Abstract
23 min readSeveral effects of the proinflammatory cytokine, interleukin-1β (IL-1β), have been described in the central nervous system, and one area of the brain where marked changes have been reported is the hippocampus. Among these changes are an IL-1β-induced inhibition of long term potentiation (LTP) in perforant path-granule cell synapses and an attenuation of glutamate release in synaptosomes prepared from the hippocampus. Evidence suggests that, at least in circulating cells, the anti-inflammatory cytokine, IL-10, antagonizes certain effects of IL-1. We investigated the effect of IL-10 on IL-1β-induced inhibition of LTP and glutamate release. The evidence presented indicates that IL-1β stimulates the stress-activated protein kinase, c-Jun-activated protein kinase (JNK), and IL-1 receptor-associated kinase, which may explain its inhibitory effect on release and LTP, and that IL-10 reversed the IL-1β-induced stimulation of JNK activity and inhibition of release and LTP. We observed that IL-10 abrogated the stimulatory effect of IL-1β on superoxide dismutase activity and reactive oxygen species production, whereas the H2O2-induced inhibition of LTP was also blocked by IL-10. We present evidence that suggests that the action of IL-10 may be mediated by its ability to induce shedding of the IL-1 type I receptor. Several effects of the proinflammatory cytokine, interleukin-1β (IL-1β), have been described in the central nervous system, and one area of the brain where marked changes have been reported is the hippocampus. Among these changes are an IL-1β-induced inhibition of long term potentiation (LTP) in perforant path-granule cell synapses and an attenuation of glutamate release in synaptosomes prepared from the hippocampus. Evidence suggests that, at least in circulating cells, the anti-inflammatory cytokine, IL-10, antagonizes certain effects of IL-1. We investigated the effect of IL-10 on IL-1β-induced inhibition of LTP and glutamate release. The evidence presented indicates that IL-1β stimulates the stress-activated protein kinase, c-Jun-activated protein kinase (JNK), and IL-1 receptor-associated kinase, which may explain its inhibitory effect on release and LTP, and that IL-10 reversed the IL-1β-induced stimulation of JNK activity and inhibition of release and LTP. We observed that IL-10 abrogated the stimulatory effect of IL-1β on superoxide dismutase activity and reactive oxygen species production, whereas the H2O2-induced inhibition of LTP was also blocked by IL-10. We present evidence that suggests that the action of IL-10 may be mediated by its ability to induce shedding of the IL-1 type I receptor. interleukin-1β IL-1 type 1 receptor long term potentiation IL-1 receptor-activated kinase c-Jun NH2-terminal kinase lipopolysaccharide tumor necrosis factor vasoactive intestinal protein extracellular signal-regulated kinase analysis of variance excitatory postsynaptic potential Interleukin-1β (IL-1β)1 is a proinflammatory cytokine that is released from antigen-presenting cells during infection or inflammation, and although its effects were originally considered to be confined to the immune system, it is now known to exert profound effects in the central nervous system. These effects include modulation of thermoregulation, sleep, and appetite, which are perhaps consistent with the relatively high expression of the signal-generating IL-1 type 1 receptors (IL-1R1) in hypothalamus (1Lechan R.M. Toni R. Clark B.D. Cannon J.G. Shaw A.R. Dinarello C.A. 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McGahon B. McBennett S. Lynch M. Neurobiol. Aging. 1997; 18: 343-348Crossref PubMed Scopus (98) Google Scholar, 8Plata-Salaman C.R. ffrench-Mullen J.M.H. Eur. J. Pharmacol. 1994; 266: 1-10Crossref PubMed Scopus (129) Google Scholar) in the hippocampus, and it has been shown to inhibit long term potentiation (LTP) in CA1, CA3, and dentate gyrus in vitro (9Bellinger F.P. Madamba S. Siggins G.R. Brain Res. 1993; 628: 227-234Crossref PubMed Scopus (312) Google Scholar, 10Katsuki H. Nakai S. Hirai Y. Akaji K. Kiso Y. Satoh M. Eur. J. Pharmacol. 1990; 181: 323-326Crossref PubMed Scopus (283) Google Scholar, 11Cunningham A.J. Murray C.A. O'Neill L.A.J. Lynch M.A. O'Connor J.J. Neurosci. Lett. 1996; 203: 1-4Crossref PubMed Scopus (342) Google Scholar) and in dentate gyrus in vivo (12Murray C. Lynch M.A. J. Neurosci. 1998; 18: 2974-2981Crossref PubMed Google Scholar, 13Murray C. Lynch M.A. J. Biol. Chem. 1998; 273: 12161-12168Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar, 14Vereker E. O'Donnell E. Lynch M.A. J. Neurosci. 2000; 20: 6811-6819Crossref PubMed Google Scholar, 15Lynch M.A. Prog. Neurobiol. (New York). 1998; 56: 1-19Crossref PubMed Scopus (144) Google Scholar); these effects are consistent with the high distribution of IL-1R1 in hippocampus (1Lechan R.M. Toni R. Clark B.D. Cannon J.G. Shaw A.R. Dinarello C.A. Reichlin S. Brain Res. 1990; 514: 135-140Crossref PubMed Scopus (331) Google Scholar, 2Ban E. Milon G. Prudhomme N. Fillion G. Haour F. Neuroscience. 1991; 43: 21-30Crossref PubMed Scopus (261) Google Scholar, 3Cunningham Jr., E.T. Wada E. Carter D.B. Tracey D.E. Battey J.F. De Souza E.B. J. Neurosci. 1992; 12: 1101-1114Crossref PubMed Google Scholar, 4Parnet P. Amindari S. Wu C. Brunke-Reese D. Goujon E. Weyhenmeyer J.A. Danzer R. Kelley K.W. Mol. Brain Res. 1994; 27: 63-70Crossref PubMed Scopus (132) Google Scholar, 5Ericsson A. Liu C. Kasckow J. Hart R.P. Sawchenko P.F. Soc. Neurosci. Abstr. 1993; 19: 95Google Scholar). The inhibitory effects of IL-1β in hippocampus have been linked with stimulation of the stress-activated kinases, p38 and JNK (14Vereker E. O'Donnell E. Lynch M.A. J. Neurosci. 2000; 20: 6811-6819Crossref PubMed Google Scholar, 16O'Donnell E. Vereker E. Lynch M.A. Eur. J Neurosci. 2000; 12: 345-352Crossref PubMed Scopus (115) Google Scholar), which have also been shown to be activated by IL-1β in other cells (17Raingeaud J. Gutpa S. Rogers J.S. Dickens M. Han J. Ulevitch R.J. Davis R.J. J. Biol. Chem. 1995; 270: 7420-7426Abstract Full Text Full Text PDF PubMed Scopus (2030) Google Scholar, 18Rizzo M.T. Carlo-Stella C. Blood. 1996; 88: 3792-3800Crossref PubMed Google Scholar, 19Uciechowski P. Saklatvala J. von der Ohe J. Resch K. Szamel M. Kracht M. FEBS Lett. 1996; 394: 273-278Crossref PubMed Scopus (33) Google Scholar, 20Derijard B. Hibi M. Wu I.-H. Barrett T. Su B. Deng T. Karin M. Davis R.J. Cell. 1994; 76: 1025-1037Abstract Full Text PDF PubMed Scopus (2941) Google Scholar). Evidence suggests that activation of IL-1 receptor-activated kinase (IRAK) is closely linked with JNK activation (21O'Neill L.A.J. Greene C. J. Leukocyte Biol. 1998; 63: 650-657Crossref PubMed Scopus (493) Google Scholar). Among the documented consequences of enhanced activity of JNK and/or p38 in some cells are growth arrest and deterioration of cell function or even cell death (22Park D.S. Stefanis L. Yan C.Y.I. Farinelli S.E. Greene L.A. J. Biol. Chem. 1996; 271: 21898-21905Abstract Full Text Full Text PDF PubMed Scopus (195) Google Scholar, 23Maroney A.C. Glicksman M.A. Basma A.N. Walton K.M. Knight Jr., E. Murphy C.A. Bartlett B.A. Finn J.P. Angeles T. Matsuda Y. Neff N.T. Dionne C.A. J. Neurosci. 1998; 18: 104-111Crossref PubMed Google Scholar). In contrast to the proinflammatory effects of IL-1β, IL-10 has been shown to possess anti-inflammatory properties. Like IL-1β, IL-10 was originally identified as a product of certain cells of the immune system, i.e. T helper cells, B cells, monocytes, and macrophages (24Fiorentino D.F. Bond M.W. Mosmann T.R. J. Exp. Med. 1989; 170: 2081-2095Crossref PubMed Scopus (2456) Google Scholar, 25Moore K.W. O'Garra A. DeWall Malefyt R. Vieira O. Mosmann T.R. Annu. Rev. Immunol. 1993; 11: 165-190Crossref PubMed Scopus (2361) Google Scholar), although more recently it has been suggested that IL-10 is produced by cells in the hypothalamus and pituitary (26Rady P.L. Smith E.M. Cadet O. Opp M.R. Tyring S.K. Huges Jr., T.K. Cell. Mol. Neurobiol. 1995; 15: 289-296Crossref PubMed Scopus (31) Google Scholar). IL-10 is co-released with IL-1β following injection of lipopolysaccharide (LPS (27Durez P. Abramowicz D. Gerard C. Van Mechelen M. Amraoui Z. Dubois C. Leo O. Velu T. Goldman M. J. Exp. Med. 1993; 177: 551-555Crossref PubMed Scopus (112) Google Scholar, 28Van der Poll T. Jansen J. Levi M. Ten Cate H. Ten Cate J.W. Van Deventer J.H. J. Exp. Med. 1994; 180: 1985-1988Crossref PubMed Scopus (170) Google Scholar)), but it has been shown to inhibit the production of IL-1β and TNFα in LPS-activated macrophages (24Fiorentino D.F. Bond M.W. Mosmann T.R. J. Exp. Med. 1989; 170: 2081-2095Crossref PubMed Scopus (2456) Google Scholar). IL-10 has also been shown to reverse the IL-1-induced fever that follows LPS injection (29Leon L.R. Kozak W. Kluger M.J. Ann. N. Y. Acad. Sci. 1998; 856: 69-75Crossref PubMed Scopus (48) Google Scholar), whereas IL-1β induces slow wave sleep (30Shoham S. Davenne S. Cady A.B. Dinarello C.A. Krueger J.M. Am. J. Physiol. 1987; 253: R142-R149PubMed Google Scholar), IL-10 reduces sleep (31Opp M.R. Smith E.M. Hughes T.K. J. Neuroimmunol. 1995; 60: 165-168Abstract Full Text PDF PubMed Scopus (109) Google Scholar). At the level of the hippocampus, it has been shown that recovery following traumatic brain injury was improved by treatment with IL-10, and this was associated with decreased concentration of IL-1 in hippocampus (32Knoblach S.M. Faden A.I. Exp. Neurol. 1998; 153: 143-151Crossref PubMed Scopus (237) Google Scholar). The evidence therefore indicates that IL-10 inhibits certain actions of IL-1β, in some cases by inhibiting IL-1β production and/or release. In an effort to examine this question further, we set out to establish whether IL-10 might antagonize the inhibitory effect of IL-1β on synaptic function in the hippocampus. The data indicate that IL-10 abrogates the IL-1β-induced inhibition of glutamate release and LTP and its stimulatory effect on JNK. We propose that this action of IL-10 may be mediated by its ability to prevent reactive oxygen species production by IL-1β. Male Wistar rats (BioResources Unit, Trinity College, Dublin, Ireland) were used in these experiments. Animals were housed in groups of 4–6 under a 12-h light schedule; ambient temperature was controlled between 22 and 23 °C, and rats were maintained under veterinary supervision. The activities of ERK (33McGahon B. Maguire C. Kelly A. Lynch M.A. Neuroscience. 1999; 90: 1167-1175Crossref PubMed Scopus (50) Google Scholar) and JNK (16O'Donnell E. Vereker E. Lynch M.A. Eur. J Neurosci. 2000; 12: 345-352Crossref PubMed Scopus (115) Google Scholar) were analyzed in P2 preparations obtained from dentate gyrus. Tissue samples were equalized for protein concentration (34Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (211983) Google Scholar) and diluted so that the same concentration of protein (1 mg/ml) was loaded onto each lane. In experiments in which the effect of vasoactive intestinal protein (VIP) was assessed, P2 preparations were made in the presence of VIP (1 μm) allowing incorporation of the peptide into synaptosomes before membranes resealed. In other experiments, samples (which were/were not prepared in the presence of VIP) were preincubated for 15 min in IL-1β (1 ng/ml), IL-10 (10 ng/ml), H2O2 (200 μm), or a combination of some of these agents; in all circumstances, control samples were incubated in vehicle (Krebs solution containing 1.8 mmCaCl2) only. In a separate series of experiments, synaptosomes were prepared from dentate gyrus of rats that were injected intracerebroventricularly with saline or IL-1β (3.5 ng/ml) or H2O2 (200 μm). Aliquots (10 μl, 1 mg/ml) were added to sample buffer (10 μl; Tris-HCl, 0.5 mm, pH 6.8; glycerol 10%; SDS, 10%; β-mercaptoethanol, 5%; bromphenol blue, 0.05% w/v), boiled for 5 min, and loaded onto gels (10% SDS for ERK; 12% for JNK). Proteins were separated by application of 30 mA constant current for 25–30 min, transferred onto nitrocellulose strips (225 mA for 75 min), and immunoblotted with the appropriate antibody. To assess ERK activity, proteins were immunoblotted overnight at 4 °C with an antibody specific for the phosphorylated form of ERK (Promega; 1:4,000 in phosphate-buffered saline/Tween (0.1% Tween 20; PBS-T) containing 2% non-fat dried milk). To assess JNK activity, proteins were immunoblotted with an antibody that specifically targets phosphorylated JNK (Santa Cruz Biotechnology; 1:2,000 in PBS-T (0.1% Tween 20) containing 2% non-fat dried milk) for 2 h at room temperature. To assess IRAK, proteins were immunoblotted with a rabbit polyclonal anti-IRAK-1 antibody (1:4,000 Tris-buffered saline/Tween (0.1% Tween 20 containing 0.1% bovine serum albumin) for 2 h at room temperature. In all cases, nitrocellulose strips were washed and incubated for 2 h at room temperature with secondary antibody (horseradish peroxidase-linked anti-rabbit antibody; 1:10,000 dilution (Amersham Pharmacia Biotech) in the case of ERK, horseradish peroxidase-linked anti-rabbit antibody; 1:1,000 dilution (Amersham Pharmacia Biotech) in the case of IRAK, and peroxidase-linked anti-mouse IgG; 1:2,000 dilution (Sigma) in the case of JNK). Protein complexes were visualized by ECL detection (Amersham Pharmacia Biotech) in the case of ERK and JNK and Supersignal (Pierce) in the case of IRAK. Immunoblots were exposed to film for 3–4 h in the case of ERK, overnight in the case of JNK, and 10 s in the case of IRAK and processed using a Fuji x-ray processor. Protein bands were quantitated by densitometric analysis. The impure synaptosomal preparation, P2, was prepared as described previously (35McGahon B. Lynch M.A. Neuroscience. 1996; 72: 847-855Crossref PubMed Scopus (52) Google Scholar) and resuspended in oxygenated Krebs solution containing 2 mm CaCl2. Synaptosomes were preincubated for 15 min at 37 °C in oxygenated Krebs solution containing 2 mm CaCl2 or Krebs solution to which 1 ng/ml IL-1β, 10 ng/ml IL-10, or both were added. In some experiments, synaptosomes were prepared in the presence of VIP (1 μm) as described above and were subsequently incubated with or without IL-1β (1 ng/ml) or H2O2 (200 μm). Tissue samples were aliquoted onto Millipore filters (0.45 mm), rinsed under vacuum, and then incubated in 250 μl of oxygenated Krebs solution at 37 °C for 3 min in the presence or absence of 40 mm KCl. The filtrate was collected and stored at −80 °C for later analysis (36Ordronneau P. Abdullah L. Petruse P. J. Immunol. Methods. 1991; 142: 169-176Crossref PubMed Scopus (41) Google Scholar). Triplicate samples (50 μl) or glutamate standards (50 μl; 50 nm to 10 μmprepared in 100 mm Na2HPO4 buffer, pH 8.0) were added to glutaraldehyde-coated 96-well plates, incubated, and washed. Ethanolamine (250 μl; 0.1 m in 100 mm Na2HPO4 buffer) was used to bind any unreacted aldehydes, and donkey serum was used to block nonspecific binding. Antiglutamate antibody (raised in rabbit; 100 μl; 1:5,000 in PBS-T; Sigma) was added, incubated, washed, and reacted with secondary antibody (anti-rabbit horseradish peroxidase-linked antibody; 100 μl; 1:10,000 in PBS-T; Amersham Pharmacia Biotech). 3,3′,5,5′-Tetramethylbenzidine liquid substrate was added as chromogen; samples were incubated for exactly 60 min at room temperature, and H2SO4 (4 m; 50 μl) was added to stop the reaction. Optical densities were determined at 450 nm using a multiwell plate reader, and values were calculated with reference to the standard curve, corrected for protein (34Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (211983) Google Scholar) and expressed as μmol of glutamate/mg of protein. Samples (dentate gyrus synaptosomes), which were preincubated for 20 min at 37 °C in IL-1β (1 ng/ml), IL-10 (10 ng/ml), or both, were assessed for IL-1R1 expression by gel electrophoresis and immunoblotting. Following incubation, samples underwent one freeze-thaw cycle and were centrifuged (10,000 × g for 10 min). The supernatant was used to assess soluble IL-1R1, and the pellet, which was resuspended in Krebs solution containing 2 mmCaCl2, was used to assess membrane-associated IL-1R1. In both cases, samples were equalized for protein concentration, and then proteins were separated by application of 30 mA constant current for 25–30 min, transferred onto nitrocellulose strips (225 mA for 75 min), and blocked overnight at 4 °C in PBS-T containing 6% non-fat dried milk. After appropriate washing (5 times 10-min washes in PBS-T), membranes were incubated in the primary antibody (rabbit anti-rat IL-1R1 IgG (Santa Cruz Biotechnology; 1:1,000 in PBS-T containing 2% non-fat dried milk)) for 45 min at room temperature and 45 min at 37 °C, washed (4 times 10-min washes in PBS-T), incubated in the secondary antibody (horseradish peroxidase-linked anti-rabbit, 1:2,000 in PBS-T containing 2% non-fat milk) for 45 min at room temperature and 45 min at 37 °C, and washed. Protein complexes were visualized by ECL detection (Amersham Pharmacia Biotech) by exposing immunoblots to film for overnight at 4 °C and processed using a Fuji x-ray processor. Protein bands were quantitated by densitometric analysis. Rats were anesthetized by intraperitoneal injection of urethane (1.5 g/kg intraperitoneal); the absence of a pedal reflex was considered to be an indicator of deep anesthesia. LTP was induced unilaterally in perforant path-granule cell synapses as described previously (12Murray C. Lynch M.A. J. Neurosci. 1998; 18: 2974-2981Crossref PubMed Google Scholar, 13Murray C. Lynch M.A. J. Biol. Chem. 1998; 273: 12161-12168Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar). Briefly, a bipolar stimulating electrode and an unpopular recording were stereotaxically positioned in the perforant path (4.4 mm lateral to λ) and dorsal cell body region of the dentate gyrus (2.5 mm lateral and 3.9 mm posterior to Bregma), respectively. Rats were injected intracerebroventricularly (2.5 mm posterior, and 0.5 mm lateral, to Bregma) with saline, IL-1β (3.5 ng/ml) alone, or together with IL-10 (35 ng/ml or 1 μg/ml) or with H2O2 (200 μm) alone, or together with IL-10 (1 μg/ml); injection volume was 5 μl in all cases. Test shocks were given at 30-s intervals and recorded for 10 min before and 40 min after tetanic stimulation (3 trains of stimuli; 250 Hz for 200 ms; 30 s intertrain interval). Tetanic stimulation was delivered 40 min after injection. The formation of reactive oxygen species was assessed by analyzing formation of the highly fluorescent 2′,7-dichlorofluorescein from the non-fluorescent probe, 2′7′-dichlorofluorescein diacetate (Molecular Probes (37Lebel C.P. Bondy S.C. Neurochem. Int. 1990; 17: 435-440Crossref PubMed Scopus (283) Google Scholar)). The synaptosomal pellet, P2, was prepared from hippocampus and resuspended in 1 ml of ice-cold 40 mm Tris buffer, pH 7.4. Samples were incubated at 37 °C for 15 min in the presence of 2′7′-dichlorofluorescein diacetate (10 μl; final concentration 5 μm; from a stock solution of 500 μm in methanol) to which IL-1β (1 μg/ml) and/or IL-10 (10 ng/ml) was added. To terminate the reaction, the dye-loaded synaptosomes were centrifuged at 13,000 × g for 8 min. The pellet was resuspended in 3 ml of ice-cold 40 mm Tris buffer, pH 7.4. Fluorescence was monitored at a constant temperature of 37 °C immediately before stimulation with IL-1β (1 ng/ml) and 15 min post-stimulation, at 488 nm excitation (bandwidth 5 nm), and 525 nm emission (bandwidth 20 nm). Reactive oxygen species formation was quantified from a standard curve of 2′,7-dichlorofluorescein in methanol (range 0.05 to 1 μm). Protein concentration was determined (34Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (211983) Google Scholar), and the results were expressed as nmol/mg protein/min. Superoxide dismutase activity was determined according to the method described previously (38Spitz D.R. Oberley L.W. Anal. Biochem. 1989; 179: 8-18Crossref PubMed Scopus (582) Google Scholar). Briefly, hippocampal slices were homogenized in Krebs solution containing CaCl2 and centrifuged at 15,000 for 10 min. Aliquots (800 μl) of incubation buffer (50 mmpotassium buffer (pH 7.8) containing 1.8 mm xanthine, 2.24 mm nitro blue tetrazolium, 40 units of catalase, 7 μl/ml xanthine oxidase, and 1.33 mm diethylenetriaminepentaacetic acid) were added to samples of supernatant (100 μl) at different dilutions (1:2, 1:5, 1:10, 1:20, 1:50, and 1:100) and analyzed by UV spectroscopy at 560 nm. In some experiments, slices were incubated for 30 min at 37 °C in IL-1β (100 pg/ml) in the presence/absence of IL-10 (10 ng/ml) to analyze the effect of the cytokines on superoxide dismutase activity. Enzyme activity was assessed as the rate of reduction of nitro blue tetrazolium, which was inhibited with increasing concentrations of protein. One unit of activity was defined as the amount of protein necessary to decrease the rate of the reduction of nitro blue tetrazolium by 50%. Fig. 1 A shows that IL-1β (1 ng/ml) increased JNK activity as indicated by an increase in the phosphorylated form of JNK; this effect is demonstrated in one sample immunoblot and also in the mean data obtained from seven experiments that indicate a statistically significant effect of IL-1β (p < 0.05; Student's t test for paired means). IL-10 reversed the stimulatory effect of IL-1β on JNK activity. In contrast to its effect on JNK, neither IL-1β alone nor in combination with IL-10 affected ERK phosphorylation (Fig.1 B). Data from previous experiments indicated that IL-1β inhibited KCl-stimulated [3H]glutamate release in hippocampus (7Murray C.A. McGahon B. McBennett S. Lynch M. Neurobiol. Aging. 1997; 18: 343-348Crossref PubMed Scopus (98) Google Scholar) and that increased JNK activity was coupled with decreased endogenous glutamate release (14Vereker E. O'Donnell E. Lynch M.A. J. Neurosci. 2000; 20: 6811-6819Crossref PubMed Google Scholar, 16O'Donnell E. Vereker E. Lynch M.A. Eur. J Neurosci. 2000; 12: 345-352Crossref PubMed Scopus (115) Google Scholar); therefore, we analyzed the effect of IL-1β (1 ng/ml) alone and in the presence of IL-10 (10 ng/ml) on endogenous glutamate release. Fig. 1 C indicates that although incubation of hippocampal synaptosomes in the presence of 40 mm KCl significantly enhanced glutamate release (**,p < 0.01; ANOVA), this effect was blocked when IL-1β was incubated in the incubation medium. The data also indicate that unstimulated release was decreased by IL-1β (+, p < 0.05; ANOVA). IL-10 completely abrogated the effects of IL-1β so that both unstimulated and KCl-stimulated release were similar to values observed under control conditions. Because JNK activation is reported to be closely coupled with IRAK phosphorylation in certain cell types (21O'Neill L.A.J. Greene C. J. Leukocyte Biol. 1998; 63: 650-657Crossref PubMed Scopus (493) Google Scholar), we addressed the question of a similar coupling in hippocampus and reported that, although IL-1β significantly increases the 100-kDa phosphorylated form of IRAK (Fig. 1 D; p < 0.05; ANOVA), IL-10 inhibits this IL-1β-associated effect. When the 80-kDa unphosphorylated form of IRAK was assessed, no significant change with IL-1β, IL-10, or the combination of both was observed (data not shown); however, analysis of the ratio of 100-kDa IRAK to 80-kDa IRAK revealed a significant increase with IL-1β which was suppressed by IL-10 (Fig. 1 E; p < 0.05; ANOVA). The data are consistent with the idea that JNK activation by IL-1β and the inhibition of this effect by IL-10 is linked with, and may be a consequence of, IRAK activation. The inhibitory effect of IL-1β on glutamate release represents one factor that might contribute to its inhibitory effect on LTP. It might be argued that, if this is the case, the effect of IL-1β on LTP may also be suppressed by IL-10. Fig. 2indicates that, in saline-treated rats, there was an immediate increase in the population epsp slope following tetanic stimulation and that this increase persisted for the duration of the experiment. The mean percentage change (± S.E.) in the last 5 min of the experiment was 140.14 ± 2.47 (compared with the value in the 5 min prior to the tetanus). Intracerebroventricular injection of IL-1β inhibited both the early and later components of LTP; in this group, the mean percentage change in population epsp slope in the last 5 min of the experiment was 96.44 ± 1.30 (Fig. 2 B;p < 0.001; ANOVA). IL-10 attenuated the effect of IL-1β in a dose-dependent manner; thus the mean percentage changes in population epsp slope (± S.E.) in the last 5 min of the experiment were 105.5 ± 0.75 and 117.3 ± 0.91, respectively, in rats treated with IL-1β and 35 ng/ml IL-10 and in rats treated with IL-1β and 1 μg/ml IL-10; these values were significantly different from the value in IL-1β-treated rats (p < 0.001; ANOVA). However, the data also show that IL-10 exerted an inhibitory effect on LTP; the mean percentage change in the last 5 min of the experiment in IL-10-treated rats was 113.1 ± 0.37, which was significantly lower than that in saline-treated controls (p < 0.001; ANOVA). We considered that one mechanism by which IL-10 might act to inhibit the effect of IL-1β was by modulating expression of IL-1R1. Fig. 3 indicates that incubation of tissue in the presence of IL-10 significantly decreased expression of IL-1R1 in membrane fractions (p < 0.05; Student'st test for paired means) and significantly increased its expression in cytosolic fractions (p < 0.05; Student's t test for paired means). Because previous data indicated that IL-1β increased reactive oxygen species production in hippocampus (12Murray C. Lynch M.A. J. Neurosci. 1998; 18: 2974-2981Crossref PubMed Google Scholar, 13Murray C. Lynch M.A. J. Biol. Chem. 1998; 273: 12161-12168Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar), it seemed reasonable to propose that IL-10 might antagonize this effect. Fig.4 A indicates that IL-1β significantly increased reactive oxygen species accumulation (p < 0.05; Student's t test for paired values), and this was reversed by co-incubation in the presence of IL-10 (10 ng/ml). In parallel, IL-1β significantly increased superoxide dismutase activity (p < 0.05; Student'st test for paired values; Fig. 4 B), and this effect was also reversed by IL-10, although it is acknowledged that the S.E. values in this case are rather large. We report that H2O2 mimicked the stimulatory effect of IL-1β on JNK; thus intracerebroventricular injection of IL-1β (3.5 ng/ml; Fig.5 A) or H2O2 (200 μm; Fig. 5 C) increased JNK activity as shown by the sample immunoblots; analysis of the mean values obtained from densitometric analysis indicated a significant stimulatory effect of both agents (p < 0.05; Student's t test for paired values). These effects of IL-1β and H2O2 were mimicked in vitro; thus incubation of hippocampal tissue in the presence of IL-1β (Fig. 5 B) or H2O2 (Fig.5 D) increased JNK as indicated by the sample immunoblots; densitometric analysis revealed that these effects were statistically significant (p < 0.05; Student'st test for paired values). If IL-1β mediates its effects by increasing reactive oxygen species production, and if IL-10 inhibits the effect of IL-1β by antagonizing this, then the inhibitory effect of IL-1β on LTP should be mimicked by H2O2, which generates reactive oxygen species, and IL-10 should suppress this effect of H2O2. Fig.6 A indicates that LTP was induced and sustained in saline-treated rats but blocked in rats that received an intracerebroventricular injection of H2O2; the mean percentage changes in population epsp slope (± S.E.) in the last 5 min of the experiment (compared with the value in the 5 min prior to the tetanus) were 141.2 ± 0.81 and 102.4 ± 0.83 in saline-treated and H2O2-treated rats, respectively (Fig.6 B). Intracerebroventricular injection of IL-10 (1 μg/ml) reversed the inhibitory effect of H2O2, but values were not completely restored to control values; thus the mean percentage change in population epsp slope in the last 5 min of the experiment was 129.9 ± 1.58 (Fig. 6 B). Both IL-1β and H2O2 induced parallel changes in JNK activation and glutamate release, but confirmation of a causal relationship between the two measures requires assessment of the effect of a JNK inhibitor on IL-1β-induced inhibition of glutamate release. Although not specific, VIP has been shown to inhibit JNK in some cells, and here we assessed the possibility that it might inhibit IL-1β- and H2O2-induced JNK activation in hippocampus. Fig. 7, A and C,shows sample immunoblot in which the stimulatory effect of IL-1β (Fig. 7 A) and H2O2 (Fig.7 C) on JNK phosphorylation are clearly shown (comparelanes 1 and 2; control and IL-1β- or H2O2-treated, respectively); these sample immunoblots also show that VIP inhibited the IL-1β- and H2O2-induced effects (lane 4), whereas VIP alone exerted no marked effect (lane 3).
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