(provided by PI): The goal of these studies is to determine the molecular mechanisms by which environmental chemicals impair immune cell development. While many studies demonstrate that environmentally ubiquitous polycyclic aromatic hydrocarbons (PAH) suppress the mature immune system, relatively few have evaluated their effects on the developing immune system. To bridge this gap, we exploited B cell development models to define how PAH affect bone marrow B cells. We demonstrated that PAH suppress B lymphopoiesis by inducing pro-B and pre-B cell apoptosis mediated indirectly through stromal cells constituting the hematopoietic microenvironment. These studies led to the hypothesis that the developing B cell compartment is exquisitely sensitive to environmental chemicals, which inappropriately activate B cell death programs. Our recent studies have begun to elucidate the cascade of signals that executes the B cell death program. The working model built on these studies proposes at least two potentially overlapping pathways, one involving caspase-8 and another involving mitochondrial activation. A combination of unique resources, including apoptosis gene-defective, nontransformed early B cell lines and a stable of knockout mice will be exploited to test and to refine this model.
Three specific aims are proposed: 1) Define PAH-induced, caspase-8-dependent, apoptosis signaling pathways in bone marrow B cells (D. 1): We will: a) determine the role of death receptors in caspase-8 activation, b) evaluate the contribution of JNK, PKR, and ASK1 kinases to caspase-8-dependent apoptosis, and c) identify components of activated caspase-8 complexes by mass spectrometry (MALDI-TOF). 2) Evaluate factors contributing to mitochondria-dependent apoptosis signaling. (D.2): The elements of a PAH-induced mitochondrial apoptosis amplification pathway will be mapped out. Specifically, we will: a) determine the roles of caspases-2 and -8 in mitochondrial activation mediated by Bid, b) assess the contribution of JNK, PKR and p53 in caspase-independent mitochondrial activation, and c) define where NF-kappaB down-regulation, a prerequisite to PAH-induced pro/pre-B cell death, interfaces with caspase signaling. 3) Investigate in vivo correlates of the apoptosis pathway mapped out in vitro. (D.3): To build on results obtained in vitro, we will use gene-knockout mice: a) to determine the relative sensitivities of bone marrow B cell subsets to AhR-dependent apoptosis signaling in vivo and b) to investigate PAH-induced, caspase-dependent death signaling in bone marrow B cell subsets in vivo. These studies will not only contribute greatly to our understanding of the molecular effects of PAH on developing B cells, but they also will validate a platform that is easily applicable to the study of other immunotoxic environmental chemicals and other hematopoietic cell subsets on the molecular level.
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