Limits...
Scavenger receptor SREC-I mediated entry of TLR4 into lipid microdomains and triggered inflammatory cytokine release in RAW 264.7 cells upon LPS activation.

Murshid A, Gong J, Prince T, Borges TJ, Calderwood SK - PLoS ONE (2015)

Bottom Line: Scavenger receptor associated with endothelial cells I (SREC-I) was shown to be expressed in immune cells and to play a role in the endocytosis of peptides and antigen presentation.We have shown here that SREC-I became associated with TLR4 on binding bacterial lipopolysaccharides (LPS) in RAW 264.7 and HEK 293 cells overexpressing these two receptors.Our experiments therefore indicated that SREC-I could bind LPS and might be involved in innate inflammatory immune responses to extracellular danger signals in RAW 264.7 cells or bone marrow-derived macrophages.

View Article: PubMed Central - PubMed

Affiliation: Molecular and Cellular Radiation Oncology, Beth Israel Deaconess Medical Center, Harvard Medical School, Center for Life Sciences, 3 Blackfan Circle, Boston, Massachusetts, United States of America.

ABSTRACT
Scavenger receptor associated with endothelial cells I (SREC-I) was shown to be expressed in immune cells and to play a role in the endocytosis of peptides and antigen presentation. As our previous studies indicated that SREC-I required intact Toll-like receptor 4 (TLR4) expression for its functions in tumor immunity, we examined potential interactions between these two receptors. We have shown here that SREC-I became associated with TLR4 on binding bacterial lipopolysaccharides (LPS) in RAW 264.7 and HEK 293 cells overexpressing these two receptors. The receptors then became internalized together in intracellular endosomes. SREC-I promoted TLR4-induced signal transduction through the NF-kB and MAP kinase pathways, leading to enhanced inflammatory cytokine release. Activation of inflammatory signaling through SREC-I/TLR4 complexes appeared to involve recruitment of the receptors into detergent-insoluble, cholesterol-rich lipid microdomains that contained the small GTPase Cdc42 and the non-receptor tyrosine kinase c-src. Under conditions of SREC-I activation by LPS, TLR4 activity required Cdc42 as well as cholesterol and actin polymerization for signaling through NF-kB and MAP kinase pathways in RAW 264.7 cells. SREC-I appeared to respond differently to another ligand, the molecular chaperone Hsp90 that, while triggering SREC-I-TLR4 binding caused only faint activation of the NF-kB pathway. Our experiments therefore indicated that SREC-I could bind LPS and might be involved in innate inflammatory immune responses to extracellular danger signals in RAW 264.7 cells or bone marrow-derived macrophages.

No MeSH data available.


Related in: MedlinePlus

Ligand-bound SREC-I colocalized with TLR4 after LPS treatment.A, SREC-I and TLR4 did not interact in the absence of LPS. Raw 264.7 cells were transfected with FLAG-SREC-I for 22 hours. Cells were then fixed and stained with anti TLR4 ab (green) and anti-FLAG ab (red). B, TLR4 colocalized with SREC-I in the presence of LPS. Raw 264.7 cells overexpressing FLAG-SREC-I were exposed to LPS (1 μg/ml) for 20–30 min at 4°C. Cells were then fixed and stained for TLR4 (green) and FLAG (red). Percent colocalization with or without LPS is shown in the adjacent histogram. C, D, LPS, TLR4 and SREC-I were internalized at 37°C. Raw 264.7 cells overexpressing FLAG-SREC-I were incubated with Alexa LPS (1 μg/ml) at 4°C for 20 mins and then medium was replaced with warm medium. Cells were then incubated at 37°C for 10–15 mins. Cells were fixed and stained for TLR4 (green, C, D) and FLAG-SREC-I (red in C, purple in D). Alexa LPS was shown in red (D). E, F, Endogenous TLR4 and SREC-I did not colocalize in the absence of LPS. Raw 264.7 cells were treated for 30 mins with LPS (1 μg/ml) and then fixed and stained for TLR4 (green) and SREC-I (red). E, TLR4 and SREC-I colocalize in the presence of LPS. Cells were labeled with LPS (1μg/ml) for 20–30 minutes at 4°C then fixed and stained for TLR4 (green) and SREC-I (red) (F). G, SREC-I expression level in Raw 264.7 cells and in cells overexpressing FLAG-SREC-I. H, SREC-I interacted with TLR4 physically in the presence of LPS (1 μg/ml). HEK 293 cells expressing FLAG-SREC-I and TLR4s were treated with or without Hsp90 or LPS. FLAG-SREC-I was then immunoprecipitated (IP) with FLAG ab. The IP complex was separated with SDS-PAGE and blotted for TLR4. The amount of FLAG-SREC-I immunoprecipitated was determined and β-actin was used as a loading control. All images were representative of 3 different planes from each sample. Each experiment was performed 3 times reproducibly. Scale bar, 5 μm.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4383338&req=5

pone.0122529.g001: Ligand-bound SREC-I colocalized with TLR4 after LPS treatment.A, SREC-I and TLR4 did not interact in the absence of LPS. Raw 264.7 cells were transfected with FLAG-SREC-I for 22 hours. Cells were then fixed and stained with anti TLR4 ab (green) and anti-FLAG ab (red). B, TLR4 colocalized with SREC-I in the presence of LPS. Raw 264.7 cells overexpressing FLAG-SREC-I were exposed to LPS (1 μg/ml) for 20–30 min at 4°C. Cells were then fixed and stained for TLR4 (green) and FLAG (red). Percent colocalization with or without LPS is shown in the adjacent histogram. C, D, LPS, TLR4 and SREC-I were internalized at 37°C. Raw 264.7 cells overexpressing FLAG-SREC-I were incubated with Alexa LPS (1 μg/ml) at 4°C for 20 mins and then medium was replaced with warm medium. Cells were then incubated at 37°C for 10–15 mins. Cells were fixed and stained for TLR4 (green, C, D) and FLAG-SREC-I (red in C, purple in D). Alexa LPS was shown in red (D). E, F, Endogenous TLR4 and SREC-I did not colocalize in the absence of LPS. Raw 264.7 cells were treated for 30 mins with LPS (1 μg/ml) and then fixed and stained for TLR4 (green) and SREC-I (red). E, TLR4 and SREC-I colocalize in the presence of LPS. Cells were labeled with LPS (1μg/ml) for 20–30 minutes at 4°C then fixed and stained for TLR4 (green) and SREC-I (red) (F). G, SREC-I expression level in Raw 264.7 cells and in cells overexpressing FLAG-SREC-I. H, SREC-I interacted with TLR4 physically in the presence of LPS (1 μg/ml). HEK 293 cells expressing FLAG-SREC-I and TLR4s were treated with or without Hsp90 or LPS. FLAG-SREC-I was then immunoprecipitated (IP) with FLAG ab. The IP complex was separated with SDS-PAGE and blotted for TLR4. The amount of FLAG-SREC-I immunoprecipitated was determined and β-actin was used as a loading control. All images were representative of 3 different planes from each sample. Each experiment was performed 3 times reproducibly. Scale bar, 5 μm.

Mentions: We first investigated the effects of LPS on SREC-I the intracellular localization of SREC-I (Fig 1). As SREC-I was shown to be expressed at low levels in resting macrophages (B. Zhou & SK Calderwood, unpublished data), we carried out overexpression of the receptor in the mouse macrophage cell line Raw 264.7 to permit effective visualization by immunofluorescence. We then incubated these cells with Escherichia coli derived LPS (1 μg/ml) at 4°C, fixed the cells and analyzed SREC-I and TLR4 localization by confocal microscopy. Prior to LPS exposure, SREC-I was detected largely in the cytosol whereas TLR4 was mostly membrane-associated; minimal overlap between these fluorescence signals was observed (Fig 1A). However, TLR4 and SREC-I became partially coincident on the cell surface in the presence of LPS as indicated by the strong overlap in fluorescence patterns in cells at 4°C (Fig 1A and 1B). We then investigated internalization of TLR4 and SREC-I in LPS treated cells after warming the medium to 37°C (Fig 1C). LPS exposure prompted internalization of both receptors at 37°C and their relocation to intracellular vesicles, with some of these structures marked both by anti TLR4 and anti-FLAG antibodies (for SREC-I) (Fig 1D). We also showed fluorescent, Alexa-labeled LPS to be localized in intracellular vesicles containing TLR4 and SREC-I at 37°C, suggesting partial co-internalization of SREC-I, LPS and TLR4 (Fig 1C and 1D). As TLR4 was not shown previously to bind directly to LPS, these results suggested SREC-I to be a recognizing receptor for LPS and that could induce the recruitment of TLR4 to SREC-I marked regions on the cell surface. Although CD14 is a well-established LPS recognizing molecule cooperating with TLR4, our experiments suggested that SREC-I was also capable of recognizing the endotoxin and interacting with TLR4.


Scavenger receptor SREC-I mediated entry of TLR4 into lipid microdomains and triggered inflammatory cytokine release in RAW 264.7 cells upon LPS activation.

Murshid A, Gong J, Prince T, Borges TJ, Calderwood SK - PLoS ONE (2015)

Ligand-bound SREC-I colocalized with TLR4 after LPS treatment.A, SREC-I and TLR4 did not interact in the absence of LPS. Raw 264.7 cells were transfected with FLAG-SREC-I for 22 hours. Cells were then fixed and stained with anti TLR4 ab (green) and anti-FLAG ab (red). B, TLR4 colocalized with SREC-I in the presence of LPS. Raw 264.7 cells overexpressing FLAG-SREC-I were exposed to LPS (1 μg/ml) for 20–30 min at 4°C. Cells were then fixed and stained for TLR4 (green) and FLAG (red). Percent colocalization with or without LPS is shown in the adjacent histogram. C, D, LPS, TLR4 and SREC-I were internalized at 37°C. Raw 264.7 cells overexpressing FLAG-SREC-I were incubated with Alexa LPS (1 μg/ml) at 4°C for 20 mins and then medium was replaced with warm medium. Cells were then incubated at 37°C for 10–15 mins. Cells were fixed and stained for TLR4 (green, C, D) and FLAG-SREC-I (red in C, purple in D). Alexa LPS was shown in red (D). E, F, Endogenous TLR4 and SREC-I did not colocalize in the absence of LPS. Raw 264.7 cells were treated for 30 mins with LPS (1 μg/ml) and then fixed and stained for TLR4 (green) and SREC-I (red). E, TLR4 and SREC-I colocalize in the presence of LPS. Cells were labeled with LPS (1μg/ml) for 20–30 minutes at 4°C then fixed and stained for TLR4 (green) and SREC-I (red) (F). G, SREC-I expression level in Raw 264.7 cells and in cells overexpressing FLAG-SREC-I. H, SREC-I interacted with TLR4 physically in the presence of LPS (1 μg/ml). HEK 293 cells expressing FLAG-SREC-I and TLR4s were treated with or without Hsp90 or LPS. FLAG-SREC-I was then immunoprecipitated (IP) with FLAG ab. The IP complex was separated with SDS-PAGE and blotted for TLR4. The amount of FLAG-SREC-I immunoprecipitated was determined and β-actin was used as a loading control. All images were representative of 3 different planes from each sample. Each experiment was performed 3 times reproducibly. Scale bar, 5 μm.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4383338&req=5

pone.0122529.g001: Ligand-bound SREC-I colocalized with TLR4 after LPS treatment.A, SREC-I and TLR4 did not interact in the absence of LPS. Raw 264.7 cells were transfected with FLAG-SREC-I for 22 hours. Cells were then fixed and stained with anti TLR4 ab (green) and anti-FLAG ab (red). B, TLR4 colocalized with SREC-I in the presence of LPS. Raw 264.7 cells overexpressing FLAG-SREC-I were exposed to LPS (1 μg/ml) for 20–30 min at 4°C. Cells were then fixed and stained for TLR4 (green) and FLAG (red). Percent colocalization with or without LPS is shown in the adjacent histogram. C, D, LPS, TLR4 and SREC-I were internalized at 37°C. Raw 264.7 cells overexpressing FLAG-SREC-I were incubated with Alexa LPS (1 μg/ml) at 4°C for 20 mins and then medium was replaced with warm medium. Cells were then incubated at 37°C for 10–15 mins. Cells were fixed and stained for TLR4 (green, C, D) and FLAG-SREC-I (red in C, purple in D). Alexa LPS was shown in red (D). E, F, Endogenous TLR4 and SREC-I did not colocalize in the absence of LPS. Raw 264.7 cells were treated for 30 mins with LPS (1 μg/ml) and then fixed and stained for TLR4 (green) and SREC-I (red). E, TLR4 and SREC-I colocalize in the presence of LPS. Cells were labeled with LPS (1μg/ml) for 20–30 minutes at 4°C then fixed and stained for TLR4 (green) and SREC-I (red) (F). G, SREC-I expression level in Raw 264.7 cells and in cells overexpressing FLAG-SREC-I. H, SREC-I interacted with TLR4 physically in the presence of LPS (1 μg/ml). HEK 293 cells expressing FLAG-SREC-I and TLR4s were treated with or without Hsp90 or LPS. FLAG-SREC-I was then immunoprecipitated (IP) with FLAG ab. The IP complex was separated with SDS-PAGE and blotted for TLR4. The amount of FLAG-SREC-I immunoprecipitated was determined and β-actin was used as a loading control. All images were representative of 3 different planes from each sample. Each experiment was performed 3 times reproducibly. Scale bar, 5 μm.
Mentions: We first investigated the effects of LPS on SREC-I the intracellular localization of SREC-I (Fig 1). As SREC-I was shown to be expressed at low levels in resting macrophages (B. Zhou & SK Calderwood, unpublished data), we carried out overexpression of the receptor in the mouse macrophage cell line Raw 264.7 to permit effective visualization by immunofluorescence. We then incubated these cells with Escherichia coli derived LPS (1 μg/ml) at 4°C, fixed the cells and analyzed SREC-I and TLR4 localization by confocal microscopy. Prior to LPS exposure, SREC-I was detected largely in the cytosol whereas TLR4 was mostly membrane-associated; minimal overlap between these fluorescence signals was observed (Fig 1A). However, TLR4 and SREC-I became partially coincident on the cell surface in the presence of LPS as indicated by the strong overlap in fluorescence patterns in cells at 4°C (Fig 1A and 1B). We then investigated internalization of TLR4 and SREC-I in LPS treated cells after warming the medium to 37°C (Fig 1C). LPS exposure prompted internalization of both receptors at 37°C and their relocation to intracellular vesicles, with some of these structures marked both by anti TLR4 and anti-FLAG antibodies (for SREC-I) (Fig 1D). We also showed fluorescent, Alexa-labeled LPS to be localized in intracellular vesicles containing TLR4 and SREC-I at 37°C, suggesting partial co-internalization of SREC-I, LPS and TLR4 (Fig 1C and 1D). As TLR4 was not shown previously to bind directly to LPS, these results suggested SREC-I to be a recognizing receptor for LPS and that could induce the recruitment of TLR4 to SREC-I marked regions on the cell surface. Although CD14 is a well-established LPS recognizing molecule cooperating with TLR4, our experiments suggested that SREC-I was also capable of recognizing the endotoxin and interacting with TLR4.

Bottom Line: Scavenger receptor associated with endothelial cells I (SREC-I) was shown to be expressed in immune cells and to play a role in the endocytosis of peptides and antigen presentation.We have shown here that SREC-I became associated with TLR4 on binding bacterial lipopolysaccharides (LPS) in RAW 264.7 and HEK 293 cells overexpressing these two receptors.Our experiments therefore indicated that SREC-I could bind LPS and might be involved in innate inflammatory immune responses to extracellular danger signals in RAW 264.7 cells or bone marrow-derived macrophages.

View Article: PubMed Central - PubMed

Affiliation: Molecular and Cellular Radiation Oncology, Beth Israel Deaconess Medical Center, Harvard Medical School, Center for Life Sciences, 3 Blackfan Circle, Boston, Massachusetts, United States of America.

ABSTRACT
Scavenger receptor associated with endothelial cells I (SREC-I) was shown to be expressed in immune cells and to play a role in the endocytosis of peptides and antigen presentation. As our previous studies indicated that SREC-I required intact Toll-like receptor 4 (TLR4) expression for its functions in tumor immunity, we examined potential interactions between these two receptors. We have shown here that SREC-I became associated with TLR4 on binding bacterial lipopolysaccharides (LPS) in RAW 264.7 and HEK 293 cells overexpressing these two receptors. The receptors then became internalized together in intracellular endosomes. SREC-I promoted TLR4-induced signal transduction through the NF-kB and MAP kinase pathways, leading to enhanced inflammatory cytokine release. Activation of inflammatory signaling through SREC-I/TLR4 complexes appeared to involve recruitment of the receptors into detergent-insoluble, cholesterol-rich lipid microdomains that contained the small GTPase Cdc42 and the non-receptor tyrosine kinase c-src. Under conditions of SREC-I activation by LPS, TLR4 activity required Cdc42 as well as cholesterol and actin polymerization for signaling through NF-kB and MAP kinase pathways in RAW 264.7 cells. SREC-I appeared to respond differently to another ligand, the molecular chaperone Hsp90 that, while triggering SREC-I-TLR4 binding caused only faint activation of the NF-kB pathway. Our experiments therefore indicated that SREC-I could bind LPS and might be involved in innate inflammatory immune responses to extracellular danger signals in RAW 264.7 cells or bone marrow-derived macrophages.

No MeSH data available.


Related in: MedlinePlus