Proinflammatory Cytokines Macrophages and Release of Leading to Augmented Phagocytosis by RIIA (CD32) on Apoptotic Neutrophils

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Immune Complexes Bind Preferentially to Fc γRIIA (CD32) on Apoptotic Neutrophils, Leading to Augmented Phagocytosis by Macrophages and Release of This information is current as Proinflammatory Cytokines of September 25, 2021. Simon P. Hart, Karen M. Alexander and Ian Dransfield J Immunol 2004; 172:1882-1887; ; doi: 10.4049/jimmunol.172.3.1882 http://www.jimmunol.org/content/172/3/1882 Downloaded from

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2004 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Immune Complexes Bind Preferentially to Fc␥RIIA (CD32) on Apoptotic Neutrophils, Leading to Augmented Phagocytosis by Macrophages and Release of Proinﬂammatory Cytokines1

Simon P. Hart,2 Karen M. Alexander, and Ian Dransﬁeld

Many human inflammatory diseases are associated with tissue deposition of immune complexes and influx of neutrophils. We show that immune complexes bind preferentially to apoptotic neutrophils via Fc␥RIIA (CD32) and that increased binding is associated with clustering of immune complexes on the plasma membrane of the apoptotic cell. Phagocytosis of immune complex-opsonized apoptotic neutrophils by human macrophages was substantially enhanced (4.4-fold increase compared with control apoptotic neutrophils) and stimulated macrophages to release the proinflammatory cytokines TNF-␣ and IL-6. Immune complexes may perturb the normal pathways for clearance of apoptotic neutrophils by augmenting their clearance at the price of proinflammatory Downloaded from cytokine release. This represents a novel mechanism by which immune complexes may modulate the resolution of inflammation. The Journal of Immunology, 2004, 172: 1882Ð1887.

mmune complexes (ICs)3 have been found in the blood and cient removal of apoptotic cells, because in situations where ex- tissues in many human inflammatory diseases, and adminis- cessive apoptotic cell load occurs development of autoimmune or tration of ICs in animal models results in inflammation and chronic inflammatory pathology may ensue (11, 12). Down-regu- I http://www.jimmunol.org/ fibrosis (1, 2). ICs containing IgG contribute to inflammation by lation of proinflammatory mediator release and production of anti- activating complement and cross-linking leukocyte surface Fc re- inflammatory mediators have been widely quoted as universal ceptors, resulting in cell activation (3). Human neutrophils express macrophage responses to phagocytosis of apoptotic cells (13, 14), two receptors for complexed IgG, Fc␥RIIA (CD32) and Fc␥RIIIB but proinflammatory sequelae to phagocytosis of apoptotic cells (CD16), both of which have low affinity for monomeric IgG, but have also been reported (15, 16). Apoptosis is associated with bind effectively to aggregates of Ab, such as ICs. Inappropriate or many alterations in the protein and carbohydrate composition of uncontrolled neutrophil activation results in the release of histo- the plasma membrane (10, 17Ð19). Some of these changes may be toxic products that may damage surrounding tissue and propagate responsible for the binding potential of opsonins, including com-

the inflammatory response, leading ultimately to tissue destruction plement proteins (20, 21), collectins (22, 23), pentraxins (24Ð26), by guest on September 25, 2021 and scarring. There is strong evidence that neutrophil activation and IgM (27). These findings are important because although the induced by ICs is fundamental in the pathogenesis of certain in- process of macrophage clearance of apoptotic cells has been stud- flammatory diseases (4). Excessive production of ICs has been ied extensively under serum-free conditions in vitro, the presence demonstrated in a variety of neutrophil-mediated diseases, such as of opsonins in the inflammatory milieu means it is unlikely that rheumatoid arthritis, idiopathic pulmonary fibrosis, inflammatory “naked” apoptotic cells would be encountered by macrophages bowel disease, and the acute respiratory distress syndrome, al- in vivo. though an etiological role for ICs in these conditions has not been We have recently reported that a murine IgG1 mAb bound spe- proven (5Ð7). cifically to apoptotic neutrophils by forming a complex with its Ag The physiological fate of the huge number of neutrophils re- in serum (28). In the present study we investigated the role of cruited to sites of inflammation is death by apoptosis (8). Apopto- Fc␥RIIA in the binding of murine and human immune complexes sis is associated with down-regulation of cellular functions with to apoptotic neutrophils and tested the hypothesis that opsonization potential to inflict host damage, such as stimulated release of gran- with immune complexes perturbs the normal anti-inflammatory ule contents (9, 10). Furthermore, surface membrane alterations on macrophage response following phagocytosis of apoptotic neutro- apoptotic cells leads to their recognition and phagocytosis by mac- phils. We report that Fc␥RIIA-mediated binding of ICs to human rophages (8). Successful resolution of inflammation requires effi- apoptotic neutrophils profoundly augments phagocytosis by macrophages, but leads to the production of proinflammatory cytokines. These data imply that opsonization of apoptotic neutrophils Medical Research Council Center for Inflammation Research, University of Edin- by ICs alters their pathway for clearance, and so influences the burgh Medical School, Edinburgh, United Kingdom resolution of inflammation and hence the likelihood of developing Received for publication June 10, 2003. Accepted for publication November chronic inflammatory or autoimmune disease. 18, 2003. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance Materials and Methods with 18 U.S.C. Section 1734 solely to indicate this fact. Reagents 1 This work was supported by a Medical Research Council Clinician Scientist ␥ Ј 3G8 (anti-Fc RIIIB) F(ab )2 were obtained from Ancell (Bayport, MN), Fellowship. and IV3 (anti-Fc␥RIIA) FabЈ were purchased from Medarex (Princeton, 2 Address correspondence and reprint requests to Dr. Simon P. Hart, Medical Re- NJ). FITC-conjugated BN34 (IgG1 anti-biotin), biotinylated BSA, human search Council Center for Inflammation Research, University of Edinburgh Medical IgG, and FITC-conjugated human IgG were obtained from Sigma-Aldrich School, Teviot Place, Edinburgh, U.K. EH8 9AG. E-mail address: [email protected] Ј (Poole, U.K.). F(ab )2 rabbit anti-human IgG was purchased from DAKO 3 Abbreviation used in this paper: IC, immune complex. (Ely, U.K.). Cell culture materials and FCS were obtained from Invitrogen

(Paisley, U.K.), and Percoll was purchased from Pharmacia (Little Chal- Cytokine measurement font, U.K.). Cytokines TNF-␣ (assay sensitivity, 3.7 pg/ml), IL-1␤ (7.2 pg/ml), IL-6 (2.5 pg/ml), IL-8 (3.6 pg/ml), IL-10 (3.3 pg/ml), and IL-12p70 (1.9 pg/ml) Cell isolation were measured in macrophage supernatants using a fluorescent bead-based sandwich assay (Human Inflammation Cytometric Bead Array, BD Bio- Leukocytes were isolated from human peripheral blood by dextran sedi- sciences, San Diego, CA) and analyzed on a FACSCalibur flow cytometer. mentation and discontinuous Percoll gradient centrifugation as previously TGF-␤ was acid-activated in undiluted supernatants and measured by described (18). All the neutrophil donors were G homozygous or G/A ELISA (assay sensitivity, 32 pg/ml; R&D Systems, Minneapolis, MN). heterozygous at position 519 in exon 4 of the Fc␥RIIA gene. These are the two most prevalent genotypes in our population and exhibit similar levels Data analysis and statistics of murine IC binding to apoptotic neutrophils (28). No donor was A519 homozygous. Neutrophils were cultured in IMEM containing 10% autol- Results are presented as the mean Ϯ SEM of at least three independent

ogous serum at 37¡C in a 95% air/5% CO2 atmosphere for 20 h, during experiments using cells from different donors. Results were compared us- which time a proportion of the cells underwent apoptosis (29). Apoptosis ing either a paired t test or repeated measures ANOVA and Tukey-Kramer was assessed by binding of PE-conjugated annexin V (Caltag, Towchester, multiple comparisons test as appropriate, using InStat version 3 (GraphPad, U.K.) and assessment of characteristic morphology on stained cytocentri- San Diego, CA). fuge preparations. Mononuclear cells were suspended in IMEM and ad- hered to 48-well cell culture plates during incubation at 37¡Cfor1h. Ͼ Results Adherent cells ( 90% CD14-positive monocytes) were washed twice in Complexes of murine IgG1 bind to apoptotic neutrophils PBS, and monocytes/macrophages were cultured 4Ð7 days in IMEM containing 10% autologous serum. For phagocytosis assays, apoptotic neutro- We generated labeled ICs by incubating biotinylated BSA with a phils were cultured for 44 h, at which time Ͼ80% had undergone apopto-

FITC-conjugated mouse IgG1 anti-biotin mAb. Binding studies Downloaded from sis. Membrane integrity was routinely tested by exclusion of propidium iodide, and all preparations were Ͼ85% (44 h) or Ͼ95% (20 h) propidium were performed on ice with aged human neutrophils, comprising iodide-negative. both apoptotic and nonapoptotic cells, and apoptotic cells were identified by counterstaining with annexin V. Analysis by two- Immune complex binding color flow cytometry revealed that ICs bound significantly more avidly to apoptotic neutrophils than nonapoptotic neutrophils over ␮ Complexes of murine IgG1 were formed by combining 500 g/ml biotin- a range of concentrations (Fig. 1a). There was no further increase ␮ http://www.jimmunol.org/ ylated BSA with 170 g/ml FITC-conjugated anti-biotin monoclonal IgG1 Ͻ (clone BN34) in PBS for 30 min on ice. ICs were then diluted in PBS in IC binding to the small proportion ( 5%) of membrane-perme- before use in neutrophil binding assays, and the concentration was ex- able postapoptotic (necrotic) cells within the aged neutrophil pop- pressed as the concentration of total IgG. ulation (data not shown). Labeled complexes of human IgG were formed by heating FITC-conjugated human IgG (20 mg/ml) at 63¡C for 20 min in PBS. Precipitates IC binding to apoptotic neutrophils is mediated by Fc␥RIIA were removed by centrifugation at 10,000 ϫ g for 2 min. In some experiments heat-aggregated IgG was ultracentrifuged at 300,000 ϫ g in an To determine the involvement of neutrophil Fc receptors in bind- Optima TLX ultracentrifuge with TLA100.3 rotor (Beckman Coulter, Ful- ing of murine IgG1 ICs, we preincubated neutrophils with anti- lerton, CA) for 60 min, which removed 80% of the ICs as assessed by cell FcR Abs that are known to specifically block IC binding. FabЈ of binding of the supernatant using flow cytometry and FITC-conjugated ␥ by guest on September 25, 2021 F(abЈ) rabbit anti-human IgG. In blocking experiments we used the well- Fc RIIA Ab IV3 completely inhibited binding of murine IgG1 ICs 2 Ј characterized, function-blocking Abs IV3 (FabЈ anti-CD32; 10 ␮g/ml) and to both apoptotic and nonapoptotic neutrophils, whereas F(ab )2 of Ј ␮ 3G8 (F(ab )2 anti-CD16; 10 g/ml), which were incubated with aged neu- Fc␥RIIIB Ab 3G8 had no effect (Fig. 1b). These results indicate trophils for 30 min on ice. FITC-labeled ICs were then incubated with that neutrophil Fc␥RIIA is wholly responsible for binding murine neutrophils for 30 min on ice. Final staining with annexin V-PE was per- IgG1 ICs and imply that Fc␥RIIA on apoptotic neutrophils exhib- formed before two-color flow cytometric analysis on an EPICS XL ma- chine (Coulter, Hialeah, FL). its increased avidity for ICs. Distribution of bound ICs on apoptotic neutrophils Immunofluorescence microscopy As Fc␥RIIA mediated IC binding, we re-examined the expression Aged neutrophils were labeled with murine ICs, fixed in 3% paraformal- of FcRs on apoptotic and nonapoptotic neutrophils. The expression dehyde, permeabilized with 0.1% Triton X-100, counterstained with TO- ␥ ␥ PRO-3 (Molecular Probes, Leiden, The Netherlands), and cytocentrifuged of Fc RIIA and Fc RIIIB was reduced on apoptotic neutrophils onto glass slides. Visualization was performed with a TCSNT confocal system (Leica Microsystems, Mannheim, Germany).

Macrophage phagocytosis assay Macrophage phagocytosis of apoptotic neutrophils was assessed using modifications of a previously described serum-free phagocytosis assay (29). Aged neutrophils were preincubated with PBS or 500 ␮g/ml heat- aggregated IgG for 30 min on ice, then washed twice. Adherent macrophages in 48-well plate were washed, and 2 ϫ 106 aged neutrophils in 250 ␮l of IMEM were added to each well. After 60-min incubation at 37¡C, the cells were washed with PBS, fixed with 1% glutaraldehyde, and stained for myeloperoxidase with 0.1 mg/ml dimethoxybenzidine and 0.03% (v/v) hy- drogen peroxide in PBS. The percentage of macrophages that had ingested one or more apoptotic neutrophils, and the number of apoptotic neutrophils ingested per 100 macrophages (phagocytic index) were quantified by ex- amination of at least five fields (minimum of 400 cells) in duplicate wells with an inverted microscope. In parallel experiments, apoptotic neutrophils FIGURE 1. Murine IgG1 ICs bind to apoptotic neutrophils via were incubated with macrophages for 6 h. The supernatants were har- ␥ vested, centrifuged at 1000 ϫ g for 5 min to remove cellular debris, and Fc RIIA. a, Preferential binding of FITC-conjugated murine IgG1 ICs to F stored at Ϫ70¡C before cytokine measurement. For comparison, macro- apoptotic human neutrophils ( ) compared with nonapoptotic neutrophils phages were stimulated with 50 ng/ml Escherichia coli 018 LPS for 4 h, (E). b, Effects of preincubation of neutrophils with FabЈ of Fc␥RIIA Ab Œ Ј ␥ ࡗ and the supernatants were harvested after an additional 6-h incubation in IV3 ( ) or F(ab )2 of Fc RIIIB Ab 3G8 ( ) before murine IgG1 IC medium. binding. 1884 IMMUNE COMPLEXES OPSONIZE APOPTOTIC NEUTROPHILS compared with nonapoptotic neutrophils by 39 and 79%, respectively (Fig. 2a). Next we examined the distributions of IC binding and Fc␥RIIA on apoptotic neutrophils. Murine ICs bound in a striking focal manner to apoptotic neutrophils (Fig. 2b). In contrast, little binding to nonapoptotic neutrophils was demonstrated. This observation is consistent with the increased lateral mobility of Fc␥RIIA in the membrane of apoptotic neutrophils, which enables IC binding through receptor clustering. In the absence of ICs, Fc␥RIIA (stained with FabЈ IV3) remained diffusely distributed in the neutrophil membrane, and apoptotic neutrophils stained more weakly than nonapoptotic cells (Fig. 2c).

Human immune complexes bind to apoptotic neutrophils We hypothesized that binding of ICs containing human IgG to apoptotic neutrophils would also be increased and mediated via Fc␥RIIA. Using the well-validated method of heat aggregation of IgG to generate ICs (30), we demonstrated that apoptotic neutrophils exhibited significantly increased binding at IC concentrations of 5Ð10 ␮g/ml compared with nonapoptotic neutrophils (mean, Downloaded from 2.1-fold increased binding at 10 ␮g/ml; p ϭ 0.0008). However, at higher IC concentrations, nonapoptotic neutrophils exhibited greater binding of ICs when the assay was performed on ice (Fig. 3a). Importantly, monomeric IgG did not bind significantly to apoptotic or nonapoptotic neutrophils (data not shown). The greater binding of human ICs to nonapoptotic cells at high IgG concen- FIGURE 3. Human ICs bind to apoptotic neutrophils via Fc␥RIIA. a, http://www.jimmunol.org/ trations can be explained by the numerical superiority of Fc␥RIIIB On ice, FITC-labeled human ICs bound more avidly to apoptotic neutro- on the nonapoptotic cells. When we repeated the binding studies at phils (F) at low concentrations (5Ð10 ␮g/ml), whereas at high concentra- E 37¡C, human IC binding to apoptotic neutrophils was increased tions binding to nonapoptotic cells ( ) predominated. b,At37¡C, apoptotic F across a wide range of concentrations (Fig. 3, b and c). In contrast, neutrophils ( ) exhibited increased human IC binding across a wide range of concentrations compared with nonapoptotic neutrophils (E). c, Repre- binding to nonapoptotic neutrophils was reduced. We further ex- sentative flow cytometry dot plot of human IC binding (10 ␮g/ml) to aged human neutrophils at 37¡C. Apoptotic cells have been colabeled with annexin V-PE. d, On ice, Fc␥RIIIB blockade revealed increased binding of human ICs to apoptotic neutrophils (F) compared with nonapoptotic neu- by guest on September 25, 2021 trophils (E). Additional blockade of Fc␥RIIA completely inhibited binding of ICs to apoptotic neutrophils (Œ), demonstrating that the enhanced IC binding is mediated by Fc␥RIIA. The effect of additional Fc␥RIIA blockade on binding to nonapoptotic neutrophils (‚) is also shown.

amined the differential roles of neutrophil FcRs by performing Ј binding studies with human ICs on ice in the presence of F(ab )2 3G8 Ab to block Fc␥RIIIB. Binding of human ICs to apoptotic neutrophils was minimally affected when Fc␥RIIIB was blocked, whereas binding to nonapoptotic cells was substantially reduced (compare Fig. 3a and Fig. 3d). Human IC binding was completely blocked by coincubation with FabЈ of Fc␥RIIA Ab IV3, indicating that these two FcRs mediate all IC binding to neutrophils. Despite reduced Fc␥RIIA surface expression, apoptotic neutrophils exhibit signiﬁcantly enhanced Fc␥RIIA-mediated IC binding compared with nonapoptotic neutrophils, which is revealed after Fc␥RIIIB blockade (Fig. 3d). These results lead us to propose that Fc␥RIIA on apoptotic neutrophils has increased avidity for human ICs, or is “enabled.”

Macrophage phagocytosis of IC-opsonized apoptotic neutrophils leads to release of proinflammatory cytokines We hypothesized that apoptotic neutrophils opsonized by ICs FIGURE 2. FcR expression and IC binding to apoptotic neutrophils. a, could present IgG-Fc to macrophage FcRs, so circumventing the The expression of Fc␥RI (CD64), Fc␥RIIA (CD32), and Fc␥RIIIB (CD16) normal anti-inflammatory clearance pathways for apoptotic cells. on apoptotic (f) and nonapoptotic (Ⅺ) human neutrophils. The expression of Fc␥RIIA and Fc␥RIIIB on apoptotic neutrophils is reduced. b, FITC- To test this hypothesis we used a well-validated in vitro phagocy- conjugated murine ICs (green) bound in clusters to the surface of apoptotic tosis assay, in which adherent human monocyte-derived macro- neutrophils (white arrows), which were identified by their characteristic phages were incubated with either “naked” control apoptotic neu- condensed chromatin (blue). c,Fc␥RIIA (green) is distributed diffusely on trophils or apoptotic neutrophils that had been opsonized by neutrophils and is down-regulated on apoptotic cells (white arrows). preincubation with human ICs. As a control for the ICs and to rule The Journal of Immunology 1885 out possible contamination of IgG, we also preincubated apoptotic neutrophils with ultracentrifuged aggregated IgG. Macrophage phagocytosis of IC-opsonized apoptotic neutrophils was vastly augmented compared with that of control neutrophils (Fig. 4, a and b). The percentage of macrophages that ingested one or more apoptotic cells (percent phagocytosis) was increased 1.9-fold over baseline (Fig. 4c), whereas the number of ingested cells per 100 macrophages (phagocytic index) was increased 4.4-fold (Fig. 4d), reflecting the substantial increase in the phagocytic capacity of each ingesting macrophage. Because FcR-mediated phagocytosis of opsonized microorgan- isms by macrophages leads to the generation of proinflammatory mediators (31), we tested whether macrophage phagocytosis of IC-opsonized apoptotic neutrophils led to up-regulation of inflammatory mediator release. We collected macrophage supernatants after interaction with control apoptotic neutrophils or apoptotic neutrophils that had been preincubated with ICs. Macrophage release of TNF-␣ and IL-6 was significantly increased after phagocytosis of IC-opsonized apoptotic neutrophils (Fig. 5). There was Downloaded from also a trend toward increased production of IL-1␤, IL-8, IL-10, and IL-12p70, but these differences did not reach statistical significance. For some cytokines there was a small increase in production after the ingestion of neutrophils incubated with ultracentrifuged aggregated IgG, which reflects the presence of residual aggregated

IgG in the ultracentrifuged material. TGF-␤ was undetectable http://www.jimmunol.org/ (Ͻ32 pg/ml) in all macrophage supernatants (data not shown). By way of comparison, supernatant concentrations of TNF-␣ and IL-6 increased to 34.9 Ϯ 4.9 and 171 Ϯ 38.9 pg/ml, respectively, after stimulation with bacterial LPS (n ϭ 3). There was no signiﬁcant cytokine release from neutrophils alone (i.e., in the absence of FIGURE 5. Cytokine release from macrophages that have phagocy- macrophages) after incubation with ICs, except for a small amount tosed IC-opsonized apoptotic neutrophils. Effect of phagocytosis of IC- of IL-8 (less than that from unstimulated macrophages; data not opsonized apoptotic neutrophils (Agg.IgG) on release of inﬂammatory cy- shown). tokines compared with phagocytosis of apoptotic neutrophils preincubated by guest on September 25, 2021 with PBS (control) or ultracentrifuged aggregated IgG (UF Agg.IgG). a, p Ͻ 0.05 vs control ,ء .TNF-␣; b, IL-6; c, IL-1␤; d, IL-8; e, IL-10; f, IL-12 and UF Agg.IgG; #, p Ͻ 0.01 vs control and UF Agg.IgG; ¤, p Ͻ 0.01 vs control (n ϭ 3).

Discussion We have shown that ICs containing either human IgG or murine IgG1 bound more avidly to apoptotic than nonapoptotic human neutrophils. Murine IC binding to apoptotic neutrophils was mediated by Fc␥RIIA, because binding could be completely inhibited by a well-characterized, function-blocking FabЈ Fc␥RIIA Ab (IV3) (32). In keeping with previous work, surface expression of Fc␥RIIA was reduced by 39% on apoptotic neutrophils in the present study. Previous studies have shown that Ab IV3 binds close to the IgG-binding region on the second Ig-like domain of Fc␥RIIA (33), so it is unlikely that there could be an apoptosis- associated change in Fc␥RIIA that leaves a functional molecule not recognized by IV3. Despite reduced expression of Fc␥RIIA and Fc␥RIIIB, human ICs bound preferentially to apoptotic neutrophils at low concentrations. Increased avidity of Fc␥RIIA on apoptotic neutrophils was unmasked at higher concentrations of ICs when the binding studies were performed at 37¡C, or by block- FIGURE 4. Macrophage phagocytosis of apoptotic neutrophils opso- ing Fc␥RIIIB with F(abЈ) mAb 3G8. We conclude that Fc␥RIIA nized with ICs. a, Control phagocytosis of apoptotic neutrophils (dark re- 2 on apoptotic neutrophils must be functionally activated or enabled. action product) by adherent human macrophages. b, Phagocytosis is substantially augmented by prior opsonization of apoptotic neutrophils with This is supported by our finding of striking clustering of ICs on the ␥ ICs. c, The percentage of macrophages that ingested one or more apoptotic surface of apoptotic neutrophils. Fc RIIA molecules on apoptotic neutrophils (percent phagocytosis). d, The number of apoptotic neutrophils neutrophils may exhibit increased lateral mobility and freedom of p Ͻ 0.05; #, p Ͻ movement in the plasma membrane that facilitates the clustering of ,ء .(ingested per 100 macrophages (phagocytic index 0.005 (compared with control; n ϭ 3). receptors necessary for efficient binding of multivalent ligands 1886 IMMUNE COMPLEXES OPSONIZE APOPTOTIC NEUTROPHILS such as ICs. The molecular events responsible for increased mem- matory cytokines. Cytokine release favors perpetuation of inflam- brane mobility of Fc␥RIIA remain to be elucidated. mation, whereas augmented phagocytosis favors resolution. An The different patterns of binding of murine and human ICs can important goal of future studies will be to determine precisely how be explained by the low avidity for murine IgG1 of Fc␥RIIIB (34, this balance of pro- and anti-inflammatory processes influences the 35), which is present at several times the density of Fc␥RIIA on final outcome of inflammation. freshly isolated nonapoptotic neutrophils (36). The situation may be very different in disease, however, because Fc␥RIIIB is shed Acknowledgments from the surface of viable neutrophils during cell activation (37). We are grateful to Linda Sharp for operating the confocal microscope and Furthermore, elastase present in the inflammatory milieu cleaves Jenny Woof for helpful discussions. Fc␥RIIIB from the neutrophil surface (38), whereas Fc␥RIIA is relatively elastase resistant (39). Selective loss of Fc␥RIIIB from References viable neutrophils means that preferential binding of ICs to apo- 1. Yamaguchi, H., H. Takeuchi, C. 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