Procedures involving the use of animals was approved by the Institutional Animal Care and Use Committee (Columbia University). For all experiments, 6–8-week-old male and female C57/BL6 mice were obtained from Jackson Laboratories (Ellsworth, ME). The following groups of mice were used: C57BL/6 (WT) mice with LPS injury alone (n = 10) and injury control (PBS) (n = 8), WT LPS mice treated with anti-RAGE ab (n = 18) with 6 PBS controls, WT LPS treated with non-immune isotype matched control IgG (n = 18) with 12 PBS controls. Mice were weighed daily.
Escherichia coli O111:B4-derived LPS (Sigma Chemical) was dissolved in sterile, endotoxin-free PBS (GIBCO) under sterile conditions. Mice are anesthetized with isoflurane, and LPS (1.5 mg/kg) is delivered via nasal instillation using a micropipette. LPS doses were repeated at day 3 and mice sacrificed on day 4. PBS was administered by nasal instillation in matching mice in treatment groups.
The anti-RAGE antibody was initially produced using an immunizing peptide that contained a unique AA sequence on the V domain of the extracellular portion of the antibody. Murine monoclonal antibodies were produced by hybridoma technology and subsequently the ab was humanized and the humanized IgG1 isotype produced using the chimeric antibody intermediary process. The antibody cDNA was cloned and expressed on mammalian cells (ExpiCHO-S) and then large-scale antibody produced using plasmid technology (Fair Journey Biologics, Porto Portugal). Testing with this antibody revealed the following. By FACS analysis the antibody bound to cells from several RAGE-expressing cell lines. It significantly reduced AKT phosphorylation induced by S100B in aortic smooth muscle cells in dose responsive manner, and inhibited S100B -induced TNFα and IL-1b secretion in dose responsive manner. It inhibited HMGB1- induced IL-6 secretion from human peripheral bone marrow cells. The peptide sequences of this antibody are the same as for the antibody referred to as CR-3 in our recent publications [11, 12].
Animals were allowed to recover for 24 h following injury before administration of any drug treatment. Dose of either anti-RAGE ab or isotype control non-immune IgG (200 µg) were divided between IV administration and IP (volume 200 µl per divided dose). Animals are closely monitored for changes in appetite, weight loss, and respiratory difficulty.
At day 4 (24 h after last LPS dose) mice were euthanized with isoflurane followed by cervical dislocation. Two hours before euthanasia, mice were injected with FITC-labeled albumin (Sigma-Aldrich, no. A9771) to assess potential pulmonary vascular leak. Lungs, serum, and BALF (Bronchoalveolar lavage fluid) are taken for analysis. Briefly, the chest was opened, the right ventricle cannulated and about 250 μL of blood removed. BALF was removed and the right lung snap-frozen for later analysis. Blood sample was taken and centrifuged to separate and collect serum for frozen storage. The left lung was preserved in 10% formalin for paraffin embedding, sectioning, and subsequent staining.
BALF samples were centrifuged (Cytospin 4 Thermo Scientific) and the supernatant frozen for cytokine analysis. The cell pellets were placed in cell counting instrument (Countess, Invitrogen Thermo fissure Scientific) to count living cells. Slides were made of the centrifuged cells, stained with DifQuik (Polyscience) and examined under microscope to confirm that they were neutrophils. Results were expressed as cells × 105/ml.
Samples of serum and BALF were loaded into well plates and fluorescence intensity readings taken using a microplate reader (Spectrum Max iD3, Molecular Devices).
Paraffin embedded lungs were sectioned into 5-micron slices and mounted on slides. Serial tissue sections were stained for hematoxylin and eosin (H&E) and for myeloperoxidase (MPO) (rabbit anti-myeloperoxidase antibody, 1:8000, abcam) and for RAGE (human anti-RAGE antibody, 50 µg/ml). Secondary staining was performed with HRP-conjugated goat anti-rabbit or goat anti-human antibody (1:200). Staining for MPO and for RAGE (brown staining) was quantified by IHC on five sections from each lung section using Image Pro Plus software (Media Cybernetics, Silver Spring, MD).
To determine the cell type expressing RAGE in lung inflammation, dual fluorescent confocal microscopy studies were performed. Briefly, lung sections (5-μm-thick) were stained for MPO and RAGE with the respective antibodies as described above. The slides were then co-stained by incubating with fluorescent tagged secondary antibodies (Texas Red and fluorescein isothiocyanate) (manufacturers). The images were examined using Nikon Eclipse 50i confocal fluorescence microscope (Nikon, Melville, NY, USA) and Image Pro Plus software (Diagnostic Instruments, Inc., Sterling Heights, MI, USA).
Frozen BALF samples from all experiments included in the final data were sent to EVE technologies (Calgary, Alberta, CA) for bead based multiplex assays using Luminex technology. From the EVE technologies 32 plex mouse panel analysis, 12 cytokine/chemokines were selected for comparative analysis.
The following variables were compared between groups: vascular leakiness, inflammatory cell counts in BALF, lung MPO and RAGE staining on IHC. Lung MPO values were plotted vs. BALF inflammatory cell counts. An unpaired, two-tailed Student’s t test was used for comparisons between experimental groups. One-way analysis of variance was used for statistical analysis of three or more groups followed by the Bonferroni post hoc test. All statistical analysis was performed using GraphPad Prism software (GraphPad Software, La Jolla, CA).