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Dissection of the Anti-Inflammatory Effect of the Core and C-
Terminal (KPV)
␣
-Melanocyte-Stimulating Hormone Peptides
STEPHEN J. GETTING, HELGI B. SCHI ¨
OTH, and MAURO PERRETTI
The William Harvey Research Institute, London, UK (S.J.G., M.P.); and Department of Neuroscience, Uppsala University, Uppsala, Sweden
(H.S.)
Received March 14, 2003; accepted May 9, 2003
ABSTRACT
In this study, we analyzed the anti-inflammatory effects of
␣
-melanocyte stimulating hormone (MSH)
11–13
(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
4
-c[Asp
5
,D-Phe
7
,Lys
10
]NH
2
ACTH4-10
(MTII) but not the selective MC1-R agonist H-Ser-Ser-Ile-Ile-Ser-
His-Phe-Arg-Trp-Gly-Lys-Pro-Val-NH
2
(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
4
-c[Asp
5
,D-
2
Nal
7
,Lys
10
]NH
2
ACTH4-10 (SHU9119). In vitro, macrophage ac-
tivation, determined as release of KC and interleukin (IL)-1

was
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

functions.
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-
tor-
B activation (Manna and Aggarwal, 1998; Kalden et al.,
1999) and protection of I
B
␣
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
http://jpet.aspetjournals.org.
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.
0022-3565/03/3062-631–637$7.00
THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 306, No. 2
Copyright © 2003 by The American Society for Pharmacology and Experimental Therapeutics 51623/1082430
JPET 306:631–637, 2003 Printed in U.S.A.
631
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Different fragments of the anti-inflammatory melanocortin
peptide
␣
-MSH have been investigated for their efficacy,
including the core region of
␣
-MSH
4–10
(MEHFRWG) and the
C-terminal peptide
␣
-MSH
11–13
(KPV).
␣
-MSH
4–10
, identical
to ACTH
4–10
, has been shown to inhibit prostaglandin E
1
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).
KPV (
␣
-MSH
11–13
) 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
factor-
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
Animals
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
2
exposure and peritoneal cavities
were washed with 3 ml of PBS containing 3 mM EDTA and 25 units
ml
⫺1
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 400g⫻10
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
4
-c[Asp
5
,D-Phe
7
,Lys
10
]NH
2
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-
Pro-Val-NH
2
(MS05; 0.66–66 nmol) (Szardenings et al., 2000; Get-
ting et al., 2003),
␣
-MSH
6–9
(HFRW; 104 nmol), or PBS (100
l) was
administered s.c. either alone or in combination with the MC3/4-R
antagonist Ac-Nle
4
-c[Asp
5
,D-
2
Nal
7
,Lys
10
]NH
2
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.
MTII, HFRW,
␣
-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
6
/well) was prepared by 2-h adherence at 37°C in 5%
CO
2
, 95% O
2
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
above.
Intracellular cAMP Accumulation. MØs (1 ⫻10
5
) 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.
Statistics
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
P⬍0.05 was taken as significant.
Results
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
6
/mouse) from 7.14 ⫾0.94 to
4.17 ⫾0.46 ⫻10
6
(n⫽11, P⬍0.05 versus PBS control). At
the effective dose of 6 nmol (Getting et al., 1999) of
␣
-MSH (6
nmol) caused 33% reduction in PMN migration (*P⬍0.05
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%, n⫽11, *P⬍0.05 versus PBS control).
A comparable degree of inhibition was observed after
␣
-MSH
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
region
␣
-MSH
4–10
, reduced PMN migration by 35% (*P⬍
0.05, n⫽6 versus PBS control) (Fig. 2C). The peptide HFRW
corresponding to
␣
-MSH
6–9
was also found to inhibit PMN
migration by ⬃50% (*P⬍0.05, n⫽6 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.
2D).
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 (0.66–66 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 n⫽6 mice/
group. ⴱ,P⬍0.05 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 n⫽8 mice/
group. ⴱ,P⬍0.05 versus control group (no
antagonist).
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%, *P⬍0.05; Fig.
3A) and IL-1

(51%, *P⬍0.05; Fig. 3B). A similar degree of
inhibition was measured after cell incubation with
␣
-MSH
(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

-Mediated
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
5
) were incubated with KPV (3–88
M,
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 n⫽4 determinations. ⴱ,P⬍0.05 versus vehicle control.
Fig. 5. Effect of KPV on MSU crystal-induced PMN migration, KC and
IL-1

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 n⫽6 mice/
group. ⴱ,P⬍0.05 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 (0.66–66
M,
filled squares),
␣
-MSH (6
M), MTII (9.3
M), or PBS (dotted line) were
added to adherent peritoneal MØ (5 ⫻10
6
) 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 n⫽4 determinations. ⴱ,P⬍0.05 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
IL-1

(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

by
56 and 38%, respectively (*P⬍0.05), and a similar degree of
inhibition was observed after treatment with the tripeptide
KPV (⫺52%; *P⬍0.05). 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%; *P⬍0.05),
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)
Discussion
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
IL-1

-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),
␣
-MSH
(6 nmol), MTII (9.3 nmol), MS05 (6.6 nmol), HFRW (104 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 (A) and KC release (B) were assessed at the 6-h
time point. Data are mean ⫾S.E. of n⫽6 mice/group. ⴱ,P⬍0.05 versus
PBS group.
TABLE 1
Anti-inflammatory effects of KPV and MS05 in IL-1

-induced
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 n⫽5
mice/group.
Treatment PMN KC
10
6
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
*P⬍0.05 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
action.
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
␥
2
-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.
Acknowledgments
We thank Drs. R. de Me´dicis and A. Lussier (University of Sher-
brooke, Sherbrooke, QC, Canada) for the supply of MSU crystals.
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Dissection of the Anti-Inflammatory Effects of KPV 637
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