Mast Cells Are Involved by the Malignant Process and Play An Important Role in the Pruritogenesis in Patients with Myeloproliferative Disorders

The Philadelphia chromosome negative (Ph⁻) myeloproliferative disorders (MPD), which include polycythemia vera (PV), essential thrombocythemia (ET) and primary myelofibrosis (PMF) are thought to result from the transformation of a multipotent hematopoietic stem cell (HSC). These Ph⁻ MPDs are characterized by trilineage bone marrow hyperplasia with increased production of red cells, granulocytes and platelets which largely determines their clinical manifestations. Pruritus is common in Ph⁻ MPDs occurring in approximately 50% of cases. The itching occurs spontaneously or appears when taking a hot shower or following other sudden environmental changes. In the present study, we explored the hypothesis that mast cells (MCs) play an important role in pruritogenesis in Ph⁻ MPDs. PB CD34⁺ cells from 18 PV, 11 PMF, and 7 G-CSF mobilized (Gmob) volunteers were cultured in the presence of SCF (100ng/ml) and IL-6 (50ng/ml) for 7–8 weeks. After 49 days of culture, Gmob PB CD34⁺ cells generated significantly greater numbers of MCs (range: 5.6–20.1×10⁶; mean±SD: 11.3±5.2×10⁶) than PV (0.5–3.8×10⁶; 1.2±0.9×10⁶; p<0.005) or PMF (0.5–18.5×10⁶; 4.9±5.5×10⁶; p<0.05) CD34⁺ cells. When MC colony forming assays were conducted with PB CD34⁺ cells from 13 PV, 7 PMF, 4 Gmob volunteers, the average number of MC colonies formed by PV (36.1±30.3/1×10³ CD34⁺ cells), PMF (28.9±20.8) or Gmob (37.6±4.5) PB CD34⁺ cells were similar. Further analysis showed that cultured PV or PMF MCs underwent increased apoptosis in vitro. By using JAK2V617F, MplW515L and chromosomal abnormalities as markers of involvement by the malignant process, we demonstrated that MPD MCs were involved by the malignant clone in Ph⁻ MPDs. In addition, JAK2V617F⁺ MC colonies were assayable from all of the 17 JAK2V617F⁺ MPD patients examined. Cultured MPD MCs exhibited an increased ability to migrate toward SCF and IL-8. To evaluate the role of MPD MCs in the pruritogenesis, we analyzed the production of pruritogenic MC mediators and cytokines including histamine, leukotrienes (LTs), prostaglandins D₂ (PGD₂), IL-4 and IL-31 by the cultured MPD and normal MCs. The release and/or production of histamine and leukotrienes (LTs) by cultured MPD MCs was significantly elevated as compared to those of normal MCs (p<0.05), while the level of an anti-pruritogenic factor, prostaglandins D₂ (PGD₂) was shown to be decreased following incubation at 42°C. The production of IL-31, a pruritogenic mediator known to be produced by activated T cells, was significantly higher in cultured MPD MCs and CD3⁺ T cells than MCs and T cells from healthy volunteers. However, the release of tryptase and IL-4 by MPD and normal MCs was shown to be similar. When the data obtained from MPD MCs were correlated with the degree of pruritus in the cohort of MPD patients studied, we found that pruritus was associated with

1. the generation of a greater number of MCs by CD34⁺ cells;

2. a greater number of MC colonies formed by CD34⁺ cells;

3. a lower percentage of apoptotic cells;

4. a higher plasma levels of IL-31; and

5. a lower levels of PGD₂ released by cultured MCs.

Erlotinib, a JAK2 inhibitor, was able to inhibit the ability of JAK2V617F⁺ PV CD34+ cells to form MC colonies, indicating that abnormal MC progenitors are a potential cellular target for JAK2 inhibitors. These studies not only provide direct evidence for the clonal involvement of MCs in MPDs, but also supply evidence suggesting that abnormal MCs play a crucial role in pruritogenesis in patients with MPDs by producing higher levels of pruritogenic and/or decreased levels of anti-pruritogenic factors. These studies might lead to the identification of cellular and molecular targets for the development anti-pruritus drugs for patients with Ph⁻ MPDs.