Fas ligand exerts its pro-inflammatory effects via neutrophil recruitment but not activation

Abstract

Fas ligand (FasL) expression induces apoptosis of activated T cells and has been suggested as a strategy to inhibit graft rejection. Unfortunately, the use of FasL to confer 'immune privilege' in this setting has been hampered by the finding that it may also provoke a destructive granulocytic response. While the Fas/FasL-mediated apoptotic pathways are well defined, the pro-inflammatory effects of FasL are poorly understood. Our aim in this study was to define in vitro the biological effects of FasL on neutrophil recruitment and activation. DAP-3 cells expressing human FasL on the cell membrane (mFasL) potently induced apoptosis in human neutrophils and in activated T lymphocytes. Recombinant human soluble FasL (sFasL), by contrast, was a very weak inducer of apoptosis, even at high concentrations. This latter observation suggests that cleavage of mFasL by naturally occurring matrix metalloproteinases may serve to down-regulate FasL activity in vivo. However, in the presence of a cross-linking antibody, the efficiency of apoptosis-induction by sFasL was greatly increased, suggesting that the lesser pro-apoptotic potency of sFasL reflects an inability to induce trimerization of the Fas receptor. With regard to pro-inflammatory effects, we found that sFasL is a potent neutrophil chemoattractant and, given that it induces little apoptosis, the dominance of sFasL over mFasL may mean that graft-infiltrating neutrophils will survive to mediate inflammation. Neither sFasL nor mFasL produced neutrophil activation as assessed by chemiluminescence assay. This suggests that neutrophils recruited to an inflammatory site by FasL will be activated by mechanisms other than Fas-FasL signalling.

Figure 1

DAP-3 cells expressing human FasL induce apoptosis in activated human T cells. DAP-3 murine fibroblasts, either untransfected (DAP-3) or transfected to express wild-type human FasL (DAP-3 hFasL) were seeded at 10⁴ cells/well in 24-well tissue culture plates and incubated overnight at 37° with 5% CO₂ to create an adherent monolayer of cells. Human CD3 cells, activated by stimulation with phytohaemagglutinin 1 μg/ml and recombinant human interleukin-2 10 μg/ml for 48 hr, were used as Fas-expressing target cells; 2 × 10⁵ CD3 cells were added to the DAP-3 monolayers and incubated for 4 hr. Apoptosis was assessed by staining the T cells with propidium iodide and annexin V-FITC and then analysing by flow cytometry. Apoptotic cells were defined as propidium iodide negative/annexin V positive. A representative example of three experiments is shown.

Figure 2

Soluble FasL (sFasL) requires cross-linking to deliver an apoptotic signal. T lymphocytes (CD3) were isolated as described above and plated out in a 24-well plate at 10⁶ cells/ml suspended in RPMI-1640/10% FCS. To render the T cells more susceptible to Fas-mediated apoptosis, they were first cultured for 48 hr in the presence of phytohemagglutinin 1 μg/ml and IL-2 10 μg/ml to induce activation and up-regulate Fas expression. For the assay, 2 × 10⁵ cells were incubated for 4 hr with either recombinant human sFasL alone (0–2000 ng/ml) or sFasL cross-linked by a murine polyclonal anti-6× histidine antibody directed against the 6× His tag present on this recombinant molecule. T-cell apoptosis was detected by double-staining with propidium iodide and annexin V-FITC followed by flow cytometry. Apoptotic cells were defined as being propidium iodide negative/annexin V positive. The typical dose–response pattern is shown above. A representative example of three experiments is shown.

Figure 3

Membrane FasL (mFasL) and cross-linked soluble FasL (sFasL), but not uncross-linked sFasL, induce apoptosis in neutrophils. To assess apoptosis induction by mFasL (a), 2 × 10⁵ PMN were added to monolayers of DAP-3 or DAP-3 hFasL and incubated for 4 hr. Neutrophil apoptosis was detected by double-staining with propidium iodide and annexin V-FITC followed by flow cytometric analysis. To assess apoptosis induction by sFasL (b), PMN were added to a 96-well plate at 2 × 10⁵ cells/well suspended in 200 μl RPMI-1640/10% FCS and incubated for 4 hr with sFasL alone (0–1000 ng/ml) or sFasL cross-linked by a murine polyclonal anti-6× histidine antibody. Again, apoptosis was detected by double-staining with propidium iodide and annexin V-FITC. Representative examples of three experiments are shown.

Figure 4

Soluble FasL (sFasL) is chemotactic for neutrophils: 10⁶ freshly isolated neutrophils were placed in the upper chamber of a transwell and varying concentrations of sFasL in 500 μl PBS were placed in the lower chamber separated by a semi-permeable membrane (3-μm pore size). We assessed neutrophil migration by counting the number of cells appearing in the lower chamber after 2 hr. fMLP 10⁻⁹ m in the lower chamber was used as a positive control. PBS in the lower chamber was used as a negative control. A representative example of three experiments is shown.

Figure 5

Soluble FasL (sFasL) does not produce neutrophil activation: 10⁵ freshly isolated neutrophils suspended in 200 μl RPMI-1640/10% FCS were added to the wells of a 96-well plate then stimulated with varying concentrations of sFasL in the presence or absence of cross-linking antibody. Neutrophil activation was assessed using a standard chemiluminescence assay read with an Anthos Lucy-1 chemiluminometer. Luminol 5 mm was used as the indicator. Readings were taken at 3-min intervals over 120 min. The intensity of luminescence (arbitrary units) produced correlates with the degree of neutrophil activation. Triplicates are shown for each condition. Figure shown is a representative example of three experiments. Time is shown on the x-axis (0–120 min for each well). Luminesence is shown on the y-axis measured in arbitrary ‘light units’ (0–100).

Figure 6

Membrane FasL does not produce neutrophil activation: 10⁵ freshly isolated neutrophils suspended in 200 μl RPMI-1640/10% fetal calf serum were added to the wells of a six-well plate coated with a monolayer of DAP-3 fibroblasts either untransfected (DAP-3) or transfected to express human FasL (DAP-3 hFasL). Neutrophil activation was assessed using a standard chemiluminescence assay read with an Anthos Lucy-1 chemiluminometer. Luminol 5 mm was used as the indicator. The intensity of luminescence produced correlates with the degree of neutrophil activation. A representative example of three experiments is shown.