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. 2004 Dec;138(3):447–452. doi: 10.1111/j.1365-2249.2004.02638.x

Divalent cation-dependent and -independent augmentation of macrophage phagocytosis of apoptotic neutrophils by CD44 antibody

S VIVERS 1, S J HEASMAN 1, S P HART 1, I DRANSFIELD 1
PMCID: PMC1809234  PMID: 15544621

Abstract

Phagocytosis of apoptotic neutrophils by macrophages is required for resolution of an inflammatory response. Removal of intact apoptotic neutrophils prevents the release of cytotoxic granules that would otherwise cause tissue damage and may lead to development of fibrosis. Importantly, macrophage phagocytosis of apoptotic neutrophils fails to induce release of proinflammatory mediators, consistent with a ‘safe’ pathway for disposal of potentially harmful inflammatory cells. One pathway for increasing phagocytosis of apoptotic cells to allow matching of tissue phagocyte capacity to apoptotic cell load in vitro is via antibody-mediated cross-linking of CD44, providing a mechanism for limiting tissue damage during resolution of inflammation. In this study, we have defined divalent cation-dependent and -independent actions of the CD44 antibody. For the divalent cation-independent CD44 antibody effect, we provide evidence that ‘enabled’ CD32 on the apoptotic neutrophil binds to intact CD44 antibody on the macrophage surface. One implication is that macrophages can phagocytose apoptotic neutrophils that are ‘tethered’ to the macrophage surface in a manner that is independent of defined apoptotic mechanisms. These data also provide an explanation for the greater efficacy of intact CD44 antibody when compared with F(ab′)2 fragments.

Keywords: apoptosis, inflammation, granulocyte, macrophage, phagocytosis

INTRODUCTION

Macrophage phagocytosis of apoptotic neutrophils is an essential process in the normal resolution of an inflammatory response [1]. Although the physiological fate of neutrophils recruited to inflammatory sites is to undergo apoptosis, a process which maintains membrane integrity and prevents the release of cytotoxic granules, uncontrolled granule content release may contribute to host tissue damage that is associated with development of disease. Apoptotic neutrophils are therefore swiftly recognized and then phagocytosed by local macrophages [1]. Importantly, ingestion of apoptotic neutrophils by macrophages does not stimulate the release of proinflammatory mediators, thereby providing a mechanism for the safe disposal of potentially harmful inflammatory cells [2]. However, if the rate of cell death by apoptosis exceeds the rate of clearance by macrophages, apoptotic cells can undergo secondary necrosis, resulting in the release of histotoxic substances and damage to surrounding tissues. Indeed, impaired clearance of apoptotic neutrophils has been linked to the development of inflammatory conditions such as acute respiratory distress syndrome (ARDS), fibrosing alveolitis, rheumatoid arthritis and ulcerative colitis [3]. We have previously identified CD44 as a potentially novel mechanism for rapidly matching tissue phagocyte capacity to apoptotic cell load that is distinct from soluble mediators or cell–cell interactions [4,5]. Ligation of CD44 may therefore determine the rate of apoptotic neutrophil clearance by macrophages, a finding supported by recent in vivo studies [6].

CD44 is a cell-surface glycoprotein expressed on many cell types such as leucocytes, erythrocytes, fibroblasts and a number of tumour cells [7]. It is heavily glycosylated and can be expressed as a number of variant isoforms that may influence its function [8,9]. CD44 acts as a receptor for a variety of extracellular matrix molecules including hyaluronan [10,11], fibronectin [12], fibrin [13] and collagen [14,15]. Many diverse functions have been attributed to CD44 including involvement in cellular adhesion and migration, activation and proliferation of lymphocytes, cytocidal activity of natural killer (NK) cells and tumour metastasis [7]. Binding of bivalent CD44 monoclonal antibody (MoAb) has been shown to augment macrophage phagocytosis of apoptotic neutrophils [16]. This effect appears to be specific for apoptotic neutrophils, as phagocytosis of apoptotic lymphocytes or erythrocytes opsonized with immunoglobulin was not augmented after MoAb-induced cross-linking of macrophage CD44. Although the mechanism of this effect remains to be fully characterized, CD44 MoAb may promote redistribution of cell surface CD44 facilitating phagocyte–apoptotic neutrophil interactions, or alternatively may transduce a signal within the macrophage that alters phagocytic capacity.

To characterize further the molecular basis for the augmentation of macrophage phagocytosis of apoptotic neutrophils following CD44 MoAb binding, we have undertaken studies of the effects of depletion of divalent cations. In previous studies using microscopy-based assay systems, vigorous washing of adherent macrophages was required to ensure removal of bound target cells leading to detachment of macrophages (approximately 20% of total cells [17]), an effect that was greater when divalent cation-free conditions were used. We have therefore used a carefully validated technique for assessment of phagocytosis by flow cytometry which obviates the need for washing, minimizing the impact of cell losses that limit microscopy-based studies. In this report, we have identified both divalent cation-independent and -dependent components to the augmentation of phagocytosis following CD44 MoAb binding to macrophages. For the divalent cation-independent effect, blockade of CD32 on the apoptotic neutrophil was found to be inhibitory. In support of this finding, F(ab′)2 fragments of CD44 MoAb fail to augment phagocytosis in a cation-deplete environment. While these findings are somewhat counter-intuitive, as both apoptotic and non-apoptotic neutrophils express CD32, our recent studies have demonstrated that CD32 is enabled on apoptotic neutrophils [18]. In summary, augmentation of macrophage phagocytosis of apoptotic neutrophils by CD44 MoAb involves two distinct effects: first, a direct effect of cross-linking that is observed with F(ab′)2 fragments of CD44 MoAb that requires the presence of divalent cations; and secondly, a divalent cation-independent tethering of apoptotic neutrophils via CD32 to the Fc portion of MoAb bound to the macrophage surface.

MATERIALS AND METHODS

Reagents and antibodies

All cell culture materials were from Invitrogen (Paisley, UK) and other reagents were from Sigma (Poole, UK) unless stated otherwise. The following MoAb were used: 5A4 (anti-CD44, a kind gift of Dr Graeme Dougherty, San Francisco, CA, USA) IV3 (anti-CD32 Fab′ fragments for function blocking studies; Medarex, Annandale, NJ, USA), IVC7 (anti-CD36; CLB, Amsterdam), 61D3 (anti-CD14 provided by Dr A. Devitt, Edinburgh, UK), DFT-1 (anti-CD43, Serotec, Oxford, UK) and T29/33 (anti-CD45, DakoCytomation, Ely, UK). IgG1 isotype control MoAb was purchased from Beckman-Coulter, High Wycombe (UK). All MoAb were used at saturating concentrations as determined by indirect immunofluorescence techniques and flow cytometric analysis (FACSCaliber, Becton-Dickinson, Oxford, UK).

Purification of CD44 F(ab)2 antibodies

CD44 MoAb (5A4) was purified from hybridoma supernatant using protein A affinity chromatography and pepsin digestion (1% wt/wt) to generate F(ab′)2 fragments. Residual intact antibody was removed by three rounds of depletion using protein A agarose affinity resin and the resulting F(ab′)2 fragments were analysed using sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) under non-reducing conditions to confirm purity. Indirect immunofluorescence analysis together with flow cytometry was used to confirm that preparation of antibody fragments did not compromise binding and that antibody fragments were used at saturating concentrations in other assays.

Cell isolation

Monocytes and neutrophils were isolated from human peripheral blood by dextran sedimentation and discontinuous Percoll™ (Amersham Pharmacia, St Albans, UK) gradient centrifugation, as described previously [19]. Mononuclear cells were suspended at 4 × 106/ml in IMDEM and allowed to adhere to cell culture plates during incubation at 37°C for 1 h. Non-adherent cells (mostly lymphocytes) were removed and adherent cells were washed three times in Hanks’ balanced salt solution without Ca2+/Mg2+. Adherent monocytes (approximately 90% CD14+) were then cultured in Iscove's modification of Dulbecco's modified Eagles’ medium (IDMEM) containing 10% autologous serum for 5 days (monocyte-derived macrophages). Neutrophils were incubated at 2 × 107/ml with 22 µm 5-chloromethylfluorescein diacetate (CMFDA, Molecular Probes, Oregon, USA) at 37°C for 15 min. Labelled neutrophils were then cultured at 4 × 106/ml in IDMEM with 10% autologous serum for 24 h, during which time they underwent spontaneous apoptosis [1]. Apoptosis was assessed either by microscopic inspection of cytocentrifuge preparations that had been stained with Diff-Quik™ (Baxter Healthcare, Glasgow, UK) as per the manufacturer's protocol, or alternatively assessed by dual immunofluorescence analysis using annexinV-FITC in combination with propidium iodide to determine the proportions of apoptotic and necrotic cells.

Phagocytosis of apoptotic neutrophils

Macrophage phagocytosis of apoptotic neutrophils was assessed using a flow cytometric assay that has been validated carefully to quantify internalization of apoptotic targets and not surface binding [17]. Monocyte-derived macrophages were washed with IDMEM and incubated with CD44 MoAb diluted in IDMEM for 15 min at 37°C. Excess MoAb was removed and monocyte-derived macrophages washed with 25 m m Hepes (pH 7·4) buffered saline (HBS) prior to adding 2 × 106 CMFDA-labelled apoptotic neutrophils in 0·5 ml of either IDMEM, chelex-treated IDMEM (divalent cation-depleted) or HBS (divalent cation-free) to each well for 60 min at 37°C. At the end of the assay, a solution of 0·25% trypsin/1 m m EDTA was added to each well and the cells incubated at 37°C for 15 min to detach all monocyte-derived macrophages and remove non-internalized neutrophils. After a further 15 min incubation on ice, the proportion of macrophages that exhibited increased fluorescence (corresponding to phagocytosis of fluorescently labelled apoptotic neutrophils) was determined by flow cytometry using a FACSCalibur analyser with post-acquisition data analysis performed using cellquest software (Becton-Dickinson).

The effects of antibody/ligand inhibitors upon phagocytosis

Monocyte-derived macrophages were washed with HBS without Ca2+/Mg2+ and incubated with CD44 MoAb diluted in HBS for 15 min at 37°C. Unbound antibody was washed off and monocyte-derived macrophages were pretreated with saturating concentrations of putative inhibitory MoAb, or 1 mg/ml fucoidan (scavenger receptor inhibitor) or 50 U/ml heparin (heparan sulphate proteoglycan inhibitor) prior to assessment of phagocytosis of apoptotic neutrophils using the flow cytometric assay described above.

RESULTS

Macrophage phagocytosis of apoptotic neutrophils in divalent-cation depleted conditions is augmented by CD44 MoAb

The requirement for the presence of either Ca2+ or Mg2+ for adhesion molecules to bind to their cognate ligands provides a simple method for discrimination of potential receptor pathways involved in CD44 MoAb-augmented phagocytosis of apoptotic neutrophils. Control (untreated) or CD44 MoAb-treated monocyte-derived macrophages were challenged with apoptotic neutrophils in the presence or absence of divalent cations and the percentage of phagocytosis was determined (Fig. 1). For control monocyte-derived macrophages, the absence of divalent cations (HBS) virtually eliminated phagocytosis. To confirm that this effect was not due to a change in media conditions used, we determined the levels of phagocytosis in IDMEM that had been depleted specifically of divalent cations using chelex resin. Phagocytosis in IDMEM was reduced from 34·2 ± 6 to 6·6 ± 2 in chelex-treated IDMEM. In contrast, CD44 MoAb-treated macrophage phagocytosis of apoptotic neutrophils was still observed in the absence of divalent cations, suggesting that CD44 MoAb binding to monocyte-derived macrophages may allow engagement of distinct divalent cation-independent apoptotic cell recognition mechanisms.

Fig. 1.

Fig. 1

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CD44 MoAb-augmented macrophage phagocytosis of apoptotic neutrophils in the presence and absence of divalent cations. Adherent human monocyte-derived macrophages were preincubated with either CD44 MoAb or medium alone for 15 min, washed and then incubated with CM-CFDA-labelled apoptotic neutrophils in the presence or absence of divalent cations as indicated for 60 min. (a) Typical flow cytometric profiles demonstrating macrophage phagocytosis of fluorescent apoptotic neutrophils are shown. (b) Phagocytosis of apoptotic neutrophils by macrophages following incubation with CD44 MoAb (grey bars) or IDMEM (black bars) was determined in duplicate samples by flow cytometry. Results are expressed as the mean ± s.e.m. of 22 independent experiments.

CD32 is required for CD44 MoAb augmented phagocytosis of apoptotic neutrophils in the absence of divalent cations

To investigate the molecular basis for the CD44 MoAb augmented phagocytosis, we tested the effects of inhibitors of putative divalent cation-independent recognition pathways. Pretreatment with fucoidan (scavenger receptor inhibitor), heparin (heparan sulphate proteoglycan inhibitor), anti-CD36 or anti-CD14 prior to incubation with apoptotic neutrophils under divalent cation-free conditions failed to inhibit the CD44 MoAb augmented phagocytosis (Fig. 2). In contrast, CD32 MoAb completely blocked CD44 MoAb-augmented macrophage phagocytosis of apoptotic neutrophils in the absence of divalent cations (Fig. 2). We did not observe inhibition of phagocytosis by CD32 MoAb in the presence of divalent cations (data not shown).

Fig. 2.

Fig. 2

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The effect of inhibitory antibodies and ligands upon CD44 MoAb-augmented phagocytosis of apoptotic neutrophils by macrophages. Macrophages were incubated with CD44 MoAb (grey bars) or IDMEM (black bars) for 15 min, washed and then incubated with the antibody/ligand indicated for a further 15 min. Macrophages were incubated with apoptotic neutrophils for 60 min in the absence of divalent cations. Macrophage phagocytosis of apoptotic neutrophils in duplicate samples was then determined by flow cytometry. Results are expressed as mean ± s.e.m. of at least three independent experiments.

Role of macrophage or apoptotic neutrophil CD32 in CD44 MoAb augmented phagocytosis

We next used MoAb preincubation strategies to determine whether  CD32  was  required on the macrophage or the neutrophil or both cell types. When macrophages were pretreated with CD32 MoAb there was no inhibitory effect upon CD44 MoAb-augmented phagocytosis in divalent cation-free conditions (Fig. 3). These data suggested that macrophage CD32 was not mediating the divalent cation-independent CD44 MoAb-augmented phagocytosis. In contrast, when neutrophils were preincubated with CD32 MoAb, divalent cation-independent phagocytosis was inhibited. Although neutrophils also express another receptor for IgG (CD16), apoptosis is associated with >90% reduction in surface expression of this receptor [19] and CD16 blockade does not prevent aggregated murine IgG binding to apoptotic neutrophils [18]. These data imply that CD32 on the apoptotic neutrophil is able to mediate binding to intact CD44 MoAb bound to macrophages. Based upon these results, one prediction would be that binding of antibody to any highly expressed macrophage surface receptor would also augment phagocytosis under divalent cation-free conditions.

Fig. 3.

Fig. 3

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Effect of pretreatment of macrophages or neutrophils with anti-CD32 MoAb. Macrophages or apoptotic neutrophils were preincubated with CD32 MoAb for 15 min and then washed with medium. Macrophages were the incubated with either CD44 MoAb (grey bars) or IDMEM (black bars) for 15 min, washed and incubated with apoptotic neutrophils in the absence of divalent cations for 60 min. Macrophage phagocytosis of apoptotic neutrophils in duplicate samples was then determined for each experiment by flow cytometry. Results shown are mean ± s.e.m. of at least three independent experiments.

Effects of MoAb binding to other highly expressed macrophage receptors

We next tested whether binding of MoAb to other highly expressed surface receptors would also augment macrophage phagocytosis of apoptotic neutrophils. Macrophages were incubated with MoAb of the same isotype (IgG1) against either CD43, CD44 or CD45, molecules that are expressed at similar levels on the macrophage surface (data not shown). We found that preincubation of macrophages with CD43 MoAb resulted in a slight augmentation of phagocytosis in the presence of divalent cations (from 18·8 ± 7% to 33·5 ± 7%, n = 3) when compared to medium alone. In a separate series of experiments, CD45 MoAb pretreatment also increased phagocytosis of apoptotic neutrophils in the presence of divalent cations from 24·4 ± 3% to 37·7 ± 6% (n = 3). In contrast, CD44 treatment resulted in a robust increase of phagocytosis in the presence of divalent cations (66·6 ± 8% and 64·4 ± 5% for the CD43 MoAb and CD45 MoAb experiments, respectively). These observations would be consistent with macrophage-bound antibody providing a ligand for neutrophil receptors for IgG and thereby contributing to apoptotic cell uptake. Based upon these observations, we would have predicted that CD43 antibodies would also augment phagocytosis under divalent cation-free conditions. However, examination of macrophage phagocytosis following treatment with CD43 MoAb did not support this assertion (Fig. 4).

Fig. 4.

Fig. 4

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Distinct effects of MoAb binding to highly expressed receptors and CD44 MoAb. Macrophages were preincubated with either CD44 MoAb or CD43 MoAb or untreated control for 15 min, washed and then incubated with apoptotic neutrophils in the absence of divalent cations for 60 min. Macrophage phagocytosis of apoptotic neutrophils in duplicate samples was then determined by flow cytometry. Results are shown as mean ± s.e.m. of six independent experiments.

Effects of CD44 F(ab′)2 fragments upon phagocytosis of apoptotic neutrophils

We next investigated the effects of F(ab′)2 fragments of CD44 MoAb. In the presence of divalent cations, treatment of macrophages with F(ab′)2 resulted in augmentation of phagocytosis of apoptotic neutrophils. In contrast, in the absence of divalent cations there was no effect (Fig. 5). These data demonstrate a specific effect of ligation of CD44 by MoAb upon macrophage phagocytic function that is independent of involvement of CD32.

Fig. 5.

Fig. 5

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Effects of CD44 F(ab′)2 fragments upon macrophage phagocytosis of apoptotic neutrophils. (a) Macrophages were preincubated with intact CD44 MoAb (grey bars), CD44 F(ab′)2 fragments (white bars) or medium alone (black bars) for 15 min, washed and then incubated with apoptotic neutrophils for 60 min in the presence of divalent cations. Macrophage phagocytosis of apoptotic neutrophils in duplicate samples was then determined by flow cytometry. Results are shown as the mean ± s.e.m. of five independent experiments. Macrophages were incubated with intact CD44 MoAb (grey bars), CD44 F(ab′)2 fragments (white bars) or medium alone (black bars) for 15 min, washed and then incubated with apoptotic neutrophils for 60 min in the absence of divalent cations. Macrophage phagocytosis of apoptotic neutrophils in duplicate samples was then determined by flow cytometry. Results are shown as the mean ± s.e.m. of five independent experiments.

DISCUSSION

In this study, we have used removal of divalent cations to eliminate the potential contribution of integrins [20] or C-type lectins [21] to CD44 MoAb-mediated augmentation of macrophage phagocytosis of apoptotic neutrophils. We have confirmed that phagocytosis of apoptotic neutrophils by untreated macrophages requires exclusively divalent cation-dependent recognition pathways. In contrast, we found that CD44 MoAb augmented macrophage phagocytosis had both divalent cation-dependent and independent components. Our data suggest that augmentation of apoptotic cell phagocytosis by macrophages following CD44 MoAb treatment in the absence of divalent cations requires tethering of the apoptotic neutrophils via CD32 to CD44 antibodies bound to the macrophage surface. This interaction may facilitate engagement of apoptotic cell recognition pathways that lead subsequently to internalization. Despite similar levels of binding of CD44 and CD43 MoAb (both IgG1) to the macrophage surface, we failed to observe increased phagocytosis by CD43 MoAb in the absence of divalent cations. At present the reason for this difference is not clear. One possibility is that this may reflect the differences in the distribution of CD44 and CD43 in the macrophage membrane.

Data from in vitro studies of macrophage recognition of apoptotic cells has implicated involvement of a number of distinct, unrelated molecular pathways (reviewed in [22]). It has been proposed recently that many of these molecules or even well-established opsonins [23] may be involved in tethering of apoptotic targets. It is possible that internalization of tethered apoptotic targets is then driven via a common phosphatidylserine receptor-dependent mechanism [24]. Recently we have demonstrated that CD32 expressed on apoptotic neutrophils becomes ‘enabled’, mediating binding to multimeric IgG [18]. Thus, despite an overall reduction in levels of expression of CD32 on apoptotic neutrophils when compared with non-apoptotic neutrophils, we have demonstrated that only apoptotic neutrophils become tethered to CD44 MoAb bound to the macrophage surface and that binding is mediated by CD32 on the neutrophil. Although it is possible that both apoptotic and non-apoptotic neutrophils are able to bind to CD44 MoAb on the macrophage surface and that our assay conditions only allow detection of phagocytosis, we suggest that our data indicate that apoptotic neutrophils tether specifically to antibody bound to the macrophage surface.

In this study we have demonstrated that anti-CD44 F(ab′)2 fragments were capable of augmenting phagocytosis of apoptotic neutrophils, eliminating possible Fc receptor-dependent effects in the augmentation of phagocytosis of apoptotic neutrophils following CD44 ligation. As found for untreated macrophages, phagocytosis requires the presence of divalent cations, raising the possibility that CD44 MoAb mediated cross-linking increases the efficiency of existing recognition pathways. One cautionary note is that promotion of apoptotic neutrophil tethering by MoAb bound to the macrophage surface (see Fig. 4) may act to counter inhibitory effects of antibodies in other studies of apoptotic targets bearing Fc receptors and potentially ‘mask’ true inhibition. Such a possibility may account for lesser inhibition of macrophage uptake of CD32 bearing cell types compared with CD32 negative apoptotic targets [25]. Finally, our data have important implications for use of antibodies to investigate apoptotic cell clearance in vivo. While the shorter in vivo half-life of proteolytic fragments of antibodies may restrict their use therapeutically and in functional studies [26], intact antibodies may have additional regulatory effects upon macrophage clearance of CD32 expressing apoptotic cells.

Acknowledgments

This work was supported by an MRC Clinician Scientist fellowship (SPH) and MRC studentship (SV). We are grateful to colleagues in the Centre for Inflammation Research for help with leucocyte preparation and helpful comments relating to this work.

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