Thorsten W. Orlikowsky

Evaluation of IL-8-Concentrations in Plasma and Lysed EDTA-Blood in Healthy Neonates and Those with Suspected Early Onset Bacterial Infection

Plasma IL-8 is a diagnostic parameter of early-onset bacterial infection (EOBI) in neonates but has a short half-life. The detergent-lysed whole-blood (DLWB) IL-8 consists of both extracellular and cell-bound IL-8. The objective of this study was to investigate kinetics of plasma and DLWB IL-8 in healthy newborns and those with suspected EOBI and to test the hypothesis that determination of DLWB IL-8 results in higher sensitivity for EOBI detection. Sixty-one neonates with clinical and serologic signs of EOBI composed the study group; 188 neonates with risk factors but without EOBI served as control subjects. IL-8 concentrations were determined in plasma and DLWB. In the control group, DLWB IL-8 concentrations were 280-fold higher (9599 pg/mL; SD 4433) up to 24 h post partum than corresponding plasma levels (34.2 pg/mL; SD 18.1). The sensitivity of DLWB versus plasma IL-8 for EOBI was 0.97 versus 0.71 after 6 h and 0.70 versus 0.32 after 24 h. Corresponding values for specificity were 0.95 versus 0.90 after 6 h and 0.92 versus 0.99 after 24 h. After 24 h, the negative predictive value for DLWB versus plasma IL-8 was 0.80 versus 0.66. DLWB IL-8 showed a higher sensitivity for EOBI within 6 h after first clinical suspicion than plasma IL-8. It also remained elevated longer. Our results suggest that DLWB IL-8 results in a higher sensitivity for EOBI.

Differential effect of surfactant and its saturated phosphatidylcholines on human blood macrophages

Blood monocyte-derived macrophages invading the alveolus encounter pulmonary surfactant, a phospholipoprotein complex that changes composition during lung development. We tested the hypothesis that characteristic phosphatidylcholine (PC) components differentially influence macrophage phenotype and function, as determined by phagocytosis of green fluorescent protein-labeled Escherichia coli and αCD3-induced T cell proliferation. Human macrophages were exposed to surfactant (Curosurf®), to two of its characteristic phosphadidylcholine (PC) components (dipalmitoyl-PC and palmitoylmyristoyl-PC), and to a ubiquituous PC (palmitoyloleoyl-PC) as control. Interaction of Curosurf and PC species with macrophages was assessed using Lissamine™-dihexadecanoyl-phosphoethanolamine-labeled liposomes. Curosurf and both saturated surfactant PC species downregulated CD14 expression and upregulated CD206. HLA-DR and CD80 were upregulated by Curosurf and palmitoylmyristoyl-PC, whereas dipalmitoyl-PC showed no effect. The latter upregulated TLR2 and TLR4 expression, whereas Curosurf and palmitoylmyristoyl-PC had no effect. PC species tested were incorporated in comparable amounts by macrophages. Curosurf and PC species inhibited phagocytosis of E. coli. Scavenger receptor CD36, CD68, SR-A, and LOX-1 mRNA expression was upregulated by Curosurf, whereas PC species only upregulated SR-A. Curosurf and palmitoylmyristoyl-PC inhibited αCD3-induced T cell proliferation by 50%, whereas dipalmitoyl-PC and palmitoyloleoyl-PC showed no effect. These data identify individual surfactant PC species as modifiers of macrophage differentiation and suggest differential effects on innate and adaptive immune functions.

Diminished Phagocytosis-Induced Cell Death (PICD) in Neonatal Monocytes upon Infection with Escherichia coli

An imbalance in apoptosis or survival of immune cells plays an essential role in the pathophysiology of sepsis. Phagocytosis-induced cell death (PICD) is a common result of the pathogen-host cell interaction mediated by reactive oxygen species (ROS). Neonatal sepsis is frequently characterized by hyperinflammation. Cord blood monocytes (CBMO) are equivalent to monocytes of adults [peripheral blood monocytes (PBMO)], both in terms of phagocytosis and killing of Escherichia coli. We investigated whether CBMO are less sensitive toward PICD compared with PBMO. Monocytes were infected with green fluorescent protein (GFP)-labeled E. coli. Phagocytic activity, cell-count, Annexin V staining, hypoploid DNA content, CD95 and CD95L expression, and caspase-8 and -9 activities were analyzed by flow cytometry, ROS production by chemiluminescence, and CD95L mRNA expression by reverse-transcriptase polymerase chain reaction. With equal phagocytic activity and ROS production, PBMO cell count was decreased by 82 ± 6% versus 28 ± 8% for CBMO after infection. Annexin V binding was enhanced fivefold on PBMO; 56 ± 15% of PBMO showed a hypodiploid DNA content compared with 9 ± 6% of CBMO. Caspases CD95L and CD95L mRNA were up-regulated in PBMO. Our results indicate that CBMO are less sensitive toward E. coli-mediated PICD than PBMO. Modifying monocyte apoptosis may be a target for future interventions in sepsis.

Expression and regulation of B7 family molecules on macrophages (MΦ) in preterm and term neonatal cord blood and peripheral blood of adults

Macrophage (MΦ) receptors of the B7 family (CD80, CD86) play a crucial role in T cell activation: the lack of costimulation leads to anergy or apoptosis of reactive T cells. MΦ may differentiate into different subsets, the balance of which defines MΦ‐dependent T cell reactions. The aim of this study was to examine neonatal and adult T cell response with respect to the costimulatory MΦ‐potential in order to identify molecular predictors for the neonatal immune defense.

Effect of Dexamethasone on B7 Regulation and T Cell Activation in Neonates and Adults

The safety of dexamethasone for neonates has been questioned, partly because of its multiple unspecific effects on the immune system. Specific effects of dexamethasone on co-stimulatory and immune suppressive functions of neonatal compared with adult macrophages (MΦ) are not known. We evaluated the effect of dexamethasone on the expression and regulation of MΦ B7 family receptors (B7-1, CD80; B7-2, CD86) and on their ability to co-stimulate T cells. Cord blood macrophages (CBMΦ) and MΦ from healthy adults (PBMΦ) were isolated, and cell surface markers were phenotyped by flow cytometry. In tissue culture, cells were exposed to dexamethasone, interferon-γ (IFN-γ), cAMP, or a T cell mitogen (αCD3) and examined for their capacity to activate or destroy T cells. CBMΦ were less able to up-regulate CD80 and CD86 than PBMΦ (p < 0.05). Dexamethasone inhibited the up-regulation of CD80, CD86, and HLA-DR on PBMΦ and even more so on CBMΦ (p < 0.05 versus PBMΦ for CD80 and CD86). In the presence of dexamethasone, stimulation with αCD3 MAb enhanced cytotoxic functions of PMBMΦ and CBΜΦ with an increase in deleted T cells, a reduced fraction of enlarged T cells, and an inhibition of T cell CD28 up-regulation, which again were more pronounced with CBMΦ (p < 0.05 versus PBMΦ). In conclusion, neonatal MΦ are exquisitely sensitive to the inhibitory effects of dexamethasone on B7 expression. Although perhaps producing the desired therapeutic effect, dexamethasone may do so in newborns at the expense of a near complete paralysis of MΦ-dependent T cell function.

Flow Cytometric Methods in the Detection of Neonatal Infection

Bacterial infection remains the main cause of neonatal morbidity and mortality. With clinical signs unspecific and little, its course is fulminant, and only early antibiotic therapy protects from death or major adverse sequelae. Methods: Conventional diagnostic tests have shortcomings and are only partly suitable to identify infected vs. non-infected newborns. There is a fast increasing demand for standardized flow cytometric techniques in the detection of neonatal infection which require minimal amounts of blood. Target cells are monocytes/ macrophages, granulocytes and NK cells. Results: The choice of receptors and interpretation of results need to be done carefully: A variety of receptors are expressed differently, basally or upon cytokine-mediated regulation, in full- or preterm neonates than in adults, and their expression is influenced by perinatal confounders. Examples are HLA-DR, CD80, CD86, CD16, CD32 receptors on monocytes/macrophages. Flow cytometric measurement of CD11b and CD64 expression on phagocytes combined with cytokine (IL-6 or IL-8) or C-reactive protein measurement in plasma or whole blood have turned out to be most sensitive and specific markers for detecting early- and late-onset bacterial infection. Conclusion: To date, no flow cytometric marker or set of markers is sensitive and specific enough to allow neonatologists to withhold antibiotic treatment of a sick neonate who is suspected to be infected.

Flow Cytometry in the Detection of Neonatal Sepsis

Neonatal sepsis remains a burden problem by showing minimal initial symptoms of subtle character, nonspecific manifestation, and diagnostic pitfalls. The clinical course can be fulminant and fatal if treatment is not commenced promptly. It is therefore crucial to establish early diagnosis and initiate adequate therapy. Besides clinical symptoms, the most reliable laboratory markers in establishing diagnosis is currently the combined measurement of CRP and a cytokine (IL-6 and IL-8). Due to their different kinetics, a diagnostic gap might occur and thus withholding antimicrobial therapy in clinical suspicion of infection is not acceptable. We therefore need parameters which unerringly differentiate between infants in need for antimicrobial therapy and those who are not. Flow cytometry promises to be a useful tool in this field, allowing the determination of different cellular, dissolved, and functional pathophysiological components of sepsis. Despite technical and methodical advances in flow cytometry, its use in clinical routine is still limited. Advantages and disadvantages of promising new parameters in diagnosis of sepsis performed by flow cytometry, particularly CD64, HLA-DR, and apoptosis, are reviewed here. The necessity of tests to be used as an “ideal” parameter is presented.

Diminished Response to Interleukin-10 and Reduced Antibody-Dependent Cellular Cytotoxicity of Cord Blood Monocyte-Derived Macrophages

Monocyte-derived macrophage (MΦ) subsets are generated by antagonistic induction pathways. A helper MΦ-type (Mh-MΦ) is induced by interferon gamma (IFN-γ), whereas a cytotoxic MΦ-type (Mc-MΦ), induced by interleukin-10 (IL-10), is a potent mediator of antibody-dependent cellular cytotoxicity (ADCC). Compared with MΦ from healthy adults [peripheral blood monocyte-derived macrophages (PBMΦ)], cord blood MΦ (CBMΦ) were found less capable of generating Mh-MΦ. Here we tested the hypothesis that their generation of Mc-MΦ via IL-10 is also impaired. MΦ surface markers were phenotyped. IL-10 protein and mRNA production were detected after stimulation [αCD3 monoclonal antibody (mAb)]. CBMΦ or PBMΦ were co-cultured with MΦ-depleted mononuclear cells of adults and CD4-targeting antibodies as models for ADCC were added. In cord blood, we found diminished αCD3-induced IL-10 protein and mRNA production (p < 0.05 versus adults). Basal CD16 and HLA-DR expressions on CBMΦ of preterm and full-term neonates were lower (p < 0.05 versus PBMΦ). IL-10 had reduced effects on CD16 up- and HLA-DR down-modulation on CBMΦ (p < 0.05 versus PBMΦ). CD4-directed receptor modulation and deletion were reduced in the presence of CBMΦ (p < 0.05 versus PBMΦ). IL-10 failed to enhance their ADCC capacity, which was in contrast to PBMΦ (p < 0.05). These data suggest that CBMΦ have an impaired cytotoxic capacity via lower sensitivity toward IL-10.

The CD95/CD95L pathway is involved in phagocytosis-induced cell death of monocytes and may account for sustained inflammation in neonates

Background:The propensity for sustained inflammation after bacterial infection in neonates, resulting in inflammatory sequelae such as bronchopulmonary dysplasia and periventricular leucomalacia, is well known, but its molecular mechanisms remain elusive. Termination of inflammatory reactions physiologically occurs early after removal of bacteria by phagocytosis-induced cell death (PICD) of immune effector cells such as monocytes. PICD from cord blood monocytes (CBMOs) was shown to be reduced as compared with that of peripheral blood monocytes (PBMOs) from adult donors in vitro.Methods:PBMOs, CBMOs, and Fas (CD95)-deficient (lpr) mouse monocytes were analyzed in an in vitro infection model using green fluorescence protein–labeled Escherichia coli (E. coli-GFP). Phagocytosis and apoptosis were quantified by flow cytometry and CD95L secretion was quantified by enzyme-linked immunosorbent assay.Results:We demonstrate the involvement of the CD95/CD95 ligand pathway (CD95/CD95L) in PICD and provide evidence that diminished CD95L secretion by CBMOs may result in prolonged activation of neonatal immune effector cells.Conclusion:These in vitro results offer for the first time a molecular mechanism accounting for sustained inflammation seen in neonates.

Differential Modulation of Cord Blood and Peripheral Blood Monocytes by Intravenous Immunoglobulin

Immunoglobulins (IVIG) have been shown to be useful in adults suffering from sepsis. In contrast, prophylactic and curative IVIG trials failed to show beneficial effects in neonates. We tested the hypothesis that IVIG, have different effects on monocytes from cord blood (CBMO) and peripheral blood monocytes from adults (PBMO) with respect to survival, phenotype, and function.