Hiroki Kawano

Matrix-embedded osteocytes regulate mobilization of hematopoietic stem/progenitor cells.

The bone marrow (BM) niche comprises multiple cell types that regulate hematopoietic stem/progenitor cell (HSPC) migration out of the niche and into the circulation. Here, we demonstrate that osteocytes, the major cellular component of mature bone, are regulators of HSPC egress. Granulocyte colony-stimulating factor (G-CSF), used clinically to mobilize HSPCs, induces changes in the morphology and gene expression of the osteocytic network that precedes changes in osteoblasts. This rapid response is likely under control of the sympathetic nervous system, since osteocytes express the β2-adrenergic receptor and surgical sympathectomy prevents it. Mice with targeted ablation of osteocytes or a disrupted osteocyte network have comparable numbers of HSPCs in the BM but fail to mobilize HSPCs in response to G-CSF. Taken together, these results indicate that the BM/bone niche interface is critically controlled from inside of the bone matrix and establish an important physiological role for skeletal tissues in hematopoietic function.

Osteocytes Regulate Primary Lymphoid Organs and Fat Metabolism

Osteocytes act as mechanosensors to control local bone volume. However, their roles in the homeostasis of remote organs are largely unknown. We show that ablation of osteocytes in mice (osteocyte-less [OL] mice) leads to severe lymphopenia, due to lack of lymphoid-supporting stroma in both the bone marrow and thymus, and complete loss of white adipose tissues. These effects were reversed when osteocytes were replenished within the bone. In contrast, neither in vivo supply of T cell progenitors and humoral factors via shared circulation with a normal parabiotic partner nor ablation of specific hypothalamic nuclei rescued thymic atrophy and fat loss in OL mice. Furthermore, ablation of the hypothalamus in OL mice led to hepatic steatosis, which was rescued by parabiosis with normal mice. Our results define a role for osteocytes as critical regulators of lymphopoiesis and fat metabolism and suggest that bone acts as a central regulator of multiple organs.

A biphenotypic transformation of 8p11 myeloproliferative syndrome with CEP1/FGFR1 fusion gene

Abstract:  We describe here the first case of 8p11 myeloproliferative syndrome (EMS) with t(8;9)(p11;q33), who unusually demonstrated B‐lymphoblastic/monoblastic biphenotypic transformation. A 57‐year‐old woman was admitted because of leukocytosis and diagnosed as EMS. Bone marrow was infiltrated with myeloperoxidase (MPO)‐, CD10+, CD19+, CD20+, CD34+, HLA‐DR+ small lymphoblasts and MPO+, CD2+, CD4+, CD13+, CD14+, CD33+, HLA‐DR+ large monoblasts. The karyotype was 46,XX,t(8;9)(p11;q33)[20] and the CEP1/FGFR1 fusion transcript between CEP1 exon 38 and FGFR1 exon 9 was detected. This case clearly indicates that the blastic transformation in EMS with t(8;9) could arise in the stem cells, which differentiate into not only myelomonocytic but also B‐lymphocytic lineages.

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.

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.

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.

Delayed neutrophil engraftment in cord blood transplantation with intensive administration of mycophenolate mofetil for GVHD prophylaxis

Delayed neutrophil engraftment in cord blood transplantation with intensive administration of mycophenolate mofetil for GVHD prophylaxis

G-CSF-induced sympathetic tone provokes fever and primes antimobilizing functions of neutrophils via PGE2

Granulocyte colony-stimulating factor (G-CSF) is widely used for peripheral blood stem/progenitor mobilization. G-CSF causes low-grade fever that is ameliorated by nonsteroidal anti-inflammatory drugs (NSAIDs), suggesting the activation of arachidonic acid (AA) cascade. How G-CSF regulated this reaction was assessed. G-CSF treatment in mice resulted in fever, which was canceled in prostaglandin E synthase (mPGES-1)-deficient mice. Mobilization efficiency was twice as high in chimeric mice lacking mPGES-1, specifically in hematopoietic cells, suggesting that prostaglandin E2 (PGE2) from hematopoietic cells modulated the bone marrow (BM) microenvironment. Neutrophils from steady-state BM constitutively expressed mPGES-1 and significantly enhanced PGE2 production in vitro by β-adrenergic stimulation, but not by G-CSF, which was inhibited by an NSAID. Although neutrophils expressed all β-adrenergic receptors, only β3-agonist induced this phenomenon. Liquid chromatography-tandem mass spectrometry traced β-agonist-induced PGE2 synthesis from exogenous deuterium-labeled AA. Spontaneous PGE2 production was highly efficient in Gr-1high neutrophils among BM cells from G-CSF-treated mice. In addition to these in vitro data, the in vivo depletion of Gr-1high neutrophils disrupted G-CSF-induced fever. Furthermore, sympathetic denervation eliminated both neutrophil priming for PGE2 production and fever during G-CSF treatment. Thus, sympathetic tone-primed BM neutrophils were identified as one of the major PGE2 producers. PGE2 upregulated osteopontin, specifically in preosteoblasts, to retain progenitors in the BM via EP4 receptor. Thus, the sympathetic nervous system regulated neutrophils as an indispensable PGE2 source to modulate BM microenvironment and body temperature. This study provided a novel mechanistic insight into the communication of the nervous system, BM niche components, and hematopoietic cells.

HIV-negative, HHV-8-unrelated primary effusion lymphoma-like lymphoma with genotypic infidelity and c-MYC expression

Dear Editor, HIVor HHV-8-related primary effusion lymphoma (PEL) is a rare and poor prognostic disease which presents as serous effusion without detectable tumor masses. PEL occasionally has both immunoglobulin (Ig) and T cell receptor (TCR) gene rearrangements, so-called genotypic infidelity [1, 2]. Recently, HIV-negative HHV-8-unrelated primary effusion lymphomalike lymphoma (PEL-LL) has been reported, and some cases are associated with c-MYC amplification. Although HHV-8 virus antigen can increase c-MYC stability in PEL [3], c-MYC expression in PEL-LL has not been confirmed so far. A 76-year-old male had suffered from esophageal cancer, which responded to chemoradiotherapy. Three years after treatment, however, he had experienced cough, general fatigue, and night sweats. Physical examination revealed no breath sounds on the right hemithorax. Chest X-ray showed severe pleural effusion in the right lung (Fig. 1a). However, no tumor mass or lymphadenopathy was detected. Gastrointestinal fiberscope showed no sign of esophageal cancer recurrence. Complete blood count and serum biochemistry studies were as follows: WBC 7,170/μl, hemoglobin 10.6 g/dL, platelet 402,000/μl, AST 20 IU/L, ALT 12 IU/L, LDH 377 IU/L, total bilirubin 0.4 mg/dL, and sIL-2R 525 U/ml. Serological tests for HIV, HTLV-1, and HCV were negative. Bone marrow was intact. In analysis of pleural effusion, there were many large atypical lymphocytes which were positive for CD19, CD20, CD30, CD79a, and Ig lambda light chain but negative for CD10 and CD56 (Fig. 1b). HHV-8 and EBVencoded RNA by immunochemistry were negative. There was a small fraction of normal T cells. Eighty percent of lymphocytes were Ki-67 positive. Southern blot analysis of tumor cells demonstrated Ig heavy chain (IgH) as well as TCR rearrangement (Fig. 1c). Karyotype analysis showed complex karyotype, including add(8)(q24) and c-MYC amplification, and these cells strongly expressed c-MYC protein by immunostaining (Fig.1d). He was diagnosed with PEL-LL and was treated with six courses of R-CHOP. He remains alive and well with slight pleural effusion without additional therapy for more than 18 months. There are only two cases of PEL-LL with both IgH and TCR rearrangements. The first case is a pleural cavity-based T cell-rich B cell lymphoma [4]. Actually, it was reported for this lymphoma to show the dual gene rearrangement [5]. It could be bi-clonal lymphoma, of which the T cell clone was derived from abundant background Tcells. The second case is post-liver transplant effusion lymphoma [6]. It demonstrated incomplete TCR-β rearrangements by PCR; wherein, these rearrangements can be found in some B cell lymphomas [7]. In our case, most cells in the effusion were B cells. In addition, we confirmed both rearrangements by southern blot analysis (Fig. 1c). Therefore, we believe that this is the first definitive case of genotypic infidelity in PEL-LL. In addition, we confirmed strong c-MYC expression on tumor cells along with T. Kashiwagi :K. Minagawa :H. Kawano : T. Hirata : S. Kashiwagi :Y. Nakagawa : S. Kusaka : T. Suzuki : T. Inagaki : M.Kishi :N.Miwa : S. Kimura :M. Takechi :M. Koide :M. Iwai : T. Matsui Department of Internal Medicine, Nishiwaki Municipal Hospital, Nishiwaki, Hyogo 677-0043, Japan