PHAGOCYTES

Extravasations and emigration of neutrophils to the inflammatory site depend onthe interaction of immune-complex with Fc� receptors and can be effectivelyblocked by decoy Fc� receptorsRangaiah Shashidharamurthy,1 Randolph A. Hennigar,1 Sebastien Fuchs,1 Purani Palaniswami,1 Melanie Sherman,1 andPeriasamy Selvaraj1

1Department of Pathology and Laboratory Medicine, Emory University School of Medicine, Atlanta, GA

Extravasation and emigration of neutro-phils to the site of inflammation are essen-tial early steps in the initiation of manyantibody-mediated autoimmune diseases.The Fc domains of cell bound autoanti-bodies or immune-complexes (IC) are ca-pable of triggering the neutrophil emigra-tion via complement and Fc�Rs-mediatedmechanisms. To define the clinical rel-evance and the relative contribution ofthese 2 pathways in IC-mediated neutro-phil emigration, we have neutralized theFc�R-binding activity of IC with a recom-binant dimeric Fc receptor, CD16A-Ig, and

investigated the early events of IC-induced inflammation in mice. Systemicadministration of purified CD16A-Igblocked IC-induced inflammation, mast-cell degranulation, and extravasation ofneutrophils in a reversed Arthus reaction.Although the binding of CD16A-Ig toIC did not alter the complement-activating properties of IC, no evidencefor complement-dependent neutrophilemigration was observed. These resultssuggest that interaction of IC with cellsexpressing Fc�Rs at the inflammatorysite results in the secretion of chemoat-

tractants, which mediate complement-independent emigration of neutrophils inthis cutaneous acute inflammation model.Furthermore, blocking the interaction ofIC to Fc�Rs expressed on inflammatorycells by administering high-avidity Fc fu-sion dimers of low-affinity Fc�Rs is aneffective way of preventing IC-inducedacute inflammation in autoimmune dis-eases. (Blood. 2008;111:894-904)

© 2008 by The American Society of Hematology

Introduction

Autoimmune diseases are heterogeneous in nature and are the mostfrequent cause of disability in adults.1 Many autoimmune diseaseslead to vital organ damage, disability and are often fatal in humans.Both the cellular and humoral arms of the immune system areinvolved in the pathogenesis of autoimmune diseases. Abnormalactivation of autoantigen specific T and B cells due to molecularmimicry, viral infections, or cytokine dysregulation are among thesuggested mechanisms for the initiation of the disease.2 In manyautoimmune diseases the binding of autoantibodies to tissuescauses tissue injury. The effector functions of the Fc domains ofthese tissue-antigen bound antibodies depend on their ability tointeract with complement components and/or Fc gamma receptors(Fc�Rs) expressed on inflammatory cells. The relative role andcontribution of these 2 effector systems to the initiation anddevelopment of antibody mediated inflammatory diseases are stillunder intense investigation.

Studies have shown that the accumulation of neutrophils at thesite of IC deposition is pivotal to the development of antibody-mediated autoimmune inflammation.3 The ICs trigger complementactivation and production of chemotactic peptides such as C5a,which directly attract neutrophils to the inflammatory site.4,5 Apartfrom attracting neutrophils, C5a also induce degranulation of mastcells resulting in further recruitment of neutrophils to the site of ICdeposition.5-7 These studies were further supported by the fact thatmice deficient in C5a receptor gene show attenuated autoantibody-

induced inflammation.4,8 Thus the complement peptides producedby IC orchestrate accumulation of neutrophils and cause tissuedamage. On the other hand, studies using Fc� knockout mice havedemonstrated the pivotal role of Fc�Rs in neutrophil recruitment9,10

and antibody-mediated autoimmune diseases. The Fc� subunit isessential for cell-surface expression of activating Fc�Rs such asCD64, Fc�RIV, and CD16A.11-14 These Fc� or CD16A geneknockout mice did not develop autoimmune diseases under experi-mental conditions that induced the diseases in wild type mice.15-18

Although these knockout mice studies have demonstratedindependent roles of complement and Fc�Rs, recent observa-tions suggest interdependency of these 2 pathways in theprogression of inflammation.19 At the inflammatory site, C5aincreases the potency of the Fc�R-dependent inflammatorypathway by up-regulating activating CD16A while down-regulating inhibitory CD32B in inflammatory cells.20 Further-more, the interaction of IC with CD16A upregulates theproduction of cell-derived C5.21 This suggests that the absenceof the complement pathway might influence the effectiveness ofthe Fc�R-mediated pathway of inflammation and vice versa.Because many of these studies used either gene knockout miceor mast cell deficient mice which might have adapted develop-mental compensatory mechanisms, the results and interpreta-tions of the relative role of these functionally interdependentpathways may or may not reflect normal conditions. Therefore,

Submitted April 16, 2007; accepted October 11, 2007. Prepublished online as BloodFirst Edition paper, October 25, 2007; DOI 10.1182/blood-2007-04-085944.

The online version of this article contains a data supplement.

The publication costs of this article were defrayed in part by page chargepayment. Therefore, and solely to indicate this fact, this article is herebymarked ‘‘advertisement’’ in accordance with 18 USC section 1734.

© 2008 by The American Society of Hematology

894 BLOOD, 15 JANUARY 2008 � VOLUME 111, NUMBER 2

to further understand the IC-induced inflammatory pathwaysleading to neutrophil emigration in mice with intact complementsystem and Fc�Rs, we analyzed acute inflammation induced byIC after administration of novel decoy Fc�R dimers. Thedimeric CD16A-Ig fusion protein we used22,23 does not blockcomplement activation by IC, but maintains its potential tocompetitively block the interaction of ICs with Fc�R-expressinginflammatory cells. Our data demonstrate that the interaction ofIC with Fc�R-expressing cells, present at the extravascular site,is critical for the production of complement-independentchemoattractants that trigger the extravasation and emigrationof neutrophils during the early phase of the cutaneousArthus reaction.

Methods

Cell lines and reagents

The ovalbumin-fluorescein isothiocyanate (FITC-Ova) and rabbit antiovalbu-min IgG were from Roche Molecular Biochemicals (Indianapolis, IN).PKH-26 labeling kit, mouse IgG subtypes, rabbit anti-DNP IgG, HRP-conjugated anti–human Fc antibody, cobra venom factor, and pepsin werefrom Sigma (St Louis, MO). Rabbit anti-DNP IgG-Alexa-fluro-448 wasfrom Molecular Probes/Invitrogen (Carlsbad, CA). The Micro BCA-proteinassay kit was from Pierce (Rockford, IL) and the horseradish peroxidase(HRP)-substrate from BioRad (Hercules, CA). FITC-conjugated F(ab�)2-goat anti–mouse IgG and HRP-conjugated F(ab�)2 goat anti–mouse IgGwere from Jackson Immunoresearch Laboratories (West Grove, PA). TheRat-on-Mouse polymer detection kit was from Biocare Medicals (Concord,CA). 2.4G2, an anti–mouse CD16A/CD32B mAb, was from BD Pharmin-gen. F(ab�)2 of 2.4G2 was generated as described.24 F4/80, rat-anti–mousemacrophage antibody, was from AbD Serotec (Oxford, United Kingdom).The mouse macrophage cell line P388D1 was from the American TypeCulture Collection (Manassas, VA). Cell culture reagents were from LifeTechnologies (Gaithersburg, MD). C57BL/6 (8- to 10-week-old) femalemice were from Jackson laboratory (Bar Harbor, Maine). The experimentswith mice were conducted following Emory University institutional animalcare and use committee protocol. Macrophages were obtained from theperitoneal cavity of C57BL/6 mice 2 days after injecting 2 mL of 3%thioglycollate broth and were purified by adherence of the cells to tissueculture plates for 2 hours at 37°C. Cells were used within 4 days for theexperiments.

Construction, expression, and purification of recombinantsoluble CD16A-Ig and CD32A-Ig

The construction and expression of the dimeric form of CD16A-Ig byligating the extracellular domain of human CD16A to the Fc domain of thehuman IgG1 heavy chain has already reported by us.22 The Fc domain hasmutations that abolish the Fc�R-binding activity of IgG1 molecules.25-27

A similar strategy was used to construct and express the R-allele of humanCD32A-Ig. The recombinant molecules were purified from CHO celltransfectants using a protein-G GammaBind Plus (Pharmacia Biotech,Piscataway, NJ) Sepharose column. The purified CD16A-Ig and CD32A-Igwere analyzed using 10% sodium dodecyl sulfate–polyacrylamise gelelectrophoresis (SDS-PAGE) under reducing and nonreducing conditions,and the protein bands were visualized by silver staining and Western blot.Protein concentration was measured using the Micro BCA protein assay kit(Pierce) with BSA as a standard.

Soluble IC binding assay

Soluble immune complex (sIC) was prepared by mixing FITC-Ova withrabbit anti-Ova IgG (1:1 molar ratio) for 4 hours at 4°C.28,29 The complexwas centrifuged at 355g for 30 minutes at 4°C and the supernatant was used

for the FITC-IC binding assay. The macrophages (50 �l of 5 � 106) werepreincubated with blocking agents for 30 minutes at 4°C and then incubatedwith FITC-IC (20 �g/mL) in binding buffer (pH 7.4) for 1 hour at 4°C. Thecells were then washed and analyzed by flow cytometry.

EA binding and phagocytosis assays

Analysis of rabbit anti-DNP IgG–opsonized sheep erythrocytes (EA)binding to macrophages was carried out by flow cytometry as describedearlier28,29 after labeling EA with PKH fluorescent dye. Briefly, sheep redblood cells (SRBC) were coated with DNP as described30 and opsonisedwith rabbit anti-DNP IgG. The phagocytosis assay was carried out usingflow cytometry as described by Zhang et al31 using sheep erythrocyteswith Alexa-flura-448–labeled rabbit anti-DNP antibodies (Alexa-EA) ata ratio of 1:50 (macrophages to Alexa-EA) in the presence or absence of2.4G2 or CD16A-Ig.

Complement-mediated hemolytic assay

Complement-mediated hemolytic activity of mouse serum was carried outas described.32 Briefly, rabbit red blood cells (rRBCs) were opsonized withrabbit anti-DNP-IgG as described for SRBCs (“EA binding and phagocyto-sis assay”). The IgG-opsonized rRBCs (100 �L of 2 � 108 cells/mL) indextrose-gelatin vernal (DGV) buffer were incubated with or withoutvarious concentrations of CD16A-Ig for 1 hour at 4°C, after which 100 �lof 1:5 diluted mouse serum was added and the samples incubated at 37°Cfor 60 minutes. The reaction was stopped by the addition of 0.8 mL ofice-cold DGV buffer. All the samples were centrifuged for 5 minutes at700g at 4°C. The supernatant was collected and read at 450 nm.

In vivo Fc�R-Ig clearance studies

The mice were injected with 100 �g/mouse of dimers intravenously.Subsequently, 3 �l of blood samples were collected at several time pointsand diluted to 300 �L in phosphate-buffered saline (PBS)/5mM EDTA(ethylene diamino tetra acetic acid) and plasma was separated by centrifuga-tion. A sandwich enzyme-linked immunosorbent assay (ELISA) wascarried out to detect the presence of dimers in the plasma using Fc�R-specific mAbs as capture antibodies and HRP-conjugated anti–humanFc–specific antibody as the detection antibody.

Reversed passive Arthus reaction (RPA)

RPA was carried out as described.18 To determine the effect of locallyadministered CD16A-Ig, mice were injected intradermally in the dorsalskin with 25 �L of PBS (control), 20 �g of anti-ovalbumin rabbit IgG(anti-Ova), or various concentrations of CD16A-Ig or CD32A-Ig mixedwith 20 �g anti-Ova at defined sites.

To determine the effect of systemically administered CD16A-Ig,CD16A-Ig was administered intravenously to the mice (n � 3) at concentra-tions of 5, 25, and 50 �g/mL of blood. The blood volume in milliliters wasestimated to be 7% of mouse body weight in grams.33 After 1 hour, micewere injected with 12.5 or 25 �g per site of anti-Ova intradermally in a totalvolume of 25 �L of PBS at different sites. At 5 minutes after injection ofanti-Ova, RPA was initiated by injecting 100 �L PBS containing 500 �g ofovalbumin and 1% Evan blue through the tail vein. The mice were killedafter 3 hours and the injection sites on the reverse side of the skin wereexamined for extravasation of the blue dye. Photographs were takenimmediately and used to quantitate the degree of inflammation elicited byRPA reaction. The intensity of each dermal lesion, shown in blue in thephotographs, was quantified using ImageJ software (National Institutes ofHealth, Bethesda) and KaleidaGraph (Synergy Software, Reading, PA).After the photographs, skin biopsies taken from injection areas weresubjected to histologic analysis. The skin biopsies were sliced horizontally,fixed in 4% buffered formalin, embedded in paraffin, and stained withhematoxylin and eosin. Immunohistochemistry of paraffin-embedded tissuewas performed to detect the accumulation of macrophage using ratanti–mouse F4/80 antibody. The antibodies were detected using Rat-on-Mouse polymer detection kit.

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In some experiments, hypocomplementemia was induced by injectingthe mice twice intraperitoneally with 150 U cobra venom factor (CVF) at8-hour intervals beginning 24 hours before the initiation of RPA reaction.18

The complement-depleting activity of CVF was determined by assaying thehemolysis of rRBC by mouse serum as described.32

Microscopes and image acquisition

Images shown in Figures 2A and 3A were taken using digital Sonycyber-shot DSC-P200, 7.2 mega pixel camera (Sony Style, Atlanta,GA). Images shown in Figures 4A,C, and 5A,C were acquired using aNikon Eclipse 80i microscope with 40�/0.95 DICM/N2 objectives.Neutrophils and macrophages were counted in the slides at 10 randomsites using 63�/1.4 DIC N2 objectives (Nikon Instruments, Melville,NY). Mast cell degranulation (Figure 4 insets) images were acquiredusing Olympus BX40F microscope (Olympus, Melville, NY) with100�/1.4 NA objectives and photographs were taken with a digitalNikon-D100 6.1 mega pixel camera. All the photos were saved as JPEGfiles. Images were processed using Microsoft Photo Editor software(Microsoft, Redmond, WA).

Statistical analysis

A statistical comparison of the control and treated samples was performedusing student t test; P less than .01 (*) considered as significant and P lessthan .001 (**) considered as highly significant.

Results

The human CD16A-Ig dimer blocks IC binding andphagocytosis by mouse macrophage Fc�Rs

Previously, we reported that purified human CD16A-Ig was able tocompete with cell surface expressed human Fc�Rs for binding toICs.22 It is known that human Fc�Rs are known to bind IgG fromother species, such as mouse and rabbit. However, before adminis-tering human dimeric CD16A-Ig and CD32A-Ig in mice, weassessed whether the purified dimeric molecules could block ICbinding functions of mouse Fc�Rs in vitro. We used the mousemacrophage cell line P388D1, which expresses all 3 types of

Figure 1. CD16A-Ig competes with cell surface Fc�Rs and blocks immune-complex binding and phagocytosis by mouse macrophage cells. (A) CD16A-Ig blockedthe binding of soluble IC to the mouse macrophage cell line P388D1 in a dose dependent manner. P388D1 cells were incubated with FITC-IC (soluble IC) in the presence andabsence of various concentrations of CD16A-Ig and the cells were analyzed for binding of FITC-IC using flow cytometry. (B) Specificity of blocking by CD16A-Ig. P388D1 cellswere incubated with soluble IC in the presence of the following reagents: anti-CD16A/32B mAb (2.4G2), CD16A-Ig and CD32A-Ig or mIgG2a. The FITC-IC binding to P388D1cells was analyzed using standard flow cytometry. (C) CD16A-Ig blocked the binding of particulate IC to the mouse macrophage cells. P388D1 cells (50 �l of 5 � 106/mL) inbinding buffer (PBS/5 mM EDTA/1% BSA) were incubated with PKH labeled EA (50 �L of 1.5 � 108) for 2 hours at 4°C. Binding assays were performed in the absence orpresence of CD16A-Ig or CD32A-Ig, or 10 �g/mL blocking mAbs for mouse CD16A/CD32B. Monomeric mIgG2a (50 �g/mL) was used to block CD64. Blocking mAbs (2.4G2),CD16A-Ig, and CD32A-Ig were preincubated for 30 minutes at 4°C and then continuously present during their incubation with EA. EA bound to the cells were analyzed by flowcytometry. Cells incubated with PKH-labeled unopsonized-E were used as a background control. The EA binding in presence of medium was taken as 100% to calculate thepercentage EA binding. Data shown are the average (mean � SD) of 3 individual experiments. (D) CD16A-Ig blocked the phagocytosis of opsonized cells by macrophages.Phagocytosis of Alexa-EA was carried out using P388D1 cells. P388D1 cells were incubated with Alexa-EA in the absence or presence of the following blocking reagents:2.4G2 (10 �g/mL) and CD16A-Ig (25, 50, and 75 �g/mL). The cells were then fixed and the fluorescence of phagocytosed Alexa-EA was measured using flow cytometry. Cellsincubated with EA at 4°C served as the background control. The phagocytic index (PI) was calculated using the formula PI � %P � MF/100.59 The experiment was repeatedtwice. Data shown are the average of 3 individual experiments. *P � .01, **P � .001.

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Fc�Rs: CD16A, CD32B, and CD64. Coincubation of the P388D1cells with CD16A-Ig blocked the binding of the soluble IC (sIC) ina dose dependent manner (Figure 1A). At 25 �g/mL, CD16A-Igblocked up to 70% of sIC binding. The combination of CD16A-Igand mIgG2a was able to block up to 80%, whereas mIgG2a aloneblocked only 20% of sIC binding (Figure 1B). Because monomericmIgG2a binds specifically to CD64,34 the results suggest that CD64contributes minimally to IC binding by macrophages in our system.This is not due to lack of CD64 functional activity. Flow cytometryanalysis showed that the macrophage cell line that we used binds tofree IgG2a (Figure S1A, available on the Blood website; see theSupplemental Materials link at the top of the online article),suggesting that either some or all of the CD64 is functionallyavailable to bind IgG. The results obtained using the F(ab�)2

fragment of 2.4G2, which specifically blocks CD16A/CD32Bmolecules, also demonstrate that 80% of the IC binding is mediatedby CD16A/CD32B-dependent mechanisms (Figure S1B). At presentwe do not know the reason for the low IC binding activity of CD64.It is possible that the high affinity CD64 molecule may be partiallyoccupied by IgG from the serum used for culturing cells or haslower affinity for rabbit IgG. The combination of CD16A-Ig andCD32A-Ig blocked up to 70% of sIC binding, which is not moreeffective than CD16A-Ig alone; suggesting that CD32A-Ig is not anefficient blocker of mouse Fc�Rs in vitro. This is further supportedby the minimal blockage observed with CD32A-Ig (Figure 1B).Some IC, such as autoantibody-coated target cells, are particulatein nature and therefore we assessed whether the soluble dimericreceptors can block the binding of particulate IC (antibody-coatedsheep erythrocytes; EA) to mouse macrophage cells. Nearly 70%of EA binding to P388D1 was blocked by CD16A-Ig at 25 �g/mL,whereas CD32A-Ig was unable to block EA binding (Figure 1C).

Results obtained using mIgG2a and mAb specific to CD16A/CD32B showed that CD64 plays little or no role in the EA binding(Figure 1C), which is consistent with the results from the sIC-binding studies described above (Figure 1B). CD16A-Ig alsoblocked EA binding to macrophages isolated from mouse perito-neal cavity in a dose dependent fashion (Figure S2A). At25 �g/mL, CD16A-Ig was able to block up to 70% of EA binding,whereas CD32A-Ig was unable to block at the doses tested(Figure S2B). Phagocytosis of antibody-coated target cells bymacrophages and neutrophils is one of the major mechanisms bywhich autoantibodies lead to tissue injury in many acute andchronic inflammatory autoimmune diseases. Therefore, wedetermined whether CD16A-Ig could block phagocytosis medi-ated by Fc�Rs expressed on mouse macrophages. Flow cytomet-ric analysis of phagocytosed Alexa-EA showed that pretreat-ment of EA with 50 �g/mL of CD16A-Ig blocked up to 60% ofthe EA phagocytosis by the P388D1 cells (Figure 1D). A similarresult was observed using primary cells such as the thioglycollate-elicited peritoneal macrophages (Figure S2C). Taken together,this data demonstrates that dimeric CD16A-Ig is capable ofcompeting with Fc�Rs expressed on mouse inflammatory cellsfor binding to IC.

CD16A-Ig inhibits antibody-mediated acute inflammation invivo in a murine reversed passive Arthus reaction (RPA) model

Next, we determined whether the in vivo administration of purifiedCD16A-Ig dimer could block the antibody mediated acute inflam-mation induced in a murine RPA model.35 RPA has been exten-sively studied as an in vivo model of immune complex-mediatedacute tissue injury and inflammation. The reaction is induced by

Figure 2. Local administration of CD16A-Ig at the inflammatory site blocked reversed passive Arthus reaction in mice with or without complement depletion.(A) The mice (n � 3) were injected intradermally with PBS (site 1), or various concentrations of CD16A-Ig (0 �g/mL at site 2, 5 �g/mL at site 3, 25 �g/mL at site 4, and 50 �g/mLat site 5) along with rabbit anti-Ova antibody in PBS, immediately after which ovalbumin with 1% Evan blue was injected through the tail vein. To deplete the complement, micewere twice injected intraperitoneally with cobra venom factor before the initiation of RPA. After 3h the mice were euthanized, and the dorsal side of the skin was photographedfor analysis. The figure shows 3 representative mice. (B) Quantitative analysis of RPA. The dermal lesion, seen blue in the photographs, was quantified using ImageJ andKaleidaGraph softwares for groups with or without CVF treatment. Data are presented as mean plus or minus the SD from 3 individual mice. *P � .01, **P � .001.

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injecting an antigen (chicken ovalbumin; Ova) intravenously alongwith a blue dye followed by intradermal administration of poly-clonal antibodies (rabbit antiovalbumin IgG; anti-Ova) specific tothe antigen. Vascular permeability and extravasation of neutrophils,a hallmark of the IC-induced immunopathologic cascade, can bevisualized by extravasation of intravenously injected Evan blue dyeand standard histologic analysis.

First, we determined the optimal concentration of anti-Ovarequired to induce inflammation by injecting various concentra-tions of anti-Ova (0-50 �g/site) intradermally. We found thatArthus reaction was induced optimally with 20 �g/site of rabbitanti-Ova (data not shown). We then determined the ability oflocally administered CD16A-Ig and CD32A-Ig to inhibit theinduction of RPA. The results show that the administration ofCD16A-Ig at the site of IC-formation significantly (P � .001)decreased the size and intensity of the inflammation compared with

the sites injected with rabbit anti-Ova alone (Figure 2A left panels).The degree of inflammation was not reduced at CD32A-Ig injectedsites, suggesting that CD32A-Ig is unable to block IC binding tomouse Fc�Rs in vivo.

The IC-mediated Arthus reaction may be triggered by eitherof two pathways: the complement system and the Fc�Rs.18,36-40

To determine the contribution of complement-mediated RPA inour system, hypocomplementemia was induced in mice usingcobra venom factor (CVF) as described by Hazenbos et al.18 Thedepletion of complement was confirmed by estimating thehemolytic activity of the serum obtained from CVF-treated miceand normal mice (Figure S3). We then studied the effectivenessof CD16A-Ig and CD32A-Ig administration in inhibiting theRPA. No decrease in the intensity of RPA was observed incomplement-depleted mice (Figure 2A right panels). CD16A-Igblocked RPA in a dose dependent manner (Figure 2B). At

Figure 3. Systemic administration of CD16A-Ig effi-ciently blocked the reversed passive Arthus reactionin vivo in mice without complement depletion. (A) Themice (n � 3) were injected with various concentrations ofCD16A-Ig intravenously (panel iv, 5 �g/mL; panel v,25 �g/mL; panel vi, 50 �g/mL of blood). After 1 hour, micewere injected intradermally with PBS (site 1), 12.5 �g(site 2), and 25 �g (site 3) of anti-Ova per site. RPA wasinitiated by injecting Ova with 1% Evan blue intravenouslythrough tail vein. For CD32A-Ig, 50 �g of CD32A-Ig/mL ofblood was injected (panel ii). The antibody (2.4G2)treated control mice (panel iii) were injected with25 �g/mL of blood (40 �g/mice) mAb. The PBS-injectedmice served as the untreated positive control (panel i).After 3 hours the mice were killed, and the dorsal side ofthe skin was photographed for analysis. The figure shows3 representative mice. (B) Quantitative analysis of RPA.The dermal lesion, shown in blue in the photographs, wasquantified using ImageJ and KaleidaGraph softwares forgroups with or without Fc�R dimer treatment. Data arepresented as the mean plus or minus the SD from3 experiments. *P � .01, **P � .001.

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50 �g/mL it was able to block up to 70% of RPA, whereasCD32A-Ig had no detectable effect under similar condi-tions (Figure 2B). Moreover, the combination of CD16A-Igand CD32A-Ig was able to block only 70% of RPA, whichis not more effective than CD16A-Ig alone; suggestingthat CD32A-Ig is not competing with murine Fc�Rs in vivo(data not shown).

We then determined the efficacy of these dimers in blocking theIC-mediated inflammation when administered systemically. Asobserved with locally administered CD16A-Ig, the systemicallyadministered CD16A-Ig also blocked RPA in a dose dependentmanner (Figure 3Aiv-vi). A dose of 50 �g/mL of blood CD16A-Igblocks more than 70% of the inflammation as measured by the

intensity of the Evan blue extravasation (Figure 3B). Under similarconditions, as seen in the previous experiments (Figure 2),CD32A-Ig was unable to block RPA (Figure 3B), demonstratingthe specificity of the inhibition of RPA by CD16A-Ig. Similarexperiments with systemically administered molecules conductedin CVF-treated mice showed that complement depletion did nothave any effect on the intensity of RPA (data not shown). Thesedata show that systemic administration of recombinant solubledimeric CD16A-Ig effectively blocks the IC-mediated inflamma-tion in vivo.

As specificity controls for RPA, groups (n � 3) of mice wereinjected intradermally at different sites with either PBS or PBScontaining various concentrations of anti-Ova antibody or Fc�R

Figure 4. Histologic examination and evaluation of neutrophil infiltration in skin biopsies from anti-Ova injected areas of mice treated with or without Fc�R dimers.(Ai) Skin biopsy from anti-Ova antibody–injected area in an untreated mouse with RPA (from Figure 3Ai site 3). The epidermis and dermis are essentially unremarkable.However, the subdermal fat is edematous and shows the margination of neutrophils along capillary walls (arrows; bottom inset) as well as neutrophilic infiltration intosurrounding soft tissue. (Aii) Skin biopsy from PBS-injected area in an untreated mouse with RPA (from Figure 3Ai site 1). No specific pathologic changes are identified.(Aiii) Skin biopsy from anti-Ova antibody injected area in a mouse treated with 50 �g/mL CD16A-Ig before the induction of RPA (from Figure 3Avii site 3). No specific pathologicchanges are identified, suggesting abrogation of the Arthus reaction. (Aiv) Skin biopsy from anti-Ova antibody injected area in a mouse treated with 50 �g/mL CD32A-Ig beforethe induction of RPA (from Figure 3Aii site 3). The epidermis and dermis are essentially unremarkable. However, the subdermal fat is edematous and shows neutrophilicinfiltrates in soft tissue (similar to Figure 3Ai), as well as scattered foci of leukocytoclastic vasculitis (arrows; bottom inset). Top inset: Mast cell degranulation was observed in10 different sites at 100x magnification and one of the cells is represented in the figure inset. (B) Neutrophils were counted in 10 random sites at 63x magnification and sum ofthe neutrophils were represented. C: To deplete the complement, mice were twice injected intraperitoneally with cobra venom factor before the initiation of RPA. The mice wereinjected with CD16A-Ig and CD32A-Ig intravenously (50 �g/mL of blood). After 1 hour, mice were injected intradermally with 25 �g of anti-Ova per site. (Ci) Skin biopsy fromanti-Ova antibody injected area in an untreated mouse with RPA. The epidermis and dermis are essentially unremarkable. However, the subdermal fat is edematous and showsthe margination of neutrophils along capillary walls (arrows; bottom inset) as well as neutrophilic infiltration into surrounding soft tissue. (Cii) Skin biopsy from PBS injected areain an untreated mouse with RPA. No specific pathologic changes are identified. (Ciii) Skin biopsy from anti-Ova antibody injected area in a mouse treated with 50 �g/mLCD16A-Ig before the induction of RPA. No specific pathologic changes are identified, suggesting abrogation of the Arthus reaction. (Civ) Skin biopsy from anti-Ova antibodyinjected area in a mouse treated with 50 �g/mL CD32A-Ig before the induction of RPA. The epidermis and dermis are essentially unremarkable. However, the subdermal fat isedematous and shows neutrophilic infiltrates in soft tissue, as well as scattered foci of leukocytoclastic vasculitis (arrows; bottom inset). Top inset: Mast cell degranulation wasobserved in 10 different sites 100x magnification and one of the cells is represented in the Figure inset. (D) Neutrophils were counted in 10 random sites at 63x magnificationsand sum of the neutrophils were represented.

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dimers, and immediately 100 �l of 1% Evan blue in PBSintravenously. These control experiments show that the intrader-mally injected Fc�R-Ig dimers or anti-Ova do not causeinflammation per se (data not shown). We have also determinedthe effect of systemically administered anti–mouse CD16A mAbon the RPA. The results show that IC-induced RPA can beeffectively blocked by anti-CD16A/CD32B mAb (Figure 3B);demonstrating IC-induced inflammation in our experiments ismediated by Fc�Rs.

Histologic analyses show that CD16A-Ig blocks IC-inducedaccumulation of inflammatory cells and tissue damagemediated by mouse Fc�Rs

To visualize the intensity and character of the inflammation in RPA,the skin biopsies taken from antibody-injected areas of untreated,CD16A-Ig–treated and CD32A-Ig–treated mice, shown in Figure3A, were subjected to histologic analysis. As a control, a PBS-injected area of untreated mice was used. Tissue sections from theanti-Ova injected area of untreated mice revealed edema and aninflammatory infiltrate composed predominantly of neutrophils ascharacterized by their multilobed nuclei (Figure 4Ai,B) and

degranulated mast cell (Figure 4Ai top inset). Histologic sectionsof anti-Ova–injected areas of skin taken from the mice treated withCD16A-Ig showed a marked reduction in neutrophil accumulation,margination around venules (Figure 4Aiii,B), and degranulation ofmast cells (Figure 4Aiii top inset) compared with the untreatedmice (Figure 4Ai), whereas the skin sections of CD32A-Ig–treatedmice (Figure 4Aiv) showed features that were similar to thoseobserved in untreated mice (Figure 4Ai). PBS-injected area ofuntreated mice did not show any evidence of inflammation(Figure 4Aii).

Because complement peptides such as C5a are known chemoat-tractants of neutrophils, we have also analyzed the tissue sectionsof mice treated with CVF to determine the role of complement ininducing cellular infiltration in RPA. The biopsies taken fromantibody-injected and PBS-injected areas of the skin of untreated,CD16A-Ig–treated, and CD32A-Ig–treated mice were subjected tohistologic analysis. As seen in Figure 4C,D, the neutrophilaccumulation, margination around venules, and mast cell degranu-lation (top insets) in the anti-Ova injected areas of untreated(Figure 4Ci), CD16A-Ig treated (Figure 4Ciii), and CD32A-Ig–treated (Figure 4Civ) mice were similar to those of mice without

Figure 5. Immunohistochemical examination to detect the macrophages in skin biopsies from anti-Ova injected areas of mice treated with or without Fc�R dimers.(A) Skin biopsies of the above mentioned sections were processed to detect the macrophages (brown cells indicated by arrow) by immunohistochemistry. (Ai) Accumulation ofmacrophages in anti-Ova antibody injected area of untreated mice. (Aii) Presence of resident macrophages in PBS injected area of untreated mice. (Aiii) Inhibition ofmacrophage accumulation in anti-Ova antibody injected area of CD16A-Ig treated mice. (Aiv) Accumulation of macrophages in anti-Ova antibody injected area of CD32A-Igtreated mice. (B) Macrophages were counted in 10 random sites at 63x magnification and the sum of the macrophages is presented. (C) Skin biopsies of the above mentionedsections from complement-depleted mice were processed to detect the macrophages (brown cells indicated by arrow) by immunohistochemistry. (Ci) Accumulation ofmacrophages in anti-Ova antibody injected area of untreated mice. (Cii) Presence of resident macrophages in PBS injected area of untreated mice. (Ciii) Inhibition ofmacrophage accumulation in anti-Ova antibody injected area of CD16A-Ig treated mice. (Civ) Accumulation of macrophages in anti-Ova antibody injected area of CD32A-Igtreated mice. (D) Macrophages were counted in 10 random sites at 63x magnification and the sum of the macrophages is presented.

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complement depletion (Figure 4Ai-Aiv,B). These data suggest thatthe interaction of IC with Fc�Rs expressed on the cells reside at thesite of IC-formation, not the complement pathway, play a majorrole in induction of RPA and neutrophil emigration from thecirculation. These histologic data further confirm the resultsobtained using dye extravasation method (Figures 2,3), and showthat recombinant CD16A-Ig dimer blocks IC-induced tissue dam-age when administered in vivo.

We have also carried out immunohistochemistry to determinethe accumulation of macrophages in RPA (Figure 5A,B).Resident macrophages were found in the PBS injected area ofthe skin (Figure 5Aii), while accumulation of macrophages wasobserved in antibody-injected area of skin in untreated mice(Figure 5Ai). The results from the dimer treated mice show thatlike neutrophils, the IC-induced accumulation of macrophageswas also inhibited by CD16A-Ig administration (Figure 5Aiii,B).CD32A-Ig treatment did not inhibit macrophage accumulation(Figure 5Aiv). Analysis of slides from complement-depletedmice demonstrated that the accumulation of macrophages is alsoindependent of the complement system (Figure 5C,D). Interest-ingly, unlike neutrophils, we did not observe margination ofmacrophages around venules, suggesting that most of thesemacrophages may have migrated from the tissues surroundingthe IC injected area (data not shown). This suggests that themechanism of accumulation of these two types of inflammatorycell populations may be distinct.

CD16A-Ig binding to IC does not block thecomplement-mediated hemolytic activity of IgG molecule

The Fc domain of IgG IC is capable of inducing inflammation usingboth Fc�Rs and complement pathways. Although complement- andFc�R-binding sites on the Fc domain of IgG are distinct, it is apossibility that the binding of CD16A-Ig molecule to the Fc domain ofIC may hinder the binding of C1q and thus prevent complementactivation. To rule out whether CD16A-Ig binding altered the comple-ment activating property of IC, we determined the hemolysis of rabbitanti-DNP IgG opsonized rRBCs by mouse serum in the presence andabsence of CD16A-Ig. Because mouse complement does not lyseIgG-opsonized sheep or mouse RBCs, we used rabbit anti-DNP IgGopsonized rRBCs for these assays.41 Titration of mouse serum showedthat 1:5 dilution caused 50% hemolysis of rRBC (Figure S4). At thesame serum dilution, the complement-mediated hemolysis of rRBCswas unaffected by the presence of CD16A-Ig (Figure 6A). Thissuggests that the lack of inflammation induced by IC during CD16A-Igadministration is not due to any alteration in complement activatingproperty of IC that might have resulted from binding of Fc domain ofIgG by CD16A-Ig.

In vivo half-life of CD16A-Ig

Analysis of the kinetics of clearance from the circulation showsthat the plasma level of both the administered dimers droppedquickly (nearly 23%) until 8 hours, maintained a steady state up to90 hours and then started declining rapidly. The reason for thesudden initial drop is not clear. It is possible that the dimers mightbe diffusing into extracellular space from the circulation. TheFc�R-Ig dimers were detected in mouse blood up to 160 hours aftertheir administration. The in vivo half-life in the circulation for bothdimeric CD16A-Ig and CD32A-Ig was estimated to be 120 hours(Figure 6B). This suggests that it is possible to maintain a plasmaconcentration of the systemically administered decoy receptorshigh enough to block of IC-mediated inflammation for several

days. The high half-life of Fc fusion proteins has been attributed tothe Fc domain; the Fc domain of IgG binds to neonatal Fc receptor(FcRn), which results in protection from degradation by theendocytic machinery of the cells.42 The Fc domain that we usedhere to make Fc�R-Ig fusion proteins has mutations that affect theFc�R binding.25-27 Because FcRn and Fc�R bind to distinct sites onIgG, we do not anticipate that the mutations in the Fc domain haveany effect on the FcRn binding and consequently the half-life of themutated IgG-Fc fusion molecules.43,44

Discussion

Autoantibody-antigen IC formed in many autoimmune diseasesamplify tissue damage by attracting inflammatory cells, such as

Figure 6. Effect of CD16A-Ig on complement-mediated hemolytic activity.(A) Rabbit anti-DNP-IgG opsonized rRBCs were incubated with or without variousconcentrations of CD16A-Ig and then 1:5 diluted mouse serum was added. Hemolyticactivity was then carried out as described in the text. Cells treated with buffer aloneserved as a control. Cells lysed with water represented 100% hemolysis. Thesupernatant was read at 450nm. Data are representative of 3 independent experi-ments. (B) Clearance of Fc�R-Ig dimers from circulation. A group of mice (n � 3)were injected with dimers and blood samples were collected at various time pointsand diluted in PBS/EDTA. Plasma was collected to detect the Fc�R-Ig dimers bysandwich ELISA. Plates were coated with 50 �L of 10 �g/mL anti-hCD16A(CLBFcgran-1) and anti-hCD32A (IV.3) mAbs overnight at 4°C. The wells were thenblocked with PBS/5mM EDTA/1% BSA. After washing, 50 �L of the plasma sampleswere added into the wells and incubated for another 1 hour. The wells were washedand 50 �L of HRP-conjugated anti–human Fc specific antibody were added andincubated for another 1 hour. HRP substrate was added to the washed wells and readat 450 nm. The plasma from a group of normal mice injected with PBS served as aspecificity control. As positive controls, purified CD16A-Ig and CD32A-Ig were usedwhile BSA coated wells served as negative controls. Purified CD32A-Ig was used asa standard to quantify the level of dimers in the blood. Data are means plus or minusSD of triplicates.

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neutrophils using both complement and Fc�R-dependent mecha-nisms. Dissecting the precise roles of these two pathways inrecruitment of neutrophils to the inflammatory site will lead to abetter understanding of the mechanisms and treatment of thedisease. We have used an experimental strategy that selectivelyneutralizes the Fc�R-binding activity of the ICs without manipu-lating the expression of the Fc�Rs or complement receptors onthe inflammatory cells. The CD16A-Ig dimer that we usedcompetitively blocked the binding of IC to inflammatory cells inin vitro assays and blocked IC-induced inflammation in vivo atcirculating concentrations as low as 25 to 50 �g/mL. Accumula-tion of neutrophils is a hallmark of IC-induced RPA.45 There-fore, the relative roles of complement-derived chemotacticpeptides and Fc�Rs in recruiting neutrophils to the extravascu-lar sites of IC formation were investigated by analyzingneutrophil infiltration in normal and dimer administered micebefore or after complement depletion. The absence of neutrophilinfiltration in the dimer-treated animals (Figure 4D) and thepresence of neutrophil infiltration at the site of IC-formationdespite the depletion of complements (Figure 4Ci) suggest thatthe chemotactic complement peptides are not involved in therecruitment of neutrophils to the site of IC deposition in themodel system used here. A similar complement-independent butFc�R-dependent accumulation of macrophages was observed inRPA. Complement peptides such as C5a have been shown notonly to recruit neutrophils via chemotaxis, but also activate mastcell degranulation. Thus, it is intriguing that complementactivation that might have been triggered by IC in CD16A-Ig–administered mice did not cause mast-cell degranulation. Theabrogation of mast-cell degranulation by neutralizing ICs withCD16A-Ig in the cutaneous Arthus reaction model presentedhere suggests that the complement activation by IC alone maynot be sufficient for mast-cell degranulation. This suggests thatinitiation of mast-cell degranulation depends essentially onIC-Fc�Rs interaction but not on the complement activatingproperty of the ICs. While this is contrary to observations madein other systems that show that mast cells degranulate inresponse to C5a,4,5 it is in agreement with studies usingFc�R-knockout mice that show that Fc�Rs on mast cells arenecessary for initiation of IC-induced inflammation.18,46 Acti-vated mast cells are known to produce compounds that are

chemotactic to neutrophils,6,47,48 and therefore it is possible thatabsence of neutrophil infiltration in the dimer-treated mice maybe a consequence of lack of the mast-cell degranulation.

However, the participation of cell-derived C5a in neutrophilrecruitment to inflammatory site cannot be ruled out. Recently, ithas been shown20,21 that engagement of activating Fc�Rsup-regulated the production of cell-derived C5 and C5a, and,subsequently, C5a up-regulated the expression of activatingFc�Rs while down-regulating inhibitory Fc�Rs,21 suggestingcell-derived C5a could play a role in IC-induced inflammationby altering the level of expression of Fc�Rs. Thus the IC canproduce the neutrophil chemoattractant C5a using two differentpathways; directly by triggering classical pathway of comple-ment activation, or indirectly by inducing production of cell-derived C5a by engaging Fc�Rs. The complement depletionstudies described here used CVF, which blocks the three majorpathways of C5a production by binding to C349,50 but not thecell-derived pathway. Therefore, although the results presentedhere show that complement activation pathways directly trig-gered by IC do not recruit neutrophils, they do not rule out therole of cell-derived C5a that are produced as a result of ICinteraction with the inflammatory cells in neutrophil recruit-ment. At present, the relative contribution of the direct and thecell-derived pathways of complement activation in the recruit-ment of neutrophils are not known.

The results show that the extravasation and emigration ofneutrophils that occur during the early phase of IC-inducedcutaneous inflammation depend on the ability of IC to interactwith Fc�Rs and are independent of the complement activationpathways that can be blocked by CVF. The administration ofCD16A-Ig also blocked mast-cell degranulation at the inflamma-tory site. Based on these observations, we hypothesize that theadministration of decoy CD16A-Ig dimer prevented IC frombinding to mast-cell Fc�Rs by occupying the Fc domain of theIC (Figure 7) and consequently prevented mast cell degranula-tion, neutrophil recruitment, and inflammation. This is inagreement with the previous observations from Fc�R knockoutmice and mast cell deficient mice, which suggested that theArthus reaction depends on Fc�Rs-triggered mast-cell degranu-lation.18,46,51 In contrast to our observations with recombinantsoluble CD16A-Ig dimer presented here, an earlier study that

Figure 7. A hypothetical model depicting the action ofin vivo administered CD16A-Ig in Arthus reaction.M� � macrophages, N� � neutrophils.

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used a recombinant soluble CD32A has concluded that parenter-ally administered soluble Fc�Rs is not a practical approach toblock IC-mediated inflammation.52 Their conclusion was basedon the observation that the systemically administered CD32Ahas a half-life of only 25 minutes and is unable to block Arthusreaction. This report also showed that soluble CD32A couldblock Arthus reaction only when administered locally alongwith the IC at a concentration of 5mg/mL. It is possible that therequirement for such a high concentration and very low in vivohalf-life may be due to the monomeric nature of soluble CD32Athat was used in their study. Interestingly, however, our resultswith CD32A-Ig dimer suggest other possibilities. We found thatCD32A is not able to block the Arthus reaction in mice evenafter dimerization. This is not due to loss of function of CD32Aafter dimerization; the purified CD32A-Ig is able to competi-tively block binding of CHO cells expressing cell surfaceCD32A to IC (data not shown). Our results suggest thatCD32A-Ig is not able to compete with mouse Fc�Rs for bindingto IC either because the affinity of CD32A-Ig may be too low, ormouse Fc�Rs and human CD32A may be binding to distinctregions of rabbit IgG. Thus our results, although supporting theobservation reported by Ireino et al52 that CD32A cannotpractically block the IC-mediated inflammation, do, however,demonstrate the feasibility of using systemic administration ofCD16A-Ig to block acute inflammation mediated by IC.

In summary, our results suggest that the interaction of ICwith the Fc�Rs expressed on cells residing in the extravascularsite triggers a pathway of neutrophil recruitment that is indepen-dent of IC-induced complement activation. Our results empha-size that the engagement of Fc�Rs by ICs is a crucial event ininitiating the inflammatory cascade. The observations reportedhere may be of clinical relevance. The interaction of Fc�Rs-

bearing inflammatory immune cells with autoantigen-antibodyICs is implicated in the pathogenesis of several autoimmunediseases such as systemic lupus erythematosus, glomerulonephri-tis, and arthritis.15,53-58 Neutrophil infiltration plays an importantrole in the initiation of inflammation in these autoimmunediseases. Because the inflammation and recruitment of neutro-phils can be blocked by systemically administered decoyFc�R-Ig dimers, this strategy could be applied to treat antibody-mediated inflammatory and autoimmune diseases.

Acknowledgments

The authors thank Dr Aaron Lukacher, Prof Aftab Ahmed Ansari,Dr Robert Swerlick, Seema Patel, and Saranya Selvaraj for criticalreading of the manuscript, and Dr Kenneth E. Bernstein for his helpin taking high-resolution mast-cell pictures.

This study was supported by a National Institutes of Healthgrant (RO1 AI 049400) to P.S.

Authorship

Contribution: R.S. designed, performed research, analyzed dataand drafting the paper; R.A.H., S.F., P.P., T.L., and M.S., performedresearch and analyzed data; P.S. designed, performed research,analyzed data, and wrote the paper.

Conflict-of-interest disclosure: The authors declare no compet-ing financial interests.

Correspondence: Periasamy Selvaraj, PhD, Associate Profes-sor, Department of Pathology, 7309 WMB, Emory University,101 Woodruff Circle, Atlanta, GA 30322; e-mail:pselvar@emory.edu.

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