Identification of neutrophil extracellular traps in the blood of patients with systemic inflammatory response syndrome

Abstract

Objective

Neutrophils are able to form ‘neutrophil extracellular traps’ (NETs), which they use to trap and kill pathogens such as bacteria and fungi at the foci of infection. This observational study investigated the presence of NETs in the blood from critically ill patients and healthy volunteers.

Methods

Fluorescent triple-colour immunocytochemical analysis of blood smears collected from patients with systemic inflammatory response syndrome (SIRS; associated with various clinical conditions) who had been hospitalized in the intensive care unit, and healthy volunteers, was undertaken to identify NETs in the blood. Blood smears were stained for DNA, histone H1 and neutrophil elastase.

Results

NETs were identified in 10 of 21 (47.6%) blood samples from the study group compared with none of the blood samples from eight healthy volunteers.

Conclusion

These data suggest that fluorescent triple-colour immunocytochemical staining of NETs in the blood could be used to simplify the early identification of critically ill patients with SIRS. Larger studies are required to clarify the pathophysiological role of NETs in this specific patient population.

Introduction

Neutrophils are the first line of defence provided by the innate immune system. ¹ A novel mechanism of action of neutrophils used to kill bacteria, called ‘neutrophil extracellular traps’ (NETs), has been identified. ² NETs are fibrous mesh-like structures that rapidly trap and kill microbial pathogens. ³ NETs are induced in activated neutrophils by a variety of proinflammatory stimuli including lipopolysaccharides, interleukin-8, tumour necrosis factor and various pathogens,^4–6 and are extruded into the extracellular space. This series of events is called ‘NETosis’. ⁵

Neutrophil extracellular traps are composed of decondensed chromatin including histones (H1, H2A, H2B, H3, and H4), covered with antimicrobial factors such as neutrophil elastase, myeloperoxidase, bactericidal permeability-increasing protein, matrix metalloproteinase 9, pentraxin 3, and LL37.^7–10

Before the discovery of NETs, increases in circulating free DNA (cf-DNA) in the blood had been reported in various diseases including several cancers,^11–13 trauma, stroke, autoimmune disorders, sepsis and septic arthritis.^14–16 This cf-DNA was formerly thought to originate from necrotic and/or apoptotic cells, ¹⁷ but several articles have suggested an association between NETs and cf-DNA.^14–18 In these reports, cf-DNA was present both in infectious and noninfectious disease conditions, including a mouse model of lipopolysaccharide-induced shock. It was also reported that NETs in capillary vessels might be related to the pathophysiology of disseminated intravascular coagulation. ¹⁹

Although NETs have been extensively studied,^2–10 it has been difficult to evaluate NET formation in clinical specimens. The authors have previously reported that purulent sputum from patients with acute respiratory infections contained abundant NETs, the amount of which gradually decreased as the clinical symptoms and inflammation subsided. ²⁰ The present study, using a similar immunocytochemical approach, investigated NET formation in the bloodstream using clinical blood smear samples from critically ill patients who were hospitalized in an intensive care unit (ICU) and from healthy volunteers.

Patients and methods

Study Population and Sample Collection

For this preliminary pilot study, patients who fulfilled the criteria for systemic inflammatory response syndrome (SIRS) associated with various critical conditions were selected from the ICU, Trauma and Acute Critical Care Centre, Osaka University Hospital, Osaka, Japan, between October 2012 and May 2011. SIRS was diagnosed on the basis of criteria defined by the American College of Chest Physicians/Society of Critical Care Medicine Consensus Committee. ²¹ Patients who died or who were extubated during the study period were excluded from this study. Healthy volunteers were recruited from Suita City, Osaka Prefecture, Japan, as controls.

Blood samples were collected at the time of admission to the ICU on an individual case basis, or from healthy controls, using commercial syringes (TERUMO, Tokyo, Japan). Blood samples were immediately applied in a thin layer to glass slides, dried and stored at –80°C until the immunocytochemical analysis was performed.

This study was approved by the Ethics Committee of Osaka University Graduate School of Medicine, and all clinical samples were obtained with verbal informed consent from each patient, in accordance with the Declaration of Helsinki.

Identification of NETs

To identify NETs, the major NET components, DNA, histone H1, and neutrophil elastase were visualized simultaneously with triple-colour fluorescent immunostaining. In brief, the sample on the glass slide was fixed with 4% paraformaldehyde for 30 min, washed with 10 mM phosphate-buffered saline (PBS; pH 7.4), then blocked with a solution containing 20% BLOCK-ACE (Dainippon-Sumitomo Seiyaku Pharma, Osaka, Japan) and 0.005% saponin in 10 mM PBS for 10 min. The samples were then incubated at room temperature for 60 min with the following primary antibodies: antihuman histone H1 mouse monoclonal antibody (ab71594; Abcam®, Cambridge, MA, USA), diluted 1 : 100 in 10 mM PBS with 5% BLOCK-ACE and 0.005% saponin; and antihuman neutrophil elastase rabbit polyclonal antibody (no. 481001; Calbiochem, Darmstadt, Germany), diluted 1 : 50, as described above. Samples were gently washed three times for 5 min each, in 10 mM PBS at room temperature. Each primary antibody was visualized with a secondary antibody diluted 1 : 500, as described above: Alexa-Fluor®-546-conjugated goat antimouse immunoglobulin (Ig) G (Invitrogen, Carlsbad, CA, USA) or Alexa-Fluor®-488-conjugated goat antirabbit IgG (Invitrogen). After incubation for 60 min at room temperature with the secondary antibody, samples were gently washed three times for 5 min each time in 10 mM PBS at room temperature, and the DNA was stained with 4′,6-diamidino-2-phenylindole (DAPI; Invitrogen) diluted in 10 mM PBS for 5 min at room temperature. Specimens were visualized using a fluorescence microscope (BZ-9000; Keyence, Osaka, Japan).

Neutrophil extracellular traps were defined as extracellular string-like structures that were simultaneously immunoreactive for DNA, histone H1 and neutrophil elastase. Samples were considered negative if no NETs were identified in 300 neutrophils. Although there was a possibility that neutrophils could be damaged during the process of smearing onto the glass slide, it was confirmed that these structures were not observed in the blood smears from the healthy control subjects.

Statistical Analyses

All statistical analyses were performed using Prism® software, version 5.02 (GraphPad Software, San Diego, CA, USA). Fisher’s exact test and Student’s t-test were used to compare differences between the study and control groups. A P-value < 0.05 was considered statistically significant.

Results

Population Characteristics

Demographic and clinical characteristics of 21 patients hospitalized in the ICU and eight healthy volunteers are shown in Table 1, together with the numbers of patients who had NETs detected in their blood smear. NETs were detected in significantly more specimens from participants in the study group, compared with the control group (P = 0.0265).

Table 1.

Demographic and clinical characteristics of the study population (n = 29) and the prevalence of circulating neutrophil extracellular traps (NETs) in the blood by immunocytochemical analysis.

Characteristic	Study group n = 21	Control group n = 8	Statistical significance a
Age, years	52.2 ± 23.0	52.9 ± 23.7	NS
Sex, males/females	15/6	6/2	NS
NETs(+) in blood	10 (47.6)	0 (0)	0.0265
NETs(+) in blood based on diagnosis b
Sepsis	12 [5]	NA
Trauma	2 [1]	NA
Burn	1 [1]	NA
Cardiopulmonary arrest	4 [2]	NA
Drug overdose	1 [0]	NA
Gastric ulcer-related haemorrhagic shock	1 [1]	NA

Data presented as mean ± SD, n or n (%) of patients, unless stated otherwise.

a

Statistical analyses undertaken using Fisher’s exact test and Student’s t-test.

b

Diagnoses included a range of diseases; all patients in the study group were clinically diagnosed with SIRS; n patients with subdiagnosis [n patients with subdiagnosis who also had NETs in blood].

NETs(+), positive for NETs; NA, not applicable; NS, no statistically significant differences (P ≥ 0.05).

Representative Cases

Case 1 was a 36-year-old woman who had been admitted to the ICU with sepsis and disturbed consciousness as a result of encephalitis. On admission, her white blood cell count was 11 050 cells/µl and her blood cultures were negative for bacteria. Fluorescent triple-colour immunostaining indentified NET components in her blood smear. Interestingly, the string-like structures extending from deformed cell bodies were triply stained for DNA (blue), histone H1 (red), and neutrophil elastase (green), as shown in Figure 1A, suggesting that these structures were NETs and that NET formation had been induced in the blood. The patient survived and was eventually discharged.

Figure 1.

Fluorescent immunocytochemical staining of blood smears from representative patients with systemic inflammatory response syndrome and healthy volunteers. (A) Case 1, and (B) cases 2 and 3 had circulating neutrophil extracellular traps (NETs) identified by three-colour immunocytochemical staining for DNA (blue; stained with 4′,6-diamidino-2-phenylindole), histone H1 (red), and neutrophil elastase (N-E, green), whereas (C) healthy controls, cases 4 and 5, did not. White arrowheads, NET structures. Scale bars = 30 µm.

Fluorescent triple-colour immunostaining also detected NETs in the blood samples of critically ill patients with haemorrhagic shock arising from a bleeding gastric ulcer (case 2, Figure 1B) and with acute subdural haematoma (case 3, Figure 1B). In contrast, no NETs were observed in blood samples taken from all eight healthy control subjects (cases 4 and 5, Figure 1C).

Discussion

Neutrophil extracellular traps are generally considered to be formed from activated neutrophils after stimulation by invading bacteria or fungi at the foci of infection. ⁴ The present study showed, however, that NETs in blood are formed not only under septic conditions, but also under noninfectious conditions including trauma and burns. All of the patients presenting with NETs in blood samples were clinically diagnosed with SIRS. This could be explained by the fact that NETs may be released into the blood in association with cytokines and inflammatory mediators. ²²

Although NETs were detected in the present study, the source of their release remains unclear. One hypothesis is that NETs are released in capillary vessels and carried to the bloodstream. ²³ A previous study reported that NETs coexist with platelets inside capillaries and form complexes with platelets, under septic conditions. ¹⁹

In the present study, NETs were identified in the blood of critically ill patients with SIRS, but not in healthy individuals. Although only one similar report has demonstrated that circulating NETs were observed in malaria smears, ²⁴ it could not confirm that the structures were NETs because only DAPI was used to stain the DNA. To ensure that a structure is a NET, three-colour fluorescent immunocytochemical staining of DNA, histone, and neutrophil elastase is required. This immunocytochemical method of using blood smears to confirm the presence of NETs in blood might be a useful diagnostic approach in critical conditions such as SIRS. As this method may be simpler than measuring inflammatory markers, it might prove to be helpful in the early diagnosis of SIRS. Once the string-like structures detected on the blood smears are confirmed as NETs, it might be logical to detect subsequent NETs using simple DAPI staining alone, as previously reported. ²⁴

In conclusion, the present study suggests that NETs are present in the blood of critically ill patients with SIRS and their detection might aid in early diagnosis. Larger studies are required to clarify the pathophysiological role of NETs in this specific patient population.