To investigate whether bone marrow (BM) stem cell compartment and/or BM microenvironment are affected by the immune insult in autoimmune cytopenias (AICs), BM stem cell reserve and function and BM stromal function were studied in 15 AIC patients. Stem cells were evaluated by means of flow cytometry, clonogenic progenitor cell assays, long-term BM cultures (LTBMCs), and limiting dilution assay for quantification of long-term–culture initiating cells (LTC-ICs). Stromal cell function was assessed with the use of preformed irradiated LTBMCs from patients and normal controls, recharged with normal CD34+ cells. AIC patients exhibited a high number of CD34+, CD34+/CD38+, and CD34+/CD38 cells; high frequency of granulocyte-macrophage colony forming units in the BM mononuclear cell fraction; high colony recovery in LTBMCs; and normal LTC-IC frequency. Patient BM stromal layers displayed normal hematopoietic-supporting capacity and increased production of granulocyte-colony stimulating factor. Data from this study support the concept that AIC patients with severe, resistant disease might be appropriate candidates for autologous stem cell transplantation.

Autoimmune cytopenias (AICs) are well-defined hematologic disorders characterized by a reduced number of circulating mature blood elements due to increased peripheral cell destruction and/or decreased cell production in the bone marrow (BM) by humoral or cellular cytotoxic mechanisms. They may present as isolated cytopenias affecting red blood cells (autoimmune hemolytic anemia, pure red cell aplasia), neutrophils (autoimmune neutropenia), or platelets (autoimmune thrombocytopenic purpura) or as a combination involving 2 (eg, Evans syndrome) or 3 lineages (autoimmune pancytopenia).1 

Although immune-mediated destruction of mature blood elements has long been recognized, mechanisms involved in the pathogenesis of AIC were better defined following the introduction of in vitro clonogenic assays.1 With the use of culture studies, it was shown that immune inhibition of hematopoietic cell production may be as important as increased peripheral cell destruction in AIC2-8 and that humoral or cellular immune mechanisms affecting not only the progenitor cells but even BM stromal cells may be involved in their pathogenesis.9 However, stem cells per se and BM microenvironment in these patients have not been extensively studied.

Because there has been much interest during the last decade in exploring the use of high-dose immunotherapy followed by autologous hematopoietic stem cell transplantation in patients with severe, resistant autoimmune diseases including AIC,10 it seems crucial to answer the question of whether stem cell compartment and/or BM microenvironment is already affected by the immune insult in these patients. The aim of the present study was to evaluate hematopoietic stem cell reserve and function and BM stromal function in terms of its capacity to support hematopoiesis in patients with AIC.

BM samples and immunophenotyping

BM mononuclear cells (BMMCs) were obtained from 15 AIC patients (Table 1) and 20 normal controls as previously described.11 CD34+ cells were isolated from BMMCs by indirect magnetic labeling by means of a MACS isolation kit (Mitenyi Biotec, Germany) according to the manufacturer's instructions. BMMCs or CD34+ cells were labeled with anti-CD34 (HPCA-2; Becton Dickinson, United Kingdom) and anti-CD38 (HIT-2; Becton Dickinson) fluorescent monoclonal antibodies and were analyzed on a FACScan flow cytometer (Becton Dickinson). Data on a minimum of 50 000 events were acquired and processed by means of CellQuest software.

Table 1.

Clinical and laboratory data of the patients studied

Patient no.SexAge (y)Disease*Duration (mo)Previous medicationsAntibody specificityBone marrowPeripheral blood counts1-153
CellularityHb (g/dL)WBC (×109/L)Neutro (×109/L)Lympho (×109/L)Plt (×109/L)Ret (×109/L)
52 Idiopathic AIN 60 G-CSF ND Increased/hypercellular myeloid series with normal maturation 12.3 1.2 0.5 0.3 165 46 
58 AIN/Sjogren 12 None ND Normal/normal myeloid series 10.8 1.9 0.8 0.8 152 45 
20 AIN/thyroiditis 15 G-CSF anti-NA1 Normal/normal myeloid series 13.8 2.0 0.2 1.2 250 55 
25 Idiopathic AIN None anti-NA1 Increased/hypercellular myeloid series with left shift 10.5 2.3 0.7 1.4 154 45 
19 Idiopathic AIN/
pancytopenia 
66 PDN/γ-globulin anti-NA1 Increased/hypercellular myeloid series with left shift, increased Meg series 8.9 3.6 0.7 1.7 140 55 
anti-GPV  
35 Idiopathic AIHA PDN/γ-globulin  Markedly increased/hypercellular erythroid and Meg series 12.9 6.1 3.3 2.2 360 114 
46 ITP/AIHA 48 PDN/AZA anti-GPIIb/IIIa Mildly increased/hyperplasia of Meg series 12.0 7.5 4.5 1.6 63 65 
γ-globulin anti-GPIb/IX 
30 ITP None anti-GPIIb/IIIa Mildly increased/hyperplasia of Meg series 16.0 5.0 2.4 1.9 94 50 
24 ITP 28 PDN/AZA/CSA anti-GPIb/IX Mildly increased/hyperplasia of Meg series 12.0 2.7 1.9 0.4 87 60 
VCR/CAMPATH-1  
γ-globulin  
10 20 ITP/AIHA/Behcet disease 84 PDN ND Normal/slightly increased Meg series 12.1 4.4 2.3 1.7 173 55 
11 65 Primary PRCA1-155 48 PDN/AZA/CSA  Normal/near absence of erythroid series, normal myeloid and Meg series 6.1 4.6 1.7 1.6 110 35 
12 48 Primary PRCA1-155 24 PDN/ALG/CSA  Normal/reduced erythroid series, normal myeloid and Meg series 11.6 5.6 3.4 1.8 255 18 
13 30 Primary PRCA1-155 36 PDN/CSA  Normal/near absence of erythroid series, normal myeloid and Meg series 8.9 4.3 2.5 1.7 333 20 
14 34 Primary PRCA 24 PDN/CSA  Normal/absence of erythroid series, normal myeloid and Meg series 5.3 5.7 3.5 2.0 205 25 
15 34 Primary PRCA 14 PDN/CSA  Normal/reduced erythroid series, normal myeloid and Meg series 8.4 5.9 3.7 1.9 335 25 
Patient no.SexAge (y)Disease*Duration (mo)Previous medicationsAntibody specificityBone marrowPeripheral blood counts1-153
CellularityHb (g/dL)WBC (×109/L)Neutro (×109/L)Lympho (×109/L)Plt (×109/L)Ret (×109/L)
52 Idiopathic AIN 60 G-CSF ND Increased/hypercellular myeloid series with normal maturation 12.3 1.2 0.5 0.3 165 46 
58 AIN/Sjogren 12 None ND Normal/normal myeloid series 10.8 1.9 0.8 0.8 152 45 
20 AIN/thyroiditis 15 G-CSF anti-NA1 Normal/normal myeloid series 13.8 2.0 0.2 1.2 250 55 
25 Idiopathic AIN None anti-NA1 Increased/hypercellular myeloid series with left shift 10.5 2.3 0.7 1.4 154 45 
19 Idiopathic AIN/
pancytopenia 
66 PDN/γ-globulin anti-NA1 Increased/hypercellular myeloid series with left shift, increased Meg series 8.9 3.6 0.7 1.7 140 55 
anti-GPV  
35 Idiopathic AIHA PDN/γ-globulin  Markedly increased/hypercellular erythroid and Meg series 12.9 6.1 3.3 2.2 360 114 
46 ITP/AIHA 48 PDN/AZA anti-GPIIb/IIIa Mildly increased/hyperplasia of Meg series 12.0 7.5 4.5 1.6 63 65 
γ-globulin anti-GPIb/IX 
30 ITP None anti-GPIIb/IIIa Mildly increased/hyperplasia of Meg series 16.0 5.0 2.4 1.9 94 50 
24 ITP 28 PDN/AZA/CSA anti-GPIb/IX Mildly increased/hyperplasia of Meg series 12.0 2.7 1.9 0.4 87 60 
VCR/CAMPATH-1  
γ-globulin  
10 20 ITP/AIHA/Behcet disease 84 PDN ND Normal/slightly increased Meg series 12.1 4.4 2.3 1.7 173 55 
11 65 Primary PRCA1-155 48 PDN/AZA/CSA  Normal/near absence of erythroid series, normal myeloid and Meg series 6.1 4.6 1.7 1.6 110 35 
12 48 Primary PRCA1-155 24 PDN/ALG/CSA  Normal/reduced erythroid series, normal myeloid and Meg series 11.6 5.6 3.4 1.8 255 18 
13 30 Primary PRCA1-155 36 PDN/CSA  Normal/near absence of erythroid series, normal myeloid and Meg series 8.9 4.3 2.5 1.7 333 20 
14 34 Primary PRCA 24 PDN/CSA  Normal/absence of erythroid series, normal myeloid and Meg series 5.3 5.7 3.5 2.0 205 25 
15 34 Primary PRCA 14 PDN/CSA  Normal/reduced erythroid series, normal myeloid and Meg series 8.4 5.9 3.7 1.9 335 25 

Hgb indicates hemoglobin; WBC, white blood cells; Neutro, neutrophils; Lympho, lymphocytes; Plt, platelets; Ret, reticulocytes; AIN, autoimmune neutropenia; AIHA, autoimmune hemolytic anaemia; ITP, autoimmune thrombocytopenic purpura; PRCA, pure red cell aplasia; Meg series, megakaryocytic series; G-CSF, granulocyte-colony stimulating factor; PDN, prednisone; AZA, azathioprine; CSA, cyclosporin A; VCR, vincristine; ALG, antilymphocyte globulin; ND, not defined; NA1, neutrophil antigen 1; GP, platelet glycoprotein.

*

AIN diagnosis was based on the presence of specific antineutrophil antibodies in patient serum detected with chemilumonescence and immunofluorescence techniques.23 ITP diagnosis was based on the presence of antiplatelet antigens or anti-GP specific antibodies in patient sera and platelets detected by means of flow cytometry (platelet flow cytometric immunofluorescence technique; PIFT) and ELISA (monoclonal antibody immobilization of platelet antigen assay; MAIPA).24 

In cases of AIN and ITP.

At diagnosis.

F1-153

Performed at the time of study.

F1-155

Bone marrow T-cell receptor and immunoglobulin gene rearrangement studies had been performed to exclude clonality.

Clonogenic progenitor cell assays

BMMCs or CD34+ cells were cultured in methylcellulose culture medium (StemCell Technologies, Vancouver, BC, Canada) for granulocyte-macrophage (CFU-GM) and erythroid-burst colony formation (BFU-E) as previously described.12 BMMCs and CD34+ cells were also assessed for megakaryocytic colony formation (CFU-Meg) by means of a commercially available kit (MegaCult-C, StemCell Technologies) according to the manufacturer's protocol.

Long-term BM cultures and cytokine measurement in culture supernatants

Long-term BM cultures (LTBMCs) from 107BMMCs were grown according to a standard technique.11-14At weekly intervals, nonadherent cells were counted and assayed for colony formation, and results were expressed as total numbers of colony forming cells (CFCs) (CFU-GM + BFU-E). At week 3, cell-free supernatants were harvested and stored at −70°C for granulocyte-colony stimulating factor (G-CSF) and granulocyte-macrophage colony stimulating factor (GM-CSF) quantification by means of commercially available ELISA kits (R&D Systems, Oxon, United Kingdom).

Limiting dilution assay for quantification of long-term culture initiating cells

Seven dilutions of a single suspension of CD34+cells were overlaid on preformed murine MS-5 stromal layers15 at concentrations ranging from 10 to 1000 cells per well in 96-well culture plates. Cultures were fed weekly by demi-depopulation and, after 5 weeks, were overlaid with methylcellulose culture medium for 2 additional weeks. The frequency of long-term culture initiating cells (LTC-ICs) was calculated by determining, by means of a Fig. P Biosoft PC program, the CD34+ cell dilution that resulted in 37% wells or fewer being negative for colonies.16 17 

Assessment of BM stromal cell function

Irradiated confluent stromal layers from patients and normal controls grown in standard LTBMCs were recharged with 5 × 104 normal allogeneic CD34+ BM cells as previously described.14 At weekly intervals, supernatants were monitored by determining the number of nonadherent cells and CFC frequency.

Data were analyzed by means of the nonparametric Wilcoxon rank test, standard 2-way variance analysis test, and 2-tailed Studentt test.

AIC patients had significantly higher proportions of CD34+ cells compared with controls owing to the higher proportion of both the committed CD34+/CD38+cells and the more primitive CD34+/CD38 cells (Figure 1). Furthermore, CFU-GM colony formation by 107 BMMCs was significantly higher in AIC patients than in the normal controls. The frequency of BFU-E/107 BMMCs and the frequency of CFU-Meg/107 BMMCs did not differ statistically between patients and normal controls (Figure1). To investigate whether the high CFU-GM colony formation in AIC patients was due to an intrinsic increased clonogenic potential of patient stem cells or was the consequence of an extrinsic effect, we tested the frequency of CFU-GM obtained by immunomagnetically sorted highly purified CD34+ BM cells. The number of colonies obtained was similar in patients (mean, 307 per 104 CD34+ cells; range, 174-537) and normal controls (mean, 262 per 104 CD34+ cells; range, 93-540; P = .429), suggesting that accessory cells may influence the colony growth in patient unfractionated samples. An alternative explanation is that the increased colony number obtained by patient BMMCs simply reflects the increased proportion of CD34+ cells in AIC BM. Similarly, no significant difference was observed between patients and normal controls in the mean number of BFU-E and CFU-Meg obtained from CD34+ cells (P = .189 and P = .591, respectively).

Fig. 1.

Bone marrow CD34+ cells and clonogenic progenitor cells in AIC patients.

The left bars represent the mean percentages (± SEM) of CD34+ cells in 15 AIC patients and 20 normal controls obtained in 2-color flow cytometric analysis of BMMCs. The right bars represent the mean colony values (± SEM) obtained from BMMCs in the clonogenic progenitor cell assays. We cultured 105BMMCs from AIC patients (n = 13) and normal controls (n = 16) in 1 mL methylcellulose 0.9% in Iscove modified Dulbecco medium supplemented with 30% fetal calf serum (PAA Laboratories GmbH, Linz, Austria), 1% bovine serum albumin (BSA; Sigma, St Louis, MO), 10−4 mol/L mercaptoethanol (Sigma), 0.075% sodium bicarbonate (GibcoBRL; Life Technologies), and 2 mmol/L L-glutamine (Sigma), in the presence of 5 ng GM-CSF, 50 ng interleukin (IL)-3 and 2 IU erythropoietin for CFU-GM and BFU-E colony formation. Colonies were enumerated on day 14. We cultured 106 BMMCs from AIC patients (n = 12) and normal controls (n = 10) in MegaCult-C medium for CFU-Meg colony growth. Colonies were scored after 10 to 12 days of incubation after fixation and staining by alkaline phosphatase antialkaline phosphatase technique with the use of anti-CD41 monoclonal antibody. Comparison between patients and normal controls was performed by means of the 2-tailed Student t test.

Fig. 1.

Bone marrow CD34+ cells and clonogenic progenitor cells in AIC patients.

The left bars represent the mean percentages (± SEM) of CD34+ cells in 15 AIC patients and 20 normal controls obtained in 2-color flow cytometric analysis of BMMCs. The right bars represent the mean colony values (± SEM) obtained from BMMCs in the clonogenic progenitor cell assays. We cultured 105BMMCs from AIC patients (n = 13) and normal controls (n = 16) in 1 mL methylcellulose 0.9% in Iscove modified Dulbecco medium supplemented with 30% fetal calf serum (PAA Laboratories GmbH, Linz, Austria), 1% bovine serum albumin (BSA; Sigma, St Louis, MO), 10−4 mol/L mercaptoethanol (Sigma), 0.075% sodium bicarbonate (GibcoBRL; Life Technologies), and 2 mmol/L L-glutamine (Sigma), in the presence of 5 ng GM-CSF, 50 ng interleukin (IL)-3 and 2 IU erythropoietin for CFU-GM and BFU-E colony formation. Colonies were enumerated on day 14. We cultured 106 BMMCs from AIC patients (n = 12) and normal controls (n = 10) in MegaCult-C medium for CFU-Meg colony growth. Colonies were scored after 10 to 12 days of incubation after fixation and staining by alkaline phosphatase antialkaline phosphatase technique with the use of anti-CD41 monoclonal antibody. Comparison between patients and normal controls was performed by means of the 2-tailed Student t test.

Close modal

The average nonadherent cell recovery, over a period of 8 weeks, was similar in patient and normal LTBMCs (F = 1.343 < F1,188 at 5%) but the CFC frequency was significantly higher in patients than in normal controls (F = 6.464 > F1,188 at 1‰), supporting the concept that the stem cell compartment is increased in our patients. However, the frequency of LTC-ICs, which represent the best available approximation of primitive stem cells,18 19 did not differ significantly between patients (mean, 12.29 per 5 × 103 CD34+ cells; range, 5.3-22.42) and normal controls (mean, 12.99 per 5 × 103CD34+ cells; median, 11.94; range, 7.4-20; P = .753).

Patient stromal function, assessed by its ability to support hematopoietic progenitor cell growth, was comparable to the normal controls as indicated by the number of nonadherent cells (F = 2.497 < F1,105 at 5%) and the CFC frequency (F = .029 < F1 105 at 5%) over a period of 5 weeks. In keeping with the fact that BM stromal cell function was normal in AIC patients were the increased G-CSF concentrations in patient supernatants (mean, 642.39 pg/mL; range, 30.5-1913; n = 10) compared with the normal supernatants (mean, 154.32 pg/mL; range, 26.85-409; n = 10; P = .0156), suggestive of a compensatory G-CSF production by patient stromal cells in response to the peripheral cytopenia.20-22 In contrast, GM-CSF levels did not differ statistically between AIC patient and normal control supernatants (P = .089).

In conclusion, our findings suggest that AIC patients exhibit normal stem cell function and high frequency of committed progenitors as indicated by the significant increase in the proportions of CD34+ cells in flow cytometric analysis, the increased numbers of CFU-GM in BMMCs, and the increased committed progenitor cell recovery in LTBMCs. Our data also suggest that AIC patients display normal BM stromal function in terms of its ability to support normal hematopoiesis. This study encourages further the concept that patients with severe, resistant AIC might be appropriate candidates for autologous stem cell transplantation following intensive immunosuppression.

The authors thank Novartis Pharmaceuticals UK Ltd and Janssen-Ciliag Ltd for their gifts of cytokines and the hematology clinical staff of St George's Hospital for aspirating bone marrow samples.

Supported by a European Molecular Biology Organization grant.

The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked “advertisement” in accordance with 18 U.S.C. section 1734.

1
Cline
MJ
Golde
DW
Immune suppression of hematopoiesis.
Am J Med.
64
1978
301
310
2
Hartman
KR
LaRussa
VF
Rothwell
SW
Atolagbe
TO
Ward
FT
Klipple
G
Antibodies to myeloid precursor cells in autoimmune neutropenia.
Blood.
84
1994
625
631
3
Currie
MS
Weinberg
JB
Rustagi
PK
Logue
GL
Antibodies to granulocyte precursors in selective myeloid hypoplasia and other suspected autoimmune neutropenias: use of HL-60 cells as targets.
Blood.
69
1987
529
536
4
Harmon
DC
Weitzman
SA
Stossel
TP
The severity of immune neutropenia correlates with the maturational specificity of antineutrophil antibodies.
Br J Haematol.
58
1984
209
215
5
Hoffman
R
Zaknoen
S
Yang
HH
et al
An antibody cytotoxic to megakaryocyte progenitor cells in a patient with immune thrombocytopenic purpura.
N Engl J Med.
312
1985
1170
1174
6
Abgrall
JF
Berthou
C
Sensebe
L
Le Niger
C
Escoffre
M
Decreased in vitro megakaryocyte colony formation in chronic idiopathic thrombocytopenic purpura.
Br J Haematol.
85
1993
803
804
7
de Alarcon
PA
Mazur
EM
Schmieder
JA
In vitro megakaryocytopoiesis in children with acute idiopathic thrombocytopenic purpura.
Am J Pediatr Hematol Oncol.
9
1987
212
218
8
Nidorf
D
Saleem
A
Immunosuppressive mechanisms in pure red cell aplasia: a review.
Ann Clin Lab Sci.
20
1990
214
219
9
Murphy
MF
Izaguirre
CA
Dhaliwal
H
Wrigley
PFM
Waters
AH
Pure red cell aplasia: evidence for an inhibitory action of the bone marrow adherent cell layer.
Clin Lab Haematol.
6
1984
61
67
10
Tyndall
A
Gratwohl
A
Blood and marrow stem cell transplants in autoimmune disease: a consensus report written on behalf of the European League Against Rheumatism (EULAR) and the European Group for Blood and Marrow Transplantation (EBMT).
Br J Rheumatol.
36
1997
390
392
11
Marsh
JC
Chang
J
Testa
NG
Hows
JM
Dexter
TM
The hematopoietic defect in aplastic anemia assessed by long-term marrow culture.
Blood.
76
1990
1748
1757
12
Gibson
FM
Gordon-Smith
EC
Long-term culture of aplastic anaemia bone marrow.
Br J Haematol.
75
1990
421
427
13
Gibson
FM
Scopes
J
Daly
S
Ball
S
Gordon-Smith
EC
Haemopoietic growth factor production by normal and aplastic anaemia stroma in long-term bone marrow culture.
Br J Haematol.
91
1995
551
561
14
Marsh
JC
Chang
J
Testa
NG
Hows
JM
Dexter
TM
In vitro assessment of marrow ‘stem cell’ and stromal cell function in aplastic anaemia.
Br J Haematol.
78
1991
258
267
15
Issaad
C
Croisille
L
Katz
A
Vainchenker
W
Coulombel
L
A murine stromal cell line allows the proliferation of very primitive human CD34++/CD38− progenitor cells in long-term cultures and semisolid assays.
Blood.
81
1993
2916
2924
16
Weaver
A
Ryder
WDJ
Testa
NG
Measurement of long-term culture initiating cells (LTC-ICs) using limiting dilution: comparison of endpoints and stromal support.
Exp Hematol.
25
1997
1333
1338
17
Fazekas de St Groth
The evaluation of limiting dilution assays.
J Immunol Methods.
49
1982
R11
R23
18
Sutherland
HJ
Eaves
CJ
Eaves
AC
Dragowska
W
Lansdorp
PM
Characterization and partial purification of human marrow cells capable of initiating long-term hematopoiesis in vitro.
Blood.
74
1989
1563
1570
19
Sutherland
HJ
Lansdorp
PM
Henkelman
DH
Eaves
AC
Eaves
CJ
Functional characterization of individual human hematopoietic stem cells cultured at limiting dilution on supportive marrow stromal layers.
Proc Natl Acad Sci U S A.
87
1990
3584
3588
20
Roberts
AW
Nicola
NA
Granulocyte colony-stimulating factor.
Colony-Stimulating Factors: Molecular and Cellular Biology.
Garland
JM
Quesenberry
PJ
Hilton
DJ
1997
203
226
Marcel Dekker
New York, NY
21
Omori
F
Okamura
S
Shimoda
K
Otsuka
T
Harada
M
Niho
Y
Levels of human serum granulocyte colony-stimulating factor and granulocyte-macrophage colony-stimulating factor under pathological conditions.
Biotherapy.
4
1992
147
153
22
Yang
FC
Tsuji
K
Oda
A
et al
Differential effects of human granulocyte colony-stimulating factor (hG-CSF) and thrombopoietin on megakaryopoiesis and platelet function in hG-CSF receptor-transgenic mice.
Blood.
94
1999
950
958
23
Lucas
GF
Prospective evaluation of the chemiluminescence test for the detection of granulocyte antibodies: comparison with the granulocyte immunofluorescence test.
Vox Sang.
66
1994
141
147
24
Brighton
TA
Evans
S
Castaldi
PA
Chesterman
CN
Chong
BH
Prospective evaluation of the clinical usefulness of an antigen-specific assay (MAIPA) in idiopathic thrombocytopenic purpura and other immune thrombocytopenias.
Blood.
80
1996
194
201

Author notes

Helen A. Papadaki, Department of Hematology of the University Hospital of Heraklion, PO Box 1352, Heraklion, Crete, Greece; e-mail: epapadak@med.uoc.gr.

Sign in via your Institution