The L-selectin adhesion system

L-selectin is a cell surface glycoprotein expressed on most leukocyte subsets that mediates leukocyte interaction with ligands on lymphoid tissue high endothelial venule cells as well as with ligands on activated endothelium at sites of inflammation in nonlymphoid organs. Similar to two other members of the selectin family, L-selectin behaves as a lectin, recognizing carbohydrate ligands in a calcium-dependent fashion. Recent in vivo studies reveal the importance of L-selectin expression in both normal and pathologic conditions.     

The changes of Mac-1 and L-selectin expression on granulocytes and soluble L-selectin level during hemodialysis

L-selectin and Mac-1 expressed on leukocytes are critical for leukocyte adhesion to inflamed endothelium. L-selectin is known to be rapidly shed from the cell surface of granulocytes after activation. In the present study the change of expressions of these adhesion molecules on granulocytes were analyzed by flow cytometry, and the serum concentration of shed L-selectin (soluble L-selectin; sL-selectin) was measured by enzyme-linked immunosorbent assay (ELISA) during hemodialysis in patients treated with regenerated cellulose membranes (RC group) versus polysulfone membranes (PS group). In the RC group, Mac-1 expression on granulocytes increased significantly at 30 min after the initiation of hemodialysis (p < 0.05) compared with predialysis values, coinciding with the nadir of dialysis-induced granulocytopenia. Granulocyte L-selectin expression decreased significantly at 15 min after the initiation of hemodialysis (p < 0.05) and remained decreased through the course of dialysis session, compared with predialysis values. Serum sL-selectin level significantly increased at 15 min after the initiation of hemodialysis (p < 0.05), compared with predialysis values. In the PS group, no significant variation in Mac-1 and L-selectin expression on granulocytes and serum sL-selectin level were detected. This reciprocal change of Mac-1 and L-selectin on granulocyte cell surface was attributed to development of granulocytopenia and subsequent reversal during dialysis with cellulose membranes. In this study, we confirmed the shedding of L-selectin during cellulosic dialysis by ELISA. The increase in sL-selectin, which has potential activity of inhibiting L-selectin-dependent adhesion of granulocyte to endothelium, might be involved in rebound granulocytosis during dialysis with cellulose membranes and impairment of the granulocyte function in patients on chronic hemodialysis.     

Effects of trauma and sepsis on soluble L-selectin and cell surface expression of L-selectin and CD11b

OBJECTIVES: To examine (1) the effects of trauma on changes in neutrophil L-selectin and CD11b expression and on the levels of soluble L-selectin and (2) whether these alterations are different on leukocyte subpopulations in those patients who develop multiple organ dysfunction syndrome. MATERIALS AND METHODS: Twenty patients with Injury Severity Score (ISS) > or = 16 and 15 patients with ISS score < 16 were studied. Arterial blood were collected serially after injury. The staining of leukocyte surface adhesion molecules was performed with antibodies against L-selectin and CD11b. Positive cell count and mean fluorescence intensity were determined by flow cytometry. Soluble L-selectin was measured using enzyme-linked immunosorbent assay. RESULTS: In patients with ISS > or = 16, neutrophil L-selectin expression showed an immediate increase, reaching peak levels between 3 to 4 hours after injury (p < 0.05 vs. patients with ISS < 16), followed by a gradual decrease. Plasma levels of soluble L-selectin reached peak levels at 6 hours after injury. However, in patients with ISS < 16, minimal changes in L-selectin expression and soluble L-selectin were observed. Neutrophil CD11b expression showed an immediate increase for the first 3 hours followed by a gradual increase up to 24 hours after injury. In patients who developed multiple organ dysfunction syndrome, CD11b both on neutrophils and lymphocytes remained elevated for 120 hours. CONCLUSIONS: These findings suggest that acute neutrophil activation is an early event after trauma and may be implicated as "a vulnerable window" for leukocyte-mediated end organ injury.     

Nicotinic receptor-induced apoptotic cell death of hippocampal progenitor cells

Nicotine has many effects on CNS functions, presumably through its action on neuronal nicotinic acetylcholine receptors (AChRs). One subclass of AChRs that binds the snake venom toxin alpha-bungarotoxin (alpha-Bgt-AChRs) has been shown to modulate neurotransmission in the brain. We now show that alpha-Bgt-AChR activation by low doses of nicotine results in apoptotic cell death of both primary and immortalized hippocampal progenitor cells. In HC2S2-immortalized hippocampal progenitors, nicotine is cytotoxic to undifferentiated cells, whereas it spares the same cells once differentiation has been induced. The activation of alpha-Bgt-AChRs by nicotine results in the induction of the tumor suppressor protein p53 and the cdk inhibitor p21. The cytotoxic effect of nicotine is dependent on extracellular calcium levels and is probably attributable to the poor ability of undifferentiated progenitors to buffer calcium loads. The major calcium buffer in these cells, calbindin D28K, is present only after differentiation has been induced. Furthermore transfection of undifferentiated cells with calbindin results in dramatic protection against the cytotoxic effects of nicotine. These results show that nicotine abuse could have significant effects on the survival of progenitor populations in the developing and adult brain and also suggest an endogenous role for alpha-Bgt-AChRs in neuronal development and differentiation.     

Expression of an L-selectin ligand on hematopoietic progenitor cells

The process of hematopoiesis is dependent on discrete cell-cell and cell-matrix interactions which are tightly regulated by expression of adhesion molecules. L-selectin, an adhesion protein best known for regulating leukocyte attachment to endothelium, is characteristically expressed on the earliest hematopoietic progenitor cells. Ligands for L-selectin have been extensively characterized on endothelial cells. We recently identified a ligand for L-selectin expressed on the human hematopoietic progenitor cell line KG1a. This molecule is an integral membrane glycoprotein which is structurally different from all ligands previously described. We hypothesize that this molecule may mediate L-selectin-specific adhesive interactions during hematopoiesis. This article discusses the biology of L-selectin and its ligands, and reviews our current understanding of the structure and distribution of the L-selectin ligand expressed on hematopoietic cells.     

Nitric oxide mediates N-methyl-D-aspartate receptor-induced activation of p21ras

N-methyl-D-aspartate (NMDA) glutamate receptor-mediated increases in intracellular calcium are thought to play a critical role in synaptic plasticity. The mechanisms by which changes in cytoplasmic calcium transmit the glutamate signal to the nucleus, which is ultimately important for long-lasting neuronal responses, are poorly understood. We show that NMDA receptor stimulation leads to activation of p21(ras) (Ras) through generation of nitric oxide (NO) via neuronal NO synthase. The competitive NO synthase inhibitor, L-nitroarginine methyl ester, prevents Ras activation elicited by NMDA and this effect is competitively reversed by the NO synthase substrate, L-arginine. NMDA receptor stimulation fails to activate Ras in neuronal cultures from mice lacking neuronal NO synthase. NMDA-induced Ras activation occurs through a cGMP-independent pathway as 1H-[1,2,4]oxadiazolo[4,3-alpha]quinoxalin-1-one (ODQ), a potent and selective inhibitor of guanylyl cyclase, has no effect on NMDA receptor-induced activation of Ras, and the cell-permeable cGMP analog, 8Br-cGMP, does not activate Ras. Furthermore, NO directly activates immunoprecipitated Ras from neurons. NMDA also elicits tyrosine phosphorylation of extracellular signal-regulated kinases, a downstream effector pathway of Ras, through a NO/non-cGMP dependent mechanism, thus supporting the physiologic relevance of endogenous NO regulation of Ras. These results suggest that Ras is a physiologic target of endogenously produced NO and indicates a signaling pathway for NMDA receptor activation that may be important for long-lasting neuronal responses.     

Functional and phenotypic characterization of monoclonal antibodies to bovine L-selectin

OBJECTIVE: To characterize 2 bovine neutrophil monoclonal antibodies (MAB) as to effects on bovine neutrophil function and their binding antigens on the cell surface of bovine neutrophils. ANIMALS: 16 healthy, lactating Holstein cattle, 1 calf with leukocyte adhesion deficiency, and 1 age-matched control calf, 2 healthy ewes, and 2 healthy human beings as neutrophil sources. PROCEDURE: Neutrophil chemotactic and respiratory burst activities and calcium influx, and binding properties of the 2 MAB were determined. Molecular mass of corresponding cell surface antigens also was determined, as was binding of human L-selectin MAB DREG56 to molecules recognized by MAB 11G10 and 2G8 on the surface of bovine neutrophils. RESULTS: MAB 11G10 and 2G8 inhibited chemotactic activity of bovine neutrophils, up-regulated amplitude of native chemiluminescence, and shortened the time to reach maximal chemiluminescence induced by serum-opsonized zymosan. Crosslinking both MAB with a second antibody induced rapid increase in intracellular free calcium concentration. Binding density of MAB 11G10 and 2G8 to bovine neutrophils treated with trypsin was increased (P < 0.05), compared with that of untreated neutrophils. Neutrophils treated with phosphatidylinositol-specific phospholipase C had decreased (P < 0.05) binding density of MAB 11G10 and 2G8. Binding of the various MAB to neutrophils from calves with bovine leukocyte adhesion deficiency was lower (P < 0.05) than binding to neutrophils from healthy calves. Expression of antigens recognized by the aforementioned MAB on the surface of bovine neutrophils was decreased (P < 0.05) within 10 minutes. CONCLUSION: MAB 11G10 and 2G8 recognized L-selectin molecules on bovine neutrophil membrane. L-Selectin (CD62L) is involved in low-affinity adhesion reactions between leukocytes and L-selectin ligand on postcapillary venular endothelial cells.     

Antigen receptor-induced B lymphocyte apoptosis mediated via a protease of the caspase family

An extensive body of data, in a variety of systems, denoted the caspase family of proteases as a key player in the execution of programmed cell death. This family consists of cysteine proteases that cleave after asparagine-containing motifs. It is well established that the caspases are essential for the apoptosis mediated by Fas (CD95) and TNF receptor p55, molecules that contain the "death domain" in the cytoplasmic tail. However, little is known about the mechanisms underlying the antigen receptor-mediated cell death in B lymphocytes, a process instrumental in negative selection of potentially autoreactive B cells. Here, we investigated the involvement of caspases in cell death triggered via the antigen receptor in B lymphocytes (BCR) by using specific inhibitors. Initially, we used a well-established cell line, CH31, which is a model of B cell tolerance, to demonstrate that these proteases indeed participate in the BCR-induced apoptotic pathway. Next, we confirmed the physiological relevance of the caspase-mediated cell death pathway in splenic B cell populations isolated ex vivo that were induced to undergo apoptosis by extensive cross-linking of their BCR. Most interestingly, our data demonstrated that caspases regulate not only the nuclear DNA fragmentation, but also the surface membrane phosphatidylserine translocation as well as the degradation of a specific nuclear substrate. Taken together, this report supports the hypothesis that regulation of the caspase family is crucial in controlling the life/death decision in B lymphocytes mediated by the antigen receptor signal transduction.     

Relationship between cleaved L-selectin levels and the outcome of acute myeloid leukemia

High plasma levels of the shed form of L-selectin (sL-selectin) are frequently detectable in acute myeloid leukemia (AML). sL-selectin can inhibit blast cell adhesion to vascular endothelium and may thereby influence the phenotype of AML. In this study, we have investigated the relationship between sL-selectin levels and clinical presentation or disease outcome in 100 patients with AML. Fifty-eight patients were found to have sL-selectin levels >/=3.12 microgram/mL (>/=3 SD above the mean of healthy controls: "increased"). Patients with extramedullary disease such as lymphadenopathies, splenomegaly, hepatomegaly, and/or muco-cutaneous infiltration had significantly increased sL-selectin levels (P < .001). sL-selectin levels were significantly heterogeneous in the French-American-British subtypes (P = .0003). Patients with "normal" sL-selectin levels had higher probability of achieving complete remission (CR) than with "increased" levels: 81% versus 64%, respectively (P = .06). When adjusting for clinically relevant covariates predictive for CR (sex, age, Auer rods), "normal" sL-selectin levels were significantly associated with CR (odds ratio, 3.08; 95% confidence interval [CI], 1.10 to 8.58; P = .03). Moreover, patients with "increased" sL-selectin levels (>/=3.12 microgram/mL) had shorter event-free survival (EFS) (median 7.3 v 12 months, P = .008) and overall survival (median 1 v 2.05 years, P = .03) than patients with sL-selectin <3.12 microgram/mL. Multivariate statistical analysis (adjusted for age and presence of Auer rods) indicated that sL-selectin was an independent prognostic factor for EFS (hazard ratio [HR], 1.96; 95% CI, 1.21 to 3.17, P = .006) and overall survival (HR, 1.80; 95% CI, 1.09 to 2.98; P = .02). Thus, plasma sL-selectin may be a useful prognostic marker in the evaluation of AML at diagnosis.     

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.     

Expression of beta 2-integrins and L-selectin on polymorphonuclear leukocytes in septic patients

Adhesion molecules on polymorphonuclear leukocytes (PMNL) play an important role in nonspecific defense mechanisms directed at invading microorganisms. When local infection, however, cannot be controlled, a systemic inflammatory response syndrome (SIRS) ensues which may progress to septic shock and multiple organ failure, these being major determinants of the patient's outcome. In the present study, the expression of beta 2-integrins and L-selectin on blood PMNL was measured on subsequent days in patients with sepsis (n = 17) and in healthy volunteers (n = 15). beta 2-Integrins and L-selectin molecules were detected by flow cytometry, using the monoclonal antibodies IB4 (anti-CD18) and Dreg200 (anti-CD62L), respectively. Adhesion molecules were determined at baseline immediately after blood collection and also 45 min after incubation of cells in vitro at body temperature to allow for spontaneous regulation. In addition, PMNL were activated by receptor-dependent and receptor-independent stimuli to characterize stimulus-specific adhesion molecule expression. In parallel with the measurement of adhesion molecules, severity of sepsis was assessed by the Elebute score. The results demonstrate significant differences in the basal, spontaneous and stimulus-induced expression of adhesion molecules between healthy volunteers, survivors (n = 11) and nonsurvivors (n = 6). Moreover, when survivors and nonsurvivors with severe sepsis (Elebute score > 12) were compared, basal expressions of both beta 2-integrins and L-selectin were significantly lower in patients who did not survive. Thus, measurement of adhesion molecules on circulating PMNL may be useful to identify septic patients at high risk for lethal outcome.     

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.     

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.