Natural human polyreactive IgM induce apoptosis of lymphoid cell lines and human peripheral blood mononuclear cells

Sooryanarayana Varambally1,3, Yaron Bar-Dayan1, Jagadeesh Bayry1, Sébastien Lacroix-Desmazes1, Michael Horn1, Marc Sorel1, Yosefa Bar-Dayan1, Giovina Ruberti2, Michel D. Kazatchkine1 and Srini V. Kaveri1

1 INSERM Unité 430 and Université Pierre et Marie Curie, Institut des Cordeliers, 75006 Paris, France 2 Institute of Cell Biology, National Research Council, Campus ‘A. Buzzati-Traverso’, 00016 Monterotondo Scalo (Rome), Italy 3 Department of Pathology, University of Michigan Medical School, Ann Arbor, MI 48109, USA

Correspondence to: S. V. Kaveri; E-mail: srini.kaveri{at}u430.bhdc.jussieu.fr
Transmitting editor: I. Pecht


    Abstract
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Natural polyreactive IgM autoantibodies, encoded by unmutated germline Ig V genes, represent a major fraction of the normal circulating IgM repertoire. We have previously shown that therapeutic preparation of pooled IgM exerts immunomodulatory effects as assessed by in vitro and in vivo studies. Here, we show that the IgM preparation induces cell death in lymphoblastoid cell lines and in human peripheral blood mononuclear cells. The IgM-induced cell death involved classical features of apoptosis such as nuclear fragmentation and activation of caspases. Treatment of leukemic cells with IgM resulted in the cleavage of poly-(A)DP ribose polymerase, a substrate of caspase, and in a reduction in mitochondrial transmembrane potential during the early period of apoptosis induction. Natural IgM-induced apoptosis was inhibited by soluble Fas molecules and affinity-purified Fas antibodies from pooled IgM preparation induced apoptosis in lymphoblastoid cells, suggesting the involvement of the Fas receptor. Our results suggest a role for normal IgM in controlling cell death and proliferation, and imply a possible therapeutic role for IgM in autoimmune and lymphoproliferative disorders.

Keywords: caspase, Fas, immunomodulation, IVIgM


    Introduction
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Programmed cell death (apoptosis) is a physiological process of selective cell deletion that occurs during embryogenesis, metamorphosis, tissue atrophy and tumor regression (14). Tissue remodeling during embryogenesis and tissue homeostasis during adult life is governed by an equilibrium between proliferation, growth and apoptosis. Any disturbance of this balance leads to pathological situations, such as cellular accumulation in cancer. Apoptosis plays a critical role in maintaining peripheral lymphocyte homeostasis and for minimizing the expansion of autoreactive lymphocytes. It is also a mechanism for the induction of tolerance in autoreactive B and T cells, and, furthermore, excess cells after an immune response are removed by the induction of apoptosis (5). Recent work has clearly demonstrated that deregulation of apoptosis may underlie the pathogenesis of autoimmune diseases by allowing abnormal autoreactive lymphocytes to survive (68).

Natural antibodies (NAb) are those that are present in the serum of healthy individuals in the absence of deliberate immunization with the target antigen (911). The importance of NAb reactive with self-antigens has long been neglected, as tolerance to self was thought to be primarily dependent on the deletion of autoreactive clones during ontogeny. However, it is now well established that autoreactive antibodies and B and T cells are present in healthy individuals, and that autoreactive repertoires are predominantly selected during fetal life (1113). Most NAb are encoded by their genes in germline configuration by B cells, which have not been subjected to somatic hypermutation and affinity maturation. It was initially considered that NAb are preferentially of the IgM isotype, since NAb were first reported in mice where IgM predominates over the other isotypes at the neonatal stage (14). It is now known that NAb belong to the IgM, IgG and IgA isotypes. In man and mouse, natural autoreactive antibodies (NAA) were shown to bind to a broad range of evolutionarily conserved cell-surface, intracellular and circulating antigens (15,16). Although several functions have been postulated for NAA under physiological conditions, the role of natural IgM antibodies remains as yet unclear.

Here, we have examined the hypothesis that natural IgM exerts a role in the maintenance of immune homeostasis by inducing apoptosis of lymphocytes. As a source of natural IgM, we have used a therapeutic preparation of pooled IgM (IVIgM). We have previously shown that the IgM preparation exerts in vitro and in vivo immunomodulatory functions. IVIgM inhibits the activity of a large panel of autoantibodies and protects susceptible strains of animals from autoimmune diseases (1720). In the present study, we have examined the apoptosis-inducing potential of IgM using different human lymphoblastoid cell lines, such as CEM, Jurkat (T cells) and Raji (B cells), as well as human peripheral blood mononuclear cells (PBMC). The apoptosis induced by natural IgM is associated with nucleosomal cleavage of DNA and expression of phosphatidylserine on the cell surface. The natural IgM-induced apoptosis is at least in part dependent on the Fas receptor and on the activation of caspases. Taken together, these observations provide novel insights into the role of normal circulating IgM in controlling proliferation and mortality of lymphocytes, and thereby a role in the maintenance of homeostasis of the immune system. Our results further imply a possible therapeutic role of IgM in autoimmune and lymphoproliferative disorders.


    Methods
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Human Ig
Normal IgM (IVIgM; a kind gift from the Laboratoire Français du Fractionnement et des Biotechnologies, Les Ulis, France) was processed from pooled plasma of over 2500 healthy donors by using a modified Deutsch–Kistler–Nitschmann’s ethanol fractionation procedure (17). The preparation contained >90% pure IgM.

Cell lines
Human lymphoblastoid cell lines CEM (T cells), Jurkat (T cells), Raji (B cells), HuT78 (Fas-sensitive human T cell lymphoma line) and HuT78.B1 (Fas-resistant subclone of the human T cell lymphoma line HuT78) (21) were maintained in RPMI 1640 medium (Gibco, Paisley, UK) supplemented with 1% L-glutamine, 50 IU/ml penicillin, 50 µg/ml streptomycin and 10% FCS. PBMC from young healthy individuals were isolated by centrifugation on a Ficoll density gradient (Pharmacia, Uppsala Sweden), washed and resuspended in RPMI 1640 medium containing 5% FCS.

Recombinant Fas molecules
DAP.3 stable cell lines expressing Fas{Delta}Exo3.4 and Fas{Delta}Exo4 Fas splicing variants were used as a source of soluble Fas proteins as previously described (22). The FasExtra–GST fusion protein, previously described (22), was expressed in BL21 Escherichia coli by induction with 1 mM isopropyl-ß-D-thiogalactopyranoside for 4 h at 37°C and purified from bacterial lysates with glutathione–Sepharose 4B (Pharmacia), according to the manufacturer’s instructions.

Affinity purification of anti-Fas antibodies from IgM
Purified human extracytoplasmic GST–Fas fusion protein was coupled to CNBr-activated Sepharose 4B. IgM (10mg/ml; 0.01 mM) was incubated with 1 ml of GST–Fas affinity matrix overnight on a rocking platform at 4°C. After extensive washing with PBS containing 0.02% NaN3 (PBS-Az), bound IgM was eluted using 0.2 M glycine–HCl, pH 2.8, and immediately neutralized with 2 M Tris and dialyzed against PBS.

Assays for apoptosis
Analysis of DNA fragmentation was performed using a previously described procedure (23). In brief, 2 x 106 cells were harvested by centrifugation, washed with PBS, and lysed in 20 mM Tris, pH 7.4, 0.4 mM EDTA and 0.4% Triton X-100. Cell lysates were centrifuged at 10,000 g for 5 min. DNA fragments in the supernatants were precipitated with 0.5 M NaCl and an equal volume of isopropanol at –70°C. Samples were centrifuged at 10,000 g and the pellets were washed with 70% ethanol. Air-dried pellets were resuspended in 10–20 µl of TE (10 mM Tris–HCl, pH 7.4 and 1 mM EDTA) containing 0.1 mg/ml RNase and incubated at 37°C for 30 min. DNA samples from equivalent numbers of cells were then analyzed in 0.8% agarose minigels.

Expression of phosphatidylserines was determined by staining the cells with Annexin-V–FITC (2.5 µg/ml; Bender-Medsystems, Biowhittaker, Verviers, France) for 30 min on ice. As negative control, non-treated or HSA-treated (Biotrans fusion, Paris, France) cells were used. Cells were washed with PBS-Az and resuspended in PBS-Az before adding propidium iodide (PI; Sigma, St Louis, MO) (50 µg/ml) to each sample (24). Samples were analyzed for green fluorescence (Annexin-V labeling) and for red fluorescence (PI uptake) using a FACScan flow cytometer (Becton Dickinson, Mountain View, CA).

Western blot analysis for poly-(A)DP-ribose polymerase (PARP)
Cells (106 cells) were treated for various times with IgM. Cell extracts were analyzed on 10% SDS–PAGE. Separated proteins were transferred onto PVDF membranes, blocked for 12 h in PBS containing 5% BSA and 0.1% Tween 20, and incubated for 2 h with rabbit anti-PARP antibody (Santa Cruz Biotechnology, Santa Cruz, CA). Membranes were washed 5 times each with PBS containing 0.1% Tween 20 (PBS-T) and PBS, and incubated with horseradish peroxidase-conjugated secondary goat anti-rabbit IgG for 2 h. After washing the membranes 5 times with PBS-T and PBS, PARP was visualized using the ECL chemiluminescence system (Amersham, Arlington Heights, IL).

Measurement of mitochondrial transmembrane potential ({Delta}{Psi}m)
IgM (0.01 mM)-treated cells (106) were incubated for 20 min with CMTMRos (150 nM; Molecular Probes, Eugene, OR) in culture medium in 5% CO2 at 37°C. The cells were washed once with PBS, centrifuged at 200 g and fixed in 2 ml PBS containing 4% paraformaldehyde for 15 min at room temperature using a horizontal shaker at 150 r.p.m. Flow cytometric analysis of the fixed cells was carried out (Becton Dickinson) using excitation of a single 488-nm argon laser (25).

Statistical analysis
An unpaired Student’s t-test was used to determine the statistical significance of the data. A value of P <0.05 was considered as the level of statistical significance.


    Results
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
IgM induces cell death in autonomously growing cell lines with features characteristic of apoptosis
Cells of the T lymphoblastoid lineage CEM and the B cell line Raji were cultured with IVIgM. A dose-dependent loss of viability was observed in the presence of IVIgM in both CEM and Raji cell lines as assessed by MTT assay and uptake of PI (data not shown). For characterization of IgM-induced cell death, we tested whether the cells show typical characteristics of apoptosis such as DNA fragmentation and cell-surface expression of phosphatidylserines. As shown in Fig. 1(A), IVIgM induced typical DNA ladder formation in a dose-dependent manner in CEM T cells after 24 h of incubation with different concentrations of IgM, indicating evidence for apoptotic cell death in these cells rather than necrosis. Exposure of phosphatidylserines on the cell surface is an early event in apoptosis (26,27) and phosphatidylserines on the surface of cells can be detected with Annexin-V. Thus, CEM cells were incubated for 12 h in the presence of IgM, anti-Fas mAb CH-11 (IgM isotype, 100 ng/ml; Upstate Biotechnology, Lake Placid, NY), HSA or culture medium and subsequently stained with Annexin-V–FITC, a ligand of phosphatidylserine, as well as with PI, in order to detect dead cells. Early apoptotic cells will be single-positively stained for Annexin-V. As shown in Fig. 1(B), cells challenged with 0.05 mM IgM or 100 ng/ml mAb CH-11 show increased staining of Annexin-V (75 and 11% respectively) compared with non-treated or several fold higher concentration of HSA (0.15 mM)-treated cells (~5%). The expression of phosphatidylserines on the surface of the dying cells demonstrated that the cell death was apoptosis rather than necrosis.



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Fig. 1. Cell death induced by IgM shows characteristic apoptotic features. (A) DNA fragmentation of IgM-treated cells. DNA isolated from CEM cells treated for 24 h with 0, 0.01 and 0.05 mM of IVIgM (lanes 2–4) or 0.15 mM HSA (lane 5) were analyzed by agarose gel electrophoresis. Lane 1: DNA mol. wt marker. (B) Annexin-V labeling of IgM-treated CEM T cells. CEM cells (106/ml) were treated with 0.05 mM IgM for 12 h. Untreated cells and cells treated with 100 ng/ml of CH-11 or 0.15 mM of HSA were used as controls. Cells were double stained with Annexin-V–FITC (FL-1) and PI (FL-2). Panel (i) shows untreated cells, (ii) cells treated with 0.15 mM HSA, (iii) cells treated with CH11 (100 ng/ml) and (iv) cells treated with 0.05 mM IgM.

 
A role for Fas in IgM-induced apoptosis
Fas, a member of the tumor necrosis factor receptor family, is constitutively expressed on tumor cells of hematopoietic and non-hematopoietic origin (28). Cross-linking of Fas by its natural ligand or by agonistic antibodies results in apoptosis of Fas+ cells (29,30). We investigated the role of Fas in IgM-induced apoptosis using two experimental approaches. In the first approach, soluble Fas molecules were used along with IgM to overcome the IgM-induced apoptosis. We used two different soluble Fas molecules obtained from cultures of DAP.3 fibroblast clones transfected with expression vectors encoding soluble Fas molecules (DAP3.Fas{Delta}Exo3.4 and DAP3.Fas{Delta}Exo4). Culture supernatants obtained from DAP3 fibroblast clones transfected with the vector alone were used as negative control. CEM T cells were challenged with IgM (0.01 mM) in the presence of DAP3 clone supernatants for 48 h. Induced cell death was measured by PI uptake. As shown in Fig. 2(A), both forms of recombinant soluble Fas molecules significantly inhibited IgM-mediated apoptosis as compared with negative control-containing supernatants obtained from DAP3 fibroblast clones transfected with the empty pCDNA3 vector.



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Fig. 2. The role of Fas in IgM-mediated apoptosis. (A) CEM cells were cultured for 48 h with 0.01 mM IgM in the presence of tissue culture supernatants of the following DAP3 clones. DAP3-Fas{Delta}Exo3.4, DAP3Fas {Delta}Exo4 and DAP3.pcDNA3 empty vector. The percentage of IgM-induced cell death [i.e. (death in the presence of IgM and the supernatant) – (spontaneous death in the presence of supernatant alone)] was measured by PI uptake and shown as mean values ± SE obtained from three separate experiments carried out in duplicates. Statistical significance as determined by Student’s t-test is indicated (*P < 0.05) (B) Fas-sensitive HuT78 and Fas-resistant HuT78.B1 lymphoblastoid T cell lines were cultured with IVIgM (0.01 mM), HSA (0.15 mM) CH-11 (100 ng/ml) or 10% FCS for 16 h. Mortality of cells was measured using the PI-uptake assay. The decrease in the level of apoptosis in HuT78.B1 is significant. (C) Induction of apoptosis by affinity-purified Fas IgM antibodies. Fas antibodies were affinity purified from IVIgM using the GST–FasExtra fusion protein as indicated in Methods. The lymphoblastoid CEM cells were treated with affinity-purified Fas IgM antibodies (2 and 0.15 µM), HSA (0.15 mM) or CH-11 (100 ng/ml) for 24 h and the mortality was assessed using PI uptake by the dead cells.

 
In a second approach to investigate the role of Fas antibodies in natural IgM-induced apoptosis, Fas-sensitive and Fas-resistant cell lines were treated with IgM, and cell death was evaluated. HuT78 is a human T cell lymphoma line, highly sensitive to Fas-mediated apoptosis, whereas HuT78.B1 cells are resistant to Fas-mediated apoptosis due to the expression of a wild-type and a truncated Fas receptor (21). There was a significant reduction in IgM-mediated apoptosis in HuT78.B1 Fas-resistant cells compared with parental HuT78 cells, as measured by PI uptake (Fig. 2B).

Fas-specific antibodies in IgM preparation
To investigate the role of Fas receptor in IgM-induced apoptosis, Fas antibodies were purified from IVIgM by affinity chromatography using GST–Fas extracytoplasmic domain fusion protein immobilized on Sepharose beads. To test the biological activity of the affinity-purified Fas antibodies, CEM cells were incubated for 24 h with the antibodies at concentrations of 2 and 0.15 µM (Fig. 2C). The concentration of affinity-purified Fas IgM required to induce mortality was significantly lower than that of unfractionated IgM, as assessed by PI uptake. Similar results were obtained when Raji cells were treated with affinity-purified Fas antibodies (data not shown). To assess the potential therapeutic in vivo activity of the Fas IgM antibodies, we used freshly isolated PBMC. Both unfractionated IVIgM (0.01 mM) and affinity-purified Fas IgM (20 µM) induced apoptosis in purified PBMC (Fig. 3). Incubation of PBMC with commercial CH-11 mAb also resulted in apoptosis (data not shown). The overall extent of apoptosis of resting PBMC was, however, lower than that observed with transformed cells and consistent with the observations that transformed cells are more sensitive to apoptosis (31,32).



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Fig. 3. Induction of apoptosis of non-transformed cells by IgM. PBMC from healthy blood donors were cultured for 40 h with IVIgM (0.01 mM, hatched bar), affinity-purified Fas IgM (2 µM, filled bar) and HSA (0.15 mM, open bar). Cell death was quantified by measuring PI uptake and expressed as the percentage of dye-positive cells. A representative result of two independent experiments conducted in duplicates.

 
Induction of apoptosis is associated with the activation of caspases, a family of cysteine proteases (3335). Here, we tested the involvement of caspase-3 in the IgM-induced apoptosis in Raji and CEM cells (Fig. 4 and data not shown). Upon activation, caspase-3 cleaves PARP, resulting in the appearance of an 85-kDa band on western blotting. Raji cells were challenged with IgM to induce apoptosis and the activity of caspase-3 in the Raji cells was evaluated by western blotting with a PARP antibody. Cleavage of PARP was detected at 18 h in the treated cells (Fig. 4, lane 2), while there was no degradation of PARP in the untreated cells (Fig. 4, lane 1). Similar results were obtained when CEM cells were subjected to IVIgM-induced apoptosis (data not shown).



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Fig. 4. Activation of caspase-3 in IgM-induced apoptosis. Raji cells (106 cells) were treated for 0, 18 or 36 h with IVIgM (lanes 1–3 respectively) or CH-11 Fas antibody (lane 4) as described earlier. Cell extracts were prepared and analyzed on 10% SDS–PAGE. Separated proteins were transferred onto PVDF membranes and the cleavage of PARP was detected by western blot with an anti-PARP polyclonal antibody. The arrow indicates the 85-kDa PARP product.

 
We then investigated whether IgM-induced apoptosis involves alteration of the {Delta}{Psi}m. Changes in the {Delta}{Psi}m occur in early phases of programmed cell death (36). To examine the role of mitochondria in IgM-mediated apoptosis, Raji cells were challenged with IgM (0.01 mM) and the {Delta}{Psi}m was monitored by the fluorescence of the cationic lipophilic dye CMTMRos. Reduced {Delta}{Psi}m was measured by the change in the fluorescence (Fig. 5). Indeed, there was a weak shift in the mean fluorescence after 2 h of incubation indicating a reduction in {Delta}{Psi}m. Prolonged incubation of Raji cells with IgM (up to 8 h) further reduced the {Delta}{Psi}m. These results indicate an immediate effect of IgM on the membrane potential of mitochondria. Mitochondrial changes associated with apoptosis include permeability transition pore formation in the mitochondrial membranes (37). The exact mechanism involved in the pore formation is still not well understood, but members of the Bcl-2 family of proteins play an important role in the pore formation (38). Similar results were obtained with IVIgM-treated CEM cells (data not shown).



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Fig. 5. Reduction in {Delta}{Psi}m upon treatment with IgM. Raji cells were cultured with IgM (0.01 mM) for the indicated time points. The cells were then treated with CMTMRos and analyzed by flow cytometry to measure changes in {Delta}{Psi}m. The percentage of cells with low {Delta}{Psi}m is depicted as a shift to weaker CMTMRos fluorescence and was quantified using CellQuest software. Cells at zero time point are indicated as control and the bottom panel represents the unlabeled cells.

 

    Discussion
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
In this study, we have characterized the apoptosis-inducing effects of normal human IgM on human lymphoid cell lines as well as freshly isolated PBMC. We have observed that the IgM-induced cell death was dose dependent. The cell death was associated with DNA ladder formation, loss of membrane integrity and cell-surface exposure of phosphatidylserine—characteristic features of apoptosis. We demonstrate that IgM-induced cell death involves the Fas receptor, activation of caspase-3 and a reduction in mitochondrial transmembrane potential during the early phase of apoptosis induction. Our results suggest a role for normal IgM in controlling cell survival and death, and imply a possible therapeutic role of IgM in autoimmune and lymphoproliferative disorders.

Defective apoptosis is associated with the pathogenesis of several autoimmune diseases (3943). It has been proposed that too little apoptosis would lead to persistence of autoreactive cells resulting in the production of autoantibodies (40). The importance of Fas-mediated apoptosis in lymphoproliferative disorders is well documented. MRL mice homozygous for lpr (lymphoproliferation) or gld (generalized lymphoproliferative disease) develop a systemic autoimmune disease resembling systemic lupus erythematosus (SLE), Sjögren’s disease and rheumatoid arthritis (44). Induction of Fas-mediated apoptosis by IVIgM preparations indicates the possibility to use IgM in the treatment of autoimmune and lymphoproliferative diseases.

We demonstrate that IgM induced apoptosis of lymphocytes at least partially mediated by Fas receptor cross-linking as suggested by two different approaches. The Fas-sensitive HuT78, a human T lymphoblastic cell line, was compared for its sensitivity to IgM-mediated apoptosis with that of a Fas-resistant variant, HuT78.B1. HuT78.B1 contains a wild-type and a truncated Fas receptor (21). In addition, these cells are defective in eliciting some early signal transduction events upon Fas cross-linking such as activation of acidic sphingomyelinase (45). Our results indicate that the Fas-resistant cell line displayed reduced sensitivity to IgM-mediated apoptosis. The latter data, together with the observation that IgM-mediated apoptosis is inhibited by soluble Fas molecules, provided strong evidence for a role of Fas cross-linking in IgM-induced apoptosis. However, induction of apoptosis of HuT78B1 cells with IgM indicates the involvement of additional apoptotic pathways.

It is possible that IgM binds to other cell-surface molecules including cytokine receptors and possibly growth factor receptors. It is conceivable that such stimulations could lead to apoptosis. Thus, cross-linking of the B cell antigen receptor with IgM antibodies initiates a cascade of events that culminates with growth arrest and cell death in B lymphocytes (46). Several studies have shown that the caspase cascade plays a central role in this process with or without the involvement of mitochondria (4749). The use of the caspase inhibitor z-VAD-fmk or overexpression of Bcl-2 prevents apoptosis, but not growth arrest induced by anti-IgM antibody treatment (47). Results from the present study suggest that IgM-mediated apoptosis involves the activation of caspase-3 and a reduction in the {Delta}{Psi}m.

Fc receptors for IgM on B lymphocytes and macrophages have been characterized (50). Although the role of Fc receptors for IgM in IgM-mediated apoptosis has not been elucidated, it is likely that the interactions initiated through the binding of Fc portions of antibodies or immune complexes to cell-surface Fc receptors on hematopoietic cells lead to a variety of immune responses that may include apoptosis (51,52). The ability of natural human IgM to induce apoptosis of neuroblastoma cells through interaction with a 260-kDa surface antigen on neuroblastoma cells has been demonstrated (53,54). Abnormal and modified glycolipid and glycoprotein synthesis by tumor cells is a common feature of malignant cells, and results in the expression of these modified structures on the surface (55,56). It is likely that natural IgM antibodies bind most likely to epitopes on carbohydrate structures of specific receptors.

It is well established that natural IgM play an important role in the first line of defense against infectious agents (57). Results from the present study demonstrate that not only transformed lymphoblastoid cells, but also resting PBMC, are sensitive to IgM-induced apoptosis, suggesting that natural IgM antibodies might have a role in the regulation of proliferation of immune cells and in the first-line defense against infections, and in the immunosurveillance against transformed malignant cells (58). However, we believe that the apoptotic effect of natural antibody preparations is not indiscriminate since all lymphocytes are not equally sensitive to antibody-mediated apoptosis. In fact, transformed cells are more sensitive to apoptosis than the non-transformed cells (31,32) and naive PBMC are less sensitive to apoptosis than activated PBMC (59). Thus, it is possible that such Ig preparations might be beneficial in restraining an immune system that is abnormally activated due to polyclonal activation.

Serum IgM plays an important role in controlling the expression of IgG autoreactivity (60,61). The addition of purified IgM to autologous IgG suppresses IgG-associated autoreactivity (61). IgM-dependent regulation of IgG autoreactivity in serum is defective in autoimmune conditions, such as autoimmune thyroiditis and SLE (61,62). In addition, loss of detectable IgG autoantibody activity in the serum of patients in remission of ANCA+ vasculitis was shown to be associated with the generation of IgM anti-idiotypes directed against the patient’s acute-phase IgG autoantibodies, indicating that IgM may suppress pathogenic autoantibodies of the IgG isotype in remission of autoimmune disease (62). Mice that lack secreted IgM developed elevated levels of IgG autoantibodies to double-stranded DNA and histones, had abundant deposits of immune complexes in the glomeruli, and succumbed to the disease at an earlier age. Similarly, the absence of secreted IgM also resulted in an accelerated development of IgG autoantibodies in normal mice. These findings suggest that secreted IgM, including IgM autoantibodies produced naturally or as part of an autoimmune response, may lessen the severity of autoimmune pathology associated with IgG autoantibodies (63). We have previously shown that infusion of (Lewis x Brown-Norway) F1 rats with IVIgM protects the animals against experimental autoimmune uveitis induced by immunization with the soluble retinal S antigen (17). IVIgM was shown to suppress anti-acetylcholine receptor (AchR) autoantibody production in vivo and to prevent the loss of AchR at the muscle endplates in a reconstituted SCID mouse model of myasthenia gravis (18). As apoptosis plays a critical role in maintaining peripheral lymphocyte homeostasis and for minimizing the expansion of autoreactive lymphocytes, our results suggest that the beneficial and protective role of IgM in various models of diseases may involve induction of apoptosis of autoreactive lymphocytes and inhibition of cellular proliferation. Thus, together with IVIg containing exclusively IgG, therapeutic IgM may be of potential benefit in several conditions, including cancer, in which defective apoptosis is implicated in the pathogenesis (6469).


    Acknowledgements
 
Supported by Institut National de la Santé et de la Recherche Médicale and Centre National de la Recherche Scientifique France, and by a grant from ZLB Bioplasma AG, Bern, Switzerland. The authors wish to thank Ms E. Bonnin and S. Delignat for technical support.


    Abbreviations
 
{Delta}{Psi}m—mitochondrial transmembrane potential

IVIgM—i.v. IgM

NAb—natural antibodies

NAA—natural autoreactive antibodies

PARP—poly(A)DP-ribose polymerase

PI—propidium iodide

PBS-Az—PBS containing 0.02% NaN3

PBS-T—PBS containing 0.1% Tween 20

PBMC—peripheral blood mononuclear cell

SLE—systemic lupus erythematosus


    References
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

  1. Tomer, Y., Sherer, Y. and Shoenfeld, Y. 1998. Autoantibodies, autoimmunity and cancer [Review]. Oncol. Rep. 5:753.[ISI][Medline]
  2. Newton, K. and Strasser, A. 2000. Cell death control in lymphocytes. Adv. Immunol. 76:179.[ISI][Medline]
  3. Strasser, A., O‘Connor, L. and Dixit, V. M. 2000. Apoptosis signaling. Annu. Rev. Biochem. 69:217.[CrossRef][ISI][Medline]
  4. Wyllie, A. H., Kerr, J. F. R. and Currie, A. R. 1980. Cell death: the significance of apoptosis. Int. Dev. Cytol. 68:251.
  5. Goodnow, C. C., Glynne, R., Mack, D., Weintraub, B., Rathmell, J., Healy, J. I., Chaudhry, S., Miosge, L., Loy, A. and Wilson, L. 1999. Mechanisms of self-tolerance and autoimmunity: from whole-animal phenotypes to molecular pathways. Cold Spring. Harb. Symp. Quant. Biol. 64:313.[ISI][Medline]
  6. Kamradt, T. and Mitchison, N. 2001. Tolerance and autoimmunity. N. Engl. J. Med. 344:655.[Free Full Text]
  7. Jackson, C. E. and Puck, J. M. 1999. Autoimmune lymphoproliferative syndrome, a disorder of apoptosis. Curr. Opin Pediatr. 11:521.[CrossRef][Medline]
  8. Mackay, I. R. 2000. Science, medicine, and the future: tolerance and autoimmunity. Br. Med. J. 321:93.[Free Full Text]
  9. Tomer, Y. and Shoenfeld, Y. 1988. The significance of natural autoantibodies. Immunol. Invest. 17:389.[ISI][Medline]
  10. Shoenfeld, Y. and Isenberg, D. 1989. The mosaic of autoimmunity. Immunol. Today 10:123.[CrossRef][ISI][Medline]
  11. Coutinho, A., Kazatchkine, M. D. and Avrameas, S. 1995. Natural autoantibodies. Curr. Opin. Immunol. 7:812.[CrossRef][ISI][Medline]
  12. Nossal, G. J. 1994. Negative selection of lymphocytes. Cell 76:229.[ISI][Medline]
  13. Hayakawa, K., Asano, M., Shinton, S. A., Gui, M., Allman, D., Stewart, C. L., Silver, J. and Hardy, R. R. 1999. Positive selection of natural autoreactive B cells. Science 285:113.[Abstract/Free Full Text]
  14. Dighiero, G., Lymberi, P., Holmberg, D., Lundquist, I., Coutinho, A. and Avrameas, S. 1985. High frequency of natural autoantibodies in normal newborn mice. J. Immunol. 134:765.[Abstract/Free Full Text]
  15. Casali, P. and Schettino, E. W. 1996. Structure and function of natural antibodies. Curr. Top. Microbiol. Immunol. 210:167.[ISI][Medline]
  16. Avrameas, S. 1991. Natural autoantibodies: from ‘horror autotoxicus’ to ‘gnothi seauton’. Immunol. Today 12:154.[ISI][Medline]
  17. Hurez, V., Kazatchkine, M. D., Ramanathan, S., Basuyaux, B., Pashov, A., Vassilev, T., De-Kozak, Y., Bellon, B. and Kaveri, S. V. 1997. Pooled normal human polyspecific IgM contains neutralizing antiidiotypes to IgG autoantibodies of autoimmune patients and protects from autoimmune disease. Blood 70:4004.
  18. Vassilev, T., Yamamoto, M., Aissaoui, A., Bonnin, E., Berrih-Aknin, S., Kazatchkine, M. D. and Kaveri, S. V. 1999. Pooled normal immunoglobulin M prevents the loss of acetylcholine receptors in experimental myasthenia gravis. Eur. J. Immunol. 29:2436.[CrossRef][ISI][Medline]
  19. Warrington, A. E., Asakura, K., Bieber, A., Ciric, B., Van Keulen, V., Kaveri, S. V., Kyle, R. A., Pease, L. R. and M., R. 2000. Human monoclonal antibodies reactive to oligodendrocytes promote remyelination in a model of multiple sclerosis. Proc. Natl Acad. Sci. USA 97:6820.[Abstract/Free Full Text]
  20. Bieber, A., Warrington, A. E., Asakura, K., Ciric, B., Van Keulen, V., Kaveri, S. V., Pease, L. R. and M., R. 2002. Human antibodies accelerate the rate of remyelination following lysolecithin-induced demyelination in mice. Glia 37:241.[CrossRef][ISI][Medline]
  21. Cascino, I., Papoff, G., De-Maria, R., Testi, R. and Ruberti, G. 1996. Fas/Apo-1 (CD95) receptor lacking the intracytoplasmic domain protects tumor cells from Fas-mediated apoptosis. J. Immunol. 156:13.[Abstract]
  22. Papoff, G., Cascino, I., Eramo, A., Starace, G., Lynch, D. H. and Ruberti, G. 1996. An N-terminal domain shared by Fas/Apo-1 (CD95) soluble variants prevents cell death in vitro. J. Immunol. 156:4622.[Abstract/Free Full Text]
  23. Tian, Q., Taupin, J. L., Elledge, S., Robertson, M. and Anderson, P. 1995. Fas-activated serine/threonine kinase (FAST) phosphorylates TIA-1 during Fas-mediated apoptosis. J. Exp. Med. 182:865.[Abstract]
  24. McGahon, A. J., Martin, S. J., Bissonnette, R. P., Mahboubi, A., Y. Shi, Mogil, R. J., Nishioka, W. K. and Green, D. R. 1995. The end of the (cell) line: methods for the study of apoptosis in vitro. Methods Cell Biol. 46:153.[ISI][Medline]
  25. Bossy-Wetzel, E., Newmeyer, D. and Green, D. 1998. Mitochondrial cytochrome c release in apoptosis occurs upstream of DEVD-specific caspase activation and independently of mitochondrial transmembrane depolarization. EMBO J. 17:37.[Abstract/Free Full Text]
  26. Fadok, V. A., D. R. Voelker, P. A. Campbell, J. J. Cohen, D. L. Bratton, and Henson, P. M. 1992. Exposure of phosphatidylserine on the surface of apoptotic lymphocytes triggers specific recognition and removal by macrophages. J. Immunol. 148:2207.[Abstract/Free Full Text]
  27. Koopman, G., Reutelingsperger, C. P., Kuijten, G. A., Keehnen, R. M., Pals, S. T. and van Oers, M. H. 1994. Annexin-V for flow cytometric detection of phosphatidylserine expression on B cells undergoing apoptosis. Blood 84:1415.[Abstract/Free Full Text]
  28. Kovacs, B., Patel, A., Hershey, J. N., Dennis, G. J., Kirschfink, M. and Tsokos, G. C. 1997. Antibodies against p53 in sera from patients with systemic lupus erythematosus and other rheumatic diseases. Arthritis Rheum. 40:980.[Medline]
  29. Nagata, S. and Golstein, P. 1995. The Fas death factor. Science 267:1449.[ISI][Medline]
  30. Maher, S., Toomey, D., Condron, C. and Bouchier-Hayes, D. 2002. Activation-induced cell death: the controversial role of Fas and Fas ligand in immune privilege and tumour counterattack. Immunol. Cell Biol. 80:131.[CrossRef][ISI][Medline]
  31. Ucker, D., Hebshi, L., Blomquist, J. and Torbett, B. 1994. Physiological T-cell death: susceptibility is modulated by activation, aging, and transformation, but the mechanism is constant. Immunol. Rev. 142:273.[ISI][Medline]
  32. Massaia, M., Borrione, P., Attisano, C., Barral, P., Beggiato, E., Montacchini, L., Bianchi, A., Boccadoro, M. and Pileri, A. 1995. Dysregulated Fas and Bcl-2 expression leading to enhanced apoptosis in T cells of multiple myeloma patients. Blood 85:3679.[Abstract/Free Full Text]
  33. Li, H. and Yuan, J. 1999. Deciphering the pathways of life and death. Curr. Opin. Cell Biol. 11:261.[CrossRef][ISI][Medline]
  34. Green, D. and Kroemer, G. 1998. The central executioners of apoptosis: caspases or mitochondrial? Cell Biol. 8:267.
  35. Shi, Y. 2002. Mechanisms of caspase activation and inhibition during apoptosis. Mol. Cell. 9:459.[ISI][Medline]
  36. Ferlini, C., Cesare, S. D., Rainaldi, G., Malorni, W., Samoggia, P., Biselli, R. and Fattorossi, A. 1996. Flow cytometric analysis of the early phases of apoptosis by cellular and nuclear techniques. Cytometry 24:106.[CrossRef][ISI][Medline]
  37. Crompton, M. 1999. The mitochondrial permeability transition pore and its role in cell death. Biochem. J. 341:233.[CrossRef][ISI][Medline]
  38. Kroemer, G., Dallaporta, B. and Resche-Rigon, M. 1998. The mitochondrial death: life regulator in apoptosis and necrosis. Annu. Rev. Physiol. 60:619.[CrossRef][ISI][Medline]
  39. George, J., Adler, Y., Zhang, J., Shoenfeld, Y. and Youinou, P. 1995. Apoptosis and its association with autoimmunity. Clin. Rheumatol. 14:505.[ISI][Medline]
  40. Rudin, C. M. and Thompson, C. B. 1997. Apoptosis and disease: regulation and clinical relevance of programmed cell death. Annu. Rev. Med. 48:267.[CrossRef][ISI][Medline]
  41. Stassi, G. and De-Maria, R. 2002. Autoimmune thyroid disease: new models of cell death in autoimmunity. Nat. Rev. Immunol. 2:195.[CrossRef][ISI][Medline]
  42. Rathmell, J. C. and Thompson, C. B. 2002. Pathways of apoptosis in lymphocyte development, homeostasis, and disease. Cell 109:S97.
  43. Debatin, K. M. 2001. Cell death in T- and B-cell development. Ann. Hematol. 80:B29.
  44. Cohen, J. J. 1993. Apoptosis. Immunol. Today 14:126.[ISI][Medline]
  45. Cifone, M. G., Roncaioli, P., Maria, R. D., Grazia, C., Santoni, A., Ruberti, G. and Testi, R. 1995. Multiple pathways originate at the Fas/Apo-1(CD95) receptor: sequential involvement of phosphatidylcholine-specific phospholipase C and acidic sphingomyelinase in the propagation of the apoptotic signal. EMBO J. 14:5859.[Abstract]
  46. Marches, R., Vitetta, E. S. and Uhr, J. W. 2001. A role for intracellular pH in membrane IgM-mediated cell death of human B lymphomas. Proc. Natl Acad. Sci. USA 98:3434.[Abstract/Free Full Text]
  47. Bras, A., Ruiz-Vela, A., Garcia-Domingo, D. and Martinez, C. 2000. Apoptosis as a scaffold for building up the B cell repertoire. Ann. NY Acad. Sci. 926:13.[Abstract/Free Full Text]
  48. Berard, M., Mondiere, P., Casamayor-Palleja, M., Hennino, A., Bella, C. and Defrance, T. 1999. Mitochondria connects the antigen receptor to effector caspases during B cell receptor-induced apoptosis in normal human B cells. J. Immunol. 163:4655.[Abstract/Free Full Text]
  49. Chen, W., Wang, H. G., Srinivasula, S. M., Alnemri, E. S. and Cooper, N. R. 1999. B cell apoptosis triggered by antigen receptor ligation proceeds via a novel caspase-dependent pathway. J. Immunol. 163:2483.[Abstract/Free Full Text]
  50. Shibuya, A., Sakamoto, N., Shimizu, Y., Shibuya, K., Osawa, M., Hiroyama, T., Eyre, H. J., Sutherland, G. R., Endo, Y., Fujita, T., Miyabayashi, T., Sakano, S., Tsuji, T., Nakayama, E., Phillips, J. H., Lanier, L. L. and Nakauchi, H. 2000. Fc alpha/mu receptor mediates endocytosis of IgM-coated microbes. Nat. Immunol. 1:441.[CrossRef][ISI][Medline]
  51. Ravetch, J. V. and Clynes, R. A. 1998. Divergent roles for Fc receptors and complement in vivo. Annu. Rev. Immunol. 16:421.[CrossRef][ISI][Medline]
  52. Daëron, M. 1997. Fc receptor biology. Annu. Rev. Immunol. 15:203.[CrossRef][ISI][Medline]
  53. Ollert, M. W., David, K., Schmitt, C., Hauenschild, A., Bredehorst, R., Erttmann, R. and Vogel, C.-W. 1996. Normal human serum contains a natural IgM antibody cytotoxic for human neuroblastoma cells. Proc. Natl Acad. Sci. USA 93:4498.[Abstract/Free Full Text]
  54. David, K., Heiligtag, S., Ollert, M. W., Teppke, M., Vogel, C. W. and Bredehorst, R. 2001. Initial characterization of the apoptosis-inducing receptor for natural human anti-neuroblastoma IgM. Med. Pediatr. Oncol. 36:251.[CrossRef][ISI][Medline]
  55. Karsten, U., Diotel, C., Klich, G., Paulsen, H., Goletz, S., Muller, S. and Hanisch, F. G. 1998. Enhanced binding of antibodies to the DTR motif of MUC1 tandem repeat peptide is mediated by site-specific glycosylation. Cancer Res. 58:2541.[Abstract]
  56. Egea, G., Franci, C., Gambus, G., Lesuffleur, T., Zweibaum, A. and Real, F. X. 1993. cis-Golgi resident proteins and O-glycans are abnormally compartmentalized in the RER of colon cancer cells. J. Cell Sci. 105:819.[Abstract/Free Full Text]
  57. Ochsenbein, A. and Zinkernagel, R. M. 2000. Natural antibodies and complement link innate and acquired immunity. Immunol. Today 21:624.[CrossRef][ISI][Medline]
  58. Brandlein, S., Pohle, T., Ruoff, N., Wozniak, E., Muller-Hermelink, H. K. and Vollmers, H. P. 2003. Natural IgM antibodies and immunosurveillance mechanisms against epithelial cancer cells in humans. Cancer Res. 63:7995.[Abstract/Free Full Text]
  59. Patki, A. H., Zielske, S. P., Sieg, S. F. and Lederman, M. M. 2000. Preferential S phase entry and apoptosis of CD4+ T lymphocytes of HIV-1-infected patients after in vitro cultivation. Clin. Immunol. 97:241.[CrossRef][ISI][Medline]
  60. Adib, M., Ragimbeau, J., Avrameas, S. and Ternynck, T. 1990. IgG autoantibody activity in normal mouse serum is controlled by IgM. J. Immunol. 145:3807.[Abstract/Free Full Text]
  61. Hurez, V., Kaveri, S. V. and Kazatchkine, M. D. 1993. Expression and control of the natural autoreactive IgG repertoire in normal human serum. Eur. J. Immunol. 23:783.[ISI][Medline]
  62. Ronda, N., Haury, M., Nobrega, A., Kaveri, S. V., Coutinho, A. and Kazatchkine, M. D. 1994. Analysis of natural and disease-associated autoantibody repertoires: anti-endothelial cell IgG autoantibody activity in the serum of healthy individuals and patients with systemic lupus erythematosus. Int. Immunol. 6:1651.[Abstract]
  63. Boes, M., Prodeus, A. P., Schmidt, T., Carroll, M. C. and Chen, J. 1998. A critical role of natural immunoglobulin M in immediate defense against systemic bacterial infection. J. Exp. Med. 188:2381.[Abstract/Free Full Text]
  64. Shoenfeld, Y. and Fishman, P. 1999. Gamma-globulin inhibits tumor spread in mice. Int. Immunol. 11:1247.[Abstract/Free Full Text]
  65. Kazatchkine, M. D. and Kaveri, S. V. 2001. Immunomodulation of autoimmune and inflammatory diseases with intravenous immune globulin. N. Engl. J. Med. 345:747.[Free Full Text]
  66. Jonsson, V., Gemmell, C. G. and Wiik, A. 2000. Emerging concepts in the management of the malignant haematological disorders. Expert Opin. Pharmacother. 1:713.[Medline]
  67. Merimsky, O., Meller, I., Inbar, M., Bar-Yehuda, S., Shoenfeld, Y. and Fishman, P. 2002. A possible role for IVIg in the treatment of soft tissue sarcoma: a clinical case and an experimental model. Int. J. Oncol. 20:839.[ISI][Medline]
  68. Sherer, Y., Levy, Y. and Shoenfeld, Y. 2002. IVIG in autoimmunity and cancer—efficacy versus safety. Expert Opin. Drug Safety 1:153.
  69. Amital, H., Rewald, E., Levy, Y., Bar-Dayan, Y., Manthorpe, R., Engervall, P., Sherer, Y., Langevitz, P. and Shoenfeld, Y. 2003. Fibrosis regression induced by intravenous gammaglobulin treatment. Ann. Rheum. Dis. 62:175.[Abstract/Free Full Text]




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