Contribution of the putative heparan sulfate–binding motif BBXB of RANTES to transendothelial migration

Simi Ali1, Sarah J. Fritchley, Benjamin T. Chaffey and John A. Kirby

The Applied Immunobiology Group, Department of Surgery, The Medical School, University of Newcastle, Newcastle upon Tyne, NE2 4HH, UK

Received on December 21, 2001; revised on May 21, 2002; accepted on May 22, 2002


    Abstract
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
The chemokines are a family of small chemoattractant proteins that have a range of functions, including activation and promotion of vectorial migration of leukocytes. Regulation on activation, normal T cell expressed and secreted (RANTES; CCL5), a member of the CC-chemokine subfamily, has been implicated in a variety of immune responses. In addition to the interaction of CC-chemokines with their cognate cell-surface receptors, it is known that they also bind to glycosaminoglycans (GAGs), including heparan sulfate. This potential for binding to GAG components of proteoglycans on the cell surface or within the extracellular matrix might allow formation of the stable chemokine concentration gradients necessary for leukocyte chemotaxis. In this study, we created a panel of mutant RANTES molecules containing neutral amino acid substitutions within putative, basic GAG-binding domains. Despite showing reduced binding to GAGs, it was found that each mutant containing a single amino acid substitution induced a similar leukocyte chemotactic response within a concentration gradient generated by free solute diffusion. However, we found that the mutant K45A had a significantly reduced potential to stimulate chemotaxis across a monolayer of microvascular endothelial cells. Significantly, this mutant bound to the CCR5 receptor and showed a potential to mobilize Ca2+ with an affinity similar to the wild-type protein. These results show that the interaction between RANTES and GAGs is not necessary for specific receptor engagement, signal transduction, or leukocyte migration. However, this interaction is required for the induction of efficient chemotaxis through the extracellular matrix between confluent endothelial cells.

Key words: cell–cell interaction/chemokines/chemotaxis/inflammation/transendothelial migration


    Introduction
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
The vascular endothelium plays a critical role in the selective recruitment of leukocytes to subendothelial tissues during inflammation by facilitating a cascade process involving intravascular arrest of certain cell types and directed extravasation of responsive cells (Butcher and Picker, 1996Go). Members of the chemokine family are implicated in the regulation of this process.

The chemokines are a family of small chemoattractant proteins capable of activating and promoting the vectorial migration of a variety of leukocytes. They can be grouped into four subfamilies on the basis of the position of conserved cysteine residues; these are termed the C, CX3C, CC, and CXC groups (Rollins, 1997Go; Zlotnik and Yoshie, 2000Go). The C and CX3C groups each have only one known member, whereas the CC and CXC groups have many members. The CC chemokines, in which the first two conserved cysteine residues are adjacent, are believed to be most important mediators of mononuclear cell migration in inflammation.

The chemokines posses two binding domains, which interact respectively with cognate, seven-transmembrane-spanning G-protein-coupled receptors and with glycosaminoglycans (GAGs) such as heparan sulfate (HS) (Tanaka and Aso, 1998Go; Witt and Lander, 1994Go). Nine receptors for CC chemokines have been identified; however, in vitro studies have shown some redundancy with more than one chemokine binding to a given receptor and several receptors being able to bind a single chemokine.

Motile cells (such as leukocytes) will respond to a chemokine concentration gradient, yet it is unclear exactly how this gradient is formed and maintained in vivo. It is unlikely that a soluble chemokine gradient could be sustained at the endothelial cell surface due to rapid blood flow. It is believed that GAGs capture secreted chemokine molecules within the extracellular matrix or on the surface of local cells. This may allow the formation of a solid-phase-stabilized concentration gradient that peaks at the source of chemokine secretion (Kuschert et al., 1999Go; Rot, 1992Go). In support of this, Gilat et al. (1994)Go have shown that MIP-1{alpha} and regulated on activation normal T cell expressed and secreted (RANTES) bind to an ex vivo extracellular matrix preparation in a heparinase-sensitive manner and that these bound chemokines are capable of stimulating T cell adhesion. However, contrary to this view, a recent report suggests that apical endothelial chemokines can promote lymphocyte transendothelial migration (TEM) even in the absence of chemotactic gradients across the endothelial barrier (Cinamon et al., 2001aGo,b).

A number of growth factors and proinflammatory cytokines have been shown to bind to GAGs (Tanaka et al., 1998Go). These include acidic and basic fibroblast growth factors (Ruoslahti and Yamaguchi, 1991Go), several chemokines (Ali et al., 2000Go; Hoogewerf et al., 1997Go; Luster et al., 1995Go), interferon (IFN)-{gamma} (Lortat-jacob and Grimaud, 1992Go), and many interleukins (ILs), including IL-2, IL-5, and IL-10 (Lipscombe et al., 1998Go; Salek-Ardakani et al., 2000Go; Tanaka and Aso, 1998Go). Although the functional implications of these interactions are not fully understood, it has been proposed that proteoglycans protect these small molecules from degradation, act as cytokine storage sites, and aid presentation to specific cell-signaling receptors (Bernfield et al., 1999Go).

RANTES (CCL5), a member of the CC-chemokine family, has received much attention pertaining to its roles in normal homeostasis (Broxmeyer and Kim, 1999Go) and in disease (Conti et al., 1999Go). RANTES has a diverse function in leukocyte trafficking as it induces chemotaxis of T cells, eosinophils, and monocytes (Appay and Rowland-Jones, 2001Go). It is thought that the N-terminus of RANTES is important for triggering leukocyte responses via G-protein-coupled receptors, because N terminally modified met-RANTES is a potent antagonist (Proudfoot et al., 1996Go).

Chemokines are highly basic (with the exception of MIP-1{alpha} and MIP-1ß) with pI values of around 9. All chemokines are able to bind heparin but show varying affinities. A conserved consensus sequence of basic amino acids is found in CC chemokines in the form of the BBXB motif, where B represents either of the basic amino acids arginine and lysine and X any other amino acid. In addition, a BBXXB sequence further upstream toward the C-terminal end is found in RANTES. These sequences may contribute to the binding of RANTES to HS (Burns et al., 1998). However, a recent study has shown that the BBXB motif (44RKNR47) plays a major role in binding RANTES to GAGs (Proudfoot et al., 2001Go)

The relationship between chemokine–GAG interactions and biological activity has only been addressed in a few studies. Maione et al. (1991)Go) have shown that a non-GAG-binding mutant of the CXC chemokine PF4, in which four basic residues in the C-terminal helix are changed to acidic or neutral residues, retains both in vitro antiangiogenic and in vivo antitumor activity. These results indicate that for PF4, heparin-binding capacity and biological activity are not directly related. By contrast, IL-8 fails to bind heparin and has impaired cell activation and receptor-binding properties following truncation of the HS-binding C-terminal helix (Webb et al., 1993Go). It is known that the heparin-binding site on human IL-8 is spatially distinct from the residues involved in receptor binding (Kuschert et al., 1998Go), which suggests that GAGs may play a role in presenting IL-8 to its receptors. However, studies using non–heparin binding mutant of the CC chemokines MIP-1{alpha} (Koopmann and Krangel, 1997Go), MIP-1ß (Koopmann et al., 1999Go), and the CXC chemokine SDF-1 (Amara et al., 1999Go) demonstrate that GAG binding is not a prerequisite for receptor ligation and signal transduction in vitro.

GAGs are known to play an important role in the activities of RANTES. For example, the removal of cell-surface GAGs from PM1 cells inhibits the antiviral effects of RANTES (Oravecz et al., 1997Go). Furthermore, Wagner et al. (1998)Go have shown that the human immunodeficiency virus inhibitory effects of RANTES are augmented when RANTES is complexed to proteoglycans. Although there is some evidence that the interaction between chemokines and GAGs can modify chemokine activity, the role of GAG interaction in leukocyte chemotaxis has not been tested directly.

To assess the importance of GAG binding for the biological activity of RANTES, a series of mutant variants of this protein were constructed by replacement of basic residues in the BBXB motif with the neutral amino acid alanine. These mutants were then used in chemotaxis assays to investigate the role of GAG binding in promoting TEM.


    Results
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
Identification of RANTES amino acids involved in heparin binding and purification of RANTES mutants
On the basis of sequence alignment of several members of the conserved CC-chemokine subfamily, a consensus sequence for the putative heparin-binding domain BBXB (Koopmann and Krangel, 1997Go) in RANTES was identified between residues 44 and 47, where B represents a basic amino acid and X represents any amino acid. A set of point mutants was created in which each of these basic residues was mutated to alanine.

Mutagenesis was achieved by an inverse polymerase chain reaction technique; the methods used are described in a previous study (Proudfoot et al., 2001Go). Wild-type (Wt) and mutant RANTES were generated as recombinant proteins in Escherichia coli.

Ability of the RANTES mutants to induce a chemotactic response.
A series of experiments was performed to determine how mutation of the GAG-binding site altered the ability of RANTES to stimulate leukocyte chemotaxis. A system was established in which two solutions were separated by a non–protein binding polyethylene terepthalate membrane perforated by 3-µm pores; this system allowed a concentration gradient to form within the pores by solute diffusion. Chemotaxis assays were performed in which Wt RANTES or either the R44A, K45A, or R47A mutants was added to the lower well at concentrations ranging between 100 pM to 1 µM and peripheral blood mononuclear cells (PBMCs) were added to the upper well. After incubation for 90 min the number of migrant cells was determined (Figure 1a). In each case it was found that optimal migration was stimulated by a 10 nM concentration of the RANTES species; at this concentration the number of cells stimulated to migrate by Wt RANTES was not significantly different from that stimulated by any of the mutants. This was always a true chemotactic response, because the cells did not migrate significantly by chemokinesis when the chemokine species were present at an equal concentration on both sides of the filter.



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Fig. 1. Biological activity of RANTES and the mutants. (a) Dose-response curves for chemotaxis of PBMCs toward RANTES and its mutants in a free diffusion chemokine gradient. Experiments were performed in duplicate on three separate occasions. Error bars show the SEM. (b) The chemotactic response promoted by 10 nM RANTES and its mutants across a monolayer of cultured HMEC-1. Experiments were performed in triplicate on three separate occasions. Error bars show the SEM.

 

Ability of RANTES mutants to induce transendothelial chemotaxis.
A series of assays was then performed to compare the potential for stimulation of transendothelial leukocyte migration of Wt RANTES with each of the point mutants (Figure 1b). In these experiments the human microvascular endothelial cell line (HMEC-1) was cultured to confluency on the porous filter used previously, each of the chemokine species were added below the filter at a concentration of 10 nM (optimal concentration in transwell chamber), and PBMCs were added to the apical surface of the cells. After incubation, it was found that the R44A and R47A mutants stimulated similar chemotaxis to Wt RANTES; however, the K45A mutant elicited a transendothelial chemotactic response that was 48% smaller than that produced by Wt RANTES under the same conditions (P < 0.025).

Dose response curve of K45A for transendothelial chemotaxis.
Transendothelial chemotaxis in these experiments was carried out using optimal chemokine concentrations defined by chemotaxis experiments performed in free solute diffusion gradients. Further experiments were performed to address the possibility that the reduced response by K45A during TEM could be due to a shift in the dose-response curve in this system.

Preliminary chemotaxis experiments were carried out using PBMCs, but these cells showed down-regulation of chemokine receptor expression with increased time in culture (unpublished data) and variability of receptor expression between different donors. For these reasons, THP-1 cells were used for more extensive chemotaxis experiments to avoid interexperiment variability. This monocyte cell line expresses chemokine receptors at a constant level (Tsou et al., 1998Go), but the cells were treated with IFN-{gamma} (300 IU/ml) for 24 h prior to using them in all experiments to produce a more responsive phenotype (Musso et al., 2001Go).

Initially, the ability of the Wt RANTES at 10 nM to stimulate TEM of monocytes was tested for different periods of time. Although significant migration of THP-1 cells was observed at 90 min, at 2 h the migration was threefold higher (data not shown). Hence, 2 h was chosen as the optimal incubation time for TEM. Wt RANTES and K45A were also tested for vectorial chemotaxis activity. This was demonstrated by showing that the migration induced by both factors was optimal when chemokine was added to the lower chamber only. When the chemokines were added to both chambers no chemokinesis was observed. Figure 2 shows the effect of increasing concentration of RANTES and K45A in stimulating TEM of monocytic cells over a period of 2 h. It was found that at concentrations 10 and 100 nM, respectively, K45A stimulated a response that was 61% (P = 0.001) and 54.3% (P = 0.012) less than the response produced by Wt RANTES under the same conditions. Thus, K45A elicited reduced TEM over a range of concentration compared to the Wt RANTES.



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Fig. 2. Dose response of Wt RANTES and K45A mutant for transendothelial migration of the human monocytic cells (THP-1). Cells were stimulated with varying concentrations of chemokine at 37°C for 2 h. Negative controls include migration in the absence of chemokines, and Wt RANTES CK and K45A CK indicates migration when the Wt RANTES and K45A were present at an equal concentration (10 nM) on both sides of the membrane. Data were normalized to 100% and expressed as means ± SEM from a representative experiment (N = 3) repeated three times with similar results.

 
Role of chemokines presented on the apical surface of endothelium on TEM
A recent report has demonstrated that apical chemokines bound to cell-surface GAGs on inflamed endothelium can trigger robust peripheral blood lymphocyte migration across the cells in the absence of chemotactic gradients (Cinamon et al., 2001bGo).

To further investigate the requirement of GAG binding for apical presentation of chemokines and its role in monocyte TEM, we compared the TEM response generated by the Wt RANTES with that produced by the K45A mutant. Monolayers of tumor necrosis factor (TNF)-{alpha} and IFN-{gamma} activated HMEC-1 cells were used as a model for inflamed vascular endothelium because these cells express the major adhesive ligands found on many inflamed venules (Luscinskas et al., 1996Go). Cytokine-activated cells were treated apically with either Wt RANTES or the K45A mutant (each at 12 nM). RANTES immobilized on the apical surface could promote limited TEM of THP-1 cells across the endothelial cells. In comparison the TEM induced by K45A was significantly lower (P = 0.0001; Figure  3). Assays performed in the absence of exogenously added chemokines (data not shown) and utilizing monolayers prewashed with heparin verified that the subendothelial chemokines endogenously produced by activated HMECs were present in amounts that were too low to exert significant chemotactic activity. These data suggest that leukocyte adhesion and migration through vascular endothelium is dependent on endothelial presentation of chemokines.



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Fig. 3. TEM induced by apical endothelial chemokines. TEM stimulated by HMEC cells (treated with TNF-{alpha} and IFN-{gamma} for 24 h) reconstituted with apically overlaid chemokines (100 ng/ml) at 37°C for 2 h. This assay was performed in triplicate, and the number of migrating cells per high-power field (x 400) was counted for each membrane. Negative controls include migration induced by monolayers prewashed with heparin (100 µg/ml). The mean (± SEM) is shown; result is representative of two independent experiments.

 
Calcium flux
One hallmark of G-protein coupled receptor–mediated chemokine signaling is a transient rise in intracellular free calcium, which is necessary for the cytoskeletal remodeling events that control motility and contractility. Because integrin-mediated leukocyte migration depends on repeated transient increases in Ca2+, we asked whether the inability of K45A to support TEM was due to the effect of mutation on its potential to stimulate Ca2+ flux. Wt Chinese hamster ovary (CHO) cells transfected separately with human chemokine receptors CCR1 (CHO-CCR1) and CCR5 (CHO-CCR5) were used to determine the extent to which these receptors could induce an intracellular Ca2+ flux following stimulation with K45A compared to Wt RANTES. CCR1 was examined in addition to CCR5, because CCR1 is the predominant receptor expressed on mononuclear cells. It was found that the response generated by Wt CHO cells stably transfected with CCR5 or CCR1 was not significantly different when treated either with 10 nM concentrations of Wt RANTES or K45A mutant (Figure 4). These results provide further evidence that interaction with GAGs on the responding cell surface is not necessary for subsequent specific receptor ligation and signal transduction.



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Fig. 4. Calcium-flux results. Representative changes in intracellular Ca2+ concentration following RANTES and the K45A mutant (10 nM) stimulation of Wt CHO cells transfected with chemokine receptor CCR5 or CCR1. Experiments were repeated at least three times and representative experiments are shown.

 
Radioligand binding assays
To determine whether modification of the capacity to interact with GAG influenced interaction between K45A and its specific chemokine receptor, a series of radioligand binding experiments was performed. For these assays, Wt CHO cells stably transfected with human CCR5 were used, and cold ligand competition was performed with Wt RANTES and the K45A mutant; representative results are shown in Figure 5. Binding studies using the Wt CHO-CCR5 cell line indicated that Wt RANTES competed effectively (IC50 1 ± 1.3 nM) with iodinated Wt RANTES. The IC50 measured for the point mutant K45A was 1.8 ± 1.5nM. Thus, the point mutant K45A showed an affinity for this receptor that was not significantly different from that of the Wt protein, indicating that the mutant RANTES binds the receptor with properties that are indistinguishable from the Wt RANTES.



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Fig. 5. Radioligand binding assays. Representative homologous cold-competition curves showing the binding by Wt RANTES and K45A to Wt CHO cells stably transfected with human chemokine receptor CCR5. The IC50 values calculated from this experiment were 1.6 nM and 1.07 nM for Wt RANTES and K45A, respectively.

 
Effect of heparin on chemotaxis by RANTES mutants
Additional experiments were performed to determine the significance for chemotactic activity of interaction between chemokines and GAGs. It was found that incubation of Wt RANTES with heparin at a concentration of 100 µg/ml reduced its potential to stimulate transendothelial chemotaxis by 90%. Significantly, treatment of the K45A mutant with heparin did not decrease further the potential of this species to stimulate transendothelial chemotaxis (Figure 6a).



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Fig. 6. Role of GAGs in supporting TEM. (a) The effect of interaction of chemokines with GAGs on their chemotactic activity was determined. The chemotactic response promoted by 10 nM RANTES and its mutants (filled columns) was compared with that observed after treatment with 100 µg/ml heparin (shaded columns). Experiments were performed in triplicate on three separate occasions. Error bars represent the SEM. These results are normalized for background migration in the absence of chemokine. (b) Results demonstrating the importance of GAGs for supporting leukocyte chemotaxis toward RANTES and K45A. This experiment measured the number of THP-1 cells induced to cross monolayers of Wt CHO and GAG deficient mutant 745-CHO cells in response to stimulation by 10 nM Wt RANTES and K45A. The experiment was performed in duplicate on three separate occasions with similar results; the error bars show the SEM of the data.

 
Investigating the potential of GAGs to support haptotactic responses to chemokines
In view of the significant reduction of transendothelial chemotaxis produced by K45A, the requirement for GAGs in gradient formation was also investigated. Wt CHO and mutant 745-CHO cells, with a defect in normal glycosaminoglycans expression, were used.

To determine the cell-surface expression of GAGs on Wt CHO cells and to verify the lack of GAGs on 745-CHO cells, these cells were stained with anti-HS antibodies. This antibody reacts with N-sulfated glucosamine residues present on the HS GAG chain. Wt CHO cells expressed a high level of cell-surface HS. By contrast, the mutant 745-CHO cells expressed only a minimal amount of HS (P < 0.001) (data not shown). These cells were then used to examine the role of cell-surface GAGs in supporting the chemotactic response toward RANTES and K45A (Figure 6b). It was found that the Wt CHO cells supported efficient migration of THP-1 cells in response to stimulation by Wt RANTES, whereas chemotaxis across the mutant cells was inhibited by 72% (P < 0.001). We further examined the migration of THP-1 cells across the Wt CHO and mutant cells in response to K45A. Chemotaxis was reduced across Wt CHO and 745 CHO by 69% and 78%, respectively, by K45A. Thus, the migration in response to K45A was not significantly different across GAG-expressing and GAG-deficient cells (P = 0.48).


    Discussion
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
A number of chemokines (Kuschert et al., 1999Go; Ali et al., 2001Go), cytokines (Balasubramanian and Ramanathan, 2000Go), and growth factors (Pellegrini et al., 2000Go) are dependent on binding to proteoglycans for proper function. Furthermore, proteoglycan binding may be important for microenvironmental sequestration and presentation of a range of important regulatory factors (Bernfield et al., 1999Go). Previous studies have shown that the RANTES BBXB motif is critical for heparin binding and that mutation within this site causes a selective effect on receptor interactions. In the current study these mutants were used to examine the role of such interactions in the TEM of leukocytes by RANTES. We believe that this allows us to address important questions concerning the relevance of GAG binding to cellular recruitment in vivo; this has been previously hypothesized but not been validated experimentally.

The basic amino acids within the BBXB domain were mutated to a neutral amino acid to nullify potential interactions with anionic GAG domains. The site identified as the proteoglycan-binding site in RANTES represents a well-conserved CC-chemokine motif that appears to have surface accessibility, as demonstrated by nuclear magnetic resonance structural analysis (Skelton et al., 1995Go).

RANTES has a comparatively high affinity for HS compared to other CC chemokines such as MIP-1{alpha} and MIP-1ß. Previous results indicate that the R44, K45, and R47 cluster of residues in the RANTES protein individually contribute to its ability to bind HS, whereas the cluster of basic residues in the K55, K56, and R59 loops preceding the carboxy terminal helix plays no role in heparin binding (Proudfoot et al., 2001Go). Though the mutant protein in which the entire 40s cluster (44RKNR47) is mutated eluted at lower NaCl concentration (0.47 M) compared to the point mutants (0.61–0.65 M), it also showed an 80-fold reduction in affinity for its receptor, CCR1. The biological significance of the loss in CCR1 affinity is shown by its failure to induce chemotaxis of monocytes, which express CCR1 as their predominant RANTES receptor. Hence, for this study we chose to use point mutants of the 44RKNR47 motif to examine the contribution of the HS-binding domain in leukocyte migration. GAG-binding sites are not essentially restricted to the BBXB motif. Though the binding site of several chemokines (such as MIP-1{alpha}, MIP-1ß, and SDF-1) conform to this motif, the main residues involved in binding IL8 (Kuschert et al., 1998Go) and MCP-1 (Chakravarty et al., 1998Go) to heparin are spatially separated.

It was found that Wt RANTES and each of the point mutants promote a similar chemotactic response in free diffusion gradients through endothelial monolayer-free porous supports. On this basis it can be concluded that interaction with HS is not required for migration within a diffusion gradient of RANTES or for receptor ligation. However, it is unlikely that a stable solute concentration gradient could occur in vivo for the periods of time required for tissue inflammation.

To address this issue, a more physiologically relevant model was developed in which chemotaxis was studied across monolayers of cultured endothelial cells. Using this system it was found that Wt RANTES and the R44A and R47A mutants were able to promote equally efficient transendothelial chemotaxis. However, the K45A mutant elicited a smaller chemotactic response in this system, suggesting that this lysine residue might play a biologically important role during the chemotactic response through tissue. To address the possibility that the reduced TEM response elicited by K45A was a consequence of a shift in the dose-response curve, an additional series of experiments was performed with this ligand. It was observed that K45A elicited reduced TEM compared to the Wt RANTES across a range of concentrations.

Significantly, mutation of the K45 residue to alanine did not effect the capacity of RANTES to induce a Ca2+ flux following stimulation of both CCR1 and CCR5 expressing Wt CHO transfectants. Furthermore, this mutation did not have a significant effect on ligand binding to CCR5: the measured IC50 was essentially identical to the value observed for the Wt protein. Proudfoot et al. (2001)Go report a threefold deviation in receptor-binding affinities of mutants K45A and R47A compared to the Wt RANTES for CCR1 using heterologous binding competition assays with 125I-MIP-1{alpha} as radiolabeled ligand. In the current study, radioligand-binding experiments were performed using 125I-RANTES and each of the three point mutants (data not shown). It was found that R44A, K45A, and R47A, respectively, had an IC50 of 3.6 ± 1.6 nM, 1.8 ± 1.5 nM, and 16 ± 2.4 nM for CCR5. Clearly, the largest deviation compared to the Wt RANTES was observed for R47A, but the other point mutants showed an affinity that was not significantly different from that of Wt RANTES. These results suggest that the reduced TEM produced by K45A is not a consequence of reduced receptor affinity.

Chemokines can be constitutively or inducibly produced by endothelial cells or transported and displayed by endothelial cell-surface GAGs. It has been suggested that TEM is triggered by chemokine gradients. However, a recent report has suggested that simple apical presentation of chemokines coupled with intravascular shear forces can promote TEM of lymphocytes (Cinamon et al., 2001bGo). We have examined the requirement for GAG to allow apical presentation by endothelial cells and have found that the number of monocytes induced to undergo TEM by K45A was significantly lower compared to Wt RANTES. In vivo, the contribution of apical and sublumenal chemokines (forming gradients) to the process of leukocyte extravasation will likely vary with the endothelial site, chemokine density and distribution, and the subset of emigrating leukocytes. However, in spite of this variation and as demonstrated by our experiments, the requirement for binding to cell surface GAGs seems essential.

These data are consistent with the finding that soluble heparin, which is known to inhibit interaction between chemokines and solid-phase HS (Kuschert et al., 1999Go), can potently inhibit TEM stimulated by RANTES, presumably by disrupting gradient formation. In contrast, heparin did not diminish further the already reduced chemotactic response produced by the K45A mutant, which already has a reduced potential to bind HS. Previous studies by our group have shown that soluble heparin-like GAGs are able to antagonize the biological activity of IFN-{gamma} (Douglas et al., 1997bGo) and MIP-1{alpha} in vitro (Ali et al., 2000Go). Furthermore, mixture of heparin and MCP-1 prior to incubation with PBMCs has been shown to block the induction of intracellular phosphorylation (Douglas et al., 1997aGo).

Monolayers of GAG-deficient mutant CHO cells were unable to support a normal transmonolayer chemotactic response toward Wt RANTES. However, migration in response to K45A was reduced but not significantly different across Wt CHO and the mutant 745-CHO. We have previously shown the equal chemotactic response toward the tripeptide formyl-methionine-leucine-phenylalanine (FMLP) across the monolayers of Wt CHO and 745 CHO cells (Ali et al., 2001Go). This provides clear evidence that both cell types are physically capable of supporting leukocyte migration. On this basis, it is likely that the reduced chemotaxis across Wt CHO and mutant cells in response to K45A is due to the failure of this chemokine to be bound and presented as a stable transmonolayer concentration gradient.

The reduced potential of K45A to support TEM is intriguing and is in apparent contradiction to the suggestion that despite similar charges, arginine residues bind more tightly than lysine residues (Hileman et al., 1998Go). One possible explanation for this observation is that RANTES, like antithrombin III (Mille et al., 1994Go) undergoes a significant conformational change on binding to GAGs. Thus the tertiary structure might play an important role and K45A could be critical in promoting specific and productive interactions. Furthermore, mutation of the second basic residue within the BBXB domain of chemokines, such as MIP-1{alpha} and MIP-1ß, has resulted in non–heparin binding chemokines (Koopmann et al., 1999Go; Koopmann and Krangel, 1997Go). Unlike MIP-1{alpha} and MIP-1ß, RANTES is a highly basic protein. However, although the 44RKNR47 motif has been identified as a principal heparin-binding site, it is known that mutation of the entire BBXB domain does not produce a complete absence of GAG-binding; hence, residues outside this domain are also thought to play a role in GAG binding.

It is reasonable to propose three modes by which RANTES may be presented to its specific receptor. The first involves presentation of the soluble chemokine to its receptor in the absence of any interaction with HS; this is unlikely to be of importance in vivo given the high affinity of chemokines for GAG molecules. Following interaction with an immobilized GAG, a chemokine can be presented either in a cis or trans fashion. In the former mode, the chemokine, the GAG, and the specific receptor are all present on the same cell surface and potentially might form a trimolecular complex of the form described for presentation of basic fibroblast growth factor (Yayon et al., 1991Go). In the latter mode of presentation, the chemokine is immobilized by GAGs within the extracellular matrix or on an adjacent cell and then presented to a second cell expressing a specific receptor.

As disruption of the interaction between RANTES and HS by site-directed chemokine mutation does not seem to alter the specific stimulation of receptors on CCR5 or CCR1 transfected cells, it can be argued that the cis form of presentation is of minimal significance for cellular chemotaxis. This is consistent with data showing that non–GAG-binding variants of the CC chemokines MIP-1{alpha} and MIP-1ß retain normal receptor binding function and can stimulate chemotaxis in a free diffusion solute gradient (Koopmann et al., 1999Go; Koopmann and Krangel, 1997Go). However, failure of the K45A mutant to stimulate efficient TEM provides evidence in favor of both the trans model for RANTES presentation and the importance of the K45 residue for this process. Further support for the role of trans chemokine presentation during chemotaxis through cell monolayers is provided by the observation that HS-deficient epithelial cell mutants support only a minimal chemotactic response compared with Wt cells.

Identification of the importance of trans presentation of RANTES by HS and of the role of the K45 residue in this process for efficient transendothelial chemotaxis may allow the design of small molecule antagonists that can specifically block chemokine-mediated recruitment of leukocytes to sites of inflammation.


    Materials and methods
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
Cell lines
The HMEC-1 cell line (Ades et al., 1992Go) was propagated in MCDB-131 medium (Sigma) supplemented with epidermal growth factor (10 ng/ml), hydrocortisone (1 µg/ml), and 10% fetal calf serum (FCS). Jeffrey Esko (University of California San Diego, Cellular and Molecular Medicine, La Jolla CA), kindly supplied the Wt CHO and 745-CHO cells. Cells stably transfected with the human chemokine receptor CCR5 and CCR1 separately were maintained in Ham F12 medium supplemented with 10% FCS and G418 (600 µg/ml, Calbiochem).

Cloning of Wt RANTES and generation of non–heparin binding mutants
Human recombinant RANTES was cloned into the vector pT7 (kindly supplied by Dr. Proudfoot), which contains a specific endoproteinase ARG-C cleavable tag (MKKKWPR-RANTES). Cleavage ensures that there is no retention of the initiating methionine, which would render recombinant RANTES protein antagonistic (Proudfoot et al., 1996Go).

Mutagenesis was achieved by an inverse polymerase chain reaction technique (Hidajat and McNicol, 1997Go). The point mutations were introduced into one of the two primers used to hybridize to RANTES in the opposite orientation. The sequences of mutagenic primers and the method used are described in a previous study (Proudfoot et al., 2001Go).

Purification of Wt and mutant RANTES
Purification of the mutant proteins was carried out in collaboration with Dr. Proudfoot (Serono), details of which have been published elsewhere (Proudfoot et al., 2001Go). The mutant proteins were made available for this study. The purity and authenticity of the proteins was verified by reverse-phase high-performance liquid chromatography and mass spectroscopy. RANTES elutes at 0.8 M NaCl on heparin-affinity chromatography, whereas mutants of the BBXB domain R44A, K45A, and R47A eluted at 0.61, 0.65, and 0.65 M NaCl, respectively.

Chemotaxis assays
The ability of Wt RANTES and mutant RANTES to stimulate chemotaxis of PBMC populations was assessed using both a standard transwell chamber assay and a modified assay whereby chemotaxis occurs across an endothelial cell layer. PBMCs were isolated from fresh anticoagulant-treated blood using Ficoll-Hypaque (Pharmacia-Biotech), washed twice in serum-free RPMI 1640 (Life Technologies), and then resuspended in RPMI 1640 containing 10% FCS.

In the transwell chamber assay, chemotaxis was performed in a 48-well micro chamber (Neuroprobe) with a pore size of 3 µm. Purified chemokines in RPMI 1640 with 10% FCS were added in triplicate to the bottom wells of the chemotaxis chamber in a volume of 800 µl. PBMCs were added at a concentration of 5 x 105/well to the upper chamber of each well. Following incubation for 90 min at 37°C, the cells that had migrated through to the lower chamber were isolated and resuspended for preparation and quantification by fluorescence-assisted cell sorting analysis. PBMCs were stained with anti-CD45 antibody (Dako) or CD14 (Dako) for 15 min and washed. Equal numbers of fluorescein isothiocyanate–conjugated flow count beads were added to each of the tubes to aid quantification. Ten thousand events were collected and the cell:bead ratio was calculated to ascertain the degree of cell migration.

In the modified chemotaxis assay (whereby a process of haptotaxis is imitated) endothelial cells (HMEC-1) were grown on transwell filters of 3.0 µm pore size (Falcon) overnight to establish a monolayer. These cells were then stimulated for 24 h with TNF-{alpha} (100 IU/ml) and IFN-{gamma} (100 IU/ml). Chemokine was placed at varying concentrations in the lower well of a 24-well transwell system (Falcon) in 800 µl RPMI 1640. PBMCs or THP-1 cells (monocytic cell line, treated with IFN-{gamma} 300 IU/ml for 24 h) were added at a concentration of 5 x 105 cells/well in a total volume of 500 µl to the upper well of the transwell system. The chamber was incubated at 37°C for 2 h. Quantification of chemotaxis was performed by counting the mean number of migrant cells per high power field. All assays were performed in triplicate.

To establish whether the cell migration was a true chemotactic response, controls included chemokine placed at equal concentrations on both sides of the membrane (CK). Negative controls included migration in the absence of chemokine; migration in the presence of FMLP was used as a positive control.

Similar experiments were performed to measure chemotactic leukocyte migration across cultured Wt CHO and 745-CHO cell lines, following stimulation with 10 nM of Wt RANTES.

The effect of soluble heparin on the chemotactic activity of RANTES was determined by pretreating Wt RANTES (10 nM) with heparin at a concentration of 100 µg/ml for 1 h at 4°C. This RANTES with heparin was then placed in the lower chamber of a 24-well transwell system in the modified chemotaxis assay as described.

Monocyte migration across vascular endothelium bearing apical chemokines
HMECs were grown on transwell filters of 3.0 µm pore size (Falcon) overnight to establish a monolayer. These cells were then stimulated for 24 h with TNF-{alpha} (100 IU/ml) and IFN-{gamma} (100 IU/ml). RANTES or mutant chemokine (12 nM) in MCDB medium-131 (Sigma) were overlaid for 1 h on the endothelial cells and washed extensively. THP-1 cells (treated with IFN-{gamma} 300 IU/ml for 24 h) (5 x 105 in a total volume of 500 µl) were added over the HMEC cells. The chamber was incubated at 37°C in 5% CO2 for 2 h. After this time, the filter was stripped to remove cells from the upper surface, and the cells on the underside were fixed with methanol and stained with hemotoxylin. Quantification of the chemotaxis was performed by counting the mean number of migrant cells per high-power field. All assays were performed in triplicate. Controls included migration in absence of chemokine (negative control) as well as prewashing the monolayers with RPMI containing 100 µg/ml of heparin for 5 min on shaker to remove any chemokines induced by the cytokine treatment. Positive control included migration in presence of FMLP (1 ng/ml).

Calcium mobilization assay
Wt CHO cells stably transfected with either human chemokine receptor CCR5 or CCR1 were detached from 75-cm2 flask with phosphate buffered saline (PBS)–ethylenediamine tetra-acetic acid (EDTA) (3 mM) and resuspended in 1 ml Ham-F12 containing 10% FCS and 3 µg/ml FURA-2 (Sigma). Cells were incubated for 30 min at 37°C and then washed and resuspended at 2 x 106 cells/ml in calcium signaling buffer, composed of 140 mM NaCl, 4.5 mM KCl, 1 mM MgCl2, 1 mM NaH2PO4, 2.5 mM CaCl2, 5 mM glucose, and 5 mM HEPES, pH 7.4. Volumes of 1.5 ml cell suspension were placed in continuously stirred cuvette at 37°C into a fluorimeter (LS-5B, Perkin-Elmer Cetus) for measurement of intracellular calcium. Cells were temperature equilibrated for 10 min, and chemokines were added at concentrations between 10 and 100 nM. Fluorescence was monitored every 1.5 s at excitation wavelengths of 340 and 380 nm and an emission wavelength of 510 nm. The data presented are the relative ratio of fluorescence excited at 340 and 380 nm.

Radioligand binding assays
Wt CHO cells expressing the human CCR5 receptor were used in this experiment. Before assay the transfectant cell line was seeded in flat-profile 96-well plates at 5000 cells/well in Ham’s F-12 medium containing 10% FCS and allowed to attach for 48 h. After this period, the cells were rinsed with PBS and the medium removed by aspiration. Cold-ligand competition assays were performed in a total volume of 150 µl (Hank’s balanced salt solution, 10 mM HEPES, 0.1% bovine serum albumin) using 100 pM 125I-RANTES and a variable concentration of unlabeled chemokine. After incubation of the cells at 37°C for 90 min, the plate was washed with Hank’s balanced salt solution containing 10 mM HEPES and 0.5 M NaCl to remove the unbound 125I chemokines. The cells were then lysed by incubation at 37°C for 2 h in a solution containing 1% SDS and 1 M NaOH. The lysed cells were transferred to test tubes and the radioactivity was measured in a {gamma}-counter. Radioligand-binding parameters were calculated using Prism 3 software (GraphPad).

Antibodies and flow cytometric analysis
The antibody used for detecting cell-surface HS expression was mouse anti-human AB-10E4 (Seikagaku, Japan). Wt CHO and 745-CHO cells were detached from flasks using PBS-EDTA (3 mM) stained with an optimal concentration of antibody (10 ng/µl) at 4°C for 1 h. Cells were then washed and counterstained with a fluorescein isothiocyanate-conjugated secondary antibody for a further 20 min. The stained cells were analyzed by flow cytometry (FACSort, Becton Dickinson). Data analysis was performed using Lysis II software (Becton Dickinson). An irrelevant isotype-matched antibody (IgG1, X931; Dako) was used as a negative control in each labeling experiment.

Statistical analysis
All results are expressed as mean ± SEM of the corresponding replicates. Significance of changes was assessed by application of Student’s t-test. All data was analyzed using Prism 3 software.


    Acknowledgments
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
We are thankful to Dr A.E.I. Proudfoot (Serono Pharmaceuticals Research Institute, Geneva, Switzerland) for her help with the purification of the mutant proteins. This work was supported by the grants from British Heart Foundation and the ROCHE Organ Transplantation Research Foundation.


    Abbreviations
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
CHO, Chinese hamster ovary; CK, chemokinesis; EDTA, ethylenediamine tetra-acetic acid; FCS, fetal calf serum; FMLP, formyl-methionine-leucine-phenylalanine; GAGs, glycosaminoglycans; HMEC-1, human microvascular endothelial cells; HS, heparan sulfate; IFN, interferon; IL, interleukin; PBMCs, peripheral blood mononuclear cells; PBS, phosphate buffered saline; RANTES: regulated on activation normal T cell expressed and secreted; RPMI, Roswell Park Memorial Institute; TEM: transendothelial migration; TNF, tumor necrosis factor; Wt: wild type.


    Footnotes
 
1 To whom correspondence should be addressed; E-mail: simi.ali@newcastle.ac.uk Back


    References
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
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