The velocity of rolling leukocytes is thought to be determined by the expression of adhesion molecules and the prevailing wall shear stress. Here, we investigate whether rapid cleavage of L-selectin may be an additional physiologic regulatory parameter of leukocyte rolling. A unique protease in the membrane of leukocytes cleaves L-selectin after activation, resulting in L-selectin shedding. The hydroxamic acid-based metalloprotease inhibitor KD-IX-73-4 completely prevented L-selectin shedding in vitro and significantly decreased the rolling velocity of leukocytes in untreated wild-type C57BL/6 mice from 55 to 35 micrometer/seconds in vivo. When E-selectin was expressed on the endothelium (tumor necrosis factor [TNF]-alpha treatment 2.5-3 h before the experiment), rolling velocity was 4 micrometer/seconds and did not change after the application of KD-IX-73-4. However, KD-IX-73-4 decreased mean rolling velocity by 29% from 23 to 16 micrometer/seconds in E-selectin-deficient mice treated with TNF-alpha. The reduction of velocity caused by KD-IX-73-4 was immediate (<5 s) after injection of KD-IX-73-4 as shown by a novel method using a local catheter. These results establish a role for L-selectin shedding in regulating leukocyte rolling velocity in vivo.

Leukocyte endothelial cell interaction is a fundamentally important process in many disease states. Current methods to analyze such interactions include the parallel-plate flow chamber and intravital microscopy. Here, we present an improvement of the traditional intravital microscopy that allows leukocyte-endothelial cell interaction to be studied from the time the leukocyte makes its initial contact with the endothelium until it adheres to or detaches from the endothelium. The leukocyte is tracked throughout the venular tree with the aid of a motorized stage and the rolling and adhesive behavior is measured off-line. Because this method can involve human error, methods to automate the tracking procedure have been developed. This novel tracking method allows for a more detailed examination of leukocyte-endothelial cell interactions.

Nicotine and several related metabolites were examined for their ability to induce leukocyte rolling and adhesion in the cerebral microcirculation of the mouse. A cranial window was surgically prepared for the visualization of the pial microcirculation using an intra-vital microscopy imaging system. Using this technique rhodamine-labeled leukocytes could be visualized and video-recorded as they traveled within the microvessels, and the quantitation of their rolling and adhesion along the pial venule walls was assessed during an off-line video playback analysis. Nicotine was found to produce significant dose-related increases in leukocyte rolling and adhesion. Cotinine, a major nicotine metabolite, did not induce the same degree of leukocyte rolling and adhesion. Mecamylamine, a nicotine antagonist, was found to inhibit the nicotine-induced leukocyte rolling and adhesion. Anti-P-selectin antibody blocked nicotine-induced leukocyte rolling, while anti-CD18 antibody effectively inhibited leukocyte adhesion, but not rolling in similar experiments. Nicotine-induced leukocyte rolling and adhesion were also inhibited by superoxide dismutase and catalase. These data suggest that nicotine, the principle pharmacological agent in cigarette smoke and related tobacco products, acts via a ganglionic-type nicotinic receptor to enhance leukocyte rolling via P-selectin and reactive oxygen radical-dependent mechanisms in cerebral microcirculation of the mouse.

Nitric oxide (NO) is a multifunctional signaling molecule that regulates important cellular events in inflammation including leukocyte recruitment. Previous studies have shown that pharmacological inhibition of NO synthesis induces leukocyte recruitment in various in vitro and animal models. However, it is not known whether NO modulation has similar effects on leukocyte-endothelial cell interactions within the human microvasculature. The present study explored the effect of systemic L-NAME treatment on leukocyte recruitment in the SCID-hu mouse model.

Sphingosine-1-phosphate (S1P) participates in inflammation; however, its role in leukocyte rolling is still unclear. Here we use intravital microscopy in inflamed mouse cremaster muscle venules and human endothelial cells to show that S1P contributes to P-selectin-dependent leukocyte rolling through endothelial S1P receptor 3 (S1P3) and Gαq, PLCβ and Ca(2+). Intra-arterial S1P administration increases leukocyte rolling, while S1P3 deficiency or inhibition dramatically reduces it. Mast cells involved in triggering rolling also release S1P that mobilizes P-selectin through S1P3. Histamine and epinephrine require S1P3 for full-scale effect accomplishing it by stimulating sphingosine kinase 1 (Sphk1). In a counter-regulatory manner, S1P1 inhibits cAMP-stimulated Sphk1 and blocks rolling as observed in endothelial-specific S1P1(-/-) mice. In agreement with a dominant pro-rolling effect of S1P3, FTY720 inhibits rolling in control and S1P1(-/-) but not in S1P3(-/-) mice. Our findings identify S1P as a direct and indirect contributor to leukocyte rolling and characterize the receptors mediating its action.

1. The main objective of this study was to analyse the role and mode of action of the mast cell mediator histamine in leukocyte-endothelium interactions in small venules in vivo. For this purpose, we used a histological approach (combined with intravital microscopy) that allows studies of rapid mediator-induced venular leukocyte accumulation, reflecting leukocyte rolling, in the undisturbed microcirculation of the rat mesentery where rolling is normally absent. 2. We first examined the relative importance of histamine and 5-hydroxytryptamine (5-HT) in acute mast cell-dependent leukocyte recruitment. The mast cell secretagogue compound 48/80 (i.p. for 15 min) induced a marked venular accumulation of polymorphonuclear leukocytes (PMNL) which was almost abolished by combined histamine1 (H1)- and histamine2 (H2)-receptor blockade. In contrast, the 5-HT-receptor antagonist methysergide was inactive in this regard. Moreover, exogenous 5-HT was less active than exogenous histamine in evoking venular PMNL accumulation (histamine response dose-dependent; 5-HT response bell shaped). Prostaglandin D2 did not cause PMNL accumulation. 3. The venular PMNL response to exogenous histamine peaked between 15 min and 1 h, was still significantly elevated at 2 h, and then returned to prechallenge values after 3 h. At all time points, the histamine-induced PMNL accumulation was nearly abolished by i.v. treatment with the polysaccharide fucoidin (which blocks rolling but not firm adhesion per se), suggesting that the PMNL response to histamine was due to rolling rather than firm adhesion over the entire 3 h period. At no time point did histamine trigger accumulation of mononuclear leukocytes (MNL). 4. To examine the role of histamine-receptors in the histamine-induced PMNL accumulation (i.e. rolling), the animals were pretreated with diphenhydramine (H1-receptor antagonist), cimetidine, or ranitidine (H2-receptor antagonists). Diphenhydramine alone inhibited the venular PMNL response to histamine by 52%, while both H2-receptor antagonists were completely inactive. However, the combination of cimetidine and diphenhydramine reduced the histamine-induced PMNL rolling by 82%. Furthermore, in contrast to an H3-receptor agonist, challenge with either the H1-receptor agonist 2-thiazolylethylamine or two different H2-receptor agonists (impromidine, dimaprit) was sufficient to provoke significant venular PMNL accumulation. 5. Treatment with the nitric oxide-synthase inhibitor L-NAME did not affect the histamine-induced PMNL rolling. On the other hand, 3 h pretreatment with dexamethasone reduced the PMNL response to histamine by 73%, and flow cytometric analysis showed that the dexamethasone treatment almost completely inhibited binding of soluble P-selectin to rat isolated PMNLs. 6. We conclude that initial leukocyte recruitment after mast cell activation in the rat mesentery is critically dependent on histamine release. The cellular response to histamine was specifically due to PMNL rolling, involved activation of both H1- and H2-receptors, and lasted for 2 3 h. Moreover, the histamine-induced PMNL rolling was not dependent on nitric oxide synthesis, but was sensitive to glucocorticoid treatment, possibly via inhibition of expression or function of leukocytic P-selectin ligand(s).

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.

Acute loss of renal function reduces leukocyte recruitment into inflamed tissues, and we studied the molecular basis of this using intravital microscopy of cremaster muscle and an autoperfused flow chamber system after bilateral nephrectomy or sham operation in mice. Acute loss of renal function resulted in cessation of selectin-induced slow leukocyte rolling on E-selectin/intercellular adhesion molecule 1 (ICAM-1) and P-selectin/ICAM-1. It also reduced in vivo neutrophil extravasation (assessed by reflected light oblique transillumination) without affecting chemokine-induced arrest. This elimination of selectin-mediated slow leukocyte rolling was associated with a reduced phosphorylation of spleen tyrosine kinase, Akt, phospholipase C-γ2, and p38 MAPK. However, the levels of adhesion molecules located on the neutrophil surface were not altered. Leukocytes from critically ill patients with sepsis-induced acute kidney injury showed a significantly higher rolling velocity on E-selectin/ICAM-1- and P-selectin/ICAM-1-coated surfaces compared with patients with sepsis alone or healthy volunteers. Thus, an acute loss of renal function significantly impairs neutrophil rolling and transmigration, both in vivo and in vitro. These effects are due, in part, to decreased phosphorylation of selectin-dependent intracellular signaling pathways.

1. Although intravital microscopy is the method of choice for observation of inflammatory leukocyte rolling and adhesion in small venules in vivo, a problem with this technique is that surgical exposure of suitable tissues per se triggers the rolling mechanism. In this study, we describe an approach to investigate induction of rolling in undisturbed microvessels. For this purpose, intravital microscopic observation of leukocyte rolling and adhesion in the rat mesentery was combined with histological determination of the intravascular concentrations of polymorphonuclear and mononuclear leukocytes (PMNL and MNL). 2. By relating the histologically determined number of intravascular leukocytes to either microvessel volume or to the erythrocyte concentration, the baseline MNL and PMNL content was found to be 3-6 fold higher in venules than in systemic blood. This increase in microvessel leukocyte concentration did not seem to be related to leukocyte-endothelium interactions, because the leukocyte concentration was similarly elevated in arterioles where rolling and adhesion did not take place. 3. Preparation of the rat mesentery for intravital microscopy time-dependently increased the venular PMNL concentration to over 100 fold the systemic PMNL concentration 45 min after exteriorization of the small intestine. The MNLs were much less responsive to the preparative manipulation. By treatment with the polysaccharide fucoidin (inhibits rolling but not firm adhesion per se), or by use of intravital microscopy immediately before tissue fixation, approximately 90% of the accumulated venular PMNLs were found to represent rolling cells. 4. Intraperitoneal injection of 10(-3) M histamine increased the venular PMNL (but not the MNL) concentration to almost 50 fold the systemic PMNL value. The histamine response did not vary with venular diameter, and the relative contribution of rolling vs firmly adherent cells to the PMNL, accumulation was again approximately 90%. Intraperitoneal injection of leukotriene C4, but not prostaglandin E2, caused a significant increase in venular PMNL concentration. 5. Systemic treatment with the anti-P-selectin monoclonal antibody PB1.3 had no effect on the histamine-induced venular PMNL accumulation (i.e. rolling) in female Wistar or male Sprague-Dawley rats. On the other hand, identical treatment with PB1.3 very effectively inhibited the histamine-induced PMNL response in the mesentery of rabbits. 6. In conclusion, we have shown that a histologically determined increase in leukocyte concentration in rat mesenteric venules may be used as an index of mediator-induced leukocyte rolling if the relative contribution of rolling and firm leukocyte adhesion is first determined, for example by the means described in this study. This relatively simple approach may be very useful for studying various aspects of leukocyte rolling when the 'spontaneous' rolling triggered by preparation of tissues for intravital microscopy is undesirable.

Flowing leukocytes roll on P-selectin that is mobilized from secretory granules to the surfaces of endothelial cells after stimulation with histamine or thrombin. Before it is internalized, P-selectin clusters in clathrin-coated pits, which enhances its ability to support leukocyte rolling. We found that thrombin and histamine induced comparable exocytosis of P-selectin on endothelial cells. However, compared with histamine, thrombin decreased the recruitment of P-selectin into clathrin-coated pits, slowed the internalization of P-selectin, and reduced the number and stability of neutrophils rolling on P-selectin. Significantly more RhoA was activated in thrombin- than in histamine-stimulated endothelial cells. Inhibitors of RhoA or its effector, Rho kinase, reversed thrombin's ability to inhibit the internalization and adhesive function of P-selectin in endothelial cells. Experiments with transfected cells confirmed that the inhibitory actions of thrombin and Rho kinase on P-selectin required its cytoplasmic domain. Thus, a signaling event affects both the function and clearance of a protein that enters the constitutive clathrin-mediated endocytic pathway.

We recently demonstrated that tyrosine sulfation is an important contributor to monocyte recruitment and retention in a mouse model of atherosclerosis. P-selectin glycoprotein ligand-1 (Psgl-1) is tyrosine-sulfated in mouse monocyte/macrophages and its interaction with P-selectin is important in monocyte recruitment in atherosclerosis. However, whether tyrosine sulfation is required for the P-selectin binding function of mouse Psgl-1 is unknown. Here we test the function of native Psgl-1 expressed in leukocytes lacking endogenous tyrosylprotein sulfotransferase (TPST) activity.

1. Anti-inflammatory actions of heparin and related glycosaminoglycans have been described in the literature. Here, we used intravital microscopy of the rat mesentery microcirculation to examine effects of locally administered heparin on leukocyte rolling and chemoattractant-induced firm adhesion. 2. It was found that topical application of heparin reduced N-formyl-methionyl-leucyl-phenylalanine (fMLP)-induced leukocyte adhesion. Notably, the inhibitory action of heparin was not dose-dependent, but rather a biphasic dose-response was found, i.e. low (2 and 20 iu ml(-1)) and high (1000 iu ml(-1)) concentrations of heparin significantly reduced adhesion, whereas an intermediate dose (200 iu ml(-1)) was less effective. 3. Heparin, 2 and 20 iu ml(-1), decreased rolling leukocyte flux, while having no effect on blood flow or total leukocyte flux. By contrast, heparin, 200 and 1000 iu ml(-1), increased total leukocyte flux in parallel with a rise in volume blood flow resulting in recovery of the rolling leukocyte flux at these doses. Thus, the biphasic inhibitory action of heparin on fMLP-induced firm adhesion could in part be attributed to changes in leukocyte delivery (i.e. blood flow) and rolling leukocyte flux induced by heparin. 4. When compensating for the influence of different rolling levels on fMLP-evoked adhesion, a dose-related inhibitory effect of heparin on the firm adhesive response per se was revealed. Similar results were obtained in a static adhesion assay in vitro where heparin 200 and 1000 iu ml(-1) (but not 2 and 20 iu ml(-1)) significantly inhibited fMLP-induced leukocyte adhesion in the absence of any modulatory influence on changes in rolling. 5. Our data show that locally administered heparin inhibits leukocyte rolling as well as chemoattractant-induced firm adhesion in vivo which thus may contribute to the postulated anti-inflammatory activity of this compound. However, because of interference with several microvascular functions, strict dose-dependent responses to heparin treatment were not found, which illustrates the complex interplay between local blood flow, leukocyte rolling and chemoattractant-induced adhesion as determinants of leukocyte recruitment to tissues in inflammation.

Bovine gamma/delta T cells and neutrophils roll on 24 h cytokine- or lipopolysaccharide-stimulated bovine fetal umbilical cord endothelial cells in assays done under physiological flow. An antibody directed against E- and L-selectin has minimal blocking effect on this rolling interaction. mAbs were raised against the stimulated bovine endothelial cells and screened for inhibition of gamma/delta T cell rolling. One mAb (GR113) was identified that recognizes an antigen (GR antigen) selectively expressed by stimulated bovine endothelial cells isolated from fetal umbilical cord, mesenteric lymph nodes, and aorta. GR113 blocked bovine gamma/delta T cell as well as neutrophil rolling on the 24 h-activated endothelial cells. The GR antigen was constitutively expressed at low levels on the cell surface of platelets and its expression was not upregulated after stimulation of these cells with thrombin or phorbol myristate acetate. However, stimulated platelets released a soluble, functionally active form of the molecule that selectively bound in solution to gamma/delta T cells in a mixed lymphocyte preparation. GR113 mAb blocked the binding of the soluble platelet molecule to the gamma/delta T cells. Soluble GR antigen also bound a subset of human lymphocytes. Cutaneous lymphocyte-associated antigen (CLA) bright human lymphocytes exhibited the greatest capacity to bind the GR antigen, though CLA was not required for binding. Subsets of both human CD4 and CD8 T cells bound the GR antigen. Immunoprecipitation experiments showed the GR antigen to be 110-120 kD Mr. The binding of soluble GR antigen was inhibited by EDTA and O-sialoglycoprotease, but not neuraminidase treatment of the target cells.

P-selectin glycoprotein ligand-1 (PSGL-1) binding to P-selectin controls early leukocyte rolling during inflammation. Interestingly, antibodies and pharmacological inhibitors (eg, rPSGL-Ig) that target the N-terminus of PSGL-1 reduce but do not abolish P-selectin-dependent leukocyte rolling in vivo whereas PSGL-1-deficient mice have almost no P-selectin-dependent rolling. We have investigated mechanisms of P-selectin-dependent, PSGL-1-independent rolling using intravital microscopy. Initially we used fluorescent microspheres to study the potential of L-selectin and the minimal selectin ligand sialyl Lewis(x) (sLe(x)) to interact with postcapillary venules in the absence of PSGL-1. Microspheres coated with combinations of L-selectin and sLe(x) interacted with surgically stimulated cremaster venules in a P-selectin-dependent manner. Microspheres coated with either L-selectin or sLe(x) alone showed less evidence of interaction. We also investigated leukocyte rolling in the presence of PSGL-1 antibody or inhibitor (rPSGL-Ig), both of which partially inhibited P-selectin-dependent leukocyte rolling. Residual rolling was substantially inhibited by L-selectin-blocking antibody or a previously described sLe(x) mimetic (CGP69669A). Together these data suggest that leukocytes can continue to roll in the absence of optimal P-selectin/PSGL-1 interaction using an alternative mechanism that involves P-selectin-, L-selectin-, and sLe(x)-bearing ligands.

Leukocyte recruitment to target tissue is initiated by weak rolling attachments to vessel wall ligands followed by firm integrin-dependent arrest triggered by endothelial chemokines. We show here that immobilized chemokines can augment not only arrest but also earlier integrin-mediated capture (tethering) of lymphocytes on inflamed endothelium. Furthermore, when presented in juxtaposition to vascular cell adhesion molecule 1 (VCAM-1), the endothelial ligand for the integrin very late antigen 4 (VLA-4, alpha4beta1), chemokines rapidly augment reversible lymphocyte tethering and rolling adhesions on VCAM-1. Chemokines potentiate VLA-4 tethering within <0.1 s of contact through Gi protein signaling, the fastest inside-out integrin signaling events reported to date. Although VLA-4 affinity is not altered upon chemokine signaling, subsecond VLA-4 clustering at the leukocyte-substrate contact zone results in enhanced leukocyte avidity to VCAM-1. Endothelial chemokines thus regulate all steps in adhesive cascades that control leukocyte recruitment at specific vascular beds.

E-selectin binding to P-selectin glycoprotein ligand-1 (PSGL-1) can activate the beta(2) integrin lymphocyte function-associated antigen-1 by signaling through spleen tyrosine kinase (Syk). This signaling is independent of G alpha(i)-protein-coupled receptors, results in slow rolling, and promotes neutrophil recruitment to sites of inflammation. However, the signaling pathways linking E-selectin engagement of PSGL-1 to Syk activation are unknown. To test the role of Src family kinases and immunoreceptor tyrosine-based activating motif (ITAM)-containing adaptor proteins, we used different gene-deficient mice in flow chamber, intravital microscopy, and peritonitis studies. E-selectin-mediated phosphorylation of Syk and slow rolling was abolished in neutrophils from fgr(-/-) or hck(-/-) lyn(-/-) fgr(-/-) mice. Neutrophils from Tyrobp(-/-) Fcrg(-/-) mice lacking both DAP12 and FcRgamma were incapable of sustaining slow neutrophil rolling on E-selectin and intercellular adhesion molecule-1 and were unable to phosphorylate Syk and p38 MAPK. This defect was confirmed in vivo by using mixed chimeric mice. G alpha(i)-independent neutrophil recruitment into the inflamed peritoneal cavity was sharply suppressed in Tyrobp(-/-) Fcrg(-/-) mice. Our data demonstrate that an ITAM-dependent pathway involving the Src-family kinase Fgr and the ITAM-containing adaptor proteins DAP12 and FcRgamma is involved in the initial signaling events downstream of PSGL-1 that are required to initiate neutrophil slow rolling.

Leukocyte rolling adhesion, facilitated by selectin-mediated interactions, is a highly dynamic process in which cells roll along the endothelial surface of blood vessel walls to reach the site of infection. The most common approach to investigate cell-substrate adhesion is to analyze the cell rolling velocity in response to shear stress changes. It is assumed that changes in rolling velocity indicate changes in adhesion strength. In general, cell rolling velocity is studied at the population level as an average velocity corresponding to given shear stress. However, no statistical investigation has been performed on the instantaneous velocity distribution. In this study, we first developed a method to remove systematic noise and revealed the true velocity distribution to exhibit a log-normal profile. We then demonstrated that the log-normal distribution describes the instantaneous velocity at both the population and single-cell levels across the physiological flow rates. The log-normal parameters capture the cell motion more accurately than the mean and median velocities, which are prone to systematic error. Lastly, we connected the velocity distribution to the molecular adhesion force distribution and showed that the slip-bond regime of the catch-slip behavior of the P-selectin/PSGL-1 interaction is responsible for the variation of cell velocity.

Selectin-ligand interactions mediate tethering and rolling of circulating leukocytes on the vessel wall during inflammation. Extensive study has been devoted to elucidating the kinetic and mechanical constraints of receptor-ligand-interaction-mediated leukocyte adhesion, yet many questions remain unanswered. Here, we describe our design of an inverted flow chamber to compare adhesions of HL-60 cells to E-selectin in the upright and inverted orientations. This new, to our knowledge, design allowed us to evaluate the effect of gravity and to investigate the mechanisms of flow-enhanced adhesion. Cell rolling in the two orientations was qualitatively similar, and the quantitative differences can be explained by the effect of gravity, which promotes free-flowing cells to tether and detached cells to reattach to the surface in the upright orientation but prevents such attachment from happening in the inverted orientation. We characterized rolling stability by the lifetime of rolling adhesion and detachment of rolling cells, which could be easily measured in the inverted orientation, but not in the upright orientation because of the reattachment of transiently detached cells. Unlike the transient tether lifetime of E-selectin-ligand interaction, which exhibited triphasic slip-catch-slip bonds, the lifetime of rolling adhesion displayed a biphasic trend that first increased with the wall shear stress, reached a maximum at 0.4 dyn/cm(2), and then decreased gradually. We have developed a minimal mathematical model for the probability of rolling adhesion. Comparison of the theoretical predictions to data has provided model validation and allowed evaluation of the effective two-dimensional association on-rate, kon, and the binding affinity, Ka, of the E-selectin-ligand interaction. kon increased with the wall shear stress from 0.1 to 0.7 dyn/cm(2). Ka first increased with the wall shear stress, reached a maximum at 0.4 dyn/cm(2), and then decreased gradually. Our results provide insights into how the interplay between flow-dependent on-rate and off-rate of E-selectin-ligand bonds determine flow-enhanced cell rolling stability.

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.

The use of in vivo videomicroscopy at the bedside has demonstrated microcirculatory flow disturbances in sepsis. The ability of in vivo videomicroscopy to detect changes in the prevalence of rolling and adhered leukocytes that occur in sepsis is not well-described in humans. We sought to (1) develop methodology for accessing and quantifying sublingual leukocyte rolling and adherence with sidestream dark field (SDF) imaging; (2) compare the number of rolling and adhered leukocytes between patients with septic shock and non-infected controls; and (3) compare the number of rolling and adhered leukocytes between survivors and non-survivors of septic shock.