Complement in Sepsis

A. BENGTSSON 1, H. REDL 2, and G. SCHLAG 2

1 Department of Anesthesiology & Intensive Care, Sahlgren's Hospital, Gothenburg, Sweden and 2Ludwig Boltzmann Institute for Clinical and Experimental Traumatology, Donaueschingenstrasse 13, Vienna, Austria

CONTENTS ______________________________________________________ _

Complement System Network ................ 447 Complement Measurement ................... 447

Sample Preparation and Storage ........... 447 Measurement Techniques .................. 448

Complement Activation by Endotoxin and Bacteria: In Vitro Evidence .............. 449 Sepsis-Induced Complement Activation ........ 450

Complement System Network

Endotoxin, bacteria, and immune complexes lead to activation of the complement cascade. As an ef­fect of complement activation the anaphylatoxins C3a and C5a and the terminal C5b-9 complement complex are formed. The anaphylatoxins increase contraction of smooth muscles, enhance vascular permeability, and release histamine from mast cells [1-4]. Once formed in the blood, the C3a and C5a molecules are converted to spasmogenically inac­tive C3adeSArginine and C5adeSArginine derivates [5]. C5a is a potent activator of leukocytes and is che­motactically active. In addition, the desArginine form is able to stimulate neutrophil chemotaxis [5, 6]. C5a induces secretion of lysosomal enzymes from macrophages and neutrophils and may also induce interleukin and prostaglandin production from macrophages [7 -9].

Studies indicate that there is a relation between high concentrations of anaphylatoxins and the de­velopment of adult respiratory distress syndrome (ARDS) or multisystem organ failure (MOF) in pa­tients with sepsis [10-13]. However, it has also been demonstrated that ARDS appears in patients with neutropenia. These findings indicate that ac­tivation of the complement cascade and of neutro­phils are not absolute prerequisites for ARDS [14-17].

The aim of the present review is to discuss the possible effects of complement activation in sepsis.

Animal Studies .......................... 450 Human Studies .......................... 451

Effects of Complement Inactivation or Inhibition ............................... 454 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 454 References ................................. 454

Complement Measurement

Sample Preparation and Storage

The risk of developing remote organ failure due to activation of complement depends on the levels of circulating anaphylatoxins and terminal C5b-9 complement complexes and the duration of exten­sive complement activation. Death due to sepsis may be caused by severe circulatory shock or by development of ARDS. Death due to circulatory insufficiency will occur early, while ARDS and MOF will develop later in sepsis. This may indicate that ARDS and MOF are dependent on a long­standing activation of the complement cascade and that circulatory shock is more dependent on initial high plasma concentrations of anaphylatoxins and terminal C5b-9 complement complexes. This hypo­thesis may explain the existing controversy regard­ing the relations between complement activation at admission and later development of ARDS or MOE However, several investigators agree that there is a close relation between complement ac­tivation due to sepsis and shock on the one hand and a fatal outcome due to circulatory insufficiency on the other. Therefore, it is important to draw serial samples for complement activation when stu­dying its association with development of ARDS or MOE

EDTA-treated blood is used in most studies of complement activation in humans. Polybrene may

G. Schlag et al. (eds.), Pathophysiology of Shock, Sepsis, and Organ Failure© Springer-Verlag Berlin Heidelberg 1993

448 A. Bengtsson et al.

be added to the blood samples to prevent activation of the contact system, which in turn may lead to ac­tivation of complement. The plasma samples have to be stored at a temperature of -70 DC to - SO DC. A higher temperature, rethaw, or the use of serum samples leads to spontaneous activation of the complement cascade, giving falsely high values of complement split products and falsely low concen­trations of complement proteins.

Measurement Techniques

Whole Complement (CHso)

Titration of whole complement is a method de­scribed by Mayer [1S]. When the hemolytic activity of the classical pathway is determined, the whole complement activity is expressed in terms of 50070 hemolytic units per milliliter of undiluted serum. Sheep erythrocytes coated with anti sheep erythro­cyte antibodies are used. By photometric deter­mination the degree of partial hemolysis is estimat­ed as a percentage of complete hemolysis. This method for the evaluation of complement activa­tion has been used in several human and animal studies [19, 20]. For titration of the hemolytic ac­tivity of the alternative pathway (APCHso) the method described by Joiner and coworkers can be used [21].

Factor B, Factor D, Factor I, C1, C1inh, C3, C4, C5, C6, C7, CS, and C9

The concentration is plasma or serum of different complement proteins (factor B, factor D, factor I, C1, C1 inh, C3, C4, C5, C6, C7, CS, and C9) can be determined with immune techniques, i.e., radial im­mune diffusion, rocket immunoelectrophoresis, double-decker rocket immunoelectrophoresis [22 - 25]. A chromogenic assay for determining ac­tivated first complement component (classical ac­tivation) has been described by Munkvad and coworkers [26]. This method, which determines the concentrations of free activated C1, is not influenc­ed by the concentrations of C1inh.

Anaphylatoxins (C3a, C4a, and C5a)

A method to determine the complement anaphyla­toxins (C3a and C5a) was described by Hugli and Chenoweth [27]. A modification of this radioim­munoassay (RIA) method is commercially avail-

able. By mixing EDTA-plasma with HCI, C3 or C5 are denaturated and precipitated. The acid stable anaphylatoxin remains in solution. Many human studies for analysis of complement activation dur­ing the 19S0s have been performed by measuring C3a or C5a. By measuring C4a it is also possible to discriminate between classical and alternative ac­tivation [2S].

New RIA and enzyme-linked immunosorbent assay (ELISA) methods for anaphylatoxin deter­minations have been developed [29 - 32]. Klos and coworkers have developed an ELISA method for determining C3a and C5a. Like the method by Hugli and Chenoweth, this method requires a pre­cipitation step to eliminate uncleaved C3 and C5 [29]. An RIA method for C3a measurements has also been described by Lamche and coworkers [30]. In this method, separation is performed using char­coal, which abolishes the need for a second an­tibody. Another modification of the original assay for anaphylatoxin determinations has been per­formed by Hack and coworkers. In their method the plasma samples are incubated with polyethy­lene glycol to prevent interference between C3a and native C3 [31]. Native C3 is precipitated in the supernatant and can effectively be removed. All the above described procedures require precipitation steps to eliminate C3 or C5. Zilow and coworkers have developed an ELISA method with monoclo­nal antibodies reacting against a determinant on C3a which is not present on the native C3. This means that it is possible to determine C3a/ C3adesArginine without separating the native C3 prior to the determination [32]. Determinations of anaphylatoxins can normally only be used for human analysis as the antibodies normally do not cross-react with animal antigen.

An ELISA method for determining C3 activa­tion in vivo has been developed by Mollnes [33]. This method determines the formation of C3d and can be used as an indicator of early phase activa­tion. Other methods for determining C3d split pro­ducts are performed by radial immunodiffusion and electrophoretic techniques [23, 24].

Terminal C5b-9 Complement Complex

Methods for detecting and quantifying the presence of the terminal C5b-9 complement com­plex in plasma with ELISA methods have been developed during the last 10 years [34 - 36]. It has been demonstrated that the terminal C5b-9 com­plement complex appears in normal plasma [37].

Anaphylatoxin Inactivator

The anaphylatoxin in activator (AI) inactivates the complement-derived anaphylatoxins as well as bradykinin by removing the basic COOH-terminal acid residue from these peptides. One method de­scribed by Corbin and coworkers is a quantitative colorimetric assay [38, 39]. Salmine is selected as substrate because of its multiple COOH-terminal arginine residues. The colorimetric values obtained are related to micromoles of arginine released. One unit of AI has the power to release one nanomole of arginine in the test solution per minute. Patients with dengue shock syndrome have a reduced anaphylatoxin inhibitor activity in their blood [39].

Complement Activation by Endotoxin and Bacteria: In Vitro Evidence

Bacteria, protein A, virus, immune complexes, and heparin-protamine complex are known activators of the classical pathway [40-46].

Incubation of plasma with lipopolysaccharide (0.1 ~g/ml) has been shown to activate complement with the formation of C3a (Fig. 1). Polysaccharide components from the cell walls of gram-negative bacteria activate the cascade nonspecifically while lipid A is able to initiate the classical pathway [41, 45]. However, Keil and coworkers have demonstrat­ed that lipopolysaccharide (Salmonella minnesota endotoxin) induces activation of the complement cascade solely via the alternative pathway [46]. In addition, Van Deventer et al. have studied activa­tion of complement and neutrophils in relation to

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Complement in Sepsis 449

the appearance of tumor necrosis factor (TNF) in the circulation. In experimental endotoxemia in humans they demonstrated marked neutropenia shortly after increase of circulating TNF levels and at a period with no signs of activation of the com­plement cascade [47].

Numerous in vivo and in vitro studies have dem­onstrated that different types of bacteria lead to ac­tivation of complement. Such studies have shown the formation of anaphylatoxins as well as terminal C5b-9 complement complexes.

Escherichia coli bacteria (06K13 strain of viable bacteria) were incubated in fresh human serum from healthy individuals at 37 DC for 15 min with different concentrations of bacteria. The amounts of bacteria incubated were 106, 107, and 108 E. coli per milliliter plasma. This incubation gave a dose­related release of C3a, C5a, and terminal C5b-9 complement complex (Figs. 2, 3).

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450 A. Bengtsson et al.

Sepsis-Induced Complement Activation

Animal Studies

Activation of complement due to infusion of bacteria or endotoxin has been demonstrated in different animal models [48 - 54]. In the last few years animal studies using immunoassays for C3a, C4a, C5a, and terminal C5b-9 complement com­plex have been performed in some different species. Investigations where the anaphylatoxins have been determined have been performed by Hangen et al. [51] and Smedegard et al. [52]. In a Macaca primate model E. coli was infused at a dosage of tOlD/kg body weight over half an hour [51]. The E. coli infusion led to septic shock and to pulmonary edema. The mortality rate was 75%. No differences in these variables were observed in the control group. Similar results were presented by Smedegard and coworkers. In their rat model infu­sion of endotoxin and of C5adeSArgmme led to hypo­perfusion and leukocytopenia [52]. Smedegard and coworkers demonstrated increase of C3adeSArginine and C5adeSArgimne after injection of endotoxin (50 mg lipopolysaccharide/kg body weight in 15 s) in rats. They were also able to demonstrate that in­fusion of 5 /lg C5adesArginine produced similar clinical effects to those induced by lipopolysac­charide. Lipopolysaccharide and C5adesArginine in­fusion caused systemic hypotension and a decreas­ed number of polymorphonuclear leukocytes, mo­nocytes, and platelets in the circulation. Others have demonstrated that anaphylatoxins and other complement protein determinations by commerci­ally available assays can be used for analysis in primates [51]. Hangen et al. infused E. coli to Macaca primates and determined C3, C4, C5, C3adesArgmme' C4adeSArgmme' and C5adesArginine dur­ing and after E. coli infusion. The E. coli infusion resulted in severe septic shock and pulmonary edema. Seventy-five per cent of the animals died. In the septic animals reduced C3, C4, and C5 levels were found. The fall in C4 indicates that the classical pathway was activated. They also found increased C3adeSArgmme and C5adeSArginine concentra­tions in plasma soon after infusion of E. coli. These experiments performed in Macaca primates and rats demonstrate that C5a and C5adeSArgmme are of importance in the development of respirato­ry failure and shock in sepsis.

In studies regarding complement activation due to sepsis in baboons it was not possible to use com­mercial anaphylatoxin assays (C3a, C4a, and C5a) but it has been demonstrated that the method for

the determination of terminal C5b-9 complement complexes in humans also can be used for baboons. Formation of terminal C5b-9 complement com­plexes were analyzed during infusion of E. coli [53]. Blood samples for complement determinations were drawn before infusion of E. coli and regularly after the start of the E. coli infusion. Six animals received tOlD and six 5 x 108 live E. coli/kg body weight over a period of 8 h. In the septic animals the plasma concentration of terminal C5b-9 com­plement complexes were significantly increased as early as 2 h after start of infusion. After 6 h the plasma terminal C5b-9 complement complexes concentrations were elevated compared to the levels found before start of infusion and 2 h after the start of E. coli infusion (Fig. 4).

It has been demonstrated that infusion of en­dotoxin (2 mg/kg body weight) intravenously (ex­tracted from E. coli) over 5 min in 14 dogs lead to activation of complement. The investigators dem­onstrated a fall in whole complement activity (CH50) by 380/0 within 2 h after infusion [48]. Zim­mermann and coworkers studied a combination of endotoxin and hypovolemic shock in dogs by deter­mination of whole complement activity and C5a­activity in the blood [50]. They found a marked de­crease in the activity of the alternative and the classical complement pathway. They also reported an increase in C5a-like activity.

Ulevitch and Cochrane injected a lethal dose of lipopolysaccharide in rabbits [55]. This injection led to hypotension. Prior depletion of C3 with cobra venom factor did not alter the degree of hy­potension of thrombocytopenia compared to nor-

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8

Fig. 4. Terminal C5b-9 complement complexes determin­ed by an enzyme-linked immunosorbent assay in septic baboons. Animals receiving 5 x 108 E. coli/kg body weight (n = 6) are indicated by filled bars and animals receiving 1010 E. coli/kg body weight (n = 6) are in­dicated by open bars

mal and non-C3-depleted animals. The authors concluded that the lethal effects of lipopolysac­charide were not reduced by prior depletion of complement (C3). Olson and coworkers induced sepsis in mice by ligation of the cecum [56]. They evaluted the differences in survival time, Pa02, in­tracapillary granulocyte trapping, and air-blood­barrier thickness between C5-sufficient and C5-de­ficient mice. They were able to demonstrate that the survival time was longer in C5-deficient mice and that the granulocyte trapping and the air-blood­barrier thickness was increased in C5-sufficient mice. The Pa02 levels were lower in C5-sufficient mice, while Pa02 levels and the air-blood-barrier thickness remained normal in the C5-deficient mice. These data suggest in agreement with the results of Tvedten and coworkers [57] that C5 and split products of C5 are of great importance in the pathogenesis of septic lung injury.

Human Studies

Activation of complement with the formation of anaphylatoxins and terminal C5b-9 complement complexes occur in many categories of patients [58-61]. The most profound activation of comple­ment has been observed in association with sepsis and septic shock, multiple injuries, acute pancreati­tis, and in patients undergoing cardiopulmonary bypass [20, 62, 63].

Most studies on the activation of complement in septic patients were earlier performed by deter­minations of changes in plasma concentrations of the complement proteins C3 and C4 or by whole complement activity [64-74]. Most of the studies demonstrate reduction of the complement proteins and whole complement activity in association with sepsis and septic shock [64-70, 72-74]. Several studies have during the last 10 years been perform­ed that demonstrate formation of C3a, C4a, C5a, and terminal C5b-9 complement complex in associ­ation with sepsis and septic shock [75 - 87].

It is important to keep in mind that the duration of septicemia and the duration of shock and hypoperfusion at the time of blood sampling may vary significantly between different investigations. This may lead to differences regarding the results of complement activation by sepsis or septic shock. It has been reported that central hemodynamics are altered as soon as 24 h before the hypotension oc­curs [88]. Activation of complement may occur in septic patients much earlier than the time at which the samples are drawn, as hemodynamics may be

Complement in Sepsis 451

altered 24 h before septic shock can be identified [88, 89].

The definition of sepsis and the criteria for sep­tic shock differ between different investigators. This may influence the results of complement determinations in sepsis and in septic shock. Treat­ment has to be started early, often before the results of bacteriological cultures are available. This means that the diagnosis of sepsis has to be con­sidered even if it can not be bacteriologically prov­ed. Studies have been performed in which the authors did not require a positive blood culture for the diagnosis of sepsis. Weinburg and coworkers in­cluded patients in their study [76] with a known or strongly suspected source of systemic infection other than the lung. These patients fulfilled at least two of five criteria: (1) history of fever and rigors, (2) rectal or core temperature> 38.3 °C or < 35°C, (3) total white blood cell count greater than 12000 cells/mm3 or polymorphonuclear leukocyte count less than 1000 cells/mm3 or immature polymor­phonuclear leukocyte count greater than 600 cells/ mm3, (4) systemic blood pressure less than 90 mmHg, not attributable to dehydration or myo­cardial injury, and (5) metabolic acidosis with an increased anion gap not attributable to renal fai­lure, ketoacidosis, or ingestion of toxins. In their study 25 patients out of 40 had positive blood cul­tures [76]. Elevated C3adeSArginine concentrations were determined in 35 of the 40 patients. However, all patients with positive blood cultures had elevated plasma anaphylatoxin concentrations. Other investigators have required bacteremia (positive blood cultures) and a known source of in­fection to include patients in a sepsis study [68, 74, 78, 79].

A third question that differ between reports and which may influence the results of complement determinations is the definition of septic shock. Most investigators consider patients to be in septic shock if their mean arterial blood pressure is < 90 mmHg or, in hypertensive patients, more than 50 mmHg lower than a previously measured systol­ic pressure, with decreased organ perfusion (altered mental status or oliguria) and continued hypoten­sion despite intravenous infusions. It is also impor­tant to have a clear definition of ARDS and MOP. ARDS is usually defined by diffuse pulmonary in­filtrations resembling pulmonary edema, pulmon­ary wedge pressure < 18 mmHg, and an aterial oxy­gen tension less than 75 mmHg at a fraction of at least 0.5 inspired oxygen [82]. MOF is usually de­fined as failure of two or more vital organ systems.

452 A. Bengtsson et al.

Several investigators have demonstrated that ac­tivation of complement occurs in patients with sep­sis and septic shock [64 - 87]. Some investigators have also found a more pronounced decrease in complement proteins in patients with a fatal out­come [66, 72, 74, 90]. However, other investigators have not found low complement protein concentra­tions in septic shock [71], nor have they been able to record any difference between patients with a fatal outcome and patients with an uneventful course [69]. Leon and colleagues determined whole complement (CH so), C3, and C4 in 48 patients and in 25 healthy individuals. They did not find any dif­ferences regarding complement values between the patients in nonseptic shock (n = 9) and the control group [69]. In patients with sepsis and normoten­sion (n = 20) CHso titers were lower than normal values, while C3 and C4 remained unchanged. In patients in septic shock CHso , C3, and C4 were markedly decreased. There were no differences in complement levels between infection due to gram­negative and gram-positive sepsis, nor could they find any differences between patients who survived and those who died. Four days after admission the complement variables had returned to the normal range again. These results were verified by Sprung and coworkers, who in 1986 published a study of 42 patients with septic shock [74]. They found lower C3, C4, and factor B levels in patients with septic shock than in controls. In agreement with Leon and coworkers, they found no differences in comple­ment variables between gram-negative and gram­positive infections. However, in their study the C3 and factor B levels were lower in patients who died than in those who survived.

Weinburg and coworkers showed that all pa­tients with sepsis (defined by positive blood cultures) had elevated C3adesArgmme and C5adeSArginine levels. Like Leon et al. and Sprung et al., Hack and coworkers studied the differences in the degree of complement activation due to gram­negative and gram-positive sepsis respectively [84]. In their study C3a formation was slightly less among patients with gram-negative than among those with gram-positive infections. In a study of 47 patients with intra-abdominal infections Solomkin and coworkers found increased plasma C5a levels compared to controls [75]. They also demonstrated a positive relation between the for­mation of C5a and chemotaxis (r = 0.56, p<0.01) and between chemotaxis and intracellular fJ­glucuronidase (r = 0.82, p < 0.001). In a recent study, Brandtzaeg and coworkers showed that in patients with systemic meningococcal disease

(n = 39) there was a positive correlation between formation of terminal C5b-9 complement com­plexes and plasma levels of endotoxin on admission [85].

Several investigators have correlated the activa­tion of complement to the occurrence of ARDS, MOF, or death in septic patients, with different results. Some authors have found a close relation between levels of complement proteins on patients' arrival in the intensive care unit and the risk of ARDS or death; others have not been able to dem­onstrate any positive relation between these vari­ables at all. Hach and coworkers demonstrated in a recent study that elevated plasma levels of the anaphylatoxins C3a and C4a are associated with a fatal outcome in sepsis [82]. They also found higher C3a levels in patients with septic shock compared to normotensive patients. However, they could not find any statistically significant differences in plasma anaphylatoxin levels on admission between patients who developed ARDS and those who did not.

Weinberg and coworkers studied 40 patients with suspected sepsis (25 patients had positive blood cultures) and failed to demonstrate a close relation between C3adesArginine functional activity or C5adesArginme or neutrophil-aggregating capacity on admission and the development of acute lung injury [76]. However, the highest C5adeSArginine levels were found in patients with hypotension and/or acidosis. Similar results concerning comple­ment activation with release of C3adeSArginine and C5adeSArginine and septic shock were presented by Slot man and coworkers [77]. They found increased plasma C3adesArginine concentrations in patients with septic shock compared to those with sepsis but without hypotension and compared to patients with hypovolemic shock. Among these different groups they did not find any significant differences regarding plasma C5adesArginine concentrations. However, granulocyte aggregation was higher in pa­tients with sepsis than in those with hypovolemic shock. Kellerman and coworkers followed C3adeSArginine every 12 h in 27 patients with sepsis. They found that patients with ARDS had higher concentrations than those without pulmonary complications [83]. However, Parsons and Giclas studied 75 patients with sepsis, trauma, hyper­transfusion, multiple fractures, aspiration, or pan­creatitis who did not develop ARDS although esti­mated at risk of ARDS and 23 patients who developed ARDS. None of the measured comple­ment variables or combination of complement pro­teins could be used as a predictor of which patients

would develop ARDS [86]. Others have demon­strated that there is a close relation between ARDS development and complement activation if the determinations are performed on a longitudinal basis [78, 79].

Zilow and coworkers studied 38 poly trauma pa­tients at risk of ARDS. They analyzed complement variables over a period of 14 days. Determinations were made every 6 h during the first 48 h [20]. They found that elevated plasma C3a concentrations on­ly a few hours after injury were associated with de­velopment of ARDS. The ratio between C3a and C3 was an even more sensitive indicator of ARDS development. They were also able to correlate C3a levels and C3a/C3 ratio to the increase of ex­travascular lung water from day 4 after injury [20].

Langlois and Gawryl demonstrated that the ter­minal C5b-9 complement complex could be used as a predictor of development of ARDS in septic pa­tients [80]. In another study, 18 patients with sepsis were also studied in regard to the formation of ter­minal C5b-9 complement complexes [78]. Both pa­tients who later developed MOF and patients with uneventful courses had elevated terminal C5b-9 complement complex values in plasma on admis­sion to the intensive care unit, compared to healthy controls. There were no significant differences in regard to terminal C5b-9 complement complexes between those who developed remote organ failure and those without signs of organ dysfunction on admission. One week after admission patients with MOF still had elevated plasma terminal C5b-9 complement complex concentrations, while those with uneventful courses had normal terminal C5b-9 complement complex concentrations.

Diogini and coworkers analzyed the relation be­tween sepsis score and complement factor B as related to death after sepsis in 66 surgical patients with severe sepsis [73]. With this combination they were able to predict patient outcome with 100070 ac­curacy. Thirty-six patients developing ARDS sec­ondary to multiple injuries, major abdominal surgery, pancreatitis, severe burns, or disseminated intravascular coagulation were studied by Ducha­teau and coworkers in regard to C3d/C3 ratio and C5a-like activity and their relation to ARDS devel­opment [70]. They found a positive relation be­tween these variables but they could not consider them as predictors of ARDS in the patients studied.

In a recent study, Parsons and coworkers evalu­ated the relation between complement activation and circulating endotoxin levels and the develop­ment of ARDS [91]. Patients were defined as hav-

Complement in Sepsis 453

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0 0 7 Days

Fig. 5. Plasma C3a in septic patients on admission to the intensive care unit and 1 week after admission. Both pa­tients developing multisystem organ failure (open bars) and those with an uneventful course (filled bars) had elevated plasma concentrations of C3a on admission. Seven days later, patients developing multisystem organ failure still had increased plasma levels of C3a, while those with no signs of multisystem organ failure had plasma C3a concentrations within the normal range

ing ARDS (n = 24) or being at high risk of develop­ing ARDS (n = 56). They could not find any cor­relation between release of C5 split products and development of ARDS. C3 split products were in­creased in 89% of ARDS patients. However, they were also increased in 62 % of patients at risk of developing ARDS. Endotoxin (lipopolysaccharide) was detected in 74% of the patients who developed ARDS. Only 22% of patients who did not develop ARDS had endotoxin in their plasma. This in­dicates that the combination of endotoxins and complement split products is of major importance in the development of ARDS.

It has been demonstrated that even low lipo­polysaccharide concentrations (1-10 ng/ml) in­crease C5a-stimulated release of superoxides and elastase from neutrophils [92, 93]. In the study by Brandtzaeg and coworkers there was a positive rela­tion between activation of complement and the presence of endotoxin [85]. They found that surviv­ing patients with septicemia and significantly lower terminal C5b-9 complement complexes in plasma than those who died [85]. Kalter and coworkers studied 45 patients during 61 episodes of bac­teremia or septic shock with determinations of total hemolytic complement activity, alternative pathway activity, and plasma C3 [72]. In 29 pa­tients with uncomplicated bacteremia no altera­tions were found in the complement determina­tions. However, during fatal and nonfatal septic shock all variables were significantly decreased. In the group of patients who died (n = 13) the activa­tion of complement was more pronounced than in

454 A. Bengtsson et al.

the group of patients with septic shock who surviv­ed (n = 19). Similar results regarding C3 values were published by McCabe as early as 1973 [64]. He did not find any significant differences regarding C3 levels between 75 control patients and 68 pa­tients with gram-negative bacteremia. However, in 26 patients with septic shock the plasma C3 con­centrations were significantly lower than in patients with sepsis without shock or control patients [64].

Effect of Complement Inactivation or Inhibition

The most important therapeutic intervention is to remove the source of complement activation. Ade­quate treatment with antibiotics and surgical drainage of abscesses result in decreased levels of circulating anaphylatoxins and terminal C5b-9 complement complexes [67, 78, 94].

Many in vivo and in vitro studies have been per­formed with corticosteroids. The main effect of corticosteroids may be inhibition of granulocyte aggregation and the release of inflammatory mediators from granulocytes [95], but the concen­trations necessary are higher than those used in vivo in the treatment of ARDS due to septic shock or trauma [96, 97]. Eighty-seven patients with sep­sis who received either methylprednisolone (30 mg/kg body weigth) or mannitol as placebo were studied by Luce and coworkers. In addition they also studied the degree of complement activa­tion (formation of C3adeSArgmme and C5adesArginine) in these patients. They did not find any differences in mortality or ARDS development between the two groups, nor did they find any differences regar­ding the degree of anaphylatoxin formation. One explanation is that in this situation methylpred­nisolone does not modify the complement cascade and therefore does not improve the clinical out­come of these patients. Similar results were found by Sprung and coworkers, who reported in 1986 that high-dose corticosteroids (30 mg methylpred­nisolone/kg or 6 mg dexametasone/kg) did not alter complement protein levels in severe septic shock, nor in their study did corticosteroid therapy improve survival [98].

A few animal studies with the use of anti-C5a antibodies have been presented [50, 99, 100-107]. Stevens and coworkers and Hangen and coworkers have evaluated the effects of anti-C5a antibodies [94, 99, 100]. In vitro experiments demonstrated that anti-C5a antibodies inhibit neutrophil chemo­taxis [106]. E. coli was infused to primates in a dose

which resulted in severe ARDS, septic shock, and a mortality rate of 750/0. However, of animals pre­treated with anti-C5a antibodies, all survived. The plasma concentration of C5adesArginine was lower in anti-C5a-treated animals than in the controls. In another study it was demonstrated that anti-C5a antibodies prevented reduced oxygenation of arterial blood and increased extravascular lung water, which was recorded in the control group given E. coli without corticosteroid treatment. Smedegard and coworkers demonstrated that pre­treatment with anti-C5a antibodies reduced the de­gree of hypotension after infusion of endotoxin in rats [50].

Conclusion

Most of the studies regarding sepsis and septic shock and its relation to activation of complement indicate that complement is activated and anaphy­latoxins and terminal C5b-9 complement com­plexes are released. Patients with septic shock and patients who die due to circulatory insufficiency seem already to have more pronounced activation of complement on admission. However, it is not possible to predict the development of ARDS or MOF by measuring different complement variables at admission. Determinations of complement ac­tivation has to be done in a longitudinal fashion to distinguish patients developing complications to sepsis from those who will not do so.

In regard to the complement system in sepsis we have the interesting situation that we have better evidence from clinical trials than from experiments that complement activation (products) play an im­portant role in sepsis-related organ failure and death. Practically the only complement-deficient animals are rodents, so studies are limited in their clinical relevance. For this reasons, the main need is for primate studies, where at least some of the human assay systems can be used and where anti­bodies are available for therapeutic studies, which could indicate that the complement system is of the highest importance in sepsis-related organ failure. However, in studies performed so far no attention has been paid to the at least equally important cytokine network, so at present there is no final answer and thus no obvious starting point for therapy.

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