The overall goal of the proposed studies is to define the role(s) of chemokines in the induction of ACAID, a mechanism of immune privilege adapted by the eye to prevent devastating intraocular inflammation. During the first six months of my training I generated data that showed that conditions empowering macrophages to become """"""""ACAID conferring"""""""" result in their production of a unique set of chemokines including MIP-2 and TCA-3. Likewise, I showed that peripheral blood monocytes from ACAID induced mice exhibited a selective and temporal up-regulation of MIP-2, coinciding precisely with the appearance and disappearance of blood borne ACID conferring cells described by others. While MIP-2 is best known as a neutrophil chemokine, my preliminary studies showed that it his highly chemotactic for NKT cells to the spleen, as well as an absolute requirement for NKT cells during ACAID laboratory showing recruitment of NKT cells to the spleen, as well as an absolute requirement for NKT cells during ACAID induction.
The first aim of the proposal will use multi-probe RNAse protection assays and multi-color flow cytometry to clearly define the chemokines produced by macrophages under conditions that induce ACAID and those that do not.
This aim will also use immuno-magnetic bead selection and flow cytometry to identity the exact phenotype of chemokine producing cells.
The second aim will use classic chemotaxis assays to define the migratory responses of NKT cells to ACAID associated chemokines, along with molecular techniques to confirm the expression of chemokine receptors on these cells. Finally, our third aim will explore the mechanisms of chemokine-NKT cell interaction during ACAID induction by reconstituting NKT cell knockout mice with NKT cells from chemokine receptor knockout mice. A local adoptive transfer assay will be used to relate the interaction to the generation of ACAID regulator cells. Finally, the third aim will explore the mechanisms of chemokine-NKT cell interaction during ACAID induction by reconstituting NKT cell knockout mice with NKT cells from chemokine receptor knockout mice. A local adoptive transfer assay will be used to related the interaction to the generation of ACAID regulator cells. We anticipate that these studies will yield new and intriguing information, concerning the contributions of chemokines to the development of ACAID, as well as implying a role for chemokines in immune privilege and immunologic tolerance in general.
| Stein-Streilein, J; Sonoda, K H; Faunce, D et al. (2000) Regulation of adaptive immune responses by innate cells expressing NK markers and antigen-transporting macrophages. J Leukoc Biol 67:488-94 |
