1Department of Chemical Engineering and 2Department of Physics and Astronomy, Ohio University, Athens, Ohio; 3Department of Veterinary and Biomedical Sciences, University of Minnesota, St. Paul, Minnesota; and 4Department of Biomedical Engineering, University of Virginia, Charlottesville, Virginia
Submitted 18 June 2004 ; accepted in final form 23 March 2005
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ABSTRACT |
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adhesion; leukocyte; inflammation
The issue of whether neutrophil PSGL-1 is a functional ligand for E-selectin has received significant attention. Although PSGL-1 isolated from human neutrophils recognizes E-selectin in static fluid phase recognition assays (32), pretreatment of human neutrophils with a MAb to PSGL-1 has been found to have no effect on the rate of tethering to E-selectin under fluid shear conditions in vitro (31). Early work (48) with the PSGL-1 knockout mouse revealed that PSGL-1 is not required for E-selectin-mediated neutrophil rolling in vivo. A later report (47) found that leukocytes from PSGL-1-deficient mice have significantly lower rates of tethering to E-selectin compared with leukocytes from wild-type mice, suggesting that PSGL-1 is an important tethering ligand for E-selectin. We note that there are differences in murine and human leukocytes (17, 19), so extrapolation from what occurs in mice to what is true for human neutrophils should be made with caution.
The role of PSGL-1 in lymphocyte adhesion to E-selectin has also received considerable attention and provides insights into the study of neutrophil PSGL-1. Hirata et al. (16) reported that Th1 cells derived from PSGL-1-deficient mice exhibited a significant decrease (89%) in their ability to bind to E-selectin under semistatic conditions and also exhibited decreased migration into the skin when injected into P-selectin-deficient mice. PSGL-1 isolated from murine CD8+ T cell clones can exist in a form able to bind E-selectin and not able to bind E-selectin and the ability to bind to E-selectin correlates with expression of HECA-452 reactive epitopes (3). Note that the presence of HECA-452 often correlates with the ability to bind to E-selectin (3), although the HECA-452 epitope may not actually bind to E-selectin (18, 44). With the recognition that the adhesion mediated by selectins is coupled to fluid shear, a recent study (13) significantly extended these studies with the use of a blot rolling assay to determine whether PSGL-1 isolated from human T cells and bearing HECA-452 reactive epitopes (CLA+ PSGL-1) could support the adhesion of cells expressing E-selectin under fluid shear conditions. Their results demonstrated, for the first time, direct real-time observation of E-selectin-mediated rolling on immobilized HECA-452-positive PSGL-1 derived from T cells (13).
Similar to the study of T cell-derived PSGL-1 (13), it is of interest to probe the interaction of human neutrophil derived PSGL-1, in isolation, with endothelial expressed E-selectin under defined fluid shear conditions. In this regard, Chinese hamster ovary cells stably expressing E-selectin have been reported to roll under fluid shear on a broad 140-kDa glycoprotein band (presumably monomeric PSGL-1) isolated from HL-60 cells (11). We (14) previously reported that microspheres coated with recombinant PSGL-1, generated in the presence of a fucosyltransferase, tether and roll on E-selectin expressing endothelium. While these studies strongly suggest that neutrophil PSGL-1 can support adhesion to E-selectin, they have important limitations. First, the PSGL-1 microsphere experiments were performed with recombinant PSGL-1 (14). This is a key point because it is predominantly the carbohydrates that bind to E-selectin and recombinant PSGL-1 may not be glycosylated in the same manner as native PSGL-1 (13). Second, the E-selectin ligands require posttranslational modifications that are important for conferring the ability to bind to E-selectin and these modifications can vary with species (e.g., mouse vs. human), cell type (e.g., neutrophil vs. lymphocyte), and maturation/activation (e.g., effector/memory T-lymphocytes) (3, 17, 19). Indeed, these arguments were elegantly made in a recent study (13) and motivated, along with recognition of the importance of fluid shear, the blot-rolling assay of T cell-derived PSGL-1.
In addition to PSGL-1, many other leukocyte surface molecules, each of which are decorated with sLex-type glycans, have been suggested as possible underlying scaffolds that present glycans for binding to E-selectin. These include L-selectin (32, 34, 49), CD11b/CD18 (8), E-selectin ligand-1 (22, 42), CD66-nonspecific cross-reacting antigens (20), CD44 (11), CD43 (24), and certain glycolipids (5). Leukocyte-sized microspheres conjugated with sLex alone tether and roll on surfaces coated with E-selectin (4). Whereas each of the named leukocyte molecules recognize E-selectin, and some have even been shown to support tethering of leukocyte-sized particles under flow (5, 8, 14), it is unclear whether each proposed ligand supports similar adhesion (e.g., similar rates of initial tethering, similar rolling velocity) to E-selectin. Cellular effects aside [e.g., the topological distribution of the ligand on the cell surface (43)], several biophysical studies (2, 7, 40) indicate that a given ligand-receptor bond must have unique biophysical properties to mediate tethering and rolling on E-selectin and these properties play a key role in determining the characteristics of the adhesion mediated by the ligand- receptor pair. Given that each ligand for E-selectin has a unique biochemistry (e.g., perhaps differences in the antigen that binds to E-selectin or the scaffold that presents the antigen for binding to E-selectin) that will presumably give rise to unique biophysical properties, we hypothesized that ligand biochemistry plays a key role in dictating the characteristics of adhesion to E-selectin.
These considerations led us to conjugate leukocyte-sized microspheres with either sLex or PSGL-1 purified from myeloid cells (neutrophils and HL-60) and compare their adhesion to endothelial expressed E-selectin under defined shear conditions. We found that PSGL-1 supports tethering and rolling to E-selectin and the rate of tethering is significantly greater than that supported by multivalent sLex. Furthermore, we found that pretreatment of the PSGL-1 and sLex microspheres with HECA-452 did not inhibit tethering to E-selectin. These results support the hypotheses that 1) PSGL-1 is a high-efficiency tethering ligand for E-selectin, 2) ligand biochemistry can significantly influence initial tethering to E-selectin, and 3) PSGL-1 tethering to E-selectin can occur via non-HECA-452 reactive epitopes.
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MATERIALS AND METHODS |
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Antibodies. Murine MAb to human E-selectin (7A9, IgG1) was generously provided by Dr. Francis W. Luscinskas (Brigham and Womens Hospital, Boston, MA). Murine MAb to human E-selectin (HEL3/2, IgG2a) was a generous gift from Dr. Raymond T. Camphausen (Wyeth Research; Cambridge, MA). Murine MAb to human PSGL-1 (KPL-1, IgG1), rat MAb HECA-452 (IgM) and fluorescein isothiocyanate (FITC)-labeled HECA-452 (6:1 Fluorophore/Protein ratio) were from BD Pharmingen (San Diego, CA). Murine MAbs to human PSGL-1 (PL1 and PL2, IgG1) were from Calbiochem (San Diego, CA) and SeroTech (Raleigh, NC), respectively. Murine MAbs to human L-selectin (LAM114, IgG1) was a generous gift from Thomas F. Tedder (Duke University, Durham, NC), to human CD18 (TS1/18, IgG1) was from Endogen (Woburn, MA), to human CD49d (BU49, IgG1) and E-selectin (HAE-1f, IgG1) were from AnCell (Bayport, MN). Nonspecific mouse IgG was from Sigma. Nonspecific rat IgM was from Zymed (San Francisco, CA). Nonspecific FITC-labeled rat IgM was from Santa Cruz Biotechnology (Santa Cruz, CA). FITC-labeled goat F(ab')2 anti-mouse IgG Fc-specific (2.4:1 fluorophore/protein ratio), and FITC-labeled goat F(ab')2 anti-rat IgM polyclonal antibodies were from Jackson ImmunoResearch Labs (West Grove, PA). Peroxidase-conjugated goat F(ab')2 anti-mouse IgG (Calbiochem) and anti-rat IgM (Jackson ImmunoResearch) polyclonal secondary antibodies were used to detect the primary MAbs in the ELISA.
Cell culture.
HUVEC were purchased from Clonetics (San Diego, CA) and cultured as described previously (9). The HUVEC were cultured within autoclave-sterilized 5-mm flexiPERM gaskets mounted at the center of 35-mm tissue culture dishes. To induce E-selectin expression, HUVEC were pretreated with 50 U/ml of IL-1 for 4 h before use in the adhesion studies. We have observed that VCAM-1 protein expression on HUVEC in response to IL-1
stimulation is considerably less compared with VCAM-1 protein expression in response to TNF-
-stimulation. Thus, we chose to activate the HUVEC with IL-1
, as opposed to TNF-
, because we wanted to limit the expression of VCAM-1 while achieving E-selectin expression. HL-60 cells, cultured as previously described (9), were withdrawn from culture, washed, and resuspended to 1 x 108 cells/ml in RPMI 1640 and held (<4 h) at 4°C until used in the assays.
Neutrophil isolation. For PSGL-1 isolation, peripheral blood was collected from normal healthy donors, using sodium heparin. This procedure was performed in accordance with a protocol approved by the Institutional Review Board Human Subjects Committee at the University of Minnesota. Neutrophils (polymorphonuclear leukocytes) were isolated similar to previous methods (25, 45). For flow cytometric analysis, neutrophils were isolated from human venous blood by a modified Ficoll density gradient centrifugation using mono-poly resolving medium (ICN Biochemicals, Aurora, OH), followed by hypotonic lysis of RBCs. This procedure was performed in accordance with a protocol approved by the Institutional Review Board Human Subjects Committee at Ohio University.
Preparation of PSGL-1 and sLex microspheres. The technique for generating PSGL-1 microspheres was similar to that described previously (8, 30). Briefly, 9.70 µm microspheres were washed in Tris buffer and incubated overnight at 4°C in a solution containing PSGL-1 diluted 1:30 in Tris buffer. The next day the microspheres were washed and resuspended to 1 x 108/ml in blocking buffer. A similar procedure was used to generate the glycophorin and asialoglycophorin microspheres. To generate sLex and biotin microspheres, 9.95-µm superavidin-coated microspheres were washed and incubated in blocking buffer. Subsequently the microspheres were incubated (1 x 107/ml) in biotinylated multivalent sLex (diluted to different concentrations in blocking buffer) or D-biotin for 1 h at room temperature. After the incubation period, the microspheres were washed and resuspended to 1 x 108/ml in blocking buffer. HECA-452 microspheres were generated by incubating 9.70-µm microspheres (1 x 107/ml) overnight in a solution containing HECA-452 (40 µg/ml in PBS). The next day, the microspheres were washed and resuspended to 1 x 108/ml in blocking buffer. Before perfusion through the parallel plate flow chamber, microspheres were diluted to 5 x 105/ml in assay buffer. The microspheres were used in the flow cytometric analysis and adhesion assays within 4 h of preparation.
Enzymatic treatments of PSGL-1 microspheres. In certain cases, the PSGL-1 microspheres were treated with enzymes before use in the assays. For these experiments, the PSGL-1 microspheres were incubated in blocking buffer (supplemented with 25 mM HEPES) containing OSGE (160 µg/ml; 30 min at 37°C), neuraminidase (0.1 U/ml; 30 min at 37°C), or no enzymes (30 min at 37°C). After the incubations, the PSGL-1 microspheres were washed and resuspended to 1 x 108/ml and used within 4 h of preparation.
ELISA. ELISA was performed similar to that described previously (10). HUVEC were washed with HBSS+, fixed in 1% formaldehyde at 4°C for 20 min, washed, and incubated in M199 containing 8% FBS. All antibodies were diluted with HBSS+, 1% BSA. Antibodies and rat IgM were added (10 µg/ml) and the HUVEC were incubated at 4°C for 20 min. After incubation, the wells were washed, and a peroxidase-conjugated polyclonal antibody to mouse IgG or rat IgM was added (diluted 1:50). After a 20-min incubation period at 4°C, the wells were washed and treated with O-phenylenediamine dihydrochloride dissolved in phosphate citrate buffer containing sodium perborate. After a 10-min incubation period, the absorbance of each well was determined at 450 nm using a micro-well plate spectrophotometer (Molecular Devices, Sunnyvale, CA).
Flow cytometric analysis.
Aliquots of 2 x 105 microspheres or neutrophils were washed with blocking buffer and incubated with unlabeled primary MAbs, FITC-labeled HECA-452 primary MAb, or FITC-labeled rat IgM (20 µg/ml). Subsequently, those microspheres or cells incubated with unlabeled primary MAbs were washed and incubated with FITC-labeled polyclonal antibodies (1:50 dilution). The microspheres or cells were finally washed and fixed in 1% formaldehyde. FITC fluorescence of microspheres or cells was determined using a FACSort flow cytometer (Becton-Dickinson Immunocytometry Systems, Mountain View, CA) and plotted on a four-decade log scale. All antibodies were diluted in blocking buffer and incubations were for 20 min. To make a relative comparison of the neutrophils, PSGL-1, and sLex microspheres, we used Quantum-26 calibration beads to generate standard curves. These curves were used to convert flow cytometric MCF data to molecules of equivalent soluble fluorochrome (MESF). The MESF of an appropriate negative control was subtracted out to arrive at the Net MESF/particle. Net MESF was divided by the surface area of the microspheres or neutrophils to get Net MESF/µm2. Net MESF/particle and Net MESF/µm2 are reported in Table 1. In making the comparison of PSGL-1 and HECA-452, we used the anti-PSGL-1 MAb KPL-1, followed by a FITC labeled polyclonal secondary (2.4:1 fluorophore/protein ratio) to detect PSGL-1 and a FITC-labeled MAb HECA-452 (6:1 fluorophore/protein ratio) to detect HECA-452 antigen. To account for the differences in f/p ratios, the PSGL-1 MESF was multiplied by 6 and divided by 2.4 to allow comparison to the HECA-452 MESF. Note that with the use of the primary, followed by polyclonal secondary, detection antibody for PSGL-1, compared with the FITC monoclonal detection used for HECA-452, gives an upper bound on the relative PSGL-1 MESF compared with HECA-452 MESF.
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Data analysis. Initial tethering was quantified by determining the number of microspheres that attached from the free stream to the HUVEC (primary attachment) during the first 2.5 min of flow. This number was normalized to the area of the field of view and the duration (2.5 min) of the observation. Secondary attachments or particles that rolled into the field of observation from the upstream region were not counted as initial tethering events. Note that the HUVEC were cultured in flexiPERM gaskets in the center of the 35-mm dishes. This resulted in 35-mm dishes that were only partially covered with HUVEC. The observations were made near the center of the flow chamber and at the first field of view that had HUVEC (i.e., substrate upstream of the field of view under observation had no HUVEC). This resulted in the acquisition of adhesion data that was not confounded by events that may have occurred upstream. To evaluate the percent detachment, the number of microspheres that tethered to the HUVEC and subsequently detached from the HUVEC and reentered the free stream during the first 2.5 min of flow was determined. This value was divided by the number of initial tethering events to yield the percent detachment. Note that microspheres that rolled out of the field of observation were not counted as detachment events. To determine the percentage of microspheres that were rolling, microspheres that moved during a 5-s period of observation were scored as rolling. This number was divided by the total number of adherent microspheres to arrive at a percent rolling. The rolling velocities, determined as described previously (39), of at least 10 rolling microspheres were averaged to give the rolling velocity for a particular experiment. Initial tethering, percent detachment, percent rolling, and average rolling velocity determined on one HUVEC monolayer represented the result for a single experiment. Multiple (n) adhesion assays were run and averaged to give the results presented in the figures.
Statistics.
Statistical differences between two means were estimated using unpaired Students t-tests. In case of multiple comparisons against a single control, a single-factor ANOVA coupled with Bonferronis test was used. P values 0.05 were considered statistically significant. All error bars represent means ± SE.
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RESULTS |
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PSGL-1 isolated from human neutrophils is more efficient than multivalent sLex at mediating initial tethering to endothelial expressed E-selectin. The above data demonstrates that PSGL-1 isolated from human neutrophils can support initial tethering of leukocyte-sized particles to endothelial expressed E-selectin under flow. We next sought to compare adhesion mediated by PSGL-1 to that mediated by sLex. For this comparison, we used sLex microspheres that have previously been shown to tether to solid supports coated with recombinant E-selectin (4). Note that the sLex is presented on a polymer scaffold to create a multivalent array (multivalent sLex). As shown in Fig. 3A, by increasing the concentration of sLex used during the coupling procedure, we were able to generate sLex microspheres with increasing HECA-452 reactivity. We used flow cytometric analysis and calibration beads to compare the microspheres. The results of this analysis are given in Table 1 where it is revealed that the HECA-452 reactivity on PSGL-1 microspheres is significantly less than all of the sLex microspheres.
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We next made a direct comparison of the adhesion mediated by PSGL-1 and sLex. To make this comparison, we used the set of sLex microspheres described in Fig. 3 and Table 1. As is true for leukocytes, ligand-coated microspheres can adhere to HUVEC via a multistep cascade involving initial tethering, rolling, and firm adhesion (8, 14). Subsequent to initial tethering, the microspheres may also release from the substrate and reenter the free stream. Thus, to compare the adhesion of the microspheres, we did a detailed analysis of the initial tethering, the detachment, the percent of adherent microspheres that were rolling, as opposed to firmly adherent, and the rolling velocity of the microspheres. As shown in Fig. 4, the rate of initial tethering of the PSGL-1 microspheres to the 4 h IL-1 activated HUVEC was significantly greater than the sLex microspheres despite the fact that all of the sLex microspheres had greater HECA-452 reactivity than the PSGL-1 microspheres (Table 1). Note also that some of the sLex microspheres (i.e., those prepared with 1.0, 0.5, and 0.25 µg/ml sLex) appeared to have significantly greater (
10x or more) ligand density (i.e., HECA-452 reactivity) than the ligand density (i.e., MAb to PSGL-1 reactivity) of the PSGL-1 microspheres (Table 1). Interestingly, ANOVA revealed that the rate of initial tethering of the sLex microspheres was not a function of the level of sLex present on the microspheres.
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DISCUSSION |
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The data presented in Fig. 4 support the hypothesis that there are distinct differences in the ability of the E-selectin ligands sLex and PSGL-1 to support initial tethering to E-selectin. Specifically, we found that PSGL-1 microspheres had a higher rate of initial tethering to endothelial expressed E-selectin compared with sLex microspheres, even though the HECA-452 reactivity of all the sLex microspheres was significantly greater than for the PSGL-1 microspheres. Similar results were observed with microspheres coated with PSGL-1 isolated from HL-60 cells (data not shown). One can use the MESF values reported in Table 1 to compare the ligand densities of PSGL-1 and sLex microspheres (i.e., comparing PSGL-1 on PSGL-1 microspheres to HECA-452 antigen on sLex microspheres). The PSGL-1 microspheres had higher rates of initial tethering than sLex microspheres with significantly higher ligand surface densities (e.g., compare 1.0, 0.5, and 0.25 µg/ml sLex microspheres to PSGL-1 microspheres in Fig. 4 and Table 1). Note that the values in Table 1 were adjusted for differences in f/p ratios of the FITC antibodies used to detect PSGL-1 and HECA-452 antigen. This adjustment, along with the fact that PSGL-1 was detected with a FITC polyclonal secondary antibody subsequent to treatment with a PSGL-1 MAb and the HECA-452 antigen was detected using a FITC labeled HECA-452 monoclonal antibody, makes this a reasonable comparison that likely overestimates the relative level of PSGL-1. Interestingly, on the PSGL-1 microspheres, the Net MESF value for PSGL-1 was higher than the Net MESF value for HECA-452 (Table 1) despite the fact that PSGL-1 can potentially express HECA-452 epitopes on multiple side chains per PSGL-1 molecule. This somewhat counterintuitive observation may be explained, in part, by the difference in the detection techniques, essentially polyclonal for PSGL-1 vs. monoclonal for HECA-452, that may lead to an overestimation of the relative level of PSGL-1. It is noteworthy that the rate of initial tethering of sLex microspheres was not a function of the level of sLex (Fig. 4), suggesting that the rate of tethering of the sLex microspheres was limited by the rate of formation of bonds to E-selectin. Interestingly, this limit was reached at a relatively low sLex surface density.
The above observations suggest that either PSGL-1 is more efficient at presenting HECA-452 reactive epitopes than sLex or that non-HECA-452 reactive epitopes on PSGL-1 bind to E-selectin. In an initial step to resolve these two possibilities, we tested the effect of pretreating the PSGL-1 microspheres with HECA-452 before the adhesion assay. Quite interestingly, we found that HECA-452 did not inhibit initial tethering of the PSGL-1 or the sLex microspheres (Fig. 8). Other investigators have found that 1) pretreatment of HECA-452 positive T-lymphoblasts with HECA-452 does not inhibit rolling on E-selectin (18) and 2) a variant HL-60 cell line that does not express HECA-452 is able to bind to E-selectin (44). These and similar observations have led to the hypothesis that HECA-452 is a marker for ability to bind to E-selectin but does not define the actual epitope that binds to E-selectin (44). The data presented in this paper supports this hypothesis. Furthermore, our results (Fig. 8B) suggest that the antigenic epitope of HECA-452 comprises structural features of sLex that are distinct from the E-selectin recognition site.
Our finding that PSGL-1 is a high-efficiency tethering ligand for E-selectin is in agreement with two recent studies. First, as noted in the introduction, leukocytes from PSGL-1 deficient mice were found to have significantly lower rates of tethering to E-selectin compared with leukocytes from wild-type mice, suggesting that PSGL-1 is an important tethering ligand for E-selectin (47) [although there are differences in murine and human leukocytes (17, 19) and thus extrapolation should be made with caution]. Second, Hanley et al. (15) recently reported that the frequency of binding events of E-selectin-coated cantilevers to human neutrophils was significantly reduced by pretreatment of neutrophils with a MAb to PSGL-1 despite the fact that the MAb presumably would not block the numerous sLex glycans present on a variety of non-PSGL-1 proteins (28) and glycolipids (5). The present study bolsters the hypothesis that PSGL-1 is a major tethering ligand for E-selectin and suggests that the biochemical/biophysical attributes of PSGL-1 (e.g., perhaps the presentation of the relevant epitope on O-linked glycans) contribute to its ability to be a highly efficient tethering ligand for E-selectin. While other studies have revealed that the topological distribution of the ligand on the cell surface can significantly influence its ability to initiate tethering (43), the relatively long PSGL-1-E-selectin bond length may also be a factor in the high tethering rate of PSGL-1 for E-selectin (33). The length scale of the microvilli effect is on the order of 0.5 µm (38). The length of the ligand, a nanometer scale issue, is not strictly controlled in our study and could have an effect. We point out, however, that the sLex construct used is multivalent sLex presented on a polymer backbone whose length may be as much as 30 nm. This length is not insignificant and compares reasonably well with 60 nm, the reported length of PSGL-1 (33). In any case, by using the microsphere approach we have eliminated the relatively large 0.5-µm scale issue that is present on neutrophils allowing a more direct comparison of ligands when presented on a relatively standardized and uniform topology.
Analysis of posttethering adhesion (Figs. 57) revealed that, in contrast to what was observed for initial tethering (Fig. 4), posttethering adhesion was a function of the level of sLex. This result demonstrates that initial tethering becomes independent of ligand density at a much lower ligand density than posttethering adhesion. With the use of Table 1 to compare the ligand densities of PSGL-1 and sLex microspheres (i.e., comparing PSGL-1 on PSGL-1 microspheres to HECA-452 on sLex microspheres), it appears that PSGL-1 microspheres are less stably adherent than sLex microspheres that have an order of magnitude greater ligand density (1.0 and 0.5 µg/ml sLex), as stably adherent as sLex microspheres that have greater ligand density but not in excess of an order of magnitude (0.25 and 0.125 mg/ml), and are as stably adherent as sLex microspheres with similar ligand densities (0.06 mg/ml). Thus, although PSGL-1 appears to be more efficient at mediating initial tethering to E-selectin than sLex, it is unclear whether PSGL-1 is more efficient than sLex at mediating posttethering rolling on E-selectin. In this regard, it is interesting to note that Xia et al. (47) found no difference between leukocytes isolated from PSGL-1-deficient mice and leukocytes isolated from wild-type mice with regard to posttethering (i.e., rolling velocity and detachment) to E-selectin. Similarly, it has been reported that chymotrypsin treatment of neutrophils, while significantly inhibiting tethering, has little impact on rolling (21).
Finally, we would like to comment on the relevance of this work to targeted drug delivery. Recently there has been an increasing focus on the development of polymeric and lipid based drug carriers that are targeted via endothelial cell adhesion chemistry (12, 35, 36). In general, the approach is to conjugate a ligand for an endothelial cell adhesion molecule that is discretely upregulated at the target site. For example, several studies suggest that E-selectin is upregulated at sites of inflammation and could be used as a target for directed drug delivery to sites of inflammation (41). A variety of ligands could be chosen to target E-selectin including MAbs, and recombinant or native forms of "natural" ligands for E-selectin. The results of the present study suggest that the choice of a ligand such as PSGL-1 could potentially enhance the binding of the carrier to E-selectin-targeted endothelium and thus the performance of the drug delivery carrier. Consequently, both the biochemistry of the ligand, as well as the particular hydrodynamics present at the site of inflammation, could play an important role in targeted drug delivery.
In summary, we have found that PSGL-1 purified from myeloid cells (neutrophils and HL-60 cells) can support significant initial tethering to endothelial-expressed E-selectin and that PSGL-1 is a better tethering ligand than multivalent sLex. Pretreatment of PSGL-1 and sLex microspheres with HECA-452 did not inhibit initial tethering to E-selectin. These results provide support for the hypotheses that 1) PSGL-1 is a high-efficiency ligand for endothelial-expressed E-selectin, 2) ligand biochemistry can significantly influence initial tethering to E-selectin, and 3) PSGL-1 tethering to E-selectin can occur via non-HECA-452 reactive epitopes.
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GRANTS |
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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REFERENCES |
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2. Alon R, Hammer DA, and Springer TA. Lifetime of the P-selectin-carbohydrate bond and its response to tensile force in hydrodynamic flow. Nature 374: 539542, 1995.[CrossRef][ISI][Medline]
3. Borges E, Pendl G, Eytner R, Steegmaier M, Zollner O, and Vestweber D. The binding of T cell-expressed P-selectin glycoprotein ligand-1 to E- and P-selectin is differentially regulated. J Biol Chem 272: 2878628792, 1997.
4. Brunk DK, Goetz DJ, and Hammer DA. Sialyl Lewis x/E-selectin-mediated rolling in a cell-free system. Biophys J 71: 29022908, 1996.[Abstract]
5. Burdick MM, Bochner BS, Collins BE, Schnaar RL, and Konstantopoulos K. Glycolipids support E-selectin-specific strong cell tethering under flow. Biochem Biophys Res Commun 284: 4249, 2001.[CrossRef][ISI][Medline]
6. Carlos TM and Harlan JM. Leukocyte-endothelial adhesion molecules. Blood 84: 20682101, 1994.
7. Chang KC, Tees DF, and Hammer DA. The state diagram for cell adhesion under flow: leukocyte rolling and firm adhesion. Proc Natl Acad Sci USA 97: 1126211267, 2000.
8. Crutchfield KL, Shinde Patil VR, Campbell CJ, Parkos CA, Allport JR, and Goetz DJ. CD11b/CD18-coated microspheres attach to E-selectin under flow. J Leukoc Biol 67: 196205, 2000.[Abstract]
9. Dagia NM and Goetz DJ. A proteasome inhibitor reduces concurrent, sequential and long term IL-1 and TNF-
induced endothelial cell adhesion molecule expression and adhesion. Am J Physiol Cell Physiol 285: C813C822, 2003.
10. Dagia NM, Harii N, Meli AE, Sun X, Lewis CJ, Kohn LD, and Goetz DJ. Phenyl methimazole inhibits TNF- induced VCAM-1 expression in an IFN regulatory factor-1-dependent manner and reduces monocytic cell adhesion to endothelial cells. J Immunol 173: 20412049, 2004.
11. Dimitroff CJ, Lee JY, Rafii S, Fuhlbrigge RC, and Sackstein R. CD44 is a major E-selectin ligand on human hematopoietic progenitor cells. J Cell Biol 153: 12771286, 2001.
12. Eniola AO, Rodgers SD, and Hammer DA. Characterization of biodegradable drug delivery vehicles with the adhesive properties of leukocytes. Biomaterials 23: 21672177, 2002.[CrossRef][ISI][Medline]
13. Fuhlbrigge RC, King SL, Dimitroff CJ, Kupper TS, and Sackstein R. Direct real-time observation of E- and P-selectin-mediated rolling on cutaneous lymphocyte-associated antigen immobilized on Western blots. J Immunol 168: 56455651, 2002.
14. Goetz DJ, Greif DM, Ding H, Camphausen RT, Howes S, Comess KM, Snapp KR, Kansas GS, and Luscinskas FW. Isolated P-selectin glycoprotein-1 dynamic adhesion to P- and E-selectin. J Cell Biol 137: 509519, 1997.
15. Hanley WD, Wirtz D, and Konstantopoulos K. Distinct kinetic and mechanical properties govern selectin-leukocyte interactions. J Cell Sci 117: 25032511, 2004.
16. Hirata T, Merrill-Skoloff G, Aab M, Yang J, Furie BC, and Furie B. P-Selectin glycoprotein ligand 1 (PSGL-1) is a physiological ligand for E-selectin in mediating T helper 1 lymphocyte migration. J Exp Med 192: 16691676, 2000.
17. Ito K, Handa K, and Hakomori S. Species-specific expression of sialosyl-Lex on polymorphonuclear leukocytes (PMN), in relation to selectin-dependent PMN responses. Glycoconj J 11: 232237, 1994.[CrossRef][ISI][Medline]
18. Knibbs RN, Craig RA, Maly P, Smith PL, Wolber FM, Faulkner NE, Lowe JB, and Stoolman LM. (1,3)-Fucosyltransferase VII-dependent synthesis of P- and E-selectin ligands on cultured T lymphoblasts. J Immunol 161: 63056315, 1998.
19. Kobzdej MM, Leppanen A, Ramachandran V, Cummings RD, and McEver RP. Discordant expression of selectin ligands and sialyl Lewis x-related epitopes on murine myeloid cells. Blood 100: 44854494, 2002.
20. Kuijpers TM, Hoogerwerf M, van der Laan LJ, Nagel G, van der Schoot CE, Grunert F, and Roos D. CD66 nonspecific cross-reacting antigens are involved in neutrophil adherence to cytokine-activated endothelial cells. J Cell Biol 118: 457466, 1992.[Abstract]
21. Lawrence MB, Bainton DF, and Springer TA. Neutrophil tethering to and rolling on E-selectin are separable by requirement for L-selectin. Immunity 1: 137145, 1994.[CrossRef][ISI][Medline]
22. Levinovitz A, Muhlhoff J, Isenmann S, and Vestweber D. Identification of a glycoprotein ligand for E-selectin on mouse myeloid cells. J Cell Biol 121: 449459, 1993.[Abstract]
23. Luscinskas FW and Gimbrone MA. Endothelial-dependent mechanisms in chronic inflammatory leukocyte recruitment. Annu Rev Med 47: 413421, 1996.[CrossRef][ISI][Medline]
24. Maemura K and Fukuda M. Poly-N-acetyllactosaminyl O-glycans attached to leukosialin. The presence of sialyl Lex structures in O-glycans. J Biol Chem 267: 2437924386, 1992.
25. Matala E, Alexander SR, Kishimoto TK, and Walcheck B. The cytoplasmic domain of L-selectin participates in regulating L-selectin endoproteolysis. J Immunol 167: 16171623, 2001.
26. Moore KL, Eaton SF, Lyons DE, Lichenstein HS, Cummings RD, and McEver RP. The P-selectin glycoprotein ligand from human neutrophils displays sialylated, fucosylated, O-linked poly-N-acetyllactosamine. J Biol Chem 269: 2331823327, 1994.
27. Moore KL, Stults NL, Diaz S, Smith DF, Cummings RD, Varki A, and McEver RP. Identification of a specific glycoprotein ligand for P-selectin (CD62) on myeloid cells. J Cell Biol 118: 445456, 1992.[Abstract]
28. Norgard KE, Moore KL, Diaz S, Stults NL, Ushiyama S, McEver RP, Cummings RD, and Varki A. Characterization of a specific ligand for P-selectin on myeloid cells. J Biol Chem 268: 1276412774, 1993.
29. Norman KE, Katopodis AG, Thoma G, Kolbinger F, Hicks AE, Cotter MJ, Pockley AG, and Hellewell PG. P-Selectin glycoprotein ligand-1 supports rolling on E- and P-selectin in vivo. Blood 96: 35853591, 2000.
30. Park EY, Smith MJ, Stropp ES, Snapp KR, DiVietro JA, Walker WF, Schmidtke DW, Diamond SL, and Lawrence MB. Comparison of PSGL-1 microbead and neutrophil rolling: microvillus elongation stabilizes P-selectin bond clusters. Biophys J 82: 18351847, 2002.
31. Patel KD and McEver RP. Comparison of tethering and rolling of eosinophils and neutrophils through selectins and P-selectin glycoprotein ligand-1. J Immunol 159: 45554565, 1997.[Abstract]
32. Patel KD, Moore KL, Nollert MU, and McEver RP. Neutrophils use both shared and distinct mechanisms to adhere to selectins under static and flow conditions. J Clin Invest 96: 18871896, 1995.[ISI][Medline]
33. Patel KD, Nollert MU, and McEver RP. P-Selectin must extend a sufficient length from the plasma membrane to mediate rolling of neutrophils. J Cell Biol 131: 18931902, 1996.[CrossRef][ISI]
34. Picker LJ, Warnock RA, Burns AR, Doerschuk CM, Berg EL, and Butcher EC. The neutrophil selectin LECAM-1 presents carbohydrate ligands to the vascular selectin ELAM-1 and GMP-140. Cell 66: 921933, 1991.[CrossRef][ISI][Medline]
35. Sakhalkar H, Dalal MK, Salem A, Ansari R, Fu J, Kiani MF, Kurjiaka DT, Hanes J, Shakesheff KM, and Goetz DJ. Leukocyte inspired biodegradable particles that selectively and avidly adhere to inflamed endothelium in vitro and in vivo. Proc Natl Acad Sci USA 100: 1589515900, 2003.
36. Sakhalkar HS, Hanes J, Fu J, Benavides U, Malgor R, Borruso CL, Kohn LD, Kurjiaka DT, and Goetz DJ. Enhanced adhesion of ligand-conjugated biodegradable particles to colitic venules. FASEB J 19: 792794, 2005.
37. Sako D, Chang XJ, Barone KM, Vachino G, White HM, Shaw G, Veldman GM, Bean KM, Ahern TJ, Furie B, Cumming DA, and Larsen GR. Expression cloning of a functional glycoprotein ligand for P-selectin. Cell 75: 11791186, 1993.[CrossRef][ISI][Medline]
38. Shao JY, Ting-Beall HP, and Hochmuth RM. Static and dynamic lengths of neutrophil microvilli. Proc Natl Acad Sci USA 95: 67976802, 1998.
39. Shinde Patil VR, Campbell CJ, Yun YH, Slack SM, and Goetz DJ. Particle diameter influences adhesion under flow. Biophys J 80: 17331743, 2001.
40. Smith MJ, Berg EL, and Lawrence MB. A direct comparison of selectin-mediated transient, adhesive events using high temporal resolution. Biophys J 77: 33713383, 1999.
41. Spragg DD, Alford DR, Greferath R, Larsen CE, Lee K, Gurtner GC, Cybulsky MI, Tosi PF, Nicolau C, and Gimbrone MA Jr. Immunotargeting of liposomes to activated vascular endothelial cells: a strategy for site-selective delivery in the cardiovascular system. Proc Natl Acad Sci USA 94: 87958800, 1997.
42. Steegmaier M, Levinovitz A, Isenmann S, Borges E, Lenter M, Kocher HP, Kleuser B, and Vestweber D. The E-selectin-ligand ESL-1 is a variant of a receptor for fibroblast growth factor. Nature 373: 615620, 1995.[CrossRef][ISI][Medline]
43. Von Andrian UH, Hasslen SR, Nelson RD, Erlandsen SL, and Butcher EC. A central role for microvillous receptor presentation in leukocyte adhesion under flow. Cell 82: 989999, 1995.[CrossRef][ISI][Medline]
44. Wagers AJ, Stoolman LM, Craig R, Knibbs RN, and Kansas GS. An sLex-deficient variant of HL60 cells exhibits high levels of adhesion to vascular selectins: further evidence that HECA-452 and CSLEX1 monoclonal antibody epitopes are not essential for high avidity binding to vascular selectins. J Immunol 160: 51225129, 1998.
45. Walcheck B, Leppanen A, Cummings RD, Knibbs RN, Stoolman LM, Alexander SR, Mattila PE, and McEver RP. The monoclonal antibody CHO-131 binds to a core 2 O-glycan terminated with sialyl-Lewis x, which is a functional glycan ligand for P-selectin. Blood 99: 40634069, 2002.
46. Wilkins PP, McEver RP, and Cummings RD. Structures of the O-glycans on P-selectin glycoprotein ligand-1 from HL-60 cells. J Biol Chem 271: 1873218742, 1996.
47. Xia L, Sperandio M, Yago T, McDaniel JM, Cummings RD, Pearson-White S, Ley K, and McEver RP. P-selectin glycoprotein ligand-1-deficient mice have impaired leukocyte tethering to E-selectin under flow. J Clin Invest 109: 939950, 2002.
48. Yang J, Hirata T, Croce K, Merrill-Skoloff G, Tchernychev B, Williams E, Flaumenhaft R, Furie BC, and Fruie B. Targeted gene disruption demonstrates that P-selectin glycoprotein ligand 1 (PSGL-1) is required for P-selectin-mediated but not E-selectin-mediated neutrophil rolling and migration. J Exp Med 190: 17691782, 1999.
49. Zollner O, Lenter MC, Blanks JE, Borges E, Steegmaier M, Zerwes H, and Vestweber D. L-Selectin from human, but not from mouse neutrophils binds directly to E-selectin. J Cell Biol 136: 707716, 1997.
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