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    View of The Suitability of BV2 Cells As Alternative Model System For Primary Microglia Cultures or For Animal Experiments Examining Brain Inflammation

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    & The Suitability of BV2 Cells as Alternative Model System for Primary Microglia Cultures or for Animal Experiments Examining Brain Inflammation Anja Henn', Soren Lund?, Maj Hedtjiirn’, André Schrattenholz*, Peter Pérzgen* and Marcel Leist! *Doerenkamp-Zbinden Chair for alternative in vitro methods to replace animal experiments, University of Konstanz, ‘onstanz, Germany; ‘Novo Nordisk A/S, Bagsverd, Denmark; *Santaris Pharma A/S, Horsholm, Denmark; “ProteoSys AG, Mainz, Germany; ‘Hawaii Pacific University, Kaneohe, USA ‘Summary The role of microglia tn neurodegeneratton, toxicology and immunity is an expanding area of biomedical research requiring large numbers of animals. Use af a mieroglia-like cell line would accelerate mary research programmes and reduce the necessity of continuous cell preparations and animal experimentation, provided that the cell line reproduces the in vivo situation or primary microglia (PM) with high fidelity The immortalised murine microglial cell line BV-2 has been used frequenaly as a substlue for PM, ‘bur recenily doubts were raised as to their suitability Here, we re-evaluated strengths and potential short comings of BV-2 cells. Their response 10 lipopolysaccharide was compared with the response of microglia in vitro and in viva. Transeriprome (480 genes) and proteome analyses after stimulation with ipopolysaccharide indicated a reaction pattern of BV-2 with many similarities to that of PM, although the average upregulation of genes was less pronounced. The cells showed a normal regulation of NO production and a functional response to IFN-y, important parameters for appropriate interaction with Teells and neurons. BV-2 were also able 1o sitmulaze other glial cells. They triggered the translocation of NF-xB. and.a subsequent production of IL-6 in astrocytes. Thus, BV-2 cells appear to be a valid substitute ‘for PM in many experimental serings, including complex cell-cell imeraction studies. Keywords: microglia, inflammation, BV.2, transcriptome, replacement 1 Introduetion Microglia are the resident macrophage-like cells of the central nervous system (CNS) with a broad role in the brain's innate immunity and in inflammatory neuropathologies (Nelson et al., 2002). They have been examined in hundreds of studies in animal models and in primary cultures. As their proliferation ‘capacity is limited, they have to be isolate freshly for each ex- ppetiment. For a typical preparation of rodent microglia, 15-20 brains are required to vield cells fora limited amount of experi- ‘ments on signalling or disease mechanisms. For biochemical ‘work or chip expression analysis, considerably higher numbers are requited. This fas a large impact on overall animal coa- sumption in biomedical research. A cel line alternative would be highly desirable, as it would save animals, time and valuable consumables, and facilitate research work. ‘Microglia show great functional plasticity when activated ‘They are equipped with a broad range of pattera-recognition receptors of the tol-ike receptor family (TLR family) to detect microbial intruders and brain damage (Lee and Lee, 2002; Fal- sig etal, 2008). Most work on microglial activation and signal. ling has been pesformed in vitro, fzequently by using cell ines such as NO (Conadia et al, 1993) and BV.2 Blasi et al, 1990). Data on primary microglia (PMD), isolated from brain cultures of azonatal pups (Giulian and Baker, 1986) or directly from adult rice (Baker etal, 2002) are more restricted, dive to the limited yield of biological matesial. In addition, some ex vivo studies have been performed with freshly isolated microalia from dis- cased bins (Baker etal, 2002). BY.2 cells were derived from rafimyc-immortalised murine aeonatal microglia and are the most frequently used substitute for peimary microslia. They have been used e.e. for pharma- colosical studies (Lund et al., 2005), sties of phagocytosis (Hirt and Leist, 2003) and for many important immunological discoveries in altogether at least 200 publications. With respect to neurodegeneration studies, itis important that BV’2, similar Feceies 15 Jarusry 2009, rosived a evee fxm and accete for pubostion 20% Fetraary 2000, ALTER 25,208 83 HENNE AL to primary microglia, express functional NADPH oxidase, an ‘enzyme frequently implicated in microgliatriggered neuronal ‘one, caspr, C486 antigen ed86, CCI2, CCIZ, CXCLS, ADCT, CCR, CCAL2, CCR2, CSF2, Endotholin 1, IL12Ab2, L-ra,1L23a, IL1, IRAK3, NGF, OSM, OSM-A, C3, CAT2, GBP, IFND, 1g, INOS, MSAI, Myeloperoxidase, NFKB-P4gip100, SODI, SOD2, TLAY, H-21 {one for class 1 MHC qlycoprotain, pBR, Proteasome SU2#-beta, PSM ave Pan Cytokines / Chemokines a | te) ah ih hemoxino igand3, MIP alpha, a8 és | 26 | ma | 02 Cherie igand 4 MIP-Tbea, Co a EE intaiouin bot 15 ero [08 Immune receptors and associated genes inter-Collular Adhesion Molecule 1, CANT ((Kappa)B(alpha), NFRB\a TNEKBI-P50/105 Tolrike receptor 2 TLR2 ‘Glycoprotein 49a, gpa (Ga20ee antigen ‘Suppress or of Gjtokine signaling 8 SOOSS et aeain Sel eukeria 2 relate pete ala Bata 3 oo [as Caspase8 52 | 25 | 16s | 56 CesnseesasneseTi za_[ se} a0 [49 ‘Stass rated ganos CGosatlenhancer bndng protein - data 2a 25 Thymidylate kinase, Ips-inducible mombor, TOK! 20 | 452 interforon-ne. prot_with teaicopephde rep. 1, it 232 inleraconand. prot_wit etralioapaphde rep iS 252 interferon regulatory facor 1, IT 8 ‘Serum amylond a2, SAAD 4a ‘Serum amyloid a3, SAAS 37 86 ALTER 26.208 & the BV-2 response was weaker and narrower than the response of PM. Only 17% of the genes detected to be significantly regulated in PM were also detected in this analysis in BV-2 (Fig. 1A). One theoretical explanation for the limited number of gene inductions may be a high basal activation state of BV-2 cells. However, the levels of inflammatory mediators (TNF-«, IL-1 and NO) in non-stimulated cultures were always under the deteetion limit of conventional detection methods, and were «greatly enhanced upon LPS stimulation. On that basis, a high basal activation state of BV-2 cells was ruled out. This is also in agreement with data from earlier work, where we found solid activation of inflammation related kinases (INK and p-38) as well as transcription factors (¢-JUN and NF-xB) when resting cells (primary or BV-2) were stimulated with LPS (Lund etal 2008). Another explanation could be thatthe rich inflammatory profile observed for primary microglia results from transcripts originating from contaminating cells in the cultures. However, microglial cultures are routinely established in our and other labs with high purity. We characterised our eultures extensively ‘with biochemical and immunocytochemical methods, and con- taminations with neurons, astrooytes or endothelial cells were overlap n=20. Fig. 1: Overiap of genes regulated by LPS in PM and in BV-2 ‘Mico (Le injection), primary microgiia (PM) or BV.2 calle wero stimulated with LPS. (A) The overiap is indicated in percent of ‘ogulatod ganes on the Neurotiame chip when comparing BV-2 to PM or vico versa (8) Comparison of hipeocameal microgiial ‘gone regulations (in vive) with BV-2. Genes ware considered ‘ogulated (n) when they excoedod 21 8 fold average induction in their respective systams at least at one time point. The percentage ‘numbers with righi-pointing atrow Indicate how many percent of the genes ta the left are algo regulatad inthe system on the ight “The percentage numbers with lel-ointing arrow indicate how ‘many percent of the ganes to th right ae also regulated in the system on the lft ALTER 25,208 HENNET AL below 5%, Furthermore, the broad transcriptional pattern iden- tified here is relatively similar to that of LPS-stimulated mac- rophages (Rosenberger et al., 2000). Microglia are believed to adopt many features of macrophages when stimulated (Kappler et al, 1997; Qin et al., 2005), which makes this overlap in re- sponse pattern very plausible 3.2 In vivo transcriptional response of microglia compared to the response of LPS-stimulated BV-2 ‘The mostrelevant comparison for the fidelity ofthe BV-2 system {snot the neonatal PM culture, but the real reaction of microglia invivo. We made use of a unique set of well-characterised data ‘on in vivo activation of microglia (Lund et al. 2006) for this, comparison. [..v. injection of LPS induced a robust expression of several inflammatory gene clusters also identified tn viro Notably, the temporal dynamics of the cerebral inflammation was distinct from that observed in vio. The response peaked after dh and then retumed almost to baseline after 16 , while a continuous up-regulation was observed for primary microslia in vio (Tab. 2). stiking correlation was found for the tran- scripts induced by LPS in virro and in vivo (after 4 h) for some iamilies of inflammatory mediators. For instance, there was a ‘very high correlation for the class of interferon regulated pro- teins when comparing in vitro with in vivo (95%). However. this did not hold trv forall genes. Ina broad analysis of several Inundred genes about 33-37% overlap has been observed over ‘multiple gene families (Lund et al .2006). In this context. it was, remarkable finding of the present study thatthe BY-2 response predicted the ix vivo response with $4% likelihood (Fig. 1B) and the genes found in vivo were also detected in BV-2 at a sate of 41°. Considering the methodological limitations and the ‘broad ranse of different 2enes, this appears to be a remarkably 200d overlap, 3.3 Confirmation of mRNA expression Chip-based transcriptome analysis ives an overview over gen- ral regulation patterns and indicated here that many eroups of ‘genes already known from PM are also regulated in the same direction in activated BV.2. Regarding exact quantification and sensitivity. PCR is superior to the chip analysis we used. ‘Therefore we analysed a subset of genes related particularly to host defence in the brain by quantitative PCR. Regulation of such genes similar to the pattern known from microglia would be essential for the use of BV-2 as a model for infection and inflammation ia the brain. Indeed we found very high overes- pression (30-200 fold) of genes such as the chemokine CCL2, interleukin-1B, inducible nitric oxide synthase (INOS), the in- flammation marker thymidylate kinase-1 or the viral defence protein Rig-I (Fig. 2A). Also, genes for the pattern recognition receptors TLR-2, TLR-3 and NOD-2, the antioxidant enzyme lutathione reductase or the macrophage inflammation mark- er IFIT-3 were clearly upregulated (Fig. 2B,C), while e-. the mRNAs for the astrocyte marker UPA or the usually constantly expressed TLR-4 were not induced. These data show that genes not detected by the broad chip approach are also regulated in BV-2 and indicate thatthe overall response of these cells may actually be broader than indicated by the chip analysis. 7 HENNET AL ‘Tab. 2: Comparison of LPS regulated genes in BV-2 with LPS regulated genes in vivo Mice were injected icv. with LPS (2.25 yg/orain) or vehicle. After 4 or 16h fatal hippocampal RNA was purified and expression profied ‘on arrays. All ganes significantly regulated on the Neuroflame array aro listed (histoncal data) The data columns indicate te rato of Up-regulation. For purposes of comparably, the table lists the data obtained in vitro from primary micragha (PM) and BY-2 cells onthe same ganes. Biological material was obtained fram atleast two independant calor animal experiments ( analysed by 4 indeponda ‘out of 4 nypriasations. ‘animalsigioup) and was chip hybridisatons. Neurofame regulations wore considered significant i a gane was regulated =1.8 fold in 3 call leukemiarymphome 2 relaled protein ata Caspase “caspase 11 Ccaatienhancer bnding protein, daa (Gaia antigen ‘Ciylin-dependant nasa inhib. Ta (p21), Cake @ ‘Ghemoxino igand 12, MCP -S, Coli2 Chemokine haand 4, MIP-1 bela, C cd (Chemoxino igand'S, RANTES. C oS (Chemokine hgand 1, Cxclt ‘Chemokina igand 11, TAC, Gxciit Chemokine iaand 2, MIP-2, Cxc (Chemoxino igand’9, Oxcio Chitinase Ske 1 Cytactvome PASO, Famaly 2 Guanvlate nucleotide binding protein 2 Tnterforon ind. prot. wit tetraticopepide rep. 1, itt Tnferforoninducble GTPase Tatton 12,112 Inferioukin 6, 16 pocalin 2 Matric netalioprotenase © MMPS Proteasome subunit, beta lype 8 Peniraxn related gene, Pha ‘Serum amylond a2, SAAB ‘Serum amyloid a3, SAAS ‘Suppressor of cytokine signaling , SOGSS Thymidylate kinase, Ipe inducible momber, TKI Talvike receptor 2 7 LR2 Zine finger protein 36 3.4 Detection of LPS-regulated proteins in BV-2 ‘When inflammatory activation is examined, proteomics analy sis is in many respects complementary to chip analysis, Typical, inflammation markers are membrane or secreted proteins. These protein types are poorly recovered by standard (2D- proteomics approaches. On the other hand, regulation of RNAs ‘of normal soluble proteins is often hard to detect because of theis high baseline expression. Therefore, we used a proteomics ap- proach to fest whether such typical soluble markers of inflam: ‘mation were detectable in activated BV-2 cells. The cells were stimolated with LPS for 24 hours and analysed by a ratiometric approach on 2D-zels. Thirty-two specifically upregulated proteins were detected. About 10 were identified by sequencing (Lund et 1.2006). For instance, manganese superoxide dismutase (SOD) (the mitochondrial inducible form of SOD; SOD-2) was clearly upregulated in LPS-stimulated BV-2, SOD.2 induction isa typi cal inflammation marker and was also detected on the transcrip. tional level in PM. Ia BV-2 the transcriptional changes were us der the detection threshold and our proteomics findings confirm that BV-2 show a broader inflammatory responce capacity than may be indicated from the chip findings with relatively hard sie nificance rules (Fig. 3). Also, Peroxiredoxin I (Pre 1) was clearly up-regulated on the protein level (Fig. 3). This protein is well ‘known to be specific for lial cells (Hattori and Oikawa, 2007) and to be an indicator of microslial activation in vivo (Krapfen bauer et al., 2003; Kim et al., 2008). It was not detected by chip analysis (neither in PM nor BV-2), but shows that BV-2 indoed behave similar to microelia in vivo HENNET AL. iy Oo £3 aT ee t wa sr Fig. 2: Analysis of transcriptional regulation of genes. associated with inflammation and host defence in BV-2 1BV.2 calls wore stimulatod with SO ngiml LPS for tho times Indicate, before MANA was extracted and analysed by ‘quantitative PCR. All amplified products were trst standardised to tho gapdh transcript, and thon the stimulation factor was calculated as relative expression of LPS treated colls and Lntroated calls Data are means #SEM of triplicate determinations from three biological samples. Abbreviations: tdk-i= inducible ‘ymidylate kinase, co-2 = chemokine (c-2 matt igand 2,11 inferloukin 1 bota,rgt = etinoc acidAnducible gone I Interforon-induced protein with tetratricopeptice repeats 3, o9r1 Glutathione reductase 1, 2 receptor 4, nod2 = nucleotide-binding oligomerieation domain containing 2 _ghPSvsControtisostecti pinto] LPS vs Controt LPS vs Control - Fig. 2. Proteomics analysis of LPS-stimulated BV-2 celle {BV-2 calls wore stimulated for 24 h with saline or LPS (100 ng/m before preparation of protein samples. which were subsequently ‘Separatad on 20 gels. For spot quantiication, samples wele analysed by tne proteotope method (aferential labeling with iodine ‘sotapes, remixing befara the 2D run and ratio-imaging of each apo, vcualised nera in blue and orange). Differentially expressed protein ‘were identified by mass spectrometric sequencing. Total protein rom four independent biological experiments was pooled and run in ttiplicates, One representative gels shown (lft) with blue spots being proteins more abundant in LPS-stimulated cals than in control cells. The enlargements to the right show examples of two regulated proteins with both reverse labelling approaches of the Proteotope technology. Protein spot 30 (top) was identified as the inducible for of superoxide dismutase (SOD-2). Protein spots 24-27 (bottom) are al variants of peroxiredoxin | (Pr« ). Both example proteins are clearly upregulated by LPS, and this is independent ofthe labelling mode, ‘Blue indicates labelling with J-125, orange indicates labelling with J-131 ALTE 25,208 HENNE AL 3.5 Interferon responsiveness For the functional integration into an immune response (e.g. in the pathology of multiple sclerosis) itis important that model cells (such as BV-2) cannot only respond to inflammatory stim- uli such as LPS, butalso to eytokines, such as interferon gamma, (IEN-) in an appropriate way. We examined here the respon- siveness to IFN-y based on the known facts that induction of ‘TNE-a by LPS is independent of IFN-y in PM, while the strong ‘expression of iNOS, leading to high production of nitrite in the medium is strictly dependent on IFN-y, Indeed, nitrite was only produced by BV-2 cells in the presence of the cytokine (Fi 4A), and this production was very pronounced. As expected. TFN-Y had no augmenting effect on the production of TNF-c. after LPS stimulation (Fig. 4B). The selectivity ofthe induction bby IFN-y was also analysed on the transcriptional level, where 2 large boost of NOS mRNA expression was observed, while IL-AP was litle affected, as expected from the literature on PM Fe 40), 3.6 Functional stimulation of astrocytes Finally, we examined the functional capacity of BV-2 in tig- zgering cell-cell interaction. Microglia constitute the first Tine ‘of defence in the brain and their acute mediators then activate astrocytes (Falsig et al., 2008). Therefore. it was essential to examine whether BV-2 were capable of activating astrocyte. We stimulated BY-2 cells with LPS and then transferred the culture supernatants (CM) to astrocytes. These supematants contained considerable amounts of TNF-c, but not IL-6 (Fis. 3A). Murine astrocytes do not react to LPS (Falsig etal. 2004 Falsig et al, 2006; Falsig et al.. 2008) with eytokine release cr activation of the inflammation master switch NF-xB Fig. 5A). However, NF-xB translocation, which controls dozens of, ‘downstream inflammation events, was triggered in astrocytes by BV-2-conditioned medium (CM) (Fig. 5B). To investigate farther whether NF-&B tyanslocation deiven by CM from BV- indeed has functional consequences in astrocytes, we measured the release of IL-6 under the different stimulatory conditions. Again, BV-2 CM triggered a significant increase in IL-6 release (Fig, $C). This indicates a capacity of BY-2 cells to take past in and to tigger a complex biological process usually observed under in vivo conditions 4 Discussion ‘We examined various functions of BV-2 cells ia selation to their potential role as substitute for primary microelia, and the pos sibility to replace in vive experiments by cell culture models, ‘What can be achieved by such approaches? At present, the ma- jor past of experimental animals (>6 million/vear in the EU) are used for basic and applied research, Thus, the stakes are very high in this area. As the biomedical research field is much less standardised than regulatory toxicology. it has been diffieult to ‘establish and validate clear 1:1 replacement systems. On the ‘other hand, the biomedical field does not require the time-con suming and cumbersome validation process that is necessary for safety evaluations. The value of a potential replacement method 90 & in basie research is mostly determined by confidence of the sei- entific community in the data that can be generated. Additional factors, much more important than in the field of toxicology, are the price of the assay and the speed of data generation. Regard- ing the latter two parameters, BV-2 colls are clearly superior to primary microglia and animal experiments. For this reason, there are several hundred selated publications, some of them involving important discoveries. These were later frequently confirmed in other cells, and would often not have been poss ble in vivo, or would have been technically virtually impossible with the use of PM. In many publications using BV-2 cells, the Bp pitrite umm 2M] ‘TNFa [ngiml +SEM] relative mRNA level [fold induction) Fig. : Interteron-y responsiveness of BV-2 celle .BV.2 cals wore stimulated with 50 ngim! LPS in the presence fF absence of murine IFN-y (5 na/m). (4) Suosrnatants were emoved trom 96-well plates afer 24h, and the concentration of nitrite was determined by the Griese assay. (B) Supernatants were removed trom 96-well plates ater 8h, and the concentration of TTNF-«.was determined by ELISA. (C) Calls wore harvested for FRNA preparation ator the times indicated. Relative mRNA levole ‘wore dotermined by real-time PCR. Data aro moans +SEM trom triplicate determinations (n = 9). :p <0.05 by Hest. inos = mRNA, of inducible nitric oxide synthase 1 = mFINA of intrleukin-1. ALTER 26.208 & data were direetly compared to some data from PM to add ered- ibility. Studies with primary microglia usually involve the use of antibiotics, at least for some time, although it is known that these can influence the outcome of experiments (Kuhlmann, 1993). We switched the BV-2 culture completely to antibiotic free conditions and also reduced the use of FBS, another source cf experimental uncertainties, to 2% during stimulations. From a purely logical point-of-view. one ean never be sure that a cell Jine will be able to replace all experiments in PM and in animals, and indeed some differences have been observed between PM and BV-2 (Haausler et al, 2002; de Jong et al 2008). However, Cytokine [nai st toca ytokine [ngiml SEM] Be ie activated cl i & i i non shmlaled asvocytes stimulated asrcytee Fig, 5: Functional capacity of BV-2 cells to tigger astrocyte activation BV-2 were stimulated with LPS (50 ng/m) and the medium ‘supernatant was removed attr different incubation times. (A) CM was characterised at ctferent ime points for its cytokine Ccontont. TNF-a and IL-6 concontrations were measured at tho times indicated by ELISA. (B) Condioned mecium (CM) from {ho 8h time point or the corresponding control medium (C, LPS _addod to modium after conditioning by non-stimulatod BV.2 for 24 ' Was added to primary astrocyte cultures. NF-x8 translocation was measured ator 60 minutos by image software capable of {quantitying the fluorescent intansilyin the nuctous and the cytosol Alleast 200 calls were imaged per data point. (C) L-6 release was measured ator h. Data are means SEM trom triplicate determinations." p 0.05 ALTER 25,208 HENNET AL in many more cases similaritias have been described. It needs to ‘be noted that also PM have disadvantages and may yield errone- ‘us data Its fr instance very dificult to obtain absolutely pore cultures of PM, and some conteabutions may arise from contam- inating cells In most cases, also the term “primary microglia’ is misleading, as these cell ae kept ina primary mixed zlal culture for about 2 weeks after isolation, uni they are re-plated and then used in secondary cultures. During the initial week Period, the ess phagocytose large amounts of neuronal debris Jeft over from the isolation procedure and may thus be modified already. On top of this, it needs to be considered thatthe cells are usally isolated from neonatal brains and may therefore not represent adult microglia Finally, mirogtia area telatively het- erogencous cell population, with marked species and regional differences within the brain (Mahe etal, 2001; Guilemin and Brew, 2004), and thus PM may not always be representative This needs to be remembered when comparing the value of BV-2 in relation to PMin vivo in practical terms. The general validity of cell fine models (Hartung, 2007) vs. animal models (artung, 2008a) has been discussed elsewhere. Conceming the validation process (Hartung, 2007a), it i impossible to deter rine the value of a cell line in general as it is impossible ta ‘compare the cell line with PM for each and every application that may have arisen in the past and may sill arse in the future. ‘An approach to this apparent dilemma is to examine the reac- tion pattem in certain application domains between cell line and primary calls or in vivo models. Ifthe overall pattern is highly overlapping for a certain domain it is ikely that also ia most cases not tested. a high degree of overlap would cecur. In this study we evaluated BV.2 forthe use i inflammation studies. This is one of the major uses of microglia, and aow doubt has been mised in this particular domain on the valve of BY.2 as a model system. In a recent study, BV.2 have been compared t primary microglia, bt the primary microglia were derived from rats, while BV-2 are murine cells. la many assays of ths study Horvath eta. 2008), BV-2 behaved very similar to PM, while there were mostly quantitative differences in oth- ess, The authors concluded fiom the relatively small data set that data from the BV.2 call line may not be credible, if not backed by PM data (Horvath etal.,2008). In contrast to this we observed in the past that BV.2 very often behaved similarly to PM. This was not only true for eytakine secretion, but also in very complex assays examining MAP kinase signalling (Lund etal., 2005) and phagocytosis (Hirt and Leist, 2003). OF course, ‘occasional differences were also observed. often of quaatitative ature, Therefore, we used a differet type of approach for the comparison here, Instead of individual assays, we looked at the coverll pattem of gene activation atthe start ofthis study Basic microglial gene expression has been described for smatine (Re etal. 2002) and rat primary cells (Duke etal. 2004) inaddition toa murine eel line (Inove et al, 1999), Changes in the transcriptional profile of primary rodent mieroglia in dif ent conditions, such as after exposure to IFN~ (Moran etal. 2004), TGF-8 Paslinawan et al., 2003) or coloay-stimlatin factors (Re et al, 2002), have been described. For inflammatory stimulation, data can be foune for human microglia (Walker et al, 2001) or the murine BV:2 (Gan et al. 2004) cell line ex- 3 HENNE AL posed to amyloid peptide and for primary mieroglia exposed to gram-positive bacteria (Kiolian etal, 2002) or LPS (Lund et al., 2006), Data on in vivo responses are still scarce, but have ‘been obtained for LPS (Lund et al 2006). The amazing finding of our study was that 90% of the genes induced in BV-2 by LPS were also found in primary microglia, and around 50% were even found in hippocampal microglia after Innvivo stimulation of mice by intmcerebroventeiulae injection ‘of LPS. This appears to usa good overlap. However, we also ‘observed a clear difference between PM and BV-2. PM reacted stronger to LPS and therefore a much large (10-fold) number cof genes was significantly regulated. This finding would arzue for the use of BY-2 only in conditions where the correlation with PM has been confirmed by pilot data, However. we went a step further and analysed by the more sensitive PCR method a ‘number of genes in BV-2 known to be regulated in PM. These genes were indzed regulated, although they were not detected in the chip study. Thus, there is probably less qualitative dif ference between PM and BV-2 than the chip study indicated. This was further confiemed by a small proteomics stady, also detecting the regulation of proteins in BV.2 that are known to bbe reuulated in PM ‘The second part of the study focused on functional assays in BY.2 cells. asithas often been claimed that cell-cell interactions ae especially difficult to model in vitro. Initially we looked at nile oxide production, as this a very frequently used param ter, and different laboratories find very different stimulation of sitic oxide synthase by LPS. This is most likely due to different

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