Date Published: January 12, 2017
Publisher: Public Library of Science
Author(s): Lawrence J. Druhan, Amanda Lance, Shimena Li, Andrea E. Price, Jacob T. Emerson, Sarah A. Baxter, Jonathan M. Gerber, Belinda R. Avalos, Arun Rishi.
Leucine-rich α2 glycoprotein (LRG1), a serum protein produced by hepatocytes, has been implicated in angiogenesis and tumor promotion. Our laboratory previously reported the expression of LRG1 in murine myeloid cell lines undergoing neutrophilic granulocyte differentiation. However, the presence of LRG1 in primary human neutrophils and a role for LRG1 in regulation of hematopoiesis have not been previously described. Here we show that LRG1 is packaged into the granule compartment of human neutrophils and secreted upon neutrophil activation to modulate the microenvironment. Using immunofluorescence microscopy and direct biochemical measurements, we demonstrate that LRG1 is present in the peroxidase-negative granules of human neutrophils. Exocytosis assays indicate that LRG1 is differentially glycosylated in neutrophils, and co-released with the secondary granule protein lactoferrin. Like LRG1 purified from human serum, LRG1 secreted from activated neutrophils also binds cytochrome c. We also show that LRG1 antagonizes the inhibitory effects of TGFβ1 on colony growth of human CD34+ cells and myeloid progenitors. Collectively, these data invoke an additional role for neutrophils in innate immunity that has not previously been reported, and suggest a novel mechanism whereby neutrophils may modulate the microenvironment via extracellular release of LRG1.
Neutrophils are traditionally viewed as the “first responders” of the innate immune system, owing to their intrinsic capacity to eliminate pathogenic organisms. An array of proteins reside within neutrophils that are packaged into cytoplasmic granules and released at sites of infection to help eliminate foreign pathogens. Four distinct subsets of neutrophil granules have been identified: primary, secondary, and tertiary granules, and secretory vesicles. Protein packaging into the various granule subsets equips neutrophils for rapid and differential release of high concentrations of proteins following neutrophil activation. Tertiary granules are released during neutrophil extravascularization, while primary and secondary granules are exocytosed upon entry of neutrophils into the extravascular space. Although a predominant antimicrobicidal function has been ascribed to neutrophil granule proteins, recent evidence suggests that neutrophil granule proteins can also modulate the microenvironment.
Previous work by our laboratory to identify differentially regulated genes expressed during neutrophilic granulocyte differentiation led to identification of the genes for murine and human LRG1 . Localization of the human gene for LRG1 by our group to a region on chromosome 19 where the genes for multiple neutrophil granule proteins also map prompted us to postulate that LRG1 might be a neutrophil granule protein. Using murine myeloid cell lines transfected with a tagged LRG1 cDNA construct, LRG1 was found to be packaged into the primary granules of differentiating cells . A drawback of these studies was the use of an overexpression system, which can lead to inappropriate targeting of proteins to subcellular compartments that differ from their native counterparts in primary cells. We therefore sought to characterize LRG1 in its native state in unmanipulated primary cells. Additionally, since our earlier studies were done in murine cells, we were interested in examining LRG1 in human cells. In the present study, we provide the first evidence that LRG1 is a genuine human neutrophil granule protein that is released upon neutrophil activation, and exhibits distinct physical and biological properties.