Platelet-activating factor enhancement of calcium influx and interleukin-6 expression, but not production, in human microglia

Calcium-sensitive fluorescence microscopy and molecular biology analysis have been used to study the effects of platelet-activating factor (PAF) on intracellular calcium [Ca2+]i and IL-6 expression in human microglia. PAF (applied acutely at 100 nM) elicited a biphasic response in [Ca2+]i consisting of an initial rapid increase of [Ca2+]i due to release from internal stores, followed by a sustained influx. The latter phase of the [Ca2+]i increase was blocked by SKF96365, a non-selective store-operated channel (SOC) inhibitor. RT-PCR analysis showed PAF treatment of microglia induced expression of the pro-inflammatory cytokine IL-6 in a time-dependent manner which was blocked in the presence of SKF96365. However, ELISA assay showed no production of IL-6 was elicited at any time point (1–24 h) for microglial exposures to PAF. These findings suggest that PAF stimulation of human microglia induces expression, but not production, of IL-6 and that SOC-mediated [Ca2+]i influx contributes to the enhanced expression of the cytokine.


Background
Microglia are resident, immunocompetent cells in the brain. They show functional plasticity and can be activated by a diversity of inflammatory stimuli including ones associated with neurodegenerative diseases [9,18]. The functional responses of microglia following activation include proliferation, phagocytosis and secretion. In the latter case microglia can secrete pro-and anti-inflammatory cytokines, chemokines, neurotrophic factors and excitotoxins such as glutamate [20].
One important inflammatory agent is platelet-activating factor (PAF), an alkyl ether phospholipid compound, which both stimulates and is produced by microglia [13]. PAF contributes to inflammatory responses in the brain and is reported to be upregulated in CNS pathophysiology [2,17]. Acute application of PAF to human microglia induces a biphasic change in levels of intracellular Ca 2+ ([Ca 2+ ] i ) with an initial rapid phase due to intracellular release from endoplasmic reticulum (ER) stores and a secondary phase due to influx through store operated channels (SOC) [15,31]. Importantly, SOC has been shown to exhibit sustained activation following stimulation of human [31] and rodent [29] microglia. Prolonged entry of Ca 2+ through SOC in stimulated microglia could constitute a coupling signal between an activating stimulus and cellular functional response. Indeed, the involvement of sustained Ca 2+ responses has been reported as a factor in the production of arachidonic acid by rat microglia [23].
The pro-inflammatory cytokine IL-6 is released from activated microglia and mediates inflammatory responses in brain. Levels of IL-6 in serum and cerebrospinal fluid have been found to be elevated in stroke patients [8,28] and the cytokine has also been implicated in the etiopathology of neurodegenerative disorders such as Alzheimer's disease (AD), Parkinson's disease (PD) and HIV encephalopathy [3,14,25]. Interestingly, some evidence is also available suggesting that under some conditions elevated levels of IL-6 in brain may actually be beneficial [27].
In this study we have examined a role for SOC mediated [Ca 2+ ] i influx in mediating actions of the inflammatory stimulus PAF to induce IL-6 in human microglia.

Preparation of cells
The procedures for the isolation of human microglia have been previously reported [24]. In brief, human embryonic brain tissues were dissected into small blocks, incubated in phosphate-buffered saline (PBS) containing 0.25% trypsin and 40 µg/ml DNase and then dissociated into single cells by repeated pipetting. Cells were plated in T75 flasks in a medium consisting of Dulbecco's modified Eagle's medium (DMEM) containing 5% horse serum, 5 mg/ml glucose, 25 µg/ml gentamicin, and 2.5 µg/ml amphotericin B. Freely floating microglia were harvested from a medium of mixed cell cultures after 7-10 days of growth in culture flasks and plated on aclar coverslips for identification, on poly-L-lysine-coated glass coverslips for calcium spectrofluorometry and plated on six-well multiplates for RT-PCR or ELISA. CD11b and ricinus communis agglutinin (RCA), specific markers for microglia, were used to confirm purity of the culture which was in excess of 98% [24,30].

Calcium spectrofluorometry
The procedures used for measurement of intracellular Ca 2+ have been reported [6,31] Following a 20 min wash in dye-free solution, coverslips were placed on the stage of a Zeiss Axiovert inverted microscope employing a ×40 quartz objective lens. Cells were exposed to alternating wavelengths of 340/380 nm at 6 s intervals and emission light passed through a 510 nm filter. An imaging system (Empix Imaging, Mississauga, ON) was used to record fluorescence ratios using a CCD camera (Retiga 1300i, Burnaby, BC). Fluorescence ratios were determined and converted to values of [Ca 2+ ]i using published procedures [11]. All experiments were done at room temperature (20-22°C).
Reverse transcription-PCR and ELISA assay IL-6 expression was detected with the reverse-transcriptase polymerase chain reaction (RT-PCR). Isolation of RNAs was performed using TRIzol (Gibco-BRL, Gaithersburg, MD, USA) and DNA contamination was eliminated using DNase. cDNA synthesis was done using M-MLV reverse transcriptase (Gibco-BRL). The sequences for the human specific primers for IL-6 as follows: sense primer: 5'-GTGTGAAAGCAGCAAAGAGGC-3'; antisense primer: 5'-CTGGAGGTACTCTAGGTATAC-3'. Human-specific IL-6 signals were generated with the GeneAmp thermal cycler and Amplitaq Gold DNA polymerase (Applied Biosystems, Foster City, CA). The conditions for PCR were as follows: initial denaturation at 95°C for 6 min followed by 28 cycles of denaturation at 95°C for 45 sec, annealing at 56°C for 1 min and extension at 72°C for 1 min. A final extension step at 72°C for 10 min was carried out. PCR products (159 bp) were identified using 1.5% agarose gels containing ethidium bromide and visualized under UV light. GAPDH was used as a reaction standard and human specific primer sequences were as follows: sense primer: 5'-CCATGTTCGTCATGGGTGTGAACCA-3'; antisense primer: 5'-GCCAGTAGAGGCAGGGATGATGTTC-3'. The intensities of each band were measured using NIH image J 1.24 software (National Institutes of Health, Bethesda, MD). Relative mRNA levels for each treatment were normalized to GAPDH.
Enzyme-linked immunosorbent assays (ELISA) were performed according to manufacturer instructions (R & D systems, Minneapolis, MN). Cells were plated on multiwell plates (≈10 5 cells/well) and treated with PAF (100 nM) in the absence or presence of SKF96365 (20 µM for 8 hr). The cell-free supernatants were used for analysis of IL-6 production (kit detects IL-6 as low as 0.7 pg/ml). Values were expressed as means ± SEM and statistical significance (p < 0.05) was determined using one-way ANOVA and Newman-Keuls multiple comparison post-test. 2+ ]i from human microglia have previously been reported [15,21,31]. Initial study showed a transient increase in SOC [31] but more recent work has shown PAF application to evoke a sustained phase of SOC following an initial component due to depletion of Ca 2+ from intracellular stores [15,21]. The differences in PAF responses is considered in the Discussion.

Effects of SKF96365 on SOC-mediated [Ca 2+ ] i influx by PAF PAF-induced changes in [Ca
A representative response to acute application of PAF (applied at 100 nM) is presented in Fig 1A (n = 18 cells). A plateau level of [Ca 2+ ]i was sustained for a duration exceeding 2 min after removal of PAF. Following establishment of a clearly defined plateau phase, the bath solution was replaced with Ca 2+ -free PSS. This procedure caused an immediate decline in [Ca 2+ ]i to baseline levels ( Fig 1A). Long durations of SOC-mediated influx of Ca 2+ have also been documented in mouse microglial cells [29].
The results of application of the SOC inhibitor SKF96365 (at 20 µM) to the plateau phase of a PAF response is shown in the representative recording of Fig 1B (n = 21  cells). SOC-mediated entry of Ca 2+ was reduced to baseline values by SKF96365. Amplitude of Ca 2+ influx through SOC was measured as the difference between baseline and plateau levels and in five independent experiments (n = 107 cells) the amplitude prior to SKF96365 was 140 ± 21 nM and after SKF96365 was at baseline levels. Previous work has shown SKF96365 pretreatment of human microglia (50 µM for 5 min) abolished a transient SOC in the cells [31].

Effects of SKF96365 on microglial expression of IL-6
We next examined effects of PAF on expression of the proinflammatory cytokine IL-6 in the absence and presence of SOC inhibition. The time-dependence of PAF stimulation (100 nM) of human microglia on IL-6 are presented in Fig 2A. The representative RT-PCR showed no constitutive expression of IL-6 in unstimulated microglia (lane 1 of Fig 2A). IL-6 was maximally expressed at 1 h of exposure to PAF then declined to lower levels at longer treatment times (longest exposure of 6 h). A similar timedependence for IL-6 expression was exhibited in a total of four experiments.
A one hour exposure of human microglia to PAF was chosen for subsequent RT-PCR analysis. As shown in Fig 2B, constitutive expression of IL-6 was absent (lane 1). PAF treatment was effective in stimulating expression of the cytokine (Fig 2B, lane 2). The expression of IL-6 was abolished when SKF96365 was included with the PAF applica-tion (Fig 2B, lane 3). No evident IL-6 expression was observed for PAF application in Ca 2+ -free PSS (Fig 2B, lane  4). SKF96365, applied alone in PSS solution, did not cause any increase in IL-6 ( Fig 2B, lane 5).
It was of interest to compare PAF as an inducer of microglial IL-6 to that of LPS (lipopolysaccharide) a potent inflammatory stimulus of cells. The results of exposure of human microglia to LPS (100 ng/ml for 6 h) is presented in Fig 2B (lane 6) showing LPS stimulation caused an intense band for IL-6. Altering the number of PCR cycles had no apparent effect on intensity (data not shown) suggesting IL-6 band saturation with LPS ( Fig 2B, lane 6). Comparison of band intensity indicated LPS was a more effective inducer of IL-6 relative to PAF. Interestingly, a partial inhibition of LPS-induced IL-6 mRNA was observed when SKF96365 was applied with LPS ( Fig 2B,  lane 7). Fig 2C  and shows PAF as an effective stimulator of IL-6 expression (n = 3). However, expression of IL-6 was considerably lower with PAF as a stimulus compared with LPS ( Fig  2B,C). Inclusion of SKF96365 with PAF or application of PAF in Ca 2+ -free PSS eliminated expression of IL-6 (n = 3). Although LPS was not the subject of this study, the decrease in LPS induction of IL-6 with SKF96365 is of interest and is discussed below.

ELISA assay for effects of PAF on microglial production of IL-6
We next investigated production of IL-6 from PAF-treated human microglia using an exposure time of 8 h. No production of IL-6 was evident in four experiments (data not shown); levels of IL-6 were below the detection levels for ELISA assay (≤ 1 pg/ml). In order to determine if the treatment time was a limiting factor in IL-6 production, a series of experiments using different microglial times of exposure to PAF were undertaken (from 1-24 h). The results are presented in Fig 3; no significant production of IL-6 (n = 4) was found for any treatment time (PAF applied for 1,2,8 or 24 h).
We also examined if a ten-fold increase in PAF concentration (to 1 µM) would be effective in producing IL-6. As shown in Fig 3, this higher concentration of PAF also had no effect to induce IL-6 production for treatment times of 8 or 24 h (n = 3 independent experiments). The effects of LPS stimulation were also determined in these experiments (using 100 ng/ml for 8 h). Microglia, treated with LPS, produced high concentrations of IL-6 to levels exceeding 400 pg/ml (n = 4 independent experiments). Expression of IL-6 in PAF treated human microglia Figure 2 Expression of IL-6 in PAF treated human microglia. A: RT-PCR analysis for different exposure times of microglia to PAF (applied at 100 nM). B: Effects of PAF, PAF plus SKF96365, PAF plus Ca 2+ -free and SKF96365 applied alone (1 h treatments). Also shown are effects of LPS and LPS plus SKF96365 (6 hr treatments). GAPDH was used as a reaction standard. C: Semi-quantitative RT-PCR for effects of the different treatments. * P < 0.05 compared with unstimulated control; # P < 0.05 compared with PAF treated microglia.

Discussion
The results from this work indicate that PAF-mediated changes in [Ca 2+ ] i are involved in the cellular expression of the pro-inflammatory agent, IL-6 in human microglia. In essence, activation of SOC acts as a transcriptional control for expression of IL-6. Our results show that inhibition of SOC with SKF96365 blocked both the influx of Ca 2+ and microglial expression of IL-6. However, PAFinduced expression of IL-6 (Fig 2) did not translate into production of the cytokine (Fig 3). This result could suggest that an additional signal or factor may be required for microglial secretion of IL-6.
As found for other types of unexcitable cells, microglia do not normally express voltage-dependent Ca 2+ channels [7]. The sustained entry of Ca 2+ through SOC is likely an important pathway for microglial responses to specific inflammatory stimuli [15,22,26]. Although opening of SOC is required for re-filling of ER stores, other roles for this influx pathway have not been well established. Activation of SOC is necessary for expression of IL-6 but an additional signal is required to produce the pro-inflammatory cytokine in human microglia. The activation state of human microglia may influence the extent of Ca 2+ influx through SOC. Microglia showing an ameboid morphology are considered representative of an activated state whereas cells with a ramified morphology are considered quiescent. We have found sustained SOC responses from PAF-stimulated microglia in cells demonstrating ameboid morphology [15,21] and also in the present work. However, an initial study using a mixture of ameboid and ramified shaped cells, showed a transient SOC response with stimulation by PAF [31]. Further work will be useful to correlate expression of SOC with cell activation.
A recent review has provided a detailed overview of ATP as an inducer of IL-6 expression and production in MG-5 microglial cell line [12]. ATP and the purinergic agonist BzATP were both effective in increasing expression of IL-6 with effects involving activation of the p38 MAPK pathway. However, ATP (activator of both metabotropic P2YR and ionotropic P2XR) but not BzATP (activator of the ionotropic subtype P2X 7 R), was found to induce production of the cytokine. The role of SOC in MG-5 cell responses is unclear since ATP evokes a monophasic change in [Ca 2+ ]i due to P2YR dependent release from intracellular stores. In human microglia we have attributed the lack of a SOC phase of [Ca 2+ ]i due to concomitant ATP binding to some P2XR (not P2X 7 R) causing cellular depolarization and block of Ca 2+ influx [6].
PAF induction of IL-6 was found to be time-dependent (Fig 2A) in addition to the dependence on the presence of extracellular Ca 2+ and SOC (Fig 2B). We observed no IL-6 expression at one-half hour and a maximal level at one hour of microglial exposure to PAF. Little or no IL-6 was expressed with longer PAF treatments of microglia. Inhibition of endoplasmic reticulum Ca 2+ ATPase (SERCA) has been reported to increase IL-6 mRNA expression in rodent macrophages within 15 min [4,19]. Blockade of SERCA, by compounds such as thapsigargin, and subsequent depletion of intracellular stores is a stimulatory protocol for activation of SOC. However, SOC-mediated entry of Ca 2+ was not determined in the rodent studies.
Although PAF was an effective stimulator of IL-6 expression in human microglia, LPS elicited a higher expression of the cytokine. Indeed, bands for IL-6 appeared saturated ( Fig 2B) and showed no change in intensity with increased number of PCR cycles (data not shown). Saturation with LPS would prevent a quantitative comparison between PAF and LPS as activating stimuli for microglial expression of IL-6 ( Fig 2C). An interesting observation was that SKF96365 partially inhibited the LPS-induced expression of IL-6 ( Fig 2C). Although LPS has been reported to act in a Ca 2+ -independent manner on macrophages [19], several studies have found the bacterial compound evokes changes in [Ca 2+ ]i in microglia/ ELISA assays for production of IL-6 in human microglia Figure 3 ELISA assays for production of IL-6 in human microglia. PAF (at 100 nM) induced no significant production of IL-6 from microglia following exposures from 1-24 h (n = 4 for each time points). PAF (at 1 µM) induced no significant production of IL-6 (following exposures for 8 h and 24 h; n = 3 for both time points); these values are near the lower limits for sensitivity of the ELISA kits. LPS was used as a positive control in these experiments (n = 4); note the change of scale for the ordinate (from 10 to 400 pg/ml). * P < 0.05 compared with unstimulated control.
The present results may have relevance to roles of IL-6 in aging. Several studies have provided evidence for agedependent increases in levels of IL-6 in rodent brain [reviewed in [10]]. For example, one finding was that brains from older mice showed considerable elevations in expression and production of IL-6 compared with brains from younger animals [33]. This result was correlated with microglial production of the cytokine [33]. It will be of interest to determine if PAF-stimulated adult human microglia are more potent producers of IL-6 compared with fetal human cells.