Application of genome-wide expression analysis to human health and disease.

The application of genome-wide expression analysis to a large-scale, multicentered program in critically ill patients poses a number of theoretical and technical challenges. We describe here an analytical and organizational approach to a systematic evaluation of the variance associated with genome-wide expression analysis specifically tailored to study human disease. We analyzed sources of variance in genome-wide expression analyses performed with commercial oligonucleotide arrays. In addition, variance in gene expression in human blood leukocytes caused by repeated sampling in the same subject, among different healthy subjects, among different leukocyte subpopulations, and the effect of traumatic injury, were also explored. We report that analytical variance caused by sample processing was acceptably small. Blood leukocyte gene expression in the same individual over a 24-h period was remarkably constant. In contrast, genome-wide expression varied significantly among different subjects and leukocyte subpopulations. Expectedly, traumatic injury induced dramatic changes in apparent gene expression that were greater in magnitude than the analytical noise and interindividual variance. We demonstrate that the development of a nation-wide program for gene expression analysis with careful attention to analytical details can reduce the variance in the clinical setting to a level where patterns of gene expression are informative among different healthy human subjects, and can be studied with confidence in human disease.

Lower levels of whole blood LPS-stimulated cytokine release are associated with poorer clinical outcomes in surgical ICU patients.

BACKGROUND: In vitro pretreatment of human monocytes with lipopolysaccharide (LPS) induces "endotoxin tolerance" with blunted TNF and IL-6 release to rechallenge with LPS. The pro-inflammatory cytokines TNF and IL-6 are important mediators in sepsis. A high IL-6 concentration has been used as a marker of infection severity, but IL-6 may also have beneficial effects as an acute-phase protein. We sought to address two questions: (a) What is the relationship between TNF and IL-6 release? (b) Is the clinical outcome different for intensive care unit (ICU) patients with ex vivo characteristics of endotoxin tolerance (low levels of ex vivo LPS-stimulated cytokine release)? MATERIALS AND METHODS: Heparinized whole blood was obtained from 62 surgical ICU patients and 15 control subjects and incubated for 3 h at 37 degrees C in the presence or absence of 10 ng/mL LPS. Concentrations of TNF and IL-6 were measured in plasma samples using an enzyme-linked immunosorbent assay (pg/mL). Clinical data on ICU length of stay (LOS), ventilator days, white blood cell count (WBC), and documented clinical infection were obtained by chart review. Outcome parameters for patients with low ex vivo LPS-stimulated cytokine release (low = IL-6 < 3000 pg/mL and TNF <2100 pg/mL) were compared to patients with Normal/High concentrations of cytokines. RESULTS: Cytokines were essentially undetectable in ICU patients or controls without LPS stimulation, however a range of values was measured for LPS-stimulated release in both ICU patients (IL-6, 7847 +/- 857 pg/mL; TNF, 4390 +/- 457 pg/mL) and controls (IL-6, 7704 +/- 793 pg/mL; TNF, 6706 +/- 715 pg/mL). There were no differences in age between High/Normal concentrations of cytokines compared to the Low cytokine group, however there were significant differences in WBC, cytokine concentrations, ICU LOS, incidence of clinical infection, and mortality. The Low group also required an average of 6.9 more days of mechanical ventilation (p < 0.05). LPS-stimulated TNF release seemed to correlate better with the observed mortality than did IL-6 release. CONCLUSION: The data suggest that ICU patients with characteristics of endotoxin tolerance (low LPS-stimulated cytokine release capacity) have significantly poorer clinical outcomes. Ex vivo LPS-stimulated whole blood cytokine production may be useful to identify ICU patients with severe sepsis.

Relative hyperoxia augments lipopolysaccharide-stimulated cytokine secretion by murine macrophages.

BACKGROUND: Increased systemic levels of inflammatory mediators are seen after open abdominal operations. Macrophages that are exposed to lipopolysaccharide secrete cytokines. Peritoneal macrophages normally reside in a pO(2) of 40 mm Hg. We hypothesize that exposure of lipopolysaccharide-stimulated macrophages to "non-physiologic" pO(2) augments cytokine secretion. METHOD: Murine macrophages were preconditioned to a pO(2) of 40 mm Hg for 24 hours. The medium then was discarded and exchanged for a medium containing a pO(2) of 40, 150, or 440 mm Hg. Macrophages were incubated in the desired pO(2) for 6 and 24 hours while stimulated with lipopolysaccharide (0 to 100 ng/mL). The effect of pO(2) was compared. Supernatant tumor necrosis factor (TNF) and interleukin-6 were measured with enzyme-linked immunosorbent assay. Statistics were performed with analysis of variance. RESULTS: We found dose-dependent lipopolysaccharide-stimulated TNF and interleukin-6 production with macrophages incubated at physiologic pO(2). Higher pO(2) did not stimulate TNF and interleukin-6 in the absence of lipopolysaccharide. However, a pO(2) of 150 and 440 mm Hg significantly (P <.05) increased lipopolysaccharide-stimulated TNF and interleukin-6 production versus 45 mm Hg. CONCLUSION: Our data suggest synergy between increased pO(2) and lipopolysaccharide for macrophage TNF and interleukin-6 production. Similar pO(2) elevations may occur with an open peritoneum or high supplemental O(2). Cytokines from peritoneal macrophages may contribute to the increased systemic inflammation after open operations.

Evidence for a CD14- and serum-independent pathway in the induction of endotoxin-tolerance in human monocytes and THP-1 monocytic cells.

Lipopolysaccharide (LPS) stimulation of macrophages or monocytes is believed to occur via a serum- and CD14-dependent signaling pathway via toll-like receptor 4 (TLR4). We sought to determine whether serum and/or CD14 are required for LPS to induce the endotoxin-tolerant state in human monocytes. LPS treatments were performed in the presence or absence of an anti-CD14 monoclonal antibody and with or without fetal bovine serum. Endotoxin tolerance was assessed after an 18-h exposure (pretreatment) to 10 ng/mL of LPS. Medium was discarded and cells were challenged with activating (1-1000 ng/mL) doses of LPS. LPS-stimulated tumor necrosis factor (TNF) secretion into culture supernatants was determined after 5 h by ELISA and p44/p42 ERK kinase activation was measured after 30 min by Western blot. Statistical analysis was by ANOVA. LPS induced endotoxin-tolerance with a significant inhibition of LPS-stimulated TNF secretion and less p44/p42 ERK kinase activation. When LPS-stimulation of naïve (nontolerant) monocytes was performed in medium with anti-CD14 antibody or without serum, there was marked blunting of TNF release. However, LPS pretreatment in medium without serum or in medium containing anti-CD14 antibody resulted in changes in monocyte activation and function characteristic of endotoxin tolerance. LPS-stimulated p44/p42 ERK kinase activation and TNF release were diminished whether or not anti-CD14 antibody was present during LPS pretreatment. LPS-stimulated TNF secretion and p44/p42 ERK kinase activation require the presence of serum and are inhibited by anti-CD14 antibody. Our findings suggest that LPS induces endotoxin tolerance in human monocytic cells via a pathway that does not require serum or cell surface CD14.

Endotoxin tolerance: A review.

Endotoxin tolerance was initially described when it was observed that animals survived a lethal dose of bacterial endotoxin if they had been previously treated with a sublethal injection. In animal models, two phases of endotoxin tolerance are described, an early phase associated with altered cellular activation and a late phase associated with the development of specific antibodies against the polysaccharide side chain of Gram-negative organisms. Recently, there has been a tremendous resurgence of interest in the mechanisms responsible for altered responsiveness to bacterial endotoxin. Host immune cells, particularly macrophages and monocytes, that are exposed to endotoxin for 3 to 24 hrs are rendered "tolerant" and manifest a profoundly altered response when rechallenged with bacterial endotoxin or lipopolysaccharide. The "lipopolysaccharide-tolerant" phenotype is characterized by inhibition of lipopolysaccharide-stimulated tumor necrosis factor production, altered interleukin-1 and interleukin-6 release, enhanced cyclooxygenase-2 activation, inhibition of mitogen-activated protein kinase activation, and impaired nuclear factor-kappaB translocation. Human monocytes and macrophages can be induced to become tolerant, and there is increasing evidence that monocytic cells from patients with systemic inflammatory response syndrome and sepsis have many characteristics of endotoxin tolerance.

Endotoxin tolerance: a review.

Endotoxin tolerance was initially described when it was observed that animals survived a lethal dose of bacterial endotoxin if they had been previously treated with a sublethal injection. In animal models, two phases of endotoxin tolerance are described, an early phase associated with altered cellular activation and a late phase associated with the development of specific antibodies against the polysaccharide side chain of Gram-negative organisms. Recently, there has been a tremendous resurgence of interest in the mechanisms responsible for altered responsiveness to bacterial endotoxin. Host immune cells, particularly macrophages and monocytes, that are exposed to endotoxin for 3 to 24 hrs are rendered "tolerant" and manifest a profoundly altered response when rechallenged with bacterial endotoxin or lipopolysaccharide. The "lipopolysaccharide-tolerant" phenotype is characterized by inhibition of lipopolysaccharide-stimulated tumor necrosis factor production, altered interleukin-1 and interleukin-6 release, enhanced cyclooxygenase-2 activation, inhibition of mitogen-activated protein kinase activation, and impaired nuclear factor-kappa B translocation. Human monocytes and macrophages can be induced to become tolerant, and there is increasing evidence that monocytic cells from patients with systemic inflammatory response syndrome and sepsis have many characteristics of endotoxin tolerance.