- Split View
-
Views
-
Cite
Cite
Edward Abraham, Alterations in Cell Signaling in Sepsis, Clinical Infectious Diseases, Volume 41, Issue Supplement_7, November 2005, Pages S459–S464, https://doi.org/10.1086/431997
- Share Icon Share
Abstract
Multiple intracellular signaling pathways involving kinases, transcriptional factors, and the expression of immunoregulatory mediators are altered in sepsis. Recent data have shown stable patterns of activation among peripheral blood mononuclear cells and neutrophils in healthy human subjects. Although polymorphisms in Toll-like receptors play a contributory role in determining cellular activation, other factors are involved as well. Increased activation of the mitogen-activated protein kinase protein 38, Akt, and nuclear factor (NF)–κB in neutrophils and other cell populations obtained at early time points in the clinical course of sepsis-induced acute lung injury or after accidental trauma is associated with a more-severe clinical course, suggesting that a proinflammatory cellular phenotype contributes to organ system dysfunction in such settings. Identification of patients with cellular phenotypes characterized by increased activation of NF-κB, Akt, and protein 38, as well as discrete patterns of gene activation, may permit identification of patients with sepsis who are likely to have a worse clinical outcome, thereby permitting early institution of therapies that modulate deleterious signaling pathways before organ system dysfunction develops, reducing morbidity and improving survival.
Multiple intracellular signaling pathways involving kinases, transcriptional factors, and the expression of immunoregulatory mediators are altered in cell populations that contribute to organ system dysfunction and mortality in sepsis. Activation of such intracellular events is initiated by interaction of microbial products with Toll-like receptors (TLRs) and other receptors, including protein—coupled receptors. In addition, circulating and locally released mediators of inflammation, including cytokines, complement fragments, and components of activated coagulation and fibrinolytic systems, that are generated in increased amounts during severe infection also interact with membrane-based receptors, leading to activation of intracellular path ways capable of further accelerating proinflammatory cascades. For example, the expression of cytokines such as TNF-α and IL-1β is increased insepsis, and engagement of TNF-α with type I(p55) and type II(p75) TNF receptors orIL-1β with IL-1 receptors belonging to the TLR/IL-1 receptor family produces activation of kinases (including Src, p38, extracellular signal—regulated kinase, and phosphoinositide 3–kinase) and transcriptional factors (such asnuclear factor [NF]–κB) important for further up-regulation of inflammatory proteins [1–9]. Similarly, association of complement fragments, such as C5a, or fibrinolytic molecules, such asurokinase plasminogen activator, with their receptors has potent proinflammatory effects [10–13].
Circulating and organ-specific cell populations are activated to produce proinflammatory mediators during sepsis. Neutrophils and PBMCs bear TLR2 and TLR4, as well as other receptors, such as protein —coupled receptor, that induce increased generation of cytokines and other immuno regulatory proteins, as well as enhance release of proinflammatory mediators, including reactive oxygen species, after engagement by microbial products from bacteria, fungi, and viruses[14–16]. Similar receptors and activation patterns are demonstrated in fixed tissue populations, such as macrophages and endothelial and epithelial cells [7, 17–20].
Genetic polymorphisms lead to alterations in TLR conformation that are accompanied by decreased cellular activation after exposure to bacterial products. In mice, mutations in tlr4, as present in the C3H/HeJ strain, are associated with resistance to challenge with endotox in[19, 21, 22]. Approximately 6% of humans have amino acid substitutions inTLR4 at As p299Gly and Thr399Ile that render them hyporesponsive to inhaled lipopolysaccharide(LPS) [23–26]. These TLR4polymorphisms also appeared to be associated with a predisposition to gram-negative bacteremia and septic shock but did not demonstrate any survival advantage or disadvantage in patients with sepsis[25, 27, 28]. However, such genetic alterations in TLR and other receptors appear to account for only a relatively small percentage of the variability demonstrated in humans when their cells are exposed to bacterial products.
Recent data have shown relatively stable patterns of cytokine production in PBMCs from some healthy humans after stimulation with LPS [29]. In that study, volunteers were identified who consistently demonstrated either decreased or increased release of TNF-α and other cytokines when their blood was cultured with LPS. We have found similar patterns of high or low response among neutrophils obtained from human subjects and stimulated with LPS, a TLR 4 lig and, peptidogly can, which interacts with TLR 2, or high mobility group box protein —1 (HMGB1), a dual TLR 2 and TLR 4 ligand. Similarly, in gene array experiments, we have also demon strated stable patterns of gene expression over time in neutrophils obtained from healthy volunteers.
Despite the stable variability in cellular activation that is present among the genetically heterogeneous human population, only a limited number of studies have examined how such patterns may correlate with clinical outcome inseps is or other critical illnesses in which the risk of organ dysfunction due to severe infection is high. Examination of such issues is hampered, in part, by the difficulty in obtaining cells for analysis at early time points in a patient' clinical course before pathophysiological events, such as hypotension or severe hypoxemia, intercede and provide clear indications of subsequent outcome. Nevertheless, a number of studies have examined the transcriptional factor NF-κB and kinases, including p38 and Akt, and provide insights into how heterogeneity in cell signaling may contribute to subsequent clinical course.
NF-kB
The transcriptional regulatory factor NF-κBis a central participant in modulating the expression of many immuno regulatory mediators involved in the acute inflammatory response [30–35]. NF-κB/rel transcription factors function as dimers held latently in the cytoplasm of cells by inhibitory IκB proteins[3, 6, 30, 31,34–39]. There are 5 known mammalian NF-κB/Rel proteins: Rel (c-Rel), p 65 (RelA), RelB, p 50, and p 52. Signaling pathways initiated by engagement of TLRs, such as TLR 2 and TLR 4, by microbial products and other inflammatory mediators lead to nuclear accumulation of NF-κB and enhanced transcription of genes responsible for the expression of cytokines, chemokines, adhesion molecules, and other mediators of the inflammatory response associated with infection. Association of NF-κB with the inhibitory protein κB-α in the cytoplasm blocks the nuclear localization sequence of NF-κB, inhibiting its movement into the nucleus [40]. Exposure of cells to inflammatory stimuli, including LPS, peptidogly can, and proinflammatory cytokines (such asTNF-α or IL-1β), results in phosphorylation of IκB-αon serines 32 and 36, leading to its subsequent ubiquitination and degradation by the 26S proteasome. Phosphorylation of IκB-α is mediated by the kinasesIKKα and IKKβ, which are catalytically active components of the IκB kinase complex (IKK) [34]. Upstream kinases that participate inthe phosphorylation and activation of IKK include those that are directly associated with TLRs, such as IL- 1 receptor —associated kinase (IRAK)- 1 and IRAK- 4, as well as kinases that can be activated through either TLR or other receptors, including p 38 and Akt [5, 6, 41–43]. Phosphorylation events, in addition to those involving IKKα/β andIκB-α, and involving NF-κB subunits (such as p 65)and nuclear coactivator proteins(such as TATA box binding protein or cAMP-responsive element—binding protein) are mediated by p 38, Akt, and other kinases and play an important role in regulating the transcriptional activity of NF-κB [44–47]. IκB-αalso is important interminating NF-κB–mediated transcription through migrating into the nucleus and associating with NF-κB,with this interaction exposing a nuclear export signal, leading to movement of NF-κB from the nucleus back to the cytoplasm.
Studies have shown that greater nuclear accumulation of NF-κBin PBMCs is accompanied by higher mortality and worse clinical course in patients with sepsis (figure 1)[48, 49]. In general, these clinical series demonstrated that persistent activation of NF-κB was found in nonsurvivors, with surviving patients having lower nuclear concentrations of NF-κB at early time points in their septic course than did nonsurvivors as well as more rapid return of nuclear accumulation of NF-κB to levels found in control, unactivated PBMCs.
Although NF-κB heterodimers consisting of the p 50:p 65 subunits are capable of trans activating genes when bound to κB promoter sequences, p 50:p 50 homodimers appear to be inhibitory and capable of blocking transcription. Endotoxin tolerance was shown to be dependent on p 50 expression, because cells from p 50-deficient mice could not be made tolerant to LPS [50]. Given the inhibitory properties of p 50:p 50 homodimers, it is interesting that nuclear concentrations of this NF-κB homodimer are increased in PBMCs from nonsurvivors of seps is relative to survivors [51]. Similarly, nonsurvivors demonstrated significantly decreased p 65;p 50/p50:p50 ratios in PBMCs, compared with those in survivors.
Although studies of patients with seps is have generally shown that nuclear concentrations of NF-κB are higher in non survivors than in survivors, an unresolved issue is whether such changes occur early and, there fore, define the subsequent course of sepsis or whether pathophysiological changes that result in poor clinical outcome also produce NF-κB activation as a secondary event, so that such changes in NF-κB are simply associated with more severe organ system dysfunction but do not contribute directly to outcome. In preclinical models of sepsis, blockade of NF-κB improves survival, indicating that NF-κB activation does contribute to cellular and organ dys function [36,52, 53]. A study of surgical patients without sepsis supports the hypothesis that neutrophil phenotypes defined by NF-κB activation patterns predict clinical outcome [54]. In that clinical series of patients undergoing repair of aortic aneurysms, higher preoperative levels of NF-κBin peripheral neutrophils were associated with death and with the development of post operative organ dysfunction [54].
Recent data from our laboratory support the contention that early alterations in NF-κB contribute to the severity of organ failure and clinical outcome [55]. In that study, peripheral blood neutrophils were collected with in the first 24 h of intubation from patients with sepsis-induced acute lung injury. Increased nuclear levels of NF-κB in unstimulated neutrophils were associated with a worse clinical outcome, as defined by death or mechanical ventilation for > 14 days, compared with surviving patients who required mechanical ventilation for ⩽14 days (figure 2). Of note, at the time neutrophils were collected, the severity of pulmonary dys function, as defined by the PaO2/FIO2ratio, as well as the overall severity of illness, as defined by APACHE II scores, was similar in the patients who subsequently had a more or less severe clinical course. In addition, neutrophils from patients whose subsequent clinical course was more severe showed increased nuclear translocation of NF-κB after being cultured with LPS. Because neutrophils play an important role in the development and severity of acute lung injury, particularly at early time points [56, 57], such data suggest that early alterations in NF-κB activation contribute to outcome insepsis. This clinical information, coupled with the above-mentioned studies that showed stable high and low responder phenotypes in the healthy population, implies that the presence of a preexistent high responder neutrophil phenotype, as characterized by increased nuclear translocation of NF-κB after stimulation with TLR 2 or TLR 4 ligands, would be associated with more severe pulmonary inflammatory response and clinical course in response to infection. Conversely, persons whose neutrophils haved iminished activation of NF-κB after stimulation would be expected to have less-intense neutrophil-driven inflammation, as wellas organ dys function.
Kinase Activation And Outcome From Sepsis
Although activation of the p 38 and phosphoinositide 3–kinase/Akt kinase path ways has been demonstrated to contribute to pulmonary neutrophil accumulation and the development of organ dysfunction in preclinical models of sepsis [58–60], there is only limited information suggesting that these kinases contribute to outcome inpatients with over whelming infection. As was the case for NF-κB, we have found that neutrophils from healthy volunteers demonstrate stable high and low responder phenotypes for phosphorylation of p38 and Akt after culture with LPS or HMGB1. Of note, persons who demonstrate consistently increased activation of p38 after LPS exposure are not necessarily the same as those who show enhanced phosphorylation of Akt.
In a study by Rosengart et al. [61] of uninfected trauma patients, increased progression to multiple organ dysfunction was found in the group with high base line levels of p 38 activation among cells obtained by bronchoalveolar lavage. Of note, there were no differences in terms of demographic characteristics, hypotension, injury severity score, number of blood transfusions, or severity of illness scores at the time samples were collected among patients with low or high base line p 38 activation patterns. Such data suggest that enhanced cellular activation, as determined byp38, at early time points after injury not only identifies patients who are likely to have a more severe subsequent clinical course but also may contribute to the development of organ system dysfunction.
To examine further the relationship between neutrophil phenotypes and subsequent clinical course in sepsis, we obtained peripheral blood neutrophils from patients with sepsis-induced acute lung injury within 24h of the initiationof mechanical ventilation and then determined baseline phosphorylation of p38 and Akt,as well as the increase in levels of phosphorylated p38 and Akt, after culture of the neutrophils with LPS for1 h [55]. Patients whose subsequent clinical course was characterized by either death or mechanical ventilation for >14 days showed significantly greater activation of Akt, but not p 38,after LPS-induced neutrophil stimulation than did the patients who required mechanical ventilation for ⩽14 days (figure 3).Of note, at the time the neutrophils were isolated, there were no differences in terms of demographics, severity of illness, or oxygenation abnormalities in the 2 groups of patients. Such results, as well as those described by Rosengart et al. [61] among trauma patients, suggest that persons whose neutrophils have a proinflammatory phenotype, as defined by increased activation of kinases, are more likely to have severe clinical course associated with their critical illness. Because the cellular alterations precede many of the organ dysfunctions that contribute to mortality in this setting, these studies also suggest that identification and correction of alterations in cellular signaling path way sat early time points in a patient' clinical course may improve outcome.
Conclusions
Modulation of intracellular signaling cascades involving kinases, such as p 38 or Akt, ortranscriptional factors, such as NF-κB, through specific inhibitory approaches has shown their pathophysiological importance in experimental models. However, the role of specific intra cellular pathways in contributing to clinical out comes in patients with sepsis remains incompletely determined, primarily because such alterations in cellular activation patterns have not been examined at early time points before the onset of multiple organ dysfunction. Recent information shows that alterations in p38, Akt, and NF-κB among neutrophils and other cell populations not only precedes the development of organ system dysfunction but also has predictive value in identifying patients with a more severe subsequent clinical course. Such results suggest that stable responder phenotypes that are independent of TLR 2 or TLR 4 polymorphisms are present among humans and may correlate with clinical out come in the setting of sepsis. Identification of patients with proinflammatory cellular phenotypes at early time points may permit institution of the rapies that interrupt deleterious signaling cascades before organ system dysfunction develops, reducing morbidity and improving survival.
Acknowledgements
Financial support. National Institutes of Health (grants HL- 62221 and HL- 068743).
Potential conflicts of interest. E.A.: no conflicts.
Comments