Regulation of LFA-1 levels in monocytic and lymphoid leukemia cell lines and toxic mediated cell death pathways by Leukothera™ (Leukotoxin)

Aggregatibacter actinomycetemcomitans protein toxin, Leukotoxin (LtxA), is being developed as a therapeutic agent for the treatment of white blood cells (WBCs) malignances and autoimmune/inflammatory diseases. LtxA targets specifically the β2 integrin, lymphocyte function antigen-1 (LFA-1) on white blood cells (WBCs) and causes cell death. Leukotoxin kills monocytes by two mechanisms involving caspases and lysosomes but lymphocytes die via apoptosis prior to activation of a lysosomal pathway. It is known that monocytic leukemia cell lines like THP-1 cells have high LFA-1 levels and lymphoid leukemia cell lines like Jurkat and RL cell lines have naturally low LFA-1 levels. Since LFA-1 is the receptor for LtxA we want to uncover if the cell death pathway that cells undergo after the toxin administration is related with the levels of LFA-1 in the cell surface or the nature of the cell line itself.   To achieve our aims, our methods are directed to manipulate LFA-1 levels, exposing the cells to LtxA and evaluate if there is a change in the cell death pathway. In order to do this, Jurkat and RL cells were incubated with Interlukin-4 (IL-4), a cytokine that is known to up regulate LFA-1 expression in WBCs. Flow cytometry analysis demonstrated a higher death rate for the IL-4 treated cells. For THP-1 cells we were able to block LFA-1 sites with an antibody specific for CD11a/CD118 to simulate a lower LFA-1 expression. Our next steps would be the toxin administration to the THP-1 LFA-1 blocked cells.