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PubMed1998

High-dose intravenous immunoglobulin treatment activates complement in vivo.

Several complement modulating effects of high-dose intravenous immunoglobulins (IVIG) have been proposed from in vitro studies and experimental animal models. However, the in vivo effects of IVIG on plasma complement in humans are yet not known. We have investigated the in vivo effects of IVIG on complement in seven women with unexplained recurrent spontaneous abortion who were without evidence of autoimmune disease. Samples were obtained before and after the very first infusion of IVIG. There was a marked increase in immunoglobulin G (IgG) from (median and range) 12.4 (9.4-15.9) to 26.8 (22.4-30.0) g/l but no change in immunoglobulin A (IgA) or immunoglobulin M (IgM). A significantly increased complement activation was demonstrated using neoepitope-specific enzyme immunoassays to the activation products C3bc (median increased from 9.8 to 31.2 AU/ml), Bb (0.66-1.66 g/ml), C5a (10.5-12.7 ng/ml), and TCC (0.81-2.19 AU/ml) (P = 0.015 for all). There were no changes in antigenic concentrations of individual complement components or regulators (C1q, C4, C3, C1-inhibitor, C4b-binding protein) and no decrease in complement haemolytic activity (classical and alternative CH50), which were all within the normal range. The classical pathway activation products C1rs/C1-inhibitor complexes, C4bc and C4d were elevated in all patients before IVIG treatment and did not change significantly during treatment. In conclusion, IVIG induced a significant activation of complement in vivo.

Abortion, Habitual↗
PubMed2005

Investigation of the mechanisms of anti-complement activity in Ixodes ricinus ticks.

The feeding success of a tick upon a host depends on its ability to suppress host anti-tick responses which include activation of the complement system. We investigated the mechanism of inhibition of the alternative pathway of complement by salivary gland extract (SGE) of the ixodid tick species, Ixodes ricinus. SGE treatment strongly inhibited C3a generation and factor B cleavage in serum when rabbit erythrocytes were used as complement activator, but not when cobra venom factor (CVF) was used as an activator. SGE treatment strongly inhibited C3b deposition on rabbit erythrocytes, and the turnover of C3 (to C3b/iC3b) in serum. However, there was no significant effect upon the formation, stability or activity of C3 convertase (C3bBb) when formed from purified C3b, factor B and factor D. SGE treatment of isolated C3 resulted in a shift in mobility of the alpha-chain (by about 5 kDa). N-terminal sequencing of this species suggests that cleavage occurs at the C-terminus of the alpha-chain of C3. Consistent with this hypothesis, the modified alpha-chain was still a substrate for pre-formed convertase. The activity was specific for the alpha-chain of C3 but not of C3(H2O) nor the alpha'-chain of C3b. It is proposed that SGE-modified C3 does not participate in convertase formation, probably having a reduced affinity for factor B.

Animals↗
PubMed1994

Activation of the alternative complement pathway by red blood cells from patients with sickle cell disease.

Increased activation of the alternative pathway of complement in serum from sickle cell disease (SCD) patients has been reported. We now show that this selective activation is not an artifact of clotting by measuring increased plasma concentrations of Bb in sickle cell patients compared to controls. Furthermore, we show that red blood cells (RBC) from SCD patients activate the alternative complement pathway more than control RBC in an in vitro system. RBC were incubated in 50% normal human serum chelated with 3.5 mM MgCl2 and 10 mM EGTA to block activation by the classical pathway, but allow alternative pathway activation. SCD RBC yielded significantly more activation, as measured by an EIA for C3, P complexes, than control RBC. Denser SCD RBC produced more activation than control RBC, unfractionated SCD RBC, and less dense SCD RBC. These findings are consistent with the hypothesis that dense irreversibly sickled cells, or membrane spicules or vesicles derived from them, may result in complement activation.

Anemia, Sickle Cell↗
PubMed1994

Classical pathway complement activation in Kawasaki syndrome.

In this study the complement breakdown products C3d, C4d, Bb and membrane attack complex were measured in plasma of patients with Kawasaki syndrome. The results suggested strong activation of the classical activation pathway. However, there was no significant decrease in hemolytic titer or in the concentrations of the intact proteins C3, C4, and B. The relationship between the serum concentrations of cytokines and complement components was examined; increased interleukin-6 concentration on the fifth day after the onset of fever was found to correlate well with the C3 and B concentrations in serum obtained 5 days later. We conclude that complement activation occurred in Kawasaki syndrome via the classical pathway but that the inflammatory reaction was accompanied by increased production of complement components. As a result, there was increased formation of activation products without changes in the serum complement levels.

Child, Preschool↗
PubMed1995

Elevated levels of iC3b and C4d, but not Bb, complement fragments from plasma of persons infected with human T cell leukemia virus (HTLV) with HTLV-I-associated myelopathy/tropical spastic paraparesis.

Plasma levels of complement (C) fragments iC3b, C4d, and Bb from human T cell leukemia virus (HTLV)-positive subjects with HTLV-I-associated myelopathy (HAM)/tropical spastic paraparesis (TSP) were analyzed by EIA. Both iC3b and C4d levels were significantly elevated in persons with HAM/TSP. These levels were similar to those in patients with human immunodeficiency virus (HIV) infection or rheumatoid arthritis (RA), who are known to have increased C fragments. Bb levels in persons with HAM/TSP wer unaffected, suggesting that C activation occurred only via the classical pathway. This differed from findings in HIV-infected or RA patients, who had elevated levels of Bb. The results showed an increase in C activation in persons with HAM/TSP and activation via the classical pathway, likely mediated by virus or immune complexes. It is possible that the C activation observed in these subjects contributed to the inflammatory pathogenesis of HAM/TSP.

Arthritis, Rheumatoid↗
PubMed1999

In vivo generation of C4d, Bb, iC3b, and SC5b-9 after OKT3 administration in kidney and lung transplant recipients.

BACKGROUND: OKT3 monoclonal antibody therapy results in an acute clinical syndrome (ACS) associated with the release of tumor necrosis factor and sequestration of neutrophils in the lungs. We have previously shown that inhibition of tumor necrosis factor does not completely eliminate OKT3-ACS, suggesting that other factors also contribute to the ACS. The current studies analyzed complement activation in vivo during the first hour after OKT3 administration. METHODS: Renal (n=4) and lung (n=4) transplant recipients received OKT3 as treatment for rejection and induction therapy, respectively. Complement activation products C4d, Bb, iC3b, and SC5b-9 were measured by ELISA. Hemodynamic parameters were also monitored in the lung transplant recipients. Neutrophil expression of CD11a, CD11b, and CD18 was monitored by flow cytometry. Controls included patients receiving methylprednisolone for rejection (n=4), two adults with adult respiratory distress syndrome who received extracorporeal membrane oxygenation, and normal volunteers (n=5). P values less than 0.05 (*) were considered significant. RESULTS: Increases in the plasma levels of C4d, Bb, iC3b, and SC5b-9 were observed in seven of eight patients after OKT3 administration. Mean values (n=8) at 0, 15, and 60 min (in microg/ml) were as follows-C4d: 1.865, 2.644*, and 2.607*; Bb: 0.245, 0.411, and 0.385; iC3b: 10.881, 17.242*, and 15.145*; and SC5b-9: 0.232, 0.269, and 0.302*. An increase in CD11b and CD18 and a decrease of CD11a on neutrophils in parallel with complement activation was observed. In lung transplant recipients, C3 activation correlated with increases in mean pulmonary and central venous pressures (P<0.05). As compared with extracorporeal membrane oxygenation, which activated classical and alternative pathways, OKT3 predominantly activated complement by the classical pathway. Methylprednisolone pulses did not activate complement. CONCLUSIONS: Complement activation is an early event after OKT3 administration and is associated with the increased expression of adhesion molecules on neutrophils and with pulmonary hemodynamic changes. Effective therapeutic approaches to the control of early monoclonal antibody side effects may require measures that limit complement activation in addition to reducing cytokine activity.

Adult↗
PubMed1994

Human liver-derived HEP G2 cells produce functional properdin.

Properdin stabilizes the alternative complement pathway C3 convertase and is synthesized by monocytes and myelomonocytic cell lines. Hepatic production of properdin has never been documented, although most other complement components are synthesized by liver. Human liver-derived Hep G2 cells were examined for the ability to produce properdin by using the polymerase chain reaction (PCR). Amplified properdin message was detected by using Southern transfer and hybridization to a murine properdin cDNA probe. Sequencing of the PCR product revealed that the Hep G2 message was nearly identical to the cDNA sequence from U937 cells. Subsequently Hep G2 cultures were stimulated with interleukin (IL-6), 25 micrograms/ml, and culture supernatants were assayed for the presence of properdin by using dot blots. Properdin concentration increased over time, and we found no obvious difference between properdin production by IL-6-stimulated and unstimulated Hep G2 cells. Finally, alternative pathway decay assays confirmed the presence of functionally active properdin in the culture supernatant. Thus, functional properdin is a product of Hep G2 cells, suggesting that biosynthesis of properdin may occur in hepatocytes. Properdin synthesis was not augmented by IL-6, a finding that is consistent with previous observations that properdin is not an acute phase reactant.

Base Sequence↗
PubMed1990

Generation of the membrane attack complex during haemodialysis: impact of classical and alternative pathway components.

1. The generation of the plasma SC5b-9 complex (the soluble form of the membrane attack complex) during haemodialysis was studied in 29 patients together with markers of the classical (C4d fragment) and alternative (Bb fragment) pathways and the common iC3b fragment. 2. In the patients dialysed with Hemophan and Cuprophan membranes, a rapid increase in the plasma levels of SC5b-9 complex, Bb fragment and iC3b fragment occurred within 15 min of the initiation of the haemodialysis procedure. The SC5b-9 complex and the Bb fragment concentrations remained at a plateau until 120 min thereafter. The iC3b fragment level showed a continual decline. 3. The C4d fragment concentrations remained unchanged, indicating that generation of the membrane attack complex is most likely due only to activation of the alternative pathway. 4. The generation of the membrane attack complex occurred throughout the full haemodialysis period. In patients treated with Polysulfon membranes no statistically significant variation in these components was noted. 5. The study shows that the membrane attack complex may be generated during the full period of the haemodialysis session and is a stable index of biocompatibility. 6. Moreover, it seems be involved directly in haemodialysis-associated phenomena.

Complement Activation↗
PubMed2002

Complement activation in chromosome 13 dementias. Similarities with Alzheimer's disease.

Chromosome 13 dementias, familial British dementia (FBD) and familial Danish dementia (FDD), are associated with neurodegeneration and cerebrovascular amyloidosis, with striking neuropathological similarities to Alzheimer's disease (AD). Despite the structural differences among the amyloid subunits (ABri in FBD, ADan in FDD, and Abeta in AD), these disorders are all characterized by the presence of neurofibrillary tangles and parenchymal and vascular amyloid deposits co-localizing with markers of glial activation, suggestive of local inflammation. Proteins of the complement system and their pro-inflammatory activation products are among the inflammation markers associated with AD lesions. Immunohistochemistry of FBD and FDD brain sections demonstrated the presence of complement activation components of the classical and alternative pathways as well as the neo-epitope of the membrane attack complex. Hemolytic experiments and enzyme-linked immunosorbent assays specific for the activation products iC3b, C4d, Bb, and C5b-9 indicated that ABri and ADan are able to fully activate the complement cascade at levels comparable to those generated by Abeta1-42. ABri and ADan specifically bound C1q with high affinity and formed stable complexes in physiological conditions. Activation proceeds approximately 70-75% through the classical pathway while only approximately 25-30% seems to occur through the alternative pathway. The data suggest that the chronic inflammatory response generated by the amyloid peptides in vivo might be a contributing factor for the pathogenesis of FBD and FDD and, in more general terms, to other neurodegenerative conditions.

Alzheimer Disease↗
PubMed2001

Regulation of in situ complement activation via the lectin pathway in patients with IgA nephropathy.

The lectin pathway, which is initiated by mannose-binding lectin (MBL) and MBL-associated serine protease (MASP), is one of the possible routes to activate the complement cascade in immunoglobulin A (IgA) nephropathy. The purpose of this study was to elucidate the regulatory mechanism of the pathway. Levels of complement activation products and regulatory proteins were measured in sera from 27 patients with IgA nephropathy, and generation of fluid-phase complement activation products in the presence of pooled normal human serum was quantified to evaluate activation in vitro. Although there were no significant differences in the serum levels and in vitro activation between the MBL-MASP positive (n = 14) and negative (n = 13) groups, there were positive correlations between complement activation products (Bb fragment and C4d fragment) and regulatory proteins (factor H, C4-binding protein, and C1 inhibitor) in the MBL-MASP-positive group. Furthermore, immunohistochemical studies demonstrated glomerular deposition of the regulatory protein (C4-binding protein, alpha2-macroglobulin, and factor H) in all patients in the MBL-MASP-positive group. These findings suggest that the regulatory proteins control in situ complement activation via the lectin pathway immediately, and continuous activation due to inadequate control will lead to the advanced glomerular injury.

Carrier Proteins↗
PubMed1998

Immunoglobulin prolongs survival of pig kidneys perfused ex vivo with human blood.

Intravenous immunoglobulin (IVIG) (Octagam), was used to determine the effect on hyperacute rejection in an ex vivo xenograft model. Six pig kidneys were perfused with IVIG and fresh human AB blood, and six control pig kidneys were simultaneously perfused with albumin and blood from the same donation. The survival of the IVIG-perfused xenografts (median, 6.5 h) was significantly (P = 0.03) longer than the albumin-perfused xenografts (median, 3.5 h). Complement was activated in both groups. The administration of IVIG to the perfused blood resulted in immediate and significantly higher complement activation in the fluid phase as compared with the albumin group. At rejection the fluid phase complement activation was higher in the IVIG group than in the albumin group for C1rs/C1inh complexes, C4bc, Bb and TCC. At the time of rejection both the albumin and the IVIG group demonstrated interstitial tubular haemorrhage, vasculitis or necrosis of glomerular capillaries and glomerular microthrombi. IgM, C1q, C3c, C4 and fibrin were located in arteries and glomeruli and IgG in the interstitium in both groups at rejection. The fluid phase findings are consistent with a modulatory effect of IVIG on complement activation by deviating the classical pathway activation towards the fluid phase. The prolonged survival of the IVIG-perfused kidneys suggests that IVIG may be useful to dampen hyperacute rejection.

Animals↗
PubMed1995

Complement activation during painful crisis in sickle cell anemia.

Previous studies documented complement activation in sickle cell disease patients and suggested that this contributes to increased risk of infection. We have demonstrated alternative pathway activation initiated by membrane phospholipid changes which occur in sickled erythrocytes. The present studies compared complement activation products in serial samples from sickle cell anemia patients at baseline and during hospitalization for painful crisis to examine the correlation between complement activation and disease activity. Plasma concentrations of Bb, C4d, and C3a were measured as well as C3 bound to erythrocytes. Patients were subdivided into those with continuous pain and those with intermittent painful episodes. In patients with intermittent pain, there was little evidence of complement activation at baseline and increased plasma concentrations of Bb and C3a during painful crisis. Elevated C3a and C4d levels were observed in patients with continuous pain regardless of hospitalization status, suggesting a continuous underlying inflammatory process in these patients.

Acute Disease↗
PubMed1987

Hypocomplementemic glomerulonephritis in an infant and mother. Evidence for an abnormal form of C3.

A mother developed hematuria during the fourth month of pregnancy, and her nursing infant son from this otherwise uncomplicated pregnancy developed hematuria at 3.5 months of age. Both had a mild glomerulonephritis characterized by mesangial prominence, focal thickening and mottling of the glomerular basement membrane and electron-dense deposits, predominantly in the intramembranous and subendothelial positions. Immunofluorescence studies revealed striking accumulations of C3 and other complement components associated with alternative complement pathway activation within glomeruli, and the presence of small or equivocal amounts of immunoglobulin. C1q, C4 and factor B were not detectable. The glomerular lesion was accompanied by hypocomplementemia. Sera of both mother and infant displayed half normal levels of C3 and factor B, increased levels of C4, and normal levels of 12 other complement proteins. High normal or slight elevation in nephritic factor-like activity was observed in serial serum samples. Studies suggested that this mother and son represent the second kindred having an abnormal form of C3 which produces an alternative complement pathway C3 convertase, C3b, Bb, resistant to control by factor H. No additional affected family members were identified. The course of the nephritis over 7 years without drug therapy has been mild with resolving hematuria and no abnormal proteinuria or decrease in creatinine clearance.

Adult↗
PubMed2000

A new apoptotic pathway for the complement factor B-derived fragment Bb.

Apoptosis is involved in both the cellular and humoral immune system destroying tumors. An apoptosis-inducing factor from HL-60 myeloid leukemia cells was obtained, purified, and sequenced. The protein found has been identified as a human complement factor B-derived fragment Bb, although it is known that factor B is able to induce apoptosis in several leukemia cell lines. Monoclonal antibodies against fragment Ba and Bb inhibited the apoptotic activity of factor B. When the purified fragment Bb was used for apoptosis induction, only the anti-Bb antibody inhibited Bb-induced apoptosis, and not the anti-Ba antibody. The apoptosis-inducing activity was found to be enhanced under conditions facilitating the formation of Bb. Blocking TNF/TNFR or FasL/Fas interactions did not interfere with the factor B-induced apoptosis. CD11c (iC3bR) acts as the main subunit of a heterodimer binding to fragment Bb in the apoptosis pathway, and the factor B-derived fragment Bb was found to possess the previously unknown function of inducing apoptosis in leukemic cells through a suicide mechanism of myeloid lineage cells during the differentiation stage.

Antibodies, Monoclonal↗
PubMed1989

Expression of the binding molecules for factor B and its fragment, Bb, of human complement on human monocytes after in vitro cultivation.

We have examined the expression of the binding molecules for Bb of human complement on the surface of resting and cultured human monocytes. Flow cytometry using biotinylated anti-B antibodies and phycoerythrin-avidin showed that although resting monocytes did not bind Bb significantly, monocytes cultivated for 24 h in the presence of fetal calf serum were capable of binding with Bb and its precursor B, but not with Ba fragment. The Bb-binding molecules were pronase-sensitive, suggesting that membrane proteins are associated with the Bb-binding molecules. In addition to human monocytes, liquid paraffin-induced guinea pig inflammatory macrophages were also found to express Bb-binding molecules on their surface. This implies that Bb receptor-like molecules become expressed during activation and differentiation of monocytes, just as observed with the C3d-receptor of monocytes.

Animals↗
PubMed1998

Studies on protein adsorption and activation of complement on hydrated aluminium surfaces in vitro.

Adsorption of human plasma and serum proteins onto hydrated aluminium was studied by ellipsometry/antibody techniques, and soluble complement components iC3b, Bb, and C4d with commercial ELISA plates. Aluminium that was incubated in plasma for 1 min bound significant amounts of anti-lipoproteins (anti-LP), no antibodies against contact activation of coagulation proteins, and no anti-fibrinogen (anti-Fib). Time course studies with serum revealed increasing deposition of anti-C3c with time. Complement factor 1q (C1q) was antibody detectable only after short-time serum incubations, but no anti-IgG and anti-properdin bound to the protein film at any time. Anti-C3c was not deposited after exposure of the surfaces to Clq-depleted serum. Intriguingly, and in spite of increasing deposition of C3 to the surface with time, the combined ellipsometry and ELISA results gave no unequivocal proof of activation of complement by hydrated aluminium.

Adsorption↗
PubMed2003

Activation of the complement system by different autologous transfusion devices: an in vitro study.

BACKGROUND: The aim of the present investigation was to study whether autologous transfusion devices activate the complement system and whether complement-activated blood is more vulnerable to further activation during processing. STUDY DESIGN AND METHODS: Forty-eight blood units were randomized to be processed by one of three different salvage systems: Group 1 underwent whole blood filtration (hemofiltration) (n=16); Group 2 underwent continuous processing, saline washing, and centrifugation (CATS, Fresenius AG ) (n=16); and Group 3 underwent saline washing and centrifugation (Cell-Saver, Haemonetics Corp.) (n=16). Eight blood units for each system were activated with cobra venom factor (CVF) at a concentration of 0.2 U per mL whole blood before processing. C activation was studied by determinations of C4d, Bb, C3a, and SC5b-9. Samples were drawn from whole blood, processed blood, and the waste bags. RESULTS: The concentrations of Bb, C3a, and SC5b-9 in whole blood after activation with CVF were significantly elevated compared to blood that was not activated (p < 0.01). Processed blood from hemofiltration contained significantly higher levels of complement-split products than techniques that use washing and centrifugation. The concentrations of SC5b-9 in blood processed by hemofiltration were higher in the experiments with CVF activation (p < 0.05). CONCLUSION: The tested autologous transfusion systems did not themselves activate the complement system, and complement-activated blood was not more vulnerable to further activation during processing. A blood-salvaging technique that used washing and centrifugation reduced elevated concentrations of complement-split products, whereas hemofiltration did not.

Blood Transfusion, Autologous↗
PubMed2001

Rejection of pig liver xenografts in patients with liver failure: implications for xenotransplantation.

The pathophysiological state of rejection in liver xenotransplantation is poorly understood. Data from clinical pig liver perfusion suggest that pig livers might be rejected less vigorously than pig hearts or kidneys. Pig livers used in clinical xenoperfusions were exposed to blood from patients with liver failure. We have shown in an animal model that transplant recipients with liver failure are less capable of initiating hyperacute rejection of a xenografted liver than a healthy transplant recipient. The goal of this report is to examine the pathological characteristics of pig livers used in 2 clinical pig liver perfusions and combine this information with in vitro studies of pig-to-human liver xenotransplantation to determine whether the findings in the perfused pig livers could be explained in part by the diminished capacity of the patient with liver failure to respond to xenogeneic tissue. Pathological analysis of the perfused pig livers showed immunoglobulin M deposition in the sinusoids with little evidence of complement activation. Our in vitro studies showed that serum from patients with liver failure caused less injury to pig liver endothelium than serum from healthy subjects. Serum from patients with liver failure had similar levels of xenoreactive antibodies as serum from healthy humans. Incubation of serum from patients with liver failure with pig hepatic endothelial cells generated less iC3b, Bb fragment, and C5b-9 than serum from healthy subjects. We conclude that the altered injury in the perfused pig livers can be attributed to the relative complement deficiency that accompanies liver failure.

Animals↗