Clinica Chimica Acta 438 (2015) 364–369
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The prognostic value of leukocyte apoptosis in patients with severe sepsis at the emergency department Chia-Te Kung a, Chih-Min Su a,b, Hsueh-Wen Chang b, Hsien-Hung Cheng a, Sheng-Yuan Hsiao a, Tsung-Cheng Tsai a, Nai-Wen Tsai c, Hung-Chen Wang d, Yu-Jih Su b,e, Wei-Che Lin f, Ben-Chung Cheng b,e, Ya-Ting Chang b,c, Yi-Fang Chiang c, Cheng-Hsien Lu b,c,⁎ a
Department of Emergency Medicine, Chang Gung Memorial Hospital-Kaohsiung Medical Center, Chang Gung University College of Medicine, Kaohsiung, Taiwan Department of Biological Science, National Sun Yat-Sen University, Kaohsiung, Taiwan c Department of Neurology, Chang Gung Memorial Hospital-Kaohsiung Medical Center, Chang Gung University College of Medicine, Kaohsiung, Taiwan d Department of Neurosurgery, Chang Gung Memorial Hospital-Kaohsiung Medical Center, Chang Gung University College of Medicine, Kaohsiung, Taiwan e Department of Medicine, Chang Gung Memorial Hospital-Kaohsiung Medical Center, Chang Gung University College of Medicine, Kaohsiung, Taiwan f Department of Radiology, Chang Gung Memorial Hospital-Kaohsiung Medical Center, Chang Gung University College of Medicine, Kaohsiung, Taiwan b
a r t i c l e
i n f o
Article history: Received 29 April 2014 Received in revised form 9 September 2014 Accepted 19 September 2014 Available online 28 September 2014 Keywords: Outcome Lymphocyte apoptosis Monocyte apoptosis Severe sepsis
a b s t r a c t Background and aim: Cell apoptosis in critically ill patients plays a pivotal role in the pathogenesis of sepsis. This study aimed to determine the prognostic value of leukocyte apoptosis in patients with severe sepsis. Methods: Leukocyte apoptosis was determined by flow cytometry. The values of annexin V, APO2.7, and 7-aminoactinomycin D (7AAD) for each subtype of leukocyte were analyzed in 87 patients with severe sepsis and 27 controls. Results: The percentages of apoptosis (APO2.7 [%]) in the leukocyte subsets were significantly higher in the patients with severe sepsis than in the controls. The percentages of APO2.7 in leukocyte apoptosis, APO2.7 in lymphocytes apoptosis, and annexin V + 7AAD in monocytes apoptosis were significantly higher in non-survivors than in survivors. Levels of APO2.7 in lymphocytes apoptosis, annexin V + 7AAD in monocytes apoptosis, and serum lactate were all independently predictive of mortality. Conclusion: Leukocyte apoptosis is significantly higher in patients with severe sepsis. The percentages of late lymphocyte and monocyte apoptosis may be predictive of outcome in such patients. Aside from serum lactate, APO2.7 level in lymphocyte apoptosis is also a useful predictor of outcome on admission to the emergency department. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Severe sepsis and sequential multiple organ failure are still the leading causes of death worldwide despite advances in medical care [1]. The pathogenesis of multiple organ failure in patients with severe sepsis is a multi-factorial process, but global tissue hypoxia due to an imbalance between systemic oxygen delivery and peripheral oxygen demand plays an important role [2,3]. There is growing evidence that aside from cellular necrosis, dysregulated leukocyte apoptosis may also influence the increasing duration and severity of systemic response to sepsis in critically ill patients, including those with acute respiratory distress syndrome, shock, and trauma [4–12]. Abbreviations: APACHE, Acute Physiology and Chronic Health Evaluation; PE, phycoerythrin; FITC, fluorescein isothiocyanate; 7-AAD, amino-actinomycin D; SOFA, sequential organ failure assessment; PBEF, pre-B cell colony-enhancing factor. ⁎ Corresponding author at: Department of Neurology, Kaohsiung Chang Gung Memorial Hospital, Chang Gung University College of Medicine, No. 123, Ta Pei Road, Niao Sung Hsiang, Kaohsiung City 833, Taiwan. Tel.: +886 7 7317123x2283. E-mail addresses: [email protected], [email protected] (C.-H. Lu).
http://dx.doi.org/10.1016/j.cca.2014.09.017 0009-8981/© 2014 Elsevier B.V. All rights reserved.
Severe sepsis and septic shock represent an over-exuberant host response to an infectious challenge. Neutrophils, monocytes, and macrophages play key roles in the initial reaction and release a variety of cytokines that marshal the immune response. Once the adaptive immune response is awakened, innate immune cells are downregulated and must be disposed of in a timely and non-injurious manner. Apoptosis represents a key mechanism in this orderly process, but it is not always good. The apoptotic cascade supervenes during the evolution of sepsis in lymphocytes, in tissue macrophages, and in intestinal epithelia and has been associated with organ dysfunction [13–15]. Moreover, the apoptosis of both alveolar epithelial cells and respiratory endothelial cells has been conclusively demonstrated in animals and humans with acute lung injury and acute respiratory distress syndrome [14]. Previous studies also reveal that lymphocyte apoptosis is rapidly increased in the blood of patients with septic shock and this leads to profound and persistent lymphopenia that is associated with poor outcome. The apoptosis of blood monocytes in septic patients is also associated with their final outcome [8,9].
C.-T. Kung et al. / Clinica Chimica Acta 438 (2015) 364–369
Previous studies have used only a single method to study apoptosis [8,9,11] or only one leukocyte subset to predict the outcome of sepsis [8,9]. Furthermore, the timing of apoptosis is important and the duration of illness before enrolment is connected to outcome. If such variations follow a standard pattern and temporal relationship, prognostication will be substantially improved. 2. Methods 2.1. Study population and definition This prospective study on patients with severe sepsis and septic shock was conducted over a 1-year period (January to December 2011). Eighty-seven adult non-traumatic and non-surgical patients at Kaohsiung Chang Gung Memorial Hospital were enrolled. The hospital's Institutional Review Committee on Human Research approved the study protocol and all of the patients provided written informed consent. All patients aged N18 years consecutively admitted from the ED were screened daily for severe sepsis and septic shock according to the specific criteria defined by the American College of Chest Physicians/Society of Critical Care Medicine. These criteria included suspected or confirmed infection, two or more manifestations of systemic inflammatory response syndrome, and at least 1 sepsis-induced acute organ dysfunction. Patients who met all three criteria were included [16]. For comparison, 27 age- and sex-matched healthy volunteers who received annual physical check-up but without clinical evidence of infection were recruited as controls. 2.2. Clinical assessment and treatment The medical records were prospectively recorded using pre-existing standardized evaluation forms that included demographic data and the Acute Physiology and Chronic Health Evaluation (APACHE) II score, which was calculated during the first 24 h of admission to assess the severity of organ dysfunction. Basic laboratory tests, lactate concentration, and inflammatory markers (i.e., plasma C-reactive protein and procalcitonin) were taken on ED admission. Data on the source of infection and use of antibiotics were also recorded. The course of various organ dysfunctions and supportive treatments, including vasoactive and ventilator therapies and renal replacement therapies, were recorded. Physicians evaluated daily the association of existing organ dysfunction and severe sepsis. It was also institutional practice to consult an infectious disease specialist for anti-microbial treatment based on treatment guidelines for different infectious etiologies during the first 24 h. 2.3. Blood sampling and assessment of leukocyte apoptosis Blood samples were collected on presentation to the ED (Day 1). Follow-up blood samples were obtained on Days 4 and 7 after admission. All blood samples were collected by venipuncture of forearm veins from both the study group and controls. All flow cytometry assays were performed within 1 h after blood extraction to ensure that the results were as close as possible to an in vivo situation. Flow cytometry assay using APO 2.7 antibody for detecting apoptosis. Fixed amounts of blood were diluted 1:5 with PBS, and 100 μL was stained with 10 μl of each of the following: fluorescence-conjugated monoclonal antibodies against CD45-phycoerythrin (PE)-Cy5 (clone J33), CD61-fluorescein isothiocyanate (FITC; clone SZ21), and APO 2.7PE (clone 2.7A6A3; Immunotech). The samples were titrated at saturating concentrations. The CD45-PE-Cy5 antibody reacted with the CD45 family of trans-membrane glycoproteins, expressed on the surface of all human leukocytes, and a pan-leukocyte marker. The CD61-FITC antibody was a pan-platelet marker that reacted with the GPIIb/IIIa complex (fibrinogen receptor).
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The APO 2.7-PE antibody reacted with a 38-kDa mitochondrial membrane protein (7A6 antigen), which was detectable on nonpermeabilized cells in the late apoptotic state [17]. Annexin staining, relevant to early apoptosis, produced similar results but was rejected for questionable reliability under fixation conditions, with formaldehyde clearly biasing the staining results. Mouse immunoglobulin G-PE was a control for non-specific staining, but it did not differ from the APO2.7-PE signal on platelets. Thus, each subject was used as its own control without changing the sample tube. After 30 min of incubation in the dark at room temperature, the stained samples were diluted with 0.5 ml of FACSFlow (Becton Dickinson). Flow cytometry was performed immediately after staining using an Epics XL flow cytometer (Beckman Coulter) and the CellQuest software. Five thousand CD45-PE-Cy5+ cells per sample were acquired in a combined forward and side scatters, and deep-red FL4 fluorescence (CD45PE-Cy5) leukocyte gate. Another 5000 CD61-FITC + cells per sample were acquired in a combined forward and side scatters, and green FL1 fluorescence (CD61-FITC) platelet gate to define the negative control threshold for the measurement of apoptosis, so that each subject was its own control. 2.3.1. Annexin V-FITC 7-AAD Fluorescence-Activated Cell Analysis Membrane phosphatidyl-serine was detected by annexin-V using a commercially available kit (Boehringer Mannheim). The PBS-washed leukocytes were incubated with annexin V-FITC and 7-amino-actinomycin D (7-AAD) for 15 min at room temperature according to the manufacturer's guidelines. Samples were transferred to 5-ml polypropylene tubes, diluted with 900 μl Hanks' balanced salt solution, and placed on ice before flow cytometry analysis. The samples were analyzed using an Epics XL flow cytometer (Beckman Coulter) and CellQuest software. Fifteen thousand events were counted per sample. Low-fluorescence debris was gated-out of the analysis. Leukocyte subtypes were identified according to their CD45 expression intensity and were divided into neutrophils, monocytes, and lymphocytes. From here on, white blood cells (WBC) represented total leukocytes. Annexin V-FITC staining was identified in fluorescent-1 and 7-AAD staining in fluorescent-4. The cells were identified as follows: early apoptotic cells if they were positive for marker annexin V-FITC but negative for 7-AAD; late apoptotic cells if they were positive for annexin V-FITC and 7-AAD; dead cells if they were negative for annexin V-FITC but positive for 7-AAD; and viable cells if they were negative for annexin V-FITC and 7-AAD. 2.3.2. Apoptosis of lymphocytes Fixed amounts of blood were diluted 1:5 with PBS and 100 μl was stained with 10 μl of each of the following: fluorescence-conjugated monoclonal antibodies against CD4-phycoerythrin (PE)-Cy5, CD19fluorescein isothiocyanate (FITC), and CD8- phycoerythrin (PE). Each sample was further stained with annexin V-FITC, 7-amino-actinomycin D (7-AAD), or APO 2.7-PE (clone 2.7A6A3; Immunotech, Marseille, France) and titrated at saturating concentrations. The samples were then transferred to 5-ml polypropylene tubes, diluted with 900 μl of Hanks balanced salt solution, and placed on ice before flow cytometry analysis. The samples were analyzed using an Epics XL flow cytometer (Beckman Coulter) and CellQuest software. Fifteen thousand events were counted per sample. Lymphocyte sub-types were identified according to their surface antigen (i.e., CD4, CD8, or CD19) expression intensity. A database coordinator was responsible for monitoring all of the data collection and entry. All of the tests were performed in a quality-controlled central laboratory at Chang-Gung Memorial Hospital. Concentrations of CRP were determined by enzyme immunoassay (EMIT; Merck Diagnostica), while PCT was measured using a time-resolved amplified cryptate emission technology assay (VIDAS; bioMerieux). Serum lactate levels were measured using a serum-based assay catalyzed by lactate oxidase (UniCel Integrated System; Beckman Coulter INS).
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2.4. Statistical analysis
Table 2 Characteristics of survivors and non-survivors among patients with severe sepsis.
Data were expressed as mean ± SD or median (inter-quartile range). Univariate analyses were compared using Student's t-test, while categorical variables were compared using χ2 test or Fisher's exact test, as appropriate. Correlation analysis was used to explore the relationship among 24-h APACHE II score, serum lactate, C-reactive protein, procalcitonin, creatinine concentration, and levels of leukocyte apoptosis of patients with severe sepsis on admission. Stepwise logistic regression was used to evaluate the relationship between significant variables and therapeutic outcomes, with adjustments for other potential confounding factors. Variables with zero cell count in a 2-by-2 table were eliminated from logistic analysis and only those strongly associated with fatality (p b 0.05) were included in the final model. Receiver operating characteristic (ROC) curves were generated for significant predictor variables of hospital mortality. All statistical analyses were conducted using the SAS software package, ver. 9.1 (2002, SAS Statistical Institute).
3. Results 3.1. Baseline characteristics of the study patients The baseline characteristics of patients with severe sepsis (n = 87) and the healthy controls (n = 27) showed no significant differences in terms of underlying disease (Table 1). The percentages of apoptosis (APO2.7 [%]) in the subsets of leukocytes, including neutrophils, monocytes, and lymphocytes, were significantly higher in the patients with severe sepsis than in the controls (p b 0.001). The characteristics of the survivors and non-survivors among patients with severe sepsis
Table 1 Baseline characteristics of patients with severe sepsis and control subjects.
Age (years) (mean ± SD) Male (%) Underlying diseases Diabetes mellitus (%) Hypertension (%) Cardiac disease (%) Systolic blood pressure (mmHg) (mean ± SD) Diastolic blood pressure (mmHg) (mean ± SD) Laboratory data (mean ± SD) White blood cells (×109/l) Platelet (×104/l) Hemoglobin(mg/dl) C-reactive protein (mg/l) Apoptosis tested by flow cytometry (mean ± SD) Leukocyte apoptosis (%) Annexin V (%) APO2.7 (%) Annexin V + 7AAD (%) Neutrophil apoptosis (%) Annexin V (%) APO2.7 (%) Annexin V + 7AAD (%) Lymphocyte apoptosis (%) Annexin V (%) APO2.7 (%) Annexin V + 7AAD (%) Monocyte apoptosis (%) Annexin V (%) APO2.7 (%) Annexin V + 7AAD (%)
Control n = 27
Study patients n = 87
p value
64.7 ± 12.0 58.6
65.3 ± 13.6 67.7
NS NS
34.5 60.6 11.1 142.7 ± 14.2
35.6 48.3 10.3 110.2 ± 42.6
NS NS NS b0.001
78.8 ± 10.1
71.5 ± 24.5
0.09
6.2 ± 2.5 234.4 ± 66.4 13.6 ± 1.7 1.2 ± 0.9
16.2 ± 11.1 140.8 ± 77.1 11.6 ± 2.9 192.1 ± 125.5
b0.001 b0.001 b0.001 b0.001
10.9 ± 2.9 0.7 ± 0.3 3.8 ± 2.0
13.7 ± 6.5 2.1 ± 1.9 7.3 ± 5.7
0.003 b0.001 b0.001
14.4 ± 5.8 0.6 ± 0.3 4.7 ± 2.6
15.2 ± 8.8 1.3 ± 1.1 7.2 ± 5.4
NS b0.001 NS
5.1 ± 1.9 0.4 ± 0.2 1.6 ± 0.7
7.8 ± 5.3 1.2 ± 0.9 2.0 ± 1.5
b0.001 b0.001 NS
13.1 ± 6.6 2.2 ± 2.0 4.9 ± 2.4
13.7 ± 7.7 7.6 ± 6.0 8.8 ± 6.1
NS b0.001 b0.001
Age (years) (mean ± SD) Male/Female Underlying diseases Diabetes mellitus Liver cirrhosis Chronic lung disease Stroke Cancer Maximum 24-h APACHE II score (mean ± SD) Bacteremia Primary site of infection Lung Urinary tract Intra-abdominal Soft tissue Unknown Causative pathogens Escherichia coli Klebsiella pneumoniae Other Gram-negative bacilli Streptococcal species Staphylococcal species Negative culture
Survivors n = 68
Non-survivors n = 19
p value
64.1 ± 14.0 44/24
69.3 ± 12.0 15/4
NS NS
22 10 10 14 13 19.4 ± 7.1
9 4 6 4 7 21.2 ± 5.6
NS NS NS NS NS NS
32
7
NS
32 24 6 1 5
11 5 2 1 0
14 8 5 7 3 31
2 3 1 2 0 11
were listed in Table 2. Among the patients, 68.2% (60/87) had septic shock within 24-h of admission and 40.2% (35/87) had ventilator treatment within 24-h of admission. Microbiology findings (Table 2) revealed positive blood cultures in 39 patients (44.8%) and negative culture results in 42 patients. Majority of the cultured isolates were Gram-negative microorganisms (n = 33), with a predominance of Escherichia coli (n = 16). The most common primary site of infection was pulmonary (n = 43). Corticosteroid treatment with low-dose hydrocortisone (300 mg/day) for 5 days was given to 38 patients (43.7%), but there was no significant difference between steroid users and non-users in terms of total leukocytes and in neutrophil, lymphocyte, and monocyte apoptosis.
Table 3 Laboratory data of the survivors and non-survivors among patients with severe sepsis. Survivors n = 68 White blood cells (×109/l) Neutrophil (×109/l) Lymphocyte (×109/l) Monocyte (×109/l) Hemoglobin (mg/dl) Platelet counts (×104/l) C-reactive protein (mg/l) Procalcitonin (ng/ml) Lactate (mg/dl) Apoptosis tested by flow cytometry Leukocyte apoptosis (%) Annexin V (%) APO2.7 (%) Annexin V + 7AAD (%) Neutrophil apoptosis (%) Annexin V (%) APO2.7 (%) Annexin V + 7AAD (%) Lymphocyte apoptosis (%) Annexin V (%) APO2.7 (%) Annexin V + 7AAD (%) Monocyte apoptosis (%) Annexin V (%) APO2.7 (%) Annexin V + 7AAD (%)
16.3 13.7 1.1 0.7 11.8 172.2 193.5 31.8 31.8
± ± ± ± ± ± ± ± ±
10.7 9.2 0.8 0.6 1.73 93.9 133.0 50.0 18.4
Non-survivors n = 19
p value
16.2 13.7 0.8 0.8 12.6 140.9 192.1 28.0 52.4
NS NS NS NS NS NS NS NS 0.001
± ± ± ± ± ± ± ± ±
11.0 9.6 0.7 0.8 1.86 77.1 125.5 45.1 38.5
13.9 ± 6.7 1.8 ± 1.5 6.8 ± 4.3
12.8 ± 5.9 3.1 ± 2.6 9.1 ± 8.9
NS 0.006 NS
15.8 ± 9.2 1.2 ± 1.4 7.2 ± 6.7
12.7 ± 6.9 1.9 ± 1.6 6.9 ± 11.5
NS 0.07 NS
7.4 ± 4.6 1.0 ± 1.1 1.8 ± 1.0
9.5 ± 7.2 1.8 ± 1.2 2.5 ± 2.5
NS 0.02 0.07
13.9 ± 7.3 6.8 ± 7.3 7.5 ± 6.3
12.7 ± 9.5 10.6 ± 9.5 14.0 ± 14.4
0NS 0.07 0.007
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3.2. Leukocyte apoptosis in survivors and non-survivors Laboratory data and the percentage of leukocyte apoptosis (Table 3) revealed significant differences in lactate concentration on admission (p b 0.001) between survivors and non-survivors. The percentages of APO2.7 in leukocyte apoptosis (p = 0.006), APO2.7 in lymphocyte apoptosis (p = 0.02), and annexin V + 7AAD in monocyte apoptosis (p = 0.007) were significantly higher among non-survivors than in survivors. 3.3. Time course of leukocyte apoptosis The time course of total leukocyte apoptosis (APO2.7), lymphocyte apoptosis (APO2.7), and monocyte apoptosis (annexin V + 7AAD) in patients with severe sepsis (Figs. 1–3) showed a clear trend that the levels of leukocyte apoptosis decreased with time among nonsurvivors, and that the first day in the ED had the highest percentages of leukocyte apoptosis (p b 0.05). Among the survivors, the highest percentage of apoptosis of total leukocytes and lymphocytes was found on Day 4. A sustained high level of lymphocyte apoptosis (APO2.7) was observed early among non-survivors from Day 1 to Day 4 (Fig. 2). There was no obvious change in monocytes apoptosis (annexin V + 7AAD) among survivors from Day1 to Day 7.
Fig. 2. Percentages of lymphocyte apoptosis (APO2.7) on different days in patients with severe sepsis and in control subjects. *p b 0.05, severe sepsis patients vs. controls; #p b 0.05, survivors vs. non-survivors.
were all independently predictive of fatality. Their areas under the ROC curve were 0.705 (p = 0.009, 95% CI: 0.563–0.848), 0.622 (p = 0.123, 95% CI: 0.455–0.790), and 0.694 (p = 0.014, 95% CI: 0.551–0.836), respectively (Fig. 4). The cutoff value for APO2.7 in lymphocyte apoptosis for predicting hospital fatality was 1.035 (61.2% sensitivity and 73.8% specificity).
3.4. Correlations between leukocyte apoptosis and inflammatory biomarkers 4. Discussion By correlation analysis, lymphocyte and monocyte late apoptosis (annexin V + 7AAD) on admission significantly correlated with the disease severity index mean sequential organ failure assessment (SOFA) score on admission (γ = 0.25, p = 0.027 and γ = 0.23, p = 0.038, respectively). There was no significant correlation between leukocyte apoptosis and C-reactive protein, procalcitonin, and lactate concentration. 3.5. Outcome and prognostic factors The case fatality rate was 21.6% (19/87). Potential prognostic factors of the 87 patients with severe sepsis were listed in Table 3. Statistical analysis of the clinical manifestations and laboratory data between the two patient groups revealed the following significant findings: APO2.7 in leukocyte apoptosis (p = 0.006), APO2.7 in lymphocyte apoptosis (p = 0.02), annexin V + 7AAD in monocyte apoptosis (p = 0.007), and serum lactate (p = 0.001). The significant univariate factors and possible confounding factors used in the stepwise logistic regression were APO2.7 in lymphocyte apoptosis, annexin V + 7AAD in monocyte apoptosis, and serum lactate. After analysis of all the above-mentioned variables, APO2.7 in lymphocyte apoptosis (p = 0.031, 95% CI: 0.378– 0.955), annexin V + 7AAD in monocyte apoptosis (p = 0.01, 95% CI: 0.855–0.979), and serum lactate (p = 0.008, 95% CI 0.944–0.992)
Fig. 1. Percentages of total leukocyte apoptosis (APO2.7) on different days in patients with severe sepsis and in control subjects. *p b 0.05, severe sepsis patients vs. controls; # p b 0.05, survivors vs. non-survivors.
The present study has several major findings. First, the percentages of APO2.7 in leukocyte apoptosis (including neutrophil, monocyte, and lymphocyte apoptosis) are significantly higher in patients with severe sepsis and septic shock than in the control subjects. Second, by correlation analysis, lymphocyte and monocyte late apoptosis (annexin V + 7AAD) on admission significantly correlate with the disease severity index mean SOFA score on admission (γ = 0.25, p = 0.027 and γ = 0.23, p = 0.038, respectively). Third, the percentages of APO2.7 in leukocyte apoptosis, APO2.7 in lymphocyte apoptosis, and annexin V + 7-AAD in monocyte apoptosis are significantly higher in non-survivors than in survivors. Fourth, APO2.7 in lymphocyte apoptosis, annexin V + 7AAD in monocyte apoptosis, and serum lactate are all independently predictive of fatality. Lastly, aside from lactate, APO2.7 level in lymphocyte apoptosis is also a useful predictor of outcome on ED admission. Sepsis is perhaps the most remarkable disease state related to abnormally increased apoptosis. Septic patients develop massive apoptosis of immune effector cells and gastrointestinal epithelial cells. The profound loss of immune effector cells in patients with sepsis inhibits their ability to eradicate the primary infection [3,12,18]. Another possible explanation for the increased apoptosis is the presence of oxidative stress. During septic shock, endothelial dysfunction is involved in impaired
Fig. 3. Percentages of monocytes apoptosis (annexin V + 7AAD) on different days in patients with severe sepsis and in control subjects. *p b 0.05, severe sepsis patients vs. controls; #p b 0.05, survivors vs. non-survivors.
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Fig. 4. Receiver operating characteristic (ROC) curve for lymphocyte apoptosis (APO2.7), monocyte apoptosis (annexin V + 7AAD), and lactate level on admission. The area under the ROC curve of lymphocyte apoptosis (APO2.7) was 0.705. (For interpretation of the references to colour in this figure, the reader is referred to the web version of this article.)
microcirculation and organ dysfunction. Reactive oxygen species (ROS) and reactive nitrogen species (RNS) have several potentially important effects on endothelial function and both ROS and RNS contribute to mitochondrial dysfunction by a range of mechanisms that induce necrotic and apoptotic cell death [19]. In the present study, the percentages of apoptosis in most subsets of leukocytes, including neutrophils, monocytes, and lymphocytes, are significantly higher in patients with severe sepsis than in controls. This phenomenon may reflect the correlation between severe inflammation and leukocyte apoptosis, such that the higher the leukocyte apoptosis, the greater the inflammatory response. Although the mechanisms contributing to increased leukocyte apoptosis in severe sepsis are not completely understood, numerous potential mechanisms have been proposed, including p38 mitogen-activated protein kinase signaling pathways, modulation of myeloid cell leukemia-1, pre-B cell colonyenhancing factor (PBEF), Fas ligand, and activation of nuclear factor-κB with a concomitant reduction in the activity of caspases 3 and 9 [20–23]. Moreover, the threshold that triggers apoptosis in different cells varies and each cell has a different pathway or different sequence of apoptotic organelles [24–26]. The present study also demonstrates that late apoptosis of the lymphocyte marker (APO2.7 on the mitochondrial membrane) is significantly higher among non-survivors and is independently predictive of mortality. Apoptosis is an important mechanism of cell death in lymphocytes and parenchymal cells in sepsis, and it occurs systemically in many organs. It may be an important cause of immunologic suppression in sepsis by inducing widespread lymphocyte depletion. A previous report has indicated that exaggerated lymphocyte apoptosis is present in the peripheral blood of patients with septic shock, in contrast to those with simple sepsis or critically ill non-septic patients. Lymphocyte apoptosis occurs rapidly, leads to profound and persistent lymphocyte loss, and is associated with poor patient outcome [5,8]. In the present study, there is a sustained high level of peripheral lymphocyte apoptosis (APO2.7) early among non-survivors, with severe sepsis from Day 1 to Day 4. This clearly demonstrates that lymphocyte apoptosis does not only appear as an early event, but is also an ubiquitous and prolonged phenomenon in severe sepsis. APO2.7 is expressed on the mitochondrial membrane and detectable lymphocyte APO2.7 may be related to mitochondrial activation and mitochondrial membrane rupture of lymphocytes during early apoptosis [27]. Two pathways have been suggested in septic lymphocyte apoptosis, i.e., the death of the receptor system and the mitochondrial pathway activation by various stimuli leading to caspase 9 activation [13,20,27]. These 2 pathways subsequently act on a final death cell program via caspase 3 cleavage [18]. In multiple
animal models of sepsis, survival rates have been remarkably improved by inhibiting lymphocyte apoptosis using selective caspase inhibitors [28], by altering pro-apoptotic/anti-apoptotic protein expression [29], by treatment with survival promoting cytokines like interleukin-7 [30], and by modulating co-stimulatory receptors [31]. Based on the present findings, the late monocyte apoptotic marker (annexin V + 7-AAD on the nuclear membrane of cells) is also significantly higher among non-survivors and is independently predictive of mortality. Although the apoptosis of blood monocytes has been reported in a limited number of patients with severe sepsis, no connection has been found with survival [32]. A previous report from a ventilatorassociated pneumonia study group reveals that early monocyte apoptosis N50% on the first day of presentation is associated with prolonged 28-day survival compared to patients with monocyte apoptosis b 50% [9]. Unlike this previous study, the present report of early monocyte apoptosis is not associated with improved survival in patients with severe sepsis. However, previous articles have used only a single method to study apoptosis (flow cytometry of annexin-V for early apoptosis). The duration of illness before enrolment in this study may introduce enough variables to make timing difficult. Moreover, sepsis is a syndrome characterized by great heterogeneity. Although all patients are ubiquitously characterized as septic, they greatly differ in terms of sex, age, co-morbid conditions, underlying infections, and offending pathogens. Monocytes play an essential role against microbial infection in the innate immune defense. Monocytes are usually regarded as contributory to the ramp up of inflammatory responses rather than triggering their cessation [33]. They rapidly exhibit an impaired production of proinflammatory cytokines in response to additional bacterial challenge [34]. During the inflammatory response, monocytes present antigens by means of expression of the human leukocyte antigen (locus) DR (HLA-DR) receptors and by secreting pro-inflammatory cytokines to amplify the immune response [35]. Many studies have demonstrated that during sepsis-induced immuno-suppression, monocytes secrete fewer cytokines and down-regulate the expression of HLA receptors. This impaired function of monocytes is generally predictive of increased risk of secondary infection and poor prognosis in critically ill patients [36].
4.1. Study limitations The strength of this study is the relative homogeneity of the study cohort (non- traumatic, non-surgical adult severe sepsis patients at the ED), use of different methods to determine leukocyte apoptosis, and prospective study design. Nonetheless, the data here should be interpreted in light of several inherent limitations. First, there are many assays that evaluate leukocyte apoptosis in vitro, so the results and interpretation of various methodologies may be partially different. The level of leukocyte apoptosis tested by flow cytometry may not necessarily reflect the real leukocyte physiologic function in vivo. Second, the choice of therapeutic strategy for sepsis (e.g., use of steroids, dosage and duration of steroids) may be different for each patient based on the preference of his/her doctor. This may cause potential bias in the statistical analysis. Lastly, the case number is small and the follow-up period is short. Large-scale prospective studies are warranted to evaluate the prognostic contribution of leukocyte apoptosis on clinical outcomes.
5. Conclusions Leukocyte apoptosis is significantly higher in patients with severe sepsis and the percentages of late apoptosis of lymphocytes and monocytes has potential use in predicting the outcome of such patients at the ED. Aside from serum lactate, APO2.7 level in lymphocyte apoptosis is also a useful predictor of outcome on ED admission.
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Acknowledgments This work was supported by grants from Chang Gung Memorial Hospital (Chang Gung Medical Research Project CMRPG891341) and NHRI-EX101-10142EI. The authors wish to thank Dr. Gene Alzona Nisperos for editing and reviewing the manuscript for English language considerations. References [1] Martin GS, Mannino DM, Eaton S, Moss M. The epidemiology of sepsis in the United States from 1979 through 2000. N Engl J Med 2003;348:1546–54. [2] Brunelle JK, Chandel NS. Oxygen deprivation induced cell death: an update. Apoptosis 2002;7:475–82. [3] Hotchkiss RS, Swanson PE, Freeman BD, et al. Apoptotic cell death in patients with sepsis, shock, and multiple organ dysfunction. Crit Care Med 1999;27:1230–51. [4] Hotchkiss RS, Tinsley KW, Karl IE. Role of apoptotic cell death in sepsis. Scand J Infect Dis 2003;35:585–93. [5] Wesche DE, Lomas-Neira JL, Perl M, Chung CS, Ayala A. Leukocyte apoptosis and its significance in sepsis and shock. J Leukoc Biol 2005;78:325–37. [6] Harter L, Mica L, Stocker R, Trentz O, Keel M. Mcl-1 correlates with reduced apoptosis in neutrophils from patients with sepsis. J Am Coll Surg 2003;197:964–73. [7] Daigneault M, De Silva TI, Bewley MA, et al. Monocytes regulate the mechanism of Tcell death by inducing Fas-mediated apoptosis during bacterial infection. PLoS Pathog 2012;8:e1002814. [8] Le Tulzo Y, Pangault C, Gacouin A, et al. Early circulating lymphocyte apoptosis in human septic shock is associated with poor outcome. Shock 2002;18:487–94. [9] Giamarellos-Bourboulis EJ, Routsi C, Plachouras D, et al. Early apoptosis of blood monocytes in the septic host: is it a mechanism of protection in the event of septic shock? Crit Care 2006;10:R76. [10] Fialkow L, Fochesatto Filho L, Bozzetti MC, et al. Neutrophil apoptosis: a marker of disease severity in sepsis and sepsis-induced acute respiratory distress syndrome. Crit Care 2006;10:R155. [11] Pelekanou A, Tsangaris I, Kotsaki A, et al. Decrease of CD4-lymphocytes and apoptosis of CD14-monocytes are characteristic alterations in sepsis caused by ventilatorassociated pneumonia: results from an observational study. Crit Care 2009;13:R172. [12] Hotchkiss RS, Schmieg Jr RE, Swanson PE, et al. Rapid onset of intestinal epithelial and lymphocyte apoptotic cell death in patients with trauma and shock. Crit Care Med 2000;28:3207–17. [13] Hotchkiss RS, Nicholson DW. Apoptosis and caspases regulate death and inflammation in sepsis. Nat Rev Immunol 2006;6:813–22. [14] Tang PS, Mura M, Seth R, Liu M. Acute lung injury and cell death: how many ways can cells die? Am J Physiol Lung Cell Mol Physiol 2008;294:L632–41. [15] Hotchkiss RS, Coopersmith CM, Karl IE. Prevention of lymphocyte apoptosis—a potential treatment of sepsis? Clin Infect Dis 2005;41:S465–9. [16] American College of Chest Physicians/Society of Critical Care Medicine Consensus Committee. Definition for sepsis and organ failures and guidelines for the use of innovative therapies in sepsis. Chest 1992;101:1658–62. [17] Koester SK, Roth P, Mikulka WR, Schlossman SF, Zhang C, Bolton WE. Monitoring early cellular responses in apoptosis is aided by the mitochondrial membrane protein-specific monoclonal antibody APO2.7. Cytometry 1997;29:306–12.
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[18] Hotchkiss RS, Tinsley KW, Swanson PE, et al. Sepsis-induced apoptosis causes progressive profound depletion of B and CD4+ T lymphocytes in humans. J Immunol 2001;166:6952–63. [19] Bayir H, Kagan VE. Bench-to-bedside review: mitochondrial injury, oxidative stress and apoptosis—there is nothing more practical than a good theory. Crit Care 2008; 12:206. http://dx.doi.org/10.1186/cc6779. [20] Ayala A, Perl M, Venet F, Lomas-Neira J, Swan R, Chung CS. Apoptosis in sepsis: mechanisms, clinical impact and potential therapeutic targets. Curr Pharm Des 2008;14:1853–9. [21] Sheth K, Friel J, Nolan B, Bankey P. Inhibition of p38 mitogen activated protein kinase increases lipopolysaccharide induced inhibition of apoptosis in neutrophils by activating extracellular signal-regulated kinase. Surgery 2001;130:242–8. [22] Jia SH, Li Y, Parodo J, et al. Pre-B cell colony-enhancing factor inhibits neutrophil apoptosis in experimental inflammation and clinical sepsis. J Clin Invest 2004;113: 1318–27. [23] Taneja R, Parodo J, Jia SH, Kapus A, Rotstein OD, Marshall JC. Delayed neutrophil apoptosis in sepsis is associated with maintenance of mitochondrial trans-membrane potential and reduced caspase-9 activity. Crit Care Med 2004;32:1460–9. [24] Le Bras M, Rouy I, Brenner C. The modulation of inter-organelle cross-talk to control apoptosis. Med Chem 2006;2:1–12. [25] Cheng JP, Lane JD. Organelle dynamics and membrane trafficking in apoptosis and autophagy. Histol Histopathol 2010;25:1457–72. [26] Bussing A, Vervecken W, Wagner M, Wagner B, Pfuller U, Schietzel M. Expression of mitochondrial Apo2.7 molecules and caspase-3 activation in human lymphocytes treated with the ribosome-inhibiting mistletoe lectins and the cell membrane permeabilizing viscotoxins. Cytometry 1999;37:133–9. [27] Sesso A, Belizario JE, Marques MM, et al. Mitochondrial swelling and incipient outer membrane rupture in pre-apoptotic and apoptotic cells. Anat Rec (Hoboken) 2012; 295:1647–59. [28] Vandenabeele P, Vanden Berghe T, Festjens N. Caspase inhibitors promote alternative cell death pathways. Sci STKE 2006;2006:pe44. [29] Matsuda N, Yamamoto S, Takano K, et al. Silencing of Fas-associated death domain protects mice from septic lung inflammation and apoptosis. Am J Respir Crit Care Med 2009;179:806–8. [30] Unsinger J, McGlynn M, Kasten KR, et al. IL-7 promotes T cell viability, trafficking, functionality and improves survival in sepsis. J Immunol 2010;184:3768–79. [31] Fife BT, Bluestone JA. Control of peripheral T-cell tolerance and autoimmunity via the CTLA-4 and PD-1 pathways. Immunol Rev 2008;224:166–82. [32 Adrie C, Bachelet M, Vayssier-Taussat M, et al. Mitochondrial membrane potential and apoptosis peripheral blood monocytes in severe human sepsis. Am J Resp Crit Care Med 2001;164:389–95. [33] Serbina NV, Jia T, Hohl TM, Pamer EG. Monocyte-mediated defense against microbial pathogens. Annu Rev Immunol 2008;26:421–52. [34] Munoz C, Carlet J, Fitting C, Misset B, Bleriot JP, Cavaillon JM. Dysregulation of in vitro cytokine production by monocytes during sepsis. J Clin Invest 1991;88:1747–54. [35] Manjuck J, Saha DC, Astiz M, Eales LJ, Rackow EC. Decreased response to recall antigens is associated with depressed co-stimulatory receptor expression in septic critically ill patients. J Lab Clin Med 2000;135:153–60. [36] Ward NS, Casserly B, Ayala A. The compensatory anti-inflammatory response syndrome (CARS) in critically ill patients. Clin Chest Med 2008;29:617–27.
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The prognostic value of leukocyte apoptosis in patients with severe sepsis at the emergency department Chia-Te Kung a, Chih-Min Su a,b, Hsueh-Wen Chang b, Hsien-Hung Cheng a, Sheng-Yuan Hsiao a, Tsung-Cheng Tsai a, Nai-Wen Tsai c, Hung-Chen Wang d, Yu-Jih Su b,e, Wei-Che Lin f, Ben-Chung Cheng b,e, Ya-Ting Chang b,c, Yi-Fang Chiang c, Cheng-Hsien Lu b,c,⁎ a
Department of Emergency Medicine, Chang Gung Memorial Hospital-Kaohsiung Medical Center, Chang Gung University College of Medicine, Kaohsiung, Taiwan Department of Biological Science, National Sun Yat-Sen University, Kaohsiung, Taiwan c Department of Neurology, Chang Gung Memorial Hospital-Kaohsiung Medical Center, Chang Gung University College of Medicine, Kaohsiung, Taiwan d Department of Neurosurgery, Chang Gung Memorial Hospital-Kaohsiung Medical Center, Chang Gung University College of Medicine, Kaohsiung, Taiwan e Department of Medicine, Chang Gung Memorial Hospital-Kaohsiung Medical Center, Chang Gung University College of Medicine, Kaohsiung, Taiwan f Department of Radiology, Chang Gung Memorial Hospital-Kaohsiung Medical Center, Chang Gung University College of Medicine, Kaohsiung, Taiwan b
a r t i c l e
i n f o
Article history: Received 29 April 2014 Received in revised form 9 September 2014 Accepted 19 September 2014 Available online 28 September 2014 Keywords: Outcome Lymphocyte apoptosis Monocyte apoptosis Severe sepsis
a b s t r a c t Background and aim: Cell apoptosis in critically ill patients plays a pivotal role in the pathogenesis of sepsis. This study aimed to determine the prognostic value of leukocyte apoptosis in patients with severe sepsis. Methods: Leukocyte apoptosis was determined by flow cytometry. The values of annexin V, APO2.7, and 7-aminoactinomycin D (7AAD) for each subtype of leukocyte were analyzed in 87 patients with severe sepsis and 27 controls. Results: The percentages of apoptosis (APO2.7 [%]) in the leukocyte subsets were significantly higher in the patients with severe sepsis than in the controls. The percentages of APO2.7 in leukocyte apoptosis, APO2.7 in lymphocytes apoptosis, and annexin V + 7AAD in monocytes apoptosis were significantly higher in non-survivors than in survivors. Levels of APO2.7 in lymphocytes apoptosis, annexin V + 7AAD in monocytes apoptosis, and serum lactate were all independently predictive of mortality. Conclusion: Leukocyte apoptosis is significantly higher in patients with severe sepsis. The percentages of late lymphocyte and monocyte apoptosis may be predictive of outcome in such patients. Aside from serum lactate, APO2.7 level in lymphocyte apoptosis is also a useful predictor of outcome on admission to the emergency department. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Severe sepsis and sequential multiple organ failure are still the leading causes of death worldwide despite advances in medical care [1]. The pathogenesis of multiple organ failure in patients with severe sepsis is a multi-factorial process, but global tissue hypoxia due to an imbalance between systemic oxygen delivery and peripheral oxygen demand plays an important role [2,3]. There is growing evidence that aside from cellular necrosis, dysregulated leukocyte apoptosis may also influence the increasing duration and severity of systemic response to sepsis in critically ill patients, including those with acute respiratory distress syndrome, shock, and trauma [4–12]. Abbreviations: APACHE, Acute Physiology and Chronic Health Evaluation; PE, phycoerythrin; FITC, fluorescein isothiocyanate; 7-AAD, amino-actinomycin D; SOFA, sequential organ failure assessment; PBEF, pre-B cell colony-enhancing factor. ⁎ Corresponding author at: Department of Neurology, Kaohsiung Chang Gung Memorial Hospital, Chang Gung University College of Medicine, No. 123, Ta Pei Road, Niao Sung Hsiang, Kaohsiung City 833, Taiwan. Tel.: +886 7 7317123x2283. E-mail addresses: [email protected], [email protected] (C.-H. Lu).
http://dx.doi.org/10.1016/j.cca.2014.09.017 0009-8981/© 2014 Elsevier B.V. All rights reserved.
Severe sepsis and septic shock represent an over-exuberant host response to an infectious challenge. Neutrophils, monocytes, and macrophages play key roles in the initial reaction and release a variety of cytokines that marshal the immune response. Once the adaptive immune response is awakened, innate immune cells are downregulated and must be disposed of in a timely and non-injurious manner. Apoptosis represents a key mechanism in this orderly process, but it is not always good. The apoptotic cascade supervenes during the evolution of sepsis in lymphocytes, in tissue macrophages, and in intestinal epithelia and has been associated with organ dysfunction [13–15]. Moreover, the apoptosis of both alveolar epithelial cells and respiratory endothelial cells has been conclusively demonstrated in animals and humans with acute lung injury and acute respiratory distress syndrome [14]. Previous studies also reveal that lymphocyte apoptosis is rapidly increased in the blood of patients with septic shock and this leads to profound and persistent lymphopenia that is associated with poor outcome. The apoptosis of blood monocytes in septic patients is also associated with their final outcome [8,9].
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Previous studies have used only a single method to study apoptosis [8,9,11] or only one leukocyte subset to predict the outcome of sepsis [8,9]. Furthermore, the timing of apoptosis is important and the duration of illness before enrolment is connected to outcome. If such variations follow a standard pattern and temporal relationship, prognostication will be substantially improved. 2. Methods 2.1. Study population and definition This prospective study on patients with severe sepsis and septic shock was conducted over a 1-year period (January to December 2011). Eighty-seven adult non-traumatic and non-surgical patients at Kaohsiung Chang Gung Memorial Hospital were enrolled. The hospital's Institutional Review Committee on Human Research approved the study protocol and all of the patients provided written informed consent. All patients aged N18 years consecutively admitted from the ED were screened daily for severe sepsis and septic shock according to the specific criteria defined by the American College of Chest Physicians/Society of Critical Care Medicine. These criteria included suspected or confirmed infection, two or more manifestations of systemic inflammatory response syndrome, and at least 1 sepsis-induced acute organ dysfunction. Patients who met all three criteria were included [16]. For comparison, 27 age- and sex-matched healthy volunteers who received annual physical check-up but without clinical evidence of infection were recruited as controls. 2.2. Clinical assessment and treatment The medical records were prospectively recorded using pre-existing standardized evaluation forms that included demographic data and the Acute Physiology and Chronic Health Evaluation (APACHE) II score, which was calculated during the first 24 h of admission to assess the severity of organ dysfunction. Basic laboratory tests, lactate concentration, and inflammatory markers (i.e., plasma C-reactive protein and procalcitonin) were taken on ED admission. Data on the source of infection and use of antibiotics were also recorded. The course of various organ dysfunctions and supportive treatments, including vasoactive and ventilator therapies and renal replacement therapies, were recorded. Physicians evaluated daily the association of existing organ dysfunction and severe sepsis. It was also institutional practice to consult an infectious disease specialist for anti-microbial treatment based on treatment guidelines for different infectious etiologies during the first 24 h. 2.3. Blood sampling and assessment of leukocyte apoptosis Blood samples were collected on presentation to the ED (Day 1). Follow-up blood samples were obtained on Days 4 and 7 after admission. All blood samples were collected by venipuncture of forearm veins from both the study group and controls. All flow cytometry assays were performed within 1 h after blood extraction to ensure that the results were as close as possible to an in vivo situation. Flow cytometry assay using APO 2.7 antibody for detecting apoptosis. Fixed amounts of blood were diluted 1:5 with PBS, and 100 μL was stained with 10 μl of each of the following: fluorescence-conjugated monoclonal antibodies against CD45-phycoerythrin (PE)-Cy5 (clone J33), CD61-fluorescein isothiocyanate (FITC; clone SZ21), and APO 2.7PE (clone 2.7A6A3; Immunotech). The samples were titrated at saturating concentrations. The CD45-PE-Cy5 antibody reacted with the CD45 family of trans-membrane glycoproteins, expressed on the surface of all human leukocytes, and a pan-leukocyte marker. The CD61-FITC antibody was a pan-platelet marker that reacted with the GPIIb/IIIa complex (fibrinogen receptor).
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The APO 2.7-PE antibody reacted with a 38-kDa mitochondrial membrane protein (7A6 antigen), which was detectable on nonpermeabilized cells in the late apoptotic state [17]. Annexin staining, relevant to early apoptosis, produced similar results but was rejected for questionable reliability under fixation conditions, with formaldehyde clearly biasing the staining results. Mouse immunoglobulin G-PE was a control for non-specific staining, but it did not differ from the APO2.7-PE signal on platelets. Thus, each subject was used as its own control without changing the sample tube. After 30 min of incubation in the dark at room temperature, the stained samples were diluted with 0.5 ml of FACSFlow (Becton Dickinson). Flow cytometry was performed immediately after staining using an Epics XL flow cytometer (Beckman Coulter) and the CellQuest software. Five thousand CD45-PE-Cy5+ cells per sample were acquired in a combined forward and side scatters, and deep-red FL4 fluorescence (CD45PE-Cy5) leukocyte gate. Another 5000 CD61-FITC + cells per sample were acquired in a combined forward and side scatters, and green FL1 fluorescence (CD61-FITC) platelet gate to define the negative control threshold for the measurement of apoptosis, so that each subject was its own control. 2.3.1. Annexin V-FITC 7-AAD Fluorescence-Activated Cell Analysis Membrane phosphatidyl-serine was detected by annexin-V using a commercially available kit (Boehringer Mannheim). The PBS-washed leukocytes were incubated with annexin V-FITC and 7-amino-actinomycin D (7-AAD) for 15 min at room temperature according to the manufacturer's guidelines. Samples were transferred to 5-ml polypropylene tubes, diluted with 900 μl Hanks' balanced salt solution, and placed on ice before flow cytometry analysis. The samples were analyzed using an Epics XL flow cytometer (Beckman Coulter) and CellQuest software. Fifteen thousand events were counted per sample. Low-fluorescence debris was gated-out of the analysis. Leukocyte subtypes were identified according to their CD45 expression intensity and were divided into neutrophils, monocytes, and lymphocytes. From here on, white blood cells (WBC) represented total leukocytes. Annexin V-FITC staining was identified in fluorescent-1 and 7-AAD staining in fluorescent-4. The cells were identified as follows: early apoptotic cells if they were positive for marker annexin V-FITC but negative for 7-AAD; late apoptotic cells if they were positive for annexin V-FITC and 7-AAD; dead cells if they were negative for annexin V-FITC but positive for 7-AAD; and viable cells if they were negative for annexin V-FITC and 7-AAD. 2.3.2. Apoptosis of lymphocytes Fixed amounts of blood were diluted 1:5 with PBS and 100 μl was stained with 10 μl of each of the following: fluorescence-conjugated monoclonal antibodies against CD4-phycoerythrin (PE)-Cy5, CD19fluorescein isothiocyanate (FITC), and CD8- phycoerythrin (PE). Each sample was further stained with annexin V-FITC, 7-amino-actinomycin D (7-AAD), or APO 2.7-PE (clone 2.7A6A3; Immunotech, Marseille, France) and titrated at saturating concentrations. The samples were then transferred to 5-ml polypropylene tubes, diluted with 900 μl of Hanks balanced salt solution, and placed on ice before flow cytometry analysis. The samples were analyzed using an Epics XL flow cytometer (Beckman Coulter) and CellQuest software. Fifteen thousand events were counted per sample. Lymphocyte sub-types were identified according to their surface antigen (i.e., CD4, CD8, or CD19) expression intensity. A database coordinator was responsible for monitoring all of the data collection and entry. All of the tests were performed in a quality-controlled central laboratory at Chang-Gung Memorial Hospital. Concentrations of CRP were determined by enzyme immunoassay (EMIT; Merck Diagnostica), while PCT was measured using a time-resolved amplified cryptate emission technology assay (VIDAS; bioMerieux). Serum lactate levels were measured using a serum-based assay catalyzed by lactate oxidase (UniCel Integrated System; Beckman Coulter INS).
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2.4. Statistical analysis
Table 2 Characteristics of survivors and non-survivors among patients with severe sepsis.
Data were expressed as mean ± SD or median (inter-quartile range). Univariate analyses were compared using Student's t-test, while categorical variables were compared using χ2 test or Fisher's exact test, as appropriate. Correlation analysis was used to explore the relationship among 24-h APACHE II score, serum lactate, C-reactive protein, procalcitonin, creatinine concentration, and levels of leukocyte apoptosis of patients with severe sepsis on admission. Stepwise logistic regression was used to evaluate the relationship between significant variables and therapeutic outcomes, with adjustments for other potential confounding factors. Variables with zero cell count in a 2-by-2 table were eliminated from logistic analysis and only those strongly associated with fatality (p b 0.05) were included in the final model. Receiver operating characteristic (ROC) curves were generated for significant predictor variables of hospital mortality. All statistical analyses were conducted using the SAS software package, ver. 9.1 (2002, SAS Statistical Institute).
3. Results 3.1. Baseline characteristics of the study patients The baseline characteristics of patients with severe sepsis (n = 87) and the healthy controls (n = 27) showed no significant differences in terms of underlying disease (Table 1). The percentages of apoptosis (APO2.7 [%]) in the subsets of leukocytes, including neutrophils, monocytes, and lymphocytes, were significantly higher in the patients with severe sepsis than in the controls (p b 0.001). The characteristics of the survivors and non-survivors among patients with severe sepsis
Table 1 Baseline characteristics of patients with severe sepsis and control subjects.
Age (years) (mean ± SD) Male (%) Underlying diseases Diabetes mellitus (%) Hypertension (%) Cardiac disease (%) Systolic blood pressure (mmHg) (mean ± SD) Diastolic blood pressure (mmHg) (mean ± SD) Laboratory data (mean ± SD) White blood cells (×109/l) Platelet (×104/l) Hemoglobin(mg/dl) C-reactive protein (mg/l) Apoptosis tested by flow cytometry (mean ± SD) Leukocyte apoptosis (%) Annexin V (%) APO2.7 (%) Annexin V + 7AAD (%) Neutrophil apoptosis (%) Annexin V (%) APO2.7 (%) Annexin V + 7AAD (%) Lymphocyte apoptosis (%) Annexin V (%) APO2.7 (%) Annexin V + 7AAD (%) Monocyte apoptosis (%) Annexin V (%) APO2.7 (%) Annexin V + 7AAD (%)
Control n = 27
Study patients n = 87
p value
64.7 ± 12.0 58.6
65.3 ± 13.6 67.7
NS NS
34.5 60.6 11.1 142.7 ± 14.2
35.6 48.3 10.3 110.2 ± 42.6
NS NS NS b0.001
78.8 ± 10.1
71.5 ± 24.5
0.09
6.2 ± 2.5 234.4 ± 66.4 13.6 ± 1.7 1.2 ± 0.9
16.2 ± 11.1 140.8 ± 77.1 11.6 ± 2.9 192.1 ± 125.5
b0.001 b0.001 b0.001 b0.001
10.9 ± 2.9 0.7 ± 0.3 3.8 ± 2.0
13.7 ± 6.5 2.1 ± 1.9 7.3 ± 5.7
0.003 b0.001 b0.001
14.4 ± 5.8 0.6 ± 0.3 4.7 ± 2.6
15.2 ± 8.8 1.3 ± 1.1 7.2 ± 5.4
NS b0.001 NS
5.1 ± 1.9 0.4 ± 0.2 1.6 ± 0.7
7.8 ± 5.3 1.2 ± 0.9 2.0 ± 1.5
b0.001 b0.001 NS
13.1 ± 6.6 2.2 ± 2.0 4.9 ± 2.4
13.7 ± 7.7 7.6 ± 6.0 8.8 ± 6.1
NS b0.001 b0.001
Age (years) (mean ± SD) Male/Female Underlying diseases Diabetes mellitus Liver cirrhosis Chronic lung disease Stroke Cancer Maximum 24-h APACHE II score (mean ± SD) Bacteremia Primary site of infection Lung Urinary tract Intra-abdominal Soft tissue Unknown Causative pathogens Escherichia coli Klebsiella pneumoniae Other Gram-negative bacilli Streptococcal species Staphylococcal species Negative culture
Survivors n = 68
Non-survivors n = 19
p value
64.1 ± 14.0 44/24
69.3 ± 12.0 15/4
NS NS
22 10 10 14 13 19.4 ± 7.1
9 4 6 4 7 21.2 ± 5.6
NS NS NS NS NS NS
32
7
NS
32 24 6 1 5
11 5 2 1 0
14 8 5 7 3 31
2 3 1 2 0 11
were listed in Table 2. Among the patients, 68.2% (60/87) had septic shock within 24-h of admission and 40.2% (35/87) had ventilator treatment within 24-h of admission. Microbiology findings (Table 2) revealed positive blood cultures in 39 patients (44.8%) and negative culture results in 42 patients. Majority of the cultured isolates were Gram-negative microorganisms (n = 33), with a predominance of Escherichia coli (n = 16). The most common primary site of infection was pulmonary (n = 43). Corticosteroid treatment with low-dose hydrocortisone (300 mg/day) for 5 days was given to 38 patients (43.7%), but there was no significant difference between steroid users and non-users in terms of total leukocytes and in neutrophil, lymphocyte, and monocyte apoptosis.
Table 3 Laboratory data of the survivors and non-survivors among patients with severe sepsis. Survivors n = 68 White blood cells (×109/l) Neutrophil (×109/l) Lymphocyte (×109/l) Monocyte (×109/l) Hemoglobin (mg/dl) Platelet counts (×104/l) C-reactive protein (mg/l) Procalcitonin (ng/ml) Lactate (mg/dl) Apoptosis tested by flow cytometry Leukocyte apoptosis (%) Annexin V (%) APO2.7 (%) Annexin V + 7AAD (%) Neutrophil apoptosis (%) Annexin V (%) APO2.7 (%) Annexin V + 7AAD (%) Lymphocyte apoptosis (%) Annexin V (%) APO2.7 (%) Annexin V + 7AAD (%) Monocyte apoptosis (%) Annexin V (%) APO2.7 (%) Annexin V + 7AAD (%)
16.3 13.7 1.1 0.7 11.8 172.2 193.5 31.8 31.8
± ± ± ± ± ± ± ± ±
10.7 9.2 0.8 0.6 1.73 93.9 133.0 50.0 18.4
Non-survivors n = 19
p value
16.2 13.7 0.8 0.8 12.6 140.9 192.1 28.0 52.4
NS NS NS NS NS NS NS NS 0.001
± ± ± ± ± ± ± ± ±
11.0 9.6 0.7 0.8 1.86 77.1 125.5 45.1 38.5
13.9 ± 6.7 1.8 ± 1.5 6.8 ± 4.3
12.8 ± 5.9 3.1 ± 2.6 9.1 ± 8.9
NS 0.006 NS
15.8 ± 9.2 1.2 ± 1.4 7.2 ± 6.7
12.7 ± 6.9 1.9 ± 1.6 6.9 ± 11.5
NS 0.07 NS
7.4 ± 4.6 1.0 ± 1.1 1.8 ± 1.0
9.5 ± 7.2 1.8 ± 1.2 2.5 ± 2.5
NS 0.02 0.07
13.9 ± 7.3 6.8 ± 7.3 7.5 ± 6.3
12.7 ± 9.5 10.6 ± 9.5 14.0 ± 14.4
0NS 0.07 0.007
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3.2. Leukocyte apoptosis in survivors and non-survivors Laboratory data and the percentage of leukocyte apoptosis (Table 3) revealed significant differences in lactate concentration on admission (p b 0.001) between survivors and non-survivors. The percentages of APO2.7 in leukocyte apoptosis (p = 0.006), APO2.7 in lymphocyte apoptosis (p = 0.02), and annexin V + 7AAD in monocyte apoptosis (p = 0.007) were significantly higher among non-survivors than in survivors. 3.3. Time course of leukocyte apoptosis The time course of total leukocyte apoptosis (APO2.7), lymphocyte apoptosis (APO2.7), and monocyte apoptosis (annexin V + 7AAD) in patients with severe sepsis (Figs. 1–3) showed a clear trend that the levels of leukocyte apoptosis decreased with time among nonsurvivors, and that the first day in the ED had the highest percentages of leukocyte apoptosis (p b 0.05). Among the survivors, the highest percentage of apoptosis of total leukocytes and lymphocytes was found on Day 4. A sustained high level of lymphocyte apoptosis (APO2.7) was observed early among non-survivors from Day 1 to Day 4 (Fig. 2). There was no obvious change in monocytes apoptosis (annexin V + 7AAD) among survivors from Day1 to Day 7.
Fig. 2. Percentages of lymphocyte apoptosis (APO2.7) on different days in patients with severe sepsis and in control subjects. *p b 0.05, severe sepsis patients vs. controls; #p b 0.05, survivors vs. non-survivors.
were all independently predictive of fatality. Their areas under the ROC curve were 0.705 (p = 0.009, 95% CI: 0.563–0.848), 0.622 (p = 0.123, 95% CI: 0.455–0.790), and 0.694 (p = 0.014, 95% CI: 0.551–0.836), respectively (Fig. 4). The cutoff value for APO2.7 in lymphocyte apoptosis for predicting hospital fatality was 1.035 (61.2% sensitivity and 73.8% specificity).
3.4. Correlations between leukocyte apoptosis and inflammatory biomarkers 4. Discussion By correlation analysis, lymphocyte and monocyte late apoptosis (annexin V + 7AAD) on admission significantly correlated with the disease severity index mean sequential organ failure assessment (SOFA) score on admission (γ = 0.25, p = 0.027 and γ = 0.23, p = 0.038, respectively). There was no significant correlation between leukocyte apoptosis and C-reactive protein, procalcitonin, and lactate concentration. 3.5. Outcome and prognostic factors The case fatality rate was 21.6% (19/87). Potential prognostic factors of the 87 patients with severe sepsis were listed in Table 3. Statistical analysis of the clinical manifestations and laboratory data between the two patient groups revealed the following significant findings: APO2.7 in leukocyte apoptosis (p = 0.006), APO2.7 in lymphocyte apoptosis (p = 0.02), annexin V + 7AAD in monocyte apoptosis (p = 0.007), and serum lactate (p = 0.001). The significant univariate factors and possible confounding factors used in the stepwise logistic regression were APO2.7 in lymphocyte apoptosis, annexin V + 7AAD in monocyte apoptosis, and serum lactate. After analysis of all the above-mentioned variables, APO2.7 in lymphocyte apoptosis (p = 0.031, 95% CI: 0.378– 0.955), annexin V + 7AAD in monocyte apoptosis (p = 0.01, 95% CI: 0.855–0.979), and serum lactate (p = 0.008, 95% CI 0.944–0.992)
Fig. 1. Percentages of total leukocyte apoptosis (APO2.7) on different days in patients with severe sepsis and in control subjects. *p b 0.05, severe sepsis patients vs. controls; # p b 0.05, survivors vs. non-survivors.
The present study has several major findings. First, the percentages of APO2.7 in leukocyte apoptosis (including neutrophil, monocyte, and lymphocyte apoptosis) are significantly higher in patients with severe sepsis and septic shock than in the control subjects. Second, by correlation analysis, lymphocyte and monocyte late apoptosis (annexin V + 7AAD) on admission significantly correlate with the disease severity index mean SOFA score on admission (γ = 0.25, p = 0.027 and γ = 0.23, p = 0.038, respectively). Third, the percentages of APO2.7 in leukocyte apoptosis, APO2.7 in lymphocyte apoptosis, and annexin V + 7-AAD in monocyte apoptosis are significantly higher in non-survivors than in survivors. Fourth, APO2.7 in lymphocyte apoptosis, annexin V + 7AAD in monocyte apoptosis, and serum lactate are all independently predictive of fatality. Lastly, aside from lactate, APO2.7 level in lymphocyte apoptosis is also a useful predictor of outcome on ED admission. Sepsis is perhaps the most remarkable disease state related to abnormally increased apoptosis. Septic patients develop massive apoptosis of immune effector cells and gastrointestinal epithelial cells. The profound loss of immune effector cells in patients with sepsis inhibits their ability to eradicate the primary infection [3,12,18]. Another possible explanation for the increased apoptosis is the presence of oxidative stress. During septic shock, endothelial dysfunction is involved in impaired
Fig. 3. Percentages of monocytes apoptosis (annexin V + 7AAD) on different days in patients with severe sepsis and in control subjects. *p b 0.05, severe sepsis patients vs. controls; #p b 0.05, survivors vs. non-survivors.
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Fig. 4. Receiver operating characteristic (ROC) curve for lymphocyte apoptosis (APO2.7), monocyte apoptosis (annexin V + 7AAD), and lactate level on admission. The area under the ROC curve of lymphocyte apoptosis (APO2.7) was 0.705. (For interpretation of the references to colour in this figure, the reader is referred to the web version of this article.)
microcirculation and organ dysfunction. Reactive oxygen species (ROS) and reactive nitrogen species (RNS) have several potentially important effects on endothelial function and both ROS and RNS contribute to mitochondrial dysfunction by a range of mechanisms that induce necrotic and apoptotic cell death [19]. In the present study, the percentages of apoptosis in most subsets of leukocytes, including neutrophils, monocytes, and lymphocytes, are significantly higher in patients with severe sepsis than in controls. This phenomenon may reflect the correlation between severe inflammation and leukocyte apoptosis, such that the higher the leukocyte apoptosis, the greater the inflammatory response. Although the mechanisms contributing to increased leukocyte apoptosis in severe sepsis are not completely understood, numerous potential mechanisms have been proposed, including p38 mitogen-activated protein kinase signaling pathways, modulation of myeloid cell leukemia-1, pre-B cell colonyenhancing factor (PBEF), Fas ligand, and activation of nuclear factor-κB with a concomitant reduction in the activity of caspases 3 and 9 [20–23]. Moreover, the threshold that triggers apoptosis in different cells varies and each cell has a different pathway or different sequence of apoptotic organelles [24–26]. The present study also demonstrates that late apoptosis of the lymphocyte marker (APO2.7 on the mitochondrial membrane) is significantly higher among non-survivors and is independently predictive of mortality. Apoptosis is an important mechanism of cell death in lymphocytes and parenchymal cells in sepsis, and it occurs systemically in many organs. It may be an important cause of immunologic suppression in sepsis by inducing widespread lymphocyte depletion. A previous report has indicated that exaggerated lymphocyte apoptosis is present in the peripheral blood of patients with septic shock, in contrast to those with simple sepsis or critically ill non-septic patients. Lymphocyte apoptosis occurs rapidly, leads to profound and persistent lymphocyte loss, and is associated with poor patient outcome [5,8]. In the present study, there is a sustained high level of peripheral lymphocyte apoptosis (APO2.7) early among non-survivors, with severe sepsis from Day 1 to Day 4. This clearly demonstrates that lymphocyte apoptosis does not only appear as an early event, but is also an ubiquitous and prolonged phenomenon in severe sepsis. APO2.7 is expressed on the mitochondrial membrane and detectable lymphocyte APO2.7 may be related to mitochondrial activation and mitochondrial membrane rupture of lymphocytes during early apoptosis [27]. Two pathways have been suggested in septic lymphocyte apoptosis, i.e., the death of the receptor system and the mitochondrial pathway activation by various stimuli leading to caspase 9 activation [13,20,27]. These 2 pathways subsequently act on a final death cell program via caspase 3 cleavage [18]. In multiple
animal models of sepsis, survival rates have been remarkably improved by inhibiting lymphocyte apoptosis using selective caspase inhibitors [28], by altering pro-apoptotic/anti-apoptotic protein expression [29], by treatment with survival promoting cytokines like interleukin-7 [30], and by modulating co-stimulatory receptors [31]. Based on the present findings, the late monocyte apoptotic marker (annexin V + 7-AAD on the nuclear membrane of cells) is also significantly higher among non-survivors and is independently predictive of mortality. Although the apoptosis of blood monocytes has been reported in a limited number of patients with severe sepsis, no connection has been found with survival [32]. A previous report from a ventilatorassociated pneumonia study group reveals that early monocyte apoptosis N50% on the first day of presentation is associated with prolonged 28-day survival compared to patients with monocyte apoptosis b 50% [9]. Unlike this previous study, the present report of early monocyte apoptosis is not associated with improved survival in patients with severe sepsis. However, previous articles have used only a single method to study apoptosis (flow cytometry of annexin-V for early apoptosis). The duration of illness before enrolment in this study may introduce enough variables to make timing difficult. Moreover, sepsis is a syndrome characterized by great heterogeneity. Although all patients are ubiquitously characterized as septic, they greatly differ in terms of sex, age, co-morbid conditions, underlying infections, and offending pathogens. Monocytes play an essential role against microbial infection in the innate immune defense. Monocytes are usually regarded as contributory to the ramp up of inflammatory responses rather than triggering their cessation [33]. They rapidly exhibit an impaired production of proinflammatory cytokines in response to additional bacterial challenge [34]. During the inflammatory response, monocytes present antigens by means of expression of the human leukocyte antigen (locus) DR (HLA-DR) receptors and by secreting pro-inflammatory cytokines to amplify the immune response [35]. Many studies have demonstrated that during sepsis-induced immuno-suppression, monocytes secrete fewer cytokines and down-regulate the expression of HLA receptors. This impaired function of monocytes is generally predictive of increased risk of secondary infection and poor prognosis in critically ill patients [36].
4.1. Study limitations The strength of this study is the relative homogeneity of the study cohort (non- traumatic, non-surgical adult severe sepsis patients at the ED), use of different methods to determine leukocyte apoptosis, and prospective study design. Nonetheless, the data here should be interpreted in light of several inherent limitations. First, there are many assays that evaluate leukocyte apoptosis in vitro, so the results and interpretation of various methodologies may be partially different. The level of leukocyte apoptosis tested by flow cytometry may not necessarily reflect the real leukocyte physiologic function in vivo. Second, the choice of therapeutic strategy for sepsis (e.g., use of steroids, dosage and duration of steroids) may be different for each patient based on the preference of his/her doctor. This may cause potential bias in the statistical analysis. Lastly, the case number is small and the follow-up period is short. Large-scale prospective studies are warranted to evaluate the prognostic contribution of leukocyte apoptosis on clinical outcomes.
5. Conclusions Leukocyte apoptosis is significantly higher in patients with severe sepsis and the percentages of late apoptosis of lymphocytes and monocytes has potential use in predicting the outcome of such patients at the ED. Aside from serum lactate, APO2.7 level in lymphocyte apoptosis is also a useful predictor of outcome on ED admission.
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