Adult Neutrophils Research Articles

Neutrophil Function in Preterm and Term Infants

After completing this article, readers should be able to: 1. List the primary cell functions of circulating neutrophils in response to microbial invasion and inflammation. 2. Describe the interaction between neutrophils and endothelial cells and how these interactions differ between neonatal and adult cells. 3. Describe differences in adhesion of phagocytes between neonatal and adult neutrophils. 4. Describe the phagocytosis-associated respiratory burst and deficiencies seen in neonatal neutrophils. Polymorphonuclear neutrophils (PMN) constitute the primary line of defense in the cellular immune system. Inborn defects in the generation of neutrophils or in neutrophil function are predisposing risk factors for life-threatening infections in infants and children. In general, newborns have an increased risk for systemic infections, and very immature preterm infants are especially prone to developing nosocomial bloodstream infections that clearly have been associated with increased morbidity and mortality. In this review, we attempt to identify possible quantitative and qualitative deficiencies observed in neonatal neutrophils and try to define, whenever possible, the underlying mechanisms of neutrophil dysfunctions in term and preterm infants. (1) ### Bone Marrow Storage Pool and Circulating Pool Neutrophils and cells of the mononuclear-phagocyte system originate in the bone marrow as a common committed progenitor cell for the granulocyte and the monocyte-macrophage pathways: the colony-forming unit-granulocyte monocyte (CFU-GM). Glycoprotein hormones termed colony-stimulating factors (G-CSF/GM-CSF) induce proliferation, maturation, and differentiation into neutrophils and monocytes. (1) The sequential development of neutrophilic granulocytes proceeds from pluripotent progenitor cell to committed stem cell to myeloblast to promyelocyte to myelocyte to metamyelocyte to band to segmented neutrophil. This sequence occurs within the bone marrow and possibly is regulated by the interaction of the hematopoietic inductive environment, CSF, and specific inhibitors of granulopoiesis. (2) Myeloblasts, promyelocytes, myelocytes, and other cells of the CFU-GM possess the possibility of cell division and form, by definition, the proliferative pool. Rodent studies have shown clearly that the total …

Effect of activators and the phosphodiesterase inhibitors Pentoxifylline and Enoximone on the deformability of neutrophils in neonates and adults

Effect of activators and the phosphodiesterase inhibitors Pentoxifylline and Enoximone on the deformability of neutrophils in neonates and adults

Effect of activators and the phosphodiesterase inhibitors Pentoxifylline and Enoximone on the deformability of neutrophils in neonates and adults

Stimulated polymorphonuclear leukocytes (PMN) are extremely rigid compared to resting PMN. They may obstruct narrow vessels and contribute to ischaemic organ injury. Deformability is a prerequisite for both active and passive movement in the microcirculation. The investigation was designed to study whether stimulators and inhibitors of stimulation show different effects on deformability of neonatal and adult PMN. Deformability of PMN was assessed by complete aspiration of a PMN into a micropipette with an internal diameter of 5 microm. Blood samples from 20 neonates and 20 adults were studied before and after stimulation with N-formyl-methionyl-leucyl-phenylalanine (fMLP), interleukin-8 (IL-8) or tumour necrosis factor-alpha (TNF-alpha). Moreover, effects of the phosphodiesterase inhibitors Pentoxifylline (PTX) and Enoximone on the deformability of stimulated PMN were investigated. fMLP, IL-8 and TNF-alpha significantly delayed aspiration times of PMN in relation to the concentrations of the stimulators. The addition of PTX or Enoximone to stimulated PMN increased the deformability up to 60% depending on the concentration of the inhibitors. No significant differences in the aspiration times were found between neonatal and adult PMN at any of the experimental conditions after activation with the three stimulators and treatment with the two inhibitors. Neonatal and adult PMN show similar reduction of passive deformability when stimulated with either fMLP, IL-8 or TNF-alpha compared to resting PMN and a similar improvement of deformability in response to PTX or Enoximone.

Comparison of L-Selectin and CD11b on Neutrophils of Adults and Neonates during the First Month of Life

The newborn infant is particularly susceptible to infection in the first weeks of life and this may be, in part, related to functional impairment of neonatal neutrophils in regard to adherence, chemotaxis, and migration. Differences in expression of the neutrophil adherence molecules, l-selectin and CD11b/CD18 (Mac-1), have been previously demonstrated in cord blood and in very young infants (≤ 48 h) compared with adults, but it is unknown for how long these differences persist. We measured surface expression of neutrophil l-selectin and CD11b using flow cytometry in healthy term human newborns from 24 h through 4 wk of age. We also measured levels of soluble l-selectin using an ELISA in neonates though the age of 4 wk. Compared with adults, neonates expressed significantly less l-selectin on resting neutrophils through 4 wk of age, with the lowest levels being at 24 h. Expression of l-selectin on the neutrophil after activation with N-formyl-methionyl-leucyl-phenylalanine was less in the neonate than in the adult (p < 0.05) for the first week of life. Soluble l-selectin showed a steady increase with age in the neonates. Soluble l-selectin was significantly lower in the 24-h neonate compared with the adult and was higher in the 4-wk neonate compared with the adult. CD11b expression was similar between neonates and adults on unstimulated neutrophils, but upon activation, the neonatal neutrophil demonstrated significantly lower up-regulation of CD11b on the neutrophil surface through 4 wk of age compared with adults (p < 0.05 for all ages except 1 wk). These findings that differences in expression of l-selectin and CD11b from that in adults persist throughout the neonatal period provide further evidence that these differences may play a role in the neutrophil defects observed during the neonatal period.

Qi-training enhances respiratory burst function and adhesive capacity of neutrophils in young adults: a preliminary study.

The main objective of this study is to examine the effect of Qi-training on the immune system, especially neutrophil bactericidal function. Nine healthy male subjects were studied for the effects of one bout of ChunDoSunBup (CDSB) Qi-training on superoxide (O2- production and adhesion capacity of neutrophils at times immediately after (Post I) and 2 hours after the Qi-training (Post II). The Qi-training enhanced O2- production, reaction velocity and neutrophil adhesion capacity and there were significant differences at Post I compared to before Qi-training (Pre). In addition, the number of white blood cells (WBC), monocytes and lymphocytes were changed significantly through Qi-training.Therefore, it seems that CDSB Qi-training may increase the resistance of trained individuals against common infection and inflammation.

The Effect of Gamma Globulin for Apoptosis of the Kawasaki Disease Patient Neutrophils

(Objective) Intravenous gamma globulin (IVGG) treatment is the most important therapy in the Kawasaki disease (KD), however there are many uncertainties about active mechanism of this treatment. This time, we examined active mechanisms of IVGG treatment affecting on apoptosis of neutrophils. (Patients and controls) Fifteen subjects (4 months ∼ 4 years old) were selected from the KD patients who had been admitted to Dokkyo University Hospital between June, 2000 and May, 2001. Healthy adults were used as controls.(Materials and methods) (1) Neutrophils were separated from heparinized blood by the specific gravity centrifugation. (2) Venoglobulin-I was used as human IgG for this experiment. (3) Apoptosis was determined with microscopic examination and flow cytometry. DNA quantity of the cells stained with PI alone and PI and anexin V was measured in flow cytometry.(Results) (1) Spontaneous apoptosis on KD patient neutrophils was more delayed than that on controls, as the picking illness day was earlier. (2) When healthy adult neutrophils was cultured in the presence of IgG (1mg/ml), apoptosis was promoted, but this effect was not observed under less than 0.1mg/ml IgG. Therefore, IgG was used at the concentration of 1mg/ml in further experiments. (3) The morphological changes unlike control neutrophils were observed on KD patient neutrophils after the culture in the presence of IgG. (4) Based on apoptosis pattern of neutrophils in the presence of IgG, samples were divided into two groups, promoted example and not. The promotion of apoptosis by the IgG addition in vitro was observed in about half of samples from the patients, in which IVGG gave more remarkable effects clinically. (Conclusions) We considered the possibility in which one part of the clinical effects of IVGG treatment had appeared by promoting the apoptotic effects to neutrophils.

Unconjugated bile acids modulate adult and neonatal neutrophil chemotaxis induced in vitro by N-formyl-met-leu-phe-peptide.

In this study, we have investigated the effect of hydrophobic and hydrophilic unconjugated bile acids (UBAs)-ursodeoxycholic acid (UDCA), chenodeoxycholic acid (CDCA), lithocholic acid, and colic acid-on chemotaxis in adult and neonatal human polymorphonuclear leukocytes (PMNs). The trypan blue exclusion dye test was preliminarily performed to determine the toxicity of the studied UBAs on PMNs. N-formyl-methionyl-leucyl-phenylalanine (100 nM) was used as a chemoattractant. Chemotaxis (1 x 10(6)cells/mL) was analyzed in the presence or absence of UBAs (10 microM) by blind well chambers. The antioxidants vitamin E and vitamin C were tested for their ability to reduce the inhibitory effect of UBAs. We found that only CDCA was able to induce damage in PMNs in the range of 1-40 microM. Both CDCA and UDCA were able to inhibit chemotaxis in PMNs, whereas lithocholic acid and colic acid were ineffective. The inhibitory effect was reversible inasmuch as PMNs incubated with either CDCA or UDCA and subsequently washed showed normal chemotaxis. Concomitant incubation of PMNs with UBAs and vitamins C or E reversed the inhibition. We did not find substantial differences between PMNs from adults or newborns. In conclusion, CDCA and UDCA are able to reduce, in a specific and reversible fashion, both adult and newborn neutrophil chemotaxis. As concomitant incubation of UBAs and electron scavengers restores PMN chemotaxis to control values, we conclude that free radicals may be involved in the mechanism of inhibition. We speculate that this defect may contribute to the impaired host response described in cholestatic patient.

A mixed population of immature and mature leucocytes in umbilical cord blood results in a reduced expression and function of CR3 (CD11b/CD18).

Neonatal neutrophils express less membrane and cytoplasmic CR3 (iC3b-receptor, Mac-1, alphaM beta2-integrin) than do adult neutrophils, and it has been suggested that this renders neonatal neutrophils deficient in diapedesis and bactericidal activity. The reason(s) for this deficiency are unknown. In this study, CR3 expression and the CR3-dependent respiratory burst activity of individual neonatal neutrophils are quantified in comparison with adult leucocytes using flow cytometry. Monocytes and neutrophils are defined as CD14highCD15low and CD14lowCD15high, respectively. Although neonatal neutrophils bore less CR3 on average than did adult neutrophils, neonatal neutrophils were more heterogeneous and many neonatal neutrophils expressed adult levels of CR3. Because of higher neutrophil concentrations in cord versus adult blood, the calculated number of neutrophils in cord blood expressing high amounts of CR3 was equivalent to that of adult blood. Similar findings were made with monocytes. The size of the CR3-dependent respiratory burst stimulated by particulate beta-glucan correlated directly with the expression of CR3 by individual neutrophils. With neonatal and adult neutrophils having comparable CR3 densities, the respiratory burst activities were equivalent. Wright-Giemsa differential staining of the subset of neonatal neutrophils with low CR3 levels isolated by fluorescence-activated cell sorting showed a higher proportion of immature cells than the sorted population expressing high CR3 levels. Therefore, higher proportions of immature cells in cord blood probably explain previous reports of deficient CR3 expression and function. The typical neutrophilia of cord blood may compensate for this apparent deficiency by providing adult concentrations of mature neutrophils.

Mechanisms underlying reduced responsiveness of neonatal neutrophils to distinct chemoattractants

Potential mechanisms underlying impaired chemotactic responsiveness of neonatal neutrophils were investigated. Two distinct chemoattractants were compared: bacterially derived N-formyl-methionyl-leucyl-phenylalanine (fMLP) and a unique chemotactic monoclonal antibody, designated DL1.2, which binds to a neutrophil antigen with an apparent molecular mass of 120 kDa. Chemotaxis of neutrophils toward fMLP, as well as DL1.2, was reduced in neonates when compared with adult cells. This did not appear to be a result of decreased fMLP receptor or DL1.2 antigen expression by neonatal neutrophils. fMLP, but not DL1.2, induced a rapid increase in intracellular calcium in adult and neonatal cells, which reached a maximum within 30 s. The calcium response of cells from neonates to fMLP was reduced when compared with adult cells, and an unresponsive subpopulation of neonatal neutrophils was identified. NF-kappaB nuclear binding activity induced by fMLP and DL1.2, as well as expression of the p65 NF-kappaB subunit and IkappaB-alpha, was also significantly reduced in neonatal cells, when compared with adult cells. In contrast, although fMLP, but not DL1.2, activated p42/44 and p38 mitogen-activated protein (MAP) kinases in neutrophils, no differences were observed between adults and neonates. Chemotaxis of adult and neonatal neutrophils toward fMLP and DL1.2 was also blocked to a similar extent by inhibitors of phosphatidylinositol 3-kinase, as well as an inhibitor of NF-kappaB. These findings indicate that reduced chemotactic responsiveness in neonatal neutrophils is a result of, at least in part, aberrations in chemoattractant-induced signaling. However, the biochemical pathways mediating this defect appear to be related to the specific chemoattractant.

Effects of granulocyte colony-stimulating factor on neutrophil adhesive molecules in neonates.

Term and preterm neonates experience quantitative and qualitative neutrophil deficiencies resulting in part from decreased production of granulocyte colony-stimulating factor (G-CSF). In adults, G-CSF improves neutrophil function by up-regulating adhesion molecules. To evaluate the effects of G-CSF on neonatal neutrophil adhesive phenotypes, cord blood samples were incubated with G-CSF or phosphate-buffered saline and stimulated with N-formyl-methionyl-leucyl-phenylalanine (FMLP), and adhesion molecules were evaluated by flow cytometry. In term and preterm neutrophils, G-CSF incubation increased beta2-integrin expression significantly compared with baseline and to a greater extent than observed in adult neutrophils. With FMLP stimulation, beta2-integrin expression increased even more in the G-CSF group. L-selectin expression decreased after G-CSF incubation and decreased even more with FMLP stimulation in the G-CSF group compared with the phosphate-buffered saline group in term and preterm samples, but not in adult samples. The data show that G-CSF increases expression of beta2-integrin and decreases expression of L-selectin on unstimulated and stimulated term and preterm neonatal neutrophils in vitro. Further study is required to determine whether G-CSF improves neonatal neutrophil function.

Hyperoxia- and hypoxia-mediated activation of polymorphonuclear leukocytes: a comparison of cord and adult venous blood

Background: Among the most prominent changes occurring in newborn infants is the exposure of tissues and blood cells to increased oxygen tension. This increase is even more pronounced in neonatal resuscitation using 100% oxygen, currently recommended in the published guidelines. Objective: To analyse the response of neonatal and adult polymorphonuclear neutrophils (PMN) to high or low oxygen tension in vitro. Materials: Neonatal cord blood and adult venous blood without previous contact to ambient air was exposed to 0, 21, or 100% oxygen for 30 min followed by incubation for up to 24 h. Methods: Flow cytometry was used to assess PMN activation as indicated by downregulation of L-selectin expression. Cell viability was quantified by the amount of propidium iodide uptake. Results: In adult PMN, L-selectin downregulation was greatly accelerated by hypoxia (PO2=27.2±3.4 mmHg) compared with both normoxia (PO2=71.0±11.0 mmHg) or hyperoxia (PO2=653.2±9.4) (P<0.05). In contrast, hyperoxia was the most potent stimulus for cord blood PMN, compared with both normoxia and hypoxia (P<0.05). Evidence of necrosis as indicated by positive staining for propidium iodide was similar in cord blood (10 h: 5.83% in oxygen) and in adult blood (10 h: 6.45% in oxygen). No differences were found between exposure to hypoxia, normoxia, or hyperoxia. Conclusion: Oxygen exposure of neonatal PMN leads to a more pronounced activation as compared with adult cells. Exposure towards high concentrations of oxygen may contribute to inflammatory processes during early neonatal life.

A comparison of foal and adult horse neutrophil function using flow cytometric techniques

Flow cytometric assays were used to compare phagocytic and oxidative burst activity of neutrophils from healthy foals less than 7 days of age with the activity of cells from healthy adult horses. The phagocytosis of Staphylococcus aureus by foal neutrophils was less than that observed for adult neutrophils when autologous serum was used as the source of opsonins in the assay. The use of adult serum did not significantly improve the ability of foal neutrophils to attach bacteria. The oxidative burst activity of foal neutrophils was equivalent to that of adult cells. However, when serum or plasma was incorporated into the oxidative burst assay, foal neutrophils demonstrated greatly reduced autofluorescence and a suppressed response to phorbol myristate acetate (PMA), relative to that demonstrated by adult cells. These results suggest that peripheral blood neutrophils from foals have a reduced ability to phagocytose bacteria relative to that exhibited by adult horse neutrophils and that the oxidative burst activity of foal neutrophils is down-regulated in response to an unidentified serum factor(s). Such changes may contribute to the increased susceptibility of foals to septic disease.

Activation of extracellular signal-related protein kinases 1 and 2 of the mitogen-activated protein kinase family by lipopolysaccharide requires plasma in neutrophils from adults and newborns.

Neutrophils exposed to low concentrations of gram-negative lipopolysaccharide (LPS) become primed and have an increased oxidative response to a second stimulus (e.g., formyl-methionyl-leucyl-phenylalanine [fMLP]). In studies aimed at understanding newborn sepsis, we have shown that neutrophils of newborns are not primed in response to LPS. To further understand the processes involved in LPS-mediated priming of neutrophils, we explored the role of extracellular signal-related protein kinases (ERK 1 and 2) of the mitogen-activated protein kinase family. We found that LPS activated ERK 1 and 2 in cells of both adults and newborns and that activation was plasma dependent (maximal at > or =5%) through LPS-binding protein. Although fibronectin in plasma is required for LPS-mediated priming of neutrophils of adults assessed by fMLP-triggered oxidative burst, it was not required for LPS-mediated activation of ERK 1 and 2. LPS-mediated activation was dose and time dependent; maximal activation occurred with approximately 5 ng of LPS per ml and at 10 to 40 min. We used the inhibitor PD 98059 to study the role of ERK 1 and 2 in the LPS-primed fMLP-triggered oxidative burst. While Western blotting showed that 100 microM PD 98059 completely inhibited LPS-mediated ERK activation, oxidative response to fMLP by a chemiluminescence assay revealed that the same concentration inhibited the LPS-primed oxidative burst by only 40%. We conclude that in neutrophils, LPS-mediated activation of ERK 1 and 2 requires plasma and that this activation is not dependent on fibronectin. In addition, we found that the ERK pathway is not responsible for the lack of LPS priming in neutrophils of newborns but may be required for 40% of the LPS-primed fMLP-triggered oxidative burst in cells of adults.

Enhancement of neonatal innate defense: effects of adding an N-terminal recombinant fragment of bactericidal/permeability-increasing protein on growth and tumor necrosis factor-inducing activity of gram-negative bacteria tested in neonatal cord blood ex vivo.

Innate defense against microbial infection requires the action of neutrophils, which have cytoplasmic granules replete with antibiotic proteins and peptides. Bactericidal/permeability-increasing protein (BPI) is found in the primary granules of adult neutrophils, has a high affinity for lipopolysaccharides (or "endotoxins"), and exerts selective cytotoxic, antiendotoxic, and opsonic activity against gram-negative bacteria. We have previously reported that neutrophils derived from newborn cord blood are deficient in BPI (O. Levy et al., Pediatrics 104:1327-1333, 1999). The relative deficiency in BPI of newborns raised the possibility that supplementing the levels of BPI in plasma might enhance newborn antibacterial defense. Here we determined the effects of addition of recombinant 21-kDa N-terminal BPI fragment (rBPI(21)) on the growth and tumor necrosis factor (TNF)-inducing activity of representative gram-negative clinical isolates. Bacteria were tested in citrated newborn cord blood or adult peripheral blood. Bacterial viability was assessed by plating assay, and TNF-alpha release was measured by enzyme-linked immunosorbent assay. Whereas adult blood limited the growth of all isolates except Klebsiella pneumoniae, cord blood also allowed logarithmic growth of Escherichia coli K1/r and Citrobacter koseri. Bacteria varied in their susceptibility to rBPI(21)'s bactericidal action: E. coli K1/r was relatively susceptible (50% inhibitory concentration [IC(50)], approximately 10 nM), C. koseri was intermediate (IC(50), approximately 1,000 nM), Klebsiella pneumoniae was resistant (IC(50), approximately 10,000 nM), and Enterobacter cloacae and Serratia marcescens were highly resistant (IC(50), >10,000 nM). All isolates were potent inducers of TNF-alpha activity in both adult and newborn cord blood. In contrast to its variable antibacterial activity, rBPI(21) consistently inhibited the TNF-inducing activity of all strains tested (IC(50), 1 to 1,000 nM). The antibacterial effects of rBPI(21) were additive with those of a combination of conventional antibiotics typically used to treat bacteremic newborns (ampicillin and gentamicin). Whereas ampicillin and gentamicin demonstrated little inhibition of bacterially induced TNF release, addition of rBPI(21) either alone or together with ampicillin and gentamicin profoundly inhibited release of this cytokine. Thus, supplementing newborn cord blood with rBPI(21) potently inhibited the TNF-inducing activity of a variety of gram-negative bacterial clinical pathogens and, in some cases, enhanced bactericidal activity. These results suggest that administration of rBPI(21) may be of clinical benefit to neonates suffering from gram-negative bacterial infection and/or endotoxemia.

Role of inflammatory mediators in priming, activation, and deformability of bovine neutrophils.

To determine the capacity of inflammatory mediators tumor necrosis factor-alpha (TNF-alpha), interleukin-8 (IL-8), platelet-activating factor (PAF), lipopolysaccharide (LPS), and leukotoxin to prime, activate, or alter deformability of adult bovine neutrophils. Blood collected from 5 healthy adult Holstein cows. Isolated neutrophils or whole blood was incubated with TNF-alpha, IL-8, PAF, LPS, or leukotoxin, and neutrophil chemiluminescence, degranulation, deformability, shape change, CD11b expression, and size distribution was measured. Incubation with TNF-alpha, IL-8, PAF, and LPS primed neutrophils for oxygen radical release but caused minimal oxygen radical release by themselves. None of the inflammatory mediators induced degranulation. Incubation with TNF-alpha and PAF resulted in a decrease in neutrophil deformability and induced shape change in neutrophils. Incubation with PAF consistently resulted in an increase in neutrophil size as measured by use of flow cytometry. Only IL-8 caused an increase in expression of CD11b by neutrophils. Inflammatory mediators tested had minimal effects on neutrophil oxygen radical production or degranulation but did prime neutrophils for oxygen radical production. Incubation with PAF and TNF-alpha caused a decrease in neutrophil deformability and altered neutrophil shape and size. Results of our study indicate that PAF- and TNF-alpha-induced changes in neutrophil deformability and size may cause integrin- and selectin-independent trapping of neutrophils in the lungs of cattle with pneumonic pasteurellosis.

Neonatal neutrophil interaction with P-selectin: contribution of P-selectin glycoprotein ligand-1 and sialic acid

Previously we had determined that neonatal neutrophils had decreased interaction with monolayers expressing P-selectin compared to adult cells. In this study we examined the function of neonatal P-selectin glycoprotein ligand-1 (PSGL-1). A rabbit polyclonal antibody directed against the amino terminus of human PSGL-1 was produced and purified (3RB-PSGL-1). Neonatal neutrophils expressed the epitope recognized by 3RB-PSGL-1 and expression was decreased compared with adult neutrophils (20%, P<0.05). In addition neonatal neutrophils had decreased interaction with Chinese hamster ovary (CHO)-P-selectin under both shear conditions and static adhesion (P<0.05). Treatment of both neonatal and adult neutrophils with 3RB-PSGL-1 similarly inhibited the interaction with P-selectin monolayers under shear conditions, effects similar to treatment with O-sialoglycoprotein endopeptidase (OSGE). Neuraminidase treatment of neonatal and adult cells also markedly inhibited the interaction. In a detachment assay marked differences were noted between neonatal and adult cells treated with either 3RB-PSGL-1 or neuraminidase. Such treatments had little effect on adult neutrophils until shear stress exceeded 2.8 dynes/cm2. Treated neonatal neutrophils were exquisitely sensitive to shear stress with a marked decrease in interaction noted at a shear stress as low as 0.6 dynes/cm2. Thus the adhesive mechanisms that remain after treatment with neuraminidase or 3RB-PSGL-1 have a relatively low avidity and function less well in neonatal neutrophils compared to adult neutrophils. We speculate that this may account for the less efficient adhesion of neonatal neutrophils to P-selectin under conditions of flow.

Effect of Fmlp, Il-8, Tnf-α, Pentoxifylline and Enoximone on Cell Deformability of Neutrophils in Neonates and Adults

Effect of Fmlp, Il-8, Tnf-α, Pentoxifylline and Enoximone on Cell Deformability of Neutrophils in Neonates and Adults

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 &lt; .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 &lt; .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.

P-Selectin Support of Neonatal Neutrophil Adherence Under Flow: Contribution of L-Selectin, LFA-1, and Ligand(s) for P-Selectin

AbstractTo 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.

Neonatal neutrophil inflammatory responses: parallel studies of light scattering, cell polarization, chemotaxis, superoxide release, and bactericidal activity.

Neutrophil dysfunction among newborn infants, especially those born prematurely, is well recognized, but the mechanism responsible for this phenomenon is yet to be clarified. In this study, we evaluated the stimulus response coupling in neutrophils from 90 healthy newborns and 96 healthy adults in an effort to establish whether defective neonatal neutrophil function is a result of impaired signal perception or immature responsiveness. Measurement of rapid- and slow-light scattering responses (LSR) to 1 microM FMLP stimulation revealed that neonatal neutrophils have about one-half the corresponding responsiveness of adult cells (rapid-LSR: 6.1 +/- 3.1 arbitrary light intensity units vs. 12.0 +/- 2.8, P < .001; and slow-LSR: 5.0 +/- 2.5 vs. 9.1 +/- 2.0; P < .001). The same markedly reduced activity was observed in newborn neutrophil chemotaxis and bactericidal activity in comparison with adult cells. Nevertheless, low FMLP concentrations (less than 1 nM) induced no difference in cell polarization between newborn and adult neutrophils, yet at higher FMLP concentrations, the newborn revealed significantly reduced cell polarization. Our data suggest that newborn infants bear a fully functional FMLP signal perception but lack the full capacity of inflammatory responsiveness.