Human sFasL aggregates in the lungs of patients with ARDS.

To further define the molecular size of sFasL in ARDS BAL fluid, we performed Western blot with nonreducing SDS-PAGE using concentrated BAL fluid from normal volunteers, patients at risk for ARDS, and patients with established ARDS. Initial experiments showed substantial cross-reactivity among several different HRP-conjugated secondary antibodies and other proteins present in the BAL fluid. To minimize cross-reactivity, the BAL fluid samples were immunodepleted of albumin and IgG. Nevertheless, bands ranging from 100 kDa to 250 kDa were present in normal plasma and BAL fluid samples from normal volunteers, patients at risk for ARDS, and patients with ARDS, even when a control antibody was used (Figure ​(Figure1G),1G), showing that these bands are not specific for sFasL. Normal plasma spiked with recombinant sFasL showed bands at 25, 50, and 75 kDa, corresponding to monomers, dimers, and trimers of sFasL (Figure ​(Figure1G,1G, lane F). Dimers of sFasL were detected in BAL fluid samples from all patients with ARDS but were not detected in the BAL fluid from normal volunteers or patients at risk for ARDS. Monomers of sFasL were not detected in any of the samples.

ARDS BAL fluid multimerizes rh-sFasL.

To determine whether substances present in the BAL fluid influence the multimerization of sFasL, rh-sFasL was incubated in PBS or BAL fluid from normal subjects, at-risk patients, or patients with established ARDS (Figure ​(Figure2).2). Western blot of the rh-sFasL incubated in PBS showed bands at 25 and 50 kDa and a less intense band at 75 kDa, consistent with the predicted molecular weight of glycosylated monomers, dimers, and trimers, respectively (Figure ​(Figure2A).2A). When rh-sFasL was incubated in BAL fluid from patients with ARDS, additional bands appeared at higher molecular weights, ranging from 100 kDa to 250 kDa, indicating the formation of SDS-resistant aggregates of sFasL.

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Figure 2

Effect of ARDS BAL fluid on multimerization and bioactivity of sFasL.

(A) Pooled BAL fluids from 3 normal volunteers, 3 at-risk patients, and 3 patients with ARDS were diluted in PBS, incubated alone or with rh-sFasL, and then analyzed by Western blot. Larger aggregates of rh-sFasL, ranging from 100 kDa to 250 kDa, were detected only when rh-sFasL was incubated in ARDS BAL fluid. A single 250-kDa band is present in the unspiked ARDS BAL fluid and represents either cross-reactivity or large aggregates of endogenous sFasL. (B) Effect of inhibitors (NaN₃ [MPO inhibitor, 1 mM], l-methionine [HOCl inhibitor, 500 μM], and catalase [H₂O₂ inhibitor, 500 U/ml]) on multimerization of rh-sFasL in ARDS BAL fluid. (C and D) Two samples of pooled ARDS BAL fluids were incubated with exogenous rh-sFasL for 90 minutes. In 1 of the ARDS BAL samples, the antioxidant l-methionine (l-met) was added after incubation with exogenous rh-sFasL (l-methionine, after). The other sample was preincubated with l-methionine, followed by incubation with exogenous rh-sFasL (l-methionine, before). Incubation of rh-sFasL in ARDS BAL fluid increased (C) multimerization and (D) cytotoxicity for Jurkat cells, which was prevented by preincubation of ARDS BAL fluid with l-methionine. (D) Low cytotoxicity of the ARDS BAL fluids is explained by dilution of the BAL fluid. Results represent mean ± SD of 3 separate experiments performed in duplicates. *P < 0.05 vs. all other samples. All Western blots were run in nonreducing conditions. Lane numbers in D correspond to lane numbers in C.

Effect of oxidants on multimerization and bioactivity of sFasL in ARDS BAL fluid.

Hypochlorous acid (HOCl), generated from H₂O₂ and Cl^– via the catalytic action of myeloperoxidase (MPO) in activated neutrophils and monocytes, is a potent oxidant that can alter the structure and biological activity of proteins (32, 35, 36). Because both MPO and H₂O₂ accumulate in the lung fluids of patients with ARDS (27, 30), we investigated the effect of oxidants on the multimerization of added rh-sFasL, using the same pooled BAL fluid samples tested previously. Aggregation of rh-sFasL was observed only when rh-sFasL was incubated in ARDS BAL fluid (Figure ​(Figure2,2, A–C). This was inhibited by the MPO inhibitor NaN₃, the H₂O₂ scavenger catalase, and the HOCl scavenger l-methionine (Figure ​(Figure2B). 2B).

To determine whether oxidants in the ARDS BAL fluid influence the biological activity of sFasL, exogenous rh-sFasL was incubated for 90 minutes in ARDS BAL fluid or PBS, and the oxidant scavenger l-methionine was added either before or after incubation with the rh-sFasL. As expected, the rh-sFasL incubated in ARDS BAL fluid showed a higher degree of multimerization in nonreducing conditions (Figure ​(Figure2C)2C) and also was more cytotoxic for Jurkat cells as compared with rh-sFasL incubated in PBS (49.8% ± 3.8% vs. 33.2% ± 4.1% cell death, respectively; P < 0.05) (Figure ​(Figure2D).2D). This increase in multimerization and cytotoxicity of rh-sFasL was prevented when the antioxidant l-methionine was added to the ARDS BAL fluid prior to incubation with rh-sFasL (27.1% ± 11.2% cell death). Thus, oxidants in ARDS BAL fluid cause multimerization/aggregation of sFasL and enhance biological activity.

To study the effects of MPO-catalyzed oxidation and nitration reactions on multimerization of human sFasL, we exposed rh-sFasL to the MPO-H₂O₂-chloride or MPO-H₂O₂-nitrite system using 2 different concentrations of H₂O₂. The rh-sFasL formed multimers when incubated with MPO, H₂O₂, and NaCl but not when incubated with MPO-H₂O₂ or MPO-NaCl alone or with the MPO-H₂O₂-NaNO₂ system (Figure ​(Figure3A).3A). The effects of H₂O₂-MPO-Cl^– or HOCl on sFasL multimerization were dose dependent (Figure ​(Figure3,3, B and C), and high concentrations of H₂O₂ and MPO with Cl^–, as well as HOCl alone, caused progressive loss of the sFasL bands, suggesting degradation or loss of reactive epitopes. Importantly, preincubation with the HOCl scavenger l-methionine completely prevented the multimerization of human sFasL as well as its degradation with the highest concentrations of H₂O₂ and MPO or HOCl (Figure ​(Figure3,3, B and C). Therefore, MPO-derived oxidants, such as HOCl, provide a mechanism for the multimerization of sFasL in the BAL fluid of patients with ARDS as well as for its degradation.

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Figure 3

Effect of oxidants on multimerization of sFasL.

(A) Western blot analysis of rh-sFasL incubated in PBS-DTPA alone or in the presence of H₂O₂ (10 μM or 20 μM) with or without MPO (50 nM) and either NaNO₂ (100 μM) or NaCl (100 mM). Reactions were terminated at 60 minutes with l-methionine (2.5 nM). Multimers of rh-sFasL (75-kDa and larger molecular weight bands) were detected when sFasL was exposed to MPO-H₂O₂-chloride. (B and C) Dose-dependent effects of MPO (nM) and H₂O₂ (μM) or HOCl (μM) on multimerization of rh-sFasL, respectively. Degradation of rh-sFasL occurred at the highest concentrations of MPO and H₂O₂ or HOCl. Western blot analyses in A–C were performed using SDS-PAGE with 2-ME and heat. (D) Lack of effect of urea (8 M) on oxidized multimers of rh-sFasL. Samples were resolved in SDS-PAGE with 2-ME (no heat). (E) Lack of effect of DTT (10 μM or 20 μM) on HOCl-oxidized multimers of rh-sFasL. Western blot was performed in SDS-PAGE with no 2-ME or heat. (F) Effect of DMS and HCl on multimerization of HOCl-oxidized multimers of rh-sFasL. Samples were filtered by centrifugation to eliminate DMS and HCl and neutralize pH and then subjected to SDS-PAGE in nonreducing conditions (no 2-ME, no heat). In D–F, the lanes were taken from the same gels. The vertical hairlines in E separate noncontiguous lanes in the same gel.

The oxidized multimers of sFasL did not dissociate in SDS-PAGE with reducing agents such as 2-ME and heat (+96°C) (Figure ​(Figure3,3, A–C) or with high concentrations of urea (8 M) or high concentrations of dithiothreitol (DTT) (10 or 20 mM) (Figure ​(Figure3,3, D and E, respectively). To explore the role of oxidized methionines in multimerization of sFasL, which could have been produced by the oxidants in the prior experiments, we incubated oxidized sFasL with dimethyl sulfide (DMS) and hydrochloric acid (HCl), which is reported to reduce methionine sulfoxide to methionine (ref. 37 and Figure ​Figure3F).3F). This resulted in the dissociation of aggregates of sFasL to monomers and lower molecular weight fragments. Taken together, these results suggest that methionine oxidation is involved in aggregation of sFasL through the formation of disulfide-independent intramolecular bonds.

ARDS BAL fluid oxidizes methionine residues in sFasL.

To investigate the role of methionine and other amino acids in the aggregation of sFasL in ARDS BAL fluid, we incubated rh-sFasL in normal, at-risk, or ARDS BAL fluid and then purified the rh-sFasL using an immunoaffinity column. The recovered rh-sFasL was digested with trypsin, and the tryptic peptides were analyzed by liquid chromatography–electrospray ionization–tandem mass spectrometry analysis (LC-ESI-MS/MS). We focused on the tryptic peptide Y²¹⁸PQDLVMMEGK, because Y²¹⁸ is in the Fas binding site, and nearby tyrosine and methionine residues are targets for modification. When sFasL was incubated with normal or at-risk BAL fluid, a major peak of material with a mass-to-charge ratio (m/z) anticipated for the doubly protonated peptide (m/z, 656.1) was detected (Figure ​(Figure4A,4A, normal and at risk). Analysis by tandem mass spectrometry (MS/MS) confirmed that the sequence of this peptide was YPQDLVMMEGK (Figure ​(Figure4B,4B, top row). In contrast, when sFasL was incubated in BAL fluid from patients with ARDS, the Y²¹⁸PQDLVMMEGK peptide almost disappeared and a major peak of new material appeared with a m/z of 672.1 (doubly charged) (Figure ​(Figure4A,4A, ARDS), suggesting that the molecular weight of the original peptide had increased by 32 Da, corresponding to the gain of 2 oxygen atoms. The MS/MS analysis of the modified peptide revealed that each of the methionine residues in the peptide gained 16 Da, indicating that they were oxidized to methionine sulfoxide (Figure ​(Figure4B,4B, bottom row). In contrast, chlorination or nitration of Y²¹⁸ were not detected, suggesting that the methionine residues of sFasL are major targets of oxidation by reactive intermediates in BAL fluid of patients with ARDS.

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Figure 4

Oxidation of methionine residues in sFasL by ARDS BAL fluid.

(A) Reconstructed ion chromatogram (RIC) of a tryptic peptide from rh-sFasL exposed to normal, at-risk, or ARDS BAL fluid. The mass-to-charge ratios of the peptide from sFasL exposed to normal and at-risk BAL fluid were 656.1. The mass-to-charge ratio of the peptide from sFasL exposed to ARDS BAL fluid was 672.1 (original peptide +32 Da, doubly charged). The increase of 32 Da corresponds to the addition of 2 oxygen atoms to the peptide. (B) MS/MS analysis of peptide Y²¹⁸PQDLVMMEGK (top row) and oxidized peptide Y²¹⁸PQDLVM(O)M(O)EGK (bottom row) in a tryptic digest of rh-sFasL exposed to normal BAL fluid (top row) or ARDS BAL fluid. Exposure to ARDS fluid resulted in oxidation of the 2 methionine residues near the receptor-ligand binding site (Y²¹⁸).

Methionine residues are involved in oxidation-mediated multimerization of sFasL.

To confirm that oxidation of methionine residues is involved in the multimerization of sFasL, we took advantage of the fact that murine sFasL contains fewer methionine residues than human sFasL. We cloned the mouse sFasL cDNA and used site-directed mutagenesis to change specific residues to methionine at the locations of methionine residues in human sFasL (Figure ​(Figure5A).5A). This “humanized” mouse sFasL contained a total of 7 methionine residues (mouse met-sFasL). When the mouse met-sFasL and the native form of mouse sFasL were subjected to mild oxidation with HOCl, the mouse met-sFasL multimerized more readily than the native sFasL (Figure ​(Figure5B),5B), and the prior addition of l-methionine prevented multimerization (Figure ​(Figure5B,5B, lanes 1 and 5).

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Figure 5

Methionine residues are involved in oxidative multimerization of sFasL.

(A) Amino acid sequence of mouse native-sFasL and met-sFasL. Native sFasL contains 2 methionine residues (underlined), whereas met-sFasL contains 7 methionine residues (underlined) in the same locations as those in human sFasL. (B) Western blot analysis of the native form of mouse sFasL (native-sFasL) and a mutated form of mouse sFasL containing methionines at the location of methionine in human sFasL (mouse met-sFasL). Native sFasL (lanes 2–4) and met-sFasL (lanes 6–8) were incubated with increasing concentrations of HOCl (μM), and the reaction was stopped with l-methionine (2.5 nM). For comparison, l-methionine was added to the reaction before incubation with mouse native sFasL (lane 1) or met-sFasL (lane 5) to prevent protein oxidation. Nonoxidized mouse native sFasL and met-sFasL showed single bands at 25 kDa, corresponding to sFasL monomers (lanes 1 and 5). When oxidized, met-sFasL (lanes 6–8) multimerized more readily than native-sFasL (lanes 2–4). (C) Biological activity of normal and oxidized forms of mouse native sFasL and met-sFasL for mouse lung epithelial cells (MLE-15). Either media alone or rh-sFasL were used as controls. Bars show the mean ± SD of 3 separate experiments. *P < 0.05 compared with all other samples. The Western blot was run in SDS-PAGE in reducing conditions (2-ME and heat).

We then compared the biological activities of the humanized mouse met-sFasL and the mouse native sFasL using MLE-15 mouse lung epithelial cells (Figure ​(Figure5C).5C). Neither mouse native sFasL nor mouse met-sFasL induced either expression of C-X-C chemokine (KC) or activation of caspase-3 in MLE-15 cells in nonoxidative or oxidative conditions, despite the high concentrations of both proteins tested (800 ng/ml), and cell death also was unaffected (data not shown). In contrast, a lower concentration of rh-sFasL (400 ng/ml) was biologically active, as shown by increased KC expression and caspase-3 activation in MLE-15 cells. Therefore, increasing the number of methionine residues in mouse sFasL promoted multimerization in oxidative conditions, but this by itself was not sufficient to increase the biological activity of this form of mouse sFasL.

Relevance of the N-terminal stalk region for bioactivity of sFasL.

Because human sFasL was biologically active in MLE-15 cells, whereas mouse met-sFasL was not, we explored the possibility that structural determinants other than methionine residues also affected the biological activity of sFasL. The mouse sFasL (both native and met-sFasL) that we used contained only the TNF domain at the C terminus, whereas the commercial form of human sFasL contained the C-terminal TNF domain as well as a stalk domain at the extracellular N terminus. Therefore, we expressed a long form of human sFasL containing both the stalk and the TNF domains (103–281 aa) and a short form of human sFasL containing the TNF domain and only 6 amino acids of the stalk region (129–281 aa) (Figure ​(Figure6A)6A) and then tested the biological activity of each form of sFasL in the lungs of C57BL/6 mice.

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Figure 6

Effect of the stalk region at the N terminus on the biological activity of human sFasL in vivo.

(A) Structure of the short and long variants of human sFasL produced in HEK cells. The short rh-sFasL contains only the TNF domain. Both proteins have a leading 15–amino acid linker peptide encoded by the expression vector. (B–H) Effects of long and short rh-sFasL in mouse lungs 16 hours after intratracheal instillation (25 ng/g body weight). (B) Representative lung tissue sections stained with hematoxylin and eosin, showing alveolar wall thickening, vascular congestion, alveolar hemorrhage, and neutrophilic infiltrates in mice treated with long rh-sFasL. h-sFasL, human sFasL. (C–F) Effect of long rh-sFasL on lung wet weight, concentration of IgM in BAL fluid and PMNs in BAL fluid, and MPO activity in lung homogenates. (G) Analysis of cell death in lung tissue sections (TUNEL, green dots) and the overlap of differential interference contrast (DIC) image and TUNEL fluorescence signal (DIC, TUNEL). (H) Measurement of TUNEL-positive cells in lung sections (mean ± SD). (I) Single section of mouse lung, dually stained with the TUNEL method, followed by staining with an antibody to cytokeratin, and then visualized by fluorescence microscopy using a red wavelength (cytokeratin) or a green wavelength (TUNEL) and light microscopy (DIC). The merged image shows cells that are dual labeled by the TUNEL method and the cytokeratin antibody (epithelial cells). (J) Caspase-3 activity in lung homogenates. Each dot represents an individual mouse. Horizontal bars represent medians. *P < 0.01; **P < 0.001. Original magnification, ×200 (B, G, and I); insets (×400).

Lung histology, permeability, and inflammation.

Intratracheal instillation of the long form of human sFasL (25 ng/g body weight) resulted in overt alveolar wall thickening, vascular congestion, alveolar hemorrhage, and neutrophilic infiltration, whereas the lungs of mice treated with the short form of sFasL were histologically normal (Figure ​(Figure6B).6B). At 16 hours after instillation, the wet weight of the lungs increased significantly in the mice treated with the long form of human sFasL but not in mice treated with the short form (Figure ​(Figure6C).6C). The concentration of IgM in BAL fluid, used as a marker of protein permeability, was significantly increased in mice treated with long human sFasL (Figure ​(Figure6D),6D), but was unaffected in mice treated with the short human sFasL. The long human sFasL caused a significant increase in the total number of polymorphonuclear leukocytes (PMNs) in BAL fluid, whereas the short form of human sFasL had no effect on the BAL cell profile (Figure ​(Figure6E).6E). Accordingly, the MPO activity in lung homogenates was significantly increased only in the lungs of mice treated with the long rh-sFasL (Figure ​(Figure6F).6F). The lungs of mice treated with the long human sFasL contained higher concentrations of the proinflammatory cytokines IL-1β, IL-6, KC, MIP-2, MCP-1, and MIP-2 and lower concentrations of the antiinflammatory cytokine IL-4 (Table ​(Table1),1), whereas cytokine concentrations were not increased in the lungs of mice treated with the short human sFasL. TNF-α, IFN-γ, IL-10, and IL-12 were not detected in any of the groups.

Table 1

Cytokine and chemokine concentrations in lung homogenates of C57BL/6 mice 16 hours after intratracheal instillation of short or long rh-sFasL

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Activation of proapoptotic pathways.

Analysis of lung tissue sections using the TUNEL method showed that the number of nuclei containing DNA strand breaks was significantly higher in mice treated with the long form of sFasL than in those treated with the short form (P < 0.05) (Figure ​(Figure6G).6G). Fluorescent double staining of DNA fragmentation and cytokeratin (marker of epithelial cells) showed that some TUNEL-positive cells in the alveolar wall were also positive for cytokeratin, indicating that alveolar epithelial cells were undergoing apoptosis (Figure ​(Figure6I).6I). Some cells were TUNEL positive but cytokeratin negative, indicating that other populations of lung cells were undergoing apoptosis. Caspase-3 activity was significantly increased only in the lungs of mice treated with long human sFasL (Figure ​(Figure6J). 6J).

All of the alterations observed in the lungs of mice treated with intratracheal instillation of the long form of sFasL were absent in lpr mice, which carry a nonfunctional Fas receptor, indicating that the long human sFasL causes lung injury and inflammation by activating the murine Fas receptor (data not shown).

The stalk region and oxidation of human sFasL influence bioactivity in human cells.

To test whether the stalk region of human sFasL also influences biological activity in human cells, we treated human Jurkat cells with the long and short forms of human sFasL (Figure ​(Figure7).7). The long human sFasL was cytotoxic at a concentration of 12.3 ng/ml (15% ± 5.6%), whereas the dose response of the short form of human sFasL was shifted significantly to the right (onset of cytotoxicity at 111.1 ng/ml). On a molar basis, this difference in cytotoxicity is even more pronounced.

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Figure 7

Cytotoxicity of long and short forms of rh-sFasL in Jurkat cells.

Jurkat cells were incubated with media supplemented with serial concentrations of the long or short forms of rh-sFasL, and cytotoxicity was determined 18 hours later using the alamarBlue assay. Cells treated with medium only were used to determine 100% survival. The long human sFasL was more cytotoxic at each concentration of sFasL. Data are the mean ± SD of 3 separate experiments performed in duplicates. *P < 0.05 for long vs. short human sFasL.

To test whether oxidation modifies the biological activity of the long and short forms of human sFasL in human lung epithelial cells, we transfected human embryonic kidney (HEK) cells with cDNA directing the expression of long or short human sFasL. The supernatants of the transfected HEK cells were treated with HOCl (100 μM) and then incubated with human SAECs for 16 hours at 50% final concentration (Figure ​(Figure8).8). Compared with short human sFasL, exposure of SAECs to long human sFasL induced higher expression of IL-8 (511.8 ± 17.6 pg/ml vs. 826.3 ± 28.7 pg/ml; P < 0.05), caspase-3 activity (33.2 ± 2.9 vs. 57.8 ± 5.9 arbitrary units; P < 0.05) and percentage of cell death (1.7% ± 2.3% vs. 32.0% ± 5.5%; P < 0.05). Oxidation increased the biological activity of long human sFasL but did not change the bioactivity of short human sFasL.

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Figure 8

Effect of oxidation on the bioactivity of long and short human sFasL for human SAECs.

Human SAECs were incubated with supernatants of transfected HEK cells that express either long or short rh-sFasL. These supernatants were pretreated with (black bars) or without (white bars) HOCl (100 μM). MO, medium only; SAECs incubated with supernatant from nontransfected HEK cells, with or without HOCl pretreatment. Expression of IL-8, caspase-3 activity, and cell death were determined after 18 hours by ELISA, enzymatic assay, and alamarBlue, respectively. SAECs treated with medium only were used to determine 100% survival. Long rh-sFasL was significantly more bioactive than short rh-sFasL, and oxidation enhanced the bioactivity of long rh-sFasL. Data are the mean ± SD of 3 separate experiments performed in duplicate. *P < 0.05 vs. medium only; ^#P < 0.05 vs. medium only and short rh-sFasL.

The stalk region and oxidation of human sFasL influence bioactivity in mouse lungs.

To determine whether oxidation modifies the biological activity of the long and short forms of human sFasL in the lungs in vivo, we treated mice with a single intratracheal instillation of HEK cell supernatant containing long human sFasL (nonoxidized or oxidized) or short human sFasL (nonoxidized or oxidized) (Figure ​(Figure9).9). In all cases, the long form was more bioactive than the short sFasL (Figure ​(Figure9).9). Oxidation increased the bioactivity of the long form of human sFasL, as shown by an increase in the number of PMNs in BAL fluid, increased caspase-3 activity in lung homogenates, higher concentrations of IgM in the BAL fluid, and higher expression of KC and IL-1β in lung homogenates, although not all the responses reached statistical significance. In contrast, oxidation did not increase the bioactivity of the short form of human sFasL. Thus, the stalk region is a critical determinant of the biological activity of human sFasL and oxidation enhances the activity of the long rh-sFasL in vivo.

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Figure 9

Effect of oxidation on the bioactivity of long and short rh-sFasL in mouse lungs in vivo.

Mice were treated with 1 intratracheal instillation of HEK cell supernatant, containing short or long human sFasL, and pretreated with or without HOCl as an oxidant and studied 16 hours later. Control refers to nontransfected HEK cell supernatant pretreated with or without HOCl (100 μM). The oxidized long rh-sFasL caused greater increases in (A) the number of PMNs in BAL fluid, (B) caspase-3 activation in lung homogenates, (C and D) concentration of total protein and IgM in BAL fluid, and (E and F) expression of KC and IL-1β in lung homogenates. Each dot represents a single mouse. Horizontal bars represent medians. *P < 0.05 vs. control supernatant and short rh-sFasL; ^#P < 0.05.

Long and short human sFasL have similar capacity to aggregate.

In order to determine whether the stalk region could cause differences in the degree of aggregation of sFasL, we compared the aggregation status of recombinant short and long human sFasL in basal or oxidative conditions (Figure ​(Figure10).10). In nonoxidative conditions, both short and long human sFasL appeared as monomers (25 kDa). After oxidation with HOCl, both short and long human sFasL showed similar degrees of dose-dependent aggregation, suggesting that the stalk region in sFasL does not enhance aggregation, even when the protein is oxidized.

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Figure 10

Oxidation-mediated multimerization of short and long sFasL.

Western blot analyses of short and long rh-sFasL incubated in PBS alone or in the presence of 2 different concentrations of HOCl (μM). After 15 minutes, the reaction was terminated with 2.5 nM l-methionine. Both short and long human sFasL have the same aggregation status in basal conditions. Following oxidation with HOCl, short and long rh-sFasL showed similar multimerization (50-kDa and larger molecular weight bands). Western blot analyses were performed using SDS-PAGE with reducing conditions (2-ME and heat).

Proteolytic cleavage of the stalk region by MMP-7 reduces the biological activity of human sFasL in vitro.

The recombinant long human sFasL contains 2 MMP-7 cleavage sites near the N terminus, 1 near the cell membrane and 1 near the beginning of the TNF homology domain (Figure ​(Figure11).11). We hypothesized that cleavage of the long sFasL protein at the Ser126-Leu127 (S-L) site by MMP-7 would release the stalk region, transforming the active long sFasL protein into the less active short sFasL. To test this, affinity-purified long human sFasL, derived from supernatant of transfected HEK cells, was incubated with different concentrations of human MMP-7 (Figure ​(Figure12A).12A). Whereas the long human sFasL showed a prominent band at 25 kDa, corresponding to monomeric sFasL, treatment with MMP-7 shifted the monomeric band to a lower molecular weight, and a new band below 10 KDa appeared, corresponding to the predicted molecular weight of the stalk region with the extra 15–amino acid linker peptide from the expression vector (Figure ​(Figure12A).12A). Importantly, treatment of sFasL with MMP-7 led to a significant reduction of cytotoxicity for Jurkat cells (Figure ​(Figure12B).12B). We further used nanoLC-ESI-MS/MS to detect cleavage of sFasL by MMP-7. The long sFasL proteins, either treated or untreated with MMP-7, were digested with trypsin, and the tryptic peptides were analyzed using nanoLC-ESI-MS/MS (Figure ​(Figure13).13). MMP-7 cleaved long human sFasL at the S-L site (about 40% of protein, Figure ​Figure13A),13A), resulting in the release of the ESTSQM¹²¹HTASS peptide in the stalk region. Together, these results show that MMP-7 cleaves the stalk region of sFasL and reduces the biological activity of the long human sFasL in vitro. Interestingly, oxidation of Met¹²¹ in the stalk region of the sFasL prevented cleavage at the S-L site by MMP-7 (Figure ​(Figure13B),13B), suggesting that oxidation of methionine residues could be a mechanism that prevents cleavage of the stalk region of human sFasL in vivo.

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Figure 11

Primary structure of FasL.

The mFasL is composed of intracellular (1–80 aa), transmembrane (81–102 aa), and extracellular (103–281 aa) domains. The extracellular domain contains the stalk region at the N terminus and the TNF homology domain (THD) at the C terminus. The amino acid sequence of the extracellular domain of FasL is shown. The stalk region is underlined, and the TNF homology domain region is italicized. Two cleavage sites for MMP-7 (1st, ELAEL; 2nd, SL) are present in the stalk region of the extracellular domain of FasL. Seven methionines (bold) are present in the TNF homology domain, and one is in the stalk region.

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Figure 12

Effect of MMP-7 on structure and cytotoxicity of human sFasL.

(A) Incubation of the long form of the rh-sFasL (0.5 μM) with serial concentrations of MMP-7 (0.16, 0.5, or 1.5 μM). The samples were analyzed by Western blot using SDS-PAGE in nonreducing conditions using a polyclonal antibody against human sFasL and (B) tested for cytotoxicity in Jurkat cells. (A) Treatment with serial concentrations of MMP-7 (lanes 2–4) resulted in a shift of the monomers (25 kDa) of sFasL to a lower molecular weight and the appearance of a new band below 10 kDa (arrow) corresponding to the predicted molecular weight of the stalk region plus the 15–amino acid linker peptide encoded by the expression vector. Numbers on the left show molecular weight. (B) Cytotoxicity of native and MMP-7–treated rh-sFasL in Jurkat cells at 18 hours using the alamarBlue assay. The white bar shows the cytotoxicity of untreated sFasL; the gray bars show the cytotoxicity of MMP-7 alone. Incubation with MMP-7 reduced the cytotoxicity of long human sFasL (black bars) as compared with the effect of MMP-7 alone (gray bars). The data are mean ± SD of 3 separate experiments performed in duplicate. *P < 0.05 vs. long human sFasL and MMP-7 alone.

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Figure 13

Effect of oxidation of Met¹²¹ on cleavage of rh-sFasL by MMP-7.

rh-sFasL was incubated with MMP-7 (0.25 μM) as in Figure ​Figure12,12, then digested with trypsin and analyzed using nanoLC-MS. (A) Reconstructed ion chromatogram of uncleaved nonoxidized peptide ESTSQM¹²¹HTASSLEK (m/z 768.5, doubly charged) and its cleaved product ESTSQM¹²¹HTASS (m/z 583.3, doubly charged), indicating that approximately 40% of the nonoxidized peptide was cleaved as detected by peak area. (B) Reconstructed ion chromatogram of uncleaved oxidized peptide ESTSQM(O)¹²¹HTASSLEK (m/z 776.5, doubly charged) and its cleaved product ESTSQM(O)¹²¹HTASS (m/z 591.3, doubly charged), indicating that only 0.5% of the oxidized peptide was cleaved.

Cloning of human and mouse sFasL cDNA and site-directed mutagenesis

Different lengths of human and mouse sFasL cDNA sequences were amplified in a standard PCR with DNA polymerase (Easy-A High-Fidelity PCR Cloning Enzyme, Stratagene), using cloned human FasL cDNA (catalog 10659567, ATCC) or cloned mouse FasL cDNA (catalog 10471123, ATCC) as templates. The sequences of PCR primers used for cDNA amplification were as follows: human long sFasL, sense 5′-CAGCTCTTCCACCTACAGAAGGAGC-3′ and anti-sense 5′-GTCCCCAAAACATCTCTCTTG-3′; human short sFasL, sense 5′-AAGCAAATAGGCCACCCCAG-3′ and anti-sense 5′-AGTTTCACCGATGGCTCAGG-3′; and mouse short sFasL, sense 5′-CCCAGTACACCCTCTGAAAAAAAAGAGCCG-3′ and anti-sense 5′-GGCTCATGATGCAGGCATTAAGGACC-3′. The PCR conditions were 95°C for 2 minutes; 95°C for 40 seconds; 55°C for 30 seconds and 72°C for 1 minute for 35 cycles; and 72°C for 7 minutes. The amplified sFasL cDNA was cloned into a pSecTag/FRT/V5-His-TOPO mammalian expression vector (Invitrogen). Mouse sFasL cDNA was mutated by site-directed mutagenesis in order to generate a humanized mouse sFasL using the mammalian expression vector encoding cloned native mouse sFasL cDNA as a template. Point mutations in a total of 5 codons in the plasmid encoding mouse sFasL cDNA were performed by using a QuikChange II Site-Directed Mutagenesis Kit (Stratagene), with the following mutagenic primers: sense 5′-GTATCCTGAGGATCTGGTGATGAGGAGGAGAAGATGATG-3′ and anti-sense 5′-CATCATCTTCTCCTCCATCATCACCAGATCCTCAGGATAC-3′ for codon 91, sense 5′-CTGCACTACTGGACAGATGTGGGCCCACAGCAGCCACCTG-3′ and anti-sense 5′-CAGGTGGCTGCTGTGGGCCCACATCTGTCCAGTAGTGCAG-3′ for codon 105, sense 5′-GCTAATGGAGGAGAAGATGATGAACTACTGCACTACTGG-3′ and anti-sense 5′-CCAGTAGTGCAGTAGTTCATCATCTTCTCCTCCATTAGC-3′ for codons 96 and 97, and sense 5′-GAACCCCCACTCAAGGTCCATGCCTCTGGAATGGGAAGAC-3′ and anti-sense and 5′-GTCTTCCCATTCCAGAGGCATGGACCTTGAGTGGGGGTTC-3′ for codon 25. The sequences of the cloned pSecTag/cDNA-FasL plasmids were confirmed using the Big Dye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems).

Expression of sFasL protein in mammalian cells

sFasL was expressed in HEK cells (FreeStyle 293-F cells, Invitrogen) transfected with pSecTag/cDNA-FasL encoding the human or mouse FasL cDNA using lipofection and incubated in a humidified incubator at 8% CO₂, 37°C, with FreeStyle293 Expression serum-free medium (Gibco), with constant shaking. After 6 days of incubation, the cell supernatant was harvested, filtered, and stored at –20°C until used. The expressed sFasL protein was purified using immunoaffinity columns (HiTrap NHS-activated HP, GE Healthcare), which were prepared according to the manufacturer’s instructions, using a mouse monoclonal anti-human FasL antibody or a rat anti-mouse FasL antibody (R&D Systems). The expressed recombinant sFasL was tested for endotoxin (<0.1 EU per 1 μg sFasL, measured by the Limulus Amebocyte Lysate, Pyrochrome). The concentrations of human and mouse sFasL proteins were measured using ELISA Kits (MBL or R&D Systems, respectively), per the manufacturer’s instructions. The lower detection limits were 75 pg/ml for human and 15 pg/ml for mouse sFasL.

Human subjects and BAL protocol

The human protocols were approved by the Human Subjects Review Committee of the University of Washington, and informed consent was obtained from the patients or their surrogates. The patient population, selection criteria, and BAL procedure and sample processing have been described (Table ​(Table22 and refs. 26, 58). The characteristics of the patients are summarized in Table ​Table2.2. Normal volunteers were healthy, nonsmoking subjects (aged 18–50 years). Each aliquot of BAL fluid was thawed and used only once. When indicated, BAL fluid samples were concentrated by centrifugation at 5,000 g at 4°C, using 5,000-Da molecular weight cut-off filter units (Millipore). When indicated, the BAL fluid samples were depleted of albumin and IgG with anti-human albumin and anti-human IgG antibodies coupled to agarose beads using the ProteoSeek Antibody-Based Albumin/IgG Removal Kit (Pierce). After 2 hours of incubation at room temperature, the beads were separated from the samples by centrifugation.

Table 2

Characteristics of the patient population from which BAL fluids were obtained

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Gel filtration of human BAL fluid

BAL fluid samples were pooled and concentrated 6-fold, as described above. The BAL fluid proteins were separated according to molecular weight using high-pressure liquid chromatography with a SynChropak GPC 100 column (250 mm × 4.6 mm; SynChrom) at a flow rate of 1 ml/min in PBS. Each pooled sample was separated into 40 fractions. The column was calibrated using alcohol dehydrogenase (150 kDa), bovine serum albumin (67 kDa), ovalbumin (43 kDa), chymotrypsinogen A (25 kDa), and ribonuclease A (13.7 kDa).

Incubation of rh-sFasL in human BAL fluid or with MPO-dependent oxidants

Exogenous rh-sFasL (Peprotech Inc.) was incubated at 37°C in BAL fluid samples for 90 minutes, or in the MPO-dependent oxidation reaction mixture for 1 hour, or with HOCl for 30 minutes. The MPO-dependent oxidation reaction was carried out in phosphate buffer (20 mM sodium phosphate, 100 μM diethylenetriaminepentaacetic acid [DTPA], pH 7.4), which was supplemented with MPO, H₂O₂, and NaCl for the MPO-H₂O₂-chloride system or NaNO₂ for the MPO-H₂O₂-nitrite system, as indicated in each experiment. At the end of the incubation (in BAL fluid, MPO reaction fluid, or HOCl), the oxidant scavenger l-methionine (2.5 mM) was added to the samples to terminate oxidation. When indicated as a control, the reaction samples were incubated with l-methionine prior to the addition of rh-sFasL to block oxidation of the protein. The concentrations of H₂O₂ and HOCl were determined spectrophotometrically (ε₂₄₀ = 39.4 M^–1cm^–1 and ε₂₉₂ = 350 M^–1cm^–1, respectively).

Western blot of sFasL

Samples were diluted in Laemmli sample buffer, with or without 2-ME (5%), as indicated in each experiment. The proteins were separated by electrophoresis in 4-12% SDS-PAGE gels (Lonza Biosciences Inc.), transferred to nitrocellulose membranes (Hybond-ECL NC, GE Healthcare), blocked with blocking buffer containing 0.2% SuperBlock (Pierce) and 0.05% Tween-20 at room temperature for 1 hour, and incubated overnight at 4°C with primary detection antibody diluted in 0.2% SuperBlock/0.05% Tween-20 buffer. After thoroughly washing with 1x PBS 0.05% Tween-20, the membranes were incubated for 1 hour at room temperature with HRP-conjugated secondary antibodies. Peroxidase activity was detected by chemiluminescence using Supersignal West Femto Maximum Sensitivity Substrate (Pierce).

Incubation of rh-sFasL with urea

The long form of rh-sFasL that was expressed in HEK cells was preoxidized by incubation with or without HOCl (50 μM) and then incubated in PBS, pH 7.4, supplemented with or without urea (8 M) at 37°C for 120 minutes. These rh-sFasL samples were diluted in Laemmli sample buffer containing 2-ME (5%), separated by electrophoresis in 4-12% SDS-PAGE gels, and analyzed by Western blotting.

Incubation of rh-sFasL with DTT

In order to determine whether disulfide bonds are involved in oxidation-mediated multimerization of human sFasL, the oxidized rh-sFasL (Peprotech Inc.) was incubated at 37°C in PBS containing DTT (10 mM or 20 mM). After 1 hour, iodoacetamide (IAM, 10 mM) was added to the reaction to prevent reformation of disulfide bonds and incubated at room temperature for another 30 minutes. As a control, rh-sFasL was diluted in PBS without either DTT or IAM. To stop the reaction and to reduce the content of DTT and IAM, the samples were diluted 1:5 in PBS and then spun in albumin-precoated centrifuge filter columns with a 3,000-Da molecular weight cut-off (Microcron Ultracel YM-3, Millipore) at 10,000 g for 25 minutes at 4°C. The molecular weight of the sFasL subspecies was determined by SDS-PAGE, without reducing sample buffer, and Western blotting.

Reduction of rh-sFasL in DMS

In order to determine whether oxidation of methionine residues is involved in oxidation-mediated multimerization of human sFasL, we incubated oxidized rh-sFasL with DMS to reduce the methionine sulfoxide (oxidized methionine) back to methionine sulfide (nonoxidized methionine) (37). A rh-sFasL (Peprotech Inc.) was incubated with 0.1 M DMS and 10 M HCl. The reaction was allowed to proceed at room temperature for 1 hour. To stop the reaction and reduce the content of DMS and HCl, the samples were diluted 1:30 in 10X PBS, pH 7.4, spun in albumin-precoated centrifuge filter columns (3,000-Da molecular weight cut-off; Microcron Ultracel YM-3, Millipore) at 14,000 g for 25 minutes at 4°C, and then diluted in ddH₂O. The molecular weight of sFasL subspecies was determined by SDS-PAGE, without reducing agents, and Western blotting.

Incubation of rh-sFasL with MMP-7

The long form of rh-sFasL was incubated at 37°C for 60 minutes with MMP-7 (0.16, 0.5, or 1.5 μM) in buffer containing 20 mM Tris, pH 7.6, 5 mM CaCl₂, and 0.1 M NaCl. After incubation, the samples were cooled to 4°C and added to Jurkat cells or analyzed by Western blotting.

LC-ESI-MS/MS

Cytotoxicity of sFasL in cultured cells

Human Jurkat T Lymphocytes (TIB-152, clone E6-1, ATCC) and MLE-15 cells (mouse lung epithelial cell line provided by J. Whitsett, Cincinnati Children’s Hospital, Cincinnati, Ohio, USA) were grown in RPMI-1640 medium supplemented with 10% or 4% heat-inactivated fetal bovine serum, respectively, at 37°C and 5% CO₂. Human SAECs (Lonza) were grown in small airway epithelial basal medium (SAGM), supplemented with SAGM SingleQuots (Lonza) at 37°C, 5% CO₂. For the bioassay experiments, Jurkat cells (2 × 10⁵ cells/well), MLE-15 cells (at 75% confluence), or SAECs (at 100% confluence) were seeded in Costar 96-well tissue culture plates (Corning Inc.) supplemented at a 50% concentration with serum-free medium containing BAL fluid samples, rh-sFasL, or medium only. After incubation for 18 hours, the medium was removed, spun at 200 g, and stored in individual aliquots at –20°C for cytokine determinations. Cell viability was assessed using the alamarBlue assay (BioSource International), which incorporates a fluorometric indicator that detects mitochondrial activity. Data are shown as the percentage of cell death, which was calculated as follows: cell death (%) = 100 × ([live cell fluorescence — experimental fluorescence]/live cell fluorescence). Untreated live cell fluorescence corresponds to the fluorescence of cells in medium only. Finally, the cells were lysed, and caspase-3 activity was measured in each well using a Caspase-3 Fluorometric Assay Kit (Biovision). The fluorescence was measured using a CytoFluor II Fluorometer (PerSeptive Biosystems).

Animals

The animal protocols were approved by the Animal Care Committee of the VA Puget Sound Health Care System (Seattle, Washington, USA). Male C57BL/6 and B6.MRL-Fas lpr/J mice (lpr mice) (The Jackson Laboratory), weighing 25–30 g, were anesthetized with inhaled isoflurane, 2%–5%, and treated once by intratracheal instillation of purified rh-sFasL (25 ng/g) or supernatants (10 ng/g) of HEK cells transfected with a cDNA encoding human sFasL. After the instillations, the mice were allowed to recover from anesthesia, returned to their cages, and provided with free access to food and water. The mice were euthanized at 16 hours after instillation with an intraperitoneal injection of pentobarbital (120 mg/kg) and exsanguinated by closed cardiac puncture. The thorax was opened rapidly, and the trachea was cannulated with a 20-gauge catheter, the left hilum was clamped, and the left lung was removed and flash frozen in liquid nitrogen. The right lung was lavaged with 5 separate 0.5 ml aliquots of 0.9% NaCl containing 0.6 mM EDTA at 37°C and fixed by intratracheal instillation of 4% paraformaldehyde at a transpulmonary pressure of 15 cm of water and then embedded in paraffin. Frozen lung sections were prepared by inflating the right lung with intratracheal instillation of Tissue-Tek OCT (Sakura Finetek USA Inc.) (50% vol/vol) in ribonuclease-free PBS. The lungs were then placed in cryomolds, covered with Tissue-Tek OCT, and frozen at –80°C. Cryosections were cut at 8 μM thickness and mounted on glass slides.

Analysis of mouse BAL fluid

The BAL fluid samples were processed immediately for total and differential cell counts. Total white cell counts were performed with a hemacytometer, and differential counts were performed on cytospin preparations stained with the Diff-quick method (Andwin Scientific). A minimum of 200 cells were counted. The remainder of the lavage fluid was spun at 200 g for 30 minutes, and the supernatant was removed aseptically and stored in individual aliquots at –80°C. The total protein concentration in BAL fluid was measured by the bicinchoninic acid method (BCA assay; Pierce), and the concentration of IgM in BAL fluid was measured using ELISA (Bethyl Laboratories) per the manufacturer’s instructions. The lower limit of detection of the IgM assay was 20 ng/ml.

Histological methods in mouse lung tissue

Paraffin-embedded murine lung tissue sections (4-μm thick) were stained with hematoxylin and eosin for light microscopy. The TUNEL fluorescent staining for detection of DNA damage in situ was performed according to the manufacturer’s instructions (Roche Diagnostics). Light, fluorescence, and differential interference contrast microscopy were performed using a Nikon Eclipse 80i microscope. Evaluation of lung tissue damage and measurement of TUNEL-positive cells were assessed in a blinded manner on 8 randomly generated visual fields at ×200 magnification. Dual immunohistochemistry was used to compare TUNEL-positive and cytokeratin-positive cells (marker of epithelial cells) in the same frozen tissue sections. Briefly, the sections were fixed in acetone for 20 minutes at 4°C, and the TUNEL method was performed first. Then, the tissues were reblocked with Dako Serum-Free Protein Block (Dako) for 30 minutes at room temperature in the dark. The tissues were incubated with A555-fluorescent labeled mouse monoclonal pan-cytokeratin antibody (from Abcam, and labeled with Alexa fluor 555 dye) overnight at 4°C in a moist chamber in the dark. After washing in PBS, the sections were covered with glass coverslips using glycerol and analyzed by light and fluorescence microscopy.

Measurements in lung homogenates

For cytokine and caspase-3 activity measurements, the left lung was homogenized in lysis buffer containing 0.5% Triton X-100, 150 mM NaCl, 15 mM Tris, 1 mM CaCl₂, and 1 mM MgCl₂ (pH 7.4) using a hand-held homogenizer. The homogenates were incubated for 30 minutes at 4°C and then spun at 10,000 g for 20 minutes. For MPO measurements, the lungs were homogenized in 50 mM potassium phosphate buffer (pH 6.0) with 0.5% hexadecyltrimethyl ammonium bromide and 5 mM EDTA and sonicated briefly at 4°C. MPO activity was measured in supernatants of lung homogenates using the Amplex Red fluorometric assay, per instructions from the manufacturer (Molecular Probes). Cytokines IL-1β, IL-4, IL-6, IL-10, IL-12, KC, MIP-2, MCP-1, TNF-α, and IFN-γ were measured in lung homogenates using Fluorokine MultiAnalyte Profiling Kits (R&D Systems) in a multiplex fluorescent bead assay (Luminex), per the manufacturer’s instructions. The sensitivities of these immunoassays were 37.9 pg/ml for IL-1β, 11.25 pg/ml for IL-4, 10.53 pg/ml for IL-6, 7.47 pg/ml for IL-10, 23.83 pg/ml for IL-12, 13.08 pg/ml for KC, 6.69 pg/ml for MIP-2, 54.15 pg/ml for MCP-1, 6.82 pg/ml for TNF-α, and 22.19 pg/ml for IFN-γ. Caspase-3 activity in lung homogenates was measured with the Caspase-3/CPP32 Fluorometric Assay Kit (Biovision), according to manufacturer’s instructions. The fluorescence signal was measured using a fluorescent microplate reader.

We thank John Ruzinski for his technical assistance with cytokine measurements and J. Whitsett for providing the MLE-15 cells. The mass spectrometry experiments were performed by X. Fu and Y. Wang in the Mass Spectrometry Resource Core, Department of Medicine, University of Washington and the Puget Sound Blood Center. This work was supported in part by the Medical Research Service of the Department of Veterans Affairs and by grants HL-081764, HL-083044, HL-075381, and P30 DK-17047 from the NIH.