Current Protocols in Immunology || Measuring Opsonic Phagocytosis via Fcγ Receptors and Complement...

UNIT 14.27Measuring Opsonic Phagocytosis via FcγγReceptors and Complement Receptors onMacrophages

David M. Mosser1 and Xia Zhang1

1University of Maryland and The Maryland Pathogen Research Institute, College Park,Maryland

ABSTRACT

Phagocytosis is a cellular process that plays crucial roles in the removal of dead or dyingcells, tissue remodeling, and host defense against invading pathogens. Most eukaryoticcells are decorated with glycoproteins containing terminal sialic acids, whose negativecharges tend to repel cells, making so-called “nonspecific” phagocytosis a relativelyinefficient process. Professional phagocytes are so designated because they express twomajor classes of receptors on their surfaces that are primarily involved in phagocytosis.Paradoxically, these receptors do not recognize microbes directly, but rather endogenousproteins that become tethered to microbes and target them for destruction. These are theFcγ receptors that bind to the Fc portion of IgG and the complement receptors (CRs),which bind primarily to cleavage products of the third component of complement, C3.This unit describes assays that are used to measure these two types of macrophagephagocytosis. Curr. Protoc. Immunol. 95:14.27.1-14.27.11. C© 2011 by John Wiley &Sons, Inc.

Keywords: phagocytosis � opson � complement receptors � macrophages

INTRODUCTION

Immunoglobulin G antibodies recognize their cognate antigens via their antigen-bindingdomains, leaving their Fc tails available to bind to FcγR on phagocytic cells. FcγRs fallinto two general classes: those that mediate effector functions and those that transport im-munoglobulin across epithelial barriers. This chapter will focus on the former receptors.There are four types of Fcγ receptors that activate effector functions, and a single receptorthat inhibits these functions. FcγRs that fall within the activation class include FcγRI(CD64), FcγRIIA (CD32a), FcγRIII (CD16), and FcγRIV (Nimmerjahn and Ravetch,2006). There is no mouse counterpart of FcγRIIA and no known human counterpart toFcγRIV. The activating FcγRs recruit an ITAM-containing γ chain to transduce acti-vating signals through the protein kinase Syk. FcγIIB (CD32b) is an inhibitory receptorthat does not transduce activating signals. This receptor contains an ITIM domain inits cytoplasmic tail that recruits phosphatases to inhibit FcγR activation. Macrophagesutilize the activating receptors to recognize antigens and pathogens opsonized with IgG.IgG-mediated uptake generally results in enhanced killing of internalized pathogens andmore efficient antigen presentation (Swanson and Hoppe, 2004). The expression of all ofthe FcγR on macrophages appears to be independently regulated, and receptor expres-sion can change dramatically with macrophage activation. Thus, the expression levels ofindividual FcγRs can sometimes be used as an indirect indicator of cellular activation.FcγR are expressed primarily on monocytes/macrophages, neutrophils, and dendriticcells. Dendritic cell maturation results in a dramatic down-regulation of FcγR expres-sion. FcγR-mediated adhesion results in rapid and efficient particle internalization, evenin resting (nonstimulated) macrophages. Thus, most of the particles targeted to FcγR

Current Protocols in Immunology 14.27.1-14.27.11, November 2011Published online November 2011 in Wiley Online Library (wileyonlinelibrary.com).DOI: 10.1002/0471142735.im1427s95Copyright C© 2011 John Wiley & Sons, Inc.

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Supplement 95

Measurement ofOpsonic

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Supplement 95 Current Protocols in Immunology

will be routed to phagolysosomes. In addition to IgG, some acute-phase proteins in thepentraxin family have been shown to bind to FcγR and promote phagocytosis.

Complement activation can result in the covalent modification of the target surface, de-positing opsonic fragments of complement that can bind to complement receptors (CRs)on phagocytic cells. Two of the three pathways by which complement can be activated(alternative and lectin) are considered “innate” and can be initiated in nonimmune indi-viduals. The third classical pathway of complement activation requires antibody. Duringcomplement activation, the third component of complement, C3, is cleaved to C3b, whichcan bind to cell surfaces. C3 is a metastable thioester that binds covalently to exposedhydroxyl and amino groups. C3 is the most abundant of the complement proteins, andthe C3 convertases represent one of the principal amplification steps in the pathway.Bound C3b is rapidly cleaved to an inactivated form that is designated iC3b. Professionalphagocytes have receptors for C3b (CR1) and iC3b (CR3, Mac-1, CD18/CD11b). TheCR1 on phagocytes can also bind to C4b. A recently described complement receptor thathas immunoglobulin-like folds (CRIg) has been reported to bind to bound C3b and iC3b(Helmy et al., 2006; He et al., 2008). This receptor has been identified on Kupffer cells.There are several other complement activation fragments that bind to specific receptorson leukocytes, but their role in mediating phagocytosis is relatively minor compared tothe receptors mentioned above, and therefore they will not be addressed in this unit.

Unlike FcγR-mediated phagocytosis, complement-mediated binding generally does notlead to efficient particle internalization by resting macrophages (Aderem and Underhill,1999). Complement-coated erythrocytes (E-IgMC) generally remain attached to the sur-face but not taken up. Treatment of macrophages with PMA or LPS, or priming withIFN-γ, can enhance complement-mediated phagocytosis. The CR Ig on liver Kupffercells, in contrast, mediates the phagocytosis of complement-opsonized particles in non-immune hosts.

This unit describes assays to quantify the binding and phagocytosis of particles op-sonized with either IgG or complement. In these assays, sheep red blood cells (SRBC)are used as the indicator particle because they are easy to visualize and count and be-cause the hemoglobin released from lysed RBCs can provide a convenient colorimetricway to quantitate SRBC binding and phagocytosis. The Basic Protocol describes lightmicroscopic and colorimetric methods to measure macrophage binding and phagocy-tosis mediated either by FcγR or CR. SRBC opsonized with IgG (IgG-SRBC) are thetargets for FcγR-mediated activity, whereas SRBC opsonized by IgM and complement(E-IgMC) are used for CR specificity. An Alternate Protocol describes a method similarto the Basic Protocol, except that inhibitors to prevent the phagocytosis of the target cellsby macrophages are added. A Support Protocol describes how to opsonize SRBC with ei-ther anti-SRBC IgG (IgG-SRBC) for the measurement of FcγR-mediated phagocytosis,or with IgM and complement (E-IgMC) to measure binding mediated by complementreceptors.

NOTE: All solutions and equipment coming into contact with living cells must be sterileand aseptic technique should be used accordingly

NOTE: All culture incubations are performed in a humidified 37◦C, 5% CO2 incubatorunless otherwise specified.

NOTE: For these assays, it is important to use fresh SRBC that should not exhibitmeasurable binding to macrophages when not opsonized. Old SRBC become “sticky”and will bind to macrophages in the absence of opsonization. Furthermore, the loss ofsialic acid on aged RBC can lead to the activation of the alternative complement pathway.Therefore, for all these assays, it is important to incorporate negative controls that should

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not bind to specific receptors in order to establish receptor-specificity of the bindingevent.

BASICPROTOCOL

MEASUREMENT OF FcγγR- OR COMPLEMENT RECEPTOR–MEDIATEDPHAGOCYTOSIS

In this Basic Protocol, adherent macrophages are incubated with IgG-opsonized SRBCand phagocytosis is measured. Following the incubation period, monolayers are typicallywashed to remove unbound erythrocytes, and then the amount of cell-associated RBC arequantitated. This can be done by visually counting the number of cell-associated SRBCon Giemsa-stained monolayers, or by spectrophotometrically quantitating the amount ofSRBC-derived hemoglobin associated with the monolayer.

Materials

Mice of interest (also see UNIT 14.1)Culture medium for macrophages (UNIT 14.1)Macrophage activating agent of choice (UNIT 14.2)Opsonized SRBC suspension (Support Protocol)ACK lysis solution (see recipe)0.1% (w/v) sodium dodecyl sulfate (SDS)2,7-diaminofluorene (DAF) working solution (see recipe)Phosphate-buffered saline (PBS; see recipe), ice cold2.5% (v/v) glutaraldehyde in PBS (see recipe for PBS), freshly prepared, ice coldGiemsa stain (Sigma; GS-500)

96-well flat-bottom tissue culture plates (e.g., Falcon or Costar)12-channel pipettor (to deliver 50- and 100-μl aliquots) and disposable reservoirs

(PGC Scientific)Inverted microscope96-well microtiter plates (e.g., Falcon or Costar)Spectrophotometer with microtiter plate reader

Additional reagents and equipment for preparation of macrophages from bonemarrow or peritoneal exudate (UNIT 14.1), activation of macrophages (UNIT 14.2),and preparation of opsonized SRBC (Support Protocol)

Prepare macrophages1a. For bone marrow–derived macrophages: Prepare bone marrow–derived macrophages

(see UNIT 14.1) and resuspend in culture medium to a concentration of ∼1 × 106

cells/ml. Seed approximately 100 μl of macrophage cell suspension in each well(∼1 × 105 cells/well) of a 96-well flat-bottom tissue culture plate (three replicatesper treatment should be prepared). Allow cells to adhere for 1 hr, wash to removenonadherent cells, and then incubate overnight.

1b. For peritoneal exudate cells: Add 100 μl of a suspension of approximately 2 × 106

cells/ml to the wells. Wash wells after 1 hr, and incubate overnight.

For macrophage-like cell lines, an amount of cells that yields a monolayer that is 50% to75% confluent after overnight incubation should be used. This will vary depending on thecell line used, but a reasonable starting concentration is approximately 5 × 105 cells/mlin 100 μl/well volume.

2. Activate macrophages with an appropriate reagent (see UNIT 14.2) that can affectphagocytosis.

Several cytokines are known to influence Fcγ R expression, including interferon-γ(IFN-γ ) and M-CSF. Cells treated with medium alone should be used as controls. Un-opsonized SRBC should be used as negative controls, and they should be included in all


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experiments. Whenever working with macrophages, it is important to avoid anycontaminating lipopolysaccharide (LPS), which is frequently found on conventionallaboratory glassware.

3. Prepare IgG-opsonized SRBC at ∼1 × 107 cells/ml for FcγR-mediated phagocytosis(E-IgG) or SRBC opsonized with IgM and complement (E-IgM-C) for complementreceptor-mediated binding (see Support Protocol).

Incubate opsonized SRBC with macrophages4. Aspirate medium from the 96-well plate containing the activated macrophages (see

step 2).

One should be careful to aspirate medium from macrophages with minimal vacuum toprevent removal of cells from the plate. Tilt the plate at an angle of ∼30◦ to 45◦ andlower the aspirator into the medium until it almost touches the bottom. The plate shouldbe microscopically examined after washing to verify that there was no loss of cells duringwashing. Cells may be lost from a small spot if the aspirator inadvertently touches theplate. For 96-well plates, a 200-μl pipet tip or a tuberculin syringe with a 25-G needleattached to a vacuum hose are convenient ways to aspirate.

5. Add 100 μl opsonized SRBC to each well containing macrophages and incubate forthe appropriate period of time.

The ratio of SRBC to macrophages should be empirically determined depending on theassay. A ratio of 10:1 is typical and will be used here. This generally results in two to threeerythrocytes bound per macrophage after a 1-hr incubation. A preliminary time coursecan determine the amount of time during which the reaction remains linear. Choose anincubation time within the linear range that allows significant phagocytosis. The linearrange may extend from 30 min to as much as 3 hr. E-IgMC bind to CR3 poorly at 4◦C.At 37◦C, resting cells will bind E-IgMC but will not initiate CR3-mediated phagocytosis.To facilitate CR3-mediated phagocytosis, PMA can be added to each well at a finalconcentration of 100 ng/ml.

Colorimetrically quantitate the phagocytosis of opsonized SRBC by macrophages6. After incubation, aspirate the medium from the macrophages and wash the monolay-

ers three times, each time with 100 μl culture medium.

7. To determine the number of SRBC that were internalized by the monolayer, aspirateculture medium and add 100 μl ACK lysis solution to each well. Gently swirl theplate in a circular motion on the laboratory bench and examine the wells under aninverted microscope for evidence of lysis.

ACK lysis solution will disrupt SRBC that have not been phagocytosed and those that arebound to the exterior of the cells. The ACK lysis solution should be allowed to remainin the wells just long enough for the SRBC to lyse: the time required for lysis generallyranges from 15 to 30 sec, and the solution should not be left on more than 1 min, as thiswill begin to lyse the macrophages.

To determine total number of monolayer-associated SRBC (bound and internalized) omitthis ACK lysis step.

8. Aspirate the ACK lysis solution and rinse each well with 100 μl culture medium.

9. Add 100 μl of 0.1% SDS to all wells and incubate 5 to 10 min at room temperatureto allow macrophages to be lysed.

10. Transfer 50 μl of macrophage lysate from each well of the original culture plate tothe corresponding well of a 96-well microtiter plate. Add 50 μl of DAF workingsolution to each well that contains macrophage lysate. Mix well and incubate for10 min at room temperature.

Negative controls should include macrophages that have incubated with unopsonizedSRBC.

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11. To set up a standard curve of OD as a function of SRBC, harvest and resuspendopsonized SRBC in sterile PBS at 1 × 108 cells/ml and dilute this SRBC suspension10-fold with 0.1% SDS to 1 × 107 cells/ml. Prepare a 2-fold serial dilution in 0.1%SDS, including 0.1% SDS blank. Transfer 50 μl of each dilution to a well of a 96-well microtiter plate. Add 50 μl of DAF working solution to each well. Mix well andincubate for 10 min at room temperature.

12. Measure the absorbance at 620 nm in a 96-well microtiter plate reader.

The absorbance is stable for 10 to 15 min.

In the presence of hydrogen peroxide, hemoglobin catalyzes the formation of fluoreneblue from DAF, which has a broad spectrum range between 500 and 690 nm. The linearrelationship between the optical density at 620 nm and the concentration of hemoglobinhas been demonstrated.

Visual quantitation of the phagocytosis of opsonized SRBC by macrophages and dataanalysis13. Wash the monolayers three times with warm medium after their incubation with

opsonized SRBC.

14. Fix monolayers by adding 2.5% fresh cold glutaraldehyde in PBS for 30 min in thecold.

15. Wash monolayers with PBS and add 100 μl Giemsa stain for 10 min.

16. Wash monolayer three times with PBS, and leave PBS in the wells.

17. Visualize monolayer on inverted microscope.

18. Perform data analysis.

The colorimetric results can reveal the total number of erythrocytes associated withthe monolayer (bound and internalized), and the number of SRBC internalized by themonolayer (after ACK lysis). For IgG-SRBC, these numbers should be fairly similar,provided the input number of SRBC is not too high. For complement-mediated SRBC,there should be much more SRBC outside the cell than inside. If this is not the case,then one should consider the possibility of LPS contamination. The number of SRBC isdetermined from the standard curve. Controls consisting of unopsonized SRBC should beused and subtracted from all values. If this control is high, then fresh new SRBC should beordered. Calculate average of triplicate determinations from individual experiments anduse an appropriate statistical test for analysis of data. The manual quantitation using lightmicroscopy can determine the number of SRBC phagocytosed per 100 phagocytes and thepercentage of macrophages with one or more SRBC associated with them. Ideal variabilityamong replicates should be below 10%. Differences between different preparations ofSRBC contribute the largest source of variability between individual experiments.

ALTERNATEPROTOCOL

MEASUREMENT OF RECEPTOR-MEDIATED BINDING ACTIVITYFOLLOWING THE ADDITION OF PHAGOCYTOSIS INHIBITORS

This alternative procedure is similar to the Basic Protocol for phagocytosis assay (above),except that inhibitors to prevent the phagocytosis of the target cells by macrophages areadded. Thus, opsonized SRBC bind to macrophages but cannot be ingested.

The phagocytosis assay employing an ACK lysis step (see Basic Protocol, step 7) mea-sures the number of erythrocytes that have been ingested, whereas the assay omittingACK lysis measures total bound and ingested RBC, as described in the annotation tothat step of the Basic Protocol. The assay described here measures total SRBC boundto the receptor but not ingested. The assay is performed exactly as described above,except that 30 min before the addition of erythrocytes, macrophages are treated with


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either cytochalasin or latrunculin to prevent phagocytosis. After the incubation period,monolayers are washed four times to remove unbound erythrocytes. Care should betaken to not wash the monolayer too vigorously, causing the bound RBC to lyse orrelease.

Additional Materials (also see Basic Protocol)

30 μM cytochalasin D (Calbiochem, cat no. 250255)10 μM latrunculin A (BIOMOL-Enzo Life Sciences, http://www.biomol.com, cat.

no. T119)24-well flat-bottom tissue culture plates (e.g., Costar or Falcon)

Prepare macrophages1. Prepare macrophages (see UNIT 14.1) and resuspend in the culture medium to a con-

centration of 1 × 106 cells/ml. Add 100 μl of suspension to each well of a 24-wellflat-bottom tissue culture plate (1 × 105 cells/well). Prepare at least duplicate wellsfor each treatment and control. Culture cells in a humidified 37◦C, 5% CO2 incubatorovernight.

2. Activate macrophages (see UNIT 14.2) to modify receptor expression if desired.

3. Prepare IgG-SRBC (see Support Protocol).

4. Add 0.1 ml of 10 μM latrunculin A or 30 μM cytochalasin D per 9.9 ml opsonizedSRBC (at ∼1-2 × 107 cells/ml).

Latrunculin A, at a final concentration of 100 nM, completely prevents the bound SRBCfrom becoming ingested via inhibition of actin polymerization and microfilament-mediatedprocesses (Oliveira et al., 1996).

Incubate opsonized SRBC with macrophages5. Aspirate medium from macrophages in the wells of the 24-well plate. Add 100 μl of

opsonized SRBC suspension to each well (∼1-2 × 106 cells/well). Incubate plate ina humidified 37◦C, 5% CO2 incubator for 30 min to 1 hr.

Measure opsonized SRBC bound to macrophages6. Aspirate SRBC suspension from macrophages carefully. With a 1-ml pipettor, gently

wash each well with 100 μl culture medium. Repeat wash procedure three moretimes.

Opsonized SRBC cells that do not bind the macrophage are washed off without beinglysed. This step demands a more thorough washing than the basic phagocytosis assay(Basic Protocol), but extra care should be taken to avoid aspirating too vigorously. Direct100 μl of medium against the side of the well so as not to dislodge the cells from theplate.

7. Add 100 μl of 0.1% SDS to each well and gently shake the plate for 10 min at roomtemperature to completely disrupt the macrophages.

8. Transfer 50 μl of macrophage lysate of each well to a 96-well microtiter plate. Add50 μl of DAF working solution to each well that contains 50 μl of macrophage lysate.Mix well and incubate for 10 min at room temperature.

9. Set up a standard curve of OD as a function of SRBC (see Basic Protocol, step 11).

10. Measure the absorbance at 620 nm in a 96-well microtiter plate reader.

The absorbance is stable for 10 to 15 min.

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Analyze data11. The results can be defined as the number of SRBC bound to the monolayer, provided

that the number of bound SRBC is determined from the standard curve. Calculateaverage replicate values from individual experiments and use an appropriate statisticaltest for analysis of data (see Basic Protocol).

SUPPORTPROTOCOL

PREPARATION OF IgG-SRBC AND E-IgMC

In this protocol, SRBC are opsonized either with anti-SRBC IgG (IgG-SRBC) for themeasurement of FcγR-mediated phagocytosis, or with IgM and complement (E-IgMC)to measure binding mediated by complement receptors. Complement is “fixed” via theclassical pathway using IgM antibody to SRBC. It is important that the antibody used toopsonize SRBC be free of IgG, because this will mediate binding to the Fcγ receptors.There should be little to no binding of IgM-opsonized SRBC to macrophages unlesscomplement is added.

Materials

Sheep red blood cells, washed and preserved (MP Biomedicals, cat no. 55876)Phosphate-buffered saline (PBS; see recipe)GVB+ buffer (see recipe)Anti-SRBC IgG (one of the following):

Rabbit Polyclonal IgG against SRBC MP Biomedicals, cat no. 55806,http://www.mpbio.com)

Mouse monoclonal IgG2a against SRBC (ATCC number TIB-111; hybridomaclone S.S-1)

Mouse monoclonal IgG2b against SRBC (ATCC number TIB-109; hybridomaclone N-S.8.1)

Anti-SRBC IgM:Rabbit IgM antibody to SRBC is obtained from Cedarlane Laboratories (cat no.

CL9000-M)Alternatively a hybridoma secreting IgM monoclonal antibody to SRBC is

obtained from the American Type Culture Collection (ATCC numberTIB-112, hybridoma clone S.S-3

Human complement component C5-deficient serum: Quidel Corporation, cat. no.A501; http://www.quidel.com, or mouse C5-deficient serum can be obtainedfrom C5-deficient mice such as AKR/J mouse (Jackson Laboratories, cat. no.000648)

50-ml conical plastic centrifuge tubes (e.g., Falcon)Refrigerated centrifuge (Eppendorf Centrifuge 5810R or equivalent)Mini-rotator (end-over-end; Glas-Col)

Additional reagents and equipment for counting cells using a hemacytometer(APPENDIX 3A)

Prepare and count SRBC1. In a 50-ml conical centrifuge tube, wash 5 ml of SRBC with 40 ml ice-cold PBS by

centrifuging 5 min at 500 × g, 4◦C. Aspirate PBS and repeat wash with an additional40 ml PBS.

2. Wash SRBC once with 40 mL ice-cold GVB+ buffer.

3. Resuspend SRBC in 20 mL GVB+ solution and transfer 100 μl of the SRBC suspen-sion for counting. Count SRBC in a hemacytometer and adjust to a concentration of2 × 108/ml.


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Alternatively, SRBC can be quantitated by measuring hemoglobin content. Resuspend100 μl SRBC in 2.9 ml of distilled water (1/30 dilution). Read the absorbance of thewater-lysed sample at OD540 versus that of water and calculate the SRBC concentration;the value of the lysed SRBC suspended at 1 ×109 cells/ml should be ∼0.350 OD540.

Preparation of IgG-SRBC4. Place 2 × 108 SRBC in a 50-ml conical tube and centrifuge 10 min at 500 × g, 4◦C.

Carefully aspirate the supernatant and resuspend the SRBC pellet to 1 ml in PBS.Add anti-SRBC IgG to the pellet at the appropriate titer (see below) and vortex.

This preparation should be enough to assay two 96-well plates of macrophages.

The optimal (subagglutinating) IgG concentration is empirically determined by addingdifferent amounts of IgG to a constant number (2 × 108) of SRBC in parallel microcen-trifuge tubes. If the agglutination titer is provided with a commercially supplied IgG, itis best to start at the agglutination titer and work downward using two-fold dilutions.The highest titer that yields minimal agglutination should be used. For convenience, theoptimal titer is recorded for future use and the antibody is aliquotted in small volumes forsubsequent experiments. The titer need only be determined once, provided that fresh SRBCare routinely used. The use of SRBC without opsonization should always be included as anegative control.

5. Gently mix SRBC with the anti-SRBC IgG mixture and incubate 30 min at roomtemperature with gentle rotation on a mini-rotator to resuspend the cells.

6. Add PBS to 25 ml final volume and centrifuge 10 min at 500 × g, room temperature.

7. Aspirate supernatant and wash cells one additional time with PBS using the techniquedescribed in step 6.

SRBC will not form a tight pellet. Be careful not to aspirate SRBC.

8. Resuspend the final I,G-SRBC pellet at a concentration of 1 × 107 SRBC /ml in PBSand add desired amount to monolayer .

Opsonized SRBC should be used within hours of completing this step.

Prepare E-IgM-C (optional)9. If measuring complement receptor–mediated phagocytosis in the Basic Protocol,

prepare E-IgM-C by placing 2 × 108 SRBC in a 50-ml conical plastic centrifugetube and centrifuging 10 min at 500 × g, 4◦C. Carefully aspirate the supernatant andresuspend in 1 ml GVB+ buffer.

10. Add anti-SRBC IgM to the SRBC and vortex. Incubate at 37◦C for 30 min withgentle agitation.

Predetermine the optimal concentration of IgM as described above for IgG.

11. Wash E-IgM in 25 ml PBS and resuspend pellet in 1 ml GVB+buffer. Add 50 μl C5Dserum (5% v/v final) and incubate at 37◦C for 30 min.

12. Resuspend the E-IgM-C to a concentration of 1 × 107 SRBC/ml and add the desiredamount to the monolayer.

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

ACK lysis solution

Dissolve 1.66 g of ammonium chloride (31 mM final), 0.2 g of potassium bicar-bonate (2 mM final), and 40 μl of 0.5 M EDTA (see APPENDIX 2A; 20 μl final) indistilled water. Adjust the volume to 1 liter with distilled water. Store up to 1 yearat room temperature.

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2,7-Diaminoflurene (DAF) stock and working solutions

DAF stock solution: Add 100 mg of DAF (Sigma-Aldrich, cat. no. D17106) to 10 mlof glacial acetic acid with vigorous vortexing at room temperature. The solutioncan be kept at 4◦C for up to 1 month in the dark.

DAF working solution: Add 1 ml of DAF stock solution and 0.1 ml of 30% hydrogenperoxide (Fisher Scientific, cat. no. H323-500) to 10 ml of 0.2 M Tris·Cl, pH 7.4(APPENDIX 2A) containing 6 M urea.

GVB+ buffer (gelatin/veronal buffer with Mg2+)

In a 1-liter graduated cylinder or volumetric flask, mix 200 ml of 5× veronal-buffered saline, pH 7.2 (see recipe), 5 ml of 200× magnesium chloride stock (seerecipe), and 2 g of gelatin powder. Heat on stirrer hot plate to dissolve gelatin. Adddistilled water to 1 liter. Check that the pH is 7.2 to 7.4 at 22◦C. Store ≤12 hr at4◦C.

If necessary, the pH may be adjusted with a small amount of 1 M HCl or 1 M NaOH. Ifstock solutions are prepared properly, little or no adjustment should be required. Largeadjustments to pH should be avoided as they may change the critical magnesium cationconcentration. This buffer may be prepared in larger quantities if it is filter sterilized toprevent bacterial contamination.

MgCl2 (magnesium chloride) stock, 200×Dissolve 2.03 g of MgCl2·6H2O in 100 ml distilled H2O. Store ≤1 year at roomtemperature.

Phosphate-buffered saline (PBS)

Dissolve 8 g of NaCl, 0.2 g of KCl, 1.44 g of Na2HPO4, and 0.24g of KH2PO4 in800 ml of distilled H2O. Adjust pH to 7.4. Adjust volume to 1 liter with additionaldistilled H2O. Sterilize by autoclaving.

Veronal-buffered saline (VBS), pH 7.2 to 7.4, 5×Dissolve 4.6 g barbituric acid in 800 ml boiling distilled water. In a separatecontainer, dissolve 2.0 g sodium barbital and 83.8 g sodium chloride in 1 literof distilled water. Mix the two solutions and add distilled water to 2 liters. Storefor ≤1 month at 4◦C. A 1/5 dilution of the stock solution with water (3.5 mMveronal buffer) should have a pH of 7.2 to 7.4. Kanamycin stock solution (1 ml of100 mg/ml kanamycin) can be added, but is not essential.

COMMENTARY

Background InformationThe macrophage FcγRs mediate phagocy-

tosis and antibody-dependent cellular cyto-toxicity (ADCC). Signals mediated by FcγRreceptors can also modulate macrophage cy-tokine production (Mosser and Edwards,2008). FcγR expression on macrophages canreflect the state of activation (Politis andVogel, 1990), and on dendritic cells FcγRcan promote DC maturation and facilitate anti-gen cross-presentation. The use of SRBC op-sonized with rabbit polyclonal IgG (describedabove) measures the total activity of all FcγRsubtypes. It is difficult to measure the activityof individual FcRs using assays such as this

because of the high avidity of IgGs within animmune complex. Similarly, the complement-dependent binding observed in the assaysdescribed above cannot be assigned to asingle complement receptor (van LookerenCampagne et al., 2007). Complement frag-ments C3b and C4b will bind to the CR1,whereas iC3b will bind to the CR3. However,the rapid cleavage of surface-bound C3b toiC3b means that the majority of complement-dependent binding to macrophages will occurvia the CR3.

Because of their uniform size and mor-phology, opsonized SRBC have been tradi-tionally counted via light microscopy. This


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technique allows an analysis of both the per-centage of macrophages in the monolayer withassociated SRBCs and the average numberof SRBC associated with each macrophage.This type of analysis enables one to discrim-inate between one monolayer in which 50%of the macrophages have bound one SRBCand another monolayer in which 25% havebound two SRBC. These two monolayerswould yield essentially the same results by thecolorimetric assay described above. Anotherconvenient way to visualize SRBC attach-ment to macrophages is to utilize fluores-cently labeled SRBC. There are now sev-eral amine-reactive fluorescent dyes that arerapidly taken up by cells and metabolizedto remain cell-associated (Greenberg andGrinstein, 2002). Macrophages are typicallycounterstained with propidium iodide and themonolayers visualized by fluorescence lightmicroscopy.

The colorimetric assay to measure SRBC-associated Hb takes advantage of the fact thathemoglobin possesses a pseudo-peroxidaseactivity. In the presence of peroxide, it actsas an enzyme to catalyze the conversionof 2,7-diaminofluorene into fluorene blue(Worthington et al., 1987). Fluorene blue hasa broad absorption spectrum from 500 nm to690 nm, and its absorption between 610 nmand 630 nm correlates in a linear fashion withthe hemoglobin concentration. Such proper-ties are applied to measure phagocytosis activ-ity and ADCC of macrophages (Montano andMorrison, 1999). An alternative way to quan-titate SRBC attachment is to measure theamount of radioactive chromium (51Cr) frompreloaded SRBC (Vogel et al., 1983). Thisassay is more sensitive, but it raises con-cerns because of the hazards associated withradioactivity.

The major difference between the bindingassay and phagocytosis assay are that in thebinding assay, a phagocytosis inhibitor is in-cluded to prevent receptor-mediated phagocy-tosis, and the hypotonic washing step withACK solution in the phagocytosis assay iseliminated in the binding assay so that the ex-tracellular membrane receptor–bound SRBCcan be measured. Latrunculin A, as a phago-cytosis inhibitor, is 10- to 100-fold strongerthan cytochalasin compounds (de Oliveiraand Mantovani, 1988; Oliveira et al., 1996).ATP-depleting agents such as iodoacetic acid(IAA) and 2,4-dinitrophenylhydrazine (2,4-DNAP) can also be used to inhibit phago-cytosis (Politis and Vogel, 1990). The proto-

cols described herein are not only for mousemacrophages but also for any adherent cellsthat express the receptors. With minor modi-fication of the wash step, the protocol is ap-plicable to nonadherent cells, which are arewashed by centrifugation and aspiration of thesupernatants. Overall, the phagocytic activityand binding assays described herein are mucheasier, faster, and cheaper, and provide a high-throughput method to accurately measure op-sonic phagocytosis and binding mediated byFcγR or complement receptors.

Critical Parameters andTroubleshooting

The optimal concentration of antibody foropsonizing SRBC needs to be determined priorto the experiment. Too much antibody cancause agglutination, whereas too little will re-sult in inefficient opsonization. High variabil-ity (>10%) among replicate wells usually in-dicates unequal numbers of macrophages inthe wells, which can be minimized by pre-cise and consistent pipetting of macrophagesuspensions into the wells of culture plate.Microscopic examination of cell viability ofmacrophages should be performed to monitorany loss of cells caused by treatment-relatedchanges to cell adherence. Another variablethat must be considered is the age of the SRBCused in the assay. In general, older SRBC be-come more sticky and adhere to macrophageseven without opsonization. Thus, controls thatinclude unopsonized SRBC should be per-formed with every assay.

Anticipated ResultsIgG-SRBCs should bind avidly to mac-

rophages and be efficiently internalized. Theengulfed SRBC will remain intact for sometime and retain hemoglobin. These cells can bevisualized under bright-field microscopy. Theefficiency of complement-mediated phago-cytosis is much lower than that of FcγR-mediated phagocytosis. Many cytokines havebeen shown to modulate receptor expressionand activity (Politis and Vogel, 1990). Thetype I interferons are able to increase FcγRexpression in mouse macrophages, whereasIFN-γ may actually decrease some FcγR ex-pression. This may not be the case with humanmacrophages (Pearse et al., 1992).

Time ConsiderationsThe generation and culture of macrophages

is the most time-consuming part of the proce-dure, which must be set up before the assays

Innate Immunity

14.27.11


are performed. Murine bone marrow–derivedmacrophages take 7 days to develop, whereasperitoneal macrophages can be used the sameday (details see UNIT 14.1). The assays per secan easily be performed within a single work-ing day. The time needed for stimulation withcytokines can vary, but generally an overnightcytokine priming step is used.

AcknowledgmentsThis work was supported by NIH Grant

AI049383.

Literature CitedAderem, A. and Underhill, D.M. 1999. Mechanisms

of phagocytosis in macrophages. Annu. Rev. Im-munol. 17:593-623.

de Oliveira, C.A. and Mantovani, B. 1988. Latrun-culin A is a potent inhibitor of phagocytosis bymacrophages. Life Sci. 43:1825-1830.

Greenberg, S. and Grinstein, S. 2002. Phagocyto-sis and innate immunity. Curr. Opin. Immunol.14:136-145.

He, J.Q., Wiesmann, C., and van LookerenCampagne, M. 2008. A role of macrophagecomplement receptor CRIg in immune clear-ance and inflammation. Mol Immunol. 45:4041-4047.

Helmy, K.Y., Katschke, K.J. Jr., Gorgani, N.N.,Kljavin, N.M., Elliott, J.M., Diehl, L., Scales,S.J., Ghilardi, N., and van Lookeren Campagne,M. 2006. CRIg: A macrophage complement re-ceptor required for phagocytosis of circulatingpathogens. Cell 124:915-927.

Montano, R.F. and Morrison, S.L. 1999. Acolorimetric-enzymatic microassay for thequantitation of antibody-dependent complementactivation. J. Immunol. Methods 222:73-82.

Mosser, D.M. and Edwards, J.P. 2008. Exploringthe full spectrum of macrophage activation. Nat.Rev. Immunol. 8:958-969.

Nimmerjahn, F. and Ravetch, J.V. 2006. Fcgammareceptors: Old friends and new family members.Immunity 24:19-28.

Oliveira, C.A., Kashman, Y., and Mantovani, B.1996. Effects of latrunculin A on immunolog-ical phagocytosis and macrophage spreading-associated changes in the F-actin/G-actincontent of the cells. Chem. Biol. Interact.100:141-153.

Pearse, R.N., Feinman, R., and Ravetch, J. 1992.Characterization of the promoter of the humangene encoding the high-affinity IgG receptor:Transcriptional induction by -interferon is medi-ated through common DNA response elements.Proc. Natl. Acad. Sci. U.S.A. 88:11305-11309.

Politis, A.D. and Vogel, S.N. 1990. Pharmacologicevidence for the requirement of protein kinaseC in IFN-induced macrophage Fc receptor andIa antigen expression. J. Immunol. 145:3788-3795.

Swanson, J.A. and Hoppe, A.D. 2004. The coordi-nation of signaling during Fc receptor-mediatedphagocytosis. J. Leukocyte Biol. 76:1093-1103.

van Lookeren Campagne, M., Wiesmann, C., andBrown, E.J. 2007. macrophage complement re-ceptors and pathogen clearance. Cell. Microbiol.9:2095-2102.

Vogel, S.N., Finbloom, D.S., English, K.E.,Rosenstreich, D.L., and Langreth, S.G. 1983.Interferon-induced enhancement of macrophageFc receptor expression: Interferon treatmentof C3H/HeJ macrophages results in increasednumbers and density of Fc receptors. J. Im-munol. 130:1210-1214.

Worthington, R.E., Bossie-Codreanu, J., and VanZant, G. 1987. Quantitation of erythroid differ-entiation in vitro using a sensitive colorimetricassay for hemoglobin. Exp. Hematol. 15:85-92.

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