Figure 3. PMNs can release soluble and MP-associated MPO.
(A) Expression of MPO under the specified activation conditions (1×106 PMN/treatment) was examined by immunoblotting of PMN cell lysates, supernatants and on PMN-MPs. Lt-B/fMLF stimulation resulted in decreased MPO levels in PMN lysates and appearance of MPO in cell supernatants and in association with PMN-MPs, consistent with its release by PMNs. Equal protein loading in cell supernatants was confirmed using Ponceau staining. (B) The presence of PMN-MPs, (particles smaller than 1µm) in supernatants of activated PMNs was confirmed by flow cytometry (left panel). PMN-MPs were further characterized by flow cytometry for the expression of membrane marker, Annexin V and MPO. (C) Transmission electron microscopy micrograph depicts a representative PMN-derived MP. Bars are 300nm and 150nm (left and right panels, respectively). For A, B, and C the images are representative of 3 independent experiments. (D–E) Assay for peroxidase activity using ABTS as the substrate was performed. (D) MPs derived from increasing numbers of Lt-B/fMLF stimulated PMNs exhibited higher MPO activity. (E) PMNs (1×106 PMN) were stimulated with fMLF, PMA or Lt-B/fMLF and MPO activity in cell lysates, supernatants and MPs was determined. * p<0.05, ** p<0.01, *** p<0.01 different from the appropriate control. ns not significantly different from control.