|
|||||||||
|
|
|
|
|
|
|
|
|
|
|
HEMATOPOIESIS
From the Division of Hematology/Oncology, Hunter Holmes
McGuire Department of Veterans Affairs Medical Center, and the Medical
College of Virginia, Virginia Commonwealth University, Richmond, VA.
When interleukin-2 (IL-2) was added to immature, low-ploidy
(greater than 80% 2N+4N) megakaryocytes generated in IL-3 and stem
cell factor (SCF)-containing liquid cultures of blood mononuclear cells highly enriched in hematopoietic progenitors, a 2- to 6-fold increase in the absolute number of polyploid (more than 8N)
megakaryocytes was noted. This effect was found to be indirect and was
mediated through natural killer (NK) cells that constitute the major
lymphoid cell contaminating day 6 megakaryocyte cell populations. IL-2 had no effect on megakaryocytes generated from CD34+ cells
stimulated with IL-3 and SCF. However, medium conditioned by
IL-2-stimulated, but not resting, NK cells (NKCM) contained a
trypsin-sensitive factor capable of increasing 2- to 5-fold the number
of polyploid megakaryocytes generated in vitro from IL-3 and
SCF-stimulated CD34+ cells. The activity in NKCM was dose
dependent and could not be neutralized by an excess of antibodies to
IL-6, IL-11, leukemia inhibitory factor (LIF), gp130, stromal
cell derived factor-1a (SDF-1a), and thrombopoietin (TPO). Addition of
IL-11, but not TPO, to NKCM-containing cultures resulted in further
augmentation of polyploidy, with the generation of 50% to 70%
polyploid megakaryocytes with a modal ploidy of 16N. This factor is
distinct from TPO because it induces endomitosis in IL-3-generated
megakaryocytes in vitro, whereas TPO does not, and its activity on
megakaryocyte ploidy is not altered by optimal concentrations of TPO.
In addition, no message for TPO is detectable in IL-2-stimulated NK
cells by reverse transcription-polymerase chain reaction. These
findings indicate that IL-2-stimulated NK cells produce a novel
peptide, distinct from TPO, IL-6, IL-11, LIF, other gp130-associated
interleukins, and SDF1a, that can induce in vitro endomitosis in
immature human megakaryocytes in the presence of IL-3 and SCF.
(Blood. 2002;99:130-136) Polyploidy is a unique feature of bone marrow
megakaryocytes. Terminal differentiation of megakaryocytes includes the
development of polyploid cells through the process of endomitosis.
Although the need for megakaryocytes to achieve polyploid status is
poorly understood, it seems related to their ability to produce
platelets because the modal ploidy of marrow megakaryocytes increases
in states of accelerated thrombopoiesis and decreases in states of suppressed thrombopoiesis.1,2 Humoral factors that may
influence the ploidy of megakaryocytes are only partially known. Among
them, thrombopoietin has been shown to be capable of inducing
endomitosis in vitro in serum-containing cultures of unseparated whole
marrow murine cells,3 but its activity on endomitosis is
limited in serum-free cultures of purified human CD34+
cells.4,5
During studies on the effects of various known growth factors,
interleukins, and cytokines on the ploidy of human blood
CFUMEG-derived megakaryocytes in vitro, it was
noted than when interleukin-2 (IL-2) was added to cultures containing
optimal concentrations of IL-3 and stem cell factor (SCF), the ploidy
of generated megakaryocytes was shifted toward higher classes. This
observation led to further experiments, described herein, by which a
unique soluble peptide was identified that is secreted by
IL-2-stimulated natural killer cells and is capable of inducing
endomitosis in human megakaryocytes generated in vitro under the
stimulation of IL-3 and SCF.
Cell separation and cell cultures
In a number of experiments in which CD34+ cells were
used for the generation of immature day 6 megakaryocytes in vitro,
cells were isolated from light-density mononuclear blood cells by
anti-CD34-coated magnetic beads (Dynabeads, Dynal, Oslo,
Norway) according to the manufacturer's protocol. They
were cultured in medium as already described for 12 to 14 days at
37°C in a highly humidified 5% CO2 atmosphere. They were
then collected, counted, and analyzed for CD41+ cells
and distribution into ploidy classes by flow cytometry.
Natural killer (NK) lymphocytes were isolated from peripheral blood
mononuclear cells by density-gradient centrifugation over Percoll (density, 1.066)9 followed by depletion of T
lymphocytes either by sheep red cell rosetting or by anti-CD3-coated
magnetic beads. In other experiments, NK cells were isolated by
anti-CD56-coated magnetic beads. After overnight incubation in
IMDM with 10% autologous serum and 4 nM (ng/mL) IL-2, were
separated from the beads and depleted of T lymphocytes with
anti-CD3-coated magnetic beads. Medium conditioned by NK lymphocytes
was prepared by culturing NK cells at a concentration of
5 × 104/mL in IMDM with 5% human serum in the presence
of 10 ng/mL IL-2 for 3 to 7 days at 37°C in a 5% CO2
highly humidified atmosphere.
Growth factors and antibodies
Measurement of megakaryocyte ploidy Measurement of DNA content of CFUMEG-derived cells was performed by 2-color flow cytometry.10 Cells from liquid cultures were removed on day 12 to 14 and were stained with fluorescein isothiocyanate (FITC)-conjugated anti-CD41 (Gen Trak, Plymouth Meeting, PA) or isotype control murine IgG1 (DAKO, Carpinteria, CA). After washing, the cells were fixed in 70% cold methanol for 30 minutes at 4°C, washed again, and treated at 37°C for 30 minutes with DNase-free RNase (Cellular DNA-Flow Cytometric Analysis Reagent Set; Boehringer Mannheim, Indianapolis, IN). After staining with propidium iodide (50 µg/mL), nuclear ploidy was measured by a FACScan (Becton Dickinson) flow cytometer. Fluorescence intensity from FITC and propidium iodide was analyzed using logarithmic display. Megakaryocytes were selected on the basis of specific membrane immunofluorescence. A threshold for GPIIb/IIIa expression (CD41) was determined using the mouse isotype control. A fluorescence gate containing 2% or less of the isotype control-positive cells was established, and the propidium iodide staining of cells within this gate was used to analyze ploidy. Ploidy distribution was determined by setting markers at the nadirs between peaks at approximately 50-channel intervals. At least 10 000 CD41+ cells were analyzed per experiment. All data were stored in list mode and then analyzed using FACScan Research Software. Results were expressed as the mean ± SEM of data obtained from 3 or more experiments. Statistical significance of ploidy distribution was determined using the 2 test.
Reverse transcription-polymerase chain reaction for thrombopoietin and SDF1a Total NK RNA was prepared with the use of guanidinium salts,11 and mRNA was purified by oligo-dT-coated magnetic beads (Dynal). NK mRNA (200 ng) and an equal amount of human liver mRNA (Clontech) were subjected to reverse transcription-polymerase chain reaction (RT-PCR). Reverse transcription was performed using the SuperScript Preamplification kit (BRL) with both oligo-dT12-18 and random hexamers according to the manufacturer's protocol. For amplification of thrombopoietin (TPO) message, the products of this reaction were subjected to PCR using two 20-mers as primers corresponding to positions 301 to 320 (5' primer) and 551 to 570 (3' primer) of the TPO complementary DNA (cDNA) sequence and Taq polymerase. The 270-bp sequence is part of the TPO cDNA that encodes for the active part of the TPO protein. As a control for the integrity of mRNA and the absence of inhibitors, a 5' and a 3' 20-mer of a 1300-bp sequence of the transferrin receptor (rTF Amplimer; Clontech) were used. After 30 cycles, the PCR products were analyzed by agarose gel electrophoresis.For amplification of SDF1a message, the products of reverse transcription were subjected to PCR using 2 oligodeoxyribonucleotide primers corresponding to positions 160 to 184 (5' primer) and 559 to 533 (3' primer) of the human SDF1a cDNA (Maxim Biotech, San Francisco, CA) in the presence of Taq polymerase. The 400-bp sequence is part of human SDF1a cDNA encoding active protein. As a control for the integrity of mRNA and the absence of inhibitors, a 5' and a 3' 20-mer of a 329-bp sequence of the enolase cDNA were used. As a positive control, 400-bp human SDF1a cDNA (Maxim Biotech) was used. After 30 cycles, the PCR products were analyzed by agarose gel electrophoresis.
Interleukin-2 induces endomitosis in immature megakaryocytes generated from blood CFUMEG in vitro When IL-2 was added to cultures of peripheral blood mononuclear cells enriched in hematopoietic progenitors containing optimal concentrations of IL-3 and SCF, the ploidy of generated megakaryocytes was shifted to higher classes (Figure 1). Among 12 experiments, the mean proportion of megakaryocytes greater than or equal to 8N increased from 26% ± 5% to 45% ± 4% (P < .0001), and the mean megakaryocyte ploidy increased from 3.3 ± 0.3N to 4.9 ± 0.2N (P < .0001). A typical histogram of megakaryocyte ploidy by flow cytometry of CD41+ cells from cultures without or with IL-2 is shown in Figure 2. Addition of IL-2 to cultures did not affect in any consistent way the number of megakaryocytes generated in vitro from blood CFUMEG.
The effect of IL-2 on endomitosis was dose dependent between 0.5 and 4 ng/mL (Figure 3). Further increases in
IL-2 concentration did not result in any additional increase of
polyploid megakaryocytes (more than 8N) in vitro. Because previous work
in this laboratory indicated that most endomitotic events occur between
days 6 and 10 in culture,6 time-course experiments were
performed in which IL-2 was added to the cultures on different days.
The effect of IL-2 was mostly pronounced when IL-2 was added on day 6 of cultures. Less pronounced effects could be seen when IL-2 was added
earlier, and its effect on ploidy of megakaryocytes disappeared on day 10 (Figure 4). In all subsequent
experiments, IL-2 was added to the culture medium on day 6.
Effect of IL-2 on megakaryocyte endomitosis is mediated by natural killer cells Day 6 megakaryocytes, the cells most responsive to IL-2, were found to have no receptors for IL-2 as tested by FACS using CD41-phycoerythrin and FITC-conjugated CD25 or CD122 for the IL-2 receptor and  , respectively. Adding IL-2 to CD34+
cells failed to cause a shifting of megakaryocyte ploidy in vitro (Figure 5). These experiments were
interpreted as evidence that the effect of IL-2 on developing
megakaryocytes is indirect.
To identify the nature of the accessory cell(s) responsible for the IL-2 effect, we analyzed by FACS and a battery of monoclonal antibodies the cell population from day 6 cultures. Seventy-five percent to 90% of the cells expressed CD33, of which 15% to 27% were positive for CD41 and the remaining for CD13. Of the remaining nonmyeloid cells, T and B cells (CD3 and CD19) made up 2% to 3% of the total cells; the remaining were CD56+, CD11+, and CD16+. Thus, the major type of accessory cells in day 6 cultures were phenotypically NK cells. It seems that the use of CD2 and CD11 for the removal of T lymphocytes, monocytes, and NK cells during the process of enrichment of peripheral blood mononuclear cells in hematopoietic progenitors by negative panning was not sufficient for the effective removal of NK cells. The role of NK cells was further confirmed by purifying and culturing
them in the presence of IL-2. After 7 to 10 days, more than 85% of
cultured cells expressed CD16 and CD56, and almost 95% of them
exhibited the morphology of large granular lymphocytes with blastic
nuclei. When these cells were washed and added to autologous
CD34+ cells grown in the presence of IL-3 and SCF, no
statistically significant change in the ploidy of megakaryocytes was
noted. However, when IL-2 was added to these cultures, a significant increase in the ploidy of megakaryocytes was observed as compared with
cultures of CD34+ cells alone (Figure
6). Similar experiments performed by
coculturing purified autologous T lymphocytes or monocytes with
CD34+ cells with or without IL-2 showed no effect on the
ploidy of day 6 megakaryocytes (data not shown). These experiments
confirmed the role of NK cells as mediators of IL-2 effects on
megakaryocyte ploidy.
Interleukin-2-stimulated natural killer cells release a soluble peptide responsible for induction of endomitosis Addressing whether NK cells release a soluble factor that mediates the effect of IL-2 or whether they require direct cell-to-cell interaction with day 6 megakaryocytes, conditioned medium from NK cells (NKCM) was tested on blood CD34+ cells grown for 6 days under the stimulation of IL-3 and SCF. This day 6 cell population was tested by FACS and was found to contain no CD56+ cells. Addition of NKCM, but not IL-2 alone, to day 6 megakaryocytes resulted in the induction of endomitosis and the generation of polyploid megakaryocytes after 6 days in culture (Figure 7). Furthermore, the effect of NKCM on the number of polyploid megakaryocytes was dose dependent (Figure 8). Medium conditioned by purified T cells or monocytes stimulated with IL-2 had no effect on the ploidy of day 6 megakaryocytes (data not shown). These experiments demonstrated that the effect of IL-2 by NK cells is mediated through a soluble factor released in the conditioned medium.
To characterize the chemical nature of this factor, NKCM was treated with trypsin immobilized on agarose in the presence of 1 mM EDTA for 1 hour at 37°C; the beads were then removed by centrifugation, and the NKCM was dialyzed in IMDM, filtered, and tested on day 6 immature megakaryocyte derived from CD34+ blood cells. Control medium was exposed to plain agarose beads and treated through the same procedures. Exposure of conditioned medium to trypsin resulted in complete loss of the activity of the soluble factor inducing megakaryocyte endomitosis compared with control-conditioned medium (Figure 7), indicating that the factor responsible for this activity is a peptide released in the culture medium. NK-released peptide is a novel lymphokine distinct from IL-6, IL-11, LIF, other gp130-related interleukins, and SDF1a Because the activation of NK cells is known to result in the release of a variety of growth factors and interleukins9,12 in subsequent experiments we investigated the effect of IL-1, -4, -6, -7, -8, -9, -10, -11, -12, tumor necrosis factor-, interferon (IFN), MIP1a, and granulocyte
macrophage-colony-stimulating factor (GM-CSF), G-CSF, and M-CSF on the
ploidy of megakaryocytes generated from CD34+ blood cells
compared with NKCM. No one of these factors was found to be able to
induce endomitosis with shifting of the megakaryocyte ploidy, as was
invariably noted with NKCM (data not shown). Therefore, the effect of
NKCM could not be attributed to the presence of either of these factors
in the conditioned medium.
Because IL-6, IL-11, and leukemia inhibitory factor (LIF) have been
proposed as factors promoting maturation and endomitosis in
megakaryocytes, additional experiments were performed in which NKCM was
incubated with an excess of neutralizing antibodies to IL-6, IL-11, and
LIF at 37°C for 1 hour and then were added to day 6 megakaryocytes.
The concentration of neutralizing antibodies in these experiments was
capable of inhibiting 5 to 10 times the amounts of IL-6, IL-11, or LIF
that have been reported to increase the percentage of megakaryocytes in
a single ploidy class. In these experiments, the shifting of
megakaryocyte ploidy toward higher classes was not affected by
any of the neutralizing antibodies (Figure 9). Similar
experiments were performed using an antibody to gp-130 to exclude the
possibility that the effects of NKCM were mediated through a
combination of gp130-related interleukins or a gp130-related
interleukin other than IL-6, IL-11, and LIF (Figure 9). In addition,
since a recent report indicated that human stromal cell derived
factor-1a (SDF-1a) increases ploidy of human megakaryocytes in
vitro,13 similar experiments were performed using a
neutralizing antibody to human SDF1a (Figure 9). Furthermore, mRNA from
IL-2-activated NK cells was subjected to RT-PCR for the amplification
of human SDF1a message showing that activated NK cells do not express
SDF1a (Figure 10).
These data indicated that NK cells activated by IL-2 release a soluble
peptide that is functionally a novel lymphokine different from GM-CSF,
M-CSF, G-CSF, erythropoietin, SCF, IL-1, -4, -6, -7 to -12, IFN Natural killer cell-released peptide is distinct from thrombopoietin To investigate whether the peptide produced by NK cells is TPO, NKCM was incubated with an excess amount of anti-TPO antibody (nonneutralizing) followed by incubation with protein G bound to agarose. After removal of the agarose beads and dialysis, the treated NKCM was tested on day 6 megakaryocytes generated from CD34+ cells. Treatment of NKCM with anti-TPO had no effect on the increase of ploidy of megakaryocytes, indicating that the peptide in NKCM is immunologically distinct from TPO (Figure 11).
In addition, NK cells were purified and cultured with IL-2, and their
mRNA was isolated and subjected to RT-PCR for the detection of TPO
message. Both the TPO 270-bp sequence and the 1300-bp rTF sequence were
amplified from liver mRNA, but only the rTF sequence was amplified from
NK mRNA, indicating lack of expression of the TPO gene
(Figure 12). Increasing the PCR cycles
to 35 and 40 resulted in a stronger signal for rTF, but the 270-bp TPO
sequence could not be amplified. Electrophoresed PCR products were
further analyzed by Southern blotting and hybridization with a
32P end-labeled 21-oligomer complementary to the 335- to
355-bp sequence of the TPO cDNA. After autoradiography, only a single band was detected in the lane containing the RT-PCR products from liver
mRNA (corresponding to 270-bp band on agarose gel), but no signal was
detected from NK cell mRNA (data not shown). Consequently, the activity
in NKCM responsible for the induction of endomitosis cannot be
attributed to TPO production by IL-2-stimulated NK cells.
NK-related peptide synergizes with IL-11 in promoting endomitosis In additional experiments, the effects of TPO and IL-11 on the ploidy of megakaryocytes generated from CD34+ cells were studied in NKCM-containing cultures. In comparison to NKCM, TPO was found to have no additive effect on the ploidy of megakaryocyte in the presence of IL-3. However, a synergy was noted between IL-11 and NKCM that resulted in the generation of polyploid megakaryocyte in vitro with a modal ploidy of 16N, a pattern similar to that found in normal bone marrow megakaryocyte (Figure 13).
Considering the ploidy distribution of megakaryocytes as an index
of human megakaryocyte maturation, it has been well established that
IL-3, though the most potent stimulator of megakaryocyte colony
formation in vitro, does not promote their terminal differentiation into polyploid cells.6,14,15 Support for this view is also provided by electron microscopy studies of megakaryocytes from IL-3-stimulated cultures showing that these cells are relatively immature and have only a few To identify factors affecting endomitosis by CFUMEG-generated megakaryocytes under the stimulation of IL-3, we performed a large number of experiments using known recombinant factors, interleukins, and cytokines, either alone or in additive combinations. In these experiments we noted that only combinations of factors containing IL-2 promoted megakaryocyte endomitosis. The experiments described in this report demonstrate that IL-2 can induce endomitosis in megakaryocytes generated from blood CFUMEG under the stimulation by IL-3 and SCF. This effect of IL-2 is indirect and is mediated through NK cells that release a peptide-inducing endomitosis in immature day 6 megakaryocytes. Unstimulated NK cells did not promote endomitosis. The peptide released by IL-2-stimulated NK cells is functionally distinct from all known and tested growth factors and interleukins and is immunologically distinct from IL-6, IL-11, LIF, other gp130-related interleukins, SDF1a, and TPO. In addition, either SDF1a or TPO cannot be held responsible for the observed effect because NK cells were shown to lack SDF1a and TPO mRNA by RT-PCR. Thus, it seems that this NK-released peptide is a novel factor capable of inducing endomitosis in megakaryocytes generated from CFUMEG stimulated by IL-3 and SCF, conditions under which TPO has only minimal or no effect on endomitosis.4,5,19 Because IL-2 is the inducer of endomitosis-promoting NK cell-related
peptide, one may assume that in vivo administration of IL-2 may result
in enhanced thrombopoiesis. However, clinical experience with IL-2
indicates the opposite. Thrombocytopenia is a recognized hematologic
side effect of IL-2 administration in high doses.20,21
This is not a paradox between in vitro and in vivo results because IL-2
administered to humans induces the release of multiple factors
including transforming growth factor The presence of thrombopoietic factors other than TPO has been suggested by a number of observations. Mice deficient in c-mpl are capable of maintaining 15% of normal platelet count despite a complete block in the action of TPO.26 In these mice residual thrombopoiesis is independent of IL-3, IL-6, IL-11, and LIF.27,28 The factor(s) responsible for maintaining residual thrombopoiesis remains unknown. In addition, in reactive thrombocytosis there is no correlation between circulating platelets and serum TPO levels, a substantial number of patients have near normal serum TPO levels, and no correlation can be established between platelet count and circulating IL-6, IL-11, or LIF serum levels.29-33 The association of NK cells with megakaryopoiesis has been previously noted. Mice injected with an anti-NK monoclonal antibody showed an almost complete abolishment of CFUMEG cycling in the marrow, whereas an increase was noted in the cycling of marrow erythroid burst-forming units (BFU-E).34 Natural killer cells have been found to enhance megakaryopoiesis in vitro,35 and CAMPATH-1G activated NK cells and their supernatant have been reported to augment BFUMEG and CFUMEG formation by canine aplastic anemia plasma.36 In addition, activated NK cells have been shown to promote granulocytic and megakaryocytic reconstitution after syngeneic bone marrow transplantation in mice.37 The current findings lend further support to older observations that NK cells may play an important role in modulating thrombopoiesis in vitro and in vivo. It is premature to hypothesize on the role these peptides may have in vivo, but because it is a product of NK cells involved in infectious and inflammatory conditions, it is tempting to postulate a possible role for it in reactive thrombocytosis. The spectrum of the activities of this NK-released peptide and its action(s) in vivo cannot be predicted until its cDNA is isolated and expressed and the appropriate experiments are completed.
Submitted March 22, 2001; accepted September 5, 2001.
Supported by a Merit Review Grant from the Research Service of the Department of Veterans Affairs and the McGuire Research Institute.
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.
Reprints: Emmanuel N. Dessypris, Division of Hematology/Oncology (111K), Hunter Holmes McGuire Veterans Affairs Medical Center, 1201 Broad Rock Blvd, Richmond, VA 23249; e-mail: edessypr{at}hsc.vcu.edu.
1. Mazur EM, Lindquist DL, De Alarcon PA, Cohen JL. Evaluation of bone marrow megakaryocyte ploidy distributions in persons with normal and abnormal platelet counts. J Laboratory Clin Med. 1988;111:194-202[Medline] [Order article via Infotrieve].
2.
Tomer A, Friese P, Conklin R, et al.
Flow cytometric analysis of megakaryocytes from patients with abnormal platelet counts.
Blood.
1989;74:594-601
3.
Broudy VC, Lin NL, Kaushansky K.
Thrombopoietin (c-mpl ligand) acts synergistically with erythropoietin, stem cell factor, and interleukin-11 to enhance murine megakaryocyte colony growth and increases megakaryocyte ploidy in vitro.
Blood.
1995;85:1719-1726 4. Wendling F, Maraskovsky E, Debili N, et al. cMpl ligand is a humoral regulator of megakaryocytopoiesis. Nature. 1994;369:571-574[CrossRef][Medline] [Order article via Infotrieve]. 5. Angchaisuksiri P, Carlson PL, Dessypris EN. Effects of recombinant human thrombopoietin on megakaryocyte colony formation and megakaryocyte ploidy by human CD34+ cells in a serum-free system. Br J Haematol. 1996;93:13-17[CrossRef][Medline] [Order article via Infotrieve]. 6. Angchaisuksiri P, Carlson PL, Day EB, Dessypris EN. Replication and endoreplication in developing megakaryocytes in vitro. Exp Hematol. 1994;22:546-550[Medline] [Order article via Infotrieve]. 7. Sawada K, Krantz SB, Kans JS, et al. Purification of human erythroid colony-forming units and demonstration of specific binding of erythropoietin. J Clin Invest. 1987;80:357-366.
8.
Ebell W, Castro-Malaspina H, Moore MAS, O'Reilly RJ.
Depletion of stromal elements in human marrow grafts separated by soybean agglutinin.
Blood.
1985;65:1105-1111 9. Trinchieri G. Biology of natural killer cells [review]. Adv Immunol. 1989;47:187-376[Medline] [Order article via Infotrieve].
10.
Jackson CW, Brown LK, Somerville BC, Lyles SA, Look AT.
Two-color flow cytometric measurement of DNA distributions of rat megakaryocytes in unfixed, unfractionated marrow cell suspensions.
Blood.
1984;63:768-778
11.
MacDonald RJ, Przybyla AE, Rutter WJ.
Isolation and in vitro translation of the messenger RNA coding for pancreatic amylase.
J Biol Chem.
1977;252:5522-5528
12.
Robertson MJ, Ritz J.
Biology and clinical relevance of human natural killer cells.
Blood.
1990;76:2421-2438
13.
Guerriero R, Mattia G, Testa U, et al.
Stromal cell-derived factor 1 increases polyploidization of megakaryocytes generated by human hematopoietic progenitor cells.
Blood.
2001;97:2587-2595
14.
Debili N, Hegyi E, Navarro S, et al.
In vitro effects of hematopoietic growth factors on the proliferation, endoreplication, and maturation of human megakaryocytes.
Blood.
1991;77:2326-2338
15.
Debili N, Issaad C, Masse JM, et al.
Expression of CD34 and platelet glycoproteins during human megakaryocytic differentiation.
Blood.
1992;80:3022-3035
16.
Kaushansky K.
Thrombopoietin: the primary regulator of platelet production.
Blood.
1995;86:419-431
17.
Williams JL, Pipia GG, Datta NS, Long MW.
Thrombopoietin requires additional megakaryocyte-active cytokines for optimal ex vivo expansion of megakaryocyte precursor cells.
Blood.
1998;91:4118-4126
18.
Debili N, Wendling F, Katz A, et al.
The Mpl-ligand or thrombopoietin or megakaryocyte growth and differentiative factor has both direct proliferative and differentiative activities on human megakaryocyte progenitors.
Blood.
1995;86:2516-2525 19. Dolzanskiy A, Hirst J, Basch RS, Karpatkin S. Complementary and antagonistic effects of IL-3 in the early development of human megakaryocytes in culture. Br J Haematol. 1998;100:415-426[CrossRef][Medline] [Order article via Infotrieve]. 20. MacFarlane MP, Yang JC, Guleria AS, et al. The hematologic toxicity of interleukin-2 inpatients with metastatic melanoma and renal cell carcinoma. Cancer. 1995;75:1030-1037[CrossRef][Medline] [Order article via Infotrieve]. 21. Paciucci PA, Mandeli J, Oleksovicz L, Ameglio F, Holland JF. Thrombocytopenia during immunotherapy with interleukin-2 by constant infusion. Am J Med. 1990;89:108-109. 22. Puolakkainen P, Twardzik D, Ranchalis J, Moroni M, Mandeli J, Paciucci PA. Increase of plasma transforming growth factor beta during immunotherapy with IL-2. Cancer Invest. 1995;13:583-589[Medline] [Order article via Infotrieve].
23.
Ishibashi T, Miller SL, Burstein SA.
Type beta transforming growth factor is a potent inhibitor of murine megakaryocytopoiesis in vitro.
Blood.
1987;69:1737-1741
24.
Kuter DJ, Gminski DM, Rosenberg RD.
Transforming growth factor beta inhibits megakaryocyte growth and endomitosis.
Blood.
1992;79:619-626 25. Lentsch AB, Edwards MJ, Miller FN. Interleukin 2 induces increased platelet-endothelium interactions: a potential mechanism of toxicity. J Lab Clin Med. 1996;128:75-82[CrossRef][Medline] [Order article via Infotrieve].
26.
Gurney AL, Karver-Moore K, de Sauvage FJ, Moore MW.
Thrombocytopenia in c-mpl deficient mice.
Science.
1994;265:1445-1447
27.
Gainsford T, Roberts AW, Kimura S, et al.
Cytokine production and function in c-mpl deficient mice: no physiologic role for interleukin-3 in residual megakaryocyte and platelet production.
Blood.
1998;91:2745-2752
28.
Gainsford T, Nandurkar H, Metcalf D, Robb L, Begley CG, Alexander WS.
The residual megakaryocyte and platelet production in c-mpl deficient mice is not dependent on the actions of interleukin 6, interleukin 11, or leukemia inhibitory factor.
Blood.
2000;95:528-534 29. Hollen CW, Henthorn J, Koziol JA, Burstein SA. Serum interleukin-6 levels in patients with thrombocytosis. Leuk Lymphoma. 1992;8:235-241[Medline] [Order article via Infotrieve]. 30. Akan H, Guven N, Aydogdu I, et al. Thrombopoietic cytokines in patients with iron deficiency anemia with or without thrombocytosis. Acta Haematol. 2000;103:152-156[CrossRef][Medline] [Order article via Infotrieve]. 31. Hsu HC, Tsai WH, Jiang ML, et al. Circulating levels of thrombopoietic and inflammatory cytokines in patients with clonal and reactive thrombocytosis. J Lab Clin Med. 1999;134:392-397[CrossRef][Medline] [Order article via Infotrieve]. 32. Wang JC, Chen C, Novetsky AD, Lichter SM, Ahmed F, Friedberg NM. Blood thrombopoietin levels in clonal thrombocytosis and reactive thrombocytosis. Am J Med. 1998;104:451-455[CrossRef][Medline] [Order article via Infotrieve]. 33. Cerutti A, Custodi P, Duranti M, Noris P, Balduini CL. Thrombopoietin levels in patients with primary and reactive thrombocytosis. Br J Haematol. 1997;99:281-284[CrossRef][Medline] [Order article via Infotrieve]. 34. Pantel K, Nakeff A. Differential effect of natural killer cells on modulating CFU-Meg and BFU-E proliferation in situ. Exp Hematol. 1989;17:1017-1021[Medline] [Order article via Infotrieve]. 35. Gewirtz AM, Xu WY, Mangan KF. Role of natural killer cells, in comparison with T lymphocytes and monocytes, in the regulation of normal human megakaryocytopoiesis in vitro. J Immunol. 1987;139:2915-2924[Abstract]. 36. Nagler A, Condiotti R, Lubina A, Deutsch VR. Enhancement of megakaryocytopoiesis by Campath-1G-treated natural killer cells. Bone Marrow Transplant. 1997;20:525-531[CrossRef][Medline] [Order article via Infotrieve].
37.
Siefer AK, Longo DL, Harrison CL, Reynolds CW, Murphy WJ.
Activated natural killer cells and interleukin-2 promote granulocytic and megakaryocytic reconstitution after syngeneic bone marrow transplantation in mice.
Blood.
1993;82:2577-2584
© 2002 by The American Society of Hematology.
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
 |
|
 |
|
  S. M. Jacobs-Helber, K.-h. Roh, D. Bailey, E. N. Dessypris, J. J. Ryan, J. Chen, A. Wickrema, D. L. Barber, P. Dent, and S. T. Sawyer Tumor necrosis factor-alpha expressed constitutively in erythroid cells or induced by erythropoietin has negative and stimulatory roles in normal erythropoiesis and erythroleukemia Blood, January 15, 2003; 101(2): 524 - 531. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
 |
 |
 |
|||||||
 |
 |
 |
 |
 |
 |
 |
 |
||
| Copyright © 2002 by American Society of Hematology Online ISSN: 1528-0020 | |||||||||
Top
Abstract
Introduction
-bearing cells were removed by rosetting with sheep erythrocytes and with sheep erythrocytes coated with goat anti-sheep red blood cell immunoglobulin (Ig)G.
5 M 
