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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

The role of MR as a receptor for HOCl-modified proteins.(A) Geometric mean fluorescence intensity of anti-MR mAb and isotype-matched control rat IgG2a binding to BM-DC, splenic DC and PEM. The graph on the right shows a representative histogram for BM-DC. (B) Effects of mannan (Man), DS and CS on AF-OVA-Cl uptake by J774 cells. (C) Binding of rMR to plate-adsorbed proteins. (D) Effects of 10 or 100 μg/ml of indicated, soluble ligands on rMR binding to plate-adsorbed TFN-Cl. (E) Effects of agents selectively blocking C-type lectin domains (EDTA, anti-MR mAb) or the cysteine-rich domain (CS) on rMR binding to plate-adsorbed OVA or OVA-Cl. (F) Uptake of indicated, fluorescently-labelled ligands by WT and MR-/- BM-DC. (G, H) IL-2 production in the co-culture of CD4+ OT-II lymphocytes with WT or MR-/- BM-DC pulsed for 3.5 h with 20 μg/ml OVA, 7 μg/ml OVA-Cl (G) or OVA-Cl + 200 ng/ml LPS (H). The results shown are averages +SEM from 4 independent experiments (A, F-H) or mean values +SEM obtained in single experiments, repeated 2–3 times with similar results (B-E). The data were analysed by the Student’s t-test (C, F-H) or by ANOVA, followed by the Tukey-Kramer post-test (B, D, E). *, p < 0.05;!, a statistically significant additive effect of two ligands; NS, non-significant.
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pone.0123293.g010: The role of MR as a receptor for HOCl-modified proteins.(A) Geometric mean fluorescence intensity of anti-MR mAb and isotype-matched control rat IgG2a binding to BM-DC, splenic DC and PEM. The graph on the right shows a representative histogram for BM-DC. (B) Effects of mannan (Man), DS and CS on AF-OVA-Cl uptake by J774 cells. (C) Binding of rMR to plate-adsorbed proteins. (D) Effects of 10 or 100 μg/ml of indicated, soluble ligands on rMR binding to plate-adsorbed TFN-Cl. (E) Effects of agents selectively blocking C-type lectin domains (EDTA, anti-MR mAb) or the cysteine-rich domain (CS) on rMR binding to plate-adsorbed OVA or OVA-Cl. (F) Uptake of indicated, fluorescently-labelled ligands by WT and MR-/- BM-DC. (G, H) IL-2 production in the co-culture of CD4+ OT-II lymphocytes with WT or MR-/- BM-DC pulsed for 3.5 h with 20 μg/ml OVA, 7 μg/ml OVA-Cl (G) or OVA-Cl + 200 ng/ml LPS (H). The results shown are averages +SEM from 4 independent experiments (A, F-H) or mean values +SEM obtained in single experiments, repeated 2–3 times with similar results (B-E). The data were analysed by the Student’s t-test (C, F-H) or by ANOVA, followed by the Tukey-Kramer post-test (B, D, E). *, p < 0.05;!, a statistically significant additive effect of two ligands; NS, non-significant.

Mentions: The above results indicate that SR-A and CD36 are the receptors responsible for the portion of chlorinated proteins uptake which is inhibited by DS, but not by mannan. The best candidate for the receptor mediating the fraction of HOCl-modified proteins uptake which is inhibited by both DS and mannan was the mannose receptor (MR/CD206). First, MR was expressed on BM-DC (Fig 10A). A small parallel shift of the histogram representing cells labelled with anti-MR mAb relative to that depicting cells incubated with an isotype control, indicates that the majority of BM-DC express MR at low level. Splenic DC expressed ~2-times more MR than BM-DC, whereas the expression level of MR on PEM was intermediate (Fig 10A). Second, OVA is a well-established ligand of MR [36], whereas the occurrence of mutual cross-competition indicates that OVA-Cl binds to the same receptors on BM-DC as OVA (Fig 5A). Third, in J774 macrophage-like cells, which express several endocytic receptors at high level, including SR-A [37] and CD36 [17], but do not express MR [38], mannan had no effect on OVA-Cl uptake (Fig 10B). Finally, although mannan is also a ligand of other receptors, MR is the only known receptor that in addition to mannan also binds sulphated saccharides [26].


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)

The role of MR as a receptor for HOCl-modified proteins.(A) Geometric mean fluorescence intensity of anti-MR mAb and isotype-matched control rat IgG2a binding to BM-DC, splenic DC and PEM. The graph on the right shows a representative histogram for BM-DC. (B) Effects of mannan (Man), DS and CS on AF-OVA-Cl uptake by J774 cells. (C) Binding of rMR to plate-adsorbed proteins. (D) Effects of 10 or 100 μg/ml of indicated, soluble ligands on rMR binding to plate-adsorbed TFN-Cl. (E) Effects of agents selectively blocking C-type lectin domains (EDTA, anti-MR mAb) or the cysteine-rich domain (CS) on rMR binding to plate-adsorbed OVA or OVA-Cl. (F) Uptake of indicated, fluorescently-labelled ligands by WT and MR-/- BM-DC. (G, H) IL-2 production in the co-culture of CD4+ OT-II lymphocytes with WT or MR-/- BM-DC pulsed for 3.5 h with 20 μg/ml OVA, 7 μg/ml OVA-Cl (G) or OVA-Cl + 200 ng/ml LPS (H). The results shown are averages +SEM from 4 independent experiments (A, F-H) or mean values +SEM obtained in single experiments, repeated 2–3 times with similar results (B-E). The data were analysed by the Student’s t-test (C, F-H) or by ANOVA, followed by the Tukey-Kramer post-test (B, D, E). *, p < 0.05;!, a statistically significant additive effect of two ligands; NS, non-significant.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0123293.g010: The role of MR as a receptor for HOCl-modified proteins.(A) Geometric mean fluorescence intensity of anti-MR mAb and isotype-matched control rat IgG2a binding to BM-DC, splenic DC and PEM. The graph on the right shows a representative histogram for BM-DC. (B) Effects of mannan (Man), DS and CS on AF-OVA-Cl uptake by J774 cells. (C) Binding of rMR to plate-adsorbed proteins. (D) Effects of 10 or 100 μg/ml of indicated, soluble ligands on rMR binding to plate-adsorbed TFN-Cl. (E) Effects of agents selectively blocking C-type lectin domains (EDTA, anti-MR mAb) or the cysteine-rich domain (CS) on rMR binding to plate-adsorbed OVA or OVA-Cl. (F) Uptake of indicated, fluorescently-labelled ligands by WT and MR-/- BM-DC. (G, H) IL-2 production in the co-culture of CD4+ OT-II lymphocytes with WT or MR-/- BM-DC pulsed for 3.5 h with 20 μg/ml OVA, 7 μg/ml OVA-Cl (G) or OVA-Cl + 200 ng/ml LPS (H). The results shown are averages +SEM from 4 independent experiments (A, F-H) or mean values +SEM obtained in single experiments, repeated 2–3 times with similar results (B-E). The data were analysed by the Student’s t-test (C, F-H) or by ANOVA, followed by the Tukey-Kramer post-test (B, D, E). *, p < 0.05;!, a statistically significant additive effect of two ligands; NS, non-significant.
Mentions: The above results indicate that SR-A and CD36 are the receptors responsible for the portion of chlorinated proteins uptake which is inhibited by DS, but not by mannan. The best candidate for the receptor mediating the fraction of HOCl-modified proteins uptake which is inhibited by both DS and mannan was the mannose receptor (MR/CD206). First, MR was expressed on BM-DC (Fig 10A). A small parallel shift of the histogram representing cells labelled with anti-MR mAb relative to that depicting cells incubated with an isotype control, indicates that the majority of BM-DC express MR at low level. Splenic DC expressed ~2-times more MR than BM-DC, whereas the expression level of MR on PEM was intermediate (Fig 10A). Second, OVA is a well-established ligand of MR [36], whereas the occurrence of mutual cross-competition indicates that OVA-Cl binds to the same receptors on BM-DC as OVA (Fig 5A). Third, in J774 macrophage-like cells, which express several endocytic receptors at high level, including SR-A [37] and CD36 [17], but do not express MR [38], mannan had no effect on OVA-Cl uptake (Fig 10B). Finally, although mannan is also a ligand of other receptors, MR is the only known receptor that in addition to mannan also binds sulphated saccharides [26].

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