News Physiol Sci 14: 194-200, 1999;
1548-9213/99 $5.00
News in Physiological Sciences, Vol. 14, No. 5, 194-200,
October 1999
© 1999 Int. Union Physiol. Sci./Am. Physiol. Soc.
Physiology of Receptor-Mediated Lymphocyte Apoptosis
F. Lang,
I. Szabo,
A. Lepple-Wienhues,
D. Siemen and
E. Gulbins
F. Lang, I. Szabo, A. Lepple-Wienhues, D. Siemen, and E. Gulbins are in the Department for Physiology of the University of Tübingen, Gmelinstrasse 5, D-72076 Tübingen, Germany.
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Abstract
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Apoptosis (programmed cell death), a physiological mechanism eliminating abundant and potentially harmful cells, is triggered by a variety of stimuli including activation of distinct receptors. The machinery mediating CD95 receptor-induced apoptosis includes caspases, ceramide, kinases, Ras and Rac, formation of O2–, mitochondrial proteins, inhibition of K+ channels, activation of Cl– channels, and osmolyte release.
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Introduction
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The maintenance of an adequate cell number requires a delicate balance between cell proliferation and cell death. This balance allows the optimal adaptation to ever-changing functional needs. For instance, proper function of the immune system requires rapid increase of lymphocyte number upon infection and their subsequent removal after recovery.
Cell death may be accomplished by at least two distinct mechanisms, necrosis and apoptosis. Necrosis is an a priori pathophysiological mechanism involving cell swelling and disruption of the cell membrane with release of intracellular proteins leading to immune response and subsequent inflammation. Among the hallmarks of apoptosis are cell shrinkage, nuclear condensation, DNA fragmentation, and a rearrangement of the cell membrane with exposure of phosphatidylserine residues at the extracellular surface (10). Eventually, the cell is decomposed into small particles that are readily taken up by macrophages. Usually, no intracellular proteins are released from the apoptotic cell, and thus inflammation is avoided.
Apoptosis serves not only in adjusting the cell number of any given tissue to the respective functional demand but also in accomplishing the removal of cells during embryogenesis and metamorphosis. Moreover, apoptosis allows the elimination of tumor cells, infected cells, and autoreactive lymphocytes (10).
Although apoptosis is a physiological mechanism, both defective and excessive apoptosis may lead to serious disease. Although defective apoptosis may account for the development of tumors and excessive function such as enhanced hormone production or autoimmune disease, excessive apoptosis may result in defective function such as neurodegeneration or immunodeficiency (10).
Important causes of necrosis include energy depletion and physical damage. Apoptosis may be triggered by a wide variety of stimuli including bacterial toxins, radiation, gross increase of extracellular osmolarity, oxidative stress, c-Myc overexpression, growth factor depletion, glucocorticoids, and the stimulation of certain cell membrane receptors, such as the tumor necrosis factor (TNF) receptor or the CD95(Fas/Apo-1) receptor. Tissue injury such as ischemia may trigger apoptosis through upregulation of the respective receptors and ligands. Thus the cells may be removed by apoptosis before necrosis and inflammation thereby avoided (10).
This brief review outlines cellular mechanisms triggered by stimulation of the CD95 receptor in lymphocytes. The CD95 receptor may not only confer apoptosis. Thus not all events triggered by the CD95 receptor necessarily serve apoptosis, and the relevance of each mechanism for apoptosis must be verified. Even though we are still far from understanding the mechanisms eventually leading to apoptotic cell death, considerable progress has been made within the past few years in disclosing the involvement of intracellular signaling molecules as well as ion channels and transporters in the cell membrane. Here we focus on CD95-induced apoptosis of Jurkat T lymphocytes. Special emphasis will be placed upon the role of transport mechanisms at the cell membrane. For more detailed information on other aspects of apoptotic cell death, the reader may consult a recent collection of reviews (10).
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Signaling of the CD95(Fas/Apo-1) receptor
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The CD95 receptor belongs to the TNF/nerve growth factor (NGF) family of receptors. Its ligand, the CD95(Fas/Apo-1) ligand (CD95L), is a member of the TNF family. A short domain in the cytoplasmic tail of the receptor has been identified by mutation experiments to be required for the induction of apoptosis and has thus been labeled the death domain. This domain is associated with several proteins including the Fas-associated death domain protein (FADD/MORT-1) and caspase 8 (FLICE1/ MACH1) (13), a cysteine protease cleaving proteins after aspartic acid (10). This complex has been named the death-inducing signal complex (DISC). Upon formation of the complex, the activation signal is transferred to further proteins including several sphingomyelinases, which cleave sphingomyelin to ceramide. Ceramide has been implied to be involved in the regulation of Jun N-terminal kinase, Raf-kinase, Bad, Src-like tyrosine kinase Lck56, the G proteins Ras and Rac, and p38 kinase, as well as formation of O2– (3, 4, 10, 13). Furthermore, activation of the CD95 receptor or cellular treatment with ceramide stimulates the release of cytochrome c from mitochondria (13). Cytochrome c associates with several cytosolic proteins, resulting in the stimulation of further caspases (10). In the terminal phase, nuclear endonucleases are activated. Apparently, a second pathway activated by the CD95 receptor leads, through regulation of caspases and p21-activated kinase (PAK) kinase, to translocation of phosphatidylserine to the outer surface of the cell membrane, allowing the binding of annexin.
The significance of most signaling elements for the apoptotic outcome has been shown by both pharmacological and genetic knockout experiments. For instance, inhibition of caspase 8 or caspase 3 prevents CD95-triggered death. Likewise, CD95-induced lymphocyte apoptosis is delayed by a deficiency in sphingomyelinase or Lck56 and restored by transfection of the deficient cells with the respective genes (1, 3, 4). Inhibition or genetic knockout of either p38 kinase or Jun kinase alone does not abolish CD95-induced apoptosis, but simultaneous knockout of both kinases prevents CD95-induced apoptosis (1).
Apoptosis is negatively regulated by several proteins, including members of the Bcl2 protein family (10). Thus Bclxl and Bcl2 prevent most forms of apoptosis. On the other hand, further members of the Bcl2 protein family, in particular Bad and Bax, promote cell death. The mode of action of these proteins is not yet clear. In lipid bilayers, Bcl2 forms ion channels, which has been taken as evidence that Bcl2 may function as an ion channel in the mitochondrial membrane (10). However, conclusive evidence is still missing that Bcl2 indeed serves as a channel in vivo. Nevertheless, Bcl2 has been shown to inhibit cytochrome c release from mitochondria.
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Activation of the anion channel ORCC and cytosolic acidosis
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Stimulation of the CD95 receptor is followed by activation of the outwardly rectifying chloride channel (ORCC) within minutes ( Fig. 1). Several lines of evidence demonstrate that activation of the channel is triggered via ceramide and the Src-like kinase Lck56 (15): inhibition of tyrosine kinases by herbimycin A abolishes the activation of ORCC by ceramide or CD95-receptor stimulation. Furthermore, in Lck56-deficient J.CaM 1.6 cells, stimulation of the CD95 receptor fails to activate ORCC, and retransfection of those cells with Lck56 restores the effect of CD95 receptor, triggering on ORCC. Most importantly, the channel can be activated by direct application of purified Lck56 in both excised cell membrane patches and whole cell recordings (8, 15). It is not entirely clear whether Lck56 phosphorylates the channel protein directly or a regulatory protein. Nevertheless, the target for Lck56 must be present in the excised cell membrane patches.
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FIGURE 1. Effect of CD95(Fas/Apo-1)-receptor stimulation on outwardly rectifying chloride channel (ORCC) in Jurkat cells. A: influence of CD95(Fas/Apo-1)-receptor stimulation (+CD95) on cytosolic pH in absence or presence of tyrosine kinase inhibitor herbimycin A (HerbA). B: activation of ORCC by stimulation of the CD95(Fas/Apo-1)-receptor before (a) and after (b) CD95-receptor stimulation. C: HerbA inhibits activation of ORCC by CD95(Fas/Apo-1)-receptor activation (a). However, in presence of HerbA, channel can still be activated by excision and subsequent depolarization (b). D: addition of purified Lck56 activates (b) and subsequent addition of antiphosphotyrosine antibody partially inhibits (c) ORCC. a, Before addition of Lck56; V(pip), pipette potential (15). From Ref. 15 with permission.
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One of the sequelae of ORCC activation is cytosolic acidosis ( Fig. 1), which may be important for the induction of cell death. Inhibition of the channel and/or the Lck56 reduces CD95-induced cytosolic acidosis. Moreover, these maneuvers impede CD95-induced DNA fragmentation and annexin binding, pointing to the functional importance of ORCC activation for apoptosis (15). However, because the available ORCC inhibitors are not very selective for this channel and the gene encoding the channel is still elusive, specific knockout of the channel is not possible and the exact role of ORCC in the apoptotic machinery remains to be shown. Beyond that, cytosolic acidification and apoptosis are not completely abolished by the chloride-channel blockers, pointing to the involvement of additional mechanisms.
Lck56 is activated and stimulates ORCC not only after CD95-receptor triggering but also during osmotic cell swelling (8). Pharmacological or genetic knockout of Lck56 prevents swelling-induced activation of ORCC as well as cell volume regulation. Thus activation of the CD95 receptor mimics the effect of osmotic cell swelling on Lck56 and ORCC.
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Inhibition of K+ channels
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Interestingly, the cells do not immediately shrink after stimulation of the CD95 receptor. Instead, they maintain a constant volume for up to 45 min (6). The failure of the cells to shrink may be at least partially due to inhibition of Kv1.3 ( Fig. 2), the cell volume regulatory K+ channel of Jurkat lymphocytes.
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FIGURE 2. CD95(Fas/Apo-1)-receptor via ceramide tyrosine phosphorylates and inhibits Kv1.3 in Jurkat cells. A: tyrosine phosphorylation of Kv1.3 upon addition of C2-ceramide or C6-ceramide. Respective blots before (0) or 10 min after (10) addition of ceramide. HC is due to reaction of heavy chain of immunoprecipitating antibody with protein G-horseradish peroxidase complex used for enhanced chemiluminescence. Kv1.3 protein is shown in bottom lanes. B: inhibition of Kv1.3 after stimulation of CD95(Fas/Apo-1)-receptor (+CD95). Shown are current traces during voltage pulses of 300 ms from a holding potential of –70 mV down to +70 mV in 20-mV steps (4, 14). From Ref. 14 with permission.
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Similar to the activation of ORCC, the inhibition of Kv1.3 requires Lck56 (4, 14). Moreover, the channel protein is tyrosine phosphorylated upon CD95-receptor stimulation, an effect abolished in cells lacking Lck56 (4, 14). On the other hand, Kv1.3 is activated by cell swelling despite simulation of Lck56, pointing to further mechanisms of channel regulation.
Besides its effect on cell volume, inhibition of Kv1.3 favors depolarization of the cell membrane, which may similarly contribute to apoptosis. Inhibition of K+ channels has been shown to foster (9) and activation of K+ channels to inhibit (7) apoptosis in other cells. Moreover, activation of K+ channels leading to hyperpolarization is one of the key events paralleling cell proliferation (2, 11). However, inhibition of K+ channels has been shown to stimulate or inhibit apoptotic cell death, and the role of K+ channels in apoptotic cell death is still far from understood.
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Release of taurine
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Possibly because of the opposing effects of ORCC activation and Kv1.3 inhibition, cell volume remains virtually constant within the first 45 min of CD95-receptor stimulation. At this time, a marked release of the osmolyte taurine is observed ( Fig. 3). Taurine release and apoptotic cell shrinkage are immediately followed by DNA fragmentation. The taurine release is not caused by nonspecific leakage of the cell membrane, because the amino acid leucine is not released after CD95-receptor triggering. However, other osmolytes are possibly released in parallel. Several chloride channel inhibitors [5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB), glibenclamide, 3'-azido-3'-deoxythymidine (AZT), tamoxifen] failed to inhibit CD95-induced taurine release (6). The differences in the time course of activation and inhibitor sensitivity indicate that the mechanism of CD95-induced taurine release is distinct from ORCC. CD95-induced taurine release is blunted by lowering the temperature to 23°C. If the cells are stimulated by CD95 at 23°C and rewarmed to 37°C after 90 min, taurine is released within a few minutes, indicating that the mechanism accounting for the delay of taurine release is not temperature sensitive. Interestingly, CD95-induced cell shrinkage and DNA fragmentation show temperature sensitivity similar to that of taurine release, i.e., the cells do not undergo cell shrinkage or DNA fragmentation after CD95 receptor stimulation at 23°C. However, rewarming after 90 min leads to rapid cell shrinkage and DNA fragmentation. These observations point to a functional link between osmolyte release, apoptotic cell shrinkage, and DNA fragmentation. It is tempting to speculate that the loss of stabilizing osmolytes participates in the apoptotic degradation of cellular macromolecules (5). However, the lack of specific means to inhibit taurine release precludes experiments to unequivocally establish the functional significance of osmolyte release.
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FIGURE 3. Effect of temperature on CD95-induced DNA fragmentation and taurine release (inset) in Jurkat cells. DNA fragmentation, as reflected by propidium iodide staining in FACS analysis, at different time points in cells treated with Fas(CD95)-antibody at room temperature up to 120 min and then exposed to 37°C (left), treated with Fas(CD95)-antibody at 370C (middle), and left untreated (right). The amount of labeled taurine in the supernatant expressed in % of original cellular taurine content [counts per min (cpm)] is shown in inset. CD95 was activated at room temperature, and cells were subsequently warmed to 37°C as indicated by arrows. CD95-induced taurine release and DNA fragmentation is blunted at room temperature, an effect rapidly reversed by subsequent increase of temperature to 37°C (5). FL2-H, fluorescence intensity. From Ref. 6 with permission. Copyright Springer-Verlag.
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The interaction of cell volume and apoptosis
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As mentioned above, a doubling of osmolarity may by itself trigger apoptosis (12). Evidence has been presented that osmotic cell shrinkage leads to clustering of TNF receptors, which allows ligand-independent activation of the postreceptor events (12). The significance of cell volume in apoptosis has further been documented by the observation that the volume regulatory ability of cells correlates with their ability to resist hypertonicity-induced apoptosis (5). Surprisingly, though, we observed an inhibition of CD95-induced DNA fragmentation after moderate (100 mM NaCl) increase of extracellular osmolarity (5). The inhibition of DNA fragmentation was paralleled by a decrease of O2– formation, as reflected by the concentration of reduced glutathione. As osmotic cell shrinkage inhibits flux of glucose through the pentose shunt pathway (5), the effect on O2– formation could have been caused by decreased availability of NADPH for NADPH oxidase. Along these lines, osmotic cell shrinkage inhibits the oxidative burst in leukocytes (5). Another cell volume-sensitive element of CD95 signaling is Lck56, which is activated during cell swelling (8). It is conceivable, albeit not yet shown, that cell shrinkage inhibits Lck56.
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Conclusions and future perspectives
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The apoptotic machinery involves a wide variety of signaling molecules, including caspases, ceramide, small G proteins, cytochrome c, tyrosine kinases, PI3 kinase, and MAP kinase pathways ( Fig. 4). Most recently, the active participation of transport processes across the cell membrane has been disclosed, such as K+ channels, anion channels, and osmolyte release mechanisms. The involvement of further signaling molecules and transport proteins is more than likely. The inhibition of K+ channels, the intracellular acidosis caused by activation of anion channels, and the cell shrinkage, caused at least in part by osmolyte release, mirror opposite events during cell proliferation, such as K+-channel activation, cytosolic alkalosis, and cell volume increase. Ample further evidence points to a major role of the transport processes for the eventual fate of the cells. Nevertheless, considerable additional information is required to fully understand the complex interplay among signaling, transport, as well as cell volume on the one side and DNA fragmentation, proteolysis, as well as translocation of phosphatidylserine in the cell membrane on the other. We hope that this brief review stimulates further experimental effort to unravel the fascinating machinery leading to apoptotic cell death.
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Acknowledgments
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Work in the authors' laboratory was supported by the Deutsche Forschungsgemeinschaft (nos. La 315/4-3, La 315/6-1, Gu 335/2-2, and Le 792/3-1) and the Bundesministerium für Bildung, Wissenschaft, Forschung und Technologie (Center for Interdisciplinary Clinical Research; no. 01 KS 9602).
Because of editorial restrictions, many pertinent papers had to be removed from the reference list. The authors apologize for not including those excellent papers and are prepared to deliver an extended literature list upon request.
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References
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Introduction
Signaling of the CD95(Fas/Apo-1)...
