ALBERT

Description: Abstract 4831 Introduction: Previously it was demonstrated that lymphopoiesis is rapidly established after transplantation of wild type stem cells into lymphopenic NODSCIDcγ−/− mice. These data were interpreted as evidence for an “empty” preformed lymphopoietic niche being replenished by lymphoid progenitors. We hypothesized that antibody-induced neutropenia might influence early post transplant fate decision to myeloid rather than lymphoid differentiation resulting in delayed lymphoid reconstitution. Materials and Methods: 25,000 flow sorted CD45.2-expressing wild type Lin-/Sca1+/c-Kit+ (LSK) cells from C57BL/6 mice were transplanted into sublethally irradiated B-/T-/NK-cell deficient NODSCIDcγ−/− mice (CD45.1). Three groups of n = 7 mice received anti-Gr1 or anti-1A8 i.p. every 48 h to induce continuous antibody-mediated neutropenia vs. PBS as control. Blood was harvested at regular intervals to monitor the engraftment. After 16, 22, and 34 days, animals were sacrificed and underwent blood and bone marrow analysis. Results: Hematopoietic regeneration started with the emergence of donor-derived monocytes in all groups as well as neutrophils in the control group as early as 9 days after transplantation. On day 14, B cells were to be detected for the first time, followed by T lymphocytes approximately 20 days after transplantation. Besides the fact that neutrophils were undetectable in the antibody treated groups, the peripheral blood revealed no significant changes between the neutropenic mice and the control group at any point of time. At the bone marrow level, an increase of LSK and granulocyte-macrophage progenitors (GMPs) at the expense of megakaryocyte erythrocyte progenitor cells (MEPs) was found in neutropenic mice. Common lymphoid progenitors (CLPs), however, were not significantly different. Conclusions: The engraftment of wild type donor cells after hematopoietic stem cell transplantation into NODSCIDcγ−/− mice started with the production of monocytes and neutrophils. B-lymphocytes were detectable by day 14 after transplantation. The production of T-cells started around day 20. Continuous antibody-mediated neutropenia did not significantly delay lymphoid regeneration. Although the marrow of neutropenic mice displayed increased proliferation of granulocyte progenitors, CLPs were unchanged. We conclude that the detection of donor-derived lymphocytes in the host peripheral blood is a relatively early event after LSK transplantation. Moreover, antibody induced neutropenia is not sufficient to induce sustainable changes in early hematopoietic fate decisions on the bone marrow level. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 4830 Introduction: G-CSF is generally accepted to be the major cytokine regulating neutrophil production, but neutrophil steady-state homeostasis has still not been fully elucidated. We have previously shown in NODSCIDcγ−/− mice that feedback G-CSF expression upon antibody-based neutrophil depletion occurs independent from lymphocytes (Bugl et al., ASH 2010) and hypothesized that there may be a sensing mechanism of neutropenia relying on the presence of microbial components, similar to emergency granulopoiesis. Therefore, germ-free wildtype mice received anti-neutrophil antibodies and underwent further analysis. Moreover, passive regulation of circulating G-CSF levels by neutrophil cell mass was examined. Methods: Anti-Ly6-G antibody (clone 1A8) was used to induce neutropenia in germ-free C57BL/6 mice. After one week of neutrophil depletion hematopoietic tissues and peripheral blood were harvested and analyzed on cellular, protein and RNA level. Moreover, neutrophil granulocytes (granulocyte-differentiation antigen-1+ Mac-1+) purified from peripheral blood of C57BL/6 mice were transfused into neutropenic wild type mice and plasma G-CSF was monitored. Results: Peripheral blood neutropenia could be effectively induced in all experimental mice by anti-1A8 antibody. Transfusion of 3.5 × 106 neutrophils into neutropenic wild type mice did not significantly change plasma G-CSF levels. Filgrastim (rhG-CSF), however, caused significant downregulation of bone marrow G-CSF at the mRNA level. Germ-free C57BL/6 mice were analyzed after 1 week of antibody-induced neutropenia and showed a shift of myeloid progenitors towards granulocyte macrophage precursors (GMP) at the expense of megakaryocyte erythrocyte progenitors (MEP) as well as significantly increased numbers of hematopoietic stem cells. In addition, G-CSF, M-CSF, and CXCL12 behaved identically in both germ-free control and wild type mice under specific-pathogen free conditions. Conclusions: Transfusion of G-CSF-receptor (CSF3R) positive neutrophils did not significantly influence G-CSF. Moreover, exogenous G-CSF downregulated marrow G-CSF on the transcriptional level. Germ-free mice are able to mount a feedback loop including G-CSF upregulation and marrow changes in progenitor cell distribution analogous to mice carrying physiological commensal bacteria. Our data are therefore consistent with G-CSF feedback regulation occurring independent from commensal germs. Moreover, our data indicate transcriptional rather than CSF3R+ cell mass-associated passive regulation of G-CSF levels. Taken together, we propose a model of myeloid bone marrow homeostasis, where feedback loops of neutrophil production upon antibody-dependent neutropenia occur through transcriptional upregulation of G-CSF. The underlying mechanisms occur independent of lymphocytes and presence of germs. Disclosures: No relevant conflicts of interest to declare.

Description: Key Points Steady-state and emergency granulopoiesis are both dependent on TLR signaling.

Description: Abstract 2613 Introduction: In contrast to red cell or platelet homeostasis, the feedback mechanisms involved in the control of peripheral neutrophil numbers are incompletely understood. Granulocyte-Colony Stimulating Factor (G-CSF) is generally accepted to be the most important determinant of neutrophil production and release from the bone marrow. Recently, a feedback loop including a specialized subset of T-lymphocytes (Tn cells) has been established to explain the correlation between peripheral neutrophil clearance and increased G-CSF production. Methods: Wild type C57bl/6 mice or NODSCIDcγ−/− received anti-Gr1 or anti-1A8 antibodies to deplete neutrophils. Hematopoietic tissues and peripheral blood were harvested at different times and analyzed on cellular, protein and RNA level. Results: Both anti-Gr1 and 1A8 antibodies reduced neutrophils effectively and persistently in vivo. The reaction on neutrophil depletion in the marrow uniformely consisted of an absolute increase in lin-/Sca1+/c-kit+ (LSK) cells and a shift of myeloid progenitors towards granulocyte-macrophage precursors (GMP) and common myeloid progenitors (CMP) at the expense of megakaryocyte-erythrocyte precursors (MEP). Of note, exogenous G-CSF resulted in identical changes. We therefore hypothesized that neutrophil depletion increases G-CSF through a feedback regulatory loop. Neutrophil depletion with anti-Gr1 antibody in wt mice increased G-CSF levels up to approximately 8-fold. While previous observations suggest that G-CSF may be passively regulated through receptor binding and internalization by mature neutrophils, we also found a 10-fold increase of G-CSF mRNA in the marrow. These findings are consistent with active feedback. Interestingly, the effects of 1A8 antibody on G-CSF were less pronounced. Instead, mice depleted of neutrophils with 1A8 antibody displayed predominant increases of M-CSF, suggesting redundant feedback pathways. Our results in wildtype mice treated with 1A8 antibody confirm previous data by Stark et al. (Immunity 2005; 22: 285–294), including increases of IL-23 and IL-17 after neutrophil depletion. However, when neutrophils were depleted in NODSCIDcγ−/− mice, who lack lymphocytes and NK-cells, both IL-23 and IL-17 remained unchanged, but G-CSF levels still increased markedly. Conclusions: Effective neutrophil depletion by antibodies directed against different neutrophil antigens uniformly results in major shifts in the hematopoietic bone marrow showing an increase in the number of LSK hematopoietic stem cells accompanied by a significant increase in myeloid progenitors at the expense of thrombopoietic, red cell, and lymphoid precursors. Our results demonstrate regulatory feedback loops of neutrophil granulopoiesis culminating in increased production of myelopoiesis stimulating cytokines such as G-CSF, GM-CSF, and M-CSF. The underlying chain of events includes IL-23 and IL-17 in wild type mice as previously described. However, additional redundant pathways exist in lymphocytopenic NODSCIDcγ−/− mice. Disclosures: No relevant conflicts of interest to declare.

Description: Trafficking of hematopoietic stem and progenitor cells (HPC) is controlled by G protein coupled receptors (GPRs), particularly by the chemokine receptor CXCR4. However, homing of HPC does not exclusively depend on CXCR4 and its ligand SDF-1. In addition to chemokine receptors, also GPRs for non-peptide lipid mediators may be involved in HPC migration. In the present study, we demonstrate that sphingosine 1-phosphate (S1P) is chemotactic for human CD34+ HPC (optimum dose 5 μM), as measured in a modified Boyden chamber system. In CD34+ cell lines, the chemotactic response was even stronger and occurred at lower S1P concentrations (e.g., optimum dose 100 nM for Jurkat cells, 2-fold stronger migration compared to HPC). By RT-PCR, we measured mRNA expression of the 5 different S1P receptors (S1P1,2,3,4,5). All CD34+ hematopoietic cell lines analyzed (KG1, KG1a, Jurkat) expressed S1P1, a S1P receptor with known chemotactic activity, while CD34- cell lines (HL-60, THP-1) were negative for S1P1. In HL-60 cells, chemotaxis in response to S1P was even reduced compared to spontaneous migration, which might be due to the expression of S1P receptors other than S1P1 with known inhibitory effects, particularly S1P2. In mobilized peripheral blood CD34+ progenitor cells from different donors, S1P1 was consistently expressed in both CD34+CD38+ and more primitive CD34+CD38- HPC, while expression of S1P2,3,4,5 was variable. S1P induced also other typical responses of GPR-mediated signaling in CD34+ cell lines and HPC, such as polymerization of filamentous actin, as measured by flow cytometry after labeling of the cells with FITC-phalloidin. We conclude that S1P is a chemotactic factor for CD34+ HPC and CD34+ cell lines due to expression of the GPR S1P1. As megakaryocytes and platelets represent an abundant source of S1P in the bone marrow, our results suggest that S1P and its receptor S1P1 may contribute to trafficking and spatial distribution of HPC in the hematopoietic microenvironment.

Description: The novel immunosuppressant FTY720 activates sphingosine 1-phosphate receptors (S1PRs) that affect responsiveness of lymphocytes to chemokines such as stromal cell-derived factor 1 (SDF-1), resulting in increased lymphocyte homing to secondary lymphoid organs. Since SDF-1 and its receptor CXCR4 are also involved in bone marrow (BM) homing of hematopoietic stem and progenitor cells (HPCs), we analyzed expression of S1PRs and the influence of FTY720 on SDF-1/CXCR4-mediated effects in human HPCs. By reverse transcriptase-polymerase chain reaction (RT-PCR), S1PRs were expressed in mobilized CD34+ HPCs, particularly in primitive CD34+/CD38- cells. Incubation of HPCs with FTY720 resulted in prolonged SDF-1-induced calcium mobilization and actin polymerization, and substantially increased SDF-1-dependent in vitro transendothelial migration, without affecting VLA-4, VLA-5, and CXCR4 expression. In nonobese diabetic-severe combined immunodeficient (NOD/SCID) mice, the number of CD34+/CD38- cells that homed to the BM after 18 hours was significantly raised by pretreatment of animals and cells with FTY720, tending to result in improved engraftment. In addition, in vitro growth of HPCs (week-5 cobblestone area-forming cells [CAFCs]) was 2.4-fold increased. We conclude that activation of S1PRs by FTY720 increases CXCR4 function in HPCs both in vitro and in vivo, supporting homing and proliferation of HPCs. In the hematopoietic microenvironment, S1PRs are involved in migration and maintenance of HPCs by modulating the effects of SDF-1. (Blood. 2004;103:4478-4486)

Description: Abstract 4012 Poster Board III-948 Introduction Platelets are important contributors to several hallmarks of tumor pathophysiology including immune escape of disseminated malignant cells, metastasis, and neoangiogenesis. Following activation, platelets release their granule content, which contains a plethora of factors that may both inhibit and stimulate angiogenesis, immunosurveillance, plasmatic coagulation, or tumor growth. Targeted deletion of thrombospondin-1 and -2 from the platelet proteome leads to altered secretion of CXCL12 and matrix metalloproteinases (MMPs) in mice (Kopp HG, Hooper AT et al., J Clin Invest 2006), and platelets derived from 25 patients with lymphoma, colorectal and ovarian cancer displayed a diminished tsp-1/VEGF-ratio (Gonzalez FJ, Rueda A et al., Int J Biol Markers, 2004). We therefore set out to analyze a carefully selected patient cohort for in vivo platelet activation and platelet contents in order to define phenotypic changes in the setting of disseminated malignancy. Patients and methods Platelet activation in percent of P-selectin positive platelets and platelet contents (i.e. absolute platelet count-corrected plasma/serum levels of VEGF-A, CXCL12 [SDF-1], CXCL4 [pf4], and thrombospondin-1) were analyzed. Patients were eligible after written informed consent if they had a first diagnosis of metastatic malignancy, were not taking medication on a regular basis, and had not taken any medication during the last 14 days before venipuncture. Healthy age- and sex-matched subjects were used as controls. Results (mean ± SEM) Tumor patients had increased platelet counts (301,558 ± 17,489/μl vs. 234,111 ± 5,946/μl) and slightly lower hematocrits. In addition, cancer patients displayed significantly elevated percentages of activated platelets as measured by CD62P expression (1.14 ± 0.20% vs. 0.24 ± 0.03%). In order to obtain absolute platelet contents per 100 million platelets, we took tumor-associated thrombocytosis and anemia in consideration and calculated as follows: ([serum VEGF/CXCL12/TSP1/CXCL4 – plasma VEGF/CXCL12/TSP1/CXCL4] * [1-hematocrit] ÷ platelets per ml whole blood) * 100×106. The results showed highly significant changes, including increased contents of VEGF (143.24 ± 15.87 pg vs. 63.02 ± 6.28 pg; p 〈 0.001) and decreased amounts of thrombospondin-1 (2,925.13 ± 217.78 ng vs. 3,764.34 ± 184.64 ng; p = 0.005), CXCL4 (1,547.60 ± 44.54 ng vs. 1,132.32 ± 55.62 ng, p