Complement Consumption Research Articles

Protection by 16,16-dimethyl prostaglandin E2 and dibutyryl cyclic AMP against complement-mediated hepatic necrosis in rats

16,16-Dimethyl prostaglandin E2, a known cytoprotective agent, was examined for its ability to protect the liver against complement-mediated necrosis induced by an intravenous injection of a monoclonal antibody against a rat liver-specific antigen in rats. The hepatic injury induced by the antibody was characterized by (a) rapid development of numerous massive hemorrhagic foci of necrotic liver cells, (b) marked increases in serum liver enzyme activities and (c) pronounced reduction in the CH50 level, presumably as a result of complement consumption in the liver. Pretreatment with 16,16-dimethyl prostaglandin E2 at intraperitoneal doses of 20 and 100 micrograms/kg suppressed the hepatic injury, as evidenced by markedly mitigated liver-cell necrosis and much smaller increases in the serum-enzyme activities compared with the values in diseased control animals. The prostaglandin analogue failed to prevent serum complement consumption in response to the antibody injection or affect the CH50 level at the preinjury stage, indicating that neither complement inactivation nor interference with the antigen-antibody reaction was involved in the hepatic protection. The hepatoprotective doses of 16,16-dimethyl prostaglandin E2 produced a significant increase in liver cyclic AMP content in a dose-related manner. In addition, intravenous dibutyryl cyclic AMP at 3 and 10 mg/kg dose-dependently prevented histological and biochemical changes in the hepatic damage without altering the rate of reduction in serum complement activity. Like 16,16-dimethyl prostaglandin E2, dibutyryl cyclic AMP did not affect the preinjury CH50 level.(ABSTRACT TRUNCATED AT 250 WORDS)

Oxidative metabolism of circulating granulocytes in adult respiratory distress syndrome

Among the different mechanisms involved, polymorphonuclear leukocytes (PMNs) may play a central role in the pathogenesis of adult respiratory distress syndrome (ARDS). PMNs were evaluated in 15 patients with ARDS, in 21 at risk of developing ARDS (AR), and in 36 controls (C). Spontaneous and opsonized zymosan (OZ), phorbol myristate acetate (PMA), and F-Met-Leu-Phe (F-M-L-P)-stimulated oxygen radical production was measured by luminol- and lucigenin-enhanced chemiluminescence (CL). Spontaneous CL activity of PMNs from ARDS patients was significantly greater than that from the PMN control (luminol CL, 2.8 ± 0.6 vs. 0.8 ± 0.1 mV, p <0.001; lucigenin CL, 2.0 ± 0.6 vs. 0.30 ± 0.04 mV, p <0.001), and the CL value from AR patients (luminol CL, 1.3 ± 0.2 mV, p <0.001 vs. C; Lucigenin CL, 0.8 ± 0.1 mV, p <0.001 vs. C) was found to be between the ARDS and C patients. The peak of PMA-stimulated CL occurred earlier and it was significantly higher in ARDS patients than in AR patients (p <0.05) and controls (p <0.001). When the CL response was elicited with F-M-L-P, no difference among the three groups was found. When stimulated with OZ, the peak CL generated by PMNs from ARDS patients was significantly depressed compared with controls (luminol CL, 26.7 ± 1.8 vs. 40.9 ± 2.3 mV, p <0.01; lucigenin CL, 5.0 ± 0.4 vs. 7.4 ± 0.5 mV, p <0.005) with a similar result being obtained from AR patients (luminol CL, 32.1 ± 2.5 mV, p <0.01 vs. C). Plasma from ARDS and AR patients showed a defective opsonizing capacity, suggesting in vivo complement consumption in both patient groups. No correlation between the severity of hypoxemia, the cause of ARDS, the outcome, and the different PMN functions could be established. Our results are in agreement with a determinant role of PMNs in the development of ARDS. The opposite metabolic responses may explain both the pulmonary injury and the increased susceptibility to infections observed in patients at risk of or with ARDS.

Endothelial cell damage in rheumatoid disease correlates with complement consumption and immune complexes

Endothelial cell damage in rheumatoid disease correlates with complement consumption and immune complexes

The role of surface charge in the activation of the classical and alternative pathways of complement by liposomes.

We have studied the complement-activating properties of liposomes. We show that surface charge is a key determinant of complement-activating liposomes. The nature of the charge, whether negative or positive, appears to dictate which pathway of the complement system is activated. Phosphatidylcholine:cholesterol (PC:CHOL, 55:45 mol/mol) liposomes were made to exhibit a positive or negative surface charge by the addition of cationic or anionic lipids, respectively. Normal human or guinea pig serum was incubated with liposomes, followed by determining the residual hemolytic activity of the serum as a measure of complement activation. Negatively charged liposomes containing phosphatidyl-glycerol, phosphatidic acid, cardiolipin, phosphatidylinositol, or phosphatidylserine activated complement in a Ca(2+)-dependent manner suggesting activation occurred via the classical pathway. Positively charged liposomes containing stearylamine or 1,2-bis(oleoyloxy)-3-(trimethylammonio)propane activated complement via the alternative pathway. Neutral liposomes, PC:CHOL (55:45) and PC:CHOL:dipalmitoylphosphatidylethanolamine (35:45:20), failed to activate complement as measured by the hemolytic assays. We show that unsaturated liposomes are more potent complement activators than saturated liposomes and that 45 mol% cholesterol promotes complement protein-liposome interactions. Immunoblot analysis of phosphatidylglycerol-containing liposomes showed that C3b and C9 were associated with these liposomes. Thus, the complement consumption measured in the hemolytic assays represents active cleavage of the complement components and not passive adsorption to the liposome surface. These studies suggest that membranes composed of net charged phospholipids can activate the complement system. This observation underlines the importance in biologic membranes of complement regulatory proteins that protect normal cells from complement attack.

The Effect of Immunoglobulin on Immune Complexes in Patients with Kawasaki Disease (MCLS)

In mucocutaneous lymph node syndrome (MCLS) IgG and IgM class immune complex (IC) showed peak levels on day 10 to 20 after the onset of the disease respectively, and a transient decrease in IgM class IC was detected at the beginning of intravenous high dose gamma-globulin therapy (IHDGT). In in vitro examination, when gamma-globulin was added to the serum samples of MCLS patients, IgG class IC increased while IgM class IC decreased. Consumption of complements was also observed in these samples. Disposition of IC in the reticuloendothelial system seemed easier by both binding of gamma-globulin to IC and increasing complement bound to IC resulting in increasing complement-binding site. These results suggest that IHDGT is effective for the treatment of IC in MCLS. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) revealed 14 bands from 300 K.D. to 42 K.D. in IC of MCLS patients in the acute phase of the disease and prior to treatment. The immunoblotting method revealed that IC contains IgG, IgM, IgE, IgA, Clq, C3, and C4.

Complement consumption in a patient with necrobiotic xanthogranuloma and paraproteinemia

Necrobiotic xanthogranuloma and paraproteinemia have been reported to be associated with acquired deficiency of the early components of the classic pathway of complement. We describe a patient in whom activation of complement was suggested by a high level of C3d, an accelerated catabolism of Clq, and a rapid consumption of C4 after an intravenous infusion of fresh-frozen plasma. This infusion was followed by an episode of pulmonary edema. Patients with complement deficiency or depletion should be monitored closely while receiving plasma, to avoid this potential complication.

In vivo anti-complementary activities of the cobra venom factors from Naja naja and Naja haje

The kinetics of complement (C) depletion and recovery of C levels upon injection of BALB/c mice with cobra venom factors (CVF), from N. naja (C3- and C5-depleting) and N. haje (selectively C3-depleting) were studied. The animals received i.p. or i.v. injections of either of the two preparations. CH50 and hemolytic C3 and C5 levels were followed as parameters of residual complement activity. N. naja CVF turned out to be as efficient in depleting total complement activity as N. haje CVF. Decreased CH50 values could largely be ascribed to C3 depletion. Complement consumption after N. naja CVF, however, lasted longer than after N. haje CVF administration. Estimated functional half-lives of N. naja and N. haje CVF were 11.5 and 4.5h, respectively. Inhibition ELISAs showed that, after in vivo administration of either of the two CVF preparations, antigenic C3 and C5 kept circulating for days.

Curdlan, a (1 → 3)-β-d-glucan from Alcaligenes faecalis var. myxogenes IFO13140, activates the alternative complement pathway by heat treatment

From the results of consumption experiments of guinea pig complement, Curdlan, a (1----3)-beta-D-glucan obtained from Alcaligenes faecalis var. myxogenes IFO13140, has been found to lack the ability to activate complement when unheated or preheated at either 40 degrees C or 50 degrees C. However, Curdlan heated at or above 60 degrees C increased complement consumption. This activation, dependent on the temperature of the Curdlan, was via the alternative complement pathway as assessed by cleavage of factor B into Ba and Bb fragments. These results suggest a substantial change must occur in Curdlan with heat treatment for alternative pathway activation.

Complement activation by individual and combinations of monoclonal antibodies to Actinomyces viscosus T14V fimbriae: A probe for epitope distribution on these polymeric proteins

The spatial requirements for IgG activation of the classical complement pathway has provided a basis for utilizing complement consumption by individual and pairs of monoclonal antibodies (mAbs) to compare the repeating epitope patterns of the type 1 and type 2 fimbriae of Actinomyces viscosus T14V and to examine the co-operative effects of mAbs against these polymeric proteins. Three of five mAbs specific for the type 1 fimbriae consumed complement when assayed individually. Four patterns of complement consumption were detected with pairs of these mAbs: inhibition, addition, enhancement or synergy. Inhibition occurred when both members of a pair reacted with the same epitope but only one consumed complement. A strictly additive effect was observed if both mAbs consumed complement and, in addition, recognized the same epitope. Complement consumption by mAbs against certain epitopes was enhanced by non-complement consuming mAbs that reacted with different epitopes. Synergy was observed with extremely low concentrations of two mAbs each of which reacted with a different epitope and consumed complement. In contrast to the anti-type 1 mAbs, only one of seven mAbs against the type 2 fimbriae consumed more than 20% of the available complement. Pairs of anti-type 2 mAbs exhibited only inhibition or synergy. The latter effect was particularly striking as pairs containing mAbs that reacted with different epitopes and failed to consume complement or were minimally active when assayed individually were extremely efficient. These data indicated that the spatial arrangements of individual mAbs bound to repeating epitopes in the type 1, but not the type 2, fimbriae were appropriate for activation of complement. Thus, the repeating epitope patterns of the two types of fimbriae apparently differ.

Consumption of complement and activation of human neutrophils by an artificial immune complex: Polyacrylic acid-IgG-polymer

An artificial immune complex consisting of IgG covalently bound to polyacrylic acid (PAIGP) was prepared and investigated for its influence on a number of immunological reactions attributed to natural immune complexes. PAIGP consumed complement in a fast reaction. Complement consumption was complete after 10 min of incubation of guinea-pig serum with PAIGP. The concentration of PAIGP for 50% consumption was 2.3 μg/ml. PAIGP induced a chemiluminescence response in human peripheral polymorphonuclear leukocytes. This response was elicited in the absence and presence of serum and in whole blood. The response was maximal for leukocytes in the absence of serum and rather low in whole blood. The induction of chemiluminescence by PAIGP was inhibited by monoclonal antibodies to one of the Fc receptors of leukocytes (anti-Leu 11B), while unrelated antibodies had no influence on the chemiluminescence induced by PAIGP. PAIGP also stimulated the production of superoxide anion by polymorphonuclear leukocytes. The efficacy of PAIGP in stimulation of superoxide production was comparable to phorbol myristate acetate (PMA) and opsonized zymosan. PAIGP induced the discharge of elastase, a constituent of the azurophile granules of PMN leukocytes. Here, PAIGP was a rather weak stimulus compared to opsonized zymosan. PMA proved unable to induce elastase release. Thus, PAIGP induced a number of biological reactions usually brought about by naturally occurring antigen antibody complexes.

A study of the necrotic actions of the venom of the wolf spider, Lycosa godeffroyi, on mouse skin

1. 1. The venom of the wolf spider, Lycosa godeffroyi, caused cutaneous necrosis when injected into mice. 2. 2. A strong inflammatory response and total loss of epidermal cellularity were features of this in vivo necrosis. 3. 3. Mouse skin envenomated while in tissue culture showed epidermal detachment and reduced cellular adhesion. 4. 4. Triprolidine and methysergide, used together, indomethacin, heparin and human and mouse sera all failed to inhibit the necrosis significantly. 5. 5. The venom caused moderate haemolysis, complement consumption and inhibition of clotting, these apparently not being the main reasons for the necrosis. 6. 6. Neither Atrax infensus venom nor hyaluronidase caused similar epithelial damage.

Biological and Immunological Properties of Sugi Basic Protein-Pullulan Conjugate

Ability of Sugi basic protein (SBP)-pullulan conjugate to elicit the Arthus reaction was found to be markedly reduced, about 100 times lower than that of native SBP. To analyze this reduced ability, activation of complement by immune complex consisting of SBP-pullulan and anti-SBP antibodies was studied. Tests for complement consumption, C3 conversion and cleavage of factor B revealed that immune complex formed with SBP-pullulan is incapable of supporting efficient activation of the complement system. Previously, we have shown data suggesting that SBP-pullulan conjugate would be a good candidate for desensitization therapy against cedar pollinosis. The results presented in this paper provide additional support for the suggestion.

Low Serum Complement Levels in Anorexia Nervosa-Reply

In Reply. —It appears that we do not disagree fundamentally with Drs Sigal and Snyder. Certainly, anorexia nervosa is not the same disease entity as malnutrition in Third-World countries. However, pairing them in the table as complement disorders, due primarily to decreased synthesis, is supported by the experimental evidence that is presently available. As we pointed out, circulating immune complexes and endotoxin in patients with malnutrition could deplete complement, but we believe that the weight of evidence at present favors decreased synthesis as the principal mechanism in both disorders. Although decreased levels of complement control proteins may lead to complement consumption in anorexia nervosa, the primary event is decreased production of these proteins. As we reported, complement abnormalities in both entities are rapidly corrected with appropriate feeding. 1,2 Perhaps this is the important point.

Deposition of complement activation products on plastic-adsorbed immunoglobulins: A simple ELISA technique for the detection of defined complement deficiencies

The activation of complement components in human serum has been studied using immunoglobulins adsorbed to microtiter plates. The sequential deposition of complement fragments was detected by a series of mono- and polyclonal antibodies in an indirect enzyme-linked immunosorbent assay (ELISA). Antibodies against C1q, C1s, C4b/d, C3b/d, factor B, C5b-9 membrane attack complex (MAC), the regulatory complement proteins C4 binding protein (C4bp) and properdin were reactive. Several lines of evidence suggest that complement activation was via the classical pathway: (1) complement activation was highly isotype-restricted with regard to the adsorbed Igs (human IgG1 and IgG3 as well as mouse IgM, IgG2a and IgG2b isotypes are strong activators in contrast to human IgG2, IgG4, IgA and mouse IgG1); (2) Ca 2+ depletion, heat treatment (56° C for 45 min), incubation with 0.5 M KSCN or heat-aggregated immunoglobulins (aggIgG) abrogated serum activity; (3) complement deficient sera (C1q def′, C2 def′, C6 def′ human sera; C2 def′, C4 def′ guinea pig sera) showed impaired deposition of the complement components that follow the missing component in the cascade of activation. In a clinical study sera from patients with systemic lupus erythematosus (SLE) were investigated in order to measure the effect of hypocomplementemia due to complement consumption. The results obtained suggest that this new and simple assay is well suited for (1) the detection of various inherited complement deficiencies, (2) the semiquantitative evaluation of sera with decreased complement levels, (3) a more detailed study of complement components bound to a solid phase.

Complement Abnormalities in Multiple Myeloma

Patients with multiple myeloma have been shown to have defective opsonization and C3 deposition. Previous studies have suggested that defective C3 deposition may be related to a failure of C3 activation in myeloma serum, the mechanism of which is unknown. We therefore decided to investigate the underlying mechanism responsible for the failure in C3 activation and deposition. The study consisted of 10 patients from whom a total of 12 serum specimens were obtained. Normal serum was prepared from a pool of serum specimens in four healthy male donors. We evaluated, in vitro, the kinetics of C3 deposition onto zymosan using radiolabeled C3 under various conditions. We also measured the serum levels of a variety of complement components using standard methods. Five of 10 patients' sera demonstrated poor C3 deposition onto zymosan at all time points, whereas an additional two showed poor C3 deposition at early time points but a rebound to normal by 30 minutes. Multiple components of the classical and alternative complement pathways were decreased in many patients, with the most striking abnormalities occurring in those with the poorest C3 deposition. No single complement component abnormality was found to be common to the group. Elevations in Bb fragment concentration strongly suggest in vivo activation as the likely mechanism for depletion of alternative pathway components; the mechanism for classical pathway abnormalities is less clear. There was an inverse correlation between paraprotein concentration and abnormal C3 deposition (p less than 0.0001) and C3 (p less than 0.0005) and C4 (p less than 0.0001) concentrations. However, no consistent evidence of fluid-phase complement consumption was present. The defect in C3 activation and deposition in multiple myeloma cannot be explained on the basis of a single complement component abnormality but rather is due to a heterogeneous group of complement abnormalities. Although no correlation between in vitro abnormalities and clinical status was identified in this small group of patients, it is likely that the described complement defects play an important role in defective host defense in multiple myeloma.

Imunodulatory compounds from Picrorhiza kurroa: Isolation and characterization of two anti-complementary polymeric fractions from an aqueous root extract

Two aqueous root extracts of Picrorhiza kurroa, one prepared by extraction at 4°C and the other by refluxing, were purified using the guidance of modulation of classical (CP) and alternative (AP) pathway complement activity. By means of methanol extraction and gel filtration chromatography, two polymeric fractions were isolated from the cold water extract. A methanol-solu- ble polymeric fraction (CS1) was highly active in inhibiting CP complement activity exclusively, whereas a methanol-insoluble polymeric fraction (CI1) exhibited an inhibitory effect on both CP and AP complement activity. Preliminary chemical analysis of the anti-complementary fractions revealed the presence of structures of carbohydrate and of peptide nature in CS1 and CI1. The modulation of CP complement activity by CS1 was studied in more detail. Its inhibitory effect was proven to be based on complement consumption rather than on chelation of Ca 2+ and/or Mg 2+ or on stabilization of the target cells in the complement-assay. The purification of the aqueous extract prepared by refluxing, resulted in the isolation of a polymeric fraction with the same qualities as CS1. However, a fraction with properties similar to CI1 could not be isolated from this extract.

Complement activation and C3 binding by serum-sensitive and serum-resistant strains of Pseudomonas aeruginosa

The relationship among complement consumption, C3 deposition, and C3 fragmentation pattern was compared for serum-sensitive (Sers) and serum-resistant (Serr) strains of Pseudomonas aeruginosa. The Sers strains, which were mucoid strains derived from patients with cystic fibrosis, had lipopolysaccharide deficient in O-antigen side chains. These organisms generally activated much less complement per organism than their Serr counterparts, characterized by the presence of lipopolysaccharide with long lipopolysaccharide O side chains. Surprisingly, however, although the Serr strains consumed more total hemolytic complement, less C3 was deposited onto the surface of these strains than onto that of the Sers strains. Maximal C3 binding required the participation of both the classical and alternative complement pathways, although classical complement pathway involvement was more important for Serr strains. Finally, while more than half of the C3 deposited on most Sers strains was in the form of C3b, most of the C3 on the Serr strains was in the form of iC3b, indicating a more rapid and extensive conversion of C3b to iC3b on the surface of these strains. Limited complement activation by Sers mucoid strains of P. aeruginosa may confer a selective survival advantage to these organisms in colonizing the airways of patients with cystic fibrosis.

Effects of CAMPATH-1 antibodies in vivo in patients with lymphoid malignancies: influence of antibody isotype

The CAMPATH-1 family of antibodies recognize an abundant glycoprotein expressed on virtually all human lymphocytes. All rat IgM and IgG antibodies of this specificity are lytic with human complement, but only IgG2b is active in antibody-dependent cell-mediated cytotoxicity (ADCC). We compared the ability of IgM, IgG2a, and IgG2b to deplete lymphocytes in vivo in two patients with prolymphocytic transformation of B-cell chronic lymphocytic leukemia (CLL). The IgM (CAMPATH-1M) produced transient depletion of blood lymphocytes with consumption of complement but had no effect on solid masses or bone marrow. Similar transient depletion of blood lymphocytes was noted with the IgG2a (YTH34.5). In contrast, the IgG2b (CAMPATH-1G) produced long-lasting depletion of lymphocytes from blood and marrow and improvement in splenomegaly but no detectable changes in complement levels. These differences probably reflect the importance of Fc receptor binding for effective clearance of target cells in vivo. We treated 16 more patients with a variety of lymphoid malignancies and noted consistent effects on blood lymphocytes, marrow infiltration, and splenomegaly. At this dose level, there was comparatively little improvement in affected lymph nodes or extranodal masses. Nevertheless, the in vivo lympholytic ability of CAMPATH-1G is very potent as compared with other monoclonal antibodies (MoAbs) and may have applications in therapy of lymphoid malignancies and as an immunosuppressive agent.

Effects of CAMPATH-1 Antibodies In Vivo in Patients With Lymphoid Malignancies: Influence of Antibody Isotype

The CAMPATH-1 family of antibodies recognize an abundant glycoprotein expressed on virtually all human lymphocytes. All rat IgM and IgG antibodies of this specificity are lytic with human complement, but only IgG2b is active in antibody-dependent cell-mediated cytotoxicity (ADCC). We compared the ability of IgM, IgG2a, and IgG2b to deplete lymphocytes in vivo in two patients with prolymphocytic transformation of B-cell chronic lymphocytic leukemia (CLL). The IgM (CAMPATH-1M) produced transient depletion of blood lymphocytes with consumption of complement but had no effect on solid masses or bone marrow. Similar transient depletion of blood lymphocytes was noted with the IgG2a (YTH34.5). In contrast, the IgG2b (CAMPATH-1G) produced long-lasting depletion of lymphocytes from blood and marrow and improvement in splenomegaly but no detectable changes in complement levels. These differences probably reflect the importance of Fc receptor binding for effective clearance of target cells in vivo. We treated 16 more patients with a variety of lymphoid malignancies and noted consistent effects on blood lymphocytes, marrow infiltration, and splenomegaly. At this dose level, there was comparatively little improvement in affected lymph nodes or extranodal masses. Nevertheless, the in vivo lympholytic ability of CAMPATH-1G is very potent as compared with other monoclonal antibodies (MoAbs) and may have applications in therapy of lymphoid malignancies and as an immunosuppressive agent.

Mechanisms of Bacterial Opsonization by Immune Globulin Intravenous: Correlation of Complement Consumption with Opsonic Activity and Protective Efficacy

To study the mechanism of bacterial opsonization by immune globulin intravenous (IGIV) complement consumption and polymorphonuclear leukocyte (PMNL) membrane receptor (FcRlo, CR1, and CR3)-mediated phagocytosis of Staphylococcus epidermidis, Klebsiella pneumoniae, and groups A and B streptococci were examined. IGIV alone did not consume complement and showed no opsonic activity by itself for these organisms. When these bacteria were preopsonized in IGIV, significant amounts of complement were consumed (44%-94%) and the uptake and killing of bacteria occurred. The in vitro opsonic activity of IGIV for these organisms was significantly correlated with the amount of complement consumed by the IGIV-opsonized bacteria (r = .85, P less than .05). The in vivo protective efficacy of IGIV also appeared to be directly associated with its ability to activate and consume complement (r = 1.0, P less than .001). Antibodies to FcRlo (Leu 11) markedly inhibited phagocytosis of bacteria opsonized in IGIV but not that of bacteria opsonized in specific IgM. Both CR1 and CR3 receptors on PMNLs were involved in uptake, but the contribution of each is different with different organisms.