Laura F. Gibson

NGF Is an Essential Survival Factor for Bronchial Epithelial Cells during Respiratory Syncytial Virus Infection

Background Overall expression of neurotrophins in the respiratory tract is upregulated in infants infected by the respiratory syncytial virus (RSV), but it is unclear where (structural vs. inflammatory cells, upper vs. lower airways) and why, these changes occur. We analyzed systematically the expression of neurotrophic factors and receptors following RSV infection of human nasal, tracheal, and bronchial epithelial cells, and tested the hypothesis that neurotrophins work as innate survival factors for infected respiratory epithelia. Methodology Expression of neurotrophic factors (nerve growth factor, NGF; brain-derived neurotrophic factor, BDNF) and receptors (trkA, trkB, p75) was analyzed at the protein level by immunofluorescence and flow cytometry and at the mRNA level by real-time PCR. Targeted siRNA was utilized to blunt NGF expression, and its effect on virus-induced apoptosis/necrosis was evaluated by flow cytometry following annexin V/7-AAD staining. Principal Findings RSV infection was more efficient in cells from more distal (bronchial) vs. more proximal origin. In bronchial cells, RSV infection induced transcript and protein overexpression of NGF and its high-affinity receptor trkA, with concomitant downregulation of the low-affinity p75NTR. In contrast, tracheal cells exhibited an increase in BDNF, trkA and trkB, and nasal cells increased only trkA. RSV-infected bronchial cells transfected with NGF-specific siRNA exhibited decreased trkA and increased p75NTR expression. Furthermore, the survival of bronchial epithelial cells was dramatically decreased when their endogenous NGF supply was depleted prior to RSV infection. Conclusions/Significance RSV infection of the distal airway epithelium, but not of the more proximal sections, results in overexpression of NGF and its trkA receptor, while the other p75NTR receptor is markedly downregulated. This pattern of neurotrophin expression confers protection against virus-induced apoptosis, and its inhibition amplifies programmed cell death in the infected bronchial epithelium. Thus, pharmacologic modulation of NGF expression may offer a promising new approach for management of common respiratory infections.

Bone marrow stromal cells regulate caspase 3 activity in leukemic cells during chemotherapy

The interaction between leukemic cells and stromal cells of the bone marrow microenvironment has been shown to enhance leukemic cell survival during exposure to chemotherapeutic agents. In the current study we investigated whether association of B lineage acute lymphoblastic leukemic cells with human bone marrow stromal cells altered caspase activation during chemotherapy treatment. Following treatment with Ara-C or VP-16 in vitro, caspase 3 activity in leukemic cells was consistently reduced by co-culture of leukemic cells with human bone marrow stromal cell layers. These observations suggest that the protective effect of the bone marrow microenvironment on leukemic cells may be due, in part, to regulation of caspase 3 activity.

Regulation of BAX and BCL‐2 expression in breast cancer cells by chemotherapy

Optimizing chemotherapeutic drug delivery strategies relies, in part, on identification of the most clinically effective sequence, dose, and duration of drug exposure. The combination of dose intensive etoposide (VP‐16) followed by cyclophosphamide has clinical efficacy in the treatment of advanced breast cancer. However, molecular mechanisms that underlie the effectiveness of this combination of chemotherapeutic agents have not been investigated. In this study we investigated regulation of BAX and BCL‐2 expression by VP‐16 and cyclophosphamide as a potential mechanism for the induction of breast cancer cell death induced by this regimen.There was a dose and time dependent increase in BAX expression in the breast cancer cell lines MCF‐7, MDA‐MB‐435S, and MDA‐MB‐A231 following in vitro treatment with 50–100 μM VP‐16. Elevation of BAX protein expression in the presence of VP‐16 alone did not correlate with reduced viability or induction of apoptosis in MCF‐7, MDA‐MB‐435S, or MDA‐MB‐A231. VP‐16 did effectively block the breast cancer cell lines evaluated (MCF‐7 and MDA‐MB‐435S) at G2/M phase of the cell cycle, confirming activity of the drug in vitro. MCF‐7 and MDA‐MB‐435S cells that were pre‐treated with VP‐16 and subsequently exposed to 1.0–12.0 μg/m1 4‐hydroperoxycyclophosphamide (4HC), an active metabolite of cyclophosphamide, had markedly reduced viability when compared to matched controls treated with either VP‐16 or 4HC individually. Consistent with this loss of viability, exposure of all three cell lines to the combination of VP‐16 and 4HC resulted in higher BAX protein levels than those observed following treatment with either single agent. This combination of chemotherapeutic agents also resulted in reduced BCL‐2 expression.These observations suggest that combination chemotherapy may derive its efficacy, in part, through coordinated regulation of specific gene products associated with apoptosis. Characterization of molecular events that underlie susceptibility of specific tumor cells to combination chemotherapeutic regimens may lead to additional improvements in treatment strategies for this disease.

Neurotrophins Regulate Bone Marrow Stromal Cell IL-6 Expression through the MAPK Pathway

Background The hosts response to infection is characterized by altered levels of neurotrophins and an influx of inflammatory cells to sites of injured tissue. Progenitor cells that give rise to the differentiated cellular mediators of inflammation are derived from bone marrow progenitor cells where their development is regulated, in part, by cues from bone marrow stromal cells (BMSC). As such, alteration of BMSC function in response to elevated systemic mediators has the potential to alter their function in biologically relevant ways, including downstream alteration of cytokine production that influences hematopoietic development. Methodology/Principal Findings In the current study we investigated BMSC neurotrophin receptor expression by flow cytometric analysis to determine differences in expression as well as potential to respond to NGF or BDNF. Intracellular signaling subsequent to neurotrophin stimulation of BMSC was analyzed by western blot, microarray analysis, confocal microscopy and real-time PCR. Analysis of BMSC Interleukin-6 (IL-6) expression was completed using ELISA and real-time PCR. Conclusion BMSC established from different individuals had distinct expression profiles of the neurotrophin receptors, TrkA, TrkB, TrkC, and p75NTR. These receptors were functional, demonstrated by an increase in Akt-phosphorylation following BMSC exposure to recombinant NGF or BDNF. Neurotrophin stimulation of BMSC resulted in increased IL-6 gene and protein expression which required activation of ERK and p38 MAPK signaling, but was not mediated by the NFκB pathway. BMSC response to neurotrophins, including the up-regulation of IL-6, may alter their support of hematopoiesis and regulate the availability of inflammatory cells for migration to sites of injury or infection. As such, these studies are relevant to the growing appreciation of the interplay between neurotropic mediators and the regulation of hematopoiesis.

Regulation of Eosinophilopoiesis in a Murine Model of Asthma

Eosinophilic inflammation plays a key role in tissue damage that characterizes asthma. Eosinophils are produced in bone marrow and recent observations in both mice and humans suggest that allergen exposure results in increased output of eosinophils from hemopoietic tissue in individuals with asthma. However, specific mechanisms that alter eosinophilopoiesis in this disease are poorly understood. The current study used a well-characterized murine animal model of asthma to evaluate alterations of eosinophil and eosinophil progenitor cells (CFU-eo) in mice during initial sensitization to allergen and to determine whether observed changes in either cell population were regulated by T lymphocytes. Following the first intranasal installation of OVA, we observed sequential temporal elevation of eosinophils in bone marrow, blood, and lung. In immunocompetent BALB/c mice, elevation of bone marrow eosinophils was accompanied by transient depletion of CFU-eo in that tissue. CFU-eo rebounded to elevated numbers before returning to normal baseline values following intranasal OVA exposure. In T cell-deficient BALB/c nude (BALB/cnu/nu) mice, CFU-eo were markedly elevated following allergen sensitization, in the absence of bone marrow or peripheral blood eosinophilia. These data suggest that eosinophilia of asthma results from alterations in two distinct hemopoietic regulatory mechanisms. Elevation of eosinophil progenitor cells in the bone marrow is T cell independent and likely results from altered bone marrow stromal cell function. Differentiation of eosinophil progenitor cells and phenotypic eosinophilia is T cell dependent and does not occur in athymic nude mice exposed to intranasal allergen.

Respiratory Syncytial Virus Infection in Human Bone Marrow Stromal Cells

Respiratory syncytial virus (RSV) is the most common respiratory pathogen in infants and young children. The pathophysiology of this infection in the respiratory system has been studied extensively, but little is known about its consequences in other systems. We studied whether RSV infects human bone marrow stromal cells (BMSCs) in vitro and in vivo, and investigated whether and how this infection affects BMSC structure and hematopoietic support function. Primary human BMSCs were infected in vitro with recombinant RSV expressing green fluorescent protein. In addition, RNA from naive BMSCs was amplified by PCR, and the products were sequenced to confirm homology with the RSV genome. The BMSC cytoskeleton was visualized by immunostaining for actin. Finally, we analyzed infected BMSCs for the expression of multiple cytokines and chemokines, evaluated their hematopoietic support capacity, and measured their chemotactic activity for both lymphoid and myeloid cells. We found that BMSCs support RSV replication in vitro with efficiency that varies among cell lines derived from different donors; furthermore, RNA sequences homologous to the RSV genome were found in naive primary human BMSCs. RSV infection disrupted cytoskeletal actin microfilaments, altered cytokine/chemokine expression patterns, decreased the ability of BMSCs to support B cell maturation, and modulated local chemotaxis. Our data indicate that RSV infects human BMSCs in vitro, and this infection has important structural and functional consequences that might affect hematopoietic and immune functions. Furthermore, we have amplified viral RNA from naive primary BMSCs, suggesting that in vivo these cells provide RSV with an extrapulmonary target.

Regulation of lymphoid and myeloid leukemic cell survival: Role of stromal cell adhesion molecules

Several laboratories have documented the necessity for direct contact of lymphoid and myeloid leukemic cells with bone marrow stromal cells for optimal survival. Subsequent studies have identified various stromal cell adhesion molecules and soluble factors that facilitate survival through leukemic cell anti-apoptotic signal transduction pathways. This report provides an overview of enhanced leukemic cell survival through adhesive interactions with bone marrow expressed molecules. In addition, we describe the establishment of cloned murine stromal cell lines engineered to constitutively express human VCAM-1 protein on their surface. These stromal cell lines will be useful in studies aimed at better understanding the specific contribution of VCAM-1 : VLA-4 signaling in maintenance of residual leukemic disease.

VEGF-induced phosphorylation of Bcl-2 influences B lineage leukemic cell response to apoptotic stimuli

Post-translational modification of Bcl-2 protein has been described in a variety of cell models with effects varying from enhanced to abrogated function. In this study, we demonstrated that Bcl-2 was constitutively phosphorylated in several hematopoietic tumor cell lines and in primary ALL cells. Increased phosphorylation of Bcl-2 protein in the JM1 ALL cell line, achieved by expression of the phosphomimetic Bcl-2 construct S70E, enhanced JM1 cell chemoresistance. In contrast, initiation of JM1 cell apoptosis was coincident with dephosphorylation of Bcl-2 and elevated protein phosphatase 2A activity. S70E expression also diminished tBid-mediated cytochrome c release and blunted chemotherapy-induced activation of caspases-9 and -3 in JM1 cells. To determine whether soluble factors produced by stromal cells in the bone marrow influence phosphorylation of Bcl-2 protein, a panel of recombinant cytokines was evaluated. Of those tested, vascular endothelial growth factor (VEGF) induced phosphorylation of Bcl-2 protein and blunted cytochrome c release during chemotherapy or tBid treatment of ALL cells. In contrast, JM1 cells transfected with S70A, resulting in expression of Bcl-2 protein that cannot be phosphorylated, were not efficiently rescued from apoptosis by VEGF. These observations suggest that optimal protection of leukemic cells by VEGF may require activation of a pathway that includes Bcl-2 phosphorylation.

Three-Dimensional Microfluidic Tri-Culture Model of the Bone Marrow Microenvironment for Study of Acute Lymphoblastic Leukemia

Acute lymphoblastic leukemia (ALL) initiates and progresses in the bone marrow, and as such, the marrow microenvironment is a critical regulatory component in development of this cancer. However, ALL studies were conducted mainly on flat plastic substrates, which do not recapitulate the characteristics of marrow microenvironments. To study ALL in a model of in vivo relevance, we have engineered a 3-D microfluidic cell culture platform. Biologically relevant populations of primary human bone marrow stromal cells, osteoblasts and human leukemic cells representative of an aggressive phenotype were encapsulated in 3-D collagen matrix as the minimal constituents and cultured in a microfluidic platform. The matrix stiffness and fluidic shear stress were controlled in a physiological range. The 3-D microfluidic as well as 3-D static models demonstrated coordinated cell-cell interactions between these cell types compared to the compaction of the 2-D static model. Tumor cell viability in response to an antimetabolite chemotherapeutic agent, cytarabine in tumor cells alone and tri-culture models for 2-D static, 3-D static and 3-D microfluidic models were compared. The present study showed decreased chemotherapeutic drug sensitivity of leukemic cells in 3-D tri-culture models from the 2-D models. The results indicate that the bone marrow microenvironment plays a protective role in tumor cell survival during drug treatment. The engineered 3-D microfluidic tri-culture model enables systematic investigation of effects of cell-cell and cell-matrix interactions on cancer progression and therapeutic intervention in a controllable manner, thus improving our limited comprehension of the role of microenvironmental signals in cancer biology.

Activation of Transforming Growth Factor-β1/p38/Smad3 Signaling in Stromal Cells Requires Reactive Oxygen Species–Mediated MMP-2 Activity During Bone Marrow Damage

Dose‐escalated chemotherapy has proven utility in a variety of treatment settings, including preparative regimens before bone marrow or hematopoietic stem cell transplantation. However, the potential damage imposed by aggressive regimens on the marrow microenvironment warrants further investigation. In the present study, we tested the hypothesis that dose‐escalated chemotherapy, with etoposide as a model chemotherapeutic agent, activates the transforming growth factor beta‐1 (TGF‐β1) signaling pathway in bone marrow stromal cells. After high‐dose etoposide exposure in vitro, Smad3 protein was phosphorylated in a time‐and dose‐dependent manner in marrow‐derived stromal cells, coincident with the release of active and latent TGF‐β1 from the extracellular matrix. Phosphorylation was modulated by p38 kinase, with translocation of Smad3 from the cytoplasm to the nucleus subsequent to its phosphorylation. Etoposide induced activation of TGF‐β1 followed the generation of reactive oxygen species and required matrix metalloproteinase‐2 (MMP‐2) protein availability. Chemotherapy effects were diminished in MMP‐2−/− knockout stromal cells and TGF‐β1 knockdown small interfering RNA–transfected stromal cells, in which phosphorylation of Smad3 was negligible after etoposide exposure. Stable transfection of a human MMP‐2 cDNA into bone marrow stromal cells resulted in elevated phosphorylation of Smad3 during chemotherapy. These data suggest TGF‐β1/p38/Smad3 signaling cascades are activated in bone marrow stromal cells after dose‐escalated chemotherapy and may contribute to chemotherapy‐induced alterations of the marrow microenvironment.