Detecting Migration and Infiltration of Neutrophils in Mice

Summary

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.

Abstract

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.

Introduction

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 cells^2,3,4, 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 molecules^3,6.

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 assays^10,11 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 inflammation^12,13. Neutrophils also modulate adaptive immune responses and can regulate B cells and T cells^14,15. 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.

Protocol

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

1. Acquisition of peritoneal exudate cells
   1. Prepare fresh 10% proteose peptone solution in ddH₂O. Calculate the volume needed according to the number of mice.
      NOTE: Set the number of mice as N, (2N+1) mL of solution must be dissolved and filtered in advance.
   2. Spray the workspace with 70% ethanol. Use an insulin injector to draw 1 mL of peptone solution and discharge bubbles.
   3. Conduct the first intraperitoneal injection of 1 mL of peptone solution per mouse.
      1. Grab the mouse in a head-down position with one hand. Disinfect the injection spot with alcohol-soaked cotton balls.
         NOTE: The preferred injection position lies in the lateral aspect of the lower left or right quadrant of the abdomen.
      2. Infuse the reagents rapidly. Inject 1 mL into the peritoneal cavity of each mouse with the insulin injector.
         NOTE: The angle between the needle and the skin should be approximately 15−30° to avoid injuring the intestine or other organs.
   4. Allow inflammatory response to develop overnight. After 12 h, conduct the second injection in the same manner as the first injection.
   5. Three hours after the second injection, fully anesthetize mice with 5% isoflurane for 5 min in a gas anesthesia chamber at a speed of 2 L/min. Remove the anesthetized mice from the chamber and sacrifice them by cervical dislocation.
      NOTE: Sufficient depth of anesthesia is ensured by pinching the toes before moving mice from the chamber to the single breathing unit. The legs should not move when the toes are pinched.
      NOTE: All the following steps should be processed in a tissue culture hood.
   6. Spray the mouse with 70% ethanol. Lay the mouse on a sterile plastic pad and fix the limbs with needles.
   7. Use a sterile set of surgical tools to make a horizontal incision (~1 cm) in the middle of the lower abdomen. Lift the skin of the upper abdomen with forceps and cut along the midline of the abdomen and expose the intact peritoneal wall.
   8. Inject 5 mL of sterile RPMI-1640 complete medium into the abdominal cavity with a 30 G x 1/2" needle. Insert the needle through the peritoneal wall with the beveled edge of the needle facing up and inject the entire volume.
   9. Shake the pad horizontally for 5 min. Massage the abdomen gently several times.
   10. Inject a 23 G x 1 1/4" needle into the lateral space of the abdomen. Extract the abdominal liquid (~5 mL) and collect it in a 50 mL centrifuge tube.
       NOTE: Place the tubes on ice as soon as possible in case of neutrophil activation.
   11. Inject another 5 mL of complete medium and repeat the procedure to remove the remaining cells from the peritoneum. Pool the peritoneal fluid in the 50 mL centrifuge tube.
   12. Centrifuge the pooled peritoneal fluid at 400 x g for 10 min at room temperature (RT).
   13. Discard the supernatant. Resuspend the cells in 1 mL of RPMI-1640 complete medium.
       NOTE: Do not vortex to avoid neutrophil activation.
2. Isolation of neutrophils
   1. Add 4 mL of freshly prepared 70.2% density gradient medium (e.g., Percoll) in a 15 mL centrifuge tube.
   2. Carefully overlay 4 mL of freshly prepared 54.8% density gradient medium on the 70.2% density gradient medium slowly along the edge of the tube with sharp pipette tips in the 15 mL centrifuge tube.
      NOTE: Exercise caution to avoid disturbing the interface between the 54.8% density gradient medium and 70.2% density gradient medium.
   3. Carefully overlay the 1 mL peritoneal cell suspension on top of the 54.8% density gradient medium layer slowly with sharp pipette tips (Figure 1A).
      NOTE: Exercise caution to avoid disturbing the interface between the cell suspension and 54.8% density gradient medium.
   4. Centrifuge at 1,500 x g for 30 min at 22 °C without braking.
   5. Collect the neutrophils at the interface of the 54.8% density gradient medium and 70.2% density gradient medium layers (Figure 1B) to a new tube.
   6. Add 1 mL of RPMI-1640 complete medium to the collected cells and carefully resuspend cells by gently pipetting several times. Centrifuge at 100 x g for 10 min at RT and carefully remove the supernatant.
   7. Repeat the wash step (step 1.2.6) once.
   8. Add 0.5 mL of culture medium to the pellet and resuspend cells by gently pipetting several times. Take a 50 µL aliquot to count the cells using an automatic hematology analyzer.

2. Neutrophil migration assay

1. Measure neutrophil migration by Transwell assay⁹ or Zigmond chamber assay as previously described^10,11.

3. Air pouch assay

1. First air injection
   1. On day 0, fully anesthetize mice with 5% isoflurane for 3 min in a gas anesthesia chamber at a speed of 2 L/min, and maintain the anesthesia of each mouse in a single breathing unit with 2% isoflurane at a speed of 0.5 L/min.
      NOTE: Sufficient depth of anesthesia is ensured by pinching the toes before moving mice from the chamber to the single breathing unit. The legs should not move when the toes are pinched.
   2. Use a 0.22 µm filter attached to a 5 mL syringe to obtain a 3 mL volume of sterilized air.
   3. Lift the back skin of the anesthetized mouse with tweezers and subcutaneously inject 3 mL of sterilized air using a 26 G x 3/8" needle.
   4. After treatment, remove the mice from the breathing unit. Monitor the mice to ensure they are alive until they start to move around.
2. Second air injection
   1. On day 3, inject an additional 3 mL of sterilized air into the previously established air pocket to sustain the air pouch as described in section 3.1.
3. Treatment
   1. On day 6, 6 h before sacrifice, inject different treatments into the air pouch. Inject 1 mL of phosphate-buffered saline (PBS) as a negative control. Inject 1 mL of 1 µg/mL LPS as the positive control to induce local inflammation.
   2. Fully anesthetize mice with 5% isoflurane for 3 min in a gas anesthesia chamber at a speed of 2 L/min, and maintain the anesthesia of each mouse in a single breathing unit with 2% isoflurane at a speed of 0.5 L/min. Prepare wash buffer according to Table of Materials.
      NOTE: Sufficient depth of anesthesia is ensured by pinching the toes before moving mice from the chamber to the single breathing unit. The legs should not move when the toes are pinched.
   3. For each air pouch, wash the air pouch with 1 mL of wash buffer and collect the inflammatory exudate in a 15 mL centrifuge tube. Wash the air pouch with 2 mL of wash buffer 2x and collect the inflammatory exudate in the same centrifuge tube.
   4. Centrifuge at 100 x g for 10 min at RT. Discard the supernatant and resuspend cells in 1 mL of wash buffer. Count the cells to quantify the neutrophil ratio using the automatic hematology analyzer.
      NOTE: See representative results in Figure 2.

4. Induction of the adjuvant-induced arthritis (AA) mouse model

1. Suspend complete Freund's adjuvant (CFA) by vortexing at least 5 s, then draw 100 µL of suspension into an insulin injector.
   NOTE: We recommend using completely new CFA in experiments to ensure sterility.
2. Anesthetize mice as described in step 3.1.1.
3. Mark the chosen paw and inject 20 µL of CFA into the ankle joint space. Inject 20 µL of suspension into four periarticular spots on the chosen paw (80 µL in total).
4. Remove mice from the breathing unit and put the processed mice in a new chamber. Monitor mice to ensure that they are breathing until they regain the ability to move.
5. Every 3 days, assess the joint diameter by measuring the ankle joint diameter using a pocket thickness gauge (Figure 3A).
6. Every 3 days, assess arthritis severity by arthritis scoring criterion (Figure 3C): 0, normal, no evidence of erythema and swelling; 1, the mildest arthritis, erythema and mild swelling confined to the tarsals or ankle joint; 2, moderate arthritis, erythema and mild swelling extending from the ankle to the tarsals; 3, severe arthritis, erythema and moderate swelling extending from the ankle to metatarsal joints; 4, the most severe arthritis, erythema and severe swelling encompass the ankle, foot and digits, or ankylosis of the limb.

5. Immunohistochemical staining of joint sections

1. Joint isolation
   1. Sacrifice the mouse in section 4 using cervical dislocation after anesthesia with isoflurane. Spray the mouse with 70% ethanol.
   2. Remove the skin and part of the muscle from the hind leg with tweezers and scissors. Spray the joint with 70% ethanol and remove the rest of the muscles using a paper towel.
   3. Fix the ankle joint in 4% paraformaldehyde for 2 days at RT. Decalcify the joint in 10% EDTA for 1 month at RT and change the medium weekly.
   4. Embed the tissue in paraffin and prepare 4-µm-thick tissue sections.
      1. Place tissue in a marked mold with certain volume of liquid paraffin. Cool briefly.
      2. Set the thickness at 4 µm and cut slices on a microtome. Float sections in a 43 °C water bath.
      3. Mount the sections onto slides and put the slides into the oven at 70 °C for 2 h. For future use, conserve the slides at -20 °C.
2. Safranin O and fast green staining of joint sections
   NOTE: The following staining steps are conducted at RT.
   1. Place the slides from step 5.1.4.3 in a rack and perform the following washes to rehydrate at RT: xylene for 5 min (3x), 100% ethanol for 2 min (2x), 95% ethanol for 2 min (2x), 70% ethanol for 2 min, and 50% ethanol for 15 min. Wash in running tap water for 3 min.
   2. Stain in 0.1% fast green solution for 5 min. Rinse in 1% acetic acid for 10 s.
   3. Stain in 0.1% safranin O staining solution for 20 min. Immerse the slides in the following washes: 95% ethanol for 2 min (2x), 100% ethanol for 2 min (2x), and xylene for 2 min (2x).
   4. Mount the tissue sections and observe the tissues under a microscope.
      NOTE: See representative images in Figure 4A.
3. Immunohistochemical staining to visualize neutrophils
   1. Bake the paraffin sections for 2 h at 78 °C. Place the slides in a rack and perform the following washes to rehydrate at RT: xylene for 15 min (2x), 100% ethanol for 5 min (2x), 95% ethanol for 5 min, 80% ethanol for 5 min, H₂O for 3 min, and PBS for 3 min.
      NOTE: Do not let slides dry at any time during this step.
   2. Add one drop of permeabilization buffer to cover the tissue. Incubate sections in a humidity-controlled tray at 37 °C.
   3. Rinse slides in PBS for 3 min (3x). Avoid rinsing the tissue directly.
   4. Perform heat-induced antigen epitope retrieval using a pressure boiler.
      1. Arrange slides in a rack. Immerse slides in the pressure boiler filled with retrieval buffer.
      2. Put the pressure boiler on a microwave oven. Set the microwave oven at 600 W and heat the slides for 10 min.
      3. After boiling, keep slides in the boiler to cool to 90 °C. Take out the slides and rinse them in PBS for 3 min (3x).
   5. Quench endogenous peroxidase activity in freshly prepared 3% H₂O₂ at RT for 15 min. Rinse slides in PBS for 3 min (3x).
   6. Outline a large circle around the sample with a hydrophobic pen, avoid touching the sample. Block with 3% bovine serum albumin (BSA) in a humidity-controlled chamber at 37 °C for 60 min.
   7. Remove blocking solution. Add 50 µL of PBS-diluted primary antibody to each section quickly. Then, incubate the slides in a humidity-controlled tray at 4 °C overnight.
      NOTE: Different dilution ratios are used for different antibodies (1:25 for MPO and 1:20 for NE).
   8. On the second day, take out the tray and let it stand at RT for 30 min. Then, rinse the slides in PBS for 3 min (3x).
   9. Add 50 µL of PBS-diluted secondary antibodies to the tissue.
      NOTE: Different dilution ratios were applied: 1:1,000 for MPO and 1:1,500 for NE.
   10. Incubate slides in a humidity-controlled tray at 37 °C for 30 min. Then, rinse the slides in PBS for 3 min (3x).
   11. Develop in diluted 3,3'-diaminobenzidine (DAB) solution for 5 min. Keep an eye on the reaction in case of the development of a dark color. Rinse the slides in distilled water.
   12. Counterstain the slides in hematoxylin for 10 s. Rinse the slides in tap water for 5 min.
   13. Rinse in acid alcohol superfast differentiation solution for 3 s. Then rinse in tap water for 10 min.
   14. Immerse the slides in the following washes at RT: 80% ethanol for 5 min, 95% ethanol for 5 min, 100% ethanol for 5 min, and xylene for 15 min (2x). Fix the coverslip with mounting solution. Observe the tissue under a microscope.
       NOTE: See representative images in Figure 4B.

Results

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).

Discussion

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 be...

Materials

Name	Company	Catalog Number	Comments
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