O05 - Thrombin-processed Ecrg4 is chemotactic and recruits inflammatory monocytes to the injury site

Jisook Lee, Stu Knowling, Raul Coimbra, Vishal Bansal, Andrew Baird, Brian Eliceiri, Todd Costantini, University of California San Diego

Background: Leukocytes utilize protease-mediated shedding of membrane-anchored factors as a mechanism to localize the release of chemotactic factors at sites of injury. We have recently shown that cell surface Ecrg4 is shed from granulocytes following LPS treatment, where shed Ecrg4 activates NF-kB signaling. These findings suggest that Ecrg4 may play an important role in recruiting inflammatory cells to sites of injury, which is consistent with our observations of loss of cell surface Ecrg4 expression in patients following trauma and burn injury. Here we further define the function of Ecrg4 in mediating the injury response by characterizing its cleavage and determining whether shed Ecrg4 can recruit inflammatory monocytes.

Hypothesis: We hypothesized that Ecrg4 is processed by thrombin and that monocyte recruitment to sites of injury will be impaired in Ecrg4 KO mice.

Methods: To determine if Ecrg4 is processed by thrombin, recombinant Ecrg4 peptide was incubated with thrombin in vitro and cleavage was assessed by immunoblotting. To evaluate the effect of Ecrg4(133-148) on the recruitment of inflammatory monocytes to sites of brain injury, we performed stereotaxic injection of the Ecrg4(133-148) peptide followed by dissociation of the brain and flow cytometry three days post-injury. To validate the role for Ecrg4 as a chemotactic factor, subcutaneous implants of polyvinyl alcohol (PVA) sponges were implanted into Ecrg4 WT and Ecrg4 KO mice, and infiltrated cells were isolated and analyzed by flow cytometry.

Results: Incubation of recombinant Ecrg4 polypeptide with thrombin confirmed that thrombin cleavage released a C-terminal fragment of Ecrg4, Ecrg4(133-148). We observed a significant Ecrg4(133-148) -mediated recruitment of CD11b+ CD45high inflammatory monocytes following stereotaxic injection in the brain, while Ecrg4(133-148) peptide did not affect recruitment of CD11b+ CD45low microglia at day 3. In Ecrg4 KO mice, there was a decreased recruitment of CD11b+ Gr-1high immature myeloid cells to the injury site in the Ecrg4 KO mice compared with Ecrg4 WT mice.

Conclusions: Together, these findings suggest a chemotactic role for thrombin-processed Ecrg4(133-148) to mediate a pro-inflammatory injury response by recruiting inflammatory myeloid cells.