The JI
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     
 

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Xu, Y.
Right arrow Articles by Kimberly, R. P.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Xu, Y.
Right arrow Articles by Kimberly, R. P.
The Journal of Immunology, 2003, 171: 562-568.
Copyright © 2003 by The American Association of Immunologists

Fc{gamma}Rs Modulate Cytotoxicity of Anti-Fas Antibodies: Implications for Agonistic Antibody-Based Therapeutics1

Yuanyuan Xu2,*, Alexander J. Szalai*, Tong Zhou*, Kurt R. Zinn{dagger}, Tandra R. Chaudhuri{dagger}, Xiaoli Li*, William J. Koopman* and Robert P. Kimberly*

* Division of Clinical Immunology and Rheumatology, Department of Medicine and {dagger} Laboratory for MultiModality Imaging Assessment, Department of Radiology, University of Alabama, Birmingham, AL 35294


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Development of anti-Fas Abs to treat diseases with insufficient Fas-mediated apoptosis has been limited by concern about hepatotoxicity. We report here that hepatotoxicity elicited by anti-Fas Ab Jo2 is dependent on Fc{gamma}RIIB. Thus, following Jo2 treatment, all Fc{gamma}RIIB-/- mice survived while 80% of wild-type and all FcR-{gamma}-/- mice died from acute liver failure. Microscopic examination suggests that Fc{gamma}RIIB deficiency protects the hepatic sinusoidal endothelium, a cell type that normally coexpresses Fas and Fc{gamma}RIIB. In vitro studies showed that Fc{gamma}RIIB, but not Fc{gamma}RI and Fc{gamma}RIII, on neighboring macrophages substantially enhanced Jo2 mediated apoptosis of Fas expressing target cells. However, Fc{gamma}RI and Fc{gamma}RIII appeared essential for apoptosis-inducing activity of a non-hepatotoxic anti-Fas mAb HFE7A. These findings imply that by interacting with the Fc region of agonistic Abs, Fc{gamma}Rs can modulate both the desired and undesired consequences of Ab-based therapy. Recognizing this fact should facilitate development of safer and more efficacious agonistic Abs.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The number of actual or proposed Ab-based therapies is increasing steadily, but growth in knowledge about in vivo mechanisms that underlie their beneficial actions and detrimental side effects has not kept pace. For example, many mechanisms have historically been proposed to account for anti-tumor activities of therapeutic Abs, including extended half-life and blockade of cell signaling. However, only recently was it established that engagement of FcR on effector cells makes the major contribution to anti-tumor activity in vivo (1). Thus, mouse Abs (and also their humanized variants) against various cell surface markers (gp75, HER2/neu, CD20), were found to be engaged by both activation (Fc{gamma}RI, Fc{gamma}RIII) and inhibitory (Fc{gamma}RIIB) FcR on effector cells in vivo (1). These were the first data to show unequivocally that Fc{gamma}R-dependent mechanisms modulate substantially the cytotoxic response of therapeutic Abs against tumors in vivo, and the first to indicate that an optimal Ab against tumor targets should bind preferentially to Fc{gamma}RIII and minimally to Fc{gamma}RIIB.

A primary aim of our research was to determine whether Fc{gamma}Rs also contribute to the agonistic activity of therapeutic apoptosis-inducing Abs. We conducted in vitro and in vivo studies using the agonistic Abs directed at Fas (CD95/APO-1), a member of the TNFR superfamily (2). Fas is a death receptor that interacts with Fas ligand (CD95L) to promote pro-apoptotic signals crucial for maintenance of homeostasis (3, 4). Most of the interest in anti-Fas Abs is directed toward understanding the consequences of Fab interaction with Fas. In comparison, only a few studies (5, 6) have considered the contribution to anti-Fas induced apoptosis made by the Fc region. The anti-Fas Abs Jo2 (7) and HFE7A (8) have been particularly well studied. Both Abs bind Fas and mimic the action of natural Fas ligand, i.e., by binding to Fas they propogate Fas-dependent cytoplasmic signals that culminate in target cell apoptosis. Importantly, because both are strong agonists, their activity in vivo does not necessarily require Ab-dependent cell-mediated cytotoxicity (ADCC)3. Of course humanized derivatives of anti-Fas Abs have clinical potential. Unfortunately, realizing this potential has been hindered by the knowledge that at least one anti-Fas Ab, Jo2, induces severe hepatotoxicity (7). Most investigations have concluded that this lethal side effect involves overwhelming apoptosis of hepatocytes, but the exact reason for hepatotoxicity remains unknown. Recent evidence suggested that apoptosis of sinusoidal endothelial cells (SECs) promotes microvascular collapse and contributes to Jo2 induced liver damage (9, 10). Accordingly, a secondary aim of our study was to measure the contribution of Fc{gamma}Rs in Jo2-induced hepatotoxicity.

Our findings show that Fc{gamma}Rs modulate the agonistic activities of anti-Fas Abs. For Jo2, Fc{gamma}RIIB is essential for target cell apoptosis in vitro but this does not depend on Fc{gamma}RIIB-mediated cell signaling. For HFE7A, apoptosis relies more on Fc{gamma}RI and Fc{gamma}RIII, and the degree of dependence on the two receptors is altered by humanization of the parent mouse Ab. Like apoptosis, Jo2-induced hepatotoxicity also depends on Fc{gamma}RIIB. The lethal effect arises because Jo2 targets and destroys hepatic sinusoidal endothelial cells, a cell type that coexpresses Fas and Fc{gamma}RIIB (10, 11). These results demonstrate that Fc{gamma}R dependent mechanisms other than classical ADCC contribute to the therapeutic and detrimental action of anti-Fas Abs. It is likely that these dualistic actions are a general property of cell surface-targeting agonistic Abs, and perhaps even Fc-fusion proteins.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals and Abs

C57BL/6 mice and congenic mutants that lack expression of the FcR common {gamma}-chain (FcR-{gamma}-/-) (12) or Fc{gamma}RIIB (Fc{gamma}RIIB-/-) (13) were from Taconic Farms (Germantown, NY). Double mutants (Fc{gamma}R-/-; Taconic Farms), generated by intercrossing FcR-{gamma}-/- with Fc{gamma}RIIB-/-, have a mixed C57BL/6 x 129 genetic background. Fc{gamma}RIII-/- mice were obtained from The Jackson Laboratory (Bar Harbor, ME) (14). All were maintained according to protocols established by the Animal Resources Program and approved by the Institutional Animal Care and Use Committee at University of Alabama at Birmingham. Mice were 8- to 12-wk old males and weighed 28.8 ± 0.69 g when used. Hamster anti-mouse Fas mAb Jo2 (IgG2) was obtained from BD PharMingen (San Diego, CA). Mouse HFE7A (muHFE7A, IgG1) and humanized HFE7A (huHFE7A, IgG1) were supplied by Sankyo (Tokyo, Japan).

Mortality studies and biochemical and histological analysis of livers

For mortality studies, mice were injected i.v. with 10 or 100 µg of Jo2 diluted in lactated Ringer’s solution (Abbott Laboratories, Abbott Park, IL). Before and after injection blood was collected hourly and survival was monitored for 48 h. Serum alanine aminotransferase (ALT) activity was determined using INFINITY ALT reagent (Sigma-Aldrich, St. Louis, MO). At specific time points, livers of select mice were removed, rinsed in PBS, and fixed in 10% buffered formalin. Paraffin embedded sections (5 µm) were stained with hematoxylin and eosin for histological examination or with a TUNEL kit (Oncogene Research Products, San Diego, CA) to visualize apoptotic cells. For transmission electron microscopy (TEM) formalin-fixed tissue was processed using a standard procedure that included postfixation in osmium tetroxide, dehydration with acetone, and embedding in Spurr’s resin. Ultrathin sections were cut on an LKB Ultrotome III, poststained with aqueous uranyl acetate and Reynold’s lead citrate, and examined on a Phillips 301 TEM.

Biodistribution of radiolabeled Jo2

Jo2 was labeled with 99mTc pertechnetate (Central Pharmacy, Birmingham, AL) as described (15). Wild-type and Fc{gamma}R-/- mice (four each) were anesthetized and injected i.v with 3 µg of 99mTc-Jo2. The mice were terminated after 15 min, and their organs were collected and weighed. The 99mTc emissions were measured in a Minaxi Auto-Gamma 5000 series gamma counter (Packard Instruments, Meriden, CT). Radioactivity in tissues was normalized to tissue weight and expressed as percent of dose of 99mTc-Jo2 administered per gram of tissue.

Confocal imaging

Jo2 Ab was conjugated with Cy5.5 (Amersham Pharmacia Biotech, Piscataway, NJ) and dialyzed into PBS for injection into mice (16). Livers from mice injected with Cy5.5-Jo2 (9 µg) were embedded in snap frozen OCT medium (Tissue Tek, Elkhart, IN) by immersion in liquid nitrogen, sectioned (5 µm), fixed in 10% formalin, and examined by confocal microscopy. Some sections were poststained with anti-CD34 (clone QBEnd/4/10, Cell Marque, Austin, TX) to visualize the sinusoidal endothelium. In this case, goat anti-mouse Alexa 594 (5 µg/ml, Molecular Probes, Eugene, OR) served as the secondary Ab and negative controls were treated with 3% goat serum. Images were acquired with Leica Laser Confocal optics (Leica Microsystems, Heidelberg, Germany). Fluorescence of Jo2 (blue) and anti-CD34 (red) was detected in Cy5.5 and Cy3 specific channels. Autofluorescence was artificially colored green to reveal background structure.

Target cell lines

The Fc{gamma}RIIB-positive murine B cell lymphoma line A20 and its Fc{gamma}RIIB-negative variant IIA1.6 were a gift from T. Wade at Dartmouth Medical Center (Lebanon, NH) and maintained as previously described (17). The human SKW6.4 B cell lymphoblast cell line was from American Type Culture Collection (Manassas, VA). IIA1.6 cells were transfected with plasmid pcDNA3 containing cDNA encoding full-length wild-type Fc{gamma}RIIB (FLFc{gamma}RIIB) or a cytoplasmic domain-truncated tailless variant (TLFc{gamma}RIIB). IIA1.6 transfectants were cultured in IMDM supplemented with 10% heat-inactivated FCS, 100 U/ml penicillin, 100 µg/ml streptomycin, 2 mM glutamine, 750 µg/ml G418, 50 µM 2-ME, and 1 mM sodium pyruvate.

Target cell apoptosis

To measure anti-Fas-Ab-induced apoptosis, the CyToxiLux fluorogenic cytoxicity assay (18) was conducted according to the manufacturer’s protocol (OncoImmunin, Gaithersburg, MD). Target cells were labeled with a cell marker (red) for 1 h at 37°C. After washing, 100 µl of target cells (2 x 105) were incubated (4 h, 37°C) with an equal volume of medium containing increasing amounts of Jo2 (0–250 ng/ml), HFE7A, or control Ab. At the end of incubation, cells were washed and incubated with 75 µl of cell-permeable caspase substrate for an additional 45 min then washed with PBS and resuspended in 200 µl of the same buffer. Target cell apoptosis was analyzed by using a FACScan flow cytometer (BD Biosciences, San Jose, CA) and WinMDI 2.8 software. The apoptotic cells containing cleaved caspase substrate were detected in the FL1 channel. The labeled target cells (T) were detected in the FL2 channel. The percentage of caspase-positive target cells in the target cell population was calculated as: % caspase staining = [(caspase+T)/(caspase+T + caspase-T)] x 100. The same assay was modified to perform mixed cell assays to determine the effect of various Fc{gamma}R-bearing bystander cells on anti-Fas-Ab-mediated apoptosis of targets. In these assays, 2 x 105 IIA1.6 or SKW6.4 target cells were sensitized with 5 µg/ml Jo2 or 2.5 µg/ml HFE7A Ab on ice for 30 min. Then, the sensitized target cells were washed and apoptosis was determined after incubating at 37°C for 4 h in medium containing various numbers of A20 cells or peritoneal macrophages (bystander:target = 1:2, 1:5, or 1:10) before addition of caspase substrate. Macrophages used as bystander cells were from peritoneal fluids of thioglycollate-inoculated mice (19).

Phagocytosis assays

Sheep erythrocytes (E) (1 x 109/ml) sensitized with polyclonal rabbit anti-sheep red blood cell IgG (A) (Sigma-Aldrich) served as target cells (EA). The assay was performed as described (20) with modification. Briefly, monolayers containing 1 x 106 peritoneal macrophages were overlaid with 5 x 107 EA and incubated (60 min, 37°C). The monolayers were then rinsed to wash away unbound EA, and macrophage-bound EA were lysed by brief hypertonic shock in 0.2% NaCl. Macrophages were examined by light microscopy to identify those that ingested at least two EA or more. Based on inspection of at least 200 macrophages the average percent of phagocytic macrophages was calculated from two separate experiments.

Statistical analysis

Kaplan-Meier survival curves were plotted and tested for significant difference using logrank Mantel-Cox tests. Differences in apoptosis and phagocytosis were compared using Student’s t tests. A value of p < 0.05 was considered significant in all cases.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Fc{gamma}RIIB promotes anti-Fas mediated apoptosis

Because A20 cells coexpress Fas and Fc{gamma}RIIB, we used these and their Fc{gamma}RIIB-negative variant IIA1.6 as target cells to investigate the contribution of Fc{gamma}RIIB to Jo2-induced apoptosis. We found that Jo2 did not induce apoptosis under the conditions tested unless the cells expressed Fc{gamma}RIIB. Thus, Fc{gamma}RIIB-positive A20 cells were sensitive to Jo2 whereas Fc{gamma}RIIB-negative IIA1.6 cells were resistant (Fig. 1A). The sensitivity of A20 cells was attributable to the presence of Fc{gamma}RIIB on those cells because pre-incubation of A20 cells with mAb 2.4G2, which blocks IgG binding to Fc{gamma}RIIB, prevented Jo2 induced apoptosis of A20 cells (Fig. 1A, inset). To determine whether the resistance of IIA1.6 cells was attributable to the absence of Fc{gamma}RIIB and if an intracellular Fc{gamma}RIIB-mediated signal was required for the apoptosis promoting effect, we compared Jo2-mediated apoptosis of IIA1.6 cells transfected with full-length vs truncated versions of human Fc{gamma}RIIB. Jo2 resistant IIA1.6 cells expressing Fc{gamma}RIIB became susceptible to Jo2-induced apoptosis even if the signaling-deficient version of the receptor was used in the assays (Fig. 1B). We conclude that coexpression of Fc{gamma}RIIB and Fas substantially increases susceptibility of target cells to Jo2. Further, because the cytoplasmic tail of Fc{gamma}RIIB is not required for the apoptosis-promoting effect, we conclude that SHIP-mediated signaling (21) is not essential. Intracellular signals transduced by the transmembrane domain of Fc{gamma}RIIB cannot be ruled out. Overall, the evidence is consistent with the extracellular domain(s) of Fc{gamma}RIIB providing a scaffold that shepherds Jo2, and thus Jo2-bound Fas, into a functional complex that promotes Fas-dependent cytotoxicity in vitro. A20 cells do not express Fc{gamma}RIII. Therefore the apoptosis promoting action is Fc{gamma}R dependent but not mediated by the mechanism of classified as ADCC.



View larger version (38K):
[in this window]
[in a new window]
  		FIGURE 1. Requirement of Fc{gamma}RIIB for Jo2 induced apoptosis of murine lymphoma cells. A, Fc{gamma}RIIB-negative cells resist killing by Jo2. Fc{gamma}RIIB-positive cells (A20) or their Fc{gamma}RIIB-negative variants (IIA1.6) were incubated with the indicated amounts of Jo2 Ab (37°C, 4 h) and the proportion of cells undergoing apoptosis was determined by flow cytometry. The inset shows that Jo2 mediated killing of A20 cells is inhibited by preincubating the cells with mAb 2.4G2 (anti-Fc{gamma}RII/Fc{gamma}RIII) but not by preincubating with control IgG. B, The cytoplasmic signaling domain of Fc{gamma}RIIB is not essential to promote Jo2 induced killing. IIA1.6 transfectants that express human Fc{gamma}RIIB either as a full-length receptor (FLFc{gamma}RIIB) or as a truncated receptor with no cytoplasmic tail (TLFc{gamma}RIIB) were incubated with the indicated amounts of Jo2 Ab and the proportion of cells undergoing apoptosis was determined. C, Fc{gamma}RIIB displayed by neighboring cells supports Jo2 mediated apoptosis of Fc{gamma}RIIB-negative targets. Labeled IIA1.6 target cells were cultured (37°C, 4 h) in the presence of A20 cells (IIA1.6:A20 = 10:1), with (+) or without (-) Jo2, and flow cytometry was performed to detect caspase activity of apoptotic target cells. The proportion of IIA1.6 target cells undergoing apoptosis is indicated (boxed region). D, Fc{gamma}RIIB displayed by mouse peritoneal macrophages supports Jo2 mediated apoptosis of Fc{gamma}RIIB-negative targets. IIA1.6 target cells (2 x 105) sensitized with Jo2 (5 µg/ml) were incubated (37°C, 4 h) with peritoneal macrophages (2 x 104) and the proportion of target cells undergoing apoptosis was determined. E, Reduced ability of Fc{gamma}RIIB-/- macrophages to support Jo2 mediated apoptosis is not due to a global defect in cell function. Macrophages were incubated (37°C, 1 h) with rabbit IgG-sensitized sheep erythrocytes (EA) and the proportion of phagocytic macrophages was determined. The statistics shown in each panel are either pooled from at least three separate experiments (A, B, D) or representative of at least two (C and E). The asterisks indicate significantly less apoptosis or phagocytosis than the wild-type group (p < 0.005, t test). ns, not significant.

 
Fc{gamma}RIIB on bystander cells supports anti-Fas mediated apoptosis of target cells

It is established that surface Ag-bound Ab can engage Fc{gamma}Rs on the same cell (22, 23). To determine whether Jo2 interacting with Fc{gamma}RIIB displayed on one cell can promote apoptosis of a neighboring Fas-bearing cell, we used a two-color FACS-based cytotoxicity assay (18), which allowed us to track apoptosis of target cells cocultured with bystander cells. Using such mixed cell populations, we found that Fc{gamma}RIIB displayed by bystander cells promoted killing of Fc{gamma}RIIB-negative targets. Thus, Fc{gamma}RIIB-deficient IIA1.6 cells that resisted Jo2-induced apoptosis in the single population assay (Fig. 1A) were killed when Fc{gamma}RIIB-positive A20 cells were supplied. Under these conditions nearly 25% of Jo2-sensitized IIA1.6 cells underwent apoptosis (Fig. 1C), compared with <4% in the absence of A20 cells (Fig. 1A). Furthermore, in the absence of Jo2 (Fig. 1C) or in the presence of an isotype-matched control hamster Ab (data not shown), apoptosis of IIA1.6 targets was reproducibly <3%. These data confirm that Jo2 bound to Fas on a target cell can induce apoptosis in a trans fashion if the Fc is available to Fc{gamma}RIIB on a bystander cell. However, Jo2 might also promote apoptosis in a cis fashion by cross-linking Fas and Fc{gamma}RIIB on a single cell.

Fc{gamma}RIIB on macrophages supports Jo2 mediated apoptosis of target cells

To determine whether other Fc{gamma}Rs also support Jo2 induced apoptosis, we repeated the cytotoxicity assays using peritoneal macrophages from wild-type, Fc{gamma}RIIB-/- and FcR-{gamma}-/- mice. Wild-type mouse macrophages promoted substantial apoptosis of Jo2-sensitized IIA1.6 target cells (Fig. 1D). This effect was not attributable to ADCC, because FcR-{gamma}-/- macrophages, which lack Fc{gamma}RIII and a substantial amount of Fc{gamma}RI, fully supported Jo2 mediated killing. Like A20 cells and IIA1.6 transfectants, macrophage-assisted killing depended on Fc{gamma}RIIB, because the apoptosis promoting ability of Fc{gamma}RIIB-/- macrophages was significantly reduced compared with wild-type (Fig. 1D). It is unlikely that the reduced ability of Fc{gamma}RIIB-/- macrophages to support Jo2 induced apoptosis was due to an unknown intrinsic defect in the cells, as Fc{gamma}RIIB-/- macrophages were fully phagocytic (Fig. 1E). Conversely, macrophages from FcR-{gamma}-/- mice, which still express Fc{gamma}RIIB, were not phagocytic yet they fully support Jo2-induced apoptosis (Fig. 1E). These data support the conclusion made earlier, i.e., Fc{gamma}RIIB on bystander cells potentiates Jo2-induced killing of Fas expressing target cells. The data also suggest that hamster Jo2 Ab preferentially engages Fc{gamma}RIIB.

Fc{gamma}RIIB is required for Jo2 induced lethality in mice

In addition to its apoptosis inducing activity to lymphocytes, Jo2 causes lethal hepatotoxicity in mice (7). To investigate the contribution of Fc{gamma}RIIB to this undesired effect, we compared the outcome of Jo2 administration in wild-type vs Fc{gamma}R-deficient mice. Nearly 80% of wild-type mice and all FcR-{gamma}-/- died within 8 h after infusion of 0.34 µg/g of Jo2 (p < 0.0001, Mantel-Cox tests) (Fig. 2A). Also all of 6 Fc{gamma}RIII-/- mice given the same treatment died within 8 h (data not shown). In contrast, the two strains of mice we tested that do not express Fc{gamma}RIIB were resistant; 9 of 9 Fc{gamma}RIIB-/- (p = 0.0006 vs wild-type, Mantel-Cox tests) and 10 of 12 Fc{gamma}R-/- mice (p = 0.0036 vs wild-type, Mantel-Cox tests) survived (Fig. 2A). In fact, 4 of 4 Fc{gamma}RIIB-/- mice given a 10-fold higher dose of Jo2 survived (data not shown). Survival of Fc{gamma}RIIB-/- and Fc{gamma}R-/- was not significantly different (Mantel-Cox tests). There was rapid and substantial increase in serum ALT level in wild-type and FcR-{gamma}-/- mice, but not in Fc{gamma}RIIB-/- and Fc{gamma}R-/- (Fig. 2B), indicating little or no hepatotoxicity occurred in the Fc{gamma}RIIB-deficient strains. One possible explanation for this outcome is that absence of one or more Fc{gamma}Rs altered the retention time of Jo2 in the circulation and/or its delivery to the liver. Biodistribution studies using 99mTc-labeled Jo2 ruled this out; neither the transit time (data not shown) nor the delivery of Jo2 to the various organs was perturbed in Fc{gamma}R-/- mice (Fig. 2B, inset). We conclude that Fc{gamma}RIIB is essential for the lethal sequela of Jo2 administration.



View larger version (33K):
[in this window]
[in a new window]
  		FIGURE 2. Fc{gamma}RIIB promotes Jo2 induced lethality. Mice were injected i.v. with 0.34 µg/g of Jo2. A, Fc{gamma}RIIB-/- and Fc{gamma}R-/- mice, which lack expression of Fc{gamma}RIIB, are resistant to the lethal effect of Jo2 whereas wild-type and FcR-{gamma}-/- mice, which express Fc{gamma}RIIB, are highly susceptible. B, In mice lacking Fc{gamma}RIIB, Jo2 treatment does not elevate serum alanine aminotransferase (ALT) activity. The inset shows that biodistribution of 99mTc-Jo2 in wild-type vs Fc{gamma}R-/- mice is not affected by absence of Fc{gamma}Rs. The number of mice used for determination of survival and ALT is indicated in A. The biodistribution data are representative of two experiments (four mice per group). Li, liver; lu, lung; sp, spleen; ki, kidneys; he, heart; bl, blood.

 
Fc{gamma}RIIB promotes Jo2 induced sinusoid destruction and hepatocyte apoptosis

To reveal possible mechanisms of action of Fc{gamma}RIIB in this system we compared and contrasted livers of Jo2-treated wild-type and Fc{gamma}RIIB-/- mice. Livers from wild-type mice were characterized by extensive and widespread sinusoidal hemorrhage (Fig. 3A); they also contained numerous apoptotic hepatocytes, which appeared to be surrounded by the damaged sinusoids (Fig. 3B). Electron microscopy (Fig. 3C) revealed complete destruction and separation of the sinusoidal endothelium from the underlying parenchyma, causing expansion of the space of Disse and infiltration of erythrocytes (see insets in Fig. 3C). Clusters of mitochondria and condensation of chromatin within hepatocytes were present, indicative of apoptosis (Fig. 3C). In stark contrast the livers of Fc{gamma}RIIB-/- mice showed no hemorrhage, no necrosis (Fig. 3D), and no apoptotic cells were present (Fig. 3E). The sinusoidal endothelium was intact in Fc{gamma}RIIB-/- mice even after a 10-fold higher dose of Jo2 was given (Fig. 3F). Images of livers from mice injected with Cy5.5-Jo2 show localization of Jo2 to the hepatic sinusoidal endothelium in both wild-type (Fig. 4A) and Fc{gamma}R-/- mice (Fig. 4B). Anti-CD34 staining confirmed physical location of Jo2 on the sinusoidal endothelium (Fig. 4B), whereas staining with the Kupffer cell marker F4/80 indicated no colocalization with this cell type (data not shown). Based on these observations, we propose that the hepatic sinusoidal endothelium is the initial target of Jo2 and that its destruction initiates the series of events culminating in death.



View larger version (127K):
[in this window]
[in a new window]
  		FIGURE 3. Hepatotoxicity, liver cell apoptosis, and destruction of the sinusoidal endothelium induced by Jo2 are Fc{gamma}RIIB dependent. Wild-type mice were injected with 0.34 µg/g of Jo2 and their livers were harvested at 1.5 h, and Fc{gamma}RIIB-/- mice were injected with 3.44 µg/g of Jo2 and their livers were harvested at 24 h. Thin sections of tissue were stained to reveal hepatic architecture (H&E) and to identify apoptotic cells (TUNEL), or processed for transmission electron microscopy to visualize ultrastructure. A–C, Livers from wild-type mice displayed extensive sinusoidal hemorrhage and widespread tissue destruction (A), and contained numerous apoptotic cells (B, brown cells). Electron microscopy (C) revealed separation of sinusoidal endothelial cells (ec, arrows) from the underlying hepatic plate, resulting in an expanded space of Disse (sd) and infiltration of erythrocytes (e) (see insets). Clustering of hepatocyte mitochondria (m) and condensation of chromatin within hepatocyte nuclei (h) are indicative of apoptosis. D–F, Despite exposure to a 10-fold higher dose of Jo2, in Fc{gamma}RIIB-/- mice the livers were not hemorrhaged (D), no apoptotic cells were seen (E), and hepatocytes and sinusoidal endothelium appeared normal (F). Each image is representative of multiple tissue sections from livers of at least 3 mice (Original magnification x40 (A, B, D, E) and x1900 (C, F)). sl, sinusoidal lumen; ec, endothelial cell.

 


View larger version (90K):
[in this window]
[in a new window]
  		FIGURE 4. Absence of Fc{gamma}Rs does not affect the localization of Jo2 at hepatic sinusoids. Mice were injected with Cy5.5-labeled Jo2 (9 µg) and their livers collected after 10 min. and processed for confocal imaging. A, A section of liver from a wild-type mouse, showing Cy5.5-labeled Jo2 (blue) localized in the hepatic sinusoids. Autofluorescence has been artificially colored green to reveal the parenchyma and erythrocytes (bright green). B, A section of liver from a Cy5.5-Jo2 injected Fc{gamma}R-/- mouse, poststained with anti-CD34 (red). Note the magenta color, indicating substantial overlap between Jo2 (blue) and the sinusoidal endothelium marker (red).

 
Modulation of cytotoxicity of anti-Fas Abs by Fc{gamma}R is not limited to Jo2

Murine HFE7A (muHFE7A) is another potent agonistic anti-Fas Ab but it is not hepatotoxic in mice (8). We used muHFE7A to determine whether Fc{gamma}R-dependence is a general property of anti-Fas Abs or a unique attribute of Jo2. In a mixed population system using mouse macrophages as bystanders and SKW6.4 cells as targets, we found that muHFE7A induced significantly less apoptosis if macrophages from Fc{gamma}RIIB-/- mice were used (Fig. 5, open bars). This observation resembles the Fc{gamma}RIIB requirement of Jo2 (Fig. 1D). However, in contrast to Jo2, macrophages from FcR-{gamma}-/- mice completely failed to support muHFE7A-mediated apoptosis (Fig. 5). Additional tests showed that Fc{gamma}RIII-/- macrophages also failed to support muHFE7A-mediated apoptosis (Fig. 5). Thus, despite the requirement of Fc{gamma}RIIB, the contribution of Fc{gamma}RIII is crucial to muHFE7A-mediated apoptosis that is not realized for Jo2. These muHFE7A data show that the modulating effect of Fc{gamma}R is not a unique property of Jo2. Furthermore, comparison of muHFE7A to huHFE7A revealed that apoptosis induction by the humanized (huIgG1) Ab is probably dependent on Fc{gamma}RI, but not Fc{gamma}RIII as shown for its parent mouse IgG1 Ab (Fig. 5, solid bars). These findings indicate that the preference of Fc{gamma}Rs that promote agonistic activity is determined by the structural and functional properties of regions other than the Ag-combining site because muHFE7Aand huHFE7A have virtually an identical Fab region (data not shown).



View larger version (19K):
[in this window]
[in a new window]
  		FIGURE 5. Apoptosis inducing activity of HFE7A Abs require different Fc{gamma}Rs. Human B lymphoblast (SKW6.4) cells (2 x 105) sensitized with murine anti-human Fas mAb HFE7A (muHFE7A) or its humanized counterpart (huHFE7A) were incubated (37°C, 4 h) with mouse peritoneal macrophages (4 x 104) and the proportion of SKW6.4 cells undergoing apoptosis was determined. The asterisks indicate significantly less apoptosis compared with the Ab-matched wild-type group (p < 0.005, t test).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Agonistic Abs like Jo2 and HFE7A are used extensively in vitro to study apoptotic-signaling pathways. The Abs are selected to target death receptors and thereby mimic naturally occurring death receptor ligands. By and large, it is assumed that the biological activities they support in vitro are faithfully recapitulated in vivo. However, in vivo studies with such agonistic Abs have produced conflicting results. On one hand, Abs against Fas and other death receptors do not always induce apoptosis as well as natural death receptor ligands, and on the other, Abs sometimes induce little or no death signal in vitro yet express significant activity in vivo (8, 24, 25). The data presented here offer a solution to this apparent paradox. In the case of anti-Fas Abs, apoptosis-inducing ability is intimately involved with engagement of Fc{gamma}Rs. In the special case of Jo2, Fc{gamma}RIIB is essential for apoptosis of A20 cells in vitro and destruction of liver cells in mice.

Our results from comparison of the effects of Jo2 on A20 vs IIA1.6 cells are in agreement with a previous report that also showed A20 cells, but not their Fc{gamma}R-negative counterparts, were sensitive to Fas-mediated death induced by Jo2 (5). In that report, three models were proposed to explain Fc{gamma}R-dependent Jo2-induced apoptosis. In model 1, two independent intracellular signals via Fas and Fc{gamma}R are required for the induction of Fas-mediated apoptosis. Our finding that IIA1.6 cells transfected with a cytoplasmic tailless variant of human Fc{gamma}RIIB refutes this model, because the tailless mutant lacks the intracellulor tyrosine-based motif required for SHIP-mediated Fc{gamma}RIIB signaling (21). In model 2, Jo2 bound Fas and Fc{gamma}R to induce apoptosis in a single cell. This model presumes cis-activation by Jo2, akin to that seen in granulocytes targeted by anti-neutrophil cytoplasmic Abs (22, 23). This possibly does occur, as treatment with mAb 2.4G2 decreases surface binding of Jo2 to A20 cells (5). Furthermore, the dissociation constants of the Jo2/Fas receptor interaction are 0.15 x 10-9 M and 1.5 x 10-9 M for Fas on A20 (Fc{gamma}RIIB-positive) and IIA1.6 (Fc{gamma}RIIB-negative) cells, respectively (K. R. Zinn, unpublished observations). Thus, coexpression of Fc{gamma}RIIB apparently increases physical association of Jo2 to A20 cells. This would be a reasonable biochemical basis for the observed acceleration of Jo2 induced apoptotic death of A20 cells. In addition, our experiments indicate clearly that Jo2 kills Fc{gamma}RIIB-negative IIA1.6 cells in the presence of A20 cells. Apparently, Jo2 interacts with Fc{gamma}RIIB on bystander A20 cells in a trans fashion (model 3) and facilitates oligomerization of Fas into biologically active apoptosis-inducing complexes (2). The apoptosis-promoting function of Fc{gamma}Rs does not require the cytoplasmic domain, which supports our proposal that Fc{gamma}RIIB acts as a scaffold for the Jo2/Fas signaling complex. Noteworthy are our observations that Fc{gamma}RIIB displayed by macrophages also supported Jo2-induced killing, but Fc{gamma}RI/RIII did not. Thus, despite its Fc{gamma}R dependence, and in direct contrast to anti-tumor Abs (1), apoptosis promoting activity of Jo2 was ADCC independent.

To our surprise, mice lacking Fc{gamma}RIIB were completely resistant to Jo2-mediated lethal hepatotoxicity. Our analysis of liver tissues showed that Jo2 localized to the hepatic sinusoidal endothelium, but in the absence of Fc{gamma}RIIB Jo2 did not destroy it. Consequently, the massive hemorrhage and parenchymal necrosis typically seen after Jo2 administration was entirely avoided. Others have suggested that destruction of hepatic sinusoidal cells (SECs) was important to the toxic effect of Jo2 (9, 26), but none predicted this might be controlled by Fc{gamma}RIIB. Vascular endothelial cells in the brain, kidney, and heart of mice also undergo apoptosis after Jo2 injection (27), but these effects cause neither hemorrhage in these organs nor lethality. Hepatic SECs possess fenestrae, lack a basement membrane, and coexpress Fas and Fc{gamma}RIIB. These unique histological features of SECs and preferential binding of Jo2 to Fc{gamma}RIIB render SECs much more sensitive to Jo2-induced apoptosis than other liver cells and ordinary endothelial cells in other organs, and their destruction is the trigger for hepatotoxicity. We do not rule out the contribution of other cell types. Others (28) recently showed that Kupffer cells, resident phagocytes that lie juxtaposed to SECs, play a major role in combating the hepatotoxic effect of Jo2. Thus depletion and/or suppression of Kupffer cells via GdCl3 treatment are associated with an increased hepatotoxic effect of Jo2 due to a compromised phagocytosis of apoptotic cells (28). Kupffer cells also help maintain morphological integrity of SECs and overall sinusoidal architecture (29). We showed that in vitro phagocytosis by FcR-{gamma}-/- macrophages was impaired, and that FcR-{gamma}-/- mice were more susceptible to lethal effect of Jo2 than wild-type animals might reflect their compromised Kupffer cell function.

Death receptors are potentially valuable targets in autoimmune or cancer therapy because they trigger apoptosis. Activation of death receptor dependent apoptotic signaling pathways requires engagement of cognate ligand or an agonistic mAb. For members of the TNFR superfamily, receptor trimerization seems to be a minimal structural unit for its apoptotic function (30, 31). An important question is how a bivalent IgG-based therapeutic, without exogenous cross-linking agent, induces potent apoptosis response in vivo. The present study indicates that Fc{gamma}R on the surface of bystander cells could serve as a cross-linking agent and provide a higher level of aggregation of Fas receptors. This endogenous cross-linking mechanism is distinct from ADCC, which was clearly demonstrated to contribute to anti-tumor activity of some therapeutic mAbs (1). Our findings and those of others also indicate that the Fc region of an agonistic mAb and the microenvironment where Ab, Ag, and Fc{gamma}Rs interact impact the efficacy and safety of anti-Fas Abs. This probably applies to any therapeutic mAb, and perhaps to Fc-fusion proteins with the potential to crosslink in vivo cell surface Ags and Fc{gamma}Rs. Predicting the in vivo outcome of Ab-based therapy remains a difficult task, but the accuracy of prediction can be increased by recognizing that Fc{gamma}Rs can contribute in ways that involve FcR-dependent mechanisms via ADCC or non-ADCC. Therapeutic Abs can thus be engineered to avoid recruitment of detrimental Fc{gamma}R-dependent pathways or to ensure recruitment of beneficial ones.


   Acknowledgments
 
We thank Zhihong Yu for her assistance with cytotoxicity assays, Mark A. McCrory and Christina Pass for their help with the animal studies, De Liu for preparing tissue sections for histochemical staining, and Christina Rodriguez-Burford for her assistance with imaging studies.


   Footnotes
 
1 This study was funded by a grant (to Y.X.) under the auspices of a program project (to W.J.K.) from the Sankyo Co. Ltd. Back

2 Address correspondence and reprint requests to Dr. Yuanyuan Xu, Division of Clinical Immunology and Rheumatology, Department of Medicine, University of Alabama, Birmingham, 1900 University Boulevard, Birmingham, AL 35294-0006. E-mail address: yuanyuan.ma{at}ccc.uab.edu Back

3 Abbreviations used in this paper: ADCC, Ab-dependent cell-mediated cytotoxicity; ALT, alanine aminotransferase; EA: Ab sensitized erythrocyte; SEC, sinusoidal endothelial cell; SHIP, Src homology-2 containing inositol 5'-phosphatase; TEM, transmission electron microscopy. Back

Received for publication February 4, 2003. Accepted for publication May 1, 2003.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Clynes, R. A., T. L. Towers, L. G. Presta, J. V. Ravetch. 2000. Inhibitory Fc receptors modulate in vivo cytotoxicity against tumor targets. Nat. Med. 6:443.[Medline]
  2. Bodmer, J.-L., P. Schneider, J. Tschopp. 2002. The molecular architecture of the TNF superfamily. Trends Biochem. Sci. 27:19.[Medline]
  3. Suda, T., H. Hashimoto, M. Tanaka, T. Ochi, S. Nagata. 1997. Membrane Fas ligand kills human peripheral blood T lymphocytes, and soluble Fas ligand blocks the killing. J. Exp. Med. 186:2045.[Abstract/Free Full Text]
  4. Siegel, R. M., F. K. Chan, H. J. Chun, M. J. Lenardo. 2000. The multifaceted role of Fas signaling in immune cell homeostasis and autoimmunity. Nat. Immunol. 6:469.
  5. Bando, M., Y. Miyake, M. Shiina, M. Wachi, K. Nagai, T. Kataoka. 2002. Actin cytoskeleton is required for early apoptosis signaling induced by anti-Fas antibody but not Fas ligand in murine B lymphoma A20 cells. Biochem. Biophys. Res. Comm. 290:268.[Medline]
  6. Jodo, S., A. M. Hohlbaum, S. Xiao, D. Chan, D. Strehlow, D. H. Sherr, A. Marshak-Rothstein, S. T. Ju. 2000. CD95 (Fas) ligand-expressing vesicles display antibody-mediated, FcR-dependent enhancement of cytotoxicity. J. Immunol. 165:5487.[Abstract/Free Full Text]
  7. Ogasawara, J., R. Watanabe-Fukunaga, M. Adachi, A. Matsuzawa, T. Kasugai, Y. Kitamura, N. Itoh, T. Suda, S. Nagata. 1993. Lethal effect of the anti-Fas antibody in mice. Nature 364:806.[Medline]
  8. Ichikawa, K., H. Yoshida-Kato, M. Ohtsuki, J. Ohsumi, J. Yamaguchi, S. Takahashi, Y. Tani, M. Watanabe, A. Shiraishi, K. Nishioka, et al 2000. A novel murine anti-human Fas mAb which mitigates lymphadenopathy without hepatotoxicity. Int. Immunol. 12:555.[Abstract/Free Full Text]
  9. Wanner, G. A., L. Mica, E. Wanner-Schmid, S. A. Kolb, H. Hentze, O. Trentz, W. Ertel. 1999. Inhibition of caspase activity prevents CD95-mediated hepatic microvascular perfusion failure and restores Kupffer cell clearance capacity. FASEB J. 13:1239.[Abstract/Free Full Text]
  10. Cardier, J. E., T. Schulte, H. Kammer, J. Kwak, M. Cardier. 1999. Fas (CD95, APO-1) antigen expression and function in murine liver endothelial cells: implications for the regulation of apoptosis in liver endothelial cells. FASEB J. 13:1950.[Abstract/Free Full Text]
  11. Nakamura, S., H. Muro, S. Suzuki, T. Sakaguchi, H. Konno, S. Baba, A. S. Syed. 1997. Immunhistochemical studies on endothelial cell phenotype in hepatocellular carcinoma. Hepatology 26:407.[Medline]
  12. Takai, T., M. Li, D. Sylvestre, R. Clynes, J. V. Ravetch. 1994. FcR {gamma}-chain deletion results in pleiotrophic effector cell defects. Cell 76:519.[Medline]
  13. Takai, T., M. Ono, M. Hikida, H. Ohmori, J. V. Ravetch. 1996. Augmented humoral and anaphylactic responses in Fc{gamma}RII-deficient mice. Nature 379:346.[Medline]
  14. Hazenbos, W. L., J. E. Gessner, F. M. Hofhuis, H. Kuipers, D. Meyer, I. A. Heijnen, R. E. Schmidt, M. Sandor, P. J. Capel, M. Daeron, et al 1996. Impaired IgG-dependent anaphylaxis and Arthus reaction in Fc{gamma}RIII (CD16) deficient mice. Immunity 5:181.[Medline]
  15. Larsen, S. K., H. F. Solomon, G. Caldwell, M. J. Abrams. 1995. [99mTc]tricine: a useful precursor complex for the radiolabeling of hydrazinonicotinate protein conjugates. Bioconjugate Chem. 6:635.[Medline]
  16. Grizzle, W. E., R. B. Myers, U. Manne, S. Srivastava. Immunohistochemical evaluation of biomarkers in prostatic and colorectal neoplasia. M. Hanausek, and Z. Walaszek, eds. Methods in Molecular Medicine In Tumor Marker Protocols 14:143. Humana Press, Totowa, NJ.
  17. Jones, B., J. P. Tite, C. A. Janeway, Jr. 1986. Different phenotypic variants of the mouse B cell tumor A20/2J are selected by antigen- and mitogen-triggered cytotoxicity of L3T4-positive, I-A-restricted T cell clones. J. Immunol. 136:348.[Abstract]
  18. Liu, L., A. Chahroudi, G. Silvestri. M. E. Wernett, W. J. Kaiser, J. T. Safrit, A. Komoriya, J. D. Altman, B. Z. Packard, M. B. Feinberg. 2002. Visualization and quantification of T cell-mediated cytotoxicity using cell-permeable fluorogenic caspase substrates. Nat. Med. 8:185.[Medline]
  19. Shiratsuchi, A., S. Osada, S. Kanazawa, Y. Nakanishi. 1998. Essential role of phophatidylserine externalization in apoptosing cell phagocytosis by macrophages. Biochem. Biophys. Res. Commun. 246:549.[Medline]
  20. Barnes, N., A. L. Gavin, P. S. Tan, P. Mottram, F. Koentgen, P. M. Hogarth. 2002. Fc{gamma}RI-deficient mice show multiple alterations to inflammatory and immune responses. Immunity 16:379.[Medline]
  21. Pearse, R. N., T. Kawabe, S. Bolland, R. Guinamard, T. Kurosaki, J. V. Ravetch. 1999. SHIP recruitment attenuates Fc{gamma}RIIB-induced B cell apoptosis. Immunity 10:753.[Medline]
  22. Mulder, A. H., P. Heeringa, E. Brouwer, P. C. Limburg, C. G. Kallenberg. 1994. Activation of granulocytes by anti-neutrophil cytoplasmic antibodies (ANCA): a Fc{gamma}RII-dependent process. Clin. Exp. Immunol. 98:270.[Medline]
  23. Porges, A. J., P. B. Redecha, W. T. Kimberly, E. Csernok, W. L. Gross, R. P. Kimberly. 1994. Anti-neutrophil cytoplasmic antibodies engage and activate human neutrophils via Fc{gamma}RIIa. J. Immunol. 153:1271.[Abstract]
  24. Huang, D. C., M. Hahne, M. Schroeter, K. Frei, A. Fontana, A. Villunger, K. Newton, J. Tschopp, A. Strasser. 1999. Activation of Fas by FasL induces apoptosis by a mechanism that cannot be blocked by Bcl-2 or Bcl-xL. Proc. Natl. Acad. Sci. USA 96:14871.[Abstract/Free Full Text]
  25. Chuntharapai, A., K. Dodge, K. Grimmer, K. Schroeder, S. A. Marsters, H. Koeppen, A. Ashkenazi, K. J. Kim. 2001. Isotype-dependent inhibition of tumor growth in vivo by monoclonal antibodies to death receptor 4. J. Immunol. 166:4891.[Abstract/Free Full Text]
  26. Cardier, J. E., C. L. Erickson-Miller. 2002. Fas (CD95)- and tumor necrosis factor-mediated apoptosis in liver endothelial cells:role of caspase-3 and the p38 MAPK. Microvasc. Res. 63:10.[Medline]
  27. Janin, A., C. Deschaumes, M. Daneshpouy, J. Estaquier, J. Micic-Polianski, P. Rajagopalan-Levasseur, K. Akarid, N. Mounier, E. Gluckman, G. Socie, J. C. Ameisen. 2002. CD95 engagement induces disseminated endothelial cell apoptosis in vivo: immunopathologic implications. Blood 99:2940.[Abstract/Free Full Text]
  28. Deaciuc, I., N. D’Souza, M. Nikolova-Karakashian, W. de Villiers, T. Sarphie, D. Hill, C. McClain. 2002. The regulation of Fas (CD95/Apo-1)-mediated liver apoptosis in Kupffer cell-depleted mice. Hepatol. Res. 24:192.[Medline]
  29. Niwano, M., S. Arii, K. Monden, S. Ishiguro, T. Nakamura, M. Mizumoto, Y. Takeda, M. Fujioka, M. Imamura. 1997. Amelioration of sinusoidal endothelial cell damage by Kupffer cell blockade during cold preservation of rat liver. J. Surg. Res. 72:36.[Medline]
  30. Ashkenazi, A., V. Dixit. 1998. Death receptors: signaling and modulation. Science 281:1305.[Abstract/Free Full Text]
  31. Banner, D. W., A. D’Arcy, W. Janes, R. Gentz, H.-J. Schoenfeld, C. Broger, H. Loetscher, W. Lesslauer. 1993. Crystal structure of the soluble human 55 kd TNF receptor-human TNF-{alpha} complex: implications for TNF receptor activation. Cell 73:431.[Medline]



This article has been cited by other articles:


Home page
 Cancer Res.Home page
H. M. Horton, M. J. Bernett, E. Pong, M. Peipp, S. Karki, S. Y. Chu, J. O. Richards, I. Vostiar, P. F. Joyce, R. Repp, et al.
Potent In vitro and In vivo Activity of an Fc-Engineered Anti-CD19 Monoclonal Antibody against Lymphoma and Leukemia
Cancer Res., October 1, 2008; 68(19): 8049 - 8057.
[Abstract] [Full Text] [PDF]

Home page
 Int ImmunolHome page
J. Nakayama, Y. Ogawa, Y. Yoshigae, Y. Onozawa, A. Yonemura, M. Saito, K. Ichikawa, T. Yamoto, T. Komai, T. Tatsuta, et al.
A humanized anti-human Fas antibody, R-125224, induces apoptosis in type I activated lymphocytes but not in type II cells
Int. Immunol., January 1, 2006; 18(1): 113 - 124.
[Abstract] [Full Text] [PDF]

Home page
 Clin. Cancer Res.Home page
K. Motoki, E. Mori, A. Matsumoto, M. Thomas, T. Tomura, R. Humphreys, V. Albert, M. Muto, H. Yoshida, M. Aoki, et al.
Enhanced Apoptosis and Tumor Regression Induced by a Direct Agonist Antibody to Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand Receptor 2
Clin. Cancer Res., April 15, 2005; 11(8): 3126 - 3135.
[Abstract] [Full Text] [PDF]

Home page
 BloodHome page
E. van Mirre, A. van Royen, C. E. Hack, A. R. Crow, S. Song, J. Freedman, C. D. Helgason, R. K. Humphries, K. A. Siminovitch, and A. H. Lazarus
IVIg-mediated amelioration of murine ITP via Fc{gamma}RIIb is not necessarily independent of SHIP-1 and SHP-1 activity
Blood, March 1, 2004; 103(5): 1973 - 1974.
[Full Text] [PDF]

Home page
 JEMHome page
K. Takeda, N. Yamaguchi, H. Akiba, Y. Kojima, Y. Hayakawa, J. E. Tanner, T. J. Sayers, N. Seki, K. Okumura, H. Yagita, et al.
Induction of Tumor-specific T Cell Immunity by Anti-DR5 Antibody Therapy
J. Exp. Med., February 17, 2004; 199(4): 437 - 448.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Xu, Y.
Right arrow Articles by Kimberly, R. P.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Xu, Y.
Right arrow Articles by Kimberly, R. P.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS