Activated Microglia Α , in TNF- 42 −1Β Peptide Receptor 2, a Receptor for Amyloid IL-4 Inhibits the Expression of Mouse

IL-4 Inhibits the Expression of Mouse Formyl Peptide Receptor 2, a Receptor for Amyloid β1−42, in TNF-α-Activated Microglia

This information is current as Pablo Iribarren, Keqiang Chen, Jinyue Hu, Xia Zhang, of September 26, 2021. Wanghua Gong and Ji Ming Wang J Immunol 2005; 175:6100-6106; ; doi: 10.4049/jimmunol.175.9.6100 http://www.jimmunol.org/content/175/9/6100 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 © 2005 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

IL-4 Inhibits the Expression of Mouse Formyl Peptide ␤ Receptor 2, a Receptor for Amyloid 1–42,in TNF-␣-Activated Microglia1,2

Pablo Iribarren,* Keqiang Chen,* Jinyue Hu,* Xia Zhang,† Wanghua Gong,‡ and Ji Ming Wang3*

Microglia are phagocytic cells in the CNS and actively participate in proinﬂammatory responses in neurodegenerative diseases. We have previously shown that TNF-␣ up-regulated the expression of formyl peptide receptor 2 (mFPR2) in mouse microglial ␤ ␤ cells, resulting in increased chemotactic responses of such cells to mFPR2 agonists, including amyloid 1–42 (A 42), a critical pathogenic agent in Alzheimer’s disease. In the present study, we found that IL-4, a Th2-type cytokine, markedly inhibited

TNF-␣-induced expression of mFPR2 in microglial cells by attenuating activation of ERK and p38 MAPK as well as NF-␬B. The Downloaded from effect of IL-4 was not dependent on Stat6 but rather required the protein phosphatase 2A (PP2A) as demonstrated by the capacity of PP2A small interfering RNA to reverse the effect of IL-4 in TNF-␣-activated microglia. Since both IL-4 and TNF-␣ are produced in the CNS under pathophysiological conditions, our results suggest that IL-4 may play an important role in the maintenance of CNS homeostasis by limiting microglial activation by proinflammatory stimulants. The Journal of Immu- nology, 2005, 175: 6100–6106. http://www.jimmunol.org/ lzheimer’s disease (AD)4 is a progressive neurodegen- leucyl-phenylalanine (fMLF) (8). FPRL1 also contributes to the ␤ erative disease, accounting for Ͼ15 million cases world- internalization of A 42 into the cytoplasmic compartment of mac- A wide. A hallmark of AD is the presence of senile plaques rophages where it forms fibrillary aggregates (9, 10). Interestingly, characterized by deposition of aberrantly produced ␤ amyloid pep- in mouse microglia the expression of the FPRL1 counterpart ␤ ␤ mFPR2 was markedly enhanced when the cells were activated by tides (A ), in particular the 42-aa form (A 42) in association with neural damage (1–4). In addition to its direct neurotoxicity (5), proinflammatory stimulants LPS and TNF-␣ (11, 12). The effect of ␤ TNF-␣ on microglia is dependent on a MAPK-mediated signaling A 42 is a potent activator of microglia, the mononuclear phagocytes in the brain, which surround and infiltrate senile plaques in pathway and involves activation of the transcription factor NF-␬B. ␤ ␣ by guest on September 26, 2021 AD. In vitro, A 42 induces chemotaxis of mouse microglial cells Since TNF- is elevated in a variety of CNS diseases, its action on ␤ activated by proinflammatory stimulants and induce the cells to microglia may promote the cell responsiveness to A 42, thus ag- release neurotoxins (6, 7) through mouse formyl peptide receptor gravating the pathogenesis of AD. In this context, agents that limit mFPR) 2, a G protein-coupled receptor. The human homolog of the stimulatory activity of TNF-␣ on microglial cells may have mFPR2/FPRL1 was originally identified as a low-affinity receptor therapeutic potential. for the bacterial chemotactic formyl peptide formyl-methionyl- IL-4 is a cytokine with diverse biological activities, including costimulation of the growth and survival of cultured T and B lym- *Laboratory of Molecular Immunoregulation and †Laboratory of Experimental Im- phocytes, differentiation of T cells to a Th2 phenotype, and down- munology, Center for Cancer Research, National Cancer Institute at Frederick, Fred- regulation of inflammatory responses of monocytes and macro- erick, MD 21702; and ‡Basic Research Program, SAIC-Frederick, Frederick, MD 21702 phages (13). In addition, IL-4 converts macrophages to a state of “alternative activation” characterized by up-regulation of the man- Received for publication February 18, 2005. Accepted for publication August 29, 2005. nose receptor, but down-regulation of NO and proinflammatory The costs of publication of this article were defrayed in part by the payment of page cytokines (13, 14). IL-4 inhibits the expression of COX2, iNOS, charges. This article must therefore be hereby marked advertisement in accordance and proinflammatory cytokines by activated microglia. In the with 18 U.S.C. Section 1734 solely to indicate this fact. CNS, IL-4 is produced by astroglial cells and its levels signifi- 1 This project has been funded in part with federal funds from the National Cancer Institute, National Institutes of Health, under Contract NO1-CO-12400. cantly decrease in the brain tissues of transgenic mice overexpress- ing human amyloid precursor protein that develop AD pathology 2 The content of this publication does not necessarily reflect the views or policies of the Department of Health and Human Services, nor does mention of trade names, (15). Moreover, IL-4 suppresses the LPS-induced TLR4 signaling commercial products, or organizations imply endorsement by the U.S. government. cascade in microglial cells, therefore reducing the expression and The publisher or recipient acknowledges the right of the U.S. government to retain a nonexclusive, royalty-free license in and to any copyright covering the article. Animal function of mFPR2 (16–18). Thus, IL-4 may participate in the care was provided in accordance with the procedures outlined in the Guide for the homeostasis of the CNS by controlling microglial cell responses to Care and Use of Laboratory Animals (NIH Publication No. 86-23, 1985). inflammatory stimulants. 3 Address correspondence and reprint requests to Dr. Ji Ming Wang, Laboratory of The aim of the present study was to examine the capacity of Molecular Immunoregulation, Center for Cancer Research, National Cancer Institute at Frederick, Building 560, Room 31-40, Frederick, MD 21702-1201. E-mail address: IL-4 to protect microglia from activation by the proinflammatory [email protected] cytokine TNF-␣. We found that IL-4 inhibits TNF-␣-induced 4 Abbreviations used in this paper: AD, Alzheimer’s disease; FPR, formyl peptide MAPK by up-regulation of the levels of the protein phosphatase receptor; siRNA, small interfering RNA; SDF-1␣, stromal cell-derived factor 1␣;CI chemotaxis index; OA, okadaic acid; PPAR, peroxisome proliferator-activated recep- 2A (PP2A). As a consequence, IL-4 reduced the transcription and tor; fMLF, formyl-methionyl-leucyl-phenylalanine; PP2A, protein phosphatase 2A. function of mFPR2 in microglial cells.

Materials and Methods evaluate the levels of PP2A utilizing a specific Ab (Upstate Biotechnol- Reagents and cells ogy). Densitometry and NIH Image software were used to calculate fold increase in phosphorylated proteins and PP2A with total p38 MAPK as fMLF and LPS were purchased from Sigma-Aldrich. Mouse TNF-␣, stro- loading controls. mal cell-derived factor 1␣ (SDF-1␣), and IL-4 were purchased from Pep- For detection of total ERK1/2 and p38, the membranes were stripped roTech. The chemotactic peptide WKYMVm (designated W peptide) was with Restore Western Blot Stripping Buffer (Pierce) followed by incuba- synthesized and purified by the Department of Biochemistry, Colorado tion with specific Abs. State University (Fort Collins, CO). The Abs against phospho-ERK1/2, ␬ total ERK1/2, phospho-p38 MAPK, and total p38 MAPK were purchased Measurement of NF- B activation from Cell Signaling Technology. Primary murine microglial cells were N9 cells cultured in 12-well plates were transfected with 1 ␮g/well of a isolated from 1-day-old newborn C57BL/6 (wild-type) mice and C57BL/6 pNFkB-luc reporter construct and 10 ng/well of a pRL-TK construct (Pro- Ϫ/Ϫ Stat6-deficient (Stat6 ) mice (a gift from Dr. M. Grusby, Harvard Uni- mega). The cells were then cultured in the presence or absence of stimu- versity, Boston, MA). The murine microglial cell line N9 was a gift from lants for different time periods. Both firefly luciferase and Renilla lucif- Dr. P. Ricciardi-Castagnoli (Universita Degli Studi di Milano-Bicocca, Mi- erase activities were measured and promoter activity was expressed as lan, Italy). The cells were grown in IMDM supplemented with 5% heat- percent increase in stimulated cells vs cells cultured in medium alone. ␮ inactivated FCS, 2 mM glutamine, 100 U/ml penicillin, 100 g/ml strep- Results are the mean Ϯ SEM of three independent experiments, each per- ␮ tomycin, and 50 M 2-ME. formed in triplicate samples. Chemotaxis assays PP2A small interfering RNA (siRNA) Chemotaxis assays for microglial cells were performed with 48-well che- N9 cells were transfected with a target-specific PP2A siRNA designed to motaxis chambers (NeuroProbe) as described previously (11). The results knockdown PP2A gene expression (Santa Cruz Biotechnology). Briefly, Ϯ are expressed as the mean SD of chemotaxis index (CI), which repre- N9 cells were seeded in 12-well plates and cultured until they reached 50% Downloaded from sents the fold increase in the number of migrated cells counted in three confluency. The cells were then transfected with target-specific PP2A high-power fields (ϫ400) in response to chemoattractants over spontane- siRNA and control siRNA (Santa Cruz Biotechnology) and cultured for an ous cell migration (to control medium). additional 24 h. The cells were evaluated by RT-PCR for the expression of PP2A mRNA using PP2A-specific primers (Santa Cruz Biotechnology). RT- PCR and real-time PCR The cells were also cultured for 6 h with IL-4 and TNF-␣ and measured for Total RNA was extracted from cells with an RNeasy Mini kit and depleted chemotactic responses to FPR2 agonists. of contaminating DNA with RNase-free DNase (Qiagen). For amplification Statistical analysis http://www.jimmunol.org/ of the mFPR2 gene, primers 5Ј-TCTACCATCTCCAGAGTTCTGTGG-3Ј (sense) and 5Ј-TTACATCTACCACAATGTGAACTA-3Ј (antisense) were All experiments were performed at least three times and the results pre- designed to yield a 268-bp product. Specific primers for mouse CXCR4 sented are from representative experiments. For cell migration, the signif- were 5Ј-GGCTGTAGAGCGAGTGTTGC-3Ј (sense) and 5Ј-GTAGAG icance of the difference between test and control groups was analyzed using GTTGACAGTGTAGAT-3Ј (antisense), which yield a product of 390 bp. the Student’s t test. RT-PCR was performed with 0.5 ␮g of total RNA for each sample (High Fidelity ProSTAR HF System; Stratagene), consisting of a 15-min reverse Results transcription at 37°C, 1 min inactivation of Moloney murine leukemia IL-4 inhibits mFPR2-mediated chemotaxis of TNF-␣-activated virus reverse transcriptase at 95°C, 40 cycles of denaturing at 95°C (45 s), annealing at 55°C (52°C for CXCR4; 45 s), and extension at 72°C (1 min), microglia with a final extension for 10 min at 72°C. Primers for murine ␤-actin gene We first examined the effect of IL-4 on the chemotactic responses by guest on September 26, 2021 were used as controls (Stratagene). Real-time PCR was performed by using ␣ an ABI Prism 7700 Sequence Detector (Applied Biosystems). Briefly, 5 ng of TNF- -activated microglia to mFPR2 agonists. Consistent with of reverse-transcribed cDNA was used in triplicate samples. The assays our previous findings (12), treatment of the murine microglial cell were initiated with 2 min at 50°C, 10 min at 95°C, and then 40 cycles of line N9 with TNF-␣ for 24 h promoted cell migration in response 15 s at 95°C and 1 min at 60°C. Primers and specific probes were obtained ␤ to a synthetic mFPR2 agonist peptide (W peptide) and A 42,an from Applied Biosystems and consisted of the following: 5Ј-CCTTA Ј Ј AD-associated peptide (Fig. 1A and data not shown). In contrast, TAGTC TTGAG AGAGC CCTGA-3 (sense), 5 -TGCAG GAGGT ␣ GAAGT AGAAC TGG-3Ј (antisense), and the probe 5Ј-FAM-TGAGG TNF- treatment down-regulated the chemotactic response of N9 ATTCT GGTCA AACCA GTGAT TCAAG C-TAMRA-3Ј. Detection of cells to SDF-1␣, a chemokine agonist for the receptor CXCR4. In mFPR2 and control 18S rRNA was performed using TaqMan Universal contrast, pretreatment of N9 cells with IL-4 inhibited the effect of PCR Master Mix (Applied Biosystems). TNF-␣ on promotion of cell chemotaxis in response to mFPR2 Flow cytometry agonists. The inhibitory effect of IL-4 was dose dependent and was blocked by a mAb against IL-4 (Fig. 1, A and B). The observations Murine microglia were examined for expression of TNFR I and TNFR II by labeling with PE-conjugated mAbs (BD Pharmingen). All staining proin the N9 cell line were corroborated with primary murine micro- cedures were completed at 4°C in Dulbecco’s PBS containing 5 mM glial cells in which TNF-␣ also up-regulated the chemotactic re- EDTA and 1% FCS. After extensive washing, the cells were analyzed sponse to mFPR2 agonists (see Fig. 5A), and the effect of TNF-␣ using a FACScan flow cytometer (BD Biosciences). was inhibited by IL-4. Interestingly, IL-4 failed to reverse the ␣ Western immunoblotting down-regulation of microglial cell responses to SDF-1 caused by TNF-␣ (Fig. 1A). This is consistent with our previous observation N9 cells were grown in 60-mm dishes until subconfluency and then were in which IL-4 did not reverse LPS-induced down-regulation of cultured overnight in FCS-free medium. After treatment with cytokines at ␣ the indicated time points, the cells were lysed with 150 ␮l of ice-cold lysis microglial responses to SDF-1 (16). Thus, IL-4 selectively abro- buffer. The cell lysate was centrifuged at 14,000 rpm (4°C) for 5 min and gated the capacity of TNF-␣ to promote the microglial response to the protein concentration of the supernatant was measured by using a BCA mFPR2 agonists. Protein Assay System (Pierce). Western blotting of phosphorylated ERK1/2 or p38 MAPK was performed according to the manufacturer’s IL-4 inhibits mFPR2 gene expression in TNF-␣-activated instruction using phospho-specific Abs. Briefly, proteins were electropho- microglia resed on a 10% SDS-PAGE precast gel (Invitrogen Life Technologies) under reducing conditions and were transferred onto Immun-Blot polyvi- We next examined the effect of IL-4 on mFPR2 mRNA expression nylidene difluoride membrane (Bio-Rad). The membranes were blocked in N9 cells induced by TNF-␣. mFPR2 mRNA was hardly detect- with 5% nonfat milk (0.1% Tween 20) in TBS overnight at 4°C and then able in unstimulated microglial cells (Fig. 1C) and IL-4 by itself were incubated with primary Abs for3hatroom temperature. After incubation with a HRP-conjugated secondary Ab, the protein bands were did not induce mFPR2 mRNA expression as measured by RT- detected with a Super Signal Chemiluminescent Substrate (Pierce) and BI- PCR. TNF-␣ significantly enhanced mFPR2 mRNA expression in OMAX-MR film (Eastman Kodak). Similar procedures were performed to the N9 cells and the effect of TNF-␣ was markedly inhibited by 6102 IL-4 INHIBITS TNF-␣-INDUCED mFPR2 IN MICROGLIA Downloaded from

FIGURE 1. Effect of IL-4 on the expression and function of mFPR2 in TNF-␣-activated microglia. A, N9 cells were incubated with different concen- trations of IL-4 at 37°C for 30 min, followed by addition of TNF-␣ (100 ng/ml) for 24 h. The cells were then examined for migration in response to the mFPR2 agonist W peptide (1 ␮M) and the chemokine SDF-1␣ (100 ng/ml). B, IL-4 (50 ng/ml) was preincubated with an anti-IL-4 mAb (10 ␮g/ml; anti-4) or a control IgG (10 ␮g/ml) for1hat37°C before being added to N9 cells. The cells were then incubated with TNF-␣ (100 ng/ml) for 24 h before evaluation of chemotaxis induced by W peptide (1 ␮M). The results are expressed as CI representing fold increase in cell migration in response to chemoattractants http://www.jimmunol.org/ Statistically significant (p Ͻ 0.01) increase in cell migration compared to unstimulated cells; #, significantly ,ء .(over the baseline migration (to medium reduced (p Ͻ 0.01) cell migration compared to cells treated with TNF-␣. C, N9 cells were cultured in the presence of IL-4 (10 ng/ml) at 37°C for 30 min, followed by addition of TNF-␣ (100 ng/ml). After incubation at 37°C for 12 h, total RNA was extracted and examined for mFPR2 gene expression by RT-PCR and real-time PCR (D). The RT-PCR products were electrophoresed on agarose gel and visualized with ethidium bromide staining. In real-time PCR, arbitrary units were used to indicate the fold difference in stimulated vs unstimulated cells after normalization with the ␤-actin transcripts. pre-exposure of the cells to IL-4 (Fig. 1C). Real-time PCR was although IL-4 was inactive in enhancing the phosphorylation of used to more quantitatively measure the changes in mFPR2 mRNA MAPKs (Fig. 3A), it significantly reduced the levels of p38 and by guest on September 26, 2021 and revealed mFPR2 mRNA to be increased in TNF-␣-stimulated ERK1/2 phosphorylation stimulated by TNF-␣. In addition, IL-4 N9 microglial cells and to be abrogated by IL-4 (Fig. 1D). As delayed the time of p38 phosphorylation induced by TNF-␣ (Fig. previously reported, N9 cells also expressed a high level of the 3A). Although IL-4 reduced the phosphorylation of both p38 and gene coding for the SDF-1␣ (CXCL12) receptor CXCR4, and the ERK1/2 in microglia stimulated by TNF-␣, its effect on p38 ap- expression of CXCR4 mRNA was not affected by TNF-␣ (Ref. 12 peared to be critical for attenuation of mFPR2 transcription, since and data not shown). The presence of IL-4 did not cause significant the p38 MAPK inhibitor SB202190, but not the MEK1/2 inhibitor changes in the levels of CXCR4 mRNA in microglial cells. These PD98059, decreased TNF-␣-induced expression of mFPR2 results suggest that in TNF-␣-activated mouse microglial cells, the mRNA (Fig. 3B and Ref. 12). selective inhibition of cell responses to mFPR2 agonists by IL-4 Activation of the transcription factor NF-␬B is the hallmark for was associated with reduction of mFPR2 mRNA transcription. the proinflammatory activity of TNF-␣ (21), and the 5Ј flanking Thus, IL-4 appears to antagonize the capacity of TNF-␣ to induce region of the mFPR2 gene contains a consensus NF-␬B motif (22). mFPR2 at both the protein and mRNA levels in microglial cells. We observed that a selective inhibitor of TNF-␣-inducible I␬B␣ phosphorylation, BAY 11-7082, blocked TNF-␣-induced expres- ␬ ␣ IL-4 inhibits activation of MAPKs and NF- B by TNF- sion of mFPR2 (Fig. 3B). Furthermore, IL-4 significantly de- To elucidate the mechanistic basis for the effect of IL-4 on TNF-␣ creased the levels NF-␬B-driven luciferase activity and the expres- signaling in microglial cells, we evaluated the capacity of IL-4 to sion of mFPR2 mRNA in N9 cells stimulated by TNF-␣ (Fig. 4A). regulate the cell surface expression of TNFRs I and II. N9 micro- IL-4 also abolished TNF-␣ induced the activity of AP-1 luciferase glial cells expressed both TNFRs I and II, with receptor II at a reporter in N9 cells stimulated by TNF-␣ (Fig. 4B). These results relatively higher level (Fig. 2). TNF-␣ time-dependently up-regu- indicate that IL-4 disrupts the TNF-␣ signaling pathway in micro- lated both TNFRs I and II on the cell surface of microglia (Fig. 2). glial cells by attenuating activation of MAPKs and important tran- In contrast, IL-4 did not cause significant changes in the expression scription factors which control the expression genes coding for of TNFRs, suggesting that IL-4 might interfere with the signaling proinflammatory proteins. cascade down stream of TNF-␣ receptors in microglial cells, thereby attenuating the induction of mFPR2. The requirement of Utilization of phosphatase, but not of Stat6, by IL-4 MAPKs, p38, and ERK1/2 in particular has been well documented Stimulation of the IL-4 receptor complex results in activation of for TNF-␣ activation of mononuclear phagocytes (12, 19, 20). We multiple signaling pathways, one of which involves Stat6 (23). We therefore evaluated whether MAPKs might be potential targets for then examined the effect of IL-4 on primary microglial cells iso- IL-4 to disrupt the TNF-␣ signaling cascade in microglial cells. lated from Stat6-deficient (Stat6 Ϫ/Ϫ) mice. In Stat6 Ϫ/Ϫ microglia Fig. 3A shows that TNF-␣ induced a rapid and transient phosphor- activated by TNF-␣, IL-4 inhibited mFPR2-mediated chemotactic ylation of p38 and ERK1/2 MAPKs in microglial cells (16), and responses equally as well as in wild-type mice (Fig. 5, A and B). The Journal of Immunology 6103 Downloaded from

FIGURE 3. Effect of MAPK activation on TNF-␣-induced mFPR2 expression and inhibition by IL-4. A, N9 cells were cultured in the presence of IL-4 (10 ng/ml) at 37°C for 30 min, followed by TNF-␣ (100 ng/ml).

The cells were lysed at the indicated time points and cell proteins were http://www.jimmunol.org/ measured for phosphorylated ERK1/2 or p38 MAPK. Anti-pan p38 Ab was used to indicate the loading of total cell protein. B, N9 cells were cultured in the presence of PD98059 (50 ␮M), SB202190 (20 ␮M), or BAY 11- 7082 (20 ␮M) for 30 min at 37°C before stimulation with TNF-␣ (100 ng/ml) for 6 h. Total cell RNA was extracted and examined for mFPR2 gene expression by RT-PCR.

signaling in microglial cells. Fig. 7A shows that although treatment by guest on September 26, 2021 of microglial cells with OA alone did not induce activation of p38, the presence of OA prevented the inhibitory effect of IL-4 on TNF- ␣-induced p38 phosphorylation. We additionally used PP2A siRNA to “knock down” the expression of PP2A in N9 cells and examined the cell response to IL-4. Transfection of PP2A siRNA FIGURE 2. Effect of IL-4 on the expression of TNF receptors on mi- decreased the levels of PP2A mRNA (Fig. 7B) in N9 cells and croglia. N9 cells, cultured for different time intervals in the presence or ␣ completely reversed the inhibitory effect of IL-4 on mFPR2-me- absence of IL-4 (10 ng/ml) and TNF- (100 ng/ml) at 37°C, were exam- ␣ ined for surface expression of TNFR I and TNFR II by flow cytometry. The diated cell migration in TNF- -activated microglia (Fig. 7C). results are presented as percentage of positive cells and mean fluorescence These results indicate that PP2A plays a key role in the “blockade” intensity (MFI) in histograms. of TNF-␣ by IL-4. Discussion Moreover, TNF-␣ significantly enhanced mFPR2 mRNA expres- In this study, we have shown that IL-4 was capable of attenuating sion in Stat6Ϫ/Ϫ microglia and the effect of TNF-␣ was markedly the TNF-␣-induced signaling cascade and the resultant functional inhibited by pre-exposure of the cells to IL-4 (Fig. 5C), suggesting expression of the chemoattractant receptor mFPR2 in microglial that Stat6 is not required for IL-4 to interfere with TNF-␣ signaling cells. We additionally demonstrated that IL-4 inhibits TNF-␣ in- in microglial cells. duced activation of MAPK and transcription factors. Furthermore, To further elucidate the nature of the molecules used by IL-4 to our study revealed that up-regulation of the phosphatase PP2A, in disrupt TNF-␣ signaling in microglial cells, we evaluated the ca- a manner independent of Stat6, may play an essential role in IL- pacity of IL-4 to activate phosphatase PP2A, which is okadaic acid 4-mediated attenuation of the TNF-␣ signaling cascade in micro- (OA) sensitive and has been implicated in the dephosphorylation glial cells and the resultant expression mFPR2, the mouse homolog and deactivation of MAPKs. We found that IL-4 rapidly (within 5 of human FPRL1, a functional receptor for the AD-associated ␤ min) increased the levels of activated PP2A in microglia (Fig. 6). A 42 peptide. In contrast, TNF-␣ only slightly increased the levels of PP2A (Fig. Microglia plays a critical role in CNS diseases. In fact, activa- 6A) at late stages of stimulation (15–30 min). In the presence of tion of microglia is an essential component in the pathogenesis of both TNF-␣ and IL-4, the peak levels of PP2A appeared at 5 min, AD, Parkinson’s disease (24), multiple sclerosis, AIDS dementia indicating that IL-4 accelerated and enhanced the activation of (25), and brain trauma caused by stroke (26). Our previous studies PP2A induced by TNF-␣ alone (Fig. 6A). In parallel experiments, showed that murine microglial cells in the resting state express IL-4 exhibited a similar capacity to modify PP2A activation in- very low levels of mFPR2 (11), a receptor that recognizes a diverse ␤ duced by LPS (Fig. 6B) in association with its attenuation of TLR4 array of chemotactic agonists, including not only A 42 but also the 6104 IL-4 INHIBITS TNF-␣-INDUCED mFPR2 IN MICROGLIA

FIGURE 4. Effect of IL-4 on TNF-␣-triggered NF-␬B and AP-1 luciferase reporter activity. N9 cells were transiently transfected with a NF-␬B(A) or AP-1 (B) reporter construct, then were treated with IL-4 (10 ng/ml) at 37°C for 30 min, followed by TNF-␣ (100 ng/ml) for 12 h. The relative NF-␬B and AP-1 promoter activity was presented as percent increase in cells cul- ,ء .(tured with cytokines vs untreated cells (medium Signiﬁcantly increased NF-␬B or AP-1 reporter activity in comparison to medium-treated cells; #, signiﬁcantly reduced NF-␬B or AP-1 reporter activity in comparison to cells treated with TNF-␣ alone.

bacterial formyl peptide fMLF, HIV-1 envelope protein-derived we could not detect increased production of major pro- or anti- peptides, and a neuroprotective peptide humanin (8, 10, 11). When inflammatory cytokines by TNF-activated microglia in the pres- stimulated with LPS or TNF-␣, microglial cells expressed high ence or the absence of IL-4, and RNase protection assays and levels of mFPR2 transcripts and became responsive to mFPR2- RT-PCR failed to identify increased cytokine transcripts in micro- Downloaded from specific agonists (11, 12), suggesting the presence of proinflam- glial cells stimulated for up to 12 h by either IL-4, TNF-␣, or both matory stimuli may promote microglial expression of mFPR2 and (data not shown). Therefore, neither TNF-␣ nor IL-5 appear to be the subsequent cell responses in CNS diseases in which agonists significant inducers of cytokines in microglial cells under our ex- for mFPR2 are elevated (27, 28). This is in agreement with the perimental condition. We however found that IL-4 potently en- notion that proinflammatory and injurious insults in the brain may hanced the levels of transcription factor peroxisome proliferator- exacerbate neurodegenerative diseases, in particular AD, in which activated receptors (PPAR) ␥ in microglial cells, which is in microglial cells are in an activated state and their responses to A␤ agreement with observations with macrophages (31). PPAR␥ and http://www.jimmunol.org/ peptides trigger the release of neurotoxins (8). Therefore, anti- its ligands are important regulators of immune and inflammatory inflammatory strategy has been used as one of the therapeutic ap- responses (32) by inhibiting genes coding for proinflammatory proaches to AD. This notion was supported by studies in which proteins, presumably through NF-␬B and AP-1 family members. selected non-steroidal anti-inflammatory drugs were documented In microglial cells, activation of PPAR␥ has been reported to retain as beneficial in retarding the onset of AD dementia by inhibiting cells in a quiescent phenotype, suggesting its potentially protective microglial cell responses to amyloid peptides as well as by reduc- role in neuroinflammation (33, 34). Whether IL-4-induced PPAR␥ ing the production of A␤ peptides by neuronal cells (29, 30). participates in reduction of TNF-␣-induced mFPR2 in microglial

IL-4 has been reported to inhibit cytokine production by LPS- cells is currently unclear and merits further investigation. by guest on September 26, 2021 activated macrophages and microglia (17, 18). In support of these Our present study revealing the capacity of the type of cytokine observations, we found that IL-4 potently reduced the production IL-4 to “disrupt” TNF-␣ signaling in microglial cells and inhibit ␤ of IL-6 by LPS-stimulated microglia (data not shown). However, the expression of a chemoattractant receptor for A 42 and other

FIGURE 5. Effect of IL-4 on mFPR2-mediated chemotaxis of primary microglial cells. Primary microglial cells from wild-type (A) or Stat6-deficient (Stat6 Ϫ/Ϫ) (B) mice were cultured in the presence of IL-4 (10 ng/ ml) for 30 min at 37°C, followed by TNF-␣ (100 ng/ml) for an additional 24 h. The cells were then examined for migration in response to mFPR2 agonists (1 ␮M W pep- ␮ ␤ ␣ tide and 17 MA 42) and the chemokine SDF-1 . The results are expressed as CI representing fold increase in cell migration in response to chemoattractants over -Statistically signifi ,ء .(baseline migration (to medium cant (p Ͻ 0.01) increase in cell migration as compared to unstimulated cells; #, significantly (p Ͻ 0.01) reduced cell migration compared with TNF-␣-treated cells. C, Primary microglial cells from Stat6 Ϫ/Ϫ mice were cultured in the presence of IL-4 (10 ng/ml) at 37°C for 30 min, followed by addition of TNF-␣ (100 ng/ml). After incubation at 37°C for 12 h, total RNA was extracted and examined for mFPR2 gene expression by RT-PCR. The RT-PCR products were electrophoresed on agarose gel and visualized with ethidium bromide staining. Fold increase in mFPR2 mRNA levels was calculated by densitometry after normalization against ␤-actin mRNA. The Journal of Immunology 6105 Downloaded from http://www.jimmunol.org/

FIGURE 6. Effect of IL-4 on PP2A levels in microglia. N9 cells were cultured in the presence of IL-4 (10 ng/ml) plus TNF-␣ (100 ng/ml; A)or FIGURE 7. LPS (300 ng/ml; B) for the indicated time points. The cells were lysed and Effect of OA and PP2A siRNA on IL-4-mediated disruption ␣ A, total protein was electrophoresed for immunoblotting of PP2A. Fold in- of TNF- signaling in microglia. After pretreatment with OA (200 nM, crease in PP2A levels was calculated by densitometry after normalization 30 min), N9 cells were cultured in the presence of IL-4 (10 ng/ml) at 37°C ␣ against total p38 as the loading control. for 30 min, followed by TNF- (100 ng/ml) for 5 min at 37°C and lysed. Equal quantities of total protein were measured for phosphorylated p38

MAPK. Anti-pan p38 Ab was used to indicate the loading of total cell by guest on September 26, 2021 peptide agonists associated with infectious diseases suggests yet protein. N9 cells were transfected with a PP2A-specific siRNA or control another anti-inflammatory strategy with a host-derived cytokine. siRNA for 24 h. Total RNA was extracted and examined for PP2A gene expression by RT-PCR (B). PP2A siRNA-transfected cells were then stim- Although a number of anti-inflammatory activities have been re- ulated with IL-4 (10 ng/ml) and/or TNF-␣ (100 ng/ml) for 10 h and ex- ported for IL-4 on activated microglia, including inhibition of the amined for migration in response to the mFPR2 agonist fMLF (10 ␮M; C). expression of cyclooxygenase 2, inducible NO synthase, and The results are expressed as CI representing fold increase in cell migration TNF-␣ as well as other proinflammatory cytokines (17, 18), the in response to fMLF over the baseline migration (to medium). *, Signifi- mechanistic basis for the inhibitory effect of IL-4 on the microglial cantly (p Ͻ 0.01) reduced cell migration compared to TNF-␣-treated cells. response to proinflammatory molecules such as TNF-␣ has not been well defined. In our study, the p38 MAPK inhibitor IL-4 significantly increased the levels of the catalytic PP2A protein SB202190 markedly diminished mFPR2 expression induced by in microglial cells. Moreover, IL-4-induced PP2A may also play a TNF-␣ in microglial cells, indicating a key role of this MAPK in major role in IL-4-mediated inhibition of LPS signaling via TLR4 the TNF-␣-mediated signaling cascade. in microglial cells (Ref. 15 and Fig. 6B). It has been suggested that A large body of studies suggests that PP2A plays a major role in OA-sensitive protein phosphatases PP1/PP2A may cause rapid de- down-regulation of the ERK MAPK pathway and probably acts at phosphorylation of MAPKs induced by proinflammatory stimu- multiple points in the signal transduction cascade. In vitro, PP2A lants to maintain a balanced cell response in stress conditions (18, dephosphorylates and inactivates MEK1 and ERK family kinases 34). Thus, IL-4 may provide protection against deleterious effects (35, 36), and the activity of both kinases are restored after treat- of proinflammatory stimuli in the CNS by maintaining a balanced ment of cells with the PP2A inhibitor OA (37, 38). It has been microglial cell response through activation of phosphatase PP2A reported that inhibition of PP2A by interaction with SV40 small in particular. Further elucidation of the mechanisms by which IL-4 t-Ag resulted in enhanced activation of the stress-activated MAPK protects microglial cells should be beneficial for the design of ther- pathways in association with increased tumor cell growth and in- apeutic approaches to neurodegenerative diseases in which inflam- vasiveness (39). Phosphatases are activated by a number of cellular matory responses exacerbate the pathogenic processes. stimulants (40, 41). For instance, IL-4 was also shown to inhibit CD40 ligand-induced activation of MAPK in macrophages (42), Acknowledgments and the effect of IL-4 was attributed to its potential activation of We thank Dr. Joost J. Oppenheim for reviewing this manuscript, phosphatases. In our study, using OA or “knocking down” the Nancy Dunlop for technical support, and Cheryl Fogle and Cheryl Nolan expression of PP2A by siRNA in microglial cells reversed the for secretarial assistance. inhibitory effect of IL-4 on TNF-␣ signaling, providing strong ev- idence for the importance of PP2A in the capacity of IL-4 to dis- Disclosures rupt TNF-␣-induced microglia activation. In support of this notion, The authors have no financial conflict of interest. 6106 IL-4 INHIBITS TNF-␣-INDUCED mFPR2 IN MICROGLIA

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