Hyper–inflammatory responses can lead to a variety of diseases including sepsis1. We now report that extracellular histones released in response to inflammatory challenge contribute to endothelial dysfunction, organ failure and death during sepsis. They can be targeted pharmacologically by antibody to histone or by activated protein C (APC). Antibody to histone reduced the mortality of mice in lipopolysaccharide (LPS), tumor necrosis factor (TNF) or cecal ligation and puncture models of sepsis. Extracellular histones are cytotoxic toward endothelium in vitro and are lethal in mice. In vivo, histone administration resulted in neutrophil margination, vacuolated endothelium, intra–alveolar hemorrhage and macro and microvascular thrombosis. Histone was detected in the circulation of baboons challenged with E. coli and the increase in histone levels accompanied the onset of renal dysfunction. APC cleaves histones and reduces their cytotoxicity. Co–infusion of APC with E. coli in baboons or histones in mice prevented lethality. Blockade of protein C activation exacerbated sublethal LPS challenge into lethality which was reversed by antibody to histone. We conclude that extracellular histones are potential molecular targets for therapeutics for sepsis and other inflammatory diseases.

Plasmacytoid dendritic cells (pDCs) competent to make type I interferon were rigorously defined as a Ly-6C ؉ and CD11c Lo subset of the B220 ؉ CD19 ؊ CD43 ؉ CD24 Lo bone marrow (BM) Fraction A. Otherwise similar Ly6C ؊ cells expressed the natural killer (NK) markers DX5 and NK1.1. pDCs represented a stable, discrete, and long-lived population. Stem cells and early lymphoid progenitors (ELPs), but not prolymphocytes, were effective precursors of pDCs, and their differentiation was blocked by ligation of Notch receptors. Furthermore, pDCs were present in the BM of RAG1 ؊/؊ , CD127/IL-7Ra ؊/؊ , and Pax5 ؊/؊ mice. pDCs in RAG1/GFP knock-in mice could be subdivided, and immunoglobulin D H -J H rearrangements, as well as transcripts for the B-lineagerelated genes Pax5, mb1/CD79a, ebf, and Bcl11a, were identified only in the green fluorescent protein-positive (GFP ؉ ) pDC1 subset. All pDCs expressed terminal deoxynucleotidyl transferase (TdT), the ETS transcription factor Spi-B, the nuclear factor-B transcription factor RelB, toll-like receptor 9 (TLR9), and interferon consensus sequence binding protein (ICSBP)/interferon regulatory factor 8 (IRF-8) transcripts; lacked CD16 and granulocyte colony-stimulating factor receptor (G-CSFR); and were uniformly interleukin-7 receptor ␣ (IL-7R␣ ؊ ) AA4.1 Lo , CD27 ؊ , Flk-2 Lo , c-Kit ؊ , DX-5 ؊ , and CD11b ؊ , while CD4 and CD8␣ were variable. GFP ؉ pDC1 subset was less potent than GFP ؊ pDC2s in T allostimulation and production of tumor necrosis factor ␣ (TNF␣), interferon ␣ (IFN␣), and interleukin-6 (IL-6), while only pDC2s made IFN␥ and IL-12 p70. Thus, 2 functionally specialized subsets of pDCs arise in bone marrow from progenitors that diverge from B, T, and NK lineages at an early stage. IntroductionPlasmacytoid dendritic cells (pDCs) are believed to play central roles in defense of viral infection and maintenance of T-cell tolerance. They represent a principal source of type I interferon and can produce inflammatory cytokines such as interleukin-12 (IL-12) p70, IL-6, and tumor necrosis factor ␣ (TNF␣). 1,2 Furthermore, pDCs have been implicated in the pathogenesis of lupus in humans. [3][4][5] While information is rapidly accumulating about pDCs, important questions remain about their origin, heterogeneity, and lifespan. The focus of our study was on pDCs that reside within bone marrow (BM).CD11c Ϫ CD123 Hi HLA class II Hi BDCA (blood dendritic cell antigen) ϩ pDCs were originally defined in human blood and distinguished from conventional CD11c ϩ CD123 Ϫ dendritic cells (DCs). [6][7][8][9] The murine counterparts of human pDCs express CD11c and CD45R/B220, but not CD19, 10,11 while murine DCs as a whole can be divided into CD8 Ϫ and CD8 ϩ subpopulations. 12 Although distinct from classical murine CD8 ϩ DCs, pDCs express variable levels of CD8. 1,13,14 They were formerly defined as CD11c Lo B220 ϩ Gr1 ϩ in spleen and as CD11c ϩ B220 ϩ CD11b Ϫ cells when derived from BM cultured with FMS-like tyrosine kinase 3 ligand (Flt3-L). 15 Expression of Ly6G/Gr1 on pDCs is controve...

CXCL9 and CXCL10 mediate the recruitment of T lymphocytes and NK cells known to be important in viral surveillance. The relevance of CXCL10 in comparison to CXCL9 in response to genital HSV-2 infection was determined using mice deficient in CXCL9 (CXCL9−/−) and deficient in CXCL10 (CXCL10−/−) along with wild-type (WT) C57BL/6 mice. An increased sensitivity to infection was found in CXCL10−/− mice in comparison to CXCL9−/− or WT mice as determined by detection of HSV-2 in the CNS at day 3 postinfection. However, by day 7 postinfection both CXCL9−/− and CXCL10−/− mice possessed significantly higher viral titers in the CNS in comparison to WT mice consistent with mortality (18–35%) of these mice within the first 7 days after infection. Even though CXCL9−/− and CXCL10−/− mice expressed elevated levels of CCL2, CCL3, CCL5, and CXCL1 in the spinal cord in comparison to WT mice, there was a reduction in NK cell and virus-specific CD8+ T cell mobilization to this tissue, suggesting CXCL9 and CXCL10 are critical for recruitment of these effector cells to the spinal cord following genital HSV-2 infection. Moreover, leukocytes from the spinal cord but not from draining lymph nodes or spleens of infected CXCL9−/− or CXCL10−/− mice displayed reduced CTL activity in comparison to effector cells from WT mice. Thus, the absence of CXCL9 or CXCL10 expression significantly alters the ability of the host to control genital HSV-2 infection through the mobilization of effector cells to sites of infection.

Hematopoietic stem and progenitor cells were previously found to express Tolllike receptors (TLRs), suggesting that bacterial/viral products may influence blood cell formation. We now show that common lymphoid progenitors (CLPs) from mice with active HSV-1 infection are biased to dendritic cell (DC) differentiation, and the phenomenon is largely TLR9 dependent. Similarly, CLPs from mice treated with the TLR9 ligand CpG ODN had little ability to generate CD19 ؉ B lineage cells and had augmented competence to generate DCs. TNF␣ mediates the depletion of late-stage lymphoid progenitors from bone marrow in many inflammatory conditions, but redirection of lymphopoiesis occurred in TNF␣ ؊/؊ mice treated with CpG ODN. Increased numbers of DCs with a lymphoid past were identified in Ig gene recombination substrate reporter mice treated with CpG ODN. TLR9 is highly expressed on lymphoid progenitors, and culture studies revealed that those receptors, rather than inflammatory cytokines, accounted for the production of several types of functional DCs. IntroductionHematopoietic stem cells (HSCs) give rise to progenitors with potential to produce blood cell types with remarkably stable characteristics. Although this process is tightly controlled, recent findings suggest that hematopoiesis is dynamic and also responsive to environmental factors. 1 The loss of differentiation options is gradual, and T lymphocytes, natural killer (NK) cells, and dendritic cells (DCs) can each be made from multiple progenitors under experimental circumstances. 1,2 Indeed, apparently similar DCs arise from distinct myeloid or lymphoid progenitors. 3 This new perspective raises the possibility that choices are made between multiple pathways to replenish effectors of the immune system. Thus, it is important to learn what normal and disease conditions favor particular differentiation routes.Several major categories of DCs have been found in murine bone marrow (BM). Conventional DCs (cDCs) are competent to present antigens, whereas plasmacytoid dendritic cells (pDCs) are potent producers of type I interferon. 3 The pDCs are divisible into 2 subtypes (pDC1 and pDC2) on the basis of RAG-1 expression and patterns of cytokine production. 4 Under experimental conditions, DCs are produced from stem cells, as well as lymphoid and myeloid progenitors. [3][4][5] Flk-2/flt-3 ligand and the associated Stat3 signaling pathway are important for DC differentiation; consequently, efficient progenitors bear the Flk-2/flt-3 receptor. 3 In our experience, the highest yields of pDCs are obtained from the primitive Lin Ϫ c-Kit hi Sca-1 ϩ (LSK) fraction of murine BM. 4 Two recent reports identified a Lin Ϫ Flt3 ϩ c-Kit lo -CD115 ϩ pro-DC population capable of generating pDCs and at least 2 categories of DCs. 6,7 However, greater yields of DCs were produced from more primitive progenitors, and some of those are already restricted to particular DC pathways. 7 Common lymphoid progenitors (CLPs) represent the main pathway to B lineage cells and include most progenitors dest...

Acute leukemias are the most common cancer in childhood and characterized by the uncontrolled production of hematopoietic precursor cells of the lymphoid or myeloid series within the bone marrow. Even when a relatively high efficiency of therapeutic agents has increased the overall survival rates in the last years, factors such as cell lineage switching and the rise of mixed lineages at relapses often change the prognosis of the illness. During lineage switching, conversions from lymphoblastic leukemia to myeloid leukemia, or vice versa, are recorded. The central mechanisms involved in these phenomena remain undefined, but recent studies suggest that lineage commitment of plastic hematopoietic progenitors may be multidirectional and reversible upon specific signals provided by both intrinsic and environmental cues. In this paper, we focus on the current knowledge about cell heterogeneity and the lineage switch resulting from leukemic cells plasticity. A number of hypothetical mechanisms that may inspire changes in cell fate decisions are highlighted. Understanding the plasticity of leukemia initiating cells might be fundamental to unravel the pathogenesis of lineage switch in acute leukemias and will illuminate the importance of a flexible hematopoietic development.

Interferon-producing killer dendritic cells (IKDCs) have only recently been described and they share some properties with plasmacytoid dendritic cells (pDCs). We now show that they can arise from some of the same progenitors. However, IKDCs expressed little or no RAG-1, Spi-B, or TLR9, but responded to the TLR9 agonist CpG ODN by production of IFN␥. The RAG-1 ؊ pDC2 subset was more similar to IKDCs than RAG-1 ؉ pDC1s with respect to IFN␥ production. The Id-2 transcriptional inhibitor was essential for production of IKDCs and natural killer (NK) cells, but not pDCs. IKDCs developed from lymphoid progenitors in culture but, unlike pDCs, were not affected by Notch receptor ligation. While IKDCs could be made from estrogen-sensitive progenitors, they may have a slow turnover because their numbers did not rapidly decline in hormonetreated mice. Four categories of progenitors were compared for IKDC-producing ability in transplantation assays. Of these, Lin ؊ Sca- IntroductionFunctionally specialized cells in the innate and adaptive immune systems are still being discovered, 1-3 and each needs to be fully understood in terms of developmental history. Replacement of immune effector cells from hematopoietic stem cells (HSCs) is an ordered process where multiple lineage potentials are gradually lost coincident with gain of specialized functions, and within the context of patterns of transcriptional activity and surface marker expression. [4][5][6][7][8][9] The focus of the present study was on recently identified interferon-producing killer dendritic cells (IKDCs). IKDCs exhibit "hybrid" phenotypic and functional characteristics of dendritic and natural killer (NK) cells. 2,3 Shared properties include expression of B220, CD11c, CD122, and NK1.1, as well as production of interferons, capability of antigen presentation, and strong cytotoxic or antitumor activities. Thus, IKDCs are unique, multifunctional cells that merit study with respect to development.HSCs and several categories of primitive progenitors reside in the lineage marker-negative Sca1 ϩ c-kit hi (LSK) fraction of BM. Up-regulation of Flk-2 and corresponding loss of erythroid/ megakaryocytic potential is an early event that sets the stage for lymphopoiesis. 10,11 LSKs include 2 overlapping subsets of lymphopoietic cells identified as Flk-2 ϩ/Ϫ Thy1.1 Ϫ L-selectin ϩ progenitors (LSPs) and RAG-1 ϩ Flk-2 ϩ CD27 ϩ early lymphoid progenitors (ELPs). [12][13][14] Although clearly B-and T-lymphoid specified, LSPs and ELPs retain some potential for myeloid, NK, and dendritic cell (DC) lineages. Firm commitment in these pathways and repression of alternative fates involve expression of key transcription factors, such as Pax5, Notch-1, and Spi-B, as well as environmental cues. 15 All lymphoid progenitors in BM are rapidly and preferentially depleted in estrogen-treated mice. 16,17 Progenitors that can more quickly give rise to lymphocytes reside in a Lin Ϫ Flk-2 ϩ c-Kit Lo prolymphocyte (Pro-L) fraction that includes Lin Ϫ c-Kit Lo Sca-1 ϩ IL-7R␣ ϩ common lymphoid proge...

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Many fundamental concepts about immune system development have changed substantially in the past few years, and rapid advances with animal models are presenting prospects for further discovery. However, continued progress requires a clearer understanding of the relationships between haematopoietic stem cells and the progenitors that replenish each type of lymphocyte pool. Blood-cell formation has traditionally been described in terms of discrete developmental branch points, and a single route is given for each major cell type. As we discuss in this Review, recent findings suggest that the process of B-cell formation is much more dynamic.