Wyrta Heagy

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-&kgr;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.

Impaired ex vivo lipopolysaccharide-stimulated whole blood tumor necrosis factor production may identify "septic" intensive care unit patients.

Currently, there is no reliable diagnostic test to identify septic intensive care unit (ICU) patients. We initiated studies to test the hypothesis that in sepsis, the in vivo exposure to endotoxin is detectable by the ex vivo analysis of lipopolysaccharide (LPS)-stimulated tumor necrosis factor (TNF) production. We obtained heparinized whole blood (WB) from 58 ICU patients and 14 healthy controls. The samples were incubated +/-10 ng/mL of LPS at 37 degrees C for 3 h. Plasma TNF levels were measured using enzyme-linked immunoassay (mean +/- standard error of the mean). Clinical data, including ICU length of stay (LOS), ventilator days (VentD), WBC, and positive cultures (Clt+), were obtained retrospectively. A wide range of LPS-stimulated WB TNF production (pg/mL) was observed in ICU patients (4481+/-469) and controls (6706+/-715). Patients were stratified into quartiles (I-IV) on the basis of the distribution of plotted LPS-stimulated TNF values (pg/mL). Patients in quartile I (N = 14) had significantly lower TNF production (< 2000 pg/mL, P < 0.05) and required increased VentD (16 vs. 10 days, P < 0.05) compared to quartiles II-IV (N = 44). Patients in quartile I also had a higher incidence of infection (79 vs. 50%) and longer LOS (18 vs. 13 d) compared to quartiles II-IV. Impaired TNF release may be a manifestation of monocyte endotoxin tolerance and may be useful to diagnose sepsis.

Impaired mitogen-activated protein kinase activation and altered cytokine secretion in endotoxin-tolerant human monocytes.

BACKGROUND Dysregulation of monocyte/macrophage cytokine production after exposure to multiple inflammatory stimuli may contribute to multiple organ failure and sepsis. Endotoxin (lipopolysaccharide [LPS]) activation of murine macrophage results in the phosphorylation of kinases in the mitogen-activated protein kinase cascade. Pretreatment of murine macrophages with LPS induces LPS-tolerance, with inhibition of LPS-stimulated activation of kinases (ERK1/2 and p38) and diminished release of tumor necrosis factor (TNF). We sought to determine whether similar alterations in LPS-dependent signal transduction are present in LPS-tolerant human peripheral blood monocytes. METHODS Human peripheral blood monocytes from healthy volunteer donors (n = 12) were incubated in RPMI 1640 culture medium +/- 10 ng/mL of LPS for 18 hours, then stimulated with 0 to 1,000 ng/mL of LPS. Supernatant TNF and interleukin-1 (IL-1) levels were measured after 5 hours by enzyme-linked immunosorbent assay. Activation of the p42/p44 kinases (ERK1/2) was measured 15 minutes after LPS with monoclonal antibodies to diphosphorylated (active) ERK1/2 using novel flow cytometric methods. RESULTS LPS-tolerant (10 ng/mL LPS pretreatment) human monocytes had significant inhibition of LPS-stimulated TNF secretion but augmented IL-1 release (p < 0.05). Nontolerant human monocytes had a dramatic increase in the percentage of ERK1/2-positive cells in response to an initial stimulation with LPS. This did not occur in the LPS-tolerant cells. Phorbol-12-myristate-13 acetate restored ERK1/2 activation in LPS-tolerant human monocytes. CONCLUSION LPS-tolerance in human monocytes is associated with inhibition of LPS-stimulated TNF secretion, augmented release of IL-1, and defective activation of mitogen-activated protein kinase cascade (ERK1/2). These results suggest a method of identifying LPS-tolerance and monocyte dysfunction in patients with sepsis.

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.