Measuring bonds between surface-associated molecules

JOURNAL OF IMMUNOLOGICAL METHOUS ELSEVIER

Journal of Immunological

Methods

196

(1996)105- 120

Review article

Measuring bonds between surface-associated Anne Pierres, Anne-Marie Laboratoire d’hnunologie,

molecules

Benoliel, Pierre Bongrand

*

INSERM U387, H6pital de Saint+Marguerite. BP 29. 13274 Marseilles, Cedex 09 France Received 5 April 1996: accepted 3 May 1996

Abstract Adhesive interactions play an essential role in immune function. Much information on these phenomena was recently obtained by applying sophisticated methods such as the surface forces apparatus, atomic force microscopy, lipid vesicle-based technology or flow chambers. In the present review it is shown that the use of hydrodynamic flow allows quantitative study

of the formation and dissociation of individual molecular bonds between receptor-bearing cells or particles and ligand-derivatized surfaces. In addition, it should be possible to determine particle-surface interaction forces with subpiconewton sensitivity and nanometer resolution. Data analysis shows that the classical concepts of bond strength, or association and dissociation rates must be reexamined in order to achieve a correct understanding of the behavior of individual molecules. Keywords: Cell adhesion;

Flow chamber;

Kinetic constant;

Molecular

1. Introduction

Nearly all functions of the immune system involve the formation and/or dissociation of molecular bonds between cells and tissues. Whereas numerous adhesion molecules have been characterized during the last 15 years, only recently was it recognized that the efficiency of these molecules was dependent on very diverse requirements. Thus, the capacity of phagocytes to engulf foreign particles (Cap0 et al., 1978) and the ability of cytotoxic T lymphocytes to kill target cells (Bongrand et al., 1983) are somewhat correlated to the formation of attachments with high

Abbreviations: histocompatibility * Corresponding 757328.

CTL, cytotoxic T lymphocyte; complex. author. Tel.: (33) 91 753906;

0022.1759/96/$15.00 Copyright PII SOO22-1759(96)00103-2

MHC,

major

Fax: (33) 91

bond

mechanical strength. The leukocyte capacity to migrate through endothelial walls towards lymphoid organs or inflamed tissues is dependent on the ability of adhesion receptors such as selectins to bind to their ligands on cells moving with a relative velocity of several hundreds of km/s, then decreasing this velocity by a factor of nearly 100 and inducing the so-called rolling phenomenon (Von Andrian et al., 1991; Lawrence and Springer, 199 I). The scanning of antigen presenting cells by circulating T lymphocytes is dependent on the latter cell capacity to detect less than 100 specific peptide/MHC complexes out of millions of membrane molecules (Harding and Unanue, 1990). This probably requires that the surfaces of interacting cells be maintained at binding distance by couples of accessory adhesion molecules such as CD4/MHC class II, CDS/MHC class I or CD2/CD58 (Springer, 1990; Van der Merwe et al., 1995).

0 1996 Elsevier Science B.V. All rights reserved.

There is a need to measure bonds between surface associated molecules. Indeed, since many adhesion receptors were characterized and produced in soluble form, it might be considered at first sight that the quantitative determination of affinity constant and kinetic parameters of association (Williams, 199 11 between soluble molecules would lead to a complete understanding of the adhesive behavior of cells bearing these molecules. However, much recent experimental evidence suggested that more information was needed to understand cell adhesion. Thus, it has long been demonstrated that the activity of integrins is highly regulated (Detmers et al., 1987; Dustin and Springer, 1989). This regulation may involve conformational changes (Van Kooyk et al., 19911, lateral redistribution of receptors on the cell membrane (Detmers et al., 1987) or association with cytoskeletal elements (Kupfer and Singer, 1989). The activity of selectin molecules was also found to depend on microvillar location (Picker et al., 1991; Kansas et al., 1993; Borregaard et al.. 1994; Von Andrian et al., 1995) and cytoskeletal interactions (Pavalko et al., 1995). Another important parameter of adhesion that cannot be studied on soluble molecules is the distdnce between interacting membranes: it has long been argued that glycocalyx elements might impair adhesion by preventing membranes from approaching at binding distance (Bell et al., 1984). The importance of this mechanism was experimentally demonstrated and it was recently shown that this repulsion was more important when adhesion occurred under dynamic (Pate1 et al., 1995: Sabri et al., 1995; Foa et al.. 1996) than static conditions. Finally, whereas soluble molecules undergoing adhesion are only subjected to thermal forces, cell adhesion may be strongly modulated by hydrodynamic forces acting on approaching surfaces. Thus, in order to understand cell adhesion. the following parameters may be of importance: (1) rate of bond formation between receptors and ligands as a function of iritermemhrane separation; (2) rate of bond dissociation as a jimction of applied force; and (3) forces exerted by cell surface components, essentially the cell coat, during adhesion. Many experimental methods are presently available to achieve this goal (see Bongrand et al., 1994 for a detailed description of different approaches). In the present review, we shall focus on the potential of

flow methods in allowing a quantitative study of several steps of cell adhesion. Methodological points will be emphasized. Theoretical data relevant to the physical basis of cell adhesion have been described elsewhere (Bongrand, 1988, 19951. However, for the reader’s convenience, some basic principles that may be unfamiliar to biological readers will first be recalled. Then we shall describe practical information that may be useful to construct a flow chamber. The principles of data analysis together with main potential artifacts will then be discussed. Finally, we shall give specific examples in order to describe the information that can be obtained with this approach as well as difficulties related to data interpretation. Due to the limited space available for this review, we shall focus on results obtained in our laboratory.

2. Basic results from fluid mechanics Most flow chambers are rectangularly shaped in order to allow convenient microscopic examination (Fig. 1). A widely used approximation is that the height (H) is much smaller than the width (W) and edge effects are neglected. When the flow rate is not too high (see, e.g.. Sommerfeld, 1969 for more details) the flow is laminar and the fluid velocity V is everywhere parallel to the pipe axis Ox. The magnitude of V is zero on the chamber wall and near the wall the following first-order approximation holds: V(z)

=Gz

(1)

where ; is the distance to the wall and G is a constant called the wall shear rate (or velocity gradient) that is expressed in s- ’ (i.e., velocity divided by

Fig. 1. Laminar shear flow. In a parallel plate flow chamber. the tluid velocity I’ is parallel to the chamber axis Ox. Near the wall, II may be approximated as a linear function of coordinate z. The derivative dr /d: is the shear rate.

A. Pierres et al. /Journal

distance). This near the wall, Many authors that is simply viscosity p: T=pxG

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parameter characterizes the flow rate which determines adhesive behavior. prefer using the wall shear stress T the product of the shear rate G and

(2)

In most papers, T is expressed in dyne/cm’ (i.e., cgs units). The viscosity of water at room temperature is very close to 0.01 poise. If T is expressed in newtons/m’ (i.e., international units), the viscosity is 0.001 Pascal X s. The wall shear rate is easily derived from the flow rate Q (in units of volume/s, e.g., cm”/s> with the following formula: G = 6Q/WH=

Methods

(3)

(Note that the same unit of length must be used for Q, W and H. Also. since G is inversely proportional to the square of H, and this latter parameter is usually quite low, it is important to measure the chamber height with high accuracy.) Corresponding formulae for flow chambers of circular section have been described elsewhere (Bongrand and Golstein, 1983; Mege et al.. 1986). Now, when studying adhesion in a flow chamber, it is important to discriminate between freely moving and bound cells. It is therefore useful to know the properties of free particles. The relevant model is the motion of a sphere moving near a plane wall in the presence of laminar flow. Although this seems a quite simple situation, the equations of motion are quite complicated. However, when the sphere size and velocity are close to the size and velocity of a flowing leucocyte, important approximations hold (corresponding to the so-called creeping flow approximation; see Happel and Brenner, 1973) and the sphere motion can be predicted with high approximation. Unfortunately, there is no simple analytic formula that can summarize the results obtained by various authors. In a series of papers, Brenner and cotleagues (Brenner, 1961; Goldman et al., 1967a,b) gave an extensive set of numerical results that are summarized in Fig. 2. Approximate analytic formulae usable on a useful range of distances between the sphere and the wall are also shown. The following points may be emphasized.

10-l

1 10-4

10-3

10-Z

10-l

1

lo &la

Fig. 2. Viscous interaction between a sphere and a plane. Results from Brenner (1961) and Goldman et al. (1967a,Goldman et al., 1967b) are summarized. Consider a sphere of radius a at distance 6 from a plane. In the presence of a laminar shear flow of wall shear rate G, the sphere velocity is U. Parameter I;x * is the dimensionless ratio between the friction coefficient along a direction parallel to the plane and the value in bulk medium (i.e., 6 apa, following Stoke’s law). Similarly, Fz * is the ratio between the friction coefficient along axis Oz (Fig. 1) and the value in bulk medium. Circles, triangles and squares represent the numerical values given by quoted authors. The lines are parabolic approximations obtained by a least square method, on an interval of interest (0.0015 5 6/a< Il. The equations of these lines are: In(U/aG) = 0.02936 ln’(6/a) + 0.4057 ln(?i/a) + 0.5829; ln(Fx’l = -0.0101 ln’(s/al0.2478 ln(?i/a) + 0.30115; ln(F:*l=0.03837 ln2(6/al-0.6609 lnt6/a)+0.6938.

(1) The essential point is that when the sphere is very close to the wall, the very thin fluid layer located between the sphere and the wall resists deformation, thus slowing the relative motion between the two surfaces. (2) When the sphere is not very close to the wall (say with a distance of at least several tenths of the radius) its velocity U is of the order of the wall shear rate times the distance between the sphere center and the wall. (3) When the width of the gap between the sphere and the wall becomes vanishingly small, the sphere velocity decreases as l/m(6). This decrease is thus quite slow: the predicted velocity will eventually be zero when contact is achieved, but the velocity is about one third of the product aG (Le., sphere radius a times the wall shear rate) when 6 is 0.01% of the sphere radius (i.e., 0.4 nm for a cell of 4 pm radius!).

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(4) When a cell is bound to the surface, it is subjected to a hydrodynamic force that tends to detach it. The force F is about 32 pa’G. Thus, a ‘standard’ leucocyte of 4 pm radius is subjected to a distractive force equal to 0.35 G, where F is expressed in piconewtons (i.e., lo-” newton) and G in s-‘. However, the force exerted on bonds linking the cell to the surface may be substantially higher due to lever effect. This lever was estimated to increase the force by a factor of four with spherical particles bound to a surface through antibody molecules (Pierres et al., 1995a). A final point of concern is the relevance of the aforementioned theory to actual cells. Indeed, cells are studded with many asperities and cannot be considered as smooth spheres. This problem was addressed by Tissot et al. (1991. Tissot et al., 1992) and Tempelman et al. (1994). First, the ratio U/UC measured on flowing cells usually ranged between about 0.55 and 0.85. Tempelman et al. (1994) found that cells moved slightly faster than spherical beads of similar radius. They ascribed this difference to the presence of microvilli on the cell surface. However, smoothing microvilli by hypotonic treatment did not result in a marked change of flowing behavior (Tissot et al., 1991). Second, when the motion of very irregularly shaped cells was studied in order to measure at the same time their translational and rotational velocities (Tissot et al.. 1992). it was concluded that an extra force had to be added to the basic equations since the relationship between translational and rotational velocities was not consistent with theoretical predictions. Thus, some caution is required when the above results are applied to actual cells.

3. Required

equipment

3. I. Building and calibrating

a flow chamber

Most authors made use of custom made flow chambers ‘. As a rule, these chambers are made of

’ We are grateful to Immunotech (and in particular to Prof. M. Delaage) for help in devising and building several flow chambers.

Methods 196 (1996) 105-120

‘c

:(gy=jg :::::::::::.:: 1

I

Tiiiiii Fig. 3. Flow chamber. The top of the chamber is a Plexiglas block where two pipes were inserted (for fluid entry and exit) and the bottom was drilled to insert a glass coverslip that was cut and stuck with silicone glue.

two separate parts. The floor is usually a removable glass coverslip that may be easily processed (for coating with cells or adhesion molecules) and allows convenient microscopical examination. We sometimes made use of plastic sheets (Thermanox ref. 5408, supplied by Miles Laboratories, Naperville: IL), but the quality of obtained images was much poorer than with glass slides. The top part must be connected to a fluid supply and exit (Fig. 3). On a theoretical basis. it would be preferable that the fluid enter the chamber through a slot rather than a circular hole in order to ensure optimal regularity of the flow (Lawrence and Springer, 1991). However, as repeatedly checked (Tissot et al., 1991, 1992) a laminar flow with shear rate consistent with predictions of Eq. 3 can be obtained with circular apertures, provided cells are studied near the middle of the chamber. The connection between the chamber parts may involve a silicone gasket provided the coverslip is applied with sufficient strength to ensure sufficient tightness. We found it convenient to simply stick the coverslip with silicone glue. This glue remains quite soft and is easily removed after the experiment. There are two drawbacks with this technique. First, the solvent is often acid. This may be a problem when the flow stops and the medium is not sufficiently buffered (note that some suppliers provide

A. Pierres et al. /Journal

of Immunological

silicone glue with neutral solvent). Second, when very shallow chambers are used, the depth may display some variations between experiments. Therefore, the chamber depth or shear rate must be controlled in each experiment. When the shear rate is very low (i.e., a few seconds- *), the shear rate is easily measured with a simple trick (Tissot et al., 1991, 1992): the chamber is filled with a suspension of low diameter (e.g., 0.7 km) beads and examined with a high magnification lens (e.g., 100 X objective). A mere stopwatch allows precise determination of the velocity of beads precisely located in the focus plane. The velocity gradient is determined by changing the focus through sequential steps of 5 or 10 pm, using the micrometer screw of the microscope. The flow is most conveniently generated by a syringe mounted on an electric syringe holder. In order that the flow be smooth and continuous, this must be driven by a synchronous motor (not a step motor). We obtained satisfactory results with a device produced by Raze1 Scientific instruments (Stamford, CT, supplied by Bioblock, Illkirch, France) with digital setting of the flow rate (with lOO-fold range).

3.2. Preparing

an adhesice

surjke

The chamber floor may be coated with cell monolayers or purified adhesion molecules. Obtaining confluent cell monolayers (e.g., with endothelial cells) obviously requires the use of a specific substratum and suitable growh factors, depending on the particular properties of used cells. An important point is that the cell shape may influence adhesion (Sabri et al., 1995). Thus, particularly at low shear rate, artefactual arrests may be generated when flowing cells or particles are trapped in immobile fluid regions constrained between neighboring cells. Also, actual cells are endowed with such a variety of adhesion molecules that it may be difficult to derive the properties of a given receptor-ligand couple from data obtained on cell-cell interactions. It is therefore not surprising that many accurate data on adhesion were obtained with experimental systems involving at least one model surface coated with one or a few molecular species.

Methods lY6 (lY961 105-120

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Several methods were used to coat surfaces with adhesion molecules. Basic requirements are (i) absence of nonspecific adhesion, (ii) high coupling yield resulting in high surface density of binding sites, (iii> absence of protein denaturation, and (iv) suitable accessibility of binding sites, which may require the incorporation of a spacer between adsorbed molecules and surfaces. The simplest procedure consists of merely adsorbing proteins on the surface (which is the basis of many immunoassays, following the first report by Catt and Tregear (1967) (see also Xia et al., 1993). Also, adsorbed molecules may be randomly oriented which sets an uncertainty on the number of available binding sites. Thus multilayer procedures were used by different authors in order to achieve fuller control of the conformation of adhesive molecules. We obtained conveniently oriented antibody molecules with the following procedure (Pierres et al., 1995a,b adapted from Michl et al., 1979). Glass coverslips were carefully cleaned with sulfuric acid (in order to remove hydrophobic contaminants). Polylysine chains were then added, leading to strong adsorption through electrostatic interactions. They were then derivatized with 2,4_dinitrobenzenesulfonic acid and coated with anti-DNP antibodies, yielding an oriented monolayer of immunoglobulin molecules with several thousands of sites per Km’. In another experiment (Pierres et al., 1994~1, polylysine-derivatized glass surfaces were treated with glutaraldehyde, then with suitable monoclonal antibodies. After neutralization of unreacted aldehyde groups with glycine, we obtained a surface coated with randomly oriented antibodies that could be used for studying adhesion under flow. The above procedure yielded a surface density of several hundreds of antibody molecules per pm’. Other procedures have been reported. Wattenbarger et al. (1990) treated glass with 3-aminopropyltriethoxysilane. This reagent was readily adsorbed, thus allowing covalent coupling of protein molecules with glutaraldehyde. Tempelman and Hammer ( 1994) prepared dinitrophenyl-derivatized polyacrylamide gels to study the adhesion of flowing cells bearing anti-DNP antibodies. Excellent results were obtained by incorporating proteins in lipid layers (Watts and McConnell, 1987; Chan et al.. 1991; Lawrence and Springer. 1991).

Purified proteins were incorporated into amphiphilic vesicles that were deposited on glass surfaces. Interestingly, in contrast with the aforementioned procedures, deposited proteins were sometimes endowed with lateral mobility, depending on their molecular structure (Ghan et al., 1991). Also, sequential deposition of lipid monolayers of suitable composition allowed convenient control of the lateral mobility of deposited molecules. Note also that suitable chemistry might be useful in allowing the insertion of spacer molecules between receptors and surface. thus increasing the binding activity (see, e.g., Leckband et al., 1995). Finally, it may be emphasized that there is presently high interest in the preparation of surfaces derivatized with immobilized ligands. This was a key component to the development of analytical methods based on surface plasmon resonance (Chaiken et al., 1992). See also Sackmann (1996) for a recent review of supported membranes.

3.3. Determination sites on dericatized

of tlze

suface

density c$ ache

surfaces

It is useful to determine the surface density of coupled molecules. This has often been done by means of radioactive antibodies (Lawrence and Springer, 1991: Xia et al., 1993). We used the following fluorescence-based method (Pierres et al.. 1994~). Coupled surfaces are labeled with fluorescent antibodies and the mean fluorescence is determined as relative units with a confocal laser microscope or a conventional fluorescence microscope equipped with a videocamera and an image analysis system (see below). There remains to calibrate fluorescence units. This was achieved by depositing a small volume (say 5 ~1) of labeled antibodies on a clean glass slide, then adding a coverslip (typically 22 X 22 mm’). This should create a thin liquid film of 5/(22’)=0.010 mm (i.e., 10 p.rn) thickness. When this was examined with a confocal microscope operated under xz mode, it was found that the actual layer was usually quite smooth with a thickness close to the theoretical value (with a coefficient of variation of the order of 30%). Determination of the average integrated fluorescence of the liquid layer yields easy calibration of relative fluorescence units.

3.4. Microscope

and related equipmer~t

The standard procedure consists of observing cells with an inverted microscope. Low magnification lenses (e.g., 20 X objective) may be sufficient, yielding larger observation fields. However. high accuracy determination of particle position requires 40 X or 100 X objectives (see below). Since real time analysis is not convenient, the microscope must be equipped with a videocamera connected to a tape recorder. Standard VHS apparatuses yield sufficient quality with minimal cost. Increased performance may be obtained with rapid videosystems (Pierres et al., 1996). An essential piece of equipment is an electronic clock (with possibly a character generator) allowing date and time incrustation (I/ 10th s accuracy is useful). The purpose is twofold. First, within an experiment, it is important to be able to retrieve the images of individual cells for repeated examination. Second. use of flow chambers rapidly leads to an accumulation of numerous videotapes, that must be accurately labeled in order to allow unambiguous identification of any past experiment. We were satisfied with a JVC Sopro 600 apparatus (supplied by Soprorep, La Valentine. Marseilles) or a VT100 videotimer (supplied by Mussetta Electronique, Marseilles). Adhesion may then be studied by manual counting of the number of flowing cells and arrests as well as arrest duration. This may be performed with a mere stopwatch (and possibly a transparent plastic sheet that may be stuck on the monitor screen to label the locations of cell arrests). It is advisable to examine a sufficient number of videotapes before making use of computer assisted methods, in order to be able to identify possible artifacts and gain an intuitive understanding of what is really measured. As will be described below. image analysis may substantially enhance the amount of information that can be drawn from flow experiments. This requires a digitizer, desk computer and suitable software. We shall briefly describe the procedure we used, with emphasis on the requirements that must be met in order to yield satisfactory results. The problem consists of recording time and particle position with optimal accuracy. We were satisfied with a PCVision + digitizer (Imaging Technology, Bedford, MA). The following features were found useful.

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of Immunological Methods 196 f 1996) 105-120

(1) Real time digitization of 5 12 X 5 12 pixel images with 8-bit accuracy (i.e., 256 gray levels) seems a minimal requirement. An essential point is the rapidity of image transfer into the host computer memory. (2) Digitized images are subjected to reverse digital-to-analog conversion and displayed on a monitor. Further, the digitizer allows individual access to separate bitplanes. Thus, it is possible to display images with 128 levels (which results in minimal decrease of contrast), keeping a bitplane available to superimpose suitable information (e.g., drawing cell trajectories). (3) Finally, a convenient feature is the possibility to change the digitizer sensitivity and contrast, which is useful in view of the difficulty to control image brightness during the experiments, when different monitors and tape recorders are used. The requirements for the computer are dependent on the software used. The software we developed was intended to minimize computer requirements, and videotapes were easily analyzed with a desk computer equipped with a 80286 microprocessor with 16 MHz frequency. However. most commercial programs required much higher capacity. The basic

1

0

M2

0

Ml

.

-

Fig. 4. Equipment. The flow chamber (fc) is deposited on the stage of an inverted microscope bearing a videocamera (C) connected to a videocassette recorder (VCR) and a videotimer (VT). The output is sent to a digitizer mounted on a computer. Ml is the computer screen. M2 is a second monitor connected to the digitizer output. The flow is generated by a syringe (s) mounted on a syringe holder (sh). Beads are inserted through the three-way stopcock (T) immediately before the experiment.

equipment we use for studying is depicted in Fig. 4.

111

adhesion

under flow

3.5. Procedures for recording particle motion We used three different procedures of increasing performance for recording cell trajectories in a flow chamber. The simplest procedure consisted of following manually individual cells by superimposing on the monitor screen live images of flowing cells and a cursor driven by the computer mouse. The cursor position was continuously recorded together with time determined on the computer clock. Time resolution is estimated to be of the order of 0.2 s. The mouse position is determined with about 4-pixel accuracy (Pierres et al., 1994a). Data are recorded as individual files for each cell trajectory and processed with a specific softw-are. Improved accuracy was achieved by developing a semi-automated methodology. A small image (e.g., 32 X 32 pixel) surrounding the cursor position is continuously transferred to the computer memory together with time (determined with the computer clock) and coordinates. Digitized images are then stored for delayed analysis. The analysis consists of examining individual images for determination of the cell position. This may be achieved by either manually determining the edge position, yielding l-pixel accuracy, or determining the cell contour (with a standard boundary-follow procedure, Andri et al., 1990) and calculating the coordinates of the image center of gravity, yielding about 0.1 -pixel accuracy for a spherical particle of 100 squared pixels (Pierres et al., 1995b). This procedure was found suitable to follow cells moving along inhomogenous surfaces (e.g., cell monolayers, Kaplanski et al., 1993) with a sampling frequency of about 10 s- ’ . Two limitations thus required the design of a more sophisticated procedure: (1) the use of the computer clock does not allow better than 0.1 s accuracy, and (2) individual examination of sequential images is quite lengthy, and is not feasible to examine more than a few hundred images per cells. The third procedure we devised (Pierres et al., 1996) was successfully applied to the monitoring of

112

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et al./

Joounml

qf Immunologicrrl

the motion of spherical particles moving along ligand-coated surfaces. This required the development of dedicated assembly language software. The basic procedure is as follows. (1) The microscope is focused in order that all particles appear as bright discs with a dark rim. (2) A vertical line parallel to the side of the microscope field is scanned in order to detect the appearance of a pixel darker than a selectable intensity level. (3) A 32 X 32 pixel image is transferred to the computer memory. An important point is to read one of every two pixels. Indeed, a video image is made of two interlaced fields (comprising odd and even lines respectively) that are built sequentially by the camera. It is therefore possible to obtain 50 or 60 separate positions out of a videotape yielding 25 or 30 interlaced images per second (following European or US standards). (4) The contour of the particle is determined, and the area and coordinates of the center of gravity are calculated and stored. In a typical experiment, 100 trajectories comprising about 200 positions each may be obtained.

4. Data processing Any study made on the interaction between flowing particles and a surface will require the measurement of particle velocity, detection of binding events and determination of the duration of these events. When only qualitative information is required, these determinations can be carried out without any difficulty. When maximal performance is required in order to analyze individual molecular events. it appears that the concepts of ‘velocity’ and ‘arrest’ require precise redefinition that cannot be independent of the measuring apparatus. This is the reason why theoretical considerations cannot be separated from methodological description. A specific software was developed in our laboratory to perform analyses. Some practical difficulties must be emphasized. 4. I. VelociQ determination The velocity of a particle may be determined using two sequential positions and dividing the spa-

Methods

196 (1996)

105-120

tial displacement by the time interval. However, accurate results can be obtained only if the error of position measurement (i.e., 6x) is much smaller than the particle displacement (note that the error of time determination is usually negligible). As a practical rule, when a particle of velocity U is followed, only its average velocity during a time interval of at least 5 6x/U can be measured (with an accuracy between 10 and 20%). This means that the velocity of a cell moving with a velocity of 10 pm/s cannot be determined during a time interval shorter than 0.2 s if the accuracy of position determination if about 1 pm. 4.2. Detection cle arrests)

of binding events (appearing

as parti-

Whereas the detection of durable adhesions is quite straightforward, there is a need for a practical definition of transient arrests. This may be achieved by defining as arrested a particle moving by less than distance Ad during a time interval At. A suitable critical threshold velocity Ad/At can be found by building the frequency histogram of particle displacements per step (Pierres et al.. 1994a). If this is bimodal, moving particles may be discriminated from arrested ones. The problem is that aforementioned random errors of position determinations may mask a transient binding event or, on the contrary, these errors may mimic such events. Therefore, the critical time interval t must be high enough that Ad be markedly higher than the uncertainty of position determination. This uncertainty may be estimated by sequentially determining the positions of particles that are obviously arrested (Pierres et al.. 1995b). Further, artefactual variations of calculated particle velocity may stem from imperfections of the image analysis procedure. If the particle brightness fluctuates. an erroneous contour can be obtained. Also, if a particle passes very near another one, the calculated contour may enclose both particles with an apparent discontinuity of the velocity. An efficient procedure for detecting these artifacts consists of observing the discontinuities of measured area. Examples of aforementioned situations are shown in Fig. 5. As a consequence, we always have a look at all trajectories where arrests have been detected and artefactual events are discarded.

A. Pierres et al. /Journal

of Immunological Methods 196 (19961 105-120 P

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Fig. 5. Representative trajectories. Bead position (top curve) and area (bottom curve) were using a rapid videosystem. Plot A represents a bead moving near the chamber floor withoug is shown in B (arrow). An artefact is shown in C (arrow): a very rapidly moving bead appeared as a short line with higher slope (top curve), and concomitant area increase (peak, _ long arrests is shown in D.

An obvious point is that an arrest is not necessarily due to the interaction between ligands and receptors that have been coupled to interacting surfaces. Therefore, it is essential to use as many controls as possible in order to check that most arrests observed may be inhibited by blocking the specific molecular interactions that are tested. of counted particles

It might seem an obvious point that any determination of binding frequency is meaningless if all studied cells or particles are not in actual contact with the chamber floor. However, there is no absolute way of defining this ‘actual contact’.

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recorded with a sampling any arrest. A short binding passed near the observed bottom curve). A particle

frequency of 200/s, event (about 40 ms) particle. This event presenting two fairly

Indeed, it is easy to recognize cells or particles that are ‘very close’ to the surface. Contours are usually sharper (depending on the location of the microscope focus plane), motion is slower, and trajectories are often more irregular due to interaction with surface asperities. However, a close examination of these criteria showed that they can be used only as approximative guidelines. (1) The optical aspect of moving particles gives information on their distance to the surface with an uncertainty of several micrometers, unless dedicated image analysis is performed (Pierres et al., 1996). (2) Closer examination of the trajectories of all moving particles may reveal irregularities that are due to thermal motion (Pierres et al., 1995b.1996).

(3) Quantitative study of the motion of regular spheres moving in a flow chamber demonstrated a significant dispersion of velocities. This may be ascribed to (i) thermal fluctuations of the distance between the particle and the surface (Pierres et al., 19961, (ii) vertical displacements due to asperities of the particle or substratum (Tissot et al.. 1992). and (iii) incomplete sedimentation. Indeed. the sedimentation velocity is expected to vanish when the distance between particle and surface decreases, due to the fluid viscosity (Brenner. 1961). The above difficulties must be borne in mind when it is attempted to quantify the rate of association between surface-bound molecules.

5. Deducing iments

bonding parameters

from flow exper-

Initial studies were performed in order to gain a better understanding of the behavior of adhering cells under dynamic conditions. We shall show that in many cases the obtained information. although of high biological relevance, was dependent on too many parameters to yield accurate information on the behavior of individual molecules. Only recently were flow chambers used to study individual binding events. 5.1. Studying adhesioil MGth cornp1e.r systems 5. I. I. Binding strength When flow methods were first used in our laboratory to study cell adhesion, our purpose was to measure the strengh of cell-cell attachment in order to gain information on the binding mechanisms. It was thus shown that the strength of phagocyte-partitle (Cap0 et al., 1978; Bongrand et al., 1979). CTL to target (Bongrand and Golstein, 1983; Bongrand et al., 1983) and phagocyte-to-surface (M&ge et al., 1986) adhesion was of the order of several tens of nanonewtons (corresponding to thousands of molecular bonds; Capo et al.. 1982). However. the significance of these figures was not clear for the following reasons: (1) the intensity of the force required to release a bound cells was dependent on the duration of application; and (2) detachment involved cell deformation (M?ge et al., 1986) and membrane rupture (Bongrand and Golstein, 1983).

It was concluded that the cell membrane was the weakest link in adhesion, and binding strength determination yielded more information on cell resistance and rigidity than on the binding process. 5.1.2. Kinetics of bond strergthening Much experimental evidence suggests that the formation of the first few bonds between a cell and a surface usually triggers a complex set of events resulting in marked strengthening of adhesion. Indeed. lateral redistribution of membrane receptors and cytoskeletal elements with concentration in contact areas (Andrt et al.. 1990. 19911, and local smoothing of cell surface asperities (Foa et al., 1988) were found to occur within 1 min following menbrane stimulation. Flow chambers should allow a convenient study of these phenomena. For this purpose. additional measurements (e.g., fluorescence determination after suitable cell labeling) will probably be required before results can be used to understand cell behavior. However. preliminary experiments suggested that active, metabolically dependent cell processes did not occur during the first second following adhesion (Pierres et al.. 1994~). 5.1.3. Kinetics of fcwnzation of the jirst bond Many authors studied cell adhesion in flow chambers (Doroszewski et al., 1979; Sakariassen et al., 1983; Forrester and Lackie, 1984; Msge et al., 1986: Lawrence et al., 1987). It might seem that reported results should give information on the kinetic properties of adhesion molecules involved in these studies. However. there are two difficulties. First. if the flow rate is too high, cell arrests will be durable enough to be detected only if a sufficient number of adhesive bonds are formed (unless adhesion molecules have especially high mechanical resistance). Thus, binding frequency may be dependent on bond strength and surface mobility of adhesion molecules (Bell. 1978). Second, binding frequency is highly dependent on the structure of cell membranes. Thus, cell surface polysaccharides probably generate repulsive forces that decrease binding efficiency (Bell et al., 1984). It was recently demonstrated that this phenomenon was more apparent under dynamic than under static conditions (Pate1 et al.. 1995: Sabri et al., 1995; Foa et al.. 1996).

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It is concluded that most results obtained with flow chambers, although highly instructive, cannot give direct information on the formation and dissocii ation of molecular bonds between surface-associated receptors and ligands. 5.2. Measuring the formation and dissociatio?l indicidual bonds with a frow chamber

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Methods 196 (1996) 105-120 BOUND FIZACTION

1

of

It appears that most aforementioned interpretation problems are notably alleviated if a flow chamber is operated under conditions allowing direct observation of the formation and dissociation of single bonds. The feasibility of this approach is supported by much theoretical (Bell, 1978) and experimental (Tha et al., 1986; Evans et al.. 1991; Danmer et al., 1995) evidence suggesting that many ‘usual’ ligand-receptor bonds may resist disruptive forces of order of tens of piconewtons. This prompted us to operate flow chambers with ultra-low shear rates of the order of a few seconds-’ (Tissot et al., 1991; Kaplanski et al., 1993; Pierres et al., 1994~). The performance of our methodology was enhanced using spherical partcles much smaller than cells (Pierres et al., 1994b,l995a.b). since applied force is inversely related to the square of particle radius. whereas particle velocity is proportional to the first power of this radius. This approach has already yielded useful information on the natural lifetime and mechanical strength of individual bonds (Kaplanski et al., 1993; Alon et al., 19951. Further refinements are required to derive kinetic parameters of association between bound molecules (Pierres et al., 1996). Problems will be considered sequentially. Then, we shall describe additional approaches yielding new information on bond length and intermolecular forces. 5.2. I. How is it possible to recognize arrests due to single molecular bonds .F As will be shown below, it is quite difficult to formally demonstrate that arrests observed in a laminar shear flow are due to single bonds. The following criteria were considered. (11 If arrests are due to single molecular events, the frequency of cell arrests must be proportional to the first power of the density of binding sites on particles or surfaces (Cap0 et al., 1982). However, if

0.1

0

1

2

’

Time (s)~

5

Fig. 6. Theoretical distribution of arrest durations. A simple two-parameter model (Pierres et al., 1994b) was used to obtain the distribution of arrest durations of beads. assuming that when the first bond occurred, the rates of bond formation and dissociation were K,, = 2 se’ and K, = 1 s-‘. Clearly. if the time resolution of determination of arrest duration is not better than 1 s. the experimental curve will mimic first order dissociation kinetics.

receptor samples contain a small proportion of aggregates that are responsible for arrests, binding frequency will also be proportional to the concentration (Van der Merwe et al.. 1993). (21 It might be expected at firt sight that the dissociation of unimolecular bonds should obey first order kinetics. Therefore, the proportion P(t) of particles remaining bound at time t after arresting at time zero should be equal to exp( - K, X t), where K, is the dissociation constant. However, as pointed out by Pierres et al. (1994b1, even if multiple bonds are involved in arrests, a semi-logarithmic plot of P(t) versus time might yield a straight line if the time resolution is not sufficient (Fig. 6). Conversely, even if single bonds are involved, single order kinetics may not be found if intermediate states are formed during the binding process (Pierres et al., 1995a). Finally, even if multiple bonds are involved in arrests, it is in principle possible to use the initial slope of the curve (i.e., dP(t)/dt,= ,)l to derive the dissociation constant. (3) As pointed out by Pierres et al. (1994b1, if arrests are due to single bonds. their lifetime should be in principle independent of the concentration of binding sites. However, if the performance of the apparatus was insufficient to detect single bonds and only arrests mediated by double bonds were re-

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ported. the initial rate of particle detachment might also be independent of the site concentration. In conclusion, it is very difficult to obtain formal proof that arrests observed in a low-shear flow chamber are mediated by single bonds. 5.2.2. Reported results OFL the duration qf short-time cell arrests Kaplanski et al. (1993) studied the motion of human blood neutrophils on endothelial cell monolayers in the presence of a shear rate of 5.25 s-‘. They observed multiple short term arrests with an initial dissociation rate of 0.5 s-‘. Arrest frequency was drastically reduced when endothelial cells were treated with anti-E-selectin antibodies (not with anti-ICAM- I). Alon et al. (1995) obtained more precise information on selectin-mediated interactions by studying neutrophil adhesion to glass slides coated with different densities of purified P-selectin molecules. The shear rate was varied between about 36 and 180 s- !. When the P-selectin density was of the order of a few sites per p_rn’, arrest frequency was proportional to the first power of this density and bond dissociation followed first order kinetics. The dissociation constant was 0.95 s-’ for the lowest shear rate and increased up to 3.5 s- ’ for a higher wall shear rate of 110 s-‘. It was important to know whether the above dissociation constants were a peculiar property of selectin-mediated interactions. as suggested in view of the special capacity of selectins to form transient bonds resulting in the rolling phenomenon (Lawrence and Springer, 199 I ). Thus, Pierres et al. (1994~) studied the interaction of CDS-positive lymphoid cells with surfaces bearing anti-CD8 antibodies. The wall shear rate was varied between 1 and 1 1 s- ’ Cells displayed numerous short-time arrests (about 1 s duration) whose frequency was drastically reduced when cells were pretreated with anti-CD8 antibodies. In other studies (Pierres et al.. 1994b. 1995a), the motion of low diameter (2.8 pm) spherical beads coated with varying dilutions of anti-rabbit immunoglobulin antibodies along immunoglobulin-coated surfaces was studied. When the site density of beads was lower than about 35 molecules per km’, arrest frequency was proportional to the site density, initial dissociation rate was fairly independent of site density and wall shear rate (between 11 and 72 s- ’ ) but

Methods

1% (3996)

105-120

bead detachment did not follow first order kinetics. The authors concluded that short-time arrests reflected the formation of incomplete bonding states (as previously described in other reports). Thus, bond formation and dissociation cannot be described by a couple of kinetic constants. It may be suspected that there might exist a wide range of intermediate bonding states between many receptors and ligands. The emphasis on binding states of about 1 s duration might thus reflect technical limitations. Indeed, using a rapid video system, we were able to detect arrests lasting a few tens of milliseconds when studying the motion of anti-immunoglobulin-coated beads along surfaces derivatized with rabbit immunoglobulin (Pierces et al.. 1996). 5.2.3. Rate of bond formation Although the frequency of arrests mediated by a few bonds is easily determined in flow chambers (Kaplanski et al., 1993; Alon et al., 1995; Pierres et al.. 1994b,c,l995a,1996). there is some difficulty in interpreting data. Indeed. when populations of neutrophils (Kaplanski et al., 1993) or homogeneous beads (Pierres et al., 1996) were studied, arrest frequency was inversely related to the velocity of individual particles. The simplest interpretation was that slower particles were closer to the surface than more rapid ones. It is concluded that the rate of bond formation is a rapidly varying function of cell-surface distance d, and it is only meaningful to estimate the ‘association function’ K,(d). This approach is currently followed in our laboratory. 5.2.4. Other chamber

informatiorz

accessible

with

a jlow

5.2.4.1. Bolld length. When a particle is bound to a surface by a simple bond, simple geometrical arguments suggest that it will display spontaneous (thermally driven) displacements with an amplitude x depending on bond length L. It is easily shown that: 6s = 2(2aL)“’

(4)

Thus. a particle of 1.4 km radius bound by a bond of 20 nm length is expected to display oscillations of about 0.5 p_rn amplitude, which is easily measurable with standard image analysis techniques. The measurement of bead displacement might thus in princi-

A. Pierres et al. / Journal

ple give direct information feasibility of this approach Pierres et al. (1995b).

of Immunological Methods I96 (19961 105-120

on bond length. The was demonstrated by

5.2.4.2. Direct determination of particle-sulfate distance without using the shear rate. When a sphere is close to a plane surface, its diffusion coefficient is expected to be markedly reduced (Brenner, 1961; Goldman et al., 1967a). The experimental determination of the brownian motion parallel to the plane (and perpendicular to the direction of the flow) may thus give direct information on the distance between the particle and the surface. The feasibility of this approach was demonstrated by Pierres et al. (1996). 5.2.4.3. Direct determination qf the interaction force between flowing particles and St&aces. Flowing particles are not always at equilibrium distance from the chamber wall for two reasons. First, when the sphere-to-surface distance d decreases, the sedimentation coefficient becomes vanishingly small (Brenner, 1961), thus, observed particles may not have undergone complete sedimentation. Second, due to thermal motion, d is expected to exhibit nonnegligible fluctuations. Indeed, in the absence of sphere-to-surface repulsion, straightforward use of Boltzmann’s formula shows that the mean value of the distance between a plane and spheres of 1.3 kg/m3 density and 1.4 pm radius would be about 120 nm (Pierres et al., 1996). Careful determination of the sedimentation velocity of flowing cells and comparison with the theoretical value may give direct information on the interaction force between the sphere and the surface. The feasibility of this approach is currently explored in our laboratory. 6. Conclusion The results we described strongly suggest that flow chamber methodology allows direct observation of the formation and dissociation of single molecular bonds between receptor-bearing cells or particles and ligand-bearing surfaces. Experimental data should therefore yield quantitative information on the behavior of isolated molecules. It is suggested that data processing might be less straightforward than was first thought. Indeed, interactions cannot be fully described with a couple of kinetic constants (i.e..

117

constants of association and dissociation). The association constant K, should be replaced with a function K,(d) involving the distance between interacting surfaces. Note that this function might depend on receptor membrane attachement, particularly lateral diffusion coefficient (Bell, 1978). The dissociation constand K,(F) is clearly dependent on the presence of a distractive force F, as predicted by Bell (1978). However, this is not the whole story since receptorligand interaction may not always be accounted for by a single binding state. Therefore, the energy/distance curve may be required to describe the interaction between receptors and ligands. Several alternative approaches were used in the past few years to obtain similar information on the interaction between surface-bound molecules. The sur$ace force apparatus allows simultaneous measurement of the interaction force and distance (with better than nanometer resolution) between cylindrical mica surfaces coated with regular arrays of adhesion molecules. This method allows excelient accuracy with respect to force and distance determinations, but the temporal resolution is sacrificed. Ligand-receptor bonds have recently been studied with this technique (Helm et al., 1991; Leckband et al., 1995). Atomic force microscopy recently allowed direct observation of very weak interactions between biological molecules (Lee et al., 1994; Florin et al., 1994; Pincet et al., 1994; Danmer et al., 1995). However, it was difficult to achieve better than piconewton sensitivity. Two highly powerful approaches were recently developed. Evans et al. (1995) recently described a dramatic improvement of the vesicle technology (Evans et al., 1991) consisting of studying the interaction between a surface and a small bead glued to a red cell or lipid vesicle. Combined use with the interferometric technique allowed a control of the bead location with about 5 nm accuracy and force determination with 0.01 pN sensitivity. A nearly comparable performance was achieved by Liebert and Prieve (1995) who used evanescent wave excitation to monitor the thermal motion of a bead maintained close to a ligand-bearing surface by a laser beam. Combined use of aforementioned techniques should bring a dramatic increase of our understanding of interactions between surface-bound molecules during the next few years.

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References Alon, R., Hammer, D.A. and Springer, T.A. (1995) Lifetime of the P-selectin-carbohydrate bond and its response to tensile force in hydrodynamic flow. Nature 374. 539-542. Andre. P., Benoliel, A.M., Capo, C., Foa, C., Buferne, M., Boyer. C., Schmitt-Verhulst. A.M. and Bongrand. P. (1990) Use of conjugates made between a cytolytic T cell clone and target cells to study the redistribution of membrane molecules in cell contact areas. J. Cell Sci. 97, 335-347. And&. P.. Gabert, J., Benoliel, A.M.. Capo, C., Boyer, C.. Schmitt-Verhulst, A.M., Malissen, B. and Bongrand, P. (1991) Wild-type and tailless CD8 display similar interaction with microfilaments during capping. J. Cell Sci. 100. 329-337. Bell. (3.1. (1978) Models for the specific adhesion of cells to cells. Science 200. 6 18-627. Bell, G.I.. Dembo, M. and Bongrand, P. (1984) Cell adhesion Competition between nonspecific repulsion and specific bonding. Biophys. J. 45, 1051-1064. Bongrand. P. (Ed.) (1988) Physical Basis of Cell-Cell Adhesion. CRC Press, Boca Raton, FL. Bongrand. P. (1995) Adhesion of cells. In: R. Lipowsky and E. Sackmann (Eds.), Structure and Dynamics of Membranes, Vol. IB. Elsevier, Amsterdam, pp. 755-803. Bongrand, P. and Golstein, P. (1983) Reproducible dissociation of cellular aggregates with a wide range of calibrated shear forces: application to cytolytic lymphocyte-target cell conjugates. J. Immunol. Methods 58. 209-224. Bongrand. P., Capo C., Benoliel. A.M. and Depieds, R. (1979) Evaluation of intercellular adhesion with a very simple technique. J. Immunol. Methods 28, 133-141. Bongrand. P., Pierres, M. and Golstein, P. (1983) T cell-mediated cytolysis: on the strength of effector-target cell interaction. Eur. J. Immunol. 13, 424-429. Bongrand. P., Claesson, P. and Curtis, A. (Eds.) (1994) Studying Cell Adhesion. Springer. Heidelberg. Borregaard, N., Kjeldsen. L.. Sengelov. H., Diamond. M.S.. Springer. T.A.. Anderson. H.C.. Kishimoto, T.K. and Bainton. D.F. (1994) Changes in subcellular localization and surface expression of L-selectin, alkaline phosphatase, and MAC-I in human neutrophils during stimulation with inflammatory mediators. J. Leukocyte Biol. 56, 80-87. Brenner, H. (1961) The slow motion of a sphere through a viscous fluid towards a plane surface. Chem. Eng. Sci. 16. 242-25 I. Capo, C.. Bongrand. P., Benoliel, A.M. and Depieds, R. (1978) Dependence of phagocytosis on strength of phagocyte-particle interaction. Immunology 35. 177-182. Capo. C., Garrouste. F.. Benoliel, A.M., Bongrand. P., Ryter, A. and Bell G.I. (1982) Concanavalin A-mediated thymocyte agglutination: a model for a quantitative study of cell adhesion. J. Cell Sci. 56. 21-48. Catt, K. and Tregear, G. ( 1967) Solid phase radioimmunoassay in antibody-coated tubes. Science 158. 1570- I57 I. Chaiken. I., Rose. S. and Karlsson, R. ( 1992) Analysis of macromolecular interactions using immobilized ligands. Anal. Biochem. 201, 197-210.

Chart, P.Y., Lawrence, M.B.. Dustin. M.L.. Ferguson. L.M., Golan, D.E. and Springer, T.A. (1991) The influence of receptor lateral mobility on adhesion strengthening between membranes containing LFA-3 and CD2. J. Cell Biol. 115, 145-255. Danmer, U.. Popescu, 0.. Wagner, P.. Anselmetti, D.. Giintherodt, H.J. and Misevic, G.N. (1995) Binding strength between cell adhesion proteoglycans measured by atomic force microscopy. Science 267. 1173- 1175. Detmers. A., Wright, S.D., Olsen. E.. Kimball, B. and Cohn. Z.A. (1987) Aggregation of complement receptors on human neutrophils in the absence of ligand. J. Cell Biol. 105. 1137- 1145. Doroszewski. .I.. Golab-Meyer, Z. and Guryn. W. (1979) Adhesion of cells in flowing suspensions: effects of shearing force and cell kinetic energy. Microvasc. Res. 18, 421-433. Dustin, M.L. and Springer, T.A. (1989) T-cell receptor cross-linking transiently stimulates adhesiveness through LFA-1. Nature 341, 619-624. Evans, E.A.. Berk, D. and Leung, A. (1991) Detachment of agglutinin-bonded red blood cells. I. Forces to rupture molecular point attachments. Biophys. J. 59, 838-848. Evans, E.. Ritchie, K. and Merkel. R. (1995) Sensitive force technique to probe molecular adhesion and structural linkages at biological interfaces. Biophys. J. 68, 2580-2587. Florin. E.L.. Moy. V.T. and Gaub. H.E. (1994) Adhesion forces between individual ligand-receptor pairs. Science 264. 415417. Foa. C.. Mege. J.L.. Capo, C., Benoliel. A.M., Galindo. J.R. and Bongrand. P. (1988) T-cell mediated cytolysis: analysis of killer and target deformability and deformation during conjugate formation. J. Cell Sci. 89, 561-573. Foa. C.. Soler. M., Benoliel. A.M. and Bongrdnd. P. (1996) Steric stabilization and cell adhesion, J. Mater. Sci. Mater. Med. 7. 141-148, Forrester. J.V. and Lackie, J.M. (1984) Adhesion of neutrophil leucocytes under conditions of flow. J. Cell Sci. 70. 93-110. Goldman, A.J.. Cox, R.G. and Brenner. H. (1967a) Slow viscous motion of a sphere parallel to a plane wall. I. Motion through a quiescent fluid. Chem. Eng. Sci. 22, 637-651. Goldman, A.J., Cox. R.G. and Brenner. H. (1967b) Slow viscous motion of a sphere parallel to a plane wall. II. Couette flow. Chem. Eng. Sci. 23, 653-660. Happel, J. and Brenner. H. (1973)Low Reynolds Number Hydrodynamics. 2nd ed. Kluwer Academinc Publ.. Dordrecht. pp. 322-33 I Harding and Unanue (1990) Helm, C.. Knoll, W. and Israelachvili. J.N. (1991) Measurement of ligand-receptor interactions. Proc. Natl. Acad. Sci. USA 88, 8169-8173. Kansas, G.S.. Ley, K.. Munro. J.M. and Tedder. T.F. (1993) Regulation of leukocyte rolling and adhesion to high endothelial venules through the cytoplasmic domain of L-selectin. J. Exp. Med. 177, 833-838. Kaplanski. G., Farnarier. C., Tissot, O., Pierres, A., Benoliel. A.M.. Alessi, M.C., Kaplanski. S. and Bongrand. P. (1993) Granulocyte-endothelium initial adhesion: analysis of transient binding events mediated by E-selectin in a laminar shear flow. Biophys. J. 64. 1922-1933.

A. Pierres et al. /Journal

of Immunological Methods 196 (19%) 105-120

Kupfer, A. and Singer, S.J. (1989) Cell biology of cytotoxic and helper T cell functions. Annu. Rev. Immunol. 7, 309-337. Lawrence, M.B. and Springer, T.A. (1991) Leukocytes roll on a selectin at physiological flow rates: distinction from and prerequisite for adhesion through integrins. Cell 65, 859-873. Lawrence, M.B.. McIntire. L.V. and Eskin. S.G. (19871 Effect of flow on polymorphonuclear leukocyte/endothelial cell adhesion Blood 70, 1284-1290. Leckband, D.E.. Kuhl, T., Wang, H.K., Herron, J., Miller, W. and Ringsdorf, H. (1995) 4-4-20 anti-fluorescyl IgG Fab’ recognition of membrane bound hapten: direct evidence for the role of protein and interfacial structure. Biochemistry 34, 1146711478. Lee, G.U., Kidwell, D.A. and Colton, R.J. (1994) Sensing discrete streptavidin-biotin interactions with atomic force microscopy. Langmuir 10, 354-357. Liebert, R.B. and Prieve, D.C. (1995) Species-specific long range interactions between receptor/ligand pairs. Biophys. J. 69, 66-73. Mege. J.L.. Capo. C., Benoliel. A.M. and Bongrand. P. (19861 Determination of binding strength and kinetics of binding initiation. A model study made on the adhesive properties of P388Dl macrophage-like cells. Cell Biophys. 8. 141-160. Michl. J., Pieczonka, M.M.. Unkeless, J.C. and Silverstein, S.C. (1979) Effects of immobilized immune complexes on Fc- and complement receptor function in resident and thioglycollateelicited mouse peritoneal macrophages. J. Exp. Med. 150. 607-62 I. Patel. K.D., Nollert, U. and McEver. R.P. (1995) P-selectin must extend a sufficient length from the plasma membrane to mediate rolling of neutrophils. J. Cell Biol. 131, 1893-1902. Pavalko, F.M.. Walker, D.M., Graham, L., Goheen. M.. Doershuk, C.M. and Kansas, G.S. (1995) The cytoplasmic domain of L-selectin interacts with cytoskeletal proteins via o-actinin: receptor positioning in microvilli does not require interaction with o-actinin. J. Cell Biol. 129. 1155-I 164. Picker, L.J., Warnock, R.A., Burns, A.R.. Doerschuk, C.M., Berg, E.L. and Butcher. E.C. (1991) The neutrophil selectin LECAM-1 presents carbohydrate ligands to the vascular selectins ELAM-1 and GMP-140. Cell 66, 921-933. Pierre% A., Tissot. 0. and Bongrand. P. (1994al Analysis of the motion of cells driven along an adhesive surface by a laminar shear flow. In: P. Bongrand. P. Claesson and A. Curtis (Eds.), Studying Cell Adhesion. Springer, Heidelberg, pp. 157-174. Pierres, A., Benoliel. A.M. and Bongrand, P. (1994bl Initial steps of cell-substrate adhesion. In: V.C. Mow, F. Guilak. R. TranSon-Tay and R.M. Hochmuth (Eds.1. Cells Mechanics and Cellular Engineering. Springer, New York, pp. 145-159. Pierres, A., Tissot. 0.. Malissen. B. and Bongrand, P. (1994~) Dynamic adhesion of CD8-positive cells to antibody-coated surfaces: the initial step is independent of microfilaments and intracellular domains of cell-binding molecules. J. Cell Biol. 125, 945-953. Pierres, A., Benoliel, A.M. and Bongrand, P. (1995a) Measuring the lifetime of bonds made between surface-linked molecules. J. Biol. Chem. 270. 26586-26592. Pierre% A., Benoliel. A.M. and Bongrand, P. (1995b) Use of

119

thermal fluctuations to study the length and flexibility of l&and-receptor bonds. C.R. Acad. Sci., Life Sci. 318, 11911196. Pierres, A., Benoliel, A.M. and Bongrand, P. (1996) Experimental study of the rate of bond formation between individual receptor-coated spheres and ligand-bearing surfaces. J. Physiol. III. 6, 807-824. Pincet. F., Perez, E.. Bryant, G.. Lebeau, L. and Mioskowski, C. (19941 Long-range attraction between nucleotides with short range specificity. Phys. Rev. Len. 73, 2780-2784. Sabri, S.. Pierres, A., Benoliel. A.M. and Bongrand, P. (1995) Influence of surface charges on cell adhesion: difference between static and dynamic conditions. Biochem. Cell Biol. 73, 4 I l-420. Sackmann, E. (19961 Supported membranes: scientific and practical applications. Science 27 I. 43-48. Sakariassen, KS.. Arts, P.A.M.M.. de Groot. P.G., Houjik. W.P.M. and Sixma, J.J. (1983) A perfusion chamber developed to investigate platelet interaction in flowing blood with human vessel wall cells, their extracellular matrix and purified components. J. Lab. Clin. Med. 102. 522-534. Sommerfeld, A. (19691 Lectures in Theoretical Physics. Vol. II. Mechanics of Deformable Bodies, Academic Press, London, Springer, T.A. (1990) Adhesion receptors of the immune system. Nature 346. 425-434. Tempelman. L.A. and Hammer. D.A. (19941 Receptor-mediated binding of IgE-sensitized rat basophilic leukemia cells to antigen-coated substrates under hydrodynamic flow. Biophys. J. 66. 1231-1243. Tempelman. L.A.. Park. S. and Hammer, D.A. (19941 Motion of model leukocytes near a wall in simple shear flow: deviation from hard sphere theory. Biotechnol. Prog. 10, 97-108. Tha. S.P.. Shuster. J. and Goldsmith. H.L. (1986) Interaction forces between red cells agglutinated by antibody. II. Measurement of hydrodynamic force of breakup. Biophys. J. 50, 1117-1126. Tissot. 0.. Foa. C.. Capo. C.. Brailly. H.. Delaage. M. and Bongrand. P. (19911 Influence of adhesive bonds and surface rugosity on the interaction between rat thymocytes and flat surfaces under laminar shear flow. J. Dispers. Sci. Technol. 12, 145-160. Tissot, 0.. Pierre& A.. Foa. C., Delaage, M. and Bongrand, P. (19921 Motion of cells sedimenting on a solid surface in a laminar shear flow. Biophys. J. 61. 204-215. Van der Merwe, P.A., Brown. M.H., Davis, S.J. and Barclay, A.N. (I 993) Affinity and kinetic analysis of the interaction of the cell adhesion molecules rat CD2 and CD48. EMBO J. 12, 4945-4954. Van der Merwe. P.A.. McNamee. P.N., Davies. E.A.. Barclay, A.N. and Davis, S.J. (1995) Topology of the CD2CD48 cell-adhesion molecule complex: implications for antigen recognition by T cells. Curr. Biol. 5. 74-84. Van Kooyk. Y.. Weder. P.. Hogervorst, F., Verhoeven. A.J.. van Seventer, G., te Velde. A.A., Borst. J., Keizer. G.D. and Figdor. C.G. (199 1) Activation of LFA- 1 through a Ca’+-dependent epitope stimulates lymphocyte adhesion, J. Cell Biol. I I?. 345-354.

120

A. Pierr-es et al. /Journal

of Immunological

Von Andrian, U.H.. Chambers, J.D., McEvoy, L.M.. Bargatze, R.F., Arfors, K.E. and Butcher, E. (1991) Two-step model of leukocyte-endothelial cell interactions in inflammation: distinct roles for LECAM-1 and the leukocyte 2-integrins in viva. Proc. Natl. Acad. Sci. USA 88, 7538-7542. Von Andrian. U.H., Hasslen, S.R.. Nelson, R.D.. Erlandsen. S.L. and Butcher, E. (1995) A central role for microvillous receptor presentation in leukocyte adhesion under flow. Cell 82, 989999. Wattenbarger. M.R.. Graves, D.J. and Lauffenburger, D.A. ( 1990)

Mrthods

196 (19961 105-120

Specific adhesion of glycophorin liposomes to a lectin surface in shear flow. Biophys. J. 57, 765-777. Watts, T.H. and McConnell, H.M. (1987) Biophysical aspects of antigen recognition by T cells. Annu. Rev. Immunol. 5. 461476. Williams. A.F. (1991) Out of equilibrium. Nature 352, 473-474. Xia. 2.. Goldsmith, H.L. and van de Ven. T.G.M. (1993) Kinetics of specific and nonspecific adhesion of red blood cells on glass. Biophys. J. 65, 1073-1083.