leucocyte adhesion under flow conditions: principles important in tissue engineering

Leucocyte adhesion under flow conditions: principles important in tissue engineering

David A. Jones*, C. Wayne Smith+ and Larry V. McIntireS * Cox Laboratory for Biomedical Engineering, institute of Biosciences and Bioengineering, Rice University, Houston, TX 77257-7892, USA; t Speros P. Martel Laboratory of leucocyte Biology, Department of Pediatrics,

Department of Microbiology and Immunology, Bay/or College of Medicine, Houston, TX 77030, USA; I Cox Laboratory for Biomedical Engineering, institute of Biosciences and Bioengineering, Department of Chemical Engineering, Rice University, Houston, TX 77251-1892, USA

An understanding of inflammatory responses is important in a wide variety of tissue engineering

applications. This review describes the current understanding of a central aspect of inflammatory

responses, the adhesion of leucocytes to blood vessel walls prior to their emigration into tissues.

These highly specific adhesive interactions are mediated by three main families of receptors: the

selectins, integrins, and members of the immunoglobulin superfamily. Under flow conditions, the

various receptors make distinct contributions to a multistep process of adhesion in which leucocytes

roll, adhere firmly, and eventually transmigrate. Two examples in which these principles are important

in tissue engineering research, lymphocyte adherence in transplant rejection and monocyte

adherence in atherosclerosis, are discussed in the last part of the paper.

Keywords: leucocytes, adhesion, integrins, selectins, rejection, atherosclerosis

Received 4 November 1994; accepted 31 January 1995

The inflammatory process normally serves to protect the body from invasion by foreign organisms. However, it can also produce unwanted effects, such as frustrating various attempts to improve health, or contributing directly to disease by causing tissue injury. For example, implantation of biomaterials or engineered tissues to replace lost function commonly leads to an inflammatory response that can destroy the foreign material, and in atherosclerosis, inflammatory cell infiltration is an important factor in development of the disease. Other tissue engineering approaches involve modifying native tissues, including insertion of genes into the endothelial cells lining blood vessels to inhibit damaging effects of inflammation. Here, too, a thorough understanding of the inflammatory response is an important prerequisite for successful therapy.

This review will discuss one important aspect of inflammatory responses, the adhesion of leucocytes to blood vessel walls prior to their emigration into tissues. This adhesion is highly specific, utilizing a wide variety of receptors and counter-receptors on endothelial cells and leucocytes. The three main families of these adhesion molecules, the selectins, integrins and members of the immunoglobulin superfamily, are reviewed in the first section of the paper. The next section describes their functions,

Correspondence to Larry V. McIntire.

reviewing the current understanding of leucocyte adhesion as a multistep process. Finally, we discuss two examples, graft rejection and atherosclerosis, as applications of the ideas of leucocyte adhesion which are relevant to current tissue engineering research.

RECEPTORS INVOLVED IN LEUCOCYTE- ENDOTHELIAL CELL ADHESIONS

Integrins

Integrins are heterodimeric cell-surface proteins consisting of one of several cc-subunits and one of several P-subunits bound non-covalently. Combinations of the 13 known cc-subunits and the seven known P-subunits produce at least 19 receptors, which have been extensively reviewedle3. This discussion focusses on those integrins which possess a p2 (CDlB), & (CD%) or Q (CD49d) subunit, since these have been most strongly implicated in specific leucocyte-endothelial cell interactions. The /S, integrins are also known as the leucocyte integrins because their distribution is limited to white blood cells. All circulating leucocytes express LFA-1 (s[~&, CDll,/CDlB), but expression of Mac-l (cL~&, CDllb/ CD18) is limited to monocytes, granulocytes and a small subset of lymphocytes. The /J’, integrins are commonly referred to as the very late activation (VLA) antigens since the first two of them discovered appear

337 Biomaterials 1996. Vol. 17 No. 3

338 Leucocyte adhesion under flow conditions: D.A. Jones et al.

2-4 weeks after stimulation of lymphocytes with antigen. p, and & integrins have an especially widespread distribution, including endothelial cells, epithelial cells and fibroblasts. This is of particular importance to the proposed primary function of these integrins, which is to guide morphogenesis and wound healing. VLA-4 (cQ/&) is an unusual & integrin in that it is expressed on resting B-cells, T-cells, monocytes and eosinophils and can bind VCAM-1 on endothelial cells. The recently characterized integrin Q& (LPAM- 1) is expressed on subsets of B- and T-lymphocytes and is involved in the recirculation of these cells through lymphoid tissues4.

Schematic diagrams of several integrins based on Ref. 1 are shown in Figure 1. cc-Subunits average approximately 1100 amino acids and /?-subunits average approximately 750 amino acids. The c(- subunits contain several divalent cation binding sites which give rise to the dependence on these cations for receptor function. Certain u-subunits also contain an I domain (for ‘inserted’, or ‘interacting’ domain), which seems to impart binding specificity to those integrins which have it. In general, integrins without the I domain bind to Arg-Gly-Asp (RGD)-containing peptide sequences in extracellular matrix proteins, whereas presence of the I domain seems to allow non- RGD binding. CI~ and MM contain an I domain, which is presumably important in the binding of leucocyte integrins to ICAM- (intercellular adhesion molecule- 1) which contains no RGD sequences. CQ appears to be an exception in that, although it lacks an I domain, VLA-4 can bind both VCAM-1 (vascular cell adhesion molecule-l] and fibronetitin at non-RGD sites. The I domain is also associated with conformational changes involved in affinity modulation5.

The regulation of integrin-mediated binding occurs at three levels: modulation of receptor synthesis,

LFA-1

Figut *e 1 Some well-characterized adhesion molecules involved in leucocyte-endothelial cell interactions. of Dr Ranga Sampath.)

mobilization of an intracellular pool of receptors and modulation of the binding affinity of individual receptors. Modulation of receptor synthesis is generally associated with cell differentiation rather than with the rapid response of terminally differen- tiated cells to particular challenges. For example, LFA- 1, VLA-4, VLA-5 and VLA-6 increase two- to four-fold upon conversion of naive to memory T- lymphocyte8’. 7, which could influence localization and recirculation of these subpopulations. Also, differentiation of myeloid lineages is associated with early expression of LFA-1 and later expression of Mac- 1, and the subsequent maturation of monocytes to tissue macrophages is associated with a decrease in Mac-l and VLA-4 expressionxZ”.

Cell adhesion can be modulated within minutes by mobilizing intracellular pools of integrin receptorslo. For example, in neutrophils and monocytes, chemotactic factors result in up to a lo-fold increase in surface expression of Mac-l”. The mechanism of this form of upregulation involves fusion of leucocyte granules containing the receptors with the cell surface”. An even more rapid mechanism of cell adhesion modulation is the ability of some integrins to increase their binding affinity through changes in molecular conformation upon stimulation of the cell. This effect has been shown for many integrins, and the details of this mechanism are an area of active research (reviewed in Ref. 13). It is also important to note that in addition to serving as effector molecules of leucocyte activation, they can also serve as sensors, triggering a variety of leucocyte activation responses upon ligand binding.

The importance of integrins is dramatically illustrated by type 1 leucocyte adhesion deficiency (LAD-l), a rare syndrome characterized by recurrent life-threatening bacterial and fungal infections14. The

L-selectin I

(Figure courtesy

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Leucocyte adhesion under flow conditions: D.A. Jones et al. 339

molecular defects underlying this disease are known to be defects in the &-subunit of the leucocyte integrins15 and pathology arises in part because neutrophils in these patients are unable to localize to sites of infection.

Immunoglobulin superfamily

The immunoglobulin superfamily of receptors is defined by the presence of the immunoglobulin domain, which is composed of 70-110 amino acids arranged in a well-characterized structure (reviewed in Refs 1, 2 and 16). This group of receptors is much more diverse than the integrins, ranging from soluble and membrane-bound immunoglobulins to the multi- receptor T-cell antigen receptor complex to single- chain cellular adhesion molecules. This review will focus on the cellular adhesion molecules ICAM- (CD54), VCAM-1 and mucosal addressing cell adhesion molecule-l (MAdCAM-I), as these have been most strongly implicated in leucocyte-endothelial cell interactions (see Figure z). Two alternatively spliced forms of the VCAM-1 molecule exist, a seven- immunoglobulin domain form and a six-domain form missing domain four.

The distribution of these members of the immunoglobulin superfamily is tightly regulated to enhance their function in specific cell-cell adhesion. ICAM- is constitutively expressed on only a few cell types, but at the site of an inflammatory response is induced on a wide variety of cells. Endothelial cells upregulate ICAM- within several hours of cytokine stimulation, and peak expression occurs after 2448 h. Since it is a counter-receptor for the leucocyte integrins LFA-1 and Mac-l, ICAM- induction provides an important means of recruiting leucocytes to inflammatory sites. ICAM-2, a closely related molecule with two immunoglobulin domains, is another ligand for LFA-1, and is constitutively expressed on endothelial cells and a variety of blood cells. VCAM-1 expression is induced by many of the same stimuli as ICAM-1, with a time course similar to ICAM-1, although expression can be somewhat selectively induced in vitro by the cytokine IL-4 (Ref. 17). VCAM-1 serves as a counter- receptor for leucocyte VLA-4 in the recruitment of leucocytes to inflammatory sites. MAdCAM- serves as the endothelial ligand for CQ/$ and appears to be important in the preferential localization of lymphocyte subsets to mucosal lymph nodes.

A fourth immunoglobulin superfamily molecule of special interest in leucocyte-endothelial cell interactions is CD31 (platelet-endothelial cell adhesion molecule-l, PECAM-l), which has six immunoglobulin homology domains and contains putative proteoglycan binding sequences. Recent in vivo studies have demonstrated that anti-PECAM-1 monoclonal antibodies inhibit recruitment of neutrophils in a peritonitis model, althou role of PECAM-1 is not yet understood 1H

h the exact . Addition-

ally, it was recently reported that this molecule is expressed on unique T-cell subsets and that CD31 binding has the capacity to induce integrin-mediated adhesion, indicating that CD31 signalling may modulate T-cell adhesionlg.

Selectins

The selectin family of adhesion receptors has three known members: L-selectin (LECCAM-1, LAM-l, murine gp90Me’14, peripheral lymph node homing receptor), E-selectin (ELAM-1) and P-selectin (GMP- 140, PADGEM, CD62). They are very similar in structure, and have been extensively reviewed1~2’20. The processed proteins have an NHz-terminal domain homologous to the type C (calcium-dependent) lectins. Following this, there is a region with homology to conserved epidermal growth (EFG) motifs and a chain of repeated domains similar to repeats found in certain complement regulatory proteins. Two of these repeats are found in L-selectin, six in E-selectin and nine in P- selectin. Each molecule ends with a hydrophobic transmembrane region followed by a short cytoplasmic tail. Splicing variants of L-selectin and P-selectin have been identified, and there are soluble forms of all three selectins which may play a role in regulating adhesion”. The lectin-like domain is the ligand binding site, and as with other lectins, ligands are carbohydrate structures (discussed in the next subsection). Binding is completely dependent on the conformational changes associated with the filling of two Ca2+ binding sites and is also sensitive to pH, with decreased binding below physiological pH.

Selectin expression is regulated by a variety of mechanisms. L-selectin is constitutively expressed on unactivated neutrophils, eosinophils and monocytes, as well as naive T-cells and a subset of memory T- cells. On neutrophils, expression of L-selectin has been shown to be localized to the tips of microvillus-like projections on the cell surface”. This striking localization should facilitate the proposed role of neutrophil L-selectin as an adhesion receptor for endothelial cell carbohydrate structures. Neutrophils and lymphocytes also have the ability to cleave L-selectin from the cell surface upon activationz3. This shedding is quite rapid, with 50% of the protein shed within five minutes of stimulation with chemotactic factors such as formyl- Met-Leu-Phe (fMLP)24.

E-selectin is present in viva only in inflamed tissues. It can be induced on endothelial cells upon stimulation by the cytokines interleukin-1, tumour necrosis factor u or lipopolysaccharide, and this induction appears to be predominantly localized to post-capillary venulesz5, the principal site of leucocyte emigration in inflammation. Expression is rapid and transient, with peak levels reached after approximately 4 h of cytokine stimulation, returning to near basal levels after 16-24 hz6. P-selectin is found constitutively within the CI granules of platelets and the Weibel-Palade bodies of endothelial cellsz7. In endothelial cells, histamine, thrombin, bradykinin and substance P cause a rapid mobilization of the Weibel- Palade bodies to the cell periphery, where they fuse with the cell membrane resulting in expression of the receptors on the cell surface28~2g. Under these conditions, expression begins within seconds of stimulation, followed by peak expression at approximately 8-10 min and endocytosis of most receptors by 45-60 min3’.

The physiological importance of selectin-mediated interactions is emphasized in the recently described

Biomaterials 1996, Vol. 17 No. 3


syndrome called leucocyte adhesion deficiency type 2 (LAD-2)31. This syndrome is characterized by impaired immunity and developmental anomalies which can be traced to defective fucose metabolism. In particular, the fucose-containing oligosaccharide moiety sialyl- Lewisx, which serves as a ligand for vascular selectins (see below), is missing in these patients.

Other adhesion molecules

Integrins, selectins and members of the immunoglobulin superfamily are not the only receptors which are necessary for specific adhesion of leucocytes to endothelial cells. Oligosaccharide ligands for selectins are the focus of considerable current research (reviewed in Refs 32 and 33). A large body of evidence suggests that the sialylated derivative of the a(l-3)fucosylated lactosaminoglycan known as LewisX (CD15) serves as a primary ligand for endothelial E- and P-selectins. Although sialyl-Lewis” (sLeX) is a widespread glycocalyx component, it appears to contribute to specificity in leucocyte- endothelial cell interactions. In particular, neutrophils, but not lymphocytes, express sLeX and are bound by endothelial E- and P-selectins”. One subset of lymphocytes, on the other hand, expresses the closely related cutaneous lymphocyte antigen (CLA), which is a sialylated oligosaccharide that can serve as an alternate ligand for E-selectin34335. Three peptide core molecules which carry oligosaccharide binding sites for L-selectin and one which is bound by P-selectin (PSGL-1, Refs 36 and 37) have recently been characterized. The L-selectin ligands are GlyCAM-1 (Sgp50, the 50 kDa component of peripheral node addressin)38, CD34 (Sgp96, the 99 kDa component of peripheral node addressin)3g and MAdCAM-14. As mentioned above, neutrophil L- selectin also carries sLeX which allows it to serve as a ligand for endothelial E- and P-selectins. These interactions account for over half of the primary adhesion of neutrophils to endothelial cells in vitro30, 40. MAdCAM- is apparently capable of serving as a ligand for both L-selectin and CQ& integrin at sites such as mucosal lymph nodes, where the necessary glycosylation takes place4.

CD44 (HCAM, the Hermes antigen) is another leucocyte adhesion molecule which can bind endothelial cell carbohydrate, but in this case binds hyaluronic acid (reviewed in Ref. 41). It appears to function in the adhesion of lymphocytes to vascular

0

endothelium, and has also been implicated as a signalling molecule. Many other recently characterized endothelial cell adhesion molecules may be important in leucocyte-endothelial cell adhesion, including VAP-142 and the IG9 antigen43.

NEUTROPHIL ADHESION UNDER FLOW CONDITIONS

The multistep process of neutrophil extravasation in acute inflammation

One of the most important aspects of leucocyte extravasation is that it is a multistep process. Figure 2 shows the current model of this process divided into several steps: initial contact, primary adhesion (often rolling), activation, secondary adhesion and transmigration. Following initial contact, leucocytes often roll slowly along the endothelium for some distance before either re-entering the free stream or establishing firm adhesion. This rolling is believed to keep the cells in close enough contact with the endothelium that they can be stimulated by substances in the local environment and engage additional binding mechanisms. Rolling requires a definite adhesive interaction, as was first demonstrated by the observation that neutrophils roll with a much slower velocity than that predicted for cells tumbling in the fluid stream adjacent to the vessel wall without any adhesion44S45. Firm adhesion can follow the rolling interaction, and is often dependent upon the leucocyte receiving an activating stimulus while in contact with the endothelium. These activating signals can be soluble substances derived from the endothelium, tissues or pathogens, factors bound to the endothelium, or possibly direct transduction of activating signals by adhesion receptors themselves (reviewed in Ref. 1). Activation of the leucocytes can bring about several changes which alter adhesive characteristics and allow the establishment of firm adhesion, including an increase in integrin binding affinities and flattening of the leucocytes to increase contact area and decrease fluid drag. Once firm adhesion has been established, the leucocytes migrate to interendothelial junctions and diapedese (reviewed in Ref. 46). It is important to note that in this multistep process of leucocyte extravasation, specificity in leucocyte recruitment can be generated at any of the steps47. For instance, a leucocyte which contains primary adhesion machinery

Figure 2 This diagram of the process of neutrophil extravasation can be viewed in two ways. Mechanistically, four adhesive interactions are observed: initial contact, rolling, firm adhesion and transmigration. Selectin-carbohydrate bonds are more important in initial contact and rolling, while integrin-peptide bonds are more important in firm adhesion and transmigration. Physiologically, the process can be divided into three steps: primary adhesion which is independent of leucocyte activation, activation of the leucocyte and activation-dependent secondary adhesion. Specificity in leucocyte localization can be gained at any of these steps.


Leucocyte adhesion under flow conditions: D.A. Jones et a/. 341

to bind a particular endothelial surface might not emigrate there due to a lack of responsiveness to local activating substances or a lack of the proper activation- dependent adhesion machinery.

Neutrophikmdothelial cell interactions as a function of wall shear stress

A cornerstone of the multistep model is the finding that different types of adhesion receptors can mediate distinct types of adhesive interactions48’4g. The initial adhesion and subsequent rolling of neutrophils along endothelial cells appear to be mediated in many cases by members of the selectin family (E- and P-selectin) binding to carbohydrate moieties of neutrophil cell surface glycoproteins. Firm adhesion and transmigration, on the other hand, generally appear to be mediated by members of the integrin family binding to members of the immunoglobulin superfamily. For neutrophils, the /I2 integrins (LFA-1 and Mac-l) and the endothelial ligand ICAM- appear to be particularly important contributors to firm adhesion and transmigration.

Quantitative studies of neutrophil adhesion under flow conditions have shown that overall leucocyte adherence decreases with increasing wall shear stress30~44*48--51. These studies generally show that leucocyte adherence is optimal in the range of post- capillary venular wall shear stresses (approximately l-

4 dyn cm-“) but decreases rapidly at higher shear stresses. Integrin-mediated adhesion in the absence of selectins, on the other hand, appears to be effective only at wall shear stresses well below 1.0 dyn cm-’ (Refs 49 and 50). These findings are summarized in Table I. Most of the data related to this idea has come horn studies of neutrophils, but many aspects of this model appear to hold for lymphocytes and monocytes as well. Lymphocyte and monocyte adhesion, along with some of their additional complexities, are discussed below.

Neutrophil rolling velocities at post-capillary venular wall shear stresses are generally reported to be of the order of 10-40 pm s-l both in vitro30940*50 and j* viv*44,51-55. Variation of rolling velocity as a function of wall shear stress has also been repo*ed30.44.4s,50,s1, as have distributions of rolling velocities of individual neutrophils30944S48. Interpreta- tion of rolling velocity data is complicated, however, by the fact that rolling neutrophils are generally subject to activating stimuli which can rapidly change

the micromechanics of rolling (as discussed in the next section). These complications have been circumvented using an in vitro system in which neutrophils roll on an artificial lipid bilayer containing purified P- selectin5’. We have also had success in our laboratory by stimulating endothelial cells with histamine, which rapidly leads to P-selectin-mediated rolling but apparently does not induce other endothelial-derived activating factors3’.

Properties of selectin-mediated adhesion

There are several possible reasons why selectin- carbohydrate bonds are more effective in the initial arrest of leucocytes from the bloodstream (see Figure 3). One of the most striking considerations is the location of L-selectin on the tips of microvillus-like projections from resting neutrophilsz2. This is especially important in light of the relatively small number of receptors on a neutrophil (approximately 2.4 x lo4 sites per ce1124S50 ) and the length of L-selectin (approximately 15 nm) relative to E- and P-selectins (approximately 30 and 40 nm, respectively56). Even though L-selectin is not a long molecule relative to other selectins, direct coupling to endothelial E- or P- selectins is predicted to form quite long tethering structures (of the order of 40 and 50 nm, respectively56). The intermembrane separation resulting from these tethers is large enough that energetically unfavourable glycocalyx interdigitation would not be necessary to form the selectin bonds57. In addition to avoiding glycocalyx steric hindrance, selectin-carbohydrate interactions avoid the steric hindrance that glycoprotein surface decorations can produce for integrin-peptide core interactions. The flexibility of long single-chain selectins relative to two- chain integrins may also be an advantage in mediating initial interactions50’58. Alternatively, some flexibility could arise in the carbohydrate chain containing the site bound by P-selectinsg. In addition to buckling of receptors themselves, flexibility could result from deformation of the microvillus-like projections on which the receptors are located5 . Any such deformations or buckling of receptors would have the effect of increasing the contact area between a spherical neutrophil and the planar endothelial cell surface, allowing more bonds to form. Flexibility might also enhanced the mobility of binding sites so that enhance diffusivity could increase the rate of formation of bonds5’. This is important because an

Table 1 Summary of leucocyte adhesion at various wall shear stress ranges

Approximate mean wall shear stress (dyn cm-*)

Location Characteristics of leucocyte adhesion on activated endothelium

g-1

l-4

>4

Only near stagnation points in arterial flow Post-capillary and high-endothelial venular flow Flow in most other blood vessels

lntegrins alone can mediate adhesion

Selectin-mediated rolling followed by integrin- mediated firm adhesion Selectin-mediated adhesion quickly becomes ineffective so leucocytes are unable to bind endothelium. However, pre-established firm adhesion mediated by integrins is shear-resistant to levels well above the physiological range



extremely rapid rate of bond formation is necessary to arrest leucocytes moving quickly in the bloodstream. The ability of selectin tethers to withstand high strain before breaking may also be important in initial adhesion and rolling, an issue addressed in two mathematical models of the rolling process5aBfi0.

Another property of selectin bonds that suits them to initial adhesion and rolling is that these bonds are functional without activation of the leucocyte. Activation-independent adhesion has been demonstrated for neutrophils, lymphocytes and monocytes, and selectins contribute to this adhesion in all cases. This makes sense physiologically since circulating leucocytes are normally unactivated, but can bind to specific endothelial sites when necessary. It is nonetheless possible that later activation can affect selectin adhesion. It has been reported, for instance, that fMLP stimulation of neutrophils induces a brief increase in L-selectin on the cell surface followed by rapid proteolytic sheddingz4, and that leucocyte activation can augment the interaction between L- selectin and a yeast-derived polysaccharide61.

Properties of integrin-mediated adhesion

An important property of integrins which makes them well suited to mediate firm adhesion and migration is the fact that they can be modulated by cell activation. As mentioned earlier, activation produces rapid conformational changes in leucocyte integrins which substantially modify their affinity for ligands. In one study”‘, the force required to break integrin bonds was found to be 2.1 x 1O-7 dyn for activated GpIIb-IIIa (Q,& integrin) and 5.7 x lop8 dyn for non-activated GpIIb-IIIa binding to fibrinogen. This modulation occurs very rapidly and has been shown to mediate increased adhesion of neutrophils to surfaces within 15-30 slO. The ability of integrins to rapidly modulate their affinity appears to be very important in neutrophil migration. Monoclonal antibodies which ‘lock’ fl, integrins in the high-affinity state inhibit neutrophil migration induced by chemotactic stimulation. The failure of integrins to return to the low-affinity state appears to tether the neutrophil to the endothelium and prevent migration63-65. Neutrophil activation also leads to mobilization of additional Mac-l receptors from an intracellular pool. However, this newly mobilized Mac- 1 does not appear to be important in mediating adhesion unless the cell is restimulated following expression of the new receptors10’6fi.

Morphological changes in the neutrophil upon activation can influence adhesion as well (Figure 3). When the neutrophil is activated, it flattens out on the endothelium, greatly increasing its contact area. Increased contact area allows many bonds to form, taking advantage of the large number of integrin receptors (50 x lo4 Mac-l receptors on an unstimulated neutrophil”) relative to selectin receptors (2-4 x 104, Refs 10 and 50). This is especially important because /& integrins are not localized to the tips of cell projections, as is L-selectin. Flattening of leucocytes also greatly decreases the fluid drag and torque leucocytes experience53. All of these factors combine to produce adhesion which is up to

Figure 3 Selectin-mediated rolling and integrin-mediated firm adhesion. Selectin-carbohydrate interactions are well suited to mediate initial leucocyte attachment and rolling for several possible reasons. These include the localization of L-selectin to tips of microvillus-like projections, the length and flexibility of receptor-ligand tethers, the ability of these tethers to withstand considerable strain before breaking and a rapid rate of bond formation. On the other hand, integrin-polypeptide interactions have characteristics which make them well suited to mediate firm adhesion and migration. Conformational changes in surface integrins can modulate binding affinity very rapidly, and on a slightly longer timescale, mobilization of intracellular stores can provide additional surface receptors. Flattening of the leucocyte upon activation greatly increases the leucocyte- endothelial cell contact area, which allows a large number of integrin bonds to form and decreases fluid forces on the cell. These characteristics combine to allow adhesion which is highly shear-resistant but can be modulated to allow migration. Further, leucocyte activation causes shedding of L-selectin, so that migration is not hindered by these bonds.

lOO-fold more shear-resistant than selectin-mediated binding5’.

As a final point of discussion, we mention the transition from a rolling state to a stationary state. Most current experimental evidence in this area is largely indirect, since studies generally aim at examining either the rolling phenomenon or aspects of secondary adhesion separately. Several lines of evidence, however, point to integrin activation as an event of central importance in the transition. First, although integrin mechanisms do not appear to be primary mediators of rolling adhesion, they do serve to decrease the average rolling velocity. This has been shown in the case of neutrophils, where the speed of P-selectin-mediated rolling on histamine-stimulated HUVECs increases by approximately 25% with antibodies blocking & and ICAM- (Ref. 30). This has also been shown for lymphocytes, where blocking either LFA-l/ICAM-1 or VLA-4/VCAM-1 increases the average rolling velocity by approximately 33% and blocking both pathways increases the rolling velocity by approximately 80%35. This indicates that integrins are capable of interacting with endothelial cells while the leucocyte is rolling, so that complete cell stopping is not a prerequisite for at least some integrin function. Secondly, a direct correlation between decreasing average rolling velocity and the fraction of rolling neutrophils which stop has been demonstratedfi7, although whether this effect results from basal affinity integrin adhesion or requires the high affinity interactions is not clear. It is clear in a different study, however, that a G-protein-mediated activating event correlates directly with lymphocyte stopping in vitrofiB. Taken together, these findings indicate that integrins are capable of slowing rolling leucocytes, and suggest that integrin activation is important for the complete stopping of rolling leucocytes.



LYMPHOCYTE ADHERENCE IN TISSUE REJECTION

Lymphocyte adhesion to endothelial cells under flow conditions appears to be considerably more complex than adhesion of neutrophils. Lymphocytes adhere at areas of inflammation, along with neutrophils and monocytes, but lymphocytes also recirculate through- out the body under normal conditions by binding to endothelial cells in lymph nodes and transmigrating into the lymphatics. There are also a wide variety of lymphocyte subsets, and their recirculation is sufficiently specific that particular subsets will preferentially localize to the most appropriate areas of the body (reviewed in Ref. 69). To accommodate this specificity, a wide variety of adhesion receptors and activating factors are believed to be involved in lymphocyte adhesion. Despite these complexities, adhesion molecule participation in transplant rejection is an area of great research activity. There may be two modes by which rejection can be modulated through adhesion molecules: the first is to simply prevent lymphocyte infiltration into the tissues by blocking adhesion to blood vessel walls, and the second mode is some form of tolerance induction by altering or preventing adhesion-dependent signalling events.

Inhibiting tissue rejection by blocking the adhesion molecules necessary for leucocyte recruitment is an obvious approach, and is already under investigation for the control of many forms of inflammation. However, since lymphocytes are believed to be the key component of a rejection response, one problem with this approach may be that the wide variety of adhesion molecules utilized by lymphocytes will require many blocking reagents. On the other hand, it is also possible that while normal localization is highly specific and utilizes many receptors, adhesion to the endothelium in rejecting sites may be controlled by the upregulation of a smaller number of high-affinity receptors which recruit a broad spectrum of lymphocyte subsets. If this turns out to be the case, then a drug which blocks one of these dominant adhesion molecules might still provide effective immunosuppression. Similar low- specificity, high-affinity recruitment is hypothesized to account for much of the lymphocyte localization to sites of chronic inflammation7’.

A first step in this approach to reducing rejection, then, is to determine which adhesion receptors are important in the rejection reaction. Several recent studies have addressed this and it appears that the ICAM- and VCAM-1 adhesion pathways are in general the most important pathways studied so far (reviewed in Refs 71 and 72), although the E-selectin pathway also contributes, particularly in early rejection73. In several experimental systems, blocking the ICAM- and VCAM-1 pathways with monoclonal antibodies following transplant significantly delayed rejection (reviewed in Refs 71 and 72) and anti-ICAM- 1 therapy with renal allograft has been tested in a phase I clinical trial with promising results. Recent data from our own laboratories using an in vitro model of T-cell adhesion in chronic inflammation suggest that the initial attachment and rolling of T-cells on endothelial cells under flow conditions is not

mediated by any of the known selectins or other well- characterized adhesion molecules35, so that anti- adhesion molecule therapy may become more effective as additional pathways are identified.

A second step toward inhibiting rejection by blocking lymphocyte adhesion is to understand any sequential or co-operative interactions in the attachment and extravasation process. As described above, neutrophil adhesion appears to follow a stepwise process, such that if any one step is blocked, the entire process is blocked. The first step in the sequence is a logical choice to block, although a full understanding of the process may indicate a better target. Lymphocytes can, at least in some instances, utilize both selectin- carbohydrate interactions and integrin-polypeptide interactions to adhere to endothelial cells, so it is appealing to think that the adhesion sequence elucidated for neutrophils (rolling, activation, then firm adhesion) may hold for lymphocytes as well. It appears, however, that a variety of adhesion sequences are possible, which may allow for greater specificity in the localization of particular lymphocyte subsets to particular endothelial beds. Our studies of lymphocyte-endothelial cell interactions in vitro show that with a mixed population of T-lymphocytes, some cells roll for extended periods without stopping while others roll briefly then adhere firmly. Still others adhere firmly as soon as they contact the endothelial cells, with no obvious period of rolling during which the lymphocytes could become activated35. This heterogeneity of binding patterns presumably reflects the heterogeneity of the lymphocyte population in terms of receptor expression and receptor activation state. Studies of lymphocyte binding in vivo also support the idea that several adhesion sequences may be utilized by lymphocyte subsets. In one study, rolling of lymphocytes along high endothelial venules (HEV) was not observed74, and in another study, a G- protein-mediated activation event was found to contribute to the establishment of firm adhesion to HEV68.

Another mechanism which may be useful in preventing tissue rejection is the induction of tolerance through modulation of immune system signals which require cell-cell adhesion. This mechanism was suggested by studies using a murine cardiac allograft model in which the combination of anti-LFA-1 and anti-ICAM- antibodies produced allospecific long- term graft survival752 76. Anti-ICAM- antibody alone delayed but did not prevent acute rejection, suggesting interactions of LFA-1 and ICAM- with more receptors than just each other. Also, the graft survival appears to have been due to a true allospecific tolerance rather than simply immunosuppression, since recipients would tolerate later skin grafts from the same donor strain, but not from a different strain.

MONOCYTE ADHERENCE IN ATHEROSCLEROSIS

Monocyte adhesion to endothelial cells under flow conditions may be similar in many respects to neutrophil and lymphocyte adhesion. The special case



of monocyte recruitment to atherosclerotic lesions, however, has several characteristics which are influenced greatly by arterial blood flow. The specific localization of monocytes at atherosclerotic lesions is likely to be determined by several factors, including receptor expression at the lesion site, activating substances released from the lesion, the low fluid shear and longer blood cell residence times noted at lesion prone sites, and flow-dependent alterations to the endothelial cells at these sites. Several recent studies have investigated the expression of adhesion receptors on the endothelial cells overlying atherosclerotic plaques. E-selectin77, P-selectin7a, ICAM- 1 78.79 and vc~~_180.8’ have all been shown to be present on atherosclerotic plaques at various times and at various levels of expression.

As with lymphocytes, it is possible but not necessary that monocyte adhesion follows a multistep adhesion cascade similar to that described for neutrophils (Figure 2). The flow field over lesion-prone arterial sites is much different than the steady flow through post-capillary venules or high endothelial venules (reviewed in Ref. 82), and this could potentially allow monocyte adhesion to atherosclerotic plaques directly via integrin mechanisms without a separate mechanism for initial attachment. Lesion-prone sites are known to be at low-shear locations within arteries, often just downstream from bifurcations. Flow can reverse at these locations, allowing for periodically alternating flow. Oscillatory wall shear stresses in this case have been shown in one study to range from -13

to 9 dyn cm ’ with a time averaged mean of only -0.5 dyn cmm2 (Ref. 83). The effect of such oscillatory flow conditions on leucocyte adhesion is likely to be very important84,85. For comparison, at non-lesion- prone arterial sites flow is continuous and wall shear stresses range from approximately 10 to 50 dyn cm-“. These shear stresses are most likely too high for even selectins to mediate adhesion”“.48.50.

In addition to affecting binding through direct forces on leucocytes, blood flow has dramatic effects on endothelial cell function which could influence leucocyte adhesion. The most readily apparent effect is the elongation and orientation in the direction of flow characteristic of endothelial cells exposed to arterial shear stresses. This has been observed in viva at high shear arterial locationsRfi and with cultured endothelial cells exposed to steady arterial wall shear stresses87.88. Endothelial cells exposed to low shear stresses, on the other hand, take on a cobblestone appearance, which correlates with the more cobblestone endothelial morphology found at lesion-prone sites. Many other differences between endothelial cells subjected to high and low shear stress have been observed82~8”~g1, which could contribute to the increased susceptibility of low shear arterial surfaces to atherosclerosis. In particular, there is evidence for a direct effect of shear stress on adhesion receptor expressiong”.

CONCLUSIONS

Inflammatory responses are a crucial consideration in the understanding of many disease processes and in

the development of tissue engineering approaches toward therapy. This review has examined current understanding of one aspect of inflammation, the binding of leucocytes to blood vessel walls while subject to the forces of flowing blood. We have seen how a variety of adhesion molecules are responsible for highly specific interactions of leucocytes with endothelial cells. These interactions are likely to be important sites for intervention in various therapeutic approaches. We have also seen how these receptors act in multiple co-ordinated steps to provide additional specificity in leucocyte-endothelial cell interactions. Understanding these steps, including selectin- mediated rolling, integrin-mediated firm adhesion and the many other possible variations should prove very useful in understanding inflammatory responses. This understanding will then hopefully lead to specific control over inflammation as well as to exploitation of the specific interactions involved in the development of new treatments for disease.

ACKNOWLEDGEMENTS

This work was supported by NIH grants HL-18672, NS- 23327, AI-233521, HL-42550 and ES-06091, and the Robert A. Welch Foundation grant c-938.

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