Timothy Bushnell

NF‐κB p50 and p52 Expression Is Not Required for RANK‐Expressing Osteoclast Progenitor Formation but Is Essential for RANK‐ and Cytokine‐Mediated Osteoclastogenesis

Expression of RANKL by stromal cells and of RANK and both NF‐κB p50 and p52 by osteoclast precursors is essential for osteoclast formation. To examine further the role of RANKL, RANK, and NF‐κB signaling in this process, we used NF‐κB p50−/−;p52−/− double knockout (dKO) and wild‐type (WT) mice. Osteoclasts formed in cocultures of WT osteoblasts with splenocytes from WT mice but not from dKO mice, a finding unchanged by addition of RANKL and macrophage colony‐stimulating factor (M‐CSF). NF‐κB dKO splenocytes formed more colony‐forming unit granulocyte macrophage (CFU‐GM) colonies than WT cells, but no osteoclasts were formed from dKO CFU‐GM colonies. RANKL increased the number of CFU‐GM colonies twofold in WT cultures but not in dKO cultures. Fluorescence‐activated cell sorting (FACS) analysis of splenocytes from NF‐κB dKO mice revealed a two‐to threefold increase in the percentage of CD11b (Mac‐1) and RANK double‐positive cells compared with WT controls. Treatment of NF‐κB dKO splenocytes with interleukin (IL)‐1, TNF‐α, M‐CSF, GM‐CSF, and IL‐6 plus soluble IL‐6 receptor did not rescue the osteoclast defect. No increase in apoptosis was observed in cells of the osteoclast lineage in NF‐κB dKO or p50−/−;p52+/− (3/4KO) mice. Thus, NF‐κB p50 and p52 expression is not required for formation of RANK‐expressing osteoclast progenitors but is essential for RANK‐expressing osteoclast precursors to differentiate into TRAP+ osteoclasts in response to RANKL and other osteoclastogenic cytokines.

Multispectral imaging of hematopoietic cells: Where flow meets morphology

Normal and abnormal blood cells are typically analyzed by either histologic or flow cytometric approaches. Histology allows morphological examination of complex visual traits but with relatively limited numbers of cells. Flow cytometry can quantify multiple fluorescent parameters on millions of cells, but lacks morphological or sub-cellular spatial detail. In this review we present how a new flow technology, the ImageStream (Amnis Corporation, Seattle, WA), blends morphology and flow cytometry and can be used to analyze cell populations in ways not possible by standard histology or flow cytometry alone. The ImageStream captures brightfield, darkfield and multiple fluorescent images of individual cells in flow. The images can then be analyzed for levels of fluorescence intensity in multiple ways (i.e. maximum, minimum, or mean) as well as the shape and size of the area of fluorescence. Combinatorial measurements can also be defined to compare levels and spatial associations for multiple fluorescent channels. We demonstrate an application of this technology to distinguish six stages of erythroid maturation which have been classically defined by morphological criteria, by measuring changes in Ter119 mean intensity and area, DNA (DRAQ5 stain) mean intensity and area, and RNA content (thiazole orange stain). Using this approach, we find that other characteristics of erythroid maturation, such as marker expression and nuclear offset, vary appropriately within the defined cell subsets. Finally, we show that additional measurements of cell characteristics not classically analyzed in cytometry, including surface unevenness and unusually high contrast in brightfield images combined with fluorescent markers allow complex discriminations of rare populations of cells.

Ontogeny of erythroid gene expression

Erythroid ontogeny is characterized by overlapping waves of primitive and definitive erythroid lineages that share many morphologic features during terminal maturation but have marked differences in cell size and globin expression. In the present study, we compared global gene expression in primitive, fetal definitive, and adult definitive erythroid cells at morphologically equivalent stages of maturation purified from embryonic, fetal, and adult mice. Surprisingly, most transcriptional complexity in erythroid precursors is already present by the proerythroblast stage. Transcript levels are markedly modulated during terminal erythroid maturation, but housekeeping genes are not preferentially lost. Although primitive and definitive erythroid lineages share a large set of nonhousekeeping genes, annotation of lineage-restricted genes shows that alternate gene usage occurs within shared functional categories, as exemplified by the selective expression of aquaporins 3 and 8 in primitive erythroblasts and aquaporins 1 and 9 in adult definitive erythroblasts. Consistent with the known functions of Aqp3 and Aqp8 as H2O2 transporters, primitive, but not definitive, erythroblasts preferentially accumulate reactive oxygen species after exogenous H2O2 exposure. We have created a user-friendly Web site (http://www.cbil.upenn.edu/ErythronDB) to make these global expression data readily accessible and amenable to complex search strategies by the scientific community.

A Purified Zinc Protease of Pea Chloroplasts, EP1, Degrades the Large Subunit of Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase.

A previously reported endopeptidase (EP1) from pea chloroplasts was purified over 11,000-fold using a four-step protocol involving ultrafiltration, sucrose gradient centrifugation, isoelectric focusing, and high performance liquid chromatography gel filtration. The enzyme was determined to be a metalloprotease requiring bound Zn2+ and added Mg2+ or Ca2+ for proper activity. Its localization in the stroma of pea chloroplasts was confirmed by demonstrating its insensitivity to thermolysin when the envelope was intact. A contaminating serine protease that attacks EP1 was found. The contaminating protease was inhibited by 4-(2-aminoethyl)-benzenesulfonyl fluoride, but not by o-phenanthroline, whereas EP1 sensitivities were the reverse. EP1 is able to hydrolyze the large subunit of native ribulose-1,5-bisphosphate carboxylase/oxygenase under physiological conditions.

Selective expression of CD44, a putative prostate cancer stem cell marker, in neuroendocrine tumor cells of human prostate cancer.

Hormonal therapy is effective for advanced prostate cancer (PC) but the disease often recurs and becomes hormone‐refractory. It is hypothesized that a subpopulation of cancer cells, that is, cancer stem cells (CSCs), survives hormonal therapy and leads to tumor recurrence. CD44 expression was shown to identify tumor cells with CSC features. PC contains secretory type epithelial cells and a minor population of neuroendocrine cells. Neuroendocrine cells do not express androgen receptor and are quiescent, features associated with CSCs. The purpose of the study was to determine the expression of CD44 in human PC and its relationship to neuroendocrine tumor cells.

Aryl Hydrocarbon Receptor Activation during Influenza Virus Infection Unveils a Novel Pathway of IFN-γ Production by Phagocytic Cells

The contribution of environmental factors is important as we consider reasons that underlie differential susceptibility to influenza virus. Aryl hydrocarbon receptor (AhR) activation by the pollutant dioxin during influenza virus infection decreases survival, which correlates with a 4-fold increase in pulmonary IFN-γ levels. We report here that the majority of IFN-γ-producing cells in the lung are neutrophils and macrophages not lymphocytes, and elevated IFN-γ is associated with increased pulmonary inducible NO synthase (iNOS) levels. Moreover, we show that even in the absence of dioxin, infection with influenza virus elicits IFN-γ production by B cells, γδ T cells, CD11c+ cells, macrophages and neutrophils, as well as CD3+ and NK1.1+ cells in the lung. Bone marrow chimeric mice reveal that AhR-mediated events external to hemopoietic cells direct dioxin-enhanced IFN-γ production. We also show that AhR-mediated increases in IFN-γ are dependent upon iNOS, but elevated iNOS in lung epithelial cells is not driven by AhR-dependent signals from bone marrow-derived cells. Thus, the lung contains important targets of AhR regulation, which likely influence a novel iNOS-mediated mechanism that controls IFN-γ production by phagocytic cells. This suggests that AhR activation changes the response of lung parenchymal cells, such that regulatory pathways in the lung are cued to respond inappropriately during infection. These findings also imply that environmental factors may contribute to differential susceptibility to influenza virus and other respiratory pathogens.

LFA-1-Mediated T Cell Costimulation through Increased Localization of TCR/Class II Complexes to the Central Supramolecular Activation Cluster and Exclusion of CD45 from the Immunological Synapse

T cell activation is associated with a dramatic reorganization of cell surface proteins and associated signaling components into discrete subdomains within the immunological synapse in T cell:APC conjugates. However, the signals that direct the localization of these proteins and the functional significance of this organization have not been established. In this study, we have used wild-type and LFA-1-deficient, DO11.10 TCR transgenic T cells to examine the role of LFA-1 in the formation of the immunological synapse. We found that coengagement of LFA-1 is not required for the formation of the central supramolecular activation cluster (cSMAC) region, but does increase the accumulation of TCR/class II complexes within the cSMAC. In addition, LFA-1 is required for the recruitment and localization of talin into the peripheral supramolecular activation cluster region and exclusion of CD45 from the synapse. The ability of LFA-1 to increase the amount of TCR engaged during synapse formation and segregate the phosphatase, CD45, from the synapse suggests that LFA-1 might enhance proximal TCR signaling. To test this, we combined flow cytometry-based cell adhesion and calcium-signaling assays and found that coengagement of LFA-1 significantly increased the magnitude of the intracellular calcium response following Ag presentation. These data support the idea that in addition to its important role on regulating T cell:APC adhesion, coengagement of LFA-1 can enhance T cell signaling, and suggest that this may be accomplished in part through the organization of proteins within the immunological synapse.

Cyclooxygenase-2 independent effects of cyclooxygenase-2 inhibitors on oxidative stress and intracellular glutathione content in normal and malignant human B-cells

We recently reported that inhibition of Cyclooxygenase-2 (Cox-2) reduced human B-CLL proliferation and survival. Herein, we investigated the mechanisms whereby small molecule Cox-2 selective inhibitors, SC-58125 (a Celebrex analog) and CAY10404 blunt survival of human B-cell lymphomas and chronic lymphocytic leukemia B-cells. SC-58125 and OSU03012 (a Celebrex analog that lacks Cox-2 inhibitory activity) both decreased intracellular glutathione (GSH) content in malignant human B-cells, as well as in Cox-2 deficient mouse B-cells. This new finding supports Cox-2 independent effects of SC-58125. Interestingly, SC-58125 also significantly increased B-cell reactive oxygen species (ROS) production, suggesting that ROS are a pathway that reduces malignant cell survival. Addition of GSH ethyl ester protected B lymphomas from the increased mitochondrial membrane permeability and reduced survival induced by SC-58125. Moreover, the SC-58125-mediated GSH depletion resulted in elevated steady-state levels of the glutamate cysteine ligase catalytic subunit mRNA and protein. These new findings of increased ROS and diminished GSH levels following SC-58125 exposure support novel mechanisms whereby a Cox-2 selective inhibitor reduces malignant B-cell survival. These observations also support the concept that certain Cox-2 selective inhibitors may have therapeutic value in combination with other drugs to kill malignant B lineage cells.

The biologic properties of leukemias arising from BCR/ABL-mediated transformation vary as a function of developmental origin and activity of the p19ARF gene

Recent reports have shown that upon expression of appropriate oncogenes, both stem cells and more differentiated progenitor populations can serve as leukemia-initiating cells. These studies suggest that oncogenic mutations subvert normal development and induce reacquisition of stem-like features. However, no study has described how specific mutations influence the ability of differentiating cell subsets to serve as leukemia-initiating cells and if varying such cellular origins confers a functional difference. We have examined the role of the tumor suppressor gene p19(ARF) in a murine model of acute lymphoblastic leukemia and found that loss of p19(ARF) changes the spectrum of cells capable of tumor initiation. With intact p19(ARF), only hematopoietic stem cells (HSCs) can be directly transformed by BCR/ABL expression. In a p19(ARF)-null genetic background expression of the BCR/ABL fusion protein renders functionally defined HSCs, common lymphoid progenitors (CLP), and precursor B-lymphocytes competent to generate leukemia stem cells. Furthermore, we show that leukemias arising from p19(ARF)-null HSC versus pro-B cells differ biologically, including relative response to drug insult. Our observations elucidate a unique mechanism by which heterogeneity arises in tumor populations harboring identical genetic lesions and show that activity of p19(ARF) profoundly influences the nature of tumor-initiating cells during BCR/ABL-mediated leukemogenesis.

Deletion of the mucin-like molecule Muc1 enhances dendritic cell activation in response to toll-like receptor ligands

Dendritic cells (DC) are potent professional antigen-presenting cells that drive primary immune responses to infections or other agonists perceived as ‘dangerous’. Muc1 is the only cell surface mucin or MUC gene product that is expressed in DC. Unlike other members of this glycoprotein family, Muc1 possesses a unique cytosolic region capable of signal transduction and attenuating toll-like receptor (TLR) activation. The expression and function of Muc1 has been intensively investigated on epithelial and tumor cells, but relatively little is known about its function on DC. We hypothesized that Muc1 would influence in vitro generation and primary DC activation in response to the TLR4 and TLR5 ligands lipopolysaccharide and flagellin. Compared with Muc1+/+ DC, we found that Muc1–/– DC were constitutively activated, as determined by higher expression of co-stimulatory molecules (CD40, CD80 and CD86), greater secretion of immunoregulatory cytokines (TNF-α and VEGF), and better stimulation of allogeneic naïve CD4+ T cell proliferation. After activation by either LPS or flagellin and co-culture with allogeneic CD4+ T cells, Muc1–/– DC also induced greater secretion of TNF-α and IFN-γ compared to similarly activated Muc1+/+ DC. Taken together, our results indicate that deletion of Muc1 promotes a heightened functional response of DC in response to TLR4 and TLR5 signaling pathways, and suggests a previously under-appreciated role for Muc1 in regulating innate immune responses of DC.