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Scleroderma autoantigens are uniquely fragmented by metal-catalyzed oxidation reactions: implications for pathogenesis.

Casciola-Rosen L, Wigley F, Rosen A - J. Exp. Med. (1997)

Bottom Line: The observation that revelation of immunocryptic epitopes in self antigens may initiate the autoimmune response has prompted the search for processes which induce novel fragmentation of autoantigens as potential initiators of autoimmunity.We demonstrate that several of the autoantigens targeted in diffuse scleroderma are uniquely susceptible to cleavage by reactive oxygen species, in a metal-dependent manner.These data suggest that the autoantibody response in scleroderma is the immune marker of unique protein fragmentation, induced by ischemia reperfusion in the presence of appropriate metals, and focus attention on abnormal metal status as a potential pathogenic principle in this disease.

View Article: PubMed Central - PubMed

Affiliation: Department of Dermatology, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, USA.

ABSTRACT
The observation that revelation of immunocryptic epitopes in self antigens may initiate the autoimmune response has prompted the search for processes which induce novel fragmentation of autoantigens as potential initiators of autoimmunity. The reversible ischemia reperfusion which characterizes scleroderma has focused attention on reactive oxygen species as molecules which might induce autoantigen fragmentation. We demonstrate that several of the autoantigens targeted in diffuse scleroderma are uniquely susceptible to cleavage by reactive oxygen species, in a metal-dependent manner. Multiple features of the fragmentation reaction and its inhibition indicate that these autoantigens possess metal-binding sites, which focus metal-catalyzed oxidation reactions (and consequent fragmentation) to specific regions of the antigens. These data suggest that the autoantibody response in scleroderma is the immune marker of unique protein fragmentation, induced by ischemia reperfusion in the presence of appropriate metals, and focus attention on abnormal metal status as a potential pathogenic principle in this disease.

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Topoisomerase I is fragmented in intact keratinocytes chronically exposed to 20 μM Cu. Confluent monolayers of human foreskin keratinocytes were cultured for 18 h (lanes 3 and 4) or 2 h (lanes 5 and 6) in  keratinocyte growth medium supplemented with 20 μM CuSO4. Control cultures were maintained in the absence of added Cu (lanes 1 and 2).  Before harvesting the cells, 2 mM H2O2 was added to some of the cultures (lanes 2, 4, and 6), but not others (lanes 1 3, and 5), for 30 min.  Cells were subsequently harvested and immunoblotted with anti-topoisomerase I serum as described in Fig. 1. Equal amounts of protein were  electrophoresed in each lane. Results are representative of those obtained  in three separate experiments.
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Figure 4: Topoisomerase I is fragmented in intact keratinocytes chronically exposed to 20 μM Cu. Confluent monolayers of human foreskin keratinocytes were cultured for 18 h (lanes 3 and 4) or 2 h (lanes 5 and 6) in keratinocyte growth medium supplemented with 20 μM CuSO4. Control cultures were maintained in the absence of added Cu (lanes 1 and 2). Before harvesting the cells, 2 mM H2O2 was added to some of the cultures (lanes 2, 4, and 6), but not others (lanes 1 3, and 5), for 30 min. Cells were subsequently harvested and immunoblotted with anti-topoisomerase I serum as described in Fig. 1. Equal amounts of protein were electrophoresed in each lane. Results are representative of those obtained in three separate experiments.

Mentions: The absolute dependence of autoantigen fragmentation on exogenous Fe or Cu in cell lysates prompted us to address whether similar autoantigen fragmentation could be induced in intact cells chronically exposed to supraphysiologic concentrations of free Cu. Human foreskin keratinocytes were cultured for 18 h in vitro in defined, serum- and albumin-free growth medium supplemented with 20 μM CuSO4. Oxidation reactions were initiated by adding 2 mM H2O2 to some cultures for an additional 30 min. This choice of cell type, metal ion and concentration, and H2O2 dose were based on preliminary studies which demonstrated that (a) loading cells with Fe was not feasible, (b) serum-free medium greatly facilitates the reproducible uptake of Cu, and (c) 20 μM Cu (with H2O2) induces very little fragmentation in the in vitro lysate system (data not shown). Only intact topoisomerase I was detected in control keratinocytes (Fig. 4, lane 1), and H2O2 treatment alone did not result in any autoantigen fragmentation (Fig. 4, lane 2). Incubation with 20 μM Cu overnight did not affect cell morphology or viability (data not shown), and did not produce any fragmentation of topoisomerase I in the absence of added H2O2 (Fig. 4, lane 3). In contrast, addition of H2O2 after overnight Cu loading resulted in the marked fragmentation of topoisomerase I into the predominant Cu-characteristic band of 95 kD (Fig. 4, lane 4). Incubation of cells in medium containing 20 μM Cu for 2 h generated no topoisomerase I fragments in the presence or absence of H2O2 (Fig. 4, lanes 5 and 6), clearly demonstrating that overnight preincubation of cells with Cu sensitizes cells for H2O2-induced fragmentation of topoisomerase I. Washing away the Cu-containing medium before adding H2O2 had little effect on the extent or characteristics of fragmentation (data not shown), indicating that the Cu effect was cell associated. Since addition of 20 μM Cu and H2O2 fails to induce autoantigen fragmentation in cell lysates, the fragmentation observed in the intact cells preincubated with this Cu concentration strongly implies that higher localized concentrations of Cu are generated in these cells, which facilitate binding of Cu to the relevant autoantigens.


Scleroderma autoantigens are uniquely fragmented by metal-catalyzed oxidation reactions: implications for pathogenesis.

Casciola-Rosen L, Wigley F, Rosen A - J. Exp. Med. (1997)

Topoisomerase I is fragmented in intact keratinocytes chronically exposed to 20 μM Cu. Confluent monolayers of human foreskin keratinocytes were cultured for 18 h (lanes 3 and 4) or 2 h (lanes 5 and 6) in  keratinocyte growth medium supplemented with 20 μM CuSO4. Control cultures were maintained in the absence of added Cu (lanes 1 and 2).  Before harvesting the cells, 2 mM H2O2 was added to some of the cultures (lanes 2, 4, and 6), but not others (lanes 1 3, and 5), for 30 min.  Cells were subsequently harvested and immunoblotted with anti-topoisomerase I serum as described in Fig. 1. Equal amounts of protein were  electrophoresed in each lane. Results are representative of those obtained  in three separate experiments.
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Related In: Results  -  Collection

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Figure 4: Topoisomerase I is fragmented in intact keratinocytes chronically exposed to 20 μM Cu. Confluent monolayers of human foreskin keratinocytes were cultured for 18 h (lanes 3 and 4) or 2 h (lanes 5 and 6) in keratinocyte growth medium supplemented with 20 μM CuSO4. Control cultures were maintained in the absence of added Cu (lanes 1 and 2). Before harvesting the cells, 2 mM H2O2 was added to some of the cultures (lanes 2, 4, and 6), but not others (lanes 1 3, and 5), for 30 min. Cells were subsequently harvested and immunoblotted with anti-topoisomerase I serum as described in Fig. 1. Equal amounts of protein were electrophoresed in each lane. Results are representative of those obtained in three separate experiments.
Mentions: The absolute dependence of autoantigen fragmentation on exogenous Fe or Cu in cell lysates prompted us to address whether similar autoantigen fragmentation could be induced in intact cells chronically exposed to supraphysiologic concentrations of free Cu. Human foreskin keratinocytes were cultured for 18 h in vitro in defined, serum- and albumin-free growth medium supplemented with 20 μM CuSO4. Oxidation reactions were initiated by adding 2 mM H2O2 to some cultures for an additional 30 min. This choice of cell type, metal ion and concentration, and H2O2 dose were based on preliminary studies which demonstrated that (a) loading cells with Fe was not feasible, (b) serum-free medium greatly facilitates the reproducible uptake of Cu, and (c) 20 μM Cu (with H2O2) induces very little fragmentation in the in vitro lysate system (data not shown). Only intact topoisomerase I was detected in control keratinocytes (Fig. 4, lane 1), and H2O2 treatment alone did not result in any autoantigen fragmentation (Fig. 4, lane 2). Incubation with 20 μM Cu overnight did not affect cell morphology or viability (data not shown), and did not produce any fragmentation of topoisomerase I in the absence of added H2O2 (Fig. 4, lane 3). In contrast, addition of H2O2 after overnight Cu loading resulted in the marked fragmentation of topoisomerase I into the predominant Cu-characteristic band of 95 kD (Fig. 4, lane 4). Incubation of cells in medium containing 20 μM Cu for 2 h generated no topoisomerase I fragments in the presence or absence of H2O2 (Fig. 4, lanes 5 and 6), clearly demonstrating that overnight preincubation of cells with Cu sensitizes cells for H2O2-induced fragmentation of topoisomerase I. Washing away the Cu-containing medium before adding H2O2 had little effect on the extent or characteristics of fragmentation (data not shown), indicating that the Cu effect was cell associated. Since addition of 20 μM Cu and H2O2 fails to induce autoantigen fragmentation in cell lysates, the fragmentation observed in the intact cells preincubated with this Cu concentration strongly implies that higher localized concentrations of Cu are generated in these cells, which facilitate binding of Cu to the relevant autoantigens.

Bottom Line: The observation that revelation of immunocryptic epitopes in self antigens may initiate the autoimmune response has prompted the search for processes which induce novel fragmentation of autoantigens as potential initiators of autoimmunity.We demonstrate that several of the autoantigens targeted in diffuse scleroderma are uniquely susceptible to cleavage by reactive oxygen species, in a metal-dependent manner.These data suggest that the autoantibody response in scleroderma is the immune marker of unique protein fragmentation, induced by ischemia reperfusion in the presence of appropriate metals, and focus attention on abnormal metal status as a potential pathogenic principle in this disease.

View Article: PubMed Central - PubMed

Affiliation: Department of Dermatology, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, USA.

ABSTRACT
The observation that revelation of immunocryptic epitopes in self antigens may initiate the autoimmune response has prompted the search for processes which induce novel fragmentation of autoantigens as potential initiators of autoimmunity. The reversible ischemia reperfusion which characterizes scleroderma has focused attention on reactive oxygen species as molecules which might induce autoantigen fragmentation. We demonstrate that several of the autoantigens targeted in diffuse scleroderma are uniquely susceptible to cleavage by reactive oxygen species, in a metal-dependent manner. Multiple features of the fragmentation reaction and its inhibition indicate that these autoantigens possess metal-binding sites, which focus metal-catalyzed oxidation reactions (and consequent fragmentation) to specific regions of the antigens. These data suggest that the autoantibody response in scleroderma is the immune marker of unique protein fragmentation, induced by ischemia reperfusion in the presence of appropriate metals, and focus attention on abnormal metal status as a potential pathogenic principle in this disease.

Show MeSH
Related in: MedlinePlus