Here, we present three methods to assess neutrophil migration and infiltration both in vivo and in vitro. These methods can be used to discover promising therapeutics targeting neutrophil migration.
Neutrophils are a major member of the innate immune system and play pivotal roles in host defense against pathogens and pathologic inflammatory reactions. Neutrophils can be recruited to inflammation sites via the guidance of cytokines and chemokines. Overwhelming infiltration of neutrophils can lead to indiscriminate tissue damage, such as in rheumatoid arthritis (RA). Neutrophils isolated from peritoneal exudate respond to a defined chemoattractant, N-formyl-Met-Leu-Phe (fMLP), in vitro in Transwell or Zigmond chamber assays. The air pouch experiment can be used to evaluate the chemotaxis of neutrophils towards lipopolysaccharide (LPS) in vivo. The adjuvant-induced arthritis (AA) mouse model is frequently used in RA research, and immunohistochemical staining of joint sections with anti-myeloperoxidase (MPO) or anti-neutrophil elastase (NE) antibodies is a well-established method to measure neutrophil infiltration. These methods can be used to discover promising therapies targeting neutrophil migration.
Neutrophils are the most abundant white blood cell and account for 50−70% of the whole white blood cell population in humans1. Neutrophils are one of the primary responders during acute inflammation. Neutrophils can be recruited to inflammation sites via the guidance of cytokines and chemokines released by tissue-resident cells2,3,4, which is mediated by the interactions between cell adhesion molecules on the surface of neutrophils and vascular endothelium cells5. Neutrophils are fundamental to host defense and play a role in pathologic inflammatory reactions due to their powerful capacity to damage tissue via the release of reactive oxygen species (ROS) and other tissue-damaging molecules3,6.
Previous studies have described several neutrophil isolation protocols from mice or humans. Oh et al. demonstrated a density gradient separation method to isolate human neutrophils from whole human blood7. However, the isolation of sufficient neutrophils from mouse blood is difficult because of the small blood volume. Alternatively, large numbers of pure and viable mouse neutrophils can be elicited from mouse peritoneal fluid, and these purified neutrophils can be used ex vivo to examine several aspects of cellular functions ex vivo, including neutrophil infiltration, migration, chemotaxis, oxidative burst, cytokine and neutrophil extracellular trap (NET) production8. Transwell assays9 or Zigmond chamber assays10,11 can be used to evaluate neutrophil migration in vitro. The air pouch model is used to evaluate the migration and infiltration of neutrophils in vivo. The subcutaneous air pouch model is a convenient in vivo animal model to study the migration of inflammatory cells.
Traditionally, neutrophils were considered as pathogen eliminators in acute phases of inflammation. However, recent findings have shown that neutrophils are complicated cells that perform a significant variety of specialized functions. Neutrophils can regulate many processes such as acute injury and repair, tumorigenesis, autoimmune response, and chronic inflammation12,13. Neutrophils also modulate adaptive immune responses and can regulate B cells and T cells14,15. Substantial shortage of neutrophils leads to mortality or severe immunodeficiency in humans and neutrophil depletion in mice leads to fatality, while excessive activation or recruitment of neutrophils in organs causes several immune diseases, such as rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE)6. Neutrophils are the most abundant cells in the synovial fluid of RA patients. Neutrophils produce excessive amounts of myeloperoxidase (MPO) and neutrophil elastase (NE) via degradation, which exacerbates cartilage erosion. MPO is a peroxidase enzyme mainly expressed in the granules of neutrophils16. NE is associated with articular cartilage destruction17. MPO and NE could be used to evaluate the status of neutrophil migration and infiltration in the tissue of RA patients.
This article provides three conventional methods to evaluate the migration of normal neutrophils induced both in vivo and in vitro, as well as the infiltration of pathological neutrophils in a mouse joint-specific inflammation model.
All experimental procedures were reviewed and approved by the Beijing University of Chinese Medicine Animal Care and Use Committee.
NOTE: C57BL/6 mice (7-8 weeks old) were used.
1. Neutrophil isolation
2. Neutrophil migration assay
3. Air pouch assay
4. Induction of the adjuvant-induced arthritis (AA) mouse model
5. Immunohistochemical staining of joint sections
Peritoneal exudate cells were collected from lavage fluid of mice. Cells were resuspended in 1 mL of RPMI-1640 complete medium, layered onto a two-step (54.8%/70.2%) discontinuous density gradient (Figure 1A), and centrifuged at 1,500 x g for 30 min. Neutrophils (≥95%, ~1 x 107 neutrophils/mouse) were recovered from the lower interface (Figure 1B).
Air pouch experiments were performed to investigate the neutrophil recruitment stimulated by LPS in vivo (Figure 2A). The leukocyte subsets in the air pouch exudates were measured (Figure 2B).
Neutrophil migration in RA was evaluated via the CFA-induced arthritis murine model. Compared with the control group, the AA group showed significant edema in the paw. In the AA group, the ankle joint diameter increased (Figure 3B) and the arthritis score rose consistently (Figure 3D).
Cartilage damage is the representative syndrome of RA, safranin O-fast green cartilage staining was performed to assess the cartilage damage in AA mouse. As shown in Figure 4A, CFA challenge induced a large amount of leukocyte infiltration, significant cartilage erosion and synovial hyperplasia. MPO and NE expression levels are representative markers of neutrophil infiltration. Immunohistochemical assays were performed to observe neutrophil infiltration in joints. MPO and NE expression was significantly upregulated in the joint section (Figure 4B).
Figure 1: Neutrophil isolation. (A) Peritoneal exudate cells resuspended in 1 mL of RPMI-1640 complete medium were layered onto a two-step (54.8%/70.2%) discontinuous density gradient. (B) After centrifugation at 1,500 x g for 30 min, neutrophils (≥95%, ~1 x 107 neutrophils/mouse) were recovered from the lower interface. Please click here to view a larger version of this figure.
Figure 2: Representative results of the air pouch assay. (A) Illustration of the air pouch assay. (B) Representative results of leukocyte subset infiltration in the air pouch assay. PBS: control; LPS: 1 µg/mL LPS. Data are presented as the mean ± SD. Please click here to view a larger version of this figure.
Figure 3: Representative results of the adjuvant-induced arthritis (AA) mouse model. (A) The ankle joint diameter was measured using a pocket thickness gauge. (B) Joint swelling was assessed based on the ankle joint diameter (n = 7). (C) Pictures of each arthritis score. (D) The severity of arthritis was graded on a scale of 0−4 points (n = 7). Data are presented as the mean ± SD. Please click here to view a larger version of this figure.
Figure 4: Representative results of safranin O-fast green cartilage staining and immunohistochemical assay of joint sections from control and AA mice. (A) Representative results of safranin O-fast green cartilage staining of joint sections. (B) Representative results of the expression level of MPO and NE in the ankle joints. Please click here to view a larger version of this figure.
Detailed protocols of highly-purified neutrophils from peripheral blood7, bone marrow and tissues18 have been available for a long time. Here we adopt a method of isolating neutrophils from peritoneal fluid19 in which mature neutrophils remain inactivated for further anti-inflammatory and antioxidant studies.
We used the air pouch experiment to explore the LPS-induced infiltration of neutrophils in vivo. This method has been proposed as a potential method for directly measuring cell infiltration in the general inflammatory environment in vivo.
It is noteworthy that the air pouch assay only demonstrates the neutrophil's function in an organ-nonspecific manner and removes several steps of the leukocyte recruitment cascade. Since neutrophil recruitment to specific organs can rely on different organ properties, adhesion molecules, and chemokines, exploring the neutrophil functional state in organ-specific conditions is of great importance for studying the role that neutrophils potentially play in certain diseases3. It has been suggested that endpoint models are required to investigate neutrophil infiltration. Therefore, performing immunohistochemical staining on the joint sections of mice in the AA model can provide an insightful perspective on the neutrophils in the joint space. According to our data, vast numbers of neutrophils are recruited into the joint tissue and serve as fundamental evidence for further research on interrupting the infiltration of neutrophils to treat RA. In addition, comprehensive assays, for example, safranin O-fast green staining, are required to evaluate the disease model for further study.
Neutrophils are the major subset of infiltrating inflammatory cells and work as the first line of defense against invading pathogens or tissue injury20,21. If neutrophils infiltrate tissues in large numbers, high levels of cytokines and NETs are secreted, which together may overwhelm the protective mechanisms in tissues and lead to tissue damage. Tissue injury further stimulates neutrophil infiltration, thus forming a vicious cycle22. Interfering with cell migration by means of trapping activated cells in lymphoid organs has been proposed as an important therapeutic approach and has been applied in clinical trials with various side effects23. Discovering a novel treatment to concurrently regulate neutrophil migration and inflammatory activity is a promising strategy for treating inflammatory diseases in the future. Furthermore, if motility-regulating agents are combined, neutrophil-mediated drug delivery24 can increase drug specificity, thus decreasing side effects.
However, further modifications can be combined to broaden the application of the above-mentioned assays. For example, in the AA mouse model, to obtain the overall impression of infiltration of inflammatory cells in the joint, researchers are encouraged to enzymatically digest joints to release the resident leukocyte populations and then apply flow cytometry to count the populations.
The protocols herein include three ways to assess neutrophil migration and infiltration. The application of these protocols is useful for discovering potential treatments for RA and other inflammatory diseases involving neutrophils.
The authors have nothing to disclose.
This work was supported by the National Natural Science Foundation of China (grant numbers 81430099 and 31500704), International Cooperation and Exchange Projects (grant number 2014DFA32950), and the research program of Beijing University of Chinese Medicine (grant numbers BUCM-2019-JCRC006 and 2019-JYB-TD013).
0.1% Fast Green Solution | Solarbio | 8348b | Transfer 20 mg of fast grene FCF in one vial into another 100 mL beaker. Add 20 mL of H2O into the beaker and dissolve the stain by stirring to make 0.1% fast green solution, and filter it using a Nalgene PES 75mm filter |
0.1% Safranin O Staining Solution | Solarbio | 8348a | Transfer 20 mg of safranin O stain in one vial into a 100 ml beaker. Add 20 mL of H2O into the beaker and dissolve the stain by stirring to make 0.1% safranin O staining solution, and filter it using a Nalgene PES 75mm filter |
0.5 M Ethylenediaminetetraacetic acid solution (EDTA), pH 8.0 | Sigma | 324506 | Sterile |
100% Ethanol | Beijing Chemical Works | ||
100% Methanol | Beijing Chemical Works | ||
15 mL Conical Polypropylene Centrifuge Tube | Falcon | 14-959-53A | |
23 G x 1 1/4" Needle | BD | 305120 | |
26 G x 3/8" Needle | BD | 305110 | |
3% bovine serum albumin (BSA) | Dissolve 0.3 g BSA in 10 mL PBS | ||
3% H2O2 | Mix 1 mL 30% H2O2 with methanol with 9 mL methanol | ||
3,3'-diaminobenzidine (DAB) kit | ZSGB-BIO | ZLI-9018 | |
30 G x 1/2" Needle | BD | 305106 | |
30% H2O2 | Beijing Chemical Works | ||
5 mL Syringe | BD | Z683574 | |
50 mL Conical Polypropylene Centrifuge Tube | Falcon | 14-432-22 | |
50% Ethanol | Mix 500 mL 100% ethanol with 500 mL dH2O | ||
54.8% Percoll | Mix 2.74 mL SIP with 2.26 mL 1×PBS, stand still | ||
70% Ethanol | Mix 700 mL 100% ethanol with 300 mL dH2O | ||
70.2% Percoll | Mix 3.51 mL SIP with 1.49 mL 1×PBS, stand still | ||
80% Ethanol | Mix 800 mL 100% ethanol with 200 mL dH2O | ||
95% Ethanol | Mix 950 mL 100% ethanol with 50 mL dH2O | ||
Acid Alcohol Superfast Differentiation Solution | Beyotime | C0165S | |
ANTIBODIES | |||
Anti-Myeloperoxidase Antibody | Abcam | ab208670 | |
Anti-Neutrophil Elastase Antibody | Abcam | ab21595 | |
Automatic Hematology Analyzer | Sysmex | XS-800i | |
Bovine Serum Albumin (BSA) | VWR | 0332-100G | |
Complete Freund's Adjuvant, 10 mg/ml | sigma | 1002036152 | |
Cover Slip | CITOGLAS | 10212432C | |
Dial Thickness Gauge | Mitutoyo | 7301 | |
Eppendorf Microtubes, 1.5 mL | Sigma | Z606340 | |
Foetal Bovine Serum (FBS) Premium | PAN | P30-1302 | |
Gas Anesthesia System | ZS Dichuang | ZS-MV-IV | |
Goat Anti-Rabbit IgG H&L (HRP) | PPLYGEN | C1309 | This is the secondary antibody used in the immunohistochemical staining. |
Hank's Balanced Salt Solution (HBSS) | Biological Industries, Beth HaEmek, Israel | 02-016-1A | Sterile |
Hematoxylin Staining Solution | ZSGB-BIO | ZLI-9609 | |
Lipopolysaccharide (LPS) | Sigma | L3012 | |
MEDIA AND SUPPLEMENTS | |||
Modified Safranin O-fast Green FCF Cartilage Stain Kit | Solarbio | G1371 | |
N-formyl-Met-Leu-Phe (fMLP) | Sigma | 47729 | |
Penicillin Streptomycin Solution, 100× | Invitrogen | 1514022 | |
Percoll | GE Healthcare | 10245207 | Density gradient medium |
Permeabilization Buffer | Mix 100 μL Triton X-100 with 1 L dH2O to get 0.01% Triton X-100 | ||
Phosphate Buffer Saline (PBS), 1× | Mix 90% ddH2O with 10% (v/v) 10×PBS, autoclaved | ||
Phosphate Buffer Saline (PBS), 10× | Dissolve 16 g NaCl, 0.4 g KCl, 2.88 g Na2HPO4·2H2O, 0.48 g KH2PO4 (anhydrous) in 200 mL ddH2O, adjust pH 7.4, autoclaved | ||
PLASTIC WARES AND EQUIPMENTS | |||
POWDER | |||
Proteose Peptone | Oxoid | 1865317 | |
Retrieval Buffer | Mix 18 mL retrieval buffer A with 82 mL retrieval buffer B, add dH2O to 1000 mL, adjust pH to 6.0 | ||
Retrieval Buffer A Stock for IHC | Dissolve 4.2 g citric acid (C6H5O7·H2O) in 200 mL dH2O | ||
Retrieval Buffer B Stock for IHC | Dissolve 5.88 g trisodium citrate dihydrate (C6H5Na3O7·2H20) in 200 mL dH2O | ||
Roswell Park Memorial Institute (RPMI)-1640 medium | Sigma | R8758 | |
RPMI-1640 Complete Medium | RPMI-1640 medium is supplemented with 10% FBS and 1% penicillin/streptomycin. | ||
Shu Rui U40 Disposable Sterile Insulin Injection Needle 1 mL | BD | 328421 | |
Slide | CITOGLAS | 10127105P-G | |
SOLUTION | |||
Stock Isotonic Percoll (SIP) | Mix 90% (v/v) of percoll with 10% (v/v) 10×PBS, stand still for 20 min | ||
Wash Buffer in Air Pouch Assay | Dilute 0.5M EDTA to 10mM with HBSS | ||
Xylene | Beijing Chemical Works |