Mirrin J. Dorresteijn

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 × 109 vs. 2.4 ± 0.1 × 109, p = .003). The increase in cytokine levels showed a more proinflammatory pattern in females as reflected by a higher increase in tumor necrosis factor-&agr; (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.

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.

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−1·d−1 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.

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.

0730. Plasma endocan levels are associated with endothelial dysfunction during experimental human endotoxemia

The endothelium plays a central role in pathophysiology of sepsis. Systemic inflammation results in endothelial dysfunction, contributing to the development of shock and multiple organ dysfunction. However, an easy to obtain, early, and clinically applicable marker of endothelial dysfunction is currently not available. Such a marker could guide early and targeted therapy such as selective iNOS inhibition. Endocan is a soluble proteoglycan secreted by endothelial cells in response to pro-inflammatory cytokines, bacterial endotoxins, and angiogenic factors, and enhances microvascular permeability. Plasma endocan levels are increased in septic patients, and have been shown to correlate with sepsis severity and mortality. However, the direct relationship between endocan and endothelial function has not yet been investigated in humans in vivo.