LETTERS C5L2 is critical for the biological activities of the anaphylatoxins C5a and C3a Nien-Jung Chen 1 , Christine Mirtsos 1 , Daniel Suh 1 , Yong-Chen Lu 1 , Wen-Jye Lin 1 , Colin McKerlie 2 , Taeweon Lee 3 , Helene Baribault 3 , Hui Tian 3 & Wen-Chen Yeh 1,3 Complement-derived anaphylatoxins regulate immune and inflam- matory responses through G-protein-coupled receptor (GPCR)- mediated signalling 1–4 . C5L2 (also known as GPR77) is a relatively new GPCR thought to be a non-signalling receptor binding to C5a, on the basis of sequence information and experimental evidence 5–7 . Here we show, using gene targeting, that C5L2 is required to facilitate C5a signalling in neutrophils, macrophages and fibroblasts in vitro. Deficiency of C5L2 results in reduced inflammatory cell infiltration, suggesting that C5L2 is critical for optimal C5a-mediated cell infiltration in certain in vivo settings. C5L2 is also involved in optim- izing C3a-induced signals. Furthermore, like mice incapable of C3a/complement 3a receptor (C3aR) signalling 4,8,9 , C5L2-deficient mice are hypersensitive to lipopolysaccharide (LPS)-induced septic shock, show reduced ovalbumin (OVA)-induced airway hyper- responsiveness and inflammation, and are mildly delayed in haem- atopoietic cell regeneration after c-irradiation. Our data indicate that C5L2 can function as a positive modulator for both C5a- and C3a-anaphylatoxin-induced responses. Proteolysis of C3 and C5 are central events of complement activa- tion. The smaller fragments, C3a and C5a, are known anaphylatoxins and induce or augment several biological responses. C3aR 10 , C5aR 11 and C5L2 share significant homology and belong to the GPCR family. C5a and C5a des-Arg bind C5L2 with high affinity 6,12,13 , but whether or not C3a and C4a are ligands for C5L2 remains debatable 12–14 . We generated C5L2-deficient mice (Supplementary Information section 1), and reverse-transcriptase–PCR (RT–PCR) analysis showed that although C5aR expression was comparable in c5l2 2/2 and wild-type livers, C5L2 messenger RNA was present only in wild-type liver (Fig. 1a). Additionally C5L2 is detectable on wild-type but not on c5l2 2/2 neutrophils (Fig. 1b) and macrophages (Supplementary Information section 1). C5aR and C3aR protein levels were compar- able in c5l2 2/2 and wild-type neutrophils, using antibodies obtained commercially as described (Supplementary Information section 1). c5l2 2/2 mice appeared healthy and displayed no obvious develop- mental abnormalities. The binding of mouse C5L2 to mouse C5a was first confirmed using COS cells overexpressing mouse C5L2 (Fig. 1c, d). To invest- igate the role of C5L2 in C5a-mediated responses, neutrophils were stimulated and the surface Mac-1 (also known as Itgam) induction was monitored; Mac-1 is normally enhanced when neutrophils are activated. c5l2 2/2 neutrophils showed impaired Mac-1 induction compared with wild-type cells in response to C5a alone (Fig. 1e, left). This defect was magnified when neutrophils were stimulated with C5a plus LPS and was also evident in a BALB/c background (Fig. 1e, right). We also examined TNF-a and IL-6 production by neutrophils after stimulation. LPS alone induced comparable levels of TNF-a (Fig. 1f, left) and IL-6 (Fig. 1f, right) in wild-type and c5l2 2/2 neu- trophils. As previously reported, C5a treatment reduced TNF-a production 15 , but enhanced IL-6 production 16 in LPS-stimulated wild-type neutrophils. In contrast, C5a was unable to affect LPS- induced TNF-a and IL-6 production in c5l2 2/2 cells. Furthermore, C5a des-Arg induced strong Mac-1 induction in wild-type neutro- phils, but not in c5l2 2/2 cells. However, neither C3a nor C3a des-Arg induced Mac-1 expression in wild-type or c5l2 2/2 neutrophils (Sup- plementary Information section 2). MAPK and PKB/Akt activation are key downstream events induced by C5a 16,17 . In wild-type neutrophils, C5a treatment alone strongly induced ERK1/2 activation, and weakly enhanced phospho- p38 expression (Fig. 2a). A low dose (20 ng ml 21 ) of LPS enhanced C5a-induced activations of various MAPKs. In contrast, c5l2 2/2 neutrophils showed only weak induction of activated ERK1/2, JNK or p38 in response to C5a, either alone or with LPS. C5a is also known to have a critical role in regulating macrophage functions 18 . We found that C5a-induced activation of ERK1/2 and AKT was severely impaired in C57BL/6 c5l2 2/2 macrophages (Fig. 2b) and BALB/c c5l2 2/2 macrophages (Fig. 2c). In addition, LPS strongly upre- gulated expression of the co-stimulation molecules CD40 and CD86 in wild-type macrophages, whereas C5a suppressed this induction. In contrast, c5l2 2/2 macrophages showed slightly higher basal CD40 expression, but a substantially lower level of LPS-induced CD40 and CD86 expression. Furthermore, C5a-mediated suppression of LPS- induced CD40 and CD86 expression was impaired in c5l2 2/2 macro- phages (Fig. 2d; Supplementary Information section 2). Unlike in neutrophils, C5a suppressed LPS-induced TNF-a and IL-6 production in wild-type macrophages: a process that is impaired in c5l2 2/2 cells (Fig. 2e). Taken together, C5L2 is an important downstream mediator of C5a functions. Furthermore, specific aspects of LPS signalling seem to depend on the presence of C5L2. To determine whether C5L2 is involved in C3a-initiated signalling, we stimulated neutrophils with C3a, and examined downstream signals. Phosphorylated ERK1/2 and AKT were readily detected in wild-type neutrophils, following stimulation, but these signals were substantially reduced in C3a-stimulated c5l2 2/2 neutrophils (Fig. 3a). Similar reductions were detected in bone marrow derived c5l2 2/2 macrophages (Supplementary Information section 3). Actin poly- merization is an early consequence of neutrophil activation induced by chemoattractants. We detected C3a-induced F-actin formation in wild-type and c5l2 2/2 neutrophils (Fig. 3b; Supplementary Infor- mation section 3). Almost 50% of wild-type cells were positive for F-actin staining after stimulation, compared with only 11% of C3a- stimulated c5l2 2/2 neutrophils. Thus, C5L2 is also required for optimal C3a signalling. 1 The Campbell Family Institute for Breast Cancer Research, Ontario Cancer Institute, University Health Network and Department of Medical Biophysics, University of Toronto, Toronto, Ontario M5G 2C1, Canada. 2 Hospital for Sick Children, Toronto, Ontario M5G 1X8, Canada. 3 Amgen San Francisco, 1120 Veterans Boulevard, South San Francisco, California 94080, USA. Vol 446 | 8 March 2007 | doi:10.1038/nature05559 203 Nature ©2007 Publishing Group