Unformatted text preview: British Journal of Rheumatology 1997;36:158–163
SCIENTIFIC REVIEW APOPTOSIS IN PERIPHERAL LYMPHOCYTES IN SYSTEMIC LUPUS ERYTHEMATOSUS: A REVIEW
L. M. ROSE, D. S. LATCHMAN and D. A. ISENBERG*
Medical Molecular Biology Unit , Department of Molecular Pathology , University College London Medical School , Cleveland Street , London W1P 6DB and *Centre for Rheumatology , Bloomsbury Rheumatology Unit , Department of Medicine , University College London Medical School , Arthur Stanley House , Tottenham Street , London W1P 9PG SUMMARY There is increasing evidence to suggest that abnormalities in apoptosis may play a part in the pathogenesis of systemic lupus erythematosus (SLE). For example, there is now considerable evidence that bcl-2 expression is enhanced in a proportion of peripheral T cells, but not in B cells, in SLE patients and correlates with overall disease activity regardless of the activity index employed. Further work is required to establish whether enhanced bcl-2 expression by some T cells in SLE patients is related to their activation or intrinsically enhanced by genetic predisposition. Mutations in Fas result in a lymphoproliferative syndrome and may play a role in accelerating autoimmune disease. A report of three children with mutations in Fas has once again focused attention on this regulator of apoptosis. The relationships between inducers and inhibitors of apoptosis may diﬀer in diﬀerent cell types, and must be elucidated before the implications of observations made in lymphocytes from SLE patients can be fully understood. K : Systemic lupus erythematosus, bcl-2, Fas, Lymphocytes, Apoptosis. P with systemic lupus erythematosus (SLE) show a broad range of immunological abnormalities. These include increased numbers of circulating activated B lymphocytes which produce large amounts of immunoglobulin and an array of autoantibodies. It has been postulated that the high levels of autoantibodies observed in SLE patients are a result, at least in part, of a polyclonal B-cell activation  and in murine models of lupus there is evidence that polyclonal B-cell activation precedes clinical expression of disease . There are several possible explanations for this activation. There may be an excess of T-cell help, by the helper/inducer subset or, as recently suggested, by a T-suppressor/inducer subset . Deﬁcient suppressor cell activity may be responsible for autoantibody overproduction as defective concanavalin A-induced suppressor cell function [4, 5] and suppressor/cytotoxic responses to Epstein–Barr virus (EBV)  have been reported in SLE patients. Finally, dysfunction of apoptosis, the process of programmed cell death which governs the lifespan of lymphocytes, must be considered. Apoptosis plays an important role in the immune system. It is thought to be the mechanism of T-cell depletion in the thymus  and is the cytotoxic mechanism induced by cytotoxic T cells , natural killer cells and cytokines such as lymphotoxin and tumour necrosis factor (TNF) [8, 9]. Apoptosis is also thought to be involved in the maturation of antibody responses through elimination of low-aﬃnity antigen receptor-bearing lymphocytes
Submitted 17 April 1996; revised version accepted 30 August 1996. Correspondence to: D. A. Isenberg, Bloomsbury Rheumatology Unit, University College London Medical School, Arthur Stanley House, 50 Tottenham Street, London W1P 9PG. . Abnormalities in apoptosis might link the defects recorded in both T and B cells in lupus patients. DEFECTS IN THE REGULATION OF LYMPHOCYTE SURVIVAL Observations in mouse models of SLE, namely that mice with mutations of the Fas gene (lpr and lpr cg ) or the Fas ligand (C3Hgld ) and mice transgenic for overexpression of bcl-2  develop prolonged B-cell survival and other features in common with SLE patients suggest that these molecules, which both play crucial roles in apoptosis, may play a role in the pathogenesis of SLE. Any alteration in the balance between clonal deletion and the induction of anergy in autoreactive cells may lead to the persistence of autoreactive lymphocytes. A high threshold for DNA damage-induced apoptosis might also explain the presence of increased DNA mutations in the T cells of SLE patients . FAS AND AUTOIMMUNITY Fas is a cell surface protein that plays a major role in the induction of apoptosis in lymphoid cells. The abnormal accumulation of lymphocytes in mice with mutations in Fas or the Fas ligand suggests that signalling via Fas is involved in the death of normal human lymphocytes. A soluble form of Fas, lacking the transmembrane region, has been detected in human sera and is found in elevated levels in sera from patients with a number of diseases, including SLE . This soluble form of Fas (sFAS) blocked anti-Fas antibody-induced apoptosis of activated human peripheral blood mononuclear cells (PBMCs) in vitro and, when injected into normal mice, caused splenomegaly and lymphadenopathy . Thus, this molecule may
= 1997 British Society for Rheumatology 158 enlargement of ROSE ET AL .: BCL-2 EXPRESSION IN SLE 159 inhibit Fas-mediated elimination of activated lymphocytes, but there is little evidence to support a signiﬁcant role for sFas in SLE. In studies of patients with laboratory evidence of autoimmune disease  and of patients with SLE and juvenile rheumatoid arthritis (JRA) , compared with healthy controls, elevated sFas levels were detected in only a minority of autoimmune sera. Soluble Fas levels were not associated with autoimmune disease or with ﬂares in autoimmune disease [16, 17]. To date, there is no evidence that patients with SLE have mutations in the genes for Fas or the Fas ligand. However, two recent reports describe a total of eight children (two siblings and six unrelated children) with mutations in Fas [18, 19]. All patients had clinical and immunological features similar to those seen in lpr mice, i.e. a lymphoproliferative syndrome and a large proportion of peripheral and splenic T cells expressing neither CD4 nor CD8. Two of these patients had deletions in the Fas gene which precluded cell surface expression of Fas, while the other six had nucleotide deletions, duplications or substitutions which resulted in the expression of both abnormal and normal Fas mRNAs [18, 19]. Fas-mediated apoptosis was defective in T cells from all eight children. Cell transfection analyses of four mutant alleles showed that they were unable to transmit a death signal and had dominant interfering eﬀects on killing mediated by non-mutant Fas . Seven of the children had clinically signiﬁcant autoimmune disorders, including haemolytic anaemia, neutropenia and thrombocytopenia. There were very few autoimmune manifestations in one child, despite the lack of Fas expression , and in parents of some of the patients carrying the same Fas mutations as their children , but the genetic dependency for autoimmune manifestation has also been noted in the murine lpr mutation. The Fas and Fas ligand mutations which produce both lymphoproliferative syndrome and autoimmune manifestations in MRL mice only result in lymphadenopathy and splenomegaly in other strains . BCL-2 EXPRESSION AND AUTOIMMUNITY Bcl-2 was identiﬁed originally because of its involvement in the majority of non-Hodgkin B-cell lymphomas where a t(14:18) interchromosomal translocation juxtaposes the bcl-2 gene with the immunoglobulin heavy chain locus, leading to transcription of high levels of bcl-2 which enhance cell survival . Bcl-2 is unique among oncogene products in that it appears to enhance lymphoid cell survival by interfering with apoptosis rather than promoting cell proliferation. It has been shown to interfere with apoptotic cell death in several experimental models . Bcl-2 protein is restricted geographically in tissues characterized by apoptotic cell death. It is, for example, conﬁned to the zones of surviving B cells in germinal centres and is restricted topographically in the thymus where the vast majority of cortical thymocytes lack bcl-2, while the mature thymocytes in the medulla express bcl-2 . This regional distribution suggests that bcl-2 is regulated diﬀerentially during T-cell maturation and is involved in the salvation of T cells. Bcl-2 protein is present in circulating, quiescent peripheral blood lymphocytes [24, 25]. Overexpression of a bcl-2 transgene in the B cells of some murine strains can result in an autoimmune syndrome resembling human lupus. Transgenic mice carrying a bcl-2 transgene expressed at high levels predominantly in the B-cell lineage showed a polyclonal expansion of B cells with a large excess of mature B cells and plasma cells [13, 26]. The mice showed hypergammaglobulinaemia with 02-fold elevations of serum IgG and IgA compared to normal litter mates. Transgenic mice in which bcl-2 was expressed mainly in the T-cell lineage had thymic and peripheral T cells which showed prolonged survival in vitro when cultured in the absence of growth factors. However, the in vivo expansion of T cells was only 60% of the magnitude of B-cell expansion in B-cell transgenics . T-cell homeostasis appeared unaﬀected as the total number of T cells and the proportions of the major subsets appeared normal in both the thymus and the periphery. Strasser et al .  maintained bcl-2 transgenic mice for longer periods, with interesting results. Mice expressing bcl-2 in T cells remained healthy for at least a year with no detectable signs of autoimmunity, but mice expressing the transgene in their B cells were prone to developing an autoimmune disease resembling SLE. After 1 yr, 60% were terminally ill with death primarily resulting from immune complex glomerulonephritis. Consistent with an autoimmune aetiology, these mice had high titres of antibodies to nuclear components including histone, double-stranded DNA (dsDNA) and Sm/ribonucleoprotein (RNP) antigens, a serological proﬁle associated with SLE . The diﬀerent in vivo eﬀects on T and B cells may be due to diﬀerences in the normal lifespan of these cells. T cells are thought to have half-lives of months to years in vivo , while B cells are estimated to live 5–7 days unless stimulated to become memory cells. Thus, T cells may be less aﬀected by bcl-2 expression because they are intrinsically long lived. Alternatively, the eﬀects observed in transgenic animals may be due to diﬀerences in the way bcl-2 expression is regulated normally. Circulating peripheral T cells normally contain substantial levels of bcl-2 protein which do not change appreciably when the cells are stimulated to proliferate. In mature B cells there is a link between signalling through the receptor for antigen and activation of apoptosis which is thought to be at the heart of the deletion process for elimination of autoreactive B cells . Bcl-2 levels in B cells decline as circulating B cells enter the germinal centres of nodes and begin to proliferate in response to antigens , making them vulnerable to death by apoptosis. Overexpression of bcl-2 may allow autoreactive B cells to survive during this period. A number of groups have now investigated bcl-2 expression in human lupus patients. 160 BRITISH JOURNAL OF RHEUMATOLOGY VOL. 36 NO. 2 BCL-2 EXPRESSION IN SLE LYMPHOCYTES One of the ﬁrst published studies of bcl-2 expression measured bcl-2 mRNA in 24 SLE patients , aged 17–68 yr, all of whom fulﬁlled the revised ARA criteria for SLE . Northern blots of RNA from total PBMCs were probed with a cDNA fragment from the bcl-2 gene. Densitometry results were normalized to the signals from a probe for glutaraldehyde-3-phosphate dehydrogenase (GAPDH). In 19 of 24 SLE patients, the concentration of bcl-2 mRNA in PBMCs was higher than the maximum value obtained from an unspeciﬁed number of healthy controls. There was no association between bcl-2 mRNA concentrations and the pattern of organ manifestation, duration of disease, concurrent therapy or serological proﬁle in these SLE patients. However, bcl-2 mRNA levels correlated with overall SLE disease activity (P Q 0.0006) assessed by the SLE Activity Index Score (SIS) . Aringer et al .  studied bcl-2 protein expression in 10 patients with SLE, ﬁve patients with rheumatoid arthritis, two chronic lymphocytic leukaemia (CLL) patients, nine healthy laboratory workers and 10 patients with severe systemic infections. SLE patients were aged 18–70 yr with a disease duration of 2–15 yr and met four or more of the ARA revised criteria for SLE . Bcl-2 was quantitated in freshly isolated PBMCs using two-colour direct immunostaining and FACS analysis. In SLE patients with active disease, assessed by the SIS score , a diﬀerent distribution pattern of the ﬂuorescence signal intensity indicated increased amounts of bcl-2 in a signiﬁcant percentage of lymphocytes. However, this pattern of bcl-2 ﬂuorescence was also observed in four patients with severe systemic infections and both CLL patients. Lymphocytes from patients with SLE had signiﬁcantly increased levels of mean ﬂuorescence intensity (MFI) of bcl-2 staining (mean 2 .. MFI 210 2 36) compared to healthy controls (mean 2 .. MFI 208 2 22) (P = 0.0002). SLE patients with increased mean expression of bcl-2 in their peripheral blood lymphocytes had a high level of disease activity. There was no association between current treatment or any type of organ involvement and MFI of bcl-2 staining in these SLE patients. In 10 patients with SLE and 10 with severe infections, lymphocytes with increased bcl-2 were all CD3+. Lymphocytes expressing high levels of bcl-2 were present in both the CD4+ and CD8+ T-cell subsets, whereas CD19+ B lymphocytes did not overexpress bcl-2 in SLE patients. This study did not examine bcl-2 expression by CD4−CD8− (double-negative) T cells. PBMCs expressing CD25 (interleukin-2 receptor) and HLA-DR (markers of activation) did not overexpress bcl-2 protein. Triple staining of lymphocytes from a further ﬁve SLE patients revealed that bcl-2 bright cells were present in both the naive (CD45RO−) and memory (CD45RO+) T cell populations. Bcl-2 mRNA quantitated by Northern blotting was elevated in PBMCs from six SLE patients compared to two healthy controls. In a study of Fas expression and bcl-2 expression in human SLE lymphocytes, Ohsako et al .  examined bcl-2 expression in freshly isolated PBMCs in ﬁve patients with active lupus, ﬁve SLE patients with inactive disease and ﬁve healthy controls. The degree of disease activity in SLE patients was determined according to the lupus activity criteria count . Using two-colour FACS analysis, the percentages of CD3 and CD22 cells expressing bcl-2 were compared. A higher percentage of CD3+ cells in SLE patients with active disease expressed bcl-2 (52–100%) (P Q 0.05) than in SLE patients with inactive disease (4–85%) or healthy controls (8–44%). Percentages of CD22+ cells expressing bcl-2 were similar in SLE patients with active disease (77–100%), inactive disease (39–95%) and in healthy individuals (62–90%). Gatenby and Irvine  studied bcl-2 protein expression in SLE patients and controls using two-colour indirect immunoﬂuorescence analysed by ﬂow cytometry. SLE patients fulﬁlled the revised ARA criteria for SLE  and disease activity was assessed by the SIS system . Forty-six SLE patients had higher percentages of bcl-2+ T cells (by a factor of 2.6) and bcl-2+ B cells (by a factor of 1.8) than 15 healthy controls. In lymphocytes from SLE patients, the percentage of cells expressing bcl-2 did not correlate with either routine serological markers of disease (ANA, anti-dsDNA antibodies, C3 or C4) or with the SIS activity score. In three patients who were followed serially, there was no apparent ﬂuctuation in the proportion of bcl-2+ B and T cells over time. Graninger  also examined bcl-2 expression in one SLE patient on two occasions. In the PBMCs from a patient with active disease, high bcl-2 mRNA concentrations were measured, while 2 months after the administration of i.v. cyclophosphamide, cells from the same patient showed no detectable bcl-2 transcription. We examined bcl-2 protein expression in freshly isolated PBMCs from 73 lupus patients and found no signiﬁcant diﬀerences in bcl-2 expression compared with cells from 40 autoimmune controls and 30 healthy individuals . However, diﬀerences in bcl-2 expression by lymphocyte subsets may be masked by measurements of unfractionated PBMCs; therefore, we have conducted a further study of bcl-2 expression in lymphocytes of 42 lupus patients, 21 healthy individuals and 35 patients with rheumatoid arthritis (unpublished observations). Freshly isolated PBMCs were ﬁxed in 1% paraformaldehyde and using direct immunoﬂuorescence cells were stained with antibodies to CD3, CD4, CD8 and CD19. After permeabilization with acetone and methanol, cells were stained for intracellular bcl-2 protein and analysed using ﬂow cytometry. In most samples, over 80% of peripheral lymphocytes expressed bcl-2. Previous studies have reported abundant bcl-2 protein in circulating normal lymphocytes [24, 25]. The mean ﬂuorescence intensity of bcl-2 staining was similar in all lymphocyte subsets in all samples tested. There were no signiﬁcant diﬀerences between lupus patients and healthy individuals or autoimmune controls in the percentages of cells ROSE ET AL .: BCL-2 EXPRESSION IN SLE
TABLE I Summary of studies to date of bcl-2 expression in lymphocytes from patients with SLE and healthy controls Authors (n SLE, n healthy controls) Graninger 1992  (24, unspeciﬁed) Ohsako et al ., 1994  (46, 5) Gatenby and Irvine, 1994  (46, 15) Aringer et al ., 1994  (10, 9) (6, 2) Rose et al ., 1995  (73, 30) NT = not tested. Correlation with disease T B Qbcl-2 activity cells cells + + + + + + + + − + NT + NT + + + NT + NT − + − NT − 161 Assay message protein (% +ve) protein (% +ve) protein mRNA protein (% +ve) expressing bcl-2 which were CD3+, CD4+, CD8+ or CD19+. However, SLE patients had a higher percentage of bcl-2+ cells in the CD4−CD8− (double-negative) T-cell subset (91.7 2 6.6%) than the healthy controls (59.0 2 7.0%) (P Q 0.05) and autoimmune controls (76.4 2 7.2%) (P Q 0.05). In the SLE patients in this study, there was a strong correlation between disease activity and the percentages of double-negative T cells expressing bcl-2 (r = 0.536, P Q 0.001). Lupus disease activity was assessed using the British Isles Lupus Assessment Group (BILAG) computerized index . This index utilizes the principle of the physician’s intention to treat, unlike the other global indices used in the other studies reported here [32, 35]. Percentages of cells expressing bcl-2 in the other lymphocyte subsets examined did not correlate with disease activity. Bcl-2 expression did not correlate with concurrent therapy or HLA status in SLE patients. DISCUSSION Most of the studies described here have reported abundant bcl-2 protein in circulating normal lymphocytes [33, 34, 37]. The high incidence of bcl-2 expression suggests that bcl-2 plays an important role in the survival of lymphocytes in the periphery. Several studies indicate that the blood of some SLE patients contains T cells that overexpress bcl-2 protein [33, 34] (summarized in Table I), but increased expression in peripheral B lymphocytes is generally not observed. Gatenby and Irvine  describe increased percentages of bcl-2+ T cells in SLE patients with active disease compared with control subjects. In our study of a large number of SLE patients (unpublished observations), a strong correlation was observed between disease activity and the proportion of the double-negative T-cell population expressing bcl-2. This suggests an important role for this T-cell population in the pathogenesis of SLE, and indicates that high levels of bcl-2 in these cells may ensure their survival. Further data supporting an important role for these doublenegative T cells comes from a study in which autologous T cells were co-cultured with B cells from patients with active lupus to study their eﬀect on the production of pathogenic IgG autoantibodies . While CD4+CD8−-enriched T cells provided maximum help for autoantibody production, double-negative (CD4−CD8−) T cells from patients with active lupus were able to function as Th cells to augment the spontaneous production of cationic IgG anti-DNA antibodies. The percentages of T and B cells staining with anti-bcl-2 were slightly higher in our study  than those published by Ohsako et al . . This may be attributable to diﬀerences in methodology, i.e. we used direct immunoﬂuorescent staining and lymphocytes were permeabilized using acetone and methanol, while Ohsako et al .  employed indirect immunostaining after permeabilization with digitonin. However, both our study and those of Ohsako et al .  and Aringer et al .  report that the majority of peripheral T and B cells express bcl-2 in both SLE patients and controls. Only one study  reports increased percentages of bcl-2+ B cells in SLE patients. The mean percentages of bcl-2+ B cells (13.6%) and T cells (8.1%) in their study in both patients and controls are considerably lower than those reported by other groups using FACS staining with the same anti-bcl-2 monoclonal antibody [33, 34]. It is also noteworthy that the authors report that there was no signiﬁcant diﬀerence between patients and controls in the proportions of either T or B cells in the periphery. SLE patients with active disease frequently have decreased numbers of T cells  and increased numbers of B cells . Although truly resting T cells are resistant to apoptosis, activation normally makes peripheral T cells (including CD4−CD8− cells) sensitive to apoptosis . A decrease in bcl-2 expression has been described in normal T lymphocytes after the transition from the CD45RO− (naive) to CD45RO+ (memory) phenotype . However, Aringer et al .  have reported bcl-2 bright cells in both the CD45RA+ and CD45RO+ T-cell fractions of SLE patients. Levels of bcl-2 do not usually change appreciably when freshly isolated, unfractionated peripheral lymphocytes are stimulated to proliferate. As yet, there are no conclusive data regarding the regulation of bcl-2 in freshly isolated lymphocyte subsets in response to proliferative stimuli. Bcl-2 expression could be aﬀected in SLE either intrinsically due to genetic predisposition or it could be upregulated as a consequence of lymphoid overreactivity , or a combination of both mechanisms may be in operation. Despite the increased rate of bcl-2 expression observed in some lymphocytes from SLE patients, the rate of in vitro apoptosis has been shown to be increased in SLE lymphocytes . This observation creates an apparent paradox whereby enhanced bcl-2 expression by lymphocytes in patients with SLE may allow the inappropriate survival of autoreactive cells while lymphocytes from SLE patients show increased apoptosis in vitro . It is not known whether this increased apoptosis occurs in vivo , but it would result in the release of nucleosomes which may provide a 162 BRITISH JOURNAL OF RHEUMATOLOGY VOL. 36 NO. 2 source of autoantigens to stimulate autoantibody production. The types of peripheral lymphocyte populations aﬀected by increased apoptosis have not yet been characterized and it is possible that both mechanisms occur in diﬀerent lymphocyte subsets. The mechanism by which bcl-2 suppresses apoptosis is not yet clear. Regulation of apoptosis in all cells is probably achieved by the balance between inducers and inhibitors of apoptosis. Bcl-2 homologues have been described which can also inﬂuence cell survival. Bax forms heterodimers with bcl-2 and can accelerate programmed cell death , while the two protein products of the bcl -x gene can either delay (bcl-xL) or accelerate apoptosis (bcl-xS) . Other members of the bcl-2 family of proteins include Bik  and Bak , which are inducers of apoptosis and can interact with other cellular survival-promoting proteins. The recent reports of eight children, with lymphoproliferative syndromes, with mutations in Fas focus attention on Fas as a regulator of apoptosis [18, 19]. Seven of these children had autoimmune disorders, suggesting that the crucial regulatory role of Fas could play a part in diseases such as SLE. The signiﬁcance of the ﬁnding of a soluble form of Fas in human sera is still unclear. In vivo , soluble Fas can induce lymphadenopathy and splenomegaly, and it has been suggested that it may contribute to the pathology of SLE by inhibiting the Fas-mediated elimination of activated lymphocytes . However, elevated levels of sFas are not a consistent feature of SLE patients and do not correlate with disease activity [16, 17]. Furthermore, a soluble form of the Fas ligand has been detected which makes it more diﬃcult to predict the eﬀect of sFas in vivo . The relationships between inducers and inhibitors of apoptosis may diﬀer in diﬀerent cell types and need to be elucidated before the implications of the observations made in lymphocytes from SLE patients can be fully understood. CONCLUSIONS There is now considerable evidence that bcl-2 expression is enhanced in a proportion of T cells, but not in B cells, in the periphery of patients with SLE. Enhanced bcl-2 expression by T cells in patients with SLE correlated with overall disease activity regardless of the activity index employed. A link between bcl-2 expression and activation in normal peripheral T cells has been proposed , although in a single study of T cells from SLE patients, cells expressing high levels of bcl-2 were present in both the naive and memory subsets . Clearly, further work must be carried out to establish whether enhanced bcl-2 expression by certain T cells in patients with SLE is related only to the activation of those cells or whether bcl-2 expression is intrinsically enhanced by genetic predisposition. It is also clear that other molecules which regulate apoptosis may contribute to the establishment, maintenance or acceleration of autoimmune diseases such as SLE. Further research to elucidate the regulation of apoptosis may lead to the development of treatments targeted towards altering the apoptotic threshold which could alleviate the progression of some autoimmune diseases. A We gratefully acknowledge the support of the Arthritis and Rheumatism council.
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