NEGATIVE INFLUENCE of IL8 and RANTES Ctyokines On CORD BLOOD CD133+ CELL SDF-1-CXCR4 Function

Abstract 1173

Recruitment of hematopoietic CD133+ cells from the bone marrow (BM) to vascular injury is mediated by the chemoattractant SDF-1 binding to the CXCR4 receptor and signaling through Akt and MAPK pathways. Synthesis of inflammatory/angiogenic cytokines IL8 and RANTES decreases with age and chronic cardiovascular disease. In contrast to cord blood (CB) CD133+ cells and young adult BM, we observed patient-derived BM CD133+ cells have reduced CXCR4 expression and reduced SDF-1 migration. We sought to determine the relationship between IL8, RANTES, SDF-1 activation and CXCR4 expression/signaling. To test this hypothesis, we performed in vitro studies on selected CB CD133+ cells to determine surface expression of CXCR4 by flow cytometry, in vitro transmigration to SDF-1, and evaluation of p42/44 MAPK phosphorylation by Western blot comparing the treatment of CB-derived CD133+ cells individually and combined with IL8 and RANTES ligands and also compared (individually and in combination) the effect of neutralizing antibodies (R&D Systems) directed against the IL8 and RANTES ligands. Selected CD133+ cells, incubated overnight in DMEM/1% HSA media, were evaluated for surface expression of CXCR4. Selected CD133+ cells were resuspended in DMEM/1% HSA media with 500 ng/ml IL8 and 50 ng/ml RANTES ligands (combined) or ligands were added individually or with 500 ng/ml anti-IL8 and 50 ng/ml anti-RANTES antibodies or antibodies added individually. After a 16–24 hour incubation at 37°C, 5 × 10⁴ CB CD133+ cells were stained for CXCR4 or were added to 5-mm Transwells and allowed to migrate (3 hr) to DMEM/1% HSA media, or 50 or 200 ng/ml SDF-1. CB CD133+ cells phosphorylated proteins after IL8 exposure (24hr) exposed to SDF-1 (200 ng/ml) for 1 minute at 37°C were evaluated for intracellular kinases MAPK p42/44. Prior to all experiments, selected CD133+ cells expressed 54 ± 11% (N=4) of CXCR4 measured after sequential gating using anti-CD45-FITC antibodies and anti-CD133-APC antibodies.CD133+ cells exposed individually to IL8 (500 ng/ml) or to RANTES (50 ng/ml) ligands or to anti-IL8 neutralizing antibody or to anti-RANTES neutralizing antibodies showed no significant differences (61.9 ± 19.4% versus 57.6 ± 25.0%). Transmigration to 200 ng/ml SDF-1 was decreased 100% with a combination of anti-IL8 and anti-RANTES neutralizing antibodies and decreased 77% when CB CD133+ cells were exposed to a combination of 500 ng/ml IL8 and 50 ng/ml RANTES ligands. Transmigration of CB CD133+ cells to 50 ng/ml SDF-1 was decreased 38% when CB CD133+ cells were exposed 16–24 hours to a combination of ligand neutralizing anti-IL8/anti-RANTES antibodies and decreased 62% when CB CD133+ cells were exposed to a combination of 500 ng/ml IL8 and 50 ng/ml RANTES ligands. When CD133+ cells were exposed individually to IL8 (500 ng/ml), or to RANTES (50 ng/ml), or to anti-IL8 neutralizing antibody (500 ng/ml), or to anti-RANTES neutralizing antibody (50 ng/ml), similar inhibition of SDF-1 transmigration results was observed when using combinations of the ligands or to combinations of antibodies. Although exposure of CD133+ cells to 200 ng/ml SDF-1 results in increased phosphorylation of p42/44 MAPK, prior exposure of CD133+ cells to 500 ng/ml IL8 blocks this phosphorylation event. These results demonstrate that the proangiogenic and chemotactic factors IL8 and RANTES negatively influence the function of SDF-1 altering signaling via the CXCR4 receptor without affecting the expression the CXCR4 receptor. We hypothesis that the ability of short-term exposure of CD133+ cells to IL8 influences SDF-1-induced activation at a site in the intracellular pathway of activation involving MAPK signaling perhaps by sequestering Gαi or Gβγ subunits critical for cell migration.