Protein phosphorylation profiling identifies potential mechanisms for direct immunotoxicity

Abstract

Signaling networks are essential elements that are involved in diverse cellular processes. One group of fundamental components in various signaling pathways concerns protein tyrosine kinases (PTK). Various toxicants have been demonstrated to exert their toxicity via modulation of tyrosine kinase activity. The present study aimed to identify common cellular signaling pathways that are involved in chemical-induced direct immunotoxicity. To this end, an antibody array-based profiling approach was applied to assess effects of five immunotoxicants, two immunosuppressive drugs and two non-immunotoxic control chemicals on the phosphorylation of 28 receptor tyrosine kinases and 11 crucial signaling nodes in Jurkat T-cells. The phosphorylation of ribosomal protein S6 (RPS6) and of kinases Akt, Src and p44/42 were found to be commonly regulated by immunotoxicants and/or immunosuppressive drugs (at least three compounds), with the largest effect observed upon RPS6. Flow cytometry and Western blotting were used to further examine the effect of the model immunotoxicant TBTO on the components of the mTOR-p70S6K-RPS6 pathway. These analyses revealed that both TBTO and the mTOR inhibitor rapamycin inactivate RPS6, but via different mechanisms. Finally, a comparison of the protein phosphorylation data to previously obtained transcriptome data of TBTO-treated Jurkat cells resulted in a good correlation at the pathway level and indicated that TBTO affects ribosome biogenesis and leukocyte migration. The effect of TBTO on the latter process was confirmed using a CXCL12 chemotaxis assay.

A comprehensive battery of flow cytometric immunoassays for the in vitro testing of chemical effects in human blood cells

“…In addition to the use of primary human immune cells, there are increasing attempts to establish rodent and human cell lines for chemical testing to avoid the use of living donors. For example, Jurkat T cell line has been used for chemical testing in reporter gene assays ( 22 ), at the level of gene expression by PCR ( 23 ) and for phosphorylation of intracellular proteins ( 24 ). However, these malignant cells are not the true equivalents of primary cells, neither in their phenotypic markers nor in their functionality.…”