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Originally published online as doi:10.1189/jlb.1006645 on January 3, 2008

Published online before print January 3, 2008
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(Journal of Leukocyte Biology. 2008;83:875-882.)
© 2008 by Society for Leukocyte Biology

CXCL4-induced migration of activated T lymphocytes is mediated by the chemokine receptor CXCR3

Anja Mueller1, Andrea Meiser, Ellen M. McDonagh, James M. Fox, Sarah J. Petit, Georgina Xanthou2, Timothy J. Williams and James E. Pease3

Leukocyte Biology Section, National Heart and Lung Institute, Faculty of Medicine, Imperial College London, London, United Kingdom

3 Correspondence: Leukocyte Biology Section, National Heart and Lung Institute, Faculty of Medicine, Imperial College London, South Kensington Campus, London SW7 2AZ, UK. E-mail: j.pease{at}imperial.ac.uk


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ABSTRACT
 
The chemokine CXCL4/platelet factor-4 is released by activated platelets in micromolar concentrations and is a chemoattractant for leukocytes via an unidentified receptor. Recently, a variant of the human chemokine receptor CXCR3 (CXCR3-B) was described, which transduced apoptotic but not chemotactic signals in microvascular endothelial cells following exposure to high concentrations of CXCL4. Here, we show that CXCL4 can induce intracellular calcium release and the migration of activated human T lymphocytes. CXCL4-induced chemotaxis of T lymphocytes was inhibited by a CXCR3 antagonist and pretreatment of cells with pertussis toxin (PTX), suggestive of CXCR3-mediated G-protein signaling via G{alpha}i-sensitive subunits. Specific binding by T lymphocytes of the CXCR3 ligand CXCL10 was not effectively competed by CXCL4, suggesting that the two are allotopic ligands. We subsequently used expression systems to dissect the potential roles of each CXCR3 isoform in mediating CXCL4 function. Transient expression of the CXCR3-A and CXCR3-B isoforms in the murine pre-B cell L1.2 produced cells that migrated in response to CXCL4 in a manner sensitive to PTX and a CXCR3 antagonist. Binding of radiolabeled CXCL4 to L1.2 CXCR3 transfectants was of low affinity and appeared to be mediated chiefly by glycosaminoglycans (GAGs), as no specific CXCL4 binding was observed in GAG-deficient 745-Chinese hamster ovary cells stably expressing CXCR3. We suggest that following platelet activation, the CXCR3/CXCL4 axis may play a role in T lymphocyte recruitment and the subsequent amplification of inflammation observed in diseases such as atherosclerosis. In such a setting, antagonism of the CXCR3/CXCL4 axis may represent a useful, therapeutic intervention.

Key Words: human • chemotaxis


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INTRODUCTION
 
Chemokines represent a large family of small peptides that signal via G-protein-coupled receptors and regulate leukocyte recruitment to inflammatory sites and lymphoid microenvironments [1 ]. Platelets act as reservoirs of some chemokines with CCL5/RANTES and CXCL4/platelet factor-4 stored within the {alpha}-granules. Following platelet activation, these chemokines are released in considerable amounts, and CXCL4 is typically deposited in micromolar concentrations [2 , 3 ]. Recent reports have shed some light on the role of the platelet in the early atherosclerotic events and have led commentators to dub them "partners in crime" with chemokines [4 ], as CCL5 and CXCL4 can act in concert to trigger monocyte arrest upon atherosclerotic endothelium [5 6 7 ]. A clinical study by Pitsilos and colleagues [8 ] found CXCL4 to be common in macrophages of early (Grade I/II) lesions and reported a correlation among CXCL4 deposition, lesion severity, and symptomatic atherosclerosis. Animal studies using apolipoprotein E (ApoE)-deficient mice reported that adhesion of platelets to the vascular endothelium of the carotid artery preceded invasion of the plaque by leukocytes and the subsequent development of the atherosclerotic lesions [9 ]. Moreover, blockade of platelet adhesion was found to significantly reduce leukocyte accumulation and attenuate the formation of lesions. Subsequent studies by Huo et al. [10 ] demonstrated that injection of activated platelets into ApoE–/– mice resulted in the formation of platelet-monocyte/leukocyte aggregates, which bind to atherosclerotic lesions, resulting in the deposition of the chemokines CCL5 and CXCL4 from the platelet to the monocyte surface and to the endothelium of atherosclerotic arteries. Abolition of platelet adhesion to the endothelium by the use of P-selectin-deficient platelets resulted in a significant reduction in the size of atherosclerotic lesions [10 ].

It was demonstrated by Lasagni et al. [11 ] that an alternatively spliced version of CXCR3, named CXCR3-B, is expressed by microvascular endothelial cells and activated T lymphocytes. CXCR3-B has an amino-terminal extension of 52 amino acids when compared with the "original" CXCR3 (renamed CXCR3-A) and in addition to binding the three CXCR3 ligands, is also a functional receptor for CXCL4. CXCR3-B mediates the apoptotic but not chemotactic responses of microvascular endothelial cells in response to micromolar concentrations of CXCL4 and interestingly, transcripts for both CXCR3 isoforms were detected in activated T lymphocytes [11 ]. A previous study described a receptor for CXCL4 on activated T lymphocytes that inhibited cell proliferation and cytokine release [12 ]. Here, we examined the possibility that activated T lymphocytes might respond chemotactically to CXCL4 and that this process might be mediated by CXCR3.


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MATERIALS AND METHODS
 
Materials
Recombinant chemokines were from PeproTech EC Ltd. (London, UK). A small molecule antagonist of human CXCR3 [N-{1-R -[3-(4-ethoxyphenyl)-4- oxo-3,4-dihydropyrido[2,3-pyrimidin-2-yl]ethyl}-N-pyridin-3-ylmethyl-2-(4- trifluoromethoxyphenyl acetamide) hydrochloride] was synthesized based on a structure published by Tularik (Patent WO02083143) and described previously [13 ]. Bordetella pertussis toxin (PTX), the mouse anti-human CXCR3 mAb (clone 49801.111), and mouse isotype-matched control antibodies were from Sigma-Aldrich (Poole, UK). Mouse anti-human CD4 and CD8 mAb were from Dako (Ely, UK). The mouse anti-human CXCR3 mAb 1C6 was a kind gift of Leukosite Inc. (Cambridge, MA, USA).

Cell preparation and maintenance
Blood was sampled from healthy, normal subjects, according to the St. Mary’s Hospital Ethics Committee’s (Paddington, London, UK) approved protocol and PBMCs isolated as described previously [14 ]. Lymphocytes were separated from monocytes by allowing the latter to adhere to a tissue-culture flask for 2 h at 37°C and were activated by culture in the presence of IL-2 (100 IU/ml) and Con A (2 µg/ml) for 7 days. The L1.2 cell line was maintained as described previously [15 ]. The Chinese hamster ovary (CHO)-K1 and CHO-745 cell lines were gifts of Dr. Simi Ali (University of Newcastle, UK) and were maintained in HAMS F12 media, supplemented with 10% FCS, as described previously [16 ].

Cell staining
Staining of transfectants and freshly isolated and activated PBMCs was carried out as described previously, using anti-human CXCR3 mAb and the irrelevant isotype-matched controls [15 ]. Staining of activated PBMCs with an anti-CD3-FITC antibody (Dako) revealed CXCR3 expression to be confined to T lymphocytes, as described previously [17 ] (data not shown).

Chemotaxis assays
Assays were carried out as described previously using 5 µm pore, 96-well chemotaxis plates (Neuro Probe Inc., Gaithersburg, MD, USA) [15 ]. Data are presented as the percentage of cells applied to the chemotaxis chamber that migrated through the filter, following incubation at 37°C, following the subtraction of basal migration to buffer alone. In assays using CHO transfectants, filters were pretreated with fibronectin to facilitate migration. Data from these assays are shown as chemotactic indices following division by the basal migratory response to buffer alone.

Intracellular calcium measurements
These were performed as described previously [18 ] using cells that were loaded with the fluorescent dye FURA-2 AM(Molecular Probes Europe BV, Leiden, The Netherlands). Following stimulation with the appropriate chemokine, real-time data were recovered using a fluorimeter (LS-50B, Perkin-Elmer, Beaconsfield, UK). Data are expressed as the relative ratio of fluorescence emitted at 510 nm after sequential stimulation at 340 and 380 nm.

Cloning of CXCR3-B and generation of CXCR3-A and -B transfectants
The CXCR3-A and CXCR3-B open-reading frames were amplified by PCR from human genomic DNA (Promega, Southampton, UK) and inserted into the vector pCDNA3. The murine pre-B cell line L1.2 was maintained and transfected transiently by electroporation, as described previously [15 ]. To enhance receptor expression and chemotactic responsiveness, L1.2 transfectants were cultured in the presence of 10 mM sodium butyrate prior to use. Mock transfections were carried out in the same fashion, except empty pCDNA3 vector was used. CHO-K1 and CHO-745 cell lines stably expressing CXCR3-A were generated by electroporation, and following selection with G418 (600 µg/ml), individual resistant colonies were selected and expanded.

Radioiodination of CXCL4 and competitive radioligand-binding assays
This was carried out as described previously using the chloramine-T method [11 ] and was separated from free iodine following passage down a Sephadex G10 column equilibrated in PBS with 1% BSA. The resulting 125I-CXCL4 had a specific activity of 8–13 µCi/µg and was used at a fixed concentration of 50 or 100 nM. Assays were performed as described previously, with separation of bound-from-free chemokine by centrifugation over oil, following washing of the cells in buffer containing 0.5 M NaCl [15 ]. Each data point was assayed in duplicate, and data are presented as the percentage of binding observed in the absence of cold competitor. Curve-fitting and subsequent data analyses were carried out using the Prism program (GraphPad Software, San Diego, CA, USA), and IC50 values were deducted from nonlinear regression analysis.

Data analysis
Unless otherwise stated, statistical analysis was performed using the unpaired Student’s t-test using Prism (GraphPad Software). P values <0.0005, <0.005, and <0.05 are represented by three, two, or one asterisks, respectively.


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RESULTS
 
CXCL4 signaling in T lymphocytes is mediated via CXCR3 coupled to PTX-sensitive G-proteins
CXCR3 protein was readily detected on the surface of resting T lymphocytes using a specific mAb and was expressed by CD4+ and CD8+ cells (Fig. 1A and 1B ), which correlates with a previous report using the same antibody [19 ]. CXCR3 cell surface expression on T lymphocytes was dramatically up-regulated following culture with IL-2 and Con A (Fig. 1C) , correlating with previous reports of increased CXCR3 expression at mRNA and protein levels following similar culture treatments [17 , 20 , 21 ]. Following culture, the cells were also found to be responsive to CXCL11 and CXCL4 (Fig. 1D) , and CXCL11 induced a typical bell-shaped migratory response from activated T lymphocytes with optimal chemotaxis at 100 nM of the chemokine. In contrast, CXCL4 was much less potent with 1 µM CXCL4 necessary to induce migration, albeit of similar efficacy. The chemotactic responses of activated T lymphocytes to CXCL4 and CXCL11 were inhibited significantly by a CXCR3 antagonist (Fig. 2A ) or by PTX pretreatment (Fig. 2B) , suggesting that one or more of the CXCR3 isoforms mediate CXCL4-induced intracellular signaling and migration via a G{alpha}i-linked pathway. This is in contrast to endothelial cells, where CXCL4 signaling events have been reported to increase cellular cAMP levels typical of G{alpha}s signaling and do not promote chemotaxis [11 ].


Figure 1
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  		Figure 1. Freshly purified and activated T lymphocytes express CXCR3 and migrate in response to CXCL11 and CXCL4. (A and B) Representative staining for CXCR3 on the CD4+ and CD8+ populations of freshly isolated PBMCs. Anti-CXCR3 staining with the mAb 1C6 is shown in the upper histograms, and staining with a suitable isotype control is below. (C) An increase in cell surface CXCR3 levels by PBMCs, as detected by staining with the anti-CXCR3 mAb clone 49801.111 following culture with IL-2 and Con A for the time periods stated. Staining with a relevant isotype control was subtracted. (D) The migratory responses of PBMCs to increasing concentrations of CXCL4 and CXCL11. Data shown are the mean ± SEM of three to four separate experiments using cells isolated from different donors.


Figure 2
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  		Figure 2. CXCL4-induced chemotaxis of activated T lymphocytes is mediated by PTX-sensitive CXCR3 signaling. The migratory responses of T lymphocytes to 50 nM CXCL11 and 1 µM CXCL4 are shown in the presence (solid bars) or absence (open bars) of a specific CXCR3 antagonist (2 µM; A) or following pretreatment (solid bars) of the cells with 100 ng/ml PTX for 18 h at 37°C (B). Data shown are the mean ± SEM of three to four separate experiments using lymphocytes from different donors.

We subsequently assessed the ability of both chemokines to induce intracellular calcium flux in activated T lymphocytes. In keeping with the chemotaxis data, high nanomolar concentrations of CXCL4 were required to induce intracellular calcium flux, in contrast to CXCL11, which mediated a more robust response with tenfold less chemokine (Fig. 3A ). In conjunction with the chemotaxis data, this suggests that binding of CXCL4 to T lymphocytes might be of lower affinity compared with that of other CXCR3 ligands. In the absence of commercially available, radiolabeled CXCL4, we assessed the ability of radiolabeled CXCL10 to be displaced by increasing concentrations of CXCL10 or CXCL4. CXCL10 displaced the homologous radiolabel with an IC50 of 0.58 nM, in contrast to CXCL4, which was unable to effectively displace the radiolabel, even when present at a 300-fold excess (Fig. 3B) . This suggests that CXCL4 and CXCL10 are allotopic ligands of CXCR3 or that CXCL4 binds to T lymphocytes with considerably lower affinity than CXCL10, beyond the level of detection of our assay.


Figure 3
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  		Figure 3. CXCL4 binds to CXCR3 at a site distinct from that of CXCL10 and induces an intracellular calcium flux in activated T lymphocytes. (A) Intracellular calcium flux obtained from activated T lymphocytes in response to CXCL11 and CXCL4. Chemokine was added to the cells at the time-point indicated by the arrowheads. The data shown are representative of three separate experiments using cells from different donors. (B) The displacement of 0.1 nM 125I-CXCL10 from activated T lymphocytes by increasing concentrations of unlabeled CXCL10 (IC50 value of 0.58 nM) and unlabeled CXCL4.

CXCL4 signaling in a pre-B cell line is mediated by coupling of the CXCR3-A and CXCR3-B isoforms to PTX-sensitive G-proteins
As CXCR3 has previously been shown to exist in A and B isoforms, we chose to further investigate the role of either CXCR3 isoform in mediating chemotaxis. cDNA constructs expressing the A or B isoform were expressed transiently in the pre-B cell line L1.2 (Fig. 4A ). As reported previously for endothelial cells [11 ], consistently higher levels of expression were obtained for CXCR3-A transfectants compared with CXCR3-B, making direct comparison difficult. Attempts to generate functional transfectants expressing similar levels of CXCR3-A and CXCR3-B by the manipulation of plasmid and butyric acid concentrations used to drive the transient expression were unfortunately unsuccessful (data not shown). L1.2 cells expressing either CXCR3 isoform were able to respond chemotactically to CXCL11, although the responses of the CXCR3-B transfectants were notably lower in terms of potency and efficacy than those of CXCR3-A transfectants (Fig. 4B and 4C) . This is in agreement with a previous report, where CXCL11 was observed to have higher affinity for the CXCR3-A isoform than for the CXCR3-B isoform, as deduced by competitive displacement of 125I-CXCL10 from microvascular endothelial transfectants [11 ].


Figure 4
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  		Figure 4. CXCR3-A and CXCR3-B transfectants are responsive to CXCL4. (A) The relative surface expression levels of CXCR3-A and CXCR3-B on transiently transfected L1.2 cells as deduced by flow cytometry following staining with the anti-CXCR3 antibody clone 49801.111. Staining with the isotype control was subtracted. (B and C) Chemotactic dose responses of CXCR3-A and CXCR3-B transfectants, respectively, in response to cell stimulation by CXCL11, CXCL10, CXCL9, and CXCL4. Responses of mock-transfected L1.2 cells to CXCL4 are also shown (C). Data shown are the mean ± SEM of three experiments. (D) Chemotactic dose responses of CXCR3-A and CXCR3-B transfectants to increasing concentrations of CXCL4. Calculated EC50 values were 750 nM (CXCR3-A) and 518 nM (CXCR3-B). Data shown are the mean ± SEM of three experiments.

However, in contrast to CXCR3-B microvascular endothelial transfectants, which are chemotactically unresponsive to CXCL4 [11 ], L1.2 transfectants expressing the A or B isoform of CXCR3 responded chemotactically to CXCL4, with migration observed in response to 500 nM chemokine (Fig. 4B and 4C) . This migration was a specific response, as cells transfected with empty vector were unresponsive to CXCL4 (Fig. 4C) . Chemotaxis was subsequently repeated for cells transiently expressing the A or B isoform using an appropriate concentration range of CXCL4 (Fig. 4D) . CXCR3-B transfectants were observed to respond to moderately lower concentrations of chemokine (EC50=518 nM) than their CXCR3-A-transfected counterparts (EC50=750 nM). Chemotactic responses of CXCR3-A and CXCR3-B transfectants to CXCL4 and CXCL11 were inhibited by a small molecule antagonist of human CXCR3 and were sensitive to PTX treatment (Fig. 5A and 5B ), suggesting that CXCL4 signaling in our model system, as in native T lymphocytes, is mediated via a G{alpha}i pathway. As CXCR3-B signaling in microvascular endothelial cells has been reported to be PTX-insensitive [11 ], the G-protein coupling of CXCR3-B appears to be dependent on the cellular context in which the receptor is expressed.


Figure 5
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  		Figure 5. CXCL4-induced chemotaxis of CXCR3-A and CXCR3-B transfectants is sensitive to PTX and a CXCR3 antagonist. (A) CXCR3-A transfectants and (B) CXCR3-B transfectants show the respective chemotactic responses to 50 nM CXCL11 and 1 µM CXCL4 in the presence (solid bars) or absence (open bars) of a specific small molecule antagonist of CXCR3 (1 µM) or following pretreatment (solid bars) of the cells with 100 ng/ml PTX for 18 h at 37°C. Data shown are the mean ± SEM of three to four separate experiments.

CXCL4 binding is of low affinity and is not displaced by a CXCR3 antagonist
We went on to assess the relative affinities of CXCL4 to bind to the CXCR3-A or -B isoforms. Recombinant CXCL4 was radiolabeled in-house and subsequently used in binding assays. As CXCL4 binds with nanomolar affinity to heparin [22 ], initial experiments were performed to reduce background binding to cell-associated glycosaminoglycans (GAGs) to the lowest possible levels by modifying the concentration of NaCl used in the salt wash. CXCL4 bound readily to naïve L1.2 cells, and despite the approximate 65% reduction in binding that was observed with concentrations of 0.4–0.8 M NaCl, significant residual binding of the chemokine remained (Fig. 6A ). Although CXCL4 was originally reported to be eluted from heparin-sepharose at concentrations of 0.9–1.0 M NaCl [23 ], such concentrations of NaCl were, unfortunately, incompatible with our binding assay methodology, which is dependent on separation of cell-bound chemokine from that in solution by centrifugation through oil. At concentrations of NaCl above 0.8 M, the supernatant was found to be denser than the oil, not allowing separation of cell-bound chemokine from free chemokine (data not shown). NaCl (0.5 M) was therefore used as a salt wash step in subsequent binding assays.


Figure 6
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  		Figure 6. CXCL4 binding to CXCR3 is of low affinity and cannot be competed with a CXCR3 antagonist. (A) The residual binding of 50 nM 125I-CXCL4 to naïve L1.2 cells following NaCl washes of varying concentration (mean±SEM of three separate experiments). (B) The displacement of 100 nM 125I-CXCL4 from CXCR3-A transfectants (IC50=1.1 µM) and CXCR3-B transfectants (IC50=860 nM), respectively, by increasing concentrations of unlabeled CXCL4. (C) The relative binding of 100 nM 125I-CXCL4 or 1 nM 125I-CXCL11 to CXCR3-A and CXCR3-B transfectants in the presence (solid bars) or absence (open bars) of a small molecule CXCR3 antagonist (2 µM). Data shown are the mean ± SEM of three separate experiments. ***, P < 0.001, as determined by two-way ANOVA followed by Bonferonni’s post-test.

L1.2 cells transiently expressing either of the CXCR3 isoforms were able to specifically bind radiolabeled CXCL4, albeit with low affinity, with IC50 values of 1.1 µM for CXCR3-A and 860 nM for CXCR3-B (Fig. 6B) . This is consistent with the moderately higher potency of CXCL4 for CXCR3-B than for CXCR3-A transfectants that we observed in chemotaxis assays (Fig. 4D) . Although the small molecule antagonist CXCR3 was able to readily displace radiolabeled CXCL11 from CXCR3-A transfectants, as described previously [13 ], no displacement of radiolabeled CXCL4 from CXCR3-A of CXCR3-B transfectants was observed (Fig. 6C) .

As proteoglycans have previously been reported to play a predominant role in CXCL4 binding to leukocytes [24 , 25 ], we attempted to try and eliminate their effects by using CHO-745 cells, a proteoglycan-deficient variant of the CHO cell line CHO-K1. The CHO-745 cell line has markedly reduced cell surface proteoglycan levels [26 ] and has been successfully used by Ali and coworkers [16 ] to determine the contribution of GAGs to the ligand-binding capabilities of CCR1 and CCR5. Although we were able to stably express our CXCR3-A construct in the CHO-K1 and CHO-745 background (Fig. 7A and 7B ), we were unable to stably express the B isoform (data not shown). CXCR3-A was able to bind CXCL11 when expressed in CHO-K1 and CHO-745 cells (Fig. 7C) , and both lines responded with typical bell-shaped dose-response curves to increasing concentrations of CXCL11, albeit with reduced maxima in the case of the CHO-745 CXCR3-A cell line (Fig. 7E) . In contrast, we were unable to observe specific binding of 125I-CXCL4 to the CXCR3-A CHO-745 line, and the data obtained were almost identical to that obtained with the parental, untransfected CHO-745 line (Fig. 7D) . Similarly, neither the CHO-K1 nor CHO-745 CXCR3-A lines responded to CXCL4 in chemotaxis assays (Fig. 7F) .


Figure 7
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  		Figure 7. CXCL4 is unable to bind to CXCR3-A in the absence of GAGs. (A and B) Representative staining profiles of CHO-K1 and CHO-745 lines stably expressing CXCR3-A. Staining is with an anti-CXCR3 antibody (open histograms) or an isotype control antibody (filled histograms). (C) Binding of 0.1 nM 125I-CXCL11 to both cell lines in the presence (solid bars) or absence (open bars) of a 1000-fold excess of unlabeled CXCL11. (D) Binding of 50 nM 125I-CXCL4 to naïve, GAG-deficient 745-CHO cells or the CHO-745 clone stably expressing CXCR3-A in the presence (solid bars) or absence (open bars) of a 200-fold excess of unlabeled CXCL4. Data shown are the mean ± SEM of four separate experiments. NS, Not significant. (E and F) The chemotactic responses of CHO-K1 and CHO-745 lines stably expressing CXCR3-A to increasing concentrations of CXCL11 (E) or CXCL4 (F). Data shown are the mean ± SEM of three separate experiments. **, P<0.01 as determined by two-way ANOVA followed by Bonferonni’s post-test; NS, not significant.


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DISCUSSION
 
In this report, we have shown that CXCL4 can induce signaling in activated T lymphocytes, which results in their chemotactic migration. This activity is in addition to the previous effects of CXCL4 on T lymphocytes reported by Fleischer and colleagues [12 ], who showed that CXCL4 was able to repress T lymphocyte proliferation via an antigen-specific stimulation model involving coculture with monocytes or by immobilized antibodies directed against CD3 and CD28. This suggests a role for CXCL4 in T cell-mediated immunoregulation. This study is further supported by a recent paper describing the ability of CXCL4 to induce CD4+CD25+ T regulatory cell proliferation and also inhibiting CD4+CD25– T cell proliferation [27 ] and another reporting CXCL4-induced differential regulation of the transcription factors T-bet and GATA-3, suggesting an ability to modulate Th1/Th2 polarization [28 ].

We conclude that the CXCL4-induced migration of T lymphocytes is mediated by CXCR3, as migration was inhibited significantly by a small molecule antagonist of CXCR3 described previously [13 ]. Notably, the migration was not completely inhibited by this molecule, despite the use of a concentration shown in pilot experiments to ablate the migration of L1.2 CXCR3-A transfectants to 50 nM CXCL11 (data not shown). According to the study by Heise et al. [13 ], in which the small molecule antagonist was first described, the compound has a threefold higher inhibitor constant for the displacement of 125I-CXCL11 from PHA/IL-2 T cells (15.8 nM) compared with the transfectant systems CXCR3-CHO and CXCR3-RBL (3.2 and 5.1 nM, respectively). This is curious and may reflect differential coupling of CXCR3 to G-proteins in these cells, which has been shown by Cox and colleagues [29 ] to markedly influence ligand binding. Notably, in assays of chemotaxis using the T cell line H9, migratory responses to CXCL11 were not ablated by this antagonist [13 ]. Interestingly, this phenomenon has also been seen for a different CXCR3 antagonist described by scientists from Pharmacopeia (Princeton, NJ, USA) and Berlex (Montville, NJ, USA) [30 ], in which the migratory responses of activated T cells to 100 nM CXCL10 were ablated by their small molecule antagonist, and responses to 100 nM CXCL11 were inhibited but remained significantly above basal levels.

CXCR3 is unusual amongst chemokine receptors, in that it exists in two distinct isoforms—CXCR3-A and CXCR3-B—as the result of the alternative splicing of mRNA transcripts [11 ]. The CXCR3-A isoform was originally described as a receptor for CXCL9, CXCL10, and CXCL11, binding all three ligands with nanomolar affinity and mediating chemotactic migration [15 , 20 , 31 ]. The alternatively spliced CXCR3-B isoform differs from the CXCR3-A isoform by the inclusion of an additional 52 amino acids at its N terminus. As this region of CXCR3 is important for activation by CXCR3 ligands [15 ], extension of this region, as observed in the B-isoform, might be expected to have an effect upon receptor function. Indeed, CXCR3-B was expressed at much lower levels in the L1.2 cell line (Fig. 4A) , and the chemotactic responses to CXCL9, CXCL10, and CXCL11 were correspondingly diminished (Fig. 4B and 4C) .

A previous study by Lasagni et al. [11 ] used a microvascular endothelial transfectant system and reported that the CXCR3-B but not the CXCR3-A isoform was able to bind CXCL4 with high affinity and that the CXCR3-B isoform was able to induce intracellular signaling but not cellular migration. In contrast to that study, when expressed in the L1.2 cell line, we found that the A and B isoforms of CXCR3 were capable of binding CXCL4, and in keeping with the apparent low affinity, transfectants expressing either receptor were chemotactic to micromolar concentrations of CXCL4. In the study by Lasagni and colleagues, the authors observed that microvascular endothelial transfectants expressing CXCR3-B were coupled to G{alpha}s proteins able to stimulate adenylate cyclase, in contrast to the PTX-sensitive G{alpha}i coupling we describe here for CXCR3 when expressed endogenously by T cells or artificially in L1.2 transfectants. As G-protein coupling has previously been shown to influence the affinity of CXCR3 for ligands [29 ], this differential coupling may explain the observed differences in affinity for CXCL4 that we report. Certainly, CXCR3 appears to interact with CXCL4 in quite a different manner to its interactions with its other ligands, as shown by the inability of CXCL4 to displace CXCL10 from activated T lymphocytes (Fig. 3B) and the finding that a specific antagonist was unable to displace CXCL4 from either of the CXCR3 isoforms, despite being able to readily displace CXCL11 from CXCR3-A (Fig. 6C) .

Our low-affinity binding data are similar to those previously reported by Petersen and coworkers [12 , 25 ], who characterized a low-affinity CXCL4 receptor on T cells and polymorphonuclear neutrophils. In both studies, the authors concluded that CXCL4 cell-surface binding was mediated predominantly by GAGs, notably those decorated with chondroitin sulfate, as treatment with chondroitinase reduced CXCL4 binding and associated responses. As the binding of CXCL4 to heparin is of nanomolar affinity [22 ], we sought to circumvent this binding by the expression of CXCR3 in a GAG-deficient cell line CHO-745. In the absence of GAGs, however, no specific binding of CXCL4 was observed to cells stably expressing CXCR3-A, in direct contrast to the ability of the same cell line to readily bind CXCL11 (Fig. 7C and 7D) . It was also noteworthy that when expressed in the L1.2 background, although both CXCR3 isoforms were able to respond in chemotaxis assays to CXCL4, neither CHO-K1 nor CHO-745 CXCR3-A transfectants were responsive to CXCL4. This might reflect a total absence of CXCL4 binding by the cells and/or an inability of the ligated receptor to transduce a signal. As CXCR3-A uncoupled from G{alpha}i proteins has previously been shown to bind CXCL11 but not CXCL10 [29 ], it may be that expression of CXCR3-A in the non-leukocyte CHO background seriously hampers its biological function. In contrast, both CHO lines migrated in response to CXCL11 with equal potencies, although the magnitude of the CHO-745 response was diminished compared with that of the CHO-K1 line, suggesting that the presence of GAGs may aid the polarization and migration of the cells by sequestering CXCL11 on the cell surface.

We conclude from our data that binding of CXCL4 to cells appears to be of relatively low affinity, especially when compared with other CXCR3 ligands such as CXCL10 and CXCL11 [15 , 31 ]. Such a low-affinity interaction of CXCL4 with CXCR3 also fits with our cell signaling data, in which near-micromolar concentrations of CXCL4 were required for migration of the CXCR3-A or CXCR3-B L1.2 transfectants (Fig. 4D) , and supports the suggestion of Fleischer and coworkers [12 ] that following binding to chondroitin sulfate proteoglycans, CXCL4 interacts with a second surface molecule or coreceptor on T lymphocytes. The evidence we present here strongly implicates CXCR3 as that coreceptor. Although Fleischer et al. [12 ] were unable to demonstrate chemotaxis or intracellular calcium signaling of T lymphocytes in response to CXCL4, this may be explained by the fact that they examined the responses of resting T cells. Although CXCR3 is expressed at the protein level by upwards of 40% of resting T cells, functional CXCR3 responses are only observed following their culture for 7–10 days with IL-2 and a mitogen such as PHA or Con A [17 ], conditions we used in the study we describe here. In the absence of a specific inhibitor of the CXCR3-A or CXCR3-B isoform, we are unable to dissect the role of each CXCR3 isoform in mediating the T lymphocyte response to CXCL4. However, the relative mRNA expression levels of both CXCR3 isoforms suggest that it is likely that the CXCR3-A isoform dominates [11 , 28 ].

Previously published reports have suggested a role for the platelet in the early pathogenesis of atherosclerosis [9 , 10 ], and the localization of CXCL4 to atherosclerotic plaques has been reported, where its presence correlates with the severity of lesions and symptomatic disease [8 ]. Supportive of a proatherogenic role for CXCL4, a recent study found that mice deficient in CXCL4 exhibited a reduced atherosclerotic burden when fed on a lipid-rich diet [32 ]. Although the immunomodulatory activities of CXCL4 on T lymphocytes have been described previously, in light of the data described here, the proinflammatory effects of CXCL4 may also play a role in disease progression. Indeed, the deposition of micromolar concentrations of CXCL4 by activated platelets onto the endothelium may be postulated to result in the recruitment of activated T lymphocytes, leading to their infiltration into the tissues and the amplification of inflammation. If this hypothesis is correct, then blockade of the CXCR3/CXCL4 axis may be an appealing target for the impediment of T lymphocyte recruitment in atherosclerosis.


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ACKNOWLEDGEMENTS
 
This work was supported by grants from the British Heart Foundation (A. M. and A. M., PG/2000055; and S. J. P., FS/05/021), the Arthritis Research Campaign (E. M. M., 17424), and the Medical Research Council (J. M. F). We are grateful to Peter Jose and Paola Romagnani for helpful discussions and to Simi Ali for her kind gift of the CHO-K1 and CHO-745 cell lines.


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FOOTNOTES
 
1 Current address: School of Chemical Sciences and Pharmacy, University of East Anglia, Norwich, NR4 7TJ, UK. Back

2 Current address: Laboratory of Cellular Immunology, Division of Cell Biology, Center of Basic Research, Foundation of Biomedical Research of the Academy of Athens, Athens, Greece. Back

Received October 27, 2007; revised November 16, 2007; accepted December 10, 2007.


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