Dulbecco's modified Eagle's medium (DMEM), penicillin, streptomycin, 0.05% (w/v) trypsin/EDTA, and phosphate-buffered saline (PBS) were obtained from GIBCO-BRL (Gaithersburg, MD, USA). Cytokines (TNFα, IL-1β, and IFNγ) were purchased from R & D Systems (Minneapolis, MN, USA). Lipopolysaccharide (LPS) (rough strains) from Escherichia coli F583 (Rd mutant) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Fetal bovine serum was from Atlanta Biologicals (Lawrenceville, GA, USA). Methylthiazolyldiphenyl-tetrazolium bromide (MTT) was from Sigma-Aldrich (St. Louis, MO, USA). Antibodies for Western blot are: (1) sPLA2-IIA human, rabbit polyclonal antibody (BioVendor, Candler, NC); (2) goat anti-rabbit IgG- horseradish peroxidase (Santa Cruz Biotechnology, Santa Cruz, CA); and (3) monoclonal anti-β-actin peroxidase (Sigma - Aldrich, St. Louis, MO). Antibodies for immunohistochemistry are: (1) anti-sPLA2-IIA polyclonal antiserum (Cayman Chemical, Ann Arbor, MI); (2) anti-GFAP monoclonal antibody for astrocytes (Millipore, Billerica, MA); (3) CD11b antibody (Abcam Inc., cat # ab63317 Inc, Cambridge, MA); (4) fluorescein isothiocyanate (FITC)-labeled goat anti-mouse and Texas red-labeled goat anti-rabbit secondary antibody (Santa Cruz Biotechnology, Santa Cruz, CA); and (5) Rhodamine-phalloidin (Molecular Probes, Eugene, OR) for F-actin.

Preparations of primary astrocytes and microglial cells involved pregnant Sprague-Dawley rats and C57BL/6 mice (Harlan, IN, USA) and 1-3 day-old pubs. All animal care and experimental protocol with post-natal pups were carried out in accordance with NIH guidelines and with the University of Missouri Animal Care and Use Committee (protocol #6728).

The immortalized mouse microglial cells (BV-2) were originally obtained from Dr. R. Donato (University of Perugia, Italy) and cultured as described previously 9 . Briefly, cells were cultured in 75 cm² flasks with DMEM (high glucose) supplemented with 5% FBS containing 100 units/ml penicillin and 100 μg/ml streptomycin, and maintained in 5% CO₂ incubator at 37°C. For subculture, cells were removed from the culture flask with a scraper, re-suspended in the culture medium and subcultured in 12-well (0.4 × 10⁶) or 6-well (1.0 × 10⁶) plates for experiments. In some experiments, cells were cultured in cover slips and used for immunostaining.

The immortalized rat microglial cell line HAPI was a generous gift from Dr. J. Hong (Laboratory of Toxicology and Pharmacology, National Institute of Environmental Health Sciences, National Institute of Health, Research Triangle Park, NC). The immortalized rat astrocytes, DITNC, were obtained from ATCC (Rockville, MD, USA). Both HAPI and DITNC cells were cultured in DMEM (high glucose), 10% FBS, 100 units/ml penicillin, and 100 μg/ml streptomycin and maintained in 5% CO2 at 37°C. To harvest HAPI microglia and DITNC astrocytes, cells were treated with 0.05% trypsin/EDTA for 2 minutes at 37°C, and centrifuged at 125 g for 10 min. The cell pellets were re-suspended in culture medium. Cell concentration was determined by counting cells with a hemocytometer. Cells were subcultured in 12-well (0.4 × 10⁶) or 6-well (1.0 × 10⁶) plates for experiments.

Primary astrocytes were prepared from the cerebral cortices of 1- 3 day-old Sprague-Dawley rats (Harlan, IN, USA) as described by McCarthy and deVellis 27 with slight modifications 28 . Briefly, cerebral cortices were dissected and meninges removed. The tissues were minced and suspended in 10 volumes 0.05% (w/v) trypsin/EDTA and incubated for 10 min at 37°C. The cell suspension was passed through a 14-gauge needle 5 times, and then filtered through 85 mm nylon mesh. The filtrate was sedimented by centrifugation at 200 g for 5 min and re-suspended in 10% FBS in DMEM containing 100 units/ml penicillin and 100 μg/ml streptomycin. Finally, cells were transferred to 75 cm² culture flasks and fresh medium was changed the next day and then every 2 days afterwards. When cells became confluent, normally within 7-9 days, flasks were shaken at 200 rpm on an orbital shaker (Fisher Scientific, St. Louis, MO) for 4 h at room temperature to remove microglial cells. After shaking, cells were rinsed three times with phosphate-buffered saline (PBS), suspended in trypsin-containing solution as above, and subcultured in 12-well plates for Griess reaction experiment and 6-well plates for Western blot analysis. These cultures contained over 95% astrocytes, as determined by immunostaining for glial fibrillary acidic protein (GFAP). For immunohistochemistry experiments, astrocytes were cultured on Poly-L-Lysine Coated Glass Coverslips (12 mm Round No. 1 German Glass) (BD Biosciences, San Jose, CA). Cells were starved for 4 h prior to experimentation in serum free DMEM medium and followed by treatments with different conditions as described.

For preparation of primary microglial cells, rat (Sprague-Dawley) or mouse (C57BL/6) pups less than 4 days of age were used. The protocol was similar to that used for preparation of primary astrocytes. Briefly, after removing the meninges, brain tissue was minced into small pieces and trypsinized by incubating tissue at 37°C for 20 min. Brain tissue was triturated with a pipet to further dissociate clumps and filtered with a 70 μm cell strainer. Cells were centrifuged at 1,200 rpm for 5 min at 4°C, and pellet was suspended in 30 ml of complete medium containing DMEM with high glucose, 10% FBS, OPI (1 mM oxaloacetate, 0.45 mM pyruvate, and 0.2 U/ml insulin), and GM-CSF (0.5 ng/ml) to enhance proliferation of microglia. The cell suspension was added to 75 cm² flasks (about 6 brains per flask of 30 ml). Cells were incubated in flasks until confluent for 7-10 days. Microglial cells were separated from astrocytes and oligodendrocytes by shaking the flasks in a rotary platform in a 37°C incubator at 200 rpm overnight. The supernatant, which was enriched with microglial cells, was then removed and centrifuged at 1200 rpm for 45 min. The microglia population was established by immunostaining with CD11b antibody. Purity for these microglial cells was determined to be around 95% (data not shown). The cells were plated for experiments using complete media without the GM-CSF.

In all experiments, cells were serum starved for 4 h prior to adding cytokines and LPS. Cell morphology was observed by using a phase contrast Nikon DIAPHOT 300 microscope attached with a CCD cool camera linked to MagnaFire 2.1C software for image processing. Representative bright field pictures were obtained using a 20× objective lens.

Our previous studies demonstrated that NO production in glial cells was mainly due to the induction of iNOS 9 . Therefore, measurement of NO was used to represent the induction process. NO released from cells was converted to nitrite in the culture medium, which was determined using the Griess reagent. In this study, cells were cultured in DMEM without phenol red. After treating cells with cytokines and LPS, aliquots (200 μl) of culture medium were transferred to test tubes and incubated with 100 μl of the reagent A (1% (w/v) sulfanilamide in 5% phosphoric acid, Sigma) for 10 minutes at room temperature in the dark. This was followed by incubation with 100 μl of reagent B (0.1%, w/v, N-1-napthylethylenediamine dihydrochloride, Sigma) for 10 minutes at room temperature in the dark. After mixing, 100 μl of the purple/magenta solution was transferred to a 96-well plate and the absorbance at 543 nm was measured within 30 minutes in a plate reader. The dilution series of sodium nitrite (0-100 μM) was used to generate the nitrite standard reference curve.

After treating cells with cytokines and LPS, cells were washed twice with ice-cold phosphate-buffered saline and harvested in lysis buffer containing 50 mM Tris-HCl (pH 7.4), 1 mM EDTA, 100 mM NaCl, 0.1% SDS, 1 mM PMSF, 1 mM sodium orthovanadate, 1 μg/ml leupeptin, 1 μg/ml pepstatin, and 10 μg/ml aprotinin. The extract was centrifuged at 10,000 × g for 15 minutes at 4°C in order to get rid of cell debris. Protein concentration was determined by using a BCA protein assay kit (Pierce Biotechnology, Rockford, IL) according to the manufacturer's instructions. Equivalent amounts of protein (25 μg) for each sample were resolved in 12% Tricine-SDS-PAGE at 120 V in duplicates. After electrophoresis, proteins were transferred to 0.2 μm PVDF membranes at 250 mA for 2 h. Membranes were incubated in Tris-buffered saline, pH 7.4 (TBS) with 0.1% Tween 20 (TBS-T) containing 5% non-fat milk for 1 h at room temperature. The blots were then incubated with sPLA2-IIA polyclonal antibody (1:2500; BioVendor, Candler, NC) overnight at 4°C. After washing with TBS-T, blots were incubated with goat anti-rabbit IgG- horseradish peroxidase (1:5000; Santa Cruz Biotechnology, Santa Cruz, CA) for 1 h at room temperature. The blots were then washed three times with TBS-T. Immunolabeling was detected by chemiluminescence (SuperSignal West Pico, Pierce, Rockford, IL). For loading control, the blots were reacted with monoclonal anti-β-actin peroxidase (1:30,000, Sigma - Aldrich, St. Louis, MO). For quantification, blots were scanned and the intensity of protein bands was measured as optical density using the Quantity One program (BioRad, Hercules, CA). sPLA2-IIA bands were detected at 15 kDa. Ratios of sPLA2-IIA to β-actin were calculated for each sample.

DITNC cells and primary astrocytes were plated onto poly-L-lysine coated glass coverslips. After treatments, cells were fixed in 4% paraformaldehyde in PBS (pH 7.4) for 15 min at room temperature. After washing three times with PBS, samples were incubated for 10 min with PBS containing 0.5% Triton-X-100. Nonspecific binding of antibodies was blocked by 5% normal goat serum (NGS) for 1 h at room temperature. Cells were then incubated overnight at 4°C in 0.5% NGS with anti-sPLA2-IIA polyclonal antiserum (1:50, Cayman Chemical, Ann Arbor, MI), anti-GFAP monoclonal antibody for astrocytes (1:50, Millipore, Billerica, MA), or anti-CD11b antibody for microglial cells (1:100, Abcam Inc, Cambridge, MA). The cells were washed with PBS and incubated for 1 h at room temperature with fluorescein isothiocyanate (FITC)-labeled goat anti-mouse and Texas red-labeled goat anti-rabbit secondary antibody (1:300, Santa Cruz Biotechnology, Santa Cruz, CA), and finally washed again with PBS. Cells were incubated for 10 min with Hoechst 33342 (1: 1000, Invitrogen, Carlsbad, CA) as a counter-stain for nuclei. Cover-slips were then mounted onto microscope slides and fluorescent intensity measurements were performed at room temperature using the Olympus X-41 fluorescence microscope and 40× objective lens.

For immunofluorescence staining of F-actin, BV-2 cells in cover-slips were fixed with 4% paraformaldehyde for 20 min and permeabilized by 0.1% Triton X-100 in PBS for 10 min. Non-specific binding was blocked with 5% normal goat serum (NGS) in PBS at room temperature for 30 min. Cells were then incubated in rhodamine-phalloidin (Molecular Probes, Eugene, OR), diluted 1:100 in PBS for 30 min, and then mounted onto microscope slides and examined using the Leica DMI4000 epifluorescence microscope with 40× objective lens.

After treating cells with cytokines and LPS, total RNA was isolated from cells using the TRIZOL reagent (Sigma-Aldrich, MO, USA). The RNA quality and concentration was evaluated by Nanodrop ND-1000 spectrophotometry (NanoDrop Technologies, Wilmington, DE). OD₂₆₀ was used for the concentration while OD₂₆₀/OD₂₈₀ and OD₂₆₀/OD₂₃₀ were used to evaluate the quality, usually ~1.8-2.2. Total RNA (0.5 μg) was used for reverse transcription to cDNA with oligo dT primers by means of the Advantage RT-for-PCR Kit (Takara Bio, Mountain View, CA) according to the manufacturer's instructions. The volume of cDNA used was 10 μl (from 50 ng RNA). Amplification was carried out in an automated thermal cycler (Eppendorf, Hauppauge, NY) with a 3-min denaturation step at 94°C, followed by 25 cycles including 45 sec at 94°C, 30 sec at 59.5°C, and 30 sec at 72°C. All PCR amplifications were submitted to a final 10-min step at 72°C. Amplified samples were separated on a 2% agarose gel containing ethidium bromide in TAE buffer. After electrophoresis, the gel was viewed by the Kodak electrophoresis documentation and analysis system (Kodak, Rochester, NY). Primers for rat sPLA₂IIA are: sense 5'-CATGGCCTTTGGCTCAATTCAGGT-3'; antisense 5'-ACAGTCATGAGTCACACAGCACCA-3'; and rat G3PDH sense 5'-TGAAGGTCGGTGTCAACGGATTTGGC-3'; antisense 5'-CATGTAGGCCATGAGGTCCACCAC-3' was used as a control.

For study to quantitate filopodia in BV-2 microglia, cells were cultured in 35 mm dish until 80% confluency. Cells were serum starved for 4 h prior to treatment with cytokines and LPS. Since thin processes (filopodia) started to appear after cytokine treatment by 2 h, a 4 h exposure time was used for quantitaion of filopodia. In each treatment condition, cells were observed under the phase contrast Nikon DIAPHOT 300 microscope and three fields with comparable dell densities were chosen. In each field, the total number of cells, as well as cells containing filopodia (processes more than 2 mm), were counted. Results are expressed as % of filopodia-containing cells against the total.

Cell viability was determined using the MTT (3-(4, 5-Dimethylthiazol-2-yl) -2, 5-diphenyltetrazolium bromide) assay protocol. Briefly, cells cultured in 12-well plates were treated with cytokines and LPS. After treatment, the medium was removed and 1 ml of MTT reagent (0.5 mg/ml) in serum free DMEM was added into each well. Cells were incubated for 4 h at 37°C, and after dissolving the formazan dye with DMSO, absorption was read at 540 nm.

Results are analyzed by one-way ANOVA followed by Dunett's multiple comparison tests, or two-way ANOVA (V4.00; GraphPad Prism Software Inc., San Diego, CA). Differences with p < 0.05 are considered significant.

Based on preliminary study and results in Table 1 treating BV-2 microglial cells with a mixture of three cytokines (TNFα, IL-1β, and IFNγ, at 10 ng/ml each) or LPS (100 ng/ml) + IFNγ (10 ng/ml) produce high levels of NO. These conditions were used to examine cell morphology and viability in different glial cell types. In this study, cells were cultured to 90% confluency, and at 4 h prior to treatment with cytokines and LPS, serum was removed from the cultures and replaced with DMEM. Bright field pictures depicting cell morphology with or without cytokine and LPS treatments were obtained at 24 h using the inverted Nikon microscope. As shown in Figure 1, control BV-2 and HAPI cells (incubated for 24 h under serum free condition) are mostly round with bright refringency and small dark nuclei; whereas, cytokine and LPS treatments for 24 h caused cells to become ramified and some are star shaped with short thick processes. Removal of serum retarded cell growth but did not cause morphological changes (data not shown). Control and treated primary mouse and rat microglial cells (derived from primary astrocytes) show similar morphology and responses as compared to immortalized microglial cells (Figure 1).

DITNC astrocytes are triangular shape with spindle-like features, and after treatment with the three cytokine mixture, they became dark with a bright refringency, but did not show obvious morphological changes as compared with microglial cells (Figure 1). Primary rat astrocytes are larger flat cells with irregular shape, and they do not show obvious morphological changes after exposure to cytokines and LPS (Figure 1).

We determined cell viability at 24 h after treating BV-2, HAPI, and DITNC astrocytes with cytokines and LPS + INFγ using the MTT assay protocol. In BV-2 cells, no change in MTT values was observed after exposure with the three cytokine mixture or LPS + INFγ for 12 h (Figure 2A). However, there are obvious decreases in MTT values in BV-2, HAPI, and DITNC cells at 24 h after exposure to cytokine and LPS + INFγ (Figures 2B-2D).

Although earlier studies had demonstrated involvement of the MEK1/2-ERK1/2 pathway in cytokine-induced sPLA2 in DITNC astrocytes 21 and iNOS in BV-2 cells 9 , a time course study to compare p-ERK1/2 activation in these two cell types was not carried out. As shown in Figure 3A, exposure of BV-2 cells to the three cytokine mixture showed a biphasic increase in p-ERK1/2; first a transient earlier phase peaking at 15 min, and then a second phase increase from 1 to 4 h. Exposure of BV-2 cells to LPS + IFNγ did not show the early phase increase, but a similar second phase of increase from 1 to 4 h (Figure 3B). Exposure of DITNC astrocytes to the three cytokine mixture indicated an early phase increase at 15 min and a second increase at 1 h (Figure 3C). Exposure of DITNC astrocytes to LPS + IFNγ also showed an early phase increase in pERK1/2 at 5 min and a subsequent phase at 2 h (Figure 3D). Unlike the BV-2 cells, DITNC astrocytes did not show a dramatic increase in p-ERK1/2 between 1 to 4 h.

We further examined the time course for morphological changes after exposing BV-2 cells to the three cytokine mixture. As shown in Figure 4A, exposure of cytokines to BV-2 cells caused the cells to become elongated with protrusion of short fine processes (filopodia) as early as 1 h. The filopodia continued to become elongated with time and by 8 h, nearly all cells showed filopodia and some have flat pancake-like structures with ruffled edges at the end (red arrows). With increasing time, filopodia started to disappear between 12 to 16 h leaving cells with stout processes as shown in Figure 1. HAPI cells show a similar time-dependent increase in filopodia as in BV-2 cells (data not shown).

Since filopodia were produced after exposing BV-2 cells to the three cytokine mixture (TNFα, IL-1β, and IFNγ) and LPS + IFNγ, we further examined filopodia formation by treating cells with individual cytokines and LPS. As shown in Figure 4B, among the three cytokines tested, filopodia were only induced by IFNγ. Although LPS alone could also induce filopodia formation, the addition of IFNγ further enhanced formation of these processes (Figure 4B).

Since ERK activation has been shown to participate in IFNγ-mediated signaling pathways and cell migration 29 30 , we tested whether p-ERK1/2 plays a role in IFNγ-induced filopodia formation. In this experiment, BV-2 cells were cultured in cover slips and serum starved for 4 h. After preincubated for 30 min with U0126 (10 μM), a specific inhibitor for MEK/ERK, they were exposure to IFNγ for 4 h. After the 4 h treatment, cells were subsequently stained for F-actin with rhodamine-phalloidin, a high-affinity F-actin probe. As shown in Figure 4C and 4D, exposing cells to IFNγ for 4 h resulted in formation of filopodia (white arrows). Treatment of cells with 10 μM of U0126 caused the cells to become round, and pretreatment of U0126 prior to exposure to IFNγ completely abrogated the formation of filopodia induced by IFNγ.

Our earlier studies demonstrated that NO production upon exposure of BV-2 cells to IFNγ and LPS is due mainly to induction of iNOS expression 9 . In this study, a time course experiment to compare NO production due to the three cytokine mixture and LPS + IFNγ indicated a detectable increase from 12 h to 24 h (Figure 5A and 5B). A similar time course for NO production was observed with the HAPI cells. In a subsequent experiment, induction of NO by individual cytokines and LPS was examined in BV-2, HAPI, DITNC and primary rat astrocytes after 24 h exposure. Similar to studies observed with BV-2 cells 9 , TNFα + IL-1β could not induce NO in any of the cell types tested (Table 1). However, IFNγ alone can induce NO in both BV-2 and HAPI microglial cells and IFNγ enhanced NO production induced by LPS (Table 1). Under similar conditions, DITNC and primary rat astrocytes did not respond to IFNγ, but low levels of NO can be observed after exposure to the three cytokine mixture (Table 1).

We further tested whether rat primary microglial cells (RPM) are capable of responding to cytokines and LPS. Due to difficulty in controlling cell numbers in the RPM preparations, data are based on the amount of proteins in the culture dish. As shown in Figure 5C, stimulation of RPM by cytokines and LPS produced similar levels of NO as compared to that in BV-2 cells.

In our previous studies, induction of sPLA2-IIA expression by cytokines had been mainly limited to assay of mRNA expression because of lacking suitable antibodies for protein detection 21 22 28 . Furthermore, information about induction of this inflammatory enzyme by microglial cells had also been lacking. In this study, we established a similar pattern for individual cytokines and LPS to induce sPLA2-IIA mRNA and protein expression in DITNC astrocytes. These results clearly indicated the capability for TNFα, IL-1β and LPS, but not IFNγ, to induce sPLA2-IIA mRNA expression (Figures 6A and 6B) and protein expression (Figures 6C and 6D) in DITNC cells. The highest level of expression was observed after treating cells with the three cytokine mixture. However, when primary astrocytes were treated with cytokines and LPS under similar conditions as for DITNC astrocytes, sPLA2-IIA protein expression was observed only after treatment with the three cytokine mixture (Figure 6E).

We further examined the ability for BV-2 and HAPI cells, as well as primary rat microglial cells, to respond to cytokines and LPS in the induction of sPLA2-IIA mRNA and protein expression. In this study, samples from DITNC astrocytes were used as a positive control. The lack of response in BV-2 cells is expected because these cells are of murine origin. However, it is surprising that cytokines and LPS could not induce sPLA2-IIA mRNA, and protein expression in HAPI cells that are of rat origin (Figure 7A and 7B). In order to further confirm that the lack of response is not due to the immortalization procedure, we tested primary mouse and rat microglial cells and showed that neither cell type could respond to cytokines and LPS to produce sPLA2-IIA (Figure 7C). These results demonstrate that despite the active response to cytokines and LPS in induction of iNOS, microglial cells lack the ability to cause induction of sPLA2-IIA mRNA and protein under cell culture conditions.

In this study, we have successfully used rabbit polyclonal antibodies against human sPLA2-IIA from BioVendor (Candler, NC) for Western blots, but these antibodies were not suitable for immunocytochemical study. Instead, testing with anti-sPLA2-IIA polyclonal antiserum from Cayman Chemical (Ann Arbor, MI) appeared to give positive immunostaining of sPLA2-IIA in DITNC cells and primary rat astrocytes. As shown in Figure 8A, DITNC cells are positive for GFAP, and an increase in sPLA2-IIA immunoreactivity can be shown upon exposing cells to the three cytokine mixture and LPS + IFNγ for 24 h (Figure 8A). Treatment with primary astrocytes with the three cytokine mixture for 48 h also showed an increase in sPLA2-IIA immunoreactivity (Figure 8B). However, double immunostaining of primary astrocytes with GFAP and sPLA2-IIA indicated variances in GFAP and sPLA2-IIA immunoreactivity after exposure to cytokines. In Figure 8B, we identified a cell (pointed by the white arrow) showing little or none immunoreactivity on GFAP, but substantial staining of sPLA2-IIA. In addition, sPLA2-IIA immunoreactivity appeared to be higher in differentiating cells containing multiple nuclei.