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The Journal of Immunology, 2002, 169: 5860-5865.
Copyright © 2002 by The American Association of Immunologists

Enhanced Recruitment of Th2 and CLA-Negative Lymphocytes by the S128R Polymorphism of E-Selectin1

Ravi M. Rao2, Dorian O. Haskard and R. Clive Landis3

BHF Cardiovascular Medicine Unit, Faculty of Medicine, Imperial College, Hammersmith Hospital, London, United Kingdom


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
E-selectin is a cytokine-inducible endothelial cell adhesion molecule that binds a restricted population of T lymphocytes consisting of Th1 memory cells bearing the cutaneous lymphocyte Ag (CLA). A serine to arginine (S128R) polymorphism in E-selectin has been reported at increased frequency in patients with systemic lupus erythematosus and atherosclerosis. Here we tested the hypothesis that the S128R substitution may contribute to increased vascular disease by altering the number and/or phenotype of lymphocytes interacting with E-selectin under shear flow. We observed that CHO cell monolayers transfected with S128R recruited significantly greater numbers of unfractionated lymphocytes than monolayers expressing an equivalent density of wild-type (WT) E-selectin. Depletion of the CLA+ subpopulation or generation of CLA- lymphoblasts abolished rolling and arrest on WT E-selectin, but left a residual population that interacted with S128R. Generation of Th subsets revealed preferential interaction of Th0 and Th2, but not Th1, cells with S128R compared with WT. However, only T cells of a memory phenotype interacted with S128R, since neither monolayer supported rolling of CD45RA+ cells. Our results demonstrate that the S128R polymorphism extends the range of lymphocytes recruited by E-selectin, which may provide a mechanistic link between this polymorphism and vascular inflammatory disease.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
E-selectin is a cytokine-inducible endothelial cell adhesion molecule that participates in the initial tethering and rolling of leukocytes before their extravasation at sites of inflammation (1). T lymphocytes differ from monocytes and neutrophils in that they exist as phenotypically distinct subsets within the circulation and are capable of tissue-specific migration (reviewed in Ref. 2). Human T lymphocytes that bind E-selectin are restricted to a subset of skin-homing cells bearing the cutaneous lymphocyte Ag (CLA),4 recognized by mAb HECA 452 (3, 4). CLA acts as the principal human T lymphocyte ligand for E-selectin (5, 6) and has been shown to be expressed as a post-translational modification of P-selectin glycoprotein ligand-1 (PSGL-1) (7). CLA+ T cells fall within the CD45RO+ memory population and coexpress the chemokine receptors CCR4 (8, 9) and CCR10 (10), which restricts their homing pattern to inflamed skin.

Differentiation of naive (CD45RA+) T lymphocytes into memory/effector subsets capable of binding E-selectin takes place in secondary lymphoid tissues and is critically dependent upon induction of {alpha},1,3-fucosyltransferase-VII (FucT-VII) activity (11, 12, 13). This, in turn, is tightly regulated by the prevailing cytokine milieu, such that FucT-VII expression is favored in the presence of the Th1-polarizing cytokine, IL-12, but inhibited in the presence of the Th2-polarizing cytokine, IL-4 (14, 15). Regional homing of Th1 vs Th2 T lymphocytes may underlie the pathogenesis of a number of autoimmune and inflammatory diseases (16).

Several polymorphisms have been described within the selectin gene cluster (17). The S128R polymorphism of E-selectin (in which an uncharged serine is replaced by a positively charged arginine at position 128 within the epidermal growth factor (EGF) domain) is of particular interest, since it has been clinically associated with early-onset atherosclerosis (18, 19, 20) and systemic lupus erythematosus (SLE) (21). In Caucasians, the allele frequency of arginine vs serine at position 128 in early-onset atherosclerosis is ~25 vs 8%, and in SLE it is 15 vs 9%. S128R is a gain-of-function mutation that binds proerythroleukemic K562 cells under static conditions, an interaction not observed with wild-type (WT) E-selectin (22). Recently we have also shown that S128R, but not WT, E-selectin supports sialic acid-independent tethering of myeloid cells under shear flow (23).

In the present study we have tested the hypothesis that S128R may bind a less-restricted subset of lymphocytes than WT E-selectin. To address this hypothesis we perfused CLA+ vs CLA-, Th1 vs Th2, and CD45RO+ vs CD45RA+ subsets over CHO cell monolayers expressing equivalent densities of WT or S128R E-selectin. We show that S128R recruits a broader phenotype of T lymphocytes than WT E-selectin and allows significant accumulation of Th2 and CLA- subsets under shear flow. Such an extended role for E-selectin in lymphocyte trafficking may be an important factor linking the S128R polymorphism to human disease.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Abs and reagents

The mAb SPLAT-1 (anti-E-selectin) was provided by Dr. M. Robinson (Celltech-Chiron Bioscience, Slough, U.K.), and HECA452 (anti-CLA) was purchased from American Type Culture Collection (Manassas, VA). Anti-PSGL-1 (PL-1 and PL-2) Abs and directly conjugated FITC:anti-CD45RO and R-PE:anti-CD45RA Abs were purchased from Serotec (Oxford, U.K.). Anti-rat Ig-conjugated microbeads and anti-CD45RO- and anti-CD45RA-conjugated microbeads were purchased from Miltenyi Biotec (Bisley, U.K.). Recombinant human IL-2 was purchased from Roche (Lewes, U.K.), and rIL-4 and rIL-12 were obtained from BioSource (Nivelles, Belgium). Human AB serum was purchased from the National Blood Service (London, U.K.), and PHA-P was obtained from Sigma-Aldrich (Poole, U.K.).

Generation of lymphocyte subpopulations

Lymphocytes were isolated from healthy volunteers by density sedimentation over Ficoll (Nycomed Amersham, Little Chalfont, U.K.), followed by panning on petri dishes to remove adherent cells. Following panning, monocyte contamination was <2%, as shown by FACS analysis. Lymphocytes were >98% viable, as assessed by trypan blue exclusion. For removal of CLA+ cells, lymphocytes were labeled with HECA452 before magnetic depletion using anti-rat Ig-conjugated microbeads (7). CD45RO+ and CD45RA+ populations were generated by magnetic depletion using anti-CD45RA- and anti-CD45RO-conjugated microbeads, respectively. The purity of undepleted, positively and negatively selected cells was assessed by flow cytometric analysis using appropriate, directly conjugated (FITC and R-PE) Abs. To generate CLA- or CLA+ lymphoblasts in culture, lymphocytes were resuspended at 3 x 106 cells/ml in either RPMI 1640 supplemented with 5% AB+ serum, or XVIVO 15 complete medium (BioWhittaker, Wokingham, U.K.). Lymphocytes were stimulated for 72 h in the presence of 2 µg/ml PHA-P (7), after which they were expanded in the presence of 2 ng/ml IL-2, with splitting and supplementation of fresh IL-2 every 48 h. Expression of CLA and PSGL-1 was monitored daily from day 6 onward, and cells were typically used in the parallel plate flow chamber between days 9–12. Th cell subsets were generated by expansion of PHA-P lymphoblasts in the presence of 2 ng/ml IL-2 (Th0 cells), 2 ng/ml IL-2 plus 2 ng/ml IL-12 (Th1 cells), or 2 ng/ml IL-2 plus 10 ng/ml IL-4 (Th2 cells) as previously described (14) Expression of CLA was monitored daily from 5 days onward, and cells were typically used between 6–9 days of culture.

The generation of CHO cell clones expressing S128R and WT E-selectin has been previously described (23). To identify a pair of precisely matched clones, >50 WT and >150 S128R stable transfectants were screened. This yielded two clones, 1B9 and 15F2, respectively, that expressed nearly identical levels of E-selectin (23). Each clone was then further subcloned, and up to six subclones of each were maintained in continuous culture to allow exactly matched pairs to be picked for each experiment. The expression of WT E-selectin was always equal to or greater than S128R expression. CHO cells were grown to confluence in 9-cm2 Nunc Slide Flaskettes (Nalge-Nunc International, Roskilde, Denmark) and mounted in a parallel plate flow chamber (channel height, 0.15 cm). Untransfected CHO cells were used as a negative control. Lymphocytes in the perfusate were labeled with 1 µg/ml Calcein-AM (Molecular Probes, Eugene, OR) and resuspended at 0.3 x 106 cells/ml in HBSS containing 2% FCS (viscosity, 0.007 Poise) before perfusion at 37°C over CHO cell monolayers at a shear stress of 1.5 dynes/cm2. Where specified, CHO cells were preincubated for 30 min at 37°C with either 50 µg/ml SPLAT-1 (anti-E-selectin) Ab or control Ig. Experiments were visualized using an inverted Diaphot 300 fluorescence microscope (Nikon, Melville, NY) connected to a JVC TK-C1360B color video camera and recorded on a Panasonic AG-6730 S-VHS video recorder (Microscope Service & Sales, Egham, U.K.). Following an initial period of perfusion (2 min) to allow the flow chamber to equilibrate, 10 random fields were recorded for 15 s each using a x10 objective. Images were acquired into a video file (In Video PCI, Focus Enhancements, Campbell, CA) at 15 frames/s, and the number of cells undergoing rolling and/or arrest and the mean rolling velocity were calculated using EML Motion Analysis software (Ed Marcus Laboratories, Brighton, MA). Rolling cells were defined as those with a mean rolling velocity <20 µm/s for at least 1 s. Mean rolling velocities were calculated from measurements of at least 20 cells/field. Arrested cells were defined as those moving <5 µm in 10 s. Since there was donor variability in the proportion of cells demonstrating rolling vs arrest, but not in the total number of adhesive interactions (i.e., rolling plus arrest), data were expressed in terms of total adhesive interactions as well as arrested cells per x20 microscopic field (0.32 mm2).

Statistics

Data are presented as the mean ± SEM number of interacting cells, calculated from at least three independent experiments, taken from at least 10 random fields each. Statistical analysis was performed using one-way ANOVA with a Bonferroni post-test. Statistical significance was assumed at p < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Unfractionated lymphocytes demonstrated significantly increased interactions when perfused at 1.5 dynes/cm2 over CHO cell monolayers bearing S128R compared with equivalent levels of WT E-selectin (Fig. 1GoA, {square}; p < 0.05). The rolling velocity on WT vs S128R was similar (12.36 ± 0.71 vs 10.03 ± 1.16 µm/s). All interactions on both S128R and WT transfectants were abrogated in the presence of the anti-E-selectin Ab, SPLAT-1 (Fig. 1Go, A and B, ), and untransfected monolayers did not support any lymphocyte interaction (data not shown), thus confirming the requirement for E-selectin in the system. The differences in accumulation between S128R and WT were greatest at 1.5 dynes/cm2, since few interactions of CLA+ cells were observed at shear stresses >2 dynes/cm2 (not shown), consistent with other reports (15).



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  		FIGURE 1. The S128R polymorphism of E-selectin enhances interaction with CLA- lymphocytes. Lymphocytes were isolated from volunteer donors and depleted of HECA452+ cells by magnetic bead selection. Fractionated and unfractionated lymphocytes were perfused over CHO cell monolayers expressing equivalent densities of WT or S128R E-selectin at 1.5 dynes/cm2. The number of interacting cells (rolling plus adherent) was calculated as described in Materials and Methods. A, There was a significant increase in the number of lymphocytes interacting with S128R compared with WT monolayers both before and after HECA452 depletion (*, p < 0.05). All interactions were abolished in the presence of the anti-E-selectin Ab SPLAT-1. B, Although no differences in mean rolling velocities were observed when comparing unfractionated lymphocytes perfused over WT or S128R monolayers (see text), there was a significant increase in the number of cells that arrested on S128R monolayers compared with WT both before and after HECA 452 depletion (*, p < 0.05). No cells arrested in the presence of the E-selectin Ab, SPLAT-1. C, Flow cytometric histograms depict the percentage of cells staining positively with HECA452 Ab before and after magnetic bead depletion. Typically, 15–40% of undepleted lymphocytes were HECA452+. D, Following depletion, <5% were HECA452+.

 
The S128R polymorphism of E-selectin allows interactions with CLA- lymphocytes

To determine whether increased recruitment was due to the capacity of S128R E-selectin to interact with CLA- lymphocytes, CLA+ cells were depleted using mAb HECA-452-conjugated magnetic beads. CLA expression varied between donors (15–40% of unfractionated lymphocytes), but depletion with mAb HECA 452 consistently reduced this to <5% the proportion of CLA+ cells regardless of starting CLA levels (Fig. 1Go, C and D). Depletion of CLA+ cells led to almost complete abolition of lymphocyte recruitment to WT E-selectin, whereas a significant residual population of CLA- lymphocytes interacted with and arrested on S128R monolayers (Fig. 1Go, A and B, {blacksquare}; p < 0.05).

Parallel experiments were carried out using cultured CLA+ and CLA- lymphoblasts, prepared by in vitro culture in serum-free or serum-supplemented medium, as previously described (7). Thus, T cells expanded under serum-free conditions (XVIVO 15 medium) have been shown to express elevated levels of FT VII, leading, in turn, to post-translational modification of PSGL-1 and expression of CLA (24). We confirmed that lymphocytes cultured in XVIVO 15 exhibited both PSGL-1 and CLA expression, whereas cells cultured in RPMI 1640 (enriched with 5% AB+ serum) expressed PSGL-1, but no detectable CLA epitope (Fig. 2Go, A and B). CLA+ and CLA- T lymphoblasts thus prepared were perfused over WT or S128R monolayers, and the total number of interactions was quantified as before. No difference in the number of CLA+ cells interacting with either WT or S128R monolayers was observed (Fig. 2GoC, {square}), and again there was no significant difference in rolling velocity (13.35 ± 0.63 vs 10.97 ± 1.09 µm/s). Comparatively few CLA- T lymphoblasts interacted with WT E-selectin, but significantly more CLA- cells were recruited to S128R (Fig. 2GoC, {blacksquare}; p < 0.01). As with unfractionated lymphocytes, there was no difference in the mean rolling velocity between CLA+ and CLA- T cells rolling on WT or S128R monolayers (15.68 ± 1.47 vs 12.84 ± 0.86 µm/s for CLA- cells). All interactions of lymphoblasts with WT or S128R E-selectin were abolished in the presence of the anti-E-selectin Ab, SPLAT-1 (data not shown).



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  		FIGURE 2. The S128R polymorphism of E-selectin enhances interaction with CLA- T lymphoblasts. PBMC were stimulated with PHA-P (2 µg/ml) and grown in either RPMI supplemented with AB+ serum and IL-2 (2 ng/ml; A) or XVIVO 15 and IL-2 (B). Cells were analyzed by flow cytometry using Ab PL-2 (against PSGL-1), HECA452 (against CLA), or control Ab ({blacksquare}). Cells grown in RPMI expressed PSGL-1, but only minimal CLA (arrows), whereas cells grown in XVIVO 15 expressed both PSGL-1 and CLA. C, Following growth in XVIVO 15, no difference was observed in the number of interacting cells on WT or S128R. In contrast, following expansion in RPMI, a significant increase in the number of interacting cells was observed on S128R compared with WT E-selectin (**, p < 0.01).

 
The S128R polymorphism of E-selectin enhances recruitment of Th2 and Th0 cells

A number of previous reports have indicated that E-selectin preferentially recruits Th1 compared with Th2 or Th0 cells (14, 15, 25, 26) To assess whether the S128R polymorphism could extend the range of Th cells recognized, Th1, Th2, or Th0 lymphoblasts were generated in culture as previously described (14). As expected, expression of the CLA epitope was promoted (42%) under Th1 culture conditions (IL-2 and IL-12), but was down-regulated (<=10%) under Th2 (IL-2 and IL-4) or Th0 (IL-2) conditions (Fig. 3Go, A–C). Th1 lymphoblasts interacted well with both WT and S128R E-selectin monolayers (Fig. 3GoD, {blacksquare}), but there was no significant difference between either monolayer and no significant difference in rolling velocity (17.78 ± 1.89 vs 13.87 ± 1.61 µm/s). Th2 and Th0 cells, on the other hand, interacted poorly on WT monolayers (Fig. 3GoD, and {square}), but showed significantly enhanced interaction on S128R (p < 0.05 and < 0.01 for Th2 and Th0, respectively) at the same rolling velocities as Th1 cells (Th2, 13.59 ± 3.43 µm/s; Th0, 12.71 ± 1.89 mm/s). All Th2 interactions on S128R were abrogated in the presence of the anti-E-selectin Ab, SPLAT-1 (Fig. 3GoD).



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  		FIGURE 3. The S128R polymorphism of E-selectin enhances the recruitment of Th2 and Th0 cells. Th1, Th0, and Th2 T lymphoblasts were prepared by stimulation in the presence of PHA-P (2 µg/ml) plus IL-12 (2 ng/ml; A), no additional cytokine (B), or IL-4 (10 ng/ml; C). All cultures received IL-2 (2 ng/ml) from day 3 onward. Percentages indicate the proportion of HECA452-positive cells by flow cytometry on day 7. There was no significant change in the expression of CLA between days 6 and 9 (data not shown). D, Elevated Th1 interactions were observed compared with Th0 or Th2, but this did not differ significantly between WT and S128R monolayers. In contrast, there were significantly more interactions of both Th0 (**, p < 0.01) and Th2 (*, p < 0.05) cells on S128R compared with WT E-selectin. All Th2 cell interactions with S128R were abolished in the presence of anti-E-selectin Ab, SPLAT-1.

 
The S128R polymorphism of E-selectin does not enhance recruitment of CD45RA+ lymphocytes

Previous work has indicated that E-selectin mediates rolling of a subset of CD45RO+ (memory) cells, but not CD45RA+ (naive) cells (8). To address whether the S128R polymorphism alters this paradigm, populations of CD45RA+ and CD45RO+ cells were isolated by positive magnetic selection. Compared with unfractionated lymphocytes, which were 37% CLA+, CD45RO+ cells were 58% CLA+, and CD45RA+ cells were 14% CLA+. When assessed in the parallel plate flow chamber, CD45RO+ cells interacted well with both WT and S128R monolayers, but significantly more cells interacted with S128R (Fig. 4GoC, {blacksquare}; p < 0.01). In contrast, CD45RA+ cells exhibited fewer interactions on WT E-selectin, and this was not enhanced on S128R monolayers (Fig. 4GoC, ). The rolling velocity of CD45RO-enriched cells (15.3 ± 4.46 µm/s on WT; 11.82 ± 1.45 µm/s on S128R) was similar to that of unfractionated lymphocytes (Fig. 1GoB).



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  		FIGURE 4. The S128R polymorphism does not enhance the interaction of CD45RA+ cells. Lymphocytes were positively selected for CD45RA+ or CD45RO+ cells by magnetic bead selection. A, The expression of CD45RA+ and CD45RO+ isoforms was determined by two-color flow cytometry and compared with that of unfractionated lymphocytes. B, Each isoform was further examined for CLA expression by HECA452 staining. The proportion of CLA+ cells increased following positive selection for CD45RO (58%) compared with that of unselected (37%) or CD45RA-selected lymphocytes (14%). C, Both unfractionated and CD45RO+ lymphocytes interacted with WT and S128R, but significantly more cells were recruited to S128R compared with WT monolayers (**, p < 0.01). Interaction with CD45RA+ lymphocytes was minimal, and this was not enhanced on S128R monolayers.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In our previous studies we demonstrated that the S128R polymorphism of E-selectin conferred a gain-of-function phenotype, leading to neuraminidase-resistant tethering of myeloid cells under flow conditions (23). In this study we have extended our characterization of the S128R polymorphism to examine whether lymphocyte recruitment may be altered, in both number and specificity.

We have shown that S128R E-selectin significantly enhances the rolling and arrest of unfractionated and CLA-depleted lymphocytes. This suggests either that non-Th1 subsets or B cells (27) were additionally recruited to S128R. To better define the T cell specificity of S128R interaction, we generated T lymphoblasts under defined culture conditions and found that Th0 and Th2 lymphoblasts were preferentially recruited to S128R compared with WT E-selectin. However, no difference was detected in the recruitment of CD45RA+ lymphocytes between WT and S128R. This is therefore the first study to demonstrate that an adhesion molecule polymorphism can alter the specificity of leukocytes recruited under shear flow. Since all interactions were abolished in the presence of an anti-E-selectin Ab, this ruled out any contribution from other rolling mechanisms, such as very late Ag-4/VCAM-1 (28, 29, 30). Rolling interactions were not fully blocked with anti-PSGL-1 Ab (PL-1) on either WT or S128R (data not shown). This is in keeping with recent observations that other WT E-selectin ligands exist on T cells (31, 32) and furthermore suggests that the augmented interaction on S128R is independent of PSGL-1. In contrast to the recently described 95-kDa WT E-selectin ligand identified in T cell lysates (32), the S128R monolayers interacted with cells that did not express CLA.

The nature of the ligands involved in the recruitment of CLA- and Th2 lymphocytes to S128R E-selectin remains unknown. Unfortunately, we were not able to examine the sialic acid requirement for the enhanced interaction with S128R E-selectin, since even the briefest treatment of lymphocytes with neuranimidase (<30 min) resulted in gross morphological change and significant cell death. Our previous study, however, demonstrated that myeloid cell interactions with S128R were fucose-independent and neuranimidase-insensitive (23). Since neither CLA- nor Th2 lymphoblasts express sufficient FucT-VII to generate adequate functional ligands for E-selectin binding (12, 14, 15), it is likely that augmented lymphocyte recruitment by S128R does not require fucosylation, although further studies would be required to prove this.

In our experimental model we consistently observed that lymphocytes not only rolled, but also arrested, on E-selectin CHO cell transfectant monolayers in the absence of any additional stimulus. Our previous studies have shown that arrest of HL60 cells is {beta}2 integrin dependent, presumably mediated through binding to hamster ICAM-1 (23). Whether contact with E-selectin is sufficient to stimulate {beta}2 integrin adhesion, as has been reported for neutrophils (33, 34, 35), remains an open question, as does the possibility that enhanced interaction with S128R may generate further signals, leading to up-regulated {beta}2 integrin function. Even if no additional signals are generated, our previous studies with {beta}2 integrin-transfected K562 cells have shown that neuraminidase-insensitive tethers on S128R are of sufficient strength and duration to be converted into firm adhesion (23), a principle that has already been established for T cells in the conversion of very late Ag-4/VCAM tethers to static adhesion in the absence of an obligate rolling step (29).

The chemokine receptors expressed by lymphocyte subsets are thought to play a key role in the regional homing of lymphocytes (36). For example, CLA+ lymphocytes recruited to skin also express the chemokine receptors, CCR4 (9), which binds thymus- and activation-regulated chemokine and plays a role in converting rolling into static interactions, and CCR10 (10), which binds cutaneous T cell-attracting chemokine, and plays a role in attracting CLA+ T cells to the epidermis (37). The ability of S128R E-selectin to bind CLA- lymphocytes raises the possibility that CLA- cells may become inappropriately exposed to regional chemokines, thus disturbing their normal homing patterns. To our knowledge, no clinical studies to date have examined the role of this polymorphism in the recruitment of T cell subsets to the affected tissues of patients with either atherosclerosis or SLE. There is some evidence that the presence of Th2 cells may worsen disease pathology in SLE (38), particularly in the chronic stages of murine lupus models (39). Furthermore, a Th1/Th2 switch is observed in severe hypercholesterolemia in the apolipoprotein E-/- mouse (40). The recruitment of CLA- or Th2 cells to skin may also impact on the pathology of diseases such as psoriasis or T cell lymphoma, which are characterized by CLA+ Th1 cell infiltration. Thus, the recruitment of Th2 cells may allow local synthesis of IL-4 and IL-5, leading to recruitment of eosinophils or, possibly, regulatory CD25+ T cells. In the absence of further clinical information, it remains to be seen what impact alterations in T cell subset recruitment would have on disease expression or response to treatment in S128R-bearing individuals.

The S128R polymorphism lies in the EGF domain of E-selectin within a sequence of three amino acids (Ser126-Cys-Ser) that is conserved among all selectins and across species. The cysteine at 127 forms part of a cysteine-rich repeat sequence described in a number of molecules containing an EGF-like domain and is thought to be structurally important due to its ability to form disulfide bonds (41, 42). Although the principal ligand contact points of the selectins lie within the lectin domain (43, 44), domain swaps between L- and P-selectins have suggested that the EGF domain can modulate the binding properties of the lectin domain to surface-immobilized ligand without affecting the equilibrium binding properties toward soluble ligand (45, 46). Thus, substitution of an uncharged serine with a positively charged arginine at residue 128 may influence E-selectin function either directly, by binding novel ligands, or indirectly, by inducing a conformational change in the lectin-EGF domains.

In summary, we have described important functional consequences of the S128R polymorphism in E-selectin that could result in imbalanced lymphocyte recruitment during inflammation and tissue-specific homing. These observations provide a possible mechanistic link between the expression of the S128R polymorphism and increased incidence of atherosclerosis and autoimmune disease.


   Footnotes
 
1 This work was supported by the Arthritis Research Campaign (to R.M.R.) and the British Heart Foundation (to D.O.H. and R.C.L.). Back

2 Current address: Vascular Research Division, Department of Pathology, Brigham and Women’s Hospital, 221 Longwood Avenue, Boston, MA 02115. Back

3 Address correspondence and reprint requests to Dr. R. C. Landis, BHF Cardiovascular Medicine Unit, Faculty of Medicine, Imperial College, Hammersmith Hospital, Du Cane Road, London, U.K. W12 0NN. E-mail address: r.landis{at}ic.ac.uk Back

4 Abbreviations used in this paper: CLA, cutaneous lymphocyte Ag; EGF, epidermal growth factor; FucT-VII, {alpha},1,3-fucosyltransferase-VII; PSGL-1, P-selectin glycoprotein ligand-1; SLE, systemic lupus erythematosus; WT, wild type. Back

Received for publication June 4, 2002. Accepted for publication September 11, 2002.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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