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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

Both SREC-I and RAGE bind HOCl-oxidised proteins with low affinities.(A) Binding of rSREC-I to proteins coated onto ELISA plates. (B) Effects of 10 or 100 μg/ml of indicated, soluble ligands on rSREC-I binding to plate-adsorbed AcLDL. (C) Binding of goat anti-mouse SREC-I Ab to untreated and LPS-pre-treated BM-DC. (D) Effects of polyclonal goat anti-mouse SREC-I Ab, relative to normal goat IgG, on the uptake of AF-AcLDL, AF-OVA-Cl and pHr-HSA-Cl by LPS-pre-treated, SR-A-deficient BM-DC. (E) Binding of rRAGE to plate-adsorbed proteins. (F) Effects of 10 or 100 μg/ml of indicated, soluble ligands on rRAGE binding to plate-adsorbed GA-BSA. The data were analysed by the Student’s t-test (A, D, E) or by ANOVA, followed by the Tukey-Kramer post-test (B, F). *, p < 0.05; NS, non-significant.
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pone.0123293.g009: Both SREC-I and RAGE bind HOCl-oxidised proteins with low affinities.(A) Binding of rSREC-I to proteins coated onto ELISA plates. (B) Effects of 10 or 100 μg/ml of indicated, soluble ligands on rSREC-I binding to plate-adsorbed AcLDL. (C) Binding of goat anti-mouse SREC-I Ab to untreated and LPS-pre-treated BM-DC. (D) Effects of polyclonal goat anti-mouse SREC-I Ab, relative to normal goat IgG, on the uptake of AF-AcLDL, AF-OVA-Cl and pHr-HSA-Cl by LPS-pre-treated, SR-A-deficient BM-DC. (E) Binding of rRAGE to plate-adsorbed proteins. (F) Effects of 10 or 100 μg/ml of indicated, soluble ligands on rRAGE binding to plate-adsorbed GA-BSA. The data were analysed by the Student’s t-test (A, D, E) or by ANOVA, followed by the Tukey-Kramer post-test (B, F). *, p < 0.05; NS, non-significant.

Mentions: Other receptors implicated in the binding of covalently-modified proteins include: SR SREC-I and FEEL-1/stabilin-1 as well as RAGE. From among these receptors, only RAGE was tested for the ability to bind HOCl-oxidised proteins and suggested to be a high-affinity receptor for HOCl-oxidised LDL and BSA, although, in our opinion, the claim concerning affinity of the observed binding was not substantiated [33,34]. When ligands were coated at 20 μg/ml, rSREC-I at 1 μg/ml bound strongly to adsorbed AcLDL and GA-BSA, used as positive controls, but no specific binding to HOCl-modified proteins was observed, except of very weak binding to TFN-Cl (S7A Fig). However, under conditions of increased concentrations of both coated proteins (40 μg/ml) and rSREC-I (1.5 μg/ml), a significant specific binding of rSREC-I to HOCl-modified proteins as well as OVA, but not to TFN or HSA, could be observed (Fig 9A). Low affinity of HOCl-modified proteins for rSREC-I has been confirmed in competition experiments. TFN-Cl and OVA-Cl produced weak, ~14% inhibition of rSREC-I binding to AcLDL only at the higher concentration of 0.1 mg/ml (Fig 9B). In contrast, rSREC-I binding was inhibited strongly and dose-dependently by DS and, less potently, by GA-BSA, but not by CS.


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)

Both SREC-I and RAGE bind HOCl-oxidised proteins with low affinities.(A) Binding of rSREC-I to proteins coated onto ELISA plates. (B) Effects of 10 or 100 μg/ml of indicated, soluble ligands on rSREC-I binding to plate-adsorbed AcLDL. (C) Binding of goat anti-mouse SREC-I Ab to untreated and LPS-pre-treated BM-DC. (D) Effects of polyclonal goat anti-mouse SREC-I Ab, relative to normal goat IgG, on the uptake of AF-AcLDL, AF-OVA-Cl and pHr-HSA-Cl by LPS-pre-treated, SR-A-deficient BM-DC. (E) Binding of rRAGE to plate-adsorbed proteins. (F) Effects of 10 or 100 μg/ml of indicated, soluble ligands on rRAGE binding to plate-adsorbed GA-BSA. The data were analysed by the Student’s t-test (A, D, E) or by ANOVA, followed by the Tukey-Kramer post-test (B, F). *, p < 0.05; NS, non-significant.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4388828&req=5

pone.0123293.g009: Both SREC-I and RAGE bind HOCl-oxidised proteins with low affinities.(A) Binding of rSREC-I to proteins coated onto ELISA plates. (B) Effects of 10 or 100 μg/ml of indicated, soluble ligands on rSREC-I binding to plate-adsorbed AcLDL. (C) Binding of goat anti-mouse SREC-I Ab to untreated and LPS-pre-treated BM-DC. (D) Effects of polyclonal goat anti-mouse SREC-I Ab, relative to normal goat IgG, on the uptake of AF-AcLDL, AF-OVA-Cl and pHr-HSA-Cl by LPS-pre-treated, SR-A-deficient BM-DC. (E) Binding of rRAGE to plate-adsorbed proteins. (F) Effects of 10 or 100 μg/ml of indicated, soluble ligands on rRAGE binding to plate-adsorbed GA-BSA. The data were analysed by the Student’s t-test (A, D, E) or by ANOVA, followed by the Tukey-Kramer post-test (B, F). *, p < 0.05; NS, non-significant.
Mentions: Other receptors implicated in the binding of covalently-modified proteins include: SR SREC-I and FEEL-1/stabilin-1 as well as RAGE. From among these receptors, only RAGE was tested for the ability to bind HOCl-oxidised proteins and suggested to be a high-affinity receptor for HOCl-oxidised LDL and BSA, although, in our opinion, the claim concerning affinity of the observed binding was not substantiated [33,34]. When ligands were coated at 20 μg/ml, rSREC-I at 1 μg/ml bound strongly to adsorbed AcLDL and GA-BSA, used as positive controls, but no specific binding to HOCl-modified proteins was observed, except of very weak binding to TFN-Cl (S7A Fig). However, under conditions of increased concentrations of both coated proteins (40 μg/ml) and rSREC-I (1.5 μg/ml), a significant specific binding of rSREC-I to HOCl-modified proteins as well as OVA, but not to TFN or HSA, could be observed (Fig 9A). Low affinity of HOCl-modified proteins for rSREC-I has been confirmed in competition experiments. TFN-Cl and OVA-Cl produced weak, ~14% inhibition of rSREC-I binding to AcLDL only at the higher concentration of 0.1 mg/ml (Fig 9B). In contrast, rSREC-I binding was inhibited strongly and dose-dependently by DS and, less potently, by GA-BSA, but not by CS.

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