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We recently reported that interleukin-18 (IL-18) is highly expressed in malignant skin tumours such as melanomas, and may play a key role in the malignancy of such tumours. This study was designed to investigate the mechanisms of IL-18 regulation by vitamin C in B16F10 murine melanoma cells. Cells were treated with vitamin C, and the expression of IL-18 was measured by reverse transcription-polymerase chain reaction and intracellular flow cytometry analysis. Decreased IL-18 production and a significant reduction in IL-18 mRNA transcript were detected in cells treated with vitamin C. The effect of vitamin C treatment was blocked by the antioxidant N-acetyl-L-cysteine, suggesting that vitamin C affects IL-18 expression by up-regulating intracellular reactive oxygen intermediate (ROI) levels. To investigate whether the mitogen-activated protein kinase (MAPK) signalling pathway is involved in the downregulation of IL-18 production, cells were pretreated with SB203580, an inhibitor of p38 MAPK, prior to the addition of vitamin C. This pretreatment blocked the decrease in IL-18 production. However, vitamin C treatment enhanced the expression of phosphorylated p38 MAPK. Taken together, we conclude that vitamin C increases intracellular ROI levels, and regulates IL-18 production through the MAPK signalling pathway.

Introduction: Protein disulfide isomerase (PDI), a cell-surface localized oxidoreductase, plays a crucial role in regulating integrin-mediated platelet functions. Although PDI is detected on the neutrophil surface, its role in neutrophil recruitment during vascular inflammation remains unknown. Objectives: To determine the physiological role of neutrophil surface PDI in integrin-mediated neutrophil recruitment during vascular inflammation. Methods and Results: Using real-time fluorescence intravital microscopy in myeloid-specific PDI knockout mice, we have demonstrated that neutrophil PDI is required for neutrophil recruitment into the site of TNF-alpha-induced cremaster venular inflammation. Infusion of wild-type PDI but not functionally-inactive PDI rapidly rescued the defect of neutrophil recruitment in the PDI knockout mice. Cell-impermeable PDI inhibitors also perturbed neutrophil recruitment to the site of TNF-alpha-inflamed venular inflammation in wild-type mice. Neutrophil recruitment was further inhibited when blocking anti-PDI antibodies were infused into alphaLbeta2 but not alphaMbeta2 knockout mice, suggesting that extracellular PDI regulates alphaMbeta2-mediated neutrophil recruitment. To further investigate the role of neutrophil surface PDI in integrin-mediated neutrophil adhesion during vascular inflammation, adhesion assays were performed using human neutrophils treated with cell-impermeable PDI inhibitors. PDI inhibition impaired human neutrophil adhesion to TNF-alpha-activated endothelial cells under venous shear and to ICAM-1-coated surfaces under static conditions. Neutrophil adhesion was further inhibited when anti-PDI was combined with anti-alphaL but not anti-alphaM antibodies. PDI inhibitors also perturbed fibrinogen binding to activated alphaMbeta2 integrin. Using PDI shRNA in neutrophil-like HL60 cells, we found that knockdown of PDI gene diminished alphaMbeta2-mediated cell adhesion to activated endothelial cells under shear. Immunoprecipitation assays and confocal microscopy revealed that surface PDI-alphaMbeta2 interaction is enhanced upon neutrophil activation and regulated by the isomerase activity. Conclusion: Our results indicate that neutrophil surface PDI regulates the alphaMbeta2-ligand interaction, thereby playing an important role in neutrophil recruitment to the activated endothelium during vascular inflammation.