- Research article
- Open Access
Dose dependent effects of platelet derived chondroitinsulfate A on the binding of CCL5 to endothelial cells
© Weingart et al; licensee BioMed Central Ltd. 2008
- Received: 13 November 2007
- Accepted: 10 December 2008
- Published: 10 December 2008
Chemokines immobilized on endothelial cells play a central role in the induced firm adhesion and transendothelial migration of leukocytes. Activation of platelets at sites of vascular injury is considered to support leukocyte adhesion and extravasation. However, activated platelets also secrete soluble glycosaminoglycans that can interfere with immobilization of chemokines. We therefore analyzed the impact of platelet derived glycosaminoglycans on the immobilization of the chemokine CCL5 (RANTES) on human microvascular endothelial cells and their influence on CCL5-CCR5 interactions.
We confirm that undiluted serum in contrast to plasma decreases binding of CCL5 to endothelial cells. However, when lower concentrations of serum were used, CCL5-presentation on endothelial cells was markedly enhanced. This enhancement was neutralized if serum was digested with chondroinitase ABC. Using different chondroitinsulfate-subtypes we demonstrate that chondroitinsulfate A mediates the enhanced presentation of CCL5 on endothelial cells, whereas chondroitinsulfate B/C even at low concentrations block CCL5 binding. CCR5 downregulation on CCR5-transfected CHO cells or human monocytes is increased by preincubation of CCL5 with serum or chondroitinsulfate A.
We show that chondroitinsulfate A released from platelets increases the binding of chemokines to endothelial cells and supports receptor internalization in a dose dependent manner. These data help to understand the proinflammatory effects of activated platelets.
- Responsible Serum Factor
- Leukocyte Recruitment
- CCR5 Expression
The adhesion and transendothelial migration of leukocytes is largely dependent on chemokines and adhesion molecules. In order to support leukocyte recruitment chemokines need to be immobilized on the luminal surface of the endothelial cell wall. Within tissues leukocytes are also directed by gradients of chemokines . By interacting with different chemokine receptors (CCR1 and CCR5) the chemokine CCL5 (RANTES) has been shown to be involved in several steps of leukocyte recruitment .
Chemokines can gain access to the luminal site of the endothelium by transcytosis through endothelial cells , after release from circulating leukocytes or after secretion from activated endothelial cells . Platelets have been identified as important source of chemoattractant factors such as CCL5 , but also release substantial amounts of chondroitinsulfate A . In vivo, platelet activation and adhesion occurs at sites of vascular injury and facilitates leukocyte recruitment [7–10].
It has been shown that membrane bound glycosaminoglycans are critically involved in immobilization and presentation of chemokines [11–14]. Different patterns of glycosaminoglycan expression on cells may favor the binding of certain chemokines and thereby influence the cellular composition of the inflammatory response. However, chemokines also interact with soluble glycosaminoglycans that compete with the binding of chemokines to cell surfaces. Heparin has the highest affinity to CCL5, followed by heparansulfate, chondroitinsulfate C, dermatansulfate (chondroitinsulfate B) and chondroitinsulfate A . We could demonstrate that human serum inhibits CCL5 binding on CHO cells and cultured human endothelial cells and could identify the responsible serum factor as chondroitinsulfate A (CSA) released from platelets after activation .
Glycosaminoglycans also alter the ability of chemokines to interact with chemokine receptors. Soluble Glycosaminoglycans have been shown to inhibit binding of IL-8 to CXCR1 and CXCR2 and CCL3 to CCR1 . It was also shown that CCL5/glycosaminoglycan complexes are able to bind to deglycated PBMC and thereby block HIV-1 infection more effectively than CCL5 alone .
Activated platelets have been identified as a major source of CSA in human serum . In addition, release of chondroitinsulfate A was shown in activated T cells [18, 19]. It is commonly thought that interaction of chemokines with soluble glycosaminoglycans reduces their ability to bind to cell surfaces and interferes with leukocyte recruitment. However, these results do not fit to the proinflammatory effects caused by intravascular activation of platelets. Therefore we analyzed in more detail the influence of serum and various glycosaminoglycans on the binding of CCL5 to endothelial cells and on the ability of CCL5 to activate CCR5.
Influence of serum and glycosaminoglycans on CCL5 binding to endothelial cells
We also investigated if CCL5 bound to the surface of endothelial cells could be removed from the cell surface by incubation with an excess of soluble glycosaminoglycans. High doses of CSC (100 μg/ml) in contrast to CSA resulted in a minor reduction (< 20%) of surface bound CCL5 (data not shown).
Enhanced downregulation of CCR5 by CCL5-CSA complexes
Activation of platelets at sites of vascular injury is considered as an important proinflammatory stimulus. Here we show that chondroitinsulfate A released from activated platelets has pronounced dose dependent effects on the immobilization of CCL5 to endothelial cells and the interaction of CCL5 with CCR5. Only at high concentrations does platelet derived CSA block CCL5 binding to endothelial cells, whereas at lower concentrations of CSA the immobilization of CCL5 and its interaction with CCR5 are markedly enhanced. Our data improve our understanding of how activation of platelets may contribute to an increased recruitment of chemokine receptor expressing leukocytes.
Cells and reagents
Human microvascular endothelial cells (Promocell, Heidelberg, Germany) were cultured in MCDB 131 medium (Invitrogen, Karlsruhe, Germany) with 15% FCS, hydrocortisone (1.2 μg/ml), epidermal growth factor (3 μg/l) and L-glutamine (4 mmol/l). Where indicated endothelial cells were stimulated for 48 h with TNF-α (10 ng/ml) and IL-1β (5 ng/ml) (PeproTech, Rocky Hill, New Jersey USA). CCR5 transfected CHO cells  were cultured in nucleotide free alpha-MEM medium containing 10% dialyzed FCS. Human PBMC were isolated from healthy human individuals by Ficoll-Paque density gradients and cultured in RPMI medium (Invitrogen, Karlsruhe, Germany) for 24 hours to induce CCR5 expression on monocytes. Human serum and plasma were obtained from healthy volunteers using standard blood sample equipment. Plasma was anticoagulated with EDTA. Where indicated serum was digested for 2 hours at 37°C with chondroinitase ABC (final concentration of 50 mU/mL; Sigma-Aldrich) in RPMI 1640 medium.
Binding of CCL5 to cell surfaces
Recombinant CCL5 (PeproTech, Rocky Hill, New Jersey USA) was preincubated for 1 h on ice with PBS or different concentrations of serum, plasma, chondroitinsulfate A (CSA), chondroitinsulfate B (CSB) or chondroitinsulfate C (CSC) (Sigma, St. Louis, USA) as indicated in the figure legends. Cells were then incubated for 1 h on ice with CCL5. After four washing steps with PBS, cells were stained with a monoclonal antibody against CCL5 (VL-1, 10 μg/ml) (kindly provided by Dr. Nelson, Munich, Germany) or a mouse IgG2b isotype control antibody (Sigma-Aldrich, St. Louis, USA) followed by a phycoerythrin-conjugated, rabbit anti-mouse F(ab)2 fragment (DAKO, Glostrup, Denmark). Alternatively staining was performed with a biotinylated antibody against CCL5 (clone VL-1, CALTAG, Burlingame, USA) followed by allophycocyanin-conjugated Streptavidin (BD-Pharmingen, Heidelberg, Germany). CCL5-binding was quantified by flow cytometry using a FACSCalibur and cell quest analysis software and is given as mean fluorescence intensity (MFI) (Becton Dickinson, Heidelberg, Germany).
Experiments to detect or block CD44 were performed with the anti-CD44 clone BRIC 235 (Bristol Institute for Transfusion Sciences, Bristol, UK) at concentrations of 10 and 20 μg/ml respectively. Expression of CD206 (mannose receptor) and CD36 was measured with monoclonal antibodies against CD206 (clone 19.2) and CD36 (clone CB 38) by flow cytometry (BD-Pharmingen, Heidelberg, Germany).
Downmodulation of CCR5
Downmodulation of CCR5 was performed as described previously with minor modification . CCL5 was preincubated with serum or CSA as described above. CCR5 transfected CHO cells or PBMC were incubated at 37°C for 2 hours with preincubated CCL5 (1 μg/ml) or with PBS (pos. control) to induce receptor downmodulation. Further steps were performed on ice to avoid receptor recycling. CCR5 expression was measured by flow cytometry using the monoclonal antibody MC-1 (10 μg/ml) or an mouse IgG1 isotype control antibody (neg. control), followed by a phycoerythrin-conjugated, rabbit anti-mouse F(ab)2 fragment. Surface expression of CCR5 was calculated as [mean fluorescence intensity (exp.) - mean fluorescence (neg. control)]/[(mean fluorescence intensity (pos. control.) - mean fluorescence (neg. control)].
All experiments were reproduced at least two times and single data points were obtained as mean values of duplicates or triplicates. Error bars indicate the standard deviation. Statistical differences of fluorescence intensities were tested by using Student's t-test where appropriate.
The authors declare that they have no competing interests.
We thank Nicole Göbel, Yvonne Talke and Kathrin Schmidbauer for technical assistance. This work was supported by a grant from the Deutsche Forschungsgemeinschaft to MM.
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