Effects of low dose GM-CSF on microglial inflammatory profiles to diverse pathogen-associated molecular patterns (PAMPs)
© Esen and Kielian; licensee BioMed Central Ltd. 2007
Received: 20 January 2007
Accepted: 20 March 2007
Published: 20 March 2007
It is well appreciated that obtaining sufficient numbers of primary microglia for in vitro experiments has always been a challenge for scientists studying the biological properties of these cells. Supplementing culture medium with granulocyte-macrophage colony-stimulating factor (GM-CSF) partially alleviates this problem by increasing microglial yield. However, GM-CSF has also been reported to transition microglia into a dendritic cell (DC)-like phenotype and consequently, affect their immune properties.
Although the concentration of GM-CSF used in our protocol for mouse microglial expansion (0.5 ng/ml) is at least 10-fold less compared to doses reported to affect microglial maturation and function (≥ 5 ng/ml), in this study we compared the responses of microglia derived from mixed glial cultures propagated in the presence/absence of low dose GM-CSF to establish whether this growth factor significantly altered the immune properties of microglia to diverse bacterial stimuli. These stimuli included the gram-positive pathogen Staphylococcus aureus (S. aureus) and its cell wall product peptidoglycan (PGN), a Toll-like receptor 2 (TLR2) agonist; the TLR3 ligand polyinosine-polycytidylic acid (polyI:C), a synthetic mimic of viral double-stranded RNA; lipopolysaccharide (LPS) a TLR4 agonist; and the TLR9 ligand CpG oligonucleotide (CpG-ODN), a synthetic form of bacteria/viral DNA.
Interestingly, the relative numbers of microglia recovered from mixed glial cultures following the initial harvest were not influenced by GM-CSF. However, following the second and third collections of the same mixed cultures, the yield of microglia from GM-CSF-supplemented flasks was increased two-fold. Despite the ability of GM-CSF to expand microglial numbers, cells propagated in the presence/absence of GM-CSF demonstrated roughly equivalent responses following S. aureus and PGN stimulation. Specifically, the induction of tumor necrosis factor-α (TNF-α), macrophage inflammatory protein-2 (MIP-2/CXCL2), and major histocompatibility complex (MHC) class II, CD80, CD86 expression by microglia in response to S. aureus were similar regardless of whether cells had been exposed to GM-CSF during the mixed culture period. In addition, microglial phagocytosis of intact bacteria was unaffected by GM-CSF. In contrast, upon S. aureus stimulation, CD40 expression was induced more prominently in microglia expanded in GM-CSF. Analysis of microglial responses to additional pathogen-associate molecular patterns (PAMPs) revealed that low dose GM-CSF did not significantly alter TNF-α or MIP-2 production in response to the TLR3 and TLR4 agonists polyI:C or LPS, respectively; however, cells expanded in the presence of GM-CSF produced lower levels of both mediators following CpG-ODN stimulation.
We demonstrate that low levels of GM-CSF are sufficient to expand microglial numbers without significantly affecting their immunological responses following activation of TLR2, TLR4 or TLR3 signaling. Therefore, low dose GM-CSF can be considered as a reliable method to achieve higher microglial yields without introducing dramatic activation artifacts.
Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a well known hematopoietic cytokine produced primarily by T cells, macrophages, endothelial cells, and fibroblasts [1–4]. GM-CSF was originally defined based on its ability to stimulate the differentiation and function of granulocytes, monocytes and macrophages . In addition, previous studies have established that GM-CSF promotes the survival and proliferation of neonatal rat, mouse, and human microglia in culture [5–9]. Based on these observations, GM-CSF is commonly used as a culture medium supplement to obtain sufficient numbers of microglia to conduct downstream in vitro experiments [10–12]. However, recent studies have suggested that microglia are not terminally differentiated and that GM-CSF can induce their functional maturation and expression of dendritic cell (DC) markers [13–15], which has raised concerns with investigators who are examining the immunological functions of primary microglia in various CNS pathologies. For example, GM-CSF has been reported to induce the transcription of genes important for T cell activation, chemotaxis, antigen processing, innate immunity, and immunosuppression, suggesting the transition of microglia into a more professional antigen presenting cell phenotype [14–16]. In addition, other studies have utilized GM-CSF to induce DC maturation from myeloid progenitor cells [17, 18]. Overall, these studies suggest that microglia can transition into a DC-like phenotype when cultured in the presence of adequate levels of GM-CSF.
During the preparation of our mouse primary mixed glial cultures, we routinely supplement culture medium with low levels of GM-CSF (0.5 ng/ml) to increase microglial yields. Despite the fact that this concentration is approximately ten-fold lower than what has been reported to modulate microglial function and transition into a DC phenotype (i.e. 5–50 ng/ml), we are often questioned regarding the consequences of microglial exposure to GM-CSF during the mixed glial culture period and whether this introduces artifacts in the activation profiles of these cells in subsequent in vitro experiments. Therefore, the primary objective of the present study was to evaluate whether low dose GM-CSF leads to alterations in microglial morphology and/or functional activation in response to a wide variety of PAMPs commonly associated with various CNS infections, namely Staphylococcus aureus (S. aureus) and its cell wall component peptidoglycan (PGN), LPS, polyI:C, and CpG-ODN. Our results demonstrate that low dose GM-CSF led to a significant expansion in microglial numbers without affecting their phagocytic activity or cytokine production profiles in response to the majority of PAMPs examined, with the exception of the TLR9 agonist CpG-ODN. However, we did observe a phenotypic transformation of microglia expanded in the presence of low dose GM-CSF, namely a transition to a DC-like morphology typified by numerous dendrites; however, the functional implication(s) of this change remain to be determined. Therefore, low dose GM-CSF can be successfully utilized as a culture medium supplement to enhance microglial recovery without overtly compromising normal responsiveness to microbial stimuli. Importantly, these findings exclude any potential GM-CSF-induced artifacts in the read-outs of microglial activation routinely used in our studies.
Primary microglia cell culture and reagents
Primary microglia were isolated from inbred neonatal C57BL/6 mice as previously described . Briefly, age-matched litters (postnatal day 2–5) were euthanized using an overdose of inhaled Halothane (Halocarbon Laboratories, River Edge, NJ) to obtain mixed glial cultures. Cerebri were collected under aseptic conditions and the meninges removed. Tissues were minced, resuspended in trypsin-EDTA (Mediatech Inc., Herndon, VA), and incubated at 37°C for 20 minutes. Subsequently, cells were resuspended in complete DMEM (4.5 g/L glucose, Mediatech Inc.) containing 10% FBS (Hyclone, Logan, UT), 200 mM L-glutamine, 100 U/ml penicillin, 0.1 mg/ml streptomycin and 0.25 μg/ml fungizone (all from Mediatech Inc.), OPI supplement (oxalacetic acid, pyruvate, insulin; Sigma, St. Louis, MO), and 0.5 ng/ml recombinant mouse GM-CSF (BD Pharmingen, San Diego, CA). The cell suspension was further triturated and filtered through a 70 μm cell strainer. Subsequently, cells were centrifuged, resuspended in complete medium, and seeded into 150 cm2 flasks. To minimize variation between microglia expanded in the presence/absence of GM-CSF, mouse pups were procured from litters born on the same day and primary cultures propagated with or without GM-CSF were derived from the same initial mixed glial population (i.e. half of the mixed glial cells recovered were cultured with GM-CSF (+) medium while the other half was cultured without GM-CSF).
Upon confluence (7–10 days), flasks were shaken overnight at 200 rpm at 37°C to recover microglia. Microglia from both (+) GM-CSF- and (-) GM-CSF-treated flasks were collected and plated in medium without GM-CSF for all subsequent experiments. The purity of microglial cultures was evaluated by immunohistochemical staining using antibodies against CD11b and GFAP to identify microglia and astrocytes, respectively, and was routinely greater than 95%. Each experiment presented in this paper was initially performed with microglia collected after the first shake and repeated with cells collected after a second shake to confirm that the responses were comparable. Based on our findings that microglial responses were similarly affected in all experiments regardless of when they were harvested, we concluded that microglial responsiveness to microbial stimuli does not significantly differ in cells collected from the first versus subsequent shakes.
Heat-inactivated S. aureus (strain RN6390) was prepared as previously described  and PGN derived from S. aureus, poly I:C, and the synthetic CpG oligonucleotide ODN1826 were obtained from InvivoGen (San Diego, CA). Escherichia coli O11:B1 LPS was purchased from List Biological Laboratories (Campbell, CA). The doses of stimuli used throughout this report were based on our previous studies that established optimal cytokine responses induced following bacterial stimulation without any evidence of toxicity [20, 21]. All non-LPS reagents were verified to have endotoxin levels < 0.03 EU/ml as determined by Limulus amebocyte lysate assay (Associates of Cape Cod, Falmouth, MA).
Enzyme linked immunosorbent assay (ELISA)
Protein levels of TNF-α, IL-12p40 (OptEIA, BD Pharmingen) and macrophage inflammatory protein (MIP-2/CXCL2, DuoSet, R&D Systems, Minneapolis, MN) were quantified in conditioned medium from PAMP-stimulated microglia using ELISA kits according to the manufacturer's instructions (level of sensitivity = 15.6 pg/ml).
Cell viability assays
To evaluate whether microglia expanded in the presence or absence of GM-CSF demonstrated similar survival profiles following bacterial activation, a standard MTT assay based upon the mitochondrial conversion of (3- [4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide (MTT) into formazan crystals was performed as previously described .
Primary microglia expanded with or without GM-CSF were seeded onto 12 mm coverslips in 24-well plates and incubated overnight. The following day, cells were treated with a heat-killed S. aureus isolate that constitutively expresses green fluorescence protein (GFP, kindly provided by Dr. Ambrose Cheung, Dartmouth Medical School) for 3 h, whereupon Hoechst 33342 (Molecular Probes, Eugene, OR) was added to visualize nuclei. Cells were washed extensively with PBS and incubated with 0.05% crystal violet in 0.15 M NaCl for 45 seconds to quench any fluorescence emitted by residual extracellular bacteria. Coverslips were viewed under fluorescence microscopy using an excitation wavelength of 460 – 490 nm (FITC filter, Olympus BX41, Tokyo, Japan).
Immunofluorescence staining and confocal microscopy
Primary microglia expanded in the presence/absence of GM-CSF were seeded onto 12 mm coverslips in 24-well plates and incubated overnight. The following day, microglia were treated with 107 heat-inactivated S. aureus for 24 h, whereupon cells were washed extensively with PBS, fixed in ice-cold methanol, and incubated with PBS/10% donkey serum to prevent non-specific antibody binding (Jackson ImmunoResearch, West Grove, PA) for 30 min at room temperature. Subsequently, microglia were incubated with a MHC class-II antibody (rat anti-mouse, BD Pharmingen) overnight at 4°C. The following day, cells were incubated with a donkey anti-rat biotinylated secondary antibody (Vector Laboratories, Burlingame, CA) and detected using a streptavidin-Alexa Fluor 568 conjugate (Molecular Probes). Subsequently, a directly conjugated CD11b-FITC antibody (BD Pharmingen) was added and cells incubated for 1 h at 37°C, whereupon Hoechst 33342 was used to visualize nuclei. Controls included microglia incubated with secondary antibodies only to assess the extent of non-specific staining. Coverslips were imaged using a Zeiss laser scanning confocal microscope (LSM 510, Carl Zeiss Microimaging). Hoechst 33342 for nuclear visualization was excited by a 405 nm diode laser, FITC to visualize CD11b immunoreactivity was excited with a 488 nm argon laser, and Alexa Fluor 568 to demonstrate MHC class II expression was excited with a 561 nm DPSS laser, with images collected using the appropriate emissions. The confocal pinhole was set to obtain an optical section thickness of 1.6 μm. To demonstrate co-localization of CD11b, MHC class II, and Hoechst 33342 signals, RGB merges of individual confocal images were performed using the ImageJ software program (NIH Image).
Primary microglia expanded in the presence/absence of GM-CSF were seeded into 6-well plates (2 × 106 cells/well), and incubated overnight. The following day, cells were treated with 107 heat-inactivated S. aureus for 24 h. At the end of the incubation period, cells were washed twice with PBS, and collected using a cell scraper. A total of 5 × 105 microglia in each group were stained for two-color flow cytometry using CD11b-FITC and CD11c-PE-Cy7 antibodies (both from BD Pharmingen). Fc receptors were blocked with the addition of Fc block™ (anti-CD16 and -CD32 cocktail, BD Pharmingen). In addition, a separate set of microglia were stained with antibodies directed against the co-stimulatory molecules CD40, CD80, and CD86, in addition to MHC class II (all from BD Pharmingen), and subsequently incubated with a donkey anti-rat IgG FITC-conjugated secondary antibody (Jackson ImmunoResearch Laboratories, West Grove, PA). Controls included microglia incubated with appropriate isotype control-matched antibodies to assess the extent of non-specific background staining. Cells were analyzed using a FACS Calibur cytometer (BD Biosciences, San Jose, CA) with settings based on the staining of microglia with isotype control antibodies alone.
Primary microglia propagated in the presence/absence of GM-CSF were seeded into 35 mm dishes (2 × 106 cells/well), and incubated overnight. The following day, cells were treated with 107 heat-inactivated S. aureus for 24 h or left unstimulated. At the end of the incubation period, cell morphology was visualized by phase-contrast microscopy and images collected using a fixed stage upright epifluorescence microscope (BX51WI, Olympus) equipped with a 40 × water immersion objective lens and a 12-bit intensified monochrome CCD camera (CoolSnap ES, Photometrics, Tucson, AZ).
Significant differences between experimental groups were determined by the t-test for unequal variances at the 95% confidence interval using Sigma Stat (SPSS Science, Chicago, IL).
Microglial recovery is enhanced following low dose GM-CSF treatment
It has been well established that GM-CSF exerts mitogenic effects on primary microglia, effectively expanding cell numbers [5, 6, 10, 12, 23]. Therefore, the use of this cytokine during the mixed glial culture period reduces the number of neonatal mice required to obtain sufficient numbers of microglia for subsequent studies. We routinely supplement our culture medium with a relatively low dose of GM-CSF; however, to date, we have not yet performed a detailed analysis regarding the efficacy of this dose on microglial expansion or more importantly, whether GM-CSF alters the subsequent responsiveness of microglia compared to cells that have never been exposed to exogenous growth factor. This became an essential issue to address since we are often questioned about the consequences of GM-CSF treatment on downstream microglial responses, thus forming the impetus for the current study. Work by others had demonstrated that GM-CSF led to the transition of microglia into macrophage and/or dendritic-like cells [13–15, 18]; however, the doses of cytokine used to drive this differentiation (i.e. 10–50 ng/ml) ranged anywhere from 20- to 50-fold higher than the concentration used to expand microglia numbers in our experiments (i.e. 0.5 ng/ml). In addition, other important factors to consider include whether microglia are procured from neonatal versus adult animals or if microglia are expanded as mixed glial cultures or as purified cells.
Low dose GM-CSF leads to morphological changes in microglia characteristic of dendritic cells (DCs)
Microglia display an ameboid morphology during embryogenesis [24, 25] and assume a ramified shape upon maturation in the normal brain under physiological conditions [24, 26, 27]. However, in response to injury or infection, microglia become activated and transition into an ameboid morphology . In vitro, ameboid microglia are rounded cells flattened to the substratum and have been reported to function as phagocytic antigen presenting cells [28, 29, 30, 31, 32]. This ameboid morphology observed in vitro is likely a consequence of the isolation procedure where, in general, ameboid characteristics are more typical of neonatal microglia, whereas adult cells normally exhibit a more quiescent ramified phenotype [33–36]. Nonetheless, ramification of ameboid microglia can be achieved by either growing cells on an astrocyte monolayer, culturing microglia in astrocyte-conditioned media, or treating with M-CSF [15, 28, 33].
Microglial expansion with low dose GM-CSF leads to differential responses to various PAMPs
Microglial phagocytosis of bacteria is not affected by the presence of GM-CSF during the mixed glial culture period
Effects of low dose GM-CSF on the expression of microglial surface markers
Primary microglia can be differentiated from macrophages based on their characteristic staining pattern of CD11bhigh and CD45low [32, 51–53]. Although it has been shown that GM-CSF used as either a culture medium supplement or a direct stimulus leads to a dramatic expansion in microglial numbers [10, 13], an effect on CD11b expression was not demonstrated . On the other hand, GM-CSF has been reported to inhibit the IFN-γ -induced expression of another surface marker, MHC class II, and as a consequence, modulate microglial APC functions [15, 54]. However, Fischer at al. (1993) have shown that MHC class II expression is not altered on microglia grown in the presence of GM-CSF, whereas its expression is induced following IFN-γ treatment .
Since neonatal microglia exhibit a partially activated phenotype in vitro, as indicated by an intermediate expression level of co-stimulatory molecules in addition to MHC class II [52, 55], we expanded our analysis to evaluate the expression of several co-stimulatory molecules including CD40, CD80 and CD86 by flow cytometry to determine the possible effects of low dose GM-CSF on microglial expression of these molecules. Similar to MHC class II, baseline CD80 and CD86 expression was not influenced by either GM-CSF or S. aureus (Figure 7). On the other hand, microglia propagated in the presence of low dose GM-CSF displayed elevated CD40 levels following S. aureus stimulation (Figure 7), whereas under resting conditions, CD40 levels were equivalent in microglia expanded with or without GM-CSF. Taken together, this data suggests that exposing microglia to low doses of GM-CSF during the mixed culture period does not lead to significant alterations in surface marker expression, with the exception of CD40, which may have an impact on microglial activation in the presence of CD40 ligands.
The adult brain parenchyma harbors a population of CD11b+ myeloid precursors [51, 56–58] that can be driven to differentiate into immature DCs by GM-CSF treatment as measured by the induction of cell surface markers such as DEC-205, CD11c, and CD80 [10, 13, 38, 40]. When Fischer and co-workers (2001) incubated primary microglia from adult mouse brain with GM-CSF (50 ng/ml) for 5 days, approximately 30% of these cells expressed CD11c compared to < 0.5% of microglia in the initial population. In our culture system, microglia are exposed to GM-CSF for a period of longer than 10 days and this was not sufficient to induce CD11c expression since, when compared to isotype control staining, none of the CD11c signal detected could be attributed to specific binding in either the "resting state" or following S. aureus exposure (Figure 8). Collectively, these findings indicate that exposure of microglia to low dose GM-CSF during the mixed glial culture period does not induce the expression of the classical DC marker CD11c.
GM-CSF, which is a potent stimulator of microglia as well as macrophages and granulocytes, is usually detected in the brain following T cell infiltration [14, 59, 60] or produced by activation of astrocytes and/or endothelial cells [7, 61]. The amount of GM-CSF released by these cells could be sufficient to induce a proinflammatory state of local microglia and/or their transformation into a DC- or macrophage-like cell. Although numerous studies have been performed examining the effects of GM-CSF on microglial morphology and function, often times these reports have produced conflicting results [10, 14, 16, 54]. This likely stems from differences in GM-CSF treatment paradigms, species of microglial origin, and/or whether adult or neonatal cells are used. All of these issues coupled with the fact that the doses of GM-CSF applied to microglia have shown tremendous variability among individual studies, makes it quite difficult to compare results and arrive at general conclusions regarding the effects of GM-CSF on primary microglia. When we reviewed the literature we found that GM-CSF was mainly evaluated in two ways. In the first, GM-CSF was provided as a supplement during the initial mixed glial culture period [10, 13, 14, 38, 40], whereas in another group of studies, GM-CSF was added as a stimulus to purified microglia that had otherwise not been previously exposed to the growth factor [6, 7, 16, 32, 37, 54]. Another variable is the dose of GM-CSF examined which has been tested over a very broad range, namely from 0.05–1 μg/ml or 1- 200 U/ml, which can be expected to have dramatic effects on the results obtained. In addition, the species from which primary microglia are procured and their maturational state, for example, mouse or rat newborn pups [6, 13, 14, 16, 40, 54], adult animals [10, 32, 38], or even human fetal microglia  have been examined. Therefore, it is essential that the results obtained from these diverse experimental settings should be evaluated within the constraints of these parameters.
One main reason why scientists studying primary microglia have supplemented culture medium with GM-CSF is to procure sufficient numbers of microglia for downstream analysis, since GM-CSF has been shown to induce microglial proliferation [6, 10]. Indeed, this modification has resulted in accelerated cell growth and increased cell yields, saving time and animal utilization. In our in vitro studies, we typically isolate primary mixed glial cells from newborn mice and culture them in medium supplemented with a relatively low dose of GM-CSF (0.5 ng/ml). Importantly, upon recovery of microglia from these cultures, cells are never exposed to exogenous GM-CSF again and are not utilized in experiments for at least 2 days following low dose GM-CSF withdrawal. With this modification, we were able to collect sufficient numbers of microglia for our experiments even after the third harvest, effectively reducing the need for additional microglia cultures. In addition, the main inflammatory responses of microglia following S. aureus stimulation, namely proinflammatory mediator production and phagocytosis, were not affected by propagating cells in the presence of GM-CSF (Figures 2 and 3). Interestingly, exposure of microglia to GM-CSF modestly influenced proinflammatory mediator production following LPS stimulation, whereas responses to polyI:C were not affected (Figure 4). It has been well established that both of these PAMPs activate microglia via TLR4 and TLR3, respectively, leading to GM-CSF production [62–66], as well as the priming ability of GM-CSF to augment LPS-induced TNF-α production in monocytes  and alveolar macrophages . However, to the best of our knowledge, the current study represents the first report investigating the effects of low dose GM-CSF on microglial responses to TLR4 and TLR3 ligands. Importantly, based on the finding that low dose GM-CSF does not dramatically alter microglial proinflammatory mediator expression in response to a diverse array of PAMPs, we propose that our cell culture procedure may be applicable to a wide range of studies investigating the immune properties of primary mouse microglia without concerns of drastically transforming cellular responsiveness. However, a word of caution must be mentioned, in that not all PAMP recognition is resilient to low dose GM-CSF, as demonstrated by our CpG-ODN findings discussed below.
Several researchers have reported that microglia express TLR9 and respond to the TLR9 agonist CpG-ODN with the robust production of numerous proinflammatory mediators as well as increased expression of immune receptors [12, 69–71]. Moreover, recent evidence demonstrating that CpG-ODN stimulated microglia induce neuron cell death in a co-culture paradigm has suggested a link between TLRs and neurotoxicity and neurodegeneration . On the other hand, we have reported that alternative TLR agonists such as S. aureus, PGN and LPS lead to a time-dependent decrease in TLR9 mRNA expression , which might be explained as a compensatory mechanism for microglia to downregulate cellular activation pathways. A recent study has shown that GM-CSF (20 ng/ml) in combination with either CpG- or non-CpG-ODN was capable of inducing several chemokines in primary human monocytes , as well as in lymphoma and neuroblastoma models [73, 74]. Although the methods used in these studies are quite distinct compared to our experimental design, they still support our findings that GM-CSF exacerbates microglial activation following CpG-ODN stimulation (Figure 4). Our results highlight the importance of experimental culture conditions and the potential effects of GM-CSF, even at very low doses, should be considered while investigating the consequences of GpG-ODN on microglial activation. The mechanism(s) responsible for this observed synergistic effect between GM-CSF and CpG-ODN remain to be clarified with further studies. Importantly, in this study microglia expanded in the presence/absence of GM-CSF expressed equivalent levels of TLR9 mRNA under resting conditions (data not shown), suggesting that the impaired responsiveness of GM-CSF (-) microglia to subsequent ODN stimulation is likely not the result of reduced TLR9 expression.
One of the main concerns reported with GM-CSF treatment of primary microglia cultures is its ability to induce microglial differentiation towards a DC phenotype [10, 15, 40]. GM-CSF can be produced by glial cells following inflammatory or infectious diseases of the brain [75–78]; therefore, it was postulated that GM-CSF may modulate the generation and maturation of DCs from microglia in the context of CNS disease . Although our experiments demonstrate that low dose GM-CSF does not lead to overt alterations in the functional properties of microglia to diverse bacterial stimuli, cytokine treatment does induce morphological changes characteristic of DC-like cells. Specifically, we found that unstimulated microglia expanded in the presence of GM-CSF displayed an increased number of dendritic processes and were enlarged and more adherent compared to cells that had not been exposed to exogenous growth factor. Despite these phenotypic differences, we were not able to uncover any functional implications for these changes and no CD11c induction on GM-CSF exposed microglia could be demonstrated. Therefore, additional studies are warranted to further investigate the consequences, if any, of this morphological transformation of microglia following low dose GM-CSF exposure. A general consensus that can be inferred from the literature, and is in partial agreement with the results presented here, is that GM-CSF can lead to the transition of microglia into a DC-like phenotype, although the magnitude of these changes appears to be influenced by the dose of GM-CSF cells are exposed to.
The expression of other surface molecules including MHC class II, CD80, and CD86 were not affected by exposure of microglia to low dose GM-CSF. The expression of these molecules has been shown by others to be elevated in neonatal microglia, where cells exhibit a partially activated phenotype in culture [15, 52, 55]. In ex vivo isolated adult microglia, GM-CSF stimulation did not alter the expression of MHC class II, CD40, or CD86 but did induce morphological changes [15, 54]. In contrast to our previous report demonstrating that S. aureus stimulation induced MHC class II and co-stimulatory molecule expression in the N9 microglial cell line , we found in the present study that the levels of MHC class II, CD80, and CD86 were not augmented in primary microglia following S. aureus stimulation. However, CD40 expression was dramatically upregulated following S. aureus stimulation in microglia that were expanded in the presence of GM-CSF. We propose that one explanation for these contradictory findings may lie in the nature of the cell types examined. For example, primary neonatal microglia in culture may inherently express higher constitutive levels of these molecules such that further activation will not result in an obvious increase in their expression. Alternatively, the length of time required to detect activation-dependent increases in surface marker expression in primary microglia may be longer than that observed for the N9 cell line; however, this possibility seems less likely. Another potential explanation is that the concentration of S. aureus required to increase MHC class II and co-stimulatory molecule expression may differ between primary microglia and the N9 cell line. Since we only examined one dose of S. aureus in the present study it remains possible that titration of higher bacterial concentrations may have elicited an increase in surface marker expression, although this possibility remains speculative. The finding that MHC class II levels did not increase in S. aureus stimulated microglia when assayed using two independent approaches (i.e. immunofluorescence and flow cytometry staining) provides additional support to establish the validity of our findings.
Another important factor to consider is the nature of how primary microglia are propagated during in vitro culture. Specifically, in our studies, microglia and astrocytes are maintained as mixed cultures during the expansion period. Therefore, it is important to acknowledge that the coordinated effects of endogenous astrocyte-derived factors in combination with exogenous GM-CSF may influence the properties of recovered microglia. As an extension, it is not surprising that different microglial phenotypes are observed when cells are directly purified from the brain parenchyma and cultured in isolation. In addition, compounded with the addition of high levels of exogenous GM-CSF used in other studies, it is not unexpected that microglia assume different biological and functional properties. We propose that the maintenance of primary microglia initially as mixed cultures with astrocytes is more reminiscent of the natural milieu these cells are exposed to in vivo and represents an acceptable model to study the immunological properties of microglia, although we clearly recognize the potential for any in vitro findings to not directly equate to in vivo responses due to the artificial nature of the former. However, the examination of microglia in isolation provides a more simplified approach to investigate the effector functions of these CNS phagocytes in the absence of confounding effects by surrounding cell types.
Our findings demonstrate that supplementation of neonatal mouse mixed glial cultures with low dose GM-CSF successfully maintained microglial expansion and did not lead to overt alterations in the functional responses of microglia following stimulation with several PAMPs of importance during CNS bacterial and viral infections including PGN, LPS, polyI:C, as well as S. aureus. This suggests that our culture paradigm may be successfully used as a method to procure larger quantities of microglia without significantly affecting their downstream responses to microbial stimuli. However, it is important to note that differences in CpG-ODN responsiveness as well as the transition towards a DC-like phenotype were observed in microglia propagated in the presence of low dose GM-CSF, the functional implications of which are currently unknown.
antigen presenting cell
cluster of Differentiation
central nervous system
Dulbecco's modified eagle medium
enzyme linked immunosorbent assay
fluorescence activated cell sorting
fetal bovine serum
green fluorescence protein
- IL-12 p40:
granulocyte-macrophage colony-stimulating factor
macrophage colony-stimulating factor
major histocompatibility complex
macrophage inflammatory protein-2
3- [45-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide
oxalacetic acid pyruvate, insulin
pathogen associated molecular pattern
phosphate buffered saline
- S. aureus :
tumor necrosis factor-alpha.
The authors would like to thank Thuang Shwe for preparation of primary glial cultures, Dr. Mohsin Md. Syed for assistance with phase-contrast and confocal microscopy, Patrick Mayes for excellent technical assistance, and Dr. Paul Drew for critical review of the manuscript. This work was supported by the NIH National Institutes of Mental Health (RO1 MH65297) and Neurological Disorders and Stroke (NINDS, RO1 NS40730) to T.K. and the NINDS supported Core facility at UAMS (P30 NS047546). Support for the Digital and Confocal Microscopy Laboratory at the University of Arkansas for Medical Sciences is provided by NIH/INBRE P20 RR6460 and NIH/NCRR S10 RR19395.
- Abbas AK, Lichtman AH: Cellular and Molecular Immunology. 2000, Philadelphia, WB Saunders, 268-269.Google Scholar
- Metcalf D: Control of granulocytes and macrophages: molecular, cellular, and clinical aspects. Science. 1991, 254: 529-533. 10.1126/science.1948028.View ArticlePubMedGoogle Scholar
- Groopman JE, Molina JM, Scadden DT: Hematopoietic growth factors. Biology and clinical applications. N Engl J Med. 1989, 321: 1449-1459.View ArticlePubMedGoogle Scholar
- Clark SC, Kamen R: The human hematopoietic colony-stimulating factors. Science. 1987, 236: 1229-1237. 10.1126/science.3296190.View ArticlePubMedGoogle Scholar
- Suzumura A, Sawada M, Yamamoto H, Marunouchi T: Effects of colony stimulating factors on isolated microglia in vitro. J Neuroimmunol. 1990, 30: 111-120. 10.1016/0165-5728(90)90094-4.View ArticlePubMedGoogle Scholar
- Giulian D, Ingeman JE: Colony-stimulating factors as promoters of ameboid microglia. J Neurosci. 1988, 8: 4707-4717.PubMedGoogle Scholar
- Lee SC, Liu W, Brosnan CF, Dickson DW: GM-CSF promotes proliferation of human fetal and adult microglia in primary cultures. Glia. 1994, 12: 309-318. 10.1002/glia.440120407.View ArticlePubMedGoogle Scholar
- Henze C, Hartmann A, Lescot T, Hirsch EC, Michel PP: Proliferation of microglial cells induced by 1-methyl-4-phenylpyridinium in mesencephalic cultures results from an astrocyte-dependent mechanism: role of granulocyte macrophage colony-stimulating factor. J Neurochem. 2005, 95: 1069-1077. 10.1111/j.1471-4159.2005.03416.x.View ArticlePubMedGoogle Scholar
- Koguchi K, Nakatsuji Y, Okuno T, Sawada M, Sakoda S: Microglial cell cycle-associated proteins control microglial proliferation in vivo and in vitro and are regulated by GM-CSF and density-dependent inhibition. J Neurosci Res. 2003, 74: 898-905. 10.1002/jnr.10829.View ArticlePubMedGoogle Scholar
- Fischer HG, Nitzgen B, Germann T, Degitz K, Daubener W, Hadding U: Differentiation driven by granulocyte-macrophage colony-stimulating factor endows microglia with interferon-gamma-independent antigen presentation function. J Neuroimmunol. 1993, 42: 87-95. 10.1016/0165-5728(93)90215-K.View ArticlePubMedGoogle Scholar
- Kielian T, Mayes P, Kielian M: Characterization of microglial responses to Staphylococcus aureus: effects on cytokine, costimulatory molecule, and Toll-like receptor expression. J Neuroimmunol. 2002, 130: 86-99. 10.1016/S0165-5728(02)00216-3.View ArticlePubMedGoogle Scholar
- Olson JK, Miller SD: Microglia initiate central nervous system innate and adaptive immune responses through multiple TLRs. J Immunol. 2004, 173: 3916-3924.View ArticlePubMedGoogle Scholar
- Santambrogio L, Belyanskaya SL, Fischer FR, Cipriani B, Brosnan CF, Ricciardi-Castagnoli P, Stern LJ, Strominger JL, Riese R: Developmental plasticity of CNS microglia. Proc Natl Acad Sci U S A. 2001, 98: 6295-6300. 10.1073/pnas.111152498.PubMed CentralView ArticlePubMedGoogle Scholar
- Re F, Belyanskaya SL, Riese RJ, Cipriani B, Fischer FR, Granucci F, Ricciardi-Castagnoli P, Brosnan C, Stern LJ, Strominger JL, Santambrogio L: Granulocyte-macrophage colony-stimulating factor induces an expression program in neonatal microglia that primes them for antigen presentation. J Immunol. 2002, 169: 2264-2273.View ArticlePubMedGoogle Scholar
- Ponomarev ED, Novikova M, Maresz K, Shriver LP, Dittel BN: Development of a culture system that supports adult microglial cell proliferation and maintenance in the resting state. J Immunol Methods. 2005, 300: 32-46. 10.1016/j.jim.2005.02.011.View ArticlePubMedGoogle Scholar
- Magnus T, Korn T, Jung S: Chronically stimulated microglial cells do no longer alter their immune functions in response to the phagocytosis of apoptotic cells. J Neuroimmunol. 2004, 155: 64-72. 10.1016/j.jneuroim.2004.06.002.View ArticlePubMedGoogle Scholar
- Delgado M, Chorny A, Ganea D, Gonzalez-Rey E: Vasoactive intestinal polypeptide induces regulatory dendritic cells that prevent acute graft versus host disease and leukemia relapse after bone marrow transplantation. Ann N Y Acad Sci. 2006, 1070: 226-232. 10.1196/annals.1317.019.View ArticlePubMedGoogle Scholar
- Vassiliou E, Jing H, Ganea D: Prostaglandin E2 inhibits TNF production in murine bone marrow-derived dendritic cells. Cell Immunol. 2003, 223: 120-132. 10.1016/S0008-8749(03)00158-8.View ArticlePubMedGoogle Scholar
- Esen N, Tanga FY, DeLeo JA, Kielian T: Toll-like receptor 2 (TLR2) mediates astrocyte activation in response to the Gram-positive bacterium Staphylococcus aureus. J Neurochem. 2004, 88: 746-758. 10.1046/j.1471-4159.2003.02202.x.View ArticlePubMedGoogle Scholar
- Esen N, Kielian T: Central role for MyD88 in the responses of microglia to pathogen-associated molecular patterns. J Immunol. 2006, 176: 6802-6811.PubMed CentralView ArticlePubMedGoogle Scholar
- Kielian T, Haney A, Mayes PM, Garg S, Esen N: Toll-like receptor 2 modulates the proinflammatory milieu in Staphylococcus aureus-induced brain abscess. Infect Immun. 2005, 73: 7428-7435. 10.1128/IAI.73.11.7428-7435.2005.PubMed CentralView ArticlePubMedGoogle Scholar
- Kielian T, McMahon M, Bearden ED, Baldwin AC, Drew PD, Esen N: S. aureus-dependent microglial activation is selectively attenuated by the cyclopentenone prostaglandin 15-deoxy-Delta12,14- prostaglandin J2 (15d-PGJ2). J Neurochem. 2004, 90: 1163-1172. 10.1111/j.1471-4159.2004.02579.x.PubMed CentralView ArticlePubMedGoogle Scholar
- Kielian T, Barry B, Hickey WF: CXC chemokine receptor-2 ligands are required for neutrophil-mediated host defense in experimental brain abscesses. J Immunol. 2001, 166: 4634-4643.View ArticlePubMedGoogle Scholar
- Giulian D, Baker TJ: Characterization of ameboid microglia isolated from developing mammalian brain. J Neurosci. 1986, 6: 2163-2178.PubMedGoogle Scholar
- Matsumoto Y, Ikuta F: Appearance and distribution of fetal brain macrophages in mice. Immunohistochemical study with a monoclonal antibody. Cell Tissue Res. 1985, 239: 271-278. 10.1007/BF00218004.View ArticlePubMedGoogle Scholar
- del Rio-Hortega P: Microglia. Cytology and Cellular Pathology of the Nervous System. Edited by: Penfield W. 1932, New York, Paul B. Hoeber Inc., 481-584.Google Scholar
- Giulian D: Ameboid microglia as effectors of inflammation in the central nervous system. J Neurosci Res. 1987, 18: 155-71, 132-3. 10.1002/jnr.490180123.View ArticlePubMedGoogle Scholar
- Rosenstiel P, Lucius R, Deuschl G, Sievers J, Wilms H: From theory to therapy: implications from an in vitro model of ramified microglia. Microsc Res Tech. 2001, 54: 18-25. 10.1002/jemt.1116.View ArticlePubMedGoogle Scholar
- Shaked I, Porat Z, Gersner R, Kipnis J, Schwartz M: Early activation of microglia as antigen-presenting cells correlates with T cell-mediated protection and repair of the injured central nervous system. J Neuroimmunol. 2004, 146: 84-93. 10.1016/j.jneuroim.2003.10.049.View ArticlePubMedGoogle Scholar
- Aloisi F, Serafini B, Adorini L: Glia-T cell dialogue. J Neuroimmunol. 2000, 107: 111-117. 10.1016/S0165-5728(00)00231-9.View ArticlePubMedGoogle Scholar
- Aloisi F, Ria F, Adorini L: Regulation of T-cell responses by CNS antigen-presenting cells: different roles for microglia and astrocytes. Immunol Today. 2000, 21: 141-147. 10.1016/S0167-5699(99)01512-1.View ArticlePubMedGoogle Scholar
- Aloisi F, De Simone R, Columba-Cabezas S, Penna G, Adorini L: Functional maturation of adult mouse resting microglia into an APC is promoted by granulocyte-macrophage colony-stimulating factor and interaction with Th1 cells. J Immunol. 2000, 164: 1705-1712.View ArticlePubMedGoogle Scholar
- Fujita H, Tanaka J, Toku K, Tateishi N, Suzuki Y, Matsuda S, Sakanaka M, Maeda N: Effects of GM-CSF and ordinary supplements on the ramification of microglia in culture: a morphometrical study. Glia. 1996, 18: 269-281. 10.1002/(SICI)1098-1136(199612)18:4<269::AID-GLIA2>3.0.CO;2-T.View ArticlePubMedGoogle Scholar
- Streit WJ: Microglia and macrophages in the developing CNS. Neurotoxicology. 2001, 22: 619-624. 10.1016/S0161-813X(01)00033-X.View ArticlePubMedGoogle Scholar
- Streit WJ, Walter SA, Pennell NA: Reactive microgliosis. Prog Neurobiol. 1999, 57: 563-581. 10.1016/S0301-0082(98)00069-0.View ArticlePubMedGoogle Scholar
- Streit WJ: Microglial senescence: does the brain's immune system have an expiration date?. Trends Neurosci. 2006, 29: 506-510. 10.1016/j.tins.2006.07.001.View ArticlePubMedGoogle Scholar
- Tambuyzer BR, Nouwen EJ: Inhibition of microglia multinucleated giant cell formation and induction of differentiation by GM-CSF using a porcine in vitro model. Cytokine. 2005, 31: 270-279. 10.1016/j.cyto.2005.05.006.View ArticlePubMedGoogle Scholar
- Fischer HG, Reichmann G: Brain dendritic cells and macrophages/microglia in central nervous system inflammation. J Immunol. 2001, 166: 2717-2726.View ArticlePubMedGoogle Scholar
- Garg S, Md Syed M, Kielian T: Staphylococcus aureus-derived peptidoglycan induces Cx43 expression and functional gap junction intercellular communication in microglia. J Neurochem. 2005, 95: 475-483. 10.1111/j.1471-4159.2005.03384.x.PubMed CentralView ArticlePubMedGoogle Scholar
- Fischer HG, Bielinsky AK: Antigen presentation function of brain-derived dendriform cells depends on astrocyte help. Int Immunol. 1999, 11: 1265-1274. 10.1093/intimm/11.8.1265.View ArticlePubMedGoogle Scholar
- Kielian T, Esen N, Bearden ED: Toll-like receptor 2 (TLR2) is pivotal for recognition of S. aureus peptidoglycan but not intact bacteria by microglia. Glia. 2005, 49: 567-576. 10.1002/glia.20144.PubMed CentralView ArticlePubMedGoogle Scholar
- Esen N, Kielian T: Recognition of Staphylococcus aureus-derived peptidoglycan (PGN) but not intact bacteria is mediated by CD14 in microglia. J Neuroimmunol. 2005, 170: 93-104. 10.1016/j.jneuroim.2005.09.003.PubMed CentralView ArticlePubMedGoogle Scholar
- Ebert S, Gerber J, Bader S, Muhlhauser F, Brechtel K, Mitchell TJ, Nau R: Dose-dependent activation of microglial cells by Toll-like receptor agonists alone and in combination. J Neuroimmunol. 2005, 159: 87-96. 10.1016/j.jneuroim.2004.10.005.View ArticlePubMedGoogle Scholar
- Zhang Z: Microglia activation in rat spinal cord by systemic injection of TLR3 and TLR7/8 agonists. J Neuroimmunol. 2005, 164: 154-160. 10.1016/j.jneuroim.2005.03.014.View ArticlePubMedGoogle Scholar
- Jordan FL, Thomas WE: Brain macrophages: questions of origin and interrelationship. Brain Res. 1988, 472: 165-178.View ArticlePubMedGoogle Scholar
- Giulian D, Li J, Bartel S, Broker J, Li X, Kirkpatrick JB: Cell surface morphology identifies microglia as a distinct class of mononuclear phagocyte. J Neurosci. 1995, 15: 7712-7726.PubMedGoogle Scholar
- Sedgwick JD, Schwender S, Imrich H, Dorries R, Butcher GW, ter Meulen V: Isolation and direct characterization of resident microglial cells from the normal and inflamed central nervous system. Proc Natl Acad Sci U S A. 1991, 88: 7438-7442. 10.1073/pnas.88.16.7438.PubMed CentralView ArticlePubMedGoogle Scholar
- Fincher EF: Microglia digest Staphylococcus aureus into low molecular weight biologically active compounds. Am J Physiol. 1996, 271: R149-56.PubMedGoogle Scholar
- Kaur C: Phagocytosis of Escherichia coli by amoeboid microglial cells in the developing brain. Acta Neuropathol (Berl). 2004, 107: 204-208. 10.1007/s00401-003-0798-7.View ArticleGoogle Scholar
- Chan A, Magnus T, Gold R: Phagocytosis of apoptotic inflammatory cells by microglia and modulation by different cytokines: mechanism for removal of apoptotic cells in the inflamed nervous system. Glia. 2001, 33: 87-95. 10.1002/1098-1136(20010101)33:1<87::AID-GLIA1008>3.0.CO;2-S.View ArticlePubMedGoogle Scholar
- Guillemin GJ, Brew BJ: Microglia, macrophages, perivascular macrophages, and pericytes: a review of function and identification. J Leukoc Biol. 2004, 75: 388-397. 10.1189/jlb.0303114.View ArticlePubMedGoogle Scholar
- Carson MJ, Reilly CR, Sutcliffe JG, Lo D: Mature microglia resemble immature antigen-presenting cells. Glia. 1998, 22: 72-85. 10.1002/(SICI)1098-1136(199801)22:1<72::AID-GLIA7>3.0.CO;2-A.View ArticlePubMedGoogle Scholar
- Ford AL, Goodsall AL, Hickey WF, Sedgwick JD: Normal adult ramified microglia separated from other central nervous system macrophages by flow cytometric sorting. Phenotypic differences defined and direct ex vivo antigen presentation to myelin basic protein-reactive CD4+ T cells compared. J Immunol. 1995, 154: 4309-4321.PubMedGoogle Scholar
- Hayashi M, Dorf ME, Abromson-Leeman S: Granulocyte-macrophage colony stimulating factor inhibits class II major histocompatibility complex expression and antigen presentation by microglia. J Neuroimmunol. 1993, 48: 23-32. 10.1016/0165-5728(93)90054-3.View ArticlePubMedGoogle Scholar
- Aloisi F: Relative efficiency of microglia, astrocytes, dendritic cells and B cells in naive CD4+ T cell priming and Th1/Th2 cell restimulation. Eur J Immunol 1999 Sep;29(9):. 1999, 29: 2705-2714.Google Scholar
- Alliot F, Lecain E, Grima B, Pessac B: Microglial progenitors with a high proliferative potential in the embryonic and adult mouse brain. Proc Natl Acad Sci U S A. 1991, 88: 1541-1545. 10.1073/pnas.88.4.1541.PubMed CentralView ArticlePubMedGoogle Scholar
- Simard AR, Soulet D, Gowing G, Julien JP, Rivest S: Bone marrow-derived microglia play a critical role in restricting senile plaque formation in Alzheimer's disease. Neuron. 2006, 49: 489-502. 10.1016/j.neuron.2006.01.022.View ArticlePubMedGoogle Scholar
- Streit WJ, Graeber MB, Kreutzberg GW: Functional plasticity of microglia: a review. Glia. 1988, 1: 301-307. 10.1002/glia.440010502.View ArticlePubMedGoogle Scholar
- Dame JB, Christensen RD, Juul SE: The distribution of granulocyte-macrophage colony-stimulating factor and its receptor in the developing human fetus. Pediatr Res. 1999, 46: 358-366.View ArticlePubMedGoogle Scholar
- Wong RL, Lingenheld EG, Fitzgerald L, Clark RB: Murine T helper cell clones secrete granulocyte-macrophage colony-stimulating factor (GmCSF) by both interleukin-2-dependent and interleukin-2-independent pathways. Cell Immunol. 1989, 123: 445-455. 10.1016/0008-8749(89)90304-3.View ArticlePubMedGoogle Scholar
- Flugel A, Berkowicz T, Ritter T, Labeur M, Jenne DE, Li Z, Ellwart JW, Willem M, Lassmann H, Wekerle H: Migratory activity and functional changes of green fluorescent effector cells before and during experimental autoimmune encephalomyelitis. Immunity. 2001, 14: 547-560. 10.1016/S1074-7613(01)00143-1.View ArticlePubMedGoogle Scholar
- Krasowska-Zoladek A: Kinetics of inflammatory response of astrocytes induced by TLR 3 and TLR4 ligation. J Neurosci Res. 2007, 85: 205-212. 10.1002/jnr.21088.View ArticlePubMedGoogle Scholar
- Park C: TLR3-mediated signal induces proinflammatory cytokine and chemokine gene expression in astrocytes: differential signaling mechanisms of TLR3-induced IP-10 and IL-8 gene expression. Glia. 2006, 53: 248-256. 10.1002/glia.20278.View ArticlePubMedGoogle Scholar
- Matsukura S, S WS: Synthetic double-stranded RNA induces multiple genes related to inflammation through Toll-like receptor 3 depending on NF-kappaB and/or IRF-3 in airway epithelial cells. Clin Exp Allergy. 2006, 36: 1049-1062. 10.1111/j.1365-2222.2006.02530.x.View ArticlePubMedGoogle Scholar
- Peck OM: Differential regulation of cytokine and chemokine production in lipopolysaccharide-induced tolerance and priming. Cytokine. 2004, 26: 202-208. 10.1016/j.cyto.2004.02.011.View ArticlePubMedGoogle Scholar
- Fan H: Differential regulation of lipopolysaccharide and Gram-positive bacteria induced cytokine and chemokine production in splenocytes by Galphai proteins. Biochim Biophys Acta. 2006, 1763: 1051-1058. 10.1016/j.bbamcr.2006.08.003.View ArticlePubMedGoogle Scholar
- Lendemans S: GM-CSF priming of human monocytes is dependent on ERK1/2 activation. J Endotoxin Res. 2006, 12: 10-20. 10.1179/096805106X89107.View ArticlePubMedGoogle Scholar
- Bozinovski S: Granulocyte/macrophage-colony-stimulating factor (GM-CSF) regulates lung innate immunity to lipopolysaccharide through Akt/Erk activation of NFkappa B and AP-1 in vivo. J Biol Chem. 2002, 277: 42808-42814. 10.1074/jbc.M207840200.View ArticlePubMedGoogle Scholar
- Iliev AI, Stringaris AK, Nau R, Neumann H: Neuronal injury mediated via stimulation of microglial toll-like receptor-9 (TLR9). Faseb J. 2004, 18: 412-414.PubMedGoogle Scholar
- Dalpke AH, Schafer MK, Frey M, Zimmermann S, Tebbe J, Weihe E, Heeg K: Immunostimulatory CpG-DNA activates murine microglia. J Immunol. 2002, 168: 4854-4863.View ArticlePubMedGoogle Scholar
- Takeshita S, Takeshita F, Haddad DE, Janabi N, Klinman DM, Dalpke AH, Schafer MK, Frey M, Zimmermann S, Tebbe J, Weihe E, Heeg K, Iliev AI, Stringaris AK, Nau R, Neumann H: Activation of microglia and astrocytes by CpG oligodeoxynucleotides Immunostimulatory CpG-DNA activates murine microglia Neuronal injury mediated via stimulation of microglial toll-like receptor-9 (TLR9). Neuroreport. 2001, 12: 3029-3032. 10.1097/00001756-200110080-00010.View ArticlePubMedGoogle Scholar
- Wang J: CpG-independent synergistic induction of beta-chemokines and a dendritic cell phenotype by orthophosphorothioate oligodeoxynucleotides and granulocyte-macrophage colony-stimulating factor in elutriated human primary monocytes. J Immunol. 2005, 174: 6113-6121.View ArticlePubMedGoogle Scholar
- Sandler AD: CpG Oligonucleotides Enhance the Tumor Antigen-specific Immune Response of a Granulocyte Macrophage Colony-stimulating Factor-based Vaccine Strategy in Neuroblastoma. Cancer Research. 2003, 63: 394-399,.PubMedGoogle Scholar
- Liu HM: Immunostimulatory CpG Oligodeoxynucleotides Enhance the Immune Response to Vaccine Strategies Involving Granulocyte-Macrophage Colony-Stimulating Factor. Blood. 1998, 92: 3730-3736.PubMedGoogle Scholar
- Hunter CA, Roberts CW, Alexander J: Kinetics of cytokine mRNA production in the brains of mice with progressive toxoplasmic encephalitis. Eur J Immunol. 1992, 22: 2317-2322. 10.1002/eji.1830220921.View ArticlePubMedGoogle Scholar
- Malipiero UV, Frei K, Fontana A: Production of hemopoietic colony-stimulating factors by astrocytes. J Immunol. 1990, 144: 3816-3821.PubMedGoogle Scholar
- Fischer HG, Nitzgen B, Reichmann G, Hadding U: Cytokine responses induced by Toxoplasma gondii in astrocytes and microglial cells. Eur J Immunol. 1997, 27: 1539-1548. 10.1002/eji.1830270633.View ArticlePubMedGoogle Scholar
- Lyons JA, Zhao ML, Fritz RB: Pathogenesis of acute passive murine encephalomyelitis II. Th1 phenotype of the inducing population is not sufficient to cause disease. J Neuroimmunol. 1999, 93: 26-36. 10.1016/S0165-5728(98)00185-4.View ArticlePubMedGoogle Scholar
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.