
	1999 Summer Scholars Program

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Effects of G-CSF Receptor Mutations on the Microbicidal Activity of Murine Neutrophils in vitro

By Alyssa D. Gregory
Mentors: Dr. Daniel C. Link, Dr. Morgan L. McLemore
Department of Pathology, Washington University School of Medicine, St. Louis, MO

In an effort to further understand and characterize the mechanisms and pathways by which neutrophils develop and function, the Link laboratory has developed various mouse models which express a genetic mutation in the granulocyte colony stimulating factor (G-CSF) receptor - a region of proven significance in neutrophil production. G-CSF is a cytokine which, through its interactions with its respective protein receptor on the cell surface of the neutrophil, regulates neutrophil proliferation and differentiation. Experimental evidence also indicates that the G-CSF receptor plays a role in neutrophil function. Mice which express a G-CSF receptor knockout mutation exhibit markedly decreased PMN production. Neutrophil mobilization and chemotaxis are similarly diminished. In order to determine what signals are initiated by specific regions of the receptor, mice have been developed to express a deletion of an intracellular portion of the G-CSF receptor. p1 mice lacking this 95 amino acid sequence have exhibited normal granulopoiesis, mobilization, and chemotaxis. This study served to further characterize the G-CSF receptor knockout, p1, and wild type mice.

Mice were injected with a glycogen solution into the peritoneal cavity in order to elicit an inflammatory response and thus neutrophil migration to the site of injection. After an incubation period neutrophils were harvested by flushing out the peritoneal cavity. Purification gradients were then utilized to obtain a high percentage of neutrophils. The PMNs were incubated for one hour with a mixture of bacteria and autologous mouse serum in order to facilitate opsonisation and bacterial killing. Cells were then lysed to release phagocytosed bacteria. The bacteria and lysed PMN mixture was then spread on agar pour plates and allowed to incubate overnight. Bacterial killing was measured by counting individual colonies of surviving bacteria.

Due to difficulties in perfecting the assay, results of this study were primarily inconclusive. In two separate experimental trials there was a suggestion of diminished microbicidal activity in the knockout neutrophils. The difference, however, is slight and may be of little significance in vivo. The next step is to simply remedy any technical error in an assay which is, in theory, very sound. More data must be collected to draw any conclusions as to a pronounced phenotype in the pl and knockout mice.

Because the G-CSF receptor plays such a significant role in various aspects of the neutrophil response, establishing a firm characterization of its structure and function can have far-reaching implications. For instance, if a region of the receptor is found to induce a proliferative response, the potential for targeting the region to increase white blood cell counts exists. Similarly, stimulating a granulopoiesis suppression region can lower WBC counts, which may offer a promising treatment for leukemia patients. Past studies have suggested, and future studies will further prove, the tremendous significance of the G-CSF receptor in both understanding granulopoiesis and in potential for widespread clinical application.

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