A phenotypic and functional characterization of NK cells in adenoids

Adenoids are part of the MALT. In the present study, we analyzed cell surface markers and cytolytic activity of adenoidal NK (A-NK) cells and compared them with NK cells derived from blood of the same donors (B-NK). NK cells comprised 0.67% (0.4-1.2%) of the total lymphoid population isolated from adenoids. The majority (median=92%) of the A-NK cells was CD56(bright)CD16(-). A-NK cells were characterized by the increased expression of activation-induced receptors. NKp44 was detected on >60%, CD25 on >40%, and HLA-DR on >50% of freshly isolated A-NK cells. Functional assays indicated that the cytotoxic machinery of A-NK is intact, and sensitive target cells are killed via natural cytotoxicity receptors, such as NKG2D. Carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1; CD66) expression was up-regulated in 23% (median) of the A-NK cells by IL-2 activation but unchanged in B-NK cells. CEACAM1 inhibited the A-NK killing of target cells. CXCR4 was expressed on more than 40% A-NK cells prior to activation. Its ligand, CXCL12, was found in endothelial cells of the capillaries within the adenoid and in cells of the epithelial lining. In addition, A-NK cells migrated in vitro toward a gradient of CXCL12 in a dose-responsive manner, suggesting a role for this chemokine in A-NK cell recruitment and trafficking. We conclude that the A-NK cells are unique in that they display an activated-like phenotype and are different from their CD16(-) B-NK cell counterparts. This phenotype presumably reflects the chronic interaction of A-NK cells with antigens penetrating the body through the nasal route.     

rhIL-15和rhIL-2诱生的LAK细胞特性比较

目的：研究ｒｈＩＬ－１５激活的ＬＡＫ细胞的增殖、细胞毒作用及相关表型的变化。方法：通过用不同浓度的ｒｈＩＬ－１５和ｒｈＩＬ－２分别诱生 ＬＡＫ细胞，研究二者诱生的 ＬＡＫ细胞的增殖状况；当 ｒｈＩＬ－２和 ｒｈＩＬ－１５为 １５００ Ｕ／ｍｌ时，ＭＴＴ法检测 ＬＡＫ细胞的细胞毒作用，利用流式细胞仪检测细胞表面ＣＤ分子表达情况。结果：ＬＡＫ细胞的增殖对ｒｈＩＬ－２和ｒｈＩＬ－１５均具有时间和剂量依赖性，且无明显差异；终浓度为 １５００ Ｕ／ｍｌ时，ｒｈＩＬ－１５诱生的 ＬＡＫ细胞杀伤 Ｋ５６２细胞的能力强于 ｒｈＩＬ－２诱生的 ＬＡＫ细胞，而对Ｒａｊｉ细胞的杀伤活性，ｒｈＩＬ－２诱生的ＬＡＫ细胞却明显高于 ｒｈＩＬ－１５诱生的 ＩＡＫ细胞，且二者杀伤活性均具有时间依赖性；同时ｒｈＩＬ－１５诱生的ＬＡＫ细胞的ＣＤ３－ＣＤ５６＋细胞、Ｃｄ９４＋细胞百分率均高于ｒｈＩＬ－２诱生的ＬＡＫ细胞。结论：ｒｈＩＬ－１５在体内对ＮＫ细胞功能可能具有较强的调节作用。     

Role of interleukin-18 in human natural killer cell is associated with interleukin-2

Human natural killer (NK) cells constitute an important cellular component of innate immunity, capable of killing infected and transformed cells. The proliferation and activation of NK cells are regulated by various cytokines. Interleukin-18 (IL-18) promotes NK cell activation; however, whether the effects of IL-18 on NK cell are associated with other cytokines is still unknown. In this study, we observed that IL-18 induced NK cell apoptosis and inhibited NK cell expansion in the presence of low concentrations of interleukin-2 (IL-2), while high concentrations of IL-2 overcame these effects of IL-18, and high concentrations of IL-2 promoted the stimulatory activity of IL-18 on NK cells. At a low concentration of IL-2, IL-18 induced NK cell apoptosis in part through activation of the FasL/Fas- and TNFα/TNFR-mediated death receptor signaling by enhancing FasL expression and inhibiting c-FLIP(long) expression. However, high concentrations of IL-2 strongly blocked IL-18-induced NK cell apoptosis through alleviating IL-18-induced FasL expression and activation of Fas-mediated death signaling and increasing anti-apoptosis molecule (Bcl-X(L)). These results reveal that the effects of IL-18 on human NK cell are associated with IL-2 concentration and suggest the importance of IL-2 level in cytokine immunotherapy.     

Modulation of the surface expression of CD158 killer cell Ig-like receptor by interleukin-2 and transforming growth factor-beta

Killer cell Ig-like receptor (KIR) binds to HLA class I molecules on the surface of target cells, and it confers inhibitory signals to NK cells. Although NK cytotoxicity can be affected by the change of the surface expression of KIR on NK cells, the effect of cytokines on the regulation of KIR expression has not been thoroughly investigated. Here in our study, we investigated the effect of several cytokines, including IL-2, TGF-beta, IFN-gamma, IL-12 and IL-18, on the surface expression of CD158 KIR, which binds to HLA-C, by the use of FACS analysis. In the isolated NK cells, IL-2 obviously increased the surface expression of CD158 KIR after 72 hr in vitro culture, and this was evidenced by the increased percentage of CD158(+) NK cells and the increased mean fluorescence intensity of CD158 in CD158(+) NK cells. In contrast, TGF-beta decreased the surface expression of CD158 KIR after 72 hr culture. However, IFN-gamma, IL-12 and IL-18 did not change the expression of CD158 KIR. The modulated expression of KIR by IL-2 and TGF-beta can be associated with the changed NK-cytotoxic target-discriminating ability of NK cells upon their exposure to IL-2 and TGF-beta.     

Role of interleukin-18 in human natural killer cell is associated with interleukin-2

Human natural killer (NK) cells constitute an important cellular component of innate immunity, capable of killing infected and transformed cells. The proliferation and activation of NK cells are regulated by various cytokines. Interleukin-18 (IL-18) promotes NK cell activation; however, whether the effects of IL-18 on NK cell are associated with other cytokines is still unknown. In this study, we observed that IL-18 induced NK cell apoptosis and inhibited NK cell expansion in the presence of low concentrations of interleukin-2 (IL-2), while high concentrations of IL-2 overcame these effects of IL-18, and high concentrations of IL-2 promoted the stimulatory activity of IL-18 on NK cells. At a low concentration of IL-2, IL-18 induced NK cell apoptosis in part through activation of the FasL/Fas- and TNFα/TNFR-mediated death receptor signaling by enhancing FasL expression and inhibiting c-FLIP_long expression. However, high concentrations of IL-2 strongly blocked IL-18-induced NK cell apoptosis through alleviating IL-18-induced FasL expression and activation of Fas-mediated death signaling and increasing anti-apoptosis molecule (Bcl-X_L). These results reveal that the effects of IL-18 on human NK cell are associated with IL-2 concentration and suggest the importance of IL-2 level in cytokine immunotherapy.     

A novel epitope of N-CAM defines precursors of human adherent NK cells

Activated, adherent natural killer (A-NK) cells represent a distinct subpopulation of interleukin (IL)-2-stimulated NK cells, which are selectively endowed with the increased expression of integrins and ability to adhere to solid surfaces, migrate into, infiltrate, and destroy cancerous tissues. The present study defines the phenotype and functions of precursors of A-NK (pre-A-NK) cells in humans. Peripheral blood pre-A-NK cells, in contrast to the rest of NK cells, express a novel epitope of CD56 neuronal cell adhesion molecule, termed ANK-1, and increased cell-surface levels of integrins. Pre-A-NK cells also express low levels of CD56 and CD161, and some express CD162 receptor, do not express CD25 or activation markers, and are effective mediators of NK cytotoxicity. Thus, pre-A-NK cells are generally similar to CD56(dim) NK cells. However, pre-A-NK cells differ from the main NK cell subpopulation by having a lower expression level of CD16 and a lower ability to mediate redirected antibody-dependent, cell-mediated cytotoxicity. More importantly, pre-A-NK cells are preferentially endowed with the ability to rapidly respond to IL-2 by integrin-mediated adherence to endothelial cells, extracellular matrix, and plastic. This early, specific response of pre-A-NK cells to IL-2 is followed by their activation, vigorous proliferation, and differentiation into phenotypically and functionally similar A-NK cells. Pre-A-NK cells represent only approximately 26% of peripheral blood NK cells but encompass the majority of NK cells in normal and cancerous, solid tissues. We conclude that pre-A-NK cells represent a distinct subset of resting, mature NK cells with the characteristics indicative of their ability to migrate and reside in solid tissues.     

CD154 stimulation of interleukin-12 synthesis in human endothelial cells

The interaction of proinflammatory type 1 T helper (Th1) cells expressing the CD40 ligand (CD154) with endothelial cells expressing the corresponding receptor (CD40) may play an important role in chronic inflammation including arteriosclerosis. Here we demonstrate that activation of CD40 in human cultured endothelial cells (e.g. by interaction with freshly isolated human T cells) not only up-regulates expression of various adhesion molecules, chemokines and cytokines, but within 12-24 h also causes the release of bioactive interleukin-12 (IL-12 p70) through induction of IL-12 p40 synthesis. IL-12 p35, on the other hand, appears to be constitutively expressed in these cells. Despite enhancing expression of the other gene products, cytokines such as interferon-gamma (IFN-gamma) or tumor necrosis factor-alpha, alone or in combination, failed to induce IL-12 p40 expression, whereas IFN-gamma markedly augmented CD154-induced IL-12 p40 and p70 release. Of note was that the magnitude of CD154-induced IL-12 synthesis in the cultured endothelial cells was comparable to that evoked in freshly isolated human monocytes. This CD40-mediated induction of endothelial IL-12 synthesis may thus lead to an enhanced activation of the adherent CD154-expressing Th1 cells, thereby fuelling the proinflammatory response.     

Interleukin-16 stimulates the expression and production of pro-inflammatory cytokines by human monocytes

Interleukin-16 (IL-16) acts as a chemoattractant for CD4+ cells, as a modulator of T-cell activation, and plays a key role in asthma. This report describes the cytokine-inducing effects of IL-16 on total peripheral blood mononuclear cells (PBMC) and PBMC subpopulations. While CD4+ T lymphocytes did not secrete cytokines in response to rhIL-16, CD14+ CD4+ monocytes and maturing macrophages secrete IL-1beta, IL-6, IL-15 and tumour necrosis factor-alpha (TNF-alpha) upon rhIL-16 stimulation. The mRNA species for these four cytokines were detected as early as 4 hr post-stimulation, with protein being secreted by 24 hr. Secretion of IL-1beta and IL-6 by total PBMC was dose dependent, with maximal secretion being observed using 50 ng/ml rhIL-16. However, for IL-15 or TNF-alpha maximal secretion by total PBMC occurred with all concentrations between 5 ng/ml to 500 ng/ml rhIL-16. Purified monocytes/macrophages secreted maximal concentrations of all four cytokines in the presence of 500 ng/ml rhIL-16, except for monocytes where maximal secretion of IL-15 was, interestingly, observed with only 50 ng/ml rhIL-16. The use of higher concentrations of rhIL-16 (1000 ng/ml) inhibited secretion of all four cytokines. While these IL-16-induced cytokines are likely to be involved in the immune system's response to antigen, the data suggest that IL-16 may play a key role in initiating and/or sustaining an inflammatory response.     

Interleukin-15 Promotes the Commitment of Cord Blood CD34^＋ Stem Cells into NK Cells

Naturalkiller(NK)cellsweresubgroupoflymphocytes thatplayedanessentialroleinthecellularbasedimmune defenseagainstvirus infectedandtransfectedcells.His torically,inteleukin 2(IL 2)wasregardedasthenatural activatorforNKcells.Recently,itwasdiscoveredthat IL …     

Enhanced survival and potent expansion of the natural killer cell population of HIV-infected individuals by exogenous interleukin-15.

The CD56+CD16+ natural killer (NK) cell population plays a crucial role in eliminating virus-infected cells and is diminished in HIV-infected individuals. This study examined the effects of exogenous interleukin (IL)-15 on proliferation and survival of CD56+ and CD16+ cells of HIV-infected individuals. When used at equivalent concentrations in vitro, IL-15 was more potent than IL-2 as a growth factor for CD56+ cells, as well as for CD16+ cells and also CD4+ and CD8+ T cells. Analysis of cell survival in etoposide-treated cultures indicated that IL-15 was also more potent than IL-2 as a survival factor for CD56+ cells by virtue of its greater ability to up-regulate bcl-2 expression. Although IL-15-induced proliferation of CD56+ cells was accompanied by increased apoptosis, IL-15 was more effective than IL-2 in increasing the representation of viable CD56+ cells in the peripheral blood mononuclear cell population, but less effective in increasing T cell representation. The immunotherapeutic potential of IL-15 appears superior to IL-2 in regard to expanding NK cell populations in HIV-infected individuals, but needs to be weighed against poorer increases in T cell populations.     

High-density oligonucleotide array analysis reveals extensive differences between freshly isolated blood and hepatic natural killer cells

Hepatic NK cells are more cytotoxic than blood NK cells against tumor cells. To understand the basis of this difference in cytotoxicity we analyzed RNA derived from freshly isolated rat blood and hepatic NK cells [high-density (HD) and low-density subpopulations] by high-density oligonucleotide arrays (Affymetrix), containing about 9,000 genes and expressed sequence tags. IL-2-treated blood NK (A-NK) cells and IL-2-treated hepatic HD cells were used as a reference of NK cell activation. About 150 genes and expressed sequence tags were differentially expressed between hepatic and blood NK cells. Surprisingly, more than half of the increased expressed genes in hepatic NK cells were not increased in A-NK cells. Differentially expressed genes like the stem cell factor receptor c-kit and the chemokine receptor CCR5 can contribute to the homing and differentiation of hepatic NK cells in the liver sinusoids. Several of the differentially expressed genes can possibly contribute to the enhanced cytotoxic activity of hepatic NK cells: cell membrane receptors like NKG2D, NKG2C, CD94, ecto-ATPase; signaling molecules like phosphatidylinositol 3-kinase; granule-associated effector molecules like granzymes and defensin NP3. Moreover, it appears that redirection of cytotoxic granules and increase in intracellular Ca2+ are convergence points of several of these genes.     

Unique phenotype of human uterine NK cells and their regulation by endogenous TGF-beta

Natural killer (NK) cells are a major population of lymphocytes in the human endometrium (EM), and NK cells can be a significant source of cytokines that alter local immune responses. The aim of this study was to determine the expression of NK cell receptors in situ and to test whether uterine NK (uNK) cells produce cytokines and how this activity may be regulated by transforming growth factor-beta (TGF-beta). We observed that human uNK cells were CD56+, CD3-, CD57-, CD9+, CD94+, killer inhibitory receptor+, and CD16+/- in situ by confocal microscopy. We examined cytokine production by uNK cells and uNK cell clones derived from human EM. Stimulation of uNK cells with interleukin (IL)-12 and IL-15, both of which are expressed in the human EM, induced interferon-gamma (IFN-gamma) and IL-10 production. IFN-gamma production by uNK cell clones was completely inhibited by TGF-beta1 in a dose-dependent manner with an inhibitory concentration 50% value of 20 pg/ml. IL-10 secretion by uNK cell clones was also inhibited by TGF-beta1 at similar concentrations. Furthermore, blocking endogenous TGF-beta in fresh human endometrial cell cultures increased the production of IFN-gamma by uNK cells. These data indicate that uNK cells have a unique phenotype that is distinct from blood NK cells. Further, data demonstrate that uNK cells can produce immunoregulatory cytokines and that inhibition of uNK cells by locally produced TGF-beta1 is a likely mechanism to regulate NK cell function in the human EM.     

重组人IL-15促人脐血CD34+造血干细胞定向分化为NK细胞

目的 探讨IL－15促CD34^ 造血干细胞定向分化为NK细胞的作用。方法 采用MACS法分离脐血中CD34^ 细胞，分别用IL－15、SCF（造血干细胞因子）和IL－15 SCF体外培养，流式细胞仪测定CD3、CD16、CD56分子，MTT法检测NK细胞杀伤活性。结果 IL－15可以促进CD34^ 细胞分化为以CD56^ CD16^-为主的NK细胞，SCF具有较强的协同作用。结论 IL－15具有促使NK细胞定向分化的作用。     

Bcl-xL is associated with the anti-apoptotic effect of IL-15 on the survival of CD56(dim) natural killer cells

Human NK cells can be distinguished into CD56(bright) and CD56(dim) subsets based on cell surface CD56 density. It has been shown that IL-2 and IL-15 have opposing effects on life and death of CD8(+) T cells. However, the roles of IL-2 and IL-15 in regulating these two NK cell subsets remain elusive. In this study, we comparatively analyzed the effects of IL-2 and IL-15 on two NK cell subsets. IL-15 improved the proliferation and activation of CD56(dim) NK cells in long-term cord blood mononuclear cell culture, but IL-2 only maintained the survival of CD56(bright) NK cells. The percentage of CD56(+)Annexin V(+) NK cells cultured with IL-15 was lower than that with IL-2; moreover, most of Annexin V(+) NK cells were primarily in the CD56(dim) NK cells. IL-15 cultured NK cells expressed higher level of Bcl-xL than IL-2 cultured cells. Furthermore, IL-15 more strongly upregulated CD25 expression and better maintained the expression of IL-15Ralpha than IL-2. These results suggest that CD56(dim) NK cells undergo apoptosis when cultured with IL-2, but IL-15 inhibits their apoptosis and Bcl-xL is associated with the anti-apoptotic effect of IL-15. So IL-15 played a crucial role in sustaining long-lasting functions of CD56(dim) NK cells.     

IL-21 induces both rapid maturation of human CD34+ cell precursors towards NK cells and acquisition of surface killer Ig-like receptors

The NK cell maturation from CD34(+) Lin(-) hematopoietic cell precursors is a complex process that requires the direct contact with stromal cells and/or the synergistic effect of different cytokines. In this study we show that IL-21 is capable of inducing an accelerated NK cell maturation when added to cultures of CD34(+) Lin(-) cells isolated from human cord blood supplemented with IL-15, Flt3-L and SCF. After 25 days of culture, 50% of CD56(+) cells expressed various NK cell markers including the NKp46 and NKp30 triggering receptors, the CD94/NKG2A inhibitory receptor and CD16. At day 35, substantial fractions of NK cells expressed KIR, CD8 and CD2, i.e. surface markers expressed by mature NK cells, that are virtually undetectable in developing NK cells cultured in the absence of IL-21. Remarkably, similar to mature NK cells all these markers were included in the CD56(dim) cell fraction, while the CD56(bright) population was only composed of CD94/NKG2A(-) and CD94/NKG2A(+) cells. Thus, IL-21 allows the induction of a full NK cell maturation in vitro and offers an important tool for dissecting the molecular mechanisms involved in different steps of NK cell maturation and in the acquisition of a mature KIR repertoire.     

The regulation and activation of CD44 by natural killer (NK) cells and its role in the production of IFN-gamma.

Natural killer (NK) cells can express high levels of CD44, and signaling through CD44 has been shown to enhance NK cell cytotoxic activity. However, little is known about the factors that regulate CD44-mediated activation of NK cells. The studies reported here reveal that resting NK cells constitutively express CD44 that is in an inactive form that does not bind to hyaluronan (HA), the principal known ligand for CD44. After infection of mice with the intracellular parasite Toxoplasma gondii, however, a population of NK cells that expressed activated CD44 emerged. To determine how expression and activation of CD44 by resting NK cells were regulated, the role of cytokines in these events was assessed. These studies revealed that whereas stimulation of resting NK cells with interleukin-12 (IL-12) or IL-18 caused increased expression of CD44, only IL-2 or IL-15 led to the upregulation and activation of CD44. The cytokine-induced upregulation and activation of CD44 was independent of NK cell proliferation. To determine the functional consequences of CD44 activation, the effects of low molecular weight HA (LMWHA) on the production of interferon-gamma (IFN-gamma) by IL-2-activated NK cells were assessed. These studies showed that HA alone had little effect on the production of IFN-gamma, but when used in combination with IL-2, IL-12, or IL-18, LMWHA was a potent enhancer of IFN-gamma production. Together, these studies indicate an important role for proinflammatory cytokines in the activation of CD44 on NK cells and identify a novel pathway to enhance the ability of activated NK cells to produce IFN-gamma.     

Long-term cultures of human peripheral blood lymphocytes with recombinant human interleukin-2 generate a population of virtually pure CD3+ CD16- CD56- large granular lymphocyte LAK cells.

It has been reported that lymphocytes from peripheral blood (PBL) cultured with interleukin-2 (IL-2) produce predominantly CD16+ lymphokine-activated killer (LAK) cells. We developed a two-step method to generate LAK cells from human PBL in long-term cultures (10-12 days) with recombinant human IL-2 (rhIL-2) and characterized the evolving LAK cell population by testing its phenotype and cytotoxic activity as a function of time. The starting PBL displayed some natural killer (NK) cytotoxicity but no LAK activity. At day 6, the cells were a mixed population of about 80% CD3+ and 6% CD16+ cells. Little proliferation was evident but strong LAK activity was detected. After 10-12 days, major cell expansion had occurred and they were essentially a pure (greater than 90%) CD3+ CD16- CD56- cell population large granular lymphocyte (LGL) by morphology that displayed strong non-MHC-restricted killing activity (greater than 200 lytic units). Over the same period of time, the CD16+ cells had almost completely regressed in these cultures. This preferential induction of CD+ LAK cells was not an effect of IL-2 concentration as 10 U/ml was as effective as 500 U/ml. Further characterization revealed a major population of CD4+ (60%) and CD8+ (30%) with a smaller fraction (less than 9%) of gamma delta + cells. These results indicate that a virtually pure CD3+ LAK cells population was produced with long-term cultures of lymphocytes from peripheral blood in rhIL-2, in which active proliferation of the CD3+ but not CD16+ cells occurred.     

Interleukin-2 activated NK cells do not use the CD95L- and TRAIL-pathways in the rapid induction of apoptosis of rat colon carcinoma CC531s cells

Natural Killer (NK) cells can induce apoptosis in target cells in at least four ways: by secretion of granzyme B/perforin (GrB/P) and via the CD95L, TRAIL and TNF-α pathways. In this study we examined the pathways used by interleukin-2 activated rat NK (A-NK) cells to induce apoptosis in the rat colon carcinoma cell line CC531s. Co-incubation of A-NK cells with CC531s cells for three hours resulted in 70% apoptosis in the latter. Addition of the GrB/P pathway-inhibitor concanamycin A reduced the number of apoptotic cells to 54%. Blockade of the CD95L, TRAIL and TNF-α pathways by specific antibodies hardly had an additional effect. However, co-incubation with transfected MEC cells that expressed CD95L or 2PK3-cells that expressed TRAIL did induce apoptosis in CC531s cells. Furthermore the A-NK cells contained CD95L and TRAIL. However, comparison of non- and permeabilized cells revealed that the majority of TRAIL was present in the cytosol of A-NK cells and was not available for induction of apoptosis. The presence of elevated levels of bcl-2 in CC531 cells reduced the sensitivity towards induction of apoptosis both by A-NK cells as well as the CD95L and TRAIL expressing cell lines. Using the caspase-inhibitors ac-IEPD-CHO, ac-DEVD-CHO and zVAD-fmk, it was shown that inhibition of the effector caspase-3 prevented A-NK cell induced apoptosis in CC531-bcl-2 cells, but not in CC531s cells. In conclusion, A-NK cells kill by secretion of GrB/P and not by the CD95L, TRAIL or TNF pathways albeit both CD95L and TRAIL are produced by the A-NK cells.