about the factors that regulate the architecture of neural fibers in lymphoid tissues. In the present study, we find that experimentally
imposed social stress can enhance the density of catecholaminergic neural fibers within axillary lymph nodes from adult rhesus ma-
caques. This effect is linked to increased transcription of the key sympathetic neurotrophin nerve growth factor and occurs predomi-
nately in extrafollicular regions of the paracortex that contain T-lymphocytes and macrophages. Functional consequences of stress-
induced increases in innervation density include reduced type I interferon response to viral infection and increased replication of the
simian immunodeficiency virus. These data reveal a surprising degree of behaviorally induced plasticity in the structure of lymphoid
Neuroanatomical studies have shown that all primary and sec-
ondary lymphoid organs receive direct innervation from the
sympathetic division of the autonomic nervous system (Madden
sympathetic nervous system (SNS) enter lymphoid organs in as-
sociation with the vasculature and subsequently radiate into pa-
presenting cells (Bellinger et al., 2001). Structural varicosities
molar concentrations of the catecholamine neurotransmitter
norepinephrine (NE) in response to stress and other stimuli
(Shimizu et al., 1994; Madden et al., 1995a). NE signals leuko-
cytes via cellular ?-adrenergic receptors, which activate the
cAMP/PKA (protein kinase A) signaling cascade to regulate a
variety of immune processes including leukocyte activation, cy-
tokine production, and cell trafficking (Kammer, 1988; Ottaway
Sanders and Straub, 2002). Pharmacologic blockade of SNS ac-
tivity can alter in vivo immune responses to model antigens and
pathogen challenges (Madden et al., 1994; Kohm and Sanders,
1999). Effects of physiological variations in SNS activity are less
tissue is believed to constitute one major pathway by which be-
havioral processes can affect immune function (Felten et al.,
1987; Madden et al., 1994, 1995b; Bellinger et al., 2001).
Most research on lymphoid innervation has presumed that
acute changes in neural activity constitute the primary mecha-
have documented structural changes in the pattern of lymphoid
innervation with aging and inflammation (Madden et al., 1997;
Kelley et al., 2003; Sloan et al., 2006). Those results suggest an
alternative model in which long-term changes in innervation
density might regulate lymph node biology independently of
short-term variations in neural activity. No studies have docu-
mented a behavioral influence on the structure of lymphoid in-
nervation, but one recent analysis found that physical (auditory)
stress could alter peptidergic innervation of the skin within a
surprisingly short period of time (?48 h) (Peters et al., 2005).
social stress might alter the density of catecholaminergic inner-
vation in secondary lymphoid tissue and, if so, what molecular
mechanisms might contribute to those dynamics. Given the key
(UCLA), the UCLA AIDS Institute, and the James B. Pendelton Charitable Trust. We thank Suzanne Stevens and
tory at California National Primate Research Center (CNPRC) for SIVmac251stock and SIV culture protocols; Harry
TheJournalofNeuroscience,August15,2007 • 27(33):8857–8865 • 8857
role that secondary lymphoid organs play in initiating immune
responses, we also sought to determine how stress-induced
changes in lymphoid innervation might impact the immune re-
sponse to a viral infection.
Social stress. Thirty-six male rhesus macaques aged 7–10 years were relo-
cated from outdoor social groups to individual housing where they lived
week for 100 min per day over 39 weeks, in cages measuring 1.8 ? 3.1 ?
2.2 m. Each animal was randomly assigned to either the stable social
condition (the same three animals met daily) or the unstable social con-
dition (two to four animals per group, with number and identity of
partners changing on a daily basis) (Capitanio et al., 1998). Unstable
hierarchies, resulting in behavioral and neuroendocrine indications of
stress (e.g., threat behavior and altered cortisol response) (Capitanio et
al., 1998). Twenty-four animals were infected with simian immunodefi-
ciency virus (SIV) during the second week of the social manipulation
serving as a challenge model to evaluate functional changes in immune
SIV infection. Randomly assigned animals were inoculated with 102.66
became infected as documented by reverse transcription (RT)-PCR de-
tection of plasma viral RNA (Sloan et al., 2006) and development of
SIV-specific antibodies (data not shown). Progression of SIV pathogen-
esis was assessed 36 weeks after inoculation by branched DNA assay of
plasma SIV viral load (Chiron, Emeryville, CA) and flow cytometric
quantification of peripheral blood CD4? T-lymphocyte levels (CD3?/
CD4? staining in lymphocytes defined by forward- versus side-scatter
gating on a FACSCalibur cytometer) (BD Biosciences, San Jose, CA). As
key sites of SIV replication in vivo (Fox et al., 1991), lymph nodes were
2 months before onset of clinical disease, except for one animal with SIV
situ hybridization of SIV replication sites within lymph node sections
were performed as previously described (Sloan et al., 2006).
In vitro analyses of SIV replication used adult male rhesus macaque
peripheral blood mononuclear cells (PBMCs) that were infected with
SIVmac251, stimulated with phytohemagglutinin (PHA) (Sigma, St.
Louis, MO), and subsequently cultured in the presence of 0.5, 5, or 50
ical, Swampscott, MA) or saline control. Viral replication at 4 and 6 d
after infection was quantified by real-time RT-PCR (see below for de-
scription) for gag and env mRNA in total cellular RNA using previously
published primer sequences (Canto-Nogues et al., 2001). Results were
normalized to expression of cellular mRNA for ?-actin (ACTB; primer
Lymph node innervation. Four axillary lymph nodes were biopsied
from each of 20 macaques after 39 weeks of stable versus unstable social
conditions (including 11 SIV? animals that remained alive 37 weeks
after virus inoculation). One to three lymph nodes from each of seven
uninfected macaques (14 nodes) and nine SIV-infected macaques (12
al., 2006). NE is believed to be released from structural varicosities situ-
ated periodically along the length of catecholaminergic fibers (Bellinger
et al., 2001). To estimate the density of functional innervation, we used
were sectioned in random orientation to provide a random two-
dimensional sample through three-dimensional space, (2) a grid of
frame to allow counting of catecholaminergic varicosities across the en-
applied to estimate the three-dimensional density of catecholaminergic
varicosities from two-dimensional varicosity frequency data. By satisfy-
ing each of these criteria, this approach gives an unbiased estimate of
relative three-dimensional innervation density from a two-dimensional
histological profile (Mouton, 2002). Tissue shrinkage may occur during
this dynamic would not differentially affect tissues from differing exper-
imental conditions. Thus, tissue shrinkage would not bias measures of
relative innervation density used in this study. Innervation density was
estimated at the level of the whole lymph node, and within functionally
distinct anatomical regions (e.g., paracortex, cortex, and medulla). Ana-
tomical subregions were mapped on adjacent hematoxylin and eosin
to localization of catecholaminergic fibers. Tissue sections were gridded
into 250 ?m2quadrates and each quadrate was assigned to a distinct
anatomical subregion based on H&E staining. Quadrates that spanned
two or more subregions were divided into eighths, and each eighth was
allocated to a specific subregion based on H&E anatomical information.
that density estimates were unbiased within distinct subregions and sta-
to information on social conditions.
isolated from 3 mg of lymph node tissue or from 106rhesus PBMCs
treated with 10?6M NE (Sigma) for 4, 8, 12, or 24 h. Total RNA was
95°C and 60 s of annealing and extension at 60°C. Triplicate determina-
tions were quantified by threshold cycle analysis of SYBR Green fluores-
cence intensity using iCycler software (Bio-Rad), and normalization to
parallel-amplified GAPDH and ACTB mRNA (Collado-Hidalgo et al.,
2006). Primer sequences and gene regions that were amplified include the
following: NGF, GenBank XM_001100522 (bp 434–601): forward (F), 5?-
GTTTTACCAAGGGAGCAGCTTTC-3?, and reverse (R), 5?-TAGTC-
CAGTGGGCTTGGGGGA-3?; IFNA (conserved 368 bp sequence of 23
IFNA family members): F, 5?-AGAATCTCTCCTTTCTCCTG-3?, and R,
5?-TCTGACAACCTCCCAGGCACA-3?; IFNB, GenBank EF064725 (bp
2263–2358): F, 5?-AAGGAGGACGCCGCATT-3?, and R, 5?-AAT-
AGTCTCATTCCAGCCAGTGC-3?; IFNG, GenBank A4376145 (bp
346–423): F, 5?-GAAAAGCTGACCAATTATTCGGTAA-3?, and R, 5?-
AGCCATCACTTGGATGAGTTCA-3?; LIF, GenBank BC093733 (bp
222–297): F, 5?-TGCCAATGCCCTCTTTATTC-3?, and R, 5?-GCCA-
CATAGCTTGTCCAGGT-3?; IFI27, GenBank BC015492 (bp 456–553): F,
5?-TCACTGGGAGCAACTGGACTC-3?, and R, 5?-GGGAGCTAGTA-
GAACCTCGCAAT-3?; GAPDH, GenBank BC083511 (bp 58–226): F, 5?-
TGGGATTC-3?; ACTB, GenBank NM_001101 (bp 549–573): F, 5?-
TCACCCACACTGTGCCCATCTACGA-3?, and R, 5?-CAGCGGAA-
NGFR and NTRK1 mRNA were amplified using TaqMan Gene Ex-
pression Assays (Hs00176787_m1 and Hs00609976_m1, respectively)
(Applied Biosystems, Foster City, CA) using Quantitect Probe RT-PCR
kit (Qiagen) and the cycling conditions described above.
Detection of NGF protein. Lymph nodes from healthy uninfected ani-
proteins were separated by SDS-PAGE on a 15% polyacrylamide gel and
transferred to nitrocellulose membrane (Hybond; Amersham Bio-
NGF polyclonal antibody (H-20; 1 ?g/ml; Santa Cruz Biotechnology,
ish peroxidase-conjugated secondary antibody and detection with ECL
Plus (Amersham Biosciences). Similar results were obtained by immu-
noprecipitation with rabbit anti-NGF serum (Cedarlane, Burlington,
NC) in 50 mM Tris-HCl, pH 8.0, 0.5 mM EDTA, 125 mM NaCl, 0.5%
8858 • J.Neurosci.,August15,2007 • 27(33):8857–8865 Sloanetal.•StressEnhancesLymphoidInnervation
NP-40, 10% glycerol, 0.1% SDS, complete Mini EDTA-free protease
inhibitors (Roche Diagnostics, Indianapolis, IN) (data not shown).
Activation of TrkA receptors in situ. Active TrkA was localized by im-
munostaining of lymph node tissue sections with polyclonal rabbit anti-
body to phosphorylated TrkA (10 ?g/ml; Upstate, Waltham, MA) using
the method detailed below (see below, Leukocyte distribution in situ).
Adjacent sections were stained with glyoxylic acid chemofluorescence
Leukocyte distribution in situ. Frozen lymph node sections were post-
fixed in 4% paraformaldehyde and permeabilized with 0.2% Triton
PBS. Sections were incubated with primary antibodies or appropriate
control antibodies in blocking solution [0.1 M Tris, pH 7.4, 0.15 M NaCl,
2% normal goat serum, 2% normal horse serum (Hyclone, Logan, UT)]
as described below) at room temperature for 1 h. Nuclei were counter-
coverslipped with fluorescent mounting medium (Dako North Amer-
ica). Antibodies used were CD3 rabbit polyclonal antibody (6.0 ?g/ml;
DakoCytomation) followed by Alexa 488-conjugated goat anti-rabbit
F(ab?)2fragments (Invitrogen, Carlsbad, CA), CD20 clone L26 (0.45
?g/ml; Zymed, South San Francisco, CA) followed by Alexa 488-
phage marker HAM56 (0.7 ?g/ml; Dako North America) or follicular
dendritic cell marker CAN.42 (1.4 ?g/ml; Dako North America) fol-
lowed by biotinylated goat anti-mouse IgM (2 ?g/ml; Vector Laborato-
ries, Burlingame, CA) and tertiary Alexa 568-conjugated streptavidin
(Invitrogen). All Alexa conjugates were used at 2 ?g/ml. For analysis of
immunofluorescence coexpression, each field was imaged at 200? mag-
nification by multichannel fluorescence microscopy for antibody stain-
ing of cell surface markers (CD3/HAM46 or CD20/FDC) and Hoechst
ware (Zeiss, Thornwood, NY). Relative frequency of T-lymphocytes was
determined by averaging the pixel density of CD3? staining in three
microscope fields selected randomly from paracortical regions in ran-
domly selected lymph nodes. Parallel analyses assessed the relative fre-
quency of macrophages (HAM56?), B-lymphocytes (CD20?), and fol-
licular dendritic cells (CAN.42?). Total cellular density was assayed by
cortex, cortex, and medulla in H&E-stained sections photographed un-
der brightfield. Total lymph node size and relative size of anatomical
rates in each lymph node (including fractional regions) (see above,
Lymph node innervation) according to the Delesse area–volume princi-
ple (Mouton, 2002).
Statistical analysis. Spatial correlation analyses were performed as de-
(Hoyle and Kenny, 1999) conducted in the context of generalized linear
Analyses conducted at the level of individual tissue quadrates controlled
ing each quadrate as a random observation nested within tissue sample,
and each tissue sample as a random effect nested within experimental
of active viral replication sites in tissue quadrate i from animal a ( yai) as
that quadrate (?ai), (2) any generalized effect of social condition not
mediated by proximity to sympathetic varicosities (?c? 1 for all quad-
quadrates in tissue from animals socialized under stable conditions), (3)
any differential functional effect of varicosities under stable versus un-
stable conditions (? ? multiplicative product of ? and ?), and (4) indi-
dent random residual) (Miller, 1986).
Animals were randomly assigned to 39 weeks of daily social in-
teraction in either stable conditions (same animals met every
day) or unstable conditions (group composition changed daily).
Unstable social conditions have been shown to induce neuroen-
docrine and behavioral indications of stress in previous studies
(Capitanio et al., 1998), and monitoring of agonistic social be-
havior confirmed those effects in the present sample. Relative to
animals socialized in stable groups, those subjected to unstable
social conditions showed a more than eightfold increase in the
frequency of threat behaviors (mean ? SE, 0.402 ? 0.130 per
animal per week vs 0.047 ? 0.014 for controls; p ? 0.041), phys-
ical aggression (0.034 ? 0.011 vs 0.000 ? 0.000; p ? 0.028), and
chases (0.028 ? 0.015 vs 0.000 ? 0.000; p ? 0.103).
1976; Guidry, 1999; Sloan et al., 2006) to map the distribution of
14 axillary lymph nodes sampled from adult male rhesus ma-
to unstable social conditions, stereological analysis revealed an
80% increase in the density of parenchymal catecholaminergic
ble conditions (Fig. 1c–e). Across 4582 individual 250 ?m2
lymph node tissue quadrates analyzed, the absolute density of
parenchymal catecholaminergic varicosities increased from an
average 1.32 ? 0.13 per quadrate under stable social conditions
to 2.38 ? 0.25 under unstable social conditions (Fig. 1e) ( p ?
renchyma, and the density of perivascular varicosities did not
change significantly ( p ? 0.332) (Fig. 1e).
the lymph node showed that social stress enhanced lymphoid
innervation specifically within the paracortex, a subregion pop-
Catecholaminergic varicosities were rarely found in the vicinity
the overall frequencies of T-lymphocytes, B-lymphocytes, mac-
rophages, or follicular dendritic cells within lymph node tissue
(all differences, p ? 0.10). Social conditions also had no detect-
able effect on the relative size of lymph nodes or their total cellu-
lar density (both ?10% difference; both p ? 0.267). All lymph
nodes analyzed in this study showed fully structured medulla,
paracortex, and cortex, with no effect of social conditions on the
relative size of these subregions within the total lymph node area
(all differences, p ? 0.643). Thus, social stress appears to focally
significantly impacting the gross morphology or cellular compo-
sition of the organ.
sion of genes known to modulate neural growth and mainte-
nance. Tissues from socially stressed animals showed signifi-
cantly higher concentrations of mRNA for NGF (mean 31%
increase over tissues from unstressed animals; p ? 0.011), which
plays a key role in the development of the SNS (Levi-Montalcini,
Sloanetal.•StressEnhancesLymphoidInnervation J.Neurosci.,August15,2007 • 27(33):8857–8865 • 8859
1987; Farinas, 1999). Parallel analyses of
two cytokines known to retract sympa-
leukemia inhibitory factor (LIF) (Guo et
al., 1997; Kim et al., 2002), showed no
stress-induced inhibition, but rather sig-
LIF mRNA and 225% increase in IFNG;
both p ? 0.01).
Consistent with the key role of NGF in
supporting peripheral sympathetic inner-
vation (Angeletti et al., 1971; Ruit et al.,
1990; Tsui-Pierchala et al., 2002), levels of
NGF mRNA showed a strong linear rela-
tionship to density of parenchymal cat-
However, tissue levels of NGF mRNA
showed no relationship to the density of
perivascular innervation (Fig. 3b). Neuro-
trophic activity is generally attributed to
al., 2000; Chao et al., 2006), although its
ral survival (Lakshmanan et al., 1989;
Chen et al., 1997; Fahnestock et al., 2004).
Western blot analysis identified both ma-
ture NGF and pro-NGF protein within
lymph node tissues (Fig. 3c). Consistent
stead et al., 1991; Klein et al., 1991), im-
phosphorylated TrkA (Fig. 3d) in cat-
echolaminergic fibers within the lymph
node parenchyma (Fig. 3e). Approxi-
mately 10% of catecholaminergic fibers
stained positive for phosphorylated TrkA.
To determine whether stress-induced
increases in NGF expression might be
quantitatively sufficient to account for ef-
fects of social conditions on parenchymal
innervation, we performed multivariate
statistical analyses of mediation (Good-
man, 1960; Hoyle and Kenny, 1999) (Fig.
3f). Variations in NGF gene expression
were sufficient to account for 86% of the
cial stress and parenchymal varicosity density (mediational path,
p ? 0.004; residual effect of stress, p ? 0.321) (Goodman, 1960;
Hoyle and Kenny, 1999). Similar analyses of IFNG and LIF indi-
cated that neither of those molecules was a plausible quantitative
ational path, p ? 0.109; residual effect of stress, p ? 0.038; LIF:
mediational path, p ? 0.154; residual effect of stress, p ? 0.073).
Statistical nonsignificance of the later results, and previous data in-
sympathetic nerve fibers (Guo et al., 1997; Kim et al., 2002), are
both consistent with the idea that these factors do not mediate
stress-induced sympathetic innervation of lymphoid tissues.
The present data are consistent with the hypothesis that up-
regulation of NGF gene expression is responsible for increased
is conceivable in which catecholamine signaling might causally
induce NGF transcription (e.g., via cAMP-responsive promoter
elements) (McCauslin et al., 2006). Using rhesus macaque PB-
node, we exposed either unstimulated or PHA-activated PBMCs
to NE concentrations comparable with those measured in sec-
ondary lymphoid organs and other solid tissues (Felten et al.,
1987; Lara and Belmar, 1991; Shimizu et al., 1994). NE did not
significantly alter NGF gene expression in either condition (see
data in supplemental Fig. 1, available at www.jneurosci.org as
supplemental material), suggesting that stress-induced NGF ex-
pression in lymphoid organs stems from other sources (Colan-
gelo et al., 2004). Together, these results underscore the key role
of NGF in structuring peripheral sympathetic innervation (Levi-
Montalcini, 1987; Carlson et al., 1995) and provide novel indica-
tions that its neurotrophic activity can be regulated by social
chemofluorescence to define structural varicosities containing the sympathetic neurotransmitter norepinephrine within the
8860 • J.Neurosci.,August15,2007 • 27(33):8857–8865Sloanetal.•StressEnhancesLymphoidInnervation
To assess the functional impact of stress-induced increases in
lymphoid innervation density, we examined the key physiologi-
cal role of the lymph node in orchestrating an immune response
to infection (von Andrian and Mempel, 2003). SIV infects
T-lymphocytes and macrophages, and replicates efficiently
within the lymph node microenvironment (McChesney et al.,
1998). To determine whether social stress impacts generalized
SIV pathogenesis, we quantified plasma SIV viral load setpoint
and circulating CD4? T-lymphocyte levels in 11 rhesus ma-
caques randomly assigned to unstable versus stable social condi-
load did not differ between groups (mean ? SE, 6.26 ? 106?
0.22 ? 106RNA copies/ml in unstable conditions vs 6.30 ? 106
ever, socially stressed animals showed significantly lower CD4?
T-lymphocyte levels at 36 weeks after infection (mean, 255 ? 23
cells/mm3in unstable conditions vs 572 ? 75 in stable condi-
tions; p ? 0.0006), indicating accelerated progression of SIV-
significantly as a function of social condition (mean, 479 ? 56 d
after infection in unstable conditions vs 436 ? 56 in stable con-
ditions; p ? 0.5292).
To determine the role of lymphoid tis-
sue viral replication dynamics in stress-
induced acceleration of SIV immuno-
pathogenesis, we harvested lymph nodes
had resolved to a stable viral replication
setpoint, but animals did not yet show
clinical illness or lymph node structural
deterioration (Sloan et al., 2006). Sites of
active viral replication were mapped by in
situ hybridization of SIV mRNA (Canto-
Nogues et al., 2001), and stereological
echolaminergic varicosities across a total
of 4300 tissue quadrates (Sloan et al.,
2006). Social stress increased the lymph
ject to unstable social conditions showed
an average 0.264 ? 0.015 active viral rep-
compared with 0.167 ? 0.022 from ani-
mals socialized in stable groups (differ-
ence, p ? 0.0001).
creased SIV replication in proximity to
catecholaminergic neural fibers within the
to determine whether stress effects on the
lymph node-wide prevalence of viral gene
expression might be specifically attribut-
able to the increased density of cat-
echolaminergic varicosities. Stereologic
analyses of in situ hybridization data
showed a twofold increase in the preva-
lence of viral replication sites within tissue
quadrates that contained one or more cat-
erage, 0.180 ? 0.012 sites per quadrate in the absence of varicos-
ities vs 0.397 ? 0.052 in varicosity-containing quadrates;
difference, p ? 0.0001). However, social stress did not signifi-
cantly influence the frequency of SIV replication sites within
unstable social conditions; difference, p ? 0.533). This suggests
that stress effects on lymph node-wide SIV replication stem pre-
dominantly from the increased overall density of sympathetic
varicosities (i.e., increased varicosities per lymph node), rather
than from altered functional activity of individual varicosities
(i.e., increased catecholamine release per varicosity). To assess
this issue more directly, we performed multivariate statistical
analyses at the level of individual tissue quadrates to quantify the
and functional influences (activity per varicosity) to stress-
induced enhancement of SIV replication. Results from this inte-
grative statistical model indicated a significant effect of presence
versus absence of catecholaminergic varicosities ( p ? 0.0002),
but did not find the quantitative magnitude of that effect to be
significantly greater under unstable social conditions ( p ?
0.664). Thus, social stress appears to increase SIV replication by
increasing the overall prevalence of sympathetic varicosities,
stable conditions. Error bars indicate SEM. b, Distribution of catecholaminergic varicosities (white arrowheads) in lymph node
Sloanetal.•StressEnhancesLymphoidInnervation J.Neurosci.,August15,2007 • 27(33):8857–8865 • 8861
without significantly altering the per varicosity support for viral
Type I interferons (IFN-? and 23 IFN-? family members) are
well documented suppressors of HIV-1 and SIV replication, and
are believed to be major physiological determinants of lentiviral
1998; Collado-Hidalgo et al., 2006). Experimental induction of
IFN-? has been shown to suppress SIV replication in vivo
(Matheux et al., 2000), and our previous work has shown that
NE-induced impairment of type I interferon expression repre-
sents a major mechanism by which NE enhances HIV-1 replica-
tion in vitro (Cole et al., 1998; Collado-Hidalgo et al., 2006). To
determine whether stress-induced increases in SIV replication
within lymphoid tissue might involve the suppression of type I
interferon genes, we quantified lymph node mRNA levels of
IFNB and a conserved region of the 23 IFNA transcripts
?80% in tissues from socially stressed animals ( p ? 0.002) (Fig.
approximately fourfold in lymph nodes from stressed animals
( p ? 0.0001) (Fig. 4a). To determine the net effect of impaired
mRNA for the downstream interferon response gene IFI27,
which is sensitive to all type I interferons. Results showed a sub-
stantial decrease in the overall type I interferon response, with
IFI27 mRNA declining by ?60% under unstable social condi-
tions ( p ? 0.0001) (Fig. 4a). The functional significance of this
ship between increased density of SIV replication sites and de-
creased expression of IFNB (Fig. 4b) and IFI27 (data not shown)
mRNA (both p ? 0.0001). Moreover, statistical mediation anal-
yses showed that impaired type I interferon response could ac-
count for 71% of the total relationship between social stress and
ities within each lymph node. c, NGF protein detected by Western blot in lymph node lysate
terstained (blue) (d) and glyoxylic chemofluorescence for catecholamines (blue–white) with
analysis indicated that 86% of the total effect of stress on the density of parenchymal cat-
echolaminergic varicosities could be attributed to variations in NGF gene expression, and no
significant residual effect of stress on innervation density remained after statistical control
lymph nodes from animals subject to stable (gray symbols) versus unstable social conditions
8862 • J.Neurosci.,August15,2007 • 27(33):8857–8865Sloanetal.•StressEnhancesLymphoidInnervation
relationship (mediational path, p ? 0.0417).
It is conceivable that stress-induced changes in SIV replication
stem from a direct effect of NGF on viral replication, rather than
an indirect effect of NGF that is mediated by sympathetic neural
suppression of antiviral cytokines. Macaque leukocytes express
mRNA for both NTRK1 (TrkA gene) and NGFR (p75NTR gene)
(supplemental Fig. 2a, available at www.jneurosci.org as supple-
mental material), providing a signaling pathway for potential
direct regulation of SIV replication by NGF. To assess this possi-
and subsequently cultured in the presence of 0.5, 5, or 50 ng/ml
measured by relative expression of env and gag viral genes (sup-
plemental Fig. 2b,c, available at www.jneurosci.org as supple-
mental material). These results are consistent with data from
human clinical trials of NGF showing no effect on HIV-1 repli-
cation (McArthur et al., 2000; Schifitto et al., 2001).
The present studies show that chronic social stress can increase
the density of sympathetic innervation within the parenchymal
These effects are associated with increased expression of the key
neurotrophic factor NGF, and do not involve changes in lymph
node size, anatomical structure, or immune cell composition.
Functional consequences include reduced type I interferon re-
sion that occurs specifically in the vicinity of catecholaminergic
neural fibers. Multivariate statistical analyses attribute the ele-
vated replication of SIV specifically to the increased density of
catecholaminergic varicosities within the lymph node paren-
chyma [as opposed to increased support for viral replication per
fects of social stress on the structural density of lymph node in-
nervation indicate an unanticipated degree of behaviorally in-
duced neuroplasticity within the primate immune system, with
mediator of behaviorally induced lymph node neuroplasticity,
defined in the lymphoid context and is an important topic for
future research. NGF can be synthesized by multiple cell types
present in the lymph node, including activated T-lymphocytes
and macrophages (Santambrogio et al., 1994; Frossard et al.,
2004). Neuronal activity can also increase NGF gene expression
(Gall and Isackson, 1989; Zafra et al., 1990; Hasan et al., 2003),
raising the possibility that the stress-induced densification of
lymphoid innervation might stem from antecedent increases in
SNS activity. Future studies assessing the longitudinal develop-
ment of stress-induced innervation and pharmacologically
blocking NGF dynamics should help clarify the basis for the be-
haviorally induced neuroplasticity observed here.
Other physiological dynamics could potentially contribute to
stress-induced upregulation of sympathetic innervation, including
glucocorticoid release from the hypothalamic–pituitary–adrenal
axis. Glucocorticoids have been shown to enhance sympathetic in-
nervation of other tissues (e.g., ovary) (Jana et al., 2005), and they
can interact with ?-adrenergic signaling to enhance the transcrip-
shown that the social stress model used here leads to a reduction in
circulating glucocorticoid levels (Capitanio et al., 1998), which
would not be able to explain the increased innervation observed in
the current study. Stress-induced decreases in neuroinhibitory fac-
tors such as IFNG and LIF are also unlikely to explain increased
lated, as would be required to permissively increase innervation).
Stress-induced upregulation of IFNG and LIF at the lymph node-
wide level is unexpected, because previous studies have shown that
?-adrenergic signaling generally reduces the expression of those
proinflammatory mediators (Cole et al., 1998; Collado-Hidalgo et
al., 2006). It is possible that these organ-level changes in IFNG and
LIF expression result from alterations in the recruitment of specific
empirically observed increase in lymph node IFNG and LIF levels
in lymph node innervation can significantly impact the primary
physiological function of the lymph node as a mediator of im-
mune response to infection. Stressful social conditions increased
SIV replication within the lymph node and accelerated systemic
immunopathogenesis, as indicated by decreased circulating
tality times did not differ significantly across conditions. It is
to correlate closely with immunologically based parameters, al-
though methodological issues may have played a role. Plasma
large individual variations in viral load within treatment groups
(?10-fold) may have undermined the statistical power to iden-
tify any existing difference. Alternatively, stress-induced changes
in nonlymphoid determinants of plasma viral load (e.g., clear-
ance rates) may have offset any stress-induced increase in viral
output from lymphoid tissue. Circulating CD4? T-lymphocyte
measures were suppressed by stress, but no parallel difference in
mortality times was observed. The latter finding may stem from
the fact that mortality in the macaque SIV model is often driven
by wasting and neurological symptoms associated with high lev-
els of inflammation during chronic infection, rather than by the
sion. For the purposes of modeling human HIV-1 pathogenesis
in lymphoid tissue, lymph node SIV replication and systemic
CD4? T-lymphocytes declines may be most relevant. The dem-
onstration in this study that social stress can alter lymph node
sympathetic innervation provides new insights into the molecu-
pathogenesis. Previous studies have identified several molecular
mechanisms by which the sympathetic neurotransmitter norepi-
nephrine can accelerate HIV-1 replication in vitro, including
enhancement of viral gene transcription (Cole et al., 2001), and
suppression of antiviral cytokine responses (Cole et al., 1998;
Collado-Hidalgo et al., 2006). In the context of recent studies
al., 2006), the current data identify a novel role of sympathetic
Sloanetal.•StressEnhancesLymphoidInnervationJ.Neurosci.,August15,2007 • 27(33):8857–8865 • 8863
molecular biology of viral replication in vivo.
Reductions in lymph node interferon-? expression, concom-
itant with stress-induced increases in sympathetic lymphoid in-
lication (Fig. 4c), because this type I interferon plays a key role in
initiating innate antiviral responses and limiting HIV-1 replica-
tion (Francis et al., 1992; Agy et al., 1995; Taylor et al., 1998;
Collado-Hidalgo et al., 2006). Indeed, recent studies suggest that
?-adrenergic inhibition of IFNB response plays a central role in
the ability of catecholamines to upregulate HIV-1 replication
(Collado-Hidalgo et al., 2006). However, the other major type I
interferon, interferon-?, was not suppressed by stress but signif-
icantly upregulated (Fig. 4a). It is unclear why these two type I
interferon species show reciprocal dynamics within the lymph
node of stressed primates. The net effect of stress on total type I
interferon activity appears to be suppressive, as indicated by the
downregulation of the IFI27 interferon response gene, which in-
tegrates the effects of both IFNA and IFNB signaling. It is tempt-
IFNA and IFNB transcription in ways that contribute to immu-
nopathogenesis. This would be consistent with the accelerated
CD4? T-lymphocyte decline observed in stressed animals, be-
beuval et al., 2005; Herbeuval and Shearer, 2007).
The reciprocal regulation of IFNA and IFNB suggests a broader
physiological rationale for dynamic innervation of lymphoid tissue
as a pathway for CNS control of peripheral cytokine activity. The
present findings are consistent with an emerging body of evidence
suggesting that the autonomic nervous system plays an important
regulating T-helper cell 1 (Th1)/Th2 cytokine profiles (Cole et al.,
1998; Kohm and Sanders, 1999) and helps resolve immune re-
and Tracey, 2005). In this context, stress-induced plasticity in the
mechanism by which chronic behavioral conditions can impose a
long-term bias on the nature of an individual’s immune response.
The teleological rationale for such a bias is unclear, but the present
ciated with individual differences in the risk of disease onset and
Molecular pathways for neural–immune interaction are well
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purpose remains unclear. The present findings suggest that it
might be possible to analyze the evolution of neural–immune
interactions from an ethological perspective that emphasizes the
vidual immune responses. Close interaction with conspecifics
have evolved as a physiological mechanism for optimizing host
resistance to infection under changing social conditions (e.g., in
the presence of socially mediated threat vs support). Previous
influence health outcomes (Seeman, 1996; Cohen, 2004). How-
ever, the quantity of social interaction was held constant in this
study. The present data show that the specific nature of social
interactions is also important and can alter the sympathetic in-
nervation of lymphoid tissue in ways that may have significant
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