Herein, we describe protocols for harvesting murine alveolar macrophages, which are resident innate immune cells in the lung, and examining their activation in response to co-culture with polyanhydride nanoparticles.
Biodegradable nanoparticles have emerged as a versatile platform for the design and implementation of new intranasal vaccines against respiratory infectious diseases. Specifically, polyanhydride nanoparticles composed of the aliphatic sebacic acid (SA), the aromatic 1,6-bis(p-carboxyphenoxy)hexane (CPH), or the amphiphilic 1,8-bis(p-carboxyphenoxy)-3,6-dioxaoctane (CPTEG) display unique bulk and surface erosion kinetics1,2 and can be exploited to slowly release functional biomolecules (e.g., protein antigens, immunoglobulins, etc.) in vivo3,4,5. These nanoparticles also possess intrinsic adjuvant activity, making them an excellent choice for a vaccine delivery platform6,7,8.
In order to elucidate the mechanisms governing the activation of innate immunity following intranasal mucosal vaccination, one must evaluate the molecular and cellular responses of the antigen presenting cells (APCs) responsible for initiating immune responses. Dendritic cells are the principal APCs found in conducting airways, while alveolar macrophages (AMɸ) predominate in the lung parenchyma9,10,11. AMɸ are highly efficient in clearing the lungs of microbial pathogens and cell debris12,13. In addition, this cell type plays a valuable role in the transport of microbial antigens to the draining lymph nodes, which is an important first step in the initiation of an adaptive immune response9. AMɸ also express elevated levels of innate pattern recognition and scavenger receptors, secrete pro-inflammatory mediators, and prime naïve T cells12,14. A relatively pure population of AMɸ (e.g., greater than 80%) can easily be obtained via lung lavage for study in the laboratory. Resident AMɸ harvested from immune competent animals provide a representative phenotype of the macrophages that will encounter the particle-based vaccine in vivo. Herein, we describe the protocols used to harvest and culture AMɸ from mice and examine the activation phenotype of the macrophages following treatment with polyanhydride nanoparticles in vitro.
1. Harvesting Alveolar Macrophages (AMɸ) from the Mouse using Lung Lavage
2. Processing of Lung Lavage AMɸ Harvest
3. Addition of Polyanhydride Nanoparticles and Control Treatments
4. Evaluating AMɸ Activation using Flow Cytometry
5. Representative Data
Nanoparticles fabricated in step 3.1 had an average diameter of 163 ± 24 nm and a morphology consistent with that obtained in previous studies5,16. Nanoparticles in solution prior to and after sonication are shown in Figure 1 to demonstrate the need for sonication to ensure adequate dispersal of the particles. Flow cytometric analysis of harvested AMɸs obtained via lung lavage is shown in Figure 2. Labeling cells with a combination of anti-mouse CD11b and anti-mouse F4/80 antibodies as well as the corresponding FMO controls allows for establishing background labeling, identifying AMɸs and gating for further analysis. Treatment of alveolar macrophages with polyanhydride nanoparticles enhances activation as shown by the increased mean fluorescence intensity of MHC II, CD40, CD86, and CIRE (Figure 3).
Figure 1. 50:50 CPTEG:CPH nanoparticles prior to (A) and after (B) 30 s of sonication.
Figure 2. Flow cytometric analysis of harvested alveolar macrophages labeled with (A) Alexa Fluor 700 anti-CD11b and the PE-Cy7 FMO Control, (B) Alexa Fluor 700 FMO Control and the PE-Cy7 anti-F4/80 and (C) Alexa Fluor 700 anti-CD11b and PE-Cy7 anti-F4/80. The number in the top right corner represents the percentage of double-positive cells.
Figure 3. Histograms demonstrating an increase in the fluorescence intensity for surface expression of MHC II (A), CD40 (B), CD86 (C) and CIRE (D) after co-culture of alveolar macrophages with 50:50 CPTEG:CPH polyanhydride nanoparticles for 48 hr. Histograms depict results for AMɸ labeled as FMO controls (), untreated AMɸ labeled with antibodies against all cell surface markers () and AMɸ cultured with nanoparticles and labeled with antibodies against all cell surface markers ().
Polyanhydride nanoparticle vaccine platforms have shown efficacy when administered intranasally in single dose regimens5. Measuring the activation of the resident phagocytic cell populations in the lungs induced by this vaccine delivery platform permits evaluation of its potential capability to ultimately promote adaptive immune responses.
Specifically, harvesting alveolar macrophages from lung lavage fluid and treating them with different formulations of the nanoparticles provides insights into the abilities of different particle chemistries to activate macrophages, leading to antigen presentation6,8. In addition, these in vitro studies are useful for assessing the capacity of these particulate adjuvant formulations to activate alveolar macrophages prior to embarking on larger and more complex in vivo studies. Experiments should always contain a positive control treatment for surface marker up-regulation, such as LPS, an agonist of Toll-like receptor 4. Care should be taken in planning experiments prior to harvesting alveolar macrophages as this protocol may not produce adequate numbers of cells needed for multiple treatments in one experiment. Lung lavages may therefore need to be performed on multiple mice to ensure adequate cell numbers (~ 5.0 x 105 cells per mouse) for larger experiments with more treatment groups. Lung lavage techniques have also been reported for other species, and the amount of fluid utilized is proportional to the species being studied (i.e., mouse lung capacity is 1 mL, rat lung capacity is 10 mL and human lung capacity is 6 L17.
Polyanhydride nanoparticles are formulated and stored in a dry powder form to prevent premature surface erosion. Because of static interactions that result in clumping of the particles, sonication is necessary before addition to cell cultures. This step allows for uniform distribution and leads to more reproducible results. The quantification of cell surface markers on alveolar macrophages using flow cytometry is complicated by strong autofluorescence signals. This obstacle can be overcome by optimizing FACS antibody concentrations, polychromatic fluorochrome combinations, and flow cytometry capture parameters (i.e., use of compensation controls). FMO controls allow the change of one fluorescent parameter at a time, are useful for setting gates for each antibody and enable correct quantification of cell surface marker expression. Differences in the strains of mice should also be considered in the selection of antibodies, particularly haplotype specificity of MHC II.
It is important to have standardized methods to determine activation of antigen presenting cells in the lung, as this will greatly facilitate comparisons of novel biomaterials between different laboratories and institutions. Generating consistent populations of alveolar macrophages through the methods presented here, provide optimal conditions for obtaining useful data regarding different formulations of nanoparticles and their immune enhancing potential.
The authors have nothing to disclose.
The authors would like to thank the U.S. Army Medical Research and Materiel Command (Grant Numbers W81XWH-09-1-0386 and W81XWH-10-1-0806) for financial support and Dr. Shawn Rigby from the Iowa State University Flow Cytometry Facility for his expert technical assistance.
Name of the reagent | Company | Catalog number | Comments |
cAM Media | |||
DMEM | Cellgro | 15-013-CV | |
50 mM 2-mercaptoethanol | Sigma | M3148-25ML | |
Penicillin/Streptomycin 10,000 μg/ mL Solution | Cellgro | 30-002-CI | |
Fetal Bovine Serum | Atlanta Biologicals | S11150 | |
FACS Buffer | |||
Sodium chloride | Fisher Scientific | S671-500 | |
Sodium phosphate | Fisher Scientific | MK7868500 | |
Potassium chloride | Fisher Scientific | P217500 | |
Potassium phosphate | Fisher Scientific | P288-200 | |
BSA (Bovine Serum Albumin) | Sigma | A7888 | |
Sodium Azide | Sigma | S2002 | |
Antibodies | |||
Rat IgG | Sigma | I4341 | |
Anti-Ms CD16/32 | eBioscience | 16-0161 | |
Anti-Ms MHC II haplotype I-A/I-E, clone M5/114.15.2, conjugated to fluorescein isothiocyanate (FITC) | eBioscience | 11-5321 | |
Anti-mouse CD86, clone GL-1, conjugated to allophycocyanin (APC)-Cy7 | Biolegend | 105030 | |
Anti-mouse CD40, clone 1C10, conjugated to APC | eBioscience | 17-0401 | |
Anti-mouse CD209, clone 5H10, conjugated to Biotin | eBioscience | 13-2091 | |
Anti-mouse CD11b, clone M1/70, conjugated to Alexa Fluor 700 | eBioscience | 56-0112 | |
Anti-mouse F4/80, clone BM8, conjugated to phycoerythrin (PE)-Cy7 | eBioscience | 25-4801 | |
PE-Texas red conjugated Streptavidin | BD Biosciences | 551487 | |
Other Supplies and Reagents | |||
Ethanol | Fisher Scientific | A405-20 | Used as 70% (v/v) |
Compressed CO2 | Linweld | 16000060 | |
1 mL Syringe | BD Biosciences | 309659 | |
Sovereign 3 ½” Fr Tom Catcatheter | Kendall | 703021 | |
Biosafety Cabinet | NUAIRE | Series 22 | |
Dissection Scissors | Fisher | 138082 | |
Forceps | Roboz | RS-8254 | |
PBS, 1X without calcium and magnesium | Cellgro | 21-040-CM | |
15 mL Centrifuge Tubes with Screw Cap | VWR International | 21008-216 | |
Six-well Tissue Culture Treated Plates | Costar | 3516 | |
Plastic Tube Racks | Nalgene | 5970 | |
Cell Scraper 24 cm | TPP | 99002 | |
5 mL Polystyrene Round-Bottom Tube | Falcon | 352008 | |
Pipet-aid XL | Drummond | 4-000-105 | |
10, 5, and 2 mL Pipettes | Fisher | 13-675 | |
200 and 10 μL micropipettors | Gilson Pipetman | F123601 | |
200 and 10 μL pipette tips | Fisher | 02-707 | |
BD Stabilizing Fixative | BD Biosciences | 338036 | |
Isoton II Diluent | Beckman-Coulter | 8546719 | |
Zap-oglobin II Lytic Reagent | Beckman-Coulter | 7546138 | |
Coulter Counter Polystyrene Vials | Beckman-Coulter | 14310-684 | |
Test Tubes | BD Biosciences | 352008 | |
Equipment | |||
Refrigerated Centrifuge | Labnet | 50075040 | |
Humidified Incubator CO2 | Nuaire | Model Autoflow 8500 | |
FACSCanto Flow Cytometer | BD Biosciences | 338960 | |
Coulter Particle Counter Z1 | Beckman-Coulter | WS-Z1DUALPC | |
Sonicator Liquid Processing Equipment with Microtip | Misonix | Model No. S-4000 |