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1.
Cytoplasmic virus-specific RNA and polyribosomes from a chronically infected feline thymus tumor cell line, F-422, were analyzed by using in vitro-synthesized feline leukemia virus (Rickard strain) (R-FeLV) complementary DNA (cDNA) probe. By hybridization kinetics analysis, cytoplasmic, polyribosomat, and nuclear RNAs were found to be 2.1, 2.6, and 0.7% virus specific, respectively. Size classes within subcellular fractions were determined by sucrose gradient centrifugation in the presence of dimethyl sulfoxide followed by hybridization. The cytoplasmic fraction contained a 28S size class, which corresponds to the size of virion subunit RNA, and 36S, 23S, and 15 to 18S RNA species. The virus-specific 36S, 23S, and 15 to 18S species but not the 28S RNA were present in both the total and polyadenylic acid-containing polyribosomal RNA. Anti-FeLV gamma globulin bound to rapidly sedimenting polyribosomes, with the peak binding at 400S. The specificity of the binding for nascent virus-specific protein was determined in control experiments that involved mixing polyribosomes with soluble virion proteins, absorption of specific gamma globulin with soluble virion proteins, and puromycin-induced nascent protein release. The R-FeLV cDNA probe hybridized to RNA in two polyribosomal regions (approximately 400 to 450S and 250S) within the polyribosomal gradients before but not after EDTA treatment. The 400 to 450S polyribosomes contained three major peaks of virus-specific RNA at 36S, 23S, and 15 to 18S, whereas the 250S polyribosomes contained predominantly 36S and 15 to 18S RNA. Further experiments suggest that an approximately 36S minor subunit is present in virion RNA. 相似文献
2.
Specificities of killing by cytotoxic lymphocytes generated in vivo and in vitro to syngeneic SV40 transformed cells. 总被引:11,自引:0,他引:11
L R Gooding 《Journal of immunology (Baltimore, Md. : 1950)》1977,118(3):920-927
Cell-mediated immunity to SV40-transformed C3H and C3H-SW cell lines was measured by using both 51Cr and 125IUdR release assays. Killing by cytotoxic cells generated on in vitro sensitization of immune spleen cells with syngeneic SV40 cells by either assay is specific for syngeneic SV40 transformants. Cytolysis mediated by in vitro sensitized cells is ablated by treatment of the effector cells with anti-theta serum and complement. Intraperitoneal immunization with syngeneic SV40 cells yields two distinct killer-cell populations in the peritoneal exudate when assayed by 125IUdR release. The first, nylon wool nonadherent and sensitive to anti-theta and complement, is indistinguishable from the killers generated in vitro. The second population, present in larger numbers and more efficient on a per-cell basis in killing of SV40 targets than the first, is nylon adherent and is not removed by treatment with anti-theta and complement. This second population will kill any SV40 transformed target, whether syngeneic or allogeneic. The possible roles of T cell and non-T cell effectors in rejection of syngeneic SV40 tumors are discussed. 相似文献
3.
Deoxyglucose transport of uninfected, murine sarcoma virus-transformed, and murine leukemia virus-infected mouse cells 总被引:3,自引:0,他引:3
P G Plagemann 《Journal of cellular physiology》1973,82(3):421-433
The initial rates of deoxy-D-glucose transport by cultures of growing and density-inhibited mouse embryo cells and lines of mouse cells transformed spontaneously or after infection by murine leukemia virus or murine sarcoma virus were investigated as a function of the deoxyglucose concentration. The apparent Km for deoxyglucose transport was about the same for all types of cells (1–2 mM). The Vmax of secondary cultures of mouse embryo cells decreased from 6 nmoles/106 cells/minute for sparse cultures to less than 1 nmole/106 cells/minute for density-inhibited cultures. The Vmax was about the same whether estimated in monolayer culture or in suspensions of cells dispersed by treatment with trypsin. The Vmax for deoxyglucose transport by the established cells, whether transformed spontaneously or by virus infection, was 4 to 25 times higher than that for density-inhibited mouse embryo cells and was independent of the cell density of the cultures. Deoxyglucose transport was competitively inhibited by Cytochalasin B, Persantin, glucose and 3-O-methyl-D-glucose and the apparent Ki values of inhibition were similar for the mouse embryo cells and the various cell lines. Similarly, the sensitivity of the glucose transport systems to inactivation by p-chloromercuribenzoate was about the same for all types of cells. The results suggest that the glucose transport system of the normal mouse embryo cells and the cells of the various established lines is qualitatively the same, but that the number of functional transport sites differs for the various cell lines and decreases markedly in mouse embryo cells with an increase in cell density of the cultures. 相似文献
4.
Presence of 5''-terminal cap structures in virus-specific RNA from feline leukemia virus-infected cells.
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The F-422 line of feline thymus tumor cells, chronically infected with the Rickard strain of feline leukemia virus (R-FeLV), was labeled with 32P, and the total cytoplasmic RNA was isolated. The RNA was centrifuged through sucrose gradients, and R-FeLV virus-specific RNA (vRNA) was located by hybridization of portions of the gradient fractions to R-FeLV complementary DNA. vRNA classes with average sedimentation coefficients of approximately 36S, 28S, 23S, and 15S were identified. Each class of RNA was recovered by hybridized with mercurated R-FeLV complementary DNA, and the hybrids were chromatographed on columns of sulfhydryl-Sepharose to separate them from unhybridized cellular RNA. Although insufficient amount of 36S and 28S vRNA were obtained for further analysis, the 23S and 15S VRNA classes were analyzed to determine the nature of their 5' termini. Each of these vRNA classes was found to contain stoichiometric amounts of cap structures per unit length of RNA, consistent with the presence of one cap per molecule. The structure of the 23S vRNA cap was found to be m7G5'ppp5'GmpAp, whereas that of the 15S vRNA cap was m7G5'ppp5'GmpGp. 相似文献
5.
Keski-Oja J Lehto VP Vartio T Badley RA Virtanen I 《European journal of cell biology》1983,30(2):191-199
The murine epithelial cell line MMC-E was used to study changes in the cytoskeletal organization associated with viral transformation of epithelial cells by two different viruses. The cells were transformed with Moloney mouse sarcoma virus (MSV) or murine leukemia virus (MuLV). The expression of actin, myosin and of intermediate filament proteins in the cells was then studied. In MMC-E cells actin and myosin were organized as belt-like structures at the edges of the border cells of the cell islands and also circumferentially in the cells inside the islands. The major change after transformation was the decrease of the actomyosin containing belt extending from cell to cell at the borders of the cell islands. Both MMC-E cells and the MSV-transformed cells contained keratin as a juxtanuclear granular aggregate whereas the MuLV-transformed cells showed bright fibrillar arrays of keratin. Both virus-transformed cell lines showed enhanced vimentin-specific fluorescence and analysis of their cytoskeletal polypeptides confirmed the result. Similar molecular forms of keratin polypeptides were seen in all cells by immunoblotting. Viral transformation of MMC-E epithelial cells thus leads to different changes in their cytoskeletal organization depending on the transforming viral or cellular gene. 相似文献
6.
Bone marrow cells cultured for 5-6 days generate cytotoxic activity against a number of natural killer (NK)-susceptible tumor cells. In this study, these bone marrow cytotoxic cells were compared to cells with NK activity obtained either from spleen cells activated in vitro with interferon (IFN-alpha/beta) or mitogen or from peritoneal exudate cells (PEC) obtained 4 days after bacillus Calmette-Guerin (BCG) infection. Splenic and PEC cytotoxic cells were shown to be Thy 1.2+, NK 1.1+, Asialo GM+1, Lyt 1.2-, Lyt 2.2-. In contrast, bone marrow cytotoxic cells were Thy 1.2+, NK 1.1-, Lyt 1.2-, Lyt 2.2- and expressed low levels of Asialo GM1 antigen (Asialo GM +/- 1). Precursor cells for bone marrow cytotoxic activity were shown to be Thy 1.2-, NK 1.1-, Lyt 1.2-, Lyt 2.2- but also expressed low levels of Asialo GM1 antigen (Asialo GM +/- 1). Cytotoxic activity for both bone marrow and spleen cells peaked in the low-density fractions of discontinuous Percoll density gradients. The cytotoxic activity of these bone marrow cells was augmented by pretreatment with IFN (-alpha/beta, -gamma) or soluble factors (IFN free) from activated EL-4 thymoma cells. Surprisingly, the ability of bone marrow cells to generate high levels of cytotoxic activity following in vitro culture appeared to be associated primarily with mice which were of the H-2b haplotype. 相似文献
7.
8.
Transforming potential of a myc-containing variant of feline leukemia virus in vitro in early-passage feline cells. 总被引:5,自引:4,他引:1
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We studied a naturally occurring variant of feline leukemia virus (FeLV) in which the oncogene myc has substituted for a portion of the viral structural genes (myc-FeLV). myc-FeLV was rescued by replication in the presence of FeLV as helper, and its biological activity was examined in early-passage feline cells in vitro. Infection of leukocytes from peripheral blood, spleen, or thymus, or of kitten fibroblasts did not immortalize these cells or alter them morphologically. Northern blot (RNA blot) analysis of virion RNA prepared from the supernatant of infected cells demonstrated the 8.2-kilobase genome of FeLV, but did not demonstrate the 5.0-kilobase genome of myc-FeLV. Apparently, the myc-FeLV genome was lost in the absence of the selective pressure of transformation. In contrast, infection of embryonic fibroblasts with myc-FeLV(FeLV) rendered these cells capable of greatly increased, if not infinite, proliferative potential. The cells were morphologically altered compared with controls and were only loosely adherent to the substrate. The cells failed to proliferate in semisolid medium and did not form tumors when inoculated subcutaneously into athymic mice. Blot analyses demonstrated the presence and expression of integrated proviral DNAs of both FeLV and myc-FeLV in these cells. They appear, then, to represent cells partially transformed by infection with myc-FeLV(FeLV). The action of feline v-myc in early-passage cells in vitro was compared to that of avian v-myc. 相似文献
9.
Suvendu Das C. Varalakshmi Leela A. Kumari Megha Patel Ashok Khar 《Cancer immunology, immunotherapy : CII》2001,50(8):428-436
This study examines the effect of fixed AK-5 tumour cells on rat NK cells. Co-culture of NK cells with fixed tumour cells augmented the cytotoxicity of NK cells against NK-sensitive targets, YAC-1 and AK-5, and induced the secretion of IFN-gamma by NK cells. Antibody against IFN-gamma suppressed the anti-tumour activity of NK cells, whereas the addition of T cells during co-culture enhanced this activity. However, macrophages and B cells had no significant effect when present during co-culture with NK cells. All the inducible cytotoxicity was contained within the NK (CD161+) and NKT (CD3+, CD161+) subsets of lymphocytes. However, in the presence of T cells, the cytolytic potential of NKT cells was higher than that of NK cells alone. The augmentation of cytotoxic activity of NK cells by AK-5 cells in presence of T cells was dependent on IL-2 and IFN-gamma secretion. NK cell activation was blocked by specific antibodies to IL-2 and IFN-gamma in the presence of T cells. Interaction between fixed AK-5 cells with NK and T cell populations induced the expression of Fas-L and perforin in NK cells. These data demonstrate that fixed AK-5 cells initiated cytokine synthesis by NK cells, and the enhanced cytotoxic activity in the presence of T cells was induced as a consequence of the products secreted by activated T lymphocytes. The present observations reflect the possible interactions taking place in vivo after the transplantation of AK-5 tumour in animals. They also suggest direct activation of NK cells after their interaction with the tumour cells. 相似文献
10.
Danuta Sosnowska Andrzej Mysliwski Krystyna Dzierzbicka Aleksander M. Kolodziejczyk 《Biotherapy》1997,10(2):161-168
The modulation of NK activity by muramyl dipeptides derivatives against Ab (amelanotic) Bomirski melanoma and human erythroleukemia
K562 cells was studied in vitro. The stimulatory effect was observed for 3 of 7 muramyl dipeptides: MDP(L-Ala)C921, MDPC857
and L18-MDP(Ala) in relation to cytotoxic activity of NK cells obtained from peripheral blood and spleen of healthy and Ab
Bomirski melanoma bearing hamsters. An increased of cytotoxic activity NK cells isolated from animals before and during the
transplantable phase of the tumor against K562 was found. A similar stimulation was received for NK cells obtained from animals
against their own melanoma cells. The most significant influence of examined MDP derivatives on the cytotoxic activity of
NK cells were obtained from animals between 10 to 12 days of tumor growth. The extent of the modulation of cytotoxic activity
of NK cells was dependent on its initial value both in healthy control and Ab Bomirski melanoma bearing hamsters. If natural
cytotoxic activity was high the stimulatory effect of the examined MDP derivatives was only slightly expressed. 相似文献
11.
Fang He Yaoying Zeng Xiaoping Wu Yuhua Ji Xianhui He Thomas Andrus Tuofu Zhu Tong Wang 《Apoptosis : an international journal on programmed cell death》2009,14(10):1212-1226
HIV-1 viral protein R (Vpr) can induce cell cycle arrest and cell death, and may be beneficial in cancer therapy to suppress
malignantly proliferative cell types, such as adult T-cell leukemia (ATL) cells. In this study, we examined the feasibility
of employing the HIV-vpr gene, via targeted gene transfer, as a potential new therapy to kill ATL cells. We infected C8166 cells with a recombinant
adenovirus carrying both vpr and GFP genes (rAd-vpr), as well as the vector control virus (rAd-vector). G2/M phase cell cycle arrest was observed in the rAd-vpr infected cells. Typical characteristics of apoptosis were detected in rAd-vpr infected cells, including sub-diploid peak exhibition in DNA content assay, the Hoechst 33342 accumulation, apoptotic body
formation, mitochondrial membrane potential and plasma membrane integrity loss. The proteomic assay revealed apoptosis related
protein changes, exhibiting the regulation of caspase-3 activity indicator proteins (vimentin and Rho GDP-dissociation inhibitor
2), mitochondrial protein (prohibitin) and other regulatory proteins. In addition, the up-regulation of anti-inflammatory
redox protein, thioredoxin, was identified in the rAd-vpr infected group. Further supporting these findings, the increase of caspase 3&7 activity in the rAd-vpr infected group was observed. In conclusion, endogenous Vpr is able to kill HTLV-1 transformed C8166 cells, and may avoid
the risks of inducing severe inflammatory responses through apoptosis-inducing and anti-inflammatory activities. 相似文献
12.
The time course of the appearance of cytotoxic cells was examined in cocultures of E-rosetting (E+) cells and EBV-infected non-T cells (4:1 ratio) from the blood of VCA-positive healthy adults. Classical HNK-1+ NK cells were present at the initiation of the cultures and they produced 76 +/- 2% specific 51Cr-release from K-562 cells, but they did not effectively lyse the NK-resistant Daudi cells, nor did they kill autologous EBV-induced lymphoblasts (LCLEBV). The NK activity decreased during the first week in culture to 40 +/- 7% cytotoxicity. At the same time, nonspecific cytotoxic cells capable of killing Daudi as well as K562 developed and persisted into the third week in culture when it declined. This later nonspecific cytotoxicity was mediated by 4F2+, T8-, HNK-1- activated E+ cells. After 10 days in culture, killing of autologous LCLEBV increased continuously, from 4 +/- 3% at Day 10 to 38 +/- 4% by Day 22. The cytotoxicity to LCLEBV was mediated by classical T8+ CTL, and it was antigen specific and at least partially HLA Class I restricted. The regression of BEBV growth that occurs in E+/BEBV cocultures coincides with the development of this CTL-mediated cytotoxicity. 相似文献
13.
Molecular cloning of Snyder-Theilen feline leukemia and sarcoma viruses: comparative studies of feline sarcoma virus with its natural helper virus and with Moloney murine sarcoma virus 总被引:15,自引:20,他引:15
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Extrachromosomal DNA obtained from mink cells acutely infected with the Snyder-Theilen (ST) strain of feline sarcoma virus (feline leukemia virus) [FeSV(FeLV)] was fractionated electrophoretically, and samples enriched for FeLV and FeSV linear intermediates were digested with EcoRI and cloned in lambda phage. Hybrid phages were isolated containing either FeSV or FeLV DNA "inserts" and were characterized by restriction enzyme analysis, R-looping with purified 26 to 32S viral RNA, and heteroduplex formation. The recombinant phages (designated lambda FeSV and lambda FeLV) contain all of the genetic information represented in FeSV and FeLV RNA genomes but lack one extended terminally redundant sequence of 750 bases which appears once at each end of parental linear DNA intermediates. Restriction enzyme and heteroduplex analyses confirmed that sequences unique to FeSV (src sequences) are located at the center of the FeSV genome and are approximately 1.5 kilobase pairs in length. With respect to the 5'-3' orientation of genes in viral RNA, the order of genes in the FeSV genome is 5'-gag-src-env-c region-3'; only 0.9 kilobase pairs of gag and 0.6 kilobase pairs of env-derived FeLV sequences are represented in ST FeSV. Heteroduplex analyses between lambda FeSV or lambda FeLV DNA and Moloney murine sarcoma virus DNA (strain m1) were performed under conditions of reduced stringency to demonstrate limited regions of base pair homology. Two such regions were identified: the first occurs at the extreme 5' end of the leukemia and both sarcoma viral genomes, whereas the second corresponds to a 5' segment of leukemia virus "env" sequences conserved in both sarcoma viruses. The latter sequences are localized at the 3' end of FeSV src and at the 5' end of murine sarcoma virus src and could possibly correspond to regions of helper virus genomes that are required for retroviral transforming functions. 相似文献
14.
T Pang R V Blanden 《The Australian journal of experimental biology and medical science》1977,55(5):539-547
The nature of infected stimulator cells in the in vitro secondary cytotoxic T cell response to ectromelia infection was investigated. It was found that macrophages were better stimulator cells than spleen cells. B cells (Ig-positive cells) were superior to T cells (Ig-negative cells) both on a relative proportion and on a cell-to-cell basis. Concanavalin A and lipopolysaccharide-stimulated lymphocytes were also effective stimulator cells but appeared to be slightly inferior to spleen cells. Spleen cells depleted of Ia-positive cells were markedly inferior to normal spleen cells as stimulators. It was also found that primary and secondary cytotoxic T cells were largely Ia-negative. These findings are discussed in relation to the likely events during T cell responses to infection in vivo. 相似文献
15.
K Sobue K Kanda I Miyamoto K Iida I Yahara R Hirai A Hiragun 《Experimental cell research》1989,181(1):256-262
With fluorescence and interference reflection microscopy (IRM), we compared the regional distribution of calspectin, its interacting proteins (nonerythroid protein 4.1 and calpactin), alpha-actinin, and vinculin in NRK cells and their avian sarcoma virus (ASV)- or temperature-sensitive (ts) Rous sarcoma virus (RSV)-transformed cells. The localization of these cytoskeletal proteins was determined with the specific antibodies. In NRK cells, alpha-actinin and vinculin were concentrated at adhesion plaques. By contrast, calspectin was distributed throughout the cytoplasm, but not concentrated at adhesion plaques. In ASV- and ts RSV-transformed cells, all three cytoskeletal proteins were concentrated at dot structures representing cellular feet. Nonerythroid protein 4.1 and calpactin were diffusely distributed throughout the cytoplasm of NRK cells and their transformed counterparts. In the case of calpactin, a part of this protein was excluded near regions of the terminal ends of stress fibers. These two proteins did not show the restricted location at the dot structures of transformed cells. From these findings, it is apparent that the accumulation of calspectin into dot structures is a specific event for cell transformation induced by the src protein. 相似文献
16.
Turnover rates of B cells,T cells,and NK cells in simian immunodeficiency virus-infected and uninfected rhesus macaques 总被引:8,自引:0,他引:8
De Boer RJ Mohri H Ho DD Perelson AS 《Journal of immunology (Baltimore, Md. : 1950)》2003,170(5):2479-2487
We determined average cellular turnover rates by fitting mathematical models to 5-bromo-2'-deoxyuridine measurements in SIV-infected and uninfected rhesus macaques. The daily turnover rates of CD4(+) T cells, CD4(-) T cells, CD20(+) B cells, and CD16(+) NK cells in normal uninfected rhesus macaques were 1, 1, 2, and 2%, respectively. Daily turnover rates of CD45RA(-) memory T cells were 1%, and those of CD45RA(+) naive T cells were 0.5% for CD4(+) T cells and approximately 1% for CD4(-)CD45RA(+) T cells. In SIV-infected monkeys with high viral loads, the turnover rates of T cells were increased approximately 2-fold, and that of memory T cells approximately 3-fold. The turnover of CD4(+)CD45RA(+) naive T cells was increased 2-fold, whereas that of CD4(-)CD45RA(+) naive T cells was marginally increased. B cells and NK cells also had increased turnover in SIV-infected macaques, averaging 3 and 2.5% per day, respectively. For all cell types studied here the daily turnover rate increased with the decrease of the CD4 count that accompanied SIV infection. As a consequence, the turnover rates of CD4(+) T cells, CD4(-) T cells, B cells, and NK cells within each monkey are strongly correlated. This suggests that the cellular turnover of different lymphocyte populations is governed by a similar process which one could summarize as "generalized immune activation." Because the viral load and the CD4 T cell count are negatively correlated we cannot determine which of the two plays the most important role in this generalized immune activation. 相似文献
17.
NK-cell cytotoxic activity and their relative distributions were studied in the spleen of female Lurcher mice with spontaneous
olivopontocerebellar degeneration (C3H) and female athymic nu/nu mice (BALB/c) influenced by 3-acetylpyridine (the neurotoxin
causing selective degeneration of cerebellar and inferior olive neurons in some rodent species). The congenital olivopontocerebellar
degeneration in Lurcher mice is followed by only an insignificant increase of NK-cell cytotoxic activity (1.2 times). On the
other hand, the congenital thymic dysgenesis in nu/nu mice is compensated by a substantial increase in cytotoxic activity
(19.4-fold). The administration of 3-acetylpyridine (including prevalent neuronal destruction particularly in Lurcher mutants)
caused a decrease of NK-cell cytotoxic activities in all groups of mice (in Lurcher and C3H controls to 60 and 50%, respectively,
and in nu/nu and BALB/c controls to 25 and 60%). Relative distributions of NK-cells in spleens of non-influenced and influenced
animals were not significantly changed. Some fundamental immune mechanisms, such as the NK-cell cytotoxic activity, were demonstrated
to be controlled by congenitally determined or artificially induced changes in both the nervous and the immune systems. 相似文献
18.
Natural killer cells vs cytotoxic T cells in the peripheral blood of virus-infected mice 总被引:2,自引:0,他引:2
L Stitz A Althage H Hengartner R Zinkernagel 《Journal of immunology (Baltimore, Md. : 1950)》1985,134(1):598-602
The cell-mediated immune response of mice against various enveloped RNA and DNA viruses expressed by immune lymphocytes from the spleen and the peripheral blood (PBL) were compared. PBL from mice of various strains infected with vaccinia virus, vesicular stomatitis virus (VSV), or lymphocytic choriomeningitis virus (LCMV) were tested on histocompatible or incompatible target cells infected with the homologous virus. PBL from immune mice showed clear H-2 restriction, but additionally, they expressed high natural killing (NK) activity on YAC-1 cells. The high NK-cytolytic activity of PBL on YAC-1 differed significantly from that expressed by splenic lymphocytes. In both lymphocyte populations lysis was detected as early as 1 day after infection; NK activity decreased in the spleen after day 4 post infection, whereas that of PBL persisted at high levels for up to 10 days after infection. Treatment of mice with anti-asialo GM1 in vivo abrogated NK activity in PBL effector cells tested in vitro. These results may explain some of the difficulties to observe MHC-restricted cytotoxic T cells in PBL from humans or primates during primary infections with virus. 相似文献
19.
Feline leukemia is a disease induced by an oncornavirus infection that inevitably causes clinically affected cats to die. It has been estimated that 40% of cats are dead within 4 weeks and 70% within 8 weeks of the onset of clinical symptoms. Acemannan is a complex carbohydrate with both immunostimulatory and direct antiviral properties. Administration of acemannan for 6 weeks intraperitoneally to clinically symptomatic cats significantly improved both the quality of life and the survival rate. Twelve weeks after initiation of treatment, 71% of treated cats were alive and in good health. 相似文献
20.
Bielawska-Pohl A Crola C Caignard A Gaudin C Dus D Kieda C Chouaib S 《Journal of immunology (Baltimore, Md. : 1950)》2005,174(9):5573-5582
Human organ-specific microvascular endothelial cells (ECs) were established and used in the present study to investigate their susceptibility to natural killer cell line (NKL)-induced lysis. Our data indicate that although IL-2-stimulated NKL (NKL2) cells adhered to the human peripheral (HPLNEC.B3), mesenteric lymph node (HMLNEC), brain (HBrMEC), and lung (HLMEC) and skin (HSkMEC.2) ECs, they significantly killed these cells quite differently. A more pronounced lysis of OSECs was also observed when IL-2-stimulated, purified peripheral blood NK cells were used as effector cells. In line with the correlation observed between adhesion pattern and the susceptibility to NKL2-mediated killing, we demonstrated using different chelators that the necessary adhesion step was governed by an Mg(2+)-dependent, but Ca(2+)-independent, mechanism as opposed to the subsequent Ca(2+)-dependent killing. To identify the cytotoxic pathway used by NKL2 cells, the involvement of the classical and alternate pathways was examined. Blocking of the Ca(2+)-dependent cytotoxicity pathway by EGTA/MgCl(2) significantly inhibited endothelial target cell killing, suggesting a predominant role for the perforin/granzyme pathway. Furthermore, using confocal microscopy, we demonstrated that the interaction between NKL2 effectors and ECs induced cytochrome c release and Bid translocation in target cells, indicating an involvement of the mitochondrial pathway in NKL2-induced EC death. In addition, although all tested cells were sensitive to the cytotoxic action of TNF, no susceptibility to TRAIL or anti-Fas mAb was observed. The present studies emphasize that human NK cell cytotoxicity toward ECs may be a potential target to block vascular injury. 相似文献