Acute respiratory distress syndrome (ARDS) is a complicated clinical syndrome characterized by non-cardiogenic pulmonary edema and acute respiratory failure (1). ARDS is life-threatening and is a major cause of intensive care units (ICU) morbidity and mortality throughout the world (2).
ARDS is initiated by excessive polymorphonuclear neutrophils (PMNs) accumulation and activation in the lungs (3, 4). PMNs release inflammatory mediators and cytokines after adhesion and activation, which lead to diffuse and uncontrolled inflammatory injury in the lungs (3–6). Human neutrophil elastase (HNE) is one of the most important organ and tissue destructive proteases that is released from PMNs (7). HNE is stored in azurophil granules of neutrophils and is used to degrade antigens that are consumed by PMNs, providing a host defense (7). Low molecular-weight inhibitors, such as elafin (PI3, peptidase inhibitor 3) and secretory leukocyte proteinase inhibitor, can sequester HNE from high molecular-weight inhibitors (8). In the lung, an imbalance between the proteinases and their inhibitors damages the alveolar-capillary barrier, resulting in leaking of protein-rich fluid into the interstitium and alveolar spaces, which is the key pathophysiological characteristic of ARDS (9) (Figure S1, http://links.lww.com/SHK/A559).
Previous studies have suggested that circulating HNE and PI3, as biomarkers, could be used to predict acute lung injury/ARDS development (10–13). However, whether they can serve as an early marker for the mortality and the severity of ARDS is still unclear. Therefore, we performed a multicenter prospective cohort study to assess the longitudinal changes in plasma levels of HNE and PI3 after ICU admission in critically ill patients, and to examine the association between HNE and PI3 with the mortality and severity of ARDS.
This was a survival and longitudinal analysis as part of a prospective cohort study conducted from January 1, 2011 to August 31, 2014 in five clinical and surgical ICUs in Beijing metropolitan area: Peking University Third Hospital in the northwest of Beijing, Beijing Friendship Hospital in the south, Beijing Shijitan Hospital in the middle, Beijing Xiyuan Hospital in the west, and China-Japan Friendship Hospital in the northeast.
Each ICU admission was screened for eligible participants. Exclusion criteria were: age <18 years; history of chronic lung diseases, such as interstitial pulmonary fibrosis or bronchiolitis; history of pneumonectomy; treatment with immunomodulating therapy other than corticosteroids, such as granulocyte colony stimulating factor, cyclophosphamide, cyclosporine, interferon, or TNF-α antagonists; presence of other immunodeficient conditions, such as HIV infection, leukemia, or neutropenia (absolute neutrophil count <1,000/μL); history of organs or bone marrow transplant other than autologous bone marrow transplant; directive to withhold intubation; ICU stay duration <72 h; or patient developed ARDS before ICU admission. Sepsis and septic shock were defined according to the Berlin definition (14). Patients at risk for developing ARDS were defined as critically ill patients with at least one predisposing condition for ARDS: sepsis; septic shock; trauma; pneumonia; aspiration; massive transfusion of packed red blood cells (PRBC; defined as > 8 PRBC units in the 24 h prior to admission); or severe pancreatitis. After enrollment, patients at risk for developing ARDS were followed daily for the development of ARDS, according to the Berlin definition (15). All patients were followed up until hospital discharge or death within 60 days after study entry, whichever occurred first.
The study protocol was approved by the institutional review boards of all participating hospitals. Written informed consent was obtained from each patient or a relative.
Plasma sample collection and measurements
Using a standardized protocol, plasma sample was collected from each patient within first day (day 1), on the third day (day 3), and on the seventh day (day 7) of ARDS development. Two milliliters peripheral blood was collected into a vacuum ethylenediaminetetraacetic acid tube and then centrifuged at 3,000 rpm for 15 min at 4°C within 30 min of collection to generate cell free plasma, which is important to prevent any HNE or PI3 that can be released from neutrophil cells after sampling. The plasma was aliquoted and stored at −80°C until assayed. Due to early discharge or death, 3 patients did not have blood specimen on day 3, and 22 patients did not have blood specimen on day 7.
Plasma HNE and PI3 levels were assayed using the Human pre-ELAFIN/SKALP enzyme-linked immunosorbent assay (ELISA) kit (# HK319-02, Hycult Biotech, Plymouth Meeting, Pa) and the Human Elastase ELISA kit (# HK318-02, Hycult Biotech), according to the manufacturer's protocol. All assays were performed in duplicate, and the mean value was used for analysis.
Assay quality control
In all assays, we randomly distributed samples based on age, gender, survival status, and sample collection date. This aspect is important to reduce batch effect from confounding variables and technical variation (16). The average intra-assay coefficients of variation (CV) of replicates were 4.3% for HNE and 12.3% for PI3, which were below the manufacturer's recommended 15% threshold and considered acceptable for duplicate samples (17). Twenty plasma samples were randomly selected as replicates for quality control of batches and showed interassay CV ranging from 5% to 15%.
Demographic and clinical variables were compared between ARDS survivors and non-survivors using the two-sided t test for parametric continuous variables, Wilcoxon–Mann–Whitney test for nonparametric continuous variables, and chi-square test for categorical variables.
We used multivariate Cox proportional regression model to assess the association between biomarker expression and overall survival with time to death. The hazard ratio (HR) was the ratio of hazards for one standard deviation change in the biomarker concentration level. Kaplan–Meier curves were used to measure the ARDS patients’ time-to-death among four groups of biomarker level: HNE≥median, PI3≥median; HNE≥median, PI3<median; HNE<median, PI3≥median; HNE<median, PI3<median. The test of equality for survival distributions was performed using the log-rank test (Mantel–Cox). Patients were excluded from day 3 or day 7 analysis if they reached to screening status (discharge from ICU or death) prior to these two time points.
Longitudinal changes of HNE and PI3 levels in ARDS survivors with non-survivors were analyzed by a mixed effect model using three time points (day 1, day 3, day 7) collected repeatedly from the same patients assuming compound symmetry covariance structure because there was a correlation among measurement and correlation was generally constant (18). Finally, we computed area under the receiver operating characteristic curve (AUC) to evaluate the prognostic performance of the biomarkers concentration level on dichotomous mortality. To assess further the incremental prognostic power that two biomarkers have when added to APACHE II and Berlin (PaO2/FIO2) categories model, we computed integrated discrimination improvement (IDI) and net reclassification index (NRI), which offer an intuitive way of quantifying improvement offered by new biomarkers (19).
A value of P < 0.05 was considered significant. In our analysis, we omitted the missing values to avoid biased results due to missing data. All analyses were performed with R software (v 3.2.3, R Foundation for Statistical Computing) and Statistical Analysis System software (v.9.4, SAS Institute, Cary, NC).
Patient enrollment and follow-up
The enrollment and follow-up of study patients are illustrated in Figure 1. During the study period, 11,829 patients were admitted to adult ICUs of 5 Tertiary hospitals. 1,147 patients were admitted to ICUs with required predisposing conditions for ARDS. Among those, we excluded 408 patients without consent, 213 patients who had stayed in ICU less than 72 h, and 200 patients who had developed ARDS based on Berlin definition before ICU admission, leaving a total of 167 patients for analysis. Of the 167 patients who developed ARDS, 62 (37.1%) died within 60 days of their diagnosis. For these patients, blood samples were collected on the first day of ARDS onset, third day, and seventh day.
Characteristics of study population
Demographic and baseline characteristics for the study population are shown in Table 1. ARDS patients who survived were younger, had higher albumin level, higher PaO2/FiO2, higher systolic blood pressure, higher platelets counts, and lower respiratory rates. Septic shock was more commonly seen in ARDS non-survivors. Other predisposing conditions, such as sepsis syndrome, pneumonia, multiple transfusions, trauma, and aspiration, were similar in both groups. There were no significant differences in gender, smoking history, diabetes, APACHE II score, or direct pulmonary injury between ARDS survivors and non-survivors.
Plasma HNE and PI3 are predictors of ARDS mortality
Of the three time measurements, HNE levels on day 1 (HR=1.76, P < 0.001 for 28-day mortality; HR=1.76, P = 0.002 for 60-day mortality) and day 7 (HR=1.70, P = 0.006 for 28-day mortality; HR=1.70, P < 0.001) were a strong risk factor for the ARDS mortality. PI3 levels on day 1 (HR = 0.49, P = 0.005 for 28-day mortality; HR = 0.50, P = 0.003 for 60-day mortality) and day 3 (HR = 0.43, P < 0.001 for 28-day mortality; HR = 0.43, P < 0.001 for 60-day mortality) were associated with higher ARDS mortality (Table S1, http://links.lww.com/SHK/A559).
Among three time points measurement, HNE levels were significantly higher in non-survivors and PI3 levels were significantly lower in non-survivors, indicating HNE as a risk factor and PI3 as a protective factor for ARDS mortality (Figure S2/S3, http://links.lww.com/SHK/A559). The Kaplan–Meier curves illustrated highest mortality was observed in patients with HNE level above the median and PI3 level below the median in all measurements (Fig. 2). Time to death is significantly shorter in patients with higher HNE level and lower PI3 level within 28 days (log-rank P = 0.002 for day 1, P < 0.001 for day 3 and P < 0.001 for day 7) from ICU admission, which showed a better ARDS mortality prediction power than APACHE II score and Berlin criteria (Figure S4/S5, http://links.lww.com/SHK/A559).
Plasma HNE and PI3 prognostic performance
Receiver-operating characteristic analysis demonstrated that combination of HNE and PI3 had the discrimination ability for 28-day and 60-day mortalities (AUC: 0.76, 95% CI: 0.69–0.83 and AUC: 0.74, 95% CI: 0.67–0.82), which was better than the combination of Berlin categories and APACHE II (AUC: 0.63, 95% CI: 0.55–0.72 and AUC: 0.61, 95%: 0.52–0.70). In order to further assess the incremental prognostic power of HNE and PI3 added to Berlin categories and APACHE II, we calculated IDI and NRI for the two models. When HNE and PI3 were combined with Berlin categories and APACH II, the ARDS mortality discrimination performances were greatly enhanced, with IDI increment of 20% and 19% for 28-day and 60-day mortality, respectively, and category-free NRI increment of 76% (P < 0.0001 for all) (Table 2/Table S2, http://links.lww.com/SHK/A559).
Plasma HNE and PI3 are associated with severity of ARDS
ARDS severity was categorized by the ARDS Berlin definition. We found plasma HNE (P < 0.0001), but not PI3 (P = 0.20), correlated with worsening Berlin categories on day 1 (Figure S6, http://links.lww.com/SHK/A559). Severe ARDS patients had significantly higher HNE levels than other ARDS patients, indicating more diffuse and uncontrolled inflammatory injury in the lungs.
Longitudinal changes of HNE and PI3 during ARDS progress
A mixed effect model was used to assess longitudinal effects of plasma HNE and PI3 in ARDS patients. As expected, we observed significant longitudinal effects of HNE and PI3 between ARDS survivors and non-survivors (Table 3). HNE levels on day 1 and day 3 were significantly higher in ARDS non-survivors than survivors, whereas PI3 level showed opposite direction (Figure S7, http://links.lww.com/SHK/A559). The mixed model predicted a decline in HNE levels beginning at ARDS onset, and PI3 levels had a delayed response to inhibit HNE (Figure S7, http://links.lww.com/SHK/A559).
To our knowledge, the current study is the first to investigate the prognostic value of HNE and PI3 in ARDS patients with measurements from multiple time points. In this multicenter prospective study, we identified the association between plasma HNE and PI3 levels at multiple time points with ARDS mortality and with severity at baseline. Berlin categories and APACHE score are currently considered effective criteria for predicting ARDS mortality (15, 20). The prognostic performance of combined HNE and PI3 is greatly better than the prognostic performance using only ARDS Berlin categories and APACHE II, and further increased when combining biomarkers with clinical criteria.
Several plasma biomarkers for ARDS endothelial and epithelial dysfunction have been found to be associated with ARDS risk and mortality (21–24). A recent meta-analysis on the association of plasma biomarkers with ARDS diagnosis or mortality showed that increased plasma levels of Ang-2 and sRAGE are most strongly associated with ARDS diagnosis in the at-risk population (25). Furthermore, recent advances on the pathophysiological mechanisms underlying ARDS have identified several additional clinical biomarkers to assess disease severity and outcome, including lung inflammatory mediators soluble suppression of tumorigenicity-2 (sST2) and IL-6 (26); products of epithelial and endothelial injury, soluble form of receptor for advanced glycation end products (sRAGE) (22, 27). However, compared with HNE and PI3, these abovementioned plasma biomarkers appear at a relatively later stage in ARDS development because they are induced by lung tissue damage mainly due to HNE destruction to the lung (21–23). HNE is the major ultimate effect cytokine that can trigger inflammation cascade, which is an important pathophysiological mechanism of ARDS. When infection, trauma, and other ARDS risk factors happen, multiple inflammatory mediators, such as TNF-α and IL-8, can stimulate neutrophils to release excess HNE at the beginning stage of ARDS development (Figure S1, http://links.lww.com/SHK/A559).
A physiological hallmark of ARDS is the clustering of activated neutrophils from circulating blood in the lungs and the release of activated material to damage lung (28). The activated materials that a neutrophil releases are a determinant of formation and severity of lung damage. Among them, HNE is one of the most destructive enzymes that can degrade extracellular matrix and proteins, and it plays a crucial role in the pathophysiology of ARDS (10). As expected, the present study found that the plasma levels of HNE were significantly higher in the ARDS non-survivor than survivors. Furthermore, we found HNE levels were correlated with PaO2/FIO2 and the severity of ARDS; the higher HNE concentration was, the more severe of ARDS was. This can help clinicians to better understand lung injury level in ARDS patients at early stage. Thus, HNE may potentially be considered to serve as a prognostic biomarker for ARDS.
On the other hand, endogenous proteinase inhibitors play important roles in protecting excessive tissue damage from inflammation cascades. HNE is rapidly inactivated by conjugating with protease inhibitors once the high-molecular-weight inhibitors including a1-antitrypsin and a2-macroglobulin are released in the circulation (10). However, PI3, a low molecular weight inhibitor, plays an important role in local protection. PI3 can sequester HNE from high molecular weight inhibitors by penetrating into the “microenvironment” created by neutrophils (8, 10). Protein-rich fluid can leak into the interstitium and alveolar spaces and cause an imbalance between proteinases and inhibitors and pulmonary parenchyma damage, which is the major mechanism for activated neutrophils initiating and propagating ARDS (29). Unsurprisingly, our study found plasma PI3 levels were significantly lower in ARDS non-survivors than survivors, indicating their protective effect on ARDS mortality. Also, we found that the altered level of PI3 was delayed compared with HNE activation, which was in consistent with the mechanism of PI3's competition with high molecular weight inhibitors. In addition, white blood cell counts were positively correlated with HNE levels, whereas negatively correlated with PI3 levels (Figure S8, http://links.lww.com/SHK/A559).
Uniquely, the present study had three time measurements during the ARDS progression ranging from day 1 to day 7 after enrollment, which is an important period in the time course of ARDS (26, 30). Utilizing this longitudinal data, we found the two biomarker levels on day 1 and day 3 are significantly different between ARDS non-survivors and survivors. This indicates the imbalance of HNE and PI3 in ARDS patients can potentially be an early signal for the mortality. Through day 1 to day 7, non-survivors’ HNE levels are constantly higher than survivors’, whereas PI3 levels are constantly lower than survivors’ (Figure S7, http://links.lww.com/SHK/A559). In addition, we found the trend of HNE level decreased steadily and PI3 level increased from day 1 to day 3 and then decreased. Previous studies have reported that the HNE levels decrease once the patients developed ARDS/acute lung injury (7). At ARDS onset, HNE attacks the lungs and is gradually consumed over time. PI3 levels may increase after disease onset because of a delayed response to HNE. Once HNE is activated, high-molecular-weight inhibitors are soon released in the circulation and are conjugated with HNE, but low-molecular-weight inhibitors, such as PI3, need to take time to accumulate and compete with high-molecular-weight inhibitors, ultimately sequestering other antagonists of HNE (8). Also, a previous randomized control trial has found Sivelestat, a neutrophil elastase inhibitor, improves the mortality rate of sepsis associated with ARDS (31).
Our study has several strengths. First, to our knowledge, the current study is the first to investigate the prognostic value of HNE and PI3 in ARDS patients. Also, we conducted repeated time measurements, which offer a more comprehensive observation and interpretation than a single-time measurement biomarker study. This is particularly important for ARDS because its rapid disease progression characteristic and alteration of biomarker level can lead to imprecise assessment of the disease and treatment. Also, our prospective enrolled cohort design in multicenter ICUs ensures clarity of temporal sequence, which is suitable for ARDS with multiple time points (32).
We also acknowledge limitations in our study. First, the selection bias may present. However, since the HNE and PI3 levels are probably not related to patient's enrollment, which reduces the possibility that the results will be biased by selecting subjects for the comparison group who may be more or less likely to have the ARDS mortality outcome. Also our population's baseline characteristics are similar to a recent large sampled ARDS study (2), which suggests our enrolled group is comparable to the other ARDS populations in terms of baseline characteristics. Finally, our study findings are limited by the sample size. Although our findings are consistent with similar previous studies (7, 10, 33), a large sample size is preferred to confirm our findings.
Inflammatory response HNE was significantly higher in ARDS non-survivors, while its conjugated inhibitor PI3 was significantly lower in ARDS non-survivors, and they were both correlated with ARDS severity. HNE concentration above the median together with PI3 concentration below the median led to the highest ARDS mortality rate. By combining these biomarkers with Berlin categories and APACHE II, prognostic power of ARDS was greatly improved. Circulation levels of HNE and PI3 may have the potential to predict ARDS mortality and better inform clinicians about mortality risk.
The authors thank Dr Elizabeth A. Loehrer from Harvard T.H. Chan School of Public Health for her editorial assistance.
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