Pivotal contributions of megakaryocytes to the biology of idiopathic myelofibrosis

Abstract

In order to investigate the biologic processes underlying and resulting from the megakaryocytic hyperplasia that characterizes idiopathic myelofibrosis (IMF), peripheral blood CD34+ cells isolated from patients with IMF, polycythemia vera (PV), and G-CSF-mobilized healthy volunteers were cultured in the presence of stem cell factor and thrombopoietin. IMF CD34+ cells generated 24-fold greater numbers of megakaryocytes (MKs) than normal CD34+ cells. IMF MKs were also shown to have a delayed pattern of apoptosis and to overexpress the antiapoptotic protein bcl-xL. MK hyperplasia in IMF is, therefore, likely a consequence of both the increased ability of IMF progenitor cells to generate MKs and a decreased rate of MK apoptosis. Media conditioned (CM) by CD61+ cells generated in vitro from CD34+ cells were then assayed for the levels of growth factors and proteases. Higher levels of transforming growth factor-beta (TGF-beta) and active matrix metalloproteinase-9 (MMP9) were observed in media conditioned with IMF CD61+ cells than normal or PV CD61+ cells. Both normal and IMF CD61+ cells produced similar levels of VEGF. MK-derived TGF-B and MMP-9, therefore, likely contribute to the development of many pathological epiphenomena associated with IMF.

Figure 1

Effect of different concentrations of TPO on the generation of CD41⁺ cells in vitro. This figure represents the percentage of CD41⁺ cells generated in suspension culture from normal and IMF CD34⁺ cells in the presence of 100 ng/mL SCF and various concentrations of TPO (0-200 ng/mL) (n = 3) after 11 days of culture. In the absence of TPO, the percentage of CD41⁺ cells was significantly greater in cultures of IMF CD34⁺ cells compared with normal (P < .05). The addition of 5 ng/mL TPO resulted in a marked increase in the percentage of normal CD41⁺ cells (> 95% of the maximal output), while causing a far smaller increment in the percentage of CD41⁺ cells in the IMF samples (38% of the maximal output). A relative plateau in the percentage of CD41⁺ cells was observed at higher doses of TPO. The * indicates significant differences between the percent of normal CD41⁺ cells in normal and IMF cultures (P < .05). Values are shown as the mean ± SD.

Figure 2

Number of cells generated from normal and IMF CD34⁺ cell cultures. (A) The absolute total number of cells generated by normal, IMF, and PV CD34⁺ cells after 15 days of culture in the presence of maximal cytokine combination (100 ng/mL TPO and 100 ng/mL SCF) favoring megakaryocyte development. Each ▴ represents the result of a single experiment with specimens from different patients. The bar represents the mean number of values for each group. The total number of cells generated from CD34⁺ cells was significantly greater in IMF samples (n = 7) compared with normal samples (n = 4) and PV samples (n = 4) (P < .05). (B) The absolute number of CD41⁺ cells generated after 15 days in the same cultures. The numbers of CD41⁺ cells generated by IMF CD34⁺ cells was statistically greater than the numbers generated by normal CD34⁺ cells (n = 4) or PV CD34⁺ cells (P < .05). No significant difference (C) in the absolute numbers of cells or (D) the absolute number of CD41⁺ cells generated by the CD34⁺ cells obtained from patients with JAK2 617V>F–positive and –negative IMF was found (P > .05).

Figure 3

MK morphology and ploidy after 15 days of culture. (A,B) Representative photomicrographs demonstrating the morphology (100 ×/0.75 NA oil objective, Olympus BH-2 microscope, Nikon digital sight DS-L1 camera) of normal and IMF MKs generated from CD34⁺ cell cultures after 15 days of incubation. (C,D) demonstrate DNA content of CD41⁺ cells generated from a representative sample of normal and IMF CD34⁺ cells as determined flow cytometrically.

Figure 4

Apoptosis of normal and IMF CD41⁺ cells. The percentages of normal, IMF, and PV CD41⁺ cells undergoing apoptosis after 8, 11, and 15 days of culture are shown here. A progressive increase in the number of CD41⁺ cells undergoing apoptosis was observed in normal, IMF, and PV CD34⁺ cell cultures. The number of IMF CD41⁺ cells undergoing apoptosis was significantly smaller than that of normal CD41⁺ cells after 11 and 15 days of culture (P < .05; n = 8-12). Values are expressed as the mean ± SD.

Figure 5

Expression of bcl-xL by normal and IMF megakaryocytes. (A) depicts a representative flow cytometric analysis of bcl-xL expression by normal and IMF CD41⁺ cells after 3 days of secondary culture (day 18). Percentages represent the percentage of CD41⁺ cells expressing Bcl-xL. (B) shows the decay of bcl-xL expression in the percentage of CD41⁺ cells present in normal, IMF, and PV cultures. After 11 days of culture, there was a marked decline in the expression of bcl-xL in the normal CD41⁺ cells, while the expression of bcl-xL remained persistently elevated until day 18 in the IMF and PV CD41⁺ cells and then decreased after that. A significant difference in the expression of bcl-xL was observed between the normal and IMF-cultured cells (P < .01) (n = 5 and 7, respectively). Values are shown as the mean ± SD. (C) represents the expression of bcl-xL by CD41⁺ cells derived from CD34⁺ cells isolated from patients with IMF. The overexpression of bcl-xL was independent of JAK2 617V>F mutational status (P > .05). Values are shown as the mean ± SD.

Figure 6

TGF-β, active MMP-9, and VEGF levels in the CM from normal and IMF cell cultures. The levels of TGF-β, active MMP-9, and VEGF were quantified in the CM after 15 days of primary cultures and 3 days of secondary cultures by ELISA. Each point represents the concentration of a growth factor or protease in the CM. The bars represent the mean values for each group. (A,B) represent the levels of TGF-β in the CM from normal, IMF, and PV samples at the 2 time points mentioned above. Significantly greater levels of TGF-β were found in the IMF CM from secondary cultures (B) (P < .01). (C,D) represent the levels of active MMP-9 in the CM from normal, IMF, and PV cultures in primary and secondary cultures. Significantly greater levels of active MMP-9 were found in the IMF CM both after 15 days of primary cultures and 3 days of secondary cultures compared with normal samples (P < .05). (E-F) VEGF levels were also measured in the CM from these cultures. In contrast to TGF-β or active MMP-9, the levels of VEGF were not found to be elevated in the IMF CM after 3 days of secondary cultures (F), however, greater levels of VEGF were found in the IMF CM from primary cultures (E) (P < .05), probably related to the greater numbers of MKs found in these cultures.