Biological Responses to Chemokine Superfamily Members

Dennis D. Taub1, Eric Schaffer1

1 National Institute on Aging, NIH, Washington, D.C.
Publication Name:  Current Protocols in Immunology
Unit Number:  Unit 6.12
DOI:  10.1002/0471142735.im0612s38
Online Posting Date:  May, 2001
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Abstract

A detailed summary of all the chemokine subfamily members including alternative names, receptor‐ligand pairs, receptor cellular distribution, and leukocyte responsiveness is presented here along with assays to measure the biological activities of chemokines on each of the major leukocyte subsets (i.e., microchemotaxis chambers, fluorescence‐based assays, use of nitrocellulose filters and membranes coated with extracellular matrix proteins, cellular adhesion assays, mobilization of intracellular free calcium, actin polymerization, degranulation assays, and the use of chemokine receptor‐specific antibodies in flow cytometric analysis and radiolabeled chemokine binding assays).

     
 
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Table of Contents

  • Basic Protocol 1: Measurement of Granulocyte Chemotaxis by Chemokines Using Microchemotaxis Assays
  • Basic Protocol 2: Measurement of Monocyte Chemotaxis by Chemokines Using Microchemotaxis Assays
  • Basic Protocol 3: Measurement of Lymphocyte Chemotaxis by Chemokines Using Microchemotaxis Assays
  • Support Protocol 1: Coating Polycarbonate Filters with Extracellular Matrix Proteins
  • Alternate Protocol 1: Measurement of Leukocyte Chemotaxis Using Nitrocellulose Filters
  • Alternate Protocol 2: Fluorescence‐Based Assay for Leukocyte Chemotaxis
  • Basic Protocol 4: Measurement of Chemokine‐Induced T Cell Adhesion to Extracellular Matrix Proteins
  • Basic Protocol 5: Measurement of Receptor Binding by Chemokines: Displacement Analysis with Unlabeled Chemokines
  • Alternate Protocol 3: Equilibrium Binding Assay of Chemokines
  • Support Protocol 2: Radioiodination of Chemokines
  • Basic Protocol 6: Measurement of Chemokine‐Induced Intracellular Free Calcium in Leukocytes
  • Basic Protocol 7: Measurement of Chemokine‐Induced Actin Polymerization and Cellular Polarization
  • Support Protocol 3: Additional Assays to Assess Chemokine Bioactivity
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Measurement of Granulocyte Chemotaxis by Chemokines Using Microchemotaxis Assays

  Materials
  • Chemotaxis medium: RPMI 1640 containing 2 nM glutamine, 25 mM HEPES, and 1% (w/v) BSA (Fraction V; Sigma)
  • Recombinant human or rodent chemokines (e.g., R & D Systems, PeproTech, PharMingen, Biosource)
  • 10−3 M formyl‐methionyl‐leucyl‐phenylalanine (fMLP; Sigma) in chemotaxis medium
  • 1 µg/ml CC chemokine RANTES (negative control for chemotaxis; Tables 6.12.1 and 6.12.2)
  • Diff‐Quik stain (Baxter)
  • Multiwell microchemotaxis chamber and accessory pack containing clips and wiper blade (Neuro Probe)
  • Polycarbonate membrane with 3‐ to 5‐µm pores (Neuro Probe), without polyvinylpyrrolidone coating (PVP‐free)
  • Zeiss Axioskop microscope (model D‐7082) with 40× and 100× oil‐immersion objectives
  • Video camera (e.g., Ikegami Electronics)
  • Image analyzer (model V; Optomax)
  • Additional reagents and equipment for isolating neutrophils (unit 7.23)

Basic Protocol 2: Measurement of Monocyte Chemotaxis by Chemokines Using Microchemotaxis Assays

  Materials
  • Chemotaxis medium: RPMI containing 2 nM glutamine, 25 mM HEPES, and 1% (w/v) BSA (Fraction V; Sigma)
  • Recombinant human or rodent chemokines (e.g., R & D Systems, PeproTech, PharMingen, Biosource)
  • 10−3 M formyl‐methionyl‐leucyl‐phenylalanine (fMLP; Sigma) in chemotaxis medium, or other positive control (Tables 6.12.1 and 6.12.2)
  • 10 µg/ml interleukin 8 (IL‐8) or platelet factor 4 (PF‐4) for negative control
  • PBS ( appendix 2A)
  • Diff‐Quik stain (Baxter)
  • Multiwell microchemotaxis chamber and accessory pack containing clips and wiper blade (Neuro Probe)
  • Polyvinylpyrrolidone (PVP)–coated polycarbonate membrane with 5‐µm pores (Neuro Probe)
  • Zeiss Axioskop microscope (model D‐7082) with 40× and 100× oil‐immersion objectives
  • Video camera (e.g., Ikegami Electronics)
  • Image analyzer (model V; Optomax)
  • Additional reagents and equipment for isolating peripheral blood mononuclear cells (unit 7.6)

Basic Protocol 3: Measurement of Lymphocyte Chemotaxis by Chemokines Using Microchemotaxis Assays

  Materials
  • Chemotaxis medium: RPMI 1640 containing 0.5% (v/v) heat‐inactivated FBS ( appendix 2A)
  • Recombinant human or rodent chemokines (e.g., R & D Systems, PeproTech, PharMingen, Biosource)
  • 10−3 M fMLP (Sigma) in chemotaxis medium for negative control
  • 10 µg/ml SDF‐1α (R & D Systems) in chemotaxis medium for positive control
  • Diff‐Quik stain (Baxter)
  • Multiwell chemotaxis chamber and accessory pack containing clips and a wiper blade (Neuro Probe)
  • ECM‐coated filter (see protocol 4)
  • Zeiss Axioskop microscope (model D‐7082) with 40× and 100× oil‐immersion objectives
  • Video camera (e.g., Ikegami Electronics)
  • Image analyzer (model V; Optomax)
  • Additional reagents and equipment for isolating T, B, or NK cells (units 7.2 7.5 & 7.7)

Support Protocol 1: Coating Polycarbonate Filters with Extracellular Matrix Proteins

  Materials
  • Extracellular matrix protein, e.g.:
  •  Human plasma fibronectin (Life Technologies)
  •  Mouse collagen type I or IV (Life Technologies)
  •  Mouse laminin (Life Technologies)
  • PBS ( appendix 2A)
  • Polycarbonate membranes with 5‐µm pores (Neuro Probe), with or without polyvinylpyrrolidone (PVP) coating

Alternate Protocol 1: Measurement of Leukocyte Chemotaxis Using Nitrocellulose Filters

  • Nitrocellulose filters (3‐ to 5‐mm pore size; NeuroProbe)
  • 18.5% (v/v) formalin: dilute 37% formalin (Sigma) 1:1 in PBS ( appendix 2A)
  • Mayer's hematoxylin (Sigma)
  • Propanol (Aldrich)
  • Xylene (Aldrich)
  • 200× oil‐immersion objective

Alternate Protocol 2: Fluorescence‐Based Assay for Leukocyte Chemotaxis

  • Calcein acetoxymethyl ester (calcein AM; Molecular Probes)
  • Chemotaxis medium: HBSS ( appendix 2A) containing 0.1% (w/v) BSA (Fraction V; Sigma)
  • Positive and negative chemotaxis controls (Tables 6.12.1 and 6.12.2)
  • 20 µM EDTA in PBS ( appendix 2A)
  • Methanol
  • 15‐ml conical centrifuge tubes
  • 48‐ or 96‐well chemotaxis chambers with microtiter plates (Neuro Probe)
  • Polycarbonate filter with 5‐µm pores (Whatman)
  • Fluorescent plate reader with a 494‐nm excitation filter and a 54‐nm emission filter
  • Centrifuge with microtiter plate holders
  • Standard microtiter plate

Basic Protocol 4: Measurement of Chemokine‐Induced T Cell Adhesion to Extracellular Matrix Proteins

  Materials
  • Extracellular matrix (ECM) protein (Life Technologies): human plasma fibronectin, mouse collagen type I or IV, or mouse laminin
  • PBS ( appendix 2A)
  • 3% (w/v) BSA in PBS
  • Adhesion medium: RPMI 1640 containing 0.5% (v/v) heat‐inactivated FBS ( appendix 2A)
  • Purified T cells (units 7.2 7.4)
  • Recombinant human or rodent chemokines (e.g., R & D Systems, PeproTech, PharMingen, Biosource)
  • 1% (v/v) Triton X‐100 solution
  • 96‐well tissue culture plates (e.g., Costar)
  • Multichannel pipettor
  • γ scintillation counter
  • Additional reagents and equipment for labeling with 51Cr (unit 3.11)

Basic Protocol 5: Measurement of Receptor Binding by Chemokines: Displacement Analysis with Unlabeled Chemokines

  Materials
  • Binding medium: RPMI 1640 containing 1% (w/v) BSA and 0.5% (w/v) sodium azide, pH 7.2 to 7.4
  • Isolated leukocytes (purified human or rodent lymphocytes, units 7.2 7.7; granulocytes, units 7.23, 7.24 & 7.31; or monocytes, unit 7.6) or tumor cells (e.g., Jurkat T cells), log phase
  • Recombinant human or rodent chemokines (e.g., R & D Systems, PeproTech, PharMingen, Biosource)
  • 125I‐labeled chemokine (see protocol 10; also available from NEN Life Sciences or Amersham Pharmacia Biotech)
  • 10% (w/v) sucrose (Sigma)/ PBS ( appendix 2A)
  • γ scintillation counter

Alternate Protocol 3: Equilibrium Binding Assay of Chemokines

  Materials
  • Bolton‐Hunter reagent (2200 Ci/mmol, mono‐iodinated; NEN Life Sciences) with charcoal trap
  • Nitrogen
  • Recombinant human or rodent chemokines (e.g., R & D Systems, PeproTech, PharMingen, Biosource)
  • 0.1 M sodium borate buffer, pH 8.5 (Sigma)
  • Termination buffer: 0.5 M glycine/0.1 M sodium borate, pH 8.5
  • 10% (v/v) glycerol (Sigma) in PBS ( appendix 2A)
  • BSA
  • Sodium azide
  • 20‐G needle
  • Sephadex G‐25 column (0.5 × 15 cm; Bio‐Rad)

Support Protocol 2: Radioiodination of Chemokines

  Materials
  • PBS ( appendix 2A)
  • Isolated leukocytes (human peripheral blood leukocytes, unit 7.1; purified human or rodent lymphocytes, units 7.2 7.7; granulocytes, units 7.23, 7.24 & 7.31; or monocytes, unit 7.6)
  • Indo‐1 acetoxymethyl ester (indo‐1 AM; Molecular Probes)
  • Pluronic F‐127, low‐UV‐absorbance grade (Molecular Probes)
  • Complete RPMI/10% FBS ( appendix 2A)
  • 10 µg/ml recombinant human or rodent chemokines (e.g., R & D Systems, PeproTech, PharMingen, Biosource)
  • 1 mM ionomycin
  • 1 µM CaCl 2
  • 10 mM MnCl 2
  • Spectrofluorometer (e.g., PE Biosystems) with thermostatically controlled cuvette, magnetic stirring attachment, excitation at 360 nm, emission at 402 and 486 nm, and output displayed on computerized monitor

Basic Protocol 6: Measurement of Chemokine‐Induced Intracellular Free Calcium in Leukocytes

  Materials
  • Isolated leukocytes from peripheral blood (purified human lymphocytes, units 7.2 7.7; monocytes, unit 7.6; or granulocytes, units 7.23, 7.24 & 7.31)
  • HBSS ( appendix 2A) with 0.1% (w/v) BSA (Fraction V; Sigma)
  • Recombinant human or rodent chemokines (e.g., R & D Systems, PeproTech, PharMingen, Biosource)
  • 3.7% (w/v) formaldehyde
  • Lysophosphatidylcholine (Sigma)
  • 7‐Nitrobenz‐2‐oxa‐1,3‐diazole‐phallacidin (NBD‐phallacidin; Sigma)
  • PBS ( appendix 2A)
  • Flow cytometer (e.g., FACScan; Becton Dickinson)
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Figures

Videos

Literature Cited

Literature Cited
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