The overall objective is to elucidate the role of tyrosine protein kinase (TPK) pathways in the induction of IgE synthesis by CD40 and IL-6 receptors and the mechanism of the effects of aromatic hydrocarbons (AH's) on TPK's in B-lymphocytes. B-lymphocyte IgE production depends on the delivery of at least three signals: Signal 1 is provided by IL-4 and permits germline transcription from the epsilon locus. Signal 2 is delivered by CD40 and induces switch recombination and production of mature epsilon transcripts. Signal 3 regulates expression of the switch epsilon gene and is delivered by IL-6R. While both CD40 and IL-6R activate TPK and ser/thr kinase activities, details about the participating kinases and their integration into a putative kinase cascade are unknown. We hypothesize that both cascades use src-like TPK's to activate downstream serine/threonine kinases (MAP kinase, ribosomal S6 kinases, c-raf,) which are involved in different aspects of B-cell differentiation and IgE synthesis. Inasmuch as AH's promote TPK and p21ras activation in B-cells, we propose that this may constitute a mechanism by which diesel exhaust particles (DEP) and tobacco smoke, which are rich in AH's, act as adjuvants in IgE production. We will employ DEP extracts and prototype AH's, benzo(a)pyrine, and 2,3,7,8- tetrachlorodibenzo-p-dioxin, to study their effect on B-cells.
Our first aim i s to study signaling by the IL-6R with specific emphasis on the role of ras and src-like protein kinases in B-cell differentiation and the mechanism of the effect of AH's on these second messengers.
The second aim i s to study signaling by the CD40 receptor with specific emphasis on the role of TPK's linked to a ser/thr kinase cascade and the mechanism of the effect of HA's on these signaling molecules. In order to accomplish these studies, we will use B-cell lines as well as normal human B-cells for in vitro activation with r.IL-6 and anti-CD40 mAB. Biochemical approaches to determine p21ras activation (GTP uptake, effect of dominant negative ras transfections), src-family kinase activation (specific enzyme and substrate identification by immune complex kinase assays and antiphosphotyrosine immunoblotting), and ser/thr kinase activation (MAP kinase, RSK, c-raf kinase assays) will be conducted to delineate the CD40 and IL-6R signaling cascade. Modulation of these activation components by AH's and DEP extracts will be studied in cells as well as mice strains which differ in the abundance of the intracellular Ah-receptor which mediates the effect of AH's. We will also attempt to disrupt IgE production by inhibitors of the signaling pathways (TPK inhibitors, rapamycin). Our long-term goals are to understand the adjuvant effect of environmental pollutants on allergic inflammation as well as defining critical biochemical target areas by which to modify IgE production in B-cells.
