Fig 1.
LRG1 is expressed during neutrophilic granulocyte differentiation and stored in lactoferrin (LF)-containing granules.
(A) HL-60 cells were induced to differentiate with ATRA (1 uM) for 6 days, and soluble proteins extracted from cells harvested at days 0, 1, 3, and 6. Samples were subjected to immunoblot analysis to detect the presence of LRG1. Pure LRG1 in lane 1 is LRG1 purified from human serum included as a positive control. Proteins isolated from unstimulated human neutrophils (PMN lysate) are shown in the last lane. (B) Confocal immunofluorescence microscopy of purified human neutrophils incubated overnight with mouse anti-LRG1 (Abnova H00116844-M01, 1:100 dilution) and goat anti-LF (Santa Cruz sc-14431, 1:250 dilution), followed by donkey anti-mouse Alexafluor 488 (green, 1:500 dilution) and donkey anti-goat Alexafluor 594 (red, 1:2000 dilution) secondary antibodies. DAPI was used to stain nuclei (blue). LRG1 is visualized as green and LF as red. The yellow/orange color in the overlay panel is the result of green and red signals merging, indicating co-localization of LRG1 (green) with LF (red). Focal areas of non-merged green and red signals can also be seen, suggesting that LRG1 may also localize to non-LF-containing compartments. The data shown are representative of 3 independent experiments.
Fig 2.
LRG1 predominantly localizes to the secondary granule compartment of neutrophils.
(A) Neutrophils were isolated from the peripheral blood of healthy donors as described, lysed by nitrogen cavitation, and the subcellular components separated by Percoll density centrifugation. The fractions are numbered 1 through 30, with fraction 1 corresponding to the most dense fraction and fraction 30 corresponding to the least dense fraction. Fractions were analyzed by ELISA for the presence of LRG1, MPO (primary granule marker), LF (secondary granule marker), and MMP9 (tertiary granule marker), and the results plotted as a function of dentisty. (B) Immunoblot analyses of fractions 1 through 21 from the same experiment presented in panel A, showing peak concentrations for MPO, LF, and MMP9 in fractions 2, 10, and 17, respectively, identical to the fractions containing the highest concentration of the indicated proteins as detected by ELISA shown in panel A. The peak concentration of LRG1 is detected in fraction 10, which corresponds to the peroxidase-negative LF-containing secondary granule compartment, consitant with the ELISA data presented in Panel A. The data shown are representative of 5 independent experiments.
Fig 3.
Activated neutrophils release LRG1.
Human neutrophils isolated from healthy donors were pre-incubated in HBSS containing calcium and magnesium at 37°C for 5 minutes, then incubated for 20 minutes at 37°C with the indicated concentrations of calcium ionophore (CaI), PMA, fMLP, or no stimulant (0, negative control). The supernatants were collected, and immunoblot and ELISA analyses performed to detect and measure the amount of granule proteins released. (A) Immunoblot analysis of each condition from a representative experiment using antibodies for myeloperoxidase (MPO), lactoferrin (LF), and gelatinase (MMP9), as markers for primary, secondary, and tertiary granule release, respectively. The pattern of stimulation for release of LRG1 is most consistent with its release from LF-containing secondary granules. The data shown are representative of three independent experiments, each with a different normal donor. (B) ELISA analyses demonstrate release of LRG1 from neutrophils following stimulation with Cal or PMA but not with fMLP, a pattern consistent with the release of LF from secondary granules. The mean of three independent experiments is shown and error bars represent standard error (n = 3).
Fig 4.
Neutrophil-derived LRG1 is differentially glycosylated but retains affinity for cytochrome c.
Neutrophil (PMN) lysates from healthy volunteers were prepared as described. PMN releasates were prepared by exposing isolated neutrophils to calcium ionophore and the resultant supernatants concentrated by ultrafiltration. (A) LRG1 purified from human serum, PMN lysate, and PMN granule releasate were left untreated (- lanes) or subjected to deglycosylation (+ lanes) using a commercially available protein deglycosylation kit, and immunoblot analysis performed using an antibody to human LRG1. Molecular weight markers (MM) are shown on the left. Neutrophil-derived LRG1 migrates with a higher molecular weight than serum-derived LRG1 (- lanes) but with the same molecular weight following deglycosylation (+ lanes), indicating different patterns of glycosylation of the native protein forms in PMNs compared to serum. The data shown are representative of three independent experiments, each with a different normal donor. (B) Purified cytochrome c was bound to a NHS-activated sepharose column. Releasate from PMNs stimulated with calcium ionophore (2 uM) was loaded onto the cytochrome c column. Bound proteins were eluted with an acetate buffer (pH 4) in 1 mL fractions, and subjected to immunoblot analysis with antibody to LRG1. Lane 1 is crude PMN releasate. The column flow through and wash is shown in lane 2. Eluate fractions are shown in lanes 3–8, with LRG1 detectable in fractions 3 and 4. Lane 9 is purified LRG1. The data shown are representative of three independent experiments.
Fig 5.
LRG1 modifies the effects of TGFβ on myeloid and hematopoietic progenitor cell growth.
(A) HL-60 cell proliferation in the presence of increasing concentrations of TGFβ alone (blue diamonds) or TGFβ plus LRG1 (800 ng/mL, red squares) was assessed using the XTT assay. A dose-response reduction in cell proliferation as measured by the decreasing A490-A650 is observed with increasing concentrations of TGFβ that is mitigated by the addition of LRG1. The Student's t-test was used to determine the significance of the differences between control and LRG1 treated samples († p < 0.05) (n = 5). (B) Purified bone marrow-derived CD34+ cells were cultured in serum-free semi-solid media in the presence of TGFβ (10 ng/mL) with or without LRG1 at the indicated concentrations, and colony numbers scored. An increase in colony numbers relative to TGFβ1 treatment alone (0) is seen with increasing concentrations of LRG1. The Student's t-test was used to determine the significance of the differences between control and LRG1 treated samples († p < 0.05) (n = 3). (C) The effects of TGFβ alone (left panel) and TGFβ plus LRG1 (right panel) on colony formation and colony composition were analyzed. Upper two images in each panel. Representative light microscopy images of methocult plates on which bone marrow-derived CD34+ cells were cultured in the presence of TGFβ alone (left) or TGFβ with LRG1 (right). Lower two images in each panel. Wright-Giemsa staining of cells plucked from individual colonies from the methocult plates in the upper images is shown. Cells were grown as described in (B) in the presence of TGFβ (10 ng/mL) with or without LRG1 (0.625 ug/mL). LRG1 mitigates the inhibitory effect of TGFβ on the growth and size of colony-forming units-granulocyte monocyte (CFU-GM). The data shown are representative of five independent experiments, each with a different normal donor.