- Email: [email protected]
TNF-α AND IL-1α INDUCE APOPTOSIS IN SUBCONFLUENT RAT MESANGIAL CELLS. EVIDENCE FOR THE INVOLVEMENT OF HYDROGEN PEROXIDE AND LIPID PEROXIDATION AS SECOND MESSENGERS
doi:10.1006/cyto.1999.0633, available online at http://www.idealibrary.com on
TNF- AND IL-1 INDUCE APOPTOSIS IN SUBCONFLUENT RAT MESANGIAL CELLS. EVIDENCE FOR THE INVOLVEMENT OF HYDROGEN PEROXIDE AND LIPID PEROXIDATION AS SECOND MESSENGERS Torsten Bo¨hler, Johannes Waiser, Helen Hepburn, Jens Gaedeke, Christine Lehmann, Pia Hambach, Klemens Budde, Hans-Hellmut Neumayer Apoptosis of mesangial cells (MC) plays a role in glomerulonephritis (GN). In this study we investigated cytokine-induced apoptosis of cultured rat MC by morphological and biochemical features. TNF- and IL-1 induced apoptosis in rat MC in a time- and concentration-dependent fashion. RT-PCR experiments revealed that MC express the TNF-receptor 1 (p60) gene constitutively. TNF- as well as IL-1 stimulated the production of reactive oxygen species (ROS) and induced lipid peroxidation. Coincubation with catalase inhibited TNF- and IL-1 induced apoptosis as well as lipid peroxidation. TNF-, but not IL-1 increased the expression of c-jun. These results provide evidence that TNF- and IL-1 induce apoptosis in rat MC with hydrogen peroxide and lipid peroxidation as second messengers. Increased c-jun expression may be a downstream intracellular signal of TNF--, but not IL-1-induced apoptosis. 2000 Academic Press
Mesangial hypercellularity caused by an imbalance between cell proliferation and cell death is a frequent feature of GN.1 During recovery from experimental proliferative GN, apoptosis plays an essential role in the resolution of mesangial hypercellularity.2 TNF- and IL-1 are secreted by infiltrating macrophages and glomerular cells in GN.3 Both cytokines induce the synthesis of ROS.4 The generation of large amounts of ROS results in tissue injury and necrosis, whereas moderate generation of ROS results in apoptosis.5 It was shown that hydrogen peroxide induces apoptosis in rat MC.6 ROS seem to be involved in cytokine-mediated signal transduction by interaction with biological macromolecules (lipids, proteins, nucleic acids and carbohydrates).7 ROS can activate, by phosphorylation, the transcription factor c-jun/ AP-1, which is involved in the signal transduction of From: Department of Internal Medicine-Nephrology, Charite´, Campus Charite´-Mitte, Humboldt-University, Berlin, Germany Correspondence to: Torsten Bo¨hler, Nephrologische Klinik, Universita¨tsklinikum Charite´, Campus Charite´-Mitte, Schumannstrasse 20/21, 10117 Berlin, Germany; E-mail: [email protected] Received 27 April 1999; received in revised form 1 September 1999; accepted for publication 20 September 1999 2000 Academic Press 1043–4666/00/070986+06 $35.00/0 KEY WORDS: apoptosis/hydrogen peroxide/IL-1/lipid peroxidation/mesangial cells/TNF- 986
apoptosis. Thus, c-jun may be a sensor protein for intracellular ROS that modulates gene expression and apoptosis.8,9 Here, we investigated the effect of TNF- and IL-1 on the delicate balance of proliferation and apoptosis in rat MC cultures.
RESULTS Growth-arrested MC were stimulated with TNF- or IL-1. Both cytokines were able to induce apoptosis dose- and time-dependently (Fig. 1). Apoptosis was predominantly found in subconfluently grown MC (Fig. 2). In untreated MC (10% FCS) the percentage of apoptotic cells was about 4%. Starving (0.5% FCS) for 24 h increased the rate of apoptotic cells to 7% (P<0.05). Both TNF-- (100 ng/ml) and IL-1(50 ng/ml) induced apoptosis of subconfluently grown MC was confirmed by DNA fragmentation (Fig. 3). Stimulation of MC with either 100 ng/ml TNF- (2.50.6103 cpm) or 50 ng/ml IL-1 (2.10.4 103 cpm) caused no significant changes in cell proliferation compared with untreated MC (2.00.3 103 cpm). TNF-receptor 1 (TNF-R1) mediates apoptosis via its so-called intracellular ‘death domain’. Using a specific primer pair for TNF-R1 (p60) cDNA, we CYTOKINE, Vol. 12, No. 7 (July), 2000: pp 986–991
TNF- and IL- induce apoptosis in rat mesangial cells / 987
Figure 1.
Detection of apoptotic mesangial cells by fluorescence microscopy.
MC were stained with AO and Etbr. Apoptotic cell morphology was assessed by fluorescence microscopy (A–C). Life (L) and apoptotic (A) cells stain green, whereas necrotic cells (N) stain orange, reflecting the loss of membrane integrity. Green apoptotic cells exert the typical morphological features of apoptosis with ‘dots’ of condensed chromatin, cell blebbing, and cell shrinkage. Confluently grown MC rarely exerted the morphological characteristics of apoptosis (D).
found that confluent and subconfluent MC constitutively express the TNF-R1 (p60) gene (Fig. 4). Catalase detoxifies hydrogen peroxide via the reaction: 2H2O2
2H2O+O2.
As H2O2 molecules are able to cross the cell membrane, extracellular catalase is able to lower the intracellular oxidative stress conditions. Coincubation with 100 ng/ ml TNF- or 50 ng/ml IL-1 and 0–100 g/ml catalase
caused a significant and dose-dependent reduction of apoptosis. However, catalase did not protect all MC from apoptosis. For TNF- we detected a reduction from 202.0% to 8.54.0% apoptosis and for IL-1treatment a decrease from 174.0% to 102.0%. Coincubation of MC with either TNF- (100 ng/ ml) or IL-1 (50 ng/ml) and nitroblue tetrazolium bromide (NBT) for 12 h resulted in blue staining of MC. The formation of blue formazan revealed ROS-production (Fig. 5). Incubation with TNF-
988 / Bo¨hler et al.
CYTOKINE, Vol. 12, No. 7 (July, 2000: 986–991)
Apoptosis (%)
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Figure 2.
(B)
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20 30 Time (hours)
40
50
60
0
Concentration dependency and kinetics of TNF- and IL-1 induced apoptosis in MC.
The percentage of apoptotic cells was determined after 24 h incubation with different concentrations of (A) TNF- (0–100 ng/ml) or (B) IL-1 (0–50 ng/ml) by counting d100 cells. Percentages represent the meanSD of three independent experiments. (C) The percentage of apoptotic cells was determined after different incubation periods (0–48 h) with (100 ng/ml) ( ) or without ( ) TNF- by counting d100 cells. Percentages represent the meanSD of three independent experiments. (D) The percentage of apoptotic cells was determined after different incubation periods (0–48 h) with (50 ng/ml) ( ) or without ( ) IL-1 by counting d100 cells. Percentages represent the meanSD of three independent experiments.
(100 ng/ml) or IL-1 (50 ng/ml) also increased lipid peroxidation as indicated by malondialdehyde (MDA) levels (TNF-: 8.51.2 M; IL-1: 7.61.4 M) as compared to controls (2.80.9 M, P<0.05). Coincubation with catalase (100 mg/ml) reduced TNF-(3.31.4 M) and IL-1- (3.80.6 M) induced MDA generation (P<0.05). Western blot experiments revealed that TNF- (100 ng/ml), but not IL-1 (50 ng/ ml), induced a 2.5-fold increased c-jun expression (Fig. 6).
Figure 3.
DNA fragmentation assay.
Incubation of MC with either TNF- (100 ng/ml) or IL-1 (50 ng/ ml) resulted in DNA cleavage as shown by gel electrophoresis of the nuclear DNA. This DNA fragmentation pattern is typical for apoptotic cells.
TNF- and IL- induce apoptosis in rat mesangial cells / 989
C on fl Su uen bc t on flu C en on t tr ol C on flu Su en bc t o C nflu on tr ent ol
Mw
(A)
280 bp
(B) TNF-R1 Figure 4.
Actin
Detection of TNF-R1 expression by RT-PCR.
Agarose gel electrophoresis of the RT-PCR products shows the amplification of cDNA from confluently and subconfluently grown MC with TNF-R1 and -actin primer pairs. We used the following sets of primers: 5 -CCTgCgATgCTgTATgCTgTg and 3 -AgAgTCTCgCggATgTTCTCCAgg for the TNF-R1 gene, 5 gTgCCACCAgACAgCACT gTgTTg and 3 -CATgAAgTCCCAg TCCTACgg for the control house-keeping gene -actin. PCR conditions included: 10 mM Tris-HCl, pH 9.0, 50 mM KCl, 1.5 mM MgCl2, 0.5 mM dNTPs, 1 M TNF-R1 primer or 0.5 M -actin primer, 0.13 U Amplitaq Gold DNA polymerase; 35 amplification cycles with the following temperature profile were carried out: (1) 95C (10 min); (2) 94C (30 s); (3) 67C (45 s) for TNF-R1 and 54C (45 s) for -actin; (4) 72C (2 min).
DISCUSSION
(C) 10
Similarly to a previous study, we showed that TNF- and IL-1 increased the percentage of apoptosis in rat MC up to 20–25%, in a time- and dosedependent manner. In contrast to other reports,8 no cotreatment with protein-synthesis inhibitors, such as actinomycin D, was necessary. However, apoptosis was almost restricted to subconfluently grown cells. This may be due to inhibitory cell–cell or cell–matrix interactions, which may also explain the contradictory reports. Expression of the IL-1 receptor in MC has already been demonstrated.11 This is the first report describing the expression of the TNF-R1 (p60) mRNA in rat MC. We suggest that TNF- and IL-1 transduce the apoptotic signal in MC via their specific receptors. We further investigated whether H2O2 is involved in the signal transduction of TNF-- and IL-1induced apoptosis. In accordance with our observation that apoptosis occurred in only 20–25% of MC, staining with NBT, a marker for ROS, was not uniformly positive. This indicates the existence of responder and non-responder cells. Catalase inhibited TNF-- and
Figure 5.
ROS production assay.
Production of ROS was visualized by staining MC with NBT: (A) no stimulation; (B) TNF- (100 ng/ml), (C) IL-1 (50 ng/ml).
IL-1-induced apoptosis, suggesting that H2O2 is involved in the pathway of TNF-- and IL-1-induced MC-apoptosis. However, catalase did not prevent all
990 / Bo¨ hler et al.
CYTOKINE, Vol. 12, No. 7 (July, 2000: 986–991)
ation of c-jun/AP-1 by c-jun NH2-terminal kinases (JNKs).9 Therefore, elevated levels of c-jun/AP-1 after H2O2-stimulation can be explained by transcriptional autoregulation of c-jun/AP-1 by activated c-jun/AP-1. In summary, our results suggest that H2O2 and lipid peroxidation act as second messengers in TNF-and IL-1- induced apoptosis in rat MC. Concerning TNF--, but not IL-1-induced apoptosis, c-jun/AP-1 seems to be another relevant second messenger.
MATERIALS AND METHODS Cell culture MC were isolated and characterized as described previously.12 For the experiments, we used MC between the fourth and fifteenth passage. Cell proliferation was determined by incorporation of [3H]-thymidine as previously described.13
Detection of apoptosis MC were grown subconfluently on rat-tail collagen, starved overnight, stimulated and stained with acridine orange (AO)/ethidium bromide (Etbr). Apoptosis was assessed as described elsewhere.14 DNA fragmentation assay was performed by using a DNA fragmentation kit (Boehringer Mannheim, Mannheim, Germany). Figure 6. Detection of c-jun expression by SDS-PAGE and immunoblot analysis.
Reverse transcription polymerase chain reaction (RT-PCR)
(A) Incubation with TNF- (100 ng/ml) increased c-jun expression in MC 2.5-fold as compared to untreated cells. (B) c-jun expression was not affected by IL-1 (50 ng/ml). Thirty micrograms of cell lysate protein per lane were separated on a 12% SDS polyacrylamide Laemmli minigel16 and directly transferred to polyvinyl membranes (Serva/Boehringer, Ingelheim, Heidelberg, Germany) in a minielectrotransfer unit (Hoefer, Freiburg, Germany) at 300 mA for 3 h in 192 mM glycine, 25 mM Tris, and 20% methanol. A monoclonal mouse IgG2a antibody against c-jun (0.25 g/ml) (Transduction Lab, Lexington, USA) and a 1:1000 diluted rabbit anti-mouse IgG1 alkaline phosphatase conjugate (Santa Cruz, Heidelberg, Germany) were used. The blots were stained with BCIP and NBT (Sigma) according to the manufacturer’s instructions. The density of protein bands was determined with a Kodak Camera Imaging System (Kodak, Rochester, USA).
RNA was isolated15 and subjected to RT-PCR with specific primers for rat TNF-R1 or -actin. PCR-products were consistent with the predicted sizes. Cloning (TACloning-Kit, Invitrogen, San Diego, CA, USA) and sequencing of the PCR-products (Replicon, Berlin, Germany) confirmed the identity of the amplified target genes.
cells from apoptostis, indicating either that extracellular catalase did not eliminate intracellular H2O2 sufficiently, or that other, H2O2-independent signal transduction pathways are involved. Catalase treatment also suppressed TNF- and IL-1 stimulated lipid peroxidation, suggesting that lipid peroxides play a role in the following signal transduction cascade. It has been reported that c-jun/AP-1 is a mediator for H2O2 initiated apoptosis in MC.8 We found increased c-jun expression in TNF-, but not in IL-1 stimulated MC. Other reports showed that the cellular redox status influences the transcription of c-jun and that H2O2 induces apoptosis in MC via phosphoryl-
Biochemistry ROS were detected by reduction of NBT (Sigma).4 MDA was used as a marker for lipid peroxidation (Bioxytech-LPO-586-kit: OXIS-International, Portland, Oregon, USA). Western blots were performed according to standard protocol. Protein concentration was determined using bicinchoninic acid.
Chemicals and reagents Recombinant rat cytokines were purchased from Pharma-Biotechnologie-Hannover (Hannover, Germany). All chemicals were purchased from Merck (Darmstadt, Germany).
Acknowledgements This study was supported by the Novartis-Stiftung fu¨ r Therapeutische Forschung and presented as a poster at the 31st annual meeting of the American
TNF- and IL- induce apoptosis in rat mesangial cells / 991
Society of Nephrology (ASN), October 25–28, 1998 in Philadelphia, PA, USA. Torsten Bo¨ hler is a recipient of a research fellowship of the Novartis-Stiftung fu¨ r Therapeutische Forschung.
REFERENCES 1. Savill J (1995) Apoptosis: will cell death add life to nephrology? Nephrol Dial Transplant 10:1977–1979 . 2. Baker AJ, Mooney A, Hughes J, Lombardi D, Johnson RJ, Savill J (1994) Mesangial cell apoptosis: the major mechanism for resolution of glomerular hypercellularity in experimental mesangial proliferative nephritis. J Clin Invest 5:2105–2116. 3. Kluth DC, Rees AJ (1996) Inhibiting inflammatory cytokines. Semin Nephrol 16:576–582. 4. Radeke HH, Meier B, Topley N, Flo¨ ge J, Habermehl GG, Resch K (1990) Interleukin 1- and tumor necrosis factor- induce oxygen radical production in mesangial cells. Kidney Int 37:767–775. 5. Sugiyama H, Kashihara N, Makino H, Yamasaki Y, Ota Z (1996) Reactive oxygen species induce apoptosis in cultured human mesangial cells. J Am Soc Nephrol 7:2357–2363. 6. Meyer TN, Gloy J, Hug MJ, Greger R, Schollmeyer P, Pavenstadt H (1996) Hydrogen peroxide increases the intracellular calcium activity in rat mesangial cells in primary culture. Kidney Int 49:388–395.
7. Shaw S, Naegeli P, Etter JD, Weidmann P (1995) Role of intracellular signaling pathways in hydrogen peroxide-induced injury to rat glomerular mesangial cells. Clin Exp Pharmacol Physiol 22:924–933. 8. Guo YL, Baysal K, Kang B, Yang LJ, Williamson JR (1998) Correlation between sustained c-jun N-terminal protein kinase activation and apoptosis induced by tumor necrosis factor- in rat mesangial cells. J Biol Chem 7:4027–4034. 9. Lo YC, Wong JM, Cruz TF (1996) Reactive oxygen species mediate cytokine activation of c-jun NH2-terminal Kinases. J Biol Chem 271:15703–15707. 10. Liu ZH, Striker GE, Stetler-Stephenson M, Fukushima P, Patel A, Striker LJ (1996) TNF-alpha and IL-1 alpha induce mannose receptors and apoptosis in glomerular mesangial but not endothelial cells. Am J Physiol 270:1595–1601. 11. Zoja C, Rambaldi A, Remuzzi G (1993) Interleukin-1 and glomerular mesangial cells. Ren Physiol Biochem 16:89–92. 12. Budde K, Neumayer HH, Salant DJ, Cybulsky AV, Coleman DL, Sterzel RB (1997) Glomerular epithelial cell products stimulate mesangial cell proliferation in culture. Kidney Int 52:733–741. 13. Budde K, Coleman DL, Lacy J, Sterzel RB (1989) Rat mesangial cells produce granulocyte-macrophage colony-stimulating factor. Am J Physiol 257:1065–1078. 14. McGahon AJ (1995) The end of the (cell) line: methods for the study of apoptosis in vitro. In Schwartz LM, Osborne BA (eds) Methods in Cell Biology. Academic Press, Boston, pp 153–185. 15. Chromczynski P, Sacchi N (1987) Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem 162:156–159.
TNF- AND IL-1 INDUCE APOPTOSIS IN SUBCONFLUENT RAT MESANGIAL CELLS. EVIDENCE FOR THE INVOLVEMENT OF HYDROGEN PEROXIDE AND LIPID PEROXIDATION AS SECOND MESSENGERS Torsten Bo¨hler, Johannes Waiser, Helen Hepburn, Jens Gaedeke, Christine Lehmann, Pia Hambach, Klemens Budde, Hans-Hellmut Neumayer Apoptosis of mesangial cells (MC) plays a role in glomerulonephritis (GN). In this study we investigated cytokine-induced apoptosis of cultured rat MC by morphological and biochemical features. TNF- and IL-1 induced apoptosis in rat MC in a time- and concentration-dependent fashion. RT-PCR experiments revealed that MC express the TNF-receptor 1 (p60) gene constitutively. TNF- as well as IL-1 stimulated the production of reactive oxygen species (ROS) and induced lipid peroxidation. Coincubation with catalase inhibited TNF- and IL-1 induced apoptosis as well as lipid peroxidation. TNF-, but not IL-1 increased the expression of c-jun. These results provide evidence that TNF- and IL-1 induce apoptosis in rat MC with hydrogen peroxide and lipid peroxidation as second messengers. Increased c-jun expression may be a downstream intracellular signal of TNF--, but not IL-1-induced apoptosis. 2000 Academic Press
Mesangial hypercellularity caused by an imbalance between cell proliferation and cell death is a frequent feature of GN.1 During recovery from experimental proliferative GN, apoptosis plays an essential role in the resolution of mesangial hypercellularity.2 TNF- and IL-1 are secreted by infiltrating macrophages and glomerular cells in GN.3 Both cytokines induce the synthesis of ROS.4 The generation of large amounts of ROS results in tissue injury and necrosis, whereas moderate generation of ROS results in apoptosis.5 It was shown that hydrogen peroxide induces apoptosis in rat MC.6 ROS seem to be involved in cytokine-mediated signal transduction by interaction with biological macromolecules (lipids, proteins, nucleic acids and carbohydrates).7 ROS can activate, by phosphorylation, the transcription factor c-jun/ AP-1, which is involved in the signal transduction of From: Department of Internal Medicine-Nephrology, Charite´, Campus Charite´-Mitte, Humboldt-University, Berlin, Germany Correspondence to: Torsten Bo¨hler, Nephrologische Klinik, Universita¨tsklinikum Charite´, Campus Charite´-Mitte, Schumannstrasse 20/21, 10117 Berlin, Germany; E-mail: [email protected] Received 27 April 1999; received in revised form 1 September 1999; accepted for publication 20 September 1999 2000 Academic Press 1043–4666/00/070986+06 $35.00/0 KEY WORDS: apoptosis/hydrogen peroxide/IL-1/lipid peroxidation/mesangial cells/TNF- 986
apoptosis. Thus, c-jun may be a sensor protein for intracellular ROS that modulates gene expression and apoptosis.8,9 Here, we investigated the effect of TNF- and IL-1 on the delicate balance of proliferation and apoptosis in rat MC cultures.
RESULTS Growth-arrested MC were stimulated with TNF- or IL-1. Both cytokines were able to induce apoptosis dose- and time-dependently (Fig. 1). Apoptosis was predominantly found in subconfluently grown MC (Fig. 2). In untreated MC (10% FCS) the percentage of apoptotic cells was about 4%. Starving (0.5% FCS) for 24 h increased the rate of apoptotic cells to 7% (P<0.05). Both TNF-- (100 ng/ml) and IL-1(50 ng/ml) induced apoptosis of subconfluently grown MC was confirmed by DNA fragmentation (Fig. 3). Stimulation of MC with either 100 ng/ml TNF- (2.50.6103 cpm) or 50 ng/ml IL-1 (2.10.4 103 cpm) caused no significant changes in cell proliferation compared with untreated MC (2.00.3 103 cpm). TNF-receptor 1 (TNF-R1) mediates apoptosis via its so-called intracellular ‘death domain’. Using a specific primer pair for TNF-R1 (p60) cDNA, we CYTOKINE, Vol. 12, No. 7 (July), 2000: pp 986–991
TNF- and IL- induce apoptosis in rat mesangial cells / 987
Figure 1.
Detection of apoptotic mesangial cells by fluorescence microscopy.
MC were stained with AO and Etbr. Apoptotic cell morphology was assessed by fluorescence microscopy (A–C). Life (L) and apoptotic (A) cells stain green, whereas necrotic cells (N) stain orange, reflecting the loss of membrane integrity. Green apoptotic cells exert the typical morphological features of apoptosis with ‘dots’ of condensed chromatin, cell blebbing, and cell shrinkage. Confluently grown MC rarely exerted the morphological characteristics of apoptosis (D).
found that confluent and subconfluent MC constitutively express the TNF-R1 (p60) gene (Fig. 4). Catalase detoxifies hydrogen peroxide via the reaction: 2H2O2
2H2O+O2.
As H2O2 molecules are able to cross the cell membrane, extracellular catalase is able to lower the intracellular oxidative stress conditions. Coincubation with 100 ng/ ml TNF- or 50 ng/ml IL-1 and 0–100 g/ml catalase
caused a significant and dose-dependent reduction of apoptosis. However, catalase did not protect all MC from apoptosis. For TNF- we detected a reduction from 202.0% to 8.54.0% apoptosis and for IL-1treatment a decrease from 174.0% to 102.0%. Coincubation of MC with either TNF- (100 ng/ ml) or IL-1 (50 ng/ml) and nitroblue tetrazolium bromide (NBT) for 12 h resulted in blue staining of MC. The formation of blue formazan revealed ROS-production (Fig. 5). Incubation with TNF-
988 / Bo¨hler et al.
CYTOKINE, Vol. 12, No. 7 (July, 2000: 986–991)
Apoptosis (%)
(A) 25
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20
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30
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Figure 2.
(B)
25
0
10
20 30 Time (hours)
40
50
60
0
Concentration dependency and kinetics of TNF- and IL-1 induced apoptosis in MC.
The percentage of apoptotic cells was determined after 24 h incubation with different concentrations of (A) TNF- (0–100 ng/ml) or (B) IL-1 (0–50 ng/ml) by counting d100 cells. Percentages represent the meanSD of three independent experiments. (C) The percentage of apoptotic cells was determined after different incubation periods (0–48 h) with (100 ng/ml) ( ) or without ( ) TNF- by counting d100 cells. Percentages represent the meanSD of three independent experiments. (D) The percentage of apoptotic cells was determined after different incubation periods (0–48 h) with (50 ng/ml) ( ) or without ( ) IL-1 by counting d100 cells. Percentages represent the meanSD of three independent experiments.
(100 ng/ml) or IL-1 (50 ng/ml) also increased lipid peroxidation as indicated by malondialdehyde (MDA) levels (TNF-: 8.51.2 M; IL-1: 7.61.4 M) as compared to controls (2.80.9 M, P<0.05). Coincubation with catalase (100 mg/ml) reduced TNF-(3.31.4 M) and IL-1- (3.80.6 M) induced MDA generation (P<0.05). Western blot experiments revealed that TNF- (100 ng/ml), but not IL-1 (50 ng/ ml), induced a 2.5-fold increased c-jun expression (Fig. 6).
Figure 3.
DNA fragmentation assay.
Incubation of MC with either TNF- (100 ng/ml) or IL-1 (50 ng/ ml) resulted in DNA cleavage as shown by gel electrophoresis of the nuclear DNA. This DNA fragmentation pattern is typical for apoptotic cells.
TNF- and IL- induce apoptosis in rat mesangial cells / 989
C on fl Su uen bc t on flu C en on t tr ol C on flu Su en bc t o C nflu on tr ent ol
Mw
(A)
280 bp
(B) TNF-R1 Figure 4.
Actin
Detection of TNF-R1 expression by RT-PCR.
Agarose gel electrophoresis of the RT-PCR products shows the amplification of cDNA from confluently and subconfluently grown MC with TNF-R1 and -actin primer pairs. We used the following sets of primers: 5 -CCTgCgATgCTgTATgCTgTg and 3 -AgAgTCTCgCggATgTTCTCCAgg for the TNF-R1 gene, 5 gTgCCACCAgACAgCACT gTgTTg and 3 -CATgAAgTCCCAg TCCTACgg for the control house-keeping gene -actin. PCR conditions included: 10 mM Tris-HCl, pH 9.0, 50 mM KCl, 1.5 mM MgCl2, 0.5 mM dNTPs, 1 M TNF-R1 primer or 0.5 M -actin primer, 0.13 U Amplitaq Gold DNA polymerase; 35 amplification cycles with the following temperature profile were carried out: (1) 95C (10 min); (2) 94C (30 s); (3) 67C (45 s) for TNF-R1 and 54C (45 s) for -actin; (4) 72C (2 min).
DISCUSSION
(C) 10
Similarly to a previous study, we showed that TNF- and IL-1 increased the percentage of apoptosis in rat MC up to 20–25%, in a time- and dosedependent manner. In contrast to other reports,8 no cotreatment with protein-synthesis inhibitors, such as actinomycin D, was necessary. However, apoptosis was almost restricted to subconfluently grown cells. This may be due to inhibitory cell–cell or cell–matrix interactions, which may also explain the contradictory reports. Expression of the IL-1 receptor in MC has already been demonstrated.11 This is the first report describing the expression of the TNF-R1 (p60) mRNA in rat MC. We suggest that TNF- and IL-1 transduce the apoptotic signal in MC via their specific receptors. We further investigated whether H2O2 is involved in the signal transduction of TNF-- and IL-1induced apoptosis. In accordance with our observation that apoptosis occurred in only 20–25% of MC, staining with NBT, a marker for ROS, was not uniformly positive. This indicates the existence of responder and non-responder cells. Catalase inhibited TNF-- and
Figure 5.
ROS production assay.
Production of ROS was visualized by staining MC with NBT: (A) no stimulation; (B) TNF- (100 ng/ml), (C) IL-1 (50 ng/ml).
IL-1-induced apoptosis, suggesting that H2O2 is involved in the pathway of TNF-- and IL-1-induced MC-apoptosis. However, catalase did not prevent all
990 / Bo¨ hler et al.
CYTOKINE, Vol. 12, No. 7 (July, 2000: 986–991)
ation of c-jun/AP-1 by c-jun NH2-terminal kinases (JNKs).9 Therefore, elevated levels of c-jun/AP-1 after H2O2-stimulation can be explained by transcriptional autoregulation of c-jun/AP-1 by activated c-jun/AP-1. In summary, our results suggest that H2O2 and lipid peroxidation act as second messengers in TNF-and IL-1- induced apoptosis in rat MC. Concerning TNF--, but not IL-1-induced apoptosis, c-jun/AP-1 seems to be another relevant second messenger.
MATERIALS AND METHODS Cell culture MC were isolated and characterized as described previously.12 For the experiments, we used MC between the fourth and fifteenth passage. Cell proliferation was determined by incorporation of [3H]-thymidine as previously described.13
Detection of apoptosis MC were grown subconfluently on rat-tail collagen, starved overnight, stimulated and stained with acridine orange (AO)/ethidium bromide (Etbr). Apoptosis was assessed as described elsewhere.14 DNA fragmentation assay was performed by using a DNA fragmentation kit (Boehringer Mannheim, Mannheim, Germany). Figure 6. Detection of c-jun expression by SDS-PAGE and immunoblot analysis.
Reverse transcription polymerase chain reaction (RT-PCR)
(A) Incubation with TNF- (100 ng/ml) increased c-jun expression in MC 2.5-fold as compared to untreated cells. (B) c-jun expression was not affected by IL-1 (50 ng/ml). Thirty micrograms of cell lysate protein per lane were separated on a 12% SDS polyacrylamide Laemmli minigel16 and directly transferred to polyvinyl membranes (Serva/Boehringer, Ingelheim, Heidelberg, Germany) in a minielectrotransfer unit (Hoefer, Freiburg, Germany) at 300 mA for 3 h in 192 mM glycine, 25 mM Tris, and 20% methanol. A monoclonal mouse IgG2a antibody against c-jun (0.25 g/ml) (Transduction Lab, Lexington, USA) and a 1:1000 diluted rabbit anti-mouse IgG1 alkaline phosphatase conjugate (Santa Cruz, Heidelberg, Germany) were used. The blots were stained with BCIP and NBT (Sigma) according to the manufacturer’s instructions. The density of protein bands was determined with a Kodak Camera Imaging System (Kodak, Rochester, USA).
RNA was isolated15 and subjected to RT-PCR with specific primers for rat TNF-R1 or -actin. PCR-products were consistent with the predicted sizes. Cloning (TACloning-Kit, Invitrogen, San Diego, CA, USA) and sequencing of the PCR-products (Replicon, Berlin, Germany) confirmed the identity of the amplified target genes.
cells from apoptostis, indicating either that extracellular catalase did not eliminate intracellular H2O2 sufficiently, or that other, H2O2-independent signal transduction pathways are involved. Catalase treatment also suppressed TNF- and IL-1 stimulated lipid peroxidation, suggesting that lipid peroxides play a role in the following signal transduction cascade. It has been reported that c-jun/AP-1 is a mediator for H2O2 initiated apoptosis in MC.8 We found increased c-jun expression in TNF-, but not in IL-1 stimulated MC. Other reports showed that the cellular redox status influences the transcription of c-jun and that H2O2 induces apoptosis in MC via phosphoryl-
Biochemistry ROS were detected by reduction of NBT (Sigma).4 MDA was used as a marker for lipid peroxidation (Bioxytech-LPO-586-kit: OXIS-International, Portland, Oregon, USA). Western blots were performed according to standard protocol. Protein concentration was determined using bicinchoninic acid.
Chemicals and reagents Recombinant rat cytokines were purchased from Pharma-Biotechnologie-Hannover (Hannover, Germany). All chemicals were purchased from Merck (Darmstadt, Germany).
Acknowledgements This study was supported by the Novartis-Stiftung fu¨ r Therapeutische Forschung and presented as a poster at the 31st annual meeting of the American
TNF- and IL- induce apoptosis in rat mesangial cells / 991
Society of Nephrology (ASN), October 25–28, 1998 in Philadelphia, PA, USA. Torsten Bo¨ hler is a recipient of a research fellowship of the Novartis-Stiftung fu¨ r Therapeutische Forschung.
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