Essential Role for the C5a Receptor in Regulating the Effector Phase of Synovial Infiltration and Joint Destruction in Experimental Arthritis Ethan P. Grant,1 Dominic Picarella,1 Timothy Burwell,1 Tracy Delaney,1 Alisa Croci,1 Nicole Avitahl,1 Alison A. Humbles,2 Jose-Carlos Gutierrez-Ramos,1 Michael Briskin,1 Craig Gerard,2 and Anthony J. Coyle1 1Millennium Pharmaceuticals, Inc., Cambridge, MA 02139 2Ina Sue Perlmutter Laboratory, Children’s Hospital, Harvard Medical School, Boston, MA 02115 Abstract A characteristic feature of rheumatoid arthritis is the abundance of inflammatory cells in the diseased joint. Two major components of this infiltrate are neutrophils in the synovial fluid and macrophages in the synovial tissue. These cells produce cytokines including tumor necrosis fac- tor and other proinflammatory mediators that likely drive the disease through its effector phases. To investigate what mechanisms underlie the recruitment of these cells into the syn- ovial fluid and tissue, we performed expression analyses of chemoattractant receptors in a re- lated family that includes the anaphylatoxin receptors and the formyl-MetLeuPhe receptor. We then examined the effect of targeted disruption of two abundantly expressed chemoattractant receptors, the receptors for C3a and C5a, on arthritogenesis in a mouse model of disease. We report that genetic ablation of C5a receptor expression completely protects mice from arthritis. Key words: arthritis • C5a receptors • granulocytes • chemoattractants • monocytes Introduction Rheumatoid arthritis (RA)* is a chronic inflammatory dis- (3). The arthritic process apparently arises through a series ease characterized by the infiltration of T cells, B cells, mac- of discrete stages. The activation and expansion of antigen- rophages, and neutrophils into the synovial lining and fluid specific T and B cells in the synovium is believed to play a of the periarticular spaces. The infiltrating cells produce role in the initiation of the disease (4). Subsequently, in the abundant cytokines, dominated by TNF and IL-1␤, “effector” phase, macrophages, neutrophils, and lympho- which stimulate infiltrating cells and resident fibroblast-like cytes are recruited to the joint where, in concert with resi- synoviocytes (1, 2). These interactions lead to fibroblast hy- dent fibroblasts, they mediate the chronic destructive events perproliferation and formation of an invasive pannus tissue through the release of cytokines, proteases, and other medi- that produces collagenases and stromelysins, ultimately re- ators (1, 2). However, the mechanisms that regulate these sulting in the destruction of neighboring cartilage and bone effector responses remain to be fully elucidated and it is not clear which chemokines and chemoattractants are primarily Address correspondence to Ethan P. Grant, Millennium Pharmaceuticals, responsible for the localization of cells into the synovium. Inc., 75 Sidney Street, Cambridge, MA 02139. Phone: 617-551-3797; Inflammatory cell recruitment into the synovial fluid and Fax: 617-225-0884; E-mail: [email protected]; or Craig Gerard, Ina Sue the synovial tissue occurs as a result of the production of Perlmutter Laboratory, Children’s Hospital, Harvard Medical School, 320 chemoattractants by activated macrophages, synovial fibro- Longwood Avenue, Boston, MA 02115. Phone: 617-355-6174; Fax: blasts, and other cells in the inflamed joint. In recent years, 617-738-1752; E-mail: [email protected] *Abbreviations used in this paper: C3aR, C3a receptor; C5aR, C5a re- attention has been focused on chemokine family members ceptor; CIA, collagen-induced arthritis; ENA, epithelial neutrophil acti- such as monocyte chemoattractant protein 1, RANTES, vator; GPCR, G protein–coupled receptor; GPI, glucose-6-phosphate macrophage inflammatory protein (MIP)-1␣, MIP-2␣, and isomerase; H&E, hematoxylin and eosin; ICAM, intercellular adhesion epithelial neutrophil activator (ENA)-78 (3). Chemokines molecule; MAC, membrane attack complex; MIP, macrophage inflam- matory protein; MMP, matrix metalloproteinase; MPO, myeloperoxi- signal through a number of related G protein–coupled re- dase; OPGL, osteoprotegerin ligand; RA, rheumatoid arthritis; VCAM, ceptors (GPCRs) that are expressed on distinct leukocyte vascular cell adhesion molecule. cell populations, including the cells recruited to the joint 1461 J. Exp. Med. The Rockefeller University Press • 0022-1007/2002/12/1461/11 $5.00 Volume 196, Number 11, December 2, 2002 1461–1471 http://www.jem.org/cgi/doi/10.1084/jem.20020205 during arthritogenesis. Given the large number of potential model. These data raise the possibility that drugs and bio- recruitment pathways that could contribute to joint inflam- therapeutics targeting the C5aR may provide a novel mation, it has been difficult to determine which, if any, strategy for therapeutic intervention to disrupt the effec- play unique roles that cannot be compensated by other tor phase of RA. chemokines and their receptors. Studies in animal models have suggested an important role for certain chemokine interactions with their receptors. For example, experimen- Materials and Methods tal arthritis can be partially ameliorated with modified Ϫ/ RANTES (met-RANTES) and monocyte chemoattractant Mice. The generation and characterization of C3aR and Ϫ/ Ϫ/Ϫ protein 1 antagonists (5–7). As these chemokines are pro- C5aR mice has been previously described (16, 18). C3aR and C5aRϪ/ mice backcrossed three and four generations, re- miscuous in binding to several distinct chemokine recep- spectively, onto a BALB/c background and littermate controls tors, the relative importance of their cognate receptors in were used for all experiments described. Animal studies were per- cell recruitment remains to be fully defined. formed according to institutional and National Institutes of A second family of chemoattractant molecules, which Health guidelines for animal use and care. signal through a related family of GPCRs, is comprised of Human Cell Isolation and Culture. Normal human peripheral complement fragments and bacterial peptides such as C3a, blood monocytes were isolated using CD14-MicroBeads and C5a, and formyl-MetLeuPhe (8–10). Like the chemokine magnetic cell separation technology according to the manufac- family, a number of these chemotactic molecules are turer’s instructions (Miltenyi Biotec). Macrophages were derived present in the synovial tissue and/or synovial fluid of RA by culturing purified monocytes for 10 d in the presence of 100 patients (9). The presence of elevated anaphylatoxin C3a ng/ml GM-CSF (Pierce Chemical Co.). Granulocytes were iso- and C5a levels is but one of several indications that the lated from peripheral blood via Ficoll density gradient separation. Isolated cell populations were cultured in RPMI 1640 medium complement system is hyperactive in RA patients. For ex- (Invitrogen) containing 10% fetal bovine serum (Invitrogen) for 4 ample, it has long been appreciated that antibody-driven or 24 h in the presence of one of the following stimuli: 1 ␮g/ml inflammatory responses, through activation of the comple- Escherichia coli 026:B6 LPS (Sigma-Aldrich), 5 ␮g/ml recombi- ment cascade or Fc-dependent cellular activation, play a nant human CD40L (PeproTech), or 100 ng/ml recombinant crucial role in arthritis. In this context, the synovial tissue human TNF (PeproTech). RNA was extracted and used in and fluid of RA patients frequently contain abundant au- TaqMan® analyses as described below. toantibodies such as rheumatoid factor (11). In addition, ar- Immunohistochemistry. Human RA synovial tissue was ob- thritis can be induced in rodents by passive transfer of anti- tained as discarded material from joint replacement surgery. 5-␮m collagen antibodies (12). The potential pathogenic role of sections of snap frozen tissue blocks were fixed in acetone autoantibodies has been further highlighted in recent years and incubated in 0.3% goat serum (Vector Laboratories)/0.3% hydrogen peroxide in PBS for 5 min. Sections were incubated in with the advent of the K/BxN arthritis model, in which PBS/5% goat serum for 30 min followed by anti-C5aR (BD disease can be induced by the transfer of anti–glucose-6- Biosciences) or control rabbit IgG (Dako) for 1 h at room tem- phosphate isomerase (GPI) autoantibodies (13). perature. The slides were developed using the VECTASTAIN® The C5a receptor (C5aR) is recognized to be a potent Elite ABC Rabbit IgG kit and the AEC Peroxidase Substrate kit chemoattractant receptor on neutrophils, monocytes, (Vector Laboratories) and counterstained in Mayer’s hematoxy- macrophages, and eosinophils (14). The liberation of C5a lin (Poly Scientific). To detect IgG deposition, mouse joints at sites of complement activation therefore leads to the re- were fixed and decalcified in 4% paraformaldehyde/10% EDTA cruitment of these cell types into sites of infection or au- (both from Sigma-Aldrich) for 14 d and embedded in optimum toantibody deposition. The C5aR is also present on a va- cutting temperature for sectioning. 6-␮m sections were incu- riety of other cell types, including bronchial epithelial and bated with FCS to block Fc receptor binding followed by bio- smooth muscle cells as well as endothelial cells and hepa- tinylated rat anti–mouse IgG (Dako). Staining was revealed using StreptABCComplex/horseradish peroxidase detection (Dako) tocytes, where expression has been implicated in cell acti- according to the manufacturer’s instructions. To detect C3 and vation and the induction