The complement system occupies a central role in innate immunity as part of the host defense and is an early, if not the earliest, part of the immune system to be activated. Excessive or uncontrolled complement activation may be detrimental and contribute to worse clinical outcomes (1). The complement system has been implicated in the pathogenesis of several inflammatory and immunological diseases, including systemic inflammatory response syndrome, sepsis, acute respiratory distress syndrome, multiple organ failure, and I/R injury (2-4), conditions typically seen after major trauma. Despite improvements in trauma resuscitation, the consequences of injury, shock, and coagulopathy are still evident in the high incidence of multiple organ failure in these patients.
Complement activation has been reported in trauma patients (5-7). However, little is known about the mechanisms of this activation and its clinical significance in the early phase after trauma (<30 min). Multiple strategies to modulate the complement system in critical illness have been developed and are currently in clinical trials (8). These may have therapeutic potential for trauma if the complement system is implicated in the pathogenesis of these critical postinjury complications.
Therefore, the first aim of the present study was to measure complement activation at this early stage and to identify the roles of injury severity and systemic hypoperfusion in this. If complement activation was identified, the second aim was to identify the predominant pathways involved. Finally, the third aim was to identify the clinical significance of early complement activation after trauma.
MATERIALS AND METHODS
This was a single-center, prospective, cohort study of major trauma patients presenting directly to a level 1 trauma center. The Institutional Review Board of the University of California at San Francisco approved the research protocol and granted a waiver of consent for the blood sampling as a minimal-risk intervention.
All adult trauma patients who met criteria for full trauma team activation were eligible for enrollment into the study. Patients less than 18 years old or transferred from other hospitals were immediately excluded.
As part of standard trauma management, one member of the trauma team is designated to gain i.v. access and draw blood for laboratory analyses as soon as the patient arrives in the trauma room. Access is via a 14- or 16-gauge cannula in the forearm or via a 7-French line in the femoral vein. A 10-mL research sample of blood was drawn from this line or as a separate venipuncture along with the standard trauma laboratory tests within 10 min of arrival in the emergency department. The sample was placed in a citrated tube and sent immediately to the hospital's central laboratory, where it was spun down, plasma-extracted, and stored in a −80°C freezer.
Samples were analyzed at the conclusion of the study by researchers who were blinded to all patients' data. Normal values for complement factors were determined by testing 10 randomly selected healthy volunteers. Plasma levels of the Bb fragments (Bb EIA, Quidel Corp., San Diego, Calif; normal volunteers, 0.6 ± 0.1 μg/mL) were measured to evaluate the activation of the alternative pathway of the complement system. To detect the early phase of the terminal complement activation via all three pathways, plasma levels of C3a-desArg were measured (C3a EIA, Quidel; normal volunteers, 358 ± 141 ng/mL). C3a-desArg is a stable split product of the very short-lived anaphylotoxin C3a. Plasma levels of soluble C5b-9 (sC5b-9) were measured to assess the late phase of terminal complement activation (sC5b-9 EIA, Quidel; normal volunteers, 193 ± 33 ng/mL). The membrane attack complex (MAC or terminal complement complex, C5b-9) is generated at the end of complement activation by the assembly of C5-C9 and mediates irreversible cell membrane damage and/or cellular activation associated with complement activation. Complexes formed in the absence of a target membrane bind to a naturally occurring regulatory serum protein, the S protein. The S protein binds to nascent C5b-9 complexes at the C5b-7 stage of assembly, resulting in sC5b-9, a stable, nonlytic form of the MAC.
Activation of the classical and lectin complement pathways was determined by measuring plasma levels of C4d fragments of C4 (C4d EIA, Quidel; normal volunteers, 1.1 ± 0.2 μg/mL). Plasma levels of mannose-binding lectin (MBL) were measured to determine a possible contribution of the lectin pathway by a novel mannan-based fluorochrome-linked immunoassay (paper submitted for review) using an Odyssey and/or Aerius infrared imaging system (Li Cor Biosciences, Lincoln, Nebr). Briefly, MBL-dependent binding to mannan-coated plates is quantified using a human MBL standardized sera sample curve and a fluorochrome-conjugated antihuman MBL monoclonal antibody. The linear portion of the standard curve ranges from 10 to 165 ng MBL/mL. As approximately 10% to 20% of the population is deficient in MBL (9, 10), for statistical analyses of the MBL data with injury severity and base deficit, we excluded the 8 patients who had low MBL levels (<100 ng/mL) with minor injury (Injury Severity Score [ISS] <9). Finally, prothrombin fragments 1 + 2 (PF 1 + 2; Enzygnost PF 1 + 2 EIA, Dade Behring, Germany) were measured to determine thrombin formation.
Data were collected prospectively on patient demographics, the injury time, mechanism (blunt or penetrating) and severity, prehospital fluid administration, time of arrival in the trauma room, and admission vital signs. The ISS was used as a measure of the degree of tissue injury (11). An arterial blood gas was drawn at the same time as the research sample as part of the standard management of major trauma patients. The base deficit was used as a measure of the degree of tissue hypoperfusion. Admission base deficit is a clinically useful early marker of tissue hypoperfusion in trauma patients, and an admission base deficit greater than 6 mmol/L has previously been identified as predictive of worse outcome in trauma patients (12, 13).
Patients were followed until hospital discharge or death. For mortality analysis, patients surviving to hospital discharge were assumed to be still alive. Secondary outcome measures were also recorded for 28-day ventilator-free days, acute lung injury (American-European Consensus Conference definition (14)) and acute renal injury (Acute Dialysis Quality Initiative Consensus Conference definition (15)) and blood transfusions required in the first 24 h.
Data analysis was performed by the investigators. Normal-quantile plots were used to test for normal distribution. Parametric data are expressed as mean ± 95% confidence intervals. Two-group analysis was performed with a two-tailed unequal variance Student t test. Correlation was assessed by Pearson method. Multiple regression was used to test for statistical independence. A P value of 0.05 or less was chosen to represent statistical significance. Data are expressed as mean and 95% confidence intervals unless otherwise stated.
We enrolled 208 trauma patients into the study over a 15-month period. Clinical characteristics of the trauma patients are shown in Table 1. Median time from injury, defined as the time from prehospital emergency medical service activation to blood sampling, was 32 min; there was no vasopressor or colloid use, and patients received an average of 150 ± 100 mL of i.v. crystalloid before the blood specimen collection.
The complement system was activated immediately after trauma and correlated with severity of injury (Fig. 1, A and B). There was a positive correlation with the Injury Severity Score and Bb fragment levels, a marker of the activation of the alternative pathway (Fig. 1A) and with the sC5b-9 (MAC), generated during the late phase of complement activation (Fig. 1B). Complement activation has been previously demonstrated in a swine model of hemorrhagic shock (16), and we therefore examined the effect systemic hypoperfusion on complement activation. Patients with higher base deficits had significantly higher levels of Bb fragments (Fig. 1) and sC5b-9 (Fig. 1D). Multiple regression analysis identified that the effects of injury severity and tissue hypoperfusion were statistically independent for both Bb fragments (r2 = 0.22; ISS P < 0.001; BD P = 0.026) and for sC5b-9 (r2 = 0.17; ISS P < 0.001; BD P = 0.007).
To examine the alternative pathway in the activation of the complement system in patients with severe trauma, we analyzed the relationship of Bb fragments with the downstream products of complement activation, C3a-desArg and sC5b-9. We found that plasma levels of Bb fragments correlated with those of C3a-desArg and sC5b-9 (Fig. 2, A and B). As expected, there was also a significant correlation between early (C3a-desArg) and late (sC5b-9) phases of complement activation (Fig. 2C).
The alternative pathway is considered to be primarily an amplification mechanism for complement activation (17). We therefore investigated activation of the classical and lectin pathways by measuring plasma levels of C4d. As expected, plasma levels of C4d were positively correlated with levels of C3a-desArg (Fig. 3A) and sC5b-9 (Fig. 3B). Furthermore, MBL levels were positively correlated with plasma levels of C4d (Fig. 3C) and C3a-desArg (Fig. 3D). Interestingly, increasing injury severity correlated with a reduction of plasma MBL levels (ISS <9, 1,440 ± 495 ng/mL; ISS >25, 748 ± 212 ng/mL, P = 0.02; upper-lower quartile comparison), and a trend was also seen with increasing base deficit, although this did not achieve significance (BD <2.2, 1,235 ± 536; BD >7.7 mEq/L, 831 ± 251 ng/mL, P = 0.19; upper-lower quartile comparison).
It has recently been shown in mice that thrombin can activate complement by bypassing the early phase and acting as a direct C5 convertase (18, 19). We examined whether this pathway might be clinically relevant in humans. There was a positive relationship between levels of sC5b-9 and thrombin generation when C3a levels were low (Fig. 4A, C3a-desArg <606 ng/mL = lower 2 quartiles). In contrast, there was no correlation between plasma levels of C3a-desArg, Bb, or C4d and PF 1 + 2 (Fig. 4, B and C). A multiple regression analysis of all measured complement components and PF 1 + 2 identified that sC5b-9 levels were independently correlated with plasma levels of Bb, C3a, and PF 1 + 2 (P = 0.001, r2 = 0.67), and not levels of MBL or C4d.
We next investigated the clinical significance of complement activation, and in particular, if activation of the alternative pathway was associated with worse clinical outcomes. We found a direct correlation between mortality rate and plasma levels of Bb fragments and sC5b-9 (Fig. 5, A and B). Nonsurvivors had significantly higher plasma levels of Bb fragments and sC5b-9 compared with survivors (Bb fragments, 0.83 ± 0.21 vs 0.66 ± 0.04 μg/mL, P = 0.02; sC5b-9, 332 ± 94 vs. 249 ± 26 ng/mL, P = 0.04). There was no significant difference in MBL, C4d, or C3a-desArg levels between survivors and nonsurvivors. Patients who later developed organ injury (acute lung injury or acute renal failure) had significantly higher plasma levels of Bb fragments and sC5b-9 (Fig. 5, C and D). These levels were also associated with fewer ventilator-free days and increased blood transfusion requirements (sC5b-9 only) (Fig. 5, C and D).
This study identifies complement activation in the early postinjury phase (within 30 min) with the degree of activation proportional to both injury severity and systemic hypoperfusion. Complement activation proceeds primarily through the alternative pathway, but there is also evidence supporting a novel C3-independent complement activation by thrombin in humans. Previous studies have reported complement activation after trauma (6, 7). However, these studies only measured C3a and C5b-9 at a later point (2 h after injury) or included a small number of patients (6, 7). Currently, the alternative pathway is understood as an amplification mechanism for the complement system, ordinarily directly activated at low levels via an enzymatic process that results in a continuous low-level production of C3b (17). Such activation of the alternative pathway does not usually cause injury to "self-cells" due to the presence of membrane-bound and fluid phase complement regulatory proteins (20).
Thus, it is likely that complement activation early after trauma is initiated via the classical and/or lectin pathways. Consistent with the standard view, we found that plasma levels of C4d, the common split product of the classical and lectin pathway activation, correlated with the generation of downstream complement products such as C3a-desArg and sC5b-9. The complement system is known to be activated both during ischemia (16) and during reperfusion (4). In vitro studies have shown that both classical and lectin pathways are activated under hypoxia in endothelial cells (21, 22). This concept has been further supported by the results of two recent studies that showed that MBL binds to a complex of circulating immunoglobulin M and self-antigens exposed at the surface of endothelial cells during ischemia. This complex then activates the MBL-dependent portion of the lectin complement pathway (21-24). Our observation of falling MBL levels with increasing injury severity and hypoperfusion is consistent with this model.
There is no unique marker of classical pathway activation, and it is possible that, although MBL binding to the endothelium does occur, initiation of the complement pathway is primarily through the classical pathway. However, in a previous mouse study, MBL-null, but not C1q-null, mice were protected from I/R injury (23-26), suggesting that the lectin pathway is the predominant initiating pathway of complement activation after ischemia. We found a positive correlation of plasma MBL levels with C4d and C3a-desArg (Fig. 3, C and D), suggesting that patients with higher levels of MBL had higher downstream complement activation. However, levels of MBL decreased with increasing injury severity and hypoperfusion. The results are complicated by the presence of a genetic MBL deficiency affecting up to 10% to 20% of patients (9, 10). Although we did try to control for this in the statistical analysis, this is clearly not as robust as a baseline sample. We also were unable to measure endothelial-bound MBL. Nevertheless, it seems that MBL is affected by trauma and shock, and the lectin pathway may be involved in the initiation of complement activation after injury.
The complement system has been shown to be far more complex than a simple cascade system: several bypass pathways have been discovered recently (17, 27), and complement is also connected to other systems such as the coagulation network. It has recently been shown that thrombin is a C5 convertase and can independently activate the complement cascade in C3-null mice (18, 19). Our data indicate a correlation between the end product of complement generation, sC5b-9, and thrombin production in trauma patients with low levels of C3-desArg (minimal upstream activation by the alternative, classical, or lectin pathways). In contrast, the other measured complement factors (Bb fragments and C4d) did not correlate with thrombin generation. Although this was proposed as a bypass mechanism in genetic C3 deficiency, it seems that thrombin cleavage of C5 may be a clinically important mechanism of complement activation in trauma patients.
Activation of the alternative complement pathway is directly related to mortality rate, and secondary outcome measures such as acute lung injury, ventilator-free days, and acute renal injury. This finding is of importance because previous studies have shown that blocking the alternative pathway can reduce injury caused by activation of the complement while maintaining the host defense afforded by the classical and/or lectin pathways. It has recently been reported that in mice null for the factor B gene, there is a dramatic reduction in neuronal cell death after experimental brain injury (28). Consistent with this, elevated factor B levels have been found in the cerebrospinal fluid of patients with traumatic brain injury (29). The importance of the alternative pathway activation has also been demonstrated in other models of I/R injury. Mice null for the factor B gene or mice pretreated with an antibody to factor B developed substantially less functional and morphological renal damage after I/R (30). Furthermore, ischemic tubular necrosis in humans is characterized by alternative pathway activation (31). Taken together with these data, our results indicate that the alternative pathway plays a critical role in complement activation after trauma, and that this pathway might be implicated in I/R-mediated organ injury.
In summary, we have shown that complement activation is observed early after trauma and correlates with injury severity, tissue hypoperfusion, and worse clinical outcomes. The alternative pathway seems to be the central pathway, although initiation may involve the lectin or classical pathways. There is also some evidence for a novel C3-independent complement activation by thrombin after trauma in humans. Several novel complement inhibitors are being developed and have shown promising results in animal models of I/R injury (1, 8, 32). For example, pretreatment with a C5 blocking antibody has recently been shown to improve responsiveness to fluid administration, thereby maintaining hemodynamic parameters with less fluid resuscitation in an experimental model of hemorrhagic shock (32), although human studies are lacking. Further investigation of the effects of injury, hypoperfusion, and coagulation activation on the complement system may lead to new therapeutic agents for reducing organ failure and mortality rate in severe trauma.
The authors thank Meghan Levee and Aimee Grush (San Francisco General Hospital) for their assistance in conducting this study.
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