UNIT 14.9Measurement of Interferon-MediatedAntiviral Activity of Macrophages

BASICPROTOCOL

Macrophages have been shown to produce interferons (IFNs) that exert an antiviraleffect—resistance to the cytopathic effect (CPE) of viral infection—on sensitive cell typessuch as fibroblasts (see UNIT 6.9). IFNs may also function as autocrine cytokines. However,measuring IFN-α/β or IFN-γ in macrophage supernatants using the protocols in UNIT 6.9

is not necessarily a reliable indicator of endogenous IFN production. In some cases, IFNlevels in macrophage supernatants may be below the limits of detection but still may besufficient to confer antiviral activity on macrophages.

This protocol provides a qualitative demonstration of the presence or absence of endo-genous IFN production in murine macrophages by measuring the resistance of macro-phages to the CPE of infection with vesicular stomatitis virus (VSV). For humanmacrophages, the choice of virus will be different (see UNIT 6.9). Macrophages are placedin 96-well tissue culture plates and allowed to adhere. They are then challenged with aviral suspension. After ∼24 hr, virus is removed and the monolayer is stained with crystalviolet, which detects cell death (CPE) as a decrease in staining intensity.

CAUTION: Vesicular stomatitis virus (VSV) is a potentially dangerous pathogen. AllVSV-infected materials (e.g., pipet tips and dilution tubes) should be placed in biohazardbags and autoclaved. Prior to formalin fixation, supernatants containing virus should beaspirated directly into flasks containing bleach.

NOTE: All reagents must be endotoxin-free and sterile. Use of aseptic technique duringculture is necessary.

Materials

Murine macrophages from peritoneal exudate, bone marrow, or other sources(UNIT 14.1)

Supplemented RPMI-10 (see recipe)Vesicular stomatitis virus stock (VSV, Indiana strain; UNIT 6.9), stored at −70°CPBS (APPENDIX 2)5% (v/v) formaldehydeCrystal violet stain: 0.05% (w/v) crystal violet in 20% ethanol100% methanol

96-well flat-bottom microtiter plate (Falcon)Aspirator with vacuum trap containing bleachMicrotiter 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% CO2 incubatorunless otherwise specified.

1. Suspend macrophages in supplemented RPMI-10 to a final concentration of 1 × 106

cells/ml.

Any source of resident or inflammatory murine macrophages can be used for these studies.Most of the common mouse strains have been used, although differences have been noted(see Vogel and Fertsch, 1987).

UNIT 14.1 describes protocols for isolating peritoneal macrophages and bone marrow–de-rived macrophages from normal mice and mice treated with inflammatory reagents.

In vivo injection of mice or in vitro treatment of macrophages with poly I:C orlipopolysaccharide (LPS) induces interferon production. Viral and bacterial infection

Copyright © 1995 by John Wiley & Sons, Inc. Supplement 13 CPI

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also increases interferon production. This procedure works well with freshly isolated orcultured cells.

2. Add 200 µl macrophage suspension to each well of a 96-well microtiter plate.Incubate 6 hr at 37°C to allow macrophages to adhere.

Set up duplicate wells for each experimental group and include wells for uninfected controlsfor each treatment. Also include one blank row with no cells for blanking the microtiterplate reader.

3. Dilute VSV stock in RPMI-10 such that 100 µl of viral suspension has a multiplicityof infection (MOI; ratio of virus to cells) of 0.1 to 1.0.

If the lower MOI does not result in efficient lysis, the higher MOI should be used.

VSV stock should be stored at −70°C and thawed just prior to dilution. For furtherinformation regarding preparation of viral stocks see UNIT 6.9.

To perform this assay with human macrophages, use EMC virus (UNIT 6.9).

4. Gently aspirate medium from adherent macrophage monolayer. Add 100 µl VSVsuspension to each experimental well of a microtiter plate. Add 100 µl supplementedRPMI-10 medium to control wells. Incubate 24 hr.

5. Aspirate supernatants and add 200 µl PBS to each well.

6. Aspirate PBS and add 200 µl of 5% formaldehyde to each well. Incubate 10 min atroom temperature.

7. Aspirate formaldehyde and add 50 µl crystal violet stain to each well. Incubate 10min at room temperature.

Crystal violet will be taken up by macrophages that were viable at the time of fixation.

8. Aspirate stain. Wash plate: fill wells with tap water from a plastic squirt bottle or thetap, then discard water by flicking plate over sink. Repeat wash five times.

Washing is performed after fixation, so cell loss due to detachment should not occur.

9. Add 100 µl methanol to each well. Gently agitate plate to elute dye. Read absorbanceat 595 nm using a microtiter plate reader.

The A595 of eluted crystal violet dye can be used to determine semiquantitatively the degreeof CPE observed in each well. Typical results of an antiviral assay are shown in Figure14.9.1.

Staining will range from intense (e.g., for uninfected control cells or samples containinghigh levels of IFNs) to very light (e.g., for cells that show complete CPE).

REAGENTS AND SOLUTIONS

Use deionized, distilled water in all recipes and protocol steps. For common stock solutions, seeAPPENDIX 2; for suppliers, see APPENDIX 5.

Supplemented RPMI-10RPMI 1640 containing:10% (v/v) FBS (heat-inactivated 1 hr at 56°C)2 mM L-glutamine30 mM HEPES (N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid)0.4% (w/v) sodium bicarbonate100 U/ml penicillin100 µg/ml streptomycinStore up to 4 weeks at 4°C

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Measurement ofInterferon-

MediatedAntiviral Activity

of Macrophages

COMMENTARY

Background InformationInterferons (IFNs) are probably best recog-

nized for their capacity to mediate an antiviralstate in IFN-sensitive cell types (Gresser,1984). This assay can be used to determinewhether cytokines or drug treatments in vitroor in vivo induce IFN production and increasethe antiviral activity in VSV-sensitive cells suchas macrophages. For IFN-sensitive fibroblasts,cytopathic effect (CPE) can be determined mi-croscopically (see UNIT 6.9). This is difficult todo for macrophages because no true plaques areobserved. Although infected macrophages mayappear more grainy, this is difficult to visualize.Therefore, examining monolayer staining is amore reliable measure of resistance to VSVinfection.

Previous studies have shown a correlationbetween the ability of macrophages to phago-cytose via FcR and their susceptibility to infec-tion with VSV (Vogel and Fertsch, 1987). Bothof these functions have been shown to be relatedto the production of endogenous IFN by macro-phages (Moore et al., 1984; Vogel and Fertsch,1984; Warren and Vogel, 1985). IFN increasesthe number of FcR (and thereby phagocytosis)

and exerts its antiviral effect in part throughactivation of RNases, which degrade viralRNA (De Maeyer and De Maeyer-Guignard,1988; Hayes and Zoon, 1994). The mecha-nism of VSV infection, however, is inde-pendent of FcR and occurs through endocy-tosis via coated pits.

It has been shown that the amount of endo-genous IFN detectable in macrophage super-natants can fall below the sensitivity of the IFNbioassay described in UNIT 6.9. Other directmethods of measuring IFNs, such as ELISA(UNIT 6.8), depend on the availability of species-specific antibodies against IFN-α/β or IFN-γ.Thus, determination of IFN production mayrequire the use of indirect measures (Vogel etal., 1986; Falk and Vogel, 1990).

The resistance of macrophages to VSV-in-duced CPE has been shown to differ amongstrains of mice and among subpopulations ofmacrophages (Vogel and Fertsch, 1987; Falkand Vogel, 1990). Demonstration of macro-phage resistance to viral-induced CPE can beused to compare these differences in IFN pro-duction. The assay can also be used to deter-mine whether certain drugs or cytokines mod-

infecteduninfected

CSF-1 (MOI = 0.1)

CSF-1 (MOI = 1.0)

rGM-CSF (MOI = 0.1)

rGM-CSF (MOI = 1.0)

Figure 14.9.1 Sensitivity of bone marrow–derived macrophages to infection with vesicular sto-matitis virus. C3H/HeJ bone marrow–derived macrophages were cultured for 7 days in liquid culturein the presence of CSF-1 or GM-CSF as described in UNIT 14.1. VSV suspensions with an MOI of0.1 or 1.0 were added to wells as indicated. After 24 hr, monolayers were fixed, stained, andexamined for virus-induced CPE.

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ulate IFN production and antiviral activity inmacrophages. This assay works equally wellwith fresh cells (such as peritoneal macro-phages) and cultured cells (such as bone mar-row–derived macrophages).

In addition to comparing different popula-tions of macrophages, this assay has also beenused to examine the ability of mouse strains toproduce IFN in vivo. Endogenous IFN produc-tion can be triggered in vivo by viral infectionor by treating mice with inflammatory agentssuch as lipopolysaccharide or polyinos-inic:polycytidylic acid (see UNIT 14.2 for primingand activation protocols). Mice should bepathogen-free and housed in clean-air units.

Peritoneal macrophages isolated fromC3H/HeJ mice are more susceptible to VSVinfection than are macrophages from C3H/HeNmice (Vogel and Fertsch, 1987). To determinewhich type of IFN is responsible for the antivi-ral response, IFN-neutralizing antibodies canbe included in the culture prior to infection withVSV. UNIT 6.9 provides details for using neutral-izing IFN antibodies, as well as a basic protocolfor measuring IFN levels in macrophage super-natants after 24 to 48 hr of culture with certainagents. IFN-γ is usually more potent than IFN-γ/β and exerts its effects at approximately 10-fold fewer units per milliliter. T cells are thepredominant IFN-γ producers; macrophagesand fibroblasts produce IFN-γ/β (Hayes andZoon, 1994).

Critical ParametersAn intact macrophage monolayer is re-

quired for this assay, so cultures should bemicroscopically inspected prior to viral infec-tion. This will make determining CPE easierand will ensure that differences in staining arenot due to gaps in the monolayer prior to infec-tion. Viral infection does not alter adhesion ofmacrophages, and cells that come off duringwashes are dying or dead cells. Increasing theviral MOI from 0.1 to 1.0 makes it easier tocompare slightly resistant cells with cells thatare highly refractory to infection.

Because all macrophage populations are notstained with equal intensity by crystal violet,mock-infected wells (treated with mediumonly) should be included as a control. Thesecells should show maximal crystal violet stain-ing. Cultures can also be treated with an IFN-neutralizing antibody prior to viral infection(see UNIT 6.9). These cells should show maximalCPE and minimal crystal violet staining. Asimple negative control would be a sample wellcontaining medium plus virus with no macro-

phages. This sample should show no crystalviolet staining.

All reagents used during isolation and cultureof the macrophages must be endotoxin-free.

Anticipated ResultsIf macrophages are producing >1 U/ml IFN,

they should be resistant to VSV-induced CPEat an MOI of 0.1 and should stain as a homo-geneous blue monolayer. Macrophages thatproduce less than this amount of IFN will besensitive to VSV-induced CPE and will sloughoff during the washing step, resulting in littleretention of crystal violet stain. Because theassay measures small amounts of endogenousIFN, it is highly sensitive and reproducible.

A typical staining pattern demonstrating viralsensitivity versus resistance is shown in Figure14.9.1. In this case, bone marrow–derivedmacrophages were cultured as described in UNIT

14.1 in either CSF-1 or GM-CSF and then as-sayed for endogenous IFN production. CSF-1-derived macrophages show little change inmonolayer integrity and staining intensitywhen infected with VSV at an MOI of 0.1. Incontrast, GM-CSF-derived macrophages ex-hibit significant loss of monolayer integrity anddecease in staining intensity following infec-tion. The lesser staining in uninfected GM-CSF-derived macrophage monolayers under-scores the fact that not all macrophages stainequally well with crystal violet dye. However,when the viral MOI is increased to 1.0, thedifference in antiviral activity of CSF-1-de-rived macrophages versus GM-CSF-derivedmacrophages is more obvious. Untreated cellsrange from 0.14 to 0.3 OD units for GM-CSF-and CSF-1-derived macrophages, respectively.A CPE of 50% corresponds to half of the ODunit values for untreated groups; 100% CPEwill be equal to background values (i.e., nocells).

Time ConsiderationsPlating of macrophages will take ∼1⁄2 hr, then

cells must incubate 4 to 6 hr for adherence. Theaddition of VSV will take another 1⁄2 hr. After a24-hr incubation, fixation, staining, and readingthe plates should take no longer than 1 hr.

Literature CitedFalk, L.A. and Vogel, S.N. 1990. Differential pro-

duction 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.

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Measurement ofInterferon-

MediatedAntiviral Activity

of Macrophages

Gresser, I. 1984. Role of interferon in resistance toviral 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 andaction of interferon: New insights into molecularmechanism of gene regulation and expression. InProgress in Drug Research. Vol. 43 (E. Jucker,ed.) pp. 240-270. Birkhauser-Verlag, Basel.

Moore, R.N., Larson, H.S., Horohov, D.W., andRouse, B.T. 1984. Endogenous regulation ofmacrophage proliferative expansion by colony-stimulating factor–induced interferon. Science223:178-180.

Vogel, S.N. and Fertsch, D. 1984. Endogenous in-terferon production by endotoxin-responsivemacrophages provides an autostimulatory differ-entiation signal. Infect. Immun. 45:417-423.

Vogel, S.N. and Fertsch, D. 1987. Macrophagesfrom endotoxin-hyporesponsive (Lpsd)C3H/HeJ mice are permissive for vesicular sto-matitis virus because of reduced levels of endo-genous interferon: Possible mechanism for natu-ral resistance to infection. J. Virol. 61:812-818.

Vogel, S.N., Havell, E.A., and Spitalny, G.L. 1986.Monoclonal antibody–mediated inhibition of in-terferon-γ-induced macrophage antiviral resis-tance and surface antigen expression. J. Immu-nol. 136:2917-2923.

Warren, M.K. and Vogel, S.N. 1985. Bone marrow–derived macrophages: Development and regula-tion of differentiation markers by colony-stimu-lating factor and interferons. J. Immunol.134:982-989.

Key ReferenceVogel, S.N. and Fertsch, D. 1987. See above.

Describes determination and verification of endo-genous interferon production by macrophages.

Contributed by Lydia A. FalkMonrovia, Maryland

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