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.

THE LOCALIZATION OF neutrophils to vascular sites of inflammation involves several processes, including cell capture, rolling, activation, and arrest.1 This coordinated series of events is mediated by three families of adhesion receptors: selectins, integrins, and the Ig gene superfamily. Selectins, consisting of L-selectin (CD62-L) on neutrophils and E-selectin (CD62-E) and P-selectin (CD62-P) on endothelial cells, have been shown to mediate capture and rolling but not arrest of neutrophils on endothelial cells under conditions of flow.2-4 Although L-selectin is constitutively present on circulating leukocytes,5,6 P-selectin and E-selectin expression is induced by several inflammatory cytokines.7-9 The ligands for the selectins have not been fully identified, although all contain specific carbohydrate moieties, such as sialylated-fucosylated lactosamines, which are critical components for binding.10,11 One ligand for P-selectin, P-selectin-glycoligand-1 (PSGL-1), has been identified as a sialomucin; ie, it has a large number of O-linked sugar chains clustered together on the polypeptide backbone.12 It is expressed on leukocytes, including neutrophils, monocytes, lymphocytes, and eosinosphils.13-15 PSGL-1 may also serve as a ligand for E-selectin and L-selectin.16-18 The CD18 integrins, LFA-1 (CD11a/CD18, αLβ2) and Mac-1(CD11b/CD18, αMβ2), mediate arrest and transmigration, but are unable to mediate capture from the flow stream at shear rates found in the postcapillary venule (>1 dynes/cm2).19,20 Intercellular adhesion molecule 1 (ICAM-1; CD54) is a member of the Ig gene superfamily. It is expressed constitutively on endothelial cells, although its expression increases when the endothelium is inflamed.21 ICAM-1 serves as a ligand for LFA-1 and Mac-1 and is required for leukocyte arrest and transmigration.22 

The susceptibility of human neonates to localized soft tissue infections as well as systemic infections due to bacterial or fungal agents has prompted extensive investigations of neonatal host defense mechanisms. Among the most consistently observed functional abnormalities are those related to leukocyte migration.23In vivo studies using Rebuck skin windows in human neonates have provided limited data suggesting that inflammatory responses, as reflected by leukocyte exudation, may differ from those in older children and adults.24 Studies in experimental animals have been more extensive. Newborn rabbits, rats, and primates have diminished leukoctye exudation into inflamed sites compared with adult animals.25-28 The basis for this appears multifactorial and includes diminished cell deformability, decreases in f-actin polymerization, abnormalities of microtubule assembly, as well as qualitative and quantitative defects in the cell surface adhesion receptors.29 

Several groups have demonstrated that the expression of Mac-1 on resting neonatal neutrophils is equal to that of adult neutrophils30-32; however, the total cell content of Mac-1 is decreased.32 In addition, neonatal neutrophils fail to upregulate Mac-1 surface expression to the same extent as adult neutrophils in response to chemotactic factor stimulation,26,30 and that which is present is functionally less active.26,30 Recently, Rebuk et al33 have reported a decrease in baseline expression of neonatal neutrophil Mac-1, but stimulated expression was equal to that of adult neutrophils. The discrepancies in these studies have not been well explained to date.33 More consistently reported is that neonatal neutrophil LFA-1 expression and function is equal to that of adult neutrophils.30,32-34 In addition, there appears to be a decreased expression of L-selectin on neonatal neutrophils.31,35 This decreased expression contributes to the diminished adherence of neonatal neutrophils to interleukin-1 (IL-1)–stimulated human umbilical vein endothelial cells (HUVECs) and monolayers of transfected cells expressing E-selectin under conditions of shear stress.4 35 

In the current study, we sought to determine if the impairment in the neonatal neutrophil's ability to localize to inflamed tissue could also be due to decreased interaction with P-selectin. Therefore, we investigated the interaction of neonatal neutrophils with monolayers expressing P-selectin under defined hydrodynamic shear stress.35 Using a parallel plate flow system, the monolayers can support neutrophil attachment, rolling, and arrest at shear rates of 2 dynes/cm2. We provide evidence here that neonatal neutrophils have markedly decreased interactions with P-selectin monolayers compared with adult neutrophils when cells must be captured from a free-flowing stream at 2 dynes/cm2. This appears to be due in part to lower levels of neonatal neutrophil L-selectin. We also demonstrate that, of the interacting neonatal neutrophils, a lower fraction is rolling, ie, they are arrested, compared with adult neutrophils. Using a detachment assay in which neutrophils attach in the absence of shear and then shear is introduced and increased in a step-wise fashion, we demonstrate that neonatal and adult neutrophils are equally able to resist detachment with increasing shear. However, the interacting neonatal cells have a lower fraction of rolling cells than adult neutrophils. Decreased rolling (and therefore increased arrest) of neonatal neutrophils is dependent on LFA-1. If the rolling fraction is increased by treatment with anti–LFA-1 monoclonal antibodies (MoAbs), neonatal cells are less able to maintain rolling interactions compared with adult neutrophils and detach. We speculate that, under these conditions, the neonatal ligand(s) for P-selectin may be functionally impaired.

Preparation of isolated neutrophils.

Venous blood was drawn from the placental cord of normal, full-term (gestational age, 38 to 41 weeks) neonates and from the peripheral veins of healthy adult donors. All neonates were products of an uncomplicated pregnancy delivered by planned caesarean section. Mothers of these neonates received epidural anesthesia for the delivery. None of the mothers were in active labor at the time of delivery. Apgar scores at 1 and 5 minute were ≥8. Blood samples were drawn immediately after birth. Informed consent was obtained from healthy adult donors. The protocol was approved by the Institutional Review Board for Human Experimentation at Baylor College of Medicine and St Luke's Episcopal Hospital.

Venous blood samples were anticoagulated with citrate phosphate dextrose (0.14 mL/mL blood: Abbot, North Chicago, IL) and sedimented in 6% (wt/vol in 0.87% NaCl) dextran (Spectrum Chemical, Gardena, CA) for 45 minutes at room temperature. Neonatal blood samples were diluted in Ca2+/Mg2+ free phosphate-buffered saline (PBS; GIBCO Laboratories, Grand Island, NY), pH 7.4, 1:1, before sedimentation. Leukocyte-rich plasma was layered on Ficoll-Hypaque gradients and centrifuged (300g for 20 minutes at room temperature), as previously described.35 The resulting granulocyte-erythrocyte pellets were washed and resuspended in PBS containing 0.2% dextrose (DPBS) at a concentration of 1 × 107 cells/mL.

In some experiments, isolated neutrophils were activated by incubation with the chemotactic tripeptide n-formyl-methionine-leucine-phenylalanine (fMLP; 10 nmol/L; Sigma, St Louis, MO) at room temperature for 15 minutes, as previously described.4 Activation of the neutrophil results in cleavage of the portion of L-selectin distal to the cell membrane and a change in cell shape from round to bipolar.5 However, bipolar cells are at a disadvantage when compared with unstimulated spherical neutrophils for adhesion and especially rolling under conditions of flow. Therefore, at the end of fMLP incubation, the cell suspension was diluted 10-fold with DPBS, washed to remove stimulant, and incubated for an additional 15 minutes in DPBS without fMLP. The abrupt decrease in concentration of chemotactic factor causes a reversal in neutrophil shape change from bipolar to slightly ruffed and spherical.4 

MoAbs.

For blocking experiments, intact antibody preparations were used. The anti–L-selectin antibody, DREG 56 (IgG1), was prepared as described and was the gift of Dr Takashi Kishimoto (Boehringer-Ingleheim Pharmaceuticals, Ridgefield, CT).36 The anti-CD11a, R7.1 (IgG1), and anti-CD18 MoAbs, R15.7 (IgG1), were prepared as described and were the gift of Dr Robert Rothlein (Boehringer-Ingleheim Pharmaceuticals).37,38 The control MoAbs, GAP8.3, an anti-CD45 (IgG1) and a nonblocking anti–LFA-1, TS2/4, were prepared from hybridoma supernatant, as was the anti-Mac-1, M1/70 (IgG2a; American Type Culture Collection [ATCC], Rockville, MD). All MoAbs directed against leukocyte adhesion markers were titered using flow cytometry (FACS-Scan; Becton Dickinson & Co, Mountain View, CA) to determine the concentration that saturated binding sites of unstimulated and stimulated cells as previously described.35 Fluorescein isothiocyanate (FITC)-labeled goat-antimouse antibody was used as second antibody (Jackson Immuno-Research Laboratories, West Grove, PA). A panel of MoAbs against ICAM-1 were used in the enzyme-linked immunosorbent assay (ELISA) and in the static adhesion assay. These included murine antihuman ICAM-1, R6.5 (IgG2a) and CA7 (IgG1) (both provided by Dr Robert Rothlein39) murine antirat ICAM-1, 1A29 (IgG1) (kind gift of M. Miyasaka, Tokyo Metropolitan Institute of Medical Science, Tokyo, Japan40); rat antimouse ICAM-1, YN-1 (IgG2b) (provided by M. Isobe, University of Tokyo, Tokyo, Japan41); anticanine ICAM-1, CL18/1 (IgG1) and CL18/6 (IgG1).42 Control antibodies included anti–P-selectin, Cytel 1747 (PB1.3, IgG143; a gift of Dr J. Paulson, Cytel Corp, San Diego, CA); antihuman L-selectin, DREG 200 (IgG1; a gift of Dr Takashi Kishimoto36); antihuman VCAM-1, CL40 (murine IgG1)44; antihuman E-selectin, CL2/6 (murine IgG2a),45 and 7A9 (murine IgG1) and antihuman HLA-A,B,C, W6/32 (IgG2a). The latter two MoAbs were produced from hybridomas purchased from ATCC. Fab fragments of R6.5 were prepared with an ImmunoPure Fab preparation kit (Pierce, Rockford, IL). Anti–L-selectin MoAb, FITC-labeled Leu-8 (FITC-Leu-8, IgG2b), and anti-CD11b, phycoerythrin (PE)-labeled Leu 15 (PE-Leu15, IgG2b), as well as isotype-matched fluorescent controls were purchased from Becton Dickinson.

Preparation of monolayers.

Endothelial cells were harvested from five to eight collagenase-treated umbilical cords, pooled, and plated on fibronectin-coated (1 mL of 5 μg/mL human plasma fibronectin for 30 minutes; GIBCO) 35-mm diameter tissue culture dishes at sufficiently high density to form a confluent monolayer without cell division, as previously reported.3Monolayers were cultured in M199 (GIBCO) supplemented with 15% fetal bovine serum (GIBCO-defined fetal bovine serum), hydrocortisone (1 μg/mL; Sigma), low molecular weight heparin (1 μg/mL; Sigma), gentamicin (25 μg/mL; Sigma), and amphotericin B (1.25 μg/mL as Fungizone; GIBCO). No growth factors were used. Cultures were maintained for 3 to 5 days at 37°C in a humidified atmosphere with 5% CO2.

CHO cells expressing a phosphatidylinositol-glycan–linked form of P-selectin (CHO-P-selectin) were a gift of Dr Christine Martens (Affymax Research Institute, Palo Alto, CA).46 These cells were plated onto coverdishes or a 96-well microtiter plate and allowed to reach confluence within 3 days. For the static adhesion assay, cells were plated onto glass coverslips that had been treated with 0.1% gelatin (Sigma) for 30 minutes.

Flow cytometry.

The CD11b and L-selectin expression levels of adult and neonatal neutrophils were determined by flow cytometry using PE-Leu-15 and FITC-Leu8. As cells were prepared for infusion into the adhesion assay flow chamber, an aliquot was reserved, immediately cooled to 4°C, and labeled with the antibodies or fluorescent isotype-matched controls. The cells were washed, and the erythrocytes were lysed and fixed (BD lysing reagent; Becton Dickinson). The mean fluorescent intensity (MFI) for 5,000 particles/sample was obtained using linear detection settings. The levels of L-selectin and CD11b for each cell type for each experiment were normalized against the value of the isotype-matched control (background).

Cell surface ELISA.

The expression of ICAM-1 or an ICAM-1–like molecule on CHO cells expressing P-selectin and nontransfected cells was determined by cell surface ELISA.47 The CHO cells were plated onto a 96-well plate. After confluence was reached, the plate was washed and fixed with 0.25% paraformaldehyde (Sigma) for 15 minutes at room temperature. The cells were then blocked with 2% bovine serum albumin (Sigma) for 2 hours at room temperature and labeled in duplicate with saturating concentrations of anticanine, antihuman, antirat, and antimurine ICAM-1; antihuman VCAM-1; antihuman L-selectin; antihuman HLA; and antihuman E-selectin MoAbs for 1 hour at 25°C. Bound antibody was detected by alkaline phosphatase-conjugated goat antimouse or antirat IgG (Sigma) with 1 mg/mL p-nitrophenyl phosphate disodium (Sigma) in 1 mol/L diethanolamine (Sigma; pH 9.8), containing 0.5 mmol/L MgCl2 (Sigma) as the substrate. The plates were read at 405 nm by an automatic microplate reader (Cambridge Technology, Waterford, MA).

Adhesion assay under static conditions.

A visual static adhesion assay has been described in detail previously.48 Briefly, CHO cell monolayers grown on 25-mm round glass coverslips were washed three times in PBS and immediately inserted into a modified Sykes-Moore Chamber. In selected experiments, monolayers were treated for 15 minutes with control or anti–ICAM-1 MoAbs and then rinsed. Untreated neutrophils or neutrophils treated with MoAb and/or chemotactic stimulus (10 nmol/L fMLP for 10 minutes at room temperature) were injected into the chamber and allowed to settle on to the monolayer for 500 seconds. The number of neutrophils in contact with the monolayer was determined by counting 2 to 3 high-power fields (40× objective). The chamber was then inverted for an additional 500 seconds so that only adherent cells remained attached to the monolayer. The number of cells was again counted in 3 to 10 fields. Results are expressed as the percentage of cells initially in contact with the monolayer that remained adherent per field.

Adherence assay under continuous flow: attachment assay.

Neutrophil interaction with histamine-stimulated HUVECs was assessed under continuous flow, as previously described.3,4 Briefly, primary seeded HUVECs were grown to confluence on fibronectin-coated 35-mm tissue culture dishes, rinsed in DPBS (with calcium and magnesium), mounted in parallel plate flow chambers, and perfused for 2 to 3 minutes with DPBS to remove all soluble factors. Histamine in PBS (final concentration, 10−4 mol/L; Sigma) was perfused for 10 minutes, at which point neutrophils were added to the feed line at a final concentration of 1 × 106/mL and perfusion was continued for an additional 10 minutes. The HUVECs were stimulated with histamine for the 20-minute duration of the experiment. This concentration of histamine resulted in maximal adhesion, with no effect on HUVEC monolayer confluence or neutrophil activation as determined by assessing neutrophil morphology. Neutrophils were either untreated or pretreated with MoAbs at saturating concentrations 15 minutes before being added to the feedline. fMLP-treated neutrophils were prepared as described earlier. Flow was maintained at a shear stress of approximately 2 dynes/cm2. A temperature-controlled Lucite box surrounding the microscope and flow chamber assured that all flow experiments were performed at 37°C. Interactions between neutrophils and the endothelial monolayer were observed by phase-contrast videomicroscopy (Diaphot-TMD microscope [Nikon, Inc, Garden City, NY] and CCD Video camera [Sony Corp, Park Ridge, NJ]) and quantified with a digital image processing system (Optimas; BioScan, Edmonds, WA). For each experiment, approximately 6 fields of view were recorded with a 20× objective at 7.5 minutes at 20 seconds per field. The total number of cells interacting with the monolayer were determined and referenced per square millimeter of the monolayer. For the purposes of the present study, interacting cells were defined as cells rolling at a velocity less than the flow stream plus those that were arrested. Rolling cells moved more than one cell diameter during a 1.0-second interval that was determined by time-lapsed digital subtraction techniques.4 The number of arrested cells was derived from the arithmetic difference between the number of interacting and rolling cells. Attachment assays with monolayers of CHO-P-selectin were performed essentially as described in the endothelial experiments, except that histamine perfusion was eliminated.

Adherence assay detachment under increasing shear stress.

To further characterize the neonatal neutrophil's adhesion to P-selectin, we assayed the strength of the neutrophil interaction with the monolayer in a detachment assay, as previously described.49 The number of neutrophils that remained interacting after a static incubation was quantified as shear stress was increased, giving a measure of the strength of adhesion to the monolayer. In this assay, cells were allowed to settle onto the CHO-P-selectin monolayer for 2 minutes in the absence of shear. The flow was begun (shear stress of 0.6 dynes/cm2) and then increased every 20 seconds to achieve stepwise increases in shear stress (1.4, 2.8, 12.2, and 22.1 dynes/cm2). The number of interacting cells were determined in three random fields in the final 10 seconds before the next increase in shear stress. Results are expressed as the percentage of neutrophils remaining interacting at that shear stress referenced to the number of neutrophils that had settled onto the monolayer in the absence of shear stress (percentage of settled cells remaining interacting). As in the attachment assay described above, interacting neutrophils included those that were rolling and those arrested (ie, not rolling greater than 1 cell diameter during a 1-second observation period). The rolling fraction of neutrophils at each shear stress was determined by dividing the number of rolling neutrophils by the total number interacting at that shear stress.

Statistical analyses.

Results are reported as mean ± SEM. Statistical assessments were performed as follows. The unpaired two-tailed Student's t-test was used to examine expression of adhesion molecules under different treatment conditions; as repeated testing was performed, significance was considered at P < .005. For static adhesion assay and attachment assays under flow conditions, a one-way analysis of variance (GraphPad Software, San Diego, CA) was performed. The probability of statistical significance between interactions of adult and neonatal neutrophils was determined by the Student-Newmann-Keuls test. Probability values less than .05 were considered significant. For detachment assays, a two-way ANOVA was performed to determine the significance of increasing shear stress on the interactions of adult and neonatal neutrophils. Significance was set at P < .05.

Neonatal neutrophils have less interaction with monolayers expressing P-selectin.

We initially examined the ability of the neonatal neutrophil to be captured by monolayers expressing P-selectin under hydrodynamic shear stress of 2 dynes/cm2. Both adult and neonatal neutrophils had minimal interaction with unstimulated HUVECs (9.8 ± 2.9v 29.3 ± 18.2 cells/mm2, respectively). Histamine causes rapid mobilization of P-selectin from the Weibel-Palade bodies to the surface of the endothelial cells and markedly increases neutrophil adhesion.3,50 In the present study, as seen in Fig 1, we also demonstrated a marked increase in the number of interacting adult neutrophils (499 ± 133 cells/mm2) to histamine-stimulated HUVECs. The peak number of interacting neutrophils occurred 5 to 10 minutes after the addition of cells and 47% ± 11% of the neutrophils exhibited rolling behavior. Arrested neutrophils did not roll for the 1-second observation period. None of the arrested neutrophils migrated through the monolayer.3There was also an increase in the number of neonatal neutrophils interacting with histamine-treated HUVECs compared with nonstimulated HUVECs, although there were significantly fewer neonatal neutrophils interacting than adult neutrophils (192 ± 63 cells/mm2, P < .05, Fig 1). The percentage of neonatal neutrophils that rolled during the observation period was 27% ± 6%. Rolling velocity was similar between neonatal and adult neutrophils (adult, 36.5 ± 5.8 μm/sec; neonate, 31.8 ± 5.4 μm/sec).

Fig. 1.

Adult (□) and neonatal (▨) neutrophil adhesion to HUVECs stimulated for 10 minutes with 10−4 mol/L histamine before the addition of neutrophils under shear stress of approximately 2 dynes/cm2. The total number of interacting neutrophils per square millimeter of monolayer includes both rolling and arrested cells. The number of interacting neutrophils was quantified beginning 7.5 minutes after the neutrophil suspension was introduced into the chamber. Neutrophils were left untreated (control) or were preincubated with anti–L-selectin MoAb, DREG 56 (50 μg/mL), or treated with 10 nmol/L fMLP as described. Data are expressed as the mean ± SEM from 7 to 11 experiments. *P < .05 compared with adult control neutrophils.

Fig. 1.

Adult (□) and neonatal (▨) neutrophil adhesion to HUVECs stimulated for 10 minutes with 10−4 mol/L histamine before the addition of neutrophils under shear stress of approximately 2 dynes/cm2. The total number of interacting neutrophils per square millimeter of monolayer includes both rolling and arrested cells. The number of interacting neutrophils was quantified beginning 7.5 minutes after the neutrophil suspension was introduced into the chamber. Neutrophils were left untreated (control) or were preincubated with anti–L-selectin MoAb, DREG 56 (50 μg/mL), or treated with 10 nmol/L fMLP as described. Data are expressed as the mean ± SEM from 7 to 11 experiments. *P < .05 compared with adult control neutrophils.

Close modal

We also examined the adhesion of adult and neonatal neutrophils to confluent monolayers of CHO cells stably transfected with a phosphatidylinositol-glycan–linked form of human P-selectin (CHO-P-selectin).46 As demonstrated with histamine-stimulated HUVECs, there were significantly fewer neonatal neutrophils interacting with the CHO-P-selectin than adult neutrophils (P < .01; Fig 2). Whereas 63% ± 6% of adult neutrophils rolled, only 32% ± 8% of neonatal neutrophils did so (P < .01). Rolling velocity was less on CHO-P-selectin than on histamine-stimulated HUVECs for both neonatal and adult neutrophils, although there was no difference between the groups (neonate, 10.9 ± 1.6 μm/sec; adult, 8.6 ± 0.7 μm/sec).

Fig. 2.

Adult (□) and neonatal (▨) neutrophil adhesion on CHO cells stably transfected with human P-selectin under shear stress of approximately 2 dynes/cm2. The total number of neutrophils per square millimeter includes both rolling and arrested cells. Neutrophil treatment and analysis were performed as outlined in Fig 1. Data are expressed as the mean ± SEM from 5 to 13 experiments. *P < .003 compared with adult control neutrophils.

Fig. 2.

Adult (□) and neonatal (▨) neutrophil adhesion on CHO cells stably transfected with human P-selectin under shear stress of approximately 2 dynes/cm2. The total number of neutrophils per square millimeter includes both rolling and arrested cells. Neutrophil treatment and analysis were performed as outlined in Fig 1. Data are expressed as the mean ± SEM from 5 to 13 experiments. *P < .003 compared with adult control neutrophils.

Close modal
Contribution of L-selectin to adult and neonatal neutrophil adhesion to P-selectin under continuous shear conditions.

We and others have published that L-selectin is important for attachment of neutrophils to P-selectin–bearing substrates.3,51 Additionally, we demonstrated that neonatal neutrophils have decreased expression of L-selectin, resulting in decreased adhesion of neonatal neutrophils to IL-1–stimulated HUVECs (which expresses both E-selectin and the L-selectin ligand35) as well as transfected cell monolayers expressing E-selectin.4 We hypothesized therefore that the decreased interaction of neonatal neutrophils with P-selectin monolayers under continuous shear conditions could at least partly be due to decreases in expression of L-selectin. Neonatal neutrophils obtained for our current studies also had significantly less L-selectin than adult neutrophils (Table 1). Neutrophil L-selectin function was inhibited by either blocking with the anti–L-selectin MoAb, DREG 56, or by stimulating with the chemotactic factor fMLP, which results in the cleavage of the extracellular portion of L-selectin.5 Incubation of either neonatal or adult neutrophils with the anti–L-selectin MoAb had no significant effect on the expression of the L-selectin epitope recognized by the MoAb, Leu8, or on the expression of Mac-1 (CD11b/CD18; Table 1). Stimulation of both neonatal and adult neutrophils with fMLP significantly decreased L-selectin and increased Mac-1 expression (P < .001v unstimulated controls for both neonatal and adult neutrophils).

Treatment with anti–L-selectin MoAb resulted in a 64% decrease in the number of adult neutrophils interacting with histamine-stimulated HUVECs (P < .05; Fig 1) and a 40% decrease in the number on CHO-P-selectin compared with untreated adult cells (P < .01; Fig 2). The number of treated adult neutrophils interacting with either histamine-stimulated HUVECs or CHO-P-selectin was equal to that of untreated neonatal neutrophils. Treatment of neonatal neutrophils with the anti–L-selectin MoAb did not decrease the number of interacting cells (Fig 1). Although there was marked loss of functional L-selectin from the surface of both neonatal and adult neutrophils with chemotactic factor stimulation (Table 1), there was no further decrease in the number of either neonatal or adult neutrophils interacting with histamine-stimulated HUVECs compared with DREG 56-treated neutrophils (Fig 1).

Neonatal neutrophils resist detachment from P-selectin with increased shear stress, but have different rolling behaviors than adult neutrophils.

We next examined the resistance to detachment of neonatal and adult neutrophils to P-selectin substrates (see detachment assay in the Materials and Methods). In this protocol, neutrophils were allowed to settle onto the CHO-P-selectin monolayer for 2 minutes in the absence of shear stress. Shear stress was then applied in a stepwise fashion from 0.6 to 22 dynes/cm2 every 20 seconds. The number of interacting neutrophils were counted at the end of each 20-second period in three fields and referenced to the number of neutrophils that had settled onto the monolayer in the absence of shear stress (percentage of settled cells remaining interacting; Fig 3). Interacting neutrophils included those that were rolling and those arrested (ie, not rolling greater than 1 cell diameter during a 1-second observation period). When neonatal cells are allowed to attach to CHO-P-selectin before shear stress is introduced and are then subjected to increased shear stress, neonatal neutrophils had an equal percentage of settled cells that remained interacting compared with adult and were able to resist detachment to the same extent as adult neutrophils (Fig 3). There was no contribution of L-selectin to this interaction, because an anti–L-selectin MoAb had no effect on either adult or neonatal neutrophils. Thus, it appears that, once neonatal and adult neutrophils interact with CHO-P-selectin, both are equally able to resist detachment from the monolayer. This interaction is not dependent on L-selectin.

Fig. 3.

The percent of adult (○, •) and neonatal (□, ▪) neutrophils initially attached to CHO cells expressing P-selectin in the absence of shear stress that remain interacting as shear stress is then applied and increased. Neutrophils are allowed to settle onto the monolayer for 2 minutes, at which point the flow is begun and increasing shear stress is applied every 20 seconds. The number of neutrophils that remain attached in the last 10 seconds before the next step up in shear stress is compared with the number of cells that had originally settled (percentage of interacting cells remaining). Neutrophils were left untreated (•, ▪) or were preincubated with anti–L-selectin MoAb, DREG 56 (○, □). Data are expressed as the mean ± SEM from 7 to 14 experiments.

Fig. 3.

The percent of adult (○, •) and neonatal (□, ▪) neutrophils initially attached to CHO cells expressing P-selectin in the absence of shear stress that remain interacting as shear stress is then applied and increased. Neutrophils are allowed to settle onto the monolayer for 2 minutes, at which point the flow is begun and increasing shear stress is applied every 20 seconds. The number of neutrophils that remain attached in the last 10 seconds before the next step up in shear stress is compared with the number of cells that had originally settled (percentage of interacting cells remaining). Neutrophils were left untreated (•, ▪) or were preincubated with anti–L-selectin MoAb, DREG 56 (○, □). Data are expressed as the mean ± SEM from 7 to 14 experiments.

Close modal

However, as with neonatal neutrophils that attached under continuous flow conditions, neonatal neutrophils demonstrated markedly different rolling behaviors compared with adult neutrophils under increasing shear conditions. The rolling fraction of neutrophils was determined by dividing the number of rolling cells by the number of interacting cells at that shear stress. As seen in Fig 4A, adult neutrophils had a significantly increased rolling fraction as shear stress increased from 0.6 dynes/cm2 (32% ± 4.7%) to 22 dynes/cm2 (69% ± 8%, P < .01, one-way ANOVA). In contrast, there was a lower fraction of neonatal neutrophils that rolled at all shear stresses. This low amount of rolling did not change with increasing shear stress (Fig 4B) and was significantly less than adult neutrophils (P < .0001, two-way ANOVA). Treatment of neonatal neutrophils with the anti–L-selectin MoAb, DREG 56, had no effect on the rolling behavior (Fig 4B). However, treatment with the anti–L-selectin MoAb resulted in a small but significant decrease in the rolling fraction of adult neutrophils (P < .05, Fig 4A).

Fig. 4.

Fraction of interacting adult (A) and neonatal (B) neutrophils that rolled on CHO-P-selectin after a 2-minute stationary contact period with increasing shear stress. Neutrophils were left untreated (•) or were preincubated with anti-CD11a MoAb, R7.1 (10 μg/mL; ▾), anti-CD18, R15.7 (10 μg/mL; ▴), or anti–L-selectin, DREG 56 (50 μg/mL; ▪). Data are expressed as the mean rolling fraction ± SEM from 7 to 14 experiments. *P < .005 compared with control neutrophils. +P < .05 compared with control. **P < .02 versus anti-CD11a–treated neutrophils.

Fig. 4.

Fraction of interacting adult (A) and neonatal (B) neutrophils that rolled on CHO-P-selectin after a 2-minute stationary contact period with increasing shear stress. Neutrophils were left untreated (•) or were preincubated with anti-CD11a MoAb, R7.1 (10 μg/mL; ▾), anti-CD18, R15.7 (10 μg/mL; ▴), or anti–L-selectin, DREG 56 (50 μg/mL; ▪). Data are expressed as the mean rolling fraction ± SEM from 7 to 14 experiments. *P < .005 compared with control neutrophils. +P < .05 compared with control. **P < .02 versus anti-CD11a–treated neutrophils.

Close modal
Adhesion of neonatal and adult neutrophils to nontransfected CHO monolayers under static conditions is CD18-dependent.

We had shown previously that, under continuous shear stress, adult neutrophil arrest on histamine-stimulated HUVECs is CD18/ICAM-1–dependent.3 To avoid CD18/ICAM interactions, we therefore used CHO cells transfected with P-selectin in the previous set of experiments. Nonetheless, only 60% to 70% of interacting adult neutrophils rolled and even fewer neonatal neutrophils did so (22% to 27%) in both the attachment assay under continuous shear and the detachment assay. We speculated that the arrest of both adult and neonatal neutrophils on CHO-P-selectin could also be CD18-ICAM-1 dependent.

We sought to determine if CHO cells have an ICAM-1–like molecule using an ELISA and a panel of MoAbs directed against mouse, human, dog, and rat ICAM-1. We were unable to detect consistent cross-reactivity between any of the anti-ICAM, E-selectin, L-selectin, VCAM-1, or HLA MoAbs on either nontransfected CHO or CHO-P-selectin, although we could consistently detect increased binding of the anti-P-selectin MoAb (Cytel 1747) on the transfected cell line. We hypothesized that, if the interaction between the ICAM-like molecule on CHO and the anti-ICAM MoAbs were of a low-affinity type, this interaction would be susceptible to the vigorous washing steps of the ELISA. Therefore, we performed a static adhesion assay in which fMLP-stimulated adult neutrophils were allowed to adhere to CHO cell monolayers pretreated with anti-ICAM or control (W6/32) MoAbs that were then gently washed (3 dips in PBS) before insertion into the Sykes-Moore adhesion chamber. Treatment with anticanine ICAM (CL18/1, CL18/6) and antihuman ICAM MoAbs (R6.5 Ig, R6.5 Fab) resulted in a 30% decrease in neutrophil adhesion compared with PBS- or W6/32-treated monolayers (Table 2). If we maintained the anti-ICAM MoAb, R6.5 Fab, or control antibody, W6/32, in the reaction mix in addition to pretreating the monolayers, adhesion was decreased further (Table 2). Thus, it appears that CHO cells express a molecule(s) that can function in cell adhesion and that this adhesion can be blocked by several anti-ICAM MoAbs.

We performed a static adhesion assay with neonatal neutrophils on nontransfected CHO cell monolayers. Baseline adhesion of neonatal neutrophils treated with a control anti-CD45 (GAP8. 3) MoAb was 18% ± 5.4%; treatment with the anti-CD11a MoAb (R7.1) decreased adhesion significantly (2.7% ± 0.7%, P < .05). In contrast, adult control neutrophils (treated with anti–LFA-1 nonblocking MoAb, TS2/4) had low baseline adhesion (3.7% ± 1.0%). Adhesion of TS2/4-treated adult neutrophils was significantly increased to 13.9% ± 1.5% (P < .01) with 10 nmol/L fMLP stimulation. Stimulated adhesion could be reduced to unstimulated levels (4.9% ± 3.8%, P < .01 compared with stimulated) only with coincubation of anti–Mac-1 (M1/70) and anti–LFA-1 (R7. 1) MoAbs and not with either individually (8.5% ± 4.3% and 8.2% ± 4.2%, respectively). Under static conditions, adult neutrophils have low baseline adhesion, although adhesion can be increased with stimulation of the neutrophils. Stimulated adhesion of adult neutrophils is dependent on LFA-1 and Mac-1 and an ICAM-1–like molecule. In contrast, unstimulated neonatal neutrophils have increased adherence to nontransfected CHO cells, and this adhesion is dependent on LFA-1.

Inhibition of CD11a/CD18 results in increased neonatal neutrophil rolling fraction and increased detachment with increased shear stress.

To assess the contribution of CD11a/CD18 to neonatal and adult neutrophil rolling on CHO-P-selectin, neutrophils were treated with MoAbs against either CD11a (R7.1) or the common CD18 subunit (R15.7) of LFA-1 and Mac-1. Such treatments had no effect on the expression of L-selectin or Mac-1 (Table 1). Neutrophils attached to CHO-P-selectin monolayers in the absence of shear stress, and then shear stress was initiated and increased every 20 seconds as outlined (detachment assay, see the Materials and Methods). The number of total interacting cells and those rolling were counted and the rolling fraction was determined. The fraction of rolling neutrophils significantly increased when either adult or neonatal cells were treated with either R7.1 or R15.7 compared with the age-matched controls (Fig 4A and B). Although the rolling fraction of adult neutrophils increased with inhibition of LFA-1, the percentage of cells remaining interacting as shear stress increased did not change significantly (Fig 5A). Therefore, with anti–LFA-1 treatment, adult neutrophils had increased rolling; however, these neutrophils were able to resist detachment with increasing shear stress and continued to roll. In contrast, as the rolling fraction of anti–LFA-1–treated neonatal neutrophils increased with shear stress (Fig 4B), the total number of interacting cells decreased significantly compared with untreated neonatal cells (Fig5B). Thus, the rolling neonatal neutrophils were less resistant to shear stress than adult neutrophils and had increased detachment.

Fig. 5.

The percentage of adult (A) and neonatal (B) of those initially attached to CHO-P-selectin in the absence of shear stress that remain interacting as shear stress is applied. Adult and neonatal neutrophils were treated with the anti-CD11a (▾) or anti-CD18 (▴) MoAb or left untreated (•). Data are expressed as the mean percentage of initially interacting cells remaining ± SEM from 7 to 14 experiments. *P < .05 compared with control neutrophils. **P < .005 compared with control neutrophils.

Fig. 5.

The percentage of adult (A) and neonatal (B) of those initially attached to CHO-P-selectin in the absence of shear stress that remain interacting as shear stress is applied. Adult and neonatal neutrophils were treated with the anti-CD11a (▾) or anti-CD18 (▴) MoAb or left untreated (•). Data are expressed as the mean percentage of initially interacting cells remaining ± SEM from 7 to 14 experiments. *P < .05 compared with control neutrophils. **P < .005 compared with control neutrophils.

Close modal

In the present study, we demonstrate that neonatal neutrophils have distinct differences compared with adult neutrophils in their interactions with monolayers expressing P-selectin. Neonatal neutrophils perfused over monolayers of P-selectin at a constant shear stress of approximately 2 dynes/cm2 demonstrated a decrease in the total number of cells that interacted with the monolayer compared with adult neutrophils. Of those cells interacting, there was a decreased fraction of neonatal neutrophils that rolled during the 1-second observation period compared with adult neutrophils. These two effects were demonstrated on both histamine-stimulated HUVECs as well as on CHO cells stably transfected with human P-selectin, although the differences in the rolling fractions was significant only on the CHO-P-selectin monolayer. When neonatal and adult neutrophils attached to CHO-P-selectin monolayers in the absence of shear stress and then shear stress was introduced, equal numbers of cells interacted with the monolayer. However, their rolling behavior again was significantly different in that neonatal neutrophils had a decreased rolling fraction compared with adult neutrophils. Treatment with anti–LFA-1 MoAbs resulted in an increase in the fraction of both neonatal and adult cells that were rolling. However, under these conditions, neonatal cells detached as shear stress increased, whereas adult neutrophils continued to roll and did not detach.

Neutrophil attachment to and rolling along the endothelia under shear flow is mediated by selectins. We have previously demonstrated that, under shear flow conditions, neonatal neutrophils have a diminished ability compared with adult neutrophils to interact with HUVECs stimulated with IL-1 (which express both E-selectin and an L-selectin ligand)35 as well as murine L cells transfected with human E-selectin.4 We and others have shown that neonatal neutrophils have diminished levels of L-selectin compared with adult neutrophils (Table 1 and previous studies33,35,52,53). This decrease in neonatal neutrophil L-selectin appears to contribute to diminished interaction with monolayers expressing the L-selectin ligand and/or E-selectin.4 35 

In the present study, we demonstrate for the first time that neonatal neutrophils also have a diminished ability to interact with monolayers expressing P-selectin under continuous shear flow. That this may also be due to decreased amounts of L-selectin on neonatal neutrophils (Table 1) is supported by the finding that treatment of adult neutrophils with the anti–L-selectin MoAb decreases the number of interacting cells compared with adult control neutrophils. Furthermore, as demonstrated previously with IL-1–stimulated HUVECs35and E-selectin monolayers,4 the number of interacting anti–L-selectin–treated adult neutrophils was equal to that seen with control neonatal neutrophils (Figs 1 and 2). L-selectin has been demonstrated to be located at the tips of the microvillus of adult51 and neonatal neutrophils (M. Mariscalco and A. Burns, unpublished observations). This location of L-selectin is particularly advantageous for capturing of the neutrophils from the free-flowing stream. However, L-selectin does not contribute to continued neutrophil interaction with the P-selectin monolayer once attachment has already occurred (Fig 3). The requirement for L-selectin in the initial capture of neutrophils from the free-flowing stream to E-selectin has been described by Lawrence et al.49 However, as with this present study, once neutrophils were attached, L-selectin was not required to maintain the interaction.

The contribution of L-selectin– to P-selectin–mediated rolled has been described by us previously.3 There has been considerable controversy as to whether L-selectin can function as a ligand for P-selectin. Picker et al51 were able to inhibit by 40% to 60% neutrophil attachment to P-selectin–transfected COS cells with the anti–L-selectin MoAb, DREG 56. In contrast, Patel et al16 were unable to inhibit adult neutrophil attachment to CHO cells expressing P-selectin at continuous shear stress with DREG 56. It is unclear as to why our findings differ from those of Patel et al,16 because our experimental procedures were remarkably similar. One explanation for our findings may be that the anti–L-selectin MoAb, DREG 56, inhibits a functional domain of P-selectin. There are reports of MoAbs which cross-react with one or more selectin molecules.4,54 55 We were unable to detect binding of DREG 56 to the transfected cell line expressing P-selectin using an ELISA, although this does not rule out low-affinity interactions. Nonetheless, pretreatment of neutrophils resulted in very low concentrations of DREG 56 in the flow assays itself, making low-affinity interactions extremely unlikely.

Others have described the contribution of leukocyte-leukocyte interactions in amplifying the capture of leukocytes from a free-flowing stream.56 This interaction appears to involve PSGL-1 on one cell and L-selectin on another.17,57 It is possible that our findings reflect a decreased L-selectin–PSGL-1 interaction due to inhibition of L-selectin (treatment of adult neutrophils with DREG 56) or decreased amount of L-selectin (neonatal neutrophils). We were unable to demonstrate leukocyte-leukocyte recruitment from a review of our videotapes, although our protocol was not designed specifically to examine these interactions.17 

We had demonstrated previously that neutrophils roll on histamine-stimulated HUVECs expressing P-selectin. In that study, some interacting neutrophils did not roll. This arrest was dependent on neutrophil CD18 and the ICAM-1 constitutively present on the HUVECs.3 CHO-P-selectin also supports rolling interactions.16 Unexpected, however, was the observation here that CHO cells can also support LFA-1–dependent arrest of both neonatal and adult neutrophils. Based on our findings, we suggest that CHO cells express a molecule that can function in a manner similar to ICAM-1.

Neonatal neutrophils have increased baseline adhesion to nontransfected CHO cells compared with adult neutrophils in the static adhesion assay. This adhesion could be blocked by treatment with anti–LFA-1 MoAbs. In addition, neonatal neutrophils attached to the CHO-P-selectin in the absence of shear had a significantly decreased rolling fraction compared with adult neutrophils when shear was introduced and then increased. Rolling fraction of neonatal neutrophils could be increased to that seen with adult neutrophils by treatment with anti–LFA-1 MoAbs. These findings suggest that neonatal LFA-1 is functionally more active than adult LFA-1, because, to date, there have been no reports of quantitative differences in LFA-1 expression between neonatal and adult neutrophils.30,32,33 Activation of the integrins leads to an increase in avidity for their respective ligands.58 This process has been described in lymphocytes for LFA-1 and in neutrophils for Mac-1.59-62 Only recently has there been evidence that activation of the neutrophil may also result in the affinity modulation of LFA-1.47 Is increased LFA-1 function due to the fact that resting neonatal neutrophils are activated compared with adult neutrophils? There are several studies that support this. Kjeldsen et al63 recently demonstrated the augmented release of both gelatinase and specific granules from neonatal neutrophils isolated from cord blood compared with adult neutrophils, suggesting that neonatal cord neutrophils appeared primed compared with control adult cells. Others have reported that neonatal cord neutrophils had increased oxidative burst activity.64It is unclear if the increased activity of LFA-1 can compensate for the decreased ability of neonatal neutrophils to be captured from the free-flowing stream. Our data suggest that it does not. To answer whether these observations will ultimately result in neutrophil emigration defects in vivo will require direct examination of leukocyte localization in neonatal animal models.

The rolling of neutrophils on purified P-selectin or P-selectin monolayers (CHO-P-selectin) has been reported to be dependent primarily on PSGL-1.13 16 Whereas inhibition of LFA-1–mediated arrests results in the increased rolling fraction of adult and neonatal neutrophils as shear stress increases, the number of interacting neonatal cells decrease. We propose that the decreased rolling of neonatal neutrophils is due to quantitative or qualitative differences in PSGL-1 (or other P-selectin ligands) compared with the adult. These results remain speculative until neonatal PSGL-1 function is evaluated directly.

Finally, we demonstrate that there is no statistical difference in the stimulated expression of Mac-1 on our sample of adult versus neonatal neutrophils. This may be interpreted as a discrepancy with previous findings.26,30,34,65 We suggest that this reflects instead the wide sampling variability in stimulated expression of Mac-1 in neonatal cord blood and adult samples (Table 1). That this may be so is supported by our retrospective review of the results of clinical testing of whole blood from infants (<2 months old) whom we had examined for the presence of Mac-1 and LFA-1. None of the infants had leukocyte adhesion deficiency type I or other known neutrophil defects.66 The average MFI of stimulated neutrophils stained for Mac-1 ± SD was 1,747 ± 657 for adults versus 1,042 ± 663 for infants (n = 14, P < .01; M. Mariscalco and R.N. Bennett, unpublished observations). Thus, any individual neonate can have adult levels of Mac-1. Nonetheless, in such an infant, Mac-1 function may still be depressed, because our previous study demonstrated that neonatal neutrophil Mac-1 function was decreased comparable to an adult group with equivalent levels of stimulated Mac-1 expression.34 

The authors thank the nurses and physicians in the Labor and Delivery Unit of St Luke's Episcopal Hospital, without whose assistance this project would not be possible. We also thank Drs Robert Rothlein and Takashei Kishimoto for supplying antibodies, Dr Christine Martens for the P-selectin transfected cell line, and Bonnie Hughes, Jia Mei, Carol Knight, and Michelle Swarthout for their continued technical and administrative assistance.

Supported by National Institutes of Health Grant No. NIH-AI-19031.

Address reprint requests to M. Michele Mariscalco, MD, Baylor College of Medicine, CNRC, 1100 Bates, Room 6014, Houston, TX 77030-2600.

The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" is accordance with 18 U.S.C. section 1734 solely to indicate this fact.

1
Albelda
SM
Smith
CW
Ward
PA
Adhesion molecules and inflammatory injury.
FASEB J
8
1994
504
2
Smith
CW
Kishimoto
TK
Abbassi
O
Hughes
BJ
Rothlein
R
McIntire
LV
Butcher
E
Anderson
DC
Chemotactic factors regulate lectin adhesion molecule 1 (LECAM-1)-dependent neutrophil adhesion to cytokine-stimulated endothelial cells in vitro.
J Clin Invest
87
1991
609
3
Jones
DA
Abbassi
O
McIntire
LV
McEver
RP
Smith
CW
P-selectin mediates neutrophil rolling on histamine-stimulated endothelial cells.
Biophys J
65
1993
1560
4
Abbassi
O
Kishimoto
TK
McIntire
LV
Anderson
DC
Smith
CW
E-Selectin supports neutrophil rolling in vitro under conditions of flow.
J Clin Invest
92
1993
2719
5
Kishimoto
TK
Jutila
MA
Berg
EL
Butcher
EC
Neutrophil Mac-1 and MEL-14 adhesion proteins inversely regulated by chemotactic factors.
Science
245
1989
1238
6
Jung
TM
Dailey
MO
Rapid modulation of homing receptors (gp90MEL-14) induced by activators of protein kinase C.
J Immunol
144
1990
3130
7
Bevilacqua
MP
Nelson
RM
Selectins.
J Clin Invest
91
1993
379
8
McEver
RP
Beckstead
JH
Moore
KL
Marshall-Carlson
L
Bainton
DF
GMP-140, a platelet alpha-granule membrane protein, is also synthesized by vascular endothelial cells and is localized in Weibel-palade bodies.
J Clin Invest
84
1989
92
9
Bevilacqua
MP
Stengelin
S
Gimbrone
Jr
, Seed B: Endothelial leukocyte adhesion molecule 1: An inducible receptor for neutrophils related to complement regulatory proteins and lectins.
Science
243
1989
1160
10
Vestweber
D
Ligand-specificity of the selectins.
J Cell Biochem
61
1996
585
11
Varki
A
Perspectives series: Cell adhesion in vascular biology. Selectin ligands: Will the real ones please stand up?
J Clin Invest
99
1997
158
12
Norgard
KE
Moore
KL
Diaz
S
Stults
NL
Ushiyama
S
McEver
RP
Cummings
RD
Varki
A
Characterization of a specific ligand for P-selectin on myeloid cells. A minor glycoprotein with sialylated O-linked oligosaccharides.
J Biol Chem
268
1993
12764
13
Moore
KL
Patel
KD
Bruehl
RE
Fugang
L
Johnson
DA
Lichenstein
HS
Cummings
RD
Bainton
DF
McEver
RP
P-selectin glycoprotein ligand-1 mediates rolling of human neutrophils on p-selectin.
J Cell Biol
128
1995
661
14
Symon
FA
Lawrence
MB
Williamson
ML
Walsh
GM
Watson
SR
Wardlaw
AJ
Functional and structural characterization of the eosinophil P-selectin ligand.
J Immunol
157
1996
1711
15
Alon
R
Rossiter
H
Wang
X
Springer
TA
Kupper
TS
Distinct cell surface ligands mediate T lymphocyte attachment and rolling on P and E selectin under physiological flow.
J Cell Biol
127
1994
1485
16
Patel
KD
Moore
KL
Nollert
MU
McEver
RP
Neutrophils use both shared and distinct mechanisms to adhere to selectins under static and flow conditions.
J Clin Invest
96
1995
1887
17
Walcheck
B
Moore
KL
McEver
RP
Kishimoto
TK
Neutrophil-neutrophil interactions under hydrodynamic shear stress involve L-selectin and PSGL-1. A mechanism that amplifies initial leukocyte accumulation on P-selectin in vitro.
J Clin Invest
98
1996
1081
18
Spertini
O
Cordey
AS
Monai
N
Giuffrè
L
Schapira
M
P-selectin glycoprotein ligand 1 is a ligand for L-selectin on neutrophils, monocytes, and CD34+ hematopoietic progenitor cells.
J Cell Biol
135
1996
523
19
Lawrence
MB
McIntire
LV
Eskin
SG
Smith
CW
Involvement of CD18 in human neutrophil adhesion to endothelium under flow conditions.
FASEB J
2
1988
A1237
20
von Andrian
UH
Chambers
JD
McEvoy
LM
Bargatze
RF
Arfors
K-E
Butcher
EC
Two step model of leukocyte-endothelial cell interaction in inflammation: Distinct roles for LECAM-1 and the leukocyte beta-2 integrins in vivo.
Proc Natl Acad Sci USA
88
1991
7538
21
Pober
JS
Lapierre
LA
Stolpen
AH
Brock
TA
Springer
TA
Fiers
W
Bevilacqua
MP
Mendrick
DL
Gimbrone MA Jr
Activation of cultured human endothelial cells by recombinant lymphotoxin: Comparision with tumor necrosis factor and interleukin 1 species.
J Immunol
138
1987
3319
22
Anderson
DC
Role of ICAM-1 in the adherence of human neutrophils to human endothelial cells in vitro
Springer
TA
Anderson
DC
Rosenthal
AS
Rothlein
R
Leukocyte Adhesion Molecules: Structure, Function, and Regulation.
1989
170
Springer-Verlag
New York, NY
23
Anderson
DC
Neonatal neutrophils.
J Lab Clin Med
120
1992
816
24
Santos
JI
Shigeoka
AO
Hill
HR
Functional leukocyte administration in protection against experimental neonatal infection.
Pediatr Res
14
1980
1408
25
Schuit
KE
Homisch
L
Inefficient in vivo neutrophil migration in neonatal rats.
J Leukoc Biol
35
1984
583
26
Fortenberry
JD
Marolda
JR
Anderson
DC
Smith
CW
Mariscalco
MM
CD18-dependent and L-selectin-dependent neutrophil emigration is diminished in neonatal rabbits.
Blood
84
1994
889
27
Martin
TR
Ruzinski
JT
Wilson
CB
Skerrett
SJ
Effects of endotoxin in the lungs of neonatal rats: Age-dependent impairment of the inflammatory response.
J Infect Dis
171
1995
134
28
Cheung
ATW
Kurland
G
Miller
ME
Ford
EW
Avin
SA
Walsh
EM
Host defense deficiency in newborn nonhuman primate lungs.
J Med Primatol
15
1986
37
29
Hill
HR
Biochemical, structural, and functional abnormalities of polymorphonuclear leukocytes in the neonate.
Pediatr Res
22
1987
375
30
Anderson
DC
Freeman
KLB
Heerdt
B
Hughes
BJ
Jack
RM
Smith
CW
Abnormal stimulated adherence of neonatal granulocytes: Impaired induction of surface Mac-1 by chemotactic factors or secretagogues.
Blood
70
1987
740
31
Torok
C
Lundahl
J
Hed
J
Lagercrantz
H
Diversity in regulation of adhesion molecules (Mac-1 and L-selectin) in monocytes and neutrophils from neonates and adults.
Arch Dis Child
68
1993
561
32
Abughali
N
Berger
M
Tosi
M
Deficient total cell content of CR3 (CD11b) in neonatal neutrophils.
Blood
83
1994
1086
33
Rebuck
N
Gibson
A
Finn
A
Neutrophil adhesion molecules in term and premature infants: Normal or enhanced leukocyte integrins but defective L-selectin expression and shedding.
Clin Exp Immunol
101
1995
183
34
Anderson
DC
Rothlein
R
Marlin
SD
Krater
SS
Smith
CW
Impaired transendothelial migration by neonatal neutrophils: Abnormalities of Mac-1 (CD11b/CD18)-dependent adherence reactions.
Blood
78
1990
2613
35
Anderson
DC
Abbassi
O
Kishimoto
TK
Koenig
JM
McIntire
LV
Smith
CW
Diminished lectin-, epidermal growth factor-, complement binding domain-cell adhesion molecule-1 on neonatal neutrophils underlies their impaired CD18-independent adhesion to endothelial cells in vitro.
J Immunol
146
1991
3372
36
Kishimoto
TK
Jutila
MA
Butcher
EC
Identification of a human peripheral lymph node homing receptor: A rapidly down-regulated adhesion molecule.
Proc Natl Acad Sci USA
87
1990
2244
37
Entman
ML
Youker
KA
Shappell
SB
Siegel
C
Rothlein
R
Dreyer
WJ
Schmalstieg
FC
Smith
CW
Neutrophil adherence to isolated adult canine myocytes: Evidence for a CD18-dependent mechanism.
J Clin Invest
85
1990
1497
38
Argenbright
LW
Letts
LG
Rothlein
R
Monoclonal antibodies to the leukocyte membrane CD18 glycoprotein complex and to intercellular adhesion molecule-1 inhibit leukocyte-endothelial adhesion in rabbits.
J Leukoc Biol
49
1991
253
39
Rothlein
R
Mainolfi
EA
Czajkowski
M
Marlin
SD
A form of circulating ICAM-1 in human serum.
J Immunol
147
1991
3788
40
Tamatani
T
Kotani
M
Toshiyuki
T
Miyasaka
M
Molecular mechanisms underlying lymphocyte recirculation. II. Differential regulation of LFA-1 in the interaction between lymphocytes and high endothelial cells.
Eur J Immunol
21
1991
855
41
Isobe
M
Yagita
H
Okumura
K
Ihara
A
Specific acceptance of cardiac allograft after treatment with antibodies to ICAM-1 and LFA-1.
Science
255
1992
1125
42
Smith
CW
Entman
ML
Lane
CL
Beaudet
AL
Ty
TI
Youker
KA
Hawkins
HK
Anderson
DC
Adherence of neutrophils to canine cardiac myocytes in vitro is dependent on intercellular adhesion molecule-1.
J Clin Invest
88
1991
1216
43
Mulligan
MS
Polley
MJ
Neutrophil dependent acute lung injury.
J Clin Invest
90
1992
1600
44
Birdsall
HH
Lane
CL
Ramser
MN
Anderson
DC
Induction of VCAM-1 and ICAM-1 on human neural cells and mechanisms of mononuclear leukocyte adherence.
J Immunol
148
1992
2717
45
Kishimoto
TK
Warnock
RA
Jutila
MA
Butcher
EC
Lane
CL
Anderson
DC
Smith
CW
Antibodies against human neutrophil LECAM-1 (LAM-1/Leu-8/DREG-56 antigen) and endothelial cell ELAM-1 inhibit a common CD18-independent adhesion pathway in vitro.
Blood
78
1991
805
46
Martens
CL
Cwirla
SE
Lee
RY-W
Whitehorn
E
Chen
EY-F
Bakker
A
Martin
EL
Wagstrom
C
Gopalan
P
Smith
CW
Tate
E
Kroller
KJ
Schatz
PJ
Dower
WJ
Barrett
RW
Peptides which bind to E-selectin and block neutrophil adhesion.
J Biol Chem
270
1995
21129
47
Gopalan
PK
Smith
CW
Lu
H
Berg
EL
McIntire
LV
Simon
SI
Neutrophil CD18-dependent arrest on ICAM-1 in shear flow can be activated through L-selectin.
J Immunol
158
1997
367
48
Smith
CW
Rothlein
R
Hughes
BJ
Mariscalco
MM
Schmalstieg
FC
Anderson
DC
Recognition of an endothelial determinant for CD18-dependent human neutrophil adherence and transendothelial migration.
J Clin Invest
82
1988
1746
49
Lawrence
MB
Bainton
DF
Springer
TA
Neutrophil tethering to and rolling on E-selectin are separable by requirement for L-selectin.
Immunity
1
1994
137
50
Hattori
R
Hamilton
KK
McEver
RP
Sims
PJ
Complement proteins C5b-9 induce secretion of high molecular weight multimers of endothelial von Willebrand factor and translocation of granule membrane protein GMP-140 to the cell surface.
J Biol Chem
264
1989
9053
51
Picker
LJ
Warnock
RA
Burns
AR
Doerschuk
CM
Berg
EL
Butcher
EC
The neutrophil selectin LECAM-1 presents carbohydrate ligands to the vascular selectins ELAM-1 and GMP-140.
Cell
66
1991
921
52
Koenig
JM
Simon
J
Anderson
DC
Smith
EO
Smith
CW
Diminished soluble and total cellular L-Selectin in cord blood is associated with its impaired shedding from activated neutrophils.
Pediatr Res
39
1996
616
53
Smith
JB
Kunjummen
RD
Kishimoto
TD
Anderson
DC
Expression and regulation of L-selectin on eosinophils from human adults and neonates.
Pediatr Res
32
1992
645
54
Abbassi
O
Lane
CL
Krater
SS
Kishimoto
TK
Anderson
DC
McIntire
LV
Smith
CW
Canine neutrophil margination mediated by lectin adhesion molecule-1 (LECAM-1) in vitro.
J Immunol
147
1991
2107
55
Jutila
MA
Watts
G
Walcheck
B
Kansas
GS
Characterization of a functionally important and evolutionarily well-conserved epitope mapped to the short consensus repeats of E-selectin and L-selectin.
J Exp Med
175
1992
1565
56
Bargatze
RF
Kurk
S
Butcher
EC
Jutila
MA
Neutrophils roll on adherent neutrophils bound to cytokine-induced endothelial cells via L-selectin on the rolling cells.
J Exp Med
180
1994
1785
57
Bennett
TA
Schammel
CMG
Lynam
EB
Guyer
DA
Mellors
A
Edwards
B
Rogelj
S
Sklar
LA
Evidence for a third component in neutrophil aggregation: Potential roles of O-linked glycoproteins as L-selectin counter-structures.
J Leukoc Biol
58
1995
510
58
Hynes
RO
Integrins: Versatility modulations and signaling in cell adhesion.
Cell
69
1992
11
59
Van Kooyk
Y
Weder
P
Hogervorst
F
Verhoeven
AJ
Van Seventer
GA
te Velde
AA
Borst
J
Keizer
GD
Figdor
CG
Activation of LFA-1 through a Ca2+-dependent epitope stimulates lymphocyte adhesion.
J Cell Biol
112
1991
345
60
Petruzzelli
L
Maduzia
L
Springer
TA
Activation of lymphocyte function-associated molecule-1 (CD11a/CD18) and Mac-1 (CD11b/CD18) mimicked by an antibody directed against CD18.
J Immunol
155
1995
854
61
Dransfield
I
Buckle
A-M
Savill
JS
McDowall
A
Haslett
C
Hogg
N
Neutrophil apoptosis is associated with a reduction in CD16 (FcγRIII) expression.
J Immunol
153
1994
1254
62
Simon
SI
Burns
AR
Taylor
AD
Gopalan
PK
Lynam
EB
Sklar
LA
Smith
CW
L-Selectin (CD62L) crosslinking signals neutrophil adhesive functions via the Mac-1 (CD11b/CD18) β2-integrin.
J Immunol
155
1995
1502
63
Kjeldsen
L
Sengelov
H
Lollike
K
Borregaard
N
Granules and secretory vesicles in human neonatal neutrophils.
Pediatr Res
40
1996
120
64
Ambruso
DR
Bentwood
B
Henson
PM
Johnston RB Jr
Oxidative metabolism of cord blood neutrophils: Relationship to content and degranulation of cytoplasmic granules.
Pediatr Res
18
1984
1148
65
Graf
JM
Smith
CW
Mariscalco
MM
Contribution of LFA-1 and Mac-1 to CD18-dependent neutrophil emigration in a neonatal rabbit model.
J Appl Physiol
80
1996
1984
66
Anderson
DC
Schmalstieg
FC
Finegold
MJ
Hughes
BJ
Rothlein
R
Miller
LJ
Kohl
S
Tosi
MF
Jacobs
RL
Waldrop
TC
Goldman
AS
Shearer
WT
Springer
TA
The severe and moderate phenotypes of heritable Mac-1, LFA-1, p150,95 deficiency: Their quantitative definition and relation to leukocyte dysfunction and clinical features.
J Infec Dis
152
1985
668
Sign in via your Institution