A p53-dependent mechanism underlies macrocytic anemia in a mouse model of human 5q- syndrome.

The identification of the genes associated with chromosomal translocation breakpoints has fundamentally changed understanding of the molecular basis of hematological malignancies. By contrast, the study of chromosomal deletions has been hampered by the large number of genes deleted and the complexity of their analysis. We report the generation of a mouse model for human 5q- syndrome using large-scale chromosomal engineering. Haploinsufficiency of the Cd74-Nid67 interval (containing Rps14, encoding the ribosomal protein S14) caused macrocytic anemia, prominent erythroid dysplasia and monolobulated megakaryocytes in the bone marrow. These effects were associated with defective bone marrow progenitor development, the appearance of bone marrow cells expressing high amounts of the tumor suppressor p53 and increased bone marrow cell apoptosis. Notably, intercrossing with p53-deficient mice completely rescued the progenitor cell defect, restoring common myeloid progenitor and megakaryocytic-erythroid progenitor, granulocyte-monocyte progenitor and hematopoietic stem cell bone marrow populations. This mouse model suggests that a p53-dependent mechanism underlies the pathophysiology of the 5q- syndrome.

Prolonged, granulocyte-macrophage colony-stimulating factor-dependent, neutrophil survival following rheumatoid synovial fibroblast activation by IL-17 and TNFalpha.

INTRODUCTION: A surprising feature of the inflammatory infiltrate in rheumatoid arthritis is the accumulation of neutrophils within synovial fluid and at the pannus cartilage boundary. Recent findings suggest that a distinct subset of IL-17-secreting T-helper cells (TH17 cells) plays a key role in connecting the adaptive and innate arms of the immune response and in regulating neutrophil homeostasis. We therefore tested the hypothesis that synovial fibroblasts bridge the biological responses that connect TH17 cells to neutrophils by producing neutrophil survival factors following their activation with IL-17. METHODS: IL-17-expressing cells in the rheumatoid synovium, and IL-17-expressing cells in the peripheral blood, and synovial fluid were examined by confocal microscopy and flow cytometry, respectively. Peripheral blood neutrophils were cocultured either with rheumatoid arthritis synovial fibroblasts (RASF) or with conditioned medium from RASF that had been pre-exposed to recombinant human IL-17, TNFalpha or a combination of the two cytokines. Neutrophils were harvested and stained with the vital mitochondrial dye 3,3'-dihexyloxacarbocyanine iodide before being enumerated by flow cytometry. RESULTS: TH17-expressing CD4+ cells were found to accumulate within rheumatoid synovial tissue and in rheumatoid arthritis synovial fluid. RASF treated with IL-17 and TNFalpha (RASFIL-17/TNF) effectively doubled the functional lifespan of neutrophils in coculture. This was entirely due to soluble factors secreted from the fibroblasts. Specific depletion of granulocyte-macrophage colony-stimulating factor from RASFIL-17/TNF-conditioned medium demonstrated that this cytokine accounted for approximately one-half of the neutrophil survival activity. Inhibition of phosphatidylinositol-3-kinase and NF-kappaB pathways showed a requirement for both signalling pathways in RASFIL-17/TNF-mediated neutrophil rescue. CONCLUSION: The increased number of neutrophils with an extended lifespan found in the rheumatoid synovial microenvironment is partly accounted for by IL-17 and TNFalpha activation of synovial fibroblasts. TH17-expressing T cells within the rheumatoid synovium are likely to contribute significantly to this effect.

Tumor necrosis factor alpha activates release of B lymphocyte stimulator by neutrophils infiltrating the rheumatoid joint.

OBJECTIVE: The tumor necrosis factor (TNF) family member B lymphocyte stimulator (BLyS) is an important regulator of B cell-dependent autoimmunity. Similar to other TNF family members, it is generally expressed as a transmembrane protein and cleaved from the surface to release its active soluble form. This study was undertaken to investigate the expression of BLyS and regulation of BLyS release from the surface of neutrophils infiltrating the rheumatoid joint. METHODS: BLyS expression was studied in neutrophils from the synovial fluid and peripheral blood of patients with rheumatoid arthritis (RA) and healthy controls, by flow cytometry, Western blotting, and immunofluorescence analyses. Peripheral blood neutrophils cultured with 50% RA synovial fluid were study for membrane expression of BLyS. Neutrophils were exposed to a range of proinflammatory cytokines to study the mechanisms of surface loss of BLyS. RESULTS: Expression of BLyS was detected on the surface of peripheral blood neutrophils from both RA patients and healthy controls, whereas BLyS expression on synovial fluid neutrophils was very low. Constitutive expression of BLyS was observed in neutrophils, both on the cell membrane and in intracellular stores; however, BLyS release from each of these sites was found to be regulated independently. Of the various cytokine stimuli, only TNFalpha triggered release of BLyS from the neutrophil membrane. This process led to release of physiologically relevant quantities of soluble BLyS, which was dependent on the presence of the pro-protein convertase furin. In contrast, stimulation of neutrophils with granulocyte colony-stimulating factor induced BLyS release from the intracellular stores. Incubation of peripheral blood neutrophils with RA synovial fluid led to TNFalpha-dependent shedding of BLyS from the cell surface. CONCLUSION: These findings indicate that as neutrophils enter the site of inflammation, they release surface-expressed BLyS in a TNFalpha-dependent manner, and thus may contribute to local stimulation of autoimmune B cell responses.

Interaction between integrin alpha9beta1 and vascular cell adhesion molecule-1 (VCAM-1) inhibits neutrophil apoptosis.

According to the prevailing paradigm, neutrophils are short-lived cells that undergo spontaneous apoptosis within 24 hours of their release from the bone marrow. However, neutrophil survival can be significantly prolonged within inflamed tissue by cytokines, inflammatory mediators, and hypoxia. During screening experiments aimed at identifying the effect of the adhesive microenvironment on neutrophil survival, we found that VCAM-1 (CD106) was able to delay both spontaneous and Fas-induced apoptosis. VCAM-1-mediated survival was as efficient as that induced by the cytokine IFN-beta and provided an additive, increased delay in apoptosis when given in combination with IFN-beta. VCAM-1 delivered its antiapoptotic effect through binding the integrin alpha9beta1. The alpha9beta1 signaling pathway shares significant features with the IFN-beta survival signaling pathway, requiring PI3 kinase, NF-kappaB activation, as well as de novo protein synthesis, but the kinetics of NF-kappaB activation by VCAM-1 were slower and more sustained compared with IFN-beta. This study demonstrates a novel functional role for alpha9beta1 in neutrophil biology and suggests that adhesive signaling pathways provide an important extrinsic checkpoint for the resolution of inflammatory responses in tissues.

Factors underlying chronic inflammation in rheumatoid arthritis.

Rheumatoid arthritis (RA)is a debilitating chronic inflammatory disease whose characteristic pathology includes swollen, painful, and deformed joints. In recent decades, both clinical and basic scientific research have tried to determine the factors involved in the pathogenesis of this common disease. Although the cause of RA is still unknown, several factors that contribute to RA have been identified. Among these are the discoveries of: susceptibility genes, disease-causing immune cells, and cytokine and signal transduction networks involved in promoting persistence of inflammation. Various therapeutic strategies, including anti-tumor necrosis factor therapy, have been developed to target one or more of these factors. Although none of these therapeutic strategies can actually cure the disease, some of these novel agents have proven to be more effective than others. This implies that the success of a therapy is very much dependent on the therapeutic targets chosen. Therefore, improved understanding of the cellular and molecular events occurring in the rheumatoid joint during the pathogenesis of the disease is particularly important if we are to better combined therapeutic strategies. In this article we summarize current understanding of the factors that contribute to disease pathogenesis in RA and identify cellular and molecular events that could drive the development of the disease and represent potential new therapeutic targets.