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Increased interleukin-6 expression by microglia from brain of aged mice
Journal of Neuroimmunology 93 Ž1999. 139–148
Increased interleukin-6 expression by microglia from brain of aged mice Shi-Ming Ye, Rodney W. Johnson
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Department of Animal Sciences, UniÕersity of Illinois, 390 Animal Sciences Laboratory, 1207 West Gregory DriÕe, Urbana, IL 61801, USA Received 30 June 1998; revised 3 September 1998; accepted 17 September 1998
Abstract Over expression of inflammatory cytokines in the brain may establish a state that is permissive to the onset of neurodegenerative disease. Because the occurrence of certain neurodegenerative diseases increases with age, in the present study we examined the expression of the inflammatory cytokine, interleukin-6 ŽIL-6., in the brain of aged mice. In an initial experiment, IL-6 was measured in crude protein extracts from brains of juvenile Ž1-month-old., adult Ž3-month-old., and aged Ž24-month-old. male BALBrc mice. The concentration of IL-6 in crude protein extracts from the cerebellum, cerebral cortex, and hippocampus increased with age. The increase in IL-6 was discrete, as levels in the hypothalamus were not age-dependent. To begin evaluating spontaneous IL-6 production in aging, glial cells were cultured from brains of neonate, adult, and aged mice. An age-associated increase in IL-6 mRNA and supernatant IL-6 concentration was evident, indicating glia from aged mice spontaneously express high levels of IL-6 relative to glia from adult and neonate mice. Flow cytometric analysis revealed that cultures established from aged brain compared to either adult or neonate brain comprised more microglia Ži.e., MAC-1-positive cells.. Furthermore, the proportion of microglia that was positive for IL-6 increased with age, whereas the proportion of astrocytes that were positive for IL-6 was not age-dependent. The present results suggest that IL-6 increases in the mouse brain with age, and that microglia cultured from aged mice spontaneously produce more IL-6 than those from neonate or adult mice. Therefore, microglia may contribute to the increased level of IL-6 present in aged brain. q 1999 Elsevier Science B.V. All rights reserved. Keywords: Aging; Brain; Interleukin-6; Astrocytes; Microglia; Mouse
1. Introduction Interleukin-6 ŽIL-6. is a pleiotropic cytokine that is involved in mediating cellular communication both in physiological and pathological states. It belongs to a subfamily of structurally related cytokines that share the same signal-transducing receptor component, gp130 ŽKishimoto et al., 1995.. Interleukin-6 is produced in the brain primarily by astrocytes and microglia, but also by neurons ŽSchoitz ¨ et al., 1992; Gadient and Otten, 1994.. Evidence indicates that IL-6 produced in the brain has an important role in central nervous system ŽCNS. development ŽQiu et al., 1995; Holliday et al., 1995; Sarder et al., 1996. and in coordinating the central component of the acute phase response ŽChai et al., 1996.. There also is evidence, however, that if IL-6 is chronically over expressed in the CNS, it establishes a state that is permissive to the onset of neurodegenerative disease. Transgenic mice that over ex-
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pressed IL-6 in the CNS exhibited neurological disease and neuropathology, both being positively related with the level of cerebral IL-6 transgene expression ŽCampbell et al., 1993.. The progressive neuropathological manifestations of IL-6 transgene expression were closely related to deficits in avoidance learning ŽHeyser et al., 1997., which is consistent with a recent study showing that IL-6 inhibited long-term potentiation in hippocampal neurons ŽLi et al., 1997.. Interleukin-6 has been detected in the CNS of patients with neurodegenerative disease, including multiple sclerosis and AIDS. Furthermore, considerable IL-6 expression is evident in senile plaques in Alzheimer’s disease ŽBauer et al., 1991.. A recent study reported IL-6 predominantly in plaques where neuritic pathology had not yet developed, indicating the cytokine may be a cause, and not just a consequence, of neuritic degeneration ŽHull et al., 1996.. Lymphoid cells isolated from old but otherwise healthy humans and mice spontaneously secrete high levels of IL-6 and consequently, a positive correlation between age and plasma IL-6 concentration has been reported ŽWei et al., 1992; Daynes et al., 1993.. If IL-6 concentration in the
0165-5728r99r$ - see front matter q 1999 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 5 - 5 7 2 8 Ž 9 8 . 0 0 2 1 7 - 3
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S.-M. Ye, R.W. Johnsonr Journal of Neuroimmunology 93 (1999) 139–148
brain reflects that in the periphery, it may explain, in part, the neuropathophysiologic phenotypes of old age, including the increased incidence of Alzheimer’s disease. Circulating cytokines are unlikely to access the CNS in large amounts, however, and it is not yet known if microglia, which like monocytes and macrophages are derived from mononuclear myeloid progenitors ŽPerry et al., 1994. spontaneously produce high levels of IL-6 in the aged brain. In the present study we cultured microglia and astrocytes from brains of neonate, adult and aged mice to determine if there exists an age-associated increase in spontaneous IL-6 production. Furthermore, we employed two-color flow cytometry to determine the effect of age on the frequency of astrocytes and microglia expressing IL-6. The present results suggest that IL-6 increases in the mouse brain with age, and that microglia cultured from aged mice spontaneously produce more IL-6 than those from neonate or adult mice.
2. Materials and methods 2.1. Animals Neonate Ž- 7-day-old., juvenile Ž3–4-week-old., and adult Ž3-6-month-old. male BALBrc mice were obtained from a breeding colony kept in barrier-reared conditions in a specific pathogen-free facility at the University of Illinois. Aged male BALBrc mice Ž24-month-old. that were reared under similar conditions were obtained from the National Institute on Aging breeding colony at Charles River Laboratories ŽKingston, NY.. Mice were kept in groups of three 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.
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 ŽELISA, Genzyme, Cambridge, MA.. 2.3. Cell culture Whole brains from mice were mechanically dissociated after a 15-min trypsinization Ž0.25% trypsin., passed through a 100-mm nylon mesh, washed twice in D-Hank’s, and plated onto poly-L-lysine-coated cover-slips, 24-well plates, or 25 cm2 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. Immunocytochemistry Cells cultured on cover slips were rinsed twice in PBS and fixed and permeabilized in methanolracetic acid Ž9:1. for 2 min at 378C. After blocking with normal swine serum, cells were incubated with rabbit anti-glial fibrillary acidic protein ŽGFAP; 1:100, Sigma. and mouse anti-
2.2. Determination of IL-6 in brain One-, three- and twenty-four-month-old mice Ž n s 6. were deeply anesthetized by halothane inhalation. Blood samples were collected from the inferior vena cava and then mice were transcardially perfused with sterile heparinized phosphate buffered saline ŽPBS; 388C. for 15 min when perfusate was found to be clear and free of cells. The brains were removed and inspected to ensure complete perfusion, and the cerebellum, cortex, hypothalamus, and hippocampus were dissected, frozen in liquid nitrogen, and stored at y808C. 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,
Fig. 1. Plasma interleukin-6 ŽIL-6. concentration increases with age. Plasma from 1-, 3-, and 24-month old male BALBrc mice Ž ns6. was assayed for IL-6 using the IL-6-dependent 7td1 cell line. The bars represent means"SE. Treatment means with different letters are significantly different from each other Ž P - 0.01..
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50% dimethylformamide ŽDMF. –20% sodium dodecyl sulfate ŽSDS. dissolved in double-deionized water were added to each well. Cells were incubated for 16 h with the 50% DMF–20% SDS solution to lyse cells and solubilize formazan crystals. Plates were read at 550 nm using an automated microplate reader ŽEL311, Bio-Tek Instruments, Winooski, VT.. Supernatants from four treatment replicates in each of three separate but identical trials Ž n s 12. were assayed. The intra-assay variation was less than 11% and the minimum detection limit of this assay was 4 pgrml. We have demonstrated specificity of this assay in that preincubation of samples Ž1 h at 378C. with a polyclonal anti-IL-6 antibody ŽR & D Systems, Minneapolis, MN. completely neutralized biological activity of the IL6-containing plasma and supernatant.
Fig. 2. Interleukin-6 ŽIL-6. increases in the brain with age. One, three and twenty-four-month old male BALBrc mice Ž ns6. were transcardially perfused to remove blood and leukocytes from the cerebral vasculature. Protein-containing supernatants extracted from the hippocampus ŽHC., cerebral cortex ŽCC., cerebellum ŽCB., and hypothalamus ŽHT. were assayed for IL-6 using an ELISA specific for murine IL-6. The bars represent means"SE. Treatment means with different letters are significantly different from each other Ž P - 0.01..
MAC-1 Ži.e., CD11b. mAb Ž1:100, Dako, Carpinteria, CA. for 40 min at room temperature. Cells were then washed extensively and GFAP-positive Žastrocytes. and MAC-1positive Žmicroglia. cells were detected using the Dako Double Staining kit ŽDako. according to the manufacturer’s instructions. 2.5. In Õitro secretion of IL-6 After confluent, glia cultured on 24-well plates were washed twice and replenished with fresh medium to achieve 1 = 10 6 cells per ml. Cells were incubated 18 h at 378C and 5% CO 2 , when cell supernatants were collected and subsequently assayed for IL-6 using the 7td1 B-cell hybridoma bioassay as previously described ŽFinck et al., 1997.. In brief, 7td1 cells were suspended in RPMI-1640 ŽGibco. with 5% FCS and seeded into 96-well plates at 10 4 cells per well. Recombinant mouse IL-6 ŽPharmingen, San Diego, CA. or diluted supernatants Ž1:6, 1:36, and 1:216. were added to triplicate wells and incubated for 72 h at 378C, 5% CO 2 , and 95% relative humidity. Proliferation was determined by adding 3-Ž4,5-dimethylthiazol-2yl.,-2,5-diphenyltetrazolium bromide ŽMTT; 2 mgrml in D-Hank’s.. Cells were incubated with MTT for 4 h, when
Fig. 3. Double-immunocytochemical staining of cultured mixed glial cells. Glial cells isolated from ŽA. neonate Ž -1-week-old., ŽB. adult Ž3-month-old., and ŽC. aged mice Ž24-month-old. were plated onto poly-L-lysine-coated cover-slips in DMEM containing 20% FCS. After 10 days, the morphology of astrocytes and microglia were determined by double-immunocytochemical staining. Cells stained red were immunoreactive to MAC-1, a microglial cell marker; whereas cells stained brown were immunoreactive to GFAP, an intracellular marker for astrocytes. Magnification: 200=.
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2.6. RNA isolation and RT-PCR Total RNA was isolated from glia according to the Tri-reagent protocol described by Chomczynski ŽChomczynski, 1993.. RT-PCR was used to detect IL-6 mRNA, similar to what we have previously described ŽYao and
Johnson, 1997.. Total RNA Ž1 mg. was reverse transcribed at 378C for 90 min with 200 U of moloney murine leukemia virus reverse transcriptase ŽGibco. and 0.2 mg of DNA random hexamers. The single-stranded cDNA or non-reverse transcribed RNA Žnegative control. was amplified Ž30 PCR cycles. with synthetic oligonucleotide primers
Fig. 4. Flow cytometric analysis of the proportion of microglia and astrocytes in glia cultures established from brains of neonate Ž- 1-week-old., adult Ž3-month-old., and aged mice Ž24-month-old.. Confluent glia cultured from mice differing in age were stained with FITC-anti-MAC-1 Žmicroglia. and FITC-anti-GFAP Žastrocytes.. ŽA. One-parameter histograms were generated from a representative experiment and statistical gates were set based on the control Žshaded area, background staining - 0.5%.. For each histogram, the percentage indicates the proportion of FITC-positive cells. ŽB. Data from four experiments where glia cultured from different mice for each age group were used each time are summarized. Values are expressed as the percentage of FITC-positive cells Žmean " SE.. For GFAP positive or MAC-1-positive cells, means with different letters are significantly different from each other Ž P - 0.05..
S.-M. Ye, R.W. Johnsonr Journal of Neuroimmunology 93 (1999) 139–148
designed to flank a 208 bp region of IL-6 wŽsense: 5X GTTCTCTGGGAAATCGTGGA. Žantisense: 5X TGTACTCCAGGTAGCTATGG.x or a 258 bp region of murine glyceraldehyde-3-phosphate dehydrogenase ŽGAPDH. wŽsense: 5X TGCATCCTGCACCACCAACT. Žantisense: 5XAACACGGAAGGCCATGCCAG.x in the presence of w a-32 PxdCTP ŽAmersham, Arlington Heights, IL.. In preliminary studies we found that under our conditions 30 cycles of PCR were the minimum needed to consistently detect IL-6 mRNA in unstimulated glia from neonate brain, and that under these same conditions GAPDH was still in the linear range. Primers were designed to span an intron so that if genomic DNA were present, amplification would produce a larger fragment containing the intronŽs.. Amplified cDNA products were loaded into a 10% polyacrylamide gel along with a 100 bp DNA ladder and size fractionated by electrophoresis. The amount of radioactivity associated with each band for each molecule was measured by phosphorimage scanning ŽImage Quant 3.3, Molecular Dynamics, Sunnyvale, CA..
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min at 48C. To ensure the specificity of the staining procedure, the binding of IL-6 Ab was blocked with molar excess of recombinant murine IL-6 ŽPharmingen.. Finally, the cells were resuspended in 500 ml SB for flow cytometric analysis. The phenotypic analysis of cells expressing IL-6 was performed using an EPICS XL MCL flow cytometer ŽCoulter Electronics, Hialeah, FL.. Dead cells were excluded by forward and side scatter gating. A minimum of 10,000 events was collected and list mode data acquired was analyzed using EPICS Elite Workstation software. 2.8. Statistical analysis Data were analyzed using General Linear Model procedures of Statistical Analysis Systems ŽSAS Institute, 1989.. They were subjected to One-way ŽAge. ANOVA. When ANOVA revealed a significant effect of Age, differences between treatments Ži.e., juvenile, adult, and aged. were tested using Duncan’s multiple range tests.
2.7. Flow cytometric analysis Glia from mice of different ages were isolated and cultured in tissue flasks as described above. Confluent cultures were washed twice and replenished with fresh medium. For intracellular IL-6 staining, cells were further incubated 12 h with 1 mgrml brefeldin A ŽPharmingen., an inhibitor of protein secretion that is used to enhance intracellular cytokine staining and detection of cytokineproducing cells ŽFujiwara et al., 1988.. Cells were then detached from the flask by addition of 0.25% trypsin– 0.02% EDTA with 40 mgrml DNase to decrease cell aggregation. Cells were washed twice in staining buffer ŽSB; D-Hank’s with 1% heat-inactivated FCS, 0.1% sodium azide, pH 7.4. and separated into two parts, one for labeling microglia ŽMAC-1., the other for labeling astrocytes ŽGFAP.. For GFAP staining, 10 6 cells were fixed in 4% paraformaldehyde for 30 min at 48C and then permeabilized by washing twice in 0.1% saponin ŽSigma. permeabilization buffer ŽPB; SB with 0.1% saponin.. Fixed and permeabilized cells in 100 ml were stained with rabbit anti-GFAP Ž1:100; Sigma. for 30 min at 48C. Cells were washed twice in SB and incubated at 48C for 30 min with 1:40 fluorescence ŽFITC.-conjugated goat anti-rabbit FŽabX . 2 fragment ŽSigma.. For microglia, 10 6 cells in 100 ml SB were incubated with 2 mgrml rat anti-mouse MAC-1 or isotype matched IgG ŽBoehringer Mannheim, Indianapolis, IN. control for 30 min at 48C. Cells were washed twice in SB and incubated at 48C for 30 min with FITC-conjugated goat anti-rat FŽabX . 2 fragment Ž1:20; Boehringer Mannheim.. After staining for MAC-1, cells were fixed in 4% paraformaldehyde for 30 min at 48C and then permeabilized by washing twice in 0.1% saponin ŽSigma. permeabilization buffer ŽPB; SB with 0.1% saponin.. Cells were then stained with 2 mgrml PE-conjugated rat anti-murine IL-6 antibody ŽPharmingen. for 30
Fig. 5. The spontaneous expression of interleukin-6 ŽIL-6. in glia cultured from neonate Ž -1-week-old., adult Ž3-month-old., and aged Ž24-monthold. mice increases with age. Glial cells grown to confluence were provided fresh medium and incubated 18 h. ŽA. Cell supernatants Ž ns12. were collected and assayed for IL-6 using the 7td1 cell line. The bars represent the treatment means"SE. Treatment means with different letters are significantly different from each other Ž P - 0.05.. B. Total cellular RNA isolated from glia was reverse transcribed and amplified by PCR. Oligonucleotide primers framed a 208 bp fragment of IL-6 and a 258 bp fragment of GAPDH. Phosphorimage scanning to quantify the radioactivity associated with each band for each molecule revealed an age-associated increase in IL-6 mRNA.
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3. Results 3.1. IL-6 is increased in the brain of aged mice We first sought to confirm that plasma IL-6 concentration in aged mice was higher than that in the plasma of adult or juvenile mice, and to determine if IL-6 levels in the brain reflect those in the plasma. One-, three- and twenty-four-month-old male BALBrc mice with no apparent signs of illness or infection were anesthetized and a blood sample was collected before perfusing the brain. Crude protein extracts from various brain regions were subjected to an ELISA specific for murine IL-6, while plasma IL-6 concentration was determined using the IL-6sensitive 7td1 cell line. One-way ANOVA of plasma IL-6 concentration revealed a significant effect of age Ž P 0.01.. Consistent with previous reports ŽWei et al., 1992; Daynes et al., 1993., aged mice had high plasma IL-6 levels compared to adult or juvenile mice ŽFig. 1.. The age-associated increase in plasma IL-6 was paralleled by an increase in IL-6 in the brain ŽFig. 2.. One-way ANOVA
of IL-6 concentration of crude protein extracts from the hippocampus, cerebral cortex, and cerebellum revealed a significant effect of age Ž P - 0.01.. The increase in IL-6 was discrete, as levels in the hypothalamus were not affected by age ŽFig. 2.. Because blood was removed from the brain by perfusion, these data are consistent with the idea that cells in the CNS of aged mice spontaneously secrete high levels of IL-6 relative to cells in the brain of young mice. 3.2. Glia from aged mice secrete high leÕels of IL-6 in Õitro To evaluate the effect of age on spontaneous IL-6 production by glial cells, astrocytes and microglia were cultured from brain of neonate, adult, and aged mice. Cells were grown to confluence in 24-well plates, washed, provided fresh medium, and incubated for 18 h before supernatants were collected for IL-6 determination. Cultures from neonate, adult, and aged mice comprised a cell population that included both astrocytes and microglia, as
Fig. 6. Two-color flow cytometric analysis of glia from neonate, adult, and aged mice expressing interleukin-6 ŽIL-6.. Glia cultured to confluence were X treated with brefeldin A and then stained with rat anti-MAC-1 or isotype matched IgG followed by FITC-goat anti-rat FŽab. 2 fragment. Cells were then fixed, permeabilized, and stained with PE-rat anti-IL-6 in the presence Žcontrol. or absence of molar excess of recombinant murine IL-6. ŽA. Two-parameter histograms were generated and quadrant statistics were set based on the control Žbackground staining for IL-6 - 0.1%.; numbers in the quadrants refer to the percentage of positive cells. ŽB. Mean fluorescence intensity ŽMFI. of PE is compared between MAC-1-positive and -negative cell populations. Data were collected from four independent studies and expressed as mean" SE. Treatment means with different letters are significantly different from each other Ž P - 0.01..
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determined by immunohistochemical staining for GFAP and MAC-1, respectively ŽFig. 3.. This also was confirmed by flow cytometry, where GFAP- and MAC-1-positive cells accounted for at least 88% of the total ŽFig. 4A.. This analysis also revealed that cultures established from brain of aged mice comprised more MAC-1-positive cells ŽFig. 4B, 52 " 1%, P - 0.05. compared to cultures established from adult Ž29 " 6%. and neonate Ž33 " 3%. mice. Accordingly, cultures from adult and neonate mice contained more GFAP-positive cells Ž61 " 4% and 57 " 5%, respectively. than cultures from aged mice ŽFig. 4B, 44 " 3%, P - 0.05.. Microglia from aged and adult mice typically had a larger cell body with more processes compared to microglia from neonates ŽFig. 3., suggesting microglia from the older mice were reactive even in the absence of any known stimulus. One-way ANOVA of supernatant IL-6 concentration revealed a significant effect of age Ž P - 0.05.. An age-associated increase in supernatant IL-6 concentration was evident, indicating glia from aged mice spontaneously secreted high levels of IL-6 relative to glia from adult and neonate mice ŽFig. 5A.. Because of the limited number of glia which could be obtained from mice, relative levels of mRNA encoding IL-6 were determined by semi-quantitative RT-PCR. Interleukin-6 mRNA was highest in glia from aged mice followed by adult and neonate ŽFig. 5B.. Collectively, these data indicate a positive association between age and IL-6 expression in the brain, and are consistent with the idea that cells in the CNS and in the periphery of aged mice spontaneously secrete copious amounts of IL-6. 3.3. The proportion of microglia expressing IL-6 increases with age To determine if aged astrocytes, microglia, or both are responsible for the increased in vitro production of IL-6, we subjected glia cultured from neonate, adult, and aged mouse brain to two-color flow cytometric analysis. Cells were stained with FITC-conjugated anti-MAC-1 mAb and PE-conjugated anti-mouse IL-6 mAb. Representative two parameter histograms plotting MAC-1 vs. IL-6 fluorescence are illustrated in Fig. 6, as is the mean fluorescence intensity ŽMFI. of IL-6 fluorescence in MAC-1-positive cells as well as in MAC-1-negative cells. The MFI was calculated from four experiments where glia cultured from different mice for each age group were used each time. More than 90% of the cells which were IL-6-positive were also MAC-1-positive, regardless of age. Staining for IL-6 was completely blocked by pre-absorbing the PE-conjugated anti-IL-6 mAb with a molar excess of recombinant mouse IL-6. The MAC-1-positive cells displayed an ageassociated increase in MFI, indicating that MAC-1-positive cells from aged mice expressed more IL-6 per cell than MAC-1-positive cells from adults or neonates. The MFI of MAC-1-positive cells derived from neonate, adult, and aged mice was 3.3 " 0.4, 5.0 " 0.2, and 8.8 " 0.9, respec-
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tively. The MFI of MAC-1-negative cells was not affected by age. Since more than 88% of the cells in the mixed cultures were either GFAP or MAC-1-positive, the MAC-1 negative cells are presumed to be astrocytes. Although we attempted to confirm this by double staining for GFAP and IL-6, double staining for two intracellular ligands did not work. Nonetheless, these data suggest that the increased IL-6 in culture supernatants from aged glia is from microglia rather than astrocytes.
4. Discussion Chronic over expression of IL-6 in the CNS may establish a state that is permissive to the onset of neurodegenerative disease. In the present study we established that normal aging is associated with increased expression of IL-6 protein in the brain. Specifically, the present study demonstrated that IL-6 is increased in the brain of aged mice, and that glia cultured from brain of aged mice spontaneously secrete copious amounts of IL-6, relative to glia from adult or neonate mice. The morphology of microglia cultured from aged mice suggested they were reactive. It was further found by flow cytometry that the proportion of microglia that were positive for IL-6 increased with age, whereas the proportion of astrocytes that were positive for IL-6 was not age-dependent. These results indicate that microglia may contribute to the increased level of IL-6 present in aged brain and be an important target for treating or abrogating the neuropathological manifestations of aging. Increased expression of IL-6 and several other inflammatory cytokines Že.g., IL-1b and TNFa . in the CNS has been documented in brain trauma ŽWoodroofe et al., 1991. and in certain diseases, including AIDS ŽGallo et al., 1989., viral ŽFrei et al., 1988. and bacteria meningitis ŽHoussiau et al., 1988., multiple sclerosis ŽHofman et al., 1989., and Alzheimer’s disease ŽBauer et al., 1991.. The emerging view is that central cytokine expression is causally related to neurodegeneration ŽPapanicolaou et al., 1998.. For instance, deposition of b amyloid within senile plaques is a classical neurohistopathological feature of Alzheimer’s disease and conditions leading to the expression of inflammatory cytokines in the CNS Že.g., focal cerebral ischemia and head injury. are associated with a rapid increase in b amyloid precursor protein ŽAbe et al., 1991; Roberts et al., 1991; Royston et al., 1992.. This may provoke a positive feedback loop, since b amyloid activates microglia which results in neuronal cell injury ŽMeda et al., 1995.. Studies with transgenic mice that overexpressed human amyloid precursor protein indicated that b amyloid was not acutely neurotoxic, but rather induced gliosis and inflammation ŽIrizarry et al., 1997.. Therefore, the suggestion that inflammatory cytokines are early primers for subsequent neuropathology that is present at the final stages of Alzheimer’s disease appears to have
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been correct ŽStanley et al., 1994.. The present study suggests that there is deterioration of the systemŽs. that controls IL-6 production in the CNS, or that events associated with aging induce chronic inflammation. Interleukin-6 is elevated in serum of normal aging humans, as well as in aged mice, which was confirmed in the present study. Lymphoid cells from aged mice spontaneously secrete more IL-6 in vitro, compared to mature adults ŽDaynes et al., 1993.. To study the effect of age on IL-6 production by brain tissue, Prechel et al. Ž1996. incubated cerebral cortical tissue slices from neonate, 2-, 6-, and 9-month-old mice and determined IL-6 levels in supernatants. The results indicated a positive relationship between age and cerebral IL-6 production ex vivo. In the present study, the effect of age on central IL-6 was examined in several brain areas, in addition to the cerebral cortex. Total protein was extracted from discrete brain areas of juvenile Ž1-month-old. and adult Ž3-month-old. mice, but also mice that were senescent Ž24-month-old.. Prior to removal, brains were perfused to displace blood and IL-6-producing leukocytes from the cerebral vasculature. We found an age-associated increase in IL-6 in the cerebellum, cortex and hippocampus, but not in the hypothalamus. Therefore, the present results are consistent with those of Prechel et al. Ž1996. and support an increase in central IL-6 production. To extend these findings, we sought to identify the cell typeŽs. whose secretion of IL-6 increases with age. We established cultures from brains of neonate, adult, and aged mice, which comprised both astrocytes and microglia. One option would have been to culture the two cell types individually to evaluate IL-6 production of each separately. However, the utility of this approach is limited because only a small number of microglia can be separated from mixed glial cell cultures. For instance, from 6–20 g of tissue dissected from the corpus callosum of postmortem human brains, Walker et al. Ž1995. obtained only 1–2.5 = 10 5 microglia. Instead, we opted to develop one- and two-color flow cytometric analysis techniques. Because most cell loss occurs when separating microglia and astrocytes, this technique allowed us to Ža. evaluate a large number of cells in an environment more indicative of in vivo conditions; Žb. determine the proportion of astrocytes and microglia in mixed cultures; and Žc. study IL-6 expression at the single-cell level. To our best knowledge this is one of the first studies to use two-color flow cytometry to investigate cytokine expression by cells of the CNS. The results suggest there is an age-related increase in IL-6 positive microglia, and that microglia are the dominant source of constitutive IL-6, since more than 90% of the cells that were IL-6 positive were also MAC-1-positive. It is unknown if, or to what extent, the in vitro manipulation affected the ratio of astrocytes and microglia isolated from neonate, adult, and aged brain, however, the age-related changes in glial cells found here is supported by several in vivo and in vitro studies. First, using cell culture tech-
niques very similar to the ones used in the present study, Rozovsky et al. Ž1998. reported that cultures from aged rats contained more microglia than those from young adult or middle-aged rats. The microglia from aged brain showed an amoeboid-like morphology and expressed MHC II, suggesting they were constitutively activated. Second, the number of large reactive microglia immunoreactive for another inflammatory cytokine, IL-1, increased in the brains of nondemented individuals over the age of 60 ŽMrak et al., 1997.. Third, in the brain of healthy aged rats, there was an increase in the number of reactive microglia as compared to juveniles ŽPerry et al., 1993.. And fourth, Lafortune et al. Ž1996. has shown that astrocytes are the dominant producers of IL-6 in the fetal brain, but that microglia are the dominant producers of IL-6 in the adult brain. In any case, the difference in supernatant IL-6 from neonate, adult, and aged cultures cannot be explained by a difference in cell population since adult cultures actually contained fewer microglia than neonate cultures Ž27% vs. 30%., but spontaneously produced more IL-6. Therefore, from the present study we believe it is reasonable to postulate that the increased IL-6 in brain of aged mice is due to an increase in reactive microglia, which spontaneously express IL-6. Additional studies are needed, however, to confirm this point. Another possible explanation for our results is that glia cultured from neonate brains are immature and incapable of producing levels of IL-6 similar to that of glia cultured from aged brain. To test this possibility in another study glial cells were incubated with various concentrations of lipopolysaccharide ŽLPS.. Glia from neonate and adult mice showed a marked increase in IL-6 secretion that was well beyond that spontaneously secreted by glia from aged mice Ždata not shown.. Although glia from aged mice also increased IL-6 secretion upon exposure to LPS, the response was less than that by glia from younger mice. Similar results were observed when cerebral cortical slices from neonate and adult mice were exposed to LPS in vitro ŽPrechel et al., 1996.. Thus, the difference in IL-6 secretion between glia from neonate, adult and aged brain is not due to an inability of glia from young animals to produce the cytokine. An important unanswered question is why expression of the IL-6 gene is higher in microglia from aged brain than from adult or neonate brain. One possibility is that the mechanisms that ordinarily inhibit IL-6 gene expression deteriorate with age. For instance, production of the steroid hormone DHEA steadily declines with age. Supplemental DHEA has been shown to decrease IL-6 in plasma of aged subjects ŽDaynes et al., 1993.. However, if it has similar effects in the brain is as yet unknown. Transforming growth factor-b, an anti-inflammatory cytokine whose concentration in the brain declines with age ŽYoung et al., 1995., decreased IL-6 secretion by cerebral cortical tissue from 9-month-old mice but not from neonates ŽPrechel et al., 1996.. Collectively, these results suggest that factors
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that inhibit central cytokine production in the young are no longer present in the old. Studies aimed at understanding how aging affects transcription factors that control IL-6 gene expression should provide new insights.
5. Conclusion In summary, the present results suggest that the increased IL-6 present in brains of aged mice is a result of an increase in reactive microglia which secrete IL-6. A chronic high level of IL-6 in the brain may create a state that is conducive to the onset of neurodegenerative disease. Therefore, microglia may represent an important target for treating or abrogating the neuropathophysiological manifestations of aging.
Acknowledgements We wish to thank Dr. Keith Kelley for helping with the flow cytometry and Dr. James Zachary for helping with the immunocytochemistry. The cost of aged mice was partially supported by the National Institute on Aging Pilot Study program. This research was supported by NIH grant DK51576 Žto R.W.J.. and by a grant Ži.e., a Future Leader Award to R.W.J.. from the North American Branch of the International Life Sciences Institute ŽILSI N.A... The opinions expressed herein are those of the authors and do not necessarily represent the views of ILSI N.A.
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Increased interleukin-6 expression by microglia from brain of aged mice Shi-Ming Ye, Rodney W. Johnson
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Department of Animal Sciences, UniÕersity of Illinois, 390 Animal Sciences Laboratory, 1207 West Gregory DriÕe, Urbana, IL 61801, USA Received 30 June 1998; revised 3 September 1998; accepted 17 September 1998
Abstract Over expression of inflammatory cytokines in the brain may establish a state that is permissive to the onset of neurodegenerative disease. Because the occurrence of certain neurodegenerative diseases increases with age, in the present study we examined the expression of the inflammatory cytokine, interleukin-6 ŽIL-6., in the brain of aged mice. In an initial experiment, IL-6 was measured in crude protein extracts from brains of juvenile Ž1-month-old., adult Ž3-month-old., and aged Ž24-month-old. male BALBrc mice. The concentration of IL-6 in crude protein extracts from the cerebellum, cerebral cortex, and hippocampus increased with age. The increase in IL-6 was discrete, as levels in the hypothalamus were not age-dependent. To begin evaluating spontaneous IL-6 production in aging, glial cells were cultured from brains of neonate, adult, and aged mice. An age-associated increase in IL-6 mRNA and supernatant IL-6 concentration was evident, indicating glia from aged mice spontaneously express high levels of IL-6 relative to glia from adult and neonate mice. Flow cytometric analysis revealed that cultures established from aged brain compared to either adult or neonate brain comprised more microglia Ži.e., MAC-1-positive cells.. Furthermore, the proportion of microglia that was positive for IL-6 increased with age, whereas the proportion of astrocytes that were positive for IL-6 was not age-dependent. The present results suggest that IL-6 increases in the mouse brain with age, and that microglia cultured from aged mice spontaneously produce more IL-6 than those from neonate or adult mice. Therefore, microglia may contribute to the increased level of IL-6 present in aged brain. q 1999 Elsevier Science B.V. All rights reserved. Keywords: Aging; Brain; Interleukin-6; Astrocytes; Microglia; Mouse
1. Introduction Interleukin-6 ŽIL-6. is a pleiotropic cytokine that is involved in mediating cellular communication both in physiological and pathological states. It belongs to a subfamily of structurally related cytokines that share the same signal-transducing receptor component, gp130 ŽKishimoto et al., 1995.. Interleukin-6 is produced in the brain primarily by astrocytes and microglia, but also by neurons ŽSchoitz ¨ et al., 1992; Gadient and Otten, 1994.. Evidence indicates that IL-6 produced in the brain has an important role in central nervous system ŽCNS. development ŽQiu et al., 1995; Holliday et al., 1995; Sarder et al., 1996. and in coordinating the central component of the acute phase response ŽChai et al., 1996.. There also is evidence, however, that if IL-6 is chronically over expressed in the CNS, it establishes a state that is permissive to the onset of neurodegenerative disease. Transgenic mice that over ex-
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Corresponding author. Tel.: q1-217-333-2118; Fax: q1-217-3338286; E-mail: [email protected]
pressed IL-6 in the CNS exhibited neurological disease and neuropathology, both being positively related with the level of cerebral IL-6 transgene expression ŽCampbell et al., 1993.. The progressive neuropathological manifestations of IL-6 transgene expression were closely related to deficits in avoidance learning ŽHeyser et al., 1997., which is consistent with a recent study showing that IL-6 inhibited long-term potentiation in hippocampal neurons ŽLi et al., 1997.. Interleukin-6 has been detected in the CNS of patients with neurodegenerative disease, including multiple sclerosis and AIDS. Furthermore, considerable IL-6 expression is evident in senile plaques in Alzheimer’s disease ŽBauer et al., 1991.. A recent study reported IL-6 predominantly in plaques where neuritic pathology had not yet developed, indicating the cytokine may be a cause, and not just a consequence, of neuritic degeneration ŽHull et al., 1996.. Lymphoid cells isolated from old but otherwise healthy humans and mice spontaneously secrete high levels of IL-6 and consequently, a positive correlation between age and plasma IL-6 concentration has been reported ŽWei et al., 1992; Daynes et al., 1993.. If IL-6 concentration in the
0165-5728r99r$ - see front matter q 1999 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 5 - 5 7 2 8 Ž 9 8 . 0 0 2 1 7 - 3
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brain reflects that in the periphery, it may explain, in part, the neuropathophysiologic phenotypes of old age, including the increased incidence of Alzheimer’s disease. Circulating cytokines are unlikely to access the CNS in large amounts, however, and it is not yet known if microglia, which like monocytes and macrophages are derived from mononuclear myeloid progenitors ŽPerry et al., 1994. spontaneously produce high levels of IL-6 in the aged brain. In the present study we cultured microglia and astrocytes from brains of neonate, adult and aged mice to determine if there exists an age-associated increase in spontaneous IL-6 production. Furthermore, we employed two-color flow cytometry to determine the effect of age on the frequency of astrocytes and microglia expressing IL-6. The present results suggest that IL-6 increases in the mouse brain with age, and that microglia cultured from aged mice spontaneously produce more IL-6 than those from neonate or adult mice.
2. Materials and methods 2.1. Animals Neonate Ž- 7-day-old., juvenile Ž3–4-week-old., and adult Ž3-6-month-old. male BALBrc mice were obtained from a breeding colony kept in barrier-reared conditions in a specific pathogen-free facility at the University of Illinois. Aged male BALBrc mice Ž24-month-old. that were reared under similar conditions were obtained from the National Institute on Aging breeding colony at Charles River Laboratories ŽKingston, NY.. Mice were kept in groups of three 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.
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 ŽELISA, Genzyme, Cambridge, MA.. 2.3. Cell culture Whole brains from mice were mechanically dissociated after a 15-min trypsinization Ž0.25% trypsin., passed through a 100-mm nylon mesh, washed twice in D-Hank’s, and plated onto poly-L-lysine-coated cover-slips, 24-well plates, or 25 cm2 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. Immunocytochemistry Cells cultured on cover slips were rinsed twice in PBS and fixed and permeabilized in methanolracetic acid Ž9:1. for 2 min at 378C. After blocking with normal swine serum, cells were incubated with rabbit anti-glial fibrillary acidic protein ŽGFAP; 1:100, Sigma. and mouse anti-
2.2. Determination of IL-6 in brain One-, three- and twenty-four-month-old mice Ž n s 6. were deeply anesthetized by halothane inhalation. Blood samples were collected from the inferior vena cava and then mice were transcardially perfused with sterile heparinized phosphate buffered saline ŽPBS; 388C. for 15 min when perfusate was found to be clear and free of cells. The brains were removed and inspected to ensure complete perfusion, and the cerebellum, cortex, hypothalamus, and hippocampus were dissected, frozen in liquid nitrogen, and stored at y808C. 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,
Fig. 1. Plasma interleukin-6 ŽIL-6. concentration increases with age. Plasma from 1-, 3-, and 24-month old male BALBrc mice Ž ns6. was assayed for IL-6 using the IL-6-dependent 7td1 cell line. The bars represent means"SE. Treatment means with different letters are significantly different from each other Ž P - 0.01..
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50% dimethylformamide ŽDMF. –20% sodium dodecyl sulfate ŽSDS. dissolved in double-deionized water were added to each well. Cells were incubated for 16 h with the 50% DMF–20% SDS solution to lyse cells and solubilize formazan crystals. Plates were read at 550 nm using an automated microplate reader ŽEL311, Bio-Tek Instruments, Winooski, VT.. Supernatants from four treatment replicates in each of three separate but identical trials Ž n s 12. were assayed. The intra-assay variation was less than 11% and the minimum detection limit of this assay was 4 pgrml. We have demonstrated specificity of this assay in that preincubation of samples Ž1 h at 378C. with a polyclonal anti-IL-6 antibody ŽR & D Systems, Minneapolis, MN. completely neutralized biological activity of the IL6-containing plasma and supernatant.
Fig. 2. Interleukin-6 ŽIL-6. increases in the brain with age. One, three and twenty-four-month old male BALBrc mice Ž ns6. were transcardially perfused to remove blood and leukocytes from the cerebral vasculature. Protein-containing supernatants extracted from the hippocampus ŽHC., cerebral cortex ŽCC., cerebellum ŽCB., and hypothalamus ŽHT. were assayed for IL-6 using an ELISA specific for murine IL-6. The bars represent means"SE. Treatment means with different letters are significantly different from each other Ž P - 0.01..
MAC-1 Ži.e., CD11b. mAb Ž1:100, Dako, Carpinteria, CA. for 40 min at room temperature. Cells were then washed extensively and GFAP-positive Žastrocytes. and MAC-1positive Žmicroglia. cells were detected using the Dako Double Staining kit ŽDako. according to the manufacturer’s instructions. 2.5. In Õitro secretion of IL-6 After confluent, glia cultured on 24-well plates were washed twice and replenished with fresh medium to achieve 1 = 10 6 cells per ml. Cells were incubated 18 h at 378C and 5% CO 2 , when cell supernatants were collected and subsequently assayed for IL-6 using the 7td1 B-cell hybridoma bioassay as previously described ŽFinck et al., 1997.. In brief, 7td1 cells were suspended in RPMI-1640 ŽGibco. with 5% FCS and seeded into 96-well plates at 10 4 cells per well. Recombinant mouse IL-6 ŽPharmingen, San Diego, CA. or diluted supernatants Ž1:6, 1:36, and 1:216. were added to triplicate wells and incubated for 72 h at 378C, 5% CO 2 , and 95% relative humidity. Proliferation was determined by adding 3-Ž4,5-dimethylthiazol-2yl.,-2,5-diphenyltetrazolium bromide ŽMTT; 2 mgrml in D-Hank’s.. Cells were incubated with MTT for 4 h, when
Fig. 3. Double-immunocytochemical staining of cultured mixed glial cells. Glial cells isolated from ŽA. neonate Ž -1-week-old., ŽB. adult Ž3-month-old., and ŽC. aged mice Ž24-month-old. were plated onto poly-L-lysine-coated cover-slips in DMEM containing 20% FCS. After 10 days, the morphology of astrocytes and microglia were determined by double-immunocytochemical staining. Cells stained red were immunoreactive to MAC-1, a microglial cell marker; whereas cells stained brown were immunoreactive to GFAP, an intracellular marker for astrocytes. Magnification: 200=.
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2.6. RNA isolation and RT-PCR Total RNA was isolated from glia according to the Tri-reagent protocol described by Chomczynski ŽChomczynski, 1993.. RT-PCR was used to detect IL-6 mRNA, similar to what we have previously described ŽYao and
Johnson, 1997.. Total RNA Ž1 mg. was reverse transcribed at 378C for 90 min with 200 U of moloney murine leukemia virus reverse transcriptase ŽGibco. and 0.2 mg of DNA random hexamers. The single-stranded cDNA or non-reverse transcribed RNA Žnegative control. was amplified Ž30 PCR cycles. with synthetic oligonucleotide primers
Fig. 4. Flow cytometric analysis of the proportion of microglia and astrocytes in glia cultures established from brains of neonate Ž- 1-week-old., adult Ž3-month-old., and aged mice Ž24-month-old.. Confluent glia cultured from mice differing in age were stained with FITC-anti-MAC-1 Žmicroglia. and FITC-anti-GFAP Žastrocytes.. ŽA. One-parameter histograms were generated from a representative experiment and statistical gates were set based on the control Žshaded area, background staining - 0.5%.. For each histogram, the percentage indicates the proportion of FITC-positive cells. ŽB. Data from four experiments where glia cultured from different mice for each age group were used each time are summarized. Values are expressed as the percentage of FITC-positive cells Žmean " SE.. For GFAP positive or MAC-1-positive cells, means with different letters are significantly different from each other Ž P - 0.05..
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designed to flank a 208 bp region of IL-6 wŽsense: 5X GTTCTCTGGGAAATCGTGGA. Žantisense: 5X TGTACTCCAGGTAGCTATGG.x or a 258 bp region of murine glyceraldehyde-3-phosphate dehydrogenase ŽGAPDH. wŽsense: 5X TGCATCCTGCACCACCAACT. Žantisense: 5XAACACGGAAGGCCATGCCAG.x in the presence of w a-32 PxdCTP ŽAmersham, Arlington Heights, IL.. In preliminary studies we found that under our conditions 30 cycles of PCR were the minimum needed to consistently detect IL-6 mRNA in unstimulated glia from neonate brain, and that under these same conditions GAPDH was still in the linear range. Primers were designed to span an intron so that if genomic DNA were present, amplification would produce a larger fragment containing the intronŽs.. Amplified cDNA products were loaded into a 10% polyacrylamide gel along with a 100 bp DNA ladder and size fractionated by electrophoresis. The amount of radioactivity associated with each band for each molecule was measured by phosphorimage scanning ŽImage Quant 3.3, Molecular Dynamics, Sunnyvale, CA..
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min at 48C. To ensure the specificity of the staining procedure, the binding of IL-6 Ab was blocked with molar excess of recombinant murine IL-6 ŽPharmingen.. Finally, the cells were resuspended in 500 ml SB for flow cytometric analysis. The phenotypic analysis of cells expressing IL-6 was performed using an EPICS XL MCL flow cytometer ŽCoulter Electronics, Hialeah, FL.. Dead cells were excluded by forward and side scatter gating. A minimum of 10,000 events was collected and list mode data acquired was analyzed using EPICS Elite Workstation software. 2.8. Statistical analysis Data were analyzed using General Linear Model procedures of Statistical Analysis Systems ŽSAS Institute, 1989.. They were subjected to One-way ŽAge. ANOVA. When ANOVA revealed a significant effect of Age, differences between treatments Ži.e., juvenile, adult, and aged. were tested using Duncan’s multiple range tests.
2.7. Flow cytometric analysis Glia from mice of different ages were isolated and cultured in tissue flasks as described above. Confluent cultures were washed twice and replenished with fresh medium. For intracellular IL-6 staining, cells were further incubated 12 h with 1 mgrml brefeldin A ŽPharmingen., an inhibitor of protein secretion that is used to enhance intracellular cytokine staining and detection of cytokineproducing cells ŽFujiwara et al., 1988.. Cells were then detached from the flask by addition of 0.25% trypsin– 0.02% EDTA with 40 mgrml DNase to decrease cell aggregation. Cells were washed twice in staining buffer ŽSB; D-Hank’s with 1% heat-inactivated FCS, 0.1% sodium azide, pH 7.4. and separated into two parts, one for labeling microglia ŽMAC-1., the other for labeling astrocytes ŽGFAP.. For GFAP staining, 10 6 cells were fixed in 4% paraformaldehyde for 30 min at 48C and then permeabilized by washing twice in 0.1% saponin ŽSigma. permeabilization buffer ŽPB; SB with 0.1% saponin.. Fixed and permeabilized cells in 100 ml were stained with rabbit anti-GFAP Ž1:100; Sigma. for 30 min at 48C. Cells were washed twice in SB and incubated at 48C for 30 min with 1:40 fluorescence ŽFITC.-conjugated goat anti-rabbit FŽabX . 2 fragment ŽSigma.. For microglia, 10 6 cells in 100 ml SB were incubated with 2 mgrml rat anti-mouse MAC-1 or isotype matched IgG ŽBoehringer Mannheim, Indianapolis, IN. control for 30 min at 48C. Cells were washed twice in SB and incubated at 48C for 30 min with FITC-conjugated goat anti-rat FŽabX . 2 fragment Ž1:20; Boehringer Mannheim.. After staining for MAC-1, cells were fixed in 4% paraformaldehyde for 30 min at 48C and then permeabilized by washing twice in 0.1% saponin ŽSigma. permeabilization buffer ŽPB; SB with 0.1% saponin.. Cells were then stained with 2 mgrml PE-conjugated rat anti-murine IL-6 antibody ŽPharmingen. for 30
Fig. 5. The spontaneous expression of interleukin-6 ŽIL-6. in glia cultured from neonate Ž -1-week-old., adult Ž3-month-old., and aged Ž24-monthold. mice increases with age. Glial cells grown to confluence were provided fresh medium and incubated 18 h. ŽA. Cell supernatants Ž ns12. were collected and assayed for IL-6 using the 7td1 cell line. The bars represent the treatment means"SE. Treatment means with different letters are significantly different from each other Ž P - 0.05.. B. Total cellular RNA isolated from glia was reverse transcribed and amplified by PCR. Oligonucleotide primers framed a 208 bp fragment of IL-6 and a 258 bp fragment of GAPDH. Phosphorimage scanning to quantify the radioactivity associated with each band for each molecule revealed an age-associated increase in IL-6 mRNA.
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3. Results 3.1. IL-6 is increased in the brain of aged mice We first sought to confirm that plasma IL-6 concentration in aged mice was higher than that in the plasma of adult or juvenile mice, and to determine if IL-6 levels in the brain reflect those in the plasma. One-, three- and twenty-four-month-old male BALBrc mice with no apparent signs of illness or infection were anesthetized and a blood sample was collected before perfusing the brain. Crude protein extracts from various brain regions were subjected to an ELISA specific for murine IL-6, while plasma IL-6 concentration was determined using the IL-6sensitive 7td1 cell line. One-way ANOVA of plasma IL-6 concentration revealed a significant effect of age Ž P 0.01.. Consistent with previous reports ŽWei et al., 1992; Daynes et al., 1993., aged mice had high plasma IL-6 levels compared to adult or juvenile mice ŽFig. 1.. The age-associated increase in plasma IL-6 was paralleled by an increase in IL-6 in the brain ŽFig. 2.. One-way ANOVA
of IL-6 concentration of crude protein extracts from the hippocampus, cerebral cortex, and cerebellum revealed a significant effect of age Ž P - 0.01.. The increase in IL-6 was discrete, as levels in the hypothalamus were not affected by age ŽFig. 2.. Because blood was removed from the brain by perfusion, these data are consistent with the idea that cells in the CNS of aged mice spontaneously secrete high levels of IL-6 relative to cells in the brain of young mice. 3.2. Glia from aged mice secrete high leÕels of IL-6 in Õitro To evaluate the effect of age on spontaneous IL-6 production by glial cells, astrocytes and microglia were cultured from brain of neonate, adult, and aged mice. Cells were grown to confluence in 24-well plates, washed, provided fresh medium, and incubated for 18 h before supernatants were collected for IL-6 determination. Cultures from neonate, adult, and aged mice comprised a cell population that included both astrocytes and microglia, as
Fig. 6. Two-color flow cytometric analysis of glia from neonate, adult, and aged mice expressing interleukin-6 ŽIL-6.. Glia cultured to confluence were X treated with brefeldin A and then stained with rat anti-MAC-1 or isotype matched IgG followed by FITC-goat anti-rat FŽab. 2 fragment. Cells were then fixed, permeabilized, and stained with PE-rat anti-IL-6 in the presence Žcontrol. or absence of molar excess of recombinant murine IL-6. ŽA. Two-parameter histograms were generated and quadrant statistics were set based on the control Žbackground staining for IL-6 - 0.1%.; numbers in the quadrants refer to the percentage of positive cells. ŽB. Mean fluorescence intensity ŽMFI. of PE is compared between MAC-1-positive and -negative cell populations. Data were collected from four independent studies and expressed as mean" SE. Treatment means with different letters are significantly different from each other Ž P - 0.01..
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determined by immunohistochemical staining for GFAP and MAC-1, respectively ŽFig. 3.. This also was confirmed by flow cytometry, where GFAP- and MAC-1-positive cells accounted for at least 88% of the total ŽFig. 4A.. This analysis also revealed that cultures established from brain of aged mice comprised more MAC-1-positive cells ŽFig. 4B, 52 " 1%, P - 0.05. compared to cultures established from adult Ž29 " 6%. and neonate Ž33 " 3%. mice. Accordingly, cultures from adult and neonate mice contained more GFAP-positive cells Ž61 " 4% and 57 " 5%, respectively. than cultures from aged mice ŽFig. 4B, 44 " 3%, P - 0.05.. Microglia from aged and adult mice typically had a larger cell body with more processes compared to microglia from neonates ŽFig. 3., suggesting microglia from the older mice were reactive even in the absence of any known stimulus. One-way ANOVA of supernatant IL-6 concentration revealed a significant effect of age Ž P - 0.05.. An age-associated increase in supernatant IL-6 concentration was evident, indicating glia from aged mice spontaneously secreted high levels of IL-6 relative to glia from adult and neonate mice ŽFig. 5A.. Because of the limited number of glia which could be obtained from mice, relative levels of mRNA encoding IL-6 were determined by semi-quantitative RT-PCR. Interleukin-6 mRNA was highest in glia from aged mice followed by adult and neonate ŽFig. 5B.. Collectively, these data indicate a positive association between age and IL-6 expression in the brain, and are consistent with the idea that cells in the CNS and in the periphery of aged mice spontaneously secrete copious amounts of IL-6. 3.3. The proportion of microglia expressing IL-6 increases with age To determine if aged astrocytes, microglia, or both are responsible for the increased in vitro production of IL-6, we subjected glia cultured from neonate, adult, and aged mouse brain to two-color flow cytometric analysis. Cells were stained with FITC-conjugated anti-MAC-1 mAb and PE-conjugated anti-mouse IL-6 mAb. Representative two parameter histograms plotting MAC-1 vs. IL-6 fluorescence are illustrated in Fig. 6, as is the mean fluorescence intensity ŽMFI. of IL-6 fluorescence in MAC-1-positive cells as well as in MAC-1-negative cells. The MFI was calculated from four experiments where glia cultured from different mice for each age group were used each time. More than 90% of the cells which were IL-6-positive were also MAC-1-positive, regardless of age. Staining for IL-6 was completely blocked by pre-absorbing the PE-conjugated anti-IL-6 mAb with a molar excess of recombinant mouse IL-6. The MAC-1-positive cells displayed an ageassociated increase in MFI, indicating that MAC-1-positive cells from aged mice expressed more IL-6 per cell than MAC-1-positive cells from adults or neonates. The MFI of MAC-1-positive cells derived from neonate, adult, and aged mice was 3.3 " 0.4, 5.0 " 0.2, and 8.8 " 0.9, respec-
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tively. The MFI of MAC-1-negative cells was not affected by age. Since more than 88% of the cells in the mixed cultures were either GFAP or MAC-1-positive, the MAC-1 negative cells are presumed to be astrocytes. Although we attempted to confirm this by double staining for GFAP and IL-6, double staining for two intracellular ligands did not work. Nonetheless, these data suggest that the increased IL-6 in culture supernatants from aged glia is from microglia rather than astrocytes.
4. Discussion Chronic over expression of IL-6 in the CNS may establish a state that is permissive to the onset of neurodegenerative disease. In the present study we established that normal aging is associated with increased expression of IL-6 protein in the brain. Specifically, the present study demonstrated that IL-6 is increased in the brain of aged mice, and that glia cultured from brain of aged mice spontaneously secrete copious amounts of IL-6, relative to glia from adult or neonate mice. The morphology of microglia cultured from aged mice suggested they were reactive. It was further found by flow cytometry that the proportion of microglia that were positive for IL-6 increased with age, whereas the proportion of astrocytes that were positive for IL-6 was not age-dependent. These results indicate that microglia may contribute to the increased level of IL-6 present in aged brain and be an important target for treating or abrogating the neuropathological manifestations of aging. Increased expression of IL-6 and several other inflammatory cytokines Že.g., IL-1b and TNFa . in the CNS has been documented in brain trauma ŽWoodroofe et al., 1991. and in certain diseases, including AIDS ŽGallo et al., 1989., viral ŽFrei et al., 1988. and bacteria meningitis ŽHoussiau et al., 1988., multiple sclerosis ŽHofman et al., 1989., and Alzheimer’s disease ŽBauer et al., 1991.. The emerging view is that central cytokine expression is causally related to neurodegeneration ŽPapanicolaou et al., 1998.. For instance, deposition of b amyloid within senile plaques is a classical neurohistopathological feature of Alzheimer’s disease and conditions leading to the expression of inflammatory cytokines in the CNS Že.g., focal cerebral ischemia and head injury. are associated with a rapid increase in b amyloid precursor protein ŽAbe et al., 1991; Roberts et al., 1991; Royston et al., 1992.. This may provoke a positive feedback loop, since b amyloid activates microglia which results in neuronal cell injury ŽMeda et al., 1995.. Studies with transgenic mice that overexpressed human amyloid precursor protein indicated that b amyloid was not acutely neurotoxic, but rather induced gliosis and inflammation ŽIrizarry et al., 1997.. Therefore, the suggestion that inflammatory cytokines are early primers for subsequent neuropathology that is present at the final stages of Alzheimer’s disease appears to have
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been correct ŽStanley et al., 1994.. The present study suggests that there is deterioration of the systemŽs. that controls IL-6 production in the CNS, or that events associated with aging induce chronic inflammation. Interleukin-6 is elevated in serum of normal aging humans, as well as in aged mice, which was confirmed in the present study. Lymphoid cells from aged mice spontaneously secrete more IL-6 in vitro, compared to mature adults ŽDaynes et al., 1993.. To study the effect of age on IL-6 production by brain tissue, Prechel et al. Ž1996. incubated cerebral cortical tissue slices from neonate, 2-, 6-, and 9-month-old mice and determined IL-6 levels in supernatants. The results indicated a positive relationship between age and cerebral IL-6 production ex vivo. In the present study, the effect of age on central IL-6 was examined in several brain areas, in addition to the cerebral cortex. Total protein was extracted from discrete brain areas of juvenile Ž1-month-old. and adult Ž3-month-old. mice, but also mice that were senescent Ž24-month-old.. Prior to removal, brains were perfused to displace blood and IL-6-producing leukocytes from the cerebral vasculature. We found an age-associated increase in IL-6 in the cerebellum, cortex and hippocampus, but not in the hypothalamus. Therefore, the present results are consistent with those of Prechel et al. Ž1996. and support an increase in central IL-6 production. To extend these findings, we sought to identify the cell typeŽs. whose secretion of IL-6 increases with age. We established cultures from brains of neonate, adult, and aged mice, which comprised both astrocytes and microglia. One option would have been to culture the two cell types individually to evaluate IL-6 production of each separately. However, the utility of this approach is limited because only a small number of microglia can be separated from mixed glial cell cultures. For instance, from 6–20 g of tissue dissected from the corpus callosum of postmortem human brains, Walker et al. Ž1995. obtained only 1–2.5 = 10 5 microglia. Instead, we opted to develop one- and two-color flow cytometric analysis techniques. Because most cell loss occurs when separating microglia and astrocytes, this technique allowed us to Ža. evaluate a large number of cells in an environment more indicative of in vivo conditions; Žb. determine the proportion of astrocytes and microglia in mixed cultures; and Žc. study IL-6 expression at the single-cell level. To our best knowledge this is one of the first studies to use two-color flow cytometry to investigate cytokine expression by cells of the CNS. The results suggest there is an age-related increase in IL-6 positive microglia, and that microglia are the dominant source of constitutive IL-6, since more than 90% of the cells that were IL-6 positive were also MAC-1-positive. It is unknown if, or to what extent, the in vitro manipulation affected the ratio of astrocytes and microglia isolated from neonate, adult, and aged brain, however, the age-related changes in glial cells found here is supported by several in vivo and in vitro studies. First, using cell culture tech-
niques very similar to the ones used in the present study, Rozovsky et al. Ž1998. reported that cultures from aged rats contained more microglia than those from young adult or middle-aged rats. The microglia from aged brain showed an amoeboid-like morphology and expressed MHC II, suggesting they were constitutively activated. Second, the number of large reactive microglia immunoreactive for another inflammatory cytokine, IL-1, increased in the brains of nondemented individuals over the age of 60 ŽMrak et al., 1997.. Third, in the brain of healthy aged rats, there was an increase in the number of reactive microglia as compared to juveniles ŽPerry et al., 1993.. And fourth, Lafortune et al. Ž1996. has shown that astrocytes are the dominant producers of IL-6 in the fetal brain, but that microglia are the dominant producers of IL-6 in the adult brain. In any case, the difference in supernatant IL-6 from neonate, adult, and aged cultures cannot be explained by a difference in cell population since adult cultures actually contained fewer microglia than neonate cultures Ž27% vs. 30%., but spontaneously produced more IL-6. Therefore, from the present study we believe it is reasonable to postulate that the increased IL-6 in brain of aged mice is due to an increase in reactive microglia, which spontaneously express IL-6. Additional studies are needed, however, to confirm this point. Another possible explanation for our results is that glia cultured from neonate brains are immature and incapable of producing levels of IL-6 similar to that of glia cultured from aged brain. To test this possibility in another study glial cells were incubated with various concentrations of lipopolysaccharide ŽLPS.. Glia from neonate and adult mice showed a marked increase in IL-6 secretion that was well beyond that spontaneously secreted by glia from aged mice Ždata not shown.. Although glia from aged mice also increased IL-6 secretion upon exposure to LPS, the response was less than that by glia from younger mice. Similar results were observed when cerebral cortical slices from neonate and adult mice were exposed to LPS in vitro ŽPrechel et al., 1996.. Thus, the difference in IL-6 secretion between glia from neonate, adult and aged brain is not due to an inability of glia from young animals to produce the cytokine. An important unanswered question is why expression of the IL-6 gene is higher in microglia from aged brain than from adult or neonate brain. One possibility is that the mechanisms that ordinarily inhibit IL-6 gene expression deteriorate with age. For instance, production of the steroid hormone DHEA steadily declines with age. Supplemental DHEA has been shown to decrease IL-6 in plasma of aged subjects ŽDaynes et al., 1993.. However, if it has similar effects in the brain is as yet unknown. Transforming growth factor-b, an anti-inflammatory cytokine whose concentration in the brain declines with age ŽYoung et al., 1995., decreased IL-6 secretion by cerebral cortical tissue from 9-month-old mice but not from neonates ŽPrechel et al., 1996.. Collectively, these results suggest that factors
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that inhibit central cytokine production in the young are no longer present in the old. Studies aimed at understanding how aging affects transcription factors that control IL-6 gene expression should provide new insights.
5. Conclusion In summary, the present results suggest that the increased IL-6 present in brains of aged mice is a result of an increase in reactive microglia which secrete IL-6. A chronic high level of IL-6 in the brain may create a state that is conducive to the onset of neurodegenerative disease. Therefore, microglia may represent an important target for treating or abrogating the neuropathophysiological manifestations of aging.
Acknowledgements We wish to thank Dr. Keith Kelley for helping with the flow cytometry and Dr. James Zachary for helping with the immunocytochemistry. The cost of aged mice was partially supported by the National Institute on Aging Pilot Study program. This research was supported by NIH grant DK51576 Žto R.W.J.. and by a grant Ži.e., a Future Leader Award to R.W.J.. from the North American Branch of the International Life Sciences Institute ŽILSI N.A... The opinions expressed herein are those of the authors and do not necessarily represent the views of ILSI N.A.
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