Regional Neural Activation Deﬁnes
a Gateway for Autoreactive T Cells
to Cross the Blood-Brain Barrier
Yasunobu Arima,1 Masaya Harada,1 Daisuke Kamimura,1 Jin-Haeng Park,1 Fuminori Kawano,2 Fiona E. Yull,3Tadafumi Kawamoto,5 Yoichiro Iwakura,6 Ulrich A.K. Betz,7 Gabriel Márquez,8,10 Timothy S. Blackwell,3,4
Yoshinobu Ohira,2 Toshio Hirano,1,9 and Masaaki Murakami1,*
1Laboratory of Developmental Immunology, JST-CREST, Graduate School of Frontier Biosciences, Graduate School of Medicine,and WPI Immunology Frontier Research Center, Osaka University, Osaka 565-0871, Japan
2Department of Health and Sports Sciences, Graduate School of Medicine, and Graduate School of Frontier Biosciences, Osaka University,
Osaka 560-0043, Japan
3Department of Cancer Biology
4Department of Medicine
Vanderbilt University, TN 37232, USA
5Radioisotope Research Institute, Department of Dental Medicine, Tsurumi University, Yokohama 230-8501, Japan
6Center for Experimental Medicine and Systems Biology, Institute of Medical Science, The University of Tokyo, Tokyo 108-8639, Japan
7Merck KGaA, Portfolio Development Merck Serono, 64293 Darmstadt, Germany
8National Center for Biotechnology (CNB-CSIC), Darwin 3, 28049 Madrid, Spain
9Laboratory of Cytokine Signaling, RIKEN Research Center for Allergy and Immunology, Yokohama 230-0045, Japan
10Present address: Genetrix SL, Plaza de la Encina, 10–11 Tres Cantos, 28760 Madrid, Spain*Correspondence: murakami@molonc.med.osaka-u.ac.jp
DOI 10.1016/j.cell.2012.01.022

SUMMARY

Although it is believed that neural activation canaffect immune responses, very little is known about
the neuroimmune interactions involved, especiallythe regulators of immune trafﬁc across the bloodbrain barrier which occurs in neuroimmune diseases
such as multiple sclerosis (MS). Using a mousemodel of MS, experimental autoimmune encephalomyelitis, we show that autoreactive T cells accessthe central nervous system via the ﬁfth lumbar spinal
cord. This location is deﬁned by IL-6 ampliﬁerdependent upregulation of the chemokine CCL20 inassociated dorsal blood vessels, which in turndepends on gravity-induced activation of sensory
neurons by the soleus muscle in the leg. Impairing
soleus muscle contraction by tail suspension is sufﬁcient to reduce localized chemokine expression andblock entry of pathogenic T cells at the ﬁfth lumbar
cord, suggesting that regional neuroimmune interactions may offer therapeutic targets for a variety ofneurological diseases.
INTRODUCTION
Neuroimmune interactions arise from at least two types of pathways (Felten, 2000; Sternberg, 2006; Thayer and Sternberg,
2010; Watkins and Maier, 1999): neurohormonal pathways that
depend on circulating hormones such as cortisol and catechol-amines including norepinephrine, and neural pathways that are
mainly mediated by direct neural connections with lymphoidorgans like the thymus, bone marrow, spleen, and lymph nodes.
Detailed anatomical analysis has suggested the lymphoidorgans are innervated by the peripheral nervous system(Sternberg, 2006), the sympathetic nervous system (Nance andSanders, 2007), and the parasympathetic nervous system(Tracey, 2007). Thus, neurohormonal pathways likely regulate
immune responses systemically, while neural pathways regulate
immune responses at a local and regional level, resulting inregional property differences even within the same organ.
Although the systemic effects of neurohormonal pathways
have been well investigated, little is known about the mechanisms used by the local neural pathways.
The central nervous system (CNS) is an immune-privileged
environment, protected by the blood-brain barrier, which isformed by speciﬁc vessels tightly attached to each other (Galeaet al., 2007; Wilson et al., 2010). This barrier, however, can becompromised upon the invasion of certain pathogens and/or
latent infections (McGavern and Kang, 2011; Salinas et al.,2010), suggesting that immune cells in the peripheral lymphoidorgans contribute to CNS related immune responses. However,
how these cells enter and/or accumulate in the CNS from peripheral lymphoid organs is unclear. Moreover, although pathogenic
CD4+ T cells, which express high levels of cytokines such as
IL-17A and IFNg and include autoreactive cells in peripheral
lymphoid organs, are believed to play an essential role in thepathogenesis of MHC class II-associated autoimmune diseases
like multiple sclerosis (MS) (Reboldi et al., 2009; Wilson et al.,2010), the initial gates of entry through the blood-brain barrier
have yet to be analyzed, particularly at the molecular level. It is
Cell 148, 447–457, February 3, 2012 ª2012 Elsevier Inc. 447

known that leukocytes like CD4+ T cells migrate into the CNS
through the choroid plexus and subarachnoid space, brain
regions that show excess chemokine levels, including the
CCL20 chemokine, speciﬁcally in late phases of disease development, suggesting that pathogenic CD4+ T cells can migratefrom the blood directly into the brain tissues during these phases(Ransohoff, 2009; Reboldi et al., 2009; Wilson et al., 2010).
Further, it has been hypothesized that barrier properties and/or
the vasculature itself differ between CNS compartments andthat these differences may inﬂuence immune cell access (Wilson
et al., 2010). We hypothesize that these differences may arise
from regional neural activation around the vessels, such that
regional neural activation can regulate immune cell/blood cell
accumulation in the CNS.
An important mediator for this regulation may be the NFkBtriggered positive-feedback-loop for IL-6 expression (IL-6 ampliﬁer) in endothelial cells, which line blood and lymphatic vessels.
We previously showed that this feedback loop is a synergistic
activator of NFkB and STAT3 in type 1 collagen+ cells after
IL-17A and IL-6 stimulation. Activation of the IL-6 ampliﬁer canlead to excess expression of various chemokines and cytokines
including CCL20 and IL-6 and the development of autoimmune
diseases like F759 arthritis and experimental autoimmune
encephalomyelitis (EAE), which is a multiple sclerosis model
induced by a myelin oligodendrocyte glycoprotein (MOG)derived autopeptide immunization (Hirano, 2010; Murakami
et al., 2011; Ogura et al., 2008). Additionally, those reports foundthat IL-6 ampliﬁer activation in mouse joints directly or indirectly
causes accumulation of various immune cell/blood cell populations including IL-17A- and IFNg-expressing pathogenic CD4+
T cells there. Knowing this, we examined whether endothelial
cell stimulation that depends on neural activity can trigger the
IL-6 ampliﬁer to cause regional pathogenic CD4+ T cell migration
into the CNS. We demonstrate that regional neural activation
deﬁnes a gateway for autoreactive T cells to cross the bloodbrain barrier via the IL-6 ampliﬁer activation in endothelial cells
at the ﬁfth lumbar cord.
RESULTS
Transfused Pathogenic CD4+ T Cells Mainly Migrate
to the CNS via the Fifth Lumbar Cord during the Early
Stages of Autoimmune Disease Development
To discover the gates of entry into the CNS for immune cells
including pathogenic CD4+ T cells, we employed a passive
transfer method for EAE induction as described previously(Ogura et al., 2008). Pathogenic CD4+ T cells were isolated
from wild-type C57BL/6 mice immunized with a MOG peptide
plus adjuvants (see the Experimental Procedures) and thencultured in vitro with bone marrow-derived dendritic cells in thepresence of the same MOG peptide plus IL-23. The resulting
pathogenic CD4+ T cells contained Th1 and Th17 cells andwere intravenously transferred into wild-type C57BL/6 mice.
EAE development peaked at about 10–12 days after the transfer(Figure S1A available online).
It has been reported pathogenic CD4+ T cells accumulate in
the choroid plexus from day 13 after EAE induction by systemic
immunizations in C57BL/6 mice (Reboldi et al., 2009). We found
448 Cell 148, 447–457, February 3, 2012 ª2012 Elsevier Inc.

no T cell accumulation around the choroid plexus during the ﬁrst
5 days after pathogenic CD4+ T cell transfer in untreated mice(data not shown). Because we have observed that pathogenic
T cells mainly accumulate in the spinal cord around day 10 of
EAE development (Sawa et al., 2009), we hypothesized that
pathogenic CD4+ T cells do not directly migrate to the choroid
plexus, but may accumulate in the spinal cord instead, making
the spinal cord a candidate for the gate in our model. We therefore investigated CD4+ T cell numbers in the spinal cord over ﬁrst
5 days. We ﬁrst investigated pathogenic CD4+ T cell accumulation in the CNS by using anterior-posterior whole mount sections
after the transfer, ﬁnding robust pathogenic CD4+ T cell accumulation in the lumbar level, but not in the thoracic and cervicallevels of the spinal cord (data not shown). We then analyzed the
lumbar level in detail. Because more than 95% of accumulatedmononuclear cells merged with a congenic marker having pathogenic CD4+ T cells, we measured pathogenic CD4+ T cellsby counting mononuclear cell numbers in ﬁve different spinal
sections (see Figure 1A legend), took the average and thencalculated the relative number of pathogenic CD4+ T cells,ﬁnding a signiﬁcant accumulation of pathogenic CD4+ T cells
in the ﬁfth lumbar cord (Figure 1A), a result conﬁrmed by ﬂow
cytometry (Figure 1B). The accumulation was much greater inthe dorsal blood vessels of the ﬁfth spinal cord than in the ventral
ones (Figure 1C), suggesting dorsal blood vessels of the ﬁfth
spinal cord may be the gate to the CNS.
Activation of the IL-6 Ampliﬁer Plays a Rolein the Accumulation of Pathogenic CD4+ T Cells
in the Fifth Lumbar Cord
Because pathogenic CD4+ T cell transfers included both Th1
and Th17 cells (Ogura et al., 2008) (data not shown), we next
investigated whether cytokines from these pathogenic CD4+
T cells contribute to the CD4+ T cell accumulation in the ﬁfth
lumbar cord. For this purpose, we employed IFNg-deﬁcientand IL-17A-deﬁcient mice and immunized them with the MOG
peptide plus CFA to obtain pathogenic CD4+ T cells lacking
IFNg or IL-17A. We then transferred these pathogenic CD4+
T cells separately into wild-type C57BL/6 mice to investigate
T cell migration. Of these, only pathogenic CD4+ T cells from
IFNg-deﬁcient mice showed an accumulation in the ﬁfth lumbar
cord like that seen in normal pathogenic CD4+ T cells (Figure 2A),
suggesting a role for IL-17A.
We have previously shown that Th17 cells accumulate in joints
in a manner dependent on activating the IL-6 ampliﬁer (Murakami et al., 2011), suggesting that the ampliﬁer might be involvedin the above accumulation. Pathogenic CD4+ T cells were intravenously transferred into mutant mice deﬁcient of IL-6 or ofeither gp130 or STAT3 in their type 1 collagen+ cells and into
wild-type C57BL/6 mice. Pathogenic CD4+ T cell accumulation
was signiﬁcantly suppressed in all mutant hosts (Figure 2B), suggesting that, indeed, IL-6 ampliﬁer activation was involved. We
also found the clinical EAE scores in these mutant mice to besigniﬁcantly suppressed (data not shown) and the IL-6 concentrations in serum to have decreased (Figure 2C). We next identiﬁed important type 1 collagen+ cell populations for pathogenic
CD4+ T cell accumulation in the ﬁfth lumbar cord, because thesecell populations include ﬁbroblasts, endothelial cells, epithelial

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Figure 1. Pathogenic CD4+ T Cells Transferredto the Blood Mainly Migrate to the CNS from theFifth Lumbar Cord during the Early Stages of EAE
Development

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DorsalvesselVentralvesselcells, astrocytes, and activated neuron cells. Pathogenic CD4+
T cells were also transferred into mutant mice with Tie2+ endothelial cells deﬁcient of gp130 and into wild-type C57BL/6
mice. Pathogenic CD4+ T cell accumulation and clinical EAE
scores were signiﬁcantly suppressed in these mutant hosts(Figures S1B and S1C), suggesting the importance of type 1
collagen+ endothelial cells in the accumulation of pathogenic
CD4+ T cell in the ﬁfth lumbar cord.
Consistent with these results, STAT3 activation was enhancedin the endothelial cells of dorsal blood vessels in the ﬁfth lumbar
cord, but neither in the fourth nor sixth lumbar cords, as determined by STAT3 phosphorylation levels (Figure 2D). These
results indicate IL-6 ampliﬁer activation in endothelial cells has
a signiﬁcant role in pathogenic CD4+ T cell accumulation in theﬁfth lumbar cord during EAE development.
CCL20 Expression Contributes to the Accumulationof Pathogenic CD4+ T Cells in the Fifth Lumbar Cord
We next attempted to identify a critical target molecule for
STAT3 and NFkB activation in type 1 collagen+ endothelial
cells during IL-6 ampliﬁer activation. We considered chemokines like CCL20, because they are synergistically expressed
via STAT3 and NFkB in type 1 collagen+ cells, are critical for
Th17 cell accumulation in an arthritis model (Murakami et al.,2011), and because pathogenic CD4+ T cells from mice deﬁcient of CCR6, a CCL20 receptor, showed minimal accumulation in the ﬁfth lumbar cord (Figure S2A). We found that
CCL20 expression in the dorsal blood vessels of the ﬁfth

L4

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(A) Pathogenic CD4+ T cell numbers in each spinal cord.
Pathogenic CD4+ T cells prepared from wild-type micetransferred into wild-type hosts. The numbers of accumulated pathogenic CD4+ T cells in the six lumber cords
were evaluated by counting mononuclear cells in six
different spinal sections (anterior-terminal, middle anterior-center, center, middle center-posterior, and posteriorterminal) and taking the average. Accumulated mononuclear cells in the ﬁfth lumber cord increased comparing
to other cords.
(B) Flow cytometry of mononuclear cells isolated fromeach spinal lumbar cord. CD4+CD90.2+ cell numbers inthe ﬁfth lumber cord increased comparing to other cords.
(C) Pathogenic CD4+ T cells isolated from EAE mice wereintravenously transferred into wild-type C57BL/6 mice.
Accumulation of pathogenic CD4+ T cells in the choroid
plexus and each spinal cord were observed in frozen
sections taken 5 days after the transfer. Left: Histologicalanalysis was performed with sections stained with hematoxylin-eosin in the central region of the ﬁfth lumber cord.
Representative data are shown. The scale bar represents
100 mm. Top right: Magniﬁcation of black rectangle. The
scale bar represents 100 mm. Bottom right: 3D picture
based on ten serial HE sections of the ﬁfth lumber cord
central region. Purple balls indicate nucleuses of pathogenic CD4+ T cell nuclei; red tubes, vessels on the ﬁfth
spinal cord.
Error bars represent the mean ± SEM. **p < 0.01. See also
Figures S1 and S5.

lumbar cord increased even in the absence of pathogenic
CD4+ T cell transfer (Figure 3A), with no such concomitanteffect in vessels of the thoracic and cervical cords (Figure S2B).
Interestingly, CCL20 expression eventually reached otherregions like the anterior lumbar cords after pathogenic CD4+
T cell transfer (Figure S2C, right), suggesting that the bloodbrain barrier is compromised broad regions after pathogenic
T cell accumulation in the CNS. Furthermore, along with
CCL20, chemokines such as CCL2, CCL3, CCL9, CCL21,
CCL24, CX3CL1, CXCL1, CXCL2, CXCL9, and CXCL11
increased in the dorsal blood vessels of the ﬁfth lumbar cord
compared to the ﬁrst lumbar cord (Figures 3A and 3B), whileanti-CCL20 antibody treatment in vivo signiﬁcantly suppressed
pathogenic CD4+ T cell accumulation and the development of
EAE (Figures 3C– 3E).
We next investigated for correlations between CCL20
expression and IL-6 ampliﬁer activation in the ﬁfth lumbar
cord. Again, mutant mice deﬁcient of IL-6 or of either gp130
or STAT3 in their type 1 collagen+ cells were employed.
CCL20 messenger RNA (mRNA) expressions in the dorsal
blood vessels of the ﬁfth lumbar cord was less in all threemutant recipients than in wild-types (Figure 3F). Together with
the importance of IL-17A and CCR6 expression in pathogenic
CD4+ T cells, these results suggest that CCL20 expression,
which is known to be regulated by IL-6 ampliﬁer activation, atleast in joints, plays a similar role in pathogenic CD4+ T cell
accumulation in the ﬁfth lumbar cord and that this leads to
EAE development.
Cell 148, 447–457, February 3, 2012 ª2012 Elsevier Inc. 449

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Figure 2. Activation of IL-6-Mediated Ampliﬁcation
Plays a Role in Pathogenic CD4+ T Cell Accumulation in the Fifth Lumbar Cord

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Sensory Nerve Activation via Soleus Muscles Is Criticalfor CCL20 Expression in the Fifth Lumbar Cord and theEnsuing Pathogenic CD4+ T Cell Accumulation
We then considered how the IL-6 ampliﬁer in dorsal bloodvessels of the ﬁfth lumbar cord is activated to express CCL20
prior to pathogenic CD4+ T cell transfer. We focused on the activation of soleus muscle-mediated sensory nerves, because theirdorsal root ganglia locate near the ﬁfth lumbar (Ohira et al., 2004).
We hypothesized that frequent stimulation of the soleus muscles
in response to a gravitational stimulus could induce activation of
the IL-6 ampliﬁer via their sensory nerves, which in turn would
directly or indirectly induce high CCL20 expression in the bloodvessels of the ﬁfth lumbar cord. For this, we employed a tailsuspension model, which is a widely accepted National Aeronautics and Space Administration ground-based model forstudying disuse-atrophy in rodents to eliminate any responseby the hind legs to the stimulus (Canu and Garnier, 2009;
De-Doncker et al., 2005; Kawano et al., 2007; Nakao et al.,2009). Tail suspensions signiﬁcantly decreased CCL20 expression in dorsal blood vessels of the ﬁfth lumbar cord (see theleft two columns in Figure 4A). Consistent with this, tail suspensions also signiﬁcantly suppressed pathogenic CD4+ T cell
accumulation in the ﬁfth lumbar cord (Figure 4B), while pathogenic CD4+ T cell accumulation and CCL20 expression in
cervical cord blood vessels increased, especially in the posteriorside (Figure 4B and Figure S3A). Moreover, c-fos expression
levels decreased in ﬁfth dorsal root ganglia (Figure 4C), but
increased in the dorsal root ganglia of the cervical spine after
450 Cell 148, 447–457, February 3, 2012 ª2012 Elsevier Inc.

L5(A) Pathogenic CD4+ T cells prepared from IL17A-deﬁcient, IFNg-deﬁcient, or wild-type mice were transferred
into wild-type hosts. Relative numbers of accumulated
pathogenic CD4+ T cells in the ﬁfth lumber cord. Accumulated mononuclear cells in the ﬁfth lumber corddecreased after transfer of IL-17A-deﬁcient T cells
comparing to that of wild-type ones.
(B) Pathogenic CD4+ T cells prepared from wild-type mice
were transferred into mutant hosts deﬁcient of IL-6 or ofeither gp130 or STAT3 in their type 1 collagen+ cells.
Relative numbers of accumulated pathogenic CD4+
T cells in the ﬁfth lumbar cord.
(C) Serum IL-6 concentrations in the mutant hosts
described in (B). Serum IL-6 decreased in all mutant micecomparing to wild-type ones.
(D) Top: Histological analysis was performed with frozen
sections stained with anti-phospho-STAT3 antibody orcontrol rabbit IgG plus secondary antibody. The fourth,ﬁfth, and sixth lumber cords are shown. Arrows indicate
endothelial cell nuclei in the dorsal blood vessels of eachlumber cord. Scale bars represent 50 mm. Bottom: Quantiﬁcation of the histological analysis (n = 300 cells for each
lumbar cord). Phospho-STAT3+ endothelial cell numbers
increased in the ﬁfth lumber cords comparing to the fourthand sixth ones.
Error bars represent the mean ± SEM, **p < 0.01 (A)–(C).
Error bars represent the mean ± SD (D), **p < 0.01. See
also Figure S1.

L6

tail suspension (Figure S3B). Finally, EAE development wassigniﬁcantly inhibited when the tail was suspended (Figure 4D).
In normal mice without pathogenic CD4+ T cell transfer, NFkB
activation, which like STAT3 activation is also critical for IL-6
ampliﬁer activation, was enhanced in the blood vessels of theﬁfth lumbar cord, but not in those of the ﬁrst lumbar or cervicalcords (Figure 4E). Tail-suspended mice, however, responded
differently, as NFkB activation was enhanced in vessels of thecervical cord, but not those of the ﬁrst or ﬁfth lumbar cords (Figure 4E). These data strongly suggest sensory nerve activation by
the soleus muscles plays a role in CCL20 expression in the dorsal
vessels of the ﬁfth lumbar cord prior to the development of autoimmune diseases that occur from an accumulation of pathogenic
CD4+ T cells. To conﬁrm this, we stimulated soleus muscles
directly by using an electronic stimulator while mice weresuspended from their tails. Electronic stimulations in the soleus
muscles increased CCL20 expressions in the dorsal bloodvessels in a manner dependent on stimulus duration (see theright four columns in Figure 4A). These stimulations also
increased the number of pathogenic CD4+ T cells in the ﬁfth
lumbar cord (Figure 4B) and c-fos expression in the ﬁfth lumbar
dorsal root ganglia (Figure 4C). All these results are consistent
with regional neural activations via soleus muscle-mediatedsensory nerve activation creating gates for pathogenic CD4+
T cell accumulation into the CNS.
For further validation of our tail suspension model, ﬁve additional experiments were done. In the ﬁrst, electrically stimulating
the quadriceps, whose afferent nerves come from the third

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Figure 3. CCL20 Expression Plays a Role in Pathogenic CD4+ T Cell Migration in the Fifth Lumbar Cord
(A) CCL20 mRNA expression in the dorsal blood vessels in each spinal cord was investigated by real time PCR in normal C57BL/6 mice. CCL20 expression in theﬁfth lumber cord increased comparing to other cords.
(B) CCL2, CCL3, CCL9, CCL21, CCL24, CX3CL1, CXCL1, CXCL2, CXCL9, and CXCL11 mRNA expression in the dorsal blood vessels of each spinal cord were
investigated by real-time PCR in normal C57BL/6 mice.
(C) Pathogenic CD4+ T cells isolated from EAE mice were intravenously transferred into wild-type C57BL/6 mice in the presence or absence of anti-CCL20
antibody administration (days 0–5 or 8–13 after the pathogenic CD4+ T cell transfer). Relative accumulation of pathogenic CD4+ T cells in the ﬁfth lumbar cord
were investigated with sections from mice 5 days after the pathogenic T cell transfer.
(D and E) Clinical EAE scores of the mice (n = 10 each) described in (C).
(F) CCL20 mRNA expression in the dorsal blood vessels of the ﬁfth lumbar cord, in wild-type C57BL/6 and mutant mice deﬁcient of IL-6 or of gp130 or STAT3 intheir type 1 collagen+ cells. CCL20 expression in the ﬁfth lumber cord decreased in all mutant mice comparing to wild-type ones.
Error bars represent the mean ± SEM, *p < 0.05, **p < 0.01, ***p < 0.005 (A and C–F). Error bars represent the mean ± SD, *p < 0.05, **p < 0.01 (B). See also
Figure S2.

lumbar dorsal root ganglia, and the epitrochlearis/triceps brachii,whose afferent nerves come from between the ﬁfth cervical andﬁfth thoracic dorsal root ganglia, caused CCL20 expression toincrease in the third lumbar vessels and between the ﬁfth cervicaland ﬁfth thoracic vessels, respectively (Figure S3C). Similarly,
these stimulations increased cfos expression (Figure S3D). We
next employed a deafferentation method where afferent ﬁbersof the ﬁfth lumbar dorsal root ganglia were resected to reduce
the soleus muscle response against gravity while only making
minimal changes to body balance (Kawano et al., 2007). We
found CCL20 expression in the ﬁfth lumbar vessels decreased
just like that in the tail suspension model, while CCL20 expression
in the ﬁrst lumbar vessels were unchanged (Figure S3E). Furthermore, cfos expression decreased in dorsal root ganglia of the ﬁfth
lumbar cords (Figure S3F). We employed tail suspensions plus
a casting, as this combination is reported to increase dorsal
root ganglion activation (Kawano et al., 2002), ﬁnding the increase
in CCL20 expression in the ﬁfth lumbar vessels was even greaterthan tail suspension alone (Figure S3G). Finally, we investigatedblood ﬂow speed in blood vessels of the ﬁfth lumbar dorsal
region, bottoms of the forefeet, palms, femoral vessels, brain

surface vessels, portal vein, and intestinal microvessels in normal
and tail suspended mice. In all cases, speeds were comparable
for all investigated regions between the two mouse types except
for the ﬁfth lumbar region (Figure S3H). These results demonstrate the viability of our tail suspension model and indicate that
regional neural activations create gates for pathogenic CD4+
T cell entry into the CNS by increasing CCL20 expression.
Soleus Muscle-Mediated Sensory Nerve Activation
at Least Partially Enhances CCL20 Expression
in the Fifth Lumbar Cord via Sympathetic Neurons
Finally, we considered how sensory nerve activations from soleus
muscles change the state of dorsal vessels of the ﬁfth lumbar
cord to cause an accumulation of pathogenic CD4+ T cells.
Continuing the blood ﬂow speed experiments described above,
we found electrically stimulating soleus muscles can change ﬁfth
lumbar blood ﬂow speed (Figure 5A). Additionally, treatment with
the norepinephrine antagonist atenolol signiﬁcantly suppressed
CCL20 mRNA expression, NFkB activation, and pathogenic
CD4+ T cell accumulation around these vessels and abrogated
EAE development (Figures 5B–5F). Similar results were obtained
Cell 148, 447–457, February 3, 2012 ª2012 Elsevier Inc. 451

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Figure 4. Sensory Nerve Activation via Soleus Muscle Stimulation Is Critical for CCL20 Expression in the Fifth Lumbar Cord and Pathogenic
CD4+ T Cell Accumulation(A) CCL20 mRNA expression in dorsal blood vessels of the ﬁfth lumbar cord in wild-type C57BL/6 mice with or without tail suspension (TS) in the presence orabsence of soleus muscle electrostimulation (ES).
(B) Accumulation of pathogenic CD4+ T cells in the ﬁfth lumbar cord or spinal cords in the cervical spines of wild-type C57BL/6 mice that received transfusions ofpathogenic CD4+ T cells isolated from EAE mice. Mice were grouped as with or without TS and ES. Relative accumulations were measured from sections 5 days
after the pathogenic T cell transfer.
(C) Top: Histological analysis with frozen sections stained with anti-cFos antibody (cFos). Representative data from the dorsal root ganglion beside the ﬁfth
lumber cords of the mice described in (B) are shown. Green, cFos molecules; blue, nuclei. Experiments were performed at least three times. Scale bars represent
50 mm. Middle: Quantiﬁcation of histological analysis (n = 300 cells each). Bottom: c-fos mRNA expression in the dorsal root ganglia of the ﬁfth lumbar cord.
Stimulations lasted 12 hr.
(D) Clinical EAE scores (n = 5 for each group).
(E) NFkB activation monitored by luciferase activity in the dorsal blood vessels of the ﬁrst lumbar cord (L1), ﬁfth lumbar cord (L5), and spinal cords in the cervicalspines (C1–C7) of C57BL/6 mice transduced with a NFkB reporter with or without TS. NFkB activation in the ﬁfth lumber cord increased comparing to otherlumber cords without tail suspension, while that in the cervical cords increased comparing to other lumber cords after tail suspension. Dorsal blood vessels wereisolated under a stereoscopic microscope.
Error bars represent the mean ± SEM, *p < 0.05, **p < 0.01 (A, B, D, and E). Error bars represent the mean ± SD, **p < 0.01 (C). See also Figure S3.

using another norepinephrine antagonist, prazosin (Figure S4).
Furthermore, about 15% of sympathetic nerves in ﬁfth lumbar
spine ganglia expressed c-fos molecules, while ﬁrst lumbar spine
ganglia did not, which is consistent with CCL20 expression in
their dorsal vessels (Figure 5G). Consistent with these in vivo
results, norepinephrine enhanced IL-6 ampliﬁer activation basedon IL-6 or CCL20 expression levels in vitro (Figures 5H and 5I).
Taken together, these results indicate that sympathetic nerve
activation induced by soleus muscles is at least partially involvedin the enhanced expression of chemokines like CCL20 in dorsal
blood vessels, and that this leads to an accumulation of pathogenic CD4+ T cells in the ﬁfth lumbar cord (Figure 5J). All theseresults demonstrate that regional neural activations via the soleus
muscle-mediated sensory nerve enable pathogenic CD4+ T cell
entry into the CNS from adjacent blood vessels.
DISCUSSION
Although it is known that autoimmune diseases can correlatewith neurological states like stress, how neural signals are phys452 Cell 148, 447–457, February 3, 2012 ª2012 Elsevier Inc.

ically transformed into an immune response in the CNS is poorly
understood. In the current study, we describe how regional
neural activations can create gates in speciﬁc blood vesselsthat allow for immune cell entry into the CNS. We employedsensory neural activations of the soleus muscles, ﬁnding sympathetic nerve activations here lead to excess CCL20 expression in
the ﬁfth lumbar cord in a manner dependent on the activation of
the IL-6 ampliﬁer. This in turn leads to the activation of NFkB and
STAT3 in type 1 collagen+ endothelial cells. Therefore, neuroimmune interactions induce regional alterations in blood vessels
and venules such that immune cells can enter the CNS. Whenpathogenic helper T cells are in the blood stream, these neuroimmune interactions risk the development of autoimmune disease.
IL-6 ampliﬁer activation has been shown to be critical for thedevelopment of autoimmune diseases like EAE and rheumatoid
arthritis (Hirano, 2010; Murakami et al., 2011; Ogura et al., 2008).
We recently proposed a mechanism that depends on local IL-6ampliﬁer activation to explain the development of MHC class
II-associated diseases as a four-step model for autoimmune
diseases. This model shows four local events that must occur

5
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Sympathetic ganglion

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A

Soleus muscle
Figure 5. Soleus Muscle-Mediated Sensory Nerve Activation at Least Partially Enhances IL-6 Ampliﬁer Activation via Sympathetic Neurons(A) Blood ﬂow in the dorsal vessel of the ﬁfth lumbar cord in wild-type C57BL/6 with or without tail suspension (TS) in the presence or absence of soleus muscle
electrostimulation (ES).
(B) CCL20 mRNA expression in vessels from wild-type C57BL/6 mice with or without atenolol treatment.
(C) NFkB activation monitored by luciferase activity in vessels from C57BL/6 mice transduced with a NFkB reporter with or without atenolol treatment.
(D) Pathogenic CD4+ T cells isolated from EAE mice were intravenously transferred into wild-type C57BL/6 mice. Relative accumulation of pathogenic CD4+
T cells in the ﬁfth lumbar cord of wild-type mice with or without atenolol treatment (days 0–5 or 8–13 after the pathogenic CD4+ T cell transfer).
(E and F) Clinical EAE scores (n = 5 for each group).
(G) Top left: Histological analysis using serial frozen sections stained with anti-tyrosine hydroxylase antibody (TH) or anti-cFos antibody (cFos). Sympathetic ﬁrst
and ﬁfth lumber cords ganglia are shown. Green, TH and cFos, respectively, in the sympathetic ganglion beside the ﬁfth lumber cord; blue, nuclei. Scale bars
represent 50 mm. Representative data are shown. Top right: Histological analysis after double staining with chicken anti-tyrosine hydroxylase antibody (TH, red)or rabbit anti-cFos antibody (cFos, green). Blue, nuclei. Scale bars represent 25 mm. Bottom: Quantiﬁcation of histological analysis based serial frozen sections(top left) (n = 300 cells for each group).
(G–I) IL-6 ampliﬁer activation was enhanced in the presence of norepinephrine. BC1 cells stimulated with human IL-6 plus soluble IL-6 receptor and/or IL-17A for
48 hr in the absence or presence of norepinephrine (G, 10–300 mM; H, 100 mM). Culture supernatants were collected and assessed using an ELISA speciﬁc formouse IL-6 (H) and CCL20 (I). IL-6 and CCL20 concentrations increased in the presence of norepinephrine in each condition.
(J) Schematic ﬁgure of neural activation-induced pathogenic T cell accumulation in the CNS. Neural activation-mediated IL-6 ampliﬁer enhancement in endothelial cells of dorsal blood vessels in the ﬁfth lumbar cord induces CCL20 followed by the accumulation of pathogenic Th17 cells, a critical event for thedevelopment of EAE.
Error bars represent the mean ± SEM, *p < 0.05, **p < 0.01, ***p < 0.005 (A–F). Error bars represent the mean ± SD, *p < 0.05, **p < 0.01, ***p < 0.005 (G–I). See also
Figure S4.

prior to chronic activation of the IL-6 ampliﬁer and subsequentchronic inﬂammation in the affected tissue: (1) activation of
CD4+ T cells regardless of the antigen-speciﬁcity, (2) triggeringfactors for local accumulation of CD4+ T cells, (3) transient activation of the IL-6 ampliﬁer in local tissues by cytokines from theCD4+ T cells, and (4) increased sensitivity to the cytokines inthese local tissues (Murakami and Hirano, 2011). Furthermore,
it is likely that each step interacts with the others, the degreeof which varies with the pathogenesis of the disease. Our current

results lead us to propose that neural activation may contributeto steps 2 or 4.
Sensory neural activation of the soleus muscles activatedsympathetic neurons. This was followed by CCL20 expression
via IL-6 ampliﬁer activation. That sympathetic nerves are
involved is consistent with other reports in which EAE wasinduced by systemic immunization in the presence or absenceof norepinephrine antagonists (Brosnan et al., 1985; Dimitrijevic
et al., 2007). We suspect increased blood ﬂow speed in the ﬁfth
Cell 148, 447–457, February 3, 2012 ª2012 Elsevier Inc. 453

lumbar cord is a direct or indirect effect of the exercise pressor
reﬂex, which is an example of sensory neuron stimulation that
leads to sympathetic neuron activation (Kaufman and Hayes,
2002; Smith et al., 2006). Activation of this reﬂex changes bloodpressure or blood ﬂow via alterations in the blood vessel, particularly those in muscle (Kaufman and Hayes, 2002; Smith et al.,2006). However, it is still an open question on how sensory
neurons via the soleus connect to sympathetic neurons andthe sympathetic ganglion. We suspect this connection occurs
at the dorsal root ganglia of the ﬁfth lumbar cord, since eventhough sympathetic axons exist in normal dorsal root ganglia
they increase in number following a sensory neuron injury (Xieet al., 2010).
Unlike mice, patients suffering from MS do not always display
symptoms in their lower body. Instead, MS symptoms are generally isolated in the upper body, particularly the thoracic cord andcerebrum. This difference might be due to different sites for thepathogenic CD4+ T cell accumulation that follows local inﬂammation. Because different regional neural activation–mediated
CCL20 expression were induced by stimulating several muscles
such as the soleus muscles, the quadriceps, and epitrochlearis/
triceps brachii (Figure 4A and Figures S3C and S3D), we believethat excess sensory stimulations in other regions like the hands,
ﬁngers, and optic nerves, and even less visceral locations likechronic mental stress may affect MS symptoms by promoting
excess neural activations and IL-6 ampliﬁer activation in vesselsof the thoracic cord and/or cerebrum. Such a theory could also
help explain the effects of acupuncture, which involves directly
stimulating peripheral nerves to change body homeostasis(Longhurst, 2010). We therefore hypothesize that acupuncture
effects may arise by changing the blood vessel status of thetarget organ.
In the rat transfer models, pathogenic T cells migrate to thespleen prior to entering the CNS (Matsumoto et al., 1988; Panitch, 1980). That we found pathogenic CD4+ T cells negligibly
accumulate in the ﬁfth lumbar cord by day 4 is consistent withthis. However, we also found that the ﬁfth lumbar cord shows
chemokine expression even in the steady state (Figures 3A
and 3B). Such an observation suggests that the transferred pathogenic CD4+ T cells need not wait until day 4, as they can instead
enter the spinal cord using this axis directly. One reason we did
not ﬁnd earlier T cell accumulation may be that the pathogenic
CD4+ T cells express relatively low levels of CCR6 after in vitro
culture. That would explain why CCR6 expression levels
increased following pathogenic CD4+ T cell division in the
superﬁcial lymph nodes, aortal lymph nodes, mescenteric lymph
nodes, and spleens on day 4 after the transfer (Figure S5) andthat deﬁcient CCR6 in pathogenic CD4+ T cells suppressed early
T cell entry into the CNS (Figure S2A). Additionally, CCL20 or
adrenergic inhibition reduced EAE development, particularly
for the ﬁrst 5 days after pathogenic CD4+ T cell transfer (Figures
3D and 5E), while anti-CCL20 or an adrenergic receptor inhibitor
from day 8 after the pathogenic CD4+ T cell transfer upon indications of slight tail polarization (Figures 3E and 5F). Theses results
suggest that both blockades have partial therapeutic effects for
multiple sclerosis.
It has been observed that the expression of chemokines like
CCL20 in the dorsal vessels of the ﬁfth lumbar cord in response
454 Cell 148, 447–457, February 3, 2012 ª2012 Elsevier Inc.

to soleus muscle stimulation can facilitate immunity against
pathogenic invasions or latent infectious states in the CNS(McGavern and Kang, 2011; Salinas et al., 2010). Consistent
with this, we found the expression of all observed chemokines
increased there. This increase could also signal the recruitmentof other cell populations, including nonimmune cells as well asother neural precursor cells including microglia ones. Therefore,
regional neural activations may maintain homeostasis of the
CNS environment by regulating the entry of various cell types
from the blood. These phenomena then may suggest the importance of the IL-6 ampliﬁer, a synergistic signal between NFkB
and STAT3, for the accumulation of various cell types including
pathogenic T cells by creating a gateway to CNS that depends
on various sets of chemokine expressions.
To summarize, we describe an entry site at the dorsal bloodvessels of the ﬁfth lumbar cord for blood cells including pathogenic CD4+ T cells into the CNS. Mechanistic experiments
demonstrated that CCL20, which we found to be positively regulated by sensory nerve activation via antigravity soleus muscles,
plays a role in this accumulation, and that its expression risks thedevelopment of autoimmune diseases when pathogenic CD4+
T cells are present in the blood stream. More importantly,
sensory nerve activation enhances CCL20 expression in thesedorsal blood vessels via the activation of sympathetic neurons,
meaning neural activation can be transformed into an inﬂammatory signal that leads to autoimmune disease. The location of thisneuroinﬂammatory transformation may prove a valuable therapeutic target for various neuroimmune diseases including autoimmune and inﬂammatory diseases and is therefore worthy of
more study.
EXPERIMENTAL PROCEDURES
Mouse Strains
C57BL/6 mice were purchased from Japan SLC. IL-6-deﬁcient mice were
provided by Dr. M. Kopf (Max Planck Institute of Immunobiology, Germany)and backcrossed with C57BL/6 mice more than ten times. Type I collagenCre mice were provided by Dr. G. Karsenty (Baylor College of Medicine,
Houston, TX) and crossed with STAT3ﬂox/ﬂox mice (provided by Dr. S. Akira,Osaka University, Japan) (Takeda et al., 1998) and gp130ﬂox/ﬂox mice (Betzet al., 1998). Tie2-Cre mice were provided by Dr. H. Kawamoto (RIKEN
Research Center for Allergy and Immunology, Yokohama, Japan). IL-17A-deﬁcient (Iwakura and Ishigame, 2006), NFkB reporter (Sadikot and Blackwell,2008), CCR6-deﬁcient (Varona et al., 2001), and IFNg-deﬁcient (Ueda et al.,2006) mice in a C57BL/6 background were also used. All mice were maintained under speciﬁc pathogen-free conditions according to the protocolsof the Osaka University Medical School. All animal experiments were
performed following the guidelines of the Institutional Animal Care and Use
Committees of the Graduate School of Frontier Bioscience and Graduate
School of Medicine, Osaka University.
Passive Transfer of Pathogenic CD4+ T Cells to Induce EAE
EAE induction was performed as described previously (Ogura et al., 2008). In
brief, C57BL/6 mice and IL-17A-deﬁcient, IFNg-deﬁcient mice were injectedwith a MOG (35–55) peptide (synthesized by Sigma) in complete Freund’s
adjuvant (Sigma-Aldrich, Tokyo) at the base of the tail on day 0 followed byintravenous injection of pertussis toxin (Sigma-Aldrich, Tokyo) on days 0, 2,
and 7. On day 10, CD4+ T cells from the resulting mice were sorted with
anti-CD4 microbeads (Milteny, Tokyo). The resulting CD4+ T cell-enriched
population (4 3 106 cells) was cocultured with rIL-23 (10 ng/ml; R&D, Minneapolis) in the presence of MOG peptide-pulsed bone marrow-derived dendritic
cells (5 3 105) for 2 days. Cells (1.5 3 107) were then injected intravenously into

wild-type or mutant mice. Clinical scores were measured as described previously (Ogura et al., 2008).

GenElute Mammalian Total RNA kit (Sigma-Aldrich, Tokyo) and DNase I(Sigma-Aldrich, Tokyo).

Mononuclear Cell Isolation from Spinal Cords
Mononuclear cells from spinal cords were isolated after cardiac perfusion with
PBS as described previously (Sawa et al., 2009).

Real-Time PCRs
A GeneAmp 5700 sequence detection system (ABI, Warrington) and SYBER
green PCR Master Mix (Sigma-Aldrich, Tokyo) were used to quantify CCL20mRNA and HPRT mRNA levels. The PCR primer pairs used for real-time
PCRs were as follows: mouse HPRT primers, 50 -GATTAGCGATGATGAACCA
GGTT-30 and 50 -CCTCCCATCTCCTTCATGACA-30 ; mouse CCL20 primers,
50 -ACAGTGTGGGAAGCAAGTCC-30 and 50 -CCGTGAACTCCTTTGACCAT-30 ;mouse CCL2 primers, 50 - CCGGCTGGAGCATCCACGTGT-30 and 50 - TGGGG
TCAGCACAGACCTCTCTCT-30 ; mouse CXCL1 primers, 50 - CACAGGGGCGC
CTATCGCCAA-30 and 50 - CAAGGCAAGCCTCGCGACCAT-30 ; mouse CXCL2
primers, 50 - ACCCCACTGCGCCCAGACAGAA-30 and 50 - AGCAGCCCAGGC
TCCTCCTTTCC-30 ; mouse CX3CL1 primers, 50 -ACGAAATGCGAAATCATG
TGC-30 and 50 -CTGTGTCGTCTCCAGGACAA-30 ; mouse CCL3 primers, 50 -TG
TACCATGACACTCTGCAAC-30 and 50 -CAACGATGAATTGGCGTGGAA-30 ;mouse CCL9 primers, 50 -CCCTCTCCTTCCTCATTCTTACA-30 and 50 -AGTCTTGAAAGCCCATGTGAAA-30 ; mouse CCL21 primers, 50 -GGGTCAGGACTGC
TGCCTTA-30 and 50 -CGGGATGGGACAGCCTAAA-30 ; mouse CCL24 primers,
50 -CCAAGAAGGGCCATAAGATCTG-30 and 50 -GCCCCTTTAGAAGGCTGG
TT-30 ; mouse CXCL9 primers, 50 -TCCTTTTGGGCATCATCTTCC-30 and 50 TTTGTAGTGGATCGTGCCTCG-30 ; mouse CXCL11 primers, 50 -GGCTTCCTTATGTTCAAACAGGG-30 and 50 -GCCGTTACTCGGGTAAATTACA-30 ; andmouse c-fos primers, 50 -CGGGTTTCAACGCCGACTA-30 and 50 -TTGGCACTAGAGACGGACAGA-30 . The conditions for real-time PCR were 40 cycles at
94 C for 15 s followed by 40 cycles at 60 C for 60 s. The relative mRNA expression levels were normalized to HPRT mRNA levels.

Frozen Section Preparations and Histological Analysis
Spines were harvested and ﬁxed in 4% paraformaldehyde for 12 hr and thendisplaced with 10% sucrose solution for 12 hr, 20% sucrose solution for
12 hr, and ﬁnally 30% sucrose solution for 12 hr (total 48 hr). The resulting
spines were embedded in SCEM compound (SECTION-LAB, Japan). The
cut surface was covered with an adhesive ﬁlm (Cryoﬁlm type IIC9,SECTION-LAB, Japan) and frozen sections (10 mm) were prepared with amacrotome (CM3600XP Leica Microsystems, Germany) according to amethod described previously (Kawamoto, 2003). The resulting sections were
stained with hematoxylin/eosin and analyzed with a BZ-9000 microscope(KEYENCE).
Intracellular Cytokine Staining
The number of Th17 cells in vivo was determined as previously described(Nishihara et al., 2007). In brief, T cells from spinal cords were stimulatedwith MOG peptide presented on LPS-stimulated bone marrow-derived
dendritic cells in the presence of GolgiPlug (BD Biosciences, Tokyo) for 6 hr.
Intracellular IL-17A and IFN-g were labeled with anti-IL-17A and anti-IFN-g
antibodies after surface staining, ﬁxation, and permeabilization.
Antibodies and ReagentsThe following antibodies were used for ﬂow cytometry analysis: APC-conjugated anti-IFN-g (eBioscience, San Diego) and control IgG1k (eBioscience,
San Diego); FITC-conjugated anti-CD8 (eBioscience, San Diego), anti-CD19(eBioscience, San Diego), anti-B220 (BD PharMingen, Tokyo), anti-NK1.1(eBioscience, San Diego), and anti-I-A/I-E (BioLegend, Tokyo); PE-conjugatedanti-IL-17A (eBioscience, San Diego), control IgG2a (eBioscience, San Diego),
and anti-I-A/I-E (BioLegend, Tokyo); PE-Cy5-conjugated anti-CD4 (BioLegend, Tokyo); PE-Cy7-conjugated anti-CD4 (BioLegend, Tokyo); PaciﬁcBlue-conjugated anti-CD44 (eBioscience, San Diego) and Streptavidin(Invitrogen, Tokyo); and Biotin-conjugated anti-CD90.2 (eBioscience, San
Diego). The following antibodies were used for immunohistochemistry: antiphospho-STAT3 (Tyr705, D3A7) (Cell signaling), anti-tyrosine hydroxylase(Abcam, Tokyo), anti-cFos (Sigma, Tokyo), and control rabbit IgG (DA1E)(Cell Signaling, Tokyo); and Alexa Fluor 488 goat anti-rabbit IgG (H + L)
and Alexa 647 goat anti-chicken IgG (Invitrogen, Tokyo). The following
antibodies were used for in vivo neutralization: puriﬁed anti-mouse CCL20
mAb (R&D, Minneapolis). Atenolol and prazosin were purchased from
Sigma-Aldrich, Tokyo. The VECTASTAIN Elite ABC Rabbit IgG Kit and theDAB Peroxidase Substrate kit were purchased from Vector Laboratories,
Burlingame.
ELISA
IL-6 and IL-17A levels in serum or cell cultures supernatant were determinedwith ELISA kits (BD PharMingen, Tokyo; eBioscience, San Diego; or R&D, Minneapolis). CCL20 levels in cell cultures supernatant were determined using
ELISA kits (R&D, Minneapolis).

Hindlimb Suspension
Hindlimb suspension was performed as described previously (Kawano et al.,2007; Ohira et al., 2002). In brief, strips of sticky tape (about 5 mm wide and3 cm long) with good cushion were placed longitudinally on the dorsal andventral sides of the midtail of the hindlimb-unloaded mouse. These strips
were further surrounded cross-sectionally by another strip. Strips wereadhesed loosely so as to keep the blood ﬂow intact. A string was inserted
through the gap between the tail and tape and fastened to the roof of thecage at a height that allowed the forelimbs to support the body weight andprevent the hindlimbs from touching any part of the cage. A mouse could reachfood and water freely by using its forelimbs. The attachment to the tail waschanged each week so as not to inhibit tail growth and blood ﬂow.
Electro Stimulations
In some experiments, hindlimb suspended mice were electrostimulated for
12 hr through the soleus muscle with electrical stimulators (Nippon Koden,Tokyo and Unique Medical, Osaka) at 0.1 mA and 1–8 ms random intervals.
Electrostimulations were done for 1 day when investigating pathogenic CD4+
T cell migration and two days when measuring CCL20 mRNA expression.
Anti-CCL20 Antibody, Atenolol Treatment, and Prazosin Treatment
Anti-CCL20 antibody (200 mg/mouse) or atenolol (500 mg/mouse) was interperitoneally injected into C57BL/6 mice (6–8 weeks old) for 5 days in which pathogenic CD4+ T cells were transferred. Atenolol (500 mg/mouse) or prazosin(500 mg/mouse) was injected into wild-type C57BL/6 mice (6–8 weeks old)for 5 days without the pathogenic CD4+ T cell transfer.

Flow Cytometry
For cell surface labeling, 106 cells were incubated with ﬂuorescence-conjugated antibodies for 30 min on ice. The cells were then analyzed with cyan
ﬂow cytometers (Beckman Coulter, Tokyo). The collected data were analyzed
with Summit software (Beckman Coulter, Tokyo) and/or Flowjo software (TreeStar, Ashland).

Luciferase Assay
Blood vessels from the dorsal side of the ﬁfth lumbar cord or ﬁrst lumbar cord
were harvested. Proteins were collected using a Luciferase Reporter Assay kit(Promega, Tokyo) and total protein amount was adjusted. Luciferase activities
were measured by a Luciferase detection device (Promega, Tokyo).

Laser Microdissection
Approximately 100 frozen sections (15 mm) were ﬁxed with acetic acid/ethyl
alcohol (1:19) for 15 min followed by PBS washing for 10 min. Tissues aroundthe dorsal blood vessels were collected by a laser microdissection device,
DM6000B (Leica, Tokyo), and prepared for total RNA measurements by the

Blood Flow Analysis
Blood ﬂow volume was measured in blood vessels from the dorsal side of theﬁfth lumbar cord in C57BL/6 mice (6–8 weeks old) in the presence or absenceof tail-suspensions and/or electrostimulations with Omegazone OZ-1 (Omega
wave, Tokyo).

Cell 148, 447–457, February 3, 2012 ª2012 Elsevier Inc. 455

Statistical Analysis
Student’s t tests (two-tailed) and ANOVA tests were used for the statisticalanalysis of differences between two groups and that of differences between
more than two groups, respectively.

SUPPLEMENTAL INFORMATION

Hirano, T. (2010). Interleukin 6 in autoimmune and inﬂammatory diseases:a personal memoir. Proc. Jpn. Acad., Ser. B, Phys. Biol. Sci. 86, 717–730.
Iwakura, Y., and Ishigame, H. (2006). The IL-23/IL-17 axis in inﬂammation.
J. Clin. Invest. 116, 1218–1222.
Kaufman, M.P., and Hayes, S.G. (2002). The exercise pressor reﬂex. Clin.
Auton. Res. 12, 429–439.

Supplemental Information includes ﬁve ﬁgures and can be found with thisarticle online at doi:10.1016/j.cell.2012.01.022.

Kawamoto, T. (2003). Use of a new adhesive ﬁlm for the preparation of multipurpose fresh-frozen sections from hard tissues, whole-animals, insects andplants. Arch. Histol. Cytol. 66, 123–143.

ACKNOWLEDGMENTS

Kawano, F., Nomura, T., Ishihara, A., Nonaka, I., and Ohira, Y. (2002). Afferent
input-associated reduction of muscle activity in microgravity environment.
Neuroscience 114, 1133–1138.

We thank Dr. M. Kopf (Max-Planck-Institute of Immunobiology, Germany) for
the IL-6-deﬁcient mice, Dr. G. Karsenty (Baylor College of Medicine, Houston,TX) for the type I collagen-Cre mice, and Dr. S. Akira (Osaka University, Osaka,
Japan) for the STAT3ﬂox/ﬂox mice. We appreciate the excellent technicalassistances provided by Dr. J-J. Jiang, Ms. A. Okuyama, and Ms. N. Kumai,
and thank Ms. R. Masuda for her excellent secretarial work. We thankDr. M. Yagi and Dr. P. Karagiannis (Osaka University) for carefully reading
the manuscript. We are grateful to Dr. Fujio Murakami (Osaka University) for
important discussions and advice for how to do the neural experiments andthank Dr. M. Miyasaka (Osaka University) for important discussions about
blood vessels for lymphocyte migration. We are also grateful to Dr. Y. Nakatsuji
and Dr. M. Kinoshita (Osaka University) for important discussions about MS
patients and thank Dr. Y. Okuyama and Dr. H. Ogura (Osaka University)
for important discussions and carefully reading this manuscript. We thankDr. Y. Oke (Osaka University) for important discussions about the tail suspension experiment. We thank Jihye Lee for her help in preparing the Graphical
Abstract. This work was supported by KAKENHI (Y.A., M.H., T.H., andM.M.), the JST-CREST program (T.H. and M.M.), and the Osaka Foundation
for the Promotion of Clinical Immunology (M.M.).
Received: June 27, 2011
Revised: September 21, 2011
Accepted: January 15, 2012
Published: February 2, 2012
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