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The Journal of Immunology, 2005, 174: 2297-2304.
Copyright © 2005 by The American Association of Immunologists

In Vitro and In Vivo Induction of Heme Oxygenase 1 in Mouse Macrophages following Melanocortin Receptor Activation1

Connie W. Lam, Stephen J. Getting and Mauro Perretti2

The William Harvey Research Institute, Bart’s and the London, Queen Mary School of Medicine and Dentistry, London, United Kingdom


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
RAW264.7 cell incubation with adrenocorticotrophin (ACTH) led to a time-dependent (4–24 h) and concentration-related (1–100 ng/ml) induction of heme oxygenase (HO)-1, and this was a specific effect, because the pattern of expression of other cellular proteins (HO-2, heat shock proteins 70 and 90) was not modified by ACTH. Combined RT-PCR and Western blot analyses revealed expression of the melanocortin receptor (MC-R) types 1 and 3, but not 4, in these cells. However, use of more selective agonists (including melanotan (MTII)) indicated a predominant role for MC3-R in the induction of HO-1 expression and activity. Relevantly, ACTH and MTII incubation with primary peritoneal macrophages (M{phi}) also induced HO-1 expression. The potential link between MC3-R dependent cAMP formation and HO-1 induction was ascertained by the following: 1) ACTH and MTII produced a concentration-dependent accumulation of cAMP in RAW264.7 cells, and 2) whereas a selective inhibitor of cAMP-dependent protein kinase A abrogated ACTH- and MTII-induced HO-1 expression, a soluble cAMP derivative promoted HO-1 induction both in RAW264.7 cells and primary M{phi}. HO-1 induction in peritoneal M{phi} was also detected following in vivo administration of MTII, and appeared to be functionally related to the antimigratory effect of this melanocortin, as determined with a specific inhibitor (zinc protoporphyrin IX). In conclusion, this study highlights a biochemical link between MC-R activation and HO-1 induction in the M{phi}, and proposes that this may be of functional relevance in determining MC-R-dependent control of the host inflammatory response.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Melanocortin peptides (e.g., {alpha}-melanocortin-stimulating hormone ({alpha}-MSH)3) have long been reported to possess anti-inflammatory effects in many experimental models of acute (1, 2) and chronic inflammation, including inflammatory bowel disease (3), allergy (4), joint arthritis (5, 6), and systemic inflammation (endotoxemia) (7). Interestingly, recent trials with {alpha}-MSH have confirmed the positive indication for this compound in controlling human disease (8), thereby reinforcing the potential impact of this line of research. Because {alpha}-MSH represents the first 13 aa within the adrenocorticotrophin (ACTH) sequence (39 aa in total) (9), it is important to recall the efficacy of the longer polypeptide in controlling rheumatoid arthritis (10).

At the molecular level, the effects of these anti-inflammatory hormones and synthetic derivatives on target cells are brought about by activation of a subgroup of G protein-coupled receptor, termed melanocortin receptors (MC-R). Five MC-Rs have been identified so far, and all of these receptors are positively coupled to adenylate cyclase such that their activation leads to increases in intracellular cAMP (9, 11). Following this early event of cAMP formation, and also perhaps partly independently from it, melanocortin peptides have been shown to down-regulate NF-{kappa}B activation and consequent cytokine synthesis (12, 13). The C terminus sequence (i.e., aa 11–13) of {alpha}-MSH outside the common core and modifications of it (14, 15) have been shown to block cytokine functions (1), rather than synthesis and release (16).

Heme oxygenase (HO)-1 is the rate-limiting enzyme in heme catabolism with consequent generation of biliverdin (then converted to bilirubin), free iron, and carbon monoxide. Three mammalian HO isoforms have been identified, one of which, HO-1, is a stress responsive protein endowed with important cytoprotective effects (for a recent review, see Ref.17). In addition, macrophage (M{phi}) HO-1 expression is part of the repairing processes that occur during resolving inflammation leading to healing and tissue repair (18). It is possible that at least some of the cytoprotective and anti-inflammatory actions of HO-1 are due to the controlled local liberation of carbon monoxide, able to signal through the cyclic GMP pathway (17) and inhibit cytokine synthesis (19). In addition, the other catabolite bilirubin is also endowed with antioxidant and anti-inflammatory effects, for instance, its application inhibits LPS-induced selectin expression in the vasculature, thus affecting leukocyte recruitment (20).

The present study was undertaken to assess a potential functional link between MC-R-dependent cAMP formation and HO-1 induction in M{phi}. Most of the experiments have been conducted with the RAW264.7 M{phi} cell line, both for data consistency and ease of manipulation (and reduction in animal sacrifice); however, crucial experiments have been repeated with primary M{phi}. Importantly, in vivo experimentation not only confirmed the biochemical link between MC-R activation and HO-1 induction, but also provided a functional relevance to this interaction. We conclude that MC3-R activation, and possibly activation of other MC-R subtype as well, can bring about anti-inflammatory effects mediated, at least in part, by HO-1 induction.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
In vitro experimental section

Cell culture and treatments. RAW264.7 cells (ECACC; Sigma-Aldrich) or primary M{phi} were cultured in DMEM or RPMI 1640, respectively, supplemented with 10% heat-inactivated FBS in a humidified atmosphere supplied with 5% CO2 and 95% air, until 80% confluency was attained for use. Before experiment, FBS in culture was reduced to 1% for 24 h, and subsequently all experiments were performed under these conditions. Cells were treated with PBS (control), ACTH (3–300 ng/ml), {alpha}-MSH (10 µg/ml), ACTH4–10 (MEHFRWG) (100 µg/ml), MTII (1–100 µg/ml), or LPS (1 µg/ml; serotype 0111:B4) over a time course up to 24 h. In some cases, cells were pretreated for 1 h with N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinoline sulfonamide (H-89; cAMP-dependent protein kinase A (PKA) inhibitor; 20 µM; Sigma-Aldrich) before treatment with ACTH or MTII. A different inhibitor was also tested, the PKA inhibitor peptide sequence 14–22 amide (Calbiochem) at the recommended concentration of 10 µM. Acetyl-(Nle4,Asp5,D-Phe7,Lys10)cyclo-{alpha}-MSH4–10 amide acetate salt (melanotan (MTII)) was purchased from Bachem. Melanocortin agonists, LPS (serotype 0111:B4), and all other materials were purchased from Sigma-Aldrich.

Generation of a polyclonal anti-MC3-R Ab. An anti-mouse MC3-R serum was generated within our laboratory using standard protocols. Briefly, New Zealand white rabbits were immunized four times with a peptide (M-NSSCCLSSVSPMLP-Y from the receptor N terminus, aa 2–15) generated from the mouse MC3-R sequence (National Center for Biotechnology Information website access code NP032587). The first three injections were at 2-wk intervals, and the fourth one came 1 mo after the last. Blood was collected and stored at –20°C before purification. The Ab was purified using a T-Gel purification column (Pierce Biotechnology) and stored at –80°C.

Western blotting analysis. Samples collected from experiments were lysed via sonication in protease inhibitory buffer containing leupeptin (10 µM), pepstatin A (1 µM), PMSF (200 µM), sodium fluoride (1 mM), and {beta}-GP (1 mM). Protein levels were determined by Bradford assay and standardized before boiling in Laemmli buffer containing 4% SDS, 20% glycerol, 10% 2-ME, EDTA (2 mM), and Coomassie brilliant blue (1 mg/ml) in 0.125 M HCl. Samples were electrophoresed in a 10% polyacrylamide gel in running buffer (0.3% Tris base, 1.44% glycine, and 0.1% SDS in distilled H2O) followed by transfer of proteins onto Hybond-C extra nitrocellulose membranes in transfer buffer (0.3% Tris base, 1.44% glycine, and 20% methanol in distilled H2O). Membranes were blocked overnight in blocking solution containing 5% nonfat milk solution in Tris buffer saline containing 0.1% Tween 20 followed by anti-MC3-R (1:500) or HO-1 (1:2000; StressGen Biotechnologies) rabbit serum incubation in blocking solution. Nonspecific Ab binding was washed before the addition of goat anti-rabbit Ab (1:2000). The membranes were further washed, and specific Ab binding was detected by ECL system. Following detection, bound Abs were removed by incubating the membranes in acidic glycine solution (100 mM glycine (pH 2.5)). The membranes were subsequently reprobed for the detection of {alpha}-tubulin (1:5,000; Sigma-Aldrich), and in selected experiments, HO-2 (1:5,000; StressGen Biotechnologies), heat shock protein (HSP)-70 (1:20,000, StressGen Biotechnologies), HSP-90 (1:2,000; Santa Cruz Biotechnology), COX-2 (1:2,000; Cayman Chemical), and inducible NO synthase (iNOS; 1:3,000; Santa Cruz Biotechnology). Images were acquired and densitometry performed using Scion Image 1.63 software (Scion Corporation).

Immunocytochemistry. Mouse peritoneal cells or RAW264.7 cells were resuspended in PBS (107 cell/ml), air-dried on microscopy slides, and stained according to classical protocols. Briefly, sample slides were washed in PBS supplemented with 0.2% Triton X-100 (PBS-T) and incubated in hydrogen peroxide (0.3% in methanol) for 15 min, and nonspecific binding was blocked by incubation with normal goat serum for 1 h. Samples were then incubated overnight at 4°C with anti-MC3-R IgG or nonimmune rabbit IgG (as negative control), at a final concentration of 3.5 µg/ml in both cases, before subsequent washes in PBS-T, and incubation with a goat anti-rabbit secondary Ab (Vector Laboratories). Staining was revealed with an avidin-biotin complex kit (Vector Laboratories). Acetate buffer (0.1 M; pH 6.0) was used to rinse the slides followed by 4-min incubation in 0.1% diamine-benzidine and 1% nickel sulfate, whereas 0.8% cobalt chloride (in 10 mM Tris buffer (pH 7.6)) was used to enhance the immunostaining. Slides were counterstained with hematoxylin.

Intracellular cAMP elevation. RAW264.7 M{phi} (1 x 105) were plated in 24-well plates in DMEM supplemented with 10% FBS and allowed to adhere for 2 h. Cells were then washed and incubated with 3 µM Forskolin (positive control), ACTH (3–300 ng/ml), or MTII (0.3–300 µg/ml) in presence of 1 mM isobutylmethylxanthine in serum-free medium and incubated for 30 min at 37°C in humidified atmosphere of 5% CO2 and 95% air. A nontreated group with serum-free medium alone was also set up under the same incubation condition as negative control. Cell supernatants were then removed, and cells were lysed. Intracellular cAMP elevation was determined with a commercially available enzyme immunoassay using a standard curve constructed with 0–3200 fmol/ml cAMP (Amersham Biosciences).

HO-1 activity assay. RAW264.7 cells (2.5 x 106 cells) were treated with PBS, LPS (1 µg/ml; positive control), ACTH (100 ng/ml), or MTII (10 µg/ml) for 8 h. Cell pellets were resuspended in magnesium supplemented potassium phosphate solution (MgCl-PBS; 100 mM potassium phosphate and 2 mM magnesium chloride; pH 7.4) and subjected to three freeze-thaw cycles for release of cytoplasmic HO-1 proteins. HO-1 enzymatic assay used a reaction mixture containing 100 mM PBS, 2 mM MgCl2, 3 mg of rat liver cytosol, 0.8 mM NADPH, 2 mM glucose-6-phosphate, 0.2 U of glucose-6-phosphate dehydrogenase, and 20 µM enzyme substrate hemin plus 400 µl of sample. The reaction was made up to a final volume of 1 ml for each sample and was incubated at 37°C for 1 h in the dark. Chloroform was added to terminate the reaction, and bilirubin was extracted following centrifugation, and measured by spectrophotometry reading the difference in absorbance between 464 and 530 nm ({epsilon} = 40/mM/cm). The protein concentration in each sample was determined with a Bradford protein assay, and the HO-1 activities were expressed in picomoles of bilirubin formed per milligram of protein per hour.

mRNA isolation and RT-PCR. mRNA of RAW267.4 cell line and mouse peritoneal M{phi} (5 x 106 cells) were isolated in accordance to the protocol in the RNeasy Mini kit (Qiagen). In brief, cells were lysed in a guanidine isothiocyanate- and 2-ME-containing RLT buffer and passed through a 20-gauge needle five times to disrupt the M{phi}. DNase was then applied onto membrane and incubated for 15 min to digest any remaining DNA contaminants in the samples. The membrane was subsequently washed with RW1 and RPE buffers supplied in the kit at 8000 x g to remove contaminants, and membrane-bound RNA was eluted with RNase-free water. RNA yield and purity were assessed spectrophotometrically at 260 and 280 nm. Aliquots of RNA (1 µg) were used in the RT-PCR preparation by denaturation at 65°C for 5 min in presence of 1 µg of random primers. The RT-PCR preparations were subsequently generated into cDNA by incubation in presence of 5x AMV-RT buffer, dNTPs, RNAsin enzyme, and AMV-RT enzymes at 42°C for 20 min. RT-PCR amplifications were performed on aliquots of cDNA (0.1 µg) in presence of 10x PCR buffer, dNTPs, 1.5 µM MgCl2, Taq polymerase, classic 18s ribosomal RNA primers (as internal control; giving a product of 324 bp), and MC-R primers (0.2 µM final concentration). 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) with fragment amplification of 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) with fragment amplification of 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) with fragment amplification of 445 bp in length (21). Primers were purchased from Applied Biosystems. Classic 18s ribosomal RNA primers and random primers were purchased from Promega. The total volume of each reaction was made up to 50 µl with RNA-free distilled water. The cycle 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. Amplified products were visualized by ethidium bromide fluorescence in 1% agarose gels.

In vivo experimental section

Male Swiss Albino mice (body weight, 20–22 g) were purchased from Bantin & Kingman. Mice were maintained on a standard chow pellet diet with tap water ad libitum using a 12-h light/dark cycle. Animal experimental work was performed according to Home Office regulations (Guidance on the Operation of Animals, Scientific Procedures Act, 1986).

For the in vitro experiments, peritoneal cells (>80% M{phi}) were collected from culled mice by injection of 3 ml of PBS supplemented with 1 mM EDTA and gentle massage of the peritoneal cavity. On average, 2–4 x 106 M{phi} were recovered from each peritoneal cavity.

In vivo, mice were treated with an anti-inflammatory dose of MTII (10 µg; Ref.22), and peritoneal cells were harvested at different times postadministration. For the inflammation model, we selected the urate crystal peritonitis (23), extensively used to investigate the anti-inflammatory actions of melanocortins (e.g., see Ref.21). In this case, at time 0, mice received 3 mg of monosodium urate crystals alone or together with MTII. Eight hours later, some animals were treated with the HO-1 inhibitor zinc protoporphyrin IX (ZnPPIX) given at the dose of 10 µmol/kg i.p (20, 24). Peritoneal cavities were washed at the 24-h time point post-crystal injection, and the number of PMN migrated was determined following staining in Turk’s and counting in a Neubauer hematocytometer as described (23).

Statistical analysis

All values are expressed as mean ± SEM, with an n number of mice for the in vivo experiments. For in vitro analysis, experiments were repeated at least with three distinct preparations (i.e., RT-PCR and Western blotting analyses). Statistical analysis was assessed either by Mann-Whitney U test (for blot analyses) or one-way ANOVA followed by Bonferroni test (for the in vivo experimentation). In all cases, a value of p < 0.05 was considered significant to reject the null hypothesis.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
RAW264.7 cells express functional MC3-R

At the beginning of the study, we confirmed that RAW264.7 cells could act as a reliable surrogate for primary M{phi}s. In line with that reported for mouse peritoneal M{phi} (21), RAW264.7 cells expressed MC3-R mRNA and protein (Fig. 1). Immunocytochemistry confirmed receptor expression on the cell surface of RAW264.7 cells and M{phi}, apparently to the same extent (Fig. 1c). RT-PCR was used to monitor the expression of other receptors of the family, concluding that RAW264.7 cells expressed MC1-R, but not MC4-R, mRNA (Fig. 1b).



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  		FIGURE 1. Detection of MC3-R mRNA and protein in RAW264.7 cells. a, Expression of MC-R mRNAs in RAW264.7 cells and primary peritoneal M{phi} as detected by RT-PCR. Specific primers, described in Materials and Methods, were used to detect mRNA expression for MC1-R (825 bp), MC3-R (820 bp), or MC4-R (445 bp), with detection of ribosomal 18s as standard. DNA molecular-mass markers are also shown. Data are representative blots for three experiments. b, MC3-R protein detection in protein extracts from RAW264.7 cells, peritoneal M{phi}, and mouse brain tissue (representative of three blots). c, Immunocytochemistry analysis shows MC3-R positivity in RAW264.7 cells as well as in mouse peritoneal M{phi}, whereas the preimmune rabbit IgG was used as negative control.

 
Addition of the mixed MC3/4-R agonist MTII to RAW264.7 cells provoked a concentration-dependent accumulation of intracellular cAMP (Fig. 2). The synthetic derivative was at least two orders of magnitude less potent than the natural agonist ACTH. Altogether, these signaling profiles are in line with those derived from experimentation with primary M{phi} (21, 22).



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  		FIGURE 2. Intracellular cAMP elevation in RAW264.7 cells by ACTH and MTII. RAW264.7 cells were incubated ACTH (3–300 ng/ml) or MTII (1–100 µg/ml) and cell-associated determined at the 30-min time point. Cell incubation with forskolin (3 µM, used as positive control) led to 950 ± 150 fmol of cAMP per well (n = 6). Data are expressed as mean ± SEM of three experiments. Dotted line refers to basal cAMP levels in unstimulated cells. *, p < 0.05.

 
MC3-R and HO-1 induction

Induction of cAMP formation has long been recognized as the major signal conveyed by MC-R activation (9), and because this signaling molecule has been linked to HO-1 induction in rat hepatocytes (25), we asked whether MC3-R could also modulate expression of this anti-inflammatory enzyme. To address this, RAW264.7 cells were incubated with different melanocortin peptides or LPS, as positive control, for longer time points. Fig. 3 reports the data obtained at 8 h, in which ACTH or {alpha}-MSH or the synthetic derivatives ACTH4–10 or MTII, caused selective induction of HO-1. In contrast, activation of MC3-R by the MTII agonist did not alter the basal expression of HO-2, HSP-70 and -90, COX-2, and iNOS (Fig. 3). Cells incubated with LPS displayed higher HO-1, iNOS, and COX-2, but not HO-2 and HSP-70/90, protein expression (Fig. 3). Cumulative densitometric analyses of these experiments are in Table I.



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  		FIGURE 3. Pattern of protein expression in RAW264.7 cells in response to melanocortin agonists. RAW264.7 cells were incubated for 8 h with PBS, ACTH (100 ng/ml), {alpha}-MSH (10 µg/ml), ACTH4–10 (100 µg/ml), MTII (10 µg/ml), or LPS (1 µg/ml; positive control) before assessment of protein expression with specific Abs. In all blots, {alpha}-Tubulin was monitored as housekeeping protein. Blots are representative of at least three experiments.

 

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  		Table I. Cumulative densitometric analysis of protein expression in RAW264.7 cells incubated with melanocortin agonistsa

 
HO-1 induction by MTII was time dependent, with a plateau at 8 h (Fig. 4). Similar findings were obtained with ACTH and RAW264.7 cells, as well as with primary M{phi} in culture (not shown for either experiment). In addition, MTII-induced HO-1 protein expression was associated with augmented enzymatic activity, particularly evident 24 h post-MTII incubation. For instance, the HO-1 activity (picomoles of bilirubin/milligram of protein/hour) was 523 ± 28 in control cells and raised to 764 ± 52 and 845 ± 45 for RAW264.7 cells treated for 24 h with 100 ng/ml ACTH or 10 µg/ml MTII, respectively (n = 4; p < 0.05) with intermediate values of enzymatic activity being measured at 8 h (data not shown).



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  		FIGURE 4. Time course of HO-1 induction in RAW264.7 cells by MTII. RAW264.7 cells were incubated for the reported time with MTII (10 µg/ml) or with LPS (positive control; 1 µg/ml, 8 h). HO-1 protein expression was assessed by Western blotting analysis. Gel is from one experiment, whereas values in the bar graph are mean ± SEM of three experiments, and are expressed as fold increase over respective control HO-1 expression (taken as 1). *, p < 0.05.

 
Finally, the functional link between early postreceptor activation signaling responses and more downstream and delayed events, such as HO-1 induction, was established with specific inhibitors and mimicking molecules. For instance, the PKA inhibitor H-89 abrogated ACTH- or MTII-mediated HO-1 induction in RAW264.7 cells and primary M{phi} (Fig. 5). Similar results were obtained with another PKA inhibitor, the structurally unrelated permeable peptide 14–22 amide (data not shown). Conversely, cell incubation with the stable cAMP analog dibutyryl-cAMP caused HO-1 protein up-regulation, as detected at 8 h, in a PKA-dependent fashion (Fig. 5).



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  		FIGURE 5. Involvement of the cAMP-PKA pathway in MTII-mediated HO-1 expression in RAW264.7 cells and primary M{phi}. a, Western blotting analysis of HO-1 protein expression in RAW264.7 cells following treatment with PBS (Control or Ctrl), Bt2cAMP (250 µM), ACTH (100 ng/ml), or MTII (10 µg/ml) for 8 h in the absence or presence of the PKA inhibitor H-89 (20 µM; –1 h) or 1% DMSO as vehicle (V). Treatment with LPS (1 µg/ml) for 8 h was used as positive control. A representative blot is show for HO-1 expression in relation to the housekeeping protein {alpha}-Tubulin. Values in the bar graph are mean ± SEM of three experiments and refer to densitometric analysis. *, p < 0.05 vs respective control values; #, p < 0.05 vs respective vehicle group. b, Densitometric analysis of three experiments performed with primary M{phi} with treatments as in a. *, p < 0.05 vs respective control values; #, p < 0.05 vs respective vehicle group.

 
In vivo relevance of MC3-R mediated HO-1 induction

Once we assessed that the cAMP-dependent HO-1 induction in RAW264.7 cells occurred also in primary cultures of mouse peritoneal M{phi}, we next sought to provide an in vivo relevance to these findings. Intraperitoneal administration of an anti-inflammatory dose of MTII (10 µg per mouse (22, 26)) provoked a time-dependent induction of HO-1 in resident M{phi}, as detected ex vivo by Western blotting analysis (Fig. 6a). Cumulative data for multiple animals as reported in Fig. 6b show a peak of induction at 8 h postinjection, reminiscent of the data obtained in vitro with RAW264.7 cells (Fig. 4) and primary M{phi} (data not shown).



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  		FIGURE 6. Time-dependent MTII-induced HO-1 protein expression in vivo. C57 mice were treated i.p. with MTII (10 µg/mouse) at time 0, and peritoneal cells were collected at the reported time points. LPS (1 µg/mouse) was used as positive control (cells collected at 8 h). a, Protein expression of HO-1 was assessed using Western analysis. The Western blotting analysis was further assessed using densitometric analysis and all HO-1 detections were standardized with the detection of {alpha}-Tubulin. Representative blot is shown on the top. b, Values in the bar graph are mean ± SEM of three experiments with cells collected from three mice each time. *, p < 0.05 vs time 0 expression.

 
The functional relevance of the MC3-R/HO-1 axis was tested by means of the HO-1 inhibitor ZnPPIX. The inflammatory response to urate crystal at 24 h (23) was used here, injecting mice with MTII at time 0 and with the HO-1 inhibitor at 8 h. Fig. 7 reports these data. As expected, MTII exerted an antimigratory effect on PMN influx as measured 24 h postcrystal injection (~50% inhibition; p < 0.05). The anti-inflammatory nature of HO-1 in this model of peritonitis was confirmed by the augmented PMN influx (70% increase; p < 0.05) measured in mice treated with ZnPPIX (Fig. 7). Importantly, when ZnPPIX was given to mice previously treated with MTII, the antimigratory effect of the melanocortin derivative was no longer evident. These in vivo data indicate a major functional role for MTII-induced HO-1 at least with respect to the delayed anti-inflammatory effect measured 24 h post-crystal inflammation.



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  		FIGURE 7. Effect of MTII with or without ZnPPIX on MSU crystal-induced inflammation. C57 mice pretreated with MTII (10 µg/mouse; i.p.) for 30 min, followed by MSU crystal (3 mg/mouse; i.p.) with or without ZnPPIX (10 µmol/kg; i.p.; 8 h post-MSU crystal injection) for 24 h. Resident M{phi} were taken from the peritoneal cavity for analysis. Aliquots were stained with Turk solution, and PMN counts were performed using a Neubauer hematocytometer. Data are expressed as mean ± SEM of five mice per group. *, p < 0.05 vs control.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
This study demonstrates a previously unknown link between M{phi} MC3-R activation and induction of the anti-inflammatory enzyme HO-1. Activation of this receptor by MTII, and the less selective agonist ACTH, produces transient alterations in intracellular cAMP that are temporally related to HO-1 up-regulation in a PKA-dependent fashion. In vivo, the MC3-R/HO-1 connection is functionally operative in bringing about the antimigratory effect of a melanocortin peptide.

Since the initial studies with {alpha}-MSH (27), melanocortin peptides have been shown to represent an important component of the counterregulatory systems that operate in the host to dampen and control the inflammatory reaction. Several studies conducted in experimental animals with models of acute and subacute inflammation (1, 28), in some cases supported by human data (29), have shown how {alpha}-MSH and other melanocortin peptides are endowed with potent inhibitory and anti-inflammatory properties (8). This field attracted much more interest once specific receptors were cloned and shown to mediate the actions of several melanocortins, including the naturally occurring ACTH and {alpha}-MSH, on different target cells. MC-R belongs to the family of G protein-coupled receptors, and their activation leads to adenylate cyclase-mediated conversion of ATP into cAMP (9, 11). Thus, accumulation of cAMP in target cells buffers cell activation with a marked effect on the production of proinflammatory cytokines (12, 13, 21). Therefore, targeting specific MC-R could certainly represent a novel strategy to develop innovative anti-inflammatory agents, as recently reviewed (30).

The similarity in mediated signaling events for the five MC-R can be problematic for drug development, impeding exploitation of specific postreceptor pathways: this aspect is made more acute by the absence of specific pharmacological tools to dissect the functions of each receptor. A few years ago, we started a project focusing on the resident M{phi}, reasoning that a certain degree of specificity could derive from narrowing down actions of a given target cell. Addition of ACTH, {alpha}-MSH, and the relatively more specific synthetic derivative MTII (31), to mouse peritoneal M{phi} inhibited the release of an array of cytokines and chemokines (21, 22). Comparison of the effects produced by agonists and antagonists, together with morphological analyses and study with mutated receptors, allowed us to pinpoint MC3-R as a major determinant for these inhibitory properties, at least at the level of the M{phi} (6).

MC3-R-mediated inhibition of cytokine synthesis and release from stimulated M{phi} in vitro occurs relatively rapid (≤2 h). In the present study, we set out to examine more delayed downstream events subsequent to MC3-R activation. RAW264.7 cells were used and initially validated for their expression of MC3-R message and protein. Confirming previous studies with monocytic cell lines (13, 32), RAW264.7 cells expressed MC1-R mRNA. However, we could not find the MC4-R mRNA, thus allowing us to use the mixed MC3/4-R agonist MTII (31) for most of the subsequent experiments. Because elevated cAMP levels have been associated with HO-1 induction in rat hepatocyte cultures (25), we monitored expression of this and other stress proteins in RAW264.7 cells following incubation with MTII or ACTH. Either peptide provoked a selective and marked up-regulation of HO-1 evident as early as 2–4 h postincubation: this effect was long lasting and fully evident even at the 24-h time point. In full agreement with the study on rat hepatocytes (25), MTII-mediated HO-1 induction was genuinely due to cAMP/PKA signaling, a fact that was confirmed also with primary M{phi} cultures. It is of interest that, during the preparation of this paper, a study showing ACTH induction of HO-1 in a mouse adrenocortical cell line was published (33). It therefore seems that activation of more than one MC-R (certainly MC3-R and MC2-R, the latter being selectively expressed in adrenal cells, and possibly all the other receptors) in target cells can lead to HO-1 up-regulation, thereby making this biochemical link a more general phenomenon. Subsequently, we investigated the potential functional consequences of this induction in experimental inflammatory settings.

M{phi} HO-1 has long been recognized to possess cytoprotective properties, and in the context of inflammation, this enzyme catabolizes heme. In fact, it has an important scavenger function during the resolution and healing phases of the acute inflammatory reaction, because HO-1 disposes excess heme of enzymatic source or from the hemoglobin lost by dying erythrocytes extravasated as part of the exudation process (17). In an acute resolving inflammatory response, that is, rat pleurisy, HO-1 is selectively expressed in M{phi}, and not PMN, during the late proresolving phase of the response (18). Interestingly, the model we used here is also acute and naturally resolves (23). Recent years have seen an appreciation of more subtle and homeostatic functions of M{phi} HO-1 together with specific networks among anti-inflammatory mediators. For instance, IL-10 acts via HO-1 to protect from excessive M{phi} activation as seen in experimental endotoxemia (34), and the scavenger receptor CD163 controls both IL-10 and HO-1 synthesis and expression in human M{phi} (35). In addition, M{phi} HO-1-derived CO can produce several biological actions, mostly protective at the concentration levels found in exudates (17, 36). The cytoprotection afforded by novel CO-releasing molecules (37) emphasizes the pivotal role that CO produced by HO-1 from heme degradation may be playing in the pathophysiological context of a resolving inflammatory process. From a mechanistic point of view, HO-1-derived carbon monoxide inhibits cytokine synthesis (19), whereas the product of the enzymatic reaction, bilirubin, is endowed with antioxidant and anti-inflammatory effects, causing, for instance, inhibition of LPS-induced selectin expression in the vasculature, thus affecting leukocyte recruitment (20).

In all cases, most of the anti-inflammatory properties of HO-1 take place in the M{phi} (18, 38). Thus, in the final part of the study, we sought to provide a functional role for MTII-induced HO-1 in an experimental model of inflammation. Our choice fell on the urate crystal peritonitis because it is characterized by a prolonged PMN influx (23) and has been shown to be sensitive to melanocortin peptide administration (21, 22). Initially, we tested whether MTII could up-regulate M{phi} HO-1 following i.p. administration: this agonist was chosen because of its selectivity, at least partial, toward MC3-R (31), long half-life (39), and efficacy in this model of peritonitis (22, 26). The profile of induction obtained was similar to that obtained following in vitro incubation with this cell type, with a peak of HO-1 expression at the 8-h time point. Next, the effect of the HO-1 inhibitor ZnPPIX on MTII-mediated inhibition of urate crystal-induced PMN recruitment into the mouse peritoneal cavity was tested, by giving the inhibitor 8 h into the inflammatory reaction. Analysis of the results produced by this set of in vivo experiments indicates that HO-1 displays anti-inflammatory properties also in this model of peritonitis (see the higher PMN numbers in the ZnPPIX group), and that in the presence of HO-1 blockade, the antimigratory effect of MTII on the 24-h PMN recruitment is abolished. Therefore, MTII-induced HO-1 expression is instrumental for this specific antimigratory effect of MTII.

In conclusion, this study indicates that HO-1 induction in M{phi} might be a major arm in the complex series of effects that are produced by melanocortin peptides acting at their MC-R. Our analyses on M{phi} function, in the present and previous studies, suggest that MC3-R is the major receptor determinant for transducing the anti-inflammatory actions of these peptides on M{phi}, although it is clear that we could also detect MC1-R in RAW264.7 cells, in analogy to Star et al. (32). However, irrespective of the specific MC-R, a more general picture is emerging in which MC-R activation on the M{phi} cell surface leads to cAMP formation and PKA activation. This signaling pathway produces at least two downstream events: inhibition of cytokine synthesis and release from stimulated M{phi} (in the presence of an inflammogen), which is evident within the first 2 h, and then up-regulation of HO-1 from ≥4 h post-MC-R activation. Importantly, the latter effect is achieved by the melanocortin peptide itself (i.e., in the absence of an inflammogen or M{phi} activator), suggesting that MC-R activation favors the acquisition of the anti-inflammatory proresolving phenotype by the M{phi} (a phenomenon originally described for glucocorticoids (40)). Fig. 8 schematizes this model of two hits, or anti-inflammatory mechanisms, activated by MC3-R agonists.



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  		FIGURE 8. Schematic representation of the anti-inflammatory effects switched on in M{phi} by MC3-R activation. Resident M{phi} express MC3-R, and agonism at this receptor leads to accumulation of cAMP with consequent activation of PKA. This results in at least two major effects: the paninhibition exerted by these peptides on stimulated cytokine synthesis and release, evident already after short incubation times (<4 h), and the induction of the stress protein HO-1, which requires longer incubation times (>4–6 h, optimal at 8 h as reported in this study). Either mechanism of action can impact on the host inflammatory response and be responsible for the potent and reproducible antimigratory effects that melanocortin peptides exert in the context of acute local and systemic inflammation. See end of Discussion for more details.

 
More and more evidence indicates the existence of networks or interconnections among several anti-inflammatory pathways. We propose that the biochemical and functional link reported in the present study for MC3-R activation and consequent HO-1 expression might be of help in the development of melanocortin peptides or derived small chemical entities endowed with potent and prolonged anti-inflammatory effects.


   Acknowledgments
 
We thank Dr. Ronald Korthuis for constructive discussion, Dr. R. Motterlini for help in performing the HO-1 enzymatic assay, and Dr. E. Solito for help in designing the MC3-R peptide for Ab production. We also thank Mr. Amilcar S. Damazo for guidance in the immunocytochemistry analysis.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by the Arthritis Research Campaign UK (ARC UK; Studentship P0575). M.P. is a senior research fellow of the ARC UK. Back

2 Address correspondence and reprint requests to Dr. Mauro Perretti, Centre of Biochemical Pharmacology, The William Harvey Research Institute, Bart’s and the London, Queen Mary School of Medicine and Dentistry, Charterhouse Square, London EC1M 6BQ, United Kingdom. E-mail address: M.Perretti{at}qmul.ac.uk Back

3 Abbreviations used in this paper: {alpha}-MSH, {alpha}-melanocortin-stimulating hormone; ACTH, adrenocorticotrophin; MC-R, melanocortin receptor; HO, heme oxygenase; M{phi}, macrophage; H-89, N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinoline sulfonamide; PKA, cAMP-dependent protein kinase A; MTII, melanotan or Ac-Nle4-c[Asp5,D-Phe7,Lys10]NH2 ACTH4–10; HSP, heat shock protein; iNOS, inducible NO synthase; ZnPPIX, zinc protoporphyrin IX. Back

Received for publication July 14, 2004. Accepted for publication November 19, 2004.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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