Neutrophil Depletion Suppresses Pulmonary Vascular Hyperpermeability and Occurrence of Pulmonary Edema Caused by Hantavirus Infection in C.B-17 SCID Mice

Virus.

Hantaan virus strain 76-118 cl-1 (53) was grown in Vero E6 cells (ATCC C1008, CRL1586) maintained in minimum essential medium with Eagle salts (MEM; Invitrogen, Carlsbad, CA) that was supplemented with 2 mM l-glutamine (Invitrogen), 5% fetal calf serum (FCS), 0.5% nonessential amino acids, 5% insulin-transferrin-selenium (Invitrogen), and 1% penicillin-streptomycin (Invitrogen). The culture supernatant was used in this experiment.

Animals.

Six- to eight-week-old female C.B-17/Icr-scid/scidJcl (SCID) mice (Nihon Clea, Tokyo, Japan) were used in the present study and were maintained at the Laboratory of Animal Experiments, School of Medicine, Hokkaido University. All animal experiments were approved by the Animal Studies Ethical Committee at Hokkaido University. All of the mice were treated according to the laboratory animal control guidelines of the Hokkaido University Institutional Animal Care and Use Committee. Experiments involving virus infections were performed in a BSL-3 facility. Mice exhibiting weight loss in excess of 25% of initial body weight were euthanized except for mice in a pilot experiment.

Infection and treatment of mice.

HTNV (4,000 focus-forming units [FFU] in MEM) was inoculated by intraperitoneal injection into adult mice. After viral challenge, the mice were observed, and body weights were measured at 0 (preinfection) and 7, 14, 21, 28, 33, 35, and 42 days postinoculation (dpi). At these time points, some of the animals were euthanized, and sera were collected and stored at −30°C. The lungs, kidneys, spleens, livers, hearts, and brains were collected and stored at −80°C for RNA extraction. The organs of some mice were fixed in 10% phosphate-buffered formalin for histological analysis. Blood samples from the remaining mice were collected at the same time course into microhematocrit capillary tubes (Fisherbrand, Pittsburgh, PA) for hematology.

Histopathology and IHC.

Tissues were fixed in 10% phosphate-buffered formalin at 4°C within 14 days and routinely embedded in paraffin, sectioned (2.0 μm), and stained with hematoxylin and eosin. To investigate the extent of pulmonary edema, the left lungs were sectioned at constant positions, and the number of alveoli with exudate was counted in more than 300 alveoli and in more than 25 high power fields (see Fig. 4F). For immunohistochemistry (IHC), the antigens were retrieved by hydrolytic autoclaving for 10 min at 121°C in 10 mM sodium citrate-sodium chloride buffer (pH 6.0). Endogenous peroxidase activity was blocked by incubation with 1% hydrogen peroxide in methanol for 30 min. The first antibody added was monoclonal antibody (MAb) clone E5/G6 (1 μg/ml; incubated overnight at 4°C) that recognizes a linear epitope common to hantavirus N (54). The Vector M.O.M. immunodetection kit (Vector Laboratories, Burlingame, CA) was used to visualize HTNV antigens in the sections.

RNA extraction and real-time PCR.

Total RNA was extracted from each tissue and serum of infected mice by using ISOGEN (Nippon Gene, Japan) and a QIAamp viral RNA minikit (Qiagen, Hilden, Germany) in accordance with the manufacturers' protocols. The RNA was then reverse transcribed by using a first-strand cDNA synthesis kit (GE Healthcare, United Kingdom). Real-time PCRs were carried out on a LightCycler 480 (Roche Diagnostics, Vienna, Austria) using TaqMan Universal master mix II (Applied Biosystems, Carlsbad, CA). The following primers and probe were used: real-time primer HTNV F (5′-TTATTGTGCTCTTCATGGTTGC-3′), real-time primer HTNV R (5′-CATCCCCTAAGTGGAAGTTGTC-3′), and probe 75 (5′-GGAGGCTG-3′) (all from Roche Diagnostics).

Recovery of lung leukocytes from mice.

Animals were euthanized at 28 dpi, and the trachea was cannulated by using a 22-gauge catheter. Bronchoalveolar lavage (BAL) was performed twice with 0.8 ml of ice-cold phosphate-buffered saline (PBS; pH 7.4) each time. After collection of BAL fluid, animals were perfused with 3 ml of ice-cold PBS via the heart. The lungs were excised, minced, and enzymatically digested for 1 h in 1 ml of RPMI 1640 medium supplemented with 10% FCS, 1% penicillin-streptomycin, 50 μM 2-mercaptoethanol, 1 mg of type VIII collagenase (Sigma-Aldrich)/ml, and 30 μg of DNase I (Sigma-Aldrich)/ml. The undigested fragment was further dispersed by double passage through 0.80- and 0.45-μm-pore-size nylon mesh filters. The total cells were pelleted, and any contaminating erythrocytes were eliminated from the cell pellet by adding 0.83% NH₄Cl in 10 mM Tris buffer (pH 7.4). After spinning, the pellet was resuspended in 10 ml of complete RPMI 1640 medium (10% FCS, 1% penicillin-streptomycin, 50 μM 2-mercaptoethanol). Cell suspensions were filtered through a 0.45-μm-pore-size nylon mesh filter and pelleted for flow cytometry.

White blood cell counts.

The total numbers of white blood cells were counted after staining with Turk's solution (Kanto Chemical Co., Tokyo, Japan) on a hemocytometer.

Flow cytometry.

All flow cytometry data were collected on a FACSCalibur (BD Biosciences, San Jose, CA) and analyzed using FlowJo software (TreeStar, Inc., San Carlos, CA). For the analysis of cell surface markers, the cells were resuspended in fluorescence-activated cell sorting (FACS) buffer (1% bovine serum albumin and 0.1% NaN₃ in PBS). The cells were stained at 4°C for 30 min, and the following MAbs were used: phycoerythrin (PE)-conjugated anti-mouse Gr-1 (clone RB6-8C5; eBioscience, San Diego, CA), fluorescein isothiocyanate (FITC)-conjugated anti-mouse CD11b (clone M1/70; BioLegend, San Diego, CA), PE-Cy5 anti-mouse CD3ε (clone 145-2C11; BioLegend), PE–anti-mouse NK1.1 (clone PK136, BD Biosciences), PE–anti-mouse CD11c (clone HL3, BD Biosciences), and FITC–anti-mouse I-A/I-E (clone M5/114.15.2; BioLegend). The cells were pretreated with anti-mouse CD16/CD32 (clone 93; BioLegend). After washing, the cells were fixed in 2% paraformaldehyde and then resuspended in FACS buffer for analysis.

In vivo depletion of neutrophils, macrophages, and NKT cells.

To deplete neutrophils, mice were treated with protein G-purified anti-Gr-1 MAb (RB6-8C5). RB6-8C5 hybridoma was provided by the Cell Resource Center for Biomedical Research Institute of Development, Aging, and Cancer, Tohoku University, and the MAb was purified from culture supernatant or ascitic fluid of mice by using a HiTrap protein G HP column (GE Healthcare). Each mouse was given 250 μg of anti-Gr-1 MAb or control IgG from rat serum (Sigma-Aldrich) by intramuscular injection every day from 19 to 33 dpi. Macrophage and monocyte depletion with liposomal clodronate was performed as previously described (55). Briefly, 10 mg of clodronate (Sigma-Aldrich)/ml in PBS was mixed with a phosphatidylcholine (Nakarai, Kyoto, Japan)-cholesterol (Nakarai) (3:1 molar ratio) solution in chloroform and incubated for 2 h at 37°C. Vesicles were formed by vacuum desiccation overnight, suspended in 5 ml of PBS, and purified by dialysis. Each mouse was given 200 μl of purified clodronate liposome PBS or control PBS by intraperitoneal injection at 18, 21, 25, and 29 dpi. To deplete NKT cells, mice were treated with anti-NK1.1 MAb (PK136), which depletes both NKT and NK cells. Each mouse was given 100 μg of anti-NK1.1 MAb or control IgG from mouse serum (Sigma-Aldrich) by intramuscular injection at 20, 25, and 30 dpi (56). Effective neutrophil depletion (>90%) at 33 dpi was confirmed by morphological analysis and flow cytometry of peripheral blood (data not shown). The extent of macrophage depletion (75 to 90%) was determined by flow cytometry of splenocytes (data not shown). Likewise, the extent of NKT cell depletion (75 to 90%) was determined by flow cytometry of peripheral blood (data not shown).

Evaluation of vascular permeability.

Vascular permeability was analyzed by the Evans blue extravasation method (57). Mice were inoculated intravenously with 200 μl of a 2% solution of Evans blue dye dissolved in normal saline (0.85% sodium chloride). After 1 h, the mice were anesthetized, and the lungs were perfused with 5 ml of normal saline through the left cardiac ventricle. The homogenized lung tissue was incubated with 1.5 ml of formamide for 16 h at 55°C and centrifuged at 4°C for 15 min at 6,000 × g. The amount of Evans blue dye in the supernatant was measured spectrophotometrically at 650 nm.

Statistical analysis.

Data were analyzed using the nonparametric Mann-Whitney U test and statistical significance was accepted when the P value was <0.05.

Body weight change after infection with HTNV.

Infected mice showed gradual, progressive weight loss and slowing of activity and they exhibited a rough coat after 21 dpi (Fig. 1). Statistically significant differences in mean body weight were observed between groups of HTNV-infected mice and uninfected mice from 21 dpi (P < 0.01). Three of the eleven infected mice had weight losses of >20% at 35 dpi. The HTNV-infected mice displayed terminal symptoms at 42 dpi with shivering and cessation of feeding and drinking and of spontaneous movement. Four of the five infected mice had weight losses of >25% at 42 dpi.

Viral loads in the organs of mice following inoculation with HTNV.

The viral genome was detected in the lungs, kidneys, heart, spleen, liver, brain, and serum from 7 dpi (Fig. 2). The viral loads were high in all organs examined at 7 dpi. The genomes in all organs except for the brain and serum were increased to a plateau level at 14 dpi. On the other hand, those of the brain and serum were gradually increased.

Histopathological examination and degree of pulmonary edema.

To examine the distribution of the virus, IHC was carried out with an E5/G6 MAb generated against the N of hantavirus. In accordance with the viral genome load, viral antigen was first observed in the spleen from 7 dpi (Table 1). In the spleen, viral antigen-positive cells were seen around white pulp at 14 dpi (data not shown). After 14 dpi, viral antigens were observed in multiple organs (Table 1). In the lungs, virus antigen-positive cells were detected, and the numbers of virus antigen-positive cells sequentially increased in the alveoli at 14, 21, 28, and 35 dpi (Fig. 3). For some reason, the number of antigen-positive cells at 42 dpi was slightly less than that at 35 dpi. In the kidney, heart, liver, and brain, virus antigen-positive cells were seen in small vessels at 14 dpi (data not shown). As shown in Fig. 5, the virus had spread from blood vessels to surrounding parenchymal cells, such as cardiac myocytes, hepatocytes, and neuronal cells, at 35 dpi. However, there was no antigen in epithelial cells of the bronchus or in renal tubule cells even at 35 dpi (Fig. 4B and 5D).

Marked histopathological change was observed in the lung. In accordance with the viral loads, cells in the lung sequentially proliferated and the rate of cellular proliferation was significantly higher at 35 dpi than at 0 dpi (Fig. 3 and Fig. 4A to D). Adhesion of neutrophils and degenerated and multilayered endothelial cells were seen in a middle-sized vein (Fig. 4A, inset). Pulmonary effusion, macrophages, and neutrophils were observed in the alveoli (Fig. 4C and D). Inflammatory cells were increased in the alveolar wall (Fig. 4C and D). No inflammatory cell infiltration was seen in those tissues except for the lung. The degree of pulmonary edema was assessed by calculation of the ratio of alveoli with exudate (Fig. 4E). Pulmonary edema transiently appeared from 28 dpi and peaked at 35 dpi. Correspondingly, the relative lung weight also increased with an almost 2-fold increase at 28 dpi and peaked with an increase of almost two and a half times at 35 dpi (data not shown).

White blood cell and neutrophil counts.

Since the present study focused on the role of neutrophils in hantavirus infection, white blood cell and neutrophil counts in peripheral blood were determined. White blood cell count in HTNV-infected SCID mice showed a significant increase from 7 dpi and was twice that in uninfected mice at 28 dpi (Fig. 6A). Neutrophil count significantly increased from 21 dpi and was three times higher than that in uninfected mice at 28 dpi (Fig. 6B). The numbers of white blood cells and neutrophils were decreased at 33 dpi compared to those in mice at 28 dpi.

Recruitment of inflammatory cells to the lung.

Since intra-alveolar edema began to be observed at 28 dpi, the change in proportion of leukocytes in the lung was investigated (Fig. 7). The percentages of CD11b⁺ Gr-1^hi (neutrophils), CD11b⁺ Gr-1^int (macrophages), and CD3⁺ NK1.1⁺ (NKT cells) in lung tissues from infected mice were significantly higher than those in lung tissues from uninfected mice (Fig. 7B).

Influence of depletion on occurrence of pulmonary edema.

Since the percentages of neutrophils, macrophages, and NKT cells were increased in lung tissues from infected mice, each subset was depleted to investigate whether these cells are involved in the occurrence of pulmonary edema. Gross observation of left lungs from HTNV-infected mice with rat IgG at 33 dpi demonstrated that the lungs were highly edematous (Fig. 8A, inset). Correspondingly, these mice had clear histopathologic changes in their lungs (Fig. 8A). On the other hand, HTNV-infected mice with anti-Gr-1 MAb showed no pulmonary edema (Fig. 8A). Moreover, the degree of pulmonary edema was significantly reduced by neutrophil depletion in HTNV-infected mice (Fig. 8B). In HTNV-infected SCID mice, the gross pulmonary edema and elevated degree of pulmonary edema were unchanged even with macrophage depletion and NKT cell depletion (Fig. 8C and D).

Pulmonary vascular permeability.

To investigate whether the pulmonary edema in the mice is caused by HTNV-induced hyperpermeability, vascular permeability in the lung was examined by measurement of the extravasation of Evans blue dye. Pulmonary vascular permeability was significantly higher in the infected mice than in uninfected mice at 33 dpi (Fig. 9). The vascular permeability at 28 dpi was slightly enhanced compared to that in control mice, but the difference was not significant. The elevated pulmonary vascular permeability conformed with the degree of pulmonary edema. Furthermore, to investigate whether neutrophils increase pulmonary vascular permeability, pulmonary vascular permeability was examined in mice with neutrophil depletion. It was found that the level of hyperpermeability was significantly reduced by neutrophil depletion at 33 dpi (Fig. 9).