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Journal of Leukocyte Biology logoLink to Journal of Leukocyte Biology
. 2008 Apr 3;84(1):191–198. doi: 10.1189/jlb.0707463

Diminishment of α-MSH anti-inflammatory activity in MC1r siRNA-transfected RAW264.7 macrophages

Dayu Li 1, Andrew W Taylor 1,1
PMCID: PMC3178503  PMID: 18388300

Abstract

The neuropeptide α-melanocyte-stimulating hormone (α-MSH) is a powerful suppressor of inflammation mediated by macrophages, which express at least two receptors, melanocortin 1 and 3 receptors (MC1r and MC3r) that bind α-MSH. Albeit, the anti-inflammatory activity of α-MSH has been well documented in macrophages, the mechanisms of α-MSH activity in macrophages are not clearly understood. This study is to investigate which of the MCr expressed on macrophages is associated with the immunosuppressive activities of α-MSH on LPS-stimulated macrophages. To address this question, we transfected RAW264.7 macrophage cells with MC1r small interfering (si)RNA, which specifically targets mouse MC1r mRNA. The diminution of MC1r mRNA expression was 82% at 24 h and 67% at 48 h after transfection. There was a significant loss in α-MSH suppression of NO generation and TNF-α production by MC1r siRNA-transfected macrophages stimulated with LPS. There was an equally diminished α-MSH suppression of LPS-stimulated intracellular activation of NF-κB and p38 phosphorylation. In addition, the diminishment of MC1r expression by siRNA transfection had no influence on MC3r expression and function in the macrophages. These findings demonstrate that α-MSH suppression of LPS-induced inflammatory activity in macrophages requires expression of MC1r. The results imply that although all of the MCr are G-coupled proteins, they may not necessarily function through the same intracellular pathways in macrophages.

Keywords: neuroimmunomodulation, immunosuppression, neuroimmunology, neuropeptides, inflammation, innate immunity

INTRODUCTION

The neuropeptide α-melanocyte-stimulating hormone (α-MSH) is a 13-aa-long peptide that plays an important role in immune homeostasis and maintenance of ocular immune privilege [1, 2]. It is derived from the proteolytic cleavage of the proopiomelanocortin hormone produced by pituitary cells, keratinocytes, macrophages, and dendritic cells [3,4,5]. The neuropeptide is constitutively expressed in various body tissues, fluids, and blood [6,7,8]. Its concentration may be regulated by acute inflammatory responses and changes in metabolic signals [6, 9]. The effects of α-MSH on immunity are well documented in that α-MSH suppresses the proinflammation activity of macrophages and neutrophils and systemic acute-phase response to infection; moreover, α-MSH suppresses the activation of autoreactive T cells and IFN-γ production and promotes the activation of regulatory T cells [1, 10, 11]. It has been demonstrated that α-MSH inhibits LPS-stimulated NO generation and TNF-α production in macrophages [5, 12,13,14,15]. The suppression of these LPS-mediated activities in macrophages may be the result of blocking TLR4 signaling through α-MSH suppression of CD14 expression or by α-MSH-mediated translocation of IL-1R-associated kinase-M to the TLR4 intracellular complex [16, 17]. Other intracellular pathways blocked by α-MSH are the activation of p38 MAPK and NF-κB in macrophages [13, 15, 18,19,20].

The receptors for α-MSH are a family of G-protein-coupled melanocortin receptors (MCr). The MC1r is the most predominate receptor for α-MSH in the body, and MC1r and MC3r are considered to be important MCr on macrophages [14, 21,22,23]. There are reports that describe α-MSH activation of the cAMP-dependent protein kinase A (PKA) signaling pathway through MC1r on endotoxin and IL-1-stimulated macrophages [12, 14, 15]. The anti-inflammatory effect of α-MSH mediated through the MC1r in the activated macrophages is through the suppression of p38 MAPK phosphorylation and NF-κB activation [13, 15]. In resting macrophages or in urate–crystal-stimulated macrophages, the MC3r may play a more important role in activating the p38 MAPK pathway and IL-10 production [18, 21, 22, 24].

The expression of multiple MCr on the immune cells has made it difficult to identify the MCr associated with specific immunomodulating activities of α-MSH. Moreover, knocking out these receptors can cause systemic, metabolic changes that can indirectly influence immunity or promote a genetic regulatory compensation, where knocking-out one of the MCr causes an overexpression of other MCr [21, 25,26,27,28,29,30]. It is assumed that knocking-out the MC1r will neutralize the anti-inflammatory activity of α-MSH on macrophages; however, it is reported that MC1r knockout mice compensate by overexpressing MC3r and show no change in their response to α-MSH treatment in suppressing inflammation [21]. Moreover, the MC1r is not truly knocked out; it is only nonfunctional [31]. If dimerization of MCr is part of their initial activation pathway, then the nonintracellular signaling MC1r can still take part by dimerizing or paring with MC3r when engaged by α-MSH [32, 33]. This dimerization of MCr can further confuse interrupting the effects of using only receptor agonists or antagonists. Therefore, we silenced the expression of the MC1r in cultured, endotoxin-stimulated macrophages using a small interfering (si)RNA approach and demonstrated neutralization of α-MSH-induced immune suppression in vitro.

MATERIALS AND METHODS

Cells and reagents

The mouse macrophage cell line RAW264.7 obtained from American Type Culture Collection (Manassas, VA, USA) was maintained in DMEM (Lonza, Walkersville, MD, USA), supplemented with 10% FCS (Hyclone, Logan, UT, USA) and 0.01 M HEPES, 1× nonessential amino acids, and 1 mM sodium pyruvate (Lonza). The cells were incubated at 37°C, 10% CO2, in a humidified incubator. The cells at passages 4–20 were used for experiments, and depending on the experiment, they were grown in six-well or 96-well culture plates (Corning, Corning, NY, USA).

The endotoxin LPS of Escherichia coli 0111:B4 was from Sigma Chemical Co. (St. Louis, MO, USA), the amidated, acetylated peptide α-MSH and the MC3r agonist melanotan II (MTII) were purchased from Bachem (King of Prussia, PA, USA), and the MC3r agonist D-Trp8-γ-MSH was purchased from Phoenix Pharmaceuticals (Burlingame, CA, USA). Antibodies to detect MC1r were obtained from Alpha Diagnostic International Inc. (San Antonio, TX, USA); antibodies to immunoprecipitate and detect MC3r were anti-MC3r antibodies H88 and C20 from Santa Cruz Biotechnology (Santa Cruz, CA, USA), to detect p38 MAPK and phosphorylated p38 MAPK, were obtained from Cell Signaling Technology Inc. (Danvers, MA, USA); and the antibody against β-actin and the secondary antibodies were purchased from Santa Cruz Biotechnology. We assayed for mouse TNF-α using a R&D Systems (Minneapolis, MN, USA) ELISA kit, and NO generation was assayed using a Griess reagent kit (Molecular Probes, Eugene, OR, USA) for nitrite determination. Transfection reagent for siRNAs and the RNeasy mini kit for total RNA isolation were purchased from Qiagen Inc. (Valencia, CA, USA). The first-strand cDNA synthesis reagents were obtained from Invitrogen (Carlsbad, CA, USA). Primers and probes for mouse MC1r, MC3r, MC5r, GAPDH, and β-actin for real-time PCR were purchased from Applied Biosystems (Foster City, CA, USA). To assay for activated NF-κB, we used an EMSA kit (Pierce Biotechnology, Rockford, IL, USA).

siRNA duplexes

Chemically synthesized, predesigned MC1r siRNA duplex against mouse MC1r mRNA duplex was obtained from Ambion (Austin, TX, USA). The sequences are shown in Table 1. FAM-labeled MC1r siRNA and Cy™3-labeled negative control siRNA (Ambion) were used to determine siRNA transfection efficiency. Mouse GAPDH siRNA (Ambion) was used to detect transfection effect and optimize transfection conditions in macrophage RAW264.7 cells. Irrelevant siRNA without sequence similarity to any known gene sequences from mouse, rat, or human was used as a negative control.

TABLE 1.

Mouse MC1r siRNA Duplex Sequences

Name Reference No. Sense sequence (5′–3′) Antisense sequence (5′–3′)
mc1r_1 NM_008559 GGGUGACAGUGAUAUCCAGtt CUGGAUAUCACUGUCACCCtc
mc1r_2 NM_008559 GGUACUCAUCCCUUCCUGAtt UCAGGAAGGGAUGAGUACCtg
mc1r_3 NM_008559 GGAUGAGCUUUAAAAUAGAtt UCUAUUUUAAAGCUCAUCCtg
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Cell culture and siRNA transfections

RAW264.7 cells were plated, depending on the experiment, into the wells of a six-well plate at 1.0 × 106 cells/well in 2 ml 10% FBS supplemented DMEM, or at 4 × 104 cells/well of a 96-well plate in 200 μl 10% FBS supplemented DMEM, the day before transfection. After 24 h of incubation, the cells were transfected with siRNA duplexes targeting mouse MC1r mRNA or GAPDH mRNA or were transfected with an irrelevant siRNA duplex. The transfection procedures were done following the manual for the RNAiFect transfection kit (Qiagen Inc.). The siRNA was diluted with kit Buffer EC-R, then mixed with kit RNAiFect transfection reagent at a ratio of siRNA:RNAiFect transfection reagent of 1 μg:6 μl. The siRNA reagent was incubated for 10 min at room temperature for formation of transfection complexes, which were added to the cell cultures at a concentration of 80 nM siRNA. The cells were incubated at 37°C, 10% CO2, for 16 h, and the media were replaced with fresh culture media. The silencing effect was assayed at 24 or 48 h post-transfection.

We optimized the transfection conditions by transfecting the RAW264.7 cells with siRNA targeting the mouse housekeeping GAPDH gene using the Qiagen RNAiFect transfection kit and assessing 24 h later GAPDH gene silencing by quantitative real-time PCR. We used endogenous β-actin mRNA levels to normalize the GAPDH mRNA expression, and the remaining GAPDH mRNA was calculated as a percentage of GAPDH mRNA detected in untreated RAW264.7 cells. The methods we described above achieved an optimal GAPDH mRNA expression knockdown of 85%. Transfection efficiency was determined by fluorescence microscopy of macrophages transfected with FAM-labeled MC1r siRNA and Cy3-labeled negative control siRNA. At 24 h after transfection, the cells were washed once with PBS and fixed with 4% formaldehyde in PBS at room temperature for 15 min. After three washes with PBS, the cells were stained with 4′, 6-diamidino-2-phenylindole for 10 min, washed three times with PBS, and observed under the fluorescence microscope to determine the percentage of transfected cells versus the total number of cells. The transfection efficiency was >95%.

Quantitative real-time PCR

Total RNA was isolated from macrophages with the RNeasy mini kit (Qiagen Inc.), according to the manufacturer’s instructions. The cells were washed three times with PBS buffer and collected. They were disrupted, lysed, and homogenized using a QIAshredder spin column (Qiagen Inc.). An equal volume of 70% ethanol was mixed with the homogenized lysates and applied to a RNeasy mini column for adsorption of total RNA to the column membrane. RNA was washed once with washing buffer. Further DNA removal was done using a column DNase digestion by the RNase-free DNase set (Qiagen Inc.). RNA was washed and eluted in RNase-free water. The RNA concentration was determined by spectrophotometry at 260 nm.

The first-strand cDNA synthesis reaction was undertaken with the SuperScript™ first-strand synthesis system for a RT-PCR kit (Invitrogen). The isolated total RNA (5 μg) was reverse-transcribed in a 20-μl reaction mixture containing 20 mM Tris-HCl (pH 8.4), 50 mM KCl, 200 μM of each dNTP, 50 ng random hexamers, 5 mM MgCl2 10 mM DTT, 40 units RNaseOUT™ recombinant RNase inhibitor, and 50 units SuperScript™ II RT. The mixture was incubated at 25°C for 10 min and transferred to 42°C for 50 min. The RT reaction was terminated by heating the mixture to 70°C for 15 min and then adding 2 units RNase H at 37°C for 20 min to remove the RNA from the cDNA:RNA hybrid molecule.

The 50-μl real-time PCR reaction mixture consisting of FAM dye-labeled TaqMan Minor Groove Binder predesigned mouse MC1r, MC3r, or MC5r probe and two unlabeled PCR primers (Applied Biosystems), 25 μl 2× TaqMan Universal PCR Master Mix containing AmpliTaq Gold DNA polymerase, AmpErase uracil N-glycosylase, dNTPs with dUTP (Applied Biosystems), and 2 μl of our sample cDNA (equivalent to 500 ng RNA). The final reaction concentration of the probes was 250 nM and 900 nM for each primer. Amplification and detection of PCR products were performed using an ABI Prism 7900HT sequence detection system with thermal cycling conditions of 2 min at 50°C for 1 cycle, 10 min at 95°C for 1 cycle, 15 s at 95°C, and 1 min at 60°C for 40 cycles. The results were analyzed with SDS 2.1 software (Applied Biosystems). Each assay was carried out in triplicate. The relative expression of MCr mRNA was normalized to the relative expression of endogenous β-actin mRNA (Applied Biosystems). The relative quantization of the MCr mRNA in each sample was measured using the comparative threshold cycle method.

Immunoblot analysis

The cells were grown in a six-well plate and transfected with MC1r siRNA duplex for 24 h. The cells were washed with cold PBS for three times and scraped off the plate surface, collected, and centrifuged. The cell pellet was washed one more time with cold PBS. The cells were then resuspended with ice-cold radioimmunoprecipitation assay lysate buffer [PBS, pH 7.4, 1% Nonidet P-40 (NP-40), 0.5% sodium deoxycholate, 0.1% SDS, 100 ug/mL PMSF, 42 μg/mL aprotinin, and 1 mM sodium orthovanadate], and the cells were homogenized by passage a couple of times through a 21-gauge needle. The sample was incubated on ice for 40 min followed by centrifuging for 20 min at 13,000 g and 4°C .The supernatant was collected and used as whole-cell lysates. The protein concentration was determined using the Bio-Rad (Hercules, CA, USA) protein assay kit. To detect MC1r protein, 20 μg protein lysate was mixed with 4× NuPAGE lithium dodecyl sulfate sample buffer (Invitrogen) and 10× NuPAGE sample-reducing agent (Invitrogen), incubated at 70°C for 10 min, loaded on a precast 4–12% NuPAGE Novex Bis-Tris gel (Invitrogen), and subjected to electrophoresis. To detect MC3r protein, 4 μg anti-MC3r antibody H88 was added to the lysate, incubated for 4 h at room temperature, followed by the addition of protein-A/G sepharose beads, and incubated overnight with end-over-end agitation at room temperature. The beads were centrifuged at 800 g, washed twice with lysing buffer, and then resuspended in the sample buffer for electrophoresis through a precast 4–12% NuPAGE Novex Bis-Tris gel (Invitrogen). The separated proteins were electrophoretically transferred from the gel onto a nitrocellulose membrane (Invitrogen). Nonspecific binding was blocked with 3% BSA, dissolved in TBS buffer (50 mM Tris, pH 7.4, 150 mM NaCl) at room temperature for 1 h. The membrane was incubated overnight at 4°C with the appropriate primary antibodies (rabbit anti-MC1r or goat anti-MC3r C20) in 3% BSA dissolved in TBS-T. Following three washes with TBS-T, the membrane was incubated with the appropriate secondary antibody, goat anti-rabbit IgG HRP for anti-MC1r, and rabbit anti-goat IgG alkaline phosphatases for anti-MC3r for 50 min at room temperature. After washing with TBS-T for three times, detection was performed with the ECL Western blotting detection reagents (Amersham Biosciences, Piscataway, NJ, USA) for MC1r or with fast 5-bromo-4-chloro-3-indolyl-phosphate/NBT substrate (Sigma-Aldrich, St. Louis, MO, USA) for MC3r detection. The immunoreactive bands were revealed, and band density was analyzed.

Measurement of NO generation

The cells were seeded at 4 × 104 cells/well in a 96-well flat-bottom culture plate and grown in 10% FBS DMEM media at 37°C, 10% CO2, the day before transfection. The cells were transfected as described above with MC1r or irrelevant siRNA and incubated for an additional 24 h. The cells were washed twice with pheno-red-free DMEM, and in pheno-red-free DMEM, the cells were stimulated with 100 ng/ml LPS and treated with 100 nM α-MSH, 10 μM MTII [18], or 15 μM D-Trp8-γ-MSH [22] and incubated for 16 h. The concentration of nitrite in the conditioned media, which is relative to the amount of NO generated by the stimulated macrophages, was assayed with a Griess reagent. The concentration of nitrite in the cell cultures was calculated by comparing the 548 nm OD of the Griess reagent color change in the sample wells to a standard curve made from the OD of the color change caused by known concentrations of sodium nitrite. Each sample was assayed on four replicates

TNF-α ELISA

The cells were cultured and transfected as we did in the NO generation experiments, except the transfected, LPS-stimulated α-MSH-, MTII-, or D-Trp8-γ-MSH-treated cells were incubated for 6 h before the conditioned media were assay for TNF-α. The concentration of TNF-α in the conditioned media was measured using a mouse TNF-α ELISA kit (R&D Systems).

Preparation of nuclear extracts and EMSA

Nuclear extracts were prepared from MC1r siRNA-transfected RAW264.7 cells that were LPS-stimulated and α-MSH-treated for 1 h using the NE-PER nuclear and cytoplasmic extraction reagent kit according to the manufacturer’s instruction. A protease inhibitor cocktail (Sigma-Aldrich) was added to the nuclear extraction reagent. Nuclear protein concentration in the nuclear extracts was measured using the Bio-Rad protein assay reagent. The EMSA was performed using a LightShift chemiluminescent EMSA kit for NF-κB (Pierce Biotechnology). Biotinylated, 3′-end-labeled and unlabeled, single-stranded sense and antisense oligonucleotide probes containing the NF-κB consensus-binding motif (underlined) 5′-AGT TGA GGG GAC TTT CCC AGG C-3′ were synthesized by Integrated DNA Technologies Inc. (Coralville, IA, USA). The double-stranded probes were produced by annealing the probes for 4 min at 95°C, then for 10 min at 70°C, followed with a 0.1°C/s decrease to 4°C and for 10 min at 4°C. Nuclear extract proteins (5 μg) were mixed with 20 μl binding-reaction solution of 10 mM Tris, pH 7.5, 50 mM KCl, 1 mM DTT, 2.5% glycerol, 0.05% NP-40, 50 ng/ul poly (dI-dC), 5 mM MgCl2, and 30 fmol of the biotin 3′-end-labeled NF-κB probe. The reaction was incubated at room temperature for 20 min. Specificity of the binding reaction was assayed by adding a 100-fold molar excess of unlabeled, native oligonucleotide to the reaction mixture to compete for the NF-κB protein in the nuclear extract. The DNA–protein complexes and unbound free probe were separated on a precast 6% DNA retardation gel (Invitrogen) with 0.5× TBE buffer (45 mM Tris, 45 mM boric acid, 1 mM EDTA, pH 8.4) at 100 V for 1 h and transferred to a nylon N+ membrane at 380 mA for 1 h. The transferred DNA was cross-linked to the membrane using a UV-light cross-linker, and the biotin 3′-end-labeled NF-κB probe was detected by treating the membrane with HRP-conjugated streptavidin and using a chemiluminescent substrate system for autoradiography film exposure of the membrane.

Assay for cAMP accumulation

The RAW264.7 macrophage cells were seeded at 2 × 105 cells/well in a 24-well tissue-culture plate and grown in 10% FBS DMEM media at 37°C, 10% CO2, for 24 h and then transfected with MC1r siRNA as described above. After a 24-h incubation, the cells were washed, and fresh media were added to the cultures with our working concentration of α-MSH (100 nM) or with the reported optimal concentration of the MC3r agonist MTII (10 μM) on RAW cells [18]. The cultures were incubated for 5 min with the α-MSH-treated cultures [5] and 30 min with the MTII-treated cultures [18]. The cells were washed and then lysed with 250 μl 0.1 M HCl for 20 min at room temperature. The lysates were centrifuged at 600 g for 5 min at room temperature. The supernatant was assayed for cAMP using a cAMP ELISA kit (Sigma-Aldrich) according to the manufacturer’s instruction.

RESULTS

Silencing of MC1r mRNA expression in macrophages

Before we attempted to silence MC1r, we assessed the relative levels of MC1r, MC3r, and MC5r mRNA expression in the monocytic leukemic cell line RAW264.7 cells. The results of quantitative real-time PCR analysis performed on the macrophage cell line showed that there was equal expression of MC1r mRNA and MC3r mRNA with the expression of MC5r mRNA no greater than background (Fig. 1). In comparison with primary macrophages from C57BL/6J mice, the ratio of MC1r and MC3r mRNA was 2:1 with no MC5r mRNA expressed (not shown); therefore, the macrophage cell line is similar to primary macrophages in the expression of MCr mRNA.

Figure 1.

Figure 1

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Expression of MC1r, MC3r, and MC5r mRNA in RAW264.7 macrophage cells. Total RNA was isolated from RAW264.7 macrophages cultured for 36 h and analyzed by quantitative real-time PCR. The results are the mean relative mRNA expression ± sd. The results are relative to β-actin mRNA expression of triplicate assays with a background set at a value of 1 and are representative of two cultures.

The macrophage cell line was transfected with one of three chemically synthesized duplex MC1r siRNA sequences, and the duplex we labeled mc1r_1 siRNA (Table 1) was effective in silencing MC1r mRNA expression when transfected into the macrophages (Fig. 2). An irrelevant siRNA with no homology to any known mammalian gene sequences was used as a nonsilencing control. Real-time PCR showed that MC1r mRNA was effectively knocked-down in MC1r siRNA-transfected macrophages compared with untransfected and irrelevantly transfected cells (Fig. 2A). Highly efficient knockdown of MC1r mRNA was achieved in the macrophages with a reduction in MC1r siRNA by 82% at 24 h after transfection and 67% at 48 h after transfection. There is no significant change in MC1r mRNA expression in the macrophages transfected with the irrelevant siRNA.

Figure 2.

Figure 2

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The effects of MC1r siRNA transfection of macrophages. The RAW264.7 macrophages were transfected with MC1r siRNA or with irrelevant siRNA. (A) After 24 and 48 h of incubation, MC1r mRNA expression was measured by real-time PCR, and the mRNA expression was normalized to endogenous β-actin mRNA expression. The results represent three independent experiments, and presented are the mean relative expression ± sd. (B) Expression of MC1r protein was assayed in the transfected macrophages by immunoblotting lysates with anti-MC1r antibody of 24 h post-transfected macrophages. The expression of β-actin protein was the loading control. The results are representative of two independent experiments. (C) The macrophages were assayed for their expression of MC3r and MC5r mRNA by quantitative real-time PCR 24 h after MC1r siRNA transfection. This experiment was conducted two times with similar results relative to β-actin mRNA expression.

Whole cell lysates were prepared from each of the transfected and untransfected macrophages, and an immunoblot analysis was done on the lysates 24 h after transfection to detect MC1r protein (Fig. 2B). There was a 72% reduction in the expression of MC1r protein in the MC1r siRNA-transfected macrophages compared with protein expression in the untransfected macrophages. This change in MC1r protein expression is proportional to the knockdown of MC1r mRNA seen in Figure 2A. The expression levels of MC1r protein in the irrelevant siRNA-transfected cells had the same level of MC1r protein as the untransfected cells (Fig. 2B).

To see whether the expression of MC3r and MC5r mRNA changed in the MC1r siRNA-transfected macrophages, the transfected macrophages were assayed by quantitative real-time PCR to measure MC3r and MC5r mRNA expression after MC1r siRNA transfection. At 24 h post-MC1r siRNA duplex transfection, there was no change in MC3r and MC5r mRNA expression; moreover, their mRNA levels did not change from the levels seen in untransfected macrophages (Fig. 2C). Therefore, transfecting the macrophages with a specific MC1r siRNA duplex is effective in specifically knocking down MC1r expression.

The effects of MC1r siRNA transfection on α-MSH suppression of macrophage proinflammatory activity

It is well known that α-MSH suppresses the expression of NO and TNF-α by LPS-stimulated macrophages [5, 12,13,14,15]. To see if this suppressive activity is dependent on the expression of MC1r, we examined the effects of α-MSH on LPS-stimulated macrophages transfected with our MC1r siRNA duplex. The macrophages were transfected with MC1r siRNA, and after 24 h incubation, the macrophages were stimulated with LPS and treated with 100 nM α-MSH. When we examined the culture supernatant for NO, we found that α-MSH suppression of NO generation was significantly neutralized in the MC1r siRNA-transfected macrophages producing almost the same amount of NO as the macrophages not treated with α-MSH (Fig. 3A). A similar neutralization of α-MSH anti-inflammatory activity was seen when we assayed the culture supernatant by ELISA for TNF-α (Fig. 3B). The anti-inflammatory activity of α-MSH on LPS-stimulated macrophages was not affected by transfecting the macrophages with an irrelevant siRNA duplex (Fig. 3). The results show that knocking down MC1r expression neutralizes the anti-inflammatory activity of α-MSH on macrophages.

Figure 3.

Figure 3

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The effects of MC1r mRNA silencing on α-MSH suppression of LPS-stimulated NO and TNF-α production in macrophages. (A) Cultures of macrophages transfected 24 h before with MC1r siRNA were treated with 100 nM α-MSH and 100 ng/ml LPS for 16 h. The culture media were assayed for NO production by the Griess reagent. (B) To assay for TNF-α, the culture media were assayed 6 h after α-MSH and LPS treatment by ELISA. The results of both assays are presented as the mean ± sem of quadruples from two distinct experiments. Significant differences (P<0.001) from the expected results of treated, untransfected macrophages with the treated MC1r siRNA-transfected macrophages are indicated. No significant difference was seen between untransfected macrophages and macrophages transfected with irrelevant siRNA.

The effects of MC1r siRNA transfection on α-MSH suppression of LPS-stimulated intracellular signaling

The mechanism of α-MSH suppression of LPS-stimulated inflammatory activity in macrophages is through preventing LPS-stimulated activation of NF-κB and p38 phosphorylation [13, 15]. The macrophages were transfected with MC1r siRNA and LPS-stimulated and treated with α-MSH as before. Nuclear extracts were prepared, and an EMSA for NF-κB DNA–protein complexes was done. As expected, the LPS-stimulated, untransfected macrophages and macrophages transfected with irrelevant siRNA were suppressed in NF-κB activation when treated with α-MSH (Fig. 4A, Lanes 4 and 8). Transfecting the macrophages with MC1r siRNA neutralized α-MSH suppression of LPS-induced NF-κB activation (Fig. 4A, Lane 6).

Figure 4.

Figure 4

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The effects of MC1r mRNA silencing on α-MSH suppression of LPS-stimulated NF-κB activation and p38 MAPK phosphorylation in macrophages. (A) Cultures of macrophages transfected 24 h before with MC1r siRNA were treated with 100 nM α-MSH and 100 ng/ml LPS for 1 h. Nuclear extracts were prepared from the cultured macrophages and were analyzed by an EMSA that probed for NF-κB. The LPS-induced NF-κB, DNA-binding complex was efficiently competed off by 100-fold excess of unlabeled NF-κB oligonucleotide (Lane C). The results shown are representative of two independent experiments. (B) To detect the level of p38 MAPK phosphorylation, cytoplasmic lysates of macrophages were extracted 30 min after α-MSH and LPS treatment. Total p38 MAPK (p38) and phosphorylated p38 (pp38) were detected by immunoblot analysis using anti-p38 MAPK or antiphospho-p38 MAPK polyclonal antibody. The results shown are representative of two independent experiments with total p38 MAPK used as the loading control. IR, irrelevant SiRNA transfection.

We examined cytoplasmic lysates for changes in p38 MAPK activation (Fig. 4B). The immunoblot analysis showed that α-MSH suppresses LPS-induced phosphorylation of p38 MAPK in untransfected and irrelevant, siRNA-transfected macrophages (Fig. 4B, Lanes 4 and 8). The suppression of p38 phosphorylation by α-MSH was strongly diminished in the α-MSH-treated, LPS-stimulated, MC1r siRNA-transfected macrophages (Fig. 4B, Lane 6). The analysis of NF-κB activation and p38 phosphorylation demonstrates that the expression of MC1r is required for α-MSH to effectively inhibit LPS-stimulated activity in macrophages.

The effects of MC1r siRNA transfection on MC3r

As there was a significant diminishment in α-MSH suppression of LPS-stimulated intracellular signaling in the MC1r siRNA-transfected macrophages, and there was still expression of MC3r mRNA in the macrophages, we wondered if the MC1r siRNA transfection affected MC3r protein expression and functionality. We lysed the MC1r siRNA-transfected macrophages and immunoblotted for MC3r protein. The transfected cells expressed similar amounts of MC3r protein as the macrophages transfected with an irrelevant siRNA (Fig. 5A). In addition, we treated the MC1r siRNA-transfected macrophages with a MC3r-specific agonist, MTII, and assayed for intracellular accumulation of cAMP. We found that the MC1r siRNA-transfected macrophages were diminished in their accumulation of cAMP in comparison with irrelevant siRNA-transfected macrophages, only when the macrophages were treated with α-MSH and not with the MC3r agonist (Fig. 5B). These results showed that MC3r is present and functional in the MC1r siRNA-transfected macrophages; however, it does not explain why the MC3r agonist is not also suppressing macrophage functionality. We assayed the possibility that MC3r stimulation is not linked to LPS-induced activity in the macrophages. The untransfected macrophages were stimulated with LPS and treated with α-MSH or the MC3r agonists MTII or D-Trp8-γ-MSH and assayed for NO generation and TNF-α production (Fig. , 5Cand 5D). We found that the MC3r agonist could not suppress LPS-induced NO generation and TNF-α production. Therefore, our results show that α-MSH suppression of LPS-stimulated functionality in the macrophages is dependent on the expression of MC1r.

Figure 5.

Figure 5

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The effects of MC1r mRNA silencing and LPS stimulation on MC3r expression and function. (A) Anti-MC3r immunoblotting of immunoprecipitated proteins from lysates of macrophages that were transfected 24 h before with MC1r siRNA or irrelevant (Irr) siRNA. Relative intensities of the bands are presented in the bar graph. (B) Cultures of macrophages transfected 24 h before with MC1r siRNA were treated with 100 nM α-MSH or 10 μM MTII for 30 min, and cAMP (pmoles/ml) accumulation in the cells was assayed. Presented are the mean ± sem of at least three independent cultures. Significant differences (P<0.001) were found between the α-MSH-treated MC1r, siRNA-transfected macrophages and α-MSH-treated, irrelevant, siRNA-transfected macrophage, but no significant difference (NS) was found between the different groups of transfected macrophages treated with the MC3r agonist MTII. (C and D) Cultures of untransfected macrophages were stimulated with 100 ng/ml LPS and treated with 100 nM α-MSH, 10 μM MTII, or 15 μM D-Trp8-γ-MSH and assayed for NO generation and TNF-α production as in Figure 3. Only α-MSH-treated, LPS-stimulated macrophages were significantly suppressed in NO generation and TNF-α production. No significant difference was found between the different groups of LPS-stimulated macrophages treated with the MC3r agonists MTII or D-Trp8-γ-MSH with the LPS-stimulated macrophages.

DISCUSSION

Our results demonstrate a requirement for MC1r expression for α-MSH to inhibit LPS-activated inflammatory activity in macrophages. Without the expression of MC1r, α-MSH could not prevent LPS-stimulated NF-κB translocation to the nucleus and p38 phosphorylation, thus permitting the production of TNF-α and NO generation. Our results correspond to earlier published literature that suggested an important role for MC1r expression by immune cells in α-MSH suppression of endotoxin-induced inflammation [12,13,14,15]; however, our results do not correspond with published work that suggests that it is through MC3r that α-MSH mediates its immunosuppressive activity on macrophages [18, 21, 22].

The importance of MC1r in α-MSH-mediated suppression of endotoxin-stimulated macrophage activity is indicated by the findings that α-MSH-treated macrophages induce their own production of α-MSH and enhance their expression of MC1r [5, 14]. This activity of α-MSH on macrophages has suggested that there is an anti-inflammatory, autoregulatory loop of α-MSH and MC1r [1]. The autoregulatory loop can only be blocked with antibodies against MC1r; moreover, the use of anti-MC1r antibodies inhibits α-MSH-mediated suppression of LPS-stimulated TNF-α production by macrophages [14]. Recent studies of α-MSH suppression of neutrophil migration have shown that anti-MC1r antibodies inhibit α-MSH suppression of neutrophil expression of CXCRs [10]. This same report demonstrated that differentiated and isolated macrophages have enhanced levels of MC1r, although MC3r and MC5r are still detected. The use of a MC1r-specific agonist 154N-5 suppressed endotoxin-induced TNF-α production by macrophages [12]. In addition, an antagonist specific to MC3r did not prevent α-MSH suppression of p38 and NF-κB activation in endotoxin-stimulated macrophages; however, a general MCr antagonist did suppress α-MSH activity in macrophages [15]. Yoon et al. [15] further discovered that α-MSH engagement of MC1r activates a cAMP-dependent PKA, which mediates intracellular signaling leading to the suppression of p38 and NF-κB activation. In our MC1r knockdown macrophages, α-MSH did not inhibit endotoxin-induced p38 and NF-κB activation intracellular signaling; therefore, the macrophages produced NO and TNF-α. Our results are similar and supportive of the literature defining MC1r as the important receptor for α-MSH suppression of endotoxin-stimulated, inflammatory activity in macrophages.

The possibility of α-MSH working through the MC3r is a more recent discovery. Mice with a natural defect that makes MC1r nonfunctional, similar to a human polymorphism linked to red hair, were still responsive to α-MSH suppression of inflammation [21]. MC3r agonists suppressed urate–crystal-stimulated, inflammatory activity in macrophages from MC1r-defective and wild-type mice. In addition, elevated cAMP levels were detected in the MC1r-defective and wild-type resting macrophages treated with a MC3r agonist [22]. Systemic treatment with a MC3r agonist suppresses urate–crystal-induced arthritis, suggesting that in wild-type mice with normal MC1r expression, α-MSH can work through MC3r to suppress inflammation. When the MC3r agonist is used on macrophages, there is an activation of an intercellular pathway, such as MC1r associated with cAMP-dependent PKA [18].

Our results demonstrating a loss of α-MSH anti-inflammatory activity on macrophages with the MC1r knocked down appear not to support a role for MC3r in α-MSH-mediated immunosuppression; however, there may be more to this than a single receptor to single function. The macrophages clearly express MC1r and MC3r, and if macrophages only need MC3r expressed to be affected by α-MSH, then we should not have seen a change in α-MSH suppression of inflammatory activity in the MC1r knocked-down macrophages. It is possible that the effect we observed is caused by how the macrophages were activated. The literature describing α-MSH mediating anti-inflammatory activity through MC1r, such as our experiments, is about endotoxin-, IL-1-, or TNF-α-stimulated macrophages [12,13,14,15], whereas studies regarding the MC3r function are about resting macrophages or macrophages that are activated through non-TLR pathways [18, 21, 22, 24]. The model systems of TLR4-activation pathways in macrophages are different from resting and urate–crystal-activated macrophages. The activation of macrophages by urate–crystals is through an undefined receptor that is linked to the Src family of tyrosine kinases, which like TLR4 intracellular signaling, converges on IκB kinase activation [34]. Therefore, it is possible that there are distinct and different pathways associated with α-MSH binding MC1r and MC3r. Such a simultaneous comparison of the two receptor pathways is yet to be done; however, our results in Figure 5 suggest that this is a possibility.

Another interesting and recent finding is that the MCr may stabilize their signal by forming homodimers and heterodimers when they bind α-MSH [32]. If the MC1r and MC3r dimerize to stabilize and transmit an intracellular signal, then in the mice with nonfunctional MC1r receptors, the MC1r is still present and could dimerize with MC3r. Although MC1r cannot function to create an intercellular signal, it may still contribute by making heterodimers with MC3r to promote MC3r signaling. The reverse may also be true when using a MC1r agonist to promote anti-inflammatory activity in macrophages; however, there is no report of the effects of using a MC1r agonist on a MC3r knockout mouse.

One of the advantages of using MC1r siRNA is that we have significantly depleted the macrophages of the MC1r protein. Such a diminishment of MC1r protein has a profound effect on α-MSH activity on endotoxin-stimulated macrophages. This experimental method allows for examining the effects of eliminating the physical presence of one MCr without the concern of the systemic effects of knocking-out the gene altogether. For studies of the MCr, this is important, as α-MSH has a role in metabolic, pigmentary, and immune homeostasis [26]. There is a potential for many unpredictable implications of systemically knocking-out a MCr. Therefore, we can further use siRNA techniques to target other specific MCr without the possible complications in development and metabolism associated with a cell isolated from a knockout mouse.

We demonstrated that knocking-down the MC1r with sequence-specific siRNA in macrophages significantly diminishes the anti-inflammatory activity of the neuropeptide α-MSH on endotoxin-stimulated macrophages. Our findings suggest that endotoxin (TLR4)-stimulated signals are suppressed by α-MSH mostly through MC1r on macrophages. The results imply that although all the MCr are G-coupled proteins, they may not necessarily function through the same intracellular pathways in macrophages.

Acknowledgments

This work was supported in part by a grant from the National Eye Institute EY010752 and a grant from the Massachusetts Lions Eye Research Foundation. We also thank David Yee for his technical assistance in maintaining the cell line and running some of the assays.

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