The accumulation of myeloid-derived suppressor cells (MDSCs) has been observed in solid tumors and is correlated with tumor progression; however, the underlying mechanism is still poorly understood. In this study, we identified a mechanism by which tumor cells induce MDSC accumulation and expansion in the bladder cancer (BC) microenvironment via CXCL2/MIF-CXCR2 signaling. Elevated expression of CXCL2 and MIF and an increased number of CD33 MDSCs were detected in BC tissues, and these increases were significantly associated with advanced disease stage and poor patient prognosis (P<0.01). A positive association was observed between CXCL2 or MIF expression and the number of tumor-infiltrating CD33 MDSCs (P<0.01). Subsequently, we demonstrated that CD45CD33CD11bHLA-DR MDSCs from fresh BC tissues displayed high levels of suppressive molecules, including Arg1, iNOS, ROS, PDL-1 and P-STAT3, and stronger suppression of T-cell proliferation. Interestingly, these CD45CD33CD11bHLA-DR MDSCs exhibited increased CXCR2 expression compared with that in peripheral blood from BC patients or healthy controls (P<0.05). Chemotaxis assay revealed that bladder cancer cell line J82 induced MDSC migration via CXCL2/MIF-CXCR2 signaling in vitro. Mechanistic studies demonstrated that J82-induced MDSC trafficking and CXCR2 expression were associated with increased phosphorylation of p38, ERK and p65. Conversely, inhibition of the phosphorylation of p38, ERK or p65 decreased J82-induced MDSC trafficking and CXCR2 expression. CXCL2/MIF-stimulated activation of the mitogen-activated protein kinase and nuclear factor kappa B pathways in MDSCs was MyD88 dependent. Overall, our results identify the CXCL2/MIF-CXCR2 axis as an important mediator in MDSC recruitment and as predictors and potential therapeutic targets in BC patients.

A variety of cytoactive factors produced during injury and inflammation are known to activate the central nervous system (CNS) macrophage, the microglia. Since extracellular potassium levels are known to rise rapidly at sites of injury in the CNS, we examined the possibility that changes in extracellular potassium could mediate changes in microglial function. The effect of an increase in potassium concentration on microglial superoxide anion production was studied in cultured neonatal rat microglia. Rather than directly inducing superoxide anion production, exposure to media containing 25 and 55 mM potassium enhanced the production of superoxide induced by phorbol 12-myristate 13-acetate. This potentiation was blocked by nifedipine, a voltage-gated calcium channel blocker. Treatment of the microglia with BAY K 8644, an agonist for voltage-gated calcium channels, produced an enhancement of superoxide levels similar to that of potassium. Because these data indicated the presence of a voltage-gated calcium channel, we also examined whole cell current in cultured microglia. A small, voltage-dependent inward calcium current was seen that was increased by exposure of the microglia to BAY K 8644. The presence of a small but finite calcium influx via these channels may be an important factor in the regulation of intracellular microglial events such as activation of the NADPH oxidase and the consequent production of superoxide anion.