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POMC Gene-Derived Peptides Activate Melanocortin Type 3 Receptor on Murine Macrophages, Suppress Cytokine Release, and Inhibit Neutrophil Migration in Acute Experimental Inflammation¹

Stephen J. Getting^*, Linda Gibbs^*, Adrian J. L. Clark, Roderick J Flower^* and Mauro Perretti^2,*

^* William Harvey Research Institute, Charterhouse Square, London, United Kingdom; and Chemical Endocrinology, St. Bartholomew’s Hospital, West Smithfield, London, United Kingdom

	Abstract

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To investigate the relevance of adrenocorticotrophic hormone (ACTH) therapy in human gouty arthritis, we have tested the effect of several ACTH-related peptides in a murine model of experimental gout. Systemic treatment of mice with ACTH_4–10 (MEHFRWG) (10–200 µg s.c.) inhibited neutrophil accumulation without altering peripheral blood cell counts or circulating corticosterone levels. A similar effect was seen with - and ß-melanocyte stimulating hormones (1–30 µg s.c.). In vivo release of the chemokine KC-(detected in the lavage fluids before maximal influx of neutrophils) was significantly reduced (-50 to -60%) by ACTH_4–10. Macrophage activation in vitro, determined as phagocytosis and KC release, was inhibited by ACTH and ACTH_4–10 with approximate IC₅₀ values of 30 nM and 100 µM, respectively. The melanocortin receptor type 3/4 antagonist SHU9119 prevented the inhibitory actions of ACTH_4–10 both in vitro and in vivo. However, melanocortin type 3, but not type 4, receptor mRNA was detected in mouse peritoneal macrophages by RT-PCR. Therefore, we propose that activation of this receptor type by ACTH_4–10 and related amino acid sequences attenuates KC release (and possibly production of other cytokines) from macrophages with consequent inhibition of the host inflammatory response, thus providing a notional anti-inflammatory mechanism for ACTH that is unrelated to stimulation of glucocorticoid release.

	Introduction

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More than 10 years ago the existence of an immunological network between pro-inflammatory cytokines and pituitary neuropeptides was identified as one of the most effective ways to control the host inflammatory response (1). Cytokines such as IL-1 release into the circulation adrenocorticotrophic hormone (ACTH),³ from the anterior pituitary, which stimulates secretion of corticosteroids (cortisol in human, corticosterone in rodents) with consequent down-regulation of the inflammatory response (2). The possibility that ACTH might be produced locally in extra-pituitary tissues has been suggested by several independent studies, which have detected either ACTH immunoreactivity or the product of the pro-opiomelanocortin (POMC) gene in peripheral organs and cells (3, 4, 5). It is unclear whether the POMC gene is expressed peripherally to release corticosteroids from the adrenal gland, or POMC-derived products produce their effects locally.

Treatment of patients with ACTH is a well-known but seldom used strategy for the clinical management of gouty arthritis. Besides being efficacious in patients who do not tolerate nonsteroidal anti-inflammatory drugs or colchicine (6), systemic treatment with ACTH was found to possess therapeutic efficacy over and above that attained with corticosteroids (7), strongly suggesting the existence of a mechanism of action distinct from that achieved by direct stimulation of the adrenal gland. Based on these clinical observations, and using a recently characterized murine model of experimental gout (8), we have sought to characterize the antimigratory profile of some POMC gene products. Only a single study has so far addressed the potential anti-inflammatory activity of these peptides, with the finding that ACTH_1–39 and a nonsteroidogenic fragment, ACTH_4–10, inhibited PGE₁ generation and edema formation in rat skin (9).

We have studied the effects of the heptapeptide ACTH_4–10, ACTH_1–39, -MSH, and ß-MSH in this model and observed novel pharmacological actions of the peptides, including inhibition of neutrophil (PMN) migration and chemokine generation. We have identified the peritoneal macrophage (M) as the principal target cell and the melanocortin type 3 receptor (MC3-R) as the receptor responsible for transducing the observed effects.

	Materials and Methods

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Male Swiss Albino mice (20–22 g body weight) were purchased from Banton & Kingsman (T.O. strain; Hull, Humberside) and maintained on a standard chow pellet diet with tap water ad libitum using a 12-h light/dark cycle. Animals were used 3–4 days after arrival. Animal work was performed according to Home Office regulations (Guidance on the Operation of Animals, Scientific Procedures Act, 1986).

In vivo experimental section

Estimation of circulating corticosterone (CCS) and peripheral blood leukocytes. Mice received i.v. injections of 20 ng (4.4 pmol) ACTH, 100 µg (104 nmol) ACTH_4–10, or 30 µg (11.3 nmol) ß-MSH 2 h before blood collection by cardiac puncture following terminal anesthesia. CCS and differential leukocyte counts in plasma aliquots were determined by RIA and light microscopy, respectively, as previously described (10).

In vivo models of PMN accumulation. Crystal-induced PMN recruitment was produced using a technique recently reported by our group (8). Briefly, mice were treated i.p. with 3 mg of monosodium urate (MSU) crystals in 0.5 ml PBS, and peritoneal cavities washed at different time points with 3 ml PBS containing 3 mM EDTA and 25 U/ml of heparin. Aliquots of the lavage fluids were then stained with Turk’s solution (0.01% crystal violet in 3% acetic acid), and differential cell counts were assessed using a Neubauer haemocytometer and a light microscope. Mononuclear cells and PMN were easily identified by their different morphology and nuclear staining. Data are reported as 10⁶ PMN per mouse. Lavage fluids were then centrifuged at 400 x g for 10 min, and supernatants were stored at -20°C for biochemical determinations (see below).

Drug treatment. The peptides reported in Table I were used in this study. ACTH_4–10 (10–200 µg ranging from 10 to 208 nmol), -MSH (3–30 µg ranging from 1.8 to 16.2 nmol), and ß-MSH (1–30 µg ranging from 0.38 to 11.3 nmol) were administered s.c. 30 min before MSU crystals. The tetrapeptide HFRW (ACTH_6–9) was administered s.c. at a dose of 80 µg (10⁴ nmol) per mouse, equimolar to 100 µg ACTH_4–10. For experiments in vitro, full-length ACTH_1–39 was used at concentrations up to 100 ng/ml (22 nM). Different batches of these peptides were obtained from Sigma (Poole, Dorset, U.K.). A scrambled ACTH_4–10 peptide (sequence MGREWFH) was prepared by solid phase step-wise synthesis at The Advance Biotechnology Centre (The Charing Cross and Westminster Medical School, London, U.K.). Purity of this peptide was more than 90% as assessed by HPLC and capillary electrophoresis (data supplied by the manufacturer).

Cytokine quantification by ELISA. Murine KC, MIP-2, and TNF- levels in the peritoneal lavage fluids were determined using commercially available ELISA kits purchased from R&D Systems (Abingdon, U.K.), whereas the murine IL-1ß ELISA Cytoscreen was from BioSource International (Canarillo, CA). In brief, lavage fluids (50 µl) were assayed for each cytokine and compared with a standard curve constructed with 0–1 ng/ml of the standard cytokine. The ELISAs showed negligible (<1%) cross-reactivity with several other murine cytokines and chemokines (data supplied by the manufacturer).

In vitro experimental section

Macrophage (M) phagocytosis. Peritoneal cells (5 x 10⁶; >80% M) were collected from untreated mice by lavage and incubated in RPMI 1640 medium supplemented with 2% FCS and different concentrations of peptides in a total volume of 1 ml at 37°C for 15 min. Cells were then diluted to 1 x 10⁶/ml in Kreb’s solution before the addition of 10 µl of the reagent Fc Oxyburst Red (Molecular Probes, Eugene, OR). Uptake of Fc oxyburst Red Complexes by the peritoneal M population was monitored in real time by use of a FACScan (Becton Dickinson, Oxford, U.K.), which not only allowed the identification of the M population by forward and side scatter characteristics, but also the quantification of the fluorescence acquired in the FL-3 channel during the 200 s of reaction. Cumulative changes in fluorescence at constant time intervals were then constructed and the area under the curve measured (13).

KC release. An enriched population (>95% pure) of peritoneal M was prepared by 2-h adherence at 37°C in 5% CO₂/95% O₂ atmosphere in RPMI 1640 + 10% FCS and 1% strep-pen (Sigma). Nonadherent cells were then washed off, and adherent cells (>95% M) were incubated with the inhibitory peptides for 15 min in RPMI 1640 medium. Cells were then stimulated with 1 mg/ml MSU crystals (a concentration chosen from preliminary experiments), and the cell-free supernatants were collected 2 h later. KC levels were measured by ELISA as described above.

cAMP formation. M (1 x 10⁵) were seeded into 96-well plates as above and incubated with serum-free RPMI 1640 medium containing 1 mM isobutylmethylxanthine and different concentrations of ACTH or ACTH_4–10. The effect of the direct adenyl cyclase stimulator forskolin (3 µM) was also tested. In selected wells, the antagonist SHU9119 was added in the presence or absence of ACTH or ACTH_4–10. After 30 min at 37°C, supernatants were removed and the cells washed and lysed. cAMP levels in the lysates were determined with a commercially available enzyme immunoassay (Amersham, Little Chalfont, Buckinghamshire, U.K.) using a standard curve constructed with 0–3.0 pmol cAMP.

RT-PCR for MCRs

Peritoneal M (5 x 10⁶) enriched by 2 h adherence at 37°C in 24-well plates were lysed in 1 ml of Trizol (Life Technologies, Paisley, U.K.), and RNA was isolated according to the manufacturer’s protocol. Briefly, RNA was extracted with chloroform and isopropanol, precipitated with ethanol, and the pellet resuspended in diethyl pyrocarbonate-treated water. The yield and purity of the RNA was then estimated spectrophotometrically at 260 nm and 280 nm. Total RNA (3 µg) was used for the generation of cDNA using the T-Primed First-Strand kit (Pharmacia Biosystems Europe; St Albans, U.K.). PCR amplification reactions were then performed on aliquots of the cDNA. All PCR reactions were performed using PCR beads (Pharmacia) in a final volume of 25 µl using a Hybaid OmniGene thermal cycler (Middlesex, U.K.). The murine MC-R primer sequences were as follows: MC1-R, 5'-GTC-CAG-TCT-CTG-CTT-CCT-GG-3' and 5'-TCT- TCA-GGA-GCC-TGT-GGT-CT-3' (forward and reverse), which amplified a fragment 825 bp in length; MC3-R, 5'-GCC-TGT-CTT-CTG-TTT- CTC-CG-3' and 5'-GCC-GTG-TAG-CAG-ATG-CAG-TA-3' (forward and reverse) which amplified a fragment 820 bp in length; MC4-R, 5'-ATC-CAT-TTG-CAG-CTT-GCT-TT-3' and 5'-ATG-AGA-CAT-GAA- GCA-CAG-ACG-C-3' (forward and reverse) which amplified a fragment 445 bp in length; MC5-R, 5'ATG-AAC-TCC-TCC-TCC-ACC-CT-3' and 5'-GCA-GTA-GAC-GTT-CTG-AGG-GC-3' (forward and reverse) which amplified a fragment 810 bp in length The cycling parameters were as follows: initial denaturation for 3 min at 94°C, followed by 30 cycles of denaturation (94°C for 45 s), annealing (60°C for 30 s), extension (72°C for 1 min), and a final extension of 72°C for 10 min. Primers for murine GAPDH (14) were also used as positive controls. Amplification products were visualized by ethidium bromide fluorescence in agarose gels. Images were inverted using the Graphic Converter software (version 2.1; Lemke Software, Peine, Germany) running on a Macintosh Performa 6200.

Statistics

Data are reported as mean ± SE of n distinct observations. Statistical differences were calculated on original data by ANOVA followed by Bonferroni test for intergroup comparisons (15), or by unpaired Student’s t test (two-tailed) when only two groups were compared. A threshold value of p < 0.05 was taken as significant.

	Results

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ACTH_4–10 inhibits PMN accumulation in experimental gout

Intraperitoneal administration of MSU crystals produced an intense and long-lasting accumulation of PMN, with maximal influx in the 6–24 h time period postinjection (Fig. 1a). The peak of cell influx was preceded by a transient release of KC in the lavage fluids, which was maximal at the 2-h time point (Fig. 1b).

View larger version (16K): [in this window] [in a new window]   FIGURE 1. Time-dependent PMN accumulation and KC release in the mouse peritoneal cavity by MSU crystals. a, Mice (n = 6) received an i.p. injection of MSU crystals (3 mg in 0.5 ml sterile PBS) at time 0. Peritoneal cavities were washed at the reported time points, and PMN influx was measured by light microscopy. b, KC protein in the inflammatory exudates collected from the peritoneal cavities treated as in a. In both cases, data are mean ± SE of n = 9–28 mice per group.

View larger version (25K): [in this window] [in a new window]   FIGURE 2. Effect of ACTH4–10 and related peptides on MSU crystal induced inflammation. a, Mice were pre-treated s.c. with 100 µg ACTH4–10, 10 µg -MSH, 10 µg ß-MSH, 80 µg peptide HFRW, 100 µg scrambled peptide, or 100 µl sterile PBS 30 min before i.p. injection of MSU crystals (3 mg in 0.5 ml sterile PBS). PMN accumulation into the peritoneal cavities was measured 6 h later. Data are mean ± SE of n mice per group. *, p < 0.05 vs PBS group. b, Dose-response curves constructed for each peptide administered as in a. Data are expressed as % of control migration (8.4 x 106 PMN per mouse). *, p < 0.05 vs control group (as calculated on original values). c, Mice were pre-treated with the reported peptides and with MSU crystals as in a, and peritoneal cavities were washed 2 or 6 h later. KC content in the cell-free lavage fluids was measured by specific ELISA. Data are mean ± SE of n = 8–10 mice per group. *, p < 0.05 vs PBS group.

ACTH_4–10 inhibits M activation

ACTH, ACTH_4–10, and ß-MSH inhibited M phagocytosis as measured by flow cytometry. Again, whereas a linear concentration-response curve was observed with both ACTH and ACTH_4–10 (the latter peptide being almost 1000 times less potent than the parent molecule) (Fig. 3a), a bell-shaped curve was obtained with ß-MSH (Fig. 3b). ACTH_4–10, -MSH, and ß-MSH were also tested on KC release from adherent M: Fig. 3c shows that a significant inhibitory effect (60%) was observed when cells were incubated with ACTH_4–10 or -MSH.

View larger version (19K): [in this window] [in a new window]   FIGURE 3. Effect of ACTH4–10 and related peptides on M activation. a, Representative graph illustrating cumulative increases in the fluorescence measured in the FL3 channel by FACS following addition of Red Oxyburst to mouse peritoneal M. ACTH (100 ng/ml), ACTH4–10 (100 µg/ml), or ß-MSH (30 µg/ml) were incubated with 5 x 106 cells for 15 min before the addition of the immunocomplexes. b, Concentration-response curves were constructed for each peptide. Data are mean ± SEM of n = 2–6 distinct experiments performed in duplicate. *, p < 0.05 vs control group (as calculated on original values). c, ACTH4–10 (100 µg/ml), -MSH (10 µg/ml), or ß-MSH (10 µg/ml) were added to adherent M for 30 min before stimulation with 1 mg/ml MSU crystals. KC levels in 2-h cell-free supernatants were determined by ELISA. Data are mean ± SEM of n = 8 distinct experiments. *, p < 0.05 vs PBS group.

The ability of ACTH_4–10 to attenuate MSU crystal-induced PMN recruitment and KC production were prevented by treatment of mice with the MC3/4-R antagonist SHU9119 (11). Both parameters were essentially unaltered by SHU9119 treatment alone, whereas the MC3/4-R antagonist reduced the inhibitory action of ACTH_4–10 in a dose-dependent manner, with total inhibition of the effect seen at the dose of 10 µg i.p. (Fig. 4). The nonselective MC-R antagonist S110 (12) did not affect ACTH_4–10 inhibition of PMN influx and KC release.

View larger version (40K): [in this window] [in a new window]   FIGURE 4. SHU9119 prevents ACTH4–10 inhibition of MSU crystal peritonitis. Mice received 100 µg s.c. ACTH4–10 with or without 3 µg or 10 µg i.p. SHU9119, or 10 µg i.p. S110, 30 min before i.p. injections of MSU crystals (3 mg in 0.5 ml sterile PBS). Peritoneal cavities were washed 6 h later, and the number of accumulated PMN (a) or the content of KC protein (b) in the lavage fluids was determined. Data are mean ± SE of n = 6–12 mice per group. *, p < 0.05 vs control group (no antagonist).

View larger version (37K): [in this window] [in a new window]   FIGURE 5. SHU9119 prevents ACTH4–10 inhibition of KC release from activated M. ACTH4–10 (100 µg/ml), ACTH (100 ng/ml), -MSH (10 µg/ml), or ß-MSH (10 µg/ml) were added to adherent M with or without 10 µg/ml SHU9119. Cells were stimulated 30 min later with 1 mg/ml MSU crystals, and KC protein was measured in the medium at 2 h by ELISA. Data are mean ± SEM of n = 4–15 distinct samples. *, p < 0.05 vs appropriate PBS group.

View larger version (30K): [in this window] [in a new window]   FIGURE 6. MC3-R expression and activation in murine M. a, Concentration-response curve for ACTH and ACTH4–10 in inducing cAMP accumulation in adherent mouse M. Also reported are the cAMP concentrations attained with forskolin and MC3/4-R agonist MTII. Some cells were coincubated with ACTH or ACTH4–10 plus 10 µg/ml (9 µM) SHU9119. Dashed line indicates basal cAMP formation in nonstimulated M. Data are mean ± SEM of n = 4–8 separate determinations. *, p < 0.05 vs basal cAMP expression (60 ± 12 fmol/well, n = 8). b, RT-PCR showing the presence of specific products for MC1-R (825 bp), MC3-R (820 bp), MC4-R (445 bp), and MC5-R (810 bp) in mouse genomic DNA (Gen) or in cDNA prepared from murine M. GAPDH (500 bp) was detected as positive controls. The arrow indicates the presence of MC3-R in the M preparation. Gel depicts a representative of three PCR runs showing identical results. m, Markers (1353, 872, 603, and 310 bp).

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
In the present study we have investigated the antimigratory action of POMC gene derived peptides containing the core ACTH_4–10 sequence. The mechanism of action we propose is that activation of MC3-R on mouse peritoneal macrophages by POMC gene-derived peptides reduces the release of pro-inflammatory cytokines and subsequent recruitment of PMN. On this basis, we suggest that agonism at MC3-R may be a novel way to control the cellular component of the host inflammatory response.

This study was prompted by the observation that treatment with ACTH possesses a unique therapeutic profile in the management of human gouty arthritis which suggest the existence of mechanism(s) of action besides glucocorticoid release from the adrenal glands (6, 7). The heptapeptide ACTH_4–10 is known to lack any glucocorticoid stimulating action (9), and we confirmed that ACTH_4–10 did not stimulate plasma CCS levels and consequent neutrophilia in our current model. We have previously reported that ACTH-induced neutrophilia is totally dependent on CCS release (10).

Intraperitoneal injection into the mouse of MSU crystals produced an intense and long-lasting accumulation of PMN into the peritoneal cavity, which we recently characterized in terms of the role of resident cells and a requirement for adhesion molecules (8). We now show that a panel of chemotactic cytokines and chemokines is also released during this inflammatory reaction, and in our study we chose to monitor the CXC chemokines KC and MIP-2 (16), and the pro-inflammatory cytokines TNF- and IL-1ß. All these mediators have been shown to be produced in other models of experimental (17, 18) and human (19, 20) gouty arthritis.

Systemic administration to mice of ACTH_4–10 inhibited MSU crystal-induced PMN accumulation, and this was associated with a reduction in KC and IL-1ß release in the inflammatory exudates. In this set of experiments the effects of -MSH and ß-MSH (which contain the ACTH_4–10 sequence) were also shown to suppress PMN influx and KC release in vivo. Whereas this is not surprising for -MSH (for a recent review, see 21), we believe this is the first time that an anti-inflammatory activity has been described for ß-MSH. Importantly, ß-MSH was also unable to modify circulating CCS and PMN levels. As for other in vitro and in vivo experimental systems (22, 23), bell-shaped dose responses were constructed for -MSH and ß–MSH. Further studies are required to investigate whether a catabolic pathway may become activated at higher doses of the melanocortins. The effect of ACTH_4–10 was validated further by using as a control a scrambled sequence: this peptide was totally ineffective on the two parameters under observation in this model of experimental gout.

The next step was to identify the cellular target(s) responsible for the observed inhibitory actions of ACTH_4–10. In view of the relatively large body of literature which relates POMC gene-derived peptides and ACTH to the M (24, 25, 26, 27, 28), we tested the hypothesis that the resident peritoneal M could be targeted by these peptides. Mouse peritoneal M have been shown to be deactivated by ACTH, such that IFN--mediated tumoricidal activity (23) and latex beads phagocytosis (24) were blocked by micromolar concentrations of this hormone. Our preliminary experiments indicated that POMC gene-derived peptides were inactive in experiments where PMN or mast cell activation was measured (data not shown). Both ACTH and ACTH_4–10 inhibited M phagocytosis as assessed by FACS analysis. Full concentration-response curves could be constructed for the two peptides, and the shorter fragment was almost 1000 times less potent than the parent molecule. ß-MSH was also effective in this assay, again producing a bell-shaped curve.

More relevant to inflammation itself, the KC release from M stimulated in vitro with MSU crystals was also determined. ACTH_4–10 also inhibited this parameter of M activation, whereas no such effect was found with ß-MSH. The reason for this discrepancy is at the moment obscure. -MSH, used as an internal positive control in view of its reported effect on M activation (21), was also found to inhibit KC release. These data complement a previous report which showed -MSH to inhibit KC mRNA expression in the mouse liver during endotoxin-induced inflammation (30), and adds KC to the list of M-derived mediators whose release is affected by this melanocortin (21).

Five MC-R have to date been identified and cloned. MC1-R binds -MSH and ACTH preferentially, whereas the MC2-R is highly selective for ACTH and is expressed predominantly in the adrenal gland (31, 32). Human and murine MC3-R, MC4-R, and MC5-R (33, 34, 35) bind to all these peptides with varying affinities (31, 32). The lack of selective drugs has hampered a full pharmacological characterization of these receptors so far. Fan et al. (11) have recently described two compounds, MTII and SHU9119, obtained by cyclization and amino acid substitution of the ACTH_4–10 sequence, as a potent MC3/4-R agonist and antagonist, respectively. A recent study has shown that dynorphin-A (6, 7, 8, 9, 10, 11, 12), or S110, acts as an nonselective antagonist to MC-R (12). When tested in our experimental systems, S110 did not affect ACTH_4–10 inhibition of PMN migration and KC release in vivo or from activated M in vitro (S.J.G., unpublished data), but the MC3/4-R antagonist SHU9119 was highly effective. When used at 10 µg i.p. (a dose already used in vivo in the mouse) (11), which is 10 times lower than ACTH_4–10 on a molar basis), SHU9119 inhibited the in vivo effects of the peptide in the MSU crystal peritonitis, whereas a partial inhibition was seen at the lowest dose of 3 µg/mouse. Importantly, SHU9119 reversed the inhibitory effect of ACTH, ACTH_4–10 and -MSH on KC release from activated M. In the latter assay we also tested the selective agonist MTII, finding a reduction in MSU crystal-induced release of the CXC chemokine. These data are strongly suggestive of an MC3-R and/or MC4-R as the molecular target(s) for the biologically activities of ACTH_4–10 and related peptides described here.

These data do not allow us to pin-point which of these two MC-R was actually responsible for the observed effects: in fact, the core sequence (ACTH_4–10) binds MC3-R and MC4-R with almost equal affinity (36). As discussed above, the efficacy of SHU9119 also does not allow receptor discrimination (11, 37). To unravel this aspect, we moved to PCR analysis of the mRNA expressed in M. The specific product for the MC3-R, but not for the MC4-R (and indeed the MC1-R or MC5-R), could be found in resting mouse peritoneal M. Thioglycollate injection before M collection did not change this profile of MC-R expression (S.J.G., unpublished observation). In addition, MC2-R mRNA was also absent in basal or elicited M (data not shown). The specificity of MC3-R primers was verified by using the murine genomic DNA preparation, and also by testing mouse brain tissue (data not shown and 38).

Finally, since MC3-R activation leads to intracellular accumulation of cAMP (34), we tested formation of this second messenger in mouse peritoneal M. Following cell incubation with ACTH or ACTH_4–10, intracellular cAMP accumulated in a concentration-dependent manner. Again ACTH was 1000 times more potent than the heptapeptide on a molar basis but, as in the case of M phagocytosis, ACTH_4–10 was able to produce a degree of inhibition similar to that attained with the parent molecule. Both ACTH and ACTH_4–10, but not forskolin, induced cAMP formation in M and was blocked by SHU9119. Overall these data indicate that MC3-R is not only expressed in murine M but is fully functional such that cAMP formation occurs after agonist activation. These observations link well with the known ability of ACTH to bind mouse leukocytes and cause intracellular accumulation of cAMP (39).

There is a resurgence of interest in the peripheral expression of the POMC gene (40); for instance, both rodent splenocytes (27, 41) and human leukocytes (42, 43) express POMC gene products. Basal expression of the POMC gene has been detected in rat M (40), and the POMC gene product seems to be normally processed to produce immunoreactive ACTH (4). We report here that peptides containing the ACTH_4–10 sequence suppress PMN accumulation in acute inflammation in a CCS-independent manner. These data identify MC3-R as the molecular target for these peptides, and together with other studies (4, 40, 41, 42, 43), may suggest the existence of a novel anti-inflammatory loop based on ACTH and MC3-R that may operate to down-regulate the acute inflammatory response or the acute phases of chronic inflammation.

	Acknowledgments

	Footnotes

 
¹ This work was supported by Grant PO537 from the Arthritis (U.K.). R.J.F. is a Principal Fellow of the Wellcome Trust, whereas M.P. is a Postdoctoral Fellow of the ARC.

² Address correspondence and reprint requests to Dr. Mauro Perretti, William Harvey Research Institute, St. Bartholomew’s and Royal London SMD, Charterhouse Square, London EC1 M 6BQ, U.K. E-mail address:

³ Abbreviations used in this paper: ACTH, adrenocorticotrophic hormone; POMC, pro-opiomelanocortin; MSH, melanocortin stimulating hormone; M, macrophage; MC-R, melanocortin receptor; CCS, corticosterone; PMN, neutrophil; MIP, macrophage inflammatory protein; MSU, monosodium urate crystal; MTII, Ac-Nle⁴-c[Asp⁵,D-Phe⁷,Lys¹⁰]NH₂ ACTH_4–10; SHU9119, Ac-Nle⁴-c[Asp⁵,D-²Nal⁷,Lys¹⁰]NH₂ ACTH_4–10; S110, p-methoxybenzoyl-Arg-Arg-Ile-Arg-Pro-Lys-D-Leu-NH₂.

Received for publication November 4, 1998. Accepted for publication April 6, 1999.

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