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Oxidation by neutrophils-derived HOCl increases immunogenicity of proteins by converting them into ligands of several endocytic receptors involved in antigen uptake by dendritic cells and macrophages.

Biedroń R, Konopiński MK, Marcinkiewicz J, Józefowski S - PLoS ONE (2015)

Bottom Line: Paradoxically, endocytic receptors involved in antigen uptake do not bind the majority of proteins, which may be the main reason why purified proteins stimulate at most weak immune responses.Other scavenger receptors, SREC-I and LOX-1, as well as RAGE were also able to bind HOCl-modified proteins, but they did not contribute significantly to these ligands uptake by dendritic cells because they were either not expressed or exhibited preference for more heavily oxidised proteins.Our results indicate that oxidation by neutrophils-derived HOCl may be a physiological mechanism of conferring immunogenicity on proteins which in their native forms do not bind to endocytic receptors.

View Article: PubMed Central - PubMed

Affiliation: Department of Immunology, Jagiellonian University Medical College, Cracow, Poland.

ABSTRACT
The initiation of adaptive immune responses to protein antigens has to be preceded by their uptake by antigen presenting cells and intracellular proteolytic processing. Paradoxically, endocytic receptors involved in antigen uptake do not bind the majority of proteins, which may be the main reason why purified proteins stimulate at most weak immune responses. A shared feature of different types of adjuvants, capable of boosting immunogenicity of protein vaccines, is their ability to induce acute inflammation, characterized by early influx of activated neutrophils. Neutrophils are also rapidly recruited to sites of tissue injury or infection. These cells are the source of potent oxidants, including hypochlorous acid (HOCl), causing oxidation of proteins present in inflammatory foci. We demonstrate that oxidation of proteins by endogenous, neutrophils-derived HOCl increases their immunogenicity. Upon oxidation, different, randomly chosen simple proteins (yeast alcohol dehydrogenase, human and bovine serum albumin) and glycoproteins (human apo-transferrin, ovalbumin) gain the ability to bind with high affinity to several endocytic receptors on antigen presenting cells, which seems to be the major mechanism of their increased immunogenicity. The mannose receptor (CD206), scavenger receptors A (CD204) and CD36 were responsible for the uptake and presentation of HOCl-modified proteins by murine dendritic cells and macrophages. Other scavenger receptors, SREC-I and LOX-1, as well as RAGE were also able to bind HOCl-modified proteins, but they did not contribute significantly to these ligands uptake by dendritic cells because they were either not expressed or exhibited preference for more heavily oxidised proteins. Our results indicate that oxidation by neutrophils-derived HOCl may be a physiological mechanism of conferring immunogenicity on proteins which in their native forms do not bind to endocytic receptors. This mechanism might enable the immune system to detect infections caused by pathogens not recognized by pattern recognition receptors.

No MeSH data available.


Related in: MedlinePlus

Roles of SR CD36 (A-E) and SR-A (G-I) as receptors for HOCl-modified proteins.(A) Uptake of fluorescently-labelled proteins by CHO cells transfected with human CD36 as compared to non-transfected cells. (B) Binding of anti-CD36 mAb and control mouse IgA to CD36-transfected and non-transfected CHO cells, determined by cellular ELISA. (C) Binding of rCD36 to plate-adsorbed proteins. (D) Effects of 10 or 100 μg/ml of indicated, soluble ligands on rCD36 binding to plate-adsorbed OVA-Cl. (E) Binding of polyclonal anti-mouse CD36 Ab and control goat IgG to BM-DC, splenic DC and PEM. A representative histogram of Ab binding to BM-DC is displayed on the left graph. (F) Binding of anti-mouse SR-A mAb and control rat IgG2b to BM-DC, splenic DC and PEM. A representative histogram of Ab binding to BM-DC is shown on the left graph. (G) Binding of SR-A present in lysates of PEM to plate-adsorbed proteins. (H) Effects of anti-SR-A 2F8 mAb and AcLDL on the uptake of fluorescently-labelled proteins by BM-DC. Shown are results of single experiments, each representative of at least 3 similar experiments performed (A-D, G, H) or averages +SEM from 4–6 independent experiments (E, F). The data were analysed with the unpaired (A-C, G) or one-sample (H) Student’s t-test or with ANOVA, followed by the Tukey-Kramer post-test (D). *, p < 0.05; ND, not done.
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pone.0123293.g007: Roles of SR CD36 (A-E) and SR-A (G-I) as receptors for HOCl-modified proteins.(A) Uptake of fluorescently-labelled proteins by CHO cells transfected with human CD36 as compared to non-transfected cells. (B) Binding of anti-CD36 mAb and control mouse IgA to CD36-transfected and non-transfected CHO cells, determined by cellular ELISA. (C) Binding of rCD36 to plate-adsorbed proteins. (D) Effects of 10 or 100 μg/ml of indicated, soluble ligands on rCD36 binding to plate-adsorbed OVA-Cl. (E) Binding of polyclonal anti-mouse CD36 Ab and control goat IgG to BM-DC, splenic DC and PEM. A representative histogram of Ab binding to BM-DC is displayed on the left graph. (F) Binding of anti-mouse SR-A mAb and control rat IgG2b to BM-DC, splenic DC and PEM. A representative histogram of Ab binding to BM-DC is shown on the left graph. (G) Binding of SR-A present in lysates of PEM to plate-adsorbed proteins. (H) Effects of anti-SR-A 2F8 mAb and AcLDL on the uptake of fluorescently-labelled proteins by BM-DC. Shown are results of single experiments, each representative of at least 3 similar experiments performed (A-D, G, H) or averages +SEM from 4–6 independent experiments (E, F). The data were analysed with the unpaired (A-C, G) or one-sample (H) Student’s t-test or with ANOVA, followed by the Tukey-Kramer post-test (D). *, p < 0.05; ND, not done.

Mentions: Stronger inhibition of AF-OVA-Cl uptake by DS than by CS suggests an involvement of SR in this process. Amongst them, the class B SR CD36 has been demonstrated to serve as a receptor for HOCl-oxidised LDL [29]. Aiming to examine the role of CD36 as a receptor for OVA-Cl, we compared uptake of AF-OVA-Cl by CHO cells transfected or not with CD36. Surprisingly, non-transfected CHO cells exhibited both the pattern and magnitude of fluorescently-labelled ligands uptake very similar to BM-DC and transfection with CD36 did not have any effect on this uptake (Fig 7A). These results indicate that either CD36 is not a receptor for OVA-Cl or that endogenous receptors of CHO cells are already sufficient to mediate the maximal uptake, which therefore cannot be further increased by overexpression of CD36. Supporting the latter possibility, we detected specific binding of anti-CD36 mAb to non-transfected CHO cells (Fig 7B). Thus, results of experiments with transfected cells are inconclusive regarding the role of CD36 as OVA-Cl receptor. We therefore resorted to a different approach, namely studying binding of isolated rCD36 to proteins adsorbed to ELISA plates. As shown in Fig 7C, rCD36 bound strongly to immobilized GA-BSA and AcLDL, used as positive controls. The binding of rCD36 to OVA-Cl, TFN-Cl and HSA-Cl was as high as to GA-BSA. In contrast, rCD36 did not exhibit specific binding to native proteins. Binding of rCD36 to adsorbed OVA-Cl was dose-dependently inhibited by soluble DS and GA-BSA, but not by CS and mannan (Fig 7D), which confirms that rCD36 retains the ligand binding specificity of natural CD36. Surprisingly, soluble OVA-Cl inhibited rCD36 binding even more potently than DS or GA-BSA, whereas OVA had no effect. These results indicates that upon oxidation by HOCl proteins gain the ability to bind to CD36 with high affinity.


Oxidation by neutrophils-derived HOCl increases immunogenicity of proteins by converting them into ligands of several endocytic receptors involved in antigen uptake by dendritic cells and macrophages.

Biedroń R, Konopiński MK, Marcinkiewicz J, Józefowski S - PLoS ONE (2015)

Roles of SR CD36 (A-E) and SR-A (G-I) as receptors for HOCl-modified proteins.(A) Uptake of fluorescently-labelled proteins by CHO cells transfected with human CD36 as compared to non-transfected cells. (B) Binding of anti-CD36 mAb and control mouse IgA to CD36-transfected and non-transfected CHO cells, determined by cellular ELISA. (C) Binding of rCD36 to plate-adsorbed proteins. (D) Effects of 10 or 100 μg/ml of indicated, soluble ligands on rCD36 binding to plate-adsorbed OVA-Cl. (E) Binding of polyclonal anti-mouse CD36 Ab and control goat IgG to BM-DC, splenic DC and PEM. A representative histogram of Ab binding to BM-DC is displayed on the left graph. (F) Binding of anti-mouse SR-A mAb and control rat IgG2b to BM-DC, splenic DC and PEM. A representative histogram of Ab binding to BM-DC is shown on the left graph. (G) Binding of SR-A present in lysates of PEM to plate-adsorbed proteins. (H) Effects of anti-SR-A 2F8 mAb and AcLDL on the uptake of fluorescently-labelled proteins by BM-DC. Shown are results of single experiments, each representative of at least 3 similar experiments performed (A-D, G, H) or averages +SEM from 4–6 independent experiments (E, F). The data were analysed with the unpaired (A-C, G) or one-sample (H) Student’s t-test or with ANOVA, followed by the Tukey-Kramer post-test (D). *, p < 0.05; ND, not done.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4388828&req=5

pone.0123293.g007: Roles of SR CD36 (A-E) and SR-A (G-I) as receptors for HOCl-modified proteins.(A) Uptake of fluorescently-labelled proteins by CHO cells transfected with human CD36 as compared to non-transfected cells. (B) Binding of anti-CD36 mAb and control mouse IgA to CD36-transfected and non-transfected CHO cells, determined by cellular ELISA. (C) Binding of rCD36 to plate-adsorbed proteins. (D) Effects of 10 or 100 μg/ml of indicated, soluble ligands on rCD36 binding to plate-adsorbed OVA-Cl. (E) Binding of polyclonal anti-mouse CD36 Ab and control goat IgG to BM-DC, splenic DC and PEM. A representative histogram of Ab binding to BM-DC is displayed on the left graph. (F) Binding of anti-mouse SR-A mAb and control rat IgG2b to BM-DC, splenic DC and PEM. A representative histogram of Ab binding to BM-DC is shown on the left graph. (G) Binding of SR-A present in lysates of PEM to plate-adsorbed proteins. (H) Effects of anti-SR-A 2F8 mAb and AcLDL on the uptake of fluorescently-labelled proteins by BM-DC. Shown are results of single experiments, each representative of at least 3 similar experiments performed (A-D, G, H) or averages +SEM from 4–6 independent experiments (E, F). The data were analysed with the unpaired (A-C, G) or one-sample (H) Student’s t-test or with ANOVA, followed by the Tukey-Kramer post-test (D). *, p < 0.05; ND, not done.
Mentions: Stronger inhibition of AF-OVA-Cl uptake by DS than by CS suggests an involvement of SR in this process. Amongst them, the class B SR CD36 has been demonstrated to serve as a receptor for HOCl-oxidised LDL [29]. Aiming to examine the role of CD36 as a receptor for OVA-Cl, we compared uptake of AF-OVA-Cl by CHO cells transfected or not with CD36. Surprisingly, non-transfected CHO cells exhibited both the pattern and magnitude of fluorescently-labelled ligands uptake very similar to BM-DC and transfection with CD36 did not have any effect on this uptake (Fig 7A). These results indicate that either CD36 is not a receptor for OVA-Cl or that endogenous receptors of CHO cells are already sufficient to mediate the maximal uptake, which therefore cannot be further increased by overexpression of CD36. Supporting the latter possibility, we detected specific binding of anti-CD36 mAb to non-transfected CHO cells (Fig 7B). Thus, results of experiments with transfected cells are inconclusive regarding the role of CD36 as OVA-Cl receptor. We therefore resorted to a different approach, namely studying binding of isolated rCD36 to proteins adsorbed to ELISA plates. As shown in Fig 7C, rCD36 bound strongly to immobilized GA-BSA and AcLDL, used as positive controls. The binding of rCD36 to OVA-Cl, TFN-Cl and HSA-Cl was as high as to GA-BSA. In contrast, rCD36 did not exhibit specific binding to native proteins. Binding of rCD36 to adsorbed OVA-Cl was dose-dependently inhibited by soluble DS and GA-BSA, but not by CS and mannan (Fig 7D), which confirms that rCD36 retains the ligand binding specificity of natural CD36. Surprisingly, soluble OVA-Cl inhibited rCD36 binding even more potently than DS or GA-BSA, whereas OVA had no effect. These results indicates that upon oxidation by HOCl proteins gain the ability to bind to CD36 with high affinity.

Bottom Line: Paradoxically, endocytic receptors involved in antigen uptake do not bind the majority of proteins, which may be the main reason why purified proteins stimulate at most weak immune responses.Other scavenger receptors, SREC-I and LOX-1, as well as RAGE were also able to bind HOCl-modified proteins, but they did not contribute significantly to these ligands uptake by dendritic cells because they were either not expressed or exhibited preference for more heavily oxidised proteins.Our results indicate that oxidation by neutrophils-derived HOCl may be a physiological mechanism of conferring immunogenicity on proteins which in their native forms do not bind to endocytic receptors.

View Article: PubMed Central - PubMed

Affiliation: Department of Immunology, Jagiellonian University Medical College, Cracow, Poland.

ABSTRACT
The initiation of adaptive immune responses to protein antigens has to be preceded by their uptake by antigen presenting cells and intracellular proteolytic processing. Paradoxically, endocytic receptors involved in antigen uptake do not bind the majority of proteins, which may be the main reason why purified proteins stimulate at most weak immune responses. A shared feature of different types of adjuvants, capable of boosting immunogenicity of protein vaccines, is their ability to induce acute inflammation, characterized by early influx of activated neutrophils. Neutrophils are also rapidly recruited to sites of tissue injury or infection. These cells are the source of potent oxidants, including hypochlorous acid (HOCl), causing oxidation of proteins present in inflammatory foci. We demonstrate that oxidation of proteins by endogenous, neutrophils-derived HOCl increases their immunogenicity. Upon oxidation, different, randomly chosen simple proteins (yeast alcohol dehydrogenase, human and bovine serum albumin) and glycoproteins (human apo-transferrin, ovalbumin) gain the ability to bind with high affinity to several endocytic receptors on antigen presenting cells, which seems to be the major mechanism of their increased immunogenicity. The mannose receptor (CD206), scavenger receptors A (CD204) and CD36 were responsible for the uptake and presentation of HOCl-modified proteins by murine dendritic cells and macrophages. Other scavenger receptors, SREC-I and LOX-1, as well as RAGE were also able to bind HOCl-modified proteins, but they did not contribute significantly to these ligands uptake by dendritic cells because they were either not expressed or exhibited preference for more heavily oxidised proteins. Our results indicate that oxidation by neutrophils-derived HOCl may be a physiological mechanism of conferring immunogenicity on proteins which in their native forms do not bind to endocytic receptors. This mechanism might enable the immune system to detect infections caused by pathogens not recognized by pattern recognition receptors.

No MeSH data available.


Related in: MedlinePlus