Adult PMNs Research Articles

Placental HTRA1 Protease Cleaves Alpha-1-Antitrypsin and Generates Neonatal NET-Inhibitory Factor

BACKGROUND: Neutrophil extracellular traps (NET) are extracellular lattices of decondensed chromatin associated with anti-microbial proteins and degradative enzymes released by polymorphonuclear leukocytes (PMN) to trap and kill invading microbes. Dysregulated NET formation, however, contributes to inflammatory tissue damage. We have identified a novel NET-inhibitory peptide, neonatal NET-Inhibitory Factor (nNIF), present in the fetal circulation. nNIF is formed as a carboxy-terminus cleavage fragment of alpha-1 antitrypsin (AAT), an abundant, circulating protease inhibitor with homologs in human and mouse blood. However, the exact mechanisms by which nNIF is generated in fetal and neonatal blood remains unknown.OBJECTIVE: High temperature requirement A 1 (HTRA1) is expressed in the placenta during fetal development and inhibits AAT. We hypothesized that placentally expressed HTRA1, a serine protease, regulates the formation of NET-inhibitory peptides, such as nNIF, through cleavage of AAT.DESIGN/METHODS: Term and preterm placenta were lysed and probed for HTRA1 expression. HTRA1 and AAT plasma expression from term and preterm infants and adults were determined by ELISA. Recombinant, bioactive HTRA1 or placenta-eluted HTRA1 were incubated with AAT and the generation of carboxy-terminus fragments of AAT was assessed using western blotting and mass spectrometry. Fragments of AAT generated by HTRA1 were incubated with LPS-stimulated PMNs and NET formation was examined qualitatively using live cell imaging and quantitatively using a high throughput fluorescence assay. The effect of the HTRA-AAT cleavage fragment on reactive oxygen species generation, neutrophil chemotaxis, phagocytosis, and bacterial killing was measured using flow cytometry, a modified Boyden chamber asssay, neutrophil labeled Escherichia coli uptake assay, and a bacterial killing assay with a pathogenic strain of Escherichia coli, respectively. Finally, NET formation was evaluated qualitatively and quantitatively in murine PMNs isolated from neonatal WT and HTRA1-/- pups between 1-3, 4-6 and 7-10 days after birth to determine when PMNs become NET-competent.RESULTS: Term and preterm infant placentas express HTRA1, and we detected significantly (p<0.05) higher levels of HTRA1 in plasma from term (465.1±71.8 µg/mL) and preterm (385.9±71.3 µg/mL) infant cord blood compared to adults (58.6±11.6 µg/mL). Recombinant, bioactive HTRA1 and placenta-derived HTRA1 incubated with AAT generate a 4kD AAT fragment based on western blot and mass spectrometry similar to the nNIF fragment found in cord blood from term and preterm infants. Pre-incubation of this fragment with LPS-stimulated PMNs significantly inhibits NET formation (p<0.05). The cleavage fragment from HTRA1-AAT, however, has no effect on reactive oxygen species generation, chemotaxis, or phagocytosis. However, incubation of this fragment with LPS-stimulated PMNs significantly (p<0.05) reduces NET-associated bacterial killing by 62% compared to a scrambled HTRA-AAT control peptide. In addition, the HTRA1-AAT fragment significantly (p<0.05) reduces nuclear decondensation by 93% compared to LPS-stimulated PMN, suggesting this fragment inhibits PAD4 activation similar to other NIFs previously examined. Neonatal murine plasma contains a 4kD AAT fragment which inhibits NET formation by adult mouse PMNs, indicating that nNIF generation is conserved in mice. Neonatal PMNs stimulated with LPS exhibit delayed NET formation following birth with PMNs becoming NET-competent by day 8 of life. However, neonatal PMNs from pups born from HTRA1-/- deficient mice generate significantly (p<0.05) more NETs between day 4-6 of life compared to WT controls, suggesting that HTRA1 regulates NET formation through nNIF production.CONCLUSIONS: Placental HTRA1 interacts with AAT to generate a carboxy-terminus cleavage fragment of AAT with identical NET-inhibitory properties to nNIF. Our data strongly indicate that placental HTRA1 generates nNIF in the fetal circulation. We speculate that nNIF participates in the required tolerance to new microbial antigens encountered during the transition to extrauterine life. DisclosuresNo relevant conflicts of interest to declare.

Diminished ROS Production in Neonatal Polymorphonuclear Leukocytes Stimulated with Escherichia coli

Bacterial infection is an important cause of death and long-term morbidity in neonates, especially in immature preterm neonates. The present study clarified the defense mechanism in neonates by measuring superoxide anion (one of reactive oxygen species: ROS) production and phagocytic activity of polymorphonuclear leukocytes (PMNs) caused by live bacterial stimulation in whole blood. In neonatal PMNs, superoxide anion production was significantly diminished when stimulated with an appropriate amount of Escherichia coli (E. coli). There was no significant difference in ROS production with stimulation of Staphylococcus aureus (S. aureus) between adult and neonatal PMNs. Both adult and neonatal PMNs showed equal activity in E. coli phagocytosis. The expression of Toll-Like Receptor 2 (TLR2), Toll-Like Receptor 4 (TLR4) and CD14 on the PMNs showed no difference between neonate and adult blood. The p38 mitogen-activated protein kinase (p38 MAPK) was also activated equally with the stimulation of live bacteria in both PMNs. Since p38 MAPK is a key molecule in lipopolysaccharide binding protein (LBP)-TLR4 mediating intracellular pathway, the receptor mediated signal transduction pathways have no difference between adult and neonatal PMNs. Diminished superoxide production of neonatal PMNs with E. coli stimulation was reconstituted, when neonatal PMNs were stimulated in adult plasma. This phenomenon was abolished by the heat inactivation of adult plasma but not by the addition of anti-interferon γ or anti-tumor necrosis factor α blocking antibody. These data suggest that some heat-labile factors in whole blood might have a role in augmentation of the ROS production of neonatal PMNs, responding to E. coli.

Different Modulating Effects of Adenosine on Neonatal and Adult Polymorphonuclear Leukocytes

Polymorphonuclear leukocytes (PMNs) are the major leukocytes in the circulation and play an important role in host defense. Intact PMN functions include adhesion, migration, phagocytosis, and reactive oxygen species (ROS) release. It has been known for a long time that adenosine can function as a modulator of adult PMN functions. Neonatal plasma has a higher adenosine level than that of adults; however, little is known about the modulating effects of adenosine on neonatal PMNs. The aim of this study was to investigate the effects of adenosine on neonatal PMN functions. We found that neonatal PMNs had impaired adhesion, chemotaxis, and ROS production abilities, but not phagocytosis compared to adult PMNs. As with adult PMNs, adenosine could suppress the CD11b expressions of neonatal PMNs, but had no significant suppressive effect on phagocytosis. In contrast to adult PMNs, adenosine did not significantly suppress chemotaxis and ROS production of neonatal PMNs. This may be due to impaired phagocyte reactions and a poor neonatal PMN response to adenosine. Adenosine may not be a good strategy for the treatment of neonatal sepsis because of impaired phagocyte reactions and poor response.

IMPAIRED NEUTROPHIL EXTRACELLULAR TRAP FORMATION IN PLATELET-ACTIVATING FACTOR- AND LIPOPOLYSACCHARIDE-STIMULATED POLYMORPHONUCLEAR LEUKOCYTES ISOLATED FROM PRETERM INFANTS.

Background Activated human polymorphonuclear leukocytes (PMNs) participate in the innate immune response precipitated by tissue injury or bacterial invasion. As a recently described response to bacterial invasion, PMNs secrete lattices of DNA and antimicrobial proteins termed neutrophil extracellular traps (NETs) to effect extracellular killing of bacteria. However, PMNs isolated from infants born prematurely exhibit in vitro and in vivo functional deficits in bacterial killing. Whether these deficits result from impaired NET formation in preterm PMNs remains unknown. Hypothesis We hypothesize that preterm PMNs fail to robustly form NETs following activation in vitro. Methods We therefore stimulated human PMNs isolated from adults, term infants, and preterm infants with platelet-activating factor (PAF) (10−8M), lipopolysaccharide (LPS) (100 ng/mL), or live bacteria (Escherichia coli, 0.1-10 MOI) for 30 to 120 minutes. We visualized NET formation of live and fixed PMNs stained for extracellular DNA and myeloperoxidase expression via confocal microscopy, with and without time-lapse photography. We also obtained high-resolution images of NET formation in all three cell types via scanning electron microscopy of fixed PMNs following stimulation with PAF, LPS, or E. coli. Results We visualized limited or absent NET formation via confocal microscopy at 30 and 120 minutes in LPS- and E. coli-stimulated preterm PMNs compared with robust NET formation at 30 and 120 minutes in term and adult PMNs following the same stimuli. PAF failed to induce NET formation in preterm PMNs but did induce NET formation in term and adult PMNs at 30 minutes. Time-lapse confocal microscopy allowed visualization of NET formation in term and adult PMNs but failed to show NET formation in preterm PMN preparations. Furthermore, scanning electron microscopy confirmed robust NET formation at 30 minutes in term and adult PMNs following stimulation with PAF, LPS, and E. coli but noted minimal NET formation under those conditions in preterm PMNs. Conclusion We conclude that in vitro NET formation is impaired in preterm PMNs when compared with term and adult PMNs. Speculation We speculate that impaired NET formation may contribute to bacterial killing deficits documented in preterm infants, thus contributing to the morbidity and mortality in preterm infants associated with neonatal sepsis.

Blood Cell Deformation in Neonates Who Have Sepsis

After completing this article, readers should be able to: 1. Describe alterations of red blood cells due to bacterial toxins. 2. Describe physical alterations of neutrophils during activation. 3. Delineate the role of red and white blood cells in the impairment of microcirculation and organ damage in sepsis. 4. List drugs that may inhibit neutrophil activation and improve their deformability and their actions. Sepsis remains a major cause of morbidity and mortality in neonates, particularly among preterm infants. (1) Impaired microcirculatory blood flow plays a pivotal role in the development of clinical manifestations and organ dysfunctions in severe sepsis and septic shock. (2)(3)(4) If not corrected, microcirculatory dysfunction can progress to organ dysfunction and subsequently to organ failure and death. Restoration of microcirculatory dysfunction is, therefore, an important step in preventing long-term sequelae (including brain damage) and death of the patient. Both red and white blood cells must deform to pass through narrow channels whose diameters are less than those of the cells. (5) Impaired deformability of red and white blood cells may contribute to impaired microcirculatory blood flow in septicemia. The membranes of neonatal red blood cells (RBCs) deform more in response to a given force than adult cells and are, therefore, more flexible. (6)(7) On the other hand, neonatal RBCs are larger and require higher pressures to enter filter pores and micropipettes that have diameters below the resting cellular diameters. (8)(9) The larger volume and increased deformability of neonatal RBCs are responsible for another favorable property, the increased Fahraeus and Fahraeus-Lindqvist effect (ie, hematocrit and viscosity reduction when going from 500- to 50-mcm tubes). (10) These favorable flow properties of neonatal RBCs suggest that their increased cellular deformability is a prerequisite for adequate flow in arterioles and capillaries. We have shown that loss of …

Dexamethasone Improves Neutrophil Mediated Killing of Group B streptococcus(GBS) † 1463

Previous reports about the effect of corticosteroids on neutrophil phagocytic-killing function are conflicting. GBS is a significant cause of infection in certain adult population and neonates. Neutrophil mediated bacterial killing is an important defense against GBS infection. We hypothesized that dexamethasone (DEX) would affect neutrophil mediated bacterial killing and studied this in vitro. Neutrophils (PMNs) were isolated from healthy adults (n=27) and term neonates (n=8) by dextran and ficoll-hypaque density centrifugation and resuspended to a concentration of 2.5 × 107 cells/ml. PMNs were then either: a) incubated for two hours with various concentrations (0 to 100 mcg/ml) of DEX in the presence of GBS (0.5 × 105 cfu/ml, serotype III, strain SS 878), complement, and human antibody; or b) incubated with the same concentrations of dexamethasone for one hour, washed and resuspended to the same above PMNs concentration then incubated for two hours with GBS, complement, and human antibody. In both intances,bacterial killing was calculated based on colony counts performed prior to and after the final two hour incubation. No bacterial killing was noted in negative controls. Results are expressed as the log antibody-1 level (log Ab-1) at which the largest reciprocal dilution promoted ≥ 90% bacterial killing. Adult PMNs that were exposed to DEX during their interaction with GBS had no change in killing activity. However, adult PMNs exposed to the same concentrations of DEXbefore their interaction to GBS demonstrated a significant increase in killing activity (log Ab-1 = 1.24 at 100 mcg/ml vs. 0.8 at zero mcg/ml). Neonatal PMNs exposed to DEX during their interaction to GBS had a significant decrease in their killing activity (log Ab-1= 1.0 at zero mcg/ml vs. 0.45 at 100 mcg/ml). Interestingly, neonatal PMNs that were exposed to DEX before their interaction to GBS tended to improve their killing activity, though this was not statistically significant. Adult and neonatal PMNs enhance their killing activity, in a dose response fashion, when they are exposed to DEX only prior to their interaction with GBS. However, neonatal PMNs exposed to DEX only at the time of their encounter with GBS will demonstrate, in a dose response fashion, a decreased killing activity against these bacteria. The interaction of DEX, PMNs and GBS is complex and we speculate it may depend upon the timing of these interactions, the population of PMNs and the concentration of DEX.

Contribution of LFA-1 and Mac-1 to CD18-dependent neutrophil emigration in a neonatal rabbit model.

Human neonatal polymorphonuclear leukocytes (PMNs) exhibit decreased mobility, adherence, and transendothelial migration in vitro compared with adult PMNs. These deficits, in part, are due to functional and quantitative defects in neonatal Mac-1 (CD11b/CD18), whereas LFA-1 (CD11a/CD18) function is similar to that found in adults (D.C.Anderson, O.Abbassi, T.K.Kishimoto, J.M.Koenig, L.V.McIntire, and C.W.Smith, J.Immunol. 146: 3372-3379, 1991; C. W. Smith, T. K. Kishimoto, O. Abbassi, B.J.Hughes, R.Rothlein, L.V.McIntire, E.Butcher, and D.C. Anderson, J. Clin. Invest. 87: 609-618, 1991). We tested the hypothesis that the primary mechanism for the neonatal PMN CD18-dependent emigration in vivo is due to LFA-1. Neutrophils from 1-day-old rabbit pups had 32 and 60% of adult rabbit levels of CD11a and CD11b, respectively. Rabbit pups or adult rabbits received the monoclonal antibody (MAb) R7.1 (anti-CD11a) or R15.7 (anti-CD18) or the vehicle phosphate-buffered saline (PBS) before the instillation of intraperitoneal thioglycollate. Six hours later peritoneal exudate was collected. The administration of MAbs R7.1 and R15.7 in adult animals resulted in 60 and 83% inhibition of leukocyte emigration, respectively, compared with PBS-treated animals (P < 0.01). In neonatal animals, R7.1 and R15.7 inhibited leukocyte peritoneal accumulation to the same extent (50 and 60%, respectively) compared with PBS-treated animals (P < 0.01). Adult animals were also treated with the anti-CD11b MAb 198. MAb 198 decreased emigration by 25%, although this was not significant compared with PBS-treated animals. We conclude that although neonatal animals have significantly less neutrophil CD11a, the diminished levels did not contribute to a reduced ability to emigrate to the peritoneum and, like adult animals, neonatal animals primarily utilize LFA-1 for accumulation in this model. The contribution of Mac-1 to neonatal leukocyte emigration remains uncertain.

Transendothelial migration of neonatal and adult bovine neutrophils in vitro.

We have compared and quantitated transendothelial migration of neonatal neutrophils (N-PMNs) and adult bovine peripheral-blood PMNs (A-PMNs) in vitro using monolayers of endothelium and a two-chamber apparatus. Bovine aortic endothelial cells were cultured to confluence on polycarbonate filters perforated with 3.0-micron-diameter pores. 51Cr-labeled PMNs were added to the upper chamber, with or without an anti-CD18 antibody (monoclonal antibody 60.3). Chemotactic stimuli in the lower chambers included recombinant human interleukin-8 (rhIL-8; 75 ng/ml), rhC5a (10(-7) M), and zymosan-activated bovine serum (ZAS; 10%). At 60 min incubation with rhIL-8, greater numbers (P < .01) of N-PMNs (24.70 +/- 5.95%) than of A-PMNs (15.77 +/- 3.66%) had migrated across the endothelial barrier, and a similar difference was present at 90 min. Migration rates of N-PMNs and A-PMNs were similar (P > .05) at all time points when using rhC5a and ZAS as stimuli. Anti-CD18 monoclonal antibody significantly decreased migration (P < .01) of both N-PMNs and A-PMNs to low levels when IL-8 and ZAS were used as stimuli. Because leukocyte integrin expression on PMNs affects transendothelial migration, we also compared surface expression of CD18, CD11a, and CD11c on PMNs from the two age groups. We found no significant quantitative differences in integrin expression between PMNs from the two age groups, regardless of whether the PMNs were incubated with buffer alone or with chemotaxins (rhIL-8, rhC5a, ZAS).

Possible roles of LFA-1 and Fc gamma receptors on the functional immaturities of cord blood polymorphonuclear leukocytes.

The functional immaturity of PMNs is one of the major causes of overwhelming sepsis in newborns. In this study, we observed functions and surface markers of PMNs to investigate what causes the functional immaturity of PMNs in newborns. As results, the percentage of EA rosette forming PMNs (58.5 +/- 15.5%) and the chemotactic movement (0.14 +/- 0.09 mm) of cord blood PMNs were significantly lower than those of adult peripheral blood PMNs (70.8 +/- 9.9%, 0.60 +/- 0.34 mm). Cord blood PMNs showed decreased glass adherence and ADCC activity. The expression of Fc gamma RII or Fc gamma RIII was a little lower than those of adult peripheral blood PMNs, but the expression of Fc gamma RI (43.1 +/- 26.8%) was significantly higher than that of adult peripheral blood PMNs (3.2 +/- 1.8%). There was a significant difference in LFA-1 expression between EA rosette forming PMNs (92.9 +/- 9.1%) and EA rosette non-forming PMNs (25.6 +/- 22.6%). From these results, it is assumed that neonatal PMNs may consist of heterogeneous populations. And the relatively high percentage of EA rosette non-forming PMNs which express a low level of LFA-1 may be responsible for the functional immaturity of cord blood PMNs.

Improved chemotactic ability of neonatal polymorphonuclear cells induced by mild membrane rigidification.

Membrane lipid fluidity of peripheral blood polymorphonuclear cells (PMNs) of 24 newborn infants, 2-4 days after birth, was determined by steady-state fluorescence polarization with 1,6-diphenyl 1,3,5-hexatriene (DPH) as a probe and compared with that of PMNs from 23 adults. Measurements with intact cells, which correspond to all cellular lipid domains, did not display any statistically significant difference between PMNs of the two groups. However, application of bixinoyl glucosamine, a membrane-impermeable fluorescence quencher, revealed that the PMN plasma membrane of the newborn is about 23% more fluid than that of the adult. Total cholesterol-to-phospholipid ratio of newborn PMNs was found to be lower by about 10% than that of the adult, which could account for the difference in their plasma membrane fluidity. The possible implication of this finding for the deficit in chemotactic ability of leukocytes from newborns was tested with neonatal PMNs that have incorporated cholesteryl hemisuccinate (CHS), an efficient plasma membrane rigidifier. In all neonatal PMNs tested a mild incorporation of CHS (0.5-1 min incubation in 50 micrograms/ml dispersion) caused a significant improvement in their net chemotaxis, from an average value of 28 +/- 7 to 43 +/- 11. Longer incubations with CHS caused a gradual decrease in chemotactic ability that approached the basal level after about 5 min incubation. The net chemotaxis in adult PMNs was significantly higher than that of neonatal PMNs (72 +/- 13) and was gradually inhibited by incorporation of CHS without any initial augmentation. Based on these results it was estimated that about 27% of the chemotactic deficit of neonatal PMNs is mediated by their immature fluid membrane.

Calcium mobilization in C5a-stimulated adult and newborn bovine neutrophils

Calcium (Ca2+) is an important second messenger central to many neutrophil (PMN) functional activities. Impaired Ca2+ mobilization in newborn PMNs following membrane perturbation could be one of the mechanisms underlying observed abnormalities in neonatal PMN function. To compare Ca2+ mobilization in bovine newborn and adult PMNs, cytosolic Ca2+ responses after stimulation with recombinant human C5a (rHC5a) were measured. PMNs from normal newborn calves (N = 6) and adult cows (N = 5) were loaded with fura-2/AM for 60 min at room temperature and the fluorescence changes monitored following stimulation with 0.1, 1, 10, or 50 nM rHC5a in Ca2(+)-containing buffer. Resting levels of Ca2+ in both newborn (54.6 +/- 1.9 nM) and adult (57.3 +/- 1.8 nM) bovine PMNs were comparable. After stimulation with rHC5a, a rapid rise of cytosolic Ca2+ was observed, which peaked within 20 sec and was followed by a sustained phase of elevated Ca2+ lasting up to 20 min. There were no significant differences (P greater than 0.05) in peak levels of cytosolic Ca2+ obtained by newborn and adult PMNs at 0.1, 10, and 50 nM rHC5a. At 1 nM rHC5a, newborn PMNs reached significantly (P less than 0.05) higher levels of cytosolic Ca2+ (217.9 +/- 21.7 nM) than did adult PMNs (158.7 +/- 7.9 nM). At 1 nM rHC5a, newborn PMNs also sustained higher levels of cytosolic Ca2+ for 3 min following the peak. At all concentrations of rHC5a tested, the time required to reach the peak and the duration of the peak were comparable in both populations. In the absence of extracellular Ca2+ (Ca2(+)-free buffer with 1 mM EGTA), resting levels of cytosolic Ca2+ were lower in both newborn (33.3 +/- 2.9 nM) and adult PMNs (27.9 +/- 2.4 nM) and the magnitude of the peak response to rHC5a was diminished at all concentrations of agonist. Additionally, in the absence of extracellular Ca2+, the return to basal cytosolic Ca2+ levels occurred rapidly and the sustained phase of increased cytosolic Ca2+ seen with rHC5a-stimulated PMNs in Ca2(+)-containing buffer was virtually eliminated. These results indicate that bovine PMNs respond well to rHC5a, that stimulated newborn bovine PMNs can mobilize Ca2+ as efficiently as adult PMNs, and that the sustained cytosolic Ca2+ response to rHC5a in both age groups requires both release of Ca2+ from intracellular stores as well as influx of extracellular Ca2+. Such data suggest that observed functional deficits in newborn bovine PMNs are probably not related to improper mobilization of Ca2+ following stimulation.

Alterations in complement-induced shape change and stimulus-specific superoxide anion generation by neonatal calf neutrophils

Increased susceptibility of neonates to infection may be related to defects in newborn neutrophil (PMN) functional activities, including altered responses to complement fragments (Cf) and defective microbicidal activity. We therefore compared the kinetics of newborn and adult bovine PMN membrane shape change responses following stimulation with zymosan-activated plasma (ZAP) as a source of Cf. Measurement of PMN membrane shape change was a rapid, sensitive, and reproducible measure of Cf stimulation within a population of PMNs; a maximum of 67-85% of the PMNs exhibited easily detectable membrane ruffling, lamellipodia formation, and polarity within 2 min. Newborn PMNs exhibited significantly increased (P less than 0.01) membrane shape change at 20, 30, 60, 120, and 300 sec after Cf stimulation. A maximum of 85.8 +/- 3.2% of newborn PMNs exhibited such Cf-induced shape changes by 120 sec. which was significantly greater (P less than 0.01) than the maximum stimulation (67.7 +/- 4.3%) attained with adult PMNs. These data indicate enhanced kinetics of induced newborn PMN membrane shape change in response to Cf stimulation. We also compared stimulus-specific superoxide anion (O2-) generation as a measure of respiratory burst activity after incubation of newborn and adult PMNs with soluble (phorbol myristate acetate, PMA) and particulate (opsonized zymosan, OZ) stimuli. When PMA was used as the stimulus, newborn PMNs generated significantly less O2- (9.3 +/- 0.5 nmol O2-/10(6) PMN, P less than 0.05) than did adult PMNs (12.4 +/- 0.3 nmol O2-/10(6) PMN). This finding was reversed when OZ was used as the stimulus; newborn PMNs generated significantly more O2- (7.7 +/- 0.4 nmol O2-/10(6) PMN, P less than 0.05) than did adult PMNs (5.5 +/- 0.5 nmol O2-/10(6) PMN). These findings collectively document biochemical and morphological differences between newborn and adult PMNs as determined by stimulus-specific O2- generation and Cf-induced membrane shape change. Such differences may be important to neonatal disease susceptibility.

Effects of Intravenous Fat Infusion on Neonatal Neutrophil and Platelet Function

Intravenous fat (Intralipid) is used extensively as a major component of parenteral nutrition for patients in the neonatal intensive care unit. Abnormalities of polymorphonuclear leukocyte (PMN) and platelet number or function related to Intralipid infusion have been reported although conflicting results exist. In order to examine potential adverse hematologic effects of Intralipid, 10 ill neonates were studied before and after a 16-hr infusion of 1 g/kilo of Intralipid. PMN count, chemokinesis, chemotaxis, and aggregation were unchanged pre- and post intralipid infusion. Platelet count, bleeding time, and platelet aggregation were also unchanged. Similar results were obtained in vitro when neonatal and adult PMNs and platelets were incubated in Intralipid and their function analyzed. These findings suggest that short-term, low-dose Intralipid has no measurable impact on neonatal PMN or platelet activity and support its use in neonates even in the presence of infection or thrombocytopenia.

Effect of pentoxifylline on developmental changes in neutrophil cell surface mobility and membrane fluidity

Polymorphonuclear leukocytes (PMNs) from human neonates respond less efficiently to chemotactic factor stimulation than do PMNs from adults. The biologic mechanisms underlying this developmental process are poorly understood. In previous studies, we have found that pentoxifylline, an agent report to enhance membrane deformability, increased the chemotactic response of neonatal PMNs. In the present studies, we have examined the effect of pentoxifylline on cell surface mobility and membrane fluidity by assessing fluorescent concanavalin A (Con A) capping and fluorescent polarization (FP). Baseline Con A capping was lower in the PMNs of neonates when compared to PMNs from adult controls. Colchicine, which increases capping by disrupting microtubules, exaggerated the differences between the adult and neonatal PMNs. Following exposure of neonatal PMNs to pentoxifylline, colchicine enhanced Con A capping to levels equivalent to those of colchicine-treated PMNs from adults. Employing a fluorescence polarization (FP) assay, we found the fluid state of the membrane of PMNs from neonates was significantly less than that of adult controls. Pentoxifylline alone significantly increased the fluidity of the cell membranes of neonatal PMNs while decreasing elevated basal levels of F-actin in the cell. These data suggest an intrinsic cytoskeletal difference in the PMNs of neonates that may be responsive to pharmacologic manipulation.

Ontogeny of inflammatory cell responsiveness

Newborn calves, like human infants, are uniquely susceptible to bacterial infections. Part of this increased susceptibility may be related to defects in newborn polymorphonuclear leukocyte (PMN) defensive functions. It remains unclear whether reported deficits in newborn PMN function represent maturational disorders or are manifestations of some form of perinatal suppression phenomenon. We therefore compared the ability of bovine newborn PMNs (less than 24 h old), newborn PMNs (7-10 days of age), fetal PMNs (210-220 days gestational age), and adult PMNs to generate superoxide anion (O2-) as an indicator of respiratory burst activity. Citrated blood was collected, and PMNs were isolated to greater than 95% purity and 98% viability. O2- generation was measured as the superoxide dismutase-inhibitable (10 micrograms/ml) reduction of ferricytochrome c (2 mg/ml) after activation of PMNs with phorbol myristate acetate (PMA, 2 micrograms/ml) to directly stimulate protein kinase C. The reaction kinetics were measured (37 degrees C, 550 nm) using a spectrophotometer and chart recorder for continuous monitoring. O2- generation was measured for 5 min after the initial lag period and the total nanomoles of O2- generated calculated using the extinction coefficient for ferricytochrome c. Newborn PMNs (N = 10) generated significantly less O2- (5.7 +/- 0.8 nmol O2-/10(6) cells/5 min, P less than 0.01) than did adult PMNs (N = 14) (9.6 +/- 2.1 nmol O2-/10(6) cells/5 min) or fetal PMNs (N = 4) (10.7 +/- 0.7 nmol O2-/10(6) cells/5 min). PMNs from 7- to 10-day-old calves (N = 9) generated almost identical amounts of O2- as newborn PMNs (5.7 +/- 1.6 nmol O2-/10(6) cells/5 min). There was no difference in measured lag time period between newborn and adult PMNs, but fetal PMNs had significantly reduced (P less than 0.01) mean lag time. The data indicated that bovine newborn PMNs have a decreased ability to generate O2- in response to PMA stimulation, which persists for at least 7-10 days, and that this functional decrement may be a manifestation of some form of perinatal PMN suppression phenomenon rather than a developmental abnormality since fetal PMNs produced O2- as well as adult PMNs.

Influence of monoclonal IgM cold agglutinins on adhesiveness, phagocytosis, and bactericidal activity on human granulocytes and monocytes.

Monoclonal IgM cold agglutinins (CA) bind and, in the presence of complement, are cytotoxic to various mammalian cells. The impact of these autoantibodies on functional capacity of phagocytes has not been studied until now. Herein we report that sera with monoclonal IgM anti-I and anti-i CA significantly reduce adhesiveness, phagocytosis, phagocytic index, and intracellular bactericidal activity of human peripheral blood polymorphonuclear cells (PMNs) at 37 degrees C and 24 degrees C. Anti-i CA were more active than anti-I. Sera with monoclonal IgMs without CA activity reduced the total number of ingested bacteria but otherwise had no effect on phagocytic functions. There was no difference in the degree of inhibition when anti-i and anti-I CA were tested against cord, maternal, and adult PMNs. Chromatographically purified a-I and a-i CA inhibited markedly phagocytosis in concentrations as low as 1 mg/ml. Phagocytic activity of peripheral blood monocytes was inhibited by CA at 18 degrees C but not at 24 degrees C or 37 degrees C. Pepsin digestion or reduction and alkylation of chromatographically pure IgM CA abolished completely their inhibitory activity. Thus, in physiological temperatures, monoclonal IgM cold agglutinins impair various phagocytic functions of human phagocytes. It may add to the susceptibility to infections in patients in which such autoantibodies are synthesized.

Increased production of superoxide anion by neonatal polymorphonuclear leukocytes stimulated with a chemotactic peptide.

In order to assess the functional property of neonatal polymorphonuclear leukocytes (PMNs), we studied their chemoattractant-stimulated superoxide anion (O2-) production using a soluble chemotactic peptide N-formyl-methionyl-leucyl-phenylalanine (FMLP) as a PMN stimulus. O2- production by neonatal PMNs was increased: when stimulated with 10(-7)M FMLP, O2- values (nanomoles; mean +/- SE) produced by 10(6) PMNs were 16.5 +/- 1.3 for neonatal PMNs and 12.6 +/- 0.6 for adult PMNs (p less than .02). When studied under various concentrations (10(-9) to 10(-6)M) of FMLP, neonatal PMNs produced more O2- than adult cells under the stimulation of a lower concentration (10(-8)M) as well as a higher concentration (10(-7)M) of FMLP. The increased O2- production by neonatal PMNs was also observed under their stimulation by phorbol myristate acetate. Neonatal and adult PMNs produced equal amounts of O2- when stimulated with a particulate stimulus of opsonized zymosan. To analyze further the mechanism for the increase in FMLP-induced O2- production by neonatal PMNs, we next studied whether there was some abnormality in their FMLP receptors. Neonatal PMNs had normal numbers of FMLP receptors; the receptor numbers (mean) per cell were 54,000 in neonatal PMNs and 56,000 in adult cells. Additionally, the FMLP receptors had normal affinity for the peptide. These results demonstrate that the increased production of O2- by FMLP-stimulated neonatal PMNs is not due to the abnormality of its binding to the receptors but is due to that of the subsequent events.

Impaired Surface Membrane Expression of C3bi but Not C3b Receptors on Neonatal Neutrophils

Because increased complement receptor expression is necessary for optimal function of adult neutrophils, we tested the hypothesis that the increased susceptibility of neonates to infection might be due to an impaired ability of neonatal neutrophils to increase expression of complement receptors in response to chemotactic stimuli. We used monoclonal antibodies and flow cytometry to compare surface expression of the receptors for the complement components C3b (CR1) and C3bi (CR3) on adult and neonatal cord blood neutrophils (PMNs). We also compared receptor expression on PMNs from infants delivered by cesarean section without labor versus infants delivered vaginally. Expression of both CR1 and CR3 was minimal on resting adult and neonatal PMNs maintained at 0 degrees C. There was a modest increase in expression of both receptors when PMNs were warmed to 37 degrees C. This increase was similar on adult and neonatal cells, both unfractionated in whole blood and after isolation with Percoll density centrifugation, with one exception. Expression of CR1 was greater on isolated PMNs from vaginally delivered infants versus adults when the cells were warmed to 37 degrees C. This difference was not observed with cells from infants delivered by cesarean section without labor, suggesting this modest increase in receptor expression may be due to factors associated with labor. When isolated cells were stimulated with either N-formyl-methionyl-leucyl-phenylalanine or zymosan-activated serum, expression of CR1 increased to the same extent in both neonatal and adult PMNs. In contrast, maximal CR3 expression on cord PMNs stimulated with N-formyl-methionyl-leucyl-phenylalanine or zymosan-activated serum was only 75% of the adult values.(ABSTRACT TRUNCATED AT 250 WORDS)

Polymorphonuclear Leukocyte Heterogeneity in Neonates and Adults

We have used a mouse monoclonal antibody (31D8) to determine whether differences in neutrophil (PMN) subpopulations might help explain decreased PMN chemotaxis in neonates compared with that of adults. 31D8 has been shown to bind heterogeneously to adult PMNs. Approximately 80% of the PMNs that strongly bind 31D8 (31D8 "bright") are the same cells that depolarize and migrate chemotactically when stimulated with the chemoattractant N-formyl-methionylleucylphenylalanine, while the 20% that weakly bind 31D8 fail to similarly respond. All neonatal PMNs bound 31D8 heterogeneously. There was a smaller population of 31D8 "bright" cells in neonates at birth (76% +/- 6%, n = 45) compared with that of neonates at three to 15 days of age (82% +/- 5%, n = 10, P less than 0.002) and both were smaller than that of adults (88% +/- 4%, n = 45, P less than 0.001 and P less than 0.001). Neonatal cord PMNs, which traversed a micropore filter in a modified Boyden chemotaxis chamber in the presence of a chemoattractant, had an increased percentage of 31D8 "bright" cells (89% +/- 7%) than did PMNs which remained above the filter (82% +/- 7%, n = 10, P = 0.034). PMN chemotaxis was less in neonates at birth (32.7 +/- 4.5 micron) than at three to six days of age (36.8 +/- 11.3 micron) and both were decreased compared with that of adults (69.1 +/- 12.4 micron, P less than 0.001 and P less than 0.001). These findings indicate that decreased PMN chemotaxis in neonates may be due in part to a smaller PMN subpopulation of highly motile cells.

Polymorphonuclear leukocyte heterogeneity in neonates and adults

We have used a mouse monoclonal antibody (31D8) to determine whether differences in neutrophil (PMN) subpopulations might help explain decreased PMN chemotaxis in neonates compared with that of adults. 31D8 has been shown to bind heterogeneously to adult PMNs. Approximately 80% of the PMNs that strongly bind 31D8 (31D8 “bright”) are the same cells that depolarize and migrate chemotactically when stimulated with the chemoattractant N-formyl-methionylleucylphenylalanine, while the 20% that weakly bind 31D8 fail to similarly respond. All neonatal PMNs bound 31D8 heterogeneously. There was a smaller population of 31D8 “bright” cells in neonates at birth (76% +/- 6%, n = 45) compared with that of neonates at three to 15 days of age (82% +/- 5%, n = 10, P less than 0.002) and both were smaller than that of adults (88% +/- 4%, n = 45, P less than 0.001 and P less than 0.001). Neonatal cord PMNs, which traversed a micropore filter in a modified Boyden chemotaxis chamber in the presence of a chemoattractant, had an increased percentage of 31D8 “bright” cells (89% +/- 7%) than did PMNs which remained above the filter (82% +/- 7%, n = 10, P = 0.034). PMN chemotaxis was less in neonates at birth (32.7 +/- 4.5 micron) than at three to six days of age (36.8 +/- 11.3 micron) and both were decreased compared with that of adults (69.1 +/- 12.4 micron, P less than 0.001 and P less than 0.001). These findings indicate that decreased PMN chemotaxis in neonates may be due in part to a smaller PMN subpopulation of highly motile cells.