Signaling of the Complement Cleavage Product Anaphylatoxin C5a Through C5aR (CD88) Contributes to Pharmacological Hematopoietic Stem Cell Mobilization.

Several mechanisms have been postulated for orchestrating the mobilization of hematopoietic stem/progenitor cells (HSPCs), and we previously proposed that activation of the complement cascade plays a crucial role in the initiation and execution of the egress of HSPCs from bone marrow (BM) into peripheral blood (PB). In support of this notion, we demonstrated that mice deficient in the mannan-binding lectin (MBL) pathway, which activates the proximal part of the complement cascade, as well as mice deficient in the fifth component of the complement cascade (C5), which is part of the distal part of the complement cascade, are poor mobilizers. To further narrow down on the exact mechanisms and the molecules involved, we performed studies in mice that do not express the receptor C5aR, which binds the C5 cleavage fragments, C5a and C5adesArg. We also employed the plasma stable nucleic acid aptamer AON-D21 that binds and neutralizes C5a and C5adesArg. We present evidence that mice deficient in C5aR or treated with AON-D21 are poor HSPC mobilizers, thereby establishing a critical role for the C5a/C5adesArg-C5aR axis in the mobilization process. While enhancing mobilization is of clinical importance for poor mobilizers, inhibition of the complement cascade could be of therapeutic importance in patients suffering from paroxysmal nocturnal hemoglobinuria (PNH) or acquired hemolytic syndrome (aHUS).

Combination of a TLR4 ligand and anaphylatoxin C5a for the induction of antigen-specific cytotoxic T cell responses.

The complement system and Toll-like receptors (TLR) are key innate defense systems which might interact synergistically on dendritic cells (DC) to reinforce adaptive immunity. In a previous work, we found that the extra domain A from fibronectin EDA (an endogenous ligand for TLR4) can favour antigen delivery to DC and induce their maturation. Given the potential of anaphylatoxins to cause inflammation and activation of myeloid cells, we hypothesized that a fusion protein between EDA, and anaphylatoxins C3a, C4a or C5a together with an antigen might improve the immunogenicity of the antigen. Naked DNA immunization with a construct expressing the fusion protein between C5a, EDA and the cytotoxic T cell epitope SIINFEKL from ovalbumin, induced strong antigen specific T cell responses. The purified recombinant fusion protein EDA-SIINFEKL-C5a induced activation of dendritic cells, the production of proinflammatory cytokines/chemokines and stimulated antigen presenting cell migration and NK cell activation. As compared to EDA-SIINFEKL, the fusion protein EDA-SIINFEKL-C5a did not induce the production of the immunosuppressive molecules IL-10, CCL17, CCL1, CXCL12 or XCL1 by DC. Moreover, EDA-SIINFEKL-C5a induced strong specific T cell responses in vivo and protected mice against E.G7-OVA tumor growth more efficiently than EDA-SIINFEKL or SIINFEKL-C5a recombinant proteins. Our results suggest that fusion proteins containing EDA, the anaphylatoxin C5a and the antigen may serve as a suitable strategy for the development of anti-tumor or anti-viral vaccines.

Preservation of antimicrobial properties of complement peptide C3a, from invertebrates to humans.

The human anaphylatoxin peptide C3a, generated during complement activation, exerts antimicrobial effects. Phylogenetic analysis, sequence analyses, and structural modeling studies paired with antimicrobial assays of peptides from known C3a sequences showed that, in particular in vertebrate C3a, crucial structural determinants governing antimicrobial activity have been conserved during the evolution of C3a. Thus, regions of the ancient C3a from Carcinoscorpius rotundicauda as well as corresponding parts of human C3a exhibited helical structures upon binding to bacterial lipopolysaccharide permeabilized liposomes and were antimicrobial against gram-negative and gram-positive bacteria. Human C3a and C4a (but not C5a) were antimicrobial, in concert with the separate evolutionary development of the chemotactic C5a. Thus, the results demonstrate that, notwithstanding a significant sequence variation, functional and structural constraints imposed on C3a during evolution have preserved critical properties governing antimicrobial activity.

Complement anaphylatoxin C3a is selectively protective against NMDA-induced neuronal cell death.

The anaphylatoxin C3a is a potent inflammatory polypeptide released at sites of complement activation. To test whether C3a might alter neuronal outcome following an ischemic insult, we determined the effects of purified human C3a on murine primary cortical cell cultures exposed to apoptotic or excitotoxic paradigms. C3a prevented neither serum deprivation-induced apoptotic neuronal death, nor AMPA/kainate-mediated excitotoxicity. However, in mixed cultures of neurons and astrocytes, C3a dose-dependently protected neurons against NMDA toxicity (47% neuroprotection using 100 nM C3a, p < 0.01, n = 12). The neuroprotective effect of C3a was observable only in the presence of astrocytes. These observations suggest that C3a is involved in excitotoxicity-mediated neuronal death through astrocyte stimulation and extend its role beyond immune functions.

Tryptophan mutants of human C5a anaphylatoxin: a fluorescence anisotropy decay and energy transfer study.

Three mutants of the anaphylatoxin C5a were prepared with positions 2, 64 and 70, respectively, substituted by tryptophan. The last mutant was additionally labelled at Cys27 for fluorescence energy transfer (FET) measurements. The structural integrity and biological activity of the molecules were not affected. Fluorescence anisotropy decay (FAD) measurements showed that the rotational correlation time for tryptophan decreases in the order: [Trp2]rhC5a > [Trp64]rhC5a > [Trp70]rhC5a, indicating an increasing mobility of the side chain. Measurements of the fluorescence energy transfer from Trp70 to the 1,5-AEDANS group at Cys27 yielded a distance distribution of 2.4 +/- 0.8 nm. This value is compatible with the C-terminal chain being arranged as a slightly stretched helix pointing away from the body of the molecule.

A recombinant hybrid anaphylatoxin with dual C3a/C5a activity.

By site-directed mutagenesis of a human complement factor C5a cDNA clone, we have designed a hybrid anaphylatoxin in which three amino acid residues in the C-terminal sequence of human C5a were exchanged to create the native C-terminal human C3a (hC3a) sequence Leu-Gly-Leu-Ala-Arg. This hybrid anaphylatoxin rC5a-(1-69)-LGLAR exhibited true C3a and C5a activity when tested in the guinea pig ileum contraction assay. Quantitative measurements of ATP release from guinea pig platelets revealed about 1% intrinsic C3a activity for this hybrid, while the C5a activity was essentially unchanged. Competitive binding assays confirmed that the rC5a-(1-69)-LGLAR mutant was able to displace radioiodinated rhC5a with a KI of approx. 40 nM and hC3a with a KI of approx. 3.7 microM from guinea pig platelets. Since the C-termini of both human C3a and C5a anaphylatoxins are known to interact with their respective receptors, we conclude that the same peptidic sequence, LGLAR, is able to bind to and activate two different receptors, the C3a receptor as well as the C5a receptor. This clone provides a novel tool for the identification of further receptor-binding residues in both anaphylatoxins, since any mutants may be tested for altered C3a and C5a activity simultaneously.

Expression cloning of a receptor for C5a anaphylatoxin on differentiated HL-60 cells.

A cDNA clone encoding the human C5a anaphylatoxin receptor has been isolated by expression cloning from a CDM8 expression library prepared from mRNA of human myeloid HL-60 cells differentiated to the granulocyte phenotype with dibutyryladenosine cyclic monophosphate. The cDNA clone was able to transfer to COS-7 cells the capacity to specifically bind iodinated human recombinant C5a. The cDNA was 2.3 kb long, with an open reading frame encoding a 350-residue polypeptide. Cross-linking of iodinated C5a to the plasma membrane of transfected COS cells revealed a complex with an apparent molecular mass of 52-55 kDa, similar to that observed for the constitutively expressed receptor in differentiated HL-60 cells or human neutrophils. Although differentiated HL-60 cells display a single class of binding sites, with a dissociation constant of approximately 800-900 pM, the C5a-R cDNA, expressed in COS cells, generates both high-affinity (1.7 nM) and low-affinity (20-25 nM) receptors. Sequence comparison established that the degree of sequence identity between the C5a receptor and the N-formylpeptide receptor is 34%.

Unequal expression of complement C4A and C4B genes in rheumatoid synovial cells, human monocytoid and hepatoma-derived cell lines.

C4A and C4B are closely related homologous complement proteins encoded in the class III region of major histocompatibility complex (MHC). The regulation of their expression is under genetic and hormonal control. In this study we investigated the synovial fluid plasma ratio of C4A and C4B of rheumatoid (RA) and osteoarthritis (OA) patients, and a predominance of the C4B gene expression by the synovial macrophages of RA patients was demonstrated. To clarify the tissue specificity of the expression of C4A and C4B genes, human monocytoid cell line U937 and hepatoma-derived HepG2 cells were studied. The gene expression of C4A and C4B were markedly different in these cells since a relative predominance of C4B mRNA in U937 cells and excess of that of C4A in HepG2 cells were detected. Recombinant interferon-gamma (IFN-gamma) up-regulated the expression of C4A gene in both cells, but had apparently no effect on the C4B gene. Our results demonstrate dissimilar expression patterns for the two human C4 genes, suggesting different tissue specific regulation of human C4A and C4B.

Gene synthesis by serial cloning of oligonucleotides.

A rapid and simple method of gene synthesis is presented. A gene is constructed by serial additions of individual gene fragments in the 5'----3' direction. The vector used as the synthesis vehicle contains a unique Bsm I site at the amino terminus of the lacZ gene. Plasmid linearized with Bsm I is recircularized in vivo by oligonucleotide-directed double-strand break repair. The synthetic oligonucleotide used to "bridge" the double-strand break carries a 70- to 100-nucleotide insert which constitutes a portion of the gene along with a BsmI site at the 3' end that regenerates the site and allows for another consecutive round of mutagenesis to extend the gene sequence. The process is repeated until the entire gene is assembled. The method uses the beta-galactosidase color assay as a means of screening for correct insert lengths. The only in vitro enzymatic step necessary is a single Bsm I restriction digest of plasmid DNA. No ligation reactions are required. Only one strand of a gene sequence needs to be synthesized chemically. The gene synthesis method presented here was used to construct the human anaphylatoxin complement factor C3a gene.

Isolation and sequence analysis of a cDNA clone encoding the fifth complement component.

We have used available protein sequence data for the anaphylatoxin (C5a) portion of the fifth component of human complement (residues 19-25) to synthesize a mixed-sequence oligonucleotide probe. The labeled oligonucleotide was then used to screen a human liver cDNA library, and a single candidate cDNA clone of 1.85 kilobase pairs was isolated. Hybridization of the mixed-sequence probe to the complementary strand of the plasmid insert and sequence analysis by the dideoxy method predicted the expected protein sequence of C5a (positions 1-12), amino-terminal to the anticipated priming site. The sequence obtained further predicted an arginine-rich sequence (RPRR) immediately upstream of the N-terminal threonine of C5a, indicating that the promolecule form of C5 is synthesized with a beta alpha-chain orientation as previously shown for pro-C3 and pro-C4. The C5 cDNA clone was sheared randomly by sonication, subcloned into M13 mp8, and sequenced at random by the dideoxy technique, thereby generating a contiguous sequence of 1703 base pairs. This clone contained coding sequence for the C-terminal 262 amino acid residues of the beta-chain, the entire C5a fragment, and the N-terminal 98 residues of the alpha'-chain. The 3' end of the clone had a polyadenylated tail preceded by a polyadenylation recognition site, a 3'-untranslated region, and base pairs homologous to the human Alu concensus sequence. Comparison of the derived partial human C5 protein sequence with that previously determined for murine C3 and human alpha 2-macroglobulin has indicated regions of pronounced sequence similarity. Examination of cytoplasmic RNA prepared from human liver and the human hepatoma cell line Hep G2 by Northern transfer has indicated a C5 mRNA species of about 5.2 kilobase pairs.