Differential Expression of EphrinB2 by Human Leukocytes: A Potential Inflammatory Mediator of Iris Endothelial Cells

Abstract

Abstract: : Purpose: EphrinB2 (B2) and its co–receptor EphB4 (B4) mediate aspects of cell movement during development. We hypothesize that they also regulate leukocyte extravasation during inflammation. We previously reported the expression of B2 in peripheral blood leukocytes (PBLs) upon stimulation with TNFα. Here, we extend our analysis to individual leukocyte subsets and evaluate the ability of B2 to activate signaling pathways in human iris endothelial cells (HIEC). Methods: Unfractionated PBLs, neutrophils, B cells, CD4⁺ and CD8⁺ T cells were treated with TNFα [10ng/ml] or the chemokine, SDF1α, [40ng/ml] for 30 min to 24 hours and then analyzed for the presence of B2 mRNA by RT–PCR. Confluent HIEC were exposed to soluble B2/IgG–Fc chimera [sB2; 2µg/ml] from zero to 1hr and assayed by immunoblot for the signaling phosphoproteins (p), pErk1/2 & pRaf. Results: B2 mRNA was undetectable in unfractionated PBLs, but was induced by 0.5 hr stimulation with TNFα, peaking at 1–6 hrs, and undetectable at 24hrs. Interestingly, the time of B2 expression was limited to ∼3hrs when stimulated with SDF1α. Leukocyte subsets demonstrated differential B2 expression. Induction of B2 in neutrophils and CD4⁺ T cells by TNFα occurred at 1.5 and 3 hrs, respectively. Unexpectedly, B2 mRNA was detectable at the zero time point in CD8⁺ and B cells isolated by either negative or positive selection, and then diminished during 1–3hrs exposure to TNFα. Preliminary results of sB2–stimulated HIEC show a transient increase in the levels of pErk1 at 15 min and a decrease in both pErk1 and 2 at 45min to 1hr. No change in pRaf was observed during the same time period. Conclusions: Expression of B2 by PBLs and its modulation by TNFα or SDF1α suggest that juxtacrine signaling by the B2/B4 system potentially contributes to leukocyte endothelial dynamics. Unique PBL subtype expression patterns may contribute to temporal cell responses during inflammation. Preliminary analyses of intracellular signaling proteins indicate that HIEC are responsive to B2 and suggest that Erk1 and 2, but not Raf, are involved in B4’s signaling cascade. Altering B2/B4 signaling in inflammation may prove useful in the management of many diseases such as anterior uveitis.

Keywords: cell–cell communication • immunomodulation/immunoregulation • cell adhesions/cell junctions