cd45 inhibits cd40l-induced microglial activation via ... · 9/7/2000 · stained positive for...

CD45 inhibits CD40L-induced Microglial Activation via negative regulation of the Src/p44/42 MAPK Pathway

Running Title: CD45 opposes CD40-mediated Microglial Activation

Jun Tan*, Terrence Town, Michael Mullan

The Roskamp Institute, Department of Psychiatry, University of South Florida, 3515 E. Fletcher Ave.,

Tampa, Florida 33316, USA

* To whom correspondence should be addressed. Tel.: 813-974-3722; Fax: 813-974-3915; E-mail:

[email protected]

Abbreviations used are: CD40L, CD40 ligand; CD40, CD40 receptor; TNF, tumor necrosis factor; mAb,

monoclonal antibody; MAPK, mitogen activated protein kinase; LPS, lipopolysaccharide; PTP, protein

tyrosine phosphatase; fluorescence-activated cell sorter, FACS; Alzheimer’s disease, AD; Multiple

Sclerosis, MS; membrane attack complex, MAC.

Copyright 2000 by The American Society for Biochemistry and Molecular Biology, Inc.

JBC Papers in Press. Published on September 7, 2000 as Manuscript M002006200 by guest on O

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SUMMARY

It has been reported that ligation of CD40 with CD40 ligand (CD40L) results in

microglial activation as evidenced by p44/42 mitogen activated protein kinase (MAPK)-

dependent tumor necrosis factor alpha (TNF-α) production. Previous studies have shown

that CD45, a functional transmembrane protein tyrosine phosphatase (PTP), is

constitutively expressed at moderate levels on microglial cells and this expression is

greatly elevated on activated microglia. To investigate the possibility that CD45 might

modulate CD40L-induced microglial activation, we treated primary cultured microglial

cells with CD40L and anti-CD45 antibody. Data show that cross-linking of CD45

markedly inhibits CD40L-induced activity of the Src-family kinases Lck and Lyn.

Further, co-treatment of microglia with CD40L and anti-CD45 antibody results in

significant inhibition of microglial TNF-α production through inhibition of p44/42

MAPK activity, a downstream signaling event resulting from Src activation.

Accordingly, primary cultured microglial cells from mice deficient in CD45 demonstrate

hyper-responsiveness to ligation of CD40, as evidenced by increased p44/42 MAPK

activation and TNF-α production. Taken together, these results show that CD45 plays a

novel role in suppressing CD40L-induced microglial activation via negative regulation of

the Src/p44/42 MAPK cascade.

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INTRODUCTION

Microglial activation, which is characterized by transformation of microglia from a

ramified to a reactive phenotype exhibiting neurotoxic properties, has been implicated as

pathological in a variety of neurodegenerative diseases, including Alzheimer’s disease

(AD), Creutzfeld-Jacob disease, and multiple sclerosis (MS) (1). As CNS-resident

professional macrophages, activated microglia produce and secrete potentially neurotoxic

pro-inflammatory cytokines including interleukin 1β and tumor necrosis factor alpha

(TNF-α) (2), both of which have been shown to promote neuronal injury (3-5).

Microglial activation is also associated with an increased expression of cell surface

molecules, including CD45, major histocompatibility complex class II antigens, protein

complement receptors such as CR4 and membrane attack complex 1 (MAC-1), and the

immunoglobulin receptors FcγRI and FcγRII (2, 6, 7). Additionally, we have recently

shown that microglial activation resulting from stimulation with Alzheimer’s β-amyloid

peptides and CD40 ligand (CD40L) results in increased CD40 expression on microglia

with resultant TNF-α secretion by these cells (8).

Intracellularly, microglial activation induced by a variety of stimuli including CD40L,

lipopolysaccharide (LPS), β-amyloid peptides and prion, has been shown to involve

activation of the mitogen activated protein kinase (MAPK) module ultimately leading to

production of neurotoxic products by these cells (9, 10). Additionally, it has been shown

that members of the Src family, including the tyrosine kinase Lyn (10), regulate

activation of MAPK in these cells. Similar regulation of MAPK by Src occurs in T cells

following mitogenic stimulation with IL-18 and anti-CD3 antibody, where the activated

Src-family member Lck has been shown to associate with and promote activation of


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MAPK (11). Yet, in microglial cells, the role of cell surface receptors in regulation of

this intracellular Src/MAPK cascade has been largely unexplored.

CD45 is a membrane-bound protein tyrosine phosphatase (PTP), which is expressed on

a variety of immune cells, including T and B lymphocytes, where it has been shown to

play a critical role in negative regulation of cellular activation (12). In addition, CD45 is

expressed on microglia at low to moderate levels, and is markedly increased following

activation of these cells (13, 14). It is generally thought that CD45 couples to Src-family

kinases, functioning to maintain Src in a dephosphorylated, and hence inactive, state (12).

This is supported by studies in T and B-lymphocytes, where CD45-deficient cell lines

demonstrate increased Src activity (15-18). Yet, the mechanism of CD45 modulation of

Src activity is complex, and it is thought that CD45 might function as both a positive and

negative regulator of Src in a site-specific manner (19).

CD40 is a 45-50 kD receptor which is a member of the TNF receptor superfamily and

is expressed on a wide range of both immune and non-immune cell types, including

dendritic cells, monocytes, macrophages, fibroblasts, endothelial cells, and smooth

muscle cells (20, 21). The CD40 pathway was initially shown to play a critical role in the

humoral and cellular immune response, as ligation of B cell CD40 induces B cell

proliferation and differentiation into antibody-secreting plasma cells (20), and the action

of Th1 cells in priming of cytotoxic T lymphocytes is mediated by CD40-CD40L

interactions (22). Recently, we and others have shown that CD40 is constitutively

expressed at low levels on microglia (N9 cells and murine primary culture; 5, 23-25), and

ligation of microglial CD40 by CD40L leads to marked TNF-α secretion by these cells

which is neurotoxic at such levels (5). CD40 signaling in T cells has been shown to be


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dependent on interaction between CD40 and Src-family kinases, in particular Lck (26,

27), and we have recently shown that the CD40-CD40L interaction on microglia leads to

activation of p44/42 MAPK in these cells (9). Based on the idea that stimulation of

CD45 might oppose the effects of CD40 ligation (28), we wished to evaluate the effects

of cross-linking CD45 in the presence of CD40L on microglial activation. Specifically,

we wished to determine the possible involvement of the Src/MAPK cascade as an early

signaling event in mediating this effect. We were particularly interested in searching for

putative negative regulators of CD40-mediated microglial activation as we have

previously shown both in vitro and in vivo in a mouse model of AD that stimulation of

this pathway results in exacerbation of microglial-mediated AD-like pathology (8).

Therefore, the identification of a molecule that could oppose this effect may provide a

molecular target for the treatment of neurodegenerative diseases with a reactive

microglial component, such as AD.

In this study, we show that cross-linking of CD45 markedly inhibits p44/42 MAPK-

dependent TNF-α production induced by CD40 ligation in murine primary culture

microglia. Furthermore, we also provide evidence that cross-linking of CD45 opposes

these effects through inhibiting CD40L-induced activation of Src-family kinases,

particularly Lck and Lyn. Finally, we demonstrate that primary culture microglia which

are deficient for CD45 are hyperresponsive to CD40 ligation, leading to marked p44/42

MAPK activation and TNF-α secretion. Taken together, our data show that CD45 plays

a novel role in mitigating against CD40L-induced microglial activation via negatively

regulating the Src/p44/42 MAPK cascade, suggesting that CD45 might be a potential


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therapeutic target for the suppression of microglial activation associated with

neurodegenerative diseases such as AD and MS.


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EXPERIMENTAL PROCEDURES

Reagents. Monoclonal antibodies (purified rat anti-mouse CD45 and purified rat IgG2b

control antibodies; FITC-conjugated rat anti-mouse CD45 and FITC-conjugated rat IgG2b

control antibodies) were purchased from PharMingen (San Diego, CA). Antibodies for

phospho-p44/42 MAPK (Thr202/Tyr204), and total p44/42 MAPK were obtained from

NEB (Beverly, MA). TNF-α antibody for Western blotting was obtained from R&D

systems (Minneapolis, MN). Human soluble recombinant CD40L protein was obtained

from Alexis Biochemicals (San Diego, CA). The CD45 phosphatase activity assay kit

was purchased from BIOMOL (Plymouth Meeting, PA). The anti-mouse alkaline

phosphatase-conjugated IgG secondary antibody was obtained from Santa Cruz

Biotechnology (Santa Cruz, CA). Immun-Blot PVDF membranes and the Immun-

Star chemiluminescence substrate were purchased from Bio-Rad Laboratories

(Hercules, CA).

Murine primary cell culture. Breeding pairs of BALB/c, CD45 deficient (CD45 def.)

(C57BL/6OlaHsd-Ptprctm1) and CD40 deficient (CD40 def.) mice (C57BL/6Ncr-

Tnfrsf5tm1Kik) were purchased from Jackson Laboratory (Bar Harbor, MA) and housed in

the animal facility at the University of South Florida Health Science Center. Murine

primary culture microglia were isolated from mouse cerebral cortices and were grown in

RPMI medium supplemented with 5% fetal calf serum, 2 mM glutamine, 100 U/mL

penicillin, 0.1 µg/mL streptomycin and 0.05 mM 2-mercaptoethanol according to

previously described methods (5, 9). Briefly, cerebral cortices from newborn mice (1-2

days old) were isolated under sterile conditions and were kept at 4° C prior to mechanical

dissociation. Cells were plated in 75 cm2 flasks and complete medium was added.


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Primary cultures were kept for 14 days so that only glial cells remained, and microglia

were isolated by shaking flasks at 200 rpm in a Lab-Line Incubator-Shaker. More than

98% of these glial cells stained positive for MAC-1 (CD11b; Boehringer Mannheim

Biochemicals, Indianapolis, IN). Additionally, between 85 and 95% of microglial cells

stained positive for CD45 by fluorescence-activated cell sorter (FACS) analysis as

previously described (5), irrespective of CD40L and/or anti-CD45 antibody treatment

(data not shown). To verify CD45 deficiency status, CD45 expression on microglia

isolated from CD45-deficient mice was also measured by FACS analysis, and CD45 was

undetectable on these cells (data not shown). To verify CD40 deficiency status in

microglia isolated from CD40 receptor-deficient mice, CD40 expression was measured

by FACS analysis, and CD40 was undetectable on these cells, either before or after IFN-γ

stimulation (data not shown).

TNF-α ELISA. Primary cultured microglial cells were plated in 24-well tissue-culture

plates (Nunclon , Nalge Nunc International, Denmark) at 5x104 cells/well and

stimulated for 24 h with CD40L protein (1 µg/mL) in the presence or absence of anti-

CD45 mAb (1:200) or appropriate controls. In some experiments, microglial cells were

pre-treated PD 98059 (5 µM, Calbiochem, La Jolla, CA) for 1 h and then incubated with

CD40L protein for 24 h. Cell-free supernatants were collected and assayed for TNF-α by

the DuoSet TNF-α ELISA kit (R&D Systems, Minneapolis, MN) in strict accordance

with the manufacturer’s instruction. The Bio-Rad protein assay (Bio-Rad Laboratories,

Hercules, CA) was performed to measure total cellular protein from each of the cell

groups under consideration just prior to quantification of cytokine release by ELISA.


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Western immunoblotting. Murine primary culture micrgolia were plated in 6-well

tissue-culture plates (Nunclon , Nalge Nunc International, Denmark) at a density of

8x105 cells/well. Cells were then incubated for 30 min (for examining p44/p42 MAPK)

or 24 h (for detecting TNF-α protein and CD40 expression) with or without CD40L

protein (1 µg/mL) in the presence or absence of anti-CD45 mAb, control antibodies

(1:200 dilution for each) or Src inhibitors (Damnacanthal, 1000 nM; PP1, 1000 nM;

obtained from CALBIOCHEM, San Diego, CA) or appropriate controls. Immediately

following culturing, microglia were washed in ice-cold phosphate buffered saline (PBS)

3x, 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 β-glycerolphosphate, 1 mM Na3VO4, 1 µg/mL leupeptin,

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 Immun-

blot PVDF membranes. Non-specific antibody binding was blocked with 5% non-fat

dry milk in TBS (20 mM Tris, 500 mM NaCl, pH 7.5) for 1 h at room temperature.

Membranes where first hybridized with a phospho-specific p44/42 MAPK antibody or rat

anti-mouse TNF-α monoclonal antibody, stripped with β-Mercaptoethanol stripping

solution (62.5 mM Tris-HCl, pH 6.8, 2% SDS, and 100 mM β-Mercaptoethanol), and

then re-probed with an antibody that recognizes total p44/42 MAPK (or actin, in the case

of TNF-α Western immunoblots). Alternatively, membranes with identical samples were

probed with either with a phospho-specific p44/42 MAPK antibody or with an antibody


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that recognizes total p44/42 MAPK. Immunoblotting was carried out with a primary

antibody followed by an anti-mouse HRP-conjugated IgG secondary antibody as a tracer.

The Immun-Star chemiluminescence substrate was used to develop the blots.

Densitometric analysis was preformed for all blots using the Flour-S MultiImager with

Quantity One software (Bio-Rad, Hercules, CA).

Flow cytometric analysis. CD40 expression was assessed by FACS analysis. Primary

cultured microglial cells were plated in 6-well tissue-culture plates (Nunclon , Nalge

Nunc International, Denmark) at 2x105 cells/well and incubated with CD40L protein in

the presence or absence of anti-CD45 mAb. Twenty-four hours after incubation,

microglial cells (approximately 1x106 cells) were re-suspended in 250 µL of ice-cold

PBS for FACS analysis, according to methods described previously (5). A minimum of

10,000 cells were accepted for FACS analysis. Cells were gated based on morphological

characteristics such that apoptotic and necrotic cells were not accepted for FACS

analysis. Percentages of positive cells (CD40 expressing) were calculated as follows: for

each treatment the mean fluorescence value for the isotype-matched control antibody was

subtracted from the mean fluorescence value for the CD40-specific antibody.

Immune complex kinase assay. Primary culture microglial cells were seeded in 6-well

tissue-culture plates at 8x105 cells/well. Thirty minutes after co-treatment with CD40L

protein (1 µg/mL) in the presence or absence of anti-CD45 mAb or appropriate controls,

microglial cells were lysed in ice-cold lysis buffer (as described above). Total cellular

protein was quantified by the Bio-Rad protein assay, and an aliquot of 50 µg of protein

for each treatment condition was separated by SDS-PAGE. Activity of p44/42 MAPK

was determined using the p44/42 MAP Kinase Assay Kit (New England BioLabs,


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Beverly, MA) in strict accordance with the manufacturer’s instruction. The

phosphorylated form of the Elk1 fusion protein was visualized by Western

immunoblotting (as described above) using a specific antibody for phosphorylated Elk1

supplied with the kit.

Immunoprecipitation and Src kinase assay. Primary culture microglial cells were

seeded at 10 x 105 cells/dish in 100 mm cell culture dishes and incubated overnight to

80% confluence. The following day, cells were treated in the presence or absence of

CD40L or anti-CD45 mAb for 30 min. Cells were then lysed in 200 µL of cell lysis

buffer as described above, and cell lysates were immunoprecipitated overnight at 4oC

with either Lyn or Lck-specific antibodies (1:50 dilution, polyclonal rabbit anti-Lyn or

anti-Lck antibodies, Pharmingen, CA). Immunoprecipitates were then immobilized with

10 µL of 50% protein sepharose beads diluted in PBS (Protein A on Sepharose CL-4B,

Sigma) for 3 h at 4oC. The resulting immobilized immunoprecipitates were pelleted and

washed 2 x in ice-cold cell lysis buffer, followed by an additional 2 x wash in ice-cold

kinase buffer (containing 25 mM Tris pH 7.5, 5 mM β-Glycerolphosphate, 2mM DTT,

0.1 mM Na3VO4 and 10 mM MgCl2), and pellets were re-suspended in 50 µL of Src

kinase reaction buffer (containing 100 mM Tris-HCl, pH 7.2, 125 mM MgCl2, 25 mM

MnCl2, 2 mM EGTA, 0.25 mM Na3VO4 and 2 mM DTT). The Src kinase assay kit

(Upstate biotechnology, NY) was used in accordance with the manufacturer’s instruction

for radioactive quantitation of immunoprecipitated Src activity based on incorporation of

[γ32P] ATP into Src kinase substrate peptide (29). Radioactivity was measured using a

1209 RACKBETA liquid scintillation counter (LKB WALLAC, Inc., Gaithersburg, MD),

and data are reported as pmol PO4/min/mg of total cellular protein.


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Statistical analysis. Data were analyzed using analysis of variance (ANOVA)

followed by post-hoc comparisons of means by Bonferroni’s or Dunnett’s T3 method,

where Levene’s test for homogeneity of variances was used to determine the appropriate

method of post-hoc comparison. Alpha levels were set at 0.05 for each analysis. All

analyses were performed using SPSS for window release 9.0.


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RESULTS

Cross-linking of CD45 results in reduction of CD40L-induced microglial TNF-a

production. We have recently shown that ligation of CD40 by CD40L induces p44/42

MAPK-dependent TNF-α production in microglia (9). It has previously been shown that

stimulation of the CD40 pathway results in T cell activation that is mediated by Src and

MAPK activation (30). CD45 is a prototypical membrane-associated PTP which

maintains Src in a dephosphorylated state resulting in its decreased kinase activity (12).

We wished to evaluate the possibility that stimulation of CD45 might mitigate against

microglial TNF-α production by decreasing Src and downstream MAPK activity induced

by CD40 ligation. In order to evaluate whether cross-linking of microglial CD45 results

in stimulation of this PTP, we measured free inorganic phosphate (Pi) in microglial cell

lysates treated in the presence or absence of anti-CD45 mAb or isotype-matched control

antibody, and find significantly higher levels of Pi in anti-CD45 mAb-treated microglia

compared to appropriate controls (data not shown). To investigate the possible functional

significance of CD45 stimulation in the presence of CD40L, we co-treated primary

culture microglia with monoclonal anti-CD45 mAb and CD40L for 24 h. Results show

that secretion of TNF-α protein is markedly increased following treatment with CD40L,

and these levels are dramatically reduced after co-treatment of these cells with anti-CD45

mAb (Fig. 1).

Cross-linking of CD45 in the presence of CD40L does not affect CD40 expression. A

previous study that focused on inhibition of CD40-mediated monocyte activation found

that such effects could be accounted for, at least in part, by decreased CD40 receptor

expression levels (31). Thus, we wished to determine whether our observed effect of


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inhibition of CD40L-induced microglial activation after cross-linking CD45 was

dependent upon decreased CD40 expression. To rule out this possibility, we examined

CD40 expression within 24 h after co-treatment with anti-CD45 mAb and CD40L. Data

show that treatment of microglia with anti-CD45 mAb in the presence of CD40L does not

affect CD40 expression compared to appropriate controls as measured by Western

immunoblotting (data not shown) and FACS analysis (Fig. 2). These data also show that

the observed effect of anti-CD45 mAb treatment on microglial activation does not

involve modulation of CD40 expression levels across the 24-h time course examined.

Interestingly, we find that treatment of microglia with CD40L alone results in a

significant increase in CD40 receptor levels on microglia, supporting the idea that

CD40L can positively regulate its receptor on microglia.

CD40L-induced increased activation of p44/42 MAPK is specific to the CD40-CD40L

interaction. We have previously shown that CD40L is able to stimulate microglial

p44/42 MAPK in a time-dependent fashion, from 30 min to 240 min, with peak activation

at 60 min. When taken together with the present data showing ~4% CD40 receptor

expression on microglia, we sought to reconcile how such a low expression level of

CD40 could mediate marked effects on increasing p44/42 MAPK phosphorylation and

activity following CD40 ligation. Thus, we sought to address the possibility that

interaction between CD40 ligand and a receptor other than CD40 may bring about these

effects. To examine this possibility, we employed murine primary culture CD40

knockout microglia, and treated them with CD40L. Data show that CD40L is unable to

elicit p44/24 MAPK phosphorylation (Fig.3A) or activity (Fig.3B) in these cells


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following stimulation with CD40L, showing that the microglial CD40-CD40L interaction

markedly elicits p44/42 MAPK activity.

Microglial CD40L-induced p44/42 MAPK and TNF-α production are dependent on

Src activation. It has previously been reported that cross-linking of CD40 by anti-CD40

mAb induces phosphorylation and activation of the Src-family kinase Lyn in B cells (32).

In addition, we and others have shown that ligation of CD40 results in TNF-α secretion

that is brought about by activation of p44/42 MAPK in monocytes and microglial cells

(9, 33). These data lead us to investigate the possibility that ligation of CD40 might

result in activation of Src-family kinases and consequent downstream activation of

p44/42 MAPK, ultimately resulting in TNF-α secretion by microglia. Thus, we co-

incubated microglial cells with CD40L and either a general inhibitor of Src-family

kinases, PP1 (1000 nM), or the Lck-specific inhibitor, Damnacanthal (1000 nM), for 30

min. In order to confirm that these agents inhibited Src kinase activity in our system, we

first assayed activity of the Src-family kinases Lck and Lyn after co-treatment of

microglia with CD40L and either PP1 or Damnacanthal. Results show that both Src

inhibitors markedly reduce CD40L-induced Src kinase activity (data not shown).

Activity of p44/42 MAPK was examined by Western blot and immune complex kinase

assay using antibodies that specifically recognize phosphorylated p44/42 MAPK or the

phosphorylated form of the Elk1 fusion protein, respectively. Data as shown in Fig. 4A

and B indicate that co-treatment of microglia with CD40L and either Src-family kinase

inhibitor results in marked reduction of p44/42 MAPK activity, suggesting that CD40L-

induced activation of p44/42 MAPK is dependent on activity of Src-family kinases. We

then assessed whether or not PP1 and Damnacanthal inhibition of CD40L-induced


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p44/42 MAPK phosphorylation and activity might be dose-dependent. Data indicate that

this is the case, with p44/42 MAPK phosphorylation (Fig. 4C) and activity (Fig. 4D)

decreasing with increasing doses of these inhibitors (from 200 nM to 1000 nM).

Furthermore, a significant reduction of TNF-α was observed after co-treatment of

microglia with CD40L and Src kinase inhibitors for 24 h, supporting the idea that

CD40L-induced microglial activation is dependent upon activation of Src and

downstream p44/42 MAPK (Fig. 4E).

Cross-linking of CD45 inhibits microglial CD40L-induced Lck and Lyn kinase activity.

It is well known that CD45 is involved in negative regulation of activity of Src-family

kinases, particularily Lck and Lyn (12). Having shown that treatment of microglia with

CD40L results in increased Src kinase activity, we wished to evaluate the possibility that

CD45 could oppose this effect by decreasing Src kinase activity. To investigate this

possibility, we co-treated microglia with CD40L and/or anti-CD45 mAb or appropriate

controls for 30 min. Phosphotransferase activity of Lck and Lyn kinases was measured

as described in Experimental Procedures. Results indicate that cross-linking of CD45

markedly inhibits Lck (Fig. 5A) and Lyn (Fig. 5B) kinase activity induced by CD40

ligation, suggesting that microglial CD40 and CD45 signalling pathways cross-modulate

each other at the level of membrane-associated Src-family kinases.

Cross-linking of CD45 suppresses CD40L-induced p44/42 MAPK activity. It has been

reported that Src kinases are involved in regulation of MAPK activation (10, 11, 34). We

and others have shown that activation of MAPK, in particular p44/42 MAPK, is involved

in TNF-α production in macrophages, monocytes, and microglia following activation of

these cells with a variety of stimuli, including LPS and CD40 ligand (9, 35, 36). Having


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shown that cross-linking CD45 inhibits CD40L-induced activity of the Src-family kinases

Lck and Lyn in microglial cells, we wished to examine whether this reduced Src kinase

activity could lead to down-regulation of p44/42 MAPK activity. To investigate this

possibility, microglial cells were co-incubated with anti-CD45 mAb and CD40L. Cell

lysates were then analyzed for phosphorylated forms of p44/42 MAPK by Western

immunobotting. Results show that cross-linking of CD45 significantly inhibits CD40L-

induced activation (phosphorylation) of p44/42 MAPK (Fig. 6A). To determine if this

effect could result in decreased MAPK activity, a direct method, immune complex kinase

assay, was performed. Results show that cross-linking of CD45 markedly reduces p44/42

MAPK activity in CD40L-treated microglia (Fig. 6B), demonstrating the functionality of

CD45 cross-linking on p44/42 MAPK activity.

Ligation of CD40 results in marked p44/42 MAPK activity and TNF-α production in

CD45-deficient microglial cells. To further substantiate the role of CD45 in negatively

regulating CD40L-induced microglial activation, microglia were obtained from CD45-

deficient or wild type mice and incubated with or without CD40L. Activity of p44/42

MAPK was then evaluated in cell lysates from these conditions 30 min post-treatment.

Data show that p44/42 MAPK activation (Fig. 7A) and activity (Fig. 7B) are markedly

enhanced in CD40L-challenged microglia that are deficient for CD45. As we have

previously shown that TNF-α release induced by CD40 ligation is dependent on p44/42

MAPK, we went on to measure TNF-α production by CD45-deficient microglia treated

with CD40L for 24 h. Results shown in Fig. 7C indicate much greater activation of

CD45-deficient microglia compared to wild-type microglia following stimulation with

CD40L, supporting that CD45 is a negative regulator of CD40-mediated microglial


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activation. Moreover, in order to evaluate whether CD45 could be a central regulator of

the p44/42 MAPK pathway, we pre-treated CD45-deficient microglial cells for 1 h with

PD 98059 (an inhibitor of MEK1/2, the upstream activator of p44/42 MAPK) and then

incubated them with CD40L for 24 h. Microglial activation was subsequently evidenced

by TNF-α production. Data show that PD 98059 notably decreases CD40L-induced

TNF-α production by CD45-deficient microglia (Fig. 7C), further suggesting that CD45

plays a major role in negative regulation of the p44/42 MAPK pathway. Yet, as PD

98059 does not completely block CD40L-induced TNF-α secretion by CD45 deficient

microglia, it seems likely that, while CD45 is not an obligatory regulator of the CD40

pathway, it does control the flux of signals emanating from CD40.


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DISCUSSION

It has previously been shown that microglia express CD45 and this expression level is

markedly enhanced following activation of these cells (13, 14). CD45 is well known to

couple to Src-family kinases, including Lyn and Lck, where it modulates Src activity via

dephosphorylation of tyrosine residues (15, 19). Yet, the role of CD45 in microglial

activation is currently speculative. We and others have shown that CD40 is also

constitutively expressed on microglia at low levels, and markedly increases after

activation of these cells (5). Ligation of microglial CD40 results in p44/42 MAPK-

dependent TNF-α production (9), and it has been shown that stimulation of Src-family

kinases results in activation of the MAPK module (11). Thus, we wished to investigate

whether CD45 might modulate CD40L-induced microglial activation through regulation

of the Src/p44/42 MAPK pathway. Our results show that cross-linking CD45 potently

inhibits microglial activation induced by CD40 ligation as evidenced by TNF-α

production. Furthermore, CD40 ligation results in marked activation of the Src-family

kinase members Lyn and Lck, with consequent downstream p44/42 MAPK activation in

activated microglia. Co-treatment of microglia with CD40L and anti-CD45 mAb results

in reduced Lck and Lyn kinase as well as p44/42 MAPK activity, showing that CD45 is a

negative regulator of CD40L-induced microglial activation and suggesting a mechanism

whereby CD45 brings about this effect by inhibiting Src kinase activity, a known

function of CD45 (12).

As we had shown that cross-linking CD45 reduces CD40L-induced microglial TNF-α

production, the possibility arose that this effect may be due, at least in part, to reduced

CD40 receptor expression on the microglial cell surface. This idea was highlighted in a


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previous report, where it was shown that pharmacological inhibition of CD40-mediated

monocyte activation was partially attributable to reduced gene expression of CD40 (31).

To rule out this possibility in our system, we treated microglia with anti-CD45 mAb in

the presence or absence of CD40L and measured CD40 expression levels on these cells

compared to appropriate controls. We did not observe a significant effect of anti-CD45

mAb on CD40 protein expression alone or in combination with CD40L (Fig. 2).

However, treatment of microglia with CD40L does result in increased CD40 receptor

expression (Fig. 2), an effect which is most likely mediated by NF-ΚB activation, as the

CD40-CD40L interaction has previously been shown to activate functional NF-ΚB (37,

38). These data suggested to us that stimulation of CD45, unlike CD40, does not effect

transcription factor-mediated gene expression of CD40, and led us to investigate the

initial intracellular mediators of CD45-mediated negative regulation of CD40L-induced

microglial activation.

CD45 is a membrane-bound PTP that is well known to couple to and directly regulate

the activity of Src-family tyrosine kinases. However, CD45-mediated dephosphorylation

of Src-family kinases is a complex and not well-understood phenomenon (15, 19, 39).

For example, CD45 can either activate or inactivate Src, depending on whether CD45

dephosphorylates inhibitory or activating sites within the SH1 kinase domain (19). It is

thought that the receptor occupation and activation status of the immune cell under

consideration (i.e., resting or antigen-associated receptor ligated) may be a critical

determinant of which sites CD45 dephosphorylates on Src (19). Thus, we considered

CD45 modulation of Src activity against a background of ligation of the CD40 receptor,

which is well known to participate in both immune cell activation and antigen-receptor


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signaling. Data show that cross-linking of microglial CD45 in the presence of CD40L

results in reduced activity of Lck and Lyn, showing negative regulation of CD40L-

induced Src activity by CD45. Interestingly, we find that cross-linking of microglial

CD45 alone results in increased Src activity (Fig. 5), supporting the hypothesis that in

non-activated, resting microglia, stimulation of CD45 results in dephosphorylation of

inhibiting regions of the Src SH1 domain. This idea is in line with the dualistic nature of

CD45-mediated Src kinase modulation proposed by Ashwell and D’Oro (15), who

concluded that CD45 can act not only as a simple “on” switch, but also as an “off” switch

depending on the activation status of the immune cell under consideration.

CD40L treatment has been shown to result in Src-family kinase activation, particularly

Lck and Lyn, on B and T cells (26, 27, 40, 41), and it has further been shown that in, B

cells deficient for Lyn, ligation of CD40 results in a decreased proliferative response

induced by IL-4 or B cell receptor stimulation (42, 43). These data suggest that CD40

may be a positive regulator of Src, and we evaluted this possibility in microglia

challenged with CD40L. Our data show that ligation of microglial CD40 results in

increased activity of the Src-family kinases Lck and Lyn (Fig. 5) as well as TNF-α

secretion by these cells (Fig. 1). We have previously shown that CD40L-induced

microglial TNF-α production is dependent on p44/42 MAPK (9), and wished to evaluate

the possibility that Src activation might bridge stimulation of microglial CD40 and

consequent p44/42 MAPK activation. Thus, we co-treated microglia with CD40L and

the Src-family kinase inhibitors PP1 or Damnacanthal, and find marked reduction in both

p44/42 MAPK activation and TNF-α secretion by these cells (Fig. 4), suggesting that

activation of Src is required to transduce p44/42 MAPK-dependent TNF-α production


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following CD40 ligation. This is particularly interesting when considered together with

stimulation of microglial CD45, where co-treatment with CD40L and anti-CD45 mAb

results in dramatic reduction of Src kinase and downstream p44/42 MAPK activities as

well as TNF-α secretion. This suggests an antagonistic system that regulates microglial

activation, whereby CD40 ligation leads to activation of these cells, while co-stimulation

with CD40L and CD45 opposes it.

Having shown that cross-linking of CD45 opposes CD40L-induced microglial

activation, we asked the question whether stimulation of CD45 with anti-CD45 mAb

could also mitigate against microglial activation induced by other pro-inflammatory

stimuli, such as LPS. To examine this possibility, we stimulated microglia with LPS in

the presence of anti-CD45 mAb, and find marked reduction in microglial p44/42 MAPK

activation and TNF-α secretion (data not shown). It has previously been reported that

LPS transduces microglial activation via activation of the MAPK module (36, 44).

Additionally, LPS-induced macrophage activation has been shown to involve one or

more Src-family kinases (45, 46), suggesting that LPS, like CD40L, stimulates the

intracellular Src/MAPK pathway in microglia. It is suggested, then, that stimulation of

CD45 is effective at blocking microglial activation induced by a variety of stimuli by

virtue of its ability to oppose Src/MAPK pathway activation. Thus, in vivo stimulation

of CD45 might be a viable therapeutic target in the treatment of neurodegenerative

diseases which involve pathological microglial activation, such as AD and MS.


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ACKNOWLEDGMENTS

The authors are grateful to Mr. and Mrs. Robert Roskamp for their generous support,

which helped to make this work possible. We would like to thank Jodi Kroeger for her

assistance in flow cytometric acquisition and analysis. We thank Yajuan Wu for her

assistance in Western immunoblotting and Demian Obregon for maintaining animals.

We would also like to thank Andon Placzek for his helpful discussion.


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Figure Legends

Fig. 1. CD45 cross-linking results in decreased CD40L-induced microglial TNF-αααα production. Graph

represents a summary of TNF-α release ELISA results (mean TNF-α pg/mg total protein ± 1 SEM) with n

= 3 for each condition presented. ANOVA revealed significant main effects of CD40L (p < .001) and anti-

CD45 (p < .01), and an interaction between them (p < .01). One-way ANOVA revealed significant

between-groups differences (p < .001), and post-hoc testing showed significant differences between control

and CD40L (p < .001) and between CD40L/anti-CD45 and CD40L/control antibody (p < .01).

Fig. 2. Microglial CD40 expression is not affected by CD45 cross-linking. Graph represents a summary

of FACS analysis results for CD40 expression on microglia (mean % of CD40-expressing cells ± 1 SEM)

with n = 3 for each condition presented. ANOVA revealed a significant main effect of CD40L (p < .001),

but not for anti-CD45 (p > .05), and no significant interaction was noted between them (p > .05). One-way

ANOVA revealed significant between-groups differences (p < .001), and post-hoc testing showed

significant differences between control and CD40L (p < .05). However, no significant differences were

noted between control and anti-CD45 (p > .05) or between CD40L/anti-CD45 and CD40L/control antibody

(p > .05).

Fig. 3. CD40L-induced increased p44/42 phosphorylation and activity are specific to the CD40-

CD40L interaction. (A) (above) Western blot showing phosphorylated p44/42 MAPK in microglia, and

(below) graph summarizing band density ratios (phospho-p42 MAPK/total p42 MAPK) (mean ± 1 SD) for

above with n = 3 for each condition presented. (B) (above) Immune complex kinase assay showing

phosphorylation of the MAPK fusion protein, Elk1, and (below) graph summarizing band densities (mean

± 1 SD) for above with n = 3 for each condition presented. For (A) and (B), one-way ANOVA revealed a

significant difference between wild-type microglia before and after CD40L treatment (p < .001), but did not

show a significant difference between CD40 def. microglia before and after CD40L treatment (p > .05),

indicating that CD40L mediates its effect on p44/42 MAPK specifically through the CD40-CD40L

interaction.


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Fig. 4. Microglial CD40L-induced p44/42 MAPK activity and TNF-αααα production are Src kinase-

dependent. (A) (above) Western blot showing phosphorylated p44/42 MAPK in microglia, and (below)

graph summarizing band density ratios (phospho-p42 MAPK/total p42 MAPK) (mean ± 1 SD) for above

with n = 3 for each condition presented. (B) (above) Immune complex kinase assay showing


± 1 SD) for above with n = 3 for each condition presented. For (C) and (D), microglia were co-treated with

CD40L (1 µg/mL) and PP1 at the doses indicated. (C) (above) Western blot showing phosphorylated

p44/42 MAPK in microglia, and (below) graph summarizing band density ratios (phospho-p42 MAPK/total

p42 MAPK) (mean ± 1 SD) for above with n = 3 for each condition presented. (D) (above) Immune

complex kinase assay showing phosphorylation of the MAPK fusion protein, Elk1, and (below) graph

summarizing band densities (mean ± 1 SD) for above with n = 3 for each condition presented. Similar

results were observed when microglia were co-treated with CD40L (1 µg/mL) Damnacanthal (dose range

from 200 to 1000 nM). (E) Summary of TNF-α release ELISA results (mean TNF-α pg/mg total protein ±

1 SEM) with n = 3 for each condition presented. For (A) and (B) and (E), ANOVA revealed a significant

main effect of CD40L (p < .001), and significant interactive terms between CD40L and either

Damnacanthal (p < .001) or PP1 (p < .001). One-way ANOVA revealed significant between-groups

differences (p < .001), and post-hoc testing showed significant differences between control and CD40L (p <

.001) as well as between CD40L and either CD40L/Damnacanthal (p < .05) or CD40L/PP1 (p < .001). For

(C) and (D), ANOVA revealed a significant main effect (p < .001) of Src kinase inhibitor dose, indicating

dose-dependent inhibition of p44/42 MAPK.

Fig. 5. CD45 cross-linking inhibits CD40L-induced Lck and Lyn kinase activity in microglia. (A)

Lck and (B) Lyn kinase activity (pmol ATP/min/mg total protein) reported as the mean ± 1 SEM with n = 3

for each condition presented. For (A) and (B), ANOVA revealed a significant main effect of CD40L (p <

.001), and significant interactive terms between CD40L and anti-CD45 (p < .001), but not between CD40L

and control antibody (p > .05). One-way ANOVA revealed significant between-groups differences (p <

.001), and post-hoc testing showed significant differences between control and either CD40L (p < .001) or

anti-CD45 (p < .001), as well as between CD40L/anti-CD45 and CD40L/control antibody (p < .001).


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Fig. 6. CD45 cross-linking suppresses CD40L-induced p44/42 MAPK activity in microglia. (A)

(above) Western blot showing phosphorylated p44/42 MAPK in microglia, and (below) graph summarizing

band density ratios (phospho-p42 MAPK/total p42 MAPK) (mean ± 1 SD) for above with n = 3 for each

condition presented. (B) (above) Immune complex kinase assay showing phosphorylation of the MAPK

fusion protein, Elk1, and (below) graph summarizing band densities (mean ± 1 SD) for above with n = 3 for

each condition presented. For (A) and (B), ANOVA revealed a significant main effect of CD40L (p <

.001), and a significant interactive term between CD40L and anti-CD45 (p < .001), but not between CD40L

and control antibody (p > .05). One-way ANOVA revealed significant between-groups differences (p <

.001), and post-hoc testing showed significant differences between control and either CD40L (p < .001) or

anti-CD45 (p = .001), as well as between CD40L/anti-CD45 and CD40L/control antibody (p < .05).

Fig. 7. CD40 ligation results in marked p44/42 MAPK activity and TNF-αααα production in microglia

deficient for CD45. (A) (above) Western blot showing phosphorylated p44/42 MAPK in microglia, and

(below) graph summarizing band density ratios (phospho-p42 MAPK/total p42 MAPK) (mean ± 1 SD) for

above with n = 3 for each condition presented. (B) (above) Immune complex kinase assay showing


± 1 SD) for above with n = 3 for each condition presented. (C) Summary of TNF-α release ELISA results

(mean TNF-α pg/mg total protein ± 1 SEM) with n = 3 for each condition presented. For (A) and (B),

ANOVA revealed significant main effects of CD40L (p < .001) and CD45 deficiency (p < .001), and a

significant interaction between them (p < .05). One-way ANOVA revealed significant between-groups

differences (p < .001), and post-hoc testing showed a significant difference between control

microglia/CD40L and CD45 def. microglia/CD40L (p < .001). For (C), one-way ANOVA revealed

significant between-groups differences (p < .001), and post-hoc testing showed a significant difference

between CD45 def. microglia /CD40L and CD45 def. microglia/CD40L/PD98059 (p < .001).


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Jun Tan, Terrence Town and Michael MullanSrc/p44/42 MAPK pathway

CD45 inhibits CD40L-induced microglial activation via negative regulation of the

published online September 7, 2000J. Biol. Chem.

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