- Open Access
The role of C1q in recognition of apoptotic epithelial cells and inflammatory cytokine production by phagocytes during Helicobacter pylori infection
© Fox et al. 2015
- Received: 26 November 2014
- Accepted: 28 August 2015
- Published: 8 September 2015
Gastric epithelial cells (GECs) undergo apoptosis during H. pylori infection and phagocytes within the mucosa engulf these cells. The recognition and clearance of apoptotic cells is a multifactorial process, enhanced by the presence of various bridging molecules and opsonins which are abundant in serum. However, it is not clear how recognition or clearance may differ in the context of H. pylori infection induced apoptosis. In addition, efferocytosis of sterile apoptotic cells is known to confer anti-inflammatory properties in the engulfing phagocyte, however it is unknown if this is maintained when phagocytes encounter H. pylori-infected cells. Thus, the ability of macrophages to bind and engulf gastric epithelial cells rendered apoptotic by H. pylori infection and the association of these interactions to the modulation of phagocyte inflammatory responses was investigated in the absence and presence of serum with a particular focus on the role of serum protein C1q.
Control (uninfected) or H. pylori-infected AGS cells were co-cultured with THP-1 macrophages in the presence or absence of serum or serum free conditions + C1q protein (40–80 μg/mL). Binding of AGS cells to THP-1 macrophages was assessed by microscopy and cytokine (IL-6 and TNF-α) release from LPS stimulated THP-1 macrophages was quantified by ELISA.
We show that macrophages bound preferentially to cells undergoing apoptosis subsequent to infection with H. pylori. Binding of apoptotic AGS to THP-1 macrophages was significantly inhibited when studied in the absence of serum and reconstitution of serum-free medium with purified human C1q restored binding of macrophages to apoptotic cells. Co-culture of sterile apoptotic and H. pylori-infected AGS cells both attenuated LPS-stimulated cytokine production by THP-1 macrophages. Further, direct treatment of THP-1 macrophages with C1q attenuated LPS stimulated TNF-α production.
These studies suggest that C1q opsonizes GECs rendered apoptotic by H. pylori. No differences existed in the ability of infected or sterile apoptotic cells to attenuate macrophage cytokine production, however, there may be a direct role for C1q in modulating macrophage inflammatory cytokine production to infectious stimuli.
- Epithelial cell
- H. pylori
Helicobacter pylori, a Gram-negative spiral bacterium, is one of the most common infections worldwide [1–3]. It colonizes the stomach of humans, usually in infancy , and has been implicated in a number of diseases including gastritis, gastroduodenal ulceration and gastric cancer [3, 5–7]. Approximately 20 % of individuals infected with H. pylori develop overt disease symptoms, the severity of which is extremely variable. Many groups have focused on identifying H. pylori virulence factors that may contribute to disease severity and a number of candidates have been proposed, including the vacuolating cytotoxin VacA [8, 9] and the cytotoxin-associated gene product CagA [9–11]. However, disease progression appears to be multi-factorial in that it also depends on a number of host factors including the immune and inflammatory response [12–15]. This includes the secretion of cytokines by the gastric epithelium [16–18] or mucosal antigen presenting cells , increased recruitment of cells such as neutrophils, a robust IgA response [20, 21] and T cell polarization [22–25]. Ultimately, this chronic, active inflammatory response contributes to tissue damage and the subsequent pathogenesis of gastroduodenal disease.
Despite the impressive advances to date, much remains to be learned about the host responses that regulate the magnitude of the inflammation and tissue damage. One mechanism that impacts inflammation involves phagocytes and their interaction and uptake of apoptotic cells, which attenuates phagocyte inflammatory responses, leading to termination of inflammation and initiation of tissue repair [26, 27]. After encountering apoptotic cells, phagocytes produce less pro-inflammatory mediators and increase their expression of anti-inflammatory, pro-resolution factors such as IL-10 and TGF-β1 [28–30]. The clearance of apoptotic cells is influenced by many soluble factors and receptor interactions. C1q, the initiating protein of the complement cascade, has been shown to be an important opsonin of apoptotic cells, enhancing their recognition and removal by phagocytes [31–33]. The importance of C1q is underscored in the autoimmune disease, systemic lupus erythematosus (SLE) where patients have a deficiency in C1q, from which the pathology has been linked to failed clearance of apoptotic cells .
In the present study, we examined the processes whereby gastric epithelial cells (AGS cells) are recognized by macrophages (THP-1 macrophages) in response to apoptosis induced by H. pylori infection. The attenuation of inflammatory responses by phagocytes following co-culture with sterile and infected apoptotic epithelial cells was also compared. As the complement protein C1q is one of several factors involved in the clearance of apoptotic cells [31, 32] and in light of recent reports of a direct role for C1q in modulation of macrophage inflammatory responses [35–39], we investigated the role of C1q in the interaction of infected gastric epithelial cells with phagocytes as well as its role in modulation of phagocyte cytokine responses.
Bacterial culture, cell lines and reagents
Helicobacter pylori strain 26695 was maintained routinely on blood agar plates containing 5 % horse blood (BD Pharmingen, San Jose, CA) at 37 °C in 10 % CO2. Prior to infection of cell cultures, bacteria were amplified in Brucella broth containing 10 % heat-inactivated FBS for 18 h. The AGS human gastric epithelial cell line and the THP-1 monocyte-like cell line were obtained from ATCC (Rockville, MD). The gastric epithelial cell line, AGS cells, were maintained in Dulbecco’s Modified Eagle’s Medium (DMEM) and THP-1 in RPMI 1640 (Gibco, NY), both containing 10 % heat-inactivated fetal bovine serum (FBS, Sigma, St. Louis, MO), at 37 oC in 5 % CO2. THP-1 cells were differentiated into a macrophage-like phenotype by treating with 600 nM phorbol myristate acetate (PMA; Sigma, St Lois, MO) for 3 days. Apoptosis was induced in AGS cells by infection with H. pylori as previously described  or by treatment with 3 μM camptothecin (Sigma, St. Louis, MO) for 24 h which proved a non-infectious means to induce apoptosis. Stimulation of apoptosis was carried out in the presence of 10 % FBS (heat-inactivated) medium after which the cells were washed twice in PBS (300 × g; 5 min) before being resuspended in the appropriate media prior to macrophage co-culture. The concentration of camptothecin or H. pylori used has previously been determined to be an optimal concentration to induce apoptosis in epithelial cells. Purified human C1q protein was purchased from Quidel (San Diego, CA).
Preparation of human monocyte-derived macrophages
Human monocytes were isolated from blood drawn from healthy volunteers using a well-established technique involving dextran sedimentation followed by Percoll gradient separation . Mononuclear cells were suspended in DMEM supplemented with 10 % autologous serum at 1 × 106 cells/ml, and then 1 ml of the suspension was added to individual wells on a 24-well plate. The plate was incubated for 1 h at 37 °C and 5 % CO2, after which time non-adherent cells were removed by washing. Maturation of the mononuclear cells into macrophages was induced by culturing for 5–7 days in DMEM with 10 % autologous serum. All procedures using human blood were approved in advance by the Institutional Review Board of the University of Virginia and University of California, San Diego.
Evaluation of phagocyte-target interactions by microscopy
Mature THP-1-derived macrophages were gently scraped and seeded onto an 8-well chamber slide (NUNC, Naperville, IL) at a density of 3 × 104 and grown overnight. The same day, epithelial cells were infected with H. pylori strain 26695 at a MOI of 300:1 or treated with camptothecin (3 μM). The following day, the macrophages were incubated with 2.5 μg/ml of the cytoplasmic dye CMFDA (Molecular Probes, Eugene, OR) for 1 h at 37 °C and 5 % CO2. Epithelial cells were trypsinized and stained in a similar manner with 2.5 μg/ml of the dye SNARF® (Molecular Probes). Both dyes freely cross the cytoplasmic membrane, where they are modified by intracellular esterases to yield a fluorescent product, which cannot cross the cell membrane. Both sets of cells were washed three times, and then apoptotic or control AGS cells were added to individual wells of the chamber slide containing macrophages at a ratio of 5:1 in media containing normal FCS that was not heat inactivated. Results with FCS were comparable to using human serum (data not shown). Interactions were allowed to proceed for 1 h at 37 °C and 5 % CO2, after which time the reaction was stopped with PBS containing 0.01 % sodium azide. The slide was washed three times with PBS, fixed with 2 % paraformaldehyde for 40 min at 37 °C, and then the plastic wells and silicon adhesive were removed and discarded. Finally the slide was mounted with a cover slip and allowed to dry. Recognition and binding of apoptotic epithelial cells by and macrophages were assessed by counting using an Axioscope fluorescent microscope (Zeiss, Thornwood, NY). For each condition, four randomly selected fields in replicate slides were photographed and the total number of green fluorescent cells (macrophages) and red fluorescent cells (AGS) were counted. The number of macrophages with one, two or three or more AGS attached was recorded.
Evaluation of cytokine responses
THP-1 cells were differentiated into a macrophage-like phenotype by treating with 600 nM phorbol myristate acetate (PMA; Sigma, St Louis, MO) for 3 days in 48-well plates. Cell numbers in the wells were approximately 100,000 and were quantified prior to the cell co-culture assay by trypsinizing cells and counting in a heamocytometer. AGS cells were made apoptotic with either camptothecin treatment (3 μM) or with H. pylori infection (MOI 100) overnight in T75 cm flasks. AGS cells were approx. 80–90 % confluent before infection and cell numbers were counted from a spare, identically seeded flask to calculate the appropriate MOI for the infection. On the day of the co-culture, AGS cells were trypsinized, washed twice in PBS (300 × g for 5 min), counted, and resuspended in THP-1 media (RPMI + 10 % FBS+ 10 mM HEPES + pen strep) or X-vivo 10 media (+2 mM L-glut) at 3 times the THP-1 cell number per ml (phagocyte:target ratio 3:1). The supernatant was removed from the THP-1 macrophages and washed using PBS to remove serum traces before addition of 1 ml of the AGS cell suspension. Where appropriate, the THP-1 macrophages were pre-treated with 5 μg/ml cytochalasin D (Sigma, St Louis, MO) for 30 min prior to and throughout AGS co-culture. The co-culture was performed for 2 h at 37 °C after which, the AGS cells were washed away from the adherent THP-1 cell monolayer using cold PBS (1 ml; × 3 washes). Media was replaced +/− LPS 100 ng/ml (from Salmonella; Sigma, St Louis, MO) for 24 h after which, the supernatant was removed, centrifuged at 10,000 × g for 15 min and stored at −80 °C until used for specific ELISA for IL-6 or TNF-α (R&D Systems, Minneapolis, MN).
Polymerase chain reaction
Total RNA was extracted according to the manufacturer’s instructions using the RNeasy kit (Qiagen, Valencia, CA), and yield estimated spectrophotometrically. RNA was reverse transcribed using the Superscript kit (Invitrogen, Carlsbad, CA) random hexamer protocol as per the manufacturer’s instructions. Amplification of gC1qR, cC1qR, C1qRp, and CD91 mRNA was performed using TaqMan® pre-designed primers from Applied Biosystems (Foster City, CA). Primers were diluted 1:20 in TaqMan® Universal PCR Master Mix and water. Real-time PCR analysis was performed on a SmartCycler® (Cepheid, Sunnydale, CA) at 50 °C for 2 min, 95 °C for 10 min, 40 cycles of 95 °C for 15 s and 60 °C for 1 min.
Mouse anti-human phycoerythrin (PE)-conjugated monoclonal antibodies (mAb) to C1qRp (product #551087), FITC-conjugated mAb to CD91 (product #550496), purified anti-calreticulin (product #612137), a FITC-rat-anti-mIgG1 secondary antibody, (product #562026) were obtained from BD Biosciences (San Diego, CA). A purified mouse anti-human mAb to gC1qR was a generous gift from Dr. Young Hahn (University of Virginia). FITC-conjugated rabbit polyclonal anti-human C1q was purchased from Abcam (product # ab4223, Cambridge, MA). PE-mIgG2b,κ, (product #556656) PE-mIgG1,κ (product #551436), FITC- mIgG1,κ (product #554679), purified mIgG1,κ (product #349040) (BD Biosciences, San Diego CA) and FITC-Rabbit IgG (product #F0382), (Sigma, St Louis, MO) served as the isotype controls.
The assessment of apoptosis was performed using the Annexin V-FITC/Propidium iodide (PI) staining kit (R&D, Minneapolis, MN) as per manufacturer’s instructions. Cells were analyzed on a FACScan flow cytometer and analysed using FlowJo software (Treestar, Ashland, OR).
For complement receptor experiments, cells were suspended at 107/ml in PBS containing 1 % BSA and 0.01 % sodium azide. Antibodies were incubated with cells for 30 min on ice, washed twice in PBS containing 0.01 % sodium azide, and resuspended in 2 % PFA. When required, secondary labeling was performed as per primary labelling, following two washes in PBS with 0.01 % azide. Cells were analysed within 24 h on a BD FACSCalibur flow cytometer, and the data subsequently analysed using FlowJo software (Treestar, Ashland, OR).
Cells were seeded and allowed to adhere for 24 h prior to treatment with H. pylori (MOI 300:1), camptothecin (3 μM) or PBS (control cells). All treatments were performed in medium containing heat-inactivated serum. After 24 h of treatment, supernatants were collected, cells were washed once with PBS and trypsinzed. Trypsinized cells were added to supernatants and the cells then washed once in PBS. Cells were resuspended in either serum-free (SF) medium, medium containing 10 % non-heat-inactivated serum, or SF medium with 40 μg/ml C1q. After 1 h, cells were washed twice in PBS and resuspended at 107 per ml in PBS with 0.01 % sodium azide. FITC-Rabbit IgG or FITC-anti-C1q was added for 30 min on ice. Cells were subsequently washed twice in PBS with 0.01 % sodium azide and resuspended in 2 % PFA until flow cytometry was performed.
C1q cytokine studies
THP-1 cells were seeded at 0.25 × 106/ml in 48 well plates in the presence of 600 nM PMA for 3 days. On the day of the assay, the number of macrophages per well was approximately 100,000 and was verified using a hemocytometer. The cells were washed with PBS and re-suspended in X-vivo 10 medium (+2 mM L-glut). C1q protein was added to the cells at 0–80 μg/ml for 30 min prior to the addition of LPS (100 ng/ml) or H. pylori (MOI 100). Unstimulated controls were run in parallel. After 24 h of treatment, supernatants were collected, centrifuged at 10,000 ×g for 10 min and stored at −80 °C until assessed for cytokine (IL-6 and TNF-α) concentration using specific ELISAs (R&D Systems, Minneapolis, MN).
Results are expressed as the mean ± SEM. Results were compared using two-tailed Student’s t-test, or one-way ANOVA with Tukey’s post-hoc correction when indicated. Statistical significance and p values are indicated in the figures.
H. pylori infected gastric epithelial cells exhibit increased binding to macrophages
The engulfment of apoptotic cells involves recognition of the apoptotic target; binding of the target to the phagocyte and subsequent internalization into the cell. To determine whether infection of epithelial cells with H. pylori affected the ability of AGS cells to be recognized and bound to macrophages, control (uninfected) or infected AGS cells were incubated for 1 h with THP-1-derived macrophages. Evidence of binding was based on the number of epithelial cells (0, 1, 2 and ≥3 or more) associated with the phagocytes.
The ability of the THP-1 cells to recognize and bind uninfected and H. pylori infected AGS cells were compared (Fig. 1b). There was no significant difference between the numbers of macrophages with a single AGS cell attached, whether or not the AGS cells had been infected. However, aggregates of three or more AGS cells were bound to macrophages at a higher frequency when the AGS cells had been infected previously with H. pylori compared with uninfected cells. Consequently, the number of macrophages with no AGS cells clearly associated was much higher in the uninfected group than the infected targets, and this was also statistically significant (50 vs. 31 % for THP-1-derived macrophages, p < 0.05).
Binding of apoptotic gastric epithelial cells to phagocytes is dependent on serum proteins and can be mediated by C1q
Induction of Apoptosis of AGS cells by camptothecin and H. pylori
C1q interacts with both the apoptotic targets and the phagocyte
It has been reported that the globular domain of C1q can bind the membrane blebs on apoptotic cells [31, 46]. In support of this notion, we set out to evaluate this in our model using apoptotic AGS. To rule out any nonspecific binding of C1q to H. pylori, camptothecin was used to induce apoptosis in AGS cells and then the cells were incubated in either serum-free medium or serum-free medium supplemented with 40 μg/ml C1q. The amount of C1q bound to the apoptotic cells was then assessed using a FITC conjugated antibody against C1q and analysed using flow cytometry. C1q was not detected in significant levels on the surface of AGS cells in the absence of serum (Fig. 4c) showing that although we induce apoptosis in serum containing conditions, this is not adequate to opsonize the cells. However, when AGS cells were incubated in medium containing additional C1q, high levels of C1q were shown to bind preferentially to the AGS cells that were stimulated to undergo apoptosis. With the data above, these observations support the notion that C1q is sufficient to bind apoptotic cells and allow detection by phagocytes expressing complement receptors which can collaborate in the clearance of the dead cells.
Sterile apoptotic and H. pylori infected AGS cells attenuate THP-1 LPS-induced pro-inflammatory cytokine production
C1q protein attenuates THP-1 macrophage production of cytokines in response to LPS and H. pylori stimulation
Engulfment of apoptotic cells is an important process for tissue remodelling and homeostasis. This response entails several steps including: recruitment of phagocytes; recognition of dead cells; binding of the corpse to a phagocyte; internalization of the target; target degradation and finally, modification of phagocyte cell function. Engulfment of apoptotic epithelial cells by macrophages or dendritic cells has been observed in the intestinal lamina propria [47–49], the stomach  and in the lung . Furthermore, epithelial cell apoptosis is highly inducible by infections [41, 52–54] and in response to inflammation [12, 55]. The engulfment of apoptotic cells does not only remove cells to prevent release of toxic cell contents, but it is now widely accepted that apoptotic cells promote an anti-inflammatory phenotype in the engulfing phagocyte, thereby limiting inflammation [26, 27]. The current report shows that C1q opsonizes apoptotic AGS cells and also confers immunomodulatory properties to the phagocyte. Our model used the human THP-1 cell line that is differentiated into macrophage like cells using PMA. Previous studies using peripheral blood derived macrophages (PBDM) have been published by our group, which show comparable levels of AGS cell binding following H. pylori infection . Furthermore, our previous work also show attenuation of PBDM cytokine responses following apoptotic AGS co-culture and are in concordance with what we observe with THP-1 macrophages. Thus, our use of the THP-1 cell line as a model macrophage is appropriate and representative of other human macrophage responses.
In healthy cells, nearly all of the phosphatidylserine (PS) is confined to the inner aspect of the plasma membrane and exposure of PS on the outer leaflet marks the cells as apoptotic. As discussed elsewhere  many receptors and adaptor molecules have been implicated in the recognition and clearance of apoptotic cells including collectin receptors, calreticulin/CD91, Fcγ receptors, c-Mer, integrins such as αvβ3, scavenger receptors thrombospondin–CD36 and phosphatidylserine receptors like BAI1 and TIM4. Some of the recognition structures that bind to PS trigger a signalling response leading to engulfment (i.e., BAI1 ) while others, such as Tim4, perform an accessory function that facilitates the PS-dependent uptake of the target [59, 60]. It has been reported that complement proteins also bind to apoptotic cells, marking them for quick removal, and a large panel of different complement proteins are involved, including C1q, mannose-binding lectin and C3bi [61, 62]. The present study revealed that the interactions between H. pylori-infected cells and macrophages were mediated by a serum component and that C1q was sufficient to restore binding of H. pylori-infected AGS cells to macrophages. Importantly, C1q also conferred an anti-inflammatory effect on macrophages stimulated with LPS or H. pylori. The concentrations of C1q used in our experiments (40–80 μg/mL) are well within the normal range found in plasma (56–276 μg/mL). In relation to H. pylori infection, Berstad and colleagues [63, 64] have detected the terminal complement complex (TCC) in the gastric mucosa of subjects infected with H. pylori showing that complement activation occurs during infection, which could regulate the interaction between APCs and apoptotic cells as well as regulate phagocyte reactivity to infection.
It has been reported that C1q can bind via its globular head to PS exposed on the surface membrane blebs of apoptotic cells [31, 32, 46]. Indeed, induction of apoptosis in AGS cells with camptothecin allowed C1q to bind the target suggesting that the interaction was through the recognition of the dead cell and no bacterial component is required for the binding by C1q to occur. The fact that THP-1 cells expressed multiple receptors for C1q (gC1qR and C1qRp), supports the feasibility of the interaction. We analyzed the expression of peripheral blood derived macrophages (PBDMs) and showed these cells also express receptors for C1q. Using siRNA approaches to inhibit individual receptor expression were successful based on assays to measure gene expression. However, they failed to significantly block the C1q-mediated binding of the apoptotic cell (data not shown). This level of redundancy is common in binding and engulfment studies due to the presence of multiple receptors and mechanisms that phagocytes utilize to clear a wide array of different targets.
Several studies have demonstrated an effect of C1q on cytokine production by monocytes, macrophages and DCs during apoptotic cell uptake [35–37]. The modulation of cytokines by C1q was variable depending on which cytokines and APC were being studied. In general, a reduction in pro-inflammatory cytokines, IL-1β and IL-1α were reported together with an increase in IL-10, IL-6 and MCP-1. In the current study, apoptotic AGS cells attenuated production of LPS-stimulated cytokines, IL-6 and TNF-α independent of target internalization or the presence of serum. Moreover, the attenuation of cytokine responses occurred regardless of the method used to induce apoptosis. It is important to note that infected cell co-cultures did not confer a net pro-inflammatory effect compared to macrophages alone. This is intriguing as there are reports of other bacterial species which can use apoptotic targets as a type of Trojan horse to invade phagocytes in order to disseminate to locations remote from the point of entry [65, 66]. Current work by our lab and others has noted the redundancy of apoptotic cell internalization for attenuated macrophage responses [56, 67]. In these studies, it has been shown that conditioned medium from apoptotic cells is insufficient to confer anti-inflammatory effects and that contact of the macrophage and apoptotic cells is required. Furthermore, we have previously shown that binding of PS residues is also not required for the attenuation response to occur . The receptors responsible for altering macrophage reactivity following apoptotic cell contact are yet to be elucidated.
No serum components were required for the apoptotic cell-induced attenuation of cytokine responses and additional C1q during co-culture did not enhance this effect (data not shown). However, C1q protein incubated directly with THP-1 macrophages in the absence of apoptotic cells did have a significant effect, demonstrating a dose-dependent effect of C1q on LPS-stimulated TNF-α. The attenuation of TNF-α was noteworthy as this suggests a role for C1q in control of host responses to infection. We observed a similar trend in attenuation of baseline (unstimulated) production of TNF-α which may also indicate that C1q could be involved in controlling the general stress response in macrophages induced, in this instance, by serum starvation. Although the mechanism of the attenuation of cytokines by C1q remains elusive in our model, a recent publication by Galvan et al. has identified activation of AMPK as a potential candidate pathway which is triggered by C1q to induce modulation of immune reactivity in murine macrophages .
We show that H. pylori-infected AGS gastric epithelial cells bind to macrophages and that this response is facilitated by the C1q component of complement. It has been confirmed that these apoptotic cells are internalized by macrophages  although no additional enhancement of this process was observed with C1q in our hands (data not shown). As mentioned previously, the reported degree of receptor redundancy in the engulfment process makes it difficult to assess the importance of one factor . Other investigations have shown that the maturation and type of APC can influence the C1q enhanced uptake of apoptotic cells . They reported that monocytes but not monocyte-derived macrophages or DCs, enhanced their phagocytic capacity for apoptotic cells in the presence of C1q. However, enhancement of phagocytosis was observed in all APCs when C1q was added in the presence of serum with active complement. These differences in assay design may explain the lack of enhancement in internalization in our hands, as we only added C1q to serum free conditions to discriminate from the involvement of other serum factors. It is also important to note the differences in the apoptotic target cells used between other studies and ours, as our epithelial cell targets are larger than the conventionally used T cell (Jurkat) targets, and therefore may be more difficult to internalize.
Epithelial cell apoptosis occurs frequently in normal physiology, where the cells are replaced from progenitor cells located at the base of the crypts. [41, 53, 54]. Apoptotic epithelial cells are shed via extrusion, however at least some of these cells are cleared by phagocytes and neighbouring epithelial cells [70–72]. In this manner, the internalization of epithelial cells provides another means by which the host can sample the environment for antigens associated with damage or infection . It has also been reported that apoptotic epithelial cells can influence inflammation [72, 74]. Juncadella et al. demonstrated that apoptotic bronchial epithelial cells can be engulfed by other epithelial cells and that these interactions are important in the control of murine airway inflammation . Relevant to H. pylori, a recent study has shown that infection by H. pylori inhibits the phagocytosis of apoptotic gastric epithelial cells . Inhibition of clearance and the concomitant increase in the release of necrotic cell contents would trigger a heightened inflammatory response, which would likely inhibit the anti-inflammatory effect conferred by apoptotic cells. Furthermore, decreased phagocytosis would result in the reduced sampling of infected cells, which could lead to reduced antigen presentation and less pathogen specific immune cell attack. These mechanisms may provide an explanation of how a predominantly luminal infection triggers such a potent immune reaction. Future studies should focus on using the more relevant tissue specific macrophages as it is becoming apparent that these cells can respond very differently to microbial insult .
We report that human epithelial cells rendered apoptotic by infection are recognized and bound by phagocytes. This response entails a role for complement proteins, with C1q being a likely candidate. The co-culture of sterile (camptothecin induced) apoptotic and H. pylori infected AGS cells with macrophages resulted in an attenuated phagocyte cytokine response to LPS compared to phagocytes that did not contact apoptotic AGS cells and the attenuation was independent of the method of apoptosis induction and internalization of the apoptotic cell. The attenuation of LPS and H. pylori induced cytokine responses following apoptotic cell co-culture was independent of the presence of serum components, as the attenuation was observed in serum free conditions. Although serum components were not required for the effect observed following apoptotic cell co-culture, we show that incubation of C1q protein itself with macrophages was able to dose-dependently diminish LPS-stimulated production of TNF-α, thus, indicating that this protein could be involved in directly controlling the host response to infection. The outcome of this process on the regulation of host responses in the gastric mucosa remains the subject for future study.
We thank Joanne Lannigan, William Ross, Mike Solga, Ron Taylor and Elizabeth Wiznerowicz for technical assistance. This work was supported by the National Institutes of Health grants DK50980, DK84063 (to P.B.E.) and DK61769 (to SEC) while the imaging facility was supported by the UCSD Cancer Center Specialized Support Grant P30 CA23100 and the UCSD Digestive Disease Research Development Center (DK080506).
Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
- World Gastroenterology Organisation Global Guideline. Helicobacter pylori in developing countries. J Clin Gastroenterol. 2011;45:383–8.View ArticleGoogle Scholar
- Hunt RH, Xiao SD, Megraud F, Leon-Barua R, Bazzoli F, van der Merwe S, et al. Helicobacter pylori in developing countries. World Gastroenterology Organisation Global Guideline. J Gastrointestin Liver Dis. 2011;20:299–304.PubMedGoogle Scholar
- Polk DB, Peek Jr RM. Helicobacter pylori: gastric cancer and beyond. Nat Rev Cancer. 2010;10:403–14.PubMed CentralView ArticlePubMedGoogle Scholar
- Ernst PB, Gold BD. Helicobacter pylori in childhood: new insights into the immunopathogenesis of gastric disease and implications for managing infection in children. J Pediatr Gastroenterol Nutr. 1999;28:462–73.View ArticlePubMedGoogle Scholar
- Ernst PB, Peura DA, Crowe SE. The translation of Helicobacter pylori basic research to patient care. Gastroenterology. 2006;130:188–206. quiz 212–183.View ArticlePubMedGoogle Scholar
- Rad R, Prinz C, Schmid RM. Helicobacter pylori and prognosis of gastric carcinoma. Lancet Oncol. 2006;7:364–5.View ArticlePubMedGoogle Scholar
- Danese S, Papa A, Gasbarrini A, Ricci R, Maggiano N. Helicobacter pylori eradication down-regulates matrix metalloproteinase-9 expression in chronic gastritis and gastric ulcer. Gastroenterology. 2004;126:369–71.View ArticlePubMedGoogle Scholar
- Ghiara P, Marchetti M, Blaser MJ, Tummuru MK, Cover TL, Segal ED, et al. Role of the Helicobacter pylori virulence factors vacuolating cytotoxin, CagA, and urease in a mouse model of disease. Infect Immun. 1995;63:4154–60.PubMed CentralPubMedGoogle Scholar
- Blaser MJ. Heterogeneity of Helicobacter pylori. Eur J Gastroenterol Hepatol. 2012;9 Suppl 1:S3–6. discussion S6-7.View ArticlePubMedGoogle Scholar
- Blaser MJ. The biology of cag in the Helicobacter pylori-human interaction. Gastroenterology. 2005;128:1512–5.View ArticlePubMedGoogle Scholar
- Yamaoka Y. Mechanisms of disease: Helicobacter pylori virulence factors. Nat Rev Gastroenterol Hepatol. 2010;7:629–41.PubMed CentralPubMedGoogle Scholar
- Ernst PB, Crowe SE, Reyes VE. How does Helicobacter pylori cause mucosal damage? The inflammatory response. Gastroenterology. 1997;113:S35–42. discussion S50.View ArticlePubMedGoogle Scholar
- Persson C, Canedo P, Machado JC, El-Omar EM, Forman D. Polymorphisms in inflammatory response genes and their association with gastric cancer: A HuGE systematic review and meta-analyses. Am J Epidemiol. 2011;173:259–70.PubMed CentralView ArticlePubMedGoogle Scholar
- Macarthur M, Hold GL, El-Omar EM. Inflammation and Cancer II. Role of chronic inflammation and cytokine gene polymorphisms in the pathogenesis of gastrointestinal malignancy. Am J Physiol Gastrointest Liver Physiol. 2004;286:G515–20.View ArticlePubMedGoogle Scholar
- El-Omar E, Carrington M, Chow WH, McColl KEL, Bream JH, Young HA, et al. Interleukin-1 polymorphisms associated with increased risk of gastric cancer. Nature. 2000;404:398–402.View ArticlePubMedGoogle Scholar
- Crabtree JE, Covacci A, Farmery SM, Xiang Z, Tompkins DS, Perry S, et al. Helicobacter pylori induced interleukin-8 expression in gastric epithelial cells is associated with CagA positive phenotype. J Clin Pathol. 1995;48:41–5.PubMed CentralView ArticlePubMedGoogle Scholar
- Crabtree JE, Shallcross TM, Heatley RV, Wyatt JI. Mucosal tumour necrosis factor alpha and interleukin-6 in patients with Helicobacter pylori associated gastritis. Gut. 1991;32:1473–7.PubMed CentralView ArticlePubMedGoogle Scholar
- Crowe SE, Alvarez L, Dytoc M, Hunt RH, Muller M, Sherman P, et al. Expression of interleukin 8 and CD54 by human gastric epithelium after Helicobacter pylori infection in vitro. Gastroenterology. 1995;108:65–74.View ArticlePubMedGoogle Scholar
- Harris PR, Ernst PB, Kawabata S, Kiyono H, Graham MF, Smith PD. Recombinant Helicobacter pylori urease activates primary mucosal macrophages. J Infect Dis. 1998;178:1516–20.View ArticlePubMedGoogle Scholar
- Wyatt JI, Rathbone BJ. Immune response of the gastric mucosa to Campylobacter pylori. Scand J Gastroenterol Suppl. 1988;142:44–9.View ArticlePubMedGoogle Scholar
- Hansson M, Hermansson M, Svensson H, Elfvin A, Hansson LE, Johnsson E, et al. CCL28 is increased in human Helicobacter pylori-induced gastritis and mediates recruitment of gastric immunoglobulin A-secreting cells. Infect Immun. 2008;76:3304–11.PubMed CentralView ArticlePubMedGoogle Scholar
- Bimczok D, Grams JM, Stahl RD, Waites KB, Smythies LE, Smith PD. Stromal regulation of human gastric dendritic cells restricts the Th1 response to Helicobacter pylori. Gastroenterology. 2011;141:929–38.PubMed CentralView ArticlePubMedGoogle Scholar
- Bamford KB, Fan XJ, Crowe SE, Leary JF, Gourley WK, Luthra GK, et al. Lymphocytes in the human gastric mucosa during Helicobacter pylori have a T helper cell 1 phenotype. Gastroenterology. 1998;114:482–92.View ArticlePubMedGoogle Scholar
- Luzza F, Parrello T, Monteleone G, Sebkova L, Romano M, Zarrilli R, et al. Up-regulation of IL-17 is associated with bioactive IL-8 expression in Helicobacter pylori -infected human gastric mucosa. J Immunol. 2000;165:5332–7.View ArticlePubMedGoogle Scholar
- Harris PR, Wright SW, Serrano C, Riera F, Duarte I, Torres J, et al. Helicobacter pylori gastritis in children is associated with a regulatory T-cell response. Gastroenterology. 2008;134:491–9.View ArticlePubMedGoogle Scholar
- Savill J, Dransfield I, Gregory C, Haslett C. A blast from the past: clearance of apoptotic cells regulates immune responses. Nat Rev Immunol. 2002;2:965–75.View ArticlePubMedGoogle Scholar
- Savill J, Fadok V. Corpse clearance defines the meaning of cell death. Nature. 2000;407:784–8.View ArticlePubMedGoogle Scholar
- Huynh ML, Fadok VA, Henson PM. Phosphatidylserine-dependent ingestion of apoptotic cells promotes TGF-beta1 secretion and the resolution of inflammation. J Clin Invest. 2002;109:41–50.PubMed CentralView ArticlePubMedGoogle Scholar
- Fadok VA, Bratton DL, Konowal A, Freed PW, Westcott JY, Henson PM. Macrophages that have ingested apoptotic cells in vitro inhibit proinflammatory cytokine production through autocrine/paracrine mechanisms involving TGF-beta, PGE2, and PAF. J Clin Invest. 1998;101:890–8.PubMed CentralView ArticlePubMedGoogle Scholar
- Ferracini M, Rios FJ, Pecenin M, Jancar S. Clearance of apoptotic cells by macrophages induces regulatory phenotype and involves stimulation of CD36 and platelet-activating factor receptor. Mediators Inflamm. 2013;950273:20.Google Scholar
- Paidassi H, Tacnet-Delorme P, Garlatti V, Darnault C, Ghebrehiwet B, Gaboriaud C, et al. C1q binds phosphatidylserine and likely acts as a multiligand-bridging molecule in apoptotic cell recognition. J Immunol. 2008;180:2329–38.PubMed CentralView ArticlePubMedGoogle Scholar
- Ogden CA, de Cathelineau A, Hoffmann PR, Bratton D, Ghebrehiwet B, Fadok VA, et al. C1q and mannose binding lectin engagement of cell surface calreticulin and CD91 initiates macropinocytosis and uptake of apoptotic cells. J Exp Med. 2001;194:781–95.PubMed CentralView ArticlePubMedGoogle Scholar
- Vandivier RW, Ogden CA, Fadok VA, Hoffmann PR, Brown KK, Botto M, et al. Role of surfactant proteins A, D, and C1q in the clearance of apoptotic cells in vivo and in vitro: calreticulin and CD91 as a common collectin receptor complex. J Immunol. 2002;169:3978–86.View ArticlePubMedGoogle Scholar
- Gaipl US, Munoz LE, Grossmayer G, Lauber K, Franz S, Sarter K, et al. Clearance deficiency and systemic lupus erythematosus (SLE). J Autoimmun. 2007;28:114–21.View ArticlePubMedGoogle Scholar
- Benoit ME, Clarke EV, Morgado P, Fraser DA, Tenner AJ. Complement protein C1q directs macrophage polarization and limits inflammasome activity during the uptake of apoptotic cells. J Immunol. 2012;188:5682–93.PubMed CentralView ArticlePubMedGoogle Scholar
- Fraser DA, Bohlson SS, Jasinskiene N, Rawal N, Palmarini G, Ruiz S, et al. C1q and MBL, components of the innate immune system, influence monocyte cytokine expression. J Leukoc Biol. 2006;80:107–16.View ArticlePubMedGoogle Scholar
- Fraser DA, Laust AK, Nelson EL, Tenner AJ. C1q differentially modulates phagocytosis and cytokine responses during ingestion of apoptotic cells by human monocytes, macrophages, and dendritic cells. J Immunol. 2009;183:6175–85.PubMed CentralView ArticlePubMedGoogle Scholar
- Verneret M, Tacnet-Delorme P, Osman R, Awad R, Grichine A, Kleman JP, et al. Relative contribution of c1q and apoptotic cell-surface calreticulin to macrophage phagocytosis. J Innate Immun. 2014;6:426–34.PubMedGoogle Scholar
- Clarke EV, Weist BM, Walsh CM, Tenner AJ. Complement protein C1q bound to apoptotic cells suppresses human macrophage and dendritic cell-mediated Th17 and Th1 T cell subset proliferation. J Leukoc Biol. 2015;97:147–60.View ArticlePubMedGoogle Scholar
- Jones NL, Day AS, Jennings HA, Sherman PM. Helicobacter pylori induces gastric epithelial cell apoptosis in association with increased Fas receptor expression. Infect Immun. 1999;67:4237–42.PubMed CentralPubMedGoogle Scholar
- Moss SF, Calam J, Agarwal B, Wang S, Holt PR. Induction of gastric epithelial apoptosis by Helicobacter pylori. Gut. 1996;38:498–501.PubMed CentralView ArticlePubMedGoogle Scholar
- Rudi J, Kuck D, Strand S, Von Herbay A, Mariani SM, Krammer PH, et al. Involvement of the CD95 (APO-1/Fas) receptor and ligand system in Helicobacter pylori -induced gastric epithelial apoptosis. J Clin Invest. 1998;102:1506–14.PubMed CentralView ArticlePubMedGoogle Scholar
- Fan XJ, Crowe SE, Behar S, Gunasena H, Ye G, Haeberle H, et al. The effect of class II MHC expression on adherence of Helicobacter pylori and induction of apoptosis in gastric epithelial cells: A mechanism for Th1 cell-mediated damage. J Exp Med. 1998;187:1659–69.PubMed CentralView ArticlePubMedGoogle Scholar
- Ghebrehiwet B, Peerschke EI. Purification of C1q receptors and functional analysis. Methods Mol Biol. 2014;1100:319–27.View ArticlePubMedGoogle Scholar
- Ghiran I, Tyagi SR, Klickstein LB, Nicholson-Weller A. Expression and function of C1q receptors and C1q binding proteins at the cell surface. Immunobiology. 2002;205:407–20.View ArticlePubMedGoogle Scholar
- Nauta AJ, Trouw LA, Daha MR, Tijsma O, Nieuwland R, Schwaeble WJ, et al. Direct binding of C1q to apoptotic cells and cell blebs induces complement activation. Eur J Immunol. 2002;32:1726–36.View ArticlePubMedGoogle Scholar
- Iwanaga T, Han H, Adachi K, Fujita T. A novel mechanism for disposing of effete epithelial cells in the small intestine of guinea pigs. Gastroenterology. 1993;105:1089–97.PubMedGoogle Scholar
- Iwanaga T, Hoshi O, Han H, Takahashi-Iwanaga H, Uchiyama Y, Fujita T. Lamina propria macrophages involved in cell death (apoptosis) of enterocytes in the small intestine of rats. Arch Histol Cytol. 1994;57:267–76.View ArticlePubMedGoogle Scholar
- Shibahara T, Sato N, Waguri S, Iwanaga T, Nakahara A, Fukutomi H, et al. The fate of effete epithelial cells at the villus tips of the human small intestine. Arch Histol Cytol. 1995;58:205–19.View ArticlePubMedGoogle Scholar
- Karam SM. Dynamics of epithelial cells in the corpus of the mouse stomach. IV. Bidirectional migration of parietal cells ending in their gradual degeneration and loss. Anat Rec. 1993;236:314–32.View ArticlePubMedGoogle Scholar
- Borges VM, Vandivier RW, McPhillips KA, Kench JA, Morimoto K, Groshong SD, et al. TNFalpha inhibits apoptotic cell clearance in the lung, exacerbating acute inflammation. Am J Physiol Lung Cell Mol Physiol. 2009;297:L586–95.PubMed CentralView ArticlePubMedGoogle Scholar
- Fan X, Gunasena H, Cheng Z, Espejo R, Crowe SE, Ernst PB, et al. Helicobacter pylori urease binds to class II MHC on gastric epithelial cells and induces their apoptosis. J Immunol. 2000;165:1918–24.View ArticlePubMedGoogle Scholar
- Jones NL, Shannon PT, Cutz E, Yeger H, Sherman PM. Increase in proliferation and apoptosis of gastric epithelial cells early in the natural history of Helicobacter pylori infection. Am J Pathol. 1997;151:1695–703.PubMed CentralPubMedGoogle Scholar
- Wagner S, Beil W, Westermann J, Logan RP, Bock CT, Trautwein C, et al. Regulation of gastric epithelial cell growth by Helicobacter pylori: offdence for a major role of apoptosis. Gastroenterology. 1997;113:1836–47.View ArticlePubMedGoogle Scholar
- Denning TL, Takaishi H, Crowe SE, Boldogh I, Jevnikar A, Ernst PB. Oxidative stress induces the expression of Fas and Fas ligand and apoptosis in murine intestinal epithelial cells. Free Radic Biol Med. 2002;33:1641–50.View ArticlePubMedGoogle Scholar
- Das S, Sarkar A, Ryan KA, Fox S, Berger AH, Juncadella IJ, et al. 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. FASEB J. 2014.Google Scholar
- Bratton DL, Henson PM. Apoptotic cell recognition: will the real phosphatidylserine receptor(s) please stand up? Curr Biol. 2008;18:R76–9.View ArticlePubMedGoogle Scholar
- Park D, Tosello-Trampont AC, Elliott MR, Lu M, Haney LB, Ma Z, et al. BAI1 is an engulfment receptor for apoptotic cells upstream of the ELMO/Dock180/Rac module. Nature. 2007;450:430–4.View ArticlePubMedGoogle Scholar
- Miyanishi M, Tada K, Koike M, Uchiyama Y, Kitamura T, Nagata S. Identification of Tim4 as a phosphatidylserine receptor. Nature. 2007;450:435–9.View ArticlePubMedGoogle Scholar
- Park D, Hochreiter-Hufford A, Ravichandran KS. The phosphatidylserine receptor TIM-4 does not mediate direct signaling. Curr Biol. 2009;19:346–51.View ArticlePubMedGoogle Scholar
- Bohlson SS, Fraser DA, Tenner AJ. Complement proteins C1q and MBL are pattern recognition molecules that signal immediate and long-term protective immune functions. Mol Immunol. 2007;44:33–43.View ArticlePubMedGoogle Scholar
- Hart SP, Smith JR, Dransfield I. Phagocytosis of opsonized apoptotic cells: roles for ‘old-fashioned’ receptors for antibody and complement. Clin Exp Immunol. 2004;135:181–5.PubMed CentralView ArticlePubMedGoogle Scholar
- Berstad AE, Brandtzaeg P, Stave R, Halstensen TS. Epithelium related deposition of activated complement in Helicobacter pylori associated gastritis. Gut. 1997;40:196–203.PubMed CentralView ArticlePubMedGoogle Scholar
- Berstad AE, Hogasen K, Bukholm G, Moran AP, Brandtzaeg P. Complement activation directly induced by Helicobacter pylori. Gastroenterology. 2001;120:1108–16.View ArticlePubMedGoogle Scholar
- Laskay T, van Zandbergen G, Solbach W. Neutrophil granulocytes as host cells and transport vehicles for intracellular pathogens: apoptosis as infection-promoting factor. Immunobiology. 2008;213:183–91.View ArticlePubMedGoogle Scholar
- Nguyen L, Pieters J. The Trojan horse: survival tactics of pathogenic mycobacteria in macrophages. Trends Cell Biol. 2005;15:269–76.View ArticlePubMedGoogle Scholar
- Lucas M, Stuart LM, Zhang A, Hodivala-Dilke K, Febbraio M, Silverstein R, et al. Requirements for apoptotic cell contact in regulation of macrophage responses. J Immunol. 2006;177:4047–54.View ArticlePubMedGoogle Scholar
- Galvan MD, Hulsebus H, Heitker T, Zeng E, Bohlson SS. Complement Protein C1q and Adiponectin Stimulate Mer Tyrosine Kinase-Dependent Engulfment of Apoptotic Cells through a Shared Pathway. J Innate Immun. 2014;17:17.Google Scholar
- Mosser DM. Receptors on phagocytic cells involved in microbial recognition. Immunol Ser. 1994;60:99–114.PubMedGoogle Scholar
- Hall PA, Coates PJ, Ansari B, Hopwood D. Regulation of cell number in the mammalian gastrointestinal tract: the importance of apoptosis. J Cell Sci. 1994;107:3569–77.PubMedGoogle Scholar
- Monks J, Smith-Steinhart C, Kruk ER, Fadok VA, Henson PM. Epithelial cells remove apoptotic epithelial cells during post-lactation involution of the mouse mammary gland. Biol Reprod. 2008;78:586–94.View ArticlePubMedGoogle Scholar
- Monks J, Rosner D, Geske FJ, Lehman L, Hanson L, Neville MC, et al. Epithelial cells as phagocytes: apoptotic epithelial cells are engulfed by mammary alveolar epithelial cells and repress inflammatory mediator release. Cell Death Differ. 2005;12:107–14.View ArticlePubMedGoogle Scholar
- Getts DR, McCarthy DP, Miller SD. Exploiting apoptosis for therapeutic tolerance induction. J Immunol. 2013;191:5341–6.View ArticlePubMedGoogle Scholar
- Juncadella IJ, Kadl A, Sharma AK, Shim YM, Hochreiter-Hufford A, Borish L, et al. Apoptotic cell clearance by bronchial epithelial cells critically influences airway inflammation. Nature. 2013;493:547–51.PubMed CentralView ArticlePubMedGoogle Scholar
- Bimczok D, Smythies LE, Waites KB, Grams JM, Stahl RD, Mannon PJ, et al. Helicobacter pylori infection inhibits phagocyte clearance of apoptotic gastric epithelial cells. J Immunol. 2013;190:6626–34.PubMed CentralView ArticlePubMedGoogle Scholar
- Smith PD, Smythies LE, Shen R, Greenwell-Wild T, Gliozzi M, Wahl SM. Intestinal macrophages and response to microbial encroachment. Mucosal Immunol. 2011;4:31–42.View ArticlePubMedGoogle Scholar