Catherine Fitting

Dysregulation of in vitro cytokine production by monocytes during sepsis.

The production by monocytes of interleukin-1 alpha (IL-1 alpha), interleukin-1 beta (IL-1 beta), interleukin-6 (IL-6), and tumor necrosis factor alpha (TNF alpha) in intensive care unit (ICU) patients with sepsis syndrome (n = 23) or noninfectious shock (n = 6) is reported. Plasma cytokines, cell-associated cytokines within freshly isolated monocytes and LPS-induced in vitro cytokine production were assessed at admission and at regular intervals during ICU stay. TNF alpha and IL-6 were the most frequently detected circulating cytokines. Despite the fact that IL-1 alpha is the main cytokine found within monocytes upon in vitro activation of cells from healthy individuals, it was very rarely detected within freshly isolated monocytes from septic patients, and levels of cell-associated IL-1 beta were lower than those of TNF alpha. Cell-associated IL-1 beta and TNF alpha were not correlated with corresponding levels in plasma. Upon LPS stimulation, we observed a profound decrease of in vitro IL-1 alpha production by monocytes in all patients, and of IL-1 beta, IL-6, and TNF alpha in septic patients. This reduced LPS-induced production of cytokines was most pronounced in patients with gram-negative infections. Finally, monocytes from survival patients, but not from nonsurvival ones recovered their capacity to produce normal amounts of cytokines upon LPS stimulation. In conclusion, our data indicate an in vivo activation of circulating monocytes during sepsis as well as in noninfectious shock and suggest that complex regulatory mechanisms can downregulate the production of cytokines by monocytes during severe infections.

Successful Cardiopulmonary Resuscitation After Cardiac Arrest as a “Sepsis-Like” Syndrome

Background—We investigated the immunoinflammatory profile of patients successfully resuscitated after cardiac arrest, representing a model of whole-body ischemia/reperfusion syndrome. Methods and Results—Plasma cytokine, endotoxin, and ex vivo cytokine production in whole-blood assays was assessed in 61, 35, and 11 patients, respectively. On admission, high levels of plasma interleukin (IL)-6, IL-8, IL-10, and soluble tumor necrosis factor (TNF) receptor type II could discriminate between survivors and nonsurvivors. Among nonsurvivors, the initial need for a vasopressor agent was associated with higher levels of IL-1 receptor antagonist, IL-10, and IL-6 on day 1. Plasma endotoxin was detected in 46% of the analyzed patients within the 2 first days. Endotoxin-induced TNF and IL-6 productions were dramatically impaired in these patients compared with healthy control subjects, whereas an unaltered production was observed with heat-killed Staphylococcus aureus. In contrast, IL-1 receptor antagonist productions were enhanced in these patients compared with healthy control subjects. The productions of T-cell–derived IL-10 and interferon-&ggr; were also impaired in these patients. Finally, using in vitro plasma exchange between healthy control subjects and patients, we demonstrated that the endotoxin-dependent hyporeactivity was an intrinsic property of patients’ leukocytes and that an immunosuppressive activity was also present in their plasma. Conclusions—Altogether, the high levels of circulating cytokines, the presence of endotoxin in plasma, and the dysregulated production of cytokines found in these patients recall the immunological profile found in patients with sepsis.

Synergistic stimulation of human monocytes and dendritic cells by Toll-like receptor 4 and NOD1- and NOD2-activating agonists

Muropeptides are degradation products of bacterial peptidoglycan (PG) sensed by nucleotide‐binding oligomerization domain 1 (NOD1) and NOD2, members of a recently discovered family of pattern recognition molecules (PRM). One of these muropeptides, muramyl dipeptide (MDP) mediates cell signaling by NOD2, exerts adjuvant activity and synergizes with lipopolysaccharide (LPS) to induce pro‐inflammatory responses in vitro and in vivo. In contrast, few and contradictory results exist about the stimulatory capacity of NOD1 agonists. Thus, the ability of NOD1 (MurNAc‐L‐Ala‐γ‐D‐Glu‐meso‐diaminopimelic acid, MtriDAP) and NOD2 (MurNAc‐L‐Ala‐D‐isoGln, MDP; MurNAc‐L‐Ala‐γ‐D‐Glu‐L‐Lys, MtriLYS) agonists to activate primary human myeloid cells was examined. We show that both CD14+ monocytes and CD1a+ immature dendritic cells (DC) express NOD1 and NOD2 mRNA. Stimulation of primary human monocytes and DC with highly purified muropeptides (MtriDAP, MDP and MtriLYS) induces release of pro‐inflammatory cytokines. We reveal here that NOD1 as well as NOD2 agonists act cooperatively with LPS to stimulate the release of both pro‐ and anti‐inflammatory cytokines in these myeloid cell subsets. Finally, we report that NOD1 as well as NOD2 agonists synergize with sub‐active doses of LPS to induce DC maturation, demonstrating that NOD agonists act cooperatively with molecules sensed by Toll‐like receptor 4 to instruct the onset of adaptive immune responses.

Circulating interleukin-8 concentrations in patients with multiple organ failure of septic and nonseptic origin.

ObjectivesInterleukin (IL)-8, a pro-inflammatory cytokine, is a potent chemoattractant factor and an activator of neutrophils produced by many cell types after stimulation by IL-1, tumor necrosis factor (TNF), or microbial products such as endotoxins. We investigated whether the presence of measurable IL-8 in plasma was associated with the clinical status of severely ill septic or nonseptic patients susceptible to the development of multiple organ failure. DesignCohort study. SettingA collaborative study between an intensive care unit and a research laboratory. SubjectsCirculating IL-8 concentrations were measured in the plasma of 27 patients with sepsis syndrome and in 16 patients with noninfectious shock because these two conditions put patients at risk for the development of multiple organ failure. Sixteen of 27 patients with severe infection and 13 of 16 patients with noninfectious pathologies developed multiple organ failure. Measurements and Main ResultsA specific enzyme-linked immunosorbent assay (ELISA) for IL-8 was set up with a monoclonal and a rabbit polyclonal antihuman IL-8 using a sandwich technique. High concentrations of circulating IL-8 were found in the plasma of patients with sepsis syndrome. Among septic patients, a significant difference was observed between concentrations of IL-8 in survivors (n = 16) and nonsurvivors (n = 11) (81 pL 13 pg/mL vs. 3326 pL 1219 pg/mL, respectively; p = .001). A correlation was noticed between plasma IL-8 and IL-6 concentrations (r2 = .42; p = .001), while no correlation was observed between IL-8 and TNF-aP values, or between IL-8 and IL-1β. Although the mortality rate of nonseptic, multiple organ failure patients was 92%, low plasma concentrations of IL-8 were found (78 pL 34 pg/mL), while high plasma concentrations were measured in septic, multiple organ failure patients (mortality rate 69%) who were sampled at a similar stage. By contrast, increased IL-6 values were observed in both septic and nonseptic, multiple organ failure patients. ConclusionsIn septic patients, high amounts of circulating IL-8 concentrations correlate with fatal outcome, whereas only low plasma concentrations of IL-8 are present in patients with nonseptic, multiple organ failure. This finding suggests that the signals involved in the exacerbation of IL-8 production are different, depending on infectious or noninfectious etiology. (Crit Care Med 1994; 22:673–679)

Interleukin 1 is released by murine macrophages during apoptosis induced by Shigella flexneri.

Peritoneal macrophages undergoing apoptosis induced by Shigella flexneri infection release the inflammatory cytokine interleukin 1 (IL-1), but not IL-6 or tumor necrosis factor alpha (TNF alpha). Wild type shigella causes a very fast and significant release of IL-1 from prestimulated peritoneal macrophages, before the cells integrity is compromised. Both IL-1 alpha and IL-1 beta are released, IL-1 beta in its mature processed form. IL-1 is released from presynthesized cytoplasmic pools. These results demonstrate that bacteria-induced apoptosis of macrophages may play an active role in vivo by releasing IL-1, which in turn mediates an early inflammatory response in epithelial tissues.

Endotoxin tolerance: is there a clinical relevance?

Beeson (1946) first defined endotoxin tolerance as a reduced endotoxin-induced fever following repeated injections of typhoid vaccine. Freudenberg and Galanos (1988) demonstrated that endotoxin tolerance that can protect against a lethal challenge of lipopolysaccharide (LPS) involves the participation of macrophages. Evans and Zuckerman (1991) reported a role for glucocorticoids in endotoxin tolerance. Prostaglandins, interleukin-(IL-)10, and transforming growth factor-β are other players of in vivo endotoxin tolerance. Dramatic reduction of plasma tumor necrosis factor (TNF) (Mathison et al. 1990) and other cytokines in response to LPS parallels endotoxin tolerance. The reduced capacity to produce TNF and other cytokines can be mimicked in vitro by pretreatment of monocytes or macrophages with LPS. It is not a specific phenomenon and can be induced by other agents or events. Cross-tolerance between LPS, TLR2 specific ligands, IL-1 and TNF has been regularly reported. A similar loss of LPS-reactivity has been repeatedly reported in leukocytes of septic patients and in patients with non-infectious systemic inflammation response syndrome (SIRS; e.g . surgery, trauma, cardiac arrest and resuscitation, etc.). Studies on cellular signaling within leukocytes from septic and SIRS patients reveal numerous alterations of the activation pathways reminiscent of those observed in endotoxin-tolerant cells. While endotoxin tolerance prevents severity of infections and ischemia-reperfusion damage, it has been suggested that the immune dysregulation observed in SIRS patients was associated with an enhanced sensitivity to nosocomial infections. In conclusion, in vitro and in vivo endotoxin tolerance, either experimental or due to clinical status, are similar but not identical.

Resilience to Bacterial Infection: Difference between Species Could Be Due to Proteins in Serum

Vertebrates vary in resistance and resilience to infectious diseases, and the mechanisms that regulate the trade-off between these often opposing protective processes are not well understood. Variability in the sensitivity of species to the induction of damaging inflammation in response to equivalent pathogen loads (resilience) complicates the use of animal models that reflect human disease. We found that induction of proinflammatory cytokines from macrophages in response to inflammatory stimuli in vitro is regulated by proteins in the sera of species in inverse proportion to their in vivo resilience to lethal doses of bacterial lipopolysaccharide over a range of 10,000-fold. This finding suggests that proteins in serum rather than intrinsic cellular differences may play a role in regulating variations in resilience to microbe-associated molecular patterns between species. The involvement of circulating proteins as key molecules raises hope that the process might be manipulated to create better animal models and potentially new drug targets.

Variable estimates of cytokine levels produced by commercial ELISA kits: results using international cytokine standards

Commercially available ELISA kits now make it possible to measure cytokines in biological samples and cell culture supernatants. We have compared the levels of IL-1 beta, IL-6, IL-8 and TNF-alpha in various pathological plasma and synovial fluids, and in supernatants of human monocytes activated by lipopolysaccharide (LPS). Measurements were performed using ELISA kits from different companies. A wide variation in values was obtained when measurements were deduced from the standard curves formed with the standard provided by the manufacturers. We also performed calibration curves for all ELISA kits, using the international standards provided by the NIBSC (UK). The coefficients of variation were then significantly improved for IL-6 and IL-8 measurements but not for IL-1 beta and TNF alpha assays. However, despite this attempt to obtain uniform measurements, none of the kits gave similar values for individual samples. These results suggest that the nature of the different pairs of monoclonal antibodies employed in each ELISA does not permit comparable recognition of cytokines in samples. Further work with the various kits is required to establish whether (i) denaturation of the recognized epitope within the natural cytokine, (ii) fragmentation of the cytokine following enzymatic cleavage, (iii) depolymerization, (iv) binding of cytokines to undefined ligands, (v) variable glycosylation of the natural cytokines (vi) recognition of precursor forms, interferes with the measurements.

Immunodepression in sepsis and SIRS assessed by ex vivo cytokine production is not a generalized phenomenon: a review.

Sepsis and non-infectious systemic inflammatory response syndrome (SIRS) are paradoxically associated with an exacerbated production of cytokines, as assessed by their presence in biological fluids, and a diminished ability of circulating leukocytes to produce cytokine upon in vitro activation. In this review, we depict that the observed cellular hyporeactivity is not a global phenomenon and that some signalling pathways are unaltered and allow the cells to respond normally to certain stimuli. Furthermore, we illustrate that during sepsis and SIRS, cells derived from tissues are either fully responsive to ex vivo stimuli or even primed, in contrast to cells derived from hematopoietic compartments (blood, spleen, etc.) which are hyporeactive. In addition to cytokine production, nuclear factor-κB (NF-κB) status within leukocytes can be used as a useful marker of hypo- or hyper-reactivity. We illustrate that the immune-depression reported in sepsis and SIRS patients, often revealed by a diminished capacity of leukocytes to respond to lipopolysaccharide, is not a generalized phenomenon and that SIRS is associated with a compartmentalized responsiveness which involves either anergic or primed cells.

Endotoxin tolerance is not a LPS-specific phenomenon: partial mimicry with IL-1, IL-10 and TGFβ

Administration of non-lethal doses of lipopolysaccharide (LPS) to experimental animals and humans results for a short period of time in a state of hyporesponsiveness to a second LPS challenge. This phenomenon, known as endotoxin tolerance, has been reproduced in vitro using human monocytes, rendered endotoxin-tolerant following a first incubation with LPS. A further activation by LPS was manifested by a dramatically reduced production of tumor necrosis factor α (TNFα). We report this low responsiveness of LPS pretreated monocytes as an endotoxin non-specific phenomenon. Indeed, TNFα release upon further activation with either killed Gram-positive bacteria (Staphylococci, Streptococci) or zymosan was also significantly diminished. This was not the case when phorbol myristate acetate (PMA) was used as a second triggering agent, suggesting that the monocyte hyporesponsiveness due to LPS does not affect all activation pathways, particularly that of protein kinase C. On the other hand, both PMA and zymosan pretreatment could reduce a further activation of monocytes by LPS. We investigated whether the first signal(s) delivered by LPS, could be mimicked by some of the LPS-induced cytokines. Preincubation of monocytes with either interleukin-1 (IL-1), IL-10 or transforming growth factor β (TGFβ) lower the TNFα production upon further activation with LPS. None of the cytokines alone was as efficient as the LPS molecule, but high levels of tolerization were obtained with combination of IL-1, IL-10 and TGFβ. Neither IL-6, IL-8 nor TNFα led to hyporeactive cells. Our data indicate that endotoxin tolerance is not an LPS-specific phenomenon and that more than one cytokine can contribute to render human monocytes hyporeactive to further activation by LPS.