Expression of the C3aR and C5aR in Human Cells and in RA Synovium.
To identify chemoattractant receptors expressed abundantly on key inflammatory cell types involved in arthritic pathogenesis, we performed a quantitative mRNA expression analysis using normal peripheral blood-derived monocytes, macrophages, and granulocytes. We extracted RNA from each of these populations before and after activation with relevant stimuli, synthesized cDNA, and performed TaqMan® real-time PCR analysis of the anaphylatoxin receptors C3aR and C5aR and the formyl-MetLeuPhe receptor-like GPCRs, FPRL1, and FPRL2. Together, these receptors form a chemoattractant receptor subfamily of GPCRs, although the biologic function and ligands for FPRL1 and FPRL2 have yet to be fully delineated. Of these receptors, C5aR had the highest levels of expression in granulocytes, monocytes, and macrophages ( A). Expression of C5aR appeared to be increased in granulocytes after a 4-h stimulation with TNFα. However, expression in monocytes and macrophages was not appreciably regulated by cell activation. C3aR expression was also notable in these three cell populations and like the C5aR, expression was augmented transiently in TNFα-stimulated granulocytes. In comparison, FPRL1 and FPRL2 were expressed at lower levels ( A). Given the abundant relative expression of C3aR and C5aR on these cell types, we performed additional expression analyses of these two receptors in diseased human tissue.
Figure 1. Expression of C5aR in human RA tissue and in a mouse model of arthritis. (A) Quantitative PCR analysis of C5aR, C3aR, FPRL1, and FPRL2 in untreated (UnTx) or activated (LPS, TNFα, IFNγ, or CD40L for 4 or 24 h) normal human peripheral blood (more ...)
To examine the distribution and expression of the C3aR and C5aR in inflamed human synovium, we performed an immunohistochemical analysis on frozen sections of human RA synovium from multiple individuals. Pronounced staining of both receptors was observed in the intimal layer, consistent with localization to macrophage-like synoviocytes, as well as scattered throughout the subintimal region ( B). Of the two receptors, C5aR was consistently more pronounced in expression, although this may in part reflect differences in avidities between the two antibodies. Immunohistochemistry with an anti-CD68 mAb on serial sections showed a very similar pattern of expression, with intense staining in the intimal layer and scattered positive subintimal cells. Thus, it is likely that the majority of C3aR+ and C5aR+ cells in the RA synovium are macrophages, although it is possible that C3aR+ and C5aR+ infiltrating neutrophils and other granulocytes are also present. In addition, C5aR was clearly detected on endothelial cells lining the blood vessels within synovial tissues ( B). The abundance of expression of these receptors in inflamed synovial tissue together with the high levels of expression on three major effector cell populations prompted us to examine the role of the C3aR and C5aR in the recruitment of inflammatory cells in a rodent model of arthritis.
Inflammatory Gene Regulation in Anticollagen Antibody-induced Arthritis.
To characterize the inflammatory changes that occur during the course of anticollagen mAb-induced arthritis, we isolated joints from mice 24, 48, and 72 h and 5, 7, 9, 11, and 13 d after intravenous injection with an arthritogenic antibody cocktail. At each time point, we assessed joint swelling and then froze isolated joints for subsequent expression analyses. We used quantitative PCR to measure the relative expression and changes in transcription of a panel of inflammatory markers, including markers for the major immune cell subsets. Most markers examined showed a consistent pattern of induction 72 h after mAb injection (24 h after an intraperitoneal LPS boost given to the animals; C). In particular, IL-1β and matrix metalloproteinase (MMP)-3 (stromelysin 1) showed marked induction from very low basal levels to high levels that were sustained to the last time point examined, 13 d after mAb injection. CD4 and CD68 mRNA levels increased substantially and with similar kinetics, reflecting the influx of T cells and macrophages, respectively, during the development of arthritis. Similarly, C3aR and C5aR expression dramatically increased 72 h after mAb administration, likely indicating the influx of neutrophils and monocyte/macrophage lineage cells, the principal immune cell subsets on which those receptors are found. In addition, the increase in C5aR expression may reflect augmented transcription in resident tissue macrophages and endothelial cells. Osteoprotegerin ligand (OPGL) message levels were also increased, presumably a reflection of the influx of T cells into the joint space, whereas osteoprotegerin, expressed mainly by resident chondrocytes and osteoclast precursors, remained relatively stable. In contrast, the expression of CD19, a marker for B lineage cells, did not change dramatically, indicating that B cells are not a major cell subset recruited into joints in this animal model (unpublished data).
C5aR-deficient Mice Are Resistant to Arthritis Induction.
As C3aR and C5aR expression were clearly up-regulated during the course of disease in this model of arthritis, we examined the effect of deleting expression of each of these receptors on disease induction and severity. Throughout the 14-d course of arthritogenesis, the paw swelling scores of C3aR−/− mice were nearly identical to those of C3aR+/+ littermates ( A). Histological analyses of joint sections taken from C3aR−/− and C3aR+/+ animals 2 wk after arthritis induction revealed similar degrees of inflammatory cell recruitment, fibroblast proliferation, and cartilage and bone destruction ( B). Thus, deletion of the C3aR appears to have no substantial effect on this animal model of arthritis. However, strikingly, in three independent experiments C5aR−/− mice were completely protected from disease induction as assessed by paw swelling (n = 5 per experiment). In contrast, wild-type animals consistently and synchronously developed arthritis 72 h after inoculation with the arthritogenic antibodies ( A). We removed joints from animals 14 d after arthritis induction and examined them for histological changes (). H&E stained sections from wild-type arthritic mice had large, obvious inflammatory cell infiltrates. Pannus formation was also evident with expansion of the synovial membrane and proliferation of the synovial lining fibroblasts. Lastly, erosion of the cartilage and bone surfaces was apparent, accompanied by invasion of the pannus tissue into the bone space, reminiscent of the severe changes associated with human RA. The most severe cases showed complete destruction of the normal joint architecture. Consistent with the clinical observation that C5aR−/− animals had reduced gross inflammation, joint sections from these animals appeared normal with thin synovial lining, only rare infiltrating inflammatory cells, and normal smooth cartilage surfaces indicating a lack of cartilage- and bone-erosive processes ( B).
Figure 2. C5aR−/− mice are protected from arthritis induction. (A) Mean clinical scores at days 0, 3, 4, 7, 9, and 14 of arthritis development in C3aR−/− (bottom) or C5aR−/− (top) mice and their littermate controls. (more ...)
Evidence for Antibody Deposition and Complement Activation.
The failure of C5aR−/− mice to develop arthritis could be due to prevention of the localization of anticollagen antibodies to the joint in the absence of the receptor. To exclude this possibility, we analyzed joint sections from C5aR+/+ and C5aR−/− animals after anticollagen antibody transfer for deposition on the cartilage surfaces. As expected, we observed no antibody deposition in joints from naive animals. In contrast, we readily detected antibody deposition in joints from both C5aR+/+ and C5aR−/− mice ( C, top). To additionally examine whether anticollagen deposition on articular surfaces in this model induces complement activation, we analyzed joint sections from naive and arthritic mice for the presence of C3, a marker of activation of both alternative and classical pathways, and C1q, a marker of classical pathway activation. We detected both C3 and C1q deposition on cartilage in diseased joints ( C, bottom). In comparison, low background staining was observed in sections stained with a control antibody ( C) and in joints from naive mice (not depicted). Thus, in this acute effector model of arthritis, the deposition of autoreactive antibodies on articular cartilage leads to the activation of the classical pathway of complement activation.
Quantitative Assessment of Joint Inflammation in C5aR-deficient Mice.
Although mice lacking the C5aR were clearly much less sensitive to arthritis induction than their wild-type littermates, we felt it important to quantitatively assess the influx of inflammatory cells into the joint as well as the induction of cytokines and proteases associated with arthritis progression. Therefore, we isolated RNA from the joints of C5aR−/− and C5aR+/+ animals 14 d after arthritis induction. We then compared the relative expression of CD4, CD68, and CD19 via quantitative PCR to assess the extent of the influx of T cells, macrophages, and B cells, respectively. C5aR+/+ mice showed clear increases in mRNA levels of CD4 and CD68 relative to naive joints, consistent with the influx of inflammatory cells into the joints () . In contrast, mRNA levels in the joints from C5aR−/− animals after arthritis induction were significantly lower than in wild-type animals. In fact, the levels of these markers remained within the range of naive animals, indicating a profound reduction in the number of T cells and macrophages recruited into the joint. Similarly, we analyzed the level of expression of several inflammatory markers, including IL-1β, TNFα, and MMP-3. The levels of mRNA for each of these genes were significantly increased in joints from arthritic wild-type mice in comparison to naive joints (). As before, mRNA levels in C5aR−/− animals remained at baseline levels 2 wk after injection of the arthritogenic mAbs. Similarly, we observed that OPGL mRNA levels increased substantially in wild-type mice during the course of disease, yet failed to change in C5aR−/− animals. This difference is likely to reflect the reduced osteoclastogenesis and bone erosion in C5aR−/− mice.
Figure 3. Quantitative assessment of inflammatory markers in C5aR+/+ and C5aR−/− mouse joints. RNA was extracted from the joints of naive wild-type or mAb-injected C5aR+/+ (+/+ A) or mAb-injected C5aR−/− (−/− A) mice (more ...)
Reduced Influx of Neutrophils into Joints of C5aR-deficient Mice.
The C5aR is expressed on and mediates chemotaxis of neutrophils, monocytes, and macrophages. In addition to C5a, other mediators of neutrophil recruitment, including the chemokines MIP-1α, MIP-2, and ENA-78, have been found at elevated levels in RA synovial tissue, synovial fluid, and peripheral blood. To assess the effect of C5aR deletion on the recruitment of neutrophils into the joint space after arthritis induction, we measured the relative expression of several chemokines known to recruit neutrophils via receptors other than the C5aR. In wild-type animals, MIP-1α, MIP-2α, and ENA-78 levels were strongly induced during the course of arthritis development. C5aR−/− animals, however, failed to show induction of any of these chemoattractants above the background levels present in joints from naive mice ( A). To directly assess the influx of neutrophils into the joints of C5aR−/− and C5aR+/+ mice, we prepared protein extracts from frozen joints 2 wk after arthritis induction. We then measured the levels of MPO enzyme activity in the joint extracts as a measure of neutrophil influx. A significant decrease (fourfold, P < 0.005) in levels of MPO was observed in the C5aR−/− animals relative to wild-type littermates, indicating a reduction in the recruitment of neutrophils into the joint as a consequence of C5aR deletion ( B). The decreased MPO enzyme levels reflect the profound reduction in the numbers of neutrophils present in the joints from C5aR−/− animals as is readily observed in joint sections at high magnification ( C).
Figure 4. Decrease in neutrophilia, chemokines, and adhesion receptor induction in C5aR−/− mice. (A) Neutrophil chemotactic factors ENA-78 (P < 0.0001, Student's t test), MIP-1α (P < 0.0001), and MIP-2 (P < 0.0001), (more ...)
Reduced Induction of Adhesion Molecule Expression in the Joints of C5aR-deficient Mice.
One mechanism by which proinflammatory cytokines and chemokines recruit leukocytes into the inflamed joint is through the induction of adhesion molecules on local endothelium and synovial fibroblasts themselves. intercellular adhesion molecule (ICAM)-1, vascular cell adhesion molecule (VCAM)-1, and E-selectin are three of the principal adhesion receptors known to be up-regulated during the course of arthritis. Therefore, we measured the quantitative induction of mRNA for these three molecules in C5aR−/− and wild-type mice during arthritogenesis. Each of these adhesion molecules was substantially up-regulated in joints from wild-type animals. However, in joints from C5aR−/− mice, VCAM-1 and E-selectin mRNA levels remained at baseline levels ( A). A slight induction of ICAM-1 was observed in C5aR-deficient joints, although the levels were significantly lower than in arthritic wild-type littermates. Therefore, the induction of the major adhesion molecules involved in the extravasation and localization of inflammatory cells in the arthritic joint is inhibited in C5aR−/− mice.