To detect the differences in macrophage differentiation,
ANOVA for paired samples was used, followed by Fisher’s least significant different test. Correlations were BGB324 purchase evaluated by Spearman’s test. The criterion of significance was set to p < 0.05. To investigate the effect of soluble Aβ-peptides on the phagocytosis of PSPs by freshly isolated human monocytes, the cells were pre-incubated with 1 μg/ml of the respective Aβ-peptide in cell culture medium. Then, 20 h after adding the fluorescent PSPs, phagocytosis was quantified by flow cytometry. The MFI of the phagocytes was used as a measure of the number of internalized fluorescent particles. The pre-incubation of monocytes with Aβ(1–42) as well as the N-terminally truncated Aβ(2–40) and Aβ(2–42), but not Aβ(1–40), induced phagocytosis at levels significantly above the control levels (p < 0.05) ( Fig. 1A). Monocytes treated with Aβ(2–40) internalized 17% more PSPs than those treated with full-length Aβ(1–40) (p < 0.05). The treatment of cells
with BSA did not influence the phagocytosis of PSPs. To assess whether the Aβ-peptides secreted http://www.selleckchem.com/products/Gefitinib.html by monocytes enhanced phagocytosis by binding to pathogens, the effect of Aβ-coated PSPs on their phagocytosis by human monocytes was examined. The phagocytosis of fluorescent particles was quantified by flow cytometry as described above (Fig. 1B). Precoating the fluorescent PSPs with all of the tested Aβ-peptides increased their phagocytosis by monocytes compared to the phagocytosis of uncoated PSP (p < 0.001). Coating the PSPs with Aβ(1–42) enhanced the amount of phagocytosed PSPs by 40% (p < 0.0001). Aβ(1–42) induced phagocytosis more effectively than Aβ(1–40) (p < 0.0001). The treatment
of monocytes with Aβ(2–42)− and Aβ(3p–42)-coated PSPs resulted in an even higher increase of the MFI values by 53% (p < 0.0001) and 56% (p < 0.0001), respectively. This result indicates that an additional Inositol monophosphatase 1 N-truncation of Aβ(1–42) further increased the phagocytosis of PSPs. In contrast to the treatment of monocytes with soluble Aβ-peptides, pre-incubation with n-truncated Aβ(2–40) did not enhance phagocytosis more effectively than Aβ(1–40). Because undifferentiated monocytes are poor phagocytes, cytochalasin D only weakly reduced phagocytosis. Phagocytosis of pHrodo Green-labeled E. coli, which is only fluorescent at an acidic pH, revealed that cytochalasin D completely inhibited phagocytosis in our setting ( Fig. 4F). Therefore, increased signal intensity after pretreatment with cytochalasin D and coincubation with permanently fluorescent prey indicated its binding to the phagocyte surface without internalization. To investigate whether the differential effects of the Aβ-peptides were due to different binding affinities for the PSPs, the amount of Aβ-peptide bound to the PSPs was quantified by immunostaining with the C- and N-terminal non-specific Aβ antibodies 6E10 and 4G8.