Guinea pigs intravenously injected with homologous serum samples previously treated with preformed immune complexes develop symptoms and tissue lesions indistinguishable from the anaphylactic shock, as persistent fall in the systemic blood pressure, constriction with obliteration of the bronchioles, and distention of alveoli [1]. The generated activity was coined anaphylatoxin to design a supposed “toxin anaphylactic”. Aliquots of serum recovered from guinea pigs intravenously injected with preformed immune complexes and suffering the anaphylactic-like symptoms when added onto isolated guinea pig ileum induced contractions, in a dose-dependent fashion, followed by tachyphylaxis after several additions of equal amounts of the treated serum. This in vitro smooth muscle contracting-activity induced by the immune complex treated serum resembles the typical anaphylactic Schultz-Dale reaction. Similar activity was also generated when serum was treated with complex polysaccharides as agar, inulin or dextran  [2-4] . The main anaphylactic symptoms induced in guinea pigs by immune complexes treated serum could be reproduced by intravenous injecting guinea pigs with histamine [5]. Histamine release from isolated guinea lungs obtained from antigen-sensitized animals upon perfusion with the antigen reinforced the hypothesis that histamine could be one of the anaphylactic shock mediators  [1,6-8] . The hypothesis that the complement system (CS) could be involved in anaphylatoxin formation was submitted to experimental approach by treating fresh serum with immune aggregates and assaying in parallel the emergence of a smooth muscle anaphylatoxin-like contraction and loss in hemolytic of the serum  [9] . However, more rigorous definition of the involvement of the CS in anaphylatoxin formation awaited purification of the different CS components and how they interplay along the activation pathways.

The CS comprises four sets of components: pro-enzymes/enzymes, stabilizers, aggregating proteins, and regulatory proteins. Pro-enzymes/enzymes: C1 complex (C1q-Ca⁺⁺-C1r₂-C1s₂), C2, C4, MASPs, factor B (fB), factor D (fD), C3. Stabilizers: properdin (P). Aggregating factors: MBL, C5, C6, C7, C8, C9. Regulatory proteins: C1-INH, H, C4Bp, DAF (CD55), MCP (CD46), CR1 (CD35), CD59, S protein, clusterin and vitronectin. This system is activated by three routes, the alternative (AP), the classical (CP) and the lectin (LP) pathways, either of which leading to formation of a cell membrane attack complex and the generation of opsonins and anaphylatoxins. Anaphylatoxins derives by enzymatic cleavage of, C3, C4 or C5 components. The “Complement. Nomenclature” was discussed and approved by a WHO sponsored committee of specialist  [10] .

C3. The native C3 molecule is a protein formed by a larger α-chain (115 kDa) and a smaller β-chain (75 kDa) held together by inter-chain disulfide bounds (Figure 1(a)). C3 derives from a pre-pro C3 native molecule after removal of the 22 N-terminus amino acid corresponding to the signal peptide and formation of the thioester bound by an intra-molecular transacylation involving the thiol group of Cys-998 and γ-amide of Gln-991 and removal

of the tetrapeptide R-R-R-R) [11] .

The protected thiol group can be exposed by hydrolysis at the slow rates of 0.005% of the C3 molecules/ minute resulting in the inactive C3 (H₂O) molecule, or by enzymatic cleavage of the peptide bound between the residues Arg-726 and Ser-727 of the C3 α-chain, resulting the free peptide C3a with 77 amino acid residues (9 kDa), and the C3b formed by the remaining α’ chain (106 kDa) attached by disulphide to intact β-chain (75 kDa). In both situations chemical energy is released after the thioester breakdown. If particles exhibiting OH⁻ or NH₂⁺ groups are sufficiently nearby ester or amide covalent bounds are constructed between C3b and the surface particles.

C4. The native C4 is a heterotrimer of 210 kDa formed by the polypeptides α, ß and γ chains held together by inter-chains disulfide bonds (Figure 1(b)). There are two isoforms of C4, C4A and C4B. This component shares with C3 the presence of the internal thioester bound also at the N-terminus of its α-chain.Activated C1 cleaves C4 molecule at the α-chain with removal of a 77-residue fragment (8.6 kDa) the C4a anaphylatoxin consisting of N-terminus of the α-chain, and C4b fragment consisting of the α-chain exposing the thioester bound and the entire ß and γ chains. C4A isoform whose thioester preferentially forms amide bonds links with surface proteins, while the isoform B whose thioester bound forms ester, attaches on carbohydrates.

C5. This component is synthesized as a single polypeptide chain (1658 amino acid residues) and posttranslationally processed resulting in two polypeptides, α (12.4 kDa) and ß (7.6 kDa) chains linked by disulfide bounds (Figure 1(c)). The resulting native protein (190 kDa) is both, the source of anaphylatoxin C5a and C5b the anchoring fragment for C6 during organization of the lytic complex MAC (Membrane Attach Complex).

The complement system is activated by alternative (AP), classical (CP) and lectin (LP) pathways (Figure 2).

AP. The AP involves the components C3, factor B (fB), factor D (fD) an active plasma protease and properdin (P) a stabilizer protein. Spontaneous reactive C3^* is created at a rate of 0.005% - 1% of total C3 per hour generating hydrolyzed C3(H₂O). Along the short hydrolysis process chemical energy is being released from the disrupted thioester bound. This C3^* is capable of binding to both the cell targets and to fluid phase native fB. Once associated to C3^* fB changes its conformation becoming susceptible to cleavage by fD into Ba an Bb. Ba is released into fluid phase and Bb remains associated to C3^*, forming the complex C3^*Bb. The exposed Bb intrinsic serine protease domain cleaves additional C3 molecules, generating C3b molecules. In a manner similar to C3^* new generated C3b interacts with the cell targets surface and with fB, forming the molecular complex C3bBb. C3^*Bb, the initiation C3 convertase, has a short life while C3bBb, upon stabilized by associating with P, resulting the trimolecular complex, C3bBbP, termed amplification C3 convertase. This enzyme is quite stable (over 10 min of life) and is responsible for saturate the target cell surface with covalently bound C3b and release C3a into the fluid phase (Figure 2).  

CP. This pathway is triggered by preformed immune complexes, C-reactive protein (CRP) and by some bacteria and virus-capsular associated components. The activation involves the complement components, C1, C4 and C2. The trigger step initiates by interactions of the activator molecules with C1q. Binding distorts strategic C1q molecules domains resulting successively in C1r, next C1s activations into esterases. C1s esterase cleaves C4 and C2. C4 is cleaved into two fragments, C4b and C4a. C4b binds covalently to hydroxyl or amino groups on cellular membranes. C2, after binding into C4b is also cleaved into C2a and C2b.The fragment C2b remains associated with C4b, resulting the molecular complex, C4b2b, the C3 convertase of the CP that cleaves C3 also in C3b and C3a. Some CP generated C3b fragments can bindfB as in AP activation generating C3bBb. AP is therefore an important “amplification loop” for the CP (Figure 2).

LP. The LP activation is initiated upon recognition of pathogen-associated mannose and N-acetyl glucosamine oligosaccharides by a serum protein, the mannose-binding lectin (MBL) [12] . The next steps following MBL binding to pathogens associated carbohydrates is followed by activation of MBL-associated serine proteases (MASPs). Activated MASPs, similarly to C1s, act on C4 and C2, assembling the C4bC2b complex essentially as in CP activation [13,14] (Figure 2). The C3 convertases, C3bBbP and C4bC2a, upon aggregating an additional C3b fragment shifts their specificity from C3 to C5, transforming into the C5 convertases C3b, Bb, P, C3b and C4b, C2a, C3b.

Lyticpathway. This pathway comprises the components C5, C6, C7. C8 and C9.These proteins are constituted by TSR, LDLR, perforin, EGF, and 2-CCPR associated in different proportions. The C5 convertases cleave C5 into C5a and C5b. C5a anaphylatoxin is released into the fluid phase. C5b expresses transient binding sites for the target cell membrane and for C6. Upon binding C6 acquires the ability to binding C7. If the complex C5b, C6, C7 is formed C8 is attached, binds C9 and promotes its polymerization. The complex C5b, C6, C7, C8 and C9 (3 - 4 molecules), dimerizes forming a macromolecule complex (around 2000 kDa) which is inserted into the cell membrane resulting in permanent pore lesions of around 100Ǻ leading to the cell lysis.

The CS includes also regulatory components aiming amplify, coordinate and confine the main steps along its activation.

C1-inhibitor. C1-INH was described as a heat labile factor in human serum that inhibited the esterolytic activity of the first complement component [15]. This protein is a member of the serpin family of protease inhibittors. C1-INH differs from other members of the terpin family in that it contains a long N-terminal domain of approximately 100 amino acid residues that is heavily glycosilated with both Nand O-linked sugars.

Factor I. fI is a serum protein (35 μg/mL), formed by two chains with molecular mass of 50 and 38 kDa and contains one serine protease domain. fI cleaves: a) C4b into iC4b and in the presence of C4-binding protein (C4bBP) with the participation of complement receptor type 1 (CR1); b) C3b into iC3b with the participation of also of CR1, factor H and membrane-cofactor protein (MCP). The protein super-family formed by CR1, factor H, Cr2, C4BPA, C4bPB, MCP and DAF are modular molecules consisting of only one type of domain, the “complement control protein repeats” (CCPRs). CCPRs are present in varying numbers in the different factors as indicated in parentheses.

CR1 (30 CCPRS). Is a cell bound protein formed by two polypeptide chains, molecular mass of 190 and 280 kDa. Serves as co-factor for Fi, participates in the clearance of immune complexes and in the phagocytosis of C3b opsonized particles.

Factor H (20 CCPRS). fH is a 150 kDa circulating protein (±480 µg/mL). Inactivates the C3 and C5 convertases by dissociating the molecular subunits, and serves as co-factor for fI.

C4bBP (3 to 8 CCPRS).C4bBP is a circulating protein (±250µg/mL), molecular mass 570 kDa.

MCP or Membrane Co-factor Protein (5 CCPRS). MCP is a two chains cell molecular bound protein, 51 - 68 kDa. Participates in inactivation of C3b and C4b.

DAF or Decay Accelerating Factor (4 CCPRS). DAF is a cell-bound protein, molecular mass 70 kDa capable of inactivate the C3 convertases C4bC2a in CC4b e C2a and C3bBb in C3b and Bb by accelerating the normal decay of these enzymes.

Along the CS cascade activation the emerging enzymes, MBL-MASP-2, C1 esterase, C3(H₂O)Bb, C3bBbP, C4bC2a, C3bBbPC3b and C4bC2aC3b, are specifically involved in anaphylatoxin generation. MBL-MASP-2 and C1 esterase, release C4a from C4; C3(H₂O)Bb, C3bBbP and C4bC2a release C3a from C3; while C3bBbPC3b and C4bC2aC3b release C5a from C5. Anaphylatoxin-like peptides can also be generated as side-products of direct proteolysis of C3, C4 and C5 components by tissues or circulating generated proteolytic enzymes.

C3a. Indication that CS could be anaphylatoxin source was the verification that purified human C1 esterase, the first complement enzyme purified to homogeneity, had the ability to increase vascular permeability in postcapillaryvenules in guinea pig skin [16]. This effect was a property of the active enzyme and was mediated by release of a histamine-like substance. In further experiments it was found that small concentrations of C1 esterase, sub-threshold for direct action in guinea pig skin, were capable of generating a permeability factor when mixed with normal guinea pig serum in vitro. The generation of this factor required heat-labile substances in serum and was correlated with consumption of hemolytic complement. The permeability factor itself was relatively heat-stable and its action in guinea pig skin was blocked by prior treatment of animals with triprolidine hydrochloride, an antihistamine drug. These observations were consistent with the hypothesis that interaction of purified C1 esterase and normal serum results in production of an anaphylatoxin like activity  [17] . This hypothesis were further reinforced in in vitro experiments by using purified human C1 esterase as activator, normal guinea pig or rat serum as the permeability source, and guinea pig ileum or rat uterus preparations as the pharmacological assays. Human C1 esterase treated but not untreated serum exhibited the following properties: a) contraction of guinea pig ileum which was made unresponsive after a few additions of the same amount of treated serum; b) failure to contract guinea pig ileum previously desensitized to agarose-anaphylatoxin or treated with antihistaminic drug; c) degranulation of mast cells and release of histamine from guinea pig tissues; d) failure to contract rat uterus. These findings indicated that the activity liberated by C1 esterase was related to anaphylatoxin. Generation was prevented by prior inactivation of C1 esterase with DFP or partially purified inhibitor of C1 esterase, prior heating of the serum at 56˚C, the presence of salicylaldoxime or phlorizin, or by selective chelating of Mg⁺⁺. A correlation between the formation of anaphylatoxin and complete consumption of hemolytic complement was also observed. The obtained dated data strongly suggested that C1 esterase generates anaphylatoxin by interacting with the subsequent complement components [18]. The Figures 3(a) and (b) show the pharmacological assay of the anaphylatoxin generated on rat serum by human purified C1 esterase.

At beginning of 1960 years the introduction of new techniques in protein chemistry allowed starting purification of some admitted complement proteins, as serum inhibitor of C1 esterase  [19] , enzyme C1 esterase  [20] , next C2 [21], C4  [22] , and C3 and C5  [23] .

Complete mixtures containing purified preparations of human C1 esterase, C4, C2 and C3, in VBS⁺⁺ buffer, pH 7.5 plus 1.0 mM MgCl₂ or incomplete mixtures lacking each onecomponent, were used as substitutes for whole serum. After incubation for 60 min, at 37˚C, aliquots were removed, and the presence of biologically active substances was assayed in vitro on isolated guinea pig ileum or rat uterus isolated preparations, and on guinea pig mesentery mast cells, or in vivo on guinea pig skin. The molecular integrity of C3 was evaluated by immunoelectrophoresis. The complete mixture, but not the incompletes exhibited: a) the ability to induce guinea pig ileum contraction followed by tachyphylaxis after two consecutive additions of the some dose (Figure 3(c)); b) it was fully blocked by the antihistaminic drug triprolidine performed in parallel assays using HA, histamine and bradikainin as agonists (Figure 3(c)); c) failed to contract rat uterus; d) induces mast cell degranulation and histamine release, and cleaves C3 (Figure 3(d)). It was concluded that anaphylatoxin is a product of the complement system  [24-26] .

In order to demonstrate the molecular origin and nature of the biologically active product, purified preparations of human C1 esterase, C4, C2, C3, and C5 were labeled with ¹²⁵I. Reaction mixtures were prepared containing a single labeled component and the other four components unlabeled. After incubation at 37˚C for 10 min at pH 7.4 in the presence of 5 × 10^–4 Mg⁺⁺, the pH was adjusted to 3.5 and subjected to sucrose density gradient ultracentrifugation and gel filtration at pH 3.5. In all cases the activity capable of contracting guinea pig ileum with tachyphylaxis was obtained in low molecular weight fractions. In addition, these fractions were labeled only when ¹²⁵I-C3 was employed, indicating that the biological activity was associated with a cleavage product of C3 (Figure 4). This C3 fragment had a molecular weight of 6800 and exhibited all biological properties attributed to anaphylatoxin as the capacity to contract guinea pig ileum with tachyphylaxis, failure to contract rat uterus,

enhancing the vascular permeability in guinea pig skin, degranulating mast cells in guinea pig mesentery, and release histamine from isolated rat mast cells. This C3 fragment did not cross-desensitize with guinea pig or rat agar anaphylatoxin and vice versa.

This result, coupled with immunoelectrophoretic conversion of C3 only in the complete reaction mixture, was highly suggestive of a mechanism involving cleavage of C3 to a biologically active fragment. Importantly, no active labeled fragment was detected in the mixtures in which ¹²⁵I-C5, instead of ¹²⁵I-C3 was used. Figure 4(a) shows these results. Generation of anaphylatoxin in HA mixture was accompanied by C3 cleavage into two fragmentsone moving faster band the other slowly (Figure 4(b)). The complete mixture HA induces skin inflammatory reaction in the injected tissue as indicated by the dilatation of small vessels, intercellular edema and afflux of neutrophils and eosinophils.

This C3 fragment was originally designed at that time F(a)C’3, latter C3a according to the WHO sponsored committee of specialist [27].

Similar conclusions were reached by [28] and by [29]. Similar results were also obtained when functional CS components were used as substitutes for the corresponding chemically isolated components. Using such preparations it was observed that the fifth component of complement that reacts hemolytically in the guinea pig or rat complement system, C’3b, is intimately associated with the generation of anaphylatoxin. Anaphylatoxin was formed 1) if this purified component reacted with a washed, intermediate complex consisting of antigen, antibody, and the first four components of complement; 2) if the component was treated with trypsin; or 3) if the component was incubated with a material isolated from

cobra venom together with a fraction from serum other than complement. The significance of this fifth component as anaphylatoxinogen is discussed and a common mechanism is proposed for the generation of anaphylatoxin in whole serum [30] . Amino acid sequence of the original human C3a peptide: CNYITELRRQHARASHLGLA.

C4a. The C4 cleavage product C4a exhibits similar function compared to C3a regarding smooth muscle contraction and enhancement of vascular permeability [31]. C4a release was demonstrated by treating purified human C4 with purified C1 esterase. The released peptide appeared to be homogeneous by electrophoresis on cellulose acetate and by polyacrylamide gel electrophoresis. Generated C4a is an 8.65 kDa peptide with an electrophoretic mobility at pH 8.6 of +2.1. One mol of Arg/mol of the generated C4a can be released by carboxypeptidase B. The partial COOH-terminal sequence was determined to be Leu-Gln-Arg-COOH. The isolated C4a was spasmogenic for guinea pig ileum at a concentration of 1 μM and it induced tachyphylaxis with respect C3a anaphylatoxin (at 0.33 μM) but not with respect to human C5a anaphylatoxin. These data suggested similar cell receptors for C3a and C4a, but distinct for C5a. The injection of 1 nmol of C4a induced immediate local erythema and edema formation. The spasmogenic, tachyphylactic, and vascular activities of C4a were abrogated by removal of the COOH-terminal Arg, a property that is characteristic also of the C3a and C5a anaphylatoxins [31].

C5a. Human C5a was next isolated from complementactivated serum and was characterized for protein and carbohydrate content, and compared with C3a. The purified C5a was judged to be homogeneous by both polyacrylamide gel electrophoresis and immunologic techniques. The polypeptide moiety of C5a contains 73 amino acid residues, which represent a molecular mass of 8.2 kDa. Analysis of the carbohydrate moiety in C5a indicated 4 moles of glucosamine, 3 to 4 moles of sialic acid, 4 moles of mannose and 2 moles of galactose. The total carbohydrate content in C5a corresponds approximately 25% of the apparent molecular mass of the C5a anaphylatoxin molecule. The protein and carbohydrate portions of C5a together equal a molecular mass of approximately 11 kDa which is considerably less than the indicated by physical measurements. Human C5a contains a COOH-terminal-Arg, which is essential for anaphylatoxin activity and a sequence of Gln-Leu-Gly-ArgCOOH at the COOH-terminus comparable with that of human C3a (Gly-Leu-Ala-Arg-COOH). Additional similarities between the C3a and C5a molecules include length of the polypeptide chain, number of disulfide bonds and an absence of Try residues. The differences amongst the two human anaphylatoxins include, carbohydrate is associated with C5a but is absent in the C3a molecule and little homology of the partial NH₂-terminal sequence of C5a NH₂-Thr-Leu-Glx-Lys-Ile-Glx-Glx-IleAla-with the corresponding NH₂-terminal sequence of C3a  [32] . C5a is cleaved from the N-terminal end of the C5 α-chain by a specific C5 convertase  [33] during C activation  [34] . Rat C5a is unusual in that the desArg derivative maintains a higher level of potency than does C5a_desArg from other species [35] . IntactC5a exhibits a number of potent biological activities primarily associated with the inflammatory response. As a spasmogensub-nanomolar concentrations of C5a can induce contraction of smooth muscle tissue from a variety of animal sources [34].

C5a promotes histamine release from mast cells and basophils and can enhance vascular permeability  [36,37] . Nanomolar concentrations of C5a also induce directed migration (i.e., chemotaxis) and degranulation of both leukocytes and monocytes  [38-40] . The C-terminal Arg that is exposed when C5a is released from C5 is rapidly removed in vivo by a serum carboxypeptidase (serum carboxypeptidase N)  [41] to produce C5adesArg. Removal of Arg and concomitant reduction in C5a potency is presumed to be a control mechanism to inactivate C5a in vivo. However, it has been shown that Arg removal reduces C5a potency to a variable extent depending both on the animal source of C5a and the biological response or assay used for measurement of C5a potency  [42,43] .

Elucidation of the primary structures of human, porcine, bovine, and mouse C5a  [32,44,45] indicate that these 74 residues peptide share a high degree of similarity with respect to size residues for human, porcine, and bovine and mouse as well as the primary structure (overall = 70% identity). In addition, the tertiary structures of human and porcineC5a are highly similar, as determined by NMR [46]. C5a consists of 4 helical bundles arranged in an anti-parallel orientation. The N-terminal and Cterminal regions are largely helical and are stabilized by hydrophobic side chain interactions with neighboring residues [47]. The internal “core” is stabilized by 3 disulfide linkages  [46,48] .

In summary, C3a, C4a and C5a are the terminal part of the corresponding C3, C4 and C5 α-chains the first two released by the action of C3 convertases and the third by the C5 convertases. C3a and C4a are the 77 amino acid peptides removed from the C3 and C4 N-terminus of C3 and C4 molecules, while C5a is 74 amino acid peptide removed from the N-terminus of C5.

The three anaphylatoxins, C3a, C4a and C5a represent the N-terminal portions of the α-chains of their respective components. They are similar in structure and functions. Anaphylatoxin C3a consists of a 74-residue polypeptide. Its N-terminal domain is intra-connected by three native disulfide bonds (Cys²²-Cys⁴⁹, Cys²³-Cys⁵⁶, and Cys³⁶- Cys⁵⁷) and a helical C-terminal domain. Its three-dimensional structure is depicted in the. Anaphylatoxin C4a consists of 74 amino acids residues. Anaphylatoxin C5a consists of a 74-residue peptide. The eight C-terminal residues, 74 to 66 aa, are disordered when the molecule is in solution. Three disulfide bounds formed by the Cys residues 21 and 47, 22 and 54, 34 and 54) are disposed around great part of the α-helix. A single carbohydrate moiety is attached to Asn 64 residue.

Circular dichroism (CD) spectra for C3a anaphylatoxins from human and porcine sources were compared in the region of 190 to 250 nm. The spectra were indistinguishable in this region, although an estimated difference of approximately 20% exists between the primary structures of human and porcine C3a. Calculations indicated a total of 40% to 45% α-helical content based on either the 208 or 222 nm extremum of the CD spectra. Biological activity was not affected upon mercaptoethanol or 6 M guanidinium chloride treatments even with the resulting decreasing in the molecule ellipicity [49]. CD spectrum distortion and biological inactivation however occur when C3a was simultaneously treated with mercaptoethanol and guanidinium chloride, both events being reversed after removal of the denaturant and reducing agents. Heat treatment or reduction and alkylation of human C3a produced biologically inactive and conformational modified anaphylatoxin. In contrast, C3a inactivated by enzymatic removal of the COOH-terminal Arg residue was structurally unchanged as judged by CD measurements. Consequently, it is proposed that in addition to the essential COOH-terminal Argresidue, a highly ordered conformation is required for biological functionality of the C3a molecule  [49] .

The circular dichroism spectra of C5a in the 200 - 250 nm region at pH 7.2 and 3.7 are nearly identical of C3a anaphylatoxin. Treatment of C5a with 2-mercaptoethanol progressively diminishes the elliptic curvature at 222 nm and its anaphylatoxic activity to limiting values. Removal of the reducing agent by dialysis completely restored both the elliptic form at 222 nm and the activity. This finding indicates that the integrity of the secondary conformation of the C5a molecule is largely dependent on disulfide bonds and is essential for its full activity ADDIN EN.CITE  ADDIN EN.CITE.DATA  [49] .

The noticeable damage caused by anaphylatoxinsare strictly in vivo controlled by naturally plasma circulating carboxypeptidase N. Once formed the anaphylatoxins C-terminal residues are removed by these enzymes yielding the C3ades-Arg derivative [50]. Carboxypeptidase N is a tetramericmetalloprotease, belonging to a family of a protein-cleaving, metal atom-containing enzymes [51]. Although C3a-desArg is completely free of inflammatory properties, C5-desArg retains around 1% - 10% of this activity [52] .

Anaphylatoxin receptors are expressed on mast cells, granulocytes, smooth muscle, endothelial cells, and monocytes-macrophages-dendriticcells.Upon interacting with the corresponding anaphylatoxins they are activated responding with release of inflammatory mediators and/or accumulation in inflammation sites.

To exert their pharmacological effects anaphylatoxins first bind to specific receptors, collectively designed ATRs. The existence of these anaphylatoxin-type recaptors wasanticipated when C3a was described by experiments showing clearly that guinea pig ileum preparations first desensitized to C3a, continues responding by the usual contraction to C5a, and vice-versa. ATRs, C3aR, C5aR and C5a-like receptor belong to a super-family of G-proteins coupled receptors GPCRs.

GPCRs exist as a superfamily of integral membrane protein receptors that contain seven transmembrane alpha-helical regions, which bind to a wide range of ligands. Upon activation by the specific ligand, the GPCR undergoes a conformational change and then activate the G proteins by promoting the exchange of GDP/GTP associated with the Galpha subunit. This leads to the dissociation of Gbeta/Ggamma dimmer from Galpha. Both these moieties then become free to act upon their downstream effectors and thereby initiate unique intracellular signaling responses. After the signal propagation, the GTP of Galpha-GTP is hydrolyzed to GDP and Galpha becomes inactive (Galpha-GDP), which leads to its reassociation with the Gbeta/Ggamma dimmer to form the inactive heterotrimeric complex. The GPCR can also transduce the signal through G protein independent pathway. GPCRs also regulate cell cycle progression [53] . Activation of GPCRs leads to the dissociation of heterotrimeric G-proteins into Gα and Gβγ subunits, which go on to regulate various effectors involved in a panoply of cellular responses. During chemotaxis, Gβγ subunits regulate actin assembly and migration, but the protein(s) linking Gβγ to the actin cytoskeleton remains unknown. The Gβγ effector, ElmoE in Dictyostelium, had demonstrated that it is required for GPCR-mediated chemotaxis [54] . As ElmoE associates with Gβγ and Dock-like proteins to activate the small GTPaseRac, in a GPCR-dependent manner, and also associates with Arp2/3 complex and F-actin, ElmoE serves as a link between chemoattractant GPCRs, G-proteins and the actin cytoskeleton. The pathway, consisting of GPCR, Gβγ, Elmo/Dock, Rac, and Arp2/3, spatially guides the growth of dendritic actin networks in pseudopods of eukaryotic cells during chemotaxis  [54] .

C3aR. This receptor is expressed on a large variety of cells as of myeloid origin like mast cells, basophils, neutrophils, eosinophils, monocytes-macrophages, dendritic cells and microglia  [55-60] , as well as in non-myeloid cells like astrocytes  [61,62] , endothelial cells  [63] , and epithelioid cells, smooth muscle cells and parenchymal cells  [64] . The presence of C3aR on activated but not non-activated T lymphocytes has also been reported  [65] . C3aR is a 482 amino acids molecule (54 kDa) protein glycosylated at the Asp-9 and Asp-195 residues and sulfated at the Tyr-174  [66,67] . The protein second extracellular loop is it the larger part accommodating the sulfated Tir-174 important molecular region involved on C3a-C3aR interactions [68]. Upon interaction the resulting signal is transduced via heterotrimeric G-proteins mobilizing Ca⁺⁺ fluxes from extra-cellular stores. G protein coupled receptors (GPCRs) comprise a superfamily of proteins involved in transducing signals emitted by the partner receptors upon it combining with the specific agonists. Over eight hundred GPCRs from various cell types have been cloned  [69] . The next signaling steps diverge with the cell type. In endothelial cells C3aR is connected with pertussis-toxin insensitive Gα16  [70] , following activation of protein-kinase C by phospholipase C. In astrocytes the protein (MAP) kinases Erk1 and Erk2 are activated  [52,71] . In mast cells C3aR binding by C3a activates cytokine production through a complex intracellular signals involving PI3K, Akt-phosohorylation and MAP kinase Erk1 and ERK2  [72] .

C5aR. C5a exerts its effects through the C5a receptor (C5aR or CD88), also a member of the rhodopsin family of seven-transmembrane(seven-TM) G protein-coupled receptors [73]. The membrane bound protein C5aR (CD88) is also expressed in many cell types. Higher expression is found in neutrophils, eosinophils, basophils, monocytes-macrophages, dendritic cells and mast cells  [38,43,74-76] . C5aR expression in lymphocytes subtype cells is controversial. Although some reports confirms its presence  [77,78] ; other indicates its absence  [79-81] , or scarcely near the negative controls in some population of lymphoid cells  [82] or even not detected by anti-C5aE antibodies [83]. GPCRs perceive many extracellular signals and transduce them to heterotrimeric G proteins, which further transduce these intracellular signals to appropriate downstream effectors allowing play important role in various signaling pathways.

The anaphylatoxins activities can be blocked by specific antibodies directed to their relevant immunogenic epitopes and by competitive antagonists with their cellular receptors accessing molecular domains.

Development of blocking monoclonal antibodies (mAb) to prevent C3, C4 or C5 cleavage and the subsequent C3a, C4a and C5a formation is a potential therapeutic approach to treat complement-induced inflammation.

C3amAb. Monoclonal Ab anti-C3a blocks in vitro and in vivo the activities of C3a.  

C5amAb. Rat anti-mouse C5 mAb (BB5.1) was utilized to investigate the effects of inhibition of C5 cleavage on disease progression and severity in experimental autoimmune uveoretinitis (EAU), a model of organ-specific autoimmunity in the eye characterized by structural retinal damage mediated by infiltrating macrophages. In vivo, systemic treatment with BB5.1 results in significantly reduced disease scores compared with control groups, while local administration results in an earlier resolution of disease. Systemic treatment with BB5.1 results in significantly reduced disease scores compared with control groups, while local administration results in an earlier resolution of disease [84] . Structural and functional studies of the interaction between the mAb F20 and C5a was used to investigate the functional epitope in C5a. Association of molecular modeling, docking methods and biological mutant experiment, were used to identify the key epitope. Based on theoretically and predicted discreet sites in antigen and antigen the key epitope in the antigen was identified. The predicted results allowed determine Lys⁶⁸ in C5a as the key C5a epitope. This epitope can be used in C5a antagonist screening and design [85] .

The central strategy used is the competitive blockage the anaphylatoxins effective interaction with their cellular receptors. Homologous peptides mimicking the anaphylatoxin peptides sequences recognized by their cell target receptors are the useful focus.

In a pioneering approach the compound N₂-[(2,2-diphenylethoxy) acetyl]-L-arginine designated SB 290157 was delineated having as model a molecule selected from 240,000 compounds synthesized by SmithKline. The molecule N₂-[(2,2-diphenylethoxy)acetic acid]-L-arginine (SK&F 63649) serving as immediate template to synthesize SB 290157 was used as control in agonist versus antagonists assays.

The ability of the modeled molecules to competitively binding with C3a for C3aR was analyzed in vitro and in vivo assays. First, membranes of RBL-C3aR cells were prebound to SPA beads mixed previously with SB 290157 or SK&F 63649 compounds, next with the ¹²⁵-C3a radioligand in appropriated buffer, and bound radiolabeled ligand measured by counting. ¹²⁵IC3a binds to RBL-C3aR cells with high affinity (K_d = 8pM) and is saturated. The detected affinities of SB 290157 and SK&F 63649 were 200 and 3000 nM, respectively. To determine whether the competitive binding reflected on functional activity of C3a, a FLIPR-based C3a-induced Ca⁺⁺ mobilization assay in RBL-C3aR measured in terms of IC₅₀S units  were more efficient with SB 290157 (30 nM) than with SK&F 63649 (308 nN). As C3a also induces mast cell chemotaxis, the ability of the SB 290157 compound to attract inflammatory cells was tested on HMC-1, a human mast cell line that naturally expresses the C3aR and for which C3a is chemotactic. At the concentration of 5μM SB 2901157 markedly inhibition of C3a-mediated chemotaxis of HMC-cells was detected. This effect was not detected on C5a-mediated chemotaxis. SB 290157 compound exhibited also potent ability to inhibit C3a-induced internalization of the C3aR [86] .  

In in vivo animals model the anti-C3a antagonist SB 290157 was also capable of inhibiting some C3a manifestation as neutrophil recruitment in a guinea pig, the LPS-induced neutrophilia in airway model and the paw edema in a rat adjuvantinduced arthritis.

The key strategy in the construction of N₂-[(2, 2- diphenylethoxy) acetyl]-L-arginine (SB290157) was the selection of Arg, the essential amino acid residue involved in the interaction of the C3a with C3aR. The compound [(2,2-Diphenylethoxyl)acetyl]-L-arginine expresses higher C3a-C3aR competitive activity with the Diphenyl radicals separately attached to the acetic-Larginine moiety.

Complement activation at local tissue sites forms a gradient of anaphylatoxins C3a, C4a and C5a which due to their properties to contract smooth muscles, to attract neutrophils, eosinophils, monocytes-macrophages and to activate these cells as well as resident mast cells and endothelial cells, unequivocally participate in the early events of the inflammatory process. Other biological anaphylatoxins attributes including regulation of cationic protein synthesis by eosinophils, adhesion of these cells to endothelial cells [87,88] , stimulation of serotonin release from platelets and modulation of the synthesis of IL-6 and TNF-α by B lymphocytes and monocytes by C3a [89-91] , expands further the anaphylatoxins roles in inflammation. In situ complement activation strongly supports the involvement of these active peptides in the pathogenesis of important lesions. Activation of the CS is also a common event along tissue homing of microbes or in non-infective lesions. The participation of anaphylatoxins have been demonstrated in the pathogenesis of several pathological conditions as in asthma  [92] , in the diarrhea-associated hemolytic syndrome and acute renal failure induced by E. coli-producing Shiga toxin  [93-95] , in the liver regenerative process  [96] , and in arthritis  [97] .