C5a regulates NKT and NK cell functions in sepsis

Complement, NKT, and NK cells play critical roles in the first line defense against pathogens. Functional roles for both C5a receptors, that is, complement receptor C5a (C5aR) and C5a receptor-like 2 (C5L2), in sepsis have been demonstrated. However, the role of C5a in innate lymphocyte activation during sepsis remains elusive. In this article, we show that naive NKT and NK cells already express high levels of C5aR and minor levels of C5L2 mRNA, but no protein. Upon Escherichia coli-induced sepsis, we found C5aR surface expression on subpopulations of NKT and NK cells, suggesting rapid translation into C5aR protein on bacterial encounter. Importantly, significantly increased survival in the absence of C5aR, NKT, and NK cells, but not of C5L2, was associated with reduced IFN-γ and TNF-α serum levels. Sepsis induction in C5aR(+)/C5aR(-) mixed bone marrow chimeras identified cognate engagement of C5aR on NKT cells as an important factor for the recruitment of NKT cells. Furthermore, we found synergistic interaction between C5aR and TLRs enhancing the production of TNF-α and IFN-γ from NKT and NK cells in cocultures with dendritic cells. Our results identify C5aR activation as a novel pathway driving detrimental effects of NKT and NK cells during early experimental sepsis.     

Complement-induced impairment of innate immunity during sepsis

This study defines the molecular basis for defects in innate immunity involving neutrophils during cecal ligation/puncture (CLP)-induced sepsis in rats. Blood neutrophils from CLP rats demonstrated defective phagocytosis and defective assembly of NADPH oxidase, the latter being due to the inability of p47(phox) to translocate from the cytosol to the cell membrane of neutrophils after cell stimulation by phorbol ester (PMA). The appearance of these defects was prevented by in vivo blockade of C5a in CLP rats. In vitro exposure of neutrophils to C5a led to reduced surface expression of C5aR and defective assembly of NADPH oxidase, as defined by failure in phosphorylation of p47(phox) and its translocation to the cell membrane, together with failure in phosphorylation of p42/p44 mitogen-activated protein kinases. These data identify a molecular basis for defective innate immunity involving neutrophils during sepsis.     

Protective effects of IL-6 blockade in sepsis are linked to reduced C5a receptor expression

IL-6 is known to be an important pro- and anti-inflammatory cytokine, which is up-regulated during sepsis. Our previous work has suggested a role for IL-6 in the up-regulation of C5aR in sepsis. We reported earlier that interception of C5a or C5aR results in improved outcomes in experimental sepsis. Using the cecal ligation/puncture (CLP) model in mice, we now demonstrate that treatment with anti-IL-6 Ab (anti-IL-6) results in significantly improved survival, dependent on the amount of Ab infused. CLP animals showed significantly increased binding of 125I-labeled anti-C5aR to organs when compared to either control mice at 0 h or CLP animals infused with normal rabbit 125I-labeled IgG. Binding of 125I-labeled anti-C5aR to lung, liver, kidney, and heart was significantly decreased in anti-IL-6-treated animals 6 h after CLP. RT-PCR experiments with mRNA isolated from various organs obtained 3, 6, and 12 h after CLP demonstrated increased C5aR mRNA expression during the onset of sepsis, which was greatly suppressed in CLP mice treated with anti-IL-6. These data suggest that IL-6 plays an important role in the increased expression of C5aR in lung, liver, kidney, and heart during the development of sepsis in mice and that interception of IL-6 leads to reduced expression of C5aR and improved survival.     

Human C5aR knock-in mice facilitate the production and assessment of anti-inflammatory monoclonal antibodies

Complement component C5a binds C5a receptor (C5aR) and facilitates leukocyte chemotaxis and release of inflammatory mediators. We used neutrophils from human C5aR knock-in mice, in which the mouse C5aR coding region was replaced with that of human C5aR, to immunize wild-type mice and to generate high-affinity antagonist monoclonal antibodies (mAbs) to human C5aR. These mAbs blocked neutrophil migration to C5a in vitro and, at low doses, both prevented and reversed inflammatory arthritis in the murine K/BxN model. Of approximately 40 mAbs generated to C5aR, all potent inhibitors recognized a small region of the second extracellular loop that seems to be critical for regulation of receptor activity. Human C5aR knock-in mice not only facilitated production of high-affinity mAbs against an important human therapeutic target but were also useful in preclinical validation of the potency of these antagonists. This strategy should be applicable to other important mAb therapeutics.     

Pharmacological targeting of anaphylatoxin receptors during the effector phase of allergic asthma suppresses airway hyperresponsiveness and airway inflammation

Airway hyperresponsiveness and airway inflammation are hallmarks of allergic asthma, the etiology of which is crucially linked to the presence of Th2 cytokines. A role for the complement anaphylatoxins C3a and C5a in allergic asthma was suggested, as deficiencies of the C3a receptor (C3aR) and of complement factor C5 modulate airway hyperresponsiveness, airway inflammation, and Th2 cytokine levels. However, such models do not allow differentiation of effects on the sensitization phase and the effector phase of the allergic response, respectively. In this study, we determined the role of the anaphylatoxins on the effector phase of asthma by pharmacological targeting of the anaphylatoxin receptors. C3aR and C5a receptor (C5aR) signaling was blocked using the nonpeptidic C3aR antagonist SB290157 and the neutralizing C5aR mAb 20/70 in a murine model of Aspergillus fumigatus extract induced pulmonary allergy. Airway hyperresponsiveness was substantially improved after C5aR blockade but not after C3aR blockade. Airway inflammation was significantly reduced in mice treated with the C3aR antagonist or the anti-C5aR mAb, as demonstrated by reduced numbers of neutrophils and eosinophils in bronchoalveolar lavage fluid. Of note, C5aR but not C3aR inhibition reduced lymphocyte numbers in bronchoalveolar lavage fluid. Cytokine levels of IL-5 and IL-13 in bronchoalveolar lavage fluid were not altered by C3aR or C5aR blockade. However, blockade of both anaphylatoxin receptors markedly reduced IL-4 levels. These data suggest an important and exclusive role for C5aR signaling on the development of airway hyperresponsiveness during pulmonary allergen challenge, whereas both anaphylatoxins contribute to airway inflammation and IL-4 production.     

The phosphatidylinositol 3-kinase signaling pathway exerts protective effects during sepsis by controlling C5a-mediated activation of innate immune functions

The PI3K/Akt signaling pathway has been recently suggested to have controversial functions in models of acute and chronic inflammation. Our group and others have reported previously that the complement split product C5a alters neutrophil innate immunity and cell signaling during the onset of sepsis and is involved in PI3K activation. We report in this study that in vivo inhibition of the PI3K pathway resulted in increased mortality in septic mice accompanied by strongly elevated serum levels of TNF-alpha, IL-6, MCP-1, and IL-10 during sepsis as well as decreased oxidative burst activity in blood phagocytes. PI3K inhibition in vitro resulted in significant increases in TLR-4-mediated generation of various proinflammatory cytokines in neutrophils, whereas the opposite effect was observed in PBMC. Oxidative burst and phagocytosis activity was significantly attenuated in both neutrophils and monocytes when PI3K activation was blocked. In addition, PI3K inhibition resulted in strongly elevated TLR-4-mediated generation of IL-1beta and IL-8 in neutrophils when these cells were co-stimulated with C5a. C5a-induced priming effects on neutrophil and monocyte oxidative burst activity as well as C5a-induced phagocytosis in neutrophils were strongly reduced when PI3K activation was blocked. Our data suggest that the PI3K/Akt signaling pathway controls various C5a-mediated effects on neutrophil and monocyte innate immunity and exerts an overall protective effect during experimental sepsis.     

Role of C5a in multiorgan failure during sepsis

In humans with sepsis, the onset of multiorgan failure (MOF), especially involving liver, lungs, and kidneys, is a well known complication that is associated with a high mortality rate. Our previous studies with the cecal ligation/puncture (CLP) model of sepsis in rats have revealed a C5a-induced defect in the respiratory burst of neutrophils. In the current CLP studies, MOF occurred during the first 48 h with development of liver dysfunction and pulmonary dysfunction (falling arterial partial pressure of O(2), rising partial pressure of CO(2)). In this model an early respiratory alkalosis developed, followed by a metabolic acidosis with increased levels of blood lactate. During these events, blood neutrophils lost their chemotactic responsiveness both to C5a and to the bacterial chemotaxin, fMLP. Neutrophil dysfunction was associated with virtually complete loss in binding of C5a, but binding of fMLP remained normal. If CLP animals were treated with anti-C5a, indicators of MOF and lactate acidosis were greatly attenuated. Under the same conditions, C5a binding to blood neutrophils remained intact; in tandem, in vitro chemotactic responses to C5a and fMLP were retained. These data suggest that, in the CLP model of sepsis, treatment with anti-C5a prevents development of MOF and the accompanying onset of blood neutrophil dysfunction. This may explain the protective effects of anti-C5a in the CLP model of sepsis.     

Different endocytosis pathways of the C5a receptor and the N-formyl peptide receptor

Two chemoattractant receptors, C5aR (the complement fragment C5a receptor) and FPR (the N-formyl peptide receptor), are involved in neutrophil activation at sites of inflammation. In this study, we found major differences in the intracellular trafficking of the receptors in transfected Chinese hamster ovary (CHO) cells. Western blot analysis showed that FPR was stable during a 3 h stimulation with ligand, but C5aR was reduced in quantity by 50%. Not all C5aR was targeted directly for degradation however; a small, but visible fraction of the receptor became re-phosphorylated upon subsequent addition of ligand, suggesting that some of the receptor had cycled to the cell surface. Light membrane fractions isolated from activated cells showed C5aR distribution at the bottom of a glycerol gradient, colocalizing with the main distribution of the late endosomal/lysosomal marker LAMP2, whereas FPR was found at the bottom of the gradient as well as in the middle of the gradient, where it cofractionated with the early/sorting endosomal marker Rab5. Using fluorescence microscopy, we observed ligand-dependent redistribution of C5aR-EGFP from the plasma membrane to LAMP2-positive compartments, whereas FPR-EGFP showed significant colocalization with the early/sorting endosomes. Analysis of endogenous C5aR and FPR in neutrophils revealed a pattern similar to the CHO transfectants: C5aR underwent degradation after prolonged ligand stimulation, while FPR did not. Finally, we confirmed the down-regulation of C5aR in a functional assay by showing reduced chemotaxis toward C5a in both CHO transfectants and neutrophils after preincubation with C5a. A similar decrease in FPR-mediated chemotaxis was not observed.     

Apoptotic neutrophils release macrophage migration inhibitory factor upon stimulation with tumor necrosis factor-alpha

Macrophage migration inhibitory factor (MIF) is an important cytokine involved in the regulation of innate immunity and present at increased levels during inflammatory responses. Here we demonstrate that mature blood and tissue neutrophils constitutively express MIF as a cytosolic protein not associated with azurophil granules. Functionally active MIF, but not proteases stored in azurophil granules, was released from apoptotic neutrophils following short term tumor necrosis factor (TNF)-alpha stimulation in a caspase-dependent manner and prior to any detectable phagocytosis by monocyte-derived macrophages. Moreover, TNF-alpha-mediated MIF release was blocked by glyburide and propenicide, both inhibitors of ATP-binding cassette-type transporters, suggesting that this transporter system is activated during neutrophil apoptosis. Taken together, apoptotic mature neutrophils release MIF upon short term TNF-alpha stimulation. Therefore, apoptosis may not always occur without the induction of pro-inflammatory mechanisms.     

Paclitaxel binds and activates C5aR1: A new potential therapeutic target for the prevention of chemotherapy-induced peripheral neuropathy and hypersensitivity reactions

Chemotherapy-induced peripheral neuropathy (CIPN) and hypersensitivity reactions (HSRs) are among the most frequent and impairing side effects of the antineoplastic agent paclitaxel. Here, we demonstrated that paclitaxel can bind and activate complement component 5a receptor 1 (C5aR1) and that this binding is crucial in the etiology of paclitaxel-induced CIPN and anaphylaxis. Starting from our previous data demonstrating the role of interleukin (IL)-8 in paclitaxel-induced neuronal toxicity, we searched for proteins that activate IL-8 expression and, by using the Exscalate platform for molecular docking simulations, we predicted the high affinity of C5aR1 with paclitaxel. By in vitro studies, we confirmed the specific and competitive nature of the C5aR1-paclitaxel binding and found that it triggers intracellularly the NFkB/P38 pathway and c-Fos. In F11 neuronal cells and rat dorsal root ganglia, C5aR1 inhibition protected from paclitaxel-induced neuropathological effects, while in paclitaxel-treated mice, the absence (knock-out mice) or the inhibition of C5aR1 significantly ameliorated CIPN symptoms—in terms of cold and mechanical allodynia—and reduced the chronic pathological state in the paw. Finally, we found that C5aR1 inhibition can counteract paclitaxel-induced anaphylactic cytokine release in macrophages in vitro, as well as the onset of HSRs in mice. Altogether these data identified C5aR1 as a key mediator and a new potential pharmacological target for the prevention and treatment of CIPN and HSRs induced by paclitaxel.Subject terms: Pathogenesis, Immunopathogenesis     

Protective effects of C5a blockade in sepsis.

Sepsis in humans is a difficult condition to treat and is often associated with a high mortality rate. In this study, we induced sepsis in rats using cecal ligation and puncture (CLP). In rats depleted of the complement factor C3, CLP led to very short survival times (about 4 days). Of the rats that underwent CLP ('CLP rats') that were C3-intact and treated with preimmune IgG, most (92%) were dead by 7 days. Blood neutrophils from these rats contained on their surfaces the powerful complement activation product C5a. This group had high levels of bacteremia, and their blood neutrophils when stimulated in vitro had greatly reduced production of H2O2, which is known to be essential for the bactericidal function of neutrophils. In contrast, when companion CLP rats were treated with IgG antibody against C5a, survival rates were significantly improved, levels of bacteremia were considerably reduced, and the H2O2 response of blood neutrophils was preserved. Bacterial colony-forming units in spleen and liver were very high in CLP rats treated with preimmune IgG and very low in CLP rats treated with IgG antibody against C5a, similar to values obtained in rats that underwent 'sham' operations (without CLP). These data indicate that sepsis causes an excessive production of C5a, which compromises the bactericidal function of neutrophils. Thus, C5a may be a useful target for the treatment of sepsis.     

The complement anaphylatoxin C5a induces apoptosis in adrenomedullary cells during experimental sepsis

Sepsis remains a poorly understood, enigmatic disease. One of the cascades crucially involved in its pathogenesis is the complement system. Especially the anaphylatoxin C5a has been shown to have numerous harmful effects during sepsis. We have investigated the impact of high levels of C5a on the adrenal medulla following cecal ligation and puncture (CLP)-induced sepsis in rats as well as the role of C5a on catecholamine production from pheochromocytoma-derived PC12 cells. There was significant apoptosis of adrenal medulla cells in rats 24 hrs after CLP, as assessed by the TUNEL technique. These effects could be reversed by dual-blockade of the C5a receptors, C5aR and C5L2. When rats were subjected to CLP, levels of C5a and norepinephrine were found to be antipodal as a function of time. PC12 cell production of norepinephrine and dopamine was significantly blunted following exposure to recombinant rat C5a in a time-dependent and dose-dependent manner. This impaired production could be related to C5a-induced initiation of apoptosis as defined by binding of Annexin V and Propidium Iodine to PC12 cells. Collectively, we describe a C5a-dependent induction of apoptotic events in cells of adrenal medulla in vivo and pheochromocytoma PC12 cells in vitro. These data suggest that experimental sepsis induces apoptosis of adrenomedullary cells, which are responsible for the bulk of endogenous catecholamines. Septic shock may be linked to these events. Since blockade of both C5a receptors virtually abolished adrenomedullary apoptosis in vivo, C5aR and C5L2 become promising targets with implications on future complement-blocking strategies in the clinical setting of sepsis.     

Changes in the novel orphan, C5a receptor (C5L2), during experimental sepsis and sepsis in humans

Sepsis is associated with extensive complement activation, compromising innate immune defenses, especially in neutrophils (PMN). Recently, a second C5a receptor (C5L2) was detected on PMN without evidence of intracellular signaling. The current study was designed to determine changes in C5L2 in blood PMN during sepsis. In vitro exposure of PMN to C5a, but not to fMLP, led to reduced content of C5L2. Following cecal ligation and puncture-induced sepsis in rats, PMN demonstrated a time-dependent decrease in C5L2. In vivo blockade of C5a during experimental sepsis resulted in preservation of C5L2. Similarly, PMN from patients with progressive sepsis showed significantly reduced C5L2 expression (n = 26), which was virtually abolished in patients who developed multiorgan failure (n = 10). In contrast, sepsis survivors exhibited retention of C5L2 (n = 12/13). The data suggest that C5L2 on PMN diminishes during sepsis due to systemic generation of C5a, which is associated with a poor prognosis.     

Complement factor C5a mediates renal ischemia-reperfusion injury independent from neutrophils

The complement system has been shown to mediate renal ischemia-reperfusion (I/R) injury. However, the contribution of complement factor C5a to I/R injury, in particular in the kidney, remains to be established. In this study, we investigated the impact of blocking the C5aR pathway on the inflammatory response and on the renal function in a murine model of I/R injury. First, we analyzed C5aR expression in kidneys of healthy mice. Intriguingly, we found expression on mesangial, as well as on tubular epithelial, cells. After I/R injury, C5aR expression was up-regulated in tubular epithelial cells. In addition, mRNA levels of CXC chemokines and TNF-alpha increased significantly and kidneys were heavily infiltrated by neutrophils. Blocking the C5aR pathway by a specific C5a receptor antagonist (C5aRA) abrogated up-regulation of CXC chemokines but not of TNF-alpha and reduced neutrophil infiltration by >50%. Moreover, application of the C5aRA significantly reduced loss of renal function. This improvement of function was independent of the presence of neutrophils because neutrophil depletion by mAb NIMP-R14 did not affect the protective effect of C5aRA treatment. Furthermore, blocking of the C5aR pathway had no influence on renal apoptosis. These data provide evidence that C5a is crucially involved in the pathogenesis of renal I/R injury by modulation of neutrophil-dependent as well as neutrophil-independent pathways, which include the regulation of CXC chemokines but not TNF-alpha or apoptotic pathways.     

ISO-1 binding to the tautomerase active site of MIF inhibits its pro-inflammatory activity and increases survival in severe sepsis

MIF is a proinflammatory cytokine that has been implicated in the pathogenesis of sepsis, arthritis, and other inflammatory diseases. Antibodies against MIF are effective in experimental models of inflammation, and there is interest in strategies to inhibit its deleterious cytokine activities. Here we identify a mechanism of inhibiting MIF pro-inflammatory activities by targeting MIF tautomerase activity. We designed small molecules to inhibit this tautomerase activity; a lead molecule, "ISO-1 ((S,R)-3-(4-hydroxyphenyl)-4,5-dihydro-5-isoxazole acetic acid methyl ester)," significantly inhibits the cytokine activity in vitro. Moreover, ISO-1 inhibits tumor necrosis factor release from macrophages isolated from LPStreated wild type mice but has no effect on cytokine release from MIFdeficient macrophages. The therapeutic importance of the MIF inhibition by ISO-1 is demonstrated by the significant protection from sepsis, induced by cecal ligation and puncture in a clinically relevant time frame. These results identify ISO-1 as the first small molecule inhibitor of MIF proinflammatory activities with therapeutic implications and indicate the potential of the MIF active site as a novel target for therapeutic interventions in human sepsis.     

An anti-inflammatory function for the complement anaphylatoxin C5a-binding protein, C5L2

C5L2 is an enigmatic serpentine receptor that is co-expressed with the C5a receptor on many cells including polymorphonuclear neutrophils. The apparent absence of coupling of C5L2 with G proteins suggests that this receptor may modulate the biological activity of C5a, perhaps by acting as a decoy receptor. Alternatively, C5L2 may affect C5a function through formation of a heteromeric complex with the C5aR, or it may utilize a G protein-independent signaling pathway. Here we show that in mice bearing a targeted deletion of C5L2, the biological activity of C5a/C5a(desArg) is enhanced both in vivo and in vitro. The biological role of C5L2 thus appears to be limiting to the pro-inflammatory response to the anaphylatoxin. Accordingly, up-regulation of C5L2 may be of benefit in inflammatory states driven by C5a, including sepsis, asthma, cystic fibrosis, and chronic obstructive lung disease.     

Chemotaxis inhibitory protein of Staphylococcus aureus binds specifically to the C5a and formylated peptide receptor

Chemotaxis inhibitory protein of Staphylococcus aureus (CHIPS) is an exoprotein produced by several strains of S. aureus, and a potent inhibitor of neutrophil and monocyte chemotaxis toward C5a and formylated peptides like fMLP. These chemoattractants act on their target cells by binding and activating the C5aR and formylated peptide receptor (FPR), respectively. In the present report, we examined the mechanism by which CHIPS affects both of these receptors. We showed that CHIPS blocked binding of anti-C5aR mAb and formylated peptide to human neutrophils as efficiently at temperatures of 0 and 37 degrees C, implying that it is independent of signal transducing systems. This was confirmed by showing that CHIPS acts completely independently of ATP. Additionally, CHIPS was not internalized upon binding to neutrophils. Furthermore, we showed that CHIPS binds specifically to the C5aR and FPR expressed on U937 cells. This binding was functional in blocking C5a- and fMLP-induced calcium mobilization in these cell lines. These results suggest that CHIPS binds directly to the C5aR and FPR, thereby preventing the natural ligands from activating these receptors. The apparent K(d) values of CHIPS for the C5aR and FPR were 1.1 +/- 0.2 nM and 35.4 +/- 7.7 nM, respectively. Moreover, after screening a wide variety of other G protein-coupled receptors, CHIPS was found to affect exclusively the C5aR and FPR. This selectivity and high-affinity binding with potent antagonistic effects makes CHIPS a promising lead for the development of new anti-inflammatory compounds for diseases in which damage by neutrophils plays a key role.     

Receptors for complement C5a. The importance of C5aR and the enigmatic role of C5L2

Complement component C5a is one of the most potent inflammatory chemoattractants and has been implicated in the pathogenesis of numerous inflammatory diseases. C5a binds two receptors, C5aR and C5L2. Most of the C5a functional effects occur through C5aR, and the pharmaceutical industry has focused on this receptor for the development of new anti-inflammatory therapies. We used a novel approach to generate and test therapeutics that target C5aR. We created human C5aR knock-in mice, and used neutrophils from these to immunize wild-type mice. This yielded high-affinity blocking mAbs to human C5aR. We tested these anti-human C5aR mAbs in mouse models of inflammation, using the human C5aR knock-in mice. These antibodies completely prevented disease onset and were also able to reverse established disease in the K/B x N arthritis model. The physiological role of the other C5a receptor, C5L2 is still unclear, and our studies with blocking mAbs to human C5L2 have failed to demonstrate a clear functional role in signaling to C5a. The development of effective mAbs to human C5aR is an alternative approach to drug development, for this highly attractive target.     

Expression of the complement anaphylatoxin C3a and C5a receptors on bronchial epithelial and smooth muscle cells in models of sepsis and asthma

The presence of the complement-derived anaphylatoxin peptides, C3a and C5a, in the lung can induce respiratory distress characterized by contraction of the smooth muscle walls in bronchioles and pulmonary arteries and aggregation of platelets and leukocytes in pulmonary vessels. C3a and C5a mediate these effects by binding to their specific receptors, C3aR and C5aR, respectively. The cells that express these receptors in the lung have not been thoroughly investigated, nor has their expression been examined during inflammation. Accordingly, C3aR and C5aR expression in normal human and murine lung was determined in this study by immunohistochemistry and in situ hybridization. In addition, the expression of these receptors was delineated in mice subjected to LPS- and OVA-induced models of inflammation. Under noninflamed conditions, C3aR and C5aR protein and mRNA were expressed by bronchial epithelial and smooth muscle cells of both human and mouse lung. C3aR expression increased significantly on both bronchial epithelial and smooth muscle cells in mice treated with LPS; however, in the OVA-challenged animals only the bronchial smooth muscle cells showed increased C3aR expression. C5aR expression also increased significantly on bronchial epithelial cells in mice treated with LPS, but was not elevated in either cell type in the OVA-challenged mice. These results demonstrate the expression of C3aR and C5aR by cells endogenous to the lung, and, given the participation of bronchial epithelial and smooth muscle cells in the pathology of diseases such as sepsis and asthma, the data suggest a role for these receptors during lung inflammation.