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Influence of neurons on lipopolysaccharide-stimulated production of nitric oxide and tumor necrosis factor-α by cultured glia
Brain Research 853 Ž2000. 236–244 www.elsevier.comrlocaterbres
Research report
Influence of neurons on lipopolysaccharide-stimulated production of nitric oxide and tumor necrosis factor-a by cultured glia Raymond C.C. Chang, Pearlie Hudson, Belinda Wilson, Lisa Haddon, Jau-Shyong Hong
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Neuropharmacology Section, MD F1-01, Laboratory of Pharmacology and Chemistry, National Institute of EnÕironmental Health Sciences, National Institutes of Health, P.O. Box 12233, Research Triangle Park, NC 27709, USA Accepted 19 October 1999
Abstract Cerebral inflammation often originates in a region where neuronal death occurs and thereafter slowly spreads outward. This study aimed to elucidate the roles of neurons in modulating the production of inflammatory factors stimulated by the bacterial endotoxin lipopolysaccharide ŽLPS.. Culturing neurons with mixed glia reduced nitrite and tumor necrosis factor-a ŽTNF-a . production compared to cultures with only mixed glia, and shifted the dose–response curve to the right. The decreased nitrite and TNF-a production were not due to the cytotoxicity of LPS. Immunocytochemical analysis of glia–neuron co-cultures revealed the morphological changes in the activated microglia. Culturing PC12 cells with rat mixed-glia also reduced nitrite production. The influence of neurons on glial inflammation was partly due to the cell–cell contacts between neurons and glia via neural cell adhesion molecules ŽNCAM. because NCAM significantly reduced LPS-stimulated nitrite production. These results demonstrate that neurons reduce the production of inflammatory factors by glia. Since cerebral inflammation is important in many neurological disorders, this study might provide insight about the role of glia–neuron interactions in inflammatory responses in the brain. q 2000 Elsevier Science B.V. All rights reserved. Keywords: Lipopolysaccharide; Neural cell adhesion molecule; Neuron–glia interaction; Microglia; Astrocyte; Cerebral inflammation
1. Introduction Inflammation occurs in many types of neurological disorders, such as multiple sclerosis, Parkinson’s disease, Alzheimer’s disease, cerebral ischemia and traumatic brain injuries, and it is one of the factors contributing to neurodegeneration wfor a review, see Ref. w23xx. In stroke and traumatic brain injuries, the primary insult activates glial cells, mainly microglia and astrocytes. The activated glia secrete various cytokines and free radicals, such as superoxide and nitric oxide ŽNO., resulting in cerebral inflammation and subsequently neuronal cell death. Glial activation is initially localized to the region of neuronal death after cerebral ischemia or traumatic brain injures. When AbbreÕiations: ABC, avidin–biotin-peroxidase complex; DMEM, Dulbecco’s modified Eagle’s medium; E, embryonic day; FBS, fetal bovine serum; GFAP, glial fibrillary acidic protein; iNOS, inducible nitric oxide synthase; LDH, lactate dehydrogenase; LPS, lipopolysaccharide; MAP2, microtubule-associated protein 2; NCAM, neural cell adhesion molecules; NO, nitric oxide; PBS, phosphate-buffered saline; TNF-a, tumor necrosis factor-a ) Corresponding author. Fax: q 1-919-541-0841; e-mail: [email protected]
progressive neuronal injury is extended, the loss of glia– neuron interactions provides an opportunity for glia to be further activated, which constitutes the second wave of glial activation. We, therefore, searched for a relationship between the neuron–glia interactions and cerebral inflammation. Studies to test this hypothesis examined whether neurons affect the production of inflammatory factors by activated glia stimulated by bacterial endotoxin lipopolysaccharides ŽLPS., and the mode of influences by neurons. We have previously reported that the presence of neurons led to the inhibition of the expression of glial fibrillary acidic protein ŽGFAP.- and enkephalin-like immunoreactivity in astrocytes w15,16x. In addition, neurons decreased the basal expression of transcription factors ŽFra, c-Jun and JunD. in co-cultures of neurons and glia w20x. Therefore, we hypothesized that neurons reduce the production of inflammatory factors by activated glia. Here, we report that the presence of neurons reduced LPS-stimulated production of nitrite and tumor necrosis factor-a ŽTNF-a . in mixed glial cultures. In addition to these secreted inflammatory factors, the morphological changes of the activated microglia were also reduced in co-cultures of glia
0006-8993r00r$ - see front matter q 2000 Elsevier Science B.V. All rights reserved. PII: S 0 0 0 6 - 8 9 9 3 Ž 9 9 . 0 2 2 5 5 - 6
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and neurons. Furthermore, our results suggested that the influences of neurons on glial inflammatory responses were, at least partly, due to the cell–cell interactions between neurons and glia via neural cell adhesion molecules ŽNCAM..
2. Materials and methods 2.1. Materials LPS Ž Escherichia coli 0111:B4. was purchased from List Biological Laboratories ŽCampbell, CA. or Calbiochem ŽLa Jolla, CA.. It was reconstituted in distilled H 2 O and kept at 48C. Dulbecco’s modified Eagle’s medium with Ham’s nutrient mixtures F12 ŽDMEMrF12., fetal bovine serum ŽFBS., Naq-pyruvate, L-glutamine, non-essential amino acids and a mixture of penicillin and streptomycin were all obtained from GIBCO-BRL ŽGaithersburg, MD.. The NCAM protein contained mainly the transmembrane forms with large and small cytoplasmic domains, and was obtained from Chemicon ŽTemecula, CA.. 2.2. Cell cultures 2.2.1. Glial cultures Primary glial cultures were prepared from postnatal day 1 CD-1 mouse brains. Briefly, the cerebral cortex was taken out aseptically and the meninges were carefully removed. Brains were dissociated by trituration in ice-cold Ca2q- and Mg 2q-free physiological buffer ŽW3 buffer. containing 145 mM NaCl, 5.4 mM KCl, 1 mM NaH 2 PO4 , 15 mM HEPES and 11 mM glucose at pH 7.4. Cell density was adjusted to 1.2 = 10 6 cellsrml and cells were seeded onto 24-well Costar tissue culture plates with an initial plating density of 0.6 = 10 6 cellsrwell. Cells were cultured with DMEMrF12 supplemented with 10% heat-inactivated FBS, 2 mM L-glutamine, 1 mM Naq-pyruvate, 0.1 mM non-essential amino acids, 50 unitsrml and 50 mgrml of a mixture of penicillin and streptomycin and 0.25 mgrml gentamicin. The medium was replenished on the second and fourth days after seeding, and was changed every 3 days thereafter. The cell density reached confluence on day 17, and cells were further cultured for 6 days prior to the treatment. Microglia in mixed glial cultures were identified with a F4r80 antibody ŽSerotec, Raleigh, NC. w6x. In the unstimulated condition, only a certain population of microglia in glial cultures was positively stained by F4r80 antibody. After stimulation with LPS, all microglia can be identified by F4r80 immunoreactivity and by their morphological changes. The staining pattern of F4r80 was similar to that observed with Mac-1 and lectin staining. Astrocytes were identified with a GFAP antibody ŽDAKO, Carpinteria, CA.. About 65% of these cells were GFAP-positive cells and 30% of the cells were
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F4r80-positive cells when the cells were stimulated with LPS, which was similar to our previous studies w8x. 2.2.2. Neuron–glia cultures The influence of neurons on glia was studied in a co-culture system that allowed cell-to-cell contacts between neurons and glia. Co-cultures were obtained by seeding dissociated embryonic day ŽE. 16 cortices Ž0.5 = 10 6 cellsrwell. onto a confluent layer of glia Žday 17 of glial cultures.. With an over 95% confluent layer of glia, neurons would be well differentiated from neuronal precursor cells; otherwise, glia will grow up instead of neurons from the precursor cells. The mixture of neurons and glia was cultured for 6 days with the same culture medium for glia and then treated with LPS. Neurons were identified with a microtubule-associated protein 2 ŽMAP2. antibody ŽBoehringer Mannheim, Indianapolis, IN. and NCAM antibody ŽChemicon.. At the time of the experiment, 15% of all the cells were positive for MAP2 and the rest of the cells were identified as glial cells. 2.2.3. Co-cultures of rat glia and the PC12 cells For the co-cultures of rat glia with the rat PC12 cells, rat glial cells were prepared from the cortex of 1-day-old Fischer 344 rats. The dissection and culture methods for rat glia were the same as those for mice glia. The cell density reached confluence on days 12 to 14. PC12 cells at 0.5 = 10 4 cellsrwell were seeded onto the glial layer. PC12 cells were co-cultured with rat glia for 24 h prior to the treatment. 2.3. Treatment of cell cultures Cultured glia Ž23 days. and neuron–glia co-cultures Ž17 q 6 days. were stimulated with concentrations of LPS from 0.001 to 5 mgrml on the same day. In some of the experiments, cells were treated and samples were collected by different persons. On the day of treatment, the culture medium was totally changed to avoid the possible effect of any secreted factors remaining in the medium. For the experiments using the NCAM, cultured glial cells Ž23 days. were incubated with 1 mgrml of the NCAM for 1 h, and were then stimulated with LPS. Samples from the culture medium were collected at 48 h for the nitrite assay. 2.4. Nitrite assay The production of NO was assessed as nitrite accumulated in the culture medium by using a colorimetric reaction with the Griess reagent w8x. Briefly, samples were collected from 24, 48 and 96 h after stimulation with LPS. An equal volume of the Griess reagent Ž0.1% N-Ž1-naphthyl.ethylenediamine dihydrochloride, 1% sulfanilamide, and 2.5% H 3 PO4 . was used. The absorbance at 540 nm was measured with an UV MAX kinetic microplate reader ŽMolecular Devices, Menlo Park, CA.. The nitrite concen-
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tration was determined from a sodium nitrite standard curve. The sensitivity of this assay was approximately 0.5 mM. 2.5. TNF-a assay The culture medium was collected after 6 h of stimulation with LPS. Based on our and other previous studies, the secretion of TNF-a reached a maximum at 6 h. The quantities of TNF-a were measured with a mouse TNF-a ELISA kit from Genzyme ŽCambridge, MA. w9x, and samples were diluted 50-fold to fit the standard curve. The detection limit for this kit was 15 pgrml. 2.6. Lactate dehydrogenase (LDH) assay Cellular cytotoxicity was assessed by measuring the LDH activity released into the culture medium. Supernatants of the cultures were collected 96 h after stimulation with LPS. LDH activity was measured with a LDH-L diagnostic kit from Sigma ŽSt. Louis, MO., and the kinetic change was examined with an UV MAX kinetic microplate reader. LDH activity was expressed as units per liter, and the results were expressed as a percentage of the control activity. 2.7. Immunocytochemical analysis of the microglial marker F4 r 80
3. Results 3.1. Neurons reduced LPS-induced increase in nitrite production in mixed glial cultures It is well known that bacterial endotoxins can induce inducible nitric oxide synthase ŽiNOS. and, in turn, the production of NO in glia. Fig. 1 shows that different concentrations of LPS stimulated the production of nitrite, a stable metabolite of NO, at 96 h by glia cultured alone or with neurons. Cultured glial cells in the resting state had only low baseline levels of nitrite production ŽFig. 1.. LPS at 1 ngrml stimulated nitrite production to 8.7 " 0.5 mM. LPS increased the production of nitrite by cultured glia in a dose-dependent manner. The addition of neurons did not change the baseline level of nitrite production ŽFig. 1.. Cultured glia–neuron stimulated with LPS at 1 ngrml showed only 5.6 " 0.5 mM nitrite production, which was significantly Ž P - 0.01. less than that of cultured glia. Increasing the concentration of LPS from 1 to 100 ngrml did not significantly increase nitrite production by glia in mixed glia–neuron cultures. The dose–response curve was shifted to the right suggesting that the sensitivity of glia to LPS was decreased in the presence of neurons. Below 1 mgrml of LPS, production of nitrite by glia–neuron cultures was only gradually increased. However, production of nitrite was dramatically stimulated by LPS from 12.2 " 0.5 mM at 1 mgrml of LPS to 23.9 " 1.1 mM at 5 mgrml of LPS. Without
The cells were fixed with a 3.7% freshly made formaldehyde solution ŽFisher Scientific, Fair Lawn, NJ. at 96 h after stimulation with LPS. Following the 20-min fixation, the cells were washed twice with phosphate-buffered saline ŽPBS.. Non-specific staining was blocked by incubating the cells with 4% horse serum for 1 h, and then 0.03% hydrogen peroxide was used to block endogenous peroxidase activity. The microglial marker F4r80 was identified with an anti-mouse F4r80 antibody. The F4r80 antibody was diluted with DAKO antibody diluent with background reducing components ŽDAKO. at 1:20. Afterwards, the cells were incubated with a biotinylated second antibody. The avidin–biotin-peroxidase complex ŽABC, Vector ABC kit; Vector Laboratories, Burlingame, CA. was used, and the color was developed with 3-amino-9-ethylcarbazole ŽAEC kit, Sigma. as the chromogen. 2.8. Statistical analysis The data were expressed as the mean " S.E. from three to six independent experiments. An analysis of variance ŽANOVA. followed by Dunnett’s multiple comparison test was used for statistical comparisons. All statistical analyses were performed according to the instruction for the statistical program Statview w . A value of P - 0.05 was considered to be significant.
Fig. 1. Concentration–response curve of LPS-induced nitrite ŽmM. production by glia with or without neurons. Mixed glia or mixed glia–neuron cultures were stimulated with LPS for 96 h. Samples from the culture medium were collected and the accumulated nitrite was detected with Greiss method Žsee Section 2.. LPS stimulated nitrite production in a concentration-dependent manner. The presence of neurons in the cultures shifted the concentration–response curve to the right. The data are expressed as the mean"S.E. from six independent experiments. Some of the error bars cannot be seen because of their small value. U P - 0.0001, a P - 0.01 vs. the corresponding nitrite produced by mixed glial cultures.
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neurons, LPS at 0.1 mgrml stimulated 12.4 " 0.7 mM nitrite by glia. To reach this level of nitrite production, the concentration of LPS in a mixture of glia and neurons had to be increased by 10-fold ŽFig. 1.. The production of nitrite in cultured glia stimulated by 100 ngrml of LPS was 2-fold higher than that in the mixture of glia and neurons. However, the difference in nitrite production by glial cultures vs. glia–neuron cultures became smaller Ž1.3-fold. when the cells were stimulated by 5 mgrml of LPS. 3.2. Time-course of neuronal inhibition on nitrite production by glia The time-course of nitrite production by glia depended on the concentration of LPS ŽFig. 2a,b.. The production of nitrite was dependent on the induction of iNOS by LPS; therefore, it could not be detected immediately after the
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application of LPS. Nitrite produced by glia was 0.035 mMrh at 0.05 mgrml of LPS from 24 to 48 h. During this period of time, the presence of neurons in cultures decreased nitrite production per hour by about 1.7-fold ŽFig. 2a.. At a low concentration of LPS Ž0.05 mgrml., nitrite production by glia increased to 0.062 mMrh from 48 to 96 h, whereas nitrite production per hour in the mixed neuron–glia cultures still remained the same as that observed from 24 to 48 h. Therefore, nitrite production per hour in the mixed neuron–glia cultures was about 2.5-fold lower than that in the glial cultures. A similar inhibition could also be observed when cells were stimulated with 1 mgrml of LPS ŽFig. 2b.. 3.3. Neurons reduced the morphological changes of actiÕated microglia Activated microglia stimulated with LPS showed an enlarged shape and an intense immunoreactivity for the F4r80 antigen, a marker for murine microglia Žas shown by the arrow in Fig. 3c., whereas untreated microglia in cultures showed a rounded shape ŽFig. 3a.. Most of the microglia in neuron–glia cultures stimulated by LPS did not show an enlarged cell size ŽFig. 3d. similar to their counterpart in resting state ŽFig. 3b.. This indicated that microglial cells were not fully activated by LPS in the presence of neurons. 3.4. Neurons also reduced the secretion of TNF-a by glia The influences of neurons on glial immune responses were not limited to the production of nitrite, but also affected the secretion of TNF-a. The secretion of TNF-a by glia stimulated with LPS reached a maximum at 6 h ŽChen and Chang, unpublished observations.. At this time, 0.01 mgrml of LPS could stimulate the secretion of TNF-a by glia to about 5606 pgrml. The presence of neurons in cultures significantly Ž P - 0.001. reduced the secretion of TNF-a by about 38% compared to glial cultures ŽFig. 4.. When the concentration of LPS was increased to 1 mgrml, neurons significantly Ž P - 0.001. reduced the secretion of TNF-a by about 24%. 3.5. Low concentrations of bacterial endotoxin did not cause cytotoxicity
Fig. 2. Time-course of LPS-induced accumulation of nitrite by glial cultures with or without neurons. Glial cultures or mixed glia–neuron cultures were stimulated with 0.05 or 1 mgrml LPS. Samples from the culture medium were collected at 24, 48 and 96 h for the nitrite assay. The data are expressed as the mean"S.E. from six independent experiments. Some of the error bars cannot be seen because of their small values. U P - 0.0001, aP - 0.05 vs. the corresponding nitrite production by mixed glia–neuron cultures.
To verify that the decreased production of nitrite and TNF-a in glia–neuron co-cultures stimulated by LPS was not due to the cytotoxicity of LPS or the inflammatory factors induced by LPS, we examined the cellular cytotoxicity by measuring the release of LDH into the culture medium. The basal level of LDH release by cells without any treatment was regarded as 100%. Fig. 5 shows that there was no significant increase in the release of LDH in low concentrations of LPS in any type of cell culture. In
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Fig. 3. Immunocytochemical analysis of the F4r80 antigen in microglia. The F4r80 antigen in microglia was identified with a monoclonal anti-F4r80 antibody and is pointed out by arrows. Ža. F4r80-like immunoreactivity showed that untreated microglial cells had a rounded shape in glial, and Žb. neuron–glial co-cultures. Žc. Stimulation with LPS Ž1 mgrml. caused an intense staining for F4r80 and an enlarged cell morphology. Žd. The presence of neurons in cultures markedly inhibited the LPS-induced morphological changes of microglia. This figure is representative of four independent experiments. The original magnification was 200 = .
fact, low concentrations of LPS from 1 ngrml to 1 mgrml decreased LDH release. However, when the concentration of LPS reached 10 mgrml, significant cytotoxicity occurred Ždata not shown.. The results indicated that the decrease of nitrite and TNF-a productions in neuron–glia co-cultures stimulated by LPS was not due to cell injury.
3.6. PC12 cells reduced nitrite production by cultured rat glia In order to make sure that the neuronal cells in the E16 cell cultures were responsible for the inhibitory influence on LPS-induced nitrite production, we have used PC12
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Table 1 Effect of PC12 cells on nitrite production by LPS-stimulated cultured rat glia Cultured rat glial cells were treated with 1 ngrml of LPS for 24 h. Samples were collected from the culture medium and were assayed for nitrite production. PC12 cells were co-cultured with glia for 24 h prior to the treatment with LPS. The results showed that the presence of PC12 cells significantly suppressed nitrite production. The data were obtained from nine wells of one representative experiment out of three independent experiments and were expressed as the mean"S.E. aP - 0.01, U P 0.0001 vs. the corresponding results from glia. Cells
Rat glia Rat gliaqPC12
Fig. 4. The presence of neurons in cultures reduced TNF-a secretion by glia. Mixed glia and glia–neuron cultures were stimulated with different concentrations of LPS for 6 h. Samples were collected from the culture medium and the secretion TNF-a was measured with a commercial assay kit. The TNF-a level was expressed as picomole per milliliter. The data are expressed as the mean"S.E. from three independent experiments. U P - 0.001 and aP - 0.01 vs. the corresponding TNF-a production by glial cultures.
cells that originated from rats. Table 1 shows the effect of PC12 cells on nitrite production by LPS-stimulated rat glia. Glial cells from Fischer 344 rats were very responsive to LPS. Therefore, 1 ngrml of LPS stimulated 20.6 " 0.8 mM nitrite production. The presence of PC12 cells significantly Ž P - 0.0001. reduced nitrite production by about 50%. Combined with the results above, the data from
Nitrite production wmMx Control
LPS w1 ngrmlx
1.3"0.4 4.9"0.9 a
20.6"0.8 10.1"0.7U
PC12 cells further support the proposal that neurons are able to reduce nitrite production by glia. 3.7. Influence of neurons on glial inflammatory responses was partly accomplished by cell–cell contacts Õia NCAM To explore the possible underlying mechanisms for the neuronal influences on glial inflammatory responses, we studied cell–cell interactions via NCAM. Glial cells were incubated with the purified NCAM proteins, which included both extra- and intracellular domains ŽChemicon., and were stimulated with LPS at 0.1 mgrml. The results ŽTable 2. showed that the presence of NCAM significantly Ž P - 0.0001. reduced nitrite production by 33%. These results indicated that cell–cell interactions via NCAM could reduce glial inflammatory factors, and suggested that cell–cell interactions between neurons and glia via NCAM could reduce glial inflammatory responses.
4. Discussion This report suggests that neurons can downregulate the production of inflammatory factors, such as accumulation of nitrite Žan index for NO production. and TNF-a by activated glia. The neurons influenced not only astrocytes, but also microglial cells since morphological changes of activated microglia were also affected. The influences of
Fig. 5. Determination of cytotoxicity with the LDH release method. Mixed glia and glia–neuron cultures were treated with different concentrations of LPS for 96 h. Cytotoxicity was determined by measuring the release of LDH into the culture medium. Samples were collected after 96 h of treatment. The results showed that there was no significant increase of LDH release from cells treated with the above concentrations of LPS. Some of the error bars cannot be seen because of their small value. The data are expressed as the mean"S.E. from six independent experiments.
Table 2 Effect of NCAM on LPS-stimulated nitrite production by cultured mixed-glia Glial cells were incubated with NCAM at 1 mgrml for 1 h, and the cells were then stimulated with 0.1 mgrml of LPS. Samples from the culture medium were taken for nitrite assay 48 h after stimulation with LPS. The results are expressed as the mean"S.E. from three independent experiments. U P - 0.0001 vs. cells with LPS treatment. Nitrite wmMx Control LPS w0.1 mgrmlxqVehicle LPS w0.1 mgrmlxqNCAM w1 mgrmlx NCAM w1 mgrmlx
1.45"0.22 8.17"0.54 5.34"0.47U 0.62"0.25
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neurons on glial inflammation are partly because of the cell–cell interactions via NCAM. 4.1. Mixed-glial cultures and LPS as tools for studying glial inflammatory responses The mixed glial cultures consist of astrocytes, microglia and a few oligodendrocytes interacting with one another. Among the various cell types in the CNS, microglia and astrocytes are the most important for the initiation of cerebral inflammation. Of these two cell types, microglia are regarded as the first line of defense in the CNS. Our and other laboratories have reported w5,8–10x that microglial cells are the major source of cytotoxic factors, such as TNF-a, following stimulation by LPS. However, interactions between these cells are important since astrocytes and microglia can modulate the inflammatory responses of each other. Therefore, studies of cerebral inflammation using a single cell type may not give real pictures because they do not take into account the interactions between microglia and astrocytes. Glial cells showed a typical dose–response curve for nitrite and TNF-a production upon stimulation by LPS, showing that cultures of mixed glial cells are an appropriate tool for studying glial inflammatory responses. This culture model has been used previously to study neuronal influences on glia in our and other laboratories w16,20,26x. LPS has long been used as an inflammatory stimulus for the immune system. LPS stimulates primarily microglia and to a lesser extent astrocytes. Microglia and astrocytes release various cytotoxic factors which initiate inflammatory responses in the CNS. These inflammatory responses resemble the situation in multiple sclerosis, Parkinson’s disease, Alzheimer’s disease, AIDS-related dementia and even cerebral ischemia and traumatic brain injuries wfor a review, see Ref. w23xx. Among the various cytotoxic factors released by glia, excessive amounts of NO and TNF-a are believed to be two of the most important substances contributing to neurodegeneration w17x. 4.2. Neurons reduced glial inflammatory responses LPS stimulated the production of nitrite by glia in a dose-dependent manner ŽFig. 1.. The presence of neurons in the glial cultures shifted the dose–response curve to the right, suggesting that the responsiveness of glia was decreased. While LPS from 1 to 100 ngrml triggered an increase of nitrite production by glia of about 27%, the same concentrations of LPS stimulated nitrite production by glia–neuron co-cultures of only about 12%. In addition, glia retained the plasticity to produce nitrite for a long period of time Žup to 96 h. when they were stimulated with low concentrations of LPS Ž50 ngrml.. In contrast, glia cultured with neurons could barely increase the production of nitrite throughout the time course of the experiment. Perry et al. w21x have observed that there are delayed responses of microglia and infiltration of neutrophils in many neurological disorders. However, it is still not fully
understood how components in the CNS microenvironment can affect glial inflammation. Our data presented here indicate that neurons decrease the production of inflammatory factors by activated glia stimulated by LPS. Besides the production of inflammatory factors, the presence of neurons markedly decreased the morphological changes of microglia ŽFig. 3d., suggesting that not only astrocytes but also microglia were affected by the presence of neurons. For the inhibitory influence of neurons on LPS-induced NO and TNF-a productions by glial cells, we have considered a possibility that there was a reduction of the number of microglia. However, all countings of the number of microglia in glia and glia–neuron cultures were the same in both control and LPS-treated groups ŽFig. 3.. This finding indicates that neurons affected the production of inflammatory factors, but not reduce the number of microglia. The influences of neurons on static functions of glial cells have been studied by several laboratories. The expression of GFAP has been the most commonly studied marker for astrocytes. Rozovsky et al. w25x, along with our laboratory w20x, had demonstrated that the presence of neurons decreased the mRNA for GFAP and DNA-binding activity for the GFAP promoter. Besides GFAP, cortical neurons inhibited basal and interleukin-1-stimulated astroglial cell secretion of nerve growth factor w26x. All these previous studies showed that neurons could actively affect the basal expression of certain genes in glial cells. Our studies extend this notion by showing that neurons could also reduce the production of inflammatory factors in glial cells activated by LPS. Neumann et al. w18x first demonstrated that neurons inhibited IFN-g-induced major histocompatibility complex ŽMHC. class II expression on microglia and astrocytes. The results are consistent with our hypothesis. In their studies, they focused on MHC expression that is most likely related to the activation of lymphocytes. The implication of their study is highly related to diseases such as multiple sclerosis. Our results demonstrated decreased production of NO and cytokines, which are related to most of the acute and chronic neurological disorders, especially cerebral ischemia and traumatic brain injuries. IFN-g does not play an important role in these types of injuries w24x. It should be noted that such neuronal inhibition could antagonize only low concentration of immune stimuli. In the case of high concentrations or potent inflammatory stimuli such as combination of LPS and IFN-g, neuronal modulation on glial inflammatory responses became ineffective and, in turn, the secreted cytotoxic factors would be harmful to neurons and progressively lead to neurodegeneration. Eventually, the inflammatory responses of glia were markedly enhanced and excessive inflammatory factors further damaged neurons in the area adjacent to the injured site. This hypothesis could be supported by reports that showed activation of glial cells after neuronal death caused by scratching the culture plate w15x or by an environmental toxin w27x.
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4.3. The modes of influences on glial inflammation by neurons Several pieces of evidence showed that neurotransmitters might be the factors affecting glial regulatory functions w2–4,13,14x. However, these studies cannot fully explain how glial inflammatory responses are modulated because of two reasons. First, most of the studies utilized high concentrations of neurotransmitters at micro molar levels that exceeded the physiological ranges. Second, neurons can release more than one type of neurotransmitters of that one can eliminate the effects of another. Based on our previous studies of gliosis w15,16,20x, we proposed that cell–cell contacts between neurons and glia play important roles in modulating glial inflammation. In fact, it was reported that astroglial proliferation is inhibited by contact inhibition with neurons w7,11,12x. We speculated that extracellular matrix or adhesion molecules could be useful agents for modulating cerebral inflammation. First, it was shown that a macrophage cell line, RAW 264 cells, preferentially adhered to neurons in an in vitro adhesion assay w1x. This suggests that there are interactions between macrophages and possibly microglia and neurons, and implies that adhesion molecules play important roles in glial inflammation w22x. Second, Krushel et al. w11,12x recently demonstrated that NCAM could inhibit normal astroglial proliferation in vitro and after stab wound injury in vivo via homophilic binding between NCAMs. Our results ŽTable 2. using NCAM proteins demonstrated that NCAM reduced nitrite production by LPS-activated glia. This finding is consistent with our result showing that PC12 cells reduced LPS-stimulated nitrite production by glia ŽTable 1., because it was found that PC12 cells express NCAM w19x. In summary, we report that the presence of neurons in cultures reduced the production of inflammatory products such as NO and TNF-a by LPS-stimulated glia. In addition, the immune modulatory properties of neurons affected not only astrocytes but also microglia. Since cerebral inflammatory responses often occur in many neurological disorders, the present results can, at least partly, explain the roles of glia–neuron interactions in progressive neuronal injury. Further studies along this line of research could pave a new road toward novel therapeutic interventions for cerebral inflammation. Acknowledgements We thank Drs. J.L. Maderdrut, K.R. Pennypacker, P.F. Maness and B. Liu for their critical comments about this manuscript. References w1x H.C. Brown, V.H. Perry, Differential adhesion of macrophages to white and grey matter in an in vitro assay, Glia 23 Ž1998. 361–373.
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w2x E.M. Frohman, T.C. Frohman, B. Vayuvegula, S. Gupta, S. van den Noort, Vasoactive intestinal polypeptide inhibits the expression of the MHC class II antigens on astrocytes, J. Neurol. Sci. 88 Ž1988. 339–346. w3x E.M. Frohman, B. Vayuvegula, S. Gupta, S. van den Noort, Norepinephrine inhibits g interferon-induced major histocompatibility class II ŽIa. antigen expression on cultured astrocytes via b 2 -adrenergic signal transduction mechanisms, Proc. Natl. Acad. Sci. U S A 85 Ž1988. 1292–1296. w4x B.D. Gitter, D. Regoli, J.J. Howbert, A.L. Glasebrook, D.C. Waters, Interleukin-6 secretion from human astrocytoma cells induced by substance P, J. Neuroimmunol. 51 Ž1994. 101–108. w5x D. Giulian, M. Corpuz, S. Chapman, M. Mansouri, C. Robertson, Reactive mononuclear phagocytes release neurotoxins after ischemic and traumatic injury to the central nervous system, J. Neurosci. Res. 36 Ž1993. 681–693. w6x S. Gordon, L. Lawson, S. Rabinowitz, P.R. Crocker, L. Morris, V.H. Perry, Antigen markers of macrophage differentiation in murine tissues, Curr. Topics Microbiol. Immunol. 181 Ž1992. 1–32. w7x M.E. Hatten, Neuronal inhibition of astroglial cell proliferation is membrane-mediated, J. Cell Biol. 104 Ž1987. 1353–1360. w8x L.Y. Kong, M.K. McMillian, R. Maronpot, J.S. Hong, Protein tyrosine kinase inhibitors suppress the production of nitric oxide in mixed glia, microglia-enriched or astrocyte-enriched cultures, Brain Res. 729 Ž1996. 102–109. w9x L.Y. Kong, C. Lai, B.C. Wilson, J.N. Simpson, J.S. Hong, Protein tyrosine kinase inhibitors decrease lipopolysaccharide-induced proinflammatory cytokine production in mixed glia, microglia-enriched or astrocyte-enriched cultures, Neurochem. Int. 30 Ž1997. 491–497. w10x G.W. Kreutzberg, Microglia: a sensor for pathological events in the CNS, Trends Neurosci. 19 Ž1996. 312–318. w11x L.A. Krushel, M.H. Tai, B.A. Cunningham, G.M. Edelman, K.L. Crossin, Neural cell adhesion molecule ŽN-CAM. domains and intracellular signaling pathways involved in the inhibition of astrocyte proliferation, Proc. Natl. Acad. Sci. U S A 95 Ž1998. 2592– 2596. w12x L.A. Krushel, O. Sporns, B.A. Cunningham, K.L. Crossin, G.M. Edelman, Neural cell adhesion molecule ŽN-CAM. inhibits astrocyte proliferation after injury to different regions of the adult rat brain, Proc. Natl. Acad. Sci. U S A 92 Ž1995. 4323–4327. w13x S.C. Lee, M. Collins, P. Vanguri, M.L. Shin, Glutamate differentially inhibits the expression of class II MHC antigens on astrocytes and microglia, J. Immunol. 148 Ž1992. 3391–3397. w14x F.C. Martin, A.C. Charles, M.J. Sanderson, J.E. Merrill, Substance P stimulates IL-1 production by astrocytes via intracellular calcium, Brain Res. 599 Ž1992. 13–18. w15x M.K. McMillian, L. Thai, J.S. Hong, J.P. O’Callaghan, K.R. Pennypacker, Brain injury in a dish: a model for reactive gliosis, Trends Neurosci. 17 Ž1994. 138–142. w16x M.K. McMillian, K.R. Pennypacker, L. Thai, G.C. Wu, H.H. Suh, K.L. Simmons, P.M. Hudson, S.B. Mullis-Sawin, J.S. Hong, Dexamethason and forskolin synergistically increase wMet 5 xenkephalin accumulation in mixed brain cell cultures, Brain Res. 730 Ž1996. 67–74. w17x J.E. Merrill, E.N. Benveniste, Cytokines in inflammatory brain lesions: helpful and harmful, Trends Neurosci. 19 Ž1996. 331–338. w18x H. Neumann, J. Boucraut, C. Hahnel, T. Misgeld, H. Wekerle, Neuronal control of MHC class II inducibility in rat astrocytes and microglia, Eur. J. Neurosci. 8 Ž1996. 2582–2590. w19x T.U. Park, L. Lucka, W. Reutter, R. Horstkorte, Turnover studies of the neural cell adhesion molecule NCAM: degradation of NCAM in PC12 cells depends on the presence of NGF, Biochem. Biophys. Res. Commun. 234 Ž1997. 686–689. w20x K.R. Pennypacker, J.S. Hong, S.B. Mullis, P.M. Hudson, M.K. McMillian, Transcription factors in primary glial cultures: changes with neuronal interactions, Mol. Brain Res. 37 Ž1996. 224–230. w21x V.H. Perry, P.B. Andersson, S. Gordon, Macrophages and inflam-
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mation in the central nervous system, Trends Neurosci. 16 Ž1993. 268–273. w22x V.H. Perry, A revised view of the central nervous system microenvironment and major histocompatibility complex class II antigen presentation, J. Neuroimmunol. 90 Ž1998. 113–121. w23x N.J. Rothwell, Neuroimmune interactions: the role of cytokines, Br. J. Pharmacol. 121 Ž1997. 841–847. w24x M. Rostworowski, V. Balasingam, S. Chabot, T. Owens, V.W. Yong, Astrogliosis in the neonatal and adult murine brain posttrauma: elevation of inflammatory cytokines and the lack of requirement for endogenous interferon-g, J. Neurosci. 17 Ž1997. 3664– 3674.
w25x Rozovsky, N.J. Laping, K. Krohn, B. Teter, J.P. O’Callaghan, C.E Finch, Transcriptional regulation of glial fibrillary acidic protein by corticosterone in rat astrocytes in Õitro is influenced by the duration of time in culture and by astrocyte–neuron interactions, Endocrinology 136 Ž1995. 2066–2073. w26x X. Vige, B. Tang, B.C. Wise, Cortical neurons inhibit basal and interleukin-1-stimulated astroglial cell secretion of nerve growth factor, Brain Res. 591 Ž1992. 345–350. w27x B. Viviani, E. Corsini, C.L. Galli, M. Marinovich, Glia increase degeneration of hippocampal neurons through release of tumor necrosis factor-a, Toxicol. Appl. Pharmacol. 150 Ž1998. 271–276.
Research report
Influence of neurons on lipopolysaccharide-stimulated production of nitric oxide and tumor necrosis factor-a by cultured glia Raymond C.C. Chang, Pearlie Hudson, Belinda Wilson, Lisa Haddon, Jau-Shyong Hong
)
Neuropharmacology Section, MD F1-01, Laboratory of Pharmacology and Chemistry, National Institute of EnÕironmental Health Sciences, National Institutes of Health, P.O. Box 12233, Research Triangle Park, NC 27709, USA Accepted 19 October 1999
Abstract Cerebral inflammation often originates in a region where neuronal death occurs and thereafter slowly spreads outward. This study aimed to elucidate the roles of neurons in modulating the production of inflammatory factors stimulated by the bacterial endotoxin lipopolysaccharide ŽLPS.. Culturing neurons with mixed glia reduced nitrite and tumor necrosis factor-a ŽTNF-a . production compared to cultures with only mixed glia, and shifted the dose–response curve to the right. The decreased nitrite and TNF-a production were not due to the cytotoxicity of LPS. Immunocytochemical analysis of glia–neuron co-cultures revealed the morphological changes in the activated microglia. Culturing PC12 cells with rat mixed-glia also reduced nitrite production. The influence of neurons on glial inflammation was partly due to the cell–cell contacts between neurons and glia via neural cell adhesion molecules ŽNCAM. because NCAM significantly reduced LPS-stimulated nitrite production. These results demonstrate that neurons reduce the production of inflammatory factors by glia. Since cerebral inflammation is important in many neurological disorders, this study might provide insight about the role of glia–neuron interactions in inflammatory responses in the brain. q 2000 Elsevier Science B.V. All rights reserved. Keywords: Lipopolysaccharide; Neural cell adhesion molecule; Neuron–glia interaction; Microglia; Astrocyte; Cerebral inflammation
1. Introduction Inflammation occurs in many types of neurological disorders, such as multiple sclerosis, Parkinson’s disease, Alzheimer’s disease, cerebral ischemia and traumatic brain injuries, and it is one of the factors contributing to neurodegeneration wfor a review, see Ref. w23xx. In stroke and traumatic brain injuries, the primary insult activates glial cells, mainly microglia and astrocytes. The activated glia secrete various cytokines and free radicals, such as superoxide and nitric oxide ŽNO., resulting in cerebral inflammation and subsequently neuronal cell death. Glial activation is initially localized to the region of neuronal death after cerebral ischemia or traumatic brain injures. When AbbreÕiations: ABC, avidin–biotin-peroxidase complex; DMEM, Dulbecco’s modified Eagle’s medium; E, embryonic day; FBS, fetal bovine serum; GFAP, glial fibrillary acidic protein; iNOS, inducible nitric oxide synthase; LDH, lactate dehydrogenase; LPS, lipopolysaccharide; MAP2, microtubule-associated protein 2; NCAM, neural cell adhesion molecules; NO, nitric oxide; PBS, phosphate-buffered saline; TNF-a, tumor necrosis factor-a ) Corresponding author. Fax: q 1-919-541-0841; e-mail: [email protected]
progressive neuronal injury is extended, the loss of glia– neuron interactions provides an opportunity for glia to be further activated, which constitutes the second wave of glial activation. We, therefore, searched for a relationship between the neuron–glia interactions and cerebral inflammation. Studies to test this hypothesis examined whether neurons affect the production of inflammatory factors by activated glia stimulated by bacterial endotoxin lipopolysaccharides ŽLPS., and the mode of influences by neurons. We have previously reported that the presence of neurons led to the inhibition of the expression of glial fibrillary acidic protein ŽGFAP.- and enkephalin-like immunoreactivity in astrocytes w15,16x. In addition, neurons decreased the basal expression of transcription factors ŽFra, c-Jun and JunD. in co-cultures of neurons and glia w20x. Therefore, we hypothesized that neurons reduce the production of inflammatory factors by activated glia. Here, we report that the presence of neurons reduced LPS-stimulated production of nitrite and tumor necrosis factor-a ŽTNF-a . in mixed glial cultures. In addition to these secreted inflammatory factors, the morphological changes of the activated microglia were also reduced in co-cultures of glia
0006-8993r00r$ - see front matter q 2000 Elsevier Science B.V. All rights reserved. PII: S 0 0 0 6 - 8 9 9 3 Ž 9 9 . 0 2 2 5 5 - 6
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and neurons. Furthermore, our results suggested that the influences of neurons on glial inflammatory responses were, at least partly, due to the cell–cell interactions between neurons and glia via neural cell adhesion molecules ŽNCAM..
2. Materials and methods 2.1. Materials LPS Ž Escherichia coli 0111:B4. was purchased from List Biological Laboratories ŽCampbell, CA. or Calbiochem ŽLa Jolla, CA.. It was reconstituted in distilled H 2 O and kept at 48C. Dulbecco’s modified Eagle’s medium with Ham’s nutrient mixtures F12 ŽDMEMrF12., fetal bovine serum ŽFBS., Naq-pyruvate, L-glutamine, non-essential amino acids and a mixture of penicillin and streptomycin were all obtained from GIBCO-BRL ŽGaithersburg, MD.. The NCAM protein contained mainly the transmembrane forms with large and small cytoplasmic domains, and was obtained from Chemicon ŽTemecula, CA.. 2.2. Cell cultures 2.2.1. Glial cultures Primary glial cultures were prepared from postnatal day 1 CD-1 mouse brains. Briefly, the cerebral cortex was taken out aseptically and the meninges were carefully removed. Brains were dissociated by trituration in ice-cold Ca2q- and Mg 2q-free physiological buffer ŽW3 buffer. containing 145 mM NaCl, 5.4 mM KCl, 1 mM NaH 2 PO4 , 15 mM HEPES and 11 mM glucose at pH 7.4. Cell density was adjusted to 1.2 = 10 6 cellsrml and cells were seeded onto 24-well Costar tissue culture plates with an initial plating density of 0.6 = 10 6 cellsrwell. Cells were cultured with DMEMrF12 supplemented with 10% heat-inactivated FBS, 2 mM L-glutamine, 1 mM Naq-pyruvate, 0.1 mM non-essential amino acids, 50 unitsrml and 50 mgrml of a mixture of penicillin and streptomycin and 0.25 mgrml gentamicin. The medium was replenished on the second and fourth days after seeding, and was changed every 3 days thereafter. The cell density reached confluence on day 17, and cells were further cultured for 6 days prior to the treatment. Microglia in mixed glial cultures were identified with a F4r80 antibody ŽSerotec, Raleigh, NC. w6x. In the unstimulated condition, only a certain population of microglia in glial cultures was positively stained by F4r80 antibody. After stimulation with LPS, all microglia can be identified by F4r80 immunoreactivity and by their morphological changes. The staining pattern of F4r80 was similar to that observed with Mac-1 and lectin staining. Astrocytes were identified with a GFAP antibody ŽDAKO, Carpinteria, CA.. About 65% of these cells were GFAP-positive cells and 30% of the cells were
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F4r80-positive cells when the cells were stimulated with LPS, which was similar to our previous studies w8x. 2.2.2. Neuron–glia cultures The influence of neurons on glia was studied in a co-culture system that allowed cell-to-cell contacts between neurons and glia. Co-cultures were obtained by seeding dissociated embryonic day ŽE. 16 cortices Ž0.5 = 10 6 cellsrwell. onto a confluent layer of glia Žday 17 of glial cultures.. With an over 95% confluent layer of glia, neurons would be well differentiated from neuronal precursor cells; otherwise, glia will grow up instead of neurons from the precursor cells. The mixture of neurons and glia was cultured for 6 days with the same culture medium for glia and then treated with LPS. Neurons were identified with a microtubule-associated protein 2 ŽMAP2. antibody ŽBoehringer Mannheim, Indianapolis, IN. and NCAM antibody ŽChemicon.. At the time of the experiment, 15% of all the cells were positive for MAP2 and the rest of the cells were identified as glial cells. 2.2.3. Co-cultures of rat glia and the PC12 cells For the co-cultures of rat glia with the rat PC12 cells, rat glial cells were prepared from the cortex of 1-day-old Fischer 344 rats. The dissection and culture methods for rat glia were the same as those for mice glia. The cell density reached confluence on days 12 to 14. PC12 cells at 0.5 = 10 4 cellsrwell were seeded onto the glial layer. PC12 cells were co-cultured with rat glia for 24 h prior to the treatment. 2.3. Treatment of cell cultures Cultured glia Ž23 days. and neuron–glia co-cultures Ž17 q 6 days. were stimulated with concentrations of LPS from 0.001 to 5 mgrml on the same day. In some of the experiments, cells were treated and samples were collected by different persons. On the day of treatment, the culture medium was totally changed to avoid the possible effect of any secreted factors remaining in the medium. For the experiments using the NCAM, cultured glial cells Ž23 days. were incubated with 1 mgrml of the NCAM for 1 h, and were then stimulated with LPS. Samples from the culture medium were collected at 48 h for the nitrite assay. 2.4. Nitrite assay The production of NO was assessed as nitrite accumulated in the culture medium by using a colorimetric reaction with the Griess reagent w8x. Briefly, samples were collected from 24, 48 and 96 h after stimulation with LPS. An equal volume of the Griess reagent Ž0.1% N-Ž1-naphthyl.ethylenediamine dihydrochloride, 1% sulfanilamide, and 2.5% H 3 PO4 . was used. The absorbance at 540 nm was measured with an UV MAX kinetic microplate reader ŽMolecular Devices, Menlo Park, CA.. The nitrite concen-
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tration was determined from a sodium nitrite standard curve. The sensitivity of this assay was approximately 0.5 mM. 2.5. TNF-a assay The culture medium was collected after 6 h of stimulation with LPS. Based on our and other previous studies, the secretion of TNF-a reached a maximum at 6 h. The quantities of TNF-a were measured with a mouse TNF-a ELISA kit from Genzyme ŽCambridge, MA. w9x, and samples were diluted 50-fold to fit the standard curve. The detection limit for this kit was 15 pgrml. 2.6. Lactate dehydrogenase (LDH) assay Cellular cytotoxicity was assessed by measuring the LDH activity released into the culture medium. Supernatants of the cultures were collected 96 h after stimulation with LPS. LDH activity was measured with a LDH-L diagnostic kit from Sigma ŽSt. Louis, MO., and the kinetic change was examined with an UV MAX kinetic microplate reader. LDH activity was expressed as units per liter, and the results were expressed as a percentage of the control activity. 2.7. Immunocytochemical analysis of the microglial marker F4 r 80
3. Results 3.1. Neurons reduced LPS-induced increase in nitrite production in mixed glial cultures It is well known that bacterial endotoxins can induce inducible nitric oxide synthase ŽiNOS. and, in turn, the production of NO in glia. Fig. 1 shows that different concentrations of LPS stimulated the production of nitrite, a stable metabolite of NO, at 96 h by glia cultured alone or with neurons. Cultured glial cells in the resting state had only low baseline levels of nitrite production ŽFig. 1.. LPS at 1 ngrml stimulated nitrite production to 8.7 " 0.5 mM. LPS increased the production of nitrite by cultured glia in a dose-dependent manner. The addition of neurons did not change the baseline level of nitrite production ŽFig. 1.. Cultured glia–neuron stimulated with LPS at 1 ngrml showed only 5.6 " 0.5 mM nitrite production, which was significantly Ž P - 0.01. less than that of cultured glia. Increasing the concentration of LPS from 1 to 100 ngrml did not significantly increase nitrite production by glia in mixed glia–neuron cultures. The dose–response curve was shifted to the right suggesting that the sensitivity of glia to LPS was decreased in the presence of neurons. Below 1 mgrml of LPS, production of nitrite by glia–neuron cultures was only gradually increased. However, production of nitrite was dramatically stimulated by LPS from 12.2 " 0.5 mM at 1 mgrml of LPS to 23.9 " 1.1 mM at 5 mgrml of LPS. Without
The cells were fixed with a 3.7% freshly made formaldehyde solution ŽFisher Scientific, Fair Lawn, NJ. at 96 h after stimulation with LPS. Following the 20-min fixation, the cells were washed twice with phosphate-buffered saline ŽPBS.. Non-specific staining was blocked by incubating the cells with 4% horse serum for 1 h, and then 0.03% hydrogen peroxide was used to block endogenous peroxidase activity. The microglial marker F4r80 was identified with an anti-mouse F4r80 antibody. The F4r80 antibody was diluted with DAKO antibody diluent with background reducing components ŽDAKO. at 1:20. Afterwards, the cells were incubated with a biotinylated second antibody. The avidin–biotin-peroxidase complex ŽABC, Vector ABC kit; Vector Laboratories, Burlingame, CA. was used, and the color was developed with 3-amino-9-ethylcarbazole ŽAEC kit, Sigma. as the chromogen. 2.8. Statistical analysis The data were expressed as the mean " S.E. from three to six independent experiments. An analysis of variance ŽANOVA. followed by Dunnett’s multiple comparison test was used for statistical comparisons. All statistical analyses were performed according to the instruction for the statistical program Statview w . A value of P - 0.05 was considered to be significant.
Fig. 1. Concentration–response curve of LPS-induced nitrite ŽmM. production by glia with or without neurons. Mixed glia or mixed glia–neuron cultures were stimulated with LPS for 96 h. Samples from the culture medium were collected and the accumulated nitrite was detected with Greiss method Žsee Section 2.. LPS stimulated nitrite production in a concentration-dependent manner. The presence of neurons in the cultures shifted the concentration–response curve to the right. The data are expressed as the mean"S.E. from six independent experiments. Some of the error bars cannot be seen because of their small value. U P - 0.0001, a P - 0.01 vs. the corresponding nitrite produced by mixed glial cultures.
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neurons, LPS at 0.1 mgrml stimulated 12.4 " 0.7 mM nitrite by glia. To reach this level of nitrite production, the concentration of LPS in a mixture of glia and neurons had to be increased by 10-fold ŽFig. 1.. The production of nitrite in cultured glia stimulated by 100 ngrml of LPS was 2-fold higher than that in the mixture of glia and neurons. However, the difference in nitrite production by glial cultures vs. glia–neuron cultures became smaller Ž1.3-fold. when the cells were stimulated by 5 mgrml of LPS. 3.2. Time-course of neuronal inhibition on nitrite production by glia The time-course of nitrite production by glia depended on the concentration of LPS ŽFig. 2a,b.. The production of nitrite was dependent on the induction of iNOS by LPS; therefore, it could not be detected immediately after the
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application of LPS. Nitrite produced by glia was 0.035 mMrh at 0.05 mgrml of LPS from 24 to 48 h. During this period of time, the presence of neurons in cultures decreased nitrite production per hour by about 1.7-fold ŽFig. 2a.. At a low concentration of LPS Ž0.05 mgrml., nitrite production by glia increased to 0.062 mMrh from 48 to 96 h, whereas nitrite production per hour in the mixed neuron–glia cultures still remained the same as that observed from 24 to 48 h. Therefore, nitrite production per hour in the mixed neuron–glia cultures was about 2.5-fold lower than that in the glial cultures. A similar inhibition could also be observed when cells were stimulated with 1 mgrml of LPS ŽFig. 2b.. 3.3. Neurons reduced the morphological changes of actiÕated microglia Activated microglia stimulated with LPS showed an enlarged shape and an intense immunoreactivity for the F4r80 antigen, a marker for murine microglia Žas shown by the arrow in Fig. 3c., whereas untreated microglia in cultures showed a rounded shape ŽFig. 3a.. Most of the microglia in neuron–glia cultures stimulated by LPS did not show an enlarged cell size ŽFig. 3d. similar to their counterpart in resting state ŽFig. 3b.. This indicated that microglial cells were not fully activated by LPS in the presence of neurons. 3.4. Neurons also reduced the secretion of TNF-a by glia The influences of neurons on glial immune responses were not limited to the production of nitrite, but also affected the secretion of TNF-a. The secretion of TNF-a by glia stimulated with LPS reached a maximum at 6 h ŽChen and Chang, unpublished observations.. At this time, 0.01 mgrml of LPS could stimulate the secretion of TNF-a by glia to about 5606 pgrml. The presence of neurons in cultures significantly Ž P - 0.001. reduced the secretion of TNF-a by about 38% compared to glial cultures ŽFig. 4.. When the concentration of LPS was increased to 1 mgrml, neurons significantly Ž P - 0.001. reduced the secretion of TNF-a by about 24%. 3.5. Low concentrations of bacterial endotoxin did not cause cytotoxicity
Fig. 2. Time-course of LPS-induced accumulation of nitrite by glial cultures with or without neurons. Glial cultures or mixed glia–neuron cultures were stimulated with 0.05 or 1 mgrml LPS. Samples from the culture medium were collected at 24, 48 and 96 h for the nitrite assay. The data are expressed as the mean"S.E. from six independent experiments. Some of the error bars cannot be seen because of their small values. U P - 0.0001, aP - 0.05 vs. the corresponding nitrite production by mixed glia–neuron cultures.
To verify that the decreased production of nitrite and TNF-a in glia–neuron co-cultures stimulated by LPS was not due to the cytotoxicity of LPS or the inflammatory factors induced by LPS, we examined the cellular cytotoxicity by measuring the release of LDH into the culture medium. The basal level of LDH release by cells without any treatment was regarded as 100%. Fig. 5 shows that there was no significant increase in the release of LDH in low concentrations of LPS in any type of cell culture. In
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Fig. 3. Immunocytochemical analysis of the F4r80 antigen in microglia. The F4r80 antigen in microglia was identified with a monoclonal anti-F4r80 antibody and is pointed out by arrows. Ža. F4r80-like immunoreactivity showed that untreated microglial cells had a rounded shape in glial, and Žb. neuron–glial co-cultures. Žc. Stimulation with LPS Ž1 mgrml. caused an intense staining for F4r80 and an enlarged cell morphology. Žd. The presence of neurons in cultures markedly inhibited the LPS-induced morphological changes of microglia. This figure is representative of four independent experiments. The original magnification was 200 = .
fact, low concentrations of LPS from 1 ngrml to 1 mgrml decreased LDH release. However, when the concentration of LPS reached 10 mgrml, significant cytotoxicity occurred Ždata not shown.. The results indicated that the decrease of nitrite and TNF-a productions in neuron–glia co-cultures stimulated by LPS was not due to cell injury.
3.6. PC12 cells reduced nitrite production by cultured rat glia In order to make sure that the neuronal cells in the E16 cell cultures were responsible for the inhibitory influence on LPS-induced nitrite production, we have used PC12
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Table 1 Effect of PC12 cells on nitrite production by LPS-stimulated cultured rat glia Cultured rat glial cells were treated with 1 ngrml of LPS for 24 h. Samples were collected from the culture medium and were assayed for nitrite production. PC12 cells were co-cultured with glia for 24 h prior to the treatment with LPS. The results showed that the presence of PC12 cells significantly suppressed nitrite production. The data were obtained from nine wells of one representative experiment out of three independent experiments and were expressed as the mean"S.E. aP - 0.01, U P 0.0001 vs. the corresponding results from glia. Cells
Rat glia Rat gliaqPC12
Fig. 4. The presence of neurons in cultures reduced TNF-a secretion by glia. Mixed glia and glia–neuron cultures were stimulated with different concentrations of LPS for 6 h. Samples were collected from the culture medium and the secretion TNF-a was measured with a commercial assay kit. The TNF-a level was expressed as picomole per milliliter. The data are expressed as the mean"S.E. from three independent experiments. U P - 0.001 and aP - 0.01 vs. the corresponding TNF-a production by glial cultures.
cells that originated from rats. Table 1 shows the effect of PC12 cells on nitrite production by LPS-stimulated rat glia. Glial cells from Fischer 344 rats were very responsive to LPS. Therefore, 1 ngrml of LPS stimulated 20.6 " 0.8 mM nitrite production. The presence of PC12 cells significantly Ž P - 0.0001. reduced nitrite production by about 50%. Combined with the results above, the data from
Nitrite production wmMx Control
LPS w1 ngrmlx
1.3"0.4 4.9"0.9 a
20.6"0.8 10.1"0.7U
PC12 cells further support the proposal that neurons are able to reduce nitrite production by glia. 3.7. Influence of neurons on glial inflammatory responses was partly accomplished by cell–cell contacts Õia NCAM To explore the possible underlying mechanisms for the neuronal influences on glial inflammatory responses, we studied cell–cell interactions via NCAM. Glial cells were incubated with the purified NCAM proteins, which included both extra- and intracellular domains ŽChemicon., and were stimulated with LPS at 0.1 mgrml. The results ŽTable 2. showed that the presence of NCAM significantly Ž P - 0.0001. reduced nitrite production by 33%. These results indicated that cell–cell interactions via NCAM could reduce glial inflammatory factors, and suggested that cell–cell interactions between neurons and glia via NCAM could reduce glial inflammatory responses.
4. Discussion This report suggests that neurons can downregulate the production of inflammatory factors, such as accumulation of nitrite Žan index for NO production. and TNF-a by activated glia. The neurons influenced not only astrocytes, but also microglial cells since morphological changes of activated microglia were also affected. The influences of
Fig. 5. Determination of cytotoxicity with the LDH release method. Mixed glia and glia–neuron cultures were treated with different concentrations of LPS for 96 h. Cytotoxicity was determined by measuring the release of LDH into the culture medium. Samples were collected after 96 h of treatment. The results showed that there was no significant increase of LDH release from cells treated with the above concentrations of LPS. Some of the error bars cannot be seen because of their small value. The data are expressed as the mean"S.E. from six independent experiments.
Table 2 Effect of NCAM on LPS-stimulated nitrite production by cultured mixed-glia Glial cells were incubated with NCAM at 1 mgrml for 1 h, and the cells were then stimulated with 0.1 mgrml of LPS. Samples from the culture medium were taken for nitrite assay 48 h after stimulation with LPS. The results are expressed as the mean"S.E. from three independent experiments. U P - 0.0001 vs. cells with LPS treatment. Nitrite wmMx Control LPS w0.1 mgrmlxqVehicle LPS w0.1 mgrmlxqNCAM w1 mgrmlx NCAM w1 mgrmlx
1.45"0.22 8.17"0.54 5.34"0.47U 0.62"0.25
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neurons on glial inflammation are partly because of the cell–cell interactions via NCAM. 4.1. Mixed-glial cultures and LPS as tools for studying glial inflammatory responses The mixed glial cultures consist of astrocytes, microglia and a few oligodendrocytes interacting with one another. Among the various cell types in the CNS, microglia and astrocytes are the most important for the initiation of cerebral inflammation. Of these two cell types, microglia are regarded as the first line of defense in the CNS. Our and other laboratories have reported w5,8–10x that microglial cells are the major source of cytotoxic factors, such as TNF-a, following stimulation by LPS. However, interactions between these cells are important since astrocytes and microglia can modulate the inflammatory responses of each other. Therefore, studies of cerebral inflammation using a single cell type may not give real pictures because they do not take into account the interactions between microglia and astrocytes. Glial cells showed a typical dose–response curve for nitrite and TNF-a production upon stimulation by LPS, showing that cultures of mixed glial cells are an appropriate tool for studying glial inflammatory responses. This culture model has been used previously to study neuronal influences on glia in our and other laboratories w16,20,26x. LPS has long been used as an inflammatory stimulus for the immune system. LPS stimulates primarily microglia and to a lesser extent astrocytes. Microglia and astrocytes release various cytotoxic factors which initiate inflammatory responses in the CNS. These inflammatory responses resemble the situation in multiple sclerosis, Parkinson’s disease, Alzheimer’s disease, AIDS-related dementia and even cerebral ischemia and traumatic brain injuries wfor a review, see Ref. w23xx. Among the various cytotoxic factors released by glia, excessive amounts of NO and TNF-a are believed to be two of the most important substances contributing to neurodegeneration w17x. 4.2. Neurons reduced glial inflammatory responses LPS stimulated the production of nitrite by glia in a dose-dependent manner ŽFig. 1.. The presence of neurons in the glial cultures shifted the dose–response curve to the right, suggesting that the responsiveness of glia was decreased. While LPS from 1 to 100 ngrml triggered an increase of nitrite production by glia of about 27%, the same concentrations of LPS stimulated nitrite production by glia–neuron co-cultures of only about 12%. In addition, glia retained the plasticity to produce nitrite for a long period of time Žup to 96 h. when they were stimulated with low concentrations of LPS Ž50 ngrml.. In contrast, glia cultured with neurons could barely increase the production of nitrite throughout the time course of the experiment. Perry et al. w21x have observed that there are delayed responses of microglia and infiltration of neutrophils in many neurological disorders. However, it is still not fully
understood how components in the CNS microenvironment can affect glial inflammation. Our data presented here indicate that neurons decrease the production of inflammatory factors by activated glia stimulated by LPS. Besides the production of inflammatory factors, the presence of neurons markedly decreased the morphological changes of microglia ŽFig. 3d., suggesting that not only astrocytes but also microglia were affected by the presence of neurons. For the inhibitory influence of neurons on LPS-induced NO and TNF-a productions by glial cells, we have considered a possibility that there was a reduction of the number of microglia. However, all countings of the number of microglia in glia and glia–neuron cultures were the same in both control and LPS-treated groups ŽFig. 3.. This finding indicates that neurons affected the production of inflammatory factors, but not reduce the number of microglia. The influences of neurons on static functions of glial cells have been studied by several laboratories. The expression of GFAP has been the most commonly studied marker for astrocytes. Rozovsky et al. w25x, along with our laboratory w20x, had demonstrated that the presence of neurons decreased the mRNA for GFAP and DNA-binding activity for the GFAP promoter. Besides GFAP, cortical neurons inhibited basal and interleukin-1-stimulated astroglial cell secretion of nerve growth factor w26x. All these previous studies showed that neurons could actively affect the basal expression of certain genes in glial cells. Our studies extend this notion by showing that neurons could also reduce the production of inflammatory factors in glial cells activated by LPS. Neumann et al. w18x first demonstrated that neurons inhibited IFN-g-induced major histocompatibility complex ŽMHC. class II expression on microglia and astrocytes. The results are consistent with our hypothesis. In their studies, they focused on MHC expression that is most likely related to the activation of lymphocytes. The implication of their study is highly related to diseases such as multiple sclerosis. Our results demonstrated decreased production of NO and cytokines, which are related to most of the acute and chronic neurological disorders, especially cerebral ischemia and traumatic brain injuries. IFN-g does not play an important role in these types of injuries w24x. It should be noted that such neuronal inhibition could antagonize only low concentration of immune stimuli. In the case of high concentrations or potent inflammatory stimuli such as combination of LPS and IFN-g, neuronal modulation on glial inflammatory responses became ineffective and, in turn, the secreted cytotoxic factors would be harmful to neurons and progressively lead to neurodegeneration. Eventually, the inflammatory responses of glia were markedly enhanced and excessive inflammatory factors further damaged neurons in the area adjacent to the injured site. This hypothesis could be supported by reports that showed activation of glial cells after neuronal death caused by scratching the culture plate w15x or by an environmental toxin w27x.
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4.3. The modes of influences on glial inflammation by neurons Several pieces of evidence showed that neurotransmitters might be the factors affecting glial regulatory functions w2–4,13,14x. However, these studies cannot fully explain how glial inflammatory responses are modulated because of two reasons. First, most of the studies utilized high concentrations of neurotransmitters at micro molar levels that exceeded the physiological ranges. Second, neurons can release more than one type of neurotransmitters of that one can eliminate the effects of another. Based on our previous studies of gliosis w15,16,20x, we proposed that cell–cell contacts between neurons and glia play important roles in modulating glial inflammation. In fact, it was reported that astroglial proliferation is inhibited by contact inhibition with neurons w7,11,12x. We speculated that extracellular matrix or adhesion molecules could be useful agents for modulating cerebral inflammation. First, it was shown that a macrophage cell line, RAW 264 cells, preferentially adhered to neurons in an in vitro adhesion assay w1x. This suggests that there are interactions between macrophages and possibly microglia and neurons, and implies that adhesion molecules play important roles in glial inflammation w22x. Second, Krushel et al. w11,12x recently demonstrated that NCAM could inhibit normal astroglial proliferation in vitro and after stab wound injury in vivo via homophilic binding between NCAMs. Our results ŽTable 2. using NCAM proteins demonstrated that NCAM reduced nitrite production by LPS-activated glia. This finding is consistent with our result showing that PC12 cells reduced LPS-stimulated nitrite production by glia ŽTable 1., because it was found that PC12 cells express NCAM w19x. In summary, we report that the presence of neurons in cultures reduced the production of inflammatory products such as NO and TNF-a by LPS-stimulated glia. In addition, the immune modulatory properties of neurons affected not only astrocytes but also microglia. Since cerebral inflammatory responses often occur in many neurological disorders, the present results can, at least partly, explain the roles of glia–neuron interactions in progressive neuronal injury. Further studies along this line of research could pave a new road toward novel therapeutic interventions for cerebral inflammation. Acknowledgements We thank Drs. J.L. Maderdrut, K.R. Pennypacker, P.F. Maness and B. Liu for their critical comments about this manuscript. References w1x H.C. Brown, V.H. Perry, Differential adhesion of macrophages to white and grey matter in an in vitro assay, Glia 23 Ž1998. 361–373.
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