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.     

Monocyte dysfunction in severe trauma: evidence for the role of C5a in deactivation

Consecutive severely traumatized patients (n = 16) requiring intensive care underwent serial monitoring of complement activation and monocyte migratory function with the chemoattractant activated serum (C5a) and formyl-methionyl-leucyl-phenylalanine (FMLP). Complement was found to be activated, and chemotaxis to C5a was correspondingly depressed maximally at a mean 5 to 7 days after injury (p = less than 0.01). The migratory response to FMLP was within the normal range throughout. Conversely, in a consecutive series of patients undergoing aortoiliac bypass grafting (n = 11), there was no evidence of complement activation, and monocyte migratory function remained normal for both C5a and FMLP. These data suggest that in patients with severe trauma, the activation of complement, particularly the fifth component (C5a), reduces the migratory responsiveness of circulating monocytes to C5a. This reduction in a host-response mechanism may explain the propensity to infection and poor wound healing seen in patients with severe trauma and also indicates that C5a, thought to be the major in vivo chemoattractant for leukocytes, has profound systemic actions.     

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.     

Suppressed neutrophil oxidative activity in sepsis: a receptor-mediated regulatory response

We have reported that neutrophils from patients with intraabdominal sepsis show suppressed superoxide production in response to soluble chemoattractants. To determine whether this could be a result of altered receptor function, neutrophil function studies and characterization of receptors were performed upon 22 patients recently operated upon for intraabdominal sepsis. Patient cells showed an absence of subagarose migratory response to activation serum (C5) but intact FMLP response. Superoxide production to FMLP was 5.3 +/- 0.1 nmole FcC compared to 11.2 +/- 0.1 nmole for laboratory normals. Addition of cytochalasin B reversed this. Scatchard analyses of patients' cells showed affinity of 55.5 +/- 0.6 as compared to 36.5 +/- 1.8 for controls. The number of receptors of cell was approximately 55,000 for both controls and patients. Internalization studies revealed significant increase in tritiated-FMLP uptake at room temperature. These results support the hypothesis that superoxide suppression is due to enhanced clearance of stimulant by enhanced internalization of the receptor-ligand complex.     

Neutrophil activation after burn injury: contributions of the classic complement pathway and of endotoxin

We attempt to elucidate the mechanisms of neutrophil (PMN) activation after burn injury. We previously reported prolonged elevations of PMN cell surface complement (C) opsonin receptor levels after burn trauma with a corresponding period of depressed PMN chemotaxis to C5a, which suggests that the C product, C5a, was responsible for PMN activation. However, a lack of direct correlation of C activation with C receptor levels soon after injury raised the possibility of a second PMN-activating substance. We therefore investigated the effect of endotoxin (LPS) on the expression of the C receptors (CR1 and CR3) by normal human PMNs. Concentrations from 0 to 50 ng/ml of LPS 026:B6 caused a dose response increase in the PMN surface expression of CR1 and CR3 as assessed by monoclonal antibody binding and indirect immunofluorescence. The relative CR1-dependent fluorescence rose from a mean of 50 to 385 and CR3 from 50 to 300. Chelation by ethylenediaminetetra acetic acid (EDTA) did not influence this dose response, thus ruling out the possibility of C activation by LPS--an inference supported by the lack of complement activation observed with these concentrations of LPS in normal serum. A similar dose response was obtained in the absence of other cell types or serum, which implies a direct effect that mimicked that of C5a. To determine the mechanism of the later, prolonged C activation after burn injury, we next examined C activation products in 22 patients with burn injuries. Elevations of plasma C3a desArg were present and persisted for 50 days. Elevations were at maximum levels on days 9 through 13 postburn (mean +/- standard error of mean [SEM], 496 +/- 47 ng/ml versus normal 113 +/- 32; p less than 0.01). These were accompanied by elevations of C4a desArg (917 +/- 154 ng/ml versus normal 424 +/- 50; p less than 0.01), which are indicative of classic pathway activation. Finally, we examined PMN function, phagocytosis and percentage killing of Staphylococcus aureus, and found PMN function to be unaltered in the 22 patients. Thus PMN activation after burn injury appears to be caused by LPS soon after injury and by C5a later after injury and affects only selected PMN functions.     

Regulation of neutrophil migratory function in burn injury by complement activation products.

Polymorphonuclear neutrophils were isolated from patients with burn injury and random mobility, chemotaxis in response to C5adesArg (as agarose-activated control serum) and to N-formyl-methionyl-leucyl-phenylalanine (F-Met-Leu-Phe) were assessed. For a group of eight patients identified as not experiencing systemic infection, all three neutrophil migratory functions were observed to fall below control levels, beginning 4 to 6 days following burn injury, and to return to control levels after 21 to 30 days of hospitalization. Over this time the chemotactic differential (distance chemotactic migration-distance random migration) for F-Met-Leu-Phe remained positive, while the chemotactic differential for activated serum became nil after postburn day 4. This temporal, specific loss of a chemotactic response to activated serum was associated with rises in immunoreactive plasma C3a and C5a. This pattern of loss of chemotactic function was associated with a selective loss of C5a but not F-Met-Leu-Phe binding activity. These results demonstrate that burn injury can alter neutrophil migratory functions generally, and specifically depress chemotactic responsiveness to activated serum. The mechanism of the latter phenomenon appears to be related to desensitization of circulating neutrophils to C5a due to complement activation.     

Surface receptor mobilization in complement-mediated neutrophil activation: characterization and effects of methylprednisolone

Complement-mediated neutrophil activation (CMNA) is an important host defense mechanism that is essential for effective neutrophil (PMN) proinflammatory activity. It has also been implicated as a pathogenic mechanism contributing toward the development of adult respiratory distress syndrome. Utilizing zymosan-activated serum pretreatment as an in vitro model for CMNA, we characterized the effects of CMNA on PMN superoxide (SO) production and N-formyl-methionyl-leucyl-phenylalanine (FMLP) receptor status. CMNA was associated with a 132 +/- 38% increase in FMLP-induced SO generation and a 110 +/- 30% increase in FMLP receptor expression. Methylprednisolone pretreatment prevented both the FMLP receptor mobilization and the SO priming effects of CMNA. These data support a concept that FMLP receptor mobilization is an important element in the PMN activation process. In addition, blocking this phenomenon may have clinical significance in attempts to modulate the potential damaging effects of the increased PMN oxidative metabolism associated with CMNA.     

Plasma fibronectin and complement activation in coronary bypass surgery

In patients with severe sepsis, plasma fibronectin concentrations are reduced and complement is activated. It is not known whether complement activation interferes with plasma fibronectin. Cardiopulmonary bypass is known to activate complement. We have therefore used this operation to study the effect of complement activation on plasma fibronectin in the absence of sepsis. After 15 min of bypass the percentage changes of plasma fibronectin and haematocrit were similar (65.9 +/- s.e.m. 4.8 and 67.0 per cent +/- s.e.m. 2.3, respectively), but the changes in C3 and C5 (58.4 +/- s.e.m. 4.8 per cent and 52.5 per cent +/- s.e.m. 2.1) were significantly greater than those of the haematocrit, indicating complement consumption. During the first 60 min of bypass there was a significant increase in the neutrophil count which is compatible with complement activation. It is unlikely that complement activation alone, in the absence of sepsis, contributes to the reduction of plasma fibronectin concentrations.     

The effects of complement activation during cardiopulmonary bypass. Attenuation by hypothermia, heparin, and hemodilution.

Complement activation was examined prospectively in 100 cardiopulmonary bypass (CPB) patients. Plasma C3a desArg (C3a) increased (cannulation: 234 +/- 33 ng/mL; 20 minutes on CPB: 622 +/- 51; 2 hours after CPB: 1143 +/- 109, p less than 0.0001). C3a at 2 hours was higher in the 13 patients requiring mechanical ventilation for longer than 1 day (1023 +/- 274) than in the 67 without respiratory complication (568 +/- 45, p less than 0.004). Five more patients were studied for neutrophil activation to confirm that a biologic effect of complement activation occurs during CPB; in these five patients C3a increased to 317% of baseline after 10 minutes on CPB with a corresponding rise in neutrophil cell surface receptors for the complement opsonin C3b (as measured by indirect immunofluorescence) to 168% (p less than 0.05). Both increases were sustained at 30 minutes. Temperature, dilution, and heparin were studied as variables relevant to CPB. Exposure of normal neutrophils to C5a in vitro caused an increase in C3b receptors which was dependent on temperature (0 specific fluorescence at 0 C, 30 at 25 C, 180 at 30 C, and 275 at 37 C). Generation of C3a and C5a in normal serum by zymosan was also temperature-dependent (ng/mL C5a generated: 0.7 at 25 C, 200 at 30 C, and 897 at 37 C; ng/mL C3a generated: 546 at 25 C, 10,872 at 30 C, and 65,667 at 37 C). Serum dilution to 33% decreased ng/mL C5a generated in the same system from 200 to 76 with no effect on C3a. Addition of heparin to 20 U/mL decreased ng/mL C3a generated from 10,872 to 913 and C5a from 200 to 8. Thus, hypothermia, dilution, and heparin protect CPB patients from complement activation by reducing both generation of C3a/C5a and the subsequent cellular response of neutrophil activation.     

Complement C5a receptor antagonist attenuates multiple organ injury in a model of ruptured abdominal aortic aneurysm

OBJECTIVE: Abdominal aortic aneurysm (AAA) rupture is associated with a systemic inflammatory response syndrome, characterized by increased microvascular permeability and neutrophil sequestration, leading to multiorgan dysfunction. We examined the role of a novel complement factor 5a (C5aR) receptor antagonist, the cyclic peptide AcF-(OpdChaWR), in attenuation of pathologic complement activation and tissue injury in a model of AAA rupture. METHODS: Anesthetized rats were randomized to sham (control) or shock and clamp (s+c) groups. Animals in the s+c group underwent 1 hour of hemorrhagic shock (mean arterial blood pressure < or =50 mm Hg), followed by 45 minutes of supramesenteric aortic clamping, then 2 hours of resuscitated reperfusion. Animals in the s+c group were randomized to receive an intravenous bolus of C5aR antagonist at 1 mg/kg or saline solution control at the end of hemorrhagic shock. Intestinal and pulmonary permeability to iodine 125-labeled albumin was measured as an indicator of microvascular permeability. Tissue myeloperoxidase activity, proinflammatory cytokine tissue necrosis factor-alpha (TNF-alpha) protein and mRNA, and C5aR mRNA levels were measured as indicators of neutrophil sequestration and inflammatory signaling, respectively. RESULTS: Lung permeability index was significantly increased in the s+c group compared with the sham group (4.43 +/- 0.96 vs 1.30 +/- 0.17; P <.01), and prevented with treatment with C5aR antagonist (1.74 +/- 0.50; P <.03). Lung myeloperoxidase activity was significantly increased in the the s+c group compared with the sham group (2.41 +/- 0.34 U/mg vs 1.03 +/- 0.29 U/mg; P <.009), and significantly attenuated with treatment with C5aR antagonist (1.11 +/- 0.09 U/mg; P <.006). Lung TNF-alpha protein levels were significantly elevated in both s+c groups, whereas lung TNF-alpha mRNA expression was significantly downregulated in both s+c groups compared with the sham group. Intestinal permeability index was significantly increased in animals in the s+c groups during reperfusion, compared with sham (P <.001), which was attenuated in early reperfusion with treatment with C5a receptor antagonist. Data represent mean +/- SEM, group comparisons with analysis of variance and post hoc Scheffé test. CONCLUSIONS: These results indicate that a potent antagonist of C5a receptor protects the rat intestine and lung from neutrophil-associated injury in a model of AAA rupture. These data suggest that complement-mediated inflammation can be modulated at the C5a receptor level, independent of proinflammatory TNF-alpha production, and prevent acute local and remote organ injury.     

Neutrophil activation in sepsis. The relationship between fmet-leu-phe receptor mobilization and oxidative activity

To elucidate further the manifestations and mechanisms of neutrophil (PMN) activation, PMNs from control and septic subjects were studied at baseline and under conditions of graded, in vitro activation. At baseline (4 degrees C PMN isolation), septic-derived PMNs were activated, as manifested by twofold increases in fmet-leu-phe (FMLP)-induced oxidative activity and concomitant FMLP surface receptor expression, compared with controls. Following degranulationlike maximal activation (phorbol myristate acetate pretreatment), both PMN populations exhibited maximal FMLP-induced oxidative priming and receptor up-regulation. However, following exudation-like moderate activation (37 degrees C pretreatment), control PMNs underwent significant receptor mobilization and oxidative priming but septic-derived PMNs exhibited oxidative deactivation (decreased FMLP-induced oxidative activity) without changes in FMLP receptor expression. Our data support the theory that while circulating PMNs in sepsis may promote oxidant-related microvascular lung injury, their oxidative deactivation following transpulmonary exudation (simulated by 37 degrees C pretreatment) may underlie the increased incidence of pulmonary infections seen in sepsis-induced adult respiratory distress syndrome.     

A single dose of endotoxin activates neutrophils without activating complement

Both complement (C) and neutrophils (PMN) are activated in critically ill patients. To evaluate the role of endotoxin in this response, we studied C activation products and PMN cell surface receptors in seven normal subjects before and after endotoxin (USRef 20 U/kg) or saline solution administered on separate occasions. By 4 hours, with endotoxin only, all subjects had myalgia, headache, an increase in body temperature and heart rate, and leukocytosis that returned to normal by 24 hours. At the same time, PMN cell surface receptors for the complement opsonin C3b increased, as measured by indirect immunofluorescence, rising to 251 +/- 44% of baseline by 4 hours (p less than 0.01) and remaining elevated at 24 hours (237 +/- 16%, p less than 0.01). PMN receptors for iC3b increased to 308 +/- 49% of baseline by 4 hours (p less than 0.02) and returned to normal by 24 hours. There was no change in plasma of C3a desArg, C4a desArg, and C5a desArg (4 hours: mean C3a: 153.4 +/- 11.5 ng/ml versus 176.2 +/- 16.2 ng/ml for saline solution, p = ns; C4a: 159.6 +/- 32 ng/ml versus 151.4 +/- 21 ng/ml, p = ns; C5a: undetectable). To confirm the lack of C activation, we examined PMN chemotaxis (CTX) to C5a for any impairment caused by prior in vivo exposure to C5a. CTX to C5a was unaffected (4 hours: 109% +/- 22% of normal versus 114% +/- 10% for saline solution, p = ns). PMN CTX to formyl-methionyl-leucine-phenylalanine and PMN phagocytosis and killing of S. aureus were also unaffected by endotoxin. Thus, a single dose of endotoxin produced a subjective febrile illness and precipitated sustained PMN activation as indicated by increased PMN cell surface complement receptor number in the absence of C activation.     

Coagulation augments neutrophil C3b receptors via formation of a protein(s) unrelated to fibrinolysis or C5 activation

The present study investigated the effect of coagulation on neutrophil complement receptors (CRs) 1 and 3, which are specific for the opsonins C3b and C3bi. Incubation of neutrophils in autologous serum, but not in plasma, increased the mean (+/- SD) expression of CR1 (x3.43 +/- 0.93) and CR3 (x3.07 +/- 0.86), in comparison with incubation in buffer. Serum also increased neutrophil superoxide production in response to opsonized zymosan from 0.48 +/- 0.21 to 1.05 +/- 0.25 nmol/10(6) cells/min. Similarly, calcium conversion of platelet-rich plasma (but not platelet-poor plasma) to serum also increased both CR1 and CR3 expression. This finding, as well as the fact that freeze-thawed platelet-rich plasma (but not platelet-poor plasma) increased CR expression, indicated that platelet constituents were the origin of this CR-inducing activity. Other nonplatelet factors formed during coagulation, such as C5a, fibrinogen degradation products, kallikrein, and factor XIIa, were shown not to be responsible for this CR-inducing activity.     

Degranulation inhibition. A potential mechanism for control of neutrophil superoxide production in sepsis

Previous studies with neutrophils from patients with intra-abdominal sepsis have provided convincing evidence of in vivo exposure to C5a. However, in contradistinction to normal cells pretreated with C5a, patient cells showed depressed superoxide response to N-formyl-methionyl-leucyl-phenyl-alanine (FMLP) and enhanced FMLP receptor affinity. To identify possible mechanisms responsible for these findings, we examined the effects of lysosomal alkalinization with the weak base clindamycin on normal neutrophils with and without C5a. Our results showed a specific suppression of FMLP-induced superoxide production and a loss of low-affinity FMLP receptors. These results occurred in the presence of clindamycin levels that did not interfere with other cellular processes. These findings suggest that regulation of neutrophil function during the course of intra-abdominal sepsis may be due to effectors active both at the cell surface (C5a) and within the lysosome. The clinical significance of our findings relates to a possible mechanism for specific pharmacologic suppression of oxide-radical production by neutrophils. Such oxide radicals are believed to be important in the capillary injury accompanying severe sepsis.     

Coagulation abnormalities associated with severe isolated traumatic brain injury: cerebral arterio-venous differences in coagulation and inflammatory markers

Although coagulopathy is known to be the major contributor to a poor outcome of traumatic brain injury (TBI), the mechanisms that trigger coagulation abnormalities have not been studied in detail. We undertook a prospective observational study at a neurosurgical ICU (NICU) in a university hospital. We examined 11 patients with severe isolated TBI, at admittance to the hospital and during the next 3 days. We collected cerebrovenous blood samples from a jugular bulb catheter, arterial blood, and cerebrospinal fluid (CSF) samples. We measured concentrations of thrombin-antithrombin complex (TAT), fibrin D-dimer (DD), prothrombin fragment 1 + 2 (F1 + 2), interleukin-6 (IL-6), and complement complex (C5b-9). All patients had some degree of consumption coagulopathy at the study start and a tendency to thrombocytopenia during the next few days. Levels of DD (3.6 +/- 2.7 mg/L), TAT (86 +/- 72 microg/L) and F1 + 2 (5.9 +/- 6.8 nmol/L) were significantly increased shortly after the trauma compared to reference values, with considerable transcranial gradients for TAT (49 microg/L) and F1 + 2 (3.2 nmol/L). Compared to controls, IL-6 levels were increased more than a hundredfold in both blood (283 +/- 192 ng/L) and CSF (424 +/- 355 ng/L) samples, with a transcranial gradient at the study start (107 ng/L). C5b-9 levels were moderately increased in blood samples, 270 +/- 114 microg/L, versus controls, 184 +/- 39 (p < 0.05). We conclude that activation of the coagulation system takes place during the passage of blood through the damaged brain, and is already evident hours after the trauma. IL-6 and activation of the complement system (C5b-9) co-vary with hemostatic parameters in TBI patients.     

Enhanced neutrophil migratory activity following major blunt trauma

BACKGROUND: Neutrophil migration into the lung is a critical, but poorly understood step in the pathogenesis of post-traumatic, acute respiratory distress syndrome (ARDS). We investigated changes in interleukin-8 (IL-8) mediated neutrophil migration and associated changes in receptor expression, of the IL-8 receptors CXCR1, the integrins CD11b/CD18 and platelet endothelial cell adhesion molecule-1 (PECAM-1) in patients sustaining major trauma. METHODS: Eleven patients with major trauma, injury severity score (ISS), median 22 (range 18-41), were followed prospectively. Eleven normal volunteers were used as controls. Blood samples were obtained within 4+/-2 h of injury, at 24 h, day 3 and day 5. Neutrophils migration was assessed by an in vitro IL-8 assay and neutrophil surface receptor expression by FACS analysis. RESULTS: IL-8 mediated neutrophil migration was significantly increased on admission following major trauma and remained elevated for 3 days (p<0.05). This was associated with up-regulation of CXCR1 (p<0.01) and down-regulation of PECAM-1 (p<0.05). CD11b and CD18 although initially unchanged, became down-regulated on day 3 (p<0.05). CONCLUSIONS: These data show that major trauma primes circulating neutrophils for increased migration in response to IL-8. This response is sustained for 72 h and is associated with changes in neutrophil surface receptor expression.     

Plasma myeloperoxidase and vitamin E levels in head injury: preliminary results related to outcome

This preliminary study was designed to assess a possible role of neutrophil activation and to determine the prognostic value of plasma myeloperoxidase (MPO) and vitamin E (Vit. E) levels in severe head injury. Plasma MPO and Vit. E levels were measured in nine severely head-injured patients (Glasgow Coma Score 相似文献   

Distinct roles for C3a and C5a in complement-induced tubulointerstitial injury

To prevent injury to host tissues, complement activation is regulated by a number of plasma and membrane-associated proteins, most of which limit C3 and C5 activation. An influx of circulating C3 from a syngeneic host into donor kidneys deficient in Crry (a membrane protein that reduces C3 convertase activity) causes spontaneous complement activation, primarily in the tubulointerstitum, leading to renal failure. To determine the roles of the C3a and C5a anaphylatoxins in tubulointerstitial inflammation and fibrosis, kidneys from Crry-/-C3-/- mice were transplanted into hosts lacking the C3a and/or C5a receptor. While unrestricted complement activation in the tubules was not affected by receptor status in the transplant recipient, C3a receptor deficiency in the recipients led to significantly reduced renal leukocyte infiltration and the extent of tubulointerstitial inflammation and fibrosis, all of which led to preserved renal function. The absence of C5a receptors in recipients was not only inconsequential, but the protective effect of C3a receptor deficiency was also eliminated, suggesting distinct roles of C3a and C5a receptor signaling in this model. There was significant infiltration of the tubulointerstitum with 7/4+F4/80+CD11b+ myelomonocytic cells and Thy1.2+ T cells along injured tubules, and interstitial collagen I and III deposition, all of which were C3a receptor dependent. Thus, blockade of C3a receptor signaling is a possible treatment to reduce renal inflammation and preserve renal function associated with complement activation.     

Role of neutrophil derived oxidants and elastase in lipopolysaccharide-mediated renal injury

Gram-negative bacterial sepsis is frequently associated with acute renal failure but the specific effects of lipopolysaccharide (LPS) and other bacterial products on kidney function are not known. Since either LPS or formyl-methionyl-leucyl-phenylalanine (FMLP)--a chemotactic peptide from bacterial cell walls--activate neutrophils (PMN) to release a number of potentially toxic factors in vitro, we determined the effect of adding PMN with LPS and/or FMLP to isolated perfused rat kidneys. Isolated rat kidneys perfused with LPS alone or LPS and normal PMN had normal glomerular filtration rates (GFR) and tubular Na reabsorption (TNa). Kidneys perfused with FMLP alone or FMLP and normal PMN also had normal GFR and TNa. In contrast, addition of PMN with both FMLP and LPS caused progressive renal dysfunction. For example, after 60 minutes of perfusion, GFR was reduced from 610 +/- 31 to 147 +/- 17 microliters/min/g and TNa from 97 +/- 1 to 72 +/- 2%, both P less than 0.01. Perfusion with the O2 metabolite scavengers catalase or dimethylthiourea afforded no protection while perfusion with the neutrophil elastase inhibitor Eglin C conferred substantial, but not complete, protection: GFR 492 +/- 34 microliters/min/g; TNa 91 +/- 3%. However, perfusion with both Eglin C and catalase completely prevented the toxic effects of LPS and FMLP-treated PMN on renal function. We conclude that in isolated kidneys, 1) the toxic effects of LPS requires FMLP-treated PMN and that 2) LPS and FMLP treated PMN cause progressive renal injury which is mediated by both O2 metabolites and neutrophil elastase.     

Novel anti-factor D monoclonal antibody inhibits complement and leukocyte activation in a baboon model of cardiopulmonary bypass

BACKGROUND: Adverse outcomes after cardiopulmonary bypass (CPB) are often related to systemic inflammation triggered by complement and leukocyte activation. To determine how inhibition of the alternative complement pathway affects systemic inflammation and tissue injury, we studied a novel monoclonal antibody (Mab), anti-human factor D murine Mab 166-32, in baboons. METHODS: Fourteen baboons (mean weight, 15 kg) underwent hypothermic CPB. The treatment group (n = 7) received a single injection of anti-factor D Mab 166-32 (5 mg/kg), and the control group (n = 7) was given saline solution. After initiation of CPB, all animals were subjected to 20 minutes of core cooling (rectal temperature, 27 degrees C), followed by 60 minutes of aortic cross-clamping, 25 minutes of rewarming, and 30 minutes of normothermic CPB. Blood samples were collected before CPB, during CPB, and 1, 2, 3, 6, and 18 hours after CPB. To measure neutrophil and monocyte activation, we performed flow cytometry for CD11b expression, ELISA for complement activation (Bb, C3a, C4d, and sC5b-9) and interleukin-6 (IL-6) production, and tissue injury studies for creatine kinase MB isoenzymes (CK-MB), creatine kinase (CK), and lactic dehydrogenase (LDH) levels. RESULTS: Anti-factor D Mab almost completely inhibited plasma Bb, C3a, and sC5b-9 production during CPB (P < .001). CD11b expression on neutrophils (129 +/- 5% vs. 210 +/- 42%; P = .0006) and on monocytes (139 +/- 14% vs. 245 +/- 43%; P = .0002) was also lower in the treatment group during CPB. The treated animals had a significantly smaller increase in plasma IL-6 concentrations than did the control animals (71 +/- 27 pg/mL vs. 104 +/- 54 pg/mL; P = .0002). CK-MB levels were also lower in the treatment group 6 hours after the end of CPB (204 +/- 30 vs. 335 +/- 59 IU/L; P = .003) and 18 hours after the end of CPB (P < .05). Creatine kinase levels (6 and 18 hours after the end of CPB) and LDH levels (3 and 6 hours after the end of CPB) showed patterns similar to those of CK-MB (P < .05). CONCLUSIONS: The alternative complement pathway plays a major role in systemic inflammation during CPB. Inhibition of complement activation via the alternative pathway by anti-factor D Mab 166-32 significantly reduces leukocyte activation and tissue injury in our baboon model.