The effect of possible inhibitors of fluid-phase pinocytosis on LDL uptake and net cholesterol accumulation in wild-type M-CSF-differentiated macrophages.

<p>Wild-type bone marrow-derived macrophages were pretreated 1 h with drug vehicle or the indicated drug. For cholesterol accumulation, macrophages then were incubated with 1 mg/ml LDL and inhibitor for 6 h. For ¹²⁵I-LDL uptake, macrophages then were incubated with 200 µg/ml ¹²⁵I-LDL and inhibitor for 6 h. All incubations were performed in serum-free medium containing 50 ng/ml M-CSF. The percent inhibition of net cholesterol accumulation compares macrophages treated with LDL alone and LDL with inhibitor (except the experiment that compares macrophages treated with LDL and dynamin peptide inhibitor with LDL and control peptide) after basal cholesterol values were subtracted from each. The percent inhibition of ¹²⁵I-LDL uptake compares macrophages treated with ¹²⁵I-LDL alone and ¹²⁵I-LDL with inhibitor. Control values for macrophages incubated with LDL alone or ¹²⁵I-LDL alone are indicated in parentheses. For cholesterol accumulation, control values are expressed as nmol net cholesterol accumulation/mg cell protein. For ¹²⁵I-LDL uptake, control values are expressed as µg ¹²⁵I-LDL uptake/mg cell protein. The range of cell-associated and degraded ¹²⁵I-LDL for all treatments except bafilomycin A1-treated macrophages was 15–19% and 82–86%, respectively. Cell-associated and degraded ¹²⁵I-LDL for bafilomycin A1-treated macrophages was 91% and 9%, respectively. * = <i>p</i><0.05. ** = <i>p</i><0.01. *** = <i>p</i><0.001. ND = not determined. “+” indicates a decrease in the number of macropinosomes and “−” indicates no effect on macropinosomes. “−” in percent inhibition columns indicates stimulation rather than inhibition.</p

Fluid-phase pinocytosis mediates LDL uptake.

<p>(A) Wild-type macrophages were incubated 6 h with either 25 µg/ml ¹²⁵I-LDL alone, or 25 µg/ml ¹²⁵I-LDL and 500 µg/ml unlabeled LDL. CD36 KO macrophages (ΔCD36) and SRA KO macrophages (ΔSRA) macrophages were incubated 6 h with 25 µg/ml ¹²⁵I-LDL alone. (B) Wild-type macrophages were incubated 6 h with either 25 µg/ml ¹²⁵I-AcLDL alone, or 25 µg/ml ¹²⁵I-AcLDL and 500 µg/ml unlabeled AcLDL. CD36 KO macrophages (ΔCD36) and SRA KO macrophages (ΔSRA) macrophages were incubated 6 h with 25 µg/ml ¹²⁵I-AcLDL alone. Incubations were performed in serum-free medium containing 50 ng/ml M-CSF. Uptake values represent the sum of cell-associated and degraded ¹²⁵I-labeled lipoprotein. The range of cell-associated and degraded ¹²⁵I-LDL was 17–21% and 79–83%, respectively. The range of cell-associated and degraded ¹²⁵I-AcLDL was 16–18% and 82–84%, respectively. ¹²⁵I-LDL uptake was not competed with excess unlabeled LDL consistent with fluid-phase pinocytosis mediating uptake. Statistical tests compare each treatment group with wild-type macrophages incubated with 25 µg/ml ¹²⁵I-AcLDL. * = <i>p</i><0.05. ** = <i>p</i><0.01. *** = <i>p</i><0.001. There was no statistical difference between macrophage groups incubated with ¹²⁵I-LDL.</p

The effect of PI3K inhibitors and cytochalasin D on LDL uptake and net cholesterol accumulation in LDLR −/− macrophages.

<p>LDLR−/− bone marrow-derived macrophages were pretreated 1 h with either drug vehicle, 50 µM LY294002, 100 nm wortmannin, or 4 µg/ml cytochalasin D. For cholesterol accumulation, macrophages then were incubated with 1 mg/ml LDL and inhibitor for 24 h. For ¹²⁵I-LDL uptake, macrophages then were incubated 5 h with 200 µg/ml ¹²⁵I-LDL and inhibitor. All incubations were performed in serum-free medium containing 50 ng/ml M-CSF. The percent inhibition of net cholesterol accumulation compares macrophages treated with LDL alone and LDL with inhibitor after basal cholesterol values were subtracted from each. The percent inhibition of ¹²⁵I-LDL uptake compares macrophages treated with ¹²⁵I-LDL alone and ¹²⁵I-LDL with inhibitor. ** = <i>p</i><0.01. *** = <i>p</i><0.001. All inhibitors showed almost complete inhibition of macropinosome formation as assessed by phase-microscopy. For LY294002 and wortmannin experiments, control macrophages incubated with LDL or ¹²⁵I-LDL alone showed net cholesterol accumulation and ¹²⁵I-LDL uptake values of 226±8 nmole/mg cell protein and 2.7±0.1 µg/mg cell protein, respectively. For cytochalasin D experiments, control macrophages incubated with LDL or ¹²⁵I-LDL alone showed net cholesterol accumulation and ¹²⁵I-LDL uptake values of 230±2 nmole/mg cell protein and 3.4±0.1 µg/mg cell protein, respectively. The range of cell-associated and degraded ¹²⁵I-LDL for all treatments was 19–33% and 67–81%, respectively.</p

Macrophage uptake of ¹²⁵I-LDL is non-saturable.

<p>A. LDLR−/− macrophages were incubated with 1 mg/ml of LDL for 0–24 h and cholesterol accumulation was then assessed. B. LDLR−/− macrophages were incubated 24 h with increasing concentrations of ¹²⁵I-LDL, and then ¹²⁵I-LDL uptake was assessed. Uptake values represent the sum of cell-associated and degraded ¹²⁵I-LDL. The range of cell-associated and degraded ¹²⁵I-LDL was 7–13% and 87–93%, respectively.</p

LDL-derived cholesterol accumulation occurs independently of class I PI3K isoforms.

<p>Cholesterol accumulation after 24 h-incubation without or with 1 mg/ml LDL was assessed in M-CSF differentiated macrophages cultured from wild-type, PI3Kγ-KI, PI3Kβ-KI, and PI3Kδ-KI mice.</p

Macropinosome formation is M-CSF dependent.

<p>A and B. Wild-type macrophages were differentiated with M-CSF for 7 days and visualized by phase-contrast microscopy following the treatments described below. Macrophages differentiated with M-CSF were pretreated 30 min with DMSO drug vehicle (A), or 5 µM of cFMS (i.e., M-CSF receptor) tyrosine kinase inhibitor, GW2580 (B). Pretreatment was carried out without either serum or M-CSF. Withdrawal of M-CSF caused disappearance of the macrophage vacuoles. Subsequently, these macrophage cultures were treated 30 min with fresh serum-free medium containing M-CSF (50 ng/ml) without (A) or with GW2580 (B). Macrophages treated with M-CSF without GW2580 showed numerous vacuoles shown to be macropinosomes in Video S1. In contrast, there was complete inhibition of macropinosome formation when macrophage cultures were treated with GW2580 (also see Video S2). Scale bar in B = 75 µm and also applies to A. (C) Wild-type macrophages were incubated 24 h with 1 mg/ml LDL without or with 5 µM GW2580, and then cholesterol accumulation was assessed. Macrophages incubated without LDL had 111±3 nmol cholesterol/mg protein. ** = <i>p</i><0.01.</p

Wild-type and LDL−/− macrophages incubated with LDL accumulate similar levels of cholesterol.

<p>Wild-type and LDLR−/− macrophages were incubated with 1 mg/ml LDL for 24 h and then total cholesterol accumulation was assessed. The baseline cholesterol levels for wild-type and LDLR−/− macrophages were 107±10 nmol cholesterol/mg protein and 122±9 nmol cholesterol/mg protein, respectively.</p

The effect of PI3K inhibitors and cytochalasin D on net cholesterol accumulation in wild-type macrophages.

<p>Wild-type bone marrow-derived macrophages were pretreated 1 h with either drug vehicle, 50 µM LY294002, 100 nm wortmannin, or 4 µg/ml cytochalasin D. Macrophages then were incubated with 1 mg/ml LDL and inhibitor for 6 h. Incubations were performed in serum-free medium containing 50 ng/ml M-CSF. The percent inhibition of net cholesterol accumulation compares macrophages treated with LDL alone and LDL with inhibitor after basal cholesterol values were subtracted from each. ** = <i>p</i><0.01. *** = <i>p</i><0.001. All inhibitors almost completely inhibited macropinosome formation as assessed by phase-microscopy. For the LY294002 experiment, control macrophages incubated with LDL alone showed net cholesterol accumulation of 31±2 nmole/mg cell protein. For the wortmannin and cytochalasin D experiments, control macrophages incubated with LDL alone showed net cholesterol accumulation of 78±2 nmole/mg cell protein.</p

Thapsigargin-induced mast cell degranulation requires PI3Kγ, but not GPCR signaling.

<p>(A) Granule release of wild type and p110γ^−/− BMMCs was determined detecting β-hexosaminidase (β-Hex) release into extracellular media. BMMC stimulation with IgE/antigen was initiated with the antigen (Ag, DNP-HSA at 10 ng/ml; 100 ng/ml IgE overnight). Alternatively, BMMCs were stimulated by the addition of thapsigargin (1 µM). Where indicated, BMMCs were preincubated for 15 min with 100 nM wortmannin. Released β-Hex was quantified 20 min after stimulation, and is expressed as mean ± standard error of the mean (SEM) (<i>n</i> = 3; <i>p</i>-values in all figures are * or &: <i>p</i><0.05, **: <i>p</i><0.005; ***: <i>p</i><0.0005; * depict here comparison with respective wild type control; & refer to comparison of untreated versus treated samples). (B) Granule release was assessed as above, but ADA (10 units/ml) was added to BMMC suspensions 1 min before stimulation where depicted. (C) Wild type or p110γ^−/− BMMCs were stimulated with adenosine (Ade; 1 µM) or thapsigargin (1 µM) for 2 min, and phosphorylation of PKB/Akt on Thr308 (pPKB), total PKB and p110γ was analyzed by Western blotting. BMMCs were incubated in starving medium (2% FCS, without IL-3) for 3 h before stimulation, and pretreated with ADA where indicated. (D) Heterotrimeric Gα_i proteins were inactivated by preincubation of wild type and p110γ^−/− BMMCs with 100 ng/ml <i>P</i>Tx for 4 h, before thapsigargin (Tg) or adenosine was added as in (C).</p

PKCβ relays thapsigargin-induced PI3Kγ activation.

<p>(A) Effect of PKC inhibitors on thapsigargin-induced PKB phosphorylation on Ser473 (S473). IL-3 starved BMMCs were preincubated with the indicated compounds for 20 min before stimulation (pan-PKC: Ro318425, Gö6983; classical PKC: PKC412 (CPG41251); classical and atypical PKC: Gö6976; Rotterlin: broad band inhibitor; see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001587#pbio.1001587.s013" target="_blank">Text S1</a>; & refers to comparison with untreated control; <i>p</i>-values see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001587#pbio-1001587-g001" target="_blank">Figure 1</a>). (B) PKB/Akt activation in response to 100 nM PMA or 1 µM thapsigargin was analyzed in wild type and PKCβ^−/− BMMCs. Cells were IL-3 deprived as in (A), and were pretreated with wortmannin (Wm, 100 nM) for 15 min before stimulation where indicated. Cells were lysed 2 min after stimulation, and analyzed for phosphorylation of PKB/Akt (T308 and S473) and MAPK (T183/Y185). (C) Wild type and PKCβ^−/− BMMCs were stimulated with 1 µM adenosine, 10 ng/ml IL3, or 10 ng/ml SCF, and processed as in (B). (D–F) PtdIns(3,4,5)<i>P</i>₃ (PI<i>P</i>₃) levels were determined in untreated (Ctrl) and classical PKC-inhibitor (PKC412)-treated wild type BMMCs and PKCβ^−/− BMMCs after stimulation with 0.5 µM thapsigargin, 200 ng/ml PMA, or 5 µM adenosine (30 s). BMMCs were metabolically labeled with [³²P]-orthophosphate, lipids were extracted, deacylated, and applied to high-pressure liquid chromatography (HPLC). (D) shows representative elution peaks of PI<i>P</i>₃ of the HPLC chromatograms. (E) Levels of PI<i>P</i>₃ in relation to PtdIns(4,5)<i>P</i>₂ (PI<i>P</i>₂) were quantified by integration of the peak areas of PI<i>P</i>₃ and PI<i>P</i>₂ and expressed as ratio of PI<i>P</i>₃/PI<i>P</i>₂ (data shown as mean ± standard error of the mean [SEM], <i>n</i>≥4–6). (F) Cellular PI<i>P</i>₃ production was measured over time in wild type BMMCs in response to PMA (200 nM) stimulation in the presence or absence of the classical PKC inhibitor PKC412 (mean ± SEM, <i>n</i> = 3). (G) Granule release and PKB activation (S473) in response to thapsigargin (1 µM) or IgE/antigen (100 ng/ml IgE overnight, 10 ng/ml DNP) was measured in the presence of increasing concentrations of the classical PKC inhibitor PKC412. Cells starved as in (A) were stimulated with IgE/antigen (IgE/Ag) or thapsigargin (Tg), and PKB phosphorylation and β-hexosaminidase release assays were performed in parallel (mean ± SEM, <i>n</i> = 3). (H) β-hexosaminidase release determined in wild type, PKCβ^−/−, and p110γ^−/− BMMCs incubated with IgE, and stimulated with the indicated antigen (Ag) concentrations (mean ± SEM, <i>n</i> = 5; * refer to comparison with wild type control. Only the higher <i>p</i>-values of the overlapping data points are indicated).</p