InVivoMAb anti-mouse C5aR (CD88)

IgG Fc receptors (FcgammaRs, especially FcgammaRIII) and complement (in particular, C5a anaphylatoxin) are critical effectors of the acute inflammatory response to immune complexes (ICs). However, it is unknown whether and how these two key components can interact with each other in vivo. We use here a mouse model of the acute pulmonary IC hypersensitivity reaction to analyze their potential interaction. FcgammaRIII and C5aR are coexpressed on alveolar macrophages (AMs), and both FcgammaRIII and C5aR mutant mice display impaired immune responses. We find that recombinant human C5a (rhC5a) can control inverse expression of various FcgammaRs, and costimulation of ICs with rhC5a results in strong enhancement of FcgammaRIII-triggered cellular activation in vitro and in vivo. Moreover, we show here that early IC-induced bioactive C5a, and its interaction with C5aR, causes induction of activating FcgammaRIII and suppression of inhibitory FcgammaRII on AMs that appears crucial for efficient cytokine production and neutrophil recruitment in lung pathology. Therefore, C5a, which is a potent chemoattractant, has a broader critical function in regulating the inhibitory/activating FcgammaRII/III receptor pair to connect complement and FcgammaR effector pathways in immune inflammation.

The anaphylatoxin C5a is a potent proinflammatory stimulus with immunomodulatory activities. Expression of its receptor C5aR (CD88) has been detected on cells of myeloid origin such as granulocytes and monocytes/macrophages. However, controversial results exist on the expression of C5aR on T and B lymphocytes as well as on mature dendritic cells (DC). The aim of the present study was to characterize expression of C5aR protein on myeloid and lymphoid cells in the mouse. For this purpose, rat monoclonal antibodies with specificity against the murine C5aR were generated. Using these reagents a distinct amount of C5aR antigen was observed on neutrophils and macrophages. In contrast, C5aR protein was not detectable on resting or stimulated murine T or B lymphocytes. Furthermore, no C5aR protein could be observed on splenic CD11c positive DC which have been classified in the literature as relatively mature. Taken together, our results suggest that in the mouse expression of C5aR protein may be restricted to leukocytes of myeloid origin whereas previous evidence for C5aR expression on lymphoid cells may be reevaluated.

Airway hyperresponsiveness and airway inflammation are hallmarks of allergic asthma, the etiology of which is crucially linked to the presence of Th2 cytokines. A role for the complement anaphylatoxins C3a and C5a in allergic asthma was suggested, as deficiencies of the C3a receptor (C3aR) and of complement factor C5 modulate airway hyperresponsiveness, airway inflammation, and Th2 cytokine levels. However, such models do not allow differentiation of effects on the sensitization phase and the effector phase of the allergic response, respectively. In this study, we determined the role of the anaphylatoxins on the effector phase of asthma by pharmacological targeting of the anaphylatoxin receptors. C3aR and C5a receptor (C5aR) signaling was blocked using the nonpeptidic C3aR antagonist SB290157 and the neutralizing C5aR mAb 20/70 in a murine model of Aspergillus fumigatus extract induced pulmonary allergy. Airway hyperresponsiveness was substantially improved after C5aR blockade but not after C3aR blockade. Airway inflammation was significantly reduced in mice treated with the C3aR antagonist or the anti-C5aR mAb, as demonstrated by reduced numbers of neutrophils and eosinophils in bronchoalveolar lavage fluid. Of note, C5aR but not C3aR inhibition reduced lymphocyte numbers in bronchoalveolar lavage fluid. Cytokine levels of IL-5 and IL-13 in bronchoalveolar lavage fluid were not altered by C3aR or C5aR blockade. However, blockade of both anaphylatoxin receptors markedly reduced IL-4 levels. These data suggest an important and exclusive role for C5aR signaling on the development of airway hyperresponsiveness during pulmonary allergen challenge, whereas both anaphylatoxins contribute to airway inflammation and IL-4 production.

Complement component C5a binds C5a receptor (C5aR) and facilitates leukocyte chemotaxis and release of inflammatory mediators. We used neutrophils from human C5aR knock-in mice, in which the mouse C5aR coding region was replaced with that of human C5aR, to immunize wild-type mice and to generate high-affinity antagonist monoclonal antibodies (mAbs) to human C5aR. These mAbs blocked neutrophil migration to C5a in vitro and, at low doses, both prevented and reversed inflammatory arthritis in the murine K/BxN model. Of approximately 40 mAbs generated to C5aR, all potent inhibitors recognized a small region of the second extracellular loop that seems to be critical for regulation of receptor activity. Human C5aR knock-in mice not only facilitated production of high-affinity mAbs against an important human therapeutic target but were also useful in preclinical validation of the potency of these antagonists. This strategy should be applicable to other important mAb therapeutics.