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The Journal of Immunology, 2004, 172: 1882-1887.
Copyright © 2004 by The American Association of Immunologists

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

Simon P. Hart2, Karen M. Alexander and Ian Dransfield

Medical Research Council Center for Inflammation Research, University of Edinburgh Medical School, Edinburgh, United Kingdom


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
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{gamma}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-{alpha} and IL-6. Immune complexes may perturb the normal pathways for clearance of apoptotic neutrophils by augmenting their clearance at the price of proinflammatory cytokine release. This represents a novel mechanism by which immune complexes may modulate the resolution of inflammation.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Immune complexes (ICs)3 have been found in the blood and tissues in many human inflammatory diseases, and administration of ICs in animal models results in inflammation and fibrosis (1, 2). ICs containing IgG contribute to inflammation by activating complement and cross-linking leukocyte surface Fc receptors, resulting in cell activation (3). Human neutrophils express two receptors for complexed IgG, Fc{gamma}RIIA (CD32) and Fc{gamma}RIIIB (CD16), both of which have low affinity for monomeric IgG, but bind effectively to aggregates of Ab, such as ICs. Inappropriate or uncontrolled neutrophil activation results in the release of histotoxic products that may damage surrounding tissue and propagate the inflammatory response, leading ultimately to tissue destruction and scarring. There is strong evidence that neutrophil activation induced by ICs is fundamental in the pathogenesis of certain inflammatory diseases (4). Excessive production of ICs has been demonstrated in a variety of neutrophil-mediated diseases, such as rheumatoid arthritis, idiopathic pulmonary fibrosis, inflammatory bowel disease, and the acute respiratory distress syndrome, although an etiological role for ICs in these conditions has not been proven (5, 6, 7).

The physiological fate of the huge number of neutrophils recruited to sites of inflammation is death by apoptosis (8). Apoptosis is associated with down-regulation of cellular functions with potential to inflict host damage, such as stimulated release of granule contents (9, 10). Furthermore, surface membrane alterations on apoptotic cells leads to their recognition and phagocytosis by macrophages (8). Successful resolution of inflammation requires efficient removal of apoptotic cells, because in situations where excessive apoptotic cell load occurs development of autoimmune or chronic inflammatory pathology may ensue (11, 12). Down-regulation of proinflammatory mediator release and production of anti-inflammatory mediators have been widely quoted as universal macrophage responses to phagocytosis of apoptotic cells (13, 14), but proinflammatory sequelae to phagocytosis of apoptotic cells have also been reported (15, 16). Apoptosis is associated with many alterations in the protein and carbohydrate composition of the plasma membrane (10, 17, 18, 19). Some of these changes may be responsible for the binding potential of opsonins, including complement proteins (20, 21), collectins (22, 23), pentraxins (24, 25, 26), and IgM (27). These findings are important because although the process of macrophage clearance of apoptotic cells has been studied extensively under serum-free conditions in vitro, the presence of opsonins in the inflammatory milieu means it is unlikely that "naked" apoptotic cells would be encountered by macrophages in vivo.

We have recently reported that a murine IgG1 mAb bound specifically to apoptotic neutrophils by forming a complex with its Ag in serum (28). In the present study we investigated the role of Fc{gamma}RIIA in the binding of murine and human immune complexes to apoptotic neutrophils and tested the hypothesis that opsonization with immune complexes perturbs the normal anti-inflammatory macrophage response following phagocytosis of apoptotic neutrophils. We report that Fc{gamma}RIIA-mediated binding of ICs to human apoptotic neutrophils profoundly augments phagocytosis by macrophages, but leads to the production of proinflammatory cytokines. These data imply that opsonization of apoptotic neutrophils by ICs alters their pathway for clearance, and so influences the resolution of inflammation and hence the likelihood of developing chronic inflammatory or autoimmune disease.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Reagents

3G8 (anti-Fc{gamma}RIIIB) F(ab')2 were obtained from Ancell (Bayport, MN), and IV3 (anti-Fc{gamma}RIIA) Fab' were purchased from Medarex (Princeton, NJ). FITC-conjugated BN34 (IgG1 anti-biotin), biotinylated BSA, human IgG, and FITC-conjugated human IgG were obtained from Sigma-Aldrich (Poole, U.K.). F(ab')2 rabbit anti-human IgG was purchased from DAKO (Ely, U.K.). Cell culture materials and FCS were obtained from Invitrogen (Paisley, U.K.), and Percoll was purchased from Pharmacia (Little Chalfont, U.K.).

Cell isolation

Leukocytes were isolated from human peripheral blood by dextran sedimentation and discontinuous Percoll gradient centrifugation as previously described (18). All the neutrophil donors were G homozygous or G/A heterozygous at position 519 in exon 4 of the Fc{gamma}RIIA gene. These are the two most prevalent genotypes in our population and exhibit similar levels of murine IC binding to apoptotic neutrophils (28). No donor was A519 homozygous. Neutrophils were cultured in IMEM containing 10% autologous serum at 37°C in a 95% air/5% CO2 atmosphere for 20 h, during which time a proportion of the cells underwent apoptosis (29). Apoptosis was assessed by binding of PE-conjugated annexin V (Caltag, Towchester, U.K.) and assessment of characteristic morphology on stained cytocentrifuge preparations. Mononuclear cells were suspended in IMEM and adhered to 48-well cell culture plates during incubation at 37°C for 1 h. Adherent cells (>90% CD14-positive monocytes) were washed twice in PBS, and monocytes/macrophages were cultured 4–7 days in IMEM containing 10% autologous serum. For phagocytosis assays, apoptotic neutrophils were cultured for 44 h, at which time >80% had undergone apoptosis. Membrane integrity was routinely tested by exclusion of propidium iodide, and all preparations were >85% (44 h) or >95% (20 h) propidium iodide-negative.

Immune complex binding

Complexes of murine IgG1 were formed by combining 500 µg/ml biotinylated BSA with 170 µg/ml FITC-conjugated anti-biotin monoclonal IgG1 (clone BN34) in PBS for 30 min on ice. ICs were then diluted in PBS before use in neutrophil binding assays, and the concentration was expressed as the concentration of total IgG.

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 were removed by centrifugation at 10,000 x g for 2 min. In some experiments heat-aggregated IgG was ultracentrifuged at 300,000 x g in an Optima TLX ultracentrifuge with TLA100.3 rotor (Beckman Coulter, Fullerton, CA) for 60 min, which removed 80% of the ICs as assessed by cell binding of the supernatant using flow cytometry and FITC-conjugated F(ab')2 rabbit anti-human IgG. In blocking experiments we used the well-characterized, function-blocking Abs IV3 (Fab' anti-CD32; 10 µg/ml) and 3G8 (F(ab')2 anti-CD16; 10 µg/ml), which were incubated with aged neutrophils for 30 min on ice. FITC-labeled ICs were then incubated with neutrophils for 30 min on ice. Final staining with annexin V-PE was performed before two-color flow cytometric analysis on an EPICS XL machine (Coulter, Hialeah, FL).

Immunofluorescence microscopy

Aged neutrophils were labeled with murine ICs, fixed in 3% paraformaldehyde, permeabilized with 0.1% Triton X-100, counterstained with TO-PRO-3 (Molecular Probes, Leiden, The Netherlands), and cytocentrifuged 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 µg/ml heat-aggregated IgG for 30 min on ice, then washed twice. Adherent macrophages in 48-well plate were washed, and 2 x 106 aged neutrophils in 250 µl 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) hydrogen 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 examination of at least five fields (minimum of 400 cells) in duplicate wells with an inverted microscope. In parallel experiments, apoptotic neutrophils were incubated with macrophages for 6 h. The supernatants were harvested, centrifuged at 1000 x g for 5 min to remove cellular debris, and stored at -70°C before cytokine measurement. For comparison, macrophages were stimulated with 50 ng/ml Escherichia coli 018 LPS for 4 h, and the supernatants were harvested after an additional 6-h incubation in medium.

Cytokine measurement

Cytokines TNF-{alpha} (assay sensitivity, 3.7 pg/ml), IL-1{beta} (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) were measured in macrophage supernatants using a fluorescent bead-based sandwich assay (Human Inflammation Cytometric Bead Array, BD Biosciences, San Diego, CA) and analyzed on a FACSCalibur flow cytometer. TGF-{beta} was acid-activated in undiluted supernatants and measured by ELISA (assay sensitivity, 32 pg/ml; R&D Systems, Minneapolis, MN).

Data analysis and statistics

Results are presented as the mean ± SEM of at least three independent experiments using cells from different donors. Results were compared using either a paired t test or repeated measures ANOVA and Tukey-Kramer multiple comparisons test as appropriate, using InStat version 3 (GraphPad, San Diego, CA).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Complexes of murine IgG1 bind to apoptotic neutrophils

We generated labeled ICs by incubating biotinylated BSA with a FITC-conjugated mouse IgG1 anti-biotin mAb. Binding studies were performed on ice with aged human neutrophils, comprising both apoptotic and nonapoptotic cells, and apoptotic cells were identified by counterstaining with annexin V. Analysis by two-color flow cytometry revealed that ICs bound significantly more avidly to apoptotic neutrophils than nonapoptotic neutrophils over a range of concentrations (Fig. 1a). There was no further increase in IC binding to the small proportion (<5%) of membrane-permeable postapoptotic (necrotic) cells within the aged neutrophil population (data not shown).



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  		FIGURE 1. Murine IgG1 ICs bind to apoptotic neutrophils via Fc{gamma}RIIA. a, Preferential binding of FITC-conjugated murine IgG1 ICs to apoptotic human neutrophils (•) compared with nonapoptotic neutrophils ({circ}). b, Effects of preincubation of neutrophils with Fab' of Fc{gamma}RIIA Ab IV3 ({blacktriangleup}) or F(ab')2 of Fc{gamma}RIIIB Ab 3G8 ({diamondsuit}) before murine IgG1 IC binding.

 
IC binding to apoptotic neutrophils is mediated by Fc{gamma}RIIA

To determine the involvement of neutrophil Fc receptors in binding of murine IgG1 ICs, we preincubated neutrophils with anti-FcR Abs that are known to specifically block IC binding. Fab' of Fc{gamma}RIIA Ab IV3 completely inhibited binding of murine IgG1 ICs to both apoptotic and nonapoptotic neutrophils, whereas F(ab')2 of Fc{gamma}RIIIB Ab 3G8 had no effect (Fig. 1b). These results indicate that neutrophil Fc{gamma}RIIA is wholly responsible for binding murine IgG1 ICs and imply that Fc{gamma}RIIA on apoptotic neutrophils exhibits increased avidity for ICs.

Distribution of bound ICs on apoptotic neutrophils

As Fc{gamma}RIIA mediated IC binding, we re-examined the expression of FcRs on apoptotic and nonapoptotic neutrophils. The expression of Fc{gamma}RIIA and Fc{gamma}RIIIB was reduced on apoptotic neutrophils compared with nonapoptotic neutrophils by 39 and 79%, respectively (Fig. 2a). Next we examined the distributions of IC binding and Fc{gamma}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{gamma}RIIA in the membrane of apoptotic neutrophils, which enables IC binding through receptor clustering. In the absence of ICs, Fc{gamma}RIIA (stained with Fab' IV3) remained diffusely distributed in the neutrophil membrane, and apoptotic neutrophils stained more weakly than nonapoptotic cells (Fig. 2c).



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  		FIGURE 2. FcR expression and IC binding to apoptotic neutrophils. a, The expression of Fc{gamma}RI (CD64), Fc{gamma}RIIA (CD32), and Fc{gamma}RIIIB (CD16) on apoptotic ({blacksquare}) and nonapoptotic ({square}) human neutrophils. The expression of Fc{gamma}RIIA and Fc{gamma}RIIIB on apoptotic neutrophils is reduced. b, FITC-conjugated murine ICs (green) bound in clusters to the surface of apoptotic neutrophils (white arrows), which were identified by their characteristic condensed chromatin (blue). c, Fc{gamma}RIIA (green) is distributed diffusely on neutrophils and is down-regulated on apoptotic cells (white arrows).

 
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{gamma}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 5–10 µg/ml compared with nonapoptotic neutrophils (mean, 2.1-fold increased binding at 10 µg/ml; p = 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 concentrations can be explained by the numerical superiority of Fc{gamma}RIIIB on the nonapoptotic cells. When we repeated the binding studies at 37°C, human IC binding to apoptotic neutrophils was increased across a wide range of concentrations (Fig. 3, b and c). In contrast, binding to nonapoptotic neutrophils was reduced. We further examined the differential roles of neutrophil FcRs by performing binding studies with human ICs on ice in the presence of F(ab')2 3G8 Ab to block Fc{gamma}RIIIB. Binding of human ICs to apoptotic neutrophils was minimally affected when Fc{gamma}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 Fab' of Fc{gamma}RIIA Ab IV3, indicating that these two FcRs mediate all IC binding to neutrophils. Despite reduced Fc{gamma}RIIA surface expression, apoptotic neutrophils exhibit significantly enhanced Fc{gamma}RIIA-mediated IC binding compared with nonapoptotic neutrophils, which is revealed after Fc{gamma}RIIIB blockade (Fig. 3d). These results lead us to propose that Fc{gamma}RIIA on apoptotic neutrophils has increased avidity for human ICs, or is "enabled."



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  		FIGURE 3. Human ICs bind to apoptotic neutrophils via Fc{gamma}RIIA. a, On ice, FITC-labeled human ICs bound more avidly to apoptotic neutrophils (•) at low concentrations (5–10 µg/ml), whereas at high concentrations binding to nonapoptotic cells ({circ}) predominated. b, At 37°C, apoptotic neutrophils (•) exhibited increased human IC binding across a wide range of concentrations compared with nonapoptotic neutrophils ({circ}). c, Representative flow cytometry dot plot of human IC binding (10 µg/ml) to aged human neutrophils at 37°C. Apoptotic cells have been colabeled with annexin V-PE. d, On ice, Fc{gamma}RIIIB blockade revealed increased binding of human ICs to apoptotic neutrophils (•) compared with nonapoptotic neutrophils ({circ}). Additional blockade of Fc{gamma}RIIA completely inhibited binding of ICs to apoptotic neutrophils ({blacktriangleup}), demonstrating that the enhanced IC binding is mediated by Fc{gamma}RIIA. The effect of additional Fc{gamma}RIIA blockade on binding to nonapoptotic neutrophils ({triangleup}) is also shown.

 
Macrophage phagocytosis of IC-opsonized apoptotic neutrophils leads to release of proinflammatory cytokines

We hypothesized that apoptotic neutrophils opsonized by ICs could present IgG-Fc to macrophage FcRs, so circumventing the normal anti-inflammatory clearance pathways for apoptotic cells. To test this hypothesis we used a well-validated in vitro phagocytosis assay, in which adherent human monocyte-derived macrophages were incubated with either "naked" control apoptotic neutrophils or apoptotic neutrophils that had been opsonized by preincubation with human ICs. As a control for the ICs and to rule 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.



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  		FIGURE 4. Macrophage phagocytosis of apoptotic neutrophils opsonized with ICs. a, Control phagocytosis of apoptotic neutrophils (dark reaction product) by adherent human macrophages. b, Phagocytosis is substantially augmented by prior opsonization of apoptotic neutrophils with ICs. c, The percentage of macrophages that ingested one or more apoptotic neutrophils (percent phagocytosis). d, The number of apoptotic neutrophils ingested per 100 macrophages (phagocytic index). *, p < 0.05; #, p < 0.005 (compared with control; n = 3).

 
Because FcR-mediated phagocytosis of opsonized microorganisms 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-{alpha} and IL-6 was significantly increased after phagocytosis of IC-opsonized apoptotic neutrophils (Fig. 5). There was also a trend toward increased production of IL-1{beta}, 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-{beta} was undetectable (<32 pg/ml) in all macrophage supernatants (data not shown). By way of comparison, supernatant concentrations of TNF-{alpha} and IL-6 increased to 34.9 ± 4.9 and 171 ± 38.9 pg/ml, respectively, after stimulation with bacterial LPS (n = 3). There was no significant cytokine release from neutrophils alone (i.e., in the absence of macrophages) after incubation with ICs, except for a small amount of IL-8 (less than that from unstimulated macrophages; data not shown).



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  		FIGURE 5. Cytokine release from macrophages that have phagocytosed IC-opsonized apoptotic neutrophils. Effect of phagocytosis of IC-opsonized apoptotic neutrophils (Agg.IgG) on release of inflammatory cytokines compared with phagocytosis of apoptotic neutrophils preincubated with PBS (control) or ultracentrifuged aggregated IgG (UF Agg.IgG). a, TNF-{alpha}; b, IL-6; c, IL-1{beta}; d, IL-8; e, IL-10; f, IL-12. *, p < 0.05 vs control and UF Agg.IgG; #, p < 0.01 vs control and UF Agg.IgG; §, p < 0.01 vs control (n = 3).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
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{gamma}RIIA, because binding could be completely inhibited by a well-characterized, function-blocking Fab' Fc{gamma}RIIA Ab (IV3) (32). In keeping with previous work, surface expression of Fc{gamma}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{gamma}RIIA (33), so it is unlikely that there could be an apoptosis-associated change in Fc{gamma}RIIA that leaves a functional molecule not recognized by IV3. Despite reduced expression of Fc{gamma}RIIA and Fc{gamma}RIIIB, human ICs bound preferentially to apoptotic neutrophils at low concentrations. Increased avidity of Fc{gamma}RIIA on apoptotic neutrophils was unmasked at higher concentrations of ICs when the binding studies were performed at 37°C, or by blocking Fc{gamma}RIIIB with F(ab')2 mAb 3G8. We conclude that Fc{gamma}RIIA on apoptotic neutrophils must be functionally activated or enabled. This is supported by our finding of striking clustering of ICs on the surface of apoptotic neutrophils. Fc{gamma}RIIA molecules on apoptotic neutrophils may exhibit increased lateral mobility and freedom of movement in the plasma membrane that facilitates the clustering of receptors necessary for efficient binding of multivalent ligands such as ICs. The molecular events responsible for increased membrane mobility of Fc{gamma}RIIA remain to be elucidated.

The different patterns of binding of murine and human ICs can be explained by the low avidity for murine IgG1 of Fc{gamma}RIIIB (34, 35), which is present at several times the density of Fc{gamma}RIIA on freshly isolated nonapoptotic neutrophils (36). The situation may be very different in disease, however, because Fc{gamma}RIIIB is shed from the surface of viable neutrophils during cell activation (37). Furthermore, elastase present in the inflammatory milieu cleaves Fc{gamma}RIIIB from the neutrophil surface (38), whereas Fc{gamma}RIIA is relatively elastase resistant (39). Selective loss of Fc{gamma}RIIIB from viable neutrophils means that preferential binding of ICs to apoptotic neutrophils may be even more apparent in inflammatory diseases. The critical role of Fc{gamma}RIIA in IC binding to apoptotic neutrophils is supported by our previous observation of markedly reduced binding to cells from individuals homozygous for an adenine nucleotide at position 519 of the Fc{gamma}RIIA gene (28). Further studies are needed to address the role of the Fc{gamma}RIIA genotype in binding of human IgG subclasses to apoptotic neutrophils, and whether this has any influence on disease.

Macrophage uptake of apoptotic cells in vitro is much less efficient than phagocytosis of opsonized particles, such as zymosan or erythrocytes coated with rabbit IgG (40). In the present study we have demonstrated that apoptotic neutrophils opsonized with human ICs were recognized by macrophages in a very different way from naked apoptotic neutrophils. There was a 4.4-fold increase in the number of IC-opsonized apoptotic neutrophils that were phagocytosed by macrophages during a 1-h assay. It is likely that macrophage FcRs conferred this augmented phagocytosis, as free IgG-Fc protruding from the surface of the IC-opsonized apoptotic neutrophils may be presented to neighboring macrophages. To test this hypothesis we examined the macrophage response to phagocytosis of IC-opsonized apoptotic neutrophils by measuring cytokine release. As a control we used aggregated human IgG that had been ultracentrifuged, which eliminated ~80% of the ICs, as assessed by cell binding in flow cytometry. Our experiments revealed a statistically significant increase in macrophage production of TNF-{alpha} and IL-6 after phagocytosis of IC-opsonized apoptotic neutrophils. A proinflammatory macrophage response was supported by increased production of other proinflammatory cytokines, but these differences did not reach statistical significance. It was noteworthy that despite dramatically increased levels of phagocytosis of IC-opsonized apoptotic neutrophils, cytokine release was still relatively low compared with levels after stimulation of macrophages with bacterial LPS. One explanation for the blunted cytokine response is that macrophage interaction with IC-coated apoptotic neutrophils engages two different uptake pathways with opposing functional consequences: an FcR-mediated mechanism that promotes proinflammatory mediator release, and an apoptotic cell receptor-mediated pathway that down-regulates proinflammatory mediator release. Blockade of either mechanism might shift the balance of cytokine production. For example, in IC-associated inflammatory diseases, elastase-mediated cleavage of the macrophage phosphatidylserine receptor (41) could result in unopposed FcR-mediated phagocytosis of IC-opsonized apoptotic neutrophils, leading to significantly increased proinflammatory cytokine production.

Many disease processes are associated with the production, circulation, and tissue deposition of ICs. We have shown that ICs opsonize apoptotic neutrophils and augment their phagocytosis by macrophages. This phenomenon may be important for the clearance of the vast numbers of neutrophils that are recruited to the tissues and die locally by undergoing apoptosis. The price to be paid is a small, but significant, increase in the release of inflammatory cytokines. Cytokine release favors perpetuation of inflammation, whereas augmented phagocytosis favors resolution. An important goal of future studies will be to determine precisely how this balance of pro- and anti-inflammatory processes influences the final outcome of inflammation.


   Acknowledgments
 
We are grateful to Linda Sharp for operating the confocal microscope and Jenny Woof for helpful discussions.


   Footnotes
 
1 This work was supported by a Medical Research Council Clinician Scientist Fellowship. Back

2 Address correspondence and reprint requests to Dr. Simon P. Hart, Medical Research Council Center for Inflammation Research, University of Edinburgh Medical School, Teviot Place, Edinburgh, U.K. EH8 9AG. E-mail address: s.hart{at}ed.ac.uk Back

3 Abbreviation used in this paper: IC, immune complex. Back

Received for publication June 10, 2003. Accepted for publication November 18, 2003.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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