Terminal complement complexes and anaphylatoxins in septic and ischemic patients

Terminal complement complex (TCC) and anaphylatoxin formation in 18 patients with sepsis and 20 patients with acute limb ischemia were studied before the start of treatment and seven days later. The septic or ischemic patients had elevated levels of plasma TCC before start of therapy. In successfully treated patients these concentrations were within the normal range one week later. Similarly, the plasma anaphylatoxin level was increased before therapy and returned to the normal range within seven days. Escherichia coli incubated in vitro in fresh human serum at body temperature started formation of TCC in a dose-related manner. As complement will induce cellular lysis via TCC and edema via anaphylatoxins, anemia and impaired respiration in these patients may be influenced by increased concentrations of terminal complement complexes and of C3a and C5a.     

Anaphylatoxin and terminal complement complexes in red cell salvage

Thirteen patients undergoing elective orthopedic surgery were studied regarding anaphylatoxin (C3a and C5a) and terminal complement complex (TCC) formation in association with red cell salvage. The auto-transfusion equipment gave a centrifuged and washed erythrocyte fraction. The concentrations of C5a and TCC were not increased but elevated C3a levels were found in the suspension. After infusion of the erythrocyte fraction to the patient, no signs of systemic complement activation were observed. Thus, plasma levels of C3a, C5a and TCC were within the normal range in all the patients before and after autotransfusion. This study indicates that the complement system is activated in the cellsaver equipment. The washing procedure, however, seems to eliminate most of the anaphylatoxins and terminal complement complexes. No extensive systemic activation of complement seems to occur in association with autotransfusion to patients undergoing elective surgery.     

Terminal complement complexes in acute poststreptococcal glomerulonephritis

Activation of the complement cascade occurs in most cases of acute poststreptococcal glomerulonephritis (APSGN) and results in the formation of the terminal complement complexes (TCC). To examine the possible role of TCC in the pathogenesis of glomerular injury in APSGN, we studied 30 patients with the clinical diagnosis of APSGN. All patients had an elevated plasma SC5b-9 concentration at the onset of clinical nephritis. Serial plasma concentrations showed an inverse linear relationship with time after onset of clinical disease (r=–0.59,P=0.0008), while plasma C3 concentrations showed a positive linear relationship (r=0.78,P=0.0001). Renal biopsies of 5 patients demonstrated co-localization of C5b-9, S-protein, and C3 deposition in a glomerular capillary loop and mesangial distribution. Urinary excretion of TCC in the acute phase of APSGN was not elevated and was not a useful marker of disease activity. These data suggest that in APSGN with terminal complement pathway activation the local generation of TCC may contribute to the pathogenesis of the disease.     

Anaphylatoxin formation in sepsis

Complement activation and anaphylatoxin formation were studied in 27 septic patients. The patients were treated with antibiotics and high-dose corticosteroids. Blood samples were drawn on admission and every week thereafter. Plasma levels of complement components C1INH, C3, C4, and C5 were low before the start of treatment but were above normal one week later in both successfully and unsuccessfully treated patients. In contrast, plasma levels of anaphylatoxins C3a/C3adesArg and C5a/C5adesArg were elevated on admission. After successful treatment, plasma levels of C3a/C3adesArg and C5a/C5adesArg returned to normal within one week. Nine patients had ongoing sepsis one week after the start of treatment and a persistent rise in anaphylatoxin concentration. They developed multisystem organ failure with respiratory, hepatic, and renal insufficiency. In vitro studies of Escherichia coli incubation in fresh serum indicated a dose-related formation of C3a/C3adesArg and C5a/C5adesArg. High concentrations of methylprednisolone inhibited the anaphylatoxin formation in vitro.     

Removal of activated complement from shed blood: comparison of high- and low-dilutional haemofiltration

Background : Perioperative blood salvage is associated with release of inflammatory mediators. Depending on type of processing, the complement system is activated to some extent in the final blood product. The aim of the present study was to evaluate a haemofiltration technique concerning complement system activation and whether the volume of added saline will have an influence on the elimination of activated complement during processing.
Methods : Sixteen patients undergoing total hip arthroplasty received wound blood salvaged intraoperatively with a haemofiltration technique. Saline was added to the reservoir for washing in a ratio of 1:1 or 5:1 of estimated blood volume. Samples for determination of the anaphylatoxins C3a and C5a, and the terminal SC5b–9 complement complex (TCC) were drawn from the patients, the collected blood, the ultrafiltrate and the processed blood.
Results : Increased concentrations of C3a, C5a and TCC were found in aspirated and processed blood. Haemofiltration did not reduce the concentrations of these factors, except that of C3a in the group where saline was added in a ratio of 5:1. There were no increased concentrations of C3a, C5a or TCC in the patient plasma after reinfusion. No differences in blood pressure, heart rate, pH, arterial oxygen tension, arterial carbon dioxide tension, or base excess were found in association with rein-fusion of the blood.
Conclusion : Collected shed blood washed through haemofiltration contained moderately elevated concentrations of C3a, C5a and TCC. Reinfusion of the blood neither led to increased systemic concentrations of complement activation products, nor to disturbances in haemodynamic or biochemical parameters.     

Anaphylatoxin generation in acute pancreatitis

Fifty-one patients with elevated serum amylase and clinical signs of acute pancreatitis were studied prospectively. The concentrations of anaphylatoxins (C3a and C5a) were measured with a radioimmunoassay and the activity of their inactivator was determined. The pancreatitis was classified as mild, moderate, or severe according to Ranson's 11 signs, appearance of peritoneal fluid, and development of multisystem organ failure (MSOF). Plasma C3a and C5a concentrations were elevated during attacks of acute pancreatitis. Anaphylatoxin levels correlated with the severity of the disease (C3a, P less than 0.001; C5a, P less than 0.05). The highest and most persistent levels were found in the group with MSOF. C3a levels decreased rapidly during recovery. In patients with complications like abscess or pseudocyst, the C3a elevation persisted until adequate treatment was instituted. In this study, no significant changes of the inactivator levels were found, except at discharge when the inactivator level of the severe group was elevated compared to that of the moderate and mild groups (P less than 0.05).     

Hypersensitivity reactions during haemodialysis: role of complement fragments and ethylene oxide antibodies

IgE-antibodies against ethylene oxide (EtO) and activation of the complement system have been suspected as causative factors for acute hypersensitivity reactions at the onset of haemodialysis. The present study was conducted to determine which of the two mechanisms is mainly responsible for the occurrence of reactions. According to clinical criteria, 13 of the 129 patients studied were identified as having suffered from at least one episode of acute hypersensitivity. Seven of them experienced severe symptoms, including five with elevated circulating serum EtO antibodies. Of the remaining six patients who experienced moderate symptoms, only one had elevated serum EtO antibodies, and of the 114 patients not exhibiting symptoms, two had enhanced serum concentrations of EtO antibodies. In-vivo complement activation was determined during haemodialysis by measurement of both C3adesarg and C5adesarg fragments. No differences in the onset levels and the degree of complement activation were found between patients suffering from severe, moderate, or no hypersensitivity reactions. In-vitro activation of the complement cascade by zymosan, measured in plasma samples obtained from patients prior to dialysis, revealed no differences in the extent of C3adesarg generation. Finally, we determined the activity of the C3a- and C5a-inactivating enzyme, carboxypeptidase N1 (CN1), in plasma samples of patients both with and without hypersensitivity reactions. No difference in the CN1 activity between the two groups of patients was found. These data lead us to conclude that sensitisation to ethylene oxide is responsible for the majority of severe hypersensitivity reactions in haemodialysis patients. However, a small subgroup of patients having anaphylactoid reactions displayed neither elevated serum EtO antibodies with excessive complement activation nor a tendency towards reduced inactivation of anaphylatoxins.     

Changes in complement breakdown products and terminal complement complex in patients with acute glomerulonephritis

In this study we examined the role of the complement system in acute glomerulonephritis (AGN). Breakdown products of complements (iC3b, C4d and Bb) and Terminal complement complex (TCC, SC5b-9 complex) in plasma samples were measured by ELISA. The microassay plates were coated with monoclonal antibodies which bind specifically to human iC3b, C4d, Bb and SC5b-9 complex. This assay accurately quantitates small amounts of in vivo complement activation. The plasma samples were drawn from patients with AGN and other glomerulonephritis. There were some patients with various glomerulonephritis whose plasma iC3b, C4d and Bb concentrations were higher than those in normal human. However specificity was not found. The ratios iC3b/C3 and C4d/C4 were increased in the early stages of AGN, but plasma Bb concentrations revealed no significant changes. Plasma SC5b-9 complex concentrations were increased in the early stages of AGN. C3c, C3d, C4d and SC5b-9 were found to be localised in the glomeruli of those AGN patients. It is suggestive that in these cases of AGN (especially case 2) complement activation is predominantly mediated through the classical pathway and TCC is formed by this activation. This complement activation in the blood and the renal tissue is presumed to be involved in the initiation and progression of AGN.     

Studies on the dose dependence of endotoxin-induced in vitro activation of the complement system

The dose and time dependence of in vitro endotoxin-induced activation of complement was studied in citrated pool plasma at 37 degrees C. C3 activation fragments (C3act) and the fluid-phase terminal complement complex (TCC) were used as indicators of initial and terminal activation, respectively. At 1 h marked elevation of C3act and TCC were found in plasma tested with the highest doses of endotoxin (2.10(9) and 2.10(8) ng/l). In test plasmas with 2.10(7) and 2.10(6) ng/l of endotoxin, no sign of activation was seen during the first 4 h, whereas both C3act and TCC values were increased at 12 h. In thest plasma with 2.10(5) ng/l of endotoxin and in control plasma no elevation of TCC values were seen during the observation period (24 h), whereas C3act values increased slightly and to the same extent in both at 24 h. Parallel to the increases in C3act and TCC concentrations, the functional C1 inhibitor (C1INH) values decreased. Changes in C1INH values, however, occurred with a time lag of approximately 6 h compared to the increases in C3act and TCC. Our in vitro study showed a dose-dependent endotoxin-induced activation of both the initial and the terminal part of the complement cascade evaluated by C3act and TCC.     

Plasma terminal complement complexes in acute poststreptococcal glomerulonephritis

In most instances of acute poststreptococcal glomerulonephritis (APSGN), activation of the complement system occurs, as reflected by decreased levels of the complement proteins C3, C5, and properdin (P). Recent studies implicate terminal complement complexes (TCC) in the pathogenesis of glomerular injury. The fluid phase TCC, SC5b-9, reflects the formation of membrane-bound C5b-9 and has been used as a clinical marker in various diseases. Plasma concentrations of SC5b-9 were measured with an enzyme immunoassay using a monoclonal antibody to a neoantigen expressed on the SC5b-9 complex in 13 children who presented with clinical and pathologic features of APSGN. SC5b-9 was significantly elevated in all plasmas obtained within 30 days after onset of clinical glomerulonephritis. Concentrations of SC5b-9 in acute plasmas were significantly higher than those of paired convalescent samples. For individual patients, as SC5b-9 concentration returned to normal there was a coincident decrease in serum creatinine concentration and urinary protein excretion, signifying clinical improvement in glomerulonephritis. Thus, TCC generation commonly occurs in the early stages of APSGN and may be of importance in the pathogenesis of the condition.     

Cardiopulmonary effects of cuprophane-activated plasma in the swine

Hemodialysis with cuprophane membrane is associated with complement activation and the formation of anaphylatoxins. Frequently, it is also complicated by various adverse reactions which include hypoxemia and hemodynamic changes. This study examined the cardiopulmonary effects of cuprophane membrane on experimental animals. To support the hypothesis that these effects were mediated by complement activation products, the effects of zymosan-activated plasma and C5adesArg challenge on the same variables were compared. We showed that intravenous infusion of autologous cuprophane-activated plasma into swine produced severe pulmonary hypertension, hypoxemia and leukopenia. In addition, mean systemic arterial pressure fluctuated and cardiac output fell. Infusion of zymosan-activated plasma produced similar results, suggesting that complement activation products are responsible for these alterations. Similar responses to porcine C5adesArg infusion suggested further that this polypeptide was the mediator. When swine were subjected to extracorporeal circulation using cuprophane membrane but without dialysis, acute pulmonary hypertension was seen preceding the onset of significant leukopenia. These data suggest that blood contact with cuprophane membrane produces both pulmonary and systemic hemodynamic changes, which are mediated by complement activation products. Furthermore, these products and/or other humoral factors, but not leukoagglutination, cause the pulmonary hypertension.     

Complement activation and clearance in acute illness and injury: evidence for C5a as a cell-directed mediator of the adult respiratory distress syndrome in man

The appearance of the adult respiratory distress syndrome (ARDS) during the course of acute illness is believed to result, in part, from intrapulmonary neutrophil sequestration and degranulation induced by circulating inflammatory mediators. To evaluate the role of complement-neutrophil interactions in the pathogenesis of ARDS in man, 34 patients suffering from intra-abdominal sepsis (seven), multisystem trauma (15), or acute pancreatitis (12) were serially studied with regard to neutrophil migratory responses to C5a and F-Met-Leu-Phe, lysosomal content of beta-glucuronidase and lysozyme, and simultaneously obtained plasma levels of immunoreactive C3adesArg and C5adesArg. Nineteen patients developed ARDS. In these patients, plasma C3adesArg levels obtained within 72 hours of admission to the hospital were elevated to 305 +/- 35 ng/ml compared with 145 +/- 16 ng/ml for patients who did not develop ARDS (p less than 0.0005). C5adesArg levels were not elevated in either group. In vitro studies showed that neutrophils from normal persons were able to clear all of the C5a/C5adesArg generated in up to 5% zymosan-activated serum, while no clearance of C3adesArg was identified. Patient migratory responses could be divided into three groups based on their initial (less than 72 hour) samples: (1) hyperresponsive to both N = formyl-methionyl-leucyl-phenylalanine (FMLP) and C5a, (2) specifically deactivated to C5a, and (3) deactivated to both C5a and FMLP. Patients in the latter two groups developed ARDS. Enzyme content of neutrophils from patients who developed ARDS showed a substantial fall in beta-glucuronidase and lysozyme levels. The finding of elevated plasma C3a levels and deactivation of migratory response to C5a support the contention that complement activation had occurred in these patients and that their neutrophils had been exposed to C5a/C5adesArg in vivo. The finding of nonspecific migratory dysfunction associated with lysozymal enzyme loss, a circumstance not reproducible in vitro by C5a exposure, suggests that other stimuli produced degranulation of neutrophils made hyperresponsive by prior exposure to C5a.     

Methylprednisolone in high doses gives different effects on the early and the late part of complement

The effects of methylprednisolone (MP) on endotoxin-induced activation of complement were studied in citrated pool plasma. Complement activation was tested in two immunoassays: one evaluating C3 activation fragments (C3act) and the other the terminal complement complex (TCC). These components are indicators of initial and terminal complement activation, respectively. Plasma samples were obtained at 1, 2, 4 and 6 h of incubation. Plasma containing endotoxin (2.10(9) ng/l) without MP revealed a marked increase of both C3act and TCC after 1 h. MP in high doses (10 mg/ml) gave an additive effect on activation of the initial part of the complement cascade compared to test plasma containing only endotoxin. In contrast, endotoxin-induced activation of the terminal part of the complement cascade was inhibited by the same dose of MP. The influence of lower doses of MP (0.1 and 1 mg/ml) on endotoxin-induced activation of complement was insignificant. Interestingly, MP without endotoxin induced activation of the initial part of complement. In test plasmas containing 5 and 10 mg/ml of MP (without endotoxin) marked increases of C3act values were seen. Despite this obvious activation of the early part of complement, only insignificant changes were found in TCC values. Test plasmas containing 0.1 and 1 mg/ml of MP revealed only minor changes in both C3act and TCC. In conclusion, the present study shows that high doses of MP activate the initial part of complement and that the endotoxin-induced activation of this cascade system was facilitated by MP. The terminal part of complement was, on the other hand, inhibited by high doses of MP.     

Isolated hyperthermic liver perfusion with cytostatic-containing perfusate activates the complement cascade.

Eight patients with advanced liver malignancy undergoing isolated hyperthermic liver perfusion with melphalan and cisplatin were studied with regard to complement activation and formation of anaphylatoxins (C3a and C5a) and terminal C5b-9 complement complexes (TCCs). Blood samples for complement variables (C1-INH, C3, C4, C5, C3a, C5a and TCCs) were taken before surgery, 1 min before the start of perfusion, 1, 2 and 3 h after the start of perfusion, and 24 h after operation. Samples were drawn from the perfusate 1 h after the start of perfusion. Activation of complement was observed during perfusion. Raised plasma concentrations of C3a and TCCs were recorded and high levels of C3a and TCCs were found in the perfusate. In vitro tests indicated that melphalan and cisplatin may activate complement. This activation occurred at 37 and 42 degrees C but was more pronounced at 42 degrees C.     

Fluid phase generation of terminal complement complex as a novel index of bioincompatibility

Blood membrane interactions in hemodialysis have been shown to trigger complement (C) activation. As indicators of C-activation the anaphylatoxins (C3a and C5a) are problematical because of methodological difficulties and their kinetic properties. We developed a sensitive and specific micro-ELISA using a monoclonal antibody against neoantigens on the terminal complement complex (TCC); highly purified human TCC served as standard. Concentrations of TCC were measured in single-path perfusion systems (in vitro) and in the blood lines (arterial inlet; venous outlet) of patients on hemodialysis using steam-sterilized or ETO-sterilized dialyzers with the following membranes: cuprophan (CU), hemophan (HE) and polysulfone F6 (PS), respectively. All dialyzers with identical geometry were run under identical conditions. All membranes tested caused continuously ongoing net generation of TCC. In vitro, contact of serum with CU minidialyzers resulted in fivefold higher net release of TCC compared with HE and PS. In vivo TCC concentration-time profiles differed significantly between membranes in the rank order CU much much greater than HE greater than PS (mean basal concentration 58 x 10(-11) M; peak increase over baseline with CU 40-fold, HE fourfold, PS threefold). In addition, more TCC was generated from the same dialyzers with ETO than steam sterilization. TCC differed from C3a and C5a in the following respects: (i) lower detection limit (4 x 10(-11) vs. less than 5 x 10(-9) M for both C-anaphylatoxins); (ii) higher relative increment (inlet) during CU dialysis (25-fold vs. eightfold and twofold, respectively); (iii) C-anaphylatoxins yielded the same ranking (CU much greater than HE greater than PS), but TCC concentrations were not a linear function of C3a or C5a concentrations, respectively. Kinetic analysis (Bateman function) showed significant differences of invasion constants between membranes, that is, CU 0.088 min-1, HE 0.09, PS 0.168. The net amount of TCC released from the dialyzer was calculated under certain assumptions. It was 75.5 mg/4 hr for CU, 7.3 for HE and 5.0 for PS. The elimination constant was also dependent on the type of membrane. Using flow cytofluorometry and immunohistochemical methods (APAAP), TCC was demonstrated on membranes of granulocytes obtained during dialysis; this is compatible with potential in vivo cell activation. Generation of PGE2 and TNF alpha by adherent monocytes induced by cuprophan was C8 dependent: levels were significantly increased by addition of C8 to C8 deficient human serum concomitantly with generation of TCC.(ABSTRACT TRUNCATED AT 250 WORDS)     

Different activation patterns in the plasma kallikrein-kinin and complement systems during coronary bypass surgery

Components of the plasma kallikrein-kinin and complement systems were determined in patients undergoing open heart surgery with cardiopulmonary bypass. Spontaneous kallikrein activity (KK), plasma prekallikrein (PKK), functional kallikrein inhibition capacity (KKI), C3 activation products (C3-act), and the terminal complement complex (TCC) were measured. A marked, transitory increase in KK and a decrease in PKK were found prior to cardiopulmonary bypass just after heparin injection. An additional decline in PKK and KKI during bypass with a return to near control levels in the postoperative period was observed. C3-act increased in all patients during bypass, reaching a peak value at wound closure. The TCC concentration also increased significantly during cardiopulmonary bypass, returned to control levels in the early postoperative period, and then increased again in the late postoperative period. It is concluded that activation of the kallikrein-kinin system started after injection of heparin, prior to cardiopulmonary bypass. Activation of both the initial and the terminal complement cascade, however, started only after onset of cardiopulmonary bypass.     

Anaphylatoxin release in association with methylmethacrylate fixation of hip prostheses

In a prospective study, thirty patients in whom a Charnley hip prosthesis was implanted with cement (methylmethacrylate) and fifteen who received a prosthesis without cement were studied. The activation of complement, as indicated by the release of anaphylatoxins (C3a and C5a), reduced activity of whole complement, and decreased levels of C3, C4, and C5 in plasma, was evaluated. Activation of complement was found when methylmethacrylate was used. In patients in whom components were fixed without cement, no formation of anaphylatoxins occurred, and only slightly reduced whole-complement activity and concentrations of C3, C4, and C5 in plasma were found. A dose-correlated release of the anaphylatoxins was found when monomethylmethacrylate was incubated in fresh serum. One explanation for the hemodynamic instability in these patients might be the biological effects of anaphylatoxins that are released in association with fixation by cement.     

Complement activation following multiple injuries

Complement activation was evaluated by assay of plasma C3dg and the terminal complement complex (TCC) in 19 patients with multiple injuries. In the nine patients with thoracic involvement, statistically significant increase of plasma TCC was found at first sampling (average 90 min post-trauma), and of C3dg after 24 hours. Such increase was not found in the ten patients without thoracic involvement. Heightened granulocyte elastase activity was found in bronchial lavage fluid 90 min after the trauma in three patients with thoracic injury. Pulmonary insufficiency (pO2/FiO2 less than 16 kPa on intermittent positive-pressure ventilation) arose in four patients. All four had raised plasma levels of TCC or C3dg on arrival at the hospital. Six patients with complement activation did not show pulmonary insufficiency. Although the series was relatively small, the results indicate that thoracic injury is particularly associated with complement activation, and that complement activation alone does not suffice to produce post-traumatic pulmonary insufficiency.     

Complement activation and lung permeability during cardiopulmonary bypass

Pulmonary dysfunction after cardiopulmonary bypass has been attributed to the damaging effects of complement activation on the lung. To further explore this phenomenon, we measured plasma levels of activated complement components (radioimmunoassay), assessed neutrophil n-formyl-methionyl-leucyl-phenylalanine (FMLP) receptor status (radioligand saturation binding assay), and quantified pulmonary epithelial permeability as radioaerosol lung clearance of technetium 99m-labeled diethylenetriamine pentaacetic acid in a series of 8 patients undergoing cardiopulmonary bypass. Significant elevations of plasma C3adesArg, C4adesArg, and C5adesArg levels were seen just after CPB, indicating activation of both the classic and alternate complement pathways. Neutrophil activation was evident as increased expression of neutrophil FMLP surface receptors after bypass. Despite the presence of complement and neutrophil activation, increased pulmonary epithelial permeability was not seen. These data support the hypothesis that complement and neutrophil activation during cardiopulmonary bypass is not associated with acute lung injury, at least not pulmonary epithelial injury. One can therefore infer that increased pulmonary epithelial permeability in patients at high risk for and experiencing sepsis-induced and trauma-induced adult respiratory distress syndrome may be due to factors other than complement and neutrophil activation.     

Clinical significance of the terminal complement complex in children with type I membranoproliferative glomerulonephritis

We measured the concentrations of terminal complement complex (TCC) in plasma (n =25) and urine (n=13) using an enzyme-linked immunosorbent assay in pediatric patients with type I membranoproliferative glomerulonephritis(MPGN). Frozen tissue from 18 renal biopsies was evaluated for the presence of TCC by direct immunoperoxidase staining. In the acute phase of the disease, TCC concentrations in plasma were elevated above 0.5 AU/ml in 14 of 25 patients (High TCC group), while the remaining 11 patients showed less than 0.5 AU/ml (Low TCC group). In the High TCC group, TCC was deposited more diffusely and intensely in the glomerulus, compared to that in the Low TCC group (p= 0.034). Furthermore, urinary TCC concentrations in the High TCC group were higher than those in the Low TCC group (p=0.0001). The High TCC group showed not only a poorer response to steroid treatment, but also a poorer prognosis than the Low TCC group. These results suggest that, in pediatric patients with type I MPGN, TCC in circulation may play a certain role in TCC formation in the glomerulus and in urine. The TCC concentration in plasma could be used as a marker of responsiveness to steroid treatment and long-term prognosis.