In Vitro Effects of Various Metals on Natural Killer Cell Activity in Cultured Human Lymphocytes

Heavy metals have been shown to have a differential effects on various aspects of immune response. Recently natural killer cells have been widely investigated due to their purported role in immune surveillance. To ascertain the immunotoxic effects of lead, cadmium, nickel and chromium on natural killer (NK) cell activity in vitro, peripheral blood lymphocytes from normal donors were examined in the presence of different concentrations (10^-5-10^-8 M) of four selected metal salts (cadmium sulphate, lead nitrate, chromium nitrate and nickel sulphate). NK cell activity was evaluated in a 4-h chromium release assay against K562 target cells. All of the metal salts were found to exert no effect on NK cell function in the human concentration range.     

Inhibition of Natural Killer Activity by Calcitonin Gene-Related Peptide

Abstract

The effect of calcitonin gene-related peptide (CGRP) on natural killer (NK) cell activity in spleen cells from Ba1b/c mice and nude mice was studied. CGRP dose-dependently (10 to 10 M) inhibited NK activity of spleen cells from both strains of mice. This inhibitory effect was observed at the effector to target ratios of 12.5:1 to 100:1. Maximum inhibition by 10^?7 M CGRP was about 60 %. The inhibition of NK activity by CGRP was also observed in anti-Thy 1.2 plus complement treated Ba1b/c spleen cells. Furthermore, when cells were treated with 10 to 10^?7 M CGRP the concentration of intracellular cyclic AMP increased in spleen cells of nude mice. The characteristics of these cells were similiar to those of NK cells, (1) being petri dish and nylon wool nonadherent, (2) expressing asialo GM₁ antigen, and (3) lacking readily detectable Thy 1 antigen and immunoglobulin. In addition, the intravenous injection of asialo GM₁ completely abolished NK activity in spleen cells from nude mice and the increase in intracellular cyclic AMP in spleen cells by CGRP was less in spleen cells from mice given an anti-asialo GM₁ injection. Our present study suggests that CGRP inhibits NK cell activity by increasing the intracellular cyclic AMP concentration. CGRP may be implicated in the regulation of NK function.     

Ultrastructure and cytochemical staining characteristics of canine natural killer cells

Background: The purpose of this work was to describe the ultrastructure and cytochemical staining characteristics of canine peripheral blood lymphocytes with natural killer (NK) cell activity, with comparison made to non-NK lymphocytes. Methods: Canine lymphocyte populations evaluated for ultrastructure, cytochemical staining, and NK function (by ⁵¹ chromium release assay) included: peripheral blood lymphocytes; lymphocytes from band 1 (NK-enriched), band 2, and the pellet of a 45/50% percoll gradient; lymphocytes from the supernatant fluid (non-conjugated lymphocytes) and pellet (lymphocytes conjugated to tumor cell targets) of a 17% percoll gradient; and null (CD4^?CD8^?) and CD4^?CD8⁺ lymphocytes. Results: NK activity was concentrated in band 1 lymphocytes of the 45/50% percoll gradient with further enhancement of activity occurring in sorted null cells. Canine NK cells were 5.5 to 6.5 μm in diameter with a reniform (kidney bean shape) nucleus, and electron-dense cytoplasmic granules. NK cells (percoll band 1 cells and null cells) had larger cell and nuclear area, and less round nuclei when compared to non-NK lymphocytes. The overall cytochemical staining (chloracetate esterase, peroxidase, sudan black B, naphthyl acetate esterase, naphthyl butyrate esterase periodic acid-Schiff stain, and acid phosphatase with and without tartrate) pattern was similar in all the lymphocyte populations evaluated. Conclusions: This work confirms the usefulness of a 45/50% percoll gradient in obtaining a NK-enriched fraction of canine lymphocytes, and shows further enhancement of NK activity in sorted CD4^?CD8^? cells. The ultrastructure of canine NK cells is similar to that reported for human NK cells, but is different from that of other canine peripheral blood lymphocytes. Standard cytochemical staining does not discriminate canine NK cells from other lymphocytes. © 1995 Wiley-Liss, Inc.     

Dissociation of natural killer and lymphocyte-activated killer cell lytic activities in human CD3− large granular lymphocytes

CD3^? large granular lymphocytes (LGL) are known to display natural killer cell (NK) activity without prior sensitization or restriction by major histocompatibility antigens. Upon short-term exposure to interleukin-2, NK cells were shown to acquire lymphocyte-activated killer cell (LAK) activity. The aim of this study was to analyze the characteristics of these lytic activities. Our data indicated that both NK and LAK activities were Ca²⁺ dependent; however, they could be dissociated by a Ca²⁺ channel blocker or a Ca²⁺ channel competitor agent. Moreover, NK activity was associated with granule exocytosis of lytic proteins spontaneously present in CD3^? LGL, the most likely candidate being the pore-forming protein perforin. By contrast, LAK activity was found to be dependent on de novo protein synthesis and distinct from granule exocytosis. Our results strongly suggest that NK and LAK activities could be defined as two distinct pathways involving different lytic mediators.     

A boy with X-linked hyper-IgM syndrome and natural killer cell deficiency

We present a boy with hyper-IgM syndrome with a previously not reported mutation in the CD40 ligand gene. He also had a concomitant natural killer (NK) cell deficiency. He had no CD56⁺ or CD16⁺ cells and no NK activity as determined in 4 h chromium release cytotoxicity assay. After 5 days in culture with IL-2-containing medium, however, his peripheral blood mononuclear cells lysed both NK-sensitive and NK-resistant targets, showing that he had lymphokine-activated killer cell precursors in the circulation. Due to the associated neutropenia, he was treated with granulocyte colony-stimulating factor (G-CSF) and responded well. In the same period we observed a transient increase in the number of NK cells. Isolated NK cell deficiencies are extremely rare. We suggest that the defect in our patient is part of the hyper-IgM syndrome, probably representing the phenotype of the new mutation described. Thus, it is possible that both the neutropenia and the NK cell deficiency are due to lack of growth-promoting signals normally delivered by the CD40 ligand.     

Developmentally regulated expression of P-glycoprotein (multidrug resistance) activity in mouse thymocytes

P-glycoprotein (P-gly) is the transmembrane efflux pump responsible for multidrug resistance in tumor cells. The activity of P-gly in mature peripheral lymphocytes is lineage specific, with CD8⁺ T cells and natural killer (NK) cells expressing high levels as compared to CD4⁺ T cells and B cells. We have now investigated P-gly activity in immature and mature subsets of mouse thymocytes. Our data indicate that P-gly activity is undetectable in immature CD4^?8^? and CD4⁺8⁺ thymocyte subsets. Among mature thymocytes, P-gly activity is absent in the CD4⁺ subset but present in the more mature (HSA^low) fraction of CD8⁺ cells. Furthermore, while thymic CD4^?8^? T cell receptor (TCR) γδ cells have little P-gly activity, a minor subset of CD4^?8^? or CD4⁺ TCR αβ⁺ thymocytes bearing the NK1.1 surface marker expresses high levels of P-gly activity. Collectively, our results indicate that P-gly activity arises late during thymus development and is expressed in a lineage-specific fashion.     

Surface Ly-5 Glycoprotein in Murine Natural Killer (NK) Cell Development,Target Binding and Cytotoxicity

The role of surface Ly-5 glycoprotein expression in the binding and lysis of susceptible tumor targets by natural killer cells was studied using NK cell-enriched splenocytes from 6–8 week old C57BL/6 mice which were reacted with anti-Ly-5 serum in the presence or absence of a source of complement. A conjugate assay was used to demonstrate that abrogation of tumor cell lysis by anti-Ly-5 serum involved the inhibition of NK cell binding to susceptible YAC-1 targets. Additionally, reconstituted membrane vesicles from NK cell-enriched splenocyte populations blocked binding of effector cells to YAC-1 lymphoma targets, a phenomenon which was abrogated by pretreatment of vesicles with anti-Ly-5 serum. Indirect immunofluorescent labeling and cell sorting were used in the physical separation of Ly-5⁺ and Ly-5^? cells to examine the effect of interferon and interleukin preparations on Ly-5 expression and NK activity. Three hour treatment of sorted Ly-5^? cells with murine α + β interferon resulted in conversion of 22% of the cells to an Ly-5⁺ phenotype, as well as a significant increase in the percent specific lysis of NK-susceptible YAC-1 targets when compared to freshly sorted Ly-5^? cells (29.5 ± 1.9 vs 2.6 ± 4.0; p < .001). In vitro proliferation of sorted Ly-5^? cells was induced by three week culture in an interferon- and interleukin-containing supernatant from ConA stimulated BALB/c splenocytes (CM), followed by repeat analysis of Ly-5 expression and cytotoxic activity. Cell sorter purified Ly-5^? cells cultured in CM acquired substantial surface Ly-5 with concomitant high levels of cytotoxic activity that remained partially susceptible to inhibition by anti-Ly-5 serum. The data presented suggest that surface Ly-5 glycoprotein expression is important for binding of freshly isolated NK cells to YAC-1 targets. In addition, Ly-5^? precursors of NK cells are present in murine splenic tissues and can be induced by CM to become highly active effector cells with increased surface Ly-5 expression. The persistent susceptibility of a subset of these cells to inhibition of cytotoxic activity by anti-Ly-5 serum provides additional evidence of an important role for the Ly-5 glycoprotein in the natural killer cell cytolytic mechanism against certain targets.     

The CD26 Antigen is Coupled to Protein Tyrosine Phosphorylation and Implicated in CD16-Mediated Lysis in Natural Killer Cells

The levels of CD26 expression, their capacity to induce protein tyrosine phosphorylation and their functional implication in natural killer (NK) cytolysis have been studied. It was found that only a small fraction (12–15%) of peripheral NK cells expresses CD26 compared with the high expression (99%) found in NK clones. The protein tyrosine phosphorylation mediated by means or CD26 activation was studied in NK cells treated with the anti-CD26 MoAb 134–2C2, and two new proteins of 50 and 21 kDa appeared phosphorylated in tyrosine residues. To study the influence of CD26 antigen in NK lysis, we analysed the lytic capacity of NK cells stimulated with different anti-CD26 MoAbs or after separation into CD26⁺ and CD26^? subsets and using K562 as target cells. Under these conditions, no differences were found in the chromium release by the target cells. Redirected lysis through CD16 was also measured by arming the effector cells (CD26⁺ and CD26^?) with anli-CD16 antibody and using K562 as target cells. It was found that CD26^? cells showed significantly less CD16-dependent lysis than CD26⁺ cells. These results indicate that CD26 is related to the CD16-dependent lysis but not to NK cytolysis which may be caused by mediation of protein tyrosine phosphorylation.     

Suppression by Human Placental Protein 14 of Natural Killer Cell Activity

ABSTRACT: Human decidua of early pregnancy contains considerable numbers of CD3^? CD56⁺ natural killer (NK) cells. In this study, two major protein products of the decidua, placental protein 14 (PP14) and placental protein 12 (PP12), were tested for the ability to regulate human NK cell activity. In vitro overnight exposure to PP14 of blood lymphocytes or purified large granular lymphocytes (LGL) resulted in suppression of cytotoxicity against K562 target cells in a 4-h ⁵¹Cr release assay. The NK inhibition was dependent on concentrations of PP14, being detectable at 5 μg/ml and reaching maximum at 50 μg/ml. Manifestation of PP14-induced NK suppression required 18-h contact with NK cells. The suppression of NK activity by PP14 was not abolished by indomethacin. In a target binding assay the number of PP14-treated LGL binding to K562 was comparable to that of untreated ones. By contrast with PP14, PP12 produced no effects on NK cells. These results indicate that PP14 suppresses the function of NK cells, which might be involved in prevention of maternal immune rejection of fetus at the fetomaternal interface.     

NBAS Variants Are Associated with Quantitative and Qualitative NK and B Cell Deficiency

Purpose

Biallelic pathogenic NBAS variants manifest as a multisystem disorder with heterogeneous clinical phenotypes such as recurrent acute liver failure, growth retardation, and susceptibility to infections. This study explores how NBAS-associated disease affects cells of the innate and adaptive immune system.

Methods

Clinical and laboratory parameters were combined with functional multi-parametric immunophenotyping methods in fifteen NBAS-deficient patients to discover possible alterations in their immune system.

Results

Our study revealed reduced absolute numbers of mature CD56^dim natural killer (NK) cells. Notably, the residual NK cell population in NBAS-deficient patients exerted a lower potential for activation and degranulation in response to K562 target cells, suggesting an NK cell–intrinsic role for NBAS in the release of cytotoxic granules. NBAS-deficient NK cell activation and degranulation was normalized upon pre-activation by IL-2 in vitro, suggesting that functional impairment was reversible. In addition, we observed a reduced number of naïve B cells in the peripheral blood associated with hypogammaglobulinemia.

Conclusion

In summary, we demonstrate that pathogenic biallelic variants in NBAS are associated with dysfunctional NK cells as well as impaired adaptive humoral immunity.

     

Pathogenesis of the natural killer cell deficiency in AIDS

Deficiency in natural killer (NK) cell activity is a common feature of acquired immune deficiency syndrome (AIDS). This is part of a general immune dysfunction in AIDS and may lead to progression of the disease, since NK cells are known to be involved in protection against tumors and against viral infections. The lack of immunological surveillance by NK cells of the growth of pathogens that activate the HIV-1 tat infectivity gene may also favor progression to AIDS. The pathogenesis of NK cell deficiency in AIDS is not known. Previous studies have shown that NK cells from AIDS patients are able to bind but not to lyse the target cell line K562. This results from an inability to rearrange the cytoskeleton microtubular (MT) system and to release the natural killer cytotoxic factor (NKCF). This report by Maria Caterina Sirianni and colleagues evaluates the possible mechanisms leading to this NK cell deficiency.     

Subsets of human natural killer cells and their regulatory effects

Human natural killer (NK) cells have distinct functions as NK^tolerant, NK^cytotoxic and NK^regulatory cells and can be divided into different subsets based on the relative expression of the surface markers CD27 and CD11b. CD27⁺ NK cells, which are abundant cytokine producers, are numerically in the minority in human peripheral blood but constitute the large population of NK cells in cord blood, spleen, tonsil and decidua tissues. Recent data suggest that these NK cells may have immunoregulatory properties under certain conditions. In this review, we will focus on these new NK cell subsets and discuss how regulatory NK cells may serve as rheostats or sentinels in controlling inflammation and maintaining immune homeostasis in various organs.     

Mechanism of tumor cells escaping from immune surveillance of NK cells

Abstract

Natural killer (NK) cells play an important role in anti-tumor and anti-infection, and perform their immune surveillance function in various ways. However, no matter what kind of cancer, the functional activity of NK cells in the tumor microenvironment (TME) is suppressed. Understanding the relationship between tumor cells and NK cells is very critical for tumor immunotherapy. This review discusses the mechanism of tumor cells escaping the immune surveillance of NK cells. These include a variety of factors that inhibit the activity of NK cells, an imbalance of activating receptors and inhibiting receptors on NK cells, abnormal binding of receptors and ligands, cross-talk of surrounding cell groups and NK cells in the TME, and other factors that affect NK cell activity. An understanding of these factors is necessary to provide new treatment strategies for tumor immunotherapy.     

Changes in NK and NKT cells in mesenteric lymph nodes after a Schistosoma japonicum infection

The mesenteric lymph node (MLN) is the main draining lymph node in mouse enterocoelia, which contains many types of immune cells. Among these cells, natural killer (NK) and natural killer T (NKT) cells belong to innate lymphoid cells (ILCs), which have potent activities for controlling a variety of pathogenic infections. In this study, C57BL/6 mice were infected with Schistosoma japonicum for 5–7 weeks. Lymphocytes were isolated from the MLN to detect changes in the phenotype and function of NK and NKT cells using a fluorescence activating cell sorter (FACS). These results demonstrated that a S. japonicum infection could significantly increase the percentage of NK cells in the mouse MLN, (P?<?0.05). We found an increase in the cell number of both NK and NKT cells. In addition, we found that NK and NKT cells from infected mice expressed higher levels of CD69 compared to normal mice (P?<?0.05). These results demonstrated that a S. japonicum infection could induce MLN NK and NKT cell activation. Moreover, we found that the expression of CD4 was increased in infected MLN NK cells (P?<?0.05). Furthermore, intracellular cytokine staining revealed that expression of IL-4 and IL-17 were significantly enhanced in both the NK and NKT cells of infected mice after phorbol 12-myristate 13-acetate (PMA) and ionomycin stimulation (P?<?0.05). Taken together, these results indicated that infection-induced MLN NK and NKT cells might play roles in modulating the classical T cell response. Finally, our results indicated that the expression of CD94 was decreased in NK cells, suggesting that the downregulation of CD94 expression might served as a mechanism in NK cell activation.     

Simultaneous Effects of Lead and Cadmium on NK Cell Activity and Some Phenotypic Parameters

Lead and cadmium are common environmental contaminants that alter the immune response. Natural killer cell (NK) cytotoxicity and phenotypic parameters are crucial immune responses, but little is known about the effects of these metals on these responses. In the present paper, we investigated the simultaneous effects of lead and cadmium on NK cell activity and CD4⁺, CD20⁺ cell percentages in the workers and compared the data with control and lead-exposed groups. We conclude that simultaneous exposure to lead and cadmium is not associated with an impairment of either NK cell function or CD4⁺ cell percentage. On the other hand, CD20⁺ cell percentage was found higher in lead+cadmium exposed group than controls.     

Natural killer cell subsets in cerebrospinal fluid of patients with multiple sclerosis

Changes in blood natural killer (NK) cells, important players of the immune innate system, have been described in multiple sclerosis (MS). We studied percentages and total cell counts of different effector and regulatory NK cells in cerebrospinal fluid (CSF) of MS patients and other neurological diseases to gain clearer knowledge of the role of these cells in neuroinflammation. NK cell subsets were assessed by flow cytometry in CSF of 85 consecutive MS patients (33 with active disease and 52 with stable MS), 16 with other inflammatory diseases of the central nervous system (IND) and 17 with non-inflammatory neurological diseases (NIND). MS patients showed a decrease in percentages of different CSF NK subpopulations compared to the NIND group. However, absolute cell counts showed a significant increase of all NK subsets in MS and IND patients, revealing that the decrease in percentages does not reflect a real reduction of these immune cells. Remarkably, MS patients showed a significant increase of regulatory/effector (CD56^bright/CD56^dim) NK ratio compared to IND and NIND groups. In addition, MS activity associated with an expansion of NK T cells. These data show that NK cell subsets do not increase uniformly in all inflammatory neurological disease and suggest strongly that regulatory CD56^bright and NK T cells may arise in CSF of MS patients as an attempt to counteract the CNS immune activation characteristic of the disease.     

Pilot Study of Natural Killer Cells in Chronic Fatigue Syndrome/Myalgic Encephalomyelitis and Multiple Sclerosis

Patients with chronic fatigue syndrome/myalgic encephalomyelitis (CFS/ME) and multiple sclerosis (MS) suffer from debilitating fatigue which is not alleviated by rest. In addition to the fatigue‐related symptoms suffered by patients with CFS/ME and MS, dysfunction of the immune system and, in particular, reduced natural killer (NK) cell cytotoxic activity has also been reported in CFS/ME and MS. The purpose of this pilot study was to compare NK cellular mechanisms in patients with CFS/ME and MS to investigate potential dysfunctions in the NK cell activity pathway. Flow cytometry protocols assessed CD56^dimCD16⁺ and CD56^brightCD16^+/? NK cell expression of adhesion molecules, NK activating and inhibiting receptors, NK cell maturation and lytic proteins. All participants in this study were female and included 14 patients with CFS/ME, nine patients with MS and 19 non‐fatigued controls. The patient groups and the non‐fatigued controls were not taking any immunosuppressive or immune‐enhancing medications. In the MS cohort, KIR2DL5 was significantly increased on CD56^brightCD16^+/? NK cells and expression of CD94 was significantly increased on CD56^dimCD16⁺ NK cells in comparison with the controls. Co‐expression of CD57 and perforin was significantly increased on CD56^dimCD16⁺ NK cells from patients with CFS/ME compared to the MS and non‐fatigued control participants. The results from this pilot study suggest that NK cells from patients with CFS/ME and MS may have undergone increased differentiation in response to external stimuli which may affect different mechanisms in the NK cell cytotoxic activity pathway.     

Allo-skin graft rejection,tumor rejection and natural killer activity in mice lacking p56lck

Mice lacking the p56^lck molecule (lck?/?) have a profound block in the maturation of thymocytes and a greatly reduced number of peripheral mature T cells. To analyze further the functions of the T cells developed in lck ?/? mice in vivo, we evaluated the ability of lck?/? mice to reject allo-skin grafts and methylcholanthrene (MCA)-induced syngeneic fibrosarcoma, and also examined the biological activity of lck ?/? natural killer (NK) cells. Mice lacking p56^lck failed to reject skin grafts from either MHC-disparate or minor-histocompatibility-different donors, even after they had been primed with donor spleen cells. They also failed to reject the MCA-induced immunogenic syngeneic fibrosarcoma, MC57X. NK activity in mice lacking p56^lck was normal, and there were no differences in the NK cell activation induced by poly(I) · poly(C) stimulation or interleukin-2 stimulation (lymphokine-activated killer induction) between mice lacking p56^lck and their immunocompetent heterozygous littermates. NK cells lacking p56^lck mediated a normal antibody-dependent cell-mediated cytotoxicity (ADCC) response. The results of this study indicate that the loss of p56^lck severely impairs the effectors of the immune system which mediate the rejection of allo-skin grafts and syngeneic tumors. The normal NK activity in lck ?/? mice suggests that p56^lck is not required for the development and activation of NK cells.     

Hybrid Resistance to EL-4 Lymphoma Cells

(C57BL/6 × DBA/2)F₁ hybrid (B6D2F₁) mice resist the growth of parental-strain (B6) EL-4 lmphoma cells inoculated intraperitoneally; that is, B6D2F₁ mice survive longer than B6 mice and do not develop uscites As compared with B6 mice, B6D2F₁ mice have higher levels of natural killer (NK) activity against ⁵¹Cr-labelled EL-4 cells in their lyphiod organs. B6D2F₁ mice treated with ⁸⁹Sr lose NK activity for certain lymphoma cell targets, e.g. YAC-1, but NK(EL-4) function is usually intact. However, ⁸⁹Sr-treated mice had hybrid resistance to EL-4 cell in vivo, as determined by survival times and the development of ascites. NK(EL-4) and NK-(YAC-1) activities were stimulated by irrudiated or unirradiated EL-4 cells, Corynebacterium parvum, or polyinosinic:polycytidylic acid (pl:pC) in spleens of normal B6D2F₁ mice, but NK(EL-4) activity was depressed Within 3 days by such treatment in B6D2F₁ mice previously injected with ⁸⁹Sr. Suppressor cells for NK(EL-4) but not for NK(YAC-1) effectors were easily detected in spleens of ⁸⁹Sr-treated mice ‘challenged’ with C. parvum. Thus, agents capable of Stimulating NK cell function in normal mice may lead to suppression of that activity in mice depleted of marrow-dependent cell function by ⁸⁹Sr. Spleen cells of ⁸⁹Sr-treated B6D2F₁ mice were also unable to generate anti-EL-4 cytotoxic T lymphocytes in a cell-mediated lympholysis system; this defect appeared also in to be mediated by suppressor cells. Lymphoid cell depleted by ⁸⁹Sr-induced marrow aplasia may have two functions in host defences against tumours (especially lymphomas): they may lyse tumour cells directly and they may ‘down-regulate’ suppressor cells capable of inhibiting other ‘natural’ or ‘induced’ immune functions.