Scientific Presentations Summer 2009 - Dana-Farber/Harvard ...

The Role of PTEN and SHIP1 in Efferocytosis of
Apoptotic Neutrophils By Macrophages
Yunwei Sun
Mentors: Hongbo R. Luo, PhD
Scientific Advisor: Subhanjan Mondal, PhD
Children’s Hospital Boston
Neutrophils are the most abundant and shortest-lived cells of the immune system
Substantial daily turnover of neutrophils occurs through spontaneous apoptosis followed
by clearance by macrophages. Defects in the clearance mechanism cause increased
necrotic cells in circulation, which may lead to generation of self antibodies
and may cause autoimmune diseases such as systemic lupus erythmatorus (SLE).
Phosphatidylinositol 3-kinase (PI3K) is important for efferocytosis,the engulfment
of apoptotic cells by phagocytes. Inhibition of PI3K reduces efferocytosis efficiency.
Phosphatidylinositol-3,4,5-triphosphate (PtdIns(3,4,5)P3), a product of PI3K, localizes
in the phagocytic cup during efferocytosis. After engulfment, PtdIns(3,4,5)P3 is
converted to PtdIns(3,4)P2 by phosphatase SHIP1 or to PtdIns(4,5)P2 by PTEN.
The focus of this study is to determine whether PTEN and SHIP1 localize to the
phagocytic cup in macrophagesduring engulfment of neutrophils. This will lead to a
greater understanding of the roles of PTEN/SHIP1 in efferocytosis.
A phagocytosis assay with immunostaining was performed by labeling apoptotic
neutrophils (isolated from mice) with snarf-1 and by transfecting RAW 264.7 macrophages
with GFP conjugated PTEN or SHIP1. Western blots were also used to
analyze PTEN and SHIP1 protein levels.
It was found that PTEN is not enriched in the phagocytic cup and is globally distributed
throughout macrophages. Therefore, it can be inferred that the little amount
of PTEN is enough for the converting PtdIns(3,4,5)P3 to PtdIns(4,5)P2. Ship1 is
found to be enriched at the phagocytic cup indicating that it is involved in the conversion
of PtdIns(3,4,5)P3 to PtdIns(3,4)P2.