ARTICLE |
Correspondence to: Yoji Ishida, Iwate Medical University, School of Medicine, 19-1, Uchimaru, Morioka 020, Japan.
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Summary |
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To test the hypothesis that the c-mpl ligand is not a primary factor in thrombocytopoiesis, we investigated the biological effects of recombinant human (rh) c-mpl ligand on differentiation of murine progenitor cells and on maturation of the cultured murine megakaryocytes under serum-free conditions on the basis of ploidy distribution, megakaryocyte/platelet-specific surface antigen CD 61 [glycoprotein (GP) IIIa], and cytoplasmic acetylcholinesterase (AchE) expression in vitro. In addition, we studied the effect of c-mpl ligand on proplatelet formation (PPF) by murine mature megakaryocytes. AchE was less strongly expressed in cultured megakaryocytic cells stimulated by c-mpl ligand than in those stimulated by recombinant murine (rm) IL-3 + rh IL-6 during the differentiation of progenitor cells. Less CD 61 was expressed by c-mpl ligand during both the differentiation of progenitor cells and the maturation of megakaryocytes compared with that by rm IL-3 + rh IL-6. Endomitosis, however, was more stimulated by c-mpl ligand than by rm IL-3 + rh IL-6 under both conditions. Furthermore, PPF of mature megakaryocytes was not stimulated by c-mpl ligand. These results indicate that c-mpl ligand stimulates the nuclear development of megakaryocytic cells but that it does not stimulate cytoplasmic maturation and PPF as much as IL-6. These data strongly suggest that c-mpl ligand is not a primary factor in platelet pro-duction. (J Histochem Cytochem 46:49-57, 1998)
Key Words: c-mpl ligand, megakaryocyte maturation and differentiation, platelet production, proplatelet formation
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Introduction |
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C-mpl LIGAND is a novel cytokine that stimulates megakaryocytopoiesis and thrombocytopoiesis (
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Materials and Methods |
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Reagents and Animals
Recombinant human (rh) c-mpl ligand and rh interleukin 6 (IL-6) were generously provided by Kirin Brewery (Tokyo, Japan) and Ajinomoto (Tokyo, Japan), respectively. Recombinant murine (rm) IL-3 and FITC-labeled anti-murine CD 61 antibody were obtained from Pharmingen (San Diego, CA). Propidium iodide (PI) was purchased from Sigma (St Louis, MO). Male ddY mice were obtained from Shizuoka Laboratory Center (Shizuoka, Japan).
Cell Culture
Preparation of Nonadherent and Nonphagocytic Cells.
Male ddY mice 6-8 weeks old were used in all studies. Nonadherent and nonphagocytic cells were obtained as described (
Culture of Nonadherent and Nonphagocytic Cells.
Nonadherent and nonphagocytic cells were cultured in IMDM supplemented with 1% Nutridoma (Behringer Mannheim Biochemica; Mannheim, Germany) with rh c-mpl ligand (20 ng/ml) or rm IL-3 (10 U/ml) + rh IL-6 (100 ng/ml) at a density of 1 x 106 cells/ml for 3 days. The cultured megakaryocytes were partially purified using an albumin gradient as described (
Nonadherent and nonphagocytic cells were cultured for 4 days under the same conditions as described above. Two-color analysis (CD 61 expression and ploidy distribution) of the cultured cells was performed as described below.
Nonadherent and nonphagocytic cells were cultured in IMDM supplemented with 1% Nutridoma and c-mpl ligand (20 ng/ml) for 3 days. The cells were washed twice with IMDM and re-cultured with various concentrations of rh c-mpl ligand (1, 20, and 100 ng/ml) or rh IL-6 (10 and 100 ng/ml) for 2 additional days at a density of 1 x 106 cells/ml. These cells were analyzed flow cytometrically as described below.
To estimate the stimulating activity of mpl ligand on DNA development of megakaryocytes, culture of nonadherent and nonphagocytic cells was performed for 4 days with various concentrations of rh c-mpl ligand (0.2, 2, and 20 ng/ml), and nonadherent and nonphagocytic cells were cultured with rh c-mpl ligand (20 ng/ml) for 3, 4, and 5 days. The ploidy distribution of the cultured megakaryocytes was analyzed flow cytometrically as described below.
AchE Staining of the Cultured Megakaryocytes.
The cells cytospun on the slides with 2% BSA were fixed with 1% glutaraldehyde for 10 min. (Nakeff et al. 1976), washed twice with phosphate buffered saline (PBS) and stained for AchE as described (
Two-color Flow Cytometry of Cultured Megakaryocytes: Analysis of Ploidy Distribution of Cultured Megakaryocytes and CD 61 Expression at each Ploidy Class.
Cultured megakaryocytes were stained with FITC-labeled anti-murine CD 61 antibody (Pharmingen) and propidium iodide (Sigma) for CD 61 and nucleus in megakaryocytes, respectively, according to the method described (
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Proplatelet Formation by the Murine Megakaryocytes
Murine megakaryocytes were partially purified from bone marrow cells using an albumin gradient as described (
Statistical Analysis
Student's t-test was used to determine statistically significant differences.
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Results |
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Culture of Nonadherent, Nonphagocytic Cells
To evaluate the stimulatory activity of rh c-mpl ligand on the cytoplasmic maturation of megakaryocytic cells during differentiation, we cultured murine bone marrow cells for 3 days. AchE expression by the cultured megakaryocytes stimulated with rh c-mpl ligand (20 ng/ml) was compared with that induced by rm IL-3 (10 U/ml) + rh IL-6 (100 ng/ml). Figure 1 shows the intensity of AchE staining and the size of each megakaryocyte. Most of the cultured megakaryocytes, including large and small cells, stimulated by rh c-mpl ligand were much less stained for AchE, whereas those cultured with rm IL-3 + rh IL-6 were stained more intensely. The megakaryocytes stimulated by rh c-mpl ligand, however, were larger than those stimulated by rm IL-3 + rh IL-6. To confirm these results, we evaluated the expression of the platelet-specific surface marker CD 61 by the cultured megakaryocytes for 4 days. In preliminary experiments, we performed two-color analysis of the cultured megakaryocytes with various concentrations of rh c-mpl ligand (0.2, 2, or 20 ng/ml) for 4 days (Figure 3). Because 20 ng/ml of rh c-mpl ligand stimulated the highest shift of ploidy distribution in megakaryocytes, the concentration of rh c-mpl ligand (20 ng/ml) was chosen in the following experiments (Table 1 and Table 2). The mean FITC fluorescence (CD 61 expression) at each ploidy (8 N, 16 N, 32 N) was approximately 1.4-1.7-fold increased (1.4-, 1.62-, and 1.74-fold increase in megakaryocytes with 8 N, 16 N, and 32 N ploidy class, respectively) in the cultured megakaryocytes stimulated by rm IL-3 + rh IL-6 over those stimulated by rh c-mpl ligand (Table 1). In addition, the maturation activity of rh c-mpl ligand or rh IL-6 on the mature megakaryocytes was evaluated. We cultured murine bone marrow cells with rh c-mpl ligand (20 ng/ml) to become mature megakaryocytes, then incubated them for 2 more days with rh c-mpl ligand (1, 20, or 100 ng/ml) or rh IL-6 (10 or 100 ng/ml). Rh IL-6 (100 ng/ml) stimulated a 1.2-, 1.25-, 1.44-, and 1.47-fold increase of CD 61 expression in the megakaryocytes with 8 N, 16 N 32 N, and 64 N ploidy class, respectively, over those stimulated by rh c-mpl ligand (20 ng/ml) (Table 3). However, c-mpl ligand stimulated a shift towards a higher ploidy class of megakaryocytes than rm IL-3 + rh IL-6 in nonadherent and nonphagocytic cells cultured for 4 days (Table 2). In addition, the ploidy distribution of megakaryocytes after 3 days of culture with rh c-mpl ligand (20 ng/ml), followed by 2 additional days of culture with rh c-mpl ligand (1, 20, or 100 ng/ml) or rh IL-6 (10 or 100 ng/ml) is shown in Table 4. The megakaryocytes with higher ploidy class were observed more frequently in the cultures with c-mpl ligand than in those with rh IL-6. To estimate the stimulatory effect of c-mpl ligand on DNA development of megakaryocytes, the culture period was extended to 5 days with rh c-mpl ligand (20 ng/ml). The ploidy distribution of the megakaryocytes is shown in Figure 4. After culture for 5 days, megakaryocytes with high ploidy class (32 N, 64 N, and 128 N) were frequently observed.
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These results indicate that c-mpl ligand plays a significant role in stimulating the endomitosis of megakaryocytes but a lesser role in the cytoplasmic maturation of megakaryocytic cells during differentiation and maturation, based on ploidy distribution and on AchE and CD 61 expressions.
Proplatelet Formation by Megakaryocytes
A total of 2 ± 0.3 x 104 (mean ± SD; n = 4) megakaryocytes were obtained from two mice. The purity of the megakaryocytes was 12.1 ± 2.0 % n = 4), determined by AchE staining. The maturation stages of the purified megakaryocytes were estimated by the measurement of the ploidy class. More than 95% of the megakaryocytes were those with 8 N, 16 N, 32 N, and greater ploidy classes. Typical ploidy distributions of the purified megakaryocytes are shown in Figure 5. After a 24-hr incubation, the number of megakaryocytes with PPF was 17.6 ± 1.4 %, 16.0 ± 2.1%, 17.3 ± 2.3%, 7.2 ± 1.1% (p<0.01), and 22.3 ± 2.5% (p< 0.05, n = 4), stimulated by medium alone (control), rh c-mpl ligand (0.2 ng/ml), rh c-mpl ligand (2 ng/ml), rh c-mpl ligand (20 ng/ml), and rh IL-6 (50 ng/ml), respectively (Figure 6). A typical megakaryocyte with PPF stimulated by rh IL-6 (50 ng/ml) is shown in Figure 7.
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Discussion |
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Many reports have described the biological effects of mpl ligand in vivo and in vitro (
At first, we focused on AchE expression of the cultured megakaryocytic cells during differentiation, stimulated by mpl ligand or IL-3 + IL-6 for 3 days. AchE has been used as a differentiation/maturation marker for megakaryocytic cells during differentiation (-granules and the demarcation membrane at the ultrastructural level under serum-free conditions.
Several groups of investigators have determined the effects of mpl ligand on the expression of platelet-specific membrane proteins.
The mpl ligand stimulated DNA development in the megakaryocytes cultured with mpl ligand for 3, 4, and 5 days (Table 2 and Table 4). Megakaryocytes of the 64 N ploidy class tended to be increased in numbers among the cultured megakaryocytes stimulated by mpl ligand (20 ng/ml) for 3 days and 2 additional days with mpl ligand (Table 4). A significant difference were not observed between the mpl ligand and the IL-6 group except for the IL-6 (100 ng/ml) group (Table 4), probably due to the biological effect of IL-6, which stimulates an increase in ploidy class of megakaryocytes in vitro (
In addition, we investigated the stimulatory activity of c-mpl ligand on proplatelet formation (PPF) by megakaryocytes. PPF is considered to be the process of functional platelet production in vitro (Figure 7). We found that c-mpl ligand did not stimulate the PPF process. However, other investigators have reported results to the contrary (
Recently,
In summary, mpl ligand stimulates the endomitosis of megakaryocytes but not cytoplasmic maturation as IL-6 does. In addition, it does not stimulate the PPF process in vitro. On the basis of these data, c-mpl ligand is not likely to be involved in the process of platelet production.
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