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The Journal of Immunology, 1999, 163: 3363-3368.
Copyright © 1999 by The American Association of Immunologists

Receptor-Mediated Modulation of Murine Mast Cell Function by {alpha}-Melanocyte Stimulating Hormone1

Shiro Adachi, Teruaki Nakano, Harrisios Vliagoftis and Dean D. Metcalfe2

Laboratory of Allergic Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The proopiomelanocortin (POMC)-derived neuropeptide {alpha}-melanocyte stimulating hormone ({alpha}-MSH) is known to modulate some aspects of inflammation through direct effects on T cells, B cells, and monocytes. To determine whether {alpha}-MSH might similarly influence mast cell responsiveness, mast cells were examined to see if they expressed the receptor for {alpha}-MSH, melanocortin-1 (MC-1), and whether {alpha}-MSH altered mast cell function. We thus first identified MC-1 on bone marrow cultured murine mast cells (BMCMC) and a murine mast cell line (MCP-5) employing flow cytometry and through detection of specific binding. Subsequent treatment of mast cells with {alpha}-MSH increased the cAMP concentration in a characteristic biphasic pattern, demonstrating that {alpha}-MSH could affect intracellular processes. We next examined the effect of {alpha}-MSH on mediator release and cytokine expression. IgE/DNP-human serum albumin-stimulated histamine release from mast cells was inhibited by ~60% in the presence of {alpha}-MSH. Although activation of BMCMC induced the expression of mRNAs for the inflammatory cytokines IL-1ß, IL-4, IL-6, TNF-{alpha}, and the chemokine lymphotactin, mRNAs for IL-1ß, TNF-{alpha}, and lymphotactin were down-modulated in the presence of {alpha}-MSH. Finally, IL-3-dependent proliferative activity of BMCMC was slightly but significantly augmented by {alpha}-MSH. Taken together, these observations suggest that {alpha}-MSH may exert an inhibitory effect on the mast cell-dependent component of a specific inflammatory response.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Neuropeptides have been implicated in the regulation of a number of immune responses, in both human and murine systems. These neuropeptides are believed to not only be synthesized by neuronal tissue, and thus to communicate neuronal signals to the immune system, but to also be produced by a variety of immunocompetent cells and, in turn, influence specific inflammatory events. Among these neuropeptides are calcitonin-related peptides, vasointestinal peptides, and proopiomelanocortin (POMC)3-derived hormones (1, 2, 3, 4).

The tridecapeptide, {alpha}-melanocyte stimulating hormone ({alpha}-MSH) is derived from POMC (5), and is of particular interest in that it has been associated with a variety of cells of the immune system. The prohormone POMC is cleaved posttranslationally into biologically active peptides, including melanocyte stimulating hormones ({alpha}-, ß-, {gamma}-MSH), adrenocorticotropic hormone, and ß-endorphin (5). Although these peptides were originally discovered in the pituitary gland and brain, they have now been detected in a variety of cells, including lymphocytes (6), monocytes/macrophages (7, 8), Langerhans cells (9), and epidermal cells (10). The receptors for POMC peptides are specific G protein-coupled receptors containing seven transmembrane helixes that activate adenylate cyclase (11). Five subtypes of this melanocortin (MC) receptor family have been recognized to date (12). MC-1 receptor has recently been discovered on immune/inflammatory cells (5).

{alpha}-MSH has been reported to influence a number of immune responses. It inhibits the activities of proinflammatory cytokines, such as IL-1ß, IL-6, and TNF-{alpha} (13, 14, 15). {alpha}-MSH down-regulates the production of IFN-{gamma} by human T cells (3) and modulates IgE synthesis by human B cells (16). {alpha}-MSH increases the production of IL-10 in human monocytes (17). An immunosuppressive role for {alpha}-MSH is supported by in vivo studies using a mouse model of contact hypersensitivity (18). Finally, increased levels of {alpha}-MSH or adrenocorticotropic hormone have been observed in chronic inflammatory conditions, such as arthritis, and with HIV and parasitic infections (19, 20, 21).

Mast cells synthesize cytokines, chemokines, and chemical mediators, including histamine and prostaglandins (22). These products are released from stimulated mast cells and contribute to the genesis of inflammation. Since {alpha}-MSH has been shown to regulate inflammatory conditions, we sought to investigate whether {alpha}-MSH also modulates mast cell responsiveness.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell culture

Bone marrow cells from 10-wk-old WBB6F1+/+ mice (The Jackson Laboratory, Bar Harbor, ME) were cultured at a density of 106 cells/ml in RPMI 1640 medium (Biofluids, Rockville, MD) supplemented with 4 mM L-glutamine, 0.1 mM nonessential amino acids, 25 mM HEPES, 1 mM sodium pyruvate, 100 mg/ml penicillin/streptomycin, 10-4 M 2-ME (Sigma, St. Louis, MO), 10% FBS (Biofluids), and 10% WEHI-3 conditioned media containing IL-3 (Collaborative Biomedical Products, Bedford, MA). Culture flasks (Nunk, Roskilde, Denmark) were incubated at 37°C in a humidified atmosphere of 5% CO2, 95% air. Half of the culture media was replaced every 7 days. Greater than 95% of the cells were identified as mast cells 4–6 wk after the initiation of the culture. MCP-5 murine mast cells (23) were cultured in the same media.

Flow cytometric analysis of MC-1 receptors on bone marrow cultured murine mast cells (BMCMC)

BMCMC were treated with 10-8 M biotinyl-[Nle4, D-Phe7]-{alpha}-MSH (NDP-{alpha}-MSH) (Peninsula, Belmont, CA) in the presence or absence of an excess of NDP-{alpha}-MSH (10-6 M) on ice for 1 h and incubated with 2 mM Bis(sulfosuccinimidyl) subetare (BS3; Pierce, Rockford, IL) on ice for 1 h to stabilize the binding between biotinyl-NDP-{alpha}-MSH and the receptor. After quenching the reaction by treating with Tris buffer for 15 min, cells were washed and stained with streptavidin-conjugated FITC (PharMingen, San Diego, CA) on ice for 40 min, and the fluorescence intensity of cells measured by FACScan (Becton Dickinson, San Jose, CA) after exclusion of dead cells. To confirm the specificity of BS3-mediated cross-linking between biotinyl-NDP-{alpha}-MSH and MC-1 protein, BMCMC were treated with biotinyl anti-murine CD3 Ab (PharMingen) in the presence of BS3 and stained with streptavidin-conjugated FITC.

Detection of specific binding of NDP-{alpha}-MSH

Cell membranes were prepared as described with modification (24). MCP-5 cells were washed twice with PBS and resuspended in ice-cold buffer containing 1 mM NaHCO3, 1 mM DTT, 0.2 mM magnesium acetate, 10 µg/ml leupeptin, 10 µg/ml soybean trypsin inhibitor, and 1 mM p-aminobenzamidine (pH 7.5). This suspension was homogenized in a Dounce-type glass homogenizer (Wheaton Science Products, Millville, NJ) (30 strokes) on ice, and the homogenate was layered on a 40% sucrose solution, prepared in the same buffer, and centrifuged at 100,000 x g for 45 min. The whitish-yellow membrane layer at the 0/40% sucrose interphase was pooled and diluted to <5% sucrose, layered on a 15% sucrose cushion, and centrifuged at 100,000 x g for 45 min. The pellet containing the cell membrane was then suspended in the same buffer (NaHCO3/DTT) and stored in liquid nitrogen until use. After thawing, the cell membranes were incubated with 10-6 M biotinyl-NDP-{alpha}-MSH (Peninsula Laboratories) in the presence or absence of an excess of NDP-{alpha}-MSH (10-5 M) at 30°C for 40 min, diluted with PBS, and centrifuged at 100,000 x g for 20 min. The pellets were suspended in 100 mM phosphate buffer (pH 7.5), incubated in the presence of BS3 (final concentration, 3 mM) at 30°C for 1 h, diluted with PBS, and centrifuged at 100,000 x g for 20 min. The membrane pellets were then dissolved in sample buffer (3% SDS, 5% 2-ME, 10% glycerol, and 0.01% bromophenol blue) and kept at 37°C for 1 h, before loading onto a 10% Tris-glycine gel. The binding complex of biotinyl-NDP-{alpha}-MSH and the receptor was detected using a BM-chemiluminescence blotting kit, according to the manufacturers instructions (Boehringer Mannheim, Mannheim, Germany).

Cell activation

For high-affinity IgE receptor-dependent activation, BMCMC were sensitized with 1 µg/ml of monoclonal murine IgE anti-DNP Ab (Sigma) for 2 h at 37°C. The sensitized BMCMC were washed with culture medium twice to remove unbound IgE and cultured either in the presence or absence of {alpha}-MSH (Sigma) for 2 h. {alpha}-MSH-treated and untreated cells were then challenged with 50 ng/ml of DNP human serum albumin (DNP-HSA) (Sigma) either in the presence or absence of {alpha}-MSH for the indicated times. For stimulation with PMA (Sigma), BMCMC were cultured either in the presence or absence of {alpha}-MSH for 2 h and stimulated with 50 ng/ml PMA for the indicated times.

Isolation of RNA and RT-PCR

Total cellular RNA was isolated from BMCMC and MCP-5 cells by the guanidine thiocyanate/phenol-chloroform extraction method (25). The first-strand cDNA was synthesized from 2 µg of total RNA using the superscript preamplification system (Life Technologies, Gaithersburg, MD), according to the manufacturer’s instructions. The first-strand cDNA was treated with RNase-free DNase (Promega, Madison, WI) to remove genomic DNA before PCR amplification. PCR was performed in a thermocycler (GeneAmp PCR system 9600; Perkin-Elmer Cetus, Norwalk, CT) as follows: 94°C for 5 min, following by 35 amplification cycles (94°C, 1 min; 60°C, 2 min; 72°C, 3 min). The sequence of the primers for MSH receptor-1 (MC-1) are as follows: upper strand, GTG AGT CTG GTG GAG AAT GTG; and lower strand, TTT TGT GGA GCT GGG CAA TGC (8).

Northern blot analysis

Twenty micrograms of total cellular RNA were electrophoresed on a 1.5% agarose-formaldehyde gel and transferred to a nylon membrane (maximum strength Nytran) (Schleicher & Schuell, Keene, NH). Blots were prehybridized at 46°C in a hybridization solution (Hybrisol; Oncor, Gaithersburg, MD) for 2 h and hybridized for 16–24 h with 32P-labeled cDNA probes at 42°C in the same solution. Hybridized blots were exposed to Kodak X-OMAT AR films using a normal screen for 4–24 h. In blots for IL-1ß of IgE/DNP-stimulated BMCMC, the blots were exposed to phosphor screen (Molecular Dynamics, Sunnyvale, CA) and visualized using a Phosphorimager 445 SI (Molecular Dynamics).

32P labeling of cDNA probes

Northern blots were probed with 32P-labeled murine IL-1ß, IL-4, IL-6, TNF-{alpha}, lymphotactin, and ß-actin cDNAs generated by RT-PCR. Fifty nanograms of a full-length murine IL-1ß, IL-4, TNF-{alpha}, lymphotactin (gift from Dr. Vanitcha Rumsaeng, National Institute of Allergy and Infectious Diseases, national Institutes of Health, Bethesda, MD), IL-6, or ß-actin cDNAs were 32P labeled by asymmetrical PCR to generate single-stranded cDNAs, as described (26). The PCR reactions (50 µl) contained cDNA templates; 1 µM antisense primer; 1.5 mM MgCl2; 2.5 U AmpliTaq DNA polymerase; 50 µM concentrations each of dATP, dGTP, and dTTP; and 50 µCi [{alpha}-32P]dCTP (3000 Ci/mmol) (DuPont NEN, Boston, MA) in reaction buffer supplied by the manufacturer (Life Technologies).

Measurement of cAMP

Mast cells were stimulated with 50 ng/ml PMA for 3 h, washed with RPMI 1640 containing 1% FBS and 1 mM 3-isobutyl-methylxanthine (Sigma), and incubated with various concentrations of {alpha}-MSH for 30 min at 37°C. After incubation, the cell suspension was treated with 1 N HCl and boiled for 2 min. The supernatants of the cell suspensions were collected and lyophilized. Intracellular cAMP concentrations were determined with the cAMP EIA system (Amersham, Buckinghamshire, England).

Histamine release assay

Sensitized BMCMC (106 cells per sample) were cultured in the presence or absence of {alpha}-MSH for 2 h and challenged with DNP-HSA for 1 h. After centrifugation, the culture supernatants were collected. The concentrations of the released histamine in the diluted supernatants were measured using an enzyme immunoassay kit (Immunotech, Marseille, France).

Cell proliferation assay

BMCMC (1 x 105 cells per well in a 96-well tissue culture plate) were cultured in the absence of IL-3 for 24 h to synchronize cells at G1 (27). Then, IL-3 (100 ng/ml) (Peprotech, Rocky Hill, NJ) and various concentrations of {alpha}-MSH were added to the culture. In another experiment, stem cell factor (SCF; 100 ng/ml) (Peprotech), in addition to IL-3, was added in the culture. After 48 h of incubation in the presence of 1 µCi/ml of [3H]thymidine, cells were harvested, and the incorporated radioactivity was measured by liquid scintillation.

Assessment of cell viability

Flow cytometric analysis was used to assess cell viability, as described (28). BMCMC were cultured in the absence of IL-3 containing WEHI-3 supernatant, either in the presence or absence of {alpha}-MSH for 24, 48, or 72 h. At the end of the incubation, cells were harvested and suspended at a concentration of 1 x 106/ml in PBS with 0.1% BSA. Propidium iodide (Sigma) was added at a final concentration of 5 µg/ml 5 min before analysis by flow cytometry.

Statistical analysis

Tukey’s test was used to compare means.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Expression of the receptor for {alpha}-MSH on mast cells

To determine whether BMCMC and MCP-5 cells might have a receptor for {alpha}-MSH (MC-1), we first attempted to detect mRNA for MC-1 by RT-PCR. An RT-PCR product specific for MC-1 with the expected length of 529 bp was detected in both BMCMC and MCP-5 cells (Fig. 1GoA). A corresponding RT product without RT when subjected to PCR did not reveal a specific product. Thus, a possible amplification of the PCR product from genomic DNA present in the RNA preparation was excluded.



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  		FIGURE 1. Expression of MC-1 by mast cells. A, Detection of MC-1 mRNA in BMCMC and MCP-5 mast cells using RT-PCR. mRNA was extracted and analyzed, as described in Materials and Methods. The experiment was performed with 5 µg of total RNA for each specimen and 35 PCR cycles. No bands were found in the control lane lacking RT, whereas a single 529-bp product was found in the lane containing MC-1 cDNA. B, Flow cytometric detection of MC-1. BMCMC were treated with biotinyl-NDP-{alpha}-MSH for 1 h. After cross-linking by BS3, cells were stained with streptavidin-conjugated FITC, and the fluorescence intensities measured using FACScan. The thick solid line shows the fluorescence intensity of biotinyl-NDP-{alpha}-MSH-treated BMCMC; the thin solid line shows biotinyl-NDP-{alpha}-MSH-treated BMCMC in the presence of an excess of NDP-{alpha}-MSH; the broken line shows BMCMC treated with biotinyl-anti-CD3 Ab; and the dotted line shows untreated BMCMC. C, Detection of MC-1 protein using biotinyl-NDP-{alpha}-MSH and BS3 on SDS-PAGE under reducing conditions. Biotinyl-NDP-{alpha}-MSH (10-6 M) was added to the plasma membrane suspension, followed by addition of BS3, and the solubilized proteins were chromatographed (lane 2). The presence of excess NDP-{alpha}-MSH (10-5 M) completely inhibited the binding of biotinyl-NDP-{alpha}-MSH to membrane proteins, even in the presence of BS3 (lane 3). Biotinyl-molecular markers are shown in lane 1.

 
To detect the expression of MC-1 protein on the BMCMC surface, and since no Abs for MC-1 protein are available, we employed biotinyl-NDP-{alpha}-MSH, a known analogue of {alpha}-MSH that binds to the receptor (29). We additionally used a cross linker, BS3, that forms a covalent bond between primary amines of adjacent proteins (30). As can be seen in Fig. 1GoB, significant binding of biotinyl-NDP-{alpha}-MSH was thus detectable on BMCMC by FACS analysis. To demonstrate specificity, we treated BMCMC with biotinyl-NDP-{alpha}-MSH in the presence of an excess of nonlabeled NDP-{alpha}-MSH. In the presence of an excess of NDP-{alpha}-MSH, the fluorescence intensity was comparable to that of the negative control. Fluorescence intensity of BMCMC treated with biotinyl-Ab against murine CD3, which was not expressed on BMCMC, was also comparable to that of the negative control, even in the presence of BS3. As confirmation of binding, biotinyl-NDP-{alpha}-MSH was next added to plasma membrane suspensions, followed by addition of BS3. Solubilized membrane proteins were then loaded on SDS-PAGE under the reducing condition (Fig. 1GoC). A significant signal was observed near the 45-kDa m.w. marker in lane 2. Addition of an excess of unlabeled NDP-{alpha}-MSH (10-fold) completely abolished the signal (lane 3).

Effect of {alpha}-MSH on cAMP accumulation

Since MC-1 is known to couple with G-proteins, we next examined the changes in cAMP in BMCMC treated with {alpha}-MSH. After 3 h of stimulation by PMA, BMCMC were incubated with various concentrations of {alpha}-MSH (10-12 to 10-6 M) for 30 min. As shown in Fig. 2Go, {alpha}-MSH at 10-12 to 10-8 M increased the cAMP concentration in mast cells. A higher concentration (10-6 M) did not increase cAMP. Similar results were obtained using IgE/DNP-HSA-stimulated BMCMC (data not shown).



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  		FIGURE 2. Effect of {alpha}-MSH on cAMP accumulation in PMA-treated mast cells. BMCMC (2 x 106 cells per sample) were stimulated with PMA (50 ng/ml) for 3 h and treated with various concentrations of {alpha}-MSH (0 to 10-6 M) for 30 min. After stopping the reaction with HCl, the cells were collected and the concentrations of cAMP measured by ELISA. Results are presented as mean ± SE of three independent experiments; *, p < 0.05; **, p < 0.01; and ***, p < 0.001, when a given result obtained in BMCMC incubated with {alpha}-MSH is compared with the result determined in BMCMC without {alpha}-MSH.

 
Down-modulation of histamine release from activated BMCMC

{alpha}-MSH is reported to down-regulate inflammation (5), and histamine is a potent proinflammatory molecule (31). These data led us to explore the effect of {alpha}-MSH on histamine release from Ag-stimulated BMCMC. As shown in Fig. 3Go, histamine release was inhibited in a concentration-dependent manner by {alpha}-MSH. The inhibition of histamine release exhibited a biphasic or "U"-shaped dose response. Maximal inhibition was observed when BMCMC were treated with 10-10 M {alpha}-MSH. The inhibitory effects of higher concentrations of {alpha}-MSH were less prominent, but still significant.



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  		FIGURE 3. Effects of {alpha}-MSH on histamine release from BMCMC. Sensitized BMCMC were incubated in the presence of {alpha}-MSH (0 to 10-6 M) for 2 h and activated with DNP-HSA for 1 h. The released histamine was measured by ELISA. Results are presented as mean ± SE of three independent experiments; *, p < 0.05; **, p < 0.01; and ***, p < 0.001, when a given result obtained in BMCMC incubated with {alpha}-MSH is compared with the result determined in BMCMC without {alpha}-MSH.

 
Effects of {alpha}-MSH on mast cell proliferation

Since {alpha}-MSH inhibits IL-1-induced proliferation of thymocytes (15) and synergistically increases the proliferation of melanocytes (32), we next examined the effects of {alpha}-MSH on the proliferative activity of BMCMC by measuring the incorporated [3H]thymidine. In the presence of IL-3, {alpha}-MSH of 10-12 to 10-6 M somewhat increased the incorporation of [3H]thymidine by BMCMC (Fig. 4Go). Since it is known that SCF synergistically increases the proliferation of BMCMC in the presence of IL-3 (33), we also examined the effect of {alpha}-MSH on SCF-induced proliferation. SCF increased the incorporation of [3H]thymidine in the presence of IL-3, and this was not altered by the presence of {alpha}-MSH (data not shown). Because BMCMC were preincubated in the absence of IL-3 for synchronization at G1 and mast cells are known to undergo apoptosis on removal of IL-3, there was a possibility that {alpha}-MSH might indirectly affect the proliferative activity through inhibiting apoptosis. Therefore, we examined the effect of {alpha}-MSH on mast cell apoptosis after IL-3 deprivation by flow cytometric analysis. Although IL-3 deprivation over 24 h induced apoptosis of mast cells (uptake of propidium iodide and decreased cell size), {alpha}-MSH did not inhibit or accelerate apoptosis of BMCMC (data not shown).



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  		FIGURE 4. Effect of {alpha}-MSH on the proliferative activity of BMCMC. BMCMC were cultured in the absence of IL-3 for 24 h, followed by incubation in the presence of IL-3 and [3H]thymidine for 48 h. The incorporated radioactivity was measured by liquid scintillation. Results are presented as mean ± SE of three independent experiments; *, p < 0.05; and **, p < 0.01, when a given result obtained in BMCMC incubated with {alpha}-MSH is compared with the result determined in BMCMC without {alpha}-MSH.

 
Selective down-modulation of cytokine and chemokine mRNA by {alpha}-MSH

{alpha}-MSH has been known to be antiinflammatory (5) and has been demonstrated to down-modulate IL-1- or TNF-{alpha}-induced reactions (13, 14, 15, 34). Because of these observations, we determined the effects of {alpha}-MSH on the expression of the cytokines IL-1ß, IL-4, IL-6, and TNF-{alpha}, and the chemokine lymphotactin (22) in BMCMC. We first examined the normal expression kinetics of cytokine mRNAs and determined the optimal conditions of stimulation. The expression of IL-1ß, IL-4, IL-6, TNF-{alpha}, and lymphotactin mRNAs were maximum at 30 min or 3 h after activation with IgE/DNP or PMA, respectively (data not shown). We then determined the effects of {alpha}-MSH on this expression. As shown in Fig. 5Go, {alpha}-MSH down-modulated the expression of IL-1ß and TNF-{alpha} mRNAs, both in IgE/DNP and in PMA-stimulated BMCMC. {alpha}-MSH also down-modulated the expression of lymphotactin mRNA in IgE/DNP-stimulated BMCMC. PMA did not induce the expression of lymphotactin mRNA, as reported (35), and {alpha}-MSH did not affect the expression of lymphotactin mRNA in PMA-stimulated BMCMC. On the other hand, {alpha}-MSH did not modulate the expression of mRNA of IL-4, or IL-6, in IgE/DNP- or PMA-stimulated BMCMC.



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  		FIGURE 5. Effects of {alpha}-MSH on expression of cytokine and chemokine mRNA in BMCMC pretreated with {alpha}-MSH and stimulated with DNP-HSA or PMA. BMCMC were treated with {alpha}-MSH (10-8 M) for 2 h and stimulated with DNP-HSA (50 ng/ml) or PMA (50 ng/ml) for 30 min or 3 h, respectively. Extracted mRNAs were analyzed on Northern blots hybridized with a 32P-labeled murine IL-1ß, IL-4, IL-6, TNF-{alpha}, or lymphotactin (Ltn) cDNA probe. The blots were stripped and rehybridized with a 32P-labeled ß-actin cDNA.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Mast cells play a major role in inflammatory processes through the generation and release of a variety of cytokines, chemokines, and chemical mediators (22). It has also been demonstrated that {alpha}-MSH has the capacity to modulate inflammation (5). Therefore, we hypothesized that {alpha}-MSH might modulate mast cell function. As expected, BMCMC and the murine mast cell line, MCP-5, expressed mRNA for one subtype of the receptors for {alpha}-MSH (MC-1) (Fig. 1GoA). To detect the receptor protein on the mast cell surface, and since no Abs against MC-1 have been established, we used biotinyl-NDP-{alpha}-MSH, a more stable analogue of {alpha}-MSH, and a cross-linker, BS3, to detect the receptor by FACS analysis (Fig. 1GoB). An excess of NDP-{alpha}-MSH completely inhibited the increase of the fluorescence intensity, and biotinyl-anti-CD3 Ab did not bind to the cell membrane of BMCMC, even in the presence of BS3. This specificity was supported by SDS-PAGE analysis (Fig. 1GoC). The m.w. of biotinyl-NDP-{alpha}-MSH/MC-1 receptor/BS3 complex was consistent with that noted in a previous report using melanoma cell lines (24). There is only one current report of the demonstration of the expression of the receptor for {alpha}-MSH on human inflammatory cells (monocytes) by FACS, without the use of a cross-linker (34). Recently, we also detected the expression of the receptor for {alpha}-MSH on a stimulated human mast cell line, HMC-1, by FACS, but without addition of BS3 (our unpublished observations). It thus seems clear that mast cells may express MC-1.

{alpha}-MSH also altered the responsiveness of mast cells. {alpha}-MSH down-modulated histamine release following Fc{epsilon}RI aggregation in BMCMC (Fig. 3Go). This inhibitory effect of {alpha}-MSH on histamine release from mast cells is consistent with a previous report that elevated levels of cAMP, which are induced by {alpha}-MSH (Fig. 2Go), inhibit histamine release (36). Histamine is known to modulate epithelial permeability, vasopermeability, and migration of inflammatory cells (31). Pretreatment with {alpha}-MSH is reported to inhibit histamine-dependent vascular permeability of i.v.-administrated dye (37). {alpha}-MSH may thus, in part, decrease histamine-dependent reactions by modulating histamine release from mast cells.

{alpha}-MSH-mediated inhibition of histamine release from activated mast cells follows a "U-shaped" dose-response (Fig. 3Go). {alpha}-MSH exerts its most profound influence on histamine release in the concentration between 10-10 and 10-8 M, and has less influence at lower or higher concentrations. This biphasic dose response has also been reported for the effect of {alpha}-MSH on melanin synthesis in melanocytes (38), for thermoregulation (39) in IL-1-dependent prostaglandin synthesis by fibroblasts with IL-1-dependent thymocyte proliferation (15), and for IL-10 production in human monocytes (17). Such biphasic responses are also known for responses due to substance P, somatostatin, and vasoactive intestinal peptide. Although the mechanism that governs the biphasic response has not been clarified, this response seems to correspond to the modulation of cAMP by {alpha}-MSH (Fig. 2Go). In our experiments, the concentration of cAMP was also modulated in a biphasic pattern. {alpha}-MSH between 10-10 and 10-8 M showed the most profound influence on the increase of cAMP. This biphasic modulation of cAMP concentration by {alpha}-MSH has also been reported in cultured spinal cord cells (40). In other reports using L cells transfected with MC-1 and RAW cells, an increase of cAMP by {alpha}-MSH occurred in a monophasic dose-dependent manner (8, 11, 41). It is possible that these responses are cell type-dependent, or that a biphasic response curve would have been seen in these studies if lower concentrations of {alpha}-MSH had been employed.

The concentration of {alpha}-MSH has been determined in synovial tissues and synovial fluid of rheumatoid arthritis patients (19), plasma of endotoxin-injected patients (42), aqueous humor (3), and plasma of patients infected with HIV (43). Concentrations ranged from 10-10 to 10-8 M in these physiological and pathological situations. These concentrations correlate with the optimal concentration of {alpha}-MSH, which induces the maximum increase of cAMP in BMCMC.

There are several reports examining the influence of {alpha}-MSH on the proliferative activity of cells. Although a number of groups have been unable to demonstrate an effect of {alpha}-MSH on human melanocyte number (38), it has been reported that {alpha}-MSH synergistically increases the proliferative activity when in combination with ß-fibroblast growth factor, hepatocyte growth factor, or SCF (44). It has also been documented that UV-irradiation induces the production of both {alpha}-MSH and SCF in epidermal keratinocytes (10, 45, 46, 47) and increases the number of melanocytes in UV-irradiated mouse skin (48). As for cells of the immune system, {alpha}-MSH did not alter the proliferative activities of IL-1-stimulated thymocytes and Ag-stimulated lymph node cells (14). {alpha}-MSH did inhibit the IL-1-induced proliferation of murine thymocytes (15). In our experiments, {alpha}-MSH slightly but significantly increased the proliferative activity of BMCMC in the presence of IL-3 (Fig. 4Go), but not when both IL-3 and SCF were present (data not shown). The significance of this modest increase in proliferation by {alpha}-MSH is unclear. The number of mast cells in intestinal tissues is known to remarkably increase in parasite infections (49). In such condition, the production of both {alpha}-MSH and IL-3 is increased (21, 50). Therefore, it is possible that {alpha}-MSH may play a role in increasing the number of mast cells in tissues in the presence of IL-3.

Mast cells synthesize and release a variety of cytokines upon stimulation (22). Previous reports have demonstrated that {alpha}-MSH inhibits proinflammatory cytokine (e.g., IL-1ß, IL-6, and TNF-{alpha})- or chemokine (e.g., IL-8)-dependent reactions both in vitro and in vivo (13, 14, 15, 51). For example, {alpha}-MSH inhibits IL-1- or TNF-{alpha}-induced neutrophil migration in vivo. {alpha}-MSH also inhibits IL-1-, TNF-{alpha}-, or pyogen-induced edema in vivo. In addition, {alpha}-MSH affects not only the responses of the effector cells, but also the synthesis of cytokines (3, 52). There is evidence that {alpha}-MSH completely abolished mRNA expression for IL-8, TNF-{alpha}, and monocyte chemoattractant protein-1 in endotoxin-induced liver inflammatory tissue (52). In our experiments, {alpha}-MSH down-modulated the transcripts of IL-1ß, TNF-{alpha}, and lymphotactin mRNAs in BMCMC (Fig. 5Go), but it did not modulate those of IL-4 and IL-6. The mechanisms of {alpha}-MSH-induced down-modulation of these cytokines are not clear yet. But, it has been known that increased intracellular cAMP is associated with selectively decreased expression of TNF-{alpha} (53, 54). In addition, it has been reported that {alpha}-MSH inhibits the activation of NF-{kappa}B, an important factor for the induced transcription of TNF-{alpha} (55). These findings may, in part, explain the mechanism for the down-modulation of TNF-{alpha} mRNA. So far, {alpha}-MSH has been reported to down-regulate the proinflammatory reactions in some cells. At least in our experiments, it did not down-modulate all proinflammatory reactions, but did selectively down-modulate the proinflammatory cytokines in mast cells. Because mast cells are a major source of cytokines, including TNF-{alpha} and IL-4, and TNF-{alpha} plays an important role in inflammatory responses, the effects of {alpha}-MSH on mast cells may be important in regulating mast cell-dependent inflammatory conditions. In summary, these findings together indicate that {alpha}-MSH affects cytokine production in, and mediator release from, mast cells through MC-1, and may thus contribute to influencing inflammatory conditions where mast cell recruitment and activation are observed.


   Acknowledgments
 
We thank Drs. Masahiro Koshiba and Hidefumi Kojima for their technical advice, Dr. Reinhard Kage for his critical review, and Ms. Cindy Pagonis for her efforts in the preparation of the manuscript.


   Footnotes
 
1 This work was supported by the National Institute of Allergy and Infectious Diseases Division of Intramural Research. Back

2 Address correspondence and reprint requests to Dr. Dean Metcalfe, National Institutes of Health/National Institute of Allergy and Infectious Diseases/Laboratory of Allergic Diseases, Building 10/Room 11C205, 10 Center Drive MSC 1881, Bethesda, MD 20892-1881. E-mail address: Back

3 Abbreviations used in this paper: POMC, proopiomelanocortin; {alpha}-MSH, {alpha}-melanocyte stimulating hormone; MC, melanocortin; BMCMC, bone marrow cultured mast cell; NDP, Nle4, D-Phe7; BS3, Bis(sulfosuccinimidyl) subetare; DNP-HSA, DNP human serum albumin; SCF, stem cell factor. Back

Received for publication August 3, 1998. Accepted for publication June 29, 1999.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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