Smokeless tobacco and nicotine bring about excessive cytokine responses of murine memory T-cells

Abstract

To determine the influence of smokeless tobacco (ST) and nicotine on the cytokine phenotype of memory T-cells, splenic mononuclear cells (SPM) were exposed to 1:10 2 or 1:10 3 dilutions of ST extract (ST-SPM), 10 or 100 μg/ml nicotine (NIC-SPM), or medium (CON-SPM) during 4 days of stimulation with anti-CD3. SPM were then washed extensively to remove residual ST or nicotine and restimulated with anti-CD3 and anti-CD28 in the absence of ST or nicotine. Expression of IL-2, IL-4, IL-10 and IFN- γ protein and mRNA levels after 4 days of primary stimulation and after 24 and 48 h of restimulation was evaluated using ELISA and RT-PCR, respectively. After 4 days of primary stimulation, SPM exposed to 100 μg/ml nicotine sustained expression of IL-2, IFN- γ, IL-10 and IL-4 mRNA as opposed to CON-SPM. Restimulation of CON-SPM resulted in maximum re-expression of cytokine mRNA at 24 h and a decline by 48 h. Restimulated NIC-SPM in the absence of nicotine delayed maximal re-expression of IL-2, IFN- γ, IL-10 and IL-4 mRNA until 48 h. Heightened expression of cytokine mRNA at 48 h was paralleled by a small but significant increase in production of IFN- γ, IL-4 and IL-10 protein by NIC-SPM as measured by ELISA. In contrast, ST-SPM did not exhibit residual expression of cytokine mRNA after 4 days of primary stimulation. Like NIC-SPM, however, restimulated ST-SPM exhibited maximum IL-2, IL-4, IFN- γ and IL-10 mRNA at 48 h. Heightened re-expression of cytokine mRNA at 48 h by ST-SPM was paralleled by increased production of IL-2, IFN- γ, IL-4 and IL-10 protein. These results indicate that exposure of T-cells to nicotine, but not ST, during a primary immune response result in inordinate cytokine expression after 4 days. In addition, memory T-cells initially exposed to nicotine or ST during a primary immune response, exhibit excessive cytokine expression when T-cells are restimulated in the absence of nicotine or ST.