Yoshio Katayama

Role for vitamin D receptor in the neuronal control of the hematopoietic stem cell niche

Hematopoietic stem/progenitor cells (HSPCs) are released from the bone marrow to the circulation by the cytokine, granulocyte colony-stimulating factor, via sympathetic nervous system (SNS)-mediated osteoblast suppression. Because the orientation of HSPCs in their osteoblastic niche is reported to be guided by [Ca(2+)], we speculated on a cooperation between the calcium-regulating hormones and SNS in the regulation of HSPC trafficking. Here, we present the severe impairment of granulocyte colony-stimulating factor-induced osteoblast suppression and subsequent HSPC mobilization in vitamin D receptor (VDR)-deficient mice. In osteoblasts, functional VDR possessing, at least in part, a transcriptional activity, was specifically induced by β2-adrenergic receptor (AR) agonists. While β2-AR agonists transiently increased mRNA expression of Vdr and its downstream gene, Rankl, 1α,25-dihydroxyvitamin-D(3) sustained the β2-AR-induced Rankl expression at high level by stabilizing VDR protein. These data suggest that VDR is essential for durable β2-AR signaling in the stem cell niche. Our study demonstrates not only a novel function of VDR as a critical modulator of HSPC trafficking, but also the presence of a SNS-mediated, bone-remodeling mechanism through VDR. VDR contributes to brain-bone-blood integration in an unanticipated way distinct from other classical calcium-regulating hormones.

Regulation of hematopoiesis in endosteal microenvironments

After birth, the hematopoietic system develops along with bone formation in mammals. Osteolineage cells are derived from mesenchymal progenitor cells, and differentiate into several types of bone-forming cells. Of the various types of cell constituents in bone marrow, osteolineage cells have been shown to play important roles in hematopoiesis. Early studies have identified osteoblasts as a hematopoietic stem cell niche component. Since that time, the role of endosteal microenvironment as a critical regulator of hematopoietic stem/progenitor cell (HSC/HPC) behavior has been appreciated particularly under stress conditions, such as cytokine-induced HSC/HPC mobilization, homing/engraftment after bone marrow transplantation, and disease models of leukemia/myelodysplasia. Recent studies revealed that the most differentiated osteolineage cells, i.e., osteocytes, play important roles in the regulation of hematopoiesis. In this review, we provide an overview of recent advances in knowledge of regulatory hematopoietic mechanisms in the endosteal area.

Ikaros expression in human hematopoietic lineages

The Ikaros gene has been implicated in lymphoid development and proliferation from the results of gene targeting studies in mice. Recently we reported that the Ikaros gene may be involved in the disease progression of chronic myelogenous leukemia (CML). In this report, we investigated Ikaros isoforms in human non-lymphoid leukemia cell lines and normal granulocyte/macrophage (CFU-GM) and erythroid (BFU-E)-derived colonies. We evaluated Ikaros gene expression by RT-PCR, Southern blotting, sequencing analysis, Northern blotting, and immunoblotting.Ikaros isoforms Ik-1 and Ik-2, 3 were predominantly expressed in human non-lymphoid leukemia cell lines. Ik-4 and Ik-8 were also detectable as a minor population. In contrast to the previous report in mice, multiple Ikaros isoforms were expressed in human CFU-GM and BFU-E-derived colonies, and the dominant-negative isoform Ik-6 was not detectable. We also showed that human Ikaros isoforms contained an additional coding sequence in the N-terminal region, which was highly homologous to the sequence reported in mice. These observations suggest that the Ikaros gene may play some role in the development of human non-lymphoid lineage hematopoiesis. Moreover, the finding that the dominant-negative isoform Ik-6, which was overexpressed in patients with blast crisis of CML, was rarely detectable in non-lymphoid lineages supports its pathogenetic role in human hematologic malignancies.

A novel feedback mechanism by Ephrin‐B1/B2 in T‐cell activation involves a concentration‐dependent switch from costimulation to inhibition

Bidirectional signals via Eph receptors/ephrins have been recognized as major forms of contact‐dependent cell communications such as cell attraction and repulsion. T cells express EphBs, and their ligands, the ephrin‐Bs, have been known as costimulatory molecules for T‐cell proliferation. Recently, another remarkable feature of ephrin‐As has emerged in the form of a concentration‐dependent transition from promotion to inhibition in axon growth. Here we examined whether this modification plays a role in ephrin‐B costimulation in murine primary T cells. Low doses of ephrin‐B1 and ephrin‐B2 costimulated T‐cell proliferation induced by anti‐CD3, but high concentrations strongly inhibited it. In contrast, ephrin‐B3 showed a steadily increasing stimulatory effect. This modulation was virtually preserved in T cells from mice simultaneously lacking four genes, EphB1, EphB2, EphB3, and EphB6. High concentrations of ephrin‐B1/B2, but not ephrin‐B3, inhibited the anti‐CD3‐induced phosphorylation of Lck and its downstream signals such as Erk and Akt. Additionally, high doses of any ephrin‐Bs could phosphorylate EphB4. However, only ephrin‐B1/B2 but not ephrin‐B3 recruited SHP1, a phosphatase to suppress the phosphorylation of Lck. These data suggest that EphB4 signaling could engage in negative feedback to TCR signals. T‐cell activation may be finely adjusted by the combination and concentration of ephrin‐Bs expressed in the immunological microenvironment.

Mycophenolate mofetil: fully utilizing its benefits for GvHD prophylaxis

Mycophenolate mofetil (MMF) has been widely used for the prophylaxis of graft-versus-host disease (GvHD) in hematopoietic stem cell transplantation (HSCT), based on clinical evidence established in organ transplantations. MMF is not a cytotoxic, but rather a cytostatic agent, and there have been several reports of significant advantages in engraftment as well as greatly reduced stomatitis compared to methotrexate (MTX). MMF has been preferred for MTX-free immunosuppression, especially in reduced intensity conditioning, but it is suitable for GvHD prophylaxis for any type of HSCT. Some clinicians doubt its effectiveness, due to the lack of advantage over MTX in acute GvHD prophylaxis, especially in myeloablative conditioning. Pharmacokinetics studies of mycophenolic acid (MPA), the active form of MMF, show large inter- and intra-patient variation, which make interpretations of its clinical usefulness difficult. Nevertheless, several studies, including ours, have demonstrated that relatively higher area under the curve (AUC) of the MPA group leads to significant suppression of acute GvHD in prophylactic use. We propose a model algorithm for optimal dose finding using therapeutic drug monitoring (TDM) for MPA. Preemptive strategies depending on plasma MPA levels could yield more effective approaches to GvHD prophylaxis, alternative to MTX.

Pharmacokinetics-based optimal dose prediction of donor source-dependent response to mycophenolate mofetil in unrelated hematopoietic cell transplantation

Mycophenolate mofetil (MMF) has been widely used for prophylaxis against graft-versus-host disease (GVHD) following allogeneic hematopoietic stem cell transplantation (allo-SCT). However, no clear advantage over methotrexate has been reported, other than reduced incidence of mucositis. We speculated that the wide inter-individual variation of plasma mycophenolic acid (MPA) levels veiled the benefits of MMF. Data from 36 unrelated allogeneic bone marrow (allo-BMT) and cord blood transplantation (CBT) were analyzed retrospectively based on MPA area under the curve (AUC0–24h). In allo-BMT, high AUC0–24h (>30 μg h/ml) resulted in no incidence of grade II–IV acute/extensive chronic GVHD and tended to show higher overall and disease-free survival, lower relapse rates, and non-relapse mortality. In CBT, AUC0–24h less than 30 μg h/ml was sufficient for low incidence of acute/chronic GVHD and high survival. Strong correlation between AUC0–24h and C2h, plasma MPA concentration at 2 h after administration was observed. Single point assessment of C2h was shown to provide a useful surrogate of AUC0–24h to predict GVHD incidence. The results of this study suggest that individualized MMF dosing in a donor source-dependent fashion may be important for maximizing the benefit of MMF in allo-SCT.

Extended Mycophenolate Mofetil Administration Beyond Day 30 in Allogeneic Hematopoietic Stem Cell Transplantation as Preemptive Therapy for Severe Graft-Versus-Host Disease

To prevent acute graft-versus-host disease (GVHD), mycophenolate mofetil (MMF) combined with calcineurin inhibitors have been used in allogeneic hematopoietic stem cell transplantation (allo-SCT). Previous studies commonly utilize MMF treatment until day 30 after allo-SCT. However, the feasibility of continuous administration after day 30 has not been well evaluated. We retrospectively assessed the safety and efficacy of extended drug administration. Twenty-five patients ceased MMF at day 30 (group A); whereas, 16 patients (group B) received extended regimens depending on individual risk factors for GVHD. No severe adverse events were observed in either group. Although the cumulative incidence (CI) of grade I to IV GVHD at day 100 was comparable between the 2 groups, the CI of grade II to IV GVHD was less among group B (12.5%) compared with group A (42.3%). Extended MMF administration may be safe and beneficial as preemptive therapy to reduce the development of moderate-to-severe acute GVHD.

Inhibition of G1 to S Phase Progression by a Novel Zinc Finger Protein P58TFL at P-bodies

We recently reported the translocation of the immunoglobulin (Ig) light chain κ locus gene with a possible tumor suppressor gene, TFL, in transformed follicular lymphoma. However, the functional significance in cell transformation remains to be elucidated. Here, we first identified two gene products, P58TFL and P36TFL, derived by alternative splicing. The expression was prominent in normal human lymphocytes but defective in some leukemia/lymphoma cell lines. Overexpression of either protein in a mouse pro-B cell line, Ba/F3, and a human leukemia cell line, Jurkat, inhibited G1 to S phase progression through suppression of retinoblastoma protein (Rb) phosphorylation. The dominant gene product, P58TFL, colocalized with mRNA-processing body markers, eukaryotic translation initiation factor 2C and DCP1 decapping-enzyme homolog A, but not with a stress granule maker, T-cell intracellular antigen 1, in the cytoplasm. Taken together with the unique CCCH-type zinc finger motif, the present study suggests that P58TFL could play an important role in the regulation of cell growth through posttranscriptional modification of cell cycle regulators, at least partially, upstream of Rb. (Mol Cancer Res 2009;7(6):880–9)

Disproportion of helper T cell subsets in peripheral blood of patients with primary Sjögren's syndrome.

We studied the proportions of Thl and Th2 cells in peripheral blood of 15 patients with primary Sjögrens syndrome (p-SS), by using a procedure to enumerate the cells synthesizing cytokines such as INF-γ or 1L-4 in cytoplasm of CD4+ lymphocytes. The frequency of Thl (INF-y containing) cells in p-SS patients was significantly reduced as compared to normal control (20.57±7.48% vs 28.78 ± 11.56%, p<0.05), while that of Th2 (IL-4 containing) cells was not different from normal control (3.33 ±0.98% vs 2.85 ± 1.88%). The ratio of Thl to Th2 cells in p-SS patients was significantly decreased as compared to normal control (6.60 ± 3.15 vs 11.55 ft 6.72,p< 0.05). There was no difference in frequency of Thl or of Th2 cells between 8 patients given small amounts of prednisolone (PSL) and 7 patients not given PSL (21.44 ±9.39% vs 19.57 ±5.05%, 3.12 ±0.80% vs 3.56 ± 1.17%). The percentage of Thl cells was not different between 7 patients with glandular symptoms (G) and 8 patients with extraglandular symptoms (EG) (18.61 ±9.63% vs 22.27 ± 5.02%). Although the frequency of Th2 cells was higher in EG-patients than that in G-patients (3.84 ± 0.78% vs 2.74 ± 0.86%) with tendency of elevated IgG level in sera, the ratio of Thl to Th2 cells was not different among them (6.26 ± 2.84 vs 6.99 ± 3.64). These results suggest that the reduced ratio of Thl to Th2 cells is essential and is related to the dysfunction of cellular immunity in p-SS.

Posttranscriptional Modulation of Cytokine Production in T Cells for the Regulation of Excessive Inflammation by TFL

Posttranscriptional machinery regulates inflammation and is associated with autoimmunity as well as tumorigenesis in collaboration with transcription factors. We previously identified the tumor suppressor gene transformed follicular lymphoma (TFL) on 6q25 in a patient with follicular lymphoma, which transformed into diffuse large B cell lymphoma. TFL families have a common RNase domain that governs macrophage-mediated inflammation. In human peripheral blood, TFL is dominantly expressed at the glycine- and tryptophan-rich cytoplasmic processing bodies of T lymphocytes, and it is persistently upregulated in activated T cells. To address its physiological role, we established TFL−/− mice in which TFL−/− lymphocytes proliferated more rapidly than TFL+/+ upon stimulation with inappropriate cytokine secretion, including IL-2, IL-6, and IL-10. Moreover, TFL inhibited the synthesis of cytokines such as IL-2, IL-6, IL-10, TNF-α, and IL-17a by 3′ untranslated region RNA degradation. Experimental autoimmune encephalitis induced in TFL−/− mice demonstrated persistent severe paralysis. CNS-infiltrated CD4+ T cells in TFL−/− mice contained a higher proportion of Th17 cells than did those in TFL+/+ mice during the resolution phase, and IL-17a mRNA levels were markedly increased in TFL−/− cells. These results suggest that TFL may play an important role in attenuating local inflammation by suppressing the infiltration of Th17 cells in the CNS during the resolution phase of experimental autoimmune encephalitis. TFL is a novel gradual and persistent posttranscriptional regulator, and the TFL-driven attenuation of excessive inflammation could contribute to recovery from T cell–mediated autoimmune diseases.