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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

OVA-Cl exhibits increased immunogenicity that may be caused by enhanced uptake by APC.(A) BM-DC were incubated with indicated concentrations of OVA or OVA-Cl for 2 h at 37°C. When indicated, 200 ng/ml LPS was additionally included. Following washing, 0.4 × 105 BM-DC were co-cultured for 2 or 3 days with 1.5 × 105 CD4+ OT-II lymphocytes. One μCi of 3H-thymidine was added for the last 20 h of co-culture and radioactivity incorporated by proliferating lymphocytes was measured by scintillation counting. (B) BM-DC were incubated for 1 h at 37°C with indicated concentrations of Alexa Fluor 647-labelled OVA (AF-OVA) or OVA-Cl (AF-OVA-Cl) and, following washing, cell-associated fluorescence was measured by flow cytometry. (C) Unfractionated splenocytes, prepared from spleens of OT-II mice, we pre-incubated with OVA, OVA-Cl and LPS, as described in A, washed, plated at 2.5 × 105/well in 0.2 ml of fresh medium and cultured for 2 days, for assessing lymphocyte proliferation, or 3 days, for assessing IFN-γ level in culture medium by ELISA. Results shown on graphs A-C are averages ± SEM of 2 (B), 3 (A) or 4 (C) replicates, obtained in single experiments which were repeated at least 3 times with similar results. (D) Expression of MHC-II and co-stimulatory molecules on the surface of BM-DC as well as splenic DC and macrophages was determined by flow cytometry. When indicated, BM-DC were pre-incubated overnight with LPS. Specific binding was calculated by subtracting binding of PE-conjugated control mAb from the total binding of specific mAb. The results shown are averages ±SEM from 4–6 independent experiments.
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pone.0123293.g001: OVA-Cl exhibits increased immunogenicity that may be caused by enhanced uptake by APC.(A) BM-DC were incubated with indicated concentrations of OVA or OVA-Cl for 2 h at 37°C. When indicated, 200 ng/ml LPS was additionally included. Following washing, 0.4 × 105 BM-DC were co-cultured for 2 or 3 days with 1.5 × 105 CD4+ OT-II lymphocytes. One μCi of 3H-thymidine was added for the last 20 h of co-culture and radioactivity incorporated by proliferating lymphocytes was measured by scintillation counting. (B) BM-DC were incubated for 1 h at 37°C with indicated concentrations of Alexa Fluor 647-labelled OVA (AF-OVA) or OVA-Cl (AF-OVA-Cl) and, following washing, cell-associated fluorescence was measured by flow cytometry. (C) Unfractionated splenocytes, prepared from spleens of OT-II mice, we pre-incubated with OVA, OVA-Cl and LPS, as described in A, washed, plated at 2.5 × 105/well in 0.2 ml of fresh medium and cultured for 2 days, for assessing lymphocyte proliferation, or 3 days, for assessing IFN-γ level in culture medium by ELISA. Results shown on graphs A-C are averages ± SEM of 2 (B), 3 (A) or 4 (C) replicates, obtained in single experiments which were repeated at least 3 times with similar results. (D) Expression of MHC-II and co-stimulatory molecules on the surface of BM-DC as well as splenic DC and macrophages was determined by flow cytometry. When indicated, BM-DC were pre-incubated overnight with LPS. Specific binding was calculated by subtracting binding of PE-conjugated control mAb from the total binding of specific mAb. The results shown are averages ±SEM from 4–6 independent experiments.

Mentions: Previously, OVA-Cl has been shown to be presented effectively by BM-DC to memory Th lymphocytes [12], known to be more easily activated than naïve lymphocytes. We have found that BM-DC, pre-incubated for 2 h with OVA-Cl, are also able to stimulate proliferation of naïve Th lymphocytes (Fig 1A). In the presence of LPS, lymphocyte proliferation during 2 days of co-culture was stimulated by lower concentrations of OVA-Cl than OVA (Fig 1A, left panel). During 3-days co-culture the difference in the sensitivity of detection between OVA and OVA-Cl disappeared, but OVA-Cl stimulated more intense proliferation of CD4+ lymphocytes than OVA in the entire range of concentrations tested (Fig 1A, right panel). In contrast, in the absence of LPS, OVA-Cl was effectively presented to lymphocytes at a concentration as low as 5 μg/ml, whereas OVA did not stimulate lymphocyte proliferation even at 20 μg/ml (Fig 1A).


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)

OVA-Cl exhibits increased immunogenicity that may be caused by enhanced uptake by APC.(A) BM-DC were incubated with indicated concentrations of OVA or OVA-Cl for 2 h at 37°C. When indicated, 200 ng/ml LPS was additionally included. Following washing, 0.4 × 105 BM-DC were co-cultured for 2 or 3 days with 1.5 × 105 CD4+ OT-II lymphocytes. One μCi of 3H-thymidine was added for the last 20 h of co-culture and radioactivity incorporated by proliferating lymphocytes was measured by scintillation counting. (B) BM-DC were incubated for 1 h at 37°C with indicated concentrations of Alexa Fluor 647-labelled OVA (AF-OVA) or OVA-Cl (AF-OVA-Cl) and, following washing, cell-associated fluorescence was measured by flow cytometry. (C) Unfractionated splenocytes, prepared from spleens of OT-II mice, we pre-incubated with OVA, OVA-Cl and LPS, as described in A, washed, plated at 2.5 × 105/well in 0.2 ml of fresh medium and cultured for 2 days, for assessing lymphocyte proliferation, or 3 days, for assessing IFN-γ level in culture medium by ELISA. Results shown on graphs A-C are averages ± SEM of 2 (B), 3 (A) or 4 (C) replicates, obtained in single experiments which were repeated at least 3 times with similar results. (D) Expression of MHC-II and co-stimulatory molecules on the surface of BM-DC as well as splenic DC and macrophages was determined by flow cytometry. When indicated, BM-DC were pre-incubated overnight with LPS. Specific binding was calculated by subtracting binding of PE-conjugated control mAb from the total binding of specific mAb. The results shown are averages ±SEM from 4–6 independent experiments.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0123293.g001: OVA-Cl exhibits increased immunogenicity that may be caused by enhanced uptake by APC.(A) BM-DC were incubated with indicated concentrations of OVA or OVA-Cl for 2 h at 37°C. When indicated, 200 ng/ml LPS was additionally included. Following washing, 0.4 × 105 BM-DC were co-cultured for 2 or 3 days with 1.5 × 105 CD4+ OT-II lymphocytes. One μCi of 3H-thymidine was added for the last 20 h of co-culture and radioactivity incorporated by proliferating lymphocytes was measured by scintillation counting. (B) BM-DC were incubated for 1 h at 37°C with indicated concentrations of Alexa Fluor 647-labelled OVA (AF-OVA) or OVA-Cl (AF-OVA-Cl) and, following washing, cell-associated fluorescence was measured by flow cytometry. (C) Unfractionated splenocytes, prepared from spleens of OT-II mice, we pre-incubated with OVA, OVA-Cl and LPS, as described in A, washed, plated at 2.5 × 105/well in 0.2 ml of fresh medium and cultured for 2 days, for assessing lymphocyte proliferation, or 3 days, for assessing IFN-γ level in culture medium by ELISA. Results shown on graphs A-C are averages ± SEM of 2 (B), 3 (A) or 4 (C) replicates, obtained in single experiments which were repeated at least 3 times with similar results. (D) Expression of MHC-II and co-stimulatory molecules on the surface of BM-DC as well as splenic DC and macrophages was determined by flow cytometry. When indicated, BM-DC were pre-incubated overnight with LPS. Specific binding was calculated by subtracting binding of PE-conjugated control mAb from the total binding of specific mAb. The results shown are averages ±SEM from 4–6 independent experiments.
Mentions: Previously, OVA-Cl has been shown to be presented effectively by BM-DC to memory Th lymphocytes [12], known to be more easily activated than naïve lymphocytes. We have found that BM-DC, pre-incubated for 2 h with OVA-Cl, are also able to stimulate proliferation of naïve Th lymphocytes (Fig 1A). In the presence of LPS, lymphocyte proliferation during 2 days of co-culture was stimulated by lower concentrations of OVA-Cl than OVA (Fig 1A, left panel). During 3-days co-culture the difference in the sensitivity of detection between OVA and OVA-Cl disappeared, but OVA-Cl stimulated more intense proliferation of CD4+ lymphocytes than OVA in the entire range of concentrations tested (Fig 1A, right panel). In contrast, in the absence of LPS, OVA-Cl was effectively presented to lymphocytes at a concentration as low as 5 μg/ml, whereas OVA did not stimulate lymphocyte proliferation even at 20 μg/ml (Fig 1A).

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