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TGF-1 Disrupts Endotoxin Signaling in Microglial Cells through Smad3 and MAPK Pathways^1,2

Yingying Le^*, Pablo Iribarren^*, Wanghua Gong, Youhong Cui^*, Xia Zhang and Ji Ming Wang^3,*

Laboratories of ^* Molecular Immunoregulation and Experimental Immunology, Center for Cancer Research, and Basic Research Program, Science Applications International Corporation-Frederick, National Cancer Institute-Frederick, Frederick, MD 21702

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
Human formyl peptide receptor-like 1 and its mouse homologue formyl peptide receptor 2 (FPR2) are G protein-coupled receptors used by a number of exogenous and host-derived chemotactic peptides, including the 42 aa form of amyloid peptide, a causative factor of Alzheimer’s disease. Functional FPR2 was induced by bacterial LPS in murine microglial cells, the resident phagocytic cells that play a pivotal role in inflammatory and immunological diseases in the CNS. To identify agents that may suppress microglial cell activation under proinflammatory conditions, we investigated the effect of TGF-1 on the expression of functional FPR2 by microglial cells activated by LPS. TGF-1 dose-dependently inhibited the mRNA expression and function of FPR2 in LPS-activated microglial cells. The inhibitory effect of TGF-1 was mediated by Smad3, a key signaling molecule coupled to the TGF- receptor, and the transcription coactivator, p300. Also, TGF-1 activates MAPKs in microglial cells that became refractory to further stimulation by LPS. These effects of TGF-1 culminate in the inhibition of LPS-induced activation of NF-B and the up-regulation of FPR2 in microglial cells. Thus, TGF-1 may exert a protective role in CNS diseases characterized by microglial cell activation by proinflammatory stimulants.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
Lipopolysaccharide (LPS, endotoxin) is a principal component of the outer membrane of Gram-negative bacteria and a potent activator of innate host immune responses. Upon stimulation by LPS, monocytic phagocytes express a variety of mediators including proinflammatory cytokines, chemokines, and reactive oxygen species, which orchestrate inflammation and activation of adaptive immunity to eliminate invading microorganisms (1). Microglia that belong to the monocytic phagocyte lineage and as resident immunocompetent cells, play a critical role in infection, inflammation, trauma, ischemia, and neurodegeneration in the CNS (2, 3). In experimental endotoxemia, LPS has been shown to enter the brain parenchyma by diffusion through regions with defective blood-brain barrier function and to activate microglia and other CNS cells (4). LPS-stimulated microglia, in addition to producing high levels of proinflammatory cytokines and chemokines (5), also increase their expression of a seven transmembrane, G protein-coupled formyl peptide receptor (FPR)2⁴ (6), which mediates the chemotactic response to both exogenous and host-derived peptide agonists including the bacterial peptide fMLP HIV-1 envelope protein-derived peptides (6), the acute phase protein serum amyloid A (7), as well as the 42 aa form of amyloid peptide (A₄₂) (6, 8), accumulated in the brain tissue of Alzheimer’s disease (AD). Although the expression of FPR2 by activated microglia may facilitate their accumulation at sites where host-derived and bacterial chemotactic agonists such as fMLP and A₄₂ are elevated, uncontrolled activation of microglia by FPR2 agonists has been shown to augment the production of reactive oxygen intermediates and inflammatory cytokines that may be toxic to neuronal cells (8, 9). In this context, agents that protect microglia from activation by proinflammatory signals may have therapeutic potential in CNS inflammatory states due to bacterial infection and neurodegenerative diseases.

TGF-1 has been appreciated as a potent anti-inflammatory cytokine and plays a pivotal role in maintaining balanced host responses in inflammatory and immunological conditions (10). Targeted disruption of mouse TGF-1 gene resulted in excessive inflammatory responses (11, 12), while administration of TGF-1 in mice prevented LPS-induced septic shock (13, 14). In vitro, TGF-1 inhibited LPS-stimulated expression of proinflammatory cytokines in macrophages (15). Based on these prominent anti-inflammatory properties of TGF-1, we investigated the capacity of this cytokine to protect microglial cells from activation by LPS by diminishing their expression of FPR2 and their capacity to respond with chemotaxis and activation to FPR2 agonists. In this study, we report that TGF-1, through Smad3-mediated pathway, is capable of inhibiting LPS-induced expression of FPR2 in microglial cells.

	Materials and Methods

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Reagents and cells

fMLP and LPS were purchased from Sigma-Aldrich (St. Louis, MO). Mouse stromal cell-derived factor (SDF)-1 and human TGF-1 were purchased from PeproTech (Rocky Hill, NJ). V3 peptide, a peptide domain derived from HIV-1 envelope protein gp120 (16), was kindly provided by National Institutes of Health AIDS Research and Reference Reagents Program (Bethesda, MD). The synthetic A₄₂ peptide was from California Peptide Research (Napa, CA). Anti-phosphorylated (p) ERK1/2 (Thr²⁰²/Tyr²⁰⁴), anti-ERK1/2, anti-p-p38 MAPK (Thr¹⁸⁰/Tyr¹⁸²), and anti-p38 Abs were from Cell Signaling Technology (Beverly, MA). MEK1/2 inhibitor PD98059, and p38 MAPK inhibitor SB202190 were from Calbiochem (Darmstadt, Germany). The expression plasmids of Smad3 and Smad3c were kindly provided by Dr. R. Derynck (University of California, San Francisco, CA) (17). p300 expression plasmid was kindly provided by Dr. N. Colburn (National Cancer Institute-Frederick, Frederick, MD) (18).

The N9 murine microglial cell line was a kind gift from Dr. P. Ricciardi-Castagnoli (Universita Degli Studi di Milano-Bicocca, Milan, Italy). These cells express typical markers of resting mouse microglia, and have been extensively used as representative of mouse microglial cells (19). The cells were grown in IMDM medium supplemented with 5% heat-inactivated FCS, 2 mM glutamine, 100 U/ml penicillin, 100 µg/ml streptomycin, and 50 mM 2-ME.

Chemotaxis assays

Chemotaxis assays were performed with 48-well chemotaxis chambers (NeuroProbe, Cabin John, MD) as described previously (6). Polycarbonate filters with 8-µm pore size and 90 min incubation at 37°C were used for measurement of microglial migration. The results are expressed as chemotaxis index that represents the fold increase in the number of migrated cells in response to chemoattractants over the spontaneous cell migration (to control medium).

RT-PCR and real-time PCR

Total RNA was extracted from cells with RNeasy Mini kit and depleted of contaminating DNA with RNase-free DNase (Qiagen, Valencia, CA) according to the manufacturer’s instructions. For amplification of FPR2 mRNA, the following primers were used to yield a 268 bp product: sense, 5'-TCT ACC ATC TCC AGA GTT CTG TGG-3', and antisense, 5'-TTA CAT CTA CCA CAA TGT GAA CTA-3'. Specific primers for mouse CXCR4 were: sense, 5'-GGC TGT AGA GCG AGT GTT GC-3', and antisense, 5'-GTA GAG GTT GAC AGT GTA GAT-3'; which yield a product of 390 bp. RT-PCR was performed with 0.5 µg total RNA for each sample (High Fidelity ProSTAR HF System; Stratagene, Kingsport, TN), consisting of a 15-min reverse transcription at 42°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 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) (20). Briefly, 5 ng of reverse-transcribed cDNA was used in triplicate samples with TaqMan Universal PCR Master Mix (Applied Biosystems) according to manufacturer’s instructions. 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 for amplification of the FPR2 cDNA were: 5'-CCTTA TAGTC TTGAG AGAGC CCTGA-3' (sense); 5'-TGCAG GAGGT GAAGT AGAAC TGG-3' (antisense); 5'-FAM-TGAGG ATTCT GGTCA AACCA GTGAT TCAAG C-BHQ-1–3' (probe). Mouse GAPDH primers and probe were from BioSource International (Camarillo, CA) and used as internal control. The comparative threshold cycle method for relative quantitation was used as per Applied Biosystems. To examine the FPR2 mRNA stability, N9 cells were treated with 10 ng/ml TGF-1 for 1 h, followed by 50 ng/ml LPS for 15 h. Actinomycin D (5 µg/ml) was then added to the cell culture (as time 0). Total RNA was collected at different time points after actinomycin D treatment, reverse transcribed, and FPR2 transcripts were measured by real-time PCR.

Transient cell transfection

To obtain transient expression of molecules coupled to TGF-1 signaling pathway, transfection was performed in N9 cells with Superfect Transfection Reagent (Qiagen) on 60-mm dishes using 5 µg plasmid DNA. Thirty hours after transfection, the cells were stimulated with LPS (50 ng/ml, 15 h), TGF-1 (10 ng/ml, 16 h), or TGF-1 (10 ng/ml) for 1 h, followed by LPS (50 ng/ml) for additional 15 h, and FPR2 expression was then examined.

Western immunoblotting

N9 cells were grown on 60-mm dishes until subconfluency and cultured overnight in FCS-free medium. After treatment with TGF-1 and/or LPS, the cells were lysed with 1x SDS sample buffer (62.5 mM Tris-HCl (pH 6.8), 2% SDS, 10% glycerol, 50 mM DTT), sonicated for 10–15 s, and heated at 100°C for 5 min. The cell lysate was centrifuged at 14,000 rpm and 4°C for 10 min, and the protein concentration of the supernatant was measured by Coomassie Protein Assay Reagent (Pierce, Rockford, IL). Western blotting of phosphorylated ERK1/2 and p38 was performed according to the manufacturer’s instruction. Briefly, proteins were electrophoresed on a 10% SDS-PAGE precast gel (Invitrogen Life Technologies, Carlsbad, CA), and transferred onto Immobilon P membranes (Millipore, Bedford, MA). The membranes were blocked with blocking buffer (1x PBS, 0.05% Tween 20 with 3% nonfat dry milk), and then were incubated with primary Abs overnight at 4°C. After incubation with a HRP-conjugated secondary Ab, the protein bands were detected with a Super Signal Chemiluminescent Substrate Stable Peroxide Solution (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.

Measurement of NF-B activation

N9 cells were seeded on 24-well plate at a density of 2 x 10⁵/well. Transient transfection of pNF-B-Luc Reporter (Promega, Madison, WI) was performed by Fugen 6 (Roche, Indianapolis, IN). pTAP-Luc was used as negative control. pRL-null vector (Promega) was used as an internal control for normalization of transfection and harvesting efficiency. After transfection, the cells were cultured in normal medium for 24–36 h, and were then treated with different concentrations of inhibitors for MAPK p38 (SB202190), ERK1/2 (PD98059), or TGF-1 for 1 h, followed by LPS (50 ng/ml) for 24 h. Cell lysis and luciferase detection were performed using the dual luciferase assay system (Promega). Promoter activity of the constructs was expressed in units relative to values measured in cells without LPS stimulation. Results are mean ± SEM of three independent experiments conducted in triplicate.

Statistical analysis

All experiments were performed at least three times and representative results were presented. Where applicable, the statistical 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

 
TGF-1 inhibits functional expression of FPR2 in LPS-activated murine microglial cells

We confirmed our earlier results (6) showing that unstimulated murine microglial cell line N9 lacked the capacity to migrate toward well-characterized FPR2 agonists V3 peptide (a chemotactic segment of HIV-1 envelope gp120), A₄₂ (AD-associated peptide), and the bacterial peptide fMLP in a micromolar concentration range. But these cells showed a potent chemotaxis response to the chemokine SDF-1 (CXCL12) that uses the receptor CXCR4 (Fig. 1A). In contrast, LPS-activated microglial cells showed considerable chemotactic responses to fMLP, V3 peptide, and A₄₂, but a remarkably decreased migration to SDF-1 (CXCL12) (Fig. 1A). Incubation of N9 cells with TGF-1 had no effect on cell response to FPR2 agonists (data not shown). However, treatment of N9 cells with TGF-1 before LPS treatment attenuated the cell response to FPR2 agonists fMLP, V3 peptide, and A₄₂, and restored the capacity of N9 cells to migrate to the chemokine SDF-1 (Fig. 1A). These results suggest that TGF-1 may interfere with the signaling of LPS in microglial cells, thereby attenuating the induction of FPR2.

View larger version (37K): [in this window] [in a new window]   FIGURE 1. TGF-1 inhibits LPS-induced functional expression of FPR2 in murine microglial cells. A, The mouse microglial cell line N9, cultured for 1 h in the absence or presence of different concentrations of TGF-1 at 37°C, was treated with 200 ng/ml LPS for 24 h, and then examined for migration in response to SDF-1 (CXCL12), fMLP, V3 peptide, and A42. The results are expressed as chemotaxis index (CI), representing fold increase in cell migration in response to chemoattractants over the baseline (migration to medium). *, Significant (p < 0.01) change of cell migration compared with cells treated with LPS alone. B, N9 cells were incubated with or without TGF-1 for 1 h at 37°C, then with LPS (50 ng/ml) for an additional 15 h. Total RNA was extracted from the cells and examined for FPR2 and CXCR4 gene expression by RT-PCR. The expression of -actin gene was used as control. The RT-PCR products at different dilutions were electrophoresed on agarose gel and visualized with ethidium bromide staining. C, N9 cells were incubated with or without 10 ng/ml TGF-1 for 1 h at 37°C, followed by LPS (50 ng/ml) for an additional 15 h. Total RNA was extracted and reverse transcribed, and FPR2 cDNA were amplified by real-time PCR. Arbitrary units were used to indicate the fold difference as compared with cells treated with control medium. D, N9 cells pretreated with 10 ng/ml TGF-1 for 1 h, followed by 50 ng/ml LPS for 15 h, were cultured with 5 µg/ml actinomycin D for the indicated time intervals. FPR2 gene was then examined by real-time PCR. The curves represent the rate of FPR2 mRNA decay in cells treated with LPS in the presence or absence of TGF-1.

The involvement of MAPKs in the effect of TGF-1

The requirement of activation of MAPK subtypes, p38 and ERK1/2, has been documented extensively in LPS-induced cytokine production in mononuclear phagocytes (1). We investigated the role of these MAPK subtypes in LPS-induced expression of FPR2. The MEK1/2 inhibitor PD98059, and p38 MAPK inhibitor SB202190 both decreased LPS-induced expression of FPR2 mRNA in N9 cells and the chemotactic response of the cells to FPR2 ligands (Fig. 2). Thus, activation of MAPKs appears to be important for the induction of functional FPR2 in microglial cells by LPS.

View larger version (29K): [in this window] [in a new window]   FIGURE 2. Functional induction of FPR2 expression by LPS is dependent on the activation of MAPKs. N9 cells cultured in the absence or presence of inhibitors for MAPK p38 (SB202190) or MEK1/2 (PD98059) for 1 h at 37°C were treated with 200 ng/ml LPS for an additional 24 h, and then were examined for migration in response to FPR2 agonists (A) and for FPR2 gene expression by RT-PCR (B). SB202190 and PD98059 had no effect on cell response to FPR2 agonists and FPR2 mRNA expression (data not shown). *, Significantly (p < 0.01) reduced cell migration compared with cells treated with LPS alone.

View larger version (47K): [in this window] [in a new window]   FIGURE 3. TGF-1 inhibits LPS-induced ERK1/2 and p38 phosphorylation. N9 cells incubated with stimulant were lysed, and the extracts were electrophoresed, transferred, and blotted. The protein bands were detected with anti-p-p38 or anti-p-ERK1/2 Abs. The membranes were also stripped and examined for total p38 (A) or ERK1/2. A, Time course with 300 ng/ml LPS and dose response with LPS for 30 min. B, Cell treatment with 10 ng/ml TGF-1 for different time periods. C, Cell treatment with different concentrations of TGF-1 for 240 min at 37°C, followed by 200 ng/ml LPS for 30 min.

TGF-1 inhibits LPS-induced NF-B activation

Activation of the transcription factor NF-B is a crucial event in the proinflammatory activity of LPS (22), and the 5' flanking region of murine FPR2 gene contains consensus NF-B motif (23). We examined the effect of TGF-1 on LPS-induced NF-B activation in microgial cells transiently transfected with an NF-B luciferase reporter construct. As shown in Fig. 4, LPS significantly increased NF-B-driven luciferase activity in N9 cells and the effect of LPS was abolished by the p38 inhibitor SB202190 and TGF-1. The MEK1/2 inhibitor PD98059 also significantly, but partially, inhibited the activation of NF-B luciferase in N9 cells stimulated by LPS. These results suggest that the inhibitory effect of TGF-1 on LPS-induced FPR2 expression in microglial cells may result from its disruption of MAPK, the p38 in particular, and NF-B axis triggered by LPS.

View larger version (33K): [in this window] [in a new window]   FIGURE 4. TGF-1 inhibits NF-B activation induced by LPS. N9 cells were transiently transfected with an NF-B reporter construct, then were treated with inhibitors for MAPK p38 (SB202190), ERK1/2 (PD98059), or TGF-1 for 60 min, followed by LPS (50 ng/ml) for 24 h. Cells were harvested for luciferase activity assay. SB202190, PD98059, and TGF-1 had no effect on NF-B activity (data not shown). Promoter activity of the constructs was expressed relative to that of the cells not treated with LPS. Results are mean ± SEM for three independent experiments conducted in triplicate. *, Statistically significant inhibition of luciferase activity.

Smad3 and transcription coactivator p300 mediate effect of TGF-1

To assess whether Smad3, a crucial signaling molecule coupled to TGF-1 receptor (24), is responsible for the inhibitory effect of TGF-1 on LPS induction of FPR2 in microglial cells, we transfected N9 cells with a Smad3 dominant-negative (Smad3c) construct (17) that lacks 39 aa on the C-terminal end, which contains three phosphorylation sites necessary for nuclear translocation of Smad3 following TGF-1 treatment. In cells transiently overexpressing Smad3c, TGF-1 completely failed to show any inhibition on LPS-induced expression of FPR2 mRNA (Fig. 5A), suggesting an essential role for Smad3 in mediating TGF-1-induced cellular events that results in inhibition of FPR2 expression in LPS-activated N9 cells.

View larger version (32K): [in this window] [in a new window]   FIGURE 5. TGF-1 inhibits LPS-induced FPR2 mRNA expression via Smad3 and transcription coactivator p300. N9 cells were transiently transfected with the expression plasmids for 30 h, then were treated with or without 10 ng/ml TGF-1 for 1 h, followed by 50 ng/ml LPS for additional 15 h. FPR2 mRNA expression was examined by RT-PCR. The expression of -actin gene was used as control. A, The effect of dominant-negative Smad3 (Smad3c). B, The effect of transcription coactivator p300 or both Smad3 and p300. C, N9 cells transfected with various constructs were examined for their chemotactic responses to fMLP (10 µM) and SDF-1 (CXCL12) (10 ng/ml). *, Significantly reduced cell response to fMLP as compared with the cells treated with both LPS and TGF-1; or significantly increased cell chemotaxis in response to SDF-1 as compared with cells treated with both LPS and TGF-1.

View larger version (23K): [in this window] [in a new window]   FIGURE 6. Smad3c reverses the inhibitory effect of TGF-1 on LPS-induced NF-B activity. N9 cells were transiently transfected with Smad3c expression vector and NF-B reporter construct, then were treated with TGF-1 for 1 h, followed by LPS (50 ng/ml) for 24 h. Cells were harvested for luciferase activity assay. Promoter activity of the constructs was expressed as fold increase relative to that of the construct in the cells not treated with LPS. Results are mean ± SEM for three independent experiments conducted in triplicate. *, Significantly reduced inhibition by TGF-1 of LPS-stimulated NF-B activity.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

Microglia play a critical role in CNS diseases. In fact, activation of microglia is an essential component in the pathogenesis of Parkinson disease (27), AD, multiple sclerosis, AIDS dementia (28), and stroke brain trauma (29). Our previous studies showed that murine microglial cells in resting state express very low levels of FPR2 (6), a homologue of human FPR-like 1 (FPRL1), which recognizes a diverse array of chemotactic agonists including the bacterial formyl peptide fMLP, HIV-1 envelope protein-derived peptides, and A₄₂, a key pathogenic agent associated with AD (6, 8). When stimulated with LPS or TNF-, microglial cells expressed high levels of FPR2 transcripts and became responsive to FPR2-specific agonists (6, 30), suggesting the presence of proinflammatory stimuli may promote microglial expression of FPR2 and subsequent cell responses in CNS diseases in which agonists for FPR2 are elevated (31, 32). This is in agreement with the notion that proinflammatory and injurious insults 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 trigger the release of neurotoxins. Therefore, anti-inflammatory strategy has been proposed as one of the therapeutic choices for AD and selected nonsteroidal anti-inflammatory drugs have been documented as beneficial in retarding the onset of AD dementia by inhibiting microglial cell response to amyloid peptides, as well as by reducing the production of A peptides by neuronal cells (33, 34).

TGF-1 is a pleiotropic cytokine involved in many pathophysiological processes including growth, development, inflammation, tissue repair, and immunity (10, 35). Yet, a prominent property of TGF-1 is its ability to inhibit proinflammatory and immune responses (10). TGF-1 signals through a heterodimeric complex of type I and type II serine/threonine kinase receptors. Upon TGF-1 binding, the type II receptor phosphorylates and activates the type I receptor, which then phosphorylates and activates Smad2 or Smad3. Smad2 or Smad3 forms heterodimers with Smad4, followed by translocation of the Smad2/3-Smad4 complex to the nucleus, where it regulates the transcription of target genes by binding directly to the promoter regions or by associating with other transcription factors or coactivators/repressors (24). Our present study suggests a critical role of Smad3 in TGF-1-mediated inhibition of LPS signaling cascade in microglial cells. Transient expression of dominant-negative Smad3, Smad3c, which was unable to translocate to the nucleus in response to TGF-1 (25), completely blocked the effect of TGF-1 on LPS-induced FPR2 expression. Because Smad proteins interact functionally with the transcriptional coactivator CREB-binding protein (CBP)/p300, a process essential to their ability to function as transcriptional effectors (25, 26), a synergistic enhancement of the inhibitory effect of TGF-1 by overexpression of Smad3 and p300 in microglia as shown in our study indicates that the transcription coactivator p300 is also an important determinant in the cross-talk between TGF-1 and LPS.

It should be pointed out that while Smads are critical to the signaling of TGF- family cytokines, TGF- also stimulate other intracellular signal transduction pathways such as ERK and p38 MAPKs (36). MAPKs have been implicated in the signaling by TGF-1 in regulation of growth, apoptosis, and gene expression (36, 37). Our study suggests that the induction of functional FPR2 expression by LPS involves the activation of ERK1/2 and p38 signaling pathways, with a more prominent effect of p38 as shown by potent inhibition of LPS-induced NF-B activation with a p38 inhibitor (Fig. 4). However, although TGF-1 also induces ERK1/2 and p38 phosphorylation in microglial cells, it failed to augment the expression of FPR2, suggesting a diverse pathway downstream of MAPK is linked to TGF-1. In contrast, although our study showed that pretreatment of N9 cells with TGF-1 inhibited LPS-stimulated phosphorylation of ERK1/2 and p38, the mechanisms involved in such inhibition remains to be elucidated. The possibility that TGF-1 may activate phosphatases that prevent MAPK phosphorylation in response to LPS was plausible as suggested by results obtained in mouse macrophages (21). However, in mouse macrophages, TGF-1 did not by itself activate p38 (21), in contrast to our observations with microglial cells. Because TGF-1-activation of MAPK reached maximum at 1 h, followed by a complete deactivation at 4 h, and a minimal 4-h pretreatment of the cells with TGF-1 was required to attenuate LPS-activation of MAPKs, it is possible that after TGF-1 treatment, MAPKs may become refractory to subsequent stimulation with LPS. These possibilities are under rigorous investigation.

Stimulation of monocytes/macrophages with LPS also activates transcription factor NF-B that mediates the induction of many genes encoding cytokines, chemokines, and chemokine receptors (38). The inhibitory effect of TGF-1 on LPS-induced NF-B activation in intestinal epithelial cells is dependent on Smad 3, as shown by a previous study (39), which was confirmed by our study in microglia. Both NF-B and Smad proteins have been demonstrated to interact with CBP/p300 (40). In endothelial cells, the inhibition of cytokine-induced E-selectin gene by TGF-1 involves a competition between TGF-1-activated Smad and cytokine-activated NF-B for binding to the transcription coactivator, CBP/p300, present in the nuclei (41). In intestinal epithelial cells, TGF-1-activated Smad mediates inhibition of LPS-induced NF-B recruitment to the IL-6 gene promoter through modulation of histone acetylation (39). Therefore, TGF-1 may disrupt LPS signaling by attenuation of MAPK cascade, as well as by inhibiting the transcription activity of NF-B through Smad3 in microglial cells, thereby abrogating the induction of FPR2 gene. However, the signaling pathways mediated by LPS and TGF-1 and their intracellular cross-talk are complex, thus more studies are warranted to fully elucidate the mechanisms by which these two important molecules regulate the cell function.

TGF-1 is one of the major cytokines detected in CNS diseases. In experimental AD models, dual roles of TGF-1 have been reported in the disease process: while overexpression of both human TGF-1 and amyloid precursor protein genes in mice causes general activation of microglial cells in the mouse brain and a reduced deposition of A peptides in parenchymal plaques (42), other reports revealed the potential of TGF-1 to promote A peptide accumulation and deposition in rodents administered intracerebroventricularly with both A peptides and TGF-1 (43). These results suggest that a tight temporal-spatial balance of TGF-1 production may determine the outcome of its influence on AD pathogenesis. FPR2 and human FPRL1 may play a role in A peptide uptake and metabolism by mononuclear phagocytes (44), and it has been suggested that such cells are actively involved in the A clearance or fibrillary deposition depending on the length of exposure (32). However, FPR2 and FPRL1 also mediate the proinflammatory activity of A in mononuclear phagocytes, which may result in the release of neurotoxic effectors. Interestingly, TGF-1 also directly inhibits the function of FPR2 already induced in microglial cells by LPS (data not shown). In human monocytes that constitutively express functional FPRL1, TGF-1 decreases the gene expression of this receptor and the subsequent cell response to FPRL1 agonists (data not show). Thus, TGF-1 can exert its capacity to modulate FPR2/FPRL1 expression and function at multiple levels and play an important role in host cell response in inflammatory diseases.

	Acknowledgments

 
We thank Dr. J. J. Oppenheim for reviewing the manuscript, N. Dunlop for technical support, and C. Fogle and C. Nolan for secretarial assistance.

	Footnotes

 
¹ This project has been funded in part with federal funds from the National Cancer Institute, National Institutes of Health, under Contract No. N01-C0-12400.

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 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, Frederick, MD 21702. E-mail address: wangji{at}mail.ncifcrf.gov

⁴ Abbreviations used in this paper: FPR, formyl peptide receptor; A₄₂, 42 aa form of amyloid peptide; AD, Alzheimer’s disease; CBP, CREB-binding protein; FPRL1, FPR-like 1; p, phosphorylated; SDF, stromal cell-derived factor.

Received for publication September 26, 2003. Accepted for publication May 10, 2004.

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