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.
No relevant conflicts of interest to declare.
Asterisk with author names denotes non-ASH members.