Enhancement of fibrinogen-triggered pro-coagulant activation of monocytes in vitro by matrix metalloproteinase-9
© Kaneider et al; licensee BioMed Central Ltd. 2010
Received: 18 September 2009
Accepted: 29 January 2010
Published: 29 January 2010
Interaction of fibrinogen with specific leukocyte integrins of monocytes may link coagulation and inflammation, however, the precise mechanism of fibrinogen leading to the pro-inflammatory and pro-coagulatory response on monocytes is yet unknown.
Fibrinogen and its digestion fragment D induced pro-coagulant activation of monocytes as assessed in a cellular coagulation assay by reductions in clotting times. Pro-coagulant activation was reversed by blocking antibodies against Mac-1 or LFA-1. Pre-exposure of monocytes to the p38 MAPK inhibitor SB 202190 and the MEK1.2 inhibitor U0126 led to significant increasees in coagulation times whereas blocking JNKII with its inhibitor had no such effect. Blocking NFκB with MG-132 also inhibited pro-coagulant activation of monocytes by fibrinogen. A selective inhibitor of matrix metalloproteinase-9 increased times to clot formation whereas other matrix metalloproteinase inhibitors did not significantly interfere with fibrinogen-augmented clot formation in this assay. Treatment of monocytes with fibrinogen increased concentrations of matrix metalloproteinase-9 immunoreactivity in their supernatants.
Fibrinogen induces monocyte pro-coagulant activation in an integrin-, nuclear factor κB-, p38 MAPK-, and MEK1.2-dependent manner. Activation of monocytes by fibrinogen increases metalloproteinase-9 secretion, metalloproteinase-9 itself enhances monocyte coagulation by an autocrine mechanism. Results provide further evidence that mediators of hemostasis have a profound impact on cells of the immune system and are closely related to inflammatory pathways.
Fibrinogen is a 45 nm long glycoprotein consisting of three pairs of polypeptide chains, Aα, Bβ and γ, symmetrically interconnected through multiple disulfide bonds forming a dimer. In addition to its well-known functions in hemostasis, over the past two decades there has been an increasing appreciation of the important function that fibrinogen exerts in the innate immune system. Studies indicate that fibrinogen plays a multifaceted role in inflammatory response, indicative of a close relationship between hemostatic and inflammatory pathways [1–4]. Acute inflammatory events are known to shift the hemostatic balance toward a pro-thrombotic state [5–7]. One established mechanism whereby inflammatory mediators can promote coagulation is the enhanced expression of tissue factor on endothelial cells and monocytes [8, 9].
The ability of fibrinogen to participate in the inflammatory response depends on its interaction with specific leukocyte integrins [10–13]. The main fibrinogen receptors on leukocytes are CD11b/CD18 (Mac-1, αmβ2) and CD11c/CD18 (αxβ2). Leukocyte emigration from the blood to the sites of inflammation is currently viewed as an adhesion cascade that involves coordinated function of a variety of adhesion receptors on leukocytes and endothelial cells . It has been shown that elevated plasma fibrinogen and fibrinogen degradation products (FgDP) inhibit several functions in neutrophils critical to the bactericidal activity of inflammatory cells . Furthermore it has been suggested that fibrinogen production may be controlled by regulatory proteins produced by monocytes in response to the fibrinogen fragments D and E . Conceivably FgDPs could stimulate monocytes to release interleukin-1, interleukin-6 and TNF-α. Moreover fibrinogen acts as a bridging ligand for the adhesion of monocytes to cultured endothelial cells by the binding of a specific sequence of its D-domain to ICAM-1 on endothelial cells [16, 17]. The N-terminal disulfide knot binds to CD11b/CD18 and CD11c/CD18 (αxβ2) on stimulated neutrophils .
Monocytes play a key role in the orchestration of the pro-inflammatory response. These cells migrate from the peripheral blood into various tissues and differentiate into macrophages. Cells of the mononuclear phagocytotic system have been linked to a variety of inflammatory diseases, particularly to atherosclerosis, where macrophages transform into foam cells and lead to the plaque formation. Moreover, elevated fibrinogen levels in young people were independently associated with subclinical atherosclerosis . Interaction of fibrinogen with specific leukocyte integrins of monocytes may link coagulation and inflammation, however, the precise mechanism of fibrinogen leading to the pro-inflammatory and pro-coagulatory response on monocytes is yet unknown.
Pro-coagulant activation of monocytes by fibrinogen
Matrix metalloproteinase-9-mediated activation of monocytes by fibrinogen
In this investigation, we provide evidence that fibrinogen-induced clot formation of monocytes dependents on Mac-1 and LFA-1 activation. Fibrinogen is thought to bind to leukocyte integrins and causes leukocyte adherence to the injured vessel wall. In this regard, recent studies demonstrated that simultaneous fibrinogen binding to leukocytes and endothelial cells enhances adhesion of monocytes to the endothelium by acting as a molecular bridge between the two cell types [16, 17, 21]. Stimulation of integrins by specific agonists activates and renders them competent to bind soluble fibrinogen, while non-stimulated integrins, however, are able to bind immobilized fibrinogen . In our case no immobilized fibrinogen was used but all experiments were performed under rotating, non-static conditions. Therefore it is very likely that mediators other than fibrinogen led to the monocyte-monocyte adhesions observed in the coagulation experiments. Our data indicate that fibrinogen initially activates pro-inflammatory pathways since blockade of NfkB prolonged the time for clot formation. It is known that fibrinogen does not only lead to adhesion of monocytes, it also induces de novo synthesis of IL-6 and TNF-α .
A strong candidate for a possible monocyte-monocyte interaction via the integrin pathway was MMP-9. Interestingly, only pharmacological inhibitors of MMP-9 increased coagulation time almost to baseline levels, suggesting an important role of MMP-9 in this context. Stimulation of monocytes with fibrinogen under rotation led to a significant increase in intra- and extracellular levels of the protease. However, MMP-9 not only binds to integrins and induces adhesion, it might also augment the pro-inflammatory response to fibrinogen via a positive feedback loop.
In vitro, fibrinogen induces monocyte pro-coagulant activation in an NFκB- and MMP-9-dependent manner by specific ligation of signal-transducing integrins that also induces TF expression in the cells. These results provide further evidence that mediators of hemostasis have a profound impact on cells of the immune system and are closely related to inflammatory pathways in which MMPs play a prominent regulatoy role.
Hanks balanced salt solution (HBSS) without Ca2+ and Mg2+ was purchased from Gibco BRL, Life Technologies (Vienna, Austria). Lymphoprep was from Nycomed Pharma (Oslo, Norway), anti-human CD14 antibody was from Miltenyi Biotec (Bergisch Gladbach, Germany), Bovine serum albumin (BSA), was purchased from Dade-Behring (Marburg, Germany). RPMI 1640 was purchased from Biological Industries (Kibbutz Beit Haemek, Israel). Fibrinogen was either from CSL Behring (Haemocomplettan) or from Haematological Technologies Inc. (Essex Junction, VT). The D- and E-fragment of fibrinogen were obtained from Haematological Technologies Inc as well. The E. coli LPS was from Sigma Aldrich. The signal transduction inhibitors SB 202190, U0126, JNK Inhibitor II and the NfkB MG-132 inhibitor were from Calbiochem (Gibbstown, NJ) as were the MMP-inhibitors. MMP-ELISA kits (proMMP-1 and MMP-9) and the blocking integrin-antibodies were purchased from R&D systems (Minneapolis, MN).
Preparation of human monocytes
Mononuclear cells were prepared from peripheral venous blood (anticoagulated with EDTA) of healthy volunteers. After Lymphoprep® density gradient centrifugation, peripheral blood mononuclear cells were collected and washed three times with normal saline. Positive selection of monocytes was performed by adding MACS colloidal superparamagnetic microbeads conjugated with monoclonal anti-human CD14 antibodies to cooled, freshly prepared peripheral blood mononuclear cell preparations in MACS buffer (PBS with 5 mM EDTA and 0.5% bovine serum albumin) according to the manufacturer's instructions. Cells and microbeads were incubated for 15 min at 4-6°C. In the meantime, the separation column was positioned in the MACS magnetic field and washed with MACS buffer at room temperature. The cells were washed with MACS buffer, resuspended, and loaded onto the top of the separation column. The eluent containing CD14- cells was withdrawn and after removal of the column from the magnet, trapped monocytes (CD14+) were eluted with the six-fold amount of cold MACS buffer, centrifuged, and resuspended in medium containing 0.5% BSA.
Monocyte coagulation assay
Based on the concept that tissue factor bearing cells localize and form the hemostatic plug, cell based models of coagulation have been developed that mimic - at least in part - in vivo coagulation reactions.
For the coagulation experiments, monocytes were incubated with the particular reagent for 4 h under continuous rotation to prevent monocyte adhesion. After washing 200 μL of re-calcified human plasma was added to the monocytes. Clotting time was measured in duplicate by using a coagulometer (Amelung; Lemgo, Germany). In some experiments monocytes were pre-treated with signal transduction inhibitors such as SB 202190 that inhibits p38 MAPK, U0126 which inhibits MEK1 and MEK2. Furthermore NF-kB was inhibited by the addition of MG-132, and JNK II was inhibited by the addition of JNK Inhibitor II. The concentration used was 3 × IC50 for each inhibitor. After washing fibrinogen was added and the cells were incubated with fibrinogen for another 4 hours.
For mouse studies animals were injected with 20 mg of fibrinogen. Four hours after injection, blood was collected by terminal cardiac puncture and the red blood cells were removed by lysis. Again, clotting time was measured by the method described above.
Determination of MMP levels
Matrix metallo proteases (MMPs) were measured either in monocyte supernatants or after treating monocytes with lysing buffer (intra-cellular levels). Monocytes were stimulated with fibrinogen for 4 h then the supernatans were collected and subjected to commercially available proMMP-1 and MMP-9 ELISA assays. After collecting the supernatants, cells were lysed and after spinning down the cell detritus, intra-cellular MMP-levels were measured as well by the same method. section.
In vivo monocyte migration assay
Six to 8 week old female CD-1 mice were given 20 mg of fibrinogen i.p. 4 days after the injection mice were sacrificed by CO2-inhalation and peritoneal lavage was performed with 10 mL of lukewarm 0.9% sodium chloride. Cells were counted in a Neubauer chamber. Peripheral leukocytes were counted in a Neubauer chamber after the lysis of red blood cells.
Results are given as mean and standard error of the mean. Statistics were calculated after Kruskal Wallis and Mann Whitney U tests using StatView (Abacus, Berkeley, CA).
The study was partially supported by a grant from CSL Behring (Marburg, Germany) to the Medical University of Innsbruck, Austria.
- Jensen T, Kierulf P, Sandset PM, Klingenberg O, Joo GB, Godal HC, Skjonsberg OH: Fibrinogen and fibrin induce synthesis of proinflammatory cytokines from isolated peripheral blood mononuclear cells. Thromb Haemost 2007, 97: 822-829.PubMedGoogle Scholar
- Smiley ST, King JA, Hancock WW: Fibrinogen stimulates macrophage chemokine secretion through toll-like receptor 4. J Immunol 2001, 167: 2887-2894.View ArticlePubMedGoogle Scholar
- Sitrin RG, Pan PM, Srikanth S, Todd RF: Fibrinogen activates NF-kappa B transcription factors in mononuclear phagocytes. J Immunol 1998, 161: 1462-1470.PubMedGoogle Scholar
- Rubel C, Fernandez GC, Rosa FA, Gomez S, Bompadre MB, Coso OA, Isturiz MA, Palermo MS: Soluble fibrinogen modulates neutrophil functionality through the activation of an extracellular signal-regulated kinase-dependent pathway. J Immunol 2002, 168: 3527-3535.View ArticlePubMedGoogle Scholar
- Esmon CT: The regulation of natural anticoagulant pathways. Science 1987, 235: 1348-1352. 10.1126/science.3029867View ArticlePubMedGoogle Scholar
- Creasey AA, Chang AC, Feigen L, Wun TC, Taylor FB, Hinshaw LB: Tissue factor pathway inhibitor reduces mortality from Escherichia coli septic shock. J Clin Invest 1993, 91: 2850-2860. 10.1172/JCI116529PubMed CentralView ArticlePubMedGoogle Scholar
- Esmon CT, Taylor FB, Snow TR: Inflammation and coagulation: linked processes potentially regulated through a common pathway mediated by protein C. Thromb Haemost 1991, 66: 160-165.PubMedGoogle Scholar
- Drake TA, Ruf W, Morrissey JH, Edgington TS: Functional tissue factor is entirely cell surface expressed on lipopolysaccharide-stimulated human blood monocytes and a constitutively tissue factor-producing neoplastic cell line. J Cell Biol 1989, 109: 389-395. 10.1083/jcb.109.1.389View ArticlePubMedGoogle Scholar
- Drake TA, Morrissey JH, Edgington TS: Selective cellular expression of tissue factor in human tissues. Implications for disorders of hemostasis and thrombosis. Am J Pathol 1989, 134: 1087-1097.PubMed CentralPubMedGoogle Scholar
- Flick MJ, Du X, Degen JL: Fibrin(ogen)-alpha M beta 2 interactions regulate leukocyte function and innate immunity in vivo. Exp Biol Med (Maywood) 2004, 229: 1105-1110.Google Scholar
- Forsyth CB, Solovjov DA, Ugarova TP, Plow EF: Integrin alpha(M)beta(2)-mediated cell migration to fibrinogen and its recognition peptides. J Exp Med 2001, 193: 1123-1133. 10.1084/jem.193.10.1123PubMed CentralView ArticlePubMedGoogle Scholar
- Ugarova TP, Yakubenko VP: Recognition of fibrinogen by leukocyte integrins. Ann N Y Acad Sci 2001, 936: 368-385.View ArticlePubMedGoogle Scholar
- Stefanidakis M, Bjorklund M, Ihanus E, Gahmberg CG, Koivunen E: Identification of a negatively charged peptide motif within the catalytic domain of progelatinases that mediates binding to leukocyte beta 2 integrins. J Biol Chem 2003, 278: 34674-34684. 10.1074/jbc.M302288200View ArticlePubMedGoogle Scholar
- Kaneider NC, Leger AJ, Kuliopulos A: Therapeutic targeting of molecules involved in leukocyte-endothelial cell interactions. FEBS J 2006, 273: 4416-4424. 10.1111/j.1742-4658.2006.05441.xView ArticlePubMedGoogle Scholar
- Ritchie DG, Levy BA, Adams MA, Fuller GM: Regulation of fibrinogen synthesis by plasmin-derived fragments of fibrinogen and fibrin: an indirect feedback pathway. Proc Natl Acad Sci USA 1982, 79: 1530-1534. 10.1073/pnas.79.5.1530PubMed CentralView ArticlePubMedGoogle Scholar
- Languino LR, Duperray A, Joganic KJ, Fornaro M, Thornton GB, Altieri DC: Regulation of leukocyte-endothelium interaction and leukocyte transendothelial migration by intercellular adhesion molecule 1-fibrinogen recognition. Proc Natl Acad Sci USA 1995, 92: 1505-1509. 10.1073/pnas.92.5.1505PubMed CentralView ArticlePubMedGoogle Scholar
- 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. 10.1016/0092-8674(93)90367-YView ArticlePubMedGoogle Scholar
- Lishko VK, Podolnikova NP, Yakubenko VP, Yakovlev S, Medved L, Yadav SP, Ugarova TP: Multiple binding sites in fibrinogen for integrin alphaMbeta2 (Mac-1). J Biol Chem 2004, 279: 44897-44906. 10.1074/jbc.M408012200View ArticlePubMedGoogle Scholar
- Minson CT, Green DJ: Measures of vascular reactivity: prognostic crystal ball or Pandora's box? J Appl Physiol 2008, 105: 398-399. 10.1152/japplphysiol.90741.2008View ArticlePubMedGoogle Scholar
- Canobbio I, Reineri S, Sinigaglia F, Balduini C, Torti M: A role for p38 MAP kinase in platelet activation by von Willebrand factor. Thromb Haemost 2004, 91: 102-110.PubMedGoogle Scholar
- Altieri DC, Duperray A, Plescia J, Thornton GB, Languino LR: Structural recognition of a novel fibrinogen gamma chain sequence (117-133) by intercellular adhesion molecule-1 mediates leukocyte-endothelium interaction. J Biol Chem 1995, 270: 696-699. 10.1074/jbc.270.2.696View ArticlePubMedGoogle Scholar
- Flick MJ, La Jeunesse CM, Talmage KE, Witte DP, Palumbo JS, Pinkerton MD, Thornton S, Degen JL: Fibrin(ogen) exacerbates inflammatory joint disease through a mechanism linked to the integrin alphaMbeta2 binding motif. J Clin Invest 2007, 117: 3224-3235. 10.1172/JCI30134PubMed CentralView ArticlePubMedGoogle Scholar
- Adams RA, Bauer J, Flick MJ, Sikorski SL, Nuriel T, Lassmann H, Degen JL, Akassoglou K: The fibrin-derived gamma377-395 peptide inhibits microglia activation and suppresses relapsing paralysis in central nervous system autoimmune disease. J Exp Med 2007, 204: 571-2582. 10.1084/jem.20061931PubMed CentralView ArticlePubMedGoogle Scholar
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