Investigating possible biological targets of Bj-CRP, the first cysteine-rich secretory protein (CRISP) isolated from Bothrops jararaca snake venom.

Cysteine-rich secretory proteins (CRISPs) are commonly described as part of the protein content of snake venoms, nevertheless, so far, little is known about their biological targets and functions. Our study describes the isolation and characterization of Bj-CRP, the first CRISP isolated from Bothrops jararaca snake venom, also aiming at the identification of possible targets for its actions. Bj-CRP was purified using three chromatographic steps (Sephacryl S-200, Source 15Q and C18) and showed to be an acidic protein of 24.6kDa with high sequence identity to other snake venom CRISPs. This CRISP was devoid of proteolytic, hemorrhagic or coagulant activities, and it did not affect the currents from 13 voltage-gated potassium channel isoforms. Conversely, Bj-CRP induced inflammatory responses characterized by increase of leukocytes, mainly neutrophils, after 1 and 4h of its injection in the peritoneal cavity of mice, also stimulating the production of IL-6. Bj-CRP also acted on the human complement system, modulating some of the activation pathways and acting directly on important components (C3 and C4), thus inducing the generation of anaphylatoxins (C3a, C4a and C5a). Therefore, our results for Bj-CRP open up prospects for better understanding this class of toxins and its biological actions.

Micrurus snake venoms activate human complement system and generate anaphylatoxins.

BACKGROUND: The genus Micrurus, coral snakes (Serpentes, Elapidae), comprises more than 120 species and subspecies distributed from the south United States to the south of South America. Micrurus snake bites can cause death by muscle paralysis and further respiratory arrest within a few hours after envenomation. Clinical observations show mainly neurotoxic symptoms, although other biological activities have also been experimentally observed, including cardiotoxicity, hemolysis, edema and myotoxicity. RESULTS: In the present study we have investigated the action of venoms from seven species of snakes from the genus Micrurus on the complement system in in vitro studies. Several of the Micrurus species could consume the classical and/or the lectin pathways, but not the alternative pathway, and C3a, C4a and C5a were generated in sera treated with the venoms as result of this complement activation. Micrurus venoms were also able to directly cleave the α chain of the component C3, but not of the C4, which was inhibited by 1,10 Phenanthroline, suggesting the presence of a C3α chain specific metalloprotease in Micrurus spp venoms. Furthermore, complement activation was in part associated with the cleavage of C1-Inhibitor by protease(s) present in the venoms, which disrupts complement activation control. CONCLUSION: Micrurus venoms can activate the complement system, generating a significant amount of anaphylatoxins, which may assist due to their vasodilatory effects, to enhance the spreading of other venom components during the envenomation process.

Dendritic polyglycerol sulfates as multivalent inhibitors of inflammation.

Adhesive interactions of leukocytes and endothelial cells initiate leukocyte migration to inflamed tissue and are important for immune surveillance. Acute and chronic inflammatory diseases show a dysregulated immune response and result in a massive efflux of leukocytes that contributes to further tissue damage. Therefore, targeting leukocyte trafficking may provide a potent form of anti-inflammatory therapy. Leukocyte migration is initiated by interactions of the cell adhesion molecules E-, L-, and P-selectin and their corresponding carbohydrate ligands. Compounds that efficiently address these interactions are therefore of high therapeutic interest. Based on this rationale we investigated synthetic dendritic polyglycerol sulfates (dPGS) as macromolecular inhibitors that operate via a multivalent binding mechanism mimicking naturally occurring ligands. dPGS inhibited both leukocytic L-selectin and endothelial P-selectin with high efficacy. Size and degree of sulfation of the polymer core determined selectin binding affinity. Administration of dPGS in a contact dermatitis mouse model dampened leukocyte extravasation as effectively as glucocorticoids did and edema formation was significantly reduced. In addition, dPGS interacted with the complement factors C3 and C5 as was shown in vitro and reduced C5a levels in a mouse model of complement activation. Thus, dPGS represent an innovative class of a fully synthetic polymer therapeutics that may be used for the treatment of inflammatory diseases.

Complement-mediated regulation of the IL-17A axis is a central genetic determinant of the severity of experimental allergic asthma.

Severe asthma is associated with the production of interleukin 17A (IL-17A). The exact role of IL-17A in severe asthma and the factors that drive its production are unknown. Here we demonstrate that IL-17A mediated severe airway hyperresponsiveness (AHR) in susceptible strains of mice by enhancing IL-13-driven responses. Mechanistically, we demonstrate that IL-17A and AHR were regulated by allergen-driven production of anaphylatoxins, as mouse strains deficient in complement factor 5 (C5) or the complement receptor C5aR mounted robust IL-17A responses, whereas mice deficient in C3aR had fewer IL-17-producing helper T cells (T(H)17 cells) and less AHR after allergen challenge. The opposing effects of C3a and C5a were mediated through their reciprocal regulation of IL-23 production. These data demonstrate a critical role for complement-mediated regulation of the IL-23-T(H)17 axis in severe asthma.

Human complement activation and anaphylatoxins generation induced by snake venom toxins from Bothrops genus.

Snake venoms are a complex mixture of components, which have a wide range of actions both on prey and human victims. The genus Bothrops causes the vast majority of snakebites in Central and South America, being responsible for 80% of snake envenomations in Brazil. Envenomations are characterized by prominent local effects, including oedema, haemorrhage and necrosis, which can lead to permanent disability. Systemic manifestations such as haemorrhage, coagulopathy, shock and acute renal failure may also occur. In the present study we have investigated the action of venoms from 19 species of snakes from the genus Bothrops, occurring in Brazil, on the complement system in in vitro studies. All venoms were able to activate the classical complement pathway, in the absence of sensitizing antibody. This activation was in part associated with the cleavage of C1-Inhibitor by proteases present in these venoms, which disrupts complement activation control. No modification of the membrane bound complement regulators, such as DAF, CR1 and CD59 was detected, after treatment of human erythrocytes with the snake venoms. Some of the Bothrops venoms were also able to activate alternative and lectin pathways, as measured in haemolytic and ELISA assays. C3a, C4a and C5a were generated in sera treated with the venoms, not only through C-activation, but also by the direct cleavage of complement components, as determined using purified C3 and C4. Metallo- and/or serine-protease inhibitors prevented cleavage of C3 and C4. These results suggest that Bothrops venoms can activate the complement system, generating a large amount of anaphylatoxins, which may play an important role in the inflammatory process presented in humans after snake envenomations, and they may also assist, due to their vasodilatory effects, to enhance the spreading of other venom components.

Generation of anaphylatoxins by human beta-tryptase from C3, C4, and C5.

Both mast cells and complement participate in innate and acquired immunity. The current study examines whether beta-tryptase, the major protease of human mast cells, can directly generate bioactive complement anaphylatoxins. Important variables included pH, monomeric vs tetrameric forms of beta-tryptase, and the beta-tryptase-activating polyanion. The B12 mAb was used to stabilize beta-tryptase in its monomeric form. C3a and C4a were best generated from C3 and C4, respectively, by monomeric beta-tryptase in the presence of low molecular weight dextran sulfate or heparin at acidic pH. High molecular weight polyanions increased degradation of these anaphylatoxins. C5a was optimally generated from C5 at acidic pH by beta-tryptase monomers in the presence of high molecular weight dextran sulfate and heparin polyanions, but also was produced by beta-tryptase tetramers under these conditions. Mass spectrometry verified that the molecular mass of each anaphylatoxin was correct. Both beta-tryptase-generated C5a and C3a (but not C4a) were potent activators of human skin mast cells. These complement anaphylatoxins also could be generated by beta-tryptase in releasates of activated skin mast cells. Of further biologic interest, beta-tryptase also generated C3a from C3 in human plasma at acidic pH. These results suggest beta-tryptase might generate complement anaphylatoxins in vivo at sites of inflammation, such as the airway of active asthma patients where the pH is acidic and where elevated levels of beta-tryptase and complement anaphylatoxins are detected.

Complement induction in spinal cord microglia results in anaphylatoxin C5a-mediated pain hypersensitivity.

Microarray expression profiles reveal substantial changes in gene expression in the ipsilateral dorsal horn of the spinal cord in response to three peripheral nerve injury models of neuropathic pain. However, only 54 of the 612 regulated genes are commonly expressed across all the neuropathic pain models. Many of the commonly regulated transcripts are immune related and include the complement components C1q, C3, and C4, which we find are expressed only by microglia. C1q and C4 are, moreover, the most strongly regulated of all 612 regulated genes. In addition, we find that the terminal complement component C5 and the C5a receptor (C5aR) are upregulated in spinal microglia after peripheral nerve injury. Mice null for C5 had reduced neuropathic pain sensitivity, excluding C3a as a pain effector. C6-deficient rats, which cannot form the membrane attack complex, have a normal neuropathic pain phenotype. However, C5a applied intrathecally produces a dose-dependent, slow-onset cold pain in naive animals. Furthermore, a C5aR peptide antagonist reduces cold allodynia in neuropathic pain models. We conclude that induction of the complement cascade in spinal cord microglia after peripheral nerve injury contributes to neuropathic pain through the release and action of the C5a anaphylatoxin peptide.

Methylation of the phosphate oxygen moiety of phospholipid-methoxy(polyethylene glycol) conjugate prevents PEGylated liposome-mediated complement activation and anaphylatoxin production.

Methoxy(polyethylene glycol), mPEG, -grafted liposomes are known to exhibit prolonged circulation time in the blood, but their infusion into a substantial percentage of human subjects triggers immediate non-IgE-mediated hypersensitivity reactions. These reactions are strongly believed to arise from anaphylatoxin production through complement activation. Despite the general view that vesicle surface camouflaging with mPEG should dramatically suppress complement activation, here we show that bilayer enrichment of noncomplement activating liposomes [dipalmitoylphosphatidylcholine (DPPC) vesicles] with phospholipid-mPEG conjugate induces complement activation resulting in vesicle recognition by macrophage complement receptors. The extent of vesicle uptake, however, is dependent on surface mPEG density. We have delineated the likely structural features of phospholipid-mPEG conjugate responsible for PEGylated liposome-induced complement activation in normal as well as C1q-deficient human sera, using DPPC vesicles bearing the classical as well as newly synthesized lipid-mPEG conjugates. With PEGylated DPPC vesicles, the net anionic charge on the phosphate moiety of phospholipid-mPEG conjugate played a key role in activation of both classical and alternative pathways of complement and anaphylatoxin production (reflected in significant rises in SC5b-9, C4d, and C3a-desarg levels in normal human sera as well as SC5b-9 in EGTA-chelated/Mg2+ supplemented serum), since methylation of the phosphate oxygen of phospholipid-mPEG conjugate, and hence the removal of the negative charge, totally prevented complement activation. To further corroborate on the role of the negative charge in complement activation, vesicles bearing anionic phospholipid-mPEG conjugates, but not the methylated phospholipid-mPEG, were shown to significantly decrease serum hemolytic activity and increase plasma thromboxane B2 levels in rats. In contrast to liposomes, phospholipid-mPEG micelles had no effect on complement activation, thus suggesting a possible role for vesicular zwitterionic phospholipid head-groups as an additional factor contributing to PEGylated liposome-mediated complement activation. Our findings provide a rational conceptual basis for development of safer vesicles for site-specific drug delivery and controlled release at pathological sites.

Overproduction of reactive oxygen species in end-stage renal disease patients: a potential component of hemodialysis-associated inflammation.

During the past decade, hemodialysis (HD)-induced inflammation has been linked to the development of long-term morbidity in end-stage renal disease (ESRD) patients on regular renal replacement therapy. Because interleukins and anaphylatoxins produced during HD sessions are potent activators for nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, an example of an enzyme that is responsible for overproduction of reactive oxygen species (ROS), this may constitute a link between leukocyte activation and cell or organ toxicity. Oxidative stress, which results from an imbalance between oxidant production and antioxidant defense mechanisms, has been documented in ESRD patients using lipid and/or protein oxidative markers. Characterization of HD-induced oxidative stress has included identification of potential activators for NADPH oxidase. Uremia per se could prime phagocyte oxidative burst. HD, far from improving the oxidative status, results in an enhancement of ROS owing to hemoincompatibility of the dialysis system, hemoreactivity of the membrane, and trace amounts of endotoxins in the dialysate. In addition, the HD process is associated with an impairment in antioxidant mechanisms. The resulting oxidative stress has been implicated in long-term complications including anemia, amyloidosis, accelerated atherosclerosis, and malnutrition. Prevention of oxidative stress in HD might focus on improving the hemocompatibility of the dialysis system, supplementation of deficient patients with antioxidants, and modulation of NADPH oxidase by pharmacologic approaches.

Hemocompatibility and anaphylatoxin formation of protein-immobilizing polyacrylonitrile hemodialysis membrane.

Plasma proteins were covalently immobilized onto polyacrylonitrile (PAN) membrane to evaluate the hemocompatibility and anaphylatoxin formation. This is used as a model to study the effect of protein-adsorption on the blood-contacting response of hemodializing membranes. The proteins used were either platelet-adhesion-promoting collagen (COL) or platelet-adhesion-inhibiting human serum albumin (HSA). The microstructure and characterization of the protein-immobilizing PAN membranes were evaluated by Coomassie dye assay, atomic force microscopy, X-ray photoelectron spectroscopy and water contact angle measurement. PAN-HSA membrane improved not only hemocompatibility including less platelet adhesion, longer blood coagulation times, and higher thrombin inactivity level, but also induced lower complement activation. On the other hand, PAN-COL membrane exhibited blood incompatibility, although induced less increase of C3, C4 antigens of serum. Overall results of this study demonstrated that the immobilization of HSA onto the surface of PAN membrane would be beneficial to improve the hemocompatibility and to reduce the anaphylatoxin formation during hemodialysis.

[Clinical applications of recombinant complement inhibitors]. / Zastosowanie kliniczne rekombinowanych inhibitorów ukladu dopelniacza.

The complement system constitutes an important part of the innate immune system. Complement activation leads to the generation of C3a, C4a and C5a anaphylatoxins and the membrane attack complex. The anaphylatoxins mediate multiple reactions in the acute inflammatory response. Membrane attack complex inserts molecules into target membranes and causes cell lysis. The complement system can not discriminate between self and non-self cells and the inappropriate complement activation may lead to host cell damage. This destructive activity is tightly regulated by family of structurally and functionally related soluble and membrane-bound proteins, which act as inhibitors of complement system. The inappropriate complement activation plays an essential role in the pathogenesis of many diseases. In the therapy of these diseases specific recombinant complement inhibitors can be used. Recombinant complement inhibitors can be produced in large amounts by different eukaryotic or prokaryotic systems. The choice of the system depends on kind of the post-translational modifications of proteins.

Complement in allergy and asthma.

The complement system is a vital link between innate and adaptive immunity. Recently, several investigators have implicated the complement anaphylatoxins C3a and C5a as potential effectors in Type 1 hypersensitivity reactions, including urticaria, rhinitis and asthma. Thus, complement activation may synergize with classical IgE mediated responses, and inhibition of complement may prove therapeutic.

Generation of anaphylatoxins through proteolytic processing of C3 and C5 by house dust mite protease.

BACKGROUND: The group 3 allergen of Dermatophagoides species (Der p 3 and Der f 3) has been identified as a 30 kd trypsin-like protease of the house dust mite. We previously showed that the 30 kd protease from Dermatophagoides farinae (Df-protease) could activate the bradykinin-generating cascade and exacerbate inflammatory reactions. OBJECTIVE: The purpose of this study was to determine whether Df-protease could enzymatically generate anaphylatoxins from complement components C3 and C5. METHODS: Df-protease was incubated with human serum C3 or C5 in a purified system, and the anaphylatoxin activity produced was assayed by measuring enhancement of vascular permeability and release of histamine from mast cells triggered by C3a and by assessing chemotaxis of polymorphonuclear cells caused by C5a. We also attempted to determine whether protease isolated from house dust could cause release of C5a from C5. RESULTS: Df-protease showed strong activation of C3 and C5, producing C3a and C5a by proteolytic cleavage of the complements. An appreciable amount of Df-protease was recovered in the house dust extract, and the house dust protease caused C5a release from C5. CONCLUSION: Df-protease activated the complement system to produce anaphylatoxins. Thus it is suggested that house dust mite proteases may contribute to the pathogenesis of allergic and inflammatory diseases caused by house dust allergens.

Neutrophil and macrophage activation and anaphylatoxin formation in orthotopic liver transplantation without the use of veno-venous bypass.

BACKGROUND: Activation of neutrophils and activation of complement may be an aetiologic factor behind circulatory insufficiency in association with reperfusion of the grafted liver. METHODS: Neutrophil and macrophage activation (determined as PMN elastase and neopterin release) and complement activation were evaluated in 15 consecutive patients undergoing orthotopic liver transplantation without the use of veno-venous bypass. RESULTS: The PMN elastase concentrations were increased at the end of the anhepatic phase, 2, 5 and 30 min after start of reperfusion and 6 and 24 h postoperatively. There were significantly higher PMN elastase concentrations in patients with circulatory instability (postreperfusion syndrome) compared with those without postreperfusion syndrome. The neopterin concentration was increased 2 min after the start of reperfusion and remained elevated until 6 h postoperatively. The plasma complement C3a concentrations were increased at the end of the anhepatic phase and 2, 5 and 30 min after the start of reperfusion. The plasma C3a levels were higher in patients with postreperfusion syndrome compared to those without. CONCLUSIONS: Activation of neutrophils and macrophages and of the complement cascade with the formation of biologically active substances may be one explanation for the circulatory instability often seen in patients undergoing orthotopic liver transplantation.

Complement-derived anaphylatoxins in human donor corneas treated with excimer laser.

BACKGROUND AND OBJECTIVE: An inflammatory response produced by excimer laser photorefractive keratectomy (PRK) may be associated with the subsequent corneal haze and regressions in refractive error observed after treatment. Complement-derived anaphylatoxins, potent mediators of inflammation, may have a role in postoperative healing. MATERIALS AND METHODS: Twenty right human donor corneas underwent a 6-D excimer laser PRK treatment. The corresponding left donor corneas served as the controls. After incubation in tissue culture media for 6 hours and elution in phosphate-buffered saline with EDTA for 24 hours, complement-derived anaphylatoxins C3a, C4a, and C5a were measured in corneal eluates by radioimmunoassay. RESULTS: Compared with control corneas, the excimer PRK corneas failed to demonstrate a significant increase in C3a, C4a, or C5a levels (P > .05). CONCLUSIONS: These results suggest that the excimer laser at this dose does not activate significant complement in the cornea.

[Fundamental and clinical studies of homologous immunoglobulin-free priming in cardiopulmonary bypass, with special reference to anaphylatoxin production].

In in-vitro study, human immunoglobulin (Ig) denatured by O2 bubbling markedly produced C4a, C3a, and C5a, whereas human albumin treated identically did not. White blood cells (WBC) treated by O2 bubbling significantly increased C3a levels alone, but at a much lesser grade than the Ig. A new priming method, i.e., homologous concentrated red cell (CRC) and human albumin was discerned from the experimental facts in-vitro, and we investigated the clinical effects of that priming method. C4a, C3a and C5a in BOG primed with homologous whole blood (HWB) were slightly higher than those in MOG during CPB. Those in the Ig-free priming group were more mildly increased than those in the HWB priming group, not only during CPB, but also after protamine administration; this tendency was clearer in BOG. It is concluded that (1) human immunoglobulin (Ig) denatured by O2 bubbling produces anaphylatoxins via the classical pathway; (2) WBC treated identically produces C3a at a far milder grade; (3) priming with CRC and human albumin reduces plasma anaphylatoxin levels; and (4) pulmonary function at an early postoperative period was improved in the homologous Ig-free priming group, especially with BOG.

[Use of cardiopulmonary bypass decreases leukocyte count and activation of the complement system]. / Zastapienie utleniania pozaustrojowego plucami chorego w krazeniu pozaustrojowym--zmniejsza aktywacje leukocytów i ukladu dopelniacza.

Platelet damage, complement activation and neutropenia during extracorporeal circulation are the result of blood contact with artificial surfaces, mainly in the oxygenator. To evaluate the biocompatibility of the ++auto-oxygenation technique of cardiopulmonary bypass (CPB) 2 techniques of extracorporeal circulation were compared in 40 patients undergoing elective coronary bypass surgery. Patients were studied in 2 groups, 20 patients in each: I (++auto-oxygenation --patients lungs used in CPB) and II (conventional technique of CPB with bubble oxygenator). Several blood samples were taken before, during and after perfusion to estimate pulmonary leukocytes sequestration in all patients and additionally complement C3a and C5a anaphylatoxins + were measured (radioimmunoassays) in 6 patients of each group. During cardiopulmonary bypass the decline in leukocyte number was observed in both groups, but leukocyte count was higher in group I then II, due to the transpulmonary leukocyte sequestration which was higher in group II. The difference between leukocytes count in group II was 1.46 +/- 0.5 x 10(3)/mm3 vs only 0.34 +/- 0.2 x 10(3)/mm3 in group I, p less than 0.001. In postoperative period an increase in circulating white blood cells was observed in both groups when compared to pre-bypass time, but the difference between groups was non significant. The level of C3a increased in group I from 244 +/- 46 ng/ml to 418 +/- 34 ng/ml, in group II from 268 +/- 46 ng/ml to 521 +/- 65 ng/ml, p less than 0.001, but in group I the levels were significantly lower, p less than 0.001. The current study confirms that cardiopulmonary bypass results in significant leukocyte and complement activation and supports the theoreticaly better biocompatibility of CPB with lung over oxygenator.

Intradialytic modulation of granulocyte C5a receptors.

Hemodialysis with new cellulosic membranes is associated with profound granulocytopenia, with a nadir 15 min after initiation, followed by a rebound leukocytosis seen 1 h after initiation and persisting up to the termination of dialysis. The rapid reversal of granulocytopenia during hemodialysis has previously been ascribed to down-regulation of granulocyte C5a receptors. In this report, a method of characterizing C5a receptors by using a novel probe consisting of C5a attached to biotin via a six-carbon spacer chain is described. Cellulose acetate electrophoresis and cation exchange HPLC demonstrated a biotin-to-C5a ratio of 1:1. Analysis of granulocyte cell surface C5a receptors were performed with the probe with a fluorescein-avidin conjugate and by using fluorescence flow cytometry. The maximum decrease in C5a receptors was measured at the 15-min sampling time, when the number of C5a receptor decreased from 189,240 +/- 24,500 predialysis to 160,740 +/- 19,380 receptors (P was not significant) at the nadir of granulocytopenia. However, during recovery from neutropenia, granulocyte cell surface C5a receptors increased to 172,140 +/- 19,380 at 30 min and 193,800 +/- 24,510 at the end of dialysis. Concentrations of C3a and C5a peaked at 15 min and declined rapidly thereafter, but both remained significantly above baseline at all times. These studies suggest that down-regulation of C5a receptors, which is seen maximally at 15 min after initiation of dialysis, does not sufficiently account for the reversal of granulocytopenia during hemodialysis.