Endothelial Cell Activation by IL-1? in the Presence of Fibrinogen Requires V?3
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动脉硬化血栓血管生物学 2005年第10期
From Hematology/Oncology Unit, Department of Medicine, University of Rochester School of Medicine & Dentistry, Rochester, NY.
Correspondence to Abha Sahni, PhD, Department of Medicine, P.O. Box 610, University of Rochester Medical Center, 601 Elmwood Ave, Rochester, NY 14642. E-mail Abha_Sahni@urmc.rochester.edu
Abstract
Objective— The purpose of this study was to investigate the receptor requirements for enhanced IL-1?–induced secretion of nitric oxide (NO) by endothelial cells (ECs) in the presence of fibrinogen.
Methods and Results— ECs were exposed to IL-1? with or without fibrinogen and NO was measured as nitrite. NO production by EC exposed to fibrinogen (0.3±0.1 μmol/L) was comparable concentration to control (0.2±0.1 μmol/L), but IL-1? significantly increased NO production (0.8±0.1 μmol/L), and the combination of both fibrinogen and IL-1? resulted in a further increase to 2.2±0.2 μmol/L (P<0.002). 7E3 or LM609, antibodies to v?3, inhibited NO production stimulated by fibrinogen-bound IL-1? to 0.2±0.1 μmol/L (P<0.001) or 0.2±0.03 μmol/L (P<0.0001), respectively. These levels were comparable to control and significantly less than with IL-1? (P<0.002). EC or fibroblasts exposed to both fibrinogen and IL-1?, but not vitronectin and IL-1?, demonstrated positive Western blotting for V?3 after immunopurification with anti- IL-1R, indicating specific association between v?3 and IL-1R. Dual immunofluorescence also revealed colocalization of v?3 and IL-1R only when the cells were exposed to both fibrinogen and IL-1?.
Conclusion— The enhanced NO production by ECs in the presence of fibrinogen-bound IL-1? requires the coordinated effects of colocalized V?3 and IL-1R.
The ability of fibrinogen-bound IL-1? to stimulate NO secretion by ECs is blocked by anti-v?3. Also, fibrinogen-bound IL-1? promotes the specific association of v?3 with IL-1R, as shown by coimmunoprecipitation or immunofluorescence staining. Fibrinogen binding enhances IL-1?–induced secretion of NO through the coordinated effects of colocalized v?3 and IL-1R.
Key Words: endothelial cells ? fibrinogen ? IL-1? ? nitric oxide
Introduction
Fibrinogen plays a central role in hemostasis, and it is also an adhesive protein supporting interaction of endothelial cells (ECs) with matrix and inflammatory cells1 and fibrin also forms the provisional matrix to support cellular processes of repair. In this role, fibrin actively directs specific receptor-mediated interactions to regulate permeability,2 EC adhesion,3 synthesis, and secretion of tissue plasminogen activator and prostacyclin,4,5 plasminogen activator inhibitor (PAI)-1,6 IL-8,7 and von Willebrand factor (vWF).8 Further, fibrinogen and fibrin are also implicated in the development, and thrombotic complications of atherosclerosis, and they are found in increased amounts in atherosclerotic vessels.9 Degradation products of fibrinogen and fibrin increase vascular permeability and induce chemotaxis at sites of inflammation.10,11
The vascular response to injury is also regulated by cytokines including those of the IL-1 family that are important in inflammation. IL-1 and IL-1? are 2 members that share structural features and act on cells through the same receptors.12 However, they differ in their promoter regions, and IL-1 is primarily cell-associated, whereas IL-1? is extracellular. IL-1 induces EC procoagulant activity,13 vWF release,14 synthesis of plasminogen activator and PAI-1,15 and inhibits the thrombomodulin-protein C anticoagulant pathway.16 On exposure to IL-1, ECs increase synthesis of NO,17 chemoattractant cytokines18 and express intercellular adhesion molecule-1 (ICAM-1)19 and vascular cell adhesion molecule (VCAM)-1.20
ECs are physiologically exposed to a high concentration of fibrinogen in blood and fibrinogen binds through V?3 and 5?1.21–23 EC adhere, spread, and proliferate on fibrinogen in vitro through binding to integrins V?3 and 5?1,21,22 and fibrinogen supports leukocyte tethering to EC through ICAM-1.1 When exposed to fibrin, EC alter their shape and adhesive properties, changes that are mediated through specific interaction of VE-cadherin with the amino terminus of the fibrin ? chain.24,25
We have previously reported that fibrin(ogen) binding to IL-1? potentiates EC secretion of NO.26 We have now investigated the involvement of specific receptors in activating EC by fibrinogen-bound IL-1?.
Methods
Fibrinogen Preparation
Human fibrinogen was obtained from Enzyme Research Laboratories and copurifying fibronectin was removed by gelatin-Sepharose chromatography and immuno-affinity chromatography as described.27 The fibronectin concentration represented <0.02 μg /mL.
Cell Culture
Primary ECs were obtained from human umbilical veins, seeded on 0.2% wt/vol gelatin-coated 25 cm2 tissue culture flasks and cultured in McCoy’s 5A medium (Flow Laboratories).28 Fibroblasts were isolated from human foreskins (HFFs) and cultured in McCoy’s 5A medium supplemented with 10% fetal bovine serum, 100 U/mL penicillin, and 0.1 mg/mL of streptomycin until they reached confluence. The cells were passaged up to 2 times before use and then placed in suspension by rinsing in Hank’s balanced salt solution followed by brief incubation with trypsin-EDTA (Gibco Invitrogen). The cells were pelleted by centrifugation for 10 minutes at 500g and resuspended in McCoy’s 5A medium in the absence of serum. This wash procedure was repeated twice before use.
Measurement of NO
Confluent ECs were incubated with 5 μg/mL of either LM609 (Chemicon International), or 7E3 antibody for 2 hours. Then, 10 ng/mL of IL-1? was added to the medium in the presence or absence of 10 μg/mL of fibrinogen. The medium was collected after 1 hour, and the NO concentration was measured as nitrite using the Nitrate/Nitrite colorimetric assay (Cayman Chemicals).
Immunoprecipitation and Western Blotting
ECs or HFFs were grown to confluence, and then medium containing 10 ng/mL of either IL-1 or IL-1? (Peprotech Inc.) was added in the presence or absence of 10 μg/mL of fibrinogen or vitronectin (Sigma Chemical Co). After 1 hour, the cells were washed 3 times with phosphate-buffered saline (pH 7.4), lysed with lysis buffer containing protease inhibitors (Promega) and immunoprecipitated using antibodies to V?3 (LM609 or 7E3), V?5 (P1F6), 5?1 (JBS5), or v (LM142) and ?1 (JB1A) (Chemicon), or control mouse IgG for 2 hours, after which protein A-Sepharose beads (Pierce) were added. The beads were centrifuged at 3000g for 10 minutes, washed twice with 0.1 mol/L sodium phosphate buffer (pH 7.4) containing 0.25 mol/L NaCl, boiled in diluent for 5 minutes. The eluates and supernatants were electrophoresed using 10% SDS-polyacrylamide gel electrophoresis (SDS-PAGE). After protein transfer, membranes were immunoblotted with antibody to IL-1R (Santa Cruz Biotechnology), and bands were detected by chemiluminescence. Similar experiments were performed using anti-IL-1R for immunoprecipitation and anti-?3 (Chemicon) to probe the blot. In some experiments, anti-V?3, anti-5?1, anti-V?5, or anti-v and anti-?1 were used for immunoprecipitation, and blots were probed using anti-IL-1R. In some experiments, blots were probed with tubulin antibody for internal loading controls.
Immunofluorescent Detection
ECs or HFFs were seeded on round glass coverslips, grown to confluence, and treated with 10 ng/mL of IL-1? added to the medium in the presence or absence of 10 μg/mL of fibrinogen. After 1 hour, cells were washed twice with cold phosphate-buffered saline, fixed with 3.7% formaldehyde in phosphate-buffered saline, incubated with 10 μg/mL of polyclonal IL-1R and 10 μg/mL of monoclonal 7E3 antibodies and Alexa Fluor 568 (red) and Alexa Fluor 488 (green; Molecular Probes) were then added. Alexa Fluor 568 was conjugated to anti-IL-1R and Alexa Fluor 488 was conjugated to 7E3. Cells were viewed using a fluorescence microscope and a color digital camera and a computer with color monitor captured images.
Data Analysis
Unless indicated otherwise, data are expressed as mean±SD. Each experiment was performed at least 3 times, and either triplicate or quadruplicate wells were used in each experiment. The significance of differences in means was determined using a 2-tailed Student t test.
Results
Specific inhibitory monoclonal antibodies were used to investigate the role of integrin and IL-1 receptors in the separate and combined effects of IL-1 and fibrinogen on NO secretion. ECs were incubated with antibodies to V?3 in the presence of IL-1? with or without fibrinogen, and NO secretion was measured as nitrite (Figure 1). In the presence of fibrinogen alone, NO concentration was 0.3±0.1 μmol/L, and this was similar to medium alone (0.2±0.1 μmol/L). IL-1? increased NO production to 0.8±0.1 μmol/L, which was significantly higher than medium alone (P<0.02). Maximum NO induction occurred in the presence of both fibrinogen and IL-1? (2.2±0.2 μmol/L) with an 11±2-fold increase. In both the presence and absence of fibrinogen, NO induction reached a maximum at an IL-1? concentration of 10 ng/mL and was higher in the presence than in the absence of fibrinogen at all IL-1? concentrations tested. Addition of LM609, a monoclonal antibody to V?3, had no effect on NO production in medium alone, or after stimulation by fibrinogen or IL-1? alone. In contrast, LM609 completely inhibited the induction of NO secretion induced by fibrinogen-bound IL-1? (0.2±0.1 μmol/L) (P<0.001), and the level of secretion was less than that induced by IL-1? alone (Figure 1). Similar results were observed with 7E3, also reactive with v?3, which inhibited secretion in response to fibrinogen-bound IL-1? (0.2±0.0.03 μmol/L) (P<0.001). These findings indicate that IL-1? has no activity when fibrinogen is present if v?3 is blocked.
Figure 1. Effect of LM609 and 7E3 on EC NO levels. EC were plated on gelatin-coated flasks in McCoy’s 5A medium supplemented with 20% fetal bovine serum (FBS), 50 μg/mL of endothelial cell growth supplement, and 100 μg/mL of heparin. At confluence, cells were washed twice with McCoy’s medium and incubated in serum-free medium containing 1% Nutridoma and 5 μg/mL of either LM609 or 7E3. After 1 hour, 10 ng/mL of IL-1?, with or without 10 μg/mL of fibrinogen was added to the medium for 1 hour. Medium was then collected, and NO was measured as nitrite concentration using the Griess reaction. The results shown are mean±SD of 3 separate experiments.
IL-1? stimulates EC NO synthase (NOS) through IL-1R. To characterize the involvement of this receptor, NO secretion was measured in the presence of an antibody to IL-1R. This reduced NO secretion to 0.3±0.04 μmol/L in the presence of IL-1? alone and to 0.2±0.03 μmol/L with fibrinogen-bound IL-1? (P<0.005 for both), and these levels were not significantly different from baseline levels (Figure 2). In control experiments, nonimmune IgG had no effect on NO secretion stimulated by either free or fibrinogen-bound IL-1?. This indicates that IL-1? requires both IL-1R and V?3 to stimulate NO production by ECs when bound to fibrinogen.
Figure 2. Effect of an IL-1R antibody on NO production. ECs were plated on gelatin-coated flasks in McCoy’s 5A medium supplemented with 20% FBS, 50 μg/mL of ECGS, and 100 μg/mL of heparin and allowed to grow to confluence. The cells were then washed twice with McCoy’s medium and incubated in serum free medium containing 1% Nutridoma and 5 μg/mL of either anti-IL-1R or control mouse IgG. After 60 minutes, 10 ng/mL of IL-1? with or without 10 μg/mL of fibrinogen, was added to the medium for an hour. Medium was then collected, and NO was measured as nitrite concentration. The results are the mean±SD of 3 separate experiments.
Immunoprecipitation studies were performed to investigate possible association of receptors. ECs or HFFs were exposed to IL-1?, fibrinogen, or to the combination of both in the medium. After 1 hour, cells were washed and lysed, after which 7E3 (Figure 3A, 3B, 3C, and 3D) or IL-1R antibody (Figure 3E and 3F) was added, and immune complexes were isolated by incubation with Protein A–coupled Sepharose beads. After centrifugation, beads were boiled in diluent and subjected to SDS-PAGE, Western blots were prepared and probed with anti–IL-1R (Figure 3A, 3B, 3C, and 3D) or anti-?3 (Figure 3E and 3F). Only cells exposed to the combination of fibrinogen and IL-1? demonstrated positive Western blotting for IL-1R after immunopurification with 7E3 (Figure 3A, lane 6). Results were similar in HFFs, with colocalization of V?3 and IL-1R only with exposure to the combination of fibrinogen and IL-1? (Figure 3C, lane 6). Notably, there was no colocalization with IL-1, which does not bind to fibrinogen (Figure 3A and 3C, lanes 4 and 5). After V?3 was immunoprecipitated the supernatants were positive for IL-1R in all samples (Figure 3B and 3D). Findings were similar when IL-1R was immunoprecipitated and the blots were probed with anti-?3 (Figure 3E). The supernatants were positive for ?3 in all samples after IL-1R was immunoprecipitated (Figure 3F). These results indicate that exposure of ECs or HFFs to the combination of IL-1? and fibrinogen promoted the association of V?3 with IL-1R.
Figure 3. Colocalization of v?3 and IL-1R using coimmunoprecipitation. Confluent ECs (A,B,E,F) or HFFs (C and D) were exposed to various conditions (as described). After 1 hour, cells were washed with phosphate-buffered saline (PBS), lysed with lysis buffer, and incubated with 5 μg/mL of either 7E3 (A, B, C, D) or anti-IL-1R (E and F). Protein A Sepharose beads were then added. After washing, the beads were incubated with diluent and electrophoresed on 10% gels. Western blotting was performed with anti-IL-1R (A, B, C, D) or with anti-?3 (E and F). B, D, and F, Supernatants after centrifugation of protein A Sepharose. Ln 1, medium alone; Ln 2, 10 μg/mL fibrinogen; Ln 3, 10 ng/mL IL-1?; Ln 4, 10 ng/mL IL-1; Ln 5, 10 ng/mL IL-1 plus 10 μg/mL fibrinogen; Ln 6, 10 ng/mL IL-1? plus 10 μg/mL fibrinogen. Arrows indicates the location of IL-1R (Mr 80 kD) in A, B, C, and D and the location of ?3 in E and F (Mr 95 kD).
Immunofluorescence studies confirmed the colocalization of the fibrinogen and IL-1? receptors (Figure 4). Confluent ECs (Figure 4A) or HFFs (Figure 4B) were exposed to medium alone (insets), (fibrinogen [Figure 4A to 4C], IL-1 [Figure 4D to 4F], IL-1 plus fibrinogen [Figure 4G to 4I], IL-1? [Figure 4J to 4L], or both IL-1? plus fibrinogen [Figure 4M to 4O]) for 1 hour. Cells were then fluorescently labeled with anti-IL-1R and 7E3. The V?3 immunofluorescence increased with fibrinogen exposure, consistent with increased binding of 7E3 to activated receptor.29 In both ECs and HFFs, dual immunofluorescent detection revealed colocalization of V?3 and IL-1R only when the cells were exposed to both fibrinogen and IL-1? (Figure 4M to 4O) as shown by yellow fluorescence. No colocalization of the receptors was observed after exposure to both fibrinogen and IL-1 (Figure 4G to 4I).
Figure 4. Colocalization of v?3 and IL-1R by immunofluorescence. Confluent ECs (A) or HFFs (B) were incubated with or without 10 ng/mL of IL-1? in the presence or absence of 10 μg/mL of fibrinogen. After 1 hour, cells were washed and fixed with 3.7% formaldehyde and stained using 10 μg/mL of IL-1R and 7E3 antibody. IL-1R was visualized as red fluorescence, V?3 visualized as green fluorescence, and colocalization of IL-1? and fibrinogen receptors as yellow fluorescence. Insets represent the staining with medium alone, in the absence of any ligands. Bars represent 25 μm.
Although V?3 is an important receptor for fibrinogen, other integrins such as 5?1 also bind to fibrinogen under certain conditions.22 To determine specificity, ECs were incubated with the combination of IL-1? and fibrinogen. Lysates were then incubated with anti-V?3, anti-5?1, anti-V?5, or anti-V and anti-?1 to immunoprecipitate various integrins, and Western blots were prepared and probed with anti-IL-1R. Positive Western blotting for IL-1R was observed only with immunoprecipitates using anti-V?3 (Figure 5A). IL-1R receptor did not coprecipitate with integrins V?5, 5?1, V, or ?1, indicating the specificity of colocalization of IL-1R with V?3. Blot probed with tubulin antibody was used as a loading control (Figure 5B).
Figure 5. Specificity of association of IL-1R with v?3. (A). ECs were incubated with 10 ng/mL IL-1? and 10 μg/mL fibrinogen for 1 hour. Cells were then lysed and incubated with 5 μg/mL of integrin antibodies: V?3, V?5, 5?1, v, or ?1. Protein-A Sepharose beads were then added and incubated further for 30 minutes. After washing, beads were boiled in electrophoresis diluent for 5 minutes and samples were electrophoresed on 10% gels. Western blotting was performed with anti- IL-1R. The result of a representative experiment is shown (B). Blot probed with tubulin antibody was used as a loading control.
To determine whether IL-1R colocalizes with V?3 activated by another ligand, ECs were incubated with vitronectin or fibrinogen with or without of IL-1?. Lysates were then incubated with anti-V?3 and Western blots were prepared and probed with anti–IL-1R (Figure 6A). Only cells exposed to the combination of fibrinogen and IL-1? demonstrated positive Western blotting for IL-1R after immunopurification with anti-V?3. No colocalization was observed with vitronectin and IL-1?, indicating the specificity of fibrinogen in promoting the association of the 2 receptors in the presence of IL-1?. Blot probed with tubulin antibody was used as a loading control (Figure 6B).
Figure 6. Specificity of fibrinogn and IL-1? in promoting the association of IL-1R with v?3. (A). ECs were incubated with 10 μg/mL fibrinogen or 10 μg/mL vitronectin in the presence or absence of 10 ng/mL IL-1? for 1 hour. Cells were then lysed and incubated with 5 μg/mL of anti-v?3. Protein-A Sepharose beads were then added and incubated further for 30 minutes. After washing, beads were boiled in electrophoresis diluent for 5 minutes and samples were electrophoresed on 10% gels. Western blotting was performed with anti-IL-1R. Ln 1, medium alone; Ln 2, IL-1?; Ln 3, fibrinogen; Ln 4, vitronectin; Ln 5, IL-1? plus vitronectin; Ln 6, IL-1? plus fibrinogen. Arrow indicates the location of IL-1R (Mr 80 kDa). B, Blot probed with tubulin antibody was used as a loading control.
Discussion
The results presented demonstrate that inhibition of the interaction of fibrinogen with V?3 blocks the ability of IL-1? to induce NO in the presence of fibrinogen and that V?3 and IL-1R colocalize when both fibrinogen and IL-1? are present. Our previous studies have shown that IL-1? binds with high affinity to fibrinogen, indicating that IL-1? would be bound to fibrinogen under these experimental conditions. Antibodies to V?3 inhibited NO production induced by fibrinogen-bound IL-1? but showed no inhibition of activity with IL-1? alone. This indicates that either IL-1R is not available for binding IL-1? in the presence of fibrinogen or that it can bind but cannot activate the receptor. This may be caused by shielding of the binding site for IL-1R when IL-1? is bound to fibrinogen. We have shown previously that IL-1? competes with FGF-2 for binding to fibrinogen, suggesting that FGF-2 and IL-1? interact with the same or related sites. The site on FGF-2 that interacts with fibrinogen has been localized by mutational analysis to 5 residues between Phe-95 and Arg-109,30 and changes in binding during plasmic degradation suggests that another interactive site may be localized in the chain.31 Further studies will be required to characterize the sites involved in the IL-1?-fibrinogen interaction.
In the presence of fibrinogen-bound IL-1?, V?3 was coimmunoprecipitated with IL-1R, indicating that V?3 and IL-1R associate in initiating the signaling pathway leading to NO activation. This colocalization did not occur with the V?3 ligand vitronectin, indicating specificity for fibrinogen. These findings link the hemostatic and matrix functions of fibrinogen with the EC regulatory activities of the cytokines and are consistent with our previous reports that VEGF, FGF-2, and IL-1? bind to fibrin(ogen) with high affinity and retain activity when bound.26,32,33 The interaction potentiates the capacity of IL-1? to stimulate NO, NF-B, and MCP-1 in ECs.26
There are several other examples of interactions between growth factors and matrix proteins with their receptors that result in altered activity, suggesting that this is a common theme in cell regulation. Wijelath et al34 found that VEGF bound to fibronectin and that this interaction enhanced its capacity to stimulate EC migration because of the association of FLK-1 and 5?1. Tsou and Isik35 demonstrated that vitronectin altered the expression of FGF and VEGF receptors and that matrix-integrin interactions regulated EC responsiveness to growth factors. Similarly, Moro et al36 showed that the interaction of fibroblasts with specific matrix proteins activated the epidermal growth factor receptor. Thus, specific interactions between growth factors and adhesive proteins are critical in regulation of cell properties.
Our finding that V?3 and IL-1R colocalize is consistent with previous reports regarding other receptors for example ligand-mediated integrin clustering in EC induces aggregation of FGFR37 and stimulates phosphorylation of PDGF-? receptors.38 Tyrosine phosphorylated PDGF-? and insulin receptors coimmunoprecipitate with v?3, and this requires growth factor stimulation of the receptor.39 In fibroblasts, EGF, PDGF-BB, and FGF-2 activate extracellular signal-regulated kinase synergistically with integrin activation and receptor aggregation.40 The V?3 and PDGF-? receptor coimmunoprecipitate in EC after activation and demonstrate synergistic stimulation.41 A direct interaction of surface-immobilized FGF-2 with v?3 was demonstrated that influenced EC adhesion, mitogenesis, and u-PA upregulation.42 This was confirmed by Tanghetti et al,43 who found that FGFR and v?3 cooperated in EC signaling, and both growth factor and integrin activation may be required for sustaining prolonged cell activation. Together, this evidence indicates that the coordinated effects of matrix proteins and growth factors in regulating cell function can be mediated by physical association of receptors.
The significance of IL-1? binding to fibrinogen must be considered in relation to both its tissue distribution and also the availability of other sites for binding. Notably, IL-1, which showed no affinity for fibrinogen, is primarily cell-associated.12 IL-1? is normally has a plasma concentration of <0.6 pmol/L, and at a normal fibrinogen concentration of 7 μmol/L and a Kd of 1.5 nmol/L, nearly all IL-1? would be fibrinogen-bound in the absence of other binding proteins. However, IL-1? also binds to other plasma proteins including 2-macroglobulin with which it interacts slowly, forming a covalent bond that inhibits receptor interaction.44 A specific IL-1? binding protein was previously identified in human plasma, and it did not interact with IL-1,45 similar to the pattern of fibrinogen binding presented. This protein was covalently crosslinked with IL-1? in vitro, and the complex had a Mr of 43 kDa under reducing conditions. This is consistent with cross-linking to the 46-kDa fibrinogen chain.31 IL-1? also binds to ECs through IL-1R and exhibits Kds of 4.0 pmol/L and 0.7 nmol/L for high- and low-affinity interactions.46,47 Fibrin forms at sites of thrombosis, tissue injury, and inflammation, and IL-1? binding may serve to localize the activation of immune responses. In support of this concept, Perez and Roman48 have shown that fibrin colocalizes with IL-1? in lung granulomas using immunohistochemical methods.
Binding to matrix molecules also affects IL-1? activity. In addition to being present in blood, fibrinogen may also serve as a matrix binding site for IL-1? as it is found in both normal and atherosclerotic vessel walls.49 IL-1? also binds to fibrin, which forms after hemostatic activation at sites of vessel injury or inflammation. Fibrin contributes to the hemostatic plug formation and also provides a provisional matrix to support local cell responses. The findings presented here demonstrate the importance of the interaction between fibrinogen and v?3 in determining the activity of IL-1? and indicate further that this is related to direct receptor interactions.
Acknowledgments
This work was supported in part by grant HL-30616 from the National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Md. The authors thank Jennifer Jodeksnis for excellent technical assistance and LiHua Rong for assistance in culturing primary endothelial cells.
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Correspondence to Abha Sahni, PhD, Department of Medicine, P.O. Box 610, University of Rochester Medical Center, 601 Elmwood Ave, Rochester, NY 14642. E-mail Abha_Sahni@urmc.rochester.edu
Abstract
Objective— The purpose of this study was to investigate the receptor requirements for enhanced IL-1?–induced secretion of nitric oxide (NO) by endothelial cells (ECs) in the presence of fibrinogen.
Methods and Results— ECs were exposed to IL-1? with or without fibrinogen and NO was measured as nitrite. NO production by EC exposed to fibrinogen (0.3±0.1 μmol/L) was comparable concentration to control (0.2±0.1 μmol/L), but IL-1? significantly increased NO production (0.8±0.1 μmol/L), and the combination of both fibrinogen and IL-1? resulted in a further increase to 2.2±0.2 μmol/L (P<0.002). 7E3 or LM609, antibodies to v?3, inhibited NO production stimulated by fibrinogen-bound IL-1? to 0.2±0.1 μmol/L (P<0.001) or 0.2±0.03 μmol/L (P<0.0001), respectively. These levels were comparable to control and significantly less than with IL-1? (P<0.002). EC or fibroblasts exposed to both fibrinogen and IL-1?, but not vitronectin and IL-1?, demonstrated positive Western blotting for V?3 after immunopurification with anti- IL-1R, indicating specific association between v?3 and IL-1R. Dual immunofluorescence also revealed colocalization of v?3 and IL-1R only when the cells were exposed to both fibrinogen and IL-1?.
Conclusion— The enhanced NO production by ECs in the presence of fibrinogen-bound IL-1? requires the coordinated effects of colocalized V?3 and IL-1R.
The ability of fibrinogen-bound IL-1? to stimulate NO secretion by ECs is blocked by anti-v?3. Also, fibrinogen-bound IL-1? promotes the specific association of v?3 with IL-1R, as shown by coimmunoprecipitation or immunofluorescence staining. Fibrinogen binding enhances IL-1?–induced secretion of NO through the coordinated effects of colocalized v?3 and IL-1R.
Key Words: endothelial cells ? fibrinogen ? IL-1? ? nitric oxide
Introduction
Fibrinogen plays a central role in hemostasis, and it is also an adhesive protein supporting interaction of endothelial cells (ECs) with matrix and inflammatory cells1 and fibrin also forms the provisional matrix to support cellular processes of repair. In this role, fibrin actively directs specific receptor-mediated interactions to regulate permeability,2 EC adhesion,3 synthesis, and secretion of tissue plasminogen activator and prostacyclin,4,5 plasminogen activator inhibitor (PAI)-1,6 IL-8,7 and von Willebrand factor (vWF).8 Further, fibrinogen and fibrin are also implicated in the development, and thrombotic complications of atherosclerosis, and they are found in increased amounts in atherosclerotic vessels.9 Degradation products of fibrinogen and fibrin increase vascular permeability and induce chemotaxis at sites of inflammation.10,11
The vascular response to injury is also regulated by cytokines including those of the IL-1 family that are important in inflammation. IL-1 and IL-1? are 2 members that share structural features and act on cells through the same receptors.12 However, they differ in their promoter regions, and IL-1 is primarily cell-associated, whereas IL-1? is extracellular. IL-1 induces EC procoagulant activity,13 vWF release,14 synthesis of plasminogen activator and PAI-1,15 and inhibits the thrombomodulin-protein C anticoagulant pathway.16 On exposure to IL-1, ECs increase synthesis of NO,17 chemoattractant cytokines18 and express intercellular adhesion molecule-1 (ICAM-1)19 and vascular cell adhesion molecule (VCAM)-1.20
ECs are physiologically exposed to a high concentration of fibrinogen in blood and fibrinogen binds through V?3 and 5?1.21–23 EC adhere, spread, and proliferate on fibrinogen in vitro through binding to integrins V?3 and 5?1,21,22 and fibrinogen supports leukocyte tethering to EC through ICAM-1.1 When exposed to fibrin, EC alter their shape and adhesive properties, changes that are mediated through specific interaction of VE-cadherin with the amino terminus of the fibrin ? chain.24,25
We have previously reported that fibrin(ogen) binding to IL-1? potentiates EC secretion of NO.26 We have now investigated the involvement of specific receptors in activating EC by fibrinogen-bound IL-1?.
Methods
Fibrinogen Preparation
Human fibrinogen was obtained from Enzyme Research Laboratories and copurifying fibronectin was removed by gelatin-Sepharose chromatography and immuno-affinity chromatography as described.27 The fibronectin concentration represented <0.02 μg /mL.
Cell Culture
Primary ECs were obtained from human umbilical veins, seeded on 0.2% wt/vol gelatin-coated 25 cm2 tissue culture flasks and cultured in McCoy’s 5A medium (Flow Laboratories).28 Fibroblasts were isolated from human foreskins (HFFs) and cultured in McCoy’s 5A medium supplemented with 10% fetal bovine serum, 100 U/mL penicillin, and 0.1 mg/mL of streptomycin until they reached confluence. The cells were passaged up to 2 times before use and then placed in suspension by rinsing in Hank’s balanced salt solution followed by brief incubation with trypsin-EDTA (Gibco Invitrogen). The cells were pelleted by centrifugation for 10 minutes at 500g and resuspended in McCoy’s 5A medium in the absence of serum. This wash procedure was repeated twice before use.
Measurement of NO
Confluent ECs were incubated with 5 μg/mL of either LM609 (Chemicon International), or 7E3 antibody for 2 hours. Then, 10 ng/mL of IL-1? was added to the medium in the presence or absence of 10 μg/mL of fibrinogen. The medium was collected after 1 hour, and the NO concentration was measured as nitrite using the Nitrate/Nitrite colorimetric assay (Cayman Chemicals).
Immunoprecipitation and Western Blotting
ECs or HFFs were grown to confluence, and then medium containing 10 ng/mL of either IL-1 or IL-1? (Peprotech Inc.) was added in the presence or absence of 10 μg/mL of fibrinogen or vitronectin (Sigma Chemical Co). After 1 hour, the cells were washed 3 times with phosphate-buffered saline (pH 7.4), lysed with lysis buffer containing protease inhibitors (Promega) and immunoprecipitated using antibodies to V?3 (LM609 or 7E3), V?5 (P1F6), 5?1 (JBS5), or v (LM142) and ?1 (JB1A) (Chemicon), or control mouse IgG for 2 hours, after which protein A-Sepharose beads (Pierce) were added. The beads were centrifuged at 3000g for 10 minutes, washed twice with 0.1 mol/L sodium phosphate buffer (pH 7.4) containing 0.25 mol/L NaCl, boiled in diluent for 5 minutes. The eluates and supernatants were electrophoresed using 10% SDS-polyacrylamide gel electrophoresis (SDS-PAGE). After protein transfer, membranes were immunoblotted with antibody to IL-1R (Santa Cruz Biotechnology), and bands were detected by chemiluminescence. Similar experiments were performed using anti-IL-1R for immunoprecipitation and anti-?3 (Chemicon) to probe the blot. In some experiments, anti-V?3, anti-5?1, anti-V?5, or anti-v and anti-?1 were used for immunoprecipitation, and blots were probed using anti-IL-1R. In some experiments, blots were probed with tubulin antibody for internal loading controls.
Immunofluorescent Detection
ECs or HFFs were seeded on round glass coverslips, grown to confluence, and treated with 10 ng/mL of IL-1? added to the medium in the presence or absence of 10 μg/mL of fibrinogen. After 1 hour, cells were washed twice with cold phosphate-buffered saline, fixed with 3.7% formaldehyde in phosphate-buffered saline, incubated with 10 μg/mL of polyclonal IL-1R and 10 μg/mL of monoclonal 7E3 antibodies and Alexa Fluor 568 (red) and Alexa Fluor 488 (green; Molecular Probes) were then added. Alexa Fluor 568 was conjugated to anti-IL-1R and Alexa Fluor 488 was conjugated to 7E3. Cells were viewed using a fluorescence microscope and a color digital camera and a computer with color monitor captured images.
Data Analysis
Unless indicated otherwise, data are expressed as mean±SD. Each experiment was performed at least 3 times, and either triplicate or quadruplicate wells were used in each experiment. The significance of differences in means was determined using a 2-tailed Student t test.
Results
Specific inhibitory monoclonal antibodies were used to investigate the role of integrin and IL-1 receptors in the separate and combined effects of IL-1 and fibrinogen on NO secretion. ECs were incubated with antibodies to V?3 in the presence of IL-1? with or without fibrinogen, and NO secretion was measured as nitrite (Figure 1). In the presence of fibrinogen alone, NO concentration was 0.3±0.1 μmol/L, and this was similar to medium alone (0.2±0.1 μmol/L). IL-1? increased NO production to 0.8±0.1 μmol/L, which was significantly higher than medium alone (P<0.02). Maximum NO induction occurred in the presence of both fibrinogen and IL-1? (2.2±0.2 μmol/L) with an 11±2-fold increase. In both the presence and absence of fibrinogen, NO induction reached a maximum at an IL-1? concentration of 10 ng/mL and was higher in the presence than in the absence of fibrinogen at all IL-1? concentrations tested. Addition of LM609, a monoclonal antibody to V?3, had no effect on NO production in medium alone, or after stimulation by fibrinogen or IL-1? alone. In contrast, LM609 completely inhibited the induction of NO secretion induced by fibrinogen-bound IL-1? (0.2±0.1 μmol/L) (P<0.001), and the level of secretion was less than that induced by IL-1? alone (Figure 1). Similar results were observed with 7E3, also reactive with v?3, which inhibited secretion in response to fibrinogen-bound IL-1? (0.2±0.0.03 μmol/L) (P<0.001). These findings indicate that IL-1? has no activity when fibrinogen is present if v?3 is blocked.
Figure 1. Effect of LM609 and 7E3 on EC NO levels. EC were plated on gelatin-coated flasks in McCoy’s 5A medium supplemented with 20% fetal bovine serum (FBS), 50 μg/mL of endothelial cell growth supplement, and 100 μg/mL of heparin. At confluence, cells were washed twice with McCoy’s medium and incubated in serum-free medium containing 1% Nutridoma and 5 μg/mL of either LM609 or 7E3. After 1 hour, 10 ng/mL of IL-1?, with or without 10 μg/mL of fibrinogen was added to the medium for 1 hour. Medium was then collected, and NO was measured as nitrite concentration using the Griess reaction. The results shown are mean±SD of 3 separate experiments.
IL-1? stimulates EC NO synthase (NOS) through IL-1R. To characterize the involvement of this receptor, NO secretion was measured in the presence of an antibody to IL-1R. This reduced NO secretion to 0.3±0.04 μmol/L in the presence of IL-1? alone and to 0.2±0.03 μmol/L with fibrinogen-bound IL-1? (P<0.005 for both), and these levels were not significantly different from baseline levels (Figure 2). In control experiments, nonimmune IgG had no effect on NO secretion stimulated by either free or fibrinogen-bound IL-1?. This indicates that IL-1? requires both IL-1R and V?3 to stimulate NO production by ECs when bound to fibrinogen.
Figure 2. Effect of an IL-1R antibody on NO production. ECs were plated on gelatin-coated flasks in McCoy’s 5A medium supplemented with 20% FBS, 50 μg/mL of ECGS, and 100 μg/mL of heparin and allowed to grow to confluence. The cells were then washed twice with McCoy’s medium and incubated in serum free medium containing 1% Nutridoma and 5 μg/mL of either anti-IL-1R or control mouse IgG. After 60 minutes, 10 ng/mL of IL-1? with or without 10 μg/mL of fibrinogen, was added to the medium for an hour. Medium was then collected, and NO was measured as nitrite concentration. The results are the mean±SD of 3 separate experiments.
Immunoprecipitation studies were performed to investigate possible association of receptors. ECs or HFFs were exposed to IL-1?, fibrinogen, or to the combination of both in the medium. After 1 hour, cells were washed and lysed, after which 7E3 (Figure 3A, 3B, 3C, and 3D) or IL-1R antibody (Figure 3E and 3F) was added, and immune complexes were isolated by incubation with Protein A–coupled Sepharose beads. After centrifugation, beads were boiled in diluent and subjected to SDS-PAGE, Western blots were prepared and probed with anti–IL-1R (Figure 3A, 3B, 3C, and 3D) or anti-?3 (Figure 3E and 3F). Only cells exposed to the combination of fibrinogen and IL-1? demonstrated positive Western blotting for IL-1R after immunopurification with 7E3 (Figure 3A, lane 6). Results were similar in HFFs, with colocalization of V?3 and IL-1R only with exposure to the combination of fibrinogen and IL-1? (Figure 3C, lane 6). Notably, there was no colocalization with IL-1, which does not bind to fibrinogen (Figure 3A and 3C, lanes 4 and 5). After V?3 was immunoprecipitated the supernatants were positive for IL-1R in all samples (Figure 3B and 3D). Findings were similar when IL-1R was immunoprecipitated and the blots were probed with anti-?3 (Figure 3E). The supernatants were positive for ?3 in all samples after IL-1R was immunoprecipitated (Figure 3F). These results indicate that exposure of ECs or HFFs to the combination of IL-1? and fibrinogen promoted the association of V?3 with IL-1R.
Figure 3. Colocalization of v?3 and IL-1R using coimmunoprecipitation. Confluent ECs (A,B,E,F) or HFFs (C and D) were exposed to various conditions (as described). After 1 hour, cells were washed with phosphate-buffered saline (PBS), lysed with lysis buffer, and incubated with 5 μg/mL of either 7E3 (A, B, C, D) or anti-IL-1R (E and F). Protein A Sepharose beads were then added. After washing, the beads were incubated with diluent and electrophoresed on 10% gels. Western blotting was performed with anti-IL-1R (A, B, C, D) or with anti-?3 (E and F). B, D, and F, Supernatants after centrifugation of protein A Sepharose. Ln 1, medium alone; Ln 2, 10 μg/mL fibrinogen; Ln 3, 10 ng/mL IL-1?; Ln 4, 10 ng/mL IL-1; Ln 5, 10 ng/mL IL-1 plus 10 μg/mL fibrinogen; Ln 6, 10 ng/mL IL-1? plus 10 μg/mL fibrinogen. Arrows indicates the location of IL-1R (Mr 80 kD) in A, B, C, and D and the location of ?3 in E and F (Mr 95 kD).
Immunofluorescence studies confirmed the colocalization of the fibrinogen and IL-1? receptors (Figure 4). Confluent ECs (Figure 4A) or HFFs (Figure 4B) were exposed to medium alone (insets), (fibrinogen [Figure 4A to 4C], IL-1 [Figure 4D to 4F], IL-1 plus fibrinogen [Figure 4G to 4I], IL-1? [Figure 4J to 4L], or both IL-1? plus fibrinogen [Figure 4M to 4O]) for 1 hour. Cells were then fluorescently labeled with anti-IL-1R and 7E3. The V?3 immunofluorescence increased with fibrinogen exposure, consistent with increased binding of 7E3 to activated receptor.29 In both ECs and HFFs, dual immunofluorescent detection revealed colocalization of V?3 and IL-1R only when the cells were exposed to both fibrinogen and IL-1? (Figure 4M to 4O) as shown by yellow fluorescence. No colocalization of the receptors was observed after exposure to both fibrinogen and IL-1 (Figure 4G to 4I).
Figure 4. Colocalization of v?3 and IL-1R by immunofluorescence. Confluent ECs (A) or HFFs (B) were incubated with or without 10 ng/mL of IL-1? in the presence or absence of 10 μg/mL of fibrinogen. After 1 hour, cells were washed and fixed with 3.7% formaldehyde and stained using 10 μg/mL of IL-1R and 7E3 antibody. IL-1R was visualized as red fluorescence, V?3 visualized as green fluorescence, and colocalization of IL-1? and fibrinogen receptors as yellow fluorescence. Insets represent the staining with medium alone, in the absence of any ligands. Bars represent 25 μm.
Although V?3 is an important receptor for fibrinogen, other integrins such as 5?1 also bind to fibrinogen under certain conditions.22 To determine specificity, ECs were incubated with the combination of IL-1? and fibrinogen. Lysates were then incubated with anti-V?3, anti-5?1, anti-V?5, or anti-V and anti-?1 to immunoprecipitate various integrins, and Western blots were prepared and probed with anti-IL-1R. Positive Western blotting for IL-1R was observed only with immunoprecipitates using anti-V?3 (Figure 5A). IL-1R receptor did not coprecipitate with integrins V?5, 5?1, V, or ?1, indicating the specificity of colocalization of IL-1R with V?3. Blot probed with tubulin antibody was used as a loading control (Figure 5B).
Figure 5. Specificity of association of IL-1R with v?3. (A). ECs were incubated with 10 ng/mL IL-1? and 10 μg/mL fibrinogen for 1 hour. Cells were then lysed and incubated with 5 μg/mL of integrin antibodies: V?3, V?5, 5?1, v, or ?1. Protein-A Sepharose beads were then added and incubated further for 30 minutes. After washing, beads were boiled in electrophoresis diluent for 5 minutes and samples were electrophoresed on 10% gels. Western blotting was performed with anti- IL-1R. The result of a representative experiment is shown (B). Blot probed with tubulin antibody was used as a loading control.
To determine whether IL-1R colocalizes with V?3 activated by another ligand, ECs were incubated with vitronectin or fibrinogen with or without of IL-1?. Lysates were then incubated with anti-V?3 and Western blots were prepared and probed with anti–IL-1R (Figure 6A). Only cells exposed to the combination of fibrinogen and IL-1? demonstrated positive Western blotting for IL-1R after immunopurification with anti-V?3. No colocalization was observed with vitronectin and IL-1?, indicating the specificity of fibrinogen in promoting the association of the 2 receptors in the presence of IL-1?. Blot probed with tubulin antibody was used as a loading control (Figure 6B).
Figure 6. Specificity of fibrinogn and IL-1? in promoting the association of IL-1R with v?3. (A). ECs were incubated with 10 μg/mL fibrinogen or 10 μg/mL vitronectin in the presence or absence of 10 ng/mL IL-1? for 1 hour. Cells were then lysed and incubated with 5 μg/mL of anti-v?3. Protein-A Sepharose beads were then added and incubated further for 30 minutes. After washing, beads were boiled in electrophoresis diluent for 5 minutes and samples were electrophoresed on 10% gels. Western blotting was performed with anti-IL-1R. Ln 1, medium alone; Ln 2, IL-1?; Ln 3, fibrinogen; Ln 4, vitronectin; Ln 5, IL-1? plus vitronectin; Ln 6, IL-1? plus fibrinogen. Arrow indicates the location of IL-1R (Mr 80 kDa). B, Blot probed with tubulin antibody was used as a loading control.
Discussion
The results presented demonstrate that inhibition of the interaction of fibrinogen with V?3 blocks the ability of IL-1? to induce NO in the presence of fibrinogen and that V?3 and IL-1R colocalize when both fibrinogen and IL-1? are present. Our previous studies have shown that IL-1? binds with high affinity to fibrinogen, indicating that IL-1? would be bound to fibrinogen under these experimental conditions. Antibodies to V?3 inhibited NO production induced by fibrinogen-bound IL-1? but showed no inhibition of activity with IL-1? alone. This indicates that either IL-1R is not available for binding IL-1? in the presence of fibrinogen or that it can bind but cannot activate the receptor. This may be caused by shielding of the binding site for IL-1R when IL-1? is bound to fibrinogen. We have shown previously that IL-1? competes with FGF-2 for binding to fibrinogen, suggesting that FGF-2 and IL-1? interact with the same or related sites. The site on FGF-2 that interacts with fibrinogen has been localized by mutational analysis to 5 residues between Phe-95 and Arg-109,30 and changes in binding during plasmic degradation suggests that another interactive site may be localized in the chain.31 Further studies will be required to characterize the sites involved in the IL-1?-fibrinogen interaction.
In the presence of fibrinogen-bound IL-1?, V?3 was coimmunoprecipitated with IL-1R, indicating that V?3 and IL-1R associate in initiating the signaling pathway leading to NO activation. This colocalization did not occur with the V?3 ligand vitronectin, indicating specificity for fibrinogen. These findings link the hemostatic and matrix functions of fibrinogen with the EC regulatory activities of the cytokines and are consistent with our previous reports that VEGF, FGF-2, and IL-1? bind to fibrin(ogen) with high affinity and retain activity when bound.26,32,33 The interaction potentiates the capacity of IL-1? to stimulate NO, NF-B, and MCP-1 in ECs.26
There are several other examples of interactions between growth factors and matrix proteins with their receptors that result in altered activity, suggesting that this is a common theme in cell regulation. Wijelath et al34 found that VEGF bound to fibronectin and that this interaction enhanced its capacity to stimulate EC migration because of the association of FLK-1 and 5?1. Tsou and Isik35 demonstrated that vitronectin altered the expression of FGF and VEGF receptors and that matrix-integrin interactions regulated EC responsiveness to growth factors. Similarly, Moro et al36 showed that the interaction of fibroblasts with specific matrix proteins activated the epidermal growth factor receptor. Thus, specific interactions between growth factors and adhesive proteins are critical in regulation of cell properties.
Our finding that V?3 and IL-1R colocalize is consistent with previous reports regarding other receptors for example ligand-mediated integrin clustering in EC induces aggregation of FGFR37 and stimulates phosphorylation of PDGF-? receptors.38 Tyrosine phosphorylated PDGF-? and insulin receptors coimmunoprecipitate with v?3, and this requires growth factor stimulation of the receptor.39 In fibroblasts, EGF, PDGF-BB, and FGF-2 activate extracellular signal-regulated kinase synergistically with integrin activation and receptor aggregation.40 The V?3 and PDGF-? receptor coimmunoprecipitate in EC after activation and demonstrate synergistic stimulation.41 A direct interaction of surface-immobilized FGF-2 with v?3 was demonstrated that influenced EC adhesion, mitogenesis, and u-PA upregulation.42 This was confirmed by Tanghetti et al,43 who found that FGFR and v?3 cooperated in EC signaling, and both growth factor and integrin activation may be required for sustaining prolonged cell activation. Together, this evidence indicates that the coordinated effects of matrix proteins and growth factors in regulating cell function can be mediated by physical association of receptors.
The significance of IL-1? binding to fibrinogen must be considered in relation to both its tissue distribution and also the availability of other sites for binding. Notably, IL-1, which showed no affinity for fibrinogen, is primarily cell-associated.12 IL-1? is normally has a plasma concentration of <0.6 pmol/L, and at a normal fibrinogen concentration of 7 μmol/L and a Kd of 1.5 nmol/L, nearly all IL-1? would be fibrinogen-bound in the absence of other binding proteins. However, IL-1? also binds to other plasma proteins including 2-macroglobulin with which it interacts slowly, forming a covalent bond that inhibits receptor interaction.44 A specific IL-1? binding protein was previously identified in human plasma, and it did not interact with IL-1,45 similar to the pattern of fibrinogen binding presented. This protein was covalently crosslinked with IL-1? in vitro, and the complex had a Mr of 43 kDa under reducing conditions. This is consistent with cross-linking to the 46-kDa fibrinogen chain.31 IL-1? also binds to ECs through IL-1R and exhibits Kds of 4.0 pmol/L and 0.7 nmol/L for high- and low-affinity interactions.46,47 Fibrin forms at sites of thrombosis, tissue injury, and inflammation, and IL-1? binding may serve to localize the activation of immune responses. In support of this concept, Perez and Roman48 have shown that fibrin colocalizes with IL-1? in lung granulomas using immunohistochemical methods.
Binding to matrix molecules also affects IL-1? activity. In addition to being present in blood, fibrinogen may also serve as a matrix binding site for IL-1? as it is found in both normal and atherosclerotic vessel walls.49 IL-1? also binds to fibrin, which forms after hemostatic activation at sites of vessel injury or inflammation. Fibrin contributes to the hemostatic plug formation and also provides a provisional matrix to support local cell responses. The findings presented here demonstrate the importance of the interaction between fibrinogen and v?3 in determining the activity of IL-1? and indicate further that this is related to direct receptor interactions.
Acknowledgments
This work was supported in part by grant HL-30616 from the National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Md. The authors thank Jennifer Jodeksnis for excellent technical assistance and LiHua Rong for assistance in culturing primary endothelial cells.
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