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 CXCL12α 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 CXCL12α the residues Lys24 and Lys27 by Ser (CXCL12α-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-α or lymphotoxin-α1β2. 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 β 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.
Highlights
Chemokines are a large family of soluble proteins involved in leukocyte activation and traffic during inflammatory responses
We have analyzed the mechanisms of interaction between exogenous CXCL12 and endothelial cells (ECs) obtained from synovial tissues, and here we show that CXCL12 binds to membrane heparan sulfate proteoglycans (HSPGs) in cultured rheumatoid arthritis (RA) ECs independently of its CXCR4 receptor
HSPGs and CXCL12 are immunodetected in RA vessels By immunoperoxidase labelling of RA (n = 9) and OA (n = 8) synovial sections, we found that CXCL12 was abundantly present on the luminal side of RA sublining vessels in contrast to OA vessels, in which it was only rarely and weakly detected (Figure 1)
Summary
Chemokines are a large family of soluble proteins involved in leukocyte activation and traffic during inflammatory responses. Chemokines induce cell-matrix or cell-cell adhesion through the activation of integrins, and studies in vivo demonstrate that the presence of chemokines immobilized on the luminal side of endothelium is a critical step for firm adhesion and transendothelial migration of rolling leukocytes [2,3,4,5,6]. This phenomenon can be reproduced in vitro in cultured endothelial cells (ECs) and depends on the addition of exogenous chemokines and the presence of fluid shear-induced mechanical stress on leukocytes [5,6]. Endothelial cells secrete a limited number of chemokines, suggesting that many of the homeostatic or inflammatory chemokines presented at the EC surface come from other cell sources by transcytosis and docking of chemokines on the EC luminal surface [7,8,9]
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