P-, E-, and L-selectin mediate migration of activated CD8+ T lymphocytes into inflamed skin

P- and E-selectin mediate CD4+ Th1 cell migration into the inflamed skin in a murine contact hypersensitivity model. In this model, not only CD4+ T cells but also CD8+ T cells infiltrate the inflamed skin, and the role of CD8+ type 1 cytotoxic T (Tc1) cells as effector cells has been demonstrated. Here we show that in mice deficient in both P- and E-selectin, the infiltration of CD8+ T cells in the inflamed skin is reduced, suggesting the role of these selectins in CD8+ T cell migration. We directly studied the role of selectins using in vitro-generated Tc1 cells. These cells are able to migrate into the inflamed skin of wild-type mice. This migration is partially mediated by P- and E-selectin, as shown by the reduced Tc1 cell migration into the inflamed skin of mice deficient in both P- and E-selectin or wild-type mice treated with the combination of anti-P-selectin and anti-E-selectin Abs. During P- and E-selectin-mediated migration of Tc1 cells, P-selectin glycoprotein ligand-1 appears to be the sole ligand for P-selectin and one of the ligands for E-selectin. P- and E-selectin-independent migration of Tc1 cells into the inflamed skin was predominantly mediated by L-selectin. These observations indicate that all three selectins can mediate Tc1 cell migration into the inflamed skin.     

The role of E-selectin, P-selectin, and very late activation antigen-4 in T lymphocyte migration to dermal inflammation

T lymphocyte infiltration into inflamed tissues is thought to involve lymphocyte rolling on vascular endothelial cells. Because both selectin and alpha(4) integrin adhesion molecules can mediate leukocyte rolling, the contribution of these receptors to lymphocyte migration to inflammation was examined. The recruitment of (111)In-labeled spleen T cells to intradermal sites injected with IFN-gamma, TNF-alpha, LPS, poly inosine-cytosine, and Con A was measured in the rat, and the effect of blocking mAbs to E-selectin, P-selectin, very late activation Ag-4 (VLA-4), and LFA-1 was determined on this T cell migration in vivo. Anti-E-selectin and anti-P-selectin mAbs each inhibited 10-40 and 20-48%, respectively, of the T lymphocyte migration to the inflammatory sites, depending on the stimulus. Blocking VLA-4 inhibited 50% of the migration to all of the lesions except Con A. Treatment with both anti-VLA-4 and anti-E-selectin mAbs inhibited up to 85% of the lymphocyte accumulation, while P-selectin and VLA-4 blockade in combination was not more effective than VLA-4 blockade alone in TNF-alpha, IFN-gamma, LPS, and poly inosine-cytosine lesions. Inhibiting E-selectin, P-selectin, and VLA-4 together nearly abolished lymphocyte migration to all inflammatory sites. Anti-LFA-1 mAb strongly inhibited lymphocyte accumulation by itself, and this inhibition was not significantly further reduced by E- or P-selectin blockade. Thus, T cell migration to dermal inflammation is dependent on E-selectin, P-selectin, and VLA-4, likely because these three receptors are required for rolling of memory T lymphocytes, but VLA-4 and E-selectin are especially important for lymphocyte infiltration in these tissues.     

PSGL-1 derived from human neutrophils is a high-efficiency ligand for endothelium-expressed E-selectin under flow

P-selectin glycoprotein ligand-1 (PSGL-1) has been proposed as an important tethering ligand for E-selectin and is expressed at a modest level on human leukocytes. Sialyl Lewis x (sLe^x)-like glycans bind to E-selectin and are expressed at a relatively high level on circulating leukocytes. It is unclear whether PSGL-1 has unique biochemical attributes that contribute to its role as an E-selectin ligand. To probe this issue, we conjugated microspheres with either sLe^x or PSGL-1 purified from myeloid cells (neutrophils and HL-60) and compared their adhesion to endothelial expressed E-selectin under defined shear conditions. We found that both sLe^x and PSGL-1 microspheres adhere to 4 h of IL-1-activated human umbilical vein endothelial cells predominantly through E-selectin. Analysis of the adhesion revealed that the rate of initial tethering of the PSGL-1 microspheres to E-selectin was significantly greater than the rate of initial tethering of the sLe^x microspheres despite the fact that the sLe^x microspheres tested had higher ligand densities than the PSGL-1 microspheres. We also found that pretreatment of the PSGL-1 or sLe^x microspheres with HECA-452 had no significant effect on initial tethering to E-selectin. These results support the hypotheses that 1) PSGL-1 is a high-efficiency tethering ligand for E-selectin, 2) ligand biochemistry can significantly influence initial tethering to E-selectin, and 3) PSGL-1 tethering to E-selectin can occur via non-HECA-452 reactive epitopes. adhesion; leukocyte; inflammation     

Leukocyte-endothelial cell interactions are enhanced in dermal postcapillary venules of MRL/fas(lpr) (lupus-prone) mice: roles of P- and E-selectin

MRL/fas(lpr) mice are affected by a systemic autoimmune disease that results in widespread leukocytic infiltration of the vasculature, including in the skin. The molecular pathways responsible for this leukocyte recruitment are poorly understood. Therefore, the aim of these experiments was to examine the mechanisms of leukocyte trafficking in the dermal microvasculature of MRL/fas(lpr) mice. Intravital microscopy was used to examine leukocyte rolling and adhesion in dermal postcapillary venules of MRL/fas(lpr) mice at 8, 12, and 16 wk of age. When compared with age-matched BALB/c and MRL(+/+) (nondiseased) mice, leukocyte rolling and adhesion in MRL/fas(lpr) mice were significantly enhanced at 12 wk of age, and remained elevated at 16 wk of age. At 8 and 12 wk, leukocyte rolling in all three strains was almost entirely inhibited by an anti-P-selectin mAb. In contrast, at 16 wk some (approximately 10%) leukocyte rolling persisted following P-selectin blockade. This residual rolling was predominantly inhibitable with an anti-E-selectin mAb; however, treatment with anti-E-selectin mAb alone had a minimal effect. P-selectin-deficient MRL/fas(lpr) mice also displayed leukocyte rolling that was significantly lower than in wild-type MRL/fas(lpr) mice. However, in these mice, leukocyte adhesion remained at the elevated levels observed in wild-type MRL/fas(lpr) mice. This adhesion was eliminated by chronic treatment with anti-E-selectin mAb. These findings indicate that leukocyte-endothelial cell interactions are enhanced in the dermal microvasculature of MRL/fas(lpr) mice above the age of 12 wk. Furthermore, the data suggest that the endothelial selectins share overlapping roles in mediating this enhanced leukocyte recruitment.     

Isolated P-selectin Glycoprotein Ligand-1 Dynamic Adhesion to P- and E-selectin

Leukocyte adhesion to vascular endothelium under flow involves an adhesion cascade consisting of multiple receptor pairs that may function in an overlapping fashion. P-selectin glycoprotein ligand-1 (PSGL-1) and L-selectin have been implicated in neutrophil adhesion to P- and E-selectin under flow conditions. To study, in isolation, the interaction of PSGL-1 with P-and E-selectin under flow, we developed an in vitro model in which various recombinant regions of extracellular PSGL-1 were coupled to 10-μm-diameter microspheres. In a parallel plate chamber with well defined flow conditions, live time video microscopy analyses revealed that microspheres coated with PSGL-1 attached and rolled on 4-h tumor necrosis factor-α–activated endothelial cell monolayers, which express high levels of E-selectin, and CHO monolayers stably expressing E-or P-selectin. Further studies using CHO-E and -P monolayers demonstrate that the first 19 amino acids of PSGL-1 are sufficient for attachment and rolling on both E- and P-selectin and suggest that a sialyl Lewis x–containing glycan at Threonine-16 is critical for this sequence of amino acids to mediate attachment to E- and P-selectin. The data also demonstrate that a sulfated, anionic polypeptide segment within the amino terminus of PSGL-1 is necessary for PSGL-1–mediated attachment to P- but not to E-selectin. In addition, the results suggest that PSGL-1 has more than one binding site for E-selectin: one site located within the first 19 amino acids of PSGL-1 and one or more sites located between amino acids 19 through 148.     

CD43 plays both antiadhesive and proadhesive roles in neutrophil rolling in a context-dependent manner

As the first step in the recruitment of neutrophils into tissues, the cells become tethered to and roll on the vessel wall. These processes are mediated by interactions between the P- and E-selectins, expressed on the endothelial cells of the vessel wall, and their ligands, expressed on the neutrophils. Recently, we reported that CD43 on activated T cells functions as an E-selectin ligand and thereby mediates T cell migration to inflamed sites, in collaboration with P-selectin glycoprotein ligand-1 (PSGL-1), a major P- and E-selectin ligand. Here, we examined whether CD43 on neutrophils also functions as an E-selectin ligand. CD43 was precipitated with an E-selectin-IgG chimera from mouse bone marrow neutrophils. A CD43 deficiency diminished the E-selectin-binding activity of neutrophils when PSGL-1 was also deficient. Intravital microscopy showed that the CD43 deficiency significantly increased leukocyte rolling velocities in TNF-alpha-stimulated venules blocked with an anti-P-selectin mAb, where the rolling was mostly E-selectin dependent, when PSGL-1 was also absent. In contrast, in venules with trauma-induced inflammation, where the rolling was largely P-selectin dependent, the CD43 deficiency reduced leukocyte rolling velocities. Collectively, these observations suggest that CD43 generally serves as an antiadhesive molecule to attenuate neutrophil-endothelial interactions, but when E-selectin is expressed on endothelial cells, it also plays a proadhesive role as an E-selectin ligand.     

Stimulation of adhesion molecule expression by Helicobacter pylori and increased neutrophil adhesion to human umbilical vein endothelial cells

Helicobacter pylori upregulates endothelial adhesion molecules but the pattern is unclear. Human umbilical vein endothelial cells (HUVEC) were exposed to control medium or H. pylori 60190. Binding of monoclonal antibodies against P-selectin, E-selectin, vascular adhesion molecule-1 (VCAM-1) and intercellular adhesion molecule-1 (ICAM-1) was determined using enzyme-linked immunosorbent assay. Binding of polymorphonuclear leukocytes to HUVEC was determined on cells exposed as above. After 6 h exposure to H. pylori, there were 30%, 124%, 167% and 100% increases in P-selectin, E-selectin, VCAM-1 and ICAM-1 levels and a 400% increase in polymorphonuclear leukocyte adhesion in HUVEC exposed to H. pylori. Effects of incubation for other intervals between 0 and 18 h are also described. H. pylori exerts some of its effects on gastric mucosa via gastric vasculature. This study gives insight into the pattern of H. pylori-associated endothelial adhesion molecule upregulation.     

Diazoxide ameliorates microcirculatory disturbances through PKC-dependent Pathway in I/R-injured rat cremaster muscles

Summary Diazoxide is a selective mitochondria ATP-sensitive potassium (K_ATP) channel opener, which has been reported to preserve the microvascular integrity of ischemia-reperfusion (I/R)-injured tissues. Our study aimed to assess diazoxide’s effects on I/R-injured cremaster muscles and to further elucidate its underlying mechanisms. Male Sprague Dawley (SD) rats were randomized (n = 8 per group) into four groups: sham-operated control group, I/R group (4 h of pudic epigastic artery ischemia followed by 2 h of reperfusion), diazoxide + I/R group, and chelerythrine (PKC inhibitor)+diazoxide+I/R group. Microscopically, we observed that I/R markedly increased the number of rolling, adhering, and transmigrating leukocytes. I/R also markedly decreased the number of functional capillaries. Biochemically, we found that I/R significantly increased TNF-α, E-selectin,L-selectin and P-selectin expressions. However, I/R did not cause significant changes in ICAM-1 and PECAM-1 expressions. On the other hand, in I/R + diazoxide group, we found that diazoxide reduced the number of rolling, adhering, and transmigrating leukocytes. Furthermore, biochemical study revealed that diazoxide caused only a decrease in L-selectin expression but had no effect on TNF-α, E-selectin, P-selectin, ICAM-1, and PECAM-1 expressions. Finally, in chelerythrine + diazoxide + I/R group, we observed that diazoxide’s protective effects were blocked by the addition of chelerythrine. Diazoxide’s ability to protect against I/R injury was confirmed by the observation that it reduced the number of rolling, adhering, and transmigrating leukocytes, and increased the number of functional capillaries. Our results indicated that diazoxide operated via a PKC-dependent pathway to achieve protection against I/R injury.     

In vivo ligand specificity of E-selectin binding to multivalent sialyl Lewisx N-linked oligosaccharides.

The in vivo specificity for E-selectin binding to a panel of N-linked oligosaccharides containing a clustered array of one to four sialyl Lewisx (SLex; NeuAcalpha2-3Gal[Fucalpha1-3]beta1-4GlcNAc) determinants was studied in mice. Following intraperitoneal dosing with lipopolysaccharide, radioiodinated tyrosinamide N-linked oligosaccharides were dosed i.v. and analyzed for their pharmacokinetics and biodistribution. Specific targeting was determined from the degree of SLex oligosaccharide targeting relative to a sialyl oligosaccharide control. Oligosaccharides targeted the kidney with the greatest selectivity after a 4-h induction period following lipopolysaccharide dosing. Unique pharmacokinetic profiles were identified for SLex biantennary and triantennary oligosaccharides but not for monovalent and tetraantennary SLex oligosaccharides or sialyl oligosaccharide controls. Biodistribution studies established that both SLex biantennary and triantennary oligosaccharides distributed to the kidney with 2-3-fold selectivity over sialyl oligosaccharide controls, whereas monovalent and tetraantennary SLex oligosaccharides failed to mediate specific kidney targeting. Simultaneous dosing of SLex biantennary or triantennary oligosaccharide with a mouse anti-E-selectin monoclonal antibody blocked kidney targeting, whereas co-administration with anti-P-selectin monoclonal antibody did not significantly block kidney targeting. The results suggest that SLex biantennary and triantennary are N-linked oligosaccharide ligands for E-selectin and implicate E-selectin as a bivalent receptor in the murine kidney endothelium.     

Molecular mechanisms underlying IL-4-induced leukocyte recruitment in vivo: a critical role for the alpha 4 integrin.

IL-4 is known to induce recruitment of eosinophils and mononuclear leukocytes. In vitro this occurs in part by selective expression of VCAM-1, the ligand for the alpha 4 integrin. The objective of this study was to determine the molecular mechanisms that underlie IL-4-induced leukocyte recruitment in vivo. Mice received an intrascrotal injection of IL-4 (100 ng). Twenty-four hours later, leukocyte rolling, adhesion, and emigration in cremasteric postcapillary venules were examined via intravital microscopy, and expression of VCAM-1 and P- and E-selectin was quantitated using a radiolabeled mAb technique. IL-4 increased VCAM-1 expression, but P-selectin and E-selectin remained at constitutive levels. IL-4 induced significant increases in leukocyte adhesion and emigration, with 50% of the emigrated cells being eosinophils and the remainder being mononuclear leukocytes. Leukocyte rolling in IL-4-treated mice was >95% inhibitable using an anti-P-selectin Ab. However, IL-4-induced leukocyte recruitment was unaltered in mice treated chronically with P-selectin Ab or mice deficient in either P-selectin or P- and E-selectin, suggesting that the residual rolling supported all of the IL-4-induced recruitment. In IL-4-treated mice following P-selectin blockade, tethering and rolling were not dependent on L-selectin, but were abolished by alpha 4 integrin blockade. These findings show that the alpha 4 integrin can initiate leukocyte-endothelial cell interactions in the absence of selectins under shear conditions in vivo, and that the absence of selectins does not affect recruitment of eosinophils and mononuclear cells to IL-4-treated tissue.     

Acquisition of P-selectin binding activity by en bloc transfer of sulfo Le(x) trisaccharide to the cell surface: comparison to a sialyl Le(x) tetrasaccharide transferred on the cell surface

Sialyl Le(x), NeuNAcalpha2 --> 3Galbeta1 --> 4(Fucalpha1 --> 3)GlcNAcbeta --> R, is known to be a ligand for E-selectin in various assays. The sulfated counterpart of sialyl Le(x), sulfo Le(x), (Sulfo --> 3) Galbeta1 --> 4 (Fucalpha1 --> 3) GlcNAcbeta --> R, was also shown to be a ligand for E-selectin in solid-phase assays employing immobilized oligosaccharides. In order to determine whether sulfo Le(x) structure on the cell surface also works as E-selectin or P-selectin ligand, a novel approach for in vitro transfer of oligosaccharides (S. Tsuboi, Y. Isogai, N. Hada, J. K. King, O. Hindsgaul, and M. Fukuda (1996) J. Biol. Chem. 271, 27213-27216) was utilized. A synthetic GDP-fucose harboring sialyl Le(x) or sulfo Le(x) oligosaccharide was enzymatically transferred to Chinese hamster ovary (CHO) cells with a milk fucosyltransferase. The resultant cells, CHO-sialyl Le(x) and CHO-sulfo Le(x) were tested for adhesion to E-selectin. IgG or P-selectin. IgG chimeric protein coated on plates. The results indicate that CHO-sialyl Le(x) adhered efficiently to E-selectin, while adhesion of CHO-sulfo Le(x) was very poor despite the fact that near equal number of the ligands had been attached to the cell surface. In contrast, CHO-sulfo Le(x) adhered efficiently to P-selectin, while CHO-sialyl Le(x) adhered modestly to P-selectin. These results demonstrate that sialyl Le(x) and sulfo Le(x) structures on the cell surface differ substantially in their ability to adhere to E- and P-selectin.     

Selective intracellular delivery of dexamethasone into activated endothelial cells using an E-selectin-directed immunoconjugate.

In chronic inflammatory diseases, the endothelium is an attractive target for pharmacological intervention because it plays an important role in leukocyte recruitment. Hence, inhibition of endothelial cell activation and consequent leukocyte infiltration may improve therapeutic outcome in these diseases. We report on a drug targeting strategy for the selective delivery of the anti-inflammatory drug dexamethasone to activated endothelial cells, using an E-selectin-directed drug-Ab conjugate. Dexamethasone was covalently attached to an anti-E-selectin Ab, resulting in the so-called dexamethasone-anti-E-selectin conjugate. Binding of the conjugate to E-selectin was studied using surface plasmon resonance and immunohistochemistry. Furthermore, internalization of the conjugate was studied using confocal laser scanning microscopy and immuno-transmission electron microscopy. It was demonstrated that the dexamethasone-anti-E-selectin conjugate, like the unmodified anti-E-selectin Ab, selectively bound to TNF-alpha-stimulated endothelial cells and not to resting endothelial cells. After binding, the conjugate was internalized and routed to multivesicular bodies, which is a lysosome-related cellular compartment. After intracellular degradation, pharmacologically active dexamethasone was released, as shown in endothelial cells that were transfected with a glucocorticoid-responsive reporter gene. Furthermore, intracellularly delivered dexamethasone was able to down-regulate the proinflammatory gene IL-8. In conclusion, this study demonstrates the possibility to selectively deliver the anti-inflammatory drug dexamethasone into activated endothelial cells, using an anti-E-selectin Ab as a carrier molecule.     

Expression of inducible cell adhesion molecules in the normal human lung: immunohistochemical study of their distribution in pulmonary blood vessels

 The distribution of cell adhesion molecules in the normal human lung was investigated using antibodies to E-selectin, P-selectin, intercellular adhesion molecule 1 (ICAM-1), and vascular cell adhesion molecule 1 (VCAM-1). Lectin staining by Ulex europaeus type I agglutinin (UEA I) and immunohistochemistry for von Willebrand factor (vWF) was used to visualize a maximum of blood vessels per section. In the bronchial mucosa, staining for P-selectin was positive in ca 90%, and staining for E-selectin, VCAM-1, and ICAM-1 was positive in 40–70% of the vessels stained with UEA I. In the pulmonary circulation (vasa publica) ca 90% of non-capillary vessels stained by anti-vWF expressed P-selectin, 54% VCAM-1, 41% E-selectin, and only ca 20% ICAM 1. The alveolar capillaries were stained consistently by UEA I, but not by the panel of antibodies tested. The alveolar epithelium and, inconstantly, basal cells of the bronchial epithelium were positive for ICAM-1. The distribution pattern of inducible adhesion molecules in normal human lung tissue suggests that a permanent low-grade endothelial activation may exist in particular in the mucosa of the airways, which could be due to the normal antigen exposure via inhaled air. Accepted: 28 April 1998     

Leukocyte and endothelial cell adhesion molecules in a chronic murine model of myocardial reperfusion injury

Expression of endothelial and leukocyte cell adhesion molecules is a principal determinant of polymorphonuclear neutrophil (PMN) recruitment during inflammation. It has been demonstrated that pharmacological inhibition of these molecules can attenuate PMN influx and subsequent tissue injury. We determined the temporal expression of alpha-granule membrane protein-40 (P-selectin), endothelial leukocyte adhesion molecule 1 (E-selectin), and intercellular cell adhesion molecule 1 (ICAM-1) after coronary artery occlusion and up to 3 days of reperfusion. The expression of all of these cell adhesion molecules peaked around 24 h of reperfusion. We determined the extent to which these molecules contribute to PMN infiltration by utilizing mice deficient (-/-) in P-selectin, E-selectin, ICAM-1, and CD18. Each group underwent 30 min of in vivo, regional, left anterior descending (LAD) coronary artery ischemia and 24 h of reperfusion. PMN accumulation in the ischemic-reperfused (I/R) zone was assessed using histological techniques. Deficiencies of P-selectin, E-selectin, ICAM-1, or CD18 resulted in significant (P < 0.05) attenuation of PMN infiltration into the I/R myocardium (MI/R). In addition, P-selectin, E-selectin, ICAM-1, and CD18 -/- mice exhibited significantly (P < 0.05) smaller areas of necrosis after MI/R compared with wild-type mice. These data demonstrate that MI/R induces coronary vascular expression of P-selectin, E-selectin, and ICAM-1 in mice. Furthermore, genetic deficiency of P-selectin, E-selectin, ICAM-1, or CD18 attenuates PMN sequestration and myocardial injury after in vivo MI/R. We conclude that P-selectin, E-selectin, ICAM-1, and CD18 are involved in the pathogenesis of MI/R injury in mice.     

Translational inhibition of E-selectin expression stimulates P-selectin-dependent neutrophil recruitment

Although known for its role in hemostasis, there is a growing body of evidence that thrombin can induce leukocyte recruitment and contribute to the inflammatory response. An in vitro parallel-plate flow chamber was used to systematically examine thrombin-induced neutrophil interactions with human endothelium. Stimulation of endothelial cells with thrombin (1 U/ml) resulted in an immediate, P-selectin-dependent increase in neutrophil rolling and adhesion that was comparable in magnitude to optimal levels of histamine (the classical inducer of P-selectin). However, thrombin, but not histamine, induced a delayed (4 h) E-selectin-dependent rolling similar to that of tumor necrosis factor-alpha, suggesting that thrombin has the unique ability to recruit neutrophils by an early P-selectin and a delayed E-selectin pathway. Surprisingly, inhibition of E-selectin expression with the general protein synthesis inhibitor cycloheximide induced P-selectin expression 4 h after thrombin stimulation. Cycloheximide and thrombin (4 h) induced sufficient P-selectin-dependent rolling to recruit as many neutrophils as were recruited with 4 h of stimulation with thrombin alone. Histamine in the presence of cycloheximide or cycloheximide alone did not evoke the P-selectin response at 4 h, suggesting that this was not due to direct cycloheximide induction of P-selectin. Treatment of endothelium with tumor necrosis factor-alpha (an E-selectin inducer) and cycloheximide also eliminated E-selectin expression but, much like thrombin, induced P-selectin expression and neutrophil recruitment. In conclusion, inhibition of E-selectin via protein synthesis inhibition activates the protein synthesis-independent pathway of P-selectin expression to support adequate leukocyte recruitment.     

The anti-inflammatory peptides, antiflammins, regulate the expression of adhesion molecules on human leukocytes and prevent neutrophil adhesion to endothelial cells.

Antiflammin-1 and antiflammin-2 are nonapeptides corresponding to the region of highest similarity between glucocorticoid-inducible proteins lipocortin-1 and uteroglobin. We have studied whether antiflammins could affect expression of adhesion molecules on human leukocytes and coronary artery endothelial cells (HCAEC) and binding of neutrophils (PMNs) to HCAEC. Although neither antiflammin-1 nor antiflammin-2 affected expression of adhesion molecules on resting PMNs, monocytes, and lymphocytes in whole blood, they attenuated changes in L-selectin and CD11/CD18 expression evoked by platelet-activating factor or interleukin-8 with IC(50) values of 4-20 micromol/l. The maximum inhibition was similar to those seen with human recombinant lipocortin-1 (100 microgram/ml). Unlike dexamethasone (100 nmol/l), the antiflammins had little effect on LPS-stimulated expression of E-selectin and ICAM-1 on HCAEC. Consistently, culture of HCAEC with dexamethasone, but not with antiflammins, decreased PMN binding to endothelial cells. Preincubation of PMNs with antiflammins markedly decreased their adhesion to LPS-activated HCAEC. Inhibition of adhesion was additive with function blocking anti-E-selectin and anti-L-selectin antibodies, but was not additive with anti-CD18 antibody. These results show that antiflammins inhibit PMN adhesion to HCAEC by attenuating activation-induced up-regulation of CD11/CD18 expression on leukocytes, and suggest that antiflammins may represent a novel therapeutic approach in blocking leukocyte trafficking in host defense and inflammation.     

P-selectin glycoprotein ligand 1 is a ligand for L-selectin on neutrophils, monocytes, and CD34+ hematopoietic progenitor cells

Selectins play a critical role in initiating leukocyte binding to vascular endothelium. In addition, in vitro experiments have shown that neutrophils use L-selectin to roll on adherent neutrophils, suggesting that they express a nonvascular L-selectin ligand. Using a L- selectin/IgM heavy chain (mu) chimeric protein as an immunocytological probe, we show here that L-selectin can bind to neutrophils, monocytes, CD34+ hematopoietic progenitors, and HL-60 and KG-1 myeloid cells. The interaction between L-selectin and leukocytes was protease sensitive and calcium dependent, and abolished by cell treatment with neuraminidase, chlorate, or O-sialoglycoprotein endopeptidase. These results revealed common features between leukocyte L-selectin ligand and the mucin-like P-selectin glycoprotein ligand 1 (PSGL-1), which mediates neutrophil rolling on P- and E-selectin. The possibility that PSGL-1 could be a ligand for L-selectin was further supported by the ability of P-selectin/mu chimera to inhibit L-selectin/mu binding to leukocytes and by the complete inhibition of both selectin interactions with myeloid cells treated with mocarhagin, a cobra venom metalloproteinase that cleaves the amino terminus of PSGL-1 at Tyr-51. Finally, the abrogation of L- and P-selectin binding to myeloid cells treated with a polyclonal antibody, raised against a peptide corresponding to the amino acid residues 42-56 of PSGL-1, indicated that L- and P-selectin interact with a domain located at the amino- terminal end of PSGL-1. The ability of the anti-PSGL-1 mAb PL-1 to inhibit L- and P-selectin binding to KG-1 cells further supported that possibility. Thus, apart from being involved in neutrophil rolling on P- and E-selectin, PSGL-1 also plays a critical role in mediating neutrophil attachment to adherent neutrophils. Interaction between L- selectin and PSGL-1 may be of major importance for increasing leukocyte recruitment at inflammatory sites.     

Mice lacking two or all three selectins demonstrate overlapping and distinct functions for each selectin.

Selectins support the capture and rolling of leukocytes in venules at sites of inflammation and in lymphocyte homing. Gene-targeted mice with null mutations at the L-, E-, or P-selectin locus develop normally and show mild (E-/-) to moderate (P-/-, L-/-) defects in inflammatory cell recruitment. Mice lacking both P- and E-selectin (E/P-/-) have severe neutrophilia and spontaneous skin infections that limit their life span. Other combinations of selectin deficiency have not been investigated. We have generated novel mice lacking L- and P-selectin (L/P-/-), L- and E-selectin (L/E-/-), or all three selectins (E/L/P-/-) by bone marrow transplantation. L/P-/- mice (only E-selectin present) show an absence of leukocyte rolling after trauma and severely reduced rolling (by approximately 90%) in inflammation induced by TNF-alpha. Residual rolling in L/P-/- mice was very slow (3.6 +/- 0.2 micrometers/s after TNF-alpha). L/E-/- mice (only P-selectin present) showed rolling similar to that of L-/- at increased velocities (15.1 +/- 0.3 micrometer/s). The number of adherent leukocytes after 2 or 6 h of TNF-alpha treatment was not significantly reduced in L/E-/- or L/P-/- mice. E/L/P-/- mice showed very little rolling after TNF-alpha, all of which was blocked by mAb to alpha4 integrin. Adherent and emigrated neutrophils were significantly reduced at 6 h after TNF-alpha. We conclude that any one of the selectins can support some neutrophil recruitment but eliminating all three selectins significantly impairs neutrophil recruitment.