Circadian Rhythms of Granzyme B, Perforin, IFN-γ, and NK Cell Cytolytic Activity in the Spleen: Effects of Chronic Ethanol

Abstract

Recent studies show that alterations in the body’s biological rhythms can lead to serious pathologies, including cancer. Acute and chronic ethanol consumption impairs the immune system by causing specific defects in the cellular components of the innate immune response and by creating increased risk and susceptibility to infections and cancer. NK cells are critical for immune surveillance against infected and malignant cells. To assess whether NK cell function follows a circadian trend and to determine ethanol effects on this rhythm, we measured, over a 24-h period, mRNA and protein levels of granzyme B, perforin, and the cytokine IFN-γ, as well as NK cell activity, in the splenocytes of ad libitum-fed, pair-fed, and ethanol-fed Sprague Dawley male rats. Circadian rhythms were found in mRNA and protein levels of granzyme B, perforin, and IFN-γ. A circadian pattern was also detected in NK cell cytolytic activity. Our data further demonstrated how chronic ethanol suppressed NK cell activity by directly disrupting the circadian rhythms of granzyme B, perforin, and IFN-γ. These findings identify the circadian functions of splenic NK cells and show the vulnerability of these rhythms to chronic ethanol.

Circadian Oscillations of Clock Genes, Cytolytic Factors, and Cytokines in Rat NK Cells

“…It is worth noting that the rhythms in protein levels of cytolytic factors and cytokines run in antiphase to those of mRNA levels. Furthermore, granzyme B, perforin, and IFN-␥ all showed a significant peak in protein levels at ZT19, exactly when the peak in NK cell cytolytic activity has been reported (25).…”

“…Although some differences exist when comparing the circadian patterns, both studies coincide in showing the highest mRNA levels of granzyme B, perforin, and IFN-␥ during the light period and the highest protein levels of granzyme B, perforin, and IFN-␥ during the dark period. The fact that the circadian rhythms in both mRNA and protein levels of granzyme B, perforin, and IFN-␥ were found to be similar (Ϯ4 h shifts) in both enriched NK cells and in the spleen (25) leads to several conclusions: 1) these circadian rhythms are conserved and reproducible in rats, 2) NK cells and other splenocytes may share similar rhythms in the expression of granzyme B, perforin, and IFN-␥, or 3) the circadian nature of the cytolytic function may be exclusive of NK cells. Regarding the latest, it would be expected for innate immune mechanisms such as NK cell function to have developed a circadian regulation to optimize their activity when most needed.…”

“…In addition, we demonstrate circadian expression of granzyme B, perforin, IFN-␥, and TNF-␣, which emphasizes the circadian nature of NK cell function. We have used previously whole rat spleens to identify the mRNA and protein rhythms of granzyme B, perforin, and IFN-␥ (25). In contrast, in this study we used enriched NK cells from rat spleens to analyze the rhythms of granzyme B, perforin, IFN-␥, and TNF-␣.…”

“…More specifically, previous works have pointed to changes in NK cell activity at different times of the day (22)(23)(24). We have recently published a conclusive study that describes the circadian rhythm in NK cell cytolytic activity in rats (25). Still, the underlying mechanisms governing NK cell circadian function are largely unknown.…”

Implementation of the EGFP‐K562 flow cytometric NK test: Determination of NK cytotoxic activity in healthy elderly volunteers before and after feeding

“…Our data also show an increasing capacity of NK cells to kill target cells. These results have prompted us to conduct further studies to analyze the number and activity of molecules involved in the mechanism of cytotoxicity, such as granzymes and perforins (35).…”