BACKGROUND
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  <cit> .
by interacting with different chemokine receptors  the chemokine ccl <dig>  has been shown to be involved in several steps of leukocyte recruitment  <cit> .
chemokines can gain access to the luminal site of the endothelium by transcytosis through endothelial cells  <cit> , after release from circulating leukocytes or after secretion from activated endothelial cells  <cit> .
platelets have been identified as important source of chemoattractant factors such as ccl <dig>  <cit> , but also release substantial amounts of chondroitinsulfate a  <cit> .
in vivo, platelet activation and adhesion occurs at sites of vascular injury and facilitates leukocyte recruitment  <cit> .
it has been shown that membrane bound glycosaminoglycans are critically involved in immobilization and presentation of chemokines  <cit> .
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 ccl <dig>  followed by heparansulfate, chondroitinsulfate c, dermatansulfate  and chondroitinsulfate a  <cit> .
we could demonstrate that human serum inhibits ccl <dig> binding on cho cells and cultured human endothelial cells and could identify the responsible serum factor as chondroitinsulfate a  released from platelets after activation  <cit> .
glycosaminoglycans also alter the ability of chemokines to interact with chemokine receptors.
soluble glycosaminoglycans have been shown to inhibit binding of il- <dig> to cxcr <dig> and cxcr <dig> and ccl <dig> to ccr <dig>  <cit> .
it was also shown that ccl5/glycosaminoglycan complexes are able to bind to deglycated pbmc and thereby block hiv- <dig> infection more effectively than ccl <dig> alone  <cit> .
activated platelets have been identified as a major source of csa in human serum  <cit> .
in addition, release of chondroitinsulfate a was shown in activated t cells  <cit> .
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 ccl <dig> to endothelial cells and on the ability of ccl <dig> to activate ccr <dig>
RESULTS
influence of serum and glycosaminoglycans on ccl <dig> binding to endothelial cells
in previous experiments we have shown a reduced binding of ccl <dig> to cell surfaces after preincubation of ccl <dig> with human serum and have identified the responsible serum factor as chondroitinsulfate a  released from activated platelets.
in these experiments we used undiluted or moderately diluted serum and high concentrations of csa  <cit> .
the use of undiluted serum or high concentrations of glycosaminoglycans may not reflect in vivo settings of local inflammation where only a minor proportion of total platelets are activated and release csa.
we therefore analyzed the effects of serum or chondroitinsulfate a over a wide range of concentrations.
ccl <dig> was preincubated with decreasing concentrations of human serum and then incubated with microvascular human endothelial cells  .
high concentrations of serum block ccl <dig> binding to the surface of microvascular endothelial cells confirming our previous results  <cit> .
however, preincubation of ccl <dig> with lower concentrations of serum resulted in a threefold higher binding of ccl <dig> to endothelial cells .
human plasma, that does not contain csa released from activated platelets, was unable to increase the binding of ccl <dig> to endothelial cells suggesting that platelet derived chondroitinsulfate a might be a crucial factor.
to demonstrate that chondroitinsulfate is responsible for the enhanced binding of ccl <dig> in the presence of serum, serum was digested with chondroinitase abc before preincubation with ccl <dig>  digested serum was no longer able to reduce or enhance the binding of ccl <dig> .
to determine which glycosaminoglycan is responsible for this effect, we compared the effect of csa, csb and csc on ccl <dig> binding to endothelial cells .
preincubation of ccl <dig> with csb and csc resulted in a strong inhibition of chemokine binding.
no increased binding of ccl <dig> was detectable even at low concentrations of these glycosaminoglycans.
in contrast, preincubation of ccl <dig> with low concentrations of csa resulted in a dose-dependent increase of the binding of ccl <dig> .
the most likely explanation for the enhanced binding of ccl <dig> after exposure to low concentrations of csa or serum is the occurrence of ccl5-csa complexes and their subsequent binding to surface glycosaminoglycans via their ccl <dig> part or to surface receptors for csa via their csa part.
among molecules that are known to bind chondroitinsulfate a  only the proteoglycan cd <dig> was strongly expressed on cultured endothelial cells.
however, neither blockade of cd <dig> with a neutralizing antibody  nor preincubation of cells with serum, csa, csb or csc altered the binding of ccl <dig> or ccl5-csa-complexes to the surface of endothelial cells .
therefore it seems unlikely that possible csa binding sites on cell surfaces play an important role in the process of chemokine immobilization.
instead, we favor the hypothesis that ccl5-csa complexes bind primarily via ccl <dig> to glycosaminoglycans on the cell surface.
the low affinity between ccl <dig> and csa may allow the binding of ccl5-csa complexes to glycosaminoglycans with high affinity for ccl <dig> .
surface heparansulfate proteoglycan expression is upregulated under proinflammatory conditions in osteosarcoma cells  <cit> .
to test the hypothesis that activation of endothelial cells leads to an increased immobilization of chemokines, cells were stimulated with tnf-α and il-1β for  <dig> hours.
however, stimulation of endothelial cells with tnf-α and il-1β did not increase ccl <dig> immobilization on primary human microvascular endothelial cells .
we also investigated if ccl <dig> 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  in contrast to csa resulted in a minor reduction  of surface bound ccl <dig> .
enhanced downregulation of ccr <dig> by ccl5-csa complexes
to assess whether the interaction of ccl <dig> with csa alters the ability of ccl <dig> to activate ccr <dig>  we performed receptor downmodulation assays with ccr <dig> transfected cho cells and human pbmc isolated from peripheral blood  <cit> .
surface expression of ccr <dig> was quantified by facs analysis.
as shown in figure 3a, downregulation of ccr <dig> on cho cells was markedly enhanced if ccl <dig> was preincubated with serum.
serum concentrations of between  <dig> % and  <dig> % resulted in the most pronounced enhancement of ccl <dig> induced ccr <dig> downregulation compared to ccl <dig> alone.
serum alone was not able to induce downregulation of ccr <dig> on transfected cho cells .
we also investigated ccl5-induced downmodulation of ccr <dig> on human pbmc and analyzed ccr <dig> surface expression on human monocytes by flow cytometry .
preincubation of ccl <dig> with csa resulted in a more pronounced downmodulation of ccr <dig> from the cell surface than preincubation of ccl <dig> with pbs.
csa alone had no significant effect on ccr <dig> expression on human monocytes.
these data demonstrate that complex formation of ccl <dig> with csa not only increases the binding of ccl <dig> to cell surfaces but also enhances the ability to interact with the chemokine receptor ccr <dig>
CONCLUSIONS
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 ccl <dig> to endothelial cells and the interaction of ccl <dig> with ccr <dig>  only at high concentrations does platelet derived csa block ccl <dig> binding to endothelial cells, whereas at lower concentrations of csa the immobilization of ccl <dig> and its interaction with ccr <dig> are markedly enhanced.
our data improve our understanding of how activation of platelets may contribute to an increased recruitment of chemokine receptor expressing leukocytes.

 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 the chemokine ccl 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 ccl, but also release substantial amounts of chondroitinsulfate a. in vivo, platelet activation and adhesion occurs at sites of vascular injury and facilitates leukocyte recruitment. it has been shown that membrane bound glycosaminoglycans are critically involved in immobilization and presentation of chemokines. 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 ccl followed by heparansulfate, chondroitinsulfate c, dermatansulfate and chondroitinsulfate a. we could demonstrate that human serum inhibits ccl binding on cho cells and cultured human endothelial cells and could identify the responsible serum factor as chondroitinsulfate a 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- to cxcr and cxcr and ccl to ccr. it was also shown that ccl5/glycosaminoglycan complexes are able to bind to deglycated pbmc and thereby block hiv- infection more effectively than ccl 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. 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 ccl to endothelial cells and on the ability of ccl to activate ccr  influence of serum and glycosaminoglycans on ccl binding to endothelial cells in previous experiments we have shown a reduced binding of ccl to cell surfaces after preincubation of ccl with human serum and have identified the responsible serum factor as chondroitinsulfate a released from activated platelets. in these experiments we used undiluted or moderately diluted serum and high concentrations of csa. the use of undiluted serum or high concentrations of glycosaminoglycans may not reflect in vivo settings of local inflammation where only a minor proportion of total platelets are activated and release csa. we therefore analyzed the effects of serum or chondroitinsulfate a over a wide range of concentrations. ccl was preincubated with decreasing concentrations of human serum and then incubated with microvascular human endothelial cells. high concentrations of serum block ccl binding to the surface of microvascular endothelial cells confirming our previous results. however, preincubation of ccl with lower concentrations of serum resulted in a threefold higher binding of ccl to endothelial cells. human plasma, that does not contain csa released from activated platelets, was unable to increase the binding of ccl to endothelial cells suggesting that platelet derived chondroitinsulfate a might be a crucial factor. to demonstrate that chondroitinsulfate is responsible for the enhanced binding of ccl in the presence of serum, serum was digested with chondroinitase abc before preincubation with ccl digested serum was no longer able to reduce or enhance the binding of ccl. to determine which glycosaminoglycan is responsible for this effect, we compared the effect of csa, csb and csc on ccl binding to endothelial cells. preincubation of ccl with csb and csc resulted in a strong inhibition of chemokine binding. no increased binding of ccl was detectable even at low concentrations of these glycosaminoglycans. in contrast, preincubation of ccl with low concentrations of csa resulted in a dose-dependent increase of the binding of ccl. the most likely explanation for the enhanced binding of ccl after exposure to low concentrations of csa or serum is the occurrence of ccl5-csa complexes and their subsequent binding to surface glycosaminoglycans via their ccl part or to surface receptors for csa via their csa part. among molecules that are known to bind chondroitinsulfate a only the proteoglycan cd was strongly expressed on cultured endothelial cells. however, neither blockade of cd with a neutralizing antibody nor preincubation of cells with serum, csa, csb or csc altered the binding of ccl or ccl5-csa-complexes to the surface of endothelial cells. therefore it seems unlikely that possible csa binding sites on cell surfaces play an important role in the process of chemokine immobilization. instead, we favor the hypothesis that ccl5-csa complexes bind primarily via ccl to glycosaminoglycans on the cell surface. the low affinity between ccl and csa may allow the binding of ccl5-csa complexes to glycosaminoglycans with high affinity for ccl. surface heparansulfate proteoglycan expression is upregulated under proinflammatory conditions in osteosarcoma cells. to test the hypothesis that activation of endothelial cells leads to an increased immobilization of chemokines, cells were stimulated with tnf-α and il-1β for hours. however, stimulation of endothelial cells with tnf-α and il-1β did not increase ccl immobilization on primary human microvascular endothelial cells. we also investigated if ccl 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 in contrast to csa resulted in a minor reduction of surface bound ccl. enhanced downregulation of ccr by ccl5-csa complexes to assess whether the interaction of ccl with csa alters the ability of ccl to activate ccr we performed receptor downmodulation assays with ccr transfected cho cells and human pbmc isolated from peripheral blood. surface expression of ccr was quantified by facs analysis. as shown in figure 3a, downregulation of ccr on cho cells was markedly enhanced if ccl was preincubated with serum. serum concentrations of between % and % resulted in the most pronounced enhancement of ccl induced ccr downregulation compared to ccl alone. serum alone was not able to induce downregulation of ccr on transfected cho cells. we also investigated ccl5-induced downmodulation of ccr on human pbmc and analyzed ccr surface expression on human monocytes by flow cytometry. preincubation of ccl with csa resulted in a more pronounced downmodulation of ccr from the cell surface than preincubation of ccl with pbs. csa alone had no significant effect on ccr expression on human monocytes. these data demonstrate that complex formation of ccl with csa not only increases the binding of ccl to cell surfaces but also enhances the ability to interact with the chemokine receptor ccr  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 ccl to endothelial cells and the interaction of ccl with ccr only at high concentrations does platelet derived csa block ccl binding to endothelial cells, whereas at lower concentrations of csa the immobilization of ccl and its interaction with ccr are markedly enhanced. our data improve our understanding of how activation of platelets may contribute to an increased recruitment of chemokine receptor expressing leukocytes. 
this enhancement was neutralized if serum was digested with chondroinitase abc. ccr downregulation on ccr5-transfected cho cells or human monocytes is increased by preincubation of ccl with serum or chondroitinsulfate a. 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. we therefore analyzed the impact of platelet derived glycosaminoglycans on the immobilization of the chemokine ccl on human microvascular endothelial cells and their influence on ccl5-ccr interactions. we confirm that undiluted serum in contrast to plasma decreases binding of ccl to endothelial cells. however, when lower concentrations of serum were used, ccl5-presentation on endothelial cells was markedly enhanced. using different chondroitinsulfate-subtypes we demonstrate that chondroitinsulfate a mediates the enhanced presentation of ccl on endothelial cells, whereas chondroitinsulfate b/c even at low concentrations block ccl binding. 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.
 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 ccl on human microvascular endothelial cells and their influence on ccl5-ccr interactions.  we confirm that undiluted serum in contrast to plasma decreases binding of ccl 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 ccl on endothelial cells, whereas chondroitinsulfate b/c even at low concentrations block ccl binding. ccr downregulation on ccr5-transfected cho cells or human monocytes is increased by preincubation of ccl 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. 