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).

Experimental design of complement component 5a-induced acute lung injury (C5a-ALI): a role of CC-chemokine receptor type 5 during immune activation by anaphylatoxin.

Excessive activation of the complement system is detrimental in acute inflammatory disorders. In this study, we analyzed the role of complement-derived anaphylatoxins in the pathogenesis of experimental acute lung injury/acute respiratory distress syndrome (ALI/ARDS) in C57BL/6J mice. Intratracheal administration of recombinant mouse complement component (C5a) caused alveolar inflammation with abundant recruitment of Ly6-G(+)CD11b(+) leukocytes to the alveolar spaces and severe alveolar-capillary barrier dysfunction (C5a-ALI; EC(50[C5a]) = 20 ng/g body weight). Equimolar concentrations of C3a or desarginated C5a (C5a(desArg)) did not induce alveolar inflammation. The severity of C5a-ALI was aggravated in C5-deficient mice. Depletion of Ly6-G(+) cells and use of C5aR1(-/-) bone marrow chimeras suggested an essential role of C5aR1(+) hematopoietic cells in C5a-ALI. Blockade of PI3K/Akt and MEK1/2 kinase pathways completely abrogated lung injury. The mechanistic description is that C5a altered the alveolar cytokine milieu and caused significant release of CC-chemokines. Mice with genetic deficiency of CC-chemokine receptor (CCR) type 5, the common receptor of chemokine (C-C motif) ligand (CCL) 3, CCL4, and CCL5, displayed reduced lung damage. Moreover, treatment with a CCR5 antagonist, maraviroc, was protective against C5a-ALI. In summary, our results suggest that the detrimental effects of C5a in this model are partly mediated through CCR5 activation downstream of C5aR1, which may be evaluated for potential therapeutic exploitation in ALI/ARDS.

Characterisation of C5a receptor agonists from phage display libraries.

C5a des-Arg(74) has a 10- to 100-fold lower receptor binding affinity than intact C5a and is only a partial agonist. We have used phage display selection from randomly mutated C5a des-Arg(74) libraries to isolate variant proteins that can activate C5a receptors with similar potency to C5a. Here we explore the interactions of three variants (V1-3) with C5aR mutated at residues involved in the differential response. The mutant Asp(282)Arg-C5aR is preferentially activated by C5a des-Arg(74), probably due to repulsion between Arg(74) of C5a and the substituent Arg(282). In accordance with this hypothesis, V2 (with a polar C-terminus which has no Arg residue) but not V1 (with a C-terminal Arg residue at position 73) could activate Asp(282)Arg-C5aR. V3, with a very hydrophobic C-terminus, was the most potent agonist at Asp(282)Arg-C5aR. Arg(175) is a potential counterion for the C-terminal carboxylate of C5a. C5aR mutated to either Ala or Asp at this position lost nearly all responsiveness to both C5a and C5a des-Arg(74), suggesting that mutation of Arg(175) caused a non-specific loss of receptor conformation and a loss of signalling capacity. However, V3 could still activate Arg(175)Asp/Ala-C5aR with the same potency as wild-type C5aR, demonstrating that the mutant receptors retained high signalling capability and showed a specific loss of responsiveness. Thus C5a des-Arg(74) variants produced by phage display are potentially useful tools for the dissection of ligand-receptor interactions.

Receptor activation by human C5a des Arg74 but not intact C5a is dependent on an interaction between Glu199 of the receptor and Lys68 of the ligand.

Despite the expression of only one type of receptor, there is great variation in the ability of different cell types to discriminate between C5a and its more stable metabolite, C5a des Arg74. The mechanism that underlies this phenomenon is not understood but presumably involves differences in the interaction with the C5a receptor. In this paper, we have analyzed the effects of a substitution mutation of the receptor (Glu199 --> Lys199) and the corresponding reciprocal mutants (Lys68 --> Glu68) of C5a, C5a des Arg74 and peptide analogues of the C-terminus of C5a on the ability of the C5a receptor to discriminate between ligands with and without Arg74. The use of these mutants indicates that the Lys68/Glu199 interaction is essential for activation of receptor by C5a des Arg74 but not for activation by intact C5a. The substitution of Asp for Arg74 of C5a [Lys68] produces a ligand with equal potency on both the wild-type and mutant receptors, suggesting that it is the C-terminal carboxyl group rather than the side chain of Arg74 that controls the responsiveness of the receptor to Lys68. In contrast, the mutation of Lys68 to Glu(68) has little effect on the ability of either C5a or C5a des Arg(74) to displace [(125)I]C5a from the receptors, indicating that binding of ligand and receptor activation are distinct but interdependent events. C5a and the truncated ligand, C5a des Arg74, appear to have different modes of interaction with the receptor and the ability of the human C5a receptor to discriminate between these ligands is at least partly dependent on an interaction with the receptor residue, Glu199.

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[A35E36R37A38S39R40], 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[Q35E36R37I38L39N40] 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.