All animals were housed and handled in accordance with the guidelines set forth by the Committee on Care and Use of Laboratory Animals of the Institute of Laboratory Animal Recourses, National Research Council. Studies were performed according to approved protocols of the Animal Care and Use Committees of the National Heart, Lung, and Blood Institute (non-human primate studies) and the National Institutes of Allergy and Infectious Diseases (murine studies).
Administration of G-CSF to mice
Mice used in this study were C57BL/6 LysM-EGFP knock-in males between 6–12 weeks of age and were chosen due to their genetically engineered EGFP-expressing populations of neutrophils and monocytes (12
). Four control mice were given a vehicle of phosphate-buffered-saline (PBS) + 0.1% bovine serum albumin (BSA) subcutaneously (SQ). Sixteen mice received 50μg/kg recombinant mouse granulocyte-colony stimulating factor (R&D Systems Inc, Minneapolis, MN, USA) SQ in 200ul PBS+ 0.1%BSA. The dose represents an equivalent surface area dosage for this species. Organs from six mice were harvested 15 and 30 minutes post G-CSF administration and organs from four mice were harvested 240 minutes post G-CSF administration. At each time point post G-CSF administration, the mice were euthanized by CO2
asphyxiation. Controls were sacrificed 30 minutes after receiving the vehicle. Lung, spleen, liver, and kidney were collected, sectioned, and fixed initially in 4% paraformaldehyde (Sigma-Aldrich, St. Louis, MO, USA) in PBS overnight, then transferred to 30% sucrose (Sigma-Aldrich) overnight in PBS at 4°C for cryo-protection, and finally placed in Tissue-Tek OCT freezing medium (Sakura Finetek, Torrance, CA, USA) and frozen in liquid nitrogen. The frozen tissues were kept frozen in a −80°C freezer until further processing was performed.
Administration of G-CSF to rhesus macaques
Four rhesus macaques (Macaca mulatta) were used in this study and were seronegative for simian T-cell leukemia, simian retrovirus type D, simian immunodeficiency virus, and herpes virus B. Blood samples were collected in 2ml ethylenediamine tetraacetic acid (EDTA) tubes prior to the subcutaneous administration of 10μg/kg of recombinant human G-CSF (Neupogen ®, Amgen, Inc. Thousand Oaks, CA) SQ, and then 15 minutes and 30 minutes post G-CSF administration. The dose represents an equivalent surface area dosage for this species. The animals were euthanized 30 minutes post administration with tissues collected shortly thereafter. A control animal was given saline and the same procedure was followed. Tissues were placed into formalin or frozen in OCT at the time of the necropsy.
Staining of C57BL/6 LysM-EGFP knock-in mouse tissue
Sections of the lung, spleen, liver, and kidney were cut 10μm thick. For staining, tissues were air-dried for 7–10 minutes and washed three times in 1x PBS (pH 7.4 for 5 minutes at room temperature. A DAPI nuclear stain (VECTASHIELD Hard set Mounting Medium with DAPI, Vector Laboratories, Burlingame, CA) was used to label nuclei of all cells. The slides were then mounted with a glass cover slip, placed in a cardboard slide tray, and stored in a 4°C refrigerator.
Immune Fluorescent Staining on frozen rhesus macaque tissue
Tissues used were of the left lower, left middle, left upper, right lower, right middle, and right upper lobes of the lung, as well as the spleen, liver, and kidney. Cryosections were cut 10μm thick.
Tissues harvested and frozen in OCT were stained using the NHLBI Pathology Core facility at NIH. For staining, tissues were dried for 7–10 minutes and washed three times in 1x PBS (pH 7.4) for 5 minutes at room temperature. The sections were then blocked with 10% normal horse serum for 1 hour at room temperature. Following this, the sections were incubated with a 1:100 dilution of the primary antibody, mouse anti-human CD18 (Clone 6.7; BD Pharmingen, San Diego, CA), overnight at 4°C. The dilution of the primary antibody for the spleen was 1:400. The sections were then washed in 1x PBS (pH 7.4) three times for 5 minutes at room temperature and incubated with a secondary antibody, an anti-mouse IgG FITC (Jackson ImmunoResearch, West Grove, PA), for 2 hours at room temperature. The dilution of the secondary antibody for the spleen was 1:200. Following incubation, the sections were washed in 1x PBS ( pH 7.4) twice for 5 minutes at room temperature and a cover slip was mounted using VECTASHIELD Hard set Mounting Medium with DAPI (Vector Laboratories). The slides were placed in a cardboard slide tray, and stored at 4°C before examination under the microscope.
Immune Fluorescent Staining on paraffin embedded rhesus macaque tissue
Tissues were collected in formalin and sent to HISTOSERV, Inc. (Germantown, MD, USA) for paraffin-embedding, sectioning, and hematoxylin and eosin staining. Double-label immunofluorescence (IFA) was performed using a 1:100 dilution of an antibody against a myeloid/histiocyte antigen clone Mac387 (M0747, DAKO, Carpinteria, CA) which labels neutrophils and monocytes and a 1:50 dilution of an anti-caveolin-1-Cy3 antibody (C3990, Sigma) to identify endothelium. The primary specific antibodies were applied sequentially followed by a secondary antibody. The secondary antibody was coupled with Alexa 488 (green) (Invitrogen, Molecular Probes). Following antibody treatment, sections were washed twice for 15 min in PBS with 0.2% fish skin gelatin. To-Pro3 (nuclear marker, Molecular Probes, Eugene, OR) was applied at 1μg/ml, incubated for five minutes and tissues were then washed in PBS. Finally, the sections were rinsed in double distilled water and mounted with aqueous mounting medium according to a protocol described in Borda et al (13
Confocal microscopy was used to observe the presence and location of neutrophils within the observed tissue. Slides of frozen tissues that underwent IHC were imaged using the Zeiss LSM-510-META (Carl Zeiss, Jena, Germany) confocal laser scanning microscope using a 20X objective. The resulting images were either of anti-CD18 immunostaining for the rhesus macaques or of EGFP expression for the mice, both imaged with a 488nm laser excitation and 505–550nm emission (displaying a green fluorescence) and DAPI nuclear stain imaged with a 405nm laser excitation and 420–480nm emission (displaying a blue fluorescence).
Slides produced from IHC staining of sections of paraffin embedded tissue underwent confocal microscopy using a Leica TCS SP2 confocal microscope (Leica Microsystems, Exton, PA) equipped with three lasers. Optical sections were collected at 512 × 512 pixels. Alexa-488 (488nm/517nm ex/em = fluorescence excitation and emission), Cy3 (514nm/540nm) and ToPro3 (642nm/661nm) were excited with the laser line 488nm, 514nm, and 633nm, respectively. NIH Image (version1.62) and Adobe Photoshop (version 7.0) were used to process and assemble images. Pl Apo objectives 20x, 40x and 63x with a NA 0.7, 1.25 and 1.4, respectively, were used to image the tissues and the resulting magnification is in the images shown as a magnification bar.
Complete blood counts (CBC) were performed using a Cell Dyn 3500 (Abbott Diagnostics, Santa Clara, CA, USA) on blood samples collected in EDTA tubes. Differentials were automated and determined based on cellular size and granularity according to parameters set by the manufacturer. Absolute neutrophil count (ANC) was calculated using the percentage of segmented neutrophils multiplied by the white blood cell count per microliter (μl) of blood.
Images were analyzed by obtaining the thresholded (to remove background) areas of the EGFP+ leukocytes or CD18+ cells and DAPI (nuclear stain) using Metamorph, software from Molecular Devices (Sunnyvale, CA, USA). By determining the ratio of the area expressing green fluorescence with that of blue fluorescence allowed us to standardize for the amount of tissue in a given field. The ratio of green to blue threshold per image was then calculated as a percentage. This volume was used to determine the normalized percentage of EGFP or CD18/FITC+ cells within the tissue. Evaluation was performed on single images from individual animals. Triplicate images per section per animal were evaluated. In the case of the non-human primates all six lobes of lung were evaluated in triplicate. Tissue sections from three non-human primates were compared with sections from one control.
Bar charts of the threshold intensity data were constructed using mean and standard deviation (SD). The random effect analysis of variance (ANOVA) models were used to compare neutrophil percentages among the tissue samples taken at different time points for both the C57BL/6 LysM-EGFP knock-in mice and rhesus macaques. The random effect models used individual image data from each animal, adjusting for the correlation among three images in each animal. A log-transformation was applied to the fluorescence data to achieve the normality if appropriate. The statistical significance was set at p < 0.05. Analyses were performed using SAS 9.2 (SAS Institute Inc., Cary, NC).