The interrelationship of complement-activation fragments and angiogenesis-related factors in early pregnancy and their association with pre-eclampsia.

OBJECTIVE: To determine the interrelationships during early pregnancy of complement-activation fragments Bb, C3a and sC5b-9, and angiogenesis-related factors placental growth factor (PiGF), soluble fms-like tyrosine kinase-1 (sFlt-1) and soluble endoglin (sEng), and their associations with pre-eclampsia. DESIGN: Prospective cohort study. SETTING: Denver complement study (June 2005-June 2008). POPULATION: A total of 668 pregnant women with singleton gestations, recruited between 10 and 15 weeks of gestation. METHODS: Using univariable and multivariable logistic regression analysis, concentrations of complement-activation fragments and angiogenesis-related factors were compared between 10 and 15 weeks of gestation in women who subsequently did or did not develop pre-eclampsia. Interrelationships between these variables were tested using the non-parametric Spearman rank correlation coefficient. MAIN OUTCOME MEASURE: Pre-eclampsia. The association of complement-activation fragments and angiogenesis-related factors with obesity was also examined. RESULTS: The mean (+/-SD) levels of complement Bb in early pregnancy among women who did and did not develop pre-eclampsia were 0.84 (+/-0.26) microg/ml and 0.69 (+/-0.2) microg/ml, respectively (P = 0.001). Concentrations of PiGF were significantly (P = 0.01) lower (31 +/- 12 pg/ml) in early pregnancy in the pre-eclamptic group of women, as compared with the normotensive group (39 +/- 32 pg/ml). The adjusted odds ratio (AOR) of Bb and PiGF were 2.1 (CI = 1.4-3.1, P < 0.0003) and 0.2 (CI = 0.07-0.7, P = 0.01), respectively. There was no significant difference in the levels of C3a, sC5b-9, sFlt-1 and sEng in early pregnancy among women who developed pre-eclampsia, compared with women who remained normotensive during pregnancy. Higher levels of Bb (P = 0.0001) and C3a (P = 0.03), and lower levels of sFlt-1 (P = 0.0002) and sEng (P = 0.0001) were found among women with obesity, compared with non-obese controls. No meaningful relationships were found between the complement-activation fragments and the angiogenesis-related factors. CONCLUSIONS: In this cohort during early pregnancy, increased concentrations of complement-activation factor Bb and lower concentrations of PiGF were associated with the development of pre-eclampsia later in pregnancy.

Characterization of plasma protein activity in riboflavin and UV light-treated fresh frozen plasma during 2 years of storage at -30 degrees C.

BACKGROUND: The Mirasol Pathogen Reduction Technology System (PRT) for Plasma (CaridianBCT) is based on a riboflavin and UV light treatment process resulting in pathogen inactivation due to irreversible, photochemically induced damage of nucleic acids. This study evaluated the in vitro protein quality of plasma products treated with riboflavin and UV light following treatment and subsequent storage for up to 104 weeks at -30 degrees C. MATERIALS AND METHODS: Apheresis and whole blood-derived plasma products were combined with riboflavin solution and exposed to ultraviolet light. Treated plasma was then flash frozen, within 8 h of collection, stored at -30 degrees C for up to 104 weeks and analysed at different stages of storage using standard coagulation assays. Results were compared with paired, untreated units stored for the same intervals. RESULTS: The average percent protein retention for all time-points in PRT-treated plasma samples after 36, 69, 87 and 104 weeks of storage at -30 degrees C in comparison with controls held under similar conditions were: Total Protein, 101%, Factor VIII, 79%, Fibrinogen, 78%, Factor II, 87%, Factor XII, 86%, Factor X, 84% and Factor IX, 81%. Anticoagulant and inhibitor proteins showed between 90% and 100% retention after 1 year (52 weeks) and 69 weeks of storage. No clinically relevant complement activation was observed in treated and stored samples. CONCLUSION: Riboflavin and UV light-treated plasma demonstrates reductions in several plasma coagulation factors following treatment. This reduction in activity levels is noted immediately after treatment and remains relatively constant during 2 years of storage at -30 degrees C.

Complement split products c3a and c4a in chronic lyme disease.

Complement split products C3a and C4a are reportedly elevated in patients with acute Lyme disease. We have now examined these immunologic markers in patients with chronic Lyme disease compared to appropriate disease controls. The study population consisted of 29 healthy controls, 445 patients with chronic Lyme disease, 11 patients with systemic lupus erythematosus (SLE) and six patients with AIDS. The Lyme disease patients were divided according to predominant musculoskeletal symptoms (324 patients) or predominant neurologic symptoms (121 patients). C3a and C4a levels were measured by radioimmunoassay. All patients with chronic Lyme disease and AIDS had normal C3a levels compared to controls, whereas patients with SLE had significantly increased levels of this marker. Patients with predominant musculoskeletal symptoms of Lyme disease and AIDS patients had significantly increased levels of C4a compared to either controls, patients with predominant neurologic symptoms of Lyme disease or SLE patients. Response to antibiotic therapy in chronic Lyme disease was associated with a significant decrease in the C4a level, whereas lack of response was associated with a significant increase in this marker. In contrast, AIDS patients had persistently increased C4a levels despite antiretroviral therapy. Lyme patients with positive single-photon emission computed tomographic (SPECT) scans had significantly lower C4a levels compared to Lyme patients with normal SPECT scan results. Patients with predominant musculoskeletal symptoms of Lyme disease have normal C3a and increased C4a levels. This pattern differs from the increase in both markers seen in acute Lyme disease, and C4a changes correlate with the response to therapy in chronic Lyme disease. C4a appears to be a valuable immunologic marker in patients with persistent symptoms of Lyme disease.

Increased serum C3 levels in Crry transgenic mice partially abrogates its complement inhibitory effects.

Complement receptor 1-related gene/protein y (Crry) is a potent murine complement regulator that inhibits C3 convertases. Transgenic mice that overexpress soluble Crry (sCrry), directed systemically by the metallothionein-I promoter, have been used as an animal model for chronic blockade of complement activation. Recently we have found that alternative pathway (AP) activity in Crry transgenic mice was not inhibited as much as expected. To elucidate the mechanism of this effect, we evaluated the AP activities and levels of sCrry and AP complement components in transgenic and non-transgenic mice. In transgenic mice, expression of sCrry was induced by feeding zinc sulphate solution to 70.1 +/- 42.7 micro g/ml mean serum level. Its corresponding level of purified sCrry inhibited 49% of AP activity of normal mice serum; however, the actual AP activities in transgenic mice were not decreased when compared to non-transgenic mice (130.2 +/- 9.0%versus 113.0 +/- 35.4%). Expressed sCrry was functional, as immunoprecipitation and removal of sCrry from transgenic sera with rabbit anti-Crry polyclonal antibody resulted in enhanced AP activity, consistent with initial levels of sCrry. We then compared the changes to C3, factor B, factor H and factor D serum levels in transgenic and non-transgenic mice after induction of sCrry expression. Of these only C3 was increased after zinc feeding in transgenic mice compared to non-transgenic mice (142.8 +/- 14.1%versus 121.4 +/- 15.1%, P = 0.023). These results suggest that the inhibitory effect of chronic exposure to sCrry is compensated by concomitant alteration in C3 levels. This result also suggests the presence of a complement regulatory protein controls the level of serum C3, which has potential importance in the design and interpretation of studies involving chronic use of complement inhibitors.

Hybridomas expressing gammadelta T-cell receptors respond to cardiolipin and beta2-glycoprotein 1 (apolipoprotein H).

Hybridomas expressing murine gammadelta T-cell receptors were found to produce cytokines in response to cardiolipin (CL) and structurally related anionic phospholipids. This response required serum at concentrations related to the amount of CL in cultures. The purified serum factor, beta2-glycoprotein 1 (beta2-GP1) (apolipoprotein H), supported the CL response alone, whereas several other serum proteins and ovalbumin did not. beta2-GP1 is known to form complexes with anionic phospholipids, particularly CL, which are often recognized by pathological autoantibodies. We speculate that gammadelta T cells also recognize such complexes and that the hybridoma response reported here reflects this specificity.

Classical pathway evaluation.

This unit describes several assay methods that can be used to determine the functional status of the classical pathway of complement and to quantitate its component proteins. The classical pathway includes C1qrs, C2, C4, C3, C5, C6, C7, C8, and C9, listed in the order in which they interact. Two CH(50) assay procedures are presented that test total classical pathway function; one is carried out in test tubes, whereas the alternate protocol describes a variation that is carried out in 96-well microtiter plates. Three support protocols describe preparing serum and erythrocyte-antibody complexes (EA) for use in CH(50) assays,and titrating of hemolysin for use in EA preparation. As an alternative to functional assays, radial immunodiffusion (RID) methods are also presented. These can be used to measure the concentrations of most circulating complement proteins and to evaluate the functional status of C1-esterase inhibitor. A fourth support protocol provides a method to determine antibody concentrations for RID assays.

Alternative pathway evaluation.

The alternative pathway of complement shares its terminal components (C3 and C5 through 9) with the classical pathway, but has several unique components, including factors D, B, and P (properdin). This unit presents methods for assaying total alternative pathway activity and the activity of factors B and D. Radial immunodiffusion (RID) can also be used to measure factor D, B, and P concentrations.

A comparative study of mammalian and reptilian alternative pathway of complement-mediated killing of the Lyme disease spirochete (Borrelia burgdorferi).

The potential bactericidal activity of the alternative complement pathway of mammalian and reptilian sera to Borrelia burgdorferi sensu stricto (s.s.) was evaluated in vitro. Complement-mediated killing was observed when cultured spirochetes were inoculated into sera from the western fence lizard (Sceloporus occidentalis) and from the southern alligator lizard (Elgaria multicarinata), but not when they were inoculated into serum from either the deer mouse (Peromyscus maniculatus) or from humans. Spirochetes were still alive after 4 hr in lizard serum that had been preheated at 56 C for 30 min to inactivate complement. Furthermore, when lizard serum was chelated with 10 mM ethylenediaminetetraacetic acid to block all complement activation, borreliacidal activity was arrested. When lizard serum was chelated with 10 mM ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid plus 4 mM MgCl2 to block only classical complement pathway activation, >85% of spirochetes were immobilized within 1 hr. Differences in B. burgdorferi s.s. mortality were not observed when chelators with or without MgCl2 were added to serum from either deer mice or humans. Proteins comprising the alternative complement pathway are responsible for the borreliacidal activity observed in the blood of S. occidentalis and E. multicarinata.

Activation of the alternative pathway of complement by a phosphorothioate oligonucleotide: potential mechanism of action.

Intravenous infusion of high doses of phosphorothioate oligonucleotides in monkeys has been associated with transient alterations in hematologic and hemodynamic parameters, which appear to be secondary to complement activation. ISIS 2302, a phosphorothioate oligonucleotide specific for human intracellular adhesion molecule-1, was used to further characterize complement activation in monkeys. Complement activation occurred selectively through the alternative pathway resulting in increased plasma concentrations of the complement split products Bb, C3a and C5a. Marked fluctuations in circulating neutrophil counts and reductions in cardiac output were closely associated with peak production of anaphylatoxins C3a and C5a. Changing both dose and infusion duration revealed that complement activation is related to plasma levels of oligonucleotide, and that there is a minimum threshold concentration of approximately 50 micrograms/ml of ISIS 2302 that is required to activate complement. Dose regimens in which plasma concentrations do not exceed this threshold do not result in complement activation. Further investigation reveals that plasma concentrations of a key regulatory component of the alternative pathway, Factor H, were also decreased after administration of ISIS 2302. Decreases in Factor H levels are suggestive of a possible mechanism of complement activation. Direct interaction between ISIS 2302 and Factor H was demonstrated in a competition assay, where increasing concentrations of ISIS 2302 eluted Factor H from a heparin-sepharose column. These data demonstrate a clear correlation between plasma oligonucleotide concentrations and complement activation. Interactions between ISIS 2302 and Factor H may lead to activation of the alternative complement pathway.

Rush immunotherapy results in allergen-specific alterations in lymphocyte function and interferon-gamma production in CD4+ T cells.

BACKGROUND: Allergen immunotherapy results in a number of changes in clinical, inflammatory, and immunologic parameters. However, the basis for the specificity of this form of therapy is unknown, especially in the context of changes in T- and B-lymphocyte function after desensitization to specific allergens. OBJECTIVE: This study was designed to determine the immunologic consequences of rush immunotherapy. METHODS: We studied 10 patients who had positive skin test responses to the house dust mite Dermatophagoides pteronyssinus (Dpt) and cat dander extract. Each received rush immunotherapy to mite, but not cat dander, over a 2- to 4-week period until maintenance was achieved. Patients were evaluated before and when maintenance was achieved for skin test and nasal reactivity to mite and cat dander; antibody levels to the allergen were monitored, as were lymphocyte proliferative responses and cytokine production. RESULTS: Rush immunotherapy to house dust mite resulted in a significant reduction in skin and nasal reactivity to mite allergen, but not to cat allergen, in 10 of 10 patients. This was accompanied by a rise in serum anti-Dpt IgE, whereas anti-cat IgE was not altered (7 of 7 patients). In seven of seven patients there was an increase in anti-Dpt IgG4 levels. T-cell proliferative responses to mite antigen were suppressed, and numbers of CD8+ T cells increased in frequency. There was a marked increase in interferon-gamma production, particularly by CD4+ T cells in 10 of 10 patients. The correlation between the increases in interferon-gamma production and the changes in cutaneous reactivity was highly significant. CONCLUSION: We show that rush immunotherapy is immunologically specific in eliciting changes in T- and B-cell responses to the desensitization antigen. The specificity and potential benefit of immunotherapy may be linked to the increase in interferon-gamma production by allergen-activated CD4+ T lymphocytes.

Age-dependent neutrophil and blood flow responsiveness in acute pulmonary inflammation in rabbits.

Diminished ability of neonatal neutrophils to orient and move in a chemotactic gradient has been linked to compromised pulmonary host defense. We investigated whether deficiency of neonatal neutrophil function in vitro was evident in acute pulmonary inflammation. Analysis of neutrophils in vitro showed impaired chemotaxis in 4-wk-old compared with adult rabbits. In vivo-directed migration of labeled neutrophils into the alveolar space of adult rabbits in response to C5f instillation was significantly less for neutrophils donated from 4-wk-old rabbits compared with those from adults. In contrast, there were no differences in the alveolar accumulation of 4-wk-old and adult labeled neutrophils in 4-wk-old rabbits in response to C5f instillation, although the response showed a shorter time course than seen in adult rabbits. Adult rabbits diverted 46% of the blood away from the right cranial lung lobe, whereas 4-wk-old rabbits showed no change in blood flow after C5f instillation. Megakaryocytes (a source of blood flow mediators) were 3.2-fold greater in adult compared with 4-wk-old lung. These data suggest that the lack of blood flow diversion from inflamed neonatal lung increases neutrophil migration into alveoli, allowing for preservation of an inflammatory response despite neutrophil deficiencies in chemotaxis.

Serum ferritin as a predictor of the acute respiratory distress syndrome.

We investigated serum ferritin levels as a predictor of the acute respiratory distress syndrome (ARDS) because: (1) proinflammatory cytokines, which are implicated in ARDS, increase ferritin synthesis; and (2) oxidative stress in patients at risk for ARDS might liberate iron from ferritin, accelerating toxic hydroxyl radical (.OH) formation. Serum ferritin levels measured by radioimmunoassay (RIA) were greater in 75 patients at risk for ARDS (women, p < 0.0001; men, p < 0.0001) and 8 patients with ARDS (women, p = 0.001; men, p = 0.0009) than in healthy control subjects. Serum ferritin levels were also greater in female (p = 0.003) and male (p = 0.003) at-risk patients who developed ARDS than in patients who did not develop ARDS. In women, a value exceeding 270 ng/ml predicted ARDS with an 83% sensitivity, 71% specificity, 67% positive, and 86% negative predictive value. In men, a value exceeding 680 ng/ml predicted ARDS with a 60% sensitivity, 90% specificity, 75% positive, and 82% negative predictive value. Serum ferritin levels did not correlate with C-reactive protein levels, were not different in medical or surgical at-risk patients, and were not accounted for by liver disease. Evaluating serum ferritin levels in at-risk patients may help predict the development of ARDS and thereby improve study and treatment of ARDS. Elevated serum ferritin levels may also regulate the participation of iron in the oxidative responses that contribute to ARDS.

Activation of complement by tissue plasminogen activator, but not acute cerebral ischemia, in a rabbit model of thromboembolic stroke.

Although complement activation is associated with tissue injury during inflammatory and ischemic states, complement activation in states of acute cerebral ischemia before and after administration of tissue plasminogen activator (TPA) has not yet been examined and is the focus of this investigation. Twenty-four New Zealand White rabbits weighing 3 to 3.5 kg were used for this study. Of these, 20 were subjected to intracranial autologous clot embolization via the internal carotid artery. Three hours postembolization, rabbits received an intravenous infusion of TPA (6.3 mg/kg, 20% bolus with the remainder infused over a 2-hour interval; 12 animals) or vehicle (eight animals). All animals were observed for a total of 7 or 8 hours postembolization. These two groups were compared to a cohort undergoing sham operation with subsequent TPA infusion (four animals). Plasma samples to quantify complement component C5 hemolytic activity (C5H5O) were obtained at the following time points: 30 minutes before and after clot embolization; 1 hour before and 1 hour after the initiation of therapy with TPA or vehicle and at the completion of the protocol; 7 to 8 hours after clot embolization. The C5 activation was not detected as the result of acute cerebral ischemia. However, animals receiving TPA with or without concomitant clot embolization exhibited C5 activation as assessed by a reduction in C5 hemolytic function, both 1 hour after initiation of TPA infusion (78.7 +/- 10.3% and 77.5 +/- 9.9% of baseline value, respectively; mean +/- standard error of the mean [SEM]) and at the end of the protocol, 2 hours after the completion of the TPA infusion (72.5 +/- 8.8% and 53.3 +/- 8.1%, respectively; mean +/- SEM, p < 0.05, each group). This study supports the conclusion that TPA, but not acute cerebral ischemia, may activate the complement cascade in this rabbit model of thromboembolic stroke.

Invasive Haemophilus influenzae type b infection in a child with familial deficiency of the beta subunit of the eighth component of complement.

A child who had had meningitis caused by Haemophilus influenzae type b, and then had meningococcal meningitis, was found to have familial deficiency of the beta subunit of the eighth component of complement. The child had not received the H. influenzae type b vaccine. If this deficiency is discovered, we recommend that family members be screened, regardless of their health status.

Complement activation and hemodynamic changes following intravenous administration of phosphorothioate oligonucleotides in the monkey.

Rapid intravenous infusion of GEM 91, a 25-mer phosphorothioate oligonucleotide complementary to the gag site of HIV, in the monkey produces transient decreases in peripheral total WBC and neutrophil counts, hemoconcentration, and a brief increase followed by a prolonged decrease in arterial blood pressure. These changes are preceded by and are likely mediated by activation of C5 complement. These effects are dose and infusion rate dependent and can be avoided by administering GEM 91 by slow intravenous infusion. Similar hemodynamic effects are produced with rapid intravenous infusion of other phosphorothioate oligonucleotides varying in length from 20- to 33-mer, and are, therefore, not sequence specific but a property of this chemical structure.

Peripheral bypass-induced pulmonary and coronary vascular injury. Association with increased levels of tumor necrosis factor.

BACKGROUND: Although cardiopulmonary bypass is associated with systemic complement activation and neutrophil sequestration, it is unclear whether bypass-induced vascular injury is localized and dependent on organ ischemia. We hypothesized that other factors perhaps related to placement of a bypass circuit or to blood perfusion of a pump-oxygenator system may produce vascular injury caused by systemically circulating mediators. In dogs, we determined whether application of a systemic venoarterial bypass circuit with pump-oxygenator perfusion but without pulmonary or cardiac flow diversion (peripheral bypass) leads to vascular injury. Since several features of the postperfusion syndrome after bypass resemble sequelae of endotoxin exposure, we also measured circulating endotoxin and tumor necrosis factor levels. METHODS AND RESULTS: Anesthetized dogs underwent 2 hours of exposure to a pump-oxygenator with peripheral venoarterial bypass. We used a double indicator measurement of pulmonary and coronary vascular permeability (protein leak index [PLI]) as indexes of vascular injury. Compared with controls (n = 7), the pulmonary PLI of dogs undergoing bypass (n = 11) increased more than threefold (18.8 +/- 2.3 vs 63.3 +/- 7.6 x 10(-4) min-1; P < .05) and the coronary PLI increased more than twofold (P < .05). The rate of disappearance of intravascular radiolabeled protein increased threefold after bypass (disappearance t1/2, 241 +/- 35 vs 84 +/- 15 minutes, control vs bypass; P < .05), suggesting a generalized increase in vascular permeability. Circulating endotoxin was detectable in blood samples from 8 of 8 bypass animals (range, 0.24 to 4.56 ng/mL) compared with 2 of 5 controls (P < .05). Tumor necrosis factor levels increased significantly with bypass (6.7 +/- 3.8 vs 146.7 +/- 33.6 U/mL, baseline vs bypass; P < .05) and were only slightly and nonsignificantly increased in controls (7.0 +/- 4.4 vs 18.2 +/- 5.9 U/mL; P = NS). Peak tumor necrosis factor but not peak endotoxin levels correlated with pulmonary and with coronary protein leak. As expected, circulating complement (CH50) levels decreased significantly during bypass, reflecting systemic complement activation. However, the levels correlated poorly with the severity of vascular injury. CONCLUSIONS: We conclude that peripheral pump-oxygenator bypass causes coronary and pulmonary vascular injury that is independent of blood flow diversion and is associated with the appearance of circulating levels of endotoxin and tumor necrosis factor, which may play a role in bypass-induced vascular injury.

A comparative study of complement activation by Denaflex, Bioflow, and BioPolyMeric vascular grafts.

This study was performed to evaluate the degree of complement activation by three bovine arterial graft materials: Bioflow (Bio-Vascular Inc., a bovine artery fixed with dialdehyde starch), BioPolyMeric (St. Jude Medical Inc., a collagen conduit of bovine arterial origin, tanned with glutaraldehyde and covered with a Dacron mesh), and Denaflex (Baxter Edwards CVS Division, a bovine artery fixed with polyepoxy compounds). The grafts were rinsed by following the manufacturer's recommended procedures and thereafter incubated with normal human serum. CH50 assays were performed on the serum after incubation, and the percentage of complement activation for each sample was calculated relative to its control serum. The results indicated that the BioPolyMeric grafts activated the most complement, with about a 48% decrease in the CH50. The BioPolyMeric graft is composed of an outer polyester mesh and an inner collagenous tubing, exhibiting a nonreversible negative surface charge. After the polyester mesh was removed, the BioPolyMeric graft showed the highest complement activation in this study, suggesting that the glutaraldehyde fixed graft is more prone to complement activation than either the polyepoxy compound or dialdehyde starch fixed grafts. The complement fragment, C5a, generated during complement activation is strongly chemotactic for polymorphonuclear leukocytes and monocytes, which likely play early and long-lasting roles in regulating tissue reaction to the implanted graft.

Immunoglobulin G, total and subclass, in children with or without recurrent otitis media.

Total IgG and subclasses IgG1, IgG2, IgG3, and IgG4 were measured in 89 subjects with recurrent otitis media. There was no significant difference between the groups with respect to the arithmetic or geometric mean levels for total IgG or subclasses IgG1, IgG2, IgG3, or IgG4.

Complement activation is a secondary rather than a causative factor in rabbit pulmonary artery ischemia/reperfusion injury.

We have previously demonstrated that reperfusion of a rabbit lung in vivo after 24 h of unilateral pulmonary artery occlusion results in edema, transient leukopenia, and intravascular leukocyte aggregation. We hypothesized that complement was activated by reperfusion and that this in turn contributed to lung injury. In the preliminary phase of the study, we found that ischemia followed by reperfusion resulted in a drop in C3 to 15 +/- 10% (mean +/- SEM) of the prereperfusion value as compared with no change in a group of control animals that had undergone an identical thoracotomy but without pulmonary artery occlusion and reperfusion (p less than 0.05). We then studied three groups of animals to determine if complement depletion with cobra venom factor (CVF) prior to ischemia and reperfusion would prevent the injury. Rabbits treated with CVF but without occlusion and reperfusion did not develop significant lung edema, with left and right lung wet/dry ratios of 5.32 +/- 0.11 and 5.26 +/- 0.12, respectively. For rabbits that were not treated with CVF but underwent ischemia and reperfusion, the comparable numbers were 6.15 +/- 0.36 and 5.19 +/- 0.32 (p less than 0.05 for right versus left). For CVF-treated rabbits that underwent ischemia and reperfusion, the right/left difference persisted (6.77 +/- 0.48 versus 5.35 +/- 0.14, p less than 0.01). Immunocytochemistry documented C3 deposition in non-CVF rabbits that underwent ischemia and reperfusion but not in CVF-treated rabbits. We conclude that ischemia/reperfusion of the lung results in complement activation, but it is not a complement-dependent injury.