Melanocortin peptides inhibit production of proinflammatory cytokines ...

Melanocortin peptides inhibit production of proinflammatory cytokines and nitric oxide by activated microglia Rene´ Delgado,*‡ Andrea Carlin,* Lorena Airaghi,* Maria Teresa Demitri,* Lucia Meda,† Daniela Galimberti,† Pierluigi Baron,† James M. Lipton,§ and Anna Catania* *III Division of Internal Medicine and †Institute of Neurology, IRCCS Ospedale Maggiore, Milano, Italy; ‡Department of Biotechnology, Center of Pharmaceutical Chemistry, Havana, Cuba; and ‡Department of Physiology, University of Texas Southwestern Medical Center at Dallas, Texas

Abstract: Inflammatory processes contribute to neurodegenerative disease, stroke, encephalitis, and other central nervous system (CNS) disorders. Activated microglia are a source of cytokines and other inflammatory agents within the CNS and it is therefore important to control glial function in order to preserve neural cells. Melanocortin peptides are pro-opiomelanocortin-derived amino acid sequences that include a-melanocyte-stimulating hormone (a-MSH) and adrenocorticotropic hormone (ACTH). These peptides have potent and broad anti-inflammatory effects. We tested effects of a-MSH (1–13), a-MSH (11–13), and ACTH (1–24) on production of tumor necrosis factor a (TNF-a), interleukin-6 (IL-6), and nitric oxide (NO) in a cultured murine microglial cell line (N9) stimulated with lipopolysaccharide (LPS) plus interferon g (IFN-g). Melanocortin peptides inhibited production of these cytokines and NO in a concentration-related fashion, probably by increasing intracellular cAMP. When stimulated with LPS 1 IFN-g, microglia increased release of a-MSH. Production of TNF-a, IL-6, and NO was greater in activated microglia after immunoneutralization of endogenous a-MSH. The results suggest that a-MSH is an autocrine factor in microglia. Because melanocortin peptides inhibit production of pro-inflammatory mediators by activated microglia they might be useful in treatment of inflammatory/degenerative brain disorders. J. Leukoc. Biol. 63: 740–745; 1998. Key Words: a-melanocyte-stimulating hormone · adrenocorticotropic hormone · tumor necrosis factor · interleukin-6 · cyclic AMP

INTRODUCTION Microglia are resident macrophages of the central nervous system (CNS) [1]. There is evidence that monocytes enter the developing CNS and are transformed into microglia [2]. These cells are widespread in the CNS and occur in highest density in substantia nigra and ventral pallidum, whereas few occur in cerebellum and brain stem [3]. Unlike brain macrophages that constitutively express MHC class II antigens, microglia are 740

Journal of Leukocyte Biology

Volume 63, June 1998

quiescent cells with down-regulated antigenic phenotype [4–5]. Activated microglia produce soluble factors, including cytokines, prostaglandins, nitric oxide (NO), and free oxygen radicals, that are crucial in host defense against CNS infections [6]. However, excessive production of pro-inflammatory mediators can be harmful. Such mediators contribute to tissue injury in CNS inflammation/injury processes of multiple sclerosis [7], HIV infection [8], and degenerative brain disorders [9–11]. Modulation of such microglial products should thus be therapeutic in CNS disorders. Melanocortin peptides are amino acid sequences derived from pro-opiomelanocortin (POMC) and include a-melanocytestimulating hormone [a-MSH (1–13)] and adrenocorticotropic hormone [ACTH (1–39)] [12]. a-MSH has anti-inflammatory influences in vivo and in vitro [13, 14]. This potent peptide reduces fever and inflammation in models of acute, chronic, and systemic inflammation [15]. Although understanding of its mechanism of action is incomplete, part of a-MSH antiinflammatory influence is exerted through reduced production of mediators by inflammatory cells [14]. a-MSH inhibits the inflammatory cascade at many sites [16–20]: it reduces production of nitric oxide [17], interleukin-1b (IL-1b), tumor necrosis factor a (TNF-a) [18, 20], interferon-g (IFN-g) [16], monocyte chemoattractant protein 1 (MCP-1), and interleukin-8 (IL-8) [18], and markedly decreases the number of inflammatory cells trafficking into sites of injury [18, 19, 21, 22]. a-MSH has little or no stimulatory effect on adrenal steroidogenesis and it is clear that its anti-inflammatory effects are exerted directly on target cells [13]. The antipyretic/anti-inflammatory message sequence of a-MSH (1–13) resides in the COOH-terminal tripeptide a-MSH (11–13) K-P-V [23–25]. This tripeptide has effects on host responses similar to those of the larger a-MSH (1–13) molecule [23–25]. The anti-inflammatory effects of

Abbreviations: CNS, central nervous system; a-MSH, a-melanocytestimulating hormone; ACTH, adrenocorticotropic hormone; NO, nitric oxide; LPS, lipopolysaccharide; IFN-g, interferon-g; POMC, pro-opiomelanocortin; MCP-1, monocyte chemoattractant protein 1; FBS, fetal bovine serum; PBS, phosphate-buffered saline; EDTA, ethylenediaminetetraacetate; ELISA, enzymelinked immunosorbent assay. Correspondence: Dr. Anna Catania, III Division of Internal Medicine (Pad. Granelli), IRCCS Ospedale Maggiore di Milano, Via F. Sforza 35, 20122 Milano, Italy. E-mail: [email protected] Received October 17, 1997; revised February 3, 1998; accepted February 4, 1998.

ACTH (1–39) and ACTH (1–24) have been traced to induction of adrenal corticosteroids. However, it is clear that ACTH peptides have extra-adrenal influences, including antipyretic [26] and nerve recovery [27] effects. Although the main source of melanotropic peptides is the pituitary and these peptides can reach target cells through the circulation [13], there is evidence that melanocortin peptides have autocrine anti-inflammatory influences in monocyte/macrophages. On stimulation of monocytes with endotoxin or cytokines, there is up-regulation of the pro-opiomelanocortin gene and both productions of a-MSH [17, 28] and ACTH [29] are increased. Monocyte expression of melanocortin receptors [17, 28] probably forms an afferent limb of autocrine circuit. The potent inhibitory influence of melanocortin peptides on monocyte products suggest that these molecules could likewise exert anti-inflammatory activity in microglia that share monocyte characteristics. The aims of the present research were to determine whether: (1) a-MSH (1–13), a-MSH (11–13), and ACTH (1–24) can inhibit production of TNF-a, IL-6, and NO by activated microglia; (2) microglia produce a-MSH that would be essential to an autocrine regulatory circuit in these cells; (3) immunoneutralization of endogenous a-MSH enhances production of proinflammatory mediators by activated microglia, thereby supporting in glial cells a functional autocrine circuit based on the peptide.

MATERIALS AND METHODS Cell cultures

blue exclusion for each experimental condition; it was consistently .98%. Dexamethasone (Sigma) and NÃ-monomethyl-L-arginine (L-NMMA; 100 µM, Cayman Chemical, Ann Arbor, MI) were used as positive controls for TNF-a and NO inhibition, respectively. Tests were repeated in at least three independent experiments and assays were performed in triplicate.

Cytokine determinations TNF-a bioactivity was measured in supernatants of cell cultures by standard cytotoxicity assay using L929 cells and recombinant human TNF-a (Sigma) as standard [31]. The detection limit of the bioassay was 20 pg/mL. IL-6 was measured using a commercial murine enzyme-linked immunosorbent assay (ELISA; RPN 2714, Amersham, Little Chalfont, UK).

Nitrite determination NO is rapidly oxidized to nitrite in culture medium, and nitrite (NO22) concentration is an indicator of NO production. Cell-free culture supernatants were mixed with equal amounts of Griess reagent (1% sulfanilamide, 0.1% naphtylethylenediamide in 2.5% phosphoric acid) in wells of 96-well ELISA plates [32]. Samples were incubated at room temperature for 10 min and absorbance was measured at 540 nm with the use of a microplate reader. Nitrite concentrations were calculated using a sodium nitrite standard curve.

cAMP accumulation cAMP accumulation in N9 cells was measured as previously described [33]. Briefly, cells in six-well plates were co-incubated at 37°C with (1) medium; (2) forskolin (100 µM); (3) LPS (10 ng/mL) 1 IFN-g (1 U/mL); (4) a-MSH (1–13) or a-MSH (11–13) (1, 10 , 50 µM); (5) LPS 1 IFN-g and either a-MSH (1–13) or a-MSH (11–13) (10 µM). Reactions were stopped after 3 min by aspirating supernatants, immediately adding 1 mL ethanol at 220°C and freezing. cAMP content in the ethanol-soluble fraction was measured using an enzyme immunoassay kit (Amersham).

Northern blot analysis

The N9 clone of murine microglial cells was obtained by immortalization of embryonic brain cultures with the 3RV retrovirus carrying an activated v-myc oncogene [30]. N9 cells were cultured in T-75-cm2 culture flasks (Corning, Cambridge, MA) and maintained at 37°C in a humidified incubator under 5% CO2 atmosphere in RPMI 1640 supplemented with 2 mM L-glutamine, 50 U/mL penicillin G, 50 µg/mL streptomycin sulfate (GIBCO-BRL, Paisley, UK), and 10% heat-inactivated fetal bovine serum (FBS, Hyclone Lab. Inc., Logan, UT) until experiments were performed. Cells were used between the first and the tenth passage.

Treatments Sub-confluent microglial cells were washed twice with phosphate-buffered saline (PBS) and incubated with trypsin 0.025% and ethylenediaminetetraacetate (EDTA) 0.02% without calcium and magnesium for 3 min at 37°C to detach the cells from the culture flask. Cells were then resuspended in medium and incubated in 24-well tissue-culture plates at a concentration of 2 3 105 cells/mL for 16 h in a humidified incubator (37°C, 5% CO2). Growth medium was removed and cell monolayers were stimulated with 10 ng/mL lipopolysaccharide (LPS, from Escherichia coli 055:B5, Sigma Chemical Co., St. Louis, MO) plus 1 U/mL murine IFN-g (Sigma). To test effects of melanocortin peptides, concentrations (1, 10, 25, 50, and 100 µM) of a-MSH (1–13), a-MSH (11–13) (both kindly provided by Dr. R. Longhi, CNR, Milano, Italy), and ACTH (1–24) (Sigma) were dissolved in medium and added to wells 10 min before treatment with LPS 1 IFN-g. In pilot experiments melanocortin peptides were tested in concentrations ranging from femtomolar to millimolar. Although production of TNF-a and NO was reduced by even much lower concentrations of the peptides, concentrations in the micromolar range had the most profound and consistent inhibitory effects. Although lower concentrations were more effective in previous research, we elected to use micromolar concentrations in the present studies because they were more effective for the experimental conditions (cell type, incubation period, concentration of the stimuli). Cell-free supernatants were harvested after 24 h incubation and assayed for TNF-a, IL-6, and NO22. Viability of cells was assessed by trypan

Total cellular RNA was extracted from 106 adherent microglial cells plated in six-well tissue culture plates (Corning) [34]. Electrophoresis of RNA samples (10 µg/lane) was performed in 1% agarose/2.2 M formaldehyde gels and the gels subsequently blotted onto nylon filters by capillary action and baked for 2 h before prehybridization. The cDNA fragments encoding murine TNF-a [20] and mouse macrophage iNOS [35] were 32P-labeled using the Ready-To-Go DNA Labeling Kit (Pharmacia, Uppsala, Sweden) before hybridization of nylon filters and autoradiography. Blots were subsequently rehybridized with human glyceraldehyde-3-phosphate dehydrogenase (G-3-PDH) cDNA probe as an internal control (Clontech Laboratories, Inc., Palo Alto, CA).

a-MSH production by microglia Microglia were plated in 24-well plates at a concentration of 2 3 105 cells/mL and a-MSH production was determined in cell-free supernatants after 24-h incubation with LPS, 10 ng/mL; IFN-g, 1 U/mL; and LPS, 10 ng/mL 1 IFN-g, 1 U/mL. a-MSH was measured with a double antibody radioimmunoassay (Euro-Diagnostica AB, Malmo¨, Sweden). The sensitivity of the assay is 0.5 pg/mL and cross-reactivity with other POMC peptides (ACTH (1–24), ACTH (1–39), b-MSH, g-MSH) is ,0.002%.

Effects of immunoneutralization of endogenous a-MSH on TNF-a, IL-6, and NO22 production by microglia Microglia as above in RPMI 1640 medium supplemented with 10% FBS were pre-incubated overnight with rabbit anti-a-MSH antibody (Euro-Diagnostica AB) diluted 1:250 with medium. Pretreatment with anti-a-MSH antibody was performed to ensure binding of a-MSH produced by resting microglia during the overnight adherence period. After preincubation, the medium was removed and cells co-incubated with LPS 10 ng/mL, IFN-g 1 U/mL, or LPS 1 IFN-g diluted in 1 mL RPMI 1640 medium (10% FBS) containing the same rabbit anti-a-MSH antibody concentration used during pre-incubation. The cell-free

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supernatants were removed after a 24-h incubation and assayed for TNF-a, IL-6, and nitrite. Control samples were treated with rabbit IgG at the same dilution.

Statistical analysis Effects of melanocortin peptides on cytokine and NO production were evaluated by repeated measures analysis of variance followed by Dunnet’s test for specific comparisons. Probability values less than 0.05 were considered significant.

RESULTS Microglia stimulated with LPS 1 IFN-g for 24 h produced substantial amounts of TNF-a, IL-6, and NO22 and micromolar concentrations of a-MSH (1–13) significantly reduced this production (Fig. 1). a-MSH 10 µM had the greatest inhibitory influence (approximately 43, 31, and 42% for TNF-a, IL-6, and NO22, respectively). The peptide concentration/inhibition pattern was U-shaped, similar to that observed in previous research on a-MSH [13, 24, 28]. The COOH-terminal tripeptide a-MSH (11–13) was likewise a very effective inhibitor of these mediators (45, 50, and 40%; Fig. 2). TNF-a, IL-6, and NO22 production was also inhibited by treatment of microglia with ACTH (1–24) (38, 65, and 41%; Fig. 3). Incubation of microglia with a-MSH (1–13) and a-MSH (11–13) caused significant increases in cAMP accumulation both in resting cells

Fig. 2. The tripeptide a-MSH (11–13) significantly reduced production of TNF-a, IL-6, and NO22 by activated microglia. Bars denote mean 6 SE.

(Fig. 4 ) and in cells co-incubated with LPS 1 IFN-g (Fig. 5). The magnitude of cAMP accumulation induced by a-MSH peptides was comparable to that caused by forskolin (Fig. 5). Northern blot analysis showed that the inhibitory influence of melanocortin peptides on TNF-a and NO22 release was caused by inhibition of mRNA for TNF-a and iNOS, respectively (Fig. 6). In addition to the pro-inflammatory agents, microglia stimulated with LPS 1 IFN-g released a-MSH (Fig. 7). Control incubation of microglia with anti-a-MSH or irrelevant antibodies did not cause mediator release. However, when microglia were stimulated with LPS 1 IFN-g after immunoneutralization of endogenous a-MSH, release of IL-6, TNF-a, and NO22 was significantly enhanced (26, 28, and 27%, respectively) compared to treatment with control IgG (Fig. 8). Measurement of a-MSH in supernatants of anti-a-MSH-treated microglia showed undetectable concentrations of the peptide to indicate successful immunoneutralization. Concentrations of a-MSH in samples from microglia co-incubated with the control IgGs were similar to those observed in cells treated with medium. Therefore, the irrelevant antibody did not alter endogenous a-MSH.

DISCUSSION 2

Fig. 1. LPS 1 IFN-g-stimulated production of TNF-a, IL-6, and NO2 by microglia was significantly inhibited by concentrations of a-MSH (1–13). Bars denote mean 6 SE. *P , 0.05; **P , 0.01.

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The results indicate that the melanocortin peptides a-MSH (1–13), a-MSH (11–13), and ACTH (1–24) all act on activated

Fig. 4. a-MSH peptides (1–13) and (11–13) induced substantial accumulation of cAMP in resting microglia. Bars denote mean 6 SE.

Fig. 3. ACTH (1–24) significantly inhibited production of TNF-a, IL-6, and NO22 by microglia stimulated with LPS 1 IFN-g. Bars denote mean 6 SE.

microglia to inhibit production of proinflammatory mediators TNF-a, IL-6, and NO. Because microglia stimulated with LPS and IFN-g also secreted a-MSH, an autocrine anti-inflammatory circuit based on this potent peptide probably occurs in brain phagocytes, much as in peripheral monocytes. This idea is supported by the observation that immunoneutralization of endogenous a-MSH enhances production of proinflammatory mediators by activated microglia. The specific melanocortin receptor subtypes involved in this autocrine circuit remain to be discovered. Recent research suggests that inflammation occurs in brain disorders, including neurodegenerative disorders. TNF-a and other proinflammatory cytokines have been linked to multiple sclerosis [7], Alzheimer’s disease [10], and HIV-1-associated cognitive-motor complex [36]. TNF-a is found in lesions of multiple sclerosis [7]; in Alzheimer’s disease it is believed to mediate neuronal damage induced by beta amyloid [10]; HIV envelope glycoprotein gp120 causes apoptotic cell death in human brain cells through induction of TNF-a and IL-6 [8]. Like TNF-a, NO is linked to neuronal toxicity [37]. NO is synthesized from L-arginine by the enzyme NO-synthase (NOS), which exists in at least three distinct isotypes. Two of them are calcium-dependent and occur mainly in neurons and endothelial cells (constitutive isoforms). Inducible, calcium-independent NO synthase (iNOS) produces much larger quantities of NO and is mainly expressed by macrophages exposed to cytokines or bacterial products [38, 39]. NO is a neurotransmit-

ter in the brain with major neuroregulatory functions, however, excessive production of NO is toxic to neurons [37]. NO interacts with superoxide anion to produce peroxynitrite anion (ONOO2), which generates highly reactive hydroxyl radical and nitrogen dioxide. Because activated microglia and brain macrophages are the main source of TNF-a and NO within the CNS, molecules that effectively reduce brain inflammation should inhibit activity of these central phagocytes.

Fig. 5. a-MSH (1–13) and a-MSH (11–13) (10 µM) induced substantial accumulation of cAMP in microglia co-incubated with LPS 1 IFN-g. cAMP accumulation was similar to that caused by forskolin (100 µM). Bars denote mean 6 SE.

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Fig. 6. Northern blot analysis showed that inhibitory influence of melanocortin peptides on TNF-a and NO22 production by activated microglia was caused by inhibition of mRNA for TNF-a and iNOS, respectively.

It is likely that the effects of one or more of the melanocortin peptides observed in the present experiments were mediated by subtypes of melanocortin receptors. At this time, five G-proteinlinked receptors (MC-1 R through MC-5 R) have been recognized and cloned [40]. Melanocortin receptors occur both in peripheral tissues and within the brain. The presence of melanocortin receptors in circumventricular organs, which lack the tight blood-brain barrier of the brain, provides direct access of circulating melanocortin hormones to central receptors [40]. Whereas melanocortin receptors on peripheral cells preferentially bind a-MSH and the MC-2 R in the adrenal glands recognizes only ACTH, melanocortin receptors in the brain show similar relative affinities for ACTH and a-MSH. So far there is no evidence that known melanocortin receptors recognize a-MSH (11–13). The idea of certain common receptors for a-MSH (1–13) and (11–13) is supported by parallel effects of the two substances in multiple in vivo and in vitro experiments [13, 14]. However, we recognize that the parallel anti-

Fig. 7. Microglia stimulated with LPS and IFN-a, alone or in combination, released substantial amounts of a-MSH. Bars denote mean 6 SE.

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Fig. 8. LPS 1 IFN-g-stimulated production of TNF-a, IL-6, and NO22 by microglia was significantly enhanced after immunoneutralization of endogenous a-MSH. Bars denote mean 6 SE.

inflammatory effects of the two peptides may indeed occur via separate receptors. We noted previously that a-MSH (11–13) did not affect binding of labeled a-MSH molecules to melanoma cells known to bear MC-1 receptors [41]. Melanocortin receptors are coupled to adenylyl cyclase and induce intracellular cAMP [40]. Increases in this mediator are believed to underlie several of the anti-inflammatory effects of melanocortin peptides, including reduced cytokine production and inhibition of chemotaxis. Furthermore, several cAMPenhancing agents including isoproterenol and forskolin inhibit iNOS expression [42] and TNF-a production [43] by microglia. Because a-MSH peptides caused marked cAMP accumulation in microglia, inhibitory influences were probably exerted by enhancing accumulation of this cell mediator. The finding that activated microglia release a-MSH is consistent with previous observations in murine and human macrophages [17, 28] and suggests that autocrine antiinflammatory influences of a-MSH occur not only in peripheral cells but also in central phagocytes. It is reasonable to believe that toxic/infective stimuli that induce proinflammatory cytokines likewise promote release of mediators such as a-MSH to modulate the inflammatory reaction. Previous research showed that a-MSH inhibits inflammation in the brain. The peptide reduced TNF-a production in brains of mice injected with LPS [20]. The present research indicates that melanocortin peptides inhibit production of proinflamma-

tory cytokines and related mediators by microglia. These observations, together with evidence of anti-inflammatory influences in macrophages [17, 28] and astrocytes [44], suggest that a-MSH and other melanocortin peptides can inhibit inflammation within the brain by acting on central phagocytes.

ACKNOWLEDGMENTS The authors thank Dr. Paola Ricciardi-Castagnoli, Centro di Citofarmacologia, CNR, Milano, Italy, for providing the N9 cell clone and Dr. Renato Longhi, Istituto di Chimica degli Ormoni, CNR, Milano, Italy for providing melanocortin peptides. This work was supported by grants 96/J/T9 Progetto Sclerosi Multipla and 9403-30 IX Progetto AIDS, from the Istituto Superiore di Sanita`, Italy; National Institute of Neurological Diseases and Stroke Grant NS10046; and NATO Collaborative Research Grant CGR 950556.

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