Lysophosphatidylcholine Stimulates IL-1β Release from Microglia via a P2X₇ Receptor-Independent Mechanism

Primary and secondary microglial cell cultures

Microglia were obtained from brain cell cultures of newborn NMRI mice, supplied by Charles River Breeding Laboratories. Mixed brain cell cultures were prepared as previously described (32). After at least 10 days of incubation, microglia were harvested by shaking the cultures for 30 min at 300 rpm. Isolated microglia were seeded in 6-well plates at a density of 5 × 10⁵/ml and maintained in DMEM supplemented with 10% FCS and 2 mM l-glutamine. Purity of microglial cell preparations was controlled by isolectin-B4 stainings (Molecular Probes). In all preparations, 98–100% of cells stained positive for this microglial/macrophage marker.

BV-2 microglial cells

BV-2 microglial cells were cultured permanently in DMEM supplemented with 10% FCS and 2 mM l-glutamine. BV-2 cells were split twice a week, and were plated on glass coverslips at a density of 1 × 10⁵/ml for subsequent patch-clamp and Ca²⁺ imaging experiments. For ELISA and caspase activity measurements, BV-2 cells were cultured in 6-well plates or 35-mm petri dishes at a density of 5 × 10⁵/ml.

Chemicals

The drugs used in this study were: LPS; synthetic l-α-LPC, palmitoyl (16:0); LPC, stearoyl (18:0); LPC, oleoyl (18:1); GdCl₃; LaCl₃; nigericin; valinomycin; BzATP (2′,3′-O-(4-benzoylbenzoyl)5′ATP); oxidized ATP (oATP); charybdotoxin (CTX; Latoxan); margatoxin (MTX; Peptide Institute); YVAD-CHO (Ac-Tyr-Val-Ala-Asp-aldehyde Bachem); and recombinant murine IL-1β (R&D Systems). If not stated otherwise, drugs were obtained from Sigma-Aldrich. YVAD-CHO was dissolved in DMSO and a 100 mM stock solution was prepared. Nigericin and valinomycin were dissolved in ethanol and stored as 20 and 10 mM stock solutions, respectively. All other drugs were dissolved in extracellular solution.

Solutions

Extracellular solution E₁ contained 130 mM NaCl, 5 mM KCl, 2 mM CaCl₂, 1 mM MgCl₂, 10 mM HEPES, and 10 mM d-glucose (pH 7.4). In some experiments CaCl₂ was omitted from the extracellular solution, while 1 mM EGTA was added and the concentration of MgCl₂ was increased to 4 mM (which became the Ca²⁺-free extracellular solution E₂). The extracellular solution E₃ contained 140 mM NaCl, 2 mM MgCl₂, and 10 mM HEPES (pH 7.4). Intracellular solution I₁ contained 125 mM CsCH₃SO₃, 2 mM MgCl₂, 10 mM HEPES, and 1 mM BAPTA (pH 7.3). Intracellular solution I₂ contained 120 mM KCl, 2 mM MgCl₂, 10 mM HEPES, and 0.1 mM BAPTA (pH 7.3).

Detection of IL-1β by ELISA

In most cases, primary cultured and BV-2 microglial cells were preincubated with 1 μg/ml LPS in DMEM/FCS culture medium at 37°C for 6 h. Immediately before the experiments, cells were washed twice with extracellular solution E₁. Thereafter, cells were treated with 1 ml of DMEM, Ca²⁺-containing extracellular solution E₁ or Ca²⁺-free extracellular solution E₂ in the absence or presence of LPC and inhibitors. After incubation of cells for 1 h at 37°C, supernatants were collected and the concentration of IL-1β was determined. Data were obtained from at least three individual microglia cultures in two to four different wells per experimental condition. For the quantitative determination of IL-1β concentration in microglial cell culture supernatants, Quantikine mouse IL-1β Immunoassay kit (R&D Systems) was used according to the manufacturer’s instructions. The sensitivity of this sandwich ELISA was <3 pg/ml.

Detection of IL-1β by immunoblot analysis

LPS-preactivated primary cultured microglial cells were washed twice with extracellular solution E₁ immediately before experiments. After a subsequent exposure for 1 h to 1 ml of extracellular solution E₁ in the presence or absence of LPC and inhibitors, cell lysates or supernatants were collected and IL-1β contents were determined by immunoblot analysis. Cell supernatants were concentrated using Microcon centrifugal filters (Millipore) with a 3-kDa cutoff. Cells were lysed with 1% Triton X-100 in the presence of a protease inhibitor mix (Complete Mini; Roche). Protein quantification was performed colorimetrically using Pierce bicinchoninic acid protein assay. The amount of total protein was determined according to the manufacturer’s protocol (Pierce). Normalized amounts of cell supernatants or lysates were run on a 10% SDS-polyacrylamide gel and then transferred onto nitrocellulose membranes (Schleicher & Schuell). To reduce nonspecific Ab binding, membranes were bathed in 5% (w/v) low-fat milk in 0.1% (v/v) Tween 20 in PBS for 1 h at room temperature and then washed with 0.1% Tween 20 in PBS. Thereafter, membranes were probed with a polyclonal goat anti-mouse IL-1β Ab (R&D Systems) at 1/250 dilution and were incubated overnight at 4°C, followed by a 1 h incubation with a HRP-conjugated polyclonal donkey anti-goat IgG (1/7500; R&D Systems) at room temperature. Immunoblots were developed using an ECL immunoblotting detection reagent kit (Pierce Supersignal West Femto, Perbio Science). Ab dilutions were prepared in 5% (w/v) low-fat milk in 0.1% Tween 20 in PBS. Quantitative analysis of background-corrected pro-IL-1β and IL-1β band densities was performed using the image processing software analySIS (Olympus Optical).

Detection of IL-1-converting enzyme (ICE)/caspase-1 activity

Activity of ICE/caspase-1 was revealed by FITC-VAD-FMK in situ (Promega) as previously described (35). Microglial cells were incubated with 10 μM FITC-VAD-FMK in extracellular solution during the 1 h of LPC stimulation. Cells were bathed in extracellular solutions E₁ or E₂. After LPC stimulation, cells were washed twice with extracellular solution and then fixed for 10 min with a fixative containing 4% paraformaldehyde, 15% of a saturated solution of picric acid and 0.1% glutaraldehyde in phosphate buffer (pH 7.4). Fluorescence intensity of cells was analyzed using an upright microscope BX51Wi (Olympus Optical) and the image processing software analySIS (Olympus Optical). The fluorescence imaging system consisted of a mercury lamp, a charge-coupled device camera (F-View II, Olympus Optical), an excitation filter of 470–490 nm wavelength, a dichroic mirror of 505 nm wavelength, and a barrier filter of 510–550 nm wavelength (all from Olympus Optical). Images of four different visual fields for three independent experiments per condition were collected and analyzed. Fluorescence intensities of all cells were corrected for background fluorescence.

Cell viability test

The influence of LPC on membrane integrity was investigated using ethidium bromide (Molecular Probes) staining as previously described (36). Cells were treated with or without 30 μM LPC and incubated in extracellular solution E₁ at 37°C for 1 h. Then, cells were washed and incubated with extracellular solution E₁ containing 10 μM ethidium bromide for 10 min. After washing, petri dishes were mounted on an Olympus IX 50 microscope and cells were excited with monochromatic light at a wavelength of 530 nm. Images of four different visual fields for five independent experiments per condition were collected. As a positive control for ethidium bromide staining cells were damaged by freezing and subsequent thawing.

Electrophysiological recordings

Whole cell membrane currents and membrane potentials were measured using an EPC-9 Patch Clamp Amplifier (HEKA Electronics). The amplifier was interfaced to an IBM computer for pulse application and data recording. Series resistance compensation was routinely used to reduce the effective series resistance by ∼70%. Patch electrodes of 2–4 MOhms were fabricated on a two-stage Narishige PP-83 puller from borosilicate glass (outer diameter at 1.5 mm and inner diameter at 1 mm; Hilgenberg). Solutions I₁ and E₃ were used for measurements of nonselective cation currents, while solutions I₂ and E₁ were used for measurements of Ca²⁺-activated K⁺ currents and of membrane potentials. All recordings were done at room temperature (20–23°C). Whole cell currents were filtered at 3 kHz and stored on computer disk for subsequent analyses. Analyses were performed on IBM computers with the Pulse/PulseFit program (HEKA Electronics). Data were corrected for liquid junction potentials. Data are presented as mean ± SEM. The number of experiments is indicated.

Ca²⁺ imaging

Microglial cells were loaded with 3 μM fura 2-AM (Molecular Probes) in extracellular solution for 30 min at room temperature (20–23°C). After washing, coverslips were mounted in a chamber on an inverted Olympus IX 50 microscope equipped with a water immersion objective ×40 UApo/340. The fluorescence imaging system consisted of a monochromator, a CCD camera and the Windows NT based image processing software (Till Photonics). Microglial cells were exposed to alternating 340 ± 5 nm and 380 ± 5 nm wavelengths of UV light and emission light was passed through a 400 nm dichroic mirror and a 420 nm long pass emission filter (both Olympus Optical) before acquisition by the CCD camera. Images were collected every 20 s. In some experiments images were collected every 5 s. The ratio of the two background corrected fluorescence intensities was converted to the intracellular Ca²⁺ concentration ([Ca²⁺]_i) of a single cell according to the equation of Grynkiewicz et al. (37): [Ca²⁺]_i = K_dβ (R−R_min)/(R_max−R), where K_d = 224 nM is the dissociation constant of fura and R is the measured ratio. R_min (0.375) and R_max (3.930) were determined using 10 μM ionomycin in extracellular solution containing zero Ca²⁺ (using 2 mM EGTA) or 10 mM Ca²⁺, respectively, and β (5.66) was calculated as the ratio of the fluorescence intensities at 380 nm in 0 and 10 mM Ca²⁺. The extracellular solution E₁ was used for Ca²⁺ imaging experiments. Some experiments were performed using Ca²⁺-free extracellular solution E₂.

Statistics

The statistical significance of differences between experimental groups was evaluated by the Mann-Whitney U test using the SPSS program. Data were considered to be statistically significant with p < 0.05.

LPC-induced release of IL-1β from microglial cells

To determine whether LPC (palmitoyl 16:0) is capable of inducing IL-1β release, microglial cells were stimulated for 1 h with LPC. Before LPC application, microglial cells were treated with 1 μg/ml LPS for 6 h. Without LPS activation IL-1β was undetectable in supernatants of unstimulated cells or of LPC-stimulated cells (n = 3 experiments in each case). In agreement with previous observations (38), levels of IL-1β released by cells maintained either in bicarbonate-buffered cell culture medium DMEM or in HEPES-buffered extracellular solution E₁ were comparable (n = 5 experiments). Therefore, if not stated otherwise, cells were maintained in extracellular solution E₁ during the time of LPC stimulation in all subsequent experiments. Both primary cultured microglial cells and BV-2 microglial cells released substantial amounts of IL-1β following stimulation with LPC. Although the total amount of IL-1β was larger in supernatants of primary microglial cells than in supernatants of BV-2 cells, the relative increases in IL-1β produced by LPC-stimulated primary cultured microglia and BV-2 microglial cells were comparable. After stimulation with 15 μM LPC, levels of IL-1β increased 9.5-fold in primary microglia (untreated, 101 ± 41 pg/ml; 15 μM LPC, 959 ± 140 pg/ml; n = 6 experiments) and 8-fold in BV-2 cells (untreated, 2.6 ± 0.6 pg/ml; 15 μM LPC, 21.1 ± 2.0 pg/ml; n = 11 experiments). Upon treatment with 30 μM LPC, the increase in IL-1β production was 17-fold in primary microglia (untreated, 101 ± 41 pg/ml; 30 μM LPC, 1746 ± 215 pg/ml; n = 3 experiments) and 25-fold in BV-2 cells (untreated, 2.3 ± 0.6 pg/ml; 30 μM LPC, 58.2 ± 6.1 pg/ml; n = 16 experiments) (Fig. 1⇓A). Similar to observations made on monocytes (39), monounsaturated LPC (oleoyl 18:1; 15 μM) did not lead to substantial IL-1β release (119 ± 21 pg/ml; n = 3 experiments) from primary microglial cells. The amount of IL-1β released from primary microglia stimulated with 15 μM LPC (stearoyl 18:0, 985 ± 247 pg/ml; n = 3 experiments) did not differ significantly from that released from microglia stimulated with 15 μM LPC (palmitoyl 16:0, 959 ± 140 pg/ml; n = 6 experiments). LPC (palmitoyl 16:0) was used in all subsequent experiments.

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FIGURE 1.

LPC-induced IL-1β release from microglial cells. A, Relative increases in IL-1β production from primary microglial cells (▪) and BV-2 microglial cells (□) stimulated with 15 or 30 μM LPC for 1 h. B, Time course of LPC-stimulated IL-1β release from microglia. BV-2 microglial cells were stimulated with 30 μM LPC (▪) or 45 μM (○) LPC for different times as indicated. Concentrations of IL-1β were normalized to the amounts of IL-1β released after LPC stimulation for 1 h. A and B, Before stimulation with LPC, all microglial cells were pretreated with 1 μg/ml LPS for 6 h in DMEM/FCS culture medium. During LPC stimulation, all cells were maintained in extracellular solution E₁. IL-1β levels (mean ± SEM) of cell supernatants were determined by ELISA.

To test whether LPC has toxic effects on microglial cells, we performed ethidium bromide stainings of microglial cells incubated for 1 h in the presence or absence of 30 μM LPC. Ethidium bromide staining was detected in 1.6 ± 0.6% (n = 5 experiments) of untreated control cells and in 4.9 ± 1.1% (n = 5 experiments) of LPC-treated microglial cells. These data suggest that LPC did not cause significant damage to microglial cells.

Fig. 1⇑B demonstrates the time-dependence of IL-1β release from LPC-stimulated microglial cells. Within the first 10–15 min of LPC stimulation, microglial cells released ∼50% of the amount of IL-1β measured after 1 h of LPC treatment. The levels of IL-1β determined for cells exposed to LPC for 45 or 60 min did not differ significantly.

Next, it was clarified whether mature IL-1β or pro-IL-1β is released from microglia following LPC stimulation. In these experiments, BV-2 microglial cells were preincubated for 1 h with 100 μM YVAD-CHO, which prevents maturation of pro-IL-1β by inhibiting ICE/caspase-1. In the presence of YVAD-CHO, IL-1β release was inhibited by 84% in cells stimulated with 15 μM LPC and by 61% in cells treated with 30 μM LPC (n = 4 experiments for each LPC concentration) (Fig. 2⇓A). These data suggest that the majority of IL-1β released from LPC-stimulated microglia is in the mature active form. Immunoblot analysis revealed the presence of the fully processed and biologically active 17-kDa IL-1β in supernatants of LPC-stimulated primary cultured microglia. Release of the 17-kDa mature IL-1β was markedly reduced upon preincubation of microglial cells with 100 μM YVAD-CHO (n = 12) (Fig. 2⇓, B1 and B2). In some experiments, small amounts of the premature and biologically inactive 33-kDa pro-IL-1β were detected in addition to the 17-kDa IL-1β (Fig. 2⇓B2). Release of pro-IL-1β was not inhibited following pretreatment of microglial cells with YVAD-CHO. The amount of extracellular pro-IL-1β of microglial cells preincubated with YVAD-CHO was 104% (n = 3 experiments) of that determined for cells kept in the absence of YVAD-CHO, while mature IL-1β was reduced to 17% in these YVAD-CHO-treated cells.

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FIGURE 2.

LPC-induced release of mature IL-1β from microglia. Before LPC stimulation, some cells were preincubated with 100 μM YVAD-CHO for 1 h in LPS-containing DMEM/FCS culture medium. Following the YVAD-CHO pretreatment, microglial cells were stimulated with 15 μM (▪) or 30 μM (□) LPC for 1 h in solution E₁. A, Effects of YVAD-CHO on LPC-induced IL-1β release from BV-2 microglia were assayed by ELISA (∗, p < 0.05; ∗∗, p < 0.01 vs LPC). B, Western blot analyses of IL-1β released from primary cultured microglial cells. The majority of LPC-stimulated cells released exclusively mature 17-kDa IL-1β (B1), which was substantially reduced upon inhibition of the ICE/caspase-1 with YVAD-CHO. Some LPC-stimulated microglial cells released both 33-kDa pro-IL-1β and 17-kDa mature IL-1β (B2). Release of pro-IL-1β was not inhibited by YVAD-CHO.

It has been demonstrated before that signaling pathways involved in IL-1β processing and release (40) as well as ion channel expression (32, 41) are similar in BV-2 microglial cells and in primary cultured microglial cells. Therefore, if not stated otherwise, BV-2 microglial cells were used in all subsequent experiments.

LPC-induced release of IL-1β from microglial cells is independent of P2X₇ receptor activation

Performing Ca²⁺ imaging and ELISA experiments, it was tested whether LPC mediates its effects by P2X₇ receptor activation. All experiments were performed on activated microglial cells that had been pretreated with 1 μg/ml LPS for 6 h. Application of 100 μM BzATP, a P2X₇ receptor agonist, caused sustained increases in [Ca²⁺]_i of microglial cells (Fig. 3⇓A). In the presence of BzATP, [Ca²⁺]_i increased from a resting level of 74 ± 2 nM to 2624 ± 124 nM (n = 123 cells). After the initial Ca²⁺ peak, [Ca²⁺]_i remained increased reaching a plateau at 732 ± 21 nM (n = 123 cells). To inhibit P2X₇ receptors, microglial cells were pretreated with 500 μM oATP for 4 h. Following pretreatment with oATP, microglial cells had a mean resting [Ca²⁺]_i of 70 ± 2 nM (n = 103 cells), which was not significantly different from that of untreated cells. In oATP-pretreated cells, 100 μM BzATP induced only small transient Ca²⁺ increases but failed to induce sustained Ca²⁺ increases (Fig. 3⇓B). The lack of sustained Ca²⁺ increases indicates complete inhibition of microglial P2X₇ receptors by oATP. The remaining small Ca²⁺ transients reflect BzATP-induced activation of other ATP receptor types.

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FIGURE 3.

No effect of oATP on LPC-induced Ca²⁺ signals. Measurements of [Ca²⁺]_i were performed in fura 2-AM-loaded microglial cells. A, Effects of 100 μM BzATP on [Ca²⁺]_i of untreated microglial cells. B, Effects of 100 μM BzATP on [Ca²⁺]_i of cells pretreated with 500 μM oATP for 4 h in LPS-containing DMEM/FCS culture medium. C, Effects of 15 μM LPC on [Ca²⁺]_i of untreated microglial cells. D, Pretreatment of microglial cells with 500 μM oATP did not change LPC-induced Ca²⁺ signals. Representative Ca²⁺ signals of individual cells are n = 32 cells (A), n = 25 cells (B), n = 17 cells (C), and n = 15 cells (D).

In additional experiments, effects of LPC on microglial cells were investigated following P2X₇ receptor inhibition. No significant differences were found between LPC-induced Ca²⁺ responses of untreated (Fig. 3⇑C) and oATP-pretreated (Fig. 3⇑D) microglial cells. Under both conditions, LPC caused sustained Ca²⁺ increases consisting of an initial peak and a subsequent plateau phase. None of the determined mean values of [Ca²⁺]_i before or during application of LPC differed markedly between untreated and oATP-pretreated microglial cells. Following application of 15 μM LPC, [Ca²⁺]_i of untreated cells (n = 158 cells) increased from 95 ± 2 nM to 2302 ± 104 nM and reached a plateau of 812 ± 16 nM. In oATP-pretreated microglial cells (n = 144 cells), resting [Ca²⁺]_i was 101 ± 2 nM, while mean values of 2182 ± 162 nM and 745 ± 23 nM were determined for LPC-induced [Ca²⁺]_i peak and plateau, respectively.

Next, we investigated whether in microglia LPC-induced IL-1β release is influenced by P2X₇ receptor inhibition with oATP. As demonstrated in Fig. 4⇓, LPC-induced IL-1β release from microglial cells was unaffected by oATP. Untreated control cells and cells pretreated with 500 μM oATP for 4 h released almost identical amounts of IL-1β following stimulation with LPC (n = 3 experiments).

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FIGURE 4.

No effect of oATP on LPC-induced IL-1β release. Some microglial cells were pretreated with 500 μM oATP for 4 h in LPS-containing DMEM/FCS culture medium. IL-1β release was determined by ELISA. Inhibition of P2X₇ receptors with oATP did not affect LPC-induced IL-1β release from microglia.

Ca²⁺ dependence of LPC-induced release of IL-1β from microglia

The LPC-induced sustained increases in [Ca²⁺]_i (Fig. 5⇓A) were exclusively caused by Ca²⁺ influx from the extracellular space. Increases in [Ca²⁺]_i could not be elicited by LPC when cells were maintained in Ca²⁺-free extracellular solution E₂ (Fig. 5⇓B). As demonstrated in Fig. 5⇓C, increases in [Ca²⁺]_i are important for LPC-induced IL-1β release from microglia. LPC-stimulated microglial cells released less IL-1β in the absence of extracellular Ca²⁺ than in the presence of extracellular Ca²⁺. In Ca²⁺-free extracellular solution E₂, IL-1β release of cells stimulated with 15 μM LPC was reduced by 80% (n = 4 experiments) compared with the levels of IL-1β released in Ca²⁺-containing extracellular solution E₁. In microglial cells stimulated with 30 μM LPC, IL-1β release was reduced by 77% (n = 4 experiments) after omission of extracellular Ca²⁺ (Fig. 5⇓C).

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FIGURE 5.

Effects of Ca²⁺-free extracellular solution on LPC-induced Ca²⁺ signals and IL-1β release. Measurements of [Ca²⁺]_i were performed in fura 2-AM-loaded microglial cells. A, LPC (15 μM) caused sustained increases in [Ca²⁺]_i. Representative Ca²⁺ signals of individual cells (n = 16 cells) are shown. B, Lack of Ca²⁺ elevation in Ca²⁺-free extracellular solution E₂. Representative Ca²⁺ signals of individual cells (n = 14 cells) are shown. C, Effects of Ca²⁺-free extracellular solution on IL-1β release from LPC-stimulated cells. Released IL-1β was determined by ELISA. Cells were stimulated with 30 μM LPC in the presence of extracellular solution E₁ containing 2 mM Ca²⁺ or in the presence of Ca²⁺-free extracellular solution E₂. In Ca²⁺-free extracellular solution E₂ (0 [Ca²⁺]_o), IL-1β release from microglia was significantly inhibited. ∗∗∗, p < 0.001 vs LPC.

Importance of nonselective cation channels for LPC-induced IL-1β release from microglia

Patch-clamp experiments were performed to identify the LPC-induced Ca²⁺ influx pathway. Application of LPC elicited nonselective cation currents in LPS-pretreated microglial cells as shown in Fig. 6⇓. Biophysical and pharmacological properties of LPC-induced nonselective cation currents of microglia have been characterized in detail elsewhere (32). Pretreatment of microglial cells with LPS did not change properties of nonselective cation currents. In microglial cells, the most effective inhibitors of LPC-induced nonselective cation currents are Gd³⁺ (Fig. 6⇓A) and La³⁺ (Fig. 6⇓B). Both lanthanides abolish microglial nonselective cation currents at a concentration of 100 μM (n = 19 cells for Gd³⁺; n = 10 cells for La³⁺). Effects of lanthanides on LPC-induced IL-1β release from microglial cells are demonstrated in Fig. 6⇓C. In the presence of either 100 μM Gd³⁺ or 100 μM La³⁺, IL-1β release from microglial cells stimulated with 15 or 30 μM LPC was completely inhibited (n = 4 experiments for each condition).

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FIGURE 6.

Nonselective cation channel pharmacology and importance for LPC-induced IL-1β release. A and B, Currents were measured in the whole cell configuration of the patch-clamp technique. Cells were held at 0 mV, and voltage ramps were applied from −90 to +60 mV for a duration of 300 ms every 10 s. Nonselective cation currents were evoked by 15 μM LPC and measured using intracellular solution I₁ and extracellular solution E₃. LPC-induced currents were recorded in the absence (LPC) and presence of 100 μM Gd³⁺ (LPC+Gd³⁺) in A or 100 μM La³⁺ (LPC+La³⁺) in B. C, Effect of Gd³⁺ and La³⁺ on LPC-stimulated IL-1β release. Released IL-1β was determined by ELISA. IL-1β release from microglia was abolished by inhibition of nonselective cation channels with either Gd³⁺ or La³⁺ (∗∗∗, p < 0.001 vs LPC).

Importance of Ca²⁺-activated K⁺ channels for LPC-induced IL-1β release from microglia

LPC-induced sustained increases in [Ca²⁺]_i caused the activation of Ca²⁺-dependent K⁺ channels in LPS-pretreated microglial cells. Ca²⁺-dependent K⁺ currents of LPS-pretreated microglia were similar to those of unstimulated microglia described before (32, 42, 43). As shown in Fig. 7⇓A, in the presence of 200 nM CTX, LPC-activated Ca²⁺-dependent K⁺ currents were abolished, whereas nonselective cation currents remained unaffected. Under control conditions before LPC stimulation, the mean resting membrane potential of microglial cells was −50.1 ± 5.2 mV (n = 7 cells). Microglial resting membrane potential was unaffected by CTX (n = 6 cells). Upon activation of Ca²⁺-dependent K⁺ channels, LPC-stimulated microglial cells reached hyperpolarized membrane potentials of −70 ± 1.6 mV (n = 7 cells). Inhibition of the channels with CTX caused membrane depolarization. In the presence of 200 nM CTX, LPC-stimulated microglial cells depolarized on average to −22.7 ± 2.3 mV (n = 7 cells) (Fig. 7⇓B). Upon LPS pretreatment, microglial cells up-regulated the expression of voltage-activated K⁺ channels, which are also CTX-sensitive. Therefore, we studied additionally effects of 1 nM MTX, which inhibits voltage-activated K⁺ channels but not Ca²⁺-activated K⁺ channels in microglia. In contrast to CTX, MTX failed to inhibit LPC-induced membrane hyperpolarization (n = 4 cells), suggesting that membrane hyperpolarization was exclusively caused by the activity of Ca²⁺-activated K⁺ channels.

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FIGURE 7.

LPC-induced activation of Ca²⁺-activated K⁺ channels. A, Currents (I) were measured in the whole cell configuration of the patch-clamp technique. Microglial cells were held at −60 mV. Voltage (V) ramps were applied from −120 to +30 mV for a duration of 300 ms every 20 s. LPC-induced Ca²⁺-activated K⁺ currents were recorded before (LPC) and during application of 200 nM CTX (LPC+CTX). B, Membrane potential (mean ± SEM) of LPC-stimulated microglial cells was determined in current clamp measurements of the patch-clamp technique in the absence (−) and presence (+) of 200 nM CTX. All values were significantly different from each other (p < 0.01). Horizontal bars are mean values of membrane potentials; error bars are SEM of the corresponding mean values. Measurements in each experiment were performed using intracellular solution I₂ and extracellular solution E₁.

The inhibitory effect of 200 nM CTX on LPC-induced IL-1β release is demonstrated in Fig. 8⇓A. In the presence of CTX, microglial cells released only 26% or 39.5% of the amount of IL-1β determined in the absence of CTX after stimulation with 15 μM (n = 3 experiments) or 30 μM (n = 4 experiments) LPC, respectively. These data determined by ELISA were supported by densitometric analysis of Western blots. Release of mature IL-1β from primary microglia stimulated with 15 μM LPC was reduced to 33.6% by 200 nM CTX (n = 3 experiments). The small bands of pro-IL-1β of LPC-stimulated cells (seen in two of three experiments) were undetectable in CTX-treated cells, suggesting that CTX inhibits release of both pro-IL-1β and mature IL-1β. Inhibition of voltage-gated K⁺ channels with 1 nM MTX did not affect LPC-induced IL-1β release (n = 3 experiments) (Fig. 8⇓B).

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FIGURE 8.

Importance of Ca²⁺-activated K⁺ channels for LPC- induced IL-1β release. A–C, Effects of K⁺ channel inhibitors on LPC-induced IL-1β release from microglia. Released IL-1β was determined by ELISA. A, Inhibition of LPC-stimulated IL-1β release by 200 nM CTX (∗, p < 0.05 vs LPC). B, Lack of effect of 1 nM MTX on LPC-stimulated IL-1β release. C, Inhibitory effects of CTX on IL-1β release from LPC-stimulated microglial cells was not reversed by 10 μM nigericin (nig.). ∗, p < 0.05 vs LPC plus nigericin.

Additional experiments were performed to test whether the CTX-induced inhibition of LPC-induced IL-1β release was due to membrane depolarization or due to inhibition of K⁺ efflux. We investigated effects of CTX on LPC-induced IL-1β release in the presence of 10 μM nigericin, which allowed K⁺ efflux, while the CTX-induced membrane depolarization remained stable. As demonstrated in Fig. 8⇑C, the K⁺ ionophore nigericin was unable to reverse CTX-induced inhibition of IL-1β release from LPC-stimulated microglial cells. Nigericin alone (10 μM) did not cause significant increases in IL-1β release from microglial cells. In its presence, LPC-stimulated IL-1β release was inhibited by CTX by 60 ± 13% (n = 3 experiments) (Fig. 8⇑C), which was almost identical with the inhibitory effect of CTX on LPC-stimulated IL-1β release (60.4 ± 16%; n = 4 experiments) (Fig. 8⇑A) determined in the absence of nigericin. These data suggest that a hyperpolarized membrane potential is required for optimal IL-1β release from LPC-stimulated microglial cells.

In additional experiments, membrane hyperpolarization was induced by 1 μM valinomycin. In the presence of valinomycin, CTX failed to inhibit the LPC-induced IL-1β release. CTX-treated microglial cells released 108% (n = 3 experiments) of the amount of IL-1β released from cells in the absence of CTX. Thus, it can be excluded that CTX has unspecific effects that are unrelated to K⁺ channel inhibition.

No importance of K⁺/Cl⁻ cotransporters for LPC-induced IL-1β release from microglia

We have demonstrated previously that K⁺/Cl⁻ cotransporters are involved in LPC-induced shape changes of microglial cells (32). Therefore, it was tested whether activity of K⁺/Cl⁻ cotransporters is also required for optimal IL-1β release from microglial cells. In these experiments, levels of IL-1β were determined in supernatants of cells stimulated with LPC in the presence or absence of 1 mM furosemide. Inhibition of K⁺/Cl⁻ cotransporters with furosemide did not affect LPC-induced IL-1β release. Following stimulation with 30 μM LPC, levels of IL-1β released from furosemide-treated microglial cells were 105.8 ± 23% (n = 4 experiments) of those released from cells in the absence of furosemide.

LPC-induced changes in caspase activity

In a final set of experiments, it was clarified whether Ca²⁺-free extracellular solution and CTX affect either processing or release of IL-1β. To investigate LPC-induced processing of pro-IL-1β to the mature IL-1β, caspase activity was determined in situ by the fluorogenic substrate FITC-VAD-FMK, which binds exclusively active caspases. As shown in Fig. 9⇓, the mean fluorescence intensity of FITC-VAD-FMK-loaded microglial cells was increased 4-fold following stimulation with 30 μM LPC (untreated, n = 1054 cells; LPC-treated, n = 1034 cells). Neither omission of Ca²⁺ from the extracellular medium nor CTX inhibited caspase activity of LPC-stimulated microglial cells. The mean fluorescence intensity of LPC-stimulated microglial cells maintained in Ca²⁺-containing solution E₁ did not differ markedly from that of LPC-stimulated cells kept in Ca²⁺-free extracellular solution E₂ (n = 765 cells). Furthermore, the mean fluorescence intensities of cells stimulated for 1 h with LPC in the absence (n = 1034 cells) or presence of 200 nM CTX (n = 1000 cells) were almost identical. Western blot analyses of intracellular pro-IL-1β did also not reveal significant differences between cells maintained in the absence or presence of 200 nM CTX. On average, the amount of intracellular pro-IL-1β of microglial cells stimulated with LPC in the presence of CTX was 114.4±15% (n = 3 experiments) of that determined in the absence of CTX. These data suggest that the LPC-induced processing of IL-1β in microglial cells was neither Ca²⁺- nor voltage-dependent.

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FIGURE 9.

Caspase activity of LPC-stimulated microglial cells. A–D, All cells were pretreated with 1 μg/ml LPS for 6 h in DMEM/FCS culture medium. Subsequently, cells were maintained in Ca²⁺-containing extracellular solution E₁ (A, B, and D) or in Ca²⁺-free extracellular solution E₂ (C) at 37°C for 1 h. Microglial cells were kept untreated (A) or were stimulated (B) with 30 μM LPC in the absence or presence of 200 nM CTX (D) or in Ca²⁺-free extracellular solution E₂ (C). Brightfield microscopy images (left column) and fluorescence images (right column) are shown. E, Relative fluorescence intensities (mean ± SEM) of FITC-VAD-FMK-loaded untreated and LPC-treated microglial cells are quantitated. Neither Ca²⁺-free solution nor CTX affected caspase activity of LPC-stimulated microglia.

LPC-induced release of IL-1β from microglia

In the present study, we provide the first evidence that LPC is capable of inducing rapid release of IL-1β from microglial cells. Furthermore, this study is the first demonstrating a novel ATP-receptor-independent mechanism of IL-1β release from microglia.

Upon stimulation with LPC, release of IL-1β occurs rapidly from microglial cells preactivated with LPS. Microglial IL-1β was undetectable in microglial cultures without LPS pretreatment. This finding is in good agreement with previous observations on ATP-stimulated microglial cells (20, 22). LPS is required for up-regulating the synthesis of pro-IL-1β and the expression of ICE/caspase-1 (44). Processing of pro-IL-1β to IL-1β requires cleavage by the enzyme ICE/caspase-1 (45). We further demonstrate that the majority of IL-1β released from microglial cells following LPC stimulation is in the mature, active 17-kDa form. Thus, similar to extracellular ATP, LPC induces both processing and secretion of IL-1β.

Activation of P2X₇ ATP receptors has been proposed to be a prerequisite for the release of IL-1β from microglial cells. Previous studies have shown that IL-1β release from microglia stimulated with ATP (20, 22), LPS (19, 20), ADP or AMP (24) is mediated by P2X₇ receptor activation. In contrast, we found that specific inhibition of microglial P2X₇ receptors with oATP (46) prevented neither LPC-stimulated Ca²⁺ signals nor LPC-induced IL-1β release. These data indicate that LPC induces IL-1β release from microglia by a mechanism independent of P2X₇ receptor activation.

Ca²⁺ dependence of LPC-induced IL-1β release from microglia

Several studies on peripheral tissue macrophages have shown that the ATP-triggered IL-1β release is a Ca²⁺-dependent process. However, controversial data exist regarding the mechanism leading to enhanced intracellular Ca²⁺ concentrations sufficient to induce IL-1β release. Ca²⁺ influx from the extracellular space through P2X₇ receptor-coupled nonselective cation channels (47, 48) or Ca²⁺ release from intracellular stores (49) have been suggested to initiate IL-1β release from ATP-stimulated macrophages. Similar to the ATP-induced IL-1β release from peripheral tissue macrophages, LPC-stimulated IL-1β release from microglia is Ca²⁺-dependent. It was inhibited following omission of extracellular Ca²⁺ or during blockade of nonselective cation channels. Thus, sustained Ca²⁺ entry from the extracellular space mediated by the activity of nonselective cation channels is required for LPC-induced microglial IL-1β release. In microglia, LPC causes exclusively Ca²⁺ influx through nonselective cation channels, but fails to evoke Ca²⁺ release from intracellular stores (Ref. 32 and the present study). Thus, it can be excluded that Ca²⁺ release from intracellular stores is a prerequisite for LPC-induced IL-1β release from microglia. LPC-induced sustained increases in [Ca²⁺]_i have also been described in human monocytes (50).

Voltage-dependence of LPC-induced IL-1β release from microglia

Microglial LPC-stimulated IL-1β release was attenuated in the presence of CTX but was unaffected by MTX. These data indicate that Ca²⁺-activated K⁺ channels but not voltage-activated K⁺ channels are important for LPC-stimulated IL-1β release. Similar to our observations, ATP-stimulated IL-1β release from peripheral tissue macrophages is independent of voltage-activated K⁺ channel activity (51). The reduction of IL-1β levels released from LPC-stimulated microglia during inhibition of Ca²⁺-activated K⁺ channels with CTX could be explained by inhibition of either IL-1β processing or IL-1β secretion. Our observation that LPC-stimulated activity of ICE/caspase-1 remained unchanged in the presence of CTX suggests that CTX inhibits secretion of IL-1β rather than the transformation process of pro-IL-1β into mature IL-1β.

What is the functional importance of Ca²⁺-activated K⁺ channels for LPC-stimulated microglial IL-1β release? Activity of Ca²⁺-activated K⁺ channels is required to keep the membrane potential of cells at negative, i.e., hyperpolarized, values. We suggest that membrane hyperpolarization is required for optimal IL-1β release and that the CTX-induced inhibition of LPC-stimulated IL-1β release is due to membrane depolarization. Our results on the inhibitory effect of CTX on LPC-stimulated IL-1β release are in line with previous studies demonstrating that high K⁺-containing solution, which also causes membrane depolarization, inhibits IL-1β release from ATP-stimulated microglia (22). However, CTX and high K⁺-containing extracellular solution also lead to reduced K⁺ efflux, which seems to be crucial for IL-1β processing (20, 22, 52, 53). CTX blocks K⁺ efflux through Ca²⁺-activated K⁺ channels, whereas high K⁺-containing extracellular solution inhibits K⁺ efflux by reducing the K⁺ gradient between extracellular solution and intracellular milieu. However, LPC-induced K⁺ efflux was not markedly affected by CTX (T. Schilling and C. Eder, unpublished observations), suggesting voltage dependence of microglial IL-1β release. To clarify further whether the CTX-induced inhibition of LPC-stimulated IL-1β release is due to membrane depolarization, effects of CTX on LPC-stimulated IL-1β release were studied in the presence of the electroneutral K⁺ ionophore nigericin. Under these conditions, cells were depolarized by CTX, whereas the large K⁺ gradient between intracellular and extracellular solutions remained, so that nigericin induced K⁺ efflux. Our observation that nigericin was unable to reverse the inhibitory effect of CTX on LPC-stimulated IL-1β release demonstrates that membrane depolarization is the crucial factor reducing IL-1β secretion from LPC-stimulated microglial cells. Additional experiments are required to identify the exact mechanism by which membrane depolarization inhibits IL-1β secretion from microglial cells. The CTX-induced inhibition of IL-1β release cannot simply be explained by a depolarization-induced reduction of the driving force for Ca²⁺ entry through nonselective cation channels. LPC-induced intracellular Ca²⁺ increases were not markedly reduced in the presence of CTX (T. Schilling and C. Eder, unpublished observations).

Microglial Ca²⁺-activated K⁺ channels are encoded by the gene K_Ca3.1 (IKCa1 or KCNN4) (43). It has been demonstrated that selective blockade of K_Ca3.1 Ca²⁺-activated K⁺ channels attenuates acute brain damage caused by traumatic brain injury (54) and ameliorates the outcome of experimental autoimmune encephalomyelitis (55, 56). In experimental autoimmune encephalomyelitis mice, reduced IL-1β levels were detected following K⁺ channel inhibition (56), which could be related to inhibition of microglial activation in vivo.

LPC-induced IL-1β release and LPC-induced deramification are mediated by different physiological mechanisms

In microglia, LPC-induced release of IL-1β is accompanied by changes in cell morphology (32). Although both IL-1β release and deramification of microglial cells occur simultaneously after stimulation with LPC, both processes are mediated by different physiological mechanisms. First, IL-1β release strongly depends on Ca²⁺-influx through nonselective cation channels, whereas microglial shape changes are Ca²⁺-independent. Second, membrane hyperpolarization induced by functional Ca²⁺-activated K⁺ channels is required for IL-1β release, whereas the transformation of microglia from ramified into ameboid morphology is unaffected upon inhibition of Ca²⁺-activated K⁺ channels. Third, KCl efflux mediated by the activity of K⁺/Cl⁻ cotransporters supports the retraction of cell extensions during microglial deramification, whereas K⁺/Cl⁻ cotransporter activity is not required for the LPC-induced IL-1β release.

We suggest a model of LPC-induced IL-1β release (Fig. 10⇓). Similar to ATP, LPC stimulates rapid processing and release of IL-1β from LPS-preactivated microglia. However, neither ATP receptors nor ATP receptor-coupled channels are affected by LPC. LPC activates nonselective cation channels. The Ca²⁺ influx through nonselective cation channels causes substantial increases in the intracellular Ca²⁺ concentration, which subsequently lead to the activation of Ca²⁺-dependent K⁺ channels. The activity of Ca²⁺-activated K⁺ channels results in sustained membrane hyperpolarization. Both intracellular Ca²⁺ elevation and membrane hyperpolarization are required for optimal release of the processed, biologically active 17-kDa IL-1β into the extracellular milieu.

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FIGURE 10.

Schematic model of IL-1β release from LPC-stimulated microglia. This model summarizes data of the present study. Details of the processes are described in Discussion.