Pro-inflammatory cytokines and lipopolysaccharide induce changes in cell morphology, and upregulation of ERK1/2, iNOS and sPLA₂-IIA expression in astrocytes and microglia

Abstract

Background: Activation of glial cells, including astrocytes and microglia, has been implicated in the inflammatory responses underlying brain injury and neurodegenerative diseases including Alzheimer's and Parkinson's diseases. Although cultured astrocytes and microglia are capable of responding to pro-inflammatory cytokines and lipopolysaccharide (LPS) in the induction and release of inflammatory factors, no detailed analysis has been carried out to compare the induction of iNOS and sPLA2-IIA. In this study, we investigated the effects of cytokines (TNF-alpha, IL-1beta, and IFN-gamma) and LPS + IFN-gamma to induce temporal changes in cell morphology and induction of p-ERK1/2, iNOS and sPLA₂-IIA expression in immortalized rat (HAPI) and mouse (BV-2) microglial cells, immortalized rat astrocytes (DITNC), and primary microglia and astrocytes.

Methods/results: Cytokines (TNF-alpha, IL-1beta, and IFN-gamma) and LPS + IFN-gamma induced a time-dependent increase in fine processes (filopodia) in microglial cells but not in astrocytes. Filopodia production was attributed to IFN-gamma and was dependent on ERK1/2 activation. Cytokines induced an early (15 min) and a delayed phase (1 ~ 4 h) increase in p-ERK1/2 expression in microglial cells, and the delayed phase increase corresponded to the increase in filopodia production. In general, microglial cells are more active in responding to cytokines and LPS than astrocytes in the induction of NO. Although IFN-gamma and LPS could individually induce NO, additive production was observed when IFN-gamma was added together with LPS. On the other hand, while TNF-alpha, IL-1beta, and LPS could individually induce sPLA₂-IIA mRNA and protein expression, this induction process does not require IFN-gamma. Interestingly, neither rat immortalized nor primary microglial cells were capable of responding to cytokines and LPS in the induction of sPLA2-IIA expression.

Conclusion: These results demonstrated the utility of BV-2 and HAPI cells as models for investigation on cytokine and LPS induction of iNOS, and DITNC astrocytes for induction of sPLA2-IIA. In addition, results further demonstrated that cytokine-induced sPLA2-IIA is attributed mainly to astrocytes and not microglial cells.

Figure 1

Cytokines (mixture of TNFα, IL-1β, IFNγ) or LPS + IFNγ alter morphology of microglial cells and astrocytes. Cells were cultured in 12-well plates and serum starved for 4 h before treatment with the three cytokine mixture (3 cyt) containing TNFα, IL-1β, and IFNγ at 10 ng/ml each respectively, or LPS (100 ng/ml) + IFNγ (10 ng/ml) for 24 h. Cell morphology was obtained by taking bright field pictures with an inverted Nikon microscope (20×) at 24 h with and without (Control) treatment with cytokines and LPS. Photomicrographs are representative pictures depicting BV-2 (murine) and HAPI (rat) microglial cells, mouse and rat primary microglial cells, and rat immortalized (DITNC) astrocytes and rat primary astrocytes.

Figure 2

Assay for cell viability after treating microglial cells and astrocytes with cytokine mixture or LPS + IFNγ. BV-2 and HAPI microglial cells and DITNC astrocytes cultured in 12-well plate were serum starved for 4 h followed by exposure to the three cytokine mixture (3 cyt) or LPS + IFNγ for the respective time indicated. Cell lysates were obtained for MTT assay. Results are mean ± SD from n = 3. **p < 0.01 vs. control (Con).

Figure 3

Time course for p-ERK1/2 and total ERK1/2 expression after treating BV-2 and DITNC cells to the three cytokine mixture or LPS + IFNγ. BV-2 and DITNC cells were serum starved for 4 h and exposed to 3 cyt or LPS + IFNγ. Cells lysates were obtained at indicated times and subjected to Western blot. Results are representative blots from three independent experiments.

Figure 4

Cytokines induce filopodia production in BV-2 cells. (A) Time course for cytokine-induced filopodia formation in BV-2 cells. Representative bright field photomicrographs were taken with an inverted Nikon microscope (20×). Red arrows show processes with a fan-like ending. (B) Counting cells containing filopodia at 4 h after exposure to individual cytokines, LPS or combination as indicated. Results are expressed as % of filopodia cells versus total cell numbers (see Methods). Results are mean ± SEM from 4 independent experiments. Results are analyzed by one-way ANOVA followed by Dunnett's multiple comparison test, **p < 0.01 vs. control. (C) Staining F-actin in BV-2 cells after treatment with IFNγ and/or MEK1/2 inhibitor U0126. BV-2 cells were cultured in coverslip and serum starved for 4 h. Cells were pretreated with U0126 for 30 min prior to exposure to IFNγ for 4 h. Cells were then fixed with 4% paraformaldehyde and permeabilized by 0.1% Triton X-100 in PBS as described in text. After blocking non-specific binding with 5% normal goat serum (NGS), cells were incubated in rhodamine-phalloidin (1:100) and then mounted onto microscope slides and examined using the Leica DMI4000 automatic epifluorescence microscope with 40× objective lens. Space bar: 20 μm. White arrows denote filopodia. (D) Bar graph representing filopodia-containing cells after incubation with/without IFNγ, U0126, and U0126 + IFNγ. Two-way ANOVA revealed a significant interaction (p = 0.009) between U0126 and IFNγ, and a significant effect of U0126 (p < 0.0001), and IFNγ (p < 0.0001).

Figure 5

Cytokines or LPS + IFNγ induce NO production in different glial cell types. (A and B) BV-2 cells were serum starved in phenol red free DMEM for 4 h prior to treatment with the three-cytokine mixture (A) or LPS + IFNγ(B). At the respective time points, culture media were collected for determination of NO using the Greiss reaction protocol as described in the text. Data are mean ± SD from three independent experiments. **p < 0.01 vs. values at 12 h. One-way ANOVA, Dunnett's multiple comparison tests. (C) Comparing NO production between BV-2 microglial cells and rat primary microglial cells (RPM) based on protein in the dish. Results on RPM preparation have been repeated.

Figure 6

Cytokines induce sPLA2-IIA mRNA and protein expression in DITNC, and primary astrocytes. After exposing DITNC astrocytes to TNFα, IL-1β, IFNγ, and LPS for 24 h, sPLA2-IIA mRNA expression was determined by RT-PCR (A), and protein expression was determined by Western blot (C). Quantitative data are expressed as relative density to G3PDH (sPLA2-IIA vs. G3PDH) (B) or to β-actin (sPLA2-IIA vs. β-actin) (D). Results are mean ± SD from three independent experiments (*p < 0.05, **p < 0.01 vs. control, One-way ANOVA, Dunnett's multiple comparison test). (E) Only the three cytokines mixture could cause induction of sPLA2-IIA in primary astrocytes.

Figure 7

Cytokines or LPS + IFNγ did not induce sPLA2-IIA in microglial cells. sPLA2-IIA mRNA (A) and protein (B) were determined by RT-PCR and Western blot in BV-2 and HAPI cells. In this experiment, sPLA2-IIA induced by DITNC astrocytes was used as positive control. (C) Rat primary microglial cells (RPM) did not produce sPLA2-IIA protein after treating with the three cytokine mixture or LPS + IFNγ. Results are from one typical experiment which has been repeated.

Figure 8

sPLA2-IIA immunoreactivity in DITNC and primary astrocytes. (A) DITNC cells were cultured on coverslips and stimulated with cytokines or LPS + IFNγ for 24 h. (B) primary astrocytes were treated with cytokines or LPS + IFNγ for 48 h. After exposure to cytokines and LPS, cells were permeabilized and double-immunostained with GFAP (left, green) and sPLA2-IIA (middle, red) with merged images (right). Scale bar represents 20 μm.