Method Article

Assessment of Lymphocyte Migration in an Ex Vivo Transmigration System

DOI:

10.3791/60060

⸱

September 20th, 2019

 , 
1Department of Internal Medicine, University of Utah, 2Research Service, VA Nebraska Iowa Health Care System, 3Department of Internal Medicine, University of Nebraska Medical Center, 4Department of Environmental, Agricultural & Occupational Health, University of Nebraska Medical Center

In This Article

Summary

$$\rightleftharpoonup{xx}$$ $$\longleftharp{xx}$$, $$\longrightharp{xx}$$,

In this protocol, lymphocytes are placed in the top chamber of a transmigration system, separated from the bottom chamber by a porous membrane. Chemokine is added to the bottom chamber, which induces active migration along a chemokine gradient. After 48 h, lymphocytes are counted in both chambers to quantitate transmigration.

Abstract

$$\rightleftharpoonup{xx}$$ $$\longleftharp{xx}$$, $$\longrightharp{xx}$$,

Herein, we present an efficient method that can be executed with basic laboratory skills and materials to assess lymphocyte chemokinetic movement in an ex vivo transmigration system. Group 2 innate lymphoid cells (ILC2) and CD4+ T helper cells were isolated from spleens and lungs of chicken egg ovalbumin (OVA)-challenged BALB/c mice. We confirmed the expression of CCR4 on both CD4+ T cells and ILC2, comparatively. CCL17 and CCL22 are the known ligands for CCR4; therefore, using this ex vivo transmigration method we examined CCL17- and CCL22-induced movement of CCR4+ lymphocytes. To establish chemokine gradients, CCL17 and CCL22 were placed in the bottom chamber of the transmigration system. Isolated lymphocytes were then added to top chambers and over a 48 h period the lymphocytes actively migrated through 3 µm pores towards the chemokine in the bottom chamber. This is an effective system for determining the chemokinetics of lymphocytes, but, understandably, does not mimic the complexities found in the in vivo organ microenvironments. This is one limitation of the method that can be overcome by the addition of in situ imaging of the organ and lymphocytes under study. In contrast, the advantage of this method is that is can be performed by an entry-level technician at a much more cost-effective rate than live imaging. As therapeutic compounds become available to enhance migration, as in the case of tumor infiltrating cytotoxic immune cells, or to inhibit migration, perhaps in the case of autoimmune diseases where immunopathology is of concern, this method can be used as a screening tool. In general, the method is effective if the chemokine of interest is consistently generating chemokinetics at a statistically higher level than the media control. In such cases, the degree of inhibition/enhancement by a given compound can be determined as well.

Introduction

$$\rightleftharpoonup{xx}$$ $$\longleftharp{xx}$$, $$\longrightharp{xx}$$,

This original transmigration method was presented by Stephen Boyden in 1962 in the Journal of Experimental Medicine1. Much of what we know about chemotaxis and chemokinetics would not be possible without the development of the Boyden chamber. Prior to the discovery of the first chemokine in 1977, ex vivo transmigration systems were used to learn about serum-factors that could arrest cellular movement in macrophages while amplifying cellular motility in neutrophils1,2. A massive wealth of knowledge has been developed regarding immune cell migration, and to date, 47 chemokines have now been discovered with 19 corresponding receptors3,4. In addition, multitudes of inhibitors/enhancers of these chemokine pathways have undergone development for therapeutic purposes5,6,7,8. Many of those compounds have been tested in similar transmigration chambers to understand direct interactions between the compounds and immune cell responsiveness to a given chemokine9.

Transmigration, or diapedesis, into inflamed tissue is an essential process to a healthy inflammatory response to clear infection10,11. A Boyden chamber, transmigration system, or transwell apparatus are generally composed of two chambers separated by a porous membrane1,12. The bottom chamber most often holds media containing the chemokine of interest, while leukocytes are placed in the top chamber. The size of the pore in the membrane can be selected based on the size of the cell of interest. For this project, we selected a 3 µm porous membrane, as lymphoid cells are 7-20 µm in size, depending on the stage of cellular development. This pore size ensures that these cells are not passively falling through the pores, but that they are actively migrating in response to the chemokine gradient.

The major advantage of this protocol is its cost effectiveness. In vivo transmigration is difficult because it requires extensive training in animal handling and surgery, and often involves high-powered microscopy that is not always available to a researcher. Cost effective screening of compounds thought to enhance or inhibit transmigration can be accomplished in advance of in vivo imaging. Because the transmigration system is tightly controlled, cells may be treated initially then added to the transwell apparatus, or, vice versa, the chemokine may be treated first with a chemokine inhibitor then cells added to the transwell apparatus. Lastly, endothelial cells and/or basement membrane proteins can be added to the bottom of the transwell insert 1-2 days prior to the transmigration experiment to understand the involvement of these barrier cells in chemokinetics. Again, these manipulations of the system provide a powerful means of determining important information about the effectiveness of a given compound in advance of more complicated in vivo studies.

Utilizing a transmigration chamber system is an effective way to assess lymphocyte mobility under various in vivo and in vitro conditions12,13,14. Herein, we describe an optimized method for assessing ex vivo lymphocyte responsiveness to chemokines in a transmigration chamber. In this example experiment, CD4+ T cells and group 2 innate lymphoid cells (ILC2) were isolated from male and female, BALB/c mice following OVA-allergen exposure. A hypothesis was generated that CCR4+ CD45+ Lineage- (LIN-) ILC2 from allergen-challenged mice would migrate more efficiently towards CCL17 and CCL22 than CCR4+ CD4+ T helper cells. CCL17 and CCL22 are chemokines commonly produced by dendritic cells and macrophages of the M2 (allergic) phenotype, among other cells, in allergy15,16. CCL17 and CCL22 can be thought of as biomarkers of allergic inflammation as they are readily detected in the lungs during airway exacerbations16,17,18. Importantly, CCR4 expression is elevated in comparison to untreated controls, as revealed in bioinformatic data generated from ILC2 isolated from house dust mite treated animals, and similarly ILC2 from naïve animals treated ex vivo with IL-33 (allergen-promoting innate cytokine) upregulates CCR419,20. Furthermore, according to data for ILC2 in the Immunological Genome Project database (www.immgen.org), CCR4 mRNA is highly expressed in these innate immune cells. To date, little is known regarding trafficking of ILC2 into tissues, but it is likely that the ILC2 and CD4+ T cells use similar chemokines and receptors for chemotaxis and chemokinetics as they express similar transcription factors and receptors. Thus, we compared CCL17 versus CCL22 responsiveness, of ILC2 and CD4+ T lymphocytes, from both male and female, OVA-challenged animals.

Access restricted. Please log in or start a trial to view this content.

Protocol

$$\rightleftharpoonup{xx}$$ $$\longleftharp{xx}$$, $$\longrightharp{xx}$$,

All methods described here were reviewed and approved by the Institutional Animal Care and Use Committees at the University of Nebraska Medical Center (UNMC) and the University of Utah.

1. Setup and Preparation of Reagents

  1. Prepare complete RPMI (Roswell Park Memorial Institute) media.
    1. Add 10 mL of heat-inactivated fetal bovine serum (FBS) to 90 mL of RPMI.
    2. Add 1 mL of 100x Penicillin-Streptomycin-Glutamine to 100 mL of 10% FBS RPMI.
  2. Prepare ILC2 Expansion Media.
    1. Add IL-2 and IL-33 (20 ng/mL each cytokine) to 10 mL of complete RPMI.
    2. If stock cytokines are 10 µg/mL, pipette 20 µL of IL-2 and IL-33 into a 15 mL tube containing 10 mL of complete RPMI media.
  3. Prepare Lung Dissociation Medium.
    1. Add 50 mg of type 1 collagenase to 250 mL of unsupplemented RPMI.
    2. Add 2.5 mL of 100x penicillin-streptomycin-glutamine to the 250 mL of media in step 1.3.1.
    3. Gently mix the media to ensure the type 1 Collagenase is completely dissolved before use.
  4. Prepare Serum-free RPMI.
    1. Dilute 1 g of lyophilized bovine serum albumin (BSA) in 200 mL of RPMI.
    2. Add 2 mL of 100x penicillin-streptomycin-glutamine.
    3. Gently mix the media to ensure the BSA is completely dissolved in the media before use.
  5. Prepare Migration Medium with CCL17.
    1. Acquire 10 mL of serum-free RPMI and add CCL17 [50 ng/mL].
      1. If stock CCL17 is 10 µg/mL, add 50 µL of CCL17 stock to 10 mL of serum-free RPMI media.
  6. Prepare Migration Medium with CCL22.
    1. Acquire 10 mL of serum-free RPMI and add CCL22 [50 ng/mL].
    2. If stock CCL22 is 10 µg/mL, add 50 µL of CCL22 stock to 10 mL of serum-free RPMI media.
  7. Prepare CCR4 Antibody Staining Cocktail.
    1. For 10 tests total, add 5 µL of each of the following antibodies to a 5 mL tube: anti-mouse CCR4, CD19, CD11b, CD45, ST2, and ICOS.
      NOTE: Example of how to make antibody cocktail for 10 samples: 0.5 µL of each antibody x 10 samples = 5 µL of each of the antibodies listed in 1.7.1.
    2. For 10 tests total, add 2.5 µL of the following antibodies to the antibody cocktail from step 1.7.1: anti-mouse CD3, CD11c, and NK1.1.
      NOTE: Example to complete the antibody cocktail for 10 samples: 0.25 µL x 10 samples = 2.5 µL of CD3, CD11c and NK1.1 antibodies.
    3. Store the CCR4 Antibody Staining Cocktail at 4 °C until ready to add to the samples. Discard antibody cocktail after 1 week if not used.
  8. Prepare 1x Stabilizing Fixative.
    1. Add 10 mL of deionized-distilled water to 5 mL of 3x stabilizing fixative concentrate

2. Preparation of Allergen-challenged BALB/c Mice

NOTE: Male and female BALB/c mice were purchased from Charles River (UNMC) or Jackson Laboratories (University of Utah) at 6 to 8 weeks of age.

  1. After acclimation (1 week), sensitize all animals to OVA.
    1. Combine 100 µg/mL OVA adsorbed to aluminum hydroxide (20 mg/mL) in a 5 mL polystyrene tube.
    2. Mix the tube and immediately draw 500 µL of the OVA-alum suspension into a 1 mL, 28 G syringe (insulin syringe).
    3. Place a mouse in a bell jar containing isoflurane (1–2 mL under a false floor, so that the animal is not standing directly in isoflurane). Allow the mouse to go under anesthesia for approximately 1–2 min, or until the rate of respiration drops.
    4. Quickly pick up the mouse by the fur on the back and shoulders and inject 100 µL of OVA-alum intraperitoneally per mouse21,22.
    5. Place the mouse back in its cage and watch to make sure they regain mobility; this should occur within 2–5 min.
  2. Seven days after sensitization, subject all animals to intranasal (i.n.) 1.5% OVA diluted in sterile saline in a nebulization chamber (Data Sciences International) for 20 min.
    1. Remove mice from their cages and place them in the animal nebulization chamber. Close the lid on the chamber.
    2. Attach the nebulization hose to the input spout on the nebulization chamber.
    3. Add 30 mL of 1.5% OVA diluted in sterile saline to the nebulization cup on the nebulizer.
    4. Turn on the nebulizer and allow the chamber to fill with mist for 20 min.
    5. Turn off the nebulizer and let the mist settle.
    6. Return animals to their cages.
  3. Repeat step 2.2 for a total of 5 times, on 5 consecutive days, to induce allergic inflammation.

3. Isolation of CD4+ T Cells from Spleens and Lungs of OVA-challenged Mice

  1. Humanely euthanize all OVA-treated male and female animals by CO2 asphyxiation according to approved IACUC protocols, using 2 to 3 animals per group, per experiment.
  2. Excise lungs and spleens from animals and place tissues in separate dissociation tubes based on the tissue type and sex of the animal23.
  3. Dissociate lung tissue in 500 µL of Lung Dissociation media (25 U/mL; collagenase, type 1) in the automated tissue dissociator using the ‘lung’ protocol.
    1. Repeat 3.2 and 3.3 a total of two times.
  4. Dissociate spleen tissue in 500 µL of complete RPMI media using the ‘spleen’ protocol on the automated tissue dissociator.
    NOTE: The remaining steps should be performed in a Biological Safety cabinet using sterile technique.
  5. Rinse the dissociation tubes containing lung and spleen homogenates with 5 mL of additional Lung Dissociation media or Complete RPMI, respectively.
  6. Filter cell suspensions through a 40 µm cell strainer and collect into 50 mL conical tubes.
  7. Incubate lung homogenates for 15–30 min in a 37 °C incubator with 5% CO2 to further dissociate lung tissue.
  8. Add 5 mL of Complete RPMI to the lung and spleen homogenates and pellet the cells at the bottom of the 50 mL tubes using centrifugation; 378 x g at room temperature (RT) for 5 min.
  9. Combine splenocytes and lung cells into a single 50 mL conical tube and determine total cell counts using the automated cell counter.
  10. Adjust male and female cell suspensions to 1 x 108 cells/mL in separation buffer and add to a 5 mL polystyrene tube.
    NOTE: The Enrichment Protocol can be adjusted up to 14 mL polystyrene tubes when more cells are acquired from spleen and lungs. This protocol was designed for tissues from 2–3 mice per group; therefore a 5 mL tube should suffice.
  11. Use approximately two thirds of the total cells for ILC2 isolation according to the ILC2 enrichment protocol.
    1. Add antibody cocktail (50 µL/mL) to the cell suspension and incubate for 5 min at RT.
    2. Vortex rapid spheres for 30 s and add to the sample at a rate of 75 µL/mL of cell suspension. Gently mix and incubate for 5 min at RT.
    3. Top the tube up to 3 mL total volume with separation buffer and place in the 8-chamber easy separation magnet. Incubate for 3 min at RT.
    4. Tip the magnet forward (away from the sphere-antibody-cell complexes adhered to the back of the tube) and pipette off the cell suspension into a clean 5 mL tube.
    5. Add 1.5 mL of complete RPMI to the tubes and centrifuge at 378 x g for 5 min at RT.
    6. Pour off the media from the cell pellet and resuspend the ILC2 at 1 x 107 cells per mL.
    7. Place 100 uL of male and female ILC2 per well in a U-bottom, 96-well plate and add 100 µL of ILC2 Expansion Media to each well.
    8. Incubate the cells for 4–5 days to expand the ILC2.
    9. Collect the ILC2 into a 5 mL tube and add up to 4.5 mL of serum-free RPMI. Centrifuge the cells at 378 x g for 5 min at RT.
    10. Count cells using a hemacytometer and resuspend at 1 x 107 ILC2 per mL in serum-free RPMI.
  12. Use the remaining cells for the CD4+ T cell isolation procedure, which is conducted according to the mouse CD4+ T cell isolation protocol with few modifications.
    1. Add rat serum (50 µL/mL) to the CD4 T cell enrichment suspension.
    2. Add Isolation cocktail (50 µL/mL) to the sample and incubate for 10 min at RT.
    3. Vortex rapid spheres for 30 s and add to the sample at a rate of 75 µL/mL.
    4. Gently mix the cell suspension and incubate for 2.5 min and RT
    5. Top the samples up to 3 mL and place the 5 mL tubes into the 8-chamber easy separation magnet and incubate for 5 min at RT.
    6. Tip the magnet forward and pipette the cell suspension into a clean 5 mL tube.
    7. Add 1.5 mL of serum-free RPMI to the tubes and centrifuge at 378 x g for 5 min at RT.
    8. Pour off the media from the cell pellet and resuspend the CD4+ T cells at 1 x 107 cells per mL in serum-free RPMI.

4. Determine CCR4 Expression on CD4+ T Cells and Group 2 Innate Lymphoid Cells (ILC2) from OVA-challenged Animals by Flow Cytometry

NOTE: The following steps may be performed on an open bench top as they are non-sterile techniques.

  1. Acquire approximately 1–2.5 x 105 ILC2 cells from step 3.11.10 and 1–2.5 x 106 CD4+ T cells from step 3.12.8 in separate 5 mL tubes.
    1. Keep an additional tube of at least 5.0 x 104 CD4+ T cells as an unstained control.
  2. Suspend the cells in 100–200 µL of FACS buffer and add 1 µL of Fc Block to every tube, then incubate on ice (or in a 4 °C refrigerator) for 10 min.
  3. Add 1–2 mL of FACS Buffer to every tube and centrifuge at 378 x g for 5 min at RT.
  4. Pour off the supernatant and resuspend the cells in 100–200 µL of FACS Buffer.
  5. Add 37.5 µL of the CCR4 Antibody Staining Cocktail to the cell suspension in every tube except the tube containing the ‘unstained’ cells.
  6. Incubate the tubes on ice, or in a 4 °C refrigerator, for 20–30 min.
  7. Add 1–2 mL of FACS Buffer to every tube and centrifuge at 378 x g for 5 min at RT.
  8. Pour off the supernatant.
  9. Repeat steps 4.7 and 4.8.
  10. Add 250–300 µL of 1x stabilizing fixative (see Table of Materials) to every tube.
  11. Prepare single-colored bead controls for each antibody in the CCR4 antibody cocktail according to the protocol provided with the compensation beads.
    1. Vortex the compensation beads.
    2. Add one drop of the beads to each single-colored control tube.
    3. Add 1 µL of each antibody in the CCR4 Antibody cocktail to its own labeled tube.
    4. Gently mix and incubate for 10 min in a 4 °C refrigerator or on ice.
    5. Add 1–2 mL of FACS Buffer to all the tubes and centrifuge at 378 x g for 5 min at RT.
    6. Pour off the supernatant and resuspend the beads in 200 µL of FACS buffer.
    7. Refrigerate the single-colored controls until all the samples are stained and ready to be analyzed on the flow cytometer.
  12. Analyze the unstained cells, single-colored controls and the experimental samples on the flow cytometer within 24 h of fixation.

5. Ex Vivo Transmigration Procedure

NOTE: The following steps should be performed in a Biological Safety Cabinet, as they require sterile technique.

  1. Acquire the ILC2 from step 3.11.10 and CD4 T cells from step 3.12.8 and determine the number of transwell inserts needed for the experiment.
    NOTE: Example: For 1.2 mL of enriched CD4 T cells from step 3.12.8, multiply 1.2 x 1,000 µL = 1,200 µL; then divide 1,200 uL by 100 µL = 12, the number of inserts needed for CD4 T transmigration.
  2. Gently move the 3 µm transwell inserts from the middle rows of a 24-well plate.
  3. Add 500 µL of migration media with CCL17 to approximately one-third of the wells.
  4. Add 500 µL of migration media with CCL22 to another one-third of the wells.
  5. Add 500 µL of serum-free RPMI containing no chemokine to the last one-third of the wells.
  6. Clearly label the lid on the plate with the appropriate transmigration media placed in the bottom wells.
  7. Place the transwell inserts back into the wells containing the various treatments.
  8. Gently add 100 µL of CD4 T cells or ILC2 to the top well of each insert. Do not mix or pipette the cell suspension up and down in the transwells, as this may confound the results of the experiment.
  9. Clearly label the lid of the plate with the cell type placed in each well and write the date and time of day that the cells were added.
  10. Repeat steps 5.2 to 5.9 until all the cells have been placed in transwell inserts with media.
  11. Gently place the plate in a 37 °C incubator with 5% CO2 for 48 h. Minimize contact with the plate over the incubation period.

6. Quantification of Ex Vivo Transmigration

  1. Gently remove the plate from the incubator and remove all the transwell inserts from the middle rows into the empty wells just above or below.
  2. Collect the cells from the bottom and top wells of the transwell inserts into tubes labeled with TOP or BOTTOM, with CCL17, CCL22, or media, with the cell type, and with the replicate number (at least 3 replicates per experiment).
  3. Rinse the bottom wells with 500 µL of 1x PBS and collect this rinse into the corresponding tube.
  4. Rinse the top wells with 250 µL of 1x PBS and collect this rinse into the corresponding tube.
  5. Pellet the cells by centrifugation at 378 x g for 5 min at RT.
  6. Gently pipette off all supernatant from the cell pellet.
  7. Resuspend T cells and ILC2 into 50 µL of 1x PBS.
  8. Take 10 µL of the cell suspensions and add to 90 µL of 0.4% trypan blue.
  9. Count the cells on the automated cell counter.
    1. Record %viability.
    2. Record the cell count per mL for each sample.
    3. Determine the total number of cells per treatment in the top and the bottom chamber; record the cell counts.

Access restricted. Please log in or start a trial to view this content.

Results

$$\rightleftharpoonup{xx}$$ $$\longleftharp{xx}$$, $$\longrightharp{xx}$$,

CCR4 expression on CD4+ T cells and ILC2.

For the success of the ex vivo transmigration experiment, it is imperative to determine whether the lymphocytes are responsive to CCL17 and CCL22 through CCR4; therefore, we determined CCR4 expression on both CD4+ T cells and ILC2 by flow cytometry. While it is well known that OVA-specific CD4+ helper T cells express CCR4, less is known of the expression of CCR4 on ILC2. Figure 1 show...

Access restricted. Please log in or start a trial to view this content.

Discussion

$$\rightleftharpoonup{xx}$$ $$\longleftharp{xx}$$, $$\longrightharp{xx}$$,

Herein, we present a well-established method for assessing chemokine-induced migration of lymphocytes in an ex vivo transmigration system. There are several critical steps in the protocol, the first of which is verifying the expression of the correct chemokine receptor on the immune cells in the experiment. In our hands, we chose CCR4 because of the body of literature that highlights the importance of CCR4 on Th2 helper T cells in allergic inflammation. Ovalbumin-induced inflammation was shown previously to be limited by...

Access restricted. Please log in or start a trial to view this content.

Disclosures

$$\rightleftharpoonup{xx}$$ $$\longleftharp{xx}$$, $$\longrightharp{xx}$$,

The authors have no financial disclosures or conflicts of interest to disclose.

Acknowledgements

$$\rightleftharpoonup{xx}$$ $$\longleftharp{xx}$$, $$\longrightharp{xx}$$,

This work was funded by the American Lung Association (K.J.W.), the Memorial Eugene Kenney fund awarded to T.A.W. and K.J.W., generous start-up support from the University of Utah for K.J.W., and a Department of Veterans Affairs award to T.A.W. (VA I01BX0003635). T.A.W. is the recipient of a Research Career Scientist Award (IK6 BX003781) from the Department of Veterans Affairs. The authors wish to acknowledge editorial assistance from Ms. Lisa Chudomelka. The authors thank the UNMC Flow Cytometry core for their support in collecting the flow cytometry data generated for this manuscript.

Access restricted. Please log in or start a trial to view this content.

Materials

List of materials used in this article
NameCompanyCatalog NumberComments
0.4% Trypan BlueSigma-Aldrich15250061
1 mL syringeBD Bioscience329424U-100 Syringes Micro-Fine 28 G 1/2" 1cc
100x Penicillin-Streptomycin, L-GlutamineGibco10378-016Dilute to 1x in RPMI media
15 mL conical tubesOlympus Plastics28-101polypropylene tubes
3 μm transwell insertsGenesee Scientific25-28824-well plate containing 12 transwell inserts
3x stabilizing fixativeBD Pharmigen338036Prepare 1x solution according to manufacturers protocol
5 mL polystyrene tubesSTEM Cell Technologies38007
50 mL conical tubesOlympus Plastics28-106polypropylene tubes
8-chamber easy separation magnetSTEM Cell Technologies18103
ACK Lysing BufferLife Technologies CorporationA1049201
Advanced cell strainer, 40 μmGenesee Scientific25-375nylon mesh, 40 μm strainers
Aluminum Hydroxide, Reagent GradeSigma-Aldrich239186-25G20 mg/mL
anti-mouse CCR4; APC-conjugatedBiolegend1312110.5 μg/test
anti-mouse CD11bBD Pharmigen5573960.5 μg/test
anti-mouse CD11c; PE eFluor 610Thermo-Fischer Scientific61-0114-820.25 μg/test
anti-mouse CD16/32, Fc blockBD Pharmigen5531410.5 μg/test
anti-mouse CD19; APC-eFluor 780 conjugatedThermo-Fischer Scientific47-0193-820.5 μg/test
anti-mouse CD3; PE Cy 7-conjugatedBD Pharmigen5527740.25 μg/test
anti-mouse CD45; PE conjugatedBD Pharmigen560870.5 μg/test
anti-mouse ICOS (CD278)BD Pharmigen5640700.5 μg/test
anti-mouse NK1.1 (CD161); FITC-conjugatedBD Pharmigen5531640.25 μg/test
anti-mouse ST2 (IL-33R); PerCP Cy5.5 conjugatedBiolegend1453110.5 μg/test
Automated Cell CounterBIORAD1450102
Automated DissociatorMACS Miltenyi Biotec130-093-235
Bovine Serum Albumin, Lyophilized PowderSigma-AldrichA2153-10G0.5% in serum-free RPMI
Cell Counter ClidesBIORAD1450015
Chicken Egg Ovalbumin, Grade VSigma-AldrichA5503-10G500 μg/mL
Collagenase, Type 1, FilteredWorthington Biochemical CorporationCLSS-1, purchase as 5 X 50 mg vials (LS004216)25 U/mL in RPMI
Compensation beadsAffymetrix01-1111-411 drop per contol tube
Dissociation TubesMACS Miltenyi Biotec130-096-335
FACS BufferBD Pharmigen5546571x PBS + 2% FBS, w/ sodium azide; stored at 4 °C
Heat Inactivated-FBSGenesee Scientific25-525H10% in complete RPMI & ILC2 Expansion Media
Mouse CCL17GenScriptZ02954-2050 ng/mL
Mouse CCL22GenScriptZ02856-2050 ng/mL
Mouse CD4+ T cell enrichment kitSTEM Cell Technologies19852
Mouse IL-2GenScriptZ02764-2020 ng/mL
Mouse ILC2 enrichment kitSTEM Cell Technologies19842
Mouse recombinant IL-33STEM Cell Technologies7804420 ng/mL
RPMILife Technologies Corporation22400071
Separation BufferSTEM Cell Technologies201441x PBS + 2% FBS; stored at 4 °C
Small animal nebulizer and chamberData Sciences International
Sterile salineBaxter2F7124; NDC 0338-0048-040.9% Sodium Chloride

References

$$\rightleftharpoonup{xx}$$ $$\longleftharp{xx}$$, $$\longrightharp{xx}$$,
  1. Boyden, S. The chemotactic effect of mixtures of antibody and antigen on polymorphonuclear leucocytes. The Journal of Experimental Medicine. 115, 453-466 (1962).
  2. Moser, B. Editorial: History of Chemoattractant Research. Frontiers in Immunology. 6, 548(2015).
  3. Borroni, E. M., Savino, B., Bonecchi, R., Locati, M. Chemokines sound the alarmin: The role of atypical chemokine in inflammation and cancer. Seminars in Immunology. 38, 63-71 (2018).
  4. Charo, I. F., Ransohoff, R. M. The many roles of chemokines and chemokine receptors in inflammation. The New England Journal of Medicine. 354 (6), 610-621 (2006).
  5. Abboud, D., Hanson, J. Chemokine neutralization as an innovative therapeutic strategy for atopic dermatitis. Drug Discovery Today. 22 (4), 702-711 (2017).
  6. Aldinucci, D., Casagrande, N. Inhibition of the CCL5/CCR5 Axis against the Progression of Gastric Cancer. International Journal of Molecular Sciences. 19 (5), E1477(2018).
  7. Chonco, L., et al. Novel DNA Aptamers Against CCL21 Protein: Characterization and Biomedical Applications for Targeted Drug Delivery to T Cell-Rich Zones. Nucleic Acid Therapy. 28 (4), 242-251 (2018).
  8. Trivedi, P. J., Adams, D. H. Chemokines and Chemokine Receptors as Therapeutic Targets in Inflammatory Bowel Disease; Pitfalls and Promise. Journal of Crohn's and Colitis. 12 (12), 1508(2018).
  9. Pietrosimone, K. M., Bhandari, S., Lemieux, M. G., Knecht, D. A., Lynes, M. A. In vitro assays of chemotaxis as a window into mechanisms of toxicant-induced immunomodulation. Current Protocols in Toxicology. 58 (Unit 18.17), (2013).
  10. Davis, D. M. How studying the immune system leads us to new medicines. Lancet. 391 (10136), 2205-2206 (2018).
  11. Culley, F. J., Pennycook, A. M., Tregoning, J. S., Hussell, T., Openshaw, P. J. Differential chemokine expression following respiratory virus infection reflects Th1- or Th2-biased immunopathology. Journal of Virology. 80 (9), 4521-4527 (2006).
  12. Denney, H., Clench, M. R., Woodroofe, M. N. Cleavage of chemokines CCL2 and CXCL10 by matrix metalloproteinases-2 and -9: implications for chemotaxis. Biochemical and Biophysics Research Communications. 382 (2), 341-347 (2009).
  13. Burrell, B. E., et al. Lymph Node Stromal Fiber ER-TR7 Modulates CD4+ T Cell Lymph Node Trafficking and Transplant Tolerance. Transplantation. 99 (6), 1119-1125 (2015).
  14. Warren, K. J., Iwami, D., Harris, D. G., Bromberg, J. S., Burrell, B. E. Laminins affect T cell trafficking and allograft fate. The Journal of Clinical Investigation. 124 (5), 2204-2218 (2014).
  15. Hirata, H., et al. Th2 cell differentiation from naive CD4(+) T cells is enhanced by autocrine CC chemokines in atopic diseases. Clinical and Experimental Allergy: Journal of the British Society for Allergy and Clinical Immunology. , (2018).
  16. Lin, R., Choi, Y. H., Zidar, D. A., Walker, J. K. L. beta-Arrestin-2-Dependent Signaling Promotes CCR4-mediated Chemotaxis of Murine T-Helper Type 2 Cells. American Journal of Respiratory Cell and Molecular Biology. 58 (6), 745-755 (2018).
  17. Zhang, Y., et al. A new antagonist for CCR4 attenuates allergic lung inflammation in a mouse model of asthma. Science Reports. 7 (1), 15038(2017).
  18. Lu, Y., et al. Dynamics of helper CD4 T cells during acute and stable allergic asthma. Mucosal Immunology. 11 (6), 1640-1652 (2018).
  19. Li, B. W. S., Beerens, D., Brem, M. D., Hendriks, R. W. Characterization of Group 2 Innate Lymphoid Cells in Allergic Airway Inflammation Models in the Mouse. Methods in Molecular Biology. 1559, 169-183 (2017).
  20. Li, B. W. S., et al. Group 2 Innate Lymphoid Cells Exhibit a Dynamic Phenotype in Allergic Airway Inflammation. Frontiers in Immunology. 8, 1684(2017).
  21. Warren, K. J., et al. Sex differences in activation of lung-related type-2 innate lymphoid cells in experimental asthma. Annals of Allergy, Asthma, Immunology: Official Publication of the American College of Allergy, Asthma, Immunology. , (2016).
  22. Warren, K. J., et al. Ovalbumin-sensitized mice have altered airway inflammation to agriculture organic dust. Respiratory Research. 20 (1), 51(2019).
  23. Poole, J. A., et al. alphabeta T cells and a mixed Th1/Th17 response are important in organic dust-induced airway disease. Annals of Allergy, Asthma, Immunology: Official Publication of the American College of Allergy, Asthma, Immunology. 109 (4), 266-273 (2012).
  24. Matsuo, K., et al. A CCR4 antagonist ameliorates atopic dermatitis-like skin lesions induced by dibutyl phthalate and a hydrogel patch containing ovalbumin. Biomedical Pharmacotherapy. 109, 1437-1444 (2019).
  25. Mikhak, Z., et al. Contribution of CCR4 and CCR8 to antigen-specific T(H)2 cell trafficking in allergic pulmonary inflammation. The Journal of Allergy and Clinical Immunology. 123 (1), 67-73 (2009).
  26. Monticelli, L. A., et al. Innate lymphoid cells promote lung-tissue homeostasis after infection with influenza virus. Nature Immunology. 12 (11), 1045-1054 (2011).
  27. Saenz, S. A., et al. IL25 elicits a multipotent progenitor cell population that promotes T(H)2 cytokine responses. Nature. 464 (7293), 1362-1366 (2010).
  28. Hoyler, T., et al. The transcription factor GATA-3 controls cell fate and maintenance of type 2 innate lymphoid cells. Immunity. 37 (4), 634-648 (2012).
  29. Nakajima, H., Shores, E. W., Noguchi, M., Leonard, W. J. The common cytokine receptor gamma chain plays an essential role in regulating lymphoid homeostasis. The Journal of Experimental Medicine. 185 (2), 189-195 (1997).
  30. Warren, K. J., et al. RSV-specific anti-viral immunity is disrupted by chronic ethanol consumption. Alcohol. , (2016).
  31. Warren, K. J., Poole, J. A., Sweeter, J. M., DeVasure, J. M., Wyatt, T. A. An association between MMP-9 and impaired T cell migration in ethanol-fed BALB/c mice infected with Respiratory Syncytial Virus-2A. Alcohol. , (2018).
  32. Molteni, R., et al. A novel device to concurrently assess leukocyte extravasation and interstitial migration within a defined 3D environment. Lab on a Chip. 15 (1), 195-207 (2015).
  33. Bersini, S., et al. Human in vitro 3D co-culture model to engineer vascularized bone-mimicking tissues combining computational tools and statistical experimental approach. Biomaterials. 76, 157-172 (2016).
  34. Jeon, J. S., et al. Human 3D vascularized organotypic microfluidic assays to study breast cancer cell extravasation. Proceedings of the National Academy of Sciences of the United States of America. 112 (1), 214-219 (2015).

Access restricted. Please log in or start a trial to view this content.

Reprints and Permissions

Request permission to reuse the text or figures of this JoVE article

Request Permission

Tags

Chemokine GradientCCR4 ExpressionILC2 IsolationCD4 T CellTranswell AssayFlow CytometryCell ViabilityChemokine Screening

Related Articles