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.     

Ischemia-induced neutrophil activation and diapedesis is lipoxygenase dependent

Ischemia and reperfusion lead to eicosanoid- and neutrophil (PMN)-dependent injury. This study tests the role of ischemia-induced lipoxygenase activity in mediating PMN activation and diapedesis. Anesthetized rabbits (n = 8) underwent 3 hours of bilateral hindlimb ischemia. At 10 minutes of reperfusion, leukotriene B4 (LTB4) levels in femoral venous effluent were 0.49 +/- 0.05 ng/ml compared with 0.04 +/- 0.07 ng/ml in sham-treated animals (n = 10) (p less than 0.05). Intracellular H2O2 production of circulating PMNs assayed flow cytometrically by dichlorofluorescein (DCF) oxidation, increased from a preischemic value of 74 +/- 14 femtomoles DCF/cell to 135 +/- 8 fmol DCF/cell (p less than 0.05). PMNs were treated with phorbol myristate acetate (PMA), 10(-7) mol/L. In contrast to a 162% increase in H2O2 production before ischemia, PMNs at 10 minutes of reperfusion had an enhanced response to PMA of 336% (p less than 0.05). Addition of authentic LTB4 (0.5 ng/ml) to PMN from sham-treated animals led to their activation, manifest by an oxidative burst, 127 +/- 12 fmol DCF/cell, and an enhanced response of 337% to PMA stimulation. To study diapedesis, plasma collected at 10 minutes of reperfusion was introduced into plastic chambers taped atop skin abrasions in rabbits (n = 8). After 3 hours, 1610 +/- 246 PMN/mm3 accumulated and LTB4 levels in blister fluid were 0.83 +/- 0.03 ng/ml, higher than values of 44 +/- 23 PMN/mm3 (p less than 0.05) and 0.04 +/- 0.03 ng LTB4/ml (p less than 0.05) with saline solution and 68 +/- 16 PMN/mm3 (p less than 0.05) and 0.19 +/- 0.02 ng/ml (p less than 0.05) with nonischemic plasma. The introduction of LTB4, 3.3 ng/ml, into the chambers resulted in an accumulation of 536 +/- 352 PMN/mm3 (p less than 0.05). Pretreatment of animals before hindlimb ischemia (n = 5) with the lipoxygenase inhibitor diethylcarbamazine abolished PMN activation (51 +/- 12 fmol DCF/cell) and ischemic plasma-induced diapedesis into the plastic chamber (38 +/- 18 PMN/mm3). Pretreatment of nonischemic animals (n = 13) used for the dermabrasion bioassay with diethylcarbamazine abolished diapedesis into the plastic chambers induced by ischemic plasma (n = 5) (32 +/- 24 PMN/mm3) or LTB4 (n = 3) (36 +/- 28 PMN/mm3). These data indicate that PMN activation after reperfusion of ischemic tissue is mediated by a lipoxygenase product, perhaps LTB4, and that both reperfusion plasma and authentic LTB4 induce diapedesis by stimulating de novo lipoxygenase activity.     

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-induced expression of cryptic receptors on the neutrophil surface: a mechanism for regulation of acute inflammation in trauma

We explored the hypothesis that identified changes in neutrophil function in patients with acute injury result from in vivo exposure to C5a. To evaluate this hypothesis, we performed a battery of tests on 26 trauma patients (14 with blunt injury, 12 with penetrating injury). Measured were plasma levels of the complement activation products C3a and C5a; neutrophil chemotaxis to C5a and N-formyl-methionyl-leucyl-phenylalanine (FMLP); neutrophil receptors for FMLP and C3b; and superoxide response to FMLP and serum-opsonized zymosan. Patient responses measured within 48 hours of admission were divided into two groups based on neutrophil migratory response to C5a. Patients unresponsive to C5a (but responsive to FMLP) showed elevated plasma C3a levels (248 +/- 6 ng/ml) compared with patients with normal C5a migratory response (104 +/- 8 ng/ml). FMLP receptor number was markedly increased in the chemotactically deactivated group (group I: 155,680 +/- 100; group II: 51,200 +/- 200) and receptor affinity was diminished. Binding activity of C3b increased in the C5a-unresponsive cells to 126% that of controls versus 94% for normally responsive patient cells. Superoxide production was found to be significantly increased in patient cells with increased receptor numbers. These results support the concept that a subgroup of trauma patients manifest plasma and neutrophil changes compatible with complement activation. The neutrophil changes identified demonstrate a state of cellular activation. The clinical significance of these results may reside in a risk of pulmonary microvascular injury if activated cells are marginated and then subsequently stimulated.     

Modulation of pulmonary permeability in vivo with agents that affect the cytoskeleton

In vitro studies show that the cytoskeleton of the microvascular barrier moderates polymorphonuclear leukocyte (PMN) diapedesis and the transport of macromolecules. This in vivo study tests indirectly whether the cytoskeleton of the pulmonary microvasculature responds similarly to agents known to reorganize the cytoskeleton to alter diapedesis and permeability in vitro. One of four agents, saline solution, cytochalasin B (which promotes actin microfilament disassembly), leukotriene (LT) B4 (PMN chemoattractant), or phalloidin (which promotes and stabilizes F-actin polymerization) was introduced into the bronchus of the anterior segment of the left lung of anesthetized rats (n = 152) through a tracheostomy and fine-bore cannula. Twenty minutes later, saline solution, cytochalasin B, LTB4, or phalloidin was similarly introduced. PMN accumulations (x 10(4) cells/ml), protein concentration in bronchoalveolar lavage fluid, and lung wet/dry weight ratios were measured 3 hours later. When the initial and secondary treatments were saline solution, PMN accumulations were 3 +/- 1 cells/ml and the protein level was 274 +/- 48 micrograms/ml. Secondary treatment of the saline-treated group with cytochalasin B or LTB4 led to increases in PMN to 18 +/- 2 and 9 +/- 2 cells/ml and protein to 960 +/- 50 and 840 +/- 40 micrograms/ml (p less than 0.05); secondary treatment with phalloidin was similar to that with saline solution. With saline solution used for both treatments, the wet/dry ratio was 3.4 +/- 0.2. Primary saline solution and secondary treatments with either cytochalasin B or LTB4 led to a similar increase in wet/dry ratios to 3.9 +/- 0.1 (p less than 0.05), whereas phalloidin was without effect (3.5 +/- 0.3). Initial cytochalasin B treatment followed by LTB4 led to increased PMN diapedesis (43 +/- 6 cells/ml and wet/dry ratio 4.3 +/- 0.2) (p less than 0.05). Secondary phalloidin treatment attenuated the cytochalasin B effect with counts of 12 +/- 2 PMN/ml, protein levels of 460 +/- 30 micrograms/ml, and a lower wet/dry ratio of 3.7 +/- 0.1 (p less than 0.05). Even more striking, phalloidin as initial treatment further reduced the cytochalasin B effect to 7 +/- 1 PMN/ml, whereas protein level was 490 +/- 60 micrograms/ml and wet/dry ratio was 3.5 +/- 0.1 (p less than 0.05). Further, phalloidin, given initially, attenuated the effect of secondary treatment with LTB4, resulting in fewer cells (4 +/- 2 PMN/ml), a lower wet/dry ratio (3.4 +/- 0.1), and protein level of 650 +/- 20 micrograms/ml (p less than 0.05).(ABSTRACT TRUNCATED AT 400 WORDS)     

Attenuation of shock-induced acute lung injury by sphingosine kinase inhibition

BACKGROUND: Prolonged elevations of cytosolic calcium concentrations ([Ca2+]i) are required for optimal neutrophil (PMN) activation responses to G-Protein coupled chemoattractants. We recently showed that the coupling of endosomal Ca2+ store depletion to more prolonged entry of external Ca2+ depends on cellular conversion of sphingosine to sphingosine 1-phosphate (S1P) by sphingosine kinase (SK). We therefore hypothesized that inhibition of SK might inhibit PMN activation and thus ameliorate lung injury after trauma and hemorrhagic shock (T/HS). METHODS: Chemotaxis (CTX) of human PMN was studied using modified Boyden chambers in the presence or absence of the selective SK inhibitor, SKI-2. After determining the concentration of SKI-2 that inhibited human PMN CTX by 50% (IC50) we subjected rats to T/HS (laparotomy, hemorrhage to 30-40 mm Hg x 90 minutes, 3 hours resuscitation). We then studied rat PMN CD11b expression using flow cytometry and lung injury using the Evans Blue dye technique in the presence of IC50 doses of SKI-2 or vehicle given in pretreatment at laparotomy. RESULTS: Human PMN CTX was suppressed slightly more than 50% by 40 micromol/L SKI-2 (233 +/- 20 vs 103 +/- 12 x 10(3) cells/well, p < 0.001). Rat PMN expression of CD11b after T/HS was decreased from 352 +/- 30 to 232 +/- 7 MFU (p < 0.001) in the presence 30 micromol/L SKI-2. Lung permeability to Evans Blue was decreased from 9.5 +/- 2 to 4.1 +/- 0.7% (p = 0.036.). SKI-2 did not cause hemodynamic instability or alter resuscitation requirements. CONCLUSION: Modulation of PMN Ca entry via SK inhibition inhibits PMN CTX in vitro, and inhibits CD11b expression in vivo without major effects on hemodynamics. These cellular changes were associated with amelioration of lung injury in vivo in a rat model of T/HS. These findings suggest that SK inhibition allows modulation of inflammation via control of [Ca2+]i without the cardiovascular compromise expected with Ca2+ channel blockade. SK inhibition therefore appears to be an important novel candidate therapy for inflammatory organ injury after shock.     

七氟醚对内毒素致急性肺损伤鼠肺泡毛细血管膜通透性的影响

目的　探讨七氟醚对内毒素致急性肺损伤鼠肺泡毛细血管膜通透性和肺泡灌洗液内炎性细胞的影响。方法　 4 8只Wistar大鼠 ,麻醉后静注伊万斯蓝 5 0mg/kg后 ,随机分为 4组 ,每组 12只。对照组 :股静脉注射生理盐水 1 2ml后机械通气 4h ;内毒素组 :股静脉注射内毒素 5mg/kg后机械通气 4h ;七氟醚 1L组和七氟醚 2L组 :股静脉注射内毒素 5mg/kg后机械通气 ,分别吸入肺泡气最低有效浓度 (MAC)为 1 0和 1 5的七氟醚 4h。 4h后取肺组织测定 :病理形态学积分 ,肺湿 /干重比 ,肺水含量 ,肺通透指数 ,伊万斯蓝含量和肺泡灌洗液内炎性细胞总数及百分比。结果　七氟醚1L组肺通透指数、伊万斯蓝含量、病理形态学积分分别由 4 6 8± 0 82 ,( 112 2 1± 11 4 4 )ng/mg ,9 17± 0 90下降到 3 98± 0 5 0 ,( 92 85± 11 80 )ng/mg ,7 5 0± 0 96 ;七氟醚 2L组下降到 3 91±0 34,( 96 33± 8 79)ng/mg ,7 6 7± 0 75。结论　 1 0和 1 5MAC七氟醚可降低内毒素所致急性肺损伤肺泡毛细血管膜通透性 ,使肺组织病理损伤减轻     

The influence of preoperative anticoagulation on heparin response during cardiopulmonary bypass.

The effect of preoperative anticoagulant therapy on intraoperative heparin response in patients undergoing cardiac operations was examined in a prospective study. The study included 45 patients with different preoperative anticoagulant treatments: 10 patients received treatment with phenprocoumon (a warfarin analogue) (group M), 12 patients received treatment with intravenous heparin (group Hiv), and 13 patients received treatment with subcutaneous heparin (group Hsc). The control group consisted of 10 patients who did not receive anticoagulant therapy before operation (group C). Preoperative antithrombin III activity was highest in group M (85% +/- 6%) and lowest in group Hiv (70% +/- 15%, p less than 0.05). The activated clotting time, determined 10 minutes after bolus injection of 250 IU (group M) or 375 IU heparin (all other groups), was 529 +/- 109 seconds in group C, greater than 1000 seconds in group M, 483 +/- 99 seconds in group Hsc, and 406 +/- 63 seconds in group Hiv (p less than 0.05). Heparin consumption during cardiopulmonary bypass varied between 4.6 +/- 1.4 IU/kg.min (group Hiv) and 2.6 +/- 0.9 IU/kg.min (group M) (p less than 0.05). Despite this increased heparin consumption, the patients who had received heparin before operation demonstrated increased activation of coagulation at the end of cardiopulmonary bypass (thrombin-antithrombin III complex, 19 +/- 4.1 ng/ml in group M and 61 +/- 7 ng/ml in group Hsc, p less than 0.05; cross-linked fibrin fragments, 257 +/- 92 ng/ml in group M and 875 +/- 152 ng/ml in group Hiv, p less than 0.05). Increased platelet activation was also found in patients with preoperative heparin therapy (beta-thromboglobulin at the end of cardiopulmonary bypass was 585 +/- 88 ng/ml in group M versus 1341 +/- 190 ng/ml in group Hsc, p less than 0.05). Drainage from the chest tube 24 hours after operation was 815 +/- 305 ml in group C, 644 +/- 238 ml in group M, 1133 +/- 503 ml in group Hsc, and 950 +/- 505 ml in group Hiv (p less than 0.05 for group M versus group Hsc). This study suggests that patients who receive heparin therapy before operation face a high risk of insufficient anticoagulation during cardiopulmonary bypass if standard heparin doses are used. Therefore, for patients who receive preoperative heparin therapy, a larger (500 IU/kg) initial bolus of heparin is recommended before cardiopulmonary bypass. On the other hand, patients who undergo preoperative treatment with phenprocoumon receive sufficient anticoagulative effect with a heparin bolus of 250 IU/kg.(ABSTRACT TRUNCATED AT 400 WORDS)     

A single dose of endotoxin increases intestinal permeability in healthy humans

To investigate the effects of endotoxin on gut barrier function, we performed paired studies of intestinal permeability in healthy humans (N = 12) receiving intravenous Escherichia coli endotoxin (4 ng/kg) or 0.9% saline solution. Two nonmetabolizable sugars, lactulose and mannitol, which are standard permeability markers, were administered orally, 30 minutes before and 120 minutes after the test injection. The 12-hour urinary excretion of these substances after endotoxin/saline solution administration was used to quantitate intestinal permeability. After endotoxin administration systemic absorption and excretion of lactulose increased almost two-fold (mean +/- SEM, 263 +/- 36 mumol per 12 hours vs 145 +/- 19 mumol per 12 hours during saline studies). Similar but less marked alterations in mannitol absorption and excretion occurred after endotoxin injection (5.7 +/- 0.3 mmol per 12 hours vs 4.9 +/- 0.3 mmol per 12 hours). When individual 12-hour lactulose excretion after endotoxin administration was related to the magnitude of systemic responses, a significant relationship occurred between lactulose excretion and elaboration of norepinephrine and between lactulose excretion and minimum white blood cell count. These data suggest that a brief exposure to circulating endotoxin increases the permeability of the normal gut. These observations are consistent with the hypothesis that during critical illness, prolonged or repeated exposure to systemic endotoxins or associated cytokines may significantly compromise the integrity of the gastrointestinal mucosal barrier.     

Neutrophil function in surgical patients: in vitro correlation of abnormal neutrophil chemotaxis by Levamisole.

Cutaneous anergy to recall skin test antigens is associated with decreased polymorphonuclear neutrophil (PMN) chemotaxis (CTX). This decreased PMN chemotaxis is mediated by factors circulating in the sera (AS) from patients with anergy. Levamisole hydrochloride will correct the chemotactic defect of neutrophils from anergic patients, in vitro, from 96.2 +/- 1.2 to 125.1 +/- 1.7 microns at concentrations of 10(-3) M to 10(-18) M. Pretreatment of normal PMN with Levamisole at 10(-4) M will protect them from the chemotactic inhibiting effect of AS. Normal PMN migrating in the normal range, 128.1+/- 2.7 microns, can be made to behave like anergic PMN by treatment with AS. These PMN which now migrate in the anergic range 92.1 +/- 1.7 microns can be converted back to normal by Levamisole treatment at 10(-3) M to 10(-18) M. In 35 surgical patients who demonstrated the spectrum of decreased PMN CTX, the majority toward the anergy level, Levamisole improved the PMN CTX toward normal levels in every instance, while not affecting the CTX of the normally migrating PMN.     

Released granulocytic elastase: an indicator of pathobiochemical alterations in septicemia after abdominal surgery

To discover the role of lysosomal enzyme release from polymorphonuclear (PMN) leukocytes during septicemia, plasma levels of PMN elastase were measured with a newly developed enzyme-linked immunosorbent assay for detection of the PMN elastase-alpha 1-proteinase inhibitor complex (E-alpha 1PI). Plasma samples from 41 patients were assayed continuously before and after major abdominal surgery. The patients were divided into a group without infection (group A) and two septicemia groups (survivors in group B and nonsurvivors in group C). The E-alpha 1PI levels of the 11 patients in group A without any signs of pre- or postoperative infection were in the normal range (a normal value of 86.5 +/- 25.5 ng/ml has been reported in 153 healthy subjects), except for a small increase to 208.8 +/- 25.6 ng/ml 12 hours after surgery. When septicemia was confirmed clinically in patients in groups B and C, the E-alpha 1PI levels rose on average to six times the norm in group B (649.9 +/- 116.3 ng/ml) and to more than 10 times the norm in group C (985.0 +/- 154.6 ng/ml). Peak values greater than 2,200 ng/ml could be measured in both groups. In patients in group B, the E-alpha 1PI levels returned to normal during recovery, while in those in group C they remained significantly elevated (560.5 +/- 174.7 ng/ml) until death. Correlations were demonstrated between the amount of elastase released into the circulation and the decrease in the activities of antithrombin III, coagulation factor XIII, and alpha 2-macroglobulin, as well as the increased C-reactive protein in plasma. We conclude that release of elastase and other lysosomal factors from PMN cells plays a major role in the pathobiochemical alterations during septicemia. In addition, significantly elevated E-alpha 1PI levels in the postoperative course seem to be a suitable indicator for onset and persistance of sepsis as well as of the severity of this disorder in patients after major surgery.     

Dexamethasone reduces the inflammatory response to cardiopulmonary bypass in children

BACKGROUND: A randomized, prospective, double-blind study of 29 children was performed to evaluate the hypothesis that dexamethasone administration prior to cardiopulmonary bypass would decrease the inflammatory mediator release and improve the postoperative clinical course. METHODS: Fifteen children received dexamethasone (1 mg/kg intravenously) and 14 (controls) received saline solution 1 hour prior to CPB. Serial blood analyses for interleukin-6, tumor necrosis factor-alpha, complement component C3a, and absolute neutrophil count were performed. Postoperative variables evaluated included temperature, supplemental fluids, alveolar-arterial oxygen gradient, and days of mechanical ventilation. RESULTS: Dexamethasone caused an eightfold decrease in interleukin-6 levels and a greater than threefold decrease in tumor necrosis factor-alpha levels after CPB (p < 0.05). Complement component C3a and absolute neutrophil count were not affected by dexamethasone. The mean rectal temperature for the first 24 hours postoperatively was significantly lower in the group given dexamethasone than in the controls (37.2 degrees +/- 0.4 degrees C versus 37.7 degrees +/- 4 degrees C; p = 0.007). Dexamethasone-treated patients required less supplemental fluid during the first 48 hours (22 +/- 28 mL/kg versus 47 +/- 34 mL/kg; p = 0.04). Compared with controls, dexamethasone-treated children had significantly lower alveolar-arterial oxygen gradients during the first 24 hours (144 +/- 108 mm Hg versus 214 +/- 118 mm Hg; p = 0.02) and required less mechanical ventilation (median duration, 3 days versus 5 days; p = 0.02). CONCLUSIONS: Dexamethasone administration prior to CPB in children leads to a reduction in the postbypass inflammatory response as assessed by cytokine levels and clinical course.     

Leukotrienes but not complement mediate limb ischemia-induced lung injury.

Reperfusion after limb ischemia leads to sequestration of polymorphonuclear leukocytes (PMN) in the lungs and to leukocyte- (WBC) and thromboxane- (Tx) dependent respiratory dysfunction. This study examines the intermediary role of the chemoattractants leukotriene (LT)B4 and complement (C) fragments. Anesthetized sheep with chronic lung lymph fistulae underwent 2 hours of tourniquet ischemia of both hind limbs. In untreated controls (n = 7), 1 minute after tourniquet release, mean pulmonary artery pressure (MPAP) rose from 13 to 38 mmHg (p less than 0.05) and returned to baseline within 30 minutes. Pulmonary artery wedge pressure was unchanged from 3.6 mmHg. There were increases in plasma LTB4 levels from 2.46 to 9.34 ng/ml (p less than 0.01), plasma TxB2 levels from 211 to 735 pg/ml (p less than 0.05), and lung lymph TxB2 from 400 to 1005 pg/ml (p less than 0.05). C3 levels were 96% of baseline values. Thirty minutes after reperfusion, lung lymph flow (QL) increased from 4.3 to 8.3 ml/30 minutes (p less than 0.05), lymph/plasma protein ratio was unchanged from 0.6, and the lymph protein clearance increased from 2.6 to 4.6 ml/30 minutes (p less than 0.05), data consistent with increased microvascular permeability. WBC count fell within the first hour from 6853 to 3793/mm3 (p less than 0.01). Lung histology showed leukosequestration, 62 PMN/10 high-power fields (HPF) and proteinaceous exudates. In contrast to this untreated ischemic group, animals treated with the lypoxygenase inhibitor diethylcarbamazine (n = 5) demonstrated a blunted reperfusion-induced rise in MPAP to 17 mmHg (p less than 0.05). There were no increases in LTB4, TxB2, QL or lymph protein clearance (p less than 0.05). WBC count was unchanged and lung leukosequestration was reduced to 40 PMN/10 HPF (p less than 0.05). Decomplementation with cobra venom factor (n = 4) resulted in plasma C3 levels, 10% of baseline, but tourniquet release still led to pulmonary hypertension, elevated LTB4, TxB2 levels, and a decline in WBC count similar to that of untreated ischemic control animals. Histology showed 46 PMN/10 HPF sequestered in the lungs. Further, bilateral hind limb ischemia in either genetically sufficient (n = 10) or deficient (n = 10) C5 mice led to significant lung leukosequestration of 108 and 106 PMN/10 HPF, respectively, compared with 42 and 47 PMN/10 HPF in sham C5(+) and C5 (-) mice (n = 20) (p less than 0.01). These results suggest that the lung leukosequestration and increased microvascular permeability after lower torso ischemia are mediated by the chemotactic agent LTB4, but not by the complement system.     

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.     

Effect of prostaglandin E in multiple experimental models. IV. Effect on resistance to endotoxin and tumor necrosis factor shock

Administration of a long-acting prostaglandin E, 16,16-dimethyl-PGE (dPGE), to rats improves their survival of bacterial peritonitis. We examined the mechanism of this protective effect with reference to its interaction with the release of cachectin (TNF). Sixty rats received saline, 20 micrograms/kg dPGE, or 80 micrograms/kg dPGE 12 hr prior to endotoxin and continuing for 48 hr. Survival rates for the saline, 20 micrograms/kg dPGE, and 80 micrograms/kg dPGE groups were 0, 40, and 85%, respectively. Forty rats received saline or 80 micrograms/kg dPGE, with the initial dose being 3 hr following endotoxin challenge and continuing for 48 hr. Survival rates for both groups were 0%. Sixty rats received saline or 80 micrograms/kg dPGE at 12 and 1 hr prior to endotoxin. Two hours after challenge, they were sacrificed and plasma TNF levels were assayed. The plasma TNF level in saline-treated rats was 22.72 +/- 0.83 ng/ml and in the dPGE-treated group, 16.03 +/- 1.13 ng/ml (P less than 0.001).     

Coronary trapping of a complement activation product (C3a des-Arg) during myocardial reperfusion in open-heart surgery

Accumulation of complement factors has been found to occur in the myocardium after infarction. We studied the possibility that the complement activation product C3a des-Arg is trapped within the coronary circulation during reperfusion of the ischemic myocardium. In 11 patients undergoing routine coronary artery bypass grafting, arterial blood was sampled before, during and after cardiopulmonary bypass. Blood was drawn from the coronary sinus concomitantly with arterial blood sampling 5 and 30 min after release of the aortic cross-clamp (n = 10). From a preoperative value of 92 +/- 13 ng/ml, C3a des-Arg rose during CPB to a maximum of 1816 +/- 393 at the end of CPB. Following reperfusion for 5 min, C3a des-Arg was 1284 +/- 232 ng/ml in arterial and 1106 +/- 100 in coronary sinus blood, a significant difference (p less than 0.05). The amount of C3a des-Arg trapped in the heart at 5-min reperfusion showed positive correlation with its arterial concentration (p less than 0.05). No significant difference was found after 30 min of reperfusion. Complement activation products trapped in the heart in the early reperfusion period may play a pathogenetic role in myocardial ischemia-reperfusion injury.     

Rapid increase in plasma levels of atrial natriuretic peptide after common bile duct ligation in the rabbit.

Previous studies have shown that common bile duct ligation in the rabbit is followed by a reduction of the extracellular water compartment. To further elucidate the mechanisms leading to volume depletion in this model, water and sodium balances and changes in plasma concentrations of atrial natriuretic peptide (ANP), vasopressin (ADH), plasma renin activity (PRA) and aldosterone (Ald) were investigated during the first 4 days after common bile duct ligation (group OJ,) or sham operation (group SO). Water and chow intakes were lower in group OJ (148 +/- 30 versus 226 +/- 40 mL/4 days; p = 0.004 and 12 +/- 9 versus 171 +/- 40 g/4 days; p = 0.0001). There were no differences in urine output. Sodium urinary losses were marginally higher in group OJ (12.4 +/- 7 versus 6.7 +/- 5 mEq/4 days; p = 0.06). Water balance was lower in group OJ (-50 +/- 56 versus 101 +/- 71 mL/4 days; p = 0.0001). At 24 hours, plasma ANP (41 +/- 7 versus 10.7 +/- 1 fmol/mL, p = 0.0001), ADH (21.8 +/- 7 versus 12.3 +/- 6 pg/mL, p = 0.008) and Ald (14.5 +/- 5 versus 3.7 +/- 3 ng/dL, p = 0.001) were higher in group OJ. These alterations persisted 72 hours after bile duct ligation, when a concomitant increase in PRA (10.7 +/- 5 versus 3 +/- 1.6 ng/dL, p = 0.006) was also observed. A group of pair-fed pair-watered sham-operated controls (group SO2, n = 13) showed a metabolic profile similar to group OJ but a low ANP concentration. Multiple venous sampling in five rabbits 24 hours after bile duct ligation showed the highest plasma levels of ANP in the aorta and infrarenal vena cava. These results suggest that common bile duct ligation in the rabbit is followed by marked hypodipsia and hypophagia, possibly mediated by ANP, leading to isotonic volume depletion and secondary activation of the water and sodium retaining hormones.     

The effect of tumor necrosis factor-alpha and interferon-gamma on neutrophil function

Tumor necrosis factor-alpha (TNF) and interferon-gamma (IFN-gamma) have been shown to regulate cell-mediated immunity and act as effective modifiers of immune function; however, their influence on neutrophil (PMN) function is not well defined. This study investigated the effect of these cytokines on PMN phagocytosis, respiratory burst, and complement receptor (C3b) expression. Human citrated whole blood was incubated with either phosphate-buffered saline (control), 20 micrograms Escherichia coli lipopolysaccharide (LPS), TNF (1, 10, or 100 units), or IFN-gamma (1, 10, or 100 units). Synergy was also assessed between TNF and IFN-gamma. Phagocytosis and respiratory burst were assayed by sequential incubation of blood with dichlorofluorescein diacetate followed by labeled Staphylococcus aureus. C3b receptor expression was assayed by labeled anti-CR3 monoclonal antibody. Measurements were expressed as the mean channel fluorescence of 2000 PMNs counted by flow cytometry. IFN-gamma alone at all doses had no effect on any of the parameters measured. TNF 1 unit/ml increased phagocytosis (666 +/- 47 vs 542 +/- 19), respiratory burst (326 +/- 33 vs 258 +/- 17), and C3b (374 +/- 42 vs 157 +/- 14; all P less than 0.05) over those of control. TNF also demonstrated dose-dependent PMN activation. The combination of TNF + IFN-gamma increased both respiratory burst and C3b compared to either agent alone. These data indicate that TNF enhances PMN function and cytokine interaction may be important in PMN activation.     

Complement activation, endothelin-1 and neuropeptide Y in relation to the cardiovascular response to endotoxin-induced systemic inflammation in healthy volunteers

BACKGROUND: Endotoxin is a major stimulus for triggering the host response in septicaemia. The pathophysiology of sepsis involves activation of the vascular endothelium and leukocytes, resulting in the release of various mediators, e.g. cytokines, nitric oxide (NO), endothelin (ET-1) and complement factors. We evaluated the blood levels of complement activation, ET-1 and neuropeptide Y (NPY) in parallel with the haemodynamic and oxygen transport response during human experimental endotoxemia. METHODS: Eleven healthy men had venous, arterial and pulmonary arterial catheters placed for continuous haemodynamic measuring. After 30 min rest endotoxin (E. Coli 4 ng kg(-1), Lot G1) was intravenously administered. Blood samples from pulmonary and arterial catheters were collected hourly over 4 h. RESULTS: Body temperature augmented significantly from baseline values (36.7 +/- 0.7 degrees C, mean +/- SEM) with a maximum after 3.5 h (39.1 +/- 0.3 degrees C, P < 0.001). Cardiac output increased by 100%, systemic vascular resistance decreased by 50%, the oxygen consumption and the tissue oxygen transport increased. Activation of the complement system was indicated by an increase in SC5b-9. Endothelin-1-like immunoreactivity (ET-1-LI) increased over time in arterial blood. NPY-like immunoreactivity (NPY-LI) did not change over time. CONCLUSION: A dose of endotoxin associated with reproducible systemic vasodilation and fever in healthy subjects causes complement activation and increased systemic levels of ET-1-LI, illustrating that the model is a useful tool for inducing moderate systemic inflammation where several mediator systems are activated.