This is a second reapplication to investigate the type and function of chemoline receptors in the rat has been greatly improved by a narrowed focus and improved probability of success. It has three specific aims which come from prior proposals, but which are smaller in scope. The first specific aims are to define the specificity of the ligand binding to the chemokine receptors CKR2, CKR5 and RBS11.
This aim has two parts. In the first part he will transect into HEK293 cells using the pcDNA3 vector. He will assess the effectiveness of the transfection by assaying for mRNA by Northern blotting. He will determine ligand binding by exposing transfected cells or their membranes to radiolabeled MCP-1. He will determine if the specificity of this binding can be competed with a variety of other CC chomolines; dissociation constants will be determined. He will also do the same for the CKR5 for MIP-1a. In the second part of this aim, he will attempt to define the selectivity for transmembrane signaling of various cytokines through these specific receptors. COS, HEK293, or K562 with the rat chemokine receptors he will study. The effectiveness of signaling will be assessed by determining the arachidonic acid production, intracellular calcium increased, inositol phosphate production and cAMP accumulation [preliminary data shows the ability of the investigator to do this]. If he fails to detect signaling, he will use these cells cotransfected with human ga14 or Ga15 for which effective signaling has been defined in these cell systems. The dose dependent response of the receptors for specific ligands will be established for a number of the chemokine ligands. In the second aim he will test the hypothesis that rat microglial cells express effective chomokine receptors in vitro. In the first part of this aim he will examine microglial responses to chemokines. Control and cytokine (INFg or LPS) stimulated microglial cells will be exposed to 4 CC chemokines and signal transduction determined by the parameters noted above. The mechanism of signaling increased intracellular calcium will be studies. The mechanism of signaling increases intracellular calcium will be studies. Microglia pre-treated with pertussis toxin (to blunt response by pre-ADP-gibosylating the G- protein's alpha subunit) will be studied to determine the source of intracellular calcium accumulating. If signaling fails in a Ca depleted medium, the source must be external. This will permit the investigator to define synergistic effects from the simultaneous binding of multiple chemolines to receptors. In the second part of the aims, the level of expression of chemokine receptors in cultured microglial cells exposed to a variety of cytokines will be studied. The chomokine concentrations most effective on hematopoetic cells will be compared to the dose and time of effect on microglial cells. The change in levels of expression will be determined by northern blotting of whole cell mRNA and/or by RNA protection assay. In the final aim, he will use an in vivo systems. This will test the hypothesis that microglial cells express functional chomokine receptors. This will be done by in situ hybridization on specific CNS tissues following defined manipulations known to activate microglial cells. The systems to be studies are facial nerve crush, or EAE. The cells modulating chemokine receptors will be defined by in situ hybridization while specific cells typed will be delineated by immunocytochemistry on adjacent croystat sections. In situ proves will be directed against specific chemokine receptor mRNAs and the mRNA for the Ga15 molecule. The expression of these in the same cell type following manipulation will be defined.
