SEARCH

SEARCH BY CITATION

Keywords:

  • apoptosis;
  • Bcl-2;
  • caspases;
  • familial haemophagocytic lymphohistiocytosis;
  • Fas/APO-1/CD95

Abstract

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. References

Familial haemophagocytic lymphohistiocytosis (FHL) is a rare and uniformly fatal disorder of early childhood characterized by fever, hepatosplenomegaly, cytopenia and widespread infiltration of vital organs by activated lymphocytes and macrophages. In order to test whether the massive accumulation of immune cells in these patients is associated with a perturbation of apoptosis, lymphocytes were isolated from eight patients and subjected to the chemotherapeutic agent etoposide or agonistic anti-Fas monoclonal antibodies in vitro. These stimuli elicited a normal apoptotic response in FHL patient cells when compared to healthy controls, as determined by phosphatidylserine exposure, DNA fragmentation, in vitro cleavage of the caspase-3-like substrate aspartate-glutamate-valine-aspartate-7-amino-4-methyl-coumarin (DEVD-AMC) and proteolysis of the anti-apoptotic protein Bcl-2. In addition, the degree of constitutive and Fas-triggered apoptosis in freshly isolated neutrophils was monitored in three children, with similar results. These studies indicate that immune cells derived from FHL patients are not inherently resistant to apoptosis induction. Specifically, etoposide-induced and Fas-triggered activation of intracellular caspases appears to remain intact in these individuals. However, the degree of spontaneous activation of caspase-3-like enzymes in activated lymphocytes was attenuated in three of the four patients tested prior to initiation of therapy, suggesting a possible biological deficiency in these individuals.

Familial haemophagocytic lymphohistiocytosis (FHL) is a rapidly fatal disease of infancy and early childhood (Farquhar & Claireaux, 1952; Janka, 1983; Henter et al, 1998). The pathogenesis of this disease remains enigmatic, but an autosomal recessive pattern of inheritance has been suggested (Gencik et al, 1984). Common clinical signs of FHL include fever, hepatosplenomegaly, cytopenia, hypertriglyceridaemia and coagulopathy (Henter et al, 1998), frequently in conjunction with neurological symptoms such as irritability and seizures (Henter & Nennesmo, 1997; Haddad et al, 1997). The most prominent histopathological feature is the multivisceral accumulation of activated lymphocytes and non-Langherhans histiocytes with active phagocytosis mainly of erythrocytes (Janka, 1983; Henter et al, 1998). Immune system derangement is a common feature, and low or absent cellular cytotoxity in these individuals (Aricòet al, 1996; Egeler et al, 1996), as well as increased levels of various pro-inflammatory cytokines (Komp et al, 1989; Henter et al, 1991b; Imashuku et al, 1991) have been reported.

Considering the importance of apoptosis (programmed cell death) for the maintenance of homeostasis within the immune system, we hypothesized that the accumulation of cells in FHL patients may be due to the defective triggering of apoptosis in these patients. We were particularily interested in the role of apoptosis mediated by the cell surface molecule Fas (APO-1/CD95), a key regulator of apoptosis in the immune system (Nagata, 1997; Ashkenazi & Dixit, 1998). A novel disorder in children, termed autoimmune lymphoproliferative syndrome (ALPS), was recently documented and found to be associated with inherited mutations in the Fas gene (Rieux-Laucat et al, 1995; Fisher et al, 1995), and these findings have therefore provided additional impetus for the present investigations. Our study was further prompted by our previous findings of elevated circulating levels of interleukin (IL)-1 receptor antagonist, but not IL-1 agonists in FHL patients (Henter et al, 1996), suggesting a possible defect of IL-1β converting enzyme (ICE), a member of the caspase family (Zhivotovsky et al, 1997; Thornberry & Lazebnik, 1998). The aim of the present study was thus to perform a functional assessment of apoptosis pathways converging on the activation of caspases in in vitro activated lymphocytes and freshly isolated neutrophils obtained from FHL patients.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. References

Clinical and laboratory features of FHL patients

Eight patients were studied, five male and three female, with a mean age at diagnosis of 6.38 months (range 1–17 months). Samples obtained prior to cytotoxic therapy were available in four cases. Clinical and laboratory data of the patients are presented in 1Table I. All patients fulfilled the diagnostic criteria for haemophagocytic lymphohistiocytosis issued by the Histiocyte Society (Henter et al, 1991a), although one patient suffered from splenic torsion in the neonatal period and splenomegaly therefore could not be evaluated. None of the subjects presented evidence for malignancy-associated haemophagocytic syndrome (Henter et al, 1991a; Janka et al, 1998).

Table 1. Table I. Clinical and laboratory features of the eight FHL patients. Abbreviations: ATG, antithymocyte globulin; Cs, corticosteroids; CSA, cyclosporin A; VP, etoposide; n.d. not determined; h, hypotonicity; i, irritability; o, opisthotonus; s, seizures; t, tiredness.* Affecting two or more of three lineages in the peripheral blood (haemoglobin <90 g/l; platelets <100 × 109/l; neutrophils <1.0 × 109/l).† Fasting triglycerides geqslant R: gt-or-equal, slanted2.0 mmol/l or geqslant R: gt-or-equal, slanted3 SD of the normal value for age.‡ Fibrinogen leqslant R: less-than-or-eq, slant1.5 g/l or leqslant R: less-than-or-eq, slant3 SD.§ Splenectomy performed due to splenic torsion.Thumbnail image of

Reagents

Agonistic anti-Fas monoclonal antibodies (clone CH-11) were purchased from Medical & Biological Laboratories, Ltd (Nagoya, Japan). Etoposide (Vepesid, VP-16) was from Bristol-Myers Squibb (Bromma, Sweden) and staurosporine was from Calbiochem (La Jolla, Calif., U.S.A.). The fluorogenic peptide DEVD-AMC was obtained from Peptide Institute Inc. (Osaka, Japan) and the peptide inhibitor zVAD-cmk (benzyloxycarbonyl-valine-alanine-aspartate-chloromethylketone) was from Enzyme Systems Products (Dublin, Calif., U.S.A.). Human recombinant interleukin-2 (IL-2) was purchased from Boerhinger Mannheim (Mannheim, Germany) and phytohaemagglutinin (PHA) was from Life Technologies (Paisley, Scotland).

Isolation and culture of lymphocytes

Peripheral blood mononuclear cells were isolated from heparinized blood obtained from healthy adult donors or affected patients by density gradient centrifugation (Lymphoprep, Nycomed Pharma AS, Oslo, Norway) according to standard protocols. After separation, cells were cultured in RPMI-1640 medium (SIGMA) supplemented with 10% heat-inactivated fetal bovine serum, 2 mm glutamine, 100 U/ml penicillin and 100 mg/ml streptomycin (Life Technologies). Cells were activated for 3 d with PHA (used at a final concentration of 0.5%) and maintained in recombinant IL-2 (50 IU/ml) for an additional 6 d prior to analysis. IL-2 medium was replenished every 48 or 72 h and assessment of cell viability was performed by trypan blue exclusion. For apoptosis induction experiments, cells were seeded at 1 × 106 cells/ml in IL-2-containing medium in 12-well culture plates. Healthy adult blood donors served as controls, since previous studies have shown no significant differences between adults and children in terms of Fas-mediated apoptotic responses (Bettinardi et al, 1997).

Isolation of neutrophils

Neutrophils were isolated from patient and donor blood by a method of dextran sedimentation and density gradient centrifugation as previously described (Fadeel et al, 1998). Freshly isolated cells were cultured at 1 × 106 cells/ml for up to 24 h in RPMI 1640 medium as above. For the purpose of morphological assessment following apoptosis induction, haematoxylin/eosin-stained cytocentrifuge preparations were prepared and scored for changes characteristic of apoptosis (diminution in cell volume, increased cytoplasmic staining, chromatin condensation).

Western blot

Immunoblot analysis of protein cleavage was performed as described previously (Samali et al, 1998). Briefly, cell lysates were electrophoresed on 12% SDS-PAGE gels under non-reducing conditions and electroblotted onto nitrocellulose membranes. Membranes were blocked in 1% bovine serum albumin and 5% non-fat dried milk and incubated with antibodies specific for Bcl-2 (1:100; DAKO, Glostrup, Denmark) and then incubated with horse radish peroxidase-conjugated goat anti-mouse Ig antibody (1:10000; Pierce, Rockford, Ill., U.S.A.). Membranes were developed using the ECL detection system (Amersham Corp., Buckinghamshire, U.K.) according to the manufacturer's instructions.

In vitro fluorogenic caspase assay

Cleavage of the fluorogenic peptide substrate DEVD-AMC was estimated in a fluorometric assay modified from Nicholson et al (1995). Briefly, lymphocytes and neutrophils were pelleted and frozen on microtitre plates at 0.5 × 106 and 1 × 106 cells per 25 μl, respectively. Substrate (50 μm) was dissolved in a standard reaction buffer (100 mm HEPES, 10% sucrose, 5 mm dithiothreitol and 0.1% CHAPS, pH 7.25) and 50 μl was added to each well. Enzyme-catalysed release of AMC was measured every 70 s for a 30 min period in a Fluoroscan II plate reader (Labsystems, Stockholm, Sweden) using 355 nm excitation and 460 nm emission wavelengths. Fluorescence units were converted to pmol of AMC using a standard curve generated with free AMC. Data from duplicate samples were analysed by linear regression.

Phosphatidylserine (PS) exposure

PS exposure was measured by the binding of annexin V using the protocol outlined in the annexin V-FITC apoptosis detection kit (Oncogene Research Products, Cambridge, Mass., U.S.A.). Cells were stained with propidium iodide (100 μg/ml) prior to analysis with a FACScan flow cytometer (Becton Dickinson, San Jose, Calif., U.S.A.) equipped with a 488 nm argon laser. Ten thousand events were collected and analysed using the CellQuest software (Becton Dickinson). Low-fluorescence debris was gated out prior to analysis. Data are presented as dot plots showing the change in mean fluorescence intensity of annexin V-FITC v propidium iodide.

DNA fragmentation assay

DNA from lymphocytes was processed as previously described (Fadeel et al, 1998). Briefly, 1.5 × 106 cells were washed and resuspended in sample buffer containing RNase (10 mg/ml) for 20 min. Samples were then loaded onto a 1.8% agarose gel with a digestion gel containing 0.8% Ultrapure agarose (Life Technologies, Paisley, Scotland), 2% SDS and 0.6 mg/ml proteinase K at the upper end. The gel was run at 20 V overnight and electrophoresis was then continued for 3 h at 90 V to separate the DNA fragments. The gel was subsequently stained with ethidium bromide and visualized under 305 nm UV illumination.

RESULTS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. References

Intact constitutive and Fas-triggered apoptosis in freshly isolated neutrophils

Neutrophils readily undergo apoptosis in vitro, and we and others have recently shown that crosslinking of the Fas receptor will markedly accelerate this process (Iwai et al, 1994; Fadeel et al, 1998). Following isolation, cells were cultured in the presence or absence of anti-Fas monoclonal antibodies (clone CH-11; 250 ng/ml), and apoptosis was estimated by the degree of phosphatidylserine (PS) exposure and morphological changes. Since several of the FHL patients enrolled in the present study were neutropenic (Table I), these analyses could be performed in only a subset of patients. As shown in Figs 1 and 2 and 2Table II, freshly isolated neutrophils from FHL patients succumbed to spontaneous and Fas-mediated apoptotic death to a similar extent as the cells from healthy controls. In addition, the level of caspase-3-like activity was determined by in vitro cleavage of the fluorogenic peptide substrate DEVD-AMC. Fig 3 depicts the maximum linear rate of AMC release at 12 h in the presence or absence of Fas stimulation. A similar two-fold induction of caspase-3-like activity was evident in all cases upon ligation of the Fas molecule.

image

Figure 1. . Analysis of spontaneous and Fas-triggered apoptosis in neutrophils obtained from FHL patients. Representative dotplots depicting the percentage of phosphatidylserine (PS) exposure in patient 1 as measured by annexin V labelling in untreated (upper panel) and Fas-treated (250 ng/ml; lower panel) samples after incubation for 12 h. Comprehensive data are presented in 2Table II.

Download figure to PowerPoint

image

Figure 2. . Analysis of spontaneous and Fas-triggered apoptosis in neutrophils obtained from FHL patients. Typical morphological changes, including nuclear pyknosis and cell shrinkage, in cytospin preparations of FHL neutrophils (patient 2) either freshly isolated (upper panel) or maintained for 12 h in culture (lower panel). Original magnification ×100.

Download figure to PowerPoint

Table 2. Table II. Phosphatidylserine (PS) exposure in neutrophils and lymphocytes from FHL patients. * Freshly isolated neutrophils were maintained in culture for 12 h in the absence or presence of anti-Fas mAb (250 ng/ml), and PS exposure was assessed by flow cytometric analysis of annexin V binding. Necrotic cells were gated out prior to analysis and the percentage PS positive (apoptotic) cells are indicated. The mean and SD of data obtained in five healthy adult controls are also shown.† IL-2-activated lymphocytes were treated with either anti-Fas mAb (250 ng/ml) or etoposide (6 μg/ml) for 12 h and PS exposure was determined as above. The mean and SD of data obtained in six healthy adult controls are also indicated.‡ Data shown are results obtained before and after initiation of chemotherapy.Thumbnail image of
image

Figure 3. . Analysis of spontaneous and Fas-triggered apoptosis in neutrophils obtained from FHL patients. Caspase-3-like activity in FHL neutrophils as determined by the cleavage of the specific peptide substrate DEVD-AMC (50 μm). Cells were incubated for 12 h in the absence (empty bars) or presence (filled bars) of anti-Fas antibodies (250 ng/ml) and the release of AMC was monitored in a continuous fluorometric assay. The maximum linear rate of AMC release (pmol/min) was estimated by linear regression (r2 > 0.99).

Download figure to PowerPoint

Normal levels of etoposide-induced and Fas-mediated apoptosis of IL-2-activated lymphocytes

We next investigated the apoptotic response of activated lymphocytes from FHL patients. Cells were primed in vitro with PHA and recombinant IL-2, and treated with the chemotherapeutic agent etoposide or anti-Fas monoclonal antibodies. Apoptosis was evaluated by the egress of PS and the rate of caspase-3-like activity in vitro. In addition, DNA fragmentation was assessed in one patient. As shown in Figs 456 and 2Table II, experiments performed in FHL lymphocytes yielded essentially normal findings compared to healthy controls. Patient 1 provided a unique opportunity to study an individual before initiation of treatment and during remission. No significant differences in the degree of PS exposure were observed under these conditions (Table II), thus conferring some validity to our experiments performed in patients undergoing therapy.

image

Figure 4. . Analysis of apoptosis induction in IL-2-activated lymphocytes from FHL patients. Representative dotplots depicting PS externalization in cells obtained from patient 4 maintained either in medium alone (upper panel), or treated with anti-Fas antibodies (250 ng/ml; middle panel) or etoposide (6 μg/ml; lower panel) for 12 h. For comprehensive data on patients and controls, see 2Table II.

Download figure to PowerPoint

image

Figure 5. . Analysis of apoptosis induction in IL-2-activated lymphocytes from FHL patients. Agarose gel electrophoresis of DNA showing the formation of oligonucleosomal DNA fragments in control and Fas-treated FHL lymphocytes (patient 1). A molecular weight marker (bp) is included.

Download figure to PowerPoint

image

Figure 6. . Analysis of apoptosis induction in IL-2-activated lymphocytes from FHL patients. Caspase-3-like activity in patient and control lymphocytes, as determined by the cleavage of the specific peptide substrate DEVD-AMC (50 μm). Experimental conditions as above (untreated, empty bars; Fas-treated, filled bars; etoposide-treated, cross-hatched bars). The maximum linear rate of AMC release (pmol/min) was estimated by linear regression (r2 > 0.99).

Download figure to PowerPoint

Decreased levels of spontaneous caspase activation in a subset of FHL patients

Déas et al (1998) reported recently that partial processing of procaspases 3 and 7, as determined by immunoblot analysis, may occur in normal activated T lymphocytes in the absence of specific apoptotic triggers. Accordingly, we consistently detected cleavage of the fluorogenic substrate DEVD-AMC in vitro in control samples from healthy individuals. However, a substantial decrease in the level of spontaneous caspase-3-like activity was observed in three out of four FHL children studied prior to initiation of cytotoxic therapy (patients 4, 6 and 8), despite similar levels of Fas- and etoposide-triggered caspase activation in cells from these patients when compared to controls (Fig 6). The data concerning patient 1 were obtained after initiation of cytotoxic therapy, since sufficient material was not available from this patient to assess DEVD-AMC cleavage prior to treatment.

Caspase-dependent cleavage of Bcl-2 in lymphocytes from normal individuals and FHL patients

To determine the level of expression of Bcl-2, an important arbiter of cell survival (Adams & Cory, 1998), activated lymphocytes from FHL patients and healthy controls were triggered to undergo apoptosis by etoposide (6 μg/ml), anti-Fas antibodies (250 ng/ml) or staurosporine (2 μm), a protein kinase C inhibitor. Bcl-2 protein levels were subsequently examined by Western blot using antibodies directed against amino acids 41–54 of human Bcl-2. As seen in Fig 77A, the level of Bcl-2 in untreated lymphocytes was similar in control and patient samples. Interestingly, a second immunoreactive band of approximately 23 kD appeared in both patient and control cells upon apoptotic triggering, indicating that Bcl-2 was proteolytically degraded during apoptosis in these cells. To gauge the role of caspases in the cleavage of Bcl-2, lymphocytes from healthy controls were incubated in the presence or absence of the general caspase inhibitor zVAD-cmk (20 μm) prior to the addition of anti-Fas antibodies (250 ng/ml) or etoposide (6 μg/ml) for 6 h. As shown in Fig 77B, preincubation with zVAD-cmk completely blocked cleavage of Bcl-2 in response to both apoptotic stimuli. These data therefore concur with recent reports on caspase-dependent cleavage of Bcl-2 in tumour cell lines triggered to undergo apoptosis (Cheng et al, 1997; Grandgirard et al, 1998).

image

Figure 7. . Cleavage of Bcl-2 in apoptotic lymphocytes. (A) Cell lysates were electrophoresed and immunoblotted with antibodies specific for the 26 kD Bcl-2 protein. In patient and control cells treated with anti-Fas antibodies (250 ng/ml), etoposide (6 μg/ml) or staurosporine (2 μm), a 23 kD immunoreactive band was evident at 12 h. Due to the scarcity of material obtained from patients 6 and 7, staurosporine treatment was not tested in cells from these individuals. (B) Preincubation of lymphocytes from healthy controls with the broad-spectrum caspase inhibitor zVAD-cmk (20 μm) completely abrogated Fas- and etoposide-induced Bcl-2 cleavage, as evidenced by the disappearance of the minor band. Similar results were obtained in four separate experiments.

Download figure to PowerPoint

DISCUSSION

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. References

FHL is a non-malignant childhood disorder with a progressive course and invariably fatal outcome in the absence of bone marrow transplantation. The present studies were undertaken in an attempt to characterize, on a functional level, specific apoptotic pathways which may be perturbed in these patients. Importantly, we were able to analyse four patients before initiation of etoposide treatment and the experimental outcome in these cases was thus unmitigated by cytotoxic therapeutic intervention. Lymphocyte death was triggered by the chemotherapeutic agent etoposide, or agonistic anti-Fas monoclonal antibodies, whereas freshly isolated neutrophils were treated either with anti-Fas antibodies or allowed to undergo spontaneous apoptosis upon ageing in culture. Apoptosis instigated by these different stimuli was found to proceed normally in both cell types. Furthermore, no significant differences were observed in etoposide-mediated and Fas-triggered apoptosis between untreated patients and patients in remission. These results demonstrate that lymphocytes and neutrophils from FHL patients express a functional Fas molecule on their surface which is capable of propagating an apoptotic signal within the cell. Moreover, it appears that FHL differs from the recently characterized condition known as ALPS (or Canale-Smith syndrome), in which autoimmunity and lymphoproliferation was found to be associated with Fas mutations and defective Fas-mediated apoptosis (Rieux-Laucat et al, 1995; Fisher et al, 1995; Drappa et al, 1996).

The aspartate-specific intracellular proteases known as caspases cleave a discrete subset of cellular substrates, including proteins involved in cytoskeleton regulation, genome function, cell-cycle progression and DNA fragmentation, leading inexorably to the systematic and orderly disassembly of the dying cell (Zhivotovsky et al, 1997; Thornberry & Lazebnik, 1998). Recent studies have shown that the anti-apoptotic molecule Bcl-2 is cleaved by caspases in tumour cell lines triggered to undergo apoptosis in response to Fas stimulation, growth factor withdrawal and alphavirus infection (Cheng et al, 1997; Grandgirard et al, 1998), and we have recently procured evidence for Bcl-2 cleavage following etoposide treatment of myeloid leukaemic cells (Fadeel et al, 1999). In the present study we demonstrated that Bcl-2 was proteolytically degraded in normal activated T lymphocytes in response to diverse stimuli such as anti-Fas antibodies, etoposide and staurosporine. This event was blocked by the general caspase inhibitor zVAD-cmk, indicating that cleavage was caspase-dependent. Lymphocytes obtained from FHL patients displayed similar basal levels of Bcl-2 when compared to healthy controls. Furthermore, a comparable pattern of Bcl-2 cleavage was observed in patient and control cells upon apoptotic triggering, demonstrating that the triggering of apoptosis in cells derived from FHL patients culminates in the caspase-dependent cleavage of appropriate cellular substrates.

Normal T lymphocytes are known to undergo spontaneous or growth factor deprivation-dependent apoptosis in vitro (Brunetti et al, 1995), and Lemaire et al (1998) showed recently that spontaneous apoptosis of B lymphocytes was accompanied by activation of caspase-3-like enzymes. Moreover, Déas et al (1998) have presented evidence for the partial processing of procaspases 3 and 7 in activated, but not in resting, human T lymphocytes. Interestingly, in the present study, the degree of spontaneous activation of caspase-3-like enzymes in IL-2-stimulated lymphocytes, as evidenced by the in vitro cleavage of the fluorogenic substrate DEVD-AMC, was markedly reduced in three of the four FHL patients sampled prior to initiation of etoposide therapy. Previous reports have described decreased proliferative responses to mitogenic stimulation in FHL patients (Ladisch et al, 1978; Egeler et al, 1996). Whether this may influence the subsequent degree of spontaneous caspase activation upon cell culture is presently unknown. A critical role of the cytokine milieu for the sensitivity of lymphocytes to apoptosis induction has previously been suggested in human immunodeficiency virus-infected persons (Clerici et al, 1994). Therefore it may prove useful to evaluate the dysregulated cytokine pattern in FHL patients in light of these considerations.

The remarkable therapeutic efficacy of etoposide in FHL patients (Henter et al, 1997), coupled with the present findings indicating that cells obtained from these patients are susceptible to apoptosis triggering in vitro, suggests that cells from these individuals are not innately resistant to apoptosis. Rather, the physiological trigger of apoptosis in vivo may be defective or absent. An attractive candidate for such a trigger is the Fas ligand. Indeed, a recent report from Hasegawa et al (1998) indicated that soluble Fas ligand (sFasL) was elevated in sera from FHL patients, although the interpretation of these data remains somewhat ambiguous. The authors suggested that sFasL may trigger apoptosis of Fas-bearing cells, yet it is presently unclear how an increased frequency of apoptosis relates to the clinical features of these patients. In contrast, recent studies have demonstrated that sFasL preferentially inhibits apoptosis mediated by the membrane-anchored form of the molecule (Suda et al, 1997; Schneider et al, 1998). Of considerable interest in this context is the fact that in FHL patients both NK activity and cytotoxic T lymphocyte activity are low or absent, despite normal numbers of cytotoxic T lymphocytes and NK cells (Aricòet al, 1996; Egeler et al, 1996). Again, these observations would appear to be compatible with the absence of Fas ligand-mediated cell death either due to defective function or lack of expression. Several cytotoxic drugs, including etoposide, were recently shown to up-regulate Fas ligand in leukaemic cells (Friesen et al, 1996), and the possibility that etoposide treatment of FHL patients may up-regulate Fas ligand in vivo and thereby restore putative defects in apoptosis triggering warrants further investigation. Other potential candidate molecules which merit further attention are components of the perforin–granzyme system, the second major effector pathway of cytotoxic T lymphocytes (Liu et al, 1996), as well as several novel Fas-related molecules including DR (death receptor) 3, DR4 and DR5, the physiological role of which has not been fully elucidated (Ashkenazi & Dixit, 1998). Yet another potential candidate is CTLA-4 (cytotoxic T lymphocyte associated-4), a cell surface receptor that mediates Fas-independent apoptosis of T lymphocytes and is a vital negative regulator of T- lymphocyte activation (Scheipers & Reiser, 1998). CTLA-4-deficient mice have been found to develop profound lymphoproliferative disease with multi-organ lymphocytic infiltration (Tivol et al, 1995), i.e. features reminiscent of those seen in FHL patients.

In conclusion, Fas-mediated and etoposide-induced activation of caspases in immune cells derived from FHL patients appears to remain intact. However, in three of the four children studied prior to initiation of cytotoxic therapy the spontaneous activation of caspase-3-like enzymes in activated lymphocytes was attenuated. The mechanism underlying these findings is presently not well understood, yet one may speculate that such a defect could be related to the massive accumulation of lymphocytes evidenced in these individuals. The present study thus represents an attempt to characterize at the molecular level the aberrations underlying FHL, and as such may serve to direct future investigations of the pathogenesis of this fatal childhood disease.

Acknowledgements

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. References

This work was supported by the Swedish Medical Research Foundation (MFR) and the Children's Cancer Foundation of Sweden. We are indebted to Drs Kirsi Jahnukainen (Department of Paediatrics, Turku University Hospital, Finland), Inga-Lill Eliasson (Department of Paediatric Oncology, Regionsjukhuset, Trondheim, Norway), Ingebjörg Storm-Mathisen (Department of Paediatrics, Rikshospitalet, Oslo, Norway), Ana Sastre Urgelles (Department of Haematology and Oncology, University Hospital ‘La Paz’, Madrid, Spain), Torben Ek (Department of Paediatrics, Sahlgrenska University Hospital/Eastern Hospital, Göteborg, Sweden), Wilfred Hurkx and Jos Bökkerink (Department of Paediatric Oncology, University Hospital, Nijmegen, The Netherlands) and Milen Minkov (Department of Paediatric Oncology, St Anna Children's Hospital, Vienna, Austria) for securing patient specimens. We also thank our colleagues Boris Zhivotovsky and David Burgess (Institute of Environmental Medicine, Karolinska Institutet) for illuminating discussions.

References

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. References
  • 1
    Adams, J.M. & Cory, S. (1998) The Bcl-2 protein family: arbiters of cell survival. Science, 281, 132213226.
  • 2
    Aricò, M., Janka, G., Fischer, A., Henter, J.-I., Blanche, S., Elinder, G., Martinetti, M. & Rusca, M.P. (1996) Hemophagocytic lymphohistiocytosis: report of 122 children from the international registry. Leukemia, 10, 197203.
  • 3
    Ashkenazi, A. & Dixit, V.M. (1998) Death receptors: signaling and modulation. Science, 281, 13051308.
  • 4
    Bettinardi, A., Brugnoni, D., Quiròs-Roldan, E., Malagoli, A., La Grutta, S., Correra, A. & Notarangelo, L.D. (1997) Missense mutations in the Fas gene resulting in autoimmune lymphoproliferative syndrome: a molecular and immunological analysis. Blood, 89, 902909.
  • 5
    Brunetti, M., Martelli, N., Colasante, A., Piantelli, M., Musiani, P. & Aiello, F.B. (1995) Spontaneous and glucocorticoid-induced apoptosis in human mature T lymphocytes. Blood, 86, 41994205.
  • 6
    Cheng, E.H-Y., Kirsch, D.G., Clem, R.J., Ravi, R., Kastan, M.B., Bedi, A., Ueno, K., & Hardwick, J.M. (1997) Conversion of bcl-2 to a bax-like death effector by caspases. Science, 278, 19661968.
  • 7
    Clerici, M., Sarin, A., Coffman, R.L., Wynn, T.A., Blatt, S.P., Hendrix, C.W., Wolf, S.F., Shearer, G.M. & Henkart, P.A. (1994) Type 1/type 2 cytokine modulation of T-cell programmed cell death as a model for human immunodeficiency virus pathogenesis. Proceedings of the National Academy of Sciences of the United States of America, 91, 1181111815.
  • 8
    Déas, O., Dumont, C., MacFarlane, M., Rouleau, M., Hebib, C., Harper, F., Hirsch, F., Charpentier, B., Cohen, G.M. & Senik, A. (1998) Caspase-independent cell death induced by anti-CD2 or staurosporine in activated human peripheral T lymphocytes. Journal of Immunology, 161, 33753383.
  • 9
    Drappa, J., Vaishnaw, A.K., Sullivan, K.E., Chu, J.-L. & Elkon, K.B. (1996) Fas gene mutations in the Canale-Smith syndrome, an inherited lymphoproliferative disorder associated with autoimmunity. New England Journal of Medicine, 335, 16431649.
  • 10
    Egeler, R.M., Shapiro, R., Loechelt, B. & Filipovich, A. (1996) Characteristic immune abnormalities in hemophagocytic lymphohistiocytosis. Journal of Pediatric Hematology/Oncology, 18, 340345.
  • 11
    Fadeel, B., Åhlin, A., Henter, J.-I., Orrenius, S. & Hampton, M.B. (1998) Involvement of caspases in neutrophil apoptosis: regulation by reactive oxygen species. Blood, 92, 48084818.
  • 12
    Fadeel, B., Hassan, Z., Hellström-Lindberg, E., Henter, J.-I., Orrenius, S. & Zhivotovsky, B. (1999) Cleavage of Bcl-2 is an early event in chemotherapy-induced apoptosis of human myeloid leukemia cells. Leukemia, 13, 719728.
  • 13
    Farquhar, J.W. & Claireaux, A.E. (1952) Familial hemophagocytic reticulosis. Archives of Disease in Childhood, 27, 519525.
  • 14
    Fisher, G.H., Rosenberg, F.J., Straus, S.E., Dale, J.K., Middleton, L.A., Lin, A.Y., Strober, W., Lenardo, M.J. & Puck, J.M. (1995) Dominant interfering Fas gene mutations impair apoptosis in a human autoimmune lymphoproliferative syndrome. Cell, 81, 935946.
  • 15
    Friesen, C., Herr, I., Krammer, P.H. & Debatin, K.-M. (1996) Involvement of the CD95(APO-1/Fas) receptor/ligand system in drug-induced apoptosis in leukaemia cells. Nature Medicine, 2, 574577.
  • 16
    Gencik, A., Signer, E. & Müller, H. (1984) Genetic analysis of familial erythrophagocytic lymphohistiocytosis. European Journal of Pediatrics, 142, 248252.
  • 17
    Grandgirard, D., Studer, E., Monney, L., Belser, T., Fellay, I., Borner, C. & Michel, M.R. (1998) Alphaviruses induce apoptosis in Bcl-2-overexpressing cells: evidence for a caspase-mediated, proteolytic inactivation of Bcl.-2. EMBO Journal, 17, 12681278.
  • 18
    Haddad, E., Sulis, M.-L., Jabado, N., Blanche, S., Fischer, A. & Tardieu, M. (1997) Frequency and severity of central nervous system lesions in hemophagocytic lymphohistiocytosis. Blood, 89, 794800.
  • 19
    Hasegawa, D., Kojima, S., Tatsumi, E., Hayakawa, A., Kosaka, Y., Nakamura, H., Sako, M., Osugi, Y., Nagata, S. & Sano, K. (1998) Elevation of the serum Fas ligand in patients with hemophagocytic syndrome and Diamond-Blackfan anemia. Blood, 91, 27932799.
  • 20
    Henter, J.-I., Andersson, B., Elinder, G., Jakobson, Å., Lübeck, P.-O. & Söder, O. (1996) Elevated circulating levels of interleukin-1 receptor antagonist but not IL-1 agonists in hemophagocytic lymphohistiocytosis. Medical and Pediatric Oncology, 27, 2125.
  • 21
    Henter, J.-I., Aricò, M., Egeler, M., Elinder, G., Favara, B.E., Filipovich, A.H., Gadner, H., Imashuku, S., Janka-Schaub, G., Komp, D., Ladisch, S. & Webb, D. (1997) HLH-94: a treatment protocol for hemophagocytic lymphohistiocytosis. Medical and Pediatric Oncology, 28, 342347.
  • 22
    Henter, J.-I., Aricò, M., Elinder, G., Imashuku, S. & Janka, G. (1998) Familial hemophagocytic lymphohistiocytosis: primary hemophagocytic lymphohistiocytosis. Hematology/Oncology Clinics of North America, 12, 417433.
  • 23
    Henter, J.-I., Elinder, G., Öst, Å. (1991a) Diagnostic guidelines for hemophagocytic lymphohistiocytosis. Seminars in Oncology, 18, 2933.
  • 24
    Henter, J.-I., Elinder, G., Söder, O., Hansson, M., Andersson, B. & Andersson, U. (1991b) Hypercytokinemia in familial hemophagocytic lymphohistiocytosis. Blood, 78, 2918.
  • 25
    Henter, J.-I. & Nennesmo, I. (1997) Neuropathologic findings and neurologic symptoms in twenty-three children with hemophagocytic lymphohistiocytosis. Journal of Pediatrics, 130, 358365.
  • 26
    Imashuku, S., Ikushima, S., Esumi, N., Todo, S. & Saito, M. (1991) Serum levels of interferon-gamma, cytotoxic factor and soluble interleukin-2 receptor in childhood hemophagocytic syndrome. Leukemia and Lymphoma, 3, 287292.
  • 27
    Iwai, K., Miyawaki, T., Takizawa, T., Konno, A., Ohta, K., Yachie, A., Seki, H. & Taniguchi, N. (1994) Differential expression of bcl-2 and susceptibility to anti-Fas-mediated cell death in peripheral blood lymphocytes, monocytes, and neutrophils. Blood, 84, 12011208.
  • 28
    Janka, G., Imashuku, S., Elinder, G., Schneider, M. & Henter, J.-I. (1998) Infection- and malignancy-associated hemophagocytic syndromes: secondary hemophagocytic syndromes. Hematology/Oncology Clinics of North America, 12, 435444.
  • 29
    Janka, G.E. (1983) Familial hemophagocytic lymphohistiocytosis. European Journal of Pediatrics, 140, 221230.
  • 30
    Komp, D.M., McNamara, J. & Buckley, P. (1989) Elevated soluble interleukin-2 receptor in childhood hemophagocytic histiocytic syndromes. Blood, 73, 21282132.
  • 31
    Ladisch, S., Poplack, D.G., Holiman, B. & Blaese, R.M. (1978) Immunodeficiency in familial erythrophagocytic lymphohistiocytosis. Lancet, i, 581583.
  • 32
    Lemaire, C., Andréau, K., Souvannavong, V. & Adam, A. (1998) Inhibition of caspase activity induces a switch from apoptosis to necrosis. FEBS Letters, 425, 266270.
  • 33
    Liu, C.-C., Young, L.H.Y. & Young, J.D.-E. (1996) Lymphocyte-mediated cytolysis and disease. New England Journal of Medicine, 335, 16511658.
  • 34
    Nagata, S. (1997) Apoptosis by death factor. Cell, 88, 355365.
  • 35
    Nicholson, D.W., Ali, A., Thornberry, N.A., Vaillancourt, J.P., Ding, C.K., Gallant, M., Gareau, Y., Griffin, P.R., Labelle, M., Lazebnik, Y.A., Munday, N.A., Raju, S.M., Smulson, M.E., Yamin, T.-T., Yu, V.L. & Miller, D.K. (1995) Identification and inhibition of the ICE/CED-3 protease necessary for mammalian apoptosis. Nature, 376, 3743.
  • 36
    Rieux-Laucat, F., Le Deist, F., Hivroz, C., Roberts, I.A.G., Debatin, K.-M., Fischer, A. & De Villartay, J.P. (1995) Mutations in Fas associated with human lymphoproliferative syndrome. Science, 268, 13471349.
  • 37
    Samali, A., Zhivotovsky, B., Jones, D.P. & Orrenius, S. (1998) Detection of pro-caspase-3 in cytosol and mitochondria of various tissues. FEBS Letters, 431, 167169.
  • 38
    Scheipers, P. & Reiser, H. (1998) Fas-independent death of activated CD4+ T lymphocytes induced by CTLA-4 crosslinking. Proceedings of the National Academy of Sciences of the United States of America, 95, 1008310088.
  • 39
    Schneider, P., Holler, N., Bodmer, J.-L., Hahne, M., Frei, K., Fontana, A. & Tschopp, J. (1998) Conversion of membrane-bound Fas (CD95) ligand to its soluble form is associated with downregulation of its proapoptotic activity and loss of liver toxicity. Journal of Experimental Medicine, 187, 12051213.
  • 40
    Suda, T., Hashimoto, H., Tanaka, M., Ochi, T. & Nagata, S. (1997) Membrane Fas ligand kills human peripheral blood T lymphocytes, and soluble Fas ligand blocks the killing. Journal of Experimental Medicine, 186, 20452050.
  • 41
    Thornberry, N.A. & Lazebnik, Y. (1998) Caspases: enemies within. Science, 281, 13121316.
  • 42
    Tivol, E.A., Borriello, F., Schweitzer, A.N., Lynch, W.P., Bluestone, J.A. & Sharpe, A.H. (1995) Loss of CTLA-4 leads to massive lymphoproliferation and fatal multiorgan tissue destruction, revealing a critical negative regulatory role of CTLA-4. Immunity, 3, 541547.
  • 43
    Zhivotovsky, B., Burgess, D.H., Vanags, D.M. & Orrenius, S. (1997) Involvement of cellular proteolytic machinery in apoptosis. Biochemical and Biophysical Research Communications, 230, 481488.