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Regulation of interleukin-6 gene expression in brain of aged mice by nuclear factor κB
Journal of Neuroimmunology 117 Ž2001. 87–96 www.elsevier.comrlocaterjneuroin
Regulation of interleukin-6 gene expression in brain of aged mice by nuclear factor k B Shi-Ming Ye, Rodney W. Johnson ) Laboratory of IntegratiÕe Biology, Department of Animal Sciences, UniÕersity of Illinois, 390 Animal Science Laboratory, 1207 West Gregory DriÕe, Urbana, IL 61801, USA Received 10 January 2001; received in revised form 4 April 2001; accepted 5 April 2001
Abstract Interleukin-6 ŽIL-6. is increased in brain of aged mice. The purpose of this study was to determine if binding of nuclear factor k B ŽNFk B. to the IL-6 promoter is responsible for the age-related increase in brain IL-6. In an initial study, the effect of age on IL-6 in brain was verified as IL-6 protein was increased in brain of aged mice compared to adult and juvenile mice. Competitive RT-PCR showed that IL-6 mRNA concentration was at least 4-fold higher in aged brain compared to adult brain. Next, binding of the transcription factor NFk B to the IL-6 promoter in brains of 1-, 6-, and 24-month-old mice was determined. Electrophoretic mobility shift assay showed that NFk B activity was increased in aged brain compared to adult and juvenile brain. Moreover, glial cells cultured from aged mice showed more NFk B DNA-binding activity and more IL-6 mRNA and protein expression than glia from adults. However, incubating glia from aged mice in the presence of k B decoy inhibited these effects of age. The same was observed in vivo as intracerebroventricular injection of k B decoy in aged mice decreased NFk B activity and IL-6 mRNA and protein in brain. These results show that the DNA-binding activity of NFk B is increased in the brain of aged mice and that at least one consequence is increased expression of IL-6. q 2001 Elsevier Science B.V. All rights reserved. Keywords: Aging; Brain; Cytokines; Interleukin-6; Mouse; Nuclear factor k B
1. Introduction Astrocytes, microglia, and neurons express inflammatory cytokines, including interleukin-6 ŽIL-6.. Under normal physiological conditions the gene encoding IL-6 is tightly regulated and expression in the brain is relatively low. However, elevated levels of IL-6 are evident in the brain in a number of central nervous system ŽCNS. diseases, and over expression of IL-6 and other inflammatory cytokines is thought to result in neuropathology. As evidence of such, inflammation and increased expression of IL-6 have been documented in brain trauma ŽWoodroofe et al., 1991. and in certain neurodegenerative diseases, including Alzheimer’s disease ŽBauer et al., 1991.. Interestingly, IL-6 is present in the early stage of b-amyloid plaque formation, a neurohistopathological feature of Alzheimer’s disease ŽHull et al., 1996.. Further evidence
) Corresponding author. Tel.: q1-217-333-2118; fax: q1-217-3338286. E-mail address: [email protected] ŽR.W. Johnson..
for a role of IL-6 in neuropathology is provided by studies with transgenic mice whose astrocytes over expressed IL-6. The neurological disease and neuropathology exhibited by these mice was positively related with the level of cerebral IL-6 transgene expression ŽCampbell et al., 1993.. Moreover, the progressive neuropathological manifestations of IL-6 transgene expression were related to deficits in avoidance learning ŽHeyser et al., 1997.. Interleukin-6 in brain also has been shown inhibit long-term potentiation in hippocampal neurons ŽLi et al., 1997., reduce motivation for food ŽPlata-Salaman, 1997., increase sensitivity to painful stimuli ŽOka et al., 1995., and exacerbate the behavioral effects of other cytokines like interleukin-1b ŽIL-1b; Kozak et al., 1997; Lenczowski et al., 1999.. Recently, elevated levels of IL-6 have been reported in brain of healthy aged animals ŽPrechel et al., 1996; Ye and Johnson, 1999.. Although IL-6 levels in aged brain do not equate to those in CNS diseases, there is evidence that chronic exposure to low levels of IL-6 cause changes in neurons that enhance the neurotoxic properties of other molecules Ži.e., NMDA; Qiu et al., 1998.. Therefore, the increase in IL-6 in aged brain may be sufficient to predis-
0165-5728r01r$ - see front matter q 2001 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 5 - 5 7 2 8 Ž 0 1 . 0 0 3 1 6 - 2
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S.-M. Ye, R.W. Johnsonr Journal of Neuroimmunology 117 (2001) 87–96
pose individuals to the onset of neurodegenerative disease or have a role in the neurophysiological manifestations of aging. Unfortunately, little is known about why IL-6 is elevated in aged brain or how aging affects transcription factors that regulate the IL-6 gene. The 5X-flanking region of the murine IL-6 gene contains a nuclear factor k B element ŽNFk B., which is important for IL-6 gene expression ŽLibermann and Baltimore, 1990; Shimizu et al., 1990; Zhang et al., 1990; Dendorfer et al., 1994.. Of note, increased DNA-binding activity of NFk B has been reported in brain of aged rats ŽKorhonen et al., 1997; Toliver-Kinsky et al., 1997.. If binding of NFk B to the IL-6 promoter is increased in brain of aged animals, it may explain the age-associated increase in IL-6 gene expression. However, this possibility has not been evaluated. Therefore, the purpose of this study was to determine if binding of NFk B to the IL-6 promoter increased in brain of aged mice relative to young adults and if an increase conferred enhanced expression of the IL-6 gene. The results show that the DNA-binding activity of NFk B is increased in the brain of aged mice and that at least one consequence of this is increased expression of the inflammatory cytokine IL-6.
2. Materials and methods 2.1. Animals One-month-old male BALBrc mice Žjuveniles. were weaned from a breeding colony that was kept in barrier-reared conditions in a specific pathogen-free facility and placed in a conventional rodent facility where they were kept until 6 months of age Žadults.. Other male BALBrc mice that had been reared under similar conditions were obtained from the National Institute on Aging breeding colony at Charles River Laboratories ŽKingston, New York. at about 15–18 months of age. These mice were kept in the same room as the adults until 24 months of age. Mice were kept in polypropylene cages and maintained at 308C under a reverse 12 h light–12 h dark cycle Žlights on at 1900 h. with ad libitum access to water and rodent chow. All procedures were in accordance with the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals and were approved by the University of Illinois Laboratory Animal Care Committee. 2.2. Measurement of brain IL-6 Six- and twenty-four-month-old mice Ž n s 6. were deeply anesthetized by halothane inhalation and were transcardially perfused with sterile heparinized phosphatebuffered saline ŽPBS; 388C. for 15 min when perfusate was found to be clear and free of cells. The brains were removed, frozen in liquid nitrogen, and stored at y808C until RNA isolation and protein preparation. For extraction
of protein, brain tissues were thawed and then homogenized in 0.5 ml lysis buffer ŽTris–NaCl, pH7.6, containing 10% glycerol, 0.5% Triton-X100, 1 mM EDTA, 2 mM PMSF, 5 mM NaF, leupeptin, antipain, aprotinin, pepstatin, 1 mgrml, and sodium azide, 0.05% wrv.. Tissue homogenates were centrifuged at 10,000 = g for 10 min at 48C. Supernatants were immediately assayed for protein content ŽBio-Rad protein assay, Richmond, CA. and IL-6 ŽGenzyme, Cambridge, MA.. The inter- and intra-assay variation was less than 10% for the IL-6 assay. 2.3. Cell culture Glial cells from adult and aged mice were isolated and cultured as described previously ŽYe and Johnson, 1999.. Briefly, whole brains from mice were mechanically and enzymatically dissociated and plated onto poly-L-lysinecoated tissue culture flasks ŽCorning-Costar, Cambridge, MA. in Dulbecco’s modified eagle medium ŽDMEM; Gibco, Grand Island, NY. containing 20% fetal calf serum ŽFCS; Sigma, St. Louis, MO. and sodium bicarbonate Ž2 grl.. Cells were maintained at 378C with 95% humidity and 7% CO 2 for 24 h, when culture medium containing non-adherent cells Ži.e., neurons and oligodendrocytes. was removed. Culture medium was replenished and adherent cells were allowed to develop morphologically for 10 d, during which medium was changed every third day until cells became confluent. 2.4. Ribonuclease protection assay Interleukin-6 mRNA levels in cultured glial cells were assessed using a ribonuclease protection assay ŽRPA.. Total RNA was isolated from glia according to the Tri-reagent protocol. RNA integrity was confirmed by denaturing agarose gel electrophoresis and RNA concentration was determined by spectrophometric absobency at two dilutions. A partial cDNA fragment for murine IL-6 was created by RT-PCR-based cloning methods ŽInvitrogen, Carlsbad, CA. and inserted into a PCRII vector ŽPCRII-IL6. as described elsewhere ŽHeyen et al., 2000.. The b-actin template was purchased from a commercial source ŽAmbion, Austin, TX. and used as an internal reference in the assay to establish the relative amount of RNA in each sample. Radiolabeled ŽUTP-w a-32 Px. anti-sense RNA probe was then generated by in vitro transcription ŽMAXIscript; Ambion.. The full-length probes for IL-6 and b-actin were 520 and 360 bases, respectively; while protected fragments were 425 and 250 bases in length, respectively. Ribonuclease protection assays were performed using the HybSpeed RPA kit ŽAmbion. with minor modification. Glial cell RNA Ž20 mg. was hybridized in solution to the gel-purified probes. Hybridization was performed for 10 min at 688C, and the mixture digested with an RNase ArT1 mixture, diluted 1:500 in RNase digestion buffer,
S.-M. Ye, R.W. Johnsonr Journal of Neuroimmunology 117 (2001) 87–96
for 30 min at 378C. Protected fragments were precipitated and resolved on a 5% acrylamider8 M urea gel. The gel was then dried and the radioactivities associated with the IL-6 mRNA and b-Actin mRNA protected fragments were measured by phosphorimage scanning ŽImage Quant 3.3, Molecular Dynamics, Sunnyvale, CA. and visualized using autoradiographic film Ž24-h exposure at y808C to Kodak BioMaxrMS film with double screen.. 2.5. CompetitiÕe RT-PCR For measurement of IL-6 mRNA transcripts in brain tissues, a competitor DNA template was created from our previously constructed PCRII-IL6 plasmid by a deletion mutation. A 52-bp fragment was deleted from the partial murine IL-6 cDNA Ž425 bp. and was replaced by a 7-bp BamHI site. A competitor RNA was then in vitro transcribed and quantified by a RT-PCR Competitor Construction Kit ŽAmbion.. To perform RT-PCR, total RNA was isolated from perfused brain tissue by the Tri-reagent method. A series of RT reactions were set up using 2- or 10-fold dilutions of RNA competitors mixed with a constant amount of total sample RNA Ž4 mg.. The reverse transcription was performed at 378C for 90 min with 200 U of moloney murine leukemia virus reverse transcriptase ŽGibco.. The singlestranded cDNA was then amplified by 35 PCR cycles by AmpliTaq Gold DNA polymerase ŽPE Biosystems, Foster City, CA. with synthetic oligonucleotide primers designed to flank a 425-bp region of IL-6 wŽsense: 5X-CCAACAGACCTGTCTATACCAC . Žantisense: 5X-CCTTCTGTGACTCCAGCTTATC.x. PCR products were analyzed by electrophoresis on 2% agarose gel stained with ethidium bromide. The RNA competicon templates produced PCR products about 10% smaller than the corresponding endogenous target Ž381 vs. 425 bp.. DNA bands were visualized with UV light and photographed. The pictures were scanned and the amount of PCR product associated with each band was analyzed using the NIH image computerized densitometry program ŽWayne Rasband, NIH, Bethesda, MD.. Molar ratios were calculated by dividing the band volume by the length of the DNA molecule. Under competitive conditions, the absolute amount of IL-6 mRNA is equal to the amount of RNA competitor added, when the molar ratio of PCR products becomes equal for the target and competitor.
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were treated with 25 mM k B decoy or control scrambled DNA for 24 h to inhibit constitutive NFk B activity in vitro. To inhibit NFk B in vivo, decoy DNA was administered by injection into a lateral cerebral ventricle as previously described ŽGrzanna et al., 1998; Yao et al., 1999. with slight modification. Mice were anesthetized by an i.p. injection of ketamine Ž125 mgrkg body weight. and xylazine Ž6 mgrkg body weight. and placed in a stereotaxic instrument ŽDavid Kopf Instrument, Tujunga, CA.. A 28gauge injection cannula was inserted into a lateral ventricle using pre-determined coordinates wŽanteroposterior y0.6 mm; lateral, 1.6 mm to the bregma; and horizontal y2.0 mm to the dura mater. Franklin and Paxinos, 1997x and k B decoy or scrambled DNA Ž10 mM. in 2 ml of saline was infused over a 1-min period using a Hamilton syringe and a syringe pump ŽWorld Precision Instruments, Sarasota, FL.. Mice were returned to their home cage, where they regained consciousness. Nine h after injection, mice were killed by CO 2 asphyxiation and brains were dissected to verify the injection site and then frozen at y808C until isolation of RNA and nuclear protein. 2.7. Nuclear extraction Mice were transcardially perfused as previously described ŽYe and Johnson, 1999. to remove blood from the cerebral vasculature. Brains were rapidly removed and homogenized in 4 volumes of lysis buffer using a Dounce homogenizer with five strokes of the A type pestle and five strokes of the B type pestle ŽKontes Glass, Vineland, NJ.. After homogenization, nuclear extracts were prepared according to the procedures described by Gorski et al. Ž1986. with only slight modification. In brief, glia or brain tissue homogenate were suspended in lysis buffer containing 10 mM HEPES–NaOH buffer ŽpH7.9., 0.25 M sucrose, 15 mM KCl, 5 mM EDTA, 1 mM EGTA, 0.15 mM
2.6. Inhibition of NFk B actiÕity The k B decoy was used to inhibit NFk B activity both in vitro and in vivo. k B decoy DNA was prepared by annealing the oligonucleotides 5X-GAGGGGACTTTCCCT-3X and 5X-AGGGAAAGTCCCCTC-3X ŽYu et al., 1999.. The scrambled control DNA was prepared by annealing the oligonucleotides 5X-GATGCGTCTGTCGCA-3X and 5X-TGCGACAGACGCATC-3X . In one experiment, glia
Fig. 1. Interleukin-6 ŽIL-6. increases in the brain with age. Subjects Ž1-, 6- and 24-month-old male BALBrc mice; ns6. were transcardially perfused to remove blood and leukocytes from the cerebral vasculature. Protein-containing supernatants extracted from whole brain tissues were assayed for IL-6 using an ELISA specific for murine IL-6. The bars represent means"S.E. Treatment means with different letters are significantly different from each other Ž p- 0.01..
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hibitors.. After centrifugation at 16,000 = g for 20 min, supernatants were assayed to determine protein concentrations and stored at y808C. 2.8. Electrophoretic mobility shift assay Purified synthetic oligonucleotide probe which corresponded to the NFk B Ž5X-ATGTGGGATTTTCCCATGAG-3X . DNA-binding sequence in the IL-6 promoter was
Fig. 2. Competitive RT-PCR indicates that the level of IL-6 mRNA increased in the brain of aged mice. Total RNA from brains of 6- or 24-month-old mice was mixed with a series of 2-fold dilutions of RNA competitors. The mixtures were reverse transcribed and amplified by PCR. The RT-PCR products were separated by agarose gel electrophoresis and stained with ethidium bromide. ŽA. A representative gel for each age is shown in the upper panel. ŽB. The amount of IL-6 mRNA in the brain of adult and aged mice Ž ns 4. was determined by the amount of RNA competitor added, when the molar ratio of PCR products became equal for the target and competitor.
spermine, 0.5 mM spermidine, 1 mM dithiothreitol ŽDTT., and protease inhibitors w0.5 mM phenylmethylsulfonyl fluoride ŽPMSF., 100 mM NaF, and 1 mgrml each of benzamidine, leupeptin, pepstatin A, and aprotininx. The cells were further lysed in cell lysis buffer containing 0.5% NP-40 for 10 min. Cell lysates were centrifuged at 12,000 = g for 5 min. The pellets were washed twice and resuspended in 2 volumes of nuclei lysis buffer Ž10 mM HEPES–NaOH, pH 7.9, 1.5 mM MgCl2, 340 mM KCl, 1 mM EDTA, 1 mM DTT, and the already mentioned protease inhibitors.. The suspensions were gently mixed for 30 min at 48C, and centrifuged at 16,000 = g for 20 min. The supernatants were desalted by centrifugal filter units ŽMillipore, Bedford, MA. against reaction buffer Ž10 mM Tris–HCl, pH 7.5, 20% glycerol, 10 mM MgCl2, 100 mM KCl, 1 mM EDTA, 1 mM DTT, and protease in-
Fig. 3. The DNA-binding activity of NFk B is increased in brains of aged mice. Nuclear protein was extracted from brains of 1-, 6- and 24-monthold mice and subjected to electrophoretic mobility shift assay. The sequence of 32 P-labeled oligonucleotide probe corresponded to the NFk B response element in the promoter region of the mouse IL-6 gene. ŽA. The DNA–protein complexes were visualized on a nondenaturing polyacrylamide gel to reveal a shift band. Each lane represents one mouse so each age group is represented three times. The binding specificity was verified by incubating nuclear extracts from LPS-stimulated N13 cells without ŽC. or with 100-fold molar excess of unlabeled specific competitor oligonucleotide ŽS., or 100-fold molar excess of unlabeled non-specific competitor oligonucleotide ŽNS.. ŽB. The shift band was quantified by phosphorimage scanning. Each bar represents the mean"S.E. Ž ns 3.. Treatment means with different letters are significantly different from each other Ž p- 0.01..
S.-M. Ye, R.W. Johnsonr Journal of Neuroimmunology 117 (2001) 87–96
purchased from Operon Technologies ŽOperon, Alameda, CA.. Single-stranded synthetic oligonucleotides were annealed according to standard methods ŽSambrook et al., 1989.. The resulting double-stranded oligos were end labeled by w g-32 Px ATP using T4 polynucleotide kinase and purified using the kit from Qiagen ŽValencia, CA.. Nuclear extracts from LPS-stimulated N13 cells Ža microglia cell line. were used as a positive control. The specific competitor was unlabelled double-stranded oligonucleotide corresponding to the NFk B binding sequence, and the nonspecific competitor was an unlabelled double-stranded oligonucleotide corresponding to the Multiple Response Element ŽMRE; 5X-ATGCTAAACGACGTCACATT-3X .. Nuclear extracts Ž15–20 mg total protein. were incubated with or without competitor DNA for 10 min at room temperature in 10 mM Tris–HCl buffer ŽpH7.5. containing 1 mg poly ŽdI-dC., 10 mM MgCl 2 , 100 mM KCl, 1 mM EDTA, 1 mM DTT, 20% glycerol, and protease inhibitors. Radio-labeled probe Ž100 fmol, 1–2 = 10 5 cpmrpmol. was then added, and the mixture was incubated at room temperature for 30 min, when bound and free probes were separated by electrophoresis on a 4% polyacrylamide gel in buffer ŽpH 8.5. containing 25 mM Tris, 0.19 M Gly, and 2 mM EDTA at a constant voltage of 200 V for 2 h in an ice bath. Gels were dried and exposed overnight at y708C or visualized by phosphorimaging. 2.9. Statistical analysis Data were analyzed using General Linear Model procedures of Statistical Analysis Systems ŽSAS Institute, 1989.. They were subjected to one-way ANOVA. When ANOVA
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revealed a significant effect of Age, differences between treatments Ži.e., juvenile, adult, and aged. were tested using Duncan’s multiple range tests.
3. Results 3.1. Interleukin-6 gene expression is increased in the brain of aged mice Before evaluating the effects of age on the DNA-binding activity of NFk B, we confirmed that IL-6 increased in brain of aged mice. Interleukin-6 was measured in crude protein extracts from brains of juvenile Ž1 month old., adult Ž6 months old., and aged Ž24 months old. male BALBrc mice. One-way ANOVA of IL-6 concentration revealed a significant effect of age Ž p - 0.01.. Consistent with a previous report ŽYe and Johnson, 1999., IL-6 concentration was higher in brain extracts of aged mice than in brain extracts of adult or juvenile mice ŽFig. 1.. To extend this finding and to find out if IL-6 gene expression might be increased in the aged brain, the effect of age on steady-state IL-6 mRNA levels was evaluated. We first tried to assess IL-6 mRNA levels using the multi-probe RNase protection assay ŽRPA.. However, IL-6 mRNA in mouse brain was undetectable by RPA Ždata not shown., mandating that competitive RT-PCR be used. Total RNA that was isolated from brains of aged Ž n s 4. and adult mice Ž n s 4. was mixed with increasing amounts of an RNA competitor. The RT-PCR products were separated by agarose gel and stained with ethidium bromide. Representative gels are shown in Fig. 2A. The steady-state levels of
Fig. 4. Increased NFk B activity in glia cultured from brains of aged mice is inhibited by k B decoy treatment. Glial cells were cultured from brains of 6- or 24-month-old mice and treated with medium, 25 mM of scrambled control DNA , or 25 mM of k B decoy DNA for 24 h. Nuclear proteins were extracted and subjected to gel mobility shift assay. The sequence of 32 P-labeled oligonucleotide probe corresponded to the NFk B response element in the promoter region of the mouse IL-6 gene. The DNA–protein complexes were visualized on a nondenaturing polyacrylamide gel.
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IL-6 mRNA transcripts in 4 mg of total RNA isolated from brains of aged mice ranged from 2 = 10 4 to 1 = 10 5 copies with a median value of 6 = 10 4 ŽFig. 2B.. In contrast, the copy number of IL-6 mRNA from brains of adult mice was lower than 5 = 10 3 per 4 mg of total RNA with a median value of 2.5 = 10 3 ŽFig. 2B.. Thus, the copy number of IL-6 transcripts was at least 4-fold higher in aged brain than in the adult brain. 3.2. The DNA-binding actiÕity of NFk B is increased in the brain of aged mice A 1.2-kb fragment of the 5X-flanking region of the IL-6 gene contains a binding site for NFk B. Because IL-6 mRNA and protein were increased in aged brain ŽFigs. 1 and 2., we hypothesized that the DNA-binding activity of NFk B would be increased. To test this hypothesis, whole brains of juvenile, adult and aged mice were collected as before and nuclear protein was extracted and subjected to
electrophoretic mobility shift assay. Synthetic probe corresponding to the NFk B DNA-binding sequence in the IL-6 promoter was used. Fig. 3A shows the results of a typical gel shift assay. Each lane represents one mouse so each age group is represented three times. The DNA-binding activity was quantified by phosphorimage scanning and mean DNA-binding activity for NFk B is shown in Fig. 3B. The DNA-binding activity of NFk B was higher in the brains of aged mice than in the brains of adult and juvenile mice Ž p - 0.05.. Thus, increased binding of NFk B to the promoter region of the IL-6 gene may contribute to the increase in IL-6 mRNA and protein in brain of aged mice. 3.3. Inhibition of NFk B actiÕity represses IL-6 expression in glia from aged mice The hypothesis that increased DNA-binding activity of NFk B results in increased IL-6 gene expression in aged brain was evaluated both in vitro and in vivo. We previ-
Fig. 5. Inhibition of NFk B DNA-binding activity by k B decoy suppresses IL-6 gene expression in glia from aged mice. Glia from 6- or 24-month-old mice were incubated with medium, 25 mM of scrambled control DNA, or 25 mM of k B decoy DNA for 24 h. ŽA. RNA was isolated and subjected to RNase protection assay. The protected bands of IL-6 and b-actin mRNA were 425 and 250 bases, respectively. Full-length probes ŽIL-6, 520 bases; b-actin, 360 bases. are shown in lane 20. ŽB. The culture supernatants were collected and assayed for IL-6 using an ELISA specific for murine IL-6. The bars represent means" S.E. Treatment means with different letters are significantly different from each other Ž p - 0.01..
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effects of k B decoy on IL-6 mRNA and protein were evaluated. Consistent with our previous report ŽYe and Johnson, 1999., steady-state levels of IL-6 mRNA were increased in glia from aged mice compared to adult mice ŽFig. 5A, lanes 1–3 vs. lanes 10–12., as was the supernatant IL-6 concentration ŽFig. 5B.. The k B decoy, which competes with the endogenous k B site for binding NFk B, decreased steady-state IL-6 mRNA levels ŽFig. 5A. and protein secretion ŽFig. 5B. in unstimulated glia from aged mice. The control scrambled DNA, however, did not affect either IL-6 mRNA ŽFig. 5A. or protein ŽFig. 5B..
Fig. 6. Intracerebroventricular administration of k B decoy decreased NFk B activity in the brains of aged mice. Nuclear protein was extracted from brains of mice that had been injected i.c.v. with scrambled control DNA or k B decoy DNA and subjected to electrophoretic mobility shift assay. The sequence of the 32 P-labeled oligonucleotide probe corresponded to the NFk B element in the promoter region of the mouse IL-6 gene. The DNA–protein complexes were visualized on a nondenaturing polyacrylamide gel. Each lane represents one mouse so each treatment is represented three times. The binding specificity was verified by incubating nuclear extracts from vehicle-treated mice with 100-fold molar excess of unlabeled non-specific competitor oligonucleotide Žlane 10., or 50 Žlane 11.- and 100 Žlane 12.-fold molar excess of unlabeled specific competitor oligonucleotide.
ously reported that mixed glial cells cultured from aged mouse brain spontaneously expressed Ži.e., expressed without stimulation. more IL-6 mRNA and secreted more IL-6 protein than glia from adult and neonate brain ŽYe and Johnson, 1999.. Therefore, this system was initially used to determine if the increased IL-6 production was due to increased activity of NFk B. Glial cells were cultured from adult or aged mice, grown to confluency, and incubated 24 h with medium alone, or medium containing k B decoy DNA or scrambled control DNA. The k B decoy competes with the DNA-binding sequences for NFk B. Fig. 4 shows the results of the gel shift assay. Each lane represents glia from one mouse so each treatment is represented three times. Consistent with the results obtained using whole brain ŽFig. 3., NFk B activity was higher in glia of aged mice compared to glia of adult mice ŽFig. 4, lanes 1–3 vs. lanes 10–12.. Treatment of glia with scrambled control DNA did not affect NFk B DNA-binding activity ŽFig. 4, lanes 4–6 and lanes 13–15.. However, treatment with k B decoy DNA decreased NFk B activity in aged glia ŽFig. 4, lanes 16–18. to a level consistent with that observed in glia from adults. The decoy molecule did not decrease NFk B activity in adult glia ŽFig. 4, lanes 7–9.. To determine if the increased NFk B activity in glia from aged mice conferred increased gene expression, the
Fig. 7. Intracerebroventricular administration of k B decoy decreased IL-6 gene expression in the brains of aged mice. Total RNA from brains of 24-month-old mice that had been injected i.c.v. with scrambled control DNA or k B decoy was mixed with a series of 10-fold dilutions of RNA competitor. The mixtures were reverse transcribed and amplified by PCR. ŽA. A representative gel for each treatment is shown in the upper panel. ŽB. The amount of IL-6 mRNA in the brain of adult and aged mice Ž ns 4. was determined by the amount of RNA competitor added, when the molar ratio of PCR products became equal for the target and competitor.
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3.4. Central injection of k B decoy represses IL-6 expression in brain of aged mice The results of the previous study indicated that NFk B DNA-binding activity is increased in aged glia and that this results in increased expression of the inflammatory cytokine IL-6. To extend this finding, we analyzed NFk B activity and IL-6 gene expression in whole brain of aged mice after intracerebroventricular Ži.c.v.. injection of k B decoy. The k B decoy DNA, scrambled control DNA, or vehicle were injected i.c.v. and brain tissue was obtained 9 h later. This protocol was adopted from Yu et al. Ž1999., who used the k B decoy in mice to inhibit the activation of NFk B and apoptosis of neurons caused by excitotoxic injury. The effect of central injection of k B decoy DNA on NFk B DNA-binding activity in brain of aged mice is shown in Fig. 6. As predicted, the NFk B DNA-binding activity was lower after i.c.v. injection of k B decoy compared to vehicle or scrambled control DNA. The decrease in NFk B activity was paralleled by a reduction in IL-6 mRNA ŽFig. 7.. In the brain of aged mice injected i.c.v. with scrambled control DNA the steady-state levels of IL-6 mRNA transcripts in 4 mg of total RNA ranged from 2 = 10 5 to 1 = 10 6 copies with a median value of 5 = 10 5 ŽFig. 7B.. This was substantially higher than what was measured previously ŽFig. 2., suggesting an inflammatory response caused by the injection ŽSchobitz et al., 1997.. In any case, k B decoy DNA reduced IL-6 mRNA transcripts. The copy number of IL-6 mRNA from brains of aged mice treated with k B decoy was 5 = 10 3 –1 = 10 5 per 4 mg of total RNA ŽFig. 7B..
4. Discussion In the present study, we confirmed that IL-6 protein is increased in brain of aged mice, and using competitive RT-PCR showed that the steady-state level of IL-6 mRNA was higher in aged brain than in adult brain. This suggested that IL-6 gene transcription was increased in aged brain, which led us to examine the effects of aging on DNA-binding of NFk B. The important finding in this study was that NFk B binding to the IL-6 gene promoter was increased in glia and brain of aged mice compared to juvenile and adult mice. Moreover, selectively inhibiting the effects of age on NFk B activity with the k B decoy, reduced IL-6 mRNA and protein to levels seen in young adult controls. Therefore, these data indicate that the DNA-binding activity of NFk B is increased in the brain of aged mice and that at least one consequence of this is increased expression of the inflammatory cytokine IL-6. The fact that NFk B DNA-binding activity increases in brain with age has been previously reported. Toliver-Kinsky et al. Ž1997. measured NFk B activity and found significantly higher levels in hippocampus and forebrain of 30-month-old rats compared to adult controls. Korhonen et
al. Ž1997. also reported increased NFk B activity in discrete brain areas of aged rats. However, neither of these studies identified a specific target gene that was affected by NFk B. In peripheral lymphoid organs of aged mice NFk B is also constitutively activated ŽSpencer et al., 1997.. In splenocytes of aged mice the increased activity of NFk B is associated with aberrant expression of several inflammatory cytokines ŽSpencer et al., 1997; Poynter and Daynes, 1998.. However, if this consequence of age is evident in cells of the CNS had not been evaluated. Thus, in the present study we sought to determine if the age-related increase in brain NFk B activity was inherently linked to the age-related increase in brain IL-6. We first evaluated this in astrocytes and microglia co-cultured from brain of adult and aged mice. Consistent with what was found in whole brain, NFk B DNA-binding activity, steady-state IL-6 mRNA level, and IL-6 protein secretion were higher in glia from aged mice than adults. In this model, comparisons between age groups must be cautiously made, however, because cultures from aged mice comprised more microglia and fewer astrocytes than the cultures established from adult brains ŽYe and Johnson, 1999.. The reliable comparisons are between treatments within an age group; and the important finding here is that k B decoy DNA decreased NFk B DNA-binding activity, steady-state IL-6 mRNA level, and IL-6 protein secretion in aged glia Žcompared to medium or scrambled control DNA.. The effect of k B decoy on these endpoints in glia from adult mice was minimal. These results can be extended to suggest that the increase in NFk B DNA-binding activity, steady-state IL-6 mRNA level, and IL-6 protein measured in whole brain is from astrocytes andror microglia. There is strong evidence that the ability of animals to effectively contend with oxidative stress declines with age. Reactive oxygen species lead to phosphorylation and ubiquitination of Ik Ba , which liberates NFk B allowing it to translocate from the cell cytoplasm to the nucleus ŽSchreck et al., 1992.. Both the DNA-binding activity of NFk B and the spontaneous secretion of IL-6 are increased in splenocytes from aged mice compared to young adults ŽPoynter and Daynes, 1998.. Supplementation of aged mice with the antioxidant vitamin E corrects the age-associated increase in NFk B activity and IL-6 production in splenocytes. Therefore, it seems likely that in the aged brain oxidative stress also initiates a cascade that results in increased NFk B DNA-binding activity and enhanced expression of cytokines like IL-6. However, this point has not yet been evaluated. Although the present study focused on IL-6, there is evidence that other cytokines such as IL-1ba are increased as well ŽMurray and Lynch, 1998.. The age-related increase in inflammatory cytokines in brain may be a factor contributing to the neuropathological effects of aging. For example, dysregulation of the IL-6 gene might establish a state that is permissive to the onset of neurodegenerative disease. This hypothesis is partially
S.-M. Ye, R.W. Johnsonr Journal of Neuroimmunology 117 (2001) 87–96
based on the work of Campbell et al. Ž1993., who produced transgenic mice whose astrocytes over expressed IL-6. Mice over expressing IL-6 in brain showed dendritic vacuolization, reduced branching of dendritic spines in CA1 neurons, and loss of hippocampal interneurons. The expression of IL-6 was also related to deficits in avoidance learning ŽHeyser et al., 1997.. Increased IL-6 expression in brain has also been documented in brain trauma ŽWoodroofe et al., 1991., AIDS dementia complex ŽGallo et al., 1991., multiple sclerosis ŽHofman et al., 1989., and Alzheimer’s disease ŽBauer et al., 1991.. Interestingly, the cytokine is present in the early stage of plaque formation ŽHull et al., 1996., suggesting it may be an early primer for neuropathological changes that occur in Alzheimer’s disease. Despite the apparent close association between IL-6 and neurodegeneration, to our best knowledge no studies have shown that IL-6 directly kills neurons. However, a recent study showed that chronic exposure of developing cerebellar granule neurons to IL-6 enhanced membrane depolarization and the intracellular calcium signal to NMDA ŽQiu et al., 1998.. Moreover, IL-6 was shown to enhance NMDA receptor-mediated neurotoxicity, indicating that it may affect neurons indirectly. The results of the present study show that the age-related increase in brain IL-6 is due to increased activity of NFk B. However, why NFk B activity increases in aged brain is not yet known and the specific effects of the age-related increase in brain IL-6 remain to be established. A better understanding of the effects of age on cytokines in brain may help explain the neurophysiologic manifestations of aging.
Acknowledgements This work was supported by grants to R.W.J. from the NIA ŽAG16710. and the NIDDKD ŽDK51576..
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Immunological markers in the cerebrospinal fluid of HIV-1-infected children. Acta Paediatr. Scand. 80, 659–666. Gorski, K., Carneiro, M., Schibler, U., 1986. Tissue-specific in vitro transcription from the mouse albumin promoter. Cell 47, 767–776. Grzanna, R., Dubin, J.R., Dent, G.W., Ji, Z., Zhang, W., Ho, S.P., Hartig, P.R., 1998. Intrastriatal and intraventricular injections of oligodeoxynucleotides in the rat brain: tissue penetration, intracellular distribution and c-fos antisense effects. Mol. Brain Res. 63, 35–52. Hofman, F.M., Hinton, D.R., Johnson, K., Merrill, J.E., 1989. Tumor necrosis factor identified in multiple sclerosis brain. J. Exp. Med. 170, 607–612. Heyser, C.J., Masliah, E., Samimi, A., Campbell, I.L., Gold, L.H., 1997. Progressive decline in avoidance learning paralleled by inflammatory neurodegeneration in transgenic mice expressing interleukin-6 in the brain. Proc. Natl. Acad. Sci. U. S. A. 94, 1500–1505. Hull, M., Berger, M., Volk, B., Bauer, J., 1996. Occurrence of interleukin-6 in cortical plaques of Alzheimer’s disease patients may precede transformation of diffuse into neuritic plaques. Ann. N. Y. Acad. Sci. 777, 205–212. Korhonen, P., Helenius, M., Salminen, A., 1997. Age-related changes in the regulation of transcription factor NF-kB in rat brain. Neurosci. Lett. 225, 61–64. Kozak, W., Poli, V., Soszynski, D., Conn, C.A., Leon, L.R., Kluger, M.J., 1997. Sickness behavior in mice deficient in interleukin-6 during turpentine abscess and influenza pneumonitis. Am. J. Physiol. 272, R621–R630. Lenczowski, M.J., Bluthe, R.M., Roth, J., Rees, G.S., Rushforth, D.A., van Dam, A.M., Tilders, F.J., Dantzer, R., Rothwell, N.J., Luheshi, G.N., 1999. Central administration of rat IL-6 induces HPA activation and fever but not sickness behavior in rats. Am. J. Physiol. 276, R652–R658. Li, A.J., Katafuchi, T., Oda, S., Hori, T., Oomura, Y., 1997. Interleukin-6 inhibits long-term potentiation in rat hippocampal slices. Brain Res. 748, 30–38. Libermann, T.A., Baltimore, D., 1990. Activation of interleukin-6 gene expression through the NF-kB transcription factor. Mol. Cell. Biol. 10, 2327–2334. Murray, C.A., Lynch, M.A., 1998. Evidence that increased hippocampal expression of the cytokine interleukin-1 beta is a common trigger for age- and stress-induced impairments in long-term potentiation. J. Neurosci. 18, 2974–2981. Oka, T., Oka, K., Hosoi, M., Hori, T., 1995. Intracerebroventricular injection of interleukin-6 induces thermal hyperalgesia in rats. Brain Res. 692, 123–128. Plata-Salaman, C.R., 1997. Anorexia during acute and chronic disease: relevance of neurotransmitter–peptide–cytokine interactions. Nutrition 13, 159–160. Poynter, M.E., Daynes, R.A., 1998. Peroxisome proliferator-activated receptor a activation modulates cellular redox status, represses nuclear factor-kB signaling, and reduces inflammatory cytokine production in aging. J. Biol. Chem. 273, 32833–32841. Prechel, M.M., Halbur, L., Devata, S., Vaidya, A.M., Young, M.R., 1996. Increased interleukin-6 production by cerebral cortical tissue of adult versus young mice. Mech. Ageing Dev. 92, 185–194. Qiu, Z., Sweeney, D.D., Netzeband, J.G., Gruol, D.L., 1998. Chronic interleukin-6 alters NMDA receptor-mediated membrane responses and enhances neurotoxicity in developing CNS neurons. J. Neurosci. 18, 10445–10456. Sambrook, J., Fritsch, E.F., Maniatis, T., 1989. Molecular Cloning: A Laboratory Manual. 2nd edn. Cold Spring Harbor Laboratory, Cold Spring Harbor. SAS Institute, 1989. 4th edn. SASrSTAT User’s Guide, Version 6, vol. 2, SAS Institute, Cary. Schobitz, B., Pezeshki, G., Probst, J.C., Reul, J.M., Skutella, T., Stohr, T., Holsboer, F., Spanagel, R., 1997. Centrally administered oligodeoxynucleotides in rats: occurrence of non-specific effects. Eur. J. Pharmacol. 331, 97–107.
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Schreck, R., Albermann, K., Baeuerle, P.A., 1992. Nuclear factor kappa B: an oxidative stress-responsive transcription factor of eukaryotic cells. Free Radical Res. Commun. 17, 221–237. Shimizu, H., Mitomo, K., Watanabe, T., Okamoto, S., Yamamoto, K., 1990. Involvement of a NF-kB-like transcription factor in the activation of the interleukin-6 gene by inflammatory lymphokines. Mol. Cell Biol. 10, 561–568. Spencer, N.F., Poynter, M.E., Daynes, R.A., 1997. Constitutive activation of NF-kappa B in an animal model of aging. Int. Immunol. 9, 1581–1588. Toliver-Kinsky, T., Papaconstatninou, J., Perez-Polo, J.R., 1997. Age-associated alterations in hippocampal and basal forebrain nuclear factor kappa B activity. J. Neurosci. Res. 48, 580–587. Woodroofe, M.N., Sarna, G.S., Wadhwa, M., Hayes, G.M., Loughlin, A.J., Tinker, A., Cuzner, M.L., 1991. Detection of interleukin-1 and interleukin-6 in adult rat brain, following mechanical injury, by in
vivo microdialysis: evidence of a role of microglia in cytokine production. J. Neuroimmunol. 33, 227–236. Yao, J.H., Ye, S., Burgess, W., Zachary, J.F., Kelley, K.W., Johnson, R.W., 1999. Mice deficient in interleukin-1beta converting enzyme resist anorexia induced by central lipopolysaccharide. Am. J. Physiol. 277, R1435–R1443. Ye, S., Johnson, R.W., 1999. Increased interleukin-6 expression by microglia from brain of aged mice. J. Neuroimmunol. 93, 139–148. Yu, Z., Zhou, D., Bruce-Keller, A.J., Kindy, M.S., Mattson, M.P., 1999. Lack of the p50 subunit of nuclear factor-kappaB increases the vulnerability of hippocampal neurons to excitotoxic injury. J. Neurosci. 19, 8856–8865. Zhang, Y., Lin, J.-X., Vilcek, J., 1990. Interleukin-6 induction by tumor necrosis factor and interleukin-1 in human fibroblasts involves activation of a nuclear factor binding to a k B-like sequence. Mol. Cell Biol. 10, 3818–3823.
Regulation of interleukin-6 gene expression in brain of aged mice by nuclear factor k B Shi-Ming Ye, Rodney W. Johnson ) Laboratory of IntegratiÕe Biology, Department of Animal Sciences, UniÕersity of Illinois, 390 Animal Science Laboratory, 1207 West Gregory DriÕe, Urbana, IL 61801, USA Received 10 January 2001; received in revised form 4 April 2001; accepted 5 April 2001
Abstract Interleukin-6 ŽIL-6. is increased in brain of aged mice. The purpose of this study was to determine if binding of nuclear factor k B ŽNFk B. to the IL-6 promoter is responsible for the age-related increase in brain IL-6. In an initial study, the effect of age on IL-6 in brain was verified as IL-6 protein was increased in brain of aged mice compared to adult and juvenile mice. Competitive RT-PCR showed that IL-6 mRNA concentration was at least 4-fold higher in aged brain compared to adult brain. Next, binding of the transcription factor NFk B to the IL-6 promoter in brains of 1-, 6-, and 24-month-old mice was determined. Electrophoretic mobility shift assay showed that NFk B activity was increased in aged brain compared to adult and juvenile brain. Moreover, glial cells cultured from aged mice showed more NFk B DNA-binding activity and more IL-6 mRNA and protein expression than glia from adults. However, incubating glia from aged mice in the presence of k B decoy inhibited these effects of age. The same was observed in vivo as intracerebroventricular injection of k B decoy in aged mice decreased NFk B activity and IL-6 mRNA and protein in brain. These results show that the DNA-binding activity of NFk B is increased in the brain of aged mice and that at least one consequence is increased expression of IL-6. q 2001 Elsevier Science B.V. All rights reserved. Keywords: Aging; Brain; Cytokines; Interleukin-6; Mouse; Nuclear factor k B
1. Introduction Astrocytes, microglia, and neurons express inflammatory cytokines, including interleukin-6 ŽIL-6.. Under normal physiological conditions the gene encoding IL-6 is tightly regulated and expression in the brain is relatively low. However, elevated levels of IL-6 are evident in the brain in a number of central nervous system ŽCNS. diseases, and over expression of IL-6 and other inflammatory cytokines is thought to result in neuropathology. As evidence of such, inflammation and increased expression of IL-6 have been documented in brain trauma ŽWoodroofe et al., 1991. and in certain neurodegenerative diseases, including Alzheimer’s disease ŽBauer et al., 1991.. Interestingly, IL-6 is present in the early stage of b-amyloid plaque formation, a neurohistopathological feature of Alzheimer’s disease ŽHull et al., 1996.. Further evidence
) Corresponding author. Tel.: q1-217-333-2118; fax: q1-217-3338286. E-mail address: [email protected] ŽR.W. Johnson..
for a role of IL-6 in neuropathology is provided by studies with transgenic mice whose astrocytes over expressed IL-6. The neurological disease and neuropathology exhibited by these mice was positively related with the level of cerebral IL-6 transgene expression ŽCampbell et al., 1993.. Moreover, the progressive neuropathological manifestations of IL-6 transgene expression were related to deficits in avoidance learning ŽHeyser et al., 1997.. Interleukin-6 in brain also has been shown inhibit long-term potentiation in hippocampal neurons ŽLi et al., 1997., reduce motivation for food ŽPlata-Salaman, 1997., increase sensitivity to painful stimuli ŽOka et al., 1995., and exacerbate the behavioral effects of other cytokines like interleukin-1b ŽIL-1b; Kozak et al., 1997; Lenczowski et al., 1999.. Recently, elevated levels of IL-6 have been reported in brain of healthy aged animals ŽPrechel et al., 1996; Ye and Johnson, 1999.. Although IL-6 levels in aged brain do not equate to those in CNS diseases, there is evidence that chronic exposure to low levels of IL-6 cause changes in neurons that enhance the neurotoxic properties of other molecules Ži.e., NMDA; Qiu et al., 1998.. Therefore, the increase in IL-6 in aged brain may be sufficient to predis-
0165-5728r01r$ - see front matter q 2001 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 5 - 5 7 2 8 Ž 0 1 . 0 0 3 1 6 - 2
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pose individuals to the onset of neurodegenerative disease or have a role in the neurophysiological manifestations of aging. Unfortunately, little is known about why IL-6 is elevated in aged brain or how aging affects transcription factors that regulate the IL-6 gene. The 5X-flanking region of the murine IL-6 gene contains a nuclear factor k B element ŽNFk B., which is important for IL-6 gene expression ŽLibermann and Baltimore, 1990; Shimizu et al., 1990; Zhang et al., 1990; Dendorfer et al., 1994.. Of note, increased DNA-binding activity of NFk B has been reported in brain of aged rats ŽKorhonen et al., 1997; Toliver-Kinsky et al., 1997.. If binding of NFk B to the IL-6 promoter is increased in brain of aged animals, it may explain the age-associated increase in IL-6 gene expression. However, this possibility has not been evaluated. Therefore, the purpose of this study was to determine if binding of NFk B to the IL-6 promoter increased in brain of aged mice relative to young adults and if an increase conferred enhanced expression of the IL-6 gene. The results show that the DNA-binding activity of NFk B is increased in the brain of aged mice and that at least one consequence of this is increased expression of the inflammatory cytokine IL-6.
2. Materials and methods 2.1. Animals One-month-old male BALBrc mice Žjuveniles. were weaned from a breeding colony that was kept in barrier-reared conditions in a specific pathogen-free facility and placed in a conventional rodent facility where they were kept until 6 months of age Žadults.. Other male BALBrc mice that had been reared under similar conditions were obtained from the National Institute on Aging breeding colony at Charles River Laboratories ŽKingston, New York. at about 15–18 months of age. These mice were kept in the same room as the adults until 24 months of age. Mice were kept in polypropylene cages and maintained at 308C under a reverse 12 h light–12 h dark cycle Žlights on at 1900 h. with ad libitum access to water and rodent chow. All procedures were in accordance with the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals and were approved by the University of Illinois Laboratory Animal Care Committee. 2.2. Measurement of brain IL-6 Six- and twenty-four-month-old mice Ž n s 6. were deeply anesthetized by halothane inhalation and were transcardially perfused with sterile heparinized phosphatebuffered saline ŽPBS; 388C. for 15 min when perfusate was found to be clear and free of cells. The brains were removed, frozen in liquid nitrogen, and stored at y808C until RNA isolation and protein preparation. For extraction
of protein, brain tissues were thawed and then homogenized in 0.5 ml lysis buffer ŽTris–NaCl, pH7.6, containing 10% glycerol, 0.5% Triton-X100, 1 mM EDTA, 2 mM PMSF, 5 mM NaF, leupeptin, antipain, aprotinin, pepstatin, 1 mgrml, and sodium azide, 0.05% wrv.. Tissue homogenates were centrifuged at 10,000 = g for 10 min at 48C. Supernatants were immediately assayed for protein content ŽBio-Rad protein assay, Richmond, CA. and IL-6 ŽGenzyme, Cambridge, MA.. The inter- and intra-assay variation was less than 10% for the IL-6 assay. 2.3. Cell culture Glial cells from adult and aged mice were isolated and cultured as described previously ŽYe and Johnson, 1999.. Briefly, whole brains from mice were mechanically and enzymatically dissociated and plated onto poly-L-lysinecoated tissue culture flasks ŽCorning-Costar, Cambridge, MA. in Dulbecco’s modified eagle medium ŽDMEM; Gibco, Grand Island, NY. containing 20% fetal calf serum ŽFCS; Sigma, St. Louis, MO. and sodium bicarbonate Ž2 grl.. Cells were maintained at 378C with 95% humidity and 7% CO 2 for 24 h, when culture medium containing non-adherent cells Ži.e., neurons and oligodendrocytes. was removed. Culture medium was replenished and adherent cells were allowed to develop morphologically for 10 d, during which medium was changed every third day until cells became confluent. 2.4. Ribonuclease protection assay Interleukin-6 mRNA levels in cultured glial cells were assessed using a ribonuclease protection assay ŽRPA.. Total RNA was isolated from glia according to the Tri-reagent protocol. RNA integrity was confirmed by denaturing agarose gel electrophoresis and RNA concentration was determined by spectrophometric absobency at two dilutions. A partial cDNA fragment for murine IL-6 was created by RT-PCR-based cloning methods ŽInvitrogen, Carlsbad, CA. and inserted into a PCRII vector ŽPCRII-IL6. as described elsewhere ŽHeyen et al., 2000.. The b-actin template was purchased from a commercial source ŽAmbion, Austin, TX. and used as an internal reference in the assay to establish the relative amount of RNA in each sample. Radiolabeled ŽUTP-w a-32 Px. anti-sense RNA probe was then generated by in vitro transcription ŽMAXIscript; Ambion.. The full-length probes for IL-6 and b-actin were 520 and 360 bases, respectively; while protected fragments were 425 and 250 bases in length, respectively. Ribonuclease protection assays were performed using the HybSpeed RPA kit ŽAmbion. with minor modification. Glial cell RNA Ž20 mg. was hybridized in solution to the gel-purified probes. Hybridization was performed for 10 min at 688C, and the mixture digested with an RNase ArT1 mixture, diluted 1:500 in RNase digestion buffer,
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for 30 min at 378C. Protected fragments were precipitated and resolved on a 5% acrylamider8 M urea gel. The gel was then dried and the radioactivities associated with the IL-6 mRNA and b-Actin mRNA protected fragments were measured by phosphorimage scanning ŽImage Quant 3.3, Molecular Dynamics, Sunnyvale, CA. and visualized using autoradiographic film Ž24-h exposure at y808C to Kodak BioMaxrMS film with double screen.. 2.5. CompetitiÕe RT-PCR For measurement of IL-6 mRNA transcripts in brain tissues, a competitor DNA template was created from our previously constructed PCRII-IL6 plasmid by a deletion mutation. A 52-bp fragment was deleted from the partial murine IL-6 cDNA Ž425 bp. and was replaced by a 7-bp BamHI site. A competitor RNA was then in vitro transcribed and quantified by a RT-PCR Competitor Construction Kit ŽAmbion.. To perform RT-PCR, total RNA was isolated from perfused brain tissue by the Tri-reagent method. A series of RT reactions were set up using 2- or 10-fold dilutions of RNA competitors mixed with a constant amount of total sample RNA Ž4 mg.. The reverse transcription was performed at 378C for 90 min with 200 U of moloney murine leukemia virus reverse transcriptase ŽGibco.. The singlestranded cDNA was then amplified by 35 PCR cycles by AmpliTaq Gold DNA polymerase ŽPE Biosystems, Foster City, CA. with synthetic oligonucleotide primers designed to flank a 425-bp region of IL-6 wŽsense: 5X-CCAACAGACCTGTCTATACCAC . Žantisense: 5X-CCTTCTGTGACTCCAGCTTATC.x. PCR products were analyzed by electrophoresis on 2% agarose gel stained with ethidium bromide. The RNA competicon templates produced PCR products about 10% smaller than the corresponding endogenous target Ž381 vs. 425 bp.. DNA bands were visualized with UV light and photographed. The pictures were scanned and the amount of PCR product associated with each band was analyzed using the NIH image computerized densitometry program ŽWayne Rasband, NIH, Bethesda, MD.. Molar ratios were calculated by dividing the band volume by the length of the DNA molecule. Under competitive conditions, the absolute amount of IL-6 mRNA is equal to the amount of RNA competitor added, when the molar ratio of PCR products becomes equal for the target and competitor.
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were treated with 25 mM k B decoy or control scrambled DNA for 24 h to inhibit constitutive NFk B activity in vitro. To inhibit NFk B in vivo, decoy DNA was administered by injection into a lateral cerebral ventricle as previously described ŽGrzanna et al., 1998; Yao et al., 1999. with slight modification. Mice were anesthetized by an i.p. injection of ketamine Ž125 mgrkg body weight. and xylazine Ž6 mgrkg body weight. and placed in a stereotaxic instrument ŽDavid Kopf Instrument, Tujunga, CA.. A 28gauge injection cannula was inserted into a lateral ventricle using pre-determined coordinates wŽanteroposterior y0.6 mm; lateral, 1.6 mm to the bregma; and horizontal y2.0 mm to the dura mater. Franklin and Paxinos, 1997x and k B decoy or scrambled DNA Ž10 mM. in 2 ml of saline was infused over a 1-min period using a Hamilton syringe and a syringe pump ŽWorld Precision Instruments, Sarasota, FL.. Mice were returned to their home cage, where they regained consciousness. Nine h after injection, mice were killed by CO 2 asphyxiation and brains were dissected to verify the injection site and then frozen at y808C until isolation of RNA and nuclear protein. 2.7. Nuclear extraction Mice were transcardially perfused as previously described ŽYe and Johnson, 1999. to remove blood from the cerebral vasculature. Brains were rapidly removed and homogenized in 4 volumes of lysis buffer using a Dounce homogenizer with five strokes of the A type pestle and five strokes of the B type pestle ŽKontes Glass, Vineland, NJ.. After homogenization, nuclear extracts were prepared according to the procedures described by Gorski et al. Ž1986. with only slight modification. In brief, glia or brain tissue homogenate were suspended in lysis buffer containing 10 mM HEPES–NaOH buffer ŽpH7.9., 0.25 M sucrose, 15 mM KCl, 5 mM EDTA, 1 mM EGTA, 0.15 mM
2.6. Inhibition of NFk B actiÕity The k B decoy was used to inhibit NFk B activity both in vitro and in vivo. k B decoy DNA was prepared by annealing the oligonucleotides 5X-GAGGGGACTTTCCCT-3X and 5X-AGGGAAAGTCCCCTC-3X ŽYu et al., 1999.. The scrambled control DNA was prepared by annealing the oligonucleotides 5X-GATGCGTCTGTCGCA-3X and 5X-TGCGACAGACGCATC-3X . In one experiment, glia
Fig. 1. Interleukin-6 ŽIL-6. increases in the brain with age. Subjects Ž1-, 6- and 24-month-old male BALBrc mice; ns6. were transcardially perfused to remove blood and leukocytes from the cerebral vasculature. Protein-containing supernatants extracted from whole brain tissues were assayed for IL-6 using an ELISA specific for murine IL-6. The bars represent means"S.E. Treatment means with different letters are significantly different from each other Ž p- 0.01..
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hibitors.. After centrifugation at 16,000 = g for 20 min, supernatants were assayed to determine protein concentrations and stored at y808C. 2.8. Electrophoretic mobility shift assay Purified synthetic oligonucleotide probe which corresponded to the NFk B Ž5X-ATGTGGGATTTTCCCATGAG-3X . DNA-binding sequence in the IL-6 promoter was
Fig. 2. Competitive RT-PCR indicates that the level of IL-6 mRNA increased in the brain of aged mice. Total RNA from brains of 6- or 24-month-old mice was mixed with a series of 2-fold dilutions of RNA competitors. The mixtures were reverse transcribed and amplified by PCR. The RT-PCR products were separated by agarose gel electrophoresis and stained with ethidium bromide. ŽA. A representative gel for each age is shown in the upper panel. ŽB. The amount of IL-6 mRNA in the brain of adult and aged mice Ž ns 4. was determined by the amount of RNA competitor added, when the molar ratio of PCR products became equal for the target and competitor.
spermine, 0.5 mM spermidine, 1 mM dithiothreitol ŽDTT., and protease inhibitors w0.5 mM phenylmethylsulfonyl fluoride ŽPMSF., 100 mM NaF, and 1 mgrml each of benzamidine, leupeptin, pepstatin A, and aprotininx. The cells were further lysed in cell lysis buffer containing 0.5% NP-40 for 10 min. Cell lysates were centrifuged at 12,000 = g for 5 min. The pellets were washed twice and resuspended in 2 volumes of nuclei lysis buffer Ž10 mM HEPES–NaOH, pH 7.9, 1.5 mM MgCl2, 340 mM KCl, 1 mM EDTA, 1 mM DTT, and the already mentioned protease inhibitors.. The suspensions were gently mixed for 30 min at 48C, and centrifuged at 16,000 = g for 20 min. The supernatants were desalted by centrifugal filter units ŽMillipore, Bedford, MA. against reaction buffer Ž10 mM Tris–HCl, pH 7.5, 20% glycerol, 10 mM MgCl2, 100 mM KCl, 1 mM EDTA, 1 mM DTT, and protease in-
Fig. 3. The DNA-binding activity of NFk B is increased in brains of aged mice. Nuclear protein was extracted from brains of 1-, 6- and 24-monthold mice and subjected to electrophoretic mobility shift assay. The sequence of 32 P-labeled oligonucleotide probe corresponded to the NFk B response element in the promoter region of the mouse IL-6 gene. ŽA. The DNA–protein complexes were visualized on a nondenaturing polyacrylamide gel to reveal a shift band. Each lane represents one mouse so each age group is represented three times. The binding specificity was verified by incubating nuclear extracts from LPS-stimulated N13 cells without ŽC. or with 100-fold molar excess of unlabeled specific competitor oligonucleotide ŽS., or 100-fold molar excess of unlabeled non-specific competitor oligonucleotide ŽNS.. ŽB. The shift band was quantified by phosphorimage scanning. Each bar represents the mean"S.E. Ž ns 3.. Treatment means with different letters are significantly different from each other Ž p- 0.01..
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purchased from Operon Technologies ŽOperon, Alameda, CA.. Single-stranded synthetic oligonucleotides were annealed according to standard methods ŽSambrook et al., 1989.. The resulting double-stranded oligos were end labeled by w g-32 Px ATP using T4 polynucleotide kinase and purified using the kit from Qiagen ŽValencia, CA.. Nuclear extracts from LPS-stimulated N13 cells Ža microglia cell line. were used as a positive control. The specific competitor was unlabelled double-stranded oligonucleotide corresponding to the NFk B binding sequence, and the nonspecific competitor was an unlabelled double-stranded oligonucleotide corresponding to the Multiple Response Element ŽMRE; 5X-ATGCTAAACGACGTCACATT-3X .. Nuclear extracts Ž15–20 mg total protein. were incubated with or without competitor DNA for 10 min at room temperature in 10 mM Tris–HCl buffer ŽpH7.5. containing 1 mg poly ŽdI-dC., 10 mM MgCl 2 , 100 mM KCl, 1 mM EDTA, 1 mM DTT, 20% glycerol, and protease inhibitors. Radio-labeled probe Ž100 fmol, 1–2 = 10 5 cpmrpmol. was then added, and the mixture was incubated at room temperature for 30 min, when bound and free probes were separated by electrophoresis on a 4% polyacrylamide gel in buffer ŽpH 8.5. containing 25 mM Tris, 0.19 M Gly, and 2 mM EDTA at a constant voltage of 200 V for 2 h in an ice bath. Gels were dried and exposed overnight at y708C or visualized by phosphorimaging. 2.9. Statistical analysis Data were analyzed using General Linear Model procedures of Statistical Analysis Systems ŽSAS Institute, 1989.. They were subjected to one-way ANOVA. When ANOVA
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revealed a significant effect of Age, differences between treatments Ži.e., juvenile, adult, and aged. were tested using Duncan’s multiple range tests.
3. Results 3.1. Interleukin-6 gene expression is increased in the brain of aged mice Before evaluating the effects of age on the DNA-binding activity of NFk B, we confirmed that IL-6 increased in brain of aged mice. Interleukin-6 was measured in crude protein extracts from brains of juvenile Ž1 month old., adult Ž6 months old., and aged Ž24 months old. male BALBrc mice. One-way ANOVA of IL-6 concentration revealed a significant effect of age Ž p - 0.01.. Consistent with a previous report ŽYe and Johnson, 1999., IL-6 concentration was higher in brain extracts of aged mice than in brain extracts of adult or juvenile mice ŽFig. 1.. To extend this finding and to find out if IL-6 gene expression might be increased in the aged brain, the effect of age on steady-state IL-6 mRNA levels was evaluated. We first tried to assess IL-6 mRNA levels using the multi-probe RNase protection assay ŽRPA.. However, IL-6 mRNA in mouse brain was undetectable by RPA Ždata not shown., mandating that competitive RT-PCR be used. Total RNA that was isolated from brains of aged Ž n s 4. and adult mice Ž n s 4. was mixed with increasing amounts of an RNA competitor. The RT-PCR products were separated by agarose gel and stained with ethidium bromide. Representative gels are shown in Fig. 2A. The steady-state levels of
Fig. 4. Increased NFk B activity in glia cultured from brains of aged mice is inhibited by k B decoy treatment. Glial cells were cultured from brains of 6- or 24-month-old mice and treated with medium, 25 mM of scrambled control DNA , or 25 mM of k B decoy DNA for 24 h. Nuclear proteins were extracted and subjected to gel mobility shift assay. The sequence of 32 P-labeled oligonucleotide probe corresponded to the NFk B response element in the promoter region of the mouse IL-6 gene. The DNA–protein complexes were visualized on a nondenaturing polyacrylamide gel.
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IL-6 mRNA transcripts in 4 mg of total RNA isolated from brains of aged mice ranged from 2 = 10 4 to 1 = 10 5 copies with a median value of 6 = 10 4 ŽFig. 2B.. In contrast, the copy number of IL-6 mRNA from brains of adult mice was lower than 5 = 10 3 per 4 mg of total RNA with a median value of 2.5 = 10 3 ŽFig. 2B.. Thus, the copy number of IL-6 transcripts was at least 4-fold higher in aged brain than in the adult brain. 3.2. The DNA-binding actiÕity of NFk B is increased in the brain of aged mice A 1.2-kb fragment of the 5X-flanking region of the IL-6 gene contains a binding site for NFk B. Because IL-6 mRNA and protein were increased in aged brain ŽFigs. 1 and 2., we hypothesized that the DNA-binding activity of NFk B would be increased. To test this hypothesis, whole brains of juvenile, adult and aged mice were collected as before and nuclear protein was extracted and subjected to
electrophoretic mobility shift assay. Synthetic probe corresponding to the NFk B DNA-binding sequence in the IL-6 promoter was used. Fig. 3A shows the results of a typical gel shift assay. Each lane represents one mouse so each age group is represented three times. The DNA-binding activity was quantified by phosphorimage scanning and mean DNA-binding activity for NFk B is shown in Fig. 3B. The DNA-binding activity of NFk B was higher in the brains of aged mice than in the brains of adult and juvenile mice Ž p - 0.05.. Thus, increased binding of NFk B to the promoter region of the IL-6 gene may contribute to the increase in IL-6 mRNA and protein in brain of aged mice. 3.3. Inhibition of NFk B actiÕity represses IL-6 expression in glia from aged mice The hypothesis that increased DNA-binding activity of NFk B results in increased IL-6 gene expression in aged brain was evaluated both in vitro and in vivo. We previ-
Fig. 5. Inhibition of NFk B DNA-binding activity by k B decoy suppresses IL-6 gene expression in glia from aged mice. Glia from 6- or 24-month-old mice were incubated with medium, 25 mM of scrambled control DNA, or 25 mM of k B decoy DNA for 24 h. ŽA. RNA was isolated and subjected to RNase protection assay. The protected bands of IL-6 and b-actin mRNA were 425 and 250 bases, respectively. Full-length probes ŽIL-6, 520 bases; b-actin, 360 bases. are shown in lane 20. ŽB. The culture supernatants were collected and assayed for IL-6 using an ELISA specific for murine IL-6. The bars represent means" S.E. Treatment means with different letters are significantly different from each other Ž p - 0.01..
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effects of k B decoy on IL-6 mRNA and protein were evaluated. Consistent with our previous report ŽYe and Johnson, 1999., steady-state levels of IL-6 mRNA were increased in glia from aged mice compared to adult mice ŽFig. 5A, lanes 1–3 vs. lanes 10–12., as was the supernatant IL-6 concentration ŽFig. 5B.. The k B decoy, which competes with the endogenous k B site for binding NFk B, decreased steady-state IL-6 mRNA levels ŽFig. 5A. and protein secretion ŽFig. 5B. in unstimulated glia from aged mice. The control scrambled DNA, however, did not affect either IL-6 mRNA ŽFig. 5A. or protein ŽFig. 5B..
Fig. 6. Intracerebroventricular administration of k B decoy decreased NFk B activity in the brains of aged mice. Nuclear protein was extracted from brains of mice that had been injected i.c.v. with scrambled control DNA or k B decoy DNA and subjected to electrophoretic mobility shift assay. The sequence of the 32 P-labeled oligonucleotide probe corresponded to the NFk B element in the promoter region of the mouse IL-6 gene. The DNA–protein complexes were visualized on a nondenaturing polyacrylamide gel. Each lane represents one mouse so each treatment is represented three times. The binding specificity was verified by incubating nuclear extracts from vehicle-treated mice with 100-fold molar excess of unlabeled non-specific competitor oligonucleotide Žlane 10., or 50 Žlane 11.- and 100 Žlane 12.-fold molar excess of unlabeled specific competitor oligonucleotide.
ously reported that mixed glial cells cultured from aged mouse brain spontaneously expressed Ži.e., expressed without stimulation. more IL-6 mRNA and secreted more IL-6 protein than glia from adult and neonate brain ŽYe and Johnson, 1999.. Therefore, this system was initially used to determine if the increased IL-6 production was due to increased activity of NFk B. Glial cells were cultured from adult or aged mice, grown to confluency, and incubated 24 h with medium alone, or medium containing k B decoy DNA or scrambled control DNA. The k B decoy competes with the DNA-binding sequences for NFk B. Fig. 4 shows the results of the gel shift assay. Each lane represents glia from one mouse so each treatment is represented three times. Consistent with the results obtained using whole brain ŽFig. 3., NFk B activity was higher in glia of aged mice compared to glia of adult mice ŽFig. 4, lanes 1–3 vs. lanes 10–12.. Treatment of glia with scrambled control DNA did not affect NFk B DNA-binding activity ŽFig. 4, lanes 4–6 and lanes 13–15.. However, treatment with k B decoy DNA decreased NFk B activity in aged glia ŽFig. 4, lanes 16–18. to a level consistent with that observed in glia from adults. The decoy molecule did not decrease NFk B activity in adult glia ŽFig. 4, lanes 7–9.. To determine if the increased NFk B activity in glia from aged mice conferred increased gene expression, the
Fig. 7. Intracerebroventricular administration of k B decoy decreased IL-6 gene expression in the brains of aged mice. Total RNA from brains of 24-month-old mice that had been injected i.c.v. with scrambled control DNA or k B decoy was mixed with a series of 10-fold dilutions of RNA competitor. The mixtures were reverse transcribed and amplified by PCR. ŽA. A representative gel for each treatment is shown in the upper panel. ŽB. The amount of IL-6 mRNA in the brain of adult and aged mice Ž ns 4. was determined by the amount of RNA competitor added, when the molar ratio of PCR products became equal for the target and competitor.
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3.4. Central injection of k B decoy represses IL-6 expression in brain of aged mice The results of the previous study indicated that NFk B DNA-binding activity is increased in aged glia and that this results in increased expression of the inflammatory cytokine IL-6. To extend this finding, we analyzed NFk B activity and IL-6 gene expression in whole brain of aged mice after intracerebroventricular Ži.c.v.. injection of k B decoy. The k B decoy DNA, scrambled control DNA, or vehicle were injected i.c.v. and brain tissue was obtained 9 h later. This protocol was adopted from Yu et al. Ž1999., who used the k B decoy in mice to inhibit the activation of NFk B and apoptosis of neurons caused by excitotoxic injury. The effect of central injection of k B decoy DNA on NFk B DNA-binding activity in brain of aged mice is shown in Fig. 6. As predicted, the NFk B DNA-binding activity was lower after i.c.v. injection of k B decoy compared to vehicle or scrambled control DNA. The decrease in NFk B activity was paralleled by a reduction in IL-6 mRNA ŽFig. 7.. In the brain of aged mice injected i.c.v. with scrambled control DNA the steady-state levels of IL-6 mRNA transcripts in 4 mg of total RNA ranged from 2 = 10 5 to 1 = 10 6 copies with a median value of 5 = 10 5 ŽFig. 7B.. This was substantially higher than what was measured previously ŽFig. 2., suggesting an inflammatory response caused by the injection ŽSchobitz et al., 1997.. In any case, k B decoy DNA reduced IL-6 mRNA transcripts. The copy number of IL-6 mRNA from brains of aged mice treated with k B decoy was 5 = 10 3 –1 = 10 5 per 4 mg of total RNA ŽFig. 7B..
4. Discussion In the present study, we confirmed that IL-6 protein is increased in brain of aged mice, and using competitive RT-PCR showed that the steady-state level of IL-6 mRNA was higher in aged brain than in adult brain. This suggested that IL-6 gene transcription was increased in aged brain, which led us to examine the effects of aging on DNA-binding of NFk B. The important finding in this study was that NFk B binding to the IL-6 gene promoter was increased in glia and brain of aged mice compared to juvenile and adult mice. Moreover, selectively inhibiting the effects of age on NFk B activity with the k B decoy, reduced IL-6 mRNA and protein to levels seen in young adult controls. Therefore, these data indicate that the DNA-binding activity of NFk B is increased in the brain of aged mice and that at least one consequence of this is increased expression of the inflammatory cytokine IL-6. The fact that NFk B DNA-binding activity increases in brain with age has been previously reported. Toliver-Kinsky et al. Ž1997. measured NFk B activity and found significantly higher levels in hippocampus and forebrain of 30-month-old rats compared to adult controls. Korhonen et
al. Ž1997. also reported increased NFk B activity in discrete brain areas of aged rats. However, neither of these studies identified a specific target gene that was affected by NFk B. In peripheral lymphoid organs of aged mice NFk B is also constitutively activated ŽSpencer et al., 1997.. In splenocytes of aged mice the increased activity of NFk B is associated with aberrant expression of several inflammatory cytokines ŽSpencer et al., 1997; Poynter and Daynes, 1998.. However, if this consequence of age is evident in cells of the CNS had not been evaluated. Thus, in the present study we sought to determine if the age-related increase in brain NFk B activity was inherently linked to the age-related increase in brain IL-6. We first evaluated this in astrocytes and microglia co-cultured from brain of adult and aged mice. Consistent with what was found in whole brain, NFk B DNA-binding activity, steady-state IL-6 mRNA level, and IL-6 protein secretion were higher in glia from aged mice than adults. In this model, comparisons between age groups must be cautiously made, however, because cultures from aged mice comprised more microglia and fewer astrocytes than the cultures established from adult brains ŽYe and Johnson, 1999.. The reliable comparisons are between treatments within an age group; and the important finding here is that k B decoy DNA decreased NFk B DNA-binding activity, steady-state IL-6 mRNA level, and IL-6 protein secretion in aged glia Žcompared to medium or scrambled control DNA.. The effect of k B decoy on these endpoints in glia from adult mice was minimal. These results can be extended to suggest that the increase in NFk B DNA-binding activity, steady-state IL-6 mRNA level, and IL-6 protein measured in whole brain is from astrocytes andror microglia. There is strong evidence that the ability of animals to effectively contend with oxidative stress declines with age. Reactive oxygen species lead to phosphorylation and ubiquitination of Ik Ba , which liberates NFk B allowing it to translocate from the cell cytoplasm to the nucleus ŽSchreck et al., 1992.. Both the DNA-binding activity of NFk B and the spontaneous secretion of IL-6 are increased in splenocytes from aged mice compared to young adults ŽPoynter and Daynes, 1998.. Supplementation of aged mice with the antioxidant vitamin E corrects the age-associated increase in NFk B activity and IL-6 production in splenocytes. Therefore, it seems likely that in the aged brain oxidative stress also initiates a cascade that results in increased NFk B DNA-binding activity and enhanced expression of cytokines like IL-6. However, this point has not yet been evaluated. Although the present study focused on IL-6, there is evidence that other cytokines such as IL-1ba are increased as well ŽMurray and Lynch, 1998.. The age-related increase in inflammatory cytokines in brain may be a factor contributing to the neuropathological effects of aging. For example, dysregulation of the IL-6 gene might establish a state that is permissive to the onset of neurodegenerative disease. This hypothesis is partially
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based on the work of Campbell et al. Ž1993., who produced transgenic mice whose astrocytes over expressed IL-6. Mice over expressing IL-6 in brain showed dendritic vacuolization, reduced branching of dendritic spines in CA1 neurons, and loss of hippocampal interneurons. The expression of IL-6 was also related to deficits in avoidance learning ŽHeyser et al., 1997.. Increased IL-6 expression in brain has also been documented in brain trauma ŽWoodroofe et al., 1991., AIDS dementia complex ŽGallo et al., 1991., multiple sclerosis ŽHofman et al., 1989., and Alzheimer’s disease ŽBauer et al., 1991.. Interestingly, the cytokine is present in the early stage of plaque formation ŽHull et al., 1996., suggesting it may be an early primer for neuropathological changes that occur in Alzheimer’s disease. Despite the apparent close association between IL-6 and neurodegeneration, to our best knowledge no studies have shown that IL-6 directly kills neurons. However, a recent study showed that chronic exposure of developing cerebellar granule neurons to IL-6 enhanced membrane depolarization and the intracellular calcium signal to NMDA ŽQiu et al., 1998.. Moreover, IL-6 was shown to enhance NMDA receptor-mediated neurotoxicity, indicating that it may affect neurons indirectly. The results of the present study show that the age-related increase in brain IL-6 is due to increased activity of NFk B. However, why NFk B activity increases in aged brain is not yet known and the specific effects of the age-related increase in brain IL-6 remain to be established. A better understanding of the effects of age on cytokines in brain may help explain the neurophysiologic manifestations of aging.
Acknowledgements This work was supported by grants to R.W.J. from the NIA ŽAG16710. and the NIDDKD ŽDK51576..
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