Measurement of Interleukin 16

David M. Center1, William W. Cruikshank1, Nereida A. Parada1, Thomas Ryan1, Arthur C. Theodore1, Gregory Viglianti2, Kaiser G. Lim3, Peter F. Weller3

1 Boston University Medical Center, Boston, Massachusetts, 2 Boston University School of Medicine, Boston, Massachusetts, 3 The Beth Israel Hospital, Boston, Massachusetts
Publication Name:  Current Protocols in Immunology
Unit Number:  Unit 6.23
DOI:  10.1002/0471142735.im0623s22
Online Posting Date:  May, 2001
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Abstract

Interleukin 16 (IL‐16) is a chemoattractant immunomodulatory cytokine that initiates its cellular responses through interaction with membrane‐expressed CD4. The protein may be detected by a number of methods; the choice of protocol will depend on the ultimate object of a particular experiment. The first method presented is the use of ELISA to measure IL‐16 in cell culture supernatants or biological fluids. For some applications, such as identification of IL‐16 in an unknown fluid or medium or direct assessment of its bioactivity, functional assays of IL‐16‐induced responses may be more appropriate. The chemotactic effects of IL‐16 on CD4+ T cells and its specific inhibition may be measured using anti‐IL‐16 antibodies; the same approach may also be applied to monocytes or eosinophils. Another effect of IL‐16 is the induction of CD25, which can be assayed using immunological staining. Finally, cell cycle progression in target cells can be measured by the incorporation of radiolabeled thymidine and confirmed by inhibition with neutralizing antibody.

     
 
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Table of Contents

  • Basic Protocol 1: Quantitation of IL‐16 Protein by ELISA
  • Basic Protocol 2: Quantitation of IL‐16 by Assessment of CD4+ T Lymphocyte Chemotaxis
  • Alternate Protocol 1: Quantitation of IL‐16 by Assessment of Monocyte Chemotaxis
  • Basic Protocol 3: Measurement of IL‐16‐Induced CD25 Expression
  • Basic Protocol 4: Measurement of Cell Cycle Progression Induced by IL‐16 in CD4+ Cell Lines
  • Commentary
     
 
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Materials

Basic Protocol 1: Quantitation of IL‐16 Protein by ELISA

  Materials
  • Capture antibody: 5 µg/ml rabbit monoclonal anti‐IL‐16 IgG (PharMingen) in recipePBS ( appendix 2A)
  • 0.2% (w/v) BSA or 2% (v/v) goat serum, prepared in recipePBS
  • IL‐16 stock solution (PeproTech, PharMingen, or Genzyme)
  • 0.5% (v/v) Tween 20 in recipePBS (PBST)
  • Samples for IL‐16 quantitation
  • Detecting antibody: 5 µg/ml murine polyclonal anti‐IL‐16 IgG in PBST (PharMingen or Genzyme)
  • 3.4 mg/ml biotinylated goat anti–mouse IgG diluted 1:1000 in PBST
  • Extravidin peroxidase diluted 1:1000 in PBST
  • 0.4 g/liter TMB (3,3′,5,5′‐tetramethyl benzidine) substrate solution
  • 96‐well flat‐bottom microtiter plates (Nunc)
  • Multichannel pipettor, pipet tips, and reagent reservoir (Fisher), or digital pipettor (e.g., Eppendorf Ultra Micro, Brinkman) and pipet tips
  • Microtiter plate reader with 490‐nm filter
  • Additional reagents and equipment for ELISA (unit 2.1)

Basic Protocol 2: Quantitation of IL‐16 by Assessment of CD4+ T Lymphocyte Chemotaxis

  Materials
  • Peripheral blood (for isolation of fresh CD4+ T lymphocytes) or motile CD4+ T cell line (e.g., SupT1)
  • Complete RPMI‐5 ( appendix 2A) containing 20 mM HEPES
  • Chemotaxis buffer: RPMI 1640 containing 20 mM HEPES and 0.4% BSA
  • Sample to be assayed for IL‐16 bioactivity
  • Neutralizing antibody: rabbit polyclonal or murine monoclonal anti‐IL‐16 IgG (PharMingen)
  • Normal rabbit IgG (Sigma)
  • 10 pM IL‐16 in chemotaxis buffer (diluted from 10 nM IL‐16 stock)
  • 50% and 100% ethanol
  • Gill's hematoxylin stain (Vector Labs)
  • 0.5% HCl
  • 1‐propanol
  • Xylenes
  • Permount or comparable mounting medium
  • 100‐µl pipettor
  • Multiwell chemotaxis chamber (Neuro Probe)
  • Nitrocellulose membrane with 8‐µm pore size (Neuro Probe)
  • Flathead forceps and printer's clips
  • Staining dishes
  • Glass slides
  • 22 × 44–mm glass cover slips
  • Additional reagents and equipment for T cell isolation (unit 7.2)
NOTE: All cell culture incubations should be in a humidified 37°C, 5% CO 2 incubator unless otherwise noted.CAUTION: When working with human blood, cells, or potentially infectious materials, biosafety practices must be followed.

Alternate Protocol 1: Quantitation of IL‐16 by Assessment of Monocyte Chemotaxis

  • Peripheral blood (for isolation of fresh monocytes) or monocyte cell line (e.g., U937, ATCC #CRL 1593; or THP1, ATCC #T1B 202)
  • Complete RPMI‐10 ( appendix 2A) containing 20 mM HEPES
  • 10−7 M fMet‐Leu‐Phe peptide (Sigma) in chemotaxis buffer
  • 3‐ml round‐bottom glass tubes, silanized ( appendix 3K)
  • Nitrocellulose membrane with 3‐µm pore size (Neuro Probe)

Basic Protocol 3: Measurement of IL‐16‐Induced CD25 Expression

  Materials
  • Peripheral blood (for isolation of fresh T cells) or cultured CD4+ T cell line (e.g., HUT 78, ATCC #T1B161)
  • Complete RPMI‐10 ( appendix 2A) containing 20 mM HEPES
  • Samples to be assayed for IL‐16 bioactivity
  • 10−7 M IL‐16 stock solution
  • T cell mitogen: Con A or phytohemagglutinin (PHA)
  • Staining buffer: recipePBS (pH 7.2) containing 1% BSA and 0.1% sodium azide, 2° to 4°C
  • Monoclonal anti‐CD25 (IL‐2Rα) antibody conjugated to either fluorescein or phycoerythrin (Becton Dickinson)
  • Monoclonal murine IgG of the same isotype, and conjugated to the same fluorochrome, as the anti‐CD25 antibody (Becton Dickinson)
  • 10% formalin in recipePBS (optional)
  • 24‐well cell culture plates (Costar)
  • Aluminum foil
  • FACScan flow cytometer (Becton Dickinson) or equivalent
  • Additional reagents and equipment for fluorescence‐based flow cytometric cell sorting (see Chapter 5)
NOTE: All cell culture incubations should be in a humidified 37°C, 5% CO 2 incubator unless otherwise noted.CAUTION: When working with human blood, cells, or potentially infectious materials, biosafety practices must be followed.

Basic Protocol 4: Measurement of Cell Cycle Progression Induced by IL‐16 in CD4+ Cell Lines

  Materials
  • CD4+ T cell line (e.g., HUT 78 or SupT1)
  • Complete RPMI‐10 ( appendix 2A) containing 25 mM HEPES buffer
  • 10−7 M IL‐16 stock solution
  • Sample to be assayed for IL‐16 bioactivity
  • Rabbit anti‐IL‐16 IgG (PharMingen)
  • Normal rabbit IgG (Sigma)
  • [3H]thymidine: 1 mCi/ml stock solution in recipePBS (NEN)
  • Liquid scintillation fluid (EcoLite or equivalent)
  • 96‐well round‐bottom culture plates (Costar)
  • Semiautomated cell harvester and appropriate filter paper
  • Scintillation counter
  • Additional reagents and equipment to measure cellular proliferation ( appendix 2A)
NOTE: All cell culture incubations should be in a humidified 37°C, 5% CO 2 incubator unless otherwise noted.CAUTION: When working with human blood, cells, or potentially infectious materials, biosafety practices must be followed.
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Literature Cited

Literature Cited
   Baier, M., Werner, A., Bannert, N., Metzner, K., and Kurth, R. 1995. HIV suppression by interleukin‐16. Nature 378:563‐564.
   Berman, J.S., Beer, D.J., Cruikshank, W.W., and Center, D.M. 1985. Chemoattractant lymphokines specific for the helper/inducer T‐lymphocyte subset. Cell. Immunol. 95:105‐112.
   Center, D.M. and Cruikshank, W. 1982. Modulation of lymphocyte migration by human lymphokines. I. Identification and characterization of chemoattractant activity for lymphocytes from mitogen‐stimulated mononuclear cells. J. Immunol. 128:2563‐2568.
   Cruikshank, W. and Center, D.M. 1982. Modulation of lymphocyte migration by human lymphokines. II. Purification of a lymphotactic factor (LCF). J. Immunol. 128:2569‐2571.
   Cruikshank, W.W., Berman, J.S., Theodore, A.C., Bernardo, J., and Center, D.M. 1987. Lymphokine activation of T4+ lymphocytes and monocytes. J. Immunol. 138:3817‐3825.
   Cruikshank, W.W., Greenstein, J.L., Theodore, A.C., and Center, D.M. 1991. Lymphocyte chemoattractant factor (LCF) induces CD4‐dependent intracytoplasmic signaling in lymphocytes. J. Immunol. 146:2928‐2934.
   Cruikshank, W.W., Center, D.M., Nisar, N., Natke, B., Theodore, A.C., and Kornfeld, H. 1994. Molecular and functional analysis of a lymphocyte chemoattractant factor: Association of biologic function with CD4 expression. Proc. Natl. Acad. Sci. U.S.A. 91:5109‐5113.
   Cruikshank, W.W., Long, A., Tarpy, R., Kornfeld, H., Carroll, M.P., Teran, L., Holgate, S., and Center, D.M. 1995. Early identification of IL‐16 (lymphocyte chemoattractant factor) and macrophage inflammatory protein 1α (MIP1α) in bronchoalveolar lavage fluid of antigen challenged asthmatics. Am. J. Respir. Cell. Mol. Biol. 13:738‐747.
   Cruikshank, W.W., Chupp, G., Keane, J., Kornfeld, H., Berman, J.S., and Center, D.M. 1996a. Biologic activities of IL‐16. Nature 382:501‐502.
   Cruikshank, W.W., Theodore, A.C., Fine, G., Lim, K.G., Weller, P.F., and Center, D.M. 1996b. Suppression of anti‐CD3 stimulated T cell proliferation by IL‐16. J. Immunol. 157:5240‐5248.
   Laberge, S., Cruikshank, W.W., Kornfeld, H., and Center, D.M. 1995. Histamine‐induced secretion of lymphocyte chemoattractant factor from CD8+ T cells is independent of transcription and translation: Evidence for constitutive protein synthesis and storage. J. Immunol. 55:2902‐2910.
   Laberge, S., Cruikshank, W.W., Kornfeld, H., Beer, D.J., and Center, D.M. 1996. Secretion of IL‐16 (lymphocyte chemoattractant factor) from serotonin‐stimulated CD8+ T cells in vitro. J. Immunol. 156:310‐315.
   Mackiewicz, C., Levy, J., Cruikshank, W., Kornfeld, H., and Center, D. 1996. Role of IL‐16 in HIV replication. Nature 383:488‐489.
   Maciaszek, J.W., Parada, N.A., Cruikshank, W.W., Center, D.M., Kornfeld, H., and Viglianti, G.A. 1997. Interleukin‐16 represses HIV‐1 promoter activity. J. Immunol. 158:5‐8.
   Parada, N.A., Ryan, T.C., Danis, H., Cruikshank, W.W., and Center, D.M. 1996. IL‐16 and other CD4 ligand‐induced migration is dependent upon protein kinase C. Cell. Immunol. 168:100‐106.
   Rand, T., Cruikshank, W.W., Center, D.M., and Weller, P.F. 1991. CD4‐mediated stimulation of human eosinophils: Lymphocyte chemoattractant factor and other CD4‐binding ligands elicit eosinophil migration. J. Exp. Med. 173:1521‐1528.
   Ryan, T., Cruikshank, W.W., and Center, D.M. 1995. Activation of CD4 associated p56 lck by the lymphocyte chemoattractant factor. Dissociation of kinase enzymatic activity with chemotactic response. J. Biol. Chem. 270:17081‐17086.
   Theodore, A.C., Center, D.M., Nicoll, J., Fine, G., Kornfeld, H., and Cruikshank, W.W. 1996. The CD4 ligand interleukin 16 inhibits the mixed lymphocyte reaction. J. Immunol. 157:1958‐1964.
Key References
   Cruikshank et al., 1991. See above.
   Description of the first transfection studies with CD4 which demonstrated that the Ca2+ and IP3 signals observed in NWNT cells can be conferred upon nonresponsive CD4− murine hybridoma cells by transfecting human CD4.
   Cruikshank et al., 1994. See above.
   Report of the cloning of the C‐terminal‐secreted biologically active IL‐16 peptide, with description of neutralizing antibodies.
   Cruikshank et al., 1996. See above.
   Definition of the differences between the precursor Pro‐IL‐16 and biologically active secreted C‐terminal peptide.
   Center, D.M., Kornfeld, H., and Cruikshank, W.W. 1996. Interleukin 16 and its functions as a CD4 ligand. Immunol. Today 17:476‐481.
  A recent review of the subject.
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