Neutrophils play an important role in antibody and cell-mediated host defense mechanisms, inflammation, and may also be involved in the rejection of tumors. Therefore, lymphokine derived neutrophil activators such as leukocyte inhibitory factor (LIF) serve as potentially important regulators of neutrophil function that are capable of augmenting their response to receptor- mediated stimuli. This revised proposal will focus on three central aspects of LIF biochemistry and biology that test the following hypotheses:
Specific Aim #1 - In order to identify whether or not LIF is a unique protein and to obtain large enough quantities of LIF for use in biochemical and biologic studies, we will test the hypothesis that the cDNA which encodes for LIF is obtainable through molecular biologic techniques. This will be accomplished through a collaboration with Dr. Steven Clark's laboratory (Genetics Institute, Cambridge, MA) which has recently developed expression cloning methods for screening cDNA libraries for desired clones by direct expression in Monkey COS-1 cells. Once the appropriate cDNA is isolated, the amino acid sequence will be deduced and recombinant DNA technology applied to the mass production of LIF.
Specific Aim #2 - We will test the hypothesis that LIF is an activator of human neutrophils and exerts its effect through its capacity to enhance neutrophil aggregation and adherence. Until recombinant LIF becomes available, we will utilize highly purified (80,000 fold) LIF that we routinely obtain from PHA-stimulated human lymphocytes for these studies. We will study the effects of LIF on adherence and aggregation by measuring the expression and function of CR1 and CR3 receptors with monoclonal antibodies and phagocytosis assays and superoxide generation; neutrophil aggregation assays; membrane depolarization; and adherence to endothelial cells.
Specific Aim #3 - We will test the hypothesis that the ability of LIF to augment receptor-mediated stimuli involves the generation of a transmembrane/intra-cellular calcium signal. This will be initially studied by measuring both calcium influx and intra- cellular calcium levels with fluorescent dyes (Quin-2/Fura-2), calcium channel blockers and calmodulin antagonists. We will then measure whether the calcium signal is generated by the production of IP3 during phosphatidyl inositol turnover; other related studies will include an indirect examination of the effects of LIF on PLC activity via the N(G) regulatory peptides utilizing the GTP analogue GTP-gamma-S, and whether LIF activates protein kinase C and down regulates cAMP production.
| Joseph, B Z; Sustiel, A M; Borish, L (1992) Neutrophils from asthmatics exhibit diminished responsiveness to 2-chloroadenosine which is reversed by theophylline. Evidence for a cyclic-AMP-independent pathway on human neutrophils. Inflammation 16:101-16 |
