Natural Killer Cell Lytic Function Research Articles

Phosphoinositide-3-Kinase Signaling in Human Natural Killer Cells: New Insights from Primary Immunodeficiency.

Human natural killer (NK) cells play a critical role in the control of viral infections and malignancy. Their importance in human health and disease is illustrated by severe viral infections in patients with primary immunodeficiencies that affect NK cell function and/or development. The recent identification of patients with phosphoinositide-3-kinase (PI3K)-signaling pathway mutations that can cause primary immunodeficiency provides valuable insight into the role that PI3K signaling plays in human NK cell maturation and lytic function. There is a rich literature that demonstrates a requirement for PI3K in multiple key aspects of NK cell biology, including development/maturation, homing, priming, and function. Here, I briefly review these previous studies and place them in context with recent findings from the study of primary immunodeficiency patients, particularly those with hyperactivating mutations in PI3Kδ signaling.

Activation of protein kinase C and protein kinase D in human natural killer cells: effects of tributyltin, dibutyltin, and tetrabromobisphenol A

Up to now, the ability of target cells to activate protein kinase C (PKC) and protein kinase D (PKD) (which is often a downstream target of PKC) has not been examined in natural killer (NK) lymphocytes. Here we examined whether exposure of human NK cells to lysis sensitive tumor cells activated PKC and PKD. The results of these studies show for the first time that activation of PKC and PKD occurs in response to target cell binding to NK cells. Exposure of NK cells to K562 tumor cells for 10 and 30 min increased phosphorylation/activation of both PKC and PKD by roughly 2-fold. Butyltins (tributyltin (TBT), dibutyltin (DBT)) and brominated compounds (tetrabromobisphenol A (TBBPA)) are environmental contaminants that are found in human blood. Exposures of NK cells to TBT, DBT, or TBBPA decrease NK cell lytic function in part by activating the mitogen-activated protein kinases (MAPKs) that are part of the NK lytic pathway. We established that PKC and PKD are part of the lytic pathway upstream of MAPKs and thus we investigated whether DBT, TBT, and TBBPA exposures activated PKC and PKD. TBT-activated PKC by 2–3-folds at 10 min at concentrations ranging from 50 to 300 nM while DBT caused a 1.3-fold activation at 2.5 µM at 10 min. Both TBT and DBT caused an approximately 2-fold increase in phosphorylation/activation of PKC. Exposures to TBBPA caused no statistically significant changes in either PKC or PKD activation.

Progression to AIDS in SIV-Infected Rhesus Macaques is Associated with Distinct KIR and MHC class I Polymorphisms and NK Cell Dysfunction.

Killer cell immunoglobulin-like receptors (KIR) regulate the activity of natural killer (NK) cells and have been shown to be associated with susceptibility to a number of human infectious diseases. Here, we analyzed NK cell function and genetic associations in a cohort of 52 rhesus macaques experimentally infected with SIVmac and subsequently stratified into high viral load (HVL) and low viral load (LVL) plasma viral loads at set point. This stratification coincided with fast (HVL) and slow (LVL) disease progression indicated by the disease course and critical clinical parameters including CD4+ T cell counts. HVL animals revealed sustained proliferation of NK cells but distinct loss of peripheral blood NK cell numbers and lytic function. Genetic analyses revealed that KIR genes 3DL05, 3DS05, and 3DL10 as well as 3DSW08, 3DLW03, and 3DSW09 are correlated, most likely due to underlying haplotypes. SIV-infection outcome associated with presence of transcripts for two inhibitory KIR genes (KIR3DL02, KIR3DL10) and three activating KIR genes (KIR3DSW08, KIR3DS02, KIR3DS05). Presence of KIR3DL02 and KIR3DSW08 was associated with LVL outcome, whereas presence of KIR3DS02 was associated with HVL outcome. Furthermore, we identified epistasis between KIR and MHC class I alleles as the transcript presence of the correlated genes KIR3DL05, KIR3DS05, and KIR3DL10 increased HVL risk when Mamu-B*012 transcripts were also present or when Mamu-A1*001 transcripts were absent. These genetic associations were mirrored by changes in the numbers, the level of proliferation, and lytic capabilities of NK cells as well as overall survival time and gastro-intestinal tissue viral load.

Brominated flame retardants, tetrabromobisphenol A and hexabromocyclododecane, activate mitogen-activated protein kinases (MAPKs) in human natural killer cells.

Natural killer (NK) cells provide a vital surveillance against virally infected cells, tumor cells, and antibody-coated cells through the release of cytolytic mediators and gamma interferon (IFN-γ). Hexabromocyclododecane (HBCD) is a brominated flame retardant used primarily in expanded (EPS) and extruded (XPS) polystyrene foams for thermal insulation in the building and construction industry. Tetrabromobisphenol A (TBBPA) is used both as a reactive and an additive flame retardant in a variety of materials. HBCD and TBBPA contaminate the environment and are found in human blood samples. In previous studies, we have shown that other environmental contaminants, such as the dibutyltin (DBT) and tributyltin (TBT), decrease NK lytic function by activating mitogen-activated protein kinases (MAPKs) in the NK cells. HBCD and TBBPA also interfere with NK cell(s) lytic function. The current study evaluates whether HBCD and/or TBBPA have the capacity to activate MAPKs and MAPK kinases (MAP2Ks). The effects of concentrations of HBCD and TBBPA that inhibited lytic function on the phosphorylation state and total levels of the MAPKs (p44/42, p38, and JNK) and the phosphorylation and total levels of the MAP2Ks (MEK1/2 and MKK3/6) were examined. Results indicate that exposure of human NK cells to 10-0.5μM HBCD or TBBPA activate MAPKs and MAP2Ks. This HBCD and TBBPA-induced activation of MAPKs may leave them unavailable for activation by virally infected or tumor target cells and thus contributes to the observed decreases in lytic function seen in NK cells exposed to HBCD and TBBPA.

Tetrabromobisphenol A decreases cell-surface proteins involved in human natural killer (NK) cell–dependent target cell lysis

Human natural killer (NK) lymphocytes are able to destroy tumor cells and virally-infected cells. Interference with their function can leave an individual with increased susceptibility to cancer development and/or viral infection. We have shown that the tumor-destroying (lytic) function of NK cells can be dramatically decreased by exposure to the environmental contaminant tetrabromobisphenol A (TBBPA). TBBPA is a flame retardant used in a variety of materials including circuit boards, carpeting, and upholstery and has been found in human blood samples. TBBPA interferes with NK cell lytic function, in part, by decreasing the ability of NK cells to bind to target cells. This study examines the effects of exposures to concentrations of TBBPA (i.e., that were able to decrease the binding capacity of NK cells) on the expression of cell-surface proteins (CD2, CD11a, CD16, CD18, and CD56) that are needed for NK cells to bind target cells. NK cells were exposed to TBBPA for 24 h, 48 h, and 6 days or for 1 h followed by 24 h, 48 h, and 6 days in TBBPA-free media. Twenty-four-hour exposures to 5 µM TBBPA caused decreases in four of the cell-surface proteins examined. CD16 was decreased by >35%. The decreases in cell-surface proteins after a 48-h exposure were similar to those seen after 24 h. The results indicate that TBBPA exposures that decrease the binding function of human NK cells do so by decreasing the expression of cell-surface proteins needed for attachment of NK cells to targets cells.

Role of p44/42 activation in TBT‐ induced losses of granzyme B and perforin in human natural killer cells

Natural killer (NK) cells are key players in innate immunity and are responsible for early defense against tumors and viruses. It is well known that both perforin and granzyme B are released from NK cells upon target cell contact. The environmental pollutant, tributyltin chloride (TBT) has been reported to decrease human NK cell lytic function with accompanying activation of p44/42 and decreased levels of granzyme B and perforin. The current studies are designed to examine the role of the mitogen activated protein kinase (MAPK), p44/42, activation in TBT‐induced losses of granzyme B and perforin. p44/42 activation was blocked by the MEK inhibitor, PD98059. NK cells were exposed to PD98059 for 1 h followed by 1 h exposure to 200 or 100 nM TBT. Following the incubation period, NK cells were washed twice with media and incubated for 24 h period in compound‐free media. Western blot analysis using an antibody to granzyme B revealed that PD98059 was able to block 37% of the 200 nM TBT‐induced loss of granzyme B. Moreover, western blot analysis using an antibody to perforin revealed that PD98059 was able to block 42 % of the 200 nM TBT‐induced loss of perforin. Thus our data show a role for TBT‐induced activation of p44/42 in the decreases in granzyme B and perforin in NK cells and that the TBT‐induced p44/42 activation is not solely responsible for the losses of granzyme B and perforin protein levels.

Activation of p44/42 MAPK plays a role in the TBT-induced loss of human natural killer (NK) cell function.

Natural killer (NK) cells destroy (lyse) tumor cells, virally infected cells, and antibody-coated cells. Previous studies indicated that exposure to the environmental contaminant tributyltin (TBT) decreases the lytic function of NK cells and activates mitogen-activated protein kinases (MAPK), including p44/42 (Aluoch and Whalen Toxicology 209:263-277, 2005). If activation of p44/42 is required for TBT-induced decreases of lytic function, then activation of p44/42 to similar extents by pharmacological agents such as phorbol 12-myristate 13-acetate (PMA) should mimic to some extent changes induced in NK cells with TBT exposures. NK cells were exposed to PMA concentrations between 0.25 and 10 nM for 10 min, 1 h, and 6 h before determining the lytic function ((51)Cr release assay) and phosphorylation state of MAPKs (Western blot). A 1-h exposure of NK cells to 5 nM PMA resulted in a loss of lytic function of 47%. Western blot analysis showed that a 1-h exposure to 5 nM PMA caused a sixfold increase in phospho-p44/42 levels. Previous studies showed a fivefold increase in phospho-p44/42 in response to a 1-h exposure to 300 nM TBT. Exposure to 300 nM TBT caused about a 40% decrease in lytic function. This study supports the hypothesis that p44/42 activation (as seen with TBT exposures) can cause a loss of NK-cell lytic function.

5-Aza-2'-Deoxycytidine Has a Biphasic Effect On Ex Vivo Expanded Natural Killer Cells.

Abstract 2664Poster Board II-640Adoptive transfer of activated autologous or allogeneic natural killer (NK) cells has emerged as a safe and potentially efficacious immunotherapy for cancer. Alloreactive HLA haploidentical NK cells in the HSCT setting have been reported to enhance engraftment, reduce GVHD and prevent relapse of leukemia. The main restrictions to developing NK cells for immunotherapy are the limited quantity of cells available for adoptive transfer and the modulation of NK cell receptors and/or NK cell ligands as a recognized mechanism of immune escape in oncogenesis. To address the first hurdle, we have developed an efficient method to expand CD3−CD56+ primary NK cells in vitro using K562 artificial APCs expressing membrane-bound IL-21. Altering the balance of activating and inhibitory signals with immunosensitizing drugs is an attractive approach to addressing the second hurdle of NK cell therapy. DNA methylation inhibitors such as 5-Aza-2'-deoxycytidine and 5-azacytidine are being tested for their activity in AML, and have been shown to sensitize AML blasts to lysis by NK cells. However, there is little information on the direct immunologic effect of these hypomethylating agents on NK cell function, reconstitution, and phenotype.Using the calcein release assay, we investigated NK cell survival and lytic function against the NK cell-sensitive HLAnull 721.221 cells after exposing expanded NK cells to 5-Aza-2'-deoxycytidine. Human peripheral blood NK cells that had been expanded ex vivo in our laboratory were incubated with a wide range of 5-Aza-2'-deoxycytidine concentrations for 3 days in the presence of 50 IU/mL IL-2. Cell viability was tested every 24 h.We found that 5-Aza-2'-deoxycytidine at higher concentrations resulted in reduced NK cell viability, but at lower concentrations inhibited NK cell killing. At these lower doses, NK cell inhibition was not associated with loss of viability, resulting in an overall U-shaped dose response curve to 5-Aza-2'-deoxycytidine with maximum inhibition in the 0.3 – 2.5 micromolar range.This biphasic response to 5-Aza-2'-deoxycytidine matches that previously described for malignant cells in which reduced proliferation at higher concentrations resulted in reduced demethylation inhibition (Qin et al, Clin Cancer Res. 2007 Jul 15;13(14):4225–32). To assess the role of demethylation in NK cells in a more dynamic setting, we are investigating 5-Aza-2'-deoxycytidine effects on NK cell proliferation and phenotype during ex vivo expansion, and assessing NK cell recovery, phenotype, and function in a prospective cross-over trial of patients receiving 5-azacytidine post HSCT. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Hexabromocyclododecane decreases the lytic function and ATP levels of human natural killer cells.

This study investigates the effect of hexabromocyclododecane (HBCD) on the lytic function of human natural killer (NK) cells and on ATP levels in NK cells. NK cells are capable of lysing tumor cells, virally infected cells, and antibody-coated cells. HBCD is a brominated cyclic alkane used primarily as an additive flame retardant. If HBCD interferes with NK cell function, this could increase risk of tumor development and/or viral infection. NK cells were exposed to various concentrations of HBCD for 24 and 48 h and 6 days before determining lytic function and ATP levels. ATP levels and lytic function were also determined in NK cells that were exposed to HBCD for 1 h followed by 24 and 48 h, and 6 days in HBCD-free media. The results indicated that exposure of NK cells to 10 microm HBCD for 24 h causes a very significant decrease in both NK cell lytic function and ATP levels (93.5 and 90.5%, respectively). Exposure of NK cells to 10 microm HBCD for 1 h followed by 24 h in HBCD-free media showed a progressive and persistent loss of lytic function (89.3%) as well as a decrease in ATP levels (46.1%). The results indicate that HBCD exposures decreased lytic function as well as ATP levels. However, a decrease in lytic function was not necessarily accompanied by a similar decrease in ATP. Importantly, these results also indicate that a brief (1 h) exposure to HBCD causes a progressive loss of lytic function over a 6 day period.

Inhibition of lytic function of human NK cells by Tributyltin: Role of Protein Kinase C

Protein kinase C (PKC) catalyzes the phosphorylation of serine/threonine residues and is involved in signal transduction associated with cell proliferation, differentiation, and apoptosis. The environmental pollutant, tributyltin chloride (TBT) has been reported to decrease human Natural killer (NK) cell lytic function. We previously showed that TBT induces activation of MAPK in NK cells. In the present study, we examined the role of PKC in TBT‐ induced inhibition of NK lysis of tumor cells and TBT‐induced activation of MAPKs by using a PKC inhibitor. A 1 h treatment with Bisindolylmaleimide I caused 85% decrease in the ability of NK cells to lyse K562 tumor cells in a 2‐h 51Cr‐release assay. Western blot analysis using an antibody to phospho‐MAPKs showed that Bisindolylmaleimide I was able to completely block TBT‐induced activation of p44/42 whereas activation of p38 and JNK were not blocked. However, when the activation of PKC was blocked by the presence of Bisindolylmaleimide I, subsequent exposure to TBT produced even greater inhibition of lytic function of NK cells than was seen with TBT alone. These results indicate the pivotal role of PKC in the TBT‐induced activation of p44/42 in NK cells, possibly by activating Raf ‐> MEK ‐> MAPKs and suggest the negative effect of TBT in NK lytic function was not prevented even when the activation of PKC was blocked.

Effect of pentachlorophenol on MAP kinases in human natural killer cells

Pentachlorophenol (PCP) is an organochlorine compound that has been used as an insecticide and a fungicide. PCP use has been restricted in the USA, it is mainly used now as a wood preservative for power‐line poles and fence poles. PCP has been detected in human tissues through inhalation and consumption of contaminated food and water. Human natural killer (NK) lymphocytes play a central role in immune defense against viral infection and tumor development by killing (lysing) virally infected and tumor cells. The lytic function of NK cells depends in part upon the activation of mitogen activated protein kinases (MAPK). In previous studies we have shown that exposure to 10‐0.5µM PCP decreases the lytic function of NK cells. In this study we evaluated the effect of these same concentrations of PCP on the phosphorylation state and total levels of p44/42, p38, and JNK ,MAPKs. Preliminary studies indicate that PCP exposure is able to increase the phosphorylation (activation) of MAPKs. This PCP‐induced phosphorylation of MAPKs may leave them unavailable for activation by virally infected or tumor target cells and thus contribute to the observed decreases in lytic function seen in NK cells exposed to PCP.Supported by NIH grant S06 GM008092‐33.

Hexabromocyclododecane decreases the lytic function and ATP levels of human natural killer cells

This study investigates the effect of Hexabromocyclododecane (HBCD) on human natural killer (NK) cell lytic function, additionally; it investigates the effects of HBCD on ATP levels in NK cells. Human NK cells are a subset of lymphocytes that are capable of lysing tumor cells, virally infected cells, and antibody‐coated cells. Hexabromocyclododecane (HBCD) is a non‐aromatic, brominated cyclic alkane used primarily as an additive flame retardant. If HBCD interferes with NK cell function, this could increase risk of tumor development and/or viral infection. NK cells were exposed to various concentrations of HBCD for 24 h, 48 h, and 6 days before determining lytic function and ATP levels. ATP levels and lytic function were also determined in NK cells that were exposed to HBCD for 1 h followed by 24 h, 48 h, and 6 days in HBCD‐free media. The results indicated that exposure of NK cells to 10 µM HBCD for 24 h causes a very significant decrease in both NK cell ATP levels and lytic function (90.5% and 93.5%, respectively). Exposure of NK cells to 10 µM HBCD for 1 h followed by 24h in HBCD‐free media also showed a very significant decrease in lytic function (89.4%) and a slight decrease in ATP levels (46.1%). The results indicate that HBCD exposures decrease ATP levels and lytic function in NK cells.Supported by NIH grant SG06 GM008092‐33

Glutathione diminishes tributyltin- and dibutyltin-induced loss of lytic function in human natural killer cells

This study investigated whether reduced glutathione (GSH) was able to alter the negative effects of tributyltin (TBT) or dibutyltin (DBT) on the lytic function of human natural killer (NK) cells. NK cells are an initial immune defense against the development of tumors or viral infections. TBT and DBT are widespread environmental contaminants, due to their various industrial applications. Both TBT and DBT have been shown to decrease the ability of NK cells to lyse tumor cells (lytic function). The results indicated that the presence of GSH during the exposure of NK cells to TBT or DBT diminished the negative effect of the butyltin on the lytic function of NK cells. This suggests that the interaction of TBT and DBT with functionally relevant sulfhydryl groups in NK cells may be part of the mechanism by which they decrease NK lytic function.

The role of p44/42 activation in tributyltin‐induced inhibition of human natural killer cells: effects of MEK inhibitors

Destruction of tumor cells is a key function of natural killer (NK) cells. Previous studies have shown that tributyltin (TBT) can significantly reduce the lytic function of the human NK cells with accompanying increases in the phosphorylation (activation) states of the mitogen activated protein kinases (MAPKs), p44/42. The current studies examine the role of p44/42 activation in the TBT-induced reduction of NK-lytic function, by using MAPK kinase (MEK) inhibitors, PD98059 and U0126. A 1 h treatment with PD98059 or U0126 or both decreased the ability of NK cells to lyse K562 tumor cells. PD98059, U0126 or a combination of both inhibitors were able to completely block TBT-induced activation of p44/42. However, when p44/42 activation was blocked by the presence of PD98059, U0126 or the combination, subsequent exposure to TBT was still able to decrease the lytic function of NK cells. These results indicate that TBT-induced activation of p44/42 occurs via the activation of its upstream activator, MEK, and not by a TBT-induced inhibition of p44/42 phosphatase activity. Additionally, as lytic function was never completely blocked by MEK inhibitors, the results indicate that activation of p44/42 pathway is not solely responsible for the activation of lytic function of freshly isolated human NK cells. Finally, the results showed that TBT-induced activation of p44/42 is not solely responsible for the loss of lytic function.

Alteration of NK Cell Signaling Pathways by Exposure of Human Natural Killer Cells to Dibutyltin

Dibutyltin (DBT) is a widespread environmental contaminant. Detectable levels have been found in human blood. Natural killer (NK) cells are able to lyse tumor cells and virally infected cells. Previous studies have shown that DBT decreases the lytic function of NK cells. In this study we assessed the effects of those concentrations of DBT that decrease NK lytic function on the status of protein tyrosine kinases (PTKs) (Syk, Zap‐70, Src and Pyk), and Mitogen‐activated protein kinases (MAPKs) (p38, p44/42, JNK). Such studies will help elucidate the mechanism by which DBT decreases NK lytic function. NK cells that were exposed to (10, 5, 2.5, 1, 0.5 μM) DBT for 10 min did not show any significant changes in the levels or activation state of Syk, Pyk, or Src. Exposure of NK cells to 10 μM DBT for 10 min resulted in a significant increase in the phosphorylation of p38 (1.8 fold) and p44/42 (1.7 fold). A 6 h exposure to 10 μM DBT caused a significant increase in phosphorylation of p44/42 but no significant change in p38, phospho‐p38, p44/42, phospho‐JNK, or JNK levels. These data indicate that in vitro exposure to DBT, caused no changes in the levels or activation state of PTKs but caused significant activation the MAPKs, p38 and p44/42 after a 10min exposure to 10 μM DBT. This activation of p44/42 maintains out to 6 h. Supported by NIH grant 2S06GM0809228.

Effects of a series of triorganotins on ATP levels in human natural killer cells

Natural killer (NK) cells are our initial immune defense against viral infections and cancer development. Thus, agents that are able to interfere with their function increase the risk of cancer and/or infection. A series of triorganotins (trimethyltin (TMT), dimethylphenyltin (DMPT), methyldiphenyltin (MDPT), and triphenyltin (TPT)) have been shown to decrease the lytic function of human NK cells. TPT and MDPT were much more effective than DMPT or TMT at reducing lytic function. This study investigates the role that decreased ATP levels may play in decreases in the lytic function of NK cells induced by these organotins (OTs). A 24 h exposure to as high as 10 μM TMT caused no decrease in ATP levels even though this level of TMT caused a greater than 75% loss of lytic function. TPT at 200 nM caused a decrease in ATP levels of about 20% while decreasing lytic function by greater than 85%. There was no association between ATP levels and lytic function for any of the compounds when NK cells were exposed for 1 or 24 h. However, after a 48 h exposure to both DMPT and TPT decreased lytic function was associated with decreased ATP levels. There was an association between decreased lytic function and decreased ATP levels after a 6-day exposure to each of the four compounds. These studies indicate that the loss of lytic function seen after 1 and 24 h exposures to this series of organotins cannot be accounted for by decreases in ATP. However, after longer exposures loss of lytic function may be in part be attributable to inadequate ATP levels.

In vitro atrazine-exposure inhibits human natural killer cell lytic granule release

The herbicide atrazine is a known immunotoxicant and an inhibitor of human natural killer (NK) cell lytic function. The precise changes in NK cell lytic function following atrazine exposure have not been fully elucidated. The current study identifies the point at which atrazine exerts its affect on the stepwise process of human NK cell-mediated lyses of the K562 target cell line. Using intracellular staining of human peripheral blood lymphocytes, it was determined that a 24-h in vitro exposure to atrazine did not decrease the level of NK cell lytic proteins granzyme A, granzyme B or perforin. Thus, it was hypothesized that atrazine exposure was inhibiting the ability of the NK cells to bind to the target cell and subsequently inhibit the release of lytic protein from the NK cell. To test this hypothesis, flow cytometry and fluorescent microscopy were employed to analyze NK cell–target cell co-cultures following atrazine exposure. These assays demonstrated no significant decrease in the level of target cell binding. However, the levels of NK intracellular lytic protein retained and the amount of lytic protein released were assessed following a 4-h incubation with K562 target cells. The relative level of intracellular lytic protein was 25–50% higher, and the amount of lytic protein released was 55–65% less in atrazine-treated cells than vehicle-treated cells following incubation with the target cells. These results indicate that ATR exposure inhibits the ability of NK cells to lyse target cells by blocking lytic granule release without affecting the ability of the NK cell to form stable conjugates with target cells.

Effect of tributyltin (TBT) on ATP levels in human natural killer (NK) cells: Relationship to TBT‐induced decreases in NK function

The purpose of this study was to investigate the role that tributyltin (TBT)-induced decreases in ATP levels may play in TBT-induced decreases in the tumor lysing (lytic) function of natural killer (NK) cells. NK cells are a subset of lymphocytes that act as an initial immune defense against tumor cells and virally infected cells. TBT is an environmental contaminant that has been detected in human blood, which has been shown to interfere with ATP synthesis. Previous studies have shown that TBT is able to decrease very significantly the lytic function of NK cells. In this study NK cells were exposed to various concentrations of TBT and to two other compounds that interfere with ATP synthesis (rotenone a complex I inhibitor and oligomycin an ATP synthase inhibitor) for various lengths of time before determining the levels of ATP and lytic function. Exposures of NK cells to 10, 25, 50 and 100 nm TBT did not significantly reduce ATP levels after 24 h. However, these same exposures caused significant decreases in cytotoxic function. Studies of brief 1 h exposures to a range of TBT, rotenone and oligomycin concentrations followed by 24 h, 48 h and 6 day periods in compound-free media prior to assaying for ATP levels or cytotoxic function showed that each of the compounds caused persistent decreases in ATP levels and lytic function of NK cells. Exposures to 0.05-5 microm rotenone or oligomycin for 1 h reduced ATP levels by 20-25% but did not have any measurable effect on the ability of NK cells to lyse tumor cells. ATP levels were also decreased by about 20-25% after 24 h or 48 h exposures to rotenone or oligomycin (0.5 microm ), and the lytic function was decreased by about 50%. The results suggest that TBT-induced decreases in ATP levels were not responsible for the loss of cytotoxic function seen at 1 h and 24 h. However, TBT-induced decreases of NK-ATP levels may be at least in part responsible for losses of NK-cytotoxic function seen after 48 h and 6 day exposures.

Effects of prolactin and cortisol on natural killer (NK) cell surface expression and function of human natural cytotoxicity receptors (NKp46, NKp44 and NKp30)

The surface density of the triggering receptors (e.g. NKp46 and NKp30) responsible for natural killer (NK) cell-mediated cytotoxicity determines the ability of NK cells to kill susceptible target cells. In this study, we show that prolactin up-regulates and cortisol down-regulates the surface expression of NKp46 and NKp30. The prolactin-mediated activation and the cortisol-mediated inhibition of natural cytotoxicity receptor (NCR) surface expression reflects gene regulation at the transcriptional level. NKp46 and NKp30 are the major receptors involved in the NK-mediated killing of K562, a human chronic myelogenous leukaemia cell line. Accordingly, the prolactin dramatically increased the NK-mediated killing of the K562 cell line, whereas cortisol abolished this activity. Our data suggest a mechanism by which prolactin activates the lytic function of NK cells, and cortisol inhibits the NK-mediated attack.

Selective loss of viability of mouse NK cells in culture is associated with decreased NK cell lytic function.

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.