Complement-derived Anaphylatoxins Research Articles

Quantifying the Sensitivity of Human Immune Cells to Chemoattractant

The efficient recruitment of immune cells is a vital cornerstone of our defense against infections and a key challenge of immunotherapeutic applications. It relies on the ability of chemotaxing cells to prioritize their responses to different stimuli. For example, immune cells are known to abandon gradients of host-cell-produced cytokines in favor of complement-derived anaphylatoxins, which then guide the cells toward nearby pathogen surfaces. The aptitude to triage stimuli depends on the cells’ specific sensitivities to different chemoattractants. We here use human neutrophils as uniquely capable biodetectors to map out the anaphylatoxic cloud that surrounds microbes in the presence of host serum. We quantify the neutrophil sensitivity in terms of the ratio between the chemoattractant concentration c and the production rate j0 of the chemoattractant at the source surface. An integrative experimental/theoretical approach allows us to estimate the c/j0-threshold at which human neutrophils first detect nearby β-glucan surfaces as c/j0 ≈ 0.0044 s/μm.

Experimental design of complement component 5a-induced acute lung injury (C5a-ALI): a role of CC-chemokine receptor type 5 during immune activation by anaphylatoxin

Excessive activation of the complement system is detrimental in acute inflammatory disorders. In this study, we analyzed the role of complement-derived anaphylatoxins in the pathogenesis of experimental acute lung injury/acute respiratory distress syndrome (ALI/ARDS) in C57BL/6J mice. Intratracheal administration of recombinant mouse complement component (C5a) caused alveolar inflammation with abundant recruitment of Ly6-G(+)CD11b(+) leukocytes to the alveolar spaces and severe alveolar-capillary barrier dysfunction (C5a-ALI; EC(50[C5a]) = 20 ng/g body weight). Equimolar concentrations of C3a or desarginated C5a (C5a(desArg)) did not induce alveolar inflammation. The severity of C5a-ALI was aggravated in C5-deficient mice. Depletion of Ly6-G(+) cells and use of C5aR1(-/-) bone marrow chimeras suggested an essential role of C5aR1(+) hematopoietic cells in C5a-ALI. Blockade of PI3K/Akt and MEK1/2 kinase pathways completely abrogated lung injury. The mechanistic description is that C5a altered the alveolar cytokine milieu and caused significant release of CC-chemokines. Mice with genetic deficiency of CC-chemokine receptor (CCR) type 5, the common receptor of chemokine (C-C motif) ligand (CCL) 3, CCL4, and CCL5, displayed reduced lung damage. Moreover, treatment with a CCR5 antagonist, maraviroc, was protective against C5a-ALI. In summary, our results suggest that the detrimental effects of C5a in this model are partly mediated through CCR5 activation downstream of C5aR1, which may be evaluated for potential therapeutic exploitation in ALI/ARDS.

Role of complement systems in IVIG mediated attenuation of cognitive deterioration in Alzheimer's disease.

Human intravenous immunoglobulin (IVIG) has been indicated as a potential therapy for autoimmune neurological disorders, as well as in many neurodegenerative diseases, with various underlying therapeutic mechanisms such as regulation of T-cell trafficking, cytokines, Fc receptor blocking, and interruption of complement activation cascade. In Alzheimer's disease (AD), IVIG presents naturally occurring antibodies against amyloid-beta (Aβ) aggregation, thus IVIG immunotherapy may increase the clearance of Aβ and protect brain function. Recently, we and others reported that besides Aβ clearance, IVIG specifically regulates the levels of complement-derived anaphylatoxins, such as C5a and C3, which play an important role in the regulation of AMPA and NMDA receptor expression in the brain and further upregulate the AMPA-PKA-CREB signaling pathway and synaptic function in AD mouse models. Since down-regulation of complement components has been linked with deficits of cognitive function in age-related dementia following the decline of innate immunity during aging, the IVIG immunotherapy could be an attractive novel AD therapeutic through its local regulation of C3, C5a component levels in brain.

Therapeutic Complement Inhibition: New Developments

Activation of the complement system significantly contributes to the pathogenesis of various acute and chronic inflammatory diseases. Current strategies to inhibit complement include the replacement or substitution of endogenous soluble complement inhibitors (e.g., C1 inhibitor [C1 inh], recombinant soluble complement receptor 1, TP10), the administration of antibodies to block key proteins of the cascade reaction (e.g., C5) or to neutralize the action of the complement-derived anaphylatoxins, or blockade of complement receptors (e.g., C5aR, CD88). The recent approvals of anti-C5 for the treatment of paroxysmal nocturnal hemoglobinuria as well as of C1 inh for the treatment of hereditary angioedema beyond European countries have provided a resurgence of interest in the potential of complement therapeutics for the treatment of disease.

Rare but Relevant Kidney Disorders

The spectrum of predisposing factors to atypical hemolytic uremic syndrome (aHUS) is widening. This syndrome includes cases of HUS that are not linked to Shiga toxin–producing bacteria. Approximately half of the patients with aHUS have mutations in genes that regulate complement system: Either loss-of-function mutations in genes that encode inhibitors of the alternative pathway of the complement system (e.g., factor H or I, CFH or CFI, membrane co-factor protein, factor H–related proteins, C4b-binding protein) or gain-of-function mutations of genes that encode factor B and C3. Antibodies against CFH have been observed in 2 to 10% of patients. Thrombomodulin is a ubiquitous transmembrane endothelial cell glycoprotein that suppresses clot formation, inhibits fibrinolysis, interferes with inflammation, and inactivates some complement-derived anaphylatoxins. Delvaeye et al. studied 152 patients who had aHUS and were recruited from the International Registry of Recurrent and Familial HUS/Thrombotic Thrombocytopenic Purpura and 380 control subjects. They sequenced the entire coding region of the thrombomodulin gene (THBD), which lacks introns. Six heterozygous mutations of THBD were identified in seven (4.6%) unrelated patients. Two of them belonged to families with aHUS. In one of these families, four heterozygous carriers (including adults) were asymptomatic. As in complement defects, the THBD mutation is not sufficient by itself to cause HUS. Low serum C3 levels were measured in four patients. The disease was evident during childhood, from 6 mo to 15 yr of age. Three of seven patients progressed to ESRD. Four had recurrent aHUS. One patient developed a first episode of aHUS after kidney transplantation at 15 yr of age. Further studies will indicate whether there is a risk for recurrence of aHUS after kidney transplantation. In vitro studies on cultured cells showed that thrombomodulin provides protection against complement activation. In addition, thrombomodulin binds to C3 and CFH and negatively regulates complement by accelerating CFI-mediated inactivation of C3b in the presence of co-factors CFH and C4b. Cultured cells that express THBD variants that are associated with aHUS had diminished capacity to inactivate C3b and to activate procarboxypeptidase B and thus were less protected from activated complement. In patients with aHUS, genetic testing should include several genes that encode proteins that regulate complement system, including thrombomodulin.

The complement‐derived anaphylatoxin C5a increases microglial GLT‐1 expression and glutamate uptake in a TNF‐α‐independent manner

Microglia can express Na+-dependent high-affinity glutamate transporters during pathological conditions in the CNS. The transporter expression seems to be activation dependent, and we therefore sought to identify factors that could induce it, in addition to the well-known effect of lipopolysaccharide (LPS) that is mediated by tumour necrosis factor-alpha (TNF-alpha). The complement-derived anaphylatoxins C3a and C5a are of potential interest as the complement system is activated in nearly all insults to the nervous system, and both C3a and C5a have been shown to protect against excitotoxicity. We have found that C5a, but not C3a, increased the expression of the microglial glutamate transporter GLT-1 in a dose-dependent manner without eliciting or modulating the release of TNF-alpha. However, the increase was not as prominent as the one induced by LPS, indicating that the microglia are in different activity states. The increase in microglial GLT-1 expression led to an increased functional uptake of glutamate without affecting the release. This suggests that C5a-stimulated microglia can be self- and neuroprotective by removing extracellular glutamate.

C5L2 is critical for the biological activities of the anaphylatoxins C5a and C3a

Complement-derived anaphylatoxins regulate immune and inflammatory responses through G-protein-coupled receptor (GPCR)-mediated signalling. C5L2 (also known as GPR77) is a relatively new GPCR thought to be a non-signalling receptor binding to C5a, on the basis of sequence information and experimental evidence. Here we show, using gene targeting, that C5L2 is required to facilitate C5a signalling in neutrophils, macrophages and fibroblasts in vitro. Deficiency of C5L2 results in reduced inflammatory cell infiltration, suggesting that C5L2 is critical for optimal C5a-mediated cell infiltration in certain in vivo settings. C5L2 is also involved in optimizing C3a-induced signals. Furthermore, like mice incapable of C3a/complement 3a receptor (C3aR) signalling, C5L2-deficient mice are hypersensitive to lipopolysaccharide (LPS)-induced septic shock, show reduced ovalbumin (OVA)-induced airway hyper-responsiveness and inflammation, and are mildly delayed in haematopoietic cell regeneration after gamma-irradiation. Our data indicate that C5L2 can function as a positive modulator for both C5a- and C3a-anaphylatoxin-induced responses.

Strategies of therapeutic complement inhibition

The involvement of complement in the pathogenesis of a great number of partly life threatening diseases defines the importance to develop inhibitors which specifically interfere with its deleterious action. Endogenous soluble complement-inhibitors, antibodies or low molecular weight antagonists, either blocking key proteins of the cascade reaction or neutralizing the action of the complement-derived anaphylatoxins have successfully been tested in various animal models over the past years. Promising results consequently led to first clinical trials. This review is focused on different approaches for the development of inhibitors, on their site of action in the cascade, on possible indications for complement inhibition based on experimental animal data, and on potential side effects of such treatment.

A blockade of complement activation prevents rapid intestinal ischaemia-reperfusion injury by modulating mucosal mast cell degranulation in rats.

We attempted to define the putative role of complement activation in association with mucosal mast cell (MMC) degranulation in the pathogenesis of rapid intestinal ischaemia-reperfusion (I/R) injury. We prepared complement activity-depleted rats by the administration of the anti-complement agent K-76COOH and the serine-protease inhibitor FUT-175. Autoperfused segments of the jejunum were exposed to 60 min of ischaemia, followed by reperfusion for various time periods, and the epithelial permeability was assessed by the 51Cr-EDTA clearance rate. The number of MMC was immunohistochemically assessed. In control rats, the maximal increase in mucosal permeability was achieved by 30-45 min of reperfusion. This increase was significantly attenuated by the administration of either K-76COONa alone or in combination with FUT-175. In contrast, the administration of carboxypeptidase inhibitor (CPI), which prevents the inactivation of complement-derived anaphylatoxins such as C5a, significantly enhanced the increase in I/R-induced mucosal permeability. These findings were confirmed morphologically by light microscopy and scanning electron microscopy. In addition, the I/R-induced mucosal injury was accompanied by a marked decrease in the number of MMC, and administration of K-76COOH significantly inhibited this change. These results indicate that complement activation and the generation of complement-derived anaphylatoxins are key events in I/R-induced mucosal injury. It is likely that intestinal I/R-induced mucosal injury may be partially mediated by MMC activation associated with the complement activation.

C5a and C5a(desArg) enhance the susceptibility of monocyte-derived macrophages to HIV infection.

Mononuclear phagocytes, which include circulating blood monocytes and differentiated tissue macrophages, are believed to play a central role in the sexual transmission of HIV infection. The ability of HIV to productively infect these cells may be influenced by action of exogenous or host-derived substances at the site of viral entry. Given the potent capacities of inflammatory mediators to stimulate anaphylatoxic and immunomodulatory functions in mucosa, the effects of complement-derived anaphylatoxins on the susceptibility of monocytes and monocyte-derived macrophages (MDM) to HIV-1 infection were examined. In our in vitro system, the susceptibility to infection was up to 40 times increased in MDM that had been exposed to C5a or C5a(desArg), but not to C3a or C3a(desArg), for 2 days before adding of virus. By contrast, the treatment with complement anaphylatoxins did not affect HIV replication in fresh monocytes. Stimulatory effect of C5a and its desArg derivative on HIV infection correlated with the increase of TNF-alpha and IL-6 secretion from MDM. All these functional effects of C5a and C5a(desArg) were reversible by treatment of cells with the mAb that functionally blocks C5aR. Taken together, these results indicate that C5a and C5a(desArg) may increase the susceptibility of MDM to HIV infection through stimulation of TNF-alpha and IL-6 secretion from these cells.

Complement-derived Anaphylatoxins in Human Donor Corneas Treated With Excimer Laser

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

Decay-accelerating factor on human umbilical vein endothelial cells. Its histamine-induced expression and spontaneous rapid shedding from the cell surface.

Decay-accelerating factor (DAF) is a membrane protein that protects host cells from attack by its own complement. Although DAF expression on endothelial cells is thought to increase pathophysiologically, little is known about the natural mediators that modulate DAF expression on endothelial cells. In this study, we evaluated the effect of histamine on DAF expression on human umbilical vein cells (HUVEC). HUVEC were cultured with histamine, and DAF expression on HUVEC was determined by flow cytometry after immunostaining with a mAb to DAF. DAF expression on HUVEC was increased at 10 microM histamine, and the final level was increased time-dependently by 150% to 200% after a 24-h incubation with 100 microM histamine. The histamine-induced DAF expression was inhibited by actinomycin D and cycloheximide and accompanied by an increase in the DAF mRNA level, indicating that both transcription and translation are required. In addition, the histamine-induced DAF expression was inhibited by pyrilamine, an H1 blocker, but not by cimetidine, an H2 blocker, indicating that histamine induces the DAF expression through H1 receptors. We also demonstrated that the turnover of DAF is faster than that of MCP and CD59, and DAF is released into the culture supernatant. DAF is a glycosylphosphatidylinositol-linked protein that is released from HUVEC by a phosphatidylinositol-specific phospholipase C. Although HUVEC also contain the glycosylphosphatidylinositol-anchored complement inhibitor CD59, this was not released during a 24-h incubation, suggesting that the shedding of DAF from HUVEC is not caused by PI-PLC but by other enzymes, possibly proteinases. These results suggest that histamine, which is released from mast cells and basophils by complement-derived anaphylatoxins, increases the complement defense ability of endothelial cells by increasing their levels of DAF expression.

Mediators of complement-independent granulocyte activation during haemodialysis: role of calcium, prostaglandins and leukotrienes

Granulocyte activation during haemodialysis using cuprophane membrane is mediated by complement-derived anaphylatoxins C3a and C5a. However, neutrophil degranulation induced by modified cellulosic membranes or synthetic membranes does not correlate with C3a or C5a concentrations. Incubation and recirculation experiments were performed to find out which messengers trigger neutrophil degranulation during blood contact with different membrane materials. During in vitro haemodialysis for 2 hours, PMMA and cuprophane induced pronounced degranulation of neutrophils. With PMMA this was associated with increased thromboxane B2 but low C3a levels, while with cuprophane membrane, marked complement activation but only little thromboxane B2 release was observed. Indomethacin (10 microM) nullified all thromboxane B2 response but could not influence elastase release, indicating that cyclo-oxygenase products are not involved in neutrophil degranulation under these conditions. During incubation of blood with dialysis membranes, inhibition of lipoxygenase by esculetin or of phospholipase A2 by hydrocortisone also had no effect on neutrophil degranulation. One messenger involved in granulocyte activation might be free cytosolic calcium. Application of different calcium channel blockers (verapamil, diltiazem, or nitrendipine) did not influence neutrophil degranulation in incubation experiments, in PMMA or cuprophane membranes. In contrast, chelation of plasmatic calcium by sodium citrate or EDTA blunted elastase release induced by these membranes. This study indicates that calcium is a key mediator required for neutrophil degranulation in complement-activating and non-complement-activating dialysis membranes, while activation of the prostaglandin or leukotriene cascade are not required.

Production of Cytokines in Hemodialysis

Tumor necrosis factor (TNF-alpha) and interleukin-1 (IL-1 beta) are cytokines primarily produced by monocytes/macrophages when stimulated by endotoxin, complement-derived anaphylatoxins and the specific antigen. In the present study, the plasma levels of TNF-alpha and IL-1 beta were evaluated before and after hemodialysis with cuprophane membrane (in 9 patients) and hemodiafiltration (in 9 patients) using three high-permeability membranes such as polymethylmethacrylate, polyacrylonitrile (AN-69) and polysulfone. In vitro spontaneous production of TNF-alpha and IL-1 beta was evaluated in the supernatants from short-term cultured monocytes obtained before and after treatment. The predialytic levels of TNF-alpha and IL-1 beta were significantly higher (p less than 0.05) in the uremic population than in 21 healthy subjects taken as controls. The analysis of the uremic population regarding the mode of therapy indicated that in hemodialysis the predialytic plasma levels of TNF-alpha and IL-1 beta did not significantly differ from those of healthy subjects. In contrast, in hemodiafiltration with polymethylmethacrylate and AN-69, but not with polysulfone, the predialytic plasma levels of both cytokines were significantly (p less than 0.05) increased. No significant variation in plasma levels of both cytokines was observed after hemodialysis with cuprophane membranes. Hemodiafiltration with polymethylmethacrylate and AN-69, but not with polysulfone, brought about a consistent reduction in plasma levels of both cytokines. Detectable amounts of TNF-alpha and IL-1 beta were spontaneously produced by peripheral-blood monocytes 6 h after the end of hemodialysis but not of hemodiafiltration. These studies suggest a possible role of TNF-alpha and IL-1 beta in the biocompatibility of different extracorporeal treatments.

Cardiac anaphylaxis. Complement activation as an amplification system.

Complement is activated, and C3a and C5a anaphylatoxins are generated during hypersensitivity reactions clinically associated with cardiopulmonary collapse. The administration of C3a or C5a to nonsensitized isolated guinea pig hearts mimics the events caused by antigen challenge of sensitized hearts (i.e., cardiac anaphylaxis) in the absence of complement. Thus, complement-derived anaphylatoxins may participate in immediate hypersensitivity reactions in which the heart is a target organ. To assess the contribution of complement activation and anaphylatoxin generation to cardiac dysfunction, we have elicited anaphylaxis in isolated guinea pig hearts in the presence of complement and found that the ensuing dysfunction is markedly enhanced. This amplification is most likely attributable to anaphylatoxin formation because 1) inactivation of C3 or selective C3 depletion, i.e., the loss of the component responsible for the formation of the anaphylatoxins C3a and C5a, prevents complement-induced exacerbation of cardiac anaphylaxis, whereas reconstitution with C3 and C5, or even only C3, restores it; in fact, the greater the C3 content at the time of antigen challenge, the more intense the anaphylactic crisis; and 2) the severity of cardiac anaphylaxis is markedly reduced by preexposure to C3a, and this reduction is directly related to the dose of C3a injected and to the amount of endogenous cardiac histamine depleted by C3a before antigen challenge. Complement-derived anaphylatoxins appear to promote the same mediator release that has been initiated by the antigen-antibody reaction; thus, complement activation functions as an amplification system in cardiac anaphylaxis.

Complement activation during systemic lupus erythematosus. C3a and C5a anaphylatoxins circulate during exacerbations of disease.

To determine whether activated complement components appear in the circulation of patients with systemic lupus erythematosus (SLE), we measured C5a and C3a by radioimmunoassay. Mean C5a concentration in the plasma of acutely ill SLE patients was 46.0 ng/ml, compared with 17.1 ng/ml in normal controls (P less than 0.01). Mean C3a concentration in patients with severe disease was 526 ng/ml, compared with 134 ng/ml in controls (P less than 0.01). In patients with moderately active SLE, the mean C3a concentration, but not the mean C5a concentration, was also elevated. In addition, C3a was elevated in 15 or 21 patients with active SLE, whereas low levels of C3 or C4 were noted in only 7 of these 21 patients. We conclude that the measurement of complement-derived anaphylatoxins may be useful in the management of patients with SLE. In addition, we suggest that these circulating mediators may contribute to the pathogenesis of vascular injury in patients with the disease.

A Single Scheme for C3a and C5a Isolation and Characterization of Chemotactic Behavior

Abstract A four-step procedure was utilized in the isolation of two complement-derived anaphylatoxins (C3a and C5a) from yeast cell activated human serum. Advantage was taken of one gel filtration and three ion exchange chromatographic steps to obtain two homogeneous products. The carboxypeptidase inhibitor epsilon-aminocaproic acid (EACA) was added to the serum to prevent inactivation of the anaphylatoxins during their generation. Purified C3a and C5a were judged to be homogeneous by sodium dodecyl sulfate polyacrylamide gel electrophoresis (PAGE), acid PAGE, and cellulose acetate strip electrophoresis. A single precipitin are was detected when each agent was challenged with the corresponding rabbit antibody after immunoelectrophoresis. Recovery of the respective anaphylatoxins from 1 liter of activated serum was 6 to 8 mg for C3a and 400 to 600 µg for C5a. A partial chemical characterization of the C5a revealed that arginine was released from the COOH-terminus by carboxypeptidase B digestion.