Expression of TNF receptors and related signaling molecules in the bone marrow from patients with myelodysplastic syndromes

Abstract

Myelodysplastic syndromes (MDS) are characterized by peripheral blood cytopenias despite hypercellularity of the bone marrow regarded as the result of ineffective hematopoiesis mainly caused by apoptosis. In this study, we examined the role of tumor necrosis factor (TNF)-induced apoptosis in the bone marrow cells of MDS patients. The constitutive expression of mRNA for TNF receptors (TNFR), including TNFRI and TNFRII, and the adapter molecules, such as the TNF receptor-associated death domain protein (TRADD), Fas-associated death domain protein (FADD), receptor interacting protein (RIP) and TNF receptor-associated factor 2 (TRAF-2) were analyzed by reverse transcriptase (RT)-PCR in bone marrow samples from control, MDS and AML cases. In bone marrow cells from refractory anemia (RA) patients, there was a significant increase in TNFRI expression as compared with control subjects. The expression of TNFRII was also up-regulated in RA cases. In contrast, RA with excess of blasts (RAEB), RAEB in transformation (RAEB-T) and AML cases revealed increased expression of TNFRII, whereas the expression of TNFRI was comparable to control subjects. Immunohistochemical staining revealed that the TNFRI, as well as TNFRII of MDS bone marrow was expressed mainly in hematopoietic cells, but not in macrophage-lineage stromal cells at the protein level. An increased constitutive expression of mRNA for TRADD, FADD and RIP and decreased expression of mRNA for TRAF-2 were observed in bone marrow cells from MDS patients, especially from RA patients, as compared with controls, although the differences were not significant. In many of the AML bone marrow samples, strong expression of TRAF-2 mRNA was marked, while expression levels of other proteins were similar to those in control subjects. These data suggested enhanced signaling by the TNFRI–TRADD–FADD pathway and suppressed signaling by the TRAF-2 pathway in RA. Thus, TNF-α-induced apoptosis may play a role in ineffective hematopoiesis in “early stage MDS” bone marrow, although the regulatory mechanisms for TNF-α-induced signaling would be complicated and not be simply explained only by these pathways.

Introduction

Myelodysplastic syndromes (MDS) are a heterogeneous group of hematological malignancies showing peripheral blood cytopenias despite bone marrow hypercellularity [1], [2], [3]. Therefore, one characteristic feature of MDS is a decrease in the number of blood cells during bone marrow hematopoiesis. This process designated as ineffective hematopoiesis is caused by frequent apoptosis of bone marrow hematopoietic cells [4], [5] and regulated by a variety of cytokines which exert dual effects, proliferative and apoptotic effects, on hematopoietic cells [6], [7]. Regarding the frequent apoptosis in bone marrow cells of MDS [8], [9], we demonstrated previously that tumor necrosis factor (TNF)-α was overexpressed in bone marrow macrophages [10], the Fas antigen was overexpressed in bone marrow hematopoietic cells [8], the Fas-ligand (Fas-L) was overexpressed in bone marrow hematopoietic cells, as well as macrophages [8], [11], and inducible nitric oxide synthase (iNOS) was overexpressed in bone marrow macrophages [12]. These findings indicated the complicated pathways regarding apoptotic signaling in bone marrow cells. Further, a possible mechanism for apoptosis [13] includes interaction between bone marrow hematopietic cells and stromal cells in an autocrine and paracrine manner.

Another distinctive feature of MDS is the etiologic aspect that the disease affects mainly elderly people [1], [2], [3]. This phenomenon was discovered during the very early period in the history of MDS research, but the reason has not yet been elucidated. Recently, the cytogenetic profile of MDS, particularly of refractory anemia with excess of blasts (RAEB)/RAEB in transformation (RAEB-T), was shown to be nearly identical to that of elderly patients with AML both in the frequency and in type of chromosomal abnormalities [14]. These results indicate that the pathogenesis of MDS may have some relationship with aging, although the cytogenetics associated with MDS are highly complex and heterogeneous [15], [16].

Aging in the hematopoietic system is characterized by various features including bone marrow hypocellularity [17], thymic involution associated with T cell lymphopenia and T cell dysfunction [18], and increased serum TNF-α level [19]. TNF-α and its receptor family are known to regulate various signaling pathways of hematopoietic cells [20], [21], [22]. In particular, TNF-α regulates two opposing activities; a destructive and protective effect on cells. The cytotoxic pathway involves interaction of death domain-containing adapter molecules and caspases leading to apoptosis, whereas the cell-protective pathway involves activation of transcription factors, including NF-κB. TNF-α exerts its effect by binding to two cell surface receptors (TNFI and TNFRII). Both receptors are present on a wide variety of cell types including bone marrow hematopoietic cells [21], [22]. TNFRI mediates most of the biological properties of TNF-α, such as programmed cell death and activation of NF-κB [23], [24]. Upon oligomerization, TNFRI binds to and recruits TRADD and indirectly binds to FADD via an interaction between the death domain of FADD and TRADD, leading to the activation of a caspase cascade resulting in apoptosis [24], [25]. In contrast, TNFRII lacks a death domain and interacts directly with TNF receptor-associated factor 2 (TRAF-2) [26], [27], [28], [29]. TRAF-2 activates both NF-κB and JNK and mediates its antiapoptotic effect [30], [31], [32]. Therefore, TNFRII is involved in the antiapoptotic effect of TNF, whereas TNFRI is involved in both apoptotic and antiapoptotic signaling.

In aged humans, the role of TNF-induced apoptosis in decrease of T cells was examined by the expression of receptors for TNF-α by peripheral blood lymphocytes [33]. Increased constitutive expression of TNFRI and TRADD and decreased expression of TNFRII and TRAF-2 were observed in lymphocytes from aged as compared with young controls. These data suggest that increased TNF-α-induced apoptosis plays a role in T cell deficiency associated with human aging. As described previously [10], TNF-α was overexpressed in the bone marrow macrophages from patients with MDS. Therefore, differential expression and selective usage of TNF receptors may also work as a switch for TNF-mediated signaling pathways on hematopoietic cells in MDS bone marrow. Thus, to determine the down-stream signaling pathways of TNF-α in the bone marrow of MDS patients, expression of TNF receptors (TNFRI and TNFRII), TRADD, FADD, RIP, and TRAF-2 were analyzed, and the mechanisms controlling apoptosis and proliferation of bone marrow cells in MDS were discussed.