Lymphokine-activated killer cells in rats: generation of natural killer cells and lymphokine-activated killer cells from bone marrow progenitor cells

The coculture of rat bone marrow cells with recombinant interleukin-2 induced the generation of cells mediating natural killer (NK) activity and subsequent lymphokine-activated killer (LAK) activity depending upon the dose of IL-2 and time of culture. NK activity was detected as early as 4 to 5 days after the addition of IL-2 and could be evoked with as little as 5 to 50 U/ml. The induced NK cells had large granular lymphocyte (LGL) morphology and expressed 0X8 and asialo GM1 surface markers but did not express 0X19 or W3/25 markers. LAK activity was detected only after 5 days of culture, and required above 100 U/ml IL-2. Cells mediating LAK activity also expressed 0X8 and asialo GM1 but not 0X19. The generation of detectable NK and subsequent LAK activity was due to induction of early progenitor cells and not contaminating mature LGL/NK cells within the bone marrow population since of removal of such mature NK cells with L-leucine methyl ester (L-LME) did not affect the subsequent generation of either activity. Moreover, the removal of actively dividing cells as well as mature NK cells from the bone marrow by treatment with 5-fluorouracil (5-FU) in vivo enriched the remaining bone marrow population for both NK and LAK progenitor cells. The phenotype of the L-LME- and 5-FU-resistant NK and LAK progenitor cells within populations of bone marrow was determined by antibody plus complement depletion analysis. Although treatment of normal bone marrow with anti-asialo GM1 + C reduced the induction of NK and LAK activity in 5-day cultures, treatment of 5-FU marrow with anti-asialo GM1 + C did not affect either activity. Treatment with a pan-T cell antibody + C did not affect the development of NK or LAK activity under any conditions. Thus, the 5-FU-resistant NK/LAK progenitors were asialo GM1 negative but became asialo GM1+ after induction by IL-2. Finally, evidence that bone marrow-derived LAK cells were generated directly from the IL-2-induced NK cells was obtained by treating the IL-2-induced LGL/NK cells with L-LME.(ABSTRACT TRUNCATED AT 400 WORDS)     

Lymphokine-activated killer cells are rejected in vivo by activated natural killer cells

A 4-h in vivo cytotoxicity assay was used to study the fate of implanted IL-2-generated, lymphokine-activated killer (LAK) cells in mice undergoing an activated NK cell response. 125Iododeoxyuridine-labeled LAK cells were rejected from selected organs of C57BL/6 mice infected with lymphocytic choriomeningitis virus or treated with IL-2 or the IFN inducer poly I:C. This rejection was abrogated by the selective depletion of NK cells with antibodies to asialo-GM1 and NK1.1 Ag. Similar results were noted when LAK cells were generated from the spleens of B and T cell-deficient severe combined immunodeficiency mice and when LAK cells were implanted into severe combined immunodeficiency mice. These data indicate that NK cells activated by virus infections or by IL-2 infusions directly or indirectly eliminate implanted LAK cells. Because LAK cells are used in the treatment of certain human cancers, the strategy of accompanying this therapy with IL-2 infusions should be reassessed in light of these results.     

Growth of tumor cells with different sensitivities for murine natural killer cells in young and adult athymic nude mice

Of four tumor cell lines, the murine YAC lymphoma, the human K562 lymphoma, and the human prostatic carcinomas PC3 and PC93, the susceptibility to murine natural killer (NK) cells as well as the tumorigenicity in young (3.5-4 weeks old) and in adult (8-10 weeks old) nude mice were studied. In young nude mice, which exhibited a lower level of NK cell activity than adult nude mice, the formation of solid tumors after inoculation of tumor cell suspensions occurred more frequently and with a shorter time lag than in adult animals. These effects were observed not only with the NK-sensitive YAC cells, but also with the relatively NK-insensitive PC3 and PC93 cells, indicating that also factors other than NK cell susceptibility may influence the growth of tumor cells in nude mice. Therefore, the use of young nude mice may enhance the rate of success of heterotransplantation of human tumors, regardless of the NK cell susceptibility of the tumor cells.     

Functional natural killer T cells in experimental mouse strains, including NK1.1- strains.

Natural killer T (NKT) cells are a newly discovered subset of lymphocytes. It appears that this subset has potential as important regulators of immune responses. But because there are relatively few NKT cells in lymphoid organs and because of technical difficulties in detecting NKT cells in most mouse strains, the roles of NKT cells have not been fully identified and little attention has been paid to the roles of NKT cells in immunological experiments in which NK1.1- strains were used. To examine the existence of functional NKT cells in various strains of experimental mice, including NK1.1- strains, we utilized alpha-galactosylceramide (KRN7000) which is thought to react specifically with NKT cells. Indeed, we could confirm that early cytokine (IL-4 and IFN-gamma) secretion at 2 h after the injection of KRN7000 was dependent on NKT cells. With this in vivo system, we have successfully detected the presence of functional NKT cells in various mouse strains, including AKR/N, BALB/c, C3H/HeJ, C3H/HeN, C57BL/6, C.B-17, CBA/N, NC, NOD, SJL, W/Wv, aly/aly and aly/+. Notable increases of serum IL-4 were detected in W/Wv and aly/+ strains, and defective response of IFN-gamma in SJL mice and that of IL-4 in NOD mice were observed. This is the first report to show the functional significance of NKT cells in cytokine secretion in various mouse strains in response to a ligand for the T cell receptor of NKT cells.     

Missing self by heterogeneous natural killer cells

Some murine (YAC, P815 and SP20) and human (Molt4, Raji and HR7) tumour cell lines were (i) treated with IFN-γ for inducing enhanced expression of MHC class I antigen, or (ii) given a brief treatment with citrate buffer (pH 3.0), which resulted in denaturation of class I MHC antigens on these tumour cells. IFL-γ or acid treated tumour cells were used as unlabelled competing targets in cold target inhibition assays. The results indicated that the competing ability of acid-treated tumour cells remained unaltered, whereas IFN-γ treated tumour cells competed with significantly less efficiency. These results have been evaluated in light of the current view of NK cell development and the expression of inhibitory receptors for MHC class I molecules (IRMs), on NK cells. A modified view on NK cell heterogeneity based upon IRM expression has been proposed which reconciles several apparently discordant observations about the activity and role of NK cells. Two classes of NK cells have been proposed. Type I NK ceils have target recognition receptors which do not recognize autologous normal cells, lack IRMs, and may participate in first line of defence against transformed cells in vivo. Type II NK cells have target recognition receptors for autologous normal cells and express at least one self-reactive IRM in order to prevent auto-killing. Type II NK cells participate in killing those transformed cells which down-regulate their MHC class I expression in order to escape cytotoxic T-cell surveillance. It is also postulated that mechanism of inverse correlation of target cell MHC class I expression levels and their susceptibility to NK cells, involves interference model of missing self hypothesis for type I NK cells and inhibitory signal model of missing self hypothesis for type II NE cells. Finally, it is proposed that acid treatment of tumour cells enhances their lysis susceptibility by making them additionally susceptible to type II NK cells, rather than enhancing their killing by type I NK cells. This proposition would explain the lack of effect of acid treatment on the competing ability of tumour cells, when target cells are only lysed by type I NE cells.     

Trafficking of natural killer cells

Natural killer (NK) cells comprise a set of lymphocytes that is capable of mediating innate immune responses to viral infections, malignancies, and allogeneic bone marrow grafts. This review summarizes what is known about the mechanisms NK cells use to arrive at their sites of action. NK cells express a wide array of adhesion molecules including alphaLbeta2, alphaMbeta2, alphaXbeta2, and alpha4beta1 integrins, ICAM-1, PSGL-1, and L-selectin. Like other immune and inflammatory cells, NK cells use the blood circulation to enter tissues and organs, which requires that they interact with the vessel wall under flow conditions, arrest, and transmigrate. NK cells are able to chemotax to a variety of cytokines and chemokines, including IL-12, IFN-(alpha/beta, CCL2, 3, 4, 5, 7, 8, CXCL8, and CX3CL1. In many cases, NK cells appear to migrate towards these soluble factors without any kind of priming. These cells also appear to distribute in secondary and tertiary lymphoid sites (i.e., spleen, bone marrow, liver, lung, and lymph nodes) both with and without stimulation. In addition to their ability to move throughout the body in an unprimed state, activated NK cells may have increased specificity in homing to sites of inflammation. NK cells not only react to, but also produce IFN-gamma, TNF-alpha, GM-CSF, CCL3, CCL4, and CCL5, enabling them to recruit various immune cells to sites of immune response.     

Homeostasis of natural killer cells

Natural killer (NK) cells are important players of innate immunity, dedicated to the host defense against viruses and also involved in the immune surveillance of tumors. NK cells are widely distributed in the body and their number may increase locally during infection. They develop mainly in the bone marrow and perhaps in other lymphoid organs. They are constantly renewed, with a half-life of about 17 days at the periphery. In this article, we review the factors that regulate the homeostasis of NK cells including their development, differentiation, export to the periphery, their turnover, their homeostatic or antigen-induced proliferation and their survival before or after activation. In addition, we discuss the homeostasis of recently described so-called "memory" NK cells.     

Self-tolerance of natural killer cells

Natural killer (NK) cells, similar to other lymphocytes, acquire tolerance to self. This means that NK cells have the potential to attack normal self cells but that there are mechanisms to ensure that this does not usually occur. Self-tolerance is acquired by NK cells during their development, but the underlying molecular and cellular mechanisms remain poorly understood. Recent studies have produced important new information about NK-cell self-tolerance. Here, we review the evidence for and against possible mechanisms of NK-cell self-tolerance, with an emphasis on the role of MHC-specific receptors.     

Natural killer cells and natural killer T cells in Lyme arthritis

Introduction

Natural killer (NK) and natural killer T (NKT) cells provide a first line of defense against infection. However, these cells have not yet been examined in patients with Lyme arthritis, a late disease manifestation. Lyme arthritis usually resolves with antibiotic treatment. However, some patients have persistent arthritis after spirochetal killing, which may result from excessive inflammation, immune dysregulation and infection-induced autoimmunity.

Methods

We determined the frequencies and phenotypes of NK cells and invariant NKT (iNKT) cells in paired peripheral blood (PB) and synovial fluid (SF) samples from eight patients with antibiotic-responsive arthritis and fifteen patients with antibiotic-refractory arthritis using flow cytometry and cytokine analyses.

Results

In antibiotic-responsive patients, who were seen during active infection, high frequencies of CD56bright NK cells were found in SF, the inflammatory site, compared with PB (P <0.001); at both sites, a high percentage of cells expressed the activation receptor NKG2D and the chaperone CD94, a low percentage expressed inhibitory killer immunoglobulin-like receptors (KIR), and a high percentage produced IFN-γ. In antibiotic-refractory patients, who were usually evaluated near the conclusion of antibiotics when few if any live spirochetes remained, the phenotype of CD56bright cells in SF was similar to that in patients with antibiotic-responsive arthritis, but the frequency of these cells was significantly less (P = 0.05), and the frequencies of CD56dim NK cells tended to be higher. However, unlike typical NKdim cells, these cells produced large amounts of IFN-γ, suggesting that they were not serving a cytotoxic function. Lastly, iNKT cell frequencies in the SF of antibiotic-responsive patients were significantly greater compared with that of antibiotic-refractory patients where these cells were often absent (P = 0.003).

Conclusions

In patients with antibiotic-responsive arthritis, the high percentage of activated, IFN-γ-producing CD56bright NK cells in SF and the presence of iNKT cells suggest that these cells still have a role in spirochetal killing late in the illness. In patients with antibiotic-refractory arthritis, the frequencies of IFN-γ-producing CD56bright and CD56dim NK cells remained high in SF, even after spirochetal killing, suggesting that these cells contribute to excessive inflammation and immune dysregulation in joints, and iNKT cells, which may have immunomodulatory effects, were often absent.     

Renewal of natural killer cells in mice having elevated natural killer cell activity

The present study was designed to examined the dynamics of splenic natural killer (NK) cells under two conditions of enhanced NK cell activity: (1) CBA/J mice given polyinosinic-polycytidylic acid (poly-I:C), an NK-cell-enhancing agent, and 62) untreated athymic nude (nu/nu) mice. The 'total NK cell activity' of the spleen (percentage specific lysis corrected for changes in organ cellularity) increased 5-fold and 2.7-fold after poly-I:C treatment for 1 day and 4 days, respectively. An injection of hydroxyurea (HU), a cell-cycle-toxic drug, given together with either poly-I:C or saline to CBA/J mice resulted in both cases in a 25% reduction in total NK cell activity 1 day later. This suggests that the renewal rate of nondividing NK cells is similar in poly-I:C-treated and saline-injected mice, and that the NK-enhancing effect of poly-I:C is not due to a stimulation of proliferation among NK cell precursors. HU administered simultaneously with poly-I:C or saline for 4 days eliminated NK cell activity in both cases, indicating that spleen NK cell activity is mediated almost entirely by newly formed (less than or equal to 4 days) cells. In nude mice, NK cell activity was assayed at various intervals after an HU depletion period of 2 days. NK depletion was initially more rapid in nu/nu mice than in control (nu/+) mice, although equally profound, and the subsequent recovery of NK cell activity after cessation of HU was also more rapid than in control (nu/+) mice.(ABSTRACT TRUNCATED AT 250 WORDS)     

Lysis of fresh murine mammary tumor cells by syngeneic natural killer cells and lymphokine-activated killer cells

Summary We have compared the ability of natural killer (NK) cells from two substrains of C3H mice that differ with respect to their susceptibility to the development of mammary adenocarcinomas to lyse fresh syngeneic mammary tumor cells. Single cell suspensions of mammary tumors from retired breeder females were used as targets in 22-h ⁵¹Cr-release cytotoxicity assays with syngeneic NK cells. Tumor cell suspensions were prepared by enzymatic digestion of finely minced tissue followed by centrifugation through a discontinuous Percoll gradient. Effector cells were prepared by passing spleen cells over nylon wool followed by centrifugation through Percoll fraction 7. Syngeneic NK cells had significant levels of lysis against 5/8 tumors studied. NK cells from low risk animals (C3Heb/FeJ) consistently demonstrated greater cytotoxicity against tumor cell preparations than did effectors from the high tumor substrain (C3H/OuJ). Study of cytocentrifuge preparations stained with Wright-Giemsa revealed that the two substrains were identical with respect to the number of azurophilic granules present in the cytoplasm of their NK cells. We have also shown that lymphokine-activated killer (LAK) cells can be generated from splenocytes in C3H mice. While LAK cells from both substrains were capable of lysing fresh syngeneic mammary tumor cells in vitro, LAK cells from the animals at high risk for the formation of mammary adenocarcinomas had greater cytotoxicity against tumor cell suspensions than LAK cells from the low tumor substrain.     

Lysis of human solid tumor cells by lymphokine-activated natural killer cells

The ability of NK cells to lyse noncultured solid tumor cells was investigated, and the results were compared with lysis of K562. Purified NK cell fractions separated by either Percoll centrifugation or a cell sorter exhibited higher level of lysis against noncultured melanoma cells than did NK-depleted cell fractions. However, the level of lysis was low (less than 10% lysis). Adding recombinant interleukin 2 (rIL 2) to the 4-hr assay induced significant lysis (more than 10%) of noncultured melanoma cells in 18 of 23 (78%) Percoll-enriched NK cell fractions and seven of 11 (64%) sorted Leu-11a+ cells at an E:T ratio of 80 and 10, respectively. In contrast, only two of 13 (14%) PBMC, five of 17 (29%) Percoll-decreased NK cell fractions, and one of 12 (8%) sorted Leu-11a- cells lysed noncultured melanomas in the presence of rIL 2. rIL 2 induced NK cells to lyse noncultured lung and breast cancer cells, as well as melanoma tumors. Exposure of NK cells to 2000 rad radiation abrogated the rIL 2-induced cytotoxicity against noncultured melanomas. Preculture of PBMC for 18 hr with recombinant interferon-gamma (rIFN-gamma) resulted in a modest level of lysis of non-cultured melanomas by sorted Leu-11a+ cells. Adding rIL 2 to the assay increased the cytotoxic activity in both rIFN-gamma-activated Leu-11a+ and Leu-7+ NK subsets. The level of noncultured tumor lysis correlated well with that of K562 lysis in all of the experiments. Purified NK cell fractions in rIL 2 cultures increased cytotoxic activity against noncultured tumor cells with incubation time for up to 3 days, and the level of NK cell-mediated lysis was dependent on both doses of rIL 2 and length of incubation. In contrast, both NK-depleted and sorted Leu-11a- cells demonstrated very low levels of solid tumor lysis after 3-day cultures with a high dose of rIL 2. Killer cell precursors induced by 3-day cultures of sorted cell fractions with rIL 2 and rIFN-gamma were found in both Leu-11a+ and Leu-7+ NK subsets, but not Leu-4+ or Leu-3a+ T lymphocytes. These results indicate that NK cells become cytotoxic for noncultured solid tumor cells by a brief contact with rIL 2, and increase cytotoxic activity after culture with rIL 2.     

Natural killer and natural killer T cells in liver fibrosis

The liver lymphocyte population is enriched with natural killer (NK) cells, which play a key role in host defense against viral infection and tumor transformation. Recent evidence from animal models suggests that NK cells also play an important role in inhibiting liver fibrosis by selectively killing early or senescence activated hepatic stellate cells (HSCs) and by producing the anti-fibrotic cytokine IFN-γ. Furthermore, clinical studies have revealed that human NK cells can kill primary human HSCs and that the ability of NK cells from HCV patients to kill HSCs is enhanced and correlates inversely with the stages of liver fibrosis. IFN-α treatment enhances, while other factors (e.g., alcohol, TGF-β) attenuate, the cytotoxicity of NK cells against HSCs, thereby differentially regulating liver fibrogenesis. In addition, the mouse liver lymphocyte population is also enriched for natural killer T (NKT) cells, whereas human liver lymphocytes have a much lower percentage of NKT cells. Many studies suggest that NKT cells promote liver fibrogenesis by producing pro-fibrotic cytokines such as IL-4, IL-13, hedgehog ligands, and osteopontin; however, NKT cells may also attenuate liver fibrosis under certain conditions by killing HSCs and by producing IFN-γ. Finally, the potential for NK and NKT cells to be used as therapeutic targets for anti-fibrotic therapy is discussed. This article is part of a Special Issue entitled: Fibrosis: Translation of basic research to human disease.     

Organ-specific features of natural killer cells

Natural killer (NK) cells can be swiftly mobilized by danger signals and are among the earliest arrivals at target organs of disease. However, the role of NK cells in mounting inflammatory responses is often complex and sometimes paradoxical. Here, we examine the divergent phenotypic and functional features of NK cells, as deduced largely from experimental mouse models of pathophysiological responses in the liver, mucosal tissues, uterus, pancreas, joints and brain. Moreover, we discuss how organ-specific factors, the local microenvironment and unique cellular interactions may influence the organ-specific properties of NK cells.     

Divergence in activation by poly I:C of human natural killer and killer cells

Summary Interferons consistently enhance spontaneous cellular cytotoxicity (SCC) mediated by natural killer (NK) cells. More controversial is the ability of interferons to enhance antibody-dependent cellular cytotoxicity (ADCC) mediated by killer (K) cells. Since NK and K cells appear to represent overlapping subpopulations of lymphocytes, the present study was undertaken to examine in greater detail the relationship between NK and K cell functional modulation by the potent interferon inducer, poly I:C. Utilizing peripheral mononuclear cells from a panel of 21 healthy individuals, treatment in vitro with poly I:C resulted in modulation of both SCC and ADCC. SCC was significantly enhanced in 52 of a series of 55 trials (95%), whereas ADCC was significantly enhanced in parallel in only 18 of the trials (33%). Cells which mediated enhanced ADCC were plastic-nonadherent, which is characteristic of K cells. SCC was consistently enhanced in all but two of the 14 individuals who were tested two or more times. By contrast, the ability of poly I:C to enhance ADCC varied between trials in 11 of these individuals. In the other three, ADCC enhancement never occurred. No correlation existed between SCC and ADCC augmentation despite use of the same target cell to assess the two lytic activities in parallel. Poly I:C exclusively enhanced SCC in 36 trials (65%) and exclusively enhanced ADCC in two trials (4%). Discordance between SCC and ADCC enhancement also occurred in three of eight trials (38%) in which lymphocytes were treated directly with interferon a. Results in long-term (18-h) ⁵¹Cr-release assays indicated that poly I:C accelerated the kinetics of ADCC without affecting the proportion of target cells lysed by K cells. By contrast, an increased proportion of target cells was killed by poly I:C-stimulated NK cells. These results suggest that the controversy concerning relative interferon effects upon NK and K cells derives from differences both quantitative and qualitative in nature. K cell activity is enhanced but at a relatively low frequency. Enhancement of NK cell activity is selective in the sense that it occurs independently of and with greater frequency than enhancement of K cell activity. Distinct biological mechanisms may, therefore, be involved in regulation and expression of NK and K cell activation by interferons.     

Neutrophil microparticles modulate cytokine production by natural killer cells

Neutrophil microparticles (NMs) are scarce in the circulation but are highly enriched at sites of inflammation and exert profound effects on immune cells. In the present study, we addressed whether NMs modulate cytokine-producing capacity of natural killer (NK) cells. NMs inhibited the production of IFN-γ and TNF-α but enhanced the release of TGF-β1 by IL-2/IL-12-activated NK cells. The inhibitory effect of NMs was strongly attenuated by blockade of phosphatidylserine exposed on NMs. Thus, NMs skew the cytokine profile of NK cells from pro-inflammatory toward anti-inflammatory, potentially favoring the resolution of inflammation.     

Recognition of vaccinia virus-infected cells by human natural killer cells depends on natural cytotoxicity receptors

Natural Killer (NK) cells are important in the immune response to a number of viruses; however, the mechanisms used by NK cells to discriminate between healthy and virus-infected cells are only beginning to be understood. Infection with vaccinia virus provokes a marked increase in the susceptibility of target cells to lysis by NK cells, and we show that recognition of the changes in the target cell induced by vaccinia virus infection depends on the natural cytotoxicity receptors NKp30, NKp44, and NKp46. Vaccinia virus infection does not induce expression of ligands for the activating NKG2D receptor, nor does downregulation of major histocompatibility complex class I molecules appear to be of critical importance for altered target cell susceptibility to NK cell lysis. The increased susceptibility to lysis by NK cells triggered upon poxvirus infection depends on a viral gene, or genes, transcribed early in the viral life cycle and present in multiple distinct orthopoxviruses. The more general implications of these data for the processes of innate immune recognition are discussed.     

Tissue-resident natural killer cells in the livers

Nature killer(NK) cells are important lymphocytes of the innate immune system,well known for their pivotal roles in immune surveillance and defense against tumor and virus-infected cells.Current studies have revealed that NK cells are not a homogeneous population,but instead consist of distinct subsets with diverse characteristics.As an organ with predominant innate immunity,the liver is enriched with NK cells,among which two distinct NK cell subsets have recently been identified:conventional NK(cNK)cells and tissue-resident NK(trNK) cells.Liver trNK cells are markedly different from cNK cells in many aspects,representing a new lineage of innate lymphoid cell(ILC) family.Here,we summarize the phenotypic and functional features of liver trNK cells,and review current knowledge regarding developmental pathway of liver trNK cells.We also overview recent advances in human liver trNK cells and discuss the striking shared hallmarks of trNK cells in different tissues.     

Further characterization of natural killer cells induced by Kunjin virus

The natural killer (NK) cell induced one to two days after Kunjin virus infection of BALB/c mice is cytotoxic for a wide range of syngeneic, allogeneic and xenogeneic cell lines. It is also weakly cytotoxic for some non-malignant cells including mouse fibroblasts, macrophages and thymocytes, but not lymph node cells. Levels of lysis of non-tumour target cells are dependent on their genotype. Furthermore, malignant cell lines may become resistant following transplantation in vivo then susceptible again after culture in vitro. The virus-induced NK cell is elicited as readily in athymic (nude) as in normal mice. X-irradiation inhibits its development if administered prior to infection. It is labile on culture at 37 degrees. The cell carries Fc receptors but its NK activity is not antibody-dependent.