Severe sepsis remains the leading cause of morbidity and mortality in intensive care units throughout the world. Sepsis is defined as systemic inflammatory response syndrome (SIRS) caused by an infection.1 Pattern recognition receptors (PRRs) are important mediators in the host immune response, culminating in SIRS. The endothelial injury caused by SIRS triggers disseminated intravascular coagulation (DIC).2 DIC involves microthrombus formation, which participates profoundly in the development of multiple organ failure.
The receptor for advanced glycation end products (RAGE) is a member of the immunoglobulin superfamily of proteins and is recognized as a PRR.3 RAGE is involved in both innate and adaptive immune systems and is widely expressed on multiple cells including endothelial cells and all types of leukocytes.4 Binding of ligands to RAGE activates several different signaling cascades for immune responses and can result in the development of SIRS. RAGE exists as both a cell membrane–bound form and a soluble form called soluble RAGE (sRAGE). Accumulated evidence suggests that the amount of circulating sRAGE reflects that of RAGE expressed in cells and the inflammatory process depending on the RAGE signaling pathway.5,6
Although elevation of sRAGE in the serum of septic patients was reported,7 no clear evidence has been presented of an association between circulating sRAGE and severity, DIC, and endothelial injury in sepsis. Therefore, the objective of this study was to investigate the sRAGE/RAGE signaling pathway in the context of the pathogenesis of sepsis and to highlight practical implications of the measurement of sRAGE.
PATIENTS AND METHODS
This cross-sectional study was conducted at the Department of Traumatology and Acute Critical Medicine, Osaka University Graduate School of Medicine, from November 2012 to September 2013. Inclusion criteria were patients with severe sepsis defined as SIRS combined with an infectious episode and dysfunction of at least one organ based on the American College of Chest Physicians/Society of Critical Care Medicine conference8 and age older than 18 years. Healthy subjects with no previous history provided blood samples as controls during the same study period.
The study, which followed the principles of the Declaration of Helsinki, was approved by the institutional review board at Osaka University. Written informed consent was obtained from the patients or their close relatives and the volunteers.
Definitions of Severity of Illness and Diagnosis of DIC
Acute Physiology and Chronic Health Evaluation II (APACHE II) and Sequential Organ Failure Assessment (SOFA) scores were assessed at the time of patient enrollment. Also, the peak SOFA score during the first 7 days after admission was evaluated. The initial SOFA score was included in the peak SOFA analysis. We used the International Society of Thrombosis and Haemostasis (ISTH) diagnostic scoring system for overt DIC9 because it has high specificity to ensure an accurate diagnosis of DIC. Fibrin/fibrinogen degradation products (FDPs) were used for fibrin-related markers and were classified into three groups:10 no increase, 0 to 9 mg/L; moderate increase, 10 to 24 mg/L; and strong increase, more than 25 mg/L.
Blood samples were collected from patients within 24 hours (median time, 14.0 [2.5–19.0] hours) after the diagnosis of severe sepsis and from the healthy volunteers. Serum or plasma was separated by centrifugation and stored at −40°C until use.
Analyses of sRAGE and Related Biologic Parameters in Blood
Total platelet counts were determined with an automated cell counter (XN9000; Sysmex Corporation, Kobe, Japan). Enzyme-linked immunosorbent assay kits were used to measure serum concentrations of interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α) (R&D Systems, Minneapolis, MN), and high-mobility group box 1 (HMGB1) (Shino-test, Kanagawa, Japan). Plasma concentrations of sRAGE, matrix metalloproteinase 9 (MMP-9), soluble vascular adhesion molecule 1 (sVCAM-1), plasminogen activator inhibitor 1 (PAI-1) (R&D Systems), endogenous secreted form of RAGE (esRAGE) (B-Bridge International, Sunnyvale, CA), advanced glycation end products (AGEs) (Cusabio Biotech, Wuhan, China), and a disintegrin and metalloproteinase domain 10 (ADAM10) (Abnova Corporation, Taipei, Taiwan) were also measured with enzyme-linked immunosorbent assay. The frozen samples were thawed and subsequently processed according to manufacturers’ instructions. Absorbance was measured with a microplate reader (SH-9000Lab; Corona Electric Co., Ltd., Ibaraki, Japan).
Continuous variables are expressed as group median with interquartile range. The nonparametric Mann-Whitney U-test was used to assess differences between sepsis patients and healthy controls. Correlations between sRAGE and other markers were investigated by means of scatter plots and Spearman rank correlation coefficients. A value of p < 0.05 was considered to indicate statistical significance. Statistical analyses were performed with JMP 11.1.1 for Windows (SAS Institute Inc., Cary, NC).
This study was composed of 24 patients with severe sepsis and 12 healthy volunteers (Table 1). There were no significant differences between sepsis patients and controls with respect to age and sex. APACHE II, SOFA, and ISTH DIC scores of the sepsis patients were 20.0 (15.0–23.5), 6.5 (3.0–10.8), and 2.5 (2.0–4.0), respectively. The overall intensive care unit-day mortality was 12.5%. Three patients died because of multiple organ dysfunction syndrome. Sources of sepsis were pulmonary (n = 11), abdominal (n = 3), soft tissue (n = 3), urinary (n = 6), and others (n = 1). The comorbidities of the patients included diabetes mellitus (n = 6), hypertension (n = 3), cerebrovascular disease (n = 2), congestive heart failure (n = 2), liver disease (n = 2), respiratory disease (n = 1), and solid tumor without metastasis (n = 1).
sRAGE in Patients With Severe Sepsis
Overall, the serum level of sRAGE was significantly increased in severe sepsis patients compared with that in healthy controls (Fig. 1A). We attempted to discern the source of sRAGE (i.e., total soluble RAGE), which is composed of two forms. One isoform, generated as a splicing variant, is called esRAGE and is secreted directly without anchoring to plasma membrane.11 The other is derived from cleavage of plasma membrane–bound RAGE by proteolytic enzymes and generally is defined as subtracted sRAGE (sRAGE – esRAGE).12 The serum level of esRAGE was not significantly different between the septic patients and the healthy controls (Fig. 1B), but the level of subtracted sRAGE was significantly increased in the septic patients (Fig. 1C). We also evaluated the fraction ratio in sRAGE to assess the balance between esRAGE and subtracted sRAGE. Compared with the ratio in the healthy controls, the esRAGE/sRAGE ratio was significantly decreased (see Figure, Supplemental Digital Content 1A, http://links.lww.com/TA/A566) and the subtracted sRAGE/sRAGE ratio was significantly increased in the septic patients (see Figure, Supplemental Digital Content 1B, http://links.lww.com/TA/A566).
Correlations Between sRAGE and Severity of Illness
To highlight the clinical relevance of sRAGE measurement, we assessed the correlation between serum level of sRAGE and disease severity using the APACHE II score, which provides a general measure of disease severity according to parameters routinely used in physiologic assessment, and the SOFA score (Fig. 2), which is used to assess organ dysfunction. Significant correlation was found between sRAGE levels and both the APACHE II (Fig. 2A) and SOFA (Fig. 2B) scores. To assess the correlation between serum level of sRAGE and subsequent development of organ dysfunctions, we evaluated the correlation between serum level of sRAGE and the peak SOFA score during the first 7 days. The serum level of sRAGE did not correlate with peak SOFA score (p = 0.09, rho = 0.354).
Correlations Between sRAGE and DIC Score or DIC Scoring System Parameters
The serum level of sRAGE was significantly increased in accord with the increase of ISTH DIC score in the severe sepsis patients (Fig. 3A). Among the parameters composing the ISTH DIC scoring system, significant negative correlation was found between the serum level of sRAGE and platelet count (Fig. 3B) but not between sRAGE and the other DIC scoring system parameters: FDP (see Figure, Supplemental Digital Content 2A, http://links.lww.com/TA/A567), fibrinogen (see Figure, Supplemental Digital Content 2B, http://links.lww.com/TA/A567), and prothrombin time (see Figure, Supplemental Digital Content 2C, http://links.lww.com/TA/A567).
Correlations Between sRAGE and Related Biochemical Parameters
To study the relation between sRAGE and the blood markers of systemic inflammation, proinflammatory cytokines IL-6 and TNF-α were measured in the serum samples. Significant correlation was found between sRAGE level and IL-6 level (Fig. 4A) but not between sRAGE level and TNF-α level (see Figure, Supplemental Digital Content 3A, http://links.lww.com/TA/A568).
To estimate the involvement of RAGE signaling in systemic endothelial injury, serum levels of sVCAM-1 were measured. VCAM-1 is induced in response to systemic inflammation and facilitates endothelial adhesion of leukocytes. sVCAM-1 is released from the endothelial cell surface on endothelial injury.13 A significant positive correlation was detected between sRAGE levels and sVCAM-1 levels (Fig. 4B). In addition, the levels of PAI-1, a serine protease inhibitor that suppresses fibrinolysis and is mainly produced from endothelium,14 were measured to evaluate endothelial activity related to coagulopathy. Significant correlation was also observed between sRAGE levels and PAI-1 levels (Fig. 4C).
Next, we estimated the serum level of the RAGE signaling pathway–inducing ligands HMGB1 and AGEs. HMGB1 is a nuclear protein present in the nucleus of all nucleated cells that acts as an inflammatory mediator once released extracellularly.15 Although sRAGE level tended to correlate with the serum level of HMGB1, no statistical significance was detected (Fig. 4D). Similarly, sRAGE level did not correlate with the levels of AGEs (see Figure, Supplemental Digital Content 3B, http://links.lww.com/TA/A568).
We also assessed the association between the level of sRAGE and proteolytic enzymes, which can produce sRAGE by cleavage of membrane-bound RAGE. The sRAGE level tended to correlate with the plasma level of ADAM10, although without significance (see Figure, Supplemental Digital Content 3C, http://links.lww.com/TA/A568) and apparently did not correlate with the serum level of MMP-9 (see Figure, Supplemental Digital Content 3D, http://links.lww.com/TA/A568).
Correlations between the components of sRAGEs and clinical and biochemical parameters are summarized in Table 2. Similar results were observed for subtracted sRAGE, but not for esRAGE, compared with total sRAGE.
The severity of sepsis increases along with the degree of coagulation disorder in association with endothelial injury.16 This is the first report, to our knowledge, to assess the relation between serum sRAGE and pathologic conditions in severe sepsis including inflammation, coagulation disorder, and endothelial injury. The results suggested that RAGE signaling may play a pivotal role in the pathogenesis of severe sepsis. The hypothetical scheme for the sRAGE/RAGE signaling pathway in sepsis, which was deduced from our findings, is presented in Supplemental Digital Content 4 (see Figure, Supplemental Digital Content 4, http://links.lww.com/TA/A569).
Hyperglycemia produces AGEs, which gradually accumulate in a living body and promote age-related diseases.17 RAGE was identified as their receptor and is recognized as one of the PRRs. Dysregulation of RAGE signaling has been implicated in the pathogenesis of diabetes complications and chronic inflammation such as Crohn’s disease.18
sRAGE is released into the circulation through cleavage of membrane-anchored RAGE or directly through alternative splicing, and circulating sRAGE reflects the summation of RAGE signaling activity in the body, which is supported by the evidence that circulating sRAGE is upregulated in diabetes and chronic inflammatory diseases.19,20
Recently, the RAGE-sRAGE axis has received considerable attention for its possible involvement in the dysregulated inflammatory response in acute illness.21 Serum sRAGE increases in septic patients with acute respiratory distress syndrome (ARDS) compared with those without ARDS, and the rise in sRAGE is associated with sepsis progression.7,22 Likewise, we showed that the serum level of sRAGE was increased in severe sepsis patients compared with that in healthy volunteers. On the basis of these results, we sought to determine the relation between the serum level of sRAGE and various biologic and clinical parameters in severe sepsis patients.
The serum level of IL-6 correlated positively with that of sRAGE. IL-6 is a representative proinflammatory cytokine whose expression is triggered by recognition of invading pathogens, and excessive production injures the body’s own tissues beyond the protection it provides against the infection.23 This machinery is evoked via binding of pathogen-derived materials to PRRs including RAGE, and the downstream intracellular signaling enhances the synthesis of IL-6 through nuclear translocation of nuclear factor-κB.24 The activation of nuclear factor-κB also induces transcription of the RAGE gene.25 This might explain the correlation between sRAGE and IL-6 in the serum of septic patients. The augmentation of RAGE expression in turn exaggerates the inflammation as a positive feedback mechanism.26 Therefore, the measurement of sRAGE may be useful to evaluate the sustained activation of systemic inflammatory response.
Damage-associated molecular patterns (DAMPs) can also bind to PRRs and induce systemic inflammation. HMGB1, AGEs, and S100A12 are representative ligands for RAGE.27–29 RAGE expression and release of sRAGE were reported to be enhanced through recognition of DAMPs by RAGE. S100A12 and RAGE are present in high concentrations in pulmonary tissue and bronchoalveolar lavage fluid within 7 days after the onset of ARDS.30 We analyzed the association of the serum level of sRAGE with that of HMGB1 or AGEs in early severe sepsis and found no correlations. The level of HMGB1 in the severe septic patients was significantly higher than that in the healthy volunteers (see Table, Supplemental Digital Content 5, http://links.lww.com/TA/A570). Although the serum level of sRAGE tended to increase in accord with the upregulation of HMGB1, HMGB1 itself seems not to be directly involved in the induction of sRAGE into the circulation, at least not in the early phase of sepsis. AGEs might be produced in a later phase of sepsis in accordance with worsening of the clinical condition including comorbid dysglycemia. Because HMGB1 is generally recognized as a late mediator in sepsis,31 it is still possible that these DAMPs aggravate inflammatory response in the later phase of sepsis through augmentation of RAGE signaling.
We investigated the source of circulating sRAGE in early sepsis using clinical samples. Interestingly, the serum level of subtracted sRAGE, which could be identical to cleaved-type sRAGE derived from the cell surface, but not that of esRAGE, was significantly upregulated in septic conditions compared with that in healthy conditions and correlated significantly with the severity of sepsis. Similarly, Uhle et al.32 reported that both sRAGE and esRAGE levels were increased in plasma within 24 hours after trauma, but esRAGE levels did not correlate with the severity of injury in contrast to sRAGE. These data suggest that proteolytic cleavage of membrane-anchored RAGE is induced and contributes to immediate upregulation of circulating sRAGE as a common early response to systemic stress such as sepsis and trauma. Unexpectedly, the levels of both MMP-9 and ADAM10, which have been identified as the proteolytic enzymes for the production of sRAGE,33 did not correlate with that of circulating sRAGE, although the induction of MMP-9 was observed in the circulation of the septic patients (see Table, Supplemental Digital Content 5, http://links.lww.com/TA/A570). The activity of MMP-9 could be inhibited by PAI-1 upregulated in the situation discussed below. It is assumed that previously unidentified enzymes might also be involved in the cleavage of plasma membrane–bound RAGE and that sepsis-induced molecules might assist the enzymatic activity in the truncation.
Endothelial injury plays a crucial role in the pathogenesis of coagulopathy in sepsis.34 RAGE is expressed abundantly in the endothelium,35 and endothelial injury augments RAGE expression.36 Recent evidence suggests that the amount of circulating sRAGE reflects RAGE expressed on injured endothelial cells.17,37 We found that the serum level of sRAGE was significantly associated with that of sVCAM-1, an early marker of endothelial activation related to systemic inflammation. The level of sRAGE also correlated with the ISTH DIC score. Interestingly, among the parameters evaluated for the DIC score, only platelet count, and not prothrombin time, FDP, or D-dimer, correlated negatively with the sRAGE level, suggesting that, in the early septic condition, platelet consumption precedes hypercoagulability and the subsequent fibrinolytic response.38 Platelet consumption is thought to progress with endothelial injury.39 The fact that the serum level of sRAGE correlated with that of PAI-1 implies that endothelium activated through RAGE signaling induced by recognition of pathogen-associated molecular patterns (PAMPs) releases PAI-1 to suppress hyperfibrinolysis in the early phase of sepsis. Injured endothelium expresses a series of adhesion molecules and chemokines and accumulates platelets and neutrophils along the vessel wall. These interactions culminate in microembolism and the development of tissue injuries, leading to multiple organ failure,40 probably at least partially because of RAGE signaling. We demonstrated a correlation between the serum level of sRAGE and the severity of sepsis as evaluated with the APACHE II and SOFA scores. RAGE signaling is thought to be involved multidirectionally in the steps comprising the progression of multiple organ failure in sepsis. However, it should be noted that commencement of the endothelial RAGE signaling system after detection of PAMPs might function primarily as a defense mechanism against invasion of pathogens by generating microthrombus in the vessel to inhibit pathogen dissemination and by producing sRAGE to act as a decoy.
We believe that sRAGE could become a biomarker with which to monitor the septic condition. Blood levels of C-reactive protein or IL-6 are generally used as representative indicators for systemic inflammation.41 In our study cohort, the level of C-reactive protein did not correlate with the APACHE II, SOFA, or ISTH DIC scores or the levels of sVCAM-1 or PAI-1 (see Table, Supplemental Digital Content 6, http://links.lww.com/TA/A571). Although the levels of both IL-6 and sRAGE did correlate with the severity of sepsis or the ISTH DIC score, we could find no correlation between IL-6 and the markers of endothelial activation. We believe that the measurement of serum sRAGE level is useful to evaluate endothelial injury related to systemic inflammation, coagulopathy, and multiple organ damage.
As one limitation of this study, it was developed partly based on the hypothesis that the serum level of circulating sRAGE reflects a summation of RAGE expression in a patient’s body. In the future, we must confirm a positive correlation between blood concentration of sRAGE and the actual histologic expression of RAGE in tissues obtained from sepsis patients. Second, we evaluated sRAGE only at the early phase of sepsis. During the progression of sepsis, for example, newly generated molecules, such as DAMPs and proteolytic enzymes, would affect the RAGE/sRAGE signaling system and chronologically alter the prognosis of sepsis. Further study addressing the chronologic changes of sRAGE on a long-term follow-up basis will be needed. Third, the numbers of patients and controls were relatively small. Also, because the 28-day mortality rate was low (12%) in this study, we could not perform any analyses of prognostic factors. Finally, the analysis developed here should also be applied to patients with sterile systemic inflammatory conditions such as trauma and heatstroke. Further studies would shed light on the essential role of the RAGE/sRAGE signaling system in the biologic reaction to acute systemic stress.
The present study is the first to report that circulating sRAGE increased with the progression of DIC and disease severity in early sepsis in association with endothelial injury. Our findings may suggest the usefulness of measuring the serum level of sRAGE as a biomarker for sepsis.
H.M. participated in study design, data acquisition, and analysis and drafted the manuscript. N.M. had a major influence on the study design, interpretation of data, and critical appraisal of the manuscript. J.S. and K.Y. made critical contributions to the development of this study and helped in drafting the manuscript. K.Y. provided advice on statistical analysis and assisted with the manuscript. H.O. and T. S. revised the manuscript and supervised the study. All authors read and approved the final manuscript.
This study was supported by a grant-in-aid for scientific research from the General Insurance Association of Japan and by a grant-in-aid from the Ministry of Education, Culture, Sports, Science and Technology, Japan.
The authors declare no conflicts of interest.
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Dr. Eileen M. Bulger (Seattle, Washington): This is a very interesting study which seeks to evaluate the use of circulating sRAGE as a biomarker for the severity of illness and inflammation in patients with severe sepsis within the first 24 hours of admission.
The study involves a single time point and evaluates correlations with initial clinical parameters such as APACHE, SOFA and a DIC score and several traditional markers of the inflammatory response.
This is an important first step in the evaluation of this novel biomarker, but there are several questions for the authors to address.
The blood sample for this study was drawn at a single time point, which was any time within the first 24 hours. What was the variability in the timing of these blood draws among the study patients? And do you think this is a sufficient assessment of this biomarker?
I would suspect that patients present at varying stages of disease and for some patients the peak RAGE levels may be later than 24 hours. Have you done any studies where you draw serial blood samples over the first several days to assess this?
You correlated sRAGE levels with score of disease severity at the time of admission. However, to be useful as a biomarker, it would be helpful to know if early increases in sRAGE correlate with the subsequent development of organ dysfunction in these patients. Do you have any data looking at the later complications?
While you identified several correlations between sRAGE and other markers of inflammation and disease severity, it appears from the scatter plots that there was considerable variability among individual patients that would make it very difficult to apply as a discrete biomarker.
Did you identify a threshold level of sRAGE that could be used clinically to predict poor outcome? And if so, how does this level correlate with disease progression and late complications?
Thank you very much for an excellent presentation and for sharing the results of this interesting study to us.
Dr. Hisatake Matsumoto (Osaka, Japan): Thank you very much for your smart and sharp questions.
Your first question concerns the validity of soluble RAGE evaluation at a single time point within 24 hours after the admission as a biomarker. It was reported that circulating soluble RAGE increased 5 hours after an endotoxin challenge against healthy volunteers, suggesting that RAGE signaling can be activated promptly following pathogen invasion in an individual body. At any rate, it is thought that the measurement of soluble RAGE in the serum on admission is useful to know the inflammatory state induced by RAGE signaling in the early condition of sepsis in the context of the recognition of PAMPs by pattern recognition receptors.
In our study, there was no significant difference in soluble RAGE between blood samples drawn before and 5 hours after the patient’s admission. Therefore, the validity of soluble RAGE within 24 hours might not act as a confounding factor.
However, we did not define when the level of soluble RAGE peaked in an individual patient. As you pointed out, serial measurement of soluble RAGE and the analysis of the values in association with a series of parameters are critically important because RAGE signaling would be modified by various sepsis-related factors in the course of sepsis. For example, DAMPs, a late mediator of sepsis, will be abundantly generated during the progression of sepsis. After their binding to RAGE, an additional reaction in RAGE signaling will occur and affect the patient’s prognosis. We would like to perform the chronological estimation of soluble RAGE in a new study.
The next is regarding the complications and prognosis after admission. Other researchers reported that the RAGE levels in pulmonary edema fluid from patients with acute lung injury were higher than those from patients with hydrostatic pulmonary edema, and the plasma levels of soluble RAGE in patients with acute lung injury were significantly higher than those in healthy volunteers in human studies.
The lung is a representative organ that abundantly expresses RAGE. Unexpectedly, the serum level of soluble RAGE did not show a significant difference between the patients complicated by acute lung injury and the patients who were not during hospitalization. As well, no significant difference was observed in terms of pneumonia complications.
On the other hand, we analyzed the correlation of the serum level of soluble RAGE with a variety of parameters estimated in this study in the patients without pulmonary complications on admission, and we found significant correlations as the results in the paper show.
We consider that induction of RAGE signaling depends on the systemic endothelial activation occurring in the early stage of sepsis. From this view point, multiple organ involvement more than lung injury should be analyzed, such as the peak of the SOFA score during hospitalization and the rate of change of the SOFA score after admission. I am sorry, but these evaluations have not been carried out so far, and according to your valuable comment, I would like to review the data after returning to Japan.
When we focused on prognosis, significant correlations were not detected between the serum level of soluble RAGE within 24 hours after admission and either the survival rate at 28 days after admission or the length of ICU stay.
In another study, both patients with sepsis or septic shock who were enrolled within the first 24 hours after onset and non-survivors showed higher levels of soluble RAGE compared with that in survivors in blood collected at day 28 after admission, suggesting that soluble RAGE is related to severity and outcome in septic patients. However, the mortality rate was 49% in that study, whereas it was 12% in our study. The difference of the cohort for evaluation of soluble RAGE might be the cause of this discrepancy in the results.
As previously mentioned, various factors that are being induced in the progression of sepsis, including DAMPs, will alter the condition of RAGE signaling, and the dynamic change in RAGE signaling will consequently affect the final prognosis.
Thank you very much for your valuable questions.