Anti-CypA single-domain antibody A1 inhibits cell migration and matrix metalloproteinase production in human monocytes/macrophages. The production of total matrix metalloproteinase (MMP)-9 and MMP-2 were tested by gelatin zymogram. (A), (C) and (E) Representative photographs of gelatin zymogram using culture supernatants of monocytes/macrophages derived from THP-1, rheumatoid arthritis (RA) patients’ peripheral blood and RA synovial fluid (SF) respectively. (B), (D) and (F) Statistical results of the density of total MMP-9 and MMP-2 produced by monocytes/macrophages derived from THP-1, RA patients’ peripheral blood and RA SF respectively. Data were results from three independent experiments. The protein expression of total MMP-9 (including Pro-MMP-9 and MMP-9) is significantly inhibited by single domain antibody (sdAb) A1 treatment. Each bar represents the mean ± standard error of the mean (SEM) of each group. (G) Cell migration mediated by cyclophilin A (CypA) is blocked by anti-CypA sdAbA1 antibody. Chemotaxis assays were set up using the peripheral mononuclear cells from RA patients incubated in the presence of a single dose of N-formyl-Met-Leu-Phe (FMLP, positive control) plus cyclosporine A (CsA) or sdAbA1, a single dose of CypA (100 ng/ml) plus sdAbA1, CsA or control antibody sdAbE2 (no binding to CypA). A chemotactic index was calculated for each group by dividing the number of migrated cells in test wells by the number of cells that migrated to medium alone. Bar graphs show mean ± SEM for each group, with n = 3 wells per group. ###P <0.001 versus control group; **P <0.01, ***P <0.001 versus CypA group. dTHP-1, differentiated THP-1; uTHP-1, undifferentiated THP-1.