Neutrophil Proteinase (PR3) Regulates Neutrophil Transendothelial Cell Migration.

Abstract 1492

Neutrophil transmigration requires the localization of neutrophils to endothelial cell junctions where receptor-ligand interactions between these cells promotes leukocyte diapedesis. Neutrophils contain several different proteases which are thought to play a role in aiding in transendothelial cell migration, either by degrading extracellular matrix components or junctional proteins, or by inducing endothelial cell activation. Proteinase 3 (PR3) is a serine protease stored in azurophil granules that is released by activated neutrophils and can rebind to the neutrophil expressed cell surface protein NB1 (CD177). The neutrophil marker NB1 is expressed on a subset of neutrophils (∼50%) and has recently been demonstrated to be a heterophilic binding partner for PECAM-1, a protein highly expressed at endothelial cell junctions. Disrupting NB1-PECAM interactions has been reported to significantly inhibit neutrophil transmigration. Because of the critical role of NB1 in neutrophil transmigration we believe that the interactions between NB1 and PECAM-1 have the potential to localize PR3 to endothelial cell junctions where it may aid in leukocyte transmigration.

For this study we sought to test the hypothesis that NB1-PR3 interactions contribute to neutrophil transmigration. Human umbilical vein endothelial cells (HUVEC) were cultured on transwell membranes, treated with IL-1β, TNFα or fMLP and then incubated with NB1+ or NB1- PMN. Using flow cytometry we observed that transmigration alone resulted in a significant increase in PR3 expression on NB1+ but not NB1- neutrophils. Using a pan-serine protease inhibitor (AEBSF) total neutrophil transmigration was significantly inhibited. However, using a highly specific PR3 inhibitor (Elafin) we observed a selective inhibition in NB1+ but not NB1- neutrophil transmigration on IL-1β stimulated HUVEC. Similarly, antibodies against the NB1 recognition site on PECAM (Ig domain 6) inhibited neutrophil transmigration of NB1+ but not NB1- cells. Interestingly, in the presence of different stimuli (TNFα, fMLP), neutrophil transmigration was significantly less dependent on NB1-PECAM interactions and inhibition of PR3 activity did not inhibit transmigration. This is despite the fact that PR3 expression was highly up-regulated on NB1+ neutrophils incubated with either of these stimuli or following neutrophil transmigration.

In conclusion, the serine protease PR3 appears to play a significant role in the transmigration of NB1+ but not NB1- neutrophils. Likewise, the contribution of NB1 and PR3 in neutrophil transmigration is regulated in a stimulus-dependent mechanism which involves NB1 interactions with PECAM. These data therefore suggest that NB1 and PR3 may regulate recruitment of a neutrophil subset in response to specific inflammatory signals and this regulation may play a role in modulating the immune response.