The role of peroxiredoxin III on late stage of proerythrocyte differentiation

Peroxiredoxin III (Prdx III), the mitochondrial peroxidase, was preferentially expressed in murine erythroleukemia (MEL) cells. However, the mechanisms by which Prdx III regulates erythroid differentiation are unknown. In this study, K562 cells were differentiated by Ara-C treatment, and Prdx III was dramatically increased until day 5. We also investigated Prdx III expression pattern on in vitro erythropoiesis of human CD34(+) cells. When human CD34(+) cells became proerythrocyte on day 7, Prdx III was diminished, and then augmented on day 12. We established the stable sublines of Prdx III overexpression (O/E), and dominant-negative (D/N). The intracellular ROS level of Prdx III O/E cell line was lower than D/N stable cell lines. Moreover, Prdx III O/E cell line was placed in G1-arrest, but not D/N cell lines. Finally, the expression level of beta-globin and GATA-1 was dramatically increased in Prdx III O/E cell line.     

Impact of sex and age on bone marrow immune responses in a murine model of trauma-hemorrhage.

Although studies have demonstrated that trauma markedly alters the bone marrow immune responses, sex and age are crucial determinants under such conditions and have not been extensively examined. To study this, 21- to 27-day-old (premature), 6- to 8-wk-old (mature), and 20- to 24-mo-old (aged) male and female (proestrus) C3H/HeN mice were sham operated or subjected to trauma (i.e., midline laparotomy) and hemorrhagic shock (30 +/- 5 mmHg for 90 min) followed by fluid resuscitation. Twenty-four hours after resuscitation, bone marrow cells were harvested. Trauma-hemorrhage induced an increased number of the early pluripotent stem cell-associated bone marrow cell subsets (Sca1(+)CD34(-)CD117(+/-)lin(+/-)) in young mice. The CD117(+) proportion of these cell subsets increased in mature proestrus females, but not in males. Aged males displayed significant lower numbers of Sca1(+)CD34(-)CD117(+/-)lin(+/-) cells compared with young male mice. Trauma-hemorrhage also increased development of granulocyte/macrophage progenitor cells (CD11b(+)Gr-1(+)). Proliferative responses to granulocyte macrophage colony-stimulating factor were maintained in mature and aged proestrus females, but decreased in young mice and mature males. Augmented differentiation into monocyte/macrophage lineage in mature and aged proestrus females was observed and associated with the maintained release of TNF-alpha and IL-6. Conversely, increased IL-10 and PGE(2) production was observed in the male trauma-hemorrhage groups. Thus, sex- and age-specific effects in bone marrow differentiation and immune responses after trauma-hemorrhage occur, which are likely to contribute to the sex- and age-related differences in the systemic immune responses under such conditions.     

Differential role for cyclic AMP response element binding protein-1 in multiple stages of B cell development, differentiation, and survival

CREB-1 is expressed in the bone marrow and in developing B cells. To determine the role of CREB-1 in developing B cells in the bone marrow, several lines of transgenic (Tg) mice overexpressing a dominant-negative Ser(119-ala) phosphomutant CREB-1 in the bone marrow were generated. Analysis of RNA and protein revealed expression of the transgene in the bone marrow. Flow cytometric analysis of bone marrow cells from Tg mice revealed approximately 70% increase in pre-B1 (CD43(+)B220(+)CD24(+(int))) and approximately 60% decreased pre-BII (CD43(+)B220(+)CD24(++(high))) cells, indicating a developmental block in pre-BI to pre-BII transition. Consistent with this, the Tg mice showed approximately 4-fold decrease in immature and mature B cells in the bone marrow. RT-PCR analysis of RNA from Tg mice revealed increased JunB and c-Jun in pre-BII cells associated with decreased S-phase entry. Adoptive transfer of bone marrow cells into RAG-2(-/-) mice resulted in reconstitution of non-Tg but not Tg bone marrow-derived CD43(+)B220(+)CD24(high) population that is normally absent in RAG-2(-/-) mice. In the periphery, the Tg mice exhibited decreased CD21(dim)CD23(high)IgM(+) follicular B cells in the spleen and increased B1a and B1b B cells in the peritoneum. While exhibiting normal Ab responses to T-independent Ags and primary response to the T-dependent Ag DNP-keyhole limpet hemocyanin, the Tg mice exhibited severely impaired secondary Ab responses. These studies provide the first evidence for a differential role for CRE-binding proteins in multiple stages of B cell development, functional maturation, and B1 and B2 B cells.     

Resistance to metastatic disease in STAT6-deficient mice requires hemopoietic and nonhemopoietic cells and is IFN-gamma dependent

Mice deficient for the STAT6 gene (STAT6(-/-) mice) have enhanced immunosurveillance against primary and metastatic tumors. Because STAT6 is a downstream effector of the IL-4R, and IL-13 binds to the type 2 IL-4R, IL-13 has been proposed as an inhibitor that blocks differentiation of tumor-specific CD8(+) T cells. Immunity in STAT6(-/-) mice is unusually effective in that 45-80% of STAT6(-/-) mice with established, spontaneous metastatic 4T1 mammary carcinoma, whose primary tumors are surgically excised, survive indefinitely, as compared with <10% of STAT(+/+) (BALB/c) mice. Surprisingly, STAT6(-/-) and BALB/c reciprocal bone marrow chimeras do not have increased immunosurveillance, demonstrating that immunity requires STAT6(-/-) hemopoietic and nonhemopoietic components. Likewise, CD1(-/-) mice that are NKT deficient and therefore IL-13 deficient also have heightened tumor immunity. However, STAT6(-/-) and CD1(-/-) reciprocal bone marrow chimeras do not have increased survival, suggesting that immunity in STAT6(-/-) and CD1(-/-) mice is via noncomplementing mechanisms. Metastatic disease is not reduced in BALB/c mice treated with an IL-13 inhibitor, indicating that IL-13 alone is insufficient for negative regulation of 4T1 immunity. Likewise, in vivo depletion of CD4(+)CD25(+) T cells in BALB/c mice does not increase survival, demonstrating that CD4(+)CD25(+) cells do not regulate immunity. Cytokine production and tumor challenges into STAT6(-/-)IFN-gamma(-/-) mice indicate that IFN-gamma is essential for immunity. Therefore, immunosurveillance in STAT6(-/-) mice facilitates survival against metastatic cancer via an IFN-gamma-dependent mechanism involving hemopoietic and nonhemopoietic derived cells, and is not exclusively dependent on counteracting IL-13 or CD4(+)CD25(+) T cells.     

Characterization of distinct conventional and plasmacytoid dendritic cell-committed precursors in murine bone marrow

The developmental pathways and differentiation relationship of dendritic cell (DC) subsets remain unclear. We report that murine CD11c(+)MHC II(-) bone marrow cells, which are immediate DC precursors of CD8 alpha(+), CD8 alpha(-), and B220(+) DC in vivo, can be separated into B220(+) and B220(-) DC precursor subpopulations. Purified B220(-) DC precursors expand, and generate exclusively mature CD11c(+)CD11b(+)B220(-) DC in vitro and after adoptive transfer. B220(+) DC precursors, which resemble plasmacytoid pre-DC, have a lower proliferative potential than B220(-) DC precursors and generate both CD11b(-) B220(+) and CD11b(+)B220(-) DC populations. Both DC precursor populations can give rise to CD8 alpha(+) and CD8 alpha(-) DC subtypes. Our findings indicate that CD11c(+)MHC II(-)B220(+) and CD11c(+)MHC II(-)B220(-) bone marrow cells are distinct DC lineage-restricted precursors.     

Accumulation of CD4+ T cells in the colon of CsA-treated mice following myeloablative conditioning and bone marrow transplantation

Syngeneic graft vs. host disease (SGVHD) was first described as a graft vs. host disease-like syndrome that developed in rats following syngeneic bone marrow transplantation (BMT) and cyclosporin A (CsA) treatment. SGVHD can be induced by reconstitution of lethally irradiated mice with syngeneic bone marrow cells followed by 21 days of treatment with the immunosuppressive agent CsA. Clinical symptoms of the disease appear 2-3 wk following cessation of CsA therapy, and disease-associated inflammation occurs primarily in the colon and liver. CD4(+) T cells have been shown to play an important role in the inflammatory response observed in the gut of SGVHD mice. Time-course studies revealed a significant increase in migration of CD4(+) T cells into the colon during CsA therapy, as well as significantly elevated mRNA levels of TNF-α, proinflammatory chemokines, and cell adhesion molecules in colonic tissue of CsA-treated animals compared with BMT controls, as early as day 14 post-BMT. Homing studies revealed a greater migration of labeled CD4(+) T cells into the gut of CsA-treated mice at day 21 post-BMT than control animals via CsA-induced upregulation of mucosal addressin cell adhesion molecule. This study demonstrates that, during the 21 days of immunosuppressive therapy, functional mechanisms are in place that result in increased homing of CD4(+) T effector cells to colons of CsA-treated mice.     

Identification of CD133-positive radioresistant cells in atypical teratoid/rhabdoid tumor

Atypical teratoid/rhabdoid tumor (AT/RT) is an extremely malignant neoplasm in the central nervous system (CNS) which occurs in infancy and childhood. Recent studies suggested that CD133 could be considered a marker for brain cancer stem-like cells (CSCs). However, the role of CD133 in AT/RT has never been investigated. Herein we report the isolation of CD133-positive cells (CD133(+)), found to have the potential to differentiate into three germ layer tissues, from tissues of nine AT/RT patients. The migration/invasion/malignancy and radioresistant capabilities of CD133(+) were significantly augmented when compared to CD133(-). The clinical data showed that the amount of CD133(+) in AT/RTs correlated positively with the degree of resistance to radiation therapy. Using cDNA microarray analysis, the genotoxic-response profiles of CD133(+) and CD133(-) irradiated with 10 Gy ionizing radiation (IR) were analyzed 0.5, 2, 6, 12 and 24 h post-IR. We then validated these microarray data and showed increased phosphorylation after IR of p-ATM, p-RAD17, and p-CHX2 as well as increased expression of BCL-2 protein in CD133(+) compared to CD133(-). Furthermore, we found that CD133(+) can effectively resist IR with cisplatin- and/or TRAIL-induced apoptosis. Immunohistochemical analysis confirmed the up-regulated expression of p-ATM and BCL-2 proteins in IR-treated CD133(+) xenotransgrafts in SCID mice but not in IR-treated CD133(-). Importantly, the effect of IR in CD133(+) transplanted mice can be significantly improved by a combination of BCL-2 siRNA with debromohymenialdisine, an inhibitor of checkpoint kinases. In sum, this is the first report indicating that CD133(+) AT/RT cells demonstrate the characteristics of CSCs. The IR-resistant and anti-apoptotic properties in CD133(+) may reflect the clinical refractory malignancy of AT/RTs and thus the activated p-ATM pathway and BCL-2 expression in CD133(+) could be possible targets to improve future treatment of deadly diseases like AT/RT.     

IL-7 enhances the responsiveness of human T cells that develop in the bone marrow of athymic mice

The beige/nude/xid/human (bnx/hu) model of human hematopoiesis provides a unique opportunity to study extrathymic human T lymphocyte development in an in vivo system. Purified human hematopoietic stem cells develop into mature T lymphocytes and immature progenitors in the bone marrow of athymic bnx mice. The human T cells are all TCR alpha beta(+) and display a restricted TCRV beta repertoire. In the current studies, we examined the effects of systemic human IL-7 (huIL-7) administration on the phenotype and the activation status of the bnx/hu T cells. In the majority of the mice that did not have huIL-7 administration, a higher frequency of human CD3(+)/CD8(+) than CD3(+)/CD4(+) T cells developed in the bone marrow. This phenomenon is also frequently observed in human bone marrow transplant recipients. Extremely low levels of IL-2 were expressed by human CD3(+) cells isolated from these mice, in response to PMA plus ionomycin and to CD3 and CD28 cross-linking. IL-4 was not expressed by cells exposed to either stimulus, demonstrating a profound inability of the bnx/hu T cells to produce this cytokine. Systemic production of huIL-7 from engineered stromal cells transplanted into the mice increased the human CD4 to CD8 ratios, and increased the ratio of memory to naive CD4(+) and CD8(+) T cells. The human CD3(+) cells recovered from mice that had systemic huIL-7 and equivalent numbers of CD3(+)/CD4(+) and CD3(+)/CD8(+) cells in the marrow were still unable to produce IL-4 in response to any condition tested, but were capable of normal levels of IL-2 production following stimulation.     

CD8(+), alphabeta-TCR(+), and gammadelta-TCR(+) cells in the recipient hematopoietic environment mediate resistance to engraftment of allogeneic donor bone marrow

Historically, conditioning for engraftment of hematopoietic stem cells has been nonspecific. In the present study, we characterized which cells in the recipient hematopoietic microenvironment prevent allogeneic marrow engraftment. Mice defective in production of alphabeta-TCR(+), gammadelta-TCR(+), alphabeta- plus gammadelta-TCR(+), CD8(+), or CD4(+) cells were transplanted with MHC-disparate allogeneic bone marrow. Conditioning with 500 cGy total body irradiation (TBI) plus a single dose of cyclophosphamide (CyP) on day +2 establishes chimerism in normal recipients. When mice were conditioned with 300 cGy TBI plus a single dose of CyP on day +2, all engrafted, except wild-type controls and those defective in production of CD4(+) T cells. Mice lacking both alphabeta- and gammadelta-TCR(+) cells engrafted without conditioning, suggesting that both alphabeta- and gammadelta-TCR T cells in the host play critical and nonredundant roles in preventing engraftment of allogeneic bone marrow. CD8 knockout (KO) mice engrafted without TBI, but only if they received CyP on day +2 relative to the marrow infusion, showing that a CD8(-) cell was targeted by the CyP conditioning. The CD8(+) cell effector function is mechanistically different from that for conventional T cells, and independent of CD4(+) T helper cells because CD4 KO mice require substantially higher levels of conditioning than the other KO phenotypes. These results suggest that a number of cell populations with different mechanisms of action mediate resistance to engraftment of allogeneic marrow. Targeting of specific recipient cellular populations may permit conditioning approaches to allow mixed chimerism with minimal morbidity and could potentially avoid the requirement for myelotoxic agents altogether.     

Age-related changes in human hematopoietic stem/progenitor cells

Adult stem cells are critical for maintaining cellular homeostasis throughout life, yet the effects of age on their regenerative capacity are poorly understood. All lymphoid and myeloid blood cell lineages are continuously generated from hematopoietic stem cells present in human bone marrow. With age, significant changes in the function and composition of mature blood cells are observed. In this study, we report that age-related changes also occur in the human hematopoietic stem cell compartment. We find that the proportion of multipotent CD34(+) CD38(-) cells increases in the bone marrow of elderly (>70 years) individuals. CD34(+) CD38(+) CD90(-) CD45RA(+/-) CD10(-) and CD34(+) CD33(+) myeloid progenitors persist at the same level in the bone marrow, while the frequency of early CD34(+) CD38(+) CD90(-) CD45RA(+) CD10(+) and committed CD34(+) CD19(+) B-lymphoid progenitors decreases with age. In contrast to mice models of aging, transplantation experiments with immunodeficient NOD/SCID/IL-2Rγ null (NSG) mice showed that the frequency of NSG repopulating cells does not change significantly with age, and there is a decrease in myeloid lineage reconstitution. An age-related decrease in the capacity of CD34(+) cells to generate myeloid cells was also seen in colony-forming assays in vitro. Thus, with increasing age, human hematopoietic stem/progenitor cells undergo quantitative changes as well as functional modifications.     

Molecular cloning of murine STAP-1, the stem-cell-specific adaptor protein containing PH and SH2 domains

To identify the novel substrate of c-kit which is important for hematopoietic stem cell self-renewal or differentiation, CD34-low/negative, Sca-1-positive, c-kit-positive, and lineage marker-negative (CD34(low/-)Sca-1(+)c-kit(+)Lin(-)) cells were sorted by a fluorescence-activated cell sorter from mouse bone marrow cells and a yeast two-hybrid cDNA library was constructed. By screening with c-kit as bait, we cloned a novel cDNA, designed STAP-1, encoding an adaptor protein with a Pleckstrin homology domain, the Src homology 2 (SH2) domain, and a number of tyrosine phosphorylation sites. RT-PCR analysis revealed that STAP-1 expression is restricted in the bone marrow cell fraction expressing c-kit. The highest expression was observed in the CD34(low/-)Sca-1(+)c-kit(+)Lin(-) stem cell-enriched fraction. The murine myeloid cell line, M1, expressed a high level of STAP-1. However, the expression was strongly repressed in response to leukemia inhibitory factor (LIF) which induced monocytic differentiation of M1 cells, suggesting that STAP-1 is associated with the undifferentiated cell type. A two-hybrid assay indicated that STAP-1 bound not only to c-kit but also to c-fms but not to JAK2 or Pyk2. In 293 cells, STAP-1 was tyrosine-phosphorylated by activated c-kit. An in vitro binding assay suggested that the STAP-1 SH2 domain interacted with several tyrosine-phosphorylated proteins including c-kit and STAT5. These suggest that STAP-1 functions as an adaptor molecule downstream of c-kit in hematopoietic stem cells.     

Hematopoietic miR155 deficiency enhances atherosclerosis and decreases plaque stability in hyperlipidemic mice

microRNA-155 (miR155) is a central regulator of immune responses that is induced by inflammatory mediators. Although miR155 is considered to be a pro-inflammatory microRNA, in vitro reports show anti-inflammatory effects in lipid-loaded cells. In this study we examined the role of miR155 in atherosclerosis in vivo using bone marrow transplantation from miR155 deficient or wildtype mice to hyperlipidemic mice. Hematopoietic deficiency of miR155 enhanced atherosclerotic plaque development and decreased plaque stability, as evidenced by increased myeloid inflammatory cell recruitment to the plaque. The increased inflammatory state was mirrored by a decrease in circulating CD4(+)CD25(+)FoxP3(+) regulatory T cells, and an increase in granulocytes (CD11b(+)Ly6G(+)) in blood of miR155(-/-) transplanted mice. Moreover, we show for the first time a crucial role of miR155 in monocyte subset differentiation, since hematopoietic deficiency of miR155 increases the 'inflammatory' monocyte subset (CD11b(+)Ly6G(-)Ly6C(hi)) and reduces 'resident' monocytes (CD11b(+)Ly6G(-)Ly6C(low)) in the circulation. Furthermore, cytokine production by resident peritoneal macrophages of miR155(-/-) transplanted hyperlipidemic mice was skewed towards a more pro-inflammatory state since anti-inflammatory IL-10 production was reduced. In conclusion, in this hyperlipidemic mouse model miR155 acts as an anti-inflammatory, atheroprotective microRNA. Additionally, besides a known role in lymphoid cell development, we show a crucial role of miR155 in myeloid lineage differentiation.     

CD40 engagement enhances antigen-presenting langerhans cell priming of IFN-gamma-producing CD4+ and CD8+ T cells independently of IL-12

The delivery of CD40 signaling to APCs during T cell priming enhances many T cell-mediated immune responses. Although CD40 signaling up-regulates APC production of IL-12, the impact of this increased production on T cell priming is unclear. In this study an IL-12-independent T cell-mediated immune response, contact hypersensitivity (CHS), was used to further investigate the effect of CD40 ligation on the phenotypic development of Ag-specific CD4(+) and CD8(+) T cells. Normally, sensitization for CHS responses induces hapten-specific CD4(+) T cells producing type 2 cytokines and CD8(+) T cells producing IFN-gamma. Treatment of mice with agonist anti-CD40 mAb during sensitization with the hapten 2,4-dinitrofluorobenzene resulted in CHS responses of increased magnitude and duration. These augmented responses in anti-CD40 Ab-treated mice correlated with increased numbers of hapten-specific CD4(+) and CD8(+) T cells producing IFN-gamma in the skin draining lymph nodes. Identical results were observed using IL-12(-/-) mice, indicating that CD40 ligation promotes CHS responses and development of IFN-gamma-producing CD4(+) and CD8(+) T cells in the absence of IL-12. Engagement of CD40 on hapten-presenting Langerhans cells (hpLC) up-regulated the expression of both class I and class II MHC and promoted hpLC migration into the T cell priming site. These results indicate that hpLC stimulated by CD40 ligation use a mechanism distinct from increased IL-12 production to promote Ag-specific T cell development to IFN-gamma-producing cells.     

Flt3 ligand expands lymphoid progenitors prior to recovery of thymopoiesis and accelerates T cell reconstitution after bone marrow transplantation

Deficient thymopoiesis and retarded recovery of newly developed CD4(+) T cells is one of the most important determinants of impaired immunocompetence after hemopoietic stem cell transplantation. Here we evaluated whether Fms-like tyrosine kinase 3 (Flt3) ligand (FL) alone or combined with IL-7 affects T cell recovery, thymopoiesis, and lymphoid progenitor expansion following bone marrow transplantation in immunodeficient mice. FL strongly accelerated and enhanced the recovery of peripheral T cells after transplantation of a low number of bone marrow cells. An additive effect on T cell recovery was not observed after coadministration of IL-7. Lineage(-)sca-1(+)c-kit(+)flt3(+) lymphoid progenitor cell numbers were significantly increased in bone marrow of FL-treated mice before recovery of thymopoiesis. Thymocyte differentiation was advanced to more mature stages after FL treatment. Improved T cell recovery resulted in better immunocompetence against a post-bone marrow transplantation murine CMV infection. Collectively, our data suggest that FL promotes T cell recovery by enhanced thymopoiesis and by expansion of lymphoid progenitors.     

Estrogen preferentially promotes the differentiation of CD11c+ CD11b(intermediate) dendritic cells from bone marrow precursors

Sex biases in autoimmunity and infection suggest that steroid sex hormones directly modulate immune cells. We show in this study that 17-beta-estradiol (E2) promotes the differentiation of functional dendritic cells (DC) from murine bone marrow precursor cells. Remarkably, ex vivo DC differentiation was inhibited in steroid hormone-deficient medium, and was restored by addition of physiological amounts of E2, but not dihydrotestosterone. DC differentiation was inhibited by the estrogen receptor (ER) antagonists ICI 182,780 and tamoxifen, and from ERalpha(-/-) bone marrow cells, indicating that E2 acted via ERs. E2 addition was most effective in promoting DC differentiation immediately ex vivo, but did not increase DC proliferation. E2 treatment specifically promoted differentiation of a CD11c(+) CD11b(int) DC population that displayed high levels of cell surface MHC class II and CD86, suggesting that E2 could augment numbers of potent APC. DC that differentiated in E2-supplemented medium were fully functional in their capability to mediate presentation of self and foreign Ags and stimulate the proliferation of naive CD4(+) T cells. The requirement for estrogen during DC differentiation suggests a mechanism by which E2 levels in peripheral tissues might modulate both the number and functional capabilities of DC in vivo, thereby influencing immune responses.     

Peroxiredoxin 6 as an antioxidant enzyme: protection of lung alveolar epithelial type II cells from H2O2-induced oxidative stress

We evaluated the antioxidant role of peroxiredoxin 6 (Prdx6) in primary lung alveolar epithelial type II cells (AEC II) that were isolated from wild type (WT), Prdx6-/-, or Prdx6 transgenic (Tg) overexpressing mice and exposed to H(2)O(2) at 50-500 microM for 1-24 h. Expression of Prdx6 in Tg AEC II was sevenfold greater than WT. Prdx6 null AEC II exposed to H(2)O(2) showed concentration-dependent cytotoxicity indicated by decreased "live/dead" cell ratio, increased propidium iodide (PI) staining, increased annexin V binding, increased DNA fragmentation by TUNEL assay, and increased lipid peroxidation by diphenylpyrenylphosphine (DPPP) fluorescence. Compared to Prdx6 null cells, oxidant-mediated damage was significantly less in WT AEC II and was least in Prdx6 Tg cells. Thus, Prdx6 functions as an antioxidant enzyme in mouse AEC II. Prdx6 has been shown previously to reduce phospholipid hydroperoxides and we postulate that this activity is a major mechanism for the effectiveness of Prdx6 as an antioxidant enzyme.     

CXCR2-Dependent Endothelial Progenitor Cell Mobilization in Pancreatic Cancer Growth

Neovascularization is essential for tumor growth. We have previously reported that the chemokine receptor CXCR2 is an important regulator in tumor angiogenesis. Here we report that the mobilization of bone marrow (BM)-derived endothelial progenitor cells (EPCs) is impaired in CXCR2 knockout mice harboring pancreatic cancers. The circulating levels of EPCs (positive for CD34, CD117, CD133, or CD146) are decreased in the bone marrow and/or blood of tumor-bearing CXCR2 knockout mice. CXCR2 gene knockout reduced BM-derived EPC proliferation, differentiation, and vasculogenesis in vitro. EPCs double positive for CD34 and CD133 increased tumor angiogenesis and pancreatic cancer growth in vivo. In addition, CD133(+) and CD146(+) EPCs in human pancreatic cancer are increased compared with normal pancreas tissue. These findings indicate a role of BM-derived EPC in pancreatic cancer growth and provide a cellular mechanism for CXCR2 mediated tumor neovascularization.     

Lineage(-)Sca1+c-Kit(-)CD25+ cells are IL-33-responsive type 2 innate cells in the mouse bone marrow

IL-33 promotes type 2 immune responses, both protective and pathogenic. Recently, targets of IL-33, including several newly discovered type 2 innate cells, have been characterized in the periphery. In this study, we report that bone marrow cells from wild-type C57BL/6 mice responded with IL-5 and IL-13 production when cultured with IL-33. IL-33 cultures of bone marrow cells from Rag1 KO and Kit(W-sh/W-sh) mice also responded similarly; hence, eliminating the possible contributions of T, B, and mast cells. Rather, intracellular staining revealed that the IL-5- and IL-13-positive cells display a marker profile consistent with the Lineage(-)Sca-1(+)c-Kit(-)CD25(+) (LSK(-)CD25(+)) cells, a bone marrow cell population of previously unknown function. Freshly isolated LSK(-)CD25(+) cells uniformly express ST2, the IL-33 receptor. In addition, culture of sorted LSK(-)CD25(+) cells showed that they indeed produce IL-5 and IL-13 when cultured with IL-33 plus IL-2 and IL-33 plus IL-7. Furthermore, i.p. injections of IL-33 or IL-25 into mice induced LSK(-)CD25(+) cells to expand, in both size and frequency, and to upregulate ST2 and α(4)β(7) integrin, a mucosal homing marker. Thus, we identify the enigmatic bone marrow LSK(-)CD25(+) cells as IL-33 responsive, both in vitro and in vivo, with attributes similar to other type 2 innate cells described in peripheral tissues.     

Ikaros isoform x is selectively expressed in myeloid differentiation

The Ikaros gene is alternately spliced to generate multiple DNA-binding and nonbinding isoforms that have been implicated as regulators of hematopoiesis, particularly in the lymphoid lineages. Although early reports of Ikaros mutant mice focused on lymphoid defects, these mice also show significant myeloid, erythroid, and stem cell defects. However, the specific Ikaros proteins expressed in these cells have not been determined. We recently described Ikaros-x (Ikx), a new Ikaros isoform that is the predominant Ikaros protein in normal human hematopoietic cells. In this study, we report that the Ikx protein is selectively expressed in human myeloid lineage cells, while Ik1 predominates in the lymphoid and erythroid lineages. Both Ik1 and Ikx proteins are expressed in early human hematopoietic cells (Lin(-)CD34(+)). Under culture conditions that promote specific lineage differentiation, Ikx is up-regulated during myeloid differentiation but down-regulated during lymphoid differentiation from human Lin(-)CD34(+) cells. We show that Ikx and other novel Ikaros splice variants identified in human studies are also expressed in murine bone marrow. In mice, as in humans, the Ikx protein is selectively expressed in the myeloid lineage. Our studies suggest that Ikaros proteins function in myeloid, as well as lymphoid, differentiation and that specific Ikaros isoforms may play a role in regulating lineage commitment decisions in mice and humans.