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Dissection of the Anti-Inflammatory Effect of the Core and C-Terminal (KPV) -Melanocyte-Stimulating Hormone Peptides


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In this study, we analyzed the anti-inflammatory effects of alpha-melanocyte stimulating hormone (MSH)11-13 (KPV) in comparison with other MSH peptides in a model of crystal-induced peritonitis. Systemic treatment of mice with KPV, alpha-MSH, the core melanocortin peptide His-Phe-Arg-Trp, and the melanocontin receptor 3/4 agonist Ac-Nle4-c[Asp5,d-Phe7,Lys10]NH2 ACTH4-10 (MTII) but not the selective MC1-R agonist H-Ser-Ser-Ile-Ile-Ser-His-Phe-Arg-Trp-Gly-Lys-Pro-Val-NH2 (MS05) resulted in a significant reduction in accumulation of polymorphonuclear leukocyte in the peritoneal cavity. The antimigratory effect of KPV was not blocked by the MC3/4-R antagonist Ac-Nle4-c[Asp5,d-2Nal7,Lys10]NH2 ACTH4-10 (SHU9119). In vitro, macrophage activation, determined as release of KC and interleukin (IL)-1beta was inhibited by alpha-MSH and MTII but not by KPV. Furthermore, macrophage activation by MTII led to an increase in cAMP accumulation, which was attenuated by SHU9119, whereas KPV failed to increase cAMP. The anti-inflammatory properties of KPV were also evident in IL-1beta-induced peritonitis inflammation and in mice with a nonfunctional MC1-R (recessive yellow e/e mice). In conclusion, these data highlight that the C-terminal MSH peptide KPV exhibits an anti-inflammatory effect that is clearly different from that of the core MSH peptides. KPV is unlikely to mediate its effects through melanocortin receptors but is more likely to act through inhibition of IL-1beta functions.
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Dissection of the Anti-Inflammatory Effect of the Core and C-
Terminal (KPV)
-Melanocyte-Stimulating Hormone Peptides
The William Harvey Research Institute, London, UK (S.J.G., M.P.); and Department of Neuroscience, Uppsala University, Uppsala, Sweden
Received March 14, 2003; accepted May 9, 2003
In this study, we analyzed the anti-inflammatory effects of
-melanocyte stimulating hormone (MSH)
(KPV) in com-
parison with other MSH peptides in a model of crystal-induced
peritonitis. Systemic treatment of mice with KPV,
-MSH, the core
melanocortin peptide His-Phe-Arg-Trp, and the melanocontin re-
ceptor 3/4 agonist Ac-Nle
(MTII) but not the selective MC1-R agonist H-Ser-Ser-Ile-Ile-Ser-
(MS05) resulted in a signif-
icant reduction in accumulation of polymorphonuclear leukocyte
in the peritoneal cavity. The antimigratory effect of KPV was not
blocked by the MC3/4-R antagonist Ac-Nle
ACTH4-10 (SHU9119). In vitro, macrophage ac-
tivation, determined as release of KC and interleukin (IL)-1
inhibited by
-MSH and MTII but not by KPV. Furthermore, mac-
rophage activation by MTII led to an increase in cAMP accumu-
lation, which was attenuated by SHU9119, whereas KPV failed
to increase cAMP. The anti-inflammatory properties of KPV
were also evident in IL-1
-induced peritonitis inflammation
and in mice with a nonfunctional MC1-R (recessive yellow
e/e mice). In conclusion, these data highlight that the C-
terminal MSH peptide KPV exhibits an anti-inflammatory
effect that is clearly different from that of the core MSH
peptides. KPV is unlikely to mediate its effects through mela-
nocortin receptors but is more likely to act through inhibition
of IL-1
The pro-opiomelanocortin gene product undergoes post-
translational processing to form the endogenous ligands of
the melanocortin receptors [
-melanocyte stimulating
hormone (MSH)] and adrenocorticotropin (ACTH), which all
contain the common amino acid motif His-Phe-Arg-Trp
(HFRW) tetrapeptide (Wikberg et al., 2000; Getting, 2002).
Five melanocortin receptors (MC-Rs) have been cloned and
are positively coupled to adenylate cyclase, thus receptor
activation leads to increases in intracellular cAMP (Wikberg
et al., 2000; Getting, 2002). These endogenous peptides are
endowed with anti-inflammatory properties, including inhi-
bition of tumor necrosis factor-
, interleukin (IL)-1, and the
CXC chemokine KC release (Getting, 2002) as well as adhe-
sion molecule expression (Kalden et al., 1999). This is possi-
bly due to their ability to inhibit nuclear transcription fac-
B activation (Manna and Aggarwal, 1998; Kalden et al.,
1999) and protection of I
degradation (Ichiyama et al.,
1999), thus affecting the humoral and cellular phases of
inflammation (Hiltz and Lipton, 1989; Lipton and Catania,
1998). These anti-inflammatory properties have been high-
lighted in several experimental models of acute and chronic
inflammation (for a recent review, see Getting, 2002).
At present, there is a lot of confusion within the field of
whether a single MC-R mediates the anti-inflammatory ef-
fects of melanocortin peptides. One of the receptors, MC1-R,
has long been regarded as the receptor responsible for the
anti-inflammatory effects of
-MSH and related peptides
(Wikberg et al., 2000), whereas more recently we have pro-
posed a central role for MC3-R (Getting et al., 1999, 2001).
The MC1-R mRNA, but not protein, expression has been
found in an array of cells, including monocytes, B-lympho-
cytes, NK cells, a subset of cytoxic T cells (Neumann
Andersen et al., 2001), dendritic cells (Becher et al., 1999) as
well as mast cells (Adachi et al., 1999). The expression of
MC3-R mRNA and protein has been detected in rodent peri-
toneal and knee joint macrophages (MØ). Importantly, the
receptor is functional because its activation leads to cAMP
accumulation. In a series of inflammatory models, the rela-
tively selective agonists have been shown to down-regulate
the host inflammatory response, and this inhibition was ab-
rogated in the presence of MC3-R, but not MC4-R antago-
nists (Getting et al., 1999, 2001, 2002).
This work was supported by the Arthritis Research Campaign UK (Grant
G0571). M.P. is a Senior Fellow of the Association pour la Recherche sur le
Cancer, UK. H.S. was supported by the Swedish Research Council (VR, medi-
cin) and Melacure Therapeutics AB.
Article, publication date, and citation information can be found at
DOI: 10.1124/jpet.103.051623.
ABBREVIATIONS: MSH, melanocyte stimulating hormone; ACTH, adrenocorticotropin; MC-R, melanocortin receptor; IL, interleukin; MØ, macrophage;
MSU, monosodium urate; PBS, phosphate-buffered saline; ELISA, enzyme-linked immunosorbent assay; PMN, polymorphonuclear leukocyte.
Copyright © 2003 by The American Society for Pharmacology and Experimental Therapeutics 51623/1082430
JPET 306:631–637, 2003 Printed in U.S.A.
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Different fragments of the anti-inflammatory melanocortin
-MSH have been investigated for their efficacy,
including the core region of
(MEHFRWG) and the
C-terminal peptide
, identical
, has been shown to inhibit prostaglandin E
generation and edema formation in rat skin (Gecse et al.,
1980) and PMN migration and IL-1
and KC release in a
model of crystal-induced inflammation (Getting et al., 1999).
) has been reported to reduce experimental
pyresis while the core region was inactive (Richards and
Lipton, 1984). KPV can also inhibit carrageenan-induced
edema formation in the mouse (Hiltz and Lipton, 1990). A
similar observation was noted by the same group in a model
of picryl chloride in the mouse (Hiltz and Lipton, 1989) and
endogenous pyrogen injected into the mouse paw (Hiltz et al.,
1992). A potential mechanism of action for KPV, in analogy to
that reported for
-MSH, is its ability to inhibit nuclear
B activation (Mandrika et al., 2001), potentially lead-
ing to inhibition of proinflammatory cytokine synthesis.
There is a great interest from pharmaceutical companies to
exploit the potent anti-inflammatory effects of the melano-
cortins. However, there has been much confusion regarding
the mechanism behind the effects of the different MSH pep-
tides and receptors. In this study, we systematically investi-
gated the anti-inflammatory effects of core and C-terminal
MSH peptides to understand the molecular mechanisms un-
derlying the efficacy of these peptides. We have used an
integrated approach taking advantage of recent selective
MC-R antagonists, as well as a strain of mice (recessive
yellow e/e; Robbins et al., 1993) without a functional MC1-R.
The effects of other melanocortin peptides were also studied
for comparative purposes.
Materials and Methods
Male C57 Bl.6 mice (20–22 g b.wt.) were purchased from Tuck
(Battlesbridge, Essex, UK) (20–22 g b.wt.), whereas the recessive
yellow (e/e) mouse strain mice (Robbins et al., 1993) was a kind gift
from Dr. Nancy Levin (Trega Bioscience, San Diego, CA). Mice were
maintained on a standard chow pellet diet with tap water ad libitum
using a 12-h light/dark cycle. Animals were used 3 to 4 days after
arrival. Animal work was performed according to Home Office reg-
ulations (Guidance on the Operation of Animals, Scientific Proce-
dures Act, 1986).
Inflammation Models
Crystal peritonitis was induced by injection of 3 mg of monoso-
dium urate (MSU) crystals in 0.5 ml of phosphate-buffered saline
(PBS) as reported previously (Getting et al., 1997). At the 6-h time
point, animals were killed by CO
exposure and peritoneal cavities
were washed with 3 ml of PBS containing 3 mM EDTA and 25 units
heparin. Aliquots of lavage fluid were then stained with Turk’s
solution, and differential cell counts were performed using a
Neubauer hemocytometer and a light microscope (B061; Olympus,
Tokyo, Japan). Lavage fluids were then centrifuged at 400g10
min, and supernatants were stored at 20°C before several biochem-
ical determinations. In another set of experiments, mice were treated
i.p. with 10 ng of murine recombinant IL-1
(provided by Dr. R. C.
Newton, DuPont, Wilmington, DE), peritoneal cavities were lavaged
4 h later and PMN accumulation quantified as described above.
ELISA Measurements
Murine IL-1
and KC levels in the lavage fluids were quantified
with Quantikine ELISA purchased fromR&DSystems (Oxford-
shire, UK). The ELISAs showed negligible (1%) cross-reactivity
with several murine cytokines and chemokines (data as furnished by
the manufacturer).
Drug Treatment
The melanocortin peptides Ac-Nle
ACTH4-10 (MTII; 9.3 nmol) (Al-Obeidi et al., 1989),
-MSH (6 nmol),
KPV (3–88 nmol), H-Ser-Ser-Ile-Ile-Ser-His-Phe-Arg-Trp-Gly-Lys-
(MS05; 0.6666 nmol) (Szardenings et al., 2000; Get-
ting et al., 2003),
(HFRW; 104 nmol), or PBS (100
l) was
administered s.c. either alone or in combination with the MC3/4-R
antagonist Ac-Nle
ACTH4-10) (SHU9119; 9
nmol) (Hruby et al., 1995). MSU crystals were given i.p. 30 min later. In
separate experiments,
-MSH (6 nmol), KPV (9 nmol), HFRW (104
nmol), MS05 (6.6 nmol), and MTII (9.3 nmol) were administered s.c. 30
min before IL-1
. Doses were selected from our previous studies and
from preliminary dose-response curves (Getting et al., 1999, 2003).
Figure 1 illustrates the primary sequences of some of the peptides used.
-MSH, and SHU9119 were purchased from
Bachem (Saffron Walden, Essex, UK), whereas MS05 and KPV were
kindly provided by Melacure Therapeutics AB (Uppsala, Sweden).
All peptides were stored at 20°C before use and dissolved in sterile
PBS (pH 7.4).
In Vitro MØ Activation
Primary MØ Culture. A rich population (95% pure) of perito-
neal MØ (5 10
/well) was prepared by 2-h adherence at 37°C in 5%
, 95% O
atmosphere in RPMI 1640 medium 10% fetal calf
serum. Nonadherent cells were then washed off, and adherent cells
(95% MØ) were incubated with the reported peptides for 15 min in
RPMI 1640 medium. Cells were then stimulated with 1 mg/ml MSU
crystals (a concentration chosen from preliminary experiments), and
the cell-free supernatants collected 2 h later (Getting et al., 1999,
Fig. 1. Amino acid sequences of selected melanocortin peptides. Structure
of KPV (A) and amino acid sequences of selected melanocortin peptides
used in the study (B).
632 Getting et al.
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2001). KC and IL-1
levels were measured by ELISA as described
Intracellular cAMP Accumulation. MØs (1 10
) were al-
lowed to adhere for2hat37°C in RPMI 1640 medium supplemented
with 10% fetal calf serum. MØs were then incubated with serum-free
RPMI 1640 medium containing 1 mM isobutylmethylxantine and
MTII (9.3
M), KPV (3–88
M), MS05 (6.6
M), or the direct ade-
nylate activator forskolin (3
M); all were dissolved in PBS. In
selected wells, MTII was incubated with the MC3/4-R antagonist
SHU9119 (9
M). In all cases after 30 min at 37°C, supernatants
were removed, and cells washed and lysed. cAMP levels in cell
lysates were determined with a commercially available enzyme im-
munoassay (Amersham Biosciences UK Ltd., Little Chalfont, Buck-
inghamshire, UK) using a standard curve constructed with 0 to 3200
fmol/ml cAMP.
Data are reported as mean S.E. of ndistinct observations.
Statistical differences were calculated on original data by analysis of
variance followed by Bonferroni’s test for intergroup comparisons
(Berry and Lindgren, 1990) or by unpaired Student’s ttest (two-
tailed) when only two groups were compared. A threshold value of
P0.05 was taken as significant.
Evaluation of the Effect of Melanocortin Peptides in
a Model of Crystal Peritonitis. The effect of KPV (3–88
nmol) and the selective MC1-R agonist MS05 (0.66 66 nmol)
was evaluated in the MSU crystal peritonitis model. KPV
inhibited PMN migration with a bell-shaped dose response. A
maximal inhibition was seen at 9 nmol with a reduction of
42% of PMN migration (10
/mouse) from 7.14 0.94 to
4.17 0.46 10
(n11, P0.05 versus PBS control). At
the effective dose of 6 nmol (Getting et al., 1999) of
-MSH (6
nmol) caused 33% reduction in PMN migration (*P0.05
versus PBS control; Fig. 2A).
Exudate levels of the CXC chemokine KC were measured
to ascertain whether the antimigratory effect was coupled to
attenuation of the release of this mediator. KPV (9 nmol)
caused a significant reduction in KC levels from 161 31 to
95 13 pg/ml (41%, n11, *P0.05 versus PBS control).
A comparable degree of inhibition was observed after
treatment, whereas higher doses of KPV did not modify KC
levels (Fig. 2B). The selective MC1-R agonist MS05 (0.66 66
nmol), which also contains a KPV region, failed to inhibit
PMN migration at any of the doses tested. However, MTII
(9.3 nmol), which is a substituted cyclic peptide of the core
, reduced PMN migration by 35% (*P
0.05, n6 versus PBS control) (Fig. 2C). The peptide HFRW
corresponding to
was also found to inhibit PMN
migration by 50% (*P0.05, n6 versus PBS control),
and this effect was blocked in the presence of the MC3/4-R
antagonist SHU9119 (Fig. 2D). At variance from several
other melanocortin peptides (Getting et al., 1999, 2001), KPV
retained anti-inflammatory activity when coinjected with an
equimolar dose of the MC3/4-R antagonist SHU9119 (Fig.
In Vitro Effects of Melanocortin Peptides on Chemo-
kine and Cytokine Release from Cultured Macro-
phages. We have previously proposed the resident MØ as
the cellular target for the action of melanocortin peptides
Fig. 2. Anti-inflammatory effects of mela-
nocortin peptides in urate induced inflam-
mation. Mice were treated s.c. with KPV
(3–88 nmol),
-MSH (6 nmol), or PBS (100
l) 30 min before i.p. injections of MSU
crystals (3 mg in 0.5 ml of sterile PBS) on
PMN migration (A) and KC release (B) as
assessed at 6-h time point. C, mice were
treated s.c. with MS05 (0.6666 nmol),
MTII (9.3 nmol), or PBS (100
l), 30 min
before i.p. injections of MSU crystals, and
PMN migration was assessed at 6-h time
point. Data are mean S.E. of n6 mice/
group. ,P0.05 versus control group. D,
lack of effect of SHU9119 on the antimigra-
tory actions of KPV. Mice were treated s.c.
with KPV (9 nmol), HFRW (104 nmol), or
PBS (100
l) alone or in combination with 9
nmol of i.p. SHU9119, 30 min before i.p.
injections of MSU crystals (3 mg), and PMN
migration was assessed at the 6-h time
point. Data are mean S.E. of n8 mice/
group. ,P0.05 versus control group (no
Dissection of the Anti-Inflammatory Effects of KPV 633
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(Getting et al., 1999). Thus, the in vivo experiments were
complemented with the analysis of KPV effects in assays of
MØ activation in vitro. Adherent cells were incubated with
KPV (3–88
M), MS05 (0.66 66
M), MTII (9.3
M), and
-MSH (6
M). KPV and MS05 failed to inhibit crystal in-
duced release of KC or IL-1
at any concentration tested. In
contrast, MTII reduced KC release (46%, *P0.05; Fig.
3A) and IL-1
(51%, *P0.05; Fig. 3B). A similar degree of
inhibition was measured after cell incubation with
(Fig. 3, A and B).
Receptor Functionality. Determination of receptor func-
tionally was quantified by measuring cAMP accumulation in
peritoneal MØ. Forskolin (3
M) and MTII (9.3
M) caused a
significant increase in cAMP accumulation with MTII caus-
ing a 450% increase above basal values (146 22 fmol/well)
(Fig. 4). This increase in cAMP was blocked in the presence of
the MC3/4-R antagonist SHU9119 (9
M) (Fig. 4). MS05 (6.6
M) and KPV (3–88
M) failed to elicit any detectable in-
crease in cAMP accumulation in MØ (Fig. 4).
Effect of KPV on MSU Crystal-Induced Inflamma-
tion in Recessive Yellow (e/e) Mice. KPV anti-inflamma-
tory effects were then investigated in mice with a nonfunctional
MC1-R (recessive yellow e/e mice). KPV inhibited PMN migra-
tion by 32 and 35% at the dose of 3 and 9 nmol, respectively (Fig.
5A). This inhibition was not associated with a reduction in
exudates levels of KC (Fig. 5B) or IL-1
(Fig. 5C).
Effect of Melanocortin Peptides in IL-1
Inflammation. Some reports have linked KPV anti-inflam-
matory actions to blockade of IL-1
effects (Uehara et al.,
Fig. 4. MC-R activation in peritoneal MØ collected from C57 Bl.6 mice.
Adherent peritoneal MØ (1 10
) were incubated with KPV (3–88
closed square), MTII (9.3
M,), alone or in the presence of the MC3/4-R
antagonist SHU9119 (9
M), MS05 (6.6
M), forskolin (3
M), and vehicle
(dotted line) for 30 min before determination of intracellular cAMP. Data are
mean S.E. of n4 determinations. ,P0.05 versus vehicle control.
Fig. 5. Effect of KPV on MSU crystal-induced PMN migration, KC and
release in recessive yellow (e/e) mice. Mice were treated s.c. with
KPV (3–88 nmol) or PBS (100
l), 30 min before i.p. injections of MSU
crystals (3 mg). PMN migration (A) was assessed at the 6-h time point,
and lavage fluids were analyzed for KC (B) and IL-1
(C) content by
commercially available ELISA. Data are mean S.E. of n6 mice/
group. ,P0.05 versus control group.
Fig. 3. Effect of melanocortin peptides on KC and IL-1
release in
primary cultured MØ. KPV (3–88
M, open circles), MS05 (0.6666
filled squares),
-MSH (6
M), MTII (9.3
M), or PBS (dotted line) were
added to adherent peritoneal MØ (5 10
) prepared from C57 Bl.6 mice,
30 min before stimulation with 1 mg/ml MSU crystals. Supernatants
were removed 2 h later and cell-free aliquots analyzed for chemokine KC
(A) and cytokine IL-1
(B) content using specific ELISA. Data are mean
S.E. of n4 determinations. ,P0.05 versus relevant PBS control.
634 Getting et al.
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1992). KPV (9 nmol), MTII (9.3 nmol), HFRW (104 nmol),
MS05 (6.6nmol), and
-MSH (6 nmol) were evaluated against
(10 ng i.p.)-induced PMN migration into the peritoneal
cavity at the 4-h time point.
-MSH and MS05 caused a
significant inhibition in PMN migration elicited by IL-1
56 and 38%, respectively (*P0.05), and a similar degree of
inhibition was observed after treatment with the tripeptide
KPV (52%; *P0.05). However, the synthetic MC3/4-R
agonist MTII and core region HFRW failed to cause a signif-
icant reduction in PMN migration (Fig. 6A). The antimigra-
tory effect of
-MSH was associated with a reduction in
release of the CXC chemokine KC (58%; *P0.05),
whereas MS05 failed to reduce this mediator (Fig. 6B). We
next evaluated the effect of KPV (9 nmol) and MS05 (6.6
nmol) on IL-1
-induced peritonitis in mice with a nonfunc-
tional MC1-R (recessive yellow e/e mice). KPV and MS05
caused a 24 and 36% reduction, respectively, in PMN migra-
tion, although this inhibition was not associated with a re-
duction in release of the CXC chemokine KC (Table 1)
There is a long-standing interest in understanding the
molecular mechanisms responsible for the melanocortin pep-
tide anti-inflammatory actions, which will potentially lead to
the development of new therapeutics (Getting, 2002). There-
fore, in this study, we have sought to determine whether the
anti-inflammatory effects of this tripeptide are mediated via
MC1-R or MC3-R, in analogy to the actions of other melano-
cortin peptides previously investigated.
In the crystal peritonitis model,
-MSH and MTII caused a
significant reduction in PMN migration and associated che-
mokine release, confirming previous findings (Getting et al.,
1999, 2001, 2002). KPV treatment caused a bell-shaped dose-
response curve with maximal inhibition of PMN migration
and KC release occurring at 9 nmol. These data are in agree-
ment with previous findings because both
-MSH and
-MSH produced a bell-shaped inhibitory effect (Getting et
al., 1999). Also, KPV inhibition of urate inflammation aug-
ments the list of models in which this tripeptide has been
shown to inhibit inflammation elicited by irritants such as
carrageenan (Hiltz and Lipton, 1990), picryl chloride (Hiltz
and Lipton, 1989) as well as by endogenous pyrogen (Hiltz et
al., 1992).
To gain some information on the MC-R potentially involved
in these actions, the effect of the selective MC1-R agonist
MS05 (Szardenings et al., 2000) was evaluated, finding that
it was inactive in this model. Interestingly, MS05 has been
found to be inactive in models of white blood cell recruitment
and consequent tissue injury (Guarini et al., 2002; Getting et
al., 2003).
To highlight a potential MC-R activation, we evaluated in
vivo the effects of KPV in the presence of receptor antago-
nists and also in recessive yellow e/e mice, which lack a
functional MC1-R (Robbins et al., 1993). KPV retained anti-
migratory activity in mice pretreated with the MC3/4-R an-
tagonist SHU9119. Importantly, the same occurred in reces-
sive yellow e/e mice. Together, these data would suggest that
KPV exhibits an anti-inflammatory effect that does not in-
volve either MC1, 3, or 4-R. This inability to function at these
MC-Rs is in agreement with previous results showing that
KPV was inactive on MC1-R expressed on a RAW264.7 MØ
cell line (Mandrika et al., 2001).
Searching for MC-R-independent effects of KPV, we used
-induced peritonitis. In fact, this tripeptide shows its
exclusive ability to interfere with IL-1
binding to its own
receptor (type I) (Mugridge et al., 1991), which drives the
neutrophil accumulation process (Perretti and Flower, 1993).
A potential explanation for this is given by the fact that the
tripeptide KPV is structurally similar to an antagonist of the
IL-1 receptor, the peptide KPT (Ferreira et al., 1988). In our
hands, MTII and HFRW failed to inhibit IL-1
-induced PMN
migration, whereas
-MSH, MS05, and KPV caused a signif-
icant reduction of cell migration. This antimigratory effect of
KPV and MS05 was also retained in recessive yellow (e/e)
mice, thus suggesting that this effect is likely to be linked to
Fig. 6. Anti-Inflammatory effects of melanocortin peptides in IL-1
induced inflammation. Mice were treated s.c. with KPV (9 nmol),
(6 nmol), MTII (9.3 nmol), MS05 (6.6 nmol), HFRW (104 nmol), or PBS
l), 30 min before i.p. injections of IL-1
(10 ng in 0.5 ml of sterile
PBS), PMN migration (A) and KC release (B) were assessed at the 6-h
time point. Data are mean S.E. of n6 mice/group. ,P0.05 versus
PBS group.
Anti-inflammatory effects of KPV and MS05 in IL-1
inflammation in recessive yellow (e/e) mice
Mice were treated s.c. with KPV (9 nmol), MS05 (6.6 nmol), or PBS (100
l) 30 min
before i.p. injections of IL-1
(10 ng in 0.5 ml of sterile PBS); PMN migration and KC
release were assessed at the 4 h timepoint. Data are mean S.E. of n5
Treatment PMN KC
per mouse pg/ml
PBS 4.2 0.4 239.2 37.7
KPV 3.2 0.3* 251.2 54.8
MS05 2.7 0.3* 255.0 26.8
*P0.05 versus PBS group.
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the KPV sequence. Interestingly, this set of data is in agree-
ment with previous work in which
-MSH was shown to
inhibit IL-1
-induced migration of neutrophils into subcuta-
neous sponges (Mason and Van Epps, 1989) whereas the
effect observed with MS05 is novel. It is of interest that the
ability of KPV to antagonize the effects of IL-1
has also been
reported in IL-1-induced anorexia (Uehara et al., 1992) and
hyperalgesia (Follenfant et al., 1989). The lack of effect of
HFRW on IL-1
experiments confirms the lack of MC-R
activation and would suggest that peptides that do not con-
tain KPV exert their anti-inflammatory effect by inhibiting
the release of chemokines and cytokines rather than their
Primary culture of murine MØ (Getting et al., 1999) was
used to measure release of the CXC chemokine KC and
cytokine IL-1
in response to urate crystal application. This
in vitro model of MØ activation is susceptible to inhibition by
the melanocortin peptides, including MTII and
-MSH (Get-
ting et al., 1999, 2001). As previously observed (Getting et al.,
1999, 2001), MTII and
-MSH caused significant reduction in
release of these proinflammatory mediators. However, KPV
and the selective MC1-R agonist MS05 failed to inhibit the
release of either mediator. This lack of effect of MS05 has
recently been reported (Getting et al., 2003), whereas the
inactivity of KPV in this experimental condition is reported
here for the first time.
The notion that the anti-inflammatory effects of KPV
might be independent from MC-R was further challenged
using an assay of receptor functionality. We have previously
shown that MC3-R is present on the MØ plasma membrane,
and here we could show that MTII caused intracellular cAMP
accumulation. These effects were abrogated in the presence
of the MC3/4-R antagonist SHU9119. KPV failed to evoke a
cAMP response in mouse peritoneal MØ, confirming that the
core region (HFRW) is required for binding and activation of
the receptor (Wikberg et al., 2000). Conversely, the MC1-R
agonist MS05, which has very low affinity for the MC3-R
(Szardenings et al., 2000), failed to cause cAMP accumula-
tion. The failure of KPV to induce cAMP has previously been
observed in RAW264.7 macrophages (Mandrika et al., 2001)
and in cells transfected with different melanocortin receptors
as reported in a recent review (Wikberg et al., 2000). It has
also been suggested using molecular modeling and ligand
docking experiments that the core region is the sequence
required to interact with MC-Rs (Prusis et al., 1997).
In conclusion, the melanocortin peptide KPV was able to
inhibit PMN migration and generation of proinflammatory
mediators in a model of urate peritonitis. This inhibition did
not seem to be associated with MC-R activation and could be
better explained by inhibition of IL-1
effects. Our results
show that at least two pharmacophores, the core region
(HFRW) involved in MC-R activation and the KPV C-termi-
nal tripeptide, which is able to counteract specific cytokines.
These findings are thus of fundamental importance for the
drug developmental strategies, including determination of
targets, exploiting the therapeutic potential attributed to
MSH peptides.
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Address correspondence to: Dr. Stephen J. Getting, Department of Bio-
chemical Pharmacology, The William Harvey Research Institute, Charter-
house Square, London EC1M 6BQ, UK. E-mail:
Dissection of the Anti-Inflammatory Effects of KPV 637
at ASPET Journals on June 14, 2017jpet.aspetjournals.orgDownloaded from
... Les trois acides aminés en C-terminal de l'α-MSH, Lys-Pro-Val ou KPV ( Figure 17), médient les activités anti-inflammatoires de l'hormone (Lipton and Catania, 1997;Luger et al., 2003). De plus, le KPV médie ses effets anti-inflammatoires indépendamment du récepteur MC1R (Dalmasso et al., 2008;Mandrika et al., 2001) mais via l'inhibition du récepteur à l'IL-1β (Getting et al., 2003b). ...
... Néanmoins, il est fort probable que les effets antiinflammatoires de l'α-MSH in vitro soient médiés non seulement par le récepteur MC1R mais également par d'autres voies effectrices. Ceci a été mis en évidence chez des patients possédant un mutant MC1R non fonctionnel (phénotype RHC) (Cooper et al., 2005) ou chez des animaux déficients pour le gène mc1r (mutation yellow) (Getting et al., 2003b;Ichiyama et al., 1999). ...
... Le KPV inhibe l'activation de NF-κB induite par le TNF-α, des MAPK, de certaines cytokines pro-inflammatoires (IL-6 et IL-12) (Dalmasso et al., 2008) et de l'H2O2 Moustafa et al., 2002) dans les kératinocytes et les mélanomes. Le KPV exerce des effets anti-inflammatoires indépendamment du récepteur MC1R (Dalmasso et al., 2008;Mandrika et al., 2001) mais interfère avec la liaison de l'IL-1β sur son récepteur de type I (Getting et al., 2003b;Mugridge et al., 1991). En effet, le KPV ressemble à un antagoniste du récepteur à l'IL-1, le peptide KPT (Ferreira et al., 1988). ...
Les ultraviolets A (UVA) sont carcinogènes et produisent des espèces réactives de l'oxygène (ERO). Le récepteur à la mélanocortine de type 1 (MC1R) est un récepteur couplé aux protéines G (RCPG) qui est impliqué dans la mélanogénèse et dans l'inflammation cutanée. Certains variants du gène sont associés à un risque accru de mélanomes et de carcinomes cutanés. Le MC1R est exprimé surtout dans les mélanocytes mais son expression peut être induite par les UV in vitro dans les kératinocytes et in vivo dans la peau. Le récepteur MC1R est activé par l'α-MSH. L'objectif de ce travail de thèse a été d'étudier les effets du récepteur MC1R sur le stress oxydatif induit par les UVA dans des lignées kératinocytaires humaines HaCaT exprimant le récepteur MC1R ou son variant non fonctionnel Arg151Cys. Nous avons montré que la production d'ERO intracellulaire induite par les UVA est fortement inhibée dans les cellules HaCaT-MC1R et que cette inhibition est renforcée en présence d'α-MSH. L'inhibition du stress oxydatif induit par les UVA dans les cellules transfectées par le MC1R est en partie dépendante de la phosphorylation de la sous-unité activatrice, NoxA1 de la NADPH oxydase. Le traitement des cellules HaCaT-MC1R par un inhibiteur du récepteur au facteur de croissance épidermique (EGFR) restaure l'habilité de ces cellules à induire un stress oxydatif après irradiation UVA. Ces résultats montrent que l'activité constitutive du récepteur MC1R dans des kératinocytes pourrait inhiber le stress oxydatif induit par les UVA via des mécanismes dépendants de l'AMPc et de l'EGFR.
... Moreover, in animal models of experimentally stimulated fever, brain inflammation, RA as well as systemic inflammation, KPV as well as KdPT was documented to decrease leukocytic pyrogen or IL-1 stimulated hyperthermia [95,103], LPS stimulated NFκB activation, picrylic acid, TNF-α, IL-1β, Or IL-6 stimulated ear swelling [104,105] ƴ-carragenan-stimulated hind paw oedema [106]. MSU crystal or IL-1β stimulated peritonitis as well as neutrophil collection [107], as well as dextran sodium sulphate stimulated Colitis with Inflammatory cell infiltration as well as myeloperoxidase activity [108,109], pointing a therapeutic role of α-MSH for therapy of inflammatory disease. ...
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After having reviewed the role of melanocortin 4 receptors in obesity here we concentrated on other role of melanocortins in inflammation, other neurological complications. Melanocortins are a collection of adrenocorticotropin hormone (ACTH), α, β as well as ƴ melanocyte stimulating hormone (MSH) along with their receptors, are a part of the old modulatory system. The clinical utility of ACTH was initiated in 1949 by Hench, who initially believed that this action was brought about by the hypothalamo-pituitary-adrenal (H-P-A) Axis and was glucocorticoid (GC)-dependent. It is now well known that the melanocortins bring about the anti-inflammatory as well as immunomodulatory actions by the activation of melanocortin receptors. Having reviewed the role of proopiomelanocortin (POMC) and its peptides in obesity here we tried to unravel the inflammatory and other neurological sequelae of utilization of these melanocortins. Thus, we conducted a systematic review on the melanocortins and their receptors utilizing the Pubmed, Google Scholar, Web of Science, search engines utilizing the MeSH terms melanocortins; melanocortin receptors 1-5; inflammation; actions in various systems; ACTH; Alpha MSH; Gamma and beta MSH; multiple sclerosis (MS); Infantile spasms; head injuries from 1940 till date on 3 st july 2020. We found a total of over 10,000 articles out of which we selected 122 articles for this review. In view of having reviewed MC4R agonism in obesity we did not use betmelinide and setmelanotide being tried for obesity in this review. Thus, we have tried to discuss comprehensively the anti-inflammatory activities that might be utilized clinically after initial enthusiasm lost in this ancient system. Adrenocorticotropin hormone (ACTH) and alpha-Melanocortin stimulating hormone (a-MSH) reduce pro-inflammatory cytokines in several pulmonary inflammatory disorders including asthma, sarcoidosis, and the acute respiratory distress syndrome. They have also been shown to reduce fibrogenesis in animal models with pulmonary fibrosis. By understanding the functions of MCR in macrophages, T-helper cell type 1, and T-helper cell type 17, we may uncover the mechanism of action of melanocortin agonists in sarcoidosis. Further translational and clinical research is needed to define the role of ACTH and a-MSH in pulmonary diseases.
... Furthermore, in animal models of experimentally induced fever, brain inflammation, skin inflammation, rheumatoid arthritis, and systemic inflammation, KPV or KdPT was reported to reduce leukocytic pyrogen-or IL-1-induced hyperthermia (170,178), LPS-induced activation of NF-κB (128), picrylic acid-, TNF-α-, IL-1β-, or IL-6-induced ear swelling (179)(180)(181), γ-carrageenan-induced hind paw edema (182), MSU crystal-or IL-1β-induced peritonitis and neutrophil accumulation (139), and dextran sodium sulfate-induced colitis with inflammatory cell infiltration and myeloperoxidase activity (183,184), indicating therapeutic potential of α-MSH for treatment of inflammatory diseases. ...
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Adrenocorticotropic hormone (ACTH), and α-, β-, and γ-melanocyte-stimulating hormones (α-, β-, γ-MSH), collectively known as melanocortins, together with their receptors (melanocortin receptors), are components of an ancient modulatory system. The clinical use of ACTH in the treatment of rheumatoid arthritis started in 1949, originally thought that the anti-inflammatory action was through hypothalamus-pituitary-adrenal axis and glucocorticoid-dependent. Subsequent decades have witnessed extensive attempts in unraveling the physiology and pharmacology of the melanocortin system. It is now known that ACTH, together with α-, β-, and γ-MSHs, also possess glucocorticoid-independent anti-inflammatory and immunomodulatory effects by activating the melanocortin receptors expressed in the brain or peripheral immune cells. This review will briefly introduce the melanocortin system and highlight the action of melanocortins in the regulation of immune functions from in vitro, in vivo, preclinical, and clinical studies. The potential therapeutic use of melanocortins are also summarized.
... A final point is that the Lys-Pro-Val used in this study was in a free acid form with no acetylation at the N-terminal. However, in the literature, some of the -MSH related peptides (e.g., KPV, KdPV, KdPT) were N-acelylated and C-amidated peptides (Hiltz et al., 1991) while others were not (Getting et al., 2003). Although no comparison has been made for the N-acelylated and C-amidated forms of the Lys-Pro-Val on antiinflammatory effect, it has been known that N-acelylated and C-amidated substrates might affect their affinity to PEPT1 . ...
The proton-coupled oligopeptide transporter PEPT1 has recently been linked to intestinal inflammation and inflammatory bowel disease (IBD) because of its ability to transport bacterial peptides (e.g., Tri-DAP, MDP, fMet-Leu-Phe) and its aberrant expression in the colon of IBD patients. Although studies have demonstrated that several bacterial peptides were substrates of PEPT1, the relative importance of this protein (as compared to other transporters and pathways) and its regional permeability have not been addressed. Therefore, the first objective of this dissertation focused on the importance of intestinal PEPT1 in transporting a bacterially-produced chemotactic peptide, fMet-Leu-Phe, using the in situ single-pass intestinal perfusion (SPIP) technique in wild-type and Pept1 knockout mice. The dynamic effect of fMet-Leu-Phe transport was examined by the activity of myeloperoxidase (MPO), a marker for neutrophil migration with a modified intestinal perfusion. These findings suggested that PEPT1 was the major transporter for the transport of fMet-Leu-Phe in the small intestine and could influence the induction of inflammation. Lys-Pro-Val has been suggested as a potential therapeutic agent for IBD due to its anti-inflammatory effects in mouse colitis models. However, some important properties (e.g., intestinal permeability and stability) have not been studied. Therefore, the second objective of this dissertation was to study the transport properties of the anti-inflammatory tripeptide Lys-Pro-Val using the intestinal perfusion technique in wild-type and Pept1 knockout mice. The results suggested that although Lys-Pro-Val may be a good candidate to treat IBD through the aberrant expression of colonic PEPT1, drug stability was a significant concern for oral administration. In conclusion, results from this dissertation suggested that PEPT1 could transport bacterial peptides, implying that aberrant expression of PEPT1 in the colon may contribute to the progression of IBD. Although colonic PEPT1 may also serve as a drug targeting (delivery) opportunity for some bioactive peptides, such as Lys-Pro-Val to treat IBD, intestinal stability of this substrate is a realistic concern.
... The antipyretic effect of α-MSH in experimentally induced fever and brain inflammation is thought to be mediated through MC3R and MC4R in the hypothalamus and brain stem (Tatro, 2000). MCR independent actions have also been described (Mugridge et al., 1991;Poole et al., 1992;Ichiyama et al., 1999;Getting et al., 2003;Cooper et al., 2005). In vitro and in vivo evidence demonstrate that α-MSH reduces binding of IL-1β hence reducing its hyperalgesic effect of IL-1β in rats (Mugridge et al., 1991;Poole et al., 1992). ...
... For poultry, this is the first report demonstrating the curcumin-induced transcriptional changes in the gut, especially those genes that are mediating the inflammatory response. Among the C. longa upregulated genes, proopiomelanocortin is known to block mouse macrophage activation through inhibition of IL-1β (Getting et al., 2003), and inositol polyphosphate-5-phosphatase suppressed the inflammatory response of macrophages following treatment with Francisella tularensis (Santic et al., 2010). Among the C. longa downregulated genes were tachykinin precursor 1, myeloperoxidase, colonystimulating factor 2 receptor α, lactotransferrin, and CD28. ...
Full-text available
The effects of dietary supplementation with an organic extract of Curcuma longa on systemic and local immune responses to experimental Eimeria maxima and Eimeria tenella infections were evaluated in commercial broiler chickens. Dietary supplementation with C. longa enhanced coccidiosis resistance as demonstrated by increased BW gains, reduced fecal oocyst shedding, and decreased gut lesions compared with infected birds fed a nonsupplemented control diet. The chickens fed C. longa-supplemented diet showed enhanced systemic humoral immunity, as assessed by greater levels of serum antibodies to an Eimeria microneme protein, MIC2, and enhanced cellular immunity, as measured by concanavalin A-induced spleen cell proliferation, compared with controls. At the intestinal level, genome-wide gene expression profiling by microarray hybridization identified 601 differentially expressed transcripts (287 upregulated, 314 downregulated) in gut lymphocytes of C. longa-fed chickens compared with nonsupplemented controls. Based on the known functions of the corresponding mammalian genes, the C. longa-induced intestinal transcriptome was mostly associated with genes mediating anti-inflammatory effects. Taken together, these results suggest that dietary C. longa could be used to attenuate Eimeria-induced, inflammation-mediated gut damage in commercial poultry production.
... The literature on tripeptide interaction with melanocortin receptor subtype 1 (MC1R) remains controversial. In vitro and in vivo studies failed to show an interaction of a-MSH (11)(12)(13) with MC1R and rather suggested, due to structural similarity, an effect by IL-1b mediated activation of IL-1 receptors that enhances anti-inflammation by anti-migratory effects of neutrophils [10]. In contrast, the tripeptide induced dependent on the presence of MC1R a calcium signal in MC1Rtransfected chinese hamster ovary (CHO)-K1 cells that led to inhibition of TNF-a-stimulated activation of nuclear factor (NF)-kB transcription factor [11]. ...
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Following traumatic brain injury (TBI) neuroinflammatory processes promote neuronal cell loss. Alpha-melanocyte-stimulating hormone (α-MSH) is a neuropeptide with immunomodulatory properties, which may offer neuroprotection. Due to short half-life and pigmentary side-effects of α-MSH, the C-terminal tripeptide α-MSH(11–13) may be an anti-inflammatory alternative. The present study investigated the mRNA concentrations of the precursor hormone proopiomelanocortin (POMC) and of melanocortin receptors 1 and 4 (MC1R/MC4R) in naive mice and 15 min, 6, 12, 24, and 48 h after controlled cortical impact (CCI). Regulation of POMC and MC4R expression did not change after trauma, while MC1R levels increased over time with a 3-fold maximum at 12 h compared to naive brain tissue. The effect of α-MSH(11–13) on secondary lesion volume determined in cresyl violet stained sections (intraperitoneal injection 30 min after insult of 1 mg/kg α-MSH(11–13) or 0.9% NaCl) showed a considerable smaller trauma in α-MSH(11–13) injected mice. The expression of the inflammatory markers TNF-α and IL-1β as well as the total amount of Iba-1 positive cells were not reduced. However, cell branch counting of Iba-1 positive cells revealed a reduced activation of microglia. Furthermore, tripeptide injection reduced neuronal apoptosis analyzed by cleaved caspase-3 and NeuN staining. Based on the results single α-MSH(11–13) administration offers a promising neuroprotective property by modulation of inflammation and prevention of apoptosis after traumatic brain injury.
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Treatment of acute gout consists of non-steroidal anti-inflammatory drugs (NSAIDs), colchicine and steroids. However, the typical patient with gout has multiple comorbidities such as cardiovascular disease, hypertension, renal dysfunction or diabetes/metabolic syndrome that represent contraindications to these therapeutic options. The aim of this study is to review the available evidence regarding the use of ACTH as an alternative therapeutic option for acute gout and explore potential mechanisms of action. We performed an electronic search (MEDLINE, Scopus and Web of Science) using the keywords ACTH or adrenocorticotropic hormone combined with gout or crystal-induced arthritis. ACTH appears suitable for patients with many comorbidities due to its good safety profile. Clinical evidence shows that ACTH is at least as effective as classic agents. The mechanism of action of ACTH in gout is not entirely known. Robust experimental evidence points to the direction that ACTH does not act solely by triggering the release of endogenous steroids but also appears to downregulate inflammatory responses by activating melanocortin receptors on innate immune cells, such as macrophages. Moreover, indirect evidence indicates that ACTH may have an IL-1 antagonistic effect. We propose that ACTH may be an alternative therapeutic option for gout in patients with multiple comorbidities. Large-scale studies assessing the efficacy and safety of ACTH compared to classic therapeutic options are needed.
Background: K(D)PT showed marked anti-inflammatory properties in preclinical studies and exhibited very low toxicity in phase I and preclinical trials. In this study, efficacy and safety of oral K(D)PT were evaluated in patients with mild-to-moderate active ulcerative colitis. Methods: A multicenter, randomized, double-blind, phase IIa trial was performed comparing add-on oral K(D)PT twice a day (20, 50, or 100 mg) with placebo in patients with mild-to-moderate active ulcerative colitis on baseline medication. The primary objective was to determine the difference in time to sustained improvement in colitis activity index (CAI) of ≥50% at week 8 between pooled K(D)PT group and placebo. Secondary endpoints included remission rates and CAI response at different time points. Results: Compared with placebo, K(D)PT (pooled group) resulted in significantly higher proportions of patients in remission at 2 and 4 weeks, (2 wk: P = 0.0349; 4 wk: P = 0.0278) and a significantly higher proportion of patients with CAI response at week 8 (P = 0.0434). K(D)PT (pooled group) met the primary endpoint after additional analyses. Because of high placebo response rates, subgroup analyses tried to identify patients with unquestionably active and more severe, but still moderate, disease (CAI score ≥9 or taking more than one concomitant medication). These subgroups showed earlier and statistically significant CAI responses to K(D)PT versus placebo. All doses of K(D)PT were well tolerated. Conclusions: Despite a very high placebo rate after week 4, study data in this preliminary trial strongly suggest that add-on K(D)PT is efficacious in patients with mild-to-moderate ulcerative colitis. Moreover, K(D)PT showed an excellent safety profile.
Subcortical ischemic vascular dementia (SIVD) caused by chronic cerebral hypoperfusion exhibits progressive white matter and cognitive impairments. However, its pathogenetic mechanisms are poorly understood. We investigated the role of interleukin-1β (IL-1β) and its receptor IL-1 receptor type 1 (IL-1R1) in an experimental SIVD model generated via right unilateral common carotid arteries occlusion (rUCCAO) in mice. We found that IL-1β expression was elevated in the corpus callosum at the early stages after rUCCAO. IL-1 receptor antagonist (IL-1Ra), when delivered at an early stage, as well as IL-1R1 knockout, rescued the downregulation of myelin basic protein (MBP) and improved remyelination at the later stage after rUCCAO. Our data suggest that the recruitment of OPCs, but not the proliferation or differentiation of OPCs, is the only compromised step of remyelination following chronic cerebral ischemia. IL-1Ra treatment and IL-1R1 knockout had no effect on the oligodendrocyte progenitor cell (OPC) proliferation, but did promote the recruitment of newly generated OPCs to the corpus callosum, which can be reversed by compensatory expression of IL-1R1 in the SVZ of IL-1R1 knockout mice. Further, we found that recruited OPCs contribute to oligodendrocyte regeneration and functional recovery. In transwell assays, IL-1β inhibited OPC migration through IL-1R1. Moreover, KdPT which can enter the brain to block IL-1R1 also showed comparable protection when intraperitoneally delivered. Our results suggest that IL-1β during the early stages following chronic cerebral hypoperfusion impedes OPC recruitment via IL-1R1, which inhibits white matter repair and functional recovery. IL-1R1 inhibitors may have potential uses in the treatment of SIVD.
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The proopiomelanocortin (POMC)-derived neuropeptide a-melanocyte stimulating hormone (a-MSH) is known to modulate some aspects of inflammation through direct effects on T cells, B cells, and monocytes. To determine whether a-MSH might similarly influence mast cell responsiveness, mast cells were examined to see if they expressed the receptor for a-MSH, melanocortin-1 (MC-1), and whether a-MSH altered mast cell function. We thus first identified MC-1 on bone marrow cultured murine mast cells (BMCMC) and a murine mast cell line (MCP-5) employing flow cytometry and through detection of specific binding. Subsequent treatment of mast cells with a-MSH increased the cAMP concentration in a characteristic biphasic pattern, demonstrating that a-MSH could affect intracellular processes. We next examined the effect of a-MSH on mediator release and cytokine expression. IgE/DNP-human serum albumin-stimulated histamine release from mast cells was inhibited by ;60% in the presence of a-MSH. Although activation of BMCMC induced the expression of mRNAs for the inflammatory cytokines IL-1 b, IL-4, IL-6, TNF-a, and the chemokine lymphotactin, mRNAs for IL-1b, TNF-a, and lymphotactin were down-modulated in the presence of a-MSH. Finally, IL-3-dependent proliferative activity of BMCMC was slightly but significantly augmented by a-MSH. Taken together, these observations suggest that a-MSH may exert an inhibitory effect on the mast cell-dependent component of a specific inflam- matory response. The Journal of Immunology, 1999, 163: 3363-3368.
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a-Melanocyte-stimulating hormone (a-MSH) is a tridecapeptide found mainly in the brain, pituitary, and circulation. It inhibits most forms of inflammation by a mechanism that is not known. As most types of inflammation require activation of NF-kB, we investigated the effect of a-MSH on the activation of this transcription factor by a wide variety of inflammatory stimuli. Electrophoretic mobility shift assay showed that a-MSH completely abolished TNF-mediated NF-kB activation in a dose- and time-dependent manner. It also suppressed NF-kB activation induced by LPS, okadaic acid, and ceramide. The effect was specific, as the activation of the transcription factor activating protein-1 by TNF was unaffected. Western blot analysis revealed that TNF-dependent degradation of the inhibitory subunit of NF-kB, IkBa, and nuclear translocation of the p65 subunit of NF-kB were also inhibited. This correlated with suppression of NF-kB-dependent reporter gene expression induced by TNF. The inhibitory effect of a-MSH appeared to be mediated through generation of cAMP, as inhibitors of adenylate cyclase and of protein kinase A reversed its inhibitory effect. Similarly, addition of membrane-permeable dibutyryl cAMP, like a-MSH, suppressed TNF-induced NF-kB activation. Overall, our results suggest that a-MSH suppresses NF-kB activated by various inflammatory agents and that this mechanism probably contributes to its anti-inflammatory effects. The Journal of Immunology, 1998, 161: 2873-2880.
: The recruitment of leukocytes from the circulation to inflamed tissue is regulated by the expression of adhesion molecules on both leukocytes and endothelial cells. The proopiomelanocortin-derived peptide α-melanocyte stimulating hormone (α-MSH) is known to modulate inflammation. Thus, we investigated the influence of α-MSH on the LPS-induced expression of the adhesion molecules E-selectin and VCAM-1 on endothelial cells. Human microvascular endothelial cells (HMEC-1) were treated with LPS (100 ng/ml) alone or in the presence of α-MSH (10−8 to 10−16 M). RT-PCR analysis showed that α-MSH significantly reduced LPS-induced expression of VCAM-1 and E-selectin. Since many adhesion molecules contain regulatory NF-κB sites in their promoter region, the role of α-MSH in the activation of the transcription factor NF-αB was also investigated. α-MSH significantly downregulated the LPS-mediated activation of NF-κB, in a dose-dependent manner. These findings indicate that modulation of the transcription factor NF-κB is a crucial molecular event, one that seems to be responsible for the antiinflammatory effects of α-MSH.
With the rise in the field of neuroimmunomodulation research, there is increased recognition of the influence of the nervous system and neuropeptides in peripheral disease. The neuropeptide α-melanocyte-stimulating hormone (α-MSH) is a neuroimmunomodulatory agent that modulates production of proinflammatory cytokines and inhibits peripheral inflammation via actions on CNS receptors. We examined whether central α-MSH operates by inhibiting activation of the nuclear factor kappa B (NF-κB) that is essential to the expression of proinflammatory cytokines and development of inflammation in the periphery. Electrophoretic mobility shift assays of nuclear extracts from the murine foot pad injected with TNF-α demonstrated that centrally administered α-MSH does inhibit NF-κB activation. Western blot analysis revealed that this inhibition was linked to central α-MSH-induced preservation of expression of IκBα protein in the peripheral tissue. The NF-κB and IκBα effects were inhibited in mice with spinal cord transection. Intraperitoneal (ip) injection of the nonspecific β-adrenergic receptor blocker propranolol, and of a specific β2-adrenergic receptor antagonist, likewise prevented these effects of central α-MSH; blockade of cholinergic, α-adrenergic, or β1-adrenergic receptors did not. Centrally administered α-MSH inhibited peripheral NF-κB activation and IκBα degradation even in mice with nonfunctional melanocortin 1 receptors (MC1R). These findings indicate that α-MSH can act centrally to inhibit NF-κB activation in peripheral acute inflammation via a descending neural pathway. The pathway involves β2-adrenergic receptors, but does not require activation of MC1R within the brain.
The neuropeptide alpha-melanocyte stimulating hormone [alpha-MSH(1-13)] occurs in the pituitary, brain, skin and other tissues and receptors for this molecule are likewise widespread. In previous research, this tridecapeptide, which shares its amino acid sequence with ACTH(1-13), was shown to have both potent antipyretic activity and a role in the endogenous control of the febrile response. alpha-MSH(1-13) and its COOH-terminal tripeptide were subsequently found to inhibit inflammation induced by general stimuli such as topical application of an irritant. The aim in the present experiments was to determine if these peptides can inhibit acute inflammatory responses induced in mice by injection of individual cytokines, endogenous pyrogen (EP), a natural cytokine mixture, and other mediators of inflammation. Inflammation induced in the mouse ear by rIL-1 beta, rIL-6 or rTNF-alpha was inhibited by alpha-MSH and a D-valine-substituted analog of alpha-MSH(11-13) whereas substantial doses of alpha-MSH(1-13) did not alter inflammation induced by LTB4, PAF and IL-8. Both peptides inhibited edema caused in the mouse paw by local injection of EP. The results indicate that alpha-MSH molecules antagonize the actions of certain cytokine mediators of inflammation, consistent with previous observations of anti-cytokine activity of these peptides. Failure to inhibit edema caused by LTB4, PAF and IL-8 suggests that, in inflammation induced by general stimuli, such as EP, the peptides act prior to the release of these mediators of the inflammatory response. Because of the anticytokine/anti-inflammatory actions of the alpha-MSH molecules they may be useful in understanding the cytokine network and for treatment of inflammatory diseases.
Interleukin-1 beta (IL-1), a cytokine released from inflammatory cells, is thought to be involved in the anorexia associated with severe infection. To assess a possible role of the amino acid sequence found in the supposed IL-1 receptor binding sites, we determined the antagonistic effects of alpha-melanocyte-stimulating hormone (MSH) and the carboxyl-terminal tripeptide of alpha-MSH-(11-13) (alpha-MSH-(11-13)) on the anorexia induced by intracerebroventricular (i.c.v.) administration of 0.5 pmol IL-1. The parent alpha-MSH molecule completely prevented the induction of anorexia by IL-1 at both doses tested, 0.5 and 5.0 pmol. In contrast, alpha-MSH-(11-13) prevented the IL-1-induced anorexia only at 5.0 pmol, but not at 0.5 pmol. Intracerebroventricular injection of 5 pmol of the parent alpha-MSH molecule alone temporarily decreased food consumption at 1-2 h; 5.0 pmol of alpha-MSH-(11-13) alone did not affect food consumption. These data indicate that alpha-MSH can antagonize the anorexic effects of IL-1. The carboxyl-terminal tripeptide portion of alpha-MSH may be important for the antagonistic action of alpha-MSH on the anorexia induced by IL-1.
Contractions elicited by CaCl2 on isolated rat stomach strip preparations have been reported to be potentiated by interleukin-1 beta (IL-1 beta). We have investigated whether this effect can be reduced by the putative IL-1 beta antagonist, alpha-melanocyte-stimulating hormone (alpha MSH). Additionally, the effects of alpha MSH on the specific binding of IL-1 beta to B- and T-cells have been investigated to further clarify its inhibitory activities. Both alpha MSH and its carboxyl terminal tripeptide concentration dependently reduced the potentiation of CaCl2-induced contractions caused by IL-1 beta but not those caused by leukotriene D4, the parent molecule being approximately 250 times more active. Additionally, both peptides potently and selectively reduced 125I-IL-1 beta binding to the T-cell sub-clone EL4-6.1 but not to the B-cell sub-clone 1H7. The results indicate that IL-1 beta effects on rat stomach may be mediated through a type-I (80 kDa) IL-1 beta receptor.
Alpha-melanocyte stimulating hormone [alpha-MSH(1-13)] occurs within the CNS, skin, circulation and in other body sites. This tridecapeptide and its COOH-terminal tripeptide, alpha-MSH (11-13), have antipyretic and anti-inflammatory actions. Studies of the anti-inflammatory effects of these molecules have been confined mainly to tests of inhibition of histamine and endogenous pyrogen-induced increases in capillary permeability in rabbits and acute inflammation of ear tissue in mice. The aim in the present experiments was to learn if alpha-MSH peptides also antagonize inflammation in two additional models: acute edema induced in the mouse paw and contact sensitivity. Significant anti-inflammatory effects were observed with MSH peptides in both models. These findings converge with previous results to indicate that alpha-MSH peptides modulate inflammation. Because circulating alpha-MSH increases after treatment of animals with endogenous pyrogen or endotoxin, administration of the peptides may simply mimic a naturally occurring modulation of host defense reactions.