Figure 4. fMLF-induced adhesion of human PMNs to IECs triggers the release of MPO.

Human PMNs (1×10⁶) were introduced to IEC monolayers (grown in 12 well tissue culture plates, ratio of 1 adherent PMN to 2 IEC cells) and allowed to adhere for 3 hours at 37°C in the presence or absence of a low concentration of fMLF (100nM) and/or PMN adhesion inhibitory anti-CD11b Ab (10µg/ml). Supernatants from the co-cultures were collected and PMN-MPs were isolated form these supernatants. (A) fMLF treatment facilitates increased PMN adhesion to IECs, which was reversed with the inhibition of CD11b. ** p<0.01. (B–D) Peroxidase activity in supernatants of co-cultured PMNs and IECs (B) and on PMN-MPs (C) following fMLF-induced PMN adhesion to IECs was quantified using an ABTS peroxidase activity assay. ** p<0.01. For panels A-C data are presented as an average of 3 independent experiments. (D) MPO presence on PMN-MPs was further confirmed using immunoblotting analysis. Blots are representative of 3 independent experiments (E) PMN-MP binding to IECs following PMN adhesion was examined using fluorescence microscopy. Representative immunofluorescence images show a robust attachment of MPO positive aggregates (green) consistent with the size of PMN-MPs (> 1µm) to IECs (visualized using DAPI nuclear stain, blue). PMNs were stained for CD11b (red). Images are representative of 3 independent experiments. The bar is 10µm.