Autografting

Normal bone marrow hematopoietic stem cell reserves and normal stromal cell function support the use of autologous stem cell transplantation in patients with multiple sclerosis

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Summary:

Bone marrow (BM) stem cell reserves and function and stromal cell hematopoiesis supporting capacity were evaluated in 15 patients with multiple sclerosis (MS) and 61 normal controls using flow cytometry, clonogenic assays, long-term BM cultures (LTBMCs) and enzyme-linked immunosorbent assays. MS patients displayed normal CD34+ cell numbers but a low frequency of colony-forming cells (CFCs) in both BM mononuclear and purified CD34+ cell fractions, compared to controls. Patients had increased proportions of activated BM CD3+/HLA-DR+ and CD3+/CD38+ T cells that correlated inversely with CFC numbers. Patient BM CD3+ T cells inhibited colony formation by normal CD34+ cells and patient CFC numbers increased significantly following immunomagnetic removal of T cells from BMMCs, suggesting that activated T cells may be involved in the defective clonogenic potential of hematopoietic progenitors. Patient BM stromal cells displayed normal hematopoiesis supporting capacity indicated by the CFC number in the nonadherent cell fraction of LTBMCs recharged with normal CD34+ cells. Culture supernatants displayed normal stromal derived factor-1 and stem cell factor/kit ligand but increased flt-3 ligand levels. These findings provide support for the use of autologous stem cell transplantation in MS patients. The low clonogenic potential of BM hematopoietic progenitors probably reflects the presence of activated T cells rather than an intrinsic defect.

Main

Intensive immunosuppression followed by autologous stem cell transplantation (ASCT) has been proposed as a therapeutic option for patients with severe refractory autoimmune diseases.1, 2, 3, 4 The driving concept is to eradicate the autoreactive cells with high-dose chemotherapy and rescue the patients from the prolonged cytopenias with stem cell reinfusion, while a more theoretical but still questionable consideration suggests the regeneration of the immune system with restoration of tolerance to self-antigens following ASCT.5, 6

Despite the encouraging early results, the procedure still remains investigational. Many transplantation-related issues need to be clarified, including the reserves and functional characteristics of bone marrow (BM) stem cells and the hematopoiesis supporting capacity of BM stromal cells. In patients with autoimmune diseases, abnormalities of the hematopoietic and immune system, primary or secondary to long-term use of immunosuppressant or immunomodulatory agents, may affect not only the composition and function of stem cell harvests but also the stem cell engraftment potential. For example, it has been reported that patients with active rheumatoid arthritis and systemic lupus erythematosus display a low frequency of BM CD34+ cells and impaired hematopoiesis supporting capacity of BM stromal cells due to the presence of autoreactive cells producing pro-inflammatory cytokines and proapoptotic mediators.7, 8, 9, 10, 11 These abnormalities may give an explanation for the recently described difficulties or failures in the stem cell collection procedure12 and occasional graft failure13 in some patients undergoing ASCT. In contrast, patients with primary autoimmune cytopenias exhibit increased numbers of hematopoietic progenitor cells and normal BM stromal cell function in terms of its capacity to support hematopoiesis.14

This interdisease heterogeneity in the reserves and functional characteristics of BM hematopoietic stem and stromal cells may reflect either the disease-related cytokine and chemokine milieu in the BM microenvironment or the effect of previous disease-specific immune-modifying treatments. In every case, BM function needs to be considered not only for potential difficulties related to the transplantation procedure but also for providing baseline data useful in the clinical outcome following engraftment. In the current study, we have evaluated the number and functional characteristics of BM stem cells and the hematopoiesis supporting capacity of BM stromal cells in patients with multiple sclerosis (MS), a T-cell-dependent autoimmune disease directed against the myelin components of the central nervous system15, 16 that has been considered among the first to be treated with ASCT on the basis of experimental data16 and clinical observations.17, 18, 19, 20, 21

Patients, materials and methods

Patients

We have studied 15 patients with clinically definite MS, aged 29–60 years (median age 32 years). Of these, 10 had the relapsing remitting (RR) form of the disease, three had secondary progressive (SP) and two primary progressive (PP) MS. For the characterization of the RR and SP forms of MS, we used the diagnostic criteria proposed by McDonald et al,22 whereas for the PP disease we used the criteria proposed by Thompson et al.23 Patients' functional status was assessed by the Kurtzke's Expanded Disability Status Scale (EDSS). Four patients were on treatment with interferon-beta (IFN-β) at the time of study but had discontinued medication for 1 week prior to BM aspiration. Specifically, three patients were receiving 30 μg IFN-β-1α (given once a week intramuscularly) and one patient 0.3 mg IFN-β-1β (administered three times weekly subcutaneously). A total of 12 patients had been treated with pulses of methylprednisone (1000 mg daily for 3 days) in the past, but had received the last cycle more than 1 year prior to the study. Three patients had never been previously exposed to immunomodulating therapy. Detailed patient characteristics are shown in Table 1. As normal controls, we studied 61 healthy volunteers, age- and sex-matched with the patients. The study has been approved by the Ethics Committee of the Hospital. Informed consent according to the Helsinki Protocol has been obtained from all subjects studied.

Table 1 Clinical and laboratory data of the MS patients studied

BM samples

BM cells obtained from posterior iliac crest aspirates were immediately diluted 1:1 in Iscove's modified Dulbecco's medium (IMDM; Gibco, Invitrogen Corporation, Paisley, Scotland), supplemented with 100 IU/ml penicillin–streptomycin (PS; Gibco) and 10 IU/ml preservative-free heparin (Sigma, St Louis, MO, USA). Diluted BM samples were centrifuged on Lymphoprep (Nycomed Pharma AS, Oslo, Norway) at 400 g for 30 min at room temperature to obtain the mononuclear cells (BMMCs).

Flow-cytometric analysis

Quantification of BM CD34+ cells

Two-color flow cytometry was used to quantitate the CD34+ cells and their subpopulations in the BMMC fraction. In brief, 1 × 106 BMMCs were stained with phycoerythrin (PE)-conjugated mouse anti-human CD34 monoclonal antibody (Mab) (QBEND-10; Beckman Coulter, Marseille, France) and fluorescein isothiocyanate (FITC)-conjugated mouse anti-human CD38 Mab (T16; Beckman Coulter) for 30 min on ice. PE- and FITC-conjugated mouse IgG isotype-matched controls were used as negative controls. Cells were washed twice in phosphate buffer saline (PBS) – 1% fetal bovine serum (FBS; Gibco) – 0.05% azide and fixed in 500 μl 2% paraformaldeyde solution (Sigma). Data were acquired and processed on 500 000 events using an Epics Elite model flow cytometer (Coulter, Miami, FL, USA). The estimation of CD34+/CD38 and CD34+/CD38+ cell populations representing the early and committed hematopoietic progenitor cells, respectively, was performed in the gate of cells with low forward (FSC) and low right-angle side scatter (SSC) properties where the BMMCs are included.7, 9, 14

Survival characteristics of BM CD34+ cells

106 BMMCs stained with PE-conjugated anti-CD34 Mab and FITC-conjugated mouse anti-human Fas (CD95) Mab (LOB 3/17; Serotec, UK), as above, were further stained, prior to fixation, with 7-amino-actinomycin D (7AAD; Calbiochem-Novabiochem, La Jolla, CA, USA) as previously described.7, 9 For the detection of Fas antigen in the CD34+ cell fraction, a scattergram was created by combining SSC with CD34 fluorescence in the gate of BMMCs and a second scattergram by combining CD34 and Fas fluorescence in the gate of CD34+ cells. Finally, a scattergram of FSC with 7AAD fluorescence was created to quantitate 7AAD-negative (live), -dim (apoptotic) and -bright (dead) cells in the gate of CD34+ cells (Figure 1).

Figure 1
figure1

Flow cytometric analysis of normal BMMCs stained with anti-CD34 mAb, anti-Fas (CD95) mAb and 7AAD. (a) Scattergram of FSC vs right-angle SSC to allow gating on the BMMCs (low FSC and low SSC properties) (R1). (b) Scattergram of CD34 fluorescence vs SSC gated on R1, to allow gating on the CD34+ cells (R2). (c) Scattergram of anti-CD34 vs anti-Fas fluorescence gated on R2 showing the Fas+ cells (R3) in the CD34+ cell fraction. (d) Scattergram of FSC vs 7AAD fluorescence gated on R2, showing the live (7AADneg) (R4), apoptotic (7AADdim) (R5) and dead (7AADbright) (R6) CD34+ cells.

Lymphocyte subsets

Two-color flow cytometry was used for the evaluation of the activation status of BM and peripheral blood (PB) lymphocytes. In brief, 100 μl aliquots of diluted BM or EDTA anticoagulated PB samples were stained for 30 min on ice with a combination of PE- or FITC-conjugated Mabs (Beckman Coulter, Marseille, France). Anti-CD3 (UCHT1) or anti-CD8 (B9.11) or anti-CD4 (13B8.2) Mab was combined with each of the following Mabs representing T-cell activation markers: anti-HLA-DR (B8.12.2), anti-CD25 (IL-2 receptor; B1.49.9) and anti-CD38 (T16). Similarly, anti-CD19 (J4.119) Mab was combined with anti-CD23 (9P25) or anti-CD69 (TP1.55.3) Mabs representing markers of B-cell activation. PE- or FITC-conjugated mouse IgG of appropriate isotype served as negative controls. After washing, contaminating red cells were lysed with 0.12% formic acid and samples were fixed in 0.2% paraformaldeyde using the Q-prep reagent system (Beckman Coulter). Analysis was performed on 10 000 events in the gate of cells with low FSC and low SSC properties where lymphocytes are included and results were expressed as proportion of cells expressing each Mab. Finally, by dividing the proportion of double-positive cells using the above described Mab combinations by the proportion of total CD3+, CD4+, CD8+ or CD19+ cells, we estimated the proportion of activated cells within each lymphocyte subpopulation (Figure 2).24

Figure 2
figure2

Flow cytometric analysis of T cells for the expression of activation cell surface markers. Total PB or BM cells were double stained with PE- or FITC-conjugated mouse antihuman anti-CD3 and anti-CD38, anti-HLA-DR or anti-CD25 Mabs. (a) Represents the scattergram of FSC vs right-angle SSC to allow gating on lymphocytes (R1). (b, c and d) Representative dot plots gated on R1 for the estimation of CD25+, CD38+ and HLA-DR+ cells, respectively, within the CD3+ cell fraction. The proportions of activated cells were calculated by dividing the double-positive CD3+/CD25+, CD3+/CD38+ and CD3+/HLA-DR+ cells (upper right quadrants) with the proportion of total CD3+ cells (upper right plus lower right quadrants; in the current case 56%). Quadrants were set on the basis of BM cells stained with PE- and FITC-conjugated mouse IgG of appropriate isotype.

Purification of BM cell subpopulation

The CD34+ and CD3+ BM cell populations were isolated from the BMMC fraction by indirect magnetic labeling (magnetic activated cell sorting; MACS isolation kit, Miltenyi Biotec GmbH, Germany) according to the manufacturer's protocol. In all experiments, purity of each positively selected population was greater than 96% as estimated by flow cytometry while the proportion of the immunomagnetically labeled cells in the negative fraction was less than 1%.

Clonogenic progenitor cell assays

BMMCs or CD34+ BM cells were cultured in 1 ml IMDM supplemented with 30% FCS, 1% bovine serum albumin (BSA; GibcoBRL, Life Technologies), 10−4 mol/l mercaptoethanol (Sigma), 0.075% sodium bicarbonate (Gibco), 2 mmol/l L-glutamine (Sigma), 0.9% methylcellulose (StemCell Technologies Inc., Vancouver, Canada), in the presence of 5 ng granulocyte-macrophage colony-stimulating factor (GM-CSF; R&D Systems, Minneapolis, MN, USA), 50 ng interleukin-3 (IL-3; R&D Systems) and 2 IU erythropoietin (EPO; Janssen-Ciliag Ltd, Athens, Greece), at a concentration of 105 BMMCs or 3 × 103 CD34+ cells/ml of culture medium. Cultures were set up in duplicate in 35-mm Petri dishes and incubated at a 37°C–5% CO2 fully humidified atmosphere. On day 14, colonies were scored and 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 both granulocyte–macrophage and erythroid elements (CFU-GEM), according to established criteria.25 Results were expressed as total number of colony-forming cells (CFCs) representing the sum of colonies. In a separate set of experiments, the CFC numbers obtained by BMMCs were also evaluated following immunomagnetic removal of T cells from the BMMC fraction and results were compared with baseline values in the respective unfractionated samples. In each case, the percentage of change in CFC number was calculated by dividing the difference between the baseline values and values obtained in the T-cell-depleted culture by the baseline values.

In another set of experiments, 1.7 × 104 normal purified CD34+ BM cells were cocultured with two different concentrations (0.85 × 105 and 1.7 × 105) of purified allogeneic CD3+ cells from healthy controls or patients with MS in 1 ml methylcellulose culture medium as described above. Cultures containing only normal CD34+ cells were used as controls in each experiment. On day 14, the number of CFCs was scored and the percentage of colony inhibition was calculated as above by dividing the difference in colony number between the control culture and test culture by the number of colonies in the respective control culture.

Long-term BM cultures

Long-term BM cultures (LTBMCs) from 107 BMMCs were grown according to the standard technique7, 9 in 10 ml IMDM supplemented with 10% FBS, 10% horse serum (Gibco), 100 IU/ml PS, 2 mmol L-glutamine and 10−6 mol hydrocortisone sodium succinate (Sigma) and incubated at a 33°C–5% CO2 fully humidified atmosphere. At weekly intervals, cultures were examined for stromal layer formation and fed by removing half of the medium and replacing it with equal volume of fresh IMDM supplemented as above. Nonadherent cells (NACs) were counted and assayed for their CFC content as described above. Furthermore, on confluence (weeks 3–4), cell-free supernatants were harvested and stored at −70°C for stromal-derived factor-1 (SDF-1), stem cell factor/kit ligand (SCF/KL) and flt-3 ligand (FL) quantification using an enzyme-linked immunosorbent assay (ELISA; R&D Systems). According to the manufacturer, the sensitivity of these assays is less than 18.0, 9.0 and 7.0 pg/ml, respectively.

Assessment of BM stromal cell function

A two-stage culture procedure was used to test the capacity of BM stromal layers to support normal hematopoiesis. Confluent stromal layers from patients and normal controls grown in standard LTBMCs were irradiated (10 Gy) and recharged with 5 × 104 normal allogeneic CD34+ BM cells as previously described.7, 9, 25 In each experiment, flasks were recharged in triplicate and the CD34+ cells from the same normal control were used to test patient and normal cultures. Cultures were fed at weekly intervals by demi-depopulation and supernatants were monitored by determining the NACs and CFCs for a period of 5 weeks as previously described.7, 9, 14

Statistical analysis

Data were analyzed in the GraphPad Prism statistical PC program (GraphPad Software, San Diego, CA, USA) by means of the nonparametric Mann–Whitney U-test and Spearman's coefficient of correlation. Comparison between CFC numbers obtained by unfractionated and T-cell-depleted BMMCs was performed by the Student's t-test for paired samples. Two-way analysis of variance (ANOVA) was used to define differences in the number of NACs and CFCs generated in LTBMCs of patients and controls. One-way ANOVA was used to compare differences between the control cultures and cultures containing different concentrations of T lymphocytes. Homogeneity of the populations was tested by the χ2 test.

Results

BM CD34+ progenitor cells

The mean proportion of CD34+ hematopoietic progenitor cells within the BMMC fraction was 2.11±1.87% in MS patients (n=15) with committed CD34+/CD38+ cells 1.71±1.67% and early CD34+/CD38 cells 0.42±0.25%. These proportions did not differ significantly from the respective values of normal controls (n=61) suggesting that patients with MS display normal reserves of BM hematopoietic progenitor cells.

In contrast, the clonogenic potential of CD34+ hematopoietic progenitor cells was found significantly reduced in our patients. As shown in Figure 3, MS patients displayed low numbers of total CFCs within the BMMC fraction and low CFC recovery from purified CD34+ cells, compared to the normal controls (P=0.0246 and 0.0348, respectively). These findings suggest either impaired survival or defective proliferative potential of hematopoietic progenitor cells in MS patients.

Figure 3
figure3

Clonogenic potential of BM hematopoietic progenitor cells. Bars represent the mean number (mean±s.e.m.) of total CFCs obtained from 107 BMMCs (left) or from 5 × 104 highly purified CD34+ progenitor cells (right) of MS patients and normal controls. Comparisons were assessed with the nonparametric Mann–Whitney U-test. P-values are indicated.

PB and BM lymphocyte subpopulations

Since hematopoiesis has been shown to be affected by a variety of immune parameters, we investigated the possible alterations and activation status of B- and T-cell lymphocyte subsets in both PB and BM.24, 26, 27, 28 As shown in Table 2, the percentages of PB CD3+ T cells and their CD4+ and CD8+ T-cell subsets did not differ statistically between MS patients (n=15) and normal controls (n=32). However, the percentages of activated T cells were significantly increased in the group of patients, compared to controls, as indicated by the increased proportions of HLA-DR+, CD25+ and CD38+ cells within the CD3+ T-cell fraction (P=0.0012, P=0.0063 and P<0.0001, respectively). This increase was concerning both, the CD4+ (P=0.0098, P=0.0032 and P<0.0001, respectively) and the CD8+ T cells (P=0.0197, P<0.0001 and P<0.0001, respectively). No significant differences could be found between patients and controls in the proportion of CD19+ cells or the percentages of CD23+ and CD69+ activated lymphocyte subsets within the CD19+ B-cell fraction.

Table 2 Flow cytometric analysis of peripheral blood T cells subsetsa

BM lymphocyte subpopulations are shown in Table 3. In the group of patients (n=15), the proportions of CD3+, CD4+ and CD8+ T cells did not differ significantly from the respective controls (n=15) but consistent with the PB findings, the percentages of activated T cells were significantly increased in the patients. Specifically, the proportions of HLA-DR+ and CD38+ but not of CD25+ BM T cells were significantly elevated within the CD3+ T cells fraction (P=0.0055 and P<0.0001, respectively). Increased proportions of HLA-DR+ and CD38+ cells were also found within the CD4+ (P=0.0344 and P<0.0001, respectively) and CD8+ (P=0.0121 and P<0.0001, respectively) T-cell subsets. In contrast, the percentage of BM CD19+ B cells did not differ significantly between patients and normal controls and the proportions of CD23+ and CD69+ cells detected within the CD19+ cell fraction ranged within the respective values of normal controls. All these findings clearly show that MS patients have increased proportions of activated T but not B cells in both the PB and BM.

Table 3 Flow cytometric analysis of bone marrow T-cell subsetsa

Activated T cells and clonogenic potential of CD34+ cells

The possible role of activated T cells in the pathogenesis of the impaired clonogenic potential of BM hematopoietic progenitor cells in our MS patients was indicated by the highly significant inverse correlations between the number of CFCs generated by patient BM CD34+ cells and the number of activated BM CD3+/HLA-DR+ and CD3+/CD38+ T cells (r=−0.8387, P<0.0001 and r=−0.5487, P=0.0342, respectively) (Figure 4). To further investigate this hypothesis, we studied the effect of patient BM CD3+ T cells on the in vitro growth of normal hematopoietic progenitor cells. Data from three sets of experiments are presented in Figure 5. The mean numbers of total CFCs obtained by 1.7 × 104 purified CD34+ cells from three different normal controls cocultured with 0.85 × 105 and 1.7 × 105 CD3+ T cells from three MS patients were elevated to 184.0±15.4 and 142±4.6, respectively, two means significantly lower than the mean number of CFCs obtained in the control cultures containing only normal CD34+ cells that were elevated to 382.0±7.6 (P=0.0007). In these experiments, the mean percentage of colony inhibition was estimated to 51.79±4.71 and 62.83±0.47%, respectively. Furthermore, in the presence of allogeneic normal BM CD3+ T cells from three normal controls, a decrease in the total CFC formation by normal CD34+ cells was noted, but the difference from the respective baseline cultures was not statistically significant. In fact, the mean percentages of CFC inhibition obtained in the presence of the above two different CD3+ T-cell concentrations were 7.26±6.68 and 9.09±5.79%, respectively.

Figure 4
figure4

Inverse correlations between the values of CFCs obtained from BMMC cultures and the proportions of CD3+/HLA-DR+ (a) and CD3+/CD38+ (b) BM cells. Data were analyzed using Spearman's correlation test. P-values are indicated.

Figure 5
figure5

Coculture experiments of normal CD34+ cells with CD3+ cells from normal controls or MS patients. 1.7 × 104 purified CD34+ BM cells from normal control subjects (n=3) were cocultured with purified CD3+ cells from allogeneic healthy individuals (n=3) or patients with MS (n=3) at a concentration of 0.85 × 105 and 1.7 × 105 cells in 1 ml methylcellulose culture medium using the clonogenic progenitor cell assay as described in the section Patients, materials and methods. Each point in the diagram represents the mean CFC number from three experiments. Comparison between cultures containing different concentrations of normal or MS CD3+ cells and control cultures containing only normal CD34+ cells was assessed with the one-way ANOVA test.

To substantiate the hypothesis that activated T lymphocytes may influence the clonogenic potential of patient hematopoietic progenitor cells, we evaluated the CFC numbers obtained in the clonogenic assay from patient (n=3) and normal (n=3) BMMCs following immunomagnetic depletion of CD3+ cells. For each subject, CFC numbers obtained from the T cells depleted BMMC samples were compared to baseline values obtained from the respective unfractionated BMMCs. Results are presented in Figure 6. The mean CFC numbers following T-lymphocyte depletion increased significantly, compared to baseline, in MS patients (128±4 per culture dish vs 97±9 per culture dish, respectively; P=0.024) but not in controls (130±20 per culture dish vs 115±11 per culture dish, respectively; P=0.353). In fact, an overall 10.25±15.87% decrease in CFC numbers was observed in the controls whereas a 32.73±12.38% increase was obtained in the patients, following T-lymphocyte depletion. These data substantiate further the effect of patient T lymphocytes on the clonogenic potential of hematopoietic progenitors.

Figure 6
figure6

Effect of removal of T lymphocytes from BMMCs on CFC numbers. We cultured 105 BMMCs from MS patients (n=3) or healthy controls (n=3) in 1 ml methylcellulose culture medium supplemented as described in the section Patients material and methods, in 35-mm Petri dishes. For each subject, cultures with the same initial BMMC concentration following immunomagnetic depletion of T cells were also grown. The bars represent the mean CFC number (mean±s.d.) obtained in patients and controls before and after T-cell depletion. Comparison between unfractionated and T-cell-depleted cultures was performed using the Student's t-test for paired sample.

To probe the possible underlying pathophysiologic mechanism of T-cell action, we studied Fas antigen expression and apoptosis of BM CD34+ cells in our patients. Such abnormalities mediated by activated T lymphocytes have already been described in certain BM failure syndromes.24, 26, 27 We found that Fas antigen expression in CD34+ cells did not differ significantly between MS patients and normal controls (10.86±6.76%, n=15 vs 7.86±4.65%, n=40; P>0.05). Nor were the proportions of either the early or the late apoptotic cells detected within the CD34+ cell fraction statistically different between MS patients and normal controls (9.42±5.87% vs 7.42±7.15%, P>0.05, and 1.31±0.29% vs 1.53±2.60%, P>0.05, respectively). Taken together all the above data suggest that a negative effect of T lymphocytes on the proliferative capacity of CFCs rather than an apoptosis-inducing effect is probably the cause of the defective clonogenic potential of hematopoietic progenitor cells in MS.

IFNs have been reported to negatively affect hemopoiesis.26, 28 To investigate whether the impaired clonogenic potential of hematopoietic progenitors in our patients was related with the previously applied therapy with IFN-β, we performed a subset analysis by excluding patients exposed to IFN-β (n=4) (Figure 3). Results have shown that the number of CFCs obtained by BMMCs (9330±3433 per 107 BMMCs) and CD34+ cells (960±506 per 5 × 104 CD34+ cells) was again significantly low in the remaining 11 MS patients never exposed to IFN-β therapy, compared to controls (P=0.0349 and P=0.0437, respectively). Additionally, no differences could be detected in the number of CFCs obtained in MS patients previously treated with IFN-β MS patients (10600±2906 CFCs per 107 BMMCs and 1097±442 CFCs per 5 × 104 CD34+ cells) compared to untreated MS patients (P=0.3606 and P=0.6952, respectively). Hence, impaired clonogenic potential of hematopoietic progenitor cells occurs in MS patients independently of treatment with IFN-β.

Evaluation of BM stromal cell function

To evaluate the hematopoiesis supporting capacity of BM stromal cells in MS patients, we initially used standard LTBMCs. Confluent stromal layers consisting of fibroblast-like cells, macrophages and cobblestone areas were formed over the first 3–4 weeks in both MS patients (n=15) and normal controls (n=24) and the mean duration of CFC colony formation by NACs did not differ significantly between patients (6.87±2.13 weeks) and normal controls (7.04±2.20 weeks, P=0.8508). However, the average number of NACs and the mean frequency of CFCs in the NAC fraction were significantly lower in the patients, compared to controls, studied over a 10-week culture period (F=19.333>F1235; P<0.001 and F=10.690>F1176; P<0.01, respectively). This might reflect either the impaired clonogenic potential of patient hematopoietic progenitor cells and/or a defect in the hematopoiesis supporting capacity of stromal cells (Figure 7). To investigate this hypothesis, we evaluated patients' stromal cell function independently of the autologous hematopoietic progenitor cells using the two-stage LTBMC system. Confluent stromal layers from all MS patients and 15 healthy controls were irradiated and recharged with allogeneic normal CD34+ cells as described above. The number of NACs and the number of CFCs obtained weekly from the NAC fraction over a period of 5 weeks did not differ significantly between patients and control subjects (F1140=0.0268, P>0.05 and F1140=1.7897, P>0.05, respectively), suggesting normal capacity of BM stroma to support hematopoiesis in patients with MS.

Figure 7
figure7

Long-term BM cultures. Bars represent the mean number of NACs (mean±s.e.m.) (a), and the mean frequency of CFCs (mean±s.e.m.) in the NAC fraction of LTBMCs (b) of MS patients (n=15) and normal controls (n=24). Comparison between patient and normal cultures was performed using the two-way ANOVA test.

Cytokines in LTBMC supernatants

Given that LTBMC stromal layers mimic in many aspects the in vivo BM microenvironment,25 we measured in LTBMC supernatants the levels of certain cytokines, namely the SDF-1, SCF/KL and FL, known to be involved in the induction of homing, survival and proliferation processes of the primitive hematopoietic progenitor cells.29 No significant differences were found between MS patients and normal controls in the levels of SDF-1 (2.09±1.71 ng/ml vs 2.19±2.89 ng/ml) or the SCF/KL (92.67±64.00 pg/ml vs 96.37±97.76 pg/ml). In contrast, a significant rise in the levels of FL was seen in the patients compared to controls (10.75±3.91 pg/ml vs 8.03±3.77 pg/ml, P=0.0442). Interestingly, the levels of FL strongly correlated with the percentages of BM CD3+ T cells (r=0.748, P=0.0068) suggesting a T-cell-related compensatory mechanism to the impaired haemopoietic progenitor cell clonogenic potential.29, 30, 31

Discussion

There is accumulating evidence suggesting that ASCT may have a beneficial effect in refractive cases of MS with respect to abrogation of brain inflammatory process or even to stabilization of the disease and disability prevention.18, 19, 20, 21, 32, 33, 34 Studies in other autoimmune diseases have already shown abnormalities in BM hematopoiesis7, 8, 9, 10 capable of affecting the mobilization and/or the engraftment of stem cells in patients undergoing ASCT.11, 12, 13 In the current study, we have evaluated the reserves and functional characteristics of BM hematopoietic progenitor cells and the hematopoiesis supporting capacity of BM stromal cells in MS patients and we have found that these patients displayed normal proportion of CD34+ hematopoietic progenitor cells in the BMMC fraction and normal proportion of early CD34+/CD38 and committed CD34+/CD38+ cells. In contrast, the clonogenic potential of hematopoietic progenitor cells was found to be significantly reduced in these patients as was demonstrated by the low CFC number obtained from BMMCs and purified CD34+ cells in short-term BM cultures and the low CFC recovery in NACs from LTBMCs.

To probe the mechanisms underlying the impaired clonogenic potential of hematopoietic progenitor cells in MS, we first investigated the activation status of patient lymphocytes. It has been previously reported that the presence of activated T cells in the BM may affect the growth and survival characteristics of BM hematopoietic progenitor cells or even the hematopoiesis supporting capacity of BM stromal cells by inducing intricate cell-to-cell interactions and proinflammatory cytokine production.26, 29, 30, 31, 32, 33, 34, 35 In agreement with previous reports, we found increased numbers of activated T cells in PB of our MS patients,15, 36, 37, 38 and also increased proportions of activated T cells in patients' BM. Interestingly, the proportion of HLA-DR+ and CD38+ cells in patient BM CD3+ cells inversely correlated with the number of CFCs in the BMMC fraction, suggesting that activated T cells may be implicated in the defective clonogenic potential of hematopoietic progenitor cells in MS. The inhibitory effect of purified BM CD3+ cells from the patients on the normal hematopoietic progenitor cell growth in a concentration-dependent manner and the significant increase of CFCs by BMMCs in the patients but not in the controls following T-cell depletion, corroborate further this assumption.

One of the main mechanisms involved in myelosuppression by T cells is thought to be the production of tumor necrosis factor-α and IFN-γ, two molecules inducing Fas-mediated apoptotic cell death of hematopoietic progenitor cells.26, 27 Fas antigen expression on CD34+ cells, as well as apoptotic progenitor cell numbers in our patients did not differ significantly from the those of normal controls. On the other hand, T-cell depletion from BMMCs resulted in a significant increase of CFC numbers in the patients who reached normal values. These findings suggest that a reversible, T-cell-mediated defect in the proliferative potential rather than apoptotic cell death accounts for the impaired clonogenic potential of hematopoietic progenitors in MS.

The impaired clonogenic potential of patient hematopoietic progenitor cells might give an explanation for the mobilization failures occasionally reported in MS patients undergoing ASCT. Specifically, in a large survey performed by Burt et al,12 who evaluated retrospectively data from 24 transplant centers to determine the outcome of the procedure in patients with a variety of autoimmune diseases including 76 patients with MS, it was identified that MS patients displayed low progenitor cell yields in comparison to rheumatoid arthritis or to systemic sclerosis patients. In particular, marginal or even failed mobilizations occurred in 9.2% of MS patients while inadequate BM progenitor cell harvests occurred in two patients.12 Difficulties in stem cell mobilization procedure have also been described by Carreras et al39 in 13.3% of cases in a series of 15 MS patients since one patient failed mobilization after two attempts while an additional patient reached the target CD34+ cell yield following three leukaphereses.

It has been proposed that the hematopoietic progenitor cell clonogenic potential and, accordingly, the CD34+ cell yield, might be compromised by prior immunosuppressive or immunomodulating medication in patients with autoimmune disorders. Corticosteroids are used for the treatment of the acute relapses of MS whereas IFN-β is shown to be of value for prevention of further attacks. Previous studies have shown that corticosteroids do not have a negative effect in progenitor cell recovery12 and may even induce a rather positive effect in the proliferation and differentiation of BM progenitor cells.40, 41 Although exposure to IFN-β therapy is thought to affect adversely the mobilization procedure,12 no conclusive evidence is currently available in this regard.39 In our study, we found defective colony formation by hematopoietic progenitor cells in MS patients never exposed to IFN-β medication. As such, our data suggest that the autoimmune process per se rather than IFN-β accounts for the impaired clonogenic potential of hematopoietic progenitors observed in MS. T-cell-derived proinflammatory cytokines may affect the proliferative potential of patient progenitor cells as previously reported in other disease states.28

The hematopoiesis supporting capacity of BM stromal cells in MS patients did not differ from that of the normal controls. MS patients had normal numbers and clonogenic potentials of NACs in irradiated patient LTBMCs recharged with normal CD34+ cells. We have also found that our patients had normal SCF/KL production by LTBMC adherent cells, a molecule mainly produced by BM stroma42 and considered as the most appropriate cytokine to evaluate when characterizing the BM stromal cell hematopoietic function.30 In contrast, the levels of FL were significantly raised in patient LTBMC supernatants and correlated positively with the proportion of activated BM T cells. This molecule is important in early stages of hematopoiesis by stimulating the proliferation and differentiation of the primitive hematopoietic progenitor cells. It is then possible that T-cell-derived compensatory FL overproduction in MS patients may give an explanation for the normal CD34+ cell numbers in the patients despite their impaired clonogenic potential. Overproduction of FL by activated T cells has also been reported as a hematopoietic rescue mechanism in response to BM failure in patients with aplastic anemia.31 On the other hand, the levels of SDF-1, a cytokine operating in the process of homing of CD34+ hematopoietic progenitors in the BM, did not differ significantly between MS patients and healthy controls, suggesting probably a normal engraftment support of BM stroma in MS patients undergoing ASCT.

In conclusion, MS patients have normal BM hematopoietic stem cell reserves and normal hematopoiesis supporting capacity of BM stroma. Hematopoietic progenitor cells, however, have impaired clonogenic potential probably due to the presence of activated T cells within the marrow microenvironment rather to an intrinsic defect. Overall, these findings provide support for the use of ASCT in patients with MS and might be proved useful in future studies exploring the safety and efficacy of ASCT in these patients.

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Acknowledgements

We thank Mrs Helen Koutala for her valuable help in performing the flow cytometric acquisition of BM and PB samples in MS patients and healthy controls. This work was supported by a grant (STEMNET) of the Hellenic General Secretariat of Research and Technology.

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Correspondence to H A Papadaki.

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Keywords

  • autoimmune diseases
  • multiple sclerosis
  • T cells
  • stem cells
  • autologous stem cell transplantation

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