Targeting the chemotactic function of CD147 reduces collagen-induced arthritis

Abstract

CD147 is a type I transmembrane glycoprotein expressed on a wide variety of cell types, including all leucocytes. While CD147 is best known as a potent inducer of matrix metalloproteinases, it can also function as a regulator of leucocyte migration through its cell surface interaction with chemotactic extracellular cyclophilins. A potential role for CD147-cyclophilin interactions during inflammatory diseases, including rheumatoid arthritis (RA), is suggested from several studies. For example, CD147 expression is increased on reactive leucocytes in the synovial fluid and tissues of patients with arthritis. In addition, the synovial fluid of patients with RA contains high levels of extracellular cyclophilin A. In the current studies we investigated the contribution of the chemotactic function of CD147-cyclophilin interactions to joint inflammation using the mouse model of collagen-induced arthritis. Our data demonstrate that proinflammatory leucocytes, specifically neutrophils, monocytes and activated CD4(+) T cells, lose their ability to migrate in response to cyclophilin A in vitro when treated with anti-CD147 monoclonal antibody. Furthermore, in vivo treatment with anti-CD147 monoclonal antibody can reduce the development of collagen-induced arthritis in mice by >75%. Such findings suggest that CD147-cyclophilin interactions might contribute to the pathogenesis of RA by promoting the recruitment of leucocytes into joint tissues.

Figure 1

Mouse CD147 expression. Neutrophils, monocytes and activated CD4⁺ T cells were stained with anti-CD147, or isotype control antibody, followed by fluorescein isothiocyanate-conjugated anti-rat immunoglobulin G. Neutrophils and monocytes were distinguished based on forward scatter/side scatter characteristics and CD4⁺ T cells were identified by costaining with Cy-anti-CD4. Histograms show CD147 staining (open) versus isotype staining (filled).

Figure 2

Presence of cyclophilin A (CypA) in collagen-induced arthritis (CIA). (a) DBA/1J mice were immunized with collagen in complete Freunds' adjuvant and then challenged 3 weeks later with collagen in phosphate-buffered saline. Starting 7 days after challenge, inflammation was scored for individual joints. Data show mean (± SE) clinical scores with n = 6 mice per time-point. (b) Groups of mice were killed when they reached specific CIA scores and proteins extracted from joints were analysed by Western blot analysis using anti-CypA antibody. Data show mean band densitometry of lysates from naïve mice versus mice with intermediate or high CIA clinical scores.

Figure 3

Leucocyte migration mediated by cyclophilin A (CypA) is blocked by RL73.2 anti-CD147. Purified neutrophils, monocytes and CD4⁺ T cells were set up in Boyden chemotaxis chambers in the presence of extracellular CypA with or without anti-CD147 or isotype control monoclonal antibody. Bar graphs show mean (± SE) chemotactic index for each group, with n = 4 to n = 6 wells per group. Horizontal dashed lines denote the cutoff chemotactic index for significant migration. Statistical differences between anti-CD147 and isotype groups were established using a Student's t-test: ***P<0·001 and **P<0·01.

Figure 4

Pro-matrix metalloproteinase 9 (pro-MMP-9) secretion is not blocked by RL73.2 anti-CD147. MC57 mouse fibroblasts and purified mouse macrophages were cocultured in the presence of RL73.2 anti-CD147, or UM-8D6 anti-CD147, or relevant isotype control monoclonal antibodies. After 72 hr, culture supernatants were tested for pro-MMP-9 by enzyme-linked immunosorbent assay. Bar graphs show mean (± SE) concentrations of pro-MMP-9 detected in cultures. Statistical differences between anti-CD147 and isotype groups were established using a Student's t-test: **P<0·01.

Figure 5

Anti-CD147 intervention reduces the severity of collagen-induced arthritis (CIA). DBA1/J mice were immunized and challenged with collagen II to induce CIA. (a) Half the mice were treated on days 21–30 with RL73.2 anti-CD147 monoclonal antibody (mAb) while the other half were treated with an isotype control mAb. Graph shows the mean (± SE) score for each group (n = 9 per group). (b) Mice were killed at the peak differential between anti-CD147 and isotype treatment and joint proteins were extracted. Myeloperoxidase levels were established using TMB substrate and tumour necrosis factor-α (TNF-α) levels were measured by enzyme-linked immunosorbent assay. A Student's t-test was used to establish statistical significance at individual time-points or between groups: **P<0·01; *P<0·05.