Monitoring of complement activation biomarkers and eculizumab in complement-mediated renal disorders.

Various complement-mediated renal disorders are treated currently with the complement inhibitor eculizumab. By blocking the cleavage of C5, this monoclonal antibody prevents cell damage caused by complement-mediated inflammation. We included 23 patients with atypical haemolytic uraemic syndrome (aHUS, n = 12), C3 glomerulopathies (C3G, n = 9) and acute antibody-mediated renal graft rejection (AMR, n = 2), treated with eculizumab in 12 hospitals in Germany. We explored the course of complement activation biomarkers and the benefit of therapeutic drug monitoring of eculizumab. Complement activation was assessed by analysing the haemolytic complement function of the classical (CH50) and the alternative pathway (APH50), C3 and the activation products C3d, C5a and sC5b-9 prior to, 3 and 6 months after eculizumab treatment. Eculizumab concentrations were determined by a newly established specific enzyme-linked immunosorbent assay (ELISA). Serum eculizumab concentrations up to 1082 µg/ml point to drug accumulation, especially in paediatric patients. Loss of the therapeutic antibody via urine with concentrations up to 56 µg/ml correlated with proteinuria. In aHUS patients, effective complement inhibition was demonstrated by significant reductions of CH50, APH50, C3d and sC5b-9 levels, whereas C5a levels were only reduced significantly after 6 months' treatment. C3G patients presented increased C3d and consistently low C3 levels, reflecting ongoing complement activation and consumption at the C3 level, despite eculizumab treatment. A comprehensive complement analysis together with drug monitoring is required to distinguish mode of complement activation and efficacy of eculizumab treatment in distinct renal disorders. Accumulation of the anti-C5 antibody points to the need for a patient-orientated tailored therapy.

Complement profile in neonates of different gestational ages.

Blood levels of regulators of the complement system in preterm babies were reported in few studies only. The aim of this study was to set up a complement profile in premature and term babies focusing on the development of blood levels of MBL, key regulatory proteins and on classical pathway activity, which may allow an estimation of potential susceptibility to infection. Complement activity (CH50), levels of mannan-binding lectin (MBL), complement regulators (factors H and I, C1 inhibitor, properdin) and C3a as marker of complement activation were assessed in three groups of healthy newborns: (1) prematures (≤34 weeks); (2) late prematures (>34-<37 weeks) and (3) term neonates (≥37 weeks). CH50 increased with gestational age with lower titres in cord blood than in day 5 post-delivery venous blood. MBL concentrations were not significantly different among groups. Quantitative and functional C1 inhibitor were below adult normal range in prematures <34 weeks and lower in cord blood as compared to day 5. Factor I, factor H and properdin remained below adult values in all groups. Low C3a levels excluded that low complement titres were due to activation-induced consumption. These results demonstrate the relative immaturity of the complement system and its regulation, especially in premature infants.

Sublytic complement protects prostate cancer cells from tumour necrosis factor-α-induced cell death.

Inflammation is a critical component of tumour progression. Although complement and tumour necrosis factor (TNF)-α potentially exert significant anti-tumour effects, both mediators may also promote tumour progression. It has been demonstrated that sublytic complement confers resistance on tumour cells not only against lytic complement, but also other danger molecules such as perforin. In low concentrations, TNF promotes survival of malignant cells rather than exerting cytotoxic activity. In this study, we tested if sublytic complement is able to interfere with TNF-mediated tumour cell killing. Our results demonstrate that either subcytotoxic concentrations of TNF or sublytic complement rescue prostate carcinoma cells (DU145) from TNF-α-mediated cell death. Upon pretreatment with low-dose TNF-α, but not upon pre-exposure to sublytic complement, TNF resistance was associated with the down-regulation of TNF receptor 1 (TNF-R1) expression. Complement-induced protection against TNF-mediated apoptosis accompanied the induction of anti-apoptotic proteins [B cell leukaemia/lymphoma (Bcl)-2 and Bcl-xL] at an early stage followed by inhibition of the TNF-induced decrease in the amount of Bcl-2 and Bcl-xL. Cell protection also accompanied the inhibition of caspase-8 activation, poly (ADP-ribose) polymerase (PARP)-1 cleavage and the activation of nuclear factor (NF)-κB. Our data extend our current view on the induction of tumour cell resistance against cytotoxic mediators supporting the role of the tumour microenvironment in mediating protection against the anti-cancer immune response.

Enhanced complement resistance in drug-selected P-glycoprotein expressing multi-drug-resistant ovarian carcinoma cells.

Multi-drug resistance (MDR) is a major obstacle in cancer chemotherapy. There are contrasting data on a possible correlation between the level of expression of the drug transporter P-glycoprotein (P-gp) and susceptibility to complement-dependent cytotoxicity (CDC). We therefore investigated the sensitivity of human ovarian carcinoma cells and their P-gp expressing MDR variants to complement. Chemoselected P-gp expressing MDR cells showed increased resistance to CDC associated with overexpression of membrane-bound complement regulatory proteins (mCRP) and increased release of the soluble inhibitors C1 inhibitor and factor I. MDR1 gene transfection alone did not alter the susceptibility of P-gp expressing A2780-MDR and SKOV3-MDR cells to CDC. However, subsequent vincristine treatment conferred an even higher resistance to complement to these cells, again associated with increased expression of mCRP. Blocking the function of P-gp with verapamil, cyclosporine A or the anti-P-gp-antibody MRK16 had no impact on their complement resistance, whereas blocking of mCRP enhanced their susceptibility to complement. These results suggest that enhanced resistance of chemoselected MDR ovarian carcinoma cells to CDC is not conferred by P-gp, but is due at least partly to overexpression of mCRP, probably induced by treatment with the chemotherapeutic agents.

Complement 4 phenotypes and genotypes in Brazilian patients with classical 21-hydroxylase deficiency.

The aim of this work was to analyse C4 genotypes, C4 protein levels, phenotypes and genotypes in patients with the classical form of 21-hydroxylase deficiency. Fifty-four patients from 46 families (36 female, 18 male; mean age 10.8 years) with different clinical manifestations (31 salt-wasting; 23 simple-virilizing) were studied. Taq I Southern blotting was used to perform molecular analysis of the C4/CYP21 gene cluster and the genotypes were defined according to gene organization within RCCX modules. Serum C4 isotypes were assayed by enzyme-linked immunosorbent assay. The results revealed 12 different haplotypes of the C4/CYP21 gene cluster. Total functional activity of the classical pathway (CH50) was reduced in individuals carrying different genotypes because of low C4 concentrations (43% of all patients) to complete or partial C4 allotype deficiency. Thirteen of 54 patients presented recurrent infections affecting the respiratory and/or the urinary tracts, none of them with severe infections. Low C4A or C4B correlated well with RCCX mono-modular gene organization, but no association between C4 haplotypes and recurrent infections or autoimmunity was observed. Considering this redundant gene cluster, C4 seems to be a well-protected gene segment along the evolutionary process.

Down-regulation of CD55 and CD46 expression by anti-sense phosphorothioate oligonucleotides (S-ODNs) sensitizes tumour cells to complement attack.

Overexpression of one or more membrane-bound complement regulatory proteins (mCRPs) protects tumour cells against complement-mediated clearance by the autologous humoral immune response and is also considered as a barrier for successful immunotherapy with monoclonal anti-tumour antibodies. Neutralization of mCRPs by blocking antibodies, enzymatic removal or cytokine-mediated down-regulation has been shown to sensitize tumour cells to complement attack. In our study we applied, for the first time, anti-sense phosphorothioate oligonucleotides (S-ODNs) to knock down the expression of the mCRPs CD55 and CD46 with the aim of exploiting complement more effectively for tumour cell damage. Potent anti-sense oligonucleotides against CD55 and CD46 were identified by screening various target sequences (n = 10) for each regulator. S-ODN anti-CD55(687) reduced CD55 protein expression up to 84% and CD46 protein expression was inhibited up to 76% by S-ODN anti-CD46(85). Reverse transcription-polymerase chain reaction (RT-PCR) analysis revealed a similar reduction of the CD55 and CD46 mRNA levels, which argues for an RNAse H-dependent anti-sense mechanism. T47D, A549 and PC3 cells, representing breast, lung and prostate carcinoma, were used for functional studies. Dependent on the particular cell line, anti-sense-based inhibition of mCRP expression enhanced complement-dependent cytolysis (CDC) up to 42% for CD55 and up to 40% for CD46, and the combined inhibition of both regulators yielded further additive effects in T47D cells. C3 opsonization of CD55/CD46-deficient tumour cells was also clearly enhanced upon mCRP suppression. Due to the clinical applicability of S-ODNs, the anti-sense approach described in this study may offer an additional alternative to improve the efficacy of antibody- and complement-based cancer immunotherapy.

Local complement activation in aqueous humor in patients with age-related macular degeneration.

PurposeTo investigate complement activation in aqueous humor and in plasma of patients with neovascular age-related macular degeneration (nAMD).Patients and methodsAqueous humor and EDTA-plasma of 31 nAMD patients and 30 age-matched controls was collected. The levels of the complement factor 3 (C3), the regulators factor H (FH), and factor I (FI), and of the complement activation products Ba, C3a, and the terminal complement complex (sC5b-9) were measured. Associations between complement levels and phenotype were determined using Mann-Whitney U-test.ResultsIn plasma, no significant differences were found between the nAMD group and the control group. In aqueous humor, significantly increased levels of Ba (P=0.002), and C3a (P=0.002) indicate local complement activation in nAMD patients and a trend for a concomitant upregulation of the complement regulators FH (P=0.02) and FI (P=0.04).ConclusionsOur findings provide strong evidence for a local complement dysregulation in nAMD patients.

Binding and activation of C4 and C3 on the red cell surface by non-complement enzymes.

We investigated the binding of C4 and C3 to red cell surfaces by non-complement enzymes. Cell bound C components were quantitated by a radioimmunoassay, the chain structure of bound components was analyzed by Western blotting and the hemolytic activity of bound components was determined. Trypsin, chymotrypsin, plasmin, elastase, thrombin, kallikrein and enzymes from Bacillus subtilis, Staphylococcus aureus and Streptomyces griseus all were found capable of binding C4b and C3b to sheep red cells. C4b bound by any of these enzymes was hemolytically active; both classical and alternate pathway activity of C3 could be demonstrated for most enzymes except plasmin and thrombin. In addition, trypsin and the bacterial enzymes were also able to generate the classical pathway C3-convertase from C4b + C2. The hemolytic efficiency of enzyme bound C4b and C3b was about the same as for these molecules bound by complement enzymes. In contrast, the process of binding by the non-complement enzymes was several hundred-fold less efficient than by cell bound complement enzymes. The results demonstrate that several enzymes can replace the C1 and C42 enzymes in the classical pathway and are able to initiate the alternative pathway by activating C3 and binding C3b to the cell surface.

Augmentation of hemolytic efficiency of anti-hapten IgG antibody by anti-IgG antibody is primarily the function of the anti-hapten IgG.

IgG anti-cell antibodies are inefficient in inducing cell lysis by complement. C mediated lysis by anti-cell IgG can be augmented by the use of C fixing anti-antibody. We have studied augmentation of rabbit anti-Forssman IgG, rabbit anti-methotrexate IgG and rabbit anti-folinic acid IgG antibodies by rabbit anti-allotype IgG antibodies. Large variability (from a low of 2 to a high of over 100) was found in augmentation efficiency even with the same anti-allotype antibody; the variability was primarily the function of the anti-hapten antibody. It was concluded that quantitative studies on anti-hapten IgG production relying on augmented hemolysis may lead to misleading conclusions.

C1 inhibitor in anti-inflammatory therapy: from animal experiment to clinical application.

Potentially life-threatening consequences due to severe inflammatory tissue destruction are often closely associated with an excessive activation of the complement system. Various clinical disorders, including capillary leak syndrome, septic shock, multiple organ failure and hyperacute graft rejection are at least in part driven by an overactivated complement system. Therapeutic support of complement regulation appears to be a logical approach to reduce undesirable inflammatory reactions. C1 inhibitor, a multifunctional regulator of all major kinin-generating protein cascade systems, is frequently observed to be reduced in patients suffering from severe inflammation, due to ligand-induced inactivation of the regulatory protein. After C1 inhibitor has for many years been proven beneficial in acute treatment of hereditary angioedema, a growing number of reports now suggests that C1 inhibitor provides an effective means to protect against complement-mediated inflammatory tissue damage. These studies not only include pathophysiologically relevant animal models but also first attempts to prove the benefits of C1 inhibitor as a novel therapeutic approach in clinical trials.

Obstacles to cancer immunotherapy: expression of membrane complement regulatory proteins (mCRPs) in tumors.

Monoclonal antibodies (mAbs) are being increasingly used in cancer therapy owing to their ability to recognize specifically cancer cells and to activate complement- and cell-mediated cytotoxicity and/or to induce growth arrest or apoptosis. The therapeutic potential of anticancer antibodies is significantly limited due to the ability of cancer cells to block killing by complement. Of the multiple resistance strategies exploited by cancer cells, the expression of membrane complement regulatory proteins (mCRPs), such as CD46 (membrane cofactor protein (MCP)), CD55 (decay-accelerating factor (DAF)), CD35 (complement receptor type-1 (CR1)) and CD59, has received most attention. CD46, CD55 and CD35 block the complement cascade at the C3 activation stage and CD59 prevents assembly of the membrane attack complex of complement (MAC). These proteins protect normal tissues from accidental injury by activated complement, but also confer resistance on cancer cells, thereby limiting the effect of complement-fixing monoclonal antibodies. Expression of mCRPs on malignant cells is highly variable, yet there is clear indication that certain tumors express higher mCRP levels than the normal tissue from which they have evolved. mCRP level of expression and cellular location may also vary during malignant transformation and between differentiated and undifferentiated tumors. Neutralizing anti-mCRP mAbs have been used in vitro to elucidate the significance of mCRP expression to the tumor complement resistance phenotype. In general, CD59 appears to be the most effective mCRP protecting tumor cells from complement-mediated lysis. Nevertheless, it acts additively, and in certain tumors even synergistically, with CD55 and CD46. It is envisaged that treatment of cancer patients with mCRP blocking antibodies targeted specifically to cancer cells in combination with anticancer complement-fixing antibodies will improve the therapeutic efficacy.

Neutrophil priming by cytokines and vitamin D binding protein (Gc-globulin): impact on C5a-mediated chemotaxis, degranulation and respiratory burst.

At the site of acute inflammation, leukocytes are confronted with multiple mediators which are expected to modulate each other with respect to cell responses to the individual ligand. Previous contact of neutrophils with pro-inflammatory cytokines, such as TNF-alpha or GM-CSF, or with the vitamin D binding protein (Gc-globulin) leads to the alteration of either multiple or rather distinct C5a-mediated neutrophil functions. Gc-globulin, the transport protein for 25-(OH)-D3, serves selectively as a cochemotactic factor for C5a/Ca(des)Arg. In contrast, TNF-alpha and GM-CSF, previously shown to modulate FMLP-induced neutrophil responses, are able to reduce C5a-mediated neutrophil chemotaxis, but augment their degranulation and respiratory burst activity. Cytokine priming was shown to be accompanied by a down-regulation of C5a receptors (CD88) whereas vitamin D binding protein had no impact on the level of neutrophil C5a receptors. C5a itself diminishes chemotaxis as well as degranulation and oxidative burst in response to a second dose of the same ligand (homologous desensitization). A similar effect, termed heterologous desensitization, occurs, if cell responses to a given mediator (e.g. to C5a) are reduced or even abolished upon the activation of another receptor of the same G-protein coupled chemoattractant receptor subfamily (e.g. receptors for FMLP or IL-8). In concert with C5a, certain molecules may either augment chemotaxis or shift neutrophil effector functions from migration to exocytosis, an essential step within the sequence of events in a coordinated inflammatory response.

Complement resistance of tumor cells: basal and induced mechanisms.

Clinical and experimental studies have suggested that complement may play a role in tumor cytotoxicity. However, the efficiency of complement-mediated tumor cell lysis is hampered by various protective mechanisms, which may be divided into two categories: basal and induced mechanisms. The basal mechanisms are spontaneously expressed in cells without a need for prior activation, whereas the induced mechanisms develop in cells subjected to stimulation with cytokines, hormones, drugs or with sublytic doses of complement and other pore-formers. Membrane-associated complement regulatory proteins, such as CD55 (DAF, Decay-Accelerating Factor), CD46 (MCP, Membrane Cofactor Protein), CD35 (CR1, Complement Receptor type 1) and CD59, which serve as an important mechanism of self protection and render autologous cells insensitive to the action of complement. appear to be over-expressed on certain tumors. Furthermore, tumor cells secrete several soluble complement inhibitors. Tumor cells may also express proteases that degrade complement proteins, such as C3, or ecto-protein kinases which can phosphorylate complement components, such as C9. Besides this basal resistance, nucleated cells resist, to some extent, complement damage by removing the membrane attack complexes (MAC) from their surface. Several biochemical pathways, including protein phosphorylation, activation of G-proteins and turnover of phosphoinositides have been implicated in resistance to complement. Calcium ion influx and activation of protein kinase C (PKC) and of mitogen-activated protein kinase (MAPK) have also been demonstrated to be associated with the complement-induced enhanced resistance to lysis. The complete elucidation of the molecular mechanisms involved in basal and induced tumor cell resistance will enable the development of strategies for interfering with these evasion mechanisms and the use of the cytotoxic complement system against tumor cells.

Inherited neuroaxonal dystrophy in C6 deficient rabbits.

We report the occurrence of a progressive neurological syndrome clinically characterized by subacute motor neuropathy in offspring of C6 deficient rabbits. On the basis of the pedigree analysis, the disease appears to be genetically transmitted, most probably with an autosomal recessive mode of inheritance. Pathological studies of affected animals revealed: transmitted, most probably with an autosomal recessive mode of inheritance. Pathological studies of affected animals revealed: 1) severe axonal degeneration in the sciatic nerve system involving mainly motor fibers; 2) occasional peripheral axonal enlargement closely associated with axonal degeneration; 3) presence of structured abnormal material in normal-size myelinated fibers of central nervous system (CNS) and peripheral nervous system (PNS); and 4) widespread occurrence of dystrophic axons and axonal spheroids in the gray matter of CNS. By ultrastructural examination, dystrophic axons are filled with tubulovesicular material, stalks of parallel membranes and dense bodies similar to what is described in human neuroaxonal dystrophies (NAD). The disease manifested by C6 deficient rabbits may represent an animal model of primary human NAD.

Protection of porcine endothelial cells from complement-mediated cytotoxicity by the human complement regulators CD59, C1 inhibitor, and soluble complement receptor type 1. Analysis in a pig-to-human in vitro model relevant to hyperacute xenograft rejection.

Inhibition of complement activation is considered a prerequisite to overcome hyperacute xenograft rejection. In the present study, we investigated the efficacy of C1 inhibitor (C1 inh) and recombinant soluble complement receptor type 1 (rsCR1) to protect xenogeneic cells against complement-mediated cytotoxicity in an in vitro xenotransplantation model. The addition of the soluble complement regulators to human serum led to a dose-dependent inhibition of complement-mediated destruction of aortic porcine endothelial cells (PEC). On a molar base, rsCR1 was more efficient than C1 inh. Transfection of PEC with cDNA of human CD59 resulted in several clones where protection against complement-mediated cell destruction correlated with the expression level of the inhibitor. Addition of low concentrations of C1 inh and rsCR1 to a CD59 (human)-positive PEC clone, expressing a suboptimal level of the membrane-bound regulator, resulted in a significant improvement of protection against complement-mediated cell destruction.

Mouse-to-rabbit xenotransplantation: a new small animal model of hyperacute rejection mediated by the classical complement pathway.

BACKGROUND: Hyperacute rejection of porcine organs transplanted into primate recipients is initiated by the binding of preformed xenoreactive natural antibodies to the vascular endothelium of the graft and activation of the classical complement pathway. Several small animal models are currently employed to study various aspects of xenograft rejection; however, none has been shown to manifest hyperacute rejection mediated by the classical pathway of complement activation. METHODS: We performed heterotopic mouse heart transplants into weanling rabbits, adult rabbits, and C6-deficient rabbits. The recipients received no immunosuppression. Rejected grafts were subjected to histologic analysis and immunofluorescence staining for rabbit IgG, IgM, and C3. Levels of preexisting cytotoxic antibodies as well as classical and alternative complement pathway activities were determined in rabbit serum using mouse red cells as targets. RESULTS: Mean graft survival was 37+/-9.6 min for mouse-to-weanling rabbit transplants (n=10), and 40+/-11.1 min for mouse-to-adult rabbit transplants (n=5). Rejected grafts showed diffuse interstitial hemorrhage, endothelial cell damage, myocyte necrosis, moderate diffuse deposition of rabbit IgG, and dense deposition of rabbit IgM and C3 on the vascular endothelium of the graft, consistent with hyperacute rejection. One mouse-to-C6-deficient rabbit transplant was rejected at 21 hr with severe interstitial hemorrhage, cellular necrosis and a moderate cellular infiltrate consisting primarily of neutrophils and some mononuclear cells. A second transplant in a C6-deficient rabbit was functioning when the recipient died at 6.5 hr as a result of complications of surgery; the graft had normal myocytes and vasculature with minimal spotty interstitial hemorrhage. Both weanling and adult rabbit serum were found to have high titers of cytotoxic IgM anti-mouse antibodies and strong classical complement pathway activity with minimal alternative pathway activity towards mouse red cells. CONCLUSIONS: The mouse-to-rabbit species combination manifests hyperacute xenograft rejection. In vitro studies suggest that this process is mediated by IgM anti-mouse natural antibodies and activation of the classical pathway of complement.

Complement inhibition by soluble complement receptor type 1 improves microcirculation after rat liver transplantation.

BACKGROUND: Recent observations provide evidence that complement is involved in the pathophysiology of ischemia/reperfusion injury. In this study, we assessed the impact of complement inhibition on hepatic microcirculation and graft function using a rat model of liver transplantation. METHODS: Arterialized orthotopic liver transplantation was performed in Lewis rats after cold preservation (University of Wisconsin solution, 4 degrees C, 24 h). Eight animals received the physiological complement regulator soluble complement receptor type 1 (sCR1) intravenously 1 min before reperfusion. Controls received Ringer's solution (n=8). Microvascular perfusion, leukocyte adhesion, and Kupffer cell phagocytic activity were studied 30-100 min after reperfusion by in vivo microscopy. RESULTS: Microvascular perfusion in hepatic sinusoids was improved in the sCR1 group (87+/-0.7% vs. 50+/-1%; P < 0.001). The number of adherent leukocytes was reduced in sinusoids (68.3+/-4.7 vs. 334.1+/-15.8 [adherent leukocytes per mm < or = liver surface]; P < 0.001) and in postsinusoidal venules after sCR1 treatment (306.6+/-21.8 vs. 931.6+/-55.9 [adherent leukocytes per mm < or = endothelial surface]; P < 0.001). Kupffer cell phagocytic activity was decreased in the sCR1 group compared to controls. Postischemic bile production reflecting hepatocellular function was increased by almost 200% (P = 0.004) after complement inhibition. Plasmatic liver enzyme activity was decreased significantly upon sCR1 treatment, indicating reduced parenchymal cell injury. CONCLUSIONS: Our results provide further evidence that the complement system plays a decisive role in hepatic ischemia/reperfusion injury. We conclude that complement inhibition by sCR1 represents an effective treatment to prevent reperfusion injury in liver transplantation.

In vitro complement activation by ragweed allergen extract in the sera of ragweed allergic and non-allergic persons.

Ragweed allergen (RWA)-induced complement activation in sera of 40 RW allergic patients and of 40 non-allergic controls was investigated. After treatment of the sera with RWA, levels of C3a, C5a, C3bBbP, C1rC1sClinh, SC5b-9 and granulocyte-aggregating activity were determined. Concentration of RW-specific IgG was also measured. After RWA treatment dose-dependent complement activation was detected in sera of RW allergic and non-allergic persons. C3a generation was observed mostly in the sera of RW allergic individuals, while levels of C3bBbP and of RW-specific IgG were significantly higher in sera of allergics, and a strong correlation was found between these two parameters. In a prospective clinical study, a significant positive correlation was observed between the extent of RWA-induced alternative pathway (AP) activation and the severity of symptoms of allergic rhinoconjunctivitis that were developed in a 4-week period subsequent to blood sampling. These observations suggest that complement activation has a role in the development of the symptoms of RW allergy.

The complement regulators C1 inhibitor and soluble complement receptor 1 attenuate acute lung injury in rabbits.

Because activation of the complement system plays a major role in the pathogenesis of acute lung injury, the availability of new specific complement inhibitors represents a promising therapeutic approach. In the present study we investigated pulmonary edema formation and pulmonary artery pressure (PAP) in acute complement-induced lung injury for possible therapeutic impact of the complement regulators C1 inhibitor and soluble complement receptor 1. Eighteen isolated and ventilated rabbit lungs were perfused with pooled normal human serum (NHS, final concentration 35%) in Krebs-Henseleit buffer in a recirculating system. Lung weight gain and PAP were continuously recorded. Complement activation was blocked by the addition of C1 inhibitor (1.0 U/mL, n = 6) or sCR 1 (2.0 microg/mL, n = 6). Lungs that received NHS without inhibitors served as controls (n = 6). This study was performed according to the Helsinki Declaration and approved by the local government. Application of NHS resulted in an increase of PAP within 20 min from 8+/-2 to 42+/-6 mmHg, which was significantly (P < 0.05) decreased by C1-Inh (25+/-5 mmHg) and sCRI (20 +/-3 mmHg). Moreover, pulmonary edema formation after NHS, as assessed by overall weight gain, was reduced by both C1-Inh and sCR1, compared with controls. These findings were paralleled with significantly decreased thromboxane release rates and reduced tissue deposition of C3c and C5b-9. C1 inhibitor and sCR1 attenuate the complement-induced pulmonary capillary leakage and PAP increase, indicating the protective effect of complement inhibition in isolated perfused rabbit lungs.

Mechanism of complement resistance of pathogenic Borrelia burgdorferi isolates.

Borrelia burgdorferi, the causative agent of Lyme disease, differ in their susceptibility to normal human serum and are consequently classified as complement-resistant, complement-sensitive and intermediate complement-sensitive. Most isolates belonging to the genospecies B. afzelii are complement-resistant, while particularly B. garinii isolates were rapidly killed by complement. In general, isolates of the genospecies B. burgdorferi sensu stricto (s.s.) are intermediate complement-sensitive. Independent of the genospecies, all Borreliae were capable to activate the classical and/or the alternative pathway. Deposition of the activation products C3, C6, and TCC is much stronger by B. burgdorferi s.s. and B. garinii isolates than by B. afzelii isolates. The mechanism(s) on how Borreliae evade complement-mediated bacteriolysis has recently been described by showing that complement-resistant B. afzelii isolates but not the complement-sensitive B. garinii isolates absorb human complement regulators FHL-1/reconectin and factor H. Surface-attached FHL-1/reconectin maintains its complement regulatory activity and supports factor I-mediated C3b cleavage to iC3b. In complement-resistant Borreliae, two outer surface proteins, the 27.5 kDa (CRASP-1, complement regulator-acquiring surface protein 1) and the 20/21 kDa (CRASP-2), are responsible for the surface attachment of the two complement regulators. CRASP-1, which is present in complement-resistant Borreliae, binds preferentially FHL-1/reconectin while CRASP-2, which is restrictively expressed, binds preferentially factor H. Thus, complement-resistant Borreliae bind human complement regulators and control complement activation on their surface and prevent the formation of toxic activation products.