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The Journal of Immunology, 1999, 163: 6614-6621.
Copyright © 1999 by The American Association of Immunologists

Ligation of Microglial CD40 Results in p44/42 Mitogen-Activated Protein Kinase-Dependent TNF-{alpha} Production That Is Opposed by TGF-ß1 and IL-10

Jun Tan1, Terrence Town, Michael Saxe, Daniel Paris, Yajuan Wu and Michael Mullan

The Roskamp Institute, Department of Psychiatry, University of South Florida, Tampa, FL 33613


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Recently, it has been demonstrated that the CD40 receptor is constitutively expressed on cultured microglia at low levels. Ligation of CD40 by CD40 ligand on these cells results in microglial activation, as measured by TNF-{alpha} production and neuronal injury. However, the intracellular events mediating this effect have yet to be investigated. We report that ligation of microglial CD40 triggers activation of p44/42 mitogen-activated protein kinase (MAPK). This effect is evident 30 min posttreatment, and progressively declines thereafter (from 30 to 240 min). Phosphorylated p38 MAPK is not observed in response to ligation of microglial CD40 across the time course examined. Inhibition of the upstream activator of p44/42 MAPK, mitogen-activated protein/extracellular signal-related kinase kinase 1/2, with PD98059, decreases phosphorylation of p44/42 MAPK and significantly reduces TNF-{alpha} release following ligation of microglial CD40. Furthermore, cotreatment of microglial cells with CD40 ligand and TGF-ß1 or IL-10, or both, inhibits CD40-mediated activation of p44/42 MAPK and production of TNF-{alpha} in a statistically interactive manner. Taken together, these data show that ligation of microglial CD40 triggers TNF-{alpha} release through the p44/42 MAPK pathway, an effect that can be opposed by TGF-ß1 and IL-10.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Previous studies have shown that the CD40 receptor is expressed on a wide range of both immune and nonimmune cell types, including dendritic cells, monocytes, macrophages, fibroblasts, endothelial cells, and smooth muscle cells (1, 2). Initially, the CD40 pathway was thought to play a critical role in the humoral and cellular immune response, because interaction between CD40 and CD40 ligand (CD40L)2 induces B cell proliferation and differentiation into Ab-secreting plasma cells (1). Recently, we and others have shown that CD40 is constitutively expressed on microglia (N9 cell line and murine primary culture) at low levels, and CD40 expression is greatly enhanced by a low dose of IFN-{gamma} (10 U/ml) (3, 4, 5, 6). Furthermore, we have shown that ligation of IFN-{gamma}-induced microglial CD40 leads to marked TNF-{alpha} production by these cells that is neurotoxic at such levels (3).

It is well known that microglia perform a key immunoregulatory role in the CNS, but the specific molecular mediators of the microglia-associated neuroimmune response are not well characterized. Because we have shown that microglia secrete TNF-{alpha} following CD40 ligation, we wished to investigate the intracellular mediators of this effect. It has been shown that engagement of the CD40 receptor on B cells results in proliferation, differentiation, and apoptosis through signaling pathways involving mitogen-activated protein kinases (MAPKs or extracellular signal-related kinases) (7, 8, 9, 10). Recent studies have indicated that p38 and p44/42 MAPK pathways mediate cytokine release in macrophages and monocytes stimulated with LPS (11, 12, 13, 14). Based on such clues as to the signal transduction cascade affected by stimulation of CD40 in other cell types, we wished to investigate the intracellular sequelae that lead to CD40-mediated TNF-{alpha} release in microglia.

We show activation of a p44/42 MAPK-associated signal transduction pathway following ligation of microglial CD40 with CD40L. We also provide evidence that microglial TNF-{alpha} production resulting from CD40 stimulation is dependent on activation of a p44/42 MAPK-associated signal transduction cascade, an effect that is markedly inhibited by blockade of the upstream activator of p44/42 MAPK (MEK1/2, which is responsible for phosphorylation and subsequent activation of p44/42 MAPK) with PD98059. Finally, we show that cotreatment of microglia with CD40L and the antiinflammatory cytokines TGF-ß1 or IL-10, alone or in combination, inhibits TNF-{alpha} release in a statistically interactive way.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Microglial cell culture and reagents

Murine primary culture microglia were isolated from mouse cerebral cortex tissue and were grown in RPMI medium supplemented with 5% FCS, 2 mM glutamine, 100 U/ml penicillin, 0.1 µg/ml streptomycin, and 0.05 mM 2-ME according to previously described methods (3). Briefly, cerebral cortices from newborn mice (1 to 2 days old) were isolated under sterile conditions and were kept at 4°C before mechanical dissociation. Cells were plated in 75-cm2 flasks, and complete medium was added. Primary cultures were kept for 14 days so that only glial cells remained, and microglia were isolated by mechanically shaking flasks at 200 rpm in a Lab-Line Incubator-Shaker. More than 98% of these glial cells stained positive for Mac-1 (Boehringer Mannheim Biochemicals, Indianapolis, IN). Human rCD40L protein was kindly provided by Dr. Jean-Yves Bonnefoy (Glaxo Institute for Molecular Biology, Geneva), and was purified according to the methods described by Mazzei and colleagues (15). The conditioned media (including CD40L, 1 µg/ml) contained <40 pg of endotoxin/ml, as determined by chromogenic Limulus Amoebocyte Lysate assay analysis (QLC-1000; BioWhittaker, Walkersville, MD). In addition, ligation of microglial CD40 with CD40L was performed in the presence or absence of the endotoxin inhibitor, polymyxin B (as described in Ref. 21 ; 1 µg/ml; Sigma, St. Louis, MO). No significant difference was detected on TNF-{alpha} production between CD40L-treated microglial cells incubated with or without polymyxin B (by t test for independent samples, p = 0.809, data not shown). Abs for phospho-p44/42 MAPK (Thr202/Tyr204), p44/42 MAPK, phospho-p38 MAPK (Thr180/Tyr182), and p38 MAPK were obtained from NEB (Beverly, MA). PD98059 (MEK1/2 inhibitor), SB203580 (p38 MAPK inhibitor), and SB202474 (a negative control for SB203580) were obtained from Calbiochem (La Jolla, CA). Anti-mouse alkaline phosphatase-conjugated IgG secondary Ab was obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Hy-bond polyvinylidene difluoride membranes and the Immun-Star chemiluminescence substrate were purchased from Bio-Rad Laboratories (Hercules, CA). Abs for anti-CD40 (IgM), anti-CD40L (IgG), and isotype-matched control Abs (IgM and IgG) were obtained from PharMingen (San Diego, CA).

TNF-{alpha} RT-PCR and ELISA

Murine primary culture microglia were plated in 6-well tissue culture plates (Nunclon, Nalge Nunc, Roskilde, Denmark) at a density of 2 x 105 cells/well and incubated with CD40L in the presence or absence of anti-CD40, anti-CD40L, or control Abs for 12 h. In a separate set of experiments, microglial cells were plated at a density of 8 x 105 cells/well and incubated with CD40L (1 µg/ml) for various time periods (30, 60, 90, 120, or 240 min), or stimulated with PD98059 (10, 5, or 1 µM) for 1 h and, in some cases, subsequently treated with CD40L (1 µg/ml) for 30 or 240 min. Some groups of microglia were harvested and prepared for Western immunoblotting for p44/42 MAPK (as described below). Cells were collected from 240-min treatment group and prepared for TNF-{alpha} RT-PCR (kit was obtained from Invitrogen, Carlsbad, CA), as previously described (16). Forward (5'-ACC ATG AGC ACA GAA AGC ATG-3') and reverse (5'-GCC CCC TCA GGG GTG TCC TTG-3') oligonucleotides were designed to produce the 562-bp murine TNF-{alpha} cDNA fragment. Cell-free supernatants were also collected from the 12-h treatment group, as well as from the other time points examined, and assayed by TNF-{alpha} ELISA kit (Genzyme, Cambridge, MA) in strict accordance with the manufacturer’s instruction.

Western immunoblotting

Murine primary culture micrgolia were plated in 6-well tissue culture plates at a density of 8 x 105 cells/well. Some of the cells were incubated with or without (control) CD40L (1 µg/ml) in the presence or absence of anti-CD40 or appropriate control Abs (1/200 dilution for each), or various cytokines for 30 min after seeding. Other groups of cells were treated with CD40L (1 µg/ml) for 30, 60, 120, and 240 min, or LPS (10 ng/ml) for 60 min, or went untreated (control) after plating. Immediately following culturing, microglia were washed in ice-cold PBS three times, scraped into ice-cold PBS, and lysed in an ice-cold lysis buffer containing 20 mM Tris (pH 7.5), 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, 2.5 mM sodium pyrophosphate, 1 mM ß-glycerophosphate, 1 mM Na3VO4, 1 µg/ml, and 1 mM PMSF. After incubating for 30 min on ice, samples were centrifuged at high speed for 15 min, and supernatants were collected. Total protein content was estimated using the Bio-Rad protein assay. An aliquot corresponding to 50 µg of total protein of each sample was separated by SDS-PAGE and transferred electrophoretically to Hy-bond polyvinylidene difluoride membranes. Nonspecific Ab binding was blocked with either 5% nonfat dry milk or 5% BSA (in the case of phospho-specific Abs) for 1 h at room temperature in TBS (20 mM Tris, 500 mM NaCl, pH 7.5). In the case of MAPK immunoblotting, membranes were first hybridized with a phospho-specific MAPK Ab, stripped with 2-mercaptoethanol stripping solution (62.5 mM Tris-HCl, pH 6.8, 2% SDS, and 100 mM 2-mercaptoethanol), and then reprobed with an Ab that recognizes total MAPK. Immunoblotting was conducted with a primary Ab followed by an anti-mouse alkaline phosphatase-conjugated IgG secondary Ab as a tracer. The Immun-Star chemiluminescence substrate was used to develop the blots. Densitometric analysis was performed for all immunoblots using the Fluor-S Multimager with Quantity One software (Bio-Rad).

Immune complex kinase assay

Primary culture microgial cells were seeded in 6-well plates at 8 x 105 cells/well. Thirty minutes following treatment with CD40L in the presence or absence of various cytokines or Abs, microglial cells were lysed in ice-cold lysis buffer (as described above). Total cellular protein was quantified with the Bio-Rad protein assay, and an aliquot of 100 µg of protein was separated by SDS-PAGE. Determination of p44/42 MAPK activity was conducted with the p44/42 MAP Kinase Assay Kit (New England Biolabs, Beverly, MA) in strict accordance with the manufacturer’s instruction. The phosphorylated form of the Elk1 fusion protein was visualized by Western blot (as described above) using a specific Ab for phosphorylated Elk1 supplied with the kit.

Statistical analysis

Data were analyzed using ANOVA followed by post hoc comparisons of means by Bonferonni’s or Dunnett’s T3 method, where appropriate. In instances of single mean comparisons, t test for independent samples was used to assess significance. {alpha} levels were set at 0.05 for each analysis. All analyses were performed using SPSS for windows release 9.0.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Ligation of microglial CD40 results in activation of p44/42 MAPK

It has been shown that activation of p44/42 MAPK is involved in TNF-{alpha} production in macrophages and monocytes following LPS stimulation (11, 12, 13, 14). These data led us to ask whether activation of the MAPK pathway may be responsible for microglial TNF-{alpha} release following CD40 ligation. To investigate this possibility, we analyzed p44/42 MAPK in microglial cell lysates following CD40L treatment or appropriate control conditions. Data show that p44/42 MAPK phosphorylation is significantly induced within 30 min after CD40 ligation compared with appropriate controls (Fig. 1GoA), an effect that can be blocked by interrupting the CD40-CD40L interaction. Accordingly, CD40L treatment results in increased CD40-CD40L interaction-dependent p44/42 MAPK enzymatic activity, as evidenced by immune complex kinase assay (Fig. 1GoB). TNF-{alpha} release is markedly enhanced by CD40 ligation, an effect that is, again, opposed by blocking the interaction between CD40 and CD40L (Fig. 1GoC). When taken together, these results suggest that ligation of microglial CD40 specifically leads to increased activity of p44/42 MAPK and TNF-{alpha} production.



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  		FIGURE 1. Ligation of microglial CD40 activates p44/42 MAPK and promotes TNF-{alpha} release. Microglia were treated with CD40L in the presence or absence of anti-CD40, anti-CD40L, or appropriate isotype-matched control Abs, or went untreated (control) for 30 min (A and B) or 12 h (C). For A, a specific Ab against phosphorylated p44/42 MAPK was used to examine phosphorylation of p44 (44 kDa) and p42 (42 kDa) MAPKs in cell lysates (above). Band density ratios to actin (phospho-p42 MAPK/actin) are represented as means ± 1 SEM (n = 3 for each condition, below). For B, a specific Ab against the phosphorylated p44/42 MAPK fusion protein (phospho-Elk1) was used to determine p44/42 MAPK activity in identical samples (100 µg of each was loaded into the gel) by immune complex kinase assay (above). Band densities are represented as means ± 1 SEM (n = 3 for each condition, below). C, Microglial TNF-{alpha} release following ligation of CD40, in which data are represented as mean TNF-{alpha} release values (pg/mg total protein) ± 1 SEM (n = 3 for each condition). For A–C, one-way ANOVA revealed significant between-groups differences (p < 0.001), and post hoc comparison showed significant differences between control and CD40L treatment (p < 0.01), and between either CD40L/anti-CD40L or CD40L/anti-CD40 cotreatment and treatment with appropriate isotype-matched control Abs (p <= 0.01).

 
To determine the temporal nature of MAPK activation following CD40 ligation, cultured microglia were treated with CD40L at various time points and assayed for MAPK activation using Abs specific for phosphorylated forms of p44/42 or p38 MAPKs. Data show that activation of p44/42 occurs in microglia within 30 min following CD40 ligation and begins to decline thereafter through 240-min posttreatment (Fig. 2GoA). In addition, when identical samples are analyzed by Western immunoblotting with anti-phosphorylated p38 Ab, data show that ligation of CD40 does not induce p38 phosphorylation within the time frame examined (Fig. 2GoB).



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  		FIGURE 2. Phosphorylation of p44/42 MAPK following ligation of microglial CD40. Microglia were stimulated with CD40L for 30, 60, 120, and 240 min; LPS for 60 min; or went untreated (control) for 60 min, as indicated. Cell lysates were analyzed by Western immunoblotting using Abs that recognize A, phosphorylated or total p44/42 MAPK, or B, phosphorylated or total p38 MAPK, as indicated. Band density ratios (A, phospho-p42/total p42 MAPK; B, phospho-p38/total p38 MAPK) are represented below Western immunoblots as means ± 1 SEM (n = 3 for each condition). For A, one-way ANOVA revealed a significant main effect of CD40L treatment time on activation of p44/42 MAPK (p < 0.001), whereas, in B, no significant effect of CD40L treatment time was observed on p38 MAPK activation (p = 0.433).

 
CD40-mediated microglial TNF-{alpha} production is MEK/p44/42 MAPK dependent

Having shown that microglial p44/42 MAPK is phosphorylated after CD40 ligation, we wished to determine whether p44/42 MAPK activation was responsible for TNF-{alpha} production. Thus, microglia were incubated with CD40L (1 µg/ml) for a time range from 30–240 min, and TNF-{alpha} RT-PCR and ELISA were performed. Results show that TNF-{alpha} mRNA signal is present within 90 min following CD40L treatment, and progressively increases between 120 and 240 min (Fig. 3GoA). In accordance with this, TNF-{alpha} protein is observed at 120 min, and increases within 240 min posttreatment (Fig. 3GoB). To determine whether activation of p44/42 MAPK was responsible for TNF-{alpha} production following ligation of the CD40 receptor, we treated microglia with PD98059, a specific inhibitor of MEK1/2, before ligating CD40. TNF-{alpha} mRNA is markedly decreased 240 min post-cotreatment with CD40L and PD98059 (see Fig. 4GoA). Most importantly, TNF-{alpha} protein expression following ligation of CD40 is dose dependently inhibited with PD98059 cotreatment within 240 min (see Fig. 4GoB). Moreover, this effect is statistically interactive, further substantiating the hypothesis that CD40 mediates TNF-{alpha} production through activation of p44/42 MAPK. As PD98059 treatment results in a significant reduction in CD40-mediated phosphorylation and activity of p44/42 MAPK within 30 min (Fig. 4Go, C and D), when taken together, these data suggest that the activity of phosphorylated p44/42 MAPK induced by CD40 ligation is crucial for TNF-{alpha} production in microglia. To further support the hypothesis that CD40L-mediated microglial activation is dependent on activation of p44/42 MAPK, we employed another specific MEK1/2 inhibitor, U0126 (200 nM; Calbiochem), and observed results similar to those obtained with PD98059 (data not shown).



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  		FIGURE 3. Analysis of microglial TNF-{alpha} production in response to CD40 stimulation. Microglia were treated with CD40L for various times, as indicated. Following stimulation, cells were harvested and prepared for TNF-{alpha} RT-PCR (A), and cell-free supernatants were collected and assayed by TNF-{alpha} ELISA (B, data shown represent mean TNF-{alpha} release values; pg/mg total protein ± 1 SEM). Data shown in A are representative of two independent experiments in which similar results were observed, and n = 3 for B. For B, ANOVA revealed significant main effects for CD40L treatment (p < 0.001) and for time (p < 0.001), and an interactive effect between these terms (p < 0.001). One-way ANOVA revealed significant between-groups differences (p < 0.001), and post hoc comparison showed significant differences between CD40L-treated and untreated microglia at 120 and 240 min (p < 0.001).

 


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  		FIGURE 4. CD40-mediated microglial TNF-{alpha} synthesis requires MEK1/2 activity. Microglia were treated with CD40L in the presence or absence of PD98059 or went untreated (control) for 240 min (A and B), or 30 min (C and D). Data shown are from TNF-{alpha} RT-PCR (A, represented as band density ratios to {gamma}-actin ± SEM), TNF-{alpha} ELISA (B, represented as mean TNF-{alpha} release values; pg/mg total protein ± SEM), p44/42 MAPK Western immunoblotting (C), and phosph-Elk1 fusion protein substrate by immune complex kinase assay (D). Data shown in A, C, and D are representative of three independent experiments in which similar results were observed. n = 4 for each condition presented in B. For A, C, and D, ANOVA revealed significant main effects of CD40L (p < 0.001) and PD98059 (p < 0.001), and a significant interaction between these terms (p < 0.001). One-way ANOVA revealed significant between-groups differences (p < 0.001), and post hoc testing showed significant differences between CD40L-treated microglia and control or PD98059-cotreated cells (p < 0.001). For B, ANOVA additionally revealed significant (p < 0.001) differences when comparing each of the CD40L and PD98059 dose cotreatment conditions with each other.

 
TGF-ß1 and IL-10 markedly inhibit CD40-mediated TNF-{alpha} production

It has been reported that TGF-ß1 and IL-10 mediate inhibitory effects on microglial activation (17, 18, 19). A recent study has shown that TGF-ß1 inhibits the induction of CD40 expression by IFN-{gamma} in cultured microglia, indicating that TGF-ß1 may play an important role in the suppression of the CD40-mediated immune response (4). In support of this, a report has shown that IL-10 reduces TNF-{alpha} and IL-1ß synthesis in monocytes by inhibiting CD40-mediated activation of a MEK1/2-dependent pathway (20). To address whether TGF-ß1 and IL-10 could modulate CD40-mediated activation of p44/42 MAPK in microglia, cultured microglia were cotreated for 30 min with CD40L (1 µg/ml) and TGF-ß1 (5 ng/ml), IL-10 (5 ng/ml), or both. Cell lysates were then analyzed for phosphorylated forms of p44/42 MAPK by Western immunoblotting. Results show that, while treatment with TGF-ß1 or IL-10 modestly inhibits activation of p44/42 MAPK induced by CD40 ligation, cotreatment with CD40L and both cytokines is much more efficient at blocking p44/42 MAPK activation than treatment with CD40L and either cytokine alone (Fig. 5Go). Inhibition of p44/42 MAPK phosphorylation by TGF-ß1 or IL-10 is statistically interactive, suggesting that these cytokines inhibit CD40-mediated p44/42 MAPK activation through a common pathway. In addition, when reprobing with an Ab that recognizes total p44/42 MAPK (both phospho- and nonphospho-forms), data show that the level of total p44/42 MAPK expression is not affected by these cytokines within 30 min, as would be expected within this relatively short time frame (Fig. 5Go).



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  		FIGURE 5. TGF-ß1 and IL-10 inhibit CD40-mediated p44/42 MAPK activation. Microglia were treated as indicated for 30 min and analyzed by Western immunoblotting for p44/42 MAPK. Band density ratio (phospho-p42/total p42 MAPK) is represented below Western immunoblots, with n = 3 for each condition presented. ANOVA revealed significant main effects of CD40L (p < 0.001), TGF-ß1 (p < 0.001), and IL-10 (p < 0.001), as well as significant interactive terms between CD40L and either TGF-ß1 (p < 0.001) or IL-10 (p < 0.001). One-way ANOVA revealed significant between-groups differences (p < 0.001), and post hoc testing showed significant differences between CD40L-treated microglia and control (p < 0.001) or cotreatment with CD40L and TGF-ß1 (p < 0.001) or IL-10 (p < 0.001). Significant differences were also noted between CD40L treatment and cotreatment with CD40L and either TGF-ß1 or IL-10 (p < 0.01), and between the CD40L, TGF-ß1, and IL-10 treatment condition and cotreatment with CD40L and either cytokine singly (p < 0.001).

 
Furthermore, to determine whether a decrease in p44/42 MAPK activation by these cytokines would result in a concomitant reduction of TNF-{alpha}, microglia were subjected to the various treatments described above for 30 and 240 min. Cells treated for 30 min were collected and prepared for immune complex kinase assay, as described in Materials and Methods. Cell-free supernatants were collected from microglia treated for 240 min and assayed by TNF-{alpha} ELISA kit. Both TGF-ß1 and IL-10 inhibit CD40-mediated p44/42 MAPK kinase activity and, when combined, display blockade that is statistically interactive (Fig. 6GoA). TNF-{alpha} ELISA data show that treatment with either TGF-ß1 or IL-10 results in a marked reduction of CD40-mediated TNF-{alpha} synthesis (~35% and 25%, respectively) as compared with control (Fig. 6GoB). Most importantly, an ~85% decrease in TNF-{alpha} production is observed in CD40L-stimulated microglia treated with a combination of these two cytokines, which is statistically interactive (Fig. 6GoB). Taken together, these data suggest that TGF-ß1 and IL-10 inhibit microglial CD40-mediated TNF-{alpha} production by modulating p44/42 MAPK activity.



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  		FIGURE 6. TGF-ß1 and IL-10 inhibit microglial CD40-mediated p44/42 MAP kinase activity and TNF-{alpha} release. Microglia were treated as indicated for 30 min, and p44/42 MAP kinase activity was assessed using an Elk fusion protein substrate (A). The band density in ODµ is represented with n = 3 for each condition presented. B, Microglia (n = 4 for each condition presented) were treated for 240 min, and mean TNF-{alpha} release was quantified by ELISA (represented as pg/mg total protein ± SEM). For A and B, ANOVA revealed significant main effects of CD40L (p < 0.001), TGF-ß1 (p < 0.001), and IL-10 (p < 0.001), as well as significant interactive terms between CD40L and either TGF-ß1 (p < 0.001) or IL-10 (p < 0.001). One-way ANOVA revealed significant between-groups differences, and post hoc testing showed significant differences between CD40L-treated microglia and control (p < 0.001) or cotreatment with CD40L and TGF-ß1 (p < 0.001) or IL-10 (p < 0.001). Significant differences were also noted between CD40L treatment and cotreatment with CD40L and either TGF-ß1 or IL-10 (p < 0.01), and between the CD40L, TGF-ß1, and IL-10 treatment condition and cotreatment with CD40L and either cytokine singly (p < 0.001).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Interaction of CD40, an important cell signaling and activation Ag, with CD40L, which is found on activated CD4+ T lymphocytes, plays a central role in both humoral and cellular immunity (1). Recently, the CD40 pathway has also been suggested to play a role in neuroimmunity, because microglia have been shown to express CD40 that can be modulated by TGF-ß1, a cytokine with numerous suppressive effects on the neuroimmune system (4). Our previous study showed that ligation of microglial CD40 results in microglial activation, as evidenced by TNF-{alpha} release (3). In this study, we provide evidence for CD40 ligation-promoted microglial activation via phosphorylation of p44/42 MAPK. It has recently been demonstrated that ligation of monocyte-derived CD40 results in p44/42 MAPK-dependent IL-1ß and TNF-{alpha} production by these cells (20). In addition, stimulation of vascular smooth muscle and endothelial cell-derived CD40 by CD40L results in IL-1ß release (21). Following CD40 ligation, we did not observe IL-1ß release from microglia (data not shown). Taken together with our results, this suggests that stimulation of CD40 results in p44/42 MAPK activation with different cell type-specific outcomes. When considering macrophages, it has been shown that TNF-{alpha} can rapidly potentiate p44/42 MAPK activation (22), suggesting that a possible feed-forward system may arise whereby ligation of CD40 could ultimately hyperpotentiate cellular activation. Our data in microglia support this notion, in which stimulation of CD40 results in p44/42 MAPK-dependent TNF-{alpha} release, which further potentiates p44/42 MAPK activation (data not shown).

As mentioned above, Suttles and colleagues showed that ligation of monocyte-derived CD40 results in IL-1ß and TNF-{alpha} release by these cells (20). Of particular interest is their finding that blockade of p44/42 MAPK, but not p38 MAPK, inhibits release of these cytokines within 18 h. Yet, inhibition of p38 MAPK in a 24-h time frame produced similar effects in their study. This result led us to investigate activation of p38 MAPK on microglial TNF-{alpha} release as a putative secondary signaling event. Inhibition of microglial p38 MAPK phosphorylation with SB203580 (5 µM) did not attenuate CD40 signaling-induced TNF-{alpha} release within 4 h, while 12 or 24 h after cotreatment with CD40L and SB203580, significant blockade of microglial TNF-{alpha} release was observed (data not shown). Additionally, when using a negative control for SB203580 (SB202474) at a similar dose, this effect was not observed (data not shown). Recently, it has been shown that p38 MAPK can modulate p44/42 MAPK activity (23, 24). To further explore this possibility in microglia, we measured p44/42 MAPK phosphorylation by Western immunoblotting and directly assessed p44/42 MAPK activity by immune complex kinase assay in microglia treated with CD40L, SB203580, or both for 30 min. Results show that, while SB203580 treatment (alone or in combination with CD40L) results in increased phosphorylation of microglial p44/42 MAPK compared with appropriate controls (Fig. 7GoA), the same is not true for p44/42 MAPK activity in these cells (Fig. 7GoB). These findings suggest that SB203580 is not an efficient modulator of p44/42 MAPK activity in microglia within the time period that we examined. Additionally, these data raise the possibility that, while our observed effect of SB203580 on CD40L-treated microglia (TNF-{alpha} reduction within 12–24 h) may be due to later inhibition of p44/42 MAPK activity, it remains likely that p38 MAPK activation is involved as a later signaling event in CD40L-stimulated microglia, possibly as a result of TNF-{alpha} autocrine stimulation.



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  		FIGURE 7. Inhibition of p38 MAPK by SB203580 results in increased phosphorylation, but not activity, of p44/42 MAPK. Microglia were treated with CD40L in the presence or absence of SB203580 or SB202474 (a negative control for SB203580), or went untreated (control) for 30 min. Cell lysates were analyzed by Western immunoblotting using Abs that recognize A, phosphorylated or total p44/42 MAPK, or B, phosphorylated Elk1 fusion protein substrate by immune complex kinase assay. Band density ratios (shown below A, phospho-p42/total p42 MAPK) and phospho-Elk1 band densities (displayed below B) are represented as means ± 1 SEM, with n = 3 for each condition. For A, ANOVA revealed significant main effects of SB203580 and CD40L (p < 0.001) on phosphorylation of p44/42 MAPK. One-way ANOVA revealed significant between-groups differences (p < 0.001), and post hoc testing showed significant differences between CD40L or SB203580 treatment and appropriate controls (p < 0.001), and between the CD40L/SB203580 and CD40L/SB202474 cotreatment conditions (p < 0.001). For B, ANOVA only showed a main effect of CD40L (p < 0.001), and one-way ANOVA revealed significant between-groups differences (p < 0.001), but post hoc testing showed only a significant difference between CD40L treatment and control (p < 0.001).

 
We choose to examine the effects of TGF-ß1 and IL-10 on CD40-mediated microglial TNF-{alpha} release, as the former cytokines have been shown to be immunosuppressive (25, 26, 27). For example, both of these cytokines have been shown to attenuate microglia and macrophage TNF-{alpha} produced by LPS (28, 29). Our data suggest that these cytokines inhibit microglial CD40-dependent TNF-{alpha} release via blockade of p44/42 MAPK activation. Yet, the possibility remained that TGF-ß1 and IL-10 could exert their effects, in part, via decreased CD40 protein expression and/or mRNA. To rule out this possibility, we examined CD40 expression within 240 min following treatment with TGF-ß1, IL-10, or both, by RT-PCR and flow-cytometric analysis. Results show that TGF-ß1, IL-10, or both do not significantly modulate CD40 expression in microglial cells (data not shown) within the time frame tested (up to 240 min posttreatment), suggesting a direct effect of these cytokines on microglial CD40-mediated signal transduction, possibly by obstructing p44/42 MAPK signaling pathway intermediates.

Previous reports have shown that IL-10 and TGF-ß1 are able to modulate MAPK-associated signal transduction pathways. For example, IL-10 has been shown to inhibit both p44/42 and p38 MAPKs stimulated by LPS, CD40 ligation, or TNF-{alpha} in monocytes (20, 30, 31). TGF-ß1 has been shown to suppress p44/42 MAPK activation induced by basic fibroblast growth factor in smooth muscle cells (32), and also to inhibit MAPKs in eosinophils and macrophages (33, 34). Yet, this cytokine has been shown to up-regulate MAPKs in T cell lines (EL4 and NOB-1) (35), and in a macrophage cell line (RAW264.7) (36). To determine the effects of these cytokines on p44/42 MAPK activation in microglia following stimulation of the CD40 pathway, we pretreated microglia with either cytokine alone or in combination, and examined phosphorylation status of p44/42 MAPK. Data show that either cytokine alone significantly reduces p44/42 MAPK activation in a statistically interactive manner, and, when added in combination, we observed an even greater blockade of p44/42 MAPK activation, which is statistically significant compared with pretreatment with either cytokine alone. These data suggest that CD40-mediated microglial activation can be attenuated by classical anti-inflammatory cytokines via modulation of p44/42 MAPK, which may be one mechanism for down-regulation of microglial activation in vivo.


   Acknowledgments
 
We are grateful to Mr. and Mrs. Robert Roskamp for their generous support, which helped to make this work possible. We extend our gratitude to Dr. Jean-Yves Bonnefoy (Glaxo Institute for Molecular Biology, Geneva) for providing human rCD40L. We also thank Jodi Kroeger for her assistance in flow-cytometric acquisition and analysis.


   Footnotes
 
1 Address correspondence and reprint requests to Dr. Jun Tan, The Roskamp Institute, Department of Psychiatry, University of South Florida, 3515 East Fletcher Avenue, Tampa, FL 33613. E-mail address: Back

2 Abbreviations used in this paper: CD40L, CD40 ligand; MAPK, mitogen-activated protein kinase; MEK, MAP/extracellular signal-related kinase kinase. Back

Received for publication May 4, 1999. Accepted for publication September 24, 1999.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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