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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

LOX-1 is capable of binding HOCl-modified proteins, but does not contribute to OVA-Cl uptake by BM-DC.(A) Binding of rLOX-1 to plate-adsorbed proteins. (B, C) Effects of 10 or 100 μg/ml of indicated, soluble ligands on rLOX-1 binding to plate-adsorbed OVA-Cl (B) or GA-BSA (C). (D) Binding of PE-conjugated anti-mouse LOX-1 mAb and control rat IgG2a to CBA BM-DC, determined by flow cytometry. (E) The effect of blocking goat anti-mouse LOX-1 polyclonal Ab, relative to normal goat IgG, on AF-OVA-Cl uptake by untreated and LPS-pre-treated CBA BM-DC. (F) LOX-1 expression on LPS-pre-treated CBA BM-DC. Results of single experiments are shown, repeated at least twice with similar results. The data were analysed by the Student’s t-test (A, E) or by ANOVA, followed by the Tukey-Kramer post-test (B, C). *, p < 0.05; NS, non-significant.
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pone.0123293.g008: LOX-1 is capable of binding HOCl-modified proteins, but does not contribute to OVA-Cl uptake by BM-DC.(A) Binding of rLOX-1 to plate-adsorbed proteins. (B, C) Effects of 10 or 100 μg/ml of indicated, soluble ligands on rLOX-1 binding to plate-adsorbed OVA-Cl (B) or GA-BSA (C). (D) Binding of PE-conjugated anti-mouse LOX-1 mAb and control rat IgG2a to CBA BM-DC, determined by flow cytometry. (E) The effect of blocking goat anti-mouse LOX-1 polyclonal Ab, relative to normal goat IgG, on AF-OVA-Cl uptake by untreated and LPS-pre-treated CBA BM-DC. (F) LOX-1 expression on LPS-pre-treated CBA BM-DC. Results of single experiments are shown, repeated at least twice with similar results. The data were analysed by the Student’s t-test (A, E) or by ANOVA, followed by the Tukey-Kramer post-test (B, C). *, p < 0.05; NS, non-significant.

Mentions: The role of LOX-1 as a receptor for HOCl-modified proteins has been suggested by a recent report demonstrating increased uptake of fluorescein-labelled OVA-Cl by CHO cells upon transfection with LOX-1 [12]. As shown in Fig 8A, rLOX-1 bound strongly to GA-BSA, used as a positive control, and only slightly less strongly to TFN-Cl. In comparison, the binding to OVA-Cl was weak, even weaker to OVA and no specific binding of rLOX-1 to BSA or TFN was observed. Binding of rLOX-1 to OVA-Cl was almost completely blocked by DS already at 10 μg/ml (Fig 8B). In contrast, CS and mannan had no effect on rLOX-1 binding to OVA-Cl, indicating that rLOX-1 preserves the ligand binding specificity of natural LOX-1. Results of additional competition experiments have confirmed low affinity of OVA and OVA-Cl for rLOX-1; whereas TFN-Cl dose-dependently inhibited rLOX-1 binding to GA-BSA, no inhibition was produced by OVA or OVA-Cl even at 0.1 mg/ml (Fig 8C). HOCl-modified lipoproteins, shown to bind to human LOX-1, were oxidised with higher concentrations of HOCl [32] than our OVA-Cl preparation. We therefore prepared a more heavily HOCl-oxidised OVA-Cl by incubating OVA with 6 mM instead of 3 mM NaOCl (OVA-ClH), and assessed its binding to rLOX-1. OVA-ClH turned out to be a higher affinity ligand of LOX-1 than OVA-Cl, inhibiting rLOX-1 binding with a similar potency as DS (Fig 8C). We then assessed involvement of LOX-1 in OVA-Cl uptake by BM-DC. LOX-1 was expressed on ~10–13% BM-DC generated from C57BL/6 or CBA mice (Fig 8D). LOX-1-positive BM-DC were among cells exhibiting the highest level of AF-OVA-Cl uptake (S6A Fig). However, blocking anti-LOX-1 Ab had no effect on AF-OVA-Cl uptake by BM-DC of either strain (Fig 8E). As the apparent lack of LOX-1 involvement in OVA-Cl uptake by untreated BM-DC might be caused by the lack of LOX-1 expression on the majority of cells, we up-regulated LOX-1 expression on BM-DC by pre-incubation with LPS. The LPS pre-treatment increased the average expression level of LOX-1 by as much as 3.4–4.3-fold, and the percentage of LOX-1-expressing BM-DC to ~39%, in case of C57BL/6 cells (S6B Fig), and to ~60%, in case of CBA cells (Fig 8F). However, also in LPS-pre-treated CBA BM-DC blocking anti-LOX-1 Ab had no effect on AF-OVA-Cl uptake (Fig 8E). Collectively, these results indicate that LOX-1 does not function as a receptor for OVA-Cl.


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)

LOX-1 is capable of binding HOCl-modified proteins, but does not contribute to OVA-Cl uptake by BM-DC.(A) Binding of rLOX-1 to plate-adsorbed proteins. (B, C) Effects of 10 or 100 μg/ml of indicated, soluble ligands on rLOX-1 binding to plate-adsorbed OVA-Cl (B) or GA-BSA (C). (D) Binding of PE-conjugated anti-mouse LOX-1 mAb and control rat IgG2a to CBA BM-DC, determined by flow cytometry. (E) The effect of blocking goat anti-mouse LOX-1 polyclonal Ab, relative to normal goat IgG, on AF-OVA-Cl uptake by untreated and LPS-pre-treated CBA BM-DC. (F) LOX-1 expression on LPS-pre-treated CBA BM-DC. Results of single experiments are shown, repeated at least twice with similar results. The data were analysed by the Student’s t-test (A, E) or by ANOVA, followed by the Tukey-Kramer post-test (B, C). *, p < 0.05; NS, non-significant.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4388828&req=5

pone.0123293.g008: LOX-1 is capable of binding HOCl-modified proteins, but does not contribute to OVA-Cl uptake by BM-DC.(A) Binding of rLOX-1 to plate-adsorbed proteins. (B, C) Effects of 10 or 100 μg/ml of indicated, soluble ligands on rLOX-1 binding to plate-adsorbed OVA-Cl (B) or GA-BSA (C). (D) Binding of PE-conjugated anti-mouse LOX-1 mAb and control rat IgG2a to CBA BM-DC, determined by flow cytometry. (E) The effect of blocking goat anti-mouse LOX-1 polyclonal Ab, relative to normal goat IgG, on AF-OVA-Cl uptake by untreated and LPS-pre-treated CBA BM-DC. (F) LOX-1 expression on LPS-pre-treated CBA BM-DC. Results of single experiments are shown, repeated at least twice with similar results. The data were analysed by the Student’s t-test (A, E) or by ANOVA, followed by the Tukey-Kramer post-test (B, C). *, p < 0.05; NS, non-significant.
Mentions: The role of LOX-1 as a receptor for HOCl-modified proteins has been suggested by a recent report demonstrating increased uptake of fluorescein-labelled OVA-Cl by CHO cells upon transfection with LOX-1 [12]. As shown in Fig 8A, rLOX-1 bound strongly to GA-BSA, used as a positive control, and only slightly less strongly to TFN-Cl. In comparison, the binding to OVA-Cl was weak, even weaker to OVA and no specific binding of rLOX-1 to BSA or TFN was observed. Binding of rLOX-1 to OVA-Cl was almost completely blocked by DS already at 10 μg/ml (Fig 8B). In contrast, CS and mannan had no effect on rLOX-1 binding to OVA-Cl, indicating that rLOX-1 preserves the ligand binding specificity of natural LOX-1. Results of additional competition experiments have confirmed low affinity of OVA and OVA-Cl for rLOX-1; whereas TFN-Cl dose-dependently inhibited rLOX-1 binding to GA-BSA, no inhibition was produced by OVA or OVA-Cl even at 0.1 mg/ml (Fig 8C). HOCl-modified lipoproteins, shown to bind to human LOX-1, were oxidised with higher concentrations of HOCl [32] than our OVA-Cl preparation. We therefore prepared a more heavily HOCl-oxidised OVA-Cl by incubating OVA with 6 mM instead of 3 mM NaOCl (OVA-ClH), and assessed its binding to rLOX-1. OVA-ClH turned out to be a higher affinity ligand of LOX-1 than OVA-Cl, inhibiting rLOX-1 binding with a similar potency as DS (Fig 8C). We then assessed involvement of LOX-1 in OVA-Cl uptake by BM-DC. LOX-1 was expressed on ~10–13% BM-DC generated from C57BL/6 or CBA mice (Fig 8D). LOX-1-positive BM-DC were among cells exhibiting the highest level of AF-OVA-Cl uptake (S6A Fig). However, blocking anti-LOX-1 Ab had no effect on AF-OVA-Cl uptake by BM-DC of either strain (Fig 8E). As the apparent lack of LOX-1 involvement in OVA-Cl uptake by untreated BM-DC might be caused by the lack of LOX-1 expression on the majority of cells, we up-regulated LOX-1 expression on BM-DC by pre-incubation with LPS. The LPS pre-treatment increased the average expression level of LOX-1 by as much as 3.4–4.3-fold, and the percentage of LOX-1-expressing BM-DC to ~39%, in case of C57BL/6 cells (S6B Fig), and to ~60%, in case of CBA cells (Fig 8F). However, also in LPS-pre-treated CBA BM-DC blocking anti-LOX-1 Ab had no effect on AF-OVA-Cl uptake (Fig 8E). Collectively, these results indicate that LOX-1 does not function as a receptor for OVA-Cl.

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