Technical Reference #1666

1666.

Focal Adhesion Kinase Regulates Pathogen-Killing Capability and Life Span of Neutrophils via Mediating Both Adhesion-Dependent and Independent Cellular Signals Anongnard Kasorn; Pilar Alcaide; Yonghui Jia; Kulandayan K. Subramanian; Bara Sarraj; Yitang Li; Fabien Loison; Hidenori Hattori;† Leslie E. Silberstein; William F. Luscinskas; and Hongbo R. Luo, Srinakharinwirot University, The Journal of Immunology, 183(1666), (2009)
Link To Paper

Abstract:
Various neutrophil functions such as phagocytosis superoxide production and survival are regulated by integrin signaling. Despite the essential role of focal adhesion kinase (FAK) in mediating this signaling pathway its exact function in neutrophils is ill defined. In this study we investigated the role of FAK in neutrophils using a myeloid-specific conditional FAK knockout mouse. As reported in many other cell types FAK is required for regulation of focal adhesion dynamics when neutrophils adhere to fibronectin or ICAM-1. Adhesion on VCAM-1-coated surfaces and chemotaxis after adhesion were not altered in FAK null neutrophils. In addition we observed significant reduction in NADPH oxidase-mediated superoxide production and complementmediated phagocytosis in FAK null neutrophils. As a result these neutrophils displayed decreased pathogen killing capability both in vitro and in vivo in a mouse peritonitis model. In adherent cells the defects associated with FAK deficiency are likely due to suppression of phosphatidylinositol (345)-trisphosphate (PtdIns(345)P3) signaling and chemoattractant-elicited calcium signaling. Disruption of FAK also reduced chemoattractant-elicited superoxide production in suspended neutrophils in the absence of cell adhesion. This may be solely caused by suppression of PtdIns(345)P3 signaling in these cells because the fMLP-elicited calcium signal was not altered. Consistent with decreased PtdIns(345)P3/Akt signaling in FAK null neutrophils we also observed accelerated spontaneous death in these cells. Taken together our results revealed previously unrecognized roles of FAK in neutrophil function and provided a potential therapeutic target for treatment of a variety of infectious and inflammatory diseases.

Materials & Methods:
Freshly prepared bone marrow-derived wild-type and FAK / mouse neutrophils in RPMI plus 2% FCS were plated onto fibronectin (10 g/ml) precoated glass-bottom dishes (MatTek) and allowed to adhere for 3 min. Cells were then uniformly stimulated with 100 nM fMLP and images were captured from multiple fields every 10 s for 20 min using a 60 objective on an inverted microscope (Olympus IX17).

Microscopic Technique
Video Microscopy, Intravital video microscopy

Cell Type(s)
Neutrophils