E T Bloom

Kanazawa Medical University, Kanazawa-shi, Ishikawa-ken, Japan

Are you E T Bloom?

Claim your profile

Publications (73)364.17 Total impact

  • [Show abstract] [Hide abstract]
    ABSTRACT: Baboons are commonly used as models for transplantation and preclinical testing of various types of therapeutic agents. For proper assessment of information gathered from these models, differences between the baboon and human immune systems need to be characterized. Natural killer (NK) cells are the first line of defense against many infectious agents and cancer and are important mediators of transplantation rejection reactions, particularly during xenotransplantation. In this study, we examined baboon NK cell function and developed methods for purifying and expanding these cells. Baboon NK cells were analyzed using a combination of extracellular and intracellular cell staining, cell sorting, interleukin (IL)-2 mediated stimulation and expansion, and 4 h cytotoxicity assays with human and pig target cell lines. Baboon peripheral blood mononuclear cell (PBMC) exert very low but detectable cytolytic activity against both human (K562) and pig (PAEC, J2) target cells, and this activity is enhanced within 4 h of treatment with IL-2. Like human NK cells, many baboon PBMC express the lytic enzymes granzyme A, granzyme B, and perforin. Based on these markers, we identified a subpopulation of CD3(-) baboon lymphocytes that are CD8(dim) and CD16(bright) that likely represents the baboon NK cells. These cells also are characterized by expression of the natural cytotoxicity receptor NKp46. Baboon CD3(-)NKp46(+) cells purified by flow cytometric cell sorting have high cytolytic capacity that can be further enhanced by IL-2 stimulation. These baboon NK cells can be expanded in vitro and retain extremely high cytolytic capacity. While fresh baboon lymphocytes express very little CD56, the expanded baboon NK cells are predominantly CD56(+); approximately 10% of the expanded NK cells are CD56(dim), and the remainder are CD56(bright). Baboon NK cells that are IL-2 responsive can be identified on the basis of a CD3(-)NKp46(+)CD8(dim)CD16(+/-) or CD3(-)CD8(dim)CD16(bright) phenotype and can be isolated and expanded in culture. These results may allow for a more accurate representation of the human innate immune system in baboon models and more accurate analyses of the role of the baboon innate immune system cells in preclinical models.
    Xenotransplantation 01/2010; 17(4):288-99. · 2.57 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: During T cell activation, TCRs cluster at the center of the T cell-antigen-presenting cell interface forming the central supramolecular activation cluster. Although it has been suggested that sphingolipid- and cholesterol-rich microdomains, termed lipid rafts, form platforms for the regulation and transduction of TCR signals, an actual role for membrane sphingomyelin (SM), a key component of lipid rafts, has not been reported. After cloning a gene responsible for SM synthesis, sphingomyelin synthase (SMS) 1, we established a SM-knockdown cell line (Jurkat-SMS1/kd) by transfection of SMS1-short-interfering RNA into Jurkat T cells, which is deficient in membrane expression of SM. Upon CD3 stimulation, expression of CD69 (the earliest leukocyte activation antigen), activation-induced cell adhesion and proliferation as well as TCR clustering was severely impaired in Jurkat-SMS1/kd cells. CD3-induced tyrosine phosphorylation and association of linker for activation of T cell with ZAP-70 and Grb2 and phosphorylation of protein kinase C (PKC) were also severely impaired in Jurkat-SMS1/kd cells. Finally, translocation of TCR, ZAP-70 and PKC into lipid rafts was markedly decreased in Jurkat-SMS1/kd cells. These findings indicate that membrane SM is crucial for TCR signal transduction, leading to full T cell activation through lipid raft function.
    International Immunology 10/2008; 20(11):1427-37. · 3.14 Impact Factor
  • Eda T. Bloom
    Xenotransplantation 06/2008; 8(3):153 - 156. · 2.57 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: It is well established that CD4(+)CD25(+) regulatory T (Treg) cells can modulate allogeneic immune responses. Xenotransplantation, proposed as a means to address the critical shortage of human organs, may also benefit from similar approaches to avert rejection. Baboons are a preferred preclinical animal model for xenogeneic organ transplantation experiments, and the characterization of baboon Treg cells will be beneficial to future tolerance studies in this animal model. We analyzed CD4(+)CD25(+) T cells from baboon lymph nodes, spleens, and blood by flow cytometry, then purified and expanded porcine antigen-specific baboon CD4(+)CD25(high) cells in vitro to evaluate their regulatory activity in the baboon anti-pig xenogeneic responses. CD4(+)CD25(high) T cells were 1.7%, 3.1%, and 1.9% of baboon splenic, lymph node, and blood T cells, respectively. The CD4(+)CD25(high) T cells expressed the Treg cell-associated transcription factor, FoxP3. Proliferation/suppression assays using irradiated pig peripheral blood mononuclear cells as stimulators showed that Treg cells suppressed the vigorous baboon CD4(+)CD25(-) T-cell anti-pig proliferation response and cytokine secretion. Expanded baboon Treg cells suppressed baboon anti-pig CD4(+)CD25(-) T-cell proliferation approximately 4- to 10-fold more than freshly isolated Treg cells. Expanded Treg cells suppressed proliferation to primary cells from the same pig used for expansion more effectively than proliferation to stimulators from a different strain of pig, suggesting a level of antigen specificity. We demonstrate that baboon Treg cells suppress immune responses to xenogeneic stimulation. These studies suggest that adoptive transfer of expanded Treg cells into transplant recipients may provide an approach to prevent cell-mediated rejection of grafts and potentially induce tolerance in the pig to baboon xenotransplantation preclinical model.
    Xenotransplantation 08/2007; 14(4):298-308. · 2.57 Impact Factor
  • Eda T Bloom
    [Show abstract] [Hide abstract]
    ABSTRACT: An overview of xenotransplantation regulatory policies in the USA was presented at the Satellite Symposium, ''Xenotransplantation--Current Standards for Clinical Trials,'' held in conjunction with the World Transplant Congress, Boston, MA, USA 2006. This article summarizes that overview.
    Xenotransplantation 08/2007; 14(4):345-6. · 2.57 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: Pluripotent human embryonic stem cells (hESCs) may provide a potential source of cellular therapies, but as allogeneic cells may require evading the recipient's immune response. Using an NIH-registry hESC line, it was found that undifferentiated hESCs induce a reduced proliferative response compared to PBMC and demonstrate that this diminished response correlates with the activity of heme oxygenase-1 (HO-1). Inhibition of HO-1 significantly increases T cell proliferation against hESC, indicating the potential suppression of these cells during transplantation of allogeneic hESC. These data suggest the hypothesis that HO-1 provides a mechanism for protecting hESCs in vivo.
    Antioxidants and Redox Signaling 07/2007; 9(6):751-6. · 7.19 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: NK cells, a component of the innate immune system, provide a first line of defense against viral infections and malignancies, interact with the adaptive immune system and have a role in rejection of allogeneic bone marrow transplants and solid allo- and xenotransplants. Immunoregulatory activity by the anti-hypercholesterolemia agents, 3-hydroxy-3-methyl-glutaryl Coenzyme A (HMG-CoA) reductase inhibitors, known as statins, has recently been reported. We analyzed the effects of three statins on human NK cell cytotoxicity. Two lipophilic statins (simvastatin and fluvastatin) suppressed the cytotoxic activity of fresh and IL-2-stimulated NK cells, while pravastatin, a hydrophilic statin, did not. Suppression was not associated with changes in intracellular perforin, granzyme A or granzyme B levels, or with changes in expression of leukocyte function-associated antigen-1, an integrin known to regulate NK activity and reported to be altered by statin treatment. Decreased cytotoxicity was associated with decreased CD107a surface expression, indicating that the exocytosis pathway was compromised by simvastatin and fluvastatin but not by pravastatin. Mevalonate, the immediate downstream product of HMG-CoA reductase, partially reversed the effect of lipophilic statins on cytotoxicity and CD107a expression. Lipophilic statins also suppressed the release of the granule component, granzyme B, by IL-2-activated NK cells following stimulation with K562. That lipophilic statins suppress NK cell activity through inhibition of the exocytosis pathway suggest an additional potential role for statins in inhibition of transplantation responses.
    International Immunology 03/2007; 19(2):163-73. · 3.14 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: Xenotransplantation of pig organs may provide an approach to alleviate the severe shortage of human organs. Natural antibodies against Galalpha(1,3)-Gal (alphaGal) epitopes cause hyperacute rejection of pig organs in primates. However, evidence for the role of alphaGal in the natural killer (NK) cell-mediated xenoresponse has been contradictory. We investigated the recognition of alphaGal by human NK cells using endo-beta-galactosidase C, an enzyme that cleaves alphaGal, and endothelial cells (EC) from alpha1,3-galactosyltransferase null pigs that do not synthesize alphaGal. Endo-beta-galactosidase C treatment variably reduced the susceptibility of porcine EC to lysis by fresh human NK cells. Removal of alphaGal from porcine EC using endo-beta-galactosidase C, produced variable results, i.e. cytotoxicity was decreased in half of the human NK cell donors tested. The two EC strains from alphaGal-/- pigs were marginally, and not significantly, less susceptible to lysis by naïve human NK cells compared with alphaGal-expressing cells obtained from animals from the same herd, but these differences were not statistically significant (P > 0.10). Treatment of porcine EC with recombinant human tumor necrosis factor (TNF)-alpha, which is known to activate porcine EC, enhanced the susceptibility of all target cells to lysis by fresh human NK cells. Surface expression of MHC or adhesion molecules on alphaGal-/- cells, compared with wild type cells, showed no consistent difference in either MHC or adhesion molecules CD106 (VCAM-1), CD31 (PECAM) or CD62E (E-selectin), either with or without TNF-alpha stimulation, that could explain the differential susceptibility to lysis. Strikingly, all alphaGal-/- and wild type EC exhibited similar susceptibility to human NK cells that had been cultured for 5 days with or without interleukin-2. These findings demonstrate that human NK cells can kill porcine targets in the absence of alphaGal, and donor variability plays a major role in whether alphaGal has a role in determining susceptibility of porcine EC to lysis. Moreover, susceptibility to lysis of alphaGal null EC is enhanced to the level of wild type EC by activation of either effector or target cells. Elimination of alphaGal alone from source pigs will be insufficient to circumvent the NK cell mediated destruction of porcine EC.
    Xenotransplantation 07/2006; 13(4):318-27. · 2.57 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: Carticel is an autologous cultured chondrocyte product that has been approved by the United States Food and Drug Administration for the repair of symptomatic cartilaginous defects of the femoral condyle that are caused by acute or repetitive trauma in patients who have been previously managed with arthroscopy or other surgical procedures. The present report describes the adverse events following Carticel implantation as reported to the Food and Drug Administration from 1996 to 2003. We reviewed adverse event reports that had been submitted to the Food and Drug Administration's MedWatch system for information on demographic characteristics, adverse events, and surgical revisions. Adverse events were categorized into sixteen non-mutually exclusive groups. Five categories were used to classify reoperations. Food and Drug Administration regulations require manufacturers to report adverse events; however, reporting by clinicians and others is voluntary. Therefore, adverse event reporting is likely to underestimate the number of event occurrences. Adverse events may be either causally or coincidentally related to the product. A total of 497 adverse events among 294 patients receiving Carticel were reported. The median interval from Carticel implantation to the diagnosis of an adverse event was 240 days (range, one to 2105 days). The median age of the patients was thirty-eight years, and 63% of the patients were male. Of the 270 events for which the anatomic site was noted, 258 (96%) involved the femoral condyles. More than one adverse event was reported for 135 patients (46%). The most commonly reported events were graft failure (seventy-three patients; 25%), delamination (sixty-five patients; 22%), and tissue hypertrophy (fifty-two patients; 18%). In addition, eighteen surgical site infections were reported, including eleven joint and seven soft-tissue infections. Surgical revision subsequent to Carticel implantation was mentioned in the records for 273 patients (93%). The reasons for the 389 revision procedures included graft-related problems (187 procedures; 48.1%), periarticular soft-tissue problems (ninety-seven procedures; 24.9%), and intra-articular problems (sixty-three procedures; 16.2%). Eight patients had a total knee replacement. Based on the manufacturer's reported distribution of 7500 Carticel lots between 1995 and 2002, 285 patients (3.8%) had an adverse event that was reported to the Food and Drug Administration. The most common adverse events reported in association with the Carticel technique involved graft failure, delamination, and tissue hypertrophy.
    The Journal of Bone and Joint Surgery 04/2006; 88(3):503-7. · 3.23 Impact Factor
  • Source
    Cynthia M Porter, Eda T Bloom
    [Show abstract] [Hide abstract]
    ABSTRACT: Due to the shortage of human organs, xenotransplantation is being explored as an alternative to allotransplantation, but immune rejection remains a major hurdle to its implementation. We tested the ability of human CD4+CD25+ T cells (Treg cells) to suppress CD4+ T cell-mediated anti-porcine xenoresponses usingin vitroassays. Human Treg cells were hyporesponsive to porcine cell stimulation and suppressed the proliferative response of CD4+CD25- T cells in a dose-dependent manner, and comparison of the allo- and xenoresponses indicated that more Treg cells might be required to suppress the xenogeneic response than the allogeneic response. Stimulation of CD4+CD25- T cells with porcine cells resulted in secretion of IFN-gamma, TNF-alpha, IL-10, IL-6 and IL-2, and Treg cells suppressed the secretion of these cytokines, as well as the CD4+CD25- T-cell cytolytic response against porcine cells. These results suggest a potential role for Treg cells in promoting xenograft survival.
    American Journal of Transplantation 09/2005; 5(8):2052-7. · 6.19 Impact Factor
  • Source
    Molecular Therapy 08/2005; 12(1):5-8. · 7.04 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Engagement of the Fas receptor (CD95) initiates multiple signaling pathways that lead to apoptosis, such as the formation of death-inducing signaling complex (DISC), activation of caspase cascades, and the generation of the lipid messenger, ceramide. Sphingomyelin (SM) is a major component of lipid rafts, which are specialized structures that enhance the efficiency of membrane receptor signaling and are a main source of ceramide. However, the functions of SM in Fas-mediated apoptosis have yet to be clearly defined, as the responsible genes have not been identified. After cloning a gene responsible for SM synthesis, SMS1, we established SM synthase-defective WR19L cells transfected with the human Fas gene (WR/Fas-SM(-)), and cells that have been functionally restored by transfection with SMS1 (WR/Fas-SMS1). We show that expression of membrane SM enhances Fas-mediated apoptosis through increasing DISC formation, activation of caspases, efficient translocation of Fas into lipid rafts, and subsequent Fas clustering. Furthermore, WR/Fas-SMS1 cells, but not WR/Fas-SM(-) cells, showed a considerable increase in ceramide generation within lipid rafts upon Fas stimulation. These data suggest that a membrane SM is important for Fas clustering through aggregation of lipid rafts, leading to Fas-mediated apoptosis.
    Journal of Experimental Medicine 08/2005; 202(2):249-59. · 13.21 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: Oxidative stress mediates positive and negative effects on physiological processes. Recent reports show that H(2)O(2) induces phosphorylation and activation of endothelial nitric oxide synthase (eNOS) through an Akt-phosphorylation-dependent pathway. In this study, we assessed activation of eNOS and Akt by determining their phosphorylation status. Whereas moderate levels of H(2)O(2) (100 microM) activated the Akt/eNOS pathway, higher levels (500 microM) did not, suggesting differential effects by differing levels of oxidative stress. We then found that two pro-oxidants with activity on sulfhydryl groups, 1-chloro-2,4-dinitrobenzene (CDNB) and diethyl maleate (DEM), blocked the phosphorylation events induced by 100 microM H(2)O(2). GSH was not a target thiol in this system because buthionine sulfoximine did not inhibit this phosphorylation. However, down-regulation of cell membrane surface and intracellular free thiols was associated with the inhibition of phosphorylation, suggesting that oxidation of non-GSH thiols inhibits the H(2)O(2)-induced phosphorylation of eNOS and Akt. DTT reversed the inhibitory effects of CDNB and DEM on Akt phosphorylation and concomitantly restored cell surface thiol levels more efficiently than it restored intracellular thiols, suggesting a more prominent role for the former. Similarly, DEM and CDNB inhibited TNF-alpha-induced Akt and eNOS phosphorylation, suggesting that thiol modification is involved in eNOS inductive pathways. Our findings suggest that eNOS activation is exquisitely sensitive to regulation by redox and that cell surface thiols, other than glutathione, regulate signal transduction leading to phosphorylation of Akt and eNOS.
    Free Radical Biology and Medicine 06/2005; 38(9):1231-42. · 5.27 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Fractalkine (now also called CX3CL1) is a unique chemokine that functions not only as a chemoattractant but also as an adhesion molecule and is expressed on endothelial cells activated by proinflammatory cytokines, such as interferon-gamma and tumor necrosis factor-alpha. The fractalkine receptor, CX3CR1, is expressed on cytotoxic effector lymphocytes, including natural killer (NK) cells and cytotoxic T lymphocytes, which contain high levels of intracellular perforin and granzyme B, and on macrophages. Soluble fractalkine causes migration of NK cells, cytotoxic T lymphocytes, and macrophages, whereas the membrane-bound form captures and enhances the subsequent migration of these cells in response to secondary stimulation with other chemokines. Furthermore, stimulation through membrane-bound fractalkine activates NK cells, leading to increased cytotoxicity and interferon-gamma production. Recently, accumulating evidence has shown that fractalkine is involved in the pathogenesis of various clinical disease states or processes, such as atherosclerosis, glomerulonephritis, cardiac allograft rejection, and rheumatoid arthritis. In addition, polymorphisms in CX3CR1, which reduce its binding activity to fractalkine, have been reported to increase the risk of HIV disease and to reduce the risk of coronary artery disease. This review will examine new concepts underlying fractalkine-mediated leukocyte migration and tissue damage, focusing primarily on the pathophysiological roles of fractalkine in various clinical conditions, especially in atherosclerosis and vascular injury.
    Arteriosclerosis Thrombosis and Vascular Biology 02/2004; 24(1):34-40. · 6.34 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: Pigs are under consideration as possible sources of organs for xenotransplantation in humans. The induction of hematopoietic microchimerism through xenotransplantation of source animal hematopoietic cells has been suggested as a means to induce tolerance in potential recipients. Because all porcine cells contain genetic information for porcine endogenous retrovirus (PERV), coculture techniques, reverse transcriptase (RT) and reverse transcriptase-polymerase chain reaction assays were used to determine whether infectious PERV is released from fresh porcine bone marrow cells cultured in the presence or absence of porcine cytokines. Human embryonic kidney cell line, HEK-293 cells cocultured with porcine bone marrow cells were positive for PERV RNA but never became positive for viral RT activity, suggesting the PERV infection was not productive. In contrast, high levels of RT activity was detected in porcine ST-IOWA cells after coculture, demonstrating that these cells became productively infected. PERV was released from cultured porcine bone marrow cells without stimulation, and combinations of the porcine hematopoietic cytokines, interleukin-3, granulocyte macrophage-colony stimulating factor and stem cell factor had no additional effect on the infectivity or in vitro tropism of released PERV virions.
    Xenotransplantation 08/2003; 10(4):337-42. · 2.57 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: Xenotransplantation, especially using porcine sources, has been proposed as a means to alleviate the shortage of human organs for transplantation. NK cells appear to be important mediators of the xenogeneic immune responses, including the human anti-pig response. Having previously established the redox regulation of NK cell activity against tumor target cells, we now report that the interaction of human NK cells with porcine target cells is also regulated by redox. Thiol-deprivation strongly diminished the capacity of IL-2-activated human NK cells to kill porcine endothelial cells. This inhibition correlated with reduced proliferation and interferon (IFN)-gamma production by IL-2-activated NK cells. For fresh NK cells, pretreatment with diethyl maleate (DEM), which was used to deplete intracellular thiols, reduced lysis of porcine and human targets. Because many adhesion molecules exhibit interspecies recognition, we further investigated whether changes in expression of adhesion molecules might explain our observations. DEM treatment reduced the expression of CD11b and CD29 on fresh NK cells. Monoclonal antibody blocking studies showed that the combination of mAb to CD11b and CD18 reduced lytic activity against both PAEC as well as K562, although other qualitative differences were observed between the porcine and human target cells. These findings suggest that the oxidative stress-induced downregulation of CD18 may be important in modulating cytotoxic activity of fresh NK cells against PAEC and K562 targets through reduced formation of the CD11b/CD18 heterodimer. Thus, the appropriate manipulation of redox status may provide a means to enhance survival of non-human animal tissues in humans through modulation of adhesion molecule expression/interactions.
    Cellular Immunology 04/2003; 222(1):35-44. · 1.74 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: Natural killer (NK) cells participate in both innate and adaptive immunity, in part by their prompt secretion of cytokines including IFN-gamma, a pro-inflammatory cytokine with an important role in Th1 polarization. To assess the involvement of fractalkine in inflammatory processes, we examined the effect of fractalkine on IFN-gamma production by NK cells. Although soluble chemokines, including MCP-1 and RANTES as well as fractalkine, had a negligible effect on IFN-gamma production, immobilized fractalkine markedly induced IFN-gamma production by NK cells in a dose-dependent manner. Pretreatment of NK cells with the phosphatidylinositol 3-kinase (PI 3-K) inhibitor, wortmannin, completely inhibited the production of IFN-gamma induced by fractalkine, and pretreatment with the protein tyrosine kinase inhibitor, herbimycin A, partially suppressed the response, suggesting that augmentation of IFN-gamma production in response to fractalkine treatment of NK cells involves signaling through PI 3-K and protein tyrosine kinases. Furthermore, co-culture of NK cells with fractalkine-transfected 293E cells markedly enhanced IFN-gamma production by NK cells compared with co-culture with control 293E cells. These findings may indicate a paracrine feedback loop system in which endothelial cells may be activated to produce more fractalkine, and also suggest a role for fractalkine expressed on endothelial cells in Th1 polarization through the stimulation of IFN-gamma production by NK cells.
    European Journal of Immunology 02/2003; 33(1):53-8. · 4.97 Impact Factor
  • Shigeru Tsuyuki, Mari Kono, Eda T Bloom
    [Show abstract] [Hide abstract]
    ABSTRACT: Endothelial cells (EC) are primary targets of the recipient's immune response to transplanted organs and constitutively express Fas (CD95) ligand (FasL) on their surface. We investigated the role of porcine FasL in the generation of the human anti-pig response in vitro. Porcine aortic endothelial cells (PAEC) lysed a Fas+ human T-cell line, Jurkat. Anti-human Fas monoclonal antibody (mAb) specifically inhibited this killing in a dose-dependent manner, suggesting that porcine FasL recognizes and binds human Fas to induce apoptosis of human Fas+ cells. We next cloned porcine FasL, identifying an open reading frame of 849 base pairs predicting a protein of 282 amino acids. The predicted amino acid sequence was 85, 76, and 75% homologous to the predicted amino acid sequences of human, mouse, and rat, respectively, and found that PAEC expressed both FasL mRNA and protein. Transient transfection was used to increase or induce porcine FasL expression in PAEC or COS-7 cells. Transfection of PAEC with a plasmid encoding porcine FasL increased their ability to induce apoptosis in Jurkat cells, fresh human T cells activated with IL-2 and anti-CD3, and fresh IL-2-activated human (natural killer) NK cells. Moreover, porcine Fas L-transfected COS-7 cells induced significant apoptosis in Jurkat cells compared with that induced by mock-transfected COS-7 cells. Finally, the overexpression of porcine FasL in PAEC reduced their susceptibility as target cells to lysis by activated human NK or T cells. These findings suggest that porcine FasL overexpression in EC of vascularized xenografts may provide protection from cellular xenograft rejection.
    Xenotransplantation 12/2002; 9(6):410-21. · 2.57 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: Natural killer (NK) cells participate in both innate and adaptive immunity through the prompt secretion of cytokines and ability to lyse virally infected cells or tumor cells. Although it has been well understood that lipid rafts (rafts) and a raft-associated linker for activation of T cells (LAT) plays a central role in TCR signal transduction, there are still great gaps in our knowledge of the molecular events involved in NK cell activation. We show here that CD2 and rafts became polarized to the site of NK cell activation by CD2 cross-linking or target cell binding using confocal microscopy, and LAT and a significant amount of CD2 colocalized in raft fractions of sucrose-density gradient from an NK cell line, NK3.3. CD2 cross-linking strongly induced tyrosine phosphorylation of LAT, resulting in increased association with phosphatidylinositol 3-kinase (PI 3-K) and phospholipase C-gamma1 (PLC-gamma1). In vitro binding studies using glutathione S-transferase fusion proteins demonstrated that a large portion of the association between LAT and PI 3-K or PLC-gamma1 was mediated through their SH2 domains in tyrosine phosphorylation-dependent manner. Furthermore, disruption of lipid rafts by cholesterol depletion from cell membranes using methyl-beta-cyclodextrin markedly reduced LAT tyrosine phosphorylation and NK cell functions, including cytotoxicity and granule exocytosis. These results document that modulation of raft integrity by aggregation of NK cell activating receptors, which leads to the formation of complexes of LAT with PI 3-K and PLC-gamma1, is essential for the NK cell lytic mechanisms.
    European Journal of Immunology 09/2002; 32(8):2188-98. · 4.97 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: Natural killer (NK) cells participate in both innate and adaptive immunity through the prompt secretion of cytokines and ability to lyse virally infected cells or tumor cells. Although it has been well understood that lipid rafts (rafts) and a raft-associated linker for activation of T cells (LAT) plays a central role in TCR signal transduction, there are still great gaps in our knowledge of the molecular events involved in NK cell activation. We show here that CD2 and rafts became polarized to the site of NK cell activation by CD2 cross-linking or target cell binding using confocal microscopy, and LAT and a significant amount of CD2 colocalized in raft fractions of sucrose-density gradient from an NK cell line, NK3.3. CD2 cross-linking strongly induced tyrosine phosphorylation of LAT, resulting in increased association with phosphatidylinositol 3-kinase (PI 3-K) and phospholipase C-γ1 (PLC-γ1). In vitro binding studies using glutathione S-transferase fusion proteins demonstrated that a large portion of the association between LAT and PI 3-K or PLC-γ1 was mediated through their SH2 domains in tyrosine phosphorylation-dependent manner. Furthermore, disruption of lipid rafts by cholesterol depletion from cell membranes using methyl-β-cyclodextrin markedly reduced LAT tyrosine phosphorylation and NK cell functions, including cytotoxicity and granule exocytosis. These results document that modulation of raft integrity by aggregation of NK cell activating receptors, which leads to the formation of complexes of LAT with PI 3-K and PLC-γ1, is essential for the NK cell lytic mechanisms.
    European Journal of Immunology 07/2002; 32(8):2188 - 2198. · 4.97 Impact Factor

Publication Stats

3k Citations
364.17 Total Impact Points

Institutions

  • 2008
    • Kanazawa Medical University
      • Department of Hematology and Immunology
      Kanazawa-shi, Ishikawa-ken, Japan
  • 1990–2007
    • U.S. Food and Drug Administration
      • • Center for Biologics Evaluation and Research
      • • Division of Cellular & Gene Therapies
      • • Laboratory of Cellular Hematology
      Washington, Washington, D.C., United States
  • 2005
    • Kyoto University
      • Graduate School of Medicine / Faculty of Medicine
      Kyoto, Kyoto-fu, Japan
  • 1997–2003
    • Osaka Dental University
      • Department of Internal Medicine
      Ōsaka, Ōsaka, Japan
  • 2001
    • KAN Research Institute
      Kōbe, Hyōgo, Japan
  • 1992
    • Juntendo University
      • Department of Immunology
      Edo, Tōkyō, Japan
  • 1985
    • University of California, Los Angeles
      Los Angeles, California, United States