hematopoietic progenitors in Diamond-Blackfan anemia In vitro growth ...

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1991 78: 2203-2210

In vitro growth and regulation of bone marrow enriched CD34+ hematopoietic progenitors in Diamond-Blackfan anemia GP Bagnara, G Zauli, L Vitale, P Rosito, V Vecchi, G Paolucci, GC Avanzi, U Ramenghi, F Timeus and V Gabutti

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In Vitro Growth and Regulation of Bone Marrow Enriched CD34’ Hematopoietic Progenitors in Diamond-Blackfan Anemia By G.P. Bagnara, G. Zauli, L. Vitale, P. Rosito, V. Vecchi, G. Paolucci, G.C. Avanzi, U. Ramenghi, F. Timeus, and V. Gabutti Diamond-Blackfan anemia (DBA) is a congenital red blood cell aplasia. No clear explanation has been given of its defective erythropoiesis, although different humoral or cellular inhibitory factors have been proposed. To clarify the nature of this defect we studied the effect of several human recombinant growth factors on an enriched CD34+ population obtained from the bone marrow of 10 DBA patients. We observed a defect underlying the early erythroid progenitors, which were unresponsiveto several growth factors (erythropoietin, interleukin-3 [IL-31, IL-6, granulocyte-macrophage colony-stimulating factor [GM-CSF], erythroid potentiating activity), either alone or in association. The production of

cytokines was not impaired, and high levels of IL-3 and GM-CSF were found in phytohemagglutinin-leukocyteconditioned medium (PHA-LCM) when tested with a sensitive biologic assay on the M-07E cell line. Hematopoietic stem cells in DBA patients may be induced to differentiate to the granulocyte megakaryocyte, but not the erythroid compartment, as shown after CD34’ cell preincubation with IL-3. Addition of the stem cell factor to IL-3 and erythropoietin induces a dramatic in vitro increase in both the number and the size of BFU-E, which also display a normal morphologic terminal differentiation. o 1991 by The American Society of Hematology.

D

ences in the experimental methods. In almost all reports, the bone marrow population was not purged of accessory cells, such as T, B, natural killer (NK) lymphocytes, or adherent cells whose involvement in the control and production of stimulating factors was crucial; moreover, recombinant human growth factorswere used in only a few instances. This article reports the effect of human recombinant growth factors on a purified CD34’ population obtained from the bone marrow of 10 DBA patients. Early erythroid progenitors failed to respond to Epo, IL-3, IL-6, GM-CSF, and erythroid potentiating activity (EPA), whether alone or in association. The defect stems from the inability of multipotent progenitors or CFU-GEMM (colony forming units: granulocyte, erythroid, monocyte, and megakaryocyte) to undergo erythroid differentiation because proliferative and differentiative activity toward granulocyte, macrophage, and megakaryocyte lineages is normal. IL-3 and GM-CSF production by peripheral mononuclear blood cells was increased in all patients. The addition of stem cell factor (SCF)”-’’ to IL-3 and Epo induced a significant increase of the number and size of BFU-E both in normal and in DBA cases.

IAMOND-BLACKFAN anemia (DBA) is a congenital red blood cell (RBC) aplasia characterized by severe normochromic-macrocytic anemia appearing in early infancy, often associated with phenotype abnormalities. The bone marrow is normocellular, with a selective deficiency of RBC precursors or, in some cases, their arrested maturation at the early stages of differentiation. The other cell lineages are normal. The exact explanation for this defective erythropoiesis has not been firmly established, and several humoral or cellular inhibitory factors have been proposed. The erythropoietin (Epo) level in DBA is generally high. Epo structure and function are normal, because the serum can support the growth of normal bone marrow erythroid progenitors or burst-forming units (BFU-E). Antibodies against Epo have never been detected.14 The number of bone marrow erythroid progenitors, as evaluated by in vitro growth of colony-forming units erythroid (CFU-E) and BFU-E, has been found to be defective both at diagnosis and throughout the disease.”’ Although the erythroid lineage seems to be the only one involved, earlier hematopoietic progenitors could be impaired, as moderate neutropenia or trombocytopenia may occur later and a higher risk of progression to leukemia has been documented.’ Cellular inhibition of erythropoiesis has sometimes been observed: T lymphocytes from DBA patients inhibited CFU-E growth when cocultured with normal bone marrow cells, and adherent-cell or T-cell depletion may improve erythroid progenitor growth.’ The meaning of these studies is controversial and their results have not been ~0nfirmed.I~ l4 Recently, the purification of progenitor cells from lymphocytes and monocytes has improved the growth of erythroid progenitors in patients responsive to steroids, but not in resistant cases.’ It has also been shown that recombinant human interleukin-3 (IL-3) is capable of supporting in vitro erythroid progenitor growth in some, but not all, DBA patients.“ Therapy with IL-3 or granulocyte-macrophage colonystimulating factor (GM-CSF) has also been experimented with transient effect.I6 This lack of uniform data may be attributable to the manifold phenotypes of DBA. It is primarily due to differBlood, Vol78, No 9 (November 1). 1991: pp 2203-2210

MATERIALS AND METHODS

Patients and methods. Bone marrow samples were obtained from 10 DBA patients and 12 normal controls of the same age after

From the Institute of Histology and General Embryology, “Giop‘o Prodi” Interdepartmental Centerfor Cancer Research, and the Department of Pediatrics III, University of Bologna; the Department of Biomedical Sciences and Human Oncology and the Department of Pediatrics, University of Turin, Italy. Submitted October 1. 1990; accepted June 21, 1991. Supported in part by an Italian Ministry of University and Scientific and Technological Research Grant, and A.I.R.C. grants to Dr L. Pegoraro and G.P.B. Address reprint requests to Gian Paolo Bagnara, MD, Institute of Histology and General Embryology, University of Bologna, Via Belmeloro 8, Bologna, Italy. The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C.section I734 solely to indicate this fact. 0 I991 by The American Society of Hematology. 0006-4971I91 17809-0032$3.00/0 2203

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BAGNARA ET AL

informed consent. Bone marrow aspirates were taken from controls for suspected hematologic disease not confirmed by subsequent analysis. Peripheral blood samples (15 mL) were collected from these patients and 10 normal donors to prepare phytohemoagglutininleukocyte-conditioned medium (PHA-LCM). The patients were four boys and six girls, ages 2 months to 16 years (mean 7 2 5 years), all diagnosed within the first 5 months of life (except for one at 2 years) on the basis of anemia with low reticulocyte count and marrow erythroid hypoplasia (erythroid nucleated cells 0.5% to 16.8%, mean 5.1% f 4.9%). In case 8, maturation had been arrested at the proerythroblast level. Clinical and hematologic data at diagnosis and the time of the study are summarized in Table 1. All patients were studied at least 6 months after suspension of steroid therapy or before its commencement (cases 5 and 8). Serum Epo levels were higher than expected for the current hemoglobin (Hb) level (mean Epo 448 f 254 mUimL, mean Hb 9.4 ? 0.77 gidL). Three patients presented congenital abnormalities (anophthalmus, ventricular septal defect, ventricular septal defect, plus pulmonary valvular stenosis). Recombinant growth factors. Recombinant human (rh) GMCSF, rhIL-6, and rhIL-3 were kindly supplied by Behring (Behring, Marburg, Germany) and stored at -70°C for no more than 3 months before use. Their specific biologic activity was determined by evaluating the number of granulocyte-macrophage colonies (CFU-GM) obtained from normal bone marrow mononuclear nonadherent cells (MNAC). rhEpo was a gift from Cilag Chemi (Cologno Monzese, Milan, Italy). Human EPA was kindly provided by Dr D. Golde (UCLA, Los Angeles, CA).*’As a source of SCF, we used the media conditioned by COS cells transfected with an expression plasmid containing cDNA encoding murine kit ligand truncated 5’ to the transmembrane domain.” The SCF was kindly supplied by Dr S.C. Clark (Genetics Institute, Cambridge, MA). The dilution 1:200 gives the half-maximal activity, as measured by the M-07E proliferation assay.

Cell separations. Heparinized bone marrow and peripheral blood samples were diluted 1:2 with Iscove’s Modified Dulbecco’s Medium (IMDM; GIBCO, Grand Island, NY), layered over Ficoll-Hypaque (1077 sd; Pharmacia, Uppsala, Sweden) and centrifuged at 1,500 rpm for 30 minutes. Light-density mononuclear cells (LD-MC) were collected and washed twice in IMDM supplemented with 10% of fetal calf serum (FCS). Mononuclear adherent cells were then removed by two steps of incubation (1 hour) in plastic flasks at 37°C in a humidified atmosphere of 5% CO, in air. Subsequently, T-lymphocyte-depleted MNAC were obtained by two steps of rosetting with neuraminidase-treated sheep RBCs. After removal of T cells, a positive reaction to the OK T11 (CD2) monoclonal antibody (MoAb) was constantly less than 1%. In cases 3, 9 and 10, the lymphocytes were negatively removed with immunomagnetic beads coated with antimouse IgG (M-450 Dynabeads; Dynal, Oslo, Norway) after incubation at 4°C with the following antibodies: CD2, CD19, CD22, CD14, CD56, and CD57. Positive selection of CD34’ cells in bone marrow samples. Bone marrow non-T MNAC, 5 x 105, were treated with 5 pL of My10 MoAb (CD34 Tecnogenetics, Milan, Italy) for 1 hour at 4”C, in a final volume of 150 to 200 KL of phosphate-buffered saline (PBS) + 1% FCS, under continuous shaking. After two washings to eliminate the excess MoAb, cells were treated with immunomagnetic beads coated with antimouse IgG for 30 minutes in ice. A ratio of 3 to 4 beads per target cell was found to provide the best recovery. CD34+cells were then collected by a magnet (MPC 1, Dynabeads; Dynal) and resuspended in IMDM + 10% FCS. After overnight incubation, they were washed and gently pipetted to facilitate their separation from the beads; 60% to 80% of the total CD34’ population was recovered. The fraction lost was mainly comprised of dead cells or cells coated too heavily with beads. CD34’ cells mainly consisted of morphologically unidentifiable blast elements on May-Griinwald-Giemsa staining, slightly contaminated by promyelocytes. In some cases, the phenotype profile was performed after positive selection, with a nondetectable reactivity (constantly under 1%) to

Table 1. Hematologic and Clinical Data 1

Patients

At presentation Sex Age (mo) Congenital anomalies* Hb g/dL Reticulocytes YO WBC x 1 0 9 / ~ Platelets x 109/L Bone marrow YOerythroid nucleated cellst Epo mU/mL

G.M

F 2 No 3.5 0.2 6.8 250

8 C.S.

P.A.

10 S.P.

M

M

F

440

1.5 Yes 4.6 0.1 11.4 492

1 No 7.6 0.1 7.2 330

5 No 5.6 0.1 8.5 320

0.4 380

16.5

400

1.8 350

-

7 10.3 0.5 4.7 36 Yes Yes 169

10 8.3 0.2 6.4 384 Yes Yes 686

2 L.D.

3 T.M.

4 C.E.

5

6

7

C.A.

M.S.

T.L.

F 5 Yes 6.8 0.3 13 200

M 24 No 3 0.2 3.5 200

F

F

2 No 3.2 0.3 9.9 150

2 No 2.4 0.1 9.9 450

F 2 No 2.3 0 10.2 200

M 1 Yes 6.6 0.2 9.7

1.5

5

5

9

5

6

-

-

-

-

-

-

16 9.5 0.2 4.5 220 Yes No 600

5 9 0.2 5.8 230 Yes No 193

4 10.5 0.1 6.7 320 Yes No 822

7 10 0.2 8.2 300 Yes No 349

2 (mo) 9.8 0.1 7.2 250 No

5

9

0.5

At the moment of study Age (vr) Hb g/dL Reticulocytes YO WBC x 1 0 9 / ~ Platelets x 109/L Regular transfusion therapy Steroid response Epo mU/mL

*Congenital anomalies: ventricular septal defect patient 2, ventricular septal defect tBM YOerythroid cells: patient 8 maturative arrest at the proerythroblast level.

-

8.3 0.1 7.3 150 Yes No

11 9.1 0.05 4.0 231 Yes No

-

-

-

7 (mol 8.9 0.05 12.8 40 1 No

320

+ pulmonary stenosis patient 8, anophthalmus patient 7.


REGULATION OF HEMATOPOIESIS IN DBA

CD4, CD2, CD16, and CD19 (Ortho Diagnostic System, Raritan, NJ). Cell culture. The colony assay for BFU-E was performed according to Iscove et al.” Bone marrow CD34+ cells, lo4, resuspended in 0.1 mL of PBS + 1% detoxified bovine serum albumin (BSA, Fraction V Chon; Sigma, St Louis, MO) were plated in a 1-mL mixture of IMDM containing 30% FCS, 2 X mol/L hemin, 5 x lo-’ mol/L p-mercaptoethanol, 1% BSA, and 0.9% methylcellulose. The cells were stimulated, in our optimal standard conditions, with rhEpo 2 U and rhIL-3 100 U or with various combinations of recombinant growth factors, as specified below. The assay for the 14-day CFU-GM was performed as previously described.24CD34’ bone marrow cells, lo4,were plated in 35-mm Petri dishes in 1 mL of IMDM containing 20% heat-inactivated FCS, 0.3% noble agar, and 200 U rhGM-CSF + 100 U of rhIL-3 as the standard source of colony-stimulating activity. For the megakaryocyte colony-forming units (CFU-MK) assay, plasma clot assay was performed according to McLeod et ala and Vainchenker et al.” CD34’ cells, 104, were seeded in IMDM, supplemented with 20 pg L-Asparagine (Sigma) 3.4 pg CaCI,, 10% BSA, 10% of a preselected batch of heat-inactivated pooled human AB serum, and 200 U rhGM-CSF. After 12 days of incubation, the plasma clot was fixed in situ with methanol-acetone 1:3 for 20 minutes, washed with PBS and double-distilled water, and then air-dried. Fixed plates were stored at -20°C until immunofluorescence staining was performed. CFU-MK colonies were scored as aggregates of 3 to 100 cells intensively fluorescent to J15 (CD41W) MoAb directed against the IIB-IIIA glycoprotein complex. Binding was shown by fluorescinated goat antimouse IgG (Ortho). To clarify whether different combinations of recombinant factors influence bone marrow progenitors, and especially BFU-E growth in DBA patients, we tested the effect of increasing concentrations of Epo 2 to 6 UimL alone or in combination with: IL-6 (1 to 100 UimL), GM-CSF (200 UlmL), IL-3 (100 UimL), EPA 1:1,OOO, IL-3 (100 U) PIUS GM-CSF (200 U), IL-3 (100 U) plus IL-6 (1 to 100 U/mL). In three patients (cases 3,9, and 10) and in three normal controls the effect of the SCF on BFU-E growth was studied on the CD34’ positive bone marrow population. The SCF has been tested at 1:100, L:200, and 1:400 in association with Epo 2 and 4 U/mL, and IL-3 (100 UimL). Pre-exposure experiments in liquid cultures. CD34t cells from four patients and four controls were either seeded immediately in semisolid media or preincubated in IMDM + 10% FCS in the presence of rhIL-3 (100 UlmL). Daily (up to the fourth day) aliquots were collected for clonogenic assays. The percentage of CD34’ cells was determined by immunofluorescence. BFU-E, CFU-GM, and CFU-MK growth is expressed as the number of colonies by lo4CD34+ cells. Preparation of PHA-LCM. LD-MC from the peripheral blood of the 10 DBA patients and 10 normal donors were seeded in IMDM, supplemented with 10% FCS and 1% phytohemoagglutinin (PHA) at a concentration of lo6cellsiml, and incubated for 72 hours. The conditioned medium was than harvested, centrifuged for 10 minutes at 2,000 rpm, filtered, and stored at -70°C for no more than 3 months, before use on the M-07E cell line. Anti-GM-CSF and anti-K-3. Murine MoAb anti-GM-CSF and anti-IL-3 were purchased from Genzyme Co (Cambridge, MA). A fixed dilution of 1:100 (PBS 1% FCS) was tested on 0.005% to 10% vol/vol concentrations of both normal and DBA patients’ PHA-LCM. Biologic assay for the production of GM-CSF and IL-3. The GM-CSF and IL-3-dependent M-07E cell line2’ was kindly provided by Dr L. Pegoraro (University of Turin, Italy). PHA-LCM was used as a source of growth factors at a final concentration of

+

1% for 96 hours. Exponentially growing M-07E cells were washed twice and seeded at 1 x 106cellsimlin IMDM with 5% FCS. After 24 hours of IL-3 deprivation, 5 x lo4cells were washed twice and seeded in 200-pL microwells in IMDM with 5% FCS in the presence of increasing concentrations of PHA-LCM (from 0.005% to 10% vol/vol). After 28 hours, triplicate wells were pulse-labeled with ’H-TdR (1 pCi/well) for 4 hours. Alcohol acid precipitated radioactivity was determined by liquid scintillation counting. The concentration of IL-3 and GM-CSF in PHA-LCM was determined by comparing the dose-response curves obtained with serial dilutions of rhIL-3 and rhGM-CSF and of PHA-LClk GM-CSF and IL-3 levels were determined by subtraction of the number of counts per minute (cpm) obtained in the presence of the anti-IL-3 or anti-GM-CSF MoAb from the total cpm amount. Epo measurement. Serum Epo levels were determined by raditlimmunoassay= using radiolabeled hormone and anti-Epo antibodies (Ingstar; Sorin, Saluggia, Italy). Statistical analysk. The results were expressed as means f 1 standard error of mean (SEM) of the data from two or more experiments performed in duplicate. Statistical analysis was per. formed using the two-tailed Student’s t-test, linear regression, and correlation tests. RESULTS

In vitro growth of enriched (CD34+) bone marrow cells. The results of the in vitro growth of 14th day CFU-Gd, CFU-MK, and BFU-E in our standard culture conditions are summarized in Table 2. Our assay did not assess CFU-E growth. Culture of CD34’ cells does not give rise to CFU-E colonies at day 7 because they are earlier progenitors, and only the 14th da$ BFU-E are detectable. The number of CFU-GM and CFU-MK colonies derived from DBA marrows was similar to that from normdl controls (P > .05). In contrast, a dramatic decrease in tHe erythroid progenitors was observed in 9 of 10 DBA patients (P < .01). Production of IL-3 and GM-CSF fromPHA-LCM of DBA patients. We investigated whether production of the cytdkines involved in erythroid differentiation accounts for impaired erythropoiesis in DBA patients. The production of IL-3 and GM-CSF by DBA patient and normal contrdl Table 2. In Vitro Growth of Granulomacrophage(CFU-GM), Megakaryocyte(CFU-MK),and Erythroid (BFU-E) Progenitors From 10’ CD34‘ Enriched Bone Marrow Samples Case No. DBA

1 2 3 4 5 6 7 8 9 10 Mean 2 SEM Controls (mean of 12 cases

CFU-GM

CFU-MK

BFU-E

20 13 16 23 27 28 25

18 22 16 10 35 32 23 17 18 16 20.7 ? 2.4

2

7 3 4 t 2.3

23.2 t 16

62 z 14

a 9 20 18.9 -+ 2.2

lr

SEM)

29

* 6.5

0

0 2 1 2 1 25


BAGNARA ET AL

peripheral blood mononuclear cells was tested on the M-07E cell line. Addition of 1:lOO vol/vol anti-GM-CSF and anti-IL-3 completely abrogated the stimulatory activity of 10 pg/mL IL-3 and 2 to 3 pg/mL GM-CSF, respectively (data not shown). However, in the experiments performed on PHALCM, incomplete neutralization with anti-GM-CSF and anti-IL-3 MoAbs was observed at the higher PHA-LCM concentrations (1% and 10%) (Fig 1).This finding may be partially explained by the presence of other cytokines (ie, IL-2, IL-4, and IL-9) to which the M-07E line is sensitive, although not dependent on them.2Y The amount of both IL-3 and GM-CSF was significantly higher (P < .01) in DBA patients: 384 ~f:27 pg IL-3 and 302 f. 12 pg GM-CSF versus 206 -r- 15 pg and 230 ~f:9.2 pg in normal controls. The difference in IL-3 production was statistically significant at the final PHA-LCM concentration of 10% (P = .034), whereas that in GM-CSF levels was only significant at 0.5% (P= .001) and 0.1% (P = .003), indicating that IL-3 production is more pronounced than that of GM-CSF in DBA.

I l l

0

0.01

0.1

10

% of PHA-LCM 40000

T

0

0.01

0.1

1

IO

X of PHA-LU1 Fig 1. Biologic assay on the M-07E cell line for the production of GM-CSF and IL-3 by PHA-LCM obtained from 10 DBA patients (A) and 10 controls (B). Data are the means f SEM of three separate controls; (-+-), experiments performed in duplicate. (A) (-m-), anti-GM-CSF; (-0-), anti-IL-3. [B) (-El-), DBA patients; (-+-), anti4WCSF; (-0-), anti-IL-3.

/1 /1

200 -

DBA patients --t

Controls

100 -

x

0

. & .

0

=

,-. 20

.

I

X

I

6

Y

.-

40

60

80

100

TIHE o f LIWID CULTURE (hours)

Fig 2. BFU-E growth after pre-exposure to IL-3. CD34‘ cells from four DBA patientsand four normal controls were pre-exposed in liquid culture to 100 U of rhlL-3. After 12, 24, 48, 72, and 96 hours, CD34’ cells were harvested and seeded in methylcellulosemedium for BFU-E growth. Data represent the means 2 SEM of the number of colonies observed in four separate experiments. Each point was performed in duplicate.

Re-exposure to ZL-3. Exposure to IL-3 increases the number of all the hematopoietic progenitors and induces their differentiati~n.~’ Therefore, we investigated the effect of a 12- to 96-hour exposure of bone marrow CD34+ cells to 100 U of IL-3 in liquid cultures in DBA and in controls. After 96 hours in controls, the number of BFU-E was twofold to threefold higher than in non-pre-exposed cultures. The number of BFU-E was constantly low in DBA patients (Fig 2). CFU-MK and CFU-GM were significantly increased after 96 hours and their growth pattern was similar in both DBA patients and normal controls (Fig 3). Effect of cytokines on the in vitro growth of BFU-E. The effect of different combinations of recombinant factors on the growth of control and DBA BFU-E is illustrated in Table 3. Hemoglobinized erythroid colonies were never detected in the absence of Epo. In CD34’ enriched normal bone marrow cultures, these combinations increased the number of BFU-E compared with Epo alone, peak stimulation being achieved by Epo (2 U) + IL-3 (100 U). DBA BFU-E were never increased. The response of the erythroid progenitors to different dilutions of SCF in three DBA cases and in normal controls is illustrated in Table 4. Addition of even the lowest SCF dilution to Epo and IL-3 induced a dramatic increase in the number of both normal and DBA BFU-E. This finding was the same when Epo was increased from 2 to 4 U/mL (data not shown). There was a striking increase of the size of each aggregate in a single BFU-E (from a mean of 30 cells to more than 200 cells) (Fig 4). DISCUSSION

Several reasons have been proposed for the erythropoietic inhibition observed in D B A a humoral mechanism involving autoimmunity or the presence of inhibiting factors,3’,’’ a cellular mechanism mediated by T lymphocytes, NK cells, or adherent cells,”” impaired cytokine production



Table 4. Number of BFU-E/ 10' CD34' Bone Marrow Cells From Three DBA Patientsand Three Normal Controls After Stimulation With Epo (2 U) IL-3 (100 U) or Epo (2 U) + IL-3 (100 U) + SCF at Three vol/vol Dilutions

+

T

T T ~

DBA Cases

Case 9

Case 10

Controls

0

0

4

34 2 2.0

32 34 33

28 24 25

36 30 36

116 8.4 81 2 14.2 96 f 13.1

Case 3

+ +

IL-3 100 U EPO 2 U IL-3 100 U EPO 2 U SCF 1:lOO 1:200 1:400

+

*

The dilution 1:200 gives the half-maximal activity on M-07E proliferation; the same half-maximal stimulation on M-07E is obtained by the addition of 5-10 ng/mL rhSCF (Santoli D, et al, manuscript in preparation). O h 96 h PRE--D(POSITION TO 100 U of IL-3 IN LIQUID CULTURES

Fig 3. CFU-GM and CFU-MK growth in agar and plasma clot media. CD34' cells from four DBA patients and four normal controls were pre-exposed to IL-3 for 96 hours. Data are the means e SEM of four separate experiments. Each point was performed in duplicate. (El), CFU-GM DBA; (W), CFU-GM controls; (E),CFU-MK DBA; (0). CFU-MK controls.

or function, or an intrinsic defect within the hematopoietic stem cells.'~' A decrease in bone marrow BFU-E and CFU-E number in DBA has been observed?.' In contrast, attempts to enhance erythroid progenitor growth and differentiation have given conflicting results. The growth defect can be partially corrected in vitro by addition of high doses of Epo or IL-3 or ~ter0ids.l~ Interestingly, the impairment of in vivo erythropoiesis seems to be more dramatic in DBA patients who do not respond to steroid the rap^."^ An increased number of reticulocytes and a decreased blood requirement in some transfusion-dependent and steroid-unresponsive DBA patients have been recently Table 3. Effect of Different Combinations of Growth Factors on the In Vitro Growth of Erythroid Progenitors (BFU-E) From Enriched Bone Marrow Samples (WCD34' cells/plate) of 10 DBA Patients and 12 Normal Controls ~

Epo 2 U Epo 4 U Epo 6 U EPO2 U+ IL-3 100 U EPO2 U+ IL-3 100 U + IL-6 1 U EPO2 U+ IL-3 100 U IL-6 10 U EPO 2 U+ IL-3 100 U + IL-6 100 U EPO 2 U+ IL-6 1 U EPO 2 U+ IL-6 10 U Epo 2 U+ IL-6 100 U EPO2 U+ GM-CSF 200 U EPO2 U+ GM-CSF 200 U + IL-3 100 U Epo 2 U+ EPA 1:lOO

+

Controls

DBA

26 f 7 30 2 13 32 2 13 62 f 14 45 13 46 f 14 60 2 15 20 6 19 f 6 19 f 7 4 5 2 13 60 f 14 55 f 14

1.2 0.7 1.3 f 0.6 2.1 f 1.1 4.3 f 2.3 4.5 f 2.6 3.4 f 1.9 3.6f 2 1.5 f 0.8 1.6 0.8 1.1 0.4 3.6 f 1.1 3.6 1.8 3.5 2 1.4

* *

* *

Data are expressed as means f 1 SEM. Each point was performed in triplicate.

reported following the administration of GM-CSF and IL-3.I6 All these findings suggest a possibility of improving erythroid growth. The results obtained have always been scanty and transient. Although this could be due to differences in the genetic defect, patient's age and clinical history, it must be underlined that the bone marrow progenitors have not always been concentrated and purified from accessory cells, and recombinant growth factors were not used by some workers. In our experiments, only CD34' positively selected progenitors, virtually free from accessory cells, were plated in the presence of well-defined recombinant growth factors. We also investigated the ability of patient's mononuclear cells to produce GM-CSF and IL-3, which are well-known regulators of erythropoiesis. Our observation that addition of various recombinant cytokines to a CD34' positively selected lymphocyte and monocyte-free marrow population always fails to support the growth of BFU-E in DBA challenges the supposition of a direct lymphocyte or monocyte involvement in the BFU-E growth defect. The inhibition of erythropoiesis reported by some investigators in cocultures with T lymphocytes from DBA patients may be ascribable to an immunologic phenomenon related to HLA antigens in subjects regularly t r a n s f ~ s e d . ~ ~ After pre-exposure to IL-3, hematopoietic progenitors were induced to differentiate to the granulocyte and the megakaryocyte, but not the erythropoietic lineage. This finding indicates that the low number of BFU-E is not due to a diminished population of multipotent stem cells, but to a defect in the passage from the CFU-GEMM to BFU-E as shown by the lack of progenitor response to the in vitro stimulating activity of Epo and IL-3, IL-6, GM-CSF, EPA, alone or in association. Moreover, impaired production of these cytokines in DBA is denied in our experiments. In a highly sensitive biologic assay, PHA-LCM produced increased amounts of IL-3 and, to a lesser extent, GM-CSF. However, if we take into account the central role played by IL-3 and by GM-CSF in normal bone marrow BFU-E growth and differentiation, it is conceivable that these increases, along


2208

BAGNARA ET AL

A

Fig 4. Size of erythroid aggregates from DBA CD34' bone marrow cells. (A) Cultures stimulated with Epo (2 UlmL) + IL-3 (100 UlmL): (8)cultures stimulatedwith Epo (2 UlmL) I IL-3 (lo0 UlmL) and SCF 1:200vol/vol.

with thc wcll-dtxumcntcd clcviition of scriim Epo in DRA. arc an attcmpt to compcnsatc for crythropoictic fiiilurc. Thc in vivo cytokinc network is still poorly tlcfincd ant1 othcr factors may hc involvctl in stem ccll expansion and erythroid diffcrcnti:ition. Thc rcccntly tlcscrihcd hcniatopoictic SCF (;ilso callcd mast ccll growth fwtor. stccl locus factor. c-kit ligand) has hccn shown to intlucc an incrcilsctl prolifcration of hcmatopoictic propnitors and to display :I potent svncrgistic activitv in combination with II.-2 m d othcr cytokincs.'-'' SCF also sccms to play ;in importiint rolc in thc ;ictiv;ition of primitivc cclls. while tlic dil~crcntiation signal is prrwidcd hv thc spccific C'SFs. Morccwcr. its in vivo administration rcvcrscs anemia in SI /SItI nititant mice."

In our cxpcrimcnts, in vitro ;idtlition of SCF to 1L-3and Epo intluccd ;I signifkint incrcasc of thc numhcr and sizc of both normal iintl DRA DFU-E. This cffcct was constant and striking. Stimulation of DRA crythroitl progcnitors was obccnrd i i t thc SCF conccntration r a n g that provides optimal growth cnh;inccmcnt o n normal CD34' cclls and M-07E cells (S.C. Clark. unpuhlishctl data), suggesting that in DRA. SCF rcccptor is normally cxprcsscd o n SCFrcsponsivc CI1.14' cclls. Thc hypothesis that othcr rcccptor molecules. such ;IS Epo rcccptors. arc involvcd in thc tlisc;isc can not hc ruled out. The ability of SCF to inducc DFU-E proliferation ;ind dilicrcntiation in DRA indiciitcs a similwity hctwccn this discmc and thc miirinc complcx



hematopoietic defect caused by mutations of S1 and W

gene^.'^ In conclusion, our findings show that SCF significantly reverses the erythropoietic impairment in DBA in vitro, suggesting a possible therapeutic utility in vivo. Further studies are needed to see whether the inability of

the bone marrow to produce or release a biologically active form of this factor is involved in the pathogenesis of DBA. ACKNOWLEDGMENT

We thank Dr S.C. Clark for kindly providing SCF and Dr L. Pegoraro for helpful discussion.

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