CXCL12 is displayed by rheumatoid endothelial cells through its basic amino-terminal motif on heparan sulfate proteoglycans - PubMed (original) (raw)

CXCL12 is displayed by rheumatoid endothelial cells through its basic amino-terminal motif on heparan sulfate proteoglycans

Begoña Santiago et al. Arthritis Res Ther. 2006.

Abstract

The chemokine CXCL12 (also known as stromal cell-derived factor, SDF-1) is constitutively expressed by stromal resident cells and is involved in the homeostatic and inflammatory traffic of leukocytes. Binding of CXCL12 to glycosaminoglycans on endothelial cells (ECs) is supposed to be relevant to the regulation of leukocyte diapedesis and neoangiogenesis during inflammatory responses. To improve our understanding of the relevance of this process to rheumatoid arthritis (RA), we have studied the mechanisms of presentation of exogenous CXCL12 by cultured RA ECs. RA synovial tissues had higher levels of CXCL12 on the endothelium than osteoarthritis (OA) tissues; in both, CXCL12 colocalized to heparan sulfate proteoglycans (HSPGs) and high endothelial venules. In cultured RA ECs, exogenous CXCL12alpha was able to bind in a CXCR4-independent manner to surface HSPGs. Desulfation of RA EC HSPGs by pretreatment with sodium chlorate, or by replacing in a synthetic CXCL12alpha the residues Lys24 and Lys27 by Ser (CXCL12alpha-K2427S), decreased or abrogated the ability of the chemokine to bind to RA ECs. Ex vivo, synovial ECs from patients with either OA or RA displayed a higher CXCL12-binding capacity than human umbilical vein ECs (HUVECs), and in HUVECs the binding of CXCL12 was increased on exposure to tumor necrosis factor-alpha or lymphotoxin-alpha1beta2. Our findings indicate that CXCL12 binds to HSPGs on ECs of RA synovium. The phenomenon relates to the interaction of HSPGs with a CXCL12 domain with net positive surface charge located in the first beta strand, which encompasses a canonical BXBB HSPG-binding motif. Furthermore, we show that the attachment of CXCL12 to HSPGs is upregulated by inflammatory cytokines. Both the upregulation of a constitutive chemokine during chronic inflammation and the HSPG-dependent immobilization of CXCL12 in EC surfaces are potential sites for therapeutic intervention.

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Figures

Figure 1

Double labelling of CXCL12/HSPGs and CXCL12/MECA-79 of rheumatoid arthritis (RA) and osteoarthritis (OA) synovial tissues. Frozen RA or OA sections were labeled simultaneously with anti-CXCL12 K15C mAb and fluorescein isothiocyanate (FITC)- conjugated anti-heparan sulfate proteoglycan (HSPG) mAb (green fluorescence) or MECA-79 mAb (red fluorescence). In CXCL12/HSPG double-labeled sections, CXCL12 was developed with immunoperoxidase (brown color) and in CXCL12/MECA-79 double-labeled sections with a secondary FITC-labeled antibody. Arrows indicate colocalization of CXCL12 to HSPG-labeled RA vessels. The same section, sequentially photographed under appropriate optics, is shown in parallel left and right panels. Original magnification × 400.

Figure 2

Binding of CXCL12 to RA ECs is independent of CXCR4. (a) Rheumatoid arthritis endothelial cells (RA ECs) were incubated with 300 nM biotinylated CXCL12α and, after extensive washing to remove free chemokine, were labeled with fluorescein isothiocyanate-conjugated avidin. Where indicated, RA ECs were simultaneously incubated with 50 μg/ml of the CXCR4 antagonist T134 or not (untreated). (b) Surface or intracellular CXCR4 was detected with 12G5 mAb. Filled histograms show isotype control IgG. Results are representative of three independent experiments with RA ECs from different donors.

Figure 3

Binding of CXCL12 peptides to HSPG on RA ECs. Rheumatoid arthritis endothelial cells (RA ECs) were incubated with 300 nM biotinylated CXCL12α and, after extensive washing to remove free chemokine, were labeled with fluorescein isothiocyanate-conjugated avidin. Where indicated, RA ECs were simultaneously incubated with soluble sodium heparin (a) or pretreated with heparitinases (b, c) or sodium chlorate (d). In (b), surface heparan sulfate proteoglycan (HSPG) was detected with 10E4 mAb in RA ECs pretreated or not with heparitinases. (e) RA ECs were incubated with 1 μM non-biotinylated wild-type CXCL12α or 2/6 CXCL12α and labelled with K15C mAb. Filled histograms show isotype control IgG. Results are representative of three to five independent experiments with RA ECs from three different donors. (f) Summary of normalized mean fluorescence intensity (MFI) data. Error bars show SD. *p < 0.05.

Figure 4

Binding of CXCL12 to synovial osteoarthritis (OA) and rheumatoid arthritis (RA) ECs and cytokine-treated HUVECs. Endothelial cells (ECs) were incubated with 300 nM biotinylated CXCL12α and, after extensive washing to remove free chemokine, were labeled with fluorescein isothiocyanate-conjugated avidin. Human umbilical-vein endothelial cells (HUVECs) were treated with tumor necrosis factor-α (TNF-α; top) or lymphotoxin α1β2 (LT-αβ; middle) for 16 hours before CXCL12α binding as indicated. Filled histograms show isotype control IgG. The bottom panel shows a summary of normalized mean fluorescence intensity (MFI) data. Error bars show SD. Results are representative of three to five independent experiments in three RA EC, four OA EC and three HUVEC lines. *p < 0.05 compared with HUVECs, **p < 0.05 compared with cytokine-untreated HUVECs.

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CXCL12 is displayed by rheumatoid endothelial cells through its basic amino-terminal motif on heparan sulfate proteoglycans - PubMed (original) (raw)