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SR-A ligand and M-CSF dynamically regulate SR-A expression and function in primary macrophages via p38 MAPK activation.

Nikolic D, Calderon L, Du L, Post SR - BMC Immunol. (2011)

Bottom Line: These changes are associated with altered SR-A expression in macrophages; however, the intracellular signal pathways involved and the extent to which SR-A ligands regulate SR-A expression are not well defined.These results demonstrate that in resident macrophages SR-A expression and function can be dynamically regulated by changes in the macrophage microenvironment that are typical of inflammatory processes.In particular, our results indicate a previously unrecognized role for ligand binding to SR-A in up-regulating SR-A expression and activating p38 MAPK.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Pathology, University of Arkansas for Medical Sciences, Little Rock, Arkansas 72205, USA.

ABSTRACT

Background: Inflammation is characterized by dynamic changes in the expression of cytokines, such as M-CSF, and modifications of lipids and proteins that result in the formation of ligands for Class A Scavenger Receptors (SR-A). These changes are associated with altered SR-A expression in macrophages; however, the intracellular signal pathways involved and the extent to which SR-A ligands regulate SR-A expression are not well defined. To address these questions, SR-A expression and function were examined in resident mouse peritoneal macrophages incubated with M-CSF or the selective SR-A ligand acetylated-LDL (AcLDL).

Results: M-CSF increased SR-A expression and function, and required the specific activation of p38 MAPK, but not ERK1/2 or JNK. Increased SR-A expression and function returned to basal levels 72 hours after removing M-CSF. We next determined whether prolonged incubation of macrophages with SR-A ligand alters SR-A expression. In contrast to most receptors, which are down-regulated by chronic exposure to ligand, SR-A expression was reversibly increased by incubating macrophages with AcLDL. AcLDL activated p38 in wild-type macrophages but not in SR-A-/- macrophages, and p38 activation was specifically required for AcLDL-induced SR-A expression.

Conclusions: These results demonstrate that in resident macrophages SR-A expression and function can be dynamically regulated by changes in the macrophage microenvironment that are typical of inflammatory processes. In particular, our results indicate a previously unrecognized role for ligand binding to SR-A in up-regulating SR-A expression and activating p38 MAPK. In this way, SR-A may modulate inflammatory responses by enhancing macrophage uptake of modified protein/lipid, bacteria, and cell debris; and by regulating the production of inflammatory cytokines, growth factors, and proteolytic enzymes.

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M-CSF induces SR-A expression and AcLDL association. (A) Cultured resident MPMs were incubated with the indicated concentrations of M-CSF for 24 hrs. Subsequently, SR-A and GAPDH proteins were detected in lysates prepared from treated cells by immunoblotting with specific antibodies. A representative immunoblot from a single experiment and the results (mean ± SEM) from at least three separate experiments are shown. (B) Cultured resident MPMs were pretreated with actinomycin D (5 μM) for 30 min, and then incubated with or without M-CSF (25 ng/ml) for the indicated times prior to assessing SR-A expression. A representative immunoblot from a single experiment and the results (mean ± SEM) from at least three separate experiments are shown. (C) Alexa488-AcLDL association with macrophages isolated from C57Bl/6 or NIH-Swiss mice was quantified by flow cytometry as described in Material and Methods. Non-specific association was determined in the presence of fucoidan (Fuc, 75 μg/ml), a blocking SR-A antibody (2F8, 10 μg/ml), or by using SR-A-/- macrophages. Shown is the mean ± SEM of three separate experiments. (D) SR-A-specific Alexa488-AcLDL association with macrophages incubated with M-CSF (25 μg/ml) for the indicated times was quantified by flow cytometry as described in Material and Methods. The results of a representative experiment and the mean ± SEM of at least three separate experiments are shown. (E) Relative changes in SR-A expression and function were plotted and the best fit line determined using a Deming linear regression model in GraphPad Prism.
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Figure 1: M-CSF induces SR-A expression and AcLDL association. (A) Cultured resident MPMs were incubated with the indicated concentrations of M-CSF for 24 hrs. Subsequently, SR-A and GAPDH proteins were detected in lysates prepared from treated cells by immunoblotting with specific antibodies. A representative immunoblot from a single experiment and the results (mean ± SEM) from at least three separate experiments are shown. (B) Cultured resident MPMs were pretreated with actinomycin D (5 μM) for 30 min, and then incubated with or without M-CSF (25 ng/ml) for the indicated times prior to assessing SR-A expression. A representative immunoblot from a single experiment and the results (mean ± SEM) from at least three separate experiments are shown. (C) Alexa488-AcLDL association with macrophages isolated from C57Bl/6 or NIH-Swiss mice was quantified by flow cytometry as described in Material and Methods. Non-specific association was determined in the presence of fucoidan (Fuc, 75 μg/ml), a blocking SR-A antibody (2F8, 10 μg/ml), or by using SR-A-/- macrophages. Shown is the mean ± SEM of three separate experiments. (D) SR-A-specific Alexa488-AcLDL association with macrophages incubated with M-CSF (25 μg/ml) for the indicated times was quantified by flow cytometry as described in Material and Methods. The results of a representative experiment and the mean ± SEM of at least three separate experiments are shown. (E) Relative changes in SR-A expression and function were plotted and the best fit line determined using a Deming linear regression model in GraphPad Prism.

Mentions: Resident (non-elicited) mouse peritoneal macrophages (MPMs) were harvested by peritoneal lavage with ice-cold sterile saline from male NIH Swiss mice (Harlan; Indianapolis, IN), and from C57Bl/6 and SR-A-/- mice on a C57Bl/6 background (Figures 1C and 4B; Jackson Laboratory, Bar Harbor, ME). Animal care and use for all procedures were done according to protocols reviewed and approved by the Institutional Animal Care and Use Committee at University of Arkansas for Medical Sciences and the University of Kentucky. Isolated macrophages were cultured as previously described [28]. Briefly, peritoneal exudates were incubated overnight at 37°C and non-adherent cells removed by gently washing with PBS. Adherent macrophages were maintained in DMEM containing antibiotics and FBS (10%) for 48 hrs prior to use in experiments. MPMs were then treated with agonists in the presence or absence of specific inhibitors as described in figure legends. Following treatment, MPMs were washed with ice-cold PBS and cell lysates prepared by incubating cells in MBST/OG buffer (25 mM MES; 150 mM NaCl; 60 mM octylglucopyranoside; 1% Triton X-100; pH 6.4) containing protease and phosphatase inhibitors (Sigma, St. Louis, MO) for 45 min on ice. Cell lysates were centrifuged at 13,000 rpm for 15 min and the pellets were discarded. Protein concentration of supernatant was determined using the BioRad DC assay using BSA as a standard (Hercules, CA).


SR-A ligand and M-CSF dynamically regulate SR-A expression and function in primary macrophages via p38 MAPK activation.

Nikolic D, Calderon L, Du L, Post SR - BMC Immunol. (2011)

M-CSF induces SR-A expression and AcLDL association. (A) Cultured resident MPMs were incubated with the indicated concentrations of M-CSF for 24 hrs. Subsequently, SR-A and GAPDH proteins were detected in lysates prepared from treated cells by immunoblotting with specific antibodies. A representative immunoblot from a single experiment and the results (mean ± SEM) from at least three separate experiments are shown. (B) Cultured resident MPMs were pretreated with actinomycin D (5 μM) for 30 min, and then incubated with or without M-CSF (25 ng/ml) for the indicated times prior to assessing SR-A expression. A representative immunoblot from a single experiment and the results (mean ± SEM) from at least three separate experiments are shown. (C) Alexa488-AcLDL association with macrophages isolated from C57Bl/6 or NIH-Swiss mice was quantified by flow cytometry as described in Material and Methods. Non-specific association was determined in the presence of fucoidan (Fuc, 75 μg/ml), a blocking SR-A antibody (2F8, 10 μg/ml), or by using SR-A-/- macrophages. Shown is the mean ± SEM of three separate experiments. (D) SR-A-specific Alexa488-AcLDL association with macrophages incubated with M-CSF (25 μg/ml) for the indicated times was quantified by flow cytometry as described in Material and Methods. The results of a representative experiment and the mean ± SEM of at least three separate experiments are shown. (E) Relative changes in SR-A expression and function were plotted and the best fit line determined using a Deming linear regression model in GraphPad Prism.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC3141791&req=5

Figure 1: M-CSF induces SR-A expression and AcLDL association. (A) Cultured resident MPMs were incubated with the indicated concentrations of M-CSF for 24 hrs. Subsequently, SR-A and GAPDH proteins were detected in lysates prepared from treated cells by immunoblotting with specific antibodies. A representative immunoblot from a single experiment and the results (mean ± SEM) from at least three separate experiments are shown. (B) Cultured resident MPMs were pretreated with actinomycin D (5 μM) for 30 min, and then incubated with or without M-CSF (25 ng/ml) for the indicated times prior to assessing SR-A expression. A representative immunoblot from a single experiment and the results (mean ± SEM) from at least three separate experiments are shown. (C) Alexa488-AcLDL association with macrophages isolated from C57Bl/6 or NIH-Swiss mice was quantified by flow cytometry as described in Material and Methods. Non-specific association was determined in the presence of fucoidan (Fuc, 75 μg/ml), a blocking SR-A antibody (2F8, 10 μg/ml), or by using SR-A-/- macrophages. Shown is the mean ± SEM of three separate experiments. (D) SR-A-specific Alexa488-AcLDL association with macrophages incubated with M-CSF (25 μg/ml) for the indicated times was quantified by flow cytometry as described in Material and Methods. The results of a representative experiment and the mean ± SEM of at least three separate experiments are shown. (E) Relative changes in SR-A expression and function were plotted and the best fit line determined using a Deming linear regression model in GraphPad Prism.
Mentions: Resident (non-elicited) mouse peritoneal macrophages (MPMs) were harvested by peritoneal lavage with ice-cold sterile saline from male NIH Swiss mice (Harlan; Indianapolis, IN), and from C57Bl/6 and SR-A-/- mice on a C57Bl/6 background (Figures 1C and 4B; Jackson Laboratory, Bar Harbor, ME). Animal care and use for all procedures were done according to protocols reviewed and approved by the Institutional Animal Care and Use Committee at University of Arkansas for Medical Sciences and the University of Kentucky. Isolated macrophages were cultured as previously described [28]. Briefly, peritoneal exudates were incubated overnight at 37°C and non-adherent cells removed by gently washing with PBS. Adherent macrophages were maintained in DMEM containing antibiotics and FBS (10%) for 48 hrs prior to use in experiments. MPMs were then treated with agonists in the presence or absence of specific inhibitors as described in figure legends. Following treatment, MPMs were washed with ice-cold PBS and cell lysates prepared by incubating cells in MBST/OG buffer (25 mM MES; 150 mM NaCl; 60 mM octylglucopyranoside; 1% Triton X-100; pH 6.4) containing protease and phosphatase inhibitors (Sigma, St. Louis, MO) for 45 min on ice. Cell lysates were centrifuged at 13,000 rpm for 15 min and the pellets were discarded. Protein concentration of supernatant was determined using the BioRad DC assay using BSA as a standard (Hercules, CA).

Bottom Line: These changes are associated with altered SR-A expression in macrophages; however, the intracellular signal pathways involved and the extent to which SR-A ligands regulate SR-A expression are not well defined.These results demonstrate that in resident macrophages SR-A expression and function can be dynamically regulated by changes in the macrophage microenvironment that are typical of inflammatory processes.In particular, our results indicate a previously unrecognized role for ligand binding to SR-A in up-regulating SR-A expression and activating p38 MAPK.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Pathology, University of Arkansas for Medical Sciences, Little Rock, Arkansas 72205, USA.

ABSTRACT

Background: Inflammation is characterized by dynamic changes in the expression of cytokines, such as M-CSF, and modifications of lipids and proteins that result in the formation of ligands for Class A Scavenger Receptors (SR-A). These changes are associated with altered SR-A expression in macrophages; however, the intracellular signal pathways involved and the extent to which SR-A ligands regulate SR-A expression are not well defined. To address these questions, SR-A expression and function were examined in resident mouse peritoneal macrophages incubated with M-CSF or the selective SR-A ligand acetylated-LDL (AcLDL).

Results: M-CSF increased SR-A expression and function, and required the specific activation of p38 MAPK, but not ERK1/2 or JNK. Increased SR-A expression and function returned to basal levels 72 hours after removing M-CSF. We next determined whether prolonged incubation of macrophages with SR-A ligand alters SR-A expression. In contrast to most receptors, which are down-regulated by chronic exposure to ligand, SR-A expression was reversibly increased by incubating macrophages with AcLDL. AcLDL activated p38 in wild-type macrophages but not in SR-A-/- macrophages, and p38 activation was specifically required for AcLDL-induced SR-A expression.

Conclusions: These results demonstrate that in resident macrophages SR-A expression and function can be dynamically regulated by changes in the macrophage microenvironment that are typical of inflammatory processes. In particular, our results indicate a previously unrecognized role for ligand binding to SR-A in up-regulating SR-A expression and activating p38 MAPK. In this way, SR-A may modulate inflammatory responses by enhancing macrophage uptake of modified protein/lipid, bacteria, and cell debris; and by regulating the production of inflammatory cytokines, growth factors, and proteolytic enzymes.

Show MeSH
Related in: MedlinePlus