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Maturation and Release of Interleukin-1β by Lipopolysaccharide-primed Mouse Schwann Cells Require the Stimulation of P2X7 Receptors

2003; Elsevier BV; Volume: 278; Issue: 33 Linguagem: Inglês

10.1074/jbc.m304534200

1083-351X

Aurore Colomar, Vincent Marty, Chantal Médina, Chantal Combe, Patricia Parnet, Thierry Amédée,

Mast cells and histamine

The P2X7 receptor, mainly expressed by immune cells, is a ionotropic receptor activated by high concentration of extracellular ATP. It is involved in several processes relevant to immunomodulation and inflammation. Among these processes, the production of extracellular interleukin-1β (IL-1β), a pro-inflammatory cytokine, plays a major role in the activation of the cytokine network. We have investigated the role of P2X7 receptor and of an associated calcium-activated potassium conductance (BK channels) in IL-1β maturation and releasing processes by Schwann cells. Lipopolysaccharide-primed Schwann cells synthesized large amounts of pro-IL-1β but did not release detectable amounts of pro or mature IL-1β. ATP on its own had no effect on the synthesis of pro-IL-1β, but a co-treatment with lipopolysaccharide and ATP led to the maturation and the release of IL-1β by Schwann cells. Both mechanisms were blocked by oxidized ATP. IL-1β-converting enzyme (ICE), the caspase responsible for the maturation of pro-IL-1β in IL-1β, was activated by P2X7 receptor stimulation. The specific inhibition of ICE by the caspase inhibitor Ac-Tyr-Val-Ala-Asp-aldehyde blocked the maturation of IL-1β. In searching for a link between the P2X7 receptor and the activation of ICE, we found that enhancing potassium efflux from Schwann cells upregulated the production of IL-1β, while strongly reducing potassium efflux led to opposite effects. Blocking BK channels actually modulated IL-1β release. Taken together, these results show that P2X7 receptor stimulation and associated BK channels, through the activation of ICE, leads to the maturation and the release of IL-1β by immune-challenged Schwann cells. The P2X7 receptor, mainly expressed by immune cells, is a ionotropic receptor activated by high concentration of extracellular ATP. It is involved in several processes relevant to immunomodulation and inflammation. Among these processes, the production of extracellular interleukin-1β (IL-1β), a pro-inflammatory cytokine, plays a major role in the activation of the cytokine network. We have investigated the role of P2X7 receptor and of an associated calcium-activated potassium conductance (BK channels) in IL-1β maturation and releasing processes by Schwann cells. Lipopolysaccharide-primed Schwann cells synthesized large amounts of pro-IL-1β but did not release detectable amounts of pro or mature IL-1β. ATP on its own had no effect on the synthesis of pro-IL-1β, but a co-treatment with lipopolysaccharide and ATP led to the maturation and the release of IL-1β by Schwann cells. Both mechanisms were blocked by oxidized ATP. IL-1β-converting enzyme (ICE), the caspase responsible for the maturation of pro-IL-1β in IL-1β, was activated by P2X7 receptor stimulation. The specific inhibition of ICE by the caspase inhibitor Ac-Tyr-Val-Ala-Asp-aldehyde blocked the maturation of IL-1β. In searching for a link between the P2X7 receptor and the activation of ICE, we found that enhancing potassium efflux from Schwann cells upregulated the production of IL-1β, while strongly reducing potassium efflux led to opposite effects. Blocking BK channels actually modulated IL-1β release. Taken together, these results show that P2X7 receptor stimulation and associated BK channels, through the activation of ICE, leads to the maturation and the release of IL-1β by immune-challenged Schwann cells. Interleukin-1 is a pro-inflammatory cytokine that mediates part of the host defense response to injury and infection. It is produced by activated monocytes and macrophages (1Dinarello C.A. FASEB J. 1998; 2: 108-115Crossref Scopus (1378) Google Scholar), but also by microglia in the central nervous system (2Hanish U.K. Glia. 2002; 40: 140-155Crossref PubMed Scopus (1331) Google Scholar). Both forms of IL-1, 1The abbreviations used are: IL, interleukin; ICE, IL-1β-converting enzyme; oATP, oxidized ATP; YVAD-CHO, Ac-Tyr-Val-Ala-Asp-aldehyde; FITC-VAD-FMK, fluoroisothiocyanate-Val-Ala-Asp-O-methlyfluoromethylketone; LPS, lipopolysaccharide; TLR4, toll-like receptor 4; LDH, lactate dehydrogenase; ELISA, enzyme-linked immunosorbent assay; AEBSF, 4-(2-aminoethyl)benzenesulfonylfluoride hydrochloride; RT, reverse transcriptase; β2mgl, β2-microglobulin; ChTx, charybdotoxin; TEA, tetraethylammonium. i.e. IL-1α and IL-1β are synthesized as precursor molecules of 31–35 kDa, which are cleaved by proteolytic enzymes into a mature form of about 17 kDa. While IL-1α is biologically active in both forms (pro-IL-1α and mature IL-1α), IL-1β is only active when converted in its mature form. The conversion of pro-IL-1β into mature IL-1β is achieved by a cysteine protease belonging to the caspase family, the IL-1β-converting enzyme (ICE) (3Kostura M.J. Tocci M.J. Limjuco G. Chin J. Cameron P. Hillman A.G. Chartrain N.A. Schmidt J.A. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 5227-5231Crossref PubMed Scopus (483) Google Scholar, 4Black R.A. Kronheim S.R. Sleath P.R. FEBS Lett. 1989; 247: 386-390Crossref PubMed Scopus (234) Google Scholar), also termed caspase-1 (5Thornberry N.A. Br. Med. Bull. 1997; 53: 478-490Crossref PubMed Scopus (202) Google Scholar). One striking feature of IL-1β is its lack of signal sequence, which normally targets proteins to the endoplasmic reticulum to be exported in the extracellular compartment (6Lomedico P.T. Gubler U. Hellmann C.P. Dukovich M. Giri J.G. Pan Y.C. Collier K. Semionow R. Chua A.O. Mizel S.B. Nature. 1984; 312: 458-462Crossref PubMed Scopus (642) Google Scholar, 7March C.J. Mosley B. Larsen A. Cerretti D.P. Braedt G. Price V. Gillis S. Henney C.S. Kronheim S.R. Grabstein K. et al.Nature. 1985; 315: 641-647Crossref PubMed Scopus (1286) Google Scholar). Despite numerous studies, the mechanisms of IL-β posttranslational processing, are still ill defined. Because of the lack of signal peptide, it was first proposed that apoptosis of IL-1β-producing cells could be responsible for the release of IL-β (8Hogquist K.A. Nett M.A. Unanue E.R. Chaplin D.D. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 8485-8489Crossref PubMed Scopus (435) Google Scholar), and this theory is still largely accepted (for review, see Ref. 9DiVirgilio F. Chiozzi P. Falzoni S. Ferrari D. Sanz J.M. Venketaraman V. Baricordi O.R. Cell Death Differ. 1998; 5: 191-199Crossref PubMed Scopus (229) Google Scholar). However, macrophages and monocytes can release IL-1β without obvious signs of cell death (10Ferrari D. Chiozzi P. Falzoni S. Dal Susino M. Melchiorri L. Baricordi O.R. Di Virgilio F. J. Immunol. 1997; 159: 1451-1458PubMed Google Scholar, 11MacKenzie A. Wilson H.L. Kiss-Toth E. Dower S.K. North R.A. Surprenant A. Immunity. 2001; 15: 825-835Abstract Full Text Full Text PDF PubMed Scopus (718) Google Scholar). This non-cytolytic production of IL-1β led to the proposal of other mechanisms like the exportation by specific transporters (12Hamon Y. Luciani M.F. Becq F. Verrier B. Rubartelli A. Chimini G. Blood. 1997; 90: 2911-2915Crossref PubMed Google Scholar), the release from endosomal vesicles (13Andrei C. Dazzi C. Lotti L. Torrisi M.R. Chimini G. Rubartelli A. Mol. Biol. Cell. 1999; 10: 1463-1475Crossref PubMed Scopus (398) Google Scholar) or by microvesicle shedding (11MacKenzie A. Wilson H.L. Kiss-Toth E. Dower S.K. North R.A. Surprenant A. Immunity. 2001; 15: 825-835Abstract Full Text Full Text PDF PubMed Scopus (718) Google Scholar). In most cases, the sole antigenic stimulation is poorly effective to release IL-1β. Indeed in generic macrophages or monocytes, pro-IL-1β tends to accumulate into the cytosol instead of being secreted, whereas mature IL-1β is absent from the intracellular compartment. It seems therefore that maturation and release processes are structurally and/or timely linked, possibly through the association of ICE subunits with the plasma membrane (14Singer I.I. Scott S. Chin J. Bayne E.K. Limjuco G. Weidner J. Miller D.K. Chapman K. Kostura M.J. J. Exp. Med. 1995; 182: 1447-1459Crossref PubMed Scopus (140) Google Scholar). Perregaux and Gabel (15Perregaux D. Gabel C.A. J. Biol. Chem. 1994; 269: 15195-15203Abstract Full Text PDF PubMed Google Scholar) were first to demonstrate that nigericin, a potassium ionophore, or ATP potentiated greatly the release of IL-1β from LPS-primed macrophages. Since this pioneer work, in vitro but also in vivo studies (16Griffiths R.J. Stam E.J. Downs J.T. Otterness I.G. J. Immunol. 1995; 154: 2821-2828PubMed Google Scholar, 17Solle M. Labasi J. Perregaux D.G. Stam E. Petrushova N. Koller B.H. Griffiths R.J. Gabel C.A. J. Biol. Chem. 2001; 276: 125-132Abstract Full Text Full Text PDF PubMed Scopus (795) Google Scholar) have shown that extracellular ATP acting on the P2X7 receptor, an ionotropic receptor that plays a pivotal role in the modulation of immune and inflammatory responses (9DiVirgilio F. Chiozzi P. Falzoni S. Ferrari D. Sanz J.M. Venketaraman V. Baricordi O.R. Cell Death Differ. 1998; 5: 191-199Crossref PubMed Scopus (229) Google Scholar), was a very potent agent to stimulate the production of large quantities of extracellular mature IL-1β. To unravel the mechanisms occurring downstream the P2X7 receptor activation and triggering the production and the release of IL-1β, we have chosen to study mouse Schwann cells for two main reasons: 1) their importance as partially immunecompetent cells within the peripheral nervous system and 2) the peculiarity of their P2X7 receptors. Indeed, in addition to their well documented roles in myelination, trophic, and metabolic support of the neuronal network, Schwann cells are able to present antigens to immunocompetent cells by expressing major histocompatibility complex class II molecules under inflammatory conditions both in vitro (18Armati P.J. Pollard J.D. Gatenby P. Muscle Nerve. 1990; 13: 106-116Crossref PubMed Scopus (72) Google Scholar) and in vivo (19Bergsteindottir K. Brennan A. Jessen K.R. Mirsky R. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 9054-9058Crossref PubMed Scopus (38) Google Scholar). They produce chemokines (macrophage chemoattractant protein-1 (MCP-1) and macrophage inflammatory protein-1α (MIP-1α); Ref. 20Taskinen H.S. Roytta M. J. Peripher. Nerv. Syst. 2002; 5: 75-81Crossref Scopus (85) Google Scholar) and cytokines (both pro- and anti-inflammatory, for review see Ref. 21Gold R. Archelos J.J. Hartung H.P. Brain Pathol. 1999; 9: 343-360Crossref PubMed Scopus (81) Google Scholar; Ref. 22Colomar, A., Marty, V., Combe, C., Médina, C., Parnet, P., and Amédée, T. (2003) J. Soc. Biol., in pressGoogle Scholar). In vitro, Schwann cells synthesize IL-1β but poorly release it when challenged by an immune stimulus such as LPS (23Bergsteindottir K. Kingston A. Mirsky R. Jessen K.R. J. Neuroimmunol. 1991; 34: 15-23Abstract Full Text PDF PubMed Scopus (94) Google Scholar). In vivo, Schwann cells in the course of experimental autoimmune neuritis the murine model for the human Guillain-Barre syndrome (24Skundric D.S. Lisak R.P. Rouhi M. Kieseier BC. Jung S. Hartung H.P. J. Neuroimmunol. 2001; 116: 74-82Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar) or Wallerian degeneration following an axonal injury (25Shamash S. Reichert F. Rotshenker S. J. Neurosci. 2002; 22: 3052-3060Crossref PubMed Google Scholar) are producing IL-1β. A specific feature of P2X7 receptors expressed by cultured mouse Schwann cells are there association with a calcium-activated potassium conductance and a chloride conductance (26Colomar A. Amédée T. Eur. J. Neurosci. 2001; 14: 927-936Crossref PubMed Google Scholar). This intriguing phenotype makes the study of P2X7 receptors expressed by Schwann cells particularly interesting as the different conductances activated by extracellular ATP could participate to an integrated regulation of inflammatory processes occuring in the peripheral nervous system. The aim of the present work was to investigate the role of the P2X7 receptor and the associated conductances in IL-1β posttranslational processing in LPS-primed Schwann cells. We found that P2X7 receptor activation, through the activation of ICE, was essential to the production and the release of IL-1β. Potassium fluxes, through the P2X7 receptor itself and through calcium-activated potassium channels, were linking the P2X7 receptor activation to IL-1β processing. These results provide new insights on IL-1β processing mechanisms and on how Schwann cells, the main glial cells of the peripheral nervous system, can sense an immune stimulation and respond to it by activating the cytokine network. Glial Cell Cultures—Schwann cells were cultured from excised dorsal root ganglia from OF1 mouse embryos (E19) as described previously (27Amédée T. Ellie E. Dupouy B. Vincent J.D. J. Physiol. (Lond.). 1991; 441: 35-56Crossref Scopus (47) Google Scholar). Briefly, mice were killed by decapitation, and dorsal root ganglia were aseptically removed from embryos and plated onto 35-mm Petri dishes (Nunc, Strasbourg, France) coated with rat tail collagen type 1 (Upstate Biotechnology, Lake Placid, NY). Cells were cultured for the first 3 days in modified Eagle's medium (Invitrogen, Paisley, UK) containing nerve growth factor (100 ng/ml, Alomone, Jerusalem, Israël) and a mixture of uridine (10 μm) and fluorodeoxyuridine (10 μm) to suppress dividing fibroblasts. Then, cells were cultured with α-modified Eagle's medium (Invitrogen) containing nerve growth factor (20 ng/ml). The medium was changed twice a week, and cells were used between 4 and 6 weeks of culture. In some experimental conditions, ganglia were excised before measuring intracellular and extracellular IL-1β to eliminate an eventual neuronal source of IL-1β. Primary glial cells from mouse brain were used as a positive control for the expression of Toll-like receptors 4 (TLR4). They were cultured from newborn mice as previously described in detail (28Pousset F. Palin K. Verrier D. Poole S. Dantzer R. Parnet P. Lestage J. Eur. Cytokine Netw. 2000; 11: 682-689PubMed Google Scholar) and were plated at a density of 5 × 104 cells/dish into Dulbecco's modified Eagle's medium containing 20% heat-inactivated fetal calf serum (Roche Molecular Biochemicals; <10 pg ml–1 endotoxins). Under these conditions, neurons do not survive the mechanical dissociation, and the low plating density prevents oligodendrocyte proliferation. Cell Treatments—Schwann cell cultures were primed for 6 h with LPS (10 μg/ml, Escherichia coli, serotype 0127B8, batch 63H4010, Sigma, St. Quentin Fallavier, France) with or without ATP (5 mm) during the last 30 min of stimulation. In some experiments, oxidized ATP (oATP) (300 μm), a P2X7 receptor antagonist, was used during the last 90 min of stimulation. High external potassium solution (90 mm K+) was obtained by adding 85 mm K+ to the culture medium during the last 30 min, while K+-free condition was obtained by replacing the culture medium by a nominal K+-free α-modified Eagle's medium medium during the last 30 min of the protocol. In some experiments, a specific inhibitor of caspase 1 (Ac-Tyr-Val-Ala-Asp-CHO (YVAD-CHO)) (50–100 μm; Bachem, Voisins-les-Bretonneux, France) was added to block IL-1β maturation. Detection of Intracellular and Extracellular IL-1β by ELISA—The concentrations of IL-1β (both pro and mature forms) released in the culture medium and present in cell lysates (i.e. intracellularly) were quantified by specific mouse IL-1β sandwich ELISAs. ELISA reagents were kindly supplied by Dr. S. Poole (National Institute for Biological Standard and Controls, Potters Bar, UK). Assay detection limits were <2 pg/ml. Following stimulation by LPS and ATP, media were collected, and a mixture of antiproteases was added to avoid protein degradation (4-(2-aminoethyl)benzenesulfonylfluoride hydrochloride (AEBSF), 0.2 mm; EDTA, 0.1 mm; bestatin, 13 μm; E64 0.14 μm; leupeptin, 0.1 μm; aprotinin, 0.03 μm). Extracellular protein contents were concentrated 20 times with Ultrafree-4 centrifugal filter units with a molecular mass cutoff of 4000 Da (Millipore, St. Quentin en Yvelines, France). Cell lysates were obtained by scratching cell cultures in a phosphate-buffered saline buffer containing 0.1% Triton X-100 and protease inhibitors. Cell debris and organelles were removed by centrifugation (12,000 × g, 10 min, 4 °C). Supernatants were assayed for intracellular IL-1β content. A sheep anti-mouse IL-1β antibody was used as a coating antibody, and a biotinylated sheep anti-mouse IL-1β antibody was used to sandwich the protein. Revelation was performed with horseradish peroxidase and o-phenylenediamine and read on a spectrophotometer. Both anti-IL1β antibodies recognize, without distinction, pro-IL-1β and mature IL-1β. Immunoblot Analysis—Because IL-1β antibodies recognized both pro and mature forms of IL-1β, Western blot analysis were performed to differentiate them according to their molecular weight. After appropriate stimulation, media were collected using the same protocol than for ELISA assays and concentrated 100 times. Cell lysates were obtained by scratching cell cultures in a lysate buffer containing (in mm): Tris-HCl, 20; EDTA, 1; MgCl2, 5; dithiothreitol, 1; aprotinin, 0.003; AEBSF, 1; sodium orthovanadate, 2 (pH 7.5) followed by mechanical trituration. Cell debris was removed by centrifugation (80 × g, 10 min, 4 °C), and supernatants were collected. Protein concentrations were determined by a colorimetric assay using bicinchoninate (MicroBCAssay, Interchim, Montluçon, France). 50 μg of protein were loaded into wells of a 13% acryl/bisacrylamide gel, and after separation, proteins were transferred to a polyvinylidene difluoride membrane (Immobilon-P, Millipore). After saturation in Tris-buffered saline-Tween (0.1%) containing 1% milk, the membrane was probed with a polyclonal sheep anti-mouse IL-1β (R&D Systems, Abingdon Oxon, UK) at 1:200 dilution and was incubated overnight at 4 °C and followed by 2-h incubation in a peroxidase-conjugated antibody-sheep IgG (1:8000). Revelation was obtained by chemoluminescence reaction (ECL, Amersham Biosciences, Orsay, France). In Situ Detection of Caspase Activity—Caspase activity was revealed by FITC-VAD-FMK assay (Promega, Charbonnières, France). FITC-VAD-FMK is a fluorogenic substrate of caspases that binds only active caspases. FITC-VAD-FMK (10 μm) was added to treated cell cultures during 45 min, and cell cultures were fixed with PFA 4% during 10 min. Fluorescence was monitored using an excitation filter (wavelength: 400–440 nm) and a barrier filter (wavelength: 480 nm). Electrophysiology—Currents were recorded from Schwann cells using the whole-cell configuration of the patch clamp technique. Patch pipettes were pulled from borosilicate glass capillaries (GF 150 TF-10, Clarck Electromedical Instruments, Pangbourne, UK) and filled with an internal solution containing (in mm): KCl, 120; CaCl2, 1; MgCl2, 2; HEPES, 10; EGTA, 10; glucose, 11; NaOH, 2; KOH, 33; (pH 7.4). In control conditions, the bathing solution (PSS) was (in mm): NaCl, 140; CaCl2, 5; MgCl2, 2; HEPES, 10; glucose, 11 and NaOH, 4 (pH 7.4). Voltage clamp protocols were applied from a holding potential of –70 mV by using a L/M-EPC-7 patch clamp amplifier (List Electronics, Darmstadt, Germany). Signals were stored on a digital audio tape recorder (DTR-1200, Biologic, Grenoble, France). Acute application of ATP was achieved to Schwann cells by a perfusion system based on electromagnetic valves controlling gravity flow (29Amédée T. Despeyroux S. Proc. R. Soc. Lond. B Biol. Sci. 1995; 259: 277-284Crossref Scopus (21) Google Scholar). Cell Viability—Cell death was assessed by colorimetric assay (Sigma), which measures the release of lactate dehydrogenase (LDH) by dying cells. Cell viability was estimated as the inverse ratio to LDH release for each experimental condition divided by total LDH release obtained by membrane permeabilization with Triton X-100 (0.01%, 15 min). RNA Isolation and Reverse Transcriptase (RT)-PCR Analysis—Total cytoplasmic RNA of mouse Schwann cells was extracted using 500 μl of RNAnow-TC extraction kit (Biogentex) according to the manufacturer's protocol. The RT-PCR was performed as follows: cDNA synthesis was carried out in a 20-μl reaction volume containing the total cytoplasmic RNA, 2.5 μm random primer (Roche Molecular Biochemicals SA, Meylan, France), 250 μm dNTPs (Amersham Biosciences, Saclay, France), 5 mm dithiothreitol, 20 units of RNase inhibitor (Promega, Paris, France), and 200 units of SuperScript™II reverse transcriptase (Invitrogen, Cergy Pontoise, France). After incubation overnight at 37 °C, samples were heated to 95 °C for 5 min and kept at –80 °C. 4 μl of cDNA were amplified in a final reaction volume of 50 μl consisting of 1× PCR buffer (Qiagen, Courtaboeuf, France) supplemented with a 50 μm concentration of each dNTP, a 0.2 μm concentration of each 5′ and 3′ specific primers, 1.5 mm MgCl2, and 2.5 units of Taq DNA polymerase (Qiagen, Courtaboeuf, France). Primer sequences were designed from Mus musculus P2X7 receptor sequence (GenBank™ accession number NM011027) and purchased from Genset (Paris, France). Primer sequences were: sense P2X7, 5′-CACATTTGGATGGTGGACCA-3′ and antisense P2X7, 5′-ACTTGAAGCCACTGTACTGC-3′. Primer sequences were designed from M. musculus TLR4 sequence (GenBank™ accession number NM021297) and purchased from Genset (Paris, France). Primer sequences were: sense TLR4, 5′-GAATTAAGCTCCATGAACTG-3′ and antisense TLR4, 5′-TCTAGATAGCTGAGACTTGG-3′. The β2-microglobulin (β2mgl) was used as an internal control and was detected using the following primers: sense β2mgl, 5′-TGACCGGCTTGTATGCTATC-3′ and antisense β2mgl, 5′-CAGTGTGAGCCAGGATATAG-3′. PCR was performed in a Mastercycler personal (Eppendorf France, Le Pecq, France) with the following parameters: denaturation at 94 °C, annealing at 61 °C for P2X7, 60 °C for TLR4, 65 °C for β2mgl, and primer extension at 72 °C for 1 min each step (35 cycles for P2X7, 30 cycles for TLR4, 29 cycles for β2mgl). The PCR products were separated by 13% acryl/bisacrylamide gel electrophoresis. The amplicons were revealed by UV illumination using ethidium bromide. The incorporation of 1 μCi of [α-32P]dCTP (3000 Ci/mmol, Amersham Biosciences, Les Ullis, France) during PCR allowed for the detection of the amplified product using a PhosphorImager screen (Amersham Biosciences, Bondoufle, France). The signal intensities of RT-PCR products were quantified by calculating the integrated volume of the band with a computing laser densitometer equipped with ImageQuant Software (Amersham Biosciences) normalized to the values for β2mgl for each experiment. The selected primers generated a predicted single PCR product of 556 bp for P2X7, 459 bp for TLR4, and 234 bp for β2mgl. Statistical Analysis—Results are expressed as mean ± S.E. Data were submitted to a normality test, and significance was tested by means of Student's paired t test and assessed at p < 0.05. When mentionned, data were analyzed by a one-way analysis of variance followed by Dunnett's method and assessed at p < 0.05. Production of IL-1β by Mouse Schwann Cells in Organotypic Cultures—Because LPS is described to act specifically through TLR4, we investigated first whether cultured mouse Schwann cells were expressing these receptors by looking at the mRNA level. As shown in Fig. 1, cultured Schwann cells expressed constitutively TLR4 mRNA. Then we studied the effectiveness of a co-treatment with LPS and ATP to induce the production of extracellular IL-1β by investigating the time of ATP treatment required to obtain a steady state level of IL-1β production. Organotypic cultures were primed with LPS during 6 h and with ATP during the last 5, 10, 15, 30, or 45 min of LPS priming. Fig. 2 shows that 5 min of stimulation by ATP led to the production of IL-1β around detection limits. The production of IL-1β became really appreciable after 10 min of stimulation by ATP and increased for longer stimulation to plateau after 30 min. However, as shown in Fig. 2B, when culture media were collected 25 min after a brief application of ATP (5 min), the concentration of IL-1β was not significantly different from that produced by 30 min of stimulation by ATP. This result suggests that if 5 min of stimulation of the P2X7 receptor is long enough to trigger IL-1β processing, mechanisms leading to the release of IL-1β need at least 10 min to yield extracellular detectable amounts of the cytokine.Fig. 2Production of IL-1b by organotypic cultures of mouse Schwann cells. Organotypic cultures were stimulated with LPS (10 μg/ml, 6 h) and with ATP (5 mm) during the indicated times. Extracellular media were collected, and IL-1β was measured by ELISA. A, the experimental protocol is described in the upper panel. Release of IL-1β became sizeable after 5 min of ATP stimulation and reached a plateau after 30 min. This time course is representative of profiles obtained in three different experiments. B, the experimental protocol is described in the upper panel. Extracellular medium was collected after 5 or 30 min of ATP stimulation or after 5 min of ATP stimulation followed by a washout of ATP and an incubation in a control medium during 25 min. Note that the concentration of IL-1β produced by a challenge with ATP of 5 min was not significantly different from that produced by 30 min ([IL-1β]5 min+25 min = 113 ± 7.8 pg/ml, n = 3; [IL-1β]30 min = 108 ± 51.5 pg/ml, n = 5; p > 0.05). Each bar represents the mean ± S.E.View Large Image Figure ViewerDownload Hi-res image Download (PPT) IL-1β Synthesis by Schwann Cells Requires LPS Priming but Not P2X 7 Stimulation—Having established the experimental conditions of a sizeable and reproducible production of IL-1β, we then studied the effectiveness of LPS and/or purinergic stimulation to induce the intracellular synthesis of IL-1β. Cultures were either treated with ATP alone for 30 min or primed by LPS for 6 h and treated with ATP during the last 30 min of the protocol. Intracellular IL-1β was assayed in cell lysates by ELISA. Fig. 3A shows that intracellular IL-1β was barely detectable in control conditions or after the sole stimulation by ATP. In contrast, LPS priming of cultures for 6 h led to the synthesis of noticeable amounts of IL-1β. Intracellular IL-1β synthesis induced by LPS priming was not significantly altered by neither a co-treatment with ATP nor the addition of oATP, a potent P2X7 antagonist. As organotypic cultures of dorsal root ganglia contain neurons and Schwann cells, we were led to consider which cell type was synthesizing IL-1β. Therefore we excised dorsal root ganglia to remove the neuronal population before assaying intracellular IL-1β content. In these conditions, IL-1β content of restricted Schwann cells lysate did not significantly differ from IL-1β content of total cells lysate (Fig. 3B). These results suggest first, that intracellular IL-1β measured in organotypic cultures was mainly, if not entirely, synthesized by Schwann cells and second, that Schwann cells did synthesize IL-1β only when challenged by an immune stimulus. Cultured Schwann Cells Release IL-1β When co-stimulated by LPS and ATP—We studied the release of IL-1β by dorsal root ganglia cultures in the same experimental conditions than for intracellular synthesis of IL-1β. In control conditions, IL-1β was barely detectable in culture medium (Fig. 4A). LPS priming or ATP stimulation alone did not induce any noticeable release of IL-1β in the culture medium, whereas a co-treatment with LPS and ATP triggered a significant release of IL-1β, which was abolished by pretreatment with oATP. Similar results were obtained in the absence of neurons (Fig. 4B). Pro- and Mature IL-1β Are Released by Schwann Cells— Western blot analysis of intracellular lysates and extracellular media was performed to investigate the production and the release processes of the pro-IL-1β (34–35 kDa) and the mature IL-1β (17 kDa) by dorsal root ganglia cultures. Fig. 5 shows that LPS priming induced the synthesis of pro-IL1β (Fig. 5A) but did not induce detectable levels of intracellular (Fig. 5A) and extracellular mature IL-1β (Fig. 5B). The co-treatment with ATP and LPS did not alter the synthesis of pro-IL-1β, but led to the production of extracellular mature IL-1β (Fig. 5B). When Schwann cells were co-treated with LPS and ATP in a potassium-free extracellular medium, mature IL-1β became detectable in intracellular lysates (Fig. 5A), and the production of extracellular IL-1β was clearly enhanced (Fig. 5B). The pretreatment with oATP (300 μm, 90 min) blocked the maturation of intracellular pro-IL-1β (Fig. 5A) and the production of extracellular mature Il-1β (Fig. 5B). These results suggest that LPS triggered the synthesis of pro-IL-1β and that ATP, most likely through P2X7 activation, induced not only the processing of pro-IL-1β into mature IL-1β, but also its release. The Processing of IL-1β by Schwann Cells Needs the Activation of ICE—To assess the involvement of ICE in the processing mechanisms of IL-1β following LPS or LPS + ATP treatments, cultured Schwann cells were primed by LPS, co-treated by ATP with or without an ICE-specific inhibitor, YVAD-CHO (100 μm, 6 h). The addition of YVAD-CHO blocked by more than 95% the production of extracellular IL-1β (Fig. 6A). Western blot analysis revealed that YVAD-CHO did not block neither the intracellular synthesis of pro-IL-1β nor its release when co-treated with LPS and ATP (Fig. 6B). The activation of ICE was revealed in cultured Schwann cells, using a caspase substrate, FITC-VAD-FMK (10 μm), which fluoresced when cleaved by activated caspases. Fig. 6C shows that ATP on its own clearly activated intracellular Schwann cells caspases and that this activation was not altered by LPS priming. The addition of YVAD-CHO strongly reduced the fluorescence, i.e. the activ