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PubMed1996

Selectins mediate eosinophil recruitment in vivo: a comparison with their role in neutrophil influx.

The role of selectins in mediating eosinophil recruitment in vivo was assessed in a model of lipopolysaccharide (LPS)-induced mouse pleurisy. LPS administration resulted in significant eosinophil influx at 24 hours, whereas neutrophil recruitment to the cavity peaked at 4 hours and persisted for 24 hours. The anti-L-selectin monoclonal antibody (MoAb) MEL-14 effectively inhibited (by 97%) eosinophil influx at 24 hours and also inhibited neutrophil recruitment at both times (75% to 95%). Eosinophil recruitment was partially reduced (54%) by the anti-P-selectin MoAb 5H1 but, in contrast, was unaffected by the anti-E-selectin MoAb 10E6. Neutrophil influx at 4 or 24 hours was not affected by the anti-P- or anti-E-selectin MoAbs. However, coadministration of anti-P-selectin and anti-E-selectin was very effective at inhibiting eosinophil influx at 24 hours (86%) and neutrophil influx at 4 (93%) and 24 hours (92%). These results show that all three selectins play a role in LPS-induced eosinophil and neutrophil recruitment in vivo, although P- and E-selectin show a degree of functional redundancy. The demonstration that P-selectin mediates eosinophil but not neutrophil influx suggests that suppressing the function of this adhesion molecule may be beneficial in blocking eosinophil accumulation in pleural inflammation.

Animals↗
PubMed1998

IL-12 promotes the adhesion of NK cells to endothelial selectins under flow conditions.

This study examined the adhesive interactions of peripheral blood NK cells with P- and E-selectin and analyzed the effect of IL-12 on the binding of NK cells to these selectins. P-selectin glycoprotein ligand-1 (PSGL-1) is expressed on most resting and IL-12-activated NK cells. However, the percentage of resting NK cells bound to P-selectin-IgG was 15%, and that of activated NK cells bound to P-selectin-IgG was 65%. Furthermore, the number of IL-12-activated NK cells bound to P-selectin-transfected Chinese hamster ovary cells was significantly higher than that of resting NK cells under flow conditions. These interactions were abolished by the incubation of these NK cells with anti-PSGL-1 (PL-1) mAb. Thus, PSGL-1/P-selectin interaction is important in the binding of resting and activated NK cells to P-selectin. NK cells express sialyl-Lewis(x) (sLe(x)) structure recognized by anti-sLe(x) mAb (KM-93), and IL-12 activation of NK cells increased the mean fluorescence intensity of KM-93-reactive NK cells. Adhesion of IL-12-activated NK cells to E-selectin-transfected Chinese hamster ovary cells was stronger than that of resting NK cells under flow conditions. These interactions were reduced markedly by incubation with anti-sLe(x) mAb. Thus, sLe(x) is the major ligand of resting and activated NK cells for E-selectin. These findings indicate that IL-12 stimulation of NK cells promotes their adhesion activity to endothelial selectins.

Animals↗
PubMed1999

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.

Animals↗
PubMed1996

Regualtion by cytokines and lipopolysaccharide of E-selectin expression by human brain microvessel endothelial cells in primary culture.

E-selectin is an adhesion molecule expressed on endothelial cells after treatment with inflammatory agents in vitro and in inflammatory diseases in vivo. Interactions between leukocytes and endothelial cells are mediated partly through this adhesion molecule. In this study, the kinetic expression of E-selectin by human cerebral endothelium was studied under standard conditions and following treatment of primary cultures with bacterial lipopolysaccharide (LPS), tumor necrosis factor-alpha (TNF-alpha), interleukin-1beta (IL-1beta), and interferon-gamma (IFN-gamma). Surface expression of E-selectin was detected by immunocytochemistry, ELISA and immunoelectron microscopy. Untreated human cerebral endothelial cells constitutively expressed low levels of E-selectin. Treatment with LPS, TNF-alpha and IL-beta increased the mean level of E-selectin expression per cell and the percentage of cells expressing E-selectin, in a time and concentration-dependent manner. E-selectin expression was maximal by 4 h post-stimulation and returned unstimulated levels by 48 h. LPS and TNF-alpha were most effective followed by IL-1beta, while the IFN-gamma had no effect on E-selectin expression. Immunoelectron microscopy demonstrated that E-selectin was preferentially expressed on the apical surface of unstimulated and TNF-alpha treated cells. Cytokine stimulation induced a several-fold increase of E-selectin expression on the apical and to a lesser extent on the basal cell surface. Modulation of E-selectin expression on cerebral endothelium by inflammatory cytokines suggests a potentially important role of this adhesion molecule in the recruitment of leukocytes in central nervous (CNS) inflammation.

Adult↗
PubMedDate not supplied

Differential effects of hydroxamate inhibitors on PMA and ligand-induced L-Selectin down-modulation: role of membrane proximal and cytoplasmic domains.

L-selectin is down-modulated from the cell surface in response to leukocyte activation or cross-linking with ligand mimetics such as anti-L-selectin antibodies or sulfatides. The down-modulation induced upon cellular activation with PMA is due to proteolytic shedding mediated by a cysteine metalloprotease, presumably the TNF-alpha converting enzyme (TALE), and is susceptible to inhibitors of the hydroxamate class. To determine if cross-linking-induced down-modulation of L-selectin is similarly affected by these inhibitors, we exposed Jurkat T cells to the anti-L-selectin antibody Dreg 200 or sulfatides in the presence of the hydroxamate TNF-alpha protease inhibitor (TAPI). TAPI completely inhibited PMA-induced downmodulation but had no effect on that induced by the anti-L-selectin antibody or sulfatides. The downmodulation induced by an anti-TCR antibody (WT31) was only partially inhibited by TAPI. An L-selectin mutant lacking the putative TACE cleavage site and resistant to PMA-induced shedding (321del.9) was expressed in L1.2 pre-B cells. Like WT L-selectin, this mutant was down-modulated when exposed to sulfatides, or Dreg 200 and this down-modulation was unaffected by TAPI. An L-selectin cytoplasmic tail deletion mutant (344del.15) expressed in L1.2 pre-B cells was down-modulated by PMA or sulfatides, but not Dreg 200. Electrophoretic analysis of L-selectin immunoprecipitated from the supernatants of Jurkat cells treated with either sulfatides or D200 revealed a proteolytic fragment of the same size as that released from the cell surface in response to PMA. Our data indicate that the down regulation of L-selectin in response to cross-linking by ligands or TCR engagement may be mechanistically distinct from that induced by PMA. Furthermore, our results with the 344del.15 mutant suggest that the down-modulation of Lselectin induced by certain sulfated carbohydrate ligands may be initiated through surface receptors other than L-selectin itself. The abbreviations used in this paper are: TAPI, TNF-alpha protease inhibitor; TACE, TNF-alpha converting enzyme; PMA, 4(3-phorbol 12-myristate 13-acetate.

Journal Article↗
PubMed2000

Role of selectins in experimental Staphylococcus aureus-induced arthritis.

The selectin family of adhesion molecules mediates the initial attachment of leukocytes to venular endothelial cells at sites of tissue injury and inflammation. To assess the role of selectin family in Staphylococcus aureus-triggered septic arthritis, we used several approaches. First, treatment with fucoidin, a carbohydrate molecule capable of binding to and blocking selectin functions, was used. In addition, we used P-selectin gene-targeted mice as well as mice pretreated with monoclonal antibody blocking L-selectin function. The P-selectin-deficient and fucoidin-treated animals initially exhibited a less severe septic arthritis both clinically and histopathologically. In the later stages of the disease no significant differences with respect to arthritis were evident. Pretreatment with L-selectin blocking antibody did not influence the severity of arthritis. High numbers of staphylococci were recovered from the kidneys of selectin-deficient mice, indicating a less efficient clearance of bacteria. Our results demonstrate a dual role for selectins in S. aureus-induced arthritis: on the one hand, blockade of these selectins leads to less severe arthritic lesions in the initial stage of the disease; on the other, delayed recruitment of phagocytes decreases the clearance of bacteria.

Animals↗
PubMed2001

Tyrosine sulfation enhances but is not required for PSGL-1 rolling adhesion on P-selectin.

P-selectin glycoprotein ligand-1 (PSGL-1) is a large (240 kDa) glycoprotein found on the surface of nearly all leukocytes. The mature molecule is decorated with multiple N- and O-linked glycans and displays copies of the tetrasaccharide sialyl-Lewis(x) (sLe(X)), as well as a cluster of three tyrosine sulfate (tyr-SO(3)) groups near the N-terminus of the processed protein. Previous studies have suggested that PSGL-1 needs to be tyrosine-sulfated, in addition to glycosylated with sLe(X), to successfully interact with P-selectin. To better understand how biochemical features of the PSGL-1 ligand are related to its adhesion phenotype, we have measured the dynamics of adhesion under flow of a series of well-defined PSGL-1 variants that differ in their biochemical modification, to both P- and E-selectin-coated substrates. These variants are distinct PSGL-1 peptides: one that possesses sLe(X) in conjunction with three N-terminal tyr-SO(3) groups (SGP3), one that possesses sLe(X) without tyrosine sulfation (GP1), and one that lacks sLe(X) but has three N-terminal tyr-SO(3) groups (SP3). Although all peptides expressing sLe(X), tyr-SO(3), or both supported some form of rolling adhesion on P-selectin, only peptides expressing sLe(X) groups showed rolling adhesion on E-selectin. On P-selectin, the PSGL-1 peptides demonstrated a decreasing strength of adhesion in the following order: SGP3 > GP1 > SP3. Robust, rolling adhesion on P-selectin was mediated by the GP1 peptide, despite its lack of tyrosine sulfation. However, the addition of tyrosine sulfation to glycosylated peptides (SGP3) creates a super ligand for P-selectin that supports slower rolling adhesion at all shear rates and supports rolling adhesion at much higher shear rates. Tyrosine sulfation has no similar effect on PSGL-1 rolling on E-selectin. Such functional distinctions in rolling dynamics are uniquely realized with a cell-free system, which permits precise, unambiguous identification of the functional activity of adhesive ligands. These findings are consistent with structural and functional characterizations of the interactions between these peptides and E- and P-selectin published recently.

Amino Acid Substitution↗
PubMed1998

The expression of P- and E-selectins in three models of middle cerebral artery occlusion.

The expression and localization of P- and E-selectins in rat brain (n=126) were examined using immunohistochemical techniques at various time points after induction of middle cerebral artery (MCA) occlusion in the suture, thrombotic and embolic models of stroke. Expression of P- or E-selectin was not observed in brain tissue of sham operated control rats (n=9). P-selectin immunoreactivity was detected as early as 15 min and decreased to control level at 1 h after the onset of the MCA occlusion in all three models. P-selectin then slightly increased at 2 h and peaked at 6 h after MCA occlusion. E-selectin immunoreactivity was first observed at 2 h and peaked at 6 h and 12 h of after MCA occlusion in all three models. P- and E-selectin immunoreactivity was colocalized with von Willebrand factor immunoreactive microvessels. 90.4+/-2.0% of all vessels expressing P-selectin immunoreactivity were 7.5 to 30.0 micron in diameter; 3.6+/-1.4% were contained in vessels smaller than 7.5 micron, and 6.0+/-1.8% were localized in vessels greater than 30.0 micron in diameter. The percent distribution of E-selectin immunoreactive vessels were 75.9+/-2.1% in vessels 7.5 to 30.0 micron in diameter; 23.6+/-2.2% were in vessels smaller than 7.5 micron, and 0.6+/-0.4% were localized in vessels greater than 30.0 micron in diameter. These findings indicate that the temporal profiles of P- and E-selectin expression are independent of these models of MCA occlusion and are consistent with the time course of selectin mediated leukocyte infiltration after focal cerebral ischemia in the rat.

Analysis of Variance↗
PubMed1998

Elevated serum levels of soluble E- and L-selectin in patients with human T-cell lymphotropic virus type I-associated myelopathy.

We compared soluble E-selectin (sE-selectin) and L-selectin (sL- selectin) levels in sera and cerebrospinal fluid (CSF) of 30 patients with human T-lymphotropic virus type I (HTLV-I)-associated myelopathy (HAM), with those of 10 patients with the relapsing-remitting form of multiple sclerosis (MS), and 16 patients with other neurological diseases (OND). Serum levels of both sE-selectin and sL-selectin, as measured by enzyme-linked immunosorbent assay, were significantly elevated in patients with HAM, compared to patients with OND. In addition, serum levels of sL-selectin were significantly elevated in HAM patients compared to MS patients. No significant difference was found in CSF levels of sL-selectin between HAM patients and controls. However, HAM patients who had received blood transfusions had significantly higher CSF levels of sL-selectin than HAM patients without a past history of transfusions, suggesting that HAM patients with past history of transfusion have a more active immunological state in the central nervous system. sE-selectin was not detected in CSF of HAM patients and controls. This finding might be based on exaggerated inflammatory conditions following increased attachment of lymphocytes to activated endothelial cells in HAM patients.

Adult↗
PubMed1998

Concentrations of circulating P-selectin are increased in patients with newly diagnosed insulin-dependent diabetes mellitus.

Endothelial cell activations and/or dysregulations of the coagulation system are crucial parameters for the prognosis of disease in patients with IDDM. Recent data suggest that expression of the adhesion molecules E-selectin and P-selectin are markers of endothelial cell activation and/or platelet activation and might modify immunologic responses after shedding from cell membranes. In patients with newly diagnosed IDDM only limited data on circulating selectins are available. This has prompted us to measure levels of soluble (s) forms of P-selectin and E-selectin in 18 patients with newly diagnosed IDDM and two years after the onset of insulin substitution therapy in comparison to 18 age-matched healthy control subjects. HbA1c and blood glucose levels were significantly higher in patients with new onset diabetes, compared to the same patients after two years of insulin therapy (HbA1c: 12 +/- 3% vs. 7.8 +/- 2%; p < 0.01; blood-glucose: 409 +/- 163 mg/dl vs. 131 +/- 23 mg/dl; p < 0.01), but no correlation between these metabolic parameters and soluble forms of E- and P-selectins were noted. Levels of sP-selectin decreased from 210 +/- 120 ng/ml in newly diagnosed IDDM patients to 127 +/- 75 ng/ml after two years of therapy (p < 0.01) and were similar to those of the control subjects (110 +/- 31 ng/ml). Serum concentrations of circulating E-selectin showed no differences in the three groups (newly diagnosed IDDM: 42 +/- 17 ng/ml; two years later: 43 +/- 19 ng/ml; control subjects: 41 +/- 14 ng/ml; n.s.). Increased levels of sP-selectin together with normal levels of sE-selectin at the onset of IDDM suggest enhanced platelet activation during the initial phase of the autoimmune process and return to baseline levels within two years of the disease.

Adult↗
PubMed2002

Synergistic effects of L- and P-selectin in facilitating tumor metastasis can involve non-mucin ligands and implicate leukocytes as enhancers of metastasis.

P-selectin facilitates human carcinoma metastasis in immunodeficient mice by mediating early interactions of platelets with bloodborne tumor cells via their cell surface mucins, and this process can be blocked by heparin [Borsig, L., Wong, R., Feramisco, J., Nadeau, D. R., Varki, N. M. & Varki, A. (2001) Proc. Natl. Acad. Sci. USA 98, 3352-3357]. Here we show similar findings with a murine adenocarcinoma in syngeneic immunocompetent mice but involving a different P-selectin ligand, possibly a sulfated glycolipid. Thus, metastatic spread can be facilitated by tumor cell selectin ligands other than mucins. Surprisingly, L-selectin expressed on endogenous leukocytes also facilitates metastasis in both the syngeneic and xenogeneic (T and B lymphocyte deficient) systems. PL-selectin double deficient mice show that the two selectins work synergistically. Although heparin can block both P- and L-selectin in vitro, the in vivo effect of a single heparin dose given before tumor cells seems to be completely accounted for by blockade of P-selectin function. Thus, L-selectin on neutrophils, monocytes, and/or NK cells has a role in facilitating metastasis, acting beyond the early time points wherein P-selectin mediates interactions of platelet with tumor cells.

Animals↗
PubMed2001

CD44 is a major E-selectin ligand on human hematopoietic progenitor cells.

E-selectin plays a critical role in mediating tissue-specific homing of T cells into skin, and of primitive hematopoietic progenitor cells (HPCs) into bone marrow (BM). Though it is known that a glycoform of PSGL-1 (CLA) functions as the principal E-selectin ligand on human T lymphocytes, the E-selectin ligand(s) of human HPCs has not been identified. We used a shear-based adherence assay to analyze and define the E-selectin ligand activity of membrane proteins from human HPCs. Our data show that PSGL-1 expressed on human HPCs is an E-selectin ligand, and that HPCs also express a previously unrecognized E-selectin ligand, CD44. The E-selectin ligand activity of CD44 is conferred by the elaboration of sialylated, fucosylated binding determinants on N-glycans. This glycoform of CD44 is expressed on primitive CD34+ human HPCs, but not on more mature hematopoietic cells. Under physiologic flow conditions, this molecule mediates E-selectin-dependent rolling interactions over a wider shear range than that of PSGL-1, and promotes human HPC rolling interactions on E-selectin expressed on human BM endothelial cells. These findings offer new insights into the structural biology and physiology of CD44, and into the molecular basis of E-selectin-dependent adhesive interactions that direct homing of human HPC to BM.

Animals↗
PubMed1998

Inhibition of L- and P-selectin by a rationally synthesized novel core 2-like branched structure containing GalNAc-Lewisx and Neu5Acalpha2-3Galbeta1-3GalNAc sequences.

The selectins interact in important normal and pathological situations with certain sialylated, fucosylated glycoconjugate ligands containing sialyl Lewisx(Neu5Acalpha2-3Galbeta1-4(Fucalpha1-3)GlcN Ac). Much effort has gone into the synthesis of sialylated and sulfated Lewisxanalogs as competitive ligands for the selectins. Since the natural selectin ligands GlyCAM-1 and PSGL-1 carry sialyl Lewisxas part of a branched Core 2 O-linked structure, we recently synthesized Galbeta1-4(Fucalpha1-3)GlcNAcbeta1-6(SE-3Galbeta1++ +-3)GalNAc1alphaOMe and found it to be a moderately superior ligand for L and P-selectin (Koenig et al. , Glycobiology 7, 79-93, 1997). Other studies have shown that sulfate esters can replace sialic acid in some selectin ligands (Yeun et al. , Biochemistry, 31, 9126-9131, 1992; Imai et al. , Nature, 361, 555, 1993). Based upon these observations, we hypothesized that Neu5Acalpha2-3Galbeta1-3GalNAc might have the capability of interacting with L- and P-selectin. To examine this hypothesis, we synthesized Galbeta1-4(Fucalpha1-3)GlcNAcbeta1-6(Neu5Acalpha2++ +-3Galbeta1-3)-GalNAc alpha1-OB, which was found to be 2- to 3-fold better than sialyl Lexfor P and L selectin, respectively. We also report the synthesis of an unusual structure GalNAcbeta1-4(Fucalpha1- 3)GlcNAcbeta1-OMe (GalNAc-Lewisx-O-methyl glycoside), which also proved to be a better inhibitor of L- and P-selectin than sialyl Lewisx-OMe. Combining this with our knowledge of Core 2 branched structures, we have synthesized a molecule that is 5- to 6-fold better at inhibiting L- and P-selectin than sialyl Lewisx-OMe, By contrast to unbranched structures, substitution of a sulfate ester group for a sialic acid residue in such a molecule resulted in a considerable loss of inhibition ability. Thus, the combination of a sialic acid residue on the primary (beta1-3) arm, and a modified Lexunit on the branched (beta1-6) arm on an O-linked Core 2 structure generated a monovalent synthetic oliogosaccharide inhibitor superior to SLexfor both L- and P-selectin.

Binding, Competitive↗
PubMed1998

Progressive vascular damage in hypertension is associated with increased levels of circulating P-selectin.

OBJECTIVE: To assess whether increased shedding of adhesion molecules in plasma provides an index for endothelial damage in hypertension. DESIGN AND METHODS: Three groups of hypertensive patients with increasing severity of vascular damage were studied: 20 essential hypertensives, 21 atherosclerotic, renovascular hypertensives and four malignant hypertensives. Twenty healthy subjects were included as a control group. Levels of P-selectin, E-selectin, intracellular adhesion molecule 1, vascular cell adhesion molecule and von Willebrand factor in venous blood were measured, using sandwich-type enzyme-linked immunosorbent assay. RESULTS: For essential hypertensives a trend for increased P-selectin and E-selectin values compared with those in controls was observed (159+/-44 versus 132+/-40 ng/ml, P = 0.062 and 40+/-13 versus 34+/-17 ng/ml, P = 0.055, respectively). P-selectin (210+/-84 ng/ml, P = 0.0021) and E-selectin (42+/-12 ng/ml, P = 0.012) levels in renovascular hypertensives were significantly higher than those in healthy controls. There were no significant increases in circulating levels of intracellular adhesion molecule 1, vascular cell adhesion molecule and von Willebrand factor either in essential hypertensives or in renovascular hypertensives. Marked increases in circulating levels of adhesion molecules and von Willebrand factor relative to those in controls were observed in malignant hypertensives (P-selectin 634+/-332 versus 132+/-40 ng/ml, P = 0.0004; vascular cell adhesion molecule 968+/-187 versus 493+/-139 ng/ml, P = 0.0004; and von Willebrand factor 259+/-75 versus 130+/-72 U/dl, P = 0.016). CONCLUSIONS: Progression of vascular damage in essential, renovascular and malignant hypertension is associated with a rise in circulating levels of P-selectins and, to a lesser extent, E-selectins, whereas levels of intracellular adhesion molecule 1, vascular cell adhesion molecule and von Willebrand factor are elevated only in diseases associated with acute severe vascular damage, including malignant hypertension. Our data suggest that selectins may be useful as indicators of vascular damage in hypertension.

Adult↗
PubMed2004

Important role of P-selectin for leukocyte recruitment, hepatocellular injury, and apoptosis in endotoxemic mice.

Leukocyte recruitment in the liver includes a two-step procedure in which selectin-dependent leukocyte rolling is a prerequisite for subsequent CD18-dependent leukocyte firm adhesion in postsinusoidal venules. However, the roles of the individual selectins in leukocyte rolling and adhesion, hepatocellular injury, and apoptosis remain elusive. Therefore, we examined the pathophysiological role of P-, E-, and L-selectin in male C57BL/6 mice challenged with lipopolysaccharide (LPS) and D-galactosamine (Gal) by use of intravital microscopy of the liver microcirculation. In control animals, administration of LPS-Gal provoked reproducible hepatic damage, including marked increases of leukocyte recruitment, liver enzymes, and hepatocyte apoptosis and reduced sinusoidal perfusion. Interestingly, pretreatment with an anti-P-selectin antibody (RB40.34) markedly reduced leukocyte rolling and firm adhesion by 65 and 71%, respectively. Moreover, interference with P-selectin function significantly improved sinusoidal perfusion and reduced the increase in liver enzymes by 49 to 84% in endotoxemic mice. Moreover, the activity of caspase-3 and the number of apoptotic hepatocytes were significantly reduced by 55 and 54%, respectively, in RB40.34-treated animals. In contrast, administration of an anti-E-selectin antibody (10E9.6) and an anti-L-selectin antibody (Mel-14) did not protect against endotoxin-induced leukocyte responses or hepatic injury. In conclusion, our novel findings document a principal role of P-selectin in mediating leukocyte rolling, a precondition to the subsequent firm adhesion of leukocytes in liver injury. Furthermore, our novel data demonstrate that inhibition of P-selectin function reduces hepatocellular injury and apoptosis, suggesting a causal relationship between leukocyte recruitment on one hand and hepatocellular injury and apoptosis on the other hand. Based on these findings, it is suggested that P-selectin may be an important therapeutic target in endotoxin-induced liver injury.

Alanine Transaminase↗
PubMed2005

Cytoskeletal interactions regulate inducible L-selectin clustering.

L-selectin (CD62L) amplifies neutrophil capture within the microvasculature at sites of inflammation. Activation by G protein-coupled stimuli or through ligation of L-selectin promotes clustering of L-selectin and serves to increase its adhesiveness, signaling, and colocalization with beta(2)-integrins. Currently, little is known about the molecular process regulating the lateral mobility of L-selectin. On neutrophil stimulation, a progressive change takes place in the organization of its plasma membrane, resulting in membrane domains that are characteristically enriched in glycosyl phosphatidylinositol (GPI)-anchored proteins and exclude the transmembrane protein CD45. Clustering of L-selectin, facilitated by E-selectin engagement or antibody cross-linking, resulted in its colocalization with GPI-anchored CD55, but not with CD45 or CD11c. Disrupting microfilaments in neutrophils or removing a conserved cationic motif in the cytoplasmic domain of L-selectin increased its mobility and membrane domain localization in the plasma membrane. In addition, the conserved element was critical for L-selectin-dependent tethering under shear flow. Our data indicate that L-selectin's lateral mobility is regulated by interactions with the actin cytoskeleton that in turn fortifies leukocyte tethering. We hypothesize that both membrane mobility and stabilization augment L-selectin's effector functions and are regulated by dynamic associations with membrane domains and the actin cytoskeleton.

Amino Acid Sequence↗
PubMed2000

Regulation of E-selectin expression in postischemic intestinal microvasculature.

Monolayers of cultured endothelial cells exposed to hypoxia-reoxygenation exhibit a transcription-dependent increase in E-selectin expression and E-selectin-dependent neutrophil-endothelial cell adhesion. The overall objectives of this study were 1) to determine whether ischemia-reperfusion (I/R) promotes upregulation of E-selectin in vivo; 2) if so, to define the mediators of this response; and 3) to assess the contribution of E-selectin to I/R-induced neutrophil recruitment. The dual-radiolabeled monoclonal antibody (MAb) technique was used to measure E-selectin expression in the intestinal vasculature. Ischemia was induced by complete occlusion (30-60 min) of the superior mesenteric artery followed by 3-24 h of reperfusion. Increasing durations of ischemia elicited progressively increasing (2- to 5-fold) levels of E-selectin expression, with the peak response noted after 45 min of ischemia and 5 h of reperfusion. Subsequent experiments revealed that I/R-induced increase in E-selectin expression (at 5 h) is significantly blunted in transgenic mice that overexpress Cu,Zn-superoxide dismutase or by treatment of wild-type mice with either a blocking antibody against tumor necrosis factor (TNF)-alpha or an inhibitor of nuclear factor-kappaB (NF-kappaB) activation (PS341). Administration of an E-selectin-specific MAb dramatically reduced I/R-induced recruitment of neutrophils in the intestine. These findings suggest that superoxide and TNF-alpha mediate gut I/R-induced E-selectin expression via an NF-kappaB-dependent mechanism; this upregulation of E-selectin contributes significantly to I/R-induced neutrophil recruitment.

Animals↗
PubMed1999

L- and P-selectin and CD11/CD18 in intracapillary neutrophil sequestration in rabbit lungs.

Infusion of complement fragments induces rapid sequestration of neutrophils within pulmonary capillaries. This study examined the mechanisms through which this sequestration occurs, as well as the effect of complement fragments on the expression of L-selectin and CD11/CD18 using ultrastructural immunohistochemistry. Studies using anti-P-selectin antibodies, fucoidin, L-selectin-depleted neutrophils, and anti-CD18 antibodies showed that selectins and CD18 were not required for neutrophil sequestration. However, maintaining the sequestered neutrophils within the pulmonary capillaries required both L-selectin and CD11/CD18. Neutrophils in the pulmonary capillaries of rabbits given complement fragments expressed 72% less L-selectin and 98% more CD11/CD18 than did those in rabbits given saline. Shedding of L-selectin occurred preferentially from the microvillar processes of the plasma membrane rather than from the flat intervening regions. About 28% of L-selectin still remained on intracapillary neutrophil membranes after 15 min and was likely available for binding. Shedding of L-selectin appeared slower in vivo than in vitro. These studies indicate that neutrophil sequestration induced by complement fragments requires at least two sequential steps, one that does not require recognized adhesion molecules followed by a second that requires L-selectin and CD11/ CD18.

Animals↗