Studies in mice indicate that α-melanocyte-stimulating hormone (αMSH) is immunosuppressive, but it is not known whether αMSH suppresses human immune responses to exogenous Ags. Human PBMCs, including monocytes, express the melanocortin 1 receptor (MC1R), and it is thought that the ability of αMSH to alter monocyte-costimulatory molecule expression and IL-10 release is mediated by this receptor. However, the MC1R gene is polymorphic, and certain MC1R variants compromise receptor signaling via cAMP, resulting in red hair and fair skin. Here, we have investigated whether αMSH can suppress Ag-induced lymphocyte proliferation in humans and whether these effects are dependent on MC1R genotype. αMSH suppressed streptokinase-streptodornase-induced lymphocyte proliferation, with maximal inhibition at 10−13–10−11 M αMSH. Anti-IL-10 Abs failed to prevent suppression by αMSH, indicating that it was not due to MC1R-mediated IL-10 release by monocytes. Despite variability in the degree of suppression between subjects, similar degrees of αMSH-induced immunosuppression were seen in individuals with wild-type, heterozygous variant, and homozygous/compound heterozygous variant MC1R alleles. RT-PCR of streptokinase-streptodornase-stimulated PBMCs for all five melanocortin receptors demonstrated MC1R expression by monocytes/macrophages, MC1R and MC3R expression by B lymphocytes, but no melanocortin receptor expression by T lymphocytes. In addition, αMSH did not significantly inhibit anti-CD3 Ab-induced lymphocyte proliferation, whereas αMSH and related analogs (SHU9119 and MTII) inhibited Ag-induced lymphocyte proliferation in monocyte-depleted and B lymphocyte-depleted assays. These findings demonstrate that αMSH, acting probably via MC1R on monocytes and B lymphocytes, and possibly also via MC3R on B lymphocytes, has immunosuppressive effects in humans but that suppression of Ag-induced lymphocyte proliferation by αMSH is independent of MC1R gene status.

The α-melanocyte-stimulating hormone (αMSH)4 is a tridecapeptide that is synthesized, through cleavage of the precursor pro-opiomelanocortin peptide, by several cell types in various organs, including the pituitary and skin (1). Although originally identified for its effects on pigmentation, αMSH has since been recognized as having anti-inflammatory and immunomodulatory actions (2, 3). For example, in animal models, central administration of αMSH can inhibit fever and other effects induced by proinflammatory molecules (IL-1, TNF-α) including increases in circulating blood neutrophils and plasma acute phase proteins, and inflammation (4, 5, 6). In addition, the epicutaneous application of αMSH can suppress the sensitization and elicitation phases of immune responses to contact sensitizers in mice and can induce hapten-specific tolerance in murine skin (7, 8, 9). αMSH exerts its effects by signaling through a family of five separate seven-pass transmembrane G protein-coupled receptors, known as melanocortin receptors (MC1R, MC2R, MC3R, MC4R, and MC5R) (10). In inflammatory/immune responses where the monocyte is of central importance, previous work has suggested that the melanocortin 1 receptor (MC1R) mediates the anti-inflammatory and immunomodulatory effects of αMSH on/via this cell. αMSH, through its binding to MC1R, can down-regulate CD86 expression on monocytes and can induce the release of IL-10 by these cells, with a maximum effect on IL-10 release at 10−13 M αMSH (11, 12). Furthermore, it has been reported that αMSH also reduces CD86 and CD40 expression on monocyte-derived dendritic cells through MC1R (13). However, the consequences of these observations remain unclear, because to date no investigations have documented whether αMSH can suppress Ag presentation/lymphocyte proliferation responses in human subjects, including those where the monocyte/macrophage act as the APC, and therefore whether αMSH is capable of inhibiting human cell-mediated immune responses to exogenous Ags.

In human skin, MC1R is expressed on melanocytes, the function of which is to synthesize melanin pigment to protect the skin from UV radiation-induced damage. However, human skin and hair color varies greatly among subjects as a result of differences in the total amount and ratio of the two types of melanin (brown/black eumelanin and red/yellow phaeomelanin) at these sites (14, 15). The human MC1R gene is highly polymorphic, with >35 genetic variants identified at present, and research into the genetic control of normal human pigmentation during the last several years has comprehensively shown that human MC1R variants are causally associated with red hair, fair skin type, and an increased susceptibility to skin cancer (16). Case control, kindred and population studies have demonstrated that, in general, two variant MC1R alleles result in red hair whereas a single variant allele gives rise to fairer skin type (17, 18, 19, 20, 21, 22). Cell transfection assays and transgenic mouse work have indicated that a number of the variant receptors, in particular the Arg151Cys, Arg160Trp, and Asp294His variants, which are frequently associated with red hair and fair skin, are significantly functionally compromised in their ability to signal intracellularly via cAMP, leading to a lighter/phaeomelanic pigmentation phenotype (23, 24, 25). It is therefore possible that human MC1R variants could have consequences on the anti-inflammatory and/or immunomodulatory actions of αMSH in humans, especially in cases where the monocyte/macrophage plays an important role.

In this study, we have investigated effects of αMSH on human PBMC-mediated Ag presentation/lymphocyte proliferation, and whether this requires wild-type/functional MC1R. The results suggest that αMSH and related analogs have potent immunosuppressive effects in humans and that signaling via MC1R on monocytes/macrophages and B lymphocytes, and possibly also via MC3R on B lymphocytes, mediates these effects.

After approval from the local research ethics committee and written informed consent by the subjects, healthy individuals between 18 and 65 years of age were recruited for the study. Individuals were excluded if they were receiving any medications (with the exception of oral contraceptives) or had significant medical illnesses.

Venous blood from healthy volunteers was collected in Vacutainer tubes containing tripotassium EDTA (BD Biosciences). PBMCs were isolated by gradient centrifugation using Lymphoprep (Axis-Shield). Cells were counted and viability determined by exclusion of 0.04% trypan blue (Sigma-Aldrich), and the cell concentration was adjusted accordingly for subsequent procedures.

PBMCs were resuspended in RPMI 1640 medium (Invitrogen Life Technologies) enriched with l-glutamine and supplemented with 5% heat-inactivated human AB serum (Sigma-Aldrich), 100 U/ml penicillin and 100 μg/ml streptomycin, and 1% sodium pyruvate (Invitrogen Life Technologies). To each well of a 24-well plate (Nunc), 106 cells were added and incubated in the presence or absence of αMSH (10−13 M, 10−11 M, 10−9 M, 10−7 M; Bachem); all cultures were performed in triplicate. Lymphocyte proliferation was stimulated by the addition of the streptococcal Ag mixture streptokinase-streptodornase (SK/SD; 0.5/0.125 U/ml; Varidase (Phoenix Pharmaceuticals). Cultures were incubated at 37°C in 5% CO2 in air, and on day 6 proliferation was assessed in triplicate by [3H]thymidine incorporation. Lymphocyte proliferation was expressed as the stimulation index (SI), calculated as follows: (cpm in presence of Ag − cpm in absence of Ag)/cpm in absence of Ag. SIs >2 were regarded as significant. In proliferation assays investigating the contribution of IL-10 to αMSH-induced suppression, IL-10 was neutralized by the addition of anti-IL-10 Abs (Abcam) at a concentration of 0.2 μg/ml; the effectiveness of the neutralizing Ab at this concentration was tested by control experiments using rIL-10 (Abcam). In cultures investigating the effects of αMSH-related analogs SHU9119 (agonist at MC1R and antagonist at MC3R/MC4R) and MTII (agonist at MC1R as well as at MC3R and MC4R) on SK/SD-induced proliferation, αMSH was used at 10−12 M, SHU9119 at 10−8 M and MTII at 10−8 M (26, 27). In the assays examining the ability of αMSH to inhibit anti-CD3-mediated T cell proliferation, anti-CD3 Abs (Abcam) were added at a final concentration of 1 μg/ml.

DNA was extracted from whole venous blood, and the MC1R gene was amplified from the purified DNA using specific primers MC1R-165 forward (fw) (5′-AGAGGGTGTGAGGGCAGATCTG-3′) and MC1R + 33 reverse (rev) (5′-CACACTTAAAGCGCGTGC-3′). Amplification was performed using BioTaq red (Bioline) 1× Optibuffer (Bioline), 2 mM MgCl2, and 200 mM dNTPs in a PerkinElmer Cetus 9700 thermal cycler and consisted of a single denaturation cycle of 94°C for 5 min followed by 35 cycles of 94°C for 1 min, 62°C for 1 min, and 72°C for 2 min with a final extension of 72°C for 7 min. The 1148-bp amplicons were purified using a Qiagen PCR cleanup column (Qiagen), and the nucleotide sequence was determined with primers MC1R332fw (5′-GCGGTGCTGCAGCAGCTGG-3′), MC1R344rev (5′-TGCTGCAGCACCGCAGCC-3′), MC1R581rev (5′-ACCACGAGGCACAGCAGG-3′), and MC1R715fw (5′-GGCGCTGTCACCCTCACC-3′). Sequencing was performed using a dye terminator cycle sequencing kit (Amersham Pharmacia Biotech) and a model ABI 377 automated DNA sequencer (Applied Biosystems).

PBMCs were purified from venous blood by centrifugation using Lymphoprep as above. To obtain the adherent and nonadherent fractions, whole PBMC (adjusted to106 cells/ml) were cultured as above in the presence of SK/SD. After 48 h of culture, the nonadherent fraction was removed by gentle washing with PBS, and the cells were pelleted at 300 × g; the adherent fraction contained 43.1% monocytes (CD14+), 6.42% B lymphocytes (CD19+), and 30.57% T cells (CD3+); the nonadherent fraction consisted of 6.74% B cells and 63.6% T lymphocytes with 6.3% monocytes. The resulting pellet and the remaining adherent fraction were lysed in lysis buffer (Stratagene), and the lysate was used for the subsequent RNA extraction.

CD14+ monocytes were positively selected from whole PBMCs using magnetic microbeads (MACS magnetic cell separation system; Miltenyi Biotech) with purities ranging from 95 to 98% as determined by flow cytometry. The enriched subset was adjusted to 106 cells/ml and cultured for 48 h in culture medium (as for PBMCs) with SK/SD as above. As part of ongoing research on contact allergic dermatitis in the same department (C. Pickard and P. Friedmann, unpublished observations), B and T cell lines were generated from PBMCs. CD19+ B cell lines (for RT-PCR experiments to ensure 100% purity) were generated from PBMC cultured in the presence of filtered supernatant of the EBV-producing marmoset cell line B95-8 (donated by G. Di Genova, Cancer Sciences) using the same medium as for PBMCs except that the human serum was replaced with 10% FCS. CD4+ and CD8+ T cell lines (for RT-PCR experiments to ensure 100% purity, and for anti-CD3 T cell proliferation) were generated from whole PBMC by limiting dilution of PBMCs challenged with PHA-protein form (2 μg/ml) in the presence of rIL-2 (Sigma-Aldrich).

CD14+ and CD19+ cells were depleted from PBMC using the MACS magnetic cell separation system, according to the manufacturer’s instructions. Briefly, 2 × 107 PBMC were incubated with either anti-CD14- or anti-CD19-conjugated MACS beads, and labeled cells were separated on a LD MACS column. The purities of the depleted fractions was determined by flow cytometry, and in all instances depleted cultures contained <0.5% contaminating CD14+ and CD19+ cells, respectively.

RNA was extracted from ∼1.5–2 × 106 cells using the Absolutely RNA microprep kit (Stratagene). Genomic DNA was removed using 2 U per reaction of RNase-free DNase 1 (Sigma-Aldrich). RNA degradation during subsequent storage at −80°C and during cDNA synthesis was prevented by the presence of a RNase inhibitor (RNAsin; Promega). First-strand cDNA was generated from total RNA using avian myeloblastosis virus reverse transcriptase (Reverse Transcription System; Promega). PCR of cDNA for each of the melanocortin receptors (product sizes: MC1R, 954 bp; MC2R, 304 bp; MC3R, 178 bp; MC4R, 568 bp; MC5R, 460 bp) was conducted by a seminested approach using the following primers with fw and rev primers in the first PCR and inner forward (infw) and rev primers in the subsequent PCR; MC1Rfw (5′-ATGGCTGTGCAGGGATCC-3′), MC1Rrev (5′-TCACCAGGAGCATGTCAGCACC-3′), MC1Rinfw (5′-AGAAGACTTCTGGGCTCC-3′); MC2Rfw (5′-CCACGTGGCAGTTTTGAAACC-3′), MC2Rrev (5′-GGAGATCTTCCTGGTGTGGG-3′), MC2Rinfw (5′-ATGACATCATCGACTCCC-3′); MC3Rfw (5′-GCCCTCACCTTGATCGTGGC-3′), MC3Rrev (5′-CTGTGGGGCCACCCCGTCGGC-3′), MC3Rinfw (5′-CATCTGGGTCTGCTGCGG-3′); MC4Rfw (5′-TACTCTGATGGAGGGTGC-3′), MC4Rrev (5′-TTGGCGGATGGCACCAGTGCC-3′), MC4Rinfw (5′-GGTGTTTGTGACTCTGGG-3′); MC5Rfw (5′-GAGGGCAACCTTTCAGGACCC-3′), MC5Rrev (5′-CCGCAGCCCGTGCAGAAAGCC-3′), and MC5Rinfw (5′-CACCATGTGAAGACATGG-3′). PCR amplification was performed using similar reagents as for PCR of MC1R from DNA, except that annealing temperatures of 60°C were used for MC1R and MC3R and lower annealing temperatures of 55°C for MC2R, MC4R, and MC5R. After agarose gel electrophoresis, correctly sized products were sequenced using the same infw and rev primers with a dye terminator cycle sequencing kit and a model ABI 377 automated DNA sequencer.

Statistical analysis was performed with Stats Direct (version 1.9.14) software for Microsoft Windows. Assessment of suppression of the SI by αMSH, the degree of suppression (expressed as area under the curve) for each MC1R genotype group, and the maximal αMSH-induced suppression for each individual in the three genotype groups were conducted using ANOVA. The effects of anti-IL-10 Ab in the proliferation assay (comparing the areas under the curve in the presence and absence of anti-IL-10 Ab), the maximal suppression of the SI by αMSH for each subject in the individual MC1R genotype groups, and the effects of αMSH on anti-CD3-mediated T lymphocyte proliferation were analyzed by the paired t test.

Previous studies in animal models have demonstrated that αMSH has immunomodulatory activity and can modify responses to exogenous Ags applied to murine skin (7, 8). However, it is not clear whether αMSH can suppress immune responses to exogenous Ags in human subjects. We therefore investigated whether αMSH could alter Ag-induced lymphocyte proliferation in humans in vitro. PBMCs from eight individuals were cultured in the presence of SK/SD (a potent streptococcal Ag mixture against which the majority of individuals manifest a T lymphocyte-mediated response) and the effect of αMSH (10−13 M, 10−11 M, 10−9 M, 10−7 M) on lymphocyte proliferation was assessed. In all cases, the addition of SK/SD resulted in a SI of >2 (range, 6–95). The addition of αMSH at each concentration did not alter the baseline lymphocyte proliferation rate in the absence of Ag. Overall, a significant reduction in the lymphocyte proliferation response to SK/SD was observed after culture with αMSH (p = 0.0074; Fig. 1), but there was considerable variability between individuals in the ability of αMSH to suppress Ag-induced lymphocyte proliferation.

FIGURE 1.

Effect of αMSH on SK/SD-induced lymphocyte proliferation in eight subjects. Overall, αMSH significantly suppressed SI (p = 0.0074), but there was variation between subjects.

FIGURE 1.

Effect of αMSH on SK/SD-induced lymphocyte proliferation in eight subjects. Overall, αMSH significantly suppressed SI (p = 0.0074), but there was variation between subjects.

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Because αMSH can cause release of IL-10 from human monocytes and IL-10 can inhibit Ag-specific T cell proliferation, we examined whether the suppression of SK/SD-induced lymphocyte proliferation by αMSH occurred through this mechanism (11, 28). PBMCs were cultured with SK/SD with and without αMSH (10−13 M, 10−11 M, 10−9 M, 10−7 M), and 0.2 μg/ml IL-10-blocking Ab. Control Ag-stimulated cultures with added 0.1 ng/ml rIL-10 in the presence and absence of anti-IL-10 Ab confirmed the ability of the Ab to inhibit completely IL-10-induced lymphocyte proliferation (data not shown). No significant abrogation of αMSH-induced suppression of SK/SD-induced lymphocyte proliferation was detected upon addition of IL-10 blocking Ab (n = 7; p = 0.3757; Fig. 2).

FIGURE 2.

αMSH-induced suppression of SK/SD-mediated lymphocyte proliferation is not inhibited by anti-IL-10 blocking Ab. Values are mean (and SEM) of suppression by αMSH at each concentration; n = 7. The effects of anti-IL-10 Ab were not significant (p = 0.3757).

FIGURE 2.

αMSH-induced suppression of SK/SD-mediated lymphocyte proliferation is not inhibited by anti-IL-10 blocking Ab. Values are mean (and SEM) of suppression by αMSH at each concentration; n = 7. The effects of anti-IL-10 Ab were not significant (p = 0.3757).

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Previous studies have indicated that MC1R is expressed by PBMCs, including human monocytes/macrophages, B lymphocytes, and a subset of CTLs (12, 29, 30). MC1R is also expressed by melanocytes in the skin, where it plays a central role in human pigmentation. MC1R variants have profound effects on human pigmentation, causing red hair and fair skin phenotypes as a result of the significantly reduced ability of variant receptors to signal via cAMP (18, 20, 22, 23, 24, 25). To investigate whether variability in αMSH-induced suppression of SK/SD-induced lymphocyte proliferation between subjects was due to genetic variability at the MC1R locus, we extended the study population to include a total of 26 subjects. Initially, pigmentation phenotype (red hair and fair skin or dark hair and tanned skin) was used to preferentially identify subjects with different MC1R genotypes to include similar numbers of individuals with wild-type MC1R, a single variant MC1R allele, and two variant MC1R alleles. Subsequently, sequencing of the MC1R coding region was performed in all 26 subjects in whom the αMSH/lymphocyte proliferation assay had been undertaken. Nine subjects were wild type at MC1R, nine had a single variant MC1R allele, and eight had two variant MC1R alleles. αMSH-induced suppression of the proliferation assay was observed in all three MC1R genotype groups, with little difference between the three groups in the mean suppression of SK/SD-induced lymphocyte proliferation at each concentration of αMSH (comparison of three groups, p = 0.7109; Fig. 3). Although the maximal αMSH-induced suppression of the SK/SD-induced lymphocyte proliferation assay ranged from 0 to 80% in the 26 subjects overall and was highly significant within each genotype group (p < 0.0001 for wild-type, single-variant, and two-variant alleles) the degree of variation in suppression was similar within each group (p = 0.4080; Table I). Maximal suppression of SK/SD-induced lymphocyte proliferation by αMSH varied from 21 to 77% in individuals who were homozygous or compound heterozygous for the Arg151Cys, Arg160Trp, and Asp294His variant alleles, which significantly impair MC1R function (23, 24, 25). Indeed, two subjects with two variant alleles and three individuals with a single MC1R variant with SIs >20 exhibited maximal αMSH-induced suppression of 46–80%, consistent with αMSH having potent immunosuppressive effects in subjects with MC1R variants in this experimental system (Table I).

FIGURE 3.

Suppression of lymphocyte proliferation assay in response to SK/SD by αMSH in different MC1R genotype groups: A, wild-type (WT/WT), n = 9; B, single-variant allele (VAR/WT), n = 9; C, two-variant alleles (VAR/VAR), n = 8. Values are mean (and SEM) suppression at each concentration of αMSH. There was no significant difference in suppression between each genotype group (p = 0.7109).

FIGURE 3.

Suppression of lymphocyte proliferation assay in response to SK/SD by αMSH in different MC1R genotype groups: A, wild-type (WT/WT), n = 9; B, single-variant allele (VAR/WT), n = 9; C, two-variant alleles (VAR/VAR), n = 8. Values are mean (and SEM) suppression at each concentration of αMSH. There was no significant difference in suppression between each genotype group (p = 0.7109).

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Table I.

Maximum percentage inhibition of SK/SD-induced lymphocyte proliferation by αMSH for each subjecta

Subject ID Number and MC1R GenotypeSI to SK/SDbSI to SK/SD at Concentration of αMSH Exhibiting Maximal InhibitionbMaximum % Inhibition of SI by αMSHbConcentration of αMSH Causing Maximal Inhibition of SI (log10M)
(17)cWT/WT 18 80 −9 
(21) WT/WT 18 53 −13 
(15) WT/WT 20 12 42 −11 
(2) WT/WT 21 14 30 −11 
(5) WT/WT 34 25 25 −9 
(20) WT/WT 15 11 22 −9 
(3) WT/WT 95 81 15 −11 
(1) WT/WT 22 20 11 −13 
(11) WT/WT N/Ad 
(24) Arg163Gln/WT 30 80 −11 
(14) Arg160Trp/WT 52 −13 
(6) Arg160Trp/WT 49 24 50 −11 
(12) Arg160Trp/WT 11 50 −11 
(16) Arg160Trp/WT 92 49 46 −13 
(4) Arg163Gln/WT 44 −7 
(13) Va192Met/WT 56 36 37 −13 
(18) Va192Met/WT 106 79 26 −13 
(10) Arg160Trp/WT 52 46 12 −11 
(26) Arg151Cys/Arg151Cys 33 77 −13 
(19) Arg151Cys/Arg160Trp 21 57 −7 
(22) Arg151Cys/Asp294His 11 46 −13 
(7) Arg160Trp/Arg160Trp 17 10 38 −11 
(23) Arg151Cys/Asp294His 13 31 −13 
(9) Arg151Cys/Asp294His 17 12 30 −13 
(25) Asp294His/Asp294His 62 49 21 −13 
(8) Pro256Ser/Asp294His 21 −11 
Subject ID Number and MC1R GenotypeSI to SK/SDbSI to SK/SD at Concentration of αMSH Exhibiting Maximal InhibitionbMaximum % Inhibition of SI by αMSHbConcentration of αMSH Causing Maximal Inhibition of SI (log10M)
(17)cWT/WT 18 80 −9 
(21) WT/WT 18 53 −13 
(15) WT/WT 20 12 42 −11 
(2) WT/WT 21 14 30 −11 
(5) WT/WT 34 25 25 −9 
(20) WT/WT 15 11 22 −9 
(3) WT/WT 95 81 15 −11 
(1) WT/WT 22 20 11 −13 
(11) WT/WT N/Ad 
(24) Arg163Gln/WT 30 80 −11 
(14) Arg160Trp/WT 52 −13 
(6) Arg160Trp/WT 49 24 50 −11 
(12) Arg160Trp/WT 11 50 −11 
(16) Arg160Trp/WT 92 49 46 −13 
(4) Arg163Gln/WT 44 −7 
(13) Va192Met/WT 56 36 37 −13 
(18) Va192Met/WT 106 79 26 −13 
(10) Arg160Trp/WT 52 46 12 −11 
(26) Arg151Cys/Arg151Cys 33 77 −13 
(19) Arg151Cys/Arg160Trp 21 57 −7 
(22) Arg151Cys/Asp294His 11 46 −13 
(7) Arg160Trp/Arg160Trp 17 10 38 −11 
(23) Arg151Cys/Asp294His 13 31 −13 
(9) Arg151Cys/Asp294His 17 12 30 −13 
(25) Asp294His/Asp294His 62 49 21 −13 
(8) Pro256Ser/Asp294His 21 −11 
a

Results for each individual, including MC1R genotype, stimulation index to SK/SD, and concentration of αMSH causing maximal inhibition, are detailed on separate lines.

b

To the nearest whole unit.

c

Numbers in parentheses, subject identification (ID) number.

d

WT, Wild type; N/A, not available.

Within the lymphocyte proliferation cultures, monocytes/macrophages and B lymphocytes present the SK/SD Ag to the Th lymphocytes, which results in clonal proliferation of the T cells. To determine which cell population is predominantly suppressed by αMSH, we first attempted to conduct the proliferation assay after initial separation of monocytes and T cells to allow them to be exposed separately to αMSH before being added back together. However, this approach was unsuccessful because the modified proliferation assay failed to achieve sufficiently potent inhibition without the continued presence of αMSH. The next approach was to see whether the different cell populations differed in their expression of melanocortin receptors. Therefore, we investigated for expression of each of the five melanocortin receptors on adherent and nonadherent PBMCs, and subsequently on purified monocytes/macrophages, B cells and T cells. After culture of PBMCs with SK/SD for 48 h, RT-PCR and subsequent sequencing of positive bands showed evidence of MC1R and MC3R expression in adherent and nonadherent fractions (Fig. 4), but no expression of MC2R, MC4R, and MC5R was detected. RT-PCR (with sequencing) demonstrated MC1R expression alone by purified monocytes/macrophages after culture with SK/SD and showed that MC1R and MC3R (but not MC2R, MC4R, and MC5R) was expressed by the B lymphocytes. There was no evidence of any melanocortin receptor expression by T cells under these culture conditions.

FIGURE 4.

RT-PCR for MC1R (954 bp) and MC3R (178 bp) expression by adherent and nonadherent PBMCs, monocytes/macrophages, B cells and T cells. AC, Adherent PBMCs; NAC, nonadherent PBMCs; Mo/Mφ, monocytes/macrophages; B, B cells; T, T cells. Lanes 1 and 12, DNA ladder (1KB Plus DNA ladder; Invitrogen Life Technologies); lanes 2, 4, 6, 8, and 10, MC1R; lanes 3, 5, 7, 9, and 11, MC3R.

FIGURE 4.

RT-PCR for MC1R (954 bp) and MC3R (178 bp) expression by adherent and nonadherent PBMCs, monocytes/macrophages, B cells and T cells. AC, Adherent PBMCs; NAC, nonadherent PBMCs; Mo/Mφ, monocytes/macrophages; B, B cells; T, T cells. Lanes 1 and 12, DNA ladder (1KB Plus DNA ladder; Invitrogen Life Technologies); lanes 2, 4, 6, 8, and 10, MC1R; lanes 3, 5, 7, 9, and 11, MC3R.

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To examine in more detail whether the effect of αMSH on SK/SD-induced lymphocyte proliferation could be due to a direct effect on T lymphocyte proliferation, the ability of αMSH to suppress anti-CD3-mediated T cell proliferation was examined. Fourteen separate T cell clones, generated from an individual who showed evidence of suppression of SK/SD-induced lymphocyte proliferation by αMSH, were stimulated with anti-CD3 Ab. Whereas αMSH consistently inhibited SK/SD-induced lymphocyte proliferation (n = 4 experiments), no suppression of anti-CD3-mediated T cell proliferation was observed (p = 0.5099; Fig. 5).

FIGURE 5.

αMSH does not suppress anti-CD3-mediated T lymphocyte proliferation. Anti-CD3 stimulated TCC, T cell clones (n = 14) with anti-CD3 Ab. SK/SD-stimulated PBMCs (n = 4) were from same individual from whom the T cell clones were generated. Percent on the y-axis indicates the responses expressed as percentage of the maximal response in the absence of αMSH.

FIGURE 5.

αMSH does not suppress anti-CD3-mediated T lymphocyte proliferation. Anti-CD3 stimulated TCC, T cell clones (n = 14) with anti-CD3 Ab. SK/SD-stimulated PBMCs (n = 4) were from same individual from whom the T cell clones were generated. Percent on the y-axis indicates the responses expressed as percentage of the maximal response in the absence of αMSH.

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The observation that MC1R is expressed by purified monocytes/macrophages after culture with SK/SD and that MC1R and MC3R are expressed by B lymphocytes under these conditions indicated that the suppressive effects of αMSH might occur via MC1R on monocytes/macrophages and/or B lymphocytes or via MC3R on B cells. To investigate this further, PBMCs from subjects in whom αMSH suppressed SK/SD-induced lymphocyte proliferation were separately depleted of monocytes (CD14+) and B lymphocytes (CD19+) before stimulation with SK/SD and the addition of αMSH or related analogs SHU9119 and MTII. SK/SD caused proliferation of monocyte-depleted and B lymphocyte-depleted cultures, and αMSH inhibited this proliferation albeit to variable degrees (Fig. 6). SHU9119 and MTII also suppressed SK/SD-induced proliferation in these depleted cultures, and in all cases to a greater extent than the suppression observed with αMSH (Fig. 6).

FIGURE 6.

αMSH and related compounds (SHU9119 and MTII) suppress SK/SD-induced lymphocyte proliferation in monocyte-depleted PBMC cultures from two subjects (a and b) and in B lymphocyte-depleted PBMC cultures from two subjects (c and d). Control, no SK/SD; SK/SD, SK/SD alone; αMSH, SK/SD plus αMSH (10−12 M); SHU, SK/SD plus SHU9119 (10−8 M); MTII, SK/SD plus MTII (10−8 M).

FIGURE 6.

αMSH and related compounds (SHU9119 and MTII) suppress SK/SD-induced lymphocyte proliferation in monocyte-depleted PBMC cultures from two subjects (a and b) and in B lymphocyte-depleted PBMC cultures from two subjects (c and d). Control, no SK/SD; SK/SD, SK/SD alone; αMSH, SK/SD plus αMSH (10−12 M); SHU, SK/SD plus SHU9119 (10−8 M); MTII, SK/SD plus MTII (10−8 M).

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It is widely accepted that αMSH has potent anti-inflammatory and immunomodulatory activity in animal models; for example, αMSH can suppress contact hypersensitivity in murine skin (7, 8). However, limited research has been conducted on the ability of αMSH to suppress immune responses in humans, and in particular responses to exogenous Ags. There is evidence for effects by αMSH on cytokine release by and costimulatory molecule expression on certain human cell types, but the effect of these alterations on responses to Ag has not been fully elucidated (11, 12). In this study, we have shown that αMSH can suppress Ag-induced proliferation of memory T lymphocytes in humans in vitro. These effects on lymphocyte proliferation are unlikely to be due to cell death secondary to αMSH toxicity because the suppressive effects were greatest at the lower concentrations of αMSH that were used in this study. This is similar to the dose-response effects by αMSH on IL-10 production from monocytes where 10−13 M αMSH is most potent, and it contrasts with the higher doses of αMSH required for maximal stimulation of cAMP/pigmentation by melanocytes/melanoma cells (11, 24, 31). The degree of suppression by αMSH varied considerably between individuals, but the results showing up to 80% suppression in several individuals with high stimulation indices to SK/SD demonstrate that αMSH is a potent modulator of human immune responses in some subjects and suggest that αMSH or related compounds may have therapeutic potential for human disease. Indeed, the results of this study in combination with previous research showing that αMSH can suppress contact hypersensitivity in mice provide support for future investigations on the ability of αMSH to treat allergic contact dermatitis in humans (7, 8, 32).

Although the exact mechanism through which αMSH can suppress SK/SD-induced lymphocyte proliferation in humans is not clear, the melanocortin receptor expression data would indicate that this could occur via MC1R on monocytes/macrophages and/or B lymphocytes or via MC3R on B lymphocytes, respectively. Although Neumann-Andersen et al. (30) have reported MC1R expression by a subset of CTLs, we did not find evidence for expression of any of the five melanocortin receptors by T lymphocytes. This difference may have been due to the presence of SK/SD in our culture system for the RT-PCR experiments, but we considered it important to examine for melanocortin receptor expression in the presence of this Ag given that environmental factors can affect expression of MC1R (29, 33). In addition to the lack of melanocortin receptor expression by T cells in this study, the fact that αMSH failed to suppress anti-CD3-mediated T lymphocyte proliferation suggests that αMSH-induced suppression of lymphocyte proliferation does not occur via a direct effect on T cells. Our attempts to culture purified monocytes in the presence of SK/SD with and without αMSH for 48 h before the addition of isolated T cells were unsuccessful because these preseparated and then reconstituted cultures gave high SIs in response to SK/SD that failed to show suppression with αMSH for this shorter period (data not shown). However, the observation that αMSH suppressed SK/SD-induced lymphocyte proliferation in separate B lymphocyte-depleted and monocyte-depleted cultures suggests that αMSH can have independent immunosuppressive effects via monocytes and B cells on Ag-induced lymphocyte proliferation. Taken together with the melanocortin receptor expression data, this indicates that some of the suppressive actions of αMSH on responses to exogenous Ag occurs via MC1R on monocytes. In addition, the use of the related compounds SHU9119 (agonist at MC1R and antagonist at MC3R) and MTII (agonist at MC1R and MC3R) in the depleted cultures indicates that MC1R on B lymphocytes as well as on monocytes is involved in this suppression, but this does not rule out a possible additional effect by αMSH and MTII through MC3R on B cells.

Although αMSH can stimulate IL-10 release by monocytes (11), our results with anti-IL-10 blocking Abs indicate that the suppressive effects of αMSH on Ag-induced lymphocyte proliferation are not via an IL-10 mechanism. In contrast to the reported Mc3r-mediated anti-inflammatory effects by αMSH in mouse peritoneal macrophages (26), we did not observe MC3R expression on monocytes/macrophages in our culture system, but our observations are similar to the findings by Bhardwaj et al. (12), who detected MC1R expression alone on human monocytes. Previous research has shown that MC1R is also expressed on mast cells and may mediate the inhibitory effects of αMSH on these cells (34). Thus, it seems likely that the wide range of anti-inflammatory and immunomodulatory actions of αMSH in mammals do not all occur via a single melanocortin receptor but that MC1R and/or MC3R may be involved to a greater or lesser extent in different types of inflammation. In addition, it is possible that a hitherto unknown melanocortin receptor or a non-melanocortin receptor mechanism (the C-terminal αMSH-related tripeptide may interact with the IL-1β receptor; Ref.35) may also mediate some of the anti-inflammatory effects of αMSH in certain situations.

The variability in the degree of immune suppression by αMSH in different subjects in this study would be consistent with an underlying genetic variability determining the effects of αMSH in this system. Logically, it would be expected that this genetic variability would be at the MC1R gene because there is much evidence that MC1R gene variants profoundly affect human pigmentation giving rise to red hair and fair skin through reduced receptor function and consequent lower cAMP signaling in pigment cells (18, 20, 22, 23, 24). This would have been seen as a lack of suppressive effect by αMSH in red haired, fair skinned individuals, whereas greatest suppression would have been expected in dark haired and tan skinned subjects. However, there was no correlation between the degree of suppression and the pigmentary phenotype. Moreover, the MC1R sequencing results demonstrate that MC1R variants (heterozygous, homozygous, and compound heterozygous) do not inhibit the ability of αMSH to suppress SK/SD-induced lymphocyte proliferation; importantly, seven of the eight subjects with two MC1R variant alleles in this study were homozygous or compound heterozygous for the Arg151Cys, Arg160Trp, and Asp294His variants, which are known to compromise receptor function and signaling through cAMP (23, 24, 25). Recent research suggests that αMSH can signal through MC1R via an alternative intracellular signaling pathway involving calcium flux (36). It is possible that MC1R variant receptors may retain this function and that the immunosuppressive effects of αMSH that we observed in this study occur through this alternative MC1R signaling pathway, but future research will be necessary to investigate this further.

In conclusion, we have demonstrated in this study that αMSH has potent suppressive effects on Ag-mediated lymphocyte proliferation in humans in vitro. These effects are likely to be mediated via MC1R on monocytes/macrophages and B lymphocytes (and possibly also via MC3R on B lymphocytes) but are independent of MC1R gene status.

The authors have no financial conflict of interest.

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

E.H. is a Medical Research Council Senior Clinical Fellow. A.C. was supported by a Wellcome Trust Entry Level Fellowship. C.L.J. is a British Skin Foundation Post-Graduate Student.

4

Abbreviations used in this paper: αMSH, α-melanocyte-stimulating hormone; MC[1–5]R, melanocortin [1–5] receptor (human, MC[1–5]R; murine, Mc[1–5]r); SK/SD, streptokinase-streptodornase; SI, stimulation index; fw, forward; rev, reverse; infw, inner forward.

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