Monocyte Differentiation and Heterogeneity: Inter-Subset and Interindividual Differences.

The three subsets of human monocytes, classical, intermediate, and nonclassical, show phenotypic heterogeneity, particularly in their expression of CD14 and CD16. This has enabled researchers to delve into the functions of each subset in the steady state as well as in disease. Studies have revealed that monocyte heterogeneity is multi-dimensional. In addition, that their phenotype and function differ between subsets is well established. However, it is becoming evident that heterogeneity also exists within each subset, between health and disease (current or past) states, and even between individuals. This realisation casts long shadows, impacting how we identify and classify the subsets, the functions we assign to them, and how they are examined for alterations in disease. Perhaps the most fascinating is evidence that, even in relative health, interindividual differences in monocyte subsets exist. It is proposed that the individual's microenvironment could cause long-lasting or irreversible changes to monocyte precursors that echo to monocytes and through to their derived macrophages. Here, we will discuss the types of heterogeneity recognised in monocytes, the implications of these for monocyte research, and most importantly, the relevance of this heterogeneity for health and disease.

Sphingosine-1-phosphate facilitates trafficking of hematopoietic stem cells and their mobilization by CXCR4 antagonists in mice.

CXCL12 and VCAM1 retain hematopoietic stem cells (HSCs) in the BM, but the factors mediating HSC egress from the BM to the blood are not known. The sphingosine-1-phosphate receptor 1 (S1P(1)) is expressed on HSCs, and S1P facilitates the egress of committed hematopoietic progenitors from the BM into the blood. In the present study, we show that both the S1P gradient between the BM and the blood and the expression of S1P(1) are essential for optimal HSC mobilization by CXCR4 antagonists, including AMD3100, and for the trafficking of HSCs during steady-state hematopoiesis. We also demonstrate that the S1P(1) agonist SEW2871 increases AMD3100-induced HSC and progenitor cell mobilization. These results suggest that the combination of a CXCR4 antagonist and a S1P(1) agonist may prove to be sufficient for mobilizing HSCs in normal donors for transplantation purposes, potentially providing a single mobilization procedure and eliminating the need to expose normal donors to G-CSF with its associated side effects.

Potentiating effects of RAD001 (Everolimus) on vincristine therapy in childhood acute lymphoblastic leukemia.

Despite advances in the treatment of acute lymphoblastic leukemia (ALL), the majority of children who relapse still die of ALL. Therefore, the development of more potent but less toxic drugs for the treatment of ALL is imperative. We investigated the effects of the mammalian target of rapamycin inhibitor, RAD001 (Everolimus), in a nonobese diabetic/severe combined immunodeficiency model of human childhood B-cell progenitor ALL. RAD001 treatment of established disease increased the median survival of mice from 21.3 days to 42.3 days (P < .02). RAD001 together with vincristine significantly increased survival compared with either treatment alone (P < .02). RAD001 induced a cell-cycle arrest in the G(0/1) phase with associated dephosphorylation of the retinoblastoma protein, and reduced levels of cyclin-dependent kinases 4 and 6. Ultrastructure analysis demonstrated the presence of autophagy and limited apoptosis in cells of RAD001-treated animals. In contrast, cleaved poly(ADP-ribose) polymerase suggested apoptosis in cells from animals treated with vincristine or the combination of RAD001 and vincristine, but not in those receiving RAD001 alone. In conclusion, we have demonstrated activity of RAD001 in an in vivo leukemia model supporting further clinical development of target of rapamycin inhibitors for the treatment of patients with ALL.

CXCR4 mediates the homing of B cell progenitor acute lymphoblastic leukaemia cells to the bone marrow via activation of p38MAPK.

The mechanisms regulating the migration of leukaemic cells between the blood and bone marrow compartments remain obscure, but are of fundamental importance for the dissemination of the disease. This study investigated the in vivo homing of human B cell progenitor acute lymphoblastic leukaemia (ALL) cells to the femoral bone marrow of non-obese diabetic severe combined immunodeficient (NOD/SCID) mice. It was demonstrated that patient ALL cells use the chemokine axis, chemokine (CXC motif) receptor 4 (CXCR4)/ chemokine (CXC motif) ligand 12 (CXCL12), to home to the femoral marrow. CXCL12-mediated signalling through p38 mitogen-activated protein kinase (MAPK) was required for optimal homing. In contrast, the homing of normal peripheral blood CD34(+) cells and the cytokine-dependent CD34(+) cell line Mo7e was independent of p38MAPK, consistent with the dependence of these cells, as well as normal CD34(+) CD19(+) B cell progenitors, on PI-3K/AKT signalling. Altogether, our data provide clarification of the direct role of CXCL12 in the bone marrow homing of ALL cells and demonstrate unique signalling molecule usage that may have therapeutic implications for this disease.

Interaction of interleukin-7 and interleukin-3 with the CXCL12-induced proliferation of B-cell progenitor acute lymphoblastic leukemia.

BACKGROUND AND OBJECTIVES: The chemokine stroma-derived factor 1a (SDF-1a or CXCL12) is essential for proliferation of B lineage acute lymphoblastic leukemia (ALL) cells in their physiological microenvironment, bone marrow stroma. CXCL12 synergizes with cytokines that stimulate myeloid cells, but its interaction with cytokines affecting lymphoid cells has not been examined. We investigated whether interleukin (IL)-7 and IL-3 interact with CXCL12 to regulate ALL proliferation. DESIGN AND METHODS: The survival of ALL cells in serum-free cultures, with or without stromal support and cytokines, was assessed by flow cytometry, and proliferation by 3H-thymidine incorporation. Signaling mechanisms were assessed by western blotting of phosphorylated forms of signaling molecules and by the use of specific inhibitors. RESULTS: CXCL12, IL-3, and IL-7 had only marginal effects on ALL cell survival under serum-free conditions. However, these molecules individually induced significant proliferative responses in stromal cultures of 11 cases of ALL. The combination of CXCL12 with IL-7 or IL-3 produced a variety of responses, with clear synergistic or additive interactions observed in four cases. Synergistic proliferation in response to CXCL12 plus IL-7 was associated with enhanced phosphorylation of the mitogen-activated protein kinases, ERK-1/2 and p38, and AKT, and was partially inhibited by pretreatment of cells with inhibitors for p38 MAPK and phosphatidylinositol 3-kinase, implicating these pathways in the proliferation in response to IL-7 plus CXCL12. INTERPRETATION AND CONCLUSIONS: These findings indicate a complex interaction between signaling from the CXCR4 receptor on ALL cells with those initiated by the cytokines IL-7 and IL-3, suggesting that CXCL12 may facilitate ALL proliferation by enhancing cytokine-signaling pathways in responsive cases.

Defective p38 mitogen-activated protein kinase signaling impairs chemotaxic but not proliferative responses to stromal-derived factor-1alpha in acute lymphoblastic leukemia.

The chemokine stromal-derived factor-1alpha (SDF-1alpha) regulates leukemic cell motility and proliferation; however, the importance of these functions in the growth and dissemination of leukemia is unclear. We examined SDF-1alpha-mediated responses of cells from 27 cases of acute lymphoblastic leukemia (ALL). Although cells from the majority of cases showed chemotactic and proliferative responses to SDF-1alpha, a subset of cases did not undergo chemotaxis in response to SDF-1alpha, while still demonstrating dependence on SDF-1alpha for proliferation in stroma-supported cultures. This chemotactic defect was associated with an absence of phosphorylation of p38 mitogen-activated protein kinase (MAPK) induced by SDF-1alpha, and of SDF-1alpha-induced augmentation of beta(1) integrin-mediated adhesion. Signaling through phosphoinositide 3-kinase and MEK was not affected. No correlation was observed between CXCR4 expression and chemotactic function, in vitro migration into bone marrow stromal layers, and engraftment of leukemic cells in nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice. This study suggests that signaling through p38 MAPK is required for ALL cell chemotaxis but not for proliferation, and that the loss of a chemotactic response to SDF-1alpha does not impede engraftment in NOD/SCID mice.

Silencer of death domains controls cell death through tumour necrosis factor-receptor 1 and caspase-10 in acute lymphoblastic leukemia.

Resistance to apoptosis remains a significant problem in drug resistance and treatment failure in malignant disease. NO-aspirin is a novel drug that has efficacy against a number of solid tumours, and can inhibit Wnt signaling, and although we have shown Wnt signaling to be important for acute lymphoblastic leukemia (ALL) cell proliferation and survival inhibition of Wnt signaling does not appear to be involved in the induction of ALL cell death. Treatment of B lineage ALL cell lines and patient ALL cells with NO-aspirin induced rapid apoptotic cell death mediated via the extrinsic death pathway. Apoptosis was dependent on caspase-10 in association with the formation of the death-inducing signaling complex (DISC) incorporating pro-caspase-10 and tumor necrosis factor receptor 1 (TNF-R1). There was no measurable increase in TNF-R1 or TNF-α in response to NO-aspirin, suggesting that the process was ligand-independent. Consistent with this, expression of silencer of death domain (SODD) was reduced following NO-aspirin exposure and lentiviral mediated shRNA knockdown of SODD suppressed expansion of transduced cells confirming the importance of SODD for ALL cell survival. Considering that SODD and caspase-10 are frequently over-expressed in ALL, interfering with these proteins may provide a new strategy for the treatment of this and potentially other cancers.

mTOR inhibition by everolimus in childhood acute lymphoblastic leukemia induces caspase-independent cell death.

Increasingly, anti-cancer medications are being reported to induce cell death mechanisms other than apoptosis. Activating alternate death mechanisms introduces the potential to kill cells that have defects in their apoptotic machinery, as is commonly observed in cancer cells, including in hematological malignancies. We, and others, have previously reported that the mTOR inhibitor everolimus has pre-clinical efficacy and induces caspase-independent cell death in acute lymphoblastic leukemia cells. Furthermore, everolimus is currently in clinical trial for acute lymphoblastic leukemia. Here we characterize the death mechanism activated by everolimus in acute lymphoblastic leukemia cells. We find that cell death is caspase-independent and lacks the morphology associated with apoptosis. Although mitochondrial depolarization is an early event, permeabilization of the outer mitochondrial membrane only occurs after cell death has occurred. While morphological and biochemical evidence shows that autophagy is clearly present it is not responsible for the observed cell death. There are a number of features consistent with paraptosis including morphology, caspase-independence, and the requirement for new protein synthesis. However in contrast to some reports of paraptosis, the activation of JNK signaling was not required for everolimus-induced cell death. Overall in acute lymphoblastic leukemia cells everolimus induces a cell death that resembles paraptosis.

Para-NO-aspirin inhibits NF-κB and induces apoptosis in B-cell progenitor acute lymphoblastic leukemia.

Although patients with acute lymphoblastic leukemia (ALL) usually achieve complete remission, disease relapse is common and difficult to treat. Para-NO-aspirin (para-NO-ASA) is a novel drug with demonstrated efficacy against a number of solid tumors and most recently chronic lymphocytic leukemia. In this study, we used ALL cell lines to assess the effects on cell viability by flow cytometry and investigated the mechanism of cell death using chemical inhibitors of key molecules and assessed the effects by flow cytometry, electrophoretic mobility shift assay, Western blotting, and quantitative reverse transcription polymerase chain reaction. Para-NO-ASA induced cell death in the pre-B ALL cell lines in association with increased reactive oxygen species, and suppression of nuclear factor-κB (NF-κB) activity. Chemical inhibitors of NF-κB similarly induced apoptosis in ALL cells, suggesting a role for suppression of NF-κB in para-NO-ASA-induced cell death. Modulation of NF-κB was not via regulation of IκB but potentially through suppression of ROCK1 and loss of reduced glutathione. Our results demonstrate that para-NO-ASA potently induces apoptosis in B-lineage ALL cells via a reactive oxygen species-dependent mechanism that is associated with suppression of NF-κB activity.