ABSTRACT
Natural Killer (NK) cells are a subset of lymphocytes that are capable of killing tumor cells, virally infected cells and antibody coated cells. Tributyltin (TBT) is a toxic chemical used for various industrial purposes such as: slime control in paper mills, disinfection of circulating industrial cooling waters, anti-fouling agents in shower curtains and the preservation of wood. TBT can be found in edible items such as dairy products and fish. This study investigates the mechanism by which TBT exposure decreases the immune function of human NK cells, in vitro. Cytotoxic function, the expression of the cytotoxic proteins (granzyme B and perforin), and cAMP response element binding protein (CREB) phosphorylation were examined. NK cells exposed to 300 nM TBT for 1 h showed no significant decrease in cytotoxic function, levels of granzyme B and perforin, or phosphorylation of CREB. However, mRNA levels for the cytotoxic proteins were significantly decreased. A 24 h exposure to 200 nM TBT caused significant decreases in cytotoxic function, levels of granzyme B and perforin, and levels of granzyme B and perforin mRNA. When NK cells were exposed to 300 nM TBT for 1h followed by a 24 h period in TBT-free media, again there were significant decreases in NK cell cytotoxic function, levels of granzyme B and perforin and their mRNA. A 1h exposure to 300 nM TBT followed by a 48 h period in TBT-free media showed similar changes in cytotoxic function and levels of granzyme B and perforin as seen after 24 h in TBT-free media. Additionally, both of these exposures showed significant decreases in phosphorylation of CREB. These results indicate that TBT exposures can disrupt the transcription of granzyme B and perforin and that this disruption cannot be entirely accounted for by a decrease in phosphorylated CREB (phosphoCREB) levels.
Subject(s)
Killer Cells, Natural/drug effects , Killer Cells, Natural/metabolism , Membrane Glycoproteins/metabolism , Serine Endopeptidases/metabolism , Trialkyltin Compounds/toxicity , Adult , Biotransformation/drug effects , Blotting, Western , Cell Separation , Cell Survival/drug effects , Cyclic AMP Response Element-Binding Protein/metabolism , Female , Granzymes , Humans , In Vitro Techniques , Male , Perforin , Phosphorylation , Pore Forming Cytotoxic Proteins , RNA, Messenger/biosynthesisABSTRACT
Cytotoxic function of human natural killer (NK) cells is modulated by a variety of cytokines. Interleukins (IL) 2 and 12 are both potent stimulators of NK cell cytotoxic function. Tributyltin (TBT) is used in a variety of consumer products and industrial applications. TBT is found in dairy products, meat, and fish. We and others have shown that there are measurable levels of TBT in human blood. Butyltins appear to increase the risk of cancer and viral infections in exposed individuals. We have demonstrated that the ability of NK cells to kill tumor cells is greatly diminished after a l-h exposure to TBT and that this inhibition persists even after removal of the compound. In the current study we examine the effects of the NK-stimulatory ILs, IL2 and IL12, on the ability of NK cells to recover from the persistent inhibitory effects of a 1-h TBT treatment. Highly purified NK cells (> 95% CD16(+)) or a lymphocyte preparation containing both T lymphocytes and NK cells were treated with 300 nM TBT and then allowed to recover for 24 h, 48 h, 4 days, and 6 days in TBT-free media containing no interleukin, 1000 U/mL IL2, 20 ng/mL IL l2, or a combination of IL2 plus IL12. Tumor killing function was then tested using a radioactive chromium release assay. As seen in our previous studies there is no recovery of NK cell cytotoxic function even after a 6-day recovery period when no interleukin is present in the medium. However, there is significant recovery of NK cytotoxic function when IL2, IL12, or the combination of IL2 plus IL12 is present in the medium during the recovery period.
Subject(s)
Cytotoxicity, Immunologic/drug effects , Interleukin-12/pharmacology , Interleukin-2/pharmacology , Killer Cells, Natural/drug effects , Recovery of Function/drug effects , Trialkyltin Compounds/toxicity , Cell Adhesion/drug effects , Cells, Cultured , Cytotoxicity Tests, Immunologic , Cytotoxicity, Immunologic/immunology , Drug Synergism , Female , Humans , K562 Cells/immunology , Killer Cells, Natural/cytology , Killer Cells, Natural/immunology , Male , Recovery of Function/immunology , T-Lymphocytes/cytology , T-Lymphocytes/drug effects , T-Lymphocytes/immunologyABSTRACT
Despite mounting evidence on butyltin (BT) contamination and related immunotoxic effects on wildlife, very little is known about BT-associated immunotoxic effects on humans, particularly the effects on human natural killer (NK) lymphocyte function. Our earlier studies demonstrated that in vitro exposure to environmentally relevant concentrations of BTs negatively affect human NK cells and that there are measurable levels of BTs in human blood. In this study we examined whether the inhibition of NK cell cytotoxic function induced by a brief exposure (1 h) to BTs is reversible when the cells are allowed to recover in BT-free media for up to 6 days. Standard methods were used in chemical preparation, blood sampling, NK cell isolation, and 51-Chromium release assay. The results revealed that exposure to 300 nM TBT for 1 h caused an approximately 65- decrease in NK cytotoxic function, whether the lymphocytes were given as long as a 6-day recovery period or no recovery period. There was no recovery (nor any further loss) of NK cytotoxic function following removal of the compound. Exposure to 5 microM DBT for 1 h showed a 41% decrease in cytotoxic function with 0-h recovery and an 83% decrease after a 24-h recovery period. Thus, not only is there no significant recovery of NK cytotoxic function when the lymphocytes are allowed to incubate in BT-free medium for up to 6 days but there is additional loss of cytotoxic function. The results indicated that short-term exposure to BTs causes persistent negative effects on NK cell ability to kill cancer cells.
