Petra Temming

Enhanced Interleukin-6 and Interleukin-8 Synthesis in Term and Preterm Infants

There is growing evidence that sepsis-related complications in neonates are crucially mediated by the action of proinflammatory cytokines. It has previously been demonstrated that elevated IL-6 and IL-8 levels can predict brain damage and chronic lung disease in preterm infants. However, it is the current view that neonates have a reduced capability to produce proinflammatory cytokines. To clarify this issue, we analyzed the inflammatory response in term and preterm infants directly at the single cell level by flow cytometry. Endotoxin challenge was performed under defined conditions on monocytes obtained from 50 healthy adults and 119 neonates, which consist of 45 term infants, 63 preterm infants (26.1–36.7 wk of gestational age), and 11 preterm infants with proven infection (24.6–29.9 wk). Our results challenge the existing view of an immature inflammatory response by demonstrating that term infants and preterm infants display a higher percentage of IL-6– and IL-8–positive cells than adults. After preincubation with dexamethasone the number of cytokine-positive cells decreased in all groups, but the number of IL-8–positive cells remained higher in term and preterm infants >32 wk compared with adults. These observations demonstrate not only a well-developed but also an enhanced inflammatory response in term and preterm infants. Under consideration of several detrimental effects of IL-6 and IL-8, our data may have major implications on the pathophysiology of inflammatory-triggered neonatal diseases.

Cytokine responses correlate differentially with age in infancy and early childhood

The functional differentiation of immune cells at early age plays a central role in immune physiology, e.g. for the sufficient eradication of pathogens. However, imbalances in effector cell responses may also have an impact in the pathophysiology of childhood diseases such as atopy and autoimmune disorders. As information on immune cell responses in infancy and early childhood is scarce, we conducted an observational, cross‐sectional study in healthy newborns (n = 18), infants and young children (n = 54) aged 1–96 months and adult controls (n = 19) to assess cytokine mRNA and protein expression upon phorbol 12‐myristate 13‐actate/ionomycin stimulation and LPS‐induced IL‐12 expression in monocytes. The intracellular expression of interferon (IFN)‐γ, tumour necrosis factor (TNF)‐α (R = 0·748, P < 0·0001; R = 0·784, P < 0·0001, respectively) and interleukin (IL)‐2 protein expression (R = 0·384, P = 0·008) was demonstrated to increase progressively with age. While a correlation between IL‐4 protein expression and age was noted (R = 0·342, P = 0·007), the levels of IL‐5 and IL‐10 protein expression tended to be regulated on an individual basis during infancy and early childhood. An age correlation was also observed for intracellular IL‐12 expression (R = 0·331, P = 0·009) in monocytes. These findings are valuable for further assessment of normal variations and maturation processes in immune cell responses and for the clinical–therapeutic monitoring of immunological status in various childhood diseases.

Immature anti-inflammatory response in neonates

The inflammatory response plays a major role in the induction of several neonatal diseases. We hypothesize that an imbalance between the pro‐ and anti‐inflammatory response is crucial for the previously shown enhanced production of proinflammatory cytokines in term and preterm infants during infection. To test this hypothesis, we compared the capacity to produce the main anti‐inflammatory cytokines IL‐10 and TGF‐β in term infants, preterm infants and adults at different levels of synthesis by quantitative real time reverse‐transcribed PCR, flow cytometry, as well as enzyme‐linked immunoassay. Term and preterm infants showed a profoundly diminished IL‐10 mRNA‐expression and IL‐10 production after stimulation. In addition, the amount of TGF‐β‐positive lymphocytes was significantly less in neonates than adults. Furthermore, there was a considerably lower inhibition of production of IL‐1α, IL‐6, IL‐8 and TNF‐α by the use of recombinant IL‐10 in term and preterm infants compared with adults. These results demonstrate not only a diminished anti‐inflammatory capacity but also a reduced response to anti‐inflammatory stimuli in term and preterm infants. From these data we conclude that neonates display an immature compensatory anti‐inflammatory response syndrome (CARS) which may predispose preterm infants to harmful effects of proinflammatory cytokines resulting in severe organ sequelae during infection.

Differential Maturation of the Innate Immune Response in Human Fetuses

Newborns and especially preterm infants show a unique susceptibility to severe bacterial infections that cause significant morbidity and mortality. As very few data are available on innate immune functions in human fetuses, we conducted a comprehensive study to investigate the expression of several adhesion molecules essentially involved in migration (CD11a, CD11b, CD11c, CD18, and CD62L). Furthermore, phagocytic activity, generation of respiratory burst products, and production of several proinflammatory cytokines were assessed. Various functions of the fetal innate immune system were demonstrated to be essentially different from those observed in term neonates or adults. Expression of several surface markers was significantly diminished on fetal granulocytes. Furthermore, a significantly reduced phagocytic activity of fetal granulocytes and monocytes was found, contrasted by an enhanced generation of reactive oxygen products. In addition, we demonstrate that significant numbers of fetal monocytes are capable of the production of proinflammatory cytokines in response to stimulation. However, the pattern of cytokine production is different from the more mature individuals: the number of IL-6 – and tumor necrosis factor-α–positive monocytes were significantly diminished, whereas more IL-8 –producing monocytes were found compared with adults. The results of our study add significantly to our understanding of the maturation and impairment of the innate immune response.

Erythropoietin inhibits cytokine production of neonatal and adult leukocytes

Background: Erythropoietin (Epo) was originally defined as a hematopoietic growth factor, but also has potent tissue‐protective properties. The cytokine‐modulating actions of Epo have received scant attention. We hypothesized that Epo significantly influences the in vitro cytokine production in both neonates and adults.

The effect of hyperglycemia on neonatal immune responses in-vitro

Introduction. Acute hyperglycemia is considered as a pro-inflammatory state and is related to an adverse outcome in critically ill adults. Neonates are susceptible to infections and systemic inflammatory response syndrome induced by pro-inflammatory cytokines. This study focuses on the interaction between neonatal glucose homeostasis and the pro-inflammatory cytokine production in term and preterm infants in-vitro. Methods. We analyzed the pro-inflammatory cytokine production in whole cord blood of term infants (n = 10), preterm infants > 32 weeks (n=16) and preterm infants ≤32 weeks of gestational age (n = 13) and in adult controls (n=14) using an in-vitro sepsis-model. Whole blood was pre-incubated with different concentrations of glucose (0–1000 mg/dl) and insulin (0–62.5 IE/l) and stimulated with lipopolysaccharide. The intracytoplasmatic TNF-α, IL-6, and IL-8 response was measured by flow cytometry. Results. In-vitro hyperglycemia induced a dose-dependent increase of IL-8 in all age groups while TNF-α was demonstrated to be stimulated by glucose in cord blood samples of preterm infants ≤32 weeks of gestational age and term infants. In contrast, insulin showed no significant effects on pro-inflammatory cytokine production in-vitro. Conclusion. Acute hyperglycemia may induce pro-inflammatory cytokine responses in neonatal whole blood in-vitro. These data provide a basis for further in-vitro signal transduction studies and in-vivo investigations about the significance of neonatal glucose homeostasis and its impact on long-term outcome of this susceptible patient cohort.

Reduced IL-10 production and -receptor expression in neonatal T lymphocytes.

Aim: To further evaluate the underlying mechanism of a formerly demonstrated immature anti‐inflammatory response in neonates ( 1 ).

Immunomodulatory Effect of Vitamin C on Intracytoplasmic Cytokine Production in Neonatal Cord Blood Cells

Background: Vitamin C (ascorbic acid) is an essential water-soluble antioxidant in cells and plasma. Besides metabolic functions, vitamin C is also known to contribute to immune homeostasis. Recently, it has been demonstrated that vitamin C has an inhibitory effect on the expression of pro-inflammatory cytokines such as interleukin (IL)-6 and tumor necrosis factor alpha (TNF-α) in adult whole blood cells in vitro. It has been postulated that vitamin C might be an interesting compound for modulation of an over-exuberant immune response, e.g., in patient cohorts susceptible for the development of systemic inflammatory response syndrome such as neonates. It was the aim of this study to investigate the modulatory effects of vitamin C on the production of inflammatory mediators in neonatal cord blood cells. Methods: The intracytoplasmic production of pro-inflammatory cytokines in neonatal cord blood cells stimulated with lipopolysaccharide or phorbol 12-myristate 13-acetate/ionomycin was assessed by flow-cytometry. Results: In contrast to our previous observations from adult whole blood cells, 20 mM vitamin C mildly stimulated the percentage of neonatal monocytes producing IL-6 after lipopolysaccharide stimulation (e.g., 11.3% increase compared to control, p = 0.005). In the presence of 20 mM vitamin C, even a stronger stimulatory effect was noted for the percentage of IL-8 (e.g., 46.7% increase, p < 0.001) and TNF-α producing neonatal monocytes (e.g., 69.2% increase, p = 0.004; n = 20). In accordance with adult data, the percentage of neonatal lymphocytes producing IL-2 after phorbol 12-myristate 13-acetate/ionomycin stimulation was dose-dependently reduced (e.g., 41.3% inhibition, p = 0.001, 20 mM vitamin C), while the percentage of TNF-α producing lymphocytes was mildly stimulated (e.g., 20.8% increase, p = 0.003, 20 mM vitamin C). Conclusions: Interestingly, vitamin C was demonstrated to enhance pro-inflammatory responses in CD14+ cord blood cells while only intracellular IL-2 production in CD3+ cells was diminished. These data suggest that vitamin C differentially influences intracytoplasmic cytokine production in adults and neonates, and further studies are needed to elucidate the underlying mechanisms of this selective immunomodulation.

Influence of Specimen Age and Use of Different Negative Controls in Determination of Intracytoplasmic Levels of Cytokines after Whole-Blood Culture Assay

ABSTRACT Intracytoplasmic detection of cytokines by flow cytometry has become a powerful tool in the characterization of cytokine-producing cells. However, it is not known to what extent specimen age and the use of various negative controls may influence the amount of cytokine-positive cells. We therefore compared different times of storage and the use of several negative controls in the determination of intracytoplasmic levels of cytokines. There was a substantial decline of interleukin-2- and gamma interferon-positive lymphocytes after 20 h and especially after 48 h of storage. The precision of intracytoplasmic interleukin-6 determination decreases after long-term storage compared to 2 h of storage, whereas the amount of interleukin-8-positive monocytes remained rather stable. Therefore, we recommend performing the analysis as fast as possible after the blood sample is drawn. Under consideration of isotype-matched antibodies and nonstimulated cells as negative controls instead of the purified antibody-blocking control, strikingly higher amounts of interleukin-2-, gamma interferon, interleukin-6-, and interleukin-8-positive cells were found. For a meaningful interpretation of data these differences have to be kept in mind. Further studies should evaluate the exact specificity of these controls.

Attenuation of monocyte proinflammatory cytokine responses to Neisseria meningitidis in children by erythropoietin

Meningococcal disease is a leading infectious cause of death in children in industrialized countries. The induction of high levels of proinflammatory cytokines has been implicated in the pathogenesis of Neisseria meningitidis‐related multi‐organ failure. Here, we demonstrate that N. meningitidis serotypes A and B induce significantly higher levels of tumour necrosis factor (TNF)‐α positive cells in vitro in infants and young children compared with adults (serotype A/B; infants: 64·9%/63·9%; children: 77·8%/64·3% versus adults: 27·7%/32%; P < 0·005). Serotype A induces also higher levels of interleukin (IL)‐6 positive cells in neonates and infants compared with adults (serotype A; newborn 55·4%; infants 58·8% versus adults 49%; P < 0·05). Treatment with human recombinant erythropoietin in vitro resulted in significant attenuation of the N. meningitidis‐induced proinflammatory response in all age groups (reduction rate of erythropoietin for IL‐6 after stimulation with serotype B: newborn 28%, infants 15%, children 23% and adults 28% and for TNF‐α after stimulation with serotype B: newborn 27%, infants 22%, children 20% and adults 28%; P < 0·05). We conclude that (i) Neisseria meningitidis induces a higher TNF‐α response in infants and children compared with adults and (ii) erythropoietin was able to attenuate IL‐6 and TNF‐α production in all investigated age groups. These data may explain the high incidence of meningococcal infection in infants and makes erythropoietin a potentially attractive candidate for interventional strategies in an otherwise devastating course of the disease.