Journal: Immunity, Inflammation and Disease
Article Title: Phosphocholine‐containing ligands direct CRP induction of M2 macrophage polarization independent of T cell polarization: Implication for chronic inflammatory states
doi: 10.1002/iid3.112
Figure Lengend Snippet: Fc receptor engagement and signaling by CRP and its ligands. Fc receptors were blocked by 10 μg/mL F(ab’) 2 preparations of antibodies against specific receptors or combinations of receptors. RI (CD64), RII (CD32, clone 41H16), and RII/III (CD16/32) represent blocks against FcγRI‐III, and human Fc block acts against all receptors as a positive control. Counts of M2 macrophages were performed after 4 days of TEM after treatment with (a) PC + CRP ( n = 4 in triplicate, * p < 0.05) or (b) liposomes (lipid) alone or with CRP ( n = 5 in triplicate, * p < 0.01). (c) To measure activation of cells via FcR, the phosphorylation of SYK was measured by an In‐cell Western assay, here expressed as the percentage of the total SYK that was phosphorylated at position 323 (pSYK323) ( n = 5, * p < 0.05). (d) Cells were treated with or without liposomes in the presence of CRP or 10 μg of IVIG or both ( n = 5, * p < 0.001).
Article Snippet: The human Fc block, isotype controls, and blocking antibodies against FcγRs CD16/32 (FcγRII/III) and CD32 (FcγRII) were obtained from BD Pharmingen (San Diego, CA), against CD64 (FcγRI) from eBioscience (San Diego, CA), against the R131 variant of FcγRII (clone 41H16 hybridoma) from Dr. Ted Zipf (MD Anderson Cancer Center, Houston, TX, retired) and their F(ab’) 2 fragments purified (Pierce F(ab’) 2 Preparation kit, Thermo Scientific, Pittsburgh, PA) before addition at 10 μg per well.
Techniques: Blocking Assay, Positive Control, Activation Assay, In-Cell ELISA