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the Renal Regeneration Laboratory and Departments of Internal Medicine (M.J.S., W.K., S.-O.M., D.H.K., S.L., K.P.K., S.K.P) and Pathology (K.Y.J.), Research Institute of Clinical Medicine, Chonbuk National University Medical School, Jeonju
Biomedical Research Center and Department of Biological Sciences (S.Y.A., C.-H.C., G.Y.K.), Korea Advanced Institute of Science and Technology, Daejeon, South Korea.
Abstract
Fractalkine is a unique chemokine that functions as a chemoattractant as well as an adhesion molecule on endothelial cells activated by proinflammatory cytokines. Alpha-lipoic acid (LA), a naturally occurring dithiol compound, is an essential cofactor for mitochondrial bioenergetic enzymes. LA improves glycemic control, reduces diabetic polyneuropathies, and mitigates toxicity associated with heavy metal poisoning. The effects of LA on processes associated with sepsis, however, are unknown. We evaluated the antiinflammatory effect of LA on fractalkine expression in a lipopolysaccharide-induced endotoxemia model. Tumor necrosis factor- (TNF-) and interleukin-1 (IL-1) significantly induced fractalkine mRNA and protein expression in endothelial cells. LA strongly suppressed TNF-eC or IL-1eCinduced fractalkine expression in endothelial cells by suppressing the activities of nuclear factor-B and specificity protein-1. LA also decreased TNF-eC or IL-1eCstimulated monocyte adhesion to human umbilical vein endothelial cells. As shown by immunohistochemistry, fractalkine protein expression was markedly increased by treatment with lipopolysaccharide in arterial endothelial cells, endocardium, and endothelium of intestinal villi. LA suppressed lipopolysaccharide-induced fractalkine protein expression and infiltration of endothelin 1-positive cells into the heart and intestine in vivo. LA protected against lipopolysaccharide-induced myocardial dysfunction and improved survival in lipopolysaccharide-induced endotoxemia. These results suggest that LA could be an effective agent to reduce fractalkine-mediated inflammatory processes in endotoxemia.
Key Words: -lipoic acid fractalkine endothelial cells inflammation
Introduction
Sepsis is a clinical syndrome that represents the systemic response to an infection and is characterized by systemic inflammation and widespread tissue injury. At the site of injury, the endothelium expresses various adhesion molecules that attract leukocytes.1 At the same time, inflammatory cells are activated and express a variety of adhesion molecules that cause the aggregation and margination of these cells to the vascular endothelium.2 When the inflammatory response is initiated, a wide variety of chemical mediators are released into circulation. These chemical mediators, including tumor necrosis factor- (TNF-) and interleukin-1 (IL-1), are associated with the continuation of the inflammatory response.3 Sepsis is caused mainly by an exaggerated systemic response to endotoxemia induced by gram-negative bacteria and their characteristic cell wall component, lipopolysaccharide (LPS).4 In mice, challenge with high doses of LPS results in a syndrome resembling septic shock in humans.5
Fractalkine (CX3CL1) is a structurally novel protein in which a soluble chemokine-like domain is fused to a mucin stalk that extends into the cytoplasm across the cell membrane.6 Fractalkine is expressed in activated endothelial cells, and its expression is upregulated by TNF-, IL-1, and LPS.7,8 As a full-length transmembrane protein, fractalkine acts as an adhesion molecule and efficiently captures cells under physiological flow conditions.9,10 Cleavage of the fractalkine mucin stalk close to the junction of the transmembrane domain, however, produces a soluble form of fractalkine that functions as a ligand of CX3CR1, a G-proteineCcoupled receptor.11 In humans, CX3CR1 is expressed predominantly in monocytes, T cells, and natural killer cells.11 Thus, fractalkine and CX3CR1 have special roles in tethering and rolling, arrest, stable adhesion, and transendothelial migration of CX3CR1-expressing leukocytes at sites of fractalkine-expressing endothelium. Endothelial cells are the primary targets of immunological attack in sepsis, and their injury can lead to vasculopathy and organ dysfunction.12 Because inflammation is a universal pathogenesis in sepsis, and LPS is a major pathogenic factor for the inflammatory response during gram-negative bacteremia, it is important to clarify the regulation of endothelial fractalkine expression in the prevention and treatment of the initial phase of endotoxemia.
Alpha-lipoic acid (1,2-dithiolane-3-pentanoic acid; LA), a disulphide derivative of octanoic acid, is known to act as an efficient antioxidant and metal-chelating agent.13,14 LA has been used to treat diabetic complications and polyneuropathies.15,16 LA also has been considered as a candidate therapeutic agent in the treatment or prevention of pathologies associated with an imbalance of oxidoreductive status, such as neurodegeneration,17 ischemiaeCreperfusion,18 and hepatic disorders.19 There is little data, however, about the effect of LA on fractalkine expression in endotoxemia.
In the present study, we examined whether fractalkine is expressed in human umbilical vein endothelial cells (HUVECs) on stimulation with TNF- or IL-1 and in arterial endothelial cells in a LPS-induced endotoxemic model in rats. We also evaluated the role of LA in TNF-eC or IL-1eCinduced expression of fractalkine in HUVECs and endothelial cells in LPS-induced endotoxemia in vivo. Our results indicated that LA decreased TNF-eC or IL-1eCinduced expression of fractalkine in HUVECs by suppressing the signaling pathways of nuclear factor-B (NF-B) and specificity protein-1 (SP-1). LA suppressed TNF-eC or IL-1eCinduced endothelial adhesiveness for monocytes. We also found that LA decreases endothelial fractalkine expression and endothelin-1 (ED-1) eCpositive cell infiltration in the intestine and myocardium in an endotoxemic rat model. LA may therefore have utility as an adjunctive agent for the treatment of fractalkine-mediated inflammation in endotoxemia.
Materials and Methods
See online data supplement at http://circres.ahajournals.org for detailed Materials and Methods.
Cell Culture
Recombinant human TNF- was purchased from R&D Systems. Anti-fractalkine antibody (fractalkine full-length) and anti-CX3CR1 were purchased from Torrey Pines BioLabs. Anti-ED-1 antibody was purchased from Serotec. LA (Thioctacid 600) was obtained from VIATRIS GmbH & Co KG. Calcein-AM was purchased from Molecular Probes. LPS, media, and other biochemical reagents were purchased from Sigma-Aldrich, unless otherwise specified. HUVECs were prepared from human umbilical cords as previously described.20
RNase Protection Assay and Western Blot Analysis
A part of the cDNA of human fractalkine (nucleotides 482 to 893, GenBank accession No. NM002996) was amplified by polymerase chain reaction and subcloned into pBluescript II KS+ (Stratagene). RNase protection assay (RPA) was performed on total RNAs using the Ambion RPA kit (Ambion). An antisense RNA probe of human cyclophilin (nucleotides 135 to 239, GenBank accession No. X52856) was used as an internal control for RNA quantification. Western blot analyses were performed as previously described.21
Flow Cytometry
For flow cytometry analysis, cells were treated as previously described.22
Electrophoretic Mobility Shift Assay
Electrophoretic mobility shift assay (EMSA) for NF-B proteins was performed as previously described.23 EMSA for SP-1 protein was performed with biotin-labeled SP-1 binding site oligomer 5'-GATCCGGTCCCCCACCATCCCCCGCCATTTCCA. Signals were detected by chemiluminescent imaging according to the manufacturer’s protocol (EMSA Gel-Shift Kit; Panomics).
Animal Experiments
Inbred male Sprague-Dawley rats (180 to 200 g) were obtained from Charles River Korea (Seoul, Korea) and were maintained on standard laboratory chow and water ad libitum. All animal studies were reviewed and approved by the Institutional Animal Care and Use Committee of Chonbuk National University Medical School. The rats (200 to 220 g) were divided into 3 groups: Control (n=6), LPS (10 mg/kg; n=6), and LPS (10 mg/kg) plus LA (10 mg · kgeC1 · deC1; n=6). Control buffer and LPS were injected intravenously through the tail vein. LA was injected intraperitoneally once per day for 3 days before LPS administration. At 12 hours after injection of vehicle or LPS, rats were anesthetized with ketamine (100 mg/kg) and xylazine (10 mg/kg) and subsequently euthanized by cervical dislocation. Heart and jejunum were harvested for Western blot analysis and immunohistochemistry of fractalkine.
After 5 days of treatment with control buffer, LPS (2.5 mg/kg), LPS plus LA (10 mg/kg), LPS plus rabbit immunoglobulin G (500 e/kg), or LPS plus antifractalkine antibody (500 e/kg), rat hearts and jejunum were harvested for immunohistochemistry of ED-1 and CX3CR1.
Cell Adhesion Assays In Vitro
Human peripheral blood monocytes were isolated from fresh blood of healthy volunteers by Ficoll-Paque gradient centrifugation. The study protocol and informed consent forms were approved by the Chonbuk National University Hospital Review Board. Monocytes were isolated by negative selection using magnetic beads (Miltenyi Biotec).24 The purity of the monocyte fraction was 93% to 95%, as determined by staining with anti-CD14, anti-CD33, anti-CD16b, and anti-CD56 mAbs and FACScan analysis (Becton Dickinson).24 Monocyte-endothelial adhesion was determined by fluorescent labeling of monocytes by a method described previously.25 The number of monocytes adhering to HUVECs was expressed as a percent calculated by the formula: % adherence=(adherent signal/total signal).
Immunohistochemical Analysis of Fractalkine, ED-1, and CX3CR1
Immunohistochemistry of fractalkine was performed as previous we used method.21 Fractalkine expression was semiquantitated by grading the degree of immunostaining (very strong=5, strong=4, moderate=3, weak=2, none=1). Three to 5 endothelial portions of each section were graded. Tissues were examined from several parts of the heart (artery, vein, endocardium, and cardiac valves) and jejunum (artery, vein, and villous endothelium). Immunofluorescent costaining of ED-1 and CX3CR1 was performed with frozen sections and was visualized using an Axio Imager microscope with AxioVision digital imaging program (Carl Zeiss).
Densitometric Analyses and Statistics
Data were expressed as mean±SD. Statistical significance was tested using Student unpaired t test or 1-way ANOVA followed by the Student-Newman-Keuls test. Statistical significance was set at P<0.05.
Results
TNF- or IL-1 Increased Expression of Fractalkine mRNA and Protein in HUVECs
We first examined the effect of TNF- and IL-1 on fractalkine expression in HUVECs. Addition of TNF- (10 ng/mL) increased the expression of fractalkine mRNA in a time-dependent manner, and maximum expression of fractalkine was observed at 4 hours (Figure 1A). The expression of fractalkine mRNA determined after 4-hour incubation was increased by TNF- in a dose-dependent manner (Figure 1B). Consistent with the increased mRNA expression, fractalkine protein was also increased by treatment with TNF-, and the level continued to be higher than in the control group for up to 24 hours (Figure 1C).
Treatment of HUVECs with IL-1 (15 ng/mL) gradually increased the expression of fractalkine mRNA for up to 4 hours, but a significant decrease in the fractalkine mRNA level was observed at 8 hours (Figure 2A). The expression of fractalkine mRNA determined after a 4-hour incubation was increased by IL-1 in a dose-dependent manner (Figure 2B). The maximum increase in fractalkine protein was observed at 4 to 6 hours, and the level continued to be higher than in the control group for up to 24 hours (Figure 2C).
LA Suppressed TNF-eC or IL-1eCInduced Expression of Fractalkine mRNA and Protein
We examined the effect of LA on TNF-eC and/or IL-1eCinduced fractalkine mRNA expression in HUVECs. LA (4 mmol/L) suppressed TNF- (10 ng/mL) or IL-1 (15 ng/mL) eCinduced expression of fractalkine mRNA in a dose-dependent manner (Figure 3A and 3B). LA suppressed approximately 70% to 80% of TNF-eC or IL-1eCinduced expression of fractalkine mRNA. Moreover, LA suppressed the expression of fractalkine mRNA induced by TNF- (10 ng/mL) and IL-1 (15 ng/mL) together (Figure 3C). LA also decreased TNF-eC and/or IL-1eCinduced expression of fractalkine protein (Figure 3D).
Flow cytometry was also used to analyze the cell surface expression of fractalkine on TNF-eC or IL-1eCstimulated HUVECs cotreated with LA. The number of fractalkine positive cells was increased &5-fold and 3.6-fold in HUVECs after stimulation with TNF- or IL-1, respectively (Figure 4A and 4B). After treatment with LA, the number of fractalkine-positive cells decreased &45% and 40%, respectively (Figure 4A and 4B). These data suggest that LA is an inhibitor of TNF-eC and/or IL-1eCinduced fractalkine expression in HUVECs.
LA Suppressed TNF-eC or IL-1eCInduced NF-B and SP-1 Binding Activities
We previously reported that NF-B is involved in TNF-eCinduced fractalkine expression in HUVECs.21 In this experiment, we used EMSA to examine whether LA inhibits NF-B activity in the nuclear extracts of TNF- (10 ng/mL)- or IL-1 (15 ng/mL)-stimulated HUVECs. NF-B (p65/p50) binding activity was increased by treatment with TNF- or IL-1, and LA (4 mmol/L) suppressed the TNF-eC or IL-1eCinduced NF-B (p65/p50) binding activity (Figure 5A). LA alone had no effect on basal NF-B (p65/p50) binding activity. These data suggest that LA suppressed TNF-eC or IL-1eCinduced fractalkine expression through suppression of NF-B activity in HUVECs.
Because TNF- increases SP-1 binding activity in HUVECs, and mithramycin, an inhibitor of SP-1, decreases TNF-eCinduced fractalkine expression in HUVECs, we examined whether LA can regulate SP-1 binding activity using nuclear extracts of TNF-eC and/or IL-1eCstimulated HUVECs.21,26 EMSA analyses revealed increased SP-1 binding activity in HUVECs treated with TNF-, IL-1, or TNF- plus IL-1. LA decreased the TNF-eC and/or IL-1eCinduced SP-1 binding activity. LA alone had no effect on basal SP-1 binding activity (Figure 5B). These data suggest that LA suppressed the TNF-eC or ILeC1-induced fractalkine expression through suppression of SP-1 activity in endothelial cells. Taken together, these data suggest that LA suppresses fractalkine expression by inhibiting NF-B and SP-1 binding activities in HUVECs.
LA Suppressed TNF-eC or IL-1eCInduced Monocyte Adhesiveness to HUVECs
Expression of fractalkine in endothelial cells induces the adhesion of CX3CR1-positive cells such as monocytes.11 We examined whether LA decreases monocyte adhesion to TNF-eC or IL-1eCstimulated HUVECs. Stimulation of HUVECs with TNF- (10 ng/mL) or IL-1 (15 ng/mL) for 6 hours induced a significant (&5 or &4-fold each) increase in the adhesion of monocytes compared with treatment with control buffer (Figure 6). Treatment of TNF-eCstimulated cells with LA, however, led to a 63% decrease in monocyte adhesion, and treatment of IL-1eCstimulated cells with LA led to a 76% decrease in monocyte adhesion (Figure 6). LA alone had no effect on HUVEC adhesiveness for monocytes. Moreover, the antibody against fractalkine decreased TNF-eC andr IL-1eCstimulated monocyte adhesion (50% and 47%, respectively). The fractalkine antibody alone had no effect on HUVEC adhesiveness for monocytes (Figure 6). These findings suggest that LA decreases monocyte adhesion to TNF-eC or IL-1eCstimulated HUVECs mainly through fractalkine expression.
LA Suppressed LPS-Induced Fractalkine Expression in Cardiac Endothelial Cells and Small Intestinal Endothelial Cells
We also examined the effect of LPS on fractalkine expression in rat heart using immunohistochemistry. Slight endogenous expression of fractalkine in a normal adult rat was observed in arterial endothelial cells, but almost no expression of fractalkine was observed in capillary endothelial cells, venous endothelial cells, endocardium, myocardium, pericardium, or cardiac valves (Figure 7A, 7B, and 7E). Intravenous injection of LPS (10 mg/kg) markedly increased fractalkine expression at 12 hours in arterial endothelial cells, endocardium, and the endocardial surface of cardiac valves, but not in venous endothelial cells (Figure 7A, 7B, and 7E). Pretreatment with LA (10 mg · kgeC1 · deC1 for 3 days) dramatically suppressed LPS-induced fractalkine expression in arterial endothelial cells, endocardium, and the endocardial surface of cardiac valves (Figure 7A, 7B, and 7E).
We further examined the effect of LPS on fractalkine expression in rat small intestine using immunohistochemistry. We observed slight endogenous expression of fractalkine mainly in arterial endothelial cells, but only slight or almost no expression of fractalkine in villous endothelial, venous, and lymphatic endothelial cells or epithelial cells (Figure 7C, 7D, and 7F). Intravenous injection of LPS increased fractalkine expression markedly at 12 hours in arterial and arteriolar endothelial cells and villous endothelium and slightly in venous endothelial cells, but not in lymphatic endothelial cells or epithelial cells (Figure 7C, 7D, and 7F). These data suggest that LPS-induced fractalkine expression is endothelial cell-specific in the small intestine. Pretreatment with LA dramatically suppressed LPS-induced fractalkine expression in arterial endothelial cells and villous endothelium. These findings suggest that LA suppressed LPS-induced fractalkine expression in cardiac endothelial cells and small intestinal endothelial cells.
LA Suppressed ED-1-Positive Cell Infiltration in Myocardium and Jejunum
Immunohistochemical examination of rat myocardium and jejunum revealed 4.6-fold and 6-fold increases, respectively, in ED-1-positive cell infiltration after treatment with LPS (Figure 8A through 8D). LA treatment prevented LPS-induced accumulation of ED-1eCpositive cells in hearts and jejunum by &46% and 61%, respectively, whereas LA alone had no effect on the numbers of positive cells. The number of infiltrated ED-1eCpositive cells in the myocardial and jejunal sections from rats treated with LPS and anti-fractalkine antibody were significantly lower than that from rats treated with LPS and control antibody (Figure 8C and 8D). Anti-fractalkine antibody alone had no effect on ED-1eCpositive cell infiltration in myocardium and jejunum. We also found that some infiltrated ED-1eCpositive cells in jejunum were also stained for CX3CR1 in the same section (Figure 8E). These findings demonstrated that infiltration of ED-1eCpositive cells can be associated with fractalkine receptors. All of these results suggest that LA decreases ED-1 infiltration into myocardium and jejunum through regulation of fractalkine expression in an endotoxemia model.
LA Has a Protective Role in Hemodynamic and Survival Studies
The preservation of hemodynamics in LA-treated rats was associated with increased mean arterial pressure (87±2.8 mm Hg in LPS versus 96±3.0 mm Hg in LPS+LA treated rats, P<0.05) and increased dP/dt (1250±210 mm Hg/sec in LPS versus 2153±420 mm Hg/sec in LPS+LA treated rats, P<0.05) (supplemental Figure I). The actuarial survival of LPS-treated rats varied from a mean of 69.2±17.9 hours (median, 70 hours) in control rats to 122±25.3 hours (median, 125 hours) in rats treated with LA (log-rank test, P<0.05) (supplemental Figure II). The survival of LPS-treated rats also varied from a mean of 72.5±19.2 hours (median, 70 hours) in control antibody treated rats to 89.4±23.3 hours (median, 85 hours) in rats treated with anti-fractalkine antibody (log-rank test, P<0.05) (supplemental Figure II). These data suggest that regulation of fractalkine with LA in LPS-induced endotoxemia can increase survival. Thus, these results suggest that fractalkine is important for the pathogenesis of endotoxemia, and LA has a protective role in the functional consequences of endotoxemia.
Discussion
Gram-negative bacterial sepsis produces a spectrum of pathophysiological alterations, including cardiopulmonary, renal, hematological, and metabolic dysfunction, leading to vascular collapse.27 The excessive production of proinflammatory cytokines is thought to contribute significantly to the lethality of sepsis. Proinflammatory TNF- and IL-1 act as initiators in the cascade of endogenous mediators that direct the inflammatory and metabolic responses, eventually leading to severe shock and organ failure.28 We found that LA decreased the LPS-induced serum level of TNF- or IL-1 (supplemental Figure III). These results suggest that LA decreased the serum level of TNF- and IL-1 during LPS-induced endotoxemia.
Vascular endothelial cells can be important pathogenic factors in inflammatory disorders such as sepsis. The important biological roles of fractalkine in endothelial inflammation and injury have been recently documented: Firm adhesion, cytotoxicity, and migration of CX3CR1-expressing cells into tissue.12,29,30 We previously reported that fractalkine is upregulated after stimulation with TNF- in HUVECs.21 Because fractalkine has important roles in inflammation, factors affecting its endothelial expression are important in regulating inflammatory processes in endotoxemia. Notably, our results indicate that LA is a strong inhibitor of TNF-eC and/or IL-1eCinduced fractalkine mRNA and protein expression in endothelial cells. We also demonstrated that LA can decrease TNF-eCstimulated human monocyte adhesiveness on HUVECs (Figure 6) and LPS-induced ED-1eCpositive cell infiltration in myocardium and jejunum (Figure 8). These results suggest that LA could be useful for preventing fractalkine-mediated vascular inflammatory injury.
Activation of NF-B could play a central role in inflammatory cytokine-induced fractalkine expression at the transcriptional level.21,28 Our previous pharmacological assays revealed that TNF-eCstimulated expression of fractalkine occurs mainly through activation of the NF-BeCdependent pathway.21 It was also reported that SP-1 nuclear activator proteins are involved in vascular injury and inflammation.31 Our EMSA indicated that LA suppressed not only NF-B binding but also SP-1 binding of TNF-eCor IL-1eCstimulated endothelial proteins to the DNA. Therefore, it is possible that LA suppresses TNF-eC or IL-1eCinduced fractalkine mRNA expression through suppression of NF-B and SP-1. Furthermore, our data demonstrated that incubation of confluent HUVECs with TNF- or IL-1 caused an almost 5-fold or 4-fold increase in adhesion of monocyte cells compared with adhesion of monocytes to unstimulated HUVECs. This increase in HUVEC adhesiveness was reduced by treatment with LA (Figure 6). Thus, LA has a regulatory role in fractalkine-mediated monocyte adhesiveness through suppression of NF-B and SP-1.
Because challenge with high doses of LPS in rats results in a syndrome resembling human sepsis, a rat model of LPS-induced endotoxemia has been used in this study.32 Our immunohistochemical analyses in the heart and intestine demonstrated that LPS increased fractalkine expression predominantly in arterial and capillary endothelial cells, whereas little or no induction was observed in venous endothelial cells. Furthermore, LPS increased fractalkine expression markedly in the endocardium of cardiac walls, the endocardial surfaces of cardiac valves, and the endothelium of intestinal villi. Considering the interaction between fractalkine-expressing endothelial cells and CX3CR1-expressing leukocytes in vivo, fractalkine must be involved in arterial inflammation rather than venous inflammation in endotoxemia. Pretreatment with LA dramatically suppressed LPS-induced fractalkine expression in arterial endothelial cells, endocardium, and the endocardial surface of cardiac valves in the heart. LA also decreased LPS-induced fractalkine expression in arterial endothelial cells and villous endothelium in the small intestine. Furthermore, LA decreased the number of LPS-induced ED-1eCpositive cells infiltrating into myocardium and jejunum (Figure 8). These data suggest that LA has a role in regulating fractalkine in arterial endothelial cells, endocardium, and the endocardial surface of cardiac valves and in subsequent macrophage infiltration in endotoxemia.
Our in vitro results have revealed that pretreatment with LA dramatically suppresses TNF-eC or IL-1eCinduced fractalkine expression in endothelial cells through suppression of NF-B and SP-1. Furthermore, LA decreases adhesiveness between cytokine-induced CX3CR1-positive leukocytes and endothelial cells through suppression of fractalkine expression. Our in vivo data also demonstrated that LA decreased LPS-induced fractalkine expression in arterial endothelial cells, endocardium, and villous endothelium. Therefore, LA warrants further evaluation as an antiinflammatory drug in endotoxemia. Because current therapy for patients with sepsis is still unsatisfactory, there have been continued efforts to find new and effective means to improve outcome by modulating inflammatory responses. Our results indicate that LA may be promising as an adjunctive treatment for endotoxemia, although the results presented here need further evaluation with other clinically relevant animal models.
Acknowledgments
This work was supported by grants from the National Research Laboratory Program of Korea Science and Engineering Foundation and a Korea Research Foundation Grant (KRF-2003-041-E00031). We thank Jennifer Macke for help in preparing the manuscript.
Both authors contributed equally to this study.
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