Selective Loss of Viability of Mouse NK Cells in Culture is Associated with Decreased NK Cell Lytic Function

Abstract

Cell culture methods can allow investigation of the mechanisms responsible for immunotoxicity. Unfortunately, natural killer (NK) cells in rodent splenic cultures rapidly lose their cytolytic function. It is not known if death of NK cells or loss of function in viable NK cells is primarily responsible for this loss. Flow cytometry and an assay of NK cell lytic function were used to address this issue and to determine if NK cell viability could be maintained by adding selected cytokines or a caspase inhibitor to the cultures. Total cells and NK cells in untreated 18 h cultures were 79 +/- 1% and 25 +/- 2% viable, respectively, and these cultured splenocytes caused only 4 +/- 1% specific release of (51)Cr from YAC-1 target cells. Cultures including polyinosinic:polycytidylic acid (poly I:C) or IL-2 had increased NK cell viability (43 +/- 2%, 47 +/- 1%) and function (58 +/- 2 and 43 +/- 1% specific release). IL-15 significantly increased NK cell viability, but not function. Previous studies demonstrated that treatment of mice with immunotoxicants such as ethanol or corticosterone diminishes NK cell activation in vitro in response to poly I:C. To determine if alterations in viability are responsible for this decreased NK cell activity, lytic function and NK activity were measured in cultures of splenocytes treated in vivo or in vitro with ethanol and/or corticosterone. Some treatments reduced IL-2 or poly I:C-enhanced lytic activity in vitro, but there was no clear relationship between these changes in function and changes in the percentage of viable NK cells. Thus, immunotoxicants that suppress NK cell activation can be investigated in vitro because commonly used activating stimuli also permit NK cell survival. However, no agents were identified that could maintain NK cell viability and function in culture (without activation) to allow investigation of the direct effects of immunotoxicants on basal NK activity in vitro.

Utilization of mouse models to decipher natural killer cell biology and potential clinical applications

“…5 Murine NK cells exhibit weak lytic capabilities when freshly isolated and can only be maintained in culture with IL-2 for 2 weeks, after which time cell death and measurable decreases in cytotoxic function and cytokine production occur. 6 The differences in initial cytotoxic capabilities can be attributed to the differences in perforin and granzyme B translation and protein expression. In mice, perforin and granzyme B mRNA are constitutively transcribed, but minimal levels of protein are detected until stimulation or activation of the NK cells that leads to rapid translation.…”

Present and future ofin vitroimmunotoxicology in drug development

“…The predictive potential of such assays after in vitro exposure has been the subject of intensive investigation (Lebrec et al, 1995;Condevaux et al, 2001). The main limitation of the use of NK cell activity for in vitro studies is the rapidly loss of cell viability and of cytolytic function once cells are isolated (Hébert and Pruett, 2001). As a possible alternative, the use of NK cell lines, such as NK-92 or IMC-1, should be explored.…”

Characterization of the Action of Drug-Induced Stress Responses on the Immune System: Evaluation of Biomarkers for Drug-Induced Stress in Rats

“…Analysis of splenocytes and thymocytes by flow cytometry was performed as previously reported in our mouse studies with the exception of antibody selection (Hebert and Pruett, 2001). from BD Pharmingen (San Diego, CA).…”