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The Journal of Immunology, 2004, 173: 3504-3513.
Copyright © 2004 by The American Association of Immunologists

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

Qiusheng Si2, Meng-Liang Zhao2, Anna C. A. Morgan3, Celia F. Brosnan and Sunhee C. Lee4

Department of Pathology, Albert Einstein College of Medicine, Bronx, NY 10461


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Regulation of cytokine and chemokine expression in microglia may have implications for CNS inflammatory disorders. In this study we examined the role of the cyclopentenone PG 15-deoxy-{Delta}12,14-PGJ2 (15d-PGJ2) in microglial inflammatory activation in primary cultures of human fetal microglia. 15d-PGJ2 potently inhibited the expression of microglial cytokines (IL-1, TNF-{alpha}, and IL-6). We found that 15d-PGJ2 had differential effects on the expression of two {alpha}-chemokines; 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 bacterial endotoxin, proinflammatory cytokines (IL-1 and TNF-{alpha}), IFN-{beta}, and HIV-1. Furthermore, IL-8/CXCL8 expression was induced by 15d-PGJ2 alone or in combination with TNF-{alpha} or HIV-1. Combined results from EMSA, Western blot analysis, and immunocytochemistry showed that 15d-PGJ2 inhibited NF-{kappa}B, Stat1, and p38 MAPK activation in microglia. Adenoviral transduction of super-repressor I{kappa}B{alpha}, dominant negative MKK6, and dominant negative Ras demonstrated that NF-{kappa}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-{kappa}B, the baseline or 15d-PGJ2-mediated IL-8/CXCL8 production was NF-{kappa}B independent. Our results demonstrate that 15d-PGJ2 has opposing effects on the expression of two {alpha}-chemokines. These data may have implications for CNS inflammatory diseases.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Prostaglandin D2, a major cyclooxygenases (COX)5 product in a variety of tissues and cells, readily undergoes dehydration to yield the bioactive cyclopentenone PGs of the J2 series, such as PGJ2, {Delta}12-PGJ2, and 15-deoxy-{Delta}12,14-PGJ2 (15d-PGJ2) (1, 2). 15d-PGJ2 is a natural ligand for the peroxisome proliferator-activated receptor {gamma} (PPAR{gamma}), a nuclear receptor involved in adipocyte differentiation, glucose metabolism, and inflammatory response (for review, see Ref. 3). PPAR{gamma} agonists have a positive regulatory role in lipid and glucose metabolism, but their role in macrophage inflammatory gene expression has been shown to be largely inhibitory. PPAR{gamma} agonists have been found to function in both a PPAR{gamma}-dependent and -independent fashion to modulate macrophage gene expression (4, 5, 6, 7). Evidence supports that 15d-PGJ2 is produced by activated macrophages in vivo and in vitro (2, 8). Furthermore, 15d-PGJ2 inhibits the expression of COX-2, the rate-limiting enzyme in PG synthesis (2), which suggests that 15d-PGJ2 can function as an endogenous feedback inhibitor of macrophage activation. In addition to inflammatory gene expression, 15d-PGJ2 has been shown to affect cell proliferation and apoptosis, indicating a potentially diverse role in inflammatory regulation and tissue repair (9, 10).

Much information regarding the role of 15d-PGJ2 is derived from rodent macrophages and cell lines. The inflammatory enzymes inducible NO synthase (iNOS) and COX-2 are among the most susceptible macrophage genes inhibited by 15d-PGJ2 (5, 11, 12, 13). However, studies of human CNS cells demonstrate that 15d-PGJ2 inhibits iNOS and COX-2 expression in astrocytes, but not in brain macrophages (14), consistent with the idea that the regulation of macrophage activation is species dependent. Similarly, results obtained for cytokine (IL-1{beta}, TNF-{alpha}, and IL-6) regulation have been mixed. Jiang et al. (15) reported that human monocytes differentially responded to 15d-PGJ2, with LPS-induced cytokine synthesis being refractory and PMA- or okadaic acid-induced synthesis being susceptible to inhibition. Chawla et al. (5) showed that 15d-PGJ2 can inhibit macrophage cytokine expression in PPAR{gamma}-deficient macrophages. More recently, Welch et al. (6) reported that PPAR{gamma} agonists inhibit selective subsets of macrophage genes (iNOS, COX-2, IL-12, and IFN-inducible protein 10 (IP-10)/CXCL10) and that cytokine genes (TNF-{alpha}, IL-1{beta}, and IL-6) were not inhibited. Taken together, the ability of PPAR{gamma} agonists to inhibit macrophage cytokine synthesis can be shown to be dependent on the type of macrophage tested, the stimulus used, and the PPAR{gamma} agonist used. In contrast to the macrophage cytokine genes, relatively little is known about the regulation of macrophage chemokine gene expression by 15d-PGJ2. In THP-1 cells, 15d-PGJ2 induces IL-8/CXCL8 while suppressing LPS-induced MCP-1 expression (16). Interestingly, 15d-PGJ2 has been shown to induce IL-8/CXCL8 expression in several cell types, including T cells and endothelial cells (16, 17, 18, 19). These results demonstrate a complex role for PPAR{gamma} agonists in inflammation and call for caution when considering 15d-PGJ2 as an anti-inflammatory agent.

Microglia are the resident brain macrophages central to the maintenance of normal homeostasis as well as the regulation of inflammatory responses within the CNS. We and others (20, 21, 22, 23, 24, 25) found that cultured human microglial cells provide a good model to study brain macrophage responses to inflammatory and pathogenic stimuli, because they recapitulate many of the attributes of microglia in vivo. In the current study we examined the role of 15d-PGJ2 in cytokine and chemokine expression in primary cultures of human microglia. We found that although 15d-PGJ2 potently inhibited LPS-induced cytokine (TNF-{alpha} and IL-1{beta}) expression, it produced differential effects on the expression of the two {alpha}-chemokines, with IL-8/CXCL8 being refractory but IP-10/CXCL10 being susceptible to inhibition after activation with a wide variety of cell stimuli (LPS, cytokines, IFN-{beta}, and HIV-1). These results suggest that 15d-PGJ2 may create a unique cytokine-chemokine environment in the CNS, tipping the balance toward an Glu-Lys-Arg (ELR)+ chemokine predominant state.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Microglial and THP-1 cell culture

Human fetal CNS cell cultures were prepared from human fetal abortuses as previously described (20, 22, 26, 27). All procedures were approved by the Albert Einstein College of Medicine institutional review board. Primary mixed CNS mixed cultures were prepared by enzymatic and mechanical dissociation of the cerebral tissue, followed by filtration through nylon meshes of 230- and 130-µm pore sizes. Single-cell suspensions were plated at 1–10 x 106 cells/ml in DMEM (Cellgro supplemented with 4.5 g/l glucose, 4 mM L-glutamine, and 25 mM HEPES) supplemented with 5% FCS (Gemini Bio-Products, Woodland, CA), penicillin (100 U/ml), streptomycin (100 µg/ml), and fungizone (0.25 µg/ml; Invitrogen Life Technologies, Gaithersburg, MD) for 2 wk, and then microglial cells were collected by aspiration of the culture medium. Monolayers of microglia were prepared in 100-mm tissue culture dishes at 106 cells/10 ml medium or in 96-well tissue culture plates at 4 x 104/0.1 ml medium. Two to four hours later, cultures were washed twice to remove nonadherent cells (neuronal and astrocytes), resulting in microglial culture that were highly pure, consisting of >98% CD 68+ cells. Microglial cells used for HIV-1 infection were kept in fungizone-free medium. THP-1 cells were obtained from Dr. W. Jacobs (Albert Einstein College of Medicine) and propagated at 106 cells/100-mm dish in RPMI 1640/10% FCS with antibiotics.

Reagents and treatment of cells

15d-PGJ2 was purchased from Cayman Chemical (Ann Arbor, MI). SB203580, SP600125, and 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidin (PP2) were purchased from Calbiochem (San Diego, CA); U0126 was obtained from Cell Signaling (Beverly, MA). Stocks of inhibitors were prepared in DMSO. LPS (Escherichia coli serotype 055:B5) and 3'-azido-deoxythymidine (AZT) were purchased from Sigma-Aldrich (St. Louis, MO). IFN-{beta} was purchased from PBL Biomedical Laboratories (New Brunswick, NJ). IL-1{beta} and TNF-{alpha} were purchased from PeproTech (Rocky Hill, NJ). Microglia or THP-1 cells were pretreated with 15d-PGJ2 for 3 h (empirically determined to give maximal inhibition) or other inhibitors for 1 h, then with cell stimuli for indicated time periods. The concentration of 15d-PGJ2 used was 20 µM for all experiments unless otherwise stated. Cytotoxicity was determined by MTT assay (Promega, Madison, WI) following the manufacturer’s protocol. For HIV-1 experiments, the 15d-PGJ2 concentration was reduced to 10 µM because of the lengthy incubation periods required for viral production (4 wk). All cell stimuli (cytokines and LPS) were used at 10 ng/ml based on previous determination for microglial activation.

HIV-1 exposure of microglia

HIV-1 isolates were obtained from the AIDS Repository and propagated in PBMC as previously described (27). Mock infection consisted of exposure to PBMC supernatants without HIV-1. Infectious viruses were generated from Nef-deficient and wild-type HIV-1ADA proviruses (28) and Vpr– (pHXB (BaL)-R–) or Vpr+ (pHXB (BaL)-R+) proviruses (29) by transfecting COS cells using Lipofectamine agents (Invitrogen Life Technologies) as previously described (30). HIV infection of microglia was performed as previously described (26, 30) by exposure to 5–20 ng/ml p24 input HIV-1 for 16 h. 15d-PGJ2 at 10 µM was added to cultures 3 h before exposure to HIV-1. Culture medium was completely changed weekly, and 15d-PGJ2 was replenished each time.

ELISA

IP-10/CXCL10 and IL-8/CXCL8 ELISA was performed using Ab pairs purchased from BD Pharmingen (San Diego, CA) and R&D Systems (Minneapolis, MN), respectively. Ab pairs for IL-1 and TNF-{alpha} were also purchased from R&D Systems. HIV-1 p24 ELISA was performed using a commercial ELISA kit (NEN Life Science Products, Boston, MA). Supernatants were diluted until the values fell within the linear range of the ELISA detection limit.

RNase protection assay (RPA)

Total RNA was extracted from microglia plated at 1 x 106 cells in 100-mm dishes using TRIzol, according to the manufacturer’s instructions. RNA was analyzed using RPA templates for human chemokines and cytokines (BD Pharmingen) according to the manufacturer’s instructions, essentially as described previously (31). Images were developed using autoradiographic film exposed to the gel at –80°C. Densitometry was performed using Image software (Scion, Frederick, MD).

EMSA

Nuclear extracts were prepared using a modified Dignam method. Buffers were supplemented with 1 mM PMSF, 1 mM DTT, and a protease inhibitor mixture (Roche, Indianapolis, IN). Cells (~1 x 106) were scraped off in 1 mM PMSF/PBS and centrifuged. Pellets were resuspended in low salt buffer (10 mM HEPES (pH 7.9), 1.5 mM MgCl2, and 10 mM KCl), and allowed to sit on ice before addition of Igepal CA-630 (Sigma-Aldrich). Samples were again pelleted and resuspended in high salt buffer (20 mM HEPES (pH 7.9), 25% glycerol, 420 mM NaCl, and 1.5 mM MgCl2); samples were rocked gently before final centrifugation. The supernatant was saved, and the protein was quantified using the Bradford assay. Oligonucleotide containing the consensus binding sequence (underlined) for NF-{kappa}B (5'-AGT TGA GGG GAC TTT CCT AGG C-3' was radiolabeled with [32P]ATP using polynucleotide T4 kinase according to the Gel Shift Assay Core System kit (Promega) instructions. Labeled probe was purified on a G-25 spin column (Roche). Three micrograms of nuclear extracts were incubated in binding buffer (4% glycerol, 1 mM MgCl2, 0.5 mM EDTA, 50 mM NaCl, 10 mM This-HCl, and 50 µg/ml poly(dI-dC)) with 1.75 pmol of specific and nonspecific competitor oligonucleotides for 15 min at room temperature before addition of labeled probe. The binding reaction (final volume, 20 µl) was allowed to proceed for another 20 min at room temperature. Samples were loaded without loading dyes that interfere with protein-DNA interactions. Gels containing 5.5% polyacrylamide, 5% glycerol, and 0.5x Tris-glycine-EDTA were electrophoresed at 150 V for ~2.5 h.

Immunocytochemistry

Immunocytochemistry was performed using rabbit Abs against p65 unit of NF-{kappa}B (Santa Cruz Biotechnology, Santa Cruz, CA) and pStat1, which specifically recognizes phosphotyrosine at residue 701 (Cell Signaling, Beverly, MA). Briefly, cells were fixed with cold methanol, then blocked with 10% normal goat serum. Primary Abs at 1/100 dilutions were applied to cells for 16 h at 4°C. This was followed by incubation with biotinylated goat anti-rabbit IgG at 1/200 for 1 h at room temperature (Vectastain ABC kit; Vector Laboratories, Burlingame, CA). The cells were then incubated with A + B solution at a 1/200 dilution for 1 h room temperature. Color was developed using diaminobenzidine.

Western blot analysis

Microglial cell lysates were prepared by scraping cells into 8 M urea. Total protein (10–50 µg) was separated in 10% SDS-PAGE, then transferred to polyvinylidene difluoride membrane. The blots were blocked in TBS/0.1% 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 in 5% BSA/TTBS, according to the manufacturer’s instructions. After overnight incubation at 4°C, the blots were washed in TTBS and then further incubated with goat anti-rabbit IgG at 1/2000 dilution in 5% nonfat milk/TTBS for 1 h at room temperature. The reaction was developed using ECL (Pierce, Rockford, IL).

Data analysis

Experiments were repeated at least three times using different brain cases with similar results. Each data point represents values from triplicate wells (mean ± SD) from a single representative experiment. For statistical analysis of the data, one-way ANOVA was performed, followed by Scheffé’s multiple comparison procedure for comparing multiple treatment conditions or Student’s t test for comparing two treatment conditions. A value of p < 0.05 was considered significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
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 proinflammatory cytokines (IL-1 and TNF-{alpha}), IFN-{beta}, and bacterial endotoxin (LPS). Cultures were pretreated with 15d-PGJ2 at 20 µM for 3 h, then stimulated with cytokines, all at 10 ng/ml. ELISA was performed to determine the levels of chemokines in the culture supernatants 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-{beta} stimulation and picogram levels were produced by IL-1 and TNF-{alpha} stimulation. 15d-PGJ2 potently inhibited IP-10/CXCL10 production by all stimuli. In contrast, IL-8/CXCL8 was induced by LPS, IL-1, and TNF-{alpha}, but not by IFN-{beta} (not shown), and 15d-PGJ2 significantly elevated the levels with all stimuli except IL-1 (p < 0.05, by Student’s t test; Fig. 1B). Dose-response experiments were performed using increasing concentrations (0.1–20 µM) of 15d-PGJ2, and the results show that 15d-PGJ2 inhibited IP-10/CXCL10 with an IC50 at ~3–5 µM (Fig. 1C). MTT assay demonstrated that inhibition of IP-10/CXCL10 was not due to cell death (Fig. 1D).



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  		FIGURE 1. Microglial IP-10/CXCL10, but not IL-8/CXCL8, production is inhibited by 15d-PGJ2. Human fetal microglia plated at 40,000 cells/well were stimulated with 20 µM 15d-PGJ2 for 3 h, then with LPS or cytokines at 10 ng/ml for an additional 24 h. ELISA was performed of the culture supernatants to determine the level of IP-10/CXCL10 (A) or IL-8/CXCL8 (B). *, p < 0.05 vs without 15d-PGJ2. 15d-PGJ2 dose response for IP-10/CXCL10 inhibition was examined as described above and showed that 15d-PGJ2 inhibited IP-10/CXCL10 at 1–20 µM, with an IC50 at ~3 µM (C). MTT assay was performed in the same culture and showed no cell toxicity (D). Values shown are the mean ± SD from triplicate wells.

 
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. Microglial cells were pretreated with 15d-PGJ2 for 3 h, then stimulated for 6 h with LPS or cytokines, all at 10 ng/ml. As shown in Fig. 2A, stimulation of microglia with LPS, IFN-{beta}, or IL-1 induced microglial IP-10/CXCL10 mRNA expression, and 15d-PGJ2 reduced 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-{beta}, and 15d-PGJ2 slightly enhanced the level of IL-8/CXCL8 mRNA (see Fig. 2C for densitometry). RANTES/CCL5, MIP-1{alpha}/CCL3, MIP-1{beta}/CCL4, and MCP-1/CXCL2 mRNA were induced by LPS, IFN-{beta}, and IL-1, but 15d-PGJ2 had little effect on these transcripts. In THP-1 cells, RPA showed robust IP-10/CXCL10 mRNA induction by LPS and IFN-{beta} 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 previously reported (16). Interestingly, 15d-PGJ2 was a stronger stimulus than LPS or IL-1 for IL-8/CXCL8 induction in THP-1 cells (see Discussion).



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  		FIGURE 2. RPA of microglia and THP-1 cells for chemokine expression. A, Microglia in 100-mm petri dish at 1 x 106 cells/dish were treated 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.

 
15d-PGJ2 inhibits microglial cytokine (IL-1 and TNF-{alpha}) expression

Microglial proinflammatory cytokines are pivotal in the establishment of inflammatory cascades in the injured CNS, including induction of chemokine expression. Thus, the ability of 15d-PGJ2 to regulate IL-1 and TNF-{alpha} expression would be critical in the overall regulation of inflammation in the CNS. We tested the effect of 15d-PGJ2 on microglial cytokine production using a human cytokine RPA probeset from BD Pharmingen and by ELISA. Cells were pretreated with 15d-PGJ2 at 20 µM for 3 h, then stimulated with IL-1 or LPS at 10 ng/ml for 6 h. As shown in Fig. 3, unstimulated microglial cells expressed IL-1R antagonist (IL-1Ra) mRNA only. After stimulation with IL-1, multiple cytokine genes were induced (TNF-{alpha}, IL-1{alpha}, IL-1{beta}, IL-1Ra and IL-6) as reported 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-{alpha} production in microglia (Fig. 3C).



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  		FIGURE 3. 15d-PGJ2 inhibits microglial cytokine expression. A, Microglia were treated as described in Fig. 2, and total RNA was analyzed for cytokine mRNA expression using a Quantikine probe set from BD Pharmingen. B, Densitometric analysis of cytokine mRNA against GAPDH mRNA shown in A. C, Cytokine (TNF-{alpha} and IL-1{beta}) protein levels were determined by ELISA in LPS-treated cultures with or without 15d-PGJ2, as described for chemokines in Fig. 1.

 
15d-PGJ2 inhibits microglial NF-{kappa}B activation

To determine which microglial activation pathways are targeted by 15d-PGJ2, we used EMSA to examine NF-{kappa}B activation by LPS or IFN-{beta}. Microglia were pretreated with 15d-PGJ2 at 20 µM for 3 h, then with LPS or IFN-{beta} at 10 ng/ml for 30 min. The nuclear extracts were prepared, and EMSA was performed with an NF-{kappa}B consensus oligonucleotide as previously described (33). As shown in Fig. 4A, nuclear NF-{kappa}B binding activity was induced in microglia treated with either LPS or IFN-{beta}; of the three complexes visible 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-{kappa}B nuclear binding activity induced by LPS or IFN-{beta}. Furthermore, 15d-PGJ2 inhibited nuclear translocation of NF-{kappa}B (p65 subunit) in microglia, as determined by immunocytochemistry (Fig. 4B). These results indicate that in primary human microglia, 15d-PGJ2 inhibits NF{kappa}B nuclear translocation and DNA binding, in contrast to results obtained in murine microglia (11).



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  		FIGURE 4. 15d-PGJ2 inhibits microglial NF-{kappa}B binding activity induced by LPS or IFN-{beta}. A, EMSA was performed with microglial cell nuclear extracts prepared by pretreatment of cells with 20 µM 15d-PGJ2 for 3 h, then for 30 min with 10 ng/ml LPS or IFN-{beta}. The top band represents the p65/p50 heterodimer. Specific competitor (SC) for EMSA consisted of a 100-fold excess of unlabeled NF-{kappa}B probe. Nonspecific competitor (NSC) was the same, except an irrelevant probe was used. B, NF-{kappa}B (p65) immunocytochemistry in microglia treated as described in A, demonstrating nuclear localization of p65 induced by LPS, which was reduced by pretreatment with 15d-PGJ2. C, 15d-PGJ2 inhibits Stat1 activation. Microglia were pretreated with 15d-PGJ2 at 20 µM for 3 h, then with 10 ng/ml IFN-{beta} for either 0.5 or 2 h. Western blot analysis was performed with an Ab specific to phosphorylated Stat1 (Y701). The blot was stripped and reprobed for total Stat1 (C). D, Immunocytochemistry for pStat1 revealed that after IFN-{beta} treatment (30 min), the majority of microglial cells had strong nuclear pStat1 immunoreactivity, which was prevented by pretreatment with 15d-PGJ2.

 
15d-PGJ2 inhibits Stat1 activation in microglia

The Jak/Stat pathways are the major signaling pathways activated by IFNs. Our results in microglia demonstrate that IFN-{beta} as well as IFN-{gamma} are potent inducers of IP-10/CXCL10, and that 15d-PGJ2 inhibits IFN-induced IP-10/CXCL10. Therefore, we examined the effects of 15d-PGJ2 on Stat1, a transcription factor essential for both IFN-{alpha}/IFN-{beta} and IFN-{gamma} signaling. We examined Stat1 phosphorylation by Western blot analysis using an Ab specific to tyrosine-phosphorylated (Y701) Stat1. As shown in Fig. 4C, IFN-{beta} induced phospho-Stat1 in microglia 30 min, but not 2 h, after stimulation, and 15d-PGJ2 reduced the amount of phospho-Stat1 in microglia. Furthermore, 15d-PGJ2 reduced the nuclear accumulation of phospho-Stat1 induced by IFN-{beta} (30 min) determined by immunocytochemistry (Fig. 4D).

Role of NF-{kappa}B in microglial IP-10/CXCL10 and IL-8/CXCL8 expression

To examine the role of NF-{kappa}B in the induction of IP-10/CXCL10 and IL-8/CXCL8, we used adenovirus-mediated expression of dominant negative (DN) vectors in primary microglia as previously described (33). Super-repressor I{kappa}B{alpha} is a proteolysis-resistant form of I{kappa}B{alpha} and, hence, functions as a DN-NF-{kappa}B. Microglial cells were infected with adenovirus for 24 h at the indicated concentrations, then stimulated with LPS for 24 h; control cultures were infected with equal amounts of adenoviruses carrying only the CMV promoter (Ad-CMV). As shown in Fig. 5A, super-repressor I{kappa}B{alpha} (Ad-I{kappa}B{alpha}) inhibited IP-10/CXCL10 production in microglia, demonstrating the involvement of NF-{kappa}B in IP-10/CXCL10 expression. The observed increase in IP-10/CXCL10 production by control adenoviral vector (Ad-CMV) is consistent with the reported immunogenic effect of adenovirus, such as activation of NF-{kappa}B and MAPKs (34, 35); however, the control adenovirus did not have the same effect on IL-8/CXCL8 production for an unknown reason (Fig. 5B; also see Fig. 6B). Similar to IP-10/CXCL10, production of IL-8/CXCL8 after LPS treatment was also inhibited by super-repressor NF-{kappa}B (Ad-I{kappa}B{alpha}) in microglia (Fig. 5B), demonstrating that NF-{kappa}B was a positive regulator of IL-8/CXCL8 induction by LPS. In contrast, the induction of IL-8/CXCL8 in control (baseline) or 15d-PGJ2-treated microglia was not inhibited by super-repressor I{kappa}B{alpha} (Fig. 5C), demonstrating that baseline or 15d-PGJ2-induced IL-8/CXCL8 was not mediated by NF-{kappa}B. Together these results suggest that different mechanisms are involved in IL-8 induction in the presence or the absence of 15d-PGJ2 and that the failure of 15d-PGJ2 to inhibit IL-8 production may be due in part to the presence of an NF-{kappa}B-independent IL-8 induction pathway activated by 15d-PGJ2 (see Discussion).



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  		FIGURE 5. Adenoviral transduction of primary human microglia with super-repressor I{kappa}B{alpha} (Ad-I{kappa}B{alpha}) or control vector (Ad-CMV) demonstrates the role of NF-{kappa}B in both IP-10/CXCL10 and IL-8/CXCL8 production induced by LPS or IL-1{beta}. A and B, Microglia were infected with Ad-CMV or Ad-I{kappa}B{alpha} at either 104 or 2.5 x 104 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, Microglia were infected with adenoviral vectors as described above, then treated with 20 µM 15d-PGJ2 for 72 h. IL-8/CXCL8 levels were determined by ELISA. Values are the mean ± SD from triplicate wells. *, p < 0.05 vs Ad-CMV.

 


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  		FIGURE 6. Roles of MAPK and Src kinase inhibitors in the induction of microglial IP-10/CXCL10 and IL-8/CXCL8. Microglia were pretreated for 1 h with inhibitors (SB203580, UO126, SP600125, and PP2) at 20 µM, 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 ± SD. *, p < 0.05 vs LPS alone.

 
Role of MAPKs in microglial IP-10/CXCL10 and IL-8/CXCL8 production

We next examined whether microglial MAPKs or Src family kinases are involved in IP-10/CXCL10 and IL-8/CXCL8 expression. Microglia were pretreated for 1 h with p38 MAPK inhibitor (SB203580 at 20 µM), ERK MAPK inhibitor (UO126 at 20 µM), JNK MAPK inhibitor (SP600125 at 0.1 µM), or the Src family kinase inhibitor PP-2 at 1 µM), based on published IC50 values reported for the specific 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 production of IL-8/CXCL8 in microglia induced by either LPS or IL-1 (Fig. 6, B and C).

Microglial IP-10/CXCL10 production is inhibited by DN-MKK6

To confirm the selective role of p38 MAPK in the induction of IP-10/CXCL10, we used adenoviral transduction of DN-MKK6 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 determined that in microglia, transduction with DN Ras inhibits phosphorylation of ERK (67). Microglia were treated first with adenoviruses, then with LPS, as described for Ad-I{kappa}B{alpha}, then chemokine levels were determined after 24 h. Fig. 7A shows that LPS-induced IP-10/CXCL10 production was inhibited by Ad-DN-MKK6, but not by Ad-DN-Ras. In contrast, neither virus affected the production of IL-8/CXCL8 in LPS-stimulated microglia (Fig. 7B). The observed increase in IP-10/CXCL10 production by control adenoviral vector (Ad-CMV) is consistent with the immunogenic effect of adenovirus (34, 35). Together, these results confirm that p38 MAPK is differentially involved in the expression of the two {alpha}-chemokines in human microglia.



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  		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 are the mean ± SD. *, p < 0.05 vs control (Ad-CMV).

 
p38 MAPK is differentially activated in primary human microglia and THP-1 cells

Because the inhibition data demonstrated that p38 is involved in IP-10/CXCL10, but not in IL-8/CXCL8, induction, we next examined whether 15d-PGJ2 inhibits p38 phosphorylation. Western blot analysis was performed with microglia pretreated 3 h with various concentrations of 15d-PGJ2, then with LPS or IFN-{beta} at 10 ng/ml for 30 min. As shown in Fig. 8A, LPS and IFN-{beta} (but not 15d-PGJ2) induced phosphorylation of p38 in microglia, and 15d-PGJ2 reduced the amount of phospho-p38 in both. However, this was seen only with high concentrations of 15d-PGJ2 (results with 30 µM shown). These results were different from those obtained in THP-1 cells. THP-1 cells were pretreated with 15d-PGJ2 for 3 h, then with LPS for the indicated time periods. Phospho-p38 was measured by Western blot analysis (Fig. 8B). In THP-1 cells, 15d-PGJ2 induced strong and sustained phosphorylation of p38 MAPK (shown up to 4 h), whereas LPS was a weak inducer of phospho-p38; 15d-PGJ2 did not show an inhibitory effect on p38 phosphorylation. Together, our results demonstrate a complex role played by 15d-PGJ2 in the microglial activation pathway and that its effects in primary microglia are different from those in THP-1 cells.



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  		FIGURE 8. The p38 MAPK activation was regulated by 15d-PGJ2. A, Microglia were pretreated with 30 µM 15d-PGJ2 for 3 h, then with 10 ng/ml LPS or IFN-{beta}. Proteins were harvested after 30 min, and Western blot was performed for phospho- and total p38. B, THP-1 cells were treated with 15d-PGJ2 and LPS as described for microglia, and Western blot analysis was performed for p38. Robust p38 phosphorylation was detected in THP-1 cells and not in microglia.

 
IP-10/CXCL10 is induced in microglia by HIV-1 in a replication- and Nef-dependent manner

To determine whether 15d-PGJ2 would inhibit IP-10/CXCL10 in the context of a disease paradigm, we used our culture system for HIV-1-infected microglia. Although HIV-1-infected monocyte-derived macrophages have been shown to produce IP-10 (40), production in microglia has not specifically been shown. In this study we confirm that microglia do indeed produce IP-10 upon infection with HIV-1. Microglia were exposed to HIV-1, and the supernatants were collected weekly, then analyzed for IP-10/CXCL10 by ELISA, as previously described (30). HIV-1 induced IP-10/CXCL10 in microglia in a time-dependent manner, with peak protein expression at 14–28 days (Fig. 9). IP-10/CXCL10 production was viral strain-dependent; R5 (ADA and BaL), but not X4 (not shown), strains induced IP-10/CXCL10 (Fig. 9A). In addition, the reverse transcriptase inhibitor AZT abolished IP-10/CXCL10 production (Fig. 9B). The roles of Nef and Vpr were tested using mutant viruses. The results showed that microglial IP-10/CXCL10 production was dependent on Nef, but not Vpr (Fig. 9, C and D).



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  		FIGURE 9. HIV-1 induction of IP-10/CXCL10 in microglia. A, Microglia were either mock-infected or exposed to R5 strains (ADA or BaL) of 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 µM 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 (Nef+) or Nef-deficient (Nef–) HIV-1ADA, as described previously. Weekly culture supernatants were examined for IP-10/CXCL10. D, Microglia were infected with Vpr+ (pHXB (BaL)-R+) or Vpr– (pHXB (BaL)-R–) virus as described previously, then IP-10/CXCL10 levels were examined in the weekly culture supernatants. Values are the mean ± SD from triplicate wells in all assays.

 
15d-PGJ2 inhibits HIV-1-induced IP-10/CXCL10 production in microglia

We then tested our HIV-infected microglial culture to determine whether 15d-PGJ2 can inhibit virus-induced IP-10/CXCL10 expression. Microglia were pretreated with 15d-PGJ2 and exposed to HIV-1 as described above. Culture medium was changed weekly, with replenishment of 15d-PGJ2. Culture supernatants were examined for the production of IP-10/CXCL10, IL-8/CXCL8, and HIV-1 p24 by ELISA. As shown in Fig. 10A, 15d-PGJ2 was a potent inhibitor of IP-10/CXCL10 induction by HIV-1 in microglia. Interestingly, IL-8/CXCL8 accumulated in control microglial cultures, and exposure to HIV-1 did not affect the expression of IL-8/CXCL8 (Fig. 10B). However, in the presence of 15d-PGJ2, microglial IL-8/CXCL8 production was increased in both control (7, 14, and 28 days) and HIV-1-exposed (21 and 28 days) cultures (Fig. 10B). The HIV-1 p24 ELISA demonstrated that 15d-PGJ2 induced mild inhibition of viral production; however, the inhibition was not statistically significant, except on day 14 (Fig. 10C).



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  		FIGURE 10. IP-10/CXCL10, but not IL-8/CXCL8, produced in HIV-1-exposed microglial cultures is inhibited by 15d-PGJ2. Microglia were exposed to HIV-1ADA as described in Fig. 9, except that they were pretreated for 3 h with 15d-PGJ2 at 10 µM before exposure to HIV-1. Values are the mean ± SD from triplicate wells. IP-10/CXCL10 and IL-8/CXCL8 were determined in the same culture supernatants (A and B). 15d-PGJ2 had differential effects in these cultures; although IP-10/CXCL10 induction was completely inhibited, IL-8/CXCL8 production in mock-infected and HIV-1-infected cultures was increased by 15d-PGJ2.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We report in this study that the cyclopentenone PG, 15d-PGJ2, modulates human microglial inflammatory gene expression. In addition to the proinflammatory cytokines (IL-1, TNF-{alpha}, and IL-6), IP-10/CXCL10 expression was consistently inhibited by 15d-PGJ2. Interestingly, the expression of IL-8/CXCL8 was not inhibited by 15d-PGJ2, but, rather, was induced by it. The IL-8/CXCL8-inducing effect of 15d-PGJ2 often showed synergism with other stimuli, such as TNF-{alpha} or HIV-1. To elucidate the mechanism underlying 15d-PGJ2-mediated cytokine and chemokine gene modulation, we determined NF-{kappa}B and Stat1 activation. We found that 15d-PGJ2 inhibited NF-{kappa}B nuclear translocation and DNA binding in human microglia. These results are similar to those reported by Rossi et al. (41) and Straus et al. (42), which demonstrated that 15d-PGJ2 directly inhibits I{kappa}B{alpha} kinase activity as well as NF-{kappa}B DNA binding, but different from those found in murine microglia, which failed to show inhibition of NF-{kappa}B-DNA binding (11). Our results with NF-{kappa}B, however, do not explain why IL-8/CXCL8 induction was not inhibited by 15d-PGJ2. IL-8/CXCL8 gene expression is regulated by several mechanisms, including transcriptional activation by NF-{kappa}B (43). In our study, adenoviral transduction of super-repressor I{kappa}B{alpha} inhibited IL-8/CXCL8 induction by LPS. Yet, 15d-PGJ2 failed to inhibit IL-8/CXCL8 expression. We also demonstrated that baseline or 15d-PGJ2-mediated IL-8/CXCL8 production was not inhibited by the super-repressor I{kappa}B{alpha}, suggesting an NF-{kappa}B-independent mechanism. Indeed, NF-{kappa}B-independent IL-8/CXCL8 expression has been demonstrated in several studies; proteasome inhibition that abolishes NF-{kappa}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 induction by 15d-PGJ2 did not include the proximal (–133 bp) promoter region, which contains cis elements for NF-{kappa}B, AP-1, and C/EBP transcription factors (16). Therefore, it is feasible that 15d-PGJ2 has opposing effects on IL-8/CXCL8 transcription: inhibition of NF-{kappa}B-dependent transcription and stimulation of NF-{kappa}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 factors belonging to the Stat or IRF family are activated by IFNs or virus and bind to the ISRE element in the promoter. We determined that 15d-PGJ2 reduced IFN-{beta}-mediated Stat1 phosphorylation 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 cytokine signaling proteins, resulting in the inhibition of Stat1 and Stat3 signaling. In microglia, pretreatment (~3 h) with 15d-PGJ2 was necessary for maximal inhibition of IP-10/CXCL10 production (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 multiple cell activation pathways, which may act synergistically.

In addition to NF-{kappa}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 adenoviral transduction of DN-MKK6 (upstream of p38). Neither IP-10/CXCL10 nor IL-8/CXCL8 production was affected by inhibitors of ERK, JNK, or Src kinases. These results contrast with those we obtained for {beta}-chemokines (RANTES/CCL5 and MIP-1{beta}/CCL4 induction by IFN-{beta} and HIV-1), in which p38 was shown to have a negative regulatory role (30, 33). The lack of inhibition by MAPK or Src kinase inhibitors was also inconsistent with the previously reported roles of these kinases in IL-8/CXCL8 induction in other cell types (43). Therefore, our results demonstrate the specificity that is required for cell-, stimulus-, and target gene-dependent control of gene expression. We also found that both LPS- and IFN-{beta}-induced p38 phosphorylation was reduced by 15d-PGJ2 in microglia, although this required a higher drug concentration (30 µM). Inhibition of Stat1 or p38 activation in combination with NF-{kappa}B inhibition may all contribute to suppression of IP-10/CXCL10 production.

In contrast to microglia, in THP-1 cells 15d-PGJ2 was a strong inducer of p38 phosphorylation. The activation was striking and paralleled the induction of IL-8/CXCL8 mRNA in THP-1 cells. In this regard, it is interesting that 15d-PGJ2 has been shown to induce robust and sustained MAPK activation in astrocytes and preadipocytes and to contribute to IL-8 induction in T cells (17, 48). Therefore, 15d-PGJ2’s ability to trigger MAPK activation and IL-8/CXCL8 induction may not be unique to THP-1 cells. Reportedly, 15d-PGJ2 has both stimulatory and inhibitory roles in MAPK and AP-1 activation (4, 49), partly dependent upon the presence or the absence of other cell stimuli (4, 17, 48). These results are not dissimilar to ours, which demonstrated that 15d-PGJ2 activated p38 phosphorylation in THP-1 cells, which, conversely, was inhibited in LPS-stimulated microglia. It is possible that low level (below detection limit) activation also occurred in microglia, which contributed to the NF-{kappa}B-independent induction of IL-8.

Because chemokines are also induced by virus, we examined whether HIV-induced chemokine expression was similarly regulated by 15d-PGJ2. First we found that HIV-1 infection induced IP-10/CXCL10 in microglia in amounts similar to those induced by LPS or IFN-{beta}, and this was inhibited by 15d-PGJ2. In contrast, IL-8/CXCL8 accumulated in microglial cultures spontaneously, with 15d-PGJ2 alone, or together with HIV-1. As Nef is an early regulatory protein positively involved in viral replication and chemokine synthesis (Refs. 28 , 30 , and 50 and this study), our data suggest that 15d-PGJ2 may have inhibited Nef signaling in microglia. In fact, Nef has been shown to trigger several cell activation pathways in macrophages, including Hck (myeloid-specific Src kinase), Stat1, and NF-{kappa}B (51, 52, 53). However, because IP-10/CXCL10 production was dependent on viral replication in microglia, these findings also raise the question of whether 15d-PGJ2 simply acted as an antiviral agent, such as that shown for PGA1 and PGA2 in macrophages (54). However, our results seem to suggest that the two events (HIV-1 and IP-10/CXCL10 expression) can be dissociated from each other. Additional mechanisms of 15d-PGJ2 action in HIV-1-infected cultures may include inhibition of IFN-{alpha}/IFN-{beta} signaling, because IFN-{alpha}/IFN-{beta} are produced after HIV-1 infection (55, 56) and are effective inducers of IP-10/CXCL10 (31).

Because 15d-PGJ2 and other PPAR{gamma} agonists exhibit macrophage-suppressive properties, they have been proposed as potential therapeutic agents for certain CNS diseases (57). For instance, beneficial effects of PPAR{gamma} agonists have been reported in the animal model for multiple sclerosis, experimental allergic encephalomyelitis (58, 59). Because microglial activation products are generally thought to be harmful, and they mediate neuronal damage in diseases such as HIV encephalitis and Alzheimer’s disease, our results (inhibition of proinflammatory cytokines) suggest that 15d-PGJ2 may prove to be beneficial in these diseases. In contrast, the significance of our findings of differential chemokine regulation by 15d-PGJ2 cannot be easily inferred. IP-10/CXCL10 is one of the first macrophage chemokines to be produced after microbial infection and is central to host innate immunity (60, 61). Increased levels of IP-10/CXCL10 protein and mRNA have been detected in cerebrospinal fluid as well as brain from patients with CNS HIV infection (40, 62); furthermore, there is evidence suggesting that IP-10/CXCL10 may contribute to neurological dysfunction and increased viral replication (62, 63). Similarly, IL-8/CXCL8 has been shown to inhibit long term potentiation in SCID mice and to increase HIV-1 replication in T cells and macrophages (64, 65). Therefore, the implication of 15d-PGJ2-induced {alpha}-chemokine imbalance in HIV encephalitis is unclear. Interestingly, due to the presence and absence of the ELR motif, IL-8/CXCL8 and IP-10/CXCL10 have been shown to possess opposing properties (66), raising the possibility that 15d-PGJ2 may create a pro-ELR+ state (angiogenesis, neurophil infiltration, etc.). However, it is not known whether other members of the ELR+ and ELR– chemokines are under similar controls by 15d-PGJ2. Clearly, more studies are needed to understand the biological significance of the observations made in this study.


   Acknowledgments
 
We thank Wa Shen for microglial culture, and the Einstein Human Fetal Tissue Repository for fetal tissue. We thank the AIDS Research and Reference Reagent Program (National Institute of Allergy and Infectious Diseases) and Drs. Mario Stevenson and Ned Landau for providing HIV-1. Drs. Richard Pestell and Sakae Tanaka provided the adenoviral vectors carrying SR-I{kappa}B{alpha} and DN-MKK6, respectively.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 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. and A.A.M.). Back

2 Q.S. and M.-L.Z. contributed equally to this study. Back

3 Current address: Department of Pediatrics, Massachusetts General Hospital, Boston, MA 02114. Back

4 Address correspondence and reprint requests to Dr. Sunhee C. Lee, Department of Pathology, F-717, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461. E-mail address: slee{at}aecom.yu.edu Back

5 Abbreviations used in this paper: COX, cyclooxygenase; Ad-CMV, adenovirus carrying only the CMV promoter; AZT, 3'-azido-deoxythymidine; DN, dominant negative; 15d-PGJ2, 15-deoxy-{Delta}12,14-PGJ2; IL-1Ra, IL-1R antagonist; IP-10, IFN-inducible protein 10; iNOS, inducible NO synthase; PPAR{gamma}, peroxisome proliferator-activated receptor {gamma}; RPA, RNase protection assay. Back

Received for publication February 12, 2004. Accepted for publication June 30, 2004.


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 Materials and Methods
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 Discussion
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