Expression of the anaphylatoxin receptors C3aR and C5aR is increased in fatal asthma

BACKGROUND: The mechanisms leading to death from asthma are not completely understood. Recent studies suggest the involvement of the anaphylatoxins C3a and C5a, generated during complement activation, and their receptors C3aR and C5aR in the pathogenesis of asthma. OBJECTIVE: The aim of our study was to investigate the expression of C3aR and C5aR in fatal asthma. METHODS: We analyzed lung tissue from 14 subjects who died of asthma (fatal asthma; FA) and 14 subjects who died of nonpulmonary causes (controls) and bronchial biopsy specimens from 16 subjects with mild intermittent asthma (MIA). C3aR and C5aR expression was evaluated by immunohistochemistry, and a semiquantitative analysis of the intensity of staining was performed according to a visual analogue scale (score, 0-3). RESULTS: C3aR was expressed on airway epithelium, smooth muscle, submucosal, and parenchymal vessels. C5aR was expressed on myeloid cells infiltrating the submucosa and on airway epithelium. Statistical analysis demonstrated higher expression of C3aR on submucosal vessels in FA compared with controls and MIA (median [minimum-maximum], controls, 0.24 [0-1.48]; MIA, 0.0 [0-1.00]; FA, 1.56 [0.13-3]; P = .002). C3aR was also increased on parenchymal vessels in FA (controls, 0.56 [0-2.00]; FA, 1.81 [0.5-3]; P = .0004). C5aR expression on airway epithelium was increased in FA compared with controls and MIA (controls, 1.25 [0.25-3]; MIA, 1.00 [0-2.00]; FA, 3.00 [1.13-3.00]; P = .001). CONCLUSION: The results of our study suggest a role of complement in FA.     

Ligand-induced phosphorylation of anaphylatoxin receptors C3aR and C5aR is mediated by "G protein-coupled receptor kinases.

Continuous stimulation of anaphylatoxin receptors C3aR and C5aR with their cognate ligands engenders, within minutes, diminished responsiveness of these receptors. We tested the hypothesis that agonist-induced desensitization involves C3aR and C5aR phosphorylation by G protein-coupled receptor kinases (GRK). When expressed in rat basophilic leukemia cells and exposed to C3a, the C3aR underwent rapid (t(1/2) approximately 15 s), dose-dependent (EC50 approximately 10 nM) and reversible phosphorylation by a kinase refractory to the effects of PKC inhibitors. Phosphoamino acid analysis revealed that the C3aR is phosphorylated on serine and threonine, but not on tyrosine residues. Overexpression of GRK2, GRK3, GRK5 or GRK6 together with C3aR in COS-7 cells enhanced the C3a-induced C3aR phosphorylation 1.5 - 1.9-fold (p < 0.05), but each kinase reduced ligand-stimulated phospholipase C activity differently. Conversely, antibody-mediated inhibition of endogenous GRK2 and GRK3 significantly inhibited C3aR phosphorylation in permeabilized cells. GRK overexpression in cells which co-expressed C5aR and were exposed to C5a resulted in the hyperphosphorylation of the C5aR. These findings are of physiological relevance, since we observed anaphylatoxin-induced phosphorylation of C3aR and C5aR endogenously expressed in human mast cells (HMC-1) which contain significant intracellular levels of GRK2 and GRK3.     

Role of histamine in the spasmogenic effect of the complement peptides C3a and C5a-desArg (classical anaphylatoxin)

The role of endogenous histamine in the spasmogenic effect of the complement-derived peptides C3a and C5a-desArg (isolated from yeast-activated hog serum) was studied in strips of terminal guinea-pig ileum. The effect of C3a is apparently histamine-independent; it neither induces histamine release from the test organ nor is its spasmogenic action inhibited by the H1-antihistaminics pheniramine and triprolidine, used in concentrations effective against histamine. However, endogenous histamine may be involved in the spasmogenic effect of C5a-desArg; when applied repeatedly C5a-desArg induces histamine release during its first and second application. Furthermore, both HI-antihistaminics inhibited C5a-desArg-induced contractions considerably, though less emciently than those of added histamine.     

Expression of complement C3, C5, C3aR and C5aR1 genes in resting and activated CD4+ T cells

Complement activation is traditionally thought to occur in the extracellular space. However, it has been suggested that complement proteins are activated and function at additional locations. T cells contain intracellular stores of C3 and C5 that can be cleaved into C3a and C5a and bind to intracellular receptors, which have been shown to be of vital importance for the differentiation and function of these cells. However, whether the origin of the complement proteins located within T cells is derived from endogenous produced complement or from an uptake dependent mechanism is unknown.The presence of intracellular C3 in T cells from normal donors was investigated by fluorescence microscopy and flow cytometry. Moreover, mRNA expression levels of several genes encoding for complement proteins with primary focus on C3, C3aR, C5 and C5aR1 during resting state and upon activation of CD4⁺ T cells were investigated by a quantitative PCR technique. Furthermore, the gene expression level was evaluated at different time points.We confirmed the presence of intracellular C3 protein in normal T-cells. However, we could not see any increase in mRNA levels using any activation strategy tested. On the contrary, we observed a slight increase in C3 and C5aR1 mRNA only in the non-activated T-cells compared to the activated T cells, and a decrease in the activated T-cells at different incubation time points.Our results show that there is a baseline intracellular expression of the complement C3, C5, C3aR and C5aR1 genes in normal CD4⁺ T cells, but that expression is not increased during T-cell activation, but rather down regulated. Thus, the pool of intracellular complement in CD4⁺ T cells may either be due to accumulated complement due low-grade expression or arise from the circulation from an uptake dependent mechanism, but these possibilities are not mutually exclusive.     

C5a anaphylatoxin as a product of complement activation up-regulates the complement inhibitory factor H in rat Kupffer cells

The 155-kDa complement regulator factor H (FH) is the predominant soluble regulatory protein of the complement system. It acts as a cofactor for the factor I-mediated conversion of the component C3b to iC3b, competes with factor B for a binding site on C3b and C3(H2O) and promotes the dissociation of the C3bBb complex. The primary site of synthesis is the liver, i.e. FH-specific mRNA and protein were identified in both hepatocytes (HC) and Kupffer cells (KC). Previous studies in rat primary HC and KC had shown that the proinflammatory cytokine IFN-gamma influences the balance between activation and inhibition of the complement system through up-regulation of the inhibitory FH. In this study we show that C5a, as a product of complement activation, stimulates the expression of FH-specific mRNA and protein in KC and thus induces a negative feedback. Quantitative-competitive RT-PCR showed an approximate threefold C5a-induced up-regulation of FH. ELISA analyses revealed a corresponding increase in FH protein in the supernatants of KC. The up-regulation of FH was completely inhibited by the C5a-blocking monoclonal antibody 6-9F. Furthermore, an involvement of LPS and IFN-gamma was excluded, which strongly indicates a direct effect of C5a on the expression of FH in KC.     

New concepts on the therapeutic control of complement anaphylatoxin receptors

The complement system is a pivotal driver of innate immunity, coordinating the host response to protect against pathogens. At the heart of the complement response lie the active fragments, C3a and C5a, acting through their specific receptors, C3aR, C5aR1, and C5aR2, to direct the cellular response to inflammation. Their potent function however, places them at risk of damaging the host, with aberrant C3a and C5a signaling activity linked to a wide range of disorders of inflammatory, autoimmune, and neurodegenerative etiologies. As such, the therapeutic control of these receptors represents an attractive drug target, though, the realization of this clinical potential remains limited. With the success of eculizumab, and the progression of a number of novel C5a-C5aR1 targeted drugs to phase II and III clinical trials, there is great promise for complement therapeutics in future clinical practice. In contrast, the toolbox of drugs available to modulate C3aR and C5aR2 signaling remains limited, however, the emergence of new selective ligands and molecular tools, and an increased understanding of the function of these receptors in disease, has highlighted their unique potential for clinical applications. This review provides an update on the growing arsenal of drugs now available to target C5, and C5a and C3a receptor signaling, and discusses their utility in both clinical and pre-clinical development.     

Plasma clearance of the human C5a anaphylatoxin by binding to leucocyte C5a receptors.

The C5a anaphylatoxin is a potent complement-derived mediator of inflammation with chemotactic activity. In this study the possible role of specific high-affinity binding sites for C5a on peripheral blood leucocytes for the removal of C5a from human blood plasma was investigated. The addition of purified granulocytes or mononuclear cells to complement-activated plasma resulted in the rapid and dose-dependent removal of up to 80% of plasma C5a, as determined by ELISA. The specific role of leucocyte C5a receptors (C5aR) in the plasma clearance of C5a was demonstrated by the inhibition of C5a uptake by the preincubation of cells with the C5aR-specific monoclonal antibody S5/1. Furthermore, U937 cells which had been induced by db-cAMP to express C5aR, but not undifferentiated U937 cells, were capable of removing C5a from plasma. The inhibition of C5aR internalization by monensin did not affect C5a uptake by leucocytes. The co-incubation with leucocytes had no effect on the plasma clearance of complement activation products C3a or terminal complement complex (TCC), as determined by this in vitro assay. The binding of the C5a anaphylatoxin to cellular receptors represents an effective control mechanism that protects the organism from systemic effects of this potent phlogistic mediator.     

Mycobacteria-primed macrophages and dendritic cells induce an up-regulation of complement C5a anaphylatoxin receptor (CD88) in CD3+ murine T cells

Complement C5a anaphylatoxin is a potent activator of macrophages, neutrophils, and dendritic cells (DC) and binds the C5a receptor (C5a-R; CD88). Although C5a is chemotactic for T cells, expression of C5a-R on murine T cells has been disputed. We report here that na?ve, Con A-activated, and cytokine (IL-12, IL-18)-stimulated murine CD3+ T cells from three strains of mice [C57Bl/6, B10.nSn (C5+/+), B10.on (C5-/-)] lacked C5a-R, as evaluated by immunophenotyping with an anti-C5a-R mAb. Ligation of CD3 induced a modest up-regulation with 3% of CD3+ T cells expressing cell surface C5a-R. T cells primed by APC differentiate into effector T cells. Activation of mycobacteria [bacillus Calmette-Guerin (BCG)]-sensitized T cells through MHC II and TCR interactions via BCG-infected macrophages enhanced the expression of C5a-R with approximately 14% of CD3+ T cells positive for C5a-R. Comparable expression was found in C5+/+ as well as C5-/- strains of mice (14% and 15%, respectively). Furthermore, anti-CD3-activated T cells were primed by BCG-infected DC, and a larger proportion of the primed T cells expressed C5a-R (30-40%). Finally, mice infected with BCG showed significant numbers of CD3+ T cells expressing C5a-R in the spleens during infection. As APC, such as macrophages and DC, can secrete C5 and cleave C5 to C5a and C5b through a peptidase, we suggest that macrophage and DC-T cell interactions can up-regulate C5a-R on T cells through MHC II-TCR and provide a C5a peptide for additional local activation of T cells via C5a-R.     

A key role of C5a/C5aR activation for the development of sepsis

In recent studies, evidence has been provided for complement activation early during the onset of experimental sepsis. Excessive production of the anaphylatoxin C5a thereby appears to elicit various harmful effects. Blockade of C5a or C5a receptor (C5aR) at the start of experimental sepsis has been demonstrated to greatly improve survival in rodents. There is evidence that C5a, during the onset of sepsis, enhances the production of various proinflammatory mediators in different cell types. Besides its known, other proinflammatory effects, recent work suggested an inhibitory role of C5a for innate-immune functions of phagocytic cells (phagocytosis, reactive oxygen species production, chemotaxis) during experimental sepsis. This review article provides an overview of the important role of C5a/C5aR activation for the onset and development of sepsis.     

Functional analysis and quantification of the complement C3 derived anaphylatoxin C3a with a monoclonal antibody.

The C3 fragment C3a belongs to the anaphylatoxins. It has immune regulatory activity and contributes to the pathogenesis of the adult respiratory distress syndrome (ARDS). The low molecular weight (9 kD) of C3a complicates the production of antibodies to C3a. We obtained a monoclonal antibody (designated H13) to human C3a. It reacts with C3a or C3a-desArg and with native C3 but not with C5 or C5a. In immunoblot analysis it reacts with the alpha- but not with beta-chain of C3 and binds to a protein with a mol. wt of about 10 kD present in zymosan-activated sera which is only marginally detectable in nonactivated serum and absent in plasma. H13 crossreacts with the analogous proteins of rabbit, guinea pig and sheep. H13 has the capacity to bind 125I-radiolabelled C3a efficiently but fails totally to react with 125I-C5a or with other C3 alpha-chain fragments. H13 blocks C3a functional activity. It markedly inhibits C3a-induced 3H-serotonin release from platelets in vitro and similarly inhibits the C3a-induced extravasation of Evans blue into the skin in vivo. H13 does not interfere with the haemolytic activity of C3. An ELISA system was established using H13 which permits quantification of C3a in sera of polytrauma patients. The antibody H13 should facilitate further functional analysis of C3a in experimental systems. It should be useful for quantification of C3a in diagnostic assays and also for application in immunopathology.     

Cloning and characterization of the guinea pig C5a anaphylatoxin receptor: interspecies diversity among the C5a receptors

The anaphylatoxin C5a receptor (C5aR, CD88 in man) plays a prominent role in mediating inflammatory and host defense processes. Direct evidence of C5aR involvement in host defense mechanisms was demonstrated recently using C5aR knockout mice. Mice deficient in C5aR were unable to clear intrapulmonary-instilled bacteria. The guinea pig system is perhaps unique for exhibiting cross-reactivity with human complement components and its high sensitivity to anaphylatoxins. Therefore, we cloned the guinea pig C5aR from a megakaryocyte cDNA library. The deduced amino acid sequence of guinea pig C5aR is 67% identical to human, 61.6% to dog, 60.2% to mouse and 63.6% to rat C5aR. Transient expression of guinea pig C5aR in COS-7 cells and stable expression on L cell fibroblasts were confirmed by FACS analysis. Competitive binding studies using [125I]C5a and stimulation of calcium mobilization by C5a proved that functional C5aR was expressed on these stably transfected L cells. The N-terminal extracellular region of guinea pig C5aR was five to seven residues shorter than the same region in C5aR from other species and sequence homology was limited to 11%. Other outer membrane loops were also poorly conserved (8-33%) when compared across five species. Transmembrane segments were highly conserved between these various species (46-86%). Guinea pig C5aR binds human C5a, therefore residues critical for C5a binding have been conserved between these species. Sequence comparison of C5aR from multiple species permits conserved elements of the ligand binding sites to be elucidated.      

Enhanced expression of human monocyte complement (C3b) receptors by chemoattractants.

The capacity of various leucoattractants to enhance, or unfold, receptors for complement (C3b) on human blood monocytes has been studied. A number of recognized monocyte chemoattractants including casein, supernatants from C. parvum 10390 and from human lymphocytes (cultured either in the presence or absence of phytohaemagglutinin) and the formyl-methionyl peptides, F-Met-Leu-Phe, F-Met-Met-Phe and F-Met-Phe, increased the percentage of monocytes which formed rosettes with IgM-sensitized sheep erythrocytes coated with complement. Comparable results were achieved irrespective of whether purified components of whole serum was used as a source of complement. In contrast, there was no significant increase in EAG (Fc) rosettes with those doses of casein which gave enhancement of C3b receptors. A small degree of complement receptor enhancement was observed with histamine but the unformylated peptides, Met-Leu-Phe and Met-Met-Phe, were without apparent effect. Maximal receptor enhancement was obtained at 30 min but when the leucoattractant was removed, enhancement was reversible, returning to normal values in approximately 120 min. Monocyte complement receptor enhancement increased with temperatures between 0 degrees C and 37 degrees C. These data (1) confirm and extend our previous findings on leucoattractant-induced enhancement of complement receptors on human monocytes; (2) indicate that the phenomenon may have potential as a clinical test for monocyte function both in health and disease.     

Electrophoresis of guinea-pig complement components (C3, C5, C6, C7, C8 and C9) on polyacrylamide gel

The electrophoretic mobilities of C3, C5, C6, C7, C8 and C9 of guinea-pig complement were studied using polyacrylamide (PAA) gel as a supporting medium. The haemolytic activity of each complement component was readily revealed as a discrete haemolytic band in blood agar containing intermediate red blood cells and appropriate reagents which was overlayed on the PAA gel plate after electrophoresis. By this method, the relative mobilities of C3, C5, C6, C7, C8 and C9 were determined as 0.19, 0.40, 0.17, 0.27, 0.31 and 0.57 respectively. An additional, distinct, haemolytic C5 band was detected when native guinea-pig serum was run. The relative mobility of the haemolytic activity which was not detected when purified C5 was electrophoresed, was 0.25.