Immunosuppressive effects of circulating bile acids in human endotoxemia and septic shock: patients with liver failure are at risk.

BACKGROUND: Sepsis-induced immunosuppression is a frequent cause of opportunistic infections and death in critically ill patients. A better understanding of the underlying mechanisms is needed to develop targeted therapies. Circulating bile acids with immunosuppressive effects were recently identified in critically ill patients. These bile acids activate the monocyte G-protein coupled receptor TGR5, thereby inducing profound innate immune dysfunction. Whether these mechanisms contribute to immunosuppression and disease severity in sepsis is unknown. The aim of this study was to determine if immunosuppressive bile acids are present in endotoxemia and septic shock and, if so, which patients are particularly at risk. METHODS: To induce experimental endotoxemia in humans, ten healthy volunteers received 2 ng/kg E. coli lipopolysaccharide (LPS). Circulating bile acids were profiled before and after LPS administration. Furthermore, 48 patients with early (shock onset within < 24 h) and severe septic shock (norepinephrine dose > 0.4 µg/kg/min) and 48 healthy age- and sex-matched controls were analyzed for circulating bile acids. To screen for immunosuppressive effects of circulating bile acids, the capability to induce TGR5 activation was computed for each individual bile acid profile by a recently published formula. RESULTS: Although experimental endotoxemia as well as septic shock led to significant increases in total bile acids compared to controls, this increase was mild in most cases. By contrast, there was a marked and significant increase in circulating bile acids in septic shock patients with severe liver failure compared to healthy controls (61.8 µmol/L vs. 2.8 µmol/L, p = 0.0016). Circulating bile acids in these patients were capable to induce immunosuppression, as indicated by a significant increase in TGR5 activation by circulating bile acids (20.4% in severe liver failure vs. 2.8% in healthy controls, p = 0.0139). CONCLUSIONS: Circulating bile acids capable of inducing immunosuppression are present in septic shock patients with severe liver failure. Future studies should examine whether modulation of bile acid metabolism can improve the clinical course and outcome of sepsis in these patients.

Parenteral bilirubin in healthy volunteers: a reintroduction in translational research.

AIMS: Preclinical results suggest therapeutic potential of mild hyperbilirubinemia in T2DM and cardiovascular disease. Translational data are limited, because an appropriate bilirubin formulation for parenteral human use is lacking. Considering its use in both clinical practice and medical research in the past, we explored the feasibility to reintroduce parenteral bilirubin for translational experiments. METHODS: We developed a preparation method in accordance with good manufacturing practice and evaluated the parenteral applicability in healthy volunteers (n = 8). Explorative pharmacokinetic and safety data were compared to the results from a literature study on the former parenteral use of bilirubin. Bilirubin was administered intra-arterially to raise the local plasma concentration in the forearm vascular bed (n = 4) and intravenously to raise the systemic plasma concentration (n = 4). Finally, pharmacokinetic characteristics were studied following a single bolus infusion (n = 3). RESULTS: During parenteral application, no side effects occurred. Adverse events mentioned during the two-week observation period were in general mild and self-limiting. Three more significant adverse events (appendicitis, asymptomatic cardiac arrhythmia and atopic eczema) were judged unrelated by independent physicians. A dose-concentration relationship appeared sufficiently predictable for both intra-arterial and intravenous administration. In line with existing knowledge, bilirubin pharmacokinetics could be described best according to a two-compartment model with a volume of distribution of 9.9 (±2.0) l and a total plasma clearance of 36 (±16) ml per minute. CONCLUSIONS: Supported by previous reports, our data suggest that it is both feasible and safe to perform translational experiments with parenteral albumin bound bilirubin.

Atazanavir-induced unconjugated hyperbilirubinemia prevents vascular hyporeactivity during experimental human endotoxemia.

Objective: Inflammation-induced free radical release is important in the pathogenesis of several diseases, including atherosclerosis and sepsis. Heme oxygenase (HO) breaks down heme into carbon monoxide, iron, and biliverdin. Biliverdin IXα is directly converted to bilirubin by biliverdin reductase. Unconjugated bilirubin is a powerful antioxidant, and elevated levels have beneficial effects in preclinical models and human cardiovascular disease. However, its impact during acute inflammation in humans is unknown. In the present study, we investigated the impact of atazanavir-induced (unconjugated) hyperbilirubinemia on antioxidant capacity, inflammation, and vascular dysfunction in human experimental endotoxemia. Approach and results: Following double-blinded four-day treatment with atazanavir 2dd300 mg (or placebo), twenty healthy male volunteers received 2 ng/kg Escherichia coli lipopolysaccharide intravenously. Blood was drawn to determine the bilirubin levels, antioxidant capacity, and cytokine response. It was demonstrated that following atazanavir treatment, total bilirubin concentrations increased to maximum values of 4.67 (95%CI 3.91-5.59) compared to 0.82 (95%CI 0.64-1.07) mg/dL in the control group (p<0.01). Furthermore, the anti-oxidant capacity, as measured by the ferric-reducing ability of plasma (FRAP), was significantly increased with 36% in hyperbilirubinemia subjects (p<0.0001), and FRAP concentrations correlated strongly to bilirubin concentrations (R2 = 0.77, p<0.001). Hyperbilirubinemia attenuated the release of interleukin-10 from 377 (95%CI 233-609) to 219 (95%CI 152-318) pg/mL (p=0.01), whereas the release of pro-inflammatory cytokines remained unaltered. In vitro, in the absence of hyperbilirubinemia, atazanavir did not influence lipopolysaccharide-induced cytokine release in a whole blood assay. Vascular function was assessed using forearm venous occlusion plethysmography after intra-arterial infusion of acetylcholine and nitroglycerin. Hyperbilirubinemia completely prevented the LPS-associated blunted vascular response to acetylcholine and nitroglycerin. Conclusions: Atazanavir-induced hyperbilirubinemia increases antioxidant capacity, attenuates interleukin-10 release, and prevents vascular hyporesponsiveness during human systemic inflammation elicited by experimental endotoxemia. Clinical trial registration: http://clinicaltrials.gov, identifier NCT00916448.

The bilirubin-increasing drug atazanavir improves endothelial function in patients with type 2 diabetes mellitus.

OBJECTIVE: In type 2 diabetes mellitus (T2DM), oxidative stress gives rise to endothelial dysfunction. Bilirubin, a powerful endogenous antioxidant, significantly attenuates endothelial dysfunction in preclinical experiments. The Gilbert syndrome is accompanied by a mild and lifelong hyperbilirubinemia and associated with only one third of the usual cardiovascular mortality risk. The hyperbilirubinemia caused by atazanavir treatment closely resembles the Gilbert syndrome. We thus hypothesized that treatment with atazanavir would ameliorate oxidative stress and vascular inflammation and improve endothelial function in T2DM. METHODS AND RESULTS: In a double-blind, placebo-controlled crossover design, we induced a moderate hyperbilirubinemia by a 3-day atazanavir treatment in 16 subjects experiencing T2DM. On the fourth day, endothelial function was assessed by venous occlusion plethysmography. Endothelium-dependent and endothelium-independent vasodilation were assessed by intraarterial infusion of acetylcholine and nitroglycerin, respectively. Atazanavir treatment induced an increase in average bilirubin levels from 7 µmol/L (0.4 mg/dL) to 64 µmol/L (3.8 mg/dL). A significant improvement in plasma antioxidant capacity (P<0.001) and endothelium-dependent vasodilation (P=0.036) and a decrease in plasma von Willebrand factor (P=0.052) were observed. CONCLUSIONS: Experimental hyperbilirubinemia is associated with a significant improvement of endothelial function in T2DM.

C1-esterase inhibitor attenuates the inflammatory response during human endotoxemia.

OBJECTIVE: Besides its role in regulation of the complement and contact system, C1-esterase inhibitor has other immunomodulating effects that could prove beneficial in patients with acute inflammation such as during sepsis or after trauma. We examined the immunomodulating properties of C1-esterase inhibitor during human experimental endotoxemia, in which the innate immune system is activated in the absence of activation of the classic complement pathway. DESIGN: Double-blind placebo-controlled study. SETTING: Research intensive care unit of the Radboud University Nijmegen Medical Centre. SUBJECTS: Twenty healthy volunteers. INTERVENTIONS: Intravenous injection of 2 ng/kg Escherichia coli lipopolysaccharide. Thirty minutes thereafter (to prevent binding of lipopolysaccharide), C1-esterase inhibitor concentrate (100 U/kg, n = 10) or placebo (n = 10) was infused. MEASUREMENTS AND MAIN RESULTS: Pro- and anti-inflammatory mediators, markers of endothelial and complement activation, hemodynamics, body temperature, and symptoms were measured. C1-esterase inhibitor reduced the release of proinflammatory cytokines as well as C-reactive protein (peak levels of: interleukin-6 1521 ± 209 vs. 932 ± 174 pg/mL [p = .04], tumor necrosis factor-α 1213 ± 187 vs. 827 ± 167 pg/mL [p = .10], monocyte chemotactic protein-1 6161 ± 1302 vs. 3373 ± 228 pg/mL [p = .03], interleukin-1ß 34 ± 5 vs. 23 ± 2 pg/mL [p < .01], C-reactive protein 39 ± 4 vs. 29 ± 2 mg/L [p = .02]). In contrast, release of the anti-inflammatory cytokine interleukin-10 was increased by C1-esterase inhibitor (peak level 73 ± 11 vs. 121 ± 18 pg/mL, p = .02). The increase in interleukin-1 receptor antagonist tended to be smaller in the C1-esterase inhibitor group, but this effect did not reach statistical significance (p = .07). Markers for endothelial activation were increased after lipopolysaccharide infusion, but no significant differences between groups were observed. The lipopolysaccharide-induced changes in heart rate, blood pressure, body temperature, and symptoms (all p < .001 over time) were not influenced by C1-esterase inhibitor. Complement fragment C4 was not increased after lipopolysaccharide challenge. CONCLUSIONS: This study is the first to demonstrate that C1-esterase inhibitor exerts anti-inflammatory effects in the absence of classic complement activation in humans.

The role of cytokines and inducible nitric oxide synthase in endotoxemia-induced endothelial dysfunction.

Sepsis is characterized by a blunted vascular responses due to impairment of endothelial function. The aim of our study was to assess endothelial function and the role of cytokines and nitric oxide (NO). Endotoxin tolerance was induced in 14 healthy volunteers by intravenous injection of 2 ng.kg.d lipopolysaccharide on 5 consecutive days. Forearm blood flow (FBF) was measured by strain-gauge plethysmography during dose-response curves of endothelium-dependent vasodilator acetylcholine and endothelium-independent vasodilator sodium nitroprusside before and 4 hours after LPS administration on days 1 and 5. In another study, 7 healthy volunteers were given selective inducible NO synthase inhibitor aminoguanidine intravenous continuously from 1 hour after a single LPS administration until 5 hours. FBF showed an attenuation of ACh-induced vasodilatory response with 67% (45%-72%) 4 hours after the first LPS administration (P = 0.01) with an unchanged dose-response curve to sodium nitroprusside. This attenuation to ACh infusion did not occur in the presence of aminoguanidine (P = 0.21) and also did not occur when tolerance was present on day 5 (P = 0.45). Our data demonstrate that endothelial dysfunction caused by endotoxemia does not occur when endotoxin tolerance develops, indicated by the absence of cytokine production and during administration of selective inducible NO synthase inhibitor aminoguanidine in vivo.

In vivo evidence for nitric oxide-mediated calcium-activated potassium-channel activation during human endotoxemia.

BACKGROUND: During septic shock, the vasoconstrictor response to norepinephrine is seriously blunted. Animal experiments suggest that hyperpolarization of smooth muscle cells by opening of potassium (K) channels underlies this phenomenon. In the present study, we examined whether K-channel blockers and/or nitric oxide (NO) synthase inhibition could restore norepinephrine sensitivity during experimental human endotoxemia. METHODS AND RESULTS: Volunteers received 2 ng/kg Escherichia coli endotoxin intravenously. Forearm blood flow (FBF) was measured with venous occlusion plethysmography. Infusion of 4 dose steps of norepinephrine into the brachial artery decreased the FBF ratio (ratio of FBF in the experimental arm to FBF in the control arm) to 84 +/- 4%, 70 +/- 4%, 55 +/- 4%, and 38 +/- 4% (mean +/- SEM) of its baseline value. After endotoxin administration, norepinephrine-induced vasoconstriction was attenuated (FBF ratio, 101 +/- 4%, 92 +/- 4%, 83 +/- 6%, and 56 +/- 7%; n = 30; P = 0.0018; pooled data). Intrabrachial infusion of the K-channel blocker tetraethylammonium (TEA) completely restored the vasoconstrictor response to norepinephrine from 104 +/- 5%, 93 +/- 7%, 93 +/- 12%, and 69 +/- 12% to 89 +/- 9%, 73 +/- 4%, 59 +/- 5%, and 46 +/- 8% (n = 6; P = 0.045). Other K-channel blockers did not affect the response to norepinephrine. The NO synthase inhibitor N(G)-monomethyl-l-arginine (L-NMMA; 0.2 mg x min(-1) x dL(-1) intra-arterially) also restored the norepinephrine sensitivity. In the presence of L-NMMA, TEA did not have an additional effect on the norepinephrine-induced vasoconstriction (n = 6; P = 0.9). CONCLUSIONS: The K-channel blocker TEA restores the attenuated vasoconstrictor response to norepinephrine during experimental human endotoxemia. Coadministration of L-NMMA abolishes this potentiating effect of TEA, suggesting that NO mediates the endotoxin-induced effect on vascular K channels. In the absence of an effect of the selective adenosine triphosphate-dependent K-channel blocker tolbutamide, we conclude that the blunting effect of endotoxin on norepinephrine-induced vasoconstriction is caused by NO-mediated activation of calcium-activated K channels in the vascular wall.

Vascular and metabolic effects of the haem oxygenase-1 inducer haem arginate in subjects with the metabolic syndrome: A translational cross-over study.

This translational randomized and vehicle-controlled cross-over study was performed to assess the impact of haem arginate treatment on haem oxygenase-1 induction, endothelial function and insulin sensitivity in subjects with the metabolic syndrome (n = 14). Both treatment periods consisted of 5 days. Haem arginate or vehicle (l-arginine) was administered intravenously on Days 1 and 3. Forearm blood flow in response to acetylcholine and nitroglycerine was measured by venous occlusion plethysmography (Day 3), insulin sensitivity by a hyperinsulinaemic clamp procedure (Day 5). Haem arginate did not improve endothelial function or insulin sensitivity but significantly reduced the vasodilator response to nitroglycerine (p < 0.01). These negative findings are in contrast to the preclinical data, which may be due to short duration of therapy and limited haem oxygenase-1 induction as well as interference by markedly elevated plasma haem levels observed after haem arginate treatment (p < 0.01). Future studies should pay attention to the delicate balance between sufficient dosing and timely normalization of plasma haem levels.

Iso-osmolar prehydration shifts the cytokine response towards a more anti-inflammatory balance in human endotoxemia.

Clinical experience suggests that the administration of fluids in human endotoxemia reduces symptoms. In the present study, the effects of a standardised fluid protocol on symptoms, inflammatory and hemodynamic parameters in human endotoxemia are determined. With approval of the local ethics committee, 16 healthy volunteers received 2 ng/kg of Escherichia coli endotoxin (O:113). After an overnight fast, nine subjects received 1.5 l of 2.5% glucose/0.45% NaCl the hour prior to the endotoxin administration and 150 ml/h during the course of the experiment ('prehydrated group'). Seven subjects only received a continuous infusion of 75 ml/h during the experiment ('non-prehydrated group'). The course of inflammatory parameters and symptoms were determined and mean arterial pressure, heart rate and forearm blood flow were measured. In the prehydrated group, TNF-alpha increased to 522 +/- 63 pg/ml (mean +/- SEM) while the maximum in the non-prehydrated group was 927 +/- 187 pg/ml (P < 0.04). IL-10 increased similarly in both groups (non-prehydrated 117 +/- 18 pg/ml and prehydrated 99 +/- 18 pg/ml; P = NS). The prehydrated group had a significantly lower (P < 0.004) symptom score and recovered sooner (P = 0.004). Endotoxin-induced changes in hemodynamics revealed no significant differences between groups. We demonstrate that prehydration in experimental human endotoxemia significantly shifts the cytokine balance towards a more anti-inflammatory pattern. This effect is associated with a reduction in symptoms, whereas the changes in hemodynamic parameters are not influenced by prehydration.

IFN-gamma is not induced through increased plasma concentrations of interleukin-12/interleukin-18 during human endotoxemia.

Endotoxin administration to animals and humans is an accepted experimental model of Gram-negative sepsis, and endotoxin is believed to play a major role in triggering the activation of cytokines. In septic patients, the IL-12/IL-18/IFN-gamma axis is activated and correlates with mortality. Our aim was to investigate the effects of endotoxin administration in humans on the activation of the IL-12/IL-18/IFN-gamma axis. Seven healthy volunteers received E. coli endotoxin (O:113). Hemodynamics, temperature and the course of plasma concentrations of TNF-alpha, IL-1beta, IL-12, IL-18 and IFN-gamma were determined. Endotoxin administration resulted in the expected flu-like symptoms, a temperature of 38.8 +/- 0.3(o)C (p < 0.003), a decrease in mean arterial blood pressure of 14.8 +/- 1.8 mmHg (p < 0.0002) and an increase in heart rate of 27.5 +/- 4.8 bpm (p < 0.002) compared to baseline values. TNF-alpha increased from 16.6 +/- 8.2 to 927 +/- 187 pg/mL (p < 0.003). IL-1beta increased from 8.6 +/- 0.5 to 25.3 +/- 2.0 pg/mL (p < 0.0001). IL-12 showed no significant increase (8.2 +/- 0.2 to 9.3 +/- 0.8 pg/mL, p = 0.13), and all IL-18 measurements remained below the level of detection. In contrast, IFN-gamma showed an increase from 106.6 +/- 57.1 to 152.7 +/- 57.8 (p < 0.005). These results indicate that pathways other than the IL-12/IL-18 axis may induce IFN-gamma production in human endotoxemia.

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

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.

Cell-type-specific downregulation of heme oxygenase-1 by lipopolysaccharide via Bach1 in primary human mononuclear cells.

Heme oxygenase (HO)-1 is the inducible isoform of the heme-degrading enzyme HO, which is upregulated by multiple stress stimuli. HO-1 has major immunomodulatory and anti-inflammatory effects via its cell-type-specific functions in mononuclear cells. Contradictory findings have been reported on HO-1 regulation by the Toll-like receptor (TLR) 4 ligand lipopolysaccharide (LPS) in these cells. Therefore, we reinvestigated the effects of LPS on HO-1 gene expression in human and murine mononuclear cells in vitro and in vivo. Remarkably, LPS downregulated HO-1 in primary human peripheral blood mononuclear cells (PBMCs), CD14(+) monocytes, macrophages, dendritic cells, and granulocytes, but upregulated this enzyme in primary murine macrophages and human monocytic leukemia cell lines. Furthermore, experiments with human CD14(+) monocytes revealed that activation of other TLRs including TLR1, -2, -5, -6, -8, and -9 decreased HO-1 mRNA expression. LPS-dependent downregulation of HO-1 was specific, because expression of cyclooxygenase-2, NADP(H)-quinone oxidoreductase-1, and peroxiredoxin-1 was increased under the same experimental conditions. Notably, LPS upregulated expression of Bach1, a critical transcriptional repressor of HO-1. Moreover, knockdown of this nuclear factor enhanced basal and LPS-dependent HO-1 expression in mononuclear cells. Finally, downregulation of HO-1 in response to LPS was confirmed in PBMCs from human individuals subjected to experimental endotoxemia. In conclusion, LPS downregulates HO-1 expression in primary human mononuclear cells via a Bach1-mediated pathway. As LPS-dependent HO-1 regulation is cell-type- and species-specific, experimental findings in cell lines and animal models need careful interpretation.

Body mass index is not associated with cytokine induction during experimental human endotoxemia.

A higher body mass index (BMI) appears to be associated with lower mortality in critically ill patients, possibly explained by an altered innate immune response. However, the precise relationship between BMI and the innate immune response in humans in vivo is unknown. We investigated the relationship between BMI and the systemic cytokine response during experimental human endotoxemia. Endotoxemia was induced in 112 healthy male volunteers by intravenous administration of 2 ng/kg Escherichia coli endotoxin. Plasma concentrations of TNF-α, IL-6, IL-10 and IL-1RA were serially determined. The relationship between BMI and the cytokine response, as well as body temperature, was investigated. The BMIs of the participants ranged from 18.3 to 33.6 kg/m(2), (median: 22.7 kg/m(2)). All participants showed a marked increase in plasma cytokine levels [median (interquartile range)] peak levels: TNF-α 509 (353-673) pg/ml; IL-6 757 (522-1098) pg/ml; IL-10 271 (159-401) pg/ml; IL-1RA 4882 (3927-6025) pg/ml; and an increase in body temperature [1.8(1.4-2.2)] during endotoxemia. No significant correlations were found between BMI and levels of any of the cytokines or body temperature. No relationship between BMI and the cytokine response was found in healthy volunteers subjected to experimental endotoxemia. These data question the relationship between BMI and cytokine responses in critical illness.

Lipopolysaccharide-stimulated whole blood cytokine production does not predict the inflammatory response in human endotoxemia.

A widely applied method to study the activation of the innate immune system is in vitro stimulation of whole blood using lipopolysaccharide (LPS). However, it is unclear if in vitro cytokine production relates to in vivo cytokine levels elicited during experimental endotoxemia or sepsis. To determine the correlation between in vitro cytokine production and the in vivo inflammatory response, blood was obtained from 15 healthy volunteers for in vitro incubation with Escherichia coli LPS, immediately followed by experimental E. coli endotoxemia. Correlations of in vitro and peak in vivo cytokine concentrations were determined using Pearson correlation coefficient. In stimulated whole blood, tumor necrosis factor (TNF)-alpha, Interleukin (IL)-1beta, IL-6, IL-10 and interferon (IFN)-gamma were induced to 279 +/- 53, 392 +/- 64, 5312 +/- 624, 83 +/- 20 and 343 +/- 85 pg/ml, respectively, whereas in vivo cytokine induction led to cytokine levels of 603 +/- 123, 11 +/- 1, 4999 +/- 1228, 167 +/- 25 and 194 +/- 40 pg/ml, respectively. Correlation coefficients between the in vitro and in vivo cytokine concentrations were for TNF-alpha, IL-1beta, IL-6, IL-10 and IFN-gamma -0.10 (P = 0.7), 0.09 (P = 0.8), 0.36 (P = 0.2), 0.19 (P = 0.5) and 0.40 (P = 0.1), respectively. Comparison between in vitro and in vivo stimulation with LPS shows no correlation between the amount of cytokines produced. In vitro cytokine production, therefore, does not predict the in vivo inflammatory response.

Gender differences in the innate immune response and vascular reactivity following the administration of endotoxin to human volunteers.

OBJECTIVE: To determine gender differences in the innate immune response and vascular reactivity during human endotoxemia. DESIGN: Clinical experimental study. SETTING: University medical center intensive care research unit. SUBJECTS: Fifteen female and 15 male volunteers. INTERVENTIONS: Intravenous injection of 2 ng/kg Escherichia coli lipopolysaccharide. MEASUREMENTS AND MAIN RESULTS: C-reactive protein, leukocytes, and cytokines were measured at regular time intervals as indicators of inflammation. Heart rate and blood pressure were continuously monitored. Forearm blood flow and the responsiveness of forearm vessels to the intrabrachial infusion of norepinephrine (1-3-10-30 ng/min/dL) were measured before and 4 hrs after the administration of endotoxin using venous occlusion plethysmography. Differences were tested with repeated-measures analysis of variance. Females showed a more proinflammatory response to lipopolysaccharide than males, illustrated by a higher rise in C-reactive protein (42 +/- 3 vs. 29 +/- 3 mg/L, p = .002) and more leukocyte sequestration (leukopenia 1.8 +/- 0.1 x 10 vs. 2.4 +/- 0.1 x 10, p = .003). The increase in cytokine levels showed a more proinflammatory pattern in females as reflected by a higher increase in tumor necrosis factor-alpha (965 +/- 193 vs. 411 +/- 35 pg/mL, p < .0001), whereas the increase of the anti-inflammatory interleukin-10 was not significantly different (95 +/- 15 pg/mL in females vs. 129 +/- 15 pg/mL in males, p = .288). Females exhibited higher baseline levels (9.9 +/- 1.1 vs. 7.0 +/- 0.8 pg/mL in males, p = .042) and an augmented increase in lipopolysaccharide-binding protein, which may explain the more pronounced inflammatory response in females. The lipopolysaccharide-induced change in heart rate was not significantly different between the genders, whereas blood pressure decreased more in females (p < .0001). Lipopolysaccharide administration significantly attenuated the norepinephrine sensitivity in males (p = .002), whereas no lipopolysaccharide-induced effect was observed in females (p = .552; difference between groups, p = .045). CONCLUSIONS: During experimental human endotoxemia, females showed a more pronounced proinflammatory innate immune response associated with less attenuation of norepinephrine sensitivity. These findings may be relevant in view of the profound and incompletely explained differences in incidence and outcome of sepsis among male and female patients.