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Immunology. Oct 2003; 110(2): 225–233.
PMCID: PMC1783033

Fas ligand-induced murine pulmonary inflammation is reduced by a stable decoy receptor 3 analogue


Fas ligand (FasL)-induced lung inflammation has recently been suggested to play an important role in the pathogenesis of acute respiratory disease syndrome (ARDS). In order to further explore this connection, we established a FasL-induced murine model of pulmonary inflammation. Instillation of recombinant FasL (rFasL) into the lung induced neutrophil infiltration and increased pulmonary permeability, as evidenced by increased total protein in the airspace; both occur in patients with ARDS. These effects were accompanied with a rapid induction of proinflammatory mediators: cytokine granulocyte–macrophage colony-stimulating factor (GM-CSF) and the chemokines macrophage inflammatory protein-2 (MIP-2) and KC. Pretreatment with a FasL antagonist, a decoy receptor 3 analogue (DcR3 analogue), reduced neutrophil infiltration into the airspace and resulted in a highly significant reduction in the levels of GM-CSF, MIP-2 and KC in bronchoalveolar lavage (BAL) fluid. We postulate that rFasL may be responsible for induction of proinflammatory chemokines and cytokines in the lung, which in turn attract neutrophil infiltration into the airspace. This proinflammatory process and the associated pulmonary permeability may, in part, explain the association of FasL with severe pulmonary inflammation, such as ARDS, and shed new light on FasL and its role in lung injury.


Fas ligand (CD95 ligand) (FasL) is a member of the tumour necrosis factor (TNF) ligand family and plays a major role in the induction of apoptosis of immune cells.13 However, recent data have shown that the Fas/FasL system is also an important mediator of inflammation. The first evidence that FasL signalling has a role in inflammation came from in vivo studies using colon carcinoma,4 heart5 and pancreatic islet6,7 graft models overexpressing FasL. Contrary to the expectation that, owing to its role in immune privilege, overexpression of FasL would protect grafted tissues, overexpression of FasL rapidly caused graft rejection by the host immune system, mainly because of a massive infiltration of host neutrophils. Subsequently, FasL treatment in vitro was shown to have a direct chemotactic activity towards neutrophils8,9 and to induce interleukin (IL)-1β secretion in peritoneal exudate cells,10 further supporting the proinflammatory function of FasL signalling.

Recent data have shown that FasL-induced apoptosis and inflammation may play an important role in the pathogenesis of acute respiratory distress syndrome (ARDS).11,12 ARDS is caused by multifactorial acute lung inflammation in which neutrophilic inflammation and severe damage to the alveolar epithelium are common histopathological findings.13 Although the mechanism of lung damage in ARDS is not fully understood, Fas/FasL signalling has been implicated. Fas and FasL immunostaining was found to be increased in airway and alveolar epithelial cells from patients who died from ARDS.11 It has been reported that elevated levels of soluble FasL have been found in the bronchoalveolar lavage (BAL) fluid of ARDS patients.12 BAL fluid obtained from ARDS patients induces apoptosis of primary pulmonary epithelial cells in vitro and is Fas/FasL dependent.12 FasL treatment in vitro induced primary human lung epithelial cells and macrophages (M[var phi]s) to secrete IL-8, a potent neutrophil chemoattractant and activator.14

Decoy receptor 3 (DcR3) is a recently discovered secreted tumour necrosis factor (TNF) receptor member lacking transmembrane and intracellular domains.15 DcR3 was subsequently shown to bind to and block the signalling of three different TNF ligand members: FasL, LIGHT and TL1A.1517 The ability of DcR3 to block FasL is of particular interest because FasL has been implicated in several diseases, including ARDS. In particular, in vivo treatment with DcR3 was shown to prevent FasL-mediated hepatic damage and lethality in a mouse model.18 This indicates a potential utility of DcR3 as a therapeutic agent in diseases in which FasL signalling causes tissue apoptosis and/or inflammation. In an effort to improve the pharmacological properties of DcR3, an analogue of DcR3 was generated, with a substitution at residue 218 from arginine to glutamine. This DcR3 analogue showed increased resistance to proteolytic degradation both in vitro and in vivo. The amino acid substitution at residue 218 lengthens the in vivo half-life of DcR3 when the protein is administered subcutaneously while retaining its ability to bind FasL.19

In this report, we investigated the mechanism of proinflammatory rFasL signalling in a mouse model of lung inflammation. We demonstrated that rFasL action in vivo leads to the rapid recruitment of neutrophils into the lung and increased total protein levels. We also showed that granulocyte–macrophage colony-stimulating factor (GM-CSF), KC and macrophage inflammatory protein-2 (MIP-2) are induced in the lung after instillation with rFasL and are closely correlated with neutrophil infiltration in the lung. These data suggest that following instillation with rFasL, rFasL-induced secretion of GM-CSF, KC and MIP-2 may be the primary effectors for neutrophil infiltration followed by vascular leakage into the alveolar space. Furthermore, we show that systemic pretreatment with a DcR3 analogue can alleviate FasL-induced lung inflammation, suggesting that the DcR3 analogue may be useful in treating lung diseases, such as ARDS, in which FasL-induced tissue damage plays a major pathological role.

Materials and methods


Female BALB/c mice were purchased from Harlan (Indianapolis, IN) and used at 7–9 weeks of age. Mice were kept in the animal facility at Lilly Research Laboratories (Indianapolis, IN) for 1 week for acclimatization before use in the current study. Mice were housed in a room of constant temperature and humidity, and were subjected to one 12-hr light/dark cycle. Mice received normal rodent chow and water ad libitum. All in vivo experiments were performed in accordance with the National Institute of Health guidelines and with the approval of the Animal Care and Use Committee of Lilly Research Laboratories.


Recombinant human ‘super’ FasL (rFasL) was purchased from Alexis Biochemicals (Carlsbad, CA) (<0·1 ng endotoxin). rFasL has a 26-amino acid linker region that encourages aggregation and therefore an enhancer is not required. Lipopolysaccharide (LPS) (Escherichia coli 055:B5) was purchased from Difco/Becton-Dickinson (Franklin Lakes, NJ) and bovine serum albumin (BSA) (Fraction V, Protease free) was purchased from Bayer (West Haven, CT). Creation and purification of a DcR3 analogue was performed according to Wroblewski et al.19

Lung inflammation models

BALB/c mice were anaesthetized with Isoflurane (Abbott Laboratories, Abbott Park, IL), prior to intratracheal instillation with recombinant human FasL, LPS or BSA. Instillation was achieved using a 24-gauge curved stainless steel feeding needle positioned just above the bifurcation of the bronchi; 50 µl of rFasL (2 µg/ml for 100 ng/mouse or 10 µg/ml for 500 ng/mouse), LPS (40 µg/ml, or 2 µg per mouse) or BSA (10 µg/ml, or 500 ng/mouse) was then instilled into the lungs, followed by a 100-µl bolus of air. The time-course studied utilized 500 ng of rFasL per mouse, and the DcR3 analogue-treatment study utilized 100 ng of rFasL per mouse. For DcR3 analogue-treatment studies, 800 µg of DcR3 analogue or BSA was given intravenously (i.v.) 1 hr prior to instillation of rFasL. At 3 hr (or as indicated) postinstillation, mice were anaesthetized, bled retro-orbitally, and exsanguinated by transection of the abdominal aorta. Mice lungs were resected and lavaged. For lavage, the diaphragm was punctured and the trachea was exposed, nicked and then cannulated with PE-90 tubing (Instech Solomon, Plymouth Meeting, PA) attached to a 20-gauge luer stub adapter (Intramedic/Becton-Dickinson, Franklin Lakes, NJ). The cannula was secured with 3-O surgical silk (Ethicon, Somerville, NJ) and the lungs infused with 0·02 ml/g body weight of 0·9% sterile saline using a 1-ml syringe. The saline was incubated in the lungs for 1 min, recovered by aspiration and the BAL fluid stored on ice until required for further processing. Five mice per group were used in the time-course study and 10 mice per group (two for naïve) were used in the DcR3-treatment study.

Intraperitoneal injection study

Eight-week-old female BALB/c mice were used in the i.p. injection study. Fresh LPS (4 µg/mouse), rFasL (500 ng/mouse) and BSA (500 ng/mouse) were diluted to appropriate concentrations in phosphate-buffered saline (PBS) immediately prior to i.p. injection, and a 200-µl volume was injected into each mouse. Four hours later, the peritoneum was washed by injecting 2·5 ml of PBS i.p.; 1 ml of wash solution was collected for measurement of cytokines and chemokines.

Determination of neutrophil levels

BAL fluid samples were centrifuged (10 min, 1000 g, 4°), after which the supernatant was removed and stored at 4°. The cell pellet was resuspended in 200 µl of PBS containing 0·1% BSA (Gibco/Invitrogen, Carlsbad, CA). A 75-µl sample of the cell suspension was placed in a 96-well V-bottom plate; the plates were centrifuged and the supernatant removed (5 min at 1000 g). Cells were resuspended in 100 µl of a 1 : 250 dilution of anti-LY-6G fluorescein isothiocyanate (FITC)-conjugated antibody (clone RB6-8C5; BD-Pharmingen, Franklin Lakes, NJ), and incubated for 30 min at 4°. Cells were washed in PBS/0·1% BSA and then resuspended in 400 µl of PBS/0·1% BSA to which 100 000 Flow-Count™ Fluorospheres (Coulter, Miami, FL) were added. Samples were analysed by FACSort (Becton-Dickinson) until 5000 fluorospheres had been counted. Cells were gated for anti-LY-6G antibody staining. Final neutrophil counts reflected the fact that 37·5% of the sample had been stained and that 5% of the stained sample had been counted. Alternatively, a total white blood cell (WBC) count was measured using a haemocytometer. Briefly, BAL cells were centrifuged at low speed (10 min, 4°, 2000 g) and resuspended in 100 µl of RPMI (Gibco/Invitrogen). Fifty microlitres of the cell suspension was mixed with 50 µl of 10% acetic acid to lyse the red blood cells. WBC were counted by microscopy, using a haemocytometer to determine the total WBC numbers in each BAL sample.

Total protein determination

Total protein in the BAL fluid (a 10-µl sample) was determined using a BCA protein assay kit (Pierce, Rockford, IL), following the manufacturer's instructions.

Cytokine determination

Cytokine levels in BAL fluid supernate or cell culture supernate (50-µl samples) were determined using a BioPlex mouse cytokine assay kit (8-plex, premixed; Bio-Rad, Hercules, CA), following the manufacturer's instructions. Cytokines measured included IL-1β, IL-2, IL-4, IL-5, IL-10, interferon-γ (IFN-γ), GM-CSF and TNF-α.

Enzyme-linked immunosorbent assays (ELISAs)

Twenty-five- or 20-fold dilutions of BAL fluid, 10-fold dilutions of peritoneal wash, or 50-µl of cell culture supernate were used to measure the concentrations of MIP-2 and KC chemokines, respectively, utilizing MIP-2 and KC Quantikine ELISA kits (R & D Systems, Minneapolis, MN).

BAL fluid was analysed (by ELISA) to determine the concentration of the immunoreactive DcR3 analogue. The immunoreactive DcR3 analogue was captured from BAL fluid by using an N-terminal anti-DcR3 analogue-specific antiserum immobilized onto a microtitre plate. The DcR3 analogue was then bound to a biotinylated antibody specific for the C-terminus of the DcR3 analogue. The biotinylated antibody was, in turn, bound by a streptavidin–horseradish peroxidase conjugate (Jackson ImmunoResearch Laboratories, West Grove, PA). Detection was completed using 3,3′,5,5′-tetramethylbenzidine (Kirkegaard & Perry Laboratories, Gaithersburg, MD) a colorimetric substrate for horseradish peroxidase. Concentrations of the DcR3 analogue were estimated from a standard curve that ranged from 0·125 to 8 ng/ml; the lower limit of quantification was determined to be 1 ng/ml. Samples were analysed in duplicate.

BAL fluid was analysed for FasL using a soluble human FasL ELISA kit (Diaclone, Besancon, France). BAL fluid samples were diluted twofold and measured following the manufacturer's instructions. This ELISA kit shows no cross-reaction with mouse FasL.

Isolation and stimulation of mouse alveolar M[var phi]

Naïve mice were killed with CO2 and exsanguinated. The diaphragm was punctured and the trachea was exposed. The trachea was nicked and cannulated with PE-90 tubing (Instech Solomon) attached to a 20-gauge luer stub adapter (Intramedic/Becton-Dickinson). The cannula was tied off with 3-O surgical silk (Ethicon) and the lungs infused with 0·75 ml of 0·9% sterile saline using a 1-ml syringe. The saline was incubated for 1 min in the lungs, recovered and the lavage repeated and samples pooled. Cells were resuspended in Dulbecco's modified Eagle's minimal essential medium (DMEM) (Gibco/Invitrogen) containing 10% fetal bovine serum (Gibco/Invitrogen) and added at 100 000 per well to a 96-well plate. rFasL (Alexis Biochemicals) was added, at 500 ng/ml, to all wells. In addition, DcR3 analogue was added, to some wells, to a final concentration of 2 µg/ml. All experiments were performed in triplicate. Cells were incubated overnight at 37° in a atmosphere of 5% CO2, and the cell culture supernate was harvested for chemokine and cytokine analysis.

Statistical analysis

Data were analysed using a one-factor analysis of variance (anova), with an overall F-test at the 0·05 significance level used to determine treatment effects. Homogeneity of variance and normality was examined to assist in the interpretation of treatment effects. In the case of heterogeneous variance, transformation was used to stabilize the variance. Least-squares means comparison was adopted at the 0·05 significance level for pairwise comparison of two treatments using the ‘LSMeans Contrast’ command in anova. The anova was implemented in the JMP software system (SAS Institute, Cary, NC).


FasL- and LPS-induced lung inflammation time-course

In order to gain a perspective on FasL action during lung inflammation, intratracheal administration of rFasL was compared with intratracheal administration of LPS, the latter being a previously well-described model of pulmonary inflammation.20 Five-hundred nanograms of rFasL, 2 µg of LPS, or 500 ng of BSA was administered to mice intratracheally and bronchoalveolar lavage was performed at 3, 6, or 24 hr. The doses of rFasL (500 ng/mouse) and LPS (2 µg/mouse) used in this study were chosen based on previous dose-titration studies and are the minimal doses that elicited the maximal response of neutrophil infiltration and protein level increases in BAL fluid (data not shown). This time-course experiment facilitated examination of the timing and progression of inflammatory end-points, as well as a comparison of the effects of rFasL and LPS administration.

rFasL induced a faster initial neutrophil infiltration compared with LPS, but LPS induced a larger infiltration at later time-points (Fig. 1a). At the 3-hr time-point, rFasL had induced neutrophil infiltration to a level threefold higher than that induced by LPS (P < 0·001) and almost 10-fold higher than that induced by BSA (P < 0·001). By 6 hr, however, LPS had induced twofold greater infiltration than rFasL (P < 0·01) and, at 24 hr, LPS had induced almost fourfold greater infiltration than rFasL (P < 0·001).

Figure 1
Pulmonary instillation of recombinant Fas ligand (rFasL) or lipopolysaccharide (LPS) increases neutrophil infiltration and total protein in bronchoalveolar lavage (BAL) fluid. rFasL (500 ng/mouse), LPS (2 µg/mouse), or bovine serum albumin (BSA) ...

rFasL induced a larger increase in BAL fluid total protein compared with LPS at all time-points (Fig. 1b). At 3 hr, rFasL treatment resulted in a twofold increase in total protein compared with BSA (P < 0·001). In contrast, LPS treatment did not result in a statistically significant increase compared with BSA at 3 hr. By 24 hr, rFasL had induced an increase in BAL total protein, a 5·8-fold increase compared with BSA (P < 0·001) and a 1·9-fold increase compared with LPS treatment (P < 0·001). Overall, rFasL treatment increased the total protein in BAL fluid more rapidly, and to a greater extent, than LPS treatment.

In order to gain a greater understanding of the mechanism and timing of neutrophil infiltration and the increase of total protein, cytokines were measured in BAL fluid. The cytokines GM-CSF, IL-1β, TNF-α, IFN-γ, IL-2, IL-4, IL-5 and IL-10 were measured using multianalyte detection technology. Again, rFasL generally induced early and potent cytokine secretion compared with LPS (Fig. 2). The GM-CSF concentration peaked at 3 hr for both rFasL and LPS treatment and decreased thereafter (Fig. 2a). Compared with LPS, rFasL treatment stimulated a higher level of GM-CSF throughout the time-course. IL-1β, IL-5 and IFN-γ showed a similar pattern of secretion (Fig. 2b, 2c, 2d); rFasL treatment resulted in a peak of these cytokines at 6 hr. In each case, LPS treatment resulted in a lower level of secretion throughout the experiment; however, by 24 hr the amounts of IL-1β, IL-5 and IFN-γ were similar for treatment with either rFasL or LPS. TNF-α was the only cytokine detected that was elicited at a higher level during LPS treatment compared with rFasL treatment (Fig. 2e). TNF-α levels induced by rFasL or LPS peaked at 3 hr and showed a decrease at subsequent time-points. The cytokines IL-2, IL-4 and IL-10 were also measured, but were not detected.

Figure 2
Pulmonary instillation of recombinant Fas ligand (rFasL) or lipopolysaccharide (LPS) increases the levels of proinflammatory cytokines in bronchoalveolar lavage (BAL) fluid. rFasL (500 ng/mouse), LPS (2 µg/mouse), or bovine serum albumin (BSA) ...

DcR3 analogue treatment alleviates FasL-induced lung inflammation

As DcR3 analogue is an antagonist of FasL, it was tested in the rFasL-induced lung inflammation model. Eight-hundred micrograms of DcR3 analogue or BSA was administered i.v. 1 hr prior to the instillation of 100 ng of rFasL into the lung. The 100-ng/mouse dose of rFasL was selected to elicit a near-maximal inflammatory response to enable the effect of DcR3 analogue to be clearly observed. The dose of DcR3 analogue, 800 µg, was chosen on the basis of the pharmacokinetic analysis of the DcR3 analogue in mice; it was determined that 4 hr after dosing there was a substantial amount of DcR3 analogue present in serum (data not shown). The animals were killed 3 hr after rFasL instillation, and BAL fluid was collected for analysis in order to examine the earliest possible response to blocking rFasL-induced inflammation.

Treatment with the DcR3 analogue significantly reduced neutrophil infiltration in comparison to treatment with BSA (P < 0·01). On average, 7000 neutrophils (absolute count) were detected in the naïve group, ≈ 60 000 neutrophils were detected in the BSA group and ≈ 22 000 neutrophils were detected in the DcR3 analogue group. Treatment with the DcR3 analogue reduced neutrophil infiltration by about 2·7-fold (Fig. 3) compared with BSA treatment.

Figure 3
Decoy receptor 3 (DcR3) analogue treatment reduces neutrophil infiltration after pulmonary instillation of recombinant Fas ligand (rFasL). Eight-hundred micrograms of DcR3 analogue or bovine serum albumin (BSA) was given intravenously 1 hr prior to the ...

Compared with BSA treatment, DcR3 analogue treatment resulted in no significant change in BAL total protein at this early time-point (data not shown). rFasL induced an ≈ 60% increase of BAL total protein compared with naïve animals. Although there was a trend towards reduction of BAL total protein, DcR3 analogue treatment did not significantly modulate this effect.

In order to further delineate the physiological mechanism by which DcR3 analogue reduces rFasL-induced neutrophil infiltration, the concentration of different cytokines and chemokines was measured in BAL fluid. Compared with GM-CSF levels in mice treated with rFasL and BSA, DcR3 analogue treatment significantly (P < 0·001) reduced GM-CSF levels (Fig. 4a) in rFasL-treated mice, by 10-fold (≈ 800 pg/ml to 80 pg/ml, respectively). TNF-α and IL-1β were also detected in BAL fluid and both were significantly reduced by DcR3 analogue treatment compared with BSA treatment. DcR3 analogue reduced TNF-α by twofold (P < 0·01) and IL-1β also by twofold (P < 0·05), compared with BSA. However, a statistically significant reduction in TNF-α and IL-1β was not seen in a repeat experiment (data not shown). The remaining cytokines that were measured – IFN-γ, IL-2, IL-4, IL-5 and IL-10 – were not detected in any group.

Figure 4
Decoy receptor 3 (DcR3) analogue treatment reduces granulocyte–macrophage colony-stimulating factor (GM-CSF), macrophage inflammatory protein-2 (MIP-2) and KC response to recombinant Fas ligand (rFasL)-induced inflammation. Eight-hundred micrograms ...

In addition to cytokines, we also measured the levels of the chemokines MIP-2 and KC in BAL fluid samples. KC and MIP-2 are the murine homologues of human growth-regulated oncogene (GRO)α and GROβ, respectively, and are potent neutrophil chemoattractants.2123 Both MIP-2 and KC were induced ≈ 10-fold by instillation of rFasL into the lung (Fig. 4b, 4c). Approximately 0·7 ng/ml MIP-2 was detected in samples from naïve, untreated mice compared with the BSA/rFasL-treated sample average of 11·4 ng/ml. DcR3 analogue treatment reduced the concentration of MIP-2 to ≈ 2·6 ng/ml, a greater than fourfold reduction (P < 0·001). KC was similarly induced by instillation of rFasL and was decreased after treatment with the DcR3 analogue. Approximately 1·6 ng/ml KC was detected in samples from naïve mice compared with 17·6 ng/ml in FasL/BSA-treated samples. DcR3 analogue treatment reduced KC to an average of 4 ng/ml, a greater than fourfold reduction (P < 0·001). Thus, DcR3 analogue treatment in the lung inflammation model reduced the levels of both MIP-2 and KC in BAL fluid by greater than fourfold.

Measurement of the DcR3 analogue in BAL fluid samples confirmed the presence of DcR3 analogue and demonstrated that an average of 18·7 ng/ml was present in BAL fluid 4 hr after i.v. administration (Fig. 5a). Measurement of FasL in the BAL fluid samples confirmed the presence of rFasL (≈ 15 ng/ml) in the lung 3 hr after instillation. The antibody used in the ELISA does not cross-react with mouse FasL; therefore, the FasL measured comes solely from the rFasL instilled into the lung (Fig. 5b).

Figure 5
Concentration of decoy receptor 3 (DcR3) analogue and Fas ligand (FasL) in bronchoalveolar lavage (BAL) fluid. Eight-hundred micrograms of DcR3 analogue or bovine serum albumin (BSA) was given intravenously 1 hr prior to the intratracheal instillation ...

Alveolar M[var phi]s respond to rFasL

In order to identify potential cell types involved in rFasL-induced lung inflammation, alveolar M[var phi]s were isolated from normal mice and incubated with rFasL in vitro. A total of 1 × 105 alveolar M[var phi]s were incubated with 500 ng/ml rFasL, with or without 2 µg/ml DcR3 analogue, for 18 hr. Treatments were performed in triplicate, and the cell culture supernate was measured for cytokines and chemokines. MIP-2 and KC were detected in the culture supernate of rFasL-treated alveolar M[var phi]s, and treatment with the DcR3 analogue reduced the levels of both (Fig. 6). MIP-2 was induced to ≈ 2800 pg/ml following treatment with rFasL and to ≈ 960 pg/ml following treatment with the DcR3 analogue, an almost threefold reduction (P < 0·001). KC was induced to ≈ 400 pg/ml following treatment with rFasL and to ≈ 230 pg/ml following treatment with the DcR3 analogue, an almost twofold reduction (P < 0·01). The secretion of MIP-2 and KC by alveolar M[var phi]s, as a result of rFasL stimulation, parallels the induction of these chemokines by rFasL-meditated lung inflammation. The cytokines IL-1β, IL-2, IL-4, IL-5, IL-10, IFN-γ, GM-CSF, and TNF-α were also measured, but not detected.

Figure 6
Recombinant Fas ligand (rFasL) stimulates alveolar macrophages (M[var phi]s) to secrete cytokines and chemokines in vitro. Isolated alveolar M[var phi]s were stimulated with 0·5 µg/ml rFasL for 18 hr, and the cell culture supernate was ...

rFasL produces little inflammation in the peritoneum

In order to determine whether all tissues are able to respond to FasL in a similar manner to that of the lung, i.p. injection of rFasL, LPS, or BSA into mice was performed. A total of 500 ng of BSA or rFasL and 4 µg of LPS were injected per mouse. After 4 hr, peritoneal wash fluid (PWF) was collected and measured for cytokines and chemokines. IL-1β was detected in the PWF of both LPS- and rFasL-stimulated mice; however, LPS stimulated about a threefold higher level of this cytokine (Fig. 7a). The cytokines IL-2, IL-4, IL-5, IL-10, IFN-γ, GM-CSF and TNF-α were measured, but not detected, in the PWF. MIP-2 and KC were also measured in the PWF. LPS stimulated a large MIP-2 and KC response of 802 pg/ml and 4·3 ng/ml, respectively. rFasL stimulated only a small chemokine response, 67- and 34-fold lower than LPS for MIP-2 and KC, respectively (Fig. 7b, 7c). No significant differences in results were observed when repeated in a subsequent experiment using 1 µg of rFasL per mouse.

Figure 7
Lipopolysaccharide (LPS), but not recombinant Fas ligand (rFasL), causes inflammation in the peritoneum. Five-hundred nanograms of bovine serum albumin (BSA), 4 µg of lipopolysaccharide (LPS), or 500 ng of rFasL were administered intraperitoneally. ...


Recent data have shown that FasL signalling may play an important role in lung diseases via its proapoptotic and proinflammatory activities.11,12,24,25 In this report, we explored the less-known proinflammatory activity of FasL signalling within the lung using an in vivo mouse model. As human FasL has been demonstrated to bind to mouse Fas,26 we used rFasL, expressed and purified from mammalian cells, in the in vivo study. In vitro, rFasL displays proapoptotic activities on Jurkat cells and A20 cells (Alexis Biochemicals) (data not shown), and has low endotoxin levels. This report shows that the activation of Fas signalling via rFasL instillation can induce rapid increases in neutrophils and total protein in the lung, two hallmarks of lung inflammation, and is the first to provide some mechanistic understanding of rFasL-induced lung inflammation through extensive cytokine and chemokine analysis. Furthermore, we showed that in vivo treatment with the DcR3 analogue can reduce FasL-mediated lung inflammation dramatically, suggesting that DcR3 may have therapeutic utility in diseases in which FasL-induced apoptosis or inflammation is involved.

LPS is a potent and broad inducer of inflammation, and has been shown previously, in mouse and rat pulmonary instillation model studies, to cause neutrophil infiltration and increased vascular permeability.27,28 We compared the proinflammatory effects of rFasL and LPS instillation in the lung. The most notable observation from the rFasL instillation study was a high and rapid infiltration of neutrophils into the lung alveolar space after rFasL instillation, which peaked as early as 3 hr after challenge with rFasL. rFasL instillation, in comparison with the LPS treatment, resulted in significantly greater acute inflammation in the lung, as demonstrated by higher protein levels, neutrophil counts and proinflammatory cytokine levels in BAL fluid after 3 hr. At the later time-points of 6 and 24 hr, higher levels of neutrophils were detected in the LPS-treatment group. However, by comparison with LPS, rFasL instillation continued to show significantly higher permeability changes at all time-points of 3, 6 and 24 hr, as determined by total protein levels in BAL fluid. This observation points to a potentially important role of Fas activation during lung inflammation, and is consistent with the data presented in other recently published reports. To investigate whether all tissue responds to FasL in a manner similar to that of the lung, an in vivo experiment was performed in which rFasL was injected into the peritoneum of mice and the secretion of proinflammatory markers was measured. In contrast to findings in the lung after rFasL instillation, there was little evidence of inflammation when the mice were injected with up to 10 times the amount of rFasL protein that was used in the lung model study. On the other hand, injection of LPS (200 µg/kg, ≈ 4 µg/mouse) into the peritoneum resulted in a higher, acute secretion of inflammatory mediators such as IL-1β, MIP-2 and KC.

To understand the underlying mechanism for rapid neutrophil recruitment and subsequent vascular damage upon FasL-induced lung inflammation, we monitored the secretion of major proinflammatory cytokines and chemokines in BAL fluid after rFasL instillation. Increased neutrophil counts were accompanied with rapid and large increases in the concentration of proinflammatory cytokines such as TNF-α, IL-1β, GM-CSF, IL-5 and IFN-γ, and chemokines such as KC and MIP-2. The time-course study showed that the concentration of GM-CSF, a potent neutrophil chemoattractant and activator,29 peaked at 3 hr, while the levels of most other cytokines peaked at the 6-hr time-point. At the 3-hr time-point, the levels of KC and MIP-2 – two prominent neutrophilic chemokines2123– were also dramatically increased upon rFasL instillation. At this time-point, there was good correlation between the decrease in neutrophilic factors, such as GM-CSF, KC and MIP-2, in BAL fluid, and the concomitant reduction of the neutrophil levels in alveolar space after treatment with the DcR3 analogue (Fig. 3, Fig. 4). Based on this data, we postulate that secreted KC, MIP-2 and GM-CSF probably play a key role in the recruitment of neutrophils into the airspace. However, FasL itself has also been shown to be a chemoattractant for neutrophils,8,9 so its contribution to neutrophil infiltration cannot be overlooked. Recruited neutrophils may then secrete secondary mediators, such as reactive oxygen species and proteases, which cause endothelial cell damage, resulting in an increased vascular permeability and production of other proinflammatory cytokines. While the infiltrating neutrophils are probably the primary causative agents for subsequent lung damage in this model of acute lung injury, we cannot rule out the possibility that rFasL directly damages lung epithelial cells, leading to increased permeability. The observation that rFasL is capable of causing apoptosis of lung epithelial cells in vitro supports this hypothesis.24,25

DcR3 is a secreted TNF receptor member that is widely expressed and has been shown to block three TNF ligand members: FasL, LIGHT and TL1A.1517 Several tumour tissues have been shown to express high levels of DcR3. It has been suggested that tumours express DcR3 in order to overcome the host immune system attack via the Fas/FasL pathway.15,29 Consistent with the hypothesis of DcR3 analogue antagonist action, both rFasL and DcR3 analogue were detected in BAL fluid at the 3-hr time-point after rFasL instillation (4 hr after DcR3 analogue treatment). Indeed, an essential property of DcR3 analogue in this model is its ability to reach the alveolar space and exert its action of blocking FasL there. Taken together, our data suggests that the DcR3 analogue may be an effective therapeutic agent in diseases where FasL-induced inflammation is a major pathology.

While blocking of the Fas/FasL pathway is the probable mechanism of DcR3 analogue action in this model, the ability of the DcR3 analogue to block the LIGHT/herpesvirus entry mediator and/or TL1A/death receptor 3 signalling pathways cannot be totally discounted because LIGHT and/or TL1A could be secondary mediators of inflammation in the lung after the initial action of rFasL. LIGHT is expressed in activated T cells and immature dendritic cells, and has a T-cell costimulatory function. LIGHT signals through the herpesvirus entry mediator and LTβR and has been implicated in several T-cell-mediated autoimmune diseases.3134 TL1A is highly expressed in endothelial cells, has a T-cell costimulatory function like LIGHT, and was shown to cause apoptosis of endothelial cells.17,35,36 Interestingly, TL1A was shown to be up-regulated by TNF treatment on endothelial cells in vitro, and it was suggested that TL1A may be the effector molecule for TNF-mediated endothelial cell apoptosis/damage.17,36 Death receptor 3, the receptor for TL1A,17 contains a death domain, further suggesting TL1A's apoptotic activity. Given that the lung is a highly vascularized organ, it is possible that TL1A is one of the downstream effector molecules that is turned on during FasL-induced lung inflammation and is involved in FasL-induced endothelial cell damage.

We were interested in exploring what cell types in the lung may be responsible for chemokine and cytokine secretion following introduction of FasL. Our in vitro studies, using alveolar M[var phi]s, showed that rFasL treatment could directly activate these cells to secrete some of the same cytokines (IL-1β and TNF-α) and chemokines (MIP-2 and KC) that had been detected in BAL fluid samples following rFasL instillation into the lungs. This suggests that alveolar M[var phi]s may be one of the key cells that are activated following rFasL introduction and may be responsible for the subsequent secretion of cytokines and chemokines. Indeed, resident peritoneal M[var phi]s have been shown to secrete neutrophil chemotactic factors in response to FasL-mediated apoptosis.37 In addition to alveolar M[var phi]s, lung epithelial cells, endothelial cells and infiltrating neutrophils may also be important components in the development of lung inflammation. These additional cell types may secrete proinflammatory cytokines and chemokines by direct FasL inflammatory signalling. Indeed, the source of GM-CSF secretion in this study was not clear, as in vitro activation of alveolar M[var phi]s with rFasL did not result in the secretion of a measurable level of GM-CSF. rFasL may directly induce lung epithelial cells, endothelial cells, or infiltrating neutrophils to secrete GM-CSF.

In summary, the mechanism for FasL-induced lung inflammation was explored by using rFasL instillation in a mouse model of lung inflammation. We showed that FasL treatment can result in rapid secretion of potent neutrophil chemoattractants such as GM-CSF, KC and MIP-2, and these agents probably initiate the rFasL-induced inflammation cascade, leading to neutrophil infiltration and lung damage. We also demonstrated that one of the cell types that could be responsible for such secretions are alveolar M[var phi]s. Our study illustrated that the lung is sensitive to FasL activation-induced inflammation, and recent data has suggested that FasL-induced apoptosis and inflammation may play an important role in the pathogenesis of ARDS.11,12 Our finding, that the presence of FasL can dramatically increase pulmonary levels of proinflammatory cytokines and chemokines, neutrophil infiltration and BAL total protein, further supports the potential role of FasL signalling in ARDS. While no therapies aimed at blunting the Fas–FasL interaction have been tested therapeutically, agents that can block FasL signalling, such as the DcR3 analogue, may be a potential therapy for ARDS.


We thank Christy McCloud for DcR3 analogue ELISA analysis of BAL fluid, Paul Atkinson and Erv Kattelman for providing DcR3 analogue for in vivo experiments, Yunfei Chen for statistical analysis support, Tim Noblitt for flow cytometry assistance, and Larry Mann for multiplexed analyte detection analysis.


acute respiratory distress syndrome
bronchoalveolar lavage
bovine serum albumin
decoy receptor 3
Fas ligand
granulocyte–macrophage colony-stimulating factor
M[var phi]
macrophage inflammatory protein-2
phosphate-buffered saline
peritoneal wash fluid
recombinant Fas ligand
tumour necrosis factor-α
white blood cells


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