Native C5a Research Articles

Unexpected Off-Target Activities for Recombinant C5a in Human Macrophages

The anaphylatoxin C5a is core effector of complement activation. C5a exerts potent proinflammatory and immunomodulatory actions through interacting with its C5a receptors, C5aR1 and C5aR2, modulating multiple signaling and functional activities of immune cells. Native C5a contains a large N-linked glycosylation site at Asn64, which accounts for up to 25% of its m.w. To date, the vast majority of published studies examining C5a are performed using Escherichia coli-generated recombinant C5a, which is readily available from numerous commercial suppliers, but lacks this glycosylation moiety. However, a plasma-purified "native" form of C5a is also commercially available. The different size and glycosylation of these two C5a versions could have functional implications. Therefore, the current study aimed to compare recombinant human C5a to purified plasma-derived human C5a in driving the signaling and functional activities of human primary macrophages. We found that both versions of C5a displayed similar potencies at triggering C5aR1- and C5aR2-mediated cell signaling, but elicited distinct functional responses in primary human monocyte-derived macrophages. Multiple commercial sources of recombinant C5a, but not the plasma-purified or a synthetic C5a version, induced human monocyte-derived macrophages to produce IL-6 and IL-10 in a C5a receptor-independent manner, which was driven through Syk and NF-κB signaling and apparently not due to endotoxin contamination. Our results, therefore, offer caution against the sole use of recombinant human C5a, particularly in functional/cytokine assays conducted in human primary immune cells, and suggest studies using recombinant human C5a should be paired with C5aR1 inhibitors or purified/synthetic human C5a to confirm relevant findings.

Development of Synthetic Human and Mouse C5a: Application to Binding and Functional Assays In Vitro and In Vivo.

The complement activation peptide C5a is a key mediator of inflammation that is associated with numerous immune disorders. C5a binds and activates two seven-transmembrane receptors, C5aR1 and C5aR2. Experimentally, C5a is utilized to investigate C5a receptor biology and to screen for potential C5aR1/C5aR2 therapeutics. Currently, laboratory sources of C5a stem from either isolation of endogenous C5a from human serum or most predominantly via recombinant expression. An alternative approach to C5a production is chemical synthesis, which has several advantages, including the ability to introduce non-natural amino acids and site-specific modifications whilst also maintaining a lower probability of C5a being contaminated with microbial molecules or other endogenous proteins. Here, we describe the efficient synthesis of both human (hC5a) and mouse C5a (mC5a) without the need for ligation chemistry. We validate the synthetic peptides by comparing pERK1/2 signaling in CHO-hC5aR1 cells and primary human macrophages (for hC5a) and in RAW264.7 cells (for mC5a). C5aR2 activation was confirmed by measuring β-arrestin recruitment in C5aR2-transfected HEK293 cells. We also demonstrate the functionalization of synthetic C5a through the introduction of a lanthanide chelating cage to facilitate a screen for the binding of ligands to C5aR1. Finally, we verify that the synthetic ligands are functionally similar to recombinant or native C5a by assessing hC5a-induced neutrophil chemotaxis in vitro and mC5a-mediated neutrophil mobilization in vivo. We propose that the synthetic hC5a and mC5a described herein are valuable alternatives to recombinant or purified C5a for in vitro and in vivo applications and add to the growing complement reagent toolbox.

Decoy nanoparticles bearing native C5a receptors as a new approach to inhibit complement-mediated neutrophil activation

Complement-activated neutrophils are integrally involved in many pathological conditions as well as in dampening the efficacy of cell-based therapies. Mesenchymal stem cells (MSCs) hold promise for regenerative medicine and inflammatory disease therapy, but current MSC-based therapies still require further improvements to ensure success. We recently reported that immediately upon delivery to the bloodstream, MSCs activate complement to produce C5a, which binds to its receptor, C5aR, on neutrophils and thus activates these cells to damage MSCs. Thus, blocking this C5a-C5aR interaction should yield improvements in MSC survival and treatment efficacy. In this project, we developed decoy nanoparticles with surface displaying native C5aR by coating membrane vesicles derived from macrophages expressing high levels of C5aR onto poly(lactic-co-glycolic acid) (PLGA) cores. These C5aR-displaying decoy nanoparticles effectively inhibited neutrophil activation and thus reduced sequential injury to MSCs upon exposure to blood both in vitro and in vivo. Consequently, survival and treatment potency of the MSCs were significantly improved by these decoy nanoparticles. This finding suggests that the C5aR-displaying decoy nanoparticles represent a unique approach toward improving current MSC-based therapies. Additionally, these decoy nanoparticles can be useful as a new reagent for the treatment of other pathological conditions that involve C5a-C5aR signaling. Statement of SignificanceComplement C5aR has been implied in the pathogenesis of many disorders and is emerging as a new target for the development of therapeutics. So far all the inhibitors of C5aR are either biologicals or small compounds with various shortcomings. Since C5aR is a G-protein coupled receptor that features a multi-loop binding interface with its ligand, C5a, soluble forms of C5aR as decoys for cell surface C5aR are unlikely. We believe this is the first evidence suggesting that C5aR decoy nanoparticles can be developed to treat various C5aR-mediated pathological conditions.

Decoy nanoparticles bearing native C5a receptor as a new approach to inhibit complement-mediated neutrophil activation

Decoy nanoparticles bearing native C5a receptor as a new approach to inhibit complement-mediated neutrophil activation

Peptidyl Arginine Deiminase from Porphyromonas gingivalis Abolishes Anaphylatoxin C5a Activity

Evasion of killing by the complement system, a crucial part of innate immunity, is a key evolutionary strategy of many human pathogens. A major etiological agent of chronic periodontitis, the Gram-negative bacterium Porphyromonas gingivalis, produces a vast arsenal of virulence factors that compromise human defense mechanisms. One of these is peptidylarginine deiminase (PPAD), an enzyme unique to P. gingivalis among bacteria, which converts Arg residues in polypeptide chains into citrulline. Here, we report that PPAD citrullination of a critical C-terminal arginine of the anaphylatoxin C5a disabled the protein function. Treatment of C5a with PPAD in vitro resulted in decreased chemotaxis of human neutrophils and diminished calcium signaling in monocytic cell line U937 transfected with the C5a receptor (C5aR) and loaded with a fluorescent intracellular calcium probe: Fura-2 AM. Moreover, a low degree of citrullination of internal arginine residues by PPAD was also detected using mass spectrometry. Further, after treatment of C5 with outer membrane vesicles naturally shed by P. gingivalis, we observed generation of C5a totally citrullinated at the C-terminal Arg-74 residue (Arg74Cit). In stark contrast, only native C5a was detected after treatment with PPAD-null outer membrane vesicles. Our study suggests reduced antibacterial and proinflammatory capacity of citrullinated C5a, achieved via lower level of chemotactic potential of the modified molecule, and weaker cell activation. In the context of previous studies, which showed crosstalk between C5aR and Toll-like receptors, as well as enhanced arthritis development in mice infected with PPAD-expressing P. gingivalis, our findings support a crucial role of PPAD in the virulence of P. gingivalis.

Complement C5a acts as molecular adjuvant in fish by enhancing antibody response to soluble antigen

C5a, the most potent anaphylatoxin generated during complement activation, has important pro-inflammatory actions and has also been shown to enhance antigen-specific antibody response in mammals, thereby acting as a molecular adjuvant. In rainbow trout, C5a has been shown to have a chemoattractant ability and its receptor has also been found on potential APCs. In this study, we tested the possible role of trout C5a as a molecular adjuvant. We demonstrated the presence of native C5a in trout serum using the antibody generated by recombinant trout C5a, and then we generated recombinant infectious hematopoietic necrosis virus glycoprotein (G), and a G-C5a fusion protein to test the adjuvant activity of trout C5a. Recombinant G-C5a displayed a potent chemoattractant activity in contrast to G alone, indicating that the C5a portion of the fusion protein was functional. Thereafter, G-C5a, partially emulsified in a small quantity of IFA, was injected into one group of trout, while the other group of trout was inoculated with the same dose of recombinant G. At four to sixteen weeks post-injection, the serum IgM antibody levels of the fish injected with recombinant G-C5a were obviously higher than those injected with G protein alone. Thus, these results suggest, for the first time, that C5a acts as molecular adjuvant in teleost fish by enhancing antibody response to a soluble antigen.

HIV‐1 induced generation of C5a attracts immature dendriticcells and promotes infection of autologous T cells

For the recruitment of dendritic cells (DC) to the site of infection, DC express several sensors for danger signals, such as receptors for C5a. This anaphylatoxin is generated upon complement activation. As HIV-1 triggers the complement cascade, we determined whether C5a is generated by the virus and tested the functional activity of C5a in migration and infection assays. The immature (i)DC responded in migration assays to recombinant C5a and native C5a, which was generated in situ upon activation of the complement system by HIV-1. In combined migration and infection assays, a C5a-dependent enhancement of HIV-1 infection in DC-T cell cocultures was observed. These results indicate that HIV induces generation of C5a and thereby attracts iDC, which in turn promote the productive infection of autologous primary T cells.

Analysis of the C5a anaphylatoxin core domain using a C5a phage library selected on differentiated U937 cells

The human anaphylatoxin C5a is a 74-amino acid comprising polypeptide with a plethora of biological functions. Site directed mutagenesis studies suggest that several residues within the core and the C-terminus mediate the interaction with the C5a receptor. However, the contribution of particular core residues to receptor binding remained to be clarified. By means of the phage display technique, the loop between positions 35–40 was randomly mutated and the resulting C5a[35–40] fusion phage library affinity selected on C5a receptor expressing U937 cells. After five rounds of affinity enrichment, residues Arg37 and Arg40 were preferably selected. Enrichment was as high as 100% for Arg37 and 79% for Arg40. No significant enrichment of consensus residues could be obtained for positions 35, 36, 38 and 39. The core mutant C5a[A 35E 36R 37A 38S 39R 40], in which only Arg37/40 and Ala38 are of the native C5a sequence, was as potent as native C5a in both receptor binding and enzyme release examined on U937 cells. In contrast, replacement of Arg40 as in the mutant C5a[Q 35E 36R 37I 38L 39N 40] resulted in a 10-fold decrease in both binding and functional activities. Thus, selected out of a multiplicity of possibilities by the natural binding partner, Arg37 as well as Arg40 appear to be anchor residues in binding to the C5a receptor.

Regulation of TNF-α Receptor Expression on Human Neutrophils by Various Cell Activating Factors

Since tumor necrosis factor-α (TNF-α) has been implicated in a wide range of inflammatory states, we sought to examine the regulation of TNF-α receptors on human neutrophils (PMNs). We were particularly interested in the relationship of TNF-α binding characteristics to various known neutrophil-priming stimuli such as temperature, N -formylmethionyl-leucyl-phenylanine (fMLP), phorbol myristate acetate (PMA), C5a des Arg, and human recombinant C5a (HrC5a). Competition binding studies indicated that there was no significant change in receptor number or affinity due to the temperature used for neutrophil isolation. PMNs isolated at 4 or 25°C had a single class of receptors with a K d of 83.1 ± 3.3 vs 90.0 ±8.3 pmole/liter and a similar number of receptors, 1575 ± 99 vs 1876 ± 279 receptors/cell. However, when either group of isolated PMNs was further incubated in buffer at 37°C for 15 min, a significant increase in K d (4-37°C, 147.9 ± 23.7 pmole/liter; 25-37°C, 165.2 ± 29.2 pmole/liter) and a decrease in receptor number (4-37°C, 1116 ± 86 receptors/cell; 25-37°C, 957 ± 71 receptors/cell) were seen. To determine the effect of other activating substances such as fMLP, PMA, C5a des Arg, HrC5a, 4°C isolated PMNs were used. Pretreatment of PMNs (15 min at 37°C) by all of these agents down-regulated TNF-α receptors by decreasing receptor number and by decreasing receptor affinity. In the course of these studies, we identified a substantial disparity in the chemotactic deactivation and TNF-α receptor down-regulation of ED 50's for native C5a des Arg compared to recombinant C5a. These results would suggest that both loss of receptors and a change in affinity of the TNF-α receptor could be a means of modulating TNF-α response in neutrophils. We speculate that activation and release of the TNF-binding portion of the receptor from a proposed trimer receptor complex explains the affinity loss.

C5a, Cutaneous Mast Cells, and Inflammation: In Vitro and In Vivo Studies in a Murine Model

To evaluate further the interactions of C5a and mast cells in cutaneous inflammation, the ability of human native C5a (nC5a) (10 to 500 ng/ml) and human recombinant C5a (rC5a) (10 ng/ml to 100 ng/ml) to induce histamine release from purified BALB/c cutaneous mast cells (CMC) and peritoneal mast cells (PMC) was analyzed. It was found that nC5a induced histamine release from CMC but not from PMC, with a maximal net release at 250 ng/ml nC5a (22.8 +/- 2.6%). Kinetic experiments demonstrated that nC5a-induced maximal net histamine release occurred 5 min after the presentation of this stimulus (25.8 +/- 6.0%). Using rC5a and CMC, dose-response studies indicated a maximal net release of 7.0 +/- 1.7% at rC5a of 10 ng/ml, and kinetic studies showed a maximal net release at 5 min of incubation (12.9 +/- 1.6%). Release induced by rC5a was calcium-dependent, and peaked at 30 degrees C. These results indicate that functional heterogeneity exists between the CMC and the PMC of BALB/c mice, that C5a is a relevant stimulus for characterization of this heterogeneity, and that CMC from these animals can serve as a convenient in vitro model for the study of human C5a-mast cell interactions. In vivo, injections of nC5a (25-100 ng) and rC5a (25-100 ng) into the skin of BALB/c mice induced an increase in cutaneous vasopermeability, as assessed by the extravasation of intravenously injected 125I-bovine serum albumin. nC5a induced a dose-dependent increase in vasopermeability, whereas alterations induced by rC5a plateaued at 50 ng. The C5a-induced vasopermeability was markedly enhanced in animals that had been previously treated with an inhibitor of serum carboxypeptidase, which converts C5a to the less potent derivative, C5a des Arg. These findings suggest that carboxypeptidase plays an important role in vivo in the modulation of C5a-induced cutaneous inflammation in murine skin.

Identification of the C5a des Arg cochemotaxin. Homology with vitamin D-binding protein (group-specific component globulin).

The chemotactic activity of human C5a des Arg is enhanced significantly by an anionic polypeptide (cochemotaxin) in normal human serum and plasma. The cochemotaxin attaches to sialic acid residues within the oligosaccharide chain of native C5a des Arg to form a complex with potent chemotactic activity for human PMN. We investigated the nature of the cochemotaxin and found that vitamin D-binding protein is the putative cochemotaxin. Vitamin D-binding protein enhanced the chemotactic activity of native C5a des Arg, but had no effect on the chemotactic activity of either native C5a or FMLP. Sialic acid prevented both enhancement by vitamin D-binding protein of the chemotactic activity of native C5a des Arg and formation of C5a des Arg-vitamin D-binding protein complexes, detected by molecular sieve chromatography. Furthermore, vitamin D-binding protein and cochemotaxin exhibited identical molecular weights, isoelectric points, antigenic reactivity, and amino acid composition.

Attachment of human C5a des Arg to its cochemotaxin is required for maximum expression of chemotactic activity.

The chemotactic activity of human C5a des Arg is enhanced significantly by an anionic polypeptide (cochemotaxin) in normal human serum and plasma. We have found that the cochemotaxin attaches to the oligosaccharide chain of native C5a des Arg to form a complex with potent chemotactic activity for human polymorphonuclear leukocytes. Although capable of enhancing the chemotactic activity of native C5a des Arg, the cochemotaxin had no effect on the chemotactic activity of either deglycosylated C5a des Arg, native C5a, or N-formyl-methionyl-leucyl-phenylalanine. Of the known components of the oligosaccharide chain, only sialic acid prevented enhancement by the cochemotaxin of the chemotactic activity exhibited by native C5a des Arg. Sialic acid also prevented the formation of C5a des Arg-cochemotaxin complexes, detected by acid polyacrylamide gel electrophoresis, molecular sieve chromatography on polyacrylamide gels, and sucrose density gradient ultracentrifugation.

Mechanism of action of the group A streptococcal C5a inactivator.

Virulent group A streptococci have been found to express a cell-surface factor that has the capability of inactivating complement-derived chemotactic factors. To determine the mechanism of action of this factor, we examined the interaction of purified inactivator with pure C5a chemotaxin. Ligand-receptor binding studies demonstrated that streptococcal chemotactic factor inactivator (SCFI)-treated C5a expressed a greatly reduced ability to bind to receptors of polymorphonuclear leukocytes as compared with native C5a. The inactivation of C5a occurred by a nonstoichiometric and temperature-dependent process. NaDodSO4/PAGE analysis indicated that SCFI mediated a small decrease in the molecular weight of C5adesArg, and sequencing of the carboxyl terminus of inactivated C5a demonstrated that a six-residue peptide was lost. The release of discrete peptide fragments from denatured bovine serum albumin upon prolonged incubation with SCFI was indicative of endoprotease activity. Although denatured bovine serum albumin was inefficiently cleaved, native bovine serum albumin and other native proteins were highly resistant to SCFI proteolysis; this indicated that activity was specific in nature.

Structure and function of human C5a anaphylatoxin. Selective modification of tyrosine 23 alters biological activity but not antigenicity.

Reaction of either human C5a or its des-Arg74 derivative (des-Arg74-C5a) with tetranitromethane under nondenaturing conditions results in selective nitration of only 1 of the 2 tyrosine residues found in these glycopolypeptides. This reactive tyrosyl residue was identified as that found in position 23 of the sequence. Nitrotyrosyl23-C5a and -des-Arg74-C5a were separated from their respective unmodified precursors by cation-exchange fast protein liquid chromatography. These purified derivatives served as reagents for the subsequent preparation of both aminotyrosyl23-C5a and -des-Arg74-C5a as well as photoreactive analogs of C5a. Radioimmunoassays performed with C5a derivatives serving as competing ligands and a murine antihuman C5a monoclonal antibody employed as first antibody demonstrated that selective modification of tyrosine23 did not produce a detectible alteration in the antigenic properties of C5a. By contrast, either nitro- or aminotyrosyl23-C5a was approximately 3-fold less active than native C5a in both bioassays and competitive ligand-receptor binding assays. Additionally, photoreactive derivatives prepared by coupling either N-succinimidyl-6-(4'-azido-2'-nitrophenylamino)-hexanoate or p-nitrophenyl-2-diazo-3,3,3-trifluoropropionate to aminotyrosyl23-C5a at pH 5.0 failed to interact with the neutrophil C5a receptor. These observations suggest that the tyrosyl23 residue of C5a may participate importantly in the binding interactions of this chemotactic factor and its granulocyte receptor.