Human Natural Killer Lymphocytes Research Articles

Role of ERK1/2 and p38 MAPKs in Tributyltin‐stimulated Interleukin 1 Beta Secretion and Production from Human Immune Cells.

Tributyltin (TBT) is an organotin compound that contaminates the environment due to its use as a biocide in a wide variety of applications. TBT has been found in human blood samples at levels as high as 265 nM. Previously, we have demonstrated that TBT interferes with the lytic function of human natural killer lymphocytes and alters the secretion of several pro‐inflammatory cytokines from lymphocytes, including interleukin 1 beta (IL‐1β). IL‐1β regulates cellular growth and immune responsiveness. Elevated levels of IL‐1β have been shown to increase the invasiveness of tumors. Our recent studies showed that TBT exposures of 5–25 nM (levels that are seen in human blood samples) caused increased secretion of IL‐1β from monocyte‐depleted peripheral blood mononuclear cells (MD‐PBMCs). In this study we examine the roles of the ERK1/2 and p38 MAPK pathways in TBT‐induced increases in IL‐1β secretion, protein production, and mRNA levels. MD‐PBMCs were treated with the MEK1/2 inhibitor PD98059 or the p38 inhibitor SB202190 for 1 h prior to exposure to TBT concentrations of 25, 10, and 5 nM for 24 h. Secretion of IL‐1β was measured using ELISA, intracellular protein levels were monitored using western blot, and mRNA expression was determined by RT‐qPCR. Results indicated that the TBT‐induced secretion of IL‐1β requires the ERK1/2 pathway. The increases in secretion are accompanied by increased protein levels in TBT‐exposed cells and this increase in protein production also utilized the ERK1/2 pathway. These data suggest that increased protein production accompanies the increases in secretion of IL‐1β seen in human immune cells exposed to TBT and these increases are dependent on MAPK pathway activation.Support or Funding InformationSupported by NIH grant 5U54CA163066.

Blocking KCa3.1 Channels Increases Tumor Cell Killing by a Subpopulation of Human Natural Killer Lymphocytes

Natural killer (NK) cells are large granular lymphocytes that participate in both innate and adaptive immune responses against tumors and pathogens. They are also involved in other conditions, including organ rejection, graft-versus-host disease, recurrent spontaneous abortions, and autoimmune diseases such as multiple sclerosis. We demonstrate that human NK cells express the potassium channels Kv1.3 and KCa3.1. Expression of these channels does not vary with expression levels of maturation markers but varies between adherent and non-adherent NK cell subpopulations. Upon activation by mitogens or tumor cells, adherent NK (A-NK) cells preferentially up-regulate KCa3.1 and non-adherent (NA-NK) cells preferentially up-regulate Kv1.3. Consistent with this different phenotype, A-NK and NA-NK do not display the same sensitivity to the selective KCa3.1 blockers TRAM-34 and NS6180 and to the selective Kv1.3 blockers ShK-186 and PAP-1 in functional assays. Kv1.3 block inhibits the proliferation and degranulation of NA-NK cells with minimal effects on A-NK cells. In contrast, blocking KCa3.1 increases the degranulation and cytotoxicity of A-NK cells, but not of NA-NK cells. TRAM-34, however, does not affect their ability to form conjugates with target tumor cells, to migrate, or to express chemokine receptors. TRAM-34 and NS6180 also increase the proliferation of both A-NK and NA-NK cells. This results in a TRAM-34-induced increased ability of A-NK cells to reduce in vivo tumor growth. Taken together, our results suggest that targeting KCa3.1 on NK cells with selective blockers may be beneficial in cancer immunotherapy.

Interleukin (IL)-2 and IL-15 have different effects on human natural killer lymphocytes

Although interleukin (IL)-2 and IL-15 share the common signal transducing receptor chains IL-2Rβ and γ(c) and give rise to the same signaling patterns in human natural killer (NK) cells in vitro, they differ in their effects on the development, activation, and proliferation of these cells in vivo. We have previously demonstrated that the activation of NK cells induces a cellular program characterized by the sequential transcription-regulated expression of IL-15 and IL-2 high-affinity receptors. We demonstrate here that these receptors induce different responses. IL-15 sustains the expression of its high-affinity receptor, leading to long-lasting STAT5 phosphorylation and BCL2 expression. By contrast, IL-2 induces the rapid disappearance of IL-2Rα and γ(c) chains when the gene transcription is downregulated, shutting down IL-2-responses as demonstrated by the absence of STAT5 phosphorylation and BCL2 expression.

Antigen Presenting Cells and Stromal Cells Trigger Human Natural Killer Lymphocytes to Autoreactivity: Evidence for the Involvement of Natural Cytotoxicity Receptors (NCR) and NKG2D

Human natural killer (NK) lymphocytes should not damage autologous cells due to the engagement of inhibitory receptor superfamily (IRS) members by HLA-I. Nevertheless, NK cells kill self cells expressing low levels or lacking HLA-I, as it may occur during viral infections (missing-self hypothesis). Herein, we show that human NK cells can be activated upon binding with self antigen presenting cells or stromal cells despite the expression of HLA-I. Indeed, NK cells can kill and produce pro-inflammatory and regulating cytokines as IFN-γ, TNF-α and IL10 during interaction with autologous dendritic cells or bone marrow stromal cells or skin fibroblasts. The killing of antigen presenting and stromal cells is dependent on LFA1/ICAM1 interaction. Further, the natural cytotoxicity receptors (NCR) NKp30 and NKp46 are responsible for the delivery of lethal hit to DC, whereas NKG2D activating receptor, the ligand of the MHC-related molecule MIC-A and the UL16 binding protein, is involved in stromal cell killing. These findings indicate that different activating receptors are involved in cell to self cell interaction. Finally, NK cells can revert the veto effect of stromal cells on mixed lymphocyte reaction further supporting the idea that NK cells may alter the interaction between T lymphocytes and microenvironment leading to autoreactivity.

Human Natural Killer Lymphocytes Alone and with an Anti-GD2/IL-2 Immunocytokine Are Cytotoxic for Multidrug Resistant Neuroblastoma Cells

s for the 19th Annual Scientific Meeting of the International Society for Biological Therapy of Cancer, San Francisco, California, November 4-7, 2004: Natural Killer Cells & Innate Immunity

Environmentally Relevant Concentrations of Butyltins Inhibit the Cytotoxic Function of Human Natural Killer Lymphocytes

Environmentally Relevant Concentrations of Butyltins Inhibit the Cytotoxic Function of Human Natural Killer Lymphocytes

Immunotoxicity of Environmentally Relevant Concentrations of Butyltins on Human Natural Killer Cells in Vitro

The widespread environmental contamination, bio-accumulation, and toxic effects of butyltins (BTs) in wildlife is well documented, but the role of BTs in debilitating human immune function mediated through natural killer (NK) lymphocytes (a primary immune defense against tumor and virally infected cells) has not been described. In this study, we assessed the effects of in vitro exposure to a range of concentrations (encompassing environmentally relevant concentrations) of MBT, DBT, and TBT on human natural killer lymphocytes obtained from adult male and female donors. TBT inhibited the tumor-killing capacity of NK cells when the NK cells were pretreated in vitro at 200 nM for as little as 1 h. Inhibition of NK cytotoxic function ranged from 40 to greater than 90%. The toxic potential of butyltins followed the order of TBT > DBT > MBT. Conjugation assays revealed that after a 24-h exposure to TBT, there was about a 50% decrease in NK cell binding to tumor cells, indicating alteration of the NK cell receptors for tumor cells. Analysis of whole-blood samples for BTs revealed the presence of detectable concentrations of MBT, DBT, and TBT in all of the donors, indicating possible exposure of NK cells to BTs in the blood. The results of this study provide evidence that butyltin compounds significantly inhibit NK cell function and possible NK cell-mediated immunotoxic potential in humans.

Occurrence of Butyltin Compounds in Human Blood

Residues of butyltin compounds, including mono- (MBT), di- (DBT) and tributyltins (TBT), were measured in human blood collected from central Michigan, U.S.A. MBT, DBT, and TBT were detected in 53, 81, and 70% of the 32 blood samples examined. Concentrations of butyltins were in the order of MBT > DBT ≥ TBT, with total butyltin concentrations ranging from less than the limit of detection to 101 ng/mL. Exposure of humans to butyltin compounds used as stabilizers or as biocides in household articles has been regarded as a source in addition to the ingestion of contaminated foodstuffs. There was no significant difference in concentrations of butyltin compounds between sexes. Concentrations of butyltin compounds did not exhibit pronounced age-dependency, which is different from those observed for persistent pollutants such as polychlorinated biphenyls (PCBs). In general, concentrations of butyltins measured in blood were less than that affected human natural killer lymphocytes (a primary immune defense against...

CD45‐mediated regulation of LFA1 function in human natural killer cells. Anti‐CD45 monoclonal antibodies inhibit the calcium mobilization induced via LFA1 molecules

The TA218 and T205 monoclonal antibodies (mAb) were selected on the basis of their ability to inhibit the non-major histocompatibility complex-restricted lysis of the murine mastocytoma P815 cell line mediated by CD3-CD16+ natural killer (NK) cells. Both mAb were found to react with CD45 molecules, as demonstrated by immunoprecipitation after surface iodination and western blot analysis. A panel of tumor target cells susceptible to lysis by polyclonal or clonal CD3-CD16+ NK cells was used to study the mAb-mediated inhibitory effect. The inhibition of cytolysis mediated by TA218 and T205 mAb was found to consistently parallel the inhibition mediated (with the same tumor target cells) by the anti-LFA1 alpha mAb TS.1.22 or by the anti-LFA1 beta mAb TS.1.18. However, different from the anti-LFA1 mAb, T205 or TA218 mAb did not inhibit the binding of activated CD3-CD16+ effector NK cells to the same tumor target cells. This finding supported the concept that the anti-CD45 mAb-mediated inhibition could occur at a post-binding stage. In polyclonal or clonal CD3-CD16+ NK cells T205 or TA218 mAb were found to reduce by 50-70% the intracellular Ca++ ([Ca++]i) mobilization induced by anti-LFA1 alpha or anti-LFA1 beta mAb. On the other hand, TA218 and T205 mAb did not inhibit the Ca++ mobilization induced by anti-CD16 mAb or phytohemagglutinin, thus suggesting that, in NK cells, CD45 molecules may exert a selective inhibitory effect on the signal transduction mediated by LFA1 molecules. In line with this hypothesis, the cytolytic activity of human NK clones was triggered in the presence of the hybridoma cells secreting either anti-CD16 or anti-LFA1 alpha mAb (as "triggering targets"). This effect of anti-LFA1 alpha, but not of anti-CD16 hybridoma was susceptible to inhibition by the anti-CD45 mAb T205 or TA218. Further, experiments on cloned NK cells indicated that T205 or TA218 mAb induced a strong decrease in the constitutive phosphorylation of the LFA1 alpha chain (but not of HLA class I antigens). Taken together, these studies suggest that in human NK lymphocytes, CD45 molecule may regulate both the activation state and the function of the LFA1 molecule.

Melatonin-induced suppression of human lymphocyte natural killer activity in vitro.

One of the important immune functions influenced by neuroendocrine factors is natural killer (NK) activity, which is directed against neoplastic and virus-infected cells. The effects of melatonin (Mel) and N-acetylserotonin (NAc-5HT) on NK activity of human peripheral blood lymphocytes were investigated. Leukocytes of healthy human subjects were used in the experiment. NK activity was estimated by measurement of radioactive chromium (51Cr) release from human leukemia cells K 562 (target cells). The previous exposure of human lymphocytes (effector cells) to Mel in concentrations of 10(-6) M and 10(-10) M resulted in an inhibition of NK activity (P less than 0.01) for all the examined effector-target cell ratios (10:1; 20:1, 40:1). NK activity was also suppressed by Mel (10(-8) M), but only if effector-target ratio equal to 20:1 was used (P less than 0.02), and by Mel (10(-12) M) for effector-target ratio equal to 40:1 (P less than 0.05). In none of the examined concentrations did NAc-5HT inhibit NK activity of human lymphocytes. On the basis of the data reported above, a direct suppressive effect of Mel (but not of NAc-5HT) on NK activity of human peripheral blood lymphocytes can be assumed.

Enhancement of human lymphocyte natural killer activity by somatostatin

The effect of somatostatin 1–14 (SS) on the natural killer (NK) activity of human peripheral blood lymphocytes was investigated. The NK activity was estimated by means of radioactive chromium ( 51Cr) assay with the use of human leukemia cells K 562 as targets. The previous exposure of lymphocytes obtained from healthy donors to somatostatin in the concentration of 10 −8 and 10 −6 M resulted in the enhancement of NK activity. The finding provides a further evidence of the immunomodulating somatostatin action.

Effects of two neuropeptides, somatoliberin (GRF) and corticoliberin (CRF), on human lymphocyte natural killer activity

The effect of corticoliberin (CRF) and somatoliberin (GRF) on the natural killer (NK) activity of human peripheral blood lymphocytes was investigated. NK activity was estimated by means of the radioactive chromium ( 51Cr) assay in which human leukemia K 562 cells serve as targets. Exposure of human lymphocytes (effector cells) to 10 −6 to 10 −10 M concentrations of CRF inhibited NK activity. NK activity was also suppressed by the same concentrations of GRF, but only when an effector:target cell ratio of 40:1 was used. In contrast, at effector:target ratios of 20:1 and 10:1, stimulatory effects of GRF were observed.

Differential effect of hemodialysis membranes on human lymphocyte natural killer function.

Lymphocytes exposed to cuprammonium cellulose membranes have been shown to exhibit depressed natural killer (NK) function. In the present study we investigated the extent to which three dialyzer membranes of different compositions suppressed human lymphocyte NK activity. Peripheral blood lymphocytes or T cells from normal donors were exposed in vitro to cuprammonium cellulose, cellulose acetate, or polycarbonate dialyzer membranes. After exposure to the membranes, NK activity of the cells was studied by using the NK-sensitive cell line K562 as targets. All three membranes adversely affected human lymphocyte NK function, with cuprammonium cellulose producing the most (70-80%) and polycarbonate producing the least (10-15%) suppression. Our results suggest that the composition of dialyzer membranes affects the extent to which the membranes impair human lymphocyte function. The use of more biocompatible membranes might lessen the potential clinical impact of abnormal NK function in hemodialysis patients.

Endorphins stimulate normal human peripheral blood lymphocyte natural killer activity

Opioid peptides are present in peripheral blood, and may bind to human lymphocytes. In order to determine their influence on human lymphocytes we studied the effect of endogenous opioid peptides on human lymphocyte natural killer function. Beta-endorphin and several analogues (i.e., γ-endorphin) are shown to enhance human peripheral blood natural killer function. The enhancement of natural killing by these opioid peptides was dose-dependent and naloxone (an opiate antagonist) reversible. In studying various analogues of β-endorphin, β-lipotropin and γ-endorphin were approximately 3–5 times more effective at enhancing peripheral blood NK function than Leu-enkephalin and -endorphin. In addition, we observed that naloxone reversed human fibroblast interferon mediated enhancements of human blood lymphocyte natural killer function. These observations suggest that circulating endogenous opioid peptides may have a physiologic role in regulating human blood lymphocyte natural killing.

Heterogeneity and mechanism of action of human natural killer lymphocytes: differential distribution of receptors for Helix pomatia haemagglutinin (HP receptors).

Neuraminidase-treated lymphocytes from the peripheral blood of normal human donors were fractionated on columns charged with Helix pomatia haemagglutinin (HP) coupled to Sepharose 4B. While lymphocytes lacking HP receptors (HP-) passed directly through the column (fraction I), lymphocytes with HP receptors (HP+) were subsequently eluted in two distinct fractions with two different concentrations of the competitive hapten N-acetyl-D-galactosamine (fraction II and III, respectively). The natural cytotoxicity of these lymphocytes to various tumour target cells (K562, T24, MANO, HCV29) was tested in a 51Cr release assay. Natural cytotoxicity was found in all three fractions recovered from the HP columns. In general, the cytotoxicity of the lymphocytes in fractions I and II was significantly enhanced over that of the unfractionated lymphocytes. Surface marker analysis and fractionation studies indicated that natural cytotoxicity in these target systems is exerted by both HP+ and HP- lymphocytes bearing Fc receptors for IgG. Since the HP receptor is considered to be a marker to T lymphocytes, the findings suggest that a significant fraction of these NK cells may be of T-cell lineage. The surface marker profiles of these NK cells are very similar to those of antibody-dependent K cells. Addition of Fab fragments of immunoadsorbent-purified rabbit antibodies to human immunoglobulin inhibited the natural cytotoxicity of HP-column-fractionated lymphocytes to various degrees, indicating that part but not all of it reflects antibody-dependent K-cell reactions. Since cytotoxicity in all three HP fractions was inhibitable in this way, the results suggest that immunoglobulin-dependent natural cytotoxicity may be displayed by both HP+ and HP- effector cells.