Leucine Rich α-2 Glycoprotein: A Novel Neutrophil Granule Protein and Modulator of Myelopoiesis.

Jacob T. Emerson,Belinda R. Avalos,Sarah A. Baxter,Amanda Lance,Jonathan M. Gerber,Lawrence J. Druhan,Andrea E. Price,Arun Rishi,Shimena Li

doi:10.1371/journal.pone.0170261

Abstract

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.

Highlights

Neutrophils are traditionally viewed as the “first responders” of the innate immune system, owing to their intrinsic capacity to eliminate pathogenic organisms
We previously reported that transcription of LRG1 is upregulated during neutrophilic granulocyte differentiation of murine 32Dcl3G cells [9]
To determine whether the observed increase in LRG1 transcription during neutrophil differentiation is accompanied by an increase in protein expression that is physiologically relevant in human cells, lysates from HL-60 cells induced to differentiate in response to all trans-retinoic acid (ATRA) as well as lysates from neutrophils from healthy human volunteer donors were subjected to immunoblot analysis for LRG1

Summary

Introduction

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.

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Results

Conclusion