Proliferation and matrix synthesis by activated pancreatic stellate cells (PSC) participate in the development of chronic pancreatitis. Apoptosis of PSC may terminate this process but has not yet been studied in this particular cell type and was the aim of the present study. PSC were isolated from rat pancreas and characterized for expression of glial fibrillary acidic protein, α-smooth muscle actin, CD95, and tumor necrosis factor-α-related apoptosis-inducing ligand (TRAIL) receptors. Apoptosis was determined by TdT-UTP nick end-labeling reaction, annexin V binding, and caspase-8 activation. Both CD95L and TRAIL induced apoptosis in PSC. The apoptotic response was minor in PSC cultured for 7 days but increased markedly thereafter. Sensitization of PSC with culture duration was accompanied by increased expression of CD95 and TRAIL receptor 2 and no alterations of Flip expression or protein kinase B phosphorylation but was paralleled by the appearance of a COOH-terminal cleavage product of receptor-interacting protein. PSC apoptosis was also induced by PK-11195, a ligand of the peripheral benzodiazepine receptor. PSC apoptosis may be important in terminating the wound-healing response after pancreas injury and exhibits features distinct from apoptosis induction in hepatic stellate cells.
- tumor necrosis factor-α-related apoptosis-inducing ligand
- receptor-interacting protein cleavage
ABOUT 30 YEARS AGO Watari and co-workers (29) identified lipid-storing cells in pancreatic tissue with marked similarities to hepatic stellate cells (HSC; see Ref. 29). Whereas HSC were intensively studied, their pancreatic counterparts (pancreatic stellate cells; PSC) were isolated in 1998 for the first time (1, 5). In normal pancreas, PSC are in a quiescent state and are characterized by the presence of vitamin A-containing lipid droplets and positive staining for glial fibrillary acidic protein (GFAP; see Ref.1). PSC are activated in both experimental and human pancreatic fibrosis and then represent the major source of collagen in chronic pancreatitis (9). Transformation of quiescent PSC to a myofibroblast-like phenotype also occurs during culture. This activation is associated with an increased expression of α-smooth muscle actin and a loss of GFAP expression (5). Cultured PSC proliferate and produce extracellular matrix proteins, such as collagens I and III, fibronectin, and laminin (5). Most of the experiments performed with isolated PSC focused on the activation mechanisms of these cells (2, 3, 5, 13, 16, 23,25), but little is known about the termination of this process. Because there is no evidence for redifferentiation of myofibroblast-like cells back into the quiescent state, apoptosis of activated cells is probably involved. However, this process has not yet been studied. Here we show that activated PSC undergo apoptosis after incubation with the two death receptor ligands CD95L and TRAIL or with the exogenous peripheral benzodiazepine receptor ligand 1-(2-chlorophenyl)-N-methyl-N-(1-methylpropyl)-isoquinoline-3-carboxamide (PK-11195).
In most cases of apoptosis, changes in mitochondrial permeability precede the major changes in cell morphology and biochemistry. This change is the result of an opening of a dynamic multiprotein pore formed in the contact side between the inner and outer mitochondrial membrane. The peripheral benzodiazepine receptor is part of this pore and seems to be involved in a wide spectrum of activities within cells (7). PK-11195 has been shown to induce apoptosis in thymocytes and more recently in HSC (6).
CD95L and tumor necrosis factor (TNF)-α-related apoptosis-inducing ligand (TRAIL) are ligands to receptors belonging to the TNF receptor family of death receptors (26). These receptors are characterized by an intracellular death domain that serves to recruit adapter proteins such as TRADD and FADD and cysteine proteases such as caspase-8. Activation of caspase-8 at the activated death receptor complex leads to apoptosis. Although the TRAIL receptors 1 and 2 are widely expressed in several tissues (4), most cell types are not sensitive to TRAIL-mediated cell killing (30).
Normal pancreatic acini, duct cells, and islet cells do not express CD95 (11), but CD95 expression is observed in chronic pancreatitis in acini and duct cells (11, 28). Various intracellular molecules affect the sensitivity of a cell to apoptotic signals. Death domain proteins are recruited to death receptors and propagate the apoptotic signal (19). This complex is known as death-inducing signaling complex (DISC). Activation of caspase-8 follows recruitment of this enzyme to the complex and leads to activation of effector caspases like caspase-3 through proteolytic cleavage and triggering mitochondrial damage (24).
Receptor-interacting protein (RIP) is another component of the DISC. The recruitment of RIP to this complex is responsible for activation of nuclear factor (NF)-κB and activator protein-1 (14, 15). Activation of NF-κB is thought to provide an antiapoptotic signal. RIP can be cleaved by caspase-8, whereby its NF-κB-inducing ability is abolished. In addition, the COOH-terminal cleavage product RIPc promotes apoptosis (12).
Nothing is known about induction and signaling pathways of apoptotic cell death in PSC. We therefore studied the expression of death receptors in quiescent and activated PSC and the potential of death receptor ligands for PSC apoptosis.
MATERIALS AND METHODS
Pronase and collagenase P were from Boehringer (Mannheim, Germany). Desoxyribonuclease 1, BSA, Percoll, propidium iodide, and rabbit anti-GFAP antibody were obtained from Sigma (Deisenhofen, Germany). Soluble CD95L with enhancer protein, TRAIL, and PK-11195 were from Alexis (Grünberg, Germany). TACS TdT Kit, Z-IETD-fluoromethylketone (FMK), Z-LHD-FMK, and Z-DEVD-FMK were purchased from R&D Systems (Wiesbaden, Germany). The enhanced chemiluminescence (ECL) kit was obtained from Amersham Pharmacia Biotech (Freiburg, Germany), and mouse anti-RIP antibody and Matrigel were from BD Transduction Laboratories (Heidelberg, Germany). Rabbit anti-phospho-PKB antibody and rabbit anti-PKB antibody were from Cell Signaling Technology. Annexin-V fluorescein isothiocyanate (FITC) was from Bender Medical Systems (Vienna, Austria). Anti-CD95 antibody and monoclonal mouse anti-FLIPS/L antibody were from Santa Cruz Biotechnology (Santa Cruz, CA), and rabbit anti-DR4 and rabbit anti-DR5 antibodies were from Serotec (Eching, Germany). All other chemicals were obtained from local sources at the highest purity available.
Isolation and culture of PSC.
For preparation of PSC, male Wistar rats (300–350 g body wt) were decapitated, and the pancreas was removed quickly. After injection of 10 ml Gey's buffered salt solution containing 5,000 units DNase 1, 12.5 units collagenase P, and 21 units pronase in the glandular parenchyma, the pancreas was minced in small fragments and incubated at 37°C (5% CO2) for 50 min. Cells were mechanically dispersed by pipetting them through a Nalgene pipette. Undigested tissue was removed with a nylon mesh (70 μm). Cells were washed (747 g; 4°C) and resuspended in DMEM (10% FCS). The cell suspension was mixed with the 1.5-fold volume of a 90% Percoll solution in 0.9% NaCl and centrifuged for 1 h at 40,000g (4°C). The upper part of the gradient, containing the PSCs and avital cells, was filtered again to remove lumping cell debris and centrifuged at 747 g for 7 min (4°C). Cells were resuspended in DMEM (10% FCS and 0.01% gentamicin) and seeded in a density of 105 cells/ml. At days 5,12, and 20 after preparation, confluent cultures were trypsinized, washed with DMEM (747 g, 10 min, 4°C), and again adjusted to 105 cells/ml. The purity of PSC preparations using the Percoll gradient was 75–85% immediately after seeding the cells. Contaminating cells were removed by carefully changing the medium 3 and 24 h after seeding, increasing the purity of the preparation to 95–99%.
Isolation of pancreatic acinar cells.
Pancreatic acinar cells (PAC) were isolated from male Wistar rats by collagenase digestion. In brief, the pancreas was removed quickly from a rat as outlined above, and 5 ml of a HEPES-Ringer (HR) solution containing 320 U/ml collagenase, 200 U/ml hyaluronidase, and 10 U/ml chymotrypsin were injected in the glandular parenchyma. The pancreas was minced in small fragments and incubated for 20 min at 37°C. The medium was replaced with 10 ml of a Ca2+- and Mg2+-depleted HR containing 1 mmol/l EDTA, and the incubation was continued for 10 min. Pancreatic fragments were returned to 5 ml HR containing 320 U/ml collagenase for a final 20-min incubation. The fragments were dispersed by repeatedly pipetting through a Nalgene pipette. The cells were filtered through a 70-μm nylon mesh, centrifuged for 5 min at 10 g, and resuspended in DMEM. For immunostaining and detection of apoptotic cell death, cells were plated on coverslips (12 mm diameter) coated with Matrigel and incubated for 30 min at 37°C before induction of apoptosis.
Immunofluorescence staining of the TRAIL receptor 2 and CD95.
Analysis of CD95 and TRAIL receptor 2 membrane trafficking was performed with living, unfixed, and unpermeabilized cells either stimulated for 24 h with 0.5 μmol/l cycloheximide alone, for 1 h with 100 ng/ml CD95L and 1 μg/ml CD95L-enhancer protein or 200 ng/ml TRAIL, or preincubated for 24 h with 0.5 μmol/l cycloheximide and then stimulated for 1 h with CD95L and enhancer or TRAIL. Control staining was performed with unstimulated cells. The receptors were stained for 45 min at 4°C with rabbit anti-CD95 antibody or rabbit anti-TRAIL receptor 2 antibody (1:500) and a Cy3-labeled goat-anti-rabbit antibody (1:500). Cells were washed in PBS supplemented with 2% FCS and 0.1% NaN3. Immunostained cells were analyzed using an Axioskop (Zeiss, Jena, Germany) with a 3CCD Camera (Intas) and a Leica TCS-NT confocal laser scanning system with an argon-krypton laser on a Leica DM IRB inverted microscope (Leica, Bensheim, Germany). Images were acquired at 568 nm wavelength to visualize Cy3. CD95 receptor or TRAIL receptor 2 membrane staining was defined as one or more intensely fluorescent spots on the surface of the cells.
Annexin V assay.
Annexin V assay was performed with cells plated on glass coverslips and incubated with various substances for the times indicated. Coverslips were transposed in a moist chamber, and cells were incubated with annexin V-FITC and propidium iodide (both diluted 1:1,000 in binding buffer provided by the manufacturer) for 15 min. Thereafter, coverslips were rinsed two times with PBS and analyzed using a Zeiss microscope equipped for fluorescence microscopy. Only fluorescein-positive cells without nuclear propidium iodide staining were regarded apoptotic and differentiated from healthy cells by phase-contrast microscopy. At least 100 cells were counted for each experiment (3–4 different preparations for each condition).
TdT nick end-labeling reaction.
Labeling and visualization of DNA fragments were performed with the TACS TdT kit using Mn2+ to enhance the reaction. Cells were counterstained with 0.5 μg/ml tetramethylrhodamine isothiocyanate (TRITC)-conjugated phalloidin for detection of cell shape. Incorporated FITC was detected using a fluorescence microscope (Zeiss) at an excitation wavelength of 488 nm. TRITC was excited at a wavelength of 568 nm. The number of apoptotic cells was determined by counting the percentage of FITC-positive cells. At least 100 cells were counted for each condition (3–4 different preparations for each experiment).
Determination of protein expression.
Cells were washed with PBS and lysed, and protein was harvested by scraping. Samples were then centrifuged at 10,000 g for 10 min, and the supernatant was collected for Western blotting. Protein content of the cell lysates was measured with the advanced protein assay reagent (Cytoskeleton) following the manufacturer's instructions using BSA as the standard. Proteins from each sample (10–40 μg/lane) were separated by gel electrophoresis using a 10% SDS-polyacrylamide gel. Known molecular weight protein standards were run with the samples. Separated proteins were transferred to a nitrocellulose membrane using a semidry blotting apparatus (Multiphor II; Pharmacia). The membrane was then incubated at 4°C with 5% BSA in Tris-buffered saline (pH 7.6) for 2 h to prevent nonspecific binding of antibodies. This was followed by an overnight incubation with the primary antibody (1:10,000) in TBS with 1% BSA. The membrane was washed three times and incubated with the secondary antibody for 2 h. Protein bands were detected by the ECL technique using the ECL kit (Amersham).
Determination of caspase-8 activity.
The caspase-8 assay from R&D Systems measures the colorimetric reaction of the cleavage of the amino acid motif IETD, thereby releasing the chromophore p-nitroanilide. The level of caspase enzymatic activity in the cell lysates is directly proportional to the color reaction that was quantified spectrophotometrically at a wavelength of 405 nm, using a microplate reader (Pharmacia). Data were corrected for background (no substrate or no cell lysate) and expressed as a percentage of the control levels (no induction of apoptosis).
Analysis of results and statistics.
Data are expressed as means ± SE. Each experiment was performed from at least three different cell preparations. Results were compared using the Student's t-test. P < 0.05 was considered statistically significant.
Characterization of isolated PSC.
PSC isolated by Percoll density gradient centrifugation exhibited the fat-storing phenotype with numerous fat droplets located in the perinuclear region of the cells (data not shown). At 2 days of culture, these cells stained positive for GFAP, whereas no α-smooth muscle actin (SMA) was detectable by Western blot analysis (Fig.1). Within the 1st wk of culture, the number and size of fat droplets and the GFAP expression decreased, and the cells developed a typical myofibroblast morphology and expressed α-SMA (Fig. 1). Therefore, PSC isolated by Percoll density centrifugation show similar characteristics as PSC isolated by other methods as Nycodenz density centrifugation or outgrowth. Similar to HSC, PSC are activated by cultivation and transform to myofibroblast-like cells.
Induction of apoptosis in PSC by CD95L.
Induction of apoptotic cell death in PSC by addition of CD95L depended strongly on the culture duration. Whereas CD95L induced neither annexin V staining (Table 1) nor DNA strand breaks [TdT nick end-labeling (TUNEL) assay; Table2] in 7-days cultured PSC, cells cultured for 14 or more days exhibited an apoptotic reaction to CD95L (Tables 1 and 2). In PSC cultured for 28 days, CD95L induced apoptotic cell death in almost one-third of the cells within 24 h. The apoptotic response to CD95L was enhanced in the presence of cycloheximide in the culture medium (Table 2). The number of TUNEL-positive cells exceeded the number of annexin V-positive cells. This might in part reflect the fact that fixed cells were used for TUNEL assay but not for the annexin V assay. In contrast to activated stellate cells, acinar cells of the pancreas showed no annexin V staining when incubated with CD95L at concentrations up to 10 ng/ml with or without cycloheximide (data not shown).
Induction of apoptosis in PSC by TRAIL.
PSC exhibited a dose- and activation-dependent sensitivity toward TRAIL-induced apoptosis, as shown by annexin V binding and TUNEL assay (Tables 3 and4). As it was true for the induction of apoptosis by CD95L, the number of apoptotic cells in cultures incubated with 100–200 ng/ml TRAIL increased with culture duration. TRAIL-induced cell death did not depend on the presence of cycloheximide (data not shown). PAC were insensitive to TRAIL when added at concentrations sufficient to induce apoptosis in activated PSC (data not shown).
Induction of apoptosis in PSC by PK-11195.
The peripheral benzodiazepine receptor ligand PK-11195 was recently shown to induce apoptosis in HSC (6). As shown in Tables 5 and6, PSC also underwent apoptosis in response to PK-11195. The effect was most pronounced in PSC cultured for up to 28 days. On the other hand, no apoptosis was induced by PK-11195 in PAC; however, at a concentration of 200 μmol/l, PK-11195 caused necrotic cell death of acinar cells within 6 h.
A general problem in determining the percentage of apoptotic cells in PSC cultures was the fact that untreated cultures showed different levels of basal apoptotic rates (2–10% in 28-day-old cells). Cultures with high apoptotic background were in general more sensitive to apoptotic stimuli independent of the method used to determine the percentage of apoptotic cells. However, in each experiment, all conditions were performed with cells from one preparation and are therefore comparable. Different experimental settings were performed with cells from different preparations and are for that reason hard to compare.
Expression of CD95 and TRAIL receptor.
Death receptor expression was studied in PSC by Western blot analysis (Fig. 2, A and B). Expression of CD95 and the TRAIL receptor 2, but not of TRAIL receptor 1, increased with increasing culture time. In unstimulated PSC, which were kept in culture for 28 days, no CD95 immunostaining was detectable in nonpermeabilized cells, indicating that CD95 was mainly localized inside the cells (Fig. 3). However, treatment of PSC with CD95L induced within 1 h intense CD95 immunostaining at the cell surface, indicating CD95 trafficking to the plasma membrane. CD95L-induced CD95 membrane targeting occurred regardless of whether cycloheximide was present or not (Fig. 3). No translocation of the CD95 receptor was observed in response to cycloheximide alone (data not shown). Treatment of PSC with TRAIL did not induce TRAIL receptor 2 translocation to the plasma membrane (Fig.3).
Studies on the mechanisms of apoptosis in PSC.
As shown in Tables 1-6, PSC in culture for 4 wk are much more sensitive to apoptosis induction by CD95L, TRAIL, or PK-11195 than PSC, which were kept in culture for 1 wk only. Although this may in part be explained by an increased expression of CD95 and TRAIL receptors with culture duration (Fig. 2), further mechanisms involving intracellular apoptosis-modulating signals may contribute. PKB is known to exert antiapoptotic effects. However, as shown in Fig. 4, PKB phosphorylation did not significantly change in PSC cultured for 1 or 4 wk, although there was a slight increase in total PKB expression. Also, expression of antiapoptotic c-Flip was largely unaffected (Fig. 4).
RIP is one of the kinases that is involved in receptor-mediated apoptosis of many cell types. As shown in Fig. 4, there is a slight increase in the amount of the uncleaved RIP (molecular mass ∼74 kDa) in PSC cultured for 4 wk compared with 1 wk of culture. However, the amount of RIPc, a cleavage product of RIP with a molecular mass of ∼50 kDa, became increasingly apparent after 4 wk of culture.
Because RIP cleavage is known to depend on caspase-8 activity, caspase-8 activity was determined in PSC cultured for 28 days. Figure5 shows that caspase-8 activity was significantly increased upon addition of CD95L or TRAIL for 24 h. CD95L-induced caspase-8 activation was further enhanced by cycloheximide. No increase in caspase-8 activity was measured after incubating PSC with 100 μM PK-11195 (Fig. 5). Inhibition of caspase-8 activity by 50 μmol/l Z-IETD-FMK completely abolished apoptosis in PSC induced by TRAIL or CD95L. This was also found upon inhibition of caspase-3 (50 μmol/l Z-DEVD-FMK) or caspase-9 (50 μmol/l Z-LEHD-FMK; Fig. 6).
Western blot analyses were performed to investigate the influence of the TRAIL and CD95L on RIP cleavage. As shown in Fig.7, both CD95L and TRAIL induced the cleavage of RIP, however, with different time courses. Whereas CD95L induced RIP cleavage within 10 min (data not shown), cleaved RIP was found only after 6 h of TRAIL treatment.
Transformation of PSC to myofibroblast-like cells was paralleled by an increased sensitivity to CD95L- and TRAIL-mediated apoptosis. This time-dependent sensitization was accompanied by an increased expression of CD95 receptor and the TRAIL receptor 2. An increasing sensitivity to CD95L parallel to cell transformation was also reported for HSC (8); however, HSC apoptosis is induced by CD95L only, when simultaneously cycloheximide is present (8). Furthermore, HSC do not undergo apoptosis in response to TRAIL (unpublished observations). This is different to PSC, which undergo apoptosis in response to CD95L or TRAIL even in the absence of cycloheximide, although cycloheximide augments CD95L-induced apoptosis. The mechanism underlying the sensitizing effect of cycloheximide is not clear (20) but may involve (8) a modulation of the p53 pathway, downregulation of Flip, and activation of stress-activated protein kinases by cycloheximide (20).
In contrast to activated PSC, PAC were not sensitive to death receptor ligand-mediated cell death. The lacking sensitivity toward CD95L can be explained by the fact that normal PAC do not express CD95 (11). The insensitivity of acinar cells toward TRAIL is due to a preferential expression of antagonistic receptors (TRAIL receptors 3 and 4) as it was shown for other cell types (22,27).
Induction of PSC apoptosis by both CD95L and TRAIL was accompanied by an activation of caspase-8 and was sensitive to inhibition of either caspase-3, -8, or -9. This finding suggests that apoptosis induction by CD95L or TRAIL may involve amplification of the death signal through the mitochondrial pathway, as suggested recently for many other cell types, including hepatocytes (10,18, 21).
As shown recently for HSC (6), PSC also are susceptible toward apoptosis induction by PK-11195, a specific ligand of the peripheral-type benzodiazepine receptor. However, HSC show a transient sensitivity to PK-11195-induced apoptosis, which is maximal at about the 7th day of culture and disappears within 4 wk (6), whereas sensitivity of PSC increased with culture over a period of 4 wk. This culture time dependence of PK-11195-induced apoptosis in PSC cannot be explained exclusively by corresponding alterations of the expression of the peripheral benzodiazepine receptor, as shown for HSC (6).
The mechanisms underlying the culture-dependent sensitization of PSC toward CD95L- and TRAIL-induced apoptosis is unclear but may involve an increased expression of CD95 and TRAIL receptors. Clearly, culture-dependent changes in PKB phosphorylation or Flip expression do not provide an explanation. Interestingly, a cleavage product of the RIP is found in PSC after 4 wk of culture, and its amount further increases in response to CD95L or TRAIL addition, as expected from the known caspase-8-triggered RIP cleavage (12, 17). RIP is thought to be antiapoptotic by activation of the transcription factor NF-κB (15). On the other hand, a COOH-terminal proteolytic fragment of RIP was shown to inhibit NF-κB activation through inhibition of inhibitory factor-κB-kinase-β (12). The COOH-terminal fragment of RIP (RIPc) also augments the association between death receptors and death domain proteins (e.g., TRADD and FADD; see Ref. 14), thereby favoring caspase-8 activation and stimulation of apoptosis. The proteolytic RIP fragment found in activated but not quiescent PSC (Fig.5) stains with an antibody specific raised again the COOH-terminal part of RIP. It exhibits a molecular mass of ∼50 kDa. This 50-kDa protein may well be the rat counterpart of the 42-kDa RIPcdescribed in HeLa and Jurkat cells (14). In these cells, the COOH-terminal cleavage product appears when the cells are incubated with TNF, CD95L, or TRAIL. A similar cleavage product with a molecular mass of 38 kDa was also detected in Jurkat T cells exposed to CD95L (17). As shown in the present study, the appearance of a COOH-terminal RIP cleavage product during the transformation of PSC to a myofibroblast-like phenotype is paralleled by an enhanced sensitivity to apoptotic signals. The amount of this protein was further enhanced by induction of apoptosis with CD95L or TRAIL. CD95L induced the protein within 10 min of incubation in the presence of cycloheximide, which argues against de novo synthesis of the protein. Probably, the 50-kDa protein is the product of cleaving native RIP by caspase-8 as it was also described by others (12, 17). In line with this, CD95L in the presence of cycloheximide increases caspase-8 activity sevenfold, and under these conditions a rapid and pronounced increase in the amount of the 50-kDa protein is found. TRAIL, however, only doubles caspase-8 activity in PSC, and this is accompanied by a less pronounced and delayed increase of the 50-kDa protein. The molecular mass of the TRAIL-induced protein is somewhat higher compared with the strong band induced by incubation with CD95L. The meaning and the mechanisms leading to this difference are not clear so far.
The increasing susceptibility of activated PSC to death receptor ligand-mediated apoptosis may be an efficient way to eliminate transformed PSC in chronic and acute pancreatitis to terminate the healing response, restore cellular homeostasis, and to prevent the persistence of cells engaged in excessive matrix production.
Address for reprint requests and other correspondence: H. Klonowski-Stumpe, Dept. of Gastroenterology, Hepatology and Infectiology, Heinrich-Heine Universität Düsseldorf, Moorenstr. 5, 40225 Düsseldorf, Germany (E-mail:).
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May 1, 2002;10.1152/ajpgi.00073.2002
- Copyright © 2002 the American Physiological Society