Complement Factor C5a Research Articles

Complement factor C5a and C5a receptor contribute to morphine tolerance and withdrawal-induced hyperalgesia in rats

Morphine is a potent opioid analgesic. However, the repeated use of morphine causes tolerance and hyperalgesia. Neuroinflammation has been reported to be involved in morphine tolerance and withdrawal-induced hyperalgesia. The complement system is a crucial effector mechanism of immune responses. The present study investigated the roles of complement factor C5a and C5a receptor (C5aR) in the development of morphine tolerance and withdrawal-induced hyperalgesia. In the present study, the levels of C5a and C5aR were increased in the L5 lumbar spinal cords of morphine-tolerant rats. The administration of C5a promoted the development of hyperalgesia and the expression of spinal antinociceptive tolerance to intrathecal morphine in both mechanical and thermal test. However, these phenomena caused by morphine were significantly attenuated by the C5aR antagonist PMX53. These results suggest that complement activation within the spinal cord is involved in morphine tolerance and withdrawal-induced hyperalgesia. C5a and C5aR may serve as novel targets for the control of morphine tolerance and withdrawal-induced hyperalgesia.

Mast cell activation via complement factor C5a modulates vein graft remodeling and accelerated atherosclerosis

Mast cell activation via complement factor C5a modulates vein graft remodeling and accelerated atherosclerosis

Complement, Attraction, and Collective Migration

Complement factor C3a and its receptor are required for the mutual attraction and collective migration of neural crest cells.

Complement-factor C5a modulates vein graft remodeling and accelerated atherosclerosis via interference in Mast cell activation

Complement-factor C5a modulates vein graft remodeling and accelerated atherosclerosis via interference in Mast cell activation

Oral treatment with complement factor C5a receptor (CD88) antagonists inhibits experimental periodontitis in rats

The complement activation product 5a (C5a) is a potent mediator of the innate immune response to infection, and may thus also importantly determine the development of periodontitis. The present study was designed to explore the effect of several novel, potent and orally active C5a receptor (CD88) antagonists (C5aRAs) on the development of ligature-induced periodontitis in an animal model. Three different cyclic peptide C5aRAs, termed PMX205, PMX218 and PMX273, were investigated. Four groups of Wistar rats (n = 10 in each group) were used. Starting 3 d before induction of experimental periodontitis, rats either received one of the C5aRas (1-2 mg/kg) in the drinking water or received drinking water only. Periodontitis was assessed when the ligatures had been in place for 14 d. Compared with control rats, PMX205- and PMX218-treated rats had significantly reduced periodontal bone loss. The findings suggest that complement activation, and particularly C5a generation, may play a significant role in the development and progression of periodontitis. Blockade of the major C5a receptor, CD88, with specific inhibitors such as PMX205, may offer novel treatment options for periodontitis.

Complement activation: a critical mediator of adverse fetal outcomes in placental malaria?

Malaria infection is a significant risk factor for low birth weight outcomes in pregnancy. Despite efforts to define the molecular mechanisms that cause low birth weight as a result of intrauterine growth restriction, the roles of inflammation and mononuclear cells in the process are incompletely understood. Data from adverse pregnancy outcomes in humans and from murine models of pathological pregnancies suggest that C5a could be an important upstream regulator of placental angiogenesis, and excessive C5a could lead to functional placental insufficiency by impairing adequate vascularization of the placenta. Based on recent evidence, we hypothesize that complement factor C5a is a central initiator of poor birth outcomes associated with placental malaria by promoting mononuclear cell migration, activation and dysregulated angiogenesis.

Low level stimulation of FcϵRI on human skin mast cells modulates expression of adenosine receptors A2aAR and A3AR. (151.5)

Abstract Adenosine is a purine nucleoside that can potentiate and inhibit IgE-induced degranulation of mast cells. Extracellular adenosine acts through four G protein-coupled receptors (A1AR, A2aAR, A2bAR and A3AR). This study shows that low level stimulation of IgE receptor (FcϵRI), but not complement factor C5a receptor (C5aR), on human skin mast cells (SMCs) differentially modulates the expression of A2aAR and A3AR. Stimulation of SMCs with the anti-FcϵRIα mAb 22E7 at a concentration that does not induce degranulation temporally induced a 5-fold increase in A2aAR and a 5-fold decrease in A3AR. A2bAR was not affected and A1AR, which is not expressed in resting SMCs, was not induced. Mechanistically, the FcϵRI-induced increase in A2aAR was dependent on the NF-κB transcription factor since it was mostly blocked by the NF-κB inhibitor BAY-11. In contrast, A3AR levels were further reduced when NF-κB was inhibited. Since A2aAR couple predominantly with Gs proteins (adenylate cyclase activation) and A3AR with Gi proteins (adenylate cyclase inhibition), cAMP levels were measured. Exposure to adenosine of SMCs incubated ±22E7 resulted in a significant increase in cAMP levels in 22E7-stimulated SMCs relative to those cultured without 22E7. These data suggest that altered expression of A2aAR and A3AR by low-level stimulation of FcϵRI which promotes increased cAMP production upon adenosine exposure might act to raise the threshold for mast cell degranulation under certain conditions.

Pathophysiological basis of surgery-linked sepsis

Infection or injury, including surgical procedures, induces an inflammatory response of the host organism. This immune response must be finely tuned and precisely regulated, because deficiencies or excesses of the inflammatory response cause morbidity and shorten the lifespan. Activated receptors of the innate immune system (pattern recognition receptors, PRRs), which recognize pathogen-associated molecular patterns (PAMPs) and damage-associated molecular patterns (DAMPs) including injured tissue-associated intracellular proteins (alarmins), lead to an exaggerated immune response. This is characterized by a complex interplay of cytokines, chemokines, complement and coagulation factors as well as inflammatory and immune regulatory cells. There is increasing recognition that the major pathophysiologic event in sepsis is the progression from the initial hyperinflammatory state to an immunosuppressive state in which the host is unable to eradicate invading pathogens and particularly prone to develop secondary nosocomial infections and organ damage. Surgical trauma-associated immune dysfunction per se predisposes the host to surgery-related sepsis. Immune suppression is mediated by massive apoptosis-induced depletion of lymphocytes and dendritic cells, decreased expression of the cell surface antigen complex HLA-DR and increased expression of negative costimulatory molecules. Besides increased numbers of regulatory T cells there is a shift from a phenotype of inflammatory Th1 cells to an antiinflammatory phenotype of Th2 cells characterized by the production of interleukin-10. Key mediators of sepsis are HMGB1, MIF and complement factor C5a. With the identification of central pathomechanistic events, e.g. amplification of the coagulation, complement and inflammation cascades, immune dysbalance and neuroimmunomodulation via the cholinergic anti-inflammatory reflex, the opportunity now exists to apply these insights to the development of new and novel therapeutics aimed at modulating rather than inhibiting the systemic host response to infection.

Treatment with the C5a receptor antagonist ADC-1004 reduces myocardial infarction in a porcine ischemia-reperfusion model

BackgroundPolymorphonuclear neutrophils, stimulated by the activated complement factor C5a, have been implicated in cardiac ischemia/reperfusion injury. ADC-1004 is a competitive C5a receptor antagonist that has been shown to inhibit complement related neutrophil activation. ADC-1004 shields the neutrophils from C5a activation before they enter the reperfused area, which could be a mechanistic advantage compared to previous C5a directed reperfusion therapies. We investigated if treatment with ADC-1004, according to a clinically applicable protocol, would reduce infarct size and microvascular obstruction in a large animal myocardial infarct model.MethodsIn anesthetized pigs (42-53 kg), a percutaneous coronary intervention balloon was inflated in the left anterior descending artery for 40 minutes, followed by 4 hours of reperfusion. Twenty minutes after balloon inflation the pigs were randomized to an intravenous bolus administration of ADC-1004 (175 mg, n = 8) or saline (9 mg/ml, n = 8). Area at risk (AAR) was evaluated by ex vivo SPECT. Infarct size and microvascular obstruction were evaluated by ex vivo MRI. The observers were blinded to the treatment at randomization and analysis.ResultsADC-1004 treatment reduced infarct size by 21% (ADC-1004: 58.3 ± 3.4 vs control: 74.1 ± 2.9%AAR, p = 0.007). Microvascular obstruction was similar between the groups (ADC-1004: 2.2 ± 1.2 vs control: 5.3 ± 2.5%AAR, p = 0.23). The mean plasma concentration of ADC-1004 was 83 ± 8 nM at sacrifice. There were no significant differences between the groups with respect to heart rate, mean arterial pressure, cardiac output and blood-gas data.ConclusionsADC-1004 treatment reduces myocardial ischemia-reperfusion injury and represents a novel treatment strategy of myocardial infarct with potential clinical applicability.

A high-throughput protein refolding screen in 96-well format combined with design of experiments to optimize the refolding conditions

Production of correctly folded and biologically active proteins in Escherichia coli 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.

A peptide mimic of the chemotaxis inhibitory protein of Staphylococcus aureus: towards the development of novel anti-inflammatory compounds

Complement factor C5a is one of the most powerful pro-inflammatory agents involved in recruitment of leukocytes, activation of phagocytes and other inflammatory responses. C5a triggers inflammatory responses by binding to its G-protein-coupled C5a-receptor (C5aR). Excessive or erroneous activation of the C5aR has been implicated in numerous inflammatory diseases. The C5aR is therefore a key target in the development of specific anti-inflammatory compounds. A very potent natural inhibitor of the C5aR is the 121-residue chemotaxis inhibitory protein of Staphylococcus aureus (CHIPS). Although CHIPS effectively blocks C5aR activation by binding tightly to its extra-cellular N terminus, it is not suitable as a potential anti-inflammatory drug due to its immunogenic properties. As a first step in the development of an improved CHIPS mimic, we designed and synthesized a substantially shorter 50-residue adapted peptide, designated CHOPS. This peptide included all residues important for receptor binding as based on the recent structure of CHIPS in complex with the C5aR N terminus. Using isothermal titration calorimetry we demonstrate that CHOPS has micromolar affinity for a model peptide comprising residues 7–28 of the C5aR N terminus including two O-sulfated tyrosine residues at positions 11 and 14. CD and NMR spectroscopy showed that CHOPS is unstructured free in solution. Upon addition of the doubly sulfated model peptide, however, the NMR and CD spectra reveal the formation of structural elements in CHOPS reminiscent of native CHIPS.Electronic supplementary materialThe online version of this article (doi:10.1007/s00726-010-0711-3) contains supplementary material, which is available to authorized users.

Kallistatin Inhibits Vascular Inflammation by Antagonizing Tumor Necrosis Factor-α–Induced Nuclear Factor κB Activation

Kallistatin is a plasma protein with anti-inflammatory properties. In this study, we investigated the role and mechanisms of kallistatin in inhibiting endothelial inflammation through its heparin-binding domain. We showed that recombinant wild-type kallistatin dose-dependently competed with tumor necrosis factor (TNF)-alpha binding to TNF-alpha receptor in endothelial cells, whereas kallistatin mutant at the heparin-binding domain had no effect. Kallistatin, but not kallistatin mutant at the heparin-binding domain, abrogated TNF-alpha-induced endothelial cell activation, as evidenced by inhibition of TNF receptor 1-associated death domain protein activation, inhibitor of nuclear factor kappaB-alpha degradation, nuclear factor kappaB translocation, and p38 mitogen-activated protein kinase phosphorylation, as well as cell adhesion molecule and cytokine expression. Moreover, kallistatin, but not kallistatin mutant at the heparin-binding domain, inhibited TNF-alpha-induced human monocytic THP-1 cell adhesion to endothelial cells and prevented vascular endothelial growth factor-induced endothelial permeability. In mice, kallistatin gene delivery prevented vascular leakage provoked by complement factor C5a, whereas delivery of kallistatin heparin mutant gene had no effect. Similarly, gene transfer of kallistatin, but not the kallistatin heparin mutant, inhibited collagen/adjuvant-induced arthritis in rats. These results indicate that kallistatin's heparin-binding site plays an essential role in preventing TNF-alpha-mediated endothelial activation and reducing vascular endothelial growth factor-induced vascular permeability, resulting in attenuation of vascular inflammation in cultured endothelial cells and animal models. This study identifies a protective role of kallistatin in vascular injury, thereby implicating the therapeutic potential of kallistatin for vascular and inflammatory diseases.

Anti-GD 2 with an FC point mutation reduces complement fixation and decreases antibody-induced allodynia

Monoclonal antibodies against GD 2 ganglioside, such as ch14.18, the human–mouse chimeric antibody, have been shown to be effective for the treatment of neuroblastoma. However, treatment is associated with generalized, relatively opiate-resistant pain. We investigated if a point mutation in ch14.18 antibody (hu14.18K332A) to limit complement-dependent cytotoxicity (CDC) would ameliorate the pain behavior, while preserving antibody-dependent cellular cytotoxicity (ADCC). In vitro, CDC and ADCC were measured using europium-TDA assay. In vivo, allodynia was evaluated by measuring thresholds to von Frey filaments applied to the hindpaws after injection of either ch14.18 or hu14.18K332 into wild type rats or rats with deficient complement factor 6. Other rats were pretreated with complement factor C5a receptor antagonist and tested following ch14.18 injection. The mutation reduces the antibody’s ability to activate complement, while maintaining its ADCC capabilities. Injection of hu14.18K322 (1 or 3 mg/kg) produced faster resolving allodynia than that engendered by ch14.18 (1 mg/kg). Injection of ch14.18 (1 mg/kg) into rats with C6 complement deficiency further reduced antibody-induced allodynia, while pre-treatment with complement factor C5a receptor antagonist completely abolished ch14.18-induced allodynia. These findings showed that mutant hu14.18 K322 elicited less allodynia than ch14.18 and that ch14.18-elicited allodynia is due to activation of the complement cascade: in part, to formation of membrane attack complex, but more importantly to release of complement factor C5a. Development of immunotherapeutic agents with decreased complement-dependent lysis while maintaining cellular cytotoxicity may offer treatment options with reduced adverse side effects, thereby allowing dose escalation of therapeutic antibodies.

Complement Factor C5a in Acute Exacerbation of Chronic Obstructive Pulmonary Disease

The complement component C5a is a potent inflammatory peptide, which may be involved in the pathogenesis of Chronic Obstructive Pulmonary Disease (COPD). We analysed the induced sputum and plasma of 28 patients with stable COPD, 12 healthy smokers and 7 non-smokers. In 13 of the patients with COPD, we also observed paired samples during acute exacerbation. The concentrations of C5a/C5a desArg and C3a/C3a desArg were measured using cytometric bead array. Both C5a and C3a concentrations in induced sputum of stable patients with COPD were significantly increased compared to the control groups of healthy smokers and non-smokers. In addition, there was a significant elevation in C5a values in exacerbation of COPD that was independent from the airway C3a levels. Airway C5a levels were negatively correlated with forced expiratory volume in first second (FEV1)% predicted and diffusing capacity of the lung (TLCO). Plasma C5a concentrations in patients with COPD were significantly higher than in healthy smokers, but no further significant systemic C5a elevation was detected with acute exacerbation of COPD. There was no important difference in local or systemic C5a concentrations between healthy smokers and non-smokers. These in vivo results clearly show that local and systemic C5a concentrations in COPD are elevated, and that the local, in contrast to systemic, C5a concentrations additionally increase in the acute exacerbation of COPD. It seems that the cigarette smoke is not related to C5a increase. The elevated local and systemic C5a levels, and additional individual local C5a increase during the exacerbation support the importance of C5a in COPD.

What has been learnt from the thrombin-activatable fibrinolysis inhibitor-deficient mouse?

Thrombin-activatable fibrinolysis inhibitor (TAFI) is a circulating zymogen that is activated physiologically by the thrombin/thrombomodulin complex to activated TAFI (TAFIa) which is a basic carboxypeptidase. Substrates include fibrin, leading to a reduction in rate of plasmin generation, and several proinflammatory mediators such as bradykinin, thrombin-cleaved osteopontin and complement factor C5a. TAFI-deficient mice have no phenotype without being challenged and TAFIa appears to play a limited role in physiological fibrinolysis in vivo. In several disease models, the TAFI-deficient mice have different outcomes from the wild type (WT), but whether the difference is beneficial or an exacerbation of the disease depends on the model. The consequences of TAFI deficiency include increased plasmin as a result of enhanced incorporation of plasminogen and tissue plasminogen activator into the fibrin clot, but also loss of its ability to degrade other substrates, with the resultant up-regulation of several proinflammatory mediators, including C5a. Criteria are recommended to demonstrate that a substrate is a physiological substrate of TAFIa.

Local complement activation in nasal polyposis

The role of the complement system in nasal polyps (CRSwNP) has so far scarcely been studied. Because nasal polyps are characterized by bacterial colonization, and the complement system is an effective defense mechanism, it might be involved in the pathogenesis of CRSwNP. This study was designed to investigate the local and systemic activation of the complement system in CRSwNP versus control mucosa in relation to the local and systemic eosinophilic and neutrophilic inflammation and local plasma exudation. Concentrations of complement factors C3a desArg and C5a desArg, and of albumin, alpha2-macroglobulin, eosinophilic cationic protein, and myeloperoxidase were determined on nasal secretions and serum from 12 CRSwNP patients and 10 control patients. Tissue cryosections were stained for the membrane attack complex (C5b9) We found a significantly higher concentration of C3a desArg and C5a desArg in nasal secretions from CRSwNP patients compared to controls, whereas the serum levels between the two groups did not differ significantly. Significant correlations were found between C5a desArg and eosinophil cationic protein in nasal secretions. Staining for the membrane attack complex revealed a deposition around blood vessels and the basal membrane exclusively in nasal polyp tissue. These data support the hypothesis that, in addition to the adaptive immune responses, the complement system is involved in the pathogenesis of CRSwNP and may contribute to typical features such as edema and granulocytic inflammation.

The complement factor C5a receptor is upregulated in NFL−/− mouse motor neurons

In NFL−/− mice, a model of motor neuron degeneration in ALS, degenerating spinal motor neurons express high levels of the receptor for the C5a anaphylatoxin (C5aR) early in the disease process. C5a is a potent in vitro neurotoxin for both Neuro2A and NGF-differentiated PC12 cells. While no interaction was observed between glutamate and C5a, both C5a and kainate upregulated the expression of activated C5aR. C5aR expression was increased in motor neurons in ALS. This data suggests that the early upregulation of C5aR may contribute to motor neuron damage that potentiates excitotoxicity in ALS.

Complement factors C3a and C5a have distinct hemodynamic effects in the rat

In the rat, C5a infusion mediates well-defined effects including hypotension and neutropenia. Conversely, the comparative effect of C3a in the rat is not yet defined. In the current study, we have investigated C3a receptor (C3aR) activation in the rat, using recombinant human C3a, the C3aR agonist WWGKKYRASKLGLAR, which is a C-terminal analogue of C3a, and a nonpeptide C3aR antagonist SB-290157, as pharmacological tools. In vitro, C3a and WWGKKYRASKLGLAR selectively bound to C3aRs and induced degranulation of C3aR-transfected RBL-2H3 cells. C3a or WWGKKYRASKLGLAR-induced degranulation was dose-dependently antagonized in a surmountable fashion by the nonpeptide C3aR antagonist. Intravenous infusion of C3a and WWGKKYRASKLGLAR to rats induced a rapid, transient and concentration-dependent hypertensive response, which was mediated by C3aR-induced prostanoid release. C3a and WWGKKYRASKLGLAR caused a small drop in circulating neutrophils, but a rise in circulating neutrophils was evident after 90–120 min. In contrast to C3a, C5a infusion resulted in hypotension, and rapid and transient neutropenia. These results demonstrate that C3a and C5a mediate distinct effects on blood pressure and circulating polymorphonuclear leukocytes in the rat.

Bicarbonate-buffered ultrafiltration during pediatric cardiac surgery prevents electrolyte and acid-base balance disturbances

Pediatric cardiopulmonary bypass is still a challenge because of electrolyte disturbances and inflammation. Many investigations deal with different types of hemofiltration to reduce these potentially harmful side effects. We tested the hypothesis of whether bicarbonate-buffered hemofiltration of the priming solution minimizes electrolyte and acid-base disturbances during the initiation of cardiopulmonary bypass and whether bicarbonate-buffered hemofiltration performed during cardiopulmonary bypass could reduce cytokine levels. Twenty children younger than 2 years of age (mean age 166 +/- 191 days; mean weight 6.42 +/- 3.22 kg) scheduled for pediatric cardiac surgery with cardiopulmonary bypass were enrolled in this prospective clinical study. Cardiopulmonary bypass circuits were primed with a bicarbonate-buffered hemofiltration solution, gelatin and 1 unit of packed red blood cells. The priming was hemofiltered using an ultrahemofilter until approximately 1000 mL of ultrafiltrate was restored with the buffered solution. Further hemofiltration was performed throughout the whole bypass time, especially during rewarming. Blood gas analyses and inflammatory mediators were monitored during the operation. Blood gas analysis results after initiation of cardiopulmonary bypass and throughout the entire study remained within the physiologic ranges. Even potassium decreased from 4.0 +/- 0.3 to 3.4 +/- 0.4 mmol l(-1) after initiation of cardiopulmonary bypass. Plasma levels of tumor necrosis factor alpha decreased significantly (47 +/- 44 vs. 24 +/- 21 pg mL(-1)) whereas complement factor C3a (5.0 +/- 2.9 vs. 16.8 +/- 6.6 ng mL(-1)) and interleukin-6 (7.3 +/- 15.2 vs. 110 +/- 173 pg mL(-1)) increased despite hemofiltration. In conclusion, this study shows that bicarbonate-buffered ultrafiltration is an efficient, simple and safe method for performing hemofiltration, both of the priming solution and during the entire bypass time. The use of a physiological restitution solution prevents electrolyte and acid-base balance disturbances. The elimination of inflammatory mediators seems to be as effective as other ultrafiltration methods.

The role of C5a in reproductive impairment in the mouse and human

Adequate dietary folate is essential for a healthy pregnancy. Folate deficiency during pregnancy has been shown to contribute to foetal demise, growth retardation, craniofacial, cardiovascular, and neural tube defects, and numerous other congenital abnormalities. The present study aimed to determine whether the complement factor C5a contributes to folate-deficiency induced reproductive impairment, given that recent investigations have demonstrated that C5a can contribute to pathology in both antibody-dependent and -independent models of pregnancy loss. Administration of a folate-deficient diet (FDD) to wildtype mice resulted in poor fertility, reduced birth weights, and a high incidence of foetal loss. In contrast, C5a receptor knockout (C5aR KO) mice were resistant to foetal loss induced by a FDD, with a comparable incidence of resorption to mice administered a folate replete diet. C5aR-deficient mice on a FDD had significantly reduced birth weights and a very high prevalence of neural tube defects (40%)—a much higher frequency than FDD-fed wildtype mice. This observation suggests that in the presence of C5a, otherwise non-viable embryos from folate-deficient dams are rescued, resulting in an increased frequency of major birth defects. We also studied the expression of complement in human placenta. Placentas from growth restricted babies exhibited significant placental deposition of the upstream complement factor C3 compared to healthy controls. Intriguingly, when foetal growth restriction was associated with preeclampsia, an upregulation of placental C5aRs was additionally observed. These results suggest a complex role for the complement system and for C5a in foetal health and survival.