Fanny Chu

Interleukin-3, but not granulocyte–macrophage colony-stimulating factor and interleukin-5, inhibits apoptosis of human basophils through phosphatidylinositol 3-kinase: requirement of NF-κB-dependent and -independent pathways

Basophils are key effector cells of allergic reactions. Although proinflammatory cytokines, such as interleukin (IL)‐3, granulocyte–macrophage colony‐stimulating factor (GM–CSF) and IL‐5, inhibit eosinophil apoptosis in vitro, little is known about basophil apoptosis, and the signalling mechanisms required for basophil survival remain undefined. To address this issue, we used a novel negative‐selection system to isolate human basophils to a purity of > 95%, and evaluated apoptosis by morphology using light and transmission electron microscopy, and by annexin‐V binding and propidium iodide incorporation using flow cytometry. In this study, we demonstrated that the spontaneous rate of apoptotic basophils was higher than that of eosinophils as, at 24 hr, 57·6 ± 4·7% of basophils underwent apoptosis compared with 39·5 ± 3·8% of eosinophils. In addition, basophil cell death was significantly inhibited when cultured with IL‐3 for 48 hr (84·6 ± 4·9% vehicle‐treated cells versus 40·9 ± 3·9% IL‐3‐treated cells). IL‐3 also up‐regulated basophil CD69 surface expression. The effects of IL‐3 on apoptosis and CD69 surface expression of human basophils were completely blocked by LY294002 (LY), a potent inhibitor of phosphatidylinositol 3‐kinase (PI3‐K), but only partially inhibited by lactacystin, a proteasome inhibitor that prevents degradation of IκB and NF‐κB translocation. These observations reveal the novel finding that IL‐3 prevents basophil apoptosis through the activation of PI3‐K, which is only partially NF‐κB dependent. As basophils are active participants in allergic reactions and IL‐3 is one of the abundant proinflammatory cytokines in secretions from allergic tissue, we suggest that IL‐3‐mediated inhibition of basophil apoptosis may exacerbate the inflammation associated with allergic disorders.

Localization of DNA and RNA in Eosinophil Secretory Granules

Background: Although the accepted paradigm is that the proteins stored in eosinophil crystalloid granules are translated from messenger RNA transcribed in the cell nucleus, recent ultrastructural evidence suggests that protein synthesis may also take place within eosinophilic granules. Methods: We used 2 different methods to detect the presence of DNA and RNA in eosinophil secretory granules. Using bromodeoxyuridine, a thymidine analogue, and bromouridine, a uracil analogue, we labeled the DNA and RNA in eosinophils in vivo in rabbits. Immunoelectron microscopy to localize these molecules was performed on ultrathin sections of blood and bone marrow eosinophils using monoclonal anti-bromodeoxyuridine antibody with IgG as a control. The immunogold grain density was measured in each subcellular compartment within the eosinophils and analyzed using image analysis software. A combination of DNA/CD63 immunofluorescence staining and a fluorescently labeled molecular probe that stains RNA was used to examine the presence of DNA and RNA in the secretory granules of human blood eosinophils. Results: The mean density of bromodeoxyuridine-labeled DNA and bromouridine-labeled RNA immunogold grains in the secretory granules of blood and bone marrow eosinophils were significantly higher (p < 0.0005) than cytoplasmic or background staining. We also demonstrated the existence of DNA and RNA in the CD63-positive secretory granules of human peripheral blood eosinophils by means of immunofluorescent staining and a fluorescently labeled molecular probe. Conclusions: These results provide evidence that eosinophil granules are the site of DNA and RNA synthesis and suggest the potential for a new role(s) for eosinophil-secretory granules.

Liver ultrastructural morphology and mitochondrial DNA levels in HIV/hepatitis C virus coinfection : no evidence of mitochondrial damage with highly active antiretroviral therapy

Liver mitochondrial toxicity is a concern, particularly in HIV/hepatitis C virus (HCV) coinfection. Liver biopsies from HIV/HCV co-infected patients, 14 ON-highly active antiretroviral therapy (HAART) and nine OFF-HAART, were assessed by electron microscopy quantitative morphometric analyses. Hepatocytes tended to be larger ON-HAART than OFF-HAART (P = 0.05), but mitochondrial volume, cristae density, lipid volume, mitochondrial DNA and RNA levels were similar. We found no evidence of increased mitochondrial toxicity in individuals currently on HAART, suggesting that concomitant HAART should not delay HCV therapy.

Loss of vascular CD34 results in increased sensitivity to lung injury

&NA; Survival during lung injury requires a coordinated program of damage limitation and rapid repair. CD34 is a cell surface sialomucin expressed by epithelial, vascular, and stromal cells that promotes cell adhesion, coordinates inflammatory cell recruitment, and drives angiogenesis. To test whether CD34 also orchestrates pulmonary damage and repair, we induced acute lung injury in wild‐type (WT) and Cd34−/− mice by bleomycin administration. We found that Cd34−/− mice displayed severe weight loss and early mortality compared with WT controls. Despite equivalent early airway inflammation to WT mice, CD34‐deficient animals developed interstitial edema and endothelial delamination, suggesting impaired endothelial function. Chimeric Cd34−/− mice reconstituted with WT hematopoietic cells exhibited early mortality compared with WT mice reconstituted with Cd34−/− cells, supporting an endothelial defect. CD34‐deficient mice were also more sensitive to lung damage caused by influenza infection, showing greater weight loss and more extensive pulmonary remodeling. Together, our data suggest that CD34 plays an essential role in maintaining vascular integrity in the lung in response to chemical‐ and infection‐induced tissue damage.