Statins are potent, cholesterol-lowering agents with newly appreciated, broad anti-inflammatory properties, largely based upon their ability to block the prenylation of Rho GTPases, including RhoA. Because phagocytosis of apoptotic cells (efferocytosis) is a pivotal regulator of inflammation, which is inhibited by RhoA, we sought to determine whether statins enhanced efferocytosis. The effect of lovastatin on efferocytosis was investigated in primary human macrophages, in the murine lung, and in human alveolar macrophages taken from patients with chronic obstructive pulmonary disease. In this study, we show that lovastatin increased efferocytosis in vitro in an 3-hydroxyl-3-methylglutaryl coenzyme A (HMG-CoA) reductase-dependent manner. Lovastatin acted by inhibiting both geranylgeranylation and farnesylation, and not by altering expression of key uptake receptors or by increasing binding of apoptotic cells to phagocytes. Lovastatin appeared to exert its positive effect on efferocytosis by inhibiting RhoA, because it 1) decreased membrane localization of RhoA, to a greater extent than Rac-1, and 2) prevented impaired efferocytosis by lysophosphatidic acid, a potent inducer of RhoA. Finally, lovastatin increased efferocytosis in the naive murine lung and ex vivo in chronic obstructive pulmonary disease alveolar macrophages in an HMG-CoA reductase-dependent manner. These findings indicate that statins enhance efferocytosis in vitro and in vivo, and suggest that they may play an important therapeutic role in diseases where efferocytosis is impaired and inflammation is dysregulated.

Statins are potent, cholesterol-lowering agents with newly recognized, broad anti-inflammatory properties (1). For example, statins suppress the innate immune response in vitro by inhibiting neutrophil migration (2), oxidative stress (3), NF-κB activation (4), proinflammatory mediator release (5, 6), expression of matrix metalloproteinases (7, 8, 9), and by increasing expression of constitutive NO synthase (10), peroxisome proliferator-activated receptor (PPAR)3α (11), PPARγ (4), and TGFβ1 (12). Statins also suppress the adaptive immune response by inhibiting IFN-γ-inducible MHC class II expression (13), decreasing expression of CD40/CD40L (14), and by direct blockade of LFA-1 (15). These pleiotropic, anti-inflammatory effects have important therapeutic implications, because 1) statins effectively treat animal models of sepsis, rheumatoid arthritis, acute lung injury, asthma, and emphysema (2, 16, 17, 18, 19), and 2) clinically, statins have a promising therapeutic role in the acute coronary syndrome, stabilization of carotid artery plaques, sepsis syndrome, lung allograft rejection, and rheumatoid arthritis (7, 20, 21, 22, 23, 24). Therefore, statins appear to be emerging as a new class of immunomodulators, surpassing their originally envisaged role as cholesterol lowering drugs.

The immunomodulatory effects of statins are largely cholesterol independent; instead, they appear to depend upon the ability of statins to posttranslationally modify an extensive array of intracellular signaling molecules, including the Rho family of GTPases (1). Rho GTPases (e.g., RhoA, Rac-1, and Cdc42) are molecular switches, which for function, depend upon the covalent attachment of lipid adducts (prenylation) that direct membrane insertion, localization, and protein:protein interaction (25, 26). Statins regulate prenylation of Rho-GTPases by blocking 3-hydroxyl-3-methylglutaryl coenzyme A (HMG-CoA) reductase, which decreases production of mevalonate, and downstream prenylation substrates, including farnesyl pyrophosphate (FPP) and geranylgeranyl pyrophosphate (GGPP) (see Fig. 1). These statin properties suggest a cholesterol-independent mechanism through which they may have far-reaching, regulatory effects.

FIGURE 1.

A, The cholesterol/prenylation pathway. Statins suppress the production of mevalonate, IPP, FPP, GGPP, and cholesterol by inhibiting HMG-CoA reductase. Farnesyltransferase and geranylgeranyltransferase prenylate Rho GTPases by catalyzing the attachment of FPP or GGPP, respectively. Statin broadly inhibit prenylation by decreasing levels of FPP and GGPP, while farnesyltransferase inhibitors (FTI) and geranylgeranyltransferase inhibitors (GGTI) more specifically affect the addition of their respective substrates. Prenylation ultimately regulates the activity of Rho GTPases and other proteins by influencing membrane insertion, localization, and protein:protein interaction. B–F, Lovastatin enhances efferocytosis, but suppresses FcγR phagocytosis, in an HMG-CoA reductase-dependent manner. HMDMs were treated with lovastatin for 6 h to 5 days, then cocultured with 2.5 × 106 apoptotic human neutrophils for an additional 40 min. Uningested neutrophils were then washed off, and a PI or binding index (BI) was determined by blinded visual inspection. The PI (or BI) is calculated by dividing the number of ingested (or bound) apoptotic cells by the total number of macrophages counted, multiplied by 100. B, Lovastatin (5 μM) increased efferocytosis of apoptotic human neutrophils at 6 h. At 24 h, lovastatin increased efferocytosis in a dose-dependent fashion, exerting a maximum effect at 10 μM. The mean PI as a percentage of control ± SEM is shown for four to five replicates per group. Control mean PI: 13.7 ± 6.3. ∗, Significantly different from control (p < 0.05); †, different from control (p = 0.08). C, Lovastatin had no effect on binding of apoptotic human neutrophils to HMDMs at 24 h. The mean BI as a percentage of control ± SEM is shown for five replicates per group. Control mean BI: 7.9 ± 3.8. D, Lovastatin increased efferocytosis of apoptotic human neutrophils at long at 5 days posttreatment. HMDMs were treated with 1 or 10 μM lovastatin for 1–5 days and assessed for neutrophil efferocytosis. A total of 10 μM lovastatin increased efferocytosis at day 1, whereas 1 μM lovastatin increased efferocytosis at day 5. The mean PI as a percentage of control ± SEM is shown for four to six replicates per group. Control mean PI: 8.5 ± 4.4. ∗, Significantly different from control (p < 0.05; Student’s t test). E, Mevalonate reversed the effect of lovastatin on uptake of apoptotic human neutrophils by HMDMs. The mean PI as percentage of control ± SEM is shown for four replicates per group. Control mean PI: 6.1 ± 1.9. ∗, Significantly different from control (p < 0.05). F, Lovastatin inhibited phagocytosis of Ig-G-opsonized erythrocytes through the FcγR, in an HMG-CoA reductase-dependent manner. Control mean PI: 81.5 ± 10.1. ∗, Significantly different from control (p < 0.05).

FIGURE 1.

A, The cholesterol/prenylation pathway. Statins suppress the production of mevalonate, IPP, FPP, GGPP, and cholesterol by inhibiting HMG-CoA reductase. Farnesyltransferase and geranylgeranyltransferase prenylate Rho GTPases by catalyzing the attachment of FPP or GGPP, respectively. Statin broadly inhibit prenylation by decreasing levels of FPP and GGPP, while farnesyltransferase inhibitors (FTI) and geranylgeranyltransferase inhibitors (GGTI) more specifically affect the addition of their respective substrates. Prenylation ultimately regulates the activity of Rho GTPases and other proteins by influencing membrane insertion, localization, and protein:protein interaction. B–F, Lovastatin enhances efferocytosis, but suppresses FcγR phagocytosis, in an HMG-CoA reductase-dependent manner. HMDMs were treated with lovastatin for 6 h to 5 days, then cocultured with 2.5 × 106 apoptotic human neutrophils for an additional 40 min. Uningested neutrophils were then washed off, and a PI or binding index (BI) was determined by blinded visual inspection. The PI (or BI) is calculated by dividing the number of ingested (or bound) apoptotic cells by the total number of macrophages counted, multiplied by 100. B, Lovastatin (5 μM) increased efferocytosis of apoptotic human neutrophils at 6 h. At 24 h, lovastatin increased efferocytosis in a dose-dependent fashion, exerting a maximum effect at 10 μM. The mean PI as a percentage of control ± SEM is shown for four to five replicates per group. Control mean PI: 13.7 ± 6.3. ∗, Significantly different from control (p < 0.05); †, different from control (p = 0.08). C, Lovastatin had no effect on binding of apoptotic human neutrophils to HMDMs at 24 h. The mean BI as a percentage of control ± SEM is shown for five replicates per group. Control mean BI: 7.9 ± 3.8. D, Lovastatin increased efferocytosis of apoptotic human neutrophils at long at 5 days posttreatment. HMDMs were treated with 1 or 10 μM lovastatin for 1–5 days and assessed for neutrophil efferocytosis. A total of 10 μM lovastatin increased efferocytosis at day 1, whereas 1 μM lovastatin increased efferocytosis at day 5. The mean PI as a percentage of control ± SEM is shown for four to six replicates per group. Control mean PI: 8.5 ± 4.4. ∗, Significantly different from control (p < 0.05; Student’s t test). E, Mevalonate reversed the effect of lovastatin on uptake of apoptotic human neutrophils by HMDMs. The mean PI as percentage of control ± SEM is shown for four replicates per group. Control mean PI: 6.1 ± 1.9. ∗, Significantly different from control (p < 0.05). F, Lovastatin inhibited phagocytosis of Ig-G-opsonized erythrocytes through the FcγR, in an HMG-CoA reductase-dependent manner. Control mean PI: 81.5 ± 10.1. ∗, Significantly different from control (p < 0.05).

Close modal

Phagocytosis of apoptotic cells (efferocytosis) is integrally involved with the regulation of the inflammatory response and maintenance of lung homeostasis by 1) removing dead cells before the onset of necrosis (27), by 2) inducing release of anti-inflammatory mediators (27) and antiproteases (28), and by 3) increasing production of growth factors (29, 30). Perhaps not surprisingly, impaired efferocytosis appears to be involved in the pathogenesis of a variety of chronic lung and systemic inflammatory diseases because it is defective in systemic lupus erythematosus, rheumatoid arthritis, cystic fibrosis, bronchiectasis, asthma, and chronic obstructive pulmonary disease (COPD) (31, 32, 33, 34, 35, 36, 37). The regulation of efferocytosis is tightly controlled by the Rho family GTPases, in that RhoA inhibits (38, 39, 40) and Rac-1/Cdc42/RhoG promotes (41, 42, 43) the process. Because statins robustly inhibit RhoA, a negative regulator of efferocytosis, we hypothesized that statins would enhance efferocytosis, and sought to determine whether statins might have therapeutic potential in COPD, a disease of impaired efferocytosis (31, 32).

Indeed, our data demonstrate that statins are potent inducers of efferocytosis in vitro and in vivo in an HMG CoA-reductase dependent manner. Statins appear to exert their effect, not by suppressing cholesterol, but by disproportionately suppressing the prenylation and membrane localization of RhoA. Finally, these data may have therapeutic implications for the treatment of chronic inflammatory diseases of the lung, because statins augment efferocytosis in the naive murine lung and in alveolar macrophages obtained from patients with COPD.

The study was approved by, and performed in accordance with, the ethical standards of the institutional review board on human experimentation at National Jewish Medical and Research Center. Written informed consent was obtained from each subject.

Mice were housed and studied under institutional animal care and use committee-approved protocols in the animal facility of National Jewish Medical and Research Center. Experiments were performed on 8- to 12-wk-old, age-matched, female ICR mice (Harlan Sprague Dawley).

Human monocyte-derived macrophages (HMDMs) and neutrophils were isolated and prepared from normal blood, as previously described (44). HMDMs were cultured in X-vivo medium (Cambrex BioScience) with 10% pooled human serum at 37°C in 10% CO2 for 7 days before use.

Human alveolar macrophages were isolated by bronchoalveolar lavage from patients with Global Initiative for Global Initiative for Chronic Obstructive Lung Disease (GOLD) stage 2 COPD as described (31, 45). The right middle lobe was lavaged with 100 ml of 0.9% saline solution at room temperature. Human alveolar macrophages were resuspended in X-vivo medium (Cambrex BioScience) and were plated on baked glass coverslips in 24-well tissue-culture plates (BD Biosciences) at 5.0 × 105 alveolar macrophages/well.

Murine thymocytes were isolated from the thymi of 3- to 4-wk-old, female ICR mice, by passing thymi through a 40-μm strainer (Fisher Scientific) to separate individual cells.

The human Jurkat leukemia T cell line was obtained from the American Type Culture Collection and cultured in RPMI 1640 with 10% FBS, supplemented with penicillin-streptomycin-glutamine, and incubated at 37°C in 10% CO2 (31, 46).

Apoptosis was induced in human neutrophils, Jurkat T cells, and murine thymocytes by exposure to UV irradiation at 312 nm (Fotodyne) for 10 min, as previously described (31). Human neutrophils were cultured in RPMI 1640 with 0.5%, low-endotoxin BSA (Sigma-Aldrich) at 37°C in 10% CO2 for 2.5 h before use. Jurkat T cells were cultured in RPMI 1640 with 10% FCS at 37°C in 5% CO2 for 3.5 h before use. Neutrophils and Jurkat T cells treated in this way were ∼80% apoptotic by nuclear condensation. Thymocytes were cultured in RPMI 1640 with 10% FCS at 2 × 106 cells/ml at 37°C in 5% CO2 for 3 h. In Fig. 5 E, thymocytes were cultured in RPMI 1640 with 0.5% low-endotoxin BSA at 4 × 106 cells/ml overnight without exposure to UV irradiation. Thymocytes exposed to UV irradiation were ∼90% annexin V positive and 30% propidium iodide positive. Thymocytes cultured overnight were ∼80% annexin V positive and 60% propidium iodide positive.

FIGURE 5.

Lovastatin increases efferocytosis in vivo. Ten million, Cell Tracker Red-stained, apoptotic murine thymocytes were instilled intratracheally into anesthetized mice. Bronchoalveolar lavage was performed 40 min later, and was assessed for (A) the alveolar macrophage PI and (B) recovery of apoptotic thymocytes. A, Representative photomicrographs of Wright-Giemsa-stained murine alveolar macrophages (magnification, ×100) with ingested apoptotic thymocytes (arrows). B, Plot on the left represents the forward-side scatter characteristics of bronchoalveolar lavage, where R1 = alveolar macrophages and R2 = recovered thymocytes + erythrocytes. Plot on the right represents side scatter-FL-2 (Cell Tracker Red) staining characteristics of R2, where FL-2-positive cells are recovered thymocytes (R4) and FL-2-negative cells are erythrocytes (R3). C, Lovastatin increased the alveolar macrophage PI (∗, significantly different from control; p = 0.035) and (D) decreased the recovery of apoptotic thymocytes (∗, different from control; p = 0.054) in vivo. Eight to 10 animals were used per group. E, Lovastatin increased the alveolar macrophage PI in the absence, but not the presence, of mevalonate, indicating that the effect of lovastatin was HMG-CoA reductase dependent in vivo. Six to 7 animals were used per group. ∗, Significantly different from control (p < 0.05).

FIGURE 5.

Lovastatin increases efferocytosis in vivo. Ten million, Cell Tracker Red-stained, apoptotic murine thymocytes were instilled intratracheally into anesthetized mice. Bronchoalveolar lavage was performed 40 min later, and was assessed for (A) the alveolar macrophage PI and (B) recovery of apoptotic thymocytes. A, Representative photomicrographs of Wright-Giemsa-stained murine alveolar macrophages (magnification, ×100) with ingested apoptotic thymocytes (arrows). B, Plot on the left represents the forward-side scatter characteristics of bronchoalveolar lavage, where R1 = alveolar macrophages and R2 = recovered thymocytes + erythrocytes. Plot on the right represents side scatter-FL-2 (Cell Tracker Red) staining characteristics of R2, where FL-2-positive cells are recovered thymocytes (R4) and FL-2-negative cells are erythrocytes (R3). C, Lovastatin increased the alveolar macrophage PI (∗, significantly different from control; p = 0.035) and (D) decreased the recovery of apoptotic thymocytes (∗, different from control; p = 0.054) in vivo. Eight to 10 animals were used per group. E, Lovastatin increased the alveolar macrophage PI in the absence, but not the presence, of mevalonate, indicating that the effect of lovastatin was HMG-CoA reductase dependent in vivo. Six to 7 animals were used per group. ∗, Significantly different from control (p < 0.05).

Close modal

Lovastatin (Sigma-Aldrich) was converted to its active form by dissolving 25 mg of the lactone form in 500 μl of 100% ethanol, heated to 50°C, alkalinized by adding 250 μl of 0.6 M NaOH, and incubated at 50°C for 2 h. After incubation, the solution was neutralized with 0.4 M HCl at pH 7.5. Aliquots of stock solution were stored frozen at −20°C until used (47, 48).

Phagocytic assays were performed on day 7 HMDMs, as previously described (31). Briefly, apoptotic human neutrophils were added to HMDMs at a 5:1 ratio (apoptotic cell to HMDM) and incubated at 37°C in 10% CO2 for 40 min in 500 μl of X-Vivo medium. HMDMs were washed gently with cold PBS to remove uningested cells, fixed, and stained with a modified Wright-Giemsa (Fisher Scientific). Phagocytosis was determined by visual inspection of samples and was expressed as the phagocytic index (PI), as described (49). Each condition was tested in duplicate and a minimum of 400 HMDMs were counted per condition. In all cases, during analysis, the reader was blinded to the sample identification.

HMDMs were pretreated with 0–10 μM lovastatin for 4–120 h before experimentation. Phagocytosis assays were then performed in the presence and absence of the following reagents at the indicated concentrations and for the indicated times: mevalonate (Sigma-Aldrich), GGPP (Sigma-Aldrich), and FPP (Sigma-Aldrich). Inhibitors of farnesyltransferase (FTI-276; Calbiochem-Novabiochem) and geranylgeranyltransferase I (GGTI-2133; Calbiochem-Novabiochem) were added to HMDMs 8 h before experimentation. FTI-276 inhibits farnesyltransferase with an IC50 = 0.5 nM, and geranylgeranyltransferase I at a much higher IC50 = 50 nM. GGTI-2133 inhibits geranylgeranyltransferase I with an IC50 = 38 nM, and farnesyltransferase at a much higher IC50 = 5.4 μM.

To test the effect of lovastatin and mevalonate on uptake of apoptotic cells, mice were divided into four groups and treated as follows: 1) control group, treated with vehicle (0.5% carboxymethylcellulose sodium, 0.9% sodium chloride, 0.4% polysorbate 80, 0.9% benzyl alcohol in deionized water) by gavage and PBS by i.p. injection; 2) lovastatin group, treated with activated lovastatin (10 mg/kg) in vehicle by gavage and PBS by i.p. injection; 3) mevalonate group, treated with vehicle by gavage and 10 mg/kg l-mevalonate by i.p. injection; 4) lovastatin/mevalonate group, treated with of lovastatin (10 mg/kg) in vehicle by gavage and mevalonate (10 mg/kg) by i.p. injection. Mice were treated three times, spaced within 30 h, before the time of experimentation.

Apoptotic thymocytes were instilled intratracheally as previously described (45). Briefly, mice were anesthetized with Avertin, following which 10 × 106 Cell Tracker Red-stained (Molecular Probes) apoptotic thymocytes, suspended in 50 μl of PBS, were instilled intratracheally using a modified animal feeding needle (Fisher Scientific). Forty minutes later, whole lung bronchoalveolar lavage was performed with a total of 5 ml of ice-cold PBS. Lavage cells were fixed and stained with modified Wright’s Giemsa (Fisher Scientific). Phagocytosis was determined by visual inspection of samples (see Fig. 5 A), as previously described (45, 50), and was expressed a PI. A minimum of 400 alveolar macrophages were counted blindly.

Recovered apoptotic thymocytes were determined as follows. Total lavage cells (including erythrocytes) were counted using a hemacytometer. The percentage of free thymocytes in the lavage was determined by FACS analysis (Fig. 5 B). Macrophages were excluded, based upon macrophage forward-side scatter characteristics, F4/80 staining, and autofluorescence. Total recovered thymocytes were calculated by multiplying total lavage cells by the percentage of Cell Tracker Red-positive cells.

FACS analysis was done as previously described (31). Briefly, HMDMs were suspended in HBSS containing 2% FCS (Gemini Bio-Products), blocked with human serum, except cells for FcγR staining, incubated with 5 μg of the primary Ab for 30 min on ice, washed twice, then incubated with the secondary Ab (1/50 dilution) on ice for 30 min. Washed macrophages were analyzed on a FACScan cytometer using CellQuest Pro (BD Biosciences) and FloJo (Tree Star) software.

Abs used in FACS analysis were as follows: BD Biosciences/BD Pharmingen Abs include mouse monoclonal anti-human CD36 IgM, anti-human CD44 IgG1, anti-human integrin β3 IgG1, anti-human CD32 (FcγRIIa) IgG2b, and mouse monoclonal IgG1, IgG2a, IgG2b, IgM, κ isotype controls (Chemicon International) Abs include mouse monoclonal anti-human integrin αVβ5 IgG1 (Affinity BioReagent) Abs include chicken polyclonal anti-human calreticulin IgY (American Diagnostica) Abs include mouse monoclonal anti-human α-chain CD91 IgG1. Mouse monoclonal anti-human PS recognition structure IgM (217) was prepared in this laboratory as previously described (27). Jackson ImmunoResearch Laboratories Abs include chicken IgY isotype control, Cy-3 goat IgG anti-mouse IgM, and Cy-3 goat IgG anti-mouse IgG, and Cy-3 goat anti-chicken IgY.

HMDM membrane fractions were prepared and separated on SDS-PAGE as described previously (51, 52). Briefly, HMDMs plated on 10-cm tissue-culture dish were harvested and resuspended in PBS. Pelleted cells were lysed by Reporter Lysis Buffer (Promega) and by repeated freeze-thaw cycles. Cell lysates were spun down at 3,000 rpm for 10 m. Supernatants were collected and spun-down at 10,000 × g for 45 m. The pellet was solubilized in lysate buffer (20 mM HEPES (pH 7.4), 150 mM NaCl, 1 mM DTT, 1% Triton X-100). Samples were run on a 7.5% SDS-PAGE gel, transferred to nitrocellulose, blocked with 3% milk, and sequentially incubated with primary and secondary Abs. Total protein was measured by the BCA Protein Assay kit (Pierce) and equivalent amounts were loaded into gels. Immunoblotting was performed using mAbs against RhoA, RhoB, and RhoC (clone 55, 3 μg/ml; Upstate Biotechnology) and Rac-1 (clone 23A8, 1 μg/ml; Upstate Biotechnology), and immunodetection was accomplished using a mouse anti-mouse HRP-conjugated secondary Ab (1/10,000). Membranes were developed using Amersham ECL system (Amersham Biosciences).

The means were analyzed using ANOVA for multiple comparisons; when ANOVA indicated significance, the Dunnett’s method was used to compare groups with an internal control. For all other experiments in which two conditions were being compared, a Student’s t test assuming equal variance was used. All data were analyzed using JMP (version 3) Statistical Software for Macintosh (SAS Institute) and are presented ± SEM.

We tested the effect of statins on efferocytosis by incubating HMDMs with lovastatin for 6–48 h. Lovastatin (5 μM) increased efferocytosis as early as 6 h (Fig. 1,B). However, at 24 h, lovastatin increased efferocytosis in a dose-dependent fashion, exerting a maximum effect at 10 μM. In contrast, lovastatin had no effect on binding of apoptotic cells to HMDMs (Fig. 1,C). Lovastatin (1 μM) also increased efferocytosis in HMDMs after 5 days of treatment compared with untreated cells, suggesting that the effect was prolonged (Fig. 1 D).

Most, but not all, statin effects are related to their ability to competitively block HMG-CoA reductase, and thereby decrease production of multiple intermediates in the cholesterol biosynthetic pathway. Mevalonate is the initial product of HMG-CoA reductase (Fig. 1,A) and mevalonate levels are decreased by statin therapy. We performed “rescue” experiments with mevalonate to determine whether lovastatin was acting through an HMG-CoA reductase-dependent pathway (Fig. 1,E). Mevalonate reversed the ability of lovastatin to potentiate efferocytosis, confirming HMG-CoA reductase dependency. In contrast, lovastatin suppressed phagocytosis of IgG-opsonized erythrocytes through the FcγR, as has recently been shown (53, 54), and this also appeared to be dependent on HMG-CoA reductase (Fig. 1 F).

We considered that lovastatin may exert its positive effect on efferocytosis by increasing expression of key uptake receptors, especially because statins were recently reported to increase the expression of CD36, a well-known efferocytosis receptor (55). Contrary to this report, we found that lovastatin had no effect on expression of HMDM efferocytosis receptors (i.e., αVβ5, β3, CD91, calreticulin, CD36, CD44, CD14) or the FcγRIIa (Fig. 2). Lovastatin also had no effect on staining by mAb 217. The protein target of mAb 217 was originally thought to be the phosphatidylserine receptor, but is now not known.

FIGURE 2.

Lovastatin had no effect on expression of HMDM efferocytosis receptors. HMDMs were treated with and without lovastatin for 24 h and examined by flow cytometry for surface expression of a variety of receptors associated with efferocytosis and phagocytosis. Histograms for each receptor are representative of three replicates per group.

FIGURE 2.

Lovastatin had no effect on expression of HMDM efferocytosis receptors. HMDMs were treated with and without lovastatin for 24 h and examined by flow cytometry for surface expression of a variety of receptors associated with efferocytosis and phagocytosis. Histograms for each receptor are representative of three replicates per group.

Close modal

Statins regulate the posttranslational modification of hundreds of proteins by controlling the production of key substrates of protein prenylation, such as FPP and GGPP. Because mevalonate is an upstream precursor of both FPP and GGPP, statins ultimately decrease their levels as well. We performed dose-response rescue experiments with GGPP and FPP, and found that both reversed lovastatin-enhanced efferocytosis, albeit with different potencies (Fig. 3, A and B). Even though these experiments suggested a role for both farnesylation and geranylgeranylation, they did not rule out the possibility that the effect of FPP was due to its conversion to GGPP and augmentation of geranylgeranylation. This is an important issue because the known Rho GTPase regulators of efferocytosis are only geranylgeranylated. Incomplete inhibition of HMG-CoA reductase could potentially allow low level synthesis of the 5-carbon isopentenyl pyrophosphate (IPP), which normally condenses with 15-carbon FPP to produce 20-carbon GGPP (Fig. 1 A), thereby creating a pathway for FPP repletion to influence geranylgeranylation.

FIGURE 3.

Lovastatin-enhanced efferocytosis is dependent on geranylgeranylation and farnesylation. A, GGPP (1, 5, and 10 μM) and (B) FPP (5 and 10 μM) prevented lovastatin from increasing efferocytosis of apoptotic human neutrophils by HMDMs, but 10 μM GGPP or FPP alone had no effect on efferocytosis. The mean PI as a percentage of control ± SEM is shown for five replicates per group. Control mean PI: 3.8 ± 0.8. ∗, Significantly different from control (p < 0.05). C, GGTI-2133 (20 and 40 nM) and (D) FTI-276 (0.5 nM) enhanced efferocytosis of apoptotic human neutrophils by HMDMs. As a positive control, HMDMs were treated with 10 μM lovastatin for 24 h. DMSO vehicle had no effect on efferocytosis. The mean PI as a percentage of control ± SEM is shown for 3–10 replicates/group. Control mean PI for C: 3.9 ± 0.3, and D: 4.9 ± 1.0. ∗, Significantly different from control (p < 0.05).

FIGURE 3.

Lovastatin-enhanced efferocytosis is dependent on geranylgeranylation and farnesylation. A, GGPP (1, 5, and 10 μM) and (B) FPP (5 and 10 μM) prevented lovastatin from increasing efferocytosis of apoptotic human neutrophils by HMDMs, but 10 μM GGPP or FPP alone had no effect on efferocytosis. The mean PI as a percentage of control ± SEM is shown for five replicates per group. Control mean PI: 3.8 ± 0.8. ∗, Significantly different from control (p < 0.05). C, GGTI-2133 (20 and 40 nM) and (D) FTI-276 (0.5 nM) enhanced efferocytosis of apoptotic human neutrophils by HMDMs. As a positive control, HMDMs were treated with 10 μM lovastatin for 24 h. DMSO vehicle had no effect on efferocytosis. The mean PI as a percentage of control ± SEM is shown for 3–10 replicates/group. Control mean PI for C: 3.9 ± 0.3, and D: 4.9 ± 1.0. ∗, Significantly different from control (p < 0.05).

Close modal

Specific inhibitors of geranylgeranyltransferase I (GGTI-2133; IC50 = 38 nM) and farnesyltransferase (FTI-276; IC50 = 0.5 nM) were used to further examine the role of these separate prenylation pathways on efferocytosis. Both inhibitors enhanced efferocytosis, but the effect of FTI-276 appeared to be more modest than GGTI-2133 (Fig. 3, C and D). It is important to note that farnesyltransferase inhibitors may increase the production of geranylgeranylated proteins, presumably by shunting excess FPP to GGPP (56). This suggests that farnesyltransferase inhibitors may not solely act by decreasing farnesylated proteins, but instead they may also change the balance in favor of geranylgeranylated proteins. Consistent with this possibility, combinations of GGTI-2133 and FTI-276 did not enhance efferocytosis more than lovastatin or GGTI-2133 alone (data not shown). Therefore, efferocytosis appears to be regulated by intracellular signaling molecules that are geranylgeranylated, and possibly by those that are farnesylated.

Statins inhibit prenylation and membrane localization of a variety of Rho GTPases, including positive (Rac-1) and negative (RhoA) regulators of efferocytosis. Yet, in HMDMs, lovastatin consistently enhanced efferocytosis, implying that lovastatin may exert a prolonged, disproportionate effect on the prenylation and membrane localization of RhoA. To address this hypothesis, HMDMs were treated with and without lovastatin for 24 h, and membrane fractions were assessed for RhoA and Rac-1 staining by Western blot. Lovastatin decreased membrane-bound RhoA greater then Rac-1 (Fig. 4, A and B), suggesting a mechanism for the positive effect of lovastatin on efferocytosis.

FIGURE 4.

Lovastatin may enhance efferocytosis by suppressing membrane-bound RhoA. A and B, HMDMs were treated with and without lovastatin for 24 h and assessed for membrane-bound RhoA and Rac-1 by Western blot and densitometry. A, Representative Western blots from five separate experiments are shown. CD71 was used as a membrane marker and control for equal loading. B, Lovastatin significantly decreased membrane-bound RhoA greater then Rac-1 (p < 0.05). C, LPA (10 μM), a potent RhoA activator, decreased ingestion of apoptotic Jurkat T cells by HMDMs in the absence, but not the presence, of lovastatin. Jurkat T cells were used in these experiments instead of neutrophils because they are ingested more avidly by HMDMs. The mean PI as a percentage of control ± SEM is shown for six replicates per group. Control mean PI: 42.2 ± 10.3. ∗, Significantly different from control (p < 0.05). †, Different from control (p = 0.051).

FIGURE 4.

Lovastatin may enhance efferocytosis by suppressing membrane-bound RhoA. A and B, HMDMs were treated with and without lovastatin for 24 h and assessed for membrane-bound RhoA and Rac-1 by Western blot and densitometry. A, Representative Western blots from five separate experiments are shown. CD71 was used as a membrane marker and control for equal loading. B, Lovastatin significantly decreased membrane-bound RhoA greater then Rac-1 (p < 0.05). C, LPA (10 μM), a potent RhoA activator, decreased ingestion of apoptotic Jurkat T cells by HMDMs in the absence, but not the presence, of lovastatin. Jurkat T cells were used in these experiments instead of neutrophils because they are ingested more avidly by HMDMs. The mean PI as a percentage of control ± SEM is shown for six replicates per group. Control mean PI: 42.2 ± 10.3. ∗, Significantly different from control (p < 0.05). †, Different from control (p = 0.051).

Close modal

We next used the potent RhoA activator, LPA to address whether lovastatin could reverse impaired efferocytosis in vitro (57). LPA-suppressed efferocytosis by HMDMs, and this suppression was prevented by lovastatin (Fig. 4 C), suggesting that lovastatin, or for that matter other Rho pathway inhibitors, could play a therapeutic role in diseases where suppression of efferocytosis contributes to disease pathogenesis.

We tested whether statins could enhance efferocytosis by treating mice with lovastatin (10 mg/kg) three times over 30 h. Ten million, Cell Tracker Red-labeled apoptotic murine thymocytes were then instilled intratracheally and clearance was assessed. This model has previously been used to evaluate efferocytosis by macrophages and epithelial cells in vivo (45, 50). Defective efferocytosis is suggested by either decreased uptake into alveolar macrophages (i.e., decreased PI; Fig. 5,A), or by increased recovery of apoptotic cells in the bronchoalveolar lavage (Fig. 5,B). Lovastatin modestly increased efferocytosis in the naive murine lung as measured by an increase in the alveolar macrophage PI (Fig. 5,C) and by a decrease in the recovery of apoptotic thymocytes (Fig. 5 D).

To examine whether the action of lovastatin on efferocytosis in vivo was HMG-CoA reductase dependent, mice were treated with lovastatin (10 mg/kg), three times over 30 h, in the presence or absence of rescue mevalonate, and clearance of apoptotic thymocytes by alveolar macrophages was assessed. Lovastatin again increased efferocytosis by alveolar macrophages in vivo, and this effect was prevented by mevalonate (Fig. 5 E). Together, these results indicate that lovastatin enhances efferocytosis in the naive murine lung in an HMG-CoA reductase-dependent manner, thus confirming in vitro results.

Accumulating evidence suggests that efferocytosis is dysregulated in chronic inflammatory lung diseases, such as COPD, and may contribute to disease pathogenesis. For example, several animal models of COPD are associated with increased accumulation (58, 59, 60, 61) and impaired removal (45) of apoptotic cells. Likewise, apoptotic cells are increased in COPD lungs (31, 62, 63, 64, 65) and efferocytosis is defective in COPD alveolar macrophages ex vivo (32). Therefore, we tested the effect of lovastatin on efferocytosis by alveolar macrophages isolated from GOLD stage 2 (66) COPD patients (Table I). Lovastatin enhanced efferocytosis in these alveolar macrophages in an HMG-CoA reductase-dependent fashion (Fig. 6). Taken together, these data suggest that statins may have therapeutic potential in diseases, such as COPD, where efferocytosis is suppressed and inflammation is dysregulated.

Table I.

Subject demographicsa

CharacteristicsValues
n 
Age (years) 57 ± 2.1 
FEV1 (% predicted) 63.3 ± 8.2 
FVC (% predicted) 80.4 ± 9.9 
FEV1/FVC (%) 60.2 ± 0.3 
Smoking history (pack-years) 47 ± 15.9 
CharacteristicsValues
n 
Age (years) 57 ± 2.1 
FEV1 (% predicted) 63.3 ± 8.2 
FVC (% predicted) 80.4 ± 9.9 
FEV1/FVC (%) 60.2 ± 0.3 
Smoking history (pack-years) 47 ± 15.9 
a

FEV, Forced expiratory volume in 1 s; FVC, forced vital capacity.

FIGURE 6.

Lovastatin reverses impaired efferocytosis in COPD alveolar macrophages ex vivo. Human alveolar macrophages were isolated from patients with GOLD stage 2 COPD, then incubated with or without lovastatin or mevalonate for 24 h before coculture with apoptotic human neutrophils. Lovastatin (Lova; 10 μM) treatment enhanced efferocytosis by GOLD stage 2 alveolar macrophages (n = 3), but not in the presence of mevalonate (l-Meva; 50 μM). Control mean PI: 5.5 ± 1.9. ∗, Significantly different from control (p < 0.05).

FIGURE 6.

Lovastatin reverses impaired efferocytosis in COPD alveolar macrophages ex vivo. Human alveolar macrophages were isolated from patients with GOLD stage 2 COPD, then incubated with or without lovastatin or mevalonate for 24 h before coculture with apoptotic human neutrophils. Lovastatin (Lova; 10 μM) treatment enhanced efferocytosis by GOLD stage 2 alveolar macrophages (n = 3), but not in the presence of mevalonate (l-Meva; 50 μM). Control mean PI: 5.5 ± 1.9. ∗, Significantly different from control (p < 0.05).

Close modal

Apoptotic cell clearance defects are increasingly recognized in diseases of chronic inflammation (31, 32) and autoimmunity (33, 34, 67, 68), suggesting that effective efferocytosis may be necessary for the maintenance of homeostasis. This concept is based on the role efferocytosis plays in suppressing both the innate (69, 70) and adaptive (68) immune response and in removing autoantigens (71). Interestingly, this concept is not new. Over 100 years ago, Metchnikoff (72) recognized this removal process and its importance to homeostasis, which he termed “physiologic inflammation.” Therefore, therapies that enhance efferocytosis may offer a unique therapeutic benefit, especially if impaired efferocytosis is a determinant of disease pathogenesis. Our findings indicate that lovastatin enhances efferocytosis in vitro and in vivo. Lovastatin’s effect on efferocytosis depends on its ability to inhibit HMG-CoA reductase, decrease prenylation substrates, and to alter the balance of Rho GTPases. Lovastatin may have therapeutic potential in chronic inflammatory diseases with impaired efferocytosis because it reverses impaired efferocytosis in vitro and in vivo, and enhances efferocytosis by alveolar macrophages from patients with COPD.

Up to 2% of expressed cellular proteins are prenylated and over 150 prenylated proteins have been identified (73, 74), suggesting that the effect of statins on efferocytosis is likely to be complex. Our findings indicate that lovastatin enhanced efferocytosis in vitro, in part, by altering the membrane balance of RhoA and Rac-1, two key regulators of efferocytosis. Geranylgeranyltransferase I prenylates both RhoA and Rac-1, yet lovastatin suppressed membrane localization of RhoA to a greater extent than Rac-1. The reason(s) for this disproportionate effect is unclear, but it suggests that enzyme kinetics favor prenylation of Rac-1 over RhoA. Because lovastatin increased efferocytosis after 5 days of treatment, the effect appears to be sustained. Possible explanations for this prolonged effect include: 1) geranylgeranyltransferase I may prenylate Rac-1 more efficiently than RhoA, or 2) prenylation of Rac-1 may be less substrate dependent. Even during lovastatin treatment, prenylation substrates (GGPP or FPP) would be expected to be present in small quantities due to incomplete blockade of HMG-CoA reductase, or due to salvage pathway activity (75). Alternatively, 3) the half-life of prenylated Rac-1 may be longer than prenylated RhoA. This possibility is less likely, because lovastatin enhanced efferocytosis as long as 5 days after treatment. We also noted that the ability of lovastatin to increase efferocytosis by HMDMs waned during days 3 and 4 of treatment, but increased again at day 5. We do not have a clear explanation for this observation, but it was consistent across both concentrations tested.

Our data suggest that farnesylated proteins might negatively regulate efferocytosis, because inhibition of farnesyltransferase modestly increased efferocytosis, and FPP repletion reversed lovastatin-enhanced efferocytosis. RhoB is an attractive candidate for this effect, because it is both farnesylated and geranylgeranylated (76) and it plays a known role in the phagocytosis of Pneumocystis (77). Whether farnesyltransferase inhibitors also shift the balance toward production of other geranylgeranylated proteins that could enhance efferocytosis, like Rac-1, Cdc42, or RhoG (43, 78), remains to be determined.

Growing evidence from animal models indicates that statins may have a role in the treatment of inflammatory lung diseases, including acute lung injury (2), asthma (18), and emphysema (19). Our data supports this notion, because lovastatin increased efferocytosis in the lungs of naive mice. In vivo, lovastatin may enhance efferocytosis by altering the balance of membrane-bound RhoA and Rac-1, as was demonstrated in vitro. Alternatively, in vivo and especially in an inflammatory environment, lovastatin may enhance efferocytosis by suppressing oxidative stress, because oxidative stress inhibits efferocytosis by activating RhoA (K. A. McPhillips, manuscript in preparation). Lovastatin also suppresses matrix metalloproteinase-9 (8), which inhibits efferocytosis in vitro (R. W. Vandivier, unpublished data), and is an important component of lung inflammation. Finally, statins increase PPARγ, which has also been shown to increase efferocytosis (79).

Lovastatin did not enhance efferocytosis by altering apoptotic cell binding or by increasing expression of efferocytosis receptors. In contrast, Ruiz-Velasco et al. (55) found that lovastatin treatment (10 μM) increased CD36 surface expression and mRNA in human monocytes at 24 h and THP-1 cells at 48 h. The disparity between our findings may relate to intrinsic differences between human monocytes and HMDMs.

Lovastatin suppressed FcγR-mediated phagocytosis in an HMG-CoA reductase-dependent manner, confirming recent reports (53, 54). Interestingly, these authors all concluded that statins suppress FcγR-mediated phagocytosis by inhibiting cholesterol biosynthesis, and not by inhibiting prenylation (53, 54). Like efferocytosis, prenylated proteins like Rac-1, Cdc42, and Rab11 are required for FcγR-mediated phagocytosis (80, 81), and a role for RhoA has been suggested (82). Therefore, we propose that under certain conditions statins might also influence FcγR phagocytosis through modulation of Rho GTPases. The effect of statins on FcγR-mediated phagocytosis is concerning because of its potential to impair host defense, but ultimately, its importance remains to be demonstrated in vivo. In a Klebsiella pneumoniae mouse model of pneumonia, lovastatin delayed bacterial clearance and enhanced dissemination (2). In contrast, statins have consistently improved survival in animal and human bacterial sepsis (16, 22, 83), implying that the beneficial effects of statins may outweigh their potential deleterious effects.

The dose and concentration of lovastatin used in the in vitro and in vivo experiments is certainly higher than is used clinically, suggesting that lovastatin may not augment efferocytosis when used in humans. In mice, however, lovastatin induces a 6- to 10-fold increase in hepatic microsomal HMG-CoA reductase after only 24 h, implying that higher doses may be required to produce clinical effects in mice compared with humans (48). In humans, prolonged administration of lovastatin at 80 mg/kg/day results in steady state concentrations ranging from 0.15 to 0.3 μM (84), which is marginally less than the lowest effective concentration of lovastatin used in our study (1 μM). The effectiveness of lovastatin in vivo, though, may depend on the clinical setting and remains to be determined in COPD. For example, it is possible that prolonged lovastatin treatment at clinically relevant doses may augment efferocytosis in humans with COPD, especially if RhoA activity is increased.

The ability of statins to enhance efferocytosis suggests a new mechanism by which statins may modulate acute and chronic inflammatory diseases, and may help direct statin clinical trials to specific diseases. For example, cystic fibrosis, bronchiectasis, and COPD are attractive targets for statin therapy because they are all associated with accumulation, and defective clearance, of apoptotic cells (31, 32). Indeed, our data provides “proof of principle” for this approach because lovastatin increased efferocytosis by human alveolar macrophages taken from patients with COPD. Lee et al. (19) have also shown that statin treatment inhibits the development of cigarette smoke-induced emphysema in rats. However, we would not suggest that potential therapeutic targets be limited to these chronic inflammatory lung diseases, because accumulation of apoptotic cells and failed efferocytosis has also been implicated in systemic inflammatory diseases, like glomerulonephritis (34), rheumatoid arthritis (36), and systemic lupus erythematosus (35).

The authors have no financial conflict of interest.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1

This work was supported by an Atorvastatin Research Award (to R.W.V.) sponsored by Pfizer, COPD Center funding (to K.M.), Pew Latin American Fellows Program in the Biomedical Sciences (to V.M.B.), and by grants from the National Institutes of Health to R.W.V. (HL072018) and P.M.H. (GM061031 and HL068864).

3

Abbreviations used in this paper: PPAR, peroxisome proliferator-activated receptor; HMG-CoA, 3-hydroxyl-3-methylglutaryl coenzyme A; FPP, farnesyl pyrophosphate; GGPP, geranylgeranyl pyrophosphate; COPD, chronic obstructive pulmonary disease; HMDM, human monocyte-derived macrophage; PI, phagocytic index; IPP, isopentenyl pyrophosphate; LPA, lysophosphatidic acid.

1
Schonbeck, U., P. Libby.
2004
. Inflammation, immunity, and HMG-CoA reductase inhibitors: statins as antiinflammatory agents?.
Circulation
109
:
II18
-II26.
2
Fessler, M. B., S. K. Young, S. Jeyaseelan, J. G. Lieber, P. G. Arndt, J. A. Nick, G. S. Worthen.
2005
. A role for hydroxy-methylglutaryl coenzyme a reductase in pulmonary inflammation and host defense.
Am. J. Respir. Crit. Care Med.
171
:
606
-615.
3
Rikitake, Y., S. Kawashima, S. Takeshita, T. Yamashita, H. Azumi, M. Yasuhara, H. Nishi, N. Inoue, M. Yokoyama.
2001
. Anti-oxidative properties of fluvastatin, an HMG-CoA reductase inhibitor, contribute to prevention of atherosclerosis in cholesterol-fed rabbits.
Atherosclerosis
154
:
87
-96.
4
Zelvyte, I., R. Dominaitiene, M. Crisby, S. Janciauskiene.
2002
. Modulation of inflammatory mediators and PPARγ and NFκB expression by pravastatin in response to lipoproteins in human monocytes in vitro.
Pharmacol. Res.
45
:
147
-154.
5
Rosenson, R. S., C. C. Tangney, L. C. Casey.
1999
. Inhibition of proinflammatory cytokine production by pravastatin.
Lancet
353
:
983
-984.
6
Pahan, K., F. G. Sheikh, A. M. Namboodiri, I. Singh.
1997
. Lovastatin and phenylacetate inhibit the induction of nitric oxide synthase and cytokines in rat primary astrocytes, microglia, and macrophages.
J. Clin. Invest.
100
:
2671
-2679.
7
Crisby, M., G. Nordin-Fredriksson, P. K. Shah, J. Yano, J. Zhu, J. Nilsson.
2001
. Pravastatin treatment increases collagen content and decreases lipid content, inflammation, metalloproteinases, and cell death in human carotid plaques: implications for plaque stabilization.
Circulation
103
:
926
-933.
8
Bellosta, S., D. Via, M. Canavesi, P. Pfister, R. Fumagalli, R. Paoletti, F. Bernini.
1998
. HMG-CoA reductase inhibitors reduce MMP-9 secretion by macrophages.
Arterioscler. Thromb. Vasc. Biol.
18
:
1671
-1678.
9
Furman, C., C. Copin, M. Kandoussi, R. Davidson, M. Moreau, F. McTaggiart, M. J. Chapman, J. C. Fruchart, M. Rouis.
2004
. Rosuvastatin reduces MMP-7 secretion by human monocyte-derived macrophages: potential relevance to atherosclerotic plaque stability.
Atherosclerosis
174
:
93
-98.
10
Laufs, U., J. K. Liao.
1998
. Post-transcriptional regulation of endothelial nitric oxide synthase mRNA stability by Rho GTPase.
J. Biol. Chem.
273
:
24266
-24271.
11
Martin, G., H. Duez, C. Blanquart, V. Berezowski, P. Poulain, J. C. Fruchart, J. Najib-Fruchart, C. Glineur, B. Staels.
2001
. Statin-induced inhibition of the Rho-signaling pathway activates PPARα and induces HDL apoA-I.
J. Clin. Invest.
107
:
1423
-1432.
12
Baccante, G., G. Mincione, M. C. Di Marcantonio, A. Piccirelli, F. Cuccurullo, E. Porreca.
2004
. Pravastatin up-regulates transforming growth factor-β1 in THP-1 human macrophages: effect on scavenger receptor class A expression.
Biochem. Biophys. Res. Commun.
314
:
704
-710.
13
Kwak, B., F. Mulhaupt, S. Myit, F. Mach.
2000
. Statins as a newly recognized type of immunomodulator.
Nat. Med.
6
:
1399
-1402.
14
Schonbeck, U., N. Gerdes, N. Varo, R. S. Reynolds, D. B. Horton, U. Bavendiek, L. Robbie, P. Ganz, S. Kinlay, P. Libby.
2002
. Oxidized low-density lipoprotein augments and 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors limit CD40 and CD40L expression in human vascular cells.
Circulation
106
:
2888
-2893.
15
Weitz-Schmidt, G., K. Welzenbach, V. Brinkmann, T. Kamata, J. Kallen, C. Bruns, S. Cottens, Y. Takada, U. Hommel.
2001
. Statins selectively inhibit leukocyte function antigen-1 by binding to a novel regulatory integrin site.
Nat. Med.
7
:
687
-692.
16
Merx, M. W., E. A. Liehn, U. Janssens, R. Lutticken, J. Schrader, P. Hanrath, C. Weber.
2004
. HMG-CoA reductase inhibitor simvastatin profoundly improves survival in a murine model of sepsis.
Circulation
109
:
2560
-2565.
17
Leung, B. P., N. Sattar, A. Crilly, M. Prach, D. W. McCarey, H. Payne, R. Madhok, C. Campbell, J. A. Gracie, F. Y. Liew, I. B. McInnes.
2003
. A novel anti-inflammatory role for simvastatin in inflammatory arthritis.
J. Immunol.
170
:
1524
-1530.
18
McKay, A., B. P. Leung, I. B. McInnes, N. C. Thomson, F. Y. Liew.
2004
. A novel anti-inflammatory role of simvastatin in a murine model of allergic asthma.
J. Immunol.
172
:
2903
-2908.
19
Lee, J. H., D. S. Lee, E. K. Kim, K. H. Choe, Y. M. Oh, T. S. Shim, S. E. Kim, Y. S. Lee, S. D. Lee.
2005
. Simvastatin inhibits cigarette smoking-induced emphysema and pulmonary hypertension in rat lungs.
Am. J. Respir. Crit. Care Med.
172
:
987
-993.
20
Cannon, C. P., E. Braunwald, C. H. McCabe, D. J. Rader, J. L. Rouleau, R. Belder, S. V. Joyal, K. A. Hill, M. A. Pfeffer, A. M. Skene.
2004
. Intensive versus moderate lipid lowering with statins after acute coronary syndromes.
N. Engl. J. Med.
350
:
1495
-1504.
21
MacMahon, S., N. Sharpe, G. Gamble, H. Hart, J. Scott, J. Simes, H. White.
1998
. Effects of lowering average of below-average cholesterol levels on the progression of carotid atherosclerosis: results of the LIPID Atherosclerosis Substudy. LIPID Trial Research Group.
Circulation
97
:
1784
-1790.
22
Almog, Y., A. Shefer, V. Novack, N. Maimon, L. Barski, M. Eizinger, M. Friger, L. Zeller, A. Danon.
2004
. Prior statin therapy is associated with a decreased rate of severe sepsis.
Circulation
110
:
880
-885.
23
Johnson, B. A., A. T. Iacono, A. Zeevi, K. R. McCurry, S. R. Duncan.
2003
. Statin use is associated with improved function and survival of lung allografts.
Am. J. Respir. Crit. Care Med.
167
:
1271
-1278.
24
McCarey, D. W., I. B. McInnes, R. Madhok, R. Hampson, O. Scherbakov, I. Ford, H. A. Capell, N. Sattar.
2004
. Trial of atorvastatin in rheumatoid arthritis (TARA): double-blind, randomised placebo-controlled trial.
Lancet
363
:
2015
-2021.
25
Maurer-Stroh, S., S. Washietl, F. Eisenhaber.
2003
. Protein prenyltransferases.
Genome Biol.
4
:
212
26
Etienne-Manneville, S., A. Hall.
2002
. Rho GTPases in cell biology.
Nature
420
:
629
-635.
27
Henson, P. M., D. L. Bratton, V. A. Fadok.
2001
. Apoptotic cell removal.
Curr. Biol.
11
:
R795
-R805.
28
Odaka, C., T. Mizuochi, J. Yang, A. Ding.
2003
. Murine macrophages produce secretory leukocyte protease inhibitor during clearance of apoptotic cells: implications for resolution of the inflammatory response.
J. Immunol.
171
:
1507
-1514.
29
Morimoto, K., H. Amano, F. Sonoda, M. Baba, M. Senba, H. Yoshimine, H. Yamamoto, T. Ii, K. Oishi, T. Nagatake.
2001
. Alveolar macrophages that phagocytose apoptotic neutrophils produce hepatocyte growth factor during bacterial pneumonia in mice.
Am. J. Respir. Cell Mol. Biol.
24
:
608
-615.
30
Golpon, H. A., V. A. Fadok, L. Taraseviciene-Stewart, R. Scerbavicius, C. Sauer, T. Welte, P. M. Henson, N. F. Voelkel.
2004
. Life after corpse engulfment: phagocytosis of apoptotic cells leads to VEGF secretion and cell growth.
FASEB J.
18
:
1716
-1718.
31
Vandivier, R. W., V. A. Fadok, P. R. Hoffmann, D. L. Bratton, C. Penvari, K. K. Brown, J. D. Brain, F. J. Accurso, P. M. Henson.
2002
. Elastase-mediated phosphatidylserine receptor cleavage impairs apoptotic cell clearance in cystic fibrosis and bronchiectasis.
J. Clin. Invest.
109
:
661
-670.
32
Hodge, S., G. Hodge, R. Scicchitano, P. N. Reynolds, M. Holmes.
2003
. Alveolar macrophages from subjects with chronic obstructive pulmonary disease are deficient in their ability to phagocytose apoptotic airway epithelial cells.
Immunol. Cell Biol.
81
:
289
-296.
33
Bratton, D. L., P. M. Henson.
2005
. Autoimmunity and apoptosis: refusing to go quietly.
Nat. Med.
11
:
26
-27.
34
Botto, M., C. Dell’Agnola, A. E. Bygrave, E. M. Thompson, H. T. Cook, F. Petry, M. Loos, P. P. Pandolfi, M. J. Walport.
1998
. Homozygous C1q deficiency causes glomerulonephritis associated with multiple apoptotic bodies.
Nat. Genet.
19
:
56
-59.
35
Baumann, I., W. Kolowos, R. E. Voll, B. Manger, U. Gaipl, W. L. Neuhuber, T. Kirchner, J. R. Kalden, M. Herrmann.
2002
. Impaired uptake of apoptotic cells into tingible body macrophages in germinal centers of patients with systemic lupus erythematosus.
Arthritis Rheum.
46
:
191
-201.
36
Firestein, G. S., M. Yeo, N. J. Zvaifler.
1995
. Apoptosis in rheumatoid arthritis synovium.
J. Clin. Invest.
96
:
1631
-1638.
37
Huynh, M. L., K. C. Malcolm, C. Kotaru, J. A. Tilstra, J. Y. Westcott, V. A. Fadok, S. E. Wenzel.
2005
. Defective apoptotic cell phagocytosis attenuates prostaglandin e2 and 15-hydroxyeicosatetraenoic acid in severe asthma alveolar macrophages.
Am. J. Respir. Crit. Care Med.
172
:
972
-979.
38
Leverrier, Y., A. J. Ridley.
2001
. Requirement for Rho GTPases and PI 3-kinases during apoptotic cell phagocytosis by macrophages.
Curr. Biol.
11
:
195
-199.
39
deCathelineau, A. M., P. M. Henson.
2003
. The final step in programmed cell death: phagocytes carry apoptotic cells to the grave.
Essays Biochem.
39
:
105
-117.
40
Tosello-Trampont, A. C., K. Nakada-Tsukui, K. S. Ravichandran.
2003
. Engulfment of apoptotic cells is negatively regulated by Rho-mediated signaling.
J. Biol. Chem.
278
:
49911
-49919.
41
Chimini, G., P. Chavrier.
2000
. Function of Rho family proteins in actin dynamics during phagocytosis and engulfment.
Nat. Cell Biol.
2
:
E191
-E196.
42
Hoffmann, P. R., A. M. deCathelineau, C. A. Ogden, Y. Leverrier, D. L. Bratton, D. L. Daleke, A. J. Ridley, V. A. Fadok, P. M. Henson.
2001
. Phosphatidylserine (PS) induces PS receptor-mediated macropinocytosis and promotes clearance of apoptotic cells.
J. Cell Biol.
155
:
649
-660.
43
deBakker, C. D., L. B. Haney, J. M. Kinchen, C. Grimsley, M. Lu, D. Klingele, P. K. Hsu, B. K. Chou, L. C. Cheng, A. Blangy, et al
2004
. Phagocytosis of apoptotic cells is regulated by a UNC-73/TRIO-MIG-2/RhoG signaling module and armadillo repeats of CED-12/ELMO.
Curr. Biol.
14
:
2208
-2216.
44
Savill, J. S., A. H. Wyllie, J. E. Henson, M. J. Walport, P. M. Henson, C. Haslett.
1989
. Macrophage phagocytosis of aging neutrophils in inflammation: programmed cell death in the neutrophil leads to its recognition by macrophages.
J. Clin. Invest.
83
:
865
-875.
45
Vandivier, R. W., C. A. Ogden, V. A. Fadok, P. R. Hoffmann, K. K. Brown, M. Botto, M. J. Walport, J. H. Fisher, P. M. Henson, K. E. Greene.
2002
. Role of surfactant proteins A, D, and C1q in the clearance of apoptotic cells in vivo and in vitro: calreticulin and CD91 as a common collectin receptor complex.
J. Immunol.
169
:
3978
-3986.
46
Gardai, S. J., Y. Q. Xiao, M. Dickinson, J. A. Nick, D. R. Voelker, K. E. Greene, P. M. Henson.
2003
. By binding SIRPα or calreticulin/CD91, lung collectins act as dual function surveillance molecules to suppress or enhance inflammation.
Cell
115
:
13
-23.
47
Jakobisiak, M., S. Bruno, J. S. Skierski, Z. Darzynkiewicz.
1991
. Cell cycle-specific effects of lovastatin.
Proc. Natl. Acad. Sci. USA
88
:
3628
-3632.
48
Kita, T., M. S. Brown, J. L. Goldstein.
1980
. Feedback regulation of 3-hydroxy-3-methylglutaryl coenzyme A reductase in livers of mice treated with mevinolin, a competitive inhibitor of the reductase.
J. Clin. Invest.
66
:
1094
-1100.
49
Fadok, V. A., J. S. Savill, C. Haslett, D. L. Bratton, D. E. Doherty, P. A. Campbell, P. M. Henson.
1992
. Different populations of macrophages use either the vitronectin receptor or the phosphatidylserine receptor to recognize and remove apoptotic cells.
J. Immunol.
149
:
4029
-4035.
50
Teder, P., R. W. Vandivier, D. Jiang, J. Liang, L. Cohn, E. Pure, P. M. Henson, P. W. Noble.
2002
. Resolution of lung inflammation by CD44.
Science
296
:
155
-158.
51
Wassmann, S., U. Laufs, A. T. Baumer, K. Muller, C. Konkol, H. Sauer, M. Bohm, G. Nickenig.
2001
. Inhibition of geranylgeranylation reduces angiotensin II-mediated free radical production in vascular smooth muscle cells: involvement of angiotensin AT1 receptor expression and Rac1 GTPase.
Mol. Pharmacol.
59
:
646
-654.
52
Liao, J. K., S. L. Clark.
1995
. Regulation of G-protein α i2 subunit expression by oxidized low-density lipoprotein.
J. Clin. Invest.
95
:
1457
-1463.
53
Hillyard, D. Z., A. G. Jardine, K. J. McDonald, A. J. Cameron.
2004
. Fluvastatin inhibits raft dependent Fcγ receptor signalling in human monocytes.
Atherosclerosis
172
:
219
-228.
54
Loike, J. D., D. Y. Shabtai, R. Neuhut, S. Malitzky, E. Lu, J. Husemann, I. J. Goldberg, S. C. Silverstein.
2004
. Statin inhibition of Fc receptor-mediated phagocytosis by macrophages is modulated by cell activation and cholesterol.
Arterioscler. Thromb. Vasc. Biol.
24
:
2051
-2056.
55
Ruiz-Velasco, N., A. Dominguez, M. A. Vega.
2004
. Statins upregulate CD36 expression in human monocytes, an effect strengthened when combined with PPAR-γ ligands putative contribution of Rho GTPases in statin-induced CD36 expression.
Biochem. Pharmacol.
67
:
303
-313.
56
Wherlock, M., A. Gampel, C. Futter, H. Mellor.
2004
. Farnesyltransferase inhibitors disrupt EGF receptor traffic through modulation of the RhoB GTPase.
J. Cell Sci.
117
:
3221
-3231.
57
Moolenaar, W. H., L. A. van Meeteren, B. N. Giepmans.
2004
. The ins and outs of lysophosphatidic acid signaling.
BioEssays
26
:
870
-881.
58
Clark, H., N. Palaniyar, P. Strong, J. Edmondson, S. Hawgood, K. B. Reid.
2002
. Surfactant protein D reduces alveolar macrophage apoptosis in vivo.
J. Immunol.
169
:
2892
-2899.
59
Kasahara, Y., R. M. Tuder, L. Taraseviciene-Stewart, T. D. Le Cras, S. Abman, P. K. Hirth, J. Waltenberger, N. F. Voelkel.
2000
. Inhibition of VEGF receptors causes lung cell apoptosis and emphysema.
J. Clin. Invest.
106
:
1311
-1319.
60
Tang, K., H. B. Rossiter, P. D. Wagner, E. C. Breen.
2004
. Lung-targeted VEGF inactivation leads to an emphysema phenotype in mice.
J. Appl. Physiol.
97
:
1559
-1566.
61
Wang, Z., T. Zheng, Z. Zhu, R. J. Homer, R. J. Riese, H. A. Chapman, Jr, S. D. Shapiro, J. A. Elias.
2000
. Interferon γ induction of pulmonary emphysema in the adult murine lung.
J. Exp. Med.
192
:
1587
-1600.
62
Kasahara, Y., R. M. Tuder, C. D. Cool, D. A. Lynch, S. C. Flores, N. F. Voelkel.
2001
. Endothelial cell death and decreased expression of vascular endothelial growth factor and vascular endothelial growth factor receptor 2 in emphysema.
Am. J. Respir. Crit. Care Med.
163
:
737
-744.
63
Imai, K., B. A. Mercer, L. L. Schulman, J. R. Sonett, J. M. D’Armiento.
2005
. Correlation of lung surface area to apoptosis and proliferation in human emphysema.
Eur. Respir. J.
25
:
250
-258.
64
Yokohori, N., K. Aoshiba, A. Nagai.
2004
. Increased levels of cell death and proliferation in alveolar wall cells in patients with pulmonary emphysema.
Chest
125
:
626
-632.
65
Hodge, S., G. Hodge, M. Holmes, P. N. Reynolds.
2005
. Increased airway epithelial and T-cell apoptosis in COPD remains despite smoking cessation.
Eur. Respir. J.
25
:
447
-454.
66
Pauwels, R. A., A. S. Buist, P. M. Calverley, C. R. Jenkins, S. S. Hurd.
2001
. Global strategy for the diagnosis, management, and prevention of chronic obstructive pulmonary disease: NHLBI/WHO Global Initiative for Chronic Obstructive Lung Disease (GOLD) Workshop summary.
Am. J. Respir. Crit. Care Med.
163
:
1256
-1276.
67
Cohen, P. L., R. Caricchio, V. Abraham, T. D. Camenisch, J. C. Jennette, R. A. Roubey, H. S. Earp, G. Matsushima, E. A. Reap.
2002
. Delayed apoptotic cell clearance and lupus-like autoimmunity in mice lacking the c-mer membrane tyrosine kinase.
J. Exp. Med.
196
:
135
-140.
68
Hoffmann, P. R., J. A. Kench, A. Vondracek, E. Kruk, D. L. Daleke, M. Jordan, P. Marrack, P. M. Henson, V. A. Fadok.
2005
. Interaction between phosphatidylserine and the phosphatidylserine receptor inhibits immune responses in vivo.
J. Immunol.
174
:
1393
-1404.
69
Fadok, V. A., D. L. Bratton, A. Konowal, P. W. Freed, J. Y. Westcott, P. M. Henson.
1998
. Macrophages that have ingested apoptotic cells in vitro inhibit proinflammatory cytokine production through autocrine/paracrine mechanisms involving TGF-β, PGE2, and PAF.
J. Clin. Invest.
101
:
890
-898.
70
Huynh, M. L., V. A. Fadok, P. M. Henson.
2002
. Phosphatidylserine-dependent ingestion of apoptotic cells promotes TGF-β1 secretion and the resolution of inflammation.
J. Clin. Invest.
109
:
41
-50.
71
Charles, P. J..
2003
. Defective waste disposal: does it induce autoantibodies in SLE?.
Ann. Rheum. Dis.
62
:
1
-3.
72
Tauber, A. I..
2003
. Metchnikoff and the phagocytosis theory.
Nat. Rev. Mol. Cell Biol.
4
:
897
-901.
73
Grunler, J., J. Ericsson, G. Dallner.
1994
. Branch-point reactions in the biosynthesis of cholesterol, dolichol, ubiquinone and prenylated proteins.
Biochim. Biophys. Acta
1212
:
259
-277.
74
Roskoski, R., Jr.
2003
. Protein prenylation: a pivotal posttranslational process.
Biochem. Biophys. Res. Commun.
303
:
1
-7.
75
Crick, D. C., D. A. Andres, C. J. Waechter.
1997
. Novel salvage pathway utilizing farnesol and geranylgeraniol for protein isoprenylation.
Biochem. Biophys. Res. Commun.
237
:
483
-487.
76
Adamson, P., C. J. Marshall, A. Hall, P. A. Tilbrook.
1992
. Post-translational modifications of p21rho proteins.
J. Biol. Chem.
267
:
20033
-20038.
77
Zhang, J., J. Zhu, X. Bu, M. Cushion, T. B. Kinane, H. Avraham, H. Koziel.
2005
. Cdc42 and RhoB activation are required for mannose receptor-mediated phagocytosis by human alveolar macrophages.
Mol. Biol. Cell
16
:
824
-834.
78
Wennerberg, K., C. J. Der.
2004
. Rho-family GTPases: it’s not only Rac and Rho (and I like it).
J. Cell Sci.
117
:
1301
-1312.
79
Asada, K., S. Sasaki, T. Suda, K. Chida, H. Nakamura.
2004
. Antiinflammatory roles of peroxisome proliferator-activated receptor γ in human alveolar macrophages.
Am. J. Respir. Crit. Care Med.
169
:
195
-200.
80
Caron, E., A. Hall.
1998
. Identification of two distinct mechanisms of phagocytosis controlled by different Rho GTPases.
Science
282
:
1717
-1721.
81
Cox, D., D. J. Lee, B. M. Dale, J. Calafat, S. Greenberg.
2000
. A Rab11-containing rapidly recycling compartment in macrophages that promotes phagocytosis.
Proc. Natl. Acad. Sci. USA
97
:
680
-685.
82
Hackam, D. J., O. D. Rotstein, A. Schreiber, W. Zhang, S. Grinstein.
1997
. Rho is required for the initiation of calcium signaling and phagocytosis by Fcγ receptors in macrophages.
J. Exp. Med.
186
:
955
-966.
83
Liappis, A. P., V. L. Kan, C. G. Rochester, G. L. Simon.
2001
. The effect of statins on mortality in patients with bacteremia.
Clin. Infect. Dis.
33
:
1352
-1357.
84
Thibault, A., D. Samid, A. C. Tompkins, W. D. Figg, M. R. Cooper, R. J. Hohl, J. Trepel, B. Liang, N. Patronas, D. J. Venzon, et al
1996
. Phase I study of lovastatin, an inhibitor of the mevalonate pathway, in patients with cancer.
Clin. Cancer Res.
2
:
483
-491. Vol. 176, No. 12.