Debra Piktel

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.

VE-cadherin Regulates Philadelphia Chromosome Positive Acute Lymphoblastic Leukemia Sensitivity to Apoptosis

The mechanisms by which the bone marrow microenvironment regulates tumor cell survival are diverse. This study describes the novel observation that in addition to Philadelphia chromosome positive (Ph+) acute lymphoblastic leukemia (ALL) cell lines, primary patient cells also express Hypoxia Inducible Factor-2α (HIF-2α) and Vascular Endothelial Cadherin (VE-cadherin), which are regulated by Abl kinase. Tumor expression of the classical endothelial protein, VE-cadherin, has been associated with aggressive phenotype and poor prognosis in other models, but has not been investigated in hematopoietic malignancies. Targeted knockdown of VE-cadherin rendered Ph+ ALL cells more susceptible to chemotherapy, even in the presence of bone marrow stromal cell (BMSC) derived survival cues. Pre-treatment of Ph+ ALL cells with ADH100191, a VE-cadherin antagonist, resulted in increased apoptosis during in vitro chemotherapy exposure. Consistent with a role for VE-cadherin in modulation of leukemia cell viability, lentiviral-mediated expression of VE-cadherin in Ph− ALL cells resulted in increased resistance to treatment-induced apoptosis. These observations suggest a novel role for VE-cadherin in modulation of chemoresistance in Ph+ ALL.

Hematopoiesis: The Role of Stromal Integrin Interactions in Pro-B Cell Proliferation.

Recent studies using long term bone marrow cultures have concluded that adherence of lymphoid precursors to the underlying stromal cells is required for normal B cell development. However, the function of specific integrin interactions in B cell development remains unresolved. In our laboratory, we observed that maximal proliferation of pro-B cells required the presence of stromal cells and that stromal cytokines alone could not replace the requirement for stromal cell contact. For that reason, we questioned whether integrin interactions play a role in regulating pro-B cell proliferation in the bone marrow. Murine pro-B cell line C1.92 expressed VLA-4, CD44, and fibronectin-receptor. Abrogation of binding of these molecules to stromal cell ligands using blocking antibodies resulted in failure of pro-B cell adherence and significant decreases in pro-B cell proliferation. Disruption of single integrin interactions did not compromise either adhesion of pro-B cells to stromal cells or IL-7 stimulated proliferation. Taken together, these data suggest that normal pro-B cells interact with stromal cells through multiple integrin interactions and that integrin mediated potentiation of pro-B cell proliferation is functionally redundant and not affected by failure of single ligand interactions.

Asthma progression to airway remodeling and bone marrow eosinophil responses in genetically distinct strains of mice

BACKGROUND Patient factors that cause long-term airway remodeling are largely unidentified. This suggests that genetic differences may determine which asthmatic patients develop airway remodeling. A murine model with repeated allergen exposure leading to peribronchial fibrosis in complement factor 5 (C5)-deficient A/J mice has been used to study asthma progression. No studies have addressed the systemic effects of allergen sensitization or chronic allergen exposure on bone marrow eosinophilopoiesis in this mouse strain. OBJECTIVE To investigate bone marrow eosinophil responses during acute sensitization and chronic allergen exposure using genetically distinct mouse strains differing in persistent airway reactivity and remodeling. METHODS The C5-sufficient BALB/c and C5-deficient A/J mice were repetitively exposed to intranasal ovalbumin for 12 weeks. Subsequently, the mice were evaluated for airway eosinophilia, mucus-containing goblet cells, and peribronchial fibrosis. Both strains of mice were also acutely sensitized to ovalbumin. Bone marrow eosinophil progenitor cells and mature eosinophils were enumerated. RESULTS BALB/c and A/J mice have similar bone marrow responses after acute allergen exposure, with elevations in bone marrow eosinophil progenitor cell and eosinophil numbers. After chronic allergen exposure, only C5-deficient A/J mice that developed peribronchial fibrosis exhibited bone marrow eosinophilia. BALB/c mice lacked peribronchial fibrosis and extinguished accelerated eosinophil production after long-term allergen challenge. CONCLUSIONS Chronic airway remodeling after repeated allergen exposure in genetically different mice correlated with differences in long-term bone marrow eosinophilopoiesis. Preventing asthma from progressing to chronic airway remodeling with fibrosis may involve identifying genetically determined influences on bone marrow responses to chronic allergen exposure.

Abstract 4793: A novel laser ablation electrospray ionization mass spectrometry (LAESI-MS) platform for biomarker discovery in cancer cells

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Introductory Sentence: LAESI is a novel technique for the direct and rapid interrogation of cancer cells by mass spectrometry (MS) that can be used to identify potential biomarkers in live cell populations. Experimental: Presented in this work, the human leukemic cell line, REH, was cultured under 3 different conditions to represent clinically relevant scenarios. These included media alone or co-culture with human bone marrow or osteoblast cells to represent conditions in which leukemic disease originates and environments in which the therapeutic response of tumors is known to differ. In previous work, REH cells co-cultured with stromal cells have shown increased resistance to chemotherapeutic treatments prompting our interest in evaluating differences in tumor cells exposed to bone marrow microenvironment conditions compared to those deprived of these survival cues. Subsequently, these unique signatures may identify therapeutic targets of interest. To identify potential therapeutic targets, the cells were pelleted using centrifugation, washed twice, and pipetted upon slides for direct LAESI-MS analysis. The LAESI-MS analysis was performed using a Protea DP-1000 LAESI system interfaced to a Waters Synapt G2 mass spectrometer. The mid-IR laser in the DP-1000 had a maximum output energy of 1 mJ, a 200 µm spot size, and an operational frequency of 10 Hz. The MS was continually calibrated using a lockmass of m/z 445.1206 resulting in mass differences typically less than 5 ppm. After LAESI-MS analysis, the data was analyzed using MarkerLynx software and subjected to a principle component analysis (PCA) and orthogonal projections to latent structures discriminant analysis (OPLS-DA) for identification of potential biomarkers from each treatment. Five replicates of each cell population were analyzed for statistical significance and potential biomarkers were searched against online databases based on accurate mass and confirmed using MS/MS where possible. Summary of Data: The LAESI-MS analysis of the REH cell pellets produced several notable differences in the cell populations that could aid in the identification of biomarkers responsible for the increased resistance to chemotherapeutic treatments. The LAESI-MS analysis was very rapid, requiring less than five minutes of analysis time per cell population to get an average “fingerprint” mass spectrum in addition to MS/MS spectra to aid in metabolite verification. Conclusions: LAESI-MS is a rapid analysis technique that can be used for cancer research as applied here to cell populations. A LAESI-MS analysis requires minimal sample pretreatment with simple washing and pelleting of the cells being the only requirement for the analysis. This method allows the direct analysis of live cells, which can ultimately lead to the determination of biomarkers of disease through the comparison of different cell populations. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4793. doi:1538-7445.AM2012-4793

Abstract 1525: Regulation of bone marrow angiogenesis by CXCL12 in the leukemia tumor microenvironment

While it is well established that angiogenesis is essential for the progression of solid tumors, its role in hematological tumors is more recently becoming fully appreciated. In the current study we have investigated the potential role of leukemic cells in regulating local angiogenesis as well as the modulation of tumor cell CXCL12 expression subsequent to interaction with bone marrow derived osteoblasts as one mechanism by which local vascular rich niches may be created. Human REH acute lymphoblastic leukemia (ALL; ATCC #CRL-8286) increased vessel density when placed on the chorioallantoic membrane (CAM) assay. Conditioned media from these same tumor cells increased human brain microvessel endothelial cell (HBMEC) motility and tube formation suggesting leukemia cell influence on surrounding vasculature. Strikingly, while REH cells express negligible CXCL12 at baseline, the chemokine is consistently induced to easily detectable levels upon co-culture with human osteoblasts indicating one mechanism by which tumor cells within the bone marrow microenvironment may be poised to enhance vascular cell recruitment even more robustly than leukemic cells alone. Preliminary evaluation of primary, patient-derived ALL leukophoresis samples demonstrated a comparable increase in tumor cell CXCL12 indicating this modulation is not limited to an established tumor cell line. Increased expression of CXCL12 by REH leukemic cells was consistent with enhanced chemotaxis of HUVECs toward the co-culture of tumor and osteoblasts when compared to either cell type alone. The inclusion of 5-Aza-deoxycytidine (Aza-C) to REH cells alone resulted in expression of CXCL12 in the absence of osteoblast co-culture, suggesting methylation may play a regulatory role in the suppression of this chemokine. As a site in which metastatic disease is often refractory to standard therapy, in addition to the anatomical niche in which hematopoietic malignancies initiate and most frequently relapse, regulation of local angiogenesis in the bone marrow microenvironment requires further investigation. Our preliminary observations suggest CXCL12 may play an important role in regulating leukemic cell niche angiogenesis, and targeting it could potentially represent a novel application of antagonists currently in clinical trials in an effort to treat refractory leukemia. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1525. doi:1538-7445.AM2012-1525

Abstract 3961: Chemotherapy treatment induces an Interleukin-6 deficit in the bone marrow microenvironment

Bone marrow stromal cells (BMSC) are a critical component of the microenvironment that supports hematopoiesis, and hematopoietic recovery following bone marrow transplantation. Aggressive chemotherapy not only affects tumor cells, but also influences structural and functional components of the microenvironment, including BMSC and osteoblasts. Successful stem cell or bone marrow transplantation following immuno-suppressive or myeloablative chemotherapy is dependent on the ability of BMSC, and other cellular components of the microenvironment, to maintain their functionality. This includes secretion of soluble factors and expression of cellular adhesion molecules that are critical for the survival, proliferation, and differentiation of immature progenitor cells. In the current study, we have investigated the effects of chemotherapy treatment on BMSC and human osteoblast (HOB) expression of Interleukin-6 (IL-6). IL-6 is a pleiotrophic cytokine which has diverse effects on hematopoietic cell development. The treatment of BMSC or HOB with Melphalan or VP-16, which are clinically relevant drugs used in pre-transplant regimens, led to decreased IL-6 protein expression. This decrease in IL-6 protein is unique to Melphalan, as treatment with other chemotherapeutic agents does not result in a similar decrease in IL-6 protein. We have also observed a decrease in gp130, the transmembrane protein necessary for IL-6 signaling, in response to Melphalan treatment, but not VP-16 treatment. Additionally, pre-treatment of BMSC and HOB with Z-VAD-FMK or Leupeptin, a broad range caspase inhibitor and lysosomal inhibitor respectively, prior to chemotherapy treatment was not able to blunt the decrease in IL-6 protein detected in cell supernatants. These data suggest chemotherapy is not enhancing the degradation of IL-6 protein through caspase or lysosomal mediated mechanisms, but rather blunting the overall expression of IL-6. Melphalan treatment is also able to decrease IL-6 mRNA and further investigation is necessary to determine the mechanism by which chemotherapy affects transcription of IL-6. Collectively, these observations suggest that chemotherapy treatment induced alteration of bone marrow microenvironment function, which results in decreased or defective hematopoietic support of human embryonic stem cells and early progenitor cells may result, in part, from an IL-6 deficit. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3961.