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The Journal of Immunology, 1999, 163: 5070-5078.
Copyright © 1999 by The American Association of Immunologists

L-Selectin Ligands Expressed by Human Leukocytes Are HECA-452 Antibody-Defined Carbohydrate Epitopes Preferentially Displayed by P-Selectin Glycoprotein Ligand-11

LiLi Tu, Patricia G. Murphy, Xuan Li and Thomas F. Tedder2

Department of Immunology, Duke University Medical Center, Durham, NC 27710


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Leukocytes express L-selectin ligands critical for leukocyte-leukocyte interactions at sites of inflammation. The predominant leukocyte L-selectin ligand is P-selectin glycoprotein ligand-1 (PSGL-1), which displays appropriate sialyl Lewis x (sLex)-like carbohydrate determinants for L-selectin recognition. Among the sLex-like determinants expressed by human leukocytes is a unique carbohydrate epitope defined by the HECA-452 mAb. The HECA-452 Ag is a critical component of L-selectin ligands expressed by vascular endothelial cells. However, HECA-452 Ag expression on human leukocyte L-selectin ligands has not been assessed. In this study, the HECA-452 mAb blocked 88–99% of neutrophil rolling on, or attachment to, adherent cells expressing L-selectin in multiple experimental systems. A function-blocking anti-PSGL-1 mAb also inhibited L-selectin binding to neutrophils by 89–98%. In addition, the HECA-452 and anti-PSGL-1 mAbs blocked the majority of P-selectin binding to neutrophils. Western blot analysis revealed that PSGL-1 immunoprecipitated from neutrophils displayed HECA-452 mAb-reactive determinants and that PSGL-1 was the predominant scaffold for HECA-452 Ag display. Leukocyte L-selectin ligands also contained sulfated determinants since culturing ligand-bearing cells with NaClO3 abrogated L-selectin binding. Consistent with this, human neutrophils expressed mRNA encoding five different sulfotransferases associated with the generation of selectin ligands: CHST1, CHST2, CHST3, TPST1, and HEC-GlcNAc6ST. Therefore, the HECA-452-defined carbohydrate determinant displayed on PSGL-1 represented the predominant L-selectin and P-selectin ligand expressed by neutrophils.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
L-selectin mediates lymphocyte binding to high endothelial venules (HEV)3 of peripheral lymph nodes and leukocyte interactions with vascular endothelial cells during immune and inflammatory responses (1). L-selectin is constitutively expressed by most leukocytes, whereas the other members of the selectin family, P- and E-selectin, are expressed by activated vascular endothelium. In addition to mediating leukocyte/endothelial cell interactions, L-selectin can also mediate intercellular interactions between leukocytes (2, 3). Neutrophil adherence to cultured endothelial cells supports the continued rolling of newly arriving neutrophils through L-selectin-dependent leukocyte-leukocyte interactions (2). Similarly, bovine {gamma}{delta} T cells bound to endothelial cell monolayers support the rolling of newly arriving {gamma}{delta} T cells (4). Subsequent studies have confirmed the requirement for L-selectin in this process (5, 6) and have identified P-selectin glycoprotein ligand-1 (PSGL-1) as the L-selectin ligand responsible for leukocyte-leukocyte interactions in vitro (7, 8, 9, 10). Although the in vivo significance and the extent that leukocytes roll on adherent leukocytes remain controversial (3), these observations suggest that leukocyte rolling on adherent leukocytes amplifies the number of effector cells accumulating at sites of inflammation. Nonetheless, the analysis of leukocyte L-selectin ligands provides an advantageous system for the biochemical characterization of functional L-selectin ligands.

L-selectin binds to PSGL-1 expressed by leukocytes (5, 7, 8, 9). It also binds to at least five different heavily glycosylated mucin-like proteins expressed by HEV: GlyCAM-1 (11), CD34 (12), MAdCAM-1 (13), Sgp200 (14), and human podocalyxin-like protein (15). Although the complete repertoire of L-selectin ligands has yet to be defined, most are heavily glycosylated mucin-like proteins (16). Sialylation and fucosylation of appropriate carbohydrate determinants are critical for L-selectin ligand generation (17), with fucosyltransferase VII dominating in selectin ligand generation (18, 19). Prototype carbohydrate ligands for the selectins include the sialyl Lewis x (sLex) tetrasaccharide that is also expressed by lymph node HEV (20, 21, 22). A specific subset of anti-sLex mAbs, HECA-452, 2F3, and 2H5, but not other anti-sLex mAbs such as CSLEX-1 and FH6, recognizes putative L-selectin ligands found on HEV (22, 23) and vascular endothelium (24, 25). The HECA-452 mAb identifies a sLex-like determinant termed the cutaneous lymphocyte-associated Ag (26, 27, 28, 29). Ag binding by the HECA-452 mAb requires both sialic acid and fucose, but is independent of sulfation. Although some structures recognized by the HECA-452 and 2H5 mAbs have been identified, the precise structure of their Ags has yet to be defined (23). Regardless, most L-selectin ligands are heavily glycosylated mucin-like proteins containing sialylated and fucosylated O-linked carbohydrate side chains that are essential for L-selectin binding (16).

L-selectin ligands also require sulfation as a post-translational modification (14, 30, 31, 32, 33). Six sulfotransferases potentially involved in the generation of selectin ligands have been recently reported (34, 35, 36, 37, 38, 39). These enzymes can sulfate either protein (tyrosylprotein sulfotransferase (TPST)) or carbohydrate (carbohydrate sulfotransferase (CHST)) moieties. L-selectin ligands induced on vascular endothelial cells require sulfation (24) and L-selectin ligands on HEV characteristically bear a sulfate-dependent carbohydrate epitope defined by the MECA-79 mAb that inhibits lymphocyte binding to peripheral lymph nodes (14). In contrast to the HECA-452 mAb, the MECA-79 mAb defines sulfate-dependent but fucosyltransferase VII- and sialic acid-independent carbohydrate epitopes. Therefore, L-selectin ligands are generated through the concerted regulation of sialyltransferases, fucosyltransferases, and appropriate sulfotransferase(s). Since little information is available on the composition of leukocyte L-selectin ligands, the current study further characterized the functional L-selectin ligands expressed by human leukocytes.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Fusion protein, Abs, and flow cytometry

A human L-selectin-mouse IgM fusion protein (L'IgM) was produced and purified as previously described (24). The LAM1-3 and LAM1-14 (anti-L-selectin) mAbs were generated as previously described (40, 41). Hybridomas producing the HECA-452, CSLEX-1 (anti-sLex), and MECA-79 mAbs were obtained from American Type Culture Collection (Manassas, VA). The PL-1 and PL-2 mAbs (anti-PSGL-1), produced as previously described (42), were provided by Dr. Kevin Moore (University of Oklahoma Health Sciences Center, Oklahoma City, OK). All mAbs were purified unless indicated otherwise. FITC-conjugated goat anti-rat IgM, anti-rat Ig(H+L), and anti-mouse IgM Abs were purchased from Southern Biotechnology Associates (Birmingham, AL). Flow cytometric analysis of cells stained with primary Abs or L'IgM fusion protein diluted to optimal concentrations for immunostaining was conducted as previously described (24). Immunofluorescent staining was analyzed on a FACScan flow cytometer (Becton Dickinson, San Jose, CA). Reactivity of the MECA-79 mAb was verified by staining HEV within lymph node tissue sections using standard immunochemistry techniques.

In some experiments, neutrophils were preincubated with O-sialoglycoprotein endopeptidase (OSGE; 80 µg/ml; Accurate Chemical and Scientific, Westbury, NY) or Vibrio cholerae neuraminidase (0.1 U/ml; Calbiochem, La Jolla, CA) in HBSS with Ca2+ and 10 mM HEPES for 1 h at 37°C before neutrophils were stained with mAbs. Untreated neutrophils were also preincubated for 1 h at 37°C before staining to serve as controls.

Cell lines and neutrophil isolation

Heparinized blood of normal volunteers was isolated using protocols approved by the Human Use Committee of Duke University. Blood neutrophils were isolated by either density gradient centrifugation using Mono-poly resolving medium (ICN Biomedicals, Costa Mesa, CA) or by dextran sedimentation followed by Ficoll-Hypaque density gradient centrifugation and hypotonic lysis of RBCs. The human progenitor cell line, KG-1a, was cultured in IMDM containing 20% FCS and glutamine. HL-60 cells were cultured as previously described (7). A mouse pre-B cell line, 300.19 cells, transfected with a nonshedding form of L-selectin (L{Delta}M-N) was generated as previously described (43) and was termed 300.19-L'. COS cells transfected with {alpha}1,3 fucosyltransferase VII cDNA (COS-FtVII cells) were generated as previously described (24).

Nonstatic cell binding assays

An in vitro adhesion assay with horizontal rotation (64 rpm) at 4°C was used as previously described (44) with minor modifications (24). To assess L-selectin binding to leukocytes, COS or COS-FtVII cells were transiently transfected with cDNA encoding the L{Delta}M-N form of L-selectin, P-selectin (from Dr. Bruce Furie, Beth Israel Hospital, Boston, MA), E-selectin (from Dr. Michael Gimbrone, Jr., Brigham and Women’s Hospital, Boston, MA), PSGL-1 (pPL85, from Dr. Dale Cumming, Genetics Institute, Cambridge, MA), CD34 (from Dr. Daniel Tenen, Beth Israel-Deaconess Hospital, Boston, MA), human podocalyxin-like protein (from Dr. David Kershaw, University of Michigan, Ann Arbor, MI), or vector alone (pMT2) as described previously (7). The COS cells were transferred onto glass slides (50 x 103 cells in a 22-mm2 area) 24 h after transfection and were cultured overnight. In some cases, the COS-FtVII cells were incubated immediately following transfection in medium containing 10 mM NaClO3. A day after transfection, the cells were transferred onto glass slides (50 x 103 cells in a 22-mm2 area) and cultured in the same medium for 24 h. Neutrophils or 300.19-L' cells were resuspended (1 x 106 in 200 µl) in cold DMEM containing 5% FCS and were then layered onto the transfected COS cells. To verify that NaClO3 treatment did not affect HECA-452 Ag or PSGL-1 expression, both untreated COS-FtVII cells and NaClO3-treated COS-FtVII cells were stained with the HECA-452 and PL-1 mAbs when the binding assays were conducted.

A full-length cDNA encoding human podocalyxin-like protein was constructed by joining two previously described (45) and partially overlapping cDNA clones (RACEN5 and NP3) by PCR. The PCR-generated podocalyxin-like protein cDNA was subcloned into the pBluescript SK+ plasmid and sequenced. Three nucleotides at the 5' end of the cDNA differed from the published consensus nucleotide sequence (45). Nucleotide 338 was a T, making aa 30 a serine instead of a proline. Nucleotide 435 was a G, making aa 62 an arginine instead of a threonine, which was consistent with the sequence of the RACEC12 cDNA (45). Nucleotide 498 was a C, making aa 83 an isoleucine instead of a threonine. The full-length podocalyxin-like protein cDNA cloned into the pMT2 expression vector (a gift from Genetics Institute, Cambridge, MA) was used to transiently transfect COS cells by the DEAE-dextran method. Indirect immunofluorescence staining of transfected COS cells with the 3D3 anti-podocalyxin-like protein mAb verified that the podocalyxin-like protein was highly expressed.

Neutrophil rolling under defined flow conditions

Neutrophil interactions with L-selectin under physiologic flow conditions were assessed using an in vitro flow chamber as previously described (46). CHO cells stably transfected with the L{Delta}M-N L-selectin cDNA were grown to confluence on 25-mm circular glass coverslips and placed in a parallel plate flow chamber. Neutrophils (1 x 106/ml) were resuspended in PBS containing 0.75 mM CaCl2, 0.75 mM MgCl2, and 0.5% (w/v) BSA, perfused through the chamber at a calculated shear stress of 1.85 dyn/cm2 via a syringe pump and videotaped. The number of rolling neutrophils that crossed a 400-µm line over a 30-s period was counted.

Immunoprecipitations and Western blot analysis

Neutrophils (5 x 107) were purified, washed three times in PBS with Ca2+ and Mg2+, and lysed in 1 ml of buffer containing 1% Nonidet P-40, 150 mM NaCl, 50 mM Tris-HCl (pH 8.0), and protease inhibitors as previously described (47). Cell lysates were either untreated or were precleared twice by incubation with control rat IgM Ab for 1 h at 4°C followed by the addition of protein G-Sepharose beads (Pharmacia, San Diego, CA) for 1 h at 4°C, with subsequent centrifugation. The lysates were precleared to reduce the levels of protein that nonspecifically bound to control mAbs during subsequent Western blot analysis. After preclearing, the cell lysates were incubated either with a control mAb or with the anti-PSGL-1 mAbs PL-1 and PL-2 (5 µg/ml) overnight at 4°C followed by the addition of protein G-Sepharose and centrifugation. Glycoproteins from the undepleted and PSGL-1-depleted neutrophil lysates were isolated using wheat germ agglutinin-agarose beads (100 µl of packed volume; Sigma, St. Louis, MO) with overnight incubations at 4°C. Following centrifugation, both the wheat germ agglutinin-agarose and anti-PSGL-1 mAb/protein G-Sepharose beads were washed four times with lysis buffer. The isolated materials were separated by SDS-PAGE under reducing conditions (7.5% gel), with subsequent Western blot analysis. The immunoblots were incubated with either the HECA-452 mAb or a control rat IgM Ab and detected using a HRP-conjugated, goat anti-rat Ig secondary Ab. The blots were developed using an enhanced chemiluminescence kit (Pierce, Rockford, IL).

PCR amplification of sulfotransferase transcripts

Cytoplasmic RNA free of DNA contamination was isolated from each cell type using a RNeasy Mini Kit (Qiagen, Chatsworth, CA) according to the manufacturer’s instructions. Equal amounts of RNA (~2 µg) were used for cDNA synthesis as previously described (48). PCR amplification of CHST1 cDNA used 5'-CAC GCG CAG CGG CTC CTC CTT CGT-3' (sense) and 5'-GCC AGG TCC TCG TAG CGC ACC G-3' (antisense) primers to generate a 713-bp fragment. CHST2 amplification used 5'-GGG CGC AAC CTC ACC ACG-3' (sense) and 5'-CCA CGA AAG GCT TGG AGG AGG-3' (antisense) primers to generate a 690-bp fragment. CHST3 amplification used 5'-CAA CCA GCA GGG CAA CAT CT-3' (sense) and 5'-CCC TAC GTG ACC CAG AAG G-3' (antisense) primers to generate a 980-bp fragment. HEC-GlcNAc6ST amplification used 5'-GTG GTG GAG AAG GCC TGC CG-3' (sense) and 5'-ACC CTC TTA GTG GAT TTG CT-3' (antisense) primers to generate a 680-bp fragment. TPST1 amplification used 5'-AAG ATG GTT GGA AAG CTG AAG C-3' (sense) and 5'-TTC TCA TCC ACC GTT CAG GAT G-3' (antisense) primers to generate a 757-bp fragment. TPST2 amplification used 5'-AGC ATG CGC CTG TCG GTG CG-3' (sense) and 5'-CAC TTG GAG AGC GCT TCC AG-3' (antisense) primers to generate a 905-bp fragment. Primers for ß-actin were 5'-ATG TTT GAG ACC TTC AAC AC-3' (sense) and 5'-CAC GTC ACA CTT CAT GAT GG-3' (antisense), which generate a 495-bp fragment. As controls, mRNA from each cell type was used in PCR reactions without RT. No PCR signal was detected in any of the control reactions, which rules out the possibility that PCR products were generated from contaminating genomic DNA. Conditions used for PCR amplification were: 94°C for 5 min, then 30 cycles of 94°C for 1 min, 55°C for 1 min, followed by 72°C for 50 s. PCR products were electrophoresed and visualized with ethidium bromide staining.

Statistical analysis

Data are expressed as the mean ± SEM. Since there was little, if any, interassay variation in the results, all data were pooled, and Student’s t test was used to determine the significance of differences in sample population means.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Myeloid cells express L-selectin ligands

L-selectin ligand expression by neutrophils and the KG-1a and HL-60 myeloid cell lines was assessed using a chimeric L-selectin–IgM fusion protein, termed L'IgM. Neutrophils, KG-1a cells, and HL-60 cells bound high levels of L'IgM when Ca2+ was present, but not in the absence of Ca2+ (Fig. 1GoA). In each case, L'IgM binding was decreased >80% by the presence of the L-selectin function-blocking mAb, LAM1-3 (Fig. 1GoA), but not by the non-function-blocking LAM1-14 mAb (data not shown). Neutrophils, KG-1a cells, and HL-60 cells also expressed high levels of PSGL-1 (Fig. 1GoB). L'IgM binding was substantially blocked by pretreatment of the cells with the PL-1 mAb that blocks both P- and L-selectin binding to PSGL-1 (48–71%; Fig. 1GoA). A non-function-blocking anti-PSGL-1 mAb, PL-2, did not affect L'IgM binding to the cells (data not shown). These results demonstrate that L-selectin ligand(s) was expressed by each of these cell types and that the predominant ligand was PSGL-1 in each case.



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  		FIGURE 1. Reactivity of the L'IgM fusion protein correlates with sLex expression. A, Cells were stained in indirect immunofluorescence assays with L'IgM (bold line; 10 µg/ml) plus FITC-labeled goat anti-mouse IgM Ab and subsequently analyzed by flow cytometry. The L'IgM protein was also preincubated with LAM1-3 mAb (50 µg/ml for neutrophils or KG-1a cells, 10 µg/ml for HL-60 cells) for 20 min and then used to stain cells as indicated. Alternatively, cells were preincubated with the PL-1 mAb (50 µg/ml for neutrophils or KG-1a cells, 10 µg/ml for HL-60 cells) for 20 min before addition of L'IgM, which diluted the PL-1 mAb by half. Dashed lines represent background staining with PBS-2% FCS, with L'IgM staining conducted in the absence of Ca2+, or when supernatant fluid from COS cells transfected with vector alone was used for staining. B, Cell surface Ag expression assessed by indirect immunofluorescence staining using the PL-1, HECA-452, CSLEX-1, and MECA-79 mAbs (bold lines). Dashed lines represent background staining with isotype-matched control mAbs.

 
Expression of sLex-like determinants by neutrophils, KG-1a cells, and HL-60 cells was assessed using the CSLEX-1 and HECA-452 mAbs. Both mAbs reacted with each cell type (Fig. 1GoB). However, neutrophils, KG-1a cells, and HL-60 cells all failed to express the MECA-79 mAb-defined carbohydrate determinant that is associated with L-selectin ligands expressed by HEV (14, 49). These results demonstrate that neutrophils, KG-1a cells, and HL-60 cells each express both L-selectin ligands and sLex-related carbohydrate determinants.

HECA-452 Ag is an essential component of neutrophil L-selectin ligands

We were unable to assess whether the CSLEX-1 or HECA-452 mAb inhibited L'IgM binding since L'IgM was generated using mouse IgM heavy chain and we were unable to identify secondary Ab reagents that distinguished clearly between mouse and rat IgM. Therefore, whether the CSLEX-1 or HECA-452 Ag contributed to L-selectin ligands expressed by myeloid cells was assessed in a nonstatic cell binding assay developed to analyze leukocyte-endothelial cell interactions (44). In this assay, L-, P-, or E-selectin transiently expressed by COS cells bound neutrophils at high levels, while very few neutrophils bound vector (pMT2)-transfected COS cells (Fig. 2Go). Preincubating neutrophils with the HECA-452 mAb reduced neutrophil binding to COS cells transfected with L-selectin by 88–92% (Fig. 2Go, A and B). Preincubating neutrophils with the PL-1 mAb also reduced neutrophil binding to COS cells transfected with L-selectin cDNA by 89–90% (Fig. 2Go, A and B). Treating neutrophils with either the HECA-452 or PL-1 mAb did not induce detectable cellular aggregation in this assay during the time period examined. Neither the CSLEX-1 nor PL-2 mAb affected L-selectin binding in this assay system (7, 24). The high degree to which the HECA-452 and PL-1 mAbs each blocked L-selectin binding suggests that the HECA-452 Ag displayed on PSGL-1 is the L-selectin ligand rather than reflecting additive effects from blocking L-selectin binding to distinct ligands.



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  		FIGURE 2. The HECA-452 Ag and PSGL-1 mediate neutrophil binding to COS cells expressing L- or P-selectin. Neutrophil binding to COS cells transiently transfected with L-selectin cDNA (COS-L'; A and B), P-selectin cDNA (COS-P; C), E-selectin cDNA (COS-E; D), or pMT2 vector (COS-pMT2) was assessed in a nonstatic binding assay at 4°C. Neutrophils were preincubated with the HECA-452 or PL-1 mAb (10 µg/ml) at 4°C for 20 min before addition to the COS cells as indicated, which diluted the mAb concentration by half. A, C, and D values represent the mean (±SEM) number of neutrophils bound to COS cells as previously described (7 ). Three slides were examined for each sample, with the number of neutrophils bound in each of 10 randomly chosen microscopic fields counted for each slide. Identical results were obtained in two independent experiments, so the numbers were pooled. Thereby, each value represents the mean number of neutrophils bound in 60 fields, except for COS-E cells treated with medium, which represents 50 microscopic fields. B, Representative neutrophil (Neut.) binding to COS-pMT2 or COS-L' cells (magnification, x400).

 
Preincubating neutrophils with the HECA-452 mAb also reduced neutrophil binding to COS cells expressing P-selectin by 78–83% (Fig. 2GoC) and E-selectin by 90–94% (Fig. 2GoD). Preincubation of neutrophils with the PL-1 mAb reduced neutrophil binding to COS cells expressing P-selectin by 80–83%, whereas the PL-1 mAb only partially inhibited neutrophil attachment to COS cells expressing E-selectin (20–33%; p < 0.02; Fig. 2Go, C and D). Thus, L-, P-, and E-selectin were similar in their requirements for neutrophil expression of the HECA-452 Ag. To verify that the majority of HECA-452 mAb-defined determinants identified by L- and P-selectin were distinct from those identified by E-selectin, neutrophils were treated with OSGE, an endopeptidase specific for glycoproteins containing closely spaced O-linked sialic acid residues. OSGE removes the amino-terminal region of PSGL-1 that interacts with P-selectin and L-selectin, but not E-selectin. Although OSGE treatment eliminated PL-1 mAb binding sites, it only reduced HECA-452 Ag expression by 39–47% in three experiments (Fig. 3Go). By contrast, neuraminidase treatment eliminated HECA-452 mAb reactivity but did not affect PL-1 mAb binding (Fig. 3Go), consistent with previous observations that the HECA-452 Ag requires sialic acid. Thus, L- and P-selectin bind the HECA-452 Ag displayed on OSGE-sensitive sites of PSGL-1, while E-selectin ligand binding is likely to include the HECA-452 Ag displayed at additional sites on PSGL-1 or on other determinants.



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  		FIGURE 3. Neutrophils predominantly display HECA-452 Ag on OSGE-resistant determinants. Neutrophils were pretreated with medium alone (top panels), OSGE (middle panels), or neuraminidase (Neur.; bottom panels) as indicated. Cell surface Ag expression was assessed by indirect immunofluorescence staining using the HECA-452 and PL-1 mAbs (bold lines). Dashed lines represent background staining with isotype-matched control mAbs. Results represent those obtained in at least three independent experiments.

 
HECA-452 mAb blocks neutrophil rolling on L-selectin under physiologic flow

The contributions of the HECA-452 and CSLEX-1 determinants to neutrophil rolling on L-selectin-bearing cells were assessed under physiologic shear flow using in vitro parallel plate flow chamber assays. High numbers of neutrophils attached to CHO cell monolayers expressing L-selectin (CHO-L' cells; Fig. 4Go). The majority of neutrophils in contact with CHO-L' monolayers rolled into the field of view, indicating that cells had attached upstream of the field under observation. The neutrophils remained in contact for their entire transit across the field of view, with little stationary adhesion. Neutrophils did not interact with untransfected CHO monolayers at detectable levels (Fig. 4Go). Preincubation of neutrophils with the HECA-452 mAb decreased neutrophil rolling on CHO-L' cells by 97–99% in two independent experiments (Fig. 4Go). L-selectin and PSGL-1 mediated most neutrophil rolling since preincubation of neutrophils with the PL-1 mAb or CHO-L' cells with the LAM1-3 mAb reduced the frequency of rolling cells by 96–100%, while the CSLEX-1, LAM1-14, or PL-2 mAb had no detectable effect. Treating neutrophils with the HECA-452 or PL-1 mAb did not induce detectable cellular aggregation. Therefore, the HECA-452 Ag and PSGL-1 were the preferred L-selectin ligands on neutrophils under physiologic flow. Since >96% of neutrophil rolling was inhibited by each mAb, these results suggest that the predominant L-selectin ligand is the HECA-452 determinant displayed by PSGL-1.



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  		FIGURE 4. The HECA-452 Ag and PSGL-1 mediate L-selectin-dependent rolling of neutrophils on CHO cells expressing L-selectin (CHO-L') under physiologic shear flow. Neutrophil rolling was assessed in a parallel plate flow chamber at a shear force of 1.85 dyn/cm2. Neutrophils or confluent monolayers of CHO-L' cells were pretreated with the indicated mAbs at 10 µg/ml for 20 min at 4°C before initiation of the assay, except the CSLEX-1 mAb was used as ascites fluid diluted 1/100. Neutrophils in either medium alone or medium containing the indicated mAbs were perfused across CHO or CHO-L' monolayers for 4 min. Values represent the mean (±SEM) number of rolling neutrophils that subsequently entered a 400-µm-wide field over a 30-s period near the centerline of the flow chamber during a 10-min period of perfusion. Identical results were obtained in two independent experiments, so the results were pooled. Thereby, each value represents the mean of results from at least 40 randomly chosen fields from two independent experiments.

 
Similar in vitro flow chamber studies were conducted with KG-1a and HL-60 cells. However, the frequency of rolling cells was undetectable compared with the frequency of rolling neutrophils in this assay system (data not shown). Whether this results from the fact that KG-1a and HL-60 cells are significantly larger in size than neutrophils and express ~4-fold lower levels of immunoreactive the HECA-452 Ag (Fig. 1GoB) is unknown. Furthermore, since L-selectin site density is important for its function in vivo (50), ligand density is also likely to be a critical factor in this assay system. However, these observations may also reflect the absence of other cellular components necessary for the generation of optimal L-selectin ligands.

Neutrophil HECA-452 Ag is displayed on PSGL-1

Whether the HECA-452 Ag was expressed on PSGL-1 and/or other proteins was assessed by Western blot analysis. The HECA-452 mAb reacted specifically with three proteins in neutrophil lysates, proteins of ~240, 160, and 120 kDa (Fig. 5Go). Most other proteins that reacted with the HECA-452 mAb also reacted with a control rat IgM mAb and are likely to represent IgM binding proteins unrelated to the Ag specificity of the HECA-452 mAb. Previous studies have shown that immunoprecipitated PSGL-1 migrates at 240, 160, and 120 kDa (42). Therefore, additional experiments were performed to determine whether these proteins were related to PSGL-1. The neutrophil lysates were depleted of PSGL-1 by immunoprecipitation with the PL-1 and PL-2 mAbs and were compared with undepleted neutrophil lysates. The bulk of the 240, 160, and 120 kDa proteins identified by the HECA-452 mAb was removed by the anti-PSGL-1 mAbs (Fig. 5Go). Additional rounds of immunodepletion reduced the protein levels in these three bands further, but detectable protein remained in each band after three rounds of immunodepletion (data not shown). The inability to immunodeplete all PSGL-1 from cellular lysates has been observed previously (51). Nonetheless, the anti-PSGL-1 mAbs immunoprecipitated 240, 160, and 120 kDa proteins equivalent in size with the proteins that were reactive with the HECA-452 mAb. These results demonstrate that the majority of the HECA-452 determinants expressed by neutrophils are displayed on PSGL-1.



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  		FIGURE 5. The HECA-452 epitope is predominantly displayed by PSGL-1 on neutrophils. Detergent lysates of neutrophils were either untreated or depleted of PSGL-1 by mAb treatment. Wheat germ agglutinin-agarose beads were then used to precipitate glycoproteins from the lysates, which were run side-by-side with the anti-PSGL-1 mAb-immunoprecipitated materials (PSGL-1) on reducing SDS-PAGE gels. The precipitated materials were transferred to nitrocellulose and immunoblotted with the HECA-452 mAb. An equivalent sample of neutrophil lysate was precipitated with wheat germ agglutinin, subjected to SDS-PAGE analysis, and immunoblotted with a control rat IgM mAb as a control (rIgM). The m.w. are shown on the left (x1000). The migration of indicated proteins is shown on the right. IgH and IgL are the heavy and light chains, respectively, of the PL-1 and PL-2 mAbs used to immunoprecipitate PSGL-1.

 
The L-selectin ligand requires PSGL-1 and sulfation

COS cells transfected with fucosyltransferase VII cDNA (COS-FtVII cells) express the HECA-452 Ag at high levels but do not support detectable L-selectin binding (24). COS-FtVII cells were therefore used to further assess whether the HECA-452 Ag required PSGL-1 to function as an L-selectin ligand. COS-FtVII cells did not support neutrophil attachment (Fig. 6GoA). However, high numbers of neutrophils attached when the COS-FtVII cells were transiently transfected with PSGL-1 cDNA (COS-FtVII/PSGL-1) but not with CD34 or podocalyxin-like protein cDNA (Fig. 6GoA). Neutrophil attachment to COS-FtVII/PSGL-1 cells was inhibited by 35–48% following treatment of these cells with the HECA-452 mAb (Fig. 6GoA) while treatment with the CSLEX-1 mAb had no detectable effect (data not shown). That the HECA-452 mAb only partly blocked neutrophil attachment to COS-FtVII/PSGL-1 cells may be explained by the high density of HECA-452 Ag and PSGL-1 expression on COS cells or the fact that neutrophils can also interact with COS cells through other adhesion receptors. Nonetheless, these results demonstrate that the HECA-452 Ag mediates L-selectin binding only when presented by a specific glycoprotein backbone.



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  		FIGURE 6. L-selectin binding to its ligand requires PSGL-1, sulfation, and HECA-452 mAb-defined epitopes. Binding of neutrophils (A) or 300.19-L' cells (B) to COS-FtVII cells transiently transfected with cDNA encoding PSGL-1, CD34, or human podocalyxin-like protein (PCLP) in a nonstatic binding assay at 4°C. Transfected COS-FtVII cells were cultured with medium alone or with medium containing NaClO3 as indicated. COS-FtVII cells expressing PSGL-1 were preincubated with the HECA-452 (HECA; 10 µg/ml) or CSLEX-1 (CSLEX; 1/100 dilution of ascites) mAbs at 4°C for 20 min before the binding assay. Values represent the mean (±SEM) of pooled results from three independent experiments representing at least 90 randomly chosen microscopic fields as previously described (7 ). C, Expression of the HECA-452 and PSGL-1 Ags on COS-FtVII cells (top panels), COS-FtVII cells expressing PSGL-1 (middle panels), and COS-FtVII cells expressing PSGL-1 after chlorate treatment (bottom panel). Bold lines represent cell staining with either the HECA-452 (10 µg/ml) or PL-1 (10 µg/ml) mAb, whereas dashed lines represent background staining with isotype-matched control mAbs.

 
Similar results were obtained when a mouse pre-B cell line transfected with L-selectin cDNA (300.19-L' cells) was used as a target cell for these experiments. 300.19-L' cells do not support detectable leukocyte-leukocyte interactions and are therefore unlikely to express functional PSGL-1 (our unpublished observations). COS-FtVII cells did not support 300.19-L' cell attachment (Fig. 6GoB). Similarly, human PSGL-1 expressed in the absence of fucosyltransferase VII did not mediate ligand binding (data not shown) (24). However, high numbers of 300.19-L' cells attached when the COS-FtVII cells were transiently transfected with PSGL-1 cDNA but not with CD34 or podocalyxin-like protein cDNA (Fig. 6GoB). 300.19-L' cell attachment to COS-FtVII/PSGL-1 cells was inhibited by 88–95% following treatment of these cells with the HECA-452 mAb (Fig. 6GoB) while treatment with the CSLEX-1 mAb had no detectable effect (Fig. 6GoB). Therefore, these results suggest that PSGL-1 serves as a specific protein scaffold for the appropriate presentation of the HECA-452 Ag to L-selectin.

Endothelial L-selectin ligands require sulfation (14, 24, 52) and P-selectin binding to PSGL-1 also requires sulfation (53, 54, 55). Whether sulfation contributes to L-selectin binding of PSGL-1 bearing HECA-452 Ag was assessed by culturing COS-FtVII cells expressing PSGL-1 in the presence of NaClO3 for 24 h. In three experiments, COS-FtVII/PSGL-1 cells expressed comparable levels of PSGL-1 after chlorate treatment as untreated COS-FtVII/PSGL-1 cells (Fig. 6GoC). However, chlorate treatment significantly reduced both neutrophil and 300.19-L' cell binding to COS-FtVII/PSGL-1 cells by 86–88 and 70–87%, respectively (Fig. 6Go, A and B). These results demonstrate that in addition to an appropriate protein scaffold, L-selectin binding to PSGL-1/HECA-452 Ag also requires sulfation.

Sulfotransferase expression by myeloid cells

Whether transcripts for sulfotransferases associated with selectin ligand generation were expressed by L-selectin ligand-bearing leukocytes was assessed by PCR amplification of cDNA from myeloid cells. Neutrophils expressed CHST1, CHST2, CHST3, and TPST1 but did not express detectable TPST2 message (Fig. 7Go). KG-1a and HL-60 cells expressed CHST2, TPST1, and TPST2 transcripts while CHST1 transcripts were barely detectable in HL-60 cells. By contrast, COS cells expressed each of these transcripts. Surprisingly, abundant transcripts for a recently described HEV-specific sulfotransferase (HEC-GlcNAc6ST) (39) were found in both HL-60 cells and neutrophils. In control experiments, PCR products were not generated from RNA samples without RT even when used at 10-fold higher concentrations (data not shown). In addition, a HEC-GlcNAc6ST PCR product amplified from HL-60 cDNA was sequenced to confirm its identity. Therefore, these results demonstrate some degree of differential regulation of CHST and TPST sulfotransferase transcription. Nonetheless, neutrophils and other L-selectin ligand-bearing cells have the capacity to generate sulfated carbohydrates and/or proteins that may serve as L-selectin ligands.



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  		FIGURE 7. Leukocytes express sulfotransferases. Cytoplasmic RNA from neutrophils, KG-1a cells, HL-60 cells, and COS cells was reverse transcripted and amplified by PCR with primers specific for the indicated sulfotransferases. The PCR products were electrophoresed and visualized by ethidium bromide staining. As controls, PCR reactions were conducted for ß-actin using cDNA (ß+cDNA) or mRNA without RT (ß+mRNA). These results represent those obtained with at least two different mRNA preparations from each cell type.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this study, the predominant L-selectin ligand on neutrophils was found to require the HECA-452 Ag; a mAb-defined subset of sLex carbohydrate determinants. The HECA-452 mAb blocked neutrophil rolling on, or attachment to, adherent cells expressing L-selectin by 88–99% (Figs. 2Go and 4Go). A function-blocking anti-PSGL-1 mAb similarly inhibited neutrophil binding to L-selectin by 89–98% (Figs. 2Go and 4Go). Therefore, it is likely that the HECA-452 Ag expressed by PSGL-1 is the preferred neutrophil L-selectin ligand. Consistent with this, PSGL-1 was the predominant neutrophil cell surface protein bearing HECA-452 mAb-defined determinants and most of the HECA-452 Ags were immunodepleted from cell lysates with anti-PSGL-1 mAbs (Fig. 5Go). HL-60 cells also express PSGL-1 that bears HECA-452 Ags (56). Both PSGL-1 and the HECA-452 Ag are likely to be required for leukocyte L-selectin ligand activity (Fig. 6Go) since independent expression of either PSGL-1 or the HECA-452 Ag is insufficient to generate functional L-selectin ligands (Fig. 6Go) (24). Therefore, the HECA-452 Ag appears essential for ligand generation but requires appropriate presentation by neutrophil PSGL-1 for L-selectin binding.

The HECA-452 Ag is specifically expressed by neutrophils, monocyte subsets, small numbers of leukocytes in tonsils and lymph nodes, and endothelium at sites of chronic inflammation (26, 27, 28, 57). PSGL-1 is expressed by neutrophils, monocytes, and lymphocytes (58). Most notable, however, is that the HECA-452 Ag has become synonymous with the cutaneous lymphocyte-associated Ag expressed by a subpopulation of skin-homing memory T cells that binds E-selectin displayed by dermal endothelium (27, 28, 29, 59, 60). The HECA-452 mAb-defined cutaneous lymphocyte-associated Ag bound by E-selectin is predominantly displayed on lymphocyte PSGL-1 (29, 56). Similarly, in this report, the HECA-452 mAb-defined L-selectin ligand on neutrophils was preferentially displayed by PSGL-1 (Fig. 5Go). Therefore, it may be appropriate to also consider the cutaneous lymphocyte-associated Ag displayed on PSGL-1 as an L-selectin ligand. Thus, HECA-452/PSGL-1 may not only provide a ligand for lymphocyte and neutrophil rolling on E-selectin, but may also facilitate leukocyte interactions with other leukocytes at sites of inflammation.

The current studies are consistent with previous observations that L- and E-selectin share similar ligands (22, 61, 62). The HECA-452 mAb has previously been shown to block lymphocyte binding to E-selectin (26, 56, 63). However, a specific protein backbone does not appear to be required for E-selectin binding to the HECA-452 Ag since overexpression of appropriate fucosyltransferases in different cell types induces HECA-452 Ag expression and functional E-selectin ligands (Fig. 2GoD) (7, 29, 64, 65, 66). In contrast to this, specific protein scaffolds are required for functional L-selectin ligand activity. The HECA-452 Ag displayed on PSGL-1 served as the preferential neutrophil L-selectin ligand (Figs. 2Go and 4Go). This was reiterated in reconstitution experiments showing that COS-FtVII cells bearing the HECA-452 Ag required PSGL-1 expression for the generation of functional L-selectin ligands while other mucins were ineffective in this assay system (Fig. 6Go). Other specific protein carriers must be able to present HECA-452 Ag for L-selectin binding on vascular endothelial cells since these cells do not express PSGL-1 (24). E-selectin appears to bind HECA-452 Ag on PSGL-1 at additional sites to those bound by L-selectin, as the PL-1 mAb completely blocked L-selectin, but not E-selectin, binding (Fig. 2GoD). L-selectin predominantly binds at or near the amino-terminal region of PSGL-1 as defined by the PL-1 mAb (7, 67). In addition, OSGE treatment failed to remove the majority of the immunoreactive HECA-452 Ag despite complete removal of the amino-terminal PL-1 mAb reactive epitopes from PSGL-1 (Fig. 3Go). This explains why the PL-1 mAb only blocked a portion of E-selectin binding to neutrophils, while the HECA-452 mAb inhibited the majority of E-selectin binding (Fig. 2GoD). Thus, the HECA-452 Ag presented by a specific region of PSGL-1 represents functional L-selectin ligands while E-selectin binding is more promiscuous.

In addition to blocking L-selectin binding to PSGL-1, the HECA-452 mAb blocked P-selectin binding to PSGL-1 (Fig. 2GoC). This is consistent with previous studies demonstrating that leukocyte PSGL-1 can serve as a ligand for both L- and P-selectin (5, 7, 8, 9). This suggests that L- and P-selectin bind neutrophils principally through HECA-452 Ags presented at similar, if not identical, amino-terminal sites on PSGL-1. The HECA-452 mAb blocked 78–83% of neutrophil binding to COS-FtVII/PSGL-1 cells (Fig. 2GoC). Others have previously shown that the HECA-452 mAb inhibits 41% of P-selectin-IgG fusion protein binding to a T cell line in flow cytometry assays (56). Thus, the HECA-452 Ag is either required for L- and P-selectin binding, or the HECA-452 carbohydrate epitope functionally or structurally overlaps with the selectin-binding carbohydrate determinant. Although the current study cannot definitively resolve this issue, it is clear that expression of fucosyltransferases that generate the HECA-452 Ag is required for generating appropriate selectin-binding carbohydrate determinants on PSGL-1 (Fig. 2Go). It is unlikely that the HECA-452 mAb binding to its carbohydrate Ag is indirectly blocking L- and P-selectin binding to PSGL-1 through steric hindrance. In support of this, the carbohydrate Ag identified by the CSLEX-1 mAb is highly expressed by neutrophils (Fig. 1GoB) and COS-FtVII cells (our unpublished observation) yet the CSLEX-1 mAb does not inhibit L-selectin binding to PSGL-1 (Fig. 3Go) (24). Therefore, the carbohydrate Ag identified by the HECA-452 mAb is a major functional component of L-, P-, and E-selectin ligands.

Sulfation was required to generate functional L-selectin ligands on PSGL-1 (Fig. 6Go). Sulfation of tyrosines within the 10 amino-terminal residues of PSGL-1 is also essential for P-selectin binding (53, 54, 55). These results and the previous demonstration that amino acid substitutions within the tyrosine sulfate motif of PSGL-1 abrogate L-selectin binding (67) argue that tyrosylprotein sulfation of PSGL-1 is a necessary component of the neutrophil L-selectin ligand. Consistent with this, neutrophils expressed the TPST1 tyrosylprotein sulfotransferase (Fig. 7Go). Neutrophils also expressed transcripts for four enzymes (CHST1, CHST2, CHST3, and HEC-GlcNAc6ST) associated with carbohydrate sulfation (Fig. 7Go) suggesting that PSGL-1 could contain both sulfated carbohydrate determinants and tyrosine residues. Despite the expression of multiple different CHST sulfotransferases (Fig. 7Go), neutrophils, HL-60 cells, KG-1a cells, and COS cells all failed to express the sulfation-dependent MECA-79 Ag at detectable levels (Fig. 1GoB and data not shown). However, neutrophils, HL-60 cells, and KG-1a cells supported significant L-selectin binding even without MECA-79 Ag expression (Figs. 1Go, 2Go, and 4Go). In fact, others have reported that L-selectin ligands on KG-1a cells are sulfate–independent (49) despite its expression of several different CHST and TPST sulfotransferases (Fig. 7Go). Therefore, whether carbohydrate sulfation is a requirement for L-selectin binding to its leukocyte ligands, as appears to be the case with L-selectin ligands expressed by HEV (32), requires further investigation. It remains possible that carbohydrate sulfation of PSGL-1 is not absolutely essential but optimizes L-selectin binding. Regardless of this, the evidence demonstrating a requirement for sulfation of L-selectin ligands suggests that carbohydrate sulfation may be an important component of the neutrophil L-selectin ligand in addition to protein sulfation. Thus, the expression of appropriate fucosyltransferases, sulfotransferases, sialyltransferases, and protein scaffolds is required for the optimal generation of functional cell surface L-selectin ligands.

In summary, the current findings demonstrate that the HECA-452 Ag provides an essential component in the expression of leukocyte L-selectin ligands that may contribute to leukocyte capture and rolling along endothelial cells during inflammation. These findings do not exclude the existence of other minor L-selectin ligands since remnants of the major HECA-452-bearing proteins remained in neutrophil lysates after extensive clearance with anti-PSGL-1 mAbs (Fig. 5Go). Others have also functionally characterized ligands on human neutrophils that appear distinct from PSGL-1. In those studies, both mucin and non–mucin-like components of neutrophil adhesion to L-selectin and L-selectin-IgG fusion proteins were observed (10, 51). Nonetheless, the current findings demonstrate that the HECA-452 Ag is a critical component of the neutrophil L-selectin ligand and that the majority of the HECA-452 Ag is displayed by PSGL-1. These studies further demonstrate those essential components of the L-, E-, and P-selectin ligands are overlapping, although each selectin demonstrates individual degrees of ligand specificity.


   Acknowledgments
 
We thank Ms. A. Miller and Drs. M. Delahunty, D. Kershaw, F. Luscinskas. J. Ross, and K. Moore for their help and for providing the reagents.


   Footnotes
 
1 This work was supported by National Institutes of Health Grants CA54464, CA81776, and HL50985. Back

2 Address correspondence and reprint requests to Dr. Thomas F. Tedder, Department of Immunology, Box 3010, Duke University Medical Center, Durham, NC 27710. E-mail address: Back

3 Abbreviations used in this paper: HEV, high endothelial venules; CHO-L', CHO cells stably transfected with a nonshedding form of L-selectin cDNA; COS-FtVII, COS cells stably transfected with fucosyltransferase VII cDNA; COS-L', COS cells transiently transfected with a nonshedding form of L-selectin cDNA; L'IgM, human L-selectin-mouse IgM fusion protein; PSGL-1, P-selectin glycoprotein ligand-1; sLex, sialyl Lewis x; CHST, carbohydrate sulfotransferase; TPST, tyrosylprotein sulfotransferase; 300.19-L', 300.19 cells stably transfected with a nonshedding form of L-selectin cDNA; OSGE, O-sialoglycoprotein endopeptidase. Back

Received for publication May 14, 1999. Accepted for publication August 20, 1999.


   References
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 Introduction
 Materials and Methods
 Results
 Discussion
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