Bicarbonate-free Hank’s solution, Ca2⁺-free Dulbecco PBS, imipramine, LPS (lipopolysaccharide from Salmonella enterica serovar Typhimurium) and E64 were obtained from Sigma (Steinheim, Germany). Ficoll-Paque was obtained from Pharmacia (Uppsala, Sweden). Fibronectin was from Calbiochem (La Jolla, United States). Coomassie Brilliant Blue G-250 was obtained from Serva, PMSF from MP Biomedical, trypan blue from Fluka AG, glutaraldehyde from Ted Pella. Trypsin was from Promega, carboxy-H₂DCF-DA from Molecular probe, United States. Analytical chromatography conditions: eluent MCI Buffer L-8800-PH-1–4 and ninhydrin coloring solution kit for Hitachi 29970501 (Wako Chemicals GmbH, United States).

Neutrophils were isolated from the blood of healthy volunteers who had not taken medication for 2 weeks. All donors gave their informed consent. The study was approved by the Bioethics Commission of M.V. Lomonosov Moscow State University, application # 6-h version 3, approved during the Bioethics Commission meeting # 131-days held on May 31, 2021.

Erythrocytes were precipitated in the presence of 3% T-500 dextran at room temperature. Neutrophils were isolated from plasma by centrifugation through Ficoll-Paque at a density of 1.077 g/ml, followed by hypotonic lysis of the remaining erythrocytes in buffer (114 mM NH₄Cl, 7.5 mM KHCO₃, 100 μM EDTA) and washing in PBS. Before experiments, neutrophils were stored in Dulbecco PBS containing 1 mg/ml glucose (no CaCl₂). Neutrophils accounted for 96–97% of the total number of cells in the preparation. The viability of neutrophils was determined by staining with trypan blue dye, which stains dead cells but does not penetrate viable cells. Neutrophils were incubated with 0.5 mM trypan blue in Hanks solution for 15 min at 37°C, washed, and the number of dead cells was counted. The percentage of dead cells did not exceed 1–2% of the total number of counted cells (3,000 cells per group).

Cellstar six-well culture plates (Frichenhausen, Germany) were coated with fibronectin for 2 h incubation with fibronectin (5 μg/ml) in Hank’s solution at room temperature and washed. Neutrophils were attached to fibronectin-coated wells (3 × 10⁶ cells in 1.3 ml/well) for 20 min incubation in Hank’s solution containing 10 mM HEPES (pH 7.35) at 37°C. LPS and imipramine were added to the cells prior to incubation. After incubation, samples of the extracellular medium were taken and mixed with inhibitors of metalloproteinase, serine and cysteine proteinases, and myeloperoxidase (EDTA, 5 mM; PMSF, 200 μM; E64, 10 μM; and sodium azide, 0.025%, respectively). Neutrophils remaining in the extracellular medium were removed by centrifugation (5 min at 400 x g at room temperature). Extracellular medium samples from three identical wells were pooled for amino acid analysis. To determine the protein content in the secretion of neutrophils, samples from six identical wells were combined.

Neutrophils (2 × 10⁵ cells/probe) in HBSS/HEPES, supplemented or not with imipramine, were incubated in fibronectin-coated 96-well plates for 30 min at 37°C in 5% CO₂. Supernatants were then carefully removed followed by double washing with warm PBS to remove free-floating or weakly attached cells. Quantification of adhesion was carried out according to the method described by Ngo and coauthors (Ngo and Lenhoff, 1980; Sud’ina et al., 2001). Briefly, hydrogen peroxide (4 mM final concentration) in permeabilizing buffer (67 mM Na₂HPO₄, 35 mM citric acid, 0.1% Triton X-100) supplemented with 5.5 mM o-Pphenylenediamine dihydrochloride (OPD) was added to substrate-bound neutrophils for 5 min. MPO-catalyzed oxidation of OPD by H₂O₂ leading to the formation of colored product 2, 3-diaminophenazine was stopped by adding of 1M H₂SO_4. The absorption was measured at a wavelength of 490 nm and compared with the calibration values.

Proteins from samples of the extracellular medium were extracted with an equal volume of chloroform-methanol (2: 1, v/v), as previously published (Galkina et al., 2012). The chloroform phase was separated by centrifugation for 20 min at 11,000 x g, collected and, after evaporation of the solvent, subjected to electrophoresis. Proteins were separated by one-dimensional electrophoresis in the presence of sodium dodecyl sulfate under non-reducing conditions on a 15% polyacrylamide gel in a Mini-PROTEAN 3 cell (Bio-Rad). Aliquots of the samples were boiled for 3 min in lysis buffer (Tris-HCl 30 mM, pH 6.8; SDS 1%; urea 3 M; glycerol 10%; bromophenol blue 0.02%) before electrophoresis. Protein bands were stained with 0.22% Coomassie brilliant blue G-250.

Matrix assisted laser desorption ionization mass spectrometry (MALDI-MS) analysis of proteins was performed with a MALDI-ToF-ToF mass spectrometer Ultraflextreme (Bruker, Karlsruhe, Germany) as previously described (Galkina et al., 2021). Protein hydrolysis with trypsin was performed directly in the gel. Gel pieces were excised from each protein band, washed, dehydrated, air dried, and subjected to trypsin digestion in the gel. The peptides resulting from hydrolysis were extracted with 0.5% trifluoroacetic acid. Aliquots were taken from each sample and mixed on a steel target with 2,5-dihydroxybenzoic acid (30 mg/ml in 30% acetonitrile and 0.5% trifluoroacetic acid), dried and subjected to mass spectrometric analysis. The [MH]⁺ molecular ions were measured in reflector mode; the accuracy of mass peak measurement was within 30 ppm. Identification of proteins was carried out by a peptide fingerprint search using Mascot software 2.5.01 (http://www.matrixscience.com, accessed on January 3, 2021), SwissProt database through the mammalian proteins. When the score was >68, protein matches were considered significant (p < 0.05).

Samples of the extracellular medium, which were combined from three identical wells, were concentrated using a Centrivap Concentrator Labconco (United States), then the proteins were precipitated with sulfosalicylic acid (4.4%). The sediments were removed by centrifugation for 30 min at 18,000 x g. Supernatants were centrifuged through Vivaspin 500 Membrane 3000 PES MWCO membrane ultrafilters (Sartorius, Germany) and subjected to amino acid analysis.

The amino acid analysis was conducted on an L-8800 amino acid analyzer (Hitachi, Tokyo, Japan) in the standard mode according to the manufacturer’s user manual (Hitachi High-Technologies Corporation, Japan, 1998) as described previously (Galkina et al., 2019). The prepared samples were separated on a 2622SC-PH ion-exchange column (Hitachi, Ltd., P/N 855-3,508, 4.6*80 mm) by step gradient of four sodium-acetate buffers at an elution rate 0.4 ml/min at 57°C. The stained products were detected by measuring the absorbance at 570 nm for all amino acids except proline and at 440 nm for proline. MultiChrom for Windows software (Ampersand Ltd., Moscow, Russia) was used for processing the chromatographic data.

For coating with fibronectin the cover slips were incubated in a buffer containing 5 μg/ml fibronectin for 2 h at room temperature and washed. Neutrophils were attached to the fibronectin-coated cover slips (3 × 10⁶ cells in 2 ml per well) during 20 min incubation in a Hanks solution containing 10 mM HEPES (pH 7.35) at 37°C. LPS (10 μg/ml) and imipramine (100 µM) were added to the cells before incubation. After incubation, attached neutrophils were fixed in 2.5% glutaraldehyde in Hanks buffer without Ca²⁺ or Mg²⁺ ions, but containing 5 mM EDTA and 0.5 mM phenylmethylsulfonyl fluoride (PMSF), metalloproteinase and serine proteases inhibitors, and 10 mM HEPES at pH 7.3. In addition, the cells were fixed with a 1% solution of osmium tetroxide in 0.1 M sodium cacodylate containing 0.1 M sucrose at pH 7.3. Then the cells were dehydrated in a series of acetones (10–100%) and dried in a Balzer apparatus at the critical point with liquid CO₂ as a transition liquid. Samples coated with gold/palladium sputtering were then examined at 15 KV with a scanning electron microscope Camscan S-2. The area occupied by the cells on the substrate was measured quantitatively using an ImageJ-win64 software in scanning electron microscopy images.

Neutrophils attached to fibronectin-coated coverslips under control conditions or in the presence of imipramine were fixed in the same way as for scanning electron microscopy. Fixed samples were dehydrated in the usual way (70% ethanol containing 2% uranyl acetate), embedded in Epon 812 (Fluka), cut into ultrathin sections with a Reichert Ultra Cut III and stained with lead citrate. The internal morphology of the cell was examined using a JEM-1400 transmission electron microscope.

Neutrophils attached to coverslips under control conditions or in the presence of imipramine were fixed in 4% paraformaldehyde in HEPES buffer free of Ca²⁺ and Mg²⁺ containing 5 mM EDTA (pH 7.3). Then the cells were treated with 0.1% Triton X-100 solution for 10 min to increase the permeability. FITC phalloidin was used to stain actin. Phase contrast and fluorescence images of neutrophils were observed using a Zeiss Axiovert 200M microscope.

The formation of intracellular ROS was monitored by measuring the green fluorescence of the oxidation product (DCF) of dichlorodihydrofluorescein diacetate (H₂DCF-DA, Molecular probe, United States). Human neutrophils were incubated with 5 μM carboxy-H₂DCF-DA for 60 min at room temperature and washed with PBS according to the manufacturer’s protocol. The cells were then plated onto fibronectin-coated 96-well plates (1 × 10⁶/ml HBSS/HEPES) during incubation according to the experimental protocol at 37°C in 5% CO₂ under control conditions and in the presence of 10 or 100 µM imipramine. DCF fluorescence signals (excitation 485 nm, emission 538 nm) were monitored at 10 min intervals on ClarioStar fluorescence microplate reader (BMG Labtech, Ortenberg, Germany).

We studied phosphatidylserine externalization and membrane integrity of neutrophils exposed to imipramine using simultaneous staining with Annexin V-Alexa Fluor 488 and nonvital dye propidium iodide (AnnV/PI) followed by flow cytometry. Neutrophils were suspended at a density of 1 × 10⁶ cells/mL in HBSS containing 10 mM HEPES under control conditions or in the presence of 10 or 100 μM imipramine and incubated for 4 h at 37°C in a 5% CO₂ incubator. After incubation, cells were sedimented by centrifugation at 270 × g and resuspended in Annexin V-Alexa Fluor 488 commercial solution (Merck, Germany) according to the manufacturer’s instructions. After 10 min on ice, propidium iodide (Merck, Germany) solution (10 μg/ml HBSS/HEPES) was added for 5 min. The samples were analyzed on CytoFLEX flow cytometer (Beckman Coulter, Krefeld, Germany) using CytExpert 2.0 software. Fluorescence was detected by photomultipliers at 525 nm (AnnV) and 620 nm (PI). Leukocyte subpopulations were plotted as a dot plot and gated according to size and granularity. 20,000 data events were collected for each acquisition.

For DNA fragmentation assessment, PMNLs were suspended at a density of 1 × 10⁶ cells/mL in RPMI 1640 medium (10% fetal bovine serum) and incubated for 18 h at 37°C in a 5% CO₂ incubator. Then cells were harvested, supplemented with ice-cold 0.05% BSA in PBS, collected by centrifugation and permeabilized in cold hypotonic PI solution (20 μg/ml PI, 0.2 mg/ml RNase in 0.1% Triton X-100 in 0.1% sodium citrate). The tubes were placed at 4°C in the dark for 10–15 min before flow cytometric analysis using CytoFLEX flow cytometer (Beckman Coulter, Krefeld, Germany) with excitation and emission wavelengths of 480 ± 10 and 585 ± 20 nm, respectively.

Each experiment to determine the amino acid or protein composition of neutrophil secretion was performed at least three times using blood from different donors. Experiments on electron microscopic determination of the morphology of neutrophils were repeated three times using the blood of different donors. Results are represented as mean ± SEM. The statistical significance was estimated using GraphPadPrism7 software.

We compared the morphology of neutrophils that were attached to the extracellular matrix protein fibronectin under control conditions and in the presence of imipramine using scanning and transmission electron microscopy. Neutrophils adhered and spread on fibronectin under control conditions (Figures 1A,C), while cell adhesion was partially inhibited in the presence of imipramine (Figures 1B,D). Imipramine reduced the area occupied by neutrophils on the substrate, which was measured in images of control and imipramine-treated cells obtained by scanning electron microscopy using ImageJ-win64 software (Figure 1E). The average area of the control cells was 232 μm² more than two times overcame the area of cells attached in the presence of imipramine 104 μm² (Figure 1E). In contrast to the well-attached control cells, the edges of imipramine-treated neutrophils were attached to the substrate only in part indicating poor attachment. The number of firmly attached control and imipramine treated neutrophils were compared after double washing with warm PBS to remove free-floating or weakly attached cells. Our data indicated that 10–100 μM imipramine statistically significant reduced cell attachment (Figure 1F). Internal morphology of neutrophils, examined by transmission electron microscopy, did not reveal specific differences in the intracellular structures of control neutrophils and neutrophils treated with imipramine (Figures 1C,D).

We also compared the organization of actin cytoskeleton in neutrophils that adhered to fibronectin under control conditions and in the presence of imipramine using fluorescent microscopy technique. The actin cytoskeleton undergoes depolymerization during the adhesion of neutrophils to fibronectin. The concentration of filamentous actin decreases, while monomeric actin increases during the first minutes of adhesion and then partial remodeling of the actin filaments occurs (Ginis et al., 1992; Wang et al., 1993). In our experiments, fluorescent actin staining showed that neutrophils placed on fibronectin under control conditions had diffuse actin staining of the entire cell with small actin filaments. There was no discernible difference in actin cytoskeleton between neutrophils that were attached to fibronectin under control conditions or in the presence of imipramine (Figure 2).

We studied the effect of imipramine on intracellular ROS production by neutrophils during adhesion to fibronectin by measuring the green fluorescence of DCF, an oxidized product of H2DCF-DA. The adhesion of neutrophils to the fibronectin-coated substrate occurs via integrin β-1 and β-2. The binding of β-2 integrin leads to the spreading of neutrophils along the substrate, the production of ROS and the outflow of chloride ions (Menegazzi et al., 1999). Assembly of the NADPH oxidase complex also occurs in response to the binding of β-1 integrin to the high-affinity binding site on fibronectin and following assembly and activation of focal adhesion complexes (Umanskiy et al., 2003). Our data demonstrated that adhesion to fibronectin itself initiated the ROS formation by neutrophils (Figure 3). Imipramine in the concentration range from 10 to 100 μM did not suppress, but significantly stimulated the production of ROS by neutrophils (Figure 3).

Neutrophils make up the main and rapidly renewing part of blood leukocytes. It is generally agreed that the physiological form of cell death in neutrophils is apoptosis (Geering and Simon, 2011). The entry of a cell into apoptosis takes a time. Neutrophils did not undergo apoptosis during 20 min adhesion (Galkina et al., 2021). To study the effect of imipramine on neutrophil apoptosis, we used standard methodological protocols, which provide for long-term incubation of cells with the test substance. In our case, neutrophils were incubated with imipramine for 4 or 18 h. We studied phosphatidylserine externalization and membrane integrity of neutrophils exposed to imipramine for 4 at 37°C using Alexa Fluor-conjugated Annexin V/propidium iodide double labeling followed by flow cytometry. Аfter 4 h incubation with 100 µM imipramine only 1.9% of cells underwent late apoptosis (Figure 4A A, region B2) and 0.2% of cells underwent necrosis (Figure 4A, region B1). Incipient apoptosis is evidenced by an increase in the number of cells with early apoptosis (Figure 4A, region B4), in which phosphatidylserine is exposed on the cell surface, but the cells retain their integrity. The effect of imipramine at a concentration of 10 μM was insignificant for early apoptosis.

To assess DNA fragmentation (Figure 4B), we incubated neutrophils with imipramine for 18 h. Neutrophils are short-lived cells. The incubation period, which lasts 18 h, seems too long for neutrophils, as a significant proportion of control cells (12.5%) die during this time (Figure 4B, control). Imipramine at concentrations of 10 or 100 μM stimulated the death of neutrophils, but the effect of imipramine was weakly concentration dependent (Figure 4B).

We studied the effect of imipramine on the composition of protein secretion by neutrophils, which adhere to fibronectin under control conditions or in the presence of LPS. Extracellular medium samples were taken from neutrophils after 20 min adhesion to fibronectin coated substrates. Proteins were extracted with a chloroform-methanol mixture. After evaporation of the solvent, the proteins of the chloroform fraction were separated by electrophoresis, subjected to hydrolysis with trypsin directly in the gel, and identified by mass spectrometric analysis (Galkina et al., 2012). Electrophoretic gels were stained with Coomassie brilliant blue, which allows the identification of proteins by mass spectrometry. Coomassie brilliant blue stained not all, but the main secreted proteins that form a stable protein profile of neutrophil secretion, specific for each treatment (Galkina et al., 2012; Galkina et al., 2017).

The protein profile of secretion of neutrophils that adhered to fibronectin under control conditions included proteins of primary (MPO) and secondary (LF, NGAL, lysozyme) granules, albumin and cytosolic S100A8 and S100A9 proteins (Figure 5A). This protein profile coincided with the protein content in the secretion of neutrophils during adhesion to fibronectin, which was previously published (Galkina et al., 2012). When neutrophils adhered to fibronectin in the presence of imipramine the protein profile of secretion did not contain NGAL and lysozyme, but was enriched with primary granule bactericide protease cathepsin G (Figure 5A, Table 1). The Mascot files obtained for all protein bands of the control + imipramine gel (Figure 5A) are presented in the supplementary file. The Mascot files contain all information about protein identification, including accuracy.

The secretion of neutrophils, which attach to fibronectin in the presence of LPS, contained the same proteins as the control cells, plus the tertiary granule component MMP-9, the primary granule components cathepsin G and defensins, and the cytoplasmic protein actin (Figure 5B). This profile coincided with the protein profile of neutrophil secretion during adhesion to fibronectin in the presence of LPS previously published (Galkina et al., 2017). The data showed that LPS stimulated the release of MMP-9, which play an important role in neutrophil adhesion and migration through its ability to modulate the extracellular matrix and remove basement membrane barriers (Dejonckheere et al., 2011). MMP-9 was secreted in parallel with NGAL, which forms complexes with MMP-9 after these components enter the extracellular environment. The NGAL complex supports allosteric activation of MMP-9 and/or protects MMP-9 from degradation by tissue metalloproteinase inhibitors, thereby maintaining enzyme activity (Tschesche et al., 2001; Yan et al., 2001).

LPS also stimulated the secretion of cathepsin G and HNP 1-3 (human neutrophil peptides 1-3 or defensins), aggressive primary granule bactericides that can initiate inflammation in surrounding tissues. In the extracellular environment, cathepsin G can interact with LF, which can serve as an allosteric enhancer of its proteolytic activity (Eipper et al., 2016).

Imipramine inhibited protein secretion by neutrophils treated with LPS upon adhesion to fibronectin. Only two major proteins, LF and S100A9, were identified in the extracellular environment of neutrophils, which attached to fibronectin in the presence of LPS and imipramine (Figure 5B, Table 1). The Mascot files obtained for all protein bands of the LPS + imipramine gel (Figure 5B) are presented in the supplementary file. Imipramine excluded MMP-9 and NGAL, cathepsin G, defensins and other proteins from neutrophil secretion during adhesion to fibronectin in the presence of LPS.

Using amino acid analysis, we compared the free amino acid composition of the extracellular environment of neutrophils taken after adhesion of cells to fibronectin under control conditions or in the presence of imipramine. Proteins from the extracellular medium were removed by precipitation with sulfosalicylic acid and subsequent centrifugation. To make sure that the presence of free amino acids in the extracellular medium is not a consequence of cell destruction, we stained neutrophils after collecting the extracellular medium with trypan blue. The percentage of stained (dead) cells was less than 1% for control or imipramine-treated cells. In the previous studies, the amino acid composition of the extracellular medium sampled after adhesion of neutrophils to fibronectin was determined. The free amino acid profile of neutrophil secretion includes branched-chain (valine, isoleucine, and leucine), aromatic (tyrosine and phenylalanine), and positively charged amino acids (hydroxylysine, ornithine, lysine, histidine, and arginine) (Galkina et al., 2019). A similar profile of free amino acids in the extracellular medium of control neutrophils was observed in this work, which indicates that the amino acid composition of the secretion is a characteristic and stable property of neutrophils (Figure 6A). Further studies showed that the secretion of only one amino acid depends on cell adhesion—hydroxylysine, and the secretion of all other amino acids is a characteristic property of neutrophils and does not depend on cell adhesion. Almost complete and selective blocking of hydroxylysine secretion occurred in experiments when neutrophils were incubated not over fibronectin, but over a non-sticky substrate (Galkina et al., 2019). In this work, imipramine selectively and almost completely suppressed the release of hydroxylysine and caused a statistically significant increase in the content of phenylalanine (Figure 6A). Imipramine had practically no effect on the release of other amino acids by neutrophils.

The secretion profile of neutrophils that adhered to the substrate in the presence of LPS did not differ significantly from that of control cells, but the release of hydroxylysine was significantly increased compared to control cells (Figure 6B). In the presence of LPS, the percentage of hydroxylysine from the total content of detected amino acids was 27 ± 4%, but 19 ± 2% (mean ± standard error of the mean) under control conditions (n = 5; p < 0.001). Imipramine caused a statistically significant decrease in the release of hydroxylysine in the presence of LPS, but did not significantly affect the release of other amino acids by neutrophils treated with LPS (Figure 6B).