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G-CSF induces E-selectin ligand expression on human myeloid cells

Abstract

Clinical use of G-CSF can result in vascular and inflammatory complications1,2,3,4,5,6,7. To investigate the molecular basis of these effects, we analyzed the adherence of G-CSF–mobilized human peripheral blood leukocytes (ML) to inflamed (TNF-α–stimulated) vascular endothelium. Studies using parallel plate assays under physiologic flow conditions and intravital microscopy in a mouse inflammation model each showed that ML take part in heightened adhesive interactions with endothelium compared to unmobilized (native) blood leukocytes, mediated by markedly increased E-selectin receptor-ligand interactions. Biochemical studies showed that ML express the potent E-selectin ligand HCELL (ref. 8) and another, previously unrecognized 65-kDa E-selectin ligand, and possess enhanced levels of transcripts encoding glycosyltransferases (ST3GalIV, FucT-IV and FucT-VII) conferring glycan modifications associated with E-selectin ligand activity. Enzymatic treatments and physiologic binding assays showed that HCELL and the 65-kDa E-selectin ligand contribute prominently to the observed G-CSF–induced myeloid cell adhesion to inflamed endothelium. Treatment of normal human bone marrow cells with a pharmacokinetically relevant concentration of G-CSF in vitro9,10 resulted in increased expression of these two molecules, coincident with increased transcripts encoding pertinent glycosyltransferases and heightened E-selectin binding. These findings provide direct evidence for a role of G-CSF in the induction of E-selectin ligands on myeloid cells, thus providing mechanistic insight into the pathobiology of G-CSF complications.

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Figure 1: ML possess enhanced binding to E-selectin relative to NL.
Figure 2: ML express multiple HECA–452-reactive E-selectin glycoprotein ligands.
Figure 3: Characterization of PSGL-1, HCELL and a newly identified 65-kDa E-selectin ligand on ML.
Figure 4: In vitro G-CSF treatment of human bone marrow cells upregulates the expression of HCELL, HECA-452–reactive 65-kDa glycoprotein, ST3GalIV, FucT-IV and FucT-VII.

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Acknowledgements

We are grateful to the staff at the Cell Manipulation Core Facility of Dana Farber Cancer Center for their assistance in procuring G-CSF–mobilized peripheral blood pheresis products and to the staff of the Cell Processing Laboratory of the Bone Marrow Transplantation Unit at the Massachusetts General Hospital for their assistance in procuring the bone marrow material. This work was supported by US National Institutes of Health grants RO1 HL060528 (R.S.), RO1 HL073714 (R.S.), the Team Jobie Leukemia Research Fund (R.S.) and RO1 EB000664 and Wellman Center Advanced Microscopy startup fund (C.P.L.).

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Authors and Affiliations

Authors

Contributions

N.M.D. designed the research, performed experiments, analyzed data and wrote the paper; S.Z.G. performed RT-PCR; C.A.K. provided technical assistance; J.A.S. and P.Z. performed intravital microscopy and analyzed data; C.P.L. oversaw intravital microscopy experiments and partially provided funding for the research; R.S. designed research, analyzed data, wrote the paper, provided funding for the research and supervised all experimentation.

Corresponding author

Correspondence to Robert Sackstein.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Fig. 1

mAbs specific to L-selectin, CD29 or CD18 have no effect on primary tethering of ML on stimulated HUVECs and there are no distinct differences in the surface expression of integrin-type homing receptors LFA-1 (CD11a/CD18; αLβ2) and VLA-4 (CD49d/CD29; α4β1) and chemokine receptor CXCR4 on ML and NL. (PDF 840 kb)

Supplementary Fig. 2

HECA-452–reactive glycoproteins of ML are sensitive to sialidase treatment. (PDF 259 kb)

Supplementary Fig. 3

E-Ig-reactive glycoproteins of ML do not stain in the presence of EDTA or with control human immunoglobulin. (PDF 270 kb)

Supplementary Fig. 4

HECA-452–reactive 65 kDa E-selectin ligand does not appear to be related to PSGL-1 or CD44. (PDF 328 kb)

Supplementary Fig. 5

G-CSF treatment significantly enhances the capability of human BM cells to adhere to endothelial E-selectin under physiologic flow conditions. (PDF 330 kb)

Supplementary Fig. 6

HCELL and 65 kDa glycoprotein are major E-selectin ligands on ML. (PDF 803 kb)

Supplementary Video 1

This movie shows ML interacting with vascular endothelium within the TNF-α–treated ear. Note the prominent presence of stable rolling interactions. (MOV 2133 kb)

Supplementary Video 2

This movie shows NL interacting with vascular endothelium within the TNF-α–treated ear. Compared to ML (Supplementary Video 1), NL display transient and faster rolling interactions. (MOV 2959 kb)

Supplementary Methods (PDF 139 kb)

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Dagia, N., Gadhoum, S., Knoblauch, C. et al. G-CSF induces E-selectin ligand expression on human myeloid cells. Nat Med 12, 1185–1190 (2006). https://doi.org/10.1038/nm1470

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