By visualizing DNA with diamidino phenylindole (DAPI), we found that hypothermal incubation followed by rewarming of human neutrophils resulted in an increased number of DAPI-positive objects representative of extensive DNA unfolding seemingly similar to neutrophil extracellular traps (NETs). In contrast to canonical NET formation, diphenylene iodonium (DPI), an NADPH oxidase inhibitor, exhibited negligible effects on formation of the DAPI-positive objects. Moreover, multiple instances of DNA damage were detected in the objects, but not in canonical NETs. Our results thus suggest the potential of hypothermia for triggering DNA structural alteration in neutrophils, which is similar to but distinct from NET formation.
Low-temperature conditions, referred to as hypothermia, are generally used for the storage of cells, tissues, organs and bodies for both scientific and clinical purposes. Hypothermia is an important means of slowing down cellular metabolism during storage, thus inhibiting injurious processes caused by the deficiency of oxygen and substrate supply. However, hypothermia can give rise to cell injury, including cell death [1,2].
Neutrophils are a main type of effector cell in the innate immune system [3,4], which circulate in the blood and engulf invading microorganisms such as bacteria and fungi by phagocytosis. In addition to such activities, Brinkmann et al. have reported that, following activation by microorganisms, neutrophils can undergo morphological changes detectable by microscopic observations [
In addition to microorganism infection, several physiological inducers of NETs have been reported [7 and references herein]. For instance, platelets activated via Tolllike receptor 4 rapidly induce NET formation [
In this study, we found that hypothermal incubation of human neutrophils at 4˚C for up to 1 h followed by incubation at 37˚C resulted in an increased number of DAPI-stainable objects similar to global DNA unfolding observed in PMA-stimulated NETs. However, our additional experimental data revealed that hypothermia/rewarming-induced DNA unfolding was regulated in a manner similar to, but biochemically and pharmacologically distinct from, canonical NETs. Although the molecular mechanism of this phenomenon is not fully understood, we inferred, based on our experimental data, the possible role of ROS, which were generated during hypothermia/rewarming-treatment in a manner independent of NADPH oxidase activity in the formation of the DAPI-positive, NET-like objects.
Human peripheral blood preparations (from two normal male donors, collected in compliance with Kumamoto Health Science University and approved by the University Oversight Committee) were enriched for neutrophils by density gradient centrifugation with HISTOPPAQUE 117 (Sigma-Aldrich) and Lymphocyte Separation Solution 1.119 (Nakarai Tesque) according to the procedures described by the supplier. Washed enriched neutrophilic fractions were counted and examined for purity using Wright Giemsa staining (Sigma-Aldrich).
Cells were incubated in culture dishes containing an immersed coverslip in RPMI 1640 (Sigma-Aldrich) supplemented with 5% fetal bovine serum (FBS), 1% penicillin/streptomycin and 0.1% gentamaycin in a humidified atmosphere containing 5% CO2. To induce NETs, PMA (Wako Pure Chemical Industries) was added to the culture medium at a concentration of 50 nM and incubated for 4 h at 37˚C. To inhibit NADPH oxidase activity, DPI (Cayman Chemical) was added to the culture medium at a concentration of 20 μM. Hypothermal treatment and rewarming of cells were performed by incubation in a humidified atmosphere containing 5% CO2. After drug and/or hypothermal/rewarming treatment, the coverslips were removed from the cultures and subjected to appropriate assays. DNA was visualized by staining with DAPI.
Cells were washed once for 5 min with ice-cold PBS and then fixed with 4% paraformaldehyde in PBS for 5 min at room temperature. After fixation, the cells were rinsed once with PBS and subjected to indirect-immunofluorescence analysis using anti-neutrophil elastase (Calbiochem), anti-histone H3 (Santa Cruz Biotechnology), and anti-histone H3 citrulline R26 (Abcam) antibodies. The secondary antibodies were obtained from Santa Cruz Biotechnology and Sigma-Aldrich.
Escherichia coli BL21 (DE3) were transformed with pET28-EGFP, a plasmid for expression of green fluorescent protein (GFP), and cultured in Luria-Bertani (LB) medium containing kanamycin at 37˚C for 16 h. 107 E. coli cells were incubated with a coverslip containing hypothermia/rewarming-induced DAPI-positive objects in RPMI 1640 supplemented with 5% FBS at 37˚C. After 20 min at room temperature, the coverslips were washed three times with PBS followed by incubation with 4% paraformaldehyde. DNA fibers were stained with DAPI. Because the E. coli expressed GFP, bacteria trapped by DNA fibers could be detected by fluorescence microscopy. For DNase I treatment, the coverslips were treated with PBS containing 100 U/ml DNase I (Takara) at 37oC for 1 hr. The numbers of E. coli with GFP signals on the coverslips were counted by fluorescent microscopy.
Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assays were performed using the MEBSTAIN Apoptosis TUNEL Kit II (MBL) according to the manufacturer’s instructions. The TUNEL-positive cells were counted under a microscope. The percentage of TUNEL-positive cells was defined by the number of positive cells among the total number of cells in each sample. For one experiment, cells were counted in at least three different microscopic fields of view.
Intracellular ROS production was monitored using the cell permeable fluorescent dye, CellROX Deep Red Reagent (Invitrogen). This agent can readily react with ROS to form a fluorescent product proportional to the amount of ROS generated in the cells. The cells were incubated with 5 μM CellROX Deep Red for 30 min and then harvested. The fluorescence intensity of the cells was measured using a FACSVerse flow cytometer (BD Biosciences).
After fixation with 4% paraformaldehyde, cultured human neutrophils were stained with the mitochondrionspecific dye, MitoTracker Red CMXRRos (Invitrogen), according to the manufacturer’s instruction. The cells were immediately analyzed using a FACSVerse flow cytometer.
Unless otherwise stated, all data are presented as the mean ± SD. Within individual experiments, data points were based on a minimum of triplicate representative samples and experiments were repeated at least three times.
After isolating human neutrophils from peripheral blood preparations (see Materials and Methods), the cells (4.5 × 106 cells) were incubated at 4˚C for 1 h followed by incubation at 37˚C for 5 h in the 6-cm culture dish supplemented with 2 ml of the culture medium, in which a coverslip was immersed (
When the materials on the coverslip were stained with DAPI without any fixative treatment, we observed bright fluorescent signals under fluorescent microscopy, many of which appeared to consist of multiple DAPI-positive strings (
We then investigated whether the requirement for DAPI-positive object production was simple exposure to hypothermia or rather the combination of hypothermia/ rewarming. When human neutrophils were maintained at a constant temperature of either 4˚C or 37˚C, significantly less DAPI-positive signals were detected as compared with cells cultured either at 4˚C, 15˚C, or 25˚C for 1 h followed by incubation at 37˚C for 5 h (
When we observed the DAPI-positive objects in hypothermia/rewarming-treated human neutrophils, we noticed that morphological similarities between the objects and DAPI-stained PMA-stimulated NETs, leading us to suspect that the DAPI-positive objects per se might represent NETs (
To further evaluate the similarities between the DAPIpositive objects and canonical NETs, we performed indirect-immunofluorescence analysis using antibodies that recognize marker proteins for NETs: anti-neutrophil
elastase (NE) and anti-histone H3 antibodies [
It should be noted, however, the experiments described above are not sufficient to conclude that the DAPI-positive objects have anti-bacterial activity. We are therefore investigating whether NE and histones on the DAPIpositive objects can indeed inactivate bacterial toxic proteins, called “virulence factors,” and inhibit bacterial growth. In addition, we wish to test whether the DAPIpositive objects can capture microorganisms besides Gram-negative bacteria (E. coli), such as fungi and parasites.
Although our results so far indicated a correlation between DAPI-positive objects and NETs, several differences were also revealed. For instance, when indirect immunofluorescence analysis was conducted using antihistone H3 citrulline R26 (anti-H3cit) antibody, we found that the antibody stained many, but not all, PMAstimulated NETs, whereas the antibody proved poor at detecting the DAPI-positive objects (
We also detected differences between the objects and NETs in TUNEL assays. As shown in
Given that ROS generation is an absolute requirement for the formation of NETs [15,16], we next assessed whether hypothermia/rewarming of neutrophils coincided with the generation of ROS. Thus, we measured ROS in hypothermia/rewarming-treated human neutrophils. As shown in
by incubation at 37˚C for 1 h or 3 h; an approximate 10- fold increase in ROS was apparent in the cells after incubation at 37˚C.
We next examined whether NADPH oxidase contributed to ROS production in hypothermia/rewarmingtreated cells. Given that PMA-induced NET formation is effectively inhibited by DPI, an inhibitor of NADPH oxidase activity [15,17,18 and see
Although where and how ROS are produced in the hypothermia/rewarming-treated cells remains unclear, it is noteworthy that the mitochondrion-specific dye, MitoTracker Red, detected structural and/or functional alterations in mitochondria in the hypothermia/rewarmingtreated cells (
DAPI-positive objects with extensive DNA unfolding were observed in human neutrophils cultured in hypothermic conditions followed by rewarming. Our experimental data indicated that such DNA structural alterations in neutrophils may be related to NET formation, but can be biochemically and pharmacologically discriminated from NET formation. We also considered that the objects might not represent apoptotic cells, given that apoptotic cells contain condensed DNA enclosed in membrane, which is not observed in the objects. We thus suggest that the hypothermia/rewarming-induced DNA unfolding is regulated in a manner distinct from either canonical NETosis or canonical apoptosis, arguing the existence of a previously unappreciated signaling pathway that alters global genomic DNA structures in eukaryotic cells. Further, the results indicate that coldtreatment followed by warming may affect NET formation, which is an important consideration because many researchers use hypothermal conditions during the isolation and culture of neutrophils.
We thank all the members of the Saitoh Laboratory for helpful discussion. This work was supported by research grant to H. S. from Astellas Foundation for Research on Metabolic Disorders, and by intramural founding in Kumamoto Health Science University to J. K.