Essential role for the C5a receptor in regulating the effector phase of synovial infiltration and joint destruction in experimental arthritis

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 factor alpha 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 synovial fluid and tissue, we performed expression analyses of chemoattractant receptors in a related 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.

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 monocytes (MONO), monocyte-derived macrophages (Mφ), and peripheral blood–derived granulocytes (GRAN). Data plotted are the mean ± SD of the relative expression of each receptor in RNA samples pooled from three different donors using β₂-microglobulin as a reference. The results are representative of three independent experiments. (B) Immunohistochemistry analysis of human RA synovial tissue stained with anti-C5aR, anti-C3aR, anti-CD68, or control IgG. Data shown are representative of expression patterns observed of four RA synovial tissues. (C) Quantitative PCR analysis of C5aR, C3aR, and inflammatory marker expression in mouse joint RNA during the course of arthritis induction. Data are the mean ± SD of the relative expression of each gene in two joints.

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 monocytes (MONO), monocyte-derived macrophages (Mφ), and peripheral blood–derived granulocytes (GRAN). Data plotted are the mean ± SD of the relative expression of each receptor in RNA samples pooled from three different donors using β₂-microglobulin as a reference. The results are representative of three independent experiments. (B) Immunohistochemistry analysis of human RA synovial tissue stained with anti-C5aR, anti-C3aR, anti-CD68, or control IgG. Data shown are representative of expression patterns observed of four RA synovial tissues. (C) Quantitative PCR analysis of C5aR, C3aR, and inflammatory marker expression in mouse joint RNA during the course of arthritis induction. Data are the mean ± SD of the relative expression of each gene in two joints.

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 monocytes (MONO), monocyte-derived macrophages (Mφ), and peripheral blood–derived granulocytes (GRAN). Data plotted are the mean ± SD of the relative expression of each receptor in RNA samples pooled from three different donors using β₂-microglobulin as a reference. The results are representative of three independent experiments. (B) Immunohistochemistry analysis of human RA synovial tissue stained with anti-C5aR, anti-C3aR, anti-CD68, or control IgG. Data shown are representative of expression patterns observed of four RA synovial tissues. (C) Quantitative PCR analysis of C5aR, C3aR, and inflammatory marker expression in mouse joint RNA during the course of arthritis induction. Data are the mean ± SD of the relative expression of each gene in two joints.

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. Each group of mice consisted of five animals. The results shown are representative of three separate experiments. (B) H&E stained joint sections from representative C3aR^−/−, C3aR^+/+, C5aR^−/−, and C5aR^+/+ mice 14 d after mAb transfer. Cartilage (C) surfaces are indicated in the images as are regions of pannus tissue (P) comprised of proliferating synoviocytes and infiltrating leukocytes. (C) Joints from naive C5aR^+/+ mice. Ab-injected C5aR^+/+ or Ab-injected C5aR^−/− mice were stained with an antibody against mouse IgG to detect deposition (brown staining, top). Inflamed joints from C5aR^+/+ mice 14 d after Ab transfer were stained with control IgG, anti-C3, or anti-C1q antibodies to detect the accumulation of complement components on the cartilage surfaces (bottom).

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. Each group of mice consisted of five animals. The results shown are representative of three separate experiments. (B) H&E stained joint sections from representative C3aR^−/−, C3aR^+/+, C5aR^−/−, and C5aR^+/+ mice 14 d after mAb transfer. Cartilage (C) surfaces are indicated in the images as are regions of pannus tissue (P) comprised of proliferating synoviocytes and infiltrating leukocytes. (C) Joints from naive C5aR^+/+ mice. Ab-injected C5aR^+/+ or Ab-injected C5aR^−/− mice were stained with an antibody against mouse IgG to detect deposition (brown staining, top). Inflamed joints from C5aR^+/+ mice 14 d after Ab transfer were stained with control IgG, anti-C3, or anti-C1q antibodies to detect the accumulation of complement components on the cartilage surfaces (bottom).

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. Each group of mice consisted of five animals. The results shown are representative of three separate experiments. (B) H&E stained joint sections from representative C3aR^−/−, C3aR^+/+, C5aR^−/−, and C5aR^+/+ mice 14 d after mAb transfer. Cartilage (C) surfaces are indicated in the images as are regions of pannus tissue (P) comprised of proliferating synoviocytes and infiltrating leukocytes. (C) Joints from naive C5aR^+/+ mice. Ab-injected C5aR^+/+ or Ab-injected C5aR^−/− mice were stained with an antibody against mouse IgG to detect deposition (brown staining, top). Inflamed joints from C5aR^+/+ mice 14 d after Ab transfer were stained with control IgG, anti-C3, or anti-C1q antibodies to detect the accumulation of complement components on the cartilage surfaces (bottom).

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 13 d after mAb transfer and analyzed via quantitative PCR analysis for the indicated genes. The levels of IL-1β, CD68, TNFα, CD4, MMP-3, and OPGL were significantly reduced in the joints from C5aR^−/− mice injected with the arthritogenic antibodies (−/− A) compared with injected C5aR^+/+ mice (+/+ A; P < 0.0001, Student's t test). Data are the mean ± SD of the relative expression of each gene in ten joints each from naive mice or from mAb-injected C5aR^−/− or C5aR^+/+ mice. The results are representative of three independent experiments.

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), and adhesion molecules ICAM-1 (P < 0.002), VCAM-1 (P < 0.0001), and E-selectin (P < 0.0001) were significantly reduced in joints from C5aR^−/− mice injected with the arthritogenic antibodies (−/− A) compared with injected C5aR^+/+ mice (+/+ A). Quantitative PCR data are expressed as described in Fig. 3. (B) Neutrophilia is reduced in joints from C5aR^−/− mice injected with the arthritogenic antibodies compared with injected C5aR^+/+ mice as assessed by an assay for MPO performed with protein extracts from five joints each from C5aR^+/+ and C5aR^−/− mice 14 d after mAb transfer (P < 0.005). (C) Neutrophilia (N) in arthritic C5aR^+/+ joints (top) is evident from examination of high powered fields of tissue sections stained with H&E. In contrast, only rare neutrophils were observed in joints from C5aR^−/− animals 14 d after mAb transfer. Arrowheads indicate region of lower power fields (left) enlarged in the high power fields (right). Cartilage (C), pannus (P), and synovial lining (S) are indicated.

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), and adhesion molecules ICAM-1 (P < 0.002), VCAM-1 (P < 0.0001), and E-selectin (P < 0.0001) were significantly reduced in joints from C5aR^−/− mice injected with the arthritogenic antibodies (−/− A) compared with injected C5aR^+/+ mice (+/+ A). Quantitative PCR data are expressed as described in Fig. 3. (B) Neutrophilia is reduced in joints from C5aR^−/− mice injected with the arthritogenic antibodies compared with injected C5aR^+/+ mice as assessed by an assay for MPO performed with protein extracts from five joints each from C5aR^+/+ and C5aR^−/− mice 14 d after mAb transfer (P < 0.005). (C) Neutrophilia (N) in arthritic C5aR^+/+ joints (top) is evident from examination of high powered fields of tissue sections stained with H&E. In contrast, only rare neutrophils were observed in joints from C5aR^−/− animals 14 d after mAb transfer. Arrowheads indicate region of lower power fields (left) enlarged in the high power fields (right). Cartilage (C), pannus (P), and synovial lining (S) are indicated.

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), and adhesion molecules ICAM-1 (P < 0.002), VCAM-1 (P < 0.0001), and E-selectin (P < 0.0001) were significantly reduced in joints from C5aR^−/− mice injected with the arthritogenic antibodies (−/− A) compared with injected C5aR^+/+ mice (+/+ A). Quantitative PCR data are expressed as described in Fig. 3. (B) Neutrophilia is reduced in joints from C5aR^−/− mice injected with the arthritogenic antibodies compared with injected C5aR^+/+ mice as assessed by an assay for MPO performed with protein extracts from five joints each from C5aR^+/+ and C5aR^−/− mice 14 d after mAb transfer (P < 0.005). (C) Neutrophilia (N) in arthritic C5aR^+/+ joints (top) is evident from examination of high powered fields of tissue sections stained with H&E. In contrast, only rare neutrophils were observed in joints from C5aR^−/− animals 14 d after mAb transfer. Arrowheads indicate region of lower power fields (left) enlarged in the high power fields (right). Cartilage (C), pannus (P), and synovial lining (S) are indicated.

Figure 5.

Model for the inflammatory cascade of arthritis. After the initiation of disease as a result of the activation of autoreactive T and B cells, rheumatoid factor and other autoantibodies are produced and deposited in the joint spaces. Immune complex formation leads to the activation of the complement cascade, ultimately yielding C5a. The C5a gradient emanating from the joint recruits and activates neutrophils and monocytes to the synovium and synovial fluid. In addition, C5a may directly activate endothelial cells to up-regulate adhesion receptor expression, promoting extravasation of inflammatory cells. Activated neutrophils liberate additional chemotactic factors that recruit additional inflammatory cell subsets, particularly monocyte/macrophage lineage cells. These latter cells, a primary source of TNFα and IL-1β, activate resident synovial fibroblasts. Activated synovial fibroblasts produce additional effector molecules, including cytokines, chemokines, and proteases. The end result is a cycle of positive feedback that amplifies the inflammation and results in cartilage and bone degradation.