15-Deoxy-∆12,14-Prostaglandin J2 Inhibits IFN-Inducible 10/CXC Ligand 10 Expression in Human Microglia: Mechanisms and Implications This information is current as of September 24, 2021. Qiusheng Si, Meng-Liang Zhao, Anna C. A. Morgan, Celia F. Brosnan and Sunhee C. Lee J Immunol 2004; 173:3504-3513; ; doi: 10.4049/jimmunol.173.5.3504 http://www.jimmunol.org/content/173/5/3504 Downloaded from

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2004 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

⌬12,14 15-Deoxy- -Prostaglandin J2 Inhibits IFN-Inducible Protein 10/CXC Chemokine Ligand 10 Expression in Human Microglia: Mechanisms and Implications1

Qiusheng Si,2 Meng-Liang Zhao,2 Anna C. A. Morgan,3 Celia F. Brosnan, and Sunhee C. Lee4

Regulation of and chemokine expression in microglia may have implications for CNS inflammatory disorders. In this ⌬12,14 study we examined the role of the cyclopentenone PG 15-deoxy- -PGJ2 (15d-PGJ2) in microglial inflammatory activation in ␣ primary cultures of human fetal microglia. 15d-PGJ2 potently inhibited the expression of microglial (IL-1, TNF- , and ؊ ␣ IL-6). We found that 15d-PGJ2 had differential effects on the expression of two -; whereas the Glu-Lys-Arg (ELR) chemokine IFN-inducible protein-10/CXCL10 was inhibited, the ELR؉ chemokine IL-8/CXCL8 was not inhibited. These findings were shown in primary human microglia and the human monocytic cells line THP-1 cells, using diverse cell stimuli such as Downloaded from bacterial endotoxin, proinflammatory cytokines (IL-1 and TNF-␣), IFN-␤, and HIV-1. Furthermore, IL-8/CXCL8 expression was ␣ induced by 15d-PGJ2 alone or in combination with TNF- or HIV-1. Combined results from EMSA, Western blot analysis, and ␬ immunocytochemistry showed that 15d-PGJ2 inhibited NF- B, Stat1, and p38 MAPK activation in microglia. Adenoviral trans- duction of super-repressor I␬B␣, dominant negative MKK6, and dominant negative Ras demonstrated that NF-␬B and p38 MAPK were involved in LPS-induced IFN-inducible protein 10/CXCL10 production. Interestingly, although LPS-induced IL-8/ ␬ ␬ CXCL8 was dependent on NF- B, the baseline or 15d-PGJ2-mediated IL-8/CXCL8 production was NF- B independent. Our http://www.jimmunol.org/ ␣ results demonstrate that 15d-PGJ2 has opposing effects on the expression of two -chemokines. These data may have implications for CNS inflammatory diseases. The Journal of Immunology, 2004, 173: 3504Ð3513.

5 rostaglandin D2, a major cyclooxygenases (COX) prod- PGJ2 can function as an endogenous feedback inhibitor of macro- uct in a variety of tissues and cells, readily undergoes de- phage activation. In addition to inflammatory expression,

hydration to yield the bioactive cyclopentenone PGs of the 15d-PGJ2 has been shown to affect cell proliferation and apoptosis, P 12 12,14 ⌬ ⌬ J2 series, such as PGJ2, -PGJ2, and 15-deoxy- -PGJ2 (15d- indicating a potentially diverse role in inflammatory regulation and PGJ2) (1, 2). 15d-PGJ2 is a natural ligand for the peroxisome pro- tissue repair (9, 10). liferator-activated receptor ␥ (PPAR␥), a nuclear receptor involved by guest on September 24, 2021 Much information regarding the role of 15d-PGJ2 is derived in adipocyte differentiation, glucose metabolism, and inflammatory from rodent and cell lines. The inflammatory en- ␥ response (for review, see Ref. 3). PPAR agonists have a positive zymes inducible NO synthase (iNOS) and COX-2 are among the regulatory role in lipid and glucose metabolism, but their role in most susceptible inhibited by 15d-PGJ2 (5, 11Ð macrophage inflammatory has been shown to be 13). However, studies of human CNS cells demonstrate that 15d- ␥ largely inhibitory. PPAR agonists have been found to function in PGJ inhibits iNOS and COX-2 expression in astrocytes, but not in both a PPAR␥-dependent and -independent fashion to modulate 2 brain macrophages (14), consistent with the idea that the regulation macrophage gene expression (4Ð7). Evidence supports that 15d- of macrophage activation is species dependent. Similarly, results PGJ is produced by activated macrophages in vivo and in vitro (2, 2 obtained for cytokine (IL-1␤, TNF-␣, and IL-6) regulation have 8). Furthermore, 15d-PGJ inhibits the expression of COX-2, the 2 been mixed. Jiang et al. (15) reported that human dif- rate-limiting enzyme in PG synthesis (2), which suggests that 15d- ferentially responded to 15d-PGJ2, with LPS-induced cytokine synthesis being refractory and PMA- or okadaic acid-induced syn- Department of Pathology, Albert Einstein College of Medicine, Bronx, NY 10461 thesis being susceptible to inhibition. Chawla et al. (5) showed that ␥ Received for publication February 12, 2004. Accepted for publication June 30, 2004. 15d-PGJ2 can inhibit macrophage cytokine expression in PPAR - The costs of publication of this article were defrayed in part by the payment of page deficient macrophages. More recently, Welch et al. (6) reported charges. This article must therefore be hereby marked advertisement in accordance that PPAR␥ agonists inhibit selective subsets of macrophage genes with 18 U.S.C. Section 1734 solely to indicate this fact. 1 (iNOS, COX-2, IL-12, and IFN-inducible protein 10 (IP-10)/ This work was supported by Grants RO1MH55477 (to S.C.L.), RO1NS40137 (to ␣ ␤ C.F.B.), AI051519 (Einstein Center for AIDS Research), and TGNS07098 (to Q.S. CXCL10) and that cytokine genes (TNF- , IL-1 , and IL-6) were and A.A.M.). not inhibited. Taken together, the ability of PPAR␥ agonists to 2 Q.S. and M.-L.Z. contributed equally to this study. inhibit macrophage cytokine synthesis can be shown to be depen- 3 Current address: Department of Pediatrics, Massachusetts General Hospital, Boston, dent on the type of macrophage tested, the stimulus used, and the MA 02114. PPAR␥ agonist used. In contrast to the macrophage cytokine 4 Address correspondence and reprint requests to Dr. Sunhee C. Lee, Department of genes, relatively little is known about the regulation of macro- Pathology, F-717, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461. E-mail address: [email protected] phage chemokine gene expression by 15d-PGJ2. In THP-1 cells, 5 Abbreviations used in this paper: COX, cyclooxygenase; Ad-CMV, adenovirus car- 15d-PGJ2 induces IL-8/CXCL8 while suppressing LPS-induced rying only the CMV promoter; AZT, 3Ј-azido-deoxythymidine; DN, dominant neg- MCP-1 expression (16). Interestingly, 15d-PGJ2 has been shown ⌬12,14 ative; 15d-PGJ2, 15-deoxy- -PGJ2; IL-1Ra, IL-1R antagonist; IP-10, IFN-induc- ible protein 10; iNOS, inducible NO synthase; PPAR␥, peroxisome proliferator- to induce IL-8/CXCL8 expression in several cell types, including activated receptor ␥; RPA, RNase protection assay. T cells and endothelial cells (16Ð19). These results demonstrate a

Copyright © 2004 by The American Association of Immunologists, Inc. 0022-1767/04/$02.00 The Journal of Immunology 3505

␥ ␮ complex role for PPAR agonists in inflammation and call for input HIV-1 for 16 h. 15d-PGJ2 at 10 M was added to cultures 3 h before exposure to HIV-1. Culture medium was completely changed weekly, and caution when considering 15d-PGJ2 as an anti-inflammatory agent. 15d-PGJ was replenished each time. Microglia are the resident brain macrophages central to the 2 maintenance of normal homeostasis as well as the regulation of ELISA inflammatory responses within the CNS. We and others (20Ð25) IP-10/CXCL10 and IL-8/CXCL8 ELISA was performed using Ab pairs found that cultured human microglial cells provide a good model purchased from BD Pharmingen (San Diego, CA) and R&D Systems (Min- to study brain macrophage responses to inflammatory and patho- neapolis, MN), respectively. Ab pairs for IL-1 and TNF-␣ were also pur- genic stimuli, because they recapitulate many of the attributes of chased from R&D Systems. HIV-1 p24 ELISA was performed using a microglia in vivo. In the current study we examined the role of commercial ELISA kit (NEN Life Science Products, Boston, MA). Super- 15d-PGJ in cytokine and chemokine expression in primary cul- natants were diluted until the values fell within the linear range of the 2 ELISA detection limit. tures of human microglia. We found that although 15d-PGJ2 po- tently inhibited LPS-induced cytokine (TNF-␣ and IL-1␤) expres- RNase protection assay (RPA) sion, it produced differential effects on the expression of the two Total RNA was extracted from microglia plated at 1 ϫ 106 cells in 100-mm ␣-chemokines, with IL-8/CXCL8 being refractory but IP-10/ dishes using TRIzol, according to the manufacturer’s instructions. RNA CXCL10 being susceptible to inhibition after activation with a was analyzed using RPA templates for human chemokines and cytokines wide variety of cell stimuli (LPS, cytokines, IFN-␤, and HIV-1). (BD Pharmingen) according to the manufacturer’s instructions, essentially as described previously (31). Images were developed using autoradio- These results suggest that 15d-PGJ2 may create a unique cytokine- graphic film exposed to the gel at Ϫ80¡C. Densitometry was performed chemokine environment in the CNS, tipping the balance toward an using Image software (Scion, Frederick, MD). ϩ Glu-Lys-Arg (ELR) chemokine predominant state. Downloaded from EMSA Materials and Methods Nuclear extracts were prepared using a modified Dignam method. Buffers Microglial and THP-1 cell culture were supplemented with 1 mM PMSF, 1 mM DTT, and a protease inhibitor mixture (Roche, Indianapolis, IN). Cells (ϳ1 ϫ 106) were scraped off in 1 Human fetal CNS cell cultures were prepared from human fetal abortuses mM PMSF/PBS and centrifuged. Pellets were resuspended in low salt as previously described (20, 22, 26, 27). All procedures were approved by buffer (10 mM HEPES (pH 7.9), 1.5 mM MgCl2, and 10 mM KCl), and the Albert Einstein College of Medicine institutional review board. Primary allowed to sit on ice before addition of Igepal CA-630 (Sigma-Aldrich). http://www.jimmunol.org/ mixed CNS mixed cultures were prepared by enzymatic and mechanical Samples were again pelleted and resuspended in high salt buffer (20 mM dissociation of the cerebral tissue, followed by filtration through nylon HEPES (pH 7.9), 25% glycerol, 420 mM NaCl, and 1.5 mM MgCl ); ␮ 2 meshes of 230- and 130- m pore sizes. Single-cell suspensions were plated samples were rocked gently before final centrifugation. The supernatant ϫ 6 at 1Ð10 10 cells/ml in DMEM (Cellgro supplemented with 4.5 g/l was saved, and the protein was quantified using the Bradford assay. Oli- glucose, 4 mM L-glutamine, and 25 mM HEPES) supplemented with 5% gonucleotide containing the consensus binding sequence (underlined) for FCS (Gemini Bio-Products, Woodland, CA), penicillin (100 U/ml), strep- NF-␬B(5Ј-AGT TGA GGG GAC TTT CCT AGG C-3Ј was radiolabeled ␮ ␮ tomycin (100 g/ml), and fungizone (0.25 g/ml; Invitrogen Life Tech- with [32P]ATP using polynucleotide T4 kinase according to the Gel Shift nologies, Gaithersburg, MD) for 2 wk, and then microglial cells were col- Assay Core System kit (Promega) instructions. Labeled probe was purified lected by aspiration of the culture medium. Monolayers of microglia were on a G-25 spin column (Roche). Three micrograms of nuclear extracts were 6 prepared in 100-mm tissue culture dishes at 10 cells/10 ml medium or in incubated in binding buffer (4% glycerol, 1 mM MgCl , 0.5 mM EDTA, 50 ϫ 4 2 by guest on September 24, 2021 96-well tissue culture plates at 4 10 /0.1 ml medium. Two to four hours mM NaCl, 10 mM This-HCl, and 50 ␮g/ml poly(dI-dC)) with 1.75 pmol later, cultures were washed twice to remove nonadherent cells (neuronal of specific and nonspecific competitor oligonucleotides for 15 min at room and astrocytes), resulting in microglial culture that were highly pure, con- temperature before addition of labeled probe. The binding reaction (final Ͼ ϩ sisting of 98% CD 68 cells. Microglial cells used for HIV-1 infection volume, 20 ␮l) was allowed to proceed for another 20 min at room tem- were kept in fungizone-free medium. THP-1 cells were obtained from Dr. perature. Samples were loaded without loading dyes that interfere with 6 W. Jacobs (Albert Einstein College of Medicine) and propagated at 10 protein-DNA interactions. Gels containing 5.5% polyacrylamide, 5% glyc- cells/100-mm dish in RPMI 1640/10% FCS with antibiotics. erol, and 0.5ϫ Tris-glycine-EDTA were electrophoresed at 150 V for ϳ Reagents and treatment of cells 2.5 h. Immunocytochemistry 15d-PGJ2 was purchased from Cayman Chemical (Ann Arbor, MI). SB203580, SP600125, and 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyra- Immunocytochemistry was performed using rabbit Abs against p65 unit of zolo[3,4-d]pyrimidin (PP2) were purchased from Calbiochem (San Diego, NF-␬B (Santa Cruz Biotechnology, Santa Cruz, CA) and pStat1, which CA); U0126 was obtained from (Beverly, MA). Stocks of specifically recognizes phosphotyrosine at residue 701 (Cell Signaling, inhibitors were prepared in DMSO. LPS (Escherichia coli serotype 055: Ј Beverly, MA). Briefly, cells were fixed with cold methanol, then blocked B5) and 3 -azido-deoxythymidine (AZT) were purchased from Sigma-Al- with 10% normal goat serum. Primary Abs at 1/100 dilutions were applied drich (St. Louis, MO). IFN-␤ was purchased from PBL Biomedical Lab- ␤ ␣ to cells for 16 h at 4¡C. This was followed by incubation with biotinylated oratories (New Brunswick, NJ). IL-1 and TNF- were purchased from goat anti-rabbit IgG at 1/200 for 1 h at room temperature (Vectastain ABC PeproTech (Rocky Hill, NJ). Microglia or THP-1 cells were pretreated kit; Vector Laboratories, Burlingame, CA). The cells were then incubated with 15d-PGJ2 for 3 h (empirically determined to give maximal inhibition) with A ϩ B solution at a 1/200 dilution for 1 h room temperature. Color or other inhibitors for 1 h, then with cell stimuli for indicated time periods. ␮ was developed using diaminobenzidine. The concentration of 15d-PGJ2 used was 20 M for all experiments unless otherwise stated. Cytotoxicity was determined by MTT assay (Promega, Western blot analysis Madison, WI) following the manufacturer’s protocol. For HIV-1 experi- ␮ ments, the 15d-PGJ2 concentration was reduced to 10 M because of the Microglial cell lysates were prepared by scraping cells into 8 M urea. Total lengthy incubation periods required for viral production (4 wk). All cell protein (10Ð50 ␮g) was separated in 10% SDS-PAGE, then transferred to stimuli (cytokines and LPS) were used at 10 ng/ml based on previous polyvinylidene difluoride membrane. The blots were blocked in TBS/0.1% determination for microglial activation. Tween 20 (TTBS) containing 5% nonfat milk, then incubated with Abs to Stat1 or p38 (total or phospho; all from Cell Signaling) at 1/1000 dilutions HIV-1 exposure of microglia in 5% BSA/TTBS, according to the manufacturer’s instructions. After overnight incubation at 4¡C, the blots were washed in TTBS and then HIV-1 isolates were obtained from the AIDS Repository and propagated in further incubated with goat anti-rabbit IgG at 1/2000 dilution in 5% nonfat PBMC as previously described (27). Mock infection consisted of exposure milk/TTBS for1hatroom temperature. The reaction was developed using to PBMC supernatants without HIV-1. Infectious viruses were generated Ϫ ECL (Pierce, Rockford, IL). from Nef-deficient and wild-type HIV-1ADA proviruses (28) and Vpr Ϫ ϩ ϩ (pHXB (BaL)-R )orVpr (pHXB (BaL)-R ) proviruses (29) by trans- Data analysis fecting COS cells using Lipofectamine agents (Invitrogen Life Technolo- gies) as previously described (30). HIV infection of microglia was per- Experiments were repeated at least three times using different brain cases formed as previously described (26, 30) by exposure to 5Ð20 ng/ml p24 with similar results. Each data point represents values from triplicate wells 3506 15d-PGJ2 INHIBITS ACTIVATION OF HUMAN MICROGLIA

(mean Ϯ SD) from a single representative experiment. For statistical anal- ysis of the data, one-way ANOVA was performed, followed by Scheffe´’s multiple comparison procedure for comparing multiple treatment condi- tions or Student’s t test for comparing two treatment conditions. A value of p Ͻ 0.05 was considered significant. Results

15d-PGJ2 potently inhibits IP-10/CXCL10, but fails to inhibit IL-8/CXCL8, production in human microglia We determined the production of IP-10/CXCL10 and IL-8/CXCL8 in primary human microglia after stimulation with proinflamma- tory cytokines (IL-1 and TNF-␣), IFN-␤, and bacterial endotoxin ␮ (LPS). Cultures were pretreated with 15d-PGJ2 at 20 Mfor3h, then stimulated with cytokines, all at 10 ng/ml. ELISA was per- formed to determine the levels of chemokines in the culture su- pernatants 24 h after cell stimulation. The results shown in Fig. 1A demonstrate that high nanogram levels of IP-10/CXCL10 were produced in microglial cultures by LPS or IFN-␤ stimulation and picogram levels were produced by IL-1 and TNF-␣ stimulation. Downloaded from 15d-PGJ2 potently inhibited IP-10/CXCL10 production by all stimuli. In contrast, IL-8/CXCL8 was induced by LPS, IL-1, and ␣ ␤ TNF- , but not by IFN- (not shown), and 15d-PGJ2 significantly elevated the levels with all stimuli except IL-1 ( p Ͻ 0.05, by Student’s t test; Fig. 1B). Dose-response experiments were per- ␮ formed using increasing concentrations (0.1Ð20 M) of 15d-PGJ2, http://www.jimmunol.org/ and the results show that 15d-PGJ2 inhibited IP-10/CXCL10 with ϳ ␮ an IC50 at 3Ð5 M (Fig. 1C). MTT assay demonstrated that inhibition of IP-10/CXCL10 was not due to cell death (Fig. 1D).

15d-PGJ2 differentially regulates microglial and THP-1 cell chemokine gene expression

To determine whether 15d-PGJ2 affects IP-10/CXCL10 and IL-8/ CXCL8 mRNA expression, microglial cultures were examined with RPA using a commercial human chemokine probe set. Mi- by guest on September 24, 2021 croglial cells were pretreated with 15d-PGJ2 for 3 h, then stimu- lated for 6 h with LPS or cytokines, all at 10 ng/ml. As shown in Fig. 2A, stimulation of microglia with LPS, IFN-␤, or IL-1 induced

microglial IP-10/CXCL10 mRNA expression, and 15d-PGJ2 re- duced the amount of mRNA in all (see Fig. 2B for densitometry). IL-8/CXCL8 was induced by LPS or IL-1, but not by IFN-␤, and

15d-PGJ2 slightly enhanced the level of IL-8/CXCL8 mRNA (see Fig. 2C for densitometry). RANTES/CCL5, MIP-1␣/CCL3, MIP- 1␤/CCL4, and MCP-1/CXCL2 mRNA were induced by LPS, ␤ IFN- , and IL-1, but 15d-PGJ2 had little effect on these transcripts. In THP-1 cells, RPA showed robust IP-10/CXCL10 mRNA in- ␤ duction by LPS and IFN- and slight induction by IL-1; 15d-PGJ2 inhibited the induction in all (Fig. 2D). Robust IL-8/CXCL8

mRNA induction was noted in THP-1 cells by 15d-PGJ2 as pre-

viously reported (16). Interestingly, 15d-PGJ2 was a stronger stim- ulus than LPS or IL-1 for IL-8/CXCL8 induction in THP-1 cells (see Discussion). ␣ 15d-PGJ2 inhibits microglial cytokine (IL-1 and TNF- ) expression Microglial proinflammatory cytokines are pivotal in the establish- ment of inflammatory cascades in the injured CNS, including in- FIGURE 1. Microglial IP-10/CXCL10, but not IL-8/CXCL8, produc- duction of chemokine expression. Thus, the ability of 15d-PGJ2 to regulate IL-1 and TNF-␣ expression would be critical in the over- tion is inhibited by 15d-PGJ2. Human fetal microglia plated at 40,000 ␮ all regulation of inflammation in the CNS. We tested the effect of cells/well were stimulated with 20 M 15d-PGJ2 for 3 h, then with LPS or 15d-PGJ on microglial cytokine production using a human cyto- cytokines at 10 ng/ml for an additional 24 h. ELISA was performed of the 2 culture supernatants to determine the level of IP-10/CXCL10 (A) or IL-8/ kine RPA probeset from BD Pharmingen and by ELISA. Cells p Ͻ 0.05 vs without 15d-PGJ . 15d-PGJ dose response for ,ء .(CXCL8 (B ␮ 2 2 were pretreated with 15d-PGJ2 at 20 M for 3 h, then stimulated IP-10/CXCL10 inhibition was examined as described above and showed with IL-1 or LPS at 10 ng/ml for 6 h. As shown in Fig. 3, un- ␮ ϳ that 15d-PGJ2 inhibited IP-10/CXCL10 at 1Ð20 M, with an IC50 at 3 stimulated microglial cells expressed IL-1R antagonist (IL-1Ra) ␮M(C). MTT assay was performed in the same culture and showed no cell mRNA only. After stimulation with IL-1, multiple cytokine genes toxicity (D). Values shown are the mean Ϯ SD from triplicate wells. The Journal of Immunology 3507 Downloaded from

FIGURE 2. RPA of microglia and THP-1 cells for chemokine expres- sion. A, Microglia in 100-mm petri dish at 1 ϫ 106 cells/dish were treated http://www.jimmunol.org/ as described in Fig. 1, except that total RNA was harvested 6 h after LPS/cytokine stimulation. Chemokine mRNA was analyzed using a BD Pharmingen probe set. B and C, Densitometric ratios of IP-10/CXCL10 and IL-8/CXCL8 mRNA to GAPDH, respectively. D, THP-1 cells were treated as described for microglia and analyzed for chemokine mRNA expression. IP-10/CXCL10 and MCP-1 were inhibited, but IL-8/CXCL8 was induced,

by 15d-PGJ2.

were induced (TNF-␣, IL-1␣, IL-1␤, IL-1Ra and IL-6) as reported by guest on September 24, 2021

previously (21, 32), and 15d-PGJ2 inhibited all except IL-1Ra mRNA (see Fig. 3B for densitometry). LPS-induced cytokine

genes were also similarly inhibited by 15d-PGJ2 (not shown), and ELISA showed that 15d-PGJ2 nearly completely inhibited LPS- induced IL-1 or TNF-␣ production in microglia (Fig. 3C). ␬ 15d-PGJ2 inhibits microglial NF- B activation To determine which microglial activation pathways are targeted by ␬ 15d-PGJ2, we used EMSA to examine NF- B activation by LPS or ␤ ␮ IFN- . Microglia were pretreated with 15d-PGJ2 at 20 Mfor3h, then with LPS or IFN-␤ at 10 ng/ml for 30 min. The nuclear extracts were prepared, and EMSA was performed with an NF-␬B consensus oligonucleotide as previously described (33). As shown in Fig. 4A, nuclear NF-␬B binding activity was induced in micro- glia treated with either LPS or IFN-␤; of the three complexes vis- ible on the gel, the top band represents the p65/p50 heterodimer, which is induced by cytokine or LPS stimulation (33). Notably, ␬ 15d-PGJ2 inhibited NF- B nuclear binding activity induced by ␤ FIGURE 3. 15d-PGJ2 inhibits microglial cytokine expression. A,Mi- LPS or IFN- . Furthermore, 15d-PGJ2 inhibited nuclear translo- cation of NF-␬B (p65 subunit) in microglia, as determined by im- croglia were treated as described in Fig. 2, and total RNA was analyzed for cytokine mRNA expression using a Quantikine probe set from BD Phar- munocytochemistry (Fig. 4B). These results indicate that in pri- mingen. B, Densitometric analysis of cytokine mRNA against GAPDH ␬ mary human microglia, 15d-PGJ2 inhibits NF B nuclear mRNA shown in A. C, Cytokine (TNF-␣ and IL-1␤) protein levels were translocation and DNA binding, in contrast to results obtained in determined by ELISA in LPS-treated cultures with or without 15d-PGJ2,as murine microglia (11). described for chemokines in Fig. 1.

15d-PGJ2 inhibits Stat1 activation in microglia

The Jak/Stat pathways are the major signaling pathways activated effects of 15d-PGJ2 on Stat1, a transcription factor essential for by IFNs. Our results in microglia demonstrate that IFN-␤ as well both IFN-␣/IFN-␤ and IFN-␥ signaling. We examined Stat1 phos- ␥ as IFN- are potent inducers of IP-10/CXCL10, and that 15d-PGJ2 phorylation by Western blot analysis using an Ab specifictoty- inhibits IFN-induced IP-10/CXCL10. Therefore, we examined the rosine-phosphorylated (Y701) Stat1. As shown in Fig. 4C, IFN-␤ 3508 15d-PGJ2 INHIBITS ACTIVATION OF HUMAN MICROGLIA Downloaded from http://www.jimmunol.org/

␬ FIGURE 4. 15d-PGJ2 inhibits microglial NF- B binding activity in- duced by LPS or IFN-␤. A, EMSA was performed with microglial cell ␮ nuclear extracts prepared by pretreatment of cells with 20 M 15d-PGJ2 by guest on September 24, 2021 for 3 h, then for 30 min with 10 ng/ml LPS or IFN-␤. The top band represents the p65/p50 heterodimer. Specific competitor (SC) for EMSA consisted of a 100-fold excess of unlabeled NF-␬B probe. Nonspecific competitor (NSC) was the same, except an irrelevant probe was used. B, NF-␬B (p65) immunocytochemistry in microglia treated as described in A, demonstrating nuclear localization of p65 induced by LPS, which was re-

duced by pretreatment with 15d-PGJ2. C, 15d-PGJ2 inhibits Stat1 activa- ␮ tion. Microglia were pretreated with 15d-PGJ2 at 20 M for 3 h, then with 10 ng/ml IFN-␤ for either 0.5 or 2 h. Western blot analysis was performed with an Ab specific to phosphorylated Stat1 (Y701). The blot was stripped FIGURE 5. Adenoviral transduction of primary human microglia with and reprobed for total Stat1 (C). D, Immunocytochemistry for pStat1 re- super-repressor I␬B␣ (Ad-I␬B␣) or control vector (Ad-CMV) demon- ␤ vealed that after IFN- treatment (30 min), the majority of microglial cells strates the role of NF-␬B in both IP-10/CXCL10 and IL-8/CXCL8 pro- had strong nuclear pStat1 immunoreactivity, which was prevented by pre- duction induced by LPS or IL-1␤. A and B, Microglia were infected with 4 4 treatment with 15d-PGJ2. Ad-CMV or Ad-I␬B␣ at either 10 or 2.5 ϫ 10 PFU/cell for 24 h, then treated with LPS at 10 ng/ml for an additional 24 h. Culture supernatants were examined for IP-10/CXCL10 and IL-8/CXCL8 levels with ELISA. C, induced phospho-Stat1 in microglia 30 min, but not 2 h, after stim- Microglia were infected with adenoviral vectors as described above, then ␮ ulation, and 15d-PGJ2 reduced the amount of phospho-Stat1 in treated with 20 M 15d-PGJ2 for 72 h. IL-8/CXCL8 levels were deter- p Ͻ ,ء .mined by ELISA. Values are the mean Ϯ SD from triplicate wells microglia. Furthermore, 15d-PGJ2 reduced the nuclear accumula- tion of phospho-Stat1 induced by IFN-␤ (30 min) determined by 0.05 vs Ad-CMV. immunocytochemistry (Fig. 4D). ␬ Role of NF- B in microglial IP-10/CXCL10 and IL-8/CXCL8 the CMV promoter (Ad-CMV). As shown in Fig. 5A, super-re- expression pressor I␬B␣ (Ad-I␬B␣) inhibited IP-10/CXCL10 production in To examine the role of NF-␬B in the induction of IP-10/CXCL10 microglia, demonstrating the involvement of NF-␬B in IP-10/ and IL-8/CXCL8, we used adenovirus-mediated expression of CXCL10 expression. The observed increase in IP-10/CXCL10 dominant negative (DN) vectors in primary microglia as previ- production by control adenoviral vector (Ad-CMV) is consistent ously described (33). Super-repressor I␬B␣ is a proteolysis-resis- with the reported immunogenic effect of adenovirus, such as acti- tant form of I␬B␣ and, hence, functions as a DN-NF-␬B. Micro- vation of NF-␬B and MAPKs (34, 35); however, the control ad- glial cells were infected with adenovirus for 24 h at the indicated enovirus did not have the same effect on IL-8/CXCL8 production concentrations, then stimulated with LPS for 24 h; control cultures for an unknown reason (Fig. 5B; also see Fig. 6B). Similar to were infected with equal amounts of adenoviruses carrying only IP-10/CXCL10, production of IL-8/CXCL8 after LPS treatment The Journal of Immunology 3509 Downloaded from http://www.jimmunol.org/

FIGURE 7. Effect of adenoviral transduction of DN-MKK6 and Ras on chemokine production. Microglia were infected with adenoviral vectors carrying DN-MKK6, DN-Ras, or CMV promoter only (control) at 100 PFU/cell for 24 h, then stimulated with LPS for an additional 24 h. Values .(p Ͻ 0.05 vs control (Ad-CMV ,ء .are the mean Ϯ SD

20 ␮M), ERK MAPK inhibitor (UO126 at 20 ␮M), JNK MAPK inhibitor (SP600125 at 0.1 ␮M), or the Src family kinase inhibitor ␮ by guest on September 24, 2021 PP-2 at 1 M), based on published IC50 values reported for the spe- cific kinases (36, 37). Cells were then stimulated with LPS at 10 ng/ml for 24 h, and ELISA was performed. As shown in Fig. 6A, the p38 inhibitor, but not ERK, JNK, or Src inhibitors, inhibited IP-10/ CXCL10 production. Interestingly, none of the inhibitors affected the FIGURE 6. Roles of MAPK and Src kinase inhibitors in the induction production of IL-8/CXCL8 in microglia induced by either LPS or of microglial IP-10/CXCL10 and IL-8/CXCL8. Microglia were pretreated IL-1 (Fig. 6,B and C). ␮ for 1 h with inhibitors (SB203580, UO126, SP600125, and PP2) at 20 M, Microglial IP-10/CXCL10 production is inhibited by DN-MKK6 then with LPS or IL-1 at 10 ng/ml. ELISA was performed to determine the levels of chemokines in 24-h culture supernatants. Values are the mean Ϯ To confirm the selective role of p38 MAPK in the induction of p Ͻ 0.05 vs LPS alone. IP-10/CXCL10, we used adenoviral transduction of DN-MKK6 ,ء .SD and DN-Ras. MKK6 is a MEK that is upstream of p38 MAPK; Ras is upstream of MEK/ERK MAPK (38, 39). We have previously was also inhibited by super-repressor NF-␬B (Ad-I␬B␣) in micro- determined that in microglia, transduction with DN Ras inhibits glia (Fig. 5B), demonstrating that NF-␬B was a positive regulator phosphorylation of ERK (67). Microglia were treated first with of IL-8/CXCL8 induction by LPS. In contrast, the induction of adenoviruses, then with LPS, as described for Ad-I␬B␣, then che- IL-8/CXCL8 in control (baseline) or 15d-PGJ2-treated microglia mokine levels were determined after 24 h. Fig. 7A shows that was not inhibited by super-repressor I␬B␣ (Fig. 5C), demonstrat- LPS-induced IP-10/CXCL10 production was inhibited by Ad-DN- ing that baseline or 15d-PGJ2-induced IL-8/CXCL8 was not me- MKK6, but not by Ad-DN-Ras. In contrast, neither virus affected diated by NF-␬B. Together these results suggest that different the production of IL-8/CXCL8 in LPS-stimulated microglia (Fig. mechanisms are involved in IL-8 induction in the presence or the 7B). The observed increase in IP-10/CXCL10 production by con- absence of 15d-PGJ2 and that the failure of 15d-PGJ2 to inhibit trol adenoviral vector (Ad-CMV) is consistent with the immuno- IL-8 production may be due in part to the presence of an NF-␬B- genic effect of adenovirus (34, 35). Together, these results confirm independent IL-8 induction pathway activated by 15d-PGJ2 (see that p38 MAPK is differentially involved in the expression of the Discussion). two ␣-chemokines in human microglia. Role of MAPKs in microglial IP-10/CXCL10 and IL-8/CXCL8 p38 MAPK is differentially activated in primary human production microglia and THP-1 cells We next examined whether microglial MAPKs or Src family kinases Because the inhibition data demonstrated that p38 is involved in are involved in IP-10/CXCL10 and IL-8/CXCL8 expression. Micro- IP-10/CXCL10, but not in IL-8/CXCL8, induction, we next ex-

glia were pretreated for 1 h with p38 MAPK inhibitor (SB203580 at amined whether 15d-PGJ2 inhibits p38 phosphorylation. Western 3510 15d-PGJ2 INHIBITS ACTIVATION OF HUMAN MICROGLIA

FIGURE 9. HIV-1 induction of IP-10/CXCL10 in microglia. A, Micro-

glia were either mock-infected or exposed to R5 strains (ADA or BaL) of Downloaded from HIV-1 for 16 h, then cultures were kept for 21 days with weekly collection of medium as described. IP-10/CXCL10 levels were determined in the weekly supernatants. B, Microglia were exposed to HIV-1ADA with or without AZT at 10 ␮M for 16 h, then cultures were kept for 12 days. IP-10/CXCL10 levels were determined by ELISA. C, Microglial cultures were mock-infected or infected with wild-type (Nefϩ) or Nef-deficient Ϫ (Nef ) HIV-1ADA, as described previously. Weekly culture supernatants http://www.jimmunol.org/ FIGURE 8. The p38 MAPK activation was regulated by 15d-PGJ . A, ϩ 2 were examined for IP-10/CXCL10. D, Microglia were infected with Vpr Microglia were pretreated with 30 ␮M 15d-PGJ for 3 h, then with 10 ϩ Ϫ Ϫ 2 (pHXB (BaL)-R )orVpr (pHXB (BaL)-R ) virus as described previ- ng/ml LPS or IFN-␤. were harvested after 30 min, and Western ously, then IP-10/CXCL10 levels were examined in the weekly culture blot was performed for phospho- and total p38. B, THP-1 cells were treated supernatants. Values are the mean Ϯ SD from triplicate wells in all assays. with 15d-PGJ2 and LPS as described for microglia, and Western blot anal- ysis was performed for p38. Robust p38 phosphorylation was detected in THP-1 cells and not in microglia. shown), strains induced IP-10/CXCL10 (Fig. 9A). In addition, the reverse transcriptase inhibitor AZT abolished IP-10/CXCL10 pro- blot analysis was performed with microglia pretreated 3 h with duction (Fig. 9B). The roles of Nef and Vpr were tested using by guest on September 24, 2021 ␤ various concentrations of 15d-PGJ2, then with LPS or IFN- at 10 mutant viruses. The results showed that microglial IP-10/CXCL10 ng/ml for 30 min. As shown in Fig. 8A, LPS and IFN-␤ (but not production was dependent on Nef, but not Vpr (Fig. 9, C and D).

15d-PGJ2) induced phosphorylation of p38 in microglia, and 15d- PGJ reduced the amount of phospho-p38 in both. However, this 15d-PGJ2 inhibits HIV-1-induced IP-10/CXCL10 production in 2 microglia was seen only with high concentrations of 15d-PGJ2 (results with 30 ␮M shown). These results were different from those obtained in We then tested our HIV-infected microglial culture to determine

THP-1 cells. THP-1 cells were pretreated with 15d-PGJ2 for3h, whether 15d-PGJ2 can inhibit virus-induced IP-10/CXCL10 ex- then with LPS for the indicated time periods. Phospho-p38 was pression. Microglia were pretreated with 15d-PGJ2 and exposed to measured by Western blot analysis (Fig. 8B). In THP-1 cells, 15d- HIV-1 as described above. Culture medium was changed weekly,

PGJ2 induced strong and sustained phosphorylation of p38 MAPK with replenishment of 15d-PGJ2. Culture supernatants were exam- (shown up to 4 h), whereas LPS was a weak inducer of phospho- ined for the production of IP-10/CXCL10, IL-8/CXCL8, and p38; 15d-PGJ2 did not show an inhibitory effect on p38 phosphor- HIV-1 p24 by ELISA. As shown in Fig. 10A, 15d-PGJ2 was a ylation. Together, our results demonstrate a complex role played potent inhibitor of IP-10/CXCL10 induction by HIV-1 in micro- by 15d-PGJ2 in the microglial activation pathway and that its ef- glia. Interestingly, IL-8/CXCL8 accumulated in control microglial fects in primary microglia are different from those in THP-1 cells. cultures, and exposure to HIV-1 did not affect the expression of

IL-8/CXCL8 (Fig. 10B). However, in the presence of 15d-PGJ2, IP-10/CXCL10 is induced in microglia by HIV-1 in a microglial IL-8/CXCL8 production was increased in both control replication- and Nef-dependent manner (7, 14, and 28 days) and HIV-1-exposed (21 and 28 days) cultures

To determine whether 15d-PGJ2 would inhibit IP-10/CXCL10 in (Fig. 10B). The HIV-1 p24 ELISA demonstrated that 15d-PGJ2 the context of a disease paradigm, we used our culture system for induced mild inhibition of viral production; however, the inhibi- HIV-1-infected microglia. Although HIV-1-infected -de- tion was not statistically significant, except on day 14 (Fig. 10C). rived macrophages have been shown to produce IP-10 (40), pro- duction in microglia has not specifically been shown. In this study Discussion we confirm that microglia do indeed produce IP-10 upon infection We report in this study that the cyclopentenone PG, 15d-PGJ2, with HIV-1. Microglia were exposed to HIV-1, and the superna- modulates human microglial inflammatory gene expression. In ad- tants were collected weekly, then analyzed for IP-10/CXCL10 by dition to the proinflammatory cytokines (IL-1, TNF-␣, and IL-6), ELISA, as previously described (30). HIV-1 induced IP-10/ IP-10/CXCL10 expression was consistently inhibited by 15d-

CXCL10 in microglia in a time-dependent manner, with peak pro- PGJ2. Interestingly, the expression of IL-8/CXCL8 was not inhib- tein expression at 14Ð28 days (Fig. 9). IP-10/CXCL10 production ited by 15d-PGJ2, but, rather, was induced by it. The IL-8/CXCL8- was viral strain-dependent; R5 (ADA and BaL), but not X4 (not inducing effect of 15d-PGJ2 often showed synergism with other The Journal of Immunology 3511

has been demonstrated in several studies; proteasome inhibition that abolishes NF-␬B activation has been shown to induce IL-8/ CXCL8 expression through the AP-1 pathway (44). Furthermore, in U937 cells, the promoter region responsible for IL-8/CXCL8 Ϫ induction by 15d-PGJ2 did not include the proximal ( 133 bp) promoter region, which contains cis elements for NF-␬B, AP-1, and C/EBP transcription factors (16). Therefore, it is feasible that

15d-PGJ2 has opposing effects on IL-8/CXCL8 transcription: in- hibition of NF-␬B-dependent transcription and stimulation of NF-␬B-independent transcription. Activation of ISRE elements has been shown to be pivotal in the induction of the IP-10/CXCL10 gene (45, 46). Transcription fac- tors belonging to the Stat or IRF family are activated by IFNs or virus and bind to the ISRE element in the promoter. We deter- ␤ mined that 15d-PGJ2 reduced IFN- -mediated Stat1 phosphoryla- tion in microglia, thereby implicating yet another mechanism for

15d-PGJ2-mediated IP-10/CXCL10 inhibition. Recently, Park et

al. (47) demonstrated that 15d-PGJ2 induces suppressor of cyto- kine signaling proteins, resulting in the inhibition of Stat1 and Downloaded from ϳ Stat3 signaling. In microglia, pretreatment ( 3 h) with 15d-PGJ2 was necessary for maximal inhibition of IP-10/CXCL10 produc- tion (data not shown), suggesting that transcriptional induction of inhibitor proteins, such as suppressor of cytokine signaling, may

be involved. Together, these results support the idea that 15d-PGJ2 inhibits microglial IP-10/CXCL10 expression by targeting multi- http://www.jimmunol.org/ ple cell activation pathways, which may act synergistically. In addition to NF-␬B and Stat proteins, MAPK and Src kinases have been shown to be involved in macrophage chemokine gene expression. In the current study we found that p38 MAPK plays a positive role in IP-10/CXCL10 expression in microglia. This was demonstrated using pharmacological inhibitors as well as adeno- viral transduction of DN-MKK6 (upstream of p38). Neither IP-10/

CXCL10 nor IL-8/CXCL8 production was affected by inhibitors of by guest on September 24, 2021 ERK, JNK, or Src kinases. These results contrast with those we obtained for ␤-chemokines (RANTES/CCL5 and MIP-1␤/CCL4 FIGURE 10. IP-10/CXCL10, but not IL-8/CXCL8, produced in HIV- induction by IFN-␤ and HIV-1), in which p38 was shown to have 1-exposed microglial cultures is inhibited by 15d-PGJ . Microglia were 2 a negative regulatory role (30, 33). The lack of inhibition by exposed to HIV-1ADA as described in Fig. 9, except that they were pre- ␮ MAPK or Src kinase inhibitors was also inconsistent with the pre- treated for 3 h with 15d-PGJ2 at 10 M before exposure to HIV-1. Values are the mean Ϯ SD from triplicate wells. IP-10/CXCL10 and IL-8/CXCL8 viously reported roles of these kinases in IL-8/CXCL8 induction in

were determined in the same culture supernatants (A and B). 15d-PGJ2 had other cell types (43). Therefore, our results demonstrate the spec- differential effects in these cultures; although IP-10/CXCL10 induction was ificity that is required for cell-, stimulus-, and target gene-depen- completely inhibited, IL-8/CXCL8 production in mock-infected and HIV- dent control of gene expression. We also found that both LPS- and 1-infected cultures was increased by 15d-PGJ2. ␤ IFN- -induced p38 phosphorylation was reduced by 15d-PGJ2 in microglia, although this required a higher drug concentration (30 ␮ stimuli, such as TNF-␣ or HIV-1. To elucidate the mechanism M). Inhibition of Stat1 or p38 activation in combination with NF-␬B inhibition may all contribute to suppression of IP-10/ underlying 15d-PGJ2-mediated cytokine and chemokine gene modulation, we determined NF-␬B and Stat1 activation. We found CXCL10 production. ␬ In contrast to microglia, in THP-1 cells 15d-PGJ was a strong that 15d-PGJ2 inhibited NF- B nuclear translocation and DNA 2 binding in human microglia. These results are similar to those inducer of p38 phosphorylation. The activation was striking and reported by Rossi et al. (41) and Straus et al. (42), which demon- paralleled the induction of IL-8/CXCL8 mRNA in THP-1 cells. In ␬ ␣ this regard, it is interesting that 15d-PGJ has been shown to in- strated that 15d-PGJ2 directly inhibits I B kinase activity as well 2 as NF-␬B DNA binding, but different from those found in murine duce robust and sustained MAPK activation in astrocytes and prea- microglia, which failed to show inhibition of NF-␬B-DNA binding dipocytes and to contribute to IL-8 induction in T cells (17, 48). (11). Our results with NF-␬B, however, do not explain why IL-8/ Therefore, 15d-PGJ2’s ability to trigger MAPK activation and IL-

CXCL8 induction was not inhibited by 15d-PGJ2. IL-8/CXCL8 8/CXCL8 induction may not be unique to THP-1 cells. Reportedly, gene expression is regulated by several mechanisms, including 15d-PGJ2 has both stimulatory and inhibitory roles in MAPK and transcriptional activation by NF-␬B (43). In our study, adenoviral AP-1 activation (4, 49), partly dependent upon the presence or the transduction of super-repressor I␬B␣ inhibited IL-8/CXCL8 absence of other cell stimuli (4, 17, 48). These results are not

induction by LPS. Yet, 15d-PGJ2 failed to inhibit IL-8/CXCL8 dissimilar to ours, which demonstrated that 15d-PGJ2 activated expression. We also demonstrated that baseline or 15d-PGJ2- p38 phosphorylation in THP-1 cells, which, conversely, was in- mediated IL-8/CXCL8 production was not inhibited by the hibited in LPS-stimulated microglia. It is possible that low level super-repressor I␬B␣, suggesting an NF-␬B-independent mech- (below detection limit) activation also occurred in microglia, anism. Indeed, NF-␬B-independent IL-8/CXCL8 expression which contributed to the NF-␬B-independent induction of IL-8. 3512 15d-PGJ2 INHIBITS ACTIVATION OF HUMAN MICROGLIA

Because chemokines are also induced by virus, we examined References whether HIV-induced chemokine expression was similarly regu- 1. Harris, S. G., J. Padilla, L. Koumas, D. Ray, and R. P. Phipps. 2002. Prostaglan- lated by 15d-PGJ . First we found that HIV-1 infection induced dins as modulators of immunity. Trends Immunol. 23:144. 2 2. Shibata, T., M. Kondo, T. Osawa, N. Shibata, M. Kobayashi, and K. Uchida. IP-10/CXCL10 in microglia in amounts similar to those induced 2002. 15-Deoxy-⌬12,14-prostaglandin J : a prostaglandin D metabolite generated ␤ 2 2 by LPS or IFN- , and this was inhibited by 15d-PGJ2. In contrast, during inflammatory processes. J Biol. Chem. 277:10459. 3. Clark, R. B. 2002. The role of PPARs in inflammation and immunity. J Leukocyte IL-8/CXCL8 accumulated in microglial cultures spontaneously, Biol. 71:388. with 15d-PGJ2 alone, or together with HIV-1. As Nef is an early 4. Ricote, M., A. C. Li, T. M. Willson, C. J. Kelly, and C. K. Glass. 1998. The regulatory protein positively involved in viral replication and che- peroxisome proliferator-activated receptor-␥ is a negative regulator of macro- phage activation. Nature 391:79. mokine synthesis (Refs. 28, 30, and 50 and this study), our data 5. Chawla, A., Y. Barak, L. Nagy, D. Liao, P. Tontonoz, and R. M. Evans. 2001. ␥ suggest that 15d-PGJ2 may have inhibited Nef signaling in micro- PPAR- dependent and independent effects on macrophage-gene expression in lipid metabolism and inflammation. Nat. Med. 7:48. glia. In fact, Nef has been shown to trigger several cell activation 6. Welch, J. S., M. Ricote, T. E. Akiyama, F. J. Gonzalez, and C. K. Glass. 2003. pathways in macrophages, including Hck (myeloid-specific Src ki- PPAR␥ and PPAR⌬ negatively regulate specific subsets of lipopolysaccharide nase), Stat1, and NF-␬B (51Ð53). However, because IP-10/ and IFN-␥ target genes in macrophages. Proc. Natl. Acad. Sci. USA 100:6712. 7. Thieringer, R., J. E. Fenyk-Melody, C. B. Le Grand, B. A. Shelton, CXCL10 production was dependent on viral replication in micro- P. A. Detmers, E. P. Somers, L. Carbin, D. E. Moller, S. D. Wright, and J. Berger. 2000. Activation of peroxisome proliferator-activated receptor ␥ does not inhibit glia, these findings also raise the question of whether 15d-PGJ2 IL-6 or TNF-␣ responses of macrophages to lipopolysaccharide in vitro or in simply acted as an antiviral agent, such as that shown for PGA1 vivo. J. Immunol. 164:1046. and PGA2 in macrophages (54). However, our results seem to 8. Bernardo, A., M. A. Ajmone-Cat, G. Levi, and L. Minghetti. 2003. 15-Deoxy- ⌬12,14 suggest that the two events (HIV-1 and IP-10/CXCL10 expres- -prostaglandin J2 regulates the functional state and the survival of micro- glial cells through multiple molecular mechanisms. J. Neurochem. 87:742. Downloaded from sion) can be dissociated from each other. Additional mechanisms 9. Wang, C., M. Fu, M. D’Amico, C. Albanese, J. N. Zhou, M. Brownlee, M. P. Lisanti, V. K. Chatterjee, M. A. Lazar, and R. G. Pestell. 2001. Inhibition of 15d-PGJ2 action in HIV-1-infected cultures may include inhi- ␣ ␤ ␣ ␤ of cellular proliferation through I␬B kinase-independent and peroxisome prolif- bition of IFN- /IFN- signaling, because IFN- /IFN- are pro- erator-activated receptor ␥-dependent repression of cyclin D1. Mol. Cell. Biol. duced after HIV-1 infection (55, 56) and are effective inducers of 21:3057. IP-10/CXCL10 (31). 10. Hortelano, S., A. Castrillo, A. M. Alvarez, and L. Bosca. 2000. Contribution of ␥ cyclopentenone prostaglandins to the resolution of inflammation through the po- Because 15d-PGJ2 and other PPAR agonists exhibit macroph- tentiation of apoptosis in activated macrophages. J. Immunol. 165:6525. age-suppressive properties, they have been proposed as potential 11. Petrova, T. V., K. T. Akama, and L. J. Van Eldik. 1999. Cyclopentenone pros- http://www.jimmunol.org/ taglandins suppress activation of microglia: down-regulation of inducible nitric- therapeutic agents for certain CNS diseases (57). For instance, ⌬12,14 oxide synthase by 15-deoxy- -prostaglandin J2. Proc. Natl. Acad. Sci. USA beneficial effects of PPAR␥ agonists have been reported in the 96:4668. animal model for multiple sclerosis, experimental allergic enceph- 12. Bernardo, A., G. Levi, and L. Minghetti. 2000. Role of the peroxisome prolif- erator-activated receptor-␥ (PPAR-␥) and its natural ligand 15-deoxy-⌬12,14- alomyelitis (58, 59). Because microglial activation products are prostaglandin J2 in the regulation of microglial functions. Eur. J. Neurosci. generally thought to be harmful, and they mediate neuronal dam- 12:2215. 13. Henson, P. 2003. Suppression of macrophage inflammatory responses by PPARs. age in diseases such as HIV encephalitis and Alzheimer’s disease, Proc. Natl. Acad. Sci. USA 100:6295. our results (inhibition of proinflammatory cytokines) suggest that 14. Janabi, N. 2002. Selective inhibition of cyclooxygenase-2 expression by 15-de- oxy-⌬12,14-prostaglandin J in activated human astrocytes, but not in human brain 15d-PGJ may prove to be beneficial in these diseases. In contrast, 2 by guest on September 24, 2021 2 macrophages. J. Immunol. 168:4747. the significance of our findings of differential chemokine regula- 15. Jiang, C., A. T. Ting, and B. Seed. 1998. PPAR-␥ agonists inhibit production of monocyte inflammatory cytokines. Nature 391:82. tion by 15d-PGJ2 cannot be easily inferred. IP-10/CXCL10 is one 16. Zhang, X., J. M. Wang, W. H. Gong, N. Mukaida, and H. A. Young. 2001. of the first macrophage chemokines to be produced after microbial Differential regulation of chemokine gene expression by 15-deoxy-⌬12,14-pros- infection and is central to host innate immunity (60, 61). Increased taglandin J2. J. Immunol. 166:7104. ⌬12,14 levels of IP-10/CXCL10 protein and mRNA have been detected in 17. Harris, S. G., R. S. Smith, and R. P. Phipps. 2002. 15-Deoxy- -PGJ2 induces IL-8 production in human T cells by a mitogen-activated protein kinase pathway. cerebrospinal fluid as well as brain from patients with CNS HIV J. Immunol. 168:1372. infection (40, 62); furthermore, there is evidence suggesting that 18. Fu, Y., N. Luo, and M. F. Lopes-Virella. 2002. Upregulation of -8 ⌬12,14 IP-10/CXCL10 may contribute to neurological dysfunction and expression by prostaglandin D2 metabolite 15-deoxy- -prostaglandin J2 (15d-PGJ2) in human THP-1 macrophages. Atherosclerosis 160:11. increased viral replication (62, 63). Similarly, IL-8/CXCL8 has 19. Jozkowicz, A., J. Dulak, M. Prager, J. Nanobashvili, A. Nigisch, B. Winter, been shown to inhibit long term potentiation in SCID mice and to G. Weigel, and I. Huk. 2001. Prostaglandin-J2 induces synthesis of interleukin-8 by endothelial cells in a PPAR-␥-independent manner. Prostaglandins Other increase HIV-1 replication in T cells and macrophages (64, 65). Lipid Mediat. 66:165. ␣ 20. Lee, S. C., W. Liu, C. F. Brosnan, and D. W. Dickson. 1992. Characterization of Therefore, the implication of 15d-PGJ2-induced -chemokine im- human fetal dissociated CNS cultures with an emphasis on microglia. Lab. Invest. balance in HIV encephalitis is unclear. Interestingly, due to the 67:465. presence and absence of the ELR motif, IL-8/CXCL8 and IP-10/ 21. Lee, S. C., W. Liu, D. W. Dickson, C. F. Brosnan, and J. W. Berman. 1993. CXCL10 have been shown to possess opposing properties (66), Cytokine production by human fetal microglia and astrocytes: differential induc- ϩ tion by LPS and IL-1b. J. Immunol. 150:2659. raising the possibility that 15d-PGJ2 may create a pro-ELR state 22. Lee, S. C., W. C. Hatch, W. Liu, W. D. Lyman, Y. Kress, and D. W. Dickson. (, neurophil infiltration, etc.). However, it is not 1993. Productive infection of human fetal microglia by HIV-1. Am. J. Pathol. ϩ Ϫ 143:1032. known whether other members of the ELR and ELR chemo- 23. Liu, J., M.-L. Zhao, C. F. Brosnan, and S. C. Lee. 1996. Expression of type II kines are under similar controls by 15d-PGJ2. Clearly, more stud- nitric oxide synthase in primary human astrocytes and microglia: role of IL-1b ies are needed to understand the biological significance of the ob- and IL-1 receptor antagonist. J. Immunol. 157:3569. 24. Dickson, D. W., S. C. Lee, L. Mattiace, S.-H. Yen, and C. F. Brosnan. 1993. servations made in this study. Microglia and cytokines in neurological disease, with special reference to AIDS and Alzheimer’s disease. Glia 7:75. 25. Lee, S. C., D. W. Dickson, and C. F. Brosnan. 1995. Interleukin-1, nitric oxide and reactive astrocytes. Brain Behav. Immun. 9:345. Acknowledgments 26. Kitai, R., M. L. Zhao, N. Zhang, L. L. Hua, and S. C. Lee. 2000. Role of MIP-1b and RANTES in HIV-1 infection of microglia: inhibition of infection and induc- We thank Wa Shen for microglial culture, and the Einstein Human Fetal tion by IFN␤. J. Neuroimmunol. 110:230. Tissue Repository for fetal tissue. We thank the AIDS Research and Ref- 27. Si, Q., M. Cosenza, M.-L. Zhao, H. Goldstein, and S. C. Lee. 2002. GM-CSF and erence Reagent Program (National Institute of Allergy and Infectious Dis- M-CSF modulate ␤-chemokine and HIV-1 expression in microglia. Glia 39:174. eases) and Drs. Mario Stevenson and Ned Landau for providing HIV-1. 28. Swingler, S., A. Mann, J. Jacque, B. Brichacek, V. G. Sasseville, K. Williams, A. A. Lackner, E. N. Janoff, R. Wang, D. Fisher, et al. 1999. HIV-1 Nef mediates Drs. Richard Pestell and Sakae Tanaka provided the adenoviral vectors lymphocyte chemotaxis and activation by infected macrophages. Nat. Med. carrying SR-I␬B␣ and DN-MKK6, respectively. 5:997. The Journal of Immunology 3513

29. Connor, R. I., B. K. Chen, S. Choe, and N. R. Landau. 1995. Vpr is required for 49. Zhang, X., and H. A. Young. 2002. PPAR and immune system: what do we efficient replication of human immunodeficiency virus type-1 in mononuclear know? Int. Immunopharmacol. 2:1029. phagocytes. Virology 206:935. 50. Sanceau, J., T. Kaisho, T. Hirano, and J. Wietzerbin. 1995. Triggering of the 30. Si, Q., M. O. Kim, M.-L. Zhao, N. R. Landau, H. Goldstein, and S. C. Lee. 2002. human interleukin-6 gene by -␥ and -a in mono- Vpr- and Nef-dependent induction of RANTES in microglial cells. Virology cytic cells involves cooperation between interferon regulatory factor-1, NF-␬B, 301:342. and Sp1 transcription factors. J. Biol. Chem. 270:27920. 31. Hua, L. L., and S. C. Lee. 2000. Distinct patterns of stimulus-inducible chemo- 51. Briggs, S. D., M. Sharkey, M. Stevenson, and T. E. Smithgall. 1997. SH3-me- kine mRNA accumulation in human fetal astrocytes and microglia. Glia 30:74. diated Hck tyrosine kinase activation and fibroblast transformation by the Nef 32. Liu, J. S. H., T. Amaral, C. F. Brosnan, and S. C. Lee. 1998. are protein of HIV-1. J Biol. Chem. 272:17899. critical regulators of IL-1b and IL-1ra expression in human fetal microglia. J. Im- 52. Federico, M., Z. Percario, E. Olivetta, G. Fiorucci, C. Muratori, A. Micheli, munol. 161:1989. G. Romeo, and E. Affabris. 2001. HIV-1 Nef activates STAT1 in human mono- 33. Kim, M. O., Q. Si, J. N. Zhou, R. G. Pestell, C. F. Brosnan, J. Locker, and cytes/macrophages through the release of soluble factors. Blood 98:2752. S. C. Lee. 2002. Interferon-␤ activates multiple signaling cascades in primary 53. Olivetta, E., Z. Percario, G. Fiorucci, G. Mattia, I. Schiavoni, C. Dennis, J. Jager, human microglia. J. Neurochem. 81:1361. M. Harris, G. Romeo, E. Affabris, et al. 2003. HIV-1 Nef induces the release of 34. Horwitz, M. S. 2001. Adenovirus immunoregulatory genes and their cellular inflammatory factors from human monocyte/macrophages: involvement of Nef ␬ targets. Virology 279:1. endocytotic signals and NF- B activation. J. Immunol. 170:1716. 35. Bhat, N. R., and F. Fan. 2002. Adenovirus infection induces microglial activa- 54. Hayes, M. M., B. R. Lane, S. R. King, D. M. Markovitz, and M. J. Coffey. 2002. ␥ tion: involvement of mitogen-activated protein kinase pathways. Brain Res. Peroxisome proliferator-activated receptor agonists inhibit HIV-1 replication in 948:93. macrophages by transcriptional and post-transcriptional effects. J. Biol. Chem. 36. Davies, S. P., H. Reddy, M. Caivano, and P. Cohen. 2000. Specificity and mech- 277:16913. ␣ anism of action of some commonly used protein kinase inhibitors. Biochem. J. 55. Gendelman, H. E., D. R. Skillman, and M. S. Meltzer. 1992. Interferon -mac- 351:95. rophage interactions in human immunodeficiency virus infection: role of IFN in 37. Bain, J., H. McLauchlan, M. Elliott, and P. Cohen. 2003. The specificities of the tempo and progression of HIV disease. Int. Rev. Immunol. 8:43. protein kinase inhibitors: an update. Biochem. J. 371:199. 56. Zagury, D., A. Lachgar, V. Chams, L. S. Fall, J. Bernard, J. F. Zagury, B. Bizzini, A. Gringeri, E. Santagostino, J. Rappaport, et al 1998. Interferon a and Tat in-

38. Brancho, D., N. Tanaka, A. Jaeschke, J. J. Ventura, N. Kelkar, Y. Tanaka, Downloaded from volvement in the immunosuppression of uninfected T cells and C-C chemokine M. Kyuuma, T. Takeshita, R. A. Flavell, and R. J. Davis. 2003. Mechanism of decline in AIDS. Proc. Natl. Acad. Sci. USA 95:3851. p38 MAP kinase activation in vivo. Genes Dev. 17:1969. 57. Feinstein, D. L. 2003. Therapeutic potential of peroxisome proliferator-activated 39. Miyazaki, T., H. Katagiri, Y. Kanegae, H. Takayanagi, Y. Sawada, receptor agonists for neurological disease. Diabetes Technol. Ther. 5:67. A. Yamamoto, M. P. Pando, T. Asano, I. M. Verma, H. Oda, et al. 2000. Re- 58. Diab, A., C. Deng, J. D. Smith, R. Z. Hussain, B. Phanavanh, A. E. Lovett-Racke, ciprocal role of ERK and NF-␬B pathways in survival and activation of oste- P. D. Drew, and M. K. Racke. 2002. Peroxisome proliferator-activated receptor-␥ oclasts. J. Cell Biol. 148:333. ⌬12,14 agonist 15-deoxy- -prostaglandin J2 ameliorates experimental autoimmune 40. Poluektova, L., T. Moran, M. Zelivyanskaya, S. Swindells, H. E. Gendelman, and encephalomyelitis. J. Immunol. 168:2508. Y. Persidsky. 2001. The regulation of ␣ chemokines during HIV-1 infection and

59. Feinstein, D. L., E. Galea, V. Gavrilyuk, C. F. Brosnan, C. C. Whitacre, http://www.jimmunol.org/ leukocyte activation: relevance for HIV-1-associated dementia. J Neuroimmunol. L. Dumitrescu-Ozimek, G. E. Landreth, H. A. Pershadsingh, G. Weinberg, and 120:112. M. T. Heneka. 2002. Peroxisome proliferator-activated receptor-␥ agonists pre- 41. Rossi, A., P. Kapahi, G. Natoli, T. Takahashi, Y. Chen, M. Karin, and vent experimental autoimmune encephalomyelitis. Ann. Neurol. 51:694. M. G. Santoro. 2000. Anti-inflammatory cyclopentenone prostaglandins are di- 60. Doyle, S., S. Vaidya, R. O’Connell, H. Dadgostar, P. Dempsey, T. Wu, G. Rao, ␬ rect inhibitors of I B kinase. Nature 403:103. R. Sun, M. Haberland, R. Modlin, et al. 2002. IRF3 mediates a TLR3/TLR4- 42. Straus, D. S., G. Pascual, M. Li, J. S. Welch, M. Ricote, C. H. Hsiang, specific antiviral gene program. Immunity 17:2513. ⌬12,14 L. L. Sengchanthalangsy, G. Ghosh, and C. K. Glass. 2000. 15-Deoxy- - 61. Toshchakov, V., B. W. Jones, P. Y. Perera, K. Thomas, M. J. Cody, S. Zhang, ␬ prostaglandin J2 inhibits multiple steps in the NF- B signaling pathway. Proc. B. R. Williams, J. Major, T. A. Hamilton, M. J. Fenton, et al. 2002. TLR4, but Natl. Acad. Sci. USA 97:4844. not TLR2, mediates IFN-␤-induced STAT1␣/␤-dependent gene expression in 43. Hoffmann, E., O. Dittrich-Breiholz, H. Holtmann, and M. Kracht. 2002. Multiple macrophages. Nat. Immunol. 3:392. control of interleukin-8 gene expression. J. Leukocyte Biol. 72:847. 62. Kolb, S. A., B. Sporer, F. Lahrtz, U. Koedel, H. W. Pfister, and A. Fontana. 1999.

44. Hipp, M. S., C. Urbich, P. Mayer, J. Wischhusen, M. Weller, M. Kracht, and Identification of a chemotactic factor in the cerebrospinal fluid of HIV-1- by guest on September 24, 2021 I. Spyridopoulos. 2002. Proteasome inhibition leads to NF-␬B-independent IL-8 infected individuals as interferon-␥ inducible protein 10. J. Neuroimmunol. transactivation in human endothelial cells through induction of AP-1. Eur. J. Im- 93:172. munol. 32:2208. 63. Lane, B. R., S. R. King, P. J. Bock, R. M. Strieter, M. J. Coffey, and 45. Cheng, G., A. S. Nazar, H. S. Shin, P. Vanguri, and M. L. Shin. 1998. IP-10 gene D. M. Markovitz. 2003. The C-X-C chemokine IP-10 stimulates HIV-1 replica- transcription by virus in astrocytes requires cooperation of ISRE with adjacent ␬B tion. Virology 307:122. site but not IRF-1 or viral transcription. J. Interferon Cytokine Res. 18:987. 64. Xiong, H., J. Boyle, M. Winkelbauer, S. Gorantla, J. Zheng, A. Ghorpade, 46. Ohmori, Y., and T. A. Hamilton. 1993. Cooperative interaction between inter- Y. Persidsky, K. A. Carlson, and H. E. Gendelman. 2003. Inhibition of long-term feron (IFN) stimulus response element and ␬B sequence motifs controls IFN␥- potentiation by interleukin-8: implications for human immunodeficiency virus- and lipopolysaccharide-stimulated transcription from the murine IP-10 promoter. 1-associated dementia. J. Neurosci. Res. 71:600. J. Biol. Chem. 268:6677. 65. Lane, B. R., K. Lore, P. J. Bock, J. Andersson, M. J. Coffey, R. M. Strieter, and 47. Park, E. J., S. Y. Park, E. H. Joe, and I. Jou. 2003. 15d-PGJ2 and rosiglitazone D. M. Markovitz. 2001. Interleukin-8 stimulates human immunodeficiency virus suppress Janus kinase-STAT inflammatory signaling through induction of sup- type 1 replication and is a potential new target for antiretroviral therapy. J. Virol. pressor of cytokine signaling 1 (SOCS1) and SOCS3 in glia. J. Biol. Chem. 75:8195. 278:14747. 66. Belperio, J. A., M. P. Keane, D. A. Arenberg, C. L. Addison, J. E. Ehlert, 48. Lennon, A. M., M. Ramauge, A. Dessouroux, and M. Pierre. 2002. MAP kinase M. D. Burdick, and R. M. Strieter. 2000. CXC chemokines in angiogenesis. cascades are activated in astrocytes and preadipocytes by 15-deoxy-⌬12Ð14-pros- J. Leukocyte Biol. 68:1. ␥ taglandin J2 and the thiazolidinedione ciglitazone through peroxisome prolifera- 67. Song, X., S. Tanaka, D. Cox, and S. C. Lee. 2004. Fc receptor signaling in tor activator receptor ␥-independent mechanisms involving reactive oxygenated primary human microglia: differential roles of PI-3K and Ras/ERK MAPK path- species. J. Biol. Chem. 277:29681. ways in phagocytosis and chemokine induction. J. Leukocyte Biol. 75:1147.