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Leukocytes play an important role in vascular inflammation prior to atherosclerosis. In particular, monocyte adhesion and migration to the endothelium contribute to the development of vascular inflammation. Previously, we showed the importance of neutrophils and complement C5a in the early phase of vascular inflammation in mice fed a high-fat diet. However, the relationship between monocytes and neutrophils is not well understood. In this study, we elucidated the involvement of neutrophils in the migration of monocytes. We observed that C5a induces CCL2 expression in neutrophil-like dHL-60 cells. To investigate the physiological significance of CCL2 secretion, we performed a chemotaxis assay. Interestingly, dHL-60 culture supernatant in the presence of C5a enhanced the migration of THP-1 in comparison with the absence of C5a. Furthermore, CCL2 expression and secretion significantly increased in C5a-stimulated dHL-60 through the phosphorylation of NF-κB p65. Actin polymerization on THP-1 was enhanced by the presence of C5a compared with the absence of C5a when stimulated by a dHL-60-cultured medium. These results suggest that crosstalk between neutrophils and monocytes via CCL2 may play an important role in vascular inflammation., (© 2021 The Authors. FEBS Open Bio published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

Emerging evidence has documented that multisystem organ failure in coronavirus disease 2019 (COVID-19) patients is strongly associated with various coagulopathies. Treatments for COVID-19-associated coagulopathy are still a clinical challenge. An advancement in the knowledge of mechanisms of the excessive or inappropriate activation of the complement cascade involved in the genesis of COVID-19-associated coagulopathy might be a fundamental approach for developing novel classes of anticoagulant drugs. In this context, there is emerging evidence indicating that C5a, a component of the complement system, and its receptors (C5aRs) play a critical role in the genesis of the COVID-19-associated hypercoagulable state. Thus, this review describes the mechanisms by which C5a/C5aR signaling participates in the cascade of events involved in the pathophysiology of COVID-19-associated coagulopathy. Furthermore, it highlights the current possibilities for the development of a novel therapeutic approach for COVID-19 patients that targets C5a/C5aRs signaling., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

This study was designed to test whether the Cronobacter sakazakii infection-impaired contextual learning and memory are mediated by the activation of the complement system; subsequent activation of inflammatory signals leads to alternations in serotonin transporter (SERT). To test this, rat pups (postnatal day, PND 15) were treated with either C. sakazakii (10 7  CFU) or Escherichia coli OP50 (10 7  CFU) or Luria bertani broth (100 μL) through oral gavage and allowed to stay with their mothers until PND 24. Experimental groups' rats were allowed to explore (PNDs 31-35) and then trained in contextual learning task (PNDs 36-43). Five days after training, individuals were tested for memory retention (PNDs 49-56). Observed behavioural data showed that C. sakazakii infection impaired contextual-associative learning and memory. Furthermore, our analysis showed that C. sakazakii infection activates complement system complement anaphylatoxin (C5a) (a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS1)) and mitogen-activated protein kinase kinase1 (MEKK1). Subsequently, MEKK1 induces pro-inflammatory signals possibly through apoptosis signal-regulating kinase-1 (ASK-1), c-Jun N-terminal kinase (JNK1/3) and protein kinase B gamma (AKT-3). In parallel, activated nuclear factor kappa-light-chain-enhancer B cells (NF-κB) induces interleukin-6 (IL-6) and IFNα-1, which may alter the level of serotonin transporter (SERT). Observed results suggest that impaired contextual learning and memory could be correlated with C5a-mediated NF-κβ and ASK1 pathways.

The novel coronavirus disease COVID-19 originates in the lungs, but it may extend to other organs, causing, in severe cases, multiorgan damage, including cardiac injury and acute kidney injury. In severe cases, the presence of kidney injury is associated with increased risk of death, highlighting the relevance of this organ as a target of SARS-CoV-2 infection. COVID-19-associated tissue injury is not primarily mediated by viral infection, but rather is a result of the inflammatory host immune response, which drives hypercytokinemia and aggressive inflammation that affect lung parenchymal cells, diminishing oxygen uptake, but also endothelial cells, resulting in endotheliitis and thrombotic events and intravascular coagulation. The complement system represents the first response of the host immune system to SARS-CoV-2 infection, but there is growing evidence that unrestrained activation of complement induced by the virus in the lungs and other organs plays a major role in acute and chronic inflammation, endothelial cell dysfunction, thrombus formation, and intravascular coagulation, and ultimately contributes to multiple organ failure and death. In this review, we discuss the relative role of the different complement activation products in the pathogenesis of COVID-19-associated tissue inflammation and thrombosis and propose the hypothesis that blockade of the terminal complement pathway may represent a potential therapeutic option for the prevention and treatment of lung and multiorgan damage., (Copyright © 2020 International Society of Nephrology. Published by Elsevier Inc. All rights reserved.)

Activation of the C5/C5a/C5a receptor 1 (C5aR1) axis during allergen sensitization protects from maladaptive T cell activation. To explore the underlying regulatory mechanisms, we analyzed the impact of C5aR1 activation on pulmonary CD11b + conventional dendritic cells (cDCs) in the context of house-dust-mite (HDM) exposure. BALB/c mice were intratracheally immunized with an HDM/ovalbumin (OVA) mixture. After 24 h, we detected two CD11b + cDC populations that could be distinguished on the basis of C5aR1 expression. C5aR1 - but not C5aR1 + cDCs strongly induced T cell proliferation of OVA-reactive transgenic CD4 + T cells after re-exposure to antigen in vitro. C5aR1 - cDCs expressed higher levels of MHC-II and CD40 than their C5aR1 + counterparts, which correlated directly with a higher frequency of interactions with cognate CD4 + T cells. Priming of OVA-specific T cells by C5aR1 + cDCs could be markedly increased by in vitro blockade of C5aR1 and this was associated with increased CD40 expression. Simultaneous blockade of C5aR1 and CD40L on C5aR1 + cDCs decreased T cell proliferation. Finally, pulsing with OVA-induced C5 production and its cleavage into C5a by both populations of CD11b + cDCs. Thus, we propose a model in which allergen-induced autocrine C5a generation and subsequent C5aR1 activation in pulmonary CD11b + cDCs promotes tolerance towards aeroallergens through downregulation of CD40., Competing Interests: The authors declare no conflict of interest.

The sequelae of diabetes include microvascular complications such as diabetic kidney disease (DKD), which involves glucose-mediated renal injury associated with a disruption in mitochondrial metabolic agility, inflammation, and fibrosis. We explored the role of the innate immune complement component C5a, a potent mediator of inflammation, in the pathogenesis of DKD in clinical and experimental diabetes. Marked systemic elevation in C5a activity was demonstrated in patients with diabetes; conventional renoprotective agents did not therapeutically target this elevation. C5a and its receptor (C5aR1) were upregulated early in the disease process and prior to manifest kidney injury in several diverse rodent models of diabetes. Genetic deletion of C5aR1 in mice conferred protection against diabetes-induced renal injury. Transcriptomic profiling of kidney revealed diabetes-induced downregulation of pathways involved in mitochondrial fatty acid metabolism. Interrogation of the lipidomics signature revealed abnormal cardiolipin remodeling in diabetic kidneys, a cardinal sign of disrupted mitochondrial architecture and bioenergetics. In vivo delivery of an orally active inhibitor of C5aR1 (PMX53) reversed the phenotypic changes and normalized the renal mitochondrial fatty acid profile, cardiolipin remodeling, and citric acid cycle intermediates. In vitro exposure of human renal proximal tubular epithelial cells to C5a led to altered mitochondrial respiratory function and reactive oxygen species generation. These experiments provide evidence for a pivotal role of the C5a/C5aR1 axis in propagating renal injury in the development of DKD by disrupting mitochondrial agility, thereby establishing a new immunometabolic signaling pathway in DKD., (© 2019 by the American Diabetes Association.)

Epigenetic mechanisms, such as DNA methylation, affect tubular maladaptive response after Acute Kidney Injury (AKI) and accelerate renal aging. Upon ischemia/reperfusion (I/R) injury, Complement activation leads to C5a release that mediates damage; however, little is known about the effect of C5a-C5a Receptor (C5aR) interaction in Renal Tubular Epithelial Cells (RTEC).Through a whole-genome DNA methylation analysis in cultured RTEC, we found that C5a induced aberrant methylation, particularly in regions involved in cell cycle control, DNA damage and Wnt signaling. The most represented genes were BCL9 , CYP1B1 and CDK6 . C5a stimulation of RTEC led to up-regulation of SA-β Gal and cell cycle arrest markers such as p53 and p21. C5a increased also IL-6 , MCP-1 and CTGF gene expression, consistent with SASP development. In accordance, in a swine model of renal I/R injury, we found the increased expression of Wnt4 and βcatenin correlating with SA-β Gal, p21, p16 and IL-6 positivity. Administration of Complement Inhibitor (C1-Inh), antagonized SASP by reducing SA-β Gal, p21, p16, IL-6 and abrogating Wnt4/βcatenin activation.Thus, C5a affects the DNA methylation of genes involved in tubular senescence. Targeting epigenetic programs and Complement may offer novels strategies to protect tubular cells from accelerated aging and to counteract progression to Chronic Kidney Disease.

Chronic lymphocytic leukemia (CLL) is the most common adult leukemia in the Western world. The therapeutic approach to CLL includes chemotherapeutic regimens and immunotherapy. Complement-mediated cytotoxicity, which is one of the mechanisms activated by the therapeutic monoclonal antibodies, depends on the availability and activity of the complement (C) system. The aim was to study the structure of circulating C components and evaluate the importance of C5 structural integrity for C activity in CLL patients. Blood samples were collected from 40 naïve CLL patients and 15 normal controls (NC). The Western blot analysis showed abnormal C5 pattern in some CLL patients, while patterns of C3 and C4 were similar in all subjects. Levels of the C activation markers sC5b-9 and C5a were quantified before and after activation via the classical (CP) and alternative (AP) pathways. In patients with abnormal C5, basal levels of sC5b-9 and C5a were increased while activities of the CP and of the CP C5-convertase, the immediate C5-upstream complex, were decreased compared to NC and to patients with normal C5. The data indicate a link between CP activation and apparent C5 alterations in CLL. This provides a potential prognostic tool that may personalize therapy by identifying a sub-group of CLL patients who display an abnormal C5 pattern, high basal levels of sC5b-9 and C5a, and impaired CP activity, and are likely to be less responsive to immunotherapy due to compromised CP activity., Competing Interests: The authors have declared that no competing interests exist.

Critical illness is an aetiologically and clinically heterogeneous syndrome that is characterised by organ failure and immune dysfunction. Mortality in critically ill patients is driven by inflammation-associated organ damage and a profound vulnerability to nosocomial infection. Both factors are influenced by the activated complement protein C5a, released by unbridled activation of the complement system during critical illness. C5a exerts deleterious effects on organ systems directly and suppresses antimicrobial functions of key immune cells. Whilst several recent reports have added key knowledge of the cellular signalling pathways triggered by C5a, there remain a number of areas that are incompletely understood and therapeutic opportunities are still being evaluated. In this review, we summarise the cellular basis for C5a-induced vulnerability to nosocomial infection and organ dysfunction. We focus on cells of the innate immune system, highlighting the major areas in need of further research and potential avenues for targeted therapies., (© 2018 Stichting European Society for Clinical Investigation Journal Foundation.)

Complement activation is considered one of the mediators of renal ischemia-reperfusion injury. Elevated levels of C5b-9, C3a, and C5a are detected in sera of deceased kidney donors. The goal of the study was to characterize the functional activity of complement pathways in donor sera and to assess their influence on transplant outcome., Materials and Methods: Sixty-four deceased kidney donors (age 45 ± 16 years; 28 female, 36 male) and 27 healthy controls (age 42 ± 12 years; 14 female, 13 male) were enrolled in the study. The results of transplantation for the respective 122 kidney recipients were included in the analysis. The functional activities of classical (CP), lectin (LP), and alternative (AP) pathways were measured using Wielisa-kit (reference normal level = 100%). In most cases, decreased functional activity reflects the activation status of the pathway., Results: The median (interquartile range) functional activities of the pathways in donor sera were CP 118 (89-150)%, LP 80 (20-127)%, and AP 74 (50-89)%, and did not differ from the control values CP 110 (102-115)%, LP 81 (26-106)%, AP 76 (61-88)%. The frequency of pathway activation observed in controls was CP 0%, LP 11%, and AP 0%. Deceased donors did not differ in activation of classical (11%) and lectin (13%) pathways, but presented a higher rate of alternative pathway activation (19%, P = .03). No significant influence of any pathway functional activity or its activation was proved to influence the transplant outcome., Conclusion: Complement activation via alternative pathway was observed in diseased donor sera. No predictive potential of donor complement functional activity on the transplant outcome could be proved., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Although the complement C5a/C5aR pathway is suggested to play a critical role in tumor pathogenesis, the underlying mechanism has yet to be fully elucidated. In the present study, we found that in patients with gastric cancer in different clinical stages (from stageⅠto stage Ⅳ), both C5aR and p-PI3K/AKT levels were significantly higher in tumoral tissues than in adjacent non-tumoral tissues. In contrast, p21/p-p21 levels were significantly lower in tumoral tissues than in adjacent non-tumoral tissues. In vitro recombinant C5a administration remarkably promoted p-PI3K/p-AKT expression, but inhibited p21/p-p21 expression. Blockage of C5a/C5aR signaling with a C5aR antagonist reversed the C5a-induced inhibitory effect on p21/p-p21 expression. C5a administration to cells pre-treated with a PI3K inhibitor also prevented this inhibitory effect, suggesting the involvement of the PI3K/AKT signaling pathway in C5a/C5aR-mediated suppression of p21/p-p21 expression. In vivo C5aR antagonist treatment caused significant reduction in tumor growth in mice, accompanied by a remarkable elevation in p21/p-p21 expression and reduction in p-PI3K/AKT activation. These results indicate that the C5a/C5aR pathway promotes gastric cancer pathogenesis by suppressing p21/p-p21 expression via activation of PI3K/AKT signaling., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Purpose of Review: Preeclampsia affects 3-4% of pregnancies with few treatment options to reduce maternal and fetal harm. Recent evidence that targeting the complement system may be an effective therapeutic strategy in prevention or treatment of preeclampsia will be reviewed., Recent Findings: Studies in humans confirm the safety and efficacy of C5 blockade in complement-mediated disorders of pregnancy, including preeclampsia. Animal models mimic the placental abnormalities and/or the maternal symptoms which characterize preeclampsia. These models in mouse and rat have defined a role for complement and its regulators in placental dysfunction, hypertension, proteinuria, endothelial dysfunction, fetal growth restriction, and angiogenic imbalance, thus informing future human studies. Targeting excessive complement activation, particularly the terminal complement complex (C5b-9) and C5a may be an effective strategy to prolong pregnancy in women with preeclampsia. Continued research is needed to identify the initiator(s) of activation, the pathways involved, and the key component(s) in the pathophysiology to allow development of safe and effective therapeutics to target complement without compromising its role in homeostasis and host defense.

Diabetic non-healing wounds are a major clinical problem. The mechanisms leading to poor wound healing in diabetes are multifactorial but unresolved inflammation may be a major contributing factor. The complement system (CS) is the most potent inflammatory cascade in humans and contributes to poor wound healing in animal models. Signal transducer and activator of transcription 4 (STAT4) is a transcription factor expressed in immune and adipose cells and contributes to upregulation of some inflammatory chemokines and cytokines. Persistent CS and STAT4 expression in diabetic wounds may thus contribute to chronic inflammation and delayed healing. The purpose of this study was to characterize CS and STAT4 in early diabetic wounds using db/db mice as a diabetic skin wound model. The CS was found to be activated early in the diabetic wounds as demonstrated by increased anaphylatoxin C5a in wound fluid and C3-fragment deposition by immunostaining. These changes were associated with a 76% increase in nucleated cells in the wounds of db/db mice vs., Controls: The novel classical CS inhibitor, Peptide Inhibitor of Complement C1 (PIC1) reduced inflammation when added directly or saturated in an acellular skin scaffold, as reflected by reduced CS components and leukocyte infiltration. A significant increase in expression of STAT4 and the downstream macrophage chemokine CCL2 and its receptor CCR2 were also found in the early wounds of db/db mice compared to non-diabetic controls. These studies provide evidence for two new promising targets to reduce unresolved inflammation and to improve healing of diabetic skin wounds., Competing Interests: The authors have declared that no competing interests exist.

Inflammatory response has been reported to contribute to the renal lesions in rat Thy-1 nephritis (Thy-1N) as an animal model of human mesangioproliferative glomerulonephritis (MsPGN). Besides C5b-9 complex, C5a is also a potent pro-inflammatory mediator and correlated to severity of various nephritic diseases. However, the role of C5a in mediating pro-inflammatory cytokine production in rats with Thy-1N is poorly defined. In the present studies, the levels of C5a, interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) were first determined in the renal tissues of rats with Thy-1N. Then, the expression of IL-6 and TNF-α was detected in rat glomerular mesangial cells (GMC) stimulated with our recombinant rat C5a in vitro. Subsequently, the activation of mitogen-activated protein kinase (MAPK) signaling pathways (p38 MAPK, ERK1/2 and JNK) and their roles in the regulation of IL-6 and TNF-α production were examined in the GMC induced by C5a. The results showed that the levels of C5a, IL-6 and TNF-α were markedly increased in the renal tissues of Thy-1N rats. Rat C5a stimulation in vitro could up-regulate the expression of IL-6 and TNF-α in rat GMC, and the activation of MAPK signaling pathways was involved in the induction of IL-6 and TNF-α. Mechanically, p38 MAPK activation promoted IL-6 production, while either ERK1/2 or JNK activation promoted TNF-α production in the GMC with exposure to C5a. Taken together, these data implicate that C5a induces the synthesis of IL-6 and TNF-α in rat GMC through the activation of MAPK signaling pathways., Competing Interests: The authors have declared that no competing interests exist.

Activation of the complement system leads to the cleavage of component factor C5 into C5a and C5b. C5a can induce chemotaxis and inflammatory responses in mammals. The function of C5a in fish is poorly understood. In this study, we report the identification and analysis of a C5 homologue, CsC5, from tongue sole (Cynoglossus semilaevis). CsC5 is composed of 1683 amino acid residues that include an anaphylatoxin homologous domain. Expression of CsC5 could be detected in a variety of tissues and was up-regulated by bacterial or viral pathogen infection. Purified recombinant CsC5a (rCsC5a) could bind to peripheral blood leukocytes (PBL) and stimulate PBL chemotaxis, proliferation, respiratory burst, acid phosphatase activity, and phagocytosis. Tongue sole administered rCsC5a exhibited enhanced resistance against bacterial and viral infections. These results indicate that CsC5a is an anaphylatoxin with a role in innate immune defense against bacterial and viral infections., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Objective: Patients with antineutrophil cytoplasmic antibody-associated vasculitis (AAV) have a high frequency of venous thromboembolic events and a hypercoagulable state. As C5a-primed neutrophils play an important role in the development of AAV, we investigated whether C5a-induced neutrophil tissue factor (TF)-expressing microparticles (MPs) and neutrophil extracellular traps (NETs) might promote hypercoagulability in AAV., Methods: TF-expressing MPs were measured by flow cytometry. TF-expressing NETs were assessed by confocal microscopy. Levels of thrombin-antithrombin complexes were determined by enzyme-linked immunosorbent assay. The effect of C5a in sera from AAV patients was evaluated by treating neutrophils with C5a receptor antagonist before incubation with sera from AAV patients with active disease., Results: Treatment of C5a-primed neutrophils with antineutrophil cytoplasmic antibody (ANCA)-positive IgG resulted in the release of TF-bearing MPs and NETs. Neutrophils from healthy donors treated with sera from patients with active AAV released TF-bearing MPs and NETs, which were abolished by treatment with C5a receptor antagonist. Involvement of TF in MP- or NET-dependent thrombin generation was indicated by the findings of antibody neutralization studies. NETs with thrombin-generating capacity were demonstrated by DNase I treatment., Conclusion: C5a-primed neutrophils produce TF-expressing MPs and NETs after stimulation with ANCAs, indicating a mechanism for hypercoagulability in AAV that was not previously recognized., (© 2015, American College of Rheumatology.)

The anaphylatoxin C5a is a chemoattractant for leukocyte migration via the C5a receptor (C5aR). We recently reported that C5aR was aberrantly expressed in a wide variety of human related cancers, while it also promotes cancer cell invasion by C5a stimulation. However, the biological significance of C5aR expression in renal cell carcinoma (RCC) has not yet been clarified. In the present study, we aimed to elucidate the biological role of C5aR in RCC progression. Clinical RCC specimens were analyzed for C5aR expression and its relationship with baseline demographic data and clinicopathological parameters was analyzed. Moreover, renal carcinoma Renca cells stably expressing C5aR were generated and used to assess the effects of C5a-C5aR axis activation on various cellular phenomena in culture. Immunohistochemistry revealed that 96.7% of the metastatic RCCs (mRCCs) showed C5aR expression, whereas only 50.5% of the non-metastatic RCCs expressed C5aR (P<0.001). Although C5a stimulation did not significantly alter anoikis of C5aR‑expressing Renca cells, C5a elicited cell morphological change and scattering of those cells accompanied with dynamic actin rearrangement, which was not observed in the Renca cells harboring the empty vector only. Moreover, C5a triggered ERK and PI3K‑dependent invasion of the C5aR-expressing renal carcinoma cells. These results are consistent with the idea that the C5a-C5aR axis plays a crucial role in renal carcinoma cell invasion, which may be one of the key steps for RCC metastasis. The present study provides proof‑of‑concept that the C5a-C5aR axis may be a useful therapeutic target for preventing RCC progression.

Complement component 5a (C5a) is a 74 amino acid glycoprotein and an important proinflammatory mediator that is cleaved enzymatically from its precursor, C5, on activation of the complement cascade. C5a is quickly metabolised by carboxypeptidases, forming the less-potent C5a desArg. C5a and C5a desArg interact with their receptors (C5aR and C5L2), which results in a number of effects which are essential to the immune response. C5a has a broad range of biological effects throughout the human body because the widespread expression of C5a receptors throughout the human organs enables C5a and C5a desArg to elicit a broad range of biological effects. Recently, accumulating evidence in humans and experimental animal models shows that the C5a-C5aR axis is involved in the development of atherosclerosis lesions. The absence or blockade of C5aRs greatly reduces the formation of atherosclerotic lesions or wire-injury-induced neointima formation in atherosclerosis-prone mice. Serum C5a level was related to the major adverse cardiovascular events in patients with advanced atherosclerosis and those with drug-eluting stent implantation. Thus, the C5a-C5aR axis may be a significant pathogenic driver of arteriosclerotic vascular disease, making C5a-C5aR inhibition an attractive therapeutic strategy.

Ischaemia-reperfusion injury (IRI) is a frequent event in kidney transplantation, particularly when the kidney comes from a deceased donor. The brain death is usually associated with generalized ischaemia due to a hyperactivity of the sympathetic system. In spite of this, most donors have profound hypotension and require administration of vasoconstrictor agents. Warm ischaemia after kidney vessels clamping and the cold ischaemia after refrigeration also reduce oxygen and nutrients supply to tissues. The reperfusion further aggravates the state of oxidation and inflammation created by ischaemia. IRI first attacks endothelial cells and tubular epithelial cells. The lesions may be so severe that they lead to acute kidney injury (AKI) and delayed graft function (DGF), which can impair the graft survival. The unfavourable impact of DGF is worse when DGF is associated with acute rejection. Another consequence of IRI is the activation of the innate immunity. Danger signals released by dying cells alarm Toll-like receptors that, through adapter molecules and a chain of kinases, transmit the signal to transcription factors which encode the genes regulating inflammatory cells and mediators. In the inflammatory environment, dendritic cells (DCs) intercept the antigen, migrate to lymph nodes and present the antigen to immunocompetent cells, so activating the adaptive immunity and favouring rejection. Attempts to prevent IRI include optimal management of donor and recipient. Calcium-channel blockers, l-arginine and N-acetylcysteine could obtain a small reduction in the incidence of post-transplant DGF. Fenoldopam, Atrial Natriuretic Peptide, Brain Natriuretic Peptide and Dopamine proved to be helpful in reducing the risk of AKI in experimental models, but there is no controlled evidence that these agents may be of benefit in preventing DGF in kidney transplant recipients. Other antioxidants have been successfully used in experimental models of AKI but only a few studies of poor quality have been made in clinical transplantation with a few of these agents and we still lack of unambiguous demonstration that pre-treatment with these antioxidants can attenuate the impact of IRI in kidney transplantation. Interference with the signals leading to activation of innate immunity, inactivation of complement or manipulation of DCs is a promising therapeutic option for the near future., (© The Author 2013. Published by Oxford University Press on behalf of ERA-EDTA. All rights reserved.)

Introduction: Acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) are the acute onset of noncardiac respiratory insufficiency associated with bilateral lung infiltrations. During the past decade, mechanical ventilation strategies using low tidal volumes have reduced the mortality of ALI/ARDS to ∼ 20 - 40%. However, ALI/ARDS continues to be a major factor in global burden of diseases, with no pharmacological agents currently available., Areas Covered: In this review, we discuss several inflammatory proteins involved in the molecular pathogenesis of ALI/ARDS. The complement cleavage product, C5a, is a peptide acting as a potent anaphylatoxin. C5a may trigger the formation of neutrophil extracellular traps (NETs) and release of histone proteins to the extracellular compartment during ALI/ARDS. NETs may activate platelets to release TGF-β, which is involved in tissue remodeling during the later phases of ALI/ARDS. Interception of C5a signaling or blockade of extracellular histones has recently shown promising beneficial effects in small animal models of ALI/ARDS., Expert Opinion: Novel protein-based strategies for the treatment of ALI/ARDS may inspire the hopes of scientists, clinicians, and patients. Although neutralization of extracellular histones/NETs, C5a, and TGF-β is effective in experimental models of ALI/ARDS, controlled clinical trials will be necessary for further evaluation in future.

Background: Recently, anti-thrombin-activatable fibrinolysis inhibitor (TAFI) mAbs selectively inhibiting plasmin-mediated TAFI activation were shown to stimulate fibrinolysis in vitro and in vivo, suggesting, in contrast to other findings, that plasmin-mediated TAFI activation plays an important role in fibrinolysis regulation., Objective: To further characterize the effects of two plasmin-specific anti-TAFI mAbs (MA-TCK11A9 and MA-TCK26D6) on TAFI-dependent inhibition of fibrinolysis., Methods and Results: Both mAbs inhibited plasmin-mediated but not thrombin/thrombomodulin-mediated TAFI activation, whereas neither inhibited the cleavage of hippuryl-arginine by activated TAFI (TAFIa). They stimulated tissue-type plasminogen activator-induced fibrinolysis in different clot lysis models through a TAFI-dependent mechanism, especially in the presence of thrombomodulin (TM), a condition in which TAFI is largely activated by the thrombin-TM complex. In a fibrinolysis-based TAFIa activity assay, both mAbs inhibited TAFIa, whereas other mAbs targeting thrombin-TM-mediated TAFI activation did not. The inhibition of TAFIa activity, however, was substrate-specific, because neither mAb inhibited the cleavage of thrombin-activated osteopontin and C5a by TAFIa, thus sparing the anti-inflammatory activity of TAFIa., Conclusions: Our anti-TAFI mAbs, by selectively inhibiting TAFIa activity on fibrin, may represent the prototype of a new class of TAFI inhibitors with improved pharmacologic activity., (© 2013 International Society on Thrombosis and Haemostasis.)

Background: Nosocomial infection occurs commonly in intensive care units (ICUs). Although critical illness is associated with immune activation, the prevalence of nosocomial infections suggests concomitant immune suppression. This study examined the temporal occurrence of immune dysfunction across three immune cell types, and their relationship with the development of nosocomial infection., Methods: A prospective observational cohort study was undertaken in a teaching hospital general ICU. Critically ill patients were recruited and underwent serial examination of immune status, namely percentage regulatory T-cells (Tregs), monocyte deactivation (by expression) and neutrophil dysfunction (by CD88 expression). The occurrence of nosocomial infection was determined using pre-defined, objective criteria., Results: Ninety-six patients were recruited, of whom 95 had data available for analysis. Relative to healthy controls, percentage Tregs were elevated 6-10 days after admission, while monocyte HLA-DR and neutrophil CD88 showed broader depression across time points measured. Thirty-three patients (35%) developed nosocomial infection, and patients developing nosocomial infection showed significantly greater immune dysfunction by the measures used. Tregs and neutrophil dysfunction remained significantly predictive of infection in a Cox hazards model correcting for time effects and clinical confounders {hazard ratio (HR) 2.4 [95% confidence interval (CI) 1.1-5.4] and 6.9 (95% CI 1.6-30), respectively, P=0.001}. Cumulative immune dysfunction resulted in a progressive risk of infection, rising from no cases in patients with no dysfunction to 75% of patients with dysfunction of all three cell types (P=0.0004)., Conclusions: Dysfunctions of T-cells, monocytes, and neutrophils predict acquisition of nosocomial infection, and combine additively to stratify risk of nosocomial infection in the critically ill.

It recently became evident that activation of the complement system also contributes to tissue regeneration after infection/injury. The complement-derived fragment C5a induces vascular modifications and attracts cells expressing its receptor (C5aR/CD88) to the site of infection and tissue injury. Besides inflammatory cells, various tissue cells express this receptor. We hypothesized that pulp progenitor cells, being exposed to local complement activation in caries lesions, may respond to C5a via the C5aR. Our work aimed at evaluating the ability of C5a to induce pulp progenitor cell migration that may link complement activation to dentin regeneration. Immunofluorescence analysis of third molar pulp sections showed perivascular localization of the mesenchymal stem cell markers STRO-1 and C5aR. RT-PCR on STRO-1-sorted pulp progenitor cells, co-expressing both STRO-1 and C5aR, revealed high C5aR mRNA levels. Experiments with the C5aR antagonist W54011 revealed that C5a specifically bound to progenitor cells via C5aR, inducing their selective migration toward the C5a gradient. Since we could also demonstrate C5b-9 formation by immunohistochemistry in carious teeth, our findings suggest that, upon local complement activation, C5a induces pulp progenitor cell migration, which may be critical in initiating the regenerative process after dentin/pulp injury.

Aims: Failure of vein graft conduits due to vein graft thickening, accelerated atherosclerosis, and subsequent plaque rupture is applicable to 50% of all vein grafts within 10 years. New potential therapeutic targets to treat vein graft disease may be found in components of the innate immune system, such as mast cells and complement factors, which are known to be involved in atherosclerosis and plaque destabilization. Interestingly, mast cells can be activated by complement factor C5a and, therefore, a direct role for C5a-mediated mast cell activation in vein graft disease is anticipated. We hypothesize that C5a-mediated mast cell activation is involved in the development and destabilization of vein graft lesions., Methods and Results: Mast cells accumulated in time in murine vein graft lesions, and C5a and C5a-receptor (CD88) expression was up-regulated during vein graft disease in apolipoprotein E-deficient mice. Mast cell activation with dinitrophenyl resulted in a profound increase in vein graft thickening and in the number of plaque disruptions. C5a application enhanced vein graft lesion formation, while treatment with a C5a-receptor antagonist resulted in decreased vein graft disease. C5a most likely exerts its function via mast cell activation since the mast cell inhibitor cromolyn totally blocked C5a-enhanced vein graft disease., Conclusion: These data provide evidence that complement factor C5a-induced mast cell activation is highly involved in vein graft disease, which identifies new targets to prevent vein graft disease.

Periodontitis is a prevalent oral chronic inflammatory disease which, in severe forms, may exert a major impact on systemic health. Clinical and histological observations, as well as experimental animal studies, suggest involvement of the complement system in periodontitis. However, the precise roles of the various complement components and pathways in periodontitis have only recently started to be elucidated. In this chapter, we review recent progress in the field and discuss the potential of complement-targeted therapeutics in the treatment of periodontitis.

When acute inflammatory states are induced by treatment with chemical mediators in C5-deficient mice, neutrophil influxes are commonly decreased. Therefore, the neutrophil C5a receptor (C5aR) is believed to be a member of the pro-inflammatory receptors. However, C5aR deficiency endows mouse neutrophils with increased sensitivity to Pseudomonas aeruginosa. We have demonstrated that C5aR accepts not only C5a but also ribosomal protein S19 (RP S19) oligomers. RP S19 oligomers released from apoptotic cells promote apoptosis or induce dual agonistic and antagonistic effects on the chemotaxis of macrophages and neutrophils in an autocrine or paracrine manner, respectively. We assumed that the function of C5aR in apoptotic cells is almost the same as that in neutrophils infiltrating acute inflammatory lesions. Therefore, we believe that RP S19 oligomers can explain the opposite response of neutrophils in C5aR-deficient mice. In the present study, we found that antihuman RP S19 rabbit IgG cross-reacted with mouse RP S19 monomers and oligomers in plasma and serum, respectively, whereas anti-human C5a rabbit IgG only cross-reacted with mouse RP S19 oligomers in serum. To examine a role of RP S19 oligomers in vivo, we injected carrageenan (50 microg/100 microL) into the thoracic cavities of mice in the simultaneous presence of rabbit IgG and antihuman C5a rabbit IgG (100 microg/100 microL). Before 4 h and after 24 h, we did not observe any inflammatory cues in pleural exudates and lung substances from control mice. However, infiltrating neutrophils were detected in pleural exudates and lung tissues at 4 h after the addition of anti-human RP S19 rabbit IgG. Moreover, anti-human C5a rabbit IgG retards the initiation phase of carrageenan-induced mouse plurality. Many of the neutrophils infiltrating the thoracic cavities of the mice remained annexin V-negative. Neutrophil infiltration into pneumonic lesions became more severe, as alveolar septal destruction and haemorrhage concomitant with increased numbers of neutrophils in the pleural exudates were observed. These in vivo data demonstrate that the neutrophil C5aR acts as a dual pro-inflammatory and pro-apoptosis receptor during the initiation and the resolution phases of acute inflammation, respectively.

In Huntington disease (HD), immune cells are activated before symptoms arise; however, it is unclear how the expression of mutant huntingtin (htt) compromises the normal functions of immune cells. Here we report that primary microglia from early postnatal HD mice were profoundly impaired in their migration to chemotactic stimuli, and expression of a mutant htt fragment in microglial cell lines was sufficient to reproduce these deficits. Microglia expressing mutant htt had a retarded response to a laser-induced brain injury in vivo. Leukocyte recruitment was defective upon induction of peritonitis in HD mice at early disease stages and was normalized upon genetic deletion of mutant htt in immune cells. Migration was also strongly impaired in peripheral immune cells from pre-manifest human HD patients. Defective actin remodeling in immune cells expressing mutant htt likely contributed to their migration deficit. Our results suggest that these functional changes may contribute to immune dysfunction and neurodegeneration in HD, and may have implications for other polyglutamine expansion diseases in which mutant proteins are ubiquitously expressed.

The complement system contributes to various immune and inflammatory diseases, including cancer. In this study, we investigated the capacity of lung cancer cells to activate complement and characterized the consequences of complement activation on tumor progression. We focused our study on the production and role of the anaphylatoxin C5a, a potent immune mediator generated after complement activation. We first measured the capacity of lung cancer cell lines to deposit C5 and release C5a. C5 deposition, after incubation with normal human serum, was higher in lung cancer cell lines than in nonmalignant bronchial epithelial cells. Notably, lung malignant cells produced complement C5a even in the absence of serum. We also found a significant increase of C5a in plasma from patients with non-small cell lung cancer, suggesting that the local production of C5a is followed by its systemic diffusion. The contribution of C5a to lung cancer growth in vivo was evaluated in the Lewis lung cancer model. Syngeneic tumors of 3LL cells grew slower in mice treated with an antagonist of the C5a receptor. C5a did not modify 3LL cell proliferation in vitro but induced endothelial cell chemotaxis and blood-vessels formation. C5a also contributed to the immunosuppressive microenvironment required for tumor growth. In particular, blockade of C5a receptor significantly reduced myeloid-derived suppressor cells and immunomodulators ARG1, CTLA-4, IL-6, IL-10, LAG3, and PDL1 (B7H1). In conclusion, lung cancer cells have the capacity to generate C5a, a molecule that creates a favorable tumor microenvironment for lung cancer progression.

Promoting complement (C) activation may enhance immunological mechanisms of anti-tumor Abs for tumor destruction. However, C activation components, such as C5a, trigger inflammation, which can promote tumor growth. We addressed the role of C5a on tumor growth by transfecting both human carcinoma and murine lymphoma with mouse C5a. In vitro growth kinetics of C5a, control vector, or parental cells revealed no significant differences. Tumor-bearing mice with C5a-transfected xenografted tumor cells had significantly less tumor burden as compared with control vector tumors. NK cells and macrophages infiltrated C5a-expressing tumors with significantly greater frequency, whereas vascular endothelial growth factor, arginase, and TNF-α production were significantly less. Tumor-bearing mice with high C5a-producing syngeneic lymphoma cells had significantly accelerated tumor progression with more Gr-1+CD11b+ myeloid cells in the spleen and overall decreased CD4+ and CD8+ T cells in the tumor, tumor-draining lymph nodes, and the spleen. In contrast, tumor-bearing mice with low C5a-producing lymphoma cells had a significantly reduced tumor burden with increased IFN-γ-producing CD4+ and CD8+ T cells in the spleen and tumor-draining lymph nodes. These studies suggest concentration of local C5a within the tumor microenvironment is critical in determining its role in tumor progression.

Objectives: Despite the experimental research data on antiphospholipid syndrome (APS), the pathogenesis of thrombosis and fetal loss remains unknown. The objective of this study was to analyze the major advances in the field of complement activation as a possible thrombosis mechanism in the APS., Methods: The authors conducted a systemic analysis of the English literature and summarized both animal and human data that indicate the inappropriate complement activation as a mechanism causing thrombosis in the APS., Results: The important role of complement activation in the pathogenesis of fetal loss was established using mice deficient in a complement regulatory protein. Further studies have shown that the infusion of human IgG antiphospholipid antibodies (aPL) induced fetal loss in pregnant mice, an effect that was abrogated by the concurrent administration of a C3 convertase inhibitor. Further studies suggested that C5a and neutrophils were the key components responsible for fetal injury. Moreover, use of F(ab)'2 fragments of aPL suggested the complement activation occurred mainly via the classical pathway. Other studies using models of induced thrombosis suggested that antibodies against β2GPI required the presence of terminal complement components to induce thrombus formation, and mice deficient in C3 or C5 were found to be resistant to aPL-induced thrombosis. Based on the aforementioned findings, it has been suggested that heparin prevents fetal loss in patients with APS by inhibiting complement activation rather than by its anticoagulant effect., Conclusions: The studies on complement are significant because they shift the focus of research in APS from thrombosis to inflammation. However, as human data are limited, more clinical research is necessary before the above findings translate in changes in the management of APS., (Copyright © 2012 Elsevier Inc. All rights reserved.)

There is accumulating evidence that the complement activation product, C5a, can orchestrate cellular immune functions. IL-27(p28/EBI3) is an emerging key player essential for regulating inflammatory responses and T cells. In this article, we report that C5a robustly suppressed IL-27(p28) gene expression and release in peritoneal macrophages. These cells from C57BL/6J mice abundantly produced IL-27(p28) after engagement of either the TLR3 (polyinosinic-polycytidylic acid) or TLR4 (LPS) receptor. Genetic deficiency of either TLR4 or LBP completely incapacitated the ability of macrophages to secrete IL-27(p28) in response to LPS. IL-27(p28)-producing macrophages also expressed the C5aR receptor, thus displaying an IL-27(p28)(+)F4/80(+)C5aR(+) phenotype. C5a suppressed IL-27(p28) in LPS-stimulated macrophages via interactions with the C5aR receptor rather than the C5L2 receptor. After endotoxemia, C5aR(-/-) mice displayed higher plasma levels of IL-27(p28) compared with C57BL/6J mice. C5a did not affect the release of IL-27(p28) or the frequency of IL-27(p28)(+)F4/80(+) macrophages after engagement of TLR3. Mechanistically, LPS activated both the NF-κB and the PI3K/Akt pathways, whereas C5a activated only the PI3K/Akt pathway. Engagement of PI3K/Akt was inhibitory for IL-27(p28) production, because PI3K/Akt pharmacologic blockade resulted in increased amounts of IL-27(p28) and reversed the suppressive effects of C5a. Blockade of PI3K/Akt in endotoxemic C57BL/6J mice resulted in higher generation of IL-27(p28). In contrast, the PI3K/Akt pathway was not involved in TLR3-mediated release of IL-27(p28). These data provide new evidence about how complement activation may selectively interfere with production of T cell regulatory cytokines by APCs in the varying contexts of either bacterial (TLR4 pathway) or viral (TLR3 pathway) infection.

We report that the bone marrow (BM) stroma-released LL-37, a member of the cathelicidin family of antimicrobial peptides, primes/increases the responsiveness of murine and human hematopoietic stem/progenitor cells (HSPCs) to an α-chemokine stromal-derived factor-1 (SDF-1) gradient. Accordingly, LL-37 is upregulated in irradiated BM cells and enhances the chemotactic responsiveness of hematopoietic progenitors from all lineages to a low physiological SDF-1 gradient as well as increasing their (i) adhesiveness, (ii) SDF-1-mediated actin polymerization and (iii) MAPK(p42/44) phosphorylation. Mice transplanted with BM cells ex vivo primed by LL-37 showed accelerated recovery of platelet and neutrophil counts by ∼3-5 days compared with mice transplanted with unprimed control cells. These priming effects were not mediated by LL-37 binding to its receptor and depended instead on the incorporation of the CXCR4 receptor into membrane lipid rafts. We propose that LL-37, which has primarily antimicrobial functions and is harmless to mammalian cells, could be clinically applied to accelerate engraftment as an ex vivo priming agent for transplanted human HSPCs. This novel approach would be particularly important in cord blood transplantations, where the number of HSCs available is usually limited.

Inflammation is a key factor in a number of neurodegenerative diseases including systemic lupus erythematosus. The complement system is an important mechanism in initiating and amplifying inflammation. Our recent studies demonstrate that C5a, a protein fragment generated during complement activation could alter the blood-brain barrier integrity, and thereby disturb the brain microenvironment. To understand the mechanism by which this occurs, we examined the effects of C5a on apoptosis, translocation of nuclear factor-κB (NF-κb) and the expression of Iκbα, MAPK, CREB and TJ protein, zona occludens (ZO-1) in mouse brain endothelial cells. Apoptosis was examined by DNA laddering and caspase 3 activity and the distribution of the ZO-1 and the p65 subunit of NF-κB were determined by immunofluorescence. Inhibition of CD88 reduced translocation of NF-κb into the nucleus, altered ZO-1 at the interfaces of neighboring cells, decreased caspase 3 activity and prevented apoptosis in these cells. Our results indicate that signaling through CD88 regulates the blood-brain barrier in a NF-κb-dependent manner. These studies suggest that the C5a receptor, CD88 is a promising therapeutic target that will reduce NF-κb-signaling cascades in inflammatory settings., (© 2011 The Authors. Journal of Neurochemistry © 2011 International Society for Neurochemistry.)

About 50% of patients with sepsis show myocardial involvement characterized by biventricular enlargement, reduced contractility and diastolic dysfunction. This increases the risk of death and leads to an extremely poor prognosis in the case of severe sepsis or septic shock, with full recovery of cardiac function seen in survivors at 7-10 days. The pathogenesis of myocardial dysfunction has long been investigated and, although it is still not fully understood, seems not to be due to reduced coronary flow, but to circulating substances released by pathogens (e.g. endotoxins) and host immuno-inflammatory responses (e.g. cytokines and mechanisms related to nitric oxide). First-line therapy is causal and consists of antibiotics plus the surgical excision of the infectious focus; in the presence of severe sepsis or septic shock, it is also necessary to promptly start circulatory and multiorgan support treatment. This review describes current knowledge concerning the instrumental and clinical characteristics, pathophysiology, prognosis and therapy of myocardial dysfunction during sepsis, and briefly considers possible future therapeutic perspectives.

There is a tight interaction of the bone and the immune system. However, little is known about the relevance of the complement system, an important part of innate immunity and a crucial trigger for inflammation. The aim of this study was, therefore, to investigate the presence and function of complement in bone cells including osteoblasts, mesenchymal stem cells (MSC), and osteoclasts. qRT-PCR and immunostaining revealed that the central complement receptors C3aR and C5aR, complement C3 and C5, and membrane-bound regulatory proteins CD46, CD55, and CD59 were expressed in human MSC, osteoblasts, and osteoclasts. Furthermore, osteoblasts and particularly osteoclasts were able to activate complement by cleaving C5 to its active form C5a as measured by ELISA. Both C3a and C5a alone were unable to trigger the release of inflammatory cytokines interleukin (IL)-6 and IL-8 from osteoblasts. However, co-stimulation with the pro-inflammatory cytokine IL-1β significantly induced IL-6 and IL-8 expression as well as the expression of receptor activator of nuclear factor-kappaB ligand (RANKL) and osteoprotegerin (OPG) indicating that complement may modulate the inflammatory response of osteoblastic cells in a pro-inflammatory environment as well as osteoblast-osteoclast interaction. While C3a and C5a did not affect osteogenic differentiation, osteoclastogenesis was significantly induced even in the absence of RANKL and macrophage-colony stimulating factor (M-CSF) suggesting that complement could directly regulate osteoclast formation. It can therefore be proposed that complement may enhance the inflammatory response of osteoblasts and increase osteoclast formation, particularly in a pro-inflammatory environment, for example, during bone healing or in inflammatory bone disorders., (Copyright © 2011 Wiley-Liss, Inc.)

Cerebral malaria is the most severe complication of Plasmodium falciparum infection and accounts for a large number of malaria fatalities worldwide. Recent studies demonstrated that C5(-/-) mice are resistant to experimental cerebral malaria (ECM) and suggested that protection was due to loss of C5a-induced inflammation. Surprisingly, we observed that C5aR(-/-) mice were fully susceptible to disease, indicating that C5a is not required for ECM. C3aR(-/-) and C3aR(-/-) × C5aR(-/-) mice were equally susceptible to ECM as were wild-type mice, indicating that neither complement anaphylatoxin receptor is critical for ECM development. In contrast, C9 deposition in the brains of mice with ECM suggested an important role for the terminal complement pathway. Treatment with anti-C9 Ab significantly increased survival time and reduced mortality in ECM. Our data indicate that protection from ECM in C5(-/-) mice is mediated through inhibition of membrane attack complex formation and not through C5a-induced inflammation.

Antineutrophil cytoplasmic autoantibodies (ANCA) are closely associated with systemic small vessel vasculitis characterized by segmental vessel wall necrotizing inflammation and a paucity of immunoglobulin deposition. Clinically, in vitro and experimental animal model observations indicate a direct pathogenic role for ANCA. This review focuses on the results of experiments utilizing a mouse model of ANCA disease induced by transfer of mouse anti-MPO IgG or anti-MPO lymphocytes into recipient mice, which causes small vessel vasculitis and glomerulonephritis that closely mimics human disease. Evidence for the following conclusion about this model, and by implication about human ANCA disease, will be summarized as follows: (1) anti-MPO IgG is sufficient even in the absence of functional T cells to cause disease and anti-MPO T lymphocytes are not sufficient to cause acute injury; (2) neutrophils are required; (3) ANCA antigens in bone marrow-derived cells are sufficient targets; (4) increased circulating pro-inflammatory cytokines and microbial products exacerbate disease, and concurrent viral infection exacerbates and modulates the phenotype of disease; (5) Fcγ receptor engagement is required for disease induction, and Fcγ receptor repertoire modulates the phenotype of disease, especially pulmonary disease; (6) activation of the alternative pathway of complement is required, complement is activated by factors released by neutrophils stimulated by ANCA IgG and engagement of C5a receptors is a primary event in complement-mediated amplification; and (7) genetic background has a marked influence on the severity and outcome of disease, and modified gene expression in bone marrow-derived cells is the primary basis for genetically determined differences in disease susceptibility. Investigations using this animal model of ANCA disease have provided important insights into the cellular, molecular and genetic factors involved in the pathogenesis of ANCA disease which are likely to lead to the identification of improved markers of disease activity and response to therapy, as well as more effective and less toxic therapies., (Copyright © 2011 S. Karger AG, Basel.)

Objective: To investigate the effect of C3a and C5a and their receptor antagonists (C3aRA and C5aRA) on the expression of BETAcatenin in renal tubular epithelial cell line HK-2., Methods: Cells were divided in into C3a group and C5a group which was further divided into four subgroups: C3a group (control, 1 micromol/L TGF-beta1, 50 nmol/L C3a, 1 micromol/L C3aRA); C5a group (control, 1 micromol/L TGF-beta1, 50 nmol/L C5a, 2.5 micromol/L C5aRA). Real time PCR and Western blot were used to detect mRNA and protein expression of beta-catenin., Results: Real-time PCR and Western blot demonstrated that 1 micromol/L TGF-beta1 could increase the expression of beta-catenin; C3a and C5a presented the similar inducible effect as TGF-beta1, which could be blocked by C3aR antagonist and C5aR antagonist (C3aRA and C5aRA)., Conclusion: Anaphylatoxin C3a and C5a can induce mRNA and protein expression of beta-catenin in renal tubular epithelial cells, which could be blocked by C3aRA and C5aRA. C3a and C5a may participate in tubular epithelial-mesenchymal transition in vitro.

Increased numbers of mast cells have been reported in explanted human hearts with dilated cardiomyopathy and in animal models of experimentally induced hypertension, myocardial infarction, and chronic volume overload secondary to aortocaval fistula and mitral regurgitation. Accordingly, mast cells have been implicated to have a major role in the pathophysiology of these cardiovascular disorders. In vitro studies have verified that mast cell proteases are capable of activating collagenase, gelatinases and stromelysin. Recent results have shown that with chronic ventricular volume overload, there is an elevation in mast cell density, which is associated with a concomitant increase in matrix metalloproteinase (MMP) activity and extracellular matrix degradation. However, the role of the cardiac mast cell is not one dimensional, with evidence from hypertension and cardiac transplantation studies suggesting that they can also assume a pro-fibrotic phenotype in the heart. These adverse events do not occur in mast cell deficient rodents or when cardiac mast cells are pharmacologically prevented from degranulating. This review is focused on the regulation and dual roles of cardiac mast cells in: (i) activating MMPs and causing myocardial fibrillar collagen degradation and (ii) causing fibrosis in the stressed, injured or diseased heart. Moreover, there is strong evidence that premenopausal female cardioprotection may at least partly be due to gender differences in cardiac mast cells. This too will be addressed.

Background: Complement 5a factor (C5a) elicits a broad range of proinflammatory effects, including chemotaxis of inflammatory cells and cytokine release. C5a is also linked to the coagulant activity in autoimmune diseases. Therefore, C5a most likely plays a crucial role in the instant blood-mediated inflammatory reaction., Methods: Intraportal transplantation of 2.5 islet equivalents/g of syngeneic rat islet grafts was performed in two groups of streptozotocin-induced diabetic rats: controls and C5a inhibitory peptide (C5aIP)-treated group., Results: The thrombin-antithrombin complex was significantly suppressed in the C5aIP group (P=0.003), and both the curative rate and the glucose tolerance were significantly improved in the C5aIP group (P<0.05 and P<0.005, respectively). Expression of tissue factor on granulocytes in recipient livers was up-regulated 1 h after islet infusion (P<0.0001), which was significantly suppressed by C5aIP (P<0.005). However, C5aIP was unable to regulate tissue factor expression on isolated islets. Furthermore, no differences were detected between the groups, regarding infiltration of CD11b-positive cells and deposition of C5b-9 on the islet grafts., Conclusions: These data suggest that C5aIP attenuates cross-talk between the complement and coagulation cascades through suppressing up-regulation of tissue factor expression on leukocytes in recipient livers but not on islet grafts, a process leading to improvement in islet engraftment. Therefore, C5aIP in combination with conventional anticoagulants could be a strong candidate strategy to control the instant blood-mediated inflammatory reaction induced in clinical islet transplantation.

Although the complement system is centrally involved in host defense, its overactivation or deregulation (e.g., due to inherent host genetic defects or due to pathogen subversion) may excessively amplify inflammation and contribute to immunopathology. Periodontitis is an oral infection-driven chronic inflammatory disease which exerts a systemic impact on health. This paper reviews evidence linking complement to periodontal inflammation and pathogenesis. Clinical and histological observations show a correlation between periodontal inflammatory activity and local complement activation. Certain genetic polymorphisms or deficiencies in specific complement components appear to predispose to increased susceptibility to periodontitis. Animal model studies and in vitro experiments indicate that periodontal bacteria can either inhibit or activate distinct components of the complement cascade. Porphyromonas gingivalis, a keystone species in periodontitis, subverts complement receptor 3 and C5a anaphylatoxin receptor signaling in ways that promote its adaptive fitness in the presence of non-productive inflammation. Overall, available evidence suggests that complement activation or subversion contributes to periodontal pathogenesis, although not all complement pathways or functions are necessarily destructive. Effective complement-targeted therapeutic intervention in periodontitis would require determining the precise roles of the various inductive or effector complement pathways. This information is essential as it may reveal which specific pathways need to be blocked to counteract microbial evasion and inflammatory pathology or, conversely, kept intact to promote host immunity., (Copyright © 2010 Elsevier Inc. All rights reserved.)

Purpose: The complement system plays a crucial role in the progression of age-related macular degeneration (AMD). In this study, the authors sought to evaluate the pathophysiologic roles of complement components C3a and C5a in the human choroid in AMD., Methods: Human RPE/choroid was assayed for the presence of C3a and C5a receptors (C3aR and C5aR) using RT-PCR and immunohistochemistry. Choroidal endothelial cell migration and proliferation were evaluated in the presence of C5a. Organ cultures of human choroid were incubated in C5a or bovine serum albumin (BSA) followed by quantitative immunohistochemistry and quantitative PCR for ICAM-1. AMD patients and controls were genotyped at SNPs in the C5R1 and C3AR1 genes., Results: C5aR, but not C3aR, was detected in human choroid. C5a did not promote endothelial cell migration or proliferation. However, choriocapillaris endothelial cells in organ culture responded to C5a by increasing ICAM-1 mRNA and protein. No significant association of SNP genotypes was detected in AMD patients at the C3AR1 and C5R1 genes., Conclusions: The generation of C5a peptides may lead to activation of choriocapillaris endothelial cells in AMD. Activation of the choroidal endothelium may affect the progression of AMD by recruitment of monocytes, leading to additional sequelae of AMD pathogenesis.

Thrombosis is a common complication of end-stage renal disease, particularly in patients on hemodialysis. Although substantial progress has been made in preventing thrombotic complications in various other groups of patients, the mechanisms of thrombosis during hemodialysis require clarification. In this report, we demonstrate that complement activation triggered by hemodialysis biomaterials, and the subsequent generation of the complement anaphylatoxin C5a, results in the expression of functionally active tissue factor (TF) in peripheral blood neutrophils. Because TF is a key initiator of coagulation in vivo, we postulate that the recurring complement activation that occurs during long-term hemodialysis contributes to thrombosis in dialyzed end-stage renal disease patients. Furthermore, we found that complement contributed to the induction of granulocyte colony-stimulating factor, which has been implicated in the pathogenesis of thrombosis in patients treated with the recombinant form of this molecule. Importantly, the inhibition of complement activation attenuated the TF expression and granulocyte colony-stimulating factor induction in blood passing through a hemodialysis circuit, suggesting that the complement system could become a new therapeutic target for preventing thrombosis in patients with chronic renal failure who are maintained on hemodialysis.

The complement system is a pivotal component of the innate immune system which protects the host from infection and injury. Complement proteins can be induced in all cell types within the central nervous system (CNS), where the pathway seems to play similar roles in host defense. Complement activation produces the C5 cleavage fragment C5a, a potent inflammatory mediator, which recruits and activates immune cells. The primary cellular receptor for C5a, the C5a receptor (CD88), has been reported to be on all CNS cells, including neurons and glia, suggesting a functional role for C5a in the CNS. A second receptor for C5a, the C5a-like receptor 2 (C5L2), is also expressed on these cells; however, little is currently known about its potential role in the CNS. The potent immune and inflammatory actions of complement activation are necessary for host defense. However, if over-activated, or left unchecked it promotes tissue injury and contributes to brain disease pathology. Thus, complement activation, and subsequent C5a generation, is thought to play a significant role in the progression of CNS disease. Paradoxically, complement may also exert a neuroprotective role in these diseases by aiding in the elimination of aggregated and toxic proteins and debris which are a principal hallmark of many of these diseases. This review will discuss the expression and known roles for complement in the CNS, with a particular focus on the pro-inflammatory end-product, C5a. The possible overarching role for C5a in diseases of the CNS is reviewed, and the therapeutic potential of blocking C5a/CD88 interaction is evaluated.

In visceral leishmaniasis, the draining LN (DLN) is the initial site for colonization and establishment of infection after intradermal transmission by the sand fly vector; however, little is known about the developing immune response within this site. Using an intradermal infection model, which allows for parasite visceralization, we have examined the ongoing immune responses in the DLN of BALB/c mice infected with Leishmania infantum. Although not unexpected, at early times post-infection there is a marked B-cell expansion in the DLN, which persists throughout infection. However, the characteristics of this response were of interest; as early as day 7 post-infection, polyclonal antibodies (TNP, OVA, chromatin) were observed and the levels appeared comparable to the specific anti-leishmania response. Although B-cell-deficient JhD BALB/c mice are relatively resistant to infection, neither B-cell-derived IL-10 nor B-cell antigen presentation appear to be primarily responsible for the elevated parasitemia. However, passive transfer and reconstitution of JhD BALB/c with secretory immunoglobulins, (IgM or IgG; specific or non-specific immune complexes) results in increased susceptibility to L. infantum infection. Further, JhD BALB/c mice transgenetically reconstituted to secrete IgM demonstrated exacerbated disease in comparison to WT BALB/c mice as early as 2 days post-infection. Evidence suggests that complement activation (generation of C5a) and signaling via the C5a receptor (CD88) is related to the disease exacerbation caused by IgM rather than cytokine levels (IL-10 or IFN-gamma). Overall these studies indicate that polyclonal B-cell activation, which is known to be associated with human visceral leishmaniasis, is an early and intrinsic characteristic of disease and may represent a target for therapeutic intervention.

Complement 5a (C5a) and Interleukin-17 (IL-17) are two important inflammatory mediators in sepsis. Here we studied the mechanisms underlying regulation of IL-17 by anaphylatoxin C5a. We found that C5a blockade increased the survival rate of mice following cecal ligation and puncture (CLP)-induced sepsis and decreased IL-17 expression in vivo. IL-17 was secreted mainly by gammadelta T cells in this model. Importantly, our data suggest that C5a participates in the regulation of IL-17 secretion by gammadelta T cells. Dendritic cells (DC) were found to act as a "bridge" between C5a and gammadelta T cells in a mechanism involving IL-6 and transforming growth factor beta (TGF-beta). These results imply that C5a affects the crosstalk between DC and gammadelta T cells during sepsis development, and this may result in a large production of inflammatory mediators such as IL-17.