This Article Abstract Full Text (PDF) All Versions of this Article: 25/10/2222    most recent 01.ATV.0000183605.27125.6fv1 Submit a response Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Sahni, A. Articles by Francis, C. W. Search for Related Content PubMed PubMed Citation Articles by Sahni, A. Articles by Francis, C. W.

(Arteriosclerosis, Thrombosis, and Vascular Biology. 2005;25:2222.)
© 2005 American Heart Association, Inc.

Thrombosis

Endothelial Cell Activation by IL-1ß in the Presence of Fibrinogen Requires _Vß₃

Abha Sahni; Sanjeev K. Sahni; Charles W. Francis

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{at}urmc.rochester.edu

	Abstract

Top Abstract Introduction Methods Results Discussion References

 
Objective— The purpose of this study was to investigate the receptor requirements for enhanced IL-1ß–induced secretion of nitric oxide (NO) by endothelial cells (ECs) in the presence of fibrinogen.

Methods and Results— ECs were exposed to IL-1ß 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-1ß significantly increased NO production (0.8±0.1 µmol/L), and the combination of both fibrinogen and IL-1ß resulted in a further increase to 2.2±0.2 µmol/L (P<0.002). 7E3 or LM609, antibodies to _vß₃, inhibited NO production stimulated by fibrinogen-bound IL-1ß 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-1ß (P<0.002). EC or fibroblasts exposed to both fibrinogen and IL-1ß, but not vitronectin and IL-1ß, demonstrated positive Western blotting for _Vß₃ after immunopurification with anti- IL-1R, indicating specific association between _vß₃ and IL-1R. Dual immunofluorescence also revealed colocalization of _vß₃ and IL-1R only when the cells were exposed to both fibrinogen and IL-1ß.

Conclusion— The enhanced NO production by ECs in the presence of fibrinogen-bound IL-1ß requires the coordinated effects of colocalized _Vß₃ and IL-1R.

The ability of fibrinogen-bound IL-1ß to stimulate NO secretion by ECs is blocked by anti-_vß₃. Also, fibrinogen-bound IL-1ß promotes the specific association of _vß₃ with IL-1R, as shown by coimmunoprecipitation or immunofluorescence staining. Fibrinogen binding enhances IL-1ß–induced secretion of NO through the coordinated effects of colocalized _vß₃ and IL-1R.

Key Words: endothelial cells • fibrinogen • IL-1ß • nitric oxide

	Introduction

Top Abstract Introduction Methods Results Discussion References

 
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-1ß 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-1ß 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 _Vß₃ and ₅ß₁.^21–23 EC adhere, spread, and proliferate on fibrinogen in vitro through binding to integrins _Vß₃ and ₅ß₁,^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 ß chain.^24,25

We have previously reported that fibrin(ogen) binding to IL-1ß potentiates EC secretion of NO.²⁶ We have now investigated the involvement of specific receptors in activating EC by fibrinogen-bound IL-1ß.

	Methods

Top Abstract Introduction Methods Results Discussion References

 
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-1ß 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-1ß (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 _Vß₃ (LM609 or 7E3)_{, V}ß₅ (P1F6), ₅ß₁ (JBS5), or _v (LM142) and ß₁ (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-ß₃ (Chemicon) to probe the blot. In some experiments, anti-_Vß₃, anti-₅ß₁, anti-_Vß₅, or anti-_v and anti-ß₁ 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-1ß 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

Top Abstract Introduction Methods Results Discussion References

 
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 _Vß₃ in the presence of IL-1ß 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-1ß 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-1ß (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-1ß concentration of 10 ng/mL and was higher in the presence than in the absence of fibrinogen at all IL-1ß concentrations tested. Addition of LM609, a monoclonal antibody to _Vß₃, had no effect on NO production in medium alone, or after stimulation by fibrinogen or IL-1ß alone. In contrast, LM609 completely inhibited the induction of NO secretion induced by fibrinogen-bound IL-1ß (0.2±0.1 µmol/L) (P<0.001), and the level of secretion was less than that induced by IL-1ß alone (Figure 1). Similar results were observed with 7E3, also reactive with _vß₃, which inhibited secretion in response to fibrinogen-bound IL-1ß (0.2±0.0.03 µmol/L) (P<0.001). These findings indicate that IL-1ß has no activity when fibrinogen is present if _vß₃ is blocked.

View larger version (13K): [in this window] [in a new window]   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-1ß, 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-1ß 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-1ß alone and to 0.2±0.03 µmol/L with fibrinogen-bound IL-1ß (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-1ß. This indicates that IL-1ß requires both IL-1R and _Vß₃ to stimulate NO production by ECs when bound to fibrinogen.

View larger version (12K): [in this window] [in a new window]   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-1ß 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-1ß, 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-ß3 (Figure 3E and 3F). Only cells exposed to the combination of fibrinogen and IL-1ß demonstrated positive Western blotting for IL-1R after immunopurification with 7E3 (Figure 3A, lane 6). Results were similar in HFFs, with colocalization of _Vß₃ and IL-1R only with exposure to the combination of fibrinogen and IL-1ß (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 _Vß₃ 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-ß3 (Figure 3E). The supernatants were positive for ß₃ in all samples after IL-1R was immunoprecipitated (Figure 3F). These results indicate that exposure of ECs or HFFs to the combination of IL-1ß and fibrinogen promoted the association of _Vß₃ with IL-1R.

View larger version (31K): [in this window] [in a new window]   Figure 3. Colocalization of vß3 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-ß3 (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-1ß; 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-1ß 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 ß3 in E and F (Mr 95 kD).

Immunofluorescence studies confirmed the colocalization of the fibrinogen and IL-1ß 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-1ß [Figure 4J to 4L], or both IL-1ß plus fibrinogen [Figure 4M to 4O]) for 1 hour. Cells were then fluorescently labeled with anti-IL-1R and 7E3. The _Vß₃ 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 _Vß₃ and IL-1R only when the cells were exposed to both fibrinogen and IL-1ß (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).

View larger version (37K): [in this window] [in a new window]   Figure 4. Colocalization of vß3 and IL-1R by immunofluorescence. Confluent ECs (A) or HFFs (B) were incubated with or without 10 ng/mL of IL-1ß 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, Vß3 visualized as green fluorescence, and colocalization of IL-1ß and fibrinogen receptors as yellow fluorescence. Insets represent the staining with medium alone, in the absence of any ligands. Bars represent 25 µm.

Although _Vß₃ is an important receptor for fibrinogen, other integrins such as ₅ß₁ also bind to fibrinogen under certain conditions.²² To determine specificity, ECs were incubated with the combination of IL-1ß and fibrinogen. Lysates were then incubated with anti-_Vß_3, anti-₅ß_1, anti-_Vß_5, or anti-_V and anti-ß₁ 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-_Vß₃ (Figure 5A). IL-1R receptor did not coprecipitate with integrins _Vß₅, ₅ß_{1, V}, or ß₁, indicating the specificity of colocalization of IL-1R with _Vß₃. Blot probed with tubulin antibody was used as a loading control (Figure 5B).

View larger version (29K): [in this window] [in a new window]   Figure 5. Specificity of association of IL-1R with vß3. (A). ECs were incubated with 10 ng/mL IL-1ß and 10 µg/mL fibrinogen for 1 hour. Cells were then lysed and incubated with 5 µg/mL of integrin antibodies: Vß3, Vß5, 5ß1, v, or ß1. 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 _Vß₃ activated by another ligand, ECs were incubated with vitronectin or fibrinogen with or without of IL-1ß. Lysates were then incubated with anti-_Vß₃ and Western blots were prepared and probed with anti–IL-1R (Figure 6A). Only cells exposed to the combination of fibrinogen and IL-1ß demonstrated positive Western blotting for IL-1R after immunopurification with anti-_Vß₃. No colocalization was observed with vitronectin and IL-1ß, indicating the specificity of fibrinogen in promoting the association of the 2 receptors in the presence of IL-1ß. Blot probed with tubulin antibody was used as a loading control (Figure 6B).

View larger version (45K): [in this window] [in a new window]   Figure 6. Specificity of fibrinogn and IL-1ß in promoting the association of IL-1R with vß3. (A). ECs were incubated with 10 µg/mL fibrinogen or 10 µg/mL vitronectin in the presence or absence of 10 ng/mL IL-1ß for 1 hour. Cells were then lysed and incubated with 5 µg/mL of anti-vß3. 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-1ß; Ln 3, fibrinogen; Ln 4, vitronectin; Ln 5, IL-1ß plus vitronectin; Ln 6, IL-1ß 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

Top Abstract Introduction Methods Results Discussion References

 
The results presented demonstrate that inhibition of the interaction of fibrinogen with _Vß₃ blocks the ability of IL-1ß to induce NO in the presence of fibrinogen and that _Vß₃ and IL-1R colocalize when both fibrinogen and IL-1ß are present. Our previous studies have shown that IL-1ß binds with high affinity to fibrinogen, indicating that IL-1ß would be bound to fibrinogen under these experimental conditions. Antibodies to _Vß₃ inhibited NO production induced by fibrinogen-bound IL-1ß but showed no inhibition of activity with IL-1ß alone. This indicates that either IL-1R is not available for binding IL-1ß 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-1ß is bound to fibrinogen. We have shown previously that IL-1ß competes with FGF-2 for binding to fibrinogen, suggesting that FGF-2 and IL-1ß 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-1ß-fibrinogen interaction.

In the presence of fibrinogen-bound IL-1ß, _Vß₃ was coimmunoprecipitated with IL-1R, indicating that _Vß₃ and IL-1R associate in initiating the signaling pathway leading to NO activation. This colocalization did not occur with the _Vß₃ 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-1ß bind to fibrin(ogen) with high affinity and retain activity when bound.^26,32,33 The interaction potentiates the capacity of IL-1ß 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 ₅ß₁. 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 _Vß₃ 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-ß receptors.³⁸ Tyrosine phosphorylated PDGF-ß and insulin receptors coimmunoprecipitate with _vß₃, 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 _Vß₃ and PDGF-ß receptor coimmunoprecipitate in EC after activation and demonstrate synergistic stimulation.⁴¹ A direct interaction of surface-immobilized FGF-2 with _vß₃ 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 _vß₃ 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-1ß 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-1ß 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-1ß would be fibrinogen-bound in the absence of other binding proteins. However, IL-1ß 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-1ß 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-1ß in vitro, and the complex had a M_r of 43 kDa under reducing conditions. This is consistent with cross-linking to the 46-kDa fibrinogen chain.³¹ IL-1ß 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-1ß 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-1ß in lung granulomas using immunohistochemical methods.

Binding to matrix molecules also affects IL-1ß activity. In addition to being present in blood, fibrinogen may also serve as a matrix binding site for IL-1ß as it is found in both normal and atherosclerotic vessel walls.⁴⁹ IL-1ß 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 _vß₃ in determining the activity of IL-1ß 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.

Received April 18, 2005; accepted August 15, 2005.

	References

Top Abstract Introduction Methods Results Discussion References

 
1. Languino LR, Plescia J, Duperray A, Brian AA, Plow EF, Geltosky JE, Altieri DC. Fibrinogen mediates leukocyte adhesion to vascular endothelium through an ICAM-1-dependent pathway. Cell. 1993; 73: 1423–1434.[CrossRef][Medline] [Order article via Infotrieve]

2. Dvorak HF, Nagy JA, Berse B, Brown LF, Yeo KT, Yeo TK, Dvorak AM, van de Water L, Sioussat TM, Senger DR. Vascular permeability factor, fibrin, and the pathogenesis of tumor stroma formation. Ann N Y Acad Sci. 1992; 667: 101–111.[Medline] [Order article via Infotrieve]

3. Chalupowicz DG, Chowdhury ZA, Bach TL, Barsigian C, Martinez J. Fibrin II induces endothelial cell capillary tube formation. J Cell Biol. 1995; 130: 207–215.[Abstract/Free Full Text]

4. Kaplan KL, Mather T, DeMarco L, Solomon S. Effect of fibrin on endothelial cell production of prostacyclin and tissue plasminogen activator. Arteriosclerosis. 1989; 9: 43–49.[Abstract/Free Full Text]

5. Ramsby ML, Kreutzer DL. Fibrin induction of tissue plasminogen activator expression in corneal endothelial cells in vitro. Invest Ophthalmol Vis Sci. 1993; 34: 3207–3219.[Abstract/Free Full Text]

6. Fukao H, Matsumoto H, Ueshima S, Okada K, Matsuo O. Effects of fibrin on the secretion of plasminogen activator inhibitor-1 from endothelial cells and on protein kinase C. Life Sci. 1995; 57: 1267–1276.[CrossRef][Medline] [Order article via Infotrieve]

7. Qi J, Kreutzer DL. Fibrin activation of vascular endothelial cells. Induction of IL-8 expression. J Immunol. 1995; 155: 867–876.[Abstract]

8. Ribes JA, Ni F, Wagner DD, Francis CW. Mediation of fibrin-induced release of von Willebrand factor from cultured endothelial cells by the fibrin beta chain. J Clin Invest. 1989; 84: 435–442.

9. Bini A, Mesa-Tejada R, Fenoglio JJ Jr, Kudryk B, Kaplan KL. Immunohistochemical characterization of fibrin(ogen)-related antigens in human tissues using monoclonal antibodies. Lab Invest. 1989; 60: 814–821.[Medline] [Order article via Infotrieve]

10. Rowland FN, Donovan MJ, Picciano PT, Wilner GD, Kreutzer DL. Fibrin-mediated vascular injury. Identification of fibrin peptides that mediate endothelial cell retraction. Am J Pathol. 1984; 117: 418–428.[Abstract]

11. Senior RM, Skogen WF, Griffin GL, Wilner GD. Effects of fibrinogen derivatives upon the inflammatory response. Studies with human fibrinopeptide B. J Clin Invest. 1986; 77: 1014–1019.

12. Dinarello CA. Interleukin-1. Cytokine Growth Factor Rev. 1997; 8: 253–265.[CrossRef][Medline] [Order article via Infotrieve]

13. Bevilacqua MP, Pober JS, Majeau GR, Cotran RS, Gimbrone MA Jr. Interleukin 1 (IL-1) induces biosynthesis and cell surface expression of procoagulant activity in human vascular endothelial cells. J Exp Med. 1984; 160: 618–623.[Abstract/Free Full Text]

14. Paleolog EM, Crossman DC, McVey JH, Pearson JD. Differential regulation by cytokines of constitutive and stimulated secretion of von Willebrand factor from endothelial cells. Blood. 1990; 75: 688–695.[Abstract/Free Full Text]

15. Schleef RR, Bevilacqua MP, Sawdey M, Gimbrone MA Jr, Loskutoff DJ. Cytokine activation of vascular endothelium. Effects on tissue-type plasminogen activator and type 1 plasminogen activator inhibitor. J Biol Chem. 1988; 263: 5797–5803.[Abstract/Free Full Text]

16. Nawroth PP, Handley DA, Esmon CT, Stern DM. Interleukin 1 induces endothelial cell procoagulant while suppressing cell-surface anticoagulant activity. Proc Natl Acad Sci U S A. 1986; 83: 3460–3464.[Abstract/Free Full Text]

17. Gross SS, Jaffe EA, Levi R, Kilbourn RG. Cytokine-activated endothelial cells express an isotype of nitric oxide synthase which is tetrahydrobiopterin-dependent, calmodulin-independent and inhibited by arginine analogs with a rank-order of potency characteristic of activated macrophages. Biochem Biophys Res Commun. 1991; 178: 823–829.[CrossRef][Medline] [Order article via Infotrieve]

18. Rollins BJ, Yoshimura T, Leonard EJ, Pober JS. Cytokine-activated human endothelial cells synthesize and secrete a monocyte chemoattractant, MCP-1/JE. Am J Pathol. 1990; 136: 1229–1233.[Abstract]

19. Bevilacqua MP, Stengelin S, Gimbrone MA Jr, Seed B. Endothelial leukocyte adhesion molecule 1: an inducible receptor for neutrophils related to complement regulatory proteins and lectins. Science. 1989; 243: 1160–1165.[Abstract/Free Full Text]

20. Natarajan M, Udden MM, McIntire LV. Adhesion of sickle red blood cells and damage to interleukin-1 beta stimulated endothelial cells under flow in vitro. Blood. 1996; 87: 4845–4852.[Abstract/Free Full Text]

21. Cheresh DA, Berliner SA, Vicente V, Ruggeri ZM. Recognition of distinct adhesive sites on fibrinogen by related integrins on platelets and endothelial cells. Cell. 1989; 58: 945–953.[CrossRef][Medline] [Order article via Infotrieve]

22. Suehiro K, Gailit J, Plow EF. Fibrinogen is a ligand for integrin alpha5beta1 on endothelial cells. J Biol Chem. 1997; 272: 5360–5366.[Abstract/Free Full Text]

23. Smith JW, Cheresh DA. Integrin (alpha v beta 3)-ligand interaction. Identification of a heterodimeric RGD binding site on the vitronectin receptor. J Biol Chem. 1990; 265: 2168–2172.[Abstract/Free Full Text]

24. Erban JK, Wagner DD. A 130-kDa protein on endothelial cells binds to amino acids 15–42 of the B beta chain of fibrinogen. J Biol Chem. 1992; 267: 2451–2458.[Abstract/Free Full Text]

25. Bach TL, Barsigian C, Chalupowicz DG, Busler D, Yaen CH, Grant DS, Martinez J. VE-Cadherin mediates endothelial cell capillary tube formation in fibrin and collagen gels. Exp Cell Res. 1998; 238: 324–334.[CrossRef][Medline] [Order article via Infotrieve]

26. Sahni A, Guo M, Sahni SK, Francis CW. Interleukin-1beta but not IL-1alpha binds to fibrinogen and fibrin and has enhanced activity in the bound form. Blood. 2004; 104: 409–414.[Abstract/Free Full Text]

27. Bunce LA, Sporn LA, Francis CW. Endothelial cell spreading on fibrin requires fibrinopeptide B cleavage and amino acid residues 15–42 of the beta chain. J Clin Invest. 1992; 89: 842–850.

28. Sahni A, Altland OD, Francis CW. FGF-2 but not FGF-1 binds fibrin and supports prolonged endothelial cell growth. J Thromb Haemost. 2003; 1: 1304–1310.[CrossRef][Medline] [Order article via Infotrieve]

29. Coller BS. A new murine monoclonal antibody reports an activation-dependent change in the conformation and/or microenvironment of the platelet glycoprotein IIb/IIIa complex. J Clin Invest. 1985; 76: 101–108.

30. Peng H, Sahni A, Fay P, Bellum S, Prudovsky I, Maciag T, Francis CW. Identification of a binding site on human FGF-2 for fibrinogen. Blood. 2004; 103: 2114–2120.[Abstract/Free Full Text]

31. Sahni A, Francis CW. Plasmic degradation modulates activity of fibrinogen-bound fibroblast growth factor-2. J Thromb Haemost. 2003; 1: 1271–1277.[CrossRef][Medline] [Order article via Infotrieve]

32. Sahni A, Odrljin T, Francis CW. Binding of basic fibroblast growth factor to fibrinogen and fibrin. J Biol Chem. 1998; 273: 7554–7559.[Abstract/Free Full Text]

33. Sahni A, Francis CW. Vascular endothelial growth factor binds to fibrinogen and fibrin and stimulates endothelial cell proliferation. Blood. 2000; 96: 3772–3778.[Abstract/Free Full Text]

34. Wijelath ES, Murray J, Rahman S, Patel Y, Ishida A, Strand K, Aziz S, Cardona C, Hammond WP, Savidge GF, Rafii S, Sobel M. Novel vascular endothelial growth factor binding domains of fibronectin enhance vascular endothelial growth factor biological activity. Circ Res. 2002; 91: 25–31.[Abstract/Free Full Text]

35. Tsou R, Isik FF. Integrin activation is required for VEGF and FGF receptor protein presence on human microvascular endothelial cells. Mol Cell Biochem. 2001; 224: 81–89.[CrossRef][Medline] [Order article via Infotrieve]

36. Moro L, Venturino M, Bozzo C, Silengo L, Altruda F, Beguinot L, Tarone G, Defilippi P. Integrins induce activation of EGF receptor: role in MAP kinase induction and adhesion-dependent cell survival. EMBO J. 1998; 17: 6622–6632.[CrossRef][Medline] [Order article via Infotrieve]

37. Plopper GE, McNamee HP, Dike LE, Bojanowski K, Ingber DE. Convergence of integrin and growth factor receptor signaling pathways within the focal adhesion complex. Mol Biol Cell. 1995; 6: 1349–1365.[Abstract]

38. Sundberg C, Rubin K. Stimulation of beta1 integrins on fibroblasts induces PDGF independent tyrosine phosphorylation of PDGF beta-receptors. J Cell Biol. 1996; 132: 741–752.[Abstract/Free Full Text]

39. Schneller MVK, Ruoslahti E. Alpha V beta 3 integrin associates with activated insulin and PDGF beta receptors and potentiates the biological activity of PDGF. EMBO J. 1997; 16: 5600–5607.[CrossRef][Medline] [Order article via Infotrieve]

40. Miyamoto S, Teramoto H, Gutkind JS, Yamada KM. Integrins can collaborate with growth factors for phosphorylation of receptor tyrosine kinases and MAP kinase activation: roles of integrin aggregation and occupancy of receptors. J Cell Biol. 1996; 135: 1633–1642.[Abstract/Free Full Text]

41. Woodard AS, Garcia-Cardena G, Leong M, Madri JA, Sessa WC, Languino LR. The synergistic activity of alphavbeta3 integrin and PDGF receptor increases cell migration. J Cell Sci. 1998; 111: 469–478.[Abstract]

42. Rusnati M, Tanghetti E, Dell’Era P, Gualandris A, Presta M. alphavbeta3 integrin mediates the cell-adhesive capacity and biological activity of basic fibroblast growth factor (FGF-2) in cultured endothelial cells. Mol Biol Cell. 1997; 8: 2449–2461.[Abstract/Free Full Text]

43. Tanghetti E, Ria R, Dell’Era P, Urbinati C, Rusnati M, Ennas MG, Presta M. Biological activity of substrate-bound basic fibroblast growth factor (FGF2): recruitment of FGF receptor-1 in endothelial cell adhesion contacts. Oncogene. 2002; 21: 3889–3897.[CrossRef][Medline] [Order article via Infotrieve]

44. Borth W, Scheer B, Urbansky A, Luger TA, Sottrup-Jensen L. Binding of IL-1 beta to alpha-macroglobulins and release by thioredoxin. J Immunol. 1990; 145: 3747–3754.[Abstract]

45. Eastgate JA, Symons JA, Duff GW. Identification of an interleukin-1 beta binding protein in human plasma. FEBS Lett. 1990; 260: 213–216.[CrossRef][Medline] [Order article via Infotrieve]

46. Cozzolino F, Torcia M, Aldinucci D, Ziche M, Almerigogna F, Bani D, Stern DM. Interleukin 1 is an autocrine regulator of human endothelial cell growth. Proc Natl Acad Sci U S A. 1990; 87: 6487–6491.[Abstract/Free Full Text]

47. Thieme TR, Hefeneider SH, Wagner CR, Burger DR. Recombinant murine and human IL 1 alpha bind to human endothelial cells with an equal affinity, but have an unequal ability to induce endothelial cell adherence of lymphocytes. J Immunol. 1987; 139: 1173–1178.[Abstract]

48. Perez RL, Roman J. Fibrin enhances the expression of IL-1 beta by human peripheral blood mononuclear cells. Implications in pulmonary inflammation. J Immunol. 1995; 154: 1879–1887.[Abstract]

49. Lu LF, Fiscus RR. Interleukin-1beta causes different levels of nitric oxide-mediated depression of contractility in different positions of rat thoracic aorta. Life Sci. 1999; 64: 1373–1381.[CrossRef][Medline] [Order article via Infotrieve]

This Article Abstract Full Text (PDF) All Versions of this Article: 25/10/2222    most recent 01.ATV.0000183605.27125.6fv1 Submit a response Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Sahni, A. Articles by Francis, C. W. Search for Related Content PubMed PubMed Citation Articles by Sahni, A. Articles by Francis, C. W.