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. 2014 May;28(5):2214–2224. doi: 10.1096/fj.13-243238

Brain angiogenesis inhibitor 1 is expressed by gastric phagocytes during infection with Helicobacter pylori and mediates the recognition and engulfment of human apoptotic gastric epithelial cells

Soumita Das *,1, Arup Sarkar *,1, Kieran A Ryan ‡,§,1, Sarah Fox *, Alice H Berger ‖,, Ignacio J Juncadella #,**, Diane Bimczok ††,‡‡, Lesley E Smythies ††,‡‡, Paul R Harris §§, Kodi S Ravichandran #,**, Sheila E Crowe †, Phillip D Smith ††,‡‡, Peter B Ernst *,2
PMCID: PMC3986834  PMID: 24509909

Abstract

After Helicobacter pylori infection in humans, gastric epithelial cells (GECs) undergo apoptosis due to stimulation by the bacteria or inflammatory cytokines. In this study, we assessed the expression and function of brain angiogenesis inhibitor 1 (BAI1) in the engulfment of apoptotic GECs using human tissue and cells. After induction of apoptosis by H. pylori or camptothecin, there was a 5-fold increase in the binding of apoptotic GECs to THP-1 cells or peripheral blood monocyte-derived macrophages as assayed by confocal microscopy or conventional and imaging flow cytometry. Binding was impaired 95% by pretreating apoptotic cells with annexin V, underscoring the requirement for phosphatidylserine recognition. The phosphatidylserine receptor BAI1 was expressed in human gastric biopsy specimens and gastric phagocytes. To confirm the role of BAI1 in apoptotic cell clearance, the functional domain of BAI1 was used as a competitive inhibitor or BAI1 expression was inhibited by small interfering RNA. Both approaches decreased binding and engulfment >40%. Exposing THP-1 cells to apoptotic cells inhibited IL-6 production from 1340 to <364 pg/ml; however, this decrease was independent of phagocytosis. We conclude that recognition of apoptotic cells by BAI1 contributes to their clearance in the human gastric mucosa and this is associated with anti-inflammatory effects.—Das, S., Sarkar, A., Ryan, K. A., Fox, S., Berger, A. H., Juncadella, I. J., Bimczok, D., Smythies, L. E., Harris, P. R., Ravichandran, K. S., Crowe, S. E., Smith, P. D., Ernst, P. B. Brain angiogenesis inhibitor 1 is expressed by gastric phagocytes during infection with Helicobacter pylori and mediates the recognition and engulfment of human apoptotic gastric epithelial cells.

Keywords: efferocytosis, mucosal immunity, stomach, bacteria, gastritis

The gastrointestinal epithelium aids in the digestion of nutrients and provides a barrier that separates the luminal contents from the underlying lamina propria. Epithelial cells turn over constantly as apoptotic/damaged cells are replaced by stem cells originating from a proliferative zone within a gland (1, 2). In fact, the entire gastrointestinal epithelium is renewed every 7−10 d, with most apoptotic cells being shed into the lumen.

In the gastric mucosa, epithelial cell apoptosis is accelerated by the bacteria or inflammatory responses associated with Helicobacter pylori infection (3−6) as well as by chemical injury due to bile reflux, drugs, or alcohol (7). H. pylori, a gram-negative spiral bacterium, colonizes the human stomach and causes gastritis, gastroduodenal ulcer, and gastric cancer (8). Electron microscopy studies show that apoptotic epithelial cells are cleared by phagocytes in the intestine (9) and by the gastric mucosa (10). Engulfment of apoptotic cells is important for tissue remodeling and the control of inflammation (11−13). In addition, engulfment of apoptotic epithelial cells provides a mechanism for antigen sampling (14). However, the mechanisms governing the recognition of apoptotic cells and their engulfment and its consequences have not been examined in detail in the stomach.

Brain angiogenesis inhibitor 1 (BAI1) is a membrane protein expressed by phagocytes that recognizes apoptotic cells via an extracellular domain containing thrombospondin repeats (TSRs; ref. 15). The intracellular domain of BAI1 interacts with engulfment and cell motility protein 1 (ELMO1), which associates with dedicator of cytokinesis 180 (Dock180). The interaction between ELMO1 and Dock180 serves as a bipartite guanine nucleotide exchange factor for small GTPase Rac1 and the activated Rac1 initiates the actin rearrangement that mediates the engulfment process (15).

In the current study, we investigated how human macrophages recognize and subsequently engulf gastric epithelial cells (GECs) induced to become apoptotic by H. pylori or the chemical camptothecin. The recognition and engulfment were assessed by confocal microscopy and conventional and imaging flow cytometry. We show that BAI1 is expressed by phagocytes in the human gastric mucosa. Further, BAI1 mediates the recognition leading to engulfment, and BAI1 is found in the phagosome after ingestion of targets. These results support the involvement of BAI1 in mediating the recognition and engulfment of apoptotic cells in the gastric mucosa and suggest a role in targeting cargo for phagosomal degradation and antigen presentation.

MATERIALS AND METHODS

Cell lines and bacterial culture

The AGS gastric epithelial cell line and the THP-1 cell line were obtained from American Type Culture Collection (Rockville, MD, USA) and maintained as described previously (16, 17). H. pylori strain 26695 was maintained routinely on trypticase soy agar plates containing 5% sheep blood (TSA II; BD Diagnostics Systems, Sparks, MD, USA) at 37°C in 10% CO2 (17, 18). Before infection, bacteria were grown in Brucella broth containing 10% heat-inactivated fetal bovine serum for 18 h. Apoptosis was induced in AGS cells chemically using 3 μM camptothecin (Sigma-Aldrich, St. Louis, MO, USA) unless otherwise indicated when apoptosis was induced by incubation with H. pylori [multiplicity of infection (MOI) of 100; ref. 3].

Preparation of human monocyte-derived macrophages (MDMs)

Human monocytes were isolated from the blood of healthy volunteers using dextran sedimentation followed by Percoll gradient separation (19). Mononuclear cells were suspended in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% human serum at 1 × 106 cells/ml, after which 1 ml was added to individual wells in a 24-well plate. The plate was incubated for 1 h at 37°C in 5% CO2; subsequently, nonadherent cells were removed by washing. Maturation of the mononuclear cells into macrophages was accomplished by culturing the cells for 5–7 d in DMEM with 10% serum and 50 ng/ml granulocyte-macrophage colony-stimulating factor (GM-CSF; R&D Systems, Minneapolis, MN, USA).

Coculture assay and staining of cells for microscopy

Monocyte-derived macrophages and THP-1-derived macrophages were seeded (3×104 cells) onto an 8-well chamber slide (Nunc, Naperville, IL, USA) and grown overnight. The same day, AGS cells were inoculated with H. pylori at an MOI of 100:1 or treated with camptothecin. The following day, the macrophages were incubated with 2.5 μg/ml of the cytoplasmic green dye CMFDA (Molecular Probes, Eugene, OR, USA) for 1 h at 37°C in 5% CO2, and epithelial cells were trypsinized and stained with 2.5 μg/ml of the red dye SNARF (Molecular Probes). After washing, apoptotic or control AGS cells were added to individual wells of the chamber slide containing macrophages at a ratio of 5:1. The ratio of 5:1 was found to be optimal after preliminary studies using various ratios of apoptotic GECs to macrophages. After incubation for 1 h at 37°C, wells were washed with 0.01% sodium azide in phosphate-buffered saline (PBS). The slide was washed 3 times with PBS and fixed with 2% paraformaldehyde and mounted with a coverslip.

Microscopy

Epithelial cells and macrophages were imaged using a Nikon Eclipse E800 microscope (Nikon, Tokyo, Japan) with a QImaging camera (QImaging, Surrey, BC, Canada) at ×40 view. Ten randomly chosen fields on 3 separate slides were photographed, and the total number of green fluorescent cells (macrophages) and red fluorescent cells (AGS cells) were counted for each condition. Counting was performed by 2 independent observers without prior knowledge of the treatment conditions. Binding was quantified as the proportion of macrophages associated with 0, 1, 2, or ≥3 epithelial cells. For confocal microscopy, Z-stack images were captured on a Zeiss LSM 510 laser-scanning microscope (Carl Zeiss Inc., Thornwood, NY, USA), and projection images with data in the x, y, and z planes were generated using Zeiss LSM Image browser software (Carl Zeiss).

Assessment of engulfment by flow cytometry

The engulfment of apoptotic cells was assessed by flow cytometry as described previously (20, 21) with modifications. In brief, macrophages were stained with 1 μM 5-(and 6)-carboxyfluorescein diacetate succinimidyl ester (CFSE; Invitrogen, Carlsbad, CA, USA), in the presence or absence of 2 μg/ml cytochalasin D (Sigma-Aldrich) for 30 min. Subsequently, cells were incubated at a ratio of 1:1 for 5 h with either untreated or camptothecin-treated AGS cells stained with 0.5 μM CypHer5E (GE Healthcare, Piscataway, NJ, USA). For TSR-BAI1 treatment, apoptotic cells were preincubated with 10 ng/μl of TSR-BAI1 (15, 22) for 15 min before coculture with macrophages. The gates were set using unstained or single-stained cells based on forward and side scatter to select the population for analysis and to determine compensations and the specificity of the fluorescence. Engulfment was detected using a FACSCalibur flow cytometer (BD Biosciences, San Jose, CA, USA), and data were analyzed with FlowJo software (Tree Star Inc., Ashland, OR, USA).

Image-based assessment of engulfment by flow cytometry

Cells were stained and cocultured as described for the detection of engulfment by flow cytometry and assayed using an ImageStream flow cytometer (Amnis Corp., Seattle, WA, USA) as described previously (23), with modifications. Collected data were saved as a raw image file and analyzed with the selecting internalization wizard of IDEAS 4.0 image data analysis software (Amnis). Single-stained samples were used as controls to compensate for between-channel images on a pixel-by-pixel basis. For the data analysis, cells were gated on the basis of their aspect ratio to differentiate the single cells from aggregates. For assaying internalization, the singlet population was gated (because this represented >90% of the particles imaged), and only the CFSE-stained singlet population was considered to exclude unstained cells. Finally, cells were gated on the basis of the maximum pixel intensity of CypHer5E and total intensity of CypHer5E to resolve the CypHer5E “high” population from the CypHer5E “low” populations.

Assessing expression of BAI1

Human biopsy specimens were obtained from subjects undergoing esophagogastroduodenoscopy for clinically indicated reasons. All studies were approved by the respective institutional review boards at the University of Virginia; University of California, San Diego; University of Alabama; or Santiago, Chile. H. pylori status was determined by the rapid urease test and microscopic evaluation, and a study subject was judged colonized if results of either test were positive.

Samples were assessed for BAI1 mRNA expression by RT-PCR or immunohistochemical analysis using modifications of techniques described previously (17, 22). BAI1 mRNA was amplified using the primers BAI1-F (5′-ACTCATCCTGCGACGGTGTG-3′) and BAI1-R (5′-TCCCTCAGGTCCTTCATGCG-3′). The change in mRNA expression was determined using the ΔΔCt method (24). To validate the mRNA expression data, slides from fresh-frozen samples were prepared for 1 h for immunohistochemical analysis. Tissue sections were stained with a rabbit polyclonal antibody to BAI1 (ab135907; Abcam, Cambridge, MA, USA) or control rabbit IgG using a Ventana Discovery Ultra system (Ventana Medical Systems; Tucson, AZ, USA). Antigen retrieval was performed using CC1 for 24 min at 95°C. BAI1 was incubated with the tissue at a dilution of 1:750 for 1 h at 37°C followed by incubation with a horseradish peroxidase-conjugated anti-rabbit antibody (760–4315; Ventana Medical Systems) for 12 min at 37°C. Subsequently, the antigen was visualized by incubation with diaminobenzidine, counterstained with hematoxylin, scanned with a NanoZoomer scanner (Hamamatsu, Hamamatsu City, Japan), and processed in Aperio image scope software (Leica Microsystems, Buffalo Grove, IL, USA).

To characterize the lineage of the mononuclear cells expressing BAI1, gastric phagocytes were prepared as described previously (25, 26) by enrichment using anti-human HLA-DR MACS beads (Miltenyi Biotech, Auburn, CA, USA). Cell purity was confirmed by fluorescence-activated cell sorter analysis, and BAI1 expression was assessed by Western blot (22). Western blots were performed as described previously (22). In brief, the primary antibody for BAI1 (dilution 1:1000) was either an from Orbigen antibody (Allele Biotechnology, San Diego, CA, USA) or a custom-made rabbit polyclonal antibody (SDIX, Newark, DE, USA) recognizing the N-terminal region of BAI1 (139−238 aa).

Down-regulation of BAI1 by small interfering RNA (siRNA)

BAI1 siRNA (On-TargetPlus SMARTpool; Dharmacon, Lafayette, CO, USA) was introduced into THP-1 cells by nucleofection (Amaxa kit V, program V-001; Lonza, Cologne, Germany). In brief, 40 nM BAI1 siRNA was added to 106 THP-1 cells resuspended in solution V. RNA and cell lysates were prepared to assess the level of BAI1 expression after siRNA by RT-PCR and Western blot analysis.

Preparation of phagosomal fractions

Phagosomal fractions were prepared from adherent macrophages following the protocol described previously by Gomez et al. (27) with modifications. In brief, the THP-1 cells were incubated with polystyrene magnetic beads (2.49 μm average diameter, 2.5% w/v suspension; Spherotech, Lake Forest, IL, USA) for different time points. After incubation, cells were washed and scraped in ice-cold PBS. Cells were then resuspended in phagosome purification buffer [PBS with 5 mM EDTA (pH 8.0), protease inhibitor (Roche Applied Science, Indianapolis, IN, USA) and phosphatase inhibitor cocktail (Sigma-Aldrich)]. Cells were lysed by gentle passing through a 22-gauge needle for ∼20 times. Lysis was considered complete when 90% of cell membranes were found to be broken as assessed by light microscopy, and lysates were transferred to an ice-cold microfuge tube. Phagosomal fractions containing magnetic beads were separated by a magnetic separator (Dynal; Life Technologies, Grand Island, NY, USA).

Effects of target cells on cytokine production by macrophages

THP-1 cells were plated at 0.5 × 106/ml in 24-well tissue culture plates and stimulated with phorbol 12-myristate 13-acetate (PMA) for 3 d to induce differentiation. Isolated human mononuclear cells were plated at 4 × 106/ml in 48-well tissue culture plates in the absence of serum. After 1 h, the nonadherent lymphocytes were removed by washing 3 times with PBS. Monocytes were differentiated to MDMs for 5–7 d using 50 ng/ml GM-CSF. Apoptotic AGS cells were incubated with THP-1 cells and MDMs at a 5:1 ratio in macrophage cell culture medium. For cytochalasin D (Sigma-Aldrich) treatments, macrophages were pretreated for 30 min (5 μg/ml) before addition of apoptotic cells. Apoptotic AGS cells were pretreated with annexin V (5 μg/ml; R&D Systems) for 30 min before addition to macrophages. Cells were cocultured for 2 h at 37°C, after which the macrophages were washed 3 times with cold PBS to remove noninternalized AGS cells. The supernatants were then replaced with macrophage cell culture medium in the absence or presence of lipopolysaccharide (LPS; 100 ng/ml, Salmonella enterica serotype Typhimurium cell culture-grade purified LPS; Sigma-Aldrich) for 24 h. Supernatants were collected and used with an interleukin-6 (IL-6) ELISA kit (R&D Systems).

Statistical analysis

Results are expressed as means ± sd. Results were compared using a 2-tailed Student's t test or 1-way ANOVA and considered significant at values of P < 0.05. Where appropriate, a correction for multiple comparisons was performed.

RESULTS

Macrophages recognize and bind apoptotic GECs

Phagocytosis entails a 3-step process whereby recognition of the apoptotic cell leads to binding, followed by the activation of signaling processes that result in target cell engulfment. The ability of phagocytes to recognize and bind apoptotic AGS cells was determined by fluorescent microscopy to compare the interaction between control or apoptotic targets incubated with macrophages. Binding was quantified on the basis of the number of epithelial targets (0, 1, 2, or ≥3) associated with the macrophages. Aggregates of ≥3 apoptotic AGS cells (induced by camptothecin) were bound to THP-1-derived macrophages 31% of the time, whereas <5% of the macrophages bound ≥3 control AGS cells (Fig. 1A). Correspondingly, the number of macrophages lacking any associated AGS cells was higher (average 48% vs. 21%) when phagocytes were incubated with the control AGS cell preparation (Fig. 1A).

Figure 1.

Figure 1.

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Macrophages preferentially recognize and bind apoptotic AGS cells. A) Macrophages were incubated with either vehicle (solid bars) or camptothecin-treated (open bars) AGS cells. Evidence of binding was based on the number of epithelial cells (0, 1, 2, or ≥3) associated intimately with the macrophages (schematic diagram at top of graph). Data shown are averages of 5 independent experiments.*P < 0.05; t test. B) MDMs were incubated for 1 h with control, uninfected (solid bars), or H. pylori-infected (open bars) AGS cells, after which the number of bound AGS cells was counted. Data are averages of 3 independent experiments.*P < 0.05. C) Confocal microscopy to show the binding of H. pylori-infected apoptotic GECs by MDMs. i) Photomicrograph of a stack series consisting of images captured 1.36 μm apart by confocal microscopy. ii) Images from i, viewed from the z axis. iii) Graphic representation of ii. iv) Second image from a different series that shows 3 AGS cells attached to one macrophage. v) Graphic representation of iv. M, macrophage; A, AGS; P, pseudopodium.

To more closely model H. pylori infection in humans, the recognition and binding of apoptotic GECs generated in response to H. pylori infection were tested using human blood MDMs (Fig. 1B). Aggregates of ≥3 AGS cells were bound to MDMs at a significantly higher frequency in the AGS cells made apoptotic by an infection with H. pylori than in uninfected cells (27 vs. 3%, P<0.05). Correspondingly, the number of macrophages lacking AGS cell targets was again higher in the uninfected group (63 vs. 38%, P<0.05).

The binding and engulfment of apoptotic GECs by THP-1 macrophages were visualized by confocal microscopy. As shown in the images in Fig. 1C, H. pylori-exposed AGS cells were surrounded by macrophage pseudopodia. Figure 1Cii, iv demonstrates the AGS target being surrounded by the phagocytes, suggesting that apoptotic cells were recognized and engulfed after H. pylori infection.

To confirm and quantify the internalization of apoptotic cells within the macrophages, we labeled apoptotic targets with CypHer5E, a pH-sensitive cyanine dye that is maximally fluorescent in acidic compartments, such as the late endosome. The region (R1) representing the internalized bright cells increased when camptothecin-treated apoptotic AGS targets were incubated with THP-1 cells (Fig. 2A) and MDMs (Fig. 2B). It has been assumed that brightly fluorescent cells confirm internalization of the labeled target, whereas dim cells are bound to the surface (20, 21). To confirm this, we used a flow cytometry-based imaging system that provides an image of each event as it passes through the flow cell. With use of the imaging flow cytometry, the bright (CypHer5E-high) targets and the dim (CypHer5E-low) targets within the phagocytes were visualized (Supplemental Fig. S1).

Figure 2.

Figure 2.

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Macrophages bind and engulf apoptotic GECs. A, B) Engulfment of AGS cells was measured with THP-1 (A) and with MDMs (B). Macrophages were stained with CFSE, pretreated with or without cytochalasin D (CytD), incubated with either untreated or camptothecin-treated AGS cells, stained with CypHer5E, and assessed for engulfment by flow cytometry. A) On the basis of the intensity of staining with CypHer5E, high/bright, low/dim, and negative intensity regions were designated as R1, R2, and R3, respectively. Representative histograms were selected from 3 independent experiments. C, D) Samples treated in the same manner were also analyzed using Amnis ImageStream. C) First window shows a flow cytometric representation of the Image Stream analysis in which the cells were focused on singlets based on their aspect ratio. In the next window, the high-intensity CFSE-stained singlet cells were selected. Histograms of CypHer5E high and low populations are shown from THP-1 cells incubated either with camptothecin-treated apoptotic AGS cells or with apoptotic AGS cells in the presence of cytochalasin D. D) Summary data from panel C showing the percentage of phagocytes with internalized apoptotic AGS cells (i.e., R1, CypHer5E bright) in the presence or absence of cytochalasin D. Data are averages ± sd of 3 independent experiments. *P < 0.05.

Another approach to demonstrate the internalization of targets is to block actin rearrangement with cytochalasin D. This treatment decreased the bright cells within both THP-1 cells (5.4 vs. 0.27% in region R1; Fig. 2A) and MDMs (8.86 vs. 0.3%; Fig. 2B) as expected. The flow cytometry-based imaging confirmed the near abrogation of internalization of AGS into THP-1 cells after treatment with cytochalasin D (Fig. 2C, D). MDMs also showed similar inhibition in internalizing camptothecin-treated apoptotic AGS cells after cytochalasin D treatment (13.1 vs. 1.35% in the CFSE-positive CypHer5E-high population; Supplemental Fig. S2A, B).

Recognition and engulfment of apoptotic GECs by macrophages are phosphatidylserine (PS) dependent

An early event in apoptosis is the flipping of membrane lipids that result in the exposure of PS on apoptotic AGS cells that, in turn, can be recognized by phagocytes. To determine whether the recognition of apoptotic AGS cells by phagocytes was PS dependent, apoptotic cells were treated with an excess of annexin V to block PS recognition. Pretreatment with annexin V abrogated the interactions between the apoptotic AGS cells and THP-1-derived macrophages (Fig. 3A).

Figure 3.

Figure 3.

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Impairment of PS recognition by annexin V abrogates interactions between apoptotic targets and phagocytes. A) THP-1 macrophages were incubated for 1 h with AGS cells treated with medium alone (open bars), camptothecin (solid bars), or camptothecin and annexin V (striped bars), after which the numbers of bound AGS cells were counted. Data are means ± sd of 5 independent experiments. *P < 0.05. B) BAI1 mRNA was detected by real-time RT-PCR in human gastric biopsy specimens. Expression of BAI1 in THP-1 cells and 3 normal gastric biopsy specimens was normalized to 18S, and relative mRNA expression was plotted after multiplying the value by 104. C) Expression of BAI1 protein in phagocytes detected by Western blot: lane 1, HLA-DR+ gastric mononuclear cells; lane 2, monocyte-derived macrophages; lane 3, THP-1 before PMA-induced differentiation; lane 4, THP-1 macrophages after PMA-induced differentiation. Top panel, BAI1 expression at 180 kDa; bottom panel, β-actin loading control. D) Immunohistochemical analysis of selected sections of gastric antrum from uninfected (n=5) and H. pylori-infected (n=3) subjects (×20). The section is a representative figure from 5 uninfected (i, iii) and 3 infected (ii, iv) specimens. i, ii) Sections stained with isotype control rabbit IgG antibody. iii, iv) Sections stained with anti-BAI1 antibody raised in rabbits. Slides were counterstained with hematoxylin. Slides were scanned with a NanoZoomer scanner and processed in Aperio image scope software.

Human gastric and blood phagocytes express the PS receptor BAI1

Park et al. (15) identified BAI1 as a phospholipid receptor that interacts directly with PS. To elucidate the biological role for BAI1 in the context of H. pylori infection, its expression was assessed in human gastric biopsy specimens using real-time RT-PCR (Fig. 3B). Because the tissue was positive for BAI1 mRNA, protein expression was assayed by Western blot and found in HLA-DR+ mononuclear phagocytes isolated from human gastric mucosa (Fig. 3C, lane 1), as well as monocyte-derived macrophages, THP-1 and THP-1-derived macrophages (Fig. 3C). The distribution of BAI1 expression was examined by immunohistochemistry using sections of human stomach obtained from uninfected and H. pylori-infected subjects (Fig. 3D). BAI1 expression was observed in the mononuclear cells in all sections.

Involvement of BAI1 in the recognition and engulfment of apoptotic GECs

To assess the ability of BAI1 to recognize and bind apoptotic GECs, a soluble BAI1 peptide fragment representing the PS-binding domain (TSR-BAI1) was used to block the recognition of the apoptotic corpse by the phagocyte. As shown in Fig. 4A, camptothecin-treated apoptotic AGS cells treated with TSR-BAI1, showed a marked reduction in binding to THP-1-derived macrophages compared with binding of apoptotic AGS cells treated with the control protein glutathione S-transferase (GST). Further, the CypHer5E bright population (high CypHer5E, R1) was reduced 50% after incubation of the targets with the TSR domain of BAI1 (Fig. 4C). The effect of TSR-BAI on engulfment of apoptotic AGS cells by THP-1 cells was confirmed by imaging flow cytometry (Fig. 4D). The TSR part of BAI1 reduced 70% of the internalization of apoptotic AGS cells by THP-1 cells (Fig. 4E). A similar reduction in the engulfment of apoptotic AGS cells by MDMs was observed when apoptotic cells were pretreated with the competitive inhibitor TSR-BAI1 (Supplemental Fig. S2A, C). These findings indicate that the TSR domain of BAI1 is sufficient to block recognition of macrophages with apoptotic GECs.

Figure 4.

Figure 4.

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Extracellular TSR domain of BAI1 impairs binding and engulfment of apoptotic AGS cells. A) Extracellular region of BAI1 containing 5 thrombospondin repeats (TSR-BAI1; 10 ng/μl) was incubated with apoptotic AGS cells before being cocultured with THP-1 macrophages. Solid bars, untreated AGS cells; open bars, AGS cells in which apoptosis was induced by camptothecin; shaded bars, apoptotic AGS cells incubated with control GST protein; striped bars, apoptotic AGS cells preincubated with TSR-BAI1 for 15 min before coculturing with macrophages. Means ± sd of 4 independent experiments of macrophages interacting with 0 or ≥3 AGS cells were determined for each group. B) TSR-BAI1 was incubated with camptothecin-treated apoptotic AGS cells, and engulfment by THP-1 cells was assayed by flow cytometry. C) Representative means ± sd of the percentage of cells within R1 from 3 experiments. D) Visual representation of image-based flow cytometry to show reduced engulfment of camptothecin-treated apoptotic AGS cells by THP-1 cells when apoptotic AGS cells were treated with TSR-BAI. E) Percentage of internalized apoptotic AGS cells by THP-1 cells in the presence or absence of TSR-BAI as calculated using Amnis ImageStream with 5000 events. Data are averages ± sd of 3 independent experiments. *P < 0.05.

To confirm the role of phagocyte BAI1 in the recognition process, BAI1 expression was down-regulated in THP-1 cells using RNA silencing before the cells were assayed for their ability to recognize and bind apoptotic cells. Nucleofection of human BAI1 siRNA into THP-1 cells caused a significant inhibition of BAI1 mRNA (Fig. 5A, B) and protein (Fig. 5C). Macrophages with knockdown of BAI1 displayed a significant reduction in the ability to bind camptothecin-treated apoptotic AGS cells compared with that in macrophages treated with control siRNA (Fig. 5D). Further, we isolated phagosomes from THP-1-derived macrophages after the uptake of polystyrene magnetic beads. As shown in Fig. 5E, BAI1 was detected in the isolated phagosomes by Western blot. Thus, BAI1 plays a key role in THP-1 macrophage recognition, binding, and internalization of apoptotic AGS cells.

Figure 5.

Figure 5.

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BAI1 is necessary for the recognition of apoptotic AGS cells by macrophages. A) BAI1 mRNA expression in THP-1-derived macrophages. Lanes 1–3, BAI1 expression (450-bp band); lanes 5–7, GAPDH expression; lane 4, 100-bp ladder. cDNA fragments amplified from untreated cells (lanes 1 and 5) and control siRNA-treated (lanes 2 and 6) and BAI1 siRNA-treated (lanes 3 and 7) THP-1 macrophages. B) Suppression of BAI1 after siRNA was quantified by real-time RT-PCR. BAI1 transcripts were normalized to 18S and compared with untreated controls. C) BAI1 protein expression (∼180 kDa) was detected by Western blot of lysates from THP-1 macrophages (top panel). α-Tubulin was used as a loading control (bottom panel). THP-1 macrophage lysates from untreated cells (lane 1), control siRNA (lane 2), and cells treated with BAI1 siRNA (lane 3). D) Recognition and binding of 0 or ≥3 apoptotic AGS cells after camptothecin treatment were determined for control siRNA-treated and BAI1 siRNA-treated THP-1 macrophages. Values are means ± sd of 3 independent experiments. *P < 0.05. E) Expression of BAI1 in phagosomes isolated by magnetic separation from THP-1-derived macrophages with magnetic beads [either alone or in presence of H. pylori (Hp)] for 2 and 4 h. Top panel, BAI1 expression at 180 kDa. Bottom panel, flotillin, a loading control of the phagosomal fraction.

Attenuation of inflammatory responses in macrophages in independent of apoptotic GEC engulfment

To investigate the consequences of apoptotic AGS cell engulfment on macrophage inflammatory responses, THP-1-derived macrophages and MDMs were cocultured with camptothecin-treated apoptotic target cells in the presence or absence of cytochalasin D or recombinant annexin V. The macrophage monolayers were washed to remove noninternalized targets, and the macrophages were stimulated with LPS. The apoptotic AGS cell coculture significantly attenuated LPS-stimulated IL-6 production by both THP-1-derived macrophages and MDMs. Although engulfment has been reported to lead to the inhibition of inflammatory responses, treatment with cytochalasin D and recombinant annexin V failed to reverse the anti-inflammatory effect (Fig. 6).

Figure 6.

Figure 6.

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Attenuation of LPS-induced IL-6 release from macrophages exposed to apoptotic targets. THP-1 cells (A) and MDMs (B) were cocultured with or without camptothecin-treated apoptotic AGS cells in the presence or absence of cytochalasin D (CytD; 5 μg/ml) to inhibit target internalization or recombinant annexin V (5 μg/ml) to prevent binding of PS residues on apoptotic cells. Macrophages were then washed to remove AGS cells and stimulated with or without LPS (100 ng/ml). Supernatants were analyzed by a specific ELISA for human IL-6. Results show IL-6 production normalized to 10,000 cells in 1 ml volume (pg/ml). Data are averages ± sd of 3 independent experiments with duplicate sets. *P < 0.05.

DISCUSSION

In the present study, we show that both H. pylori infection and chemically induced apoptosis of GECs enhance the binding of apoptotic targets to macrophages, leading to their subsequent engulfment. Furthermore, the data show that BAI1 is expressed in the human gastric mucosa by gastric antigen-presenting cells. BAI1 mediates the recognition that leads to target engulfment, and it is found in the phagosome of macrophages after phagocytosis of the targets. Apoptotic cells exerted an anti-inflammatory effect on the phagocyte that is independent of their engulfment. These results show that BAI1 mediates the recognition and engulfment of apoptotic cells in the human gastric mucosa.

The gastrointestinal epithelium has an enormous capacity for self-renewal, because it is replaced every 4–5 d (28). This turnover is tightly controlled; otherwise, excessive cell death would create a defective barrier and the uncontrolled access of bacteria into the underlying layers (29–32). Although most apoptotic epithelial cells appear to be shed into the lumen (33), a substantial portion of the intestinal (9) and gastric (10) epithelial cells undergoing apoptosis are phagocytosed by macrophages residing in the mucosa. In addition, apoptotic DNA can be detected in phagocytes in the lamina propria and Peyer's patches (34) as well as in phagocytes studied ex vivo (35). Engulfment of infected epithelial cells may provide a Trojan horse (36), whereby microbial material associated with the apoptotic cells could regulate host responses (37) or be processed and presented to helper T cells (9, 14). The biological significance was further established in a study in which virus-infected epithelial cells become apoptotic and then engulfed by dendritic cells in the murine Peyer's patches. Subsequently, viral proteins were processed and presented to T cells (14). However, the molecular basis for this recognition and engulfment in the human gut has not been defined in detail.

The findings presented in the current report extend previous studies on the role of BAI1 in apoptotic cell engulfment in Caenorhabditis elegans and murine cell lines to the human gastric mucosa. Our data provide several lines of evidence that this pathway plays a role in humans. First, BAI1 mRNA and protein were detected in the human gastric mucosa. Second, phagocytes isolated from the human gastric mucosa expressed mRNA and protein for BAI1. Finally, in vitro approaches using human cells lines, including cells isolated from the peripheral blood, showed that the optimal engulfment of apoptotic targets was dependent on PS recognition and BAI1 expression.

Although the current study indicates a role for BAI1 in the recognition and engulfment of targets by human gastric phagocytes, there is significant cooperation and redundancy in this biological process. Other phagocytic receptors recognize PS including members of the TIM family (TIM-1, TIM-3 and TIM-4; refs. 38, 39) and stabilin-2 (40). Whereas BAI1 actively signals (15), it has been suggested that Tim4 does not mediate signaling (41) but rather acts as a tethering molecule. Tim4 is involved in tethering the engulfment signaling by αvβ3 through a Rac1-dependent manner (42).

As a consequence of apoptotic cell engulfment, phagocyte inflammatory cytokine production is inhibited (37). Previous studies have documented an apoptotic cell-dependent attenuation of LPS-induced proinflammatory cytokine production, as well as up-regulation of anti-inflammatory mediators such as transforming growth factor-β1 (43–45). These data led to the hypothesis that dead cells critically influence inflammation, tissue repair, and autoimmunity (45–47). Recently, a study by Juncadella et al. (21) reported that apoptotic bronchial epithelial cells influence inflammation by modulating the cytokine release by adjacent cells (21). We have confirmed that apoptotic GECs attenuate LPS-induced proinflammatory IL-6 release. When phagocytes were exposed to sterile apoptotic targets, they produced less IL-6 in response to LPS, suggesting that this process contributes to the hyporesponsive phenotype of mucosal macrophages. As reported previously (48), the current results suggested that engulfment was not required for the decrease in inflammation because blocking recognition with annexin V or cytochalasin D had little effect. It is possible that the redundancy in receptors that recognize, bind, and mediate signaling events after exposure to cellular debris can also contribute to the regulation of cytokine production. Although sterile targets may inhibit inflammatory responses, infection may change this outcome. For example, engulfment of infected targets may deliver bacterial ligands to reactive phagocytes, whereas H. pylori-infected macrophages generate tumor necrosis factor-α, which inhibits the clearance of H. pylori-infected apoptotic GECs (35). Thus, in the context of infection, clearance of apoptotic cells may be impaired, and the hyporesponsive tone imparted in the absence of inflammation may be lost.

Because the assays used to study engulfment vary in the depth of the evidence they provide, we compared several approaches. First, simple immunofluorescence showed that apoptotic cells preferentially interacted with phagocytes in a PS- and BAI1-dependent manner. Although fluorescence microscopy detects binding, it lacks the resolution to document internalization. To achieve that, we first used confocal imaging, which provided evidence of internalization, but this approach is challenging to quantify. Subsequently, we used an indirect assay of internalization with a pH-sensitive dye (CyPher5E) to label the target cells. CyPher5E is minimally fluorescent in basic pH, and the fluorescent is maximum at acidic pH, more specifically when the target enters the acidic endosome (20, 21). With this approach, a reproducible, quantifiable, and cytochalasin D-sensitive population of brightly fluorescent cells could be identified (Fig. 2), suggesting that the targets were indeed internalized. Moreover, image-based flow cytometry showed that CyPher5E high cells contained the apoptotic targets. It is worth noting that simple fluorescence to detect the association of an apoptotic target with a phagocyte had a high predictive value for engulfment as detected with the most specific assays.

A comparison of the 2 flow cytometry-based assays showed that THP-1 cells had 3 distinct degrees of CyPher5E fluorescence, whereas MDMs only had the 2 distinct populations. This result is probably attributable to the higher phagocytic capacity of primary macrophages (49), Notably, whereas cytochalasin D blocked the accumulation of the bright cells, it did not eliminate the dim cells detected by flow cytometry. This result suggests that cytochalasin D, at the concentrations used, inhibits internalization to a degree and/or it impairs the actin-dependent cytoskeletal motors that may facilitate the maturation of late endosomes (50). Cytochalasin D inhibited internalization as detected by the flow cytometry-based imaging. The virtual absence of bound cells detected with this imaging technology could reflect the gating on singlets. However, few aggregates were found, possibly because of a higher sheer force in this technology.

In healthy cells, nearly all of the PS is confined to the inner leaflet of the plasma membrane but after the onset of apoptosis, PS is exposed on the outer leaflets (51). In response to PS and other so-called “eat me” signals displayed on the apoptotic cell surfaces (11, 12), phagocytes respond. BAI1 is expressed by phagocytes and binds PS directly to promote internalization of the apoptotic corpses (15). After engagement with PS, BAI1 interacts with the cytoplasmic engulfment promoting protein ELMO1 which, in association with Dock180, acts as a bipartite guanine nucleotide exchange factor for the small GTPase Rac (52). The active GTP-bound Rac subsequently promotes actin organization, cytoskeletal rearrangement, and various signaling cascades involved in target cell internalization.

Because H. pylori were used to induce apoptosis, it is possible that surface bacteria contributed to the recognition and subsequent engulfment of the targets. Indeed, we noted that BAI1 recognizes LPS on gram-negative bacteria (22). Although H. pylori-induced apoptosis of AGS cells in vitro, we could not find any evidence of GFP-expressing bacteria associated with the cells once they became apoptotic (data not shown). Moreover, inducing apoptosis chemically with camptothecin created targets that were also recognized and engulfed, indicating that bacterial recognition by BAI1 was not necessary. Some control AGS cells were bound by phagocytes, which is consistent with the fact that a small percentage of the cells harvested from tissue culture were apoptotic in the absence of any additional stimulation (53).

The results presented in this study demonstrate a role for BAI1-mediated engulfment of apoptotic cells in the human gastric mucosa and show that the interaction with apoptotic targets decreases the sensitivity of the phagocyte to LPS. Notably, BAI1 is recruited to the phagosome of macrophages after ingestion of a meal and after infection with H. pylori. These findings suggest that BAI1 and its signaling partners may be involved in the processes regulating phagosomal degradation and could facilitate antigen presentation. Experiments are in progress to identify the other elements of the engulfment machinery expressed by gastric mononuclear phagocytes and to characterize their function in engulfment and the regulation of host responsiveness.

Supplementary Material

Supplemental Data
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Acknowledgments

The authors thank Amber Ablack for technical assistance in the maintenance of AGS cells and H. pylori strains; Drs. Scott Vandenberg and Donald Pizzo [University of California, San Diego (UCSD) histology core facility] for the immunohistochemical analysis; Joanne Lannigan, William Ross, Mike Solga, and Elizabeth Wiznerowicz (University of Virginia) for technical assistance; and the University of Texas Medical Branch Protein Biosynthesis and Biomarker Core Laboratory (Galveston, TX, USA) for protein purification.

This work was supported by U.S. National Institutes of Health grants AI079145, DK084063, and AI070491 (to P.B.E.), DK61769 (to S.E.C.), and DK54495, DK84063, AI83539, and RR20136; as well as grants to the University of Alabama at Birmingham Mucosal HIV and Immunobiology Center (Center for Clinical and Translational Science and Immunology, Autoimmunity and Transplantation Pilot Program; DK64400), the Research Service of the Veterans Administration (to P.D.S.), the University of Virginia Digestive Health Research Center Immunology and Cell Isolation Core and Morphology/Imaging Core (DK67629), the UCSD Neuroscience Microscopy Shared Facility (P30 NS047101), the UCSD Moores Cancer Center Biorepository Tissue Technology Shared Resources (2P30 CA023100), and the UCSD Digestive Diseases Research Development Center (DK80506).

The authors declare no conflict of interest.

This article includes supplemental data. Please visit http://www.fasebj.org to obtain this information.

BAI1
brain angiogenesis inhibitor 1
CFSE
5-(and 6)-carboxyfluorescein diacetate succinimidyl ester
DMEM
Dulbecco's modified Eagle's medium
Dock180
dedicator of cytokinesis 180
ELMO1
engulfment and cell motility protein 1
GEC
gastric epithelial cell
GM-CSF
granulocyte-macrophage colony-stimulating factor
GST
glutathione S-transferase
IL-6
interleukin-6
LPS
lipopolysaccharide
MDM
monocyte-derived macrophage
MOI
multiplicity of infection
PBS
phosphate-buffered saline
PMA
phorbol 12-myristate 13-acetate
PS
phosphatidylserine
siRNA
small interfering RNA
TSR
thrombospondin repeat

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