Modifying the mechanical property and shear threshold of L-selectin adhesion independently of equilibrium properties

Interactions between adhesion molecules on two different cells differ from interactions between receptors and soluble ligands in that the adhesion molecule interaction (bond) is often subjected to force. It is widely assumed by cell biologists that the 'strength' of a bond is a simple function of the affinity of one adhesion molecule for the other, whereas biophysicists suggest that bonds have 'mechanical properties' that affect their strength. Mechanical properties are a function of the shape of the energy landscape related to bond formation and dissociation, whereas affinity is related only to the net energy change. Mechanical properties determine the amount by which the kinetics and affinity of bonds are altered by applied force. To date there has been no experimental manipulation of an adhesion molecule that has been shown to affect mechanical properties. L-selectin is an adhesion molecule that mediates lymphocyte binding to, and rolling on, high endothelial venules; these are prerequisites for the emigration of lymphocytes from the bloodstream into lymph nodes. Here we report a selective and reversible chemical modification of a mucin-like ligand that alters the mechanical properties of its bond with L-selectin. The effect of force on the rate of bond dissociation, that is, on a mechanical property, is altered, whereas there is little or no effect of the modification on the rate of bond dissociation in the absence of force. Moreover, the puzzling requirement for hydrodynamic shear flow above a threshold level for L-selectin interactions is dramatically altered.     

L-selectin binds to P-selectin glycoprotein ligand-1 on leukocytes: interactions between the lectin, epidermal growth factor, and consensus repeat domains of the selectins determine ligand binding specificity

The selectins mediate cellular interactions by binding carbohydrate determinants present on a limited number of glycoprotein ligands. L-selectin binds multiple ligands expressed on endothelial cells, while P-selectin interacts exclusively with P-selectin glycoprotein ligand-1 (PSGL-1) on leukocytes. In this study, L-selectin was shown to bind leukocytes through the P-selectin ligand, PSGL-1, although at lower levels than P-selectin. L-selectin binding to PSGL-1 is specific since it was blocked by Abs to L-selectin or PSGL-1, required appropriate glycosylation of PSGL-1, and was Ca2+ dependent. The contributions of the extracellular domains of the selectins to ligand binding was assessed using a panel of chimeric selectins created by exchange of domains between L-selectin and P- or E-selectin. The lectin and epidermal growth factor domains of L- and P-selectin contributed significantly to binding through similar, if not identical, regions of PSGL-1. The different chimeric selectins revealed that the lectin domain was the dominant determinant for ligand binding, while cooperative interactions between the lectin, epidermal growth factor, and short consensus repeat domains of the selectins also modified ligand binding specificity. L-selectin binding to PSGL-1 expressed by leukocytes may mediate neutrophil rolling on stationary leukocytes bound to cytokine-induced endothelial cells, which was previously reported to be a L-selectin-dependent process.     

Affinity and kinetic analysis of L-selectin (CD62L) binding to glycosylation-dependent cell-adhesion molecule-1

The selectin family of cell adhesion molecules mediates the tethering and rolling of leukocytes on blood vessel endothelium. It has been postulated that the molecular basis of this highly dynamic adhesion is the low affinity and rapid kinetics of selectin interactions. However, affinity and kinetic analyses of monomeric selectins binding their natural ligands have not previously been reported. Leukocyte selectin (L-selectin, CD62L) binds preferentially to O-linked carbohydrates present on a small number of mucin-like glycoproteins, such as glycosylation-dependent cell adhesion molecule-1 (GlyCAM-1), expressed in high endothelial venules. GlyCAM-1 is a soluble secreted protein which, following binding to CD62L, stimulates beta2-integrin-mediated adhesion of lymphocytes. Using surface plasmon resonance, we show that a soluble monomeric form of CD62L binds to purified immobilized GlyCAM-1 with a dissociation constant (Kd) of 108 microM. CD62L dissociates from GlyCAM-1 with a very fast dissociation rate constant (>/=10 s-1) which agrees well with the reported dissociation rate constant of CD62L-mediated leukocyte tethers. The calculated association rate constant is >/=10(5) M-1 s-1. At concentrations just above its mean serum level (approximately 1.5 microg/ml or approximately 30 nM), GlyCAM-1 binds multivalently to immobilized CD62L. It follows that soluble GlyCAM-1 may cross-link CD62L when it binds to cells, suggesting a mechanism for signal transduction.     

Leukocyte Adhesion Deficiency Type II is a generalized defect of de novo GDP-fucose biosynthesis. Endothelial cell fucosylation is not required for neutrophil rolling on human nonlymphoid endothelium

Leukocyte Adhesion Deficiency Type II (LAD II) is a recently described syndrome and the two patients with this defect lack fucosylated glycoconjugates. These glycoconjugates include the selectin ligand, sialyl LewisX, and various fucosylated blood group antigens. To date, the molecular anomaly in these patients has not been identified. We localized the defect in LAD II to the de novo pathway of GDP-fucose biosynthesis, by inducing cell-surface expression of fucosylated glycoconjugates after exposure of lymphoblastoid cell lines from the LAD II patients to exogenous fucose. This defect is not restricted to hematopoietic cells, since similar findings were elicited in both human umbilical vein endothelial cells (HUVEC) and fibroblasts derived from an affected abortus. We have used these LAD II endothelial cells to examine the consequence of fucosylation of endothelial cells on the rolling of normal neutrophils in an in vitro assay. Neutrophil rolling on LPS-treated normal and LAD II HUVEC was inhibited by an E-selectin monoclonal antibody at both high and low shear rates. LAD II HUVEC lacking fucosylated glycoproteins supported leukocyte rolling to a similar degree as normal HUVEC or LAD II cells that were fucose-fed. At low shear rates, an L-selectin antibody inhibited neutrophil rolling to a similar degree whether the LAD II cells had been fucose-fed or not. These findings suggest that fucosylation of nonlymphoid endothelial cells does not play a major role in neutrophil rolling and that fucose is not a critical moiety on the L-selectin ligand(s) on endothelial cells of the systemic vasculature.     

Comparison of tethering and rolling of eosinophils and neutrophils through selectins and P-selectin glycoprotein ligand-1

We compared the abilities of selectins and the selectin ligand, P-selectin glycoprotein ligand-1 (PSGL-1), to support tethering and rolling of eosinophils and neutrophils under physiologic flow conditions. Eosinophils and neutrophils accumulated on P-selectin at similar site densities and rolled at similar velocities, but fewer eosinophils than neutrophils accumulated at any P-selectin density. Compared with neutrophils, few eosinophils accumulated on E-selectin except at high densities, and those cells that did accumulate rolled faster than neutrophils. Examination of the mechanisms for accumulation revealed that eosinophils and neutrophils formed similar numbers of primary tethers to P-selectin, whereas eosinophils formed fewer primary tethers to E-selectin than did neutrophils. Compared with neutrophils, adherent eosinophils also supported fewer leukocyte-leukocyte interactions, resulting in diminished secondary tethers to either P- or E-selectin. Studies with mAbs to L-selectin and PSGL-1 demonstrated that neither cell type used L-selectin to form primary tethers to P- or E-selectin. Both eosinophils and neutrophils used the NH2 terminus of PSGL-1 to form primary tethers to P-selectin, but not to E-selectin. Both cell types used L-selectin and PSGL-1 to promote leukocyte-leukocyte interactions and secondary tethers to P- or E-selectin. However, eosinophils developed significantly fewer secondary interactions, probably because they express less L-selectin than do neutrophils.     

Core 2 oligosaccharide biosynthesis distinguishes between selectin ligands essential for leukocyte homing and inflammation

Mammalian serine/threonine-linked oligosaccharides (O-glycans) are commonly synthesized with the Golgi enzyme core 2 beta-1,6-N-acetylglucosaminyltransferase (C2 GlcNAcT). Core 2 O-glycans have been hypothesized to be essential for mucin production and selectin ligand biosynthesis. We report that mice lacking C2 GlcNAcT exhibit a restricted phenotype with neutrophilia and a partial deficiency of selectin ligands. Loss of core 2 oligosaccharides reduces neutrophil rolling on substrata bearing E-, L-, and P-selectins and neutrophil recruitment to sites of inflammation. However, the diminished presence of L-selectin ligands on lymph node high endothelial venules does not affect lymphocyte homing. These studies indicate that core 2 oligosaccharide biosynthesis segregates the physiologic roles of selectins and reveal a function for the C2 GlcNAcT in myeloid homeostasis and inflammation.     

Regulation of L-selectin-mediated rolling through receptor dimerization

L-selectin binding activity for its ligand expressed by vascular endothelium is rapidly and transiently increased after leukocyte activation. To identify mechanisms for upregulation and assess how this influences leukocyte/endothelial cell interactions, cell-surface dimers of L-selectin were induced using the coumermycin-GyrB dimerization strategy for cross-linking L-selectin cytoplasmic domains in L-selectin cDNA-transfected lymphoblastoid cells. Coumermycin- induced L-selectin dimerization resulted in an approximately fourfold increase in binding of phosphomanan monoester core complex (PPME), a natural mimic of an L-selectin ligand, comparable to that observed after leukocyte activation. Moreover, L-selectin dimerization significantly increased (by approximately 700%) the number of lymphocytes rolling on vascular endothelium under a broad range of physiological shear stresses, and significantly slowed their rolling velocities. Therefore, L-selectin dimerization may explain the rapid increase in ligand binding activity that occurs after leukocyte activation and may directly influence leukocyte migration to peripheral lymphoid tissues or to sites of inflammation. Inducible oligomerization may also be a common mechanism for rapidly upregulating the adhesive or ligand-binding function of other cell-surface receptors.     

Identification of podocalyxin-like protein as a high endothelial venule ligand for L-selectin: parallels to CD34

The leukocyte adhesion molecule, L-selectin, mediates the recruitment of lymphocytes to secondary lymphoid organs via interactions with specific ligands presented on high endothelial venules (HEV). Although the HEV-derived ligands for L-selectin are still incompletely defined, they share a common sialomucin-like structure which is thought to present clustered oligosaccharides to the lectin domain of L-selectin. Podocalyxin-like protein (PCLP) is a transmembrane sialomucin that is similar in structure to the well-characterized L-selectin ligand CD34. PCLP has been shown previously to be expressed on the foot processes of podocytes in the kidney glomerulus as well as on vascular endothelium at some sites. We have determined that PCLP is present on HEV, where it binds to both recombinant L-selectin and the HEV-specific monoclonal antibody MECA-79. Furthermore, purified HEV-derived PCLP is able to support the tethering and rolling of lymphocytes under physiological flow conditions in vitro. These results suggest a novel function for PCLP as an adhesion molecule and allow the definition of conserved structural features in PCLP and CD34, which may be important for L-selectin ligand function.     

Sulphation requirement for GlyCAM-1, an endothelial ligand for L-selectin

L-selectin participates in the initial attachment of leukocytes to the vascular endothelium. On lymphocytes, it mediates binding to high endothelial venules of lymph nodes. As a selectin it functions as a calcium-dependent lectin recognizing carbohydrate-bearing ligands on endothelial cells. Two lymph node ligands for L-selectin have been identified as sulphated glycoproteins of M(r) approximately 50K and approximately 90K, called Sgp50 and Sgp90 (ref. 10). The recently cloned Sgp50 (ref. 12), now designated GlyCAM-1, is a high endothelial venule-associated, mucin-like glycoprotein containing predominantly O-linked carbohydrate chains. Sialylation of GlyCAM-1 is necessary for its ligand activity and a role for fucosylation is suspected. We have used chlorate as a metabolic inhibitor of sulphation, and report here that GlyCAM-1 has an additional requirement for sulphate.     

L-selectin-mediated lymphocyte rolling on MAdCAM-1

The L-selectin, a cell surface C-type lectin, directs lymphocyte traffic to lymph nodes, and contributes to lymphocyte homing to Peyer's patches and to leukocyte interactions with inflamed venules. Here we report that the mucosal vascular addressin MAdCAM-1, a mucosal endothelial adhesion molecule with immunoglobulin- and mucin-like domains, is a facultative ligand for L-selectin. MAdCAM-1 isolated from mesenteric lymph nodes, but not from cultured endothelioma cells, bears N-glycanase-resistant sialic acid-containing carbohydrate which supports adhesion of L-selectin-transfected lymphoid cells under shear. Interacting lymphoid cells display a 'rolling' behaviour similar to the selectin-dependent rolling of neutrophils observed in inflamed venules. MAdCAM-1 is also a ligand for the lymphocyte integrin homing receptor for Peyer's patches, alpha 4 beta 7 (ref. 7), and may be uniquely adapted to support both selectin-mediated lymphocyte rolling and integrin-mediated adhesion and arrest in vivo.     

The role of adhesion molecules in human leukocyte attachment to porcine vascular endothelium: implications for xenotransplantation

Many obstacles still prevent successful xenotransplantation of porcine donor organs. When hyperacute rejection is averted, transplanted pig organs are subject to acute vascular and cellular rejection. In autologous systems, leukocyte recruitment into inflamed tissues involves selectins, integrins, and Ig family members. To determine whether these mechanisms allow human leukocytes to effectively enter porcine grafts, the pathways by which human leukocytes adhere to TNF-alpha-stimulated porcine aortic endothelium were examined under static and physiologic flow conditions. L-selectin and E-selectin had overlapping functions in neutrophil capture and rolling, whereas Ab blockade of E-selectin and the beta2 integrins inhibited firm arrest of rolling neutrophils. Combined blockade of selectins and beta2 integrins resulted in negligible human neutrophil attachment to pig endothelium. Lymphocyte attachment to porcine endothelium was primarily L-selectin mediated, whereas beta2 integrin and VCAM-1/very late Ag-4 (VLA-4) interactions promoted static adhesion. Concurrent beta2 integrin, VLA-4, VCAM-1, and L-selectin blockade completely inhibited lymphocyte attachment. Thus, interactions between leukocyte-endothelial cell adhesion receptor pairs remained remarkably intact across the human-porcine species barrier. Moreover, disrupting the adhesion cascade may impair the ability of human leukocytes to infiltrate a transplanted porcine organ during rejection.     

Leukocyte kinetics in the pulmonary microcirculation: observations using real-time confocal luminescence microscopy coupled with high-speed video analysis

To quantitatively assess blood cell kinetics in the intact pulmonary microcirculation, in which arterioles, venules, and capillaries are exceedingly intricate and densely convoluted, we recently developed a real-time confocal laser luminescence microscope with a high-speed analysis component. The system has the capacity to yield confocal images of rapidly moving cells at a rate of 1000 frames/second and at sufficiently high degrees of magnification. Applying this novel method to isolated perfused rat lungs, we estimated the endothelial distributions of constitutively expressed intercellular adhesion molecule-1 (ICAM-1) and P-selectin and also studied leukocyte hemodynamic behavior in the pulmonary microvasculature under conditions in which ICAM-1, P-selectin, and L-selectin were inhibited, respectively, by 1A29 (monoclonal antibody to rat ICAM-1), ARP2-4 (monoclonal antibody to rat P-selectin), and fucoidin (competitive inhibitor of both P- and L-selectin). The results were compared with those obtained with a nonconfocal microscope using conventional epiluminescence. Intertwined microvessel networks in the lung were clearly distinguishable in confocal images but not in conventional nonconfocal views. ICAM-1 was perceptibly expressed along venular and capillary but not arteriolar endothelium, whereas P-selectin was undetectable in all microvessels examined. Leukocytes were not firmly adhered to venular or arteriolar endothelial cells. Leukocyte rolling was recognized more frequently along arteriolar walls than along venular walls and was suppressed in arterioles by L-selectin inhibition but not by either ICAM-1 or P-selectin inhibition. In capillaries, transient and sustained arrest of leukocytes occurred at physiologic shear rates. Inhibition of ICAM-1 or P-selectin had no remarkable effect upon either transient or sustained entrapment of leukocytes in capillaries. In conclusion, physiologic and biologic characteristics of pulmonary microvessels appear to be quite different from those of the systemic microcirculation.     

Pulmonary disease in the immunocompromised child

Adhesion receptors that are known to initiate contact (tethering) between blood-borne leukocytes and their endothelial counterreceptors are frequently concentrated on the microvilli of leukocytes. Other adhesion molecules are displayed either randomly or preferentially on the planar cell body. To determine whether ultrastructural distribution plays a role during tethering in vivo, we used pre-B cell transfectants expressing L- or E-selectin ectodomains linked to transmembrane/intracellular domains that mediated different surface distribution patterns. We analyzed the frequency and velocity of transfectant rolling in high endothelial venules of peripheral lymph nodes using an intravital microscopy model. Ectodomains on microvilli conferred a higher efficiency at initiating rolling than random distribution which, in turn, was more efficient than preferential expression on the cell body. The role of microvillous presentation was less accentuated in venules below 20 micrometers in diameter than in larger venules. In the narrow venules, tethering of cells with cell body expression may have been aided by forced margination through collision with erythrocytes. L-selectin transfected cells rolled 10-fold faster than E-selectin transfectants. Interestingly, rolling velocity histograms of cell lines expressing equivalent copy numbers of the same ectodomain were always similar, irrespective of the topographic distribution. Our data indicate that the distribution of adhesion receptors has a dramatic impact on contact initiation between leukocytes and endothelial cells, but does not play a role once rolling has been established.     

Chemoattractant receptor-induced phosphorylation of L-selectin

The selectin adhesion molecules and chemoattractant receptors synergistically regulate leukocyte migration into lymphoid tissues and sites of inflammation, but little is known about how these families of receptors modulate each other's function. In this study, L-selectin was found to be phosphorylated in lymphoblastoid cell lines, and phosphorylation was enhanced by phorbol ester (phorbol 12-myristate 13-acetate (PMA)) treatment. Interactions between L-selectin and chemoattractant receptors were therefore examined using transfected rat basophilic leukemia cell lines (RBL-2H3) that expressed human L-selectin along with human leukocyte chemoattractant receptors. L-selectin was rapidly phosphorylated in cells treated with chemoattractants, thrombin, IgE receptor agonists, or PMA. Pertussis toxin or the protein kinase C inhibitor, staurosporine, completely blocked chemoattractant receptor-induced phosphorylation of L-selectin. PMA-induced phosphorylation was on serine residues within the cytoplasmic tail of L-selectin that have been well conserved during recent evolution. Although L-selectin phosphorylation was not essential for basal levels of adhesion through L-selectin in transformed cell lines, the rapid increase in ligand binding activity of L-selectin that occurs following leukocyte activation was blocked by staurosporine. These results demonstrate that L-selectin can be phosphorylated following engagement of chemoattractant receptors and suggest that this may be a physiologically relevant mechanism for the synergistic regulation of these receptors during leukocyte migration.     

The multistep process of homotypic neutrophil aggregation: a review of the molecules and effects of hydrodynamics

Homotypic adhesion of neutrophils stimulated with chemoattractant is analogous to capture on vascular endothelium in that both processes are supported by L-selectin and beta 2-integrin adhesion receptors. Under hydrodynamic shear, cell adhesion requires that receptors bind sufficient ligand over the duration of intercellular contact to withstand the hydrodynamic stresses. Using cone and plate viscometry to apply a uniform linear shear field to suspensions of neutrophils and flow cytometry to quantitate the size distribution of aggregates formed over the time course of formyl peptide stimulation, we conducted a detailed examination of the affect of shear rate and shear stress on the kinetics of cell aggregation. The efficiency of aggregate formation was fit from a mathematical model based on Smoluchowski's two-body collision theory. Over a range of venular shear rates (400-800 s-1), approximately 90% of the single cells are recruited into aggregates ranging from doublets to grouping larger than sextuplets. Adhesion efficiency fit to the kinetics of aggregation increased with shear rate from approximately 20% at 100 s-1 to a maximum level of approximately 80% at 400 s-1. This increase to peak adhesion efficiency was dependent on L-selectin and beta 2-integrin, and was resistant to shear stress up to approximately 7 dyn/cm2. When L-selectin was blocked with antibody, beta 2-integrin (CD11a,b) supported adhesion at low shear rates (< 400 s-1). Aggregates formed over the rapid phase of aggregation remain intact and resistant to shear up to 120 s. At the end of this plateau phase of stability, aggregates spontaneously dissociate back to singlets. The rate of cell disaggregation is linearly proportional to the applied shear rate. The binding kinetics of selectin and integrin appear to be optimized to function within discrete ranges of shear rate and stress, providing an intrinsic mechanism for the transition from neutrophil tethering to firm but reversible adhesion.     

An automatic braking system that stabilizes leukocyte rolling by an increase in selectin bond number with shear

Wall shear stress in postcapillary venules varies widely within and between tissues and in response to inflammation and exercise. However, the speed at which leukocytes roll in vivo has been shown to be almost constant within a wide range of wall shear stress, i.e., force on the cell. Similarly, rolling velocities on purified selectins and their ligands in vitro tend to plateau. This may be important to enable rolling leukocytes to be exposed uniformly to activating stimuli on endothelium, independent of local hemodynamic conditions. Wall shear stress increases the rate of dissociation of individual selectin-ligand tether bonds exponentially (, ) thereby destabilizing rolling. We find that this is compensated by a shear-dependent increase in the number of bonds per rolling step. We also find an increase in the number of microvillous tethers to the substrate. This explains (a) the lack of firm adhesion through selectins at low shear stress or high ligand density, and (b) the stability of rolling on selectins to wide variation in wall shear stress and ligand density, in contrast to rolling on antibodies (). Furthermore, our data successfully predict the threshold wall shear stress below which rolling does not occur. This is a special case of the more general regulation by shear of the number of bonds, in which the number of bonds falls below one.     

L-selectin from human, but not from mouse neutrophils binds directly to E-selectin

L-Selectin on neutrophils as well as inducible E- and P-selectin on endothelium are involved in the recruitment of neutrophils into inflamed tissue. Based on cell attachment assays, L-selectin was suggested to function as a carbohydrate presenting ligand for E- and P-selectin. However, previous affinity isolation experiments with an E-selectin-Ig fusion protein had failed to detect L-selectin among the isolated E-selectin ligands from mouse neutrophils. We show here that L-selectin from human neutrophils, in contrast to mouse neutrophils, can be affinity-isolated as a major ligand from total cell extracts using E-selectin-Ig as affinity probe. Binding of human L-selectin to E-selectin was direct, since purified L-selectin could be reprecipitated with E-selectin-Ig. Recognition of L-selectin was abolished by sialidase-treatment, required Ca2+, and was resistant to treatment with endoglycosidase F. Binding of L-selectin to a P-selectin-Ig fusion protein was not observed. In agreement with the biochemical data, the anti-L-selectin mAb DREG56 inhibited rolling of human neutrophils on immobilized E-selectin-Ig but not on P-selectin-Ig. No such inhibitory effect was seen with the anti-mouse L-selectin mAb MEL14 on mouse neutrophils. Rolling of E-selectin transfectants on purified and immobilized human L-selectin was inhibited by mAb DREG56. We conclude that L-selectin on human neutrophils is a major glycoprotein ligand among very few glycoproteins that can be isolated by an E-selectin affinity matrix. The clear difference between human and mouse L-selectin suggests that E-selectin-binding carbohydrate moieties are attached to different protein scaffolds in different species.     

Kinetics of beta2-integrin and L-selectin bonding during neutrophil aggregation in shear flow

Activated neutrophils aggregate in a shear field via bonding of L-selectin to P-selectin glycoprotein ligand-1 (PSGL-1) followed by a more stable bonding of LFA-1 (CD11a/CD18) to intercellular adhesion molecule 3 (ICAM-3) and Mac-1 (CD11b/CD18) to an unknown counter receptor. Assuming that the Mac-1 counter receptor is ICAM-3-like in strength and number, rate processes were deconvoluted from neutrophil homoaggregation data for shear rates (G) of 100-3000 s-1 with a two-body hydrodynamic collision model (. Biophys. J. 73:2819-2835). For integrin-mediated aggregation (characteristic bond strength of 5 microdynes) in the absence of L-selectin contributions, an average forward rate of kf = 1.57 x 10(-12) cm2/s predicted the measured efficiencies for G = 100-800 s-1. For a selectin bond formation rate constant equal to the integrin bond formation rate constant, the colloidal stability of unactivated neutrophils was satisfied for a reverse rate of the L-selectin-PGSL bond corresponding to an average bond half-life of 10 ms at a characteristic bond strength of 1 microdyne. Colliding neutrophils initially bridged by at least one L-selectin-PSGL-1 bond were calculated to rotate from 8 to 50 times at G = 400 to 3000 s-1, respectively, before obtaining mechanical stability in sheared fluid of either 0.75 or 1.75 cP viscosity. Thus for G > 400 s-1, the interaction time needed for the rotating aggregates to become stable was relatively constant at 52.5 +/- 8.5 ms, largely independent of shear rate or shear stress. Aggregation data and the colloidal stability criterion can provide a consistent set of forward and reverse rate constants and characteristic bond strengths for a known time-dependent stoichiometry of receptors on cells interacting in a shear flow field.     

Selective requirements for leukocyte adhesion molecules in models of acute and chronic cutaneous inflammation: participation of E- and P- but not L-selectin

Adhesion molecules borne by both endothelial cells and circulating leukocytes are in large measure responsible for guiding the process of extravasation. The selectin family has been primarily associated with the early stages of adhesion involving initial contact and rolling. A significant body of evidence has accumulated indicating a fundamental role for the endothelial members of this family, E- and P-selectin, in a variety of inflammatory states and models. Although originally identified as the lymph node-specific lymphocyte homing receptor, L-selectin has also been suggested to play an important role in leukocyte recruitment to sites of inflammation. We have recently demonstrated, using L-selectin-deficient mice, that defects in contact hypersensitivity (CHS) responses are in essence due to the inability of T cells to home to and be sensitized within peripheral lymph nodes, whereas nonspecific effector cells are fully capable of entry into sites of cutaneous inflammation (Catalina et al, J Exp Med 184:2341, 1996). In the present study, we perform an analysis of adhesion molecule usage in two models of skin inflammation and show in both L-selectin-deficient as well as wild-type mice that a combination of P- and E-selectin is crucial for the development of both acute (croton oil) and chronic (contact hypersensitivity) inflammation at sites of the skin, whereas L-selectin does not appear to play a significant role. Moreover, alpha4 integrins are shown to be integral to a CHS but not an acute irritant response, whereas CD44 does not significantly contribute to either. These results provide a systematic examination in one study of major adhesion molecules that are critical in acute and chronic skin inflammation. They reinforce the essential role of the collaboration of E- and P-selectin in both specific and nonspecific skin inflammatory responses and the importance of alpha4 in the specific response only. In addition, they substantiate only a limited role, if any, for L-selectin in these cutaneous effector mechanisms and demonstrate the essential equivalence in this analysis of L-selectin-deficient mice compared with normal mice treated with blocking antibodies.     

Polymorphisms and linkage analysis for ICAM-1 and the selectin gene cluster

Genetic polymorphisms in leukocyte and endothelial cell adhesion molecules may be important variables with regard to susceptibility to multifactorial disease processes that include an inflammatory component. For this reason, polymorphisms were sought for intercellular adhesion molecule-1 (ICAM-1; gene symbol ICAM1) and for the three genes in the selectin cluster, P-selectin, L-selectin, and E-selectin (gene symbols SELP, SELL, and SELE, respectively). Two amino acid polymorphisms were identified for ICAM-1; Gly or Arg at codon 241 and Lys or Glu at codon 469. Dinucleotide repeat polymorphisms were identified in the 3'-untranslated region for ICAM-1 and in intron 9 for P-selectin. Restriction fragment length polymorphisms were found using cDNAs for each of the three selectin genes as probes; E-selectin with BglII, P-selectin with ScaI, and L-selectin with HincII. Linkage analysis was performed for the selectin gene cluster and for ICAM-1 using the CEPH families; ICAM-1 is very tightly linked to the LDL receptor on chromosome 19, and the selectin cluster is linked to markers at chromosome 1q23.