Journal of Leukocyte Biology BioLegend: Treg, Th17, Stem Cell
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Originally published online as doi:10.1189/jlb.0904536 on October 4, 2005

Published online before print October 4, 2005
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
jlb.0904536v1
78/5/1052    most recent
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
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 reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Bonardelle, D.
Right arrow Articles by Bobé, P.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Bonardelle, D.
Right arrow Articles by Bobé, P.
(Journal of Leukocyte Biology. 2005;78:1052-1059.)
© 2005 by Society for Leukocyte Biology

B lymphocytes mediate Fas-dependent cytotoxicity in MRL/lpr mice

Danielle Bonardelle*, Karim Benihoud{dagger},{ddagger}, Nicole Kiger§ and Pierre Bobé*,{ddagger},1

* CNRS UPR 9045, Villejuif, France;
{dagger} CNRS/Institut Gustave Roussy, UMR 8121, Villejuif, France;
{ddagger} Université Paris-Sud, Orsay, France; and
§ INSERM U 267, Villejuif, France

1Correspondence: CNRS UPR 9045, Laboratoire d’Oncologie Virale, 7 rue Guy Môcquet, 94801 Villejuif, Cedex, France. E-mail: bobe{at}infobiogen.fr


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The Fas/Fas ligand (FasL) pathway is one of the two major effector mechanisms of T cell-mediated cytotoxicity. To prevent nonspecific killing by lymphoid cells, FasL expression on the cell surface of immune effector cells is strictly regulated. However, MRL/lpr autoimmune-prone mice massively overexpress FasL on their T lymphocytes, which render them able to kill Fas+ targets in vitro and in vivo. It is surprising that we show in the present work that B lymphocytes purified from MRL/lpr spleen cells express FasL to the same extent as T cells at the mRNA and protein level. These B cells are potent cytotoxic effectors against Fas+ but not Fas– targets. The B lymphocyte effectors were used ex vivo without any in vitro activation by B cell stimuli. Furthermore, we found that MRL/lpr B lymphocytes have the same cytotoxic potential as natural killer cells, which have been characterized as potent, Fas-mediated, cytotoxic effectors. The level of membrane-anchored FasL increases with the size of the B cell and cell-surface activation marker CD69 expression, indicating that the expression of FasL is up-regulated in parallel with the activation state of the B cell. The activated B cell population contained the major cytotoxic activity, and a minor part was associated with CD138/Syndecan-1+ plasma cells.

Key Words: activated B cell • Fas ligand • CD69 • autoimmunity


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Fas and its ligand FasL are cell-surface proteins whose interaction triggers a cascade of subcellular events that result in apoptosis of the Fas-bearing cell [1 , 2 ]. In lymphoid tissue, FasL is expressed on the cell surface of activated T lymphocytes and natural killer (NK) cells [3 ]. FasL expression on B lymphocytes is more disputed, as prior studies by Suda et al. [4] claimed that activated B cells do not express FasL. However, several other reports have demonstrated that activated B cells, like activated T cells, can express FasL [5 6 7 8 ], which on the cell surface of immune effector cells, is strictly regulated compared with Fas [4 , 9 ]. Once at the cell surface, FasL molecules are cleaved rapidly by a metalloprotease to prevent nonspecific killing by lymphoid cells [10 , 11 ]. The Fas/FasL system plays an essential role in the cytotoxic activity of T and NK cells and in downsizing the pool of activated T lymphocytes that has expanded during an immune response [2 , 3 ]. Engagement of Fas by FasL is also involved in the maintenance of peripheral B cell tolerance and prevention of inopportune T cell help [12 , 13 ]. The functional role of FasL on activated B cells is a matter of controversy. In vivo experiments with normal/FasL-deficient mixed bone marrow chimeras suggested that FasL controls the expansion of lymphocytes only when it is expressed on T cells [14 ]. In contrast, using a model of Schistosoma mansoni infection, FasL-expressing splenic B cells were shown to mediate CD4+ T cell apoptosis [7 ]. The occurrence of FasL-mediated B cell-B cell killing remained uncertain until Zuniga et al. [8] investigated the mechanisms involved in B lymphocyte apoptosis during Trypanosoma cruzi infection. They demonstrated that B lymphocyte apoptosis is mediated by Fas/FasL interaction and preferentially affects highly activated B cells specific for parasite antigens.

Dysfunctional Fas signaling, the underlying defect in humans with an autoimmune lymphoproliferative syndrome and the so-called lymphoproliferation (lpr) phenotype in mice, results in a massive accumulation of T cell receptor (TCR){alpha}ß+ CD4–CD8– [double-negative (DN)] T cells in peripheral lymphoid organs [15 , 16 ]. However, lpr mutation cannot account for the entire autoimmune syndrome of MRL/lpr mice, and other genes of pathologic importance in the MRL background have been mapped [17 , 18 ].

The DN T cells are derived from CD4+CD8– or CD4–CD8+ [single-positive (SP)] T lymphocytes [19 , 20 ], which despite antigen stimulations, have not undergone activation-induced cell death because of their defective fas gene [21 , 22 ]. In lpr mice, DN T cells express the B220 molecule. Activated T cells normally up-regulate B220 before undergoing apoptosis. When the apoptotic mechanisms are defective, T cells down-regulate their CD4 or CD8 molecules but retain the B220 expression while residing in lymphoid organs [23 ]. These DN T cells massively overexpress FasL, which renders them able to kill Fas+ targets in vitro [24 25 26 ]. MRL/lpr SP T cells are also endowed with Fas-mediated cytotoxic potential [27 ]. Furthermore, we have shown that interferon-{alpha} (IFN-{alpha}) plays an essential role in the overexpression of FasL on MRL/lpr lymphocytes [28 ]. The lpr mutation is leaky, and low but detectable levels of transcription [29 , 30 ] and translation [31 ] of the wild-type Fas molecule have been reported in this strain, as illustrated by the detection of significant amounts of Fas on MRL/lpr hepatocytes, in contrast to hepatocytes from mice rendered completely Fas-deficient by gene targeting [32 ]. Therefore, the overexpression of FasL by activated MRL/lpr lymphocytes could be responsible for a chronic, nonantigen-specific autoimmune attack on organs expressing low levels of the Fas receptor. The ability of FasL overexpressing T cells to induce a nonantigen-specific autoimmune attack was shown in hematopoietic chimeras created by grafting lethally irradiated wild-type MRL-+/+ mice with MRL/lpr lymphoid cells [27 , 33 ].

In this study, we report that highly purified B lymphocytes from autoimmune MRL/lpr spleen cells express high levels of FasL, comparable with MRL/lpr T lymphocytes and NK cells. Furthermore, they present a cytotoxic activity against Fas+ but not Fas– targets. These B lymphocytes were used ex vivo without any in vitro activation by B cell stimuli. Within cells of the B lineage, activated B lymphocytes and not plasma cells appear to be responsible for most of the killer activity. To the best of our knowledge, this is the first report of a killer B cell activity in an autoimmune pathology. The contribution of killer B cells to the pathology of the autoimmune MRL/lpr strain is discussed.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Mice
Wild-type MRL/MpJ (MRL-+/+) and MRL/MpJ-Tnfrsf6lpr/J mutant mice (MRL/lpr), originally from the Jackson Laboratory (Bar Harbor, ME), were maintained in our animal facilities. All experiments were performed in accordance with the institutional animal research committee guidelines.

Cell preparations
Spleen cell suspensions prepared from several 3-month-old mice were pooled, depleted of erythrocytes by osmotic shock, and deposited onto nylon-wool columns. Effluents, mostly containing T lymphocytes, were enriched further by a second passage through another nylon-wool column. Nylon-wool adherent cells, predominantly containing B lymphocytes and plasma cells, were recovered from the column by adding cold RPMI 1640 and forcibly pushing them out with a plunger. The enriched B cell population was incubated with anti-Thy-1.2 monoclonal antibody [mAb; hybridoma 30-H12, American Type Culture Collection (ATCC), Manassas, VA] at 4°C for 30 min. After washing, the cells were incubated with Low-Tox-M rabbit complement (Cedarlane, Ontario, Canada) at 37°C for 45 min to deplete T cells. Residual contaminating T lymphocytes and NK cells were eliminated from the enriched B cell population by magnetic cell sorter (MACS) technology (Miltenyi Biotec GmbH, Bergisch Gladbach, Germany), according to the manufacturer’s instructions. Briefly, enriched B cells were stained with anti-DX5-fluorescein isothiocyanate (FITC; anti-NK cells; PharMingen, San Diego, CA) and anti-Thy-1.2-FITC (30-H12, PharMingen) antibodies and secondarily, incubated with anti-FITC microbeads; NK and T cells were eliminated by MACS. Similarly, B lymphocytes and plasma cells were separated by MACS. Plasma cells were labeled with a biotinylated anti-CD138/Syndecan-1 antibody (PharMingen), then streptavidin-FITC, and positively selected with anti-FITC microbeads.

CD4+CD– and CD4–CD8+ SP T cells and CD4–CD8– DN T cells were purified from T lymphocyte populations enriched using nylon-wool columns. SP T cells were positively selected by MACS technology using L3T4 (anti-mouse CD4) and Ly-2 (anti-mouse CD8{alpha}) microbeads. Effluents of the columns containing mainly DN T cells were then treated with anti-CD4 and anti-CD8 mAb (respectively, hybridomas GK1-5 and 53-6-72, ATCC) and rabbit complement to avoid any SP T cell contamination.

Cytofluorometric analysis
The expression of different cell-surface antigens on spleen cells, previously incubated with a mAb to mouse Fc receptor for immunogloblulin G (IgG; 2.4G2, PharMingen) to inhibit nonspecific binding, was analyzed by flow cytometry using FITC-, phycoerythrin (PE)-, allophycocyanin (APC)-, or biotin-conjugated mAb or Ig: goat anti-mouse IgM (Sigma Chemical Co., St. Louis, MO) or anti-mouse Ig (PharMingen), rat anti-Thy-1.2 (30-H12, PharMingen), anti-CD3 (CT-CD3, Caltag Laboratories, South San Francisco, CA), anti-CD4 (RM4-5, PharMingen), anti-CD8 (53-6.7, PharMingen), anti-CD49b/Pan-NK cells (DX5, PharMingen), anti-CD45R/B220 (RA3-6B2, PharMingen), anti-CD19 (1D3, PharMingen), armenian hamster anti-CD69 (H1.2F3, PharMingen), and rat IgM, IgG2a, armenian hamster IgG1, and goat Ig as isotype controls (Serotec France, Cergy Saint-Christophe). The expression of FasL on the cell surface of lymphoid cells was analyzed using a soluble, dimeric Fas:Fc{gamma}1 fusion protein and FITC-conjugated rat mAb anti-mouse IgG1 (A85-1, PharMingen) and mouse IgG1 as a control isotype (PharMingen).

Recombinant mouse Fas:Fc{gamma}1 fusion protein
The soluble, dimeric Fas:Fc{gamma}1 fusion protein consists of the mouse Fas extracellular domain (ligand-binding domain) fused to the Fc portion of mouse IgG1. Fas:Fc{gamma}1 recombinant molecules were produced using a recombinant Adenovirus 5 and purified from the infected HeLa cell-conditioned medium by ammonium sulfate precipitation and affinity chromatography on protein A-Sepharose.

Semiquantitative reverse transcriptase-polymerase chain reaction (RT-PCR) and real-time RT-PCR
Total RNA was extracted with RNA-Plus (Qbiogene, Illkirch, France) from spleen cells and spleen-cell subpopulations, and 1 µg each sample was reverse-transcribed into cDNA using a first-strand cDNA synthesis kit (Amersham Pharmacia Biotech, Orsay, France). Semiquantitative PCR were conducted for 25 cycles with the following primer pairs: FasL forward primer (5'-CGT GAG TTC ACC AAC CAA AGC-3') and reverse primer (5'-CCC AGT TTC GTT GAT CAC AAG-3'); CD 40 ligand (CD40L) forward primer (5'-AAA GAT GAG AAG CCA ACT CTG TG-3') and reverse primer (5'-ATT GGA TAA GGT CGA AGA GGA AG-3'); and hypoxanthine-guanine phosphoribosyl transferase (HGPRT) forward primer (5'-GTA ATG ATC AGT CAA CGG GGG AC-3') and reverse primer (5'-CCA GCA AGC TTG CAA CCT TAA CCA-3'). PCR were performed in a total volume of 25 µl in the presence of 2 U Taq polymerase (Promega, Madison, WI); 50 mM KCl; 10 mM Tris-HCl, pH 9; 2.5 mM MgCl2; 0.1% Triton X-100; 0.2 mM each deoxy-unspecified nucleoside 5'-triphosphate; 50 pmol forward and reverse primers; and 5 µl cDNA at a dilution, which gave similar signals for a cDNA produced with HGPRT primers after electrophoresis on 1% agarose gels in the presence of ethidium bromide and ultraviolet illumination. Amplification conditions were as follows: 1 min at 95°C; 1 min at 50°C for Fas or 60°C for HGPRT, FasL, and CD40L; and 1 min at 72°C for 25 cycles. After PCR, 20 µl of the amplified products of each cDNA resolved on 1% agarose gels was blotted and allowed to hybridize with the corresponding radiolabeled oligonucleotide probes: 5'-GCT CTG ATC TCT GGA GTG AAG TAT AAG AAA-3' for FasL; 5'-GGA GTG TTT GAA TTA CAA GCT GG-3' for CD40L, and 5'-GCT TTC CCT GGT TAA GCA GTA CAG CCC C-3' for HGPRT to verify that the bands were specific. Semiquantitative determination (PhosphoImager System, Molecular Dynamics, Sunnyvale, CA) of FasL cDNA, present in each of the various samples, was normalized with respect to the concentration of HGPRT cDNA detected in the same sample, and FasL/HGPRT cDNA ratios were calculated.

Real-time quantitative PCR was conducted with the LightCycler system using the DNA binding dye SYBR Green for the detection of PCR products according to the manufacturer’s instructions (Roche Diagnostics, Mannheim, Germany). In brief, total RNA was isolated and reverse-transcribed as for conventional RT-PCR. Quantitative PCR was performed in a total volume of 20 µl in the presence of 2 µl cDNA, 2 µl 10x LightCycler DNA Master SYBR Green I, 1.25 mM MgCl2, 25 pmol each primer. Primers for FasL and HGPRT have been described above. After being heated at 95°C for 8 min, cDNA was amplified for 35 cycles, each cycle consisting of 95°C for 15 s, 60°C for 5 s, and 72°C for 8 s. For melting curve analysis, samples were heated to 95°C at a transition rate of 0.1°C/s with continuous fluorescence readings. The specificity of PCR products was confirmed by melting curve analysis and by running samples on agarose gels. Specific cDNA was quantified by standard curves based on known amounts of product. Standards were generated from amplified cDNA purified on agarose gels by QiaQuick gel extraction (Qiagen, Courtaboeuf, France) and quantified by spectrophotometry (optical density of 260). The standards and the samples were simultaneously amplified using the same reaction master mixture.

Cytotoxicity assay
Murine target cells consisted of sodium [51Cr]chromate-labeled thymocytes distributed in U-shaped wells (5x104 per well) of microtiter plates with spleen cells or subpopulations purified from spleen cells as effector cells at the effector-to-target (E:T) ratios indicated in 200 µl medium containing 5% fetal calf serum supplemented or not with a mixture of phorbol 12-myristate 13-acetate (PMA; final concentration, 5 ng/ml; P-8139, Sigma Chemical Co.) plus ionomycin (final concentration, 0.5 µg/ml; 407952, Calbiochem, San Diego, CA). After a 4-h incubation, microplates were processed as described previously [26 ], and the SEM (±SE) for specific 51Cr release of triplicates were always <5%. In some experiments, cytotoxicity was assessed in the presence of soluble, dimeric Fas:Fc{gamma}1 fusion protein (1 µM).

Statistical analysis
Data are reported as the mean ± SE. Comparisons were made by Student’s t-test. Statistical difference was accepted at P < 0.05.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
T and B cell lymphoproliferation in MRL/lpr mice
Flow cytometric analyses indicated that in MRL/lpr mice, splenic B cell lymphoproliferation starts at 2 months. At 5 months, analyses obtained for 20 animals show that the IgM+B220+ B cell population is four-to fivefold larger than in wild-type MRL-+/+ mice (mean B cell numbersx107±SE: 9.02±0.92 vs. 1.9±0.3, respectively; P<0.001). This is in parallel to the massive (200-fold increase), well-documented CD4–CD8– DN T cell proliferation (mean DN T cell numbersx107±SE: 25.8±4.9 vs. 0.13±0.03, respectively; P<0.001).

Fas-mediated killing by MRL/lpr T and B cells
According to our previously published data [26 ], total spleen cells and nylon-wool nonadherent T lymphocytes from MRL/lpr mice trigger the death of Fas+ thymocytes upon stimulation with a mixture of PMA plus ionomycin, which mimics TCR activation (Fig. 1 ). This cytotoxicity was Fas-mediated, as shown by the absence of killing of Fas– targets (lpr thymocytes) by MRL/lpr splenocyte effectors (data not shown). Flow cytometry analyses showed that this T cell population contained: 96.6 ± 1.7% CD3+Ig–CD19– (B220– or B220+) T cells, 1.9 ± 0.36% CD3–Ig+CD19+ B cells, and 2.3 ± 0.05% CD3–Ig–CD19– non-T, non-B cells (means±SE for n=6). It is most surprising that nylon-wool adherent B lymphoctes from MRL/lpr mice, depleted of contaminating Thy-1.2high T lymphocytes by antibody plus complement, also killed Fas+ (but not Fas–; data not shown) thymocytes (Fig. 1) . Flow cytometry analyses showed that this B cell population contained: 92.5 ± 2.5% CD3–Ig+CD19+ B cells, 3.73 ± 1.15% CD3+Ig–CD19– (B220– or B220+) T cells, and 5.3 ± 0.7% CD3–Ig–CD19– non-T, non-B cells (means±SE for n=6). In contrast, no killing was obtained when the T cell (containing 96.2±1.3% CD3+Ig–CD19– T cells, 2.9±0.1% CD3–Ig+CD19+ B cells, and 2.8±0.45% CD3–Ig–CD19– non-T, non-B cells) and the B cell population (containing 94±0.25% CD3–Ig+CD19+ B cells, 2.25±0.75% CD3+Ig–CD19– T cells, and 5.12±1.7% CD3–Ig–CD19– non-T, non-B cells) from MRL-+/+ mice (n=8) were activated by PMA plus ionomycin and subsequently used as effector cells (Fig. 1) .



View larger version (10K):
[in this window]
[in a new window]
  		Figure 1. Fas-mediated killing by MRL/lpr spleen cell subpopulations. MRL/lpr (left panel) but not MRL-+/+ effector cells (right panel) trigger the death of MRL-+/+ thymocyte targets in the standard 4-h 51Cr-release assay. Effector cells were total spleen cells ({blacksquare}), nylon-wool nonadherent T cells ({square}), or nylon-wool adherent B cells treated with anti-Thy-1.2 mAb plus complement to deplete T cells ({blacktriangleup}). Results are representative of at least four independent experiments.

 
FasL expression by T and B cells
FasL expression was determined, in parallel to the expressions of CD40L and HGPRT, using semiquantitative RT-PCR analysis on mRNA isolated from the different spleen cell subpopulations of MRL/lpr and wild-type MRL-+/+ mice. As we previously reported [26 ], the nylon-wool nonadherent T cell population overexpressed FasL when purified from MRL/lpr spleen cells (Fig. 2A ). CD4+ and CD8+ SP and CD4–CD8– DN T cells (purified by MACS) from the MRL/lpr T lymphocyte population overexpressed FasL. The level of FasL transcripts in SP and DN MRL/lpr T lymphocytes, quantified with a PhosphoImager, showed an increase of 9.5- and 15-fold, respectively, when compared with their counterparts from MRL-+/+ mice. We compared the FasL RNA level in MRL/lpr DN T lymphocytes versus MRL-+/+ SP T cells, as abnormal DN T cells are absent from MRL-+/+ mice. A peculiar characteristic of the MRL/lpr strain, never found in the MRL-+/+, was the unusual overexpression of FasL by the nylon-wool adherent B cell population. The level of FasL transcripts is increased sevenfold in the MRL/lpr B lymphocyte population compared with MRL-+/+.



View larger version (36K):
[in this window]
[in a new window]
  		Figure 2. Quantification of FasL transcripts in total or purified spleen cells from MRL-+/+ and MRL/lpr mice. (A) Semiquantitative RT-PCR analysis. FasL expression in total (Spleen) or purified spleen cells: nylon-wool nonadherent SP T cells, DN T cells, and nylon-wool adherent cells treated with anti-Thy-1.2 mAb plus complement (B cells); cDNA were amplified from equal quantities of RNA using FasL-, CD40L-, or HGPRT-specific oligonucleotide primers and subsequently analyzed by Southern blotting using FasL-, CD40L-, or HGPRT-specific, internal oligonucleotide probes. These results are representative of at least eight independent experiments. (B) Real-time RT-PCR analysis. FasL expression in purified spleen cells: SP T cells, DN T cells, and nylon-wool adherent cells treated with anti-Thy-1.2 mAb plus complement (B cells). Specific FasL cDNA was quantified by standard curves based on known amounts of product, which are 4 pg, 0.4 pg, 0.04 pg, and 0.004 pg FasL. The results are expressed as amount (pg) of FasL RNA per cell. These results are representative of four independent experiments.

 
The FasL mRNA level in the different spleen cell subpopulations was quantified accurately by real-time RT-PCR (Fig. 2B) . The level of FasL transcripts in SP and DN MRL/lpr T lymphocytes showed an increase of 15- and 200-fold, respectively, when compared with MRL-+/+ SP T cells. The level of FasL transcripts was increased 180-fold in the MRL/lpr B lymphocyte population compared with MRL-+/+. It is surprising that in MRL/lpr but not MRL-+/+ mice, we repeatedly found that the FasL mRNA level in B cells is greater than in SP T cells (4.5-fold increase; 1.25 and 0.28 pg/cell, respectively), similar to the level observed in DN T cells (3.8 pg/cell). In addition, CD40L is up-regulated in MRL/lpr SP T lymphocytes reflecting their state of activation in vivo. Expression of CD40L was restricted to MRL/lpr SP as opposed to DN T cells (thereby explaining the weak band in whole T cells), and a faint signal was observed in MRL-+/+ SP T lymphocytes (Fig. 2A) .

The surface expression of FasL on MRL/lpr and MRL-+/+ spleen cell subpopulations was determined by flow cytometry using soluble, recombinant Fas:Fc{gamma}1 fusion protein, anti-CD3, and anti-CD19 mAb. As expected, the FasL molecule is overexpressed on the MRL/lpr T cell population, especially on DN T cells (Fig. 3A and data not shown). In line with the FasL mRNA quantification, the FasL molecule is overexpressed also on the surface of MRL/lpr CD19+ B cells. Moreover, MRL/lpr CD19+ B cells with the highest FSC value showed the highest expression of FasL (Fig. 3B) , suggesting that the FasL molecule is expressed by activated B lymphocytes. Indeed, lymphocyte activation is associated with an increase in size, and this can be detected by an increase in the FSC profile. It is important that we excluded the possibility that FasL+ T cell-forming doublets with B cells are the source of FasL-expressing cells in the CD19+ B cell population. Indeed, we only found 2% of B:T cell doublets in the CD19+ B cell populations with intermediate to high FSC. Likewise, we looked for the presence of doublets of B cells and FasL+ NK cells in the flow cytometry analysis, and we only found 0.4% of B:NK doublets. However, as in MRL/lpr mice, FasL+ DN T cells express the B220 B cell marker, it may be postulated that CD19 is also expressed on some variable number of FasL+ DN T cells. To test this hypothesis, we analyzed MRL/lpr spleen cells by flow cytometry using anti-CD3, anti-CD19, and anti-Ig antibodies and soluble, recombinant Fas:Fc{gamma}1 fusion protein. Although the CD19 molecule is detected on the surface of all Ig+ (FasL– or FasL+) B cells, CD19-expressing CD3+ (FasL– or FasL+) T cells were never detected (data not shown). In addition, we examined the surface expression of the early activation marker CD69, which has been shown to be up-regulated after B cell receptor stimulation in primary lymphocytes. The percentage of CD69+ cells in the B cell population of MRL/lpr mice was significantly higher than that of wild-type MRL-+/+ mice (means±SE from 12 mice: 27±0.95% vs. 6.8±2%, respectively; P<0.001). Furthermore, the surface expression of FasL on CD69+CD19+ B lymphocytes was determined by three-color flow cytometry using soluble, recombinant Fas:Fc{gamma}1 fusion protein, anti-CD19, and anti-CD69 mAb. The FasL molecule is detected mainly on CD69+CD19+ B cells (Fig. 3C) . Hence, FasL expression is associated with B cell activation status.



View larger version (43K):
[in this window]
[in a new window]
  		Figure 3. FasL expression on MRL/lpr T and B cells by flow cytometry. (A) Spleen cells were stained for CD3 and FasL. CD3 versus a forward light scatter (FSC) dot plot was used to define bitmap gates for T cells. FasL expression on CD3+ T cells is shown as filled histograms. Staining with control isotype is shown as open histograms. M1 indicates the percentage of FasL+CD3+ T cells. M1 is defined as the difference between the positive-control and negative-control histograms, which correspond to the percentage of cells stained with the anti-FasL antibody and isotype control, respectively. The data shown are representative of five independent experiments. (B) Spleen cells were stained for CD19 and FasL. CD19 versus FSC dot plot was used to define bitmap gates for B cells with different sizes (FSC). FasL expression on CD19+ B cells with different FSC properties is shown as filled histograms. Staining with control isotype is shown as open histograms. M1 indicates the percentage of FasL+CD19+ B cells. The data shown are representative of five independent experiments. (C) Cells were stained for CD19, CD69, and FasL. CD19 versus CD69 dot plot was used to define bitmap gates for B cells (minimum of 10,000 events) with different activation states. FasL expression on CD69–CD19+ and CD69+CD19+ B cells is shown as filled histograms. Staining with control isotype is shown as open histograms. M1 indicates the percentage of FasL+ B cells. The data shown are representative of five independent experiments.

 
Fas-mediated killing by MRL/lpr T, NK, and B cells
The nylon-wool adherent MRL/lpr spleen cells depleted of Thy-1.2 lymphocytes by antibody plus complement (mostly B lymphocytes) contained, as shown by flow cytometry analysis (Fig. 4A ): 26% large granular cells in the FSChigh side-scatterhigh (SSC) gate; 16–22% of these cells expressed the NK epitope, DX5, and the B220 B cell marker, also found on some NK1.1+ cells [34 ]. The DX5+ NK cell population represents 0.1–1% of unfractionated spleen cells in MRL/lpr and MRL-+/+ strains. Therefore, a difference in the number of NK cells contaminating nylon-wool adherent spleen cells cannot account for the different cytotoxic abilities of these subpopulations (Fig. 1) . Nevertheless, to determine whether these NK cells could be responsible for the killing capacity of nylon-wool adherent cells, DX5- and Thy-1.2-positive cells were depleted immunomagnetically. Purified B cells, contaminated with <2% cells labeled with Thy1.2 and DX5 antibodies, as shown by cytofluorometric analysis, still retained a substantial Fas-dependent cytotoxic activity (Fig. 4B , upper). The cytotoxicity of B lymphocytes was dependent strictly on Fas/FasL interaction, as the soluble Fas:Fc{gamma}1 fusion protein inhibited specifically, in a dose-dependent manner, the B cell cytotoxicity on Fas+ thymocytes, and the control fusion protein (secreted alkaline phosphatase Fc) had no effect (data not shown). As expected, the positively selected Thy-1.2+ or DX5+ populations (T and NK cells, respectively) were potent effectors of Fas-mediated cytotoxicity (Fig. 4B , upper).



View larger version (41K):
[in this window]
[in a new window]
  		Figure 4. Phenotypic characterization of nylon-wool adherent cells and Fas-mediated killing by MRL/lpr T, NK, and B cells. (A) Flow cytometry analysis of the adherent population showed that 26% (within the bitmap) were large granular cells. The scatter plot gated on these cells shows DX5 versus B220 expression. Numbers refer to the percentages of positive cells. (B) Cytotoxicity assay by 51Cr release. (Upper) Fas-mediated cytotoxicity of B cells ({blacktriangleup}) depleted of Thy-1.2+ T and DX5+ NK cells by MACS, Thy-1.2+ MACS-purified T cells ({square}), and DX5+ MACS-purified NK cells ({blacksquare}). (Lower) Fas-mediated cytotoxicity of B cells ({blacktriangleup}) depleted of Thy-1.2+ T and DX5+ NK cells by MACS. This B cell population represents a mixture of B220+ B lymphocytes plus B220– plasma cells. Syndecan-1–B220+ B lymphocytes ({Delta}) and Syndecan-1+B220– plasma cells ({circ}) were separated by MACS using anti-CD138/Syndecan-1 mAb. Results are representative of five independent experiments.

 
As plasma cells (Syndecan-1+) were present along with B cells in the nylon-wool adherent population, Syndecan-1+ (90% of which were B220–) and Syndecan-1– (81% B220+) cells were separated immunomagnetically. Syndecan-1 is a member of the Syndecan family of integral membrane proteoglycans expressed by pre-B cells and plasma cells but absent from circulating, mature B cells [35 ]. Figure 4B (lower) shows that the B cell (Syndecan-1–) population was responsible for most of the cytotoxic activity, and plasma cells exerted a much lower capacity.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Following the studies by Suda et al. [4], it was considered that only T-tropic mitogens (concanavalin A or anti-CD3) induced FasL mRNA expression, whereas B-tropic mitogens [lipopolysaccharide (LPS) or anti-IgM] did not. In addition, among cytokines, only a high concentration of interleukin-2 substantially induced FasL mRNA expression. However, Hahne et al. [5] subsequently found that a T cell-depleted spleen cell population expressed FasL and became Fas-dependent cytotoxic effectors when subjected to extensive stimulation by LPS (3–4 days) followed by 14 h of incubation with medium containing a mixture of PMA plus ionomycin, which mimics B cell receptor activation. Thereafter, additional reports (including the present report) demonstrated that activated B cells, like activated T cells, can express functional FasL [6 7 8 , 36 ].

The present work shows, using a semiquantitative RT-PCR assay, that T-depleted spleen cells from MRL/lpr mice expressed FasL when tested ex vivo in the absence of any further stimulation. The level of FasL mRNA is comparable with or higher than the one measured in MRL/lpr splenic SP or DN T lymphocytes. The accuracy of the method’s semiquantitative evaluations was demonstrated by the specific detection of CD40L on MRL/lpr SP T lymphocytes but not on DN T cells, a finding in agreement with the transient expression of this costimulatory molecule following T cell antigen receptor activation [37 ]. Indeed, DN T cells are persistent, previously activated SP cells, which have failed to undergo apoptosis [19 , 20 ]. It is unexpected that we found by quantitative real-time RT-PCR that B lymphocytes from MRL/lpr mice, but not from MRL-+/+, express more FasL RNA than SP T lymphocytes. Moreover, in some MRL/lpr mice, the level of FasL in B cells reached the one found in DN T cells. Then, we checked whether the overexpression of FasL mRNA in B lymphocytes was also found at the protein level. Our analyses were focused on the membrane-anchored FasL. To this end, the expression of FasL was analyzed by flow cytometry using a soluble, recombinant Fas:Fc{gamma}1 fusion protein, anti-CD3, and anti-CD19 mAb. We observed that B and T lymphocyte populations from MRL/lpr mice overexpressed membrane FasL. Furthermore, we found that the level of membrane-anchored FasL increased with the size of the B cell and up-regulation of the early activation marker CD69, suggesting that the expression of FasL might be controlled by the activation state of the B cell. Our data are reminiscent of recent findings, which showed that FasL is expressed on large, highly activated B cells during a T. cruzi infection [8 ].

Using primers that amplify Fas receptor cDNA products beyond the transposon insertion and therefore detect the predominant mutated and the minor wild-type transcripts, we have observed a Fas overexpression in all MRL/lpr spleen subpopulations (data not shown). This observation could explain the differences observed between MRL/lpr and Fas knockout strains [32 ]. Indeed, in MRL/lpr mice, the lpr-mutated and minor (~2%) wild-type Fas transcripts [29 ] might be augmented with age to reach significant levels. This could have pathological consequences, because of the FasL overexpression by MRL/lpr lymphocyte subpopulations.

The T cell-depleted spleen population, mostly comprising B cells, was able to kill Fas-expressing thymocytes specifically. Flow cytometry analyses revealed that this population contained 4–6% large granular (DX5+) NK cells. When isolated immunomagnetically, these DX5+ NK cells were potent, in vitro, cytotoxic effectors. It is surprising that under the same conditions, the remaining B220+Thy-1.2–DX5–cells still exerted cytotoxicity against Fas+ targets. Isolation of the cells lacking or expressing Syndecan-1, an integral membrane proteoglycan expressed by pre-B cells and plasma cells but absent from mature peripheral B cells [35 ], indicated that this cytotoxicity was predominantly a result of mature B lymphocytes expressing the B220+Syndecan-1– phenotype.

FasL expression on B cells may have important immunopathological implications. Systemic lupus erythematosus (SLE), a prototype of systemic autoimmune disease, is generally thought to be solely antibody-mediated and characterized by deposits of autoantibodies and immune complexes in kidneys, vessels, skin, and salivary glands [38 ]. However, lymphoid cell infiltration of organs, such as lung, kidney, and skin, are also prominent features in SLE patients as well as their murine models, and the relative role of humoral and cellular immunities has not been defined clearly yet. In addition to autoantibody production, the autoimmune disease of MRL/lpr mice is characterized by skin lesions [39 ], glomerulonephritis [40 ], and vasculitis [41 ]. B cells are necessary for the development of nephritis and vasculitis [42 , 43 ]. Indeed, the T cell-restricted expression of transgenic Fas in MRL/lpr mice blocks the lymphoproliferation but not the glomerulonephritis [44 ]. Furthermore, it has been shown that mice with B cells but lacking serum antibodies still develop these pathologies, underlining an antibody-independent mechanism for renal and vascular disease in SLE. The mechanism suggested by Chan et al. [45] implicated the APC function of B cells. In the MRL/lpr SLE model, an alternative explanation could be that expanding B cells, which overexpress FasL but cannot be eliminated properly, may contribute, together with T cells, to the cytotoxic destruction of Fas-bearing tissues. This expression of Fas could be constitutive or result from induction by proinflammatory cytokines, such as IFN-{gamma}, which is also required for the development of MRL/lpr glomerulonephritis [46 , 47 ].

In conclusion, our data demonstrate the cytotoxic activity of MRL/lpr activated B cells on Fas+ targets. This finding suggests a new role for activated B lymphocytes in the autoimmune destruction of Fas-bearing inflammatory tissues, which could lead to new therapeutic approaches to B cell-dependent nephritis and vasculitis in SLE patients.


   ACKNOWLEDGEMENTS
 
This work was supported in part by a grant from Association de la Recherche sur la Polyarthrite. We thank Dr. Colette Kanellopoulos-Langevin (Institut Jacques Monod, CNRS UMR 7592, Paris, France) for critical review of the manuscript.

Received September 20, 2004; revised July 9, 2005; accepted July 15, 2005.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Lenardo, M., Chan, K. M., Hornung, F., McFarland, H., Siegel, R., Wang, J., Zheng, L. (1999) Mature T lymphocyte apoptosis-immune regulation in a dynamic and unpredictable antigenic environment Annu. Rev. Immunol. 17,221-253[CrossRef][Medline]
  2. Krammer, P. H. (2000) CD95’s deadly mission in the immune system Nature 407,789-795[CrossRef][Medline]
  3. Russell, J. H., Ley, T. J. (2002) Lymphocyte-mediated cytotoxicity Annu. Rev. Immunol. 20,323-370[CrossRef][Medline]
  4. Suda, T., Okazaki, T., Naito, Y., Yokota, T., Arai, N., Ozaki, S., Nakao, K., Nagata, S. (1995) Expression of the Fas ligand in cells of T cell lineage J. Immunol. 154,3806-3813[Abstract]
  5. Hahne, M., Renno, T., Schroeter, M., Irmler, M., French, L., Bornard, T., MacDonald, H. R., Tschopp, J. (1996) Activated B cells express functional Fas ligand Eur. J. Immunol. 26,721-724[Medline]
  6. Tanner, J. E., Alfieri, C. (1999) Epstein-Barr virus induces Fas (CD95) in T cells and Fas ligand in B cells leading to T-cell apoptosis Blood 94,3439-3447[Abstract/Free Full Text]
  7. Lundy, S. K., Lerman, S. P., Boros, D. L. (2001) Soluble egg antigen-stimulated T helper lymphocyte apoptosis and evidence for cell death mediated by FasL+ T and B cells during murine Schistosoma mansoni infection Infect. Immun. 69,271-280[Abstract/Free Full Text]
  8. Zuniga, E., Motran, C. C., Montes, C. L., Yagita, H., Gruppi, A. (2002) Trypanosoma cruzi infection selectively renders parasite-specific IgG+ B lymphocytes susceptible to Fas/Fas ligand-mediated fratricide J. Immunol. 168,3965-3973[Abstract/Free Full Text]
  9. Bossi, G., Griffiths, G. M. (1999) Degranulation plays an essential part in regulating cell surface expression of Fas ligand in T cells and natural killer cells Nat. Med. 5,90-96[CrossRef][Medline]
  10. Schneider, P., Holler, N., Bodmer, J. L., Hahne, M., Frei, K., Fontana, A., Tschopp, J. (1998) Conversion of membrane-bound Fas (CD95) ligand to its soluble form is associated with downregulation of its proapoptotic activity and loss of liver toxicity J. Exp. Med. 187,1205-1213[Abstract/Free Full Text]
  11. Tanaka, M., Itai, T., Adachi, M., Nagata, S. (1998) Downregulation of Fas ligand by shedding Nat. Med. 4,31-36[CrossRef][Medline]
  12. Rothstein, T. L., Wang, J. K., Panka, D. J., Foote, L. C., Wang, Z., Stanger, B., Cui, H., Ju, S. T., Marshak-Rothstein, A. (1995) Protection against Fas-dependent Th1-mediated apoptosis by antigen receptor engagement in B cells Nature 374,163-165[CrossRef][Medline]
  13. Rathmell, J. C., Townsend, S. E., Xu, J. C., Flavell, R. A., Goodnow, C. C. (1996) Expansion or elimination of B cells in vivo: dual roles for CD40- and Fas (CD95)-ligands modulated by the B cell antigen receptor Cell 87,319-329[CrossRef][Medline]
  14. MacDonald, G. C., Kakkanaiah, V. N., Sobel, E. S., Cohen, P. L., Eisenberg, R. A. (1995) In vivo depletion of Thy-1-positive cells originating from normal bone marrow abrogates the suppression of gld disease in normal-gld mixed bone marrow chimeras J. Immunol. 154,444-449[Abstract]
  15. Siegel, R. M., Chan, F. K., Chun, H. J., Lenardo, M. J. (2000) The multifaceted role of Fas signaling in immune cell homeostasis and autoimmunity Nat. Immunol. 1,469-474[CrossRef][Medline]
  16. Rieux-Laucat, F., Le Deist, F., Fischer, A. (2003) Autoimmune lymphoproliferative syndromes: genetic defects of apoptosis pathways Cell Death Differ 10,124-133[CrossRef][Medline]
  17. Kono, D. H., Theofilopoulos, A. N. (2000) Genetics of systemic autoimmunity in mouse models of lupus Int. Rev. Immunol. 19,367-387[Medline]
  18. Benihoud, K., Bonardelle, D., Soual-Hoebeke, E., Durand-Gasselin, I., Emilie, D., Kiger, N., Bobé, P. (2002) Unusual expression of LINE-1 transposable element in the MRL autoimmune lymphoproliferative syndrome-prone strain Oncogene 21,5593-5600[CrossRef][Medline]
  19. Landolfi, M. M., Van Houten, N., Russell, J. Q., Scollay, R., Parnes, J. R., Budd, R. C. (1993) CD2–CD4–CD8– lymph node T lymphocytes in MRL lpr/lpr mice are derived from a CD2+CD4+CD8+ thymic precursor J. Immunol. 151,1086-1096[Abstract]
  20. Laouar, Y., Ezine, S. (1994) In vivo CD4+ lymph node T cells from lpr mice generate CD4–CD8–B220+TCR-ß low cells J. Immunol. 153,3948-3955[Abstract]
  21. Chu, J. L., Drappa, J., Parnassa, A., Elkon, K. B. (1993) The defect in Fas mRNA expression in MRL/lpr mice is associated with insertion of the retrotransposon, ETn J. Exp. Med. 178,723-730[Abstract/Free Full Text]
  22. Nagata, S. (1994) Mutations in the Fas antigen gene in lpr mice Semin. Immunol. 6,3-8[CrossRef][Medline]
  23. Renno, T., Hahne, M., Tschopp, J., MacDonald, H. R. (1996) Peripheral T cells undergoing superantigen-induced apoptosis in vivo express B220 and upregulate Fas and Fas ligand J. Exp. Med. 183,431-437[Abstract/Free Full Text]
  24. Chu, J. L., Ramos, P., Rosendorff, A., Nikolic-Zugic, J., Lacy, E., Matsuzawa, A., Elkon, K. B. (1995) Massive upregulation of the Fas ligand in lpr and gld mice: implications for Fas regulation and the graft-versus-host disease-like wasting syndrome J. Exp. Med. 181,393-398[Abstract/Free Full Text]
  25. Watanabe, D., Suda, T., Hashimoto, H., Nagata, S. (1995) Constitutive activation of the fas ligand gene in mouse lymphoproliferative disorders EMBO J 14,12-18[Medline]
  26. Benihoud, K., Bonardelle, D., Bobé, P., Kiger, N. (1997) MRL/lpr CD4–CD8– and CD8+ T cells, respectively, mediate Fas-dependent and perforin cytotoxic pathways Eur. J. Immunol. 27,415-420[Medline]
  27. Bobé, P., Bonardelle, D., Reynès, M., Godeau, F., Mahiou, J., Joulin, V., Kiger, N. (1997) Fas-mediated liver damage in MRL hemopoietic chimeras undergoing lpr-mediated graft-versus-host disease J. Immunol. 159,4197-4204[Abstract]
  28. Hadj-Slimane, R., Chelbi-Alix, M. K., Tovey, M. G., Bobé, P. (2004) An essential role for IFN-{alpha} in the overexpression of Fas ligand on MRL/lpr lymphocytes and on their spontaneous Fas-mediated cytotoxic potential J. Interferon Cytokine Res. 24,717-728[Medline]
  29. Wu, J., Zhou, T., He, J., Mountz, J. D. (1993) Autoimmune disease in mice due to integration of an endogenous retrovirus in an apoptosis gene J. Exp. Med. 178,461-468[Abstract/Free Full Text]
  30. Kobayashi, S., Hirano, T., Kakinuma, M., Uede, T. (1993) Transcriptional repression and differential splicing of Fas mRNA by early transposon (ETn) insertion in autoimmune lpr mice Biochem. Biophys. Res. Commun. 191,617-624[CrossRef][Medline]
  31. Mariani, S. M., Matiba, B., Armandola, E. A., Krammer, P. H. (1994) The APO-1/Fas (CD95) receptor is expressed in homozygous MRL/lpr mice Eur. J. Immunol. 24,3119-3123[Medline]
  32. Adachi, M., Suematsu, S., Kondo, T., Ogasawara, J., Tanaka, T., Yoshida, N., Nagata, S. (1995) Targeted mutation in the fas gene causes hyperplasia in peripheral lymphoid organs and liver Nat. Genet. 11,294-300[CrossRef][Medline]
  33. Bonardelle, D., Bobé, P., Reynès, M., Amouroux, J., Tricottet, V., Godeau, F., Kiger, N. (2001) Inflammatory arthropathy in MRL hematopoietic chimeras undergoing Fas-mediated graft-versus-host syndrome J. Rheumatol. 28,956-961[Medline]
  34. Koyasu, S. (1994) CD3+CD16+NK1.1+B220+ large granular lymphocytes arise from both {alpha}-ß TCR+CD4–CD8– and {gamma}-{delta} TCR+CD4–CD8– cells J. Exp. Med. 179,1957-1972[Abstract/Free Full Text]
  35. Sanderson, R. D., Lalor, P., Bernfield, M. (1989) B lymphocytes express and lose syndecan at specific stages of differentiation Cell Regul 1,27-35[Medline]
  36. Strater, J., Mariani, S. M., Walczak, H., Rucker, F. G., Leithauser, F., Krammer, P. H., Moller, P. (1999) CD95 ligand (CD95L) in normal human lymphoid tissues: a subset of plasma cells are prominent producers of CD95L Am. J. Pathol. 154,193-201[Abstract/Free Full Text]
  37. Roy, M., Waldschmidt, T., Aruffo, A., Ledbetter, J. A., Noelle, R. J. (1993) The regulation of the expression of gp39, the CD40 ligand, on normal and cloned CD4+ T cells J. Immunol. 151,2497-2510[Abstract]
  38. Kotzin, B. L. (1996) Systemic lupus erythematosus Cell 85,303-306[CrossRef][Medline]
  39. Kanauchi, H., Furukawa, F., Horiguchi, Y., Horio, T., Imamura, S. (1988) Distribution of Langerhans cells in skin lesions of MRL/l mice Arch. Dermatol. Res. 280,327-331[CrossRef][Medline]
  40. Andrews, B. S., Eisenberg, R. A., Theofilopoulos, A. N., Izui, S., Wilson, C. B., McConahey, P. J., Murphy, E. D., Roths, J. B., Dixon, F. J. (1978) Spontaneous murine lupus-like syndromes. Clinical and immunopathological manifestations in several strains J. Exp. Med. 148,1198-1215[Abstract/Free Full Text]
  41. Moyer, C. F., Strandberg, J. D., Reinisch, C. L. (1987) Systemic mononuclear-cell vasculitis in MRL/Mp-lpr/lpr mice. A histologic and immunocytochemical analysis Am. J. Pathol. 127,229-242[Abstract]
  42. Chan, O., Shlomchik, M. J. (1998) A new role for B cells in systemic autoimmunity: B cells promote spontaneous T cell activation in MRL-lpr/lpr mice J. Immunol. 160,51-59[Abstract/Free Full Text]
  43. Shlomchik, M. J., Madaio, M. P., Ni, D., Trounstein, M., Huszar, D. (1994) The role of B cells in lpr/lpr-induced autoimmunity J. Exp. Med. 180,1295-1306[Abstract/Free Full Text]
  44. Fukuyama, H., Adachi, M., Suematsu, S., Miwa, K., Suda, T., Yoshida, N., Nagata, S. (1998) Transgenic expression of Fas in T cells blocks lymphoproliferation but not autoimmune disease in MRL-lpr mice J. Immunol. 160,3805-3811[Abstract/Free Full Text]
  45. Chan, O. T., Hannum, L. G., Haberman, A. M., Madaio, M. P., Shlomchik, M. J. (1999) A novel mouse with B cells but lacking serum antibody reveals an antibody-independent role for B cells in murine lupus J. Exp. Med. 189,1639-1648[Abstract/Free Full Text]
  46. Haas, C., Ryffel, B., Le Hir, M. (1997) IFN-{gamma} is essential for the development of autoimmune glomerulonephritis in MRL/lpr mice J. Immunol. 158,5484-5491[Abstract]
  47. Schwarting, A., Wada, T., Kinoshita, K., Tesch, G., Kelley, V. R. (1998) IFN-{gamma} receptor signaling is essential for the initiation, acceleration, and destruction of autoimmune kidney disease in MRL-Faslpr mice J. Immunol. 161,494-503[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 BloodHome page
P. Bobe, D. Bonardelle, K. Benihoud, P. Opolon, and M. K. Chelbi-Alix
Arsenic trioxide: a promising novel therapeutic agent for lymphoproliferative and autoimmune syndromes in MRL/lpr mice
Blood, December 15, 2006; 108(13): 3967 - 3975.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
jlb.0904536v1
78/5/1052    most recent
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
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 reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Bonardelle, D.
Right arrow Articles by Bobé, P.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Bonardelle, D.
Right arrow Articles by Bobé, P.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS