α-Melanocyte-stimulating hormone modulates lipopolysaccharide plus interferon-γ-induced tumor necrosis factor-α expression but not tumor necrosis factor-α receptor expression in cultured hypothalamic neurons

Journal of Neuroimmunology 227 (2010) 52–59

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Journal of Neuroimmunology j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / j n e u r o i m

α-Melanocyte-stimulating hormone modulates lipopolysaccharide plus interferon-γ-induced tumor necrosis factor-α expression but not tumor necrosis factor-α receptor expression in cultured hypothalamic neurons Carla Caruso a,⁎,1, Mónica Sanchez b,1, Daniela Durand a, María de la Cruz Perez a, Patricia V. Gonzalez c, Mercedes Lasaga a, Teresa N. Scimonelli c a b c

Instituto de Investigaciones en Reproducción, Facultad de Medicina, Universidad de Buenos Aires, Buenos Aires, Argentina Unidad CEPROCOR, Agencia Córdoba Ciencia, Córdoba, Argentina IFEC CONICET Departamento de Farmacología, Facultad de Ciencias Químicas, Universidad Nacional de Córdoba, Córdoba, Argentina

a r t i c l e

i n f o

Article history: Received 12 January 2010 Received in revised form 9 June 2010 Accepted 10 June 2010 Keywords: α-MSH TNF-α IL-1β Hypothalamic neurons CREB NF-κB

a b s t r a c t In a previous work we showed that the melanocortin alpha-melanocyte-stimulating hormone (α-MSH) exerts anti-inflammatory action through melanocortin 4 receptor (MC4R) in vivo in rat hypothalamus. In this work, we examined the effect of α-MSH on the expression of tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) and their receptors in primary cultured rat hypothalamic neurons. We also investigated α-MSH's possible mechanism/s of action. α-MSH (5 μM) decreased TNF-α expression induced by 24 h administration of a combination of bacterial lipopolysaccharide (LPS, 1 μg/ml) plus interferon-γ (IFN-γ, 50 ng/ml). Expression of TNF-α and IL-1β receptors TNFR1, TNFR2 and IL-1RI, was up-regulated by LPS + IFN-γ whereas α-MSH did not modify basal or LPS + IFN-γ-induced-TNFRs or IL-1RI expression. Both α-MSH and LPS + IFN-γ treatments increased CREB activation. α-MSH did not modify NF-κB activation induced by LPS + IFN-γ in hypothalamic neurons. In conclusion, our data show that α-MSH reduces TNF-α expression in hypothalamic neurons by a mechanism which could be mediated by CREB. The regulation of inflammatory processes in the hypothalamus by α-MSH might help to prevent neurodegeneration resulting from inflammation. © 2010 Elsevier B.V. All rights reserved.

1. Introduction Glial cells are believed to be the primary source of proinflammatory agents in the inflammatory response, while neuron participation in this process has been less extensively studied. Bacterial lipopolysaccharide (LPS) is a major inflammatory molecule that triggers the production of pro-inflammatory cytokines such as interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) in activated immune cells, and these cytokines play a critical role in mediating the inflammatory response of the host to infection and tissue injury (Hawiger, 2001). Although the production of proinflammatory cytokines has an important role in host defence against invading microbes, overexpression of cytokines may lead to inflammatory disorders (Han and Ulevitch, 2005). In the central nervous system (CNS), IL-1β and TNF-α are released from several cell types including astrocytes and microglia after brain injury. In addition, both

⁎ Corresponding author. Instituto de Investigaciones en Reproducción, Facultad de Medicina, Universidad de Buenos Aires, Paraguay 2155 piso 10, Ciudad de Bs As (1121ABG), Argentina. Tel.: + 54 11 5950 9500x2158; fax: + 54 11 5950 9612. E-mail address: [email protected] (C. Caruso). 1 Both authors contributed equally to this work. 0165-5728/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.jneuroim.2010.06.013

IL-1β and TNF-α have direct influence on neurodegeneration (Thornton et al., 2006; Zhao et al., 2001), and inhibition of endogenous IL-1β and TNF-α protects against neuronal injury that occurs after cerebral ischemia (Meistrell et al., 1997; Relton and Rothwell, 1992). They are also highly expressed in Alzheimer's (Fillit et al., 1991; Griffin et al. 1998) and Parkinson's (Mogi et al., 1994, 1996) diseases. TNF-α can activate two different receptors: TNFR1 and TNFR2. TNFR1 is constitutively expressed in dentate granule neurons and TNFR2 was also detected in these neurons (Harry et al., 2008). On the other hand, the biological effects of IL-1β are mediated by type I IL-1 receptor (IL-1RI) which has been detected in neurons after injury (Ericsson et al., 1995). Type II IL-1 receptor (IL-1RII) acts as a decoy receptor because it can bind IL-1β but does not lead to intracellular signalling (Sims et al., 1993). The signalling pathways that regulate cytokine production have been studied intensively. The best described pathway involves a kinase cascade leading to activation of nuclear factor-κB (NF-κB) transcription factor (Li and Verma, 2002). Many signalling pathways have also been shown to activate the cyclic AMP responsive element binding protein (CREB) (Lonze and Ginty, 2002). This transcription factor is activated by cyclic AMP (cAMP)/protein kinase A (PKA)

C. Caruso et al. / Journal of Neuroimmunology 227 (2010) 52–59

pathway by phosphorylation, activation that is sufficient to induce transcriptional activity of CREB. In addition, Ca++ signalling as well as ERK and p38 mitogen-activated protein kinase (MAPK) pathways can lead to CREB activation (Lonze and Ginty, 2002). The neuropeptide α-melanocyte-stimulating hormone (α-MSH) is a 13 amino acid cleavage product of pro-opiomelanocortin hormone. The effects of α-MSH on immunity are well documented (Catania et al., 2004). Specifically, in the CNS, an immunoprivileged environment, it clearly has anti-cytokine effects and can modulate the production and action of pro-inflammatory cytokines (Catania et al., 2004; Lasaga et al., 2008). Centrally administered α-MSH is a potent antipyretic (Catania et al., 2004) and can also prevent damage in brainstem ischemia and reperfusion injury (Giuliani et al. 2006). A variety of effects of central IL-1β administration are blocked by α-MSH such as activation of the hypothalamic–pituitary–adrenal axis (Cragnolini et al., 2004; 2006a; 2006b) and the anxiogenic behaviour and memory impairment induced by this cytokine (Gonzalez et al., 2009). Two of the five melanocortin receptor subtypes, melanocortin 3 receptor (MC3R) and MC4R, are extensively expressed in the brain and are thought to be the primary mediators of behavioural and immunomodulating effects of melanocortin peptides (Catania et al., 2004; Lasaga et al., 2008). We previously reported that α-MSH through MC4R reduced in vivo hypothalamic expression of inducible nitric oxide synthase and cyclooxygenase-2 induced by LPS (Caruso et al., 2004). We recently demonstrated that α-MSH decreases the release of nitric oxide and prostaglandins induced by LPS and interferon-γ (IFN-γ) in astrocytes through MC4R activation, reducing inflammatory response and also preventing apoptosis induced by LPS and IFN-γ in these cells (Caruso et al., 2007). In the present study, we demonstrate the presence of MC3R and MC4R receptors in cultured hypothalamic neurons. We also determined that pro-inflammatory cytokine TNF-α and its receptors are up-regulated in hypothalamic neurons by treatment with LPS + IFN-γ whereas α-MSH reduces LPS + IFN-γ-induced TNF-α expression but failed to modify cytokine receptor expression. Although the underlying signalling mechanisms involved in this anti-inflammatory action are not clear, we suggest that α-MSH effects could be mediated by CREB activation. 2. Materials and methods

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dissecting microscope and the ventromedial hypothalamic region (delimited by the optic chiasm, lateral hypothalamic sulcus and mammillary bodies) was dissected and stripped of meninges. At this early stage of development the ventromedial hypothalamic region contains the differentiating neurons for the ventromedial hypothalamic nucleus and the arcuate nucleus (Bayer and Altman, 1995). Tissue blocks were placed in 3 ml of 0.25% trypsin (Invitrogen Life Technologies) in phosphate-buffered saline (PBS) for 15 min at 37 °C. Cells were dissociated by repeated passage through a small-bored Pasteur pipette. The resulting cell suspension was centrifuged for 1 min at 1500 rpm and the cell pellet resuspended in 3 ml of DMEM-S (DMEM with Streptomycin 50 μg/ml, Penicillin 50 U/ml, 10% horse serum with N2 and B27 supplements). Cells were plated on poly-Llysine coated culture dishes and maintained with DMEM plus 10% horse serum for 1 h. Then, the medium was replaced entirely by serum-free medium plus N2 mixture. Cell culture was kept in a humidified 37 °C incubator with 5% CO2, then neurons were incubated with LPS 1 μg/ml + IFN-γ 50 ng/ml with or without α-MSH (5 μM) for 1 or 24 h. Purity of cultures was 91.68% ± 1.70 determined by immunofluorescence detection of neurons with MAP2 antibody in three different experiments with a total of 2500 cells (Fig. 1), as previously described by Ferreira and Cáceres (1991).

2.3. Immunocytochemistry Neurons were identified by a polyclonal antiserum raised against anti-microtubule-associated protein 2 antibody (MAP2, 1:1000), and astroglial cells were identified by a monoclonal anti-glial fibrillary acidic protein antiserum (GFAP, 1:200). Cells were fixed for 30 min at room temperature with 4% (wt/vol) paraformaldehyde in PBS containing 4% (wt/vol) sucrose. Cells were washed with PBS, permeabilized with 0.2% (vol/vol) Triton X-100 in PBS for 5 min, and washed again with PBS. Cell were incubated with primary antibodies (1–3 h at room temperature), washed with PBS, and then incubated with Alexa 488 (dilution 1:600) or Alexa 564 (dilution 1:600) conjugated secondary antibodies (1 h at 37 °C), then washed with PBS and the coverslips mounted using FluorSave (Calbiochem, La Jolla, CA). Cells were visualized with an inverted Zeiss microscope, and images (8 bites) were collected using a CCD camera (Orca 1000, Hamamatsu Corp., Middlesex, NJ) and Metamorph software (Molecular Devices).

2.1. Reagents LPS (Escherichia coli, serotype O127:B8) was purchased from Sigma-Aldrich Corporation (MO, USA). α-MSH was obtained from Bachem California Inc. (CA, USA). Interferon-γ (IFN-γ) was purchased from Boehringer Ingelheim, Argentina. Horse serum was obtained from PAA laboratories GmBH (Pasching, Austria). DMEM, antibiotics, N2 and B27 supplements and all RT-PCR reagents were purchased from Invitrogen Life Technologies (CA, USA) unless specified otherwise. Biotinylated donkey anti-mouse and anti-rabbit antibodies were obtained from Chemicon International Inc. (CA, USA). AntiTNFR1, anti-TNFR2 and anti-IL-1R1 antibodies were purchased from Abcam (MI, USA). Anti-NF-κB p65 was obtained from BD Biosciences (CA, USA) and anti-IκBα was from Cell Signaling Technology (MA, USA). TNF-α, TNFR1, TNFR2, IL-1β, IL-1R1, and β-actin primers were purchased from Invitrogen Life Technologies (CA, USA). MC3R and MC4R primers were obtained from Integrated DNA Technologies Inc. (IA, USA). All other media and supplements were obtained from Sigma-Aldrich Corporation unless specified otherwise. 2.2. Cell culture Pregnant Wistar rats were anesthetized and 16 day old fetuses were aseptically removed. Cerebral hemispheres were placed under a

Fig. 1. Hypothalamic neurons in culture. Hypothalamic neurons were cultured in DMEM-S. Cells were identified using MAP2 (green) as neurons and using GFAP (red) antibody as glial cells.

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2.4. Reverse transcriptase-polymerase chain reaction (RT-PCR)

2.6. Preparation of nuclear extracts

Total RNA from cultured hypothalamic neurons was extracted using TRIZOL reagent (Invitrogen Life Technologies) according to the manufacturer's protocol. 5 μg of total RNA was treated with 1 U RNase-free DNase (Promega Corporation, WI, USA) at 37 °C for 10 min and reverse transcribed as described previously (Caruso et al., 2007). Amplification was performed with 2 μl of cDNA as template in 50 μl PCR reaction volume containing 1–2 mM MgCl2, 0.2 mM of each dNTP, 50 pmol of each primer and 2.5 U of Taq DNA polymerase in the buffer provided by the manufacturer. Temperature cycles always had an initial denaturation step at 94 °C for 5 min and a final extension period of 7 min at 72 °C. Synthetic oligonucleotides used for PCR, annealing temperature, number of cycles and product size are listed in Table 1. 15 μl of each reaction was analyzed on 2% agarose gels, stained with ethidium bromide and visualized using UV light. RT-PCR products were quantitatively analyzed using SCION Image software. Results were normalized to the internal control β-actin. Values were expressed as relative increments of respective controls. Experiments always included non-reverse transcribed DNAse treated RNA samples as negative controls. PCR of these RT-controls never showed amplification, indicating that the RNA was free of DNA.

Neurons were treated for 1 h with the drugs and then nuclear extracts were prepared using Dignam's method (Dignam et al., 1983) with slight modifications. Cells were harvested by adding 0.05% Trypsin–EDTA (Gibco), washed twice with ice-cold PBS and lysed into buffer A (10 mM HEPES, pH 7.9, 10 mM KCl, 2 mM MgCl2, 0.5 mM dithiothreitol, 0.1% nonidet p-40, 1 mM phenylmethylsulfonyl fluoride, 5 μg/ml aprotinin, 5 μg/ml pepstatin A, 5 μg/ml leupeptin, 500 μM sodium orthovanadate, and 1 mM sodium fluoride) for 10 min on ice and centrifuged at 14,000 rpm for 5 min. The supernatant containing cytoplasmatic proteins was removed and stored at −70 °C until use. The pellet was resuspended in buffer B (20 mM HEPES, pH 7.9, 25% glycerol, 0.42 M NaCl, 1.5 mM MgCl2, 0.5 mM dithiothreitol, 0.2 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 5 μg/ml aprotinin, 5 μg/ml pepstatin A, 5 μg/ml leupeptin, 500 μM sodium orthovanadate and 1 mM sodium fluoride). After incubation on ice for 30 min, lysates were centrifuged at 14,000 rpm for 15 min and the supernatants containing nuclear proteins were stored at −70 °C until use.

2.5. Western blot analysis Neurons were lysed in a buffer containing 1% Igepal, 1% sodium dodecyl sulphate, 150 mM NaCl, 0.02% sodium azide, 10 μg/ml aprotinin, 10 μg/ml leupeptin, 5 μg/ml pepstatin A and 2 mM phenylmethylsulfonyl fluoride in Tris–HCl 50 mM (pH 7.4). Following centrifugation at 12,000 rpm for 30 min, the supernatant was used for immunoblot assay. The protein concentration of samples was determined by Bradford protein assay. 25–30 μg of protein was sizefractionated in an SDS-polyacrylamide gel and then electrotransferred to polyvinylidene difluoride membrane. Blots were blocked for 2 h in 5% non-fat dry milk–TBS–0.1% Tween 20 and incubated overnight with the appropriate primary antibody (TNFR1 1:1000, TNFR2 1:500, IL-1RI 1:1000, NF-κB p65 1:400 and IκBα 1:2000) in 5% milk–TBS– 0.1% Tween 20 at 4 °C. This was followed by 1 h incubation with the respective biotinylated secondary antibody. Immunoreactivity was detected by enhanced chemiluminescence (ECL plus, Amersham Biosciences, GE Healthcare). Results were normalized to the internal control β-actin. Values were expressed as relative increments of respective controls.

2.7. Electrophoretic mobility shift assay Nuclear extracts from treated hypothalamic neurons were used for the electrophoretic mobility shift assay (EMSA) which was effected essentially as previously described (Garner and Revzin, 1981). CREB consensus sequence was end labeled with [α-32P]dATP. The 32P endlabeled CRE oligonucleotide (0.5 ng) was incubated with nuclear extracts (5 μg) in a total volume of 20 μl containing 10 mM Tris, pH 7.5, 50 mM NaCl, 1 mM EDTA, 1 mM dithiothreitol, 1 μg poly d(I–C), 1 μg BSA and 10% glycerol. After 20 min incubation at room temperature, binding reactions were analyzed by electrophoresis on a 5% polyacrylamide gel in 0.25× TBE buffer (TBE: 90 mM Tris, 90 mM boric acid, 2 mM EDTA) and visualized by autoradiography. Competition experiments were performed by incubating nuclear proteins with the appropriate unlabeled oligonucleotide for 10 min prior to addition of the labeled probe.

2.8. Statistical analysis Data were expressed as mean ± SEM and were analyzed by one sample t test or one-way analysis of variance (ANOVA) followed by Bonferroni Multiple Comparisons Test. Differences with p b 0.05 were considered statistically significant. All experiments were performed at least twice.

Table 1 Oligonucleotide sequences of PCR primers. Sequence

MC4R

F 5′-GGCTTCACATTAAGAGGATCGCT-3′ R 5′-TTTATGGAACTCCATAGCGCCC-3′ MC3R F 5′-AGCAACCGGAGTGGCAGT-3′ R 5′-GGCCACGATCAAGGAGAG-3′ TNF-α F 5′-TACTGAACTTCGGGGTGATTGGTCC-3′ R 5′-CAGCCTTGTCCCTTGAAGAGAACC-3′ TNFR1 F 5′-GACTGGTTCCTTCTCTTGGT-3′ R 5′-GGTGTTCTGTTTCTCCTTAC-3′ TNFR2 F 5′-GTTCTCTGACACCACATCATCC-3′ R 5′-GTCAATAGGTGCTGCTGTTCAA-3′ IL-1β F 5′-GACCTGTTCTTTGAGGCTGAC-3′ R 5′-TCCATCTTCTTCTTTGGGTATTGTT-3 IL-1R1 F 5´-CGAAGACTATCAGTTTTTGGAAC-3′ R 5′-GTAACCTCGATGGTATCTTCCC-3′ β-actin F 5´-ACCACAGCTGAGAGGGAAATCG–3′ R 5′-AGAGGTCTTTACGGATGTCAACG-3′

Number Size Annealing of cycles (bp) temperature (°C) 33

595

65

30

421

63

35

295

65

29

407

62

35

456

60

31

578

62

35

495

60

22-24

289

60–65

Fig. 2. MC4R and MC3R are expressed in hypothalamic neurons. Hypothalamic neurons were cultured in DMEM-S. Total RNA isolated from neurons was processed for RT-PCR as described in “Materials and methods”. mRNA levels of MC4R (595 bp) and MC3R (421 bp) were determined. Lanes: 1 hypothalamic neurons and 2 hypothalamic neurons RT negative control.

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Fig. 3. α-MSH reduces TNF-α expression induced by LPS + IFN-γ in hypothalamic neurons. RNA was isolated from hypothalamic neurons treated with LPS/IFN-γ in the presence or absence of α-MSH for 24 h and processed for RT-PCR as described in “Materials and methods”. A) TNF-α mRNA levels were determined and expressed as mean ± SEM of 3–5 determinations per group. Values represent the relative increment of TNF-α/β-actin ratio and were expressed relative to control group. 3 independent experiments were analyzed. **p b 0.01 vs. control group and ^p b 0.05 vs. LPS + IFN-γ. B–C) TNFR1 and TNFR2 mRNA levels were determined and expressed as mean ± SEM of 3–4 determinations per group. Values represent the relative increment of TNFR1/β-actin (B) or TNFR2/β-actin (C) ratio and were expressed relative to control group. 3 independent experiments. *p b 0.05 and **p b 0.01 vs. control group. D–E) Cell lysates from hypothalamic neurons treated for 24 h with LPS/IFN-γ in the presence or absence of α-MSH were assayed by Western blot. Membranes were probed with antibodies to TNFR1 (D), TNFR2 (E) and β-actin. Figure shows blots of one representative experiment of 2 independent ones for each receptor.

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3. Results 3.1. MC4R and MC3R are expressed by hypothalamic neurons We recently demonstrated that MC4R was expressed in cultured rat astrocytes and that this receptor was involved in α-MSH antiinflammatory effects, whereas MC3R expression was not found in these cells (Caruso et al., 2007). Since MC4R and MC3R are the predominant subtypes expressed in the brain, we determined the presence of these receptors in cultured hypothalamic neurons. As expected, MC4R was expressed in hypothalamic neurons and MC3R expression was also found in these cells (Fig. 2). 3.2. α-MSH reduces LPS + IFN-γ-induced TNF-α expression but not TNFR expression in hypothalamic neurons Treatment with LPS + IFN-γ significantly increased TNF-α mRNA levels in hypothalamic neurons (Fig. 3A). α-MSH (5 μM) decreased the stimulatory effect of LPS + IFN-γ on TNF-α expression (Fig. 3A). α-MSH by itself did not significantly modify TNF-α expression (Fig. 3A). Since inflammation could be inducing the expression of cytokine receptors, α-MSH could possibly inhibit cytokine action not only by reducing their release but also by modulating the expression of their receptors. Therefore, we determined the expression of TNFR1 and TNFR2 in hypothalamic neurons. LPS + IFN-γ significantly increased TNFR1 and TNFR2 mRNA and protein levels in neurons (Fig. 3B–E). α-MSH failed to modify LPS + IFN-γ stimulatory effects on TNFR1 or TNFR2 expression (Fig. 3B–E) whereas by itself it did not significantly modify the expression of any of the receptors tested. 3.3. LPS + IFN-γ does not modify IL-1β expression in hypothalamic neurons Since IL-1β is another cytokine involved in inflammatory processes we determined its expression in hypothalamic neurons. LPS + IFN-γ did not modify IL-1β mRNA levels in these neurons and neither did α-MSH (data not shown). LPS + IFN-γ increased IL-1R1 mRNA levels in hypothalamic neurons but α-MSH failed to significantly modify basal or LPS + IFN-γ-induced IL-1R1 expression (Fig. 4).

Fig. 4. α-MSH does not modify IL-1RI expression. RNA was isolated from hypothalamic neurons treated with LPS/IFN-γ in the presence or absence of α-MSH and processed for RTPCR as described in “Materials and methods”. (A) IL-1RI mRNA levels were determined and expressed as mean ± SEM of 3–4 determinations per group. Values represent the relative increment of IL-1RI/β-actin ratio and were expressed relative to control group. 3 independent experiments were analyzed. *pb 0.05 and **p b 0.01 vs. control group. (B) Cell lysates from hypothalamic neurons treated for 24 h with LPS/IFN-γ in the presence or absence of α-MSH were assayed by Western blot. Membranes were probed with antibodies to IL-1RI and β-actin. Figure shows blots of one representative experiment of 2 independent ones.

3.4. α-MSH effects on NF-κB and CREB activation α-MSH has been shown to modulate NF-κB activation by inflammatory stimuli in several cell lines (Manna and Aggarwal, 1998). Activation of NF-κB signalling was determined by p65 subunit translocation to the nucleus and degradation of IκBα, which is necessary for nuclear translocation of NF-κB. We found a decrease in cytoplasmatic levels of IκBα protein with a subsequent increase in NF-κB translocation to the nucleus after treatment with LPS + IFN-γ (Fig. 5A). α-MSH had no effect on NF-κB activation in our experimental model (Fig. 5A). We also examined the activation of CREB, a transcription factor present in many types of neurons in the CNS (Sarkar et al., 2002; Srinivasan et al., 2004). Besides MCRs are known to increase cAMP production, it is possible that α-MSH may activate CREB via cAMP-PKA pathway. To investigate this possibility we measured CREB activation by EMSA. LPS + IFN-γ and α-MSH treatments increased CREB activation in hypothalamic neurons whereas both treatments together increased it further (Fig. 5B), indicating that both treatments had additive effects. 4. Discussion Neuron participation in the inflammatory response is considered less important than that of glia. Here, we show that neurons respond to an inflammatory stimulus such as LPS + IFN-γ by releasing TNF-α. We also show that melanocortins can exert anti-inflammatory action in hypothalamic neurons which express MC3R and MC4R. Since the

major site of α-MSH synthesis in the brain is the arcuate nucleus of the hypothalamus (O´Donohue and Dorsa, 1982) and it is also released in the hypothalamus in response to LPS (Caruso et al., 2004; Huang et al., 1997) this neuropeptide could be acting on hypothalamic neurons as a natural countermeasure to inflammation. Melanocortins are an ancient regulatory system with a variety of influences on the host including anti-inflammatory, immunomodulatory and anti-microbial effects. α-MSH is known to reduce the release of cytokines, chemokines and other pro-inflammatory factors produced by immune cells (Catania et al., 2004). In a previous work we showed that α-MSH reduces the expression of inducible nitric oxide synthase and cyclooxygenase-2 induced in vivo by LPS in rat hypothalamus via MC4R (Caruso et al., 2004). We also demonstrated that α-MSH attenuates the release of prostaglandins induced by IL-1β during hypothalamic–pituitary–adrenal axis activation, also acting on MC4R (Cragnolini et al., 2004). Our results show that TNF-α expression in hypothalamic neurons is increased by LPS + IFN-γ. Supporting this, TNF-α release is induced by LPS in hippocampal neurons (Chiou et al., 2006) whereas its expression was up-regulated by LPS in dopaminergic neurons (Kim et al., 2009). Our data also show that α-MSH may exert anti-inflammatory action in hypothalamic neurons since it attenuates LPS + IFN-γ-induced TNF-α expression in these cells. Concordantly, α-MSH diminishes TNF-α expression after cerebral ischemia (Huang and Tatro, 2002) and TNF-α secretion from microglia (Delgado et al., 1998).

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Fig. 5. α-MSH activates CREB but fails to modify LPS + IFN-γ induced NF-κB activation in hypothalamic neurons. (A) Neurons were treated with LPS/IFN-γ in the presence or absence of α-MSH for 1 h. Nuclear cell extracts were prepared as described in Materials and methods. Nuclear and cytoplasmatic proteins were assayed by Western blot. Membranes with nuclear proteins were probed with anti-NF-κB and β-actin, and cytoplasmatic proteins were probed with anti-IκBα and β-actin. Values represent the relative increment of NF-κB/β-actin ratio or IκBα/β-actin and were expressed relative to control group. Bars represent the media± SEM of 2 independent experiments. *pb 0.05 vs. control group. (B) Semiquantification of CREB activation. Neurons were treated with or without LPS/IFN-γ in the presence or absence of α-MSH for 1 h and then harvested for isolation of nuclear proteins and subsequent detection of CRE/CREB complexes by EMSA. CRE/CREB complexes were not detected when only the probe (CRE*) is present, showing assay specificity. Values represent the increment of CREB and were expressed relative to control group. Bars represent the media± SEM of 2 independent experiments. ^pb 0.05 vs. α-MSH.

Recognition and cloning of melanocortin receptors (MCR1 to 5) have greatly improved understanding of melanocortin effects on target organs. MC1R and MC3R are present in immune cells (Catania et al., 2004) and are candidates to mediate systemic anti-inflammatory actions of melanocortins. MC1R is expressed in only a few neurons (Xia et al., 1995) while MC4R expression is found in several brain areas such as the hypothalamus (Mountjoy et al., 1994), along

with that of MC3R (Roselli-Rehfuss et al, 1993). Also, α-MSH exerts anti-inflammatory action by decreasing NF-κB activation in brains of mice with nonfunctional MC1Rs (Ichiyama et al., 1999a), thereby suggesting that this receptor subtype is not involved in the antiinflammatory effects of α-MSH in the CNS. Here, we demonstrated the presence of MC3R and MC4R in hypothalamic neurons in culture. Although MC3R and MC4R activation could prevent IL-1β-induced

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activation of hypothalamic–pituitary–adrenal axis (Cragnolini et al., 2004), MC3R agonists failed to be neuroprotective in some experimental models (Giuliani et al., 2006; Sharma et al., 2006). Since MC4R has neuroprotective and anti-apoptotic actions in the CNS (Caruso et al., 2007; Giuliani et al., 2006) and its activation is antiinflammatory in astrocytes (Caruso et al., 2007) it is the best candidate to mediate the anti-inflammatory actions of α-MSH in hypothalamic neurons. Although IL-1 has low constitutive expression in the brain, it has been reported that LPS can induce up-regulation of IL-1 expression in cultured hippocampal neurons (Chiou et al., 2006). However, in our experimental conditions LPS + IFN-γ did not induce IL-1β expression in hypothalamic neurons. The reason for this is not clear. Since peripheral LPS administration increases IL-1β in the hypothalamus (Nguyen et al., 1998), we expected that LPS + IFN-γ would induce IL1β expression in hypothalamic neurons, but this was not the case. While there are several studies of IL-1β actions in the hypothalamus (Cragnolini et al., 2004; 2006a) no reports show IL-1β expression in vitro in hypothalamic neurons. Therefore, it is possible that IL-1β production cannot be up-regulated by LPS + IFN-γ in cultured hypothalamic neurons and that inflammation-induced IL-1β may derive from glial cells in the hypothalamus. Biological responses to TNF-α are thought to reflect the balance of multiple signals delivered via both TNFR1 and TNFR2. In this study we show that LPS + IFN-γ increase TNFR1 and TNFR2 expression in hypothalamic neurons. Both TNFRs are present in the brain, in dentate granule neurons (Harry et al., 2008), and in spinal cord injury their expression was reported to be up-regulated in rats (Yan et al., 2007). Our data shows that an inflammatory stimulus like LPS + IFN-γ increases TNFR1 and TNFR2 expression. To our knowledge ours is the first report showing TNFR1 and TNFR2 expression in cultured hypothalamic neurons. Previous studies showed that IL-1RI is expressed in hypothalamic neurons (Ericsson et al., 1995). Our present results also indicate that cultured hypothalamic neurons express IL-1RI and that LPS + IFN-γ increases this expression. Concordantly, IL-1RI expression is increased by LPS in mouse brain (Gabellec et al., 1996). Interestingly, our data show that α-MSH does not modify the expression of any of the cytokine receptors studied. Also, α-MSH was reported to fail to modify cytokine receptors expression in macrophages (Sarkar et al., 2003). The present results suggest that different mechanisms may be involved in the control of cytokine and cytokine receptor expression in hypothalamic neurons. During damage cause by vascular, inflammatory or traumatic brain injury there is production of effector molecules including cytokines and adhesion molecules. A feature common to these detrimental mediators is that their production is controlled by NF-κB. NF-κB is present in neurons where it is involved in synaptic activity and longterm potentiation (Meberg et al., 1996), but it is not yet clear whether this factor has a neuroprotective or neurodegenerative function. Here, we show that LPS + IFN-γ activates NF-κB in hypothalamic neurons. However, cell type and stimulus specificities have been described. It was reported that inflammatory stimuli do not activate NF-κB in hippocampal (Srinivasan et al., 2004) or cortical (Jarosinski et al., 2001) neurons, although activation of NF-κB by cytokines in sensory neurons (Yang et al., 2009) and by global ischemia in vivo in CA1 neurons (Clemens et al., 1997) has been reported. α-MSH and other natural or synthetic melanocortin receptor ligands protect IκBα protein from phosphorylation and consequently inhibit NF-κB activation in peripheral tissues and in the brain (Ichiyama et al., 1999a; 1999b). Although α-MSH inhibits activation of NF-κB in different cell lines (Manna and Aggarwal, 1998), we found that α-MSH does not affect NF-κB activation in hypothalamic neurons. While there are no reports on α-MSH effects on NF-κB activation specifically in neurons, there are controversial reports on α-MSH action on NF-κB activation in the CNS. Sarkar et al. (2003) reported that α-MSH did not modify NF-κB activation in H4 glioma cells while

Ichiyama et al. found that α-MSH reduced NF-κB activation by LPS in A-172 human glioma cells and in brain inflammation (1999b). However, unlike other molecules that completely abolish NF-κBmediated transcription, α-MSH only reduces it and therefore modulates the inflammatory response. This could have beneficial consequences since NF-κB signalling also exerts protective action in the CNS (Simard and Rivest, 2007). Since α-MSH (Yoon et al., 2003) and the melanocortin analogue NDP-MSH (Giuliani et al., 2006) were reported to inhibit LPS-induced activation of p38 kinase, it can be suggested that the suppressant effect of α-MSH on LPS + IFN-γinduced TNF-α production could be mediated by inhibition of p38 MAPK. CREB activation is known to occur in response to many types of stimuli in the same cell type (Lonze and Ginty, 2002). Aside from the classical cAMP-PKA pathway, factors such as mitogen- and stressactivated protein kinase-1 and the mitogen-activated protein kinases ERK and p38 can also activate CREB (Lonze and Ginty, 2002). Since MCRs are G protein coupled receptors that activate adenylate cyclase, α-MSH was expected to induce CREB activation in hypothalamic neurons as we observed in this work. Concordantly, α-MSH in vivo induces phosphorylation of CREB in neurons of the paraventricular nucleus in rats (Sarkar et al., 2002). In our model, α-MSH activates CREB and therefore promotes transcription of CREB-target genes, most likely via cAMP pathway, which may be involved in α-MSH antiinflammatory effects. On the other hand, we showed that LPS + IFN-γ treatment also increases CREB activation in neurons. Supporting this, some studies show that CREB is activated by LPS in hippocampal neurons (Chiou et al., 2006). Since inhibitors of ERK and p38 MAPKs blocked CREB phosphorylation induced by LPS in astrocytes (Buzas et al., 2002), we may speculate that LPS + IFN-γ activation of CREB in hypothalamic neurons could be mediated by activation of MAPKs. This could lead to the activation of a completely different subset of CREBtarget genes that are probably involved in the inflammatory response. In summary, the anti-inflammatory effect of α-MSH in the CNS can be exerted in hypothalamic neurons. α-MSH can reduce cytokine levels without affecting cytokine receptor expression, probably by activating CREB and not involving the NF-κB pathway. However, much work remains to elucidate mechanisms of the anti-inflammatory effects of α-MSH in the CNS and to further the development of therapeutic strategies involving melanocortins. Acknowledgments This work was supported by grants from the Agencia Nacional de Promoción Científica y Tecnológica, University of Buenos Aires, Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET) and Secretaría de Ciencia y Técnica de la Universidad Nacional de Córdoba (SeCYT), Argentina. References Bayer, S.A., Altman, J., 1995. Neurogenesis and neuronal migration. In: Paxinos, G. (Ed.), The Rat Nervous System. Academic Press, San Diego, pp. 1041–1078. Buzas, B., Rosenberger, J., Kim, K.W., Cox, B.M., 2002. Inflammatory mediators increase the expression of nociciptin/orphanin FQ in rat astrocytes in culture. Glia 39, 237–246. Caruso, C., Mohn, C., Karara, A.L., Rettori, V., Watanobe, H., Schiöth, H.B., Seilicovich, A., Lasaga, M., 2004. Alpha-melanocyte-stimulating hormone through melanocortin 4 receptor inhibits nitric oxide synthase and cyclooxygenase expression in the hypothalamus of male rats. Neuroendocrinology 79, 278–286. Caruso, C., Durand, D., Schiöth, H.B., Rey, R., Seilicovich, A., Lasaga, M., 2007. Activation of melanocortin 4 receptors reduces the inflammatory response and prevents apoptosis induced by lipopolysaccharide and interferon-γ in astrocytes. Endocrinology 148, 4918–4926. Catania, A., Gatti, S., Colombo, G., Lipton, J.M., 2004. Targeting melanocortin receptors as a novel strategy to control inflammation. Pharmacol. Rev. 56, 1–29. Chiou, S.H., Chen, S.J., Peng, C.H., Chang, Y.L., Ku, H.H., Hsu, W.M., Ho, L.L., Lee, C.H., 2006. Fluoxetine up-regulates expression of cellular FLICE-inhibitory protein and inhibits LPS-induced apoptosis in hippocampus-derived neural stem cell. Biochem. Biophys. Res. Commun. 343, 391–400.

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