Impaired defense mechanism against inflammation, hyperalgesia, and airway hyperreactivity in somatostatin 4 receptor gene-deleted mice
ABSTRACT We have shown that somatostatin released from activated capsaicin-sensitive nociceptive nerve endings during inflammatory
processes elicits systemic anti-inflammatory and analgesic effects. With the help of somatostatin receptor subtype 4 gene–deleted
mice (sst4−/−), we provide here several lines of evidence that this receptor has a protective role in a variety of inflammatory disease
models; several symptoms are more severe in the sst4 knockout animals than in their wild-type counterparts. Acute carrageenan-induced paw edema and mechanical hyperalgesia, inflammatory
pain in the early phase of adjuvant-evoked chronic arthritis, and oxazolone-induced delayed-type hypersensitivity reaction
in the skin are much greater in mice lacking the sst4 receptor. Airway inflammation and consequent bronchial hyperreactivity elicited by intranasal lipopolysaccharide administration
are also markedly enhanced in sst4 knockouts, including increased perivascular/peribronchial edema, neutrophil/macrophage infiltration, mucus-producing goblet
cell hyperplasia, myeloperoxidase activity, and IL-1β, TNF-α, and IFN-γ expression in the inflamed lung. It is concluded that
during these inflammatory conditions the released somatostatin has pronounced counterregulatory effects through sst4 receptor activation. Thus, this receptor is a promising novel target for developing anti-inflammatory, analgesic, and anti-asthmatic
- SourceAvailable from: Erika Pintér[show abstract] [hide abstract]
ABSTRACT: 1Neurogenic plasma extravasation evoked by topical application of 1% vv−1 mustard oil on the skin of the acutely denervated rat hindleg (primary reaction) inhibited the development of a subsequent oil-induced plasma extravasation induced in the skin of the contralateral hindleg by 49.3±7.06% (n=9) and in the conjunctival mucosa due to 0.1% wv−1 capsaicin instillation by 33.5±10.05% (n=6). The primary reaction also inhibited the non-neurogenic hindpaw oedema evoked by s.c. injection of 5% wv−1 dextran into the chronically denervated hindpaw by 48.0±4.6% (n=5).2Capsaicin injection (100 μg ml−1 in 50 μl, s.c.) into the acutely denervated hindleg caused 56.5±4.0% (n=5) inhibition in the intensity of plasma extravasation elicited by 1% vv−1 mustard oil smearing on the contralateral side. After chronic denervation, subplantar injection of 5% wv−1 dextran elicited a non-neurogenic inflammatory response with intensive tissue oedema without causing any systemic anti-inflammatory effect. Bilateral adrenalectomy did not inhibit the mustard oil-induced anti-inflammatory effect in the contralateral hindleg.3Pretreating the rats with polyclonal somatostatin antiserum (0.5 ml rat−1, i.v.) or with the somatostatin depleting agent cysteamine (280 mg kg−1, s.c.) prevented the inhibitory action of mustard oil-induced inflammation on subsequent neurogenic plasma extravasation and strongly diminished the inhibition of non-neurogenic oedema formation evoked by dextran.4Exogenous somatostatin (10 μg kg−1, i.p.) caused a 30.3±8.3% (n=6) inhibition of plasma extravasation caused by mustard oil smearing on the acutely denervated hindleg and this inhibitory effect was abolished by somatostatin antiserum (0.5 ml rat−1, i.v.). The plasma level of somatostatin-like immunoreactivity (SST-LI) increased by 40.03±6.8% (n=6) 10 min after topical application of 1% vv−1 mustard oil on the acutely denervated hindpaws compared to the paraffin oil treated control group. Chronic denervation of the hindlegs or cysteamine (280 mg kg−1, s.c.) pretreatment prevented the mustard oil-induced elevation of SST-LI in plasma.5It is concluded that chemical excitation of the capsaicin-sensitive sensory receptors not only induces local neurogenic plasma extravasation but also inhibits the development of a subsequent inflammatory reaction at remote sites of the body in the rat. A role for somatostatin in this systemic anti-inflammatory effect is suggested.British Journal of Pharmacology (1998) 125, 916–922; doi:10.1038/sj.bjp.0702144British Journal of Pharmacology 09/1998; 125(4):916 - 922. · 5.07 Impact Factor
- [show abstract] [hide abstract]
ABSTRACT: The present review focuses on promising new opportunities for anti-inflammatory and analgesic therapy. The theoretical background is an original observation based on our own experimental results. These data demonstrate that somatostatin is released from capsaicin-sensitive, peptidergic sensory nerve endings in response to noxious heat and chemical stimuli such as vanilloids, protons or lipoxygenase products. It reaches distant parts of the body via the circulation and exerts systemic anti-inflammatory and analgesic effects. Somatostatin binds to G-protein-coupled membrane receptors (sst(1)-sst(5)) and diminishes neurogenic inflammation by prejunctional action on sensory-efferent nerve terminals, as well as by postjunctional mechanisms on target cells. It decreases the release of pro-inflammatory neuropeptides from sensory nerve endings and also acts on receptors of vascular endothelial, inflammatory and immune cells. Analgesic effect is mediated by an inhibitory action on peripheral terminals of nociceptive neurons, since circulating somatostatin cannot exert central action. Somatostatin itself is not suitable for drug development because of its broad spectrum and short elimination half life, stable, receptor-selective agonists have been synthesized and investigated. The present overview is aimed at summarizing the physiological importance of somatostatin and sst receptors, pharmacological significance of synthetic agonists and their potential in the development of novel anti-inflammatory and analgesic drugs. These compounds might provide novel perspectives in the pharmacotherapy of acute and chronic painful inflammatory diseases, as well as neuropathic conditions.Pharmacology [?] Therapeutics 12/2006; 112(2):440-56. · 7.79 Impact Factor
- Progress in brain research 02/1995; 104:161-73. · 4.19 Impact Factor
Impaired defense mechanism against inflammation,
hyperalgesia, and airway hyperreactivity in
somatostatin 4 receptor gene-deleted mice
Zsuzsanna Helyesa, Erika Pinte ´ra, Katalin Sa ´ndora, Krisztia ´n Elekesb, A´gnes Ba ´nvo ¨lgyia, Da ´niel Keszthelyia, E´va Szo ˝kea,
Da ´niel M. To ´thc, Zolta ´n Sa ´ndorc, La ´szlo ´ Kereskaid, Ga ´bor Pozsgaia, Jeremy P. Allene, Piers C. Emsone,
Adrienn Markovicsa, and Ja ´nos Szolcsa ´nyia,1
aDepartment of Pharmacology and Pharmacotherapy,bInstitute of Pharmacognosy,cAnalgesic Research Laboratory of Gedon Richter Plc.,
anddDepartment of Pathology, Faculty of Medicine, University of Pe ´cs, H-7624 Pe ´cs, Hungary; andeLaboratory of Molecular
Neuroscience, The Babraham Institute, Babraham Research Campus, Babraham, Cambridge CB22 3AT, United Kingdom
Edited by David Julius, University of California, San Francisco, CA, and approved June 3, 2009 (received for review January 21, 2009)
We have shown that somatostatin released from activated capsa-
icin-sensitive nociceptive nerve endings during inflammatory pro-
cesses elicits systemic anti-inflammatory and analgesic effects.
With the help of somatostatin receptor subtype 4 gene–deleted
mice (sst4?/?), we provide here several lines of evidence that this
receptor has a protective role in a variety of inflammatory disease
models; several symptoms are more severe in the sst4 knockout
animals than in their wild-type counterparts. Acute carrageenan-
induced paw edema and mechanical hyperalgesia, inflammatory
pain in the early phase of adjuvant-evoked chronic arthritis, and
oxazolone-induced delayed-type hypersensitivity reaction in the
skin are much greater in mice lacking the sst4 receptor. Airway
inflammation and consequent bronchial hyperreactivity elicited by
intranasal lipopolysaccharide administration are also markedly
enhanced in sst4knockouts, including increased perivascular/peri-
bronchial edema, neutrophil/macrophage infiltration, mucus-pro-
TNF-?, and IFN-? expression in the inflamed lung. It is concluded
tin has pronounced counterregulatory effects through sst4recep-
tor activation. Thus, this receptor is a promising novel target for
allergic contact dermatitis ? arthritis ? capsaicin-sensitive afferents ?
endotoxin-induced pneumonitis ? inflammatory cytokines
ings in response to chemical and antidromic electrical stimula-
tion of these fibers (1). Several lines of further functional
evidence have also indicated that somatostatin derived from
these nerves reaches the circulation and elicits systemic anti-
inflammatory and antinociceptive effects (1–4). Calcium influx
into the nerve terminals elicits the release of both proinflam-
matory (substance P and calcitonin gene-related peptide) and
anti-inflammatory neuropeptides without the involvement of
voltage-gated channels (3). Nevertheless, these peptides are only
partially colocalized in most primary afferents (5, 6), and
whereas substance P? and calcitonin gene-related peptide–
immunoreactive fibers are present in the superficial areas,
somatostatin-containing ones are only seen in deeper layers (7).
Therefore, it is likely that when noxious stimuli reach deeper
tissues—besides the local inflammatory response—the released
somatostatin elicits systemic anti-inflammatory actions too. This
as well as in endotoxin-induced airway inflammation and poly-
neuropathic conditions (9).
Somatostatin is widely distributed throughout the body in 14
aa- and 28 aa-containing forms (7, 10, 11). Besides its localiza-
e and others have previously reported that somatostatin is
released from capsaicin-sensitive nociceptive nerve end-
produced by neuroendocrine cells (gastrointestinal intrinsic neu-
rons, pancreas, and thyroid C cells), as well as inflammatory and
immune cells (lymphocytes, macrophages, thymic epithelial
cells, activated synovial cells, and fibroblasts) (4, 11, 13). Soma-
tostatin exerts a wide range of effects in the central nervous
system and in the periphery (for review see ref. 11), which are
mediated via 5 different Gi protein–associated somatostatin
receptor subtypes (sst1–sst5). They can be divided into 2 main
groups on the basis of sequence similarities and binding of
synthetic somatostatin analogues: the somatostatin release-
inhibiting factor 1 (SRIF1) group comprises sst2, sst3, and sst5
receptors, and the SRIF2group contains sst1and sst4receptors
(14). Each receptor is coupled to the inhibition of adenylate
cyclase and the cAMP/protein kinase A via pertussis toxin
sensitive GTP binding proteins (4, 10, 11). These inhibitory
actions of somatostatin might provide the link between sst4
receptor activation and modulation of other receptors and/or ion
channels (e.g., opioid or cannabinoid receptors). Furthermore,
sst receptor activation opens various K?channels and inhibits
voltage-gated Ca2?channels, which results in inhibition of both
ref. 4). Several data indicate that receptors in the SRIF1group
mediate the endocrine and antiproliferative effects, whereas the
SRIF2group, predominantly the sst4receptor, is responsible for
the anti-inflammatory and antinociceptive actions (3, 15, 16).
On the basis of these data we have raised the possibility that
this receptor may be a promising target for anti-inflammatory
and analgesic agents with a novel mechanism of action. Because
specific sst4receptor antagonists are not available, generation of
sst4receptor gene–deficient mice is a useful tool to elucidate the
role of these receptors in inflammatory processes.
The present article describes the generation of sst4receptor
knockout mice and investigates the involvement of the sst4
receptor in different acute and chronic inflammatory disease
Generation of sst4Receptor Gene-Deficient Mice. The generation of
the sst4lacZallele was essentially carried out as described for
sst2lacZanimals (17). In the sst4targeting cassette the Ura3/Neo
selection cassette was flanked by LoxP sites to enable its removal
Author contributions: Z.H., E.P., and J.S. designed research; Z.H., K.S., K.E., A.B., D.K., E.S.,
Z.S., L.K., G.P., and A.M. performed research; J.P.A. and P.C.E. contributed new reagents/
analytic tools; K.S., K.E., and D.M.T. analyzed data; and Z.H., E.P., and J.S. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
1To whom correspondence should be addressed. E-mail: email@example.com.
This article contains supporting information online at www.pnas.org/cgi/content/full/
August 4, 2009 ?
vol. 106 ?
after targeting (Fig. 1A). The vector was transfected into 129/
Ola-derived E-14 embryonic stem cells by electroporation, fol-
lowed by G418 selection. Correctly targeted clones carrying a
sst4?/lacZUra3neoallele were identified by Southern blot analysis
using probes A and B and microinjected into C57BL/6 blasto-
cysts to produce chimeric mice. Chimeric males were mated to
C57BL/6 to generate heterozygous animals. Heterozygous in-
tercross of sst4lacZ/Ura3/Neomice produced viable and fertile litters
without obvious abnormalities at the expected Mendelian ratio.
The genotype was determined by PCR and Southern blot
analysis (Fig. 1B). Heterozygotes were then mated with Cre-
deletor mice (18) that express Cre recombinase in germ cells.
Deletion of the loxP-flanked selection cassette in F1animals to
create the allele sst4lacZwas verified by Southern blot using probe
B. The sst4lacZallele was then backcrossed to C57BL/6 mice for
a further 10 generations using males and females sst4?/lacZ
alternately. Homozygous sst4lacz/lacZand WT (sst4?/?) littermates
were generated by intercrossing sst4?/lacZmice from the 10th
generations of backcross mice. No sst4mRNA was detectable in
the sst4lacZmice, whereas lacZ was expressed (Fig. 1C). Animals
were produced in accordance with United Kingdom Home
Office guidelines and licensed under the Animals (Scientific
Procedures) Act of 1986.
Increased Carrageenan-Induced Acute Mechanical Hyperalgesia and
Paw Edema in sst4?/?Mice. The control paw volumes (sst4?/?:
0.67 ? 0.02 cm3; sst4?/?: 0.68 ? 0.02 cm3) and the mechanono-
ciceptive thresholds (sst4?/?: 8.3 ? 0.16 g; sst4?/?: 8.0 ? 0.14 g)
of the 2 groups did not differ significantly. Carrageenan admin-
istration caused inflammation of the treated paw with a marked
swelling, redness, and decrease of the mechanonociceptive
threshold. Six hours after the injection both edema and the drop
of the nociceptive threshold were significantly greater in the
knockout group. The results of WT and sst4, receptor-deficient
littermates produced from heterozygote mice were essentially
the same as data obtained in sst4?/?and sst4?/?mice bred as
separate lines. Pretreatment with the selective peptidomimetic
sst4 receptor agonist J-2156 (100 ?g/kg, i.p.) (19–21) 10 min
before carrageenan significantly inhibited mechanical hyperal-
gesia in WT mice but not in the knockouts. Surprisingly, J-2156
did not inhibit paw swelling in either group (Fig. 2 A and B).
Adjuvant-Induced Chronic Paw Inflammation, Mechanical Hyperalge-
sia, and Impaired Spontaneous Weight Bearing in sst4?/?Mice. In-
flammatory mechanical hyperalgesia was greater in sst4?/?mice
than in their WT counterparts throughout the whole 21-day
experimental period, but the difference was more pronounced
and shown to be significant up to the 13th day of the study.
Similarly, until this time significantly less weight was distributed
on the treated hindlimb in the sst4?/?than in the sst4?/?group,
but no difference could be observed later. Edema did not differ
significantly in the 2 groups: paw swelling was between 80% and
100% throughout the whole period in both sst4?/?and sst4?/?
Greater Endotoxin-Evoked Inflammatory Changes in the Lung and
Enhanced Airway Responsiveness in sst4?/?Mice. On the histologic
slides marked peribronchial/perivascular edema, granulocyte
accumulation, mononuclear cell infiltration, and hyperplasia of
mucus-producing goblet cells were seen in response to LPS
administration in both groups. In sst4?/?mice the extent of the
edema was greater, and the destruction of the alveolar spaces by
the large number of infiltrating leukocytes and the hyperplasia
of goblet cells were much more pronounced. The detailed scores
sequence of sst4resides on a single exon (white box). A replacement vector was designed to delete the entire sst4coding sequence and replace it with a cassette
initiation codon of lacZ is inserted, in frame, into the initiation codon of sst4. (B) Southern blot analysis of genomic DNA prepared from tail biopsies of an sst4
mutant pedigree shows correct targeting. DNAs were digested with BclI and hybridized with probe A or digested with SacI and hybridized with probe B (sst4?/?:
?/?; sst4?/lacZUra3neo: ?/-). (C) RT-PCR analysis of sst4and lacZ transcription. RT-PCR from total brain RNA using multiplexed sst4and lacZ primer sets demonstrates
sst4?/?animals (sst4?/?: ?/?; sst4?/lacZ: ?/-; sst4lacZ/lacZ: -/-). No amplification was observed when RT-PCR was performed in the absence of reverse transcriptase (No
RT Control) or when PCR was performed in the absence of cDNA template (No DNA).
Generation of sst4receptor gene–deficient mice. (A) Targeting strategy used for gene disruption of the mouse sst4genomic locus. The 1155-bp coding
Helyes et al.PNAS ?
August 4, 2009 ?
vol. 106 ?
no. 31 ?
are shown in the supporting information (SI) Text; the repre-
sentative light micrographs and composite values are presented
in Fig. 3 A and B. In agreement with the histologic pictures,
myeloperoxidase (MPO) activity increased in response to LPS
instillation in both groups. This quantitative biochemical marker
of accumulated granulocytes in the inflamed tissue was signif-
icantly (approximately 3-fold) greater in the sst4?/?than in the
sst4?/?mice (Fig. 3C). Furthermore, LPS induced a 12–30-fold
increase of IL-1?, IFN-?, and TNF-? concentrations in the lung
of WT mice, which was almost double in the sst4gene–deleted
group (Fig. 3D).
Flow cytometric analysis of the bronchoalveolar lavage fluid
(BALF) samples showed a remarkable accumulation of macro-
phages and lymphocytes in response to LPS in both groups, and
the number of these cells was significantly greater in sst4?/?mice
knockouts, which is in agreement with the predominantly peri-
bronchiolar/perivascular infiltration of these cells seen on the
histologic slides. Similarly to the lung homogenates, the concen-
tration of all of the 3 measured inflammatory cytokines mark-
edly increased in the BALF of LPS-treated mice compared with
noninflamed samples. Deletion of the sst4receptor significantly
enhanced IFN-? and TNF-? levels, but not IL-1? in the washing
fluid (Fig. S1).
Both baseline enhanced pause (Penh) and lung resistance
(RL) significantly increased after intranasal LPS instillation
compared with PBS-treated, noninflamed control values, with
no difference between the 2 mouse groups (SI Text). Carbachol
inhalation evoked a concentration-dependent bronchoconstric-
tion shown by both the Penh and RL curves. There was no
significant difference between the carbachol-evoked responses
of PBS-treated sst4?/?and sst4?/?animals. Responses demon-
strated as percentage increase of Penh and RL above baseline
were markedly enhanced in the LPS-treated groups compared
of the paw volume 6 h after intraplantar carrageenan (50 ?L, 3%) injection in
sst4?/?and sst4?/?mice. Percentage changes are calculated by comparing the
data with the initial self-control values. Results obtained in sst4?/?and sst4?/?
mice bred as separate lines, as well as littermate WT and knockouts, are
indicated. The effect of the selective sst4receptor agonist J-2156 (100 ?g/kg
i.p.) is also shown in both group (means ? SEM of n ? 6–8 experiments; *P ?
0.05,**P?0.01vsthesst4?/?,group,?P ? 0.05vssolventtreatedsst4?/?mice
determined by one-way ANOVA followed by Bonferroni’s modified t test).
(A) Decrease of the mechanonociceptive thresholds and (B) increase
kine concentrations of the lung in LPS-induced inflammation. (A) Represen-
tative histopathologic pictures of the lung samples of sst4?/?and sst4?/?mice
(periodic acid-Schiff staining; ?200; Br, bronchioles; V, vessels; open arrows
black arrowheads indicate granulocyte accumulation). (B) Semiquantitative
evaluation and scoring of the lung on the basis of perivascular edema,
and alveolar mononuclear cell infiltration. Columns represent medians with
upper and lower quartiles of n ? 8–10 mice;?P ? 0.01 LPS-treated inflamed
vs. respective PBS-treated noninflamed mice; *P ? 0.05 LPS-treated sst4?/?vs.
LPS-treated sst4?/?group (Kruskal-Wallis test plus Dunn’s posttest). (C) MPO
activity, as a quantitative indicator of the number of accumulated granulo-
cytes, and (D) concentrations IL-1?, IFN-?, and TNF-? determined from ho-
0.01 vs. the respective noninflamed mice;??P ? 0.01 vs. the sst4?/?group
(one-way ANOVA plus Bonferroni’s t test).
Histopathologic examination, MPO activity, and inflammatory cyto-
www.pnas.org?cgi?doi?10.1073?pnas.0900681106 Helyes et al.
with the respective noninflamed controls, showing the develop-
ment of inflammatory bronchial hyperresponsiveness. The max-
imal values were higher, the duration of bronchoconstriction was
longer, and therefore the percentage increase of the responses
above baseline was greater in mice lacking the sst4 receptor
(original records in Fig. S2). Comparison of the Penh responses
determined in unrestrained mice and RL values measured in
anesthetized and mechanically ventilated animals provided very
similar results. This supports our previously published conclu-
sions (9, 21) that although the validity of Penh in asthma models
can be questioned, this is a reliable parameter to determine
airway reactivity in the endotoxin-induced subacute airway
inflammation model (Fig. 4 A and B).
More Severe Oxazolone-Induced Allergic Contact Dermatitis in sst4?/?
Mice. In WT mice, oxazolone induced an approximately 80–
100% increase of ear thickness 24–48 h after its topical appli-
cation. In knockout animals the edema was approximately
double, 180–200% (Fig. 5A). Histologic analysis also showed
greater swelling and even more intensive accumulation of in-
flammatory cells in the oxazolone-treated ears of sst4?/?mice
compared with their sst4?/?controls (Fig. S3). Slightly raised
levels of IL-4 and IL-5 cytokines and a remarkable elevation of
IL-1? were detected in the ear samples 24 h after oxazolone
smearing, but there was no difference between the 2 groups. At
48 h TNF? and IL-1? concentrations were higher in sst4?/?mice
than in WT animals (Fig. 5B).
The present article describes the generation and the first in vivo
data on sst4receptor knockout mice and provides several lines
hyperreactivity in sst4?/?and sst4?/?mice. The panels demonstrate the per-
centage increases of (A) Penh and (B) RL above baseline [(mean values in
response to the respective carbachol concentration- baseline values)/baseline
values ? 100] calculated in each 15-min period after respective carbachol
stimulations. (C) Inflammatory cells accumulated in the BALF 24 h after
intranasal endotoxin administration in sst4?/?and sst4?/?littermate mice.
plus Bonferroni’s modified t test).
Carbachol-evoked bronchoconstriction and inflammatory airway
concentrations of the ear of sst4?/?and sst4?/?mice. Edema data points
represent means ? SEM percentage increase of ear diameter compared with
mice. Columns showing cytokine concentrations in the ear homogenates 48 h
vs. sst4?/?(one-way ANOVA followed by Bonferroni’s modified t test).
Oxazolone-induced (A) swelling and (B) inflammatory cytokine
Helyes et al. PNAS ?
August 4, 2009 ?
vol. 106 ?
no. 31 ?
inflammatory and nociceptive processes. Because sst4-selective
antagonists are not available, experiments with these genetically
manipulated mice are particularly important to elucidate the
role of sst4receptors in physiologic and pathophysiologic con-
ditions. The inhibitory actions of sensory nerve–derived soma-
tostatin in these well-established experimental models of inflam-
mation have been previously shown (1, 8, 9, 22–24). This unique,
systemic efferent protective function of these afferents was
strongly supported in disease models such as adjuvant-induced
chronic arthritis (8), endotoxin-evoked lung inflammation (9),
oxazolone-induced allergic contact dermatitis (23), and bleomy-
cin-evoked scleroderma (25). Direct evidence for the involve-
ment of somatostatin of neural origin in the observed anti-
inflammatory and analgesic actions was also provided (1–3, 8, 9).
Results obtained with synthetic somatostatin receptor agonists
suggested that these inhibitory effects were mediated by recep-
tors of the SRIF2 family (sst4/sst1) (8, 11, 15, 16, 19–21, 25).
However, because of the lack of sst4-selective antagonists, the
precise receptor mechanisms of the somatostatin-mediated
‘‘sensocrine’’ inhibitory actions on inflammation and nocicep-
tion have remained to be elucidated until the generation of sst4
receptor gene–deficient mice.
Exogenous administration of somatostatin diminishes neuro-
genic vasodilatation, plasma protein extravasation, and the
release of substance P from nerve endings (2). Inhibition of
proliferation, infiltration, chemotaxis, and phagocytosis, as well
as the release of reactive oxygen radicals and cytokines of
inflammatory cells, are also well-documented effects of soma-
tostatin (13, 16, 26–33). Sst4 immunoreactivity was shown on
vascular endothelial and smooth muscle cells (34), and its
activation exerted protective effects (35). Sst4receptors are also
present on peripheral blood monocytes, lymphocytes, fibro-
blasts, and endothelial cells (36, 37). In experimental rat arthritis
predominantly sst3and sst4are expressed on immune cells, and
in agreement with our earlier data, others also found that
octreotide did not exert immunomodulatory actions in this
model (11). The presence and inflammation-induced upregula-
tion of sst4were observed in the human kidney and on capsaicin-
sensitive afferents (15, 19). Opioid and cannabinoid receptors on
nerves and inflammatory cells also linked to Giproteins (38, 39)
have been described as being involved in somatostatin signaling,
which raises the possibility that knocking out the sst4receptor
gene might also influence their function in inflammatory and
released during different inflammatory processes exerts a potent
endogenous protective mechanism against edema formation and
pathomorphologic and immunologic changes, as well as inflam-
matory pain and airway hyperresponsiveness. The edema-
reducing effect of sst4 receptor activation, however, was ob-
served in the carrageenan-evoked acute inflammation and the
allergic contact dermatitis models but not in adjuvant-induced
chronic paw swelling. Although the increase of somatostatin-like
complete Freund’s adjuvant (CFA) model of rats (8), its sst4
receptor–mediated inhibitory action on swelling is likely to be
overridden by other immune cell–derived mediators, such as
bradykinin, prostaglandins, histamine, serotonin, or leukotri-
enes. This seems to be the explanation for the disappearance of
13th day of the experiment, by which time macrophages and
lymphocytes have accumulated in the inflamed area (8).
Our previous data revealed that synthetic sst4receptor ago-
nists—the heptapeptide TT-232 and the peptidomimetic
with neurogenic and nonneurogenic mechanisms, such as mus-
tard oil-, dextran-, carrageenan-, or bradykinin-induced acute
plasma protein extravasation (15, 16, 19), adjuvant-induced
acute and ovalbumin-evoked chronic lung inflammation and
hyperresponsiveness (21). In most models the extent of the
inhibition achieved by these agonists was similar to the differ-
experiment J-2156 inhibited carrageenan-evoked mechanical
hyperalgesia, but in mice unlike in rats it did not alter paw
In conclusion, with the help of genetically manipulated mice
we describe evidence for a previously uncharacterized counter-
regulatory mechanism mediated by sst4receptors during inflam-
matory and nociceptive processes. On the basis of these results,
the sst4 receptors localized on vascular endothelial, smooth
muscle, inflammatory, and immune cells, as well as synoviocytes,
may serve as promising new targets for the development of
broad-spectrum anti-inflammatory and analgesic drugs.
Animals. Heterozygous mice (sst4?/?) were paired in the Laboratory Animal
Center of Pe ´cs University, and the genotype of their offsprings was deter-
mined by PCR. Sst4receptor gene–deficient (sst4?/?) and WT mice (sst4?/?)
were then successfully bred on; animals used for the experiments were males
strictly within the first 3 inbred generations. In the carrageenan- and endo-
toxin-evoked inflammation models sst4?/?and sst4?/?littermates were also
tested for comparison. In the behavioral tests the experimenter was blind to
separate lines were tested simultaneously with littermate controls in this
model. Carrageenan (3%, 50 ?L) was injected intraplantarly into the left
hindpaw to induce subacute inflammation. The mechanonociceptive thresh-
old of the paw was determined with dynamic plantar esthesiometry (Ugo
Basile 37400) and the volume with plethysmometry (Ugo Basile 7140) before
and 6 h after carrageenan injection. Separate groups of sst4?/?and sst4?/?
littermates were pretreated with the selective sst4receptor agonist J-2156
(100 ?g/kg i.p. 10 min before carrageenan). Data were expressed as percent-
age changes compared with the self-control values obtained before the
induction of inflammation (20).
Adjuvant-Evoked Chronic Inflammation Model. Inflammation of the tibiotarsal
joints was evoked by s.c. injection of complete CFA (killed Mycobacteria
and the root of the tail. Paw volume was measured by plethysmometry,
mechanical touch sensitivity by esthesiometry, and weight bearing with an
incapacitance tester (Linton Instrumentation) before and 9 times after CFA
administration during a 21-day period.
Endotoxin-Induced Airway Inflammation Model. Subacute pneumonitis was
evoked by 60 ?L Escherichia coli LPS (167 ?g/mL dissolved in sterile PBS)
applied intranasally 24 h before measurement (9, 21, 22).
Airway responsiveness was determined by whole-body plethysmography
(6) and anesthetized, tracheotomized, mechanically ventilated mice. In unre-
strained animals the Penh, calculated as [(expiratory time/relaxation time) ?
1]/(maximum expiratory flow/maximum inspiratory flow), was determined as
an indicator of bronchoconstriction (9, 21, 22). In separate groups of sst4?/?
and sst4?/?littermates RL (?cmH2O * sec/mL) was directly measured. The
muscarinic receptor agonist carbachol (carbamylcholine; 5.5, 11, and 22 mM,
50 ?L per mouse for 50 sec) was nebulized to induce bronchoconstriction 24 h
after LPS instillation. Mean Penh and RL of the 15-min measurement periods
after each carbachol were expressed as percentage increases above baseline
(for details see SI Text). At the end of the study bronchoalveolar lavage was
performed in 1 series of experiments, and the lungs were excised for further
(histologic, cytokine, and myeloperoxidase) examinations in the other series
Semiquantitative scoring of the inflammatory changes in the lung sections
producing goblet cells) was performed by an expert pathologist blind to the
experimental design, on the basis of perivascular edema, perivascular/
peribronchial acute inflammation, goblet cell hyperplasia/metaplasia of the
bronchioles, and mononuclear cell infiltration into the alveolar spaces (9, 21,
www.pnas.org?cgi?doi?10.1073?pnas.0900681106 Helyes et al.
22, 40). The composite inflammation scores (ranging from 0 to 10) were
obtained by adding these values. MPO activity indicating the number of
accumulated granulocytes and macrophages in the lung was determined by
In a separate series of experiments, the number of inflammatory cells
(granulocytes, lymphocytes, and macrophages) in the BALF (21) was deter-
mined with a Partec CyFlow space flow cytometer. Differentiation was made
on the basis of the size and granulation; cell numbers were calculated by
FloMax software (Partec). The concentrations of 3 inflammatory cytokines—
IL-1?, TNF-?, and IFN-?—were measured with ELISA (BD Sciences and R&D
Systems) from both the lung homogenates and BALF samples (all technical
details are described in SI Text).
Oxazolone-Induced Allergic Contact Dermatitis Model (Delayed-Type Hypersen-
sitivity Reaction). Animals were sensitized on 2 consecutive days by smearing
2% oxazolone dissolved in 96% ethanol (50–50 ?L) on the shaved abdomen.
Six days later oxazolone was applied on the right ears (15–15 ?L on both
surfaces) to elicit allergic contact dermatitis; the ethanol-treated left ears
served as controls (23). Ear diameter was measured with an engineer’s micro-
smearing. Data were expressed as percentage increase of ear thickness com-
the histologic slides (6-?m cross-sections, hematoxylin and eosin staining).
Inflammatory cytokines (IL-2, IL-4, IL-5, IFN-?, and TNF-?) were determined by
cytokine cytometric bead array (Becton Dickinson Biosciences), and IL-1? was
measured with ELISA (for details see SI Text).
Statistical Analysis. Most results were expressed as means ? SEM of n ? 8–10
mice in each group and evaluated by one-way ANOVA followed by Bonfer-
roni’s modified t test. Histologic scores were demonstrated as box plots and
analyzed with Kruskal-Wallis test followed by Dunn’s test. In all cases P ? 0.05
was considered significant.
Act of the Hungarian Parliament on Animal Protection (243/1988), complied
the Ethics Committee on Animal Research of Pe ´cs University (license no.
ACKNOWLEDGMENTS. The authors thank Aniko ´ Perkecz for the histologic
slides and Helga Rabovszky for the expert breeding of the animals. This work
was sponsored by Hungarian Grants OTKA K73044, OTKA NK78059, RET-008/
2005, ETT-06–284/2006, ETT-287/2006, and GVOP-3.2.1–2004-04–0420/3.0.
Z.H. was supported by a Janos Bolyai Postdoctoral Fellowship.
1. Szolcsa ´nyi J, Helyes Z, Oroszi G, Ne ´meth J, Pinte ´r E (1998) Release of somatostatin and
its role in the mediation of the anti-inflammatory effect induced by antidromic
stimulation of sensory fibres of rat sciatic nerve. Br J Pharmacol 123:936–942.
2. Szolcsa ´nyi J, Pinte ´r E, Helyes Z, Oroszi G, Ne ´meth J (1998) Systemic anti-inflammatory
effect induced by counter-irritation through a local release of somatostatin from
nociceptors. Br J Pharmacol 125:916–922.
3. Szolcsa ´nyi J, Pinte ´r E, Helyes Z (2004) Sensocrine function of capsaicin-sensitive noci-
Hyperalgesia: Molecular Mechanisms and Clinical Implications, eds Handwerker HO,
Brune K (IASP Press, Seattle), pp 113–128.
4. Pinte ´rE,HelyesZ,Szolcsa ´nyiJ(2006)Inhibitoryeffectofsomatostatinoninflammation
and nociception. Pharmacol Ther 112:440–456.
5. Hokfelt T, et al. (1976) Immunohistochemical evidence for separate populations of
somatostatin-containing and substance P-containing primary afferent neurons in the
rat. Neuroscience 1:131–136.
6. Lawson SN (1995) Neuropeptides in morphologically and functionally identified pri-
mary afferent neurons in dorsal root ganglia: Substance P, CGRP and somatostatin.
Prog Brain Res 104:161–173.
7. Dux M, et al. (1999) Changes in fibre populations of the rat hairy skin following
selective chemodenervation by capsaicin. Cell Tissue Res 296:471–477.
from capsaicin-sensitive sensory nerve terminals in a Freund’s adjuvant-induced
chronic arthritis model in the rat. Arthritis Rheum 50:1677–1685.
9. Helyes Z, et al. (2007) Role of transient receptor potential vanilloid 1 receptors in
endotoxin-induced airway inflammation in the mouse. Am J Physiol Lung Cell Mol
10. Patel YC, et al. (1995) The somatostatin receptor family. Life Sci 57:1249–1265.
11. ten Bokum AM, Hofland LJ, van Hagen PM (2000) Somatostatin and somatostatin
receptors in the immune system: A review. Eur Cytokine Netw 11:161–176.
12. Maggi CA (1995) Tachykinins and calcitonin gene-related peptide (CGRP) as co-
transmitters released from peripheral endings of sensory nerves. Prog Neurobiol
A review. Eur J Clin Invest 24:91–99.
14. Hoyer D, et al. (1995) Classification and nomenclature of somatostatin receptors.
Trends Pharmacol Sci 16:86–88.
the rat. Br J Pharmacol 134:1571–1579.
16. Pinte ´r E, et al. (2002) Pharmacological characterisation of the somatostatin analogue
TT-232: Effects on neurogenic and non-neurogenic inflammation and neuropathic
hyperalgesia. Naunyn Schmiedebergs Arch Pharmacol 366:142–150.
17. Allen JP, et al. (2003) Somatostatin receptor 2 knockout/lacZ knockin mice show
impaired motor coordination and reveal sites of somatostatin action within the
striatum. Eur J Neurosci 17:1881–1895.
18. Schwenk F, Baron U, Rajewsky K (1995) A cre-transgenic mouse strain for the ubiqui-
tous deletion of loxP-flanked gene segments including deletion in germ cells. Nucleic
Acids Res 23:5080–5081.
19. Helyes Z, et al. (2006) Effects of the somatostatin receptor subtype 4 selective agonist
J-2156 on sensory neuropeptide release and inflammatory reactions in rodents. Br J
20. Sa ´ndor K, et al. (2006) Analgesic effects of the somatostatin sst4receptor selective
agonist J-2156 in acute and chronic pain models. Eur J Pharmacol 539:71–75.
21. Elekes K, et al. (2008) Inhibitory effects of synthetic somatostatin receptor subtype 4
agonists on acute and chronic airway inflammation and hyperreactivity in the mouse.
Eur J Pharmacol 578:313–322.
22. Elekes K, et al. (2007) Role of capsaicin-sensitive afferents and sensory neuropeptides
in endotoxin-induced airway inflammation and consequent bronchial hyperreactivity
in the mouse. Regul Pept 141:44–54.
23. Ba ´nvo ¨lgyi A´, et al. (2005) Evidence for a novel protective role of the vanilloid TRPV1
24. Pinte ´r E, Szolcsa ´nyi J (1996) Systemic anti-inflammatory effect induced by antidromic
stimulation of the dorsal roots in the rat. Neurosci Lett 212:33–36.
25. Szabo ´ A´, et al. (2008) Investigation of sensory neurogenic components in a bleomycin-
induced scleroderma model using transient receptor potential vanilloid 1 receptor-
and calcitonin gene-related peptide-knockout mice. Arthritis Rheum 58:292–301.
26. Elliott DE, et al. (1999) SSTR2A is the dominant somatostatin receptor subtype ex-
pressed by inflammatory cells, is widely expressed and directly regulates T cell IFN-
gamma release. Eur J Immunol 29:2454–2463.
27. Muscettola M, Grasso G (1990) Somatostatin and vasoactive intestinal peptide reduce
interferon gamma production by human peripheral blood mononuclear cells. Immu-
28. Berman AS, Chancellor-Freeland C, Zhu G, Black PH (1996) Substance P primes murine
peritoneal macrophages for an augmented proinflammatory cytokine response to
lipopolysaccharide. Neuroimmunomodulation 3:141–149.
29. Niedermuhlbichler M, Wiedermann CJ (1992) Suppression of superoxide release from
human monocytes by somatostatin-related peptides. Regul Pept 41:39–47.
30. Chowers Y, et al. (2000) Somatostatin through its specific receptor inhibits spontane-
ous and TNF-alpha- and bacteria-induced IL-8 and IL-1 beta secretion from intestinal
epithelial cells. J Immunol 165:2955–2961.
31. Sener G, Cetinel S, Erkanli G, Gedik N, Yegen BC (2005) Octreotide ameliorates
sepsis-induced pelvic inflammation in female rats by a neutrophil-dependent mecha-
nism. Peptides 26:493–499.
32. Partsch G, Matucci-Cerinic M (1992) Effect of substance P and somatostatin on migra-
tion of polymorphonuclear (PMN) cells in vitro. Inflammation 16:539–547.
33. Kolasinski SL, Haines KA, Siegel EL, Cronstein BN, Abramson SB (1992) Neuropeptides
and inflammation. A somatostatin analog as a selective antagonist of neutrophil
activation by substance P. Arthritis Rheum 35:369–375.
34. Torrecillas G, Medina J, Diez-Marques ML, Rodriguez-Puyol D, Rodriguez-Puyol M
(1999) Mechanisms involved in the somatostatin-induced contraction of vascular
smooth muscle cells. Peptides 20:929–935.
35. Tigerstedt NM, Aavik E, Aavik S, Savolainen-Peltonen S, Hayry P (2007) Vasculoprotec-
tive effects of somatostatin receptor subtypes. Mol Cell Endocrinol 279:34–38.
36. ten Bokum AM, et al. (1999) Somatostatin receptor subtype expression in cells of the
rat immune system during adjuvant arthritis. J Endocrinol 161:167–175.
37. Taniyama Y, et al. (2005) Systemic distribution of somatostatin receptor subtypes in
human: An immunohistochemical study. Endocr J 52:605–611.
selectivity toward mu opioid receptors. Life Sci 38:2221–2229.
39. Va ´squez C, Lewis DL (1999) The CB1 cannabinoid receptor can sequester G-proteins,
making them unavailable to couple to other receptors. J Neurosci 19:9271–9280.
40. Zeldin DC, et al. (2001) Airway inflammation and responsiveness in prostaglandin H
synthase-deficient mice exposed to bacterial lipopolysaccharide. Am J Respir cell Mol
Helyes et al.PNAS ?
August 4, 2009 ?
vol. 106 ?
no. 31 ?