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IL-4 Down-Regulates Lipopolysaccharide-Induced Formyl Peptide Receptor 2 in Murine Microglial Cells by Inhibiting the Activation of Mitogen-Activated Protein Kinases ^1,2

Pablo Iribarren^*, You-Hong Cui^*,, Yingying Le^*, GuoGuang Ying^*, Xia Zhang, Wanghua Gong and Ji Ming Wang^3,*

^* Laboratory of Molecular Immunoregulation, Biochemistry Section, Lanzhou Military Medical University, Lanzhou, People’s Republic of China; Laboratory of Experimental Immunology, Center for Cancer Research, National Cancer Institute, Frederick, MD 21702; and Basic Research Program, SAIC-Frederick, Frederick, MD 21702

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
Microglial cells actively participate in proinflammatory responses in the CNS. Upon stimulation with the bacterial LPS, microglial cells express a functional formyl peptide receptor 2 which mediates the chemotactic and activating effects of a variety of polypeptide agonists including amyloid (A_1–42), a critical pathogenic agent in Alzheimer’s disease. In the present study, we found that LPS-induced expression and function of formyl peptide receptor 2 in microglial cells was markedly inhibited by IL-4, a Th2-type cytokine. Our effort to elucidate the mechanistic basis revealed that IL-4 attenuated LPS-stimulated activation of NF-B, extracellular signal-regulated kinase, and p38 mitogen-activated protein kinase, and the effect of IL-4 was associated with a phosphoinositide 3-kinase pathway-dependent increase in serine/threonine phosphatase activity. These results suggest that IL-4 may play an important role in the maintenance of homeostasis of CNS and in the regulation of the disease process characterized by microglial activation in response to proinflammatory stimulants.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
Microglial cells in the CNS share many characteristics of tissue macrophages (1). Under normal conditions, microglial cells are in a resting state, but they become rapidly activated upon contact with proinflammatory signals and together with infiltrating macrophages participate in CNS responses to infection, inflammation, injury, and neurodegeneration (2). Microglial cells are regularly distributed in close vicinity to neurons in the gray matter and between fiber tracts in the white matter in healthy CNS. Under inflammatory conditions, they accumulate at the sites of lesions, presumably in response to locally generated chemotactic agents (3). Microglial cells, similar to infiltrating macrophages, express a number of chemoattractant receptors, including many chemokine receptors and the receptor for activated complement component 5 (C5aR) (4, 5). Therefore, these cells migrate in response to selected chemoattractants in vitro and, in inflammatory brain lesions, increased numbers of infiltrating microglia are associated with elevated levels of chemotactic factors (6). Activated microglial cells and macrophages are essential for the clearance of invading microorganisms and injured tissue. However, they can also be stimulated to express a variety of proinflammatory cytokines such as IL-1, TNF-, and IL-6, as well as superoxide and NO, which are neurotoxic (2, 7).

Some of the chemoatractant receptors on microglial cells are induced by proinflammatory stimuli. For instance, microglial cells activated by the Gram-negative bacterial endotoxin LPS express increased levels of a G protein-coupled chemotactic peptide receptor, formyl peptide receptor (FPR) ⁴ 2 (8). FPR2 is a homologue of human FPRL1 and both recognize a diverse array of chemotactic agonists, including the bacterial formylated peptide N-formyl-Met-Leu-Phe (fMLF), HIV-1 envelope protein-derived peptides (9), as well as the 42-aa form of A peptide (A_1–42), a key pathogenic agent associated with the brain lesions of Alzheimer’s disease (AD) (10, 11). Activated FPR2 or FPRL1 transduces a cascade of signals in phagocytic leukocytes and microglia, resulting in chemotaxis, Ca²⁺ mobilization, phagocytosis, and release of reactive oxygen intermediates and proinflammatory cytokines (9, 12).

Although the up-regulation of FPR2 in activated microglial cells is believed to represent an innate host defense against potential pathogens (13), it may also amplify inflammatory responses which cause detrimental consequences in AD and AIDS dementia, both associated with the production of FPR2 agonists (9, 10, 14). Therefore, development of agents capable of inhibiting microglial activation may have therapeutic potential for inflammatory diseases of the CNS. In this study, we investigated the effect of IL 4, a type 2 cytokine with a multiplicity of anti-inflammatory properties, on the expression and function of the chemotactic receptor FPR2 in LPS-activated microglial cells. We report that IL-4 markedly down-regulates the expression and function of FPR2 in microglial cells induced by LPS. We further demonstrate that IL-4 inhibits LPS activation of the mitogen-activated protein kinase (MAPK) cascade in microglial cells and the effect of IL-4 is dependent on activation of the phosphoinositide 3-kinase (PI3K) pathway and is associated with an increase in okadaic acid (OA)-sensitive phosphatase activity.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Reagents and cells

fMLF, LPS, and OA were purchased from Sigma-Aldrich (St. Louis, MO). Mouse stromal cell-derived factor 1 (SDF-1) and IL-4 were purchased from PeproTech (Rocky Hill, NJ). The chemotactic peptide WKYMVm (designated W peptide) was synthesized and purified by the Department of Biochemistry, Colorado State University (Fort Collins, CO). The PI3K inhibitor LY294002 and Abs against phospho-extracellular signal-regulated kinase (ERK) 1/2, total ERK1/2, phospho-p38 MAPK, total p38 MAPK, and phospho-activating transcription factor 2 (ATF-2) were purchased from Cell Signaling Technology (Beverly, MA). Primary murine microglial cells were isolated from 1-day-old newborn C57BL/6 (wild type) mice and C57BL/6 Stat6-deficient (Stat6^-/-) mice (a kind gift from Dr. M. Grusby, Harvard University, Boston, MA). The murine microglial cell line N9 was a kind gift from Dr. P. Ricciardi-Castagnoli (Universita Degli Studi di Milano-Bicocca, Milan, Italy). The cells were grown in IMDM supplemented with 5% heat-inactivated FCS, 2 mM glutamine, 100 U/ml penicillin, 100 µg/ml streptomycin, and 50 µM 2-ME.

Animal care was provided in accordance with the procedures outlined in the Guide for the Care and Use of Laboratory Animals (National Institutes of Health Publication No. 86-23, 1985).

Flow cytometry

Murine microglia were examined for CD14 and Toll-like receptor 4 (TLR4) expression by direct immunofluorescence using FITC- and PE-conjugated mAbs (BD PharMingen, San Diego, CA and eBioscience, San Diego, CA, respectively). All staining procedures were completed at 4°C in Dulbecco’s PBS containing 5 mM EDTA and 1% FCS. After extensive washing, the cells were analyzed using a FACScan flow cytometer (BD Biosciences, Mountain View, CA).

Chemotaxis assays

Chemotaxis assays for microglial cells were performed with 48-well chemotaxis chambers (NeuroProbe, Cabin John, MD) with an incubation period of 90 min at 37°C (8). The results are expressed as the mean (± SD) of the chemotaxis index (CI), which represents the fold increase in the number of migrated cells, counted in three high-power fields (x400), in response to chemoattractants over spontaneous cell migration (to control medium).

RT-PCR and real-time PCR

Total RNA was extracted from cells with an RNeasy Mini kit and depleted of contaminating DNA with RNase-free DNase (Qiagen, Valencia, CA). For amplification of the FPR2 gene, primers 5'-TCT ACC ATC TCC AGA GTT CTG TGG-3' (sense) and 5'-TTA CAT CTA CCA CAA TGT GAA CTA-3' (antisense) were designed to yield a 268-bp product. Specific primers for mouse CXCR4 were: 5'-GGC TGT AGA GCG AGT GTT GC-3' (sense) and 5'-GTA GAG GTT GAC AGT GTA GAT-3' (antisense), which yield a product of 390 bp. RT-PCR was performed with 0.5 µg of total RNA for each sample (High Fidelity ProSTAR HF System; Stratagene, La Jolla, CA), consisting of a 15-min reverse transcription at 37°C, 1 min inactivation of Moloney murine leukemia 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), with a final extension for 10 min at 72°C. Primers for the murine -actin gene were used as controls (Stratagene). Real-time PCR was performed by using an ABI Prism 7700 Sequence Detector (Applied Biosystems, Foster City, CA). Briefly, 5 ng of reverse-transcribed cDNA was used in triplicate samples. The assays were initiated with 2 min at 50°C, 10 min at 95°C, and then 40 cycles of 15 s at 95°C and 1 min at 60°C. Primers and specific probes were obtained from Applied Biosystems and consisted of the following: 5'-CCTTA TAGTC TTGAG AGAGC CCTGA-3' (sense); 5'-TGCAG GAGGT GAAGT AGAAC TGG-3' (antisense) and the probe 5'-FAM- TGAGG ATTCT GGTCA AACCA GTGAT TCAAG C-TAMRA-3'. Detection of FPR2 and control 18S rRNA was performed using TaqMan Universal PCR Master Mix (Applied Biosystems).

Measurement of NF-B activation

N9 cells cultured in 12-well plates were transfected with 1 µg/well pNFB-luc reporter construct and 10 ng/well pRL-TK construct. The cells were then cultured in the presence or absence of stimulants for different time periods. Both firefly luciferase and Renilla luciferase activities were measured and promoter activity was expressed as percent increase in stimulated cells vs cells cultured in medium alone. Results are the mean ± SEM of three independent experiments, each performed in triplicate samples.

Western immunoblotting

N9 cells were grown in 60-mm dishes until subconfluency and then were cultured overnight in medium in the absence of FCS. After treatment with LPS and/or IL-4 at the indicated time points, the cells were lysed with 150 µl of ice-cold lysis buffer. The cell lysate was centrifuged at 14,000 rpm and 4°C for 5 min, and the protein concentration of the supernatant was measured using the BCA Protein Assay (Pierce, Rockford, IL). Western blotting of phosphorylated ERK1/2 or p38 MAPK was performed according to the manufacturer’s instruction using phospho-specific Abs. Briefly, proteins were electrophoresed on a 10% SDS-PAGE precast gel (Invitrogen, Carlsbad, CA) under reducing conditions and transferred onto Immun-Blot Polyvinylidene Membrane (Bio-Rad, Hercules, CA). The membranes were blocked with 5% nonfat milk, 0.1% Tween 20 in TBS overnight at 4°C, and then were incubated with primary Abs for 3 h at room temperature. After incubation with a HRP-conjugated secondary Ab, the protein bands were detected with a Super Signal Chemiluminescent Substrate (Pierce) and BIOMAX-MR film (Eastman Kodak, Rochester, NY). For detection of total ERK1/2 and p38, the membranes were stripped with Restore Western Blot Stripping Buffer (Pierce) followed by incubation with specific Abs.

Statistical analysis

All experiments were performed at least three times and the results presented are from representative experiments. For cell migration, the significance of the difference between test and control groups was analyzed using Student’s t test.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
IL-4 inhibited FPR2-mediated migration of LPS-activated microglia

We first examined the effect of IL-4 on the function of FPR2 in LPS-stimulated microglial cells. As reported earlier (8), treatment of murine microglial cell line N9 with LPS for 24 h promoted cell migration in response to FPR2 agonists, including the bacterial formyl peptide fMLF, the HIV-1 envelope-derived peptide V3, A_1–42, and a synthetic small peptide (W peptide). In contrast, LPS treatment down-regulated the chemotactic response of N9 cells to SDF-1, a chemokine agonist for the receptor CXCR4 (Fig. 1A). IL-4 dose-dependently inhibited the effect of LPS on promotion of chemotactic responses of N9 cells to FPR2 agonists (Fig. 1, B and D) and the activity of IL-4 was blocked by a mAb against IL-4 (Fig. 1C). Treatment of primary murine microglial cells with LPS for 24 h also up-regulated the chemotactic response to FPR2 agonists (Fig. 1E), and the effect of LPS was inhibited by IL-4. Although as we previously reported, LPS concomitantly down-regulated the function of CXCR4 in microglial cells, this effect of LPS was not reversed by treatment of the cells with IL-4 (Fig. 1A), suggesting that IL-4 selectively block the induction of FPR2 activity by LPS in microglia.

View larger version (31K): [in this window] [in a new window]   FIGURE 1. The effect of IL-4 on chemotactic response of LPS-activated microglia. N9 cells (A–D) or primary microglia (E) cultured in the presence or absence of LPS (300 ng/ml) and IL-4 (50 ng/ml) at 37°C for 24 h were examined for migration in response to FPR2 agonists fMLF (10 µM), W peptide (1 µM), A1–42 (17 µM), V3 peptide (9 µM), or the chemokine SDF-1 (10 nM). IL-4 (50 ng/ml) was also preincubated with an anti-IL-4 mAb (10 µg/ml) or a control IgG (10 µg/ml) for 1 h at 37°C before being added to N9 cell culture for 24 h at 37°C (C). The results are expressed as CI, representing fold increase of cell migration in response to chemoattractants over the baseline migration (to medium). *, Statistically significant (p < 0.01) increase of cell migration compared with unstimulated cells. **, Statistically significant (p < 0.01) inhibition of cell migration compared with LPS-treated cells.

We next examined the capacity of IL-4 to modulate the expression of the gene coding for FPR2 in LPS-activated microglia. As shown in Fig. 2A, although IL-4 did not induce the FPR2 gene in N9 cells, LPS potently up-regulated the mRNA transcript for FPR2 as measured by RT-PCR. Simultaneous addition of IL-4 along with LPS in the N9 cell culture resulted in a marked decrease in FPR2 gene expression in comparison with cells treated with LPS alone. The effect of IL-4 on LPS-induced FPR2 gene expression was confirmed by real-time PCR (Fig. 2B) in which the number of FPR2 transcripts in microglial cells induced by LPS was significantly reduced in the presence of IL-4. In contrast to their effect on the FPR2 gene, neither LPS nor IL-4 exhibited an appreciable capacity to modulate CXCR4 gene expression in N9 cells (Fig. 2A). These results suggest that IL-4 interferes with the capacity of LPS to modulate FPR2 gene transcription in microglial cells.

View larger version (22K): [in this window] [in a new window]   FIGURE 2. The effect of IL-4 on FPR2 gene expression by microglia. N9 cells were cultured in the presence or absence of LPS (300 ng/ml) and IL-4 (50 ng/ml) at 37°C for 12 h and total RNA was extracted and examined for receptor gene expression by RT-PCR. A, The RT-PCR products at different dilutions were electrophoresed on agarose gel and visualized with ethidium bromide staining. B, The transcripts for FPR2 were determined by real-time PCR and normalized against rRNA. Arbitrary units were used to indicate the fold difference in stimulated cells vs unstimulated cells.

We then asked whether IL-4 might also affect LPS-induced changes in microglial cell surface markers. N9 cells expressed high levels of the mononuclear phagocyte marker CD11b and the key LPS signaling receptor TLR4, but moderate levels of CD14 (Ref.8 and Fig. 3). Incubation of the cells with LPS for 24 h increased the surface expression of CD14 but decreased TLR4 (Fig. 3). IL-4, which did not affect the baseline expression of CD11b, CD14, or TLR4 on N9 cells, decreased the effect of LPS on modulation of the CD14 and TLR4 levels on the cell surface. Thus, IL-4 is capable of attenuating LPS-induced changes in the expression of both LPS binding and signaling receptors on microglial cells.

View larger version (26K): [in this window] [in a new window]   FIGURE 3. The effect of IL-4 on the expression of surface markers by microglia. N9 cells cultured in the presence or absence of LPS (300 ng/ml) and IL-4 (50 ng/ml) at 37°C for 24 h were examined for surface expression of CD14 and the LPS signaling molecule, TLR4, by flow cytometry. The results are represented as mean fluorescence intensity (MFI) and percentage of positive cells in histograms.

IL-4 inhibited LPS-induced NF-B activation

Activation of the transcription factor NF-B is a crucial event in the proinflammatory activity of LPS (15). We therefore examined the capacity of IL-4 to inhibit LPS-induced NF-B activation in N9 cells transiently transfected with the NF-B luciferase reporter construct. LPS significantly increased NF-B-driven luciferase activity in N9 cells (Fig. 4). However in the presence of IL-4, LPS-induced NF-B activation was markedly reduced. IL-4 alone did not affect NF-B luciferase reporter activity in N9 cells.

View larger version (20K): [in this window] [in a new window]   FIGURE 4. The effect of IL-4 on NF-B-luciferase reporter gene activity. N9 cells were transiently transfected with a NF-B reporter construct, then were treated with LPS (300 ng/ml) and IL-4 (50 ng/ml) at 37°C for 12 h. The relative promoter activity was presented as percent increase in stimulated cells vs untreated cells. *, Significantly increased NF-B reporter activity in comparison to untreated cells. **, Significantly reduced NF-B reporter activity in comparison to cells treated with LPS alone.

It has been reported that the MAPK pathway is central to the biological activities of LPS (15). We confirmed that LPS-induced NF-B activation in microglial cells was dependent on MAPK activation, which is also essential for LPS-induced up-regulation of FPR2 in microglia. This was evidenced by a rapid and transient increase in phosphorylation of both ERK and p38 in LPS-stimulated N9 cells (Fig. 5A) and cells pretreated with either a p38 inhibitor, SB202190, or a mitogen-activated protein/ERK kinase 1/ERK inhibitor, PD98059, followed by LPS failed to express enhanced FPR2 transcripts (data not shown) in association with a failure of the cells to migrate in response to FPR2 agonists (Fig. 5, B and C). These observations prompted our evaluation of the effect of IL-4 on LPS-induced MAPK activation in microglial cells. IL-4 at different time points did not induce phosphorylation of ERK and p38 in N9 cells (Fig. 5A). However, it potently inhibited LPS-induced phosphorylation of ERK and p38 and appeared to shift the maximal level of ERK phosphorylation from 60 to 30 min, as compared with LPS alone (Fig. 5A). Moreover, IL-4 reduced LPS activation of the transcription factor ATF-2, which is downstream of MAPK (16). Our results thus provide evidence that the capacity of IL-4 to down-regulate LPS-induced FPR2 in microglia is associated with the inhibition of LPS-stimulated MAPK activation.

View larger version (24K): [in this window] [in a new window]   FIGURE 5. The effect of IL-4 on LPS-mediated MAPK activation in microglia. A, N9 cells were cultured in the presence or absence of LPS (300 ng/ml) and IL-4 (50 ng/ml) at 37°C and lysed at the indicated time points. Equal amounts of total proteins were electrophoresed and blotted and protein bands were detected with anti-phospho-ERK1/2, anti-phospho-p38, and anti-phospho-ATF-2 Abs, respectively. The membranes were stripped and examined for total ERK1/2 or p38 MAPK. N9 cells were also preincubated with p38 inhibitor SB202190 (B) or mitogen-activated protein/ERK kinase 1 inhibitor PD98059 (C) for 60 min at 37°C, followed by incubation with LPS for additional 24 h. Then the cells were examined for migration in response to FPR2 agonists fMLF (10 µM), V3 peptide (9 µM), W peptide (1 µM), or the chemokine SDF-1 (10 nM). The results are expressed as CI, representing fold increase in cell migration in response to chemoattractants over the baseline migration (to medium). *, Statistically significant (p < 0.01) increase of cell migration compared with unstimulated cells. **, Statistically significant (p < 0.01) inhibition of cell migration compared with LPS-treated cells.

Serine/theronine phosphatases such as protein phosphatase 1 (PP1) and protein phosphatase 2A (PP2A) are among the key regulators of MAPK phosphorylation and these phosphatases are sensitive to a selective inhibitor, OA (17, 18). To elucidate the mechanisms involved in IL-4 inhibition of microglial activation by LPS, we studied the possibility that IL-4 might increase intracellular phosphatase activity, which rapidly dephosphorylates MAPKs activated by LPS. Fig. 6A shows that although treatment of microglial cells with OA alone did not induce activation of p38, the presence of OA prevented the inhibitory effect of IL-4 on LPS-induced p38 phosphorylation. Similarly, OA reversed the inhibitory effect of IL-4 on LPS-induced ERK phosphorylation (Fig. 6B). In addition, in the presence of OA, IL-4 was unable to inhibit FPR2 gene expression in LPS-stimulated N9 cells (Fig. 6E). Our results suggest that IL-4 increases OA-sensitive phosphatase activity, which negatively regulates LPS-mediated MAPK activation and FPR2 expression.

View larger version (36K): [in this window] [in a new window]   FIGURE 6. The effects of IL-4 on LPS-stimulated MAPK phosphorylation and FPR2 expression in microglia are dependent on activation of PI3K and phosphatase. After pretreatment with OA (200 nM, 30 min; A and B) or LY294002 (50 µM, 60 min; D), N9 cells were cultured in the presence or absence of LPS (300 ng/ml) and IL-4 (50 ng/ml) for 60 min at 37°C and lysed. Equal quantities of total protein were electrophoresed and blotted, and protein bands were detected with anti-phospho-p38 or anti-phospho-ERK Abs. The membranes were then stripped and examined for total p38 or total ERK MAPK. C, Primary microglial cells from Stat6-deficient (Stat6 -/-) mice were cultured in the presence or absence of LPS (300 ng/ml) and IL-4 (50 ng/ml) at 37°C for 24 h and examined for migration in response to FPR2 agonists fMLF (10 µM), W peptide (1 µM), A1–42 (17 µM), and V3 peptide (9 µM). The results are expressed as CI, representing fold increase of cell migration in response to chemoattractants over the baseline migration (to medium). *, Statistically significant (p < 0.01) inhibition of cell migration compared with LPS-treated cells. E, After pretreatment with OA (200 nM, 30 min) or LY294002 (50 µM, 30 min), N9 cells were cultured in the presence or absence of LPS (300 ng/ml) and IL-4 (50 ng/ml) for 6 h at 37°C and total RNA was extracted and examined for receptor gene expression by RT-PCR. The RT-PCR products were electrophoresed on agarose gel and visualized with ethidium bromide staining. Fold increase in FPR2 expression was calculated taking -actin gene expression as reference.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
We have shown that IL-4 inhibited the expression and function of FPR2 in microglial cells stimulated with LPS. In addition, IL-4 attenuated LPS-induced phenotypic changes of microglial cells. The effect of IL-4 on microglia is associated with the inhibition of MAPK and NF-B activation induced by LPS. Moreover, the inhibitory effect of IL-4 on LPS-induced MAPK activation and FPR2 expression was dependent on PI3K and on increased activity of OA-sensitive phosphatases. To our knowledge, this is the first demonstration that IL-4 exerts a protective role in microglial cells by inhibiting LPS-induced functional expression of FPR2, a chemoattractant receptor that recognizes multiple agonistic peptides associated with pathological conditions (8, 9, 10, 11).

Although sharing many features with macrophages present in other tissues, the ramified microglia in normal CNS display a typical resting phenotype characterized by lack of endocytic activity and low levels of activation markers shown on tissue macrophages and peripheral blood monocytes (21). However, microglia respond rapidly to endogenous (e.g., damaged cells, cytokines, and tumors), as well as exogenous (infectious agents and endotoxin) stimuli, and actively participate in immune responses, inflammation, and tissue repair in the CNS (22, 23). During these processes, microglia acquire markers shown on differentiated macrophages and display effector functions, including secretion of proinflammatory and neurotoxic mediators (24). Activated microglial cells also accumulate at sites of inflammation in CNS diseases, including multiple sclerosis (21), AIDS encephalitis (2), prion diseases, and AD (4). The accumulation of microglial cells in CNS lesions has been attributed to the local production of chemotactic agents, which activate specific receptors on microglia.

Despite the expression of a number of chemoattractant receptors by microglia, our previous studies showed that murine microglial cells in resting state express low levels of FPR2 (8, 9, 10, 13). Only when stimulated with LPS or TNF-, microglial cells expressed high levels of FPR2 transcripts and became responsive to FPR2-specific peptide agonists (8, 25). Thus, the presence of proinflammatory stimuli may promote microglial responses in CNS diseases in which agonists for FPR2 are elevated. This is in agreement with the notion that an injurious insult in the brain may exacerbate neurodegenerative diseases, in particular AD, in which microglial cells are in an activated state and their responses to A peptides promote the release of neurotoxins (3, 26). In this context, anti-inflammatory strategy has been proposed as one of the therapeutics for AD and indeed some non-steroid anti-inflammatory drugs have been recorded as beneficial in retarding the onset of AD dementia by inhibiting microglial cell response to A peptides as well as reducing the production of these peptides by neuronal cells (27, 28).

IL-4 is a Th2 cytokine with diverse biological activities in many cell types, including costimulation for growth and promotion of survival of cultured T and B lymphocytes, differentiation of T cells to the Th2 phenotype, and down-regulation of inflammatory functions of monocytes and macrophages (29). It has been reported that exposure of macrophages to IL-4 may induce an "alternative activation state" characterized by up-regulation of mannose receptor but down-regulation of NO and proinflammatory cytokine production (29, 30). Our present study revealing the capacity of IL-4 to "disrupt" LPS signaling in microglial cells and inhibiting the expression of a chemoattractant receptor for A_1–42 and other peptide agonists associated with infectious diseases suggests yet another anti-inflammatory strategy with a host-derived cytokine. Although a number of anti-inflammatory activities have been reported for IL-4 on LPS-activated microglia, including inhibition of the expression of cyclooxygenase 2, inducible NO synthase, and TNF- and other proinflammatory cytokines (31, 32), the mechanistic basis for the inhibitory effect of IL-4 on microglial response to LPS has not been well defined.

Our observation that MAPK inhibitors markedly inhibited FPR2 expression and function induced by LPS in microglial cells indicates a key role of ERK1/2 and p38 MAPKs in LPS-mediated signaling cascade. The regulation of MAPK activation involves complex mechanisms and participation of several types of phosphatases (33), including the OA-sensitive protein phosphatases PP1/PP2A, which cause rapid dephosphorylation of MAPKs and thus may maintain a balanced cell response to proinflammatory stimulants (34, 35). Phosphatase activation is associated with signaling pathways triggered by many stimuli (17, 18). In a previous report, IL-4 was shown to rapidly inhibit CD40 ligand-induced activation of MAPK in macrophages (36), and the effect of IL-4 was attributed to its potential activation of phosphatases. In our study, pretreatment of microglial cells with OA reversed the inhibitory effect of IL-4 on LPS-induced MAPK phosphorylation and FPR2 expression, suggesting that OA-sensitive serine/threonine phosphatases may be responsible for the capacity of IL-4 to disrupt the LPS-induced signaling cascade. In support of this notion, our preliminary experiments indicated that IL-4 significantly promoted activation of PP2A in microglial cells cultured in the presence of LPS (P.I., unpublished data).

Our present study also implicated the role of PI3K in activation of phosphatases by IL-4. Activation of IL-4R leads to the binding of the p85 subunit of PI3K to phosphorylated insulin receptor substrate-1/2. The complex formation results in a conformational change in the PI3K that leads to the activation of its p110 catalytic subunit (19). Our results showing that PI3K inhibitor LY294002 blocked the effect of IL-4 on LPS-induced p38 MAPK phosphorylation and FPR2 expression in microglia support a key role of PI3K in IL-4-induced phosphatase activation and subsequent inhibition of LPS-stimulated signaling cascade in microglial cells. This signaling cascade of IL-4 is not dependent on Stat6 because IL-4 maintained its capacity to down-regulate LPS-induced functional expression of FPR2 in Stat6-deficient (Stat6 ^-/-) microglia.

The balance between pro- and anti-inflammatory signals in the CNS is important in the pathogenic process of inflammation and neurodegenerative diseases. For instance, LPS has been reported to be able to diffuse into the CNS through areas where blood-brain barrier functions are defective, thereby stimulating microglial cells (37). On the other hand IL-4 is detectable in the CNS and its levels are significantly down-regulated in amyloid precursor protein transgenic mice that develop CNS pathology similar to human AD (38). Thus, IL-4 may provide protection against the deleterious effect of proinflammatory stimuli in the CNS. Elucidating the mechanisms of IL-4 protection against microglial activation by endotoxin should be beneficial for the development of therapeutic approaches to inflammatory and neurodegenerative diseases.

	Acknowledgments

 
We thank Drs. Joost J. Oppenheim and Howard A. Young for reviewing this manuscript, Nancy Dunlop for technical support, and Cheryl Fogle and Cheryl Nolan for secretarial assistance.

	Footnotes

 
¹ This work was supported in part by federal funds from the National Cancer Institute, National Institutes of Health, Contract NO1-CO-12400.

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, commercial products, or organizations imply endorsement by the U.S. government. 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.

³ Address correspondence and reprint requests to Dr. Ji Ming Wang, Laboratory of Molecular Immunoregulation, Center for Cancer Research, National Cancer Institute, Frederick, Building 560, Room 31-40, Frederick, MD 21702-1201. E-mail address: wangji{at}mail.ncifcrf.gov

⁴ Abbreviations used in this paper: FPR, formyl peptide receptor; AD, Alzheimer’s disease; A, amyloid ; OA, okadaic acid; PI3K, phosphoinositide-3-kinase; MAPK, mitogen-activated protein kinase; ERK, extracellular signal-regulated kinase; ATF-2, activating transcription factor 2; TLR4, Toll-like receptor 4; CI, chemotaxis index; SDF, stromal cell-derived factor 1; PP, protein phosphatase.

Received for publication May 22, 2003. Accepted for publication September 11, 2003.

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