Targeted label‐free quantification of interleukin‐8 in PMA‐activated U937 cell secretome by nanoLC‐ESI‐MS/MS‐sSRM

Abstract

Monocytes are a part of the innate immune system. Their differentiation into macrophages changes their cellular proteome and secretome. Particularly secretome components such as cytokines are crucial for immune response and inflammation in many diseases. Differentiation of human lymphoma cell line U937 can be triggered by phorbol 12-myristate 13-acetate (PMA). Screening of the cytokine release in U937 upon PMA stimulation by cytometric bead array almost exclusively showed interleukin-8 (IL-8). Next, a label-free nanoLC-ESI-MS/MS-sSRM method for quantification of IL-8 in the cell secretome was established and applied to monitor the time kinetics of PMA treatment in different concentrations. Targeted secretome analysis was achieved by scheduled SRM-MS using one proteotypic peptide as precursor ion and four mass transitions. Label-free quantification was performed by external calibration using IL-8 standard. Validation results indicated that the method was suited for the quantification of IL-8 in the secretome. The maximal IL-8 release of 62.4 ng/mL was observed after incubating cells treated by 50 ng/mL PMA for 48 h. The method can now be used for quantification of IL-8 release from different cells under various conditions. Furthermore, it can be easily expanded to other secreted proteins detected by untargeted screening methods.

Proteomic Response of Human Umbilical Vein Endothelial Cells to Histamine Stimulation

“…The supernatants were collected and stored at −80 °C. The conditioned media (CM) were analyzed by CBA . Cytokine concentrations were analyzed following the manufacturer's instructions.…”

“…The conditioned media (CM) were analyzed by CBA. [15] Cytokine concentrations were analyzed following the manufacturer's instructions. The BD TM CBA Human Inflammatory Cytokine Kit was used for quantitative measurement of the protein levels of the human cytokines IL-8, IL-1β, IL-6, IL-10, TNF, and IL-12p70 in supernatants of cultured cells.…”

The effects of Phorbol 12-myristate 13-acetate concentration on the expression of miR-155 and miR-125b and their macrophage function-related genes in the U937 cell line

“…PMA also activates RhoA/ROCK signaling during monocyte to macrophage differentiation resulting in morphological changes and increased cell adherence (Yang et al, 2017). However, there is a wide range of PMA concentrations being used in the reported experiments, and this wide range could affect the macrophage status through mechanisms such as the increased transcription levels of adhesion molecules CD44, CD14, ICAM-1 or the extracellular matrix FN1 in high-dose PMA treatments (Song et al, 2015;Emirbayer et al, 2017). A previous study found that THP-1 macrophages differentiated in high concentrations of PMA rap-idly died following infection whereas those differentiated in low concentrations of PMA survived and were able to control the intracellular Salmonella Typhimurium bacteria similar to primary human macrophages (Starr et al, 2018).…”