Fig. 4

Effects of MPO-ANCA on CD206 expression in macrophages. A The indirect immunofluorescence (IF) showed that a representative of Mpo^−/− mice immunized with muMPO developed muMPO ANCA in neutrophils. B The presence of muMPO ANCA was determined by ELISA. The results showed that compared with control mice (n = 8), muMPO ANCA were positive in the immunized mice (n = 8). C Mouse bone marrow cells (BMCs) differentiated to BMDMs and total mRNA was extracted for validation of macrophages differentiation. We used F4/80 as the marker for mouse macrophages. The real-time PCR result showed that compared with untreated group, the expression of F4/80 in BMDMs increased with statistical significance (p < 0.001). The results confirmed the successful differentiation of bone marrow derived macrophages. D The MPO activity assay showed that MPO activity in BMDMs with muMPO ANCA positive serum group increased significantly in comparison with that in BMDMs with control serum (p < 0.05) or muMPO ANCA positive serum without BMDMs (p < 0.05). Such effects could be reversed by MPO inhibitor AZD5904 (p < 0.05). E, F BMDMs were treated with muMPO ANCA positive serum or control serum. The expression of MPO in BMDMs were determined by western blot (E) and analyzed by densitometry (F). Western blot showed the results from one of three independent preparations. The results were showed in cropped blots and densitometrical analysis were results from three independent cell preparations. G–I BMDMs were treated with serums from different groups of mice. The expression of CD206 was determined by western blot (G) and immunofluorescence (I). For the western blot, the membrane proteins of BMDMs were extracted and Na–K ATPase was used to verify equivalent loading (G) and shown in cropped blots. For the immunofluorescence, the slides of cells were stained for CD206 and DAPI (I). Densitometrical analysis (H) results shown were results from three independent cell preparations. Immunofluorescence and western blot showed the results from one of three independent preparations. J, K PBMC derived macrophages from healthy donors were treated with control IgG or MPO-ANCA enriched IgG. The expression of MPO were determined by western blot (J) and analyzed by densitometry (K). Western blot showed the results from one of three independent preparations. The results were showed in cropped blots and densitometrical analysis were results from three independent cell preparations. L PBMC derived macrophages from healthy donors were treated with control IgG or MPO-ANCA enriched IgG with or without AZD5904. The real-time PCR result showed that CD206 mRNA was significantly increased in the presence of MPO-ANCA enriched IgG and such effects could be counteracted by the presence of AZD5904. #p < 0.001, ##p < 0.05