Joyce M Koenig

Incidence, Neutrophil Kinetics, and Natural History of Neonatal Neutropenia Associated with Maternal Hypertension

Abstract Neutropenia occurs often among the newborns of women with hypertension, but its cause, mechanism, and clinical consequences have not been adequately studied. Of 72 infants whose mothers had hypertension during pregnancy, 35 (49 percent) had neutropenia, which persisted from 1 hour to 30 days. The disorder was more prevalent among newborns whose growth had been retarded in utero (P<0.01), those who had been delivered prematurely (P<0.001), and those whose mothers had had severe hypertension (P<0.002) or hypertension and the HELLP syndrome (hemolysis, elevated liver enzymes, and low platelets) (P<0.01). Kinetic investigations of circulating, marginated, storage, and proliferative neutrophils and their progenitors suggested that the neutropenia was the result of diminished neutrophil production. Nosocomial infections occurred during the first 2 1/2 weeks of life in eight (23 percent) of the newborns with neutropenia, but in only one (3 percent) of those without this disorder (P<0.01). We conclude th...

Anthrax Lethal Toxin Paralyzes Neutrophil Actin-Based Motility

Bacillus anthracis causes high-level bacteremia, strongly suggesting paralysis of the innate immune system. We have examined the effects of anthrax lethal toxin (LT) on human neutrophil chemotaxis, a process that requires actin filament assembly. Polymorphonuclear neutrophils (PMNs) treated with a sublethal concentration of LT (50 ng/mL) for 2 h demonstrated insignificant apoptosis or necrosis. However, this same concentration slowed human PMN formylmethionylleucylphenylalanine (FMLP)-stimulated chemokinesis by >60%, markedly reduced polar morphology, and rendered PMNs incapable of responding to a chemotactic gradient. These changes were accompanied by a >50% reduction in FMLP-induced actin filament assembly. One hour of exposure to LT failed to impair polarity or actin assembly, and the effects of LT were independent of mitogen-activated protein kinase kinase 1 inhibition. We conclude that 2 h of exposure to LT markedly impairs PMN actin assembly, and reductions in actin filament content are accompanied by a profound paralysis of PMN chemotaxis.

Production of interleukin-6 by fetal and maternal cells in vivo during intraamniotic infection and in vitro after stimulation with interleukin-1

ABSTRACT: Amniotic fluid samples were obtained by transabdominal amniocentesis from 20 women in preterm labor (≤34 wk gestation). Concentrations of IL-6 in culture-positive amniotic fluids (mean 8706 pg/mL, range 5100–14 446 pg/mL) were higher than those in culture-negative fluids (mean 1133 pg/mL, range ≤5–6534 pg/mL, p < 0.0001) or fluids from healthy term pregnancies (mean 196 pg/mL, range ≤5–790 pg/mL, p < 0.001). To assess possible sources of the IL-6 in amniotic fluid, we tested the ability of a variety of fetal and maternal cells to produce IL-6 in vitro after stimulation with IL-1, a cytokine known to stimulate IL-6 production. Very low concentrations of IL-6 were present in supernatants of cells not stimulated with IL-1; however, high concentrations were observed in supernatants of stimulated umbilical venous endothelial cells, decidual cells, and fetal and maternal blood mono-nuclear cells. To determine whether cells from adults produce IL-6 with kinetics similar to those of neonates, we incubated mononuclear cells obtained from blood of adults and term and preterm neonates with IL-1. After 6 h, IL-6 was detected in supernatants of adult cells and term neonatal cells, but not in supernatants of preterm cells. Concentrations at 18, 24, and 48 h were similar for adult and term cell supernatants, but were lower in supernatants of preterm cells. We also observed considerably more IL-6 mRNA accumulation in circulating mononuclear cells from adults than in those from neonates.

The mechanism responsible for diminished neutrophil production in neonates delivered of women with pregnancy- induced hypertension

The neonatal neutropenia after pregnancy-induced hypertension is a function of diminished neutrophil production. These studies test the hypothesis that this diminution is due to decreased production of neutrophilic growth factors, reduced responsiveness of neutrophil progenitors to these factors, or the presence of an inhibitor. While the concentrations of placentally derived colony-stimulating factors were similar in normotensive and hypertensive gestations, bioassay demonstrated less colony-stimulating activity in placental conditioned media from hypertensive gestations. Evaluation of the responsiveness of progenitors to recombinant factors revealed no differences between those from normotensive and hypertensive gestations. However, neutrophilic colony formation in vitro was significantly inhibited after the addition of conditioned media or sera from hypertensive gestations, whereas the addition of these from normotensive gestations had no inhibitory effect. Thus this common maternal-fetal disorder is associated with an inhibitor of neutrophil production, which is elaborated by the placenta and present in cord blood serum.

Spontaneous and Fas-mediated apoptosis are diminished in umbilical cord blood neutrophils compared with adult neutrophils.

Apoptosis mediates neutrophil (PMN) phagocytosis and is influenced by cytokines and the Fas/Fas ligand pathway. To determine whether apoptosis of cord blood PMN differs from those of adults, cultured PMN were evaluated by morphological analysis, flow cytometry (TUNEL assay), and DNA gel electrophoresis. In addition, we studied the effect of anti‐Fas IgM or cycloheximide on induction of PMN apoptosis. Spontaneous apoptosis (24 h) was less in cord blood PMN (mean ± SD; 29 ± 9 vs. adults, 56 ± 14%, P < 0.001). Treatment (6 h) with anti‐Fas IgM induced less apoptosis in cord blood PMN (24 ± 6 vs. adults, 63 ± 7%, P < 0.001), as did treatment with cycloheximide (13 ± 10 vs. adults, 55 ± 16%, P < 0.01). These data suggest the pre‐existence of proteins that inhibit apoptosis or the absence of those that promote apoptosis in cord blood PMN. J. Leukoc. Biol. 64: 331–336; 1998.

Neonatal Neutrophils with Prolonged Survival Exhibit Enhanced Inflammatory and Cytotoxic Responsiveness

Apoptosis is critical to the resolution of inflammation, as it promotes the removal of neutrophils (PMN) by the reticuloendothelial system. In contrast, PMN persistence characterizes the early stages of chronic inflammation. Adult PMN with delayed senescence retain some functionality, although this has not been described for neonatal PMN. We hypothesized that neonatal PMN with prolonged survival retain cytotoxic and inflammatory function. To test one aspect of inflammatory function, we determined surface CD11b expression on 0-h and 24-h PMN after chemotactic formyl-methionine-leucine-phenylalanine (fMLP) stimulation. Although fMLP induced a greater percentage up-regulation of CD11b on 0-h adult PMN, this was similar between nonapoptotic cord blood and adult PMN at 24 h. Furthermore, percentage up-regulation of CD11b was more robust for 24-h than for 0-h cord blood PMN. In contrast, there was no difference in responsiveness between 0-h and 24-h adult PMN. In studies of cytotoxic potential, we determined the expression of reactive oxygen intermediates (ROI) in phorbol 12-myristate 13-acetate–stimulated cord blood and adult PMN at 0 h and in 24-h nonapoptotic PMN, using the dihydrorhodamine 123 assay. Stimulated cord blood PMN generated more ROI than did adult PMN at both 0 h and 24 h; in addition, ROI levels in 24-h cord blood PMN were similar to those of 0-h adult PMN. We conclude that PMN with prolonged survival retain specific cytotoxic and inflammatory functions, and these are enhanced in cord blood PMN. We speculate that neonatal PMN with prolonged survival have the functional capacity to contribute to the pathogenesis of inflammatory disorders.

Diminished soluble and total cellular L-selectin in cord blood is associated with its impaired shedding from activated neutrophils.

We have previously shown that surface levels of the adhesive glycoprotein, L-selectin, are diminished on cord blood neutrophils (polymorphonuclear leukocytes, PMN) and associated with impaired adherence to endothelium under flow conditions. To test the hypothesis that diminished surface levels reflect a total cellular deficiency, we measured L-selectin in PMN lysates and plasma from cord and adult blood. L-selectin content was decreased in cord blood PMN lysates compared with those of adults by both Western blot analyses and ELISA(cord blood, 1195 ± 160 pg/mL; adult, 1870 ± 260 pg/mL;X ± SEM; p < 0.05). Soluble L-selectin levels were also decreased in cord blood plasma (324 ± 24 ng/mLversus 537 ± 28 ng/mL in adult plasma, p < 0.01). To evaluate L-selectin function, we next compared the dose dependent effect of several chemoattractants on shedding of L-selectin from cord blood and adult PMN. Adult PMN showed greater overall shedding of L-selectin as compared with cord blood PMN after stimulation with fMet-Leu-Phe (p< 0.03) and granulocyte-macrophage colony-stimulating factor (p< 0.02). In contrast, shedding of L-selectin was similar between groups after IL-8 tested stimulation. We conclude that cord blood PMN have a decreased cellular content of L-selectin in addition to an impaired ability to shed surface L-selectin in response to specific inflammatory mediators.

Fas and Fas ligand expression in maternal blood and in umbilical cord blood in preeclampsia

The Fas-Fas ligand (FasL) pathway of apoptosis is abnormally activated in diseases associated with impaired immune tolerance or chronic inflammation. Pregnancy-related hypertension is a spectrum of disease that commonly causes significant morbidity in women and in their newborn infants, is associated with generalized inflammation, and may be causally related to impaired maternal-fetal tolerance. Our recent observation of enhanced trophoblast expression of FasL in one form of pregnancy-related hypertension led us to hypothesize that this group of disorders might be associated with abnormal activation of the Fas-FasL pathway. To test this hypothesis, we prospectively quantified soluble and leukocyte-associated Fas receptor and FasL in the maternal and umbilical cord blood (CB) sera of 20 gestations complicated by preeclampsia and of 18 normal control gestations, using ELISA and flow cytometric analyses. We determined higher soluble FasL levels in paired maternal and CB sera of hypertensive gestations compared with control gestations (p < 0.01); in contrast, soluble Fas levels were similar between groups. Surface expression of FasL was lower on maternal (p < 0.01) and CB (p < 0.05) neutrophils from affected gestations, whereas surface Fas expression was lower on maternal (p < 0.02), but not CB, neutrophils and lymphocytes. We conclude that expression of Fas and FasL in sera and on leukocytes is altered in gestations complicated by preeclampsia, and speculate that activation of the Fas-FasL pathway mediates associated pathologic processes in affected women and in their neonates.

L-selectin expression enhances clonogenesis of CD34+ cord blood progenitors.

L-selectin, a surface adhesion glycoprotein expressed on leukocytes, has a well-established role in mediating inflammation and lymphocyte recirculation. Recent evidence suggests that L-selectin may also influence hematopoiesis. We observed that a greater proportion of CD34+ cells express L-selectin in cord blood compared with adult bone marrow, and we hypothesized that L-selectin expression is associated with enhanced clonogenic properties. To test this, we compared CD34+/L-selectin+ cells with CD34+/L-selectin- cells in hematopoietic clonogenic assays. From CD34+/L-selectin+ cell cultures, we observed a 3-fold increase of d 12-14 colony-forming unit-granulocyte/macrophage and multipotent progenitor cells, and a 5-fold enhancement of primitive d 21 high proliferative potential colony-forming cells compared with the progeny of CD34+/L-selectin-cells. We conclude that CD34+ cord blood cells expressing L-selectin are enriched in their clonogenic activity compared with cell fractions lacking L-selectin expression.

Effect of Erythropoietin on Granulocytopoiesis: In Vitro and in Vivo Studies in Weanling Rats

ABSTRACT: In clonogenic assays, high concentrations of erythropoietin (epo) result in reduced generation of neutrophils from progenitors. Fetal progenitors appear to be more sensitive to this effect than are those of adults. Neutropenia has not been observed, however, as a complication of epo administration to anemic adults. Nevertheless, we remained concerned that diminutions in the neutrophil proliferative or storage pools might occur in epo-treated neonates. Therefore, after establishing that epo induces down-modulation of neutrophil generation in vitro from progenitors of weanling rats, we administered high doses of epo and subsequently performed neutrophil kinetic studies. Six pairs of 8-9-d-old rats were given three daily injections of 2000 IU/kg of either epo or a control. Fortyeight h later, the epo recipients had elevations in reticulocytes, circulating normoblasts, and hematocrits, as well as in splenic and femoral normoblasts and mature erythroid progenitors. However, no changes were observed in concentrations of circulating neutrophils, monocytes, or lymphocytes. In addition, no changes were observed in the splenic or marrow neutrophil storage or proliferative pools, or the pools of granulocyte-macrophage progenitors or multipotent progenitors. Thus, although in vitro high concentrations of epo resulted in diminished generation of neutrophils from progenitors, no reductions were observed in vivo using epo doses 4- to 40-fold higher than those generally administered to humans.