The Effects of Oxidative Stress on Dendritic Cell Migration and T-Cell Interaction
Ibrahim Batal, Jamil Azzi, Marwan Mounayar, Bechara Mfarrej, Reza Abdi. Brigham and Women's Hospital, Boston, MA; Brigham and Women's, Boston, MA
Background: In ischemia reperfusion injury (IRI), donor-derived dendritic cells (DDDCs) play a pivotal role in accelerating rejection. The specific mechanisms by which DDDCs increase allogenicity have not been systematically investigated.
Design: Bone marrow-derived dendritic cells (DCs) generated from C57BL/6 (B6) mice were utilized. H2O2, a well-accepted In Vitro model for IRI, was used to induce oxidative stress (OXS). H2O2-treated DCs (OXS-DCs) were compared to non-treated DCs (NT-DCs).
Results: The ability of DDDCs to mount rejection depends on their efficient migration. OXS-DCs upregulated CCR7, a specific receptor for CCL21 expressed on lymphatics and lymphoid tissues. Compared to NT-DCs, OXS-DCs revealed increased CCL21-induced Transwell migration (TWM) (p=0.006). This increased migration is dependent on OXS and protein synthesis because it was abrogated by pretreatment with Catalase and Cycloheximide. Furthermore, OXS-DCs showed pAKT and pNFKB activation. This prompted us to study the role of PI3Kγ and NF-KB in DCs migration. OXS-DCs from PI3Kγ-/- mice showed decreased TWM compared to OXS-DCs from wild-type (p=0.04). The latter also revealed decreased TWM when pretreated with NF-KB inhibitor (p=0.004).
Besides migration, DDDCs need to activate host T-cells to trigger rejection. Compared to NT-DCs, OXS-DCs upregulated CD80 and CD86 costimulatory receptors and increased allogeneic splenocyte proliferation measured by thymidine uptake. To explore whether this lymphocytic proliferation is cytotoxic vs. helper cell, we utilized transgenic OTI and OTII mice. TCR of these mice react specifically with OVA MHC-I and II restricted residues, respectively. DCs were incubated with OVA-restricted MHC-I and II and co-cultured with CD8+OTI and CD4+OTII cells, respectively. CD4+OTII incubated with OXS-DCs revealed increased proliferation assessed by CFSE (p=0.02) and IFN-G secretion assessed by ELISPOT (p=0.009) compared to these incubated with NT-DCs. The results for CD8+OTI were less significant. Finally, proinflammatory cytokines of DCs supernatant were measured by Luminex. OXS-DCs showed increased IL-6 secretion. T-regulatory (T-reg) cells were then assessed since IL-6 is a potential inhibitor of T-reg formation. In a fully allogeneic MLR, decreased CD4+ Treg:effector ratio was observed following incubation with OXS-DC compared to NT-DCs (p<0.001).
Conclusions: OXS increases DCs migration via PI3Kγ and NF-KB and alloactivation of effector but not T-reg CD4+ cells. These findings might help guiding future therapy to induce tolerance by targeting DDDCs.
Category: Kidney (does not include tumors)
Monday, March 19, 2012 9:30 AM
Poster Session I Stowell-Orbison/Surgical Pathology/Autopsy Awards Poster Session # 250, Monday Morning