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Objective: The complement cascade as major fluid phase innate immune system is activated during progression of osteoarthritis (OA). Generated anaphylatoxins and the corresponding receptors C3aR and C5aR1 are associated with the calcification of blood vessels and involved in osteogenic differentiation. This study aims on elucidating whether complement activation products contribute to cartilage calcification of OA cartilage., Method: Human articular chondrocytes were osteogenically differentiated in vitro in the presence or absence of C3a, C5a, and bone morphogenetic protein (BMP) 2. Furthermore, macroscopically intact (OARSI grade ≤ 1) and highly degenerated human cartilage (OARSI grade ≥ 3) was used for C3aR and C5aR1 histochemistry. Calcification of the cartilage was assessed by Alizarin Red S and von Kossa staining., Results: C3a and C5a amplified matrix mineralization during in vitro osteogenesis, while inhibition of the corresponding receptors impaired calcium deposition. Moreover, C3aR and C5aR1 expression was upregulated during osteogenic differentiation and also in degenerated cartilage. Additionally, anaphylatoxin receptor expression was positively associated with calcification of native cartilage tissue and calcium deposition during osteogenic differentiation. Finally, the pro-hypertrophic growth factor BMP2 induced the expression of C5aR1., Conclusions: Our findings indicate that anaphylatoxins and their receptors play a decisive role in cartilage calcification processes during OA progression., (Copyright © 2024 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Recent advances in cryo-electron microscopy (Cryo-EM) have revolutionized our understanding of the complement C5a/C3a receptors that are crucial in inflammation. A recent report by Yadav et al. has elucidated the activation, ligand binding, selectivity, and signaling bias of these receptors, thereby enhancing structure-guided drug discovery. This paves the way for more effective anti-inflammatory therapies that target these receptors with unprecedented precision., Competing Interests: Declaration of interests F.J.F. is an employee and a shareholder of Abvance Biotech SL. M.C.V. is a founder of Abvance Biotech SL and a member of its scientific advisory board., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

The complement system is a critical part of our innate immune response, and the terminal products of this cascade, anaphylatoxins C3a and C5a, exert their physiological and pathophysiological responses primarily via two GPCRs, C3aR and C5aR1. However, the molecular mechanism of ligand recognition, activation, and signaling bias of these receptors remains mostly elusive. Here, we present nine cryo-EM structures of C3aR and C5aR1 activated by their natural and synthetic agonists, which reveal distinct binding pocket topologies of complement anaphylatoxins and provide key insights into receptor activation and transducer coupling. We also uncover the structural basis of a naturally occurring mechanism to dampen the inflammatory response of C5a via proteolytic cleavage of the terminal arginine and the G-protein signaling bias elicited by a peptide agonist of C3aR identified here. In summary, our study elucidates the innerworkings of the complement anaphylatoxin receptors and should facilitate structure-guided drug discovery to target these receptors in a spectrum of disorders., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

Allergic conditions are associated with canonical and noncanonical activation of the complement system leading to the release of several bioactive mediators with inflammatory and immunoregulatory properties that regulate the immune response in response to allergens during the sensitization and/or the effector phase of allergic diseases. Further, immune sensors of complement and regulator proteins of the cascade impact on the development of allergies. These bioactive mediators comprise the small and large cleavage fragments of C3 and C5. Here, we provide an update on the multiple roles of immune sensors, regulators, and bioactive mediators of complement in allergic airway diseases, food allergies, and anaphylaxis. A particular emphasis is on the anaphylatoxins C3a and C5a and their receptors, which are expressed on many of the effector cells in allergy such as mast cells, eosinophils, basophils, macrophages, and neutrophils. Also, we will discuss the multiple pathways, by which the anaphylatoxins initiate and control the development of maladaptive type 2 immunity including their impact on innate lymphoid cell recruitment and activation. Finally, we briefly comment on the potential to therapeutically target the complement system in different allergic conditions., (© 2023 The Authors. European Journal of Immunology published by Wiley-VCH GmbH.)

Polyphosphates are linear polymers of inorganic phosphates that exist in all living cells and serve pleiotropic functions. Bacteria produce long-chain polyphosphates, which can interfere with host defense to infection. In contrast, short-chain polyphosphates are released from platelet dense granules and bind to the chemokine CXCL4. Here, we report that long-chain polyphosphates induced the release of CXCL4 from mouse bone marrow-derived macrophages and peritoneal macrophages in a dose-/time-dependent fashion resulting from an induction of CXCL4 mRNA. This polyphosphate effect was lost after pre-incubation with recombinant exopolyphosphatase (PPX) Fc fusion protein, demonstrating the potency of long chains over monophosphates and ambient cations. In detail, polyphosphate chains >70 inorganic phosphate residues were required to reliably induce CXCL4. Polyphosphates acted independently of the purinergic P2Y1 receptor and the MyD88/TRIF adaptors of Toll-like receptors. On the other hand, polyphosphates augmented LPS/MyD88-induced CXCL4 release, which was explained by intracellular signaling convergence on PI3K/Akt. Polyphosphates induced Akt phosphorylation at threonine-308. Pharmacologic blockade of PI3K (wortmannin, LY294002) antagonized polyphosphate-induced CXCL4 release from macrophages. Intratracheal polyphosphate administration to C57BL/6J mice caused histologic signs of lung injury, disruption of the endothelial-epithelial barrier, influx of Ly6G + polymorphonuclear neutrophils, depletion of CD11c + SiglecF + alveolar macrophages, and release of CXCL4. Long-chain polyphosphates synergized with the complement anaphylatoxin, C5a, which was partly explained by upregulation of C5aR1 on myeloid cells. C5aR1 -/- mice were protected from polyphosphate-induced lung injury. C5a generation occurred in the lungs and bronchoalveolar lavage fluid (BALF) of polyphosphate-treated C57BL/6J mice. In conclusion, we demonstrate that polyphosphates govern immunomodulation in macrophages and promote acute lung injury., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Roewe, Walachowski, Sharma, Berthiaume, Reinhardt and Bosmann.)

Mas-related G protein-coupled receptor-X2 (MRGPRX2) expressed on mast cells (MCs) contributes to hypersensitivity reactions to cationic US-Food and Drug Administration (FDA) approved drugs such as the neuromuscular blocking agent, rocuronium. In addition, activation of MRGPRX2 by the neuropeptide substance P (SP) and the pro-adrenomedullin peptide (PAMP-12) is associated with a variety of cutaneous conditions such as neurogenic inflammation, pain, atopic dermatitis, urticaria, and itch. Thus, small molecules aimed at blocking MRGPRX2 constitute potential options for modulating IgE-independent MC-mediated disorders. Two inverse MRGPRX2 agonists, named C9 and C9-6, have recently been identified, which inhibit basal G protein activation and agonist-induced calcium mobilization in transfected HEK293 cells. Substance P serves as a balanced agonist for MRGPRX2 whereby it activates both G protein-mediated degranulation and β-arrestin-mediated receptor internalization. The purpose of this study was to determine if C9 blocks MRGPRX2's G protein and β-arrestin-mediated signaling and to determine its specificity. We found that C9, but not its inactive analog C7, inhibited degranulation in RBL-2H3 cells stably expressing MRGPRX2 in response to SP, PAMP-12 and rocuronium with an IC 50 value of ~300 nM. C9 also inhibited degranulation as measured by cell surface expression of CD63, CD107a and β-hexosaminidase release in LAD2 cells and human skin-derived MCs in response to SP but not the anaphylatoxin, C3a or FcϵRI-aggregation. Furthermore, C9 inhibited β-arrestin recruitment and MRGPRX2 internalization in response to SP and PAMP-12. We found that a G protein-coupling defective missense MRGPRX2 variant (V282M) displays constitutive activity for β-arrestin recruitment, and that this response was significantly inhibited by C9. Rocuronium, SP and PAMP-12 caused degranulation in mouse peritoneal MCs and these responses were abolished in the absence of MrgprB2 or cells treated with pertussis toxin but C9 had no effect. These findings suggest that C9 could provide an important framework for developing novel therapeutic approaches for the treatment of IgE-independent MC-mediated drug hypersensitivity and cutaneous disorders., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Bawazir, Amponnawarat, Hui, Oskeritzian and Ali.)

Anaphylatoxin C3a is a small signaling polypeptide that is generated during complement activation. C3a is involved in the regulation of various innate and adaptive immune system processes; however, the role of C3a in macrophage differentiation and polarization is poorly elucidated. Here we showed that C3a impairs alternative M2 polarization of human macrophages and suppressed CD206, IL1Ra and CCL22 expression. C3a leads to a decrease of nuclear receptor PPARγ expression via the ERK1/2 signaling pathway, resulting in repressed PPARγ-dependent activation of CD36, FABP4 and LXRα genes and blunted response to an LXR ligand TO901317. Using small interfering RNA and agonist/antagonist approaches we showed that C3a decreases CD206, IL1Ra and CCL22 transcription at least partly in a PPARγ-dependent manner in M2 macrophages. Moreover, C3a impairs efferocytosis by M2 macrophages and inhibits their migratory activity. By contrast, macrophages treated with C3a during differentiation show blunted response to lipopolysaccharide stimulation owing to downregulation of TLR4 and lipid raft content. At the same time, differentiation of macrophages with C3a does not change M1 polarization in interferon gamma (IFNγ) and IFNγ + lipopolysaccharide-treated macrophages. These data provide a novel role of complement system and C3a in the regulation of M2 macrophage polarizations and suggest crosstalk between C3a, TLR4, PPARγ and LXR signaling pathways., (© 2022 Australian and New Zealand Society for Immunology, Inc.)

Objectives/hypothesis: The objective of this study was to determine the role of thrombin-activatable fibrinolysis inhibitor (TAFI) as a candidate biomarker for therapeutic efficacy of sublingual immunotherapy (SLIT) and to identify the role of TAFI in the pathogenesis of allergic rhinitis (AR)., Study Design: Retrospective cohort study and laboratory study., Methods: Serum was collected from patients with allergies to Japanese cedar pollen before, during, and after treatment with SLIT. We measured the levels of immunoreactive TAFI, C3a, and C5a in serum by enzyme-linked immunosorbent assay (ELISA) and assessed their relative impact on a combined symptom-medication score. We also examined the impact of TAFI on mast cells and fibroblasts in experiments performed in vitro., Results: Serum levels of TAFI increased significantly in response to SLIT. By contrast, serum C3a levels decreased significantly over time; we observed a significant negative correlation between serum levels of TAFI versus C3a and symptom-medication score. Mast cell degranulation was inhibited in response to TAFI, as it was the expression of both CCL11 and CCL5 in cultured fibroblasts., Conclusions: High serum levels of TAFI may be induced by SLIT. TAFI may play a critical protective role in pathogenesis of AR by inactivating C3a and by inhibiting mast cell degranulation and chemokines expression in fibroblasts., Level of Evidence: 4 Laryngoscope, 131:2413-2420, 2021., (© 2021 The American Laryngological, Rhinological and Otological Society, Inc.)

T cells play a fundamental role in the early control and clearance of many viral infections of the respiratory system. In SARS-CoV-2-infected individuals, lymphopenia with drastically reduced CD4 + and CD8 + T cells correlates with Coronavirus disease 2019 (COVID-19)-associated disease severity and mortality. In this study, we characterized cellular and humoral immune responses induced in patients with mild, severe and critical COVID-19. Peripheral blood mononuclear cells of 37 patients with mild, severe and critical COVID-19 and 10 healthy individuals were analyzed by IFNγ ELISpot and multi-color flow cytometry upon stimulation with peptide pools covering complete immunodominant SARS-CoV-2 matrix, nucleocapsid and spike proteins. In addition SARS-CoV-2 antibody levels, neutralization abilities and anaphylatoxin levels were evaluated by various commercially available ELISA platforms. Our data clearly demonstrates a significantly stronger induction of SARS-CoV-2 specific CD8 + T lymphocytes and higher IFNγ production in patients with mild compared to patients with severe or critical COVID-19. In all patients SARS-CoV-2-specific antibodies with similar neutralizing activity were detected, but highest titers of total IgGs were observed in critical patients. Finally, elevated anaphylatoxin C3a and C5a levels were identified in severe and critical COVID-19 patients probably caused by aberrant immune complex formation due to elevated antibody titers in these patients. Crucially, we provide a full picture of cellular and humoral immune responses of COVID-19 patients and prove that robust polyfunctional CD8 + T cell responses concomitant with low anaphylatoxin levels correlate with mild infections. In addition, our data indicates that high SARS-CoV-2 antibody titers are associated with severe disease progression., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Lafon, Diem, Witting, Zaderer, Bellmann-Weiler, Reindl, Bauer, Griesmacher, Fux, Hoermann, Miller, Zabernigg, Wöll, Wilflingseder, Lass-Flörl and Posch.)

Anaphylaxis is a rapidly evolving, acute, life-threatening reaction that occurs rapidly on contact with a trigger. Anaphylaxis is classically defined as an allergen-driven process that induces specific IgE and the activation of mast cells and basophils through the cross-linking of IgE receptors. However, it is clear that non-IgE-mediated pathways can induce symptoms indistinguishable from those of classic anaphylaxis, and their activation could explain the severity of IgE-mediated anaphylaxis. Indeed, mast cells and basophils can be activated by antibodies against IgE or their receptors, by molecules such as anaphylatoxins, or through G-coupled receptors. Some other allergens can induce antibodies of class IgG that can activate neutrophils to produce a molecule similar to histamine to induce anaphylaxis. Finally, some inflammatory mediators such as bradykinin or prostaglandin can also modulate mast cell and basophil activation as well as directly cause vasodilation and bronchoconstriction, resulting in anaphylaxis-like reactions., (Copyright © 2021 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.)

The zoonotic intracellular bacterium Chlamydia psittaci causes life-threatening pneumonia in humans. During mouse lung infection, complement factor C3 and the anaphylatoxin C3a augment protection against C. psittaci by a so far unknown mechanism. To clarify how complement contributes to the early, innate and the late, specific immune response and resulting protection, this study addresses the amount of C3, the timing when its presence is required as well as the anaphylatoxin receptor(s) mediating its effects and the complement-dependent migration of dendritic cells. Challenge experiments with C. psittaci on various complement KO mice were combined with transient decomplementation by pharmacological treatment, as well as the analysis of in vivo dendritic cells migration. Our findings reveal that a plasma concentration of C3 close to wildtype levels was required to achieve full protection. The diminished levels of C3 of heterozygote C3 +/- mice permitted already relative effective protection and improved survival as compared to C3 -/- mice, but overall recovery of these animals was delayed. Complement was in particular required during the first days of infection. However, additionally, it seems to support protection at later stages. Migration of CD103 + dendritic cells from the infected lung to the draining lymph node-as prerequisite of antigen presentation-depended on C3 and C3aR and/or C5aR. Our results provide unique mechanistic insight in various aspects of complement-dependent immune responses under almost identical, rather physiological experimental conditions. Our study contributes to an improved understanding of the role of complement, and C3a in particular, in infections by intracellular bacteria., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Kohn, Lanfermann, Laudeley, Glage, Rheinheimer and Klos.)

Background: Thrombosis and coagulopathy are highly prevalent in critically ill patients with COVID-19 and increase the risk of death. Immunothrombosis has recently been demonstrated to contribute to the thrombotic events in COVID-19 patients with coagulopathy. As the primary components of immunothrombosis, neutrophil extracellular traps (NETs) could be induced by complement cascade components and other proinflammatory mediators. We aimed to explore the clinical roles of NETs and the regulation of complement on the NET formation in COVID-19., Methods: We recruited 135 COVID-19 patients and measured plasma levels of C5, C3, cell-free DNA and myeloperoxidase (MPO)-DNA. Besides, the formation of NETs was detected by immunofluorescent staining and the cytotoxicity to vascular endothelial HUVEC cells was evaluated by CCK-8 assay., Results: We found that the plasma levels of complements C3 and MPO-DNA were positively related to coagulation indicator fibrin(-ogen) degradation products (C3: r = 0.300, p = 0.005; MPO-DNA: r = 0.316, p = 0.002) in COVID-19 patients. Besides, C3 was positively related to direct bilirubin (r = 0.303, p = 0.004) and total bilirubin (r = 0.304, p = 0.005), MPO-DNA was positively related to lactate dehydrogenase (r = 0.306, p = 0.003) and creatine kinase (r = 0.308, p = 0.004). By using anti-C3a and anti-C5a antibodies, we revealed that the complement component anaphylatoxins in the plasma of COVID-19 patients strongly induced NET formation. The pathological effect of the anaphylatoxin-NET axis on the damage of vascular endothelial cells could be relieved by recombinant carboxypeptidase B (CPB), a stable homolog of enzyme CPB2 which can degrade anaphylatoxins to inactive products., Conclusions: Over-activation in anaphylatoxin-NET axis plays a pathological role in COVID-19. Early intervention in anaphylatoxins might help prevent thrombosis and disease progression in COVID-19 patients.

The complement system may be activated in the posterior segment of the eye with chorioretinal disease, which may be reflected to the concentration of anaphylatoxins in the aqueous humor. Little is known about the distribution of anaphylatoxins in the aqueous and vitreous humor. The aim of the present study was to investigate the distribution of anaphylatoxin concentration in the aqueous and vitreous humor of the eyes with idiopathic epiretinal membrane or idiopathic macular hole. This was an experimental, observational case series. This study included 43 eyes from 43 patients; 29 eyes with idiopathic epiretinal membrane, and 14 eyes with idiopathic macular hole. All 43 eyes underwent cataract surgery and vitrectomy. The aqueous and vitreous humor were collected at the surgery. The anaphylatoxin concentrations were measured by using a cytometric beads array, and the respective C3a, C4a, and C5a concentrations were 2.003 ± 0.679 (mean ± standard deviation) ng/ml, 1.389 ± 0.419 ng/ml, and 0.003 ± 0.004 ng/ml in the aqueous humor, and 1.236 ± 0.642 ng/ml, 1.250 ± 0.542 ng/ml, and 0.048 ± 0.069 ng/ml in the vitreous humor. The mean C3a concentration in the aqueous humor was significantly higher than in the vitreous humor in 43 eyes of iMH and iERM (P < 0.001). The mean C4a concentration showed no significant difference between the aqueous humor and vitreous humor (P = 0.282), and the mean C5a in the aqueous humor was significantly lower than in the vitreous humor overall (P < 0.001). The C3a concentration in the aqueous humor strongly correlated with that in the vitreous humor (R = 0.510, P < 0.001). The concentrations of C4a and C5a in the aqueous humor moderately correlated with those in the vitreous humor (C4a; R = 0.356, P = 0.019, C5a; R = 0.464, P = 0.022). In conclusion, the anaphylatoxin concentrations measured by cytometric beads array in the aqueous humor may be associated with those measured in the vitreous humor., (Copyright © 2020. Published by Elsevier Ltd.)

Stromal-epithelial interactions dictate cancer progression and therapeutic response. Prostate cancer (PCa) cells were identified to secrete greater concentration of mitochondrial DNA (mtDNA) compared to noncancer epithelia. Based on the recognized coevolution of cancer-associated fibroblasts (CAF) with tumor progression, we tested the role of cancer-derived mtDNA in a mechanism of paracrine signaling. We found that prostatic CAF expressed DEC205, which was not expressed by normal tissue-associated fibroblasts. DEC205 is a transmembrane protein that bound mtDNA and contributed to pattern recognition by Toll-like receptor 9 (TLR9). Complement C3 was the dominant gene targeted by TLR9-induced NF-κB signaling in CAF. The subsequent maturation complement C3 maturation to anaphylatoxin C3a was dependent on PCa epithelial inhibition of catalase in CAF. In a syngeneic tissue recombination model of PCa and associated fibroblast, the antagonism of the C3a receptor and the fibroblastic knockout of TLR9 similarly resulted in immune suppression with a significant reduction in tumor progression, compared to saline-treated tumors associated with wild-type prostatic fibroblasts. Interestingly, docetaxel, a common therapy for advanced PCa, further promoted mtDNA secretion in cultured epithelia, mice, and PCa patients. The antiapoptotic signaling downstream of anaphylatoxin C3a signaling in tumor cells contributed to docetaxel resistance. The inhibition of C3a receptor sensitized PCa epithelia to docetaxel in a synergistic manner. Tumor models of human PCa epithelia with CAF expanded similarly in mice in the presence or absence of docetaxel. The combination therapy of docetaxel and C3 receptor antagonist disrupted the mtDNA/C3a paracrine loop and restored docetaxel sensitivity., Competing Interests: Competing interest statement: Part of the work has been submitted for patent protection (S.H. and N.A.B.)., (Copyright © 2020 the Author(s). Published by PNAS.)

In the present study, we sought to elucidate the mechanisms by which monocytes migrate into the pleural space in the presence of anaphylatoxins in tuberculous pleural effusion (TPE). Monocytes in both pleural effusion and blood were counted, and their phenotypic characteristics were analyzed. Activation of the complement system was detected in TPE. The effects of Mpt64 and anaphylatoxins on the production of chemokines in pleural mesothelial cells (PMCs) were measured. The chemoattractant activity of chemokines produced by PMCs for monocytes was observed. Levels of CD14 + CD16 + monocytes were significantly higher in TPE than in blood. Three pathways of the complement system were activated in TPE. C3a-C3aR1, C5a-C5aR1, CCL2-CCR2, CCL7-CCR2, and CX3CL1-CX3CR1 were coexpressed in PMCs and monocytes isolated from TPE. Moreover, we initially found that Mpt64 stimulated the expression of C3a and C5a in PMCs. C3a and C5a not only induced CCL2, CCL7, and CX3CL1 expression in PMCs but also stimulated production of IL-1β, IL-17, and IL-27 in monocytes. C3a and C5a stimulated PMCs to secrete CCL2, CCL7, and CX3CL1, which recruited CD14 + CD16 + monocytes to the pleural cavity. As a result, the infiltration of CD14 + CD16 + monocytes engaged in the pathogenesis of TPE by excessive production of inflammatory cytokines.

Background: Understanding of iron oxide nanoparticles (IONP) interaction with the body milieu is crucial to guarantee their efficiency and biocompatibility in nanomedicine. Polymer coating to IONP, with polyethyleneglycol (PEG) and polyvinylpyrrolidone (PVP), is an accepted strategy to prevent toxicity and excessive protein binding., Aim: The aim of this study was to investigate the feature of IONP adsorption of complement proteins, their activation and consequent inflammatory response as a strategy to further elucidate their biocompatibility., Methods: Three types of IONP with different surface characteristics were used: bare (IONP-bare), coated with PVP (IONP-PVP) and PEG-coated (IONP-PEG). IONPs were incubated with human plasma and adsorbed proteins were identified. BALB/c mice were intravenously exposed to IONP to evaluate complement activation and proinflammatory response., Results: Protein corona fingerprinting showed that PEG surface around IONP promoted a selective adsorption of complement recognition molecules which would be responsible for the complement system activation. Furthermore, IONP-PEG activated in vitro, the complement system and induced a substantial increment of C3a and C4a anaphylatoxins while IONP-bare and IONP-PVP did not. In vivo IONP-PEG induced an increment in complement activation markers (C5a and C5b-9), and proinflammatory cytokines (IL-1β, IL-6, TNF-α)., Conclusion: The engineering of nanoparticles must incorporate the association between complement proteins and nanomedicines, which will regulate the immunostimulatory effects through a selective adsorption of plasma proteins and will enable a safer application of IONP in human therapy., Competing Interests: Disclosure The authors report no conflicts of interest in this work.

The anaphylatoxin C5a is generated upon activation of the complement system, a crucial arm of innate immunity. C5a mediates proinflammatory actions via the C5a receptor C5aR1 and thereby promotes host defence, but also modulates tissue homeostasis. There is evidence that the C5a/C5aR1 axis is critically involved both in physiological bone turnover and in inflammatory conditions affecting bone, including osteoarthritis, periodontitis, and bone fractures. C5a induces the migration and secretion of proinflammatory cytokines of osteoblasts. However, the underlying mechanisms remain elusive. Therefore, in this study we aimed to determine C5a-mediated downstream signalling in osteoblasts. Using a whole-genome microarray approach, we demonstrate that C5a activates mitogen-activated protein kinases (MAPKs) and regulates the expression of genes involved in pathways related to insulin, transforming growth factor-β and the activator protein-1 transcription factor. Interestingly, using coimmunoprecipitation, we found an interaction between C5aR1 and Toll-like receptor 2 (TLR2) in osteoblasts. The C5aR1- and TLR2-signalling pathways converge on the activation of p38 MAPK and the generation of C-X-C motif chemokine 10, which functions, among others, as an osteoclastogenic factor. In conclusion, C5a-stimulated osteoblasts might modulate osteoclast activity and contribute to immunomodulation in inflammatory bone disorders., (© 2018 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.)

Background and aims: Chronic ethanol exposure results in inflammation in adipose tissue; this response is associated with activation of complement as well as the development of alcohol-related liver disease (ALD). Adipose communicates with other organs, including liver, via the release of soluble mediators, such as adipokines and cytokines, characterized as the "adipose secretome." Here we investigated the role of the anaphaylatoxin receptors C3aR and C5aR1 in the development of adipose tissue inflammation and regulation of the adipose secretome in murine ALD (mALD). Methods: Wild-type C57BL/6 (WT), C3aR -/- , and C5aR1 -/- mice were fed Lieber-DeCarli ethanol diet for 25 days (6% v/v, 32% kcal) or isocaloric control diets; indicators of inflammation and injury were assessed in gonadal adipose tissue. The adipose secretome was characterized in isolated adipocytes and stromal vascular cells. Results: Ethanol feeding increased the expression of adipokines, chemokines and leukocyte markers in gonadal adipose tissue from WT mice; C3aR -/- were partially protected while C5aR1 -/- mice were completely protected. In contrast, induction of CYP2E1 and accumulation of TUNEL-positive cells in adipose in response to ethanol feeding was independent of genotype. Bone marrow chimeras, generated with WT and C5aR1 -/- mice, revealed C5aR1 expression on non-myeloid cells, likely to be adipocytes, contributed to ethanol-induced adipose inflammation. Chronic ethanol feeding regulated both the quantity and distribution of adipokines secreted from adipocytes in a C5aR1-dependent mechanism. In WT mice, chronic ethanol feeding induced a predominant release of pro-inflammatory adipokines from adipocytes, while the adipose secretome from C5aR1 -/- mice was characterized by an anti-inflammatory/protective profile. Further, the cargo of adipocyte-derived extracellular vesicles (EVs) was distinct from the soluble secretome; in WT EVs, ethanol increased the abundance of pro-inflammatory mediators while EV cargo from C5aR1 -/- adipocytes contained a greater diversity and more robust expression of adipokines. Conclusions: C3aR and C5aR1 are potent regulators of ethanol-induced adipose inflammation in mALD. C5aR1 modulated the impact of chronic ethanol on the content of the adipose secretome, as well as influencing the cargo of an extensive array of adipokines from adipocyte-derived EVs. Taken together, our data demonstrate that C5aR1 contributes to ethanol-mediated changes in the adipose secretome, likely contributing to intra-organ injury in ALD.

The complement system (CS) has recently been recognized as a bridge between innate and adaptive immunity that constitutes a very complex mechanism controlling the clearance of pathogens, cellular debris, and immune complexes. Out of three known pathways of complement activation, the alternative pathway (AP) plays a critical role in host defense by amplifying the complement response, independently of initiation pathway and continuously maintaining low-level activity in a process called 'thick-over.' A key molecule of the CS is C3, in which the AP is constantly activated. To prevent host cell destruction, a group of the AP regulators tightly controls this pathway of the CS activation. Acquired and genetic abnormalities of the CS may alter the delicate balance between enhancing and inhibiting the AP cascade. These can lead to the uncontrolled CS activation, inflammatory response, and subsequent tissue damage. Since complement components are locally produced and activated in the kidney, the abnormalities targeting the AP may cause glomerular injury. C3 glomerulopathy is a new entity, in which the AP dysregulation has been well established. However, recent studies indicate that the AP may also contribute to a wide range of kidney pathologies, including immune-complex-mediated glomerulonephritis (GN), pauci-immune GN, and primary membranous nephropathy (PMN). This article provides insight into current knowledge on the role of the AP in the pathogenesis of glomerular diseases, focusing mainly on various types of primary and secondary GN and PMN.

The mechanisms that connect complement system activation and basal deposit formation in early stages of age-related macular degeneration (AMD) are insufficiently understood, which complicates the design of efficient therapies to prevent disease progression. Using human fetal (hf) retinal pigment epithelial (RPE) cells, we have established an in vitro model to investigate the effect of complement C3a on RPE cells and its role in the formation of sub-RPE deposits. The results of these studies revealed that C3a produced after C3 activation is sufficient to induce the formation of sub-RPE deposits via complement-driven proteasome inhibition. C3a binds the C3a receptor (C3aR), stimulates deposition of collagens IV and VI underneath the RPE, and impairs the extracellular matrix (ECM) turnover by increased MMP-2 activity, all mediated by downregulation of the ubiquitin proteasome pathway (UPP). The formation of basal deposits can be prevented by the addition of a C3aR antagonist, which restores the UPP activity and ECM turnover. These findings indicate that the cell-based model can be used to test potential therapeutic agents in vitro. The data suggest that modulation of C3aR-mediated events could be a therapeutic approach for treatment of early AMD.

Thoracic aortic dissection (TAD), once ruptured, is devastating to patients, and no effective pharmaceutical therapy is available. Anaphylatoxins released by complement activation are involved in a variety of diseases. However, the role of the complement system in TAD is unknown. We found that plasma levels of C3a, C4a, and C5a were significantly increased in patients with TAD. Elevated circulating C3a levels were also detected in the developmental process of mouse TAD, which was induced by β-aminopropionitrile monofumarate (BAPN) treatment, with enhanced expression of C1q and properdin in mouse dissected aortas. These findings indicated activation of classical and alternative complement pathways. Further, expression of C3aR was obviously increased in smooth muscle cells of human and mouse dissected aortas, and knockout of C3aR notably inhibited BAPN-induced formation and rupture of TAD in mice. C3aR antagonist administered pre- and post-BAPN treatment attenuated the development of TAD. We found that C3aR knockout decreased matrix metalloproteinase 2 (MMP2) expression in BAPN-treated mice. Additionally, recombinant C3a stimulation enhanced MMP2 expression and activation in smooth muscle cells that were subjected to mechanical stretch. Finally, we generated MMP2-knockdown mice by in vivo MMP2 short hairpin RNA delivery using recombinant adeno-associated virus and found that MMP2 deficiency significantly reduced the formation of TAD. Therefore, our study suggests that the C3a - C3aR axis contributes to the development of TAD via regulation of MMP2 expression. Targeting the C3a-C3aR axis may represent a strategy for inhibiting the formation of TAD., (Copyright © 2018 by The American Association of Immunologists, Inc.)

The complement system plays important biological roles, including the activation of inflammatory processes in response to the generation of proteolytic fragments of its components. Here we evaluated the effects of Bothrops atrox venom and two of its toxins (the P-I metalloprotease Batroxase and the acidic phospholipase A 2 BatroxPLA 2 ) on the human complement system, evaluating their effects on the classical (CP), lectin (LP) and alternative (AP) pathways, as well as on different complement components associated to the generation of anaphylatoxins. Primarily, the venom and both toxins modulated the hemolytic activity of the complement CP, with the venom and Batroxase reducing this activity and BatroxPLA 2 increasing it. ELISA deposition assays indicated that B. atrox venom and Batroxase were also capable of modulating all three activation pathways (CP, LP and AP), reducing their activity after incubation with normal human serum (NHS), while BatroxPLA 2 apparently only interfered with AP. Additionally, the venom and Batroxase, but not BatroxPLA 2 , promoted significant degradation of the components C3, C4, Factor B and C1-Inhibitor, as shown by Western blot and SDS-PAGE analyses, also generating anaphylatoxins C3a, C4a and C5a. Therefore, B. atrox venom and Batroxase were able to activate the complement system by direct proteolytic action on several components, generating anaphylatoxins and affecting the activation pathways, while BatroxPLA 2 only interfered with the hemolysis induced by CP and the C3 deposition related to AP. Our results indicate that Batroxase and possibly other metalloproteases should be the main toxins in B. atrox venom to induce pronounced effects on the complement system., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Anaphylatoxin C5a, a potent pro-inflammatory peptide produced in the process of complement activation, was proved to have a vital role in tumor initiation and progession by previous investigations. However whether it could act as a prognostic marker remains unknown. Here we retrospectively enrolled 272 ccRCC patients undergoing nephrectomy in Zhongshan Hospital, Shanghai between 2005 and 2007. C5a level was assessed by immunohistochemistry and its association with clinicopathologic features and prognosis were evaluated. Our results indicated that high tumoral C5a level was associated with poor overall survival (OS) (hazard ratio = 1.753, 95% CI 1.068-2.878, P = 0.026). In addition, tumoral C5a could significantly stratify patients' prognosis both in advanced stage (TNM III + IV) and intermediate/high risk group (SSIGN score ≥4) (P < 0.001 and = 0.008, respectively). Furthermore, incorporating tumoral C5a with other parameters could improve the predicting accuracy, compared with TNM and SSIGN system (c-index = 0.789, 0.713 and 0.727, respectively). In conclusion, tumoral C5a is an independent adverse prognostic biomarker for clinical outcome of ccRCC patients after nephectomy.

Acquired demyelinating syndromes (ADS) in children evolve either as a monophasic disease diagnosed as acute demyelinating encephalomyelitis (ADEM), transverse myelitis (TM) or optic neuritis (ON), or a multiphasic one with several relapses most often leading to the diagnosis of multiple sclerosis (MS) or neuromyelitis optica (NMO). These neuroinflammatory disorders are increasingly associated with autoantibodies against proteins such as aquaporin-4 in rare instances, and more frequently against myelin oligodendrocyte glycoprotein (MOG). Recently, in adult NMO patients, C5a levels were shown to be elevated in cerebrospinal fluid (CSF) during acute exacerbation. We investigated the CSF levels of anaphylatoxins and pro-inflammatory cytokines, and plasma MOG antibodies in onset samples from children with ADS. Thirty four children presenting with a first episode of ADS, 17 with monophasic ADS (9 with ADEM, 4 with TM and 4 with ON) and 17 with MS, who had paired blood and CSF samples at onset were included and compared to 12 patients with other non-inflammatory neurological disorders (OND). Cytokines and anaphylatoxins in CSF were measured by Cytometric Bead Array immunoassay. MOG antibody titers in plasma were tested by flow cytometry using a stable cell line expressing full-length human MOG. We found a significant increase in C5a levels in the CSF of patients with monophasic ADS (n=17) compared to OND (n=12, p=0.0036) and to MS (n=17, p=0.0371). The C5a levels in MS were higher than in OND without reaching significance (p=0.2). CSF IL-6 levels were significantly increased in monophasic ADS compared to OND (p=0.0027) and to MS (p=0.0046). MOG antibody plasma levels were significantly higher in monophasic ADS (p<0.0001) and, to a lesser extent, in MS compared to OND (p=0.0023). Plasma MOG antibodies and CSF IL-6 levels were significantly correlated (r=0.51, p=0.018). CSF C5a and IL-6 levels are increased in monophasic ADS but not in MS when compared to OND, suggesting that these markers may help to predict monophasic or relapsing fate of ADS at onset. MOG antibody titers, which were higher in monophasic ADS than in MS, correlated with IL-6 levels, but not with C5a, suggesting an association between MOG antibodies and neuroinflammation in pediatric ADS., (Copyright © 2015 Elsevier B.V. All rights reserved.)

The aim of this study was to investigate the potential relationship between acylation-stimulating protein (ASP), insulin resistance, lipometabolism, the intrauterine metabolic environment and fetal growth in well-controlled gestational diabetes mellitus (GDM) women. A total of 55 well-controlled GDM women, 66 pregnant women with normal glucose tolerance (NGT) and their newborns, were included in this study. Fasting maternal and cord blood ASP, serum lipid profiles, glucose level, insulin level, HOMA-IR, in addition to neonatal anthropometry data, were measured. Maternal blood ASP in GDM is higher than that in NGT. In the GDM group, maternal blood ASP has a positive correlation with TG, FFA and HOMA-IR. Maternal and cord blood ASP levels of LGA fetuses correlate with elevated birth weight and SF4. Similarly, cord blood ASP levels of LGA fetuses also correlate with birth weight and SF4 in the NGT group. The maternal blood ASP level of GDM mothers is associated with lipometabolism, insulin resistance and LGA fetal growth. Nevertheless, the cord blood ASP level correlates with FFA of GDM mothers, LGA fetal growth of GDM and NGT mothers. ASP may be a biomarker for evaluating insulin resistance of GDM and LGA fetal growth.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease, characterized by the progressive loss of motor neurons within the central nervous system. Neural degeneration and inflammatory processes, including activation of the complement system are hallmarks of this pathology. Our past work in ALS animal models (hSOD1 G93A rodents) has revealed that blockade of the receptor for complement activation fragment C5a (C5aR), improves ALS-like symptoms and extends survival. We now show that the levels of C5a and C5b-9, but not C3a nor C4a, are significantly elevated in plasma from ALS patients compared to healthy controls. C5a was also elevated within leukocytes from ALS patients suggesting heightened C5a receptor interaction. Overall, these findings indicate that there is enhanced peripheral immune complement terminal pathway activation in ALS, which may have relevance in the disease process., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Activation of the classical pathway (CP) of complement is often associated with autoimmune disorders in which disease pathology is linked to the presence of an autoantibody. One such disorder is cold agglutinin disease (CAD), an autoimmune hemolytic anemia in which autoantibodies (cold agglutinins) bind to red blood cells (RBCs) at low temperatures. Anemia occurs as a result of autoantibody-mediated CP activation on the surface of the erythrocyte, leading to the deposition of complement opsonins that drive extravascular hemolysis in the liver. Here we test the effects of TNT003, a mouse monoclonal antibody targeting the CP-specific serine protease C1s, on CP activity induced by cold agglutinins on human RBCs. We collected 40 individual CAD patient samples and showed that TNT003 prevented cold agglutinin-mediated deposition of complement opsonins that promote phagocytosis of RBCs. Furthermore, we show that by preventing CP activation, TNT003 also prevents cold agglutinin-driven generation of anaphylatoxins. Finally, we provide evidence that CP activity in CAD patients terminates prior to activation of the terminal cascade, supporting the hypothesis that the primary route of RBC destruction in these patients occurs via extravascular hemolysis. Our results support the development of a CP inhibitor for the treatment of CAD., (© 2014 by The American Society of Hematology.)

Background: Increasing evidence demonstrates a phenotypic plasticity of endothelial cells (ECs). Endothelial-to-mesenchymal transition (EndMT) contributes to the development of tissue fibrosis. However, the pathogenic factors and signalling pathways regulating this process in ischaemia/reperfusion (I/R) injury are still poorly understood., Methods: We investigated the possible role of complement in the induction of this endothelial dysfunction in a swine model of renal I/R injury by using recombinant C1 inhibitor in vivo., Results: Here, we showed that I/R injury reduced the density of renal peritubular capillaries and induced tissue fibrosis with generation of CD31(+)/α-SMA(+) and CD31(+)/FPS-1(+) cells indicating EndMT. When we inhibited complement, the process of EndMT became rare, with preserved density of peritubular capillaries and significant reduction in renal fibrosis. When we activated ECs by anaphylatoxins in vitro, C3a and C5a led to altered endothelial phenotype with increased expression of fibroblast markers and decrease expression of specific endothelial markers. The activation of Akt pathway was pivotal for the C3a and C5a-induced EndMT in vitro. In accordance, inhibition of complement in vivo led to the abrogation of Akt signalling, with hampered EndMT and tissue fibrosis., Conclusions: Our data demonstrate a critical role for complement in the acute induction of EndMT via the Akt pathway. Therapeutic inhibition of these systems may be essential to prevent vascular damage and tissue fibrosis in transplanted kidney.

Hyperglycemia from diabetes is associated with increased risk of infection from S. aureus and increased severity of illness. Previous work in our laboratory demonstrated that elevated glucose (>6 mM) dramatically inhibited S. aureus-initiated complement-mediated immune effectors. Here we report in vivo studies evaluating the extent to which a hyperglycemic environment alters complement-mediated control of S. aureus infection in a rat peritonitis model. Rats were treated with streptozocin to induce diabetes or sham-treated and then inoculated i.p. with S. aureus. Rats were euthanized and had peritoneal lavage at 2 or 24 hours after infection to evaluate early and late complement-mediated effects. Hyperglycemia decreased the influx of IgG and complement components into the peritoneum in response to S. aureus infection and decreased anaphylatoxin generation. Hyperglycemia decreased C4-fragment and C3-fragment opsonization of S. aureus recovered in peritoneal fluids, compared with euglycemic or insulin-rescued rats. Hyperglycemic rats showed decreased phagocytosis efficiency compared with euglycemic rats, which correlated inversely with bacterial survival. These results suggest that hyperglycemia inhibited humoral effector recruitment, anaphylatoxin generation, and complement-mediated opsonization of S. aureus, suggesting that hyperglycemic inhibition of complement effectors may contribute to the increased risk and severity of S. aureus infections in diabetic patients.

Complement receptors are expressed on cells of the innate and the adaptive immune system. They play important roles in pathogen and danger sensing as they translate the information gathered by complement fluid phase sensors into cellular responses. Further, they control complement activation on viable and apoptotic host cells, clearance of immune complexes and mediate opsonophagocytosis. More recently, evidence has accumulated that complement receptors form a complex network with other innate receptors systems such as the Toll-like receptors, the Notch signaling system, IgG Fc receptors and C-type lectin receptors contributing to the benefit and burden of innate and adaptive immune responses in autoimmune and allergic diseases as well as in cancer and transplantation. Here, we will discuss recent developments and emerging concepts of complement receptor activation and regulation with a particular focus on the differentiation, maintenance and contraction of effector and regulatory T cells., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

In the tunicate Ciona intestinalis, the ciliated pharynx, which connects the external environment to a highly developed and compartmentalized gastrointestinal system, represents the natural portal of entry for a vast and diverse, potentially pathogenic microbial community. To address the role of the pharynx in immune surveillance in Ciona, we asked whether C3, the key component of the complement system, was expressed in this organ and whether the encoded protein was functionally active. We found by real-time PCR that C3, constitutively expressed in the pharynx, is up-regulated by LPS injection. Using two specific anti-CiC3 and anti-CiC3a polyclonal antibodies in immunohistochemical staining of pharynx sections, we found that the gene product was localized to hemocytes of the pharyngeal bars (identified as granular amoebocytes) and in stigmata ciliated cells. Use of the same antibodies in Western blot analysis indicated that CiC3 and its activation products CiC3b and CiC3a are present in pharynx homogenates. Our observation that the amount of the bioactive fragment CiC3a increased in the pharynx of LPS-treated animals provides the first molecular and functional evidence for complement-mediated immunological activity in the tunicate pharynx., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Reperfusion of an organ following prolonged ischemia instigates the pro-inflammatory and pro-coagulant response of ischemia / reperfusion (IR) injury. IR injury is a wide-spread pathology, observed in many clinically relevant situations, including myocardial infarction, stroke, organ transplantation, sepsis and shock, and cardiovascular surgery on cardiopulmonary bypass. Activation of the classical, alternative, and lectin complement pathways and the generation of the anaphylatoxins C3a and C5a lead to recruitment of polymorphonuclear leukocytes, generation of radical oxygen species, up-regulation of adhesion molecules on the endothelium and platelets, and induction of cytokine release. Generalized or pathway-specific complement inhibition using protein-based drugs or low-molecular-weight inhibitors has been shown to significantly reduce tissue injury and improve outcome in numerous in-vitro, ex-vivo, and in-vivo models. Despite the obvious benefits in experimental research, only few complement inhibitors, including C1-esterase inhibitor, anti-C5 antibody, and soluble complement receptor 1, have made it into clinical trials of IR injury. The results are mixed, and the next objectives should be to combine knowledge and experience obtained in the past from animal models and channel future work to translate this into clinical trials in surgical and interventional reperfusion therapy as well as organ transplantation.

Oxidatively modified low-density lipoproteins (Ox-LDL) and complement anaphylatoxins C3a and C5a are colocalized in atherosclerotic lesions. Anaphylatoxin C3a also binds and breaks bacterial lipid membranes and phosphatidylcholine liposomes. The role of oxidized lipid adducts in C3a binding to Ox-LDL and apoptotic cells was investigated. Recombinant human C3a bound specifically to low-density lipoprotein and bovine serum albumin modified with malondialdehyde (MDA) and malondialdehyde acetaldehyde (MAA) in chemiluminescence immunoassays. No binding was observed to native proteins, LDL oxidized with copper ions (CuOx-LDL), or phosphocholine. C3a binding to MAA-LDL was inhibited by two monoclonal antibodies specific for MAA-LDL. On agarose gel electrophoresis, C3a comigrated with MDA-LDL and MAA-LDL, but not with native LDL or CuOx-LDL. C3a bound to apoptotic cells in flow cytometry. C3a opsonized MAA-LDL and was taken up by J774A.1 macrophages in immunofluorescence analysis. Complement-activated human serum samples (n=30) showed increased C3a binding to MAA-LDL (P<0.001) and MDA-LDL (P<0.001) compared to nonactivated samples. The amount of C3a bound to MAA-LDL was associated with total complement activity, C3a desArg concentration, and IgG antibody levels to MAA-LDL. Proteins containing MDA adducts or MAA adducts may bind C3a in vivo and contribute to inflammatory processes involving activation of the complement system in atherosclerosis., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Aim: The aim of this study was to determine the effect of gestational diabetes mellitus on maternal circulating levels of C3a, C4a and C5a., Methods: Anaphylatoxin C3a, C4a and C5a levels were measured in maternal and cord plasma from 42 women with normal glucose tolerance and 40 women with gestational diabetes at the time of term elective Caesarean section. Maternal plasma C3a, C4a and C5a concentrations were determined by enzyme-linked immunoassay., Results: Maternal C3a and C4a concentrations were significantly lower in women with gestational diabetes compared with women with normal glucose tolerance at the time of term delivery (P < 0.05, Student's t-test). There was, however, no difference in maternal circulating C5a levels between the two groups. Additionally, there was no difference in C3a, C4a and C5a levels in cord plasma obtained from women with normal glucose tolerance and those with gesational diabetes. However, a significant positive correlation was observed between maternal and cord complement split levels., Conclusions: Gestational diabetes is characterized by lower levels of C3a and C4a in the maternal circulation at the time of term Caesarean delivery., (© 2011 The Author. Diabetic Medicine © 2011 Diabetes UK.)

Objective: To investigate the possible role of anaphylatoxin(C3a) in chronic obstructive pulmonary disease (COPD)., Methods: Sixty-four elderly patients with COPD (including 51 males, 13 females, 31 with acute exacerbation of COPD and 33 with stable COPD) and 25 healthy controls were recruited in the study. The levels of C3a in the serum of centrifugated induced sputum and cell differential count in sendiment were determined., Results: The level of C3a [44.2 (13.2-48.5) μg/L] in induced sputum in acute exacerbation of COPD was significantly higher than those in the stable COPD [5.5 (3.6-20.7) μg/L, Z=-2.974, P=0.003] and the controls [1.7 (0.1-5.9) μg/L, Z=-3.145, P=0.002]. The level of C3a in the stable COPD was higher than that in the healthy subjects, but the statistical difference was not significant. The C3a levels in smokers and non-smokers of the healthy controls were 1.97 (0.12-6.27) μg/L and 1.36 (0.09-5.57) μg/L, respectively, and had no difference (P>0.05). The level of C3a in induced sputum was positively correlated with the number of total leukocytes (r=0.543, P<0.05)., Conclusion: C3a in induced sputum is significantly increased in COPD, and its high level correlates with the advanced stage of the disease, which suggests a possible role of C3a in the pathogenesis of COPD.

Background: The activation of complement during platelet activation is incompletely understood., Objectives: We sought to explore the formation of C5b-9 and anaphylatoxins binding to collagen-activated platelets., Methods: C5b-9, anaphylatoxins C3a, C4a and C5a, and anaphylatoxin receptors C3aR1 and C5aR were measured by flow cytometry and/or confocal microscopy. Platelet microparticles were quantified by flow cytometry, and their C5b-9 content was determined by western blot analyses. In all experiments, sodium citrate was used for blood anticoagulation., Results: C5b-9 rapidly formed on the platelet surface following activation with collagen, TRAP, ADP or A23187, but was surprisingly restricted to a subset of platelets (1 to 15%) independently of P-selectin or phosphatidylserine exposure. Following collagen activation, C5b-9-positive platelets in thrombi were found associated with collagen fibres. C5b-9 formation was obliterated by Mg(2+)-EGTA and significantly reduced by the thrombin inhibitor hirudin (-37%, p<0.05), but was unaffected by chondroitinase, compstatin, SCH79797 (PAR-1 inhibitor), or in the PRP of a MBL-deficient donor. Compstatin and Mg(2+)-EGTA, but not hirudin, SCH79797 or chondroitinase, inhibited the formation of collagen-induced microparticles (-71% and -44%, respectively, p<0.04). These microparticles contained greater amounts of C5b-9 compared with the other agonists. Platelet activation by collagen or convulxin resulted in the strong binding of anaphylatoxins and the exposure of receptors C3aR1 and C5aR (CD88) on their surface., Conclusions: C5b-9 formation on collagen-activated platelets is i) partially controlled by thrombin, ii) restricted to a subset of platelets, and iii) can occur without P-selectin expression or phosphatidylserine exposure. Activated platelets bind anaphylatoxins on their surface and express C3a and C5a receptors, which may contribute to the localization of inflammatory processes during thrombosis.

Production of correctly folded and biologically active proteins in Escherichiacoli can be a challenging process. Frequently, proteins are recovered as insoluble inclusion bodies and need to be denatured and refolded into the correct structure. To address this, a refolding screening process based on a 96-well assay format supported by design of experiments (DOE) was developed for identification of optimal refolding conditions. After a first generic screen of 96 different refolding conditions the parameters that produced the best yield were further explored in a focused DOE-based screen. The refolding efficiency and the quality of the refolded protein were analyzed by RP-HPLC and SDS-PAGE. The results were analyzed by the DOE software to identify the optimal concentrations of the critical additives. The optimal refolding conditions suggested by DOE were verified in medium-scale refolding tests, which confirmed the reliability of the predictions. Finally, the refolded protein was purified and its biological activity was tested in vitro. The screen was applied for the refolding of Interleukin 17F (IL-17F), stromal-cell-derived factor-1 (SDF-1α/CXCL12), B cell-attracting chemokine 1 (BCA-1/CXCL13), granulocyte macrophage colony stimulating factor (GM-CSF) and the complement factor C5a. This procedure identified refolding conditions for all the tested proteins. For the proteins where refolding conditions were already available, the optimized conditions identified in the screening process increased the yields between 50% and 100%. Thus, the method described herein is a useful tool to determine the feasibility of refolding and to identify high-yield scalable refolding conditions optimized for each individual protein., (Copyright © 2010 Elsevier Inc. All rights reserved.)

The generation of anaphylatoxins, particularly C5a, is important in extracorporeal circulation therapies such as granulocyte/monocyte apheresis, which activates the complement system and elevates C5a levels. However, no side effects of granulocyte/monocyte apheresis using cellulose acetate beads have been reported. To investigate the mechanism of complement activation, we prepared plasma from cellulose acetate bead-treated blood (P-CAB) and compared it with zymosan-activated plasma (ZAP). Anaphylaxis activity was measured by skin test, and the activity of carboxypeptidase, which inactivates C5a, was measured by colorimetric assay. Pro-carboxypeptidase R and neutrophil elastase concentrations were measured by enzyme-linked immunosorbent assay. Although C5a was generated in P-CAB, the anaphylaxis activity of P-CAB was lower than that of ZAP. Carboxypeptidase activity and pro-carboxypeptidase R levels were suppressed in P-CAB, but not in ZAP. Furthermore, neutrophil elastase levels increased in P-CAB. The decreases in carboxypeptidase activity and inactivation of anaphylatoxin were inhibited by a neutrophil elastase inhibitor. These results suggest that cellulose acetate beads initiate the activation of carboxypeptidase R via elastase release, thereby inducing the inactivation of anaphylatoxin., (© 2010, Copyright the Authors. Artificial Organs © 2010, International Center for Artificial Organs and Transplantation and Wiley Periodicals, Inc.)

The complement system as well as the coagulation system has fundamental clinical implications in the context of life-threatening tissue injury and inflammation. Associations between both cascades have been proposed, but the precise molecular mechanisms remain unknown. The current study reports multiple links for various factors of the coagulation and fibrinolysis cascades with the central complement components C3 and C5 in vitro and ex vivo. Thrombin, human coagulation factors (F) XIa, Xa, and IXa, and plasmin were all found to effectively cleave C3 and C5. Mass spectrometric analyses identified the cleavage products as C3a and C5a, displaying identical molecular weights as the native anaphylatoxins C3a and C5a. Cleavage products also exhibited robust chemoattraction of human mast cells and neutrophils, respectively. Enzymatic activity for C3 cleavage by the investigated clotting and fibrinolysis factors is defined in the following order: FXa > plasmin > thrombin > FIXa > FXIa > control. Furthermore, FXa-induced cleavage of C3 was significantly suppressed in the presence of the selective FXa inhibitors fondaparinux and enoxaparin in a concentration-dependent manner. Addition of FXa to human serum or plasma activated complement ex vivo, represented by the generation of C3a, C5a, and the terminal complement complex, and decreased complement hemolytic serum activity that defines exact serum concentration that results in complement-mediated lysis of 50% of sensitized sheep erythrocytes. Furthermore, in plasma from patients with multiple injuries (n = 12), a very early appearance and correlation of coagulation (thrombin-antithrombin complexes) and the complement activation product C5a was found. The present data suggest that coagulation/fibrinolysis proteases may act as natural C3 and C5 convertases, generating biologically active anaphylatoxins, linking both cascades via multiple direct interactions in terms of a complex serine protease system.

Objective: The activation of the complement system results in the generation of split products with pro-inflammatory properties. The objective of this study was to determine whether preeclampsia and small-for-gestational age (SGA) are associated with changes in the maternal plasma concentrations of anaphylatoxins C3a, C4a and C5a., Methods: A cross-sectional study was conducted in the following groups: (a) normal pregnant women (n = 134); (b) women who delivered an SGA neonate (n = 53); (c) preeclampsia with (n = 52) and without SGA (n = 54). Maternal plasma anaphylatoxin concentrations were determined by enzyme-linked immunoassay., Results: (1) Women with preeclampsia with or without SGA had a significantly higher median plasma C5a concentration than that of normal pregnant women and those with SGA alone (all P < 0.01); (2) women with SGA alone did not have an increase in plasma C5a concentration; (3) in contrast, the median maternal plasma concentration of C4a was lower in women with preeclampsia and SGA than that of those with a normal pregnancy (P = 0.001); (4) no changes in C3a were observed among the study groups., Conclusion: Preeclampsia is associated with increased plasma concentration of C5a, regardless of the presence or absence of an SGA fetus. In contrast, there was no difference in the plasma C3a, C4a and C5a concentration in patients with SGA.

The incidence rate of anaphylaxis is increasing, particularly during the first 2 decades of life. Common triggers include foods, medications, and insect stings. Clinical diagnosis is based on a meticulous history of an exposure or event preceding characteristic symptoms and signs, sometimes but not always supported by a laboratory test such as an elevated serum total tryptase level. Physician-initiated investigation of patients with anaphylaxis whose symptoms and signs are atypical sometimes leads to important insights into previously unrecognized triggers and mechanisms. In idiopathic anaphylaxis, in which no trigger can be confirmed by means of skin testing or measurement of specific IgE, the possibility of mastocytosis or a clonal mast cell disorder must be considered in addition to the possibility of a previously unrecognized trigger. Long-term risk reduction in patients with anaphylaxis focuses on optimal management of relevant comorbidities such as asthma and other respiratory diseases, cardiovascular disease, and mastocytosis or a clonal mast cell disorder; avoidance of the relevant confirmed allergen trigger; and relevant immunomodulation such as medication desensitization, venom immunotherapy, and possibly in the future, immunotherapy with food. Emergency preparedness for recurrence of anaphylaxis in community settings includes having epinephrine (adrenaline) autoinjectors available, knowing when and how to use them, and having a written, personalized anaphylaxis emergency action plan and up-to-date medical identification. Randomized controlled trials of the pharmacologic interventions used in an acute anaphylaxis episode are needed.

The anaphylatoxin (AT) C3a, C5a and C5a-desArg are generally considered pro-inflammatory polypeptides generated after proteolytic cleavage of C3 and C5 in response to complement activation. Their well-appreciated effector functions include chemotaxis and activation of granulocytes, mast cells and macrophages. Recent evidence suggests that ATs are also generated locally within tissues by pathogen-, cell-, or contact system-derived proteases. This local generation of ATs is important for their pleiotropic biologic effects beyond inflammation. The ATs exert most of the biologic activities through ligation of three cognate receptors, i.e. the C3a receptor, the C5a receptor and the C5a receptor-like, C5L2. Here, we will discuss recent findings suggesting that ATs regulate cell apoptosis, lipid metabolism as well as innate and adaptive immune responses through their impact on antigen-presenting cells and T cells. As we will outline, such regulatory functions of ATs and their receptors play important roles in the pathogenesis of allergy, autoimmunity, neurodegenerative diseases, cancer and infections with intracellular pathogens.

Problem: The lack of a reliable method for early non-invasive detection of endometriosis often results in delayed diagnosis. The aim of this study was to test the hypothesis that the plasma concentration of complement factor C3a (anaphylatoxin) can be used as a non-invasive test in the diagnosis of endometriosis., Method of Study: The C3a concentration was analyzed using ELISA in 160 patients with (n = 109) or without (n = 51) endometriosis during menstruation (n = 49), follicular phase (n = 55), and luteal (n = 56) phase., Results: Plasma C3a concentration was comparable between patients with [102 (27-2213) ng/mL] and without [105 (32-2340) ng/mL] (P = 0.84) endometriosis, also when assessed separately during menstruation, follicular phase, and luteal phase., Conclusion: We found no difference in C3a levels between women with and without endometriosis and did not confirm our hypothesis that plasma C3a levels can be used as diagnostic test for endometriosis.

The mobilization of hematopoietic stem/progenitor cells (HSPCs) from bone marrow into peripheral blood (PB) is still not fully understood. Different chemokines, cytokines, growth factors, and neurotransmitters have been described that facilitate this process. However, mounting evidence suggests that mobilization of HSPCs is a part of the immune response and is mediated by innate immunity. We discuss evidence showing that complement system cleavage fragments play a crucial role in both the retention and mobilization of HSPCs by modulating their responsiveness to stromal-derived growth factor-1 (SDF-1) gradient (by C3-derived anaphylatoxins) and by modulating the release of granulocytes into PB that subsequently facilitate the egress of HSPCs (by C5-derived anaphylatoxins).

Activation of the complement system occurs in a variety of neuroinflammatory diseases and neurodegenerative processes of the CNS. Studies in the last decade have demonstrated that essentially all of the activation components and receptors of the complement system are produced by astrocytes, microglia, and neurons. There is also rapidly growing evidence to indicate an active role of the complement system in cerebral ischemic injury. In addition to direct cell damage, regional cerebral ischemia and reperfusion (I/R) induces an inflammatory response involving complement activation and generation of active fragments, such as C3a and C5a anaphylatoxins, C3b, C4b, and iC3b. The use of specific inhibitors to block complement activation or their mediators such as C5a, can reduce local tissue injury after I/R. Consistent with therapeutic approaches that have been successful in models of autoimmune disorders, many of the same complement inhibition strategies are proving effective in animal models of cerebral I/R injury. One new form of therapy, which is less specific in its targeting of complement than monodrug administration, is the use of immunoglobulins. Intravenous immunoglobulin (IVIG) has the potential to inhibit multiple components of inflammation, including complement fragments, pro-inflammatory cytokine production and leukocyte cell adhesion. Thus, IVIG may directly protect neurons, reduce activation of intrinsic inflammatory cells (microglia) and inhibit transendothelial infiltration of leukocytes into the brain parenchyma following an ischemic stroke. The striking neuroprotective actions of IVIG in animal models of ischemic stroke suggest a potential therapeutic potential that merits consideration for clinical trials in stroke patients.

Purpose of Review: This review emphasizes new information concerning the role of anaphylatoxins in the regulation of the immune response to allografts. Its timeliness relates the growing concept of the innate immune response as a regulator of the adaptive immune system and to how this concept lends itself to therapeutic advance., Recent Findings: Recent work has extended our understanding of the role of local complement synthesis and how this facilitates the interaction between antigen-presenting cells and alloreactive T cells, resulting in a potent effector response. In particular, this work has identified new roles for anaphylatoxins as regulators of antigen presentation, T-cell proliferation and T-cell longevity., Summary: Strategies for comprehensive blockade of complement at the site of action, or for more selective blockade of specific complement components, are not only possible but merit further exploration based on these results.