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Interleukin-10 (IL-10), an anti-inflammatory cytokine, may have therapeutic potential in the fetal inflammatory response syndrome and its sequelae such as bronchopulmonary dysplasia (BPD). Our aim was to compare the effects of IL-10 versus dexamethasone (DEX) on important PMN functions of the newborn. PMNs were isolated into culture medium from cord blood after elective cesarean section deliveries. IL-10 and DEX were compared on an equimolar basis corresponding to previously measured plasma levels of DEX from infants treated for BPD. The endotoxin (LPS)-stimulated release of the pro-inflammatory cytokines, tumor necrosis factor (TNFalpha) and IL-1 beta, were markedly inhibited equally by IL-10 and DEX; the anti-inflammatory cytokine IL-4 was not released and IL-1 receptor antagonist (IL-1ra) was released less with DEX compared with IL-10. PMNs exposed to LPS, N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP), or S. aureus did not show a significant difference between control, DEX and IL-10 for apoptosis, respiratory burst, phagocytosis or killing respectively. Chemotaxis to fMLP or IL-8 was unaffected by DEX or IL-10. The principal effects of both IL-10 and DEX, on the PMN functions studied, are related to the control of pro- and anti-inflammatory cytokine release.

Increased activation of the transcription factor NFkappaB in the neutrophils has been associated with the pathogenesis of sepsis, acute lung injury (ALI), bronchopulmonary dysplasia (BPD), and other neutrophil-mediated inflammatory disorders. Despite recent progress in analyzing early NFkappaB activation in human neutrophils, activation of NFkappaB in persistently stimulated neutrophils has not been previously studied. Because it is the persistent NFkappaB activation that is thought to be involved in the host response to sepsis and the pathogenesis of ALI and BPD, we hypothesized that continuously stimulated human neutrophils may exhibit a late phase of NFkappaB activity. The goal of this study was to analyze the NFkappaB activation and expression of IkappaB and NFkappaB proteins during neutrophil stimulation with inflammatory signals for prolonged times. We demonstrate that neutrophil stimulation with lipopolysaccharide (LPS) and tumor necrosis factor-alpha (TNFalpha) induces, in addition to the early activation at 30-60 min, a previously unrecognized late phase of NFkappaB activation. In LPS-stimulated neutrophils, this NFkappaB activity typically had a biphasic character, whereas TNFalpha-stimulated neutrophils exhibited a continuous NFkappaB activity peaking around 9 h after stimulation. In contrast to the early NFkappaB activation that inversely correlates to the nuclear levels of IkappaBalpha, however, in continuously stimulated neutrophils, NFkappaB is persistently activated despite considerable levels of IkappaBalpha present in the nucleus. Our data suggest that NFkappaB is persistently activated in human neutrophils during neutrophil-mediated inflammatory disorders, and this persistent NFkappaB activity may represent one of the underlying mechanisms for the continuous production of proinflammatory mediators.

Background: Neutrophils followed by monocytic cells are recruited into the lung during the early development of bronchopulmonary dysplasia (BPD)., Objectives: We determined: (1) the capacity of polymorphonuclear leukocytes (PMNs) and peripheral blood monocytic cells (PBMCs) of the newborn to produce and release the anti-inflammatory cytokine, interleukin (IL)-10, after stimulation by lipopolysaccharide (LPS) or tumor necrosis factor (TNF), and (2) the levels of exogenous IL-10 and/or dexamethasone (DEX) needed to inhibit the release of the pro-inflammatory chemokine IL-8 from stimulated cells., Methods: PMNs and PBMCs were isolated from cord blood of healthy term infants. RT-PCR and ELISA were used to detect mRNA and cytokine levels from culture media, respectively., Results: We found that PMNs did not produce IL-10 mRNA or release IL-10 but did produce IL-8 mRNA by 1 h. PBMCs did produce IL-10 mRNA after 4 h (with IL-8 mRNA expression by 1 h). LPS-stimulated PBMCs released IL-10 to a maximum of 1,038 pg/ml/5 million cells (56 femtomolar). Equimolar doses of exogenous IL-10 or DEX produced up to 83% inhibition of IL-8 from PMNs. Exogenous IL-10 was more potent than DEX, on an equimolar basis, with regard to IL-8 release from PBMCs (90 vs. 33% respectively at a 10 nanomolar level). No inhibition of IL-8 release by IL-10 or DEX was observed at 100 femtomolar level. IL-10 and DEX did not have an additive inhibitory effect on IL-8 release., Conclusions: We conclude that for the newborn: (1) PBMCs produce IL-10 far below the level needed to inhibit a submaximal release of IL-8 from PMNs or PBMCs, and (2) exogenous IL-10 was equipotent or more potent than therapeutic levels of DEX on inhibition of IL-8 from these cells. Further studies are needed to determine if exogenous IL-10 may be useful in the treatment of BPD or other inflammatory disorders of the newborn.

Tumor necrosis factor-alpha (TNFalpha) is a potent pro-inflammatory cytokine that plays a major role in the pathogenesis of acute and chronic inflammatory disorders such as septic shock and arthritis, respectively. Leukocytes stimulated with inflammatory signals such as lipopolysaccharide (LPS) are the predominant producers of TNFalpha, and thus control of TNFalpha release from stimulated leukocytes represents a potential therapeutic target. Here, we report that leptomycin B (LMB), a specific inhibitor of CRM1-dependent nuclear protein export, inhibits TNFalpha release from LPS-stimulated human peripheral blood neutrophils and mononuclear cells. In addition, immunofluorescence confocal microscopy and immunoblotting analysis indicate that TNFalpha is localized in the nucleus of human neutrophils and mononuclear cells. This study demonstrates that the cellular release of TNFalpha from stimulated leukocytes is mediated by the CRM1-dependent nuclear export mechanism. Inhibition of CRM1-dependent cellular release of TNFalpha could thus provide a novel therapeutic approach for disorders involving excessive TNFalpha release.

Objective: Despite advances in the management of sepsis and acute respiratory distress syndrome, the mortality rate remains high. Delayed apoptosis of neutrophils is associated with multiple organ failure under those conditions. Thus, development of nontoxic neutrophil apoptosis regulating molecules may provide a novel therapeutic strategy. Curcumin is a promising dietary supplement for cancer prevention. However, the effect of curcumin on human neutrophil apoptosis remains unknown. We therefore hypothesized that curcumin would produce a proapoptotic effect on neutrophils., Design: Prospective, controlled, and randomized in vitro study., Setting: Research institute laboratory., Subjects: Human peripheral neutrophils obtained from normal subjects., Interventions: None., Measurements and Main Results: In the presence or absence of curcumin, both spontaneous neutrophil apoptosis and apoptosis of neutrophils following transmigration across a human lung endothelium-epithelium bilayer were studied by morphology and terminal dUTP nucleotide end labeling analyses, respectively. Myeloperoxidase activity and migration assays were performed to determine the impact of curcumin on neutrophil function. To elucidate the potential mechanism, the p38 mitogen-activated protein kinase pathway and caspase-3 activity were examined by Western blotting and enzymatic analyses. The data demonstrate that curcumin increased constitutive neutrophil apoptosis and abrogated the transbilayer migration-induced delay in neutrophil apoptosis. Neutrophil activation was reduced by curcumin treatment as evidenced by a decrease in migration and myeloperoxidase release. A marked increase in p38 phosphorylation and caspase-3 activity was observed following curcumin exposure. In addition, inhibition of p38 mitogen-activated protein kinase with SB203580 suppressed apoptosis and caspase-3 activation induced by curcumin. Thus, activation of p38 mitogen-activated protein kinase or an increase in caspase-3 activity appears to contribute to the proapoptotic effect of human neutrophil apoptosis by curcumin., Conclusion: The characteristics of curcumin, including its proapoptotic effect and antidegranulation effect, make it a potential candidate for the therapy of neutrophil-induced lung injury and sepsis.

An increase in polymorphonuclear leukocytes (PMNs) and proinflammatory chemokines, such as IL-8 and macrophage inflammatory protein-1 alpha (MIP), are found in the airways during early stages of bronchopulmonary dysplasia. We determined whether IL-10 produces a dose-related inhibition of proinflammatory chemokine release from stimulated neutrophils of the newborn and whether the mechanism involves the pivotal transcription factor, nuclear factor-kappa B. PMNs isolated from the cord blood of healthy newborns were stimulated submaximally with either lipopolysaccharide (n = 5) or tumor necrosis factor (n = 4), with and without IL-10 (0.01-1000 ng/mL). IL-8 and MIP release were measured in cell culture supernatants at 18 h. The presence or absence of nuclear factor-kappa B activity and inhibitor-kappa B alpha degradation was measured at 30 min and 3 h after PMN stimulation began. During lipopolysaccharide stimulation, IL-10 significantly reduced IL-8 levels from 50 +/- 16 ng/mL to 7 +/- 3 ng/mL, and MIP levels from 14 +/- 5 to 0.7 +/- 0.1 ng/mL (mean +/- SEM, p < 0.01). IL-10 produced an insignificant reduction in IL-8 and MIP levels after stimulation of PMNs with tumor necrosis factor. IL-10 did not inhibit nuclear factor-kappa B activation and inhibitor-kappa B alpha degradation in PMNs stimulated with tumor necrosis factor or lipopolysaccharide for 30 min. After PMN stimulation for 3 h, inhibitor-kappa B alpha cytoplasmic levels were restored; however, they were unaffected by IL-10. We conclude that IL-10 is a potent inhibitor of lipopolysaccharide-stimulated release of IL-8 and MIP from neutrophils of the newborn via a mechanism not involving nuclear factor-kappa B activity. Further work is needed to determine whether exogenous IL-10 may be useful for suppressing inflammation in bronchopulmonary dysplasia.

Human neutrophils differ from other cells by containing high amount of IkappaBalpha in the nucleus, and this increased nuclear IkappaBalpha accumulation is associated with the inhibition of NFkappaB activity and increased apoptosis. However, the mechanisms regulating NFkappaB activation and IkappaBalpha degradation in human neutrophils are little understood. The objective of this study was to provide a further insight into the mechanisms regulating NFkappaB activity and IkappaBalpha degradation in human neutrophils. We show that okadaic acid (OA), an inhibitor of protein phosphatases PP1 and PP2A, induces sustained activation of NFkappaB and degradation of the nuclear IkappaBalpha, and increases interleukin-8 expression in the neutrophils. Furthermore, inhibitors of protein kinase C-delta (PKCdelta) and IkappaB kinase (IKK) inhibit the OA-induced activation of NFkappaB. Collectively, our results indicate that in human neutrophils, the sustained activation of NFkappaB is regulated by a continuous phosphorylation and degradation of the nuclear IkappaBalpha.

Neutrophils are among the first circulating leukocytes involved in acute inflammatory processes. Transcription factor NF-kappaB plays a key role in the inflammatory response, regulating the expression of proinflammatory and anti-apoptotic genes. Recently we have shown that human neutrophils contain a significant amount of NF-kappaB inhibitor, IkappaBalpha, in the nucleus of unstimulated cells. The present objective was to examine the mechanisms controlling the nuclear content of IkappaBalpha in human neutrophils and to determine whether increased accumulation of IkappaBalpha in the nucleus is associated with increased neutrophil apoptosis. We show for the first time that neutrophil stimulation with pro-inflammatory signals results in degradation of IkappaBalpha that occurs in both cytoplasm and nucleus. Prolonged (2-h) stimulation with TNF and LPS induces resynthesis of IkappaBalpha that is again translocated to the nucleus in human neutrophils, but not in monocytic cells. Leptomycin B, a specific inhibitor of nuclear export, increases nuclear accumulation of IkappaBalpha in stimulated neutrophils by blocking the IkappaBalpha nuclear export, and this is associated with inhibition of NF-kappaB activity, induction of caspase-3 activation, and apoptosis. Based on our data we present a new model of NF-kappaB regulation in human neutrophils by nuclear IkappaBalpha. Our results demonstrate that the NF-kappaB activity in human neutrophils is regulated by mechanisms clearly different from those in monocytes and other human cells and suggest that the increased nuclear content of IkappaBalpha in human neutrophils might represent one of the underlying mechanisms for the increased apoptosis in these cells.

The objective of this study was to determine the doses of dexamethasone (DEX), betamethasone (BET), and hydrocortisone (HC) that effectively inhibit the release of two potent proinflammatory chemokines, interleukin 8 (IL-8) and macrophage inflammatory protein alpha (MIP), from polymorphonuclear neutrophils (PMNs) of the newborn. Human PMNs were isolated from cord blood (n = 18). Chemokines were measured from PMN cell culture supernatants after 18 h of stimulation using tumor necrosis factor (1 ng/ml), with and without pretreatment by DEX (10(-10) to 10(-6) M) versus HC or BET (10(-10) to 10(-5) M). Maximal inhibitions of IL-8 release by BET, DEX, and HC were 97, 91, and 91%, respectively. For MIP, the maximal inhibitions by BET, DEX, and HC were 88, 69, and 70%, respectively. The 50% inhibitory concentrations by DEX, BET, and HC for IL-8 release were 3.4 +/- (SE) 1.6 x 10(-9), 1.8 +/- 7.4 x 10(-8), and 1.8 +/- 0.5 x 10(-7) M, respectively. The 50% inhibitory concentrations by DEX, BET, and HC for MIP release were 1.0 +/- (SE) 0.5 x 10(-8), 3.8 +/- 3.1 x 10(-8), and 4.8 +/- 1.6 x 10(-7) M, respectively. In vitro, these corticosteroids effectively inhibited the release of two structurally different chemokines that are found in the airway lavage fluids of infants developing bronchopulmonary dysplasia. When compared to plasma DEX levels previously reported during the treatment of bronchopulmonary dysplasia, our results suggest that the doses of DEX, and potentially BET, needed to treat chronic lung disease may be more than five to ten times lower than those of current DEX regimens., (Copyright 2002 S. Karger AG, Basel)

Introduction: Increasing evidence now supports the association between the fetal inflammatory response syndrome (FIRS) with the pathogenesis of preterm labor, intraventricular hemorrhage and bronchopulmonary dysplasia. Polymorphonuclear leukocyte (PMNs) and mononuclear cell (MONOs) infiltration of the placenta is associated with these disorders. The aim of this study was to reveal cell-specific differences in gene expression and cytokine release in response to endotoxin that would elucidate inflammatory control mechanisms in the newly born. Methods: PMNs and MONOs were separately isolated from the same cord blood sample. A genome-wide microarray screened for gene expression and related pathways at 4 h of LPS stimulation (n = 5). RT-qPCR and ELISA were performed for selected cytokines at 4 h and 18 h of LPS stimulation. Results: Compared to PMNs, MONOs had a greater diversity and more robust gene expression that included pro- inflammatory (PI) cytokines, chemokines and growth factors at 4 h. Only MONOs had genes changing expression (all up regulated including interleukin-10) that were clustered in the JAK/STAT pathway. Pre-incubation with IL-10 antibody, for LPS-stimulated MONOs, led to up regulated PI and IL-10 gene expression and release of PI cytokines after 4 h. Discussion: The present study suggests a dominant role of MONO gene expression in control of the fetal inflammatory response syndrome at 4 hrs of LPS stimulation. LPS-stimulated MONOs but not PMNs of the newborn have the ability to inhibit PI cytokine gene expression by latent IL-10 release. [ABSTRACT FROM AUTHOR]