anlmation. This dissertation demonstrates that PPARa also plays an active role in regulating host adaptive immune responses. PP ARa is expressed by a nunlber of cells in the immune system, including T and B lymphocytes. The PP ARa that is expressed in lymphocytes is transactivation and transrepression competent, both processes being illlportant for its regulation of lynlphocyte function. Expression of PPARa in CD4+ T cells regulates the expression and production of IL-2 and IFN-y following activation of these cells. The regulation of cytokine production by PPARa is mediated, in part, through its ability to regulate the expression of T-bet. PPARa regulates T -bet through a novel IFN-y independent signaling nlechanism that does not require DNA binding or ligand activation of the nucJear hornlone receptor. PPARa was subsequently found to suppress T-bet expression in activated CD4+ T cells through its ability to inhibit the activation of p38 MAP kinase. Additional studies in this dissertation demonstrate that the temporal regulation of activated CD4+ T cells by PPARa is inlportant for the development of normal humoral immune responses. PPARa-/- mice immunized with foreign protein antigens produced markedly lower levels of antigen-specific antibodies when compared to identically immunized PPARa +/+ mice. Polyclonal activation of B cells in vitro and inlmunization of PP ARa-/- and PI> ARa+/+ mice with T-cell independent antigens demonstrated that antibody production by B lymphocytes is not directly atTected by the expression of PPARu in these cells. Adoptive transfer of PPARu+/+ B cel1s and PPARu+/+ C04+ T cells into Rag2-/- mice produced good titers of antigen-specific antibodies fbllowing immunization with a T-cell dependent antigen. Conversely, identically immunized Rag2-/- l11ice reconstituted with PPARu+/+ B cells and PPARu-/- C04+ T cells were severely compromised in their ability to generate antigen-specific antibodies. From the findings presented in this dissertation, it is proposed that PPARu expression in C04+ T cells is important in the generation of hUll lora I imnlune responses to foreign protein antigens. v To AnlY and Chandler TABLE OF CONTENTS AI3STR.I\C'T ................................................................................................................... iv LJS1' ()f·' I·'J(jlJRf:S ........................................................................................................ ix A('KN()WIJF~D(JEMEN·rS ............................................................................................ xi Chapter 1. JNrrR()DLJC.'l'IC)N .................................................................................................... I PeroxiSOlne Proliferator-Activated Receptors ....................................................... 2 PP AR: Structure and Function ............................................................................. 3 Transcriptional Repression by PPARs ................................................................... 7 Inilarnnlation Control by the PPARs ..................................................................... 9 PPARs and Innate Immunity ............................................................................... 12 PPARs and Adaptive Immunity .......................................................................... 14 Overview of Work in this Dissertation ................................................................ 18 I"{eferences ......................................................................................................... 21 2. NUCLEAR RECEPTOR PEROXISOME PROLIFERATOR-ACTIV ATED RECEPTORa, IS EXPRESSED IN RESTING MURINE L YMPHOYCTES ......... 28 Experilnental Procedures .................................................................................... 30 Results ............................................................................................................... 3 1 Discussi()n .......................................................................................................... 34 References ......................................................................................................... 36 3. PPARu NEGATIVELY REGULATES T-BET TRANSCRIPTION THOUGH SUPPRESSION OF P38 MAP KINASE ACTIVATION ........................................ 37 Abstract ............................................................................................................. 38 Introduction ....................................................................................................... 38 Materia1 and Methods ........................................................................................ 40 Results ............................................................................................................... 44 l)iscussitln .......................................................................................................... 59 I{cterences ......................................................................................................... 6() 4. EXPRESSION OF PPARu IN CD4+ T CELLS IS ESSENTIAL FOR THE GENERATION OF HUMORAL IMMUNE RESPONES TO FOREIGN PROTEIN ANTIGENS .................................................................. 71 Abstract ............................................................................................................. 72 Il1troduction ....................................................................................................... 72 Materials and Methods ....................................................................................... 74 Results ............................................................................................................... 77 I)iscussion .......................................................................................................... 88 References ......................................................................................................... 96 5. ROLE FOR REDOX IMBALANCE IN THE MOLECULAR MECHANISMS RESPONSIBLE FOR Irvn"1uNoSENESCENCES ................................................. 99 Abstract ........................................................................................................... 100 Intr()duction ..................................................................................................... 100 Material and Methods ...................................................................................... 108 I{esults ............................................................................................................. 11 ] [)iscussioll ........................................................................................................ 123 References ....................................................................................................... 126 6. I)IS('lJSSIC)N ......................................................................................................... 133 ReferellceS ....................................................................................................... 144 VIII LIST OF FIGURES 2. I Lymphocytes express PPARcx, mRNA and protein ................................................ 31 2.2 Ratio of PPARex and PPARy mRN A in various hematopoietic cells ..................... 31 2.3 PPARa InRNA levels decrease after polyclonal T-cell activation ......................... 32 2.4 Analysis of PPARcx. nlRNA levels in lYIllphocytes isolated frOnl various secondary lymphoid organs ...................................................................... 32 2.5 Dexamethasone induces PP ARa expression in lymphocytes ................................ 32 2.6 Treatment of the murine T-cell line, TK. L with GW9578 fails to induce expression of several PPARa-regulated genes ...................................... 33 2.7 Luciferase assays of TK.l T cells transiently transfected with reporter constructs containing the Aco PPRE sequence ........................................ 33 2.8 GW9578 up-regulates expression ofPPARa regulated genes in T cells pretreated with a HDAC inhibitor ............................................................. 34 2.9 Ligand activation ofPPARa decreases NF-K8's ability to bind DNA and to transactivate gene expression ............................................................ 35 3.1 Dysregulated production of IFN-y and IL-2 in PPARa-/- T cells .......................... 46 3.2 Kinetic induction ofT-bet mRNA and protein is accelerated in 1)1) ARcx.-/- T cells ............................................................................................. 48 3.3 T-bet is expressed and Il:N-y produced in PPARo,-/- T cells activated in the presence 0 f ant i-I FNy ................................................................................. 51 3.4 PPARa mediated inhibition ofT-bet expression does not require PPARa ligand activation ...................................................................................... 54 3.5 Inhibition of the p38 MAP kinase inhibits activation-induced expression ofT-bet in T cells ............................................................................... 57 3.6 Phosphorylation of p38 MAP kinase in activated T cells is su ppressed by I) I) ARct .......................................................................................... 6() 4.1 Depressed response to HBsAg vaccination in PP ARa-/- nlice .............................. 80 4.2 Proliferation and antibody production by WT and PPARa-/- B cells .................... 83 4.3 Anti-pneumococcal polysaccharide antibody levels in immunized PI) ARu-l- and W'l' nlice , ................... , .................................... , .............. , .............. 86 4.4 Adoptive transfer ofT and B lymphocytes into Rag2-1- mice .... , ...... , .. " ... , ..... , .. , .. 89 5.1 T cells isolated from aged animals exhibit dysregulated production of activation-induced IL-2 and IFNy that correlates with decreased basal levels of PPARu expression ........................ , ............................................. 112 5.2 Kinetic induction ofT-protein is accelerated in aged 0011.10 T cells ................ 116 5.3 Dietary supplementation of aged mice with vitamin E increases PPARu levels in T cells and restores control over 'r-bet expression ...................... , ....... , ..................... , .................................... 119 5.4 T cells isolated fronl vitamin E supplemented PPARu animals exhibit dysregulated production of activation-illduced I L-2 and IFNy .................................... , ... " ........ , .............. , ..................... 121 x ACKNOWLEDGEMENTS I thank Dr. Ray Oaynes for providing daily advice and guidance that helped nlove this project t()rward. I also thank members of nly committee for their support and helpful suggestions. I also wish to thank the Hematology Department for financial support during my training. I thank the members of the Daynes Lab, especially Bernadette Manning, who taught me how to survive the "refiner's fire'. I also appreciate the many melnbers of the Pathology Department who provided nUl11erOUS ideas and reagents over the years. I thank the American Society for Biochemistry and Molecular Biology for permission to reprint previously published material in Chapter 2 of this dissertation. lowe my sincerest thanks to nly wife Amy for her unconditional support and f{)r making numerous sacrifices during this journey. I am especially grateful to my son Chandler, who relninded me on a daily basis what is most important in life. Without the love and support of AnlY and Chandler this accomplishnlent would have never been possible. CHAPTER 1 INTRODUCTION P~J:oxisolne Proliferator-Activated Receptors Most species of higher multicellular organisms have evolved complex homeostatic lTIechanis111S that allow cells to sense and respond to a diverse range of endogenous and exogenous signals. One such mechanism that has received considerable experimental attention in recent years involves the biochemical, genetic, and epigenetic events that f()llow activation of the peroxisome proliferator-activated receptors (PPARs). The PPARs are men1bers of the nuclear hornl0ne receptor supert~lll1ily, that transduce a wide variety of signals, including environmental, nutritional, and inflammatory events, into a delined and ordered set of cellular responses at the level of gene transcription (1-2). Presently. three PPAR isofc)rms. a. pIE. and y, have been identified and cloned. Although encoded by distinct genes on dine-rent chromoson1es, each PPAR receptor isoform exhibits a high degree of sequence and structural homology_ How'ever, the PPAR isoforms are unique in their quantitative patterns of tissue distribution, exhibit important differences in their regulatory activities, and n10dulate specific responses subsequent to activation. Various fatty acid species, including unsaturated, saturated. and branched-chain, can hind to and activate PPARs with some degree of isoform specificity. In addition, some of the eicosanoids derived fi'on1 the n1etabolisn1 of arachidonic acid. including leukotrienes (LTs). hydroxyeicosatetraenoic acids (HETEs). and prostaglandins (PGs), can also be ef1e-ctive ligand agonists t()r specific PPAR isoforms. Recent evidence has now uncovered an important role for PPARs in the control of various types of inflamnlatory responses. These functions are largely mediated through the abilities of the PPARa and PPARy isof()rms, using agonist-dependent mechanisms. to transrepress the activities of nUlnerous activated transcription factors, including nuclear 1ilctor KB (NF-KB). signal transducer and activator of transcription (ST AT)s, activator protein-l (AP-I), and the nuclear factor of activated T-cells (NF-A T)s. PPARy and PP ARa are now known to be expressed within macrophages, dendritic cells. as well as in --,. Band T lynlphocytes (1 10). These recent i-indings have led to the working hypothesis that PPARs are actively involved in aspects of immunoregulation, through their ability to regulate energy homeostasis, membrane lipid composition, cell proliferation, sensitivity to apoptosis, and the various transcription i-actors listed above, that are known to be involved 111 Immune processes. The prilllary o~jective of this introduction is to discuss the current understanding of the roles played by the PPARs in the mamnlalian immune system. First, PPARs are briefly introduced, including the diflerent isoi-orms and their known regulatory activities as transcriptional activators in numerous biochemical and physiologic processes. This is f()llowed by a more detailed description of how PPARs regulate gene expression using various ligand-dependent and ligand-independent molecular processes. Finally, a detailed analysis is presented of the established and projected roles for the various PP AR isoforms as regulators of both innate and adaptive immune responses. PPARs: Structure and Function The first PPAR (PPARu) was originally cloned from a mouse liver cDNA library as the nuclear receptor that mediates the effects of a group of endogenous and xenobiotic compounds tenned peroxisollle prolifcrators (1]). This chenlically diverse group of substances was so nanled for their conlnl0n property of increasing both the number and activities of liver peroxisolnes following chronic high dose administration to rodents. Chronic peroxisollle proliferation in the livers of treated rodents is accompanied by significant liver pathology that ultinlately progresses into hepatocellular carcinoma (1 13), indicating that chemical agents with signiticant peroxisome proliferating properties would be undesirable as potential therapeutics. lqentification and structural organization The three structurally-related PPAR isofornls identified in vertebrates (including hUl1lan, mouse, rat, hamster, and Xenopus) were named PPARc.( (N RIC 1), PPAR~/8 4 (NRIC2)~ and PPARy (NRIC3) when originally characterized in )(enopus (14). Phylogenetically, the PPARs are classified as a subfamily 0 f the nuclear hornl0ne receptors, that includes the receptors for vitamin D, thyroid hormone, retinoic acids, ecdysone and some additional orphan receptors (15). All of the receptors in this subgroup share the property of forming heterodimers with another nuclear receptor in the same subgroup, the 9-cis-retinoic acid receptor (RXR) (NR2B). Most biochemical functions ascribed to the PPARs require that the receptor is part of a heterodimeric complex with RXR ( 16). All three of the PPAR isoforms, when complexed to RXR, bind to the same DNA response elements fol1owing activation. These gene promoter associated elements contain direct repeats of the sequence AGGTCA separated by a single or 2 nucleotides (called DR-lor DR-2 response elements). This sequence has been termed the Peroxisome Prolilerator Response Element (PPRE) and has been found in the promoter regions of most PPAR regulated target genes ( 17 -21). Agonist-induced activation of many nlenlbers of the nuclear receptor superfamily is acconlpanied by nlinor structural changes of the receptor, conferring the ligand-bound complex with sonle additional properties. These could include an ability to shed corepressor complexes and associate with appropriate coactivators, binding to DNA, and the gaining of transactivation capabilities. The DNA-binding domain 0 f the PPARs is formed by two zinc-finger-like motifs, that fold to lDrm a globular three-dimensional structure. This domain is highly conserved between PPAR isotorms, consistent with each isoform showing restricted binding to similar PPRE sequences (22). When in nonliganded states in solution, all three isoforms of PPAR can bind various species of transcription corepressors and histone deacetylases in a DNA independent manner (23-25). Unliganded PPARP/8, however. has recently been found to nlaintain corepressor binding function when PPRE-associated as well (26). Agonist activation promotes receptor-DNA association and corepressor dissociation, allowing various coactivators (e.g., ('REB binding protein (CBP)/p300, steroid receptor coactivator (SRC)-l and members of the 5 DRIP/TRAP complex) to interact with conserved LXXLL amino acid motifs residing in the ligand binding domain (LBO) of the activated PPAR. Several of the coactivator complexes possess histone acetyltransferase activity that facilitates remodeling of chronlatin structure (22, 27-30). It is believed that such coactivator complexes bound to an agonist-activated. PPRE-associated PPAR/RXR complex, are able to disrupt nucleosonles and promote transcription-pron10ting changes in chromatin structure near regulatory regions of PP AR target genes. Secondary complexes can then form with proteins sLich as DRJP/TRAP plus the protein complexes associated with basal transcription machinery, so that transcription can be initiated. PPAR, activating ligands Naturally occurring fatty acids and eicosanoids bind and activate all three isoforms of PPARs. The PPARy isoiorn1 is activated by endogenous agonists composed of polyunsaturated fatty acids (e.g., a-linoleic (CI8:3), y-linolenic (CI8:3), arachidonic (C20:4) and eicosapentaenoic acid (C20:5», although these endogenous ligands are considered weak activators of PPARy (31). The PPARa isoform can also be activated by sin1ilar polyunsaturated fatty acids and can additionally be activated by some medium chain saturated and monounsaturated fatty acids (e.g., palnlitic (C 16:0) and oleic (C 18: ] » (32). The con1position of pPARr~/o agonists appears to reside somewhere intermediate, and like PPAR(x. PPAR~/o can interact productively with some saturated, monounsaturated and unsaturated tatty acids (31). Diflerences also exist in the nature of the eicosanoid species having PPAR-activating activities. PPARy can best be stimulated by 9-110DE. 13-110DE. and 15-deoxy-t112 J 4-prostaglandin J~ (l5dPGJ~) (33), although there is now considerable controversy as to whether 15dPGJ:: is actually a biologically relevant PP ARy activator. However. it has recently been reported that 15dPGJ~ is produced in vivo and is also produced in large quantities by macrophages in vitro (34). PPARa can be selectively activated by leukotriene B4 (LTB4) and 8(S) HETE, although 6 tissue levels of this latter agonist are not high enough to classify this lipoxygenase metaholite as a natural ligand. PPAR~/() can be activated hy various species of eicosanoids, including PGA L PGD2, and a biologically stahle synthetic prostacyclin, suggesting that naturally induced prostacyclins rnay represent endogenous PPAR~/() agonists (35). All of the naturally occurring PPAR agonists hind and activate speciiic receptor isof()fms at microll1olar levels, suggesting that the PPARs evolved to he activated naturally by low aflinity ligands. Melgpolic rol~l'iar!dJLansactivatiQD-inducible targetMnes Agonist activation of the PPARs, facilitated by low atlinity binding to natural lipid ligands, stimulates an array of molecular responses designed to maintain lipid homeostasis. PPARo:. functions as a glohal regulator of tatty acid catabolism hy upregulating the transcription of genes that transport intracellular fatty acids into peroxisomes and mitochondria for their f)-oxidation (1). PP ARo: activation also upregulates the expression of a numher of the catabolic enzymes involved in mitochondrial and peroxisomal ~­oxidation . and micro so 111al m-oxidation , as well as in the transcriptional regulation of genes necessary for the maintenance of redox halance during the oxidative catabolisn1 of fatty acids ( I ). PPARy regulates adipogenesis. and is also involved in a diverse array of biochetllical processes, including insulin sensitization and cellular diflerentiation. PPARy activation also promotes the storage of fat by stimulating CD36 expression, increasing adipocyte diflcrentiation. and enhancing the transcription of genes important 101' lipogenesis ( 1). The activation of either PPARo: or PPARy in macrophages promotes the cellular efflux of phospholipids and cholesterol into high density lipoproteins by upregulating liver-x-receptor alpha, an oxysterol-activated nuclear hormone receptor that augn1ents expression of the lipid transporter ABCA-] (36). 7 The PP ARrV8-speci1ic activation increases high-density lipoprotein levels and can beneficially alter serum lipid pro111es (37-38). PPARf)/() activation in macrophages additionally upregulates the ABCA-I transporter (38). Recent evidence indicates that PPARf)/8 can also pr0l11ote ceBular lipid accumulation by increasing expression of genes involved in lipid uptake and repressing key genes involved in lipid nletabolism and e1l1ux (39). Very importantly, PPARf)/8, unlike PPARu and PPARy, associates with PPRE­containing DNA sequences while still complexed to corepressors containing histone deacetylase activity (26). This competitive inhibition for available PPRE sites represents an agonist-independent event, and is able to negatively regulate agonist-inducible transactivation activities by both PPARcx. and PPARy. Additionally, this mechanisnl provides a unique role f()r the ubiquitously expressed PPARf)/8 as a tonic suppressor of all PPAR-induced activities under physiologic conditions where PPARf)/8 is maintained in a nonliganded state. Tran~criQtional Repression byJJP ARs Regulation of gene transcription by nuc1ear hornlone receptors extends beyond the boundaries or their ability to transactivate specific target genes in a ligand-agonist dependent nlanner. It is now appreciated that many menlbers of the nuclear hormone receptor superfamily, once activated by an agonist, can physically interact with other types of transcription factors and int1uence their fllllctional properties. This interaction can result in either an inhibition or enhancement of transcriptional activities by the individual interacting proteins. These protein-protein interactions, as well as their biologic consequences, have been best described mechanistically with ligand-activated glucocorticoid receptors (GR). The vast nlajority of the anti-inf1amlnatory properties that have been ascribed to glucocorticoid ellects. result from the ability of activated (iRs to transreprcss the enhancing activities on inflammatory gene expression by other transcription factors, like AP-L NF-KB, STATs, and NF-AT in a DNA independent 8 manner (1, 2, 40-42). It is now appreciated that many members of the nuclear hOrIl1one receptor superfamily. including the PPARs. have evolved mechanisms to functionally modulate downstream responses following activation and physical association with other transcription factors. M~chanisms oLtransrepression~hLthe PPARs Through a variety of mechanisms, PPARs can suppress the activities of Inany distinct falnilies of transcription factors. In most cases, agonist activation of the PPAR receptor is required f()r efiective transrepression to occur, regardless of which specific controlling n1echanism is being induced. Although not inclusive, there are at least three primary ways in which ligand activated PPAR:RXR con1plexes can negatively regulate the activities of other transcription factors. The tirst involves the sequestration of essential and shared coactivators by activated PPAR:RXR complexes under conditions where specific coactivator levels are rate-limiting. Activities by other transcription factors that employ the same coactivators are suppressed under these situations of coactivator competition (43). The second, through a process termed '''receptor mutual antagonism" or "cross-coupling" is mediated through the capacity of activated PPAR:RXR heterodimers to physically complex with other types of activated transcription tactors (e.g., AP-l, NF­KB. NF-AT. or STATs). thereby resulting in a functional cross-inhibition of transcription factor activities by both participants. Agonist-activated PPARa can eflectively antagonize the NF-KB and AP-I signaling pathways in a bi-directional manner through a physical interaction with p65 via its ReI homology domain and with the amino-terminal of c-Jun, respectively (44-45). PPARa activators are additionally able to upregulate mRNA and protein expression of inhibitors of NF-KS (lKS) in multiple ceJl types (46). A third mechanism of transrepression, one that involves regulation of the mitogen activated protein (MAP) kinase cascade, involves the ability of activated PPAR:RXR heterodin1crs to inhibit the phosphoryJation/activation of certain Inen1bers of the MAP kinase cascade. 9 PPARy agonists can suppress the activation of both c-Jun N-terminal Kinase (JNK) and p38 MAP kinase (47). The involvement of PPARs in the control of inflammation and inflammatory gene expression is largely mediated through their transrepression capabilities, although the transactivation of certain target genes can also be involved. lI111ammation Con1rol by the~PARs It has now been denlonstrated experinlentally that all three PPAR isof()rms can participate in the regulation of int1ammatory responses. Depending upon the atleeted tissue, and which PPAR isoforms are involved, these nuclear hormone receptors can modulate the intensity, duration, and consequences of inllammatory events. While the n1ajority of published evidence has concentrated on the anti-int1aIlln1atory activities by PP ARu and PPARy, critical roles by PP ARf3/8 in intlammation control have also been recently reported (48). rrJ\Ru The first published report to directly implicate a role for PPARs in controlling aspects of inflammation, described ditlerences in the duration of leukotriene B4 (LTB4)­induced ear swelling responses in wild type and PPARu-dcficient mice (49). The inflammatory response in wild-type l11ice to this lipid mediator was found to be of shorter duration than that t()und in nlice lacking a tlmctional PPARu. It was concluded that an association existed with the ability of LTB4 to directly activate PPARu and upregulate the expression of genes that regulated the ~)- and ill-oxidation of lipids, some of which are involved in I,TB4 catabolisnl. In the literature there exist nunlerous historical reports describing potent anti­inilammatory eflects caused by the dietary supplementation of experimental animals with (J)-3 iatty aeids or t()llowing the administration of moderate doses of dehydroepiandrosterone (DHEA) or related steroids (50-53). It is now fu1ly appreciated that both ())-3 fatly acids and the DI-lEA-Iike steroids are able to etlectively activate 10 PPARu (41, 54-55). '1'0 date. however, only a few follow-up experiments have been perfi)rmed to fi)fn1ally implicate an involvement hy PPARn in the anti-inflan1matory activities mediated by DHEA, employing an animal model of aging (41). Similarly, only fairly recently have investigators characterizing the role(s) played by dietary lipids on immune and inflammatory processes, studied the potential involvement of PPARs in these 11lechanisms (54). Isoform specific PPAR agonists and PPAR-deticient mice have been employed to investigate, both in vitro and in vivo. whether PPARs have an influence on the development and intensity of intlammatory responses. The vast majority of the studies peri(Jrmed have come to the common conclusion that PPAR activation. whether it he PPARu or PPARy specific, can negatively regulate the induction of inflammatory responses. The demonstration that known PPARa-agonists have little or no effect in systen1s where tissues H'om PPARa deficient animals arc employed, provides strong support for a PPARu-dependency of the observed anti-inflammatory responses (49, 56). The role of PPARy is less clear than that for PPARa. A rapidly expanding literature suggests a role for PPARy in regulating induced inflan1n1atory responses (42, 57- 60). PPARy-specific ligands have heen den10nstrated to inhibit the production of many inflammatory cytokines such as TNFa, IL-l~, and IL,-6, to inhibit inducible nitric oxide production. and to inhibit 11letalloproteinase-9 and scavenger receptor-A expression on various cell types. including monocytcs, lnacrophages, and epithelial cells (40, 61). Unf()rtunately, no PPARy-deficient animals are available, as the knock out of this gene is un emhryonic lethal event. Consequently, it is not yet possible to conclusivcly demonstrate whether all of the reported etlects arc truly being mediated through PPARy­dependent processes. While the recent reports that PPARy+ /- animals have an enhanced susceptibility to experin1entally induced arthritis and inflammatory bowel disease are 1 1 supportive of anti-inflammatory properties by this PPAR isoform (8, 47). an additional caveat exists that needs to be carefuHy considered. This comes from the recent experiments by Chawla et a!., in which homologous recombination was used to create embryonic ste111 cells having a null mutation for the PP ARy gene (62). These cells were dil1erentiated in vitro into macrophages and subsequently evaluated. It was determined that PPARy represents an essential regulator of inducible CD36 expression. However. PPARy was not essential f()r elicitation of the anti-inHammatory eflects mediated by treatment with the known PP ARy-agonists 15dPGJ2 or rosiglitazone. I t was already known that 15dPGJ2 can repress NF-KB activation in a PPARy-independent manner by directly inhibiting activity of the lKB-kinase con1plex and/or directly alkylating p50/p65 heterodimers (63-64). These data leave open to question whether al1 of the anti­inflammatory eflects being ascribed to agonist-activated PPARy are, in tact PPARy dependent. PPAR(lL<'2 The possible role of PPARP/8 in the control over inflammatory responses has not yet been fully investigated, largely due to a lack of isoi()flll-specitic agonists. This problem has now been partially resolved, and recent studies suggest that PPARP/8 has an influence. or might be influenced by, inflammatory processes (48). It is now appreciated that PPARJ3/8 agonist treatment of endothelial cells inhibits a stimulus induced upregulation of vascular cell adhesion molecule-l (VCAM-l) expression. and that PPARf)/8 treatment of murine keratinocytes reduces their susceptibility to apoptosis (48. 65). This observation is supported by the analysis of tissues from PPARP/8 deficient mice. that had already been reported to have an in1paired wound healing response similar to that observed in Pi> ARa-deJicient animals (66). A PPARJV8-deticiency was originally reported to negatively affect growth, adipose tissue stores, and 111yelination of the corpus callosum (67). 12 Recent findings indicate that PPARP/& can playa very inlportant role in the host response to inflammation as it is transcriptionaJJy upregulated in keratinocytcs exposed to intlamnlatory stimulL in parallel with an inflan1l11atory cytokine-induced increase in the production of endogenous PPARP/8 agonists. In addition to regulating the expression of genes associated with apoptosis, (upregulating antiapoptotic and down-regulating proapoptotic genes) inflan1nlation-activated PP ARB/8 also contro Is keratinocyte ditlerentiation and cell cycle arrest. It has been suggested that the inflammation-induced upregulation of PP ARB/8 activities in the skin is important f()r eflective wound repair of this tissue (48). Overall the story concerning lhe involvement of PPARs in inflatnmatory responses IS far fron1 conlplete at the present time. It is possible that the temporal relationship between agonist activation of a particular PPAR isoi(Hm, and the inducing conditions that initiated the stimulation of inflammation will markedly inlluence response outcome. For example, it is known that the treatment of nlacrophages with known PP ARy agonists will inhibit the subsequent intlammatory responses initiated by IFNy (43). It has also been reported that the preactivation of macrophages with IFNy can abrogate any subsequent anti-inllammatory eflects mediated by PPARy agonists (7). PPARs and Innate Immunity Innate inlnlunity represents the first line of defense against infections with microbial pathogens. Collectively, it consists of many interacting systems, including epithelial barriers (skin and mucosal epitheJiU111). antinucrobial peptides having potent broad-spectrum antibiotic activities (e.g., defensins and cryptidins), and circulating etlcctor cells (neutrophils, Ino no 11 uclear phagocytes and natural killer (NK) cells). Microbes that have successfully breached the epithelial barriers can be attacked by the circulating eflector cells. These cells display members of the toll-like tanlily of pattern recognition receptors that recognize conserved molecular patterns con1l11only found 13 associated with microbes, but not f()und on intact self tissues. Receptor activation rapidly stimulates inf1amnlatory cytokine production, that can promote nlacrophage activation and enhance microbial killing nlechanisnls. Recent evidence indicates that certain toll-like pattern recognition receptors can also be activated by molecules displayed by necrotic cells, but not apoptotic cells. suggesting that innate defense mechanisms can also be rapidly mobilized without infection in response to tissue traunla or i11jury (68). The PPARs may be playing a variety of regulatory roles in the mechanisms that collectively define the innate imlTIUne system. With regards to the integrity of epithelial barrier function, both PPARa and PPARP/8 are involved in the complex process of wound healing (48,58. 69). PPAR isof()rm deficient aninlals (PPARu or PPARB/8) show similar dc1iciencies in wound healing responses 66. Further, most species of antimicrobial peptides are not constitutively expressed, with the NF-KB signaling system being important t()r their upregulated production t()lIowing an intlammatory insult (70, 71). It is possible that transcriptional regulation mediated through PPARa or PPARy are involved in regulating the production of these peptide antibiotics. Hased on the numerous observations Inade concerning the possible roles being played by the PPARs in regulating inflammatory responses in many tissues. and the molecular nlechanisms responsible, one could easily hypothesize a nunlber of roles f()r these nuclear hormone receptors in innate inlmunity. Through its ability to upregulate expression of the scavenger receptor CD36. PPARy has recently been found to enhance Lhe innate-immune mediated phagocytosis by macrophages of malarial parasites (72). An llpreglliated expression of CD36 could also be used to hypothesize a role tt)r PPARy in enhancing the removal of apoptotic cells t()llowing uptake through CD36 recognition. thereby reducing the chances fi)r progression tow'ards necrosis and the dysregulated cellular activation 0 f an inf1amnlatory response through toll-like 2 receptor signaling (68). Finally. recent evidence indicates that both PPARa and PPARy can function as natural suppressors of the enzyme 11-f) hydroxysteroid dehydrogenase I (11 BHSOI), a 14 widely expressed enzyme that eftectively converts biologically inactive cortisone to the functiona1 glucocorticoid cortisol (73-74). A dysregulation of 1 qH-ISDI in various tissues has been implicated in obesity, insulin sensitivity and cognition deflcits (75-77). Based on the widespread ilTIlTIUnOn10dulatory effects 111ediated by glucocorticoids on both innate and adaptive immune responses, it is easy to ascribe importance to the findings of PPAR control over 1 1 PHSD] synthesis and activities. PPARs and Adaptive Immunity The presence of PPARs within cells of the adaptive Immune system has only recently been uncovered. Therefore, little is known about the specitic imnlune responses that are regu1ated by the PPARs. The majority of the reports that have investigated potential functions for the PPARs in the adaptive immune system have focused primarily on PPARy, with limited data on PPARa and PP ARP roles in the adaptive immune system. The data that have been provided thus far suggest that the PPARs are playing important roles in the regulation of the adaptive immune response at lTIany steps, and that depressed expression or activities by these receptors may lead to the inability to mount an etlective immune response or possibly to an exacerbation of autoimnlune disease. PPAlb' and Des The dendritic cell (DC) has been labeled as a 'sentinel' of the imnlune system due to its normal residence in n10st tissues throughout the body. Its primary role in this tissue associated and lo'inllnature" state is to sample its surrounding environn1ent i()r infection by potential pathogens (78). Because the DC is the only known cell type that can stimulate an immunologically naIve T celt DCs are critical for the generation of etlective prin1ary inl111une responses. While residing in the periphery the DC is in an "immature state', characterized by its phagocytic ability and by its cell surface phenotype. When DCs encounter a pathogen, they respond by undergoing a tightly controlled maturation process. with an upregulated expression of the costill1ulatory molecules CD80. C086, CD40. and 15 l\IlHC class II molecules. Maturing DCs leave the tissue microenvironn1ent and initiate migration to a draining IYll1phoid organ, ditlcrentiating along the way Irom cells that can etlcctively pinocytose or phagocytize, into cells that can et1cctively present antigen peptides to T cells (79). Recent reports have demonstrated that DCs predominantly express the PP ARy isotorm of PP ARs and that agonist activation of PP ARy can influence the l11aturation process of the DC (5). It was repo11ed that the presence of the PPARy ligand, rosiglitazone, during DC maturation with LPS or CD40 ligand could alter the membrane phenotype of these cells. The cell surface expression of the class B scavenger receptor CI)36 and the costimulatory molecule CD86 were both increased when Des where stin1Lllated in the presence ofrosiglitazone. Interestingly, the cell sur1ace expression of another costimulatory molecule CD80 was decreased in the presence of rosiglitazone. It as also reported that DCs that are activated in the presence of PPARy ligands are inhibited trom producing the chemokines IP- I 0, RANTES, and MIP-l (X as well as the cytokine 11.-12 (5, 80). 'The authors of these reports suggest that PP ARy activation within Des might influence effector T-cell diflerentiation through the ability of PPARy to alter the cell surface phenotype of the DC as well as down regulating the expression of those chemokines and cytokines that are important in ThI T-cell developn1ent. Although the n1echanism through which PPARy is able to mediate these ellects was not elucidated, it has been reported that the expression of many of these genes, including IL-12, MI P- I (1., RANTES, and CD80, are regulated by NF-KB. Therefore. it is possible that PPARy may be regulating the expression of these genes through its ability to antagonize this signaling pathway. PPARy and B __ ~ells While PPARs are expressed in B lymphocytes, possible roles of the PP ARs in B­cell physiology have not been extensively studied. However. the linlited data that have been reported on PPAR etlects on B-cell physiology have been very intriguing. In the 16 original report describing the presence of PPARy in B cells. it was demonstrated that 15dPGJ2• as well as other synthetic PPARy ligands. have an antiproliferative and cytotoxic ef1ect on both norn1al and tnalignant B cells (10. 86). It was further demonstrated that an exposure to the prostaglandin PGF2a would abrogate the ability of the PPARy ligands to induce apoptosis in B cells. 'rhe authors of these reports suggested that PP ARy ligands n1ay antagonize the stitnulating eitects by other prostaglandins in regulating B-ccll tlmction ( 10. 86). Recently. PPARy+ /- Inice have been used to study the role of PPARy in B-cell physiology (8). The authors reported that B cells isolated out of PPARy+/- 111ice are hyperproliterative and demonstrate increased viability t()llowing exposure to LPS. or f()lIowing the cross-linking of their antigen receptors. when con1pared to B cells isolated from WT (+/+) anin1als. In investigating the mechanisms behind these observations. it was further established that NF-KB was being constitutively localized to the nuclei of B cells isolated frOn1 the PPARy+/- mice. A constitutive activation of NF-KB may explain why these PPARy+/- nlice were found to have higher levels of circulating JgG and IgM when compared to WT controls. Furthermore. it was reported that the PPARy+/- lnice developed a more severe t()rm of experimentally generated antigen-induced arthritis than the \VT controls. Pl? ARy an~t~[ celh; The expression of PPARy in T lymphocytes has recently been reported (4. 6. 10 81-86). One of the first reported observations of the PPARy in T cells demonstrated that activation of this receptor was able to inhibit the expressionofIL-2 post activation (6,81. g4). The ability of PPARy to mediate this eitect was demonstrated to arise through the activated receptor's ability to physically interact with the transcription factors NF-AT and NF-KB thereby blocking these transcription factors downstream etlects (81. 87). Therd()re. by inhibiting the ability ofN F-A T and NF-KB to bind to the I L-2 promoter and 17 drive transcription of this important T-cell growth factor. PPARy would be playing a suppressive role in in1mune response development. Another report suggests that agonist­induced decrease in IL-2 production postactivation n1ay be due to an increase in apoptosis in T cells being activated in the presence of PP ARy ligands (6). Since these initial observations. it has been further demonstrated that the presence of PPARy ligands can inhibit the activation-induced production of a nunlber T -ccll cytokines, including the hallmark Th 1 T-cell cytokine I FNy (83). The ability of ligand-activated PPARy to inhibit It-I2 production in DC as well as its ability to inhibit IFNy production in T cells would suggest that this nuclear horn10ne receptor might be playing a role in the dif1erentiation of naive T cells into their etlector subsets. Furthermore, it was recently demonstrated that the presence of IL-4. a crucial cytokine f'Or the devclopn1ent of Th2 cells. can induce an upregulation or PPARy in T cells. as well as providing a potential PPAR-y-specific ligand through I L-4 's ability to up-regulate the expression of the enzyme 12115 lipoxygenase in n10nocytes (87). When this enzynle is expressed in monocytes. it converts arachidonic acid into several metabolic products including the PPARy ligand 13-HODE (88). The generation of 13-HODE within the monocyte would provide an endogenous ligand f()f the PPARy within the monocyte, thereby inhibiting this cell's ability to produce I L-12. It was also deillonstrated lhat 13-HODE could be detected in the culture supernatant of monocytes f()lIowing treatment with IL-4 (87). The authors of this study hypothesized that the 13-HODE. secreted by the n10nocytes. could potentially be taken up by neighboring T cells and activate the PPARy within the lymphocyte. I n further support f(Jr a role by PPARy in the development of Th2 ef1ector T cells. a recently published report demonstrated through the use of micro-array technology. that PPARy2 is expressed at high levels within Th2 T cells (89). While a role flU PP ARy in the regulation of adaptive immunity is starting to emerge, the role of PPARa in the adaptive inll11Une response is unknown. The work conducted in Chapters 2, 3, and 4 of this dissertation den10nstrates that PPARa an IR important role in the regulation of lymphocyte physiology. That is. the presence of PPARa with in T cells indirectly regulates the expression of the transcription factor T-bet through PPARo:s ability to transiently suppress the activation of the p38 MAP kinase. The tenlporal regulation of p38 MAP kinase and T -bet expression by PP ARa allows the T cells to produce I L-2 i()r a extended period before the cell shifts into producing the specific eflcctor cytokine I FN-y. The sustained production to I L-2 allows increases the clonal expansion of those T Iymphoctes that are required for et1ective cellular and humoral inlmunity. Qy~view of the Work in this Dissertation It was the goal of this dissertation to elucidate the role(s) that the nuclear hormone receptor PPARa plays in regulating lymphocyte physiology. In Chapter it is demonstrated that T and B lymphocytes constitutively express a functional PPARa. PPARa was J(Hll1d to be expressed at its highest level in the cytoplasmic compartnlent of resting T cells. The expression of PPARa within T cells was down regulated t()llowing cellular-activation. supporting the hypothesis that PPARa is involved in the regulation of T cell responses that occur early postactivation. The PP ARa expressed in lynlphocytes was additionally fbund to be both transactivation and transrepression competent. Exposure to specific ligand determined that PPARa in lynlphocytes could etlectively transactivate a peroxisome proiiferator response element (PPRE) reporter construct. PPARa' s ability to up-regulate the transcription of endogenous genes. however. required a cotreatment with specific ligands and histone deacetylase inhibitors. Finally, ligand-activation of PPARo. in lymphocytes antagonized NF-KB and suppressed the expression of a nunlber of genes under the direct transcriptional control of NF-KB. Although these observations failed to elucidate what physiological responses might be controlled by PPARa. they did delTIOnstrate that functional PPARa exists within T and B lymphocytes. 19 Chapter 3 f()CllSeS on understanding whether PPARu plays a definahle role in T cell biology. To address this question, activation induced responses were analyzed in T IYll1phocytes purified tron1 PPARu-/- and wild-type (WT) control mice. Through the work with PI> ARu-/- Inice, a novel Inechanisnl was uncovered that allows PPARu to exert control over activation-induced T-cell cytokine production. It was deternlined that splenic CD4' T cells, isolated rron1 PPARa-/- mice, produce more IFNy and less 11.,-2 than splenic CD4 T cells isolated from WT mice. These data suggest that the ability of PPARu to control synthesis of these two cytokines arises through its capacity to temporally regulate the transcription of T-bet. This T-box transcription factor was recently demonstrated to be a key regulator of Th 1 T-cell diflerentiation through its ability to transactivate the I FNy gene while simultaneously repressing the transcription of 11..-2. The ability of PPARfl to regulate T-bct transcription does not involve a DNA-binding dependant mechanism, but rather appears to work through irs ability to ten1porarily repress the activation of the p38 MAP kinase f()JJowing T-cell stimulation through the T-cell receptor. Activation of p38 MAP kinase was found to be necessary f()r initiating T-bet transcription. Although T-bet may be one target down stream of p38 MAP kinase activation, it could be hypothesized that if PPARu does regulate activation of the p38 MAP kinase, then other eHects that are down- stream of this signaling n10lecule may also be affected. Although the data presented in Chapter 3 demonstrated that the PPARa in lymphocytes regulates important T cell functions in vilro, the focus of Chapter 4 was to question whether CD4+ T cell functions are regulated by the presence of PPARa in vivo. I t has now determined, using a number of ditlerent vaccination strategies, that the effective generation of humoral immune responses to foreign protein antigens requires the presence of PPARcx within CD4+ T lymphocytes. Immunization of PPARet-/- mice with thymic-dependent antigens, like Hepatitis B surface antigen (HBsAg). resulted in a significant decrease in the level of antigen-specific IgG antibodies generated when con1pared to identically immunized WT animals. Interestingly. immunization of PPARcx-/- 20 mice with pneumococcal polysaccharide, a classical T-cell independent antigen, resulted in the production of anti-polysaccharide antibodies to levels that where conlparable to those observed in vaccinated WT nlice. These results suggested that the impaired humoral response in PP ARa-/- mice t(JIlowing vaecination with f()reign protein antigens may result from an inadequate amount or alteration in antigen-specific, T cell help. To accurately determine the ef1ect of PPARa-/- T cells have on the humoral response, WT or PPARo,-/­CD4+ T cells were adoptively transferred, along with WT B cells, into syngeneic Rag2-/­mice. The adoptive recipients were then vaccinated with HBsAg and their humoral immune responses were analyzed. Interestingly, the Rag2-/- mice reconstituted with PPARo.-/- CD4+ T cells and WT B cells exhibited a marked decrease in the levels of IIBsAg-specific antibodies at all time points tested following vaccination with IIBsAg. when conlpared to Rag2-/- nlice that received the WT ('04+ T cells and WT B cells. These results strongly suggest that the presence of PPARa with CD4+ T cells plays an important role in etlective humoral in1nlune responses to fc)reign protein antigens. Chapter 5 presents data denl0nstrating that the dysregulations in cytokine synthesis that are C0l11JTIonly observed in CD4' T cells from aged hosts. n1ight be similar in n1echanisl11 to those responsible f(x the altered cytokine synthesis by CD4' T cells 1'ronl PPARo. donors. It is demonstrated the depressed basal level of PP ARcI expression within (1)4+ T cells n1ay contribute to dysregulated production of IL-2 and IFN-y by these cells t()llowing activation. CD4+ T cells from aged donors further demonstrate an acceleration in T-bet expression {()llowing activation, a phenotype that is strikingly similar to what is observed in (1)4+ T cells isolated from PPARa-/- mice. As has been reported previously (41), it was confirn1ed that providing vitamin E supplen1entation to aged Juice increases the levels of PPARfl mRNA in their T cells to levels similar to those observed in T cells from normal young donors. Dietary supplementation of old J11ice with vitamin E additionally restored normal temporal control over T -bet expression, and largely eliminated the age-related dysregu)ations in IL-2 and IFNy production. 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Regulation of f)-amyloid stimulated pro inflamnlato ry responses by peroxisome proliferator-activated receptor cc Neurochemisll:V International 39, 449-457 (2001). 60. Gupta~ R.A., et a/. Activation of peroxisonle proJiferator-activated receptor y suppresses nuclear factor K B-mediated apoptosis induced by Helicobacter pylori in gastric epithelial cells. Journal (?l Biological Chemisfl}' 276, 31059-31066 (200 I). 61. Willson, T.M.~ Brown~ P.J., Sternbach, D.D. & Henke~ B.R. 'rhe PPARs: from orphan receptors to drug discovery. Journal (~llv/edicinal Chemistl:V 4], 527-550 (2000). 62. Chawla. A., ct al. PPAR-y dependent and independent effects on macrophage­gene expression in lipid metabolisnl and inflammation. Nature lv/edit-ine 7, 48-52 (20t)l ). 26 63. Castril1o. A .. Diaz-Guerra. MJ., Hortelano. S .. Martin-Sanz. P. & Bosca. L. Inhibition of IKB kinase and IKB phosphorylation by 15-deoxy-L'i12,14- prostaglandin J2 in activated murine nlacrophages. Afo/ecu/ar and Cellu/ar Biology 20. 1692-1698 (2000). 64. Straus. D.S., et 01. 15-deoxy-L'i12.14_prostaglandin J2 inhibits tnultiple steps in the NF-KB signaling pathway. Proceeding\' (?l the National Academy (?l Sciences. U .. \'.A. 97. 4844-4849 (2000). 65. Rival. Y., et 01. PPARa and PPARo activators inhibit cytokine-induced nuclear translocation of NF-KB and expression of VCAM-l in EAhy926 endothelial cells. European Journal (?lPharmac%gy 435. 143-151 (2002). 66. Michalik. L., el al. ltnpaired skin wound healing in peroxison1e proliferator­activated receptor (PPAR)(t and PPARP mutant mice. Journal (?l Cell Rio log,}' 154. 799-814 (2001). 67. Peters. J.M., et al. Growth, adipose, brain, and skin alterations resulting trom targeted disruption of the mouse peroxisorne proliferator-acLivated receptor rHo). J"Yfolecular and ('eUu/ar Biology 20, 5119-5128 (2000). 68. I j. M .. el al. An essential role of the NF-KB/Toll-like receptor pathway in induction of inilanunatory and tissue-repair gene expression by necrotic cells. Journal (?llmmunology 166, 7128-7135 (2001). 69. Komuves. I "G., et al. Keratinocyte diflerentiation in hyperproliferative epidermis: topical application of PPARu activators restores tissue homeostasis. Journal (~l Inl'esligative Dermatology 115, 361-367(2000). 70. Tsutsumi-Ishii, Y. & Nagaoka, I. NF-KB-mediated transcriptional regulation of human J~-dclcnsin-2 gene following lipopolysaccharide stirllulation. Journal (~( Leuko(vte Biology 7 L ) 54-) 62 (2002). 71. Wada. A., et al. Helicobacter pylori-mediated transcriptional regulation of the hun1an r1-detensin 2 gene requires NF-KB. Cellular MicrohioloKY 3. 11 123 C~OO 1 ). 72. Serghides, L,. & Kain~ K.C. Peroxisome proliterator-activated receptor y-retinoid X receptor agonists increase CD36-dependent phagocytosis or Plasmodiurn fhlciparum-parasitized erythrocytes and decrease malaria-induced TNF-(1. secretion by monocytes/macrophages. Journal (~( Immunolof..,TY 166, 6742-6748 (2001 ). 73. Hennanowski-Vosatka, A., el al. PPARa agonists reduce 11 rl-hydroxysteroid dehydrogenase type 1 in the liver. Biochemical and Biophysical Research ('ommunications 279, 330-336 (2000). 74. Bcrger..1., et al. Peroxisome proliferator .. activated receptor-y ligands inhibit adipocyte II f) -hydroxysteroid dehydrogenase type 1 expression and activity. Journal (~l Biological Chemistry 276, 12629-12635 (2001). 75. Yau. J.L .. el al. Lack of tissue glucocorticoid reactivation in 11 J)-hydroxysteroid dehydrogenase type 1 knockout rnice ameliorates age-related learning impairn1ents. Proceedings qllhe Nationa/ Academy (~l/""ciences, U.S.A. 98, 47) 6- 4721 (2001). 27 76. Morton. N.M., el al. Improved lipid and lipoprotein profile, hepatic insulin sensitivity~ and glucose tolerance in 1 10-hydroxysteroid dehydrogenase type 1 null mice. Journal (~lBiological ( 'hemislry 276, 41293-41300 (2001). 77. Sandeep, T.C. & Walker, B.R. Pathophysiology of modulation of local glucocorticoid levels by 11 p-hydroxysteroid dehydrogenases. Trend\' in EndocrinoloJ.,'Y and Aletaholism 12, 446-453 (2001). 78. Steinnlan. R.M., Pack. M. & Inaba, K. Dendritic cell developnlent and nlaturation. Advances in l:~'(perimental Alet/kine and Biology 417, 1-6 (1997). 79. l\1ellman, I., Turley, SJ. & Steinman. R.M. AnLigen processing for amateurs and professionals. Trendv in Cell Biology 8, 231-237 (1998). 80. Faveeuw, C., et al. Peroxisome proliferator-activated receptor y activators inhibit interleukin-12 production in nlurine dendritic cells. federation ql European Biochenlical Socielies J.el1ers 486, 261-266 (2000). 81. Yang, X.Y., et (II. Activation of human T lymphocytes is inhibited by peroxisome prolifcrator-activated receptor gamma (PP ARy) agonists. PP ARy co-association with transcription factor NF AT. Journal (?l Biological ('hemistr,V 275. 4541-4544 (2000). 82. Wang, P., el al. Inhibition of the transcription factors AP-1 and NF-KB in CD4 T cells by peroxisome proliferator-activated receptor y ligands. International ImmunophurmClcoloK)l 1, 803-812 (2001). 83. Cunard. R., et 01. Regulation of cytokine expression by ligands of peroxisome proliferator activated receptors. Journal l?llmmunolo{O' 168, 2795-2802 (2002). 84. Clark, R.B., el {II. The nuclear receptor PPARy and immunoregulation: PPARy mediates inhibition of helper T cell responses. Journal (?llmmunology 164, 1364- 1371 (2000). 85. Harris, S.G., Smith, R.S. & Phipps, R.P. 15-deoxy-~ 12J4_PGJ2 induces IL-8 production in human T ceUs by a nlitogen-activated protein kinase pathway. Journal l?llmmunolo{O' 168, 1372-1379 (2002). 86. Padilla, l, Leung, E. & Phipps, R.P. Human B lymphocytes and B lyn1phomas express PPAR-y and arc kilJed by PPAR-y agonists. Clinical Inllnul1olof.,ry 103, 22-33 (2002). 87. Yang, X.Y., e/ al. Interleukin OL)-4 indirectly suppresses lL-2 production by hU111an T Iynlphocytcs via peroxisonle prolifcrator-activated receptor y activated hy tnacrophage-derived 12/15-lipoxygenase ligands. Journal (?t' Biological ('hemistl}' 277, 3973-3978 (2002). 88. Huang, .l.T., et al. 1 nterleukin-4-dependent production of PPARy ligands in nlacrophages hy 121l5-1ipoxygenase. iValure 400, 378-382 (1999). 89. Chtanova, '1'., Kemp, R.A.~ Sutherland, A.P .. Ronchcsc. F. & Mackay, C.R. Gene microarrays reveal extensive diHerential gene expression in hoth CD4! and CD8-1 type I and type 2 'I' cells. Journal qllmmunology 167. 3057-3063 (2001). CHAPTER 2 NUCLEAR RECEPTOR PEROXISOME PROIJFERATOR­ACTIV A'fED RECEPTOR (1 IS EXPRESSED IN RESTlNG MURINE LYMPHOCYTES 29 Nuclear Receptor Peroxisome Proliferator-activated Receptor 0' (PPARa) Is Expressed in Resting Murine Lymphocytes l'IIE J'I'AHu IN T AND B LYMPHOCYTES IS BOTII THANSACTIVATJON AND THANSHEPHESSION COMPETENT:' R()eQjv(~d for puhlicntioJl, July 20.2001, and in rcvis<,d [!wm, Odobm' ~:l, 2001 Published, JDC Pal)erS in Pres~, November 28,2001. DOllO.1074/jbc.M106908200 Dallas C. Jones*~, Xiaohong Ding:j:, and Raymond A. Daynes:j:'l Fmlll the :~[)ejJ(trtlllt'lJt o/,Patlwlo{!y, Ullit,t'/ .. ~ity 01 Ut(jh School u(Medicill(l, Salt Lulu' City, Utah 8-JJ:32 (1/1(/ the 'tIGeriatrie Rt'smrc/i, Edu('(Ii ilill , (tlld C{flliml Cellil'r, Veterall8 Amlirs Afediml Cell tel'. Salt Lol/(' City. Utah tUII2 Peroxisome proIiferator-activated receptors (PPARs) are transcription factors that belong to the nuclear hor­mone receptor superfamily. PPARa and PPARy ligands have been demonstrated to exert anU-Inflammatory ac­tivities in macrophages by repressing the activities of several tr.mscription factors. PPARy is expressed in T Jymphocytes and may play a role in cytokine produc­tion, cellular proliferation, and susceptibility to apo­ptosis. Ilerein, we demonstrate that T and B lympho­cyt. es (~onstitutively express PPARlt. PPARlr represents the predominant isoform expressed in 1ymphocytes, whereas PPARl' dominates in all cell types of the mye­loid lineage. PPARa expression was down-regulated fol­lowing T-cell activation while PPARy expression in­creaspd under the same activating conditions. PPARn expression in T cells may be regulated by microenviron­mental fa(~tors. because Peyer's patch T cells expressed fur gl'(~atcr levels of PPAR(~ than T cdls isolated from peripheral lymphoid organs. Exposure to specific ligand determined that PPARa in lymphocytes can effectively Lntnsaclhait~ a peroxisullle prulirt~ratul' rcsvuul'iC de­ment reporter construct. PPARa's ability to regulate endogenous genes, however, required treatment with histone deacetylase inhibitors. Finally, ligand activa­tion of lymphocyte PPAR(y antagonized N~~-KB. Our ob­ser\' ation that a functional PPARa exists within T cells and B lymphocytes suggests an expanding role for this nuclear receptor in cells of the immune system. Tht' PPAl{:'\ I are ligand-inducible transcription Juctors that Iwiong to thE: nucle:w hormOlw receptor superfamily. To date, TIll!'; work was :,upportHI in part h~' ~atiol1al lnstilllte~ of Health CraB!": ('A25917 anc! DK!)!i:Wl. hy H Browning FOlll1rbtlOll and hy 1)f'partllH'll[ nfVetertlll's Amlir~ Medical He~!'llreh Funds, co"ts of Jlllhlication of thi- arudl' werl' d,·fi'ay,~d in part by the payment of pag!' charge:<. This article lllu~t lllPrt>i()re he herehy murkt·d "(ldlfrli:w­III!' II(' in u('mrdalH'e wilh 1,S U.S.!' ~t·(t/(m 17:14 soldy t() indicute tllli; llu't. ~ Slipporjpd h~ UH HS/I\ 1I)])K ~n[ionaJ 11I~titllt,·s of Ileallh, Hema­H.' ·;it"urdl Trailllllg (; ram '1'.;2 I )f\071lfl whOJl} (',.lrrespondtcllc(' ~Jwllld bl' addn-s,.;ed: Dept. of Pathology. I)f Utah. :10 North 190(1 Ea~t. Salt Luke City. UT 841:12- :!£)O1. ROI-SHl·:UIl:l: Fnl<:: »Ol-liRI-RfJ.16; E·mail: d:lyJw~,()me~.ri>: pHth.lll..th.Nlll. I The abbreviations lI"ed :\re: PPAH, peroxisonw proliferawr-udi-vnll, d A('o, (leyl-CnA oxidnse: CI'T-l. cHl'Ilitint' Jxdmitoyl trans-i\' ra~I'-1: dt'Xallll'thar'one; El\lSA, dectl'ophol't'ti(' mobility shift H"~!lV: IIW\C, histone de<lcetyla:le: l\i"-fI'B, nuclear fa<:tor KB: PL.\:. !wril~!lpr<111.vlllph !lode: P;.t:,W: phenylllldhyl~lIifimyi fluoride: PP, Pl'Y­" r't' patfh: PPH:I':, pel'oxi:-;ome prolifl'rawr l'e~llollse eleml'lH: PH., pm­gf- St.!:,J'{J1W rpcejltor: TSA, lril'h()~tatin A; STAT. signal trun,;dm'l'l':< dlld a('tl\'atHl~ of transl'riptioll: FCS. fptal calf' s!'rum: FITe tluorescein isolhioc·VL1I1'lt{.: InT, dilhiothl't'itol; THS. '1'l'I",-bllff~l'ed ~alille; ELIRA. ~'nzYlllt'~lilll\ed inllnullo,;orlwnt as,;ay: IL-Il, interleukill-fi; PMA. phil 1'- three PPAR rmbtypc:, hnvc been identified: PPAR<Y, PPAR8 ial~D known UR PPAR,B or NUC-1 I, and PPARy( 1-:3), '1'he PPAR i~ofimn" exhibit a high levpl of sequenc\:' and structural homol­ogy, but each dIsplays a dIVergent pattern of tISSutL"flt~CI1IC expret'sion and ligand-binding specificit.y (4, [)I, PPARa is ex­pre~" ed at relatively high levels in tissues that utiliz\' fatty acid~ as the primary energy source, including hepatocytes, cardiae myocytes. and proximal tubular epithelial cells or tilt' kidney (5, 6,. PPAR)' expre;:sion is highest in adipo;:e tissues and is moderately expreRsed in colonie muco~al epithelium 17, 81. PPARt'') is ubiquitously pxpressed in both embryonic and adu1t tissnes with a higher leve1 01' expression seen in the plaeenta and large intestine U'i, 9, 1OJ. In contrast to other nudear horuwfil' receptors, the ligand­binding domain of Lhe PPAHs can accommodate a variety or natural and synthelic ligands, Several ligands, including cer­Lain polyunsaturated hltt.v acids and eicosanoids. have hel'll reported to be pan-agonisls thaL can activate all three PPAR isoJimn" (} 1-14i. Recent stndies have indicated that 11 number or Iigand~ exi:'<t with t'pecificity for di"tinct PPAIl :>ubtype:>, The Iipoxygenuse metabolite 8(S)-hydroxyeicosatelraenoic acid, as well as the natural Rieroid hormone dehydroepiandr04erone sullilte, have been demonstrated to he ~peci1ie PPARn ncti\<1- L()r~ 115, 161, Furthermore. numerous synthetic compoundR exist that are capable of aetivating PPARH. 'Phest' indude the hypolipidemic agents WY-14,64:l and c1olibrate, phthalate e;;;­ter plaf.lticizers. herbicides, and a recently described, highly ~pecific murine PPARu agoni!';l. GW9578 (1, G, 17, 18i. The PPARs are able to positively regulat.e gene expre~sion b,Y' binding to ~pecific DNA sequences known a" a PPRE as H heterodimer with t.he 9-cis-J'etinoic acid receptor. In the unli­ganded state, evidence indicate~ thnt the PPARs are associated with a nuclear receptor co-repre::<sor (19" Upon activation, the PPARs undergo a conformat.ional change that results in lhe dissociat.ion from the co-repres"or. enabling the PPAR to hind lllJdl'Hf I'Pcpptnr (,O-Hrt.iV}l/or,-: TI)p~p en-;'l('1 ivnlors j hpl1 net In reorganize the chromatin templatps allowing the hasal t.rnn­;;; cription machinery to gain access to the promoter regions driving trall:scriptiull Dr target genes CW-~;l). III addiLil)lI Lu positively regulating gene expression, activated PPAH" have recently been demonstrated to exert anti-inflammatory activi­tie:'> through their ability 1.0 antagonize an array of important signaling pathways, including those associated wit.h STATs, AP-l, and NF-KB (11. 24-281. We have recently demonstrated that NF-KB is pre~ent in an active stat.e in both macrophages and lymphocytes, which re­" ide in the spleen nnd other secondary lymphoid orgun', of l1gcd Ilill 12-lIlyn;;latel:l-lI(,ptatt.>; PH:\', phytnheJllllgglutinin; IFN-l', intpl'­feroJl y. 1ll1CI" 12!-}L This act.ive NF'-KB \VHS 1l1rt.hf'1' dl"mllnstrnit'fj to correlate with the normal col1i'iLitulive exprei'isioll of a number of NV-KB-regulaled genes !2Hl. WI' sllbseqlH'nlly reported that llw administration of Sjl(eCil'ic PPARu activators to aged rodents elfecl ively redueHd the elevaiNt levels of active NF -J<-B in the splet'IH of ll)('~·w animal" and re-eslablislwcl conlrol over a nUlIluer of NF-KR-regulaLed gt'lles Lhl'Ough a PPARn-depeud­en! pmce:-;s (:JOI. The:-;e findings sug-gest lhat the cell Lypes residing within the spleen may be direct cellular targets fIn these PPAR([ activators. or Lhe major celJ pnpulations that [,('Flelt' WJthlll the spJc.('Il, only mucrophngcs have been reported to l'xpre"s PPAH.H I :H-;~:~ t. We lherefbre questioned if other celJ t.vIU!S residing within lhe spleen, including '1' ctdls and B Iym­phocyle:-;, lIonnally exprc,.;,.; thi::.; nuclear hormone receptor, Our re:'lIltfi indicate, [i,l' till' 1il'st tillie, that lymphocytes express PPAHH. We hlrLher demnl1straLt' thelt PPARcr is the Iwedmuinant PPAR iso/(}fJn pref:ent wiLhin lymphocytes. This is in contrast to what if; obHervcd with macrophages, where PPARy l'epl'eSHnl.s the major PPAR ,;mbtype, UlIl' findings also .suggf's\ that mil'rol'l1VironmentaJ variations within sE;eond.'1l'Y lymphoid organs can influence the felluJar level of' PPARu t'xpl'e:-;:-;ilJlt ill '1' edl but Hot B edl:-. FiuuJlv, we wen: able 10 dt'moll:-;trate that treatment of T ('elb wit h highly specific PPAR/! ad ivai nrs ('an IIp-rl''l,,''llate Ihp pxpressiol1 of pndng€'­! lOllS PJ'AH,r eoniI'olled gene!'l when hi "tIme deaedylase inhib­itors are utilized. EXPERllllEI'TAL P({()('EDPRE1' J.;.I!wrfll/(,!1l(/I,\lIflllflis· ·A ('ololl) of J)Oll.lO Tel{ lralli:igt:nic mice W,It' ('stabli:.;lwd fromlm .. edil1/i purchased from ,Jacksoll Lalxll'ilLOI it'S (lbr liarhul'. ;\IE 1. ami pheuuLypil eilal'1lc-ten:-: tHs ofthl~se 1I1l1111nls hm',. preVIOusly Il!:'en r!'ported 1:141. CHifJiel\ Illic(' wen' pllnhu~ed from ('harI!':.; Hiver L.ahor<lwriei' '. W!llllillgtun, :\1J\ I. l't>mal!' illItE' Wt'['P uspd tor a11 ui t1w eXPenll1ent~ reported herelll. All 1I11l'(; wen~ hou;;erl in ,1 sj1t'('ific Ilarhog-en-fre(; barrier facility tlH' l·uiv!'!';.;it \' of Utah Animal H.t'sotll'l'e Center, which Uses ;.;pntllwl <1111- mu!;.. to n;onitm' for the llltl"t p/,l'vnlt:"llt [lJul'ine pathogen;! ilnd gUIll'an­: l'(~" ;.;trid complwllce with l'egubtions t';;tablislwd by the Animal W,.l­fa!'!:' Act. :\nillwls u;;eci were hetween fi and R weeks IIf age, hOllsed in fll1!'I'-lll'llt"(,].pn ('a~;('s WII h ;1 1 t-h light-nark colllroll"rl ('yelp, and pt'o­nded WIth !llOIl~,,=, dlOW and waler wlli i,i(l/Il1. MIce were allesthetized wllh .']ptofanl' llnd saerifi('ed hy (,p['vieal dil'Joeation. ('1'/1 LII/r'.'; (flld ('II/luI'!' COl/dflll!lm·-TK.l, :2[;,1. "i ('('~b We 1'(' obtained from the Am('ricHIl Type ('ulkct.ioll l!\lallaS""f'. VA). The mllrine H-('ellmye)om<l ('ellUnf', P:~X6:I-Ai\~:U),j:1. was OhtHlIlPd fl'lllll Dr .Jprry Spnngrurll' II 'nivp('sity ofllluhl. Th!> T-<,tll hvhndll!1ln, DO 11.1.U. from obta1l1Hl f1'om Dr. .Jerold Woodward 'LTllin~r"it.v of Kelltllcky' Cell line:-: were maintained in HilMI L640 'Inntrogen. (;atthershurg. :'IlL>' ~lIppltlll("tltt.>d with lW" ~'CS (HyClone LuhorHwriei<. Log!lll UTi, 200 IllM L-g-Iut<lllline, ulltihiotieo'. (1m! ii 10;' \1 :2-tnf'r('nptnHilHllol Tlw murine dplHlritil' cpll line. XS-!l2. wa:; "h­tuilH: o frolll A. 'rulwt4hima (University of Texu>l Southw('sil:rn MC'rlicnl ('PlltL'r! and maillt'linpll a~ liesenhl'd ebewhere 1:1:")1. \VhpJ'e imlJ(·ated. {'I'll" were tl'tat.(>d with lO 11.11 t.ridw4alin A ISigma t'helllll'al Co .. :it. Luui,. ;\101 "lUll!' fur 18 h, or with lndmsLatill A for Iil h fc)lIowed hy II 6-h treatment with the l>pedJlc Illurine 1'(,ABet ligalld, (;WD;'j7H I;) gpllerOlil" gifi 1'10111 Dr. Pdp! Brown, (;Ia:w \\!'eIlcolllPI. For 'j'-l'd l aCtlVailOl1. ('linchI'd T rell" were re~lIspellded at ('Ollct'lltraLJon of !j 10" rellsfllli amI were actIvated in llIultiweli plale;; mated wilh a ~()llltion 01':2 ltg/Ill! antl-Cltl or 2 !l.gfm! lIJlt,i-Cln <lnd 2 fl.g/llll ill111Wl)ilized anti-CD2H : Pb<lI'Mingen, San lhegn. CAl. lh!lo/Jl'Ild (',.1/ Hl/ric/lln(,lI! Forthe preparation of:o;plpnic, PL:J and pp iympho(')i('''' i""lutl d lymphoid ceil" w('n~ ,'w;pcflilcd lit n ('ouepllt I'IIl1011 01''2 (,plls/mllll HPl\ll 11140 ('IH}t:lining !')';, }'BS. The present in the l'1'I1 sll"pln~ion \'.'('1'(' Iy;:;cd hy !)fid' treat, lllPlIl stt'rilt, aqlleous 0.1";1'1 '\\,(\') amllloniullJ chloride. For T-cell bolatlOl1, :-:mgle cell sl1spen~ion~ w(~re meuhHted \nth ~ p.glml hlOllllY­lllt{" ti tllltl-( 'IM:i allli anti-eDIlb ;llltinoriit's: PharMingen I t!1f 211 min 011 I(,t'. For H-('~II enrichment. the {',,]I susp(mHion was lllcubateci With u mixtlll'P ('on~i"tillg of 2 ,llg/mllw)tin.vlutetl anll-C 1>4, biotinylated lInt.l­{' D.'!. ;Ind bjotlll~'la1. .. d eDllh Hlltlhodies for 20 min ,11\ il'!'. \\',ISIII'11; with pho;:phntt~-l)11ff('I'i'l1 snlillP, ('I'll,. WPI'f' I'l'fHlSppnripn 1\1-21'0 lllllgnetie n.vllah{'ad~ cnH((,d with ~tl'ept.avidill I Dynal. ;'Jew 30 York, NY) incubated at a Iwad:eeli ratio of 1:1 for ~I) min with agItatIOn at 4 T. ('rll" bOllnd to anlihnrlH's Well' depipi ed hy two rOLlnd~ 1)1' f'XpOKUre to a magnetic fIeld The l'e~Hilial {'ell~ wen· col1e{'t~d, wa;.h~d. amI ~e]Jarated ftl!' liSt-' in culture or for mH.NA allaly~is. The level 'Jf jlurity of the cell preparatwHf' was Ibsessed hy ~tail\JJlg ('plls wlt.h F!'l'C-ilnti-lllou~(:, CD4, FITC-anti-nwu:,\(' CD8. und FITC-antl-moll';f" 1:1220. The level of ('ell purity was routinely ·9!i'·; qu(tlllilali((', Rca/,lillie PCH---.lit\'er"t' tnlll~(TiptiIlIl wa,q Pt'rl(lJ'Jlltd ;IS prpviolhly Ot's!'l'i1wo 1;1()1 mRI\/\ W;I:O; [snlilleli hy Hw ITwthod rof Chomezynski and SaCl'hi ':161, and PCl{ wa:; perf()l'Jlled in 11 Illioreseencp It't11IJenlLul't' ryder! Light Cydt'!. Idaho Tedlllolog)' t as fully t1e,;cnb,'d ebewhere (;J71. The Light Cyder monitor::; tIlt' cycle·by-(·'v( Ie aLX'ulllula­tion oft1uorescentl,V labeled product,. The cycle at Whl~h the PnJdllct i~ first del,eclI'd is used (I" an llldi(,Htor of rdaUve st,artlllg copy. i\leItlllg l'urve" wen' a('qllired to detprllline ::olp,.ciflcity of the PUt (;'IHI. PCB produtls for e<teh nfthe primer I'ets wen' ('()tlt1rllled hy' rUllllillg samples on an agarost' gel. The peH. reaction was carried out IfI 11 1O-p.1 fin:! I volume ('ontallling :1 111.'11 MgCl" 0.2 !lI~1 df\'1'1',. l:;~O.OOO dilution or SYBH (:J'{~en J, Ii V\I (em'hl primer, n.o;; unit. of Tn" p()lymt~l'1u<e. und 11 Ill-: IlfTaqStart :ll1t.ihody. Olignllllcipotirip,.; lI~pd fill' tlwsc' al1:1Iy~ps :IrE' as follow:;: }]urinl' 11uctin. :i'GC:C TCA GAA CCA eTC eTA TC T and :)'-(;TA A(:A ATG eCA TGT TeA AT-:\': Illllrillt' PPAHn, !i'-(;TC (;CT ncr ATA AT'!' TCC 'reT G-T and ri',(uM eel en: ATe 'I'm; AT(; <:(;T-:\,: murine 1'PAHy. ,)'-('A,\ CAe TAe ceT T1AA(;,1' CAA-:l' and ;j'-CTA (''1'1 TCA Tee CAe TTT (;(;'1'-:)': IllUflllt' CPT-I, Ij'-ACT Tee ATl\ TIT crT rCA ACT ')'('')' ('<I' and :')'-'I'(,C AGe AA\ 'rCT (;(;1\ eTC AAA 'reT G-:3: llIurilW ;\('0. !i'-CCA CCT 'reT Tee CCc AAC TGA (lee ce-:!' and ;)-(;CA (;(~T CAG ACe TeT CCC Ace r;<l (:I,Artin tran:;(,l'lpt levpls wpn, ll~ed to nOJ'lllalizp flip amount of cJ):-IA eHch sumple. and Ac(), CP'i'l. und PP,\H.(, Ll'albcript It'veb were n­potted relative to levels found ill the colltl'nl ~ample. i'rcfI(Jrllli(l11 Ii/ :Vuc!('ur f;.'·II'({cl»~-Nlidenr (!xtrnl'to; w('rt> preplu'pri frolll P:IXB~I-Agti.(l;j:1 and EL-4 (,pH line" tremment /(l[ 24 h wlth (;WfJ[)',f; Ill' "duele (\.1.1"1 1\1e)'O I. Bnen::, were washed tWIC!;' with in·-cold pho~phate buffered ~a!ine eontaining 1 1ll~1 PI\1S!<' and 111 40n J.l-I of buffer A 110 n1:\1 HI~Pt;S. pH 7.1:1.0.1 tlnl 10 111:,1 KCL and 1 n\:11 l\1"J{]2 10 /.tWm! aprotinin, 100 ~l:,f leupt'ptin. 1 111M DTT, and I lllM P~ISF and o.!)(~ Nonidet P-4(1) and itll'ubated lIll ire for Iii min. ;.iudei were thell mlleeted hy cenLnfugatHlIl 20,000 .< f! fbI' l:i s at 4 cc, 50 J.l-I of huffer r I fin 1Il:l! HEPES. pH i' .~. nn: KC!. :100 mM ~aCl. 0.1 lll~I EDTA, 10 J.l-giml aproi.l1lin. JIll) J.l-:\l leUIJelllill. 1 1lI~1 D'1'T. til 1(1 1 nnl l'lIlSF I waK a<ldPd lo !lucid awl i11t'uhated for 20 mill on ice. ~udpar debris was removed bv ('elltrifu· gation at H.II00 :< g for ao K. Tht: l'Upernlltant wa~ then rel~lO\'ecl. and pmtein ("Intent Waf; dett'rmined by Bradford :I"",t)' ::Hh l!:MS1\-I:;qual amounts of nudear extt';\('h' (~ f1.g of protem as dt'ter­mined by Bradford as~ay; " .. ere incubated With :10.000 ('pm of :;~I'­bhded N F·"B-speClfie probe Il'rol1ll'ga. Madison. WII. HeadJOI1~ were peJformed ill a 20-/.t1 towl voilime mntaining :! fig of nuclear extract. fll of!) ': gel "hift hinding huffer (20 lllM TJ'is-HCL pH 7.!), !'i !ll)'ll\lI!Cle, Ii.,i nGI D1''1'. OJi m~1 J:<:JJTA, and 2W; glycerol), 1.5 /.If!: of polyldl-dCi. and I /11 (If probe', For :,;uper:,;hdl <I~~UyH. 2 J11 of an apllropriate anti f\F-KH ~L1bllnit antibody (Santa Cruz Biotl'chn()logy, 111('.1 Wei;; added to <'<teh reil<:tion. TIlt' l'<'llctltlJ1 W<l~ JIl('ubated at mom telllJJerawl'P I()f l:i lIlill.loaded 'HI a 4', nativt:" jJolyanylaJllidl' gel. and lUll ill 0.:; THE buffpr 14!'i mM Tl'i~, 4!'i m:'] Borie Acid I mM EIYL\I. TllP gel wa:; dried clnd i'ubjert.pd to autoradIOgraphy. :-\F-"I:l-sptt'ific b,wd, wpn' con, fimlPd hy competition with a lon·filkt f')(ct'S' IIf all u!llahplt'd .'J~·- .. H prohe, whieh re~LlltPd m Illl shifted hand. or by preparing thp reDctJ()Jl With excess labpled nOlhpeeifit' pmbe, which did not redlH'p the inten, of the f\F-"B hand. isolutt>d IY11lphoeytes werp plated Oil lX-nUll llimnete!' cover·dlp, that were pretreated with 1 mgillli polyly, sinf'. Cp\ls wert' then fixed for :W min at room relllppratllre in 2(; paratormaldehyde, wm,hed WIth TBS. and permeabJlllpd III THSfO.2' ( Triton X- JOO for 5 min at room tempE!ratlll'p. Th(, ('oVPlosiips were then incubated ill Ion J.l-I of rabhit. anti-lllnu"e PPAHn polyclonai antihody • Affinity HlOreagent.':l, Colden, COlor a rabbit Ib,>{] iHotyflt' control dllti­hody ldilut,..d l:wn ill TBSJF; hm'inlo' ~E'rLlm alhumin' at room Ik'llIl:lPl'- Lltill-t foJ' 60 min, After in('uhalJOn, vi;;uulizeci H"ing Alt'xll ,)94 I Molt'C'ulnr Pmhps, r:ll![f'lw. Ihill!,: a J. .. itt' D:\lH /lIHlI'!'SI'PIH'P microscope. l'musir'n( Trans/i'llioll (lwl Assay (II' LlIl'ij;'m~r RI'{Jor/{'j' ('01/­sll'lld,~ ·--·Trani'iff'('tillnl' were peri(>rlll€d w; pre\'Jou~ly deH{:rihed (·101. BriHfiy, Ii < 10" cell~ were reHu~pPllded in (}.I1!i IllI of 1U'l\lI H;1() wilh Ifl1·f FCB and. 10 IJg ofpCL:Hbsic luciferase reporter ('clIlslnJrt (Pfl\· I1Wg;11 was lI~NI III t l'1Il1sli,cting TI<-1 n,lls ane! :zn ilf[ of \TF-Kl111('iff~raSf' reporter eonstruet ;1)1'. Andrew Thorburn. University of Utah, with 10 /1g or 1ll1l1'llH' PFAHl, plmmud Illr. Non 1':\'[10:'>, Salk IJl4itlltt~1 was tran:-.iertf:'d ,Jurkut T (·,~Ils. Olle microgram of pHL-TK Renilla luciferase reporter plasmid! I'mnwga I wa" added to control f(Jr tran;;f!,ctiofl dlieielwy. Ct'lls were lll['uhated with pla:-mid:-; feJr' ;1 min in 0.4-1'111 1'11'1'1 rod" f;ap t'lIVt'ttps ,I nvitrogPll, Carlshad, fA) llnd ut room t!'ll1pe!'nt lire tilling tht' CC'JlC PUifH'l' I Bio Had. CA I s('I. at 2HO V and !J60 microfarads (/1/<') for TK-I and at ~4(J V and H60 ILl" for .Iurkat. l 'ells were I11cubnted for r; min ut room 1.elllpenllure t1wn Iran;;fi'ITHI to 100- x 20·mm ti~sllt· culture dishes containing 10 ml of HilMI 16,10 with 10(,; FCS and in('uhaled at :J7 0(' for 24 h, Lucifl'r;lse assays wpn' pl'rformed using the Dual Lllcift'ra~e Ne'porter Ai'say sy~l{>m IPronwgai. HndIy, cells wen' han'esled, centn­fll;! l'iI at ~oo g for Ii mill, w;u;hed Lwiee in pilm;phlltp·hufTpl'pd .;aiille, rp~usIH:ndpd in :200 III 1)1' 1'< Iy"is buffi'!' i Promeg,n lind incubated at !'Hom tl'l1lpprattlfP for l!i min. ('pll delll'is was Jlf'llplt'd hy I~pnt.rifllv.al ion at 1.1.0()() jilr:1 min, and 180111 of lysate wa .. renwvpd. Cell lysate 110 Ill! was into the well of 11 white opaque microliter plate, and the dual IU('lfera,;e a~"a.v was performed automatically using the l\lLX mJ(~r{)tjj('r plate IUlllinnmder iDyn!'x. ('hulltiHy. VA •. Mel'iul injl'l'tll)IJ (If substrate;; and monitoring of light emiSSIOn fol' 10 S Wi:rl' perfnrmed for both {irelly and Rl'nilla ltleift'ra;;e, Computer ~()ftware (i)ym:x 1 auto­mati(~ ill1y ~llhtr;lc1pd h;H'kgl'Hll1Id :Hlrl normalizNl raw dm.il hy 1);)II'Illal­illg the ratio of ilrefly:1i(,lIIlla light emis .. ion valul's. 8U,,,'l\ ._( :ylnkilH' ('IHll'l'Jllraliolli'i were lIIea~lIrl'd by t:LlSA, as ue­I't'rih( ·d prpvlOu:;/y :4.1 i. I\!olloc!ollal rat Ilnti-lllllrllle 1L-2 <lnlihodier' and murine rt'CDmblllllllt 11,-2 cytokill(' 4unclard Wl'I'I' purchased from I'lwr11ingen (San Di('go, CAL Stulil<!l('t/{ /tll{(/y;;is~Statl,;tieal analy,';it' "as pt'rfonne{llI~illg Stu­dellt's I test wltil jJ' tUb depil1pd as ,~tat1l'tically slg'lllfieant. HESPLTS PPARH Is Expressed in Normal Mur;ne LYll1phocytes alld li(,lIwtopoil'fic Cdl Lines-To determine whether 111l1rine Iym­phocyLes expre~F. PPARo, we utilized quantitative, reul-Lime peR to nnalYlt PPARfI' mRNA levels in murine T <mci B lym­phocytes, We initially analyzed primary CD4 and CDS T edls and B cell,: isolatpd from the i':plecn of normal C:lH/HeN mice and compared these to splenic macl'OphageH, a Cl~JJ Lype already known to expreSH PPARcr I:H-:~a). The resuils of this Hludy (Fig, lA) dt'lIlollHlraLt' that tiplellic T cellH (uoth CD4· and CD8 I ) and Hplcnic B cells express PPARil mRNA allevels that are high!.'}' than what is Ilormally ohserVt'd in splenic macrophage;:;. In the lymphocyte populations, B cellt;; were con­sistently l(mnd to constitutively t~xpreSfl higher levels ofmRNA ((II' PPABn than T cells. We also evaluated two murine T-cell lilleH, TK.] and DO 11.1 (J, as well ttH the B-cdllllyeJolIla p:JX6a­Ag8,6!): 3. pef{ analysis revealed t.hat these T- and B-cell lines nl"o f'xprl',"sf,d PPAHIV trill!;, 1101 shown 1 We slilJl'ieqllently uLililed illllUll1w(]uoreHcence allal),H!;; to idt~nti(v the suhcellular localizutinn of PP AR(~ within splenic: T and B cells. As shown in fig. In. PPAHu protein was excluded Ii'om the nuclt>lIH in the llla,jority or T cell~. A similar localiza­tion of PPARI! was also observed in B ceJls (data not shown!. '111e rytopiuHnuc localization of PPAlllt in Jymphoeyte~ is anal­ogom; Lo what has been reported jew unactivaled macrophage:.:; L\l 1. Similarly, PPARy is also found in the cytoplasm in resting T tells but iH shuttlt.od to the nucleus upon T cell activation (4~L [t Ims alrt'ady lJeeu delllollHtraled that I,YlIIphocytes ex­preH!'> PPARy and that macrophag-eH can eXJlre~H bolh PPARn and PPARy (;1J l. We, theref(lft~, compared the relative levels of PPAl{(f ancl PI'AKy mH.NA within the dif1erent lymphoid Ct'li populatiolls. ACl ~h(Jwn in Fig. ~, the levell-i oj' PPARH mHNA were thre(~ to live time;; greater than the Jevel of PPAH)' mR:\,A in all lymphocyte populations tested, Thi~ is in contrast to what was observed in cellH of the myeloid lilwagl' Imaerophnges, dpndrit.ic eells, alld mast. 1'1'lh,p wlwre PPAH)· fl'I)J'esented the predominant iHOfilJ'll1. Cell IllaI' A.ctl!'oti(J1I DOiCl/·rt't!ulatl!s PPA.Ru mRNA llnd Pm· lein E.l]Jre8sio/l ill T LYl11plwcyles-lt has previously been re­] 1111tHI that PPAH-v Lranscripts in T cells IllCreailes j()Uowing eellular netivation (4:3). To determine if cellular activation al- 31 A /<'11:.1. Lymphocytes express PI'ARn mRNA and protein. A. freshir i~oiated splenic CU4 I T celi;;. CDR I T ('elk and B220 B ('ells. and macrophages. W!,!'l' analyzed for PPAH(( 1l1PSi:>\age hy quantitati\'e pef{ using primerH ,;pecific filr l'PARu and GAPDH, The !t'\,eb oj' !'PAHu were determined hy normaliZing thE' CAPDti Il'v!:'ls in paLlI ;;ample. iI, immllllotluore~('!:'nce dn,tly;;i:-; of PPAH" protein ill CD4 T ('ells reveals that PPAJVr lo('(dization is predominantly {'j'toplasmic. l\inn"pf'l'ifil' ,,!aining wm; a;:;,;es"'f'd lI"ing a t'ahhii 1/:,,(; j .. ",typP r:nntrnl l!:xperiments were repeated at least tJlft'e tiIllP!;. The reHults of a rep· rel'>entath-e txpenmt'nL are shown Connective Tissue Mast Cell - Mucosal Mast Cell Macrophage Dendribc Gels -XS-S2 AAW267 CD3- T-cells 8220- B·cells 0011.10 TI(.1 P3X,63Ag 20 15 10 5 0 2 4 6 8 Ratio PPARy:PPARa Ratio PPARC'l:PPARy Ftn. 2. Ratio of I'PARu and Pl'AR'l' mRNA in various hemato· poietic cells. mi{KA VI'llS isolated from primary lymphocyte and lIlye­loid eells as well as i'everaJ ('ell line,. Heal-time quantItative pel{ wa~ performed with specific primel'i'i f(ll' I'I'AH". Pi'AU" and !f1~Tt'lr';li,rlf" hyde-;J·pllllsphate dehydrogenuse. The relnth'p message Wl're nlltnirlPd hy IHlI'malizilHJ f'lIch s:lInplp 10 gIY(,f'ndd('hydp-:l-pho~ph;ltp dehydrogenase. PCtt products were c'll1f1rmed by running l'ampJe~ un an agaroKe gPi Idata !lot ,hOW111. ~;xpenll1ellt" Wl're repeu1.E'd t!lrep time;:. The results of 11 reprel'entaliw l'xpt'rimellt are ;;hOWI1. leI'S PPARH ill T cells. ('ref-Illy isulated splenic CD4 T cells were activated with immobilized anti-CD;] or immobilized anLi­ems plus anti-CD28, PPARu mR:,\A and PPARy mRNA levels were then analyzed river the Hllbsequent ~4-h period, A" shown in Fig. :.5, PPARiV tramlcripl levelt> were clownregulntHd in splenic T cdls stimulated with immobilized anti-CD3 plus anLi­CD28 as early :3 h post activation and declined further Mer lhe next 24·h period. The decline in PPARIl' expre~sion was rllntrasjpd hy an onsprvf>o inrrf'tlSe in PPAH:y mess::lgf> ovpr lilt' same 24-h time period. as hail been reported previouHly (4:jl. T Cells Isoloted /hml Dif!;'renl Secondw), Lymphoid ()rt!aw; Express VQ/~villg Levds oj'PPAR(i-'!'O further characterizE:' tlw presence of PPAHu in lymphocytes, we questIOned whether the microenvironment of' the secondary lymphoid organ in which 120y---------;=======iI 20 o~--o- ----3- h-r- ---6- h~r ----12- h-r- --2-4- h-r ~ I'll:. :1. 1)I'ARn mR:"JA levels dpcrease after polyclonal 'j'·cE'1I activation. Primary CD'l T ll·ll" wen' activated with imllwbillzed :lll1.i-Cl):l pluM allti-(~:llZH_ lllH:'JA wa" isolilL(~d from ('dis prior to stim-ulation iI!lei :1. fi. 12. and 2411 activatioll, quuntitat.ive I'CH wat' IItiliwd to detefllllJ1P the relative of PPAR(r and PPAth ~;xJlPrimpnr~ WPI'l-' I'<'Pf'iltNi thr'('I' tinwR.. '1'111' rpsi II t." 01'11 f'PI1r"~(>Ilt,lIfiv~' i~~<J1f'ril1wtll llff' ,.;1111 ..... n. I~mphoeytpll reside might inlluenee the exprellsion or PPARu mHNA To address this que"tinll, mRI\A wn~ isolated from T l'l'lls aIld B lymph()c~leg purified Ihllll the PLN, spleen, and PP of normal C;JI I/lIeN c!onol'l'i. Quant ilative, real-Lime pen wa~ a~ain u,,(·d 10 analyze Ihe relative PPAHH tranficripl. levels in the various cell popuJations. As l'ihown in ~'ig. 4. PPAHu mliNA Il'vpl.;; \Vl:'fl' quill> similar from R ce/ls iHola!{'ri frnm nil tilE' st·condary Jymphoid organs tested. In contrnst. T cells i:-mlated frolll theRe snnw org-ani; expreRspd varying l!:'velR of PPAHn IIIHNA. T cells isolatt~tl fbllll the PP wt:re {huml to express approximatdy eighl times the amollnl ofPPARH mRI\A than T ('ells isola Led from the fipJeen or PLN, To enfilll'e that till' differences in T-cell PPARn mRNA levels observed WPI'(' not due tn dil1erellee:-; in the percentages of I1wmol',Y :1nd naiVl~ T cells residing wilhin distinct "ncnndary lymphoid organs. an experiment Wfl:-; conducted ll:~rjng CD4 T Iymphnc,vte:-; i:'loluled [rom Lhe secondary l.vmphoid organs of DO 11.10 'pen II afl1'lg-l-'nic mice, Vi. tually allllH~ 'I' ndb ill t11t:1'it' mice t'xpres:-; single T-cdl receptor wil h specificity f()r an ov,dhllmin pepl.idp and retain n naive phenotype. T cells 1';0- lated from vanolls secondary lymphOid organs of tlwse TeR lransgenic: mice showed a simjJar pattern oj' PPARn mRNA t~xprc;;:-;jon to whnt wn", obfiervcd in wild type anilllab. with the CD4' T cpU..., isolated from the PP expres:-;Ing the greatest levels of PPARo' mRNA (data not shown J. utllt'IJ(,()I'timid TI'mtmf'llt Ellhml('f'!i PPARn Transcript Lf.!I­ds iii Both Band T Cd/s-It ha::.; pn'violl'ily been reported that transcription of the l'PARn gene h positively regulated by ~lu('o{'orlicoidK if! rill'/) and ill r,'i!'o (,1-1), In addition, we have pn)vjnll~ly rt'porLed that the level or glucocorticoid::; is higher in the PP than in the other secondary lymphoid organs due to a Jt!l'lCC1l'ied acti\' il)' ill lhe PP of 11 li-hydrrlxy.'iteroid deh)drogen­ase. an enzynw that eUectively converts glu('ocorlieoids to an inadivl' t l-kpjn f'Orm (4;) Wp therplhrp qllPstiOlWrl if gillrocor­tH~ Olds mIght elllllnlmte to the (hncrC'nel'~ 111 PPAH.(( Ic\'cds Iw\wpel1 T I'ell:, residmg \vllhin the PP and other secondary lymphoid organs. In an attempt to addrf's;.; thill, we treater! Fn'fihly isolated CD4 I T cell" and B cellfi with 10 7-10 ') M Dex {(II' () and 24 h. Following glucot:mticoid treatment, the cellfi were harvpsted. and I'PAlin transcript levels were measurer! bv real-time quantitative peR. Fig. i')/\ "hows Lhat treatment with I 0 ~J Dex wa" able to increase the kvd or PPARn in T Ct'!/." by :J-foJd over vehide alone. Hnwevpr, a "'imilnr enhance­ment was abo obtailwd with B cells (Fig. flBI tHlggesting that an eleva lion in glucocortieoid inJluences within the PP might !lot Le H'siJOlIl'iilde lin' IIH~ illt:real'it'd PPAHu levels o!Jl'ierveu 100 90 J 80 j a 70 C 60 III C .!..!.! 50 ~ 40 i 30 a: 20 10 0 Peripheral Spleen !=layer's Patch lymph Node 1"1(;.4. Analysi.s ofPPAUtt mUNA ill lymphocytes isolated fl'om various secondary lymphoid organs. cn:) T eelb and B220 B cell;,; were Isolated t1'OIl1 pt'1'1plleraI lymph node~, splec-Il, uno Peyer's patches using positive ,wleetion. Fullnwing isolation. mR:-JA waR. t'X­traded from the \"ariU\I!>; cell population;; and real-time quantitative pel{ was lI-L'Il to IIwasure the relative Ipwl~ of PIJAHn mRl'\A in !:'lH'h of the cell populatIOns_ Experiments were repeatpd t,hree linw" The l'e"ull~ uf a reprerlenwti,'e l'xpl'riment are -l1o\\'n, A 35,-----------------------?-----. 030 ~ ~25 ~20 e .... 15 ~ ~ 10 a: 5 o B Untreated DEX1 ()-9M DEX10-BM DEX1 !r7M 5Or-------------------------~----n 1; 45 j 40 ~35 ~ 30 ~ 25 ~ 20 ~ 15 a::: 10 5 o U'ltreated OEX 1 O'9M DEX 1 O·8M DEX 1 0-7M FI<:. !i, nexamethasone induces PPARn expression in lympho­cytes. Vl'e"hly isnlllte(j CD4 T (,(,lis IA; or B l'pll" {ll) wt'r(' treuted i;)1" G and 24 h with 10', If} " and 10 ., :,l dpxalllPthasone OJ" veillde. I"nllowing tn~atlllent. PPAU" mH:'.1A level" were by teal-time lllHllHitativp PC'l{ ExperillH'J1tf' Wi.'re repp;j[ed thrp" 'l'Ill'restilts of;1 n>pl·p~ental ive Experiment are ;;\11.>' .... 11. ouly ij\ T edlto re:o;idillg within this secolJdary lymphoid organ, Lij!(lnd i\ctimtiol/ I){ PPARa Stimulates Trallscriptioll oj' a PPRE Reporter COllstntct Bllt Is Unable t() Directly Indu('i' tI,e ExpressiON 4 Emlogelwus PPAHl! regulated Gen('s ill T Cell;;-In an attempt to :::IsseI's the function of PPARn within lymphncytes, we employed a highly specific PPARu ligand, GW9!)78. to question whether re('eptor activation would up­rt! gulale tlte e1l.fJlt!ssiuJJ or kuuwlI t'lldogellolls PPARH-f't'gU- 1 50 .Q.J) 40 a ~ 30 ~ I- 20 CD .~ III 10 "ii a: o Unlrealed GW 10 nM GW 100 nM GW 1 pM 1"lfL Ii. Treatment of the murine T-cell line, TK.I, with GW9578 fails to induce expression of several PPARlr-reguiated genes. TJC 1 T cells WPl'e tn'lltr=d with 10 lUI. ](I() 11'1. or ] 1-':\1 nWL1,:;7.>i Of v('hieh 10.1'· M~~SO) li)!':24 h. Folll)wing trPlltmcnL the l'elativ{' levt:l~ of PPAH(l, fyr· L and ACt) mHNA wen' del.t'rmint'd by qllantitatiw pelt mHNA wm; j"olated fl1lm each {If the ctll population., and mIC'lA Je\'els fill' tIlt' ahove statl,d PPAH«-fl'guluted gene,: wpre analyzed, Experilllt'lltl-' Wf'l't' repeated thl'et' time~. Tltt' re1'ulb ora l'!'prt'sentatJve t'xpt'riment eire showll, luted gPIWS The CDS T-cell line, TK 1. was lreated li}f (1 24-h period with variOLIR levels of GW9578. Following treatmenL mH:\A WiiS isolated frOllt llie treated amI COJlL)'{l1 cells, alld Lhe mH2'A levels or CPT-L Aco, and PPAHa were analyzed by qllantitative, real-t1mp P(,R A" shown in Fig, (). exposure of TK-l T cpJls 10 GW907tl. \Vlli-l unable tn sLimulate expression in any or the PPARn-regulaLed ~enes above the levels €xpr€sRed in the vehicle-treated T celh;, To ensure that the inability to UP-l'Pgulale tlw t'xpressioll of these gelles was !lO!. sped fie to TK 1 eell~, Lhi" pxperiment was repeated with the CD4 ' T-cell lymphf,mn nOll In, as well <ls witb frp,.:hly lsnbted ,;;plenir T etdls. Similar til what was ohseI'vt:t1 witiL TKl, Ln~allliellt wilh GWH!)7H did not incn:uHe L1w levels ()f these PPARcr-driven w'ne,~ in any elf these '{'-cdl populations I data nol Hhown I. To question if the inability of activated PPARn to dri\'e the C'xpresRion of certain endogenous gl'nes is possibly dUt1 to duo­malin repression, we uti1izpc\ a reporter con;;trucl thuL cnn­tained Lhe Aeo PPHE St'quence. This reporter eOnf'Lruct waH cu-Lranslcetf'd into TKI aJong with a pRL-TK ReI/ilia lucifer­Il!,;£' repnrll'1' plaHmid to control fflr LransleeLion efficiency. The transfeded cdb were then treated with GW9!l7H or vehide lin 2411. A.., shown ill Fig, 7. treatment ofTK.l cells \\'ith GW9:)7H waH able to induce a dOlle-dependent increase in the amount of rela\.iw luciieJ'af'e activity, This suggests that the PPARo within l.vmphoeytc'l it! funcLiol1uinnd docs ponf;cnn thc ability to induce gene transaetivatioll. Requirement (or Histone Deaeetvlas(' inhibitor,'; to Facilitate the indU('{ioll oj' Gell(,s lInder PPARn Conti'll! in Lymp!w­(' yles-It Ilas prf'vj()u~ly been reported that the ability of li­gmHI-< lctivated Iludear hurmone receptors Ln drin~ gene tran­Hcriptiou n·yuires the reeeptnr to dist-lOciatll from nueh2uI' recf'pl()l' eo-rt'jJrCf!f!urs and rlubReqllcnlly complex wilh nuclear reCl'ptor eO-Hctivators 120-2;);' This new complex promotei' chromatin llceL,vlation and chromatin remodeling, thereby al lowing t'ssential basal transcriptionaJ machinery to gain acceHH tl' the promoter region of genes LInder control of' the aClivaLed I1lleit'ar rtoceptor !2()-2:l). We therernre questioned whelher the inability oI'ligand-adiv1:lted PPARo to up-regulate gene expres- 1"Iilll1 within T cd!." might be due to an inability to properly aef'tylate histune core". In an attempt to addl'es~ thirl l[lJestion, TK 1 ct'lls wert' prt'l.reated with TSA, a known HDAC inhibitor. ThiS was rollow!:'d by a suh:,;equent exposul'l' to varymg dOHes 01 GW9!)7H, Trentment of TKl cellR with TSA alone, alLlwllgh capable of ... timulnLing an inereW:le in the levels oj PPARn transcrIpts, had no elIed on CPT-lor Aco mRNA levels IFig, 81. The eXpORUI't) of TSA-treated TK 1 to GW9fi78 resulted in a c\o!';p-dept'llllpnt inereAsf' in mHNA t'xprt'ssion of' variollR PPAHn-regulated gene" I Fig. Hl. Similar re~ulls were observed .,., _L) 4 3 QI <II C!I ~ .5 2 :s u. o Vehicle lnM 10nM 100nM 111M F](:, 7, Luciferase assays of TK.l T cells transiently trans­fected with reporter constructs containing the Aco PPIU<~ se­quence. TKl T cell~ wen: tranliientl,Y tra11;;ft'cted with 10 p.g of the ('PRE reporter construct lb opscrihed Linder "Experilllt'!ltal PnH'P' dUl'es," Cd!:; were rested [or ~4 11 j()lIowmg tl'anSfectlOl1. The et.'lls were then treated fot' 24 h with 10 In], 100 11.\1. or 1 I1.M GWH578 or \'t·hide I Me~Sn 01', l. Thf' data aI'f" the fmlll thrpp inoppenrlpnt p'<fll'r-iments. The a:;len.;;ks indicate ~lgl1lfi<anr diflerem:e" 'f/ (J.O'l1 bu;.;ed on SlUdellt'~ I test. when sodium butyraLe employed as the BDAC inhibiLor (data not shown) PPARn Actiuaiors Decrease the Amount olNI/deoI' NF'KB ill Tmnsj(ml1ed T-cell (lnd B-cefl Ull(',~-To further define possi­ble fi.metioJlR for PPAR(v \vit.hin lymphocytes, WI:' questioned whetllt:.1' tile activation ofPPARIt in T alld B lymplJocyte:-; would lead to the lmnBrepression oj' XF-KB. We previously reported that the sllpplementation or ::Igpd mc\pntf': with specific PPAR" activalor;; effectively reduced lite dysregul::lLed levels uf ill'Live NF-KB in the spleel1,';; of lhese animal!' ,;30), FllIihermore, treut­men!- of macro phages wilh PPAIln activators in i'itm is known to suppress interfl'ul?in·(j (fL·m gene trallf':criptioll by interfhring with NF-KB-driven promoter LransaclivaLiol1 i321. To address wheLher the aetivation ofPPAful III lymphocyLes would facilitate the transrepressinn ofNF-KB. we evaluated the murine B-cellmyeloma, P3X63-Ag8,G::m, wbich expresses con­slitutive nuclear NF-KB, and the murine T-cell thymoma, EL-4, which can be induced to expres~ nuclear NF-KB j(lJLowing lreal­ment with PMA and iOl1omyein. As preRented in Fig. 9.4., the level or nuclear NF-KB present in the B-cell myeloma was reduced following a 24-h treatment with GW9:l78 as deter­mined by E:\{SA, Similnf rCfmlt.r1 were observed in E:L 4 eell,; that had been pretreated with the PPARn agoni~t GW9!)78 and adivated with Pf\lA and ionomycin for 24 h (Fig. 9BL To ,BHeSf) if treatment of T celli; with a PPARtr aclivator leadH Lo 11 functional decrease in ~F-ld3, we first analyzed whether GW91178 inhibiled prnductioll or tlte NF-KB-regulated cytokine IL-2. The level of IL2 was measured in the superna tant nfEL-4 cells that were stimulated wit.h Pl\lA and irmomy­cin for ~4 11, follO\'\'ing a 2-11 prelreat.ment with G\V~)578. As shown in Fig. 9C, treatment orElA T celb with GW9578 Jed io a significant decrea,<;e in lL·2 production compared with control cell", GW9;)78-Ln~ated EL-4 cells were additionaHy evaluatHd 'ill' cell proliferation. Treatment with the PPARH agonist waH 'c)lInd to reduce the proliferation of EL-4 T cells in a dOHe­dependant J1laJll1el' (data not shown!. To specifically demonBtraLe thai ligand activation of PPARu can inhibit NF-I,R t.ransactivaLion, W(~ utilized a Nl'-KH lucllerase reporter ('on"tfuct that wa,.; lranslenlly transrocied into ,Jurkat T cells along with a murine PPARu expression plasmid, The co-trnnHfecLed T cells wert' then tn~atpd with variOllS concentration of CW9!)7H prior to actio vating the cells with PMA and PHA as described previously (Mil, As shown in Fig. 90, pn·t.l'enLnlPnt of.Turkat l' cdls with GW9G7H led to a do~e-dependant inhibition of luciferase ex- PPARa. OO~~======~------------~ .-TSA 10 nM .+TSA 10nM 50 1'---------' 0 Untreated GW10nM GW 100nM GW lJ.1M CPT·1 100 00 .-TSA 10 oM ii II+TSA 10 nM > Ell til .a...J. 70 '§ 60 c:: t!! 50 I- ~ 40 :::: '11fit 00 II: 20 10 o Untreated OW 10 nM OW 100 nM OW1~M Aco OOn=========~----------------, .-TSA 10 nM a; .>s 50 :g. 40 o ~ ..t.!. !30 Ql .~ 20 j £ 10 B+TSA 10 nM o Untreated OW 10 nM OW 100 nM GW 1 f./M 1"1(;, K, GW9578 up'I'egulates expression of PIJARH regulated genes in T eells prl'treated with a BDAC inhibitor. The TK·} T-tt,1I line wa~ treated wah HI n~1 IS/> tflf IH 11 followed Iw all addi­linll: t1 h-I! tl'f';lln1l'1lt with JI) Illf, Ion 11\1, or 1 I'~I c;wm;7H'or l'F>hide IOJ'; r'ollmnng tn'atmenl. the cells were harve~ted, m1-t:-lA IVal' and quantitative Pc({ wal' performed, EX[Jerill1entc were I'l'pemt'd !lIn'!' timp" 'I'hl' I't'l"lIll..; of (I n'presenta/n'p l'xpprinwilt are :<110WII. pres::;inn, TngpLher, t1ws(' l'xperiments HUgg<'st that ligand acLi\ atiull ur PPARn CUll ;-'lIIJjJn~~l- NF-KB lnlll~(:I{.:ti\'alioll, possibly b~1 interfering with NF -KB's ability to hind to its ~rH:,('ifit' respnn-;p f'lenwnt Il!S('[rSSIO:l PPAH,n \\'a:-; lirst dpscribed in the early 1990s as a hormone rpcpplor that could induct' pt:'roxi.:;onw prolifpraLinI1 in thl' Jivpr 34 of high dose agonist-treated rodents ((j l. Over the pa8t decade, the PPAR su bfamily oftranscription factors has been described to play an aClive role in many important physiological pro­Ceg8e~, including adipogenesis, fatly aeld metabolism, and in­I1mnmHlion ffi-7, 471 Thp Pl'PSPIWf' and rolp!; or PPARs in hpmat.opoif't.ir cplI8, however, hnvp ollly rpcl'I1t,ly lwl-'n exam­ined, Monocytes and macrophageR were the lirst cell" of tilt' immune ilYi'ltem in which the phy"ical pre:'lence and anti-ill­f1amm3lOry propenie;:; of PPAR" were iirst deHcribed. Onlv over the past year has PPARy been reported lo exist in other nnmune cell types of hematopmetIc nngm, mcJuchng-, dendnLlc cells. Band T lymphocytes. and mHst cells (42, 4:1, 4R-!il., In this ~tlldy, we demonstrate for the first time that IllUl'ine lym­phocytes (both Band T cells) expreRS PPARn ln1J1fi{.Tipt~, Band l' lYl1lphocytes were also found to contain PPARH protein, TIlt' liSP of rpHI-l.inw IlIHl1lti1::JliH' peR provirlpfl n I11P;ms In qmlJ1- titatiwly evnll1ate the relative lev(>\s of PPAl{H and PPAH:y mRNA in lymphocytes and in variolls cell typeK from t1w my­eloid lincnge. We determined that PPARo i~ the predominant PPAR subtype present within all testtod types of mnrine Ivm­phocytes, whereas PPARy iH the major subtype expressed in ('plls of myeloid nngin. It has previously been reported that, within resting lympho­cytes, PPARy if' excluded from the !lUciellS 142, Thii-! is in contrast to what has been reported for ft~Rling macrophages, were the mujority of PPARy is located in tbe nucleus, We thpfefhrp l1ti lized iml11l1110fluorpscPl1cp t.o dplprmi rw Uw slIhcpl­lular localization of PPAH(L Similar to what has been reported J~)r macrophages. the PPARn within lymphocyLes is pff'c1omi­mmUy cytopli:1~mic, Cellular localization of the PPAHu and PPARy iso/()rm1'l mig-ht represenL a reflection of the (listincl Junctions of these protems. It has been reponed that lympho­cyte!; undergo apoptnsis fiJlJnwing Llwil' trent nwnt with PPARy agonisttl but are not atlecled by trentment with PPAR(r-speci(j(~ Jigand::; 142~, Likewise. agonist activation or the cytoso\ic PPAHiV does not induce apoptosis in macrophages unless the cells i:ln~ treuted ill the pfe~ellce of tlllllor lwcrosis IllcLOT'-iI and IF,\-y f;·\1 L This SIJgl!psLs t.hat eyloplBsmic Incalil,ntinn or PPARn WIthin lymphocytes might preclude this protein from functioning in a way that renders the cells sllf'ceplible to apo­ptoili" /()J1owing agonist treatment alone, In addition to chang-ing the t'ubcellular lotalization o[ PPARy. T cell activatIOn ha;;; also been reporied to e1led the It'vels ofPP.ARYPxpfI'sRion 14:-11. TnLprPRlingly, we rCl1Inrl Ihl1l PPARn mRNA wai' markedl:>r' decreased with '['-cell activation whereas PPARy mRNA WHF- Iimnd io increase, The dynamic nux ofPPARn expression within T lymphocytes might suggt'sL that thii' protein is fUlIctiollul whereas T cells are ill u resting state, Activation of PPARn within resting lymphocytes could occur from the presence of an endogenous ltg-and, or posKibly through the ability of PPARu to complex with anotht'r protpin like Et8-1. The Etl-l-l proLein i:'l highly expre:-l:ied in rC:"ting- l' cells and hus recently been demollfltrated to adi\'ate PPARn in a ligand-independenL manner 1;'2), Expression on~ts-l is also rapidly clown-regulated upon T-cell acti\'ation {f);H and thul'l Its ability to interact with PPARn {iJJlowing activation would be limiLed. Likewise, it has been demonstrated that PPAH(, iR inactivated via phosphorylation provided by the mitogen-acti, vaLed proteiu ki1lH~e, extracellular iiignal-regulated kinase (54L This kinase iil rapidly up-regulated (c)lIowing T-cell acti­vation and might result in PPARa being inadivated. W(' have also determined that the levelil of PPARH tran­HcriptH in T lymphocytcH vuric:l :~ignifict1nlly between ccll;~ ifl()­la/ eel from dillerent secondary lymphoid organs, whereas 11'1111- script levels of PPARn within B cells remainH unchanged, Expression levels within T cells residing in Peyer's pnl.rhes me 35 A B r--·--·----------------------------------~--------------~ ,----------,----------------_. lii\1 101.\1 c I ~iHHI • II .'! i' HII. (I -------.- .....• -"'-... ,--.-.-----------------~ II{,\I Ii hi!,! (iW1)'1T, D I(li) _ ;';0 () \ "iih:h." I u\1 10(ln\1 Ill\l [I.it \1 1"11,. 9, Ligand activation of PI' ARer decreases NF-KB's ability to bind DI\'A and to transactivate gene expression. A. P:lX6:l-AgR6,i:l celll< were treutRd with lor 10 /1:'! GW9578 for a 24-11 period, Jj, the murine T-ceillim\ EtA, was treated with lor 10 U\V9!)78 for it period pnor to activatioll fill' ~4 h willl liO ng/ml PMA and 1 /J.:'l ionomycin, In both experiment", ('eli:: were harve",ted, extracts were prt'pan~d, und 1l11l\F "B EMS;\ wu,< peri{lnllulw;ing:l ILl!: of l1uclcur c)ltmet;i, Ext1'l.1Ct" were incubated with llutibodie" lTcognizing the f\F t<B :;ubllnit:; p:iO and p6:'i prior to performing the EMSA ttl confirm .l\F-Kl~-speeifi(' hands. C. l';L-4 t:ells were pretreated With I;\V9,)78 or velJide iIl.l", :\If' ~SO) fOl ~ h followed hy u 24-h treatment with flO Ilg/ml P~\1A and 1 !l~l ionomycin. The leveJ~ ()f IL-2 were then mea;,;ured in tbe sUp!:'l'Ilatllnts hy I~LlSi\, 0, .Iulkat T cellI> tran~lt'ntly lfall;;!\;,(,tt'd with a pb;"lllid expre~sing PPAHn and a .'JJ<'-KilluClfera"e-n:purter construct wert:' stimulai.t:'d with ,iO lIg/ml Pi\L\ and 1 fIg/ml PHA for;j h tlllluwing a ~-h preir!:'ittmtJl[ ,,,ttll (;\\,9678 or vehicle (0.1';; flle),OL Lllciferase expre;;~1\)1l j:,; "hOWIl as the pprc!:ntagl:' o)f ('()ntroL t;xpl:'l'Illwnb were repeated three tin:es. The re~1I1tt' of a represt'lltative t'xperiment ilJ'e shown. The asterisks indicait' stntJsticuJly ;;ignilieant C\iHi"relwl's {P' n,On, ba~ed Oil Stud('ut's ! test. 1()U[' to six times that seen in the PLNs Of spleen. The mecha­nism nh,;pollsiblt> fill' I his phl'nnnwilon has Hilt ypi IWf'n eluci­dated. Presently. only !ClUI' Indors are known to pO:'llively n'/:\,ulatp PPA.Rev gene expresKion, These inclnde the hormnne ul'tiOI1H of glucoeorticnid;;. "latinH, leplin. nnd ligand-mediated PPAR([ adivation 1t"ell' (44. 55, fifi I. Glu('ocorticoidii and PPARn al'tivatinll dn 1I0t appear to bt, rCHpon;;ible ('or tlw nb­,; erved diIfen'llcl':'! in the level or PPARo mRNA observed be­tween tbe T-cell populations residing in lhe PP and other seCOncillfV lymphoid organs. Although glucocorticOId treatment (1fT ('Pil" ill citro doeR I'l,~mlt in un up regulation PPAR(\, mRNA ex Pl'PS 5-' 10 ll, a similar up-regulation waH ob,;erved in glucocor­ticoid- treated H Ct>lJ;: elS weil. Treatment ofT cells with PPARo­spet'iJk ligamls wa,..; lIllalJle tu illduce all Llv-regululed t'xprt's­sion ofPPAltll itself and waii unable to up-regulate the levpls of otllPr pndngPllolls PPARn-n'guh1t.f'd g(>nes TI1I:'sl:' findings sug­gest that gene transadivation hy PPAl<a may not lw respollsi­hlp Ill!' the /luduutiol1H in PPARH mRNA level;.; spell in T cells n;'liding in ltw dineJ'(~nt secondary lymphoid orgall;;, The inability til induce Hgand-activated PPARn trnnsactiva­lion in lymphrH'Yll:'s could hl> ovt'rcomt' by employing \wo elif­li'n~ 1I1 approaches_ The first ut.i]izt'd transient tralliil'ection uJ' a lI1urint' T-cell lint' with a reporter construct containing the Aco PPHE sequence. Treatment ofthese cells with specific PPAHu ljgnnd~; lip rt·gulutcd expn'Ht;ioll of the rcportpr gmul, The sec ond set of condit inns capable of inducing efTecllve transactiva­tioll by PPAHH employed an HDAC inhibitor in conjunction with Jigand netimllon, T cells treated with TSA or "odium butyrate, both known HDAC inhibitors, were rendpred Sllseep­tible I.n 8t'1ivat.ion by spp.l'ifir PPAH(r agonistf'. Hoi h of thp"p situations suggest that hgand actlV3L10n of PI'AHa may not be able to ell'ectively induce expression of certain endogenOlll" gene:'! within lymphocytes. due to an inability to inil.inte proper chromatin remodeling. It has previously been demon:<trated. with cells containillg the PR, that ligand treatment dT'eclively induced expression or transiently Lransfeeted rer)orter COll­struet but failed to induce up-regulation of endogenollsly ex­pressed genes (fi7, fiHl. This phenomenon was reportedly due to chromatin packing of the endoA'enou::; genes, not nllowinA' the PR to gain access to the promoter region. Transiently trans­feeled reporter constmets are not a:.;"nciated with chromatin and therefore call be dl'edivt'ly LntllSaclivdled by PR upon ligand activation. At present. we have only studied a narrow set or PPARll'-regulated g(>Ilt:'S invnlved in fnUy (lrid metabo­lism. It if; highly pOf;sibJe that activation of PPAHrv can ellec­liveJy transactivute other genet:' within l' cells thnl wt' have nol yet examined. Although direct ligand activation 0(' PPARn in lymphocytPii JiJiled to lip-regulate thl:' I:'xpre~hion of sevel'al PPAHI1-fPiitl­bled gelles, ligand adi valiou of PPAR{\' did re:"1111 ill tlll' dli:!t:­tive trangrepressiol1 of NF -KB. in both tranSf()fmed l' cell:.; and B lymphocytes. We have yet to establish whether the ahilily or PPAR( to trun~1rCpretii' NF /,B ir; nehi(wed through the dired interaction ofPPARn with NF-KB or through the up-regulation of the IKE gene. Both processes represent recently described mechanisms through which aclivmed PPARu can e1Tenively control lhe IE·vel of aclive N}<'-"H 111 cell;; (fJ!:IL It has been previoul"'ly demOnf.1lrated that the ability of activatnd PPARR t.o trnniu'cpreHs the activilies of NF-"B, as well ail a number of other tnmscriptioll fado!'''!, has important physiological conge­qlH'lWf'~ within !lwsp rellf.;" indlHling anti-inflammatory arl iv­ities. transcriplioo repression. and cell de<lth ! 24. :n. GO, 61 J. Although we have shoWIl that limctional PPARn receplor is presl>nt within '1' and H lymphocytes, we have yet to eFilab1J~b tl roIL. {'OJ' this r{)cljptor in lymphnc,Ylo biology. However, we ha\'p recenlly Jenmu that T cell~ isolated [rolll mice taking a functional PPAHli receptor 1 PPARli I ) t'xhibit dysregulation ill lldiv,JI illll-illtltwt'd IFN-y JII'IUllldiulI, ",Ilt~n' PPARIY I T cpils produce much higher levels of IF)J-y than T C('lIs /i'om PI'Alul :' mice,:: The~t' obRervatinos were made in the abRence 01 added exogenous hgand. further suggestmg that the en dog­pnou;; PPAHH protein in lymphocytes may already be in an adive state. Clearly, additional experimentation ig net~ded to elucidate tlw mechaniRm(sl through which prARH might reg-­IlIHII" 1111' pxprpssion or inoll('ihh-. I'ylokinps in adivat.ecl lym­phocytes and any addiLional role( s) lhi~ nuclear hormone may play in l.vmpiloc,Yte biolog,v, ,kkllOl!'I"rif,{lIIuih · .. We thank Dr. Peter Brown for providing tht' (:W9fi7H ('ompollnd.lk To)m l\klntyre !f)!' providing the PPH1:: reporter (,(11l:4run. Dr. Andrpw Tborburn il»)' p/'Ovidillg Uw 1\1"-1\11 reporter mn­;; 1 fuel. and Dr. Hon r~\'aJl;; for providing Ult' I'PAHn expreksion l'IJ01ltrueL REFERENCE'" DI'l·v,'1. j' KI'!'~' I.:,. KI'i:t'1', H .. (JiVl'1. l" .. Hdnl·nl'l·in, (;" uud Wahli. W , l!)~l~ (",II 118. S 79, R" I ('h','n, I'" Law, :-" \V, .un1 (l':\klk\', l\, W, 'HJ!Ja, iJuw/iI,1It B;"uln'"" 1\"" CIlIII/'iillt. 196, ~ Kllt'w,,!'. S ;\" fo 1'111;] 11 , L, ]1.1 Hllll!li>{'r!!. 1.1. ()n~ K ~" l'. ~lall!;,"h'd,,,I'f. D ,J" I 'llIl'''OJ:". K .. <In,J Eva:I", H,':'1. 119fH' l'mt' 8('1 S ,I. 91. 7;,;):")- 7:lr)~J i\mi"sant. 0 .. F,)uklk Sl'Oltp, C, llilllGl ~1.. ;til.! \\,lhli, W, d~!961 Elido 137, :I:}·l ~:c.ijli :llld C;r"'n, S, ,19fU)1 Xutlll,(, 347, G4:; I;"i) '1';'111111101 P H" I': ,""I Il \l '1'1!):11(",//7!1.1147 'I,m j\,LU1~"IL A" (;u"tn:ioli,J)i:1Z Bath·)', ,1.. I!wlllln~, (:,. Iud (Jtlt<till~h"n. J, l\, ,H19i'. t/lflrfl!'lIi l1illld',s ii", ('''/)/11'''" ~t4,- H:; 1 9, Bnu"sHnt. 0 .. and Wnhli, W. I I Hf!1'! 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J), ./, , 1~,19Hj Mol. 1'1,(lrllww!, 50. oJ7/4 lAulidllll. .\l.. Lr'lllwnl. .J, .\1.. 'llhl'l. 13, H .. l{ill~()lcl, (l .\1.. ami K,l·\\I'J'. ~ ,\ 119~17 i.J. Hi,,/, ('I"'II! 272. :3406-:iHU 1M, P,.\, I'lunKet. K IJ" !'Ilo'll'l'. L, 11.. Lt'wb,.\'1. C, W.ll'UlI. WtI, Z" L'lupman. ,1. 1\1.. Ll'lIllWllll, J, .\1.. KII(,\\,(,r. ~, A" ,Iud \hbdll. T. M I Hlllll.,J. Jlc(/, ('fILIII 42, IH lliHl'llZO. ,J,. ::. .. d"l',trom, lit" Krm.~awa, II.. ~l. FL. Hi('(lt". 1\1.. In:'I"'Y S IIndt'in. :\, Il""'llli lei 1\1. (;, :In<l (' K 11997111,,1 (-..II. HI,.I 17, :)11)(1 10 f)]""." S ,\ 'ls,,; S l' Tsa;, M. ,I and! rl\bl1c'Y, B W i 19!!i'iI.'\('fCJl<'<' 270. ]:J".4···I:,1'17 c .lone:->, Il C" lJlllg-, X., Zhang. 1'., and llllynl''';. K A. Il1tU1l1"('npt in pn"llar:ltion, 36 :!l Kallll'i. I" Xu, L Hl'illll·!. 'L TilJ'rhlil. ,/" Klirokawa, H" (a,)~,;, 1.1.. Lin. ~, (,,, Ht'\IlHlJ1, H. A .. Ho~(·. D, W , GIa~" C, K, aurll{oH'llti,ld \1. C, 1199lj! (,,,if 85, ,lQ;,\- 4 14 2e:! TUI'"hia, ({nl"", 11, \Ii,. l",x,tJ'l)z.l, ,J KUllWi, y" \\\·"tin, S" (:la 'I<, ( , K. and )(",'>'n1<'I(\, M (" It!)!'/' ;\;a/lIl'c :UI7. Iii 1-0;;4 2:1 Hannis!,,!', A ,J, and J("uzuml">I. T. ,IH06Il1,',,/ul'(, 384. 641- (,,1:1 2,1 Jiang, ( .. Till;!. A, ,\,,, .111d Sr~·.!, B. 1199>;l XUI:I/,<' 391. 1\2 2i'i Hil'ott:, 11,1.. Li.A C Will"OIl, '1',:\1.. I\.,·lh,(', ,J.. and Gb"". C', K 1189~! ;Vu I d!'!' :l!l1. 7~IH~ 2f; (;"ttlkhL'J', .\L Wit!;nark. lL L1 and (;u4af~~'IlL .J, j\, I W92i /'uJ>'. /\a/I, :itad ".'0, K ,t\, 89, 21 K. S" S E .. l'atl·L H It;',dlllhin~ki. n, A., and ( ap"ll'·,.! I' .1. 271, 2(:\ :;1:1<'1,. B" KOI'Eig, \V .. Hahib. l\." ,\'It·lya!. It, Ll,bn't.l\1 Turra, I 1'" lld"l'i\'l', \,,, Fa(M. t\" Chi:wtti. U .. Jirudl;lI't..J ('" :\a,:ih. ,J,. .\'lm'j"'IL ,J.. <',m! 'I\'d;!lll, i\, 1~9,- N(I/urc 393.i!J(l :W Sp('llcer, 1\. 1"" Poyntl'!', .\1. E, 1m. S, y" ,lI1t1 lJ"YIII'S It.\ 'I~J(171 111/, /1I11111IllU/, 9, lfi8l-1:)8~ '\1. K