Involvement of lysosomal cathepsins in the cleavage of DNA topoisomerase I during necrotic cell death

Authors


  • Presented in part at the 67th Annual Scientific Meeting of the American College of Rheumatology, Orlando, FL, October 2003.

Abstract

Objective

Autoantibodies to DNA topoisomerase I (topo I) are associated with diffuse systemic sclerosis (SSc), appear to be antigen driven, and may be triggered by cryptic epitopes exposed during in vivo topo I fragmentation. These autoantibodies recognize topo I and fragments of this autoantigen generated during apoptosis and necrosis. We undertook this study to determine whether lysosomal cathepsins are involved in topo I fragmentation during necrosis.

Methods

Topo I cleavage during necrosis was assessed by immunoblotting of lysates from L929 fibroblasts exposed to tumor necrosis factor α (TNFα) and the broad caspase inhibitor Z-VAD-FMK, and by immunoblotting of lysates from endothelial cells treated with HgCl2. Purified topo I and L929 nuclei were incubated with cathepsins B, D, G, H, and L, and topo I cleavage was detected by immunoblotting. The intracellular localization of cathepsin L activity and topo I in necrotic cells was examined using fluorescence microscopy.

Results

Treatment of L929 cells with TNFα and Z-VAD-FMK induced caspase-independent cell death with necrotic morphology. This cell death involved topo I cleavage into fragments of approximately 70 kd and 45 kd. This cleavage profile was reproduced in vitro by cathepsins L and H and was inhibited by the cathepsin L inhibitor Z-FY-CHO. During necrosis, cathepsin L activity diffused from lysosomes into the cytoplasm and nucleus, whereas topo I partially relocalized to the cytoplasm. Z-FY-CHO delayed necrosis and partially blocked topo I cleavage. The topo I cleavage fragments were also detected in necrotic endothelial cells and recognized by SSc sera containing anti–topo I antibodies.

Conclusion

These results implicate cathepsins, particularly cathepsin L, in the cleavage of topo I during necrosis. This cleavage may generate potentially immunogenic fragments that could trigger anti–topo I immune responses in SSc.

Autoantibodies to DNA topoisomerase I (topo I) are associated with diffuse cutaneous involvement and pulmonary fibrosis in patients with systemic sclerosis (SSc) (1, 2). Although the pathogenic role of these autoantibodies remains unclear, there is evidence that their serum levels correlate positively with disease severity and activity (2). The original molecular target of these autoantibodies was designated Scl-70 (scleroderma-associated autoantigen of 70 kd) because of its migration as a 70-kd band on sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) (3, 4). Subsequently, it was demonstrated that this 70-kd band was a proteolytic fragment corresponding to the catalytic C-terminal domain of topo I (4–6). Anti–topo I autoantibodies from SSc patients recognize epitopes in the central and C-terminal portions of the protein, but not in the N-terminus, which suggests that the 70-kd fragment is processed by antigen-presenting cells (APCs) to initiate an immune response to topo I in vivo (1). Consistent with this, fragmented topo I presented by dendritic cells (DCs) elicited a vigorous T cell response in vitro more efficiently than full-length topo I (7). These observations strongly suggest that cryptic epitopes generated by in vivo proteolytic fragmentation of topo I might drive the generation of anti–topo I responses in SSc.

There is compelling evidence indicating that dying cells, both apoptotic and necrotic, are reservoirs of fragmented or cleaved forms of intracellular autoantigens (8–12). The excessive accumulation of these cleavage products, which could potentially expose cryptic epitopes, might break immune tolerance in a proinflammatory microenvironment and elicit specific humoral and cellular immune responses in patients with systemic autoimmune diseases. Topo I appears to be highly susceptible to proteolytic fragmentation during cell death. In apoptotic cells, the protein is cleaved by caspases to generate C-terminal fragments of 70–80 kd which are recognized by autoantibodies from SSc patients and are catalytically active (12, 13). Topo I fragments of 72–75 kd are also produced by granzyme B both in vitro and during cell death induced by cytotoxic T lymphocytes (14). Our group demonstrated previously that topo I is also cleaved into fragments of approximately 70 kd and 45 kd in cells undergoing primary or secondary necrosis (10, 11).

The proteases responsible for topo I cleavage during necrosis have not been identified. We hypothesized that cathepsins, which are released from lysosomes during both apoptosis and necrosis (15–19), might be involved in this cleavage. In the present study, we show that topo I is cleaved into fragments of approximately 70 kd and 45 kd in mouse L929 fibroblasts and human endothelial cells undergoing necrotic cell death, and that cathepsins, particularly cathepsin L, are capable of generating these fragments. Furthermore, these fragments are recognized by most SSc sera containing anti–topo I antibodies.

MATERIALS AND METHODS

Cells and reagents.

L929 cells were obtained from American Type Culture Collection (Rockville, MD) and cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 2 mML-glutamine, 100 units/ml of penicillin, 100 μg/ml of streptomycin (all obtained from Cellgro, Herndon, VA), and 10% fetal bovine serum (Omega Scientific, Tarzana, CA). Human dermal microvascular endothelial cells (HDMECs) and bovine coronary artery endothelial cells (BCAECs) were obtained from Cambrex (Walkersville, MD) and cultured in EGM-MV and EGM-2 MV media (Cambrex), respectively. Actinomycin D, trypan blue, CA-074, acridine orange, 4′,6-diamidino-2-phenylindole (DAPI), and human and mouse tumor necrosis factor α (TNFα) were from Sigma (St. Louis, MO). Purified cathepsins B, D, G, H, L, and S, and the cathepsin L–specific inhibitor Z-FY-CHO were from Calbiochem (San Diego, CA). LysoSensor Green DND-189 was from Molecular Probes (Eugene, OR). The broad caspase inhibitor Z-VAD-FMK was from Biomol International (Plymouth Meeting, PA). Boc.D-FMK (broad caspase inhibitor), Z-YVAD-FMK (inhibitor of caspases 1 and 4), Z-VDVAD-FMK (caspase 2 inhibitor), Z-DEVD-FMK (caspase 3 inhibitor), Z-VEID-FMK (caspase 6 inhibitor), Z-IETD-FMK (caspase 8 inhibitor), and Z-LEHD-FMK (caspase 9 inhibitor) were from Enzyme Systems Products (Livermore, CA).

The cathepsin L fluorogenic substrate Magic Red MR-(FR)2 and the DNA fluorescent binding dye Hoechst 33342 were from Immunochemistry (Bloomington, MN). Mouse anti-human topo I monoclonal antibody clone C-21 and recombinant caspase 3 were from BD PharMingen (San Diego, CA). Anti–lamin B goat polyclonal antibody C-20 and anti–cathepsin L goat polyclonal antibody M-19 were from Santa Cruz Biotechnology (Santa Cruz, CA). Anti–poly(ADP-ribose) polymerase (anti-PARP) monoclonal antibody C2-10 was from R&D Systems (Minneapolis, MN). Nitrocellulose membrane was from VWR (Brisbane, CA). Human sera containing autoantibodies to topo I and lamin B were a kind gift from Drs. Eng M. Tan and Michael Pollard (The Scripps Research Institute, La Jolla, CA). Baculovirus-expressed human topo I was a kind gift from Dr. James J. Champoux (University of Washington, Seattle) and was purified as described previously (20).

Induction of cell death.

Caspase-independent cell death with necrotic morphology was induced in L929 cells by preincubation with the broad caspase inhibitor Z-VAD-FMK (100 μM) for 1 hour followed by exposure to 10 ng/ml of human or mouse TNFα. There was no difference in the levels of cell death induced by human versus mouse TNFα. Necrosis was induced in HDMECs and BCAECs by treatment with 80 μM and 40 μM HgCl2, respectively, for up to 12 hours. Apoptosis was induced in L929 cells by preincubation with 1 μg/ml actinomycin D 1 hour before exposure to TNFα. In some experiments, cells were pretreated with the cathepsin L inhibitor Z-FY-CHO (150 μM) or with individual caspase inhibitors 1 hour prior to addition of TNFα. Quantification of cytoplasmic membrane rupture, indicative of necrosis, was performed by trypan blue exclusion (11). Morphologic analysis of cells was performed using an Olympus IX70 inverted microscope (Olympus, Melville, NY) equipped with Hoffman modulation contrast.

Preparation of cell lysates and immunoblotting procedures.

After treatment with cell death–inducing agents, cells were harvested for preparation of total cell lysates. Floating cells in the culture medium were pooled and combined with the attached cells and resuspended directly in lysis buffer containing 100 mM Tris HCl (pH 6.8), 4% SDS, 10% (volume/volume) glycerol, and the CØMPLETE protease inhibitor cocktail (Roche Diagnostics, Mannheim, Germany). This cocktail inhibits a broad spectrum of serine, cysteine, and metallo proteases, and calpains (www.roche-applied-science.com). Lysates were passed sequentially through 18–27-gauge needles to shear DNA. The protein concentration was determined using the DC Protein Assay (Bio-Rad, Hercules, CA). Prior to electrophoresis, lysates were heated for 5 minutes at 95°C in the presence of 4% mercaptoethanol and 0.04% bromphenol blue. Twenty micrograms of lysates was loaded onto each lane in 12% SDS-PAGE Ready Gels (Bio-Rad) and transferred to nitrocellulose membrane.

Immunoblotting was performed as described previously (10). Topo I was detected using human and mouse antibodies to topo I (1:500). Lamin B was detected using human and goat antibodies to lamin B (1:500). Cathepsin L was detected using a goat polyclonal antibody to cathepsin L (1:100). Bound primary antibodies were detected using horseradish peroxidase–conjugated goat anti-human IgG, goat anti-mouse IgM, or rabbit anti-goat IgG secondary antibodies (Zymed, South San Francisco, CA; Santa Cruz Biotechnology) in combination with enhanced chemiluminescence (Perkin Elmer Life Sciences, Boston, MA).

In vitro cleavage of recombinant topo I.

Two hundred nanograms of baculovirus-purified recombinant topo I, resuspended in phosphate buffered saline (PBS; pH 5.5 or 7.5), was incubated with individual cathepsins B, D, G, H, and L for 1 hour at 37°C. Reactions were stopped with lysis buffer and heated at 65°C for 15 minutes. In some experiments, cathepsin L was preincubated for 30 minutes with Z-FY-CHO (150 μM) prior to mixing with topo I. Samples were analyzed in NuPAGE 4–20% Bis-Tris gels (Invitrogen, Carlsbad, CA) or 12% SDS-PAGE Ready Gels, followed by immunoblotting.

Treatment of nuclei with individual cathepsins.

Untreated L929 cells were rinsed with ice-cold PBS, trypsinized, centrifuged, and washed twice in cold PBS. Cells were resuspended in hypotonic buffer (20 mM HEPES [pH 7.5], 10 mM KCl, 1.5 mM MgCl2, 1 mM dithiothreitol, 0.05 mM phenylmethylsulfonyl fluoride, 0.05 μg/ml aprotinin) for 30 minutes at 4°C and then disrupted with 40–50 strokes in a Dounce homogenizer in the presence of 0.03% Nonidet P40. Homogenates were transferred to a 1.5-ml tube layered with hypotonic buffer containing 35% sucrose and were then centrifuged at 800g for 15 minutes at 4°C. Pelleted nuclei were resuspended in PBS (pH 7.5) and used immediately for cleavage assays or stored in small aliquots at −80°C. Isolated nuclei were incubated with individual cathepsins for up to 6 hours at 37°C. In some experiments, cathepsin L was preincubated for 30 minutes with Z-FY-CHO (150 μM) prior to mixing with nuclei. To stop the cleavage reactions, samples were centrifuged, and the pelleted nuclei were resuspended in lysis buffer (100 mM Tris HCl [pH 6.8], 4% SDS, 10% glycerol) supplemented with the CØMPLETE protease inhibitor cocktail to prevent further proteolysis. Nuclear lysates were passed through 21–30-gauge needles to shear the DNA, sonicated briefly, and processed for SDS-PAGE and immunoblotting.

Determination of lysosomal integrity, detection of intracellular cathepsin L activity, and visualization of intracellular topo I by fluorescence microscopy.

L929 cells were exposed to 5 μg/ml acridine orange in complete DMEM for 15 minutes at 37°C, rinsed twice with medium, and directly examined under an Olympus BX50 epifluorescence microscope using a LUMPlanPl 60X/0.90W water immersion objective (Olympus). Images were acquired using a digital Spot camera system (Diagnostic Instruments, Sterling Heights, MI). Alternatively, cells were exposed to 1 μM LysoSensor Green DND-189 in complete medium for 2 hours at 37°C, rinsed twice in medium, and examined directly by fluorescence microscopy as indicated above.

Cathepsin L activity was detected using the fluorogenic substrate Magic Red MR-(FR)2. Briefly, cultured L929 cells were exposed for 30 minutes to the cathepsin L substrate MR-(FR)2, rinsed twice with medium, and directly examined under the fluorescence microscope. Cells were counterstained with Hoechst 33342 for nuclear visualization. CA-074 (20 μM), a specific inhibitor of cathepsin B (21), was added to cells to increase the specificity of the cleavage of the MR-(FR)2 substrate by cathepsin L.

For intracellular visualization of topo I, L929 cells growing in glass coverslips were fixed and permeabilized with ice-cold methanol/acetone (3/1 [v/v]), rinsed with PBS, and incubated with a highly monospecific human anti–topo I autoimmune serum (10, 11). After washing with PBS, cells were then incubated with a rhodamine-labeled goat anti-human IgG secondary antibody. Cells were counterstained with DAPI in PBS for chromatin visualization. Coverslips were mounted onto glass slides, and cells were examined by fluorescence microscopy.

Statistical analysis.

All statistical analyses were done using GraphPad Prism, version 2.0 (GraphPad Software, San Diego, CA). The unpaired t-test was used in all cases. SDs were calculated based on at least 3 independent experiments performed in triplicate.

RESULTS

Induction of L929 cell death by TNFα.

Treatment of L929 murine fibroblasts with TNFα induces a relatively slow death in which both apoptotic and necrotic cells can be observed (22–25). Blockade of caspase activation in these cells with Z-VAD-FMK dramatically potentiates TNFα-mediated necrosis (22), whereas inhibition of transcription or translation with actinomycin D or cycloheximide, respectively, in the presence of TNFα shifts cell death toward apoptosis (22–25). Incubation of L929 cells with TNFα (10 ng/ml) led to a gradual loss of cytoplasmic membrane integrity, characteristic of necrosis, as measured by the ability of cells to take up trypan blue (Figure 1A). Necrosis was dramatically enhanced in the presence of 100 μM Z-VAD-FMK, resulting in >90% of cells taking up trypan blue by 6 hours. Treatment with Z-VAD-FMK alone did not exert any cytotoxic effects (Figure 1A). This enhancement was reproduced with the broad caspase inhibitor Boc.D-FMK, the caspase 3 inhibitor Z-DEVD-FMK, and the caspase 8 inhibitor Z-IETD-FMK, all used at high concentrations (100 μM) to ensure caspase inhibition (data not shown). However, it was not observed with inhibitors of caspases 1, 2, 4, 6, 9, and 10 (data not shown).

Figure 1.

Induction of cell death in L929 fibroblasts by tumor necrosis factor α (TNFα) in the presence of caspase inhibitors. A, Time-dependent induction of cell death in L929 cells treated with 10 ng/ml of TNFα, alone or in combination with 100 μM Z-VAD-FMK (Z-VAD). Controls included untreated cells (Control) and cells treated only with 100 μM Z-VAD-FMK. Cell viability was measured by trypan blue exclusion (11). Values are the mean ± SD. B, Immunoblots showing cleavage of poly(ADP-ribose) polymerase into its signature apoptotic 85-kd fragment in L929 cells exposed to 10 ng/ml TNFα, alone or in combination with either actinomycin D (Act-D; 1 μg/ml) or Z-VAD-FMK. Line indicates band corresponding to intact protein; arrow indicates band corresponding to proteolytic fragments generated during cell death. C, Time-dependent morphologic changes in L929 fibroblasts treated with 10 ng/ml of TNFα alone, in combination with either 100 μM Z-VAD-FMK or 1 μg/ml actinomycin D, or in combination with both. In all panels, cells were preincubated with either Z-VAD-FMK or actinomycin D for 1 hour prior to exposure to TNFα. Images were acquired using an inverted Olympus IX70 microscope equipped with Hoffman modulation contrast (original magnification × 40).

To confirm that L929 cells treated with TNFα/Z-VAD-FMK died in a caspase-independent manner, we performed an immunoblotting analysis of PARP cleavage. Figure 1B shows that PARP underwent limited cleavage into its caspase 3–generated 85-kd fragment in cells treated with TNFα alone, and extensive cleavage in cells treated with TNFα/actinomycin D. However, in the presence of Z-VAD-FMK, cleavage was minimal, suggesting that caspase 3 activity was significantly blocked. This was confirmed in a caspase activity assay using the colorimetric DEVD–paranitroanilide substrate, which revealed that while there was significant caspase 3 and caspase 7 activity in cells treated with TNFα alone or in combination with actinomycin D, this activity was reduced to background levels in cells treated with TNFα/Z-VAD-FMK (results not shown).

Cells treated with TNFα/Z-VAD-FMK displayed necrosis-like morphologic features such as condensed nuclei and swelled cytoplasms, with no cytoplasmic blebbing (Figure 1C). In contrast, cells treated with TNFα/actinomycin D displayed the typical blebbing associated with apoptosis. Cultures treated with TNFα alone contained both apoptotic and necrotic cells, with the latter predominating after 24 hours (Figure 1C and results not shown).

Cleavage of topo I into 70-kd and 45-kd fragments during L929 necrosis mediated by TNFα.

Our group showed previously that topo I is cleaved into a fragment of ∼70 kd in both apoptotic and necrotic cells, and into a fragment of 45 kd in cells undergoing primary or secondary necrosis (10, 11). To determine whether topo I was cleaved into these fragments during TNFα-mediated cell death, we analyzed its proteolysis in L929 cells treated with HgCl2, TNFα alone, TNFα/Z-VAD-FMK, TNFα/actinomycin D, or TNFα/actinomycin D/Z-VAD-FMK.

Lysates from L929 cells exposed to TNFα alone or to TNFα/actinomycin D for 6 hours showed the appearance of the 70-kd fragment, as detected by immunoblotting (Figure 2A). After 12 hours, the 45-kd fragment was already visible in lysates from cells treated with TNFα/actinomycin D, indicating that cells were making the transition from apoptosis to secondary necrosis (11) (Figures 2A and B). A 70-kd fragment, in addition to the 45-kd fragment, was also detected in lysates from cells treated with HgCl2, TNFα/Z-VAD-FMK, or TNFα/actinomycin D/Z-VAD-FMK (Figures 2A and B), consistent with the necrotic morphology of these cells (Figure 1C). The 70-kd fragment was abundant by 6 hours (Figure 2A) and usually appeared by 3 hours after induction of necrosis (results not shown). The amount of the 45-kd fragment increased as the amount of the necrotic 70-kd fragment decreased (Figures 2A and B), suggesting that it is a late cleavage product of the 70-kd fragment. In control experiments, we used human and goat antibodies to lamin B, which is cleaved during apoptosis but not necrosis (10, 11). Cleavage of lamin B into its signature 45-kd apoptotic fragment (10, 11) was observed in cells treated with TNFα (detected with the goat anti–lamin B antibody but not with the human antibody) or TNFα/actinomycin D, but not in cells treated with HgCl2, TNFα/Z-VAD-FMK, or TNFα/actinomycin D/Z-VAD-FMK (Figures 2A and B).

Figure 2.

Immunoblots showing the time-dependent progression of the cleavage of topoisomerase I (topo I) and lamin B in L929 cells treated with 10 ng/ml of TNFα alone, in combination with either 100 μM Z-VAD-FMK or 1 μg/ml actinomycin D, or in combination with both. Equal amounts (20 μg) of total cellular protein were loaded onto each lane in 12% sodium dodecyl sulfate polyacrylamide gels, separated by electrophoresis, and transferred to nitrocellulose membranes. Lysates from cells induced to die by primary necrosis upon exposure to 40 μM HgCl2 for 12 hours were included as positive controls for the necrotic cleavage of topo I. Protein bands were detected by chemiluminescence after exposing the membranes to appropriate dilutions of A, human autoimmune sera containing high-titer, highly specific autoantibodies to topo I (hAb-topo I) and lamin B (hAb-lamin B), and B, a mouse monoclonal antibody to topo I (mAb-topo I) and a goat polyclonal antibody to lamin B (gAb-lamin B). Lines indicate bands corresponding to intact proteins; arrows indicate bands corresponding to proteolytic fragments generated during cell death. Blots are representative of at least 3 independent experiments. See Figure 1 for other definitions.

Generation by cathepsin L of signature necrotic cleavage fragments of topo I in vitro.

We hypothesized that lysosomal cathepsins might be responsible for the cleavage of topo I into 70-kd and 45-kd fragments during TNFα/Z-VAD-FMK–induced necrosis in L929 cells. To determine whether cathepsins can generate the signature necrotic cleavage fragments of topo I, we incubated purified topo I or L929 nuclei at a neutral pH of 7.5 with various concentrations of cathepsins B, D, G, H, and L.

Figure 3A shows that only cathepsins L and H generated prominent bands of approximately 70 kd and 45 kd when incubated with purified topo I. Minor bands migrating below 45 kd were often observed after cathepsin treatment. Some of these have been observed previously in necrotic Jurkat T cells (10). The band generated by cathepsins L and H around the 70-kd region appeared as a doublet or triplet of ∼65–80 kd, suggesting that the protein might be cleaved at several different but closely spaced sites. These immunoblots were obtained using NuPAGE 4–20% Bis-Tris gels for optimal resolution of bands around this region. In contrast, immunoblots obtained using 12% SDS-PAGE Ready Gels showed only single bands of 70 kd and 45 kd (Figure 2 and results not shown). There was no difference in the cleavage profile of topo I produced by cathepsin L when the cleavage reactions were conducted at pH 5.5 (Figure 3B). The other cathepsins either did not cleave topo I or cleaved it without generating the necrotic 45-kd fragment. For instance, cathepsin B generated fragments of approximately 80 kd and 70 kd, whereas cathepsin G efficiently generated a single fragment of ∼70 kd (Figure 3A).

Figure 3.

Immunoblots showing in vitro cleavage of topoisomerase I (topo I) by cathepsins (Cat). A, Approximately 200 ng of purified human topo I was incubated in phosphate buffered saline (pH 7.5) for 1 hour at 37°C with various milliunits of individual cathepsins. B, Cleavage of topo I by cathepsin L at pH 5.5. C, Inhibition of cathepsin L–mediated topo I cleavage at pH 7.5 after preincubation of cathepsin L with its inhibitor, Z-FY-CHO (150 μM), for 30 minutes. D, Nuclei from L929 cells were incubated with 5 milliunits of individual cathepsins for various periods of time (0–6 hours). Lysates from untreated cells and cells treated with TNFα/Z-VAD-FMK were included as controls for the detection of topo I cleavage in immunoblots. Lines indicate bands corresponding to intact proteins; arrows indicate bands corresponding to cleavage fragments. Bands were recognized using a human autoantibody to topo I. Blots are representative of at least 3 independent experiments. See Figure 1 for other definitions.

In isolated L929 nuclei, only cathepsin L efficiently generated a prominent topo I band migrating in the 65–80-kd range, as well as the 45-kd cleavage product (Figure 3D). Given the prominence of the band around the 70-kd region (consistently observed in repeated experiments) (Figure 3D), it appears that cleavage of nuclear topo I by cathepsin L into fragments migrating around this region exposed epitopes that were highly reactive with the human anti–topo I autoantibody used for these experiments. The specific cathepsin L inhibitor Z-FY-CHO inhibited cathepsin L–mediated cleavage of purified and nuclear topo I (Figures 3C and D). The concentration ranges of cathepsins and the incubation conditions used in these experiments were based on reported studies (26, 27). It should be noted that untreated topo I, either in purified form or in the isolated nuclei, underwent limited degradation into a 70-kd product during protein or nuclear preparation (Figure 3), consistent with previous reports (3–5). Nuclear preparations were done under limited protease inhibitory conditions to prevent inhibition of cathepsins.

Intracellular relocalization of cathepsin L and topo I during L929 necrosis mediated by TNFα.

The rupture of lysosomes during necrosis causes extensive leakage of cathepsins into the cytoplasmic compartment (17). We hypothesized that if cathepsin L plays a role in the cleavage of topo I in L929 cells exposed to TNFα/Z-VAD-FMK, it should be possible to detect leakage of this enzyme into the cytosolic compartment and perhaps relocalization into the nucleus, and/or relocalization of topo I into the cytoplasm. To explore this, we first analyzed the integrity of lysosomes in L929 cells exposed to acridine orange in the presence and absence of TNFα/Z-VAD-FMK. Due to proton trapping, this vital dye accumulates mainly in the acidic vacuolar apparatus, preferentially in lysosomes (15). When excited by blue light, acridine orange emits red/orange fluorescence at high (lysosomal) concentrations and green fluorescence at low (nuclear and cytosolic) concentrations. Disruption of lysosomal integrity leading to leakage of acridine orange into the cytosol produces a bright yellow fluorescence.

Fluorescence microscopic analysis of lysosomes in untreated L929 cells showed intact lysosomal compartments as indicated by clusters of orange fluorescence separated from green fluorescence, whereas cells treated with TNFα/Z-VAD-FMK for 4 hours either lacked the orange clusters or displayed bright yellow fluorescence, indicative of acridine orange leakage into the cytosol (Figure 4A). This leakage was partially inhibited by preincubation with Z-FY-CHO. After 6 hours of treatment with TNFα/Z-VAD-FMK, however, Z-FY-CHO was no longer able to block acridine orange release (results not shown). Similar results (not shown) were obtained with LysoSensor Green DND-189, another specific probe for lysosome integrity.

Figure 4.

Intracellular relocalization of cathepsin L activity and topoisomerase I (topo I) during TNFα/Z-VAD-FMK–induced necrosis in L929 cells. A, Determination of lysosomal integrity in L929 cells treated with TNFα/Z-VAD-FMK. Cells were exposed to acridine orange for lysosome visualization. Untreated cells showed localized granular orange fluorescence corresponding to lysosomal staining. In the cytoplasm of intact cells, acridine orange gives a green fluorescent color. In the TNFα/Z-VAD-FMK–treated cells, a decrease or absence of orange fluorescence, with increased yellow fluorescence (indicative of lysosomal leakage), was observed in some cells (arrows). Lack of orange or yellow fluorescence indicates leakage of lysosomal acridine orange into the extracellular milieu due to loss of cytoplasmic membrane integrity. Cells treated with TNFα after preincubation with Z-VAD-FMK and Z-FY-CHO for 1 hour displayed delayed loss of lysosomal integrity. Corresponding phase-contrast images are shown in the lower panel. B, Detection of intracellular cathepsin L activity in L929 cells, using the fluorogenic substrate MR-(FR)2. Cells treated with TNFα/Z-VAD-FMK show diffuse cathepsin L activity (red fluorescence, white arrows), whereas in untreated cells, the activity is localized mainly in cytoplasmic granules corresponding to lysosomes. The yellow arrow indicates a necrotic cell with very little cathepsin L activity that appeared to have lost cytoplasmic membrane integrity (see corresponding phase-contrast image at right). C, Intracellular visualization of topo I in L929 cells, using a highly specific human anti–topo I serum (10, 11) as primary antibody and a rhodamine-conjugated goat anti-human IgG as secondary antibody. In untreated cells, the rhodamine fluorescence is confined to the nucleus, whereas in cells treated with TNFα/Z-VAD-FMK, the nuclei appear pyknotic and the fluorescence is localized both in the nucleus and cytoplasm. All representative images were acquired using an Olympus BX50 epifluorescence microscope 4–5 hours after exposure of cells to TNFα/Z-VAD-FMK. DAPI = 4′,6-diamidino-2-phenylindole (see Figure 1 for other definitions). (Original magnification × 40.)

To determine whether cathepsin L is released from lysosomes in L929 cells treated with TNFα/Z-VAD-FMK, we sought to examine changes in the intracellular localization of this enzyme by fluorescence microscopy. We used the fluorogenic substrate–based assay Magic Red MR-(FR)2, which allows the localization of cathepsin L activity in live cells. To augment the specificity of this localization, we simultaneously exposed the L929 cells to the cathepsin B inhibitor CA-074 to avoid nonspecific cleavage of the fluorogenic substrate by cathepsin B. Cells were counterstained with Hoechst 33342 to visualize nuclei. As expected, the red fluorescence was localized in untreated cells in cytoplasmic granules around the nuclei, corresponding to lysosomes, where cathepsin L is normally stored (Figure 4B). In cells treated with TNFα/Z-VAD-FMK, the red fluorescence was diffused in the cytosol and even appeared to colocalize with nuclei (Figure 4B). It should be cautioned, however, that since these experiments were done in live cells, it is not entirely clear whether cathepsin L actually penetrated the nuclear compartment. We also examined the localization of topo I in fixed L929 cells, using a highly specific human anti–topo I serum. In untreated cells, topo I was confined to the nucleus, whereas in cells treated with TNFα/Z-VAD-FMK, both nuclear and cytoplasmic staining were evident (Figure 4C).

Inhibition of cathepsin L activity delays necrosis and partially inhibits topo I cleavage.

To determine whether cathepsin L activity is required for topo I fragmentation in necrotic L929 cells, we preincubated cells for 1 hour in the presence of the specific cathepsin L inhibitor Z-FY-CHO (150 μM) and Z-VAD-FMK (100 μM) prior to exposure to TNFα. We first examined whether cathepsin L is processed into the mature 29-kd form during TNFα/Z-VAD-FMK–induced necrosis. A time course study of cathepsin L processing revealed an increase in the appearance of the 29-kd mature form of this protease during necrosis (Figure 5A). This increase was not observed in cells preincubated with Z-FY-CHO, which instead consistently showed smaller amounts of the 39-kd proenzyme and a slight accumulation of an ∼52-kd band. This band might correspond to an immature or complexed form of the enzyme that is not efficiently processed or released in the inhibited cells. These results suggested that Z-FY-CHO blocked the autocatalytic processing of cathepsin L into its mature 29-kd form (28). Preincubation of cells with Z-FY-CHO for 1 hour delayed necrosis during the first 6 hours of exposure to TNFα/Z-VAD-FMK (P < 0.05) (Figure 5B). However, the majority of cells eventually (after >12 hours) died by necrosis (data not shown). Consistent with these results, we detected partial inhibition of topo I cleavage into the 70-kd and 45-kd fragments in cells preincubated with Z-FY-CHO for 1 hour prior to exposure to TNFα/Z-VAD-FMK (Figure 5C).

Figure 5.

The cathepsin L inhibitor Z-FY-CHO prolongs L929 viability and delays cleavage of topoisomerase I (topo I) in the presence of TNFα/Z-VAD-FMK. A, Immunoblots showing the processing of cathepsin L into its mature 29-kd form in L929 cells treated with TNFα/Z-VAD-FMK. Processing was partially inhibited by Z-FY-CHO (150 μM). B, Loss of viability in L929 cells treated with TNFα/Z-VAD-FMK in the presence or absence of Z-FY-CHO. Cell viability was determined using trypan blue exclusion. Values are the mean ± SD. C, Immunoblots showing partial inhibition of topo I cleavage by Z-FY-CHO in L929 cells treated with TNFα/Z-VAD-FMK. In AC, cells were preincubated with Z-VAD-FMK or Z-FY-CHO for 1 hour prior to exposure to TNFα. Arrows in A and C indicate bands corresponding to proteolytic fragments generated during cell death; line in C indicates band corresponding to intact protein. The x-axis in B represents time after this preincubation. See Figure 1 for other definitions.

SSc sera containing autoantibodies to topo I recognize the 70-kd and 45-kd cleavage fragments in necrotic L929 and endothelial cells.

To determine whether the necrotic cleavage fragments of topo I are recognized by the majority of SSc sera containing anti–topo I autoantibodies, we used immunoblotting to analyze 38 SSc sera containing autoantibodies reactive with a band of ∼100 kd and displaying an immunofluorescence staining pattern characteristic of anti–topo I autoantibodies. We assumed that this 100-kd band corresponded to topo I, although we could not rule out the possibility that some of the sera contained autoantibodies against the ∼100-kd subunits of the SSc-associated autoantigens PM-Scl or RNA polymerase I.

Analysis of the reactivity of the 38 sera against whole cell lysates from L929 cells exposed to TNFα/Z-VAD-FMK revealed that 34 sera (89%) recognized the signature 70-kd and 45-kd necrotic cleavage fragments, indicating that these fragments display epitopes recognized by the majority of SSc sera containing anti–topo I autoantibodies. Representative results with sera reacting with the 70-kd and 45-kd necrotic cleavage fragments are shown in Figure 6A. In this particular experiment, the control lysates from L929 cells also showed moderate reactivity against the 70-kd fragment (Figure 6A), a phenomenon that was occasionally observed when lysates were stored at −80°C for prolonged periods or when the viability of the untreated L929 cell cultures did not exceed 95% prior to lysate preparation for immunoblotting. Unlike previous reports on the U1–70-kd autoantigen (29), we did not observe preferential antibody reactivity against any of the cleavage fragments.

Figure 6.

Representative immunoblots showing detection of topoisomerase I (topo I) necrotic cleavage fragments by autoimmune sera from 5 different patients with systemic sclerosis (SSc) in A, L929 cells, B, human dermal microvascular endothelial cells (HMVEC-d), and C, bovine coronary artery endothelial cells (BCAEC). Necrosis was induced in endothelial cells by treatment with 40 μM HgCl2 for 12 hours. D, Control immunoblots showing reactivity of the 5 SSc sera against purified topo I. Lines indicate bands corresponding to intact proteins; arrows indicate bands corresponding to proteolytic fragments generated during cell death. C = control; N = necrotic.

Since endothelial cell death has been implicated in the pathology of SSc (30), we next determined whether SSc sera also recognize the 70-kd and 45-kd cleavage fragments in endothelial cells undergoing necrosis. Figures 6B and C show representative results with SSc sera reacting with the 70-kd and 45-kd fragments in whole lysates from HDMECs and BCAECs, respectively, induced to die by necrosis with HgCl2. To confirm that the representative sera were recognizing topo I, we included control blots showing their reactivity against purified recombinant topo I (Figure 6D).

DISCUSSION

The results presented here provide evidence that cathepsins, particularly cathepsin L, are involved in the cleavage of topo I during necrotic cell death. Our previous analysis of topo I cleavage during nonapoptotic cell death revealed that this protein is cleaved into fragments of approximately 70 kd and 45 kd that are recognized by autoantibodies in Jurkat T cells dying by primary or secondary necrosis (10, 11). In the present study, we detected these fragments in L929 fibroblasts undergoing caspase-independent death with necrotic morphology in response to treatment with TNFα/Z-VAD-FMK. We also detected these fragments in human and bovine endothelial cells undergoing necrosis induced with HgCl2. These observations suggest that the generation of the topo I 70-kd and 45-kd cleavage fragments is mediated by a conserved proteolytic mechanism that is activated in necrotic cell death. Hence, anti–topo I antibodies recognizing these fragments could be used to distinguish apoptosis from necrosis by immunoblotting. This distinction could be enhanced in combination with antibodies to lamin B, which is selectively cleaved in apoptotic cells but not in necrotic cells (10, 11).

Cathepsins participate in apoptosis by cleaving a number of intracellular proteins (31, 32), but knowledge of their substrates in necrosis is scarce. A previous report implicated cathepsins B and G in the necrotic cleavage of PARP, a lupus-associated autoantigen (26). Our results indicated that cathepsins B, G, H, and L can induce topo I fragmentation in vitro. However, only cathepsins L and H generated both 70-kd and 45-kd necrotic cleavage fragments. When incubated with purified nuclei, cathepsin L (but not cathepsin H) generated both fragments, suggesting that cathepsin L may access topo I more efficiently in the nucleus. It should be noted that the cleavage reactions were conducted at a neutral pH of 7.5. Cleavage reactions with cathepsin L conducted at an optimal pH of 5.5 yielded a cleavage profile identical to that observed at neutral pH. Although optimal enzymatic activity by cathepsins is achieved at pH 5.5, Goulet et al (27) demonstrated recently that cathepsin L is active and can process substrates at neutral pH. Those investigators suggested that the suboptimal pH of 7.5 should not be taken as an obstacle but as a key element that enables cathepsin L and perhaps other cathepsins to play an important role in the limited protein cleavage outside the lysosomes, particularly in the nucleus.

We detected cathepsin L activity outside the lysosomes and in the nucleus during TNFα/Z-VAD-FMK–mediated cell death. We also showed by immunoblotting a gradual increase in the levels of processed 29-kd cathepsin L during necrosis, most likely arising via autocatalytic processing (28). These results suggested that cathepsin L was active even in the presence of 100 μM Z-VAD-FMK, a high concentration that is known to inhibit cathepsins B and H in cultured cells (33). Interestingly, topo I was also detected in the cytoplasm of necrotic cells, suggesting that a portion of it may leak from the nucleus into the cytoplasm during necrosis. Thus, cathepsin L may encounter topo I both in the nucleus and in the cytoplasm.

While results of our studies suggest that cathepsin L is involved in topo I cleavage during necrosis, other cathepsins are also likely to generate topo I cleavage in necrotic L929 cells because it was not completely blocked in cells preincubated with the specific cathepsin L inhibitor Z-FY-CHO. However, Z-FY-CHO may block cathepsin L activity only when the protease leaks from the lysosomes into the cytosol, and, conceivably, it may not completely block all the released cathepsin L. Our data also suggest that cathepsin L plays a role in the progression of L929 necrosis mediated by TNFα/Z-VAD-FMK, because preincubation of cells with Z-FY-CHO delayed but did not inhibit cell death. This indicates that other cathepsins are involved, although late, in this necrosis process. Additional insights into the role of cathepsins in necrosis and in the necrotic cleavage of intracellular autoantigens could be obtained in future studies using cathepsin-deficient cell lines and mouse models. Cell lines have been described in which cathepsin L has been genetically knocked out (16), silenced via RNA inhibition (34), or rendered inactive by persistent viral infection (35). In preliminary experiments, we observed that the L929-derived mutant cell line LX, which was obtained by persistent reovirus infection and lacks activity of both cathepsin B and cathepsin L (35), showed a slight delay in TNFα/Z-VAD-FMK–mediated necrosis and topo I cleavage compared with L929 cells (Pacheco FJ, et al: unpublished observations). However, LX cells abundantly express cathepsin H (35), which could compensate for the absence of cathepsin L.

There is increasing interest in necrotic cells, particularly when they arise as the result of defective clearance of apoptotic cells, as reservoirs of altered autoantigens and danger signals that could contribute to the generation of autoantibody responses in rheumatic diseases (36–39). A current hypothesis is that impaired phagocytic removal of apoptotic cells may cause accumulation of secondary necrotic cells in germinal centers of secondary lymphoid organs, leading to exposure by the immune system to high concentrations of altered forms of autoantigens for which tolerance has not been previously achieved (36, 37). Consistent with this hypothesis, our group demonstrated previously that the progression from apoptosis to secondary necrosis is associated with fragmentation of topo I and other intracellular autoantigens into forms not normally generated during apoptosis (11). In the presence of appropriate environmental adjuvants, danger signals, or inflammatory cytokines that can act as maturation factors for DCs, fragmented autoantigens revealing cryptic epitopes and released by dying cells may initiate autoantibody responses (40).

Cathepsins and caspases both produce an ∼70-kd topo I cleavage fragment that is recognized by the SSc autoantibodies, which indicates strongly that this fragment corresponds to the immunogenic C-terminal domain of the protein. Because these enzymes have different amino acid specificities (aspartic acid for caspases and various nonaspartic amino acids for cathepsins), their cleavage sites in topo I must be different. However, it is possible that these sites could be present within a protease-sensitive region separating the nonimmunogenic N-terminus from the immunogenic C-terminus. Studies by Hu and colleagues have shown that the N-terminal domain of topo I comprising the first 200 amino acids of the protein is not recognized by anti–topo I–positive sera from SSc patients (1). Those investigators demonstrated that in all anti–topo I–positive SSc patients, the autoantibodies recognize both linear and conformational epitopes and have the same molecular recognition pattern, with the core central region of the protein primarily targeted. The nonantigenic N-terminal region of the protein appears to be irrelevant for the initiation of anti–topo I responses, and it might be removed from the whole molecule before topo I is taken up and processed by APCs (1). This N-terminal region is removed by caspases during apoptosis (13) and, most likely, by cathepsins during necrosis.

There is evidence that autoantibody responses to topo I in SSc are driven by cryptic determinants and that a very specific combination of fragmented forms of topo I and the type of APCs that process these forms may be involved in breaking tolerance to topo I in the early stages of development of this disease (1, 2, 7, 41). It is conceivable that immunogenic fragmented forms of topo I could arise in vivo via caspase- or cathepsin-mediated cleavage of the protein during apoptosis or secondary necrosis. Endothelial cell death appears to play an important role in the pathogenesis of SSc (30), and our observation that cleavage of topo I into 70-kd and 45-kd fragments can occur in cultured endothelial cells undergoing necrotic cell death suggests that dying endothelial cells (both apoptotic and necrotic) could serve as reservoirs of potentially immunogenic fragments of topo I in SSc patients.

Acknowledgements

We are very grateful to Drs. Eng M. Tan and Michael Pollard (The Scripps Research Institute, La Jolla, CA) and to Dr. Bernhard Hildebrandt (Technische Universitat, Munich, Germany) for the kind gift of human autoimmune sera from scleroderma patients. We are grateful to Dr. James J. Champoux (University of Washington, Seattle) for the valuable gift of purified human recombinant topo I. We thank Dr. Hansel Fletcher and Shaun Sheets (Loma Linda University, Loma Linda, CA) for providing technical assistance in the use of the primary endothelial cells (HDMECs and BCAECs). Special thanks are due to Drs. Ulf T. Brunk (Linkoping University, Linkoping, Sweden) and Peter Vandenabeele (Ghent University, Ghent, Belgium) for helpful discussions and suggestions.

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