Peroxynitrite Exposure of CXCL12 Impairs Monocyte, Lymphocyte and Endothelial Cell Chemotaxis, Lymphocyte Extravasation in vivo and Anti-HIV-1 Activity.

Rik Janssens,Sam Noppen,Anneleen Mortier,Ghislain Opdenakker,Flávio A Amaral,Dominique Schols,Daiane Boff,Olav Larsen,Pieter Ruytinx,Sandra Liekens,Viktorija Daugvilaite,Vincent Vanheule,Mette M Rosenkilde,Sofie Struyf,Mauro M Teixeira,Ingrid De Meester,Paul Proost

doi:10.3389/fimmu.2018.01933

Abstract

CXCL12 is a chemotactic cytokine that attracts many different cell types for homeostasis and during inflammation. Under stress conditions, macrophages and granulocytes produce factors such as peroxynitrite as a consequence of their oxidative response. After short incubations of CXCL12 with peroxynitrite, the gradual nitration of Tyr7, Tyr61, or both Tyr7 and Tyr61 was demonstrated with the use of mass spectrometry, whereas longer incubations caused CXCL12 degradation. Native CXCL12 and the nitrated forms, [3-NT61]CXCL12 and [3-NT7/61]CXCL12, were chemically synthesized to evaluate the effects of Tyr nitration on the biological activity of CXCL12. All CXCL12 forms had a similar binding affinity for heparin, the G protein-coupled chemokine receptor CXCR4 and the atypical chemokine receptor ACKR3. However, nitration significantly enhanced the affinity of CXCL12 for chondroitin sulfate. Internalization of CXCR4 and β-arrestin 2 recruitment to CXCR4 was significantly reduced for [3-NT7/61]CXCL12 compared to CXCL12, whereas β-arrestin 2 recruitment to ACKR3 was similar for all CXCL12 variants. [3-NT7/61]CXCL12 was weaker in calcium signaling assays and in in vitro chemotaxis assays with monocytes, lymphocytes and endothelial cells. Surprisingly, nitration of Tyr61, but not Tyr7, partially protected CXCL12 against cleavage by the specific serine protease CD26. In vivo, the effects were more pronounced compared to native CXCL12. Nitration of any Tyr residue drastically lowered lymphocyte extravasation to joints compared to native CXCL12. Finally, the anti-HIV-1 activity of [3-NT7]CXCL12 and [3-NT7/61]CXCL12 was reduced, whereas CXCL12 and [3-NT61]CXCL12 were equally potent. In conclusion, nitration of CXCL12 occurs readily upon contact with peroxynitrite and specifically nitration of Tyr7 fully reduces its in vitro and in vivo biological activities.

Highlights

CXCL12 is a homeostatic CXC chemokine that exists in six splice variants and is expressed in most tissues [1]
Since CXCR4 is a main co-receptor for HIV-1 infection and CXCL12 functions as a natural competitor for HIV-1, we tested the effects of nitration on the anti-HIV-1 capacity of CXCL12 [9]
Using multiple reaction monitoring (MRM), the eluate of the peroxynitriteincubated sample that was loaded on the HPLC was scanned by automated MS2 for CXCL12 variants with none, one, two or three additional nitro compounds

Summary

Introduction

CXCL12 is a homeostatic CXC chemokine that exists in six splice variants and is expressed in most tissues [1]. CXCL12 was discovered as a pre-B cell growth factor of crucial importance for lymphopoiesis and embryogenesis and was shown to be a co-stimulator for CD4+ T cell activation [2, 3]. CXCL12 was purified from its main source, the bone marrow stroma, based on its ability to attract lymphocytes and monocytes [4]. CXCL12 prevents the release of hematopoietic progenitor cells into the circulation [5]. CXCL12 is an angiogenic chemokine and attracts endothelial progenitor cells in response to hypoxia [6]. CXCL12 activity has been shown in several inflammatory conditions, such as rheumatoid arthritis, inflammatory bowel disease and cancer growth and metastasis [3, 7, 8]

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