The Journal of Clinical Investigation http://www.jci.org Volume 114 Number 1 July 2004
Ghrelin inhibits leptin- and activation-induced
proinflammatory cytokine expression
by human monocytes and T cells
Vishwa Deep Dixit,1 Eric M. Schaffer,1 Robert S. Pyle,1 Gary D. Collins,1 Senthil K. Sakthivel,2
Ravichandran Palaniappan,2 James W. Lillard Jr.,2 and Dennis D. Taub1
1Laboratory of Immunology, National Institute on Aging, NIH, Baltimore, Maryland, USA. 2Department of Microbiology and Immunology,
Morehouse School of Medicine, Atlanta, Georgia, USA.
Ghrelin, a recently described endogenous ligand for the growth hormone secretagogue receptor (GHS-R), is
produced by stomach cells and is a potent circulating orexigen, controlling energy expenditure, adiposity, and
growth hormone secretion. However, the functional role of ghrelin in regulation of immune responses remains
undefined. Here we report that GHS-R and ghrelin are expressed in human T lymphocytes and monocytes,
where ghrelin acts via GHS-R to specifically inhibit the expression of proinflammatory anorectic cytokines
such as IL-1β, IL-6, and TNF-α. Ghrelin led to a dose-dependent inhibition of leptin-induced cytokine expres-
sion, while leptin upregulated GHS-R expression on human T lymphocytes. These data suggest the existence of
a reciprocal regulatory network by which ghrelin and leptin control immune cell activation and inflammation.
Moreover, ghrelin also exerts potent anti-inflammatory effects and attenuates endotoxin-induced anorexia in
a murine endotoxemia model. We believe this to be the first report demonstrating that ghrelin functions as a
key signal, coupling the metabolic axis to the immune system, and supporting the potential use of ghrelin and
GHS-R agonists in the management of disease-associated cachexia.
Ghrelin is a 28-amino-acid acylated polypeptide secreted predomi-
nantly from X/A-like enteroendocrine cells of the stomach (1, 2).
Several lines of evidence implicate ghrelin in growth hormone
(GH) release, energy balance, food intake, and long-term regula-
tion of body weight in rodents (3, 4) and humans (5). The ghrelin
gene encodes a 117-amino-acid peptide, preproghrelin, for which
there is an 82% homology between rat and human (1). Ghrelin is
presently regarded as the only known circulating orexigen and
exerts antagonistic effects on the leptin-induced decrease in food
intake through activation of the hypothalamic neuropeptide Y–Y1
(NPY-Y1) pathway (3, 6). The effects of ghrelin are mediated via a
7-transmembrane G protein–coupled receptor (GPCR) called growth
hormone secretagogue receptor (GHS-R) (7). This receptor is evolu-
tionarily conserved from puffer fish to humans (8), which suggests
that ghrelin may play a fundamental role in organism growth and
development. The GHS-R type 1a has been implicated in GH release,
and a nonspliced, nonfunctional receptor mRNA variant identified
as GHS-R type 1b has recently been identified within a wide variety
of tissues including lymphoid organs (9). Hexarelin is a synthetic
analogue that binds GHS-R to induce GH secretion from porcine
and bovine PBMCs, which suggests that GHS-R ligands may exert
some direct effects on the immune system (10). In addition, the
wide tissue distribution of GHS-R in the lymphoid system suggests
that ghrelin and GHS-R ligands may function as signal modulators
among the endocrine, nervous, and immune systems.
Inflammatory cytokines released by immune cells have been
shown to act on the CNS to control food intake and energy
homeostasis (11). Decrease in food intake and anorexia are among
the most common symptoms of illness, injury, or inflammation
(12). Cytokines such as IL-1β, IL-6, and TNF-α have been implicat-
ed in wasting associated with inflammation (13), chronic low-grade
inflammation in aging (14, 15), and atherosclerosis (16). Regula-
tion of inflammatory cytokine production by endogenous factors
holds promise in the amelioration of a wide variety of ailments and
disease conditions. In the present report, we describe a novel func-
tion of ghrelin in the immune system and on proinflammatory
cytokine expression by human T cells and mononuclear cells upon
cellular activation or leptin exposure. These results have implica-
tions in the potential use of ghrelin as a therapeutic target associ-
ated with a host of inflammatory diseases.
GHS-R is a functional receptor expressed on the surface of human T cells.
While previous results have described only the mRNA expression
of GHS-R in lymphoid organs (9), our initial studies focused on
the expression and spatial localization of GHS-R protein in puri-
fied human T cells. GHS-R displayed a heterogeneous subcellular
expression pattern in resting human T cells, ranging from crescent
to punctate or diffuse phenotypes (Figure 1A, upper panel, and
Supplemental Figure 1B; supplemental material available at http://
www.jci.org/cgi/content/full/114/1/57/DC1). In resting T cells,
the majority of ghrelin receptors were segregated from the GM1+
lipid rafts (Figure 1A, upper panel). However, upon activation of
T cells via T cell receptor (TCR) ligation, we observed a dramatic
subcellular reorganization of GHS-R, demonstrating a polarized
capped phenotype and aggregation in lipid rafts (Figure 1A, lower
panel). Flow cytometric analysis revealed that up to 30% of high-
ly purified resting human T cells exhibited specific staining for
Nonstandard abbreviations used: Alexa Fluor (AF); G protein–coupled receptor
(GPCR); growth hormone (GH); growth hormone secretagogue receptor (GHS-R);
neuropeptide Y (NPY); stromal cell–derived factor-1α (SDF-1α); T cell receptor (TCR).
Conflict of interest: The authors have declared that no conflict of interest exists.
Citation for this article: J. Clin. Invest. 114:57–66 (2004).
58 The Journal of Clinical Investigation http://www.jci.org Volume 114 Number 1 July 2004
GHS-R as demonstrated via the use of a blocking peptide (Figure
1B). In contrast, in human PBMCs, no preferential expression pat-
tern of GHS-R was observed on CD3+, CD3+CD4+, and CD3+CD8+
T cells (data not shown). In highly purified human T cells, GHS-R
expression significantly increased upon cellular activation (Figure
1C), and in the presence of antibody-specific blocking peptide, this
GHS-R labeling was almost completely ablated. Moreover, upon
T cell activation, there was also a marked upregulation of GHS-R
gene expression, as demonstrated by quantitative analysis of PCR
products using Agilent gene chip technology (Agilent Technolo-
gies, Palo Alto, California, USA) and real time RT-PCR (Figure 1D).
GHS-R mRNA was found to be upregulated in T cells as early as
6–12 hours after activation, with colocalization in GM1+ domains.
The presence of GHS-R within lipid rafts and specific upregulation
of the GHS-R gene upon T cell activation strongly supports a role
for these receptors in T cell function. We observed a similar staining
pattern for ghrelin receptors on activated T cells utilizing a second
antibody recognizing amino acid residues 186–265 proximal to the
C terminal region of human GHS-R (Supplemental Figure 1A).
Ligation of seven transmembrane GPCRs typically results in cal-
cium mobilization from the intracellular stores by generation of
inositol triphosphate (1, 17). Ghrelin has previously been shown
to induce intracellular calcium release in GHS-R–transfected CHO
cells (1). Here, using cultured human T cells, we demonstrate a sig-
nificant and specific rise in intracellular [Ca2+] in response to both
full-length ghrelin peptide (Figure 1E) as well as ghrelin 1–18 frag-
ment, while the des-acyl ghrelin did not elicit calcium release (data
not shown). This ghrelin-induced calcium flux was found to be
GHS-R specific, as pretreatment with [D-Lys-3]-GHRP-6, a highly
selective GHS-R antagonist, markedly attenuated the ghrelin-medi-
ated intracellular calcium release from T cells (Figure 1E). Inter-
estingly, the intracellular calcium mobilization induced by ghrelin
treatment was similar in magnitude to that observed in response
to our positive control, stromal cell–derived factor-1α (SDF-1α),
a potent T cell chemokine ligand that specifically binds and sig-
nals through the cell surface GPCR, CXCR4 (18). In addition to
calcium mobilization, ligation of GPCRs is often accompanied by a
dramatic remodeling of the actin cytoskeleton and cell surface mol-
ecules and leads to polarization and, in many cases, the directional
migration of immune cells (19, 20). Here, ghrelin induced a marked
increase in broad membrane structures characteristic of lamellipo-
dia with typical polarization of F-actin in a manner quite similar to
the SDF-1α–treated cells (Figure 1F). Together, these data demon-
strate the presence of functional GHS-R on the surface of human
Expression of functional GHS-R in human
T cells. (A) Primary human T cells were
labeled for GHS-R, and subcellular
localization in lipid rafts was visualized
in resting and anti-CD3–activated cells.
Arrowheads indicate colocalization of
GHS-R with polarized lipid rafts. (B) Flow
cytometric analysis of GHS-R on highly
purified resting human T cells. Specific
T cell labeling (green) was abolished in
presence of antibody-specific blocking
peptide (blue). T cells stained with con-
trol IgG demonstrated no specific label-
ing. (C) Flow analysis of GHS-R expres-
sion on highly purified (>96%) activated
CD3+ human T cells. Staining specific-
ity was demonstrated through the use of
antibody-specific blocking peptide. (D)
GHS-R mRNA is upregulated upon T cell
activation as assessed using Agilent gene
chip quantitation and real time RT-PCR;
values are expressed as mean ± SEM
(*P < 0.05). (E) Ghrelin induces intra-
cellular calcium mobilization in cultured
human T cells. T cells were stimulated
with ghrelin (100 ng/ml; blue), or SDF-1
(100 ng/ml; green) at 60 seconds. T cells
were also treated with the GHS-R antago-
nist, [D-Lys-3]-GHRP-6 (10–4 M), at 60
seconds followed by ghrelin (100 ng/ml;
red) at 180 seconds. FL, fluorescence.
(F) Ghrelin causes actin-polymerization
in human T cells. Cells were treated with
ghrelin (100 ng/ml) and positive control
SDF-1α (100 ng/ml) for 20 minutes and
labeled for F-actin with phalloidin AF-594.
Arrowheads indicate polymerized F-actin
associated with lamellipodia of cells.
The Journal of Clinical Investigation http://www.jci.org Volume 114 Number 1 July 2004
T cells and mononuclear cell subsets and support a biological role
for ghrelin and GHS-R within the immune system.
GHS-R is expressed in human monocytes, and ghrelin inhibits inflam-
matory cytokines. Among the mononuclear cells, monocytes con-
stitute an important source of proinflammatory cytokines, which
prompted us to examine the GHS-R expression on monocytes.
Flow cytometric analysis revealed that approximately 21% of CD14+
cells expressed GHS-R (Figure 2A). Using immunofluorescence
microscopy, we detected diffuse GHS-R expression on the cell sur-
face of purified monocytes (Figure 2B, upper panel), and control
IgG demonstrated no specific labeling (Figure 2B, lower panel).
Similarly, GHS-R expression was observed in immature and
mature monocyte–derived dendritic cells (data not shown). Real
time RT-PCR analysis also demonstrated the presence of GHS-R
type 1a mRNA in monocytes with similar expression levels to pri-
mary human T cells (data not shown). Furthermore, ghrelin led
to a dose-dependent inhibition of IL-1β and IL-6 (Figure 2, C and
D) via a GHS-R–specific pathway, as concomitant treatment with
GHS-R antagonist blunted these effects.
Ghrelin selectively inhibits proinflammatory cytokine expression. The
classical proinflammatory cytokines, IL-1α, IL-1β, IL-6, and
TNF-α, are known to play a critical role in development of anorex-
ia-cachexia syndrome (21). Anorexia-cachexia syndrome is a com-
plex multifactorial metabolic condition associated with altered
protein, carbohydrate, and fat metabolism resulting in anorexia,
negative energy balance, weight loss, and muscle wasting (13). It
has been hypothesized that ghrelin is involved in mealtime hunger
and long-term regulation of body weight (4, 5). Considering the
critical role played by proinflammatory cytokines in controlling
metabolic activity, we next examined the ability of ghrelin to regu-
late the production of IL-1β, IL-6, and TNF-α by activated PBMCs
and T cells. Human PBMCs derived from healthy male subjects
were stimulated with the polyclonal mitogen phytohemagglutinin
and incubated in the presence or absence of ghrelin and GHS-R
antagonist for 24 hours, after which supernatants were collected
and examined for cytokine levels. Interestingly, ghrelin treatment
resulted in a significant inhibition of IL-1β, IL-6, and TNF-α pro-
duction by PBMCs at ghrelin levels ranging from 1 to 100 ng/ml
(Figure 3, A–C); however, ghrelin treatment failed to alter TGF-β
production by these PBMCs at any concentration tested (Figure
3D). This effect was found to be GHS-R specific, as the addition of
GHS-R antagonist to these cultures attenuated this ghrelin-medi-
ated inhibition, and similar ghrelin effects on cytokine produc-
tion were observed using concanavalin A–stimulated PBMCs (data
not shown) and LPS-treated monocytes. In addition, the primary
human T cells stimulated with immobilized anti-CD3 antibody
in the presence of ghrelin for 24 hours demonstrated a signifi-
cant dose-dependent inhibition of IL-1β and IL-6 (Figure 3, E–G).
TNF-α secretion by T cells also demonstrated a reproducible
declining trend in response to ghrelin, as was observed in the
PBMC studies; however, due to individual variations in TNF-α
production among these donors, this inhibition failed to reach
statistical significance (P > 0.05). It should also be noted that this
ghrelin-mediated inhibition was not due to any cytolytic effects
of the hormone on T cells or PBMCs, as measurement of lactate
dehydrogenase and cell counts using trypan blue exclusion failed
to demonstrate any significant difference between control and
hormone-treated cells. Furthermore, ghrelin had no significant
effect on proliferation or IL-2 (Supplemental Figure 1C) and
IFN-γ secretion (data not shown) from human T cells. Using real-
time RT-PCR analysis, we further demonstrate that ghrelin sig-
nificantly inhibits IL-1β. A ghrelin dose of 10–100 ng/ml inhibited
IL-6 and TNF-α mRNA expression in all the donors, demonstrat-
ing a reduction in cytokine production (Figure 3H). These results
strongly support a role for ghrelin in the transcriptional regula-
tion of inflammatory cytokine expression.
Ghrelin inhibits leptin-mediated proinflammatory cytokine expression.
As leptin and ghrelin exert antagonistic effects on food intake
at the hypothalamic level (4, 6), we next sought to determine the
mechanism by which these metabolic hormones regulate inflam-
matory cytokine production. Recent studies have demonstrated
that leptin-deficient mice are protected from T cell–mediated
hepatotoxicity (22) and that leptin exerts proinflammatory effects
in these mice (23). In humans, leptin has been recently shown
to increase IL-6 and TNF-α protein production by PBMCs and
monocytes (24). Human T cells and porcine PBMCs have also
been shown to express leptin receptor (Ob-R) mRNA (25, 26). In
support of these findings, we demonstrate here the diffuse expres-
sion of Ob-R protein on the surface of human T cells (Figure 4A).
Moreover, we have shown, we believe for the first time, that leptin
directly induces a significant dose-dependent increase in IL-1β
(Figure 4B), IL-6 (Figure 4C), and TNF-α (Figure 4D) protein
and mRNA expression by primary human T cells (Figure 4E) and
PBMCs (data not shown). Upon concomitant addition of ghrelin
to these cultures, a dose-dependent inhibition of leptin-induced
cytokine protein and gene expression by T cells was observed in
response to various concentrations of ghrelin (Figure 4, B–E). This
strongly suggests that ghrelin and leptin, similar to their mutually
antagonistic effects on food intake in hypothalamus, also exert
reciprocal regulatory effects on inflammatory cytokine expression
Ghrelin receptors are expressed on human monocytes. (A) Human
PBMCs were double stained with CD14 PE and GHS-R AF-488. (B)
Immunofluorescence labeling revealed GHS-R expression on cell sur-
face of purified monocytes (upper panel); negative control failed to
show any specific staining (lower panel). Ghrelin inhibits IL-1β (C) and
IL-6 (D) secretion from LPS-treated (10 ng/ml) monocytes.
60 The Journal of Clinical Investigation http://www.jci.org Volume 114 Number 1 July 2004
in the immune system. Thus, the variations in circulating levels
of leptin and ghrelin may significantly influence the production
of various cytokines by immune cell populations. Such reciprocal
immunoregulatory effects may be critical in maintaining immune
cell homeostasis, thereby preventing aberrant cytokine produc-
tion, which may result in or amplify illness and pathology.
Human T cells express and actively secrete ghrelin. Ghrelin is current-
ly thought to be produced exclusively by the stomach and sub-
sequently secreted into the peripheral circulation (1). However,
recent reports have demonstrated that peripheral ghrelin levels
gradually increase after gastrectomy, suggesting that additional
cellular sources of ghrelin compensate for stomach-derived ghrelin
(27). Lymphocytes are known to produce many well-characterized
hormones, such as GH (26), which exerts a number of regulatory
effects on the immune system (28). Given the potent effect of ghre-
lin on cytokine expression, the possible presence of ghrelin endog-
enously produced by immune cells was hypothesized. Our results
demonstrate the presence of immunoreactive ghrelin and GHS-R
(Figure 5A, upper panel) in resting human T cells with a broad dis-
tribution phenotype, with areas of colocalization suggesting pos-
sible ligand-receptor interaction and an autocrine role for ghrelin
in T cells. Interestingly, upon TCR ligation, a distinct change in
the spatial localization of the endogenous immunoreactive ghre-
lin was observed, resulting in a polarized expression phenotype.
Ghrelin appeared to specifically associate within GM1+ lipid rafts
(Figure 5A, middle panel), suggesting that, upon activation, ghre-
lin may be produced and specifically targeted toward lipid rafts
and its own specific receptor. In further support of ghrelin syn-
thesis by human T cells, we found that the 117-amino-acid pre-
pro form of ghrelin is also coexpressed and colocalized within the
Golgi apparatus (Figure 5A, lower panel), where the preproghrelin
is presumably cleaved and processed to its mature form prior to
secretion. Both ghrelin and preproghrelin staining in primary T
cells was abolished upon addition of antibody-specific blocking
peptide (Supplemental Figure 1D).
These findings are further supported by flow cytometric analy-
sis of various T cell subsets for the mature ghrelin protein: the
majority of T cells appeared to be ghrelin positive with no pref-
erential expression in CD3+CD4+ or CD3+CD8+ T cell subsets. In
addition to expression of intracellular ghrelin by T cells, TCR liga-
tion of these cells resulted in the secretion of substantial levels of
ghrelin protein into the culture supernatant, with levels peaking
at 48 hours and declining thereafter (Figure 5B). Furthermore, T
cell activation induced a greater than fivefold increase in ghrelin
mRNA expression, as demonstrated by real time RT-PCR analysis
(Figure 5C). We also demonstrate that acylated (active) ghrelin is
coexpressed in human PBMCs with total ghrelin in 30% of cells
(Supplemental Figure 2).
Given the presence and production of ghrelin by T cells, it is possi-
ble that ghrelin concentrations within the local microenvironment
may reach significantly high levels without undergoing the clas-
sic dilution effect typically seen upon the release of ghrelin from
stomach into the peripheral circulation. Thus, T cell–derived
ghrelin may serve an important role in regulating cell function
within an immune microenvironment or organ. Considering the
specific antagonistic effect of ghrelin on leptin-mediated inflam-
matory cytokine expression and the previous studies demonstrat-
ing leptin-induced ghrelin inhibition in the stomach (29), we
next examined the possible cross-regulatory effects of leptin on
ghrelin and GHS-R expression in T cells. In our current studies,
leptin failed to exert any significant effects on ghrelin protein pro-
duction or gene expression within human T cell cultures (Figure
5D). More interestingly, we noted that leptin treatment resulted
in a significant increase in GHS-R mRNA expression by human
T cells as measured by real-time RT-PCR (Figure 5E). Hence, the
downregulation of leptin-induced cytokine expression by ghrelin
may constitute a reciprocal regulatory signaling pathway by which
these hormones control each other’s activities within the immune
system (Figure 5F). In addition, real-time PCR analysis revealed
that ghrelin expression levels in human stomach were 11-fold
higher than in lymphoid organs (T cells, spleen, and thymus) (Fig-
Ghrelin inhibits inflammatory cytokine expression from human
PBMCs and T cells. Human PBMCs (n = 6) were stimulated with
phytohemagglutinin (PHA) (1 μg/ml) (A–D), or T cells were activated
via immobilized anti-CD3 antibody (E–H) in presence or absence of
various doses of ghrelin (closed circles) and concomitantly with GHS-R
antagonist, [D-Lys-3]-GHRP-6 (10–4 M; open circles) for 24 hours. The
harvested supernatants were subsequently assayed for IL-1β (A and
E), IL-6 (B and F), and TNF-α (C and G) and TGF-β (D). The cytokine
protein data is expressed as the mean ± SEM representing six healthy
adult donors (*P < 0.05). (H) Fold change in IL-1β, IL-6, and TNF-α
mRNA expression in T cells after normalization with GAPDH, mea-
sured by real time RT-PCR.
The Journal of Clinical Investigation http://www.jci.org Volume 114 Number 1 July 2004
ure 5G). Lymphoid organs and small intestines expressed ghrelin
mRNA levels fivefold higher than did placenta.
Ghrelin downregulates inflammatory cytokine expression and anorexia
in response to endotoxin challenge. Bacterial LPS, the principal com-
ponent in the pathogenesis of endotoxic shock, acts primarily on
monocytes and evokes an acute phase response in vivo, resulting in
excessive production of IL-1β, IL-6, and TNF-α. The amplification
of these proximal cytokines has a broad array of proinflammatory
and anorexigenic effects (12, 13), contributing to pathogenesis of
sepsis and multiple organ failure (30, 31). In an effort to examine
the ability of ghrelin to modulate inflammatory cytokine expres-
sion in vivo, we treated mice with ghrelin prior to and after LPS
administration. As shown in Figure 6, ghrelin exerted a potent anti-
inflammatory effect on LPS-induced endotoxemia, with inhibition
of IL-1β, IL-6, and TNF-α expression in vivo. Real-time PCR analy-
sis of mRNA derived from the spleen and liver of these endotoxin-
treated mice revealed a strong induction of these cytokine genes 4
hours after LPS administration (Figure 6, A–C), with a significant
diminishment in mRNA expression by 24 hours (Figure 6, D–F).
Mice treated with ghrelin and challenged with endotoxin demon-
strated an attenuation of IL-1β and IL-6 mRNA expression in both
spleen and liver after 4 and 24 hours (Figure 6, A–F). Attenuation
of TNF-α mRNA was observed in both spleen and liver at 4 hours
(Figure 6C); TNF-α expression was also inhibited in liver 24 hours
after LPS administration and remained unchanged in spleen (Fig-
ure 6F). Similar inhibition of proinflammatory cytokines was
observed in lungs and mesenteric lymph nodes of ghrelin-treated
mice 4–24 hours after LPS challenge (data not shown).
To measure circulating serum cytokine levels, mice were treated
with LPS and then with ghrelin for either 4 or 24 hours. Analysis of
the serum cytokine levels revealed a significant change in circulat-
ing TNF-α (Figure 7C), but not in IL-1β (Figure 7A) or IL-6 (Figure
7B), levels at 4 hours after ghrelin treatment; however, a significant
inhibition of IL-1β and IL-6 was observed 24 hours after LPS chal-
lenge (Figure 7, D and E). TNF-α levels were undetectable in the
serum 24 hours after LPS challenge. To examine the effects of ghre-
lin on endotoxin-induced anorexia, food intake was also assessed
at 24 hours after ghrelin and/or LPS administration. While the
LPS-challenged mice demonstrated a dramatic diminishment in
food consumption compared with sham-treated mice (80%), prior
ghrelin treatment resulted in a significant attenuation of this LPS-
induced anorexia (Figure 7F). As expected, ghrelin-treated control
mice in the absence of LPS challenge also demonstrated a signifi-
cant increase in food intake (30%) compared with sham-treated
controls (data not shown). Interestingly, serum IL-1β and IL-1α lev-
els were also significantly inhibited in these mice infused with ghre-
lin alone when compared with sham-treated control mice (Figure 7,
G–H), and serum IL-1α levels were inhibited 24 hours after LPS and
ghrelin treatment (Figure 7H). Neither LPS nor ghrelin treatment
altered serum leptin levels in these mice (data not shown).
We believe this to be the first report to demonstrate that ghre-
lin, via functional cell surface GHS-R, exerts both specific and
selective inhibitory effects on the expression and production of
the inflammatory cytokines IL-1β, IL-6, and TNF-α by human
PBMCs and T cells. GHS-Rs on primary and cultured human T
cells, similar to other classical GPCRs, elicit a potent intracellular
calcium release upon ligation with their natural ligand, ghrelin,
and are preferentially associated with GM1+ lipid rafts upon cel-
lular activation. We also observed that, consistent with expres-
sion of functional GHS-R ghrelin on T cells, ghrelin actively
induces actin polymerization within human T cells. Similar to
treatment with chemokines (SDF-1α), ghrelin treatment led to
the cellular polarization of leukocytes and actin distribution
changes from a linear cortical pattern in resting lymphocytes
to more concentrated patterns at the leading edge and contact
zones in polarized and activated T cells (19, 20). These GPCR-like
redistribution patterns also support a potential role for GHS-R
in immune cell signaling and trafficking.
There is increasing evidence that the immune system — in par-
ticular the production of inflammatory cytokines by leukocytes
— may play an important role in the development of anorexia-
cachexia syndrome (11–13). The cytokines considered to be the
most relevant to inflammatory anorexia include IL-1β, IL-6, and
TNF-α. Peripherally administered ghrelin has been shown to
block IL-1β–induced anorexia (29) and produces positive energy
balance by promoting food intake and decreasing energy expendi-
ture. Our current data demonstrates an inhibitory effect of ghrelin
on proinflammatory cytokine expression, supporting a possible
Ghrelin inhibits leptin-induced increase in inflammatory cytokines.
(A) The localization of the leptin receptor (Ob-R) on the surface of
human T cells. (B–D) Anti-CD3 mAb-activated T cells from human
adult donors (n = 6) were incubated with various concentration of leptin
or coincubated with various doses of ghrelin with a biologically opti-
mal concentration of leptin (100 nM). Cytokine production and mRNA
expression was evaluated after 24 hours of culture. The cytokines
examined were (B) IL-1β, (C) IL-6, and (D) TNF-α. (E) Fold change
in IL-1β, IL-6, and TNF-α mRNA expression after normalization with
GAPDH and measured by real-time RT-PCR. Values are expressed
as mean ± SEM (*P < 0.05).
62 The Journal of Clinical Investigation http://www.jci.org Volume 114 Number 1 July 2004
regulatory role for ghrelin and GHS-R in controlling cytokine-
induced anorexia. Moreover, the combination of IL-1β and leptin
has also been shown to inhibit ghrelin expression in stomach (29),
and stomach ghrelin expression is increased in leptin-deficient
mice. Leptin and ghrelin are considered to exert mutually antago-
nistic effects on the food intake at the hypothalamic level (4, 6).
Leptin, a member of gp130 family of cytokines, induces a strong
Th1 response (25) and is regarded as a proinflammatory inducer
(22–26). Leptin’s actions on food intake are controlled, in part, by
an increase in the level of IL-1β in the hypothalamus (32). Simi-
larly, anorectic effects of IL-1 are mediated via increasing leptin
levels (33). However, the relationship between leptin and ghrelin
at the level of immune cells is completely unknown.
We demonstrate here that leptin can directly induce the mRNA
expression and secretion of IL-1β, IL-6, and TNF-α by human T
cells and PBMCs. Leptin and several other gp130 ligands includ-
ing LIF, CNTF, and IL-6 all appear to exert similar effects on host
metabolism (34, 35). Moreover, IL-6–deficient mice, in a fashion
similar to leptin-deficient mice, develop obesity (36). While leptin
has been shown to be associated with cachexia, leptin levels are
not elevated in many cancer-associated wasting conditions (37),
most likely due to a systemic decline in adipose tissue. However,
cachexia seen in chronic heart failure patients is associated with
hyperleptinemia (38). In contrast, ghrelin attenuates cachexia
associated with chronic heart failure in rats (39), and the GHS-R
analogue, GHRP-2, counteracts protein hypercatabolism, skel-
etal muscle proteolysis, and osteoporosis in critically ill patients
with wasting condition (40). It has recently been reported that
an increase in the level of circulating leptin within a murine MS
model regulates inflammatory anorexia and disease susceptibil-
ity (41). Moreover, fasting-induced suppression of leptin levels
dramatically attenuates the onset of experimental autoimmune
Ghrelin is expressed and secreted from human T cells. (A) Ghrelin and GHS-R coexpression in resting T cells (upper row); activated T cells dem-
onstrating that ghrelin is strongly colocalized in GM1+ lipid rafts (middle row); preproghrelin colocalizes in Golgi bodies in activated human T cells
(lower row). (B) Kinetics of ghrelin secretion from anti-CD3 mAb–stimulated T cells. (C) Fold change in ghrelin mRNA levels upon T cell activation
as assessed by real time RT-PCR analysis. Values are expressed as mean ± SEM (*P < 0.05). (D) Ghrelin expression was quantitated in T cells
stimulated in presence of immobilized anti-CD3 antibody and in the presence or absence of different concentrations of leptin after 24 hours in
culture. Fold change in ghrelin mRNA expression (black bars) after normalization with GAPDH and measured by real time RT-PCR. Ghrelin protein
production was determined by EIA (white bars). (E) Fold change in GHS-R gene expression after normalization with GAPDH (n = 6), with values
expressed as mean ± SEM (*P < 0.05). (F) Hypothetical model for functional role of ghrelin as a signal linking the immune and endocrine systems
in control of food intake. (G) Comparative ghrelin mRNA expression in stomach as compared to lymphoid organs. SI, small intestine.
The Journal of Clinical Investigation http://www.jci.org Volume 114 Number 1 July 2004
encephalomyelitis (EAE) in this model (41). Given that fasting is
associated with a decrease in serum leptin and a strong increase in
circulating ghrelin levels (5, 6), it seems feasible that the observed
anti-inflammatory effects of fasting in this murine MS model
may also be mediated, in part, by ghrelin. Furthermore, it has
recently been demonstrated that NPY attenuates Th1-mediated
induction of EAE (42). Given that fasting and ghrelin induce NPY,
it seems likely that the orexigenic axis might play a critical role in
regulating endogenous inflammatory responses. Ghrelin has now
been recognized to have pleiotropic functions in a variety of organ
systems; however, studies by Smith and colleagues (43) have dem-
onstrated no physiological abnormalities in a ghrelin knockout
mouse, suggesting involvement of some possible compensatory
mechanisms regulated by other orexigens.
Because regulation of hunger is most critical for the survival
of species, a complex circuitry of compensatory and overlapping
mechanisms has evolved to protect the host against deficiency in
one or more of these regulators. Similar genetic approaches to
study the other potent orexigens such as NPY and agouti-related
peptide have also failed to yield a definite phenotype (44). There-
fore, additional controlled studies in orexigens or their receptor
knockout mouse models in response to stress, inflammation, or
pathogenic challenge might shed more light and reveal additional
unique and overlapping functions of these orexigens.
To date, ghrelin has only been reported to be produced by endo-
crine-like cells in the stomach and subsequently released into the
peripheral circulation. Through a number of analytical techniques,
we demonstrate here that ghrelin is endogenously produced and
secreted by both T cells and PBMCs in a fashion similar to many
immune-derived cytokines. The majority of T cells examined from
human donors were found to constitutively express low levels of
endogenous ghrelin, which is significantly increased upon cellular
activation. This high percentage of ghrelin-positive cells may also
be due to the fact that our anti-ghrelin antibody recognized both
the mature as well as the 117-amino-acid preproghrelin forms.
However, the preproghrelin antibody does not bind the mature
peptide, and subcellular localization revealed tight colocalization
of preproghrelin in the Golgi apparatus. Activated T cells express
and secrete the ghrelin protein, which strongly suggests that pre-
pro peptide must be actively cleaved in T cells to yield the active
ghrelin peptide. Similar to several cytokines (e.g., TGF-β) and
hormones (e.g., thyroid stimulating hormone), these precursor
proteins are synthesized and subsequently stored for immediate
cleavage and use when needed. Furthermore, we also demonstrate
the expression and secretion of the mature form of ghrelin from
T cells after activation via TCR ligation. Gastrectomy results in
only a 35–50% decline in circulating ghrelin, and ghrelin levels
increase to two thirds of pre-gastrectomy levels in human subjects,
which suggests that other tissues compensate for maintaining the
peripheral ghrelin levels (27). Secretion of ghrelin from T cells
suggests that immune cell–derived ghrelin might make up part of
residual concentration of circulating ghrelin. In addition, ghrelin
is also regarded as the only known hormone where the hydroxyl
group of the third serine residue is acylated by n-octanoic acid,
and this acylation is critical for some of the biological activities
of this polypeptide (1). N-terminal acylated peptides are known
to preferentially aggregate in cholesterol rich microdomains (45),
and, interestingly, we observed that ghrelin immunoreactivity in
activated T cells is highly colocalized within cholesterol-rich GM1+
domains. These results suggest that ghrelin may be selectively tar-
geted to the plasma membrane to facilitate interaction with its
own transmembrane receptor to optimally mediate receptor-ligand
interactions. Such a pathway would have strong implications
regarding the role of ghrelin in the control of immune responses.
In addition, it seems likely that localized production of ghrelin
may play a critical role in the immediate control of ongoing and
leptin-mediated responses within the local microenvironment.
LPS-induced endotoxemia in mice is a well-recognized model
for inducing septic shock and is also associated with anorexia
due to excessive production of proinflammatory mediators. In
spite of a large body of data, the causes of systemic inflamma-
tory response syndrome (SIRS) remain unknown, and various
therapeutic approaches have yielded minimal beneficial results
(30, 31). LPS directly acts on mononuclear cells, but the resultant
Ghrelin inhibits inflammatory cytokine expression and anorexia in a
murine endotoxemia model. Real-time PCR analysis of inflammatory
cytokine mRNA in spleen and liver 4 and 24 hours after LPS and ghre-
lin administration in BALB/c mice. Ct values for cytokines were normal-
ized with GAPDH and expressed as fold change over collapsed values
for sham-treated, control mice (n = 6). At 4 and 24 hours post LPS
injection, ghrelin inhibits IL-1β (A and D) and IL-6 (B and E) transcrip-
tion in both spleen and liver. TNF-α mRNA expression was attenuated
at 4 hours after LPS in spleen, but ghrelin failed to further inhibit TNF-α
in spleen at 24 hours. However, ghrelin continued to significantly sup-
press TNF-α mRNA in liver (C and F).
64 The Journal of Clinical Investigation http://www.jci.org Volume 114 Number 1 July 2004
endotoxemia also affects a wide variety of cells and systems and
is associated with a refractory catabolic state. Because ghrelin
receptors are ubiquitously expressed in monocytes (Figure 2), B
cells, and dendritic cells (data not shown) and that ghrelin inhib-
its inflammatory cytokines in human mononuclear cells as well
as monocytes, we utilized an established murine model of LPS-
induced endotoxemia (46). We demonstrate that ghrelin infu-
sions in LPS-challenged mice lead to a significant inhibition of
proinflammatory cytokines IL-1α and IL-β, IL-6, and TNF-α in
circulation as well as in liver, spleen, lungs, and mesenteric lymph
nodes. In addition, LPS-induced endotoxemia results in inhibi-
tion of ghrelin secretion (47), and ghrelin infusion increases body
weight in septic animals (48). Considering the data presented
herein, it seems plausible that inhibition of ghrelin secretion after
LPS challenge might exacerbate the ongoing inflammatory insult
and promote development of a catabolic state. Furthermore, we
demonstrated that LPS-induced inflammatory anorexia is also
significantly reduced in ghrelin-treated mice. These data strongly
support possible inclusion of ghrelin and synthetic GHS as poten-
tial candidates in treatment of SIRS. Ghrelin might also have a
regulatory role in chronic conditions such as Helicobacter pylori
infection, where persisting gastric inflammation is associated with
lower ghrelin levels (49) and correction of infection leads to up
regulation of ghrelin secretion.
Our current studies suggest that ghrelin functions as a vital
counterregulatory signal in the immune system, controlling
not only activation-induced cytokine expression but also leptin-
induced expression of these same inflammatory mediators. The
reciprocal regulatory effects of these hormones on expression of
IL-1β, IL-6, and TNF-α by immune cells may have widespread
implications in the development of wasting diseases, aging, and
frailty. Proposed interventions to lower ghrelin levels or to block
GHS-R for treatment of obesity may result in a potentiation of
ongoing inflammatory insults or lead to immune dysregulation.
On the contrary, the novel anti-inflammatory actions of ghrelin
within the immune system may have potential benefits in manage-
ment of anorexia-cachexia syndrome associated with a wide range
of inflammatory conditions and cancer.
Human subjects. Pheresis packs were prepared from six healthy male
donors between 22 and 37 years age for the isolation of PBMCs
and T cells. The average BMI of our donors is 24.6 and is within
the normal range (18.5–24.9).
Mice. Male BALB/c mice (Taconic, Germantown, New York, USA),
8–10 weeks old and weighing 20–22 g, were used. The guidelines
proposed by the committee for the Care and Use of Laboratory
Animal Resources, Commission of Life Sciences, National Research
Council, were followed to minimize animal pain and distress. Each
animal received rodent laboratory chow and ad libitum water.
LPS-induced inflammation. Endotoxin shock in mice was induced
by intraperitoneal (i.p.) injection with 10 μg of LPS (E. coli serotype
055:B5; Sigma-Aldrich, St. Louis, Missouri, USA) as described pre-
viously (46). Animals also received a single i.p. injection of ghrelin
in PBS at 24 hours 30 minutes prior to LPS administration. Mice
were sacrificed 4 and 24 hours after LPS challenge, and visceral
organs and serum were collected.
T cell isolation and culture. PBMCs were obtained by Ficoll-Hypaque
density centrifugation. T cells were purified from PBMCs using
human T cell enrichment columns (R&D Systems, Minneapolis,
Minnesota, USA) via high-affinity negative selection according to
the manufacturer’s instructions. Flow cytometric analysis typically
revealed greater than 90% purity. T cells were stimulated with plate
bound anti-human CD3 antibody (BD Pharmingen, San Diego,
California, USA) (200 ng/ml) at a concentration of 3 × 106 cells/
ml in AIM-V (Gibco-BRL, Carlsbad, California, USA) serum-free
media for 24 hours. Ghrelin 1–18 octanoylated fragment (Peptide
International, Louisville, Kentucky, USA) was used to treat the cell
in culture; this fragment was found to elicit biological effects com-
parable to the intracellular calcium release and cytokine expres-
sion induced by full-length octanoylated peptide.
Immunofluorescence staining. Cellular staining was performed
as described previously (50). Briefly, cells were incubated with
different combinations of human anti–GHS-R goat IgG, anti–
GHS-R rabbit IgG recognizing 186–202 amino acids near the
C terminus of human GHS-R (Santa Cruz Biotech, Santa Cruz,
California, USA), anti–total ghrelin rabbit IgG, anti–preproghre-
Cytokine levels in the serum of treated mice after LPS and ghrelin
treatment. Cytokines tested were IL-1β (A), IL-6 (B), and TNF-α (C) at
4 hours and IL-1β (D) and IL-6 (E) at 24 hours. Ghrelin stimulates food
intake in LPS challenged mice (F). Ghrelin treatment inhibits basal
IL-1β and IL-1α secretion in periphery (G and H). Ghrelin also inhib-
its serum IL-1α levels 24 hours after LPS challenge (I). Values are
expressed as mean ± SEM (*P < 0.05).
The Journal of Clinical Investigation http://www.jci.org Volume 114 Number 1 July 2004
lin rabbit IgG (Phoenix Peptides, Belmont, California, USA),
anti–acylated guinea pig antibody (Linco Research Inc., St.
Charles, Missouri, USA) overnight at 4°C. Lipid rafts were visu-
alized using cholera toxin–Alexa Fluor–594 (AF-594) (Molecu-
lar Probes, Eugene, Oregon, USA) at 20 μg/ml for 45 minutes.
Golgi bodies were stained with goat anti-mouse Golgin-97, a
marker for Golgi bodies (Molecular Probes). Cells were thereaf-
ter labeled with appropriate secondary antibodies conjugated to
AF-488 and AF-594. Nuclei were counterstained using DAPI (1
μg/ml). Images were acquired by Spot Advanced software (Diag-
nostic Instruments Inc., Sterling Heights, Michigan, USA) on
a Zeiss Axiovert S100 microscope under a ×100 objective lens
(Carl Zeiss, Thornwood, New York, USA).
Flow cytometric analysis. Human PBMCs (1 × 106) in PBS contain-
ing 2% heat-inactivated FBS were fixed using 1% paraformaldehyde
and stained for CD3 APC–, CD4 PE–, CD8 PE–, and CD14 PE–con-
jugated antibodies (BD Pharmingen) and incubated for 30 min-
utes on ice. Cells were washed with PBS, stained for GHS-R and
ghrelin, and labeled with specific secondary antibodies conjugated
to AF-488; then analyzed on a FACScan cytometer (BD, Franklin
Lakes, New Jersey, USA).
Intracellular calcium mobilization. Measurement of intracellular
calcium release in response to ghrelin and SDF-1 was performed
as described previously (17). Purified human T cells were activated
via TCR ligation for 24 hours and expanded in the presence of
IL-2 (10 U/ml) for 3–4 days in RPMI 1640 supplemented with 10%
FCS, 2% human serum, 5 × 10–5 M 2-mercapthoethanol, 1 mM
L-glutamine, 1 mM sodium pyruvate, 0.1 mM nonessential amino
acids, penicillin (100 U/ml), and streptomycin (100 μg/ml) (Sigma-
Aldrich). Cells were incubated in PBS containing 5 mM Fura-2
acetoxymethyl ester (Molecular Probes) for 30 minutes at room
temperature. The cells were subsequently washed and then resus-
pended at 1 × 106/ml in PBS. A total of 2 ml of the cell suspension
was placed in a continuously stirring cuvette at room temperature
in an LS50B spectrophotometer (Perkin-Elmer, Wellesley, Mas-
sachusetts, USA). Fluorescence was monitored at λex1 = 340 nm,
λex2 = 380 nm, and λem = 510 nm. The data are presented as the
relative ratio of fluorescence excited at 340 and 380 nm.
Actin polymerization. Human T cells were incubated either with
ghrelin (100 ng/ml) or positive control SDF-1 (100 ng/ml) for
20 min. Thereafter, cells were fixed and permeabilized in 2%
paraformaldehyde plus 0.1% Triton-X 100 and stained for actin
using phalloidin AF-594 and nucleus by DAPI.
Cytokine estimation. IL-1β, IL-6, and TNF-α were estimated in
T cell supernatants after 24 hours using commercial ELISA kits
according to manufacturer’s instructions (Biosource, Camarillo,
California, USA). Serum cytokines were analyzed using Bio-
Plex Mouse Cytokine 18-Plex Panel according to manufacturers
instructions (Biorad Laboratories, Hercules, California, USA).
Ghrelin estimation. Immunoreactive ghrelin was measured in dupli-
cate with an ELISA using a rabbit polyclonal antibody against full-
length, octanoylated human ghrelin that recognizes the acylated
and des-acyl forms of the hormone (3, 5) (Phoenix Pharmaceuticals,
Belmont, California, USA). Active ghrelin was estimated using RIA
(Linco Research Inc.) according to manufacturer’s instruction.
Real Time RT-PCR analysis. RT-PCR was performed as described
previously (51). Total RNA (2 μg) and oligo-dT primers were used
to synthesize single-stranded cDNA using the Reverse Transcrip-
tion kit (Life Technologies, Gaithersburg, Maryland, USA) accord-
ing to manufacturer’s instructions. The PCR was set up using
SYBR green Master Mix (Applied Biosystems, Foster City, Califor-
nia, USA), 1 μl cDNA, and gene-specific primers at a final concen-
tration of 0.3 μM. Thermal cycling was carried out on the Applied
Biosystems GeneAmp 7700 Sequence Detector, and SYBR green
dye intensity was analyzed using GeneAmp 7700 SDS software.
Primers for human IL-1β, IL-6, and TNF-α genes and GAPDH as
control were purchased from Biosource International (Camaril-
lo, California, USA); human GHS-R 1a and ghrelin were used as
described previously (9). Mouse IL-1β, IL-6, TNF-α, GAPDH, and
human GHS-R 1a primers were designed using ABI prism software
(Applied Biosystems). The PCR product of the GHS-R 1a amplifi-
cation was quantitated using the Agilent 2100 Bioanalyzer (Agi-
lent Technologies). Primers are available upon request. No PCR
products were generated from genomic versus cDNA template.
Statistical analysis. Results were expressed as the mean ± SEM. Sta-
tistical analysis was carried out by one-way ANOVA. Significant dif-
ferences between treatment groups were determined by the Student-
Newman-Keuls test; statistical significance was inferred at P < 0.05.
We thank the NIA Flow Cytometry Laboratory and Pheresis Unit
for their assistance. We also thank Dan Longo and Ashani Weera-
ratna (NIA), and Nahid Parvizi (Institut für Tierzucht und Tierver-
halten, Neustadt, Germany), for valuable input and discussions.
We thank Igor Espinoza’s laboratory at NIA for providing us some
of the purified monocytes utilized in our studies.
Received for publication January 21, 2004, and accepted in revised
form April 27, 2004.
Address correspondence to: Dennis D. Taub, Laboratory of Immu-
nology, National Institute on Aging, NIH, 5600 Nathan Shock
Drive, Baltimore, Maryland, 21224, USA. Phone: (410) 558-8181;
Fax: (410) 558-8284; E-mail: TaubD@grc.nia.nih.gov.
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