Bart P. Ramakers

Functional heterogeneity and differential priming of circulating neutrophils in human experimental endotoxemia

Neutrophils play an important role in host defense. However, deregulation of neutrophils contributes to tissue damage in severe systemic inflammation. In contrast to complications mediated by an overactive neutrophil compartment, severe systemic inflammation is a risk factor for development of immune suppression and as a result, infectious complications. The role of neutrophils in this clinical paradox is poorly understood, and in this study, we tested whether this paradox could be explained by distinct neutrophil subsets and their functionality. We studied the circulating neutrophil compartment immediately after induction of systemic inflammation by administering 2 ng/kg Escherichia coli LPS i.v. to healthy volunteers. Neutrophils were phenotyped by expression of membrane receptors visualized by flow cytometry, capacity to interact with fluorescently labeled microbes, and activation of the NADPH‐oxidase by oxidation of Amplex Red and dihydrorhodamine. After induction of systemic inflammation, expression of membrane receptors on neutrophils, such as CXCR1 and ‐2 (IL‐8Rs), C5aR, FcγRII, and TLR4, was decreased. Neutrophils were also refractory to fMLF‐induced up‐regulation of membrane receptors, and suppression of antimicrobial function was shown by decreased interaction with Staphylococcus epidermis. Simultaneously, activation of circulating neutrophils was demonstrated by a threefold increase in release of ROS. The paradoxical phenotype can be explained by the selective priming of the respiratory burst. In contrast, newly released, CD16dim banded neutrophils display decreased antimicrobial function. We conclude that systemic inflammation leads to a functionally heterogeneous neutrophil compartment, in which newly released refractory neutrophils can cause susceptibility to infections, and activated, differentiated neutrophils can mediate tissue damage.

Functional and genetic evidence that the Mal/TIRAP allele variant 180L has been selected by providing protection against septic shock.

Adequate responses by our innate immune system toward invading pathogens were of vital importance for surviving infections, especially before the antibiotic era. Recently, a polymorphism in Mal (Ser180Leu, TIRAP rs8177374), an important adaptor protein downstream of the Toll-like receptor (TLR) 2 and 4 pathways, has been described to provide protection against a broad range of infectious pathogens. We assessed the functional effects of this polymorphism in human experimental endotoxemia, and we demonstrate that individuals bearing the TIRAP 180L allele display an increased, innate immune response to TLR4 and TLR2 ligands, but not to TLR9 stimulation. This phenotype has been related to an increased resistance to infection. However, an overshoot in the release of proinflammatory cytokines by TIRAP 180L homozygous individuals suggests a scenario of balanced evolution. We have also investigated the worldwide distribution of the Ser180Leu polymorphism in 14 populations around the globe to correlate the genetic makeup of TIRAP with the local infectious pressures. Based on the immunological, clinical, and genetic data, we propose that this mutation might have been selected in West Eurasia during the early settlement of this region after the out-of-Africa migration of modern Homo sapiens. This combination of functional and genetic data provides unique insights to our understanding of the pathogenesis of sepsis.

Differential ex vivo and in vivo endotoxin tolerance kinetics following human endotoxemia.

Objectives:Endotoxin (lipopolysaccharide) tolerance is characterized by a transient refractory state to a subsequent lipopolysaccharide challenge. Following human endotoxemia, ex vivo tolerance of circulating leukocytes to lipopolysaccharide resolves within 24 hrs. However, the duration of in vivo tolerance, assumed to be primarily mediated by tissue-resident macrophages, is unknown. Design, Setting, Subjects, and Interventions:Clinical experimental study in 16 healthy male volunteers at an intensive care research unit. To compare ex vivo and in vivo tolerance kinetics, whole blood from healthy volunteers was stimulated with lipopolysaccharide before, 4 hrs after, and 1, 2, 3, and 4 wks following in vivo endotoxin (2 ng/kg; lipopolysaccharide derived from Escherichia coli O:113) administration. Furthermore, we compared the inflammatory response during two subsequent endotoxemia experiments in healthy volunteers with an interval of 2 wks. The cytokines tumor necrosis factor-&agr;, interleukin-6, interleukin-10, interleukin-1 receptor antagonist, and transforming growth factor-&bgr; were measured. Measurements and Main Results:Four hours after in vivo lipopolysaccharide administration, production of tumor necrosis factor-&agr;, interleukin-6, and interleukin-10, but not interleukin-1 receptor antagonist in ex vivo lipopolysaccharide-stimulated whole blood was diminished. Ex vivo lipopolysaccharide tolerance completely resolved within 1 week. In contrast, in vivo lipopolysaccharide tolerance was still apparent after 2 wks. Compared to the first lipopolysaccharide administration, plasma peak levels of tumor necrosis factor-&agr;, interleukin-6, interleukin-10, interleukin-1 receptor antagonist, and transforming growth factor-&bgr; were attenuated by 46%, 36%, 45%, 10%, and 14%, respectively (all p < .05). Conclusions:While ex vivo lipopolysaccharide tolerance quickly resolves, in vivo lipopolysaccharide tolerance persists for at least 2 wks. These findings strengthen the notion that the in vivo response to lipopolysaccharide is mediated by tissue-resident macrophages and that ex vivo stimulation does not accurately reflect the in vivo innate immune response. Intervention studies utilizing the human endotoxemia model should be performed using parallel groups rather than a crossover design.

Oral therapy with dipyridamole limits ischemia-reperfusion injury in humans

Adenosine receptor stimulation induces several effects that could limit ischemia‐reperfusion injury. We hypothesize that treatment with the nucleoside uptake inhibitor dipyridamole increases endogenous adenosine and limits ischemia‐reperfusion injury in humans.

Endotoxemia-induced inflammation and the effect on the human brain

IntroductionEffects of systemic inflammation on cerebral function are not clear, as both inflammation-induced encephalopathy as well as stress-hormone mediated alertness have been described.MethodsExperimental endotoxemia (2 ng/kg Escherichia coli lipopolysaccharide [LPS]) was induced in 15 subjects, whereas 10 served as controls. Cytokines (TNF-α, IL-6, IL1-RA and IL-10), cortisol, brain specific proteins (BSP), electroencephalography (EEG) and cognitive function tests (CFTs) were determined.ResultsFollowing LPS infusion, circulating pro- and anti-inflammatory cytokines, and cortisol increased (P < 0.0001). BSP changes stayed within the normal range, in which neuron specific enolase (NSE) and S100-β changed significantly. Except in one subject with a mild encephalopathic episode, without cognitive dysfunction, endotoxemia induced no clinically relevant EEG changes. Quantitative EEG analysis showed a higher state of alertness detected by changes in the central region, and peak frequency in the occipital region. Improved CFTs during endotoxemia was found to be due to a practice effect as CFTs improved to the same extent in the reference group. Cortisol significantly correlated with a higher state of alertness detected on the EEG. Increased IL-10 and the decreased NSE both correlated with improvement of working memory and with psychomotor speed capacity. No other significant correlations between cytokines, cortisol, EEG, CFT and BSP were found.ConclusionsShort-term systemic inflammation does not provoke or explain the occurrence of septic encephalopathy, but primarily results in an inflammation-mediated increase in cortisol and alertness.Trial registrationNCT00513110.

Annexin A5 Scintigraphy of Forearm as a Novel In Vivo Model of Skeletal Muscle Preconditioning in Humans

Background—Nonlethal ischemia and reperfusion reduce ischemia-reperfusion–induced cell death, a phenomenon called ischemic preconditioning. In animal models, this potent endogenous protection is mimicked in vivo by administration of adenosine. In humans, exploitation of ischemic preconditioning is hindered by the lack of an appropriate in vivo model to study this phenomenon. To solve this problem, we aimed to set up an easy-to-use human in vivo model to study ischemic or pharmacological preconditioning. Methods and Results—Healthy male volunteers performed unilateral ischemic handgrip. At reperfusion, we intravenously injected technetium-99m–labeled Annexin A5, a presumed marker of ischemic injury, and we imaged both forearms and hands simultaneously with a gamma camera. Region of interest analysis (counts per pixel) and subsequent calculation of the percentage difference in radioactivity between experimental and control hands (thenar muscle; mean±SE) revealed significant uptake to the ischemically exercised tissue (26±3% at 4 hours after reperfusion; P<0.05). This selective localization of Annexin A5 was reduced by ischemic preconditioning (10 minutes of ischemia plus reperfusion before ischemic exercise) or by infusion of adenosine into the brachial artery to 6±1% and 10±3%, respectively (P<0.05 versus ischemic exercise alone), resembling observations in animal models with infarct size as an end point. Appropriate control experiments supported our conclusion. Conclusions—Annexin A5 scintigraphy can be applied to test pharmacological or physiological interventions for their ability to prevent ischemia-reperfusion injury.

Modulation of innate immunity by adenosine receptor stimulation

In the past decades, increased concentrations of the signaling molecule adenosine have been shown to play an important role in the prevention of tissue damage evoked by several stressful circumstances. During systemic inflammation, the circulating adenosine concentration increases rapidly, even up to 10-fold in septic shock patients. By binding to specific adenosine receptor subtypes, designated A1, A2a, A2b, and A3, adenosine exerts a wide variety of immunomodulating and (cyto)protective effects. Only recently, several specific adenosine receptor agonists and other drugs that modulate adenosine metabolism have been developed for human use. Importantly, correct interpretation of the effects of adenosine is highly related to the model of inflammation used, e.g., administration of endotoxin or live bacteria. This review will discuss the potential role for adenosine as an immunomodulating and cytoprotective signaling molecule and will discuss its potential role in the treatment of the patient suffering from sepsis.ABBREVIATIONS-AMP-adenosine monophosphate; 5NT-5&vprime;-nucleotidase; ADA-adenosine deaminase; AK-adenosine kinase; ENT-equilibrative nucleoside transporter; cAMP-5&vprime; cyclic adenosine monophosphate; CLP-cecal ligation and puncture; NF-&kgr;B-nuclear factor &kgr;B

Inflammation-induced increases in plasma endocan levels are associated with endothelial dysfunction in humans in vivo.

ABSTRACT Although endothelial dysfunction is central to the pathogenesis of sepsis, no specific and clinically applicable marker for endothelial dysfunction is currently available. Endocan, a proteoglycan excreted by endothelial cells in response to inflammatory cytokines, may serve as such a marker. Our objective was to investigate the kinetics of endocan and its relationship with inflammation-induced endothelial dysfunction during experimental human endotoxemia. Endothelial function was assessed in 17 healthy male volunteers before and 4 h after the administration of 2 ng/kg lipopolysaccharide (LPS) by determination of the vasodilatory response of forearm blood vessels to intra-arterial infusion of endothelium-dependent (acetylcholine) or endothelium-independent (nitroglycerin/sodium nitroprusside) vasodilators using venous occlusion plethysmography. Plasma levels of endocan, inflammatory cytokines, intercellular adhesion molecule (ICAM), and vascular cell adhesion molecule (VCAM) were measured, and correlations with endothelial dysfunction were explored. Plasma levels of all measured cytokines, endocan, ICAM, and VCAM concentrations significantly increased after LPS administration. Furthermore, LPS administration resulted in a significantly blunted response to acetylcholine (mean ± SD increase in forearm blood flow [FBF] of 383% ± 320% before LPS vs. 173% ± 134% after LPS, P = 0.03), whereas the response to nitroglycerin/sodium nitroprusside was not affected (mean ± SD increase in FBF of 174% ± 120% before LPS vs. 110% ± 82% after LPS, P = 0.11). Furthermore, there was a significant correlation between the increase in plasma endocan levels and the attenuation of vasodilatory responses to acetylcholine (r = −0.48, P < 0.05). No correlation existed between plasma levels of ICAM or VCAM and the attenuation of the acetylcholine-induced vasodilatory response. Endocan levels are related to endothelial dysfunction in humans in vivo during systemic inflammation evoked by experimental endotoxemia. Therefore, this study suggests that endocan could be a novel marker of endothelial dysfunction in inflammatory conditions.

Continuous administration of enteral lipid- and protein-rich nutrition limits inflammation in a human endotoxemia model

Objective:An overzealous inflammatory response is an important cause of morbidity and mortality in surgical, trauma, and critically ill patients. Enteral administration of lipid-rich nutrition was previously shown to attenuate inflammation and reduce organ damage via a cholecystokinin-1 receptor–mediated vagovagal reflex in animal studies. The current preclinical study investigates the immunomodulatory potential of a custom-made enteral nutrition during systemic inflammation in man. Design:Double-blind, randomized controlled trial. Setting:Intensive care research unit. Subjects:Male volunteers. Interventions:After an overnight fast, 18 healthy male subjects received an IV bolus of Escherichia coli lipopolysaccharide (2 ng/kg). Subjects in the fasted group (n = 6) were deprived of food throughout the study, while subjects in the intervention groups were fed either custom-made lipid- and protein-rich nutrition (n = 6) or isocaloric control nutrition (n = 6) via nasojejunal tube, starting 1 hour prior to lipopolysaccharide administration until 6 hours afterward. Measurements and Main Results:Bolus lipopolysaccharide administration resulted in a marked inflammatory response. Continuous postpyloric administration of nutrition significantly increased plasma cholecystokinin levels throughout the lipopolysaccharide-induced inflammatory response. Lipid- and protein-rich nutrition attenuated circulating levels of the proinflammatory cytokines tumor necrosis factor-&agr; and interleukin-6 and the interleukin-1 receptor antagonist compared with control nutrition (all p < 0.05) and fasted subjects (all p < 0.05). In additional, lipid- and protein-rich nutrition augmented the anti-inflammatory response, reflected by increased plasma levels of interleukin-10 compared with fasted subjects (p < 0.0001). Conclusions:The current preclinical study expands the immunomodulating effects of enteral nutrition as previously observed in rodents to man. Continuous administration of enteral nutrition resulted in a rapid anti-inflammatory effect. Furthermore, enrichment of the nutritional composition with lipid and protein was shown to enhance the anti-inflammatory potential. Therefore, continuous enteral administration of lipid- and protein-rich nutrition is a promising intervention to modulate the immune response in the early course of systemic inflammation in man.

Interplay between the acute inflammatory response and heart rate variability in healthy human volunteers.

The autonomic nervous system and the inflammatory response are intimately linked. Heart rate variability (HRV) analysis is a widely used method to assess cardiac autonomic nervous system activity, and changes in HRV indices may correlate with inflammatory markers. Here, we investigated whether baseline HRV predicts the acute inflammatory response to endotoxin. Second, we investigated whether the magnitude of the inflammatory response correlated with HRV alterations. Forty healthy volunteers received a single intravenous bolus of 2 ng/kg endotoxin (LPS, derived from Escherichia coli O:113). Of these, 12 healthy volunteers were administered LPS again 2 weeks later. Heart rate variability was determined at baseline (just before LPS administration) and hourly thereafter until 8 h after LPS administration. Plasma cytokine levels were determined at various time points. Baseline HRV indices did not correlate with the magnitude of the LPS-induced inflammatory response. Despite large alterations in HRV after LPS administration, the extent of the inflammatory response did not correlate with the magnitude of HRV changes. In subjects who were administered LPS twice, inflammatory cytokines were markedly attenuated after the second LPS administration, whereas LPS-induced HRV alterations were similar. Heart rate variability indices do not predict the acute inflammatory response in a standardized model of systemic inflammation. Although the acute inflammatory response results in HRV changes, no correlations with inflammatory cytokines were observed. Therefore, the magnitude of endotoxemia-related HRV changes does not reflect the extent of the inflammatory response.