Isolation and characterization of porcine natural killer (NK) cells

The morphologic and biological properties of porcine cells mediating natural killer (NK) activity were determined. In a previous study, we demonstrated that lymphocytes from the peripheral blood of pigs greater than 1 week of age possessed NK activity to K562 tumor cells and that lymphocytes from the blood and spleen of pigs greater than 1 day of age were able to mediate natural cytotoxicity against parainfluenza-3 (PI3) virus-infected Vero cells (Yang and Schultz, 1986a). Discontinuous density gradients were used to enrich NK cells. NK cytotoxicity was mainly present in high-density Percoll fractions (50 to 55% and 55 to 60%); little or no NK activity was present in lower density fractions. The NK cell enriched lymphocytes responded to the mitogens PHA, ConA and PWM. NK cells were sensitive to the suppressive effect of corticosteroid, but Protein A did not affect NK activity. The amount of cytotoxicity directly corresponded to the degree of binding that occurred between the NK enriched lymphocyte population and the target cells. Cytochemical and morphological studies demonstrated that these bond cells which are believed to be responsible for the NK activities, were mainly small to medium lymphocytes lacking azurophilic cytoplasmic granules. These findings were confirmed by ultrastructural studies of effector and "target-binding" cells. The results of the present study suggested that the cells mediating NK activity in pigs have the morphological and density characteristics of small and medium sized lymphocytes; findings that differ from those described for NK cells in human and other animal species.     

重组腺病毒Ad－HP对肝癌细胞及肝癌肺转移模型的抑制效应

为探讨重组腺病毒Ad－HP对人肝癌细胞BEL－7402的影响及其对鼠肝癌肺转移模型的抑制作用,本研究利用MTT染色法进行了Ad－HP的体外抑瘤研究,并应用AnnexinV检测、Caspase活性检测、AO／EB染色和DAPI染色等方法分析了Ad－HP的抑瘤方式。另外,本研究利用鼠肝癌细胞H22,建立了肝癌肺转移动物模型,并检测了Ad－HP对肝癌肺转移的抑制作用及其对模型动物免疫的影响。结果表明,Ad－HP能够有效抑制BEL－7402细胞的生长,且其作用具有一定时效和剂效关系趋势;Ad－HP感染导致BEL7402细胞呈现磷脂膜外翻、细胞核皱缩、细胞膜通透性增加和Caspase酶活性增强等典型凋亡特征;应用Ad－HP治疗肿瘤模型能够有效延长模型动物平均生存期,控制转移灶形成,增强NK和CTL活性,上调Thl类细胞因子水平。总之,Ad－HT能够通过诱导细胞凋亡抑制BEL－7402肿瘤细胞增殖,并且能够有效控制肝癌肺转移灶形成,延长模型动物平均生存期。     

白细胞介素21及其研究进展

白细胞介素21(IL-21)是一种新发现的细胞因子,与IL-2有高度同源性,可调控T细胞、B细胞增殖,增强NK细胞的活性,并促进机体由先天性免疫向适应性免疫的转变.已有人、鼠、牛、猪等的IL-21分子被克隆.IL-21有希望在免疫缺陷病以及肿瘤的治疗中发挥重要的作用.文章就IL-21的生物学作用、信号传导机制、分子结构做一综述.     

日本大耳白兔红细胞调控淋巴细胞和NK细胞活性研究

应用红细胞促淋巴细胞转化和促NK细胞活性试验,对不同生理发育时期日本大耳白兔红细胞调控淋巴细胞和NK细胞活性进行研究。结果表明：青年组日本大耳白兔红细胞调控淋巴细胞能力明显高于幼年兔和老年兔（P〈0．01）;老年组红细胞调控NK活性能力明显高于青年组和幼年组（P〈0．01）,青年组高于幼年组（P〈0．01）。日本大耳白兔红细胞调控淋巴细胞能力在青年时期达到了高峰．随后逐渐降低：而红细胞调控NK活性随年龄增长而显著增强,这提示老年兔的免疫活性细胞较多地处于预激活状态,并对刺激显示良好的免疫应答能力。     

重组鸡白细胞介素18(rChIL-18)对MDV感染SPF鸡细胞免疫的影响

用马立克氏病病毒（MDV）感染5日龄SPF鸡后,取21日龄和35日龄鸡的淋巴细胞,运用3H-TdR掺入法检测T淋巴细胞对ConA和重组鸡白细胞介素18（rChIL-18）的反应,并用MTT法检测NK细胞和CTL对MD肿瘤细胞系CU147的杀伤活性及rChIL-18和IFN-γ对它们的作用。结果显示,SPF鸡感染MDV后淋转水平显著下降,NK细胞、CTL杀伤活性在感染后21日龄时升高,而在35日龄时NK细胞杀伤活性显著下降。rChIL-18对对照组和感染组SPF鸡的淋转水平和杀伤活性均有提高作用,同样IFN-γ也具有提高NK细胞和CTL杀伤活性的作用。     

Biological effects of leptospiral lipopolysaccharide to mouse B, T and NK cells

Leptospiral lipopolysaccharides (LPSs) extracted from Leptospira interrogans serovars copenhageni and hebdomadis were tested for the biological effect to mouse B, T and NK cells. Each leptospiral LPS was a potent mitogen for spleen B cells. Activation of the cells was also expressed by polyclonal B cell activation. In contrast, mitogenicity for T cells, induction of interleukin-2 (IL-2) secretion in T cells and increase of tumor-killing activity and chemiluminescence in NK cells were not observed after stimulation with leptospiral LPS. After intravenous injection of leptospiral LPS in mice, the spleen and lymphnodes were examined by histocytochemical technique. Increase of Ig-bearing lymphocytes was recognized while decrease of T cells was observed in the lymphoid organs. Mitogenic response to PHA, Con A and PWM decreased with relation to the T cell depletion. In conclusion, it is apparent that leptospiral LPS possess marked immunological potencies on B cells but not T and NK cells. The biological effects of leptospiral LPS were common ones as LPS but the level was considered to be different from classical LPS such as Escherichia coli LPS.     

Interleukin-2 corrects defective NK activity of patients with leukemia

NK cells of patients with leukemia display low cytotoxic potential. Since the NK cells have been suggested to play a role in natural resistance to leukemia, we considered it of importance to investigate the approaches leading to the correction of NK defect of leukemic patients. Our studies demonstrate that culture of effector cells with interleukin-2 (IL-2) resulted in restoration of cytotoxic defect. This was indicated by normalization of tumor-binding as well as lytic NK activity, by normal frequency of cytotoxic cells and their ability to recycle. The NK cell nature of cytotoxic cells was shown by abrogation or depletion of cytotoxicity by antibody directed against NK cell-associated, but not T cell-associated antigen. The generation of NK cell activity against fresh leukemic cells suggests that adoptive transfer of IL-2 activated NK cells may be a new approach to leukemic treatment.     

Characterization of NCR1+ cells residing in lymphoid tissues in the gut of lambs indicates that the majority are NK cells

Natural killer (NK) cells are important for immune protection of the gut mucosa. Previous studies have shown that under pathologic conditions NK cells, T cells and dendritic cells are found co-localised in secondary lymphoid organs where their interaction coordinates immune responses. However, in the gut-associated lymphoid tissues (GALTs), there are few detailed reports on the distribution of NK cells. Sheep harbour several types of organised lymphoid tissues in the gut that have different functions. The ileal Peyer’s patch (IPP) functions as a primary lymphoid tissue for B cell generation, while the jejunal Peyer’s patches (JPPs) and colon patches (CPs) are considered secondary lymphoid tissues. In the present study, we analysed tissues from healthy lambs by flow cytometry and in situ multicolour immunofluorescence, using recently described NCR1 antibodies to identify ovine NK cells. Most NCR1+ cells isolated from all tissues were negative for the pan T cell marker CD3, and thus comply with the general definition of NK cells. The majority of NCR1+ cells in blood as well as secondary lymphoid organs expressed CD16, but in the GALT around half of the NCR1+ cells were negative for CD16. A semi-quantitative morphometric study on tissue sections was used to compare the density of NK cells in four compartments of the IPPs, JPP and CPs. NCR1+ cells were found in all gut segments. Statistical analysis revealed significant differences between compartments of the primary lymphoid organ IPP and the secondary lymphoid organs of the JPPs and CP. NK cells co-localised and made close contact with T cells, dendritic cells and other NK cells, but did not show signs of proliferation. We conclude that NK cells are present in all investigated segments of the sheep gut, but that presence of other innate lymphoid cells expressing NCR1 cannot be excluded.     

Host environment as a modulating factor of swine natural killer cell activity

The large granular lymphocyte (LGL) population includes such heterologous effector cells as the natural killer (NK), lymphokine activated killer (LAK), antibody dependent cellular cytotoxic (ADCC) and non-MHC restricted T cells. These LGL subpopulations have all been associated with NK activity. In some species, enhanced NK activity is correlated with exposure to viral, bacterial and parasitic agents. Consequently, the host environment could serve as a modulatory factor of NK activity in laboratory animals. During our investigation of tumor regression in melanoma swine, we observed marked differences in the NK activity of peripheral blood lymphocytes collected from two separate groups of Sinclair melanoma miniature swine maintained under different conditions. Group A pigs were vaccinated and extensively treated for endo- and ectoparasites while group B swine were not. In addition, chronic exposure to infectious and parasitic diseases have been documented in the group B swine. Peripheral blood NK activity was assessed by standard in vitro 4-h chromium release assays. The NK activity of group B swine was markedly exaggerated when compared to group A swine. Thus, the significance of NK activity may be distorted as a result of the modulating effect of pathogen exposure.     

Bovine natural killer cells

Natural killer (NK) cells have received much attention due to their cytotoxic abilities, often with a focus on their implications for cancer and transplantation. But despite their name, NK cells are also potent producers of cytokines like interferon-gamma. Recent discoveries of their interplay with dendritic cells and T-cells have shown that NK cells participate significantly in the onset and shaping of adaptive cellular immune responses, and increasingly these cells have become associated with protection from viral, bacterial and parasitic infections. Furthermore, they are substantially present in the placenta, apparently participating in the establishment of normal pregnancy. Consequently, NK cells have entered arenas of particular relevance in veterinary immunology. Limited data still exist on these cells in domestic animal species, much due to the lack of specific markers. However, bovine NK cells can be identified as NKp46 (CD335) expressing, CD3(−) lymphocytes. Recent studies have indicated a role for NK cells in important infectious diseases of cattle, and identified important bovine NK receptor families, including multiple KIRs and a single Ly49. In this review we will briefly summarize the current understanding of general NK cell biology, and then present the knowledge obtained thus far in the bovine species.     

Infection with foot-and-mouth disease virus (FMDV) induces a natural killer (NK) cell response in cattle that is lacking following vaccination

Natural killer (NK) cells play a role in innate antiviral immunity by directly lysing virus-infected cells and producing antiviral cytokines such as interferon gamma (IFN-γ). We developed a system for characterizing the bovine NK response to foot-and-mouth disease virus (FMDV), which causes a disease of cloven-hoofed animals and remains a threat to livestock industries throughout the world. IL-2 stimulation of PBMC resulted in poor killing of human K562 cells, which are often used as NK target cells, while lysis of the bovine BL3.1 cell line was readily detected. Depletion of NKp46-expressing cells revealed that 80% of the killing induced by IL-2 could be attributed to NKp46⁺ cells. In order to characterize the response of NK cells against FMDV in vivo, we infected groups of cattle with three different strains of the virus (A24 Cruzeiro, O1 Manisa, O Hong Kong) and evaluated the cytolytic ability of NK cells through the course of infection. We consistently observed a transient increase in cytolysis, although there was variation in magnitude and kinetics. This increase in cytolysis remained when CD3⁺ cells were removed from the preparation of lymphocytes, indicating that cytolysis was independent of MHC-T cell receptor interaction or γδ T cell activation. In contrast, animals monitored following vaccination against FMDV did not exhibit any increase in NK killing. These data suggest that NK cells play a role in the host immune response of cattle against FMDV, and contrast with the suppression of NK activity previously observed in swine infected with FMDV.     

Effect of Bacillus subtilis spore administration on activation of macrophages and natural killer cells in mice

The effect of Bacillus subtilis (strain A102) spores on the activation of murine macrophages and natural killer cells (NK) was examined. The macrophage activity and NK activity were enhanced by oral administration of A102 spores, and slightly enhanced by oral administration of culture supernatant. There was no difference in the results of macrophage activity and NK activity using other live or dead spores. The NK activity and macrophage activity were increased with increments of concentration up to 0.1 g per mouse, and both activities were decreased at concentration of more than 0.15 g per mouse. The NK activity was increased 1 and 2 days after oral administration of A102 spores, and the activity level 2 days after administration was about 3-fold higher than the level prior to treatment. Macrophage activity was also increased from 1 to 3 days after oral administration of A102 spores, and the activity level 3 days after administration was about 3-fold higher than the level prior to treatment. The induction of interferons at 1 day after oral administration in mouse serum was 5-fold higher than that in controls. These findings indicate that oral administration of A102 gave rise to the induction of interferons, and it is likely that macrophages and NK cells were activated by interferons.     

Bovine neonate natural killer cells are fully functional and highly responsive to interleukin-15 and to NKp46 receptor stimulation

Natural killer (NK) cells are key components of the innate immune system with their killing and cytokine producing abilities. Bovine NK cells have been characterized as NKp46⁺/CD3⁻ lymphocytes, but little is known about these cells in neonatal calves. As the newborn calf, with an insufficiently developed acquired immunity, has to employ the innate immune system, we wanted to investigate whether neonate NK cells had the same characteristics as cells from older calves. Freshly isolated neonate and calf NK cells presented the same resting CD2⁺/CD25^low/CD8^−/low phenotype. Neonates less than 8 days old had one third of the circulating NKp46⁺ cells of older calves, but the NK cells proliferated more actively in vitro in the presence of interleukin (IL)-2 or IL-15. Moreover, neonate NK cells were more cytotoxic both in an NKp46 mediated redirected lysis assay and in direct killing of a bovine cell line MDBK when cultured in the presence of IL-15. Neonate and calf NK cells cultured in the presence of IL-2 and then stimulated with IL-12 produced similar dose-dependent interferon (IFN)-γ amounts, while IL-15 cultured NK cells did not give such a response whatever the age. However, neonatal NK cells cultured in IL-15 and stimulated by IL-12 concomitantly with cross-linking of NKp46, produced 4 to 5 times more IFN-γ than calf NK cells. These data suggest that although present in lower number at birth, neonate NK cells are fully functional and are more responsive to IL-15 and activation through the NKp46 receptor than NK cells from older calves.     

The antiproliferative effect of bovine lactoferrin on canine mammary gland tumor cells

Lactoferrin has several biological activities, including antitumor activities in some human and animal tumor cells. Clinical trials have been carried out in human medicine based on these effects. However, the antitumor effects of lactoferrin in veterinary medicine remain unknown. In this in vitro study, we demonstrated that co-incubation of canine mammary gland tumor cells (CIPp and CHMp) and bovine lactoferrin induced growth arrest of tumor cells. This growth arrest was associated with induction of G1 arrest. Furthermore, this effect was stronger in tumor cells than in normal cells. These findings demonstrate that bovine lactoferrin has anti-tumor activity in canine mammary tumors and has the potential for use in tumor-bearing dogs.     

Active-oxygen involvement in canine NK-mediated cytotoxicity

We examined the relationship between natural killer (NK) cell-mediated cytotoxicity, the produced active-oxygen and cytotoxic factor (CF) release in co-culturing canine NK cells with tumor cells (CL-1 target cells). In co-culturing, the adding of n-propyl gallate (active-oxide scavenger) removed the produced active-oxygen, which inhibited NK cell-mediated cytotoxicity and the CF release. Moreover, adding of this agent inhibited the tyrosine phosphorylation of NK intracellular protein which observed in co-culturing. Therefore, the active-oxygen produced from canine NK cells are thought to relate the signal transduction in NK-mediated cytotoxicity.     

Bovine NK cells acquire cytotoxic activity and produce IFN-gamma after stimulation by Mycobacterium bovis BCG- or Babesia bovis-exposed splenic dendritic cells

Early interactions of innate immune cell populations, such as dendritic cells (DC) and natural killer (NK) cells, can affect the ability of the acquired immune response to control infection of intracellular microorganisms. In this study, we investigated the activation of bovine NK cells by CD13(+) splenic DC stimulated with either Mycobacterium bovis BCG or Babesia bovis merozoites. Splenic DC were used either immediately after selection (cytokine(-)) or after exposure to GM-CSF, IL-4 and Flt3L for 72 h (cytokine(+)). Phenotypic analyses showed up-regulation of MHCII, CD80 and CD86 on cytokine(+) DC when compared to cytokine(-) DC. Purified NK cells (CD335(+)CD3(-)CD2(+/-)CD8alpha(+/-)) were co-cultured with microbial-exposed cytokine(-) DC or cytokine(+) DC in either transwell or cell-to-cell format and NK cell IFN-gamma production and cytotoxicity were assessed. NK cell IFN-gamma production was dependent on cell-to-cell contact. Microbial-stimulated cytokine(+) DC induced significantly more IFN-gamma production from NK cells than cytokine(-) cells. In contrast, cytotoxicity and perforin up-regulation were more pronounced in NK cells cultured with cytokine(-) DC than cytokine(+) DC. Therefore, activation of bovine NK cells by microbial-stimulated CD13(+) splenic DC is influenced by the maturation state of the DC suggesting different roles for the splenic DC during disease-induced maturation.     

Natural immunity against ovarian tumors

We have analysed natural killer (NK) cytotoxic activity in peripheral blood and ascitic fluids of patients with advanced stage of ovarian epithelial carcinoma. All patients displayed low NK activity in peripheral blood and virtually no cytotoxicity in ascitic fluids. NK activity in ascitic fluids could be substantially augmented after regional administration of virus-modified tumor cell extracts (VMTE), and that in peripheral blood after culture of effector cells with interleukin-2 (IL-2) in vitro. Activated NK cells displayed cytotoxic activity against NK-sensitive and NK-resistant tumor cell lines as well as against fresh ovarian tumors. Parallelism was found between regional NK augmentation and regression of malignant ascites. The latter observation suggests possible NK cell role in defense against ovarian tumors.     

Evaluation of the hepatic NK cell response during the early phase of Fasciola hepatica infection in rats

The in situ distribution of NK cells in rat liver during the first 28 days of an experimental infection with F hepatica was investigated. NK cells were distributed homogeneously throughout the hepatic parenchyma in uninfected animals. The total number of hepatic mononuclear cells increased significantly following infection, but the proportion of NK cells did not change. After infection, these cells were found around the portal space, around the centrolobular vein, in the periportal fibrosis and in the band of collagen. However, no NK cells could be detected in or around the granuloma during infection. The frequency of both I L-2- and IFNgamma-producing NK cells was higher on day 7 postinfection (pi) but only the percentage of IFNgamma -CD161+ subsets remained elevated thereafter, whereas the percentage of both IL-2+CD161+ and IL-4+CD161+ subsets returned to the baseline. The number of CD161+IL10+ cells did not change significantly. These results suggest that NK cells could be another source for the early production of IFNgamma but provide no evidence that these cells are involved in early events associated with granuloma formation.     

Canine CD8 T cells showing NK cytotoxic activity express mRNAs for NK cell-associated surface molecules

Natural killer (NK) cells have been considered to be a group of lymphocytes lacking clonally distributed receptors for antigens typical of T cells and B cells. In some mammalian species, including humans, a subpopulation of CD8⁺ peripheral blood lymphocytes (PBLs) exhibits NK activity. This NK subpopulation has not been well characterized in mammals and its characterization is particularly poor in the dog. In this study, we demonstrated that a subset of canine CD8⁺ cells derived from PBLs and lymphokine (IL-2)-activated killers (LAKs) of PBLs that was CD3⁺, CD4^?, CD21^?, CD5^lo, α/βTCR⁺, and γ/δTCR^? contained substantially higher levels of mRNAs for NK cell-related receptors (NKp30, NKp44, NKG2D, 2B4, and CD16 for PBL, and NKG2D and CD56 for LAK) than the corresponding CD8^? cells. This subset of CD8⁺ lymphocytes derived from LAKs also displayed significantly higher NK cytotoxic activity than the corresponding CD8^? cells. In contrast, CD8⁺ cells derived from nonstimulated PBLs showed very low levels of NK cytotoxic activity. Our results indicate that, in IL-2-stimulated PBLs, canine CD8⁺ cells are an important subset associated with NK cytotoxic activity.