Culture of HIV in Monocytes and Macrophages

Suzanne Crowe1, Clare Maslin1, Philip Ellery1, Katherine Kedzierska2

1 Macfarlane Burnet Institute for Medical Research and Monash University, Melbourne, Australia, 2 University of Melbourne, Melbourne, Australia
Publication Name:  Current Protocols in Immunology
Unit Number:  Unit 12.4
DOI:  10.1002/0471142735.im1204s70
Online Posting Date:  January, 2006
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Abstract

Monocytes and macrophages constitute important cellular targets for macrophage‐tropic (M‐tropic) strains of HIV‐1 and are believed to play a major role in the pathogenesis of AIDS. This updated unit provides procedures for culturing HIV in these target cells under suspension and adherent cell culture conditions. The unit also provides methods for expanding M‐tropic primary isolates of HIV and determining the 50% tissue culture infectious dose (TCID50) of HIV stocks. Curr. Protoc. Immunol. 70:12.4.1‐12.4.13. © 2005 by John Wiley & Sons, Inc.

     
 
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Table of Contents

  • Basic Protocol 1: HIV Infection of Cultured Monocyte‐Derived Macrophages Adherent to Plastic
  • Alternate Protocol 1: HIV Infection of Cultured Monocyte‐Derived Macrophages in Suspension
  • Support Protocol 1: Isolation of Monocytes from PBMC by Adherence
  • Support Protocol 2: Isolation of Monocyte Subsets
  • Support Protocol 3: Expansion of Laboratory‐Adapted Macrophage‐Tropic HIV Strains
  • Support Protocol 4: Expansion of Macrophage‐Tropic Primary Isolates
  • Support Protocol 5: TCID50 Quantification of HIV in MDM
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: HIV Infection of Cultured Monocyte‐Derived Macrophages Adherent to Plastic

  Materials
  • Macrophage‐tropic strain of HIV‐1 (laboratory‐adapted stock, protocol 5, or primary isolate, protocol 6)
  • Monocyte‐derived macrophages (MDM), day 5 to 7 following isolation, purified by either adherence ( protocol 3) or elutriation (unit 7.6), cultured in 24‐ or 96‐well plates or 25‐ or 80‐cm2 flasks
  • Phosphate buffered saline (PBS; e.g., Life Technologies) supplemented with 1% FBS (PBS/FBS)
  • Supplemented Iscove's/RPMI‐1640 medium (see recipe)
  • 2‐ml plastic transfer pipets or pipet attached to vacuum system
  • 1.8‐ml screw‐top cryovials (e.g., Nunc)
  • Additional reagents and equipment for antibody sandwich, kinetic p24 ELISA, or reverse transcriptase assay for HIV (unit 12.5) and counting cells ( appendix 3A)

Alternate Protocol 1: HIV Infection of Cultured Monocyte‐Derived Macrophages in Suspension

  • MDM, days 5 to 7 following isolation, purified by either adherence ( protocol 3) or elutriation (unit 7.6), cultured in 15‐, 30‐, 60‐ml Teflon jars (e.g., Savillex)
  • 15‐ and 50‐ml conical polypropylene tubes
  • Beckman centrifuge with GH‐3.8 rotor, or equivalent

Support Protocol 1: Isolation of Monocytes from PBMC by Adherence

  Materials
  • PBMC isolated from peripheral blood or buffy packs (obtained from the blood bank) by Ficoll‐gradient centrifugation (unit 7.1)
  • Adherence medium: Supplemented Iscove's/RPMI‐1640 medium (see recipe) containing 10% to 20% heat‐inactivated human serum, prewarmed to 37°C
  • Phosphate‐buffered saline (PBS; e.g., Life Technologies) with Ca2+ and Mg2+, prewarmed to 37°C, and without Ca2+ and Mg2+, prechilled to 4°C
  • FBS
  • Supplemented Iscove's/RPMI‐1640 medium (see recipe)
  • PBS (see above) supplemented with 1% (v/v) FBS (PBS/FBS), prechilled to 4°C
  • Anti‐CD14 mouse monoclonal antibody (MAb) conjugated to phycoerythrin (CD14‐PE; Becton Dickinson)
  • Anti‐CD3 mouse MAb conjugated to fluorescein (CD3‐FITC; Becton Dickinson)
  • 1% (v/v) formaldehyde, ultrapure
  • 150‐cm2 plastic tissue culture plates (e.g., Nunc)
  • Inverted phase‐contrast microscope
  • Cell scrapers
  • 50‐ml conical centrifuge tubes
  • 24‐well tissue culture plates, 80‐cm2 tissue culture flasks, or Teflon jars (see protocol 2 for details on the jars)
  • 5‐ml polypropylene round‐bottom tubes (Becton Dickinson)
  • Beckman centrifuge with GH‐3.8 rotor, or equivalent
  • Additional reagents for counting cells with a hemacytometer ( appendix 3A) and flow cytometry (unit 5.4)

Support Protocol 2: Isolation of Monocyte Subsets

  Materials
  • Monocytes isolated from PBMC by elutriation (unit 7.6) for FACS or PBMC isolated from small sample of whole blood by Ficoll‐gradient centrifugation (unit 7.1) for MACS
  • PBS/FBS: phosphate‐buffered saline (PBS; e.g., Life Technologies) without Ca2+ or Mg2+, containing 1% (v/v) FBS or 0.1% (w/v) bovine serum albumin and 2 mM EDTA, prechilled to 4°C
  • Anti‐CD14 mouse monoclonal antibody (MAb) conjugated to phycoerythrin (CD14‐PE; Becton Dickinson)
  • Anti‐CD16 mouse MAb conjugated to fluorescein (CD16‐FITC; Becton Dickinson)
  • Fetal bovine serum (FBS)
  • Supplemented Iscove's/RPMI‐1640 (see recipe)
  • Monocyte Isolation Kit II (Miltenyi Biotec) containing:
    • Monocyte Biotin‐Antibody Cocktail (cocktail of biotin‐conjugated monoclonal antibodies against CD3, CD7, CD16, CD19, CD56, CD123 and glycophorin A)
    • Anti‐biotin (mouse IgG1; clone Bio3‐18E7.2) microbeads
    • Fc receptor blocking reagent (human immunoglobulin) to block nonspecific binding of detection antibodies to monocytes
  • Anti‐CD16 MAb–coated microbeads (Miltenyi Biotec)
  • 5‐ml polypropylene round‐bottom tubes (Becton Dickinson)
  • Beckman refrigerated centrifuge with GH‐3.8 rotor, or equivalent
  • High‐speed fluorescence‐activated cell sorter (FACS, e.g., MoFlo, Dako Cytomation; also see unit 5.4)
  • MACS columns and MACS separator (e.g., LS columns in the Midi‐MACS setup; Miltenyi Biotec)
  • Additional reagents for counting cells using a hemocytometer ( appendix 3A) and flow cytometry (units 5.1& 5.4)

Support Protocol 3: Expansion of Laboratory‐Adapted Macrophage‐Tropic HIV Strains

  Materials
  • Monocytes isolated from PBMC by adherence method ( protocol 3)
  • Stock of laboratory‐adapted M‐tropic strain of HIV‐1 in culture medium, e.g., HIV‐1 Ba‐L, HIV‐1 JR‐FL (AIDS Research and Reference Reagent Program; http://www.aidsreagent.org)
  • Supplemented Iscove's/RPMI‐1640 medium (see recipe)
  • HIV‐1 p24 ELISA kit (unit 12.5)
  • 80‐cm2 flasks
  • Beckman centrifuge with GH‐3.8 rotor, or equivalent
  • 0.22‐µm filters
  • 1.8‐ and 5‐ml cryovials (e.g., Nunc)
  • 50‐ml conical tubes
Additional reagents and equipment for antibody sandwich, kinetic p24 ELISA, or reverse transcriptase assay for HIV (unit 12.5), counting cells ( appendix 3A), and determination of MOI by TCID 50 assay ( protocol 7)

Support Protocol 4: Expansion of Macrophage‐Tropic Primary Isolates

  Materials
  • PBMC isolated by density gradient centrifugation from two HIV‐seronegative donors (unit 7.1).
  • Stock of M‐tropic primary isolate (amplified from PBMC obtained from HIV‐seropositive individuals as in unit 12.2).
  • Complete RPMI medium ( appendix 2A) containing 10% FBS
  • Phytohemagglutinin (PHA; e.g., Murex Diagnostics)
  • Macrophage colony‐stimulating factor (M‐CSF; e.g., Genzyme).
  • Interleukin‐2 (IL‐2; e.g., Boehringer Mannheim)
  • 25‐cm2 flasks
  • 14‐ml polypropylene round‐bottom tubes (e.g., Nunc)
  • 15‐ml conical tubes
  • Anti‐CD8 magnetic beads (Dynal)
  • Magnetic particle concentrator (e.g, Dynal)
  • Sample mixer (e.g., Dynal)
  • 1.8‐ml cryovials (e.g., Nunc)
  • Additional reagents and equipment for stimulating PBMC with PHA (unit 12.2), counting cells with a hemacytometer ( appendix 3A), and antibody sandwich or kinetic p24 ELISA for HIV antigens (unit 12.5)

Support Protocol 5: TCID50 Quantification of HIV in MDM

  Materials
  • HIV‐1 virus stock to be titrated (AIDS Research and Reference Reagent Program; http://www.aidsreagent.org)
  • MDM isolated by either adherence ( protocol 3) or elutriation (unit 7.6) and cultured for 5 to 7 days in a Teflon jar (see protocol 2)
  • Supplemented Iscove's/RPMI‐1640 medium (see recipe)
  • 96‐well flat‐bottom plates (e.g., Nunc)
  • HIV‐p24 ELISA kit (unit 12.5)
  • Additional reagents and equipment for antibody sandwich or kinetic p24 ELISA for HIV antigens (unit 12.5), and counting cells ( appendix 3A)
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Figures

Videos

Literature Cited

   Alkhatib, G., Combadiere, C., Broder, C., Feng, Y., Kennedy, P., Murphy, P., and Berger, A. 1996. CC CKR5: A RANTES, MIP‐α, MIP‐β receptor as a fusion cofactor for macrophage‐tropic HIV‐1. Science 272:1955‐1958.
   Biggs, B., Hewish, M., Kent, S., Hayes, K., and Crowe, S. 1995. HIV‐1 infection of human macrophages impairs phagocytosis and killing of Toxoplasma gondii. J. Immunol. 154:6132‐6139.
   Chowdhury, M., Munakata, T., Koyanagi, Y., Arai, S., and Yamamoto, N. 1994. Mycoplasma stimulates HIV‐1 expression from acutely and dormantly infected promonocyte/monoblastoid cell lines. Arch. Virol. 139:431‐438.
   Crowe, S. 1995. Role of macrophages in the pathogenesis of human immunodeficiency virus (HIV) infection. Aust. NZ J. Med. 25:777‐783.
   Crowe, S., Mills, J., and McGrath, M. 1987. Quantitative immunocytofluorographic analysis of CD4 surface antigen expression and HIV infection of human peripheral blood monocyte/macrophages. AIDS Res. Hum. Retroviruses 3:135‐145.
   Fear, W., Kesson, A., Naif, H., Lynch, G., and Cunningham, A. 1998. Differential tropism and chemokine receptor expression of human immunodeficiency virus type 1 in neonatal monocytes, monocyte‐derived macrophages, and placental macrophages. J. Virol. 72:1334‐1344.
   Kedzierska, K., Mak, J., Mijch, A., Cooke, I., Rainbird, M., Roberts, S., Paukovics, G., Jolley, D., Lopez, A., and Crowe, S. 2000. Granulocyte‐macrophage colony‐stimulating factor augments phagocytosis of Mycobacterium avium complex by human immunodeficiency virus type 1‐infected monocytes/macrophages in vitro and in vivo. J. Infect. Dis. 181:390‐394.
   Kornbluth, R., Oh, P., Munis, J., Cleveland, P., and Richman, D. 1986. Interferons and bacterial lipopolysaccharide protect macrophages from productive infection by human immunodeficiency virus in vitro. J. Exp. Med. 169:1137‐1151.
   Lewin, S., Sonza, S., Irving, L., McDonald, C., Mills, J., and Crowe, S. 1996. Surface CD4 is critical to in vitro HIV infection of human alveolar macrophages. AIDS Res. Hum. Retroviruses 12:877‐883.
   Locksley, R., Crowe, S., Sadick, M., Heinzel, F., Gardner, K., McGrath, M., and Mills, J. 1988. Release of interleukin‐1 inhibitory activity (contra‐IL‐1) by human monocyte‐derived macrophages infected with human immunodeficiency virus in vitro and in vivo. J. Clin. Invest. 82:2097‐2105.
   Perno, C., Yarchoan, R., Cooney, D., Hartman, N., Gartner, S., Popovic, M., Hao, Z., Gerrard, T., Wilson, Y., Johns, D., and Broder, S. 1988. Inhibition of human immunodeficiency virus (HIV‐1/HTLV‐IIIBa‐L) replication in fresh and cultured human peripheral monocyte/macrophages by azidothymidine and related 2′,3′‐dideoxynucleosides. J. Exp. Med. 168:1111‐1125.
   Sonza, S., Maerz, A., Deacon, N., Meanger, J., Mills, J., and Crowe, S. 1996. Human immunodeficiency virus type 1 replication is blocked prior to reverse transcription and integration in freshly isolated peripheral blood monocytes. J. Virol. 70:3863‐3869.
   Stent, G. and Crowe, S. 1997. Effects of monocyte purification and culture on integrin expression. APMIS 105:663‐670.
Key References
   Biggs et al., 1995. See above.
  A detailed description of HIV infection and maintenance of monocyte‐derived macrophages cultured adhered to plastic.
   Crowe et al., 1987. See above.
  A detailed description of HIV infection and maintenance of monocyte‐derived macrophages cultured in suspension.
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