Bench-to-bedside review: The role of C1-esterase inhibitor in sepsis and other critical illnesses

The purpose of this bench-to-bedside review is to summarize the literature relating to complement activation in sepsis and other critical illnesses and the role of C1-esterase inhibitor (C1 INH) as a potential therapy.

thrombin will aggravate the excessive infl ammatory response and further complement system activation and may be involved in the increased rate of apoptosis of lymphocytes and splenocytes, causing a marked decrease in cell numbers and resulting immunoparesis. Para doxically, the apoptosis of macrophages and neutrophils seems to be unaff ected or even delayed. Complement may also interact with the autonomic nervous system, Figure 1. The classic and alternative pathways of the complement system. The squiggly line arrows indicate that proteolytic cleavage of the molecule at the tip of the arrow has occurred; a line over a complex indicates that it is enzymatically active. Note that the nomenclature of the cleavage products of C2 is undecided. Some call the large fragment C2a and others call it C2b. Here, all small fragments are labeled 'a' and all large fragments are labeled 'b' . Hence, the C3 convertase is depicted as C4b,2b. Note that proteases associated with the mannose-binding lectin cleave C4 as well as C2. Reprinted with permission from McGraw-Hill Medical [67].
which is also dysregulated in sepsis. Further investigations are needed to establish the signifi cance of these various interactions and the identifi cation of new therapeutic targets.

C1 inhibitor
As stated above, the biological activities of C1 INH include the inactivation of a variety of proteases, includ ing complement system proteases (C1r, C1s, and MASP2), contact system proteases (factor XII, plasma, and kalli krein), intrinsic coagulation proteases (factor XI), and fi brino lytic proteases (plasmin and tissue plasminogen activator) ( Table 1) [8]. In addition to inhibiting proteases, C1 INH interacts with extracellular matrix components, the comple ment component C3, endothelial cells and leukocytes, and endotoxin. C1 INH is licensed in Europe and the US for the management of hereditary angioedema (including acute attacks and routine prophylaxis). In addition, over the past 20 years , administration of C1 INH has been associated with improved survival or correction (or both) of biochemical parameters in a variety of clinical scenarios. However, these have been predominantly lab-based studies; human investigations have been mainly at the level of individual anecdote, small case series, or small randomized controlled trials. Table 2 summarizes the research and clinical studies included in the present review.

Animal models
Sepsis and endotoxic shock C1 INH improves outcomes in animal models of sepsis. Croner and colleagues [18] reported a reduction in hepatic leukocyte-endothelial interactions in post-sinusoidal venules and an improved hepatic perfusion in lipopolysaccharide-induced sepsis in rats. Decreased leukocyte adherence and suppression of the increase in vascular permeability were also demon strated in both rats and mice treated with C1 INH [19,20]. Fischer and colleagues [21] reported improved survival in C3-deficient mice challenged with endotoxin and also demonstrated the role of complement in endotoxin clearance. In a rabbit model, combined anti-thrombin and C1 INH treatment during early endotoxic shock decreased organ fi brin deposition and improved cardiovascular stability [22]. Treatment with C1 INH also prevented the hypoxemia induced by endotoxic shock in dogs [23] and decreased cytokine production in both primates [24] and mice [25].
In a cecal ligation and puncture (CLP) models of sepsis, treatment with a single dose of C1 INH improved survival [26]. Th is improvement was enhanced by the administration of multiple doses. However, survival was not improved with a single dose at 6 hours rather than immediately after puncture but was improved when two doses were given at 3 and 9 hours after CLP. Th is fi nding led to the suggestion that the eff ectiveness of C1 INH treatment in sepsis may relate, at least in part, to its ability to enhance phagocytosis rather than inhibit the complement and contact systems.

Ischemia-reperfusion injury and acute pancreatitis
Animal studies have shown that administration of C1 INH at or just prior to reperfusion results in a reduction in myocardial injury; reduced plasma levels of creatine  kinase, troponin T, C3a, and C5a; and suppression of the endothelial expression of P-selectin and ICAM-1 (intercellular adhesion molecule-1) [27][28][29][30]. C1 INH also suppressed ischemia-reperfusion-induced apoptosis in rat myocardial cells with a possible eff ect on myocardial C3 expression [31,32].
Benefi cial eff ects of C1 INH have also been described in a number of other animal ischemia-reperfusion models, including skeletal muscle, liver, cerebral artery, and mesenteric artery occlusion. Th ese studies were reviewed in detail by Davis and colleagues [8]. More recently, C1 INH treatment reduced intestinal tissue injury, attenuated leukocyte infi ltration, and improved survival in a murine model of intestinal ischemia-reperfusion injury [33]. Th e authors proposed that C1 INH may play a direct role in the suppression of leukocyte transmigration into reperfused tissue.
Complement and kinin pathways are also activated in acute pancreatitis [34]. C1 INH treatment ameliorated the degree of histological alterations in animal models of  acute pancreatitis but did not improve survival [35]. Murine models demonstrate that C5a exerts an antiinfl ammatory eff ect in pancreatitis [36], but, to date, there is only limited evidence for the use of C1 INH in acute pancreatitis [35].

Transplantation
Antibody-mediated complement activation is implicated in transplant rejection [8,37]. C1 INH inhibited the action of porcine aortic cells incubated with human serum [38] and improved perfusion and survival of pig kidneys [39]. C1 INH has also been reported to provide some protection against injury in ex vivo mouse lung or pig lung whole human blood models [40][41][42]. C1 INH has also suppressed hyperacute and acute vascular rejection in porcine models [43,44].

Thermal injury
Th ermal injury is associated with profound immunologic alterations and activation of the complement system [45]. Th is association may play an important role in the development of capillary leak syndrome and infl am matory tissue destruction. In a porcine model, C1 INH treatment reduced complement activation by 50% and reduced edema formation [45]. Th ese data suggest that C1 INH therapy may have a potential therapeutic role in the management of burn injury.

Pneumococcal meningitis
Complement-mediated opsonic activity may appear in the cerebrospinal fl uid of patients who recover completely from bacterial meningitis and may infl uence the outcome of the disease [46]. In a rat model of meningitis, treatment with C1 INH was associated with reduced clinical severity, a less pronounced infl ammatory infi ltrate around the meninges, and lower brain levels of proinfl ammatory cytokines and chemokines. C1 INH treatment also increased bacterial clearance, possibly through an upregulation of CR3 [47]. Th ese data suggest that C1 INH may be a useful agent in the treatment of pneumococcal meningitis. However, further clinical studies are needed in light of data from clinical studies of the monoclonal antibody eculizumab (that binds with high affi nity to the complement protein C5) suggesting an increased risk of sepsis necessitating meningococcal vaccination 2 weeks prior to treatment [48].

Clinical studies Sepsis and endotoxic shock
Th ere are limited clinical data on the use of C1 INH in human sepsis. Early studies in small numbers of patients with septic shock reported that C1 INH treatment suppressed activation of the complement and contact systems [49,50]. Th ese investigators then performed a double-blind study in 40 patients with severe sepsis or septic shock [51]. C1 INH was infused intravenously over the course of an hour, starting with 6,000 IU, followed by 3,000, 2,000, and 1,000 IU at 12-hour intervals. Patients treated with C1 INH had less severe multiple organ dysfunction and improved renal function, but there was no diff erence in mortality compared with the control group. Th e same group also reported that C1 INH reduced circulating elastase-α 1 -antitrypsin complex levels as well as complement activation and interleukin-8 release [52]. As neutrophils may contribute to the development of organ dysfunction during sepsis, the reduction in neutrophil count after C1 INH treatment may account in part for its favorable eff ect. A positive eff ect on mortality and an inhibitory eff ect of C1 INH (dose of 12,000 IU) on complement was also reported in an open-label Russian study of patients with sepsis [53].
In a recently published study, Tapisiz and colleagues [54] measured levels of C1 INH in 18 newborns who had had neonatal sepsis and who were blood culture-positive. Th ese levels were repeated 48 hours later and compared against 23 healthy controls of the same gestational age. Th e authors reported no diff erence between the two groups and no diff erence in edema formation and concluded that, in neonates, other factors are responsible for vascular leak and edema [54].

Ischemia-reperfusion injury
Bauernschmitt and colleagues [55] reported on the use of C1 INH as salvage therapy in three patients following emergency surgical revascularization after failed percutaneous transluminal coronary angioplasty. Initially, a 2,000-unit bolus of C1 INH was administered, followed by 1,000 units at 12 and 24 hours after surgery. Th e authors reported rapid hemodynamic stabilization of all three patients. C1 INH therapy instituted after thrombolytic therapy was reported by de Zwaan and colleagues [56] to diminish complement activation and reduce troponin T and creatine kinase levels in patients with myocardial infarction by 36% and 57%, respectively (P = 0.001). In a double-blind study of 57 patients undergoing emergency coronary artery bypass surgery, Th ielmann and colleagues [57] also reported a reduction in cardiac troponin levels in the subgroup of patients treated with C1 INH before 6 hours.
Th e cardioprotective eff ects of C1 INH in ST segment elevation myocardial infarction in 80 patients undergoing emergent reperfusion with coronary artery bypass grafting were investigated in an Italian double-blind study [58]. Th e authors reported statistically signifi cant diff erences between the C1 INH-treated patients and controls for cardiopulmonary bypass support (P = 0.04), administration of high-dose inotropes (P = 0.001), time to intubation (P = 0.03), and intensive care and hospital lengths of stay (P = 0.04 and 0.03, respectively). Stroke volume (P = 0.03) and cardiac index (P = 0.02) were also signifi cantly improved in the C1 INH group.

Transplantation
Nurnberger and colleagues [59] reported on the use of C1 INH in 15 patients with severe capillary leakage syndrome. Cumulative dosage was 180 units/kg body weight (initial dose: 60 units/kg followed by two doses at 30 units/kg and four doses at 15 units/kg every 12 hours). One year after bone marrow transplantation, the survival rates were 57% in the C1 INH treatment group and 14% in the control group (7 patients not treated with C1 INH) (P = 0.008). After infusion of C1 INH, the plasma levels of C5a and C4d normalized and the activity of C1 INH rose to 139% ± 10% of normal human plasma (NHP) pool (mean ± standard deviation).
Strüber and colleagues [60] reported the use of highdose C1 INH given over 2 days in two patients with graft failure either due to reperfusion injury immediately after transplantation or due to an acute rejection 2 months after double-lung transplantation. In both cases of graft failure, a capillary leak syndrome occurred with pleural eff usions of from 7 L per day to more than 10 L per day. In one case, the disturbance of gas exchange during severe reperfusion injury could not be treated eff ectively with other treatment modalities such as nitric oxide ventilation or surfactant administration. With the use of C1 INH, pleural eff usions decreased within 12 hours, leading to normal graft function within 4 days. In the second recipient, acute rejection forced the use of extracorporeal membrane oxygenation (ECMO) within 24 hours despite immunosuppressive therapy. After administration of C1 INH, pleural eff usions decreased from 19 L per day to 300 mL within 3 days of treatment. ECMO was discontinued after C1 INH treatment and the patient was extubated 2 weeks later [60].

Thermal injury
Kowal-Vern and colleagues [61] reported signifi cant decreases in C1 INH levels (P = 0.0001) in 60 patients with thermal injuries and sepsis on the fi rst day of admission, although this had increased to near-normal levels by day 5.

Conclusions
C1 INH is a multifaceted anti-infl ammatory protein that exerts its eff ects through a variety of mechanisms that may or may not depend on protease inhibition via covalent bonding [62] or other non-covalent interactions with proteins, cell surfaces, or lipids. C1 INH binds to a variety of extracellular matrix components, including type IV collagen, laminin, entactin, and fi brinogen. A non-covalent interaction with C3b inhibits formation of the alternative pathway C3 convertase, whereas an interaction with E and P selectins on endothelial cells is mediated by the Lewis(x) tetrasaccharides expressed on C1 INH. Th ese interactions result in suppression of leukocyte rolling and transmigration. A fourth interaction is the binding of C1 INH to Gram-negative bacterial endotoxin and this results in suppression of endotoxic shock by interfering with the interaction of endotoxin with its receptor complex on macrophages. Lastly, C1 INH binds directly to Gram-negative bacteria, leading to suppression of the development of sepsis, as demonstrated in the CLP model. It has been suggested that pharmacological inhibition of C5a or C5aR may be protective in sepsis [63].
As discussed above, C1 INHs are currently licensed for routine prophylaxis and treatment of angioedema attacks in adolescents and adults. C1 INHs have also been used for the treatment of RAAS (renin-angiotensin-aldosterone system) blocker-induced angioedema and acquired angioedema. Other therapeutic options for bradykinin-mediated angioedema include inhaled epi nephrine, corticosteroids, bradykinin receptor antago nists (icatibant), and the plasma kallikrein inhibitor ecallantide [9,64,65]. C1 INHs have been shown to be benefi cial not only in sepsis and Gram-negative endo toxic shock but also in vascular leak syndrome, transplant rejection ischemia-reperfusion injury, intestinal ischemia and reperfusion, pancreatitis, and burns. However, serious thrombogenesis has been reported following high-dose use of human C1 INH in neonatal patients undergoing cardiac surgery using an extracorporeal circu lation [64]. C1 INHs may also have a role in the treatment of cerebral malaria [66]. Clinical studies and well-designed dose-titrating studies are needed to clarify the levels of C1 INH needed to produce a therapeutic eff ect and to establish whether treatment with C1 INH has a place in the management of these conditions. Other infections in which the infl am matory response contri butes to mortality, such as pneumonia and peritonitis, may also be worthy of examination.