Is the In Vitro Observed NETosis the Favored Physiological Death of Neutrophils or Mainly Induced by an Isolation Bias?

Centrifugation steps are regularly used for neutrophil isolation. Thereby, the influences of applied g-forces on the functionality of PMNs have hardly been analyzed and could consequently have been overlooked or led to biased results. We now hypothesize that blood PMNs-when gently isolated-can be long-lived cells and they physiologically become apoptotic rather than NETotic. Neutrophils were isolated from whole blood without centrifugation using a sedimentation enhancer (gelafundin). PMNs were analyzed via live-cell imaging for migratory activity and vitality condition by fluorescent staining. Native neutrophils showed still relevant migratory activity after more than 6 days ex vivo. The percentage of cells that were annexin V+ or PI+ increased successively with increasing ex vivo time. In addition, the characteristics of DAPI staining of gently isolated granulocytes differed markedly from those obtained by density gradient separation (DGS). We conclude that NETosis occurring after DGS is the consequence of applied g-forces and not a physiological phenomenon. Future studies on neutrophils should be performed with most native cells (applied g-time load as low as possible).

Predictors of delirium after cardiac surgery in patients with sleep disordered breathing.

INTRODUCTION: Delirium ranks among the most common complications after cardiac surgery. Although various risk factors have been identified, the association between sleep disordered breathing (SDB) and delirium has barely been examined so far. Here, our objectives were to determine the incidence of post-operative delirium and to identify the risk factors for delirium in patients with and without SDB. METHODS: This subanalysis of the ongoing prospective observational study CONSIDER-AF (ClinicalTrials.gov identifier NCT02877745) examined risk factors for delirium in 141 patients undergoing cardiac surgery. The presence and type of SDB were assessed with a portable SDB monitor the night before surgery. Delirium was prospectively assessed with the validated Confusion Assessment Method for the Intensive Care Unit on the day of extubation and for a maximum of 3 days. RESULTS: Delirium was diagnosed in 23% of patients: in 16% of patients without SDB, in 13% with obstructive sleep apnoea and in 49% with central sleep apnoea. Multivariable logistic regression analysis showed that delirium was independently associated with age ≥70 years (OR 5.63, 95% CI 1.79-17.68; p=0.003), central sleep apnoea (OR 4.99, 95% CI 1.41-17.69; p=0.013) and heart failure (OR 3.3, 95% CI 1.06-10.35; p=0.039). Length of hospital stay and time spent in the intensive care unit/intermediate care setting were significantly longer for patients with delirium. CONCLUSIONS: Among the established risk factors for delirium, central sleep apnoea was independently associated with delirium. Our findings contribute to identifying patients at high risk of developing post-operative delirium who may benefit from intensified delirium prevention strategies.

Paralytic Impact of Centrifugation on Human Neutrophils.

Centrifugation is a common step in most of the popular protocols for the isolation of neutrophils from whole blood. Inconsistent results from previous studies on neutrophils may originate from an underestimation of the centrifugation effect, as in consequence impaired, not native cells, being investigated. We hypothesize, that centrifugation significantly impairs major neutrophil functions. However, there is no data yet whether the application of g-force itself or the product of g-force and duration of centrifugation (="g-time") defines the impact on neutrophils. Neutrophils were isolated from whole blood via centrifugation with different g-times and subsequently analyzed via live cell imaging for migration, as well as via flow cytometry for oxidative burst and surface antigen expression. Chemotactic migration was significantly reduced with increasing g-time. Oxidative burst decreased likewise the higher the g-time applied. Expression of CD11b was no longer upregulated in response to an n-formylmethionine-leucyl-phenylalanine (fMLP) stimulus in neutrophils having experienced high g-time during the isolation process. We conclude that centrifugation "paralyzes" neutrophils in the form of a significant decrease in functionality. Future investigations on neutrophil granulocytes should reduce the g-time load as far as possible.

Enhanced Cardiac CaMKII Oxidation and CaMKII-Dependent SR Ca Leak in Patients with Sleep-Disordered Breathing.

BACKGROUND: Sleep-disordered breathing (SDB) is associated with increased oxidant generation. Oxidized Ca/calmodulin kinase II (CaMKII) can contribute to atrial arrhythmias by the stimulation of sarcoplasmic reticulum Ca release events, i.e., Ca sparks. METHODS: We prospectively enrolled 39 patients undergoing cardiac surgery to screen for SDB and collected right atrial appendage biopsies. RESULTS: SDB was diagnosed in 14 patients (36%). SDB patients had significantly increased levels of oxidized and activated CaMKII (assessed by Western blotting/specific pulldown). Moreover, SDB patients showed a significant increase in Ca spark frequency (CaSpF measured by confocal microscopy) compared with control subjects. CaSpF was 3.58 ± 0.75 (SDB) vs. 2.49 ± 0.84 (no SDB) 1/100 µm-1s-1 (p < 0.05). In linear multivariable regression models, SDB severity was independently associated with increased CaSpF (B [95%CI]: 0.05 [0.03; 0.07], p < 0.001) after adjusting for important comorbidities. Interestingly, 30 min exposure to the CaMKII inhibitor autocamtide-2 related autoinhibitory peptide normalized the increased CaSpF and eliminated the association between SDB and CaSpF (B [95%CI]: 0.01 [-0.1; 0.03], p = 0.387). CONCLUSIONS: Patients with SDB have increased CaMKII oxidation/activation and increased CaMKII-dependent CaSpF in the atrial myocardium, independent of major clinical confounders, which may be a novel target for treatment of atrial arrhythmias in SDB.

Potential Impact of Local Anesthetics Inducing Granulocyte Arrest and Altering Immune Functions on Perioperative Outcome.

INTRODUCTION: Local anesthetics (LAs) are frequently used during anesthesia; however, they may influence granulocyte function which in turn could modify immune responses in the perioperative period. Therefore, the aim of this study was to investigate the impact of clinically used doses of bupivacaine and lidocaine on granulocyte function with regard to migration, reactive oxygen species (ROS) production, neutrophil extracellular traps (NETosis) formation, and viability. METHODS: A total of 38 granulocyte-enriched samples from healthy subjects were obtained by whole blood lysis. Polymorphonuclear neutrophil (PMN) samples were incubated simultaneously with different concentrations of either bupivacaine (0.03-3.16 mmol/L) or lidocaine (0.007-14.21 mmol/L), or without drug (control). Live cell imaging was conducted in order to observe granulocyte chemotaxis, migration, ROS production, and NETosis. Flow cytometry was used to analyze viability and antigen expression. RESULTS: The track length (TL) of PMNs exposed to bupivacaine concentrations of 0.16 mmol/L and above significantly decreased compared to the control. Low concentrations of lidocaine were associated with slight but significant increases in TL, whereas this changed with concentrations above 1.4 mmol/L, showing a significant decrease in TL. PMN incubated with bupivacaine concentrations of 1.58 mmol/L and above or lidocaine concentrations of at least 3.6 mmol/L showed no migration or chemotaxis at all. Time to onset of maximal ROS production and time for half-maximal NETosis decreased in a dose-dependent manner for both substances. Equipotency in NETosis induction was reached by bupivacaine (1.1 mmol/L) at significantly lower concentrations than lidocaine (7.96 mmol/L). Cell viability and oxidative burst were unaffected by LAs. CONCLUSION: Local anesthetics in clinically used doses ameliorate granulocyte defense mechanisms, thus indicating their potentially decisive effect during the perioperative period.

Anaesthesia-related complications and side-effects in TAVI: a retrospective study in Germany.

OBJECTIVES: This study was performed to analyse anaesthesia-related complications and side effects in patients undergoing transcatheter aortic valve implantation (TAVI) under general anaesthesia. DESIGN: Retrospective study. SETTING: The study was performed as a single-centre study in a hospital of tertiary care in Germany. PARTICIPANTS: All 853 patients, who underwent TAVI at the Universitätsklinikum Regensburg between January 2009 and July 2015, were included. 52.5% were female patients. PRIMARY AND SECONDARY OUTCOME MEASURES: We gathered information, such as recent illness, vital parameters and medication administered during the intervention and postoperatively for 12 hours. We analysed all anaesthesia-related complications and anaesthesia-related side effects that occurred during the intervention and entire hospital stay. RESULTS: We analysed all 853 TAVI procedures. The mean patient age was 79 ± 6 years. In 99.5% of cases, we used volatile-based anaesthesia. 2.8% (n=24; transfemoral (TF): n=19 [3.8%]; transapical (TA): n=5 [1.4%]) of all cases suffered from anaesthesia-related complications. 819 (TF: n=447; TA: n=372) anaesthesia-related side effects occurred in 586 (68.7%, TF: n=325 [64.2%], TA: n=261 [75.2%]) patients. Neither the complications nor the side effects had any serious consequences. Intraoperative hypothermia in 44% of cases (TF: n=202 [39.9%]; TA: n=173 [49.9%]) and postoperative nausea and vomiting in 27% (n=232; TF: n=131 [25.9%], TA: n=101 [29.1%]) of cases were the most common anaesthesia-related side effects. CONCLUSION: In this study, serious anaesthesia-related complications were rarely seen, and non-critical anaesthesia-related side effects could have been avoided through consistent prophylaxis and management. Therefore, despite their high anaesthetic risk, general anaesthesia is justifiable in patients who underwent TAVI.

Complications after transcatheter aortic valve implantation using transfemoral and transapical approach in general anaesthesia.

BACKGROUND: Transcatheter aortic valve implantation (TAVI) is a minimally invasive procedure used to treat degenerative heart valve disease. The implantation requires a highly specific and interdisciplinary management approach. Currently, TAVI is performed with the patient under local or general anaesthesia. METHODS: This study was a retrospective analysis of all TAVI procedures performed at the University Hospital of Regensburg between January 2009 and July 2015. All pre-, intra and postoperative data focusing on perioperative complications were recorded. RESULTS: A total of 853 transfemoral- and transapical-TAVI patients were included in the study. All patients underwent general anaesthesia. The ASA classifications were primarily 3-4. The average logistic EuroScores for the transfemoral- and transapical-TAVI patients were 18 ± 12% and 21 ± 15% (p = 0.002), respectively. The anaesthesia coverage time was 170 ± 49 min., including 37 ± 12 minutes for anaesthetic management. Overall, 458 complications were recorded; with pneumonia, acute renal failure, indication for a permanent pacemaker and non-extubation in the operating theatre the most frequently recorded complications. CONCLUSION: In the present study, we showed that our patients' outcomes are comparable to those reported in the available literature. Compared to TF, TA patients show an overall worse physical condition as well as a higher perioperative morbidity and mortality. Consequently TA patients need additional care and should only be operated in appropriately experienced medical centres.

Sleep-disordered breathing is associated with disturbed cardiac repolarization in patients with a coronary artery bypass graft surgery.

BACKGROUND: The development of malignant ventricular arrhythmias due to abnormal cardiac repolarization is a major complication after coronary artery bypass graft surgery (CABG). Sleep-disordered breathing (SDB) is linked to prolonged cardiac repolarization in non-surgical patients. This study evaluates cardiac repolarization in patients with and without SDB who underwent CABG. METHODS: 100 patients who had received CABG (84% men, age 68 ± 10 years, body-mass-index [BMI] 28.7 ± 4.2 kg/m2) were retrospectively evaluated. Polygraphy was recorded the night before CABG. SDB was defined as an apnea-hypopnea index (AHI) of ≥15/h and differentiated into central (CSA) and obstructive (OSA) sleep apnea. Cardiac repolarization was assessed by means of T-peak-to-end (TpTe) and QTc-intervals and TpTe/QT-ratios derived from 12-lead electrocardiography (ECG). RESULTS: 37% of patients had SDB, 14% CSA and 23% OSA. Before CABG, patients with CSA and OSA had longer TpTe intervals than those without SDB (TpTe: CSA 100 ± 26 vs. OSA 97 ± 19 vs. no SDB 85 ± 14 ms, p = 0.013). QTc intervals and TpTe/QT ratios differed between the two groups (QTc: 444 ± 54 vs. 462 ± 36 vs. 421 ± 32 ms, p < 0.001; TpTe/QT ratio: 0.24 ± 0.04 vs. 0.23 ± 0.05 vs. 0.21 ± 0.03, p = 0.045). SDB was associated with abnormal cardiac repolarization independent of known risk factors for cardiac arrhythmias, such as age, sex, BMI, N-terminal-pro-brain-natriuretic-peptide (NT-proBNP), and heart failure (TpTe: B-coefficient [95%CI]: 16.0, [7.6-24.3], p < 0.001; QTc: 27.2 [9.3-45.1], p = 0.003; TpTe/QT ratio: 2.9 [1.2-4.6], p < 0.001). CONCLUSION: Independent of known risk factors for cardiac arrhythmias, SDB was significantly associated with abnormal cardiac repolarization before CABG. Data suggest that SDB may contribute to an increased risk of ventricular arrhythmias after CABG.

Postoperative complications after elective coronary artery bypass grafting surgery in patients with sleep-disordered breathing.

BACKGROUND: Sleep-disordered breathing (SDB) may increase the risk of postoperative complications in patients after cardiac surgery. This study evaluated the length of hospital stay as well as postoperative cardiac, respiratory, and renal complications after elective coronary artery bypass grafting (CABG) in patients without SDB, with central sleep apnea (CSA), or with obstructive sleep apnea (OSA). METHODS: The presence and type of SDB had been assessed with polygraphic recordings in 100 patients the night before elective CABG surgery. SDB was defined as an apnea-hypopnea index (AHI) of ≥ 15/h. Prolonged length of hospital stay (LOS) and postoperative hemodynamic instability due to any cause were retrospectively evaluated as primary endpoints and cardiac, respiratory, and renal complications as secondary endpoints. RESULTS: 37% of patients had SDB, 14% CSA, and 23% OSA. LOS differed significantly between patients without SDB and those with CSA and OSA [median (25;75. percentile): 8.0 days (7.5;11.0) vs. 9.5 days (7.0;12.5) vs. 12.0 days (9.0;17.0), Kruskal-Wallis test between three groups: p = 0.023; OSA vs. no SDB: p = 0.005]. AHI was significantly associated with prolonged LOS [> 9 days; odds ratio (OR) (95% confidence interval): 1.047 (1.001;1.095), p = 0.044]. Prolonged need of vasopressors (≥ 48 h) was observed in 36% of patients without SDB, in 64% with CSA, and in 62% with OSA (p = 0.037). AHI was significantly associated with prolonged (≥ 48 h) need of vasopressors [OR (95% CI): 1.052 (1.002;1.104), p = 0.040], independent of any confounders. CONCLUSIONS: SDB, particularly OSA, is associated with prolonged LOS after CABG, independent of known confounders. Prolonged LOS in patients with SDB may be due to increased postoperative hemodynamic instability due to any cause.

Perforation of myocardial wall and great vessels after cardiovascular interventions-a 5-year analysis.

BACKGROUND: Less invasive procedures have replaced open surgical treatment in many cardiovascular disorders. During these interventions, iatrogenic cardiac perforation may ensue, which is a severe complication and requires immediate diagnostic assessment and treatment. METHODS: From March 2011 to April 2016, all patients referred to the Dept. of Cardio-thoracic Surgery with the diagnosis of iatrogenic perforation of myocardial wall or great vessels were included into the retrospective study. Complications during transapical transcutaneous aortic valve replacements (TAVR) procedures and percutaneous coronary intervention (PCI) were excluded from analysis. Symptoms, therapeutic strategy, intraoperative findings, and outcome were evaluated. RESULTS: Forty-four patients suffered from myocardial wall or vessel perforation. Most common site of perforation were right (n=26; 59.1%) and left (n=8; 18.2%) ventricle. Other structures were involved in ten cases (22.7%). Open surgical treatment was required in 27 cases (61.4%). Mortality after left and right ventricular laceration was 75.0% and 11.5%, respectively. Most common cause of death was cardiocirculatory failure (n=5). CONCLUSIONS: Iatrogenic perforation of myocardial wall or central vessels during percutaneous interventional procedures is a rare but life-threatening complication. Despite immediate treatment efforts, mortality is high, particularly after left ventricular laceration.

Anaesthetic sensitivity of fMLP-induced cell signalling in Xenopus oocytes.

FMLP stimulation of Xenopus oocytes expressing fMLP receptors leads to a concentration-dependent biphasic inward current. To identify the evolution of these currents we have examined the effects of blocking various cell signalling pathways. In addition we have analysed the effects of three intravenous anaesthetics on these fMLP-induced currents. Xenopus oocytes were microinjected with cRNA encoding the fMLP receptor and fMLP-stimulated (100 nM) currents measured, using two-electrode voltage-clamp (-70 mV), before and after injection of heparin (120 ng ml-1), wortmannin (1 microM), U73122 (5 microM) or buffer. Concentration-response curves were established for the action on fMLP-stimulated currents of thiopentone (5-500 microM), methohexitone (0.2-200 microM) and propofol (0.5-500 microM). Heparin significantly enhanced the fast current (p<0.05). Wortmannin had no effect on either current. U73122 inhibited only the slow current (p<0.05). All anaesthetics inhibited both currents, with the maximum inhibition for the fast/slow currents 70%/100%, 60%/60% and 100%/100% for thiopentone (IC50 147/120 microM), methohexitone (IC50 4.7/2.2 microM) and propofol (IC50 33/8 microM), respectively. We suggest (a) the slow current arises via the PLC/PKC pathway because it is reduced by the PLC inhibitor U73122, (b) the PI3K- and PLD-mediated pathways are not involved because wortmannin had no effect and (c) activation of the two conductance channels must be different because U73122 reduced the slow but not the fast current. Since both currents are decreased by all three anaesthetics, their inhibition might be mediated through an action at the agonist/receptor, although, since the slow current is consistently more sensitive than the fast, there may be additionally an action on cell signalling.

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

1. 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, Galpha-16. 2. Stimulation of voltage-clamped oocytes (-70 mV) with fMLP produced a dose-dependent biphasic inward current with fast and slow components. Analysis using GTP-gamma-S and cholera and pertussis toxins suggested these currents are mediated by an endogenous G-protein of the Gq family. 3. 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 Ca(2+)-dependent and blocked by Cd(2+), 4-AP (4-aminopyridine) and haloperidol, suggesting activation of a mixed population of cation channels. 4. 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 Cd(2+), 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 Cd(2+), TEA, 4-AP or haloperidol. 5. 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.

Differential effects of clindamycin on neutrophils of healthy donors and septic patients.

Antibiotics are frequently administered to ICU patients in case of bacterial infections. Little is known, however, about the interference of antibiotics with neutrophil host defence mechanisms in patients with sepsis and multiple organ dysfunction syndrome (MODS). With our study, evidence for differential clindamycin effects on neutrophils in healthy donors and septic patients without or with MODS was sought. Functional parameters (oxidative response and phagocytosis) and fMLP receptor expression were analysed. The study was approved by the local ethical board. Venous blood was drawn from healthy donors and septic patients. Neutrophils in PBS were incubated with 0, 5, 25 or 125 microg/ml clindamycin and analysed flow cytometrically. Neutrophils of patients with sepsis and MODS showed a significantly higher basal activation compared to healthy donors. Clindamycin application led to a dose-dependent significant suppression of the fMLP-induced oxidative response in patients with sepsis and MODS, but not in healthy donors or septic patients in the absence of MODS. In patients with sepsis and MODS, phagocytosis of Escherichia coli and Staphylococcus aureus was significantly suppressed by clindamycin 125 microg/ml. In both other treatment groups, clindamycin did not affect phagocytosis. fMLP receptor expression was not altered by clindamycin. High-dose clindamycin selectively suppresses functional responses of neutrophils in septic patients with MODS. Simultaneously applied drugs, such as general anaesthetics, may potentiate this modulation of antibacterial defence and inflammation.

Significance of intraoperative testing in right-sided implantable cardioverter-defibrillators.

BACKGROUND: Implantation of implantable cardioverter-defibrillators (ICD) from the left pectoral region is the standard therapeutical method. Increasing numbers of system revisions due to lead dysfunction and infections will consecutively increase the numbers of right-sided implantations. The reliability of devices implanted on the right pectoral side remains controversially discussed, and the question of testing these devices remains unanswered. METHODS: In a prospectively designed study all 870 patients (60.0 ± 14 years, 689 male) who were treated with a first ICD from July 2005 until May 2012 and tested intraoperatively according to the testing protocol were analyzed. The indication for implantation was primary prophylactic in 71.5%. Underlying diseases included ischemic cardiomyopathy (50%), dilative cardiomyopathy (37%), and others (13%). Mean ejection faction was 27 ± 12%. Implantation site was right in 4.5% and left in 95.5%. RESULTS: Five patients supplied with right-sided ICD (13%, p = 0.02 as compared to left-sided) failed initial intraoperative testing with 21 J. 3 patients were male. The age of the patients failing intraoperative testing with right-sided devices appeared higher than of patients with left-sided devices (p = 0.07). The ejection fraction was 28 ± 8%. All patients reached a sufficient DFT ≤ 21 J after corrective procedures. CONCLUSION: Implantation of ICDs on the right side results in significantly higher failure rate of successful termination of intraoperatively induced ventricular fibrillation. The data of our study suggest the necessity of intraoperative ICD testing in right-sided implanted ICDs.

The anticatabolic effect of neuraxial blockade after hip surgery.

UNLABELLED: 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 microg/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 micromol.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. IMPLICATIONS: We studied the effect of combined spinal/epidural blockade (CSE) on protein and glucose metabolism during and after hip surgery. In comparison to general anesthesia followed by intravenous patient-controlled analgesia, CSE inhibits the increase in glucose plasma concentration during surgery and prevents protein loss on the first postoperative day.

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 degrees-41 degrees 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)-alpha 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 wi thopsonized bacteria. Hypothermia slightly increased N-formyl-L-methionyl-L-leucyl-phenylalanine receptors on neutrophils: hyperthermia decreased expression, especially with TNF-alpha. N-formyl-L-methionyl-L-leucyl-phenylalanine-induced H2O2 production was inversely related to temperature, especially in the presence of TNF-alpha. 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.