The long term goals are to isolate and characterize a unique human chemotactic factor for lymphocytes (LCF); determine how it and other chemotactic factors modulate intracellular events in lymphoctes; and determine if they will recruit lymphocytes and alter growth of tumor cells in vivo.
The first aim i s to isolate and characterize the LCF found in supernatants from Con A stimulated human mononuclear cells. Isolation will be achieved by molecular seive and reversed phase chromatography using HPLC and amino acid and sequence analysis will be performed.
The second aim i s to determined if purified LCF and other lymphocyte chemotactic factors (IL-2 and IL-1) will induce microfilament assembly, Ca++ influx and membrane depolarization in lymphocytes. Responses will be evaluted using specific fluorescent probes and quantitative flow cytometry and will allow us to determine which lymphocyte subsets respond to LCF.
The third aim of the proposal will determine if LCF alters the expression of lymphocyte function associated antigen (LFA1) and if it is involved in the response of lymphocytes to LCF.
The fourth aim i s to determine if purified LCF, IL-2, or IL-1 will recruit lymphocytes in vivo, and if so what phenotypes of lymphocytes respond. These studies will use a mouse model system where a collagen sponge is implanted and serves as a focus for chemotactic factor injection. The sponge can be removed, digested, and infiltrating cells quantitated and analyzed for number and cell phenotype using flow cytometry.
The final aim of this proposal is to determine if chemotactic factors can be used in vivo to specifically recruit leukocytes to an EMT 6 tumor and alter the growth of that tumor. Chemotactic factors will be injected into a collagen sponge with tumor cells. Tumor cells and host cells will be quantitated afer removal of the sponge/tumor complex and analyzed using flow cytometry and specific fluorochromes that discriminate host and tumor cells. In summary, this project will define the characteristics of human LCF and the action of this and other lymphocyte chemotactic factors Ca++ mobilization, membrane depolarization and microfilament assembly. These studies will also determine if LCFs are chemotactic in vivo. LFA1 is involved in T cell chemotaxis and if IL-1, IL-2 and LCF which are chemotactic in vitro are also chemotactic for lymphocytes in vivo. Finally we will determine if chemotactic factors injected locally will modify tumor growth in vivo by modulating the influx of host leukocytes into that tumor.
| Van Epps, D E; Potter, J; Vachula, M et al. (1989) Suppression of human lymphocyte chemotaxis and transendothelial migration by anti-LFA-1 antibody. J Immunol 143:3207-10 |
