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Keywords:

  • CD34+ cells;
  • apoptosis;
  • stromal cells;
  • systemic lupus erythematosus (SLE);
  • long-term bone marrow cultures (LTBMCs)

Abstract

  1. Top of page
  2. Abstract
  3. Patients and methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References

The changes in bone marrow (BM) stem cell reserve and function and stromal cell function in patients with active systemic lupus erythematosus (SLE) were investigated. The study was carried out on seven SLE patients and 28 healthy controls using flow cytometry and in vitro cell culture assays. We found that patients had low CD34+ cells, compared with the control group, reflecting the decrease of both CD34+/CD38 and CD34+/CD38+ cells. Patient CD34+/Fas+ but not CD34/Fas+ cells were significantly increased. Apoptotic (7AADdim) cells were higher among CD34+/Fas+ than among CD34+/Fas cells, and individual values of apoptotic CD34+ cells strongly correlated with the number of CD34+/Fas+ cells. These findings are suggestive of a Fas-mediated apoptosis accounting for the low CD34+ cells in SLE patients. Moreover, we found that patients had low numbers of granulocyte-macrophage colony-forming units (CFU-GM) and erythroid burst-forming units (BFU-E), compared with the control group, and that the generation of colony-forming cells in long-term BM cultures was significantly reduced. Patient BM stroma failed to support allogeneic progenitor cell growth. In one patient, CD34+ cells were increased, apoptotic CD34+/Fas+ cells were normalized and defective stromal cell function was restored after autologous stem cell transplantation. We concluded that defective haemopoiesis in SLE patients is probably caused, at least in part, to the presence of autoreactive lymphocytes in BM.

High-dose chemotherapy with immunoablative intent followed by autologous stem cell transplantation (ASCT) has been proposed recently as a putative treatment modality for severe autoimmune diseases including systemic lupus erythematosus (SLE) refractory to conventional medications (Tyndall & Gratwohl, 1997; van Bekkum, 1999; Burt et al, 2000; Traynor et al, 2000). The rationale for this therapeutic approach relies on the fact that a vigorous preparative regimen may eliminate autoreactive immunocompetent lymphocytic clones leading to immune deregulation, whereas the subsequent ASCT may rescue the patient from prolonged cytopenias. It has also been suggested that immune reconstitution after stem transplantation, even by autologous stem cells, may lead to ontogenic re-organization of the immune system with re-induction of tolerance to self-antigens (Emmons & Quesenberry, 1999).

Immune deregulation in autoimmune diseases may affect bone marrow (BM) progenitor cell development at several stages of differentiation and/or BM stromal cell function (Otsuka et al, 1988; Atta et al, 1994). Ikehara (1998a), based on animal models, suggested that autoimmune diseases may be seen as primary stem cell disorders. In humans, serum autoantibodies capable of inhibiting granulocyte and erythroid colony formation in vitro were detected in a number of SLE patients (Bailey et al, 1989; Liu et al, 1995). T-lymphocyte-mediated suppression of colony growth, even at the level of multipotential haemopoietic progenitors, as well as antibodies directed against fibronectin affecting cell-to-matrix interactions, have also been reported (Otsuka et al, 1988; Atta et al, 1994). However, little is known about the number and functional characteristics of BM stem cells, and even less about the capacity of BM microenvironment to support progenitor cell growth in SLE patients. In the current study, we present data suggestive of the existence of significant changes in BM stem cell reserve and function and BM stromal cell function in SLE patients. The data from a patient studied before and after ASCT are also discussed.

Patients and methods

  1. Top of page
  2. Abstract
  3. Patients and methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References

Patients Seven patients with active SLE, one man and six women aged 24–59 years (median age 37 years) were enrolled in the study. All fulfilled the diagnostic criteria of SLE proposed by the American College of Rheumatology (Tan et al, 1982). In all patients, BM samples were drawn 24 h after steroid administration. As the control group, 28 healthy subjects, five men and 23 women aged 24–62 years (median age 38 years), were used. No statistically significant differences could be demonstrated in age and sex distribution between patients and control subjects by applying the χ2-test. Informed consent was obtained from all subjects included in the study. Some clinical and laboratory characteristics of the patients studied are presented in Table I.

Table I.  Some clinical and laboratory characteristics of the patients studied.
 Patient 1Patient 2Patient 3Patient 4Patient 5Patient 6Patient 7
  1. WBC, white blood cells; ANA, antinuclear antibodies; Anti-dsDNA, antibodies against double stranded DNA; PDN, prednisone; CyS, cyclosporine; CPM, cyclophosphamide; AZP, azathioprine; IVIG, intravenous immunoglobulin; n.d., not done. *Anti-PMN antibodies were tested in the sera using leucoagglutination and immunofluorescent tests against a panel of normal neutrophils. †Anti-platelet antibodies were tested in the sera using the MAIPA method (monoclonal antibody immobilization of platelet antigen assay).

Age (years)24323756594234
SexFMFFFFF
Disease duration (years)676122464
Previous medicationPDNPDN/CySPDN/CPMPDNPDN/AZPPDNPDN/IVIG
Current medicationPDN (5 mg/d)PDN (10 mg/d)PDN (20 mg/d)PDN (8 mg/d)PDN (30 mg/d)PDN (5 mg/d)PDN (10 mg/d)
Haemoglobin (g/dl)12·310·99·911·810·711·811·6
Reticulocytes ( × 109/l)28385635556745
WBC ( × 109/l)6·203·403·103·604·304·107·30
Neutrophils ( × 109/l)3·721·951·902·081·201·834·75
Lymphocytes ( × 109/l)2·321·240·851·422·921·982·10
Platelets ( × 109/l)23187154134174818
Coombs testNegativePositivePositiveNegativePositiveNegativePositive
Anti-PMN Abs*n.d.PositivePositiven.d.NegativeNegativen.d.
Anti-Plts AbsPositiveNegativeNegativeNegativePositivePositivePositive
ANA1:3201:12801:6401:1601:3201:3201:160
Anti-dsDNANegative1:160Negative1:801:801:40Negative
C3 (mg/dl)1073060785011387
C4 (mg/dl)12·33·09·010·36·010·49·3
Creatinine (µmol/l)79·198·265·579·274·587·384·5

Preparation of bone marrow samples BM samples obtained from posterior iliac crest aspirates were immediately diluted 1:1 in Iscove's modified Dulbecco's medium (IMDM; Gibco BRL, Life Technologies, Paisley, Scotland) supplemented with 100 IU/ml penicillin-streptomycin (PS; Gibco BRL, Life Technologies) and 10 IU/ml preservative-free heparin (Leo). Diluted samples were centrifuged on Ficoll–Paque 1·077 g/cm3 density (Amersham Pharmacia Biotech, Uppsala, Sweden) at 400 g for 35 min at room temperature. Separated mononuclear cells (BMMCs) were removed from the interphase, washed twice with medium, viability tested with trypan blue, and resuspended in complete culture medium at a concentration of 107/ml.

Immunophenotyping and 7-amino-actinomycin D (7AAD) staining An indirect immunofluorescence technique was used for quantification of BM CD34+ cells. Briefly, 106 BMMCs washed twice in phosphate-buffered (PBS) supplemented with 1% fetal calf serum (FCS; PAA Laboratories GmbH, Linz, Austria) and 0·05% azide (PBS-1%FCS) were incubated with 40 µl of human gamma-globulin for 10 min on ice. Next, 10 µl of each, fluorescein isothiocyanate (FITC)-conjugated mouse anti-human CD34 monoclonal antibody (HPCA-2; Becton Dickinson, Oxford, UK) and phycoerythrin (PE)-conjugated mouse anti-human CD38 monoclonal antibody (HIT-2; PharMingen, Becton Dickinson) were added, and cells were incubated for 30 min on ice. FITC-and PE-conjugated mouse IgG isotype-matched controls were used as negative controls. Cells were washed twice in PBS-1%FCS and fixed in 500 µl of 2% paraformaldeyde solution (Sigma; St Louis, MO, USA). Samples were analysed in a FACScan flow cytometer within 24 h of fixation.

In parallel, 106 BMMCs stained with 10 µl of PE-conjugated anti-CD34 monoclonal antibody (HPCA-1; Becton Dickinson) and 20 µl of FITC-conjugated mouse anti-human Fas (CD95) monoclonal antibody (LOB 3/17; Serotec, UK), were further stained before fixation, with 7AAD (Calbiochem-Novabiochem, La Jolla, CA, USA) as previously described (Philpott et al, 1996). Briefly, 100 µl of 7AAD solution (7AAD was dissolved in acetone and diluted in PBS at a concentration of 200 mg/ml) were added to cells suspended in 1 ml PBS, and incubated for 20 min on ice protected from the light. After centrifugation, supernatant fluid was discarded and cells were fixed in paraformaldeyde as above. Unstained fixed cells were used as negative controls. Samples were analysed within 30 min of fixation. Data on 200 000 events were acquired and processed using cellquest software. After creating a scattergram combining forward and right-angle light scatter for the whole population, a region was drawn around BMMCs (low forward and low right scatter properties) for CD34+ and CD38+ cell estimation. When cells were stained for CD34 antigen, Fas antigen and 7AAD, a scattergram was created by combining right-angle light scatter with CD34 fluorescence for the whole nucleated cell population excluding cell debris, and a second scattergram was generated by combining CD34 and Fas fluorescence for the CD34-positive cell population. Finally, a scattergram was generated by combining forward light scatter with 7AAD fluorescence to quantify 7AAD-neg, -dim, -bright cells in the gate of CD34+, CD34+/Fas+ and CD34+/Fas cells. Regions were drawn around clear-cut populations, and the proportion of cells within each region was calculated (Fig 1).

image

Figure 1. Normal BMMCs stained with anti-CD34 antibody, anti-Fas (CD95) antibody and 7AAD: (A) Scattergram of FSC versus SSC to allow gating on the whole nucleated cell population (R1). (B) Scattergram of FL2 versus SSC gated on RI to allow gating on CD34+ CELLS (R2). (C) Scattergram of FL3 versus FSC gated on R2 showing 7AADbright (dead), 7AADdim (apoptotic), and 7AADneg (live) cells. (D) Scattergram of FL2 versus FL1 gated on R2 showing Fas+ and Fas CD34+ cells. FSC, forward light scatter; SSC, right angle light scatter; FL1, anti-Fas fluorescence; FL2, anti-CD34 fluorescence; FL3, 7AAAD fluorescence. Similar staining patterns were obtained for SLE bone marrow samples.

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Clonogenic progenitor cell assays BMMCs (105) were cultured in 1 ml of IMDM supplemented with 30% pre-selected FCS, 1% pre-selected bovine serum albumin (BSA; Sigma), 10−4M mercaptoethanol (Sigma), 0·075% sodium bicarbonate (Gibco BRL, Life Technologies), 2 mmol/l l-glutamine (Sigma), 0·9% methylcellulose (StemCell Technologies, Vancouver, Canada), in the presence of 5 ng of granulocyte-macrophage colony-stimulating factor (GM-CSF; Novartis Pharm., Camberley, Surrey, UK), 50 ng Interleukin 3 (IL-3; Novartis) and 2I IU erythropoietin (EPO; Janssen-Cilag, Buckinghamshire, UK). Cultures were set up in duplicate in 35 mm Petri dishes (Nunclon) and incubated in a humid atmosphere at 37°C 5%CO2. On d 14, colonies were scored using an inverted microscope and were classified as granulocyte colony-forming units (CFU-G), macrophage colony-forming units (CFU-M), granulocyte-macrophage colony-forming units (CFU-GM), erythroid burst-forming units (BFU-E) and colonies containing granulocyte, macrophage and erythroid elements (CFU-GEM), according to previously established criteria (Coutinho et al, 1993). Results were expressed as total number of CFU-GM (CFU-G plus CFU-M plus CFU-GM) and total number of BFU-E (BFU-E plus CFU-GEM).

Long-term bone marrow cultures (LTBMCs) BMMCs (107) were cultured into 25-cm2 plastic flasks in 10 ml of IMDM (340mOsm/Kg) supplemented with 10% FCS, 10% horse serum (HS; LabTech International Limited, Ringmer, East Sussex, UK), 100 IU/ml penicillin, 100 µg/ml streptomycin, 2 mmol/l l-glutamine and 10−6mol/l hydrocortisone sodium succinate (Sigma), in a humid incubator of 33°C 5%CO2 according to a standard technique (Gibson & Gordon-Smith, 1990; Marsh et al, 1990). At weekly intervals, cultures were examined for stromal cell layer formation using an inverted microscope and fed by removing half of the medium and replacing it with an equal volume of fresh IMDM supplemented as above. Non-adherent cells were counted, resuspended in IMDM and assayed for CFU-GM and BFU-E progenitor cells as described. Colony results were expressed as total numbers of colony-forming cells (CFC), i.e. CFU-GM plus BFU-E.

Assessment of BM stromal cell function A two-stage culture procedure was used to test the capacity of BM stromal layers to support normal haemopoiesis. Confluent stromal layers from patients and normal controls, grown in standard LTBMC (after 4–6 weeks of culture) were irradiated (10 Gy) and recharged with the same normal allogeneic CD34+ BM cells (5 × 104/10 ml IMDM) (Marsh et al, 1991). CD34+ cells were isolated from BMMCs by magnetic labelling using a MACS isolation kit (Miltenyi Biotec, Bergisch Gladbach, Germany) according to the manufacturer's instructions. At weekly intervals, half of the medium and non-adherent cells were removed and replaced by fresh medium as described. Culture supernatants were monitored by determining the total number of non-adherent cells and the frequency of CFC generated weekly in the culture.

Statistical analysis Data were analysed using the GraphPad Prism statistical PC program (San Diego, CA. USA). Standard two-way analysis of variance was used to define differences in the number of non-adherent cells and CFCs generated in LTBMCs. All other numerical data were analysed with the non-parametric Mann–Whitney U-test, and the Spearman's coefficient of correlation test. A probability ≤ 5% was considered as statistically significant.

Results

  1. Top of page
  2. Abstract
  3. Patients and methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References

Flow-cytometric analysis of BM CD34+ cells

The data from the flow-cytometric analysis of BM CD34+ cells are shown in Table II. The mean proportion of CD34+ cells among BMMCs was significantly lower in the group of patients than in the group of control subjects (P = 0·0003, Mann–Whitney test). This decrease reflected the reduction of both the committed CD34+/CD38+ and the more primitive CD34+/CD38 stem cells compared with the controls (P = 0·0008 and P = 0·0118 respectively; Mann–Whitney test). Interestingly, the degree of reduction was of the same magnitude in both cell populations (58·4% and 61·7% respectively, P = 0·7938, anova test).

Table II.  Flow cytometric analysis of BM cells.
 SLE patients (n = 7)Normal controls (n = 28)
  1. BMMCs, bone marrow mononuclear cells; 7AAD, 7-amino-Actinomycin D. Statistical analysis was performed using the non-parametric Mann–Whitney U-test. *Denotes comparison between patients and normal controls. **Denotes comparison between the percentages of apoptotic CD34+/Fas+ and apoptotic CD34+/Fas cells in each group studied. †All values are expressed as means ±1SD.

BMMC fraction
 % CD34+ cells0·89 ± 0·54†2·17 ± 0·79
 Median (range)0·98 (0·11–1·61)2·07 (1·10–3·83)
 P-value*0·0003 
 % CD34+/CD38+ cells0·71 ± 0·441·71 ± 0·67
 Median (range)0·79 (0·08–1·32)1·70 (0·80–2·99)
 P-value*0·0008 
 % CD34+/CD38 cells0·18 ± 0·100·47 ± 0·28
 Median (range)0·19 (0·03–0·29)0·44 (0·10–1·18)
 P-value*0·0118 
CD34+ cell fraction
 % Fas+ cells42·20 ± 24·808·79 ± 4·05
 Median (range)44·51 (18·80–58·62)9·22 (1·59–17·8)
 P-value*< 0·0001 
 % 7AADdim cells38·38 ± 11·615·39 ± 3·21
 Median (range)37·87 (23·80–58·23)4·82 (1·20–11·04)
 P-value*< 0·0001 
CD34+/Fas+ cell fraction
 % 7AADdim cells51·66 ± 10·6223·75 ± 13·26
 Median (range)51·02 (33·07–69·34)25·10 (1·05–46·73)
 P-value*0·0002 
CD34+/Fascell fraction
 % 7AADdim cells12·27 ± 3·736·67 ± 6·87
 Median (range)12·45 (8·16–19·30)5·16 (0·92–32·00)
 P-value*0·0032 
 P-value**< 0·001< 0·0001

To investigate whether the decrease in CD34+ cells may be caused by increased apoptotic cell death, we first determined the expression of Fas antigen on CD34+ cells, a surface molecule frequently involved in apoptosis-related pathway (Nagata & Goldstein, 1995). We assumed that the time required for cell death and disintegration was the same for SLE and normal cells, although this assumption may need validation. We found that patient CD34+ cells contained significantly higher numbers of Fas+ cells than the normal controls (P < 0·0001, Mann–Whitney test). In contrast, no significant difference could be demonstrated between patients and normal controls in the proportion of Fas+ cells detected within the non-CD34+ BM cell fraction (P = 0·2930, Mann–Whitney test). Moreover, we found that the compartment of patient CD34+ cells contained significantly higher numbers of apoptotic (7AADdim) cells than the respective one of controls (P < 0·0001, Mann–Whitney test). A highly significant correlation was noted between the proportion of apoptotic cells and the proportion of Fas+ cells within the CD34+ cell population (r = 0·5921, P = 0·0002, Spearman's r-test). Notably, the proportion of apoptotic cells was higher among the CD34+/Fas+ cells than among the CD34+/Fas cells in both SLE patients and normal controls (P = 0·0006 and P < 0·0001 respectively; Mann–Whitney test). These data suggest that Fas-mediated apoptosis may account for the decreased number of CD34+ cells in SLE patients.

Clonogenic progenitor cell assays

Quantitative and qualitative evaluation of clonogenic progenitor cells was performed by enumerating CFU-GM and BFU-E colonies after a 14 d BMMC culture (Coutinho et al, 1993). Results are shown in Table III. SLE patients had significantly lower CFU-GM and lower BFU-E colonies per 107 BMMCs than the normal controls (P = 0·0012 and P = 0·0011 respectively; Mann–Whitney test). The degree of reduction was approximately the same in both cell populations (58·7% and 48·7% respectively; P = 0·3182, anova test). In the entire group of subjects studied, the proportion of CD34+ cells strongly correlated with the number of both CFU-GM and BFU-E (r = 0·6033, P = 0·0001 and r = 0·5436, P = 0·0007 respectively; Spearman's r-test), suggesting that the decreased number of these clonogenic cells found in our patients, probably reflected the low number of CD34+ cells included in the BMMC cell suspensions tested. Moreover, the fact that the degree of reduction in all four cell populations, CD38+/CD38+, CD34+/CD38, CFU-GM and BFU-E, was of the same magnitude (F = 0·5309 < inline image at 5%; one-way variance analysis test) indicates that defective haemopoiesis in SLE patients is not stage-specific.

Table III.  Clonogenic potential of BMMCs.
 Patients (n = 7)Controls (n = 28)P-value
  1. *All values are expressed as means ±1 SD per 107 BMMCs; median and range are shown in parentheses. †Comparison of values between patients and control subjects was performed with the non-

  2. parametric Mann–Whitney U-test; statistically significant at P ≤ 0·05.

CFU-GM colonies3411 ± 1678* (3800, 980–5900)8269 ± 3735 (7350, 2100–16200)P = 0·0012
BFU-E2157 ± 700 (2300, 1100–3200)4179 ± 173 3900, 2000–9800)P = 0·0011

Long-term bone marrow cultures

To evaluate the primitive stem cell compartment and the interactions existing between BM haemopoietic and stromal cells, we set up LTBMCs by allowing the formation of adherent cell layers that are structurally and functionally similar although not identical to the in vivo BM microenvironment. This culture system provides the ability to evaluate the capacity of BM microenvironment to support the growth and differentiation of primitive stem cell for up to 8–12 weeks (Coutinho et al, 1993). In LTBMCs initiated from patient BMMCs, typical stromal layers containing fibroblast-like cells, fat cells, macrophages, endothelial cells and closely packed haemopoietic foci known as ‘cobblestone areas’ were regularly formed over the first 3 weeks of culture, as in LTBMCs initiated from normal BMMCs (data not shown). The average number of nucleated cells generated in the non-adherent cell fraction over a period of 5 weeks did not differ significantly between patients and normal controls (P > 0·05; two-way analysis of variance). However, the frequency of colony-forming cells (CFCs) in the non-adherent cell fraction was significantly reduced in the group of patients compared with controls (P < 0·001; two-way analysis of variance). Cultures from SLE patients failed to produce colonies beyond week 7 (median 5 weeks, range 3–7 weeks), whereas cultures from normal subjects continued to produce colonies even 12 weeks after the initiation of culture (median 9 weeks, range 6–12 weeks).

Assessment of BM microenvironment function

To assess BM microenvironment function independently of autologous stem cell function, confluent stromal layers from SLE patients and normal controls were irradiated to eliminate endogenous haemopoiesis and recharged by allogeneic CD34+ BM cells derived from the same normal donor. Data from these experiments are shown in Fig 2. Patient LTBMC adherent layers failed to support progenitor cell growth in a manner similar that of the control group, as indicated by the low number of non-adherent cells recovered (P < 0·001; two-way analysis of variance) and the low frequency of CFCs detected in the non-adherent cell fraction (P < 0·05; two-way analysis of variance) over a period of 5 weeks after CD34+ cell inoculum.

image

Figure 2. Bone marrow stromal cell function of SLE patients in a two-stage LTBMC system. Irradiated LTBMC stromal layers from patients (n = 7) and normal controls (n = 7) were recharged with 5 × 104 allogeneic normal CD34+ bone marrow cells (week 0). The total number of non-adherent cells and the clonogenic progenitor cell content of the non-adherent cell fraction were evaluated over the time after the CD34+ cell inoculum. Each point in the upper diagram represents the mean ±SEM number of non-adherent cells from patient and normal cultures, and in the lower diagram the mean ±SEM frequency of CFCs. Differences between the distribution were tested with the two-way analysis of variance.

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Post-transplantation data

In one patient undergoing ASCT (Patient 3, Table I), CD34+ cells were analysed at d +90 and results were compared with those found before transplantation. The proportion of CD34+ cells among BMMCs was increased after ASCT (1·08% post- versus 0·39% pre-ASCT), but it remained below the range of the respective values found in the controls. This increase reflected mainly the rise in the proportion of committed CD34+/CD38+ cells (0·99% post- versus 0·30% pre-ASCT) because the proportion of the more primitive CD34+/CD38 cells remained at low levels (0·09% post- versus 0·09% pre-ASCT). Interestingly, the proportion of CD34+ cells expressing the Fas antigen was normalized after transplantation (2·9% post- versus 49·3% pre-ASCT), and it was associated with a significant decrease in the proportion of apoptotic cells within the CD34+ cell compartment (6·0% post- versus 65·7% pre-ASCT).

Colony formation by 107 patient BMMCs was significantly reduced after ASCT. The number of CFU-GM decreased from 5900 pre-ASCT to 4300 post-ASCT, and that of BFU-E from 2400 pre-ASCT to 1100 post-ASCT. In contrast, defective patient stromal cell function in terms of its inability to sustain allogeneic haemopoiesis was normalized after ASCT, as indicated by the normal number of non-adherent cells and the normal frequency of CFCs recovered from LTBMCs initiated from normal stem cells plated on patient-irradiated stromal cell layers.

Discussion

  1. Top of page
  2. Abstract
  3. Patients and methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References

There is increasing evidence that haemopoiesis may be severely disturbed in patients with SLE. It has been suggested that many of these disturbances could be attributed to the presence of autoreactive immunocompetent lymphocytes and the action of a variety of pro-inflammatory cytokines (Otsuka et al, 1993; Tsokos, 1994). However, BM stem cell reserve and function and BM microenvironment function have not extensively been studied in SLE patients. The present study describes a stem cell defect in SLE patients indicated by the low number of CD34+ cells in flow cytometry, the low frequency of CFU-GM and BFU-E in clonogenic assays, the low progenitor cell recovery in LTBMCs and the increased proportion of apoptotic cells within the CD34+ cell compartment. from a theoretical point of view, these abnormalities could be attributed either to a primary stem cell defect (Ikehara, 1998a) or to a stem cell damage closely related to immune deregulation in SLE (Tsokos, 1994) or even to an abnormal function of BM microenvironment (Otsuka et al, 1993). The possibility for a drug-related effect cannot be ruled out for certain, but it seems unlikely here as only two of our patients had ever been exposed to cytotoxic drugs such as azathioprine or cyclophosphamide. Corticosteroids and cyclosporine can induce apoptosis in mitogen-stimulated peripheral blood mononuclear cells (Horigome et al, 1997) but, to our knowledge, they do not damage the haemopoietic stem cells. In contrast, there is strong evidence for a positive effect of corticosteroids on the proliferation and differentiation of BM progenitor cells (Rinehart et al, 1997; Von Lindern et al, 1999; Clausen et al, 2000).

A primary stem cell defect in autoimmune diseases has been suggested by Ikehara (1998a, b) in animal models, but there is no evidence for such a defect in humans. In keeping with the latter aspect is the observation that both increased proportion of Fas+ cells and increased proportion of apoptotic cells within the CD34+ cell population returned to normal range and were associated with a significant rise in the number of CD34+ cells after ASCT in one of the patients studied. On the other hand, the defective colony formation found in our patient after ASCT does not necessarily indicate a primary stem cell defect in SLE given that significant reduction in the clonogenic potential of CD34+ cells may be seen for several years after the recovery from autologous or allogeneic stem cell transplantation performed for unrelated reasons (Novitzky & Mohammed, 1997).

The role of autoreactive immunocompetent lymphocytes in the pathogenesis of SLE has been studied previously by several investigators. Because these cells are present in patient BMMCs and LTBMC adherent layers (Berneman et al, 1989; Otsuka et al, 1993), one could postulate that a mechanism of immune-mediated inhibition of colony formation, or even an immune-mediated apoptotic stem cell depletion, may be operative in SLE patients. Indeed, removal of T lymphocytes from SLE marrow samples has been reported to result in a significant rise in the progenitor cell clonogenic potential (Kiely et al, 1995). Moreover, it has been shown that Fas-mediated induction of apoptosis by immune cytokines is normally involved in the maintenance of homeostasis in BM haemopoietic progenitor cell population (Nagafuji et al, 1995). In the present study, Fas antigen was significantly upregulated in patient CD34+ cells, and a highly significant correlation was noted between the proportion of apoptotic cells and the proportion of Fas+ cells within the CD34+ cell compartment. Although a non-functional accumulation of Fas in patient CD34+ cells cannot be ruled out, this hypothesis seems unlikely because the proportion of apoptotic cells was much higher in the Fas-positive than in the Fas-negative stem cell population in the subjects studied. This suggests that the Fas pathway is actively involved in the induction of apoptosis in BM progenitor cells in SLE patients. Although Fas expression does not always lead to apoptotic cell death (Miyawaki et al, 1992), involvement of Fas antigen in the induction of apoptosis within the stem cell compartment has already been well documented in a number of other disease states (Maciejewski et al, 1995; Saheki et al, 2000).

The mechanisms leading to upregulation of Fas in CD34+ cells in SLE patients are unknown. Normal BM CD34+ cells do not express Fas antigen on their surface, or they express the molecule at a proportion < 10% (Maciejewski et al, 1995; Saheki et al, 2000). It has been reported that interferon-gamma (IFN-γ) and tumour necrosis factor-alpha (TNF-α) can induce the expression of functional Fas molecules on cultured normal haemopoietic progenitor cells (Nagafuji et al, 1995). Although IFN-γ and TNF-α have rarely been found significantly increased in the sera of SLE patients (Aderka et al, 1993; Lacki et al, 1997), they can be produced in increased amounts at sites of tissue inflammation such as in the kidney (Lacki et al, 1997; Kelley & Wuthrich, 1999). An aberrant TNF-α mRNA expression in BM has also been reported in SLE patients (Alvarado-de la Barrera et al, 1998). Fas-mediated apoptosis of haemopoietic stem cells caused by increased production of TNF-α and IFN-γ by activated BM cytotoxic T lymphocytes has been well documented in patients with aplastic anaemia (Maciejewski et al, 1995), a disease usually caused by immune-mediated inhibition of haemopoiesis (Zoumbos et al, 1985; Young, 1995).

The present study also describes a defect in BM microenvironment in SLE patients, in terms of the inability of patient LTBMC adherent layers to support haemopoietic progenitor cell growth in a two-stage cell culture system. The fact that this defective function has been restored by ASCT provides additional evidence for the secondary cause of the disturbance, which may be caused by the presence of autoreactive lymphocytes within the stromal cell population. The observation that isolated BM fibroblasts from SLE patients gave normal fibroblast colonies in vitro (Otsuka et al, 1993) is in agreement with this suggestion. On the other hand, abnormally produced cytokines in SLE (Tsokos, 1994; Horwitz et al, 1998), may also affect the ability of BM microenvironment to support haemopoiesis.

In conclusion, BM primitive CD34+/CD38 and committed CD34+/CD38+ stem cells were both reduced in our SLE patients. Patients had also increased apoptosis within the CD34+ cell compartment, reduced frequency of CFC and defective haemopoiesis supporting capacity of LTBMC adherent layers. The cause and the underlying pathogenetic mechanisms of these abnormalities are presently unknown, but it seems to be related to a defective function of BM microenvironment rather than a primary stem cell defect. The role of autoreactive lymphocytes and the changes in the cytokine network within the BM microenvironment remain to be elucidated. Our data also provide some evidence that an intensive immunosuppression to eradicate autoreactive lymphocytic clones, followed by an autologous stem cell graft to offer probably a more normal set of haemopoietic cells may restore, at least in part, some of the abnormalities of haemopoiesis found in SLE patients.

Acknowledgments

  1. Top of page
  2. Abstract
  3. Patients and methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References

This study was supported by the University Hospital of Heraklion (Greece), the European Molecular Biology Organization (EMBO), and the Marrow Environment Fund (UK).

References

  1. Top of page
  2. Abstract
  3. Patients and methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References
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