Activation of melanocortin type 3 receptor as a molecular mechanism for adrenocorticotropic hormone efficacy in gouty arthritis




To test the hypothesis that local activation of melanocortin receptor(s) by adrenocorticotropic hormone (ACTH) could be responsible, at least in part, for its efficacy in human gouty arthritis.


Monosodium urate monohydrate (MSU) crystals were administered into rat knee joints either alone or with ACTH or a selective melanocortin type 3 receptor (MC3-R) agonist. Neutrophil migration, arthritis score, increases in joint size, and cytokine levels were measured over time. MC3-R expression on rat knee joint macrophages was monitored by electron microscopy and intracellular accumulation of cyclic adenosine monophosphate.


MSU crystals produced a knee joint inflammation that was time dependent and was characterized by cell influx and cytokine release that was sensitive to treatment with classic anti-arthritic drugs (indomethacin, colchicine, dexamethasone). Local, but not systemic, ACTH had an antiinflammatory effect in normal rats, a dose that did not alter circulating corticosterone (5 μg). This treatment was also effective in adrenalectomized rats. Rat knee joint macrophages expressed functional MC3-R. The MC3-R antagonist (SHU9119, 10 μg) blocked ACTH antiinflammatory actions, whereas antiinflammatory activity was retained with a selective MC3-R agonist (γ2-melanocyte–stimulating hormone).


This research provides evidence for a separate mechanism of action of ACTH in experimental gouty arthritis and points to a novel antiinflammatory target (selective agonists at MC3-R) for clinical management of human gouty arthritis and possibly other chronic inflammatory conditions.

Gouty arthritis is a disease caused by the deposition of monosodium urate monohydrate (MSU) crystals in the joint (1, 2). Its incidence is higher in men than in women younger than the age of 60 (3), a finding that was first observed by Hippocrates, who described its rarity in premenopausal women and eunuchs. Clinically, gout is associated with edema and erythema of the joints, together with severe pain; characteristically, urate crystals are present in lavage fluids aspirated from gouty joints (2). There is intense infiltration of bloodborne neutrophils into the joint space (2, 4), likely due to the release of an array of cytokines and chemokines, including interleukin-1 (IL-1) and IL-8 (5–7).

Gout is currently managed either with nonsteroidal antiinflammatory drugs (NSAIDs) or with colchicine. NSAIDs partially relieve the symptoms of gout and provide an analgesic action. However, NSAIDs produce characteristic side effects, especially in patients with preexisting renal insufficiency, peptic ulceration, and gastric problems. It is not yet known if selective cyclooxygenase inhibitors will have a better safety profile in gout patients. Colchicine, a compound that binds to microtubular proteins and interferes with the function of mitotic spindles, potently decreases leukocyte migration and phagocytosis (8). It is also effective in controlling short-term episodes of human gouty arthritis. However, the safety margin with this drug is low and the side effects associated with colchicine treatment (nausea, vomiting, profuse diarrhea, and gastrointestinal hemorrhage) (9) can be so intense as to limit its appeal to patients.

Studies conducted in the late 1940s characterized the efficacy of systemic treatment with adrenocorticotropic hormone (ACTH1–39, full-length polypeptide) in human gouty and rheumatoid arthritis (10, 11). A more recent and controlled clinical trial showed that intramuscular administration of ACTH1–39 controlled human gout. The efficacy of this treatment was so pronounced that Ritter and colleagues suggested the existence of other mechanism(s) of action, besides direct activation of the adrenal gland and consequent glucocorticoid release (12). A separate study has also demonstrated the benefits of intravenous administration of ACTH in gout patients in whom colchicine and NSAIDs were contraindicated (13). All these clinical observations suggest the existence of another molecular mechanism for ACTH antigout effects.

ACTH1–39 is the prototype of melanocortin peptide, produced by the cleavage of the pro-opiomelanocortin gene product (14). Several studies have suggested that melanocortins (in particular, α-melanocyte–stimulating hormone [α-MSH]) are endogenous down-regulators of the inflammation process (for review, see ref. 15). Melanocortin peptides exert their effects via 5 distinct melanocortin receptors (MC-Rs), which have been cloned and partially characterized (16). All MC-Rs belong to 7 transmembrane domain G-protein–linked receptors and are positively coupled to adenylate cyclase such that their activation causes intracellular accumulation of cAMP (17, 18). In a murine model of MSU crystal–induced peritonitis, we demonstrated the presence of MC3-R, but not MC1-R, MC2-R, MC4-R, or MC5-R messenger RNA in peritoneal macrophages. We have proposed that activation of this receptor by ACTH4–10 and other melanocortins devoid of adrenal-stimulating properties (19) reduced cytokine release and attenuated peritoneal inflammation (19–21). The present study was undertaken to test the hypothesis that a specific MC-R could be targeted in the joint by ACTH1–39 to reduce experimental arthritis.


In vivo experiment.

Naive and adrenalectomized male Sprague-Dawley rats (220–270 gm body weight) were purchased from Charles River (Kent, UK) and maintained on a standard chow pellet diet. Rats with intact adrenal glands drank tap water, whereas adrenalectomized animals received 0.9% NaCl + 1% glucose. All animals were exposed to a 12-hour light/dark cycle. Animal work was carried out under license from the Home Office in accordance with the Animals (Scientific Procedures) Act of 1986.

Experimental model of gouty arthritis.

MSU crystals were prepared using a previously described method (22). Briefly, 0.03M MSU was prepared at pH 7.5 by dissolving uric acid and sodium hydroxide and filtering with an Acropor membrane filter (AN-3000, 3 μM; Gelman, Ann Arbor, MI). Sodium chloride (0.1M) was added to accelerate and improve the uniformity of the crystals. The crystals were characterized by x-ray diffraction (Rigaku Geirflex D/MAX; Rigaku, Tokyo, Japan), by examination under phase and polarizing microscopy, and by scanning electron microscopy. The crystals showed triclinic morphologic characteristics, and the dimensions were 10 × 1 × 1 μM to 25 × 1.5 × 1.5 μM. The preparation was free of endotoxin, as determined by the Limulus amebocyte cell lysate assay (Whittaker Bioproducts, Walkersville, MD).

Rats were anesthetized with halothane, and MSU crystals (10–50 mg/ml) were injected into the synovial space of the right knee in a volume of 50 μl sterile phosphate buffered saline (PBS) (equivalent to 0.5–2.5 mg/joint). The left knee was injected with sterile PBS alone. At 2–96 hours, the animals were killed and the knee joint was exposed to determine the arthritis score and the size of the joint according to the method of Yang et al (23). Both knee joints were excised and lavaged with 1 ml PBS containing EDTA (3 mM) and heparin (25 units/ml). In a final set of experiments, rats received MSU crystals or PBS at time 0 and at 72 hours. Animals were killed 24 hours later (96 hours after the first injection) and the joint was lavaged as described before. Lavage fluids were then centrifuged at 400g for 10 minutes and supernatants were stored at −20°C before biochemical determinations (see below). The resulting pellet was stained with Turk's solution, and leukocytes were identified as >95% neutrophils by light microscopy determination of characteristic nuclear morphology in a Neubauer hemocytometer. For each rat, data are reported as 105 neutrophils per joint, corrected for the PBS value as measured in the left knee joint. Similarly, joint size is reported as the increase in the MSU-injected joint above the value measured in the PBS-treated joint.

Drug treatment.

Melanocortin peptides were purchased from either Sigma-Aldrich (Poole, UK) or Bachem (Saffron Walden, UK), stored at −20°C prior to use, and freshly diluted in sterile PBS (pH 7.4). The γ2-MSH (YVMGHFRWDRFG; 25 μg) and ACTH1–39 (1–100 μg) were administered either subcutaneously (SC) or intraarticularly (IA). Systemic treatments were given 30 minutes prior to MSU crystal injection, whereas local treatment was given in combination with MSU crystals to avoid unnecessary risk of joint damage through repetitive injections. In some experiments, the melanocortin agonists were tested in the presence of the MC3/4-R antagonist SHU9119 (Ac-Nle4-c[Asp5,D-2Nal7,Lys10]NH2 ACTH4–10) (24), of which 9 nmoles (10 μg) was injected into the joint. The doses of melanocortin agonists and antagonist used were extrapolated from our previous studies of the mouse (19–21). Dexamethasone and indomethacin were suspended in polyethylene glycol 400 (BDH Chemicals, Poole, UK) and given orally at doses of 1 mg/kg and 3 mg/kg, respectively. Colchicine was given intraperitoneally at 3 mg/kg, with vehicle being sterile PBS. Local MSU crystal injection was done 60 minutes later for dexamethasone and indomethacin, and 30 minutes later for colchicine.

Rat IL-1β and IL-6 levels in the knee joint lavage fluids were determined using commercially available enzyme-linked immunosorbent assay (ELISA) kits purchased from R&D Systems (Abingdon, UK). Briefly, lavage fluid aliquots (50 μl) were assayed for each cytokine and compared with a standard curve constructed with 0–2 ng/ml for IL-1β and 0–1 ng/ml for IL-6. The ELISA showed negligible cross-reactivity with several other rat cytokines (data supplied by the manufacturer).

Naive rats received ACTH1–39 either at 100 μg SC or 5 μg IA 2 hours before blood collection by cardiac puncture under terminal anesthesia. Corticosterone (CCS) concentration in plasma aliquots was determined by radioimmunoassay, using a commercially available kit (ICN Pharmaceuticals, Basingstoke, UK) as previously described (19).

In vitro experiment.

We used a protocol recently validated with human neutrophils (25). Knee joints of naive rats were washed as described above, and cells were fixed with a mixture of freshly prepared 3% (weight/volume) paraformaldehyde and 0.05% (volume/volume) glutaraldehyde in PBS (pH 7.2) for 4 hours at 4°C, washed briefly in PBS, and transferred to a solution of 2.3M sucrose (in PBS) at 4°C overnight. The cryoprotected cells were slam-frozen (Reichert MM80E; Leica, Milton Keynes, UK), freeze-substituted at −80°C in methanol for 48 hours, and embedded at −20°C in LRGold acrylic resin (London Resin, Reading, UK) in a Reichert freeze-substitution system. Ultrathin sections (50–80 nm) were prepared using a Reichert Ultracut-S ultratome and incubated at room temperature for 2 hours with a polyclonal goat anti–MC3-R antibody (dilution 1:200; Santa Cruz Biotechnology, Santa Cruz, CA) or goat anti–MC3-R preabsorbed with MC3-R blocking peptide (20 μg; Santa Cruz Biotechnology), followed by protein A linked to 15 nm gold (British Biocell, Cardiff, UK) for 1 hour. As an additional negative control, sections were incubated with nonimmune goat serum (1:200; Sigma-Aldrich) in place of the primary antibody. The serum and antiserum were diluted in 0.1M phosphate buffer containing 0.1% egg albumin. After immunolabeling, sections were lightly counterstained with uranyl acetate and lead citrate and examined with a JEOL 1010 transmission electron microscope (JEOL, Peabody, MA).

Intracellular accumulation of cAMP.

Knee joint macrophages (2 × 105) or rat fibroblast-like synoviocytes (2 × 105; immortalized by retrovirus SV40-U19tsA58 infection) (a generous gift of Professor P. G. Winyard, Bone & Joint Unit, Bart's and The London, Queen Mary University of London, London, UK) were seeded into 24-well plates. Macrophages were enriched by a 2-hour adhesion step at 37°C followed by a wash with warm RPMI 1640 medium. Cells were then incubated in serum-free RPMI 1640 medium containing 1 mM isobutylmethylxanthine (Sigma-Aldrich) and with different concentrations of ACTH1–39 (2.2–66 nM or 10–300 ng/ml) or the direct adenylate cyclase activator forskolin (3 μM; Sigma-Aldrich). In some cases, the mixed MC3/4-R antagonist SHU9119 was also added at 9 μM. As in the in vivo studies, drug concentrations were selected from previous experiments conducted with murine macrophages (19–21). After 30 minutes at 37°C, cells were washed and lysed, and cAMP levels in cell lysates were determined with an enzyme immunoassay (Amersham, Little Chalfont, UK) using a standard curve constructed with 0–3,200 fmoles/ml cAMP.

Statistical analysis.

Values for the in vivo experiments were reported as the mean ± SEM of n distinct observations. In vitro experiments were repeated n times in triplicate or quadruplicate. Statistical differences among means and SEM were calculated on original data by analysis of variance followed by the Bonferroni adjustment for intergroup comparisons or by Student's unpaired t-test when only 2 groups were compared (26). P values less than 0.05 were considered significant.


Characterization and validation of MSU crystal–induced joint inflammation.

IA injection of MSU crystals into the rat knee joint was used to mimic the etiologic cause of human gouty arthritis (27). MSU crystal injection produced an intense and reproducible accumulation of bloodborne neutrophils into the rat joint. Neutrophil migration was found to occur in a dose- and time-dependent manner (Figures 1 and 2). The dose-response curve was relatively steep, with a maximum neutrophil migration of 3.48 ± 0.42 × 105 (mean ± SEM; n = 10) at the 1-mg dose. A lesser effect was observed at the highest dose tested (2.5 mg) (Figure 1A), probably due to the toxic action of the crystals. Arthritis scores and changes in joint thickness followed a similar pattern (Figures 1B and C). The MSU crystal dose of 1 mg was selected for subsequent experiments.

Figure 1.

Dose-response characterization of monosodium urate monohydrate (MSU) crystal–induced inflammation into rat knee joint cavities. At time 0, rats received 50 μl intraarticularly of sterile phosphate buffered saline (PBS) alone in the left joint (PBS group) or PBS supplemented with different doses of MSU crystals in the right joint. Neutrophil influx (A), arthritis score (B), and increase in joint size (C) were quantified 16 hours later. Values are the mean and SEM of 10 rats per group. All doses of MSU produced significant (∗ = P < 0.05) neutrophil accumulation and increase in arthritis score and joint size when compared with the PBS-injected joint.

Figure 2.

Time course characterization of MSU crystal–induced inflammation in rat knee joint cavities. At time 0, rats received 50 μl intraarticularly of sterile PBS alone in the left joint (PBS group; open circles) or PBS supplemented with 1 mg MSU crystals (solid squares) in the right joint. Neutrophil influx (A), arthritis score (B), increase in joint size (C), and cytokine levels (D; open circles for interleukin-1β [IL-1β] and solid squares for IL-6) were quantified at the reported time points. Values are the mean ± SEM of 10 rats per group. MSU crystals produced significant (∗ = P < 0.05) neutrophil accumulation, increase in arthritis score, joint size, and cytokine levels when compared with the PBS-injected joint. See Figure 1 for other definitions.

In the time course experiments, a rapid influx of neutrophils was observed in response to MSU crystal injection, with the highest rate of influx between 2 hours and 16 hours (∼0.2 × 105 neutrophils per hour) (Figure 2A). PBS injection provoked a negligible cell influx (less than one-tenth at 6 hours and 16 hours). Arthritis scores and changes in joint swelling exhibited a similar, although not identical, profile (Figures 2B and C).

The peak of cell influx was preceded by a transient release of IL-1β and IL-6 in the lavage fluids, which was maximum at the 6-hour time point, with 107 ± 37 and 123 ± 37 pg/ml for IL-1β and IL-6, respectively (mean ± SEM; n = 10) (P < 0.05 compared with the 2-hour time, since at time 0 values were below detection limits for either cytokine; Figure 2D). PBS-injected joints yielded cytokine levels below the detection limit (data not shown). All changes in inflammation parameters subsided by 48–72 hours after MSU crystal injection. The 16-hour time point was chosen for subsequent experiments since it was characterized by high values for all markers of inflammation; hence, it was more suitable to study potential drug modulation.

The effect of known antiarthritis drugs was tested next. Table 1 shows data obtained with systemic treatment of rats with active doses (see Materials and Methods) of dexamethasone, indomethacin, or colchicine. Dexamethasone, indomethacin, and colchicine significantly reduced neutrophil accumulation by 86%, 49%, and 48%, respectively, with a similar effect on arthritis score (n = 5). The first 2 treatments (steroids and NSAIDs) significantly attenuated the increase in joint size, with a reduction of 62% and 44% (P < 0.05; n = 5), while colchicine caused a nonsignificant 20% reduction in joint size. However, colchicine produced a significant reduction (55%) of IL-1β content and a near-abrogation of IL-6 levels (Table 1).

Table 1. Effect of known antirheumatic drugs on markers of inflammation in monosodium urate monohydrate (MSU) crystal–induced rat knee joint inflammation*
TreatmentNeutrophils, 105 per jointArthritis score, unitsChange in joint size, mmIL-1β, pg/mlIL-6, pg/ml
  • *

    Rats were pretreated with either vehicle (PEG400), dexamethasone (1 mg/kg), or indomethacin (3 mg/kg) orally 1 hour prior to treatment with MSU crystals (1 mg) intraarticularly (IA). In separate experiments, phosphate buffered saline or colchicine (3 mg/kg) was given intraperitoneally 30 minutes prior to 1 mg MSU crystals given IA. Knee joint cavities were lavaged 16 hours later and cytokine contents in cell-free supernatants were analyzed using specific enzyme-linked immunosorbent assays. Values are the mean ± SEM of 5 rats per group; values in parentheses are the percent inhibition compared with vehicle. IL-1β = interleukin-1β; NT = not tested.

  • P < 0.05 versus vehicle group.

Vehicle4.24 ± 0.722.90 ± 0.330.77 ± 0.09NTNT
Dexamethasone0.60 ± 0.27 (86)0.80 ± 0.37 (72)0.29 ± 0.15 (62)NTNT
Indomethacin2.16 ± 0.44 (49)2.0 ± 0.32 (31)0.43 ± 0.05 (44)NTNT
PBS3.0 ± 0.622.75 ± 0.250.75 ± 0.10470 ± 66145 ± 38
Colchicine1.55 ± 0.55 (48)1.38 ± 0.13 (50)0.60 ± 0.18 (20)213 ± 24 (55)25 ± 9 (83)

Local and systemic ACTH inhibits neutrophil accumulation into rat knee joints, with a divergent effect of CCS.

Treatment of rats with SC injection of ACTH1–39 significantly reduced neutrophil influx, from 1.93 ± 0.11 × 105 (mean ± SEM; n = 10) to 0.49 ± 0.11 × 105 (−74%; P < 0.05) (mean ± SEM; n = 10) at a dose of 100 μg (22 nmoles). A lower dose of ACTH (20 μg SC; equivalent to 4.4 nmoles) caused a more modest inhibition of neutrophil accumulation (41%; P < 0.05) (Figure 3A). Treatment with 100 μg ACTH SC also attenuated the increase in joint size (−70%) (Figure 3B) and arthritis score (−55%; P < 0.05) (data not shown) produced by MSU crystals. Local IA administration of ACTH1–39 (5 μg; equivalent to 1.1 nmoles) was also effective in reducing neutrophil influx (42% inhibition) and joint swelling (52% inhibition) (Figures 3A and B), as well as arthritis score (−38%; P < 0.05) (data not shown).

Figure 3.

Systemic and local adrenocorticotropic hormone (ACTH) efficacy in joint inflammation in naive rats. Rats received ACTH1–39 subcutaneously (SC) 30 minutes prior to, or intraarticularly (IA) at the same time as, MSU crystals (1 mg). A, Neutrophil accumulation in rat knee joints, assessed at 16 hours. B, Increase in right knee joint size at 16 hours. Values are the mean and SEM of 10 rats per group. ∗ = P < 0.05 versus vehicle group. See Figure 1 for other definitions.

To dissect the mechanism of the ACTH1–39 antiinflammatory effect, we next compared the effect of the 2 routes of administration on blood CCS concentration. Consistent with results of previous studies (28, 29), systemic ACTH1–39 caused a marked increase in circulating CCS as measured at the 2-hour time point, while IA injection of 5 μg ACTH1–39 was inactive (Figure 4). Finally, whereas antiinflammatory activity in adrenalectomized rats was retained with 5 μg IA ACTH1–39, this was not the case with 100 μg SC ACTH1–39. Table 2 illustrates the results of these experiments, in which 5 μg IA ACTH1–39 was found to be particularly effective, with inhibitions of 82%, 88%, and 75% on cell influx, joint swelling, and arthritis score, respectively. Similar degrees of inhibition were also noted with respect to IL-1β and IL-6 levels (Table 2). To investigate this putative novel mechanism that operates at a local level, we used IA ACTH1–39 injection as a route of administration in subsequent experiments.

Figure 4.

Effect of ACTH on plasma corticosterone levels in naive rats. Rats received 100 μg SC or 5 μg IA of ACTH1–39 at time 0. Two hours later, blood was obtained from untreated rats as well as ACTH-treated rats to measure plasma corticosterone concentration. Values are the mean and SEM of 5–10 rats per group. ∗ = P < 0.05 versus untreated group. See Figure 3 for definitions.

Table 2. Divergent efficacy of local and systemic administration of adrenocorticotropic hormone 1–39 (ACTH1–39) on monosodium urate monohydrate (MSU) crystal–induced knee joint inflammation in adrenalectomized rats*
TreatmentNeutrophils, 105 per jointArthritis score, unitsChange in joint size, mmIL-1β, pg/mlIL-6, pg/ml
  • *

    Adrenalectomized rats received ACTH1–39 subcutaneously (SC) 30 minutes prior to, or intraarticularly (IA) at the same time as, MSU crystals (1 mg). Knee joint cavities were lavaged 16 hours later and cytokine levels in cell-free supernatants were analyzed using enzyme-linked immunosorbent assays. Values are the mean ± SEM of 5 rats per group; values in parentheses are the percent inhibition compared with vehicle. IL-1β = interleukin-1β.

  • P < 0.05 versus vehicle group.

Vehicle2.08 ± 0.462.0 ± 0.160.51 ± 0.1616.5 ± 3.3129.3 ± 2.1
ACTH, 5 μg IA0.38 ± 0.07 (82)0.5 ± 0.16 (75)0.06 ± 0.04 (88)5.5 ± 2.2 (67)46.1 ± 3.2 (64)
ACTH, 100 μg SC1.72 ± 0.57 (17)1.8 ± 0.3 (10)0.33 ± 0.05 (35)42.1 ± 14.3 (0)82.4 ± 14.7 (36)

Identification of the MC-R involved in local ACTH actions.

When knee joint cells were analyzed using electron microscopy, positive immunoreactivity was consistently detected on the macrophages. MC3-R immunolabeling was predominantly located on the plasma membrane, possibly with particular gold particles accumulating on membrane protrusions (Figure 5A). Gold immunolabeling was specific for MC3-R insofar as it was abrogated when the primary antibody was preabsorbed with the blocking peptide (Figure 5B). As a final control, cells stained with nonimmune goat IgG did not display gold immunolabeling on their plasma membrane (Figure 5C).

Figure 5.

Expression of melanocortin type 3 receptor (MC3-R) on rat knee joint macrophages as detected by electron microscopy. A, Rat knee joint macrophages were stained with a selective anti–MC3-R antibody (final dilution 1:200) and analyzed by electron microscopy. Immunoreactivity was predominantly localized on the plasma membrane, with a minor degree of staining also detected in the cytosol. Characteristic clusters of gold particles (diameter 15 nm) were visualized on plasma membrane protrusions (arrowheads). B, Lack of gold immunolabeling on rat knee joint macrophages when the anti–MC3-R antibody was preabsorbed with the blocking peptide. C, Similar negative staining was obtained when cells were stained with nonimmune goat IgG (1:200). Photomicrographs are representative of 10 distinct cells. (Original magnification × 16,000.)

To ascertain if MC3-R on knee joint macrophages was functionally active, the effect of ACTH1–39 on cAMP accumulation was determined. Macrophage incubation with ACTH1–39 induced a concentration-dependent intracellular accumulation of cAMP, with an ∼50% maximum response concentration (EC50) of 12.5 nM. ACTH1–39 (22 nM) caused an increase of 616% in cAMP compared with vehicle, with 696.7 ± 78.3 fmoles/well being detected (mean ± SEM; n = 4) (P < 0.05) (Figure 6A). This effect was blocked by the mixed MC3/4-R antagonist SHU9119, which reduced the level to 183.9 ± 38.4. This compound did not affect cAMP accumulation produced by the direct adenylate cyclase activator forskolin (Figure 6A). ACTH1–39 was inactive in the rat fibroblast-like synoviocyte cell line, whereas forskolin retained its stimulatory activity (Figure 6B).

Figure 6.

Effect of adrenocorticotropic hormone 1–39 (ACTH1–39) on intracellular cAMP accumulation. A, cAMP accumulation in rat knee joint macrophages stimulated with ACTH1–39 (solid squares) or 3 μM forskolin. The effect of the antagonist SHU9119 (SHU; 9 μM) (open square) was also determined. Values are the mean ± SEM of 4 experiments. ∗ = P < 0.05 versus basal values (55 ± 25 fmoles/well). SHU effect on ACTH1–39 was also significant (∗ = P < 0.05). B, Same as in A, but ACTH1–39 and forskolin were incubated with rat fibroblast-like synoviocytes. Values are the mean ± SEM of 2 experiments performed in triplicate. ∗ = P < 0.05 versus basal values (180 ± 15 fmoles/well).

Next, we determined the potential inhibitory action of the antagonist SHU9119 in this model of gout. As in the previous set of experiments, IA treatment with ACTH1–39 (5 μg) reduced neutrophil recruitment into the MSU crystal–treated joint (Figure 7A), together with a significant attenuation of the synthesis and/or release of the cytokines IL-1β and IL-6 (Figure 7B). Coinjection of SHU9119 (10 μg; equivalent to 9 nmoles) abrogated the inhibitory actions of ACTH1-39 (Figures 7A and B). Similar effects were measured for joint size and arthritis score (data not shown).

Figure 7.

Modulation of knee joint inflammation by melanocortin type 3 receptor (MC3-R) activation. A, Rats received 5 μg adrenocorticotropic hormone 1–39 (ACTH1–39) or 25 μg γ2-melanocyte–stimulating hormone (γ2-MSH) intraarticularly (IA) at the same time as monosodium urate monohydrate (MSU) crystals (1 mg IA) alone or in conjunction with the MC3/4-R antagonist SHU9119 (SHU) (10 μg). Effects on neutrophil influx (A) and cytokine levels (B) were determined at the 16-hour time point. Values are the mean and SEM of 6 rats. ∗ = P < 0.05 versus phosphate buffered saline (PBS) control group.

As a further support to the hypothesis that MC3-R is involved in these biologic effects, we used γ2-MSH, a putative endogenous agonist of MC3-R (30). Local (IA) application of this hormone (25 μg; equivalent to 63 nmoles) produced a reduction in cell influx, and this was again absent when it was coadministered with SHU9119 (Figure 7A). Modulation of MSU crystal–induced neutrophil extravasation was mirrored by changes in joint size (0.60 ± 0.11 mm and 0.22 ± 0.04 mm in vehicle- and γ2-MSH–treated rats (mean ± SEM; n = 5) (P < 0.05) and arthritis score (2.50 ± 0.37 and 0.80 ± 0.40 arbitrary units in vehicle- and γ2-MSH–treated rats; P < 0.05). After coinjection with SHU, γ2-MSH inhibitory effects on knee joint size and arthritis score were no longer present (joint size 0.54 ± 0.05 mm in SHU9119 alone– and 0.62 ± 0.09 mm in γ2-MSH + SHU9119–treated rats; arthritis score 1.90 ± 0.30 in SHU9119 alone– and 1.90 ± 0.30 in γ2-MSH + SHU9119–treated rats).

ACTH and γ2-MSH inhibit neutrophil accumulation following repeated MSU crystal administrations.

It has long been known that attacks of gouty arthritis are interspersed with periods of apparent quiescence (31). To mimic more closely this clinical situation, MSU crystals were injected at time 0 and also at 72 hours, when the first influx of cells had subsided. Treatment of inflamed joints with ACTH1–39 (5 μg) or γ2-MSH (25 μg), together with the second MSU crystal injection, significantly reduced neutrophil accumulation in the joint as measured at the 96-hour time point (Figure 8A). A similar degree of inhibition with ACTH1–39 and γ2-MSH, respectively, was also seen in the increase of joint size (65% and 54%) and in the overall arthritis score (63% and 62%). The antagonist SHU9119 prevented these inhibitory actions, not only on neutrophil influx (Figure 8B) but also on cytokine release (data not shown).

Figure 8.

Effect of ACTH on recurrent MSU crystal–induced neutrophil migration. A, MSU crystals were injected twice (1 mg IA) (arrows), and PBS (solid squares), ACTH1–39 (solid circles), or γ2-MSH (open squares) was given together with the second MSU crystal injection. Neutrophil influx was measured 24 hours later, i.e., 96 hours after the first MSU crystal administration. Values are the mean ± SEM of 5 rats per group. ∗ = P < 0.05 versus 96-hour PBS group. B, SHU blocking of the antimigratory effect of ACTH1–39 and γ2-MSH as measured at 96 hours, i.e., 24 hours after the second injection of MSU crystals. Values are the mean and SEM of 5 rats per group. ∗ = P < 0.05 versus proper PBS group; # = P < 0.05 versus the same group in the absence of SHU treatment. See Figure 7 for definitions.


This study was prompted by the fact that parenteral administration of ACTH1–39 was reported, more than 50 years ago, to be clinically effective in controlling the symptoms of gouty arthritis (10, 31). However, ACTH1–39 treatment was rarely used because of the intense suppression of the hypothalamic–pituitary–adrenal (HPA) axis observed following repeated administrations (32). ACTH1–39 activation of its own specific receptor on the adrenal glands (recently cloned and termed MC2-R) (18) produces rapid synthesis and release of glucocorticoid hormones in the circulation (33). In view of the potent antiinflammatory profile that adrenal gland–derived glucocorticoids display, it is not surprising that this mechanism of action was initially proposed to explain the antiinflammatory properties of ACTH1–39. However, recent controlled clinical studies have confirmed the efficacy of ACTH1–39 itself, suggesting the involvement of a mechanism separate from adrenal gland activation (12). These clinical data highlight the antiinflammatory properties of ACTH but do not allude to a specific mechanism of action.

Here we have set up an experimental model of gouty arthritis. IA administration of MSU crystals caused a remarkable accumulation of neutrophils. It is noteworthy that the neutrophil influx was not followed by migration of monocytes into the inflamed joint, making this model similar to the situation in humans (34). Neutrophil influx peaked at 16 hours and was mirrored by changes in other parameters of joint inflammation, including joint size and arthritis score. Consistent with our previous study of MSU crystal–induced mouse peritonitis (35), cell influx was preceded by elevation of IL-1β and IL-6 release, peaking at 6 hours, just prior to migration of neutrophils.

The potential for MSU crystal–induced rat joint inflammation as a valid experimental model of gouty arthritis was determined by testing classic antirheumatic and antigout drugs. As expected (36–38), systemic treatment of animals with either the glucocorticoid dexamethasone, the cyclooxygenase inhibitor indomethacin, or the microtubule dissociator colchicine led to inhibition of cell influx. Interestingly, colchicine produced a pronounced inhibition of IL-1β release. This is a novel finding, since colchicine has previously been shown to inhibit IL-1 activity but not IL-1 production (39), and it may contribute to the overall efficacy of this drug.

Systemic administration of ACTH1–39 produces a dose-dependent reduction of several parameters of MSU crystal–induced joint inflammation. In a sense, this observation experimentally confirms the validity of the clinical data mentioned above. Interestingly, IA ACTH1–39 inhibits inflammation in a corticosterone-independent manner, as demonstrated by its failure to increase circulating corticosterone and its retained efficacy in adrenalectomized rats. Thus, local ACTH1–39 must activate an MC-R other than MC2-R, which is selectively expressed in adrenal glands (40) and adipocytes (41).

The next step was the identification of the MC-Rs potentially involved. Because of findings in our previous study of the mouse (19), as well as in vitro studies from other groups (42, 43), we focused our attention on the resident knee joint macrophage. In fact, macrophage-derived cytokines have a causal role in crystal-induced inflammation and this has been demonstrated not only in an experimental setting but also in humans (6, 44–46). Of relevance, ACTH1–39 produces a marked inhibition of cytokine release from mouse macrophages and other leukocytes (18, 19). Of the 5 MC-Rs cloned to date, MC1-R is expressed by melanocytes and binds preferentially to α-MSH, whereas MC2-R is highly selective for ACTH1–39 (17). Human and rodent MC3-R, MC4-R, and MC5-R bind all melanocortin peptides with varying affinities (47–49). We have shown by reverse transcriptase–polymerase chain reaction (19) and Western blotting (21) that MC3-R is expressed on murine macrophages but not on neutrophils or mast cells (20).

The presence of MC3-R on rat knee joint macrophages was visualized using electron microscopy, and this showed a predominant distribution of the receptor on the plasma membrane and its microvilli. Since MC3-R activation leads to intracellular cAMP accumulation (18–20, 30) it was important to check receptor functionality in this cell type. In addition, we also tested ACTH1–39 on a rat fibroblast-like synoviocyte cell line. ACTH1–39 caused a concentration-dependent increase in this secondary mediator selectively in rat knee joint macrophages. The EC50 for the hormone calculated in this assay (∼12 nM) is in line with the affinity determined with experiments of binding to MC3-R (47). Together, these data indicate that MC3-R is not only expressed on joint macrophages, but is fully functional such that cAMP formation occurs after agonist activation. The lack of selective agonists and/or antagonists has hampered a full pharmacologic characterization of melanocortin receptors. Fan et al recently described a mixed antagonist to the MC3/4-R, termed SHU9119, modified from the ACTH4–10 sequence (24). ACTH1–39–induced, but not forskolin-induced, cAMP formation in rat macrophages was blocked in the presence of SHU9119.

The in vivo relevance of MC3-R was addressed by treatment of rats with the putative selective MC3-R agonist γ2-MSH (21, 30), and the results showed that it attenuates joint inflammation. Similar results have also been obtained with the synthetic mixed MC3/4-R agonist termed MTII (ref. 24, and Getting SJ: unpublished observations). Coinjection with the antagonist SHU9119 abrogates the inhibitory actions not only of γ2-MSH but also of ACTH1-39. These pharmacologic data substantiate the in vitro observations, linking MC3-R function with melanocortin inhibition of neutrophil migration elicited by MSU crystals.

Since gouty arthritis is a disease in which the acute and painful attacks are followed by periods of remission, it is important to determine the impact of ACTH and γ2-MSH treatment after MSU crystal–induced joint inflammation. Both melanocortins retain activity when tested against a second injection of MSU crystals, and their inhibitory properties are blocked by coinjection with SHU9119. These data indicate that an initial joint inflammation does not alter MC3-R expression, and may have clinical relevance because they suggest that there is no loss of response after repeated administrations. Moreover, it is important to note that IA administration of antirheumatic drugs is used in the clinical management of chronic inflammatory conditions. This route of administration could be used for ACTH1–39 itself and would have the advantage of minimal suppression of HPA axis function.

The data here may give a fresh impulse to drug discovery, since a selective MC3-R agonist would mimic the antiinflammatory actions displayed by ACTH1–39 and γ2-MSH in this experimental setting, without the ACTH1–39–induced side effects that are mediated by MC2-R. Such a selective compound should not cross the blood–brain barrier, since studies with MC3-R gene-deficient mice suggest that within the central nervous system this receptor may control subtle mechanisms of energy expenditure (50, 51).


The authors wish to thank Drs. A. Lussier and R. de Médicis (University of Sherbrooke, Sherbrooke, Quebec, Canada) for the generous supply of MSU crystals, and Professor P. G. Winyard (Queen Mary, University of London, London, UK) for the rat fibroblast-like synoviocyte cell line.