Gene expression profile of endotoxin-stimulated leukocytes of the term new born: control of cytokine gene expression by interleukin-10.

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.

Okadaic acid induces sustained activation of NFkappaB and degradation of the nuclear IkappaBalpha in human neutrophils.

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.

Interleukin-10 inhibits proinflammatory chemokine release by neutrophils of the newborn without suppression of nuclear factor-kappa B.

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.

Patterns of colonization with Ureaplasma urealyticum during neonatal intensive care unit hospitalizations of very low birth weight infants and the development of chronic lung disease.

BACKGROUND: Ureaplasma urealyticum and its association with chronic lung disease (CLD) of prematurity has remained a controversial topic. To readdress this question, we performed a longitudinal study using culture and polymerase chain reaction to detect U urealyticum in the respiratory tract of very low birthweight infants throughout their neonatal intensive care unit hospitalizations. METHODS: We screened 125 infants weighing <1500 g and/or <32 weeks' gestational age over a 12-month period, collecting endotracheal, nasopharyngeal, and throat specimens on days of age 1, 3, 7, and weekly thereafter. CLD was defined as dependency on supplemental oxygen at 28 days and at 36 weeks' postconceptional age. RESULTS: Forty infants (32%) had 1 or more positive specimens by culture or polymerase chain reaction. We identified 3 patterns of U urealyticum colonization: persistently positive (n = 18), early transient (n = 14), and late acquisition (n = 8). We compared the rates of CLD in each of the 3 colonized groups with the rate of CLD in the noncolonized group. We found a significantly higher rate of CLD at 28 days of age (odds ratio: 8.7; 95% confidence interval: 3.3, 23) and at 36 weeks' postconception (odds ratio: 38.5, 95% confidence interval: 4.0, 374) only for infants with persistently positive colonization. CONCLUSIONS: This study demonstrates that the risk of developing CLD varies with the pattern of U urealyticum colonization. Only the persistently positive colonization pattern, which accounted for 45% of the U urealyticum-positive infants, was associated with a significantly increased risk of development of CLD.

NF-kappa B regulation in human neutrophils by nuclear I kappa B alpha: correlation to apoptosis.

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.