Sigrid Wittmann

Cytokine upregulation of surface antigens correlates to the priming of the neutrophil oxidative burst response

Neutrophil activation is strongly related to organ dysfunction that occurs during systemic inflammatory responses. The aim of our study was to analyze the oxidative burst response in correlation to the up‐ and downregulation of N‐formyl‐L‐methionyl‐L‐leucyl‐phenylalanine (fMLP) receptors and the surface antigens CD11b, CD62L, and CD66b as potential surrogate markers of the degree of neutrophil priming for an increased oxidative burst response induced by proinflammatory cytokines.

Mild hyperthermia down-regulates receptor-dependent neutrophil function

Mild hypothermia impairs resistance to infection and, reportedly, impairs phagocytosis and oxidative killing of unopsonized bacteria. We evaluated various functions at 33°–41°C in neutrophils taken from volunteers. Adhesion on endothelial cells was determined using light microscopy. Adhesion molecule expression and receptors, phagocytosis, and release of reactive oxidants were assessed using flow cytometric assays. Adhesion protein CD11b expression on resting neutrophils was temperature-independent. However, up-regulation of CD11b with tumor necrosis factor (TNF)-&agr; was increased by hypothermia and decreased with hyperthermia. Neutrophil adhesion to either resting or activated endothelial cells was not temperature-dependent. Bacterial uptake was inversely related to temperature, more so with Escherichia coli than Staphylococcus aureus. Temperature dependence of phagocytosis occurred only with opsonized bacteria. Hypothermia slightly increased N-formyl-l-methionyl-l-leucyl-phenylalanine receptors on neutrophils: hyperthermia decreased expression, especially with TNF-&agr;. N-formyl-l-methionyl-l-leucyl-phenylalanine-induced H2O2 production was inversely related to temperature, especially in the presence of TNF-&agr;. Conversely, phorbol-13-myristate-12-acetate, an activator of protein kinase C, induced an extreme and homogenous release of reactive oxidants that increased with temperature. In contrast to nonreceptor-dependent phagocytosis and oxidative killing, several crucial receptor-dependent neutrophil activities show temperature-dependent regulation, with hypothermia increasing function. The temperature dependence of neutrophil function is thus more complicated than previously appreciated.

The anticatabolic effect of neuraxial blockade after hip surgery

Although the protein-sparing effect of neuraxial blockade after abdominal surgery is well established, its metabolic effect after operations on the lower extremities remains unclear. In this study, we tested the hypothesis that combined spinal and epidural blockade (CSE) inhibits amino acid oxidation after hip surgery. Sixteen patients undergoing hip replacement surgery received either general anesthesia followed by IV patient-controlled analgesia with piritramide (control; n = 8) or CSE using bupivacaine 0.5% for spinal anesthesia and ropivacaine 0.2% with 0.5 &mgr;g/mL of sufentanil for postoperative epidural analgesia (CSE; n = 8). Glucose and protein kinetics were assessed by stable isotope tracer technique ([6,6-2H2]glucose, L-[1-13C]leucine) on the day before and one day after surgery. Plasma concentrations of glucose, lactate, free fatty acids, cortisol, glucagon, and insulin were also determined. CSE prevented the increase in plasma glucose concentration during and immediately after the operation (60 min after skin incision: CSE 4.9 ± 0.7 versus control 6.2 ± 0.7 mmol/L; P < 0.05; postanesthesia care unit: CSE 5.0 ± 0.9 versus control 7.3 ± 1.1 mmol/L; P < 0.05). Intraoperative cortisol plasma concentrations were smaller in the CSE group than in the control group. One day after the operation, however, glucose plasma concentration, glucose production, and glucose clearance were comparable in both groups. CSE inhibited the postoperative increase in leucine oxidation rate (CSE 30 ± 12 versus control 43 ± 8 &mgr;mol·kg−1·h−1; P < 0.05). There were no differences between the groups in protein breakdown, whole body protein synthesis, and plasma concentrations of lactate, free fatty acids, insulin, and glucagon. In conclusion, CSE prevents hyperglycemia during hip surgery and inhibits protein catabolism thereafter.

Nitrous oxide impairs the neutrophil oxidative response

Background Nitrous oxide has been shown inconsistently to impair the oxidative function of neutrophils. The choice of the stimulus, receptor agonists, or stimuli acting independent of receptors seems to determine whether nitrous oxide impairs the oxidative functions, suggesting an interference with the cytosolic signaling of neutrophils. Methods Production of hydrogen peroxide by neutrophils was assessed using flow cytometric analysis. N‐formyl‐methionyl‐leucyl‐phenylalanine (FMLP), C5a, dioctanylglycerol, and phorbol‐12‐myristate‐13‐acetate were used as stimuli. In addition, the expression of receptors for FMLP and the cytosolic‐free calcium response of cells were measured. Results Nitrous oxide depresses C5a‐ or FMLP‐induced generation of reactive oxygen derivatives in a concentration‐dependent manner. However, the response with direct activation of protein kinase C was unaffected. Further, the number of FMLP receptors and the cytosolic calcium response were unaffected. Inhibition of the oxidative response was not reversible within the observation period of 4 h. Conclusions Nitrous oxide inhibited the intracellular signaling of the investigated G‐protein‐coupled receptors for chemotactic peptides. No interference of nitrous oxide with reduced nicotinamide adenine dinucleotide phosphate oxidase, the oxidative enzyme system of neutrophils, nor with its activation through protein kinase C was detected.

Characterization of the human fMLP receptor in neutrophils and in Xenopus oocytes

N‐formyl peptides (e.g. fMLP; N‐formyl‐L‐methionyl‐L‐leucyl‐phenylalanine) are potent mediators for inflammatory reactions. We report functional expression in Xenopus oocytes of human fMLP‐R98 cDNA, without co‐expression of the promiscuous G‐protein subunit, Gα‐16. Stimulation of voltage‐clamped oocytes (−70 mV) with fMLP produced a dose‐dependent biphasic inward current with fast and slow components. Analysis using GTP‐γ‐S and cholera and pertussis toxins suggested these currents are mediated by an endogenous G‐protein of the Gq family. The fast current reversed at −25 mV and was blocked by SITS (4‐acetamido‐4′‐isothiocyanatostilbene‐2,2′‐disulphonic acid), suggesting the current is carried by Cl−. The slow current showed weak inward rectification, was Ca2+‐dependent and blocked by Cd2+, 4‐AP (4‐aminopyridine) and haloperidol, suggesting activation of a mixed population of cation channels. Comparative experiments with human neutrophils using flow cytometric analysis showed that the proportion of neutrophils activated by fMLP was reduced in the presence of SITS, in the absence of external calcium and in the presence of Cd2+, TEA (tetraethylammonium) and haloperidol but not 4‐AP. In addition, the oxidative burst from activated neutrophils was reduced by SITS and by the absence of external calcium but not by Cd2+, TEA, 4‐AP or haloperidol. We suggest that in human neutrophils activation by fMLP is dependent on store‐operated calcium influx that appears to be regulated by Cl− channels and linked, in part, to non‐selective cation channels.

Thiopental impairs neutrophil oxidative response by inhibition of intracellular signalling.

BACKGROUND AND OBJECTIVE Thiopental in clinically relevant concentrations inhibits the oxidative function of neutrophils, whereas only very high, non-therapeutic concentrations of methohexital induce a similar effect. The study characterized the molecular basis of this differential action of oxy- and thiobarbiturates on neutrophils. METHODS Neutrophils were incubated in vitro with thiopental or methohexital using concentrations within the therapeutic range. Neutrophil responses were induced using different stimuli: N-formyl-L-methionyl-L-leucyl-L-phenylalanine (FMLP), C5a and 1,2-dioctanoyl-sn-glycerol (DiC8-DAG). FMLP and C5a bind to specific G-protein-coupled receptors that share the same second messenger cascade. In contrast, DiC8-DAG, an activator of protein kinase C, bypasses the signal transduction pathway downstream of the receptors. Hydrogen peroxide production by neutrophils was assessed using flow cytometry. To characterize the localization of the interaction site, FMLP receptor expression and cytosolic-free calcium were further analysed. RESULTS FMLP and C5a-induced hydrogen peroxide production were both significantly impaired by thiopental, but not by methohexital. When postreceptor signalling was bypassed, by stimulation with DiC8-DAG, neither thiopental nor methohexital affected hydrogen peroxide production. Additionally, neither of the barbiturates impaired the cytosolic Ca2+ response. CONCLUSIONS We conclude that neither protein kinase C nor the hydrogen peroxide-generating enzymes are affected by thiopental or methohexital. The unimpaired Ca2+ response suggests that the function of the receptors and G-proteins were also unimpaired. Taken together, this indicates that the site of action of thiopental is in the cellular signalling upstream of protein kinase C.

Inhibition of the neutrophil oxidative response by propofol: preserved in vivo function despite in vitro inhibition.

Background and objective: Propofol has been shown to inhibit a variety of functions of neutrophils in vitro, but there is a lack of in vivo data. To analyse the effects of propofol on neutrophil function in vivo we chose to investigate cataract surgery since it represents a small surgical procedure with minimal immunomodulatory effects induced by surgery. We sought to analyse any immunosuppressive effects of propofol after short‐term administration in vivo in comparison to local anaesthesia as well as to in vitro effects of propofol. Methods: The study was designed as an open randomized trial enrolling 20 patients undergoing general or local anaesthesia. The neutrophil oxidative response and propofol plasma concentration were assessed prior, during and after anaesthesia. Neutrophil function was determined flow cytometrically based on dihydrorhodamine 123 oxidation. Results: Propofol concentrations which yielded a marked suppression in vitro did not alter the neutrophil oxidative response during cataract surgery in vivo. However, after local anaesthesia the neutrophil oxidative response declined to 37%, compared to the control response prior to anaesthesia. Conclusions: Although we could detect the well established suppression of neutrophil function by propofol in vitro it was not evident in vivo. This may be due to compensating effects on neutrophil function during surgery in vivo. The decline in the neutrophil oxidative response in the local anaesthesia group might be due to increased stress and catecholamine concentrations or a direct interaction of local anaesthetics with neutrophil intracellular signalling.