Membrane-bound Fas ligand only is essential for Fas-induced apoptosis

Abstract

Fas ligand (FasL), an apoptosis-inducing member of the TNF cytokine family, and its receptor Fas are critical for the shutdown of chronic immune responses and prevention of autoimmunity. Accordingly, mutations in their genes cause severe lymphadenopathy and autoimmune disease in mice and humans. FasL function is regulated by deposition in the plasma membrane and metalloprotease-mediated shedding. Here we generated gene-targeted mice that selectively lack either secreted FasL (sFasL) or membrane-bound FasL (mFasL) to resolve which of these forms is required for cell killing and to explore their hypothesized non-apoptotic activities. Mice lacking sFasL (FasL(Deltas/Deltas)) appeared normal and their T cells readily killed target cells, whereas T cells lacking mFasL (FasL(Deltam/Deltam)) could not kill cells through Fas activation. FasL(Deltam/Deltam) mice developed lymphadenopathy and hyper-gammaglobulinaemia, similar to FasL(gld/gld) mice, which express a mutant form of FasL that cannot bind Fas, but surprisingly, FasL(Deltam/Deltam) mice (on a C57BL/6 background) succumbed to systemic lupus erythematosus (SLE)-like autoimmune kidney destruction and histiocytic sarcoma, diseases that occur only rarely and much later in FasL(gld/gld) mice. These results demonstrate that mFasL is essential for cytotoxic activity and constitutes the guardian against lymphadenopathy, autoimmunity and cancer, whereas excess sFasL appears to promote autoimmunity and tumorigenesis through non-apoptotic activities.

Figure 1. Generation of mutant mice that specifically lack either secreted FasL or membrane-bound FasL

a, Schematic diagram of the wt mouse fasl gene, the mutation for creating the FasL^Δs allele, in which the metalloproteinase recognition site (middle sequence) was altered to prevent FasL shedding and the mutation for creating the FasL^Δm allele, in which sequences encoding the human G-CSF signal sequence were fused in frame with those for the extracellular region of mouse FasL (lower sequence) to preclude insertion of FasL into the plasma membrane but allowing secretion of FasL (sFasL). P, CYTO, TM and EXT represent the promoter, cytoplasmic, transmembrane and extracellular regions, respectively. The positions of PCR primers used for genotyping of the gene-targeted mice are indicated. b, Immunofluorescent staining and confocal microscopy to demonstrate intracellular localisation of FasL (green) in activated T cells from wt, FasL^Δs/Δs and FasL^Δm/Δm mice. DAPI (blue) was used to label nuclei. c, ELISA to quantify the levels of FasL in the supernatants of activated T cells from wt, FasL^Δs/Δs and FasL^Δm/Δm mice. Each dot represents a single mouse and the bar indicates the mean +/- SD. FasL^Δs/Δs T cells had significantly (p<0.03) less FasL in their supernatants compared to wt T cells. d,e, Cell surface immunofluorescent staining and FACS analysis to measure expression of membrane-bound FasL on activated T cells from wt, FasL^Δs/Δs (d) and FasL^Δm/Δm (e) mice. Solid lines show staining with an anti-FasL antibody and dotted lines show staining with an isotype-matched control antibody. Values represent mean +/- SD of mFasL⁺ T cells from 3 independent experiments (p<0.05 wt vs FasL^Δm/Δm mice at 6 and 24 h; p<0.05 wt vs FasL^Δs/Δs mice at 6 h). For all experiments (b,c,d,e) T cells were stimulated with ConA for 3 days, rested for 2 days in IL-2 (0 h) and then restimulated with PMA plus ionomycin for 6 or 24 h.

Figure 2. Membrane-bound but not secreted FasL is essential for target cell killing and AICD

a,b, CH1 target cells (FasL sensitive) were co-cultured with activated T cells from wt (blue squares), FasL^Δs/Δs (black stars) or FasL^gld/gld (purple triangles) mice (a) or from wt (blue squares), FasL^Δm/Δm (red circles) or FasL^gld/gld (purple triangles) mice (b) at the indicated effector:target ratio. The percentage of CH1 target cell killing was measured by FACS analysis after 24 h. c,d, Activated T cells from wt (blue squares), FasL^Δs/Δs (black stars) or FasL^gld/gld (purple triangles) mice (c) or from wt (blue squares) FasL^Δm/Δm (red circles) or FasL^gld/gld (purple triangles) mice (d) were either cultured in medium plus IL-2 (broken lines) or restimulated for 6, 24 or 48 h with antibodies to CD3 (solid lines). Cell survival was measured by staining with PI plus FITC-coupled annexin V and FACS analysis. Values in graphs in a,b,c,d represent mean +/- SEM from 3 independent experiments. * p<0.05 a,c FasL^Δs/Δs vs wt, b,d FasL^Δm/Δm vs wt.

Figure 3. Membrane-bound FasL but not secreted FasL is essential to prevent lymphadenopathy, splenomegaly and hyper-gammaglobulinemia with anti-nuclear autoantibodies

a, Lymph nodes (axillary, brachial, inguinal and mesenteric) and b, spleens from wt (blue), FasL^gld/gld (purple), FasL^Δm/wt (green) or FasL^Δm/Δm (red) mice of the ages indicated were weighed. c,d, The percentages of the ‘unusual’ CD3⁺B220⁺ T cells in the lymph nodes of these mice were determined by FACS analysis (numbers indicate % of cells in each quadrant). e, The levels of the indicated immunoglobulin isotypes in the sera of these mice (age 5 months) were determined by ELISA, mean +/- SD. f, The levels of anti-nuclear autoantibodies (ANA) in the sera of these mice, aged 3 (left panel) or 5 months (right panel) were quantified by indirect immunofluorescence staining (1/100 serum dilution) of slides covered with human HEp-2 epithelial cells and scoring of brightness of fluorescence intensity on a scale of 0: no fluorescence to 3+: maximal fluorescence. g, Examples of ANA quantification in sera of mice of the indicated genotypes. Immunofluorescence intensity score is indicated in brackets. Values in graphs in a,b represent mean +/-SEM and in c,e,f they represent mean +/-SD from a minimum of 10 to 52 FasL^Δm/Δm mice. h, The levels of anti-DNA auto-antibodies in sera of mice of the indicated genotypes and ages were measured by ELISA (each dot represents the value for a single mouse). Bar at 12.5 IU/ml indicates the level diagnostic for SLE.

Figure 4. FasL^Δm/Δm mice die considerably earlier than FasL^gld/gld mice due to SLE-like fatal glomerulonephritis and histiocytic sarcoma

a, Kaplan-Meyer survival curves for control (blue line, wt and FasL^Δm/wt combined), FasL^Δm/Δm (red line) and FasL^gld/gld mice (purple line) (control vs FasL^gld/gld: p<0.0001; control vs FasL^Δm/Δm: p<0.0001; FasL^Δm/Δm vs FasL^gld/gld: p<0.0001). b, Incidence of severe autoimmune kidney disease (glomerulonephritis (GN) score ≥3 in FasL^Δm/Δm (red line) and FasL^gld/gld (purple line) mice. (FasL^Δm/Δm vs FasL^gld/gld: p<0.0175). c, H&E stained sections of kidneys from mice of the indicated genotypes and ages were examined for pathological changes, such as hypercellularity, cellular crescents, dilated tubules or sclerotic glomeruli and the % of total glomeruli affected scored on a scale of 0-4 (0=normal, 1=0-25%, 2=25-50%, 3=50-75%, 4=>75% affected; scores indicated in brackets). d, Incidence of histiocytic sarcoma in FasL^Δm/Δm, FasL^gld/gld and control wt mice (FasL^Δm/Δm vs FasL^gld/gld: p=0.03), ** . e. Pictures of two FasL^Δm/Δm mice with histiocytic sarcoma (aged 329 and 417 days). Macroscopic lesions are readily seen in the livers and spleens. f, H&E stained sections of livers, spleens and lungs of histiocytic sarcoma-burdened FasL^Δm/Δm mice (aged 329 and 417 days; * indicates histiocytic lesions). Scale bar represents 155 μm.