Measurement of Interferon‐Mediated Antiviral Activity of Macrophages

Lydia A. Falk1

1 null, Monrovia, Maryland
Publication Name:  Current Protocols in Immunology
Unit Number:  Unit 14.9
DOI:  10.1002/0471142735.im1409s13
Online Posting Date:  May, 2001
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Macrophages have been shown to produce interferons (IFNs) that exert an antiviral effect‐‐resistance to the cytopathic effect (CPE) of viral infection‐‐on sensitive cell types such as fibroblasts. In some cases, IFN levels in macrophage supernatants may be below the limits of detection but still may be sufficient to confer antiviral activity on macrophages. This unit provides an assay for the qualitative demonstration of the presence or absence of endogenous IFN production in murine macrophages by measuring the resistance of macrophages to the CPE of infection with vesicular stomatitis virus (VSV).

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Table of Contents

  • Reagents and Solutions
  • Commentary
  • Figures
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Basic Protocol 1:

  • Murine macrophages from peritoneal exudate, bone marrow, or other sources (unit 14.1)
  • recipeSupplemented RPMI‐10 (see recipe)
  • Vesicular stomatitis virus stock (VSV, Indiana strain; unit 6.9), stored at −70°C
  • PBS ( appendix 2A)
  • 5% (v/v) formaldehyde
  • Crystal violet stain: 0.05% (w/v) crystal violet in 20% ethanol
  • 100% methanol
  • 96‐well flat‐bottom microtiter plate (Falcon)
  • Aspirator with vacuum trap containing bleach
  • Microtiter plate reader (Bio‐Tek or equivalent)
  • Additional reagents and equipment for counting cells ( appendix 3A)
NOTE: All culture incubations are performed in a humidified 37°C, 5% CO 2 incubator unless otherwise specified.
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Literature Cited

Literature Cited
   Falk, L.A. and Vogel, S.N. 1990. Differential production of IFN‐alpha/beta by CSF‐1‐and GM‐CSF‐derived macrophages. J. Leukocyte Biol. 48:43‐49.
   De Maeyer, E. and De Maeyer‐Guignard, J. 1988. Interferons and Other Regulatory Cytokines. John Wiley and Sons, New York.
   Gresser, I. 1984. Role of interferon in resistance to viral infection in vivo. In Interferon, Vol. 2 (J. Vilchek and E. De Maeyer, eds.) pp. 221‐247. Elsevier Science Publishing, New York.
   Hayes, M.P. and Zoon, K.C. 1994. Production and action of interferon: New insights into molecular mechanism of gene regulation and expression. In Progress in Drug Research. Vol. 43 (E. Jucker, ed.) pp. 240‐270. Birkhauser‐Verlag, Basel.
   Moore, R.N., Larson, H.S., Horohov, D.W., and Rouse, B.T. 1984. Endogenous regulation of macrophage proliferative expansion by colony‐stimulating factor–induced interferon. Science 223:178‐180.
   Vogel, S.N. and Fertsch, D. 1984. Endogenous interferon production by endotoxin‐responsive macrophages provides an autostimulatory differentiation signal. Infect. Immun. 45:417‐423.
   Vogel, S.N. and Fertsch, D. 1987. Macrophages from endotoxin‐hyporesponsive (Lpsd) C3H/HeJ mice are permissive for vesicular stomatitis virus because of reduced levels of endogenous interferon: Possible mechanism for natural resistance to infection. J. Virol. 61:812‐818.
   Vogel, S.N., Havell, E.A., and Spitalny, G.L. 1986. Monoclonal antibody–mediated inhibition of interferon‐γ‐induced macrophage antiviral resistance and surface antigen expression. J. Immunol. 136:2917‐2923.
   Warren, M.K. and Vogel, S.N. 1985. Bone marrow–derived macrophages: Development and regulation of differentiation markers by colony‐stimulating factor and interferons. J. Immunol. 134:982‐989.
Key Reference
   Vogel, S.N. and Fertsch, D. 1987. See above.
  Describes determination and verification of endogenous interferon production by macrophages.
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