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Urocortin 1 and Urocortin 2 induce macrophage apoptosis via CRFR2
FEBS 29764
FEBS Letters 579 (2005) 4259–4264
Urocortin 1 and Urocortin 2 induce macrophage apoptosis via CRFR2 Christos Tsatsanisa,*, Ariadne Androulidakia, Erini Dermitzakia, Ioannis Charalampopoulosb, Joachim Spiessc, Achille Gravanisb, Andrew N. Margiorisa,* a
Department of Clinical Chemistry-Biochemistry, School of Medicine, University of Crete, Heraklion, Crete GR-710 03, Greece b Department of Pharmacology, School of Medicine, University of Crete, Heraklion, Crete GR-710 03, Greece c Department of Molecular Neuroendocrinology, Max Planck Institute for Experimental Medicine, 37075 Goettingen, Germany Received 5 January 2005; revised 11 June 2005; accepted 19 June 2005 Available online 18 July 2005 Edited by Michael R. Bubb
Abstract Macrophages undergo apoptosis as a mechanism of regulating their activation and the inflammatory reaction. Macrophages express the Corticotropin-Releasing Factor Receptor-2 (CRFR2) the endogenous agonists of which, the urocortins, are also present at the site of inflammation. We have found that urocortins induced macrophage apoptosis in a dose- and time-dependent manner via CRFR2. In contrast to lipopolysaccharide (LPS)-induced apoptosis, the pro-apoptosis pathway activated by urocortins involved the pro-apoptotic Bax and Bad proteins and not nitric oxide, JNK and p38MAPK characteristic of LPS. In conclusion, our data suggest that endogenous CRFR2 ligands exert an anti-inflammatory effect via induction of macrophage apoptosis. 2005 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. Keywords: Macrophage; Urocortin; CRFR2; Apoptosis; Bax
1. Introduction Following their activation, macrophages undergo apoptosis as a mechanism to contain inflammation [1]. Bacterial lipopolysaccharide (LPS) activates macrophages and promotes their apoptosis via induction of the nitric oxide NO pathway [2,3]. The corticotropin-releasing factor (CRF) family of peptides and their receptors are expressed in neural and non-neural cells including immune cells [4,5]. Urocortin 1 (UCN1), a member of the CRF family, is detected in lymphoid organs including spleen and thymus, the gastro-intestinal tract and heart [6,7] while Urocortin 2 (UCN2) [8] and Urocortin 3 (UCN3) [9] are mainly expressed in skin and muscle. Urocortins utilize two different receptors, CRFR1 and CRFR2 [10,11]. UCN1 has high affinity towards CRFR2 but it also binds strongly to CRFR1, while UCN2 and UCN3 are exclusive ligands of CRFR2 [8,9]. Several published reports suggest that urocortins may play an anti-inflammatory role. Indeed, in gastric biopsies of patients with Helicobacter pylori-induced gastritis, UCN1 is
*
Corresponding authors. Fax: +30 2810 394581. E-mail addresses: [email protected] (C. Tsatsanis), [email protected] (A.N. Margioris).
significantly elevated during the stage of eradication of H. pylori and the amelioration of the inflammatory response while it fails to do so in non-responders [12]. Furthermore, UCN1 tested in a systemic inflammation model appears to exert a direct effect on the cascade of events taking place in inflammation suppressing LPS-induced TNF-a production independently of corticosterone [13]. UCN1 also suppresses skin edema induced by thermal injury [14]. It thus appears that urocortins may act as endogenous anti-inflammatory agents. Aim of this work was to test the hypothesis that the anti-inflammatory effect of urocortins involves induction of macrophage apoptosis.
2. Materials and methods 2.1. Reagents and antibodies UCN1, Escherichia coli-derived LPS (serotype 0111:B4), a-helical CRF[9–41], propidium iodide were purchased from Sigma Chemicals Co. (St. Louis, MO) and 7-amino-actinomycinD (7-AAD) was provided by ICN Biomedicals, Inc. (Costa Mesa, CA). UCN2 and antisauvagine30 were chemically synthesized. Thermoscript RT and Platinum Taq polymerase were purchased from Invitrogen (NY, NY), anti-Bax from Santa Cruz Biotechnology Inc. (Santa Cruz, CA), anti-Bad from Cell Signalling Technology Inc. (Beverly, MA) and anti-actin from Chemicon (Temecula, CA). All other chemicals and reagents were obtained from Sigma, if not stated otherwise. 2.2. RAW 264.7 cell culture RAW 264.7 cells (derived from ATCC) were cultured in DulbeccoÕs modified EagleÕs medium supplemented with 10% fetal calf serum (FCS), 10 mM L -glutamine, 100 U/ml penicillin, 0.1 mg/ml streptomycin (all purchased from Invitrogen), at 5% CO2 and 37 C. Cells were plated in 25 cm2 flasks at a concentration of 4 · 105/ml and growth medium was replaced with DMEM that does not contain FCS 12 h prior to stimulation. Cells were incubated with LPS plus/minus the specified neuropeptides for 24 h. 7AAD staining or pro-apoptotic protein expression measured apoptosis. In the experiments where inhibitors were used, cells were treated with the inhibitor 1 h prior to stimulation with each neuropeptide. Parallel control cells treated only with the diluent were collected at the same time points. 2.3. Isolation and stimulation of thioglycollate-elicited macrophages A 4% thioglycollate solution was prepared and autoclaved 2 days prior to administration. 1.5 ml of the thioglycollate solution was injected intraperitoneally in C57BL/6 mice and peritoneal macrophages were isolated by lavage of the peritoneal cavity with DulbeccoÕs modified medium [15]. Cells were then cultured in DMEM supplemented with 10% FCS, 10 mM L -glutamine, 100 U/ml penicillin, 0.1 mg/ml streptomycin (Gibco).
0014-5793/$30.00 2005 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.febslet.2005.06.057
4260 2.4. Quantitation of nucleosome fragmentation Cells were plated in 96-well plates at an initial concentration of 10 000 cells per well for 24 h. Apoptosis was measured by direct determination of nucleosomal DNA fragmentation with the ‘‘cell death detection’’ ELISA plus kit (Cat No. 1 774 425, Boehringer Mannheim GmbH, Mannheim, Germany). Briefly, cells were harvested and lysed according to the manufacturerÕs protocol. The mono- and oligo-nucleosomes contained in cell lysates were determined using an anti-histonebiotin antibody, which binds to histones and reacts with single- and double-stranded DNA. Data were expressed in photometric units. Each unit corresponds to approximately 12 500 apoptotic cells. The concentration of the nucleosomes was determined photometrically using 2,2 0 -azino-di[3-ethylbenzthiazolin-sulfonate] as substrate for the biotin present on the antibody. The optical density was read on a Dynatech MicroElisa Reader (Chantilly, VA) at 405 nm. Each experiment was repeated three times and each sample in triplicate.
C. Tsatsanis et al. / FEBS Letters 579 (2005) 4259–4264
ratio 1:1 between control and treated cells. Control cells were exposed to the diluent only, for the same period as treated cells. Basal apoptosis was assigned as 100%. UCN1 and UCN2 induced apoptosis reaching a peak between 108 and 1010 M (Fig. 1A). Subsequently, cells were exposed to UCN1 or UCN2 at 109 M and apoptosis was measured at several time points. UCN1 and UCN2 induced apoptosis, which became detectable at 3 h reaching a peak at 24 h (Fig. 1B). It should be noted that modest changes of macro-
2.5. Quantitation of apoptotic cells The APOPercentage Apoptosis Assay (Biocolor Ltd., Belfast, N. Ireland) uses a dye that stains red the apoptotic cells undergoing the membrane flip-flop event when phosphatidylserine is translocated to the outer leaflet. Apoptosis was quantified by measuring the dye incorporated in apoptotic cells following cell lysis, at 550 nm (reference filter 620 nm) using a color filter microplate colorimeter (Dynatech MicroElisa reader Chantilly, VA) as previously described [16,17]. Each sample was measured in triplicate and each experiment was repeated three times. 2.6. Flow cytometric analysis of apoptotic cells Cells were treated in 6-well plates as described above and harvested in culture medium containing 7-AAD. Following 15-min incubation, cells were washed in cold PBS and re-suspended in PBS containing propidium iodide (PI) (1 lg/ml) to detect both necrotic and late apoptotic cells that were excluded from the analysis. Cells were visualized and analyzed on a flow cytometer (Epics Elite Coulter, UK). The percentage of 7AAD positive/PI negative cells that represent apoptotic population was compared to live cells that were 7AAD negative/PI negative. 2.7. RT-PCR Primers for actin were sense, 5 0 -TCA GAA GAA CTC CTA TGT GG-3 0 ; antisense, 5 0 -TCT CTT TGA TGT CAC GCA CG-3 0 , giving a 499 bp product. Primers for CRFR2 were: 5 0 -CCGGAATGCCTACAGAGAGT-3 0 and 5 0 -AGGTGGTGATGAGGTTCCAG-3 0 giving a 250 bp product. Total cellular RNA was isolated using Trizol reagent (Invitrogen). Following reverse transcription, 1 ll of the cDNA product was amplified by PCR, at 33 cycles, annealing to temperature of 55 C. 10 ll of the amplified products were separated on a 3% agarose gel and visualized by ethidium bromide staining using the BioRad Molecular Analyst System. 2.8. Western blot analysis Cell lysates were electrophoresed through a 12% SDS–polyacrylamide gel, then proteins were transferred to nitrocellulose membranes, which were processed according to standard Western blotting procedures, as previously described [16]. 2.9. Statistical analysis Apoptosis data are presented as optical density (OD) or as normalized ratios to parallel controls or actin content. For statistical evaluation analysis of variance, post hoc comparisons of means followed by the FisherÕs least significance difference test were used. For data expressed as percent changes the non-parametric Kruskal–Wallis test for several independent samples was used.
3. Results 3.1. Urocortins promoted macrophage apoptosis Raw264.7 cells were treated with increasing concentrations of UCN1 or UCN2 and apoptosis was quantified by measuring nucleosomal fragmentation. Apoptosis was presented as
Fig. 1. UCN1 and UCN2 promoted Raw264.7 macrophage apoptosis (nucleosomal fragmentation data). (A) Dose–response of UCN1 and UCN2-induced apoptosis. (B) UCN1 or UCN2 (109 M)-induced apoptosis was measured at different time points. (C) Raw264.7 cells were treated with UCN1 or UCN2 at 107 M and apoptosis was measured at different time points. Results represent the average of three independent experiments where each sample was tested in triplicate. \P < 0.05, \\P < 0.01 or \\\P < 0.001 denote statistically significant differences compared to parallel controls.
C. Tsatsanis et al. / FEBS Letters 579 (2005) 4259–4264
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phage apoptosis should be regarded as highly significant since the remaining functional macrophages tend to clear apoptotic neighbors by phagocytosis not allowing them to accumulate in the medium hiding net apoptosis. No effect on apoptosis was detectable at higher than 107 M concentrations of UCN1 or UCN2. To confirm this lack of effect and to rule out effect at other time points, cells were treated with UCN1 or UCN2 at 107 or 106 M at apoptosis was measured at multiple time points. No effect was observed at any of the time points tested (Fig. 1C and data not shown). Flow cytometry following staining with 7-AAD confirmed the nucleosomal fragmentation data (Fig. 2A and B). LPS induced a similar to urocortins degree of macrophage apoptosis, supporting the significant role of the latter. In thioglycollate-elicited primary mouse macrophages (TEPM), UCN1 and UCN2 induced a similar degree of apoptosis, which peaked at 6 h (Fig. 3A). It should be noted that the observed earlier apoptosis in primary cultures was expected because of the well-known higher sensitivity of primary cells compared to cell lines. Finally, in agreement with the cell line data, exposure of primary macrophages to UCN1 or UCN2 at higher concentrations, i.e. 107 or 106 M did not show any significant effect on apoptosis (Fig. 3B and data not shown).
Fig. 3. UCN1 and UCN2 promoted thioglycollate-elicited peritoneal macrophage (TEPM) apoptosis. TEPMs were exposed to UCN1 (109 M), UCN2 (109 M) or LPS (10 lg/ml) (A) or UCN1 (107 M) or UCN2 (107 M) (B) for different time periods. Results represent the average of three independent experiments where each sample was tested in triplicate. \P < 0.05 denotes statistically significant differences compared to parallel controls.
Fig. 2. UCN1 and UCN2 promoted Raw264.7 macrophage apoptosis (Flow cytometry data). Raw264.7 cells were treated with the CRFR2 agonists UCN1 or UCN2 (109 M), or LPS (10 lg/ml) and apoptotic cells were stained with 7-AAD and visualized by Flow Cytometry. (A) Apoptotic cells are shown as percentage of the total population. (B) Representative raw data of three independent experiments.
3.2. Urocortins induced macrophage apoptosis via CRFR2 Raw264.7 and TEPM cells express the CRFR1 and CRFR2 receptors (Fig. 4A and B). Specific CRFR1 and CRFR2 receptors antagonists were used to assess their role in UCN-induced apoptosis using the inversion of phosphatidylserine located at cell membranes (Apo-percentage assay), a method detecting apoptosis at a much earlier stage. Exposure of Raw264.7 or TEPM cells to CRFR2 specific antagonist anti-sauvagine30 at 107 M completely abolished the pro-apoptotic effect of UCN1 and UCN2 (Fig. 4C and D). Exposure to CRFR1 antagonist a-helical CRF[9-41] (ah), at 107 M had no effect on the apoptosis induced by UCN1 (Fig. 4C and D) suggesting that the pro-apoptotic effect is mediated solely by CRFR2. UCN1 exhibits high affinity to CRFR2 and 10 times lower affinity to CRFR1, while UCN2 is a selective CRFR2 agonist which can bind to CRFR1 only at very high concentrations (IC50 350 nM) [18]. We, therefore, hypothesized that the proapoptotic effect observed at 109 M of the neuropeptides may be masked at higher concentrations through their binding on both CRF receptors. Raw264.7 cells and TEPM were treated with UCN1 or UCN2 at 107M together with anti-sauvagine30 or ahCRF[9–41] at 5 · 106 M, which may unmask such effect. No statistically significant difference on apoptosis was observed (Fig. 5).
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Fig. 4. The pro-apoptotic effect of UCN1 and UCN2 was mediated by CRFR2. (A) Expression of CRFR1 and CRFR2 in Raw264.7 macrophages (A) and TEPM (B) as detected by RT-PCR. Raw264.7 cells (C) or TEPMs (D) were treated with vehicle (control), UCN1 or UCN2 (109 M) for 24 h in the presence or absence anti-sauvagine30 (asvg-30) or a-helical CRF[9–41] (ah). Apoptosis was detected by measuring the inversion of membrane phosphatidyl-serine (Apop-percentage assay). \P < 0.05 or \\P < 0.01 denote statistically significant differences compared to parallel controls. # P < 0.05 compared to UCN1 or UCN2 treated cells, respectively.
Fig. 5. Inhibition of CRFR1 or CRFR2 signals did not alter macrophage apoptosis in the presence of high concentrations of UCN1 or UCN2. Raw264.7 cells (A,B) or TEPMs (C,D) were treated with UCN1 or UCN2 at 107 M in the presence of ahCRF[9–41] or asvg-30 at 106 M and apoptosis was measured at different time points. No statistically significant difference was observed.
C. Tsatsanis et al. / FEBS Letters 579 (2005) 4259–4264
3.3. The signaling pathway in urocortin-induced apoptosis is different from that of LPS Raw264.7 cells were treated with UCN1 for different time periods and cells were lysed to detect expression of pro-apoptotic molecules. We found that UCN1, and to a greater extent UCN2 induced Bax expression (2.4 and 3.2-fold, respectively) while LPS induces Bax but at lower levels than urocortins (1.5fold). The effect became evident at 4 h following treatment (Fig. 6A) and Bax remained at high levels 12 h following stimulation. Both LPS and CRFR2 signals induced Bad in Raw264.7 cells, being 4.8-fold for UCN1, 5.3-fold for UCN2 and 5.5-fold for LPS (Fig. 6B). Thus, induction of Bad confirmed the involvement of pro-apoptotic Bcl2-related proteins in CRFR2-induced apoptosis and appears to be a common pathway activated by UCN1, UCN2 and LPS. LPS-induced apoptosis of Raw264.7 macrophages requires activation of JNK and p38MAPK. CRFR2 signals had no effect on JNK or p38MAPK phosphorylation while LPS, which induced their activation (Fig. 7A and B). LPS-induced apoptosis in macrophages is tightly associated to NO production [1,3,19]. CRFR2 signals did not affect NO production at any concentration or time point (data not shown).
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Fig. 7. In contrast to LPS, CRFR2 signals did not affect p38MAPK or JNK. Raw264.7 cells were treated with UCN1 (109 M), UCN2 (109 M) or LPS (10 lg/ml) for 10 min and the phosphorylated form of p38MAPK (A) or JNK (B) was measured by Western blot analysis. Results are representative of three independent experiments.
4. Discussion CRF is a paracrine modulator of inflammation. It exerts a direct effect on macrophages augmenting their production of pro-inflammatory cytokines [15,20]. CRF and UCN1 are detected at the site of inflammation in humans [21,22] although the role of urocortins is unclear. Evidence suggests that urocortins at inflammation sites may counteract the pro-inflammatory effects of CRF [14,23]. Indeed, UCN1 present in the mucosa of patients suffering from H. pylori-induced gastritis increases during the process of microbe eradication and the amelioration of inflammation while in patients resistant to treatment its levels remain low [12]. In the present study, we
Fig. 6. CRFR2 signals activated Bax and Bad. Raw264.7 cells were treated for 4 h with vehicle (control), UCN1 (109 M), UCN2 (109 M) or LPS (10 lg/ml) and the expression levels of Bax (A) or Bad (B) was estimated by Western blot. Results are representative of three independent experiments.
propose that urocortins may act as paracrine anti-inflammation agents by accelerating macrophage apoptosis. CRFR1 signals have been associated with anti-apoptotic or pro-apoptotic actions in several cell types [16,24]. This is the first report associating CRFR2 signals to apoptosis. We present evidence that although both CRF receptors, the CRFR1 and CRFR2 are expressed in primary macrophages and in RAW264.7 cells the pro-apoptotic effect of urocortins is mediated only by the CRFR2 receptors. The pro-apoptotic effect of urocortins was detectable only at low concentrations ranging (1010–108 M). Indeed, we did not observe any significant pro- or anti-apoptotic effect of urocortins at higher concentrations suggesting that their proapoptotic action takes places in a limited window of concentrations, which may reflect tissue-specificity based on CRF receptor distribution. Blocking one or the other of CRF receptors did not unmask any pro- or anti-apoptotic effect suggesting that in macrophages UCN1 affects apoptosis only via CRFR2. We thus hypothesize that at higher concentrations urocortins may either induce activation of different signaling molecules that do not affect macrophage homeostasis or bind to different sites of the CRF receptors resulting in altered intracellular signals and biological effects. Our data also suggest that in macrophages, activation of the CRFR2 receptor induces apoptosis via the pro-apoptotic proteins Bax and Bad without affecting p38MAPK, JNK or the production of NO characteristics of LPS-induced apoptosis. Thus, it appears that urocortins promote macrophage apoptosis via a direct effect on pro-apoptotic Bcl2-related proteins in contrast to LPS that utilizes an indirect pathway. Indeed, CRFR2-induced apoptosis appears to be faster than that triggered by LPS and comparable in magnitude. In conclusion, our data suggest that urocortins, the endogenous ligands of the CRFR2 receptor, play an anti-inflammatory role via their pro-apoptotic effect on macrophages via a different to LPS signaling pathway.
4264 Acknowledgments: C.T. and A.A. contributed equally to this work. This project was supported by the Hellenic Secretariat for Research and Technology (PENED 01ED386), the Association for International Cancer Research (Grant # AICR03-061) and Medicon Hellas Co.
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C. Tsatsanis et al. / FEBS Letters 579 (2005) 4259–4264 [12] Chatzaki, E., Charalampopoulos, I., Leontidis, C., Mouzas, I.A., Tzardi, M., Tsatsanis, C., Margioris, A.N. and Gravanis, A. (2003) Urocortin in human gastric mucosa: relationship to inflammatory activity. J. Clin. Endocrinol. Metab. 88, 478– 483. [13] Agnello, D., Bertini, R., Sacco, S., Meazza, C., Villa, P. and Ghezzi, P. (1998) Corticosteroid-independent inhibition of tumor necrosis factor production by the neuropeptide urocortin. Am. J. Physiol. 275, E757–E762. [14] Turnbull, A.V., Vale, W. and Rivier, C. (1996) Urocortin, a corticotropin-releasing factor-related mammalian peptide, inhibits edema due to thermal injury in rats. Eur. J. Pharmacol. 303, 213–216. [15] Agelaki, S., Tsatsanis, C., Gravanis, A. and Margioris, A.N. (2002) Corticotropin-releasing hormone augments proinflammatory cytokine production from macrophages in vitro and in lipopolysaccharide-induced endotoxin shock in mice. Infect. Immun. 70, 6068–6074. [16] Dermitzaki, E., Tsatsanis, C., Gravanis, A. and Margioris, A.N. (2002) Corticotropin-releasing hormone induces Fas ligand production and apoptosis in PC12 cells via activation of p38 mitogen-activated protein kinase. J. Biol. Chem. 277, 12280– 12287. [17] Charalampopoulos, I., Tsatsanis, C., Dermitzaki, E., Alexaki, V.I., Castanas, E., Margioris, A.N. and Gravanis, A. (2004) Dehydroepiandrosterone and allopregnanolone protect sympathoadrenal medulla cells against apoptosis via antiapoptotic Bcl-2 proteins. Proc. Natl. Acad. Sci. USA 101, 8209–8214. [18] Jahn, O., Tezval, H., van Werven, L., Eckart, K. and Spiess, J. (2004) Three-amino acid motifs of urocortin II and III determine their CRF receptor subtype selectivity. Neuropharmacology 47, 233–242. [19] Singhal, P.C., Bhaskaran, M., Patel, J., Patel, K., Kasinath, B.S., Duraisamy, S., Franki, N., Reddy, K. and Kapasi, A.A. (2002) Role of p38 mitogen-activated protein kinase phosphorylation and Fas–Fas ligand interaction in morphine-induced macrophage apoptosis. J. Immunol. 168, 4025–4033. [20] Venihaki, M., Dikkes, P., Carrigan, A. and Karalis, K.P. (2001) Corticotropin-releasing hormone regulates IL-6 expression during inflammation. J. Clin. Invest. 108, 1159–1166. [21] Kawahito, Y., Sano, H., Mukai, S., Asai, K., Kimura, S., Yamamura, Y., Kato, H., Chrousos, G.P., Wilder, R.L. and Kondo, M. (1995) Corticotropin releasing hormone in colonic mucosa in patients with ulcerative colitis. Gut 37, 544– 551. [22] Crofford, L.J., Sano, H., Karalis, K., Friedman, T.C., Epps, H.R., Remmers, E.F., Mathern, P., Chrousos, G.P. and Wilder, R.L. (1993) Corticotropin-releasing hormone in synovial fluids and tissues of patients with rheumatoid arthritis and osteoarthritis. J. Immunol. 151, 1587–1596. [23] Torpy, D.J., Webster, E.L., Zachman, E.K., Aguilera, G. and Chrousos, G.P. (1999) Urocortin and inflammation: confounding effects of hypotension on measures of inflammation. Neuroimmunomodulation 6, 182–186. [24] Radulovic, M., Hippel, C. and Spiess, J. (2003) Corticotropinreleasing factor (CRF) rapidly suppresses apoptosis by acting upstream of the activation of caspases. J. Neurochem. 84, 1074– 1085.
FEBS Letters 579 (2005) 4259–4264
Urocortin 1 and Urocortin 2 induce macrophage apoptosis via CRFR2 Christos Tsatsanisa,*, Ariadne Androulidakia, Erini Dermitzakia, Ioannis Charalampopoulosb, Joachim Spiessc, Achille Gravanisb, Andrew N. Margiorisa,* a
Department of Clinical Chemistry-Biochemistry, School of Medicine, University of Crete, Heraklion, Crete GR-710 03, Greece b Department of Pharmacology, School of Medicine, University of Crete, Heraklion, Crete GR-710 03, Greece c Department of Molecular Neuroendocrinology, Max Planck Institute for Experimental Medicine, 37075 Goettingen, Germany Received 5 January 2005; revised 11 June 2005; accepted 19 June 2005 Available online 18 July 2005 Edited by Michael R. Bubb
Abstract Macrophages undergo apoptosis as a mechanism of regulating their activation and the inflammatory reaction. Macrophages express the Corticotropin-Releasing Factor Receptor-2 (CRFR2) the endogenous agonists of which, the urocortins, are also present at the site of inflammation. We have found that urocortins induced macrophage apoptosis in a dose- and time-dependent manner via CRFR2. In contrast to lipopolysaccharide (LPS)-induced apoptosis, the pro-apoptosis pathway activated by urocortins involved the pro-apoptotic Bax and Bad proteins and not nitric oxide, JNK and p38MAPK characteristic of LPS. In conclusion, our data suggest that endogenous CRFR2 ligands exert an anti-inflammatory effect via induction of macrophage apoptosis. 2005 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. Keywords: Macrophage; Urocortin; CRFR2; Apoptosis; Bax
1. Introduction Following their activation, macrophages undergo apoptosis as a mechanism to contain inflammation [1]. Bacterial lipopolysaccharide (LPS) activates macrophages and promotes their apoptosis via induction of the nitric oxide NO pathway [2,3]. The corticotropin-releasing factor (CRF) family of peptides and their receptors are expressed in neural and non-neural cells including immune cells [4,5]. Urocortin 1 (UCN1), a member of the CRF family, is detected in lymphoid organs including spleen and thymus, the gastro-intestinal tract and heart [6,7] while Urocortin 2 (UCN2) [8] and Urocortin 3 (UCN3) [9] are mainly expressed in skin and muscle. Urocortins utilize two different receptors, CRFR1 and CRFR2 [10,11]. UCN1 has high affinity towards CRFR2 but it also binds strongly to CRFR1, while UCN2 and UCN3 are exclusive ligands of CRFR2 [8,9]. Several published reports suggest that urocortins may play an anti-inflammatory role. Indeed, in gastric biopsies of patients with Helicobacter pylori-induced gastritis, UCN1 is
*
Corresponding authors. Fax: +30 2810 394581. E-mail addresses: [email protected] (C. Tsatsanis), [email protected] (A.N. Margioris).
significantly elevated during the stage of eradication of H. pylori and the amelioration of the inflammatory response while it fails to do so in non-responders [12]. Furthermore, UCN1 tested in a systemic inflammation model appears to exert a direct effect on the cascade of events taking place in inflammation suppressing LPS-induced TNF-a production independently of corticosterone [13]. UCN1 also suppresses skin edema induced by thermal injury [14]. It thus appears that urocortins may act as endogenous anti-inflammatory agents. Aim of this work was to test the hypothesis that the anti-inflammatory effect of urocortins involves induction of macrophage apoptosis.
2. Materials and methods 2.1. Reagents and antibodies UCN1, Escherichia coli-derived LPS (serotype 0111:B4), a-helical CRF[9–41], propidium iodide were purchased from Sigma Chemicals Co. (St. Louis, MO) and 7-amino-actinomycinD (7-AAD) was provided by ICN Biomedicals, Inc. (Costa Mesa, CA). UCN2 and antisauvagine30 were chemically synthesized. Thermoscript RT and Platinum Taq polymerase were purchased from Invitrogen (NY, NY), anti-Bax from Santa Cruz Biotechnology Inc. (Santa Cruz, CA), anti-Bad from Cell Signalling Technology Inc. (Beverly, MA) and anti-actin from Chemicon (Temecula, CA). All other chemicals and reagents were obtained from Sigma, if not stated otherwise. 2.2. RAW 264.7 cell culture RAW 264.7 cells (derived from ATCC) were cultured in DulbeccoÕs modified EagleÕs medium supplemented with 10% fetal calf serum (FCS), 10 mM L -glutamine, 100 U/ml penicillin, 0.1 mg/ml streptomycin (all purchased from Invitrogen), at 5% CO2 and 37 C. Cells were plated in 25 cm2 flasks at a concentration of 4 · 105/ml and growth medium was replaced with DMEM that does not contain FCS 12 h prior to stimulation. Cells were incubated with LPS plus/minus the specified neuropeptides for 24 h. 7AAD staining or pro-apoptotic protein expression measured apoptosis. In the experiments where inhibitors were used, cells were treated with the inhibitor 1 h prior to stimulation with each neuropeptide. Parallel control cells treated only with the diluent were collected at the same time points. 2.3. Isolation and stimulation of thioglycollate-elicited macrophages A 4% thioglycollate solution was prepared and autoclaved 2 days prior to administration. 1.5 ml of the thioglycollate solution was injected intraperitoneally in C57BL/6 mice and peritoneal macrophages were isolated by lavage of the peritoneal cavity with DulbeccoÕs modified medium [15]. Cells were then cultured in DMEM supplemented with 10% FCS, 10 mM L -glutamine, 100 U/ml penicillin, 0.1 mg/ml streptomycin (Gibco).
0014-5793/$30.00 2005 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.febslet.2005.06.057
4260 2.4. Quantitation of nucleosome fragmentation Cells were plated in 96-well plates at an initial concentration of 10 000 cells per well for 24 h. Apoptosis was measured by direct determination of nucleosomal DNA fragmentation with the ‘‘cell death detection’’ ELISA plus kit (Cat No. 1 774 425, Boehringer Mannheim GmbH, Mannheim, Germany). Briefly, cells were harvested and lysed according to the manufacturerÕs protocol. The mono- and oligo-nucleosomes contained in cell lysates were determined using an anti-histonebiotin antibody, which binds to histones and reacts with single- and double-stranded DNA. Data were expressed in photometric units. Each unit corresponds to approximately 12 500 apoptotic cells. The concentration of the nucleosomes was determined photometrically using 2,2 0 -azino-di[3-ethylbenzthiazolin-sulfonate] as substrate for the biotin present on the antibody. The optical density was read on a Dynatech MicroElisa Reader (Chantilly, VA) at 405 nm. Each experiment was repeated three times and each sample in triplicate.
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ratio 1:1 between control and treated cells. Control cells were exposed to the diluent only, for the same period as treated cells. Basal apoptosis was assigned as 100%. UCN1 and UCN2 induced apoptosis reaching a peak between 108 and 1010 M (Fig. 1A). Subsequently, cells were exposed to UCN1 or UCN2 at 109 M and apoptosis was measured at several time points. UCN1 and UCN2 induced apoptosis, which became detectable at 3 h reaching a peak at 24 h (Fig. 1B). It should be noted that modest changes of macro-
2.5. Quantitation of apoptotic cells The APOPercentage Apoptosis Assay (Biocolor Ltd., Belfast, N. Ireland) uses a dye that stains red the apoptotic cells undergoing the membrane flip-flop event when phosphatidylserine is translocated to the outer leaflet. Apoptosis was quantified by measuring the dye incorporated in apoptotic cells following cell lysis, at 550 nm (reference filter 620 nm) using a color filter microplate colorimeter (Dynatech MicroElisa reader Chantilly, VA) as previously described [16,17]. Each sample was measured in triplicate and each experiment was repeated three times. 2.6. Flow cytometric analysis of apoptotic cells Cells were treated in 6-well plates as described above and harvested in culture medium containing 7-AAD. Following 15-min incubation, cells were washed in cold PBS and re-suspended in PBS containing propidium iodide (PI) (1 lg/ml) to detect both necrotic and late apoptotic cells that were excluded from the analysis. Cells were visualized and analyzed on a flow cytometer (Epics Elite Coulter, UK). The percentage of 7AAD positive/PI negative cells that represent apoptotic population was compared to live cells that were 7AAD negative/PI negative. 2.7. RT-PCR Primers for actin were sense, 5 0 -TCA GAA GAA CTC CTA TGT GG-3 0 ; antisense, 5 0 -TCT CTT TGA TGT CAC GCA CG-3 0 , giving a 499 bp product. Primers for CRFR2 were: 5 0 -CCGGAATGCCTACAGAGAGT-3 0 and 5 0 -AGGTGGTGATGAGGTTCCAG-3 0 giving a 250 bp product. Total cellular RNA was isolated using Trizol reagent (Invitrogen). Following reverse transcription, 1 ll of the cDNA product was amplified by PCR, at 33 cycles, annealing to temperature of 55 C. 10 ll of the amplified products were separated on a 3% agarose gel and visualized by ethidium bromide staining using the BioRad Molecular Analyst System. 2.8. Western blot analysis Cell lysates were electrophoresed through a 12% SDS–polyacrylamide gel, then proteins were transferred to nitrocellulose membranes, which were processed according to standard Western blotting procedures, as previously described [16]. 2.9. Statistical analysis Apoptosis data are presented as optical density (OD) or as normalized ratios to parallel controls or actin content. For statistical evaluation analysis of variance, post hoc comparisons of means followed by the FisherÕs least significance difference test were used. For data expressed as percent changes the non-parametric Kruskal–Wallis test for several independent samples was used.
3. Results 3.1. Urocortins promoted macrophage apoptosis Raw264.7 cells were treated with increasing concentrations of UCN1 or UCN2 and apoptosis was quantified by measuring nucleosomal fragmentation. Apoptosis was presented as
Fig. 1. UCN1 and UCN2 promoted Raw264.7 macrophage apoptosis (nucleosomal fragmentation data). (A) Dose–response of UCN1 and UCN2-induced apoptosis. (B) UCN1 or UCN2 (109 M)-induced apoptosis was measured at different time points. (C) Raw264.7 cells were treated with UCN1 or UCN2 at 107 M and apoptosis was measured at different time points. Results represent the average of three independent experiments where each sample was tested in triplicate. \P < 0.05, \\P < 0.01 or \\\P < 0.001 denote statistically significant differences compared to parallel controls.
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phage apoptosis should be regarded as highly significant since the remaining functional macrophages tend to clear apoptotic neighbors by phagocytosis not allowing them to accumulate in the medium hiding net apoptosis. No effect on apoptosis was detectable at higher than 107 M concentrations of UCN1 or UCN2. To confirm this lack of effect and to rule out effect at other time points, cells were treated with UCN1 or UCN2 at 107 or 106 M at apoptosis was measured at multiple time points. No effect was observed at any of the time points tested (Fig. 1C and data not shown). Flow cytometry following staining with 7-AAD confirmed the nucleosomal fragmentation data (Fig. 2A and B). LPS induced a similar to urocortins degree of macrophage apoptosis, supporting the significant role of the latter. In thioglycollate-elicited primary mouse macrophages (TEPM), UCN1 and UCN2 induced a similar degree of apoptosis, which peaked at 6 h (Fig. 3A). It should be noted that the observed earlier apoptosis in primary cultures was expected because of the well-known higher sensitivity of primary cells compared to cell lines. Finally, in agreement with the cell line data, exposure of primary macrophages to UCN1 or UCN2 at higher concentrations, i.e. 107 or 106 M did not show any significant effect on apoptosis (Fig. 3B and data not shown).
Fig. 3. UCN1 and UCN2 promoted thioglycollate-elicited peritoneal macrophage (TEPM) apoptosis. TEPMs were exposed to UCN1 (109 M), UCN2 (109 M) or LPS (10 lg/ml) (A) or UCN1 (107 M) or UCN2 (107 M) (B) for different time periods. Results represent the average of three independent experiments where each sample was tested in triplicate. \P < 0.05 denotes statistically significant differences compared to parallel controls.
Fig. 2. UCN1 and UCN2 promoted Raw264.7 macrophage apoptosis (Flow cytometry data). Raw264.7 cells were treated with the CRFR2 agonists UCN1 or UCN2 (109 M), or LPS (10 lg/ml) and apoptotic cells were stained with 7-AAD and visualized by Flow Cytometry. (A) Apoptotic cells are shown as percentage of the total population. (B) Representative raw data of three independent experiments.
3.2. Urocortins induced macrophage apoptosis via CRFR2 Raw264.7 and TEPM cells express the CRFR1 and CRFR2 receptors (Fig. 4A and B). Specific CRFR1 and CRFR2 receptors antagonists were used to assess their role in UCN-induced apoptosis using the inversion of phosphatidylserine located at cell membranes (Apo-percentage assay), a method detecting apoptosis at a much earlier stage. Exposure of Raw264.7 or TEPM cells to CRFR2 specific antagonist anti-sauvagine30 at 107 M completely abolished the pro-apoptotic effect of UCN1 and UCN2 (Fig. 4C and D). Exposure to CRFR1 antagonist a-helical CRF[9-41] (ah), at 107 M had no effect on the apoptosis induced by UCN1 (Fig. 4C and D) suggesting that the pro-apoptotic effect is mediated solely by CRFR2. UCN1 exhibits high affinity to CRFR2 and 10 times lower affinity to CRFR1, while UCN2 is a selective CRFR2 agonist which can bind to CRFR1 only at very high concentrations (IC50 350 nM) [18]. We, therefore, hypothesized that the proapoptotic effect observed at 109 M of the neuropeptides may be masked at higher concentrations through their binding on both CRF receptors. Raw264.7 cells and TEPM were treated with UCN1 or UCN2 at 107M together with anti-sauvagine30 or ahCRF[9–41] at 5 · 106 M, which may unmask such effect. No statistically significant difference on apoptosis was observed (Fig. 5).
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Fig. 4. The pro-apoptotic effect of UCN1 and UCN2 was mediated by CRFR2. (A) Expression of CRFR1 and CRFR2 in Raw264.7 macrophages (A) and TEPM (B) as detected by RT-PCR. Raw264.7 cells (C) or TEPMs (D) were treated with vehicle (control), UCN1 or UCN2 (109 M) for 24 h in the presence or absence anti-sauvagine30 (asvg-30) or a-helical CRF[9–41] (ah). Apoptosis was detected by measuring the inversion of membrane phosphatidyl-serine (Apop-percentage assay). \P < 0.05 or \\P < 0.01 denote statistically significant differences compared to parallel controls. # P < 0.05 compared to UCN1 or UCN2 treated cells, respectively.
Fig. 5. Inhibition of CRFR1 or CRFR2 signals did not alter macrophage apoptosis in the presence of high concentrations of UCN1 or UCN2. Raw264.7 cells (A,B) or TEPMs (C,D) were treated with UCN1 or UCN2 at 107 M in the presence of ahCRF[9–41] or asvg-30 at 106 M and apoptosis was measured at different time points. No statistically significant difference was observed.
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3.3. The signaling pathway in urocortin-induced apoptosis is different from that of LPS Raw264.7 cells were treated with UCN1 for different time periods and cells were lysed to detect expression of pro-apoptotic molecules. We found that UCN1, and to a greater extent UCN2 induced Bax expression (2.4 and 3.2-fold, respectively) while LPS induces Bax but at lower levels than urocortins (1.5fold). The effect became evident at 4 h following treatment (Fig. 6A) and Bax remained at high levels 12 h following stimulation. Both LPS and CRFR2 signals induced Bad in Raw264.7 cells, being 4.8-fold for UCN1, 5.3-fold for UCN2 and 5.5-fold for LPS (Fig. 6B). Thus, induction of Bad confirmed the involvement of pro-apoptotic Bcl2-related proteins in CRFR2-induced apoptosis and appears to be a common pathway activated by UCN1, UCN2 and LPS. LPS-induced apoptosis of Raw264.7 macrophages requires activation of JNK and p38MAPK. CRFR2 signals had no effect on JNK or p38MAPK phosphorylation while LPS, which induced their activation (Fig. 7A and B). LPS-induced apoptosis in macrophages is tightly associated to NO production [1,3,19]. CRFR2 signals did not affect NO production at any concentration or time point (data not shown).
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Fig. 7. In contrast to LPS, CRFR2 signals did not affect p38MAPK or JNK. Raw264.7 cells were treated with UCN1 (109 M), UCN2 (109 M) or LPS (10 lg/ml) for 10 min and the phosphorylated form of p38MAPK (A) or JNK (B) was measured by Western blot analysis. Results are representative of three independent experiments.
4. Discussion CRF is a paracrine modulator of inflammation. It exerts a direct effect on macrophages augmenting their production of pro-inflammatory cytokines [15,20]. CRF and UCN1 are detected at the site of inflammation in humans [21,22] although the role of urocortins is unclear. Evidence suggests that urocortins at inflammation sites may counteract the pro-inflammatory effects of CRF [14,23]. Indeed, UCN1 present in the mucosa of patients suffering from H. pylori-induced gastritis increases during the process of microbe eradication and the amelioration of inflammation while in patients resistant to treatment its levels remain low [12]. In the present study, we
Fig. 6. CRFR2 signals activated Bax and Bad. Raw264.7 cells were treated for 4 h with vehicle (control), UCN1 (109 M), UCN2 (109 M) or LPS (10 lg/ml) and the expression levels of Bax (A) or Bad (B) was estimated by Western blot. Results are representative of three independent experiments.
propose that urocortins may act as paracrine anti-inflammation agents by accelerating macrophage apoptosis. CRFR1 signals have been associated with anti-apoptotic or pro-apoptotic actions in several cell types [16,24]. This is the first report associating CRFR2 signals to apoptosis. We present evidence that although both CRF receptors, the CRFR1 and CRFR2 are expressed in primary macrophages and in RAW264.7 cells the pro-apoptotic effect of urocortins is mediated only by the CRFR2 receptors. The pro-apoptotic effect of urocortins was detectable only at low concentrations ranging (1010–108 M). Indeed, we did not observe any significant pro- or anti-apoptotic effect of urocortins at higher concentrations suggesting that their proapoptotic action takes places in a limited window of concentrations, which may reflect tissue-specificity based on CRF receptor distribution. Blocking one or the other of CRF receptors did not unmask any pro- or anti-apoptotic effect suggesting that in macrophages UCN1 affects apoptosis only via CRFR2. We thus hypothesize that at higher concentrations urocortins may either induce activation of different signaling molecules that do not affect macrophage homeostasis or bind to different sites of the CRF receptors resulting in altered intracellular signals and biological effects. Our data also suggest that in macrophages, activation of the CRFR2 receptor induces apoptosis via the pro-apoptotic proteins Bax and Bad without affecting p38MAPK, JNK or the production of NO characteristics of LPS-induced apoptosis. Thus, it appears that urocortins promote macrophage apoptosis via a direct effect on pro-apoptotic Bcl2-related proteins in contrast to LPS that utilizes an indirect pathway. Indeed, CRFR2-induced apoptosis appears to be faster than that triggered by LPS and comparable in magnitude. In conclusion, our data suggest that urocortins, the endogenous ligands of the CRFR2 receptor, play an anti-inflammatory role via their pro-apoptotic effect on macrophages via a different to LPS signaling pathway.
4264 Acknowledgments: C.T. and A.A. contributed equally to this work. This project was supported by the Hellenic Secretariat for Research and Technology (PENED 01ED386), the Association for International Cancer Research (Grant # AICR03-061) and Medicon Hellas Co.
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