Molecular Immunology 61 (2014) 185–190
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The role and regulation of complement activation as part of the thromboinflammation elicited in cell therapies夽 Bo Nilsson a,∗ , Yuji Teramura a,b , Kristina N. Ekdahl a,c a b c
Dept. of Immunology, Genetics and Pathology (IGP), Rudbeck Laboratory, Uppsala University, SE-751 85 Uppsala, Sweden Department of Bioengineering, The University of Tokyo, 7-3-1 Hongo, Bunkyo-Ku, Tokyo 113-8656, Japan Linnæus Center of Biomaterials Chemistry, Linnæus University, SE-391 82 Kalmar, Sweden
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Article history: Received 19 May 2014 Accepted 9 June 2014 Available online 3 July 2014 Keywords: Thromboinflammation Instant blood-mediated inflammatory reaction (IBMIR) Contact system Complement Coagulation Platelets
a b s t r a c t Cell therapies in which the cells come into direct contact with blood and other body fluids are emerging treatment procedures for patients with various diseases, such as diabetes mellitus, liver insufficiency, and graft-versus-host disease. However, despite recent progress, these procedures are associated with tissue loss caused by thromboinflammatory reactions. These deleterious reactions involve the activation of the complement and coagulation cascades and platelet and leukocyte activation, ultimately resulting in clot formation and damage to the implanted cells. In this concept review, we discuss the basic mechanisms underlying the thrombininflammatory process, with special reference to the engagement of complement and emerging strategies for the therapeutic regulation of these reactions that include the use of selective systemic inhibitors and various procedures to coat the surfaces of the cells. The coating procedures may also be applied to other treatment modalities in which similar mechanisms are involved, including whole organ transplantation, treatment with biomaterials in contact with blood, and extracorporeal procedures. © 2014 Published by Elsevier Ltd.
1. Introduction
1.1. The concept of thromboinflammation
Treatments with cells or cell clusters that are infused into the circulation are emerging therapies in modern medicine. These treatments include transplantation of islets of Langerhans, hepatocyte transplantation, and treatment involving mesenchymal stem cells (MSCs) and immune cells. However, all these treatments are associated with cell damage and a significant loss of cells, leading to poor engraftment and cell function. One of several explanations for this problem is that the cells are recognized by the innate immune system, triggering complement activation and induction of thromboinflammation that damages the cells. These reactions occur whether the treatment is allogeneic, autologous, or xenogeneic. In this concept review, we will describe these three kinds of reactions and discuss in detail the role of complement in this context.
Physiologically, thromboinflammation is part of the repair process after damage and is triggered by the cascade systems and cells of the blood. The humoral innate immune system, which primarily consists of the cascade systems of the blood, plays a leading role in this context. These cascade systems consist of the complement, contact, coagulation and fibrinolysis systems. They subsequently activate platelets, leukocytes, and endothelial cells (EC), finally resulting in thrombotic and inflammatory reactions. Thromboinflammation is also an important pathophysiological mechanism in several clinical conditions, in treatments such as cell and cell cluster transplantation and therapies, and in ischemia–reperfusion injury in whole-organ transplantation. In addition, thrombotic events such as cardiac infarction, stroke and other cardiovascular conditions; rheumatic disorders (scleroderma, SLE, and antiphospholipid syndrome); and procedures involving pharmacological delivery systems (e.g., lipid micelles, polymers, and virus vectors), biomaterial implants (e.g., joint replacements and scaffolds for tissue engineering), and extracorporeal treatments (hemodialysis, cardiopulmonary bypass) are also associated with thromobinflammation. As part of the innate immune system, these cascade systems include several recognition molecules that can distinguish self
夽 This article belongs to SI: XXV ICW Rio 2014. ∗ Corresponding author. Tel.: +46 18 6113977/+46 709423977; fax: +46 18 553149. E-mail address: [email protected] (B. Nilsson). http://dx.doi.org/10.1016/j.molimm.2014.06.009 0161-5890/© 2014 Published by Elsevier Ltd.
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Fig. 1. The concept of thromboinflammation. Recognition molecules initiate activation of the different cascade systems of the blood, resulting in the generation of thrombin (which elicits platelet activation and fibrin formation) and the anaphylatoxins C3a and C5a (which activate PMNs and monocytes). The ultimate result is a thrombus, in which activated leukocytes and platelets are trapped in a fibrin network. FXII = coagulation factor XII; TF = tissue factor; FVII = coagulation factor VII; MBL = mannan-binding lectin; CRP = C-reactive protein.
from non-self (Fig. 1) (Ricklin et al., 2010). Mannan-binding lectin (MBL) and Ficolins-1, -2 and -3 recognize carbohydrates and other non-self patterns, thereby initiating the lectin pathway of complement (LP). C1q of the classical pathway of complement (CP) binds to immunoglobulins; negatively charged molecules such as DNA, LPS, and heparin; and to pentraxins such as C-reactive protein (CRP) and pentraxin-3, which recognize phospholipid structures. Recognition can also be achieved by properdin in the alternative pathway (AP). The primary recognition molecule in the contact system is factor XII (FXII), which is contact-activated by negatively charged molecules such as LPS and glycose-aminoglycans or negatively charged surfaces of foreign (bio)materials. Indirect recognition molecules that signal altered self are chemo/cytokines and tissue factor (TF), expressed by cells under stress (e.g., during ischemia/hypoxia and inflammation) (Fig. 1). We have recently reviewed the crosstalk between the different cascade systems and discussed how this crosstalk leads to thromboinflammation in various diseases and treatment modalities (Nilsson et al., 2010, 2011; Ricklin et al., 2010). There are many pathways of the various cascade systems that have been reported to interact with other systems. For instance, it was recently shown
that coagulation factor FXa and thrombin are able to cleave a number of complement components (albeit under extreme conditions; Amara et al., 2010). FXIIa of the contact system has been reported to cleave C1r (Ghebrehiwet et al., 1981), and C1-INH is a shared protease inhibitor of the contact and complement systems. However, the most abundant crosstalk between the cascade systems occurs via cell activation. The complement system triggers leukocyte activation and a number of activation products, such as the anaphylatoxins C3a and C5a, the terminal pathway complex sC5b9, and C3 fragments C3b, iC3b, and C3dg. Leukocytes and EC are also activated by bradykinin, produced by the contact system, and platelets are activated by thrombin, the major activation product of the contact/coagulation systems. Examples of crosstalk are: activated platelets triggering complement activation by chondroitin sulfate (Hamad et al., 2008); soluble C5b-9 activating platelets via immune complexes, for example (Sims and Wiedmer, 1991); C5a up-regulating tissue factor (TF) on monocytes and PMNs (Osterud and Bjorklid, 2012; Ritis et al., 2006); and platelets eliciting contact system activation by activating FXII via fibrin formation (Back et al., 2009, 2010, 2013) and the coagulation system via the LP (Gulla et al., 2010). In summary, one can state that thromboinflammation
Fig. 2. The PEG-phospholipid construct that inserts into the membrane of cells after incubation of the construct (micelle) with the target cells.
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engaging all of the described components can be triggered by each of the individual cascade system pathways alone.
1.2. The instant blood-mediated inflammatory reaction (IBMIR) in cell therapies We have previously characterized a thromboinflammatory reaction, the instant blood-mediated inflammatory reaction (IBMIR), which is elicited both in vitro and in vivo when pancreatic islets come in direct contact with blood during clinical intraportal islet transplantation (Bennet et al., 1999; Johansson et al., 2005; Moberg et al., 2002a). This reaction is characterized by rapid activation of the coagulation and complement systems, activation and binding of platelets to the islet surface, and infiltration of leukocytes into the islets. After 15 min, the islets are encapsulated in clots, and after an hour, most of the islets are infiltrated by numerous leukocytes (mainly PMNs), resulting in a disruption of islet integrity and islet loss (Bennet et al., 1999; Moberg et al., 2005). The occurrence of thromboinflammation in vivo was originally demonstrated in the allogeneic pig models of intraportal islet transplantation (Bennet et al., 1999; Lamblin et al., 2001). Allogeneic IBMIR occurring during clinical islet transplantation was also established in patients who had undergone clinical islet transplantation (Johansson et al., 2005; Moberg et al., 2002b). A time-restricted peak in the levels of thrombin-antithrombin (TAT) complexes and generation of D-dimer after islet infusion reflects an ongoing clotting process that is followed by an increase in C-peptide (a proinsulin marker), indicating that the islet cells have been damaged. The IBMIR is also responsible for tissue loss in xenogeneic islet transplantation. Its damaging effects have been established in both in vitro and in vivo models of intraportal islet transplantation: pig islets to mice (Goto et al., 2004) and pig to monkey (Bennet et al., 2000; Kirchhof et al., 2004). The reaction is very similar to that in the allogeneic setting. As in allogeneic IBMIR, the key factor seems to be TF, which has been demonstrated by Ji et al. (2011) using neonatal islet cell clusters (NICC) and siRNA. A reduction in TF expression leads to a significant reduction in the formation of blood clots, platelet activation, thrombin generation, and complement activation after exposure of the NICC to human ABO-compatible blood in vitro. Interestingly, the IBMIR also occurs in autologous islet transplantation (Naziruddin et al., 2014). A reaction identical to that occurring in allogeneic clinical islet transplantation (CIT) was recently described in patients with pancreatitis who were treated with resection of the pancreas followed by autologous islet transplantation. The occurrence of this reaction shows that the eliciting mechanism of thromboinflammation is not the result of any specific individual difference between the donor and recipient, such as in HLA phenotype. Recent studies have shown that the IBMIR occurs not only in CIT but also in clinical hepatocyte transplantation and during mesenchymal stem/stromal cell (MSC) therapy to attenuate graftversus-host disease. The inflammatory and thrombotic reaction in hepatocyte transplantation is very similar to that during clinical islet transplantation (Gustavson et al., 2005). We saw all the hallmarks of the IBMIR: coagulation and complement activation, followed by activation of platelets and leukocytes both in vitro and during the infusion of hepatocytes into a newborn child with FVII deficiency. Similarly, human MSCs have been shown to trigger complement activation when exposed to ABO-compatible human blood in a whole-blood model and in vivo (Moll et al., 2011, 2012), suggesting that the IBMIR is a reaction common to most cell therapies in which cells that normally do not come into contact with blood are infused into the circulation.
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1.3. Mechanisms underlying thromboinflammation in cell therapies Several mechanisms are involved in thromboinflammation induced by cell therapies: (1) The cells express structures that can be recognized by natural antibodies, leading to complement activation (Titus et al., 2003; Tjernberg et al., 2008). Direct antibody-mediated complement activation on the cell surface (C1q, C4, C3, C9) of pancreatic islets has been demonstrated in vitro by several investigators (Titus et al., 2003; Tjernberg et al., 2008). When exposed to ABOcompatible human plasma, human islets release C peptide. This release is attenuated by the complement C3 inhibitor compstatin, indicating that complement attack is a major cause of the damage to the islets that occurs during the IBMIR (Tjernberg et al., 2008).Antibodies have an even greater impact on xenogeneic islet transplantation, despite the fact that islets do not express very high levels of the Gal antigen. Both antibodies and complement components (C1q, C4, C3, C9) are found on the surface of porcine islets after exposure to non-human primate or human plasma (Goto et al., 2008; Kirchhof et al., 2004). In a study employing non-human primates, infusion of porcine islets was immediately followed by a remarkable release of insulin from the islets that could be totally blocked by complement inhibitor TP-10 (sCR1). This result clearly demonstrated that the porcine islets were severely damaged by complement attack (Bennet et al., 2000). (2) The cells lack membrane-bound regulators (e.g., DAF, MCP, CD59, heparan sulfate), facilitating coagulation and complement activation (Bennet et al., 2000; Moll et al., 2011; Tjernberg et al., 2008). The lack of DAF (CD55) and MCP (CD46) in particular, as well as low levels of CD59, on cells not normally exposed to blood makes these cells extremely vulnerable to blood contact and contributes to AP amplification. Also, the lack of a glycocalyx containing heparan sulfate leads to an increased sensitivity to contact and coagulation activation. (3) The cells expose extracellular matrix proteins and have an inflammatory phenotype, which leads to clotting (Johansson et al., 2003). An inflammatory phenotype is a common feature of grafts in whole-organ transplantation and cell transplantation. In whole-organ transplantation, this phenotype gives rise to reperfusion injury, and in cell transplantation it contributes to the IBMIR. Of great importance for the induction of this phenotype is the brain death of the donor, in combination with warm ischemia. The graft is also subject to considerable stress during its procurement, transportation, isolation, and culture, during which hypoxia often occurs. Several groups have studied the inflammatory response in a brain-death model in rats (Contreras et al., 2003; Saito et al., 2010) and found a significant up-regulation of TF, MCP-1, TNF-alpha, IL-1-beta, and IL-6, concomitant with higher nuclear activities of NF-kappaB, p50, c-Jun, and ATF-2. Blockade of TF inhibits most of the immediate thromboinflammatory reactions induced by the pancreatic islets in contact with blood. Complement activation is also elicited by the clotting process induced by the therapeutic cells and can be inhibited by thrombin inhibitors (Ozmen et al., 2002). The cause of this complement activation has been suggested to be the release of chondroitin sulfate from the activated platelets (Hamad et al., 2008) as well as plateletbound P-selectin and/or properdin (Del Conde et al., 2005; Saggu et al., 2013). In addition to directly damaging cells, complement activation leads to the formation of the anaphylatoxins C3a and C5a (the more potent), which trigger inflammation. Tokodai et al. showed that after intraportal transplantation of syngeneic rat islet
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grafts, TAT complex generation was significantly suppressed in animals given C5a inhibitory peptide (C5aIP), and both the curative rate and glucose tolerance were significantly improved. Expression of TF on granulocytes in recipient livers was up-regulated 1 h after islet infusion and significantly suppressed by C5aIP, indicating crosstalk between leukocytes and the complement and coagulation cascades in the recipients’ livers (Tokodai et al., 2010).
stimulation, even after 7 days. The polymer membrane structure surrounding the islets and cells was well maintained for at least 30 days. In addition, the membrane formed showed much lower thrombogenicity and inhibited complement activation upon exposure to human whole blood and serum.
1.4. Therapeutic regulation of thromboinflammation
Few attempts have been made to reduce thrombinflammation in clinical cell therapies. The most rigorous evaluation thus far was a recent study that evaluated peritransplant intensive insulin and heparin treatment in clinical islet transplantation. The use of insulin and intensive heparin infusions peritransplant emerged as significant factors associated with insulin independence, likely reflecting a mitigation of the IBMIR’s effects (Koh et al., 2010). In xenotranplantation, more evidence is available to indicate that attenuating the IBMIR via anti-coagulant and anti-complement treatment preserves the graft: Rood and coworkers initiated IBMIRspecific treatment in two groups of monkeys, one in which cobra venom factor completely inhibited complement activity, and a second given low molecular weight dextran sulfate (LMW-DS). A significant effect was obtained with both treatments, but the authors concluded that a combination was needed (Rood et al., 2007). This result supports our previous proposal that combining low molecular dextran sulfate with an anti-complement drug (e.g., compstatin) would constitute an optimal approach to preventing the IBMIR in xenogeneic islet transplantation (Goto et al., 2008).
1.4.1. Systemic administration A wide variety of procedures and techniques to inhibit thromboinflammation has been reported. An obvious target for intervention is TF. Consistent with this approach, a monoclonal anti-TF antibody (CNTO859) has been shown to enhance engraftment in a non-human primate marginal mass model (Berman et al., 2007). In addition to direct inhibition of TF, inhibition of the coagulation system at various stages of activation, for example by using the thrombin inhibitor Melagatran (Ozmen et al., 2002), active siteinactivated FVII (iFVIIa) (Moberg et al., 2002a), N-acetylcysteine (Beuneu et al., 2007), activated protein C (APC) (Contreras et al., 2004), anti-GP IIb/IIIa in combination with APC (Akima et al., 2009) or thrombomodulin (Cui et al., 2009), is able inhibit thromboinflammation in vitro or in vivo. As already mentioned, the complement inhibitor compstatin inhibits the release of C-peptide from pancreatic cells exposed to human plasma (Tjernberg et al., 2008). 1.4.2. Cell-surface coatings A novel technique for conjugating macromolecular heparin complexes to cell surfaces has been described: It is based on the dual affinity of avidin-expressing binding sites for both biotin and a macromolecular complex of heparin (Cabric et al., 2008). This heparin coating has been shown to provide protection against the IBMIR, both in vitro and in an allogeneic porcine model of clinical islet transplantation (Cabric et al., 2007). The coating can also be used to bind heparin-binding growth factors before transplantation in order to improve angiogenesis and islet engraftment (Cabric et al., 2010). A new technique for PEG modification of the islets of Langerhans has been shown to improve graft survival immediately after intraportal transplantation into mice with streptozotocininduced diabetes (Teramura and Iwata, 2009). The graft survival of PEG-modified islets, as compared to bare islets, was significantly prolonged in the livers of diabetic mice. This technique has been used to conjugate proteins and cells to the islet surface. For instance, urokinase, thrombomodulin, and soluble complement receptor 1 (sCR1) have been bound to the islet surface (Chen et al., 2011; Luan et al., 2011). We have taken this technique further by using a PEG-lipid construct and conjugating a factor H-binding peptide and an ADP-degrading enzyme to cell surfaces (Nilsson et al., 2013) (Fig. 2). When exposed to human whole blood, factor H was specifically recruited to the modified surfaces and inhibited complement attack. In addition, activation of platelets and coagulation was efficiently attenuated as a result of ADP degradation. Thus, by inhibiting thromboinflammation with a multicomponent approach, we created a hybrid surface with the potential to greatly reduce incompatibility reactions involving biomaterials and transplantation. A further advancement was to encapsulate the islets and cells within a stable ultra-thin polymer membrane using poly(ethylene glycol)-conjugated phospholipid bearing a maleimide group (MalPEG-lipids) and multiple interactive polymers (e.g., 4-arm PEG-Mal and 8-arm PEG-SH) (Teramura et al., 2013). Microencapsulation of islets with the polymer membranes, which showed semipermeability, did not impair insulin release in response to glucose
1.5. Attenuation of thrombinflammation in clinical cell therapies
2. Conclusion and future perspectives As shown in vitro and in vivo, thromboinflammation can severely damage therapeutic cells exposed to a recipient’s blood. In addition to the direct damage caused by the thrombinflammation (particularly complement activation), inflammation at the implantation site propagates cell destruction and provides a powerful “danger signal” to enhance antigen presentation, accelerating and reinforcing cell-mediated immune responses. Thus, the thromboinflammation is a target for attenuation in the presently available cell therapies and for further investigation and evaluation in other future clinical cell-based therapies, such as the infusion of stem cell-derived cells. Acknowledgments This work was supported by the European Union’s Seventh Framework Programme FP7/2007-2013/ under RTD grant agreement No. 602699 (DIREKT) and the strategic program StemTherapy, and by grants from the Swedish Research Council (VR) 201365X-05647-34-4, 2009-4675, and from the Swedish Foundation for International Cooperation in Research and Higher Education (STINT). The authors have no financial disclosures to report. References Akima, S., Hawthorne, W.J., Favaloro, E., Patel, A., Blyth, K., Mudaliar, Y., Chapman, J.R., O’Connell, P.J., 2009. Tirofiban and activated protein C synergistically inhibit the Instant Blood Mediated Inflammatory Reaction (IBMIR) from allogeneic islet cells exposure to human blood. Am. J. Transplant. 9, 1533–1540 (official journal of the American Society of Transplantation and the American Society of Transplant Surgeons). Amara, U., Flierl, M.A., Rittirsch, D., Klos, A., Chen, H., Acker, B., Bruckner, U.B., Nilsson, B., Gebhard, F., Lambris, J.D., Huber-Lang, M., 2010. Molecular intercommunication between the complement and coagulation systems. J. Immunol. 185, 5628–5636. Back, J., Lang, M.H., Elgue, G., Kalbitz, M., Sanchez, J., Ekdahl, K.N., Nilsson, B., 2009. Distinctive regulation of contact activation by antithrombin and
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Teramura, Y., Oommen, O.P., Olerud, J., Hilborn, J., Nilsson, B., 2013. Microencapsulation of cells, including islets, within stable ultra-thin membranes of maleimide-conjugated PEG-lipid with multifunctional crosslinkers. Biomaterials 34, 2683–2693. Titus, T.T., Horton, P.J., Badet, L., Handa, A., Chang, L., Agarwal, A., McShane, P., Giangrande, P., Gray, D.W., 2003. Adverse outcome of human islet-allogeneic blood interaction. Transplantation 75, 1317– 1322.
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Molecular Immunology journal homepage: www.elsevier.com/locate/molimm
The role and regulation of complement activation as part of the thromboinflammation elicited in cell therapies夽 Bo Nilsson a,∗ , Yuji Teramura a,b , Kristina N. Ekdahl a,c a b c
Dept. of Immunology, Genetics and Pathology (IGP), Rudbeck Laboratory, Uppsala University, SE-751 85 Uppsala, Sweden Department of Bioengineering, The University of Tokyo, 7-3-1 Hongo, Bunkyo-Ku, Tokyo 113-8656, Japan Linnæus Center of Biomaterials Chemistry, Linnæus University, SE-391 82 Kalmar, Sweden
a r t i c l e
i n f o
Article history: Received 19 May 2014 Accepted 9 June 2014 Available online 3 July 2014 Keywords: Thromboinflammation Instant blood-mediated inflammatory reaction (IBMIR) Contact system Complement Coagulation Platelets
a b s t r a c t Cell therapies in which the cells come into direct contact with blood and other body fluids are emerging treatment procedures for patients with various diseases, such as diabetes mellitus, liver insufficiency, and graft-versus-host disease. However, despite recent progress, these procedures are associated with tissue loss caused by thromboinflammatory reactions. These deleterious reactions involve the activation of the complement and coagulation cascades and platelet and leukocyte activation, ultimately resulting in clot formation and damage to the implanted cells. In this concept review, we discuss the basic mechanisms underlying the thrombininflammatory process, with special reference to the engagement of complement and emerging strategies for the therapeutic regulation of these reactions that include the use of selective systemic inhibitors and various procedures to coat the surfaces of the cells. The coating procedures may also be applied to other treatment modalities in which similar mechanisms are involved, including whole organ transplantation, treatment with biomaterials in contact with blood, and extracorporeal procedures. © 2014 Published by Elsevier Ltd.
1. Introduction
1.1. The concept of thromboinflammation
Treatments with cells or cell clusters that are infused into the circulation are emerging therapies in modern medicine. These treatments include transplantation of islets of Langerhans, hepatocyte transplantation, and treatment involving mesenchymal stem cells (MSCs) and immune cells. However, all these treatments are associated with cell damage and a significant loss of cells, leading to poor engraftment and cell function. One of several explanations for this problem is that the cells are recognized by the innate immune system, triggering complement activation and induction of thromboinflammation that damages the cells. These reactions occur whether the treatment is allogeneic, autologous, or xenogeneic. In this concept review, we will describe these three kinds of reactions and discuss in detail the role of complement in this context.
Physiologically, thromboinflammation is part of the repair process after damage and is triggered by the cascade systems and cells of the blood. The humoral innate immune system, which primarily consists of the cascade systems of the blood, plays a leading role in this context. These cascade systems consist of the complement, contact, coagulation and fibrinolysis systems. They subsequently activate platelets, leukocytes, and endothelial cells (EC), finally resulting in thrombotic and inflammatory reactions. Thromboinflammation is also an important pathophysiological mechanism in several clinical conditions, in treatments such as cell and cell cluster transplantation and therapies, and in ischemia–reperfusion injury in whole-organ transplantation. In addition, thrombotic events such as cardiac infarction, stroke and other cardiovascular conditions; rheumatic disorders (scleroderma, SLE, and antiphospholipid syndrome); and procedures involving pharmacological delivery systems (e.g., lipid micelles, polymers, and virus vectors), biomaterial implants (e.g., joint replacements and scaffolds for tissue engineering), and extracorporeal treatments (hemodialysis, cardiopulmonary bypass) are also associated with thromobinflammation. As part of the innate immune system, these cascade systems include several recognition molecules that can distinguish self
夽 This article belongs to SI: XXV ICW Rio 2014. ∗ Corresponding author. Tel.: +46 18 6113977/+46 709423977; fax: +46 18 553149. E-mail address: [email protected] (B. Nilsson). http://dx.doi.org/10.1016/j.molimm.2014.06.009 0161-5890/© 2014 Published by Elsevier Ltd.
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Fig. 1. The concept of thromboinflammation. Recognition molecules initiate activation of the different cascade systems of the blood, resulting in the generation of thrombin (which elicits platelet activation and fibrin formation) and the anaphylatoxins C3a and C5a (which activate PMNs and monocytes). The ultimate result is a thrombus, in which activated leukocytes and platelets are trapped in a fibrin network. FXII = coagulation factor XII; TF = tissue factor; FVII = coagulation factor VII; MBL = mannan-binding lectin; CRP = C-reactive protein.
from non-self (Fig. 1) (Ricklin et al., 2010). Mannan-binding lectin (MBL) and Ficolins-1, -2 and -3 recognize carbohydrates and other non-self patterns, thereby initiating the lectin pathway of complement (LP). C1q of the classical pathway of complement (CP) binds to immunoglobulins; negatively charged molecules such as DNA, LPS, and heparin; and to pentraxins such as C-reactive protein (CRP) and pentraxin-3, which recognize phospholipid structures. Recognition can also be achieved by properdin in the alternative pathway (AP). The primary recognition molecule in the contact system is factor XII (FXII), which is contact-activated by negatively charged molecules such as LPS and glycose-aminoglycans or negatively charged surfaces of foreign (bio)materials. Indirect recognition molecules that signal altered self are chemo/cytokines and tissue factor (TF), expressed by cells under stress (e.g., during ischemia/hypoxia and inflammation) (Fig. 1). We have recently reviewed the crosstalk between the different cascade systems and discussed how this crosstalk leads to thromboinflammation in various diseases and treatment modalities (Nilsson et al., 2010, 2011; Ricklin et al., 2010). There are many pathways of the various cascade systems that have been reported to interact with other systems. For instance, it was recently shown
that coagulation factor FXa and thrombin are able to cleave a number of complement components (albeit under extreme conditions; Amara et al., 2010). FXIIa of the contact system has been reported to cleave C1r (Ghebrehiwet et al., 1981), and C1-INH is a shared protease inhibitor of the contact and complement systems. However, the most abundant crosstalk between the cascade systems occurs via cell activation. The complement system triggers leukocyte activation and a number of activation products, such as the anaphylatoxins C3a and C5a, the terminal pathway complex sC5b9, and C3 fragments C3b, iC3b, and C3dg. Leukocytes and EC are also activated by bradykinin, produced by the contact system, and platelets are activated by thrombin, the major activation product of the contact/coagulation systems. Examples of crosstalk are: activated platelets triggering complement activation by chondroitin sulfate (Hamad et al., 2008); soluble C5b-9 activating platelets via immune complexes, for example (Sims and Wiedmer, 1991); C5a up-regulating tissue factor (TF) on monocytes and PMNs (Osterud and Bjorklid, 2012; Ritis et al., 2006); and platelets eliciting contact system activation by activating FXII via fibrin formation (Back et al., 2009, 2010, 2013) and the coagulation system via the LP (Gulla et al., 2010). In summary, one can state that thromboinflammation
Fig. 2. The PEG-phospholipid construct that inserts into the membrane of cells after incubation of the construct (micelle) with the target cells.
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engaging all of the described components can be triggered by each of the individual cascade system pathways alone.
1.2. The instant blood-mediated inflammatory reaction (IBMIR) in cell therapies We have previously characterized a thromboinflammatory reaction, the instant blood-mediated inflammatory reaction (IBMIR), which is elicited both in vitro and in vivo when pancreatic islets come in direct contact with blood during clinical intraportal islet transplantation (Bennet et al., 1999; Johansson et al., 2005; Moberg et al., 2002a). This reaction is characterized by rapid activation of the coagulation and complement systems, activation and binding of platelets to the islet surface, and infiltration of leukocytes into the islets. After 15 min, the islets are encapsulated in clots, and after an hour, most of the islets are infiltrated by numerous leukocytes (mainly PMNs), resulting in a disruption of islet integrity and islet loss (Bennet et al., 1999; Moberg et al., 2005). The occurrence of thromboinflammation in vivo was originally demonstrated in the allogeneic pig models of intraportal islet transplantation (Bennet et al., 1999; Lamblin et al., 2001). Allogeneic IBMIR occurring during clinical islet transplantation was also established in patients who had undergone clinical islet transplantation (Johansson et al., 2005; Moberg et al., 2002b). A time-restricted peak in the levels of thrombin-antithrombin (TAT) complexes and generation of D-dimer after islet infusion reflects an ongoing clotting process that is followed by an increase in C-peptide (a proinsulin marker), indicating that the islet cells have been damaged. The IBMIR is also responsible for tissue loss in xenogeneic islet transplantation. Its damaging effects have been established in both in vitro and in vivo models of intraportal islet transplantation: pig islets to mice (Goto et al., 2004) and pig to monkey (Bennet et al., 2000; Kirchhof et al., 2004). The reaction is very similar to that in the allogeneic setting. As in allogeneic IBMIR, the key factor seems to be TF, which has been demonstrated by Ji et al. (2011) using neonatal islet cell clusters (NICC) and siRNA. A reduction in TF expression leads to a significant reduction in the formation of blood clots, platelet activation, thrombin generation, and complement activation after exposure of the NICC to human ABO-compatible blood in vitro. Interestingly, the IBMIR also occurs in autologous islet transplantation (Naziruddin et al., 2014). A reaction identical to that occurring in allogeneic clinical islet transplantation (CIT) was recently described in patients with pancreatitis who were treated with resection of the pancreas followed by autologous islet transplantation. The occurrence of this reaction shows that the eliciting mechanism of thromboinflammation is not the result of any specific individual difference between the donor and recipient, such as in HLA phenotype. Recent studies have shown that the IBMIR occurs not only in CIT but also in clinical hepatocyte transplantation and during mesenchymal stem/stromal cell (MSC) therapy to attenuate graftversus-host disease. The inflammatory and thrombotic reaction in hepatocyte transplantation is very similar to that during clinical islet transplantation (Gustavson et al., 2005). We saw all the hallmarks of the IBMIR: coagulation and complement activation, followed by activation of platelets and leukocytes both in vitro and during the infusion of hepatocytes into a newborn child with FVII deficiency. Similarly, human MSCs have been shown to trigger complement activation when exposed to ABO-compatible human blood in a whole-blood model and in vivo (Moll et al., 2011, 2012), suggesting that the IBMIR is a reaction common to most cell therapies in which cells that normally do not come into contact with blood are infused into the circulation.
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1.3. Mechanisms underlying thromboinflammation in cell therapies Several mechanisms are involved in thromboinflammation induced by cell therapies: (1) The cells express structures that can be recognized by natural antibodies, leading to complement activation (Titus et al., 2003; Tjernberg et al., 2008). Direct antibody-mediated complement activation on the cell surface (C1q, C4, C3, C9) of pancreatic islets has been demonstrated in vitro by several investigators (Titus et al., 2003; Tjernberg et al., 2008). When exposed to ABOcompatible human plasma, human islets release C peptide. This release is attenuated by the complement C3 inhibitor compstatin, indicating that complement attack is a major cause of the damage to the islets that occurs during the IBMIR (Tjernberg et al., 2008).Antibodies have an even greater impact on xenogeneic islet transplantation, despite the fact that islets do not express very high levels of the Gal antigen. Both antibodies and complement components (C1q, C4, C3, C9) are found on the surface of porcine islets after exposure to non-human primate or human plasma (Goto et al., 2008; Kirchhof et al., 2004). In a study employing non-human primates, infusion of porcine islets was immediately followed by a remarkable release of insulin from the islets that could be totally blocked by complement inhibitor TP-10 (sCR1). This result clearly demonstrated that the porcine islets were severely damaged by complement attack (Bennet et al., 2000). (2) The cells lack membrane-bound regulators (e.g., DAF, MCP, CD59, heparan sulfate), facilitating coagulation and complement activation (Bennet et al., 2000; Moll et al., 2011; Tjernberg et al., 2008). The lack of DAF (CD55) and MCP (CD46) in particular, as well as low levels of CD59, on cells not normally exposed to blood makes these cells extremely vulnerable to blood contact and contributes to AP amplification. Also, the lack of a glycocalyx containing heparan sulfate leads to an increased sensitivity to contact and coagulation activation. (3) The cells expose extracellular matrix proteins and have an inflammatory phenotype, which leads to clotting (Johansson et al., 2003). An inflammatory phenotype is a common feature of grafts in whole-organ transplantation and cell transplantation. In whole-organ transplantation, this phenotype gives rise to reperfusion injury, and in cell transplantation it contributes to the IBMIR. Of great importance for the induction of this phenotype is the brain death of the donor, in combination with warm ischemia. The graft is also subject to considerable stress during its procurement, transportation, isolation, and culture, during which hypoxia often occurs. Several groups have studied the inflammatory response in a brain-death model in rats (Contreras et al., 2003; Saito et al., 2010) and found a significant up-regulation of TF, MCP-1, TNF-alpha, IL-1-beta, and IL-6, concomitant with higher nuclear activities of NF-kappaB, p50, c-Jun, and ATF-2. Blockade of TF inhibits most of the immediate thromboinflammatory reactions induced by the pancreatic islets in contact with blood. Complement activation is also elicited by the clotting process induced by the therapeutic cells and can be inhibited by thrombin inhibitors (Ozmen et al., 2002). The cause of this complement activation has been suggested to be the release of chondroitin sulfate from the activated platelets (Hamad et al., 2008) as well as plateletbound P-selectin and/or properdin (Del Conde et al., 2005; Saggu et al., 2013). In addition to directly damaging cells, complement activation leads to the formation of the anaphylatoxins C3a and C5a (the more potent), which trigger inflammation. Tokodai et al. showed that after intraportal transplantation of syngeneic rat islet
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grafts, TAT complex generation was significantly suppressed in animals given C5a inhibitory peptide (C5aIP), and both the curative rate and glucose tolerance were significantly improved. Expression of TF on granulocytes in recipient livers was up-regulated 1 h after islet infusion and significantly suppressed by C5aIP, indicating crosstalk between leukocytes and the complement and coagulation cascades in the recipients’ livers (Tokodai et al., 2010).
stimulation, even after 7 days. The polymer membrane structure surrounding the islets and cells was well maintained for at least 30 days. In addition, the membrane formed showed much lower thrombogenicity and inhibited complement activation upon exposure to human whole blood and serum.
1.4. Therapeutic regulation of thromboinflammation
Few attempts have been made to reduce thrombinflammation in clinical cell therapies. The most rigorous evaluation thus far was a recent study that evaluated peritransplant intensive insulin and heparin treatment in clinical islet transplantation. The use of insulin and intensive heparin infusions peritransplant emerged as significant factors associated with insulin independence, likely reflecting a mitigation of the IBMIR’s effects (Koh et al., 2010). In xenotranplantation, more evidence is available to indicate that attenuating the IBMIR via anti-coagulant and anti-complement treatment preserves the graft: Rood and coworkers initiated IBMIRspecific treatment in two groups of monkeys, one in which cobra venom factor completely inhibited complement activity, and a second given low molecular weight dextran sulfate (LMW-DS). A significant effect was obtained with both treatments, but the authors concluded that a combination was needed (Rood et al., 2007). This result supports our previous proposal that combining low molecular dextran sulfate with an anti-complement drug (e.g., compstatin) would constitute an optimal approach to preventing the IBMIR in xenogeneic islet transplantation (Goto et al., 2008).
1.4.1. Systemic administration A wide variety of procedures and techniques to inhibit thromboinflammation has been reported. An obvious target for intervention is TF. Consistent with this approach, a monoclonal anti-TF antibody (CNTO859) has been shown to enhance engraftment in a non-human primate marginal mass model (Berman et al., 2007). In addition to direct inhibition of TF, inhibition of the coagulation system at various stages of activation, for example by using the thrombin inhibitor Melagatran (Ozmen et al., 2002), active siteinactivated FVII (iFVIIa) (Moberg et al., 2002a), N-acetylcysteine (Beuneu et al., 2007), activated protein C (APC) (Contreras et al., 2004), anti-GP IIb/IIIa in combination with APC (Akima et al., 2009) or thrombomodulin (Cui et al., 2009), is able inhibit thromboinflammation in vitro or in vivo. As already mentioned, the complement inhibitor compstatin inhibits the release of C-peptide from pancreatic cells exposed to human plasma (Tjernberg et al., 2008). 1.4.2. Cell-surface coatings A novel technique for conjugating macromolecular heparin complexes to cell surfaces has been described: It is based on the dual affinity of avidin-expressing binding sites for both biotin and a macromolecular complex of heparin (Cabric et al., 2008). This heparin coating has been shown to provide protection against the IBMIR, both in vitro and in an allogeneic porcine model of clinical islet transplantation (Cabric et al., 2007). The coating can also be used to bind heparin-binding growth factors before transplantation in order to improve angiogenesis and islet engraftment (Cabric et al., 2010). A new technique for PEG modification of the islets of Langerhans has been shown to improve graft survival immediately after intraportal transplantation into mice with streptozotocininduced diabetes (Teramura and Iwata, 2009). The graft survival of PEG-modified islets, as compared to bare islets, was significantly prolonged in the livers of diabetic mice. This technique has been used to conjugate proteins and cells to the islet surface. For instance, urokinase, thrombomodulin, and soluble complement receptor 1 (sCR1) have been bound to the islet surface (Chen et al., 2011; Luan et al., 2011). We have taken this technique further by using a PEG-lipid construct and conjugating a factor H-binding peptide and an ADP-degrading enzyme to cell surfaces (Nilsson et al., 2013) (Fig. 2). When exposed to human whole blood, factor H was specifically recruited to the modified surfaces and inhibited complement attack. In addition, activation of platelets and coagulation was efficiently attenuated as a result of ADP degradation. Thus, by inhibiting thromboinflammation with a multicomponent approach, we created a hybrid surface with the potential to greatly reduce incompatibility reactions involving biomaterials and transplantation. A further advancement was to encapsulate the islets and cells within a stable ultra-thin polymer membrane using poly(ethylene glycol)-conjugated phospholipid bearing a maleimide group (MalPEG-lipids) and multiple interactive polymers (e.g., 4-arm PEG-Mal and 8-arm PEG-SH) (Teramura et al., 2013). Microencapsulation of islets with the polymer membranes, which showed semipermeability, did not impair insulin release in response to glucose
1.5. Attenuation of thrombinflammation in clinical cell therapies
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