The Journal of Neuroscience, April 14, 2010 • 30(15):5437–5450 • 5437

Neurobiology of Disease

Neuregulin-ErbB Signaling Promotes Microglial Proliferation

and Chemotaxis Contributing to Microgliosis and Pain after
Peripheral Nerve Injury
Margarita Calvo,1 Ning Zhu,1 Christoforos Tsantoulas,1 Zhenzhong Ma,2 John Grist,1 Jeffrey A. Loeb,2
and David L. H. Bennett1
1Wolfson Center for Age Related Diseases, King’s College London, London SE1 1UL, United Kingdom, and 2Center for Molecular Medicine and Genetics
and Department of Neurology, Wayne State University School of Medicine, Detroit, Michigan 48201

A key component in the response of the nervous system to injury is the proliferation and switch to a “proinflammatory” phenotype by
microglia (microgliosis). In situations where the blood– brain barrier is intact, microglial numbers increase via the proliferation and
chemotaxis of resident microglia; however, there is limited knowledge regarding the factors mediating this response. After peripheral
nerve injury, a dorsal horn microgliosis develops, which directly contributes to the development of neuropathic pain. Neuregulin-1
(NRG-1) is a growth and differentiation factor with a well characterized role in neural and cardiac development. Microglia express the
NRG1 receptors erbB2, 3, and 4, and NRG1 signaling via the erbB2 receptor stimulated microglial proliferation, chemotaxis, and survival,
as well as interleukin-1␤ release in vitro. Intrathecal treatment with NRG1 resulted in microglial proliferation within the dorsal horn, and
these cells developed an activated morphology. This microglial response was associated with the development of both mechanical and
cold pain-related hypersensitivity. Primary afferents express NRG1, and after spinal nerve ligation (SNL) we observed both an increase in
NRG1 within the dorsal horn as well as activation of erbB2 specifically within microglia. Blockade of the erbB2 receptor or sequestration
of endogenous NRG after SNL reduced the proliferation, the number of microglia with an activated morphology, and the expression of
phospho-P38 by microglia. Furthermore, consequent to such changes, the mechanical pain-related hypersensitivity and cold allodynia
were reduced. NRG1-erbB signaling therefore represents a novel pathway regulating the injury response of microglia.

Introduction kines, complement components, and purines. These molecules

Microglia are the resident myeloid cells of the CNS and respond regulate different components of the injury response (a process
rapidly to injury of the nervous system. This injury response (or that can be thought of as a continuum rather than an “all-or-
“microgliosis”) has a number of different components including: nothing event”). The microglial response to peripheral nerve injury
microglial cell proliferation (Ajami et al., 2007; Echeverry et al., contributes to the generation of neuropathic pain, and these cells are
2008), migration to the site of injury (Yao et al., 1990; Peterson et al., also implicated in disease processes as diverse as traumatic brain
1997; Honda et al., 2001; Ifuku et al., 2007); phagocytosis of cellular injury, neurodegeneration, and demyelination (Hanisch and Ket-
debris (Neumann et al., 2009); antigen presentation (Fischer and tenmann, 2007; Scholz and Woolf, 2007; Ransohoff and Perry,
Reichmann, 2001); adoption of an amoeboid morphology; and the 2009). It has recently been demonstrated using chimeric animals
release of a broad range of cytokines and chemokines (Milligan and obtained by parabiosis or bone marrow transplantation and se-
Watkins, 2009). Microglia express receptors for and respond to a lective irradiation that in situations where the blood– brain bar-
range of injury-related molecules including cytokines, chemo- rier is intact the principal means by which microglial numbers
increase is through the proliferation of resident microglia (rather
than recruitment of blood-borne, bone marrow-derived micro-
Received Oct. 17, 2009; revised Feb. 25, 2010; accepted March 1, 2010.
D.B. is a Wellcome Trust Clinical Scientist and a member of the London Pain Consortium. M.C. is sponsored by the
glial progenitors) (Ajami et al., 2007; Mildner et al., 2007). In this
Chilean National Scholarship Program for Graduate Studies. C.T. is a student of the Wellcome Trust-funded London study, we focus on the role of neuregulin-1 (NRG1) in modulat-
Pain Consortium. J.L. is supported by National Multiple Sclerosis Society Grant RG 3410B3. We thank Genentech for ing microglial proliferation chemotaxis and cytokine release.
provision of NRG1. We thank M. Malcangio and S.B. McMahon for helpful advice. We thank G. Mallucci (MRC NRG1 is one of a family of growth factors (NRG1– 4). Nrg1
Toxicology Unit, Leicester University, Leicester, UK) for provision of Nfh Cre mice and C. Birchmeier (Max Delbr ück
undergoes alternative splicing and differential promoter use to
Center for Molecular Medicine, Berlin, Germany) for provision of Nrg1f1/f1 mice. D.B. conceived of and coordinated
this program of work. M.C. and N.Z. designed experiments, and performed animal surgery, microglial cultures, and produce at least 15 isoforms, which include both secreted and
histological analysis. J.G. and M.C. performed behavioral testing. C.T. performed ISH. Z.M. and J.A.L. developed and transmembrane forms (Falls, 2003; Esper et al., 2006). NRG1 has
provided information on the use of the NRG antagonist. M.C. and D.B. performed data analysis and wrote the a key role in neural and cardiac development (Lee et al., 1995); it can
manuscript. All authors discussed the results. modulate synaptic plasticity (Mei and Xiong, 2008), and it can stim-
Correspondence should be addressed to David L. H. Bennett at the above address. E-mail: David.Bennett@
kcl.ac.uk.
ulate the proliferation, survival, and motility of a number of different
DOI:10.1523/JNEUROSCI.5169-09.2010 cell types (Falls, 2003). All Nrg1 isoforms contain an epidermal
Copyright © 2010 the authors 0270-6474/10/305437-14$15.00/0 growth factor (EGF)-like domain, which is critical for mediating
5438 • J. Neurosci., April 14, 2010 • 30(15):5437–5450 Calvo et al. • Neuregulin-ErbB Signaling Promotes Microglial Proliferation and Chemotaxis

biologic activity and binds to the tyrosine

kinase receptors erbB3 and 4. These re-
ceptors subsequently heterodimerize with
erbB2, which lacks a ligand-binding do-
main but is a key coreceptor in mediating
signal transduction (Carraway and Cant-
ley, 1994). ErbB3 lacks an active tyrosine
kinase domain, and so its interaction with
erbB2 is essential for signaling. ErbB4 can
signal via homodimers but dimerizes
preferentially with erbB2 (Graus-Porta et
al., 1997). Microglia are reported to ex-
press erbB2, 3, and 4 receptors (Gerecke et
al., 2001; Dimayuga et al., 2003); however,
very little is known regarding the function
of this signaling pathway within micro-
glia. We therefore studied the effects of
NRG1 on microglial survival, prolifera-
tion, chemotaxis, and cytokine release in
vitro, and subsequently tested the hypoth-
esis that this signaling pathway contrib-
utes to microgliosis and neuropathic pain
after peripheral nerve injury in vivo.

Materials and Methods Figure 1. ErbB receptor expression in primary cultured microglia. a, Using RT-PCR analysis, we found that primary cultured rat
microglia express the erbB2, 3, and 4 receptors. The 2% agarose gels stained with ethidium bromide and photographed under
Animals and surgery. Adult male Wistar rats
ultraviolet light are shown for the PCR products of each of the genes amplified. Pos, Positive control (E16 embryonic rat brain cDNA);
were used in accordance with UK Home Office
MG, microglia; Neg, negative control (water). b, ErbB receptors expression is shown in microglial cells by immunohistochemistry
regulations. In one group of animals, the left L5
(IB4 labels microglia green, the erbB receptors are in red). Scale bar: 20 ␮m. c, Western blot analysis showing a 185 kDa band for
spinal nerve was ligated and transected. Sham
erbB2, 3, and 4 in lysates from microglial cells.
surgery animals were prepared in an identical
manner but without injuring the nerve. To la-
bel dividing cells, rats were injected with 5-bromo-2⬘-deoxyuridine measured the time that the animal spent licking, shaking, or lifting the
(BrdU; Sigma) dissolved in 0.007N NaOH/PBS (100 mg/kg body weight, paw during the following 2 min (Kontinen and Dickenson, 2000).
i.p.) once daily for 3 consecutive days before perfusion and fixation. Histology. After defined survival times, animals were killed by terminal
Drugs and delivery. PD 168393 (Calbiochem), an irreversible erbB anesthetization and transcardially perfused with 4% paraformaldehyde
inhibitor, was dissolved in 5% DMSO and delivered intrathecally at 1.25, plus 1.5% picric acid in 0.1% phosphate buffer. The lumbar spinal cords
2.5, 5, or 10 ␮g/d by an Alzet osmotic pump (model 2002). Control and L5 dorsal root ganglia were excised, cryoprotected in 20% sucrose,
animals were given the same vehicle solution, lacking the active com- cryostat cut (10 or 20 ␮m), and thaw-mounted onto glass slides. Spinal
cord sections were incubated overnight with the primary antibody, rab-
pound. Anti-erbB2 antibodies [monoclonal antibody (mAb) 7.16.4 from
bit anti-phospho-p38 mitogen-activated protein kinase (MAPK) (1:100;
Calbiochem and mAb 9 from Thermo Scientific, without azide] were
Cell Signaling Technology) or rabbit anti-phospho-erbB2 [p-Neu (Tyr
delivered intrathecally via lumbar puncture 1 h before nerve injury (2.5,
1248), 1:200; Santa Cruz Biotechnology], both of which were viewed by
5 or 10 ␮g diluted in 25 ␮l of sterile PBS). For perineural application of
tyramide amplification (TSA Biotin System, PerkinElmer) or with anti-
mAb 7.16.4 to the site of nerve injury, a 0.5 ⫻ 0.5 cm strip of oxidized
rabbit neuregulin-1 (H-210), Polyclonal Antibody (1:100; Santa Cruz
regenerated cellulose (Surgicell, Ethicon) soaked in 10 ␮g of the antibody
Biotechnology). For colocalization studies, the slides were then incu-
diluted in sterile PBS was applied directly over the site of spinal nerve
bated with rabbit anti-Iba1 (1:1000; WAKO), mouse anti-OX42 (1:200)
ligation. Control animals received a nonimmune IgG2a antibody (Cal- (Serotec), rabbit anti-GFAP (1:1000; Dako), or mouse anti-NeuN (1:
biochem) via the same method (intrathecally or perineurally). To se- 1000; Millipore Bioscience Research Reagents). DRGs sections were in-
quester endogenous NRG, we used a fusion protein (HBD-S-H4) that cubated overnight with the calcitonin gene-related peptide antibody
was injected intrathecally every 3 d (3 ␮g in 20 ␮l of sterile saline per (rabbit anti-CGRP, 1:2000; Sigma) and neurofilament 200 antibody
injection). The drug doses were selected on the basis of previous reports (mouse anti-NF200, 1:1000; Millipore Bioscience Research Reagents) or
and our preliminary studies. Neuregulin-␤1 EGF domain (rHRG␤1 with anti-rabbit Neuregulin-1 (H-210). After primary antibody incuba-
aa177-244) was administered intrathecally via lumbar puncture (0.4 or 4 tion, sections were washed and incubated for 1.5 h with corresponding
ng in a volume of 20 ␮l). To label dividing cells after NRG1 treatment, secondary antibody solution. Isolectin B4 (IB4) detection was performed
rats were injected with BrdU (Sigma) dissolved in 0.007N NaOH/PBS, using biotin-conjugated IB4 (1:50; (Sigma) and ExAvidin-AMCA (1:400;
100 mg/kg body weight, i.p. 24 h before perfusion and fixation. Before Vector Laboratories). Slides were washed, coverslipped with Vectashield
surgery, animals were randomly allocated into experimental study mounting medium with or without DAPI (Vector Laboratories), and
groups (computer-generated randomization schedules). An indepen- visualized under a Zeiss Axioplan 2 fluorescent microscope. The speci-
dent investigator prepared the drugs individually and labeled them for ficity of immunostaining was checked by the omission of the appropriate
each animal according to the randomization schedule. Operators and primary antibody or preincubation with the corresponding blocking
data analysts were blinded throughout the study. peptide. Antibody detection of BrdU incorporated into DNA requires
Behavioral testing. Mechanical withdrawal thresholds were tested us- pretreatment of the tissue to expose the BrdU epitope. For this purpose,
ing the Dynamic Plantar Aesthesiometer (Ugo Basile), which is an auto- we used the antigen retrieval method described previously (Tang et al.,
mated version of the von Frey hair assessment. A maximum cutoff of 50 g 2007). The primary antibody solution contained mouse anti-BrdU (1:
was used. The withdrawal threshold is calculated as the average of three 200; BD Biosciences) plus rabbit anti-Iba-1 (1:1000; Wako), and the
consecutive tests with at least 10 min between each test. To measure cold secondary antibody solution contained corresponding IgG-conjugated
allodynia, we applied a drop of acetone to the plantar hindpaw and FITC 1:200 plus IgG-conjugated Cy3 1:400 (both from Stratech).
Calvo et al. • Neuregulin-ErbB Signaling Promotes Microglial Proliferation and Chemotaxis J. Neurosci., April 14, 2010 • 30(15):5437–5450 • 5439

35
S-dATP (PerkinElmer Life Sciences) using ter-
minal deoxynucleotidyl transferase (Promega).
After prehybridization treatments (acetylation,
dehydration, and delipidation), the radioactive
probe was added on the sections and hybridiza-
tion was performed overnight at 37°C. The next
day, slides were washed in standard saline citrate
solutions with increasing stringencies, rapidly de-
hydrated through graded alcohols, air-dried,
dipped in autoradiographic emulsion (LM1; GE
Healthcare), and developed for 3– 4 weeks.
Quantification and analysis. Quantitative as-
sessment was performed by determining the
numbers of immunoreactive cells within four ar-
eas of 10,000 ␮m 2 in the superficial dorsal horn
on five to seven randomly selected L5 spinal sec-
tions from each animal. For BrdU staining, the
whole dorsal horn was analyzed. Microglia in
which process length was less than double the
soma diameter were classified as presenting an
activated (or effector) morphology. Microglia in
which the process length was double the soma
diameter were classified as resting (or surveying)
cells (Stence et al., 2001) (see Fig. 7). Cells were
sampled only if the nucleus was visible within the
plane of section and if cell profiles exhibited dis-
tinctly delineated borders. For quantification of
dorsal root ganglia ISH, cells exhibiting grain
density twice that of background levels were
counted as exhibiting a positive hybridization
signal. All analyses were performed with the op-
erator blinded to treatment groups.
Detection of NRG1-␤1 by ELISA. Ipsilateral
dorsal horns were homogenized in lysate buffer
Figure 2. NRG1 stimulates microglial survival, proliferation, and chemotaxis in vitro. Survival of microglia in vitro was assessed (20 mM Tris, pH 8, 137 mM NaCl, 1% NP-40, 1
by incubating cultured microglia for 3 d in serum-free medium, which was supplemented with increasing doses of NRG1 (0.5–10 mM sodium orthovanadate, and protease in-
nM) or GM-CSF (1 nM) (as a positive control). a, b, Representative wells are shown in which microglia are identified by Iba1 hibitor mixture), and NRG1 was detected from
immunostaining after no treatment (a) or addition of 10 nM NRG1 (b). NRG1 promoted microglial survival, and erbB2 inhibition lysates using a NRG1-␤1-specific ELISA kit
using PD168393 (INH, 10 ␮M) or mAb 7.16.4 (AB, 4 ␮g/ml) blocked this effect. c, Quantification of microglial numbers is shown. (detection range: 250 – 40,000 pg/ml; DuoSet
d–f, Cultured microglia were incubated for 1 day in serum-free medium with (e) or without (d) the addition of NRG1. DAPI staining ELISA DY377, R&D Systems). NRG1-␤1 stan-
(blue) demonstrated nuclei, and TUNEL (yellow) revealed those undergoing apoptosis. Treatment with NRG1 (10 nM) significantly dards (250 – 40,000 pg/ml) and 100 ␮l of dorsal
decreased apoptosis, an effect that was prevented when microglia were treated with the erbB inhibitor PD168393 in combination horn lysate (50 ␮g of protein) were run in du-
with NRG1 (quantification in f ). g–i, Proliferation was assessed by incubating microglia in medium supplemented with 5% FBS for plicate following the manufacturer instructions.
3 d and pulse-labeling with BrdU (yellow). NRG1 treatment significantly increased the proportion of BrdU-positive microglial Samples were read at 450 nm with wavelength
nuclei, an effect that was erbB2-dependent. j–l, The effects of NRG1 on microglial migration were studied using a Boyden chamber. correction at 540 nm. Samples were considered
The addition of NRG1 to the lower well of the chamber (k) increased microglial migration to the inner membrane surface compared with NRG1-␤1-positive if the signal was higher than
control(j).InhibitionoferbB2blockedthisaction.I,Quantificationofmigratedcells.Scalebars:100 ␮m.Errorbarsrepresent⫾SEM(three background and within the range of the standard
to five independent experiments). The statistical tests used were one-way ANOVA with Bonferroni post hoc analysis for all comparisons curve.
exceptfortheproliferationexperimentinwhichthedatawerenotnormallydistributedandANOVA on ranks with Dunn’s post hoc test Primary microglia cell culture. Mixed glial
was used instead. *p ⬍ 0.05, **p ⬍ 0.005. CON, Control; INH, inhibitor (PD168393); AB, neutralizing antibody (7.16.4). cultures were isolated from cortex of P3 Wistar
rats according to the method of Giulian and
Microglia cells and macrophages were 4% PFA-fixed for immunohis- Baker (1986). After mechanical and chemical
tochemistry. Microglia were double-stained with erbB receptors anti- dissociation, cells were seeded in DMEM with 10% fetal bovine serum
bodies [erbB 2/Neu (C-18), erbB3 (C-17), and erbB4 (C-18) rabbit, (FBS) at a density of 500,000 cells/ml and cultured at 37°C in humidified
1:100; Santa Cruz Biotechnology) and isolectin B4 biotin-conjugated (to 5% CO2/95% air. All reagents used were purchased from Invitrogen.
label microglia, 1:50; Sigma) and visualized with corresponding second- Medium was replaced every 2–3 d, and confluency was achieved after 5 d
ary antibodies [Cy3, 1:400, for erbB receptors (Stratech); extra-avidin in vitro. Confluent mixed glial cultures were manually shaken for 5 min, and
FITC, 1:200, for IB4 (Sigma)]. For detecting class II major histocompat- the floating cells were pelleted and subcultured. After 15 min of plating, the
ibility complex (MHC-2), we used the monoclonal mouse anti-rat RT1B medium was changed to discharge all nonadherent cells. This method re-
(clone OX-6) antibody from Serotec (1:100). We used the ApopTag Flu- sulted in 96 –99% purity as assessed by Iba1 and DAPI staining. For prolif-
orescein In Situ kit (Millipore Bioscience Research Reagents) to identify eration assays, granulocyte monocyte colony stimulating factor (GM-CSF)
apoptotic nuclei by terminal deoxynucleotidyl transferase-mediated bi- (Cell Sciences, 1 nM) was used as a positive control (Giulian and Ingeman,
otinylated UTP nick end labeling (TUNEL). The purity of macrophages 1988). Neuregulin-␤1 EGF domain (rHRG␤1 aa177-244; Genentech) was
and microglial cultures was assessed by staining cells with Iba1 and DAPI. used (0.5–10 nM). NRG1, GM-CSF (1 nM), and negative control (only me-
In situ hybridization. In situ hybridization (ISH) was performed after im- dium) were applied in triplicate. For blocking the erbB2 receptor, we used
munohistochemistry using 34-nucleotide-long probes as previously de- PD168393 at 2.5, 5, or 10 ␮M, or mAb 7.16.4 at 1, 2, or 4 ␮g/ml. BrdU 10 ␮M
scribed (Michael et al., 1997). The sequence and specificity of the NRG1 was administered 15 h before fixation to label proliferating cells.
probe has been described before (Fricker et al., 2009). Oligonucleotides were Peritoneal macrophage preparation. Adult rats were killed with in-
chemically synthesized (Sigma-Genosys) and radioactively end-labeled with creasing CO2 concentrations and immediately after they were injected
5440 • J. Neurosci., April 14, 2010 • 30(15):5437–5450 Calvo et al. • Neuregulin-ErbB Signaling Promotes Microglial Proliferation and Chemotaxis

intraperitoneally with 20 ml of sterile HBSS. The elicited peritoneal

exudate cells were harvested and resuspended in DMEM supple-
mented or not with 10% FBS as required. Cells were plated at a density
of 300,000 cells/ml and cultured at 37°C in humidified 5% CO2/95%
air. For chemotaxis assays, cells were used immediately after
harvesting.
Chemotaxis assay. Chemotaxis was assessed using the Boyden chamber
(Neuroprobe). Polycarbonate filters (5 ␮m pore) were installed in the
chamber, whose bottom wells were filled with serum-free DMEM or
NRG1 at various concentrations. Freshly prepared microglia or macro-
phages were suspended in serum-free DMEM and were placed into the
top wells (50,000 cells/well). The chamber was kept in a CO2 incubator at
37°C for 3 h. The filter was removed and stained with RapiDiffII (Bio-
stain Ready Reagents). The cells on the top side of the filter were wiped Figure 3. The migration of microglial cells in response to NRG1 is a true chemotactic re-
off, and the number of cells that had migrated to the bottom side were sponse. In a Boyden chamber, microglial cells migrate from an upper to a lower well through a
counted. polycarbonate filter in response to a concentration gradient of the putative chemotactic agent.
Reverse transcriptase-PCR. Reverse transcriptase (RT)-PCR was used A checkerboard analysis of three independent experiments assessing the chemotactic response
for assessing mRNA expression of erbB receptors in microglial cultures. to NRG1 performed in triplicate and normalized to the unstimulated control is shown. Microglial
Total RNA was isolated using an RNeasy Mini Kit (Qiagen) and first- cells were suspended in medium alone or with 0.1, 1, or 10 nM NRG1 and then allowed to
strand cDNA was reverse-transcribed. The sequences for primers used in migrate for 3 h at 37°C toward different concentrations of NRG1 in the lower compartments.
this study were: erbB2, CCTGCCTCCACTTCAATCAT (forward), Highlighted in gray boxes are the results achieved by using the same concentration of NRG1 in
CAGGATCCCACTTCCGTAGA (reverse); erbB3 TGAAGATGTGT- upper and lower wells. Note that in this circumstance NRG1 increases microglial migration,
GAGCCTTGC (forward), GGTCCAGTGCGGGTATCTTA (reverse); indicating chemokinesis. When there is an increasing concentration gradient from the upper to
and erbB4 ATGGCCTTCCAACATGACTC (forward), CACCTGCCAT- the lower well (values in gray), migration is clearly enhanced, indicating a true chemotactic
CACATTGTTC (reverse) (all 5⬘–3⬘). Amplification of templates was de- response. *p ⬍ 0.05 for migration across a gradient vs migration when the NRG1 concentration
tected using SYBR Green 1 dye (Roche) on a Rotor-Gene thermal cycler is the same in both wells. Numbers represent the mean ⫾ SEM.
(Corbett Life Science). Melting curve analysis and sequencing confirmed the
specificity of the products. PCR products were visualized in a 2% agarose gel.
Detection of interleukin-1␤ by ELISA. We used the Rat IL-1␤/IL-1F2
Quantikine ELISA Kit (RLB00, R&D Systems), which mainly detects the
Results
mature or released form of interleukin-1␤ (IL-1␤). IL-1␤ standards NRG1 promotes the survival, proliferation, and chemotaxis
(31.2–2000 pg/ml) and 100 ␮l of microglia culture supernatants were run of microglia
in duplicate following manufacturer instructions. Samples were read at Using RT-PCR analysis, we found that cultured rat microglia
450 nm with wavelength correction at 540 nm. Samples were considered express erbB2, 3, and 4 receptor mRNA, and expression was con-
IL-1␤-positive if the signal was higher than background and within the firmed by immunocytochemistry and Western blot analysis (Fig
range of the standard curve. The supernatants of microglia were collected 1). When microglial cells are kept in serum-free conditions, ap-
after the cells were primed with lipopolysaccharide (LPS) (1 ␮g/ml; optotic cell death is observed within 24 h (Koyama et al., 2000).
Sigma for 3 h) and treated with ATP (1 mM; Sigma) or NRG1 (10 nM) for We found that NRG1 significantly promoted the survival of mi-
30 min. croglia, which had been cultured in such conditions for 72 h (Fig.
Western blots. Microglial and macrophage cultures or spinal cord dor-
2a– c). At NRG1 concentrations of 10 nM, the number of micro-
sal horn were homogenized in lysate buffer (20 mM Tris, pH 8, 137 mM
NaCl, 1% NP-40, 1 mM sodium orthovanadate, and protease inhibitor glia was more than five times higher than seen in controls ( p ⫽
mixture). The lysate was spun at 13,000 rpm at 4°C for 15 min, and the 0.001, one-way ANOVA, four independent experiments). NRG1
protein concentration of supernatant was determined using a BCA Pro- application (10 nM) resulted in a threefold reduction in the num-
tein Assay kit (Pierce). Proteins (50 ␮g/sample) were separated using 8% ber of apoptotic microglial nuclei after 24 h in serum-free condi-
SDS-PAGE and transferred to nitrocellulose membranes. Membranes tions, as seen by TUNEL ( p ⫽ 0.008, one-way ANOVA, three
were then blocked in 10% skimmed milk for 1 h at room temperature and independent experiments) (Fig. 2d–f ). This indicates that inhibi-
washed in PBS-T three times for 5 min each. Membranes were incubated tion of apoptosis was responsible for the survival-promoting ef-
with primary antibody, rabbit erbB2 (1:200), rabbit erbB3 (1:100), and fects of NRG1. Because in many instances the erbB2 receptor is a
rabbit erbB4 (1:200; all from Santa Cruz Biotechnology) overnight at key mediator of NRG1 signaling, we tested the consequences of
4°C. After several PBS-T washes, as described before, membranes were
erbB2 receptor blockade. We used either an irreversible specific
incubated with donkey anti-rabbit HRP-conjugated secondary antibody
(1:10,000; GE Healthcare) for 1 h at room temperature. As a loading
erbB inhibitor, PD168393 (Bose et al., 2006), which blocks the
control, we used mouse ␤-actin (1:10,000, Sigma), and the secondary ATP binding site of the receptor, or mAb 7.16.4, which specifi-
antibody used was anti-mouse HRP-conjugated secondary antibody (1: cally binds to the extracellular domain of erbB2 (sharing anti-
10,000; GE Healthcare). Membranes were revealed using ECL-plus re- genic epitopes with Herceptin, Genentech) (Katsumata et al.,
agent for 5 min (GE Healthcare) for detection by autoradiography. 1995; Zhang et al., 1999). Both of these agents, which themselves
Protein bands for all erbB receptors (185 kDa) and ␤-actin (42 kDa) were had no effect on cell viability, could completely prevent the
quantified using a model GS-700 imaging densitometer (Bio-Rad Labo- survival-promoting effects of NRG1 in a dose-dependent manner
ratories). All quantifications were normalized against ␤-actin to ensure (there was no significant difference comparing treatment with
equal sample loading. NRG1 combined with PD168393 or mAb 7.16.4 and control, p ⫽
Statistical analysis. Sample sizes for experiments were based on results 1, one-way ANOVA, three independent experiments) (Fig. 2c,f;
from pilot studies. Datasets were tested for normality using the Kolmog-
supplemental Fig. 1, available at www.jneurosci.org as supple-
orov–Smirnov test, and for homogeneity of variance using Levene’s test.
Parametric or nonparametric tests were used accordingly. Behavioral mental material).
data were analyzed using repeated measures (RM) two-way ANOVA. We subsequently investigated the effects of NRG1 on microglial
When the assumptions of sphericity were violated (Mauchly’s test; p ⬍ proliferation. In serum-free conditions, we observed virtually no
0.05), the Greenhouse–Geisser correction was applied. p ⬍ 0.05 was basal proliferation and very little response to either GM-CSF or
considered as significant. Data are presented as the mean ⫾ SEM. NRG1 (data not shown). Microglia were therefore cultured in me-
Calvo et al. • Neuregulin-ErbB Signaling Promotes Microglial Proliferation and Chemotaxis J. Neurosci., April 14, 2010 • 30(15):5437–5450 • 5441

( p ⬍ 0.05, ANOVA on ranks, three to

four independent experiments). This ef-
fect was again dependent on activation of
the erbB2 receptor as it could be inhibited
by PD168393 or mAb 7.16.4 in a dose-
dependent manner (both were not signifi-
cantly different from control, p ⫽ 0.215 and
p ⫽ 0.516, respectively, for NRG1 plus
PD168393 or mAb 7.16.4, t tests, three inde-
pendent experiments) (supplemental Fig. 1,
available at www.jneurosci.org as supple-
mental material). The effect of NRG1 was
comparable to that elicited by GM-CSF, a
previously well described microglial mito-
gen (Giulian and Ingeman, 1988).
Microglia are motile cells and demon-
strate directed migration toward regions
of injury. Using a Boyden chamber, in
which microglia migrate through pores in
a polycarbonate filter across a concentra-
tion gradient, NRG1 was found to signif-
icantly increase microglial chemotaxis in a
dose-dependent manner (Fig. 2j–l ). A
“checkerboard analysis” (Martinet et al.,
1994) in which different concentrations of
NRG1 were used in the upper and lower
wells was performed. This demonstrated
that, although there was migration when
NRG1 was added to both upper and lower
wells in equal concentrations, migration
was greatest when the concentration gradi-
ent between the lower and upper well was
maximal (Fig. 3). This is consistent with a
true chemotactic response (i.e., directed mi-
gration along a concentration gradient)
rather than a solely chemokinetic response
(i.e., random migration) produced by
NRG1. The chemotactic action of NRG1
was dose-dependently inhibited by erbB2
receptor blockade (NRG1 plus PD168393
or mAb 7.16.4, p ⫽ 1 and p ⫽ 0.36 vs con-
trol, respectively, t tests, three independent
experiments) (supplemental Fig. 1, available
at www.jneurosci.org as supplemental ma-
terial). Note that neither of these erbB2
receptor-blocking agents had any effect on
Figure 4. NRG1 promotes the release of IL-1␤ from LPS-primed microglia but does not stimulate MHC-2 expression. a, An ELISA basal migration (PD168393 vs control, p ⫽
was used to quantify IL-1␤ released into the medium of cultured microglia. Neither ATP (1 mM) nor NRG1 (10 nM) alone produced 0.3; mAb 7.16.4 vs control, p ⫽ 0.6, Mann–
IL-1␤ release from naive microglia; however, both significantly promoted IL-1␤ release from LPS (1 ␮g/ml)-primed microglia in Whitney rank sum test). Microglia are
a dose-dependent manner. Error bars: ⫾ SEM. Statistical test: ANOVA on ranks. *p ⬍ 0.05, n ⫽ 3. b, Another aspect of the closely related to tissue macrophages, and
activation (or effector) response of microglia is that they increase expression of MHC-2. To investigate wherever NRG1 treatment indeed we found that peritoneal macro-
leads to such a response in microglia, we incubated primary cultures in serum-free medium and treated them with LPS (1 ␮g/ml), phages also express erbB 2, 3, and 4 recep-
NRG1 (10 nM), or medium alone (CON). Microglia were immunostained with OX-6 (which labels MHC-2), Iba1 (microglial marker), tors and demonstrate enhanced motility in
and DAPI (nuclear staining). LPS treatment led to a fivefold increase in OX-6 expression compared with control ( p ⬍ 0.001), while
response to NRG1 (supplemental Fig. 2,
NRG1 treatment did not elicit such a response ( p ⫽ 1). Treatment with LPS also resulted in microglia adopting an amoeboid
morphology, unlike NRG1. Scale bars: 100 ␮m. Error bars: ⫾ SEM (three independent experiments) Statistical test: one-way
available at www.jneurosci.org as supple-
ANOVA, Bonferroni post hoc analysis. **p ⬍ 0.001. mental material).
In response to CNS injury, microglia
adopt an effector (also termed “acti-
dium supplemented with 5% FBS, and cell proliferation in re- vated”) state, which is associated with development of an amoe-
sponse to NRG1 was quantified using pulse labeling with BrdU. boid morphology, proinflammatory cytokine expression, and
NRG1 treatment increased the proliferation of microglia in a expression of MHC-2 (Hanisch and Kettenmann, 2007). NRG1
dose-dependent manner (Fig. 2g,i) such that a dose of 10 nM had no effect on IL-1␤ release from naive microglia; however, it
increased the proportion of BrdU-labeled nuclei from 11 to 60% significantly enhanced IL-1␤ release from microglia that had
5442 • J. Neurosci., April 14, 2010 • 30(15):5437–5450 Calvo et al. • Neuregulin-ErbB Signaling Promotes Microglial Proliferation and Chemotaxis

Figure 5. Intrathecal administration of NRG1 results in dorsal horn microgliosis associated with mechanical and cold pain-related hypersensitivity. NRG1␤ was administered intrathecally (0.4 or
4 ng given daily for 3 d). a, b, Dorsal horn of animals treated with saline (control in a) or NRG1 (4 ng in b) 3 d after the first injection, immunostained with Iba1. Note the increase in the numbers of
microglia with an activated morphology. c, Shows quantification of this response at 1 and 3 d after NRG1 injections. *p ⬍ 0.05, **p ⬍ 0.001 (comparing NRG1 doses vs control). d–l, We assessed
proliferation (pulse labeling with BrdU) after NRG1 injections (Iba1 is shown in red, BrdU in green, DAPI in blue, and in the last panel merged images are shown). d– g, Dorsal horn microglia from
a saline-treated animal (note that no BrdU is present). h– k, Dorsal horn microglia from a NRG1 (4 ng)-treated animal. l, Quantification of all BrdU-positive microglia in the dorsal horn of all groups
at days 1 and 3 after injections were started. *p ⬍ 0.05 (comparing 4 ng vs control). Mechanical (m) and cold (n) pain-related hypersensitivity developed after NRG1 injections in a dose-dependent
manner. *p ⬍ 0.05, **p ⬍ 0.001 for 4 ng vs control; #p ⬍ 0.05 for 0.4 ng versus control. Scale bars: (in b) a, b: 100 ␮m; (in k) d– k: 10 ␮m.

been primed with LPS [a molecule known to activate microglia daily for 3 d), after which we assessed both the response of dorsal
via the Toll-like receptor 4 (Lehnardt et al., 2003); p ⬍ 0.05 vs horn microglia as well as pain-related behavior. Intrathecal
control, one-way ANOVA on ranks] (Fig. 4). The administration NRG1 produced a dose-dependent increase in the number of
of LPS results in process retraction and increased OX6 (a marker microglia within the lumbar dorsal horn, and many of these cells
of MHC-2 expression) immunoreactivity of microglia. NRG1, were noted to develop an activated morphology with hypertro-
however, did not elicit such changes (the percentage of OX6 im- phy of the cell body and process retraction (Fig. 5a– c). Although
munoreactivity in LPS-treated cells was five times higher than a significant dorsal horn microgliosis was observed at day 1 (0.4
that in the control, p ⬍ 0.001; the NRG1-treated cells were no or 4 ng vs control, p ⫽ 0.02 and p ⫽ 0.002, respectively, one-way
different from control, p ⫽ 1; one-way ANOVA, three indepen- ANOVA, n ⫽ 4 per group), it was much more apparent at day 3
dent experiments) (Fig. 4). (Fig. 5a– c) (0.4 and 4 ng vs control, p ⬍ 0.05, ANOVA on ranks,
n ⫽ 4). Furthermore, the number of microglia undergoing pro-
Intrathecal NRG1 produces dorsal horn microgliosis associated liferation as assessed by pulse labeling with BrdU also signifi-
with mechanical and cold pain-related hypersensitivity cantly increased (Fig. 5d–l ) ( p ⬍ 0.05, 4 ng vs control, ANOVA
To investigate the effects of NRG1 on microglia in vivo, we ad- on ranks, n ⫽ 4 per group). The microglial response within the
ministered this molecule intrathecally (0.4 or 4 ng administered dorsal horn after NRG1 treatment was mirrored by the develop-
Calvo et al. • Neuregulin-ErbB Signaling Promotes Microglial Proliferation and Chemotaxis J. Neurosci., April 14, 2010 • 30(15):5437–5450 • 5443

astrocyte marker glial fibrillary acidic pro-

tein (GFAP) (Fig. 6b-g). The time course
of phosphorylated erbB2 (p-erbB2) ex-
pression, which was maximal at days 3–7,
closely mirrored the temporal profile of
the development of microgliosis (Fig. 7d–
h). Using Western blot analysis, we saw a
significant increase in erbB3 receptor ex-
pression in the dorsal horn after SNL. No
change was observed in erbB2 and erbB4
expression ( p ⫽ 0.02, naive vs day 3 after
SNL for erbB3; p ⫽ 0.34 and p ⫽ 1, respec-
tively, for erbB2 and erbB4; Mann–Whit-
ney rank sum test, n ⫽ 4) (supplemental
Fig. 3, available at www.jneurosci.org as
supplemental material).
The principal isoforms of NRG1 ex-
pressed within the nervous system are
those with a ␤ EGF domain (Meyer and
Birchmeier, 1994; Shinoda et al., 1997).
To identify cells expressing NRG1 in the
adult, we used an oligonucleotide probe that
recognizes a sequence common to all ␤ iso-
forms (Fricker et al., 2009). In dorsal root
ganglia, as previously noted, ␤ EGF mRNA
is expressed by both small-diameter
(identified through the expression of the
neuropeptide CGRP or binding of isolec-
tin B4) and large-diameter [identified by
the expression of phosphorylated neuro-
filament heavy chain (NF200)] DRG cells.
Figure 6. Peripheral nerve injury leads to increased phospho-erbB2 expression within dorsal horn microglia. a, Three days after Expression was highest in large-diameter
L5 SNL, the expression of the phosphorylated form of the erbB2 receptor was increased in the ipsilateral dorsal horn. b– d, DRG cells, which is consistent with the
Phospho-erbB2 immunostaining (green) colocalized with Iba1 (microglial marker in red, b), but not with GFAP (astrocyte marker high level of expression of the type III ␤
in red, c) or NeuN (neuronal marker in red, d). e, f, P-erbB2 (green, e) also colocalized with OX-42 (a marker of activated microglia; NRG isoform in these cells. In the naive
red, f ). Scale bars: a, 200 ␮m; b– d, 50 ␮m; e– g, 10 ␮m. situation, 67 ⫾ 3.7% of DRG cells ex-
pressed ␤ EGF mRNA, and at 3 d after
ment of a dose-dependent mechanical and cold pain-related hy- SNL (when microgliosis is well established) 62 ⫾ 1% of DRG cells
persensitivity (Fig. 5m) ( p ⫽ 0.009 and p ⬍ 0.001, respectively, at expressed ␤ EGF mRNA (Fig. 8a–f ). This probe demonstrated
days 1 and 3 for 4 ng vs control; p ⫽ 0.04 at day 3 for 0.4 ng vs neuronal expression within the spinal cord, particularly in mo-
control) (Fig. 5n) ( p ⬍ 0.001 at day 3 for 4 ng vs control, RM toneurons and occasionally in neurons in the deeper laminae of
two-way ANOVA, n ⫽ 7– 8 per group). As with the morpholog- the dorsal horn (data not shown). We did not see expression in
ical changes in microglia, pain-related hypersensitivity was much astrocytes either in the naive state or after injury. The most likely
more marked at day 3 than at day 1. source of NRG within the dorsal horn is therefore likely to be
primary afferent terminals.
ErbB2 receptor inhibition or sequestration of endogenous The H 210 antibody that recognizes the N terminus of NRG1
NRG reduces microgliosis after peripheral nerve injury was used to localize NRG1 immunoreactivity. To confirm anti-
We next explored the role of NRG1-erbB signaling in microglia in body specificity in tissue from mice with conditional Nrg1, gene
a nerve injury model [L5 spinal nerve ligation (Kim and Chung, ablation in sensory neurons was used [mice expressing Cre under
1992)], which results in a robust microgliosis. To visualize the control elements of the neurofilament heavy chain gene Nfh Cre
location of erbB2 in its activated state, a phosphorylation state- (Mallucci et al., 2002) were crossed with mice with loxp sites
specific antibody was used (Guertin et al., 2005). After L5 SNL, a flanking the essential EGF domain of Nrg1 (Nrgf1/f1) (Yang et al.,
significant increase in expression of the phosphorylated (i.e., ac- 2001)]. In Nfh Cre, Nrg1f1/f1 mice Nrg1 is ablated in sensory
tivated) form of erbB2 receptor was seen in the ipsilateral dorsal neurons in the late embryonic period, resulting in a severe
horn of the spinal cord (Fig. 6a). No immunostaining was ob- dysmyelinating neuropathy and shortened survival (D.L.H. Ben-
served after preincubation of the antibody with the peptide used nett, unpublished observations). Note that normal NRG1 immu-
as the immunogen. Phospho-erbB2 expression was entirely re- noreactivity is observed in DRG cells of Nrgf1/f1 (i.e., control)
stricted to microglia; phospho-erbB2 colocalized with the micro- animals whereas no immunoreactivity is present DRG cells of
glial markers Iba-1 (ionized calcium-binding adapter molecule Nfh Cre, Nrg1f1/f1 animals (Fig. 8g– h). NRG1 immunoreactivity
1) and OX-42 (which recognizes complement receptor type 3). was observed within DRG cells of all cell sizes (in naive situation:
Of the 518 phospo-erbB2-positive cells counted, 99% coex- 61.9 ⫾ 1.2% of all cells were positive; at 3 d after SNL: 58.4 ⫾
pressed Iba-1. There was no colocalization with either the neuro- 2.1% of all cells were positive) (Fig. 8i–j). NRG1 immunore-
nal marker neuronal-specific nuclear protein (NeuN) or the activity was also seen within the dorsal horn of the spinal cord
5444 • J. Neurosci., April 14, 2010 • 30(15):5437–5450 Calvo et al. • Neuregulin-ErbB Signaling Promotes Microglial Proliferation and Chemotaxis

(especially within superficial laminae)

(Fig. 8k), and this increased at 3 d after SNL
(ipsilateral to lesion). An ELISA that recog-
nizes NRG1 isoforms containing ␤ EGF and
IgG domains demonstrated a significant in-
crease in the level of this protein within the
dorsal horn at 3 d after SNL (Fig. 8l) ( p ⫽
0.04, one-way ANOVA Bonferroni post hoc
test, n ⫽ 3 per group).
To test whether activation of the erbB2
receptor contributes to microgliosis in vivo,
we inhibited it using PD168393, the erbB2
receptor-blocking antibodies mAb7.16.4
(discussed above), or mAb 9 (B10), which
causes erbB2 homodimerization without
activation of downstream signaling path-
ways and accelerates receptor endocytosis
(Sawyer et al., 2002; Pugatsch et al., 2006).
At day 3 after SNL, when the microglial re-
sponse is well established, we found that
blocking the erbB2 receptor could dose-
dependently reduce the number of micro-
glia within the ipsilateral dorsal horn with
an activated morphology [using PD168393,
5 ␮g/d, i.t. infusion ( p ⫽ 0.002); mAb
7.16.4, 5 ␮g, i.t. injection ( p ⬍ 0.001); or
mAb 9, 5 ␮g, i.t. injection ( p ⬍ 0.001), com-
pared with control, one-way ANOVA, n ⫽
3– 4 per group) (Fig. 9a– c) (supplemental
Fig. 1d, available at www.jneurosci.org as
supplemental material). The number of mi-
croglia within the dorsal horn that were
phospho-p38 MAPK-positive was also sig-
nificantly reduced after erbB2 inhibition
( p ⬍ 0.001, after treatment with PD168393,
5 ␮g/d, i.t. infusion; mAb 7.16.4, 5 ␮g, i.t.
injection; or mAb 9, 5 ␮g, i.t. injection, com-
pared with control, one-way ANOVA, n ⫽
3– 4 per group) (Fig. 9f–j).
To sequester endogenous NRG, we
used a fusion protein (HBD-S-H4) con-
sisting of the soluble ectodomain of
erbB4, which has high affinity for NRG,
and a heparin-binding domain, which
helps target the molecule to the same
heparan sulfate-rich cell surfaces that
bind NRG. This has been shown to be Figure 7. The time course of phospho-erbB2 expression is coincident with the development of microgliosis. The microglial
highly effective in vitro and blocks the response to nerve injury develops during the first week after injury (demonstrated by immunostaining with the microglial marker
survival-promoting activity of NRG on Iba1). a, An increase in microglial numbers is observed in the ipsilateral dorsal and ventral horn of the spinal cord of L5 SNL animals.
Schwann cells in vivo (Ma et al., 2009). b, c, A closer view shows that microglia change their morphology from having long processes and a small soma (b, naive animal,
Intrathecal administration of HBD-S-H4 termed surveying microglia) to having retracted processes and a hypertrophic soma (c, 3 d after L5 SNL, termed an effector or
(3 ␮g, i.t. injection) significantly reduced activated morphology). Simultaneously with this microgliosis, we observed an increased expression of the p-erbB2 receptor
both the number of microglia with an ac- (green) in the ipsilateral dorsal horn microglia (labeled with Iba1 in red). d– g, Naive (d), 1 d after SNL (e), 3 d after SNL (f ), and 21 d
tivated morphology within the dorsal after SNL (g). There was a significant increase in both total microglial number and the proportion that were p-erbB2-positive
horn and phospho-p38 expression after within the dorsal horn after nerve injury (quantification shown in h). *p ⬍ 0.05 (n ⫽ 3– 4; one-way ANOVA on ranks, post hoc
SNL (in both cases, p ⬍ 0.01, after treat- Student–Newman–Keuls method). Scale bars: a, 200 ␮m; b, c, 10 ␮m; d– g, 100 ␮m.
ment with HBD-S-H4 vs control, un-
paired t test, n ⫽ 3– 4 per group) (Fig. 9). ⬃60 –70% after blockade of erbB2 [p ⬍ 0.001, after treatment
Pulse labeling with BrdU showed a marked increase in the rate with PD168393 (5 ␮g/d, i.t. infusion), mAb 7.16.4 (5 ␮g/d, i.t.
of cell proliferation within the dorsal horn of the spinal cord after infusion), or mAb 9 (5 ␮g/d, i.t. infusion) compared with con-
SNL and, as previously reported (Echeverry et al., 2008), the vast trol, one-way ANOVA, n ⫽ 3– 4 per group] (Fig. 10).
majority of these cells (94.6 ⫾ 1.2%) were microglia. The number Inhibition of erbB2 significantly reduced the number of mi-
of proliferating microglia within the dorsal horn was reduced by croglia with activated morphology, the expression of phospho-
Calvo et al. • Neuregulin-ErbB Signaling Promotes Microglial Proliferation and Chemotaxis J. Neurosci., April 14, 2010 • 30(15):5437–5450 • 5445

role in signaling between axons and

Schwann cells in peripheral nervous sys-
tem (Chen et al., 2003; Nave and Salzer,
2006; Fricker et al., 2009). There is there-
fore the theoretical possibility that these
changes could be secondary to changes in
axoglial signaling in the periphery if the
inhibitor or antibody were to spread out-
side of the intrathecal space. To exclude
this possibility, we administered mAb
7.16.4 directly to the site of spinal nerve
ligation and found that in contrast to in-
trathecal treatment this peripheral ad-
ministration had no effect on dorsal horn
microgliosis ( p ⫽ 0.18, t test, n ⫽ 3– 4)
(supplemental Fig. 6, available at www.
jneurosci.org as supplemental material).
A week after SNL, we observed a signif-
icant increase in astrocytes within the
ipsilateral spinal cord ( p ⫽ 0.004, t test,
n ⫽ 4), which is in accordance with previ-
ous reports of a delayed astrocytosis after
nerve injury (Colburn et al., 1999). How-
ever, unlike the microgliosis this was not
reduced by erbB2 inhibition ( p ⫽ 0.88, t
test, n ⫽ 4) (supplemental Fig. 7, available
at www.jneurosci.org as supplemental
material).

ErbB2 receptor inhibition or

sequestration of endogenous NRG
reduces mechanical and cold
pain-related hypersensitivity after
nerve injury
There is increasing evidence that the mi-
croglial activation observed within the
dorsal horn of the spinal cord after nerve
injury contributes to the development of
neuropathic pain (Tsuda et al., 2003; Mil-
ligan and Watkins, 2009). Intrathecal in-
fusion of PD168393 over 7 d had no effect
Figure 8. NRG1 expression after L5 spinal nerve ligation. a–f, In situ hybridization images using a probe directed against the on mechanical withdrawal thresholds in
␤EGF domain of NRG1 in L5 DRG of naive animals (a– c) and 3 d after SNL (d–f ). Expression is highest in large-diameter DRG cells naive animals ( p ⫽ 0.25, RM two-way
positive for NF 200 (green) but is also observed in small DRG cells positive for either IB4 (blue) or CGRP (red). Arrows provide ANOVA, PD168393, 5 ␮g/d, vs vehicle;
illustrative examples of double labeled cells. There was no significant change in the proportion of DRG cells expressing EGF mRNA
data not shown). We therefore monitored
at 3 d after SNL. g– k, IHC images. g, h, We used the H210 antibody, which is raised against the N-terminal extracellular domain of
f1/f1
NRG1. To confirm specificity, we tested it in Nrg1 knock-out tissue (Nfh Cre Nrg1 mice in which Nrg1 is ablated in sensory
pain-related behavior over 14 d after L5
neurons in the late embryonic period). Note that normal NRG1 immunoreactivity is observed in DRG cells of Nrgf1/f1 mice (i.e., SNL in animals that were treated with the
control), whereas no immunoreactivity is present DRG cells of Nfh Cre Nrg1f1/f1 mice. i– k, Immunohistochemistry confirmed that erbB2 inhibitor or vehicle delivered via
NRG1 was present in ⬃60% of DRG cells (i, j) and within the dorsal horn of the spinal cord (especially within superficial laminae, continuous intrathecal infusion. In both
k). l, NRG1 expression was also assessed using a NRG1␤-specific ELISA. Lysate from dorsal horn spinal cord of naive, and 1 and 3 d groups of animals, a clear mechanical hy-
after SNL animals were run (50 ␮g of protein for each sample). All sample values were within the range of the ELISA detection. A persensitivity was observed at day 1 after
significant increase in NRG1 was detected at 3 d after SNL ( p ⫽ 0.04, one-way ANOVA, Bonferroni post hoc test, n ⫽ 3). SNL; however, at subsequent time points
Scale bars: f, h, j, 50 ␮m; k, 200 ␮m. erbB2 receptor inhibition resulted in a
significant dose-dependent reduction in
p38 MAPK, and microglial proliferation at a later time point after mechanical pain hypersensitivity on the side ipsilateral to the
nerve injury, i.e., 7 d after SNL (supplemental Fig. 4, available at lesion (Fig. 11a) ( p ⬍ 0.05, RM two-way ANOVA, PD168393, 10
www.jneurosci.org as supplemental material). In a further set of or 5 ␮g/d, vs vehicle, n ⫽ 7–11/group) with no effect on the
experiments, we assessed dorsal horn microgliosis at 7 d after contralateral paw (data not shown). Once the pump had emp-
pump emptying (i.e., 21 d after SNL). We found no significant tied, the mechanical withdrawal thresholds of the group receiving
difference in dorsal horn microgliosis at this time point compar- PD168393, 5 ␮g/d, returned to those of control (Fig. 11b). De-
ing intrathecal treatment with PD168393 and control ( p ⫽ 0.57, layed treatment with PD168393, 5 ␮g/d, from day 3 onward (by
t test, n ⫽ 3– 4) (supplemental Fig. 5, available at www.jneurosci. which time microgliosis is well established) was not effective at
org as supplemental material). Neuregulin is known to have a key reversing mechanical pain-related hypersensitivity (Fig. 11c)
5446 • J. Neurosci., April 14, 2010 • 30(15):5437–5450 Calvo et al. • Neuregulin-ErbB Signaling Promotes Microglial Proliferation and Chemotaxis

( p ⫽ 0.75, RM two-way ANOVA, n ⫽ 7

per group). Cold allodynia (as assessed by
nociceptive behavior in response to ace-
tone application to the paw) also devel-
oped after SNL and was significantly
attenuated in a dose-dependent fashion
by erbB2 inhibition (Fig. 11d) ( p ⬍ 0.05
PD168393, 10 or 5 ␮g/d, vs vehicle, RM
two-way ANOVA, n ⫽ 7–11/group). In-
trathecal injection of HBD-S-H4 (two 3
␮g intrathecal injections administered on
the day of surgery and on day 3 after sur-
gery), which sequesters endogenous NRG,
also significantly attenuated mechanical
pain-related hypersensitivity (Fig. 11e)
( p ⬍ 0.001 vs control, RM two-way
ANOVA, n ⫽ 8 per group) and cold allo-
dynia (Fig. 11f ) ( p ⬍ 0.05 vs control, RM
two-way ANOVA, n ⫽ 8 per group).

Discussion
Microglia closely resemble tissue macro-
phages and are key sensors of injury to the
CNS produced by many diverse patholo-
gies. NRG1 has not previously been impli-
cated in regulating microglial function.
Here, we demonstrate that NRG1 is a
survival, proliferative, and chemotactic
factor for microglia in vitro, and that
NRG1-erbB signaling is activated specifi-
cally within microglia after peripheral Figure 9. ErbB2 receptor blockade or sequestration of endogenous NRG inhibits the development of microgliosis after spinal
nerve injury, contributing to the develop- nerve ligation. The effect of erbB2 receptor blockers (PD168393, 5 ␮g/d, i.t.; or mAb 9 or 7.16.4, 5 ␮g, i.t.) or a NRG-sequestering
molecule (HBD-S-H4, 3 ␮g, i.t.) was determined at 3 d after SNL. a– e, ErbB2 receptor blockade (b and e, low- and high-power
ment of microgliosis and consequently
photomicrographs, respectively) resulted in a reduction in both p-erbB2 (green) expression and in the number of microglia in the
neuropathic pain. dorsal horn (Iba1 immunostaining, red) compared with vehicle (a and d, low- and high-power photomicrographs, respectively).
Microglia express the NRG1 recep- Quantification (c) demonstrates that SNL results in an increased number of microglia with activated morphology compared with
tors erbB2, 3, and 4. This factor pro- sham surgery animals. This increase was significantly attenuated after treatment with erbB2 inhibitor, erbB2 receptor-blocking
motes the survival of microglia in vitro antibodies, or the NRG1 antagonist (HBD-S-H4) with respect to control. f–j, SNL results in increased numbers of microglia express-
through inhibition of apoptosis and is a ing phospho-p38. The number of microglia (red) expressing p-p38 MAPK (green) after SNL was significantly reduced after erbB2
potent proliferative factor for these cells receptor blockade or sequestration of NRG using HBD-S-H4. h, Quantification. Error bars represent ⫾ SEM (n ⫽ 3– 4 per group).
in an erbB2-dependent manner. In addi- SH, Sham; VH, vehicle; INH, inhibitor (PD168393); IgG2a, nonimmune IgG (control); ANT, antagonist (HBD-S-H4). Statistical test: t
tion to proliferation, the other means by test (vehicle vs inhibitor and control vs antagonist) or one-way ANOVA, Bonferroni post hoc test (IgG2a vs erbB2 receptor-blocking
which microglia can accumulate at the antibodies).**p ⬍ 0.005. Scale bars: a, b, f, g, 100 ␮m; d, e, i, j, 50 ␮m.
site of injury is through directed migra-
tion in response to the release of chemotactic agents, which Because of the potent effects we observed of NRG1 on cul-
include the following: purines (Honda et al., 2001), comple- tured microglia, we explored the relevance of this signaling path-
ment components (Yao et al., 1990), bradykinin (Ifuku et al., way in vivo. Intrathecal administration of NRG1 resulted in a
2007), and chemokines (Peterson et al., 1997). We have used dorsal horn microgliosis and also produced mechanical and cold
the Boyden chamber chemotaxis assay and by manipulating the pain-related hypersensitivity [in agreement with a previous study
concentration gradient in the upper and lower wells have shown by Lacroix-Fralish et al. (2008)]. Spinal nerve ligation provides a
that NRG1 is a chemotactic agent for microglia acting in an well characterized model of nerve injury associated with a robust
erbB2-dependent manner. Neuregulin has previously been microgliosis (Tsuda et al., 2003; Scholz and Woolf, 2007) and the
shown to increase the motility of a number of different cell types development of neuropathic pain (Kim and Chung, 1992). We
including malignant cell lines (Hijazi et al., 2000), keratinocytes used an antibody that recognizes phosphorylated erbB2 as a mea-
(Schelfhout et al., 2002), and Schwann cells (Lyons et al., 2005). sure of receptor activation (Guertin et al., 2005). In naive and
NRG1 could also enhance the release of interleukin-1␤ from sham surgery animals, there was only a low level of p-erbB2 ex-
LPS-primed microglia. Unlike LPS, however, NRG1 application pression in resting microglia. After SNL, p-erbB2 expression in-
did not promote the development of amoeboid morphology or creased, and it was localized specifically within microglia,
MHC class II expression in microglia. We have found that mac- especially those with an activated morphology. The time course
rophages express erbB2, 3, and 4 receptors. Therefore, in situa- of p-erbB2 expression within the dorsal horn was coincident with
tions where there is major disruption of the blood– brain barrier the development of microgliosis being detectable at day1 after
(e.g., traumatic brain injury) resulting in macrophage infiltration injury and peaking at day 3–7 after SNL.
into the CNS the function of macrophages may also be modu- For NRG1 to act as a proliferative and chemotactic agent for
lated by NRG1. microglia, it needs to be released at the site of injury. All NRG1
Calvo et al. • Neuregulin-ErbB Signaling Promotes Microglial Proliferation and Chemotaxis J. Neurosci., April 14, 2010 • 30(15):5437–5450 • 5447

CSF (Pankonin et al., 2009). In culture

systems, NRG1 activity has also been
shown to be released from DRG cell axons
(Esper and Loeb, 2004; Taveggia et al.,
2005); this release is enhanced by BDNF
[which is itself released by microglia after
nerve injury (Coull et al., 2005)]. The
rapid response of microglia after SNL sug-
gests that increased NRG1 activity within
the dorsal horn after nerve injury is due to
the following: altered NRG1 protein traf-
ficking or release from primary afferent
terminals, or, possibly, release from the
extracellular matrix. We suggest that such
NRG1 release provides an injury-related
signal from primary afferents triggering a
microglial response within the dorsal
horn.
We tested the hypothesis that inhibi-
tion of NRG-erbB signaling would reduce
the microgliosis produced by SNL. We
used two strategies for erbB2 receptor in-
hibition: intrathecal treatment with either
an irreversible specific erbB inhibitor
(PD168393), which blocks the ATP bind-
ing site of the receptor or monoclonal an-
tibodies (mAb 7.16.4 and mAb 9). mAb
7.16.4 specifically binds to the extracellu-
Figure 10. Microglial proliferation after spinal nerve ligation is significantly reduced after erbB2 receptor inhibition. a– h, lar domain of erbB2, downregulates sur-
Three days after L5 SNL, a proliferative response is seen in microglia within the dorsal horn, as shown here by labeling newly face expression of erbB2, and blocks
dividing cells with BrdU (yellow) and microglia with Iba1 (red). DAPI is shown in blue to delineate nuclei. The BrdU-labeled nuclei activation of erbB2/3 and erbB2/4 het-
are almost exclusively within microglial cells (as seen in the merge image). Blocking the erbB2 receptor with PD168393 (5 ␮g/d, erodimers; this antibody has previously
i.t.) or with the blocking antibodies (mAb 9 or 7.16.4, 5 ␮g, i.t.) resulted in a significant reduction in microglial proliferation been shown to have efficacy in vivo (Kat-
(quantification in i). Scale bars: 100 ␮m. Error bars represent ⫾ SEM (n ⫽ 3– 4 per group). SH, Sham; VH, vehicle; INH, inhibitor sumata et al., 1995; Yarden and Sli-
(PD168393); IgG2a, nonimmune IgG (control). Statistical test: one-way ANOVA, Bonferroni post hoc test. **p ⬍ 0.005. wkowski, 2001). Inhibition of the erbB2
receptor after nerve injury resulted in a
isoforms contain an EGF domain, which is required for biological significant reduction in the microgliosis within the dorsal horn as
activity and can be classified into subgroups according to the well as reducing the expression of phospho-p38 (a component of
structure of their N termini. Type I and II isoforms have Ig-like the MAPK signaling cascade), which is specifically expressed
domains and either can be directly secreted or alternatively trans- within activated microglia (Jin et al., 2003; Kobayashi et al.,
membrane isoforms can undergo metalloprotease-mediated 2008). This effect on p38 signaling is likely to be indirect as NRG1
cleavage to be released as soluble proteins from the cell surface. did not stimulate the p38 MAPK pathway in cultured microglia,
These isoforms bind avidly to the extracellular matrix, which can although it does activate the extracellular signal-regulated kinase
act as a source of localized release (Loeb and Fischbach, 1995). kinase– extracellular signal-regulated kinase (MEK–ERK) and
Type III isoforms possess a cysteine-rich domain and undergo phosphatidylinositol-3 kinase–Akt pathways (M. Calvo, N. Zhu,
proteolytic cleavage by ␤-secretase enzyme (Willem et al., 2006); and D.L.H. Bennett, unpublished observations). Interestingly,
however, the EGF domain remains membrane tethered, and so ERK previously has been shown to be activated in microglia after
this molecule signals in a juxtacrine fashion. DRG cells have peripheral nerve injury (Zhuang et al., 2005). We have also used a
been reported to principally express type I and III isoforms molecule (HBD-S-H4) to sequester endogenous NRG1. This is a
(Bermingham-McDonogh et al., 1997). We found clear expres- fusion protein consisting of the soluble ectodomain of erbB4,
sion of NRG1 by small- and large-diameter DRG cells using in which has high affinity for NRG1, and a heparin-binding do-
situ hybridization and a probe that recognizes all ␤ EGF isoforms main, which helps target the molecule to the same heparan-
(the nervous system principally expresses ␤ rather than ␣ iso- sulfate rich cell surfaces that bind NRG1. This molecule could
forms). ␤ EGF mRNA was expressed at low levels by occasional also effectively reduce dorsal horn microgliosis and pp38 expres-
dorsal horn neurons, and so we propose that the most likely sion after SNL.
source of NRG1 within the dorsal horn is from primary afferent Given the lack of major disruption to the blood– brain barrier
terminals. We did not observe a change in ␤ EGF mRNA expres- after SNL (Abram et al., 2006; Lu et al., 2009), proliferation of
sion in DRG cells or the dorsal horn at 3 d after SNL, a time point resident microglia is likely to be the principal means by which
when microgliosis is well established. We did find significantly microglial numbers increase after SNL. We found a clear increase
increased ␤ EGF containing NRG1 protein within the dorsal horn in microglial cell proliferation within the dorsal horn in the first
using an ELISA after SNL. Cultured neurons have been shown to 3 d after peripheral nerve injury. ErbB2 receptor blockade signif-
release soluble NRG1 in an activity-dependent manner (Ozaki et icantly reduced this proliferative response. Inhibition of micro-
al., 2004), and NRG1 activity has also been detected in human glial proliferation provides one explanation for the reduction in
5448 • J. Neurosci., April 14, 2010 • 30(15):5437–5450 Calvo et al. • Neuregulin-ErbB Signaling Promotes Microglial Proliferation and Chemotaxis

microgliosis observed after erbB2 recep-

tor inhibition, although impaired chemo-
taxis of local microglia may also play a
role. ErbB2 inhibition specifically reduced
microgliosis after nerve injury and did not
prevent the development of astrocytosis.
There is now increasing evidence that
glial cells (both microglia and astrocytes)
play an important role in the generation
of neuropathic pain. A number of mole-
cules have been implicated in the recruit-
ment and activation of microglia within
the dorsal horn after nerve injury includ-
ing cytokines, chemokines (such as CCL2
and Fractalkine), complement compo-
nents, and purines (Jin et al., 2003; Tsuda
et al., 2003; Clark et al., 2007; Scholz and
Woolf, 2007; Zhang et al., 2007; Milligan
and Watkins, 2009; Thacker et al., 2009).
After activation, microglia release cyto-
kines and BDNF, which enhance the ex-
citability of dorsal horn neurons (Coull et
al., 2005; Kawasaki et al., 2008). Inhibition
of the microglial response to injury has
been shown to effectively reduce the de-
velopment of neuropathic pain (Tsuda et
al., 2003; Coull et al., 2005; Ledeboer et al.,
2005; Clark et al., 2007). ErbB2 receptor
inhibition or sequestration of endogenous
NRG1 could ameliorate (but not com-
pletely reverse) both mechanical and cold
pain-related hypersensitivity after SNL.
After peripheral nerve injury, the proin-
flammatory microglial response is clearly Figure 11. ErbB2 receptor inhibition or sequestration of endogenous NRG reduced mechanical pain hypersensitivity and cold
deleterious as it contributes to neuropathic allodynia after spinal nerve ligation. Animals underwent L5 SNL and received a continuous intrathecal infusion of the erbB2
inhibitor (PD168393, 1.25–10 ␮g/d) or vehicle for 14 d. a, Mechanical hypersensitivity developed at day 1 after SNL in both
pain; however, in other contexts there is ev-
groups and after day 2 was significantly attenuated in animals receiving the erbB2 inhibitor in a dose-dependent fashion. *p ⬍
idence that these cells may be neuroprotec-
0.05, **p ⬍ 0.001, for 10 ␮g/d vs vehicle; #p ⬍ 0.05 for 5 ␮g/d vs vehicle; n ⫽ 7–11/group. b, Once the pump had emptied, the
tive (Hanisch and Kettenmann, 2007). withdrawal thresholds of the group receiving the erbB2 inhibitor (5 ␮g/d) returned to those of control. c, Delayed treatment with
NRG-erbB signaling represents a novel the erbB2 inhibitor (5 ␮g/d) from day 3 onward (by which time microgliosis is well established) was not effective at reversing
pathway stimulating microglial prolifera- mechanical pain-related hypersensitivity ( p ⫽ 0.75) d, Cold allodynia was also significantly reduced in a dose-dependent fashion
tion and chemotaxis. We have shown that by inhibiting the erbB2 receptor. **p ⬍ 0.001 for 10 ␮g/d vs vehicle; #p ⬍ 0.05 for 5 ␮g/d vs vehicle, n ⫽ 7–11/group.
this contributes to the development of e, f, Intrathecal administration of HBD-S-H4 (3 ␮g) at days 0 and 4 after SNL (shown by arrows) significantly reduced mechanical
neuropathic pain, and it will be of great ( p ⬍ 0.001) (e) and cold ( p ⬍ 0.05) (f ) pain-related hypersensitivity. Error bars represent ⫾ SEM. Statistical tests: two-way
interest in the future to determine the role ANOVA, Bonferroni test, or Fischer LSD post hoc analysis. When the assumptions of sphericity were violated (Mauchly’s test; p ⬍
of NRG-erbB signaling in mediating the 0.05), the Greenhouse–Geisser correction was applied and independent two-tailed t tests were used to determine differences
microglial response to other forms of CNS between groups. INH, Inhibitor (PD168393); NRG ANT, NRG antagonist (HBD-S-H4). The lines in a– d denote the period of pump
injury. infusion. The arrows in e and f denote the days of intrathecal injections.

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