Neutrophils Promote Hematopoiesis Via a Novel Mechanism Involving Leucine Rich α-2 Glycoprotein (LRG)

Leucine-rich α2 glycoprotein (LRG), the founding member of the leucine-rich repeat superfamily of proteins, was initially identified in serum more than 30 years ago, but its biologic function has remained elusive. A role for LRG has been implicated in inflammation and angiogenesis. Our laboratory previously identified cDNA and genomic clones for human and murine LRG, and showed that ectopically expressed LRG localizes to the granule compartment in transfected myeloid cell lines and promotes their granulocytic differentiation. We also demonstrated that expression of LRG is transcriptionally regulated during neutrophil granulocyte differentiation in a manner similar to that reported for genes encoding the different subsets of neutrophil granule proteins. The presence of LRG in primary neutrophils and a role for LRG in hematopoiesis, however, have not been previously described. Based on our prior studies in transfected myeloid cell lines, we considered the tantalizing possibility that LRG is a novel neutrophil granule protein that is secreted extracellularly upon neutrophil activation to modulate hematopoiesis. To investigate this, we examined LRG in primary human neutrophils isolated from healthy volunteers. Immunoblot analysis of whole cell lysates from neutrophils (97% purity) identified a higher molecular weight LRG species in neutrophils (62 kDa) compared to serum (50 kDa); our data demonstrate the difference in apparent molecular weight is due to differential glycosylation. Immunofluorescence microscopy using antibodies to human LRG and antibodies to the neutrophil granule proteins myeloperoxidase (MPO), lactoferrin (LF), and matrix metalloproteinase 9 (MMP-9, also known as gelatinase), along with fluorescently-labeled secondary antibodies, demonstrated the presence of LRG in the cytoplasm of neutrophils in a compartment corresponding to LF. ELISA and immunoblot analyses of subcellular fractions from isolated neutrophils prepared by nitrogen cavitation demonstrated the presence of LRG in LF-containing fractions as well as some MMP-9-containing fractions, consistent with localization of LRG to the secondary/tertiary granule compartment. Neutrophil exocytosis assays using ionomycin, phorbol-12-myristate 13-acetate, and f-Met-Leu-Phe as stimulants also indicated that LRG is co-released with LF and MMP9, but not with MPO. Notably, LRG secreted from activated neutrophils could bind cytochrome c as reported for LRG purified from serum. Recent reports that LRG can also bind to the TGFβR1 receptor on endothelial cells prompted us to investigate the effects of LRG on TGFβ signaling in hematopoietic cells. LRG significantly antagonized the inhibitory effect of TGFβ on HL-60 cell proliferation (n=3; p<0.05) and also on colony growth of human hematopoietic progenitor cells. When LRG was added to hematopoietic progenitor cells cultured in TGFβ-containing Methocult (SF H4436, serum free), a 50% increase in CFU-GMs was observed. Collectively, these data suggest a novel mechanism whereby neutrophils modulate hematopoiesis in the microenvironment via extracellular release of LRG, and invoke an additional role for neutrophils in innate immunity that has not previously been reported