Published online before print October 7, 2008
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* UCD School of Medicine and Medical Sciences, UCD Conway Institute, University College Dublin, Dublin, Ireland; and
Professor Eoin O’Malley National Centre for Cardiothoracic Surgery, Mater Misericordiae University Hospital, Dublin, Ireland
1 Correspondence: UCD School of Medicine and Medical Sciences, UCD Conway Institute and Mater Misericordiae University Hospital, University College Dublin, Dublin, Ireland. E-mail: belinda.maher{at}ucd.ie
ABSTRACT
The ability of neutrophils to sense and migrate toward damaged tissue is a vital component of the innate immune response. Paradoxically, this same migration serves as the hallmark of a number of inflammatory conditions, including ischemic reperfusion injury, atherosclerosis, arthritis, and Crohn’s disease. More recent evidence suggests that neutrophil infiltration into the cardiac allograft following transplantation is a contributing factor in allograft rejection. We have demonstrated previously a positive correlation between the degree of neutrophil migration and subsequent rejection grades in a cohort of cardiac transplant recipients. Intracellular signaling pathways that are intimately involved in neutrophil migration thus offer potential targets of manipulation in the treatment of such conditions. 3-Hydroxy-3-methylyglutaryl-coenzyme A reductase inhibitors or statins are emerging as potential anti-inflammatory agents and have a proven survival benefit in the transplant population. Yet, little is known about their ability to modulate neutrophil function and their subsequent mechanism of action. We demonstrate here that pravastatin, simvastatin, and atorvastatin significantly reduce neutrophil transendothelial migration toward the chemoattractant fMLP. This effect is independent of any change in neutrophil adhesion or adhesion molecule expression but is related to the ability of statins to reduce fMLP-induced Rho activity in neutrophils. This was confirmed by the ability of the Rho precursor geranylgeranyl pyrophosphate to rescue the statin-mediated reduction in neutrophil transendothelial migration. Understanding the mechanisms of action of statins in the neutrophil allows for their use in targeting excessive migration in inappropriate inflammatory conditions.
Key Words: ischemic reperfusion injury • atherosclerosis • arthritis • Crohn’s disease • reductase
INTRODUCTION
Neutrophils are the most abundant granulocyte in the circulation and are invaluable for host defense. However, they are also implicated in the pathogenesis of several conditions such as atherosclerosis [1 ], ischemic reperfusion injury [2 , 3 ], and more recently, cardiac allograft rejection, where a growing body of evidence implicates their role in the early stages of acute rejection [4 ]. Neutrophils have been found in murine myocardium within the first hour post-transplant [5 ]. In addition, inhibition of neutrophil infiltration is associated with enhanced allograft survival and reduced rejection rates. These findings have been supported by other studies [6 , 7 ]. Within our own group, the significance of neutrophils in human transplant pathology was demonstrated by a positive correlation between the degree of neutrophil infiltration, assessed by myeloperoxidase (MPO) expression, and rejection severity at the first postoperative biopsy [8 ]. Finding strategies that could reduce neutrophil infiltration may be of tremendous benefit in the management of not only allograft rejection but also many other inflammatory diseases.
Statins or 3-hydroxy-3-methylyglutaryl-coenzyme A (HMG-CoA) reductase inhibitors are proven pharmaceutical agents for the treatment of hypercholesterolemia and ischemic heart disease. Their efficacy in reducing morbidity and mortality associated with cardiovascular events has been demonstrated in a number of clinical trials [9 10 11 ]. Statins are also administered routinely to cardiac transplant recipients for hypercholoesterolemia associated with immunosuppressive agents. In an 8-year retrospective analysis, benefits were observed that were greater than that expected with atherosclerosis prevention alone. Simvastatin and pravastation use was shown to improve overall survival, reduce the incidence of graft vasculopathy, and reduce the severity of rejection events in cardiac allografts [12 , 13 ]. In addition, statin use following lung transplantation is associated with fewer epiodes of acute rejection, lower neutrophil counts in the bronchoalveolar lavage, and improved 6-year survival [14 ]. It is now widely recognized that statins also possess pleiotropic or anti-inflammatory effects independent of their lipid-lowering capabilities. Although research has begun to focus on these lipid independent actions, in vitro studies about the ability of statins to modulate neutrophil function have been relatively neglected. Clinical observations have been noted with regard to statins and neutrophils. For example, atorvastatin administered to patients prior to cardiopulmonary bypass significantly reduced neutrophil CD11b expression and neutrophil-endothelial adhesion [15 ]. Neutrophils obtained from coronary artery bypass surgery patients pretreated with simvastatin demonstrated decreased apoptotic rates [16 ] and a reduced perioperative rise in CD11b expression [17 ]. Simvastatin has also been found to attenuate fMLP-stimulated IL-8 release from neutrophils in addition to significantly reducing reactive oxygen species (ROS) generation [18 ]. Cerivastatin and pravastatin have been shown to inhibit neutrophil transendothelial migration [19 , 20 ]. All of these data point to an immunomodulatory action of statins, independent of their lipid-lowering capabilities, yet the mechanism of this action and the direct signaling pathways they alter remain poorly understood.
Statins specifically mediate their effects through inhibition of HMG-CoA reductase, preventing the conversion of HMG-CoA to mevalonate with the subsequent downstream inhibition of cholesterol synthesis [21 , 22 ]. However, intermediate products of the cholesterol synthesis pathway are used in various other cell-signaling pathways. For example, the isoprenoids geranylgeranyl pyrophosphate (GGP) and farnesyl pyrophosphate (FPP), metabolites of the cholesterol biosynthesis pathway, are involved in the prenylation and hence, activation of the small GTPase family of signaling molecules that includes RhoA [23 ], which with its effector Rho kinase, are thought to control distinct cellular functions such as cytoskeleton rearrangement, migration, and ROS generation [24 ].
We hypothesize that statins modulate neutrophil function through an inhibition of RhoA activity. We aimed therefore to determine the in vitro effect of pravastatin, simvastatin, and atorvastatin on neutrophil migration and adhesion and to correlate this with a direct inhibition of Rho activity. An inhibition of neutrophil functional activity would advocate the use of statins prior to cardiac surgery and transplantation as effective treatments reducing neutrophil infiltration, therefore attenuating the severity of rejection episodes.
MATERIALS AND METHODS
Reagents
DMEM, penicillin, and streptomycin solution, L-glutamine, and PBS were purchased from Gibco-Life Technologies Ltd. (Gaithersburg, MD, USA). Dextran and Ficoll were purchased from Amersham Biosciences (Piscataway, NJ, USA). Pravastatin and simvastatin sodium salts were purchased from Merck Biosciences (San Diego, CA, USA). Atorvastatin salt was purchased from Pfizer (Groton, CT, USA). PE-conjugated CD11b and PE-conjugated CD62 ligand (CD62L) were purchased from BD Biosciences (San Jose, CA, USA), and PE-conjugated CD162 [P-selectin glycoprotein ligand 1 (PSGL-1)] was purchased from Fitzgerald Industries International (Concord, MA, USA). Transwell polycarbonate membrane filters (6.5 mm diameter, 3.0 µM pore size) were purchased from Corning Costar (Cambridge, MA, USA). FCS, fMLP, human placental type IV collagen, ABTS, GGP, and FPP were all purchased from Sigma-Aldrich (Dorset, UK). Rho-associated kinase (ROCK) inhibitor Y-27632 was purchased from Merck Biosciences.
Neutrophil isolation
Venous blood was taken from six healthy individuals who were free from infection and medication at the time of collection. Each individual served as his/her own control. Neutrophils were isolated by Dextran (3%) sedimentation and centrifuged through a discontinuous Ficoll gradient. Remaining RBCs were lysed using 0.8% NH4Cl. Neutrophils were resuspended in DMEM supplemented with 10% FCS (heat-inactivated), 1% L-glutamine, and 1% penicillin/streptomycin solution at a concentration of 1 x 106 per ml. Cell purity was >95%, as assessed by flow cytometry.
Detection of neutrophil surface adhesion molecules
Surface molecules were detected on neutrophils isolated as described above. Briefly, isolated neutrophils (0.5x106 in 500 µl medium) were incubated with or without pravastatin, simvastatin, or atorvastatin, giving final concentrations ranging from 0.5 µM to 50 µM at 37°C in a humidified CO2 incubator for 3 h. Subsets of neutrophils were stimulated additionally with LPS (1 µg/ml) and fMLP (1 ng/ml) for 1 h further, after which time, neutrophils were incubated with 10 µl PE-labeled CD11b antibody, 10 µl PE-labeled CD62L antibody, or 10 µl PE-labeled PSGL-1 antibody for 20 min in the dark at 4°C. Surface expression in the form of mean channel fluorescence was determined by flow cytometry using a Coulter ELITE cytofluorometer. A minimum of 5000 events was collected and analyzed.
Collagen migration assays
The collagen assay used in these experiments was modified from that developed by Mackarel et al. [25
]. Briefly, lyophilized type IV collagen (Sigma-Aldrich) was diluted one in 10 with 0.25% acetic acid solution to give a working stock solution of 50 µg/ml. Of this solution, 50 µl was added to each Transwell polycarbonate membrane filter (6.5 mm diameter, 3.0 µm pore size, Corning Costar). The Transwell filter inserts were suspended in 24-well culture plates so that the filter separated the upper and lower compartments. The collagen was allowed to air-dry overnight in a Laminar airflow cabinet with the airflow switched on. The following day, filters were sterilized and allowed to air-dry once again. Isolated neutrophils (10x106cells/ml) were treated with or without increasing concentrations of pravastatin and simvastatin (0.5 µM–50 µM) for 3 h at 37°C, as mentioned previously. Following incubation, cells were centrifuged at 1200 rpm for 10 min and resuspended in fresh media (10x106cells/ml). Subsequently, 100 µl neutrophils were added to the filters. fMLP (10–8 M) was added to the lower compartment, and the neutrophils were allowed to migrate for 3 h.
Cell culture
Human pulmonary artery endothelial cells (HPAECs; Cambrex, East Rutherford, NJ, USA) were cultured in 100% humidity and 5% CO2 at 37°C in endothelial growth medium supplemented with 5% FBS, epidermal growth factor (10 ng/ml), hydrocortisone (1 µg/ml), bovine brain extract (10 µg/ml), and gentamicin (50 ng/ml, Cambrex). When 
80% confluent, cells were harvested, resuspended in fresh medium, and seeded at a concentration of 0.1 x 106 cells in 200 µl media onto Transwell polycarbonate membrane filters (6.5 mm diameter, 3.0 µm pore size, Corning Costar) that had been coated with human type IV collagen. Culture medium, 600 µl, was added to the lower compartment, and the cells were cultured for a further 4 days. All experiments were carried out on HPAECs between passages 6 and 10.
Transendothelial migration assay
The transmigration assay used in these experiments was developed by Mackarel et al. [25
] and resembles the collagen assay described above. Briefly, on the HPAECs’ 4th day of culture on the filters, neutrophils were isolated, resuspended at a concentration of 10 x 106cells/ml, and incubated with pravastatin, simvastatin (10 µM), or atorvastatin (0.5 µM–50 µM) for 3 h at 37°C. In addition, subsets of neutrophils were stimulated further with LPS (1 µg/ml) for an additional 1 h. As above, neutrophils resuspended in fresh media (0.1x106cells/100 µl) were added to the upper compartment, and fMLP (10–8M) was added to the lower compartment. Neutrophils were allowed to migrate for 3 h at 37°C. After the 3-h incubation time, the plate was placed on ice, and nonmigrated neutrophils (upper compartment) and migrated neutrophils (lower compartment) were collected and centrifuged at 1200 rpm for 10 min. The neutrophils were then lysed by suspension in HBSS containing Ca2+ and Mg2+ (cHBSS) containing 0.25% (w/v) Brij-35. The filter containing the HPAEC monolayer with associated adherent neutrophils was removed by carefully cutting the filter membrane out of the insert and lysed by addition of cHBSS containing 0.25% Brij-35.
MPO assay
The protocol used to measure MPO levels in these migration studies was developed from that used by Madara et al. [26
]. Briefly, the pH of neutrophil lysates was adjusted to 4.2 by the addition of 100 mM citrate buffer. Aliquots of each sample were then transferred to a 96-well plate and incubated for 10 min with 100 µl 2 mM ABTS in 100 mM citrate buffer, pH 4.2, containing 0.06% H2O2. The reaction was stopped by the addition of 2% SDS. Absorbance was measured at 405 nm using a Microplate EL309 autoreader (Bio-Tek Instruments, Inc., Winooski, VT, USA). A standard curve of the MPO standards was plotted against neutrophil numbers. Using this standard curve, the number of neutrophils in each sample fraction was determined, and the percentage of neutrophils, which had migrated, was calculated.
Rho kinase pull-down assay
Isolated neutrophils were treated with fMLP (10–8) for increasing time periods in the presence or absence of pravastatin, simvastatin (10 µM), and atorvastatin (50 µM). Cells were resuspended in ice-cold 1x assay/lysis buffer with standard protease inhibitors (1 µl leupeptin and aproptinin, 2 µl pepstatin, 1 µl 1 mM DTT, 5 µl 10 mM PMSF), and protein was extracted. Protein quantification was assessed by the Bradford assay. Protein, 25 µg, from each sample was run on a separate gel for whole cell Rho determination. Assay lysis buffer (1x) was then added to the remainder of each sample, so each was of an equal volume (protein content normalized to the control). The final concentration of the samples before proceeding was 0.5 mg/ml. The Rhotekin Rho-binding domain (RBD) agarose bead slurry was resuspended thoroughly by vortexing. Resuspended bead slurry, 40 µl, was quickly added per ml sample to each tube and incubated at 4°C for 1 h with gentle agitation. The beads were subsequently pelleted by centrifugation for 10 s at 14,000 g. The supernatant was discarded, and the beads were washed three times with 200 µl 1x assay buffer, centrifuging and aspirating each time. After the last wash, the beads were resuspended in 40 µl loading dye per ml initial sample. The samples were boiled for 5 min and centrifuged for 10 s at 14,000 g. Pull-down supernatant (20 µl/well) was then loaded to a polyacrylamide gel (including a prestained MW standard). SDS-PAGE was performed as per usual, and the gel proteins were transferred to a nitrocellulose membrane, which was blocked with 5% nonfat dry milk in TBST for 1 h at room temperature, after which time, it was incubated with anti-Rho antibody, freshly diluted 1:1000 in 5% nonfat dry milk/TBST for 1–2 h at room temperature with constant agitation. The blotted membrane was washed three times with TBST, 5 min each time. Finally, the membrane was incubated with a secondary antibody (anti-mouse), freshly diluted in 5% nonfat dry milk/TBST for 1 h at room temperature, after which time, the membrane was washed and developed.
Incubation with isoprenoid precursors
Briefly, isolated neutrophils (10x106 cells/ml) were coincubated with pravastatin, simvastatin (10 µM), or atorvastatin (50 µM) and 10 µM GGP or FPP for 3 h at 37°C. Following this, neutrophils (1x106) were added to HPAEC-coated filters and allowed to migrate for 3 h toward fMLP (10–8) as described above. In addition, coincubated neutrophils were lysed and subjected to a Rho pull-down assay as described above.
Statistical analysis
Statistical analysis was performed using Minitab Release 13 statistical software package (Minitab Inc., State College, PA, USA). Comparison of groups was performed using a paired Student’s t-test. A P value of <0.05 was considered to be significant.
RESULTS
The effect of statins on neutrophil migration
We first aimed to determine if statins could modulate the ability of neutrophils to migrate toward the chemoattractant fMLP and at what concentration this effect was maximal. We demonstrated that there was a dose-dependent reduction in the percentage of neutrophils that had migrated across collagen-coated filters following preincubation with pravastatin (70.4±6.3% control, 66.1±8.2% 0.5 µM, 59.1±4.6% 5 µM, 58±7.2% 10 µM, n=5) and simvastatin (67.6±4.4% control, 60.7±3.9% 0.5 µM, 59.0±4.6% 5 µM, 54.5±3.1% 10 µM, n=5). The reduction in the percentage of neutrophils migrating was significant with 5 µM and 10 µM pravastatin pretreatment (P=0.034 and 0.014, respectively) and with 10 µM simvastatin pretreatment (P=0.018). Although atorvastatin pretreatment resulted in reduced neutrophil migration, this was not achieved in a dose-dependent manner. However, at the 50-µM concentration, atorvastatin reduced neutrophil migration significantly (49.5±14.7.1% control, 37.4±15.21% 50 µM, n=5, P=0.000). In vivo, neutrophils must adhere to and migrate through endothelial cells to reach their target site. These endothelial cells act as an additional barrier that neutrophils must negotiate. Taking the optimal doses of each statin as determined above, we investigated if statins could exert a similar effect in a more realistic in vivo setting. Transmigration assays were performed using HPAECs. Neutrophils were preincubated with pravastatin (10 µM), simvastatin (10 µM), and atorvastatin (50 µM) for 3 h, after which time, the cells were washed to remove any remaining statin before transfer to the endothelial monolayer. This ensured that any change in migration was a result of the effects of the statin on the neutrophil and not the HPAEC monolayer. We found that all three statins could significantly reduce the transmigration of neutrophils toward fMLP as compared with untreated cells (47.5±11.1% untreated; 33.7±12.0%, P=0.019 pravastatin; 36.0±6.9%, P=0.031 simvastatin, 33.5±18.8%, P=0.000, atorvastatin n=6; Fig. 1A
). Most migrating neutrophils in vivo are primed prior to their negotiation with endothelial cells; thus, we stimulated statin-pretreated neutrophils with LPS to determine if any of the three statins could attenuate the percentage of primed neutrophils that would migrate. Figure 1B
demonstrates that LPS resulted in a significant increase in the percentage of neutrophils migrating toward fMLP (P=0.002); however, neither statin drug could reduce LPS-stimulated migration significantly.
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Figure 1. Effect of statin treatment on neutrophil migration. (A) Neutrophils were preincubated with pravastatin (Prav) and simvastatin (Simv; 10 µM) and atorvastatin (Atorv; 50 µM) at 37°C for 3 h. They were then allowed to migrate for 3 h across HPAEC-coated filters to fMLP (10–8 M). (B) Finally, neutrophils preincubated with statins were additionally stimulated with LPS (1 µg/ml) for 1 h and transmigrated across endothelial cells. The percentage of migrated neutrophils was determined by comparison of MPO in neutrophils recovered from the lower compartment of the system with total MPO content of neutrophils added to the system. Results are mean ± SD for a maximum of six independent experiments. *, P < 0.05, versus control.
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Table 1. Effect of Statin Treatment on Neutrophil Adhesion
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Table 2. Effect of Statin Treatment on Adhesion Molecule Expression
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Figure 2. Effect of Rho inhibitor Y-27632 on neutrophil migration. Neutrophils were preincubated at 37°C for 1 h with 10 µM of the Rho kinase inhibitor Y-27632. Treated neutrophils were allowed to migrate for 3 h across collagen-coated Transwell filters toward fMLP (10–8 M), as described in Materials and Methods. The percentage of migrated neutrophils was determined by comparison of MPO in neutrophils recovered from the apical compartment with total MPO content of neutrophils added to the system. Results are expressed as mean ± SD for four independent experiments. * P < 0.05, versus control.
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Figure 3. The effect of statin treatment on Rho kinase activity. (A–C) Neutrophils were treated with or without pravastatin and simvastatin (10 µM) or atorvastatin (50 µM) for 3 h at 37°C, followed by stimulation with fMLP (10–8M) for 5, 15, and 30 min. Protein was extracted, and active GTP-bound RhoA was determined as described previously. (D) Neutrophils were treated with pravastatin, simvastatin, and atorvastatin for 3 h at 37°C, after which time, they were stimulated with LPS alone or LPS in combination with fMLP for a further 1 h (LPS) and 30 min (fMLP). Protein was extracted, and active GTP-bound RhoA was determined using a Rhotekin RBD agarose assay, as described in Materials and Methods. The amount of RBD-bound RhoA was normalized to the total amount of RhoA in cell lysates. Western blots represent one of three independent experiments.
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Figure 4. The effect of isoprenoid precursors on neutrophil migration. (A) Neutrophils were coincubated with pravastatin (10 µM), simvastatin (10 µM), or atorvastatin (50 µM) with GGP (10 µM) or FPP (10 µM) for 3 h at 37°C. Protein was extracted, and active GTP-bound RhoA was determined using a Rhotekin RBD agarose assay, as described in Materials and Methods. The amount of RBD-bound RhoA was normalized to the total amount of RhoA in cell lysates. Western blots represent one of three independent experiments (B). Neutrophils were coincubated with pravastatin (10 µM), simvastatin (10 µM), or atorvastatin (50 µM) with GGP (10 µM) or FPP (10 µM) for 3 h at 37°C. Neutrophils were allowed to migrate for 3 h across HPAEC-coated filters to fMLP (10–8 M). The percentage of migrated neutrophils was determined by comparison of MPO in neutrophils recovered from the lower compartment of the system with total MPO content of neutrophils added to the system. Results are mean ± SD for a maximum of three independent experiments. *, P < 0.05, versus control; **, P < 0.05 ggp versus statin treatment alone.
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Neutrophil migration into the subendothelial matrix is a critical event in the progression of a number of inflammatory diseases. We have demonstrated the ability of three HMG-CoA reductase inhibitors, pravastatin, atorvastatin, and simvastatin, to attenuate neutrophil migration through an inhibition of RhoA activity.
Migrating neutrophils are key regulators of infection and inflammation and thus, are integral components of the innate immune system. Paradoxically, neutrophils are also implicated in the pathogenesis of a number of inflammatory diseases, including atherosclerosis, ischemic reperfusion injury, systemic inflammatory response syndrome, and more recently, allograft rejection [2 3 4 ]. Given this double-edged role of neutrophils in host defense and injury, research has intensified on identifying strategies by which to prevent or reduce their recruitment and migration to a site of inflammation. Statins or HMG-CoA reductase inhibitors have proven protective during coronary events. The survival benefits associated with these drugs are not only attributed to cholesterol-lowering but also to various anti-inflammatory effects on the vascular wall, which include improved endothelial function, altered adhesiveness, as well as antioxidant activity [29 30 31 ]. However, survival benefits associated with direct modulation of inflammatory cell function are less-characterized. The ability of statins to modulate neutrophil migration, a vital contributing step in the pathogenesis of many inflammatory conditions, is relatively unexplored. Cerivastatin and simvastatin have been shown previously to reduce neutrophil chemotaxis [15 , 16 ]. We sought to determine the direct in vitro effects of pravastatin, atorvastatin, and simvastatin on neutrophil transendothelial migration. Using collagen-coated filters and HPAEC monolayers, we demonstrated that all three statins could reduce neutrophil transendothelial migration significantly. This finding is supported by a previous study that demonstrated that preincubation of neutrophils with pravastatin resulted in significant reduction in the number of neutrophils that transmigrated an endothelial monolayer [20 ]. We demonstrate here for the first time the ability of atorvastatin and simvastatin to similarly alter neutrophil migration.
Adhesion of neutrophils to the vascular endothelium precedes their eventual diapedises into the underlying extracellular matrix. In this study, it was demonstrated that pretreatment of neutrophils with pravastatin, atorvastatin, or simvastatin did not significantly alter the percentage of neutrophils adhered to collagen-coated filters or HPAEC monolayers. This was supported by the fact that the statins could not modulate neutrophil surface expression of CD11b. These findings are in contrast to several other studies, where chronic simvastatin use in patients undergoing coronary artery bypass surgery attenuated the intraoperative rise in CD11b expression and significantly reduced neutrophil-endothelial cell interactions [14 ]. In a separate study, atrovastatin was found to significantly reduce neutrophil adhesion to the venous endothelium in patients undergoing coronary artery bypass grafting with cardiopulmonary bypass, and this was associated with reduced neutrophil CD11b expression [12 ]. Observations in monocytes and macrophages have demonstrated the ability of lovastatin to inhibit CD11b/CD18 expression and prevent CD11b-dependent adhesion [32 ]. Although these studies advocate the ability of statins to alter neutrophil adhesion and adhesion molecule expression, it must be noted that these studies were carried out in clinical settings, where individuals or animals were orally taking statin medication. Thus, the clinical observation may not reflect a direct effect of statins on neutrophil adhesion molecule expression but rather, a suppression of the signals in in vivo whole blood that stimulates their expression. In support of our findings, an isolated cell analysis of neutrophil adhesion found that pravastatin could not attenuate neutrophil- endothelial adhesion [33 ]. Thus, although the literature suggests that statins can modulate the ability of neutrophils to express adhesion molecules and subsequently adhere to endothelial cells, this may well be an indirect whole system effect. In our in vitro model, we have demonstrated that pravastatin, atorvastatin, and simvastatin can reduce neutrophil migration, but this effect is not a result of an alteration in the ability of neutrophils to adhere.
The cholesterol synthesis pathway produces many intermediate products that function in separate cellular signaling pathways. These pathways are therefore susceptible to modulation by statins. Considerable attention has focused on the isoprenoid proteins, FPP and GGP. As biproducts of the cholesterol pathway, these proteins are responsible for the post-translational prenylation of a number of small GTPase molecules such as Rho, Rac, and CDC42 [34 ]. These GTPases control a number of cellular functions including cell adhesion, migration, apoptosis, and ROS generation [24 ]. Within the neutrophil, Rho activity is required for necessary tail retraction during migration [35 ]. In support of this and in line with previous reports, the present study found that inhibition of Rho using the specific ROCK inhibitor Y-27632 significantly prevented neutrophils from migrating. We demonstrated here for the first time that fMLP-stimulated neutrophils preincubated with all three statins exhibited a reduction in the levels of fMLP-induced, active GTP-bound Rho. To confirm that statins were mediating their effects through a Rho-dependent pathway, neutrophils were coincubated with FPP and GGP. These isoprenoid proteins are intermediate products generated during the cholesterol synthesis pathway that have various roles in separate molecular signaling pathways. They function in the prenylation of smaller molecular proteins such as Rho, where prenylation is necessary for activation. Previous studies investigating simvastatin and monocyte function found that the statin inhibited cellular migration, and this was reversed through addition of exogenous GPP and not FPP [36 ]. In support of this, we provide new evidence that coincubation with GGP only and not FPP could reverse the effects of pravastatin, simvastatin, and atorvastatin on neutrophil migration. This suggests that statin-mediated effects are through a depletion of the GGP arm of the cholesterol synthesis pathway, making this pathway rather than FPP more vital for effective neutrophil migration. We conclude that the ability of all three statins to reduce neutrophil migration is related directly to their ability to reduce GGP and hence, Rho activity within these cells. It is also worth noting, however, that Rac and CDC42 are crucial for effective neutrophil migration. The importance of Rac was highlighted with the identification of an individual with a naturally occurring, dominant-negative Rac2 mutation [37 , 38 ]. This individual showed markedly reduced neutrophil migration and was susceptible to severe infections. CDC42 has also been implicated as a key regulator of neutrophil polarity, maintaining correct orientation of the leading edge of the migrating neutrophil [39 ]. Like Rho, these proteins rely on prenylation to become functionally active; thus, the reduction in migration observed in our statin-treated neutrophils could also reflect a partial reduction in the active levels of these additional proteins. Statins have been found previously to inhibit the activity of these molecules in other cell types such as endothelial cells [40 ].
The ability of pravastatin, simvastatin, and atorvastatin to suppress basal migration without any effect on LPS-primed migration advocates their potential use in chronic inflammatory conditions, where one might expect low but continuous levels of neutrophil infiltration. Therefore, in such conditions, statins could, in effect, "dampen" down the chronic activity of neutrophils, and their inability to modulate neutrophil function in response to major stimuli would allow those neutrophils to perform an effective, antimicrobial role.
In conclusion, we have demonstrated that pravastatin, simvastatin, and atorvastatin are effective at reducing neutrophil transendothelial migration. This action is independent of an alteration in neutrophil adhesion capabilities. Instead, we demonstrate for the first time that statins mediate their antimigratory effects in the neutrophil through a reduction in fMLP-induced Rho activity. This was confirmed by the ability of GGP, a precursor in the Rho activation pathway, to restore the statin-mediated reduction in neutrophil-transendothelial migration. These results present fresh evidence of and the mechanism of statin anti-inflammatory actions, which could explain their survival benefits in a number of disease states. Statins, therefore, should also be considered as new, therapeutic agents in the fight against chronic inflammatory conditions.
Received June 25, 2008; revised September 10, 2008; accepted September 10, 2008.
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