α--Stimulating Hormone Suppresses Antigen-Induced Lymphocyte Proliferation in Humans Independently of 1 Status This information is current as of September 24, 2021. Ashley Cooper, Samantha J. Robinson, Chris Pickard, Claire L. Jackson, Peter S. Friedmann and Eugene Healy J Immunol 2005; 175:4806-4813; ; doi: 10.4049/jimmunol.175.7.4806 http://www.jimmunol.org/content/175/7/4806 Downloaded from

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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 © 2005 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

␣-Melanocyte-Stimulating Hormone Suppresses Antigen-Induced Lymphocyte Proliferation in Humans Independently of Gene Status1

Ashley Cooper,2 Samantha J. Robinson,2 Chris Pickard,2 Claire L. Jackson, Peter S. Friedmann, and Eugene Healy3

Studies in mice indicate that ␣-melanocyte-stimulating hormone (␣MSH) is immunosuppressive, but it is not known whether ␣MSH 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 ␣MSH 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 hair and fair skin. Here, we have investigated whether ␣MSH can suppress Ag-induced Downloaded from lymphocyte proliferation in humans and whether these effects are dependent on MC1R genotype. ␣MSH suppressed streptokinase- streptodornase-induced lymphocyte proliferation, with maximal inhibition at 10؊13–10؊11 M ␣MSH. Anti-IL-10 Abs failed to prevent suppression by ␣MSH, 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 ␣MSH-induced immunosuppression were seen in individuals with wild-type, heterozygous variant, and homozygous/compound heterozygous variant MC1R . RT-PCR of streptokinase- streptodornase-stimulated PBMCs for all five melanocortin receptors demonstrated MC1R expression by monocytes/macro- http://www.jimmunol.org/ phages, MC1R and MC3R expression by B lymphocytes, but no expression by T lymphocytes. In addition, ␣MSH did not significantly inhibit anti-CD3 Ab-induced lymphocyte proliferation, whereas ␣MSH and related analogs (SHU9119 and MTII) inhibited Ag-induced lymphocyte proliferation in monocyte-depleted and B lymphocyte-depleted assays. These findings demonstrate that ␣MSH, acting probably via MC1R on monocytes and B lymphocytes, and possibly also via MC3R on B lym- phocytes, has immunosuppressive effects in humans but that suppression of Ag-induced lymphocyte proliferation by ␣MSH is independent of MC1R gene status. The Journal of Immunology, 2005, 175: 4806–4813.

he ␣-melanocyte-stimulating hormone (␣MSH)4 is a induce hapten-specific tolerance in murine skin (7–9). ␣MSH ex- by guest on September 24, 2021 tridecapeptide that is synthesized, through cleavage of the erts its effects by signaling through a family of five separate seven- T precursor pro-opiomelanocortin , by several pass transmembrane G -coupled receptors, known as mela- types in various organs, including the pituitary and skin (1). Al- nocortin receptors (MC1R, MC2R, MC3R, MC4R, and MC5R) though originally identified for its effects on pigmentation, ␣MSH (10). In inflammatory/immune responses where the monocyte is of has since been recognized as having anti-inflammatory and immu- central importance, previous work has suggested that the melano- nomodulatory actions (2, 3). For example, in animal models, cen- cortin 1 receptor (MC1R) mediates the anti-inflammatory and im- tral administration of ␣MSH can inhibit fever and other effects munomodulatory effects of ␣MSH on/via this cell. ␣MSH, through induced by proinflammatory molecules (IL-1, TNF-␣) including its binding to MC1R, can down-regulate CD86 expression on increases in circulating blood neutrophils and plasma acute phase monocytes and can induce the release of IL-10 by these cells, with , and inflammation (4–6). In addition, the epicutaneous a maximum effect on IL-10 release at 10Ϫ13 M ␣MSH (11, 12). ␣ application of MSH can suppress the sensitization and elicitation Furthermore, it has been reported that ␣MSH also reduces CD86 phases of immune responses to contact sensitizers in mice and can and CD40 expression on monocyte-derived dendritic cells through MC1R (13). However, the consequences of these observations re- main unclear, because to date no investigations have documented Dermatopharmacology Unit, University of Southampton, Southampton, United ␣ Kingdom whether MSH can suppress Ag presentation/lymphocyte prolif- Received for publication October 8, 2004. Accepted for publication July 18, 2005. eration responses in human subjects, including those where the The costs of publication of this article were defrayed in part by the payment of page monocyte/macrophage act as the APC, and therefore whether charges. This article must therefore be hereby marked advertisement in accordance ␣MSH is capable of inhibiting human cell-mediated immune re- with 18 U.S.C. Section 1734 solely to indicate this fact. sponses to exogenous Ags. 1 E.H. is a Medical Research Council Senior Clinical Fellow. A.C. was supported by In human skin, MC1R is expressed on , the function a Wellcome Trust Entry Level Fellowship. C.L.J. is a British Skin Foundation Post- Graduate Student. of which is to synthesize pigment to protect the skin from 2 A.C., S.J.R., and C.P. contributed equally to the project. UV radiation-induced damage. However, human skin and hair color varies greatly among subjects as a result of differences in the 3 Address correspondence and reprint requests to Dr. Eugene Healy, Mailpoint 825, Level F, South Block, Southampton General Hospital, Southampton, SO16 6YD, total amount and ratio of the two types of melanin (brown/black United Kingdom. E-mail address: [email protected] eumelanin and red/yellow phaeomelanin) at these sites (14, 15). 4 Abbreviations used in this paper: ␣MSH, ␣-melanocyte-stimulating hormone; The human MC1R gene is highly polymorphic, with Ͼ35 genetic 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; variants identified at present, and research into the genetic control infw, inner forward. of normal human pigmentation during the last several years has

Copyright © 2005 by The American Association of Immunologists, Inc. 0022-1767/05/$02.00 The Journal of Immunology 4807

comprehensively shown that human MC1R variants are causally MgCl2, and 200 mM dNTPs in a PerkinElmer Cetus 9700 thermal cycler associated with , fair skin type, and an increased suscep- and consisted of a single denaturation cycle of 94°C for 5 min followed by tibility to (16). Case control, kindred and population 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 studies have demonstrated that, in general, two variant MC1R al- using a Qiagen PCR cleanup column (Qiagen), and the nucleotide sequence leles result in red hair whereas a single variant gives rise to was determined with primers MC1R332fw (5Ј-GCGGTGCTGCAG fairer skin type (17–22). Cell transfection assays and transgenic CAGCTGG-3Ј), MC1R344rev (5Ј-TGCTGCAGCACCGCAGCC-3Ј), mouse work have indicated that a number of the variant receptors, MC1R581rev (5Ј-ACCACGAGGCACAGCAGG-3Ј), and MC1R715fw (5Ј-GGCGCTGTCACCCTCACC-3Ј). Sequencing was performed using a in particular the Arg151Cys, Arg160Trp, and Asp294His variants, dye terminator cycle sequencing kit (Amersham Pharmacia Biotech) and a which are frequently associated with red hair and fair skin, are model ABI 377 automated DNA sequencer (Applied Biosystems). significantly functionally compromised in their ability to signal intracellularly via cAMP, leading to a lighter/phaeomelanic pig- Generation of adherent and nonadherent PBMC fractions, mentation phenotype (23–25). It is therefore possible that human monocytes/macrophages, B and T cell lines, and monocyte- MC1R variants could have consequences on the anti-inflammatory depleted and B lymphocyte-depleted cultures ␣ and/or immunomodulatory actions of MSH in humans, especially PBMCs were purified from venous blood by centrifugation using Lym- in cases where the monocyte/macrophage plays an important role. phoprep as above. To obtain the adherent and nonadherent fractions, whole In this study, we have investigated effects of ␣MSH on human PBMC (adjusted to106 cells/ml) were cultured as above in the presence of PBMC-mediated Ag presentation/lymphocyte proliferation, and SK/SD. After 48 h of culture, the nonadherent fraction was removed by gentle washing with PBS, and the cells were pelleted at 300 ϫ g; the whether this requires wild-type/functional MC1R. The results sug- adherent fraction contained 43.1% monocytes (CD14ϩ), 6.42% B lympho- gest that ␣MSH and related analogs have potent immunosuppres- cytes (CD19ϩ), and 30.57% T cells (CD3ϩ); the nonadherent fraction con- Downloaded from sive effects in humans and that signaling via MC1R on monocytes/ sisted of 6.74% B cells and 63.6% T lymphocytes with 6.3% monocytes. macrophages and B lymphocytes, and possibly also via MC3R on The resulting pellet and the remaining adherent fraction were lysed in lysis B lymphocytes, mediates these effects. buffer (Stratagene), and the lysate was used for the subsequent RNA extraction. CD14ϩ monocytes were positively selected from whole PBMCs using Materials and Methods magnetic microbeads (MACS magnetic cell separation system; Miltenyi

After approval from the local research ethics committee and written in- Biotech) with purities ranging from 95 to 98% as determined by flow http://www.jimmunol.org/ 6 formed consent by the subjects, healthy individuals between 18 and 65 cytometry. The enriched subset was adjusted to 10 cells/ml and cultured years of age were recruited for the study. Individuals were excluded if they for 48 h in culture medium (as for PBMCs) with SK/SD as above. As part were receiving any medications (with the exception of oral contraceptives) of ongoing research on contact allergic dermatitis in the same department or had significant medical illnesses. (C. Pickard and P. Friedmann, unpublished observations), B and T cell lines were generated from PBMCs. CD19ϩ B cell lines (for RT-PCR ex- Isolation of PBMCs periments to ensure 100% purity) were generated from PBMC cultured in the presence of filtered supernatant of the EBV-producing marmoset cell Venous blood from healthy volunteers was collected in Vacutainer tubes line B95-8 (donated by G. Di Genova, Cancer Sciences) using the same containing tripotassium EDTA (BD Biosciences). PBMCs were isolated by medium as for PBMCs except that the human serum was replaced with gradient centrifugation using Lymphoprep (Axis-Shield). Cells were 10% FCS. CD4ϩ and CD8ϩ T cell lines (for RT-PCR experiments to counted and viability determined by exclusion of 0.04% trypan blue ensure 100% purity, and for anti-CD3 T cell proliferation) were generated by guest on September 24, 2021 (Sigma-Aldrich), and the cell concentration was adjusted accordingly for from whole PBMC by limiting dilution of PBMCs challenged with PHA- subsequent procedures. protein form (2 ␮g/ml) in the presence of rIL-2 (Sigma-Aldrich). ϩ ϩ ␣ CD14 and CD19 cells were depleted from PBMC using the MACS Lymphocyte proliferation assay/effects of MSH magnetic cell separation system, according to the manufacturer’s instruc- 7 PBMCs were resuspended in RPMI 1640 medium (Invitrogen Life Tech- tions. Briefly, 2 ϫ 10 PBMC were incubated with either anti-CD14- or nologies) enriched with L-glutamine and supplemented with 5% heat-in- anti-CD19-conjugated MACS beads, and labeled cells were separated on a activated human AB serum (Sigma-Aldrich), 100 U/ml penicillin and 100 LD MACS column. The purities of the depleted fractions was determined ␮ by flow cytometry, and in all instances depleted cultures contained Ͻ0.5% g/ml streptomycin, and 1% sodium pyruvate (Invitrogen Life Technolo- ϩ ϩ gies). To each well of a 24-well plate (Nunc), 106 cells were added and contaminating CD14 and CD19 cells, respectively. incubated in the presence or absence of ␣MSH (10Ϫ13 M, 10Ϫ11 M, 10Ϫ9 M, 10Ϫ7 M; Bachem); all cultures were performed in triplicate. Lympho- RNA extraction, RT-PCR, and sequencing of products cyte proliferation was stimulated by the addition of the streptococcal Ag RNA was extracted from ϳ1.5–2 ϫ 106 cells using the Absolutely RNA mixture streptokinase-streptodornase (SK/SD; 0.5/0.125 U/ml; Varidase microprep kit (Stratagene). Genomic DNA was removed using2Uper (Phoenix Pharmaceuticals). Cultures were incubated at 37°C in 5% CO in 2 reaction of RNase-free DNase 1 (Sigma-Aldrich). RNA degradation during air, and on day 6 proliferation was assessed in triplicate by [3H]thymidine subsequent storage at Ϫ80°C and during cDNA synthesis was prevented by incorporation. Lymphocyte proliferation was expressed as the stimulation the presence of a RNase inhibitor (RNAsin; Promega). First-strand cDNA index (SI), calculated as follows: (cpm in presence of Ag Ϫ cpm in absence was generated from total RNA using avian myeloblastosis virus reverse of Ag)/cpm in absence of Ag. SIs Ͼ2 were regarded as significant. In transcriptase (Reverse Transcription System; Promega). PCR of cDNA for proliferation assays investigating the contribution of IL-10 to ␣MSH-in- each of the melanocortin receptors (product sizes: MC1R, 954 bp; MC2R, duced suppression, IL-10 was neutralized by the addition of anti-IL-10 Abs 304 bp; MC3R, 178 bp; MC4R, 568 bp; MC5R, 460 bp) was conducted by (Abcam) at a concentration of 0.2 ␮g/ml; the effectiveness of the neutral- a seminested approach using the following primers with fw and rev primers izing Ab at this concentration was tested by control experiments using in the first PCR and inner forward (infw) and rev primers in the subsequent rIL-10 (Abcam). In cultures investigating the effects of ␣MSH-related an- PCR; MC1Rfw (5Ј-ATGGCTGTGCAGGGATCC-3Ј), MC1Rrev (5Ј-TCAC alogs SHU9119 ( at MC1R and antagonist at MC3R/MC4R) and CAGGAGCATGTCAGCACC-3Ј), MC1Rinfw (5Ј-AGAAGACTTCTGG MTII (agonist at MC1R as well as at MC3R and MC4R) on SK/SD-in- Ϫ Ϫ GCTCC-3Ј); MC2Rfw (5Ј-CCACGTGGCAGTTTTGAAACC-3Ј), MC2Rrev duced proliferation, ␣MSH was used at 10 12 M, SHU9119 at 10 8 M and Ϫ (5Ј-GGAGATCTTCCTGGTGTGGG-3Ј), MC2Rinfw (5Ј-ATGACATCATC MTII at 10 8 M (26, 27). In the assays examining the ability of ␣MSH to GACTCCC-3Ј); MC3Rfw (5Ј-GCCCTCACCTTGATCGTGGC-3Ј), MC3Rrev inhibit anti-CD3-mediated T cell proliferation, anti-CD3 Abs (Abcam) (5Ј-CTGTGGGGCCACCCCGTCGGC-3Ј), MC3Rinfw (5Ј-CATCTGGGTCT were added at a final concentration of 1 ␮g/ml. GCTGCGG-3Ј); MC4Rfw (5Ј-TACTCTGATGGAGGGTGC-3Ј), MC4Rrev (5Ј- DNA extraction, PCR, and MC1R sequencing TTGGCGGATGGCACCAGTGCC-3Ј), MC4Rinfw (5Ј-GGTGTTTGTGA CTCTGGG-3Ј); MC5Rfw (5Ј-GAGGGCAACCTTTCAGGACCC-3Ј), DNA was extracted from whole venous blood, and the MC1R gene was MC5Rrev (5Ј-CCGCAGCCCGTGCAGAAAGCC-3Ј), and MC5Rinfw (5Ј- amplified from the purified DNA using specific primers MC1R-165 for- CACCATGTGAAGACATGG-3Ј). PCR amplification was performed using sim- ward (fw) (5Ј-AGAGGGTGTGAGGGCAGATCTG-3Ј) and MC1R ϩ 33 ilar reagents as for PCR of MC1R from DNA, except that annealing temperatures reverse (rev) (5Ј-CACACTTAAAGCGCGTGC-3Ј). Amplification was of 60°C were used for MC1R and MC3R and lower annealing temperatures of performed using BioTaq red (Bioline) 1ϫ Optibuffer (Bioline), 2 mM 55°C for MC2R, MC4R, and MC5R. After agarose gel electrophoresis, correctly 4808 ␣MSH SUPPRESSES HUMAN IMMUNE RESPONSE TO Ag sized products were sequenced using the same infw and rev primers with a dye whether the suppression of SK/SD-induced lymphocyte prolifera- terminator cycle sequencing kit and a model ABI 377 automated DNA sequencer. tion by ␣MSH occurred through this mechanism (11, 28). PBMCs ␣ Ϫ13 Statistics were cultured with SK/SD with and without MSH (10 M, 10Ϫ11 M, 10Ϫ9 M, 10Ϫ7 M), and 0.2 ␮g/ml IL-10-blocking Ab. Statistical analysis was performed with Stats Direct (version 1.9.14) soft- Control Ag-stimulated cultures with added 0.1 ng/ml rIL-10 in the ware for Microsoft Windows. Assessment of suppression of the SI by ␣MSH, the degree of suppression (expressed as area under the curve) for presence and absence of anti-IL-10 Ab confirmed the ability of the each MC1R genotype group, and the maximal ␣MSH-induced suppression Ab to inhibit completely IL-10-induced lymphocyte proliferation for each individual in the three genotype groups were conducted using (data not shown). No significant abrogation of ␣MSH-induced ANOVA. The effects of anti-IL-10 Ab in the proliferation assay (compar- suppression of SK/SD-induced lymphocyte proliferation was de- ing the areas under the curve in the presence and absence of anti-IL-10 Ab), tected upon addition of IL-10 blocking Ab (n ϭ 7; p ϭ 0.3757; the maximal suppression of the SI by ␣MSH for each subject in the indi- vidual MC1R genotype groups, and the effects of ␣MSH on anti-CD3- Fig. 2). mediated T lymphocyte proliferation were analyzed by the paired t test. Suppression of SK/SD-induced lymphocyte proliferation is Results unrelated to MC1R genotype ␣MSH suppresses SK/SD-induced lymphocyte proliferation in Previous studies have indicated that MC1R is expressed by humans PBMCs, including human monocytes/macrophages, B lympho- Previous studies in animal models have demonstrated that ␣MSH cytes, and a subset of CTLs (12, 29, 30). MC1R is also expressed has immunomodulatory activity and can modify responses to ex- by melanocytes in the skin, where it plays a central role in human ogenous Ags applied to murine skin (7, 8). However, it is not clear pigmentation. MC1R variants have profound effects on human pig- Downloaded from whether ␣MSH can suppress immune responses to exogenous Ags mentation, causing red hair and fair skin phenotypes as a result of in human subjects. We therefore investigated whether ␣MSH the significantly reduced ability of variant receptors to signal via could alter Ag-induced lymphocyte proliferation in humans in cAMP (18, 20, 22–25). To investigate whether variability in ␣ vitro. PBMCs from eight individuals were cultured in the presence MSH-induced suppression of SK/SD-induced lymphocyte prolif- of SK/SD (a potent streptococcal Ag mixture against which the eration between subjects was due to genetic variability at the majority of individuals manifest a T lymphocyte-mediated re- MC1R , we extended the study population to include a total http://www.jimmunol.org/ sponse) and the effect of ␣MSH (10Ϫ13 M, 10Ϫ11 M, 10Ϫ9 M, of 26 subjects. Initially, pigmentation phenotype (red hair and fair 10Ϫ7 M) on lymphocyte proliferation was assessed. In all cases, skin or dark hair and tanned skin) was used to preferentially iden- the addition of SK/SD resulted in a SI of Ͼ2 (range, 6–95). The tify subjects with different MC1R genotypes to include similar addition of ␣MSH at each concentration did not alter the baseline numbers of individuals with wild-type MC1R, a single variant lymphocyte proliferation rate in the absence of Ag. Overall, a signif- MC1R allele, and two variant MC1R alleles. Subsequently, se- icant reduction in the lymphocyte proliferation response to SK/SD quencing of the MC1R coding region was performed in all 26 ␣ was observed after culture with ␣MSH ( p ϭ 0.0074; Fig. 1), but there subjects in whom the MSH/lymphocyte proliferation assay had was considerable variability between individuals in the ability of been undertaken. Nine subjects were wild type at MC1R, nine had by guest on September 24, 2021 ␣MSH to suppress Ag-induced lymphocyte proliferation. a single variant MC1R allele, and eight had two variant MC1R alleles. ␣MSH-induced suppression of the proliferation assay was Suppression of SK/SD-induced lymphocyte proliferation occurs observed in all three MC1R genotype groups, with little difference via an IL-10-independent mechanism between the three groups in the mean suppression of SK/SD-in- ␣ Because ␣MSH can cause release of IL-10 from human monocytes duced lymphocyte proliferation at each concentration of MSH and IL-10 can inhibit Ag-specific T cell proliferation, we examined

FIGURE 2. ␣MSH-induced suppression of SK/SD-mediated lympho- FIGURE 1. Effect of ␣MSH on SK/SD-induced lymphocyte prolifera- cyte proliferation is not inhibited by anti-IL-10 blocking Ab. Values are tion in eight subjects. Overall, ␣MSH significantly suppressed SI (p ϭ mean (and SEM) of suppression by ␣MSH at each concentration; n ϭ 7. 0.0074), but there was variation between subjects. The effects of anti-IL-10 Ab were not significant (p ϭ 0.3757). The Journal of Immunology 4809

(comparison of three groups, p ϭ 0.7109; Fig. 3). Although the Expression of melanocortin receptors by maximal ␣MSH-induced suppression of the SK/SD-induced lym- monocytes/macrophages and B and T lymphocytes phocyte proliferation assay ranged from 0 to 80% in the 26 sub- Within the lymphocyte proliferation cultures, monocytes/macro- jects overall and was highly significant within each genotype phages and B lymphocytes present the SK/SD Ag to the Th lym- group ( p Ͻ 0.0001 for wild-type, single-variant, and two-variant phocytes, which results in clonal proliferation of the T cells. To alleles) the degree of variation in suppression was similar within determine which cell population is predominantly suppressed by each group ( p ϭ 0.4080; Table I). Maximal suppression of SK/ ␣MSH, we first attempted to conduct the proliferation assay after SD-induced lymphocyte proliferation by ␣MSH varied from 21 to initial separation of monocytes and T cells to allow them to be 77% in individuals who were homozygous or compound heterozy- exposed separately to ␣MSH before being added back together. gous for the Arg151Cys, Arg160Trp, and Asp294His variant al- However, this approach was unsuccessful because the modified leles, which significantly impair MC1R function (23–25). Indeed, proliferation assay failed to achieve sufficiently potent inhibition two subjects with two variant alleles and three individuals with a without the continued presence of ␣MSH. The next approach was single MC1R variant with SIs Ͼ20 exhibited maximal ␣MSH- induced suppression of 46–80%, consistent with ␣MSH having to see whether the different cell populations differed in their ex- potent immunosuppressive effects in subjects with MC1R variants pression of melanocortin receptors. Therefore, we investigated for in this experimental system (Table I). expression of each of the five melanocortin receptors on adherent Downloaded from http://www.jimmunol.org/ by guest on September 24, 2021

FIGURE 3. Suppression of lym- phocyte proliferation assay in re- sponse to SK/SD by ␣MSH in differ- ent MC1R genotype groups: A, wild- type (WT/WT), n ϭ 9; B, single- variant allele (VAR/WT), n ϭ 9; C, two-variant alleles (VAR/VAR), n ϭ 8. Values are mean (and SEM) sup- pression at each concentration of ␣MSH. There was no significant dif- ference in suppression between each genotype group (p ϭ 0.7109). 4810 ␣MSH SUPPRESSES HUMAN IMMUNE RESPONSE TO Ag

Table I. Maximum percentage inhibition of SK/SD-induced lymphocyte proliferation by ␣MSH for each subjecta

SI to SK/SD at Concentration of Concentration of Maximum % ␣MSH Causing Subject ID Number and SI to ␣MSH Exhibiting Inhibition of Maximal Inhibition b b ␣ b MC1R Genotype SK/SD Maximal Inhibition SI by MSH of SI (log10 M) (17)c WT/WT 18 3 80 Ϫ9 (21) WT/WT 18 8 53 Ϫ13 (15) WT/WT 20 12 42 Ϫ11 (2) WT/WT 21 14 30 Ϫ11 (5) WT/WT 34 25 25 Ϫ9 (20) WT/WT 15 11 22 Ϫ9 (3) WT/WT 95 81 15 Ϫ11 (1) WT/WT 22 20 11 Ϫ13 (11) WT/WT 9 9 0 N/Ad (24) Arg163Gln/WT 30 6 80 Ϫ11 (14) Arg160Trp/WT 7352Ϫ13 (6) Arg160Trp/WT 49 24 50 Ϫ11 (12) Arg160Trp/WT 11 5 50 Ϫ11 Ϫ (16) Arg160Trp/WT 92 49 46 13 Downloaded from (4) Arg163Gln/WT 6344Ϫ7 (13) Va192Met/WT 56 36 37 Ϫ13 (18) Va192Met/WT 106 79 26 Ϫ13 (10) Arg160Trp/WT 52 46 12 Ϫ11 (26) Arg151Cys/Arg151Cys 33 8 77 Ϫ13 (19) Arg151Cys/Arg160Trp 21 9 57 Ϫ7

(22) Arg151Cys/Asp294His 11 6 46 Ϫ13 http://www.jimmunol.org/ (7) Arg160Trp/Arg160Trp 17 10 38 Ϫ11 (23) Arg151Cys/Asp294His 13 9 31 Ϫ13 (9) Arg151Cys/Asp294His 17 12 30 Ϫ13 (25) Asp294His/Asp294His 62 49 21 Ϫ13 (8) Pro256Ser/Asp294His 6421Ϫ11

a Results for each individual, including MC1R genotype, stimulation index to SK/SD, and concentration of ␣MSH 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. by guest on September 24, 2021

and nonadherent PBMCs, and subsequently on purified mono- ␣MSH and related analogs suppress SK/SD-induced lymphocyte cytes/macrophages, B cells and T cells. After culture of PBMCs proliferation in monocyte-depleted and B lymphocyte-depleted with SK/SD for 48 h, RT-PCR and subsequent sequencing of pos- cultures itive bands showed evidence of MC1R and MC3R expression in The observation that MC1R is expressed by purified monocytes/ adherent and nonadherent fractions (Fig. 4), but no expression of macrophages after culture with SK/SD and that MC1R and MC3R MC2R, MC4R, and MC5R was detected. RT-PCR (with sequenc- ing) 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 re- ceptor expression by T cells under these culture conditions.

␣MSH does not suppress anti-CD3-mediated T lymphocyte proliferation To examine in more detail whether the effect of ␣MSH on SK/ SD-induced lymphocyte proliferation could be due to a direct ef- fect on T lymphocyte proliferation, the ability of ␣MSH to sup- press 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 ␣MSH, were stimulated with anti-CD3 Ab. FIGURE 4. RT-PCR for MC1R (954 bp) and MC3R (178 bp) expres- Whereas ␣MSH consistently inhibited SK/SD-induced lympho- sion by adherent and nonadherent PBMCs, monocytes/macrophages, B cyte proliferation (n ϭ 4 experiments), no suppression of anti- cells and T cells. AC, Adherent PBMCs; NAC, nonadherent PBMCs; Mo/ ␾ Lanes 1 12 CD3-mediated T cell proliferation was observed ( p ϭ 0.5099; M , monocytes/macrophages; B, B cells; T, T cells. and , DNA ladder (1KB Plus DNA ladder; Invitrogen Life Technologies); lanes 2, 4, Fig. 5). 6, 8, and 10, MC1R; lanes 3, 5, 7, 9, and 11, MC3R. The Journal of Immunology 4811

study, we have shown that ␣MSH can suppress Ag-induced pro- liferation of memory T lymphocytes in humans in vitro. These effects on lymphocyte proliferation are unlikely to be due to cell death secondary to ␣MSH toxicity because the suppressive effects were greatest at the lower concentrations of ␣MSH that were used in this study. This is similar to the dose-response effects by ␣MSH on IL-10 production from monocytes where 10Ϫ13 M ␣MSH is most potent, and it contrasts with the higher doses of ␣MSH re- quired for maximal stimulation of cAMP/pigmentation by mela- FIGURE 5. ␣MSH does not suppress anti-CD3-mediated T lymphocyte proliferation. Anti-CD3 stimulated TCC, T cell clones (n ϭ 14) with anti- nocytes/ cells (11, 24, 31). The degree of suppression by ␣ CD3 Ab. SK/SD-stimulated PBMCs (n ϭ 4) were from same individual MSH varied considerably between individuals, but the results from whom the T cell clones were generated. Percent on the y-axis indi- showing up to 80% suppression in several individuals with high cates the responses expressed as percentage of the maximal response in the stimulation indices to SK/SD demonstrate that ␣MSH is a potent absence of ␣MSH. modulator of human immune responses in some subjects and sug- gest that ␣MSH or related compounds may have therapeutic po- tential for human disease. Indeed, the results of this study in com- are expressed by B lymphocytes under these conditions indicated bination with previous research showing that ␣MSH can suppress that the suppressive effects of ␣MSH might occur via MC1R on contact hypersensitivity in mice provide support for future inves- monocytes/macrophages and/or B lymphocytes or via MC3R on B Downloaded from ␣ cells. To investigate this further, PBMCs from subjects in whom tigations on the ability of MSH to treat allergic contact dermatitis ␣MSH suppressed SK/SD-induced lymphocyte proliferation were in humans (7, 8, 32). ␣ separately depleted of monocytes (CD14ϩ) and B lymphocytes Although the exact mechanism through which MSH can sup- (CD19ϩ) before stimulation with SK/SD and the addition of press SK/SD-induced lymphocyte proliferation in humans is not ␣MSH or related analogs SHU9119 and MTII. SK/SD caused pro- clear, the melanocortin receptor expression data would indicate that this could occur via MC1R on monocytes/macrophages and/or

liferation of monocyte-depleted and B lymphocyte-depleted cul- http://www.jimmunol.org/ tures, and ␣MSH inhibited this proliferation albeit to variable de- B lymphocytes or via MC3R on B lymphocytes, respectively. Al- grees (Fig. 6). SHU9119 and MTII also suppressed SK/SD- though Neumann-Andersen et al. (30) have reported MC1R ex- induced proliferation in these depleted cultures, and in all cases to pression by a subset of CTLs, we did not find evidence for ex- a greater extent than the suppression observed with ␣MSH pression of any of the five melanocortin receptors by T (Fig. 6). lymphocytes. This difference may have been due to the presence of SK/SD in our culture system for the RT-PCR experiments, but we Discussion considered it important to examine for melanocortin receptor ex- It is widely accepted that ␣MSH has potent anti-inflammatory and pression in the presence of this Ag given that environmental fac- immunomodulatory activity in animal models; for example, tors can affect expression of MC1R (29, 33). In addition to the lack by guest on September 24, 2021 ␣MSH can suppress contact hypersensitivity in murine skin (7, 8). of melanocortin receptor expression by T cells in this study, the However, limited research has been conducted on the ability of fact that ␣MSH failed to suppress anti-CD3-mediated T lympho- ␣MSH to suppress immune responses in humans, and in particular cyte proliferation suggests that ␣MSH-induced suppression of responses to exogenous Ags. There is evidence for effects by lymphocyte proliferation does not occur via a direct effect on T ␣MSH on cytokine release by and costimulatory molecule expres- cells. Our attempts to culture purified monocytes in the presence of sion on certain human cell types, but the effect of these alterations SK/SD with and without ␣MSH for 48 h before the addition of on responses to Ag has not been fully elucidated (11, 12). In this isolated T cells were unsuccessful because these preseparated and

FIGURE 6. ␣MSH and related compounds (SHU9119 and MTII) suppress SK/SD-induced lymphocyte prolifer- ation in monocyte-depleted PBMC cultures from two sub- jects (a and b) and in B lymphocyte-depleted PBMC cul- tures from two subjects (c and d). Control, no SK/SD; SK/SD, SK/SD alone; ␣MSH, SK/SD plus ␣MSH (10Ϫ12 M); SHU, SK/SD plus SHU9119 (10Ϫ8 M); MTII, SK/SD plus MTII (10Ϫ8 M). 4812 ␣MSH SUPPRESSES HUMAN IMMUNE RESPONSE TO Ag then reconstituted cultures gave high SIs in response to SK/SD that eration in humans in vitro. These effects are likely to be mediated failed to show suppression with ␣MSH for this shorter period (data via MC1R on monocytes/macrophages and B lymphocytes (and not shown). However, the observation that ␣MSH suppressed SK/ possibly also via MC3R on B lymphocytes) but are independent of SD-induced lymphocyte proliferation in separate B lymphocyte- MC1R gene status. depleted and monocyte-depleted cultures suggests that ␣MSH can have independent immunosuppressive effects via monocytes and B Disclosures cells on Ag-induced lymphocyte proliferation. Taken together with The authors have no financial conflict of interest. the melanocortin receptor expression data, this indicates that some of the suppressive actions of ␣MSH on responses to exogenous Ag References occurs via MC1R on monocytes. In addition, the use of the related 1. Lipton, J. M., and A. Catania. 1997. Anti-inflammatory actions of the neuroim- compounds SHU9119 (agonist at MC1R and antagonist at MC3R) munomodulator ␣-MSH. Immunol. Today 18: 140–145. 2. Lerner, A. B., and J. S. McGuire. 1961. Effect of ␣- and ␤melanocyte stimulating and MTII (agonist at MC1R and MC3R) in the depleted cultures hormones on the skin colour of man. Nature 189: 176–179. indicates that MC1R on B lymphocytes as well as on monocytes is 3. Luger, T. A., T. E. Scholzen, T. Brzoska, and M. Bohm. 2003. New insights into involved in this suppression, but this does not rule out a possible the functions of ␣-MSH and related in the immune system. Ann. NY Acad. Sci. 994: 133–140. additional effect by ␣MSH and MTII through MC3R on B cells. 4. Daynes, R. A., B. A. Robertson, B. H. Cho, D. K. Burnham, and R. Newton. Although ␣MSH can stimulate IL-10 release by monocytes (11), 1987. ␣-Melanocyte-stimulating hormone exhibits target cell selectivity in its capacity to affect interleukin 1-inducible responses in vivo and in vitro. J. Im- our results with anti-IL-10 blocking Abs indicate that the suppres- munol. 139: 103–109. sive effects of ␣MSH on Ag-induced lymphocyte proliferation are 5. Lipton, J. M., A. Macaluso, M. E. Hiltz, and A. Catania. 1991. Central admin- not via an IL-10 mechanism. In contrast to the reported Mc3r- istration of the peptide ␣-MSH inhibits inflammation in the skin. Peptides 12: Downloaded from ␣ 795–798. mediated anti-inflammatory effects by MSH in mouse peritoneal 6. Robertson, B., K. Dostal, and R. A. Daynes. 1988. regulation of macrophages (26), we did not observe MC3R expression on mono- inflammatory and immunologic responses. The capacity of ␣-melanocyte-stim- cytes/macrophages in our culture system, but our observations are ulating hormone to inhibit tumor necrosis factor and IL-1-inducible biologic re- sponses. J. Immunol. 140: 4300–4307. similar to the findings by Bhardwaj et al. (12), who detected 7. Rheins, L. A., A. L. Cotleur, R. S. Kleier, W. B. Hoppenjans, D. N. Saunder, and MC1R expression alone on human monocytes. Previous research J. J. Nordlund. 1989. ␣-Melanocyte stimulating hormone modulates contact hy- persensitivity responsiveness in C57/BL6 mice. J. Invest. Dermatol. 93: 511–517. has shown that MC1R is also expressed on mast cells and may http://www.jimmunol.org/ 8. Grabbe, S., R. S. Bhardwaj, K. Mahnke, M. M. Simon, T. Schwarz, and mediate the inhibitory effects of ␣MSH on these cells (34). Thus, T. A. Luger. 1996. ␣-Melanocyte-stimulating hormone induces hapten-specific it seems likely that the wide range of anti-inflammatory and im- tolerance in mice. J. Immunol. 156: 473–478. ␣ 9. Dai, R., and J. W. Streilein. 1997. Ultraviolet B-exposed and soluble factor-pre- munomodulatory actions of MSH in do not all occur incubated epidermal Langerhans cells fail to induce contact hypersensitivity and via a single melanocortin receptor but that MC1R and/or MC3R promote DNP-specific tolerance. J. Invest. Dermatol. 108: 721–726. may be involved to a greater or lesser extent in different types of 10. Gantz, I., and T. M. Fong. 2003. The melanocortin system. Am. J. Physiol. En- docrinol. Metab. 284: E468–E474. inflammation. In addition, it is possible that a hitherto unknown 11. Bhardwaj, R. S., A. Schwarz, E. Becher, K. Mahnke, Y. Aragane, T. Schwarz, melanocortin receptor or a non-melanocortin receptor mechanism and T. A. Luger. 1996. Pro-opiomelanocortin-derived peptides induce IL-10 pro- (the C-terminal ␣MSH-related tripeptide may interact with the duction in human monocytes. J. Immunol. 156: 2517–2521. 12. Bhardwaj, R., E. Becher, K. Mahnke, M. Hartmeyer, T. Schwarz, T. Scholzen, by guest on September 24, 2021 IL-1␤ receptor; Ref. 35) may also mediate some of the anti- and T. A. Luger. 1997. Evidence for the differential expression of the functional inflammatory effects of ␣MSH in certain situations. ␣-melanocyte-stimulating hormone receptor MC-1 on human monocytes. J. Im- ␣ munol. 158: 3378–3384. The variability in the degree of immune suppression by MSH 13. Becher, E., K. Mahnke, T. Brzoska, D. H. Kalden, S. Grabbe, and T. A. Luger. in different subjects in this study would be consistent with an un- 1999. Human peripheral blood-derived dendritic cells express functional mela- derlying genetic variability determining the effects of ␣MSH in nocortin receptor MC-1R. Ann. NY Acad. Sci. 885: 188–195. 14. Prota, G., and R. H. Thomson. 1976. Melanin pigmentation in mammals. En- this system. Logically, it would be expected that this genetic vari- deavour 35: 32–38. ability would be at the MC1R gene because there is much evidence 15. Thody, A. J., E. M. Higgins, K. Wakamatsu, S. Ito, S. A. Burchill, and that MC1R gene variants profoundly affect human pigmentation J. M. Marks. 1991. Pheomelanin as well as eumelanin is present in human epi- dermis. J. Invest. Dermatol. 97: 340–344. giving rise to red hair and fair skin through reduced receptor func- 16. Rees, J. L. 2003. Genetics of hair and skin color. Annu. Rev. Genet. 37: 67–90. tion and consequent lower cAMP signaling in pigment cells (18, 17. Box, N. F., J. R. Wyeth, L. E. O’Gorman, N. G. Martin, and R. A. Sturm. 1997. Characterization of melanocyte stimulating hormone receptor variant alleles in 20, 22–24). This would have been seen as a lack of suppressive twins with red hair. Hum. Mol. Genet. 6: 1891–1897. effect by ␣MSH in red haired, fair skinned individuals, whereas 18. Flanagan, N., E. Healy, A. Ray, S. Philips, C. Todd, I. J. Jackson, greatest suppression would have been expected in dark haired and M. A. Birch-Machin, and J. L. Rees. 2000. Pleiotropic effects of the melanocortin 1 receptor (MC1R) gene on human pigmentation. Hum. Mol. Genet. 9: tan skinned subjects. However, there was no correlation between 2531–2537. the degree of suppression and the pigmentary phenotype. More- 19. Harding, R. M., E. Healy, A. J. Ray, N. S. Ellis, N. Flanagan, C. Todd, C. Dixon, over, the MC1R sequencing results demonstrate that MC1R vari- A. Sajantila, I. J. Jackson, M. A. Birch-Machin, and J. L. Rees. 2000. Evidence for variable selective pressures at MC1R. Am. J. Hum. Genet. 66: 1351–1361. ants (heterozygous, homozygous, and compound heterozygous) do 20. Healy, E., N. Flannagan, A. Ray, C. Todd, I. J. Jackson, J. N. Matthews, not inhibit the ability of ␣MSH to suppress SK/SD-induced lym- M. A. Birch-Machin, and J. L. Rees. 2000. Melanocortin-1-receptor gene and sun sensitivity in individuals without red hair. Lancet 355: 1072–1073. phocyte proliferation; importantly, seven of the eight subjects with 21. Smith, R., E. Healy, S. Siddiqui, N. Flanagan, P. M. Steijlen, I. Rosdahl, two MC1R variant alleles in this study were homozygous or com- J. P. Jacques, S. Rogers, R. Turner, I. J. Jackson, M. A. Birch-Machin, and pound heterozygous for the Arg151Cys, Arg160Trp, and J. L. Rees. 1998. Melanocortin 1 receptor variants in an Irish population. J. In- vest. Dermatol. 111: 119–122. Asp294His variants, which are known to compromise receptor 22. Valverde, P., E. Healy, I. Jackson, J. L. Rees, and A. J. Thody. 1995. Variants of function and signaling through cAMP (23–25). Recent research the melanocyte-stimulating hormone receptor gene are associated with red hair suggests that ␣MSH can signal through MC1R via an alternative and fair skin in humans. Nat. Genet. 11: 328–330. 23. Healy, E., S. A. Jordan, P. S. Budd, R. Suffolk, J. L. Rees, and I. J. Jackson. 2001. intracellular signaling pathway involving calcium flux (36). It is Functional variation of MC1R alleles from red-haired individuals. Hum. Mol. possible that MC1R variant receptors may retain this function and Genet. 10: 2397–2402. that the immunosuppressive effects of ␣MSH that we observed in 24. Robinson, S. J., and E. Healy. 2002. Human melanocortin 1 receptor (MC1R) gene variants alter melanoma cell growth and adhesion to extracellular matrix. this study occur through this alternative MC1R signaling pathway, Oncogene 21: 8037–8046. but future research will be necessary to investigate this further. 25. Schioth, H. B., S. R. Phillips, R. Rudzish, M. A. Birch-Machin, J. E. Wikberg, ␣ and J. L. Rees. 1999. Loss of function of the human melanocortin 1 In conclusion, we have demonstrated in this study that MSH receptor are common and are associated with red hair. Biochem. Biophys. Res. has potent suppressive effects on Ag-mediated lymphocyte prolif- Commun. 260: 488–491. The Journal of Immunology 4813

26. Getting, S. J., H. C. Christian, C. W. Lam, F. N. Gavins, R. J. Flower, tively expressed on leucocyte subpopulations with antigen presenting and cyto- H. B. Schioth, and M. Perretti. 2003. Redundancy of a functional melanocortin 1 toxic functions. Clin. Exp. Immunol. 126: 441–446. receptor in the anti-inflammatory actions of melanocortin peptides: studies in the 31. Hunt, G., P. D. Donatien, J. Lunec, C. Todd, S. Kyne, and A. J. Thody. 1994. recessive yellow (e/e) mouse suggest an important role for melanocortin 3 re- Cultured human melanocytes respond to MSH peptides and ACTH. Pigm. Cell ceptor. J. Immunol. 170: 3323–3330. Res. 7: 217–221. 27. Hruby, V. J., D. Lu, S. D. Sharma, A. L. Castrucci, R. A. Kesterson, 32. Friedmann, P. S. 1998. Allergy and the skin. II. Contact and atopic eczema. BMJ F. A. al Obeidi, M. E. Hadley, and R. D. Cone. 1995. Cyclic lactam ␣-mela- 316: 1226–1229. notropin analogues of Ac-Nle4-cyclo[Asp5, D-Phe7,Lys10] ␣-melanocyte-stim- 33. Schiller, M., T. Brzoska, M. Bohm, D. Metze, T. E. Scholzen, A. Rougier, and ulating hormone-(4-10)-NH2 with bulky aromatic amino acids at position 7 show T. A. Luger. 2004. Solar-simulated ultraviolet radiation-induced upregulation of high antagonist potency and selectivity at specific melanocortin receptors. J Med. the melanocortin-1 receptor, , and ␣-melanocyte-stimulating Chem. 38: 3454–3461. hormone in human epidermis in vivo. J. Invest. Dermatol. 122: 468–476. 28. de Waal, M. R., J. Haanen, H. Spits, M. G. Roncarolo, V. A. te, C. Figdor, 34. Adachi, S., T. Nakano, H. Vliagoftis, and D. D. Metcalfe. 1999. Receptor-me- K. Johnson, R. Kastelein, H. Yssel, and J. E. de Vries. 1991. Interleukin 10 diated modulation of murine mast cell function by ␣-melanocyte stimulating (IL-10) and viral IL-10 strongly reduce antigen-specific human T cell prolifera- hormone. J. Immunol. 163: 3363–3368. tion by diminishing the antigen-presenting capacity of monocytes via downregu- 35. Haddad, J. J., R. Lauterbach, N. E. Saade, B. Safieh-Garabedian, and S. C. Land. lation of class II major histocompatibility complex expression. J Exp. Med. 174: 2001. ␣-Melanocyte-related tripeptide, Lys-D-Pro-Val, ameliorates endotoxin-in- 915–924. duced nuclear factor ␬B translocation and activation: evidence for involvement of ␤ 29. Salazar-Onfray, F., M. Lopez, A. Lundqvist, A. Aguirre, A. Escobar, A. Serrano, an interleukin-1 193–195 receptor antagonism in the alveolar epithelium. Biochem C. Korenblit, M. Petersson, V. Chhajlani, O. Larsson, and R. Kiessling. 2002. J. 355: 29–38. Tissue distribution and differential expression of melanocortin 1 receptor, a ma- 36. Elliott, R. J., M. Szabo, M. J. Wagner, E. H. Kemp, S. MacNeil, and lignant melanoma marker. Br. J Cancer 87: 414–422. J. W. Haycock. 2004. ␣-Melanocyte-stimulating hormone, MSH 11-13 KPV and 30. Neumann, A. G., O. Nagaeva, I. Mandrika, R. Petrovska, R. Muceniece, adrenocorticotropic hormone signalling in human keratinocyte cells. J. Invest. L. Mincheva-Nilsson, and J. E. Wikberg. 2001. MC(1) receptors are constitu- Dermatol. 122: 1010–1019. Downloaded from http://www.jimmunol.org/ by guest on September 24, 2021