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Keywords:

  • CLIC3;
  • CRIg;
  • Listeria;
  • Phagosome;
  • Signaling

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgments
  8. Conflict of interest
  9. References
  10. Supporting Information

Macrophages provide a first line of defense against bacterial infection by engulfing and killing invading bacteria, but intracellular bacteria such as Listeria monocytogenes (LM) can survive in macrophages by various mechanisms of evasion. Complement receptor of the immunoglobulin (CRIg), a C3b receptor, binds to C3b on opsonized bacteria and facilitates clearance of the bacteria by promoting their uptake. We found that CRIg signaling induced by agonistic anti-CRIg mAb enhanced the killing of intracellular LM by macrophages, and that this occurred in LM-containing phagosomes. Chloride intra-cellular channel 3 CLIC3, an intracellular chloride channel protein, was essential for CRIg-mediated LM killing by directly interacting with the cytoplasmic domain of CRIg, and the two proteins colocalized on the membranes of LM-containing vacuoles. CLIC3−/− mice were as susceptible to LM as CRIg−/− mice. These findings identify a mechanism embedded in the process by which macrophages take up opsonized bacteria that prevents the bacteria from evading cell-mediated killing.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgments
  8. Conflict of interest
  9. References
  10. Supporting Information

Phagosomes serve as the first line of anti-microbial defense by macrophages. Phagsomes form by invagination of the plasma membrane, and endomembrane compartments such as the endoplasmic reticulum and endosomes contribute additional components to the phagosome [1]. Phagosomes undergo constant remodeling and fuse with lysosomes to form phagolysosomes [2].

Listeria monocytogenes (LM) has been widely used to study the interaction between intracellular pathogens and cellular lytic systems such as phagosomes and autophagosomes [3]. LM produces a major virulence factor called listeriolysin O (LLO), a cholesterol-dependent pore-forming toxin that blocks phagosome-lysosome fusion by generating small pores that uncouple pH and calcium gradients across the phagosome membrane [4]. By delaying phagosome maturation and its own degradation, LM prolongs its survival inside phagosome/endosomes as a prelude to escaping into the cytoplasm [5].

Complement receptor of the immunoglobulin (CRIg) is a 50 kD type I transmembrane receptor that belongs to the Ig superfamily and is expressed specifically on macrophages and mature dendritic cells [6, 7]. The corresponding gene was discovered independently by other workers and designated Z39Ig [8] and VSIG4 [9]. CRIg is a novel receptor for C3b and iC3b. It promotes rapid phagocytosis of C3b-opsonized intracellular bacteria and efficiently sequesters pathogens within macrophages. Moreover, inflammatory diseases such as experimental arthritis and ischemia/reperfusion injury are ameliorated by exposure to recombinant CRIg-Fc, which inhibits harmful humoral and cellular reactions [10, 11]. Hence, CRIg is believed to be involved in negative regulation of immune responses [9].

Fusion between phagosomes and lysosomes is accelerated when the phagosomal lumen becomes acidic. This acidification is essential for the killing of bacteria engulfed by macrophages, and is known to require H+-ATPase activity [12]. The cystic fibrosis transmembrane conductance regulator Cl channel is reported to regulate phagosome acidification in alveolar macrophages [13], but it is not clear whether other chloride-related channel proteins, such as chloride intracellular channel (CLIC) proteins, are involved in the acidification of macrophage phagosomes [14]. Of the seven CLIC proteins, CLIC1 and CLIC4 have been shown to be essential molecular components of anion channels, but the channel activities of the other forms are less well characterized [15, 16].

CRIg signaling appears to be triggered during the uptake of opsonized bacteria by direct interaction of CRIg with C3b on the bacteria, and regulates killing of the bacteria by macrophages. By employing the yeast two-hybrid screen, we discovered that CLIC3 binds to the cytoplasmic domain of CRIg and is essential for phagosomal acidification in macrophages.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgments
  8. Conflict of interest
  9. References
  10. Supporting Information

CRIg signaling eliminates the intracellular pathogen LM

To examine whether triggering of CRIg enhances the killing of intracellular LM by macrophages, THP-1 cells, which constitutively express CRIg on their surface, were infected with LM for 1 h and treated with agonistic anti-human (h) CRIg mAb, or mouse (m) IgG as a control. LM began to grow after an initial 4–6 h of incubation in THP-1 cells (Supporting Information Fig. 1A). Treatment of the THP-1 cells with agonistic anti-hCRIg (10 μg/mL or more) rapidly eliminated the intracellular LM (Fig. 1A). Heat-inactivated anti-hCRIg was ineffective, indicating that the effect of anti-hCRIg was not due to contamination with LPS (Supporting Information Fig. 1B). Anti-hCRIg was also effective in LPS-stimulated THP-1 cells (Supporting Information Fig. 1C).

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Figure 1. CRIg signaling facilitates clearance of L. monocytogenes (LM) in macrophages. (A and B) LM killing by (A) the indicated doses of anti-hCRIg or (B) the natural ligand C3b in THP-1 cells is shown. (C) Growth curves of LM and opLM in J774 cells is shown. (D) LM killing in WT or CRIg−/− macrophages is shown. LM or opLM-infected WT and CRIg−/− BMDMs were treated with 10 μg/mL of rIgG or anti-mCRIg for 6 h. (A–D) Data are shown as mean + SD of three samples and are representative of three experiments. (E) THP-1 or J774 cells were infected with 10 MOI of TMR-labeled LM for 1 h and incubated with FITC-conjugated anti-hCRIg or anti-mCRIg mAb for 30 min at 4°C. The antibody-labeled cells were then split; half were incubated at 4°C for a further 30 min — while the other half were incubated at 37°C for 30 min. Samples were then immediately fixed in paraformaldehyde, mounted, and visualized with a confocal microscope. CRIg and LM colocalization events were calculated based on LM in macrophage and are shown as mean + SD of n = 15 (right). Scale bar = 5 μm. Data shown are representative of three experiments performed. *p < 0.05; **p < 0.01; ***p < 0.001, Student's t-test.

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Since C3b is the natural ligand of CRIg [6], we tested whether C3b stimulated LM killing in THP-1 cells. We found that C3b reduced the multiplication of intracellular LM (Fig. 1B). In addition, since macrophages efficiently internalize complement-opsonized LM (opLM) as a result of interaction between CRIg and complement C3b [6], we tested whether opLM triggered CRIg signaling and enhanced the elimination of phagocytosed LM. As expected, opLM were more efficiently phagocytosed by J774 cells than LM since CFU at 0 h were significantly higher in the opLM-infected cells than in those infected with LM (Fig. 1C). CFU gradually increased in the J774 cells infected with LM, whereas they declined rapidly in the cells infected with opLM before later increasing. Statistical analysis indicated that CFU were significantly lower in the cells infected with opLM than in those infected with LM until 2 h after infection (Fig. 1C), indicating that opLM-mediated CRIg signaling enhances LM killing but may fail to kill escaped LM. We next generated bone marrow-derived macrophages (BMDMs) from wild type (WT) and CRIg−/− mice and confirmed that CRIg was expressed on more than 72% of the WT BMDMs but not on CRIg−/− BMDMs (Supporting Information Fig. 1D). Following 1 h of exposure to LM, BMDMs were treated with agonistic anti-mCRIg or rat (r) IgG. In the WT BMDMs, the growth of both LM and opLM was efficiently inhibited by anti-mCRIg mAb. However, the anti-mCRIg mAb was completely in-effective in the CRIg−/− BMDMs (Fig. 1D). Moreover, we found that the membrane-bound CRIg was internalized and became colocalized with intracellular LM following cross-linking with anti-CRIg mAb at 37°C but not at 4°C. Approximately 50% of the LM bacteria were surrounded by CRIg (Fig. 1E).

These observations indicate that the activation of CRIg by an agonistic mAb enhances the killing of intracellular LM.

CRIg signaling kills LM via interaction with CLIC3

We used inhibitors to test whether CRIg-dependent LM killing was mediated by the production of nitric oxide (NO) or reactive oxygen species (ROS). Anti-hCRIg was still effective in killing LM when NO and ROS production were blocked, indicating that they were not involved in the killing (Supporting Information Fig. 2).

To understand the signals leading to LM killing, we searched for molecules associated with the cytoplasmic domain of hCRIg. In a yeast two-hybrid screen, we isolated a previously known protein, CLIC3, which interacted with the cytoplasmic domain of hCRIg (Supporting Information Fig. 3A). We found that CLIC3 coimmunoprecipitated with hCRIg in CLIC3- and hCRIg-transfected HeLa cells, pointing to an interaction between the two molecules (Fig. 2A). In addition, analysis of serial deletions and point mutations of CLIC3 indicated that hCRIg interacted with amino acid 212 (E) of CLIC3 (Supporting Information Fig. 3B and C). Mouse (m) CLIC3 also interacted with the cytoplasmic domain of mCRIg (Supporting Information Fig. 3D).

image

Figure 2. CRIg interacts with CLIC3 to suppress LM infection. (A) Coimmunoprecipitation of hCRIg with hCLIC3 is shown. HA-tagged CLIC3 (CLIC3-HA) and control vector were overexpressed in HeLa cells or FLAG-tagged hCRIg (hCRIg-FLAG)-expressing HeLa cells. Human CRIg was immunoprecipitated with anti-FLAG mAb and coprecipitated CLIC3 was detected with anti-HA mAb. Data shown are from one experiment representative of four experiments performed. (B) J774 mouse macrophage cells were incubated with TMR-conjugated anti-mCRIg mAb for 30 min at 4°C. The antibody-labeled J774 cells were then split and half of the cells were incubated at 4°C for 30 min and the other half at 37°C for 30 min. The cells were then fixed with paraformaldehyde, stained with anti-mCLIC3-FITC and visualized by confocal microscopy. Scale bar = 10 μm. Cells showing colocalization of mCLIC3 and mCRIg were counted and shown as mean + SD of 20 cells/fields of view/samples (right). Data shown are representative of three experiments performed. (C) Cells incubated with control siRNA or siCLIC3 THP-1 cells were infected with LM and incubated with mIgG or anti-hCRIg for 4 h. LM killing was then assayed. (D) CRIg-mediated LM killing is inhibited by about 200 μm of R(+)-IAA-94. (C and D) Data are shown as mean + SD of three samples and are representative of three (C) or four (D) independent experiments. ***p < 0.001, Student's t-test.

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CLIC3 has been reported to be located in nuclei, endosomes, and lysosomes when overexpressed in COS, CV-1, or A2780 human ovarian cancer cells [17, 18]. We therefore examined the expression pattern of CLIC3 in various cell types such as CV-1, 293, NIH-3T3, and J774 cells as well as peritoneal macrophages, and observed a punctate distribution of CLIC3 in the cytosol of J774 cells and peritoneal macrophages (data not shown). Moreover, CLIC3 colocalized with the internalized CRIg but not with cell membrane-bound CRIg in J774 cells (Fig. 2B). These findings indicate that CLIC3 interacts with the cytoplasmic domain of CRIg.

We generated siRNA (siCLIC3) to silence CLIC3 and subsequently selected a THP-1 clone that expressed siCLIC3 and tested its ability to kill LM. Anti-hCRIg-mediated LM killing was completely absent in the siCLIC3 THP-1 cells (Fig. 2C). CLIC3 is a member of the family of CLIC proteins, but its role as a chloride channel is not well established. We therefore examined anti-hCRIg-mediated LM killing in the presence of a broad specificity chloride channel blocker R(+)-IAA-94 and found that the blocker indeed inhibited anti-hCRIg-mediated LM killing in a dose-dependent manner. R(+)-IAA-94 was not toxic to the THP-1 cells up to a concentration of 200 μM, and it did not inhibit LM multiplication (Fig. 2D).

Chloride channels permit entry of Cl into the phagosome lumen [13]. The vacuolar (V)-type H+-ATPase is known to be an integral component of phagosomes and functions to pump H+ into the phagosomes to acidify them [19]. However, hCRIg triggering was not coupled to a V-Type H+-ATPase because bafilomycin did not inhibit anti-hCRIg-mediated killing (Fig. 3B). CRIg-mediated LM killing was completely blocked by NH4Cl (which alkalinizes the cytoplasm and vacuoles) and ouabain (an Na+/K+ ATPase blocker), and partially by amiloride, Na+/K+ exchanger 1 (NHE1) inhibitor (Fig. 3C and D), indicating that Na+/K+ ATPase activity is required for CRIg-mediated LM killing.

image

Figure 3. Na+/K+-ATPase is required for CRIg-mediated LM killing. LM-infected J774 cells were incubated with mIgG or anti-CRIg for 0, 2, 4, and 6 h in the presence of (A) DMSO, (B) bafilomycin A (100 nM), (C) NH4Cl (10 mM), (D) ouabain (10−6 M), or (E) amiloride (0.01 mM). LM killing was assayed as described in Materials and methods. Data are shown as mean + SD of three samples and are representative of three independent experiments. *p < 0.05; **p < 0.01, Student's t-test.

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These results demonstrate that CLIC3 interacts with CRIg on phagosomal membranes and is required for CRIg-mediated LM killing.

CRIg signaling increases the chloride concentration in LM vacuoles and acidifies them

We measured the chloride concentration in LM vacuoles using dual-labeled chloride-sensing LM [20]. 6-Methoxyquinoline-N-6-hexanoic acid (MQHA) is a fluorescent pH-insensitive but Cl-sensitive indicator whose fluorescence is progressively quenched by increasing [Cl]. LM was labeled with MQHA and tetramethylrhodamine (TMR), a red chloride-insensitive fluorophore to provide a constant fluorescence standard. When THP-1 cells were infected with the dual-labeled LM, green and red LM vacuoles were visible by confocal microscopy in control mIgG-treated samples (Fig. 4A). Anti-hCRIg treatment reduced the MQHA fluorescence while not affecting TMR staining (Fig. 4A, THP-1). It also reduced MQHA fluorescence in the presence of concentrations of NH4Cl that cause alkalization of the cytoplasm and vacuoles [21], indicating that the CRIg-mediated increase of [Cl] can occur without phagosome acidification. Anti-hCRIg mAb did not quench MQHA fluorescence in siCLIC3 THP-1 cells, showing that the anti-hCRIg-mediated Cl influx was dependent on CLIC3 (Fig. 4A, siCLIC3 THP-1).

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Figure 4. CRIg signals increase the chloride concentration and acidification of LM vacuoles. (A–C) THP-1 or siCLIC3 THP-1 cells were infected with MQHA-TMR-LM for 1 h, and incubated with mIgG or anti-hCRIg in the presence or absence of 10 mM NH4Cl. (A) Confocal images of MQHA and TMR fluorescence were taken after 30 min incubation with 10 μg/mL mIgG or anti-hCRIg mAb. Scale bar = 5 μm. (B) Standard curve of the red/green fluorescence ratio versus [Cl]. At each chloride concentration, 30 dual-labeled LM in THP-1 cells were randomly selected and their red/green ratios determined. (C) [Cl] in LM vacuoles. THP-1 or siCLIC3 THP-1 cells were infected with dual-labeled LM and treated with mIgG or anti-hCRIg with or without 10 mM NH4Cl. The red/green ratios of 15 randomly selected LM vacuoles were determined for each treatment. [Cl] was then determined from the standard curve. (D) Anti-hCRIg-mediated acidification of LM vacuoles is dependent on CLIC3 and inhibited by ouabain. THP-1 cells (left), siCLIC3 THP-1 cells (middle), and 10−6 M ouabain-treated THP-1 cells (right) were infected with FITC/TMR dual-labeled LM, and treated with mIgG or anti-hCRIg for 30 min. Cell images were scanned by confocal microscope. Scale bar = 10 μm. (E) Standard curve of the excitation ratio of FITC/TMR as a function of buffer pH. For each pH point, 30 dual-labeled LM vacuoles in THP-1 cells were randomly selected and the ratios of green/red fluorescence were determined. (F) The pH of vacuoles was determined after THP-1 or siCLIC3 THP-1 cells were infected with dual-labeled LM, then treated with mIgG or anti-hCRIg in the presence of 100 nM bafilomycin A (baf), 10 mM NH4Cl, or 10−6 M ouabain for 1 h. The pH of randomly selected LM vacuoles was determined for each treatment. (A, D) Data shown are representative of three experiments performed, (B, E) shown as mean + SD of 30 samples and are representative of three experiments. (C, F) Each symbol represents an individual LM vacuole and the bars represent the means. Data shown are from one experiment representative of three performed. ***p < 0.001, Student's t-test.

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To measure [Cl] in LM vacuoles, we constructed a standard curve of the fluorescence ratio of the dual fluorophore-labeled probe versus chloride concentration in iso-osmotic solutions. We obtained a linear relationship, and the Stern–Volmer constant (Ksv) in our conditions was approximately 20 M−1 (Fig. 4B). When we measured the effect of CRIg signals on [Cl], we found that LM vacuole [Cl] increased from ∼30 mM to ∼100 mM. The CRIg-mediated increase of [Cl] occurred independently of NH4Cl, and was abrogated in the absence of CLIC3 (Fig. 4C). These data confirm that CLIC3 is required for the CRIg-mediated increase of [Cl] in LM vacuoles.

To examine whether the increased [Cl] leads to acidification of LM vacuoles, LM were conjugated with pH-sensitive carboxyfluorescein (fluorescein) and pH-insensitive TMR [20]. Fluorescein quenching occurs at or near the acidic pH and thus allows accurate measurement of phagosomal pH by two-color imaging. This ratiometric approach can measure local pH rather accurately, minimizing the influence of probe concentration. When the dual-labeled LM was introduced into THP-1 cells, anti-hCRIg treatment resulted in quenching of the pH-sensitive fluorescein in 30 min, while control mIgG had minimal effect. Merging of the green and red fluorophores yielded a yellow image in the case of the control mIgG, while red images were detected when the cells were exposed to anti-hCRIg, due to quenching of the green fluorophore (Fig. 4D, THP-1). When CLIC3 expression was knocked down (Fig. 4D, siCLIC3 THP-1) or ouabain was added to the THP-1 cells (Fig. 4D, THP-1 + ouabain), anti-hCRIg treatment did not affect the fluorescence intensity. We constructed a standard curve of pH versus degree of quenching (the 488/543 nm ratio) to estimate the pH of the LM vacuoles (Fig. 4E). The median pH of the anti-hCRIg-treated LM vacuoles was approximately 4.5, and CRIg-dependent acidification did not occur when CLIC3 was knocked down, or in the presence of ouabain (Fig. 4F). Bafilomycin did not block the CRIg-mediated acidification. CRIg-mediated acidification was, however, prevented by the addition of NH4Cl [21] and was dependent on expression of CLIC3 (Fig. 4F). These results indicate that CRIg signals cause acidification of LM vacuoles in a CLIC3-dependent manner.

CRIg signals promote fusion of LM vacuoles and lysosomes

The acidification of phagosomes is known to facilitate their fusion with lysosomes [12]. We therefore tested whether anti-hCRIg treatment promoted fusion of LM vacuoles and lysosomes. We used TMR-conjugated dextran as a lysosome marker, and fluorescein-conjugated LM, to determine the kinetics of fusion. Both the dextran and LM were introduced into THP-1 and siCLIC3 THP-1 cells, and the fusion events were observed at various time points. Fusion of red lysosomes and green LM occurred in less than 10% of the LM vacuoles by 60 min of exposure of THP-1 cells to control mIgG (Fig. 5A, upper panels). Anti-hCRIg, however, induced LM vacuole-lysosome fusion, as indicated by the yellow-merged images (Fig. 5A, middle panels). Fusion did not take place in the absence of CLIC3 (Fig. 5A, lower panels). The frequencies of colocalization events in the three treatment groups are shown in Fig. 5B. Anti-hCRIg-induced fusion was detected as early as 5 min after antibody addition and affected 50% of the LM vacuoles by 60 min, whereas only about 10% of the LM vacuoles underwent fusion by 60 min in the presence of control mIgG (Fig. 5C). Again, the fusion of LM vacuoles and lysosomes was inhibited by NH4Cl but not by bafilomycin (Fig. 5D). Therefore, we conclude that CRIg triggering induces the fusion of LM vacuoles and lysosomes by promoting phagosomal acidification.

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Figure 5. CRIg signaling induces LM vacuole-lysosome fusion in a CLIC3-dependent manner. (A) TMR-dextran-loaded THP-1 or siCLIC3 THP-1 cells were infected with FITC-LM for 1 h. The cells were treated with 10 μg/mL anti-hCRIg mAb or mIgG. The localization of dextran (red) and LM (green) was analyzed by confocal microscope. Scale bar = 10 μm. Data shown are representative of three experiments performed. (B) The frequency of colocalization events was determined for one hundred cells randomly selected in each group; the number of cells containing fused LM vacuole-lysosomes was counted. Yellow LMs were counted as “fused” and green LM as “nonfused”. (C) Kinetics of the% LM vacuole-lysosome fusion. Colocalization events were calculated as the number of yellow LM vacuoles among 150 randomly selected LM vacuoles for each time point. (D) Effects of bafilomycin A (baf) and NH4Cl on LM vacuole-lysosome fusion. TMR-dextran-loaded THP-1 cells were infected with FITC-LM for 1 h. The THP-1 cells were treated with mIgG and anti-hCRIg in the presence or absence of baf or NH4Cl. Colocalization was calculated as described in (B). (B–D) Data are shown as the means ± SDs of four samples and are representative of three experiments. *p < 0.05; **p < 0.01, Student's t-test.

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Impairment of CRIg-mediated LM killing in CLIC3−/− macrophages

To confirm the role of CLIC3 in CRIg-mediated killing of LM in vitro and in vivo, we generated CLIC3−/− mice (Supporting Information Fig. 4) and compared the susceptibilities to LM infection of WT, CRIg−/−, and CLIC3−/− mice. The CLIC3−/− mice were as susceptible to LM as CRIg−/− mice, indicating that CLIC3 is required to control LM infection (Fig. 6A). We next tested whether C3-opLM activated the CRIg-mediated signaling pathway and required CLIC3 for LM killing. WT, CRIg−/−, and CLIC3−/− BMDMs were incubated with TMR labeled-unopsonized LM and opLM for various times (0–60 min) and their uptake was analyzed by fluorescence microscopy. We detected an approximately twofold increase in the rate of uptake of opLM in WT and CLIC3−/− cells compared with unopsonized LM, whereas there was no difference in uptake of opLM and unopsonized LM in the CRIg−/− BMDMs (Fig. 6B). These results indicate that CLIC3 is not required for the uptake of opLM.

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Figure 6. Impairment of CRIg-mediated LM killing in CLIC3−/− macrophages. (A) WT, CRIg−/−, and CLIC3−/− mice were infected i.v. with LM (5 × 103 CFU) and the survival of the mice was determined (n = 8). (B) CRIg-dependent uptake of opLM by macrophages in vitro. BMDMs from WT, CRIg−/−, and CLIC3−/− mice were incubated with a MOI of five of TMR labeled-LM or -opLM for various periods of time. Each group of cells was harvested, fixed, and mounted with DAPI. Numbers of LM per cell were counted by fluorescence microscopy. (C) LM killing by WT and CLIC3−/− macrophages. LM- and opLM-infected WT and CLIC3−/− BMDMs were treated with 10 μg/mL of rIgG or anti-mCRIg for 6 h. (D) [Cl] in LM vacuoles in WT or CLIC3−/− BMDMs treated with rIgG or anti-mCRIg. (E) The pH of LM vacuoles in WT and CLIC3−/− BMDMs after treatment with rIgG or anti-mCRIg. (F) CRIg+/+, CRIg−/−, CLIC3+/−, and CLIC3−/− C57B6 mice were injected i.v. with 2 × 107 LM. Ten min after the injection, their blood, spleens, and livers were harvested. Blood and serial dilutions of spleen and liver homogenates were plated on BHI agar plates. Numbers of LM are expressed as CFU per 0.1 mL of blood, per 100 mg of spleen, or per 100 mg of liver. (B, C, F) Data are shown as mean + SD of 15 (B) or 3 (C, F) samples and are representative of three (B, F) or four (C) experiments; (D, E) each symbol represents an individual mouse and the bars represent the means. *p < 0.05**p < 0.01; ***p < 0.001, Student's t-test.

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We next compared the efficacy of in vitro LM killing in BMDMs from WT and CLIC3−/− mice in response to opLM and anti-CRIg stimulation. Antibody-mediated hyper-stimulation of CRIg only inhibited intracellular LM growth significantly in the WT macrophages (Fig. 6C). CRIg-mediated Cl influx and acidification of the LM vacuoles were also impaired in CLIC3−/− BMDMs (Fig. 6D and 6E).

We next compared the in vivo uptake of LM in WT, CRIg−/−, CLIC3+/−, and CLIC3−/− mice. The mice were intravenously (i.v.) injected with 2 × 107 LM; they were killed after 10 min and LM was counted in blood, spleen, and liver. There were no significant differences in LM CFU in blood, spleen, and liver in the CLIC3−/− versus CLIC+/− mice, indicating again that CLIC3 is not required for uptake of LM. However, as reported previously [6], uptake of LM in the liver was impaired in the CRIg−/− mice, and resulted in higher titers of LM in their blood and spleens (Fig. 6F). Taken together these findings indicate that CLIC3 is not required for uptake of LM by liver Kupffer cells (KCs) but is essential for CRIg-mediated LM killing.

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgments
  8. Conflict of interest
  9. References
  10. Supporting Information

We have described a cellular process in which an extracellular signal regulates phagosomal activities, and we suggest that this process could be exploited clinically to control intracellular pathogens. We propose that CRIg signaling activates the CLIC3 to increase [Cl] in the phagosomal lumen and induces the phagosomal acidification through the coactivation of Na+/K+ ATPase and Na+/H+ exchangers. This creates a window of opportunity to prevent the escape of LM from the phagosomes so that they can be killed by subsequent phagosome-lysosome fusion. It may provide a platform for developing new drugs against multiple drug resistant intracellular pathogens. Therefore, it will be important to establish whether this type of phagosome-mediated killing mechanism operates against other intracellular pathogens such as Mycobacterium tuberculosis, Salmonella typhimurium, Brucella suis, and Yersinia pestis.

CRIg has been extensively characterized as a receptor for C3b, and was found to bind C3-opsonized Listeria and particles and to be translocated to the phagosomal membrane. van Lookeren Campagne et al. [6] believe that CRIg separates from the phagosome and reenters the endosomal compartment prior to phagosome-lysosome fusion because they did not find CRIg on lysosomes. These authors also speculate that CRIg plays an active role in complement-mediated phagocytosis over and above its role as a simple receptor for C3-opsonized pathogens [6]. We found that CRIg signaling mediated by anti-CRIg mAb in macrophages led to the elimination of LM (Fig. 1A and 6C), but that C3-opsonized bacteria only delayed the multiplication of the LM even when CRIg signaling was triggered in vitro (Fig. 1C and 6C). Because the opLM-mediated CRIg signals are restricted to regions of the plasma membrane forming phagosomes, CRIg signaling appears unable to kill the LM if they escape from the phagosomes into the cytosol. On the other hand, many of the cross-linked CRIg molecules may become integrated into the LM-containing phagosomes and enforces phagosomal activities, killing LM before escaping into cytosol (Fig. 1E) [6].

CRIg differs in structure and function from the complement C3 fragment receptors, CR1 (CD35), CR2 (CD21), CR3 (CD11b/CD18), and CR4 (CD11c/CD18) [22]. CR1–CR4 are expressed on various types of cell [23, 24], recognize C3b, and promote phagocytosis, leukocyte migration and production of reactive oxygen species [25]. CRIg is primarily expressed on resting macrophages rather than activated ones, facilitates the phagocytosis of pathogens, and has a role as a negative T-cell regulator [9]. Therefore, the role of CRIg seems to stimulate the development of adaptive immunity against pathogens by maximizing their uptake and processing by immature macrophages.

Acidification of phagosomes plays an important role in the clearance of pathogens; a low (pH < 5) phagosomal pH is optimal for the activity of degradative enzymes [26], and is required for phagosome fusion with lysosomes [12]. LLO produces pores in phagosome membranes at pH 5–6.5, but is absolutely inactive at pH 4.5 or 7 [27]. Under natural conditions, phagosomes are weakly acidic with a pH of 5–6 [19]; at such a pH the phagosome membrane is destroyed by LLO, and LM can easily escape into the cytosol. However, LLO may not be active in forming pores in phagosomal membranes following CRIg-mediated acidification because of the low pH (Fig. 4F).

Chloride has an important role in the acidification of intracellular organelles, because Cl is a counterion of the H+ transported into the lumen [13]. We found that CRIg interacts directly with CLIC3 (Fig. 2A and B) and that CRIg-mediated LM killing is abolished when CLIC3 activity is blocked by a siRNA or inhibitor (Fig. 2C and D), or by gene deletion (Fig. 6C). This indicates that CLIC3 is required for the CRIg-mediated accumulation of Cl in the phagosomal lumen. Although the primary structures of CLICs lack well-defined membrane-spanning domains [28], previous reports suggest that they nevertheless form ion channels. For example, CLIC1 has been shown to form channels in phospholipid vesicles and artificial lipid bilayers [15], and the dimerization of CLIC1 increases its stability in vesicles [29]. Since the GST domain of CLIC3 is C-terminal [28], it is possible that CLIC3 is still able to insert into lipid bilayers and vesicles. Indeed, recent report showed that CLIC3 located on the late endosome and lysosome in A2780 human ovarian carcinoma cell line [18].

CRIg-mediated LM killing was not dependant on the well-known V-type H+-ATPase, but required essentially the Na+/K+-ATPase and partly Na+/H+ exchanger 1 (Fig. 3). Macrophages are able to alternatively induce the acidification of phagosome using Na+/K+-ATPase and NHE1 in the absence of V-type ATPase [12, 30] and CRIg signals appear to intensify the Na+/K+-ATPase and NHE1-mediated phagosomal acidification through the increase of [Cl] into the phagosomal lumen by CLIC3.

In summary, our results demonstrate that CRIg signaling not only promotes the uptake of LM, but also enhances killing of intracellular LM by modulating phagosomal activities, which processes require CLIC3. CRIg signaling provides a mechanism by which macrophages prevent intracellular bacteria from evading cellular killing.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgments
  8. Conflict of interest
  9. References
  10. Supporting Information

Cells, flow cytometry, and hybridomas

THP-1, J774, and HeLa cells were purchased from the ATCC. Expression of CRIg on THP-1, J774, and transfected HeLa cells was detected with a FACS Calibur (BD Bioscience). Hybridomas producing monoclonal antibodies to hCRIg and mCRIg were generated by immunizing BALB/c and Sprague Dawley rats with hCRIg- and mCRIg-transfected HeLa cells, respectively, as described previously [7].

Animals

All animal studies were approved by the Institutional Animal Care and Use Committee (IACUC) review board of National Cancer Center (NCC-12–045D) and conducted under the guidelines of the National Cancer Center IACUC. C57BL/6 mice were purchased from Orient, Inc., Korea and CRIg−/− mice in a C57BL/6 background were a kind gift of Dr. M. van Lookeren Campagne (Genentech, South San Francisco, CA) [6]. All mice were maintained in an SPF facility.

Antibodies and chemicals

The following additional antibodies were used: donkey anti-rabbit-HRP (Dako), goat anti-mouse-HRP (Jackson Immunoresearch), mouse anti-FLAG (Millipore), mouse anti-HA-HRP (Miltenyi Biotec), mouse anti-CLIC3 (Abnova), anti-transferrin receptor mAb (BD Biosciences), anti-LAMP1 mAb (BD Biosciences. Sources of reagents were as follows: A/G agarose beads (Santa Cruz), 4-(2-aminoethyl) benzenesulfonyl fluoride (AEBSF) (Calbiochem), ampicillin (Sigma), bafilomycin A (Abcam), 5(6)-carboxyfluorescein N-hydroxysuccinime ester (fluorescein) (Sigma), 5(6)-carboxytetramethylrhodamine N-hydroxysuccinimide ester (TMR) (Sigma), 2-ethyl-2-thioseudourea (S-Ethyl-ITU, HBr) (Calbiochem), Griess reagent (Sigma), hygromycin B (Sigma), Hyperfect (Adipogen), lipopolysacchrides (LPS) (Sigma), MQHA (Sigma), NBT solution (Sigma), N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide (Sigma), neomycin (Sigma), N-hydroxy-succinimide (Sigma), nigericin (Sigma), tributyltin (Sigma), N-monomethyl-l-arginine (L-NMMA) (Calbiochem), N-nitro-l-arginine methyl ester (L-NAME) (Calbiochem), ouabain (Fluka), amiloride (Calbiochem), paraformaldehyde (PFA) (Sigma), poly-l-lysine (Sigma), protease inhibitor mixture (Roche), TMR-dextran (Molecular probe), and phalloidin-rhodamine (Invitrogen). Human C3b monomer (Calbiochem) was used to produce C3b dimers by incubation with bis-maleimido-hexane (Pierce) and purification over a Superdex S-200 10/300GL gel filtration column (Amersham) [6].

Primer sequences

The coding sequence for human CRIg was PCR amplified from THP-1 cells using the following primers: forward primer: 5′-CGG GAT CCC GTC CCA TCC TGG AAG TGC CAG AG-3′nd reverse primer: 5′-AAG GAA AAA AGC GGC CGC TTA ACA GAC ACT TTT GCC CTC AGT-3′. The coding sequence for mouse CRIg was PCR amplified from mouse placenta cDNA using forward primer: 5′-AGC TCA AGC TTG CCA CCA TGG AGA TCT CAT CAG GCT TGC-3′ and reverse primer: 5′-GCC CGG GAT CCC GGC AGG CAG GAA TAG ACA TTG TTG–3′. CLIC3 cDNA was amplified by PCR from THP-1 cells using forward primer: 5′-AAG CTT CCA TGG CGG AGA CC-3′ and reverse primer: 5′-GGA TCC CTA GCG GGG GTG CA-3′. HA-tagged CLIC3 was PCR amplified from a human leukocyte cDNA library in pACT2 using forward primer: 5′-AAA GAG ATC TGT ATG GCT TAC CCA-3′ and reverse primer: 5′-TGC GGG GTT TTT CAG TAT CTA CGA-3′. SiCLIC3 oligomers were synthesized by COSMO Ltd. and their sequences were forward: 5′-ACC TCG CTG CAG ATC GAG GAC TTT CTT CAA GAG AGA AAG TCC TCG ATC TGC AGC TT-3′ and reverse: 5′-CAA AAA GCT GCA GAT CGA GGA CTT TCT CTC TTG AAG AAA GTC CTC GAT CTG CAG CG-3′. 3′ deletion mutants of CLIC3 were PCR amplified from CLIC3/pEGFP-N1 using forward primer: 5′-GAA TTC CCA TGG CGG AGA CC-3′ and reverse deletion 1 primer: 5′-CTC GAG CTA GCG GGG GTG CAC-3′, reverse deletion 2 primer: 5′-CTC GAG CGG TAG GCC GCC AGG A-3′, reverse deletion 3 primer: 5′-CTC GAG TCG GCG CTG TGC GGA-3′, reverse deletion 4 primer: 5′-CTC GAG CTA TTT GAA CTC TTT-3′, reverse deletion 5 primer: 5′-CTC GAG ATC GCG CTG TCC AGG TA-3′, reverse deletion 6 primer: 5′-CTC GAG GGC GTA CGC CGC GCA G-3′, reverse deletion 7 primer: 5′-CTC GAG GCG CAG CTC CG-3′, reverse deletion 8 primer: 5′-CTC GAG CGG GGA TGG GCG CCT-3′, reverse deletion 9 primer: 5′-CTC GAG GCT GTC CAG CCT G-3′, or reverse deletion 10 primer: 5′-CTC GAG CTC CCT GTA ACG AGG-3′. Single point mutants of CLIC3 were PCR amplified from CLIC3/pEGFP-N1 using forward primer: 5′-GGG GAT CCG AAT TCC GAG TGA GGA CGG GGA G-3′, reverse primer of amino acid (a.a) 211 mutation: 5′-ATA GAT CTC TCG AGT GTA TTT GAA CTC TTT CTC CGC CAT CGC GCT GTC-3′, reverse primer of a.a 212 mutation: 5′-ATA GAT CTC TCG AGT GTA TTT GAA CTC TTT CGC CTG CAT CGC GCT-3′, reverse primer of a.a 213 mutation: 5′-ATA GAT CTC TCG AGT GTA TTT GAA CTC CGC CTC CTG CAT CGC-3′, reverse primer of a.a 214 mutation: 5′-ATA GAT CTC TCG AGT GTA TTT GAA CGC TTT CTC CTG CAT CGC-3′, reverse primer of a.a 215 mutation: 5′-ATA GAT CTC TCG AGT GTA TTT CGC CTC TTT CTC CTG CAT-3′, reverse primer of a.a216 mutation: 5′-ATA GAT CTC TCG AGT GTA CGC GAA CTC TTT CTC CTG-3′, reverse primer of a.a 217 mutation: 5′-ATA GAT CTC TCG AGT CGC TTT GAA CTC TTT CTC CTG-3′.

Plasmid constructs, siRNA, and transfected cell lines

The coding sequences of mature human or mouse CRIg were cloned in CD5L-Flag-pcDNA3.0 vectors and used to transfect HeLa cells. Stably transfected cell lines were selected by incubation with 1500 μg/mL neomycin for 2 months. CLIC3 (with or without an HA tag) was cloned in pIRES2-EGFP or pEGFP-N1 (Clontech). The siCLIC3 sequence and scrambled siCLIC3 (control siRNA) were designed with siRNA wizard (www.sirnawizard.com) and cloned in psiRNA-h7SKhygro (Invivogen). THP-1 clones stably expressing siCLIC3 were selected by incubation with 300 μg/mL hygromycin B for 2 months.

Killing of Listeria monocytogenes

LM (strain 10403S, ATCC) was passaged in mice to maintain virulence, and cultured in Brain Heart Infusion (BHI) broth (Difco Laboratories). LM was opsonized using normal serum as previously described [31]. For in vitro LM killing assays, THP-1 cells, peritoneal macrophages, and BMDMs were incubated with LM (10 MOI) for 1 h at 37°C, washed with PBS, and exposed to a high dose of ampicillin (300 μg/mL) for 15 min to remove free LM. The infected cells were then incubated with 10 μg/mL of anti-CRIg or control IgG at 37°C in a CO2 incubator for 0, 2, 4, or 6 h. The cells were lysed in water at the times indicated, and serial dilutions of the lysates were plated on BHI agar. LM-infected cells were incubated with blocking agents such as R(+)-IAA- for 1 h followed by anti-CRIg mAb or control IgG.

Measurements of NO and ROS production

NO production was measured according to the protocol provided by Promega. In brief, 100 μL of cell culture supernatant was mixed with one volume of Griess reagent and incubated for 15 min at room temperature. NO was determined at 532 nm (Wallac 1420). ROS production was determined as follows. THP-1 cells were cultured on poly-L-lysine-coated 96-well culture plates. Attached THP-1 cells were washed with PBS, 100 μL of NBT solution (1 mg/mL, in PBS containing 0.25% glucose) was added, and the cells were incubated for 3 h at 37°C. The wells were washed 3 times with 70% methanol, dried, and 100 μL of 2 M KOH and 100 μL of DMSO were added. ROS were determined at 630 nm. The culture medium used was phenol red free.

Yeast two-hybrid screening and coimmunoprecipitation

A matchmaker Gal4 Two-Hybrid System 3 (Clontech) was used to identify proteins that bound to the cytoplasmic domain of hCRIg. The interaction between hCRIg and hCLIC3 was confirmed by coimmunoprecipitation. HA-tagged CLIC3 and control vector were transfected into Hela cells and Hela cells expressing FLAG-tagged hCRIg. The cells were pelleted and lysed in lysis buffer (50 mM HEPES (pH = 7.5), 1% Nonidet P-40, 0.5% sodium deoxycholate, 150 mM NaCl, 1 mM EDTA, 0.1 mM sodium vanadate, protease inhibitor mixture, and phosphatase inhibitor cocktail) and spun at 14 000 rpm for 15 min at 4°C. hCRIg and CLIC3 were precipitated with anti-FLAG and anti-HA mAb, respectively, cross-linked with Dynabeads® protein G (Invitrogen), resolved by 10% SDS PAGE, and transferred to PVDF membranes. CRIg and CLIC3 were detected with the anti-FLAG and anti-HA mAbs, and secondary Ab-HRP. Bound Abs were detected by ECL (Amersham Pharmacia Biotech, Little Chalfont, UK).

Serial deletions and point mutations of CLIC3

To determine the amino acids of CLIC3 to which the cytoplasmic domain of CRIg binds, serial deletion mutants of CLIC3, and single point mutants of amino acids 211–217 were cloned in pACT2 and the cytoplasmic domain of hCRIg (hCRIg-cyto) was cloned in pGBT9. The interactions between the CLIC3 mutants and hCRIg-cyto were examined in Matchmaker Gal4 Two-Hybrid System 3.

Generation of CLIC3−/− mice

Targeting CLIC3 locus gDNA: 129S7/AB2.2 mouse bacterial artificial chromosome (BAC) clones (bMQ-24G11, Geneservice) containing a portion of the mouse CLIC3 locus. Targeting vector: a 0.9 kb region including exon 2 and 3 was replaced by the PGK-Puro selection marker. A 6.8 kb HindIII fragment that includes the 5′ upstream sequence of Clic3 and exon 1 was used as the left homology arm, and a 2.4 kb EcoRI/EcoRV fragment spanning Clic3 intron 3 and BC029214 exon 5 was used as the right homology arm. These arms of homology were inserted into the HindIII and NotI sites, respectively, of PGK-Puro vector. The diphtheria toxin A chain cassette (DT-A) was employed as a negative selection marker.

Targeted CLIC3 locus: E14 ES cells (IGTC, International Gene Trap Consortium) in a 129 background were used to generate CLIC3+/− embryonic stem cells, and homologous recombination was confirmed by Southern blot analysis. The 3′ external probe (296 bp) recognized a 16 kb XbaI fragment of the targeted CLIC3 gene and a 20 kb XbaI fragment of the wt CLIC3 gene. Two targeted ES clones were injected into C57BL/6 blastocysts to generate chimeric mice. Chimeric males were bred with C57BL/6 females, and germ-line transmission of the Clic3+/− allele was verified by PCR and Southern blot analysis. For routine genotyping, we employed a PCR-based strategy using a common forward primer 5′-CTT CAC TTT GGC TCG TGG ACC-3′ and a WT-specific (5′-CTC TGA GCA GAC CCA GGA ATC-3′) and knockout-specific (5′-GAG TAG AAG GTG GCG CGA AG-3′) reverse primers, amplifying a 523 bp fragment from the WT allele and a 340-bp fragment from the mutant allele.

Measurement of LM vacuole [Cl]

Dual-labeled fluorescent LM was used to measure [Cl] in phagosomes, as described for human neutrophils [20]. LM was conjugated with chloride-sensitive MQHA and chloride-insensitive TMR by standard succinimidyl ester chemistry. Fluorescence measurements were performed with an LSM 510 Meta (Zeiss) confocal microscope. MQHA-derived fluorescence was measured at excitation wavelength 360 nm and emission wavelength 450 nm, and TMR fluorescence was measured at excitation wavelength 543 nm and emission wavelength 560–615 nm; [Cl] was determined from the ratio of intensities of TMR (red) and MQHA (green) using a standard curve. In brief, chloride-containing Ringer's buffer (150 mM sodium chloride, 1.2 mM magnesium gluconate, 4 mM calcium gluconate, 2.4 mM K2HPO4, 0.6 mM KH2PO4, 10 mM dextrose, 20 mM HEPES, pH 7.4) and chloride-free Ringer's buffer (sodium chloride replaced by sodium gluconate) were prepared. THP-1 cells infected with TMR/MQHA dual-labeled LM were incubated in mixtures of various ratios of chloride-containing and chloride-free Ringer′s buffer together with ([Cl] from 0 to 150 mM) plus ionophores (10 μM nigericin and 10 μM tributyltin) for 1 h at 37°C. The fluorescence ratio of TMR to MQHA was determined for each [Cl] by confocal microscopy. [Cl] (X axis) was plotted versus the red/green fluorescence ratio (Y axis), and a standard curve for [Cl] was constructed using the Stern–Volmer equation [20], F0/F = 1 + Ksv[Q], where F0 is the unquenched fluorescence intensity of MQHA; F is the fluorescence intensity at [Q], and [Q] is [Cl]. At each chloride concentration, 30 dual-labeled LM in THP-1 cells were randomly selected and their red/green ratios determined.

Synthesis of a fluorescent pH probe and determination of the pH of LM vacuoles

A dual fluorophore probe was synthesized by conjugating LM to both 5(6)-carboxyfluorescein and TMR. In brief, 0.1 mg of 5(6)-carboxyfluorescein N-hydroxysuccinime ester and 0.1 mg of 5(6)-carboxytetramethylrhodamine N-hydroxysuccinimide ester were dissolved together in 10 μL of DMSO. Meanwhile, the LM was washed three times with PBS and resuspended in 500 μL of 0.1 M NaHCO3 (pH 8.2). The mixture of fluorescein and TMR was added to the LM and the resulting suspension was incubated in the dark at room temperature for 1 h. The dual-labeled LM was washed with PBS three times and suspended in RPMI 1640 medium. To measure the pH of LM vacuoles, THP-1, or siCLIC3 THP-1 cells were incubated with the dual-labeled LM for 1 h, followed by 10 μg/mL of anti-hCRIg or control mIgG for 30 min in a CO2 incubator, and analyzed by confocal microscopy. The pH of LM vacuoles was measured from the ratio of the intensitities of fluorescein (green) and TMR (red), using a standard pH curve.

To construct a standard curve for pH, fluorescein/TMR dual-labeled LM-infected THP-1 cells were incubated in pH 0 to 9 buffers containing the ionophores nigericin (10 μM) and tributyltin (10 μM) for 1 h at 37°C. 5(6)-Carboxyfluorescein-derived fluorescence was measured at excitation wavelength 488 nm and emission bandwidth 505–550 nm, and TMR-derived fluorescence was measured at excitation wavelength 543 nm and emission bandwidth of 560–516 nm using a confocal microscope, and changes of pH of LM vacuoles were determined from the intensity ratios of fluorescein (green) and TMR (red), using the standard pH curve; pH (X axis) versus ratio of green to red fluorescence (Y axis) was plotted, and the curve was completed using the Boltzmann sigmoidal equation [13], Y = Bottom + (Top-Bottom)/(1+exp((V50-X)/S)) where Y is the excitation ratio at 488/543; bottom is the lowest excitation ratio; top is the highest excitation ratio; V50 is the pH at the midpoint of the curve; X is the pH, and S is the slope of the pH dependence of the excitation ratio. Thirty double-labeled LM were randomly selected at each pH point.

Detection of LM vacuole-lysosome fusion

THP-1 cells were incubated with 1 mg/mL of TMR-dextran (Molecular Probes) at 37°C for 1 h. The cells were washed with PBS and reincubated in fresh medium for another 2 h to allow the dextran to translocate into the lysosomes. The dextran-loaded THP-1 cells were infected with fluorescein-labeled LM for 1 h, washed and treated with 10 μg/mL of anti-hCRIg or control mIgG for 1 h at 37°C. They were then fixed in MeOH (pH 8.0, adjusted by NaOH) to restore the fluorescence of the carboxyfluorescein quenched by low pH in the phagolysosomes. Fluorescein/TMR-double positive cells were counted under a confocal microscope.

LM infection model

C57BL/6 mice were infected i.v. with LM and subsequently injected intraperitoneally (i.p.) with rIgG or agonistic anti-mCRIg mAb (500 μg per mice) (n = 5). Spleens, livers, and lungs were collected from the mice on the indicated days PI, homogenized in PBS, and plated on BHI agar plates.

Uptake of LM by Kupffer cells in vivo

C57BL/6 mice were infected i.v. with 2 × 107 cfu of TMR-labeled LM and subsequently injected i.p. with 500 μg of anti-mCRIg or rIgG. Frozen sections were prepared from the livers of each group of mice 1, 2, 4, and 6 h after the LM infection and stained with FITC-anti-F4/80 mAb. Numbers of LM per KC were counted with a confocal microscope in 30 randomly selected KCs.

Data analysis

Images were analyzed with LSM 5 Image Browser (Zeiss), Image J (National Institutes of Health, Bethesda), and AxioVision LE Rel. 4.5 (Zeiss).

Statistical analysis

All data were analyzed with the statistical program, Prism 4.0 GraphPad (San Diego, CA). Student's t-test was used to determine the statistical significance of differences between groups.

Acknowledgments

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgments
  8. Conflict of interest
  9. References
  10. Supporting Information

We thank Dr. Menno van Lookeren Campagne for CRIg−/− mice; Mi-Ae Kim for help with confocal microscopy; Sang-Hyun Park for frozen and paraffin-embedded sections, Jung-Dae Kim for yeast two-hybrid screening, and Mun-Sun Kim for animal experiments. This work was supported by a research grant from the National Cancer Center, Korea (NCC-1210370-1) and a National Research Foundation of Korea (NRF) grant funded by the Korean government (MEST) (2005-084-E00001 and 2006-2004212).

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  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgments
  8. Conflict of interest
  9. References
  10. Supporting Information
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Abbreviations
BMDM

bone marrow-derived macrophage

CLIC

chloride intracellular channel

CRIg

complement receptor of the immunoglobulin superfamily

KC

Kupffer cell

LLO

listeriolysin O

LM

Listeria monocytogenes

opLM

complement-opsonized LM

MQHA

methoxyquinoline-N-6-hexanoic acid

NHE1

Na+/K+ exchanger 1

TMR

tetramethylrhodamine

Supporting Information

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgments
  8. Conflict of interest
  9. References
  10. Supporting Information

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Figure S1. CRIg signaling facilitates clearance of LM in monocytes.

Figure S2. Killing of intracellular LM by CRIg signaling is NO- and ROS-independent.

Figure S3. CLIC3 binds to the cytoplasmic domain of CRIg.

Figure S4. Generation of CLIC3−/− mice.

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