The role of glycolipids in mediating cell adhesion: a £ow chamber … · 2017. 1. 4. · The role of glycolipids in mediating cell adhesion: a £ow chamber study1 Jan Vogel a;*,

The role of glycolipids in mediating cell adhesion: a £ow chamber study1

Jan Vogel a;*, Gerd Bendas a, Udo Bakowsky a, Gerd Hummel b,Richard R. Schmidt b, Ursula Kettmann c, Ulrich Rothe c

a Department of Pharmacy, Martin Luther University Halle, Wolfgang-Langenbeck-Str. 4, 06120 Halle, Germanyb Department of Chemistry, University Konstanz, 78457 Konstanz, Germany

a Institute of Physiological Chemistry, Medical Faculty, Martin Luther University Halle, Hollystrasse 1, 06097 Halle, Germany

Received 9 March 1998; accepted 26 March 1998

Abstract

Selectins constitute a family of proteins that mediate leukocyte tethering and rolling along the vascular endothelium byrecognizing various carbohydrate ligands in response to inflammation. To test the hypothesis that multivalent binding ofselectins to their ligands is the molecular basis for achieving sufficient binding forces, we have performed this flow chamberstudy. Selectin-containing Chinese hamster ovarial cells (CHO-E) bind and roll along a support-fixed phospholipidmembrane containing a defined concentration of a synthetic Sialyl Lewisx (sLex) glycolipid ligand. Ligands are eitherhomogeneously distributed, or arranged in defined lateral clusters, as illustrated here for the first time. The lateral glycolipidclusters which appear as recognition motifs are essential for mediating cell rolling. Furthermore, the transition from firm celladhesion to cell rolling depends on the site density of ligands. Rolling velocity shows little dependence on shear forces withina broad range. As we found out that cells do not roll along the model membranes with homogeneous ligand distribution, ourresults therefore support the hypothesis of multivalent binding events. Since these investigations suggest that lipid-anchoredsLex, functionally embedded in a lipid matrix, can mediate cell rolling, this study demonstrates the relationship betweendynamic glycolipid binding to selectins with the hypothesis of multivalency of binding for the first time. ß 1998 ElsevierScience B.V. All rights reserved.

Keywords: Selectin; Cell adhesion; Glycolipid; Sialyl Lewisx ; Cell rolling; Planar bilayer, supported

1. Introduction

The receptor-mediated recruitment of leukocytesto sites of injury, or infection is essential for thedevelopment of an appropriate immune response.Leukocyte rolling along endothelial cells under theshear force in postcapillary venules represent the ¢rststep in a sequence of adhesive interactions that leadto ¢rm attachment and subsequent emigrationthrough the venular wall [1]. The selectins, a familyof three adhesion molecules, are thought to mediaterolling by binding carbohydrate presenting ligands

0005-2736 / 98 / $19.00 ß 1998 Elsevier Science B.V. All rights reserved.PII: S 0 0 0 5 - 2 7 3 6 ( 9 8 ) 0 0 0 5 8 - 3

Abbreviations: AFM, atomic force microscopy; CHO-E, Chi-nese hamster ovarial cells (containing E-selectin) ; CLA, cutane-ous lymphocyte associated antigen; DSPC, 1,2-distearoyl-sn-glyc-ero-3-phosphocholine; FITC, £uorescein-5-isothiocyanate; NBD-PC, 1-palmitoyl-2-[12-[(7-nitro-2-1,3 benzoxadiazol-4-yl)amido]-dodecanoyl]-sn-glycero-3-phosphocholine; NBD-PE, 1,2-dipalmi-toyl-sn-glycero-3-phosphoethanolamine-N-(7-nitro-2-1,3-benzoxa-diazol-4-yl) ; PBS, phosphate-bu¡ered saline; POPC, 1-palmitoyl,2-oleoyl-sn-glycero-3-phosphocholine; sLex, sialyl Lewisx

* Corresponding author. Fax: +49 (345) 552-7022;E-mail : [email protected]

1 This paper is dedicated to Professor H.W. Meyer (Universityof Jena) on the occasion of his 65th birthday.

BBAMEM 77384 13-7-98 Cyaan Magenta Geel Zwart

Biochimica et Biophysica Acta 1372 (1998) 205^215

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with rapid association and dissociation rates con-stants [2]. E-selectin and P-selectin can be expressedon endothelial cells by in£ammatory signals, andboth bind to carbohydrate ligands on subsets of leu-kocytes [3]. L-selectin, which is constitutively ex-pressed on leukocytes and shed after activation,binds inducible ligands on the endothelium [4]. Allselectins share a highly conserved N-terminal lectindomain that can interact with sialylated and fucosyl-ated polylactosaminoglycans presented by mucin-type glycoproteins, where sLex epitopes (NeuAcK2^3GalL1^4(FucK1^3)GlcNAc) are of key importance.Although numerous studies performed under staticconditions have con¢rmed fundamental ¢ndingsabout selectin binding characteristics and structure^activity relations of their binding epitopes [5^7], theydo not provide information on how ligands interactwith selectins under dynamic conditions.

Springer and coworkers have performed variousstudies to investigate the role of dynamic parametersand the contribution of the individual selectins in therolling process [8,9]. Therefore, they immobilized de-¢ned amounts of E-selectin or P-selectin into planarmembranes or onto plastic and analyzed the rollinginteraction of neutrophils under de¢ned shear £owconditions. They could show that tethering, rollingvelocity and shear force resistance of neutrophils areregulated by the kinetics of bond formation and dis-sociation, which are di¡erent for each of the individ-ual selectins [10].

These studies had been focused on modifying se-lectins for the characterization of their binding prop-erties. Several questions concerning the carbohydrateligands remained unanswered. Whereas selectins bindthe identi¢ed single carbohydrate epitopes with lowa¤nity, they bind, however, with much higher a¤n-ity to the sialomucins [11]. Multiple protein^carbo-hydrate interactions are therefore thought to be thereason for a higher binding e¤ciency mediated by aspecial molecular arrangement of binding epitopes[12]. Various groups prepared synthetic oligomericsLex derivatives, which could only in part supportthis hypothesis of slowly increased a¤nities [13,14].Furthermore, the role of the carbohydrate presentinganchor group is not fully elucidated. Contrary tothe natural occurring glycoproteins, various bind-

ing studies have been performed with glycolipids,which were disorderedly immobilized onto solidsurfaces. But in our opinion, these surfaces cannotadequately simulate conditions at the membrane sur-face.

We have performed this study to investigate themolecular features of ligands such as structure, den-sity and lateral clustering, which are fundamental forthe rolling process. Therefore we incorporated vari-ous concentrations of glycolipid ligands into planarsupport-¢xed phospholipid membranes to get a wellde¢ned model membrane, which could simulate theconditions at cell surfaces. By using the Langmuir^Blodgett technique, glycolipids can be organized asde¢ned clusters in the matrix, and may serve thus asrecognition determinants. We wanted to illuminate iflateral clusters are the basis for the discussed higherbinding a¤nity of sialomucins by multivalent bind-ing. Therefore, the model membrane was assembledinto a £ow chamber to interact with selectin-contain-ing cells under de¢ned shear £ow conditions. Usingtransparent support materials, direct detection of cellbinding events with an inverted confocal laser scan-ning microscope was made possible.

To examine whether carbohydrates presentedby lipid anchor groups in a de¢ned membrane areable to mediate cell rolling, we choose a syntheticderivative where the sLex moiety is directly linkedto a ceramide anchor (NeuAcK2^3GalL1^4(FucK1^3)GlcNAc-ceramide). By modifying the density andthe appearance of clustered lateral arrangementof this ligand in the phospholipid matrix, the roleof multiple ligand^receptor bonds should be detect-able.

We were able to show that sLex ceramide can actas ligand which mediates rolling or ¢rm adhesion ina concentration-dependent manner, when laterallyorganized in clusters. The absolute ligand concentra-tion was further reduced, when ligand clusters werediluted by non-reactive lipids. When the glycolipidwas homogeneously distributed into a phospholipidmatrix, cells adhered selectin-induced, but did notroll upon diluting the ligand density.

Thus, our results support the hypothesis of multi-valent ligand receptor interactions as prerequisite forrolling and higher binding constants.


J. Vogel et al. / Biochimica et Biophysica Acta 1372 (1998) 205^215206

2. Materials and methods

2.1. Materials

1,2-Distearoyl-sn^glycero-3-phosphocholine(DSPC), 1-palmitoyl, 2-oleoyl-sn^glycero-3-phospho-choline (POPC) were purchased from Sigma (Deisen-hofen, Germany). The purity of these lipids was an-alyzed by HPTLC and HPLC and regarded asgreater than 99%. POPC was frequently analyzedduring this study and did not show any oxidativedegradation products. 1,2-Dipalmitoyl-sn^glycero-3-phosphoethanolamine-N-(7-nitro-2-1,3-benzoxadia-zol-4-yl) (NBD-PE) and 1-palmitoyl-2-[12-[(7-nitro-2-1,3 benzoxadiazol-4-yl)amido]dodecanoyl]-sn^glyc-ero-3-phosphocholine (NBD-PC) were purchasedfrom Avanti Polar Lipids (Alabaster, AL, USA).The glycolipid was synthesized as previously pub-lished [15] and used with a purity of greater than99%. The substances were used without further pu-ri¢cation.

2.2. Cell cultivation

Mouse E-selectin-transfected CHO-cells (CHO-Ecells) were a generous gift of P. Vasalli. Cells weregrown in MEM-K media containing 10% fetal calfserum, 2 mM L-glutamine and 100 nM penicillin/streptomycin. Flasks seeded with 5U104 CHO-Ecells were incubated at 37³C in 5% CO2 for 3 or 4days until cells were grown nearly con£uent. Aftertrypsinization for 3 min with 2 ml 0.25% trypsin inEDTA, cells were transferred to slowly rotating plas-tic tubes. Cells stayed in suspension for up to 4 h.Within this time, rolling experiments had to be per-formed in the £ow chamber.

2.3. Immunolabeling and embedding for electronmicroscopy

Cells (approximately 1.8U107) transferred andwashed in PBS were ¢xed with 2.5% formaldehydein PBS for 5 min at room temperature and washedwith PBS, afterwards with PBS containing 50 mMglycine (blocking free aldehyde groups) and ¢nallywith PBS containing 0.5% bovine serum albuminand 0.2% gelatine (PBG). Cells were incubated over-night at 4³C with speci¢c anti-E-selectin antibody

from rat. After six times washing with PBG, cellswere incubated for 1 h with 12 nm gold-labeled sec-ondary antibody (anti-rat IgI from goat; Dianova)and afterwards carefully washed with PBG and PBS(all centrifugation steps at 1200 rpm = 120Ug).

For embedding, the cells were post¢xed for 2 hwith 4% formaldehyde/0.5% glutaraldehyde, thenwashed in PBS and suspended in 1.25% agar. Smallcell^agar pieces were conventionally dehydrated inacetone and embedded in Durcupan (Fluka, Neu-Ulm, Germany), hardened at 65 and 100³C. Ultra-thin sections were stained with UO2 acetate and Pbcitrate.

2.4. Film balance and £uorescence ¢lm balancetechnique

A ¢lm balance system of type R and K (Rieglerand Kirstein, Germany) was used with MilliQ wateras subphase. The lipids were dissolved in freshly dis-tilled chloroform/methanol (2:1, v/v) at 40^60 mM.After spreading the lipid solution at the water sur-face, an equilibrium period of 10 min followed.Then, the ¢lm was compressed at 25³C with a com-pression speed of 0.01 nm2 min31 molecule31.

To simultaneously measure the isotherms and do-main growth, the trough was integrated into a £uo-rescence microscope (Olympus, Japan) with a motordriven xy stage, using the dye NBD-PC at 0.5 mol%,that preferentially partitioned into disordered liquid-expanded (LE) phases.

2.5. Preparation of supported planar bilayers

Supported planar bilayers were prepared using theLangmuir^Blodgett technique. Microscope slides(glass, diameter of 18 mm, thickness of 0.2 mm)were used as transparent supports. Slides were ¢rstcleaned to achieve a highly homogeneous surface.Therefore, slides were treated with concentratedH2SO4/H2O2 mixture (7/3) at 80³C for 30 min underultrasonic conditions, and were then rinsed with ul-trapure water for 30 min. To increase the density ofsilanole groups at the surface, a cleaning procedurewith NH3/H2O2/H2O (1/1/5) followed, before ¢nallyrinsing with ultrapure water and drying the slides.The ¢rst step in forming a supported bilayer is thecovalent binding of monochlordimethyloctadecyl-si-


J. Vogel et al. / Biochimica et Biophysica Acta 1372 (1998) 205^215 207

lane (Sigma, Deisenhofen, Germany) at 50³C for 30min to produce a ¢rst monolayer at the slide. Thebilayer was completed by transferring the preformedlipid ¢lm at the Langmuir trough.

The lipid mixtures were transferred at a lateralpressure of 38 mN/m and a speed of 0.5 mm/minto hydrophobic substrates as a X-type monolayer.The transfer ratios were between 0.95 and 1. Freshlyprepared supported bilayers were immediately usedfor experiments in the £ow chamber.

2.6. Atomic force microscopy

The Langmuir^Blodgett ¢lms for AFM weretransferred at an applied surface pressure of 38mN/m to a hydrophobized silicon wafer (Wacker,Mu«nchen, Germany, rectangular 15U7 mm, thick-ness 2 mm) as a hydrophobic X-type monolayer.The samples were examined within 6 h of prepara-tion. AFM measurements were performed with aNanoscope IIIa (Digital Instruments, Santa Barbara,USA) under full hydrated conditions in a specialwater cell (Digital Instruments, Santa Barbara,USA). The contact modus AFM was used with com-mercial Si3N4 cantilevers with a nominal force con-stant of 0.12 N/m.

2.7. Laminar £ow experiments

The parallel plate £ow chamber used in these stud-ies has been described in detail in our previous in-vestigations [16]. The £ow apparatus was mountedonto an inverted £uorescent microscope Axiovert135 of a Laser Scanning Microscope (LSM 410 in-vert, Carl Zeiss, Germany).

Adhesion experiments were performed at 25³C in atemperature-controlled manner to maintain the later-al structure of the model membrane. MEM-K me-dium (Section 2.2) was used as £ow medium at shearrates between 200 and 1000 s31 powered by hydro-static pressure. For the £ow experiments, 106 £uores-cently labeled CHO-E cells in 100 Wl medium wereinjected into the streaming medium without dilution.Either, cell adhesion or rolling was analyzed imme-diately, or, the £ow was stopped for 5 min to allowcells to interact with the supported membrane. Afterthis time, £ow with the desired shear force was con-tinued and adhesion behavior of cells was monitored

by a sequence of images taken each 2 s. To charac-terize the cell movement, 50^150 cells within an areaof 630U630 Wm were analyzed throughout 20 s. Onlythose cells observed to directly contact the membranein absence of prior contacts with adherent cells werecounted and analyzed. The experiments for the pre-sented data were repeated four times under similarconditions.

3. Results and discussion

3.1. Detection of selectins at the CHO-E cell surface

The aim of the present study was to characterizethe in£uence of molecular features of synthetic selec-tin ligands on cell adhesion or cell rolling. In order tofocus on ligand e¡ects more distinctly, constant con-ditions on receptor site had to be employed. There-fore we choose CHO-E cells that constitutively ex-press high levels of E-selectin without the need offurther activation. Despite the fact that CHO-E cellsare widely used for various selectin-experiments, westarted to illustrate the selectin density and distribu-tion at the cell surface by immunogold labeling to getan impression of surface topology and for a bettercomparison of our results with previous binding

Fig. 1. Immunogold labeling (12 nm gold) of E-selectins (blackpoints) at the surface of a CHO-E cell.



studies using leukocytes. The electron microscopepicture in Fig. 1 demonstrates a plain cell surfacewith a homogeneously distributed high level of E-selectin. This appearance of selectins remains con-stant at the cell surface upon activating the cellwith reagents, such as TNFK, IL1K or -L (data notshown). Therefore, CHO-E cells appear as excellentmodels in our investigations to focus on ligand be-havior.

3.2. Preparation and characterization of the supportedplanar model membranes

The naturally occurring selectin ligands are mucin-like glycoproteins which present their carbohydrateside chains in a special molecular arrangement,which has been hypothesized to be responsible forhigher a¤nity [12]. We introduce sLex ceramide asa glycolipid ligand with a common lipid anchor inour model membranes. By using the Langmuir^Blodgett technique, the glycolipids will be embeddedin a phospholipid matrix similar to the conditions atthe cell membrane surface. Furthermore, accordingto temperature, lateral pressure and the kind ofphospholipid in the membrane, glycolipids can eitherbe homogeneously distributed in the matrix or bearranged in lateral local clusters. Since such clustersdisplay concentrated multiple copies of a bindingepitope, their in£uence on cell binding could be

checked to support the multivalent binding hypoth-esis in comparison to the membranes with homoge-neous ligand distribution.

We started our work establishing optimal experi-mental conditions for the preparation of highly re-producible membranes. Therefore we ¢rst analyzedthe lipid monolayers at the air/water interface, whichshould thereupon be transferred to the solid support.We found optimal conditions for glycolipid-inducedDSPC-clustering using ceramide concentrations be-low 10 mol%. The appearance of sLex-enriched clus-ters is illustrated by £uorescence microscopy for thepreparation with 10% as shown in Fig. 2.

In comparison, we prepared monolayers with ho-mogeneous ceramide distribution. Since clusteringhad been the result of a phase separation of thematrix lipid and the coexistence of £uid expandedand crystalline areas and subsequent accumulationof the ceramide in one of these phases, we chosePOPC as matrix lipid to avoid phase transitionsand therefore cluster generation in our experiments.As illustrated in Fig. 3, the sLex ceramide is homo-geneously distributed in the POPC matrix, when em-ployed below 10 mol%.

The retention of the membrane structures aftertransfer to the glass surface is shown in Fig. 4.Whereas the POPC membrane shows a homogeneousappearance (not shown), glycolipid clusters (0.025%)are detectable in a DSPC matrix. Since clusters ofthose low ligand concentrations could not be de-tected with £uorescently labeled lipids, the structure

Fig. 2. Fluorescence microscope image of a sLex glycolipid-con-taining DSPC monolayer at air water interface. The pictureshows £uorescently marked distribution of sLex lipid (brightareas) in the DSPC matrix (dark) at a Langmuir trough at lat-eral pressure of 41 mN/m. Diagonal: 110 Wm.

Fig. 3. Fluorescence microscope image of a sLex glycolipid-con-taining POPC monolayer at air/water interface. The sLex lipidis homogeneously distributed in the POPC matrix at a Lang-muir trough at lateral pressure of 41 mN/m. Diagonal: 110 Wm.



was proved by binding FITC-conjugated anti-sLex

antibodies to the clusters in the membrane.Furthermore, using Atomic Force Microscopy of

transferred ¢lms we could analyze the geometry ofclusters and the in£uence of glycolipid reduction oncluster formation in the membrane. Analyzing di¡er-ent DSPC membranes with varying glycolipidamounts, we could see that a glycolipid reductionleads to a decrease in the number of clusters withobviously no changes in their size (diameter of about20 nm corresponding to about 500 sLex molecules).An example of an AFM picture of a membrane with1% sLex is shown in Fig. 5. Furthermore, this picture

illustrates that in membranes with higher glycolipidconcentrations (above 0.5%), clusters tend to organ-ize in greater aggregates consisting of about 10^20single clusters.

In analogy to natural membranes, supported pla-nar bilayers show lateral lipid di¡usion [17]. To guar-antee that the transferred structure is preserved dur-ing cell binding studies, rather than changed bydi¡usion processes, we analyzed the time range ofthe lateral di¡usion. Therefore, we employed a modi-¢ed £uorescence recovery after photobleaching(FRAP) technique, where the time of di¡usion of£uorescence markers into a previously bleachedmembrane area of de¢ned size is analyzed. The ex-perimental data of a POPC matrix in Fig. 6 show,that the lipid di¡usion into an area of 30U30 Wmtook more than 120 min for total recovery. There-fore, we conclude that the freshly prepared and an-alyzed model membranes maintain their clusteredstructure when used within 30 min.

3.3. In£uence of clustered ligand concentration on celladhesion and rolling

The amount of selectin-binding epitopes in thenatural sialomucins is low and their number is notwell characterized [18^20]. On the other hand, thenumber of sLex lipids at neutrophil surfaces is esti-mated, but it has not been proved whether all theseglycolipids are involved in selectin binding. There-fore, we started our cell adhesion experiments ana-lyzing the in£uence of ligand concentration in theclustered ligand membranes on cell behavior. Thecells were allowed to interact with the model mem-brane under static conditions for 5 min and the num-

Table 1Characterization of the cell adhesion or rolling behavior in dependence on the ligand concentration in a DSPC matrix at a shear rateof 200/s

Ligand concentration (%) Remaining bound cell fraction (%) Rolling cell fraction (%) Detached cell fraction (%)

10 91.7 0 8.31 92.4 0 7.60.1 90.1 0 9.90.05 27.4 61.2 11.40.015 9.3 74.6 16.10.01 5.0 77.2 17.8

0.005 4.7 0 95.3

Data represent means of at least 4 measurements.

Fig. 4. Laser scanning microscope image of transferred sLex

glycolipid clusters in a DSPC matrix. The model membranewas treated with FITC-conjugated rat anti-human CLA mAbs,that speci¢cally bind to sLex moieties. CLA, cutaneous lympho-cyte-associated antigen; a carbohydrate domain shared by sial-yl-Lewisx and sialyl-Lewisa antigens.



ber of sedimented cells was counted. After that time,the cell adhesion and rolling was analyzed at a shearrate of 200 s31 and illustrated by a sequence of mi-croscopic pictures within the ¢rst 60 s. In this way,the fraction of cells which remained bound, the num-ber of rolling cells and their velocity could be de-tected. At ligand concentrations between 10 and0.05%, a fraction of approximately 90% of cellsstayed adhered in the shear £ow and cell rollingdid not occur. Further reducing the ligand concen-tration in the membrane resulted in the appearanceof a great rolling cell fraction between 60 and 80%under the same conditions. Despite the di¡erent lig-and concentrations between 0.05 and 0.01%, therewere hardly any di¡erences in number of rolling cellsas described in Table 1 and their velocity. At glyco-

lipid concentrations below 0.01% in the membrane, asmall fraction of cells roll with higher velocity,whereas further reduction of ligand density abolishedcell^membrane interactions. The pure DSPC matrixshows no interaction with the selectin-containingcells under £ow conditions, as control experiments.The cell interaction with the ceramides could beblocked totally by an E-selectin antibody, or by theaddition of EDTA, which suggests that rolling is aselectin-dependent process.

To further adapt these measurements to physiolog-ical conditions, we repeated these experiments with-out preincubating the cells at the membrane understatic conditions. In general, we could detect similarcell behavior in response to ligand concentration, butthe absolute number of interacting cells at the mem-

Fig. 5. Atomic force microscopic image of a DSPC membrane containing 1% sLex ceramide. The glycolipid clusters appear as brightelevations.



brane was reduced to about one-third (about 30 cellsper analyzed area versus about 100 cells after set-tling). This should be explained with the geometricconditions of our £ow chamber. The model deviceshould o¡er because of its greater dimensions worseopportunities for an initial cell-membrane contactcompared to postcapillary venules. Furthermore,the surface of the CHO-E cells, which has no micro-villi presentation of selectins, will also contribute tothe reduced interaction. Nevertheless, these resultsdemonstrate, in principle, the functionality of ourglycolipid-based model system under these morephysiological conditions. In order to get more celladhesion data allowing a better statistical evaluation,we continued our further studies with such experi-ments, where cells can interact with the membranebefore rinsing.

From this point, we could conclude that functionalorganized sLex glycolipids can mediate cell rolling,implying that the ability to mediate rolling doesnot depend on sialomucin structures. The interplaybetween ¢rm cell adhesion and rolling movement isligand density controlled, whereas only a very smallligand fraction is necessary for mediating rolling.

Our study gives an impression about the ratio ofligand number and area which is necessary for medi-ating cell rolling. The ligand concentrations of 0.05or 0.01% correspond to site densities of about 800 toabout 20/Wm2; respectively, assuming an area ofabout 60 Aî 2 for sLex tetrasaccharide headgroupand 45 Aî 2 for DSPC at a transfer pressure of 40mN/m. The range of site densities of sLex capable

to mediate rolling is below the amount of sLex atleukocytes, which is thought to be about 50 000/Wm2 [21]. Assuming an idealized round cell shapeof CHO-E cells (Fig. 1) with a diameter of 10 Wmand a selectin density of about 1000/Wm2, these datacorrespond to ligand/receptor densities of about 4:5or 1:5, respectively, in the contact zone. Therefore,glycolipid clustering is essential to enable cell-mem-brane interactions by drastically increasing the localligand/receptor ratio.

Contrary to our glycolipid, where the tetrasaccha-ride is closely connected to the hydrophobic moiety,naturally occurring sLex lipids seem to be more com-plex, which leads to higher a¤nity and tethering ca-pacity. Our results show that glycolipids, which haverecently been discussed as physiological selectin lig-ands [22,23], can act as functional ligands, mediatingrolling in our in vitro investigations, when arrangedin clustered forms.

3.4. Cell rolling at various shear force conditions

To examine the dynamic characteristics of rollingprocess, we performed further experiments with aconstant ligand concentration of 0.025% in theDSPC matrix at di¡erent shear force conditions.Therefore, after sedimentation of cells within the ¢rst5 min, shear rate was increased in steps every 10 suntil a shear rate of 1000 s31 was reached. Up to ashear rate of 50 s31 the cells adhered ¢rmly and roll-ing could not be observed, representing the criticalshear threshold for initiating rolling. Between shear

Fig. 6. Laser scanning microscope image of a transferred POPC monolayer in topographic version (NBD-PE-labeled). Pictures aretaken after 0, 60, 120 min (from left). The lipid di¡usion into an area of 30U30 Wm is recovered totally after more than 120 min.



rates of 50 and 170 s31 cells roll slowly without de-pendence on shear rate. Increasing shear levels up to450 s31 rolling velocity increased proportionally toshear stress and reached a near-plateau at about 27Wm/s with further increase in shear force, as illus-trated in Fig. 7. These ¢ndings totally agree withprevious results of neutrophil rolling on immobilizedselectins [8]. This balanced rolling behavior uponchanged shear force conditions was hypothesized tobe a special feature of selectins contrary to otherreceptor proteins [10], whereas the molecular basisof this phenomenon is still not clear.

Considering the fraction of rolling cells at di¡erentshear rates, it is evident that above the shear thresh-old of 50 s31 an equal cell fraction of about 75% isrolling similarly to the experiments with changed lig-and concentrations. Accordingly, the fraction of cellsthat remain bound is nearly constant at a very lowlevel at di¡erent shear rates above the threshold.

Despite the individual characteristics, in general,all cells of the rolling fraction rolled with relativesimilar velocities at the corresponding shear rates.Actually, cells rolled with jerky motion. They repeat-edly rolled a distance and than interrupted rolling bybrief pauses which should be attributed to a move-ment along the ligand clusters.

3.5. Cell adhesion at membranes with homogeneousligand distribution

In order to investigate the importance of ligandclusters for cell interactions we repeated the experi-ments under modi¢ed conditions. Employing the

POPC membranes with the homogeneous ligand dis-tribution, di¡erences in the cell adhesion or rollingshould be evident compared to the DSPC results.Similar to the DSPC experiments we reduced theligand concentration starting from 10 mol% and an-alyzed the cell behavior. The results (shown in Fig. 8)suggest that analogous to the previous results, cellsstrictly adhered at the membrane, at ligand concen-trations above 0.05%. This cell adhesion could beinhibited by a blocking E-selectin antibody whichdemonstrates the speci¢c nature of interactions.However, a small fraction of cells (about ¢ve cellscorresponding to 5% of total adhered fraction) re-main bound after blocking. Experiments with lowerligand concentrations show only the adhesion of asimilar small cell fraction (about ¢ve cells) adheredboth at these membranes and at a pure POPC ma-trix. This should be attributed to unspeci¢c interac-tions at the membrane surface, because this cell bind-ing could not be blocked with E-selectin antibodies.Contrary to the DSPC matrix, a certain ligand con-centration which mediates both speci¢c cell bindingand cell rolling could not be achieved in POPC ma-trices. The remaining cells show only a slow move-ment with descending velocity of about 2 Wm/s athigher shear rates which should not be de¢ned ascell rolling.

Since CHO-E cells have a strong tendency to at-tach at surfaces within a short time, this may be thereason for the unspeci¢c attachment of some cells.One can assume that the liquid expanded POPC

Fig. 8. Dependence of cell rolling velocity on ligand concentra-tion. Rolling velocity was detected at a shear rate of 200 s31 atdecreasing ligand concentrations in dependence on variousphospholipid matrices. Velocities below 5 Wm/s has not been de-¢ned as rolling movement.

Fig. 7. Velocity of the rolling fraction of CHO-E cells above aDSPC matrix containing 0.025% sLex, in dependence on shearstress.



membrane gives better opportunities for the cells tospread on it than the crystalline DSPC.

The biophysical basis for the di¡erences in celladhesion at DSPC and POPC matrices are likely tobe complex, requiring consideration of di¡erences inphase separations, microdomain formations andphase transitions. Because this goes beyond the scopeof this study, it should be described separately.Nevertheless, we conclude that the appearance ofclustered glycolipids in this study is a prerequisitefor mediating rolling.

3.6. Studies at membranes with mixedPOPC/glycolipid clusters

To further support this thesis and to verify that theinability to mediate cell rolling is not a POPC e¡ectfor itself, we further modi¢ed the lipid matrix. Inorder to maintain ceramide clusters in combinationwith POPC, £uid expanded POPC clusters, that con-tain the similar £uid glycolipid molecules, were pre-pared in a crystalline DSPC matrix. This should leadto glycolipid-containing clusters, which have a lowerglycolipid concentration per area compared to theclusters in the pure DSPC experiments. We inter-preted this fact from confocal laser scanning micros-cope pictures of these membranes (not shown), whichshowed greater, but less £uorescent, glycolipid clus-ters opposite to Fig. 4. To maintain an overall cer-amide concentration of 0.025%, POPC mixed with0.5% ceramide was incorporated into a DSPC matrixwith 5% in order to create clusters. If the assumptionof this experiment is true, the resulting rolling behav-ior should be similar as described for DSPC inTable 1.

We could see that this membrane was able to me-diate cell rolling under the described conditions. Itwas evident that cells with a average velocity of 10Wm/s rolled slower than at the DSPC membrane withan equal glycolipid amount. The results demonstratethat this form of glycolipid clustering is practicableand that POPC does not abolish cell rolling in gen-eral.

This slower cell movement should be an indicatorfor a more e¡ective cell binding compared to thesimilar glycolipid concentration in the case of thepure DSPC matrix. If one assumes that in the mixedDSPC/POPC matrix the glycolipids are more diluted

within the clusters because of their greater areas, oneshould conclude that the absolute amount of glyco-lipids in these membranes could be decreased with-out loss of binding ability. To try this out, we furtherreduced the ceramide concentration in the membraneto 0.005 and 0.002%, concentrations which are notable to mediate rolling in the pure DSPC matrix.

Surprisingly, we could see that cells rolled nor-mally along these membranes as in the case of higherglycolipid densities in the DSPC membrane as dem-onstrated in Fig. 8. We interpret these results as fol-lows: despite the fact that a clustered arrangement ofglycolipids in the membrane is necessary to inducerolling, the glycolipid density within the clusters canbe modi¢ed. As shown here for a distinct concentra-tion range, the mixed clusters of POPC and glycolip-id are as e¡ective as the higher local glycolipid con-centration within the clusters in the DSPCmembrane. The range of glycolipid tolerance media-ting rolling can thus be shifted to lower concentra-tions without loss of functionality.

Leaving out this tolerance range with lower con-centrations (6 0.002%), no cell membrane interac-tions could be detected again. Furthermore, contraryto the pure POPC membrane the amount of attachedcells is very low, which proves that adhesive POPCe¡ects are not involved in the described cell behavior.

Summarizing, these results support the hypothesisthat a lateral clustered arrangement of glycolipid lig-ands is necessary for mediating cell rolling. Further-more, it could be shown that ligand clusters, oncedeveloped, retain their ability to induce rollingupon diluting their glycolipid concentration withinan appropriate concentration range.

4. Conclusions

In this study, we focused on the molecular featuresof glycolipid ligands for selectins in the cell rollingprocess. Therefore we introduced an in vitro model,where selectin-containing cells adhere or roll along asupport ¢xed, ligand-containing membrane in a sim-ulated shear £ow of vasculature. These model mem-branes, which were created using the Langmuir^Blodgett technique, allow the incorporation of sev-eral concentrations of sLex ceramide in either de¢nedlateral clusters, or homogeneously distributed in the



matrix lipid. This was the basis to investigate theimportance of multivalent ligand^receptor interac-tions.

In membranes with glycolipid clusters, the transi-tions from ¢rm attachment to rolling to detachmentis glycolipid density-controlled. Since membraneswith homogeneous ligand distributions have beenshown to be unable to mediate rolling, lateral ligandclustering appears as an essential prerequisite forrolling. Furthermore, we could show that the re-quired ligand concentration can further be reducedupon diluting the glycolipid concentration within theclusters without loss of functionality. These resultsclearly support the hypothesis of multivalent selec-tin^carbohydrate interactions for getting su¤cientbinding strength.

Since this study demonstrates that carbohydratespresented by a lipid carrier rather than by a polypep-tide backbone are able to mediate rolling, it repre-sents the ¢rst report which combines dynamic glyco-lipid binding studies with the multivalent bindinghypothesis.

Acknowledgements

This work was supported by the Deutsche For-schungsgemeinschaft (Sonderforschungsbereich 197).The authors would like to thank Prof. Vestweber,University Mu«nster for supplying a CHO-E cell-line.

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The role of glycolipids in mediating cell adhesion: a £ow chamber … · 2017. 1. 4. · The role of glycolipids in mediating cell adhesion: a £ow chamber study1 Jan Vogel a;*,