P-Selectin support of neonatal neutrophil adherence under flow: contribution of L-selectin, LFA-1, and ligand(s) for P-selectin

To further define the neonatal neutrophil's ability to localize to inflamed tissue compared with adult cells, we examined the neonatal neutrophil interactions with P-selectin monolayers under two conditions: (1) attachment under constant shear stress and flow and (2) detachment where cells were allowed to attach in the absence of shear stress and then shear stress is introduced and increased in step-wise increments. Cord blood and adult neutrophils had minimal interactions with unstimulated human umbilical vein endothelial cells (HUVECs) at a constant shear stress of 2 dynes/cm2. There was a marked increase in the number of both neonatal and adult cells interacting (interacting cells = rolling + arresting) with HUVECs after histamine stimulation, although the neonatal value was only 40% of adult (P < .05). Neonatal neutrophils also had significantly decreased interaction with monolayers of Chinese hamster ovary (CHO) cells transfected with human P-selectin (CHO-P-selectin; 60% of adult values, P < .003). Of the interacting cells, there was a lower fraction of neonatal cells that rolled compared with adult cells on both stimulated HUVECs and CHO-P-selectin. That neonatal neutrophil L-selectin contributes to the diminished attachment to P-selectin is supported by the following: (1) Neonatal neutrophils had significantly diminished expression of L-selectin. (2) Anti-L-selectin monoclonal antibody reduced the number of interacting adult neutrophils to the level seen with untreated neonatal neutrophils, but had no effect on neonatal neutrophils. In contrast, L-selectin appeared to play no role in maintaining the interaction of either neonatal or adult neutrophils in the detachment assay. Once attachment occurred, the neonatal neutrophil's interaction with the P-selectin monolayer was dependent on LFA-1 and to other ligands to a lesser degree based on the following: (1) Control neonatal neutrophils had decreased rolling fraction compared with adult neutrophils, although the total number of interacting neutrophils was equal between groups. (2) Anti-LFA-1 treatment resulted in an increase in the rolling fraction of both neonatal and adult neutrophils. However, whereas the number of interacting adult neutrophils remained unchanged, the number of neonatal neutrophils decreased with increased shear stress. We speculate that this increased detachment of neonatal cells is due to differences in neutrophil ligand(s) for P-selectin.     

L-selectin-mediated lymphocyte rolling on MAdCAM-1

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

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

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

Regulation of L-selectin-mediated rolling through receptor dimerization

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

In vivo neutrophil and lymphocyte function studies in a patient with leukocyte adhesion deficiency type II

We investigated in vivo neutrophil and lymphocyte function in a patient who lacks Sialyl-Lewis-X, a ligand for the selectin family of leukocyte adhesion molecules (leukocyte adhesion deficiency II, LAD II). As assessed by skin chamber and skin window techniques, in vivo chemotaxis of neutrophils was markedly impaired (less than 6% of normal values). A marginal pool was present as determined by an increase in circulating neutrophils after epinephrine injection and calculated recovery of infused radiolabeled autologous neutrophils. Kinetic studies showed a reduced half-life of 3.2 hours (normal 7 hours) and markedly increased turnover rate (cells/kg/d) of approximately eight times the normal value. A normal antibody response to the T-cell-dependent antigen bacteriophage phi X174 showed that T/B-cell interaction is not affected in LAD II. These findings provide direct evidence that the selectin family and its ligands play an important role in neutrophil function.     

L-selectin-dependent leukocyte adhesion to microvascular but not to macrovascular endothelial cells of the human coronary system

To characterize L-selectin-dependent cell adhesion to human vascular endothelium, human cardiac microvascular endothelial cells (HCMEC) and human coronary endothelial cells (HCEC) were isolated from explanted human hearts. The adhesion behavior of human (NALM-6) and mouse (300.19) pre-B cells transfected with cDNA encoding for human L-selectin was compared with that of the respective nontransfected cells in a flow chamber in vitro. More than 80% of the adhesion to tumor necrosis factor-alpha (TNF-alpha)-stimulated HCMEC at shear stresses >2 dyne/cm2 was L-selectin dependent and could be equally well blocked by an anti-L-selectin antibody or a L-selectin-IgG-chimera. No L-selectin dependent adhesion to HCEC could be shown. The L-selectin dependent adhesion to HCMEC was insensitive to neuraminidase, but greatly inhibited by addition of NaClO3, which inhibits posttranslational sulfation and remained elevated for at least 24 hours of stimulation. E-selectin dependent adhesion of HL60 cells to HCMEC was blocked by neuraminidase, but not by NaClO3 and returned to control levels within 18 hours of HCMEC stimulation. It is concluded that microvascular, but not macrovascular endothelial cells express TNF-alpha-inducible sulfated ligand(s) for L-selectin, which differ from known L-selectin ligands, because sialylation is not required. The prolonged time course of L-selectin dependent adhesion suggests a role in sustained leukocyte recruitment into inflammatory sites in vivo.     

The Fcgamma receptor-mediated respiratory burst of rolling neutrophils to cytokine-activated, immune complex-bearing endothelial cells depends on L-selectin but not on E-selectin

Intracellular H2O2 generation, as a measure of the respiratory burst, was determined after stimulation of neutrophils by immune complex (IC)-bearing human umbilical vein endothelial cells. Under static conditions, neutrophils basically responded to the immune deposits on resting endothelial cells. The rotating shear forces of approximately 0.7 dynes/cm2, corresponding to the physiological flow in postcapillary venules, completely abolished this basal H2O2 generation. After activation of the IC-bearing endothelial layers with interleukin-1 (IL-1) or tumor necrosis factor (TNF), or both, for 4 hours, rolling adhesion of the neutrophils was induced, accompanied by considerable H2O2 production. The neutrophil respiratory burst was prominently inhibited by anti-FcgammaRIII MoAb 3G8 (72.4%), and partially by MoAb 2E1 against FcgammaRII (38.5%). Both MoAbs together inhibited the Fc-mediated H2O2 generation by 93. 4%. The respiratory burst and rolling adhesion were markedly blocked by MoAb LAM1-3 against L-selectin (91.3%), whereas the nonfunctional anti-L-selectin MoAb LAM1-14 was ineffective. F(ab)2' fragments of MoAb 7A9 against E-selectin inhibited neutrophil rolling by 98.6%, but not the respiratory burst. Moreover, rolling adhesion of neutrophils and the related oxidative burst were CD11b/CD18- independent. In summary, L-selectin has a unique auxiliary function in triggering the FcgammaR-mediated respiratory burst of rolling neutrophils to IC-bearing endothelial cells, thereby substituting CD11b/CD18 under conditions of flow.     

The kinetics and shear threshold of transient and rolling interactions of L-selectin with its ligand on leukocytes

The kinetics of rolling and transient adhesions through selectins may depend on the kinetics and mechanical properties of the selectin:ligand bond, as well as on cellular properties including receptor-anchoring to the cell membrane and cytoskeleton. Kinetics are known to depend on the selectin and may also be ligand dependent. Here, we study the kinetics of transient and rolling interactions of leukocytes with L-selectin immobilized on a substrate. Remarkably, all properties examined are similar to those seen when the sidedness is opposite, i.e., when the L-selectin ligand is on the substrate and when the ligand is isolated from HEV rather than present on leukocytes. The similar properties include rolling velocity, a threshold shear stress above 0.4 dyn/cm2 required to support rolling, a k degreesoff of 7.0 to 6.8 s-1 for the L-selectin tether bond, and a mechanical bond length of 0.24 to 0.20 A. Our results argue against a model in which L-selectin shedding mediates rolling. Furthermore, the fast and force-resistant kinetic properties suggest that L-selectin is specialized dynamically for tethering leukocytes to vessel walls and adherent leukocytes.     

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

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

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

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

Laboratory values improve predictions of hospital mortality

Migration of circulating neutrophils occurs in several steps: capture and rolling adhesion are followed by activation of beta 2-integrins and immobilisation, and then neutrophils move over and through the endothelium. However, it is not clear how the underlying mechanisms and completion of each step depend on the concentration of stimulatory cytokines such as tumour necrosis factor-alpha (TNF). We therefore perfused neutrophils over human umbilical vein endothelial cells (HUVEC) which had been cultured with varying concentration of TNF (1-1000 U/ml) for 4 h, and recorded adhesion and migration by videomicroscopy. The number of adherent neutrophils increased with increasing TNF up to 5 U/ml, but changed little at higher concentrations. Interestingly, rolling adhesion at first predominated, but an increasing proportion of adherent cells became immobilised and migrated through the HUVEC monolayer over the complete TNF range. Immobilisation was inhibited by treating neutrophils with antibody against CD18, so that the major change in adhesive behaviour at higher levels of TNF occurred because the surface of the HUVEC presented agent(s) able to activate neutrophil beta 2-integrins. It was also evident that the selectins initiating capture of flowing neutrophils varied with concentration of TNF. At 100 U/ml TNF, both E-selectin and P-selectin supported capture and rolling adhesion, and antibody blockade of both receptors was required to inhibit adhesion. At lower dose (10 U/ml TNF), stable adhesion was blocked by antibody against E-selectin, although short-lived attachments could still be seen which were inhibited by antibody against P-selectin. Expression of sclectins increased with increasing concentration of TNF, judging from surface ELISA and reduction in the velocity of rolling adherent cells. Thus the efficiency of capture, the selectins mediating capture and the proportion of captured cells immobilised and migrating all depend on the concentration of TNF to which endothelial cells are exposed. These results suggest a model in which highly localised and efficient migration of neutrophils is achieved if a concentration gradient of TNF exists around an inflammatory locus.     

Novel anti-inflammatory compounds induce shedding of L-selectin and block primary capture of neutrophils under flow conditions

Leumedins are small molecules that inhibit neutrophil movement into inflamed tissues. These compounds have been shown to inhibit the adherence of neutrophils in static adhesion assays mediated by beta2-integrins. We now report that leumedins, like activating agents, induce the loss of L-selectin from the neutrophil surface. The loss of L-selectin is unrelated to the inhibition of static adhesion, since neutrophils that have been pretreated with leumedins to cause shedding of L-selectin, followed by removal of drug, adhere normally in a static adhesion assay, and this adhesion is inhibited upon readdition of leumedin. In an assay of adhesion to endothelial cells under conditions of physiologic wall shear stress, leumedins prevent both primary capture of neutrophils mediated by L-selectin and firm adherence mediated by beta2-integrins.     

Characterization of an adhesion molecule that mediates leukocyte rolling on 24 h cytokine- or lipopolysaccharide-stimulated bovine endothelial cells under flow conditions

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.     

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

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

POP66, a paraneoplastic encephalomyelitis-related antigen, is a marker of adult oligodendrocytes

L-selectin on lymphocytes reacts with glycosylated ligands on high endothelial venule walls in lymphoid organs. Through this carbohydrate-dependent interaction, rolling and initial attachment of lymphocytes to endothelium is mediated. Here we have studied an earlier described L-selectin-induced homotypic aggregation, to further elucidate the events that occur after engagement of L-selectin. It was found that the interaction of L-selectin with fucoidan, but not with other carbohydrates, or with monoclonal antibodies directed against the carbohydrate recognition domain of L-selectin, resulted in homotypic aggregation among both B- or T lymphocytes. Importantly, this aggregation was shown to be both lymphocyte function-associated antigen-1 (LFA-1) and calcium-independent. Furthermore, for aggregation metabolic energy was required, and signalling via protein tyrosine kinase appeared to be involved. Neither de novo protein synthesis, protein kinase C mediated signalling, Gi-protein mediated signal transduction, nor calcium mobilization were required for aggregation. During aggregation, L-selectin was not shed from the lymphocyte's cell surface. Finally, it was found that the lymphocyte binding capacity to high endothelial venules on cryostat sections was not altered upon triggering these lymphocytes via L-selectin. Interestingly, L-selectin-triggered cells showed increased binding to paracortical areas in peripheral lymph nodes. Our data suggest that signals via L-selectin, might lead to altered expression of cell surface molecules, important in interactions other than the first stage of lymphocyte rolling.     

Simultaneous determination of primidone and its three major metabolites in rat urine by high-performance liquid chromatography using solid-phase extraction

Sialosyl-fucosyl poly-LacNAc without sialosyl-Lex epitope in myeloid cell line HL60 was shown to be the ligand for E-selectin-dependent adhesion, particularly under dynamic flow conditions, in our previous study (Handa K, Stroud MR, Hakomori S, Biochemistry 36, 12412-12420, 1997). HL60 cells express only fucosyl-transferase (FT) IV and VII. X3NeuAcVII3FucnLc10, a representative component showing E-selectin-dependent binding under dynamic flow conditions, is not alpha 1-->3 fucosylated at the penultimate GlcNAc catalyzed by FT-VII, but is alpha 1-->3 fucosylated at the internal GlcNAc catalyzed by FT-IV. VI3NeuAcnLc6 is converted to VI3NeuAcIII3FucnLc6 by FT-IV, but is also converted to VI3NeuAcV3FucnLc6 by FT-VII. Thus, penultimate fucosylation catalyzed by FT-VII is not restricted for nLc6 backbone, but is highly restricted for nLc10 backbone. The cooperative effect of FT-IV and FT-VII for synthesis of poly-LacNAc having sialosyl-Lex with internal fucosylation may be blocked or highly restricted in poly-LacNAc having more than two LacNAc units, because preferential alpha 1-->3 fucosylation by FT-IV takes place at internal GlcNAc, inhibiting penultimate fucosylation by FT-VII.     

Core fucosylation of high-mannose-type oligosaccharides in GlcNAc transferase I-deficient (Lec1) CHO cells

During studies on the fucosylation of endogenous proteins in parental (Pro5) and N-acetyl-D-glucosamine (GlcNAc) transferase I-deficient (Lec1) Chinese hamster ovary (CHO) cells, we observed that Lec1 cells incorporate approximately 10-fold less [3H]fucose into macromolecules than Pro5 cells. Interestingly, most of the labelled oligosaccharides from both cell types could be released from the macromolecules by digestion with peptide N-glycosidase F (PNGase F). This was unexpected for Lec1 cells because they do not synthesize complex- or hybrid-type N-glycans. Structural analyses of the fucosylated oligosaccharides from Lec1 cells showed the fucose to be in an alpha 1,6 linkage to the core GlcNAc of relatively small oligomannose N-glycans (Man4GlcNAc2 and Man5GlcNAc2, where Man is D-mannose). Comparing the sizes of oligomannose N-glycans from Pro5 and Lec1 cells demonstrated a much higher proportion of the small (Man4GlcNAc2 and Man5GlcNAc2) oligomannose species in Lec1 cells. These results suggest that the core alpha 1,6 fucosyltransferase will fucosylate small (Man4-Man5GlcNAc2), but not large (Man8-Man9GlcNAc2) oligomannose N-glycans.     

CD24 mediates rolling of breast carcinoma cells on P-selectin

P-selectin mediates rolling of neutrophils and other leukocytes on activated endothelial cells and platelets through binding to P-selectin glycoprotein ligand-1 (PSGL-1). Certain PSGL-1 negative tumor cell lines can bind P-selectin under static conditions through the GPI-linked surface mucin, CD24, but the physiological significance of this interaction and whether it can occur under flow conditions is not known. Here, we show that CD24+ PSGL-1- KS breast carcinoma cells attach to and roll on recombinant P-selectin under a continuous wall shear stress, although at a lower density and higher velocity than CD24+ PSGL-1+ cells, such as HL-60. Adding excess soluble CD24 or removing CD24 from the cell surface with phosphatidylinositol-phospholipase C (PI-PLC) significantly reduced KS cell rolling on P-selectin. The ability of KS cells to roll on P-selectin was positively correlated with the CD24 expression level. Comparison with three other CD24+ cell lines established that expression of sialyl-Lewis(x) antigen was also necessary for CD24-mediated rolling on P-selectin. CD24 purified from KS cells supported rolling of P-selectin transfectants, but not L-selectin transfectants. Finally, KS cells rolled on vascular endothelium in vivo in a P-selectin-dependent manner. Together our data show that CD24 serves as a ligand for P-selectin under physiological flow conditions. Interaction of tumor cells with P-selectin via CD24 may be an important adhesion pathway in cancer metastasis.     

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

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