Involvement of granzyme B and perforin gene expression in the lytic potential of human natural killer cells

Natural killer (NK) cells (CD3⁻) or large granular lymphocytes (LGL) spontaneously kill K562 targets but are unable to kill Daudi cells in the absence of IL-2 stimulation. IL-4 is reported to prevent or inhibit the IL-2-driven lymphokine-activated killer (LAK) generation in NK cells. Therefore, we wished to determine whether the antagonistic effect of IL-4 on IL-2-induced LAK activity might regulate the expression of genes encoding proteins involved in lysis, such as perforin, the pore-forming protein, or which are associated with lysis, such as granzymes A and B. By using in situ hybridization, we showed that, in addition to inducing LAK activity, IL-2 stimulation increased the amount of perforin and granzyme B mRNA at the single-cell level in 40 to 100% of the total CD3⁻ LGL cell population. In addition, our results indicated that the stimulatory effect of IL-2 can be downregulated by IL-4 for both LAK activity and granzyme B and perforin gene expression. Here again, a decrease in the amount of specific mRNA per cell was noted. These findings suggest that modulation of the lytic machinery via lymphokines might be associated with regulation of the lytic potential of NK cells.     

Differential expression of granzyme A and granzyme B proteases and their secretion by fresh rat natural killer cells (NK) and lymphokine-activated killer cells with NK phenotype (LAK-NK).

Granzymes A-G are a family of serine proteases localized in the cytoplasmic granules of cytotoxic T lymphocytes (Masson, D. et al. Cell 1987. 49: 679) and granzyme A is secreted by T lymphocytes in response to antigenic stimulation. Granzyme A is also expressed by natural killer (NK) and lymphokine-activated killer (LAK) cells. Here we show that fresh rat NK cells constitutively express granzyme B and that granzyme A and granzyme B are differentially regulated in unstimulated NK cells vs. LAK cells with NK phenotype (LAK-NK cells). We also show that both granzymes A and B are secreted in a calcium-dependent manner, by NK and LAK-NK cells in response to stimuli which trigger NK cell functions.     

Decreased perforin and granzyme B expression in senescent HIV-1-specific cytotoxic T lymphocytes

Cytotoxic T lymphocyte (CTL) senescence may be an important mechanism of immune failure in HIV-1 infection. We find that senescence of HIV-1-specific CTL clones causes loss of killing activity, preventable by transduction with telomerase. Furthermore, senescence is associated with reduced expression of the effector molecules granzyme and perforin, suggesting CTL "exhaustion" can result in hypofunction. These results agree with other studies showing that HIV-1-specific CTL exhibit abnormal phenotypes in vivo, and suggest the possibility that chronic turnover is an important mechanism of antiviral failure in HIV-1 infection.     

人外周血NK细胞在细胞因子刺激下扩增后穿孔素、颗粒酶B基因表达的变化

目的 探讨经过纯化及在多种细胞因子的作用下扩增后的NK细胞,其穿孔素(per-furin)、颗粒酶B(granzyme B)表达水平的改变与细胞杀伤率的关系.方法 应用竞争性定量RT-PCR方法 检测了8例供者纯化、扩增后NK细胞的穿孔素、颗粒酶B基因表达水平,同时检测其对K562细胞的杀伤率.结果 经纯化、扩增后的NK细胞,在多种细胞因子作用下穿孔素和颗粒酶B的基因表达水平明显提高,且.IL-2+IL-15组、IL-2+IL-12+IL-15组基因的表达量均显著高于其他组,NK细胞对K562的杀伤率结果 与基因表达水平一致.结论 应用细胞因子后可使NK细胞的穿孔素、颗粒酶B基因表达水平明显提高,同时提高了NK细胞的杀伤功能.     

Delivery and therapeutic potential of human granzyme B

Summary: Granzyme B (GzmB) is used by cytotoxic lymphocytes as a molecular weapon for the defense against virus-infected and malignantly transformed host cells. It belongs to a family of small serine proteases that are stored in secretory vesicles of killer cells. After secretion of these cytolytic granules during killer cell attack, GzmB is translocated into the cytosol of target cells with the help of the pore-forming protein perforin. GzmB has adopted similar protease specificity as caspase-8, and once delivered, it activates major executioner apoptosis pathways. Since GzmB is very effective in killing human tumor cell lines that are otherwise resistant against many cytotoxic drugs and since GzmB of human origin can be recombinantly expressed, its use as part of a ‘magic bullet’ in tumor therapy is a very tempting idea. In this review, we emphasize the peculiar characteristics of GzmB that make it suited for use as an effector domain in potential immunoconjugates. We discuss what is known about its uptake into target cells and the trials performed with GzmB-armed immunoconjugates, and we assess the prospects of its potential therapeutic value.     

年龄对人细胞毒性T淋巴细胞和自然杀伤细胞中穿孔素表达的影响

目的　通过研究健康儿童、成人和老年人外周血淋巴细胞 (PBL)中细胞毒效应分子穿孔素 (PFP)的表达水平的变化 ,了解细胞毒性T细胞 (CTL )的杀伤活性随年龄增长而下降的分子机制。方法　利用抗人PFP、抗人CD3和抗人CD5 6单克隆抗体 (mAb) ,采用单标记免疫组化染色法 ,检测外周血单个核细胞 (PBMC)中PFP的表达。采用双标记免疫组化染色法 ,检测上述细胞表面标志CD3或CD5 6的表达和细胞内PFP的表达。结果　儿童组、成人组及老年组PBL中表达PFP阳性细胞的百分率 (% ) ,分别为 (2 6 .4± 2 .7) % ,(2 1.6± 3.2 ) %和 (13.4± 2 .0 ) % ,老年组明显下降 ,与其他两组相比较P <0 .0 0 1。 3个年龄组CD3+ T细胞表达PFP的阳性率 (% ) ,分别为 (30 .1± 4 .8) %、(2 1.4± 7.3) %和 (18.8± 4 .2 ) % ,随年龄的增长而显著下降。3个年龄组CD5 6 +NK细胞PFP的表达差别不显著。结论　健康人PBL中PFP的表达随年龄增长而递减 ,CD3+ T细胞亚群PFP表达水平的下降尤其显著。这可能是老年人CTL杀伤活性下降的重要原因之一     

Differential expression of perforin and granzyme B in the liver of patients with chronic hepatitis C

T lymphocytes have been reported to be the predominant inflammatory cells in the liver of patients with chronic hepatitis C. On activation, CD8+ T lymphocytes can exert their cytolytic activity by releasing their granule components, notably perforin and granzyme B. The aim of the present study was to assess whether the granule cytolytic pathway was used by liver-infiltrating CD8+ T lymphocytes. Immunostaining for perforin and granzyme B was performed in 25 patients with chronic hepatitis C, according to the disease activity and their virologic status. Cells stained for perforin and for granzyme B represented 0.15% and 0.10% of the total liver-infiltrating CD8+ T lymphocytes, respectively. Perforin was expressed mainly by activated CD8+ T lymphocytes located in liver lobules. In contrast, granzyme B was expressed mainly by activated CD8+ T lymphocytes located in interface hepatitis and portal tracts. The results were similar in the different groups of patients, whatever the disease activity. In conclusion, this is the first study showing a differential expression of granule components of CD8+ T lymphocytes in the same tissue in vivo. Perforin and granzyme B may be differently expressed by liver-infiltrating CD8+ T lymphocytes, according to their localization in the different specific compartments of the liver, in patients with chronic hepatitis C.     

穿孔素与颗粒酶B在鼻NK/T细胞淋巴瘤诊断中的意义

目的　检测鼻NK/T细胞淋巴瘤中的穿孔素、颗粒酶B表达分布情况 ,为临床治疗和预后判断提供依据。方法　运用免疫组化S P法检测 11例鼻NK/T细胞淋巴瘤中穿孔素、颗粒酶B的表达和分布情况 ;同时与组织芯片中其他类型淋巴瘤进行比较研究 ,10例淋巴组织反应性增生作为对照。结果　 11例鼻NK/T细胞淋巴瘤中均见穿孔素和颗粒酶B过度表达。以 6 0例淋巴瘤制成组织芯片的 4 2例B细胞淋巴瘤中见 11例少许穿孔素和 14例少许颗粒酶B阳性表达细胞散在分布于瘤细胞间 ,10例外周T细胞淋巴瘤中 8例少许表达穿孔素和 9例少许表达颗粒酶B。 2例间变性大细胞淋巴瘤见少许穿孔素和颗粒酶B阳性表达细胞。 2例霍奇金淋巴瘤阴性。NK/T细胞淋巴瘤的穿孔素和颗粒酶B表达程度与T、B淋巴瘤比较 ,差异有显著性 (P <0 0 1)。结论　穿孔素和颗粒酶B是鉴定活化的细胞毒性细胞的免疫标记物 ,可作为NK/T细胞淋巴瘤的诊断性标记物 ;其在外周T细胞淋巴瘤和B细胞淋巴瘤中的表达 ,反映了机体存在抗肿瘤的免疫反应机制。     

A central role for phosphoinositide hydrolysis in activating the lytic mechanism of human natural killer cells

Using neomycin, which inhibits phosphoinositide breakdown, cytotoxicity mediated by natural killer (NK) cells was suppressed in a dose-dependent manner, with complete inhibition at 16 mM. Generation of inositol phosphates in effector cells after target cell binding was inhibited in the presence of neomycin. The formation of effector to target cell conjugates was not affected. Neomycin-induced inhibition of NK killing was abolished when TPA was added to the cytotoxic assays. This reconstitution was dependent upon extracellular Ca2+. When the intracellular free Ca2+ level in effector cells was reduced from 73 +/- 11 nM to 43 +/- 3 nM (n = 4) using the Ca2+ indicator dye, Quin 2, NK killing was markedly reduced. Inhibiting the enzyme diacylglycerol (DG) kinase in effector cells with 10 microM R59022 (DG kinase inhibitor) potentiates NK killing, suggesting an increase in protein kinase C (PKC) activity due to accumulation of DG. The PKC inhibitor, H-7, suppressed NK killing in a concentration-dependent manner. These results demonstrate that phosphoinositide metabolism is an early event and its derived second messengers play a central role in activating the lytic mechanism of NK cells.     

Pentoxifylline inhibits granzyme B and perforin expression following T-lymphocyte activation by anti-CD3 antibody

Pentoxifylline (PTX), a methylxanthine derivative, is known to inhibit the production of the Th 1 cytokines interleukin-2, tumour necrosis factor-α and interferon-y. Because these cytokines play an important role in promoting the development of cell-mediated immunity, we hypothesized that PTX would also interfere with the generation of cytotoxic effector cells in response to an immunological stimulus. In this study we used a mouse model system to investigate the effect of PTX on the induction of non-specific killer lymphocytes by anti-CD3 monoclonal antibody. Anti-CD3-induced T-cell proliferation and the generation of anti-CD3-activated killer (AK) cells was inhibited in a dose-dependent fashion by PTX (25–100 gg/ml). The inhibitory effect of PTX could not be attributed to a defect in the recognition/adhesion phase of cytolysis because AK cells generated in the presence of PTX conjugated normally with P815 tumour target cells. However, AK cell expression of the cytoplasmic granule-associated cytolytic effector molecules granzyme B and perforin was markedly reduced when AK cells were induced in the presence of PTX. In contrast, PTX had no effect on AK cell expression of Fas ligand, a cell-surface cytolytic effector molecule which is involved in granule-independent cytotoxicity. PTX thus has a profound inhibitory effect in vitro on the induction of granule-dependent cytolytic effector mechanisms in a mouse model system.     

Involvement of natural killer cells in endogenous biological retranslation

Analysis of functional properties of natural killer cells displayed in reactions of natural cytotoxicity and noncytotoxic regulatory intercellular interactions suggests that this population of lymphocytes is involved in endogenous biological retranslation. In the immune system, retranslation is the production of regulatory immunoactive cytokines by a cell, cellular complex, or functional complex. The substances produced are identical to those affecting these structures. Various forms of endogenous biological retranslation in humans and higher animals, as well as its phylogenetic and ontogenetic manifestations (on the basis of noncytotoxic regulatory interactions of natural killer cells with cells of lymphoid or nonlymphoid nature) during evolution of the complex of immunobiological surveillance are considered. The axiomatic basis of retranslation realized through the system of natural cytotoxicity was established. Prospects for application of the methodology of endogenous biological retranslation to experimental and clinical studies of functioning of natural killer cells are considered. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 126, No. 8, pp. 124–135, August, 1998     

PFP、GrB真核共表达载体的构建及表达分析

目的：体外扩增出PFP、GrB基因的全片段并构建共表达重组体pVAX1-PIG（即pVAX1-PFP-IRES-GrB）,分析其在人喉癌细胞Hep-2中的表达情况。方法：利用RT-PCR的方法从人的喉癌组织浸润淋巴细胞中扩增全长PFP、GrB的cDNA片段并构建共表达重组体pVAX1-PIG。利用脂质体LipofectamineTM2000将重组真核表达载体pVAX1-PIG转染入人的Hep-2细胞株中,采用RT-PCR、间接免疫荧光法分析其在人Hep-2中的表达情况。结果：经双酶切、测序法证实已成功扩增PFP、GrB基因的全长cDNA,并构建共表达重组体pVAX1-PIG,在荧光显微镜下可见已转染pVAX1-PIG的人Hep-2细胞的胞浆发出苹果绿色荧光。结论：成功扩增PFP、GrB全片段、构建共表达重组体pVAX1-PIG,并在人Hep-2细胞中检测到PFP、GrB蛋白的表达,穿孔素（PFP）/颗粒酶B共同表达能够介导细胞的凋亡,为其在喉癌治疗中的应用研究奠定了基础。     

2B4-mediated activation of human natural killer cells

2B4 is a member of the CD2 subset of the immunoglobulin superfamily of cell surface receptors. Other members of this family include CD2, CD48, CD58, CD84, signaling lymphocytic activation molecule and Ly-9. Some of these molecules are activating structures expressed by natural killer cells and T cells. We have recently cloned and characterised the human homologue of 2B4 and found that the cytoplasmic domain of 2B4 can interact with SAP, a signaling adaptor protein that is mutated in the immunodeficiency X-linked lymphoproliferative disease (XLP). Additionally, the natural ligand of 2B4 has been identified as CD48. These findings have facilitated the investigation of the functional role of this receptor-ligand pair, and associated signal transduction pathways, on immune cells. In this study, it was found that the interaction between 2B4 on effector cells and CD48 on target cells induced NK-cell activation, as evidenced by increased cytotoxicity and secretion of IFN-gamma. The responses induced by ligation of 2B4 could be reduced by the co-ligation of inhibitory receptors expressed by NK cells, demonstrating that activation signals delivered via 2B4 can be regulated by the action of certain inhibitory receptors. Because the signalling pathway of 2B4 involves SAP, it is possible that 2B4-mediated NK-cell activation may be compromised in patients with XLP due to mutations in SAP. This may contribute to the phenotype and progression of this disease.     

Functional expression of CD43 on human natural killer cells

CD43 is the major leukocyte sialoglyco-protein that plays important functional roles in neutrophils and lymphocytes. However, the expression of CD43 on human natural killer (NK) cells and its participation in the regulation of NK activity has not been studied. We have therefore investigated the expression of CD43 isoforms on human NK cell subpopulations as well as the role of this molecule in NK cell activation and cytotoxicity. We found that CD56bright and CD56dim NK cells express different sialylated forms of CD43, observing that activation of the CD56bright NK cells induces the change of tetrasaccharide O-glycans to hexasaccharide O-glycans on CD43. Cross-linking of the molecule with mAbs results in a metalloprotease-dependent loss of CD43 from the NK cell surface, whereas soluble anti-CD43 mAbs induce a vigorous NK cell proliferation. This property is distinct from T cells, which proliferate after CD43 cross-linking only in the presence of monocytes. Occupancy of the CD43 receptor on NK cells transduces specific signals, leading to enhanced killing activity and tyrosine phosphorylation and de-phosphorylation of several substrates. We therefore propose that CD43 significantly contributes to the regulation of the NK cell function by participating in the control of effector/target interactions and, if pertinent, by transducing activation signals.     

The relationship of natural killer cell counts, perforin mRNA and CD2 expression to post-exercise natural killer cell activity in humans

The aim of this study was to further investigate the mechanism of suppression of natural killer (NK) cell cytotoxic activity in peripheral blood following strenuous exercise. Blood was collected for analysis of NK cell concentration, cytotoxic activity, CD2 surface expression and perforin gene expression from runners (RUN, n=6) and resting controls (CONTROL, n=4) pre-exercise, 0, 1.5, 5, and 24 h following a 60-min treadmill run at 80% of VO2 peak. Natural killer cytotoxic activity, measured using a whole blood chromium release assay, fluctuated minimally in the CONTROL group and increased by 63% and decreased by 43% 0 and 1.5 h post-exercise, respectively, in the RUN group (group x time, P < 0.001). Lytic index (cytotoxic activity per cell) did not change. Perforin mRNA, measured using quantitative real-time polymerase chain reaction (QRT-PCR) decreased from pre- to post-exercise and remained decreased through 24 h. The decrease from pre- to 0 h post-exercise was seen predominately in the RUN group and was inversely correlated (r=- 0.95) to pre-exercise perforin mRNA. The NK cell surface expression of CD2 (lymphocyte function-associated antigen-2) was determined using fluorescent antibodies and flow cytometry. There was no change in the proportion of NK cells expressing CD2 or CD2 density. We conclude that (1) numerical redistribution accounted for most of the change in NK cytotoxic activity following a strenuous run, (2) decrease in perforin gene expression during the run was inversely related to pre-exercise levels but did not parallel changes in cytotoxic activity, and (3) CD2 surface expression was not affected by exercise.     

Involvement of fibronectin and interacting serum components in the regulation of activity of human natural killer cells

The cytotoxic activity of natural killer cells and the intensity of conjugate formation are studiedin vitro in the natural cytotoxicity reaction against³H-uridine-labeled human erythromyeloleukotic cells K-562 in the presence of fibronectin, γ-globulin, and fibronectin/γ-globulin combination. It is demonstrated that fibronectin does not change natural cytotoxicity, γ-globulin increases the activity of human natural killer cells, and the fibronectin — γ-globulin combination increases both the intensity of conjugate formation and the cytotoxic activity of natural killer cells. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 118, N ^o 7, pp. 54–59, July, 1994