This information is current as
of June 16, 2017.
Redundancy of a Functional Melanocortin 1
Receptor in the Anti-inflammatory Actions of
Melanocortin Peptides: Studies in the
Recessive Yellow (e/e) Mouse Suggest an
Important Role for Melanocortin 3 Receptor
Stephen J. Getting, Helen C. Christian, Connie W. Lam,
Felicity N. E. Gavins, Roderick J. Flower, Helgi B. Schiöth
and Mauro Perretti
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The Journal of Immunology is published twice each month by
The American Association of Immunologists, Inc.,
1451 Rockville Pike, Suite 650, Rockville, MD 20852
Copyright © 2003 by The American Association of
Immunologists All rights reserved.
Print ISSN: 0022-1767 Online ISSN: 1550-6606.
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J Immunol 2003; 170:3323-3330; ;
doi: 10.4049/jimmunol.170.6.3323
http://www.jimmunol.org/content/170/6/3323
The Journal of Immunology
Redundancy of a Functional Melanocortin 1 Receptor in the
Anti-inflammatory Actions of Melanocortin Peptides: Studies
in the Recessive Yellow (e/e) Mouse Suggest an Important Role
for Melanocortin 3 Receptor1
Stephen J. Getting,2* Helen C. Christian,† Connie W. Lam,* Felicity N. E. Gavins,*
Roderick J. Flower,* Helgi B. Schiöth,‡ and Mauro Perretti*
elanocortin peptides, (e.g., ␣-melanocyte stimulating
hormone (␣-MSH)3) are derived from a larger precursor called the pro-opiomelanocortin gene product and
are characterized by a common amino acid motif (HFRW). These
endogenous peptides have long been reported to possess antiinflammatory effects in many experimental models of acute and
chronic inflammation, including experimental bowel disease, allergy, and chronic (mycobacterium-induced arthritis) and systemic
inflammation (endotoxemia) (1, 2). Melanocortins act at a subgroup of G-protein coupled receptor, termed melanocortin receptors (MC-R), of which five members have been identified so far.
M
*The William Harvey Research Institute, London, United Kingdom; †Department of
Human Anatomy and Physiology, University of Oxford, Oxford, United Kingdom;
and ‡Department of Neuroscience, Uppsala University, Uppsala, Sweden
Received for publication October 15, 2002. Accepted for publication January
16, 2003.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1
This work was supported by the Arthritis Research Campaign U.K. (Grant G0571).
M.P. is a Senior Fellow of the Arthritis Research Campaign, U.K. R.J.F. is a Principal
Research Fellow of the Wellcome Trust U.K. H.S.S. was supported by the Swedish
Research Council (VR, medicin) and Melacure Therapeutics, A.B. (Uppsala,
Sweden).
2
Address correspondence and reprint requests to: Dr. Stephen J. Getting, Department
of Biochemical Pharmacology, The William Harvey Research Institute, Bart’s and
The London, Queen Mary School of Medicine and Dentistry, Charterhouse Square,
London EC1 M 6BQ, U.K. E-mail address: [email protected]
3
Abbreviations used in this paper: MSH, melanocyte stimulating hormone; MC-R,
melanocortin receptors; M␾, macrophage; MSU, monosodium urate.
Copyright © 2003 by The American Association of Immunologists, Inc.
MC-Rs have a wide and varied distribution throughout the body
(3). All MC-Rs are positively coupled to adenylate cyclase, and
agonism at these receptors leads to increases in intracellular cAMP
(3). Some specific actions have been attributed to specific members
of the receptor family; examples being MC1-R mediated skin pigmentation, MC4-R control of obesity and MC2-R stimulation of
adrenal steroidogenesis (3). It remains still unclear if the antiinflammatory actions of melanocortin peptides are mediated by a
single MC-R.
The MC1-R has long been regarded as the receptor responsible
for the anti-inflammatory effects of ␣-MSH and related peptides
(3). MC1-R mRNA, but not protein, expression has been found on
monocytes, B lymphocytes, NK cells, a subset of cytoxic T cells
(4), dendritic cells (5), and more recently mast cells (6). MC1-Rmediated anti-inflammatory effects appear to occur via inhibition
of NF-␬B activation (7, 8) and protection of I␬B␣ degradation (9).
These intracellular events would produce a reduction in the expression of proinflammatory cytokines (10) and adhesion molecules (8), thereby affecting the humoral and cellular phases of inflammation (11, 12).
Recently, our own studies have identified a putative role for
MC3-R in modulating experimental inflammation (13). Selective
agonists at the MC3-R (the natural ␥2-MSH) (14) and the synthetic
peptide MTII (15)) displayed inhibitory activity in a murine model
of monosodium urate (MSU) crystal-induced peritonitis (16). The
in vivo data have been supported by the detection of both MC3-R
mRNA and protein on mouse and rat peritoneal macrophages
(M␾) as well as on rat knee joint M␾ (13, 16, 17). In addition,
MC3-R activation on M␾ caused cAMP accumulation and inhibition of cytokine release (13, 16, 17).
0022-1767/03/$02.00
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The issue of which melanocortin receptor (MC-R) is responsible for the anti-inflammatory effects of melanocortin peptides is still
a matter of debate. Here we have addressed this aspect using a dual pharmacological and genetic approach, taking advantage of
the recent characterization of more selective agonists/antagonists at MC1 and MC3-R as well as of the existence of a naturally
defective MC1-R mouse strain, the recessive yellow (e/e) mouse. RT-PCR and ultrastructural analyses showed the presence of
MC3-R mRNA and protein in peritoneal macrophages (M␾) collected from recessive yellow (e/e) mice and wild-type mice. This
receptor was functional as M␾ incubation (30 min) with melanocortin peptides led to accumulation of cAMP, an effect abrogated
by the MC3/4-R antagonist SHU9119, but not by the selective MC4-R antagonist HS024. In vitro M␾ activation, determined as
release of the CXC chemokine KC and IL-1␤, was inhibited by the more selective MC3-R agonist ␥2-melanocyte stimulating
hormone but not by the selective MC1-R agonist MS05. Systemic treatment of mice with a panel of melanocortin peptides inhibited
IL-1␤ release and PMN accumulation elicited by urate crystals in the murine peritoneal cavity. MS05 failed to inhibit any of the
inflammatory parameters either in wild-type or recessive yellow (e/e) mice. SHU9119 prevented the inhibitory actions of ␥2melanocyte stimulating hormone both in vitro and in vivo while HS024 was inactive in vivo. In conclusion, agonism at MC3-R
expressed on peritoneal M␾ leads to inhibition of experimental nonimmune peritonitis in both wild-type and recessive yellow (e/e)
mice. The Journal of Immunology, 2003, 170: 3323–3330.
3324
MELANOCORTINS’ ANTI-INFLAMMATORY EFFECTS IN MC1-R-DEFICIENT MICE
This study was planned to address the apparent dichotomy between MC1-R and MC3-R in mediating the anti-inflammatory actions of melanocortins. We have taken advantage of the recent
characterization of more selective MC-R agonists, and from the
availability of the recessive yellow (e/e) mouse strain. A frameshift
mutation in the MC1-R gene in these animals results in a single
deletion of a nucleotide at position 549; the outcome is a receptor
protein with a premature termination in the fourth trans-membrane
domain, thus unable to couple to adenylate cyclase and activate
cAMP synthesis (18, 19). Interestingly, these mice have altered
pigmentation (yellow mice) without clear defects in the immune
system (18, 19), thus resembling the lack of reported phenotype
associated with red hair as studied in humans (20). The results
produced here with an experimental model of peritonitis show that
a functional MC1-R is not necessary to elicit the anti-inflammatory
actions of melanocortin peptides.
Materials and Methods
In vitro experimental section
Primary culture of M␾: detection of KC and IL-1␤ release. An enriched
population of peritoneal M␾ (⬎95% pure) was prepared by 2 h adherence
at 37°C in 5% CO2/95% O2 atmosphere in RPMI 1640 supplemented with
10% FCS, by culturing 5 ⫻ 106 M␾ in 24-well plates. Nonadherent cells
were washed off using warm medium, and adherent cells (⬎95% M␾) were
then incubated with ␥2-MSH (95 ␮M) alone or in combination with
SHU9119 (9 ␮M) for 15 min in RPMI 1640 medium. Cells were then
stimulated with 1 mg/ml MSU crystals (a concentration chosen from previous experiments (13)), and cell-free supernatants were collected 2 h later.
KC and IL-1␤ levels were measured by ELISA as described below.
cAMP formation. M␾s (1 ⫻ 105) were allowed to adhere in 24-well
plates as above, and incubated with serum-free RPMI 1640 medium containing 1 mM isobutylmethylxanthine with ␥2-MSH (30 ␮g/ml equivalent
to 95 ␮M), MTII (10 ␮g/ml equivalent to 9.3 ␮M), MS05 (30 ␮g/ml
equivalent to 22 ␮M), or the direct adenylate cyclase activator forskolin (3
␮M). In some experiments the effect of these peptides in the presence of 9
␮M of the MC3/4-R antagonist SHU9119 or the selective MC4-R antagonist HS024 were investigated. After 30 min at 37°C, supernatants were
removed and cells were washed and lysed. cAMP levels in cell lysates were
determined with a commercially available enzyme immunoassay (Amersham Life Sciences, Little Chalfont, U.K.) using a standard curve constructed with 0–3,200 fmol/ml cAMP.
RT-PCR for MC3-R message. Peritoneal M␾ (5 ⫻ 106) enriched by 2 h
adherence at 37°C in 24-well plates and lysed in 1 ml of Trizol reagent
(lysis buffer for RNA preparation) from Life Technologies (Paisley, U.K.)
and RNA was isolated according to manufacturer’s protocol. RNA was
extracted with chloroform and isopropanol, precipitated with ethanol, and
the pellet was resuspended in diethyl pyrocarbonate-treated water. The
yield and purity of the RNA was then estimated spectrophotometrically at
260 and 280 nm. Total RNA (3 ␮g) was used for the generation of cDNA.
PCR amplification reactions were then performed on aliquots of the cDNA.
All PCR were performed using PCR beads (Pharmacia Biosystems Europe,
St Albans, U.K.) in a final volume of 25 ␮l using a Hybaid OmniGene
thermal cycler (Middlesex, U.K.). The murine MC3-R primer sequences
were used as previously described (13): MC3-R, 5⬘-GCC TGT CTT CTG
TTT CTC CG-3⬘ and 5⬘-GCC GTG TAG CAG ATG CAG TA-3⬘ (forward
and reverse) which amplified a fragment of 820 bp in length. The cycling
parameters were as follows: after an initial denaturation for 3 min at 94°C,
30 cycles of annealing at 60°C (30 s), extension at 72°C (1 min), and
denaturation at 94°C (45 s), and a final further extension of 72°C for 10
min. Amplification products were visualized by ethidium bromide fluorescence in agarose gels. Images were inverted using the Graphic Converter
software (version 2.1) running on a Macintosh Performa 6200 (Reine,
Germany).
Western blotting analysis. We used a protocol recently validated for rat
and mouse peritoneal M␾ (16). Protein was isolated from samples of wild-
In vivo experimental section
MSU crystal-induced peritonitis. PMN recruitment into the peritoneal
cavity was elicited by MSU crystals as recently reported (21). Mice were
treated i.p. with 3 mg of MSU crystals in 0.5 ml PBS, and peritoneal
cavities lavaged at 2–96 h post challenge with 3 ml PBS containing EDTA
(3 mM) and heparin (25 U/ml). Aliquots of the lavage fluids were then
stained with Turk’s solution (0.01% crystal violet in 3% acetic acid), and
differential cell counts were performed by light microscopy using a
Neubauer hematocytometer. Data are reported as 106 PMN per mouse.
Lavage fluids were then centrifuged at 400 ⫻ g for 10 min and supernatants
were stored at ⫺20°C before biochemical determinations (see below).
Drug treatment. The natural hormone ␣-MSH (10 ␮g per mouse equivalent to 6 nmol), the relatively selective MC3-R agonist ␥2-MSH
(YVMGHFRWDRFG) (30 ␮g per mouse equivalent to 95 nmol) (14), the
mixed MC3/4-R agonist MTII (Ac-Nle-cyclo[-Asp-His-D-Phe-Arg-TrpLys-NH2) (10 ␮g per mouse equivalent to 9.3 nmol) (15), the pan-MC-R
agonist HP228 (Ac-Nle-Gln-His-D-Phe-Arg-D-Trp-Gly-NH2; 15 ␮g per
mouse equivalent to 15.2 nmol) (22), and the selective MC1-R agonist
MS05
(H-Ser-Ser-Ile-Ile-Ser-His-Phe-Arg-Trp-Gly-Lys-Pro-Val-NH2;
1–100 ␮g per mouse equivalent to 0.66–66 nmol) (23) were administered
s.c. 30 min before MSU crystals. In some experiments, agonist effect was
tested in the presence of the MC3/4-R antagonist SHU9119 (Ac-Nle-cyclo[-Asp-His-D-2-Nal-Arg-Trp-Lys-NH2) or selective MC4-R antagonist
HS024 (Ac-Cys-Nle-Arg-D-2-Nal-Arg-Trp-Lys-Cys-NH2) (16) of which 9
nmol were given i.p. 30 min before MSU crystals. MTII, ␣-MSH, ␥2-MSH,
HP228, SHU9119, and HS024 were purchased from Bachem (Saffron Walden, Essex, U.K.), stored at ⫺20°C before use, and dissolved in sterile PBS
(pH 7.4). MS05 was kindly provided by Melacure Therapeutics (Uppsala,
Sweden).
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Male C57BL.6 mice (20 –22 g body weight) were purchased from Tuck
(Battlesbridge, Essex, U.K.) and recessive yellow (e/e) mice (18) were a
kind gift from Dr. N. 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. Animal experimental work was performed according to Home Office regulations (Guidance on the Operation of Animals,
Scientific Procedures Act, 1986).
type and recessive yellow (e/e) mice peritoneal M␾ and spleens in PBS
containing EDTA (3 mM), leupeptin (0.39 mg/ml) and PMSF (10 mM).
Protein levels were then determined (Bio-Rad protein assay; Bio-Rad, Hercules, CA) and 50 ␮g of protein was mixed with 0.125 M Tris-HCl (pH
6.8), 2 mM EDTA, 4% sodium dodecyl sulfate (SDS), 10% mercaptoethanol, 20% glycerol and boiled for 10 min before loading and running on an
10% polyacrylamide gel (Protogel; National Diagnostics, Ashby De La
Zouche, Leicestershire, U.K.) for 60 min at 100 V. Protein was transferred
onto polyvinylidine difluoride membranes (Amersham Life Sciences) by
semidry blotting (Bio-Rad) for 60 min using a Tris/glycine buffer containing 20% methanol. Membranes were then blocked overnight at 4°C by
immersion in a 5% nonfat dried milk solution made up in PBS containing
0.1% Tween 20. Membranes were then incubated for 2 h at 4°C in a 5%
nonfat dried milk solution with an affinity-purified goat polyclonal Ab (1/
200 final dilution; Autogen Bioclear, Mile Elm Calne, U.K.) raised against
a peptide mapping human MC3-R. This goat polyclonal MC3-R Ab
showed cross-reactivity with mouse and rat, but did not cross-react with
MC1-R, MC2-R, MC4-R, and MC5-R of any species (data supplied by the
manufacturer). Following one 15 min and three 5 min washes in PBS and
Tween 20 (0.1%), the membrane was incubated for 1 h with an HRPconjugated donkey anti-goat IgG secondary Ab (1/5000 in 0.1% BSA in
PBS and 0.1% Tween). After another 15 min and three 5 min washes in
PBS/Tween), blots were incubated with ECL solution (Amersham Life
Sciences) for 1 min and then exposed to autoradiographic film for detection
of chemiluminescence. Cruz m.w. markers were also used (Autogen
Bioclear).
Electron microscopy analysis. We used a protocol recently validated for
rat knee joint M␾ (17). Peritoneal M␾ from wild-type and recessive yellow
mice (e/e) were lavaged as described above, and cells were fixed with a
mixture of freshly prepared 3% (w/v) paraformaldehyde and 0.05% (v/v)
glutaraldehyde in PBS, pH 7.2, for 4 h at 4°C, washed briefly in PBS, and
transferred to a solution of 2.3 M sucrose (in PBS) at 4°C overnight. The
cryoprotected cells were slam-frozen (Reichert MM80E; Leica, Milton
Keynes, U.K.), freeze-substituted at ⫺80°C in methanol for 48 h, and
embedded at ⫺20°C in LRGold acrylic resin (London Resin Company,
Reading, U.K.) in a Reichert freeze-substitution system. Ultrathin sections
(50–80 nm) were prepared by use of a Reichert Ultracut-S ultratome and
incubated at room temperature for 2 h with a polyclonal goat anti-MC3-R
Ab (dilution 1/200; Autogen Bioclear) or goat anti-MC3-R preabsorbed
with MC3-R blocking peptide (20 ␮g/ml; Autogen Bioclear) followed by
protein A linked to 15 nm gold (British Biocell, Cardiff, U.K.) for 1 h. As
additional negative control sections were incubated with nonimmuno goat
serum (1/200, Sigma-Aldrich, Poole, Dorset, U.K.) in place of the primary
Ab. The serum and antiserum were diluted in 0.1 M phosphate buffer
containing 0.1% egg albumin. After immuno-labeling sections were lightly
counterstained with uranyl acetate and lead citrate and examined with a
JEOL 1010 transmission electron microscope (JEOL, Peabody, MA).
The Journal of Immunology
Cytokine quantification by ELISAs. Murine KC and IL-1␤ levels in peritoneal lavage fluids were determined using commercially available ELISA
purchased from R&D Systems (Abingdon, U.K.). In brief, lavage fluids (50
␮l) were assayed for each cytokine and compared with a standard curve
constructed with 0–1 ng/ml of the standard cytokine. The ELISAs showed
negligible (⬍1%) cross-reactivity with several murine cytokines and chemokines (data as furnished by manufacturer).
Statistics. Data are reported as mean ⫾ SE of n distinct observations.
Statistical differences were calculated on original data by ANOVA followed by Bonferroni test for intergroup comparisons (24), or by unpaired
Student’s t test (two-tailed) when only two groups were compared. A
threshold value of p ⬍ 0.05 was taken as significant.
Results
Identification and functionality of the MC-R on peritoneal M␾
RT-PCR, Western blotting, and electron microscopy analyses were
used to monitor MC-R expression in resident peritoneal M␾ and as
a positive control spleen (Western blotting only) from both wildtype and recessive yellow (e/e) mice. RNA extracted from murine
peritoneal M␾ showed the presence of MC3-R by RT-PCR in
either mouse strain (Fig. 1a). To determine whether this message
was translated to protein we used Western blotting and electron
microscopy. Expression of MC3-R in wild-type and recessive yellow (e/e) mice peritoneal M␾ and spleen was monitored by Western blotting analysis. Western blotting confirmed the presence of
the MC3-R in protein extracts prepared from mouse peritoneal M␾
and spleen, with a band of the right m.w. (43 kDa) being obtained
(Fig. 1b). Electron microscopy was then used to highlight MC3-R
gold immunolabeling which was predominantly located on the
plasma membrane with a particular higher density in correspondence of membrane protrusions in wild-type (Fig. 1c) and recessive yellow (e/e) mice (Fig. 1d). This immunostaining was specific
for MC3-R insofar as it was absent when the primary Ab was
preabsorbed with the blocking peptide in wild-type (Fig. 1e) or
recessive yellow (e/e) mice (Fig. 1f). As a final control, cells
stained with nonimmune goat IgG did not display immuno-gold
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FIGURE 1. Expression of MC3-R on wildtype and recessive yellow (e/e) mice peritoneal
M␾ as detected by RT-PCR and electron microscopy. a, RT-PCR analysis demonstrates the presence of specific products for MC3-R (820 bp) in
M␾ taken from wild-type and recessive yellow
(e/e) mice. Genomic DNA (Gen) was used as a
positive control. The arrows indicate the presence of MC3-R message and 18s RNA detected
as control. Gel depicts a representative of four
distinct experiments with identical results. M,
markers shown as base pair. (b) Western blot
analysis demonstrates protein expression of
MC3-R in peritoneal M␾ and spleen from wildtype and recessive yellow (e/e) mice. c—h, Electron microscopy analysis: expression of MC3-R
on murine peritoneal MØ taken from wild-type
(c) and recessive yellow (e/e) (d) mice; immunoreactivity was predominantly localized on the
plasma membrane, with a minor degree of staining also detected in the cytosol; arrowheads,
characteristic clusters of gold particles on plasma
membrane protrusions. Lack of immuno-gold labeling on murine peritoneal M␾ when the antiMC3-R Ab was preabsorbed with the blocking
peptide in wild-type (e) and recessive yellow
(e/e) (f) cells. Similar negative results were obtained following nonimmune goat IgG staining
of M␾ from wild-type (g) and recessive yellow
(e/e) (h) mice. Microphotographs are representative of 10 distinct cells. n, nucleus; magnification
⫻16,000.
3325
3326
MELANOCORTINS’ ANTI-INFLAMMATORY EFFECTS IN MC1-R-DEFICIENT MICE
labeling on their plasma membrane in wild-type (Fig. 1g) or recessive yellow (e/e) mice (Fig. 1h).
Receptor functionality was determined quantifying cAMP accumulation in peritoneal M␾. In C57 wild-type mice the natural
and synthetic MC3-R agonists, ␥2-MSH and MTII, caused significant increases in cAMP accumulation with a 450% and 420%
increase above basal levels (73 ⫾ 12 fmol/well). A similar observation was noted with respect to the recessive yellow (e/e) mice in
which a 436% and 400% increase above basal levels (80 ⫾ 11
fmol/well) was measured for ␥2-MSH and MTII, respectively (Fig.
2a). These increases in both wild-type and recessive yellow (e/e)
mice were blocked in the presence of the MC3/4-R antagonist
SHU9119 (Fig. 2b) but not the selective MC4-R antagonist HS024
(Fig. 2c). The effect of forskolin was retained in the presence of
either antagonist. The selective MC1-R agonist MS05 failed to
elicit any increase in cAMP in either type of M␾ (Fig. 2, a–c).
MSU crystal-induced peritonitis
Anti-inflammatory effect of nonselective MC-R agonists in
experimental peritonitis
Nonselective MC-R agonists, the synthetic peptide HP228, and the
naturally occurring hormone, ␣-MSH, modulated the inflammatory response in wild-type mice, with inhibitions in PMN migration of 35% and 47%, for HP228 and ␣-MSH, respectively (Fig.
4a). The peptides were then administered to recessive yellow (e/e)
mice and similar inhibitory responses (⬃50%) were observed (Fig.
4a). HP228 and ␣-MSH caused a reduction in IL-1␤ and KC levels
measured in peritoneal lavages collected from both wild-type and
recessive yellow (e/e) mice (Fig. 4, b and c). In the absence of
MSU crystal injection, the number of PMN and levels of IL-1␤
and KC were below the detection limits in either mouse strain
(data not shown).
Anti-inflammatory effect of selective agonists MC-R in
experimental peritonitis
Next we evaluated the effect of more selective melanocortin peptides in this model of peritonitis. The natural (␥2-MSH, 30 ␮g/
mouse) and synthetic (MTII, 10 ␮g/mouse) MC3-R agonists inhibited both the cellular and humoral response produced by urate
FIGURE 2. MC3-R activation in peritoneal M␾ collected from wildtype and recessive yellow (e/e) mice. Adherent peritoneal M␾ were incubated with ␥2-MSH (95 ␮M), MTII (9.3 ␮M), MS05 (22 ␮M), forskolin (3
␮M), alone or together with the given MC-R antagonist, for 30 min before
determination of intracellular cAMP. a, Melanocortin agonists alone; b, in
the presence of 9 ␮M SHU9119; c, in the presence of 9 ␮M HS024. Data
are mean ⫾ SE of four mice per group. ⴱ, p ⬍ 0.05 vs vehicle control.
FIGURE 3. Time dependency of MSU crystal-induced PMN accumulation and KC release in the mouse peritoneal cavity. Mice received an
injection of MSU crystals (3 mg in 0.5 ml sterile PBS i.p.) at time 0.
Peritoneal cavities were washed at the reported time points before measurement of PMN influx (a) or KC protein in the cell-free exudates (b).
Data are mean ⫾ SE of eight mice per group. ⴱ, p ⬍ 0.05 vs time 0 group.
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Intraperitoneal injection of MSU crystals produced a time dependent accumulation of PMN with a similar profile in wild-type and
recessive yellow (e/e) mice. MSU crystal-induced PMN accumulation was maximal at 6 h postinjection (with an approximate influx rate of 1.1 ⫻ 106 PMN per hour in the 2- to 6-h time interval)
(Fig. 3a). PMN recruitment plateaued between 6 and 24 h, remaining at the same level at the later time point in wild-type mice
(⬎6 ⫻ 106 PMN per mouse). There appears to be no difference in
the cell emigration at 6 and 24 h in recessive yellow (e/e) mice
compared with wild type following injection of urate crystals into
the peritoneal cavity. Very few resident lymphocytes or M␾ could
be detected in the lavage fluids following challenge (data not
shown). The maximal PMN accumulation occurred at the 6-h time
point and was preceded by a marked release of the proinflammatory CXC chemokine KC, maximal at 2 h (Fig. 3b). Again no
difference in the release of KC was measured between wild-type
and recessive yellow (e/e) mice.
The Journal of Immunology
crystals. Fig. 5a shows the data for C57 wild-type mice, in which
MTII and ␥2-MSH significantly ( p ⬍ 0.05) inhibited MSU crystalinduced PMN recruitment by 54% and 45%, respectively. Similar
degrees of inhibition were observed in the recessive yellow (e/e)
mice with MTII and ␥2-MSH causing a 63% and 42% reduction in
PMN migration ( p ⬍ 0.05). The selective synthetic MC1-R agonist MS05 (1–100 ␮g) did not modify the inflammatory response
in either wild-type or recessive yellow (e/e) mice (Fig. 5a).
In view of our previous studies with intact mice (16), exudate
IL-1␤ levels were measured in recessive yellow (e/e) mice. Treatment of these mice with MTII or ␥2-MSH caused a significant
reduction in IL-1␤ release with 62% and 40% of inhibition, respectively (Fig. 5b).
Effect of MC-R antagonists
The attenuation of MSU crystal-induced inflammation by ␥2-MSH
was prevented by the MC3/4-R antagonist SHU9119 but not by the
selective MC4-R antagonist HS024, both antagonist being essentially inactive when administered on their own (Fig. 6a). In this set
of experiments, ␥2-MSH inhibited MSU crystal-induced PMN recruitment by 33% and 31% in wild-type and recessive yellow (e/e)
mice, respectively; this effect was abrogated by SHU9119 but not
HS024 (Fig. 6a). ␥2-MSH inhibition of PMN migration was associated with lower IL-1␤ levels in both wild-type and recessive
FIGURE 5. Effect of ␥2-MSH, MTII, and MS05 on MSU crystal-induced inflammation in wild-type and recessive yellow (e/e) mice. Mice
were pretreated s.c. with sterile PBS (100 ␮l), 30 ␮g ␥2-MSH, 10 ␮g MTII,
or with 1–100 ␮g MS05 30 min before i.p. injection of MSU crystals (3 mg
in 0.5 ml sterile PBS). PMN accumulation (a) and IL-1␤ levels in cell-free
exudates (b) were measured 6 h later. Data are mean ⫾ SE of eight mice
per group. ⴱ, p ⬍ 0.05 vs PBS group.
yellow (e/e) mice and this action of the peptide was again blocked
in the presence of the antagonist SHU9119 (Fig. 6b).
In vitro effects of ␥2-MSH on cytokine release from cultured M␾
Cytokine and chemokine release from adherent M␾ in vitro was
evaluated as marker cell activation. C57 wild-type M␾ incubation
with ␥2-MSH significantly reduced MSU crystal-elicited KC and
IL-1␤ release (Table I). A similar profile was observed in M␾
taken from recessive yellow (e/e) mice. In line with the in vivo
data, ␥2-MSH inhibition of KC and IL-1␤ release by M␾ collected
from either wild-type and recessive yellow (e/e) mice was abolished by coincubation with 9 ␮M SHU9119 (Table I).
Discussion
In this study we have used an integrated approach with ultrastructural in vitro and in vivo analyses to demonstrate that a functional
MC1-R is not necessary for the anti-inflammatory efficacy of melanocortin peptides. This does not appear to be the case for MC3-R,
thus possibly explaining why MC1-R deficiency or lack of function does not cause clear immunological defects in rodents or
humans.
This study was prompted by the unclear role that distinct MC-Rs
might play in mediating the anti-inflammatory properties of melanocortin peptides. We have previously shown that MC3-R is
present on rodent resident peritoneal M␾ by Western blotting and
RT-PCR, and proposed that its activation down-regulated the experimental inflammatory response (13, 16). However, it has been
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FIGURE 4. Effect of HP228 and ␣-MSH on MSU crystal-induced inflammation in wild-type and recessive yellow (e/e) mice. Mice were pretreated s.c. with sterile PBS (100 ␮l), 15 ␮g HP228, or 10 ␮g ␣-MSH 30
min before i.p. injection of MSU crystals (3 mg in 0.5 ml sterile PBS).
PMN accumulation (a) and IL-1␤ and KC levels in cell-free exudates (b
and c) were measured 6 h later. Data are mean ⫾ SE of eight mice per
group. ⴱ, p ⬍ 0.05 vs PBS group.
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suggested by other studies (3, 25) that MC1-R could be responsible
for the anti-inflammatory effects displayed by ␣-MSH and other
melanocortin peptides. Here we sought to address this discrepancy
using a panel of more selective melanocortin agonists and antagonists and, more importantly, mice with a defected MC1-R. The
recessive yellow (e/e) mouse has a frameshift mutation that leads
to the expression of a nonfunctional MC1-R (18).
Initially, we monitored the basal conditions in the recessive yellow (e/e) mouse. We have previously shown that MC3-R is expressed on mouse resident peritoneal M␾ by RT-PCR and Western
blotting (13, 16) and on rat knee joint M␾ by electron microscopy
(17). Thus, we confirmed first the presence of MC3-R mRNA in
Table I. Effect of ␥2-MSH on MSU crystal stimulated KC and IL-1␤ release in vitroa
Agonist
Antagonist
KC (pg/ml)
IL-1␤ (pg/ml)
Wild type
PBS
␥2-MSH
PBS
␥2-MSH
PBS
PBS
SHU9119
SHU9119
390 ⫾ 92
188 ⫾ 12ⴱ
438 ⫾ 94
494 ⫾ 143
355 ⫾ 24
142 ⫾ 11ⴱ
307 ⫾ 14
339 ⫾ 23
Recessive yellow (e/e)
PBS
␥2-MSH
PBS
␥2-MSH
PBS
PBS
SHU9119
SHU9119
109 ⫾ 11
81 ⫾ 6
210 ⫾ 63
137 ⫾ 23
398 ⫾ 34
203 ⫾ 12ⴱ
394 ⫾ 27
343 ⫾ 24
Mice
a
PBS or SHU9119 (9 ␮M) were added to adherent M␾ (5 ⫻ 106) prepared from wild-type or recessive yellow (e/e) mice, 10 min prior to PBS or 95 ␮M ␥2-MSH. M␾ were
stimulated 15 min later with 1 mg/ml MSU crystals. Supernatants were removed 2 h later and cell-free aliquots were analyzed for chemokine and cytokine content using specific
ELISA. Data are mean ⫾ SE of three or four determinations.
ⴱ, p ⬍ 0.05 vs relevant PBS control.
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FIGURE 6. SHU9119 prevents ␥2-MSH inhibition of MSU crystal peritonitis in wild-type and recessive yellow (e/e) mice. Mice received PBS
(100 ␮l s.c.) or ␥2-MSH (30 ␮g s.c.) with or without 9 nmol i.p. SHU9119
or HS024 30 min before MSU crystals (3 mg in 0.5 ml sterile PBS i.p.).
Peritoneal cavities were washed 6 h later, and the number of accumulated
PMN (a) or the content of IL-1␤ in cell-free exudates (b) were measured
6 h later. Data are mean ⫾ SE of eight mice per group. ⴱ, p ⬍ 0.05 vs
appropriate PBS group.
M␾ taken from these mice using RT-PCR. Western blotting analysis confirmed that message was translated to protein in both wildtype and recessive yellow (e/e) mice peritoneal M␾ and, as a positive control, spleens. We then used electron microscopy to
visualize a punctuate distribution of the receptor on the M␾ plasma
membrane and its microvilli, in cells taken from wild type as well
as from the mice bearing a nonfunctional MC1-R. Finally, MC3-R
was functionally intact on these cells, since melanocortin agonists
elicited equal levels of intracellular cAMP. Together these data
indicate that MC3-R is not only expressed on peritoneal M␾ from
wild-type and recessive yellow (e/e) mice but is fully functional,
such that cAMP formation occurs after agonist activation. In addition, the recessive yellow (e/e) mouse did not display any signs
of on-going inflammatory response; analysis of mesenteries of
wild-type and recessive yellow (e/e) mice by intravital microscopy
did not show an augmented interaction between circulating white
blood cells and the postcapillary endothelium, nor higher numbers
of leukocytes in the interstitium (data not shown). A lack of difference between the two mouse strains was also observed in terms
of MSU crystal-induced peritonitis (assessed as degree of PMN
migration and release of KC). The crystals produced an intense and
long-lasting accumulation of blood-borne PMN into the peritoneal
cavity, previously characterized (21), with essentially no difference
between wild-type and recessive yellow (e/e) mice, not only with
regards to the maximal responses, but also for the time-course
profiles. Together these data indicate that an alteration in MC1-R
functionality does not lead to subtle changes in animal homeostasis
as well as in the host response to crystal injury.
Satisfied with the background conditions, we then tested the
effect of several melanocortin peptides in these two mouse strains.
Systemic administration of the nonselective melanocortin agonists
␣-MSH (7–10) and HP228 (22) inhibited MSU crystal-induced
PMN migration and this was associated with a reduction in IL-1␤
and KC in the inflammatory exudates. These peptides were equally
active in wild-type and recessive yellow (e/e) mice. Another study
has reported the anti-inflammatory effect of ␣-MSH in a model of
LPS induced brain inflammation in recessive yellow (e/e) mice (9),
strongly indicating that involvement of another MC-R also in this
experimental condition. Whereas ␣-MSH anti-inflammatory actions have been reported in several studies (13, 25–28) this is the
first study that the nonselective peptide HP228 has been shown to
inhibit PMN migration, adding HP288 to the growing list of melanocortins able to down-regulate experimental inflammation. Similarly to ␣-MSH, HP228 fully retained its anti-inflammatory efficacy in recessive yellow (e/e) mice.
The Journal of Immunology
Acknowledgments
We thank Drs. R de Médicis and A Lussier (University of Sherbrooke,
Sherbrooke, Canada) for the supply of MSU crystals. Recessive yellow
(e/e) mice were a kind gift from Dr. Nancy Levin (Trega Bioscience).
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The lack of involvement of MC1-R in the experimental peritonitis was further substantiated with the use of more selective melanocortin peptides, and we chose ␥2-MSH (putative endogenous
agonist at MC3-R (14)), MTII (long lasting MC3-R activator (15))
and MS05 (selective MC1-R activator (23)). Systemic administration of MTII and ␥2-MSH before MSU crystal injection attenuated
PMN migration equally in wild-type and recessive yellow (e/e)
mice and this was associated with a reduction in IL-1␤ exudates
levels. This data is in agreement with previous studies in this
model (16). As mentioned in the introduction, melanocortin inhibition of IL-1␤ is not surprising, and it likely is due to cAMPmediated inhibition of transcription factor functions (3, 29). Aside
from our previous study (16), ␥2-MSH inhibition of LPS-induced
IL-1␤ gene expression has also been documented (30). Also, our
own in vitro data and those published previously all agree for an
exquisite inhibitory action of melanocortins on cytokine/chemokine synthesis and release (3, 13, 16, 26 –28, 31).
The proposition that MC3-R is the predominant antiinflammatory receptor for melanocortins (13, 16, 17) is supported
by the fact that the selective MC1-R agonist MS05 (23) was inactive in both wild-type and recessive yellow (e/e) mice. This selective agonist has been reported to down-regulate TNF-␣-induced
E-selectin, VCAM and ICAM mRNA, and protein expression in
human dermal vascular endothelial cells (32). These in vitro data
have been extrapolated to explain a potential anti-inflammatory
role for MC1-R. In our experimental conditions, though, MC1-R
does not appear to be active. A lack of involvement of MC1-R has
also been observed when assessing the protective effects of melanocortins in a model of myocardial ischemia/reperfusion-induced
arrhythmias (33). The central role of MC3-R was also supported
by the experiments with the MC-R antagonists. The MC3/4-R antagonist SHU9119, but not the selective MC4-R antagonist HS024,
abrogated cAMP accumulation produced by the different agonists
on M␾ in vitro and PMN accumulation in vivo. These results were
equally obtained in wild-type and recessive yellow (e/e) mice.
It is worth noting a potential extrapolation of the data here presented to the human system. Several human MC1-R single mutation have been reported within the Northern European population
with 75% of individuals showing some allelic variants (3) and
similar single nucleotide polymorpisms as well as frame shift mutations have been identified in other mammals (3). The importance
of these mutations lies in the fact that these receptors are nonfunctional and in turn lead to a red or blond hair coloration, lighter skin
types and less ability to tan (34, 35). To date, no clear affection of
the immune system has been reported in these subjects. Similarly,
no correlation between this phenotype and a higher risk of inflammatory disorders has been made (3).
In conclusion, we have demonstrated here that MC3-R activation modulates the host inflammatory response in this experimental
model of peritonitis, and that this role is not solely played in mice
with a nonfunctional MC1-R (recessive yellow (e/e) but also in
intact wild-type mice. The apparent lack of involvement of MC1-R
in this specific model was reinforced by the experiment with the
selective MC1-R agonist (MS05). These findings highlight the involvement of MC3-R in modulating the inflammatory response.
With the development of more selective compounds and the use of
knockout mice for the MC3-R (36, 37) this scientific challenge can
be addressed clearly in a conclusive manner.
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