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:
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
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 (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
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. (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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