Open Access

Neutrophil apoptosis: a marker of disease severity in sepsis and sepsis-induced acute respiratory distress syndrome

  • Léa Fialkow1, 2Email author,
  • Luciano Fochesatto Filho1,
  • Mary C Bozzetti3,
  • Adriana R Milani1,
  • Edison M Rodrigues Filho2, 4, 5,
  • Roberta M Ladniuk1,
  • Paula Pierozan6,
  • Rafaela M de Moura7,
  • João C Prolla1,
  • Eric Vachon8 and
  • Gregory P Downey8
Critical Care200610:R155

DOI: 10.1186/cc5090

Received: 18 July 2006

Accepted: 8 November 2006

Published: 8 November 2006

Abstract

Introduction

Apoptosis of neutrophils (polymorphonuclear neutrophils [PMNs]) may limit inflammatory injury in sepsis and acute respiratory distress syndrome (ARDS), but the relationship between the severity of sepsis and extent of PMN apoptosis and the effect of superimposed ARDS is unknown. The objective of this study was to correlate neutrophil apoptosis with the severity of sepsis and sepsis-induced ARDS.

Methods

A prospective cohort study was conducted in intensive care units of three tertiary hospitals in Porto Alegre, southern Brazil. Fifty-seven patients with sepsis (uncomplicated sepsis, septic shock, and sepsis-induced ARDS) and 64 controls were enrolled. Venous peripheral blood was collected from patients with sepsis within 24 hours of diagnosis. All surgical groups, including controls, had their blood drawn 24 hours after surgery. Control patients on mechanical ventilation had blood collected within 24 hours of initiation of mechanical ventilation. Healthy controls were blood donors. Neutrophils were isolated, and incubated ex vivo, and apoptosis was determined by light microscopy on cytospun preparations. The differences among groups were assessed by analysis of variance with Tukeys.

Results

In medical patients, the mean percentage of neutrophil apoptosis (± standard error of the mean [SEM]) was lower in sepsis-induced ARDS (28% ± 3.3%; n = 9) when compared with uncomplicated sepsis (57% ± 3.2%; n = 8; p < 0.001), mechanical ventilation without infection, sepsis, or ARDS (53% ± 3.0%; n = 11; p < 0.001) and healthy controls (69% ± 1.1%; n = 33; p < 0.001) but did not differ from septic shock (38% ± 3.7%; n = 12; p = 0.13). In surgical patients with sepsis, the percentage of neutrophil apoptosis was lower for all groups when compared with surgical controls (52% ± 3.6%; n = 11; p < 0.001).

Conclusion

In medical patients with sepsis, neutrophil apoptosis is inversely proportional to the severity of sepsis and thus may be a marker of the severity of sepsis in this population.

Introduction

Sepsis is a leading cause of death in intensive care unit (ICU) patients [1], with an estimated incidence of 700,000 cases per year in the United States resulting in more than 200,000 deaths annually [2, 3]. Acute respiratory distress syndrome (ARDS) is a frequent complication of sepsis [46]. The mortality rate of ARDS remains high, ranging between 20% and 60% [4, 713]. Leucocytes, including neutrophils and macrophages, are believed to contribute to inflammatory tissue injury in sepsis and ARDS. It is hypothesised that unrestrained release of leucocyte-derived cytotoxic products contributes to injury of lungs and other organs [1416]. A better understanding of the pathophysiology of sepsis and ARDS is essential for the treatment or prevention of these devastating conditions.

Apoptosis is involved in removal of senescent cells and is thought to be essential for the non-injurious resolution of inflammation [1727]. The role of apoptosis in the pathophysiology of sepsis and multiple organ dysfunction syndrome (MODS) has been the focus of recent studies. There is evidence of an association between apoptosis and outcomes of patients with MODS [15, 20, 22, 23, 25, 28]. Recent studies suggest that neutrophil apoptosis is decreased in systemic inflammatory response syndrome (SIRS) [28, 29], sepsis [3037], and ARDS [12, 14, 16, 26, 3840]. The increased life span of neutrophils may be associated with increased tissue injury in these syndromes [12, 1416, 20, 22, 29]. Currently, information on the relationship between neutrophil apoptosis and the severity of sepsis and sepsis-induced ARDS is incomplete [22, 23, 3235, 41]. Accordingly, the objective of the current study was to determine whether neutrophil apoptosis correlates with the severity of sepsis and sepsis-induced ARDS.

Materials and methods

Patient selection and study protocol

A prospective cohort study enrolled patients at three tertiary teaching hospitals in Porto Alegre city, southern Brazil, from January 2000 to December 2004. Patients were included in the study if they met criteria for sepsis and ARDS.

Sepsis

Sepsis and its subsets were defined according to the Consensus Conference of the American College of Chest Physicians and the Society of Critical Care Medicine [1]. Sepsis, a systemic inflammatory response secondary to infection, was defined by two or more of the following criteria: (a) body temperature greater than 38°C or less than 36°C, (b) heart rate greater than 90 beats per minute, (c) respiratory rate greater than 20 breaths per minute or a PaCO2 (arterial partial pressure of carbon dioxide) less than 32 mm Hg, and (d) leucocytes greater than 12,000 cells per cubic millimetre, less than 4,000 cells per cubic millimetre, or greater than 10% bands. Septic shock was defined as sepsis-induced hypotension, despite adequate fluid resuscitation, along with the presence of hypoperfusion abnormalities or organ dysfunction. In our study, the term 'uncomplicated sepsis' was used for patients with sepsis according to the Consensus criteria instead of the more frequently used, but ambiguous, term 'sepsis.'

ARDS

ARDS was defined according to criteria of the 1994 American-European Consensus Conference on ARDS [42]. These included acute hypoxemia, ratio of PaO2 (arterial partial pressure of oxygen) to FiO2 (fraction of inspired oxygen) of 200 mm Hg or less, bilateral infiltrates on chest x-ray, pulmonary artery wedge pressure less than or equal to 18 mm Hg, or no clinical evidence of left atrial hypertension.

Control groups

1. Healthy controls were healthy blood donors (more than 18 years old) at the Hospital de Clínicas de Porto Alegre.

2. Surgical controls were patients submitted for elective surgery who had no evidence of infection, sepsis, or ARDS. Studies suggest that surgery itself has an influence on neutrophil apoptosis [4346].

3. The mechanical ventilation (MV) group consisted of patients submitted to MV but without evidence of infection, sepsis, or ARDS. The objective was to verify whether the MV itself influenced neutrophil apoptosis. All patients of this group were on MV for a period of 24 hours.

Exclusion criteria

Exclusion criteria were congestive heart failure, ARDS secondary to factors other than sepsis (for example, pancreatitis, burns, and multiple trauma), interstitial lung disease, use of immunosuppressive drugs (for example, corticosteroids), AIDS, malignancies, chronic inflammatory diseases (for example, rheumatoid arthritis), and transfusion of blood or blood products within the preceding 24 hours.

Ethical issues

The study was approved by the hospitals' ethics committees, and informed consent was obtained from the patient or a surrogate and from the healthy volunteers.

Sample and data collection

The venous blood sampling of medical patients was performed within 24 hours of diagnosis of sepsis and its subsets, ARDS, and for patients on MV. All surgical groups, including controls, had their blood drawn 24 hours after surgery. For healthy controls, a blood sample was obtained at the time of blood donation. The investigators followed each patient admitted to the ICU to identify patients who fulfilled the entry criteria. For each patient, a data record was completed and stored in a data bank.

Study variables

Outcome variables

The primary outcome variable was mean percentage of neutrophil apoptosis.

Independent variables

Independent variables were age, gender, medical/surgical patient status, Acute Physiology and Chronic Health Disease Classification System II (APACHE II) score, total maximum sequential organ failure assessment (SOFA) score, organ system failure based on the SOFA score, and 28-day mortality from the time of entry into the study. If the patient was discharged from the hospital, mortality was assessed by telephone or mail.

Study procedures

Neutrophil isolation

Human neutrophils (more than 98% pure) were isolated from whole blood using dextran sedimentation and discontinuous plasma-Percoll (Amersham Biosciences AB, now part of GE Healthcare, Little Chalfont, Buckinghamshire, UK) gradients as described previously [47]. The separation procedure required two hours, and the cells were used immediately after isolation for the experiments described. The functional integrity and non-activated state of isolated neutrophils have been validated in previous reports [47, 48]. Neutrophil viability was greater than 97% using Trypan blue exclusion.

Neutrophil apoptosis

After isolation, neutrophils were washed twice and resuspended at a density of 1 × 106 cells per millilitre in RPMI 1640 with 10% foetal bovine serum, L-glutamine (2 mM), penicillin (100 mg/ml), and streptomycin (100 μg/ml) (Gibco, now part of Invitrogen Corporation, Carlsbad, CA, USA). Cells were then incubated at 37°C in a 5% CO2 atmosphere for 24 hours in polypropylene tubes to prevent adherence. Cell viability assessed by Trypan blue exclusion exceeded 97%. After 24 hours, neutrophils were sedimented by cytocentrifugation on a glass microscope slide as described below.

Quantification of neutrophil apoptosis

Neutrophil apoptosis was assessed by light microscopy (×200) analysis of cytospun cells stained with Wright's Giemsa method and identification of nuclear changes (condensation of chromatin and simplification of nuclear structure) characteristic of apoptosis [17, 49, 50]. Two blinded investigators assessed the percentage of neutrophil apoptosis on cytospun preparations by analysing 500 cells per slide each. The analysis was performed on two different slides from the same patient. Data were reported as the percentage of apoptotic cells. The percentage was obtained by using the mean value obtained by the two investigators.

To validate the light microscopic method of assessment of neutrophil apoptosis, we used a second independent method in healthy donors, annexin V binding with quantification by flow cytometry [51]. In brief, neutrophils (1 × 106) were washed with ice-cold phosphate-buffered saline (PBS) and then incubated with fluorescein isothiocyanate (FITC)-conjugated annexin V (R&D Systems, Inc., Minneapolis, MN, USA) in the presence of propidium iodide (PI) for 30 minutes at 4°C. Cells were washed, resuspended in PBS, and analysed by flow cytometry (FACStar; Becton Dickinson, Mountain View, CA, USA). Cells that were FITC-positive and PI-negative were considered to be apoptotic. The extent of neutrophil apoptosis was compared with the percentage of neutrophil apoptosis determined by nuclear morphology and light microscopy (linear regression slope 0.87 R2 = 0.968, n = 6). These results confirm the validity of Wright's Giemsa staining to assess apoptosis.

Sample size

The sample size was calculated using data from the study patients because there was no information in the literature to help sample size estimation. The study power for the study comparisons was 90%.

Data quality control

A database coordinator was responsible for monitoring all data collection and entry. All data were checked for any inconsistencies. A random sample of 20% of the records was selected and compared with the original data-collection forms to detect any data-entry errors.

Statistical analysis

A stratified analysis was performed considering the status of medical or surgical patients. For each strata, the percentage of neutrophil apoptosis measured in the different groups was compared using one-way analysis of variance (ANOVA), considering that the study variables were normally distributed and that the variances were equal. All comparisons with a p value less than 0.05 were considered statistically significant. A post hoc Tukey test was used. Continuous variables, other than the percentage of neutrophil apoptosis, were also compared using ANOVA and the post hoc Tukey tests. For continuous variables comparing two groups, the Student t test was used. Categorical variables were compared using the χ2 test. Correlation analysis (Pearson) was performed between the main outcome of neutrophil apoptosis and other continuous variables, including age and APACHE II and SOFA scores, stratified for medical and surgical status. All analyses were performed using the Statistical Package for Social Sciences, version 12 (SPSS Inc., Chicago, IL, USA).

Results

A total of 57 patients and 64 controls were included in the study (see Table 1 for population characteristics). A detailed description of the diagnoses, sites of infection, microbiology, and sources of materials for culture from all patients is included in Table 2 (medical patients) and Table 3 (surgical patients).
Table 1

Characteristics of the study population according to group allocation

Variables

Uncomplicated sepsis (n = 16)

Septic shock (n = 23)

Sepsis-induced ARDS (n = 18)

Mechanical ventilation (n = 20)

Controls

(n = 44)

P valuea

Age (years, mean ± SEM)

57 ± 3.3

57 ± 4.5

46 ± 4.4

54 ± 3.5

43 ± 1.8

0.002

Male/Female (percentage)

62.5/37.5

52.2/47.8

50/50

55/45

50/50

0.93

Medical/Surgical (percentage)

50/50

52.2/47.8

50/50

55/45

75/25

-

aAnalysis of variance or χ2 test. ARDS, acute respiratory distress syndrome; SEM, standard error of the mean.

Table 2

Detailed description of the medical patients

Patient

Group

Uncomplicated sepsis (n = 8)

 

Diagnosis

Site of infection

Microorganism

Material

1

Pneumonia/COPD

Respiratory

Not identified

Sputum/Blood

2

Pneumonia/COPD

Respiratory

Staphylococcus aureus

Blood

3

Pneumonia/Stroke

Respiratory

Not identified

Sputum/Blood

4

Pneumonia/Guillain-Barre syndrome

Respiratory

Enterobacter sp

Sputum

5

Pneumonia/Subarachnoid hemorrhage

Respiratory

Staphylococcus sp

Blood

6

Pneumonia/DM/Pickwick syndrome

Respiratory

Not identified

Sputum/Blood

7

Pneumonia/Head trauma

Respiratory

Pseudomonas aeruginosa

Sputum

8

Pneumonia/Intracerebral hemorrhage

Respiratory

S. aureus

Sputum

Septic shock (n = 12)

 

Diagnosis

Site of infection

Microorganism

Material

1

Pneumonia/COPD

Respiratory

S. aureus

Blood

2

Pneumonia

Respiratory

S. aureus

Sputum

3

Pneumonia/COPD

Respiratory

S. aureus

Blood

4

Pneumonia/COPD

Respiratory

P. aeruginosa/Haemophilus influenzae

Sputum

5

Pneumonia/UTI

Respiratory/Urinary

Not identified/Klebsiella pneumoniae

Sputum/Urine

6

Pneumonia/UTI/DM

Respiratory/Urinary

Not identified/Candida sp

Sputum/Blood and urine

7

UTI/SBP/Cirrhosis

Abdominal/Urinary

S. aureus and Streptococcus viridans/Enterococcus faecium, S. viridans, and Escherichia coli

Ascites/Urine

8

Pneumonia

Respiratory

Not identified

Sputum/Blood

9

Pneumonia/COPD

Respiratory

Not identified

Sputum/Blood

10

Meningitis

CNS

Neisseria meningitis

Liquor/Blood

11

UTI/Lyell syndrome

Urinary/Skin

Enterococcus sp/Acinetobacter sp

Urine/Skin secretion

12

Pneumonia

Respiratory

S. aureus

Blood

Sepsis-induced ARDS (n = 9)

 

Diagnosis

Site of infection

Microorganism

Material

1

Pneumonia/Leptospirosis

Respiratory

Enterobacter sp

Sputum

2

Pneumonia/Suicide attempt (glicosate ingestion)

Respiratory

P. aeruginosa

Sputum

3

Pneumonia/UTI/Diarrhea

Respiratory, Urinary, and Intestinal

Not identified/K. pneumoniae/E. coli OH157

Sputum/Urine/Feces

4

Pneumonia/UTI/DM

Respiratory/Urinary

Streptococcus agalactiae, S. aureus/Staphylococcus sp, E. coli

Blood/Urine

5

Pneumonia

Respiratory

Haemophilus sp

Sputum

6

Septic arthritis

Joint

S. aureus

Sinovial liquid

7

Pneumonia

Respiratory

Not identified

Sputum/Blood

8

Pneumonia

Respiratory

E. coli, Moraxella sp

Sputum

9

Pneumonia/COPD

Respiratory

Not identified

Sputum/Blood

Mechanical ventilation (n = 11)

 

Diagnosis

   

1

Anaphylaxis (anaesthesia)

   

2

Head trauma

   

3

Spinal cord trauma

   

4

Subarachnoid hemorrhage

   

5

Subarachnoid hemorrhage

   

6

Intracerebral hemorrhage

   

7

Anaphylaxis (anaesthesia)

   

8

Guillain-Barre syndrome

   

9

Intracerebral hemorrhage

   

10

Anaphylaxis (anaesthesia)

   

11

Epilepsy

   

ARDS, acute respiratory distress syndrome; CNS, central nervous system; COPD, chronic obstructive respiratory disease; DM, diabetes mellitus; SBP, spontaneous bacterial peritonitis; UTI, urinary tract infection.

Table 3

Detailed description of the surgical patients

Patient

Group

Uncomplicated sepsis (n = 8)

 

Diagnosis

Site of infection

Microorganism

Material

Surgery

1

Pneumonia/Intracerebral haemorrhage

Respiratory

Pseudomonas aeruginosa

Sputum

Craniotomy

2

Pneumonia/COPD

Respiratory

P. aeruginosa

Sputum

Aortic-femoral bypass

3

Pneumonia/Perforated ulcer

Abdominal

Candida albicans

Ascites

Laparatomy

4

Cholangitis/UTI

Abdominal/Urinary

Enterococcus sp/Escherichia coli

Blood/Urine

Exploratory laparotomy

5

Pneumonia/Peritonitis/Colonic perforation due to colonoscopy

Respiratory/Abdominal

Enterobacter sp/Not identified

Sputum/Blood

Exploratory laparotomy

6

Cholecystitis

Abdominal

Enterococcus sp

Blood

Cholecystectomy

7

Pneumonia/Intracerebral haemorrhage

Respiratory

Enterobacter sp, Haemophilus sp, and Staphylococcus aureus

Sputum

Craniotomy

8

Pneumonia/Stroke/UTI/Celulitis

Respiratory/Urinary/Skin

Not identified/Enterobacter sp/S. aureus

Sputum/Urine/Skin secretion

Abdominal aortic aneurysm repair

Septic shock (n = 11)

 

Diagnosis

Site of infection

Microorganism

Material

Surgery

1

Pneumonia

Respiratory/Catheter

P. aeruginosa/S. aureus

Sputum/Catheter

Carotid aneurysm repair

2

Diverticulitis/UTI

Abdominal/Urinary

Not identified/Candida sp

Blood/Urine

Small bowel resection with anastomosis

3

Perforated peptic ulcer/Cirrhosis/Alcohol abuse

Abdominal

S. aureus

Blood/Ascites

Laparatomy

4

Septic arthritis (Hip)

Joint

Streptococcus agalactiae

Joint fluid

Surgical drainage

5

Pneumonia/Head trauma (subdural haematoma)

Respiratory

Not identified

Sputum/Blood

Craniotomy

6

Pyelonephritis/Nephrolitiasis/Neurogenic bladder

Urinary

Staphylococcus sp/P. aeruginosa

Blood/Urine

Nephrectomy and abscess drainage

7

Perforated peptic ulcer

Abdominal

S. aureus, Streptococcus viridans, and Enterobacter sp

Ascites

Exploratory laparotomy

8

Pneumonia/Endocarditis/Intracerebral haemorrhage

Respiratory/Heart

Pseudomonas sp/Not identified

Sputum/Blood

Craniotomy

9

Pneumonia/COPD/UTI Oesophageal laceration

Respiratory/Urinary

S. aureus and Acinetobacter sp/E. coli

Sputum/Urine

Oesophageal laceration repair

10

Cholangitis

Abdominal

E. coli

Blood

Exploratory laparotomy

11

Peritonitis/Perforated peptic ulcer

Abdominal

Enterococcus sp, Candida sp, and S. aureus

Ascites

Laparotomy

Sepsis-induced ARDS (n = 9)

 

Diagnosis

Site of infection

Microorganism

Material

Surgery

1

Cholangitis

Abdominal

Klebsiella pneumoniae

Ascites

Hepatic artery aneurysm ligation

2

Diverticulitis

Abdominal

E. coli

Blood

Small bowel resection with anastomosis

3

Pneumonia

Respiratory

P. aeruginosa

Sputum

Pleurostomy closure

4

Pneumonia

Respiratory

Staphylococcus coag neg

Blood

C-section

5

Septic arthritis

Hip joint

Staphylococcus haemolyticus

Blood

Hip drainage

6

UTI/Intestinal fistula

Urinary

Candida sp and Enterococcus sp

Blood and urine

Intestinal fistula closure

7

Pneumonia/UTI/Peripheral vascular disease

Respiratory/Urinary/Skin

Not identified/Candida sp/S. aureus

Sputum/Urine/Skin secretion

Above-knee amputation

8

Septic arthritis

Hip joint

Stenotroptomonas maltophilia and Staphylococcus coag neg/S. agalactiae

Blood and joint fluid

Hip drainage

9

Cholecystitis

Abdominal/Catheter

E. coli/S. aureus

Blood/Catheter

Cholecystectomy

Mechanical ventilation (n = 9)

 

Diagnosis

Surgery

   

1

Head trauma (subdural haematoma)

Craniotomy

   

2

Intracerebral haemorrhage

Craniotomy

   

3

Head trauma (subdural haematoma)

Craniotomy

   

4

Head trauma (subdural haematoma)

Craniotomy

   

5

Head trauma (epidural haematoma)

Craniotomy

   

6

Uterine leyomioma

Hysterectomy

   

7

Abdominal trauma

Exploratory laparotomy

   

8

Head trauma (subdural haematoma)

Craniotomy

   

9

Intracerebral haemorrhage

Craniotomy

   

Controls (n = 11)

 

Surgery

    

1

Humeral prostheses

    

2

Inguinal hernia repair

    

3

Septoplasty

    

4

Inguinal hernia repair

    

5

Arthrodesis (tibia-tarsus)

    

6

Tibial osteosynthesis

    

7

Septoplasty

    

8

Diaphragmatic hernia repair and laparoscopic fundoplication

    

9

Iliofemoral bypass

    

10

Incisional hernia repair

    

11

Septoplasty

    

ARDS, acute respiratory distress syndrome; COPD, chronic obstructive respiratory disease; UTI, urinary tract infection.

The comparison of the percentage of neutrophil apoptosis was significantly different among all groups (p < 0.001; ANOVA). A stratified analysis was performed considering surgical/medical status. The mean percentage of neutrophil apoptosis (± standard error of the mean [SEM]) was significantly lower in the surgical controls (52% ± 3.6%) when compared with healthy controls (69% ± 1.1%; p = 0.001; Student t test).

In medical patients, a significant difference was observed in the age variable (Table 4). The control group was younger than the MV group (p = 0.02; Tukey test). A Pearson correlation test showed a weak and negative correlation (p = 0.35) between age and neutrophil apoptosis, suggesting that age did not have a major effect on the percentage of neutrophil apoptosis in this study (data not shown).
Table 4

Characteristics of the medical patients

 

Uncomplicated sepsis

(n = 8)

Septic shock

(n = 12)

Sepsis-induced ARDS

(n = 9)

Mechanical ventilation

(n = 11)

Controls

(n = 33)

P value

Age (years, mean ± SEM)

50.8 ± 4.9

56 ± 5.6

43.2 ± 5.8

57.5 ± 4.9

37.1 ± 1.7

<0.008a

Male/Female (percentage)

50.0/50.0

58.3/41.7

44.4/55.6

36.4/63.6

48.5/51.5

0.7b

APACHE II score (percentage)

13.6 ± 2.2

21 ± 2.2

21.5 ± 1.2

-

-

0.1

Maximum SOFA score (percentage)

4.7 ± 0.7

9.9 ± 1.0

12 ± 0.8

-

-

<0.001a

Organ dysfunction (percentage)

      

0

50

25

0

-

-

-

1

0

16.7

77.8

-

-

-

2

25

41.7

0

-

-

-

3

25

16.7

22.2

-

-

-

Organ failure (percentage)

      

0

25

0

0

-

-

-

1

62.5

25

0

-

-

-

2

12.5

41.7

55.6

-

-

-

3

0

33.3

33.3

-

-

-

4

0

0

11.1

-

-

-

Mortality in 28 days (percentage)

37.5

75

77.8

-

-

-

Neutrophil apoptosis (mean percentage ± SEM)

57 ± 3.2

38 ± 3.7

28 ± 3.3

53 ± 3.0

69 ± 1.1

<0.001a

aP value from the comparisons using analysis-of-variance test; bp value from the comparisons using χ2 test. APACHE II, Acute Physiology and Chronic Health Disease Classification System II; ARDS, acute respiratory distress syndrome; SEM, standard error of the mean; SOFA, sequential organ failure assessment.

Neutrophil apoptosis differed significantly among the groups of medical patients. Figure 1 shows images of neutrophil apoptosis in Wright's Giemsa-stained slides obtained from a healthy control (a) and from a patient with ARDS (b). The percentage of neutrophil apoptosis (± SEM) was lower in ARDS (28% ± 3.3%; n = 9) compared with uncomplicated sepsis (57% ± 3.2%; n = 8; p < 0.001), MV (53% ± 3.0%; n = 11; p < 0.001), and with healthy controls (69% ± 1.1%; n = 33; p < 0.001). However, it did not differ from septic shock (38% ± 3.7%; n = 12; p = 0.13) (Tukey test; Figure 2). In the septic shock group, the mean percentage of neutrophil apoptosis was significantly lower than in uncomplicated sepsis, MV, and healthy controls (p < 0.001; Tukey test). The mean percentage of neutrophil apoptosis was significantly lower in patients with uncomplicated sepsis (p = 0.02; Tukey test) and in the MV group (p < 0.001; Tukey test) compared with healthy controls. There was no difference in the mean percentage of neutrophil apoptosis between the uncomplicated sepsis and the MV groups (p = 0.8; Tukey test). These observations suggest that in medical patients, the severity of sepsis is inversely proportional to the mean percentage of neutrophil apoptosis (Figure 2).
https://static-content.springer.com/image/art%3A10.1186%2Fcc5090/MediaObjects/13054_2006_Article_4611_Fig1_HTML.jpg
Figure 1

Apoptosis of neutrophils in a healthy donor and in a patient with sepsis-induced acute respiratory distress syndrome (ARDS). (a) Apoptosis of neutrophils in a healthy donor. Wright's Giemsa staining of cytocentrifuge smear shows predominance of cells in apoptosis. Inset shows morphological detail of an apoptotic cell, with loss of chromatin fine granularity (condensation) and karyorrhexis. (b) Apoptosis of neutrophils in a patient with sepsis-induced ARDS. Wright's Giemsa staining of cytocentrifuge smear shows predominance of normal-looking cells. Inset shows morphological detail of a normal cell, with fine granularity of chromatin and normal lobulated nucleus. Magnifications ×200 (insets ×500).

https://static-content.springer.com/image/art%3A10.1186%2Fcc5090/MediaObjects/13054_2006_Article_4611_Fig2_HTML.jpg
Figure 2

Mean percentage of neutrophil apoptosis in medical patients. There was a statistically significant difference among the groups (p < 0.001; analysis of variance). The differences between individual groups as determined by a post hoc Tukey test are illustrated. ARDS, acute respiratory distress syndrome.

Variables such as 28-day mortality and APACHE II and SOFA scores were also analysed in the medical groups (Table 4). Twenty-eight-day mortality was higher in the ARDS and septic shock groups when compared with the group with uncomplicated sepsis (Table 4). ARDS and septic shock groups had a higher mean SOFA score when compared with the other groups (p < 0.001; Tukey test) (Table 4). However, no statistical difference was observed between the ARDS and septic shock groups (p = 0.3; Tukey test).

Detailed data regarding number of organ dysfunctions/failures, according to SOFA score, are summarised in Table 4. Many patients with uncomplicated sepsis developed organ failure after blood sampling and during their hospitalisation in the ICU.

In surgical patients, the mean percentage of neutrophil apoptosis in all groups (uncomplicated sepsis [p = 0.04], septic shock [p = 0.04], ARDS [p < 0.002], and MV [p = 0.007] groups [Tukey test]) was significantly lower than in controls (Figure 3). No statistical difference was found among the mean percentage of neutrophil apoptosis of uncomplicated sepsis, septic shock, ARDS, and MV groups. Other variables were also analysed in surgical groups (Table 5).
https://static-content.springer.com/image/art%3A10.1186%2Fcc5090/MediaObjects/13054_2006_Article_4611_Fig3_HTML.jpg
Figure 3

Mean percentage of neutrophil apoptosis in surgical patients. There were statistically significant differences among the groups (p < 0.001; analysis of variance). Post hoc Tukey test results are illustrated. ARDS, acute respiratory distress syndrome.

Table 5

Characteristics of the surgical patients

 

Uncomplicated sepsis (n = 8)

Septic shock (n = 11)

Sepsis-induced ARDS (n = 9)

Mechanical ventilation (n = 9)

Controls (n = 11)

P value

Age (years, mean ± SEM)

64.0 ± 3.3

58 ± 7.4

49.5 ± 6.9

51 ± 5

45.9 ± 5.5

0.1

Male/Female (percentage)

75/25

45.5/54.5

55.6/44.4

77.8/22.2

54.5/45.5

0.5a

APACHE II score (percentage)

15.3 ± 1.8

21.1 ± 1.9

21.7 ± 3.9

-

-

0.1

Maximum SOFA score (percentage)

6.8 ± 1.1

10 ± 1.3

12.2 ± 0.8

-

-

<0.001b

Organ dysfunction (percentage)

      

0

0

9.1

11.1

-

-

-

1

50

18.2

44.4

-

-

-

2

37.5

36.4

22.2

-

-

-

3

12.5

18.2

22.2

-

-

-

4

0

18.2

0

   

Organ failure (percentage)

      

0

12.5

9.1

0

-

-

-

1

62.5

36.4

11.1

-

-

-

2

25

36.4

66.7

-

-

-

3

0

18.2

22.2

-

-

-

Mortality in 28 days (percentage)

62.5

45.5

66.7

-

-

-

Neutrophil apoptosis (mean percentage ± SEM)

35 ± 3.2

36 ± 5.2

29 ± 2.1

32 ± 3.9

52 ± 3.6

<0.001b

aP value from the comparisons using χ2 test; bp value from the comparisons using analysis-of-variance test. APACHE II, Acute Physiology and Chronic Health Disease Classification System II; ARDS, acute respiratory distress syndrome; SEM, standard error of the mean; SOFA, sequential organ failure assessment.

We attempted to perform a subgroup analysis based on the different degrees of severity of sepsis in medical and surgical patients to ascertain whether there was an association between neutrophil apoptosis and mortality. This was not successful, probably due to the small sample size studied. A moderate and negative correlation between the mean SOFA score and the percentage of neutrophil apoptosis in medical patients was observed (R = -0.56; p < 0.001), indicating that the lower the mean percentage of apoptosis, the higher the mean SOFA score. However, in surgical patients, this correlation was weak and not statistically significant.

Discussion

The primary observation of the current study is that the extent of neutrophil apoptosis correlates inversely with the severity of sepsis and sepsis-induced ARDS in medical patients. Neutrophils from medical patients with uncomplicated sepsis, septic shock, and ARDS displayed lower degrees of apoptosis as compared with controls. Furthermore, we observed a progressive decrease in neutrophil apoptosis as the severity of sepsis increased. This is the first study to correlate the extent of apoptosis of peripheral blood neutrophils with the severity of sepsis and ARDS.

Our study confirms and extends the previous reports of decreased neutrophil apoptosis in patients with sepsis and ARDS with or without sepsis [3033, 35, 36, 3840]. One study reported that neutrophil apoptosis was decreased in patients with sepsis compared with healthy controls [33]. However, that study combined patients with different degrees of severity of sepsis into one large group (labelled 'sepsis') that was compared with healthy controls but did not correlate the extent of neutrophil apoptosis with the severity of sepsis. Other studies that examined apoptosis of circulating neutrophils from septic patients assessed only one level of severity of sepsis (for example, only severe sepsis [3032] or MODS [35]). Another study examined the rates of apoptosis of neutrophils in bronchoalveolar lavage fluid (BALF) of septic patients and demonstrated decreased apoptosis when all cells (including neutrophils) from the BALF were analysed ex vivo [36].

In patients with ARDS, our study is in agreement with previous studies that have demonstrated decreased neutrophil apoptosis in patients with ARDS, including those with sepsis-induced ARDS [3840]. Several studies have documented that BALF recovered from patients during the early stages of both septic and non-septic ARDS is able to prolong the life span of neutrophils incubated ex vivo and that this effect may be ascribable to elevated levels of cytokines such as granulocyte-colony stimulating factor, granulocyte macrophage-colony stimulating factor (GM-CSF), and interleukin (IL)-2 [3840]. Interestingly, Matute-Bello and colleagues [39] reported that higher GM-CSF levels in BALF correlated with survival in patients with ARDS. The authors suggested that this effect may not be related to modulation of neutrophil apoptosis but rather due to effects on other cells such as alveolar macrophages and epithelial cells. Lesur and colleagues [40] also demonstrated that exposure of normal blood neutrophils to BALF from patients with ARDS delayed apoptosis in vitro. In general, these results are in agreement with our observations and indicate that modulation of apoptosis of neutrophils and other lung cells is an early phenomenon in the inflammatory milieu of the lung in sepsis. It is noteworthy that our study is the first to evaluate apoptosis of peripheral blood neutrophils specifically from patients with sepsis-induced ARDS.

The mechanisms responsible for the decreased neutrophil apoptosis in sepsis and ARDS are incompletely understood. One potential mechanism involves activation of nuclear factor-κB with a concomitant reduction of the activity of caspases 3 and 9, and maintenance of mitochondrial transmembrane potential [33]. Other possible mechanisms involve modulation of Mcl-1 (myeloid cell leukaemia-1) [32], PBEF (pre-B cell colony-enhancing factor) [35], and p38 mitogen-activated protein kinase (p38 MAPK) [41] signalling pathways.

The current study was stratified (medical/surgical status) because previous studies have suggested that surgery per se may influence neutrophil apoptosis [4346]. Additionally, because MV has been shown to affect apoptosis in other cell types [5257], we included a control group of patients (medical and surgical) submitted to MV but who had no history of infection, sepsis, or ARDS.

We observed that the extent of neutrophil apoptosis was significantly lower in the surgical controls when compared with medical controls, an effect that has been reported by others [4345]. Indeed, we observed a decrease in neutrophil apoptosis in all surgical groups. However, there was no statistical difference between these groups. Therefore, the correlation between neutrophil apoptosis and the severity of sepsis observed in medical patients was not observed in the surgical groups. There are several factors that might account for the decreased neutrophil apoptosis in surgical patients, including effects of anaesthesia and of the localised tissue trauma related to the surgical procedure with release of cytokines such as IL-6 [43] and IL-8 [45]. In this regard, a recent study [58] examined the effects of surgery on Fas-induced neutrophil apoptosis and reported that the anti-apoptotic action of plasma was not affected by the addition of neutralising antibodies to GM-CSF, IL-6, or IL-8, indicating that these cytokines are not a dominant factor mediating the anti-apoptotic effects on Fas-induced apoptosis in surgical patients. However, the anti-apoptotic effect of plasma was attenuated by pharmacological inhibitors of either PI3 kinase or extracellular signal-regulated kinase (ERK), but not by a p38 MAPK inhibitor, implicating PI3 kinase and ERK in the signalling pathway mediating the anti-apoptotic effect of plasma under the conditions described above. Another study demonstrated a decrease in apoptosis of exudative neutrophils obtained from peritoneal fluid from patients with recent gastrointestinal surgery [44]. In contrast, a recent report describes enhanced apoptosis of peripheral blood neutrophils of patients undergoing elective surgery under general anaesthesia [46]. Taken together, alterations in neutrophil function which occur in the post-operative period may predispose to untoward outcomes via modulation of the complex inflammatory response to surgery.

Previous studies support the concept that injurious modes of MV per se may result in release of inflammatory mediators that lead to inflammatory lung injury [52, 53, 59, 60]. In support of this notion, we observed that neutrophil apoptosis was diminished in the group of patients subject to MV but without evidence of infection, sepsis, or ARDS. However, our results also indicate that MV per se did not account for the low percentage of neutrophil apoptosis observed in the group of patients with more complicated sepsis. The effect of MV extends beyond the lungs to other organs and has been termed 'biotrauma' [54, 55]. Imai and collaborators [52] documented effects of MV on epithelial cell apoptosis in the lung as well as in the kidneys and small intestine, the former accompanied by biochemical evidence of organ dysfunction. A previous study from our group demonstrated that BALF obtained from ARDS patients ventilated with injurious MV activated neutrophil oxidant production and release of elastase, effects that correlated to the degree of lung injury and systemic inflammatory response and to multiple organ failure [61]. Although the effect of BALF on neutrophil apoptosis was not assessed in this study, we predict that it would decrease apoptosis. The 'biotrauma' hypothesis is supported by evidence from experimental models, including humans [59], animals [62], isolated lung [54], and stressed cell systems [63].

We observed that the mortality rate was higher in medical patients with ARDS, followed by septic shock, when compared with the uncomplicated sepsis group. To understand the significance of these mortality rates, we used instruments such as the total maximum SOFA score to quantify the severity of illness. From a correlation test evaluating the association between the mean percentage of neutrophil apoptosis and the mean SOFA score, two correlations merit further consideration: (a) the correlation between the severity of sepsis and the percentage of neutrophil apoptosis and (b) the association among the severity of sepsis, percentage of neutrophil apoptosis, and mortality. The correlation analysis suggests an inverse association between disease severity and the percentage of neutrophil apoptosis. Because the mean SOFA score correlates with mortality [64, 65], our findings suggest that there is an association between the severity of sepsis, the extent of neutrophil apoptosis, and mortality.

We did not observe an association between neutrophil apoptosis and mortality in the current study. One limitation in this regard is that the sample size was not sufficient to assess such an association. However, the observed results of the mean percentage of neutrophil apoptosis, the mean SOFA score, and the mortality rates suggest that the higher the mortality rate (and disease severity), the lower the percentage of neutrophil apoptosis. In future studies with a larger sample size, it will be important to evaluate whether the percentage of neutrophil apoptosis is associated with mortality within the different degrees of severity of sepsis.

Study limitations

The decrease in the percentage of neutrophil apoptosis may not be specific for sepsis and ARDS. In fact, it appears that any event resulting in SIRS (such as sepsis) has the potential to affect the immune system, including neutrophil survival and function. However, the patients included in our study, including the controls groups, were carefully selected to allow us to study the specific correlation between neutrophil apoptosis and sepsis. Our results demonstrate that in medical patients with sepsis, neutrophil apoptosis is inversely proportional to the severity of sepsis. The correlations of neutrophil apoptosis with other causes of SIRS, if any, require further study.

The observations in the current study represent an important first step to a better understanding of the influence of sepsis on neutrophil apoptosis by defining the clinical associations of differing degrees of neutrophil apoptosis in this milieu. However, the observational design of the current study did not allow us to explore the possible mechanism(s) such as the role of specific receptors and intracellular signalling pathways in modulation of neutrophil apoptosis during sepsis. Further studies will be important to address these issues and will provide important information on the signal transduction pathways modulating neutrophil apoptosis during sepsis.

Conclusion

We observed that in medical patients with sepsis, neutrophil apoptosis is inversely proportional to the severity of this syndrome, including ARDS. In surgical patients, the mean percentage of neutrophil apoptosis for all sepsis groups was significantly lower than in the control group, but was not proportional to the severity of sepsis. These observations suggest that in medical patients, neutrophil apoptosis may be a marker of the severity of sepsis. We speculate that an influx of long-lived neutrophils may contribute to enhanced inflammatory injury to the lungs and other organs. The identification of specific mechanisms of neutrophil apoptosis in sepsis, including sepsis-induced ARDS, may lead to new strategies to improve the survival of those patients and patients with other inflammatory disorders in which neutrophils have been directly implicated.

Key messages

  • In medical patients with sepsis, neutrophil apoptosis is inversely proportional to the severity of sepsis.

  • In surgical patients with sepsis, the rate of apoptosis was lower than in controls but was not proportional to the severity of sepsis.

  • These observations suggest that in medical patients, neutrophil apoptosis may be a marker of the severity of sepsis.

Abbreviations

ANOVA: 

analysis of variance

APACHE II: 

Acute Physiology and Chronic Health Disease Classification System II

ARDS: 

acute respiratory distress syndrome

BALF: 

bronchoalveolar lavage fluid

ERK: 

extracellular signal-regulated kinase

FITC: 

fluorescein isothiocyanate

GM-CSF: 

granulocyte macrophage-colony stimulating factor

ICU: 

intensive care unit

IL: 

interleukin

MODS: 

multiple organ dysfunction syndrome

MV: 

mechanical ventilation

p38 MAPK: 

p38 mitogen-activated protein kinase

PBS: 

phosphate-buffered saline

PI: 

propidium iodide

SEM: 

standard error of the mean

SIRS: 

systemic inflammatory response syndrome

SOFA: 

sequential organ failure assessment.

Declarations

Acknowledgements

We thank Dr. Vinícius Duval da Silva for his contribution in digital imaging of Wright's Giemsa slides and Ada Regina Schenini Diehl for her orientation on cytopathology techniques. LF and MCB are recipients of a Research Award from the National Council of Scientific and Technological Development (CNPq). GPD holds a Tier 1 Canada Research Chair. This study received funding from Fundação de Amparo à Pesquisa do Rio Grande do Sul (FAPERGS), Fundo de Incentivo à Pesquisa do Hospital de Clínicas de Porto Alegre (FIPE/HCPA), Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), and the Canadian Institutes of Health Research.

Authors’ Affiliations

(1)
Department of Internal Medicine, Faculty of Medicine, Federal University of Rio Grande do Sul
(2)
Intensive Care Unit, Intensive Care Division, Hospital de Clínicas de Porto Alegre
(3)
Department of Social Medicine, Faculty of Medicine, Federal University of Rio Grande do Sul
(4)
Intensive Care Unit of Trauma and Neurosurgery, Hospital Cristo Redentor, Grupo Hospitalar Conceição
(5)
Intensive Care Unit, Hospital Dom Vicente Scherer, Complexo Hospitalar Santa Casa de Porto Alegre
(6)
Faculty of Pharmacy, Federal University of Rio Grande do Sul
(7)
Faculty of Pharmacy, Pontifícia Universidade Católica do Rio Grande do Sul
(8)
Division of Respirology, Department of Medicine, Toronto General Hospital Research Institute of the University Health Network and University of Toronto,Toronto General Hospital

References

  1. American College of Chest Physicians/Society of Critical Care Medicine Consensus Conference: Definitions for sepsis and organ failure and guidelines for the use of innovative therapies in sepsis. Crit Care Med 1992, 20: 864-874.View ArticleGoogle Scholar
  2. Martin GS, Mannino DM, Eaton S, Moss M: The epidemiology of sepsis in the United States from 1979 trough 2000. N Engl J Med 2003, 348: 1546-1554. 10.1056/NEJMoa022139View ArticlePubMedGoogle Scholar
  3. Angus DC, Linde-Zwirble WT, Lidicker J, Clermont G, Carcillo J, Pinsky MR: Epidemiology of severe sepsis in the United States: analysis of incidence, outcome, and associated costs of care. Crit Care Med 2001, 29: 1303-1310. 10.1097/00003246-200107000-00002View ArticlePubMedGoogle Scholar
  4. Rubenfeld GD, Caldwell E, Peabody E, Weaver J, Martin DP, Neff M, Stern EJ, Hudson LD: Incidence and outcomes of acute lung injury. N Engl J Med 2005, 353: 1685-1693. 10.1056/NEJMoa050333View ArticlePubMedGoogle Scholar
  5. Udobi KE, Childs ED, Touijer K: Acute respiratory distress syndrome. Am Fam Phys 2003, 67: 315-322.Google Scholar
  6. Dreyfuss D, Ricard JD: Acute lung injury and bacterial infection. Clin Chest Med 2005, 26: 105-112. 10.1016/j.ccm.2004.10.014View ArticlePubMedGoogle Scholar
  7. Amato MBP, Barbas CSV, Medeiros DM, Magaldi RB, Schettino GPP, Lorenzi-Filho G, Kairalla RA, Deheinzelin D, Munoz C, Oliveira R, et al.: Effect of a protective-ventilation strategy on mortality in the acute respiratory distress syndrome. N Engl J Med 1998, 338: 347-354. 10.1056/NEJM199802053380602View ArticlePubMedGoogle Scholar
  8. Stewart TE, Meade MO, Cook DJ, Granton JT, Hodder RV, Lapinsky SE, Mazer D, McLean RF, Rogovein TS, Schouten D, et al.: Evaluation of a ventilation strategy to prevent barotrauma in patients at high risk for acute respiratory distress syndrome. Pressure- and Volume-Limited Ventilation Strategy Group. N Engl J Med 1998, 338: 355-361. 10.1056/NEJM199802053380603View ArticlePubMedGoogle Scholar
  9. Brochard L, Roudot-Thoraval F, Roupie E, Delclaux C, Chastre J, Fernandez-Mondéjar E, Clémenti E, Mancebo J, Factor P, Matamis D, et al.: Tidal volume reduction for prevention of ventilator-induced lung injury in acute respiratory distress syndrome. The multicenter trial group on tidal volume reduction in ARDS. Am J Respir Crit Care Med 1998, 158: 1831-1838.View ArticlePubMedGoogle Scholar
  10. Brower RG, Shanholtz CB, Fessler HE, Shade DM, White P Jr, Wiener CM, Teeter JG, Dodd-o JM, Almog Y, Piantadosi S: Prospective, randomized, controlled clinical trial comparing traditional versus reduced tidal volume ventilation in acute respiratory distress syndrome patients. Crit Care Med 1999, 27: 1492-1498. 10.1097/00003246-199908000-00015View ArticlePubMedGoogle Scholar
  11. The Acute Respiratory Distress Syndrome Network: Ventilation with lower tidal volumes as compared with traditional tidal volumes for acute lung injury and the acute respiratory distress syndrome. N Engl J Med 2000, 342: 1301-1308. 10.1056/NEJM200005043421801View ArticleGoogle Scholar
  12. Ware LB, Matthay MA: Clinical progress: the acute respiratory distress syndrome. N Engl J Med 2000, 342: 1334-1349. 10.1056/NEJM200005043421806View ArticlePubMedGoogle Scholar
  13. Eichacker PQ, Gerstenberger EP, Banks SM, Cui X, Natanson C: Meta-analysis of acute lung injury and acute respiratory distress syndrome trials testing low tidal volumes. Am J Respir Crit Care Med 2002, 166: 1510-1514. 10.1164/rccm.200208-956OCView ArticlePubMedGoogle Scholar
  14. Lee WL, Downey GP: Neutrophil activation and acute lung injury. Curr Opin Crit Care 2001, 7: 1-7. 10.1097/00075198-200102000-00001View ArticlePubMedGoogle Scholar
  15. Hotchkiss RS, Karl IE: The pathophysiology and treatment of sepsis. N Engl J Med 2003, 348: 138-148. 10.1056/NEJMra021333View ArticlePubMedGoogle Scholar
  16. Abraham E: Neutrophils and acute lung injury. Crit Care Med 2003,31(4 Suppl):S195-9. 10.1097/01.CCM.0000057843.47705.E8View ArticlePubMedGoogle Scholar
  17. Savill JS, Wyllie AH, Henson JE, Walport MJ, Henson PM, Haslett C: Macrophage phagocytosis of aging neutrophils in inflammation. J Clin Invest 1989, 83: 865-875.PubMed CentralView ArticlePubMedGoogle Scholar
  18. Savill J, Haslett C: Granulocyte clearance by apoptosis in the resolution of inflammation. Seminars Cell Biol 1995, 6: 385-393. 10.1016/S1043-4682(05)80009-1View ArticleGoogle Scholar
  19. Haslett C: Granulocyte apoptosis and its role in the resolution and control of lung inflammation. Am J Respir Crit Care Med 1999, 160: S5-S11.View ArticlePubMedGoogle Scholar
  20. Mahidhara R, Billiar TR: Apoptosis in sepsis. Crit Care Med 2000, 28: N105-N113. 10.1097/00003246-200004001-00013View ArticlePubMedGoogle Scholar
  21. Giles KM, Hart SP, Haslett C, Rossi AG, Dransfield I: An appetite for apoptotic cells? Controversies and challenges. Br J Haematol 2000, 109: 1-12. 10.1046/j.1365-2141.2000.01805.xView ArticlePubMedGoogle Scholar
  22. Oberholzer C, Oberholzer A, Clare-Salzler M, Moldawer LL: Apoptosis in sepsis: a new target for therapeutic exploration. FASEB J 2001, 15: 879-892. 10.1096/fj.00-058revView ArticlePubMedGoogle Scholar
  23. Power C, Fanning N, Redmond HP: Cellular apoptosis and organ injury in sepsis: a review. Shock 2002, 18: 197-211. 10.1097/00024382-200209000-00001View ArticlePubMedGoogle Scholar
  24. Martin TR, Nakamura M, Matute-Bello G: The role of apoptosis in acute lung injury. Crit Care Med 2003,31(4 Suppl):S184-S188. 10.1097/01.CCM.0000057841.33876.B1View ArticlePubMedGoogle Scholar
  25. Riedemann NC, Guo R-F, Ward PA: The enigma of sepsis. J Clin Invest 2003, 112: 460-467. 10.1172/JCI200319523PubMed CentralView ArticlePubMedGoogle Scholar
  26. Maianski NA, Maianski AN, Kuijpers TW, Roos D: Apoptosis of neutrophils. Acta Haematol 2004, 111: 56-66. 10.1159/000074486View ArticlePubMedGoogle Scholar
  27. Steven HW: To die or not to die: an overview of apoptosis and its role in disease. JAMA 1998, 279: 300-307. 10.1001/jama.279.4.300View ArticleGoogle Scholar
  28. Papathanassoglou EDE, Moynihan JA, McDermott MP, Ackerman MH: Expression of Fas (CD95) and Fas ligand on peripheral blood mononuclear cells in critical illness and association with multiorgan dysfunction severity and survival. Crit Care Med 2001, 29: 709-718. 10.1097/00003246-200104000-00002View ArticlePubMedGoogle Scholar
  29. Jimenez MF, Watson WG, Parodo J, Evans D, Foster D, Steinberg M, Rotstein OD, Marshall JC: Dysregulated expression of neutrophil apoptosis in the systemic inflammatory response syndrome. Arch Surg 1997, 132: 1263-1270.View ArticlePubMedGoogle Scholar
  30. Keel M, Ungethüm U, Steckholzer U, Niederer E, Hartung T, Trentz O, Ertel W: Interleukin-10 counterregulates proinflammatory cytokine-induced inhibition of neutrophil apoptosis during severe sepsis. Blood 1997, 90: 3356-3363.PubMedGoogle Scholar
  31. Ertel W, Keel M, Infanger M, Ungethüm U, Steckholzer U, Trentz O: Circulating mediators in serum of injured patients with septic complications inhibit neutrophil apoptosis through up-regulation of protein-tyrosine phosphorylation. J Trauma 1998, 44: 767-776.View ArticlePubMedGoogle Scholar
  32. Härter 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: 964-973. 10.1016/j.jamcollsurg.2003.07.008View ArticlePubMedGoogle Scholar
  33. Taneja R, Parodo J, Jia SH, Kapus A, Rotstein OD, Marshall JC: Delayed neutrophil apoptosis in sepsis is associated with maintenance of mitochondrial transmembrane potential and reduced caspase-9 activity. Crit Care Med 2004, 32: 1460-1469. 10.1097/01.CCM.0000129975.26905.77View ArticlePubMedGoogle Scholar
  34. Sayeed MM: Delay of neutrophil apoptosis can exacerbate inflammation in sepsis patients: cellular mechanisms. Crit Care Med 2004, 32: 1604-1606. 10.1097/01.CCM.0000130997.85379.0FView ArticlePubMedGoogle Scholar
  35. Jia SH, Li Y, Parodo J, Kapus A, Fan L, Rotstein OD, Marshall JC: Pre-B cell colony-enhancing factor inhibits neutrophil apoptosis in experimental inflammation and clinical sepsis. J Clin Invest 2004, 113: 1318-1327. 10.1172/JCI200419930PubMed CentralView ArticlePubMedGoogle Scholar
  36. Liacos C, Katsaragakis S, Konstadoulakis MM, Messaris EG, Papanicolaou M, Georgiadis GG, Menenakos E, Vasiliadi-Chioti A, Androulakis G: Apoptosis in cells of bronchoalveolar lavage: a cellular reaction in patients who die with sepsis and respiratory failure. Crit Care Med 2001, 29: 2310-2317. 10.1097/00003246-200112000-00013View ArticlePubMedGoogle Scholar
  37. Wesche DE, Lomas-Neira JL, Perl M, Shung C-S, Ayala A: Leukocyte apoptosis and its significance in sepsis and shock. J Leukoc Biol 2005, 78: 325-337. 10.1189/jlb.0105017View ArticlePubMedGoogle Scholar
  38. Matute-Bello G, Liles WC, Radella F II, Steinberg KP, Ruzinski JT, Jonas M, Chi EY, Hudson LD, Martin TR: Neutrophil apoptosis in the acute respiratory distress syndrome. Am J Respir Crit Care Med 1997, 156: 1969-1977.View ArticlePubMedGoogle Scholar
  39. Matute-Bello G, Liles WC, Radella F II, Steinberg KP, Ruzinski JT, Jonas M, Chi EY, Hudson LD, Martin TR: Modulation of neutrophil apoptosis by granulocyte colony-stimulating factor and granulocyte/macrophage colony-stimulating factor during the course of acute respiratory distress syndrome. Crit Care Med 2000, 28: 1-7. 10.1097/00003246-200001000-00001View ArticlePubMedGoogle Scholar
  40. Lesur O, Kokis A, Hermans C, Fülöp T, Bernard A, Lane D: Interleukin-2 involvement in early acute respiratory distress syndrome: relationship with polymorphonuclear neutrophil apoptosis and patient survival. Crit Care Med 2000, 28: 3814-3822. 10.1097/00003246-200012000-00010View ArticlePubMedGoogle Scholar
  41. 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: 242-248. 10.1067/msy.2001.115902View ArticlePubMedGoogle Scholar
  42. Bernard GR, Artigas A, Brigham KL, Carlet J, Falke K, Hudson L, Lamy M, Legall JR, Morris A, Spragg R, et al.: The American-European consensus conference on ARDS: definitions, mechanisms, relevant outcomes and clinical trial coordination. Am J Respir Crit Care Med 1994, 149: 818-824.View ArticlePubMedGoogle Scholar
  43. Fanning NF, Porter J, Shorten GD, Kirwan WO, Bouchier-Hayes D, Cotter TG, Redmond HP: Inhibition of neutrophil apoptosis after elective surgery. Surgery 1999, 126: 527-534.View ArticlePubMedGoogle Scholar
  44. Matsuda T, Saito H, Fukatsu K, Han I, Inoue T, Furukawa S, Ikeda S, Hidemura A: Cytokine-modulated inhibition of neutrophil apoptosis at local site augments exudative neutrophil functions and reflects inflammatory response after surgery. Surgery 2001, 129: 76-85. 10.1067/msy.2001.109060View ArticlePubMedGoogle Scholar
  45. Suzuki R, Iwase M, Miyaoka K, Kondo G, Watanabe H, Ohashi M, Nagumo M: Modulation of neutrophil apoptosis in plasma of patients after orthognathic surgery. J Surg Res 2006, 130: 110-118. 10.1016/j.jss.2005.08.001View ArticlePubMedGoogle Scholar
  46. Delogu G, Moretti S, Famularo G, Antonucci A, Signore L, Marcellini S, Lo Bosco L, De Simone C: Circulating neutrophils exhibit enhanced apoptosis associated with mitochondrial dysfunctions after surgery under general anaesthesia. Acta Anaesthesiol Scand 2001, 45: 87-94. 10.1034/j.1399-6576.2001.450114.xView ArticlePubMedGoogle Scholar
  47. Haslett C, Guthrie LA, Kopaniak MM, Johnston RB Jr, Henson PM: Modulation of multiple neutrophil functions by preparative methods or trace concentrations of bacterial lipopolysaccharide. Am J Pathol 1985, 119: 101-110.PubMed CentralPubMedGoogle Scholar
  48. Downey GP, Chan CK, Lea P, Takai A, Grinstein S: Phorbol ester-induced actin assembly in neutrophils: role of protein kinase C. J Cell Biol 1992, 116: 695-706. 10.1083/jcb.116.3.695View ArticlePubMedGoogle Scholar
  49. Newman SL, Henson JE, Henson PM: Phagocytosis of senescent neutrophils by human monocyte-derived macrophages and rabbit inflammatory macrophages. J Exp Med 1982, 156: 430-442. 10.1084/jem.156.2.430View ArticlePubMedGoogle Scholar
  50. Cox G: IL-10 enhances resolution of pulmonary inflammation in vivo by promoting apoptosis of neutrophils. Am J Physiol 1996,271(Lung Cell Mol Physiol 15):L566-L571.PubMedGoogle Scholar
  51. Brown S, Bailey K, Savill J: Actin is cleaved during constitutive apoptosis. Biochem J 1997, 323: 233-237.PubMed CentralView ArticlePubMedGoogle Scholar
  52. Imai Y, Parodo J, Kajikawa O, Perrot M, Fischer S, Edwards V, Cutz E, Liu M, Keshavjee S, Martin TR, et al.: Injurious mechanical ventilation and end-organ epithelial cell apoptosis and organ dysfunction in an experimental model of acute respiratory distress syndrome. JAMA 2003, 289: 2104-2112. 10.1001/jama.289.16.2104View ArticlePubMedGoogle Scholar
  53. Plötz FB, Slutsky AS, van Vught AJ, Heijnen CJ: Ventilator-induced lung injury and multiple system organ failure: a critical review of facts and hypotheses. Intensive Care Med 2004, 30: 1865-1872. 10.1007/s00134-004-2363-9View ArticlePubMedGoogle Scholar
  54. Tremblay L, Valenza F, Ribeiro SP, Li J, Slutsky AS: Injurious ventilatory strategies increase cytokines and c- fos m-RNA expression in an isolated rat lung model. J Clin Invest 1997, 99: 944-952.PubMed CentralView ArticlePubMedGoogle Scholar
  55. Slutsky AS, Tremblay LN: Multiple system organ failure: is mechanical ventilation a contributing factor? Am J Respir Crit Care Med 1998, 157: 1721-1725.View ArticlePubMedGoogle Scholar
  56. Lionetti V, Recchia FA, Ranieri VM: Overview of ventilator-induced lung injury mechanisms. Curr Opin Crit Care 2005, 11: 82-86. 10.1097/00075198-200502000-00013View ArticlePubMedGoogle Scholar
  57. Kuiper JW, Groeneveld ABJ, Slutsky AS, Plötz FB: Mechanical ventilation and acute renal failure. Crit Care Med 2005, 33: 1408-1415. 10.1097/01.CCM.0000165808.30416.EFView ArticlePubMedGoogle Scholar
  58. Iwase M, Kondo G, Watanabe H, Takaoka S, Uchida M, Ohashi M, Nagumo M: Regulation of Fas-mediated apoptosis in neutrophils after surgery-induced acute inflammation. J Surg Res 2006, 134: 114-123. 10.1016/j.jss.2005.10.013View ArticlePubMedGoogle Scholar
  59. Ranieri VM, Suter PM, Tortorella C, De Tullio R, Dayer JM, Brienza A, Bruno F, Slutsky AS: Effect of mechanical ventilation on inflammatory mediators in patients with acute respiratory distress syndrome: a randomized controlled trial. JAMA 1999, 282: 54-61. 10.1001/jama.282.1.54View ArticlePubMedGoogle Scholar
  60. dos Santos DD, Slutsky AS: Mechanotransduction, ventilator-induced lung injury and multiple organ dysfunction syndrome. Intensive Care Med 2000, 26: 638-642. 10.1007/s001340051217View ArticlePubMedGoogle Scholar
  61. Zang H, Downey GP, Suter PM, Slutsky AS, Ranieri VM: Conventional mechanical ventilation is associated with bronchoalveolar lavage-induced activation of polymorphonuclear leukocytes: a possible mechanism to explain the systemic consequences of ventilator-induced lung injury in patients with ARDS. Anesthesiology 2002, 97: 1426-1433. 10.1097/00000542-200212000-00014View ArticleGoogle Scholar
  62. Chiumello D, Pristine G, Slutsky AS: Mechanical ventilation affects local and systemic cytokines in an animal model of acute respiratory distress syndrome. Am J Respir Crit Care Med 1999, 160: 109-116.View ArticlePubMedGoogle Scholar
  63. Vlahakis NE, Schroeder MA, Limper AH, Hubmayr RD: Stretch induces cytokine release by alveolar epithelial cells in vitro. Am J Physiol 1999,227(1 Pt 1):L167-73.Google Scholar
  64. Moreno R, Vincent JL, Matos R, Mendonça A, Cantraine F, Thijs L, Takala J, Sprung C, Antonelli M, Bruining H, et al.: The use of maximum SOFA score to quantify organ dysfunction/failure in intensive care. Results of a prospective, multicenter study. Intensive Care Med 1999, 25: 686-696. 10.1007/s001340050931View ArticlePubMedGoogle Scholar
  65. Ferreira FL, Bota DP, Bross A, Mélot C, Vincent J-L: Serial evaluation of the SOFA score to predict outcome in critically ill patients. JAMA 2001, 286: 1754-1758. 10.1001/jama.286.14.1754View ArticlePubMedGoogle Scholar

Copyright

© Fialkow et al.; licensee BioMed Central Ltd. 2006

This article is published under license to BioMed Central Ltd. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.