Skip Navigation


International Immunology Advance Access originally published online on December 16, 2005
International Immunology 2006 18(1):113-124; doi:10.1093/intimm/dxh353
This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow All Versions of this Article:
18/1/113    most recent
dxh353v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrow Search for citing articles in:
ISI Web of Science (3)
Right arrowRequest Permissions
Google Scholar
Right arrow Articles by Nakayama, J.
Right arrow Articles by Ohtsuki, M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Nakayama, J.
Right arrow Articles by Ohtsuki, M.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?


© The Japanese Society for Immunology. 2005. All rights reserved. For permissions, please e-mail: journals.permissions@oxfordjournals.org

A humanized anti-human Fas antibody, R-125224, induces apoptosis in type I activated lymphocytes but not in type II cells

Junichi Nakayama1, Yukie Ogawa1, Yasushi Yoshigae2, Yoshiko Onozawa1, Akiko Yonemura1, Motoko Saito2, Kimihisa Ichikawa3, Takashi Yamoto4, Tomoaki Komai1, Toru Tatsuta1 and Masahiko Ohtsuki5

1 Biological Research Laboratories, 2 Drug Metabolism and Pharmacokinetics Research Laboratories, and 3 Core Technology Research Laboratories, Sankyo Co., Ltd, Tokyo 140-8710, Japan
4 Medicinal Safety Research Laboratories, Sankyo Co., Ltd, Fukuroi 437-0065, Japan
5 Global Project Management, Sankyo Pharma Development, Sankyo Pharma Inc., 399 Thornall Street, Edison, NJ 08837, USA

Correspondence to: M. Ohtsuki; E-mail: mohtsuki{at}sankyopharma.com


   Abstract
Top
 Abstract
Introduction
 Methods
 Results
 Discussion
 References
 
Fas-mediated apoptosis plays an important role in the immune system, including the elimination of autoreactive lymphoid cells. The Fas-mediated signaling pathway is classified into two types, type I and type II, in human lymphoid cell lines. We investigated whether a humanized anti-human Fas mAb, R-125224, has cell selectivity in induction of apoptosis. R-125224 induced apoptosis in H9 cells, SKW6.4 cells and activated human lymphocytes when cross-linked with anti-human IgG. On the other hand, R-125224 did not induce apoptosis in HPB-ALL cells, Jurkat cells or human hepatocytes. By analysis of death-inducing signaling complex formation, it was demonstrated that R-125224 induced apoptosis selectively in type I cells but not in type II cells. Type I cells also expressed more Fas and had more Fas-clustering activity than type II cells. Moreover, co-localization of these clusters and GM1, which is an sphingoglycolipid associated with lipid rafts, was detected. It was also shown that R-125224 treatment could reduce the number of activated human CD3+Fas+ cells in a SCID mouse model in vivo. Thus, we demonstrated that R-125224 induces apoptosis specifically in type I cells in vitro and in vivo.

Keywords: activated human lymphocytes, anti-autoimmune disease therapy, DISC, lipid rafts


   Introduction
Top
 Abstract
 Introduction
Methods
 Results
 Discussion
 References
 
Ligation of Fas (CD95) with agonistic antibodies or Fas ligand (FasL) induces apoptosis in several types of cells (1–3). Fas-mediated apoptosis plays an important role in the immune system, including the elimination of autoreactive lymphoid cells (4, 5). In mice, lymphoproliferation disorder is caused by a mutation of the Fas gene (6), and impaired Fas signaling is partly responsible for defective T cell apoptosis in autoimmune disease models (7, 8). Moreover, mutations of the human Fas gene led to impaired apoptosis observed in human autoimmune lymphoproliferative syndrome (9).

Among members of the death receptor family, such as Fas, tumor necrosis factor (TNF) receptor-1 and TNF-related apoptosis-inducing ligand receptor-1 and -2, the Fas-mediated apoptosis-signaling pathway has been well studied (10). Upon binding of FasL to Fas on the cell surface, Fas is oligomerized, and Fas-associated death domain (FADD) and caspase-8 are recruited to form a death-inducing signaling complex (DISC) (11). Death signals are then transduced through the caspase and/or mitochondrial pathways. Recently, human lymphoid cell lines and tumor cell lines were classified into two types, type I and type II cells, by the modes of Fas-mediated apoptosis-signaling pathway (12, 13). For type I cells, caspase-8 is recruited to form DISC, resulting in a release of activated caspase-8, which can activate caspase-3. In type II cells, not enough DISC is found. The efficiency of DISC formation is very important for classification into the two cell types. As apoptosis in type II cells is blocked by over-expression of Bcl-2, the Fas-mediated apoptosis-signaling pathway in type II cells is considered to occur mainly through the mitochondrial amplification loop (12). On the other hand, it has been reported that Bcl-2 did not protect mouse lymphocytes and hepatocytes from Fas-mediated apoptosis in vivo (14) and Bid (a protein of the Bcl-2 family)-deficient mice were resistant to Fas-mediated hepatocellular apoptosis in vivo (15), indicating that blocking effects of Bcl-2 to Fas-mediated apoptosis in vivo may vary according to the cell type and conditions. In this regard, the efficiency of DISC formation is important for classification of type I and type II cells. However, the precise characterization of the two types in apoptosis induction and application in human primary cells has not been fully elucidated.

Apoptosis-inducing therapy using anti-Fas antibody was shown to be a powerful strategy in mouse autoimmune disease models (7, 16). However, administration of anti-mouse Fas antibody Jo-2 caused fulminant hepatitis in mice, indicating that certain anti-Fas antibodies may cause adverse effects in the liver (17). Therefore, it is necessary for therapeutic anti-Fas antibodies to induce apoptosis selectively in pathogenic cells including autoreactive lymphoid cells. We obtained a novel anti-human Fas antibody, m-HFE7A (18), and succeeded in humanizing m-HFE7A to create R-125224 (h-HFE7A) by Complementarity Determining Region (CDR) grafting (19). R-125224, which induced apoptosis in not only human Fas-expressing cells but also synovial lymphocytes from rheumatoid arthritis (RA) patients when cross-linked, had a therapeutic effect in an in vivo RA model (20), and suppressed osteoclastogenesis in RA synovial tissues (21). In the present study, we report that R-125224 showed apoptosis-inducing activity in type I cells including activated lymphocytes, but not in type II cells including hepatocytes both in vitro and in vivo.


   Methods
Top
 Abstract
 Introduction
 Methods
Results
 Discussion
 References
 
Anti-human Fas antibodies and soluble FasL
R-125224, a humanized anti-human Fas mAb, was produced from NS0 cells harboring R-125224 cDNA and purified by column chromatography (19). Anti-human Fas antibodies, CH-11 (IgM type) and APO-1-3 (IgG3 type), were purchased from MBL (Nagoya, Japan) and Alexis, respectively. Soluble Fas ligand (sFasL) was purchased from Kamiya Biomedical Company. This recombinant protein contains the extracellular domain of human FasL fused to a linker peptide and a FLAG tag at the N-terminus. Anti-human IgG (anti-hIgG), protein A and anti-FLAG M2 antibody (anti-FLAG) were purchased from Biosource, Wako Pure Chemical and Stratagene, respectively.

Cell cultures
H9 (human T cell line, HTB-176), SKW6.4 (human B cell line, TIB-215) and Jurkat (human T cell line, CRL-2570) cells were purchased from American Type Culture Collection. HPB-ALL (human T cell line) and WR19L12a (murine cell line into which human Fas cDNA has been introduced) cells were kindly provided by S. Yonehara (Kyoto University). All cell lines were maintained in RPMI 1640 medium containing 10% fetal bovine serum (FBS), penicillin (50 units ml–1)–streptomycin (50 µg ml–1) and 55 µM of ß-mercaptoethanol (R10F) at 37°C.

Human PBMCs from healthy volunteers were isolated by layering on a Ficoll–Paque density gradient followed by centrifugation at 18°C for 30 min. PBMCs were diluted to 2.0 x 106 cells ml–1 in R10F, and incubated in tissue culture flasks for 1 h at 37°C. Non-adherent cells were collected and incubated in R10F with 20 ng ml–1 anti-human CD3 antibody (BD PharMingen) at 37°C for 3 days. After medium was changed, the cells were incubated in R10F with 10 ng ml–1 human IL-2 (Genzyme) at 37°C for another 4 days to obtain activated human lymphocytes. Non-adherent cells obtained above were used as non-activated lymphocytes.

Human hepatocytes were purchased from Tissue Transformation Technologies. Frozen human hepatocytes were thawed quickly at 37°C. After centrifuging the hepatocyte suspension at 4°C for 3 min, the supernatant was aspirated and the precipitated hepatocytes were re-suspended in ice-cold complete nutrient culture medium. These hepatocytes were incubated in a flask coated with collagen (CL) type I (Asahi Techno Glass, Funabashi, Japan), and maintained in complete nutrient culture medium at 37°C. The use of human lymphocytes and hepatocytes was approved by the Sankyo Ethical Committee.

Preparation of activated cynomologus monkey lymphocytes was as follows. Each sample of peripheral blood was treated with 6% dextran and the mixture was incubated at room temperature for 80 min, purified and activated using the same procedure as that for the preparation of the activated human lymphocytes.

Cell viability measurement
The human lymphoid cell lines (5.0 x 104 cells per well) and activated human lymphocytes and non-activated human lymphocytes (2.0 x 105 cells per well) were treated with sFasL and anti-FLAG or R-125224 on a 96-well plate at 37°C for 20 h. For the R-125224 treatment group, wells were pre-coated with anti-hIgG. Human hepatocytes (7.5 x 104 cells per well) were incubated on a 96-well plate coated with CL type I at 37°C for 4 h to adhere the cells to the bottom of the wells. For the sFasL treatment groups, the cells were further incubated with sFasL and anti-FLAG. For the R-125224 treatment groups, R-125224 was added and the adherent cells were incubated at 37°C for 1 h. After removing the media by very gentle aspiration, R10F or anti-hIgG in R10F was added and the cells were further incubated at 37°C for 20 h. Cell viability was determined by the XTT method (20, 22). Data are expressed as the mean and SD of triplicate determinations. The statistical analysis of the cell viabilities of treatment groups compared with those of control groups was performed by the Student's t-test. A P value of <0.01 was considered to be statistically significant.

Western blotting analysis and DISC formation
Cells were lysed in lysis buffer [20 mM HEPES, pH 7.4, 150 mM NaCl, 5 mM EDTA, 10% glycerol, 0.5% Triton X-100, 1 µg ml–1 Pefabloc SC (Roche), 2 µg ml–1 aprotinin, 2 µg ml–1 leupeptin and 2 µg ml–1 pepstatin A]. Protein concentrations of cell lysates were determined using a BCA protein assay reagent kit (Pierce). Twenty micrograms of protein was separated by SDS-PAGE. These membranes were incubated with each primary antibody and HRP-conjugated secondary antibody, and specific bands were detected using ECL kit (Amersham Bioscience). Anti-human Fas (clone: C-20; Santa Cruz) and anti-human Bid (Santa Cruz), anti-human FADD and anti-human caspase-3 (BD PharMingen) and anti-human caspase-8 (MBL) were purchased.

For the detection of DISC, H9 and HPB-ALL cells (2.0 x 107), activated human lymphocytes (5.0 x 107 cells) and human hepatocytes (1.0 x 107 cells) were treated with 100 ng ml–1 of sFasL with anti-FLAG for 30 min at 37°C or incubated for 30 min at 37°C (control samples). Cell lysates from each sample were incubated with R-125224 for 1 h, and were further incubated with protein-G-coupled sepharose beads (Amersham Bioscience) for 2 h at 4°C and washed four times with the lysis buffer (23). The samples of the immunoprecipitates or total cell lysates were separated by SDS-PAGE and subjected to western blotting analysis.

Mitochondrial membrane potential
Variations in the mitochondrial transmembrane potential ({Delta}{Psi}m) during apoptosis were studied using 3,3'-dihexyloxacarbocyanine iodide (DiOC6; Molecular Probes). This cyanin dye accumulates in the mitochondrial matrix under the influence of {Delta}{Psi}m. H9 and HPB-ALL cells (1.0 x 106) were treated with 1000 ng ml–1 R-125224 in an anti-hIgG-coated 12-well dish for 2–4 h at 37°C or were incubated at 37°C in a non-coated dish (control). These cells were further treated with DiOC6 for 15 min and analysis was performed using a flow cytometer (FACSCaliburTM).

Detection of Fas expression
To determine the expression of Fas, 5 x 105 cells were incubated with FITC anti-human Fas (clone: DX-2; BD PharMingen) at 4°C for 30 min. FITC mouse IgG1 was used as a negative control. After washing the cells twice with D-PBS containing 3% FBS and 0.1% sodium azide (FACS buffer), the cells were suspended in FACS buffer containing propidium iodide (PI). Analysis was performed using a flow cytometer. Data in the PI-negative fraction were obtained and the mean fluorescent intensity was calculated.

[125I]-R-125224 was prepared using [125I]p-iodobenzoate as a labeling agent (24). Tested cells (final: 1.5 x 106 cells ml–1) were incubated with various concentrations of [125I]-labeled R-125224 and 0.2% BSA (v/v) on ice for 1 h. After incubation, the cell mixture was transferred into tubes containing mixed oils, and was centrifuged to separate the supernatants and cell pellets. The supernatants and pellets were separately collected and subjected to radioactivity measurement with a {gamma}-counter using a RIASTAR (Packard). The experiments using human lymphoid cell lines were conducted in duplicate for three independent experiments. Those using activated human lymphocytes and hepatocytes were conducted from each of three individuals. The affinity (Kd) and maximum binding (Bmax) were calculated using Scatchard plot analysis.

Detection of Fas clustering
H9 and HPB-ALL cells (5.0 x 105) and non-activated or activated human lymphocytes (1.0 x 106 cells) were treated with Cell TrackerTM Orange CMTMR (CMTMR; Molecular Probes) for 30 min at 37°C and washed twice (25). The cells were treated with R-125224 for 1 h on ice and washed twice. The cell pellet was treated with FITC anti-hIgG (Biosource) for 1 h on ice. The cells were incubated at 37°C for the indicated times (stimulated group) or kept on ice (control group) and washed twice. The cell pellet was fixed for 20 min on ice and washed once. Drops of these cell solutions were placed onto slides coated with poly-L-lysine and these slides were viewed using an OLYMPUS confocal laser scanning microscope, FV500, with x20 objective lens. Fas clustering was judged as positive when more than two concentrated fluorescent yellow signals or when one concentrated signal that was larger than one-eighth of the cell surface of a single cell was observed. Total cell numbers (CMTMR-positive cells) and cells with Fas clustering were counted in a blinded manner. Data are expressed as the mean and SD. The statistical analysis of Fas-clustering cells of the treatment groups compared with those of the control groups and of the treatment groups in H9 cells compared with those of HPB-ALL cells was performed by the Student's t-test. A P value of <0.01 was considered to be statistically significant. To study Fas clusters formed onto lipid rafts, H9 cells were treated with R-125224 and cholera toxin B subunit conjugated to rhodamine (List Biological Laboratories) for 30 min on ice and washed. The cell pellet was treated with FITC anti-hIgG for 30 min on ice and further incubated at 37°C for 15 min.

Studies of human PBMCs reconstituted in mice
Male C·B-17/Icr Crj-scid bgBR (SCID-bg) mice were purchased from Charles River Japan, Inc., and used at the age of 7 weeks. For detection of Fas and CD69 on human PBMCs in a SCID-Hu PBMC model, 2 days before human PBMC transfer, SCID-bg mice were irradiated. Human PBMCs (5.0 x 107) were transferred to mice on day 0, then the PBMCs themselves were analyzed for expression of human CD3, Fas and CD69. On day 14 after PBMC transfer, cells from mouse spleen were analyzed.

Human PBMCs prepared from seven volunteers were transferred to mice intravenously from the tail vein, and then the mice were allocated randomly to each dosing group (control; D-PBS, R-125224: 0.008, 0.04 and 0.2 mg kg–1) while avoiding allocating the mice to which the same PBMC had been transferred to the same dosing group. One day after the human PBMC transfer, each dose of R-125224 or D-PBS was administered intravenously. The number of mice in each group was as follows: control, five; R-125224 0.008 mg kg–1, five; R-125224 0.04 mg kg–1, five, and R-125224 0.2 mg kg–1, four. On day 5 after human PBMC transfer, the mice were sacrificed by exsanguination, and the spleens were obtained and minced. After bursting the RBCs, single-cell suspensions were washed twice and counted. Cells were incubated with purified anti-mouse CD16/CD32 to block mouse FcRs at 4°C for 10 min, and stained with FITC anti-human Fas and PE anti-human CD3 at 4°C for 30 min. Analysis was performed using a flow cytometer, and data are expressed as the percentage of the control and SE.

For the statistical analysis of the number of human CD3+Fas+ cells in the spleens of SCID-bg mice, the linear mixed model was applied with ‘dose’ as a fixed effect and ‘human’ as a random effect. Dose denotes the variable for the dose level of R-125224 (control, 0.008, 0.04, 0.2 mg kg–1) and human denotes the variable for the blood of volunteers used in this experiment. The means of the number of human CD3+Fas+ cells of each dosing group were calculated. Multiple adjustment was by the non-parametric Dunnett test. A P value of <0.05 was considered to be statistically significant.

Studies of cynomologus monkeys
Male cynomologus monkeys (n = 6) were used at the age of 21–27 months. R-125224 at 6 mg kg–1 was administered intravenously to each monkey (day 0). Blood sampling was done on day –3 (control) and day 15. Aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels detected in the serum samples were analyzed with a Hitachi 917 clinical chemistry analyzer. Data are expressed as the mean and SD. The statistical analysis of AST and ALT levels in the serum samples was performed by the Student's t-test. A P value of <0.05 was considered to be statistically significant.


   Results
Top
 Abstract
 Introduction
 Methods
 Results
Discussion
 References
 
Cell selectivity of apoptosis induction by R-125224 treatment but not sFasL or other anti-human Fas antibodies in human lymphoid cell lines
R-125224 induced apoptosis in human Fas-expressing cells, WR19L12a (XTT method) (20), detected by the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate–biotin nick end-labeling and Annexin V/PI methods (26) by flow cytometer (data not shown). To characterize Fas-mediated apoptosis in human lymphoid cell lines, we used four human lymphoid cell lines (H9, SKW6.4, HPB-ALL and Jurkat cells). R-125224 induced apoptosis in H9 and SKW6.4 cells at 1000 ng ml–1 when cross-linked with anti-hIgG (Fig. 1A). On the other hand, R-125224 induced almost no apoptosis when cross-linked with anti-hIgG in HPB-ALL or Jurkat cells. In separate experiments, we tested up to 10 µg ml–1 of R-125224 with no apparent apoptosis (data not shown). These cell lines are sensitive to Fas-mediated apoptosis because sFasL at 100 ng ml–1 cross-linked with anti-FLAG induced apoptosis in allthe four lymphoid cell lines (Fig. 1B). We also examined the apoptosis sensitivity to other anti-human Fas antibodies, CH-11 and APO-1-3 (1, 2). In all four lymphoid cell lines, both CH-11 alone and APO-1-3 cross-linked with protein A induced apoptosis (Fig. 1C). These results suggest that R-125224 is a novel anti-human Fas antibody with the cell selectivity different from CH-11 and APO-1-3 in human lymphoid cell lines. In apoptosis induction by R-125224, different cross-linkers such as anti-hIgG and protein A did not change the cell selectivity profile (data not shown).



View larger version (41K):
[in this window]
[in a new window]
  		Fig. 1. Apoptosis induction by R-125224 treatment in human lymphoid cell lines. H9, SKW6.4, HPB-ALL and Jurkat cells were incubated with R-125224 or R-125224 cross-linked with anti-hIgG (A), or incubated with 100 ng ml–1 of sFasL cross-linked with anti-FLAG (B). (A) Cells were incubated with 10, 30, 100, 300 or 1000 ng ml–1 of R-125224 (filled circle) or R-125224 cross-linked with anti-hIgG (filled triangle). (C) Cells were incubated with 1, 10, 100 or 1000 ng ml–1 of CH-11 (filled circle) or APO-1-3 cross-linked with protein A (filled triangle). The viability of each cell line was determined by the XTT method. Data are expressed as the mean and SD of triplicate determinations. The statistical analysis was performed by the Student's t-test. A P value of <0.01 was considered to be statistically significant (*).

 
Apoptosis induction by R-125224 treatment in human primary cells
We examined whether R-125224 induces apoptosis in human primary cultured cells, activated human lymphocytes, non-activated lymphocytes and human hepatocytes. First, we examined the sensitivity of Fas-mediated apoptosis in human lymphocytes activated with anti-human CD3 antibody and IL-2. sFasL reduced the viability of activated lymphocytes when cross-linked with anti-FLAG, indicating that these cells were sensitive to Fas-mediated apoptosis under these conditions (Fig. 2A). R-125224 reduced the viability of activated lymphocytes at 1000 ng ml–1 when cross-linked, but R-125224 did not reduce the viability when not cross-linked (Fig. 2A). Neither sFasL cross-linked with anti-FLAG nor R-125224 cross-linked with anti-hIgG reduced the cell viability of non-activated human lymphocytes (Fig. 2B). So activation of lymphocytes converts to sFasL-sensitive and R-125224-sensitive status. Next, we examined the sensitivity to R-125224-induced apoptosis of human primary hepatocytes. Reduction in cell viability of hepatocytes was observed after the treatment of sFasL with anti-FLAG, indicating that these cells were sensitive to Fas-mediated apoptosis (Fig. 2C). On the other hand, R-125224 at 1000 ng ml–1 with or without anti-hIgG did not reduce the cell viability of hepatocytes (Fig. 2C). In separate experiments, we tested apoptosis induction in non-activated lymphocytes and hepatocytes up to 10 µg ml–1 of R-125224 with no apparent apoptosis (data not shown). These results indicate that activated lymphocytes are sensitive to sFasL-induced and R-12522-induced apoptosis, whereas human hepatocytes are sensitive to sFasL-induced apoptosis but insensitive to R-125224-induced apoptosis.



View larger version (37K):
[in this window]
[in a new window]
  		Fig. 2. Apoptosis induction by R-125224 treatment in activated human lymphocytes and hepatocytes. Activated human lymphocytes (A) and non-activated human lymphocytes (B) were incubated with 100 ng ml–1 of sFasL cross-linked with anti-FLAG (left panels), and incubated with R-125224 or R-125224 cross-linked with anti-hIgG (right panels). The cells were incubated with R-125224 (filled square) or R-125224 cross-linked with anti-hIgG (filled circle). (C) Human hepatocytes were incubated with 100 ng ml–1 of sFasL cross-linked with anti-FLAG (left panels), and incubated with R-125224 or R-125224 cross-linked with anti-hIgG (right panels). The viability of each cell was determined by the XTT method. Data are expressed as the mean and SD of triplicate determinations. The statistical analysis was performed by the Student's t-test. A P value of <0.01 was considered to be statistically significant (*).

 
DISC analysis in various human cells
We examined the formation of DISC in H9 and HPB-ALL cells during the treatment with sFasL. Recruitment of FADD and caspase-8 to the Fas molecule was detected in sFasL-treated H9 cells, but not in the non-treated control (Fig. 3A and B). On the other hand, recruitment of FADD and caspase-8 to Fas was barely observed in HPB-ALL cells (Fig. 3A and B). These results indicate that H9 cells are type I cells and that HPB-ALL cells are type II cells according to classification based on the efficiency of DISC formation (12). Next, we examined the formation of DISC in activated human lymphocytes and human hepatocytes. In each kind of primary cell, Fas was almost equally detected in the control cells or sFasL-stimulated cells (Fig. 3C). Caspase-8 was detected in the DISC of the activated human lymphocytes (Fig. 3C). On the other hand, little binding of caspase-8 to activated Fas was detected in any sample of human hepatocytes under the present conditions (Fig. 3C). Moreover, proteolytic cleavage forms of caspase-8 (p43 and p41), which is formed after Fas stimulation, were included in the DISC components of activated human lymphocytes as well as H9 cells. These results indicate that activated human lymphocytes are type I cells and that human hepatocytes are type II cells.



View larger version (39K):
[in this window]
[in a new window]
  		Fig. 3. Analysis of DISC formation. H9 (left panels) and HPB-ALL (right panels) cells were treated with sFasL and anti-FLAG for 30 min at 37°C (right part) or incubated for 30 min at 37°C (control; left part). The bands were detected using anti-FADD (A) and anti-caspase-8 (B) antibodies. IgG L represents the IgG light chain, and p41 and p43 represent the proteolytic cleavage forms of caspase-8. (C) Activated human lymphocytes (5.0 x 107 cells) and hepatocytes (1.0 x 107 cells) were treated with sFasL and anti-FLAG for 30 min at 37°C (right part) or incubated for 30 min at 37°C (control; left part). The thick arrows indicate Fas (upper panel) or caspase-8 and proteolytic cleavage forms of caspase-8 (lower panel).

 
Effect of Fas-mediated apoptosis-signaling pathways by R-125224 treatment on type I and type II cells
To study the mechanism for the cell selectivity in apoptosis induction by R-125224, we examined the cleavage of caspase-8, caspase-3 and Bid that is the main mediator in apoptosis signal transduction between the caspase and mitochondrial pathways (27, 28) in H9 and HPB-ALL cells. Decreases in caspase-8, caspase-3 and Bid and appearance of activated forms of caspase-3 were detected in H9 cells but not HPB-ALL cells by R-125224 treatment from 2 to 4 h (Fig. 4A). Decreases in caspase-8 and Bid were detected in HPB-ALL cells by sFasL and anti-FLAG treatments, thus Fas-mediated apoptosis-signaling pathways worked normally in HPB-ALL cells (Fig. 4B). We also detected a reduction in {Delta}{Psi}m in H9 cells by R-125224 cross-linked with anti-hIgG (Fig. 4C). On the other hand, in HPB-ALL cells, almost no reduction in {Delta}{Psi}m was observed (Fig. 4C). These results indicate that a difference in the R-125224-induced signaling pathway between H9 and HPB-ALL cells exists upstream of caspase-8 activation.



View larger version (20K):
[in this window]
[in a new window]
  		Fig. 4. Effect of Fas-mediated apoptosis-signaling pathways by R-125224 treatment on type I and type II cells. (A) H9 and HPB-ALL cells were treated with R-125224 in an anti-hIgG-coated flask for 2 or 4 h at 37°C, or placed on ice in a non-coated flask (0 h). The thick arrows indicate caspase-8, caspase-3 or Bid. The thin arrows indicate the proteolytic cleavage forms of caspase-8 or active form of caspase-3. (B) HPB-ALL cells were treated with sFasL and anti-FLAG for 2 or 4 h at 37°C, or placed on ice (0 h). (C) H9 and HPB-ALL cells were treated with R-125224 in an anti-hIgG-coated dish for 2 h (dotted lines) or 4 h (thin lines) at 37°C or incubated at 37°C in a non-coated dish (control, thick lines) and further treated with DiOC6 for 15 min.

 
Fas expression in various human cells
We examined the Fas expression by flow cytometry using FITC-labeled anti-human Fas in four human lymphoid cell lines. H9 and SKW6.4 cells expressed more Fas than HPB-ALL and Jurkat cells (Fig. 5). Fas expression levels were a little higher in activated lymphocytes than in non-activated lymphocytes and it was not possible to detect Fas expression in human hepatocytes as these cells had a high background (data not shown). For quantitative measurement of Fas expression, we examined the binding of [125I]-labeled R-125224 to Fas. The binding was specifically inhibited by non-labeled R-125224 but not by control IgG, and [125I]-labeled R-125224 bound to the four human lymphoid cell lines with high affinity (data not shown). The Bmax values to H9 and SKW6.4 cells were larger than those of HPB-ALL and Jurkat cells (Fig. 5) and consistent with flow cytometry results. Thus, it was shown that H9 and SKW6.4 cells (type I cells) expressed more Fas than HPB-ALL and Jurkat cells (type II cells). These results also indicate that the Fas expression levels correlate with R-125224-mediated apoptosis selectivity in the four human lymphoid cell lines.



View larger version (13K):
[in this window]
[in a new window]
  		Fig. 5. Fas expression in human lymphoid cell lines. Each cell line was stained with FITC anti-human Fas (thick lines) or FITC mouse IgG1 (control, thin lines) and analyzed with a flow cytometer. Data in the PI-negative fraction were analyzed and mean fluorescent intensity (MFI) was calculated. Various concentrations of [125I]-labeled R-125224 were incubated with each cell line, and the mixtures were separated into cell-bound and -free fractions. The radioactivity in each fraction was measured, and the Bmax was calculated. The MFI and Bmax are indicated under each quadrant.

 
We further examined the binding of [125I]-labeled R-125224 to Fas in activated human lymphocytes and hepatocytes. [125I]-Labeled R-125224 bound to activated human lymphocytes and human hepatocytes with high affinity, and the Bmax of sites per human hepatocyte was similar to that per activated human lymphocyte (Table 1). In general, the diameter of hepatocytes is ~20–30 µm and that of lymphocytes is ~10 µm, and the hepatocytes, we used in our experiments, were clearly larger than the activated lymphocytes under an optical microscope (data not shown). These results indicate that the density of Fas expression over a cell-surface area in activated lymphocytes is higher than that in hepatocytes.


View this table:
[in this window]
[in a new window]
  		Table 1. Binding of [125I]-labeled R-125224 to Fas in activated human lymphocytes and hepatocytes

 
Fas-clustering activity in human lymphoid cell lines and lymphocytes
Fas has been reported to form clusters at the cell surface in a ligand-dependent fashion in type I cells (29). To examine the correlation between the apoptosis-inducing activity and the formation of Fas clustering by R-125224 treatment, we compared the activity of Fas clustering in H9 and HPB-ALL cells using a confocal laser microscope. In H9 cells, Fas clustering was detected after the treatment with R-125224 and FITC anti-hIgG for 30 min at 37°C but not on ice (Fig. 6A and B). On the other hand, in HPB-ALL cells, a little Fas clustering was observed (Fig. 6A and B). Similarly, Fas clustering was detected in SKW6.4 cells as in H9 cells but not in Jurkat cells (data not shown).



View larger version (23K):
[in this window]
[in a new window]
  		Fig. 6. Fas clustering in human lymphoid cell lines and lymphocytes. (A) H9 and HPB-ALL cells were treated with CMTMR, and further treated with R-125224. The cell pellet was treated with FITC-labeled anti-hIgG on ice. The cells were incubated at 37°C for 30 min (stimulated group) or kept on ice for 30 min (control group). Representative data of the stained cells are shown. (B) The percentage of Fas clustering in each field was calculated. Data are expressed as the mean and SD of eight fields. The statistical analysis of Fas-clustering cells of the treatment groups compared with those of the control groups and of the treatment groups in H9 cells compared with those of HPB-ALL cells was performed by the Student's t-test. A P value of <0.01 was considered to be statistically significant. (*). (C) H9 cells were treated with R-125224, FITC anti-hIgG and cholera toxin B subunit conjugated to rhodamine. (D) Non-activated lymphocytes (left panel) and activated lymphocytes (right panel) were treated with CMTMR, R-125224 and FITC anti-hIgG. The cells were incubated at 37°C for 15 or 60 min or kept on ice for 60 min. Data are expressed as the mean and SD of five fields. The statistical analysis of Fas-clustering cells of the treatment groups compared with those of control groups was performed by the Student's t-test. A P value of <0.01 was considered to be statistically significant (*).

 
Lipid rafts, which are liquid-ordered microdomains enriched with sphingolipids and cholesterol, serve as signaling platforms for TCR and BCR (30). The multivalent binding of ligands to these receptors induces the oligomerization of the receptors; the oligomer has a higher affinity for lipid rafts. Many reports also indicate that these microdomains are important for the efficiency of signaling and composition of receptor signaling complex in Fas-mediated apoptosis (31–34). To study Fas clusters formed onto lipid rafts, we examined co-localization of Fas clusters and the raft-associated sphingoglycolipid GM1 in H9 cells. Fas clusters of H9 cells by R-125224 cross-linked with FITC anti-hIgG were co-localized with patches of cholera toxin B subunit conjugated to rhodamine (30, 35), which bound to GM1 (Fig. 6C).

Next, we examined the ability of Fas clustering in primary cultured cells, which are non-activated and activated human lymphocytes. Although Fas clustering was detected with R-125224 and FITC anti-hIgG in non-activated and activated human lymphocytes (Fig. 6D), more Fas clusters in activated lymphocytes were observed than in non-activated lymphocytes. Non-activated lymphocytes, in which apoptosis had not been induced by R-125224 with anti-hIgG (Fig. 2B), were reported to be type II cells (36). These results also suggest that type I cells form Fas clusters by R-125224 treatment more easily than type II cells do.

Effects of R-125224 on human activated T cells reconstituted in mice
Next, we examined apoptosis induction by R-125224 in vivo using a functional human immune system reconstituted in SCID mice (37). SCID-bg mice were used as xenograft recipients, and human PBMCs were transferred intravenously (SCID-Hu PBMC model). SCID-bg is a strain of double-mutant mice which has impaired lymphoid development and reduced NK cell activity (38) and is considered to be useful as a xenograft recipient. In this model, increased expression of Fas and/or CD69, the activation marker of lymphocytes, was observed in human PBMCs 14 days after transfer as compared with the original PBMCs themselves (Fig. 7A). Using this model, the effect of R-125224 on the percentage of human CD3+Fas+ cells in the spleen of SCID-bg mice in which the PBMCs transferred was determined. R-125224 treatment significantly reduced the number of human CD3+Fas+ cells at doses of 0.04 mg kg–1 (P = 0.0291) and 0.2 mg kg–1 (P = 0.0049) as compared with the control group (Fig. 7B). These results indicate that R-125224 treatment reduced the number of activated human Fas+ T cells in a SCID mouse model even at the low dose of 0.04 mg kg–1 in vivo.



View larger version (16K):
[in this window]
[in a new window]
  		Fig. 7. Effects of R-125224 on human activated T cells reconstituted in mice. (A) Human PBMCs were transferred to mice on day 0, and were analyzed for the expression of CD3, Fas and CD69 (upper parts). Fourteen days after PBMC transfer, samples of cells from mouse spleen were analyzed for the expression of CD3, Fas and CD69 (lower parts). (B) One day after the PBMC transfer, each dose of R-125224 or D-PBS was administered intravenously. Five days after PBMC transfer, samples of cells from mouse spleen were analyzed for the expression of CD3 and Fas. The means of the number of CD3+Fas+ cells of each dosing group were calculated. Data are expressed as the mean and SE of the number of CD3+Fas+ cells. Multiple adjustment was performed by the Dunnett test. A P value of <0.05 was considered to be statistically significant (*).

 
Effect of R-125224 on liver damage in cynomologus monkeys
To examine the hepatotoxic effects of R-125224 in vivo, we selected the cynomologus monkey as an animal model. The reason was that R-125224 binds to cynomologus monkey Fas (data not shown), and sFasL cross-linked with anti-FLAG and R-125224 cross-linked with anti-hIgG also induced apoptosis in activated lymphocytes of cynomologus monkeys as well as those of humans (Figs 2A and 8A). AST and ALT levels as liver damage markers in serum samples of 6 mg kg–1 R-125224 treatment group did not change to those of the control group up to a 15-day observation period (Fig. 8B).



View larger version (11K):
[in this window]
[in a new window]
  		Fig. 8. Effect of R-125224 against liver damage in cynomologus monkeys. (A) Activated cynomologus monkey lymphocytes were incubated with sFasL and anti-FLAG (left panel), and incubated with R-125224 or R-125224 cross-linked with anti-hIgG (right panel). In the right panel, the cells were incubated with R-125224 (filled squre) or R-125224 cross-linked with anti-hIgG (filled circle). The viability of each cell was determined by the XTT method. Data are expressed as the mean and SD of triplicate determinations. The statistical analysis was performed by the Student's t-test. A P value of <0.01 was considered to be statistically significant (*). (B) R-125224 at 6 mg kg–1 was administered intravenously to each cynomologus monkey (n = 6; day 0). Blood sampling was performed on day –3 (control) and day 15. White bars indicate the control group, and black bars indicate the R-125224 treatment group. Data are expressed as the mean and SD. The statistical analyses of AST and ALT levels in serum samples were performed by the Student's t-test. A P value of <0.05 was considered to be statistically significant (*). NS indicates not statistically significant.

 

   Discussion
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
References
 
We show three important findings in this study. The first finding is that sFasL-induced apoptosis-sensitive cells, both cell lines and primary cells, were classified into two types based on their sensitivity to R-125224-induced apoptosis and this classification is consistent with type I and type II cells. The second finding is that activated lymphocytes are classified as type I cells and R-125224-sensitive cells, whereas hepatocytes are classified as type II cells and R-125224-insensitive cells. The third finding is that these selectivities for activated lymphocytes and hepatocytes were also shown in animal models.

H9 and SKW6.4 cells and activated human lymphocytes were sensitive to R-125224-induced apoptosis, but HPB-ALL and Jurkat cells and human hepatocytes were insensitive. Interestingly, the former cells were classified as type I cells, and the latter cells were classified as type II cells (Figs 1–3GoGo). These data suggested that the cells which are sensitive to R-125224-induced apoptosis corresponded to type I cells, and the insensitive cells corresponded to type II cells, not only in cell lines but also in primary cells. To our knowledge, this is the first study in which Fas-mediated apoptosis selectivity between type I and type II cells has been applied in different human primary cells by DISC formation analysis.

Many reports indicated that lipid rafts are important for the efficiency of signaling and composition of receptor signaling complex in Fas-mediated apoptosis, although the requirement for lipid rafts varied widely according to the cell type and/or experimental conditions in each study (29, 31–34). In our experiments, three pieces of evidence suggested the importance of lipid rafts in Fas-mediated apoptosis signaling of type I cells (Fig. 6). First, Fas clusters of H9 cells formed by R-125224 were co-localized with the raft-associated sphingoglycolipid, GM1. Second, Fas clustering was detected at 37°C but not on ice in H9 cells. As the temperature of phase transition in biological membranes is thought to occur between 10 and 40°C, our experimental conditions of incubation on ice did not allow translocation of Fas by R-125224 treatment. Third, more Fas clusters in activated lymphocytes were observed than in non-activated lymphocytes. These data also may support that Fas clustering of type I cells by R-125224 treatment depends on lipid raft formation.

We demonstrated that R-125224, when cross-linked, reduced the viability of human lymphocytes activated with anti-human CD3 antibody and IL-2 but not that of non-activated lymphocytes. Moreover, activated lymphocytes have more Fas-clustering activity by R-125224 than non-activated lymphocytes. Re-stimulation with anti-human CD3 antibody in activated human CD4+ T cells was associated with a rapid distribution of Fas into lipid rafts and the sensitivity of Fas-mediated apoptosis (33). Upon cross-linking of TCRs associated with rafts, lipid rafts become larger, microscopic and more stable structures, often attached to the actin cytoskeleton (30). As treatment of cytochalasin D, inhibitor of actin polymerization, blocked R-125224-induced apoptosis in H9 cells (data not shown) and CH-11-induced apoptosis in activated human lymphocytes (39), the stable structures of lipid rafts may be an important place for Fas-mediated human T cell apoptosis.

How can the apoptosis selectivity of R-125224 be explained between type I cells and type II cells? One explanation could be the different binding sites of Fas between R-125224 and FasL. FasL likely binds to trimerized Fas from the inside based on the predicted three-dimensional structure, which is modeled based on the crystal structure of TNFß and TNFR-1 complex (19, 40, 41). On the other hand, R-125224 likely binds to Fas from the outside as a linear epitope of R-125224 (41). These results suggest that the portion of the binding sites of the Fas clusters may influence the cross-linking effects of R-125224, and the distance between Fas clusters affects the efficiency of apoptosis induction. In fact, Fas expression levels correlated with R-125224-mediated apoptosis selectivity in all tested cells (Figs 1, 2 and 5 and Table 1). Another explanation may be that conformational changes of Fas by R-125224 treatment lead to translocation into lipid rafts easily. A recent report by Muppidi and Siegel (33) suggested this possibility. They reported that pre-associated Fas is preferentially distributed within lipid rafts, and the presence of Fas in lipid rafts enhances apoptosis-signaling efficiency in type I cells but not in type II cells, although the precise mechanisms of Fas translocation into rafts have not been fully clarified. More recently, Gajate et al. (42) reported that the synthetic ether phospholipid ET-18-OCH3 induces clustering of Fas into lipid rafts with intracellular triggering and apoptosis. The finding in this report may also support that R-125224 cross-linked with anti-hIgG induces clustering of Fas into lipid rafts with extracellular triggering.

We demonstrated that the effect of R-125224 treatment was not limited to in vitro but was also shown in vivo (SCID-Hu PBMC model). In this model, activated phenotype cells (high expression of Fas and/or CD69) were increased. Because antigen-presenting cells (APCs) are included in human PBMCs, human lymphocytes may be activated and proliferated by human APCs, which recognize peptides derived from mouse components.

An anti-hIgG for cross-linking of Fas seems to be unnecessary for R-125224-induced apoptosis activity in vivo. We reported that R-125224 with human Fc{gamma}R-positive THP-1 cells induced apoptosis against WR19L12a cells in vitro (20). Probably, inflammatory macrophages and/or neutrophils in SCID mice work as Fc{gamma}R-positive cells to cross-link and to reduce activated human Fas+ T cells in vivo. To support this hypothesis, Xu et al. (43) also reported that R-125224 with mouse peritoneal macrophages but not those cells derived from FcR-common {gamma}-chain–/– mice could induce apoptosis against SKW6.4 cells (type I cells).

We also demonstrated that R-125224 did not affect liver damage markers, AST and ALT, in cynomologus monkeys. These results are consistent with the results that hepatocytes (type II cells) were not induced apoptosis by R-125224 in vitro. Recently, Eichhorst et al. (44) reported the anti-apoptotic effect of suramine, whose effects seemed to be only in type II and not in type I cells, and its inhibitory effect on Fas-induced fulminant liver failure in mice. Fas-mediated apoptosis-inducing therapy using R-125224, which has type I specificity, would be an effective treatment against autoimmune diseases such as RA without side effects in the liver.


   Acknowledgements
 
We thank H. Haruyama and A. Takasaki for helpful discussions, T. Oda and M. Yamashita for critical reading of the manuscript and T. Kubo for discussions and help with confocal microscope analysis. We also thank M. Uemori for statistical analysis.


   Abbreviations
 
ALT   alanine aminotransferase
anti-FLAG   anti-FLAG M2 antibody
anti-hIgG   anti-human IgG
APC   antigen-presenting cell
AST   aspartate aminotransferase
Bmax   maximum binding
CL   collagen
CMTMR   Cell TrackerTM Orange CMTMR
DiOC6   3,3'-dihexyloxacarbocyanine iodide
DISC   death-inducing signaling complex
FADD   Fas-associated death domain
FasL   Fas ligand
PI   propidium iodide
RA   rheumatoid arthritis
SCID-bg   C·B-17/Icr Crj-scid bgBR
sFasL   soluble Fas ligand
TNF   tumor necrosis factor
{Delta}{Psi}m   mitochondrial transmembrane potential

   Notes
 
Transmitting editor: K. Yamamoto

Received 25 May 2005, accepted 14 October 2005.


   References
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

  1. Yonehara, S., Ishii, A. and Yonehara, M. 1989. A cell-killing monoclonal antibody (anti-Fas) to a cell surface antigen co-downregulated with the receptor of tumor necrosis factor. J. Exp. Med. 169:1747.[Abstract/Free Full Text]
  2. Trauth, B. C., Klas, C., Peters, A. M. et al. 1989. Monoclonal antibody-mediated tumor regression by induction of apoptosis. Science 245:301.[Abstract/Free Full Text]
  3. Suda, T., Takahashi, T., Golstein, P. and Nagata, S. 1993. Molecular cloning and expression of the Fas ligand, a novel member of the tumor necrosis factor family. Cell 75:1169.[CrossRef][ISI][Medline]
  4. Nagata, S. 1997. Apoptosis by death factor. Cell 88:355.[CrossRef][ISI][Medline]
  5. Siegel, R. M., Chan, F. K., Chun, H. J. and Lenardo, M. J. 2000. The multifaceted role of Fas signaling in immune cell homeostasis and autoimmunity. Nat. Immunol. 1:469.[CrossRef][ISI][Medline]
  6. Watanabe-Fukunaga, R., Brannan, C. I., Copeland, N. G., Jenkins, N. A. and Nagata, S. 1992. Lymphoproliferation disorder in mice explained by defects in Fas antigen that mediates apoptosis. Nature 356:314.[CrossRef][Medline]
  7. Kishi, S., Saijyo, S., Arai, M. et al. 1997. Resistance to fas-mediated apoptosis of peripheral T cells in human T lymphocyte virus type I (HTLV-I) transgenic mice with autoimmune arthropathy. J. Exp. Med. 186:57.[Abstract/Free Full Text]
  8. Zhang, J., Bardos, T., Mikecz, K., Finnegan, A. and Glant, T. T. 2001. Impaired Fas signaling pathway is involved in defective T cell apoptosis in autoimmune murine arthritis. J. Immunol. 166:4981.[Abstract/Free Full Text]
  9. Fisher, G. H., Rosenberg, F. J., Straus, S. E. et al. 1995. Dominant interfering Fas gene mutations impair apoptosis in a human autoimmune lymphoproliferative syndrome. Cell 81:935.[CrossRef][ISI][Medline]
  10. Schulze-Osthoff, K., Ferrari, D., Los, M., Wesselborg, S. and Peter, M. E. 1998. Apoptosis signaling by death receptors. Eur. J. Biochem. 254:439.[ISI][Medline]
  11. Muzio, M., Chinnaiyan, A. M., Kischkel, F. C. et al. 1996. FLICE, a novel FADD-homologous ICE/CED-3-like protease, is recruited to the CD95 (Fas/APO-1) death-inducing signaling complex. Cell 85:817.[CrossRef][ISI][Medline]
  12. Scaffidi, C., Fulda, S., Srinivasan, A. et al. 1998. Two CD95 (APO-1/Fas) signaling pathways. EMBO J. 17:1675.[CrossRef][ISI][Medline]
  13. Algeciras-Schimnich, A., Pietras, E. M., Barnhart, B. C. et al. 2003. Two CD95 tumor classes with different sensitivities to antitumor drugs. Proc. Natl Acad. Sci. USA 100:11445.[Abstract/Free Full Text]
  14. Huang, D. C., Hahne, M., Schroeter, M. et al. 1999. Activation of Fas by FasL induces apoptosis by a mechanism that cannot be blocked by Bcl-2 or Bcl-x(L). Proc. Natl Acad. Sci. USA 96:14871.[Abstract/Free Full Text]
  15. Yin, X. M., Wang, K., Gross, A. et al. 1999. Bid-deficient mice are resistant to Fas-induced hepatocellular apoptosis. Nature 400:886.[CrossRef][Medline]
  16. Nishimura-Morita, Y., Nose, M., Inoue, T. and Yonehara, S. 1997. Amelioration of systemic autoimmune disease by the stimulation of apoptosis-promoting receptor Fas with anti-Fas mAb. Int. Immunol. 9:1793.[Abstract/Free Full Text]
  17. Ogasawara, J., Watanabe-Fukunaga, R., Adachi, M. et al. 1993. Lethal effect of the anti-Fas antibody in mice. Nature 364:806.[CrossRef][Medline]
  18. Ichikawa, K., Yoshida-Kato, H., Ohtsuki, M. et al. 2000. A novel murine anti-human Fas mAb which mitigates lymphadenopathy without hepatotoxicity. Int. Immunol. 12:555.[Abstract/Free Full Text]
  19. Haruyama, H., Ito, S., Miyadai, K. et al. 2002. Humanization of the mouse anti-Fas antibody HFE7A and crystal structure of the humanized HFE7A Fab fragment. Biol. Pharm. Bull. 25:1537.[CrossRef][ISI][Medline]
  20. Matsuno, H., Yudoh, K., Nakazawa, F. et al. 2002. Antirheumatic effects of humanized anti-Fas monoclonal antibody in human rheumatoid arthritis/SCID mouse chimera. J. Rheumatol. 29:1609.[ISI][Medline]
  21. Ogawa, Y., Ohtsuki, M., Uzuki, M. et al. 2003. Suppression of osteoclastogenesis in rheumatoid arthritis by induction of apoptosis in activated CD4+ T cells. Arthritis Rheum. 48:3350.[CrossRef][ISI][Medline]
  22. Scudiero, D. A., Shoemaker, R. H., Paull, K. D. et al. 1988. Evaluation of a soluble tetrazolium/formazan assay for cell growth and drug sensitivity in culture using human and other tumor cell lines. Cancer Res. 48:4827.[Abstract/Free Full Text]
  23. Imai, Y., Kimura, T., Murakami, A., Yajima, N., Sakamaki, K. and Yonehara, S. 1999. The CED-4-homologous protein FLASH is involved in Fas-mediated activation of caspase-8 during apoptosis. Nature 398:777.[CrossRef][Medline]
  24. Hadley, S. W. and Wilbur, D. S. 1990. Evaluation of iodovinyl antibody conjugates: comparison with a p-iodobenzoyl conjugate and direct radioiodination. Bioconjug. Chem. 1:154.[CrossRef][Medline]
  25. Cumberledge, S. and Krasnow, M. A. 1993. Intercellular signalling in Drosophila segment formation reconstructed in vitro. Nature 363:549.[CrossRef][Medline]
  26. Coumans, B., Thellin, O., Zorzi, W. et al. 1999. Lymphoid cell apoptosis induced by trophoblastic cells: a model of active foeto-placental tolerance. J. Immunol. Methods 224:185.[CrossRef][ISI][Medline]
  27. Li, H., Zhu, H., Xu, C. J. and Yuan, J. 1998. Cleavage of BID by caspase 8 mediates the mitochondrial damage in the Fas pathway of apoptosis. Cell 94:491.[CrossRef][ISI][Medline]
  28. Madesh, M., Antonsson, B., Srinivasula, S. M., Alnemri, E. S. and Hajnoczky, G. 2002. Rapid kinetics of tBid-induced cytochrome c and Smac/DIABLO release and mitochondrial depolarization. J. Biol. Chem. 277:5651.[Abstract/Free Full Text]
  29. Algeciras-Schimnich, A., Shen, L., Barnhart, B. C., Murmann, A. E., Burkhardt, J. K. and Peter, M. E. 2002. Molecular ordering of the initial signaling events of CD95. Mol. Cell. Biol. 22:207.[Abstract/Free Full Text]
  30. Dykstra, M., Cherukuri, A., Sohn, H. W., Tzeng, S. J. and Pierce, S. K. 2003. Location is everything: lipid rafts and immune cell signaling. Annu. Rev. Immunol. 21:457.[CrossRef][ISI][Medline]
  31. Hueber, A. O., Bernard, A. M., Herincs, Z., Couzinet, A. and He, H. T. 2002. An essential role for membrane rafts in the initiation of Fas/CD95-triggered cell death in mouse thymocytes. EMBO Rep. 3:190.[CrossRef][ISI][Medline]
  32. Scheel-Toellner, D., Wang, K., Singh, R. et al. 2002. The death-inducing signaling complex is recruited to lipid rafts in Fas-induced apoptosis. Biochem. Biophys. Res. Commun. 297:876.[CrossRef][ISI][Medline]
  33. Muppidi, J. R. and Siegel, R. M. 2004. Ligand-independent redistribution of Fas (CD95) into lipid rafts mediates clonotypic T cell death. Nat. Immunol. 5:182.[CrossRef][ISI][Medline]
  34. Eramo, A., Sargiacomo, M., Ricci-Vitiani, L. et al. 2004. CD95 death-inducing signaling complex formation and internalization occur in lipid rafts of type I and type II cells. Eur. J. Immunol. 34:1930.[CrossRef][ISI][Medline]
  35. Kozak, S. L., Heard, J. M. and Kabat, D. 2002. Segregation of CD4 and CXCR4 into distinct lipid microdomains in T lymphocytes suggests a mechanism for membrane destabilization by human immunodeficiency virus. J. Virol. 76:1802.[Abstract/Free Full Text]
  36. Schmitz, I., Krueger, A., Baumann, S., Schulze-Bergkamen, H., Krammer, P. H. and Kirchhoff, S. 2003. An IL-2-dependent switch between CD95 signaling pathways sensitizes primary human T cells toward CD95-mediated activation-induced cell death. J. Immunol. 171:2930.[Abstract/Free Full Text]
  37. Mosier, D. E., Gulizia, R. J., Baird, S. M. and Wilson, D. B. 1988. Transfer of a functional human immune system to mice with severe combined immunodeficiency. Nature 335:256.[CrossRef][Medline]
  38. Berney, T., Molano, R. D., Pileggi, A. et al. 2001. Patterns of engraftment in different strains of immunodeficient mice reconstituted with human peripheral blood lymphocytes. Transplantation 72:133.[CrossRef][ISI][Medline]
  39. Parlato, S., Giammarioli, A. M., Logozzi, M. et al. 2000. CD95 (APO-1/Fas) linkage to the actin cytoskeleton through ezrin in human T lymphocytes: a novel regulatory mechanism of the CD95 apoptotic pathway. EMBO J. 19:5123.[CrossRef][ISI][Medline]
  40. Banner, D. W., D'Arcy, A., Janes, W. et al. 1993. Crystal structure of the soluble human 55 kd TNF receptor-human TNF beta complex: implications for TNF receptor activation. Cell 73:431.[CrossRef][ISI][Medline]
  41. Ito, S., Takayama, T., Hanzawa, H. et al. 2002. Crystal structure of the antigen-binding fragment of apoptosis-inducing mouse anti-human Fas monoclonal antibody HFE7A. J. Biochem. (Tokyo) 131:137.[Abstract/Free Full Text]
  42. Gajate, C., Del Canto-Janez, E., Acuna, A. U. et al. 2004. Intracellular triggering of Fas aggregation and recruitment of apoptotic molecules into Fas-enriched rafts in selective tumor cell apoptosis. J. Exp. Med. 200:353.[Abstract/Free Full Text]
  43. Xu, Y., Szalai, A. J., Zhou, T. et al. 2003. Fc gamma Rs modulate cytotoxicity of anti-Fas antibodies: implications for agonistic antibody-based therapeutics. J. Immunol. 171:562.[Abstract/Free Full Text]
  44. Eichhorst, S. T., Krueger, A., Muerkoster, S. et al. 2004. Suramin inhibits death receptor-induced apoptosis in vitro and fulminant apoptotic liver damage in mice. Nat. Med. 10:602.[CrossRef][ISI][Medline]

Add to CiteULike CiteULike   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us    What's this?


This article has been cited by other articles:


Home page
 J. Virol.Home page
C. Le Clorennec, T.-S. Ouk, I. Youlyouz-Marfak, S. Panteix, C.-C. Martin, J. Rastelli, E. Adriaenssens, U. Zimber-Strobl, J. Coll, J. Feuillard, et al.
Molecular Basis of Cytotoxicity of Epstein-Barr Virus (EBV) Latent Membrane Protein 1 (LMP1) in EBV Latency III B Cells: LMP1 Induces Type II Ligand-Independent Autoactivation of CD95/Fas with Caspase 8-Mediated Apoptosis
J. Virol., July 1, 2008; 82(13): 6721 - 6733.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow All Versions of this Article:
18/1/113    most recent
dxh353v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive