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(Journal of Leukocyte Biology. 2000;68:65-72.)
© 2000 by Society for Leukocyte Biology

Sulfatide binding and activation of leukocytes through an L-selectin-independent pathway

Ziqiang Ding, Hiroto Kawashima and Masayuki Miyasaka

Department of Bioregulation, Biomedical Research Center, Osaka University Medical School, Japan

Correspondence: Dr. Ziqiang Ding, Department of Pediatrics, IWK Grace Health Center, 5850 University Avenue, Halifax, NS, B3J 3G9, Canada. E-mail: zding{at}is.dal.ca


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Sulfatide has been reported to activate leukocytes through L-selectin. Here we provide evidence that sulfatide binds to and activates leukocytes through both L-selectin-dependent and -independent pathways. Rat leukocytes of various sources shed surface L-selectin after phorbol myristate acetate (PMA) treatment, however, these cells retained the ability to bind sulfatide. In addition, sulfatide also bound to an L-selectin-negative cell line EL-4, and the binding was up-regulated by PMA. Sulfatide induced aggregation of L-selectin-positive lymphocytes, which was highly dependent on divalent cations, protein tyrosine kinases (PTK), and protein kinase C (PKC), but was independent of ß1 and ß2 integrins. In contrast, sulfatide-induced EL-4 cell aggregation required an LFA-1/ICAM-1 adhesion pathway but not PTK and PKC. A sulfatide receptor of 65 kDa was isolated from EL-4 cells. Taken together, this study suggests that sulfatide can bind to and activate leukocytes through an L-selectin-independent molecule and triggers signal transduction pathways different from those induced by L-selectin activation.

Key Words: adhesion molecules • lymphocytes • signal transduction • phorbol ester


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Sulfatide, 3-sulfated galactosylceramide [Gal(3-SO4)ß1-1Cer], is a glycolipid found at high concentrations in the white matter of the brain as well as in the kidney, spleen, digestive tract, serum and on granulocytes, red blood cells, and platelets [1 , 2 ]. Sulfatide has been reported to be involved in neuronal development [3 ], ion-exchange upon distal tubules of the kidney [1 ], modulation of blood coagulation [4 ], and tumor cell metastasis [5 ]. As a cell membrane component, sulfatide may be used as a specific site for the initial attachment and establishment of infection by malaria [6 ], Helicobacter pylori [7 ], and several kinds of viruses [8 ]. Sulfatide can also bind to L- and P-selectins [9 10 11 ]. Intravenous administration of sulfatide decreases L-/P-selectin-dependent leukocyte recruitment and alleviates tissue damage in animal models of acute inflammatory diseases [12 13 14 ]. On the other hand, activated granulocytes can also secrete sulfatide. This has been hypothesized to relieve leukocyte binding to endothelium, which facilitates leukocyte migration [15 ].

Sulfatide has recently been used as an L-selectin ligand to study post-receptor signal transduction after L-selectin engagement. Sulfatide can activate leukocytes [16 17 18 ] and trigger cytoplasmic calcium fluxes, tyrosine kinase activation, superoxide anion release, and cytokine production [19 20 21 ]. Sulfatide has also been reported to regulate lymphocyte proliferation and immunoglobulin production [22 ]. These sulfatide-induced events in leukocytes have been thought to be mediated via L-selectin [9 , 23 , 24 ]. However, our group has previously demonstrated that a 65-kDa sulfatide receptor (SulfR), distinguishable from L-selectin by its resistance to PMA-induced down-regulation, is also involved in lymphocyte activation [25 ].

In this study, our previous findings are extended by using various leukocyte subsets expressing different levels of L-selectin and SulfR. The signal transduction pathways activated by L-selectin and SulfR are also compared. The results demonstrate that SulfR is ubiquitously expressed by these leukocytes. From an L-selectin-negative mouse T lymphoma cell line, EL-4, a SulfR of 65 kDa has been identified. L-selectin engagement induces LFA-1-independent lymphocyte aggregation via protein tyrosine kinase (PTK) and protein kinase C (PKC). In contrast, SulfR triggers a PTK and PKC-independent signal transduction pathway that can promote LFA-1 binding to ICAM-1 and induce aggregation of EL-4 cells.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Animals
Eight- to 16-week-old male specific-pathogen-free Wistar rats were provided by the Shionogi Pharmaceutical Co. (Osaka, Japan), and used in accordance with the guidelines of the Animal Ethics Committee of Osaka University Medical School.

Cells
Rat mesenteric lymph node (MLN) lymphocytes were obtained by dicing lymph nodes with a scalpel and then gently grinding between glass slides. The cell preparation was passed through a nylon mesh to obtain single cell suspension. Peritoneal neutrophils were induced by intraperitoneal injection of 10 mL of 1% casein and recovered by peritoneal lavage 14–16 h later. The purity of neutrophils obtained was greater than 90% by crystal violet staining. Peripheral blood leukocytes were isolated by dextran sedimentation. Briefly, rat peripheral blood was withdrawn by cardiac puncture and was centrifuged at 200 g for 10 min to remove platelet-rich plasma. The pellet was mixed with two volumes of phosphate-buffered saline (PBS) and one-third final volume of 3% Dextran T-500. The mixture was settled for sedimentation at room temperature. The supernatant was retrieved as leukocyte-rich plasma and centrifuged at 300 g for 5 min. The pellet was washed and the contaminating red blood cells were lysed with 3 mL of 0.2% NaCl at room temperature for 20 s. The osmolarity was recovered immediately by adding 3 mL of 1.6% NaCl. The cells were centrifuged and washed with PBS. Granulocytes and lymphocytes were discriminated on the basis of their characteristic forward and side light-scattering patterns in the flow cytometer. EL-4 cells (mouse T lymphoma) were cultured in RPMI 1640 medium supplemented with 10% fetal calf serum (FCS), HEPES (10 mM), penicillin (100 U/mL), streptomycin (100 µg/mL), L-glutamine (2 mM), sodium pyruvate (1 mM), nonessential amino acid (0.1 mM), and 2-mercaptoethanol (2-ME; 50 µM).

Antibodies and reagents
GS5 [26 ], a mouse anti-sulfatide mAb (IgM), was used as culture supernatant. HRL2 and HRL3 are nonblocking and blocking hamster anti-rat L-selectin mAbs (both IgG), respectively, prepared in our laboratory [27 ]. WT.1 [27 ], OX50 [28 ], 1A29 [29 ], and TA-2 [30 ] are mouse IgG mAbs to rat CD11a (LFA-1), CD44, ICAM-1, and {alpha}4 integrin, respectively. MEL-14 [31 ], KBA [32 ], KAT-1 [33 ], KM201 [34 ], and M301 [14 ] are rat IgG mAbs to mouse L-selectin, LFA-1, ICAM-1, CD44, and ß7 integrin, respectively. HMß1 [35 ] is a hamster IgG mAb against mouse ß1 integrin (PharMingen). Fluorescein isothiocyanate (FITC)-conjugated goat anti-hamster IgG was purchased from Organon Teknika (Durham, NC). FITC-conjugated donkey anti-mouse IgM was obtained from Jackson ImmunoResearch Laboratories (West Grove, PA). FITC-conjugated goat anti-mouse Ig (IgG + IgM) and FITC-conjugated goat anti-rat Ig were from Southern Biotechnology Associates (Birmingham, AL). Phorbol myristate acetate (PMA), herbimycin A, genistein, staurosporine, cytochalasin B, EGTA, sulfatide, fucoidan, and bovine serum albumin (BSA; fraction V) were all obtained from Sigma (St. Louis, MO). Galactosylceramide was from Funakoshi (Tokyo, Japan). 2’, 7’-Bis-(2-carboxyethyl)-5-carboxyfluorescein acetoxymethyl ester (BCECF-AM) was from Dojindo (Osaka, Japan). Dextran T-500 (mol wt 523,000) was from Pharmacia (Uppsala, Sweden).

Flow cytometry
Sulfatide binding to leukocytes was detected by immunofluorescence flow cytometry with anti-sulfatide mAb GS5. Sulfatide was first dissolved in dimethyl sulfoxide (DMSO) at 50 mg/mL and diluted with PBS to appropriate concentrations just before use. Leukocytes were either left untreated or stimulated with 50 ng/mL PMA at 37°C for 10–30 min, unless otherwise indicated. Cells were washed and incubated with 50–300 µg/mL sulfatide in Ca2+-, Mg2+-free PBS on ice for 30 min. Then cells were washed twice with PBS and incubated with anti-sulfatide mAb GS5, using a 1:50 dilution of culture supernatant, on ice for 30 min. Cells were then stained with FITC-conjugated donkey anti-mouse IgM at 5 µg/mL on ice for 30 min. Finally, the cells were washed twice again, and resuspended in PBS for flow cytometric analysis in an Epics XL flow cytometer (Coulter Electronics, Hialeah, FL).

Adhesion assay
Adhesion of EL-4 cells to immobilized sulfatide or galactosylceramide (GalCer) was determined on 96-well enzyme-linked immunosorbent assay (ELISA) plates (SUMILON, H type, Japan). Sulfatide or GalCer dissolved in methanol was immobilized onto plates by drying at 37°C for 1.5 h. The nonspecific binding sites were then blocked with 3% BSA in PBS overnight at 4°C. The wells were washed three times with 0.05% Tween 20 in PBS. Cultured EL-4 cells were washed twice with RPMI 1640 medium, labeled with 5 µM BCECF-AM at 37°C for 30 min. Then the cells were washed three times and resuspended in RPMI medium containing 10% FCS. Cells were left untreated or preincubated with 100–1000 µg/mL sulfatide on ice for 20 min, washed, and resuspended in RPMI + 10% FCS at 2 x 106 cells/mL. Next, 50 µL of the cell suspension was added in quadruplicate to wells coated with sulfatide or galactosylceramide. After 40 min incubation at 7°C, the wells were filled with RPMI medium and sealed with parafilm. The plates were inverted at 7°C for 40 min to allow the nonadherent cells to detach from the bottom surface of the wells. The medium containing unbound cells was aspirated. Bound cells were lysed with 50 µL of 1% NP-40/PBS, and fluorescence intensity was determined in a Fluoroskan II microplate fluorometer (Labsystem, Tokyo, Japan) at an excitation wavelength of 485 nm and an emission wavelength of 538 nm. Percent bound cells represented the percentage of fluorescence intensity of bound cells over the fluorescence intensity of total added cells. The results were expressed as means ± standard deviation of quadruplicate determinations.

Affinity purification and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis of SulfR
Sulfatide was hydrophobically conjugated to octyl-Sepharose CL-4B (Pharmacia) as reported previously [25 ]. Nonspecific binding sites on Sepharose beads were blocked by incubation with 1% BSA for 1 h at room temperature. EL-4 cells were surface-biotinylated with 50 µg/mL NHS-LC-biotin (Pierce, IL) and lysed with 0.5% NP-40 in Ca2+-, Mg2+-free PBS supplemented with 1 mM phenylmethylsulfonyl fluoride (PMSF). The cell lysate was applied first to a nonconjugated octyl-Sepharose column for preclearance. The pass-through fraction was subsequently applied to a sulfatide-conjugated octyl-Sepharose column. The column was washed consecutively with PBS, 1 M NaCl in PBS, and 3 M NaSCN in PBS as described previously [25 ]. The NaSCN eluate was dialyzed against PBS overnight and precipitated first with galactosylceramide-conjugated octyl-Sepharose and then with sulfatide-conjugated octyl-Sepharose. Precipitated proteins were eluted by boiling the Sepharose beads for 3 min in SDS sample buffer supplemented with 2-ME. The eluted proteins were then resolved by SDS-PAGE in 4–20% gradient gels (Daiichi Pure Chemicals, Tokyo, Japan), transferred to IPVH membrane (Millipore, Japan), and detected by a horseradish peroxidase-conjugated avidin-biotin complex kit (ABC kit, Funakoshi, Tokyo, Japan) and enhanced chemiluminescence (ECL, Amersham), according to the manufacturer’s instructions.

Aggregation assay
MLN lymphocytes and EL-4 cells were suspended at 2 x 106 cells/mL in RPMI medium containing 10% FCS. Next, 100 µL of cells were added in duplicate wells to 96-well culture plates (IWAKI, Japan). Reagents for cell pretreatment were added at the indicated concentrations 30 min before cell stimulation with anti-L-selectin mAbs (1 µg/mL) or sulfatide (100 µg/mL). Ten microliters of MEL-14 (anti-mouse L-selectin mAb) culture supernatant was used for EL-4 cell stimulation. Cell aggregation was allowed to proceed at 37°C for 3 h and was scored under a phase-contrast microscope (Olympus, Japan) on a scale of 0–5, according to Rothlein and Springer [36 ], as modified by Ruegg et al. [37 ]. Zero indicates no cells were aggregated; 1 indicates up to 20% of the cells were in aggregates; 2, 20–40%; 3, 40–60%; 4, 60–80%; and 5, 80–100%.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Both L-selectin and non-L-selectin receptors mediate sulfatide binding to leukocytes
Sulfatide was previously demonstrated to bind to PMA-treated lymphocytes lacking surface L-selectin via a novel binding site [25 ]. To examine whether various leukocyte subsets also express sulfatide binding sites other than L-selectin, the sulfatide-binding capacity of these cells before and after PMA treatment was studied. As shown in Figure 1 , 10 min treatment with PMA completely abrogated L-selectin expression on peripheral blood leukocytes, MLN lymphocytes, and peritoneal neutrophils. The ability of these cells to bind sulfatide, however, was still retained, although the binding was decreased compared with that in untreated cells. The decreased binding apparently corresponded to complete shedding of surface L-selectin, which has also been demonstrated to bind sulfatide [9 ]. This decrease in sulfatide binding was much smaller in inflammation-induced peritoneal neutrophils in agreement with the much lower expression of surface L-selectin on these neutrophils (Figs. 1 and 2) . By extending the time of PMA treatment to 30 min, L-selectin expression on these neutrophils remained undetectable but the level of sulfatide binding was up-regulated substantially. In peripheral blood granulocytes, lymphocytes, and peritoneal neutrophils, sulfatide binding was increased to levels even higher than the initial values. This increase in sulfatide binding was observed in the whole cell population, as can be seen in the representative histograms in peritoneal neutrophils (Fig. 2 ). In MLN lymphocytes, L-selectin seemed to account for most of the sulfatide binding, and the PMA-induced increase of sulfatide binding was less remarkable than that seen in peripheral blood lymphocytes. Ca2+ and Mg2+ were not required for sulfatide binding because EDTA did not affect the binding (data not shown).



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  		Figure 1. Sulfatide binds to leukocytes through L-selectin-dependent and -independent pathways. Peripheral blood leukocytes, peritoneal neutrophils, and MLN lymphocytes were left untreated or stimulated with 50 ng/mL PMA at 37°C for 10–30 min. Cells were washed and incubated with 50–300 µg/mL sulfatide on ice for 30 min. Sulfatide binding was detected by anti-sulfatide mAb GS5 and FITC-conjugated anti-mouse IgM with a flow cytometer. Expression of L-selectin was also assessed at the same time. The ordinates represent relative mean channel fluorescence intensity in log values. A representative result of three similar experiments is presented. The mean fluorescence intensity was lower than 0.5 when an irrelevant antibody was used.

 


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  		Figure 2. L-selectin expression and sulfatide binding on peritoneal neutrophils. Peritoneal infiltrating neutrophils were induced by casein injection. Neutrophils were unstimulated or stimulated with 50 ng/mL PMA at 37°C for 10–30 min. Sulfatide binding at 100 µg/mL and L-selectin expression were detected as described in Figure 1 . Solid lines represent the flow cytometric profiles obtained by GS5 (anti-sulfatide mAb) and by HRL2 (anti-rat L-selectin mAb), respectively. Dotted lines represent the flow cytometric profiles obtained by control irrelevant Ab. A representative result from three similar experiments is presented.

 
We then screened a number of leukemic cell lines and found that a mouse lymphoma cell line, EL-4, did not express L-selectin (Fig. 3A ) but exhibited readily detectable sulfatide binding (Fig. 3B) . Sulfatide binding to EL-4 cells was increased to twofold the resting level after PMA stimulation for 60 min (Fig. 3B) . These observations together indicate that a sulfatide binding site distinguishable from L-selectin was expressed on leukocytes of various sources.



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  		Figure 3. Sulfatide binds to L-selectin-negative EL-4 cells. (A) Representative histograms of L-selectin expression on EL-4 cells (a) and mouse lymph node lymphocytes (b). Solid and dotted lines represent the flow cytometric profiles obtained by MEL-14 (anti-L-selectin) and control irrelevant Ab, respectively. (B) Effect of PMA on sulfatide binding to EL-4 cells. EL-4 cells were incubated with or without 50 ng/mL PMA at 37°C for 10–60 min. Cells were washed and incubated with 50–300 µg/mL sulfatide on ice for 30 min. Sulfatide binding was detected by monoclonal anti-sulfatide mAb GS5 and FITC-conjugated anti-mouse IgM with a flow cytometer. The ordinate represents relative mean channel fluorescence intensity in log values. A representative result of three similar experiments is presented.

 
The non-L-selectin binding sites for sulfatide display the essential characteristics of a receptor
To further characterize the sulfatide binding site, we next examined the binding of L-selectin-negative EL-4 cells to immobilized sulfatide. EL-4 cells bound to immobilized sulfatide but not to desulfated analogous glycolipid, galactosylceramide (Fig. 4A ). The adhesion was dependent on the amount of immobilized sulfatide, peaking at 300 ng/well of sulfatide. Preincubation of the cells with soluble sulfatide resulted in inhibition of cell binding in a concentration-dependent manner (Fig. 4B) .



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  		Figure 4. Adhesion of EL-4 cells to immobilized sulfatide is specific and saturable. (A) Adhesion of EL-4 cells to immobilized sulfatide (Sulf) and galactosylceramide (GalCer). Various concentrations of sulfatide and GalCer were adsorbed to 96-well plates in quadruplicate. BCECF-labeled EL-4 cells were added to the wells and incubated at 7°C for 40 min. The fluorescence intensity of the bound cells was determined in a microplate fluorometer. (B) Inhibition of EL-4 cell adhesion to immobilized sulfatide by preincubating cells with sulfatide in a concentration-dependent fashion. Sulfatide was immobilized in 96-well plates at 300 ng/well. Fluorescence-labeled EL-4 cells were preincubated with 0.1–1.0 mg/mL sulfatide in an ice bath for 20 min, washed, and resuspended in RPMI medium containing 10% FCS. The subsequent procedures for adhesion assay were the same as in panel A. Each column is expressed as mean ± SE of quadruplicate experiments. Results from one of three experiments are presented.

 
We next examined the kinetics of sulfatide binding to EL-4. As shown in Figure 5A , sulfatide binding to EL-4 was rapid, with maximal binding occurring at 10 min. Sulfatide binding was not further increased even if the incubation time was extended over 10 min. Sulfatide binding to EL-4 was concentration-dependent and was almost saturated at the presence of 1 mg/mL of sulfatide. Half-maximal binding was seen at about 0.3 mg/mL of sulfatide. These results suggest that the L-selectin-independent sulfatide binding sites on EL-4 cells are specific and saturable, which represent the essential characteristics of a receptor.



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  		Figure 5. Sulfatide binding to L-selectin-negative EL-4 cells is rapid, dose-dependent, and saturable. (A) Sulfatide binding to EL-4 cells as a function of time. EL-4 cells were incubated with 300 µg/mL of sulfatide on ice for 5–60 min. Cells were washed and sulfatide binding was detected by mAb GS5 and FITC-conjugated anti-mouse IgM with a flow cytometer. (B) Concentration-dependent binding of sulfatide by EL-4 cells. EL-4 cells were incubated with 0.05–2 mg/mL sulfatide on ice for 30 min, washed, and stained by GS5 and FITC-conjugated anti-mouse IgM. The ordinate represents relative mean channel fluorescence intensity in log values. A representative result from three experiments is presented.

 
Identification of the SulfR as a 65-kDa protein
EL-4 cells lack surface L-selectin expression yet display a readily detectable level of sulfatide binding under resting conditions. Therefore, we used EL-4 cells for the isolation and purification of the novel sulfatide receptor (SulfR). Using affinity column chromatography and ligand-receptor precipitation with sulfatide-conjugated octyl-Sepharose, a protein of about 65 kDa apparently specific for sulfatide binding was obtained (Fig. 6 ). The displayed single band was not seen in the precipitate obtained with control octyl-Sepharose beads conjugated with galactosylceramide. In addition, this band became remarkably faint when soluble sulfatide was used to saturate the binding material in the affinity-isolated fraction from sulfatide-conjugated Sepharose column. Fucoidan, which binds L-selectin as well as several non-L-selectin-type sulfatide-binding proteins [2 , 38 ], did not block precipitation of this 65-kDa material. These results indicate that the 65-kDa molecule represents a specific receptor for sulfatide.



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  		Figure 6. A 65-kDa molecule is detected as a sulfatide binding protein from EL-4 cells. EL-4 cells were surface biotinylated and lysed with NP-40 in PBS with PMSF. The cell lysate was first precleared by passing through an octyl-Sepharose column, then applied to an affinity column with sulfatide-conjugated octyl-Sepharose CL-4B. The column was washed consecutively with PBS, 1 M NaCl, and 3 M NaSCN. The NaSCN eluate was dialyzed, and precipitated with galactosylceramide (GalCer)-conjugated octyl-Sepharose beads (lane 1). The supernatant after GalCer-beads precipitation was separated into three aliquots. One aliquot was directly precipitated with sulfatide-conjugated Sepharose beads (lane 2). The other two were first preincubated with either 0.5 mg/mL sulfatide (lane 3) or 0.5 mg/mL fucoidan (lane 4) on ice for 30 min, then precipitated with sulfatide-conjugated beads. The precipitates were subjected to SDS-PAGE under reducing conditions, transferred to IPVH membranes, and detected by ABC kit and ECL as described in Materials and Methods. The molecular mass markers (kDa) are indicated on left.

 
Both L-selectin and SulfR transduce signals for cell aggregation, but the signal transduction pathways and adhesion molecules involved appear to be different
L-selectin and SulfR can both bind sulfatide. Post-receptor signal transduction pathways activated by L-selectin and SulfR were examined and compared. Anti-L-selectin mAb induced aggregation of lymphocytes expressing L-selectin, but did not induce aggregation of EL-4 cells (Table 1 ). In contrast, sulfatide induced aggregation of both cell types, which did not occur at 4°C, and was stringently dependent on the presence of serum, active metabolism, and an intact cytoskeleton (Table 1 and Fig. 7 ). Sulfatide-induced aggregation of both cell types was abolished by the deprivation of FCS from culture medium, pretreatment of cells with sodium azide (NaN3) or cytochalasin B. Sulfatide-induced aggregation of MLN lymphocytes was strongly inhibited by EDTA, EGTA, herbimycin A, and staurosporine, indicating the essential requirement of Ca2+, tyrosine kinases, and PKC (Table 1) . As for adhesion molecules, LFA-1, ICAM-1, CD44, and {alpha}4 integrin did not appear to be required for the aggregation of lymphocytes, because none of the blocking mAbs to these adhesion molecules was inhibitory for either anti-L-selectin mAb or sulfatide-induced lymphocyte aggregation. Combined blockade of LFA-1 and ICAM-1 or LFA-1 and {alpha}4 integrin also did not inhibit the aggregation of MLN lymphocytes. Compared to lymphocyte aggregation, EL-4 cell aggregation was not inhibited by herbimycin A and staurosporine, but the size of individual aggregation was substantially inhibited by EDTA and EGTA, although the percentage of cells in aggregates was not substantially altered (Fig. 7) . EL-4 cell aggregation was also strongly inhibited by anti-LFA-1 and partially by anti-ICAM-1 (Table 1 and Fig. 8 ), suggesting that LFA-1/ICAM-1 interaction is activated by sulfatide stimulation and that this activation is essential in sulfatide-induced EL-4 cell aggregation. LFA-1 activation on EL-4 cells was also suggested by the finding that sulfatide-treated EL-4 cells adhered more efficiently than untreated cells to immobilized ICAM-1 (data not shown).


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  		Table 1. Mechanisms Involved in Sulfatide-Induced Lymphocyte Aggregation

 


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  		Figure 7. Effect of various inhibitors on sulfatide-induced EL-4 cell aggregation. EL-4 cells resuspended in RPMI medium containing 10% FCS were added in duplicate to 96-well culture plates. Reagents for cell pretreatment at concentrations described in Table 1 were added 30 min before sulfatide (100 µg/mL) stimulation. Photographs were taken under a phase-contrast microscope after 3 h of incubation. Original magnification x100.

 


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  		Figure 8. Adhesion molecules involved in sulfatide-induced EL-4 cell aggregation. EL-4 cells resuspended in RPMI medium containing 10% FCS were added in duplicate to 96-well culture plates. Blocking mAbs to LFA-1 (KBA), ICAM-1 (KAT-1), ß1 integrin (HMß1), ß7 integrin (M301), and CD44 (KM201) at 30 µg/mL were added to the cells 30 min before sulfatide (100 µg/mL) stimulation. Photographs were taken under a phase-contrast microscope after 3 h of incubation. Original magnification x100.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
L-selectin has been reported to bind sulfatide and mediate sulfatide-induced neutrophil activation [19 , 20 , 23 ]. Therefore, L-selectin has been generally considered to be the receptor for sulfatide. We previously reported that lymph node lymphocytes express a novel receptor for sulfatide (SulfR) [25 ], which is apparently distinct from L-selectin by its resistance to PMA-induced down-regulation of surface expression. In the current study, we extended our previous observations to peripheral blood lymphocytes, granulocytes, peritoneal neutrophils, and an L-selectin-negative lymphoma cell line, EL-4. SulfR was expressed on all of these cells as assessed by sulfatide binding, which was measured by flow cytometry with anti-sulfatide mAb GS5. Stimulation of L-selectin and SulfR can both induce lymphocyte aggregation, however, the signal transduction pathways involved in the aggregation appeared to be different.

To more clearly distinguish SulfR from L-selectin, leukocytes of various sources were treated with PMA for an extended time course. Peripheral blood lymphocytes and granulocytes expressed comparable levels of L-selectin, which was almost completely lost after 10 min treatment with PMA and was not reexpressed within 30 min. In the context of a dramatic decline in L-selectin expression, sulfatide binding detected by GS5 was only moderately decreased after 10 min treatment with PMA. It is of note that sulfatide bindings recovered to levels as high as the baselines after 30-min stimulation with PMA when L-selectin expression was still undetectable. These findings suggest that L-selectin can mediate sulfatide binding to peripheral blood leukocytes but L-selectin appears to be only one of the binding sites for sulfatide. The dissociation between the levels of L-selectin expression and sulfatide binding was further demonstrated in peritoneal infiltrating neutrophils, which expressed much less surface L-selectin compared with blood neutrophils. Sulfatide binding to peritoneal neutrophils was accordingly less decreased after 10 min treatment with PMA. This decrease in sulfatide binding corresponded to the loss of L-selectin expression. However, sulfatide binding to peritoneal neutrophils that had been treated with PMA for 30 min was increased to approximately twofold the initial level. These findings indicate that an L-selectin-independent molecule was also involved in sulfatide binding to leukocytes. This novel sulfatide binding receptor (SulfR) can be up-regulated by PMA in expression or affinity.

L-selectin-independent sulfatide binding was also demonstrated in L-selectin-negative EL-4 cells. In addition, sulfatide binding to PMA-treated EL-4 cells did not show the initial decrease as seen in the other leukocytes, but rather was gradually increased as the time of PMA stimulation was extended. This result further supports our conclusion that the newly identified SulfR is distinct from L-selectin.

The protein structure of SulfR and its relationship with other cell adhesion molecules remains to be determined. Several proteins have been reported to bind sulfatide in vitro. The adhesive proteins in extracellular matrix, laminin, thrombospondin, and von Willebrand factor (vWF) have been shown to bind sulfatide [2 ]. In addition, antistasin [39 ] and properdin [40 ] also bind sulfatide. Antistasin is a leech salivary protein and an inhibitor of coagulation and metastasis. Properdin is a complement alternative pathway protein. Based on the structure of these proteins, a consensus sequence (Cys-Ser-Val-Thr-Cys-Gly-X-Gly-X-X-X-Arg-X-Arg) has been proposed to represent a sulfatide binding domain [39 ]. However, there are no reports indicating that proteins bearing this consensus sequence are also expressed on the leukocyte surface and are up-regulated by PMA. Therefore, it is unlikely that these proteins are candidates for our proposed SulfR. Several other proteins lacking the above consensus sequence have also been reported to bind to sulfatide. For instance, P-selectin can bind to sulfatide in vitro [41 ]. Sulfatide can block P-selectin-mediated leukocyte adhesion and migration in vivo [11 , 12 ]. However, P-selectin is only expressed on activated endothelial cells and platelets but not on neutrophils and lymphocytes. Amphoterin [42 ] and cytotactin [43 ] have also been demonstrated to be sulfatide binding proteins. These two proteins are present in the neural tissues and seem to be important in neural cell adhesion, differentiation, and myelin formation. Although it is not known whether these proteins are also expressed on the leukocyte surface, their reported molecular weights are different from that of SulfR we have demonstrated in this study.

It is of interest to assume that sulfatide, which is widely expressed in the extravascular tissue, can promote leukocyte locomotion, on the basis of haptotaxis, in the extravascular space. Leukocytes that migrate through the vascular wall have undergone activation and shed surface L-selectin. Therefore, SulfR may become more important in leukocyte interactions with sulfatide in the tissue. More studies are required to address the importance of tissue-associated sulfatide in leukocyte activation and interstitial movement.

Sulfatide can activate MLN lymphocytes and induce intercellular aggregation. This process was dependent on temperature, energy, and an intact cytoskeleton. At 4°C, sulfatide did not induce cellular proximity and aggregation, suggesting that sulfatide-induced cell aggregation is an active process requiring signal transduction and active metabolism. Mechanisms for the aggregation of MLN lymphocytes and EL-4 cells were apparently different. Sulfatide-induced aggregation of lymphocytes was substantially but not completely inhibited after PMA treatment, which removed cell-surface L-selectin [25 ]. Therefore, both L-selectin and SulfR appeared to be involved in lymphocyte aggregation. PTK and PKC were also involved in this aggregation. In contrast, sulfatide-induced aggregation of EL-4 cells was mediated solely through the stimulation of SulfR because L-selectin was not expressed by EL-4 cells. PTK and PKC did not seem to play a role. These results together suggest that the intracellular signals induced by L-selectin and SulfR are different.

Adhesion molecules involved in the aggregation of MLN lymphocytes and EL-4 cells also appeared to be different. Blocking one of the adhesion molecules LFA-1, ICAM-1, and VLA-4 was not inhibitory to sulfatide-induced aggregation of MLN lymphocytes. Blocking both LFA-1 and ICAM-1, or both LFA-1 and VLA-4, also did not inhibit this aggregation. However, EDTA almost completely blocked lymphocyte aggregation, suggesting that divalent cation-dependent molecular interactions or Ca2+ influx are essential. In contrast to the aggregation of MLN lymphocytes, EL-4 cell aggregation was substantially inhibited by blocking LFA-1 and ICAM-1 adhesion pathway. It is interesting that EDTA decreased the size of EL-4 cell aggregates but did not alter the percentage of cells in aggregates, suggesting that cation-independent molecules were also involved in the initiation of EL-4 cell aggregation. These results together suggest that lymphocytes and EL-4 cells may have different mechanisms for sulfatide-induced signal transduction and cellular interaction. However, it cannot be excluded that the signal transduction pathways in normal MLN lymphocytes can be different from those in EL-4 cells.

An unexpected finding in the present study was the prerequisite of serum for intercellular aggregation. For both MLN lymphocytes and EL-4 cells, sulfatide-induced intercellular aggregation failed to occur without fetal calf serum in the medium. The molecular mechanism involved in this phenomenon is currently not clear. It has been reported that fibrinogen can promote leukocyte adhesion to endothelium by bridging CD11b/CD18 and ICAM-1 in vitro and also bridging two ICAM-1 molecules on the opposing lymphocytes [44 ]. However, in our experiment, addition of albumin, fibrinogen, fibronectin, or hyaluronic acid to the serum-free culture medium did not increase sulfatide-induced cell aggregation (data not shown), suggesting that some other serum components may be important in the intercellular aggregation.

In conclusion, we have demonstrated a novel sulfatide receptor of 65 kDa on leukocytes, which is distinct from the sulfatide binding proteins identified to date. Signals from SulfR engagement can activate LFA-1, promoting LFA-1-dependent aggregation of EL-4 cells. The molecular structure and function of the SulfR are currently under investigation.


   ACKNOWLEDGEMENTS
 
This work was supported in part by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science, and Culture of Japan, and a grant from the Ministry of Science and Technology of Japan. We thank Dr. Thomas Issekutz (Dalhousie University, Canada) for the gift of mAb TA2 and Dr. Yasuo Suzuki (University of Shizuoka) for stimulating discussions.

Received September 18, 1999; revised February 16, 2000; accepted February 17, 2000.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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